A Short History of Nutritional Sciences, Carpenter K.J. (2003)

History of Nutrition
A Short History of Nutritional Science: Part 1 (17851885)

Kenneth J. Carpenter1
Department of Nutritional Sciences, University of California, Berkeley, CA 97420-3104
This is the first of four invited articles planned to provide a

short introduction to the history of our science and a possible
text for courses in the subject. Given the space limitations, I
have concentrated on work most directly related to discovering nutritional needs and the qualities of foods in supplying
them. Our science has greatly relied on developments in
analytical chemistry and general physiology, but there are
already histories that cover these subjects.
It would have been possible to give brief references to more
names and papers, but it would, I believe, have made for more
tedious reading. I have preferred to select topics that were
breaking new ground, and seemed to inspire other work. No
two authors would make the same choices in this situation.
I have also tried to portray the problems as they were seen
by workers at the time, and to follow a chronological course,
without referring prematurely to modern explanations of phenomena. In most instances the original historical reference to
a paper is given, but it is often supplemented with a more
easily available review of the subject that also contains additional references. Where a quotation comes from a book or a
long article, the Editors have given special permission for the
exact page(s) on which it occurs to be listed.
Before 1785 many scholars had published their ideas about
how the food we ate was used in our bodies, but it was only
with the so-called Chemical revolution in France at the end
of the eighteenth century, with its identification of the main
elements and the development of methods of chemical analysis, that old and new ideas began to be tested in a quantitative, scientific way. There is one exception to this generalization that we will return to later. It is understandable that
modern workers should have little knowledge of the work of
the late eighteenth century scientists who carried out this
revolution, and therefore little appreciation of its quality.
But we should remember that they were the leaders, and ahead
of us in time in making the first inroads into what has been
called the dark forest of animal chemistry.
Take, for example, the finding with important implications
that was reported to the French Academy of Sciences in 1785
by Claude Berthollet. He had found that the vapor that came
from decomposing animal matter was ammonia, and that this
gas was composed of three volumes of hydrogen and one
volume of nitrogen, or around 17% hydrogen and 83% nitrogen by weight, for which the modern values are 17.75 and
1
To whom correspondence should be addressed.
E-mail: kcarp@uclink.berkeley.edu
0022-3166/03 $3.00 2003 American Society for Nutritional Sciences. J. Nutr. 133: 638 645, 2003.
Manuscript received 18 December 2002. Revision accepted 27 December 2002.
638
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82.25%, respectively (1). This was impressive work and one

wonders how many of todays researchers would be able to
repeat this finding, especially if they could use only the equipment available at the time.
Others confirmed the presence of nitrogen in animal matter
and its absence from sugars, starch and fats. It had been
realized for some time that wheat flour contained a fraction
(that we know as gluten) that seemed to have the properties of
animal matter, including the evolution of alkaline vapor when
a sample was allowed to rot. It had been a matter of debate as
to whether this was what made wheat such a good food, and
whether the more newly introduced potatoes, which seemed to
contain nothing comparable to gluten, could be considered to
be an adequate substitute for wheat (2).
Many of the chemists involved in the Chemical revolution in France, including its most famous member Antoine
Lavoisier, also had an interest in metabolism. In collaboration
with his assistant Armand Seguin, he measured human respiratory output of carbonic acid (that we now know as carbon
dioxide), both at rest and when lifting weights, and showed
how it increased with activity (3,4) (Fig. 1). This, in itself, was
an important advance because it had previously been supposed
that the sole purpose of respiration was the cooling of the
heart, and that the bodily balance of adults required that the
weight of ingested material that was not recovered in stools or
urine must have been lost through insensible perspiration.
Lavoisier also collaborated with the mathematician PierreSimon Laplace in comparing the heat produced by the guinea
pig with its production of carbon dioxide, and comparing those
results with the heat produced by a lighted candle or charcoal.
Heat production was measured in an ice calorimeter, in which
the heat evolved was related to the weight of water released
from the melting of the ice surrounding the inner chamber
where the animal or burning material was housed (5,6). Although not precise, the results were consistent, they believed,
with at least most of the animal heat coming from slow
combustion of organic compounds within the guinea pigs
tissues. The further progress in calorimetry will be discussed in
Part 2.
Lavoisier had returned to further studies on respiration
when he was arrested in 1793 during the Reign of Terror and
kept in prison. On the day of his trial in 1794 he pleaded for
a short stay of execution that would allow him to do one more
experiment, but the judge is believed to have replied that the
Republic had no need of savants, and he was guillotined the
same afternoon.
In addition to the scientific progress during the time of the
French revolution, there also seemed to be a new spirit at
work, a feeling that it was a time to begin again with none of
Preface
HISTORY OF NUTRITIONAL SCIENCE (17851885)
639
FIGURE 1 Schematic drawing by

Mme. Lavoisier of her husband measuring the carbonic acid output of his collaborator Armand Seguin, while she
noted down the results. (Wellcome Institute, London)
Everyone knows that dogs can live very well on bread alone,
but later, when he actually put this to the test he found that
a dog does not live above fifty days. His final conclusion, still
echoed in present-day dietary guidelines, was that diversity
and multiplicity of aliments is an important rule of hygiene;
which is, moreover, indicated to us by our instincts (9).
At this time there was a controversy as to whether gelatin,
obtained by boiling bones, and which was nitrogen-rich, could
be used as an economical substitute for meat in French hospitals. Magendie was asked by the Academy of Sciences to
carry out further trials to investigate the question. After 10 y
of research, which yielded apparently paradoxical results, he
had to report that: As so often in research, unexpected results
had contradicted every reasonable expectation. It was clear
that gelatin was not a complete food for dogs, but neither was
meat after it had been extracted with water. He suggested that
chemists investigate what essential material it was that was
leached out of meat: It could perhaps be iron or other salts,
fatty material or lactic acid (10). In fact, there was to be a gap
of another 75 y before this type of question began to be
re-explored in the United States by E. V. McCollum, using the
young rat as a more convenient model.
An important, unmentioned assumption behind Magendies work was that an animal species could be used as a model
for humans; in other words, that our bodies were essentially of
the same general character as those of animals. This may have
arisen, at least in part, as a result of an interest in France for
studies in comparative anatomy.
Another active investigator in France in the 1830s, with a
quite different background from that of Magendie, was also
studying the source of an animals nitrogen-rich tissues. This
was Jean Baptiste Boussingault, who had learned his chemistry
in a school for mining engineers. After a period of adventurous
geological exploration in South America, he returned, married
a farm owners daughter and put his mind to agricultural
science. He obtained a position at the Sorbonne in Paris,
where he collaborated with J. B. Dumas, one of the leading
French chemists, and divided his year between Paris and the
farm (11).
Working first with plant crops, he was able to show that
leguminous plants, but not cereal grains, were able to utilize
atmospheric nitrogen during growth. He then turned to cows
the old assumptions being taken for granted. The period certainly marked a new beginning for nutritional science, and the
chemical revolution had provided the necessary tools for its
development. A young French pioneer commented that: Nutrition has often been the subject of conjectures and ingenious
hypotheses but our actual knowledge is so insufficient that
their only use is to try to satisfy our imagination. If we could
arrive at some more exact facts they could well have applications in medicine.
The writer was Franc ois Magendie, who had grown up in
revolutionary Paris and practiced as a surgeon before changing
to physiology (7). His first work in the field was reported to the
Academy of Sciences in 1816, and addressed directly the
question as to whether animals could use atmospheric nitrogen
to animalize ingested foods of low nitrogen content. There
was, of course, a plentiful supply of nitrogen in the air, and
some chemists had suggested that this kind of combination
must occur during an animals digestion of plant foods so as to
give the ingesta the characteristics that would allow them to
be incorporated into the animals own tissues either for growth
or replacement of worn-out materials.
Magendies famous experiment was a very simple one, so
simple that one wonders at its never having been tried before.
It was to take a single food that was accepted as being nutritious, even though it did not contain nitrogen, and to feed it
to dogs, a species that would eat both plant and animal foods.
Sugar was the food that he tested with his first dog. It continued to eat well for about 2 wk, but then began to lose weight
and to develop a corneal ulcer. After a month it died. He
repeated the experiment, and then tried using olive oil, gum or
butter as the sole foods for his dogs, in each case with the same
result, except that no ulceration was seen in the dog receiving
olive oil (8).
His conclusions were that none of these foods was preeminently nutritive (which I take to mean providing all the
dogs needs), even though they were well absorbed, and,
second, that at least the majority of the nitrogen in a dogs
tissue must come from the food that it has consumed. With
hindsight, we can see the gap in his reasoning; there may have
been other deficiencies in the foods tested apart from nitrogenous material, and he had no positive control, such as sugar
plus albumin or gluten. In his 1816 paper he had written:
CARPENTER
640
TABLE 1
Summarized results from a pioneering balance trial,
comparing the daily nitrogen intake of a dairy cow from
its feed with that of its output in milk and excreta,
as reported by Boussingault in 18391
Input
Hay, 7.5 kg
Potatoes, 15 kg
Total in
Output
Milk, 8.54 kg
Urine, 8.20 kg
Dung, 8.42 kg
Dry weight
Nitrogen
Nitrogen
kg
6.31
4.17
2.4
1.2
151
50
201
1.15
0.97
4.00
Total out
46
37
92
175
26
1 Ref. 12.
and horses, whose common feeds had the reputation of being

exceptionally low in nitrogen. His approach was first to find
the level of feeding that kept his animals at constant weight,
and then for 3 d to record the animals feed, excreta and, in the
case of the cow, its milk, and also to analyze all these for their
nitrogen content. With the horse, receiving altogether some
8.5 kg hay and oats per 24 h, the daily nitrogen intake was
139 g, and the nitrogen recovered in urine and dung came to
only 116 g. The cow, fed on hay and potatoes, had a daily
intake of 201 g nitrogen and the recovered output, including
46 g from milk, was only 175 g (Table 1). He concluded that
the animals feed provided sufficient nitrogen to meet their
needs and that there was no need to hypothesize that they had
to obtain nitrogen from the atmosphere (12,13).
These seem to have been the first of the many thousands of
balance trials that would continue to be carried out until the
present day. Unfortunately, the only method of analysis for
nitrogen that had been developed at that time required him
first to dry his samples, which could be expected to result in
loss of ammonia when he was drying urine and dung. This
could explain the apparent positive balance in these animals
that were assumed to be in a steady state.
Why the concentration on nitrogen?
Even before carrying out his balance experiments with
herbivores, Boussingault had proposed that the relative nutritional values of plant foods could be assessed from their contents of nitrogen (14). His justification for this went roughly as
follows: Magendie has shown that foods that do not contain
nitrogen cannot continue to support life, therefore the nutritional value of a vegetable substance resides principally in the
gluten and vegetable albumin that it contains. Investigators
at this time certainly knew that animal bodies also contained
minerals that they must have obtained from their food. Even
earlier, two workers had written that: Beans are so nourishing
because they contain starch, an animal matter, phosphate,
lime, magnesia, potash and iron. They yield at once the
aliments and the materials proper to form and color the blood
and to nourish the bones (15). Perhaps in response to such
Synthesis only by plants

In light of the results considered above, Boussingaults
colleague, the chemist Dumas, concluded that the plant kingdom alone was capable of synthesizing the kinds of nitrogenous
compounds abundant in animal tissues. Then, from the observation that the overall reactions of animals were characterized
by oxidation, he made the further generalization that the
animal kingdom was only capable of oxidizing the materials
that it obtained from its plant food (19).
The leading German organic chemist of the time, Justus
Liebig, now comes into the picture. He too had become
interested in the subject of animal chemistry, and wrote that
Dumas must be wrong because it was well known that pigs
would fatten when fed on potatoes that were rich in starch, but
contained only a negligible level of fat. This meant that
animals must be able to convert carbohydrates to fat even
though the conversion required reduction rather than oxidation.
This was a challenge to the French workers who had been
the undisputed authorities in the field, and Boussingault put
the matter to the test in another pioneering study. He killed
and analyzed the carcass of a young pig, while feeding a
littermate of the same starting weight on measured amounts of
feed for an additional 3 mo. Carcass analysis of the second pig
showed that it contained an additional 13.6 kg fat, whereas the
feed it had eaten had only contained 6.8 kg (20).
This careful work had therefore shown that the French
school was in the wrong on this point. Boussingault and
Dumas both retired from working with animals, and Liebig
became the new authority, even though he had never actually
carried out a feeding trial. He continued to push his ideas on
physiology and nutrition. Most of these were gradually shown
to have been completely wrong, but at least they stimulated
others to do research, putting them to the test.
The atomic theory
While the work described above was in progress there was
another important advance in chemistry that would be put to
use in subsequent nutritional studies. John Dalton, a poor and
largely self-educated schoolmaster in the north of England,
had an important idea. This was that all elements are made up
of indivisible particles, or atoms, and that for each element
Apparent daily balance
4.0
3.8
2.3
criticism, Boussingault explained, I am far from regarding

nitrogenous materials alone as sufficient for the nutrition of
animals; but it is a fact that where nitrogenous materials are
present at high levels in vegetables they are generally accompanied by the other organic and inorganic substances which
are also needed for nutrition (16). It is clear from the context
that the organic substances to which he is referring are
starches and not any hypothetical trace nutrients.
Was there any reason at this period for investigators to
suspect that other nutrients might also be needed to constitute
a complete diet? One might think that the problem of scurvy
appearing among sailors and the evidence for the value of
fruits and green foods in the prevention of the disease, would
have suggested it. However, even James Lind, famous for his
controlled clinical trial of different potential antiscorbutics,
believed that they were active in countering the bad effects of
sea air, and were not required by people living on land any
more than quinine would be of any value for people not living
in a malarious area (17,18). Also, it was clear that dogs, the
animals being used by the French workers, thrived without
such supplementary food items.
The composition of animal substance

Up until then it had been recognized that so-called animal
substances came in different forms that were named albumin,
fibrin, casein and so forth, and that they differed in solubility
and other physical properties, but all contained 16% of
nitrogen. In 1839, it was suggested by a Dutch worker, Gerrit
Mulder, that these substances were all compounds of a
common radical combined with different proportions of phosphorus, sulfur or both; and the hypothetical radical was named
protein, from a Greek term implying that it was the primary material of the animal kingdom. He further proposed,
using the symbol Pr for the radical, that egg albumin could
be expressed as Pr10 SP and serum albumin as Pr10 S2P,
and that the radical itself had the molecular formula
C40H62N10O12 (25).
Liebig received these ideas enthusiastically, and reported
that the comparable materials that he had isolated from plant
tissues also had exactly 4 atoms of carbon to 1 atom of
nitrogen. He went further and suggested that, although it was
only plants that could make the protein radical, animals had
the power to add or subtract the added elements, thus converting albumin to fibrin, etc. (26). Dumas and Cahours,
working in Paris, wrote that they too had found a 4:1 ratio of
C:N in both casein and serum albumin. However, legumin
extracted from peas and beans, and which Liebig had called
vegetable casein, had only a 3.25:1 ratio. This was a problem
because there was reason to believe that legume crops had a

high nutritive value, although legumin clearly could not be
converted into albumin just by addition or subtraction of sulfur
and/or phosphorus (27).
Liebig too was beginning to regret having adopted Mulders
ideas. Workers in his laboratory had been unable to obtain the
protein radical by removing the sulfur from egg albumin in
the way described by Mulder; nor could they find the expected
proportions of sulfur and phosphorus in different materials.
Mulder was enraged by the tone of the criticism from Liebig,
who was now denying what he himself had previously asserted.
In any case, the concept of a protein radical now disappeared
from the literature and the term Protein gradually began to
be applied to all the materials previously described as animal
substance.
Protein the only true nutrient
Meanwhile, Liebig had published a widely read book entitled Animal Chemistry or Organic Chemistry in its Application to
Physiology and Pathology. In it he argued that, because his
analyses of muscles failed to show the presence of any fat or
carbohydrate, the energy needed for their contraction must
come from an explosive breakdown of the protein molecules
themselves, resulting in the production and excretion of urea.
Protein was therefore the only true nutrient, providing both
the machinery of the body and the fuel for its work (28).
If that was true, what role was left for the other constituents
of the diet, and why did carbonic acid production increase so
greatly during exercise? Liebigs explanation was that increased
respiration was needed to keep the heart and other tissues from
overheating. However, this unfortunately led to more oxygen
gaining access to the tissues, which could cause oxidative
damage and loss of protein tissue. It was the function of the
fats and carbohydrates to mop up this excess by being themselves preferentially oxidized.
Liebigs book was at first generally regarded as a giant
intellectual synthesis, and many people were converted to his
ideas. For example, when the Professor of Medicine at Edinburgh University was called in to investigate a serious and
unexpected outbreak of scurvy in a Scottish prison, his immediate conclusion was that it must be the result of an inadequate
intake of protein (29). However, his calculations indicated
that the average daily protein intake was an ample 135 g. But
only 15 g of this quantity were from animal sources and 102 g
were from gluten. He suggested that the power of the body to
convert gluten to animal protein was limited, and that the
level of milk in the diet should be increased so as to raise the
intake of animal protein. Another Scottish physician replied
that the value of lemon juice in the prevention of scurvy was
well established and could not possibly be attributed to its
protein content, given that a curative dose contained only a
negligible amount of nitrogen (30).
Another difficulty in believing that muscular work required
the breakdown of protein was that the traditional diet of
laborers was of lower protein content that that of the less
active rich. Edward Smith, a British physician and physiologist
who was interested in the welfare of prisoners, and was concerned at the stressfulness of their having to work on a treadmill, measured their urea excretion in the 24 h during and
after their 8 h of work, and again on their subsequent rest days,
and found no difference (31) (Fig. 2). This was, of course,
quite contrary to what Liebig would have predicted on the
basis that the energy expended all came from the breakdown
of protein that resulted in the production of urea.
every atom is identical. Chemical combination occurs when

two or more different atoms form a firm union (21). These
ideas were supported by the proportions of different elements
in any compound being fixed and by the different compounds
between the same two elements being in simple ratios by
weight. Thus the gas we call carbon dioxide has exactly
twice the weight of oxygen (per unit weight of carbon) that is
present in the other gas called carbon monoxide. Finally,
gases were found to combine in simple relations by volume.
Thus 3 volumes of hydrogen combine with 1 volume of nitrogen to form exactly 2 volumes of ammonia gas (22). From this
it also follows that equal volumes of different gases contain the
same numbers of molecules, once one accepts that many
elements, such as hydrogen, oxygen and nitrogen, have two
atoms combined together to form a single molecule.
For some years there was controversy as to whether carbon
and oxygen each had one-half of the atomic weights that are
now assigned to them, although it is easy to correct molecular
formulas obtained in that period. Thus Prout, in England,
subjected urea to improved methods of analysis, and obtained
a molecular formula of C2H4N2O2, which agrees with the
modern formula of CH4N2O when we double the atomic
weights for C and O (23). In the following decade, Friedrich
Wo hler in Germany found that he had obtained urea by
heating silver cyanate with ammonium chloride. He wrote
excitedly to his former professor: I can make urea without the
use of kidneys. Admittedly, urea was only an excretion product, but the synthesis was one small step in demonstrating that
an organic compound produced in living systems could also be
produced in the laboratory without the aid of any vital force.
Wo hler, in collaboration with Liebig, also developed an
important concept in organic chemistry. This was the idea of
a common radical that would combine with other reagents,
but still retain its own nature and be recoverable by further
reactions. The first example was the benzoyl radical. Starting
with benzaldehyde, one could oxidize it to benzoic acid or form
a chlorinated derivative, and so on, and then reproduce the
original benzaldehyde by appropriate reduction (24).
641
642
CARPENTER
FIGURE 2 Picture of the treadwheel in a London prison of the type

used by Edward Smith to compare urea
excretion on work and rest days, and
to measure his own increase in carbon
dioxide output when climbing a known
distance on the treads. Both sets of
measurements were used to advance
knowledge of the fuels used by muscles
and their efficiency (British Register,
1823).
The next stage in this story involves another line of basic

work, the development of the concept of the conservation of
energy in its different forms. This advance cannot be attributed to any one person, but James Joule, a young Englishman
working on the problem in his spare time, was the first to
establish a good value for the mechanical equivalent of heat
(32). This was then used to calculate the efficiency of human
muscular effort in relation to heat production. Edward Smith,
already mentioned for his work on the urea excretion of
prisoners, had also developed portable equipment for measuring carbon dioxide output under different conditions. On the
basis of the additional carbon dioxide that Smith had himself
exhaled when working on the convicts treadmill, Hermann
Helmholtz estimated that the human engine functioned with
about 25% efficiency (33).
A critical experiment was then designed by two Swiss
scientists, the physiologist Adolf Fick and the chemist Johannes Wislicenus, to test Liebigs belief that protein constituted the sole muscle fuel. They traveled to the base of a
mountain in Switzerland with a path to the top that was fairly
easy to climb and a hotel at the top. They ate a very low
nitrogen diet before and during their experiment, and collected their urine during and for 6 h after their climb (34).
Analysis of the urine samples showed that they had excreted,
on average, a quantity of nitrogen equivalent in nitrogen
content to 35.0 g protein, using the usual N 6.25 conversion factor. They calculated, as best they could, the energy
that could have been obtained from the combustion of this
quantity of protein, but had values only for the combustion of
carbon and hydrogen as such, that yielded a high value of 6.73
kcal/g protein. Even with this value they calculated that the
energy obtainable was less than the work that they had done
against gravity in their climbing.
In the same period, Edward Frankland, Ficks brother-in-
law in England, was developing a technique for measuring

directly the heat of combustion of foods and of urea. For
protein, with an allowance for the gross energy remaining in
the excreted urea, he obtained a metabolizable energy value of
4.37 kcal/g. Using this factor, the energy obtained from the
average quantity of protein metabolized (i.e., 35 g) was 153
kcal. With the mechanical equivalent of heat being taken as
423 kg m against gravity per kcal, the mechanical work
obtainable from 153 kcal was 64,700 kg m.
In the climb the two men had risen 1956 m against the
force of gravity and, with an average weight of 71 kg, had done
an absolute minimum of 138,900 kg m of work per head
(Table 2). Because this was more than twice the energy that
could have come from their breakdown of body protein, even
assuming 100% efficiency of the muscles and neglecting the
work of the heart and so forth, much of the fuel consumed
must have come from other sources, presumably fat and/or
carbohydrate (35). Frankland drew the analogy of a muscle to
a steam engine in which the engine did not consume itself
when working, but remained intact while using an entirely
different fuel.
Liebig was unwilling to accept this conclusion, even though
he and his colleagues obtained comparable results with dogs.
They tried to avoid it by suggesting that living systems might
be able to obtain more energy from a reaction than was
obtainable in vitro, or that proteins released their energy
gradually so that even resting muscles gradually gained potential mechanical energy comparable to that of the coiled spring
in a watch (36). We will see in the following chapter that the
German enthusiasm for high protein diets continued well
beyond the evidence for their value.
Digestion
During the period under consideration, very little original
work that related to nutrition was carried out in the United
The conservation of energy
TABLE 2
Calculation of results from the FickWislicenus climbing
experiment relating the net work done to the metabolizable
energy obtainable from the protein broken down to urinary
nitrogenous compounds1
Fick
1965 m
66
76
129,700
149,300
5.68
5.52
35.5
34.5
4.37
155.1
150.8
423
65,600
63,800
1 Ref. 34.
States. However, two names are remembered from this time.

John Young, a medical student who died tragically at 21 from
tuberculosis, described in 1803 in his M.D. thesis numerous
experiments on digestion. He had found that regurgitated
stomach contents did not undergo acetous fermentation,
which was contrary to the current opinion. He also followed
the fate of bagged samples of foods that he subjected to gastric
digestion in frogs, snakes and birds, and showed that animals
that were normally carnivorous could at least dissolve plant
foods, and vice versa (37).
Some 20 y later, a U.S. Army surgeon, William Beaumont,
had the opportunity to become a pioneering physiologist. At a
remote trading post a young man was accidentally shot in the
stomach and the wound left a permanent fistula through which
food samples could be introduced and removed. Because the
victim was destitute, Beaumont took him into his house and
used him as a subject intermittently for almost 10 y. He
observed that gastric juice, which always contained hydrochloric acid, was secreted only in response to eating. He also saw
that oily food was only slowly digested, but that it was speeded
by minuteness of division (38).
At that time the stomach was thought of as the major site
of digestion. However, in the 1850s, Claude Bernard discovered that the secretions into the small intestine from the
pancreas, together with the emulsifying effect of the bile, were
of the greatest importance for the digestion of fat into glycerol
and free fatty acids, and its absorption (39). This and the later
discoveries of the proteolytic activity in the small intestine, to
be discussed in Part 2, made the study of purely gastric digestion seem less important.
Scurvy and other diseases
In 1842 George Budd, Professor of Medicine at Kings
College, London, gave a memorable lecture titled Disorders
resulting from defective nutriment, from which these are
some of his opening comments: There is no subject of more
interest to the physiologist or of more practical importance to
the physician . . . than the disorders resulting from defective
nourishment. . . . These disorders are, no doubt, frequently

presented to us by the destitute poor in our large towns;
but . . . from our not being acquainted with all the circumstances in which they arise, their real cause escapes us. It is
onlyas in ships, garrisons, prisons and asylumswhen large
numbers of men . . . become affected with one disease, that our
attention is fixed upon it, and that we can succeed in discovering its cause by considering what is peculiar in their circumstances (40).
There is one exception to the generalization at the beginning of the chapter that no systematic work relevant to nutrition had been carried out before 1785, and this must now be
described. It is the pioneering controlled clinical trial of the
various therapies recommended for the disease of scurvy,
which was carried out in 1746 by James Lind on sailors at sea.
Lind was, at that time, a 30-y-old ships surgeon in the British
navy, with no academic education, but with a special interest
in the problem of scurvy. He took 12 sailors, all with a similar
severity of the disease, divided them into pairs and, for 2 wk,
gave each pair one of the many treatments that had been
recommended for the condition. His trial is described in more
detail elsewhere, but the salient point for modern readers is
that the pair receiving lemons and oranges were almost recovered after only 6 d, whereas those receiving either dilute
sulfuric acid or vinegar had shown no improvement after 2 wk
(41,42).
The importance of Linds trial has often been described as
showing that citrus fruit was a cure, or preventive, for scurvy.
This had, in fact, been known already for some 200 y but could
not always be made use of. Neither oranges and lemons nor
fruit juice could be stored on long voyages before the days of
refrigeration because they went moldy. Because of this, the
College of Physicians in London had reasoned that other acids
could act as substitutes, given that it was thought that scurvy
was a putrid disease, and animal tissues that went putrid
became alkaline. It seemed therefore to follow that citrus juice
acted as it did as a result of its acidity, and that other more
stable acids like sulfuric acid (diluted before use!) or vinegar
could be used equally well. As a consequence, ships surgeons
were issued with sulfuric acid for many years without its actual
value having been put to a critical test.
In 1753, after Lind had qualified as a university-trained
physician, he wrote in his treatise on scurvy: The Channel
fleet for many years was supplied with vitriol [sulfuric acid]. Yet
it often had a thousand men miserably over-run with the
disease. . . . Of theory in physic [medicine] . . . it is indeed
absolutely necessary yet, by carrying it too far, it may be
doubted whether it has done more good or hurt in the world
(43).
If other acids could not replace lemon juice, and if lemons
or their juice were too unstable for carriage on long voyages,
what could be done? Lind himself suggested that the juice
could be slowly concentrated in shallow bowls over boiling
water until they had condensed to a thick syrup, or rob as he
called it. This was tried but found to be of little value in
practice. A more effective product, and one welcome to sailors, was to preserve the juice with a proportion of rum or
brandy. Another approach was to extract citric acid from the
juice and to issue that to ships. Unfortunately, many writers,
assuming that citric acid was the active factor, would refer to
administering the citric acid, even when they were actually
giving lemon juice. In some instances this is clear, but in
others not. Finally, after many years of uncertainty it was
agreed that true pure citric acid was not antiscorbutic (44).
Lind had believed that the true value of citrus fruit was that
it had a saponaceous, attenuating and resolving virtue [or
Height of climb, m
Body weight plus equipment, kg
net work done against gravity,
kg m
Excretion of urinary N during the
climb and for an additional 6 h, g
Protein (N 6.25) equivalent to
urinary N, g
Metabolizable energy of protein,
kcal/g
Energy yield from metabolized
protein, kcal
Mechanical equivalent of heat,
kg m/kcal
Net work from metabolized protein,
kg m
Wislicenus
643
644
CARPENTER
Arctic scurvy
As the nineteenth century progressed, ship travel became
faster, so that long voyages without the opportunity of collecting fresh food at ports of call became rarer, as did sea scurvy.
However, arctic exploration proved to be the exception to this
generalization. In 1875 the British navy mounted an expedition that would attempt to get farther north than had been
achieved by earlier explorers. The navy was confident that
scurvy would not be a problem. In the Napoleonic wars they
had been able to blockade French ports and to remain at sea
for long periods as a result of the well-organized supply of
lemons from Sicily. Now the authorities had changed to using
limes from the West Indies, believing that, because they were
more acidic, they would also be even better antiscorbutics.
The juice was bottled in England, preserved with spirits.
The two ships sailed in May 1875 with 122 on board,
wintered in the ice at 82N and sent out sledging expeditions
in the following spring. By June there had been 60 cases of
scurvy with four deaths and the ships returned home. This was
considered to have been a major scandal and a thorough
inquiry was begun. Everyone on board had received daily
rations that included 4 oz of preserved vegetables, 1 oz of
pickles and 1 oz of lime juice. One critic argued that Liebig
had made physiology a new science and that the doctrine of
antiscorbutics had been given a death blow. Others urged that
attention should be given to how the Eskimos managed to
remain healthy in the far North without the use of fruit or
fresh vegetables (47). As we will see in the following chapter,
this problem led to the adoption of new theories that were to
mislead explorers for a considerable time, and to provide an

example of knowledge apparently going backward for at least
20 y.
Another problem encountered on long voyages, sometimes
in conjunction with scurvy, was night blindness. Some ships
surgeons considered it be an early sign of developing scurvy,
and both conditions were found to respond to the addition of
fresh green vegetables to the diet. However, most believed it to
be a separate disease because the two conditions did not always
appear together, and sufferers from night blindness frequently
went on to develop ulcers on their corneas (48).
There are several reports by physicians of their successful
treatment of the condition with fish or cod liver oil early in the
nineteenth century (49,50). It was also a very old folk treatment for the eye problems to give patients cooked liver from
any of a variety of animals. This was put to the test in the
1850s on a round-the-world voyage organized by the Austrian
navy. On the last long leg of the voyage, from the Cape of
Good Hope to Gibraltar, 60 of the 350 on board developed
night blindness. The ships surgeon, who had been asked to
carry out the test if an opportunity presented itself, obtained
ox liver at Gibraltar, gave it to all 60 and reported that the
result was a true miracle (51). Nevertheless, he was attacked
in the medical press for his frivolous conclusion that it was a
nutritional disease, which could only be regarded as selfaggrandizement by someone ignorant of the literature on the
subject.
In 1863 P. Bitot, a French physician whose name has been
given to the white spots on the cornea that he recognized as
being associated with night blindness, made no mention of
being able to cure his patients with any food supplement, and
described the condition as being purely vital or nervous in
nature (52).
In 1881 a British physician reported that the condition
responded well in patients being dosed with cod liver oil and
suggested that they were possibly suffering from some want of
tone or nutrition (53). However, this was still not the generally established conclusion. In 1884 a German physician
who had seen the condition at an orphanage where he had
medical responsibilities concluded that it must be the result of
an infection, given that the children were receiving a good
diet (54).
Goiter and cretinism
Goiter, seen as a swelling in the front of the neck, had been
recognized as existing in some specific areas for millennia. In
the same areas there was also a smaller proportion of babies
born with cretinism, characterized by stunting and low mentality. For sufferers from goiter it had been a long-standing folk
treatment to give them dried seaweed and sponges, or the ash
prepared by burning them. In 1812 the element iodine was
discovered in this kind of ash, and French chemists suggested
that it be used for the treatment of goiter. However, it was
frequently found to be toxic in the doses given and the
treatment was largely abandoned (55). At the end of the
period covered by this presentation, Hirsch, in a wide-ranging
scholarly review, wrote that the iodine-deficiency theory was
a short-lived opinion . . . endemic goiter and cretinism have
to be reckoned among the infective diseases (56).
We have seen in the examples of Arctic scurvy, night
blindness and goiter, the growing belief that more and more
diseases were going to be explained in terms of either direct
infection with microorganisms or indirectly by the power of
these organisms to produce toxins. Undoubtedly, the development of the germ theory of disease made an enormous contri-
detergent action] that helped to free perspiration pores in

the skin that had become clogged in sea air so that poisons
accumulated without being able to escape. He believed that
the disease did not occur on land, so that land dwellers did not
therefore require an antiscorbutic as sailors did. But this was
not the case. It was clear by 1843 that there had, from time to
time, been at least 20 outbreaks of scurvy in British prisons.
The condition seen in the prisoners was exactly the same as
that seen at sea. The only common factor that could be found
to explain these outbreaks was that, for some time previous to
the outbreaks, potatoes had been omitted from the diet; and
when these were added back to the diets, the disease disappeared (45).
The importance of potatoes as antiscorbutics was confirmed
in the period from 1845 to 1848, when successive European
potato harvests failed because of fungal attack. In Ireland,
where potatoes had become the major source of energy for
much of the population, there was disastrous starvation on top
of the expected scurvy. In England, where more grain was
grown and there was no overall shortage of energy, the major
effect was again a series of outbreaks of scurvy, this time in the
general population as well as in prisons. The serious outbreak
in a Scottish prison has already been discussed in relation to
the belief of a disciple of Liebig that protein was the only true
nutrient and therefore, if a diet was inadequate in quality, the
deficiency must be in the supply of protein. In practice, green
vegetables were found to be effective alternative antiscorbutics
when neither potatoes nor fruit were available.
Land scurvy would continue to be a problem whenever food
supplies were limited by supply problems. Thus it occurred
among prospectors during the California gold rush, soldiers
during the Crimean War, prisoners in the American Civil War
and ordinary civilians during the Siege of Paris in 1871 (46).
In every case, the problems were resolved when either fresh
vegetables or fruit juice became available again.
bution to reducing human suffering but, at least for a period,

some well-established facts regarding other diseases were to be
treated as no more than old wives tales. To take a further
quotation from George Budd: Large numbers of men . . . have
been kept on a diet insufficient in quantity and variety . . . diseases of strange kind have appearedthe cause has been
recognized, and the remedy applied . . . but the lesson has been
forgottenand at a short interval of time, and in a different
place, a knowledge of the imperious necessity of nourishment
more abundant or more varied is again dearly bought by the
experience of wholesale sickness (57).
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[English transl. in Leicester, H. M. & Klickstein, H. S. (1965) A Source Book in
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25. Mulder, G. J. (1839) U ber die Zusammensetzung einiger thierischen
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26. Liebig, J. (1841) Ueber die stickstoffhaltingen Nahrungsmittel des
Pflanzenreichs. Ann. Chem. Pharm. 39: 129 160.
27. Dumas, J. B. & Cahours, A. (1842) Me moire sur les matie`res azote es
neutres de lorganisation. C. R. Hebdomadaires Acad. Sci. Paris 15: 976 1000.
28. Liebig, J. (1842) Animal Chemistry or Organic Chemistry in its Application to Physiology and Pathology (Gregory, W., transl.). Owen, Cambridge, MA.
29. Christison, R. (1847) Account of an epidemic of scurvy which prevailed in the general prison at Perth in 1846. Monthly J. Med. Sci. 7: 873 891.
30. Anderson, A. (1847) On the recent differences of opinion as to the
cause of scurvy. Monthly J. Med. Sci. 8: 176 181.
31. Smith, E. (1862) On the elimination of urea and urinary water. Philos.
Trans. R. Soc. London 151: 747 834.
32. Joule, J. P. (1843) On the calorific effects of magneto-electricity, and
on the mechanical value of heat. Philos. Mag. London (ser. 4) 23: 435 443.
33. Helmholtz, H. (1861) On the application of the law of the conservation
of force to organic nature. R. Inst. Proc. 3: 347357.
34. Fick, A. & Wislicenus, J. (1866) On the origin of muscular power.
Philos. Mag. London (ser. 4) 31: 485503.
35. Frankland, E. (1866) On the origin of muscular power. Philos. Mag.
London (ser. 4) 32: 182199.
36. Liebig, J. (1870) The source of muscular power. Pharm. J. Trans. (ser.
3) 1: 161163, 182185.
37. Young, J. R. (1803) An Experimental Inquiry into the Principles of
Nutrition. Eaken & Mecum, Philadelphia, PA.
38. Beaumont, W. (1833) Experiments and Observations on the Gastric
Juice. Allen, Plattsburgh, NY. [Facsimile edition published in 1959, Dover, New
York, NY.]
39. Bernard, C. (1985) Memoir on the Pancreas (Henderson, J., transl.).
Academic Press, London, UK. [Transl. of Me moire sur le Pancre as (1856)
Bailliere, Paris, France.]
40. Budd, G. (1842) Lectures on the disorders resulting from defective
nutriment. Lond. Med. Gaz. 2: 632 636, 712716, 743749, 906 915.
41. Lind. (1753) See cit. no. 17, pp. 145148.
42. Carpenter. (1986) See cit. no. 18, pp. 5254.
43. Lind. (1753) See citation no. 17, pp. 145148.
44. Bryson, A. (1850) On the respective values of limejuice, citric acid, and
the nitrate of potash, in the treatment of scurvy. Med. Times Gaz. (Lond.) 21:
212214, 435 436.
45. Baly, W. (1843) On the prevention of scurvy in prisoners, pauper
lunatic asylums etc. Lond. Med. Gaz. [new ser.] 1: 699 703.
46. Carpenter. (1986) See cit. no. 18, pp. 109 112.
47. Ibid., pp. 140 145.
48. Ibid., pp. 119 121.
49. Budd. (1842) See cit. no. 40, pp. 746 748.
50. Wolf, G. (1998) M. Moris definitive recognition of vitamin A deficiency
and its cure in children. Nutrition 14: 481 484.
51. Wolf, G. (1997) Eduard Schwarz, a neglected pioneer in the history of
nutrition. Nutrition 13: 844 846.
52. Bitot, P. (1863) Me moire sur une lesion conjonctivale, non encore
decrite, concident avec lhe me ralopie. Gaz. Me d. Paris 435 443.
53. Snell, S. (1881) On nyctalopia with peculiar appearances on the conjunctivae. Trans. Ophthalmol. Soc. UK 1: 207215.
54. Kuschbert, S. (1884) Die Xerosis conjunctivae und ihre Begleiterscheinungen. Dtsche. Med. Wochenschr. 10: 321324.
55. Guggenheim, K. Y. (1981) Nutrition and Nutritional Diseases: The
Evolution of Concepts, pp. 277289. The Collamore Press, Lexington, MA.
56. Hirsch, A. (1885) Handbook of Historical and Geographical Pathology,
vol. 2, p. 196. New Sydenham Society, London, UK.
57. Budd. (1842) See cit. no. 40, p. 632.
1. Berthollet, C. L. (1785) Analyse de lalkali volatil. Me m. Acad. Sci. Paris

316 326.
2. Carpenter, K. J. (1994) Protein and Energy, p. 14. Cambridge University Press, New York, NY.
3. Seguin, A. & Lavoisier, A. L. (1789) Premier me moire sur la transpiration des animaux. Me m. Acad. Sci. Paris 601 661.
4. Holmes, F. L. (1985) Lavoisier and the Chemistry of Life, pp. 440 446.
University of Wisconsin Press, Madison, WI.
5. Lavoisier, A. L. & Laplace, P. S. (1780) Me moire sur la chaleur. Me m.
Acad. Sci. Paris 355 408.
6. Holmes. (1985) See cit. no. 4, pp. 162170.
7. Grmek, M. D. (1974) Franc ois Magendie. Dict. Sci. Biogr. 9: 6 11.
8. Magendie, F. (1816) Sur les proprie te s nutritives des substances qui
ne contiennent pas dazote. Ann. Chim. (ser. 2) 3: 66 77, 408 410.
9. Magendie, F. (1831) An Elementary Compendium of Physiology (Milligan, E., transl.). John Carfrae, Edinburgh, UK.
10. Magendie, F. (1841) Rapport fait a` lAcade mie des Sciences au nom
de la Commission dite de la ge latine. C. R. Acad. Sci. Paris 237283.
11. McCosh, F.W.J. (1984) Boussingault, Chemist and Agriculturalist. Reidel, Dordrecht, The Netherlands.
12. Boussingault, J. B. (1839) Analyses compare es des aliments consomme s et des produits rendus par une vache laitie`re. Ann. Chim. (ser. 2) 71:
113127.
13. Boussingault, J. B. (1839) Analyses compare es des aliments consomme s et des produits rendus par un cheval soumis a` la ration dentretien. Ann.
Chim. (ser. 2) 71: 128 136.
14. Boussingault, J. B. (1836) Recherches sur la quantite dazote contenue dans les fourrages, et sur leurs equivalents. Ann. Chim. (ser. 2) 63: 225244.
15. Vauquelin, L. N. & Fourcroy, A. N. (1806) Memoir upon the germination
and fermentation of grains and farinaceous substances. Philos. Mag. 25: 176
182.
16. Boussingault, J. B. (1845) Rural Economy (Law, G. transl.). Orange
Judd, New York, NY.
17. Lind, J. (1753) A Treatise of the Scurvy, p. 60. Millar, Edinburgh, UK
(Reprinted 1953 by University of Edinburgh Press).
18. Carpenter, K. J. (1986) The History of Scurvy and Vitamin C, pp.
57 61. Cambridge University Press, New York, NY.
19. Dumas, J. B. (1841) On the chemical statics of organized beings.
Philos. Mag. 19: 337347, 456 469.
20. Boussingault, J. B. (1845) Recherches expe rimentales sur le de veloppement de la graisse pendant lalimentation des animaux. Ann. Chim. (ser. 3)
14: 41.
21. Dalton, J. (1808) A New System of Chemical Philosophy (repr. 1964).
Philosophical Library, New York, NY.
22. Ihde, A. J. (1964) The Development of Modern Chemistry. Harper &
Row, New York, NY.
23. Prout, W. (1819) Propriete s chimiques et composition de quelquesuns des principes imme diats de lurine. Ann. Chim. Phys. (ser. 2) 10: 369 388.
645
A Short History of Nutritional Science: Part 2 (18851912)1
Department of Nutritional Sciences, University of California, Berkeley, CA 97420 3104
recommending diets only on the basis of the economic provision of protein and energy was that fruits and green vegetables
became dispensable luxuries. At this period, the purchase of
food typically took 50% of a working familys income.
The challenge to high protein standards came finally from
Russell Chittenden, Yale Universitys Professor of Physiological Chemistry. He had found some relief from what may have
been a rheumatic condition after he had deliberately reduced
his general intake of food, and particularly that of meat, and
was greatly impressed by having fully maintained both his
physical and mental activity, although his intake of protein
had not been 40 g/d (equivalent to 48 g for someone of the
standard weight of 150 lb).
Chittenden then organized three controlled trials using low
protein diets. In the first, Chittenden and three scientific
colleagues remained healthy and in nitrogen balance for 6 mo
on daily diets containing 62 g protein on average, after adjustment to standard body weight. The second trial used 11
corpsmen from the U.S. army who also remained in good
health and physical condition with a standardized daily intake
of 61 g protein (Fig. 1). In the final trial, a group of 7 Yale
student athletes consumed 64 g protein (standardized) per
day, maintained their levels of athletic performance and said
that they felt better for it (5).
Others were reluctant to accept Chittendens recommendation of such diets as representing physiological economy,
and argued that the almost universal consumption of high
protein diets in prosperous countries showed an important
relationship that might not become apparent in short-term
trials. He replied that his critics were reversing cause and
effect; people did not become rich because they ate more
protein, but ate meat and other more expensive high protein
foods because they had already attained an income sufficient to
afford them (6). Later studies have only confirmed Chittendens findings.
Before 1885, nearly all of the nutritional studies had been

carried out in Western Europe and most were concerned with
the need for either protein or energy. These lines of work
continued in the 1880s but in the next 25 y, and in many more
parts of the world, important new lines of work were being
developed that would, in the long run, greatly broaden our
understanding of nutritional requirements.
Protein research continued
Until this time, there had been little significant work in
nutritional science in the United States, but Wilbur Atwater,
born in 1844 in New England and by 1885, a professor of
chemistry at Wesleyan University, was determined to change
that. He had already spent several months in Munich studying
the nitrogen balance procedures in use at the laboratory of
Carl Voit, who had been Liebigs protege. Voit believed that
people with sufficient income to choose the diet that they
preferred would instinctively select a diet containing the
amount of protein that they needed to remain healthy and
productive. His estimate was that the average German workman doing moderate physical work chose to eat 118 g protein/d, and this became his standard (1). Atwater found that
American workmen were generally better off and ate more.
They also, he thought, worked harder and he set his standard
at 125 g/d (2).
With hindsight, it seems ironic that he should not have
been more questioning concerning whether they really needed
so much of this relatively expensive ingredient. Apparently he
looked to the German school of nutritionists as the authorities
in a field in which he was only a newcomer. Voit accepted that
vegetarians who lived on a much lower protein intake could
remain in nitrogen balance, but he remained convinced that
such people exposed themselves to disadvantages (3). The
American group suggested that even if protein was not directly
used as the fuel for muscular contraction, it provided the
nervous energy required to wish to make the effort (4).
The main thrust of Atwaters work in this period was to
analyze foods by the proximate system (nitrogen, fiber, ash,
ether extract, moisture and carbohydrate by difference) and
to use these values to teach the poor how they could obtain
their requirement for protein, the most expensive of their
needs, more economically (Table 1). An unfortunate effect of
Protein digestion and interconversion

Throughout the writings of Voit, Atwater and Chittenden,
there was the unstated assumption that all proteins were of
equal quality. Thus, Atwater had no doubt that meat protein
in the diet could safely be replaced by the same quantity of
protein from beans. With hindsight, this is surprising because
Mulders hypothesis that all proteins contained the same radical had collapsed, and even the ratio of carbon:nitrogen had
been reported to differ between legumin extracted from
beans and some animal proteins (7).
For most of the 19th century, even after the breakdown of
Mulders theory, it had been assumed by workers in nutrition
1
This is the second of four invited papers on the history of nutritional science.
The first paper in this series was published in the March 2003 issue of The Journal
of Nutrition (7).
2
E-mail: kcarp@uclink.berkeley.edu.
0022-3166/03 $3.00 2003 American Society for Nutritional Sciences. J. Nutr. 133: 975984, 2003.
Manuscript received 3 January 2003. Revision accepted 8 January 2003.
975
CARPENTER
976
TABLE 1
Examples of Atwaters calculations of the relative economy
of different foods in supplying protein and energy1
25 cents will pay for:
Food item
Animal
Oysters
Beef sirloin
Cheese
Beef liver
Vegetable
Potatoes
Wheat bread
Wheat flour
Dried beans
Fruit
Oranges
Total weight
Protein
Energy
lb
kcal
1.21
1.27
1.67
3.13
41
86
213
286
285
1120
3420
2095
20
4.3
7.1
5.0
163
170
360
520
5900
5500
11750
8070
2.5
375
1 Source: (2).
that proteins ingested in foods were absorbed almost intact and

then modified in some slight ways, if necessary, to convert
them from fibrin to albumin, for example. However, other
workers studying the physiology of digestion first showed the
existence of a substance (pepsin), secreted by the stomach
wall, that converted proteins into more soluble derivatives.
Liebig regarded this as being no more than breaking up aggregations of molecules, allowing them to pass through the gut
more easily. A few years later, the pancreas was found to
secrete another substance (trypsin) that further broke down
the products of treating proteins with pepsin to produce materials that were noncoagulable, diffusible through parchment
and included the chemicals tyrosine and leucine. This subject
has been thoroughly reviewed, with full references, by Greenstein and Winitz in an easily available volume (8).
Now, tyrosine and leucine were already known as two of the
compounds, first called amino-bodies and then amino acids, that chemists had obtained by boiling proteins in strong
acids. These breakdown products had not been considered of
interest to nutritionists because the kind of destruction effected by strong, boiling acids had been assumed to be quite
different from what happened under the mild conditions in the
gut. However, the discovery of amino acids as products in a
biological system was obviously highly relevant, especially
because analysts had already reported that proteins appeared to
differ in the relative quantities of different amino acids that
they yielded on treatment with acids.
There always seems to be a way around unwelcome findings
and in 1895 Chittenden wrote: We may well consider the
formation of these amino acids in pancreatic proteolysis as a
means of quickly ridding the body of any excess of ingested
protein food, with the least possible expenditure of energy on
the part of the system (9). Thus, he was suggesting that the
proteins that the body needed were still being absorbed pretty
well intact, and it was just the unwanted surplus that was being
broken down before its disposal. Even in 1902, a German textbook was saying essentially the same thing: such a profound
decomposition would be a waste of chemical potential energy,
and a reunion of such products is highly improbable (10).
However, other workers in Germany and Denmark were
studying whether animals could use mixtures of amino acids as
substitutes for dietary protein. Most found that meat proteins

treated with pepsin and trypsin for long periods, and apparently
free of intact protein, did serve as nutritional substitutes, when fed
to adult dogs, but that acid hydrolysates of protein, even after
neutralization and removal of excess salts, did not (11).
It had been suspected that strong acid treatment was destroying some component of the protein because proteins, and
even enzymic digests, gave a color reaction suggesting the
presence of an indole derivative, but acid hydrolysates did not.
Finally, in 1902, F. G. Hopkins and S. W. Cole, working in
Cambridge, isolated the amino acid tryptophan, which contains an indole ring, from an enzymic digest and showed that
it was destroyed by conditions of acid hydrolysis (12). Then in
1906, Hopkins and another colleague reported that mice receiving zein (which contains no tryptophan) as their sole
protein source, lived longer if they also received a supplement
of tryptophan (13). And in 1909, Abderhalden found that
adult dogs could remain in nitrogen balance if the acidhydrolysates of protein that they were receiving were supplemented with this amino acid (14). These results did not yet
prove that tryptophan was utilized for protein synthesis because there was no growth, but they did show that this organic
compound had some essential function.
FIGURE 1 A photograph used by Chittenden to demonstrate that

his subjects remained in good physical condition while consuming a
relatively low protein diet. Reproduced from (5).
Calorimetry
After the work of Lavoisier and Seguin at the end of the
18th century, several workers in France and Germany gradually improved the equipment for measuring the respiratory
exchange of animals and their heat output under different
conditions (15,16). Finally in 1894, Max Rubner was able to
demonstrate with a dog that its heat output did exactly match
the heat of combustion of the foods that it was metabolizing,
as measured by urea output and gas exchange measured at the
same time (17).
We return now to the work of Wilbur Atwater, who also
had an interest in the energy value of foods; his group established estimates for the metabolizable energy of the carbohydrates, protein and fat in mixed diets as 4, 4 and 9 kcal/g,
respectively. These Atwater factors were slightly different from
those proposed by Rubner, but have stood the test of time (18).
However, Atwaters real ambition was to make a fundamental contribution to nutritional science by building and
conducting research with a respiration calorimeter that would
hold a human subject over long periods and also measure their
heat output directly. Figure 2 illustrates the kind of equipment
needed for a human respiration calorimeter, but to measure
the heat output at the same time is much more complex. This
was an ambitious and expensive machine that took Atwaters
group five years to build and test. The first aim was to confirm
that the heat produced by the human body was the same as
that produced outside the body (i.e., in vitro) by the combustion of the same quantity of nutrients. This they were able to
do with considerable precision (19).
They then made the controversial finding that ethanol,
given to a subject in a series of small doses, could also serve as
a source of useful energy (20). This was controversial because
the liquor trade made use of it in its advertising, whereas their
university (Wesleyan) was supported by the Methodist church,
which recommended total abstinence from alcohol, and its
members circulated pamphlets that described it as nothing but
poison. Atwater responded that the Almighty would not wish
moral teaching to be based on untruths (21).
After Atwater suffered a disabling stroke in 1904, his colleagues prepared for publication the further work demonstrating that the energy from combustion of either fats or carbohydrates could be used for mechanical work with at least
similar efficiency (22). The equipment was then moved away
from Atwaters laboratory, and there is a sad story of his
977
daughter having to make up accounts of what it was still being

used for to her bedridden father, who had been kept unaware
of his groups dispersal.
Wilbur Atwater is remembered as the father of nutritional
science in the United States. This is justified not solely by his
own research, but at least equally by his administrative work at
the U.S. Department of Agriculture where he organized food
consumption studies in many parts of the country. He also set
in place a policy of encouraging long-term basic work in
nutrition at agricultural experiment stations. The fruit of this
policy will be seen, for example, in part 3 of this sequence, in
which we will review Thomas Osbornes long-term work at the
Connecticut experiment station on the amino acid composition of proteins, and his collaboration with Mendel on their
nutritional significance.
Anemia
Anemia, or chlorosis (literally the green disease), had
long been a common problem among young women in their
teens. By 1885, it was agreed that the blood of an anemic
patient would show a decreased blood cell count and proportionally an even greater fall in hemoglobin level. It was also
generally agreed that the condition would respond to the
administration of pills containing ferrous sulfate. However, it
was not generally accepted that this response was the straightforward result of the iron being absorbed through the wall of
the gut and incorporated into molecules of hemoglobin. There
was first the theoretical objection that: it was just as unlikely
that the animal kingdom could make hemoglobin from inorganic iron as that it could make protein from potassium nitrate
and starch (23,24). Of course we have already seen the 19th
century belief that only the plant kingdom was capable of
synthesizing protein.
Second, German workers had found that when they added
a supplement of ferrous sulfate to a dogs diet of meat, the
additional iron recovered in the feces was at least nearly equal
to that ingested and, given the inherent variability in such
determinations, they concluded that inorganic iron was essentially indigestible. Typical results from one study are summarized in Table 2 (25).
At this time, investigators had the most experience in
studying the digestibility of proteins, which had been found to
be equally well digested at any level of intake. There was no
reason therefore to suspect that the digestibility of another
FIGURE 2 A diagram used by Atwater to illustrate the kind of equipment

needed for a human respiration calorimeter. Reproduced from (15).
CARPENTER
978
TABLE 2
Iron balance in a dog, used as evidence for the unavailability
of inorganic iron1
Period
Diet
Fe
intake
Fecal
Fe
d
06
721
Urine
Fe
(Net
balance)
mg/d
Meat alone
Meat FeSO4
Difference between
treatments
25.0
57.0
24.5
51.3
3.2
3.9
(2.7)
(1.8)
32.0
26.8
0.7
(4.5)
1 The calculated net balance was not included in the original

paper (25).
nutrient would depend on the level ingested or on the prior

status of the test animal.
Looking at the data in Table 2 through modern eyes, one
wonders why the authors in the earlier period missed the
equally obvious point that the values obtained with meat
alone indicated that organic iron was also indigestible. It
illustrates how easy it is to see what one wants to see in a set
of data, and to be blind to anything that does not fit in with
ones preconceptions.
Human nature, as we have said before, seems to allow us to
explain our way out of almost anything, and this was no
exception. Here the argument was that there is never really a
shortage of organic iron in the diet, but that some people
unfortunately have a dyspepsia accompanied by the production of hydrogen sulfide in their small intestine, and that this
compound has such a strong affinity for iron (and some other
metals) that it takes even organic iron into an indigestible
complex and makes it nutritionally unavailable. The value of
ferrous sulfate is explained by the hydrogen sulfide reacting
with it still more readily, and leaving no excess to react with
the organic iron that therefore remains available for use. It was
also believed that nontoxic compounds of other metals, such
as bismuth and manganese that also formed insoluble sulfides,
should be equally effective (24).
In the 1890s, these ideas were challenged by Ralph Stockman, a physician at Edinburgh University. He first tested the
usefulness of subcutaneous injections of small quantities of
ferrous citrate into anemic patients and found good responses
in both red cell counts and hemoglobin levels in the blood. He
then gave other patients keratin-coated capsules containing
ferrous sulfide. According to the prevailing theory, these
should have been inactive when released by digestion of the
coating in the small intestine but, in fact, they too resulted in
an improved blood picture. On the other hand, giving either
bismuth oxide or manganese dioxide (both supposedly able to
bind and neutralize hydrogen sulfide as well as ferrous salts)
proved ineffective with anemia patients (26). All of these
results were contrary to what would be expected in terms of
the dyspepsia theory.
Stockman went on to investigate the assumption that there
was always plenty of iron in ordinary human diets and found
that there was a source of interference in the standard method
of iron analysis in use in that period (27). When a sample
contained a large proportion of carbohydrates, some survived
the initial ashing step, and in the final stage, in which the
extract was titrated with potassium permanganate that oxidized ferrous to ferric ions, an additional quantity of permanganate was reacting with carbohydrate breakdown products.
Because of this, bread had been considered to be as rich in iron

as meat. With Stockmans revised procedure, bread was found
to have only 5 mg iron/kg, whereas meat had 40 mg/kg (28).
Analyses of five actual daily diets, consisting mainly of tea,
milk, bread and butter, which were being consumed in surprisingly small quantities by anemic young women, showed an
average intake of only 3 mg iron. In contrast, the more varied
and abundant diets of healthy nurses were all found to contain
9 10 mg iron. He concluded that the combination of low iron
intake and menstrual losses was sufficient to explain the occurrence of chlorotic anemia in young women, and also that
when red blood corpuscles break down, the great bulk of the
released iron is retained and reutilized so that both the excretion and the requirement for the element are quite small.
Stockmans work illustrates the importance of analytical procedures being specific and accurate, and how an inefficient
procedure may seriously mislead investigators concerning the
cause of a clinical problem.
Beriberi
By the 1880s, Japan had developed a navy using warships
built in Europe and employing the general practices of Western navies. However, an alarming proportion of the sailors was
succumbing to a disease known in Japan as kakke, but soon
recognized to be the same as that called beriberi elsewhere in
Asia and classified as a polyneuritis (Fig. 3). It was characterized by initial weakness and loss of feeling in the legs, and
then the development of heart failure and breathlessness,
together with edema in some cases (29). Kanehiro Takaki, a
naval surgeon who had received postgraduate training in England, was made responsible for eliminating the problem. The
only difference that he could find in the conditions of the
Japanese sailors compared with those in European navies in
which the problem did not exist, was that their diet was
considerably lower in protein content and well below the
Voit standards accepted then. (This was before the work of
Chittenden, discussed above.)
One voyage of a cadet training ship, to New Zealand and
back, had been particularly stricken, with over one half sick
with beriberi and 25 dead. Takaki persuaded the authorities to
repeat the voyage with the rations modified to include more
meat, condensed milk, bread and vegetables at the expense of
rice, so as to increase the proportion of protein. This time
there were no deaths and only 14 cases of the disease, all
among men who had not been willing to eat the full extra
rations. Takaki believed that he had confirmed that the disease resulted from a deficiency of protein and, indeed, the
problem virtually disappeared from the navy after the rations
were changed throughout the fleet (30,31).
In the same period, there was a similar problem of beriberi
in the native army, recruited by the Dutch in their East Indian
colony (now Indonesia), which had been sent into the field to
suppress a local uprising. In view of recent successes by Robert
Koch and others in identifying the microorganisms responsible
for several diseases, the Dutch government dispatched a small
team led by Professor Pekelharing, who was trained in bacteriology, to spend 8 mo investigating the beriberi outbreak. He
had no knowledge of Takakis work at that time.
Their examinations of autopsy material showed evidence of
neural degeneration. No bacteria were found in the blood of
beriberi patients in a base hospital, but they were found in
both sick and healthy soldiers in Atjeh where the fighting was
in progress. It was also of interest that soldiers posted there
would develop the disease only after 60 d in the area. There,
Pekelharing also found that a single injection of blood from a
979
FIGURE 3 Victims of beriberi, with

difficulty in walking, in one of the Javanese prisons surveyed by Vorderman.
Reproduced from (38).
beriberi patient into a dog had no bad effect, but that some
dogs that had received as many as 20 repeated injections over
a period of 6 wk, did become sick and gave indications of
nervous degeneration (32).
In his final report, Pekelharing wrote that he believed the
disease to be the result of an unusual type of bacterial infection, but that more work was required to confirm this, and he
recommended that this be carried out by Christian Eijkman, a
young army physician who had been assisting him, and was
permanently stationed in the colony. Eijkman was therefore
relieved of military duties and put in charge of a modest
research unit under civilian control, but adjacent to an army
hospital on the outskirts of Batavia (now Djakarta) that contained many beriberi patients.
Chicken polyneuritis
Eijkman decided, in view of the variability of individual
animals, that he would need to use large numbers, and he
chose chickens because they were readily available and cheap
to buy, as well as to house and feed. This remains a surprising
choice for an animal model considering that they are not even
mammals, but it turned out to be a lucky one. He began by
trying to infect birds by injecting them with blood from
hospital patients and, after a few months, he did begin to see
birds with an unsteady gait, somewhat reminiscent of beriberi
cases. However, the same condition was seen in uninjected
control animals kept in the same compound. This could, of
course, have resulted from the infection jumping from bird to
bird. Examination of autopsied birds showed the presence of
degenerated nerves and this encouraged him to hope that he
had indeed induced a condition comparable to the human
disease. He therefore started more trials with the controls
further separated from those injected, but now he failed to see
the problem developing in any of his chickens (33).
I believe that at this point most of us would have abandoned trying to use chickens that were proving so inconsistent, but Eijkman thought that there must be some explanation. From the local servant responsible for the maintenance
of the birds he discovered that, at the time when the birds
were showing leg weakness, the man had, for some months,
been able to beg leftover cooked rice from the hospital kitchen
and to use that to feed the birds. But then a new cook had
been appointed who had said that he was not going to give
military rice to civilian chickens (34).
Eijkman at once began tests with the leftover cooked rice
and found that its use did lead to leg weakness in his chickens
after 3 8 wk, whereas, at the same time, chickens fed either
uncooked hospital rice or rough, feed-grade rice, remained
healthy for the 3 mo of the trial. He then looked into the
technology of rice preparation and learned that the white
rice used in the hospital had had the grains polished to
remove the bran layer, after the usual preliminary removal of
the husks. He also learned that the local peoples in their
villages would pound rice grains each day, to remove just the
husk by winnowing, and then they would cook and eat the
brown rice with its bran still attached. This was no problem
when it was consumed fresh but, for supplying an army for
which foodstuffs often had to be shipped and stockpiled for a
period, brown rice was unsuitable because it would become
rancid under tropical conditions and hence unpalatable.
He considered the disease in the birds to be a variety of
peripheral polyneuritis, on the basis of his finding damaged
peripheral nerves on autopsy examination. From reading the
literature, he learned that in humans it appeared usually to be
caused by some kind of poisoning, perhaps only indirectly
through the production of toxins by bacteria. His first thought
therefore was that: the cooked hospital rice favored conditions for the development of micro-organisms of an unknown
nature in the intestinal tract, and hence for the formation a
poison that caused nerve degeneration. One aspect of the
disease in chickens that differed from human beriberi was that
the birds uniformly lost weight, but giving birds a reduced
amount of brown rice so that they too lost weight did not
result in leg weakness.
Eijkman now began a long series of feeding trials, partly
interrupted by his suffering bouts of malaria, and it was to be
six years before he made a progress report on his work. One of
his first findings had been that the disease would, after all,
appear in birds receiving uncooked white rice, although usually only after a longer period than with the cooked material.
He therefore had to abandon his first idea that the disease was
caused by pathogenic microorganisms that had thrived in the
cooked rice during its overnight storage. He also discovered
that sick birds could be cured by switching them to a diet of
980
CARPENTER
brown rice. He therefore concentrated on what factor in the

bran coat, still present in brown rice, could be responsible for
its protective effect. He wondered if it could be the fiber
content of the bran, but he found that giving ground-up bran
was still effective, whereas giving ground-up husks, as an
alternative source of fiber, was not.
In parallel experiments, he had found that feeding birds
either sago or tapioca starch also resulted in both their losing
weight and developing the characteristic leg weakness, so that
the effect was not peculiar to rice. Then he tried feeding a bird
each day 500 g tapioca and 25 g raw meat, which contained at
least as much protein as did a brown rice diet. The bird gained
weight but, after 4 wk, developed the usual leg weakness. This
impressed Eijkman as showing that chicken polyneuritis was
not inevitably accompanied by emaciation. When the bird was
changed to a diet of meat alone it gradually recovered, and
other birds fed meat alone from the beginning also remained
healthy. He concluded that the only common factor in diets
that produced this condition was starch, and that starch was
probably subject to fermentation in the intestines by microorganisms that produce a toxin and further, that the bran surrounding the rice grain provided an antidote to the toxin (35).
Even this account of his work has omitted many experiments, some of them with other species that yielded inconclusive results. It is no wonder that he used as the text for a
lecture given after his return to the Netherlands: Simplicity is
not characteristic of truth (36). One must feel for this man.
His wife had died on his first tour in Indonesia. He himself was
still suffering from bouts of malaria and would soon have to
leave the tropics for good, and he knew that others were
skeptical whether the disease in chickens, and the new kind of
phenomenon into which he had stumbled, had any real relation to beriberi. Indeed one critic, after reading his report, was
to write that: It must be considered the most inadequate
product which can be found in the literature from the Director
of a scientific institute (37).
One finding was to console him, however, after his return
to the Netherlands in 1896. Before leaving Batavia he had
talked about his work with Adolphe Vorderman, the medical
inspector of prisons in Java. It occurred to them that there
might already be a natural experiment in progress with different prisons using different kinds of rice. Surprisingly, there
were 101 prisons and 250,000 prisoners in this island, roughly
the size of Greece. Vorderman made a thorough investigation
and found that, indeed (as seen in Fig. 4), the incidence of
beriberi was vastly greater in the prisons in which mostly white
rice was in use (38,39). It appeared that less than one prisoner
in 10,000 developed beriberi in the brown rice prisons, and
one in 39 in the prisons using mostly white rice. Most of the
prisoners had very short sentences, but among the long-term
prisoners receiving white rice, 1 in 4 had developed beriberi.
Moreover, other factors that had been suggested as being
conducive to the disease, such as overcrowding and poor
ventilation, showed no evidence of having any adverse effect.
This was a strong indication therefore that Eijkmans work did
have relevance to the human disease.
The man who succeeded Eijkman in Batavia was Gerrit
Grijns, whose background was similar to that of his predecessor. His first work was to try to fractionate rice bran to discover
the character of the active material that it contained; at first,
he was disappointed to find that his manipulations seemed to
destroy the activity. Then he realized that this gave him the
opportunity to test Eijkmans idea that the appearance of the
chicken disease depended on the presence of starch. He autoclaved meat and fed it as the sole food for 8 cockerels and they
all died, with all but one showing characteristic paralysis. The
FIGURE 4
Vordermans figure illustrating the contrasting incidence of beriberi in Javanese prisons according to the type of rice in
use. Reproduced from (38).
evidence for starch being responsible for the condition, perhaps through its stimulating a toxic fermentation, was therefore discredited. Grijns also showed that several varieties of
beans were even more effective than rice bran in supplementing a chickens diet of white rice, and he ended his 1901 paper
with these historic sentences:
There occur in natural foods, substances, which cannot
be absent without serious injury to the peripheral nervous
system. The distribution of these substances in different foodstuffs is very unequal. . . . The separation of these substances
meets with the difficulty that they are so easily disintegrated. . . . They cannot be replaced by simple compounds (40).
Grijns, too, had to return to the Netherlands in 1902 to
spend two years recovering from another tropical disease, but
his work was immediately pursued by others. Hulshoff Pol, the
physician in charge of a mental hospital in Indonesia in which
there had been a serious problem of beriberi, heard of his
experiments with beans and decided to test their value for his
subjects. He had the men who had already developed the
disease transferred to a hospital unit. For the trial, the subjects,
all initially healthy, were housed in six separate buildings. His
control groups, housed in three buildings, consumed their
standard rations, and some in each building developed the
disease over the next 9 mo (altogether 19 out of a total of 58).
Those in three other houses, chosen at random, received the
standard daily ration supplemented with 150 g mung beans,
and none of the 78 men in these houses developed the disease.
Those who had developed the disease with consumption of the
standard diet were then given the same supplement of beans
and they were cured (41). This result was a further confirmation of the relevance of work with a small animal model for the
cure and prevention of a human disease.
By 1905, the Dutch workers in Indonesia had demonstrated
fairly convincingly that this disease was the results of white
rice lacking some unknown, heat-labile component. However,
this was not yet accepted in other parts of Asia in which the
disease was a problem. During the Russo-Japanese war of
1904 1905, many Japanese soldiers serving in Manchuria suffered from beriberi (the estimates ranged from 90,000 to
200,000) and many were brought back to temporary hospitals

in Japan and treated on the basis that it was an infection (42).
It was a chronic problem in Malaysia also, and Leonard
Braddon, who had served there as a British colonial medical
officer became convinced, without knowing of the recent
Dutch research, that white rice was the culprit. But he believed that the polishing of the grain left it with a porous
surface that allowed it to pick up pathogenic fungi that had
accumulated in the milling machinery; in addition, during
storage, the fungi continued to proliferate in the white rice
and were responsible for the signs of beriberi (43).
Workers at the Malaysian Institute for Medical Research,
who had learned of the Dutch work and begun their own
studies with chickens, found that adding alcoholic extracts
from brown rice to a diet of white rice prevented the appearance of polyneuritis in their birds, whereas feeding the brown
rice from which the alcoholic extract had been taken now
induced the disease (44). These findings could be explained
only in terms of the white rice being deficient, rather than
toxic.
The United States, which had become the occupying power
in the Philippine Islands after the Spanish-American war of
1898, was also faced with the problem of beriberi among its
native troops, and the authorities there called an international
meeting in 1910 to discuss the problem. This was well attended, with delegates from Japan, Java, Malaysia, Thailand,
Sri Lanka and French delegates from the area that is now
Vietnam. It was fairly generally agreed that the disease was
restricted to those who had white rice as their staple food, and
the American delegation suggested that the public health
approach should be to make its production illegal, or to tax it
so highly that it could be afforded only by those better off who
would, in any cause, eat a more varied diet containing other
items that would make up for the deficiency of the rice. Other
delegates thought this to be impracticable because of the
problem of brown rice going rancid during storage in the
tropics (45).
In Japan it had been noticed some years earlier that breastfed infants were subject to a disease called taon that was
characterized by vomiting, edema and stoppage of urine secretion. The death rate was very high, and it seemed as if their
mothers milk was poisonous to them because they could
recover if given cows milk. Physicians working with the U.S.
Army Medical Corps in the Philippines made similar observations; they suspected that the condition was analogous to adult
beriberi, and found that infants would also recover if breastfeeding was supplemented with extracts made from rice bran,
using alcohol, which was then evaporated (46).
By this time, workers in Java and elsewhere were already
trying to concentrate and isolate the active factor from rice
bran, in the distant hope of being able, eventually, to identify
and even synthesize it. It was discovered in Germany that
pigeons, which were easier to house, could be used instead of
chickens and showed a characteristic head retraction when
deficient in the antiberiberi factor (Fig. 5). It now became
fairly simple for this problem to be pursued in established
laboratories in the West just as easily as in countries in which
beriberi existed. For example, because rice polishings had a
relatively high concentration of phosphorus, a worker in Germany tested phytin, phosphonucleic acids and other phosphorus-containing compounds for their potency. These were
found to be inactive, but dried yeast was discovered to be
highly active (47).
In 1911, Casimir Funk, a Polish chemist working at the
Lister Institute in London, was the first to claim to have
isolated active crystals from rice polishings: they consisted of
981
FIGURE 5 A pigeon showing characteristic head retraction after it

has been fed white rice for 23 wk (upper panel), and the same bird 3 h
after receiving a yeast extract (lower panel). Reproduced from (65).
an organic base, and 50 mg was sufficient to cure a deficient

pigeon (48). In the following year, a group of Japanese workers
obtained even more active material (49). It was later realized
that these were both still mixtures and highly competitive
attempts at purification were to continue for many more years.
Rickets
Rickets in young children, characterized by inadequate
calcification of the bones with bowed legs on walking and
deformities of the ribs, was becoming a more-and-more common problem in large cities in Western Europe and the United
States in this period. It could not be the direct result of an
inadequate intake of calcium salts because it was as rife in areas
in which the water had a high concentration of lime as
elsewhere. It also occurred in well-to-do families in which the
child was plump and well fed. Walter Cheadle, in his review of
the problem, concluded that the common factor was the child
not being breast-fed, but receiving either skim milk or newly
patented artificial foods that were high in starch, but con-
982
CARPENTER
tained much less fat than breast milk. He referred also to the
problem in the London Zoo, where the lion cubs were ignored
by their dams and had been dying of rickets until their diet of
horse meat was supplemented with cod liver oil and ground
bones (50).
Theobald Palm, who had been a medical missionary in
Japan, had been surprised at the complete absence of rickets
there. He organized a world-wide survey by fellow missionaries
of the incidence of the disease and concluded that it was
always absent where people were in long hours of good sunlight, not blocked by industrial smoke (51). Putting the work
of the two authors together it appeared that the disease appeared only when two conditions were met: sunlight was
limited and the child was fed an inadequate substitute for
breast milk. In the following period, these ideas were to be
subjected to further tests with animal models, but there was
already sufficient evidence concerning how the disease could
be avoided.
Infantile scurvy
By 1885, a London pediatrician, Thomas Barlow, had observed in some children with rickets an additional problem
reminiscent of adult scurvy (52). Postmortem examinations
had shown effusion of blood around the ends of the long bones
and the separation of the rib bones from their connecting
cartilage. These were characteristic effects of scurvy in adults
as seen by Lind and other early investigators, but not at all
characteristic of rickets. In France and Germany the same
condition was recognized as becoming common and was called
Barlows disease.
It was being seen increasingly in the United States also,
and by 1897, over fifty papers had been published on Barlows
disease. In the following year the American Pediatric Society
had completed an enquiry into 356 cases in which the method
of feeding was known. Of these, only 12 had been receiving
breast milk, and the great majority had been receiving either
cows milk that had been sterilized or condensed, or
proprietary powders reconstituted with water. It had also
been found that giving children orange juice together with raw
cows milk, or even raw cows milk alone would result in their
recovery (53).
A major cause of death in young children before this time
had been summer diarrhea, which was believed to be caused
in large part by infection due to gross bacterial contamination
of the milk brought into large cities. There had therefore been
an active and successful program in many cities to make
sterilized milk available for feeding to young children. Pediatricians were therefore understandably reluctant to incriminate this product, which was so beneficial in other respects.
The problem was also debated in Paris and Berlin. One idea
was that the heat process resulted in changes to some of the
milk proteins that made them less easily digestible, so that
indigestible residues putrefied in the large intestine and caused
autointoxication. This, of course, was an analogous idea to
Eijkmans explanation for rice starch producing beriberi in
chickens. But the idea was not supported by the condition
being cured, even when sterilized milk continued to be given,
by supplementing it with either potato gruel or orange juice.
No animal model of the disease was available as had been the
case with beriberi.
Adult scurvy
During this period, examples continued to occur of people
on some expeditions in the Arctic succumbing to scurvy even
though they regularly took one ounce of lime juice each day,
whereas on other expeditions in which people were stranded
on land, they kept healthy without lime juice, but did eat raw
or lightly cooked meat and blood (54). Frederick Jackson, the
leader of one successful expedition, concluded: The use of
lime juice neither prevents nor cures scurvy . . . [it] is a disease
developed through eating tainted food. . . . No scientific study
of scurvy has been prosecuted since the discoveries of Pasteur
have shown us the havoc produced by bacteria as a cause of
disease. He put the blame specifically on canned meat, which
had replaced the traditional salt meat that had been taken on
earlier expeditions. He suspected that, before the canning
process, the meat had deteriorated and the bacteria multiplying in it had produced ptomaines and other toxic materials
that survived autoclaving, even though the bacteria themselves were killed by the procedure. He quoted his own experience of surviving by eating fresh game and also the experience of the Hudsons Bay Company who had found that when
scurvy had broken out in one of their depots, it was enough to
send them a good huntsman so that their diet could be
supplemented with fresh meat (55).
On Jacksons return to London, he obtained the collaboration of the Professor of Physiological Chemistry at London
University, and they fed monkeys meat from either freshly
opened cans or from cans that had been left open for several
days, so that the meat had become sour. Unfortunately, the
monkeys had been newly imported and were not acclimatized
to their conditions; all developed diarrhea and died within 8
wk. However, the observers believed that they had seen
spongy gums in 5 of the 8 animals receiving soured meat, and
in none of those eating from the freshly opened cans (56).
Their work was presented to a prestigious audience at a meeting of the Royal Society in London and it was to have
considerable influence.
The next British expedition, this time to the Antarctic, was
provisioned in terms of the ptomaine theory. Before it sailed in
1901 the senior surgeon said: the benefit of the so-called
antiscorbutic is an illusion . . . . An animal food is scorbutic if
bacteria have been able to produce ptomaines in it, . . . otherwise it is not (57). After a winter during which they lived
largely on canned meat that was inspected and approved by
the surgeon, sledging began and, within a very few weeks,
scurvy became a serious problem. The policy was now reversed:
lime juice was placed on the tables at meal times, although still
not made a standard issue, seals were killed to provide fresh
meat and the surgeon began to grow mustard and cress. Gradually, most of the men recovered their health (58).
Guinea pig scurvy
In 1902, Axel Holst, a Norwegian professor of bacteriology
and hygiene who had been concerned at the appearance of
what had been diagnosed as beriberi in the crews of Norwegian
sailing ships, seized an opportunity to visit Grijns in Batavia
and to see his work on chicken polyneuritis. On his return to
Oslo, he attempted to obtain a closer model of ship-beriberi
by using a mammal as his experimental species, and chose
guinea pigs. He fed them grains, either whole or milled, and
found that they all died within 30 d. When the carcasses
were opened he saw pronounced hemorrhages and looseness
of the molar teeth. Theodor Fro lich, a pediatrician with experience of infantile scurvy, confirmed that the condition
appeared to be scurvy with no evidence of any kind of polyneuritis. The two men then found that the condition was not
produced by semistarvation, and that it was prevented by
giving two traditional antiscorbutics, lemon juice and fresh
cabbage (59). They also confirmed that cows milk lost most of
its antiscorbutic activity when it had been autoclaved to
sterilize it.
This was important work, providing an animal model for
scurvy, analogous to that of chicken polyneuritis for beriberi,
and supplying supplementary evidence that the disease was a
deficiency, rather than the result of some kind of intoxication.
TABLE 3
Summarized results from the Wisconsin single grain
experiment, with 4 heifers per group1
Total calves in 2 y
Grain
used
Night blindness and xerophthalmia

The most important work related to these conditions that
was published in this period was a study by Masamichi Mori in
Japan of 1500 cases of the disease known locally as hikan. He
recognized it to be identical to the condition of night blindness and xerophthalmia seen in the West, where some cases
progressed to keratomalacia and even blindness, as did some of
his cases. He described the diets of the weaned young children
as lacking in fat. He found that cod liver oil was the most
effective treatment, olive oil inactive and sea-lamprey oil of
intermediate activity. He surmised that the cod liver oil was
probably the best absorbed of the three. He also considered
animal milk to be protective, but that breast-fed infants could
develop the condition if the mother was also showing signs of
the disease (60). Despite the preexisting knowledge of the
value of animal liver and cod liver oil in the treatment of night
blindness, some textbooks were still failing to recommend
them (61).
Goiter
During this period, there was little advance in the understanding of this condition. In 1895, a leading German surgeon
reported successes from feeding animal thyroid glands to patients, and suggested that the hypertrophy of their own glands
might be their bodys response to receiving too little of their
secretions. However, this treatment fell out of favor again,
perhaps due to its being given in too large quantities, which
had unpleasant side effects (62). The true nature of the problem was not to be established for another 20 years.
Cattle fed single-grain diets
Stephen Babcock, the agricultural scientist famous for developing in 1890 a convenient apparatus for determining the
fat content of milk, was skeptical concerning the usefulness of
the proximate analysis determinations (nitrogen, ether extract, crude fiber, moisture and ash) for the nutritional evaluation of foods and feedstuffs. He is supposed to have irritated
Atwater by telling him that if he really believed in the system,
he should be willing to recommend using cow dung, with its
favorable proximate analysis, as feed for cows. At Wisconsin,
on his official retirement, he was succeeded as Professor of
Agricultural Chemistry by Edwin Hart, and Babcock challenged him to feed breeding heifers ingredients all from a
single cereal grain, and to compare the result with a diet made
up from mixed cereals.
Hart agreed and, with a group of colleagues, used sixteen
6-mo-old heifers and constructed three rations each based
entirely on either corn, oat or wheat products, and balancing
the proportions of ground grain, gluten and straw to obtain the
same energy value and proximate analysis. A fourth ration was
a mix of the other three. The trial was begun in 1906 and
continued for two full reproductive periods; the results are
summarized in Table 3. The heifers receiving the all-wheat
ration quickly lost condition and performed extremely badly,
with none of their calves surviving and two of the cows also
983
Mean weight gain

of heifers in 1 y
Born
lb
Wheat
Mixed
Oats
Corn
353
410
408
471
Healthy
n
5
6
8
8
Mean milk
yield
lb/d
0
3
6
8
12.1
20.6
24.7
26.0
1 Sources: (63,64).
dying before the end of the trial. In contrast, the corn-fed

heifers maintained their condition and had healthy, strong
calves, with the results from the other treatments being intermediate (63,64). This was an expensive trial for the Experiment Station, and the authors, at the conclusion of the final
report, wrote: We have no adequate explanation of our results. However, in the long run it was to prove highly productive because it provided the launch point for the research
programs that were to make the University of Wisconsin a
leading international center for nutritional science.
One man recruited by Hart to work on the single grain
project was a young chemist, E.V. McCollum, who had previously been working with Mendel at Yale. At the end of the
trial, Hart assigned him the task of finding out what was wrong
with (or perhaps deficient in) an all-wheat diet. McCollum felt
that the quantities of feed eaten by cattle were too great for
him to be able to control their quality, and that he must try to
work with a smaller species that would also have a shorter life
cycle. Despite his deans displeasure, he started on work with
rats, and that was the beginning of a considerable saga to be
described in Part 3 of this history.
LITERATURE CITED
1. Voit, C. (1881) Physiologie des allgemeinen stoffwechsels und der
Erna hrung. In: Handbuch der Physiologie (Hermann, L., ed.), vol. 6, Pt. 1, pp.
1575. Vogel, Leipzig, Germany.
2. Atwater, W. O. (1887) How food nourishes the body. Century Mag. 34:
237251.
3. Voit, C. (1889) Ueber die Kost eines Vegetariers. Z. Biol. 25: 232288.
4. Atwater, W. O. & Bryant, A. P. (1900) Dietary studies of university boat
crews. USDA Office of Experiment Stations, Bulletin 75. Government Printing
Office, Washington, DC.
5. Chittenden, R. H. (1904) Physiological Economy in Nutrition. Stokes,
New York, NY.
6. Chittenden, R. H. (1911) The merits of a relatively low protein diet. Br.
Med. J. ii: 656 662.
7. Carpenter, K. J. (2003) A short history of nutritional science: Part 1
(17851885). J. Nutr. 133: 638 645.
8. Greenstein, J. P. & Winitz, M. (1961) Chemistry of the Amino Acids, pp.
249 251. John Wiley & Sons, New York, NY.
9. Chittenden, R. H. (1895) On Digestive Proteolysis, p. 113. Tuttle,
Morehouse & Taylor, New Haven, CT.
10. Bunge, G. (1902) Textbook of Physiological and Pathological Chemistry (Starling, F. A. & Starling, E. H., translators) Blakirton, Philadelphia, PA.
11. Greenstein, J. P. & Winitz, M. (1961) See cit. no. 8, p. 254.
12. Hopkins, F. G. & Cole, S. W. (1902) A preliminary study of a hitherto
undescribed product of tryptic digestion. J. Physiol. 27: 418 428.
13. Willcock, E. G. & Hopkins, F. G. (1906) The importance of individual
amino-acids in metabolism. J. Physiol. 35: 88 102.
14. Abderhalden, E. (1909) Weiterer Beitrag zur Frage nach der Verwertung von tief abgebautem Eiweiss im tierischen Organismus. Z. Physiol. Chem.
61: 194 199.
15. Atwater, W. O. & Bryant, A. P. (1900) Availability and fuel value of food
materials. Storrs Station Report for 1899, pp. 73100. Storrs Agricultural Experiment Station, Storrs, CT.
984
CARPENTER
16. Lusk, G. (1922) A history of metabolism. In: Endocrinology and Metabolism (Barker, L. F., ed.) vol. 3, pp. 378. Appleton, New York, NY. [Reprinted
in Milestones in Nutrition (1964) (Goldblith, S. A. & Joslyn, M. A., eds.), pp. 19 94.
Avi, Westport, CT.]
17. McLean, J. A. & Tobin, G. (1987) Animal and Human Calorimetry, pp.
123. Cambridge University Press, New York, NY.
18. Rubner, M. (1894) Die Quelle der thierischen Wa rme. Z. Biol. 30:
73142.
19. Atwater, W. O. & Benedict, F. G. (1902) Experiments on the metabolism of matter and energy in the human body. USDA Office of Experiment
Stations, Bulletin 109. Government Printing Office, Washington, DC.
20. Atwater, W. O. & Benedict, F. G. (1902) An experimental inquiry
regarding the nutritive value of alcohol. Mem. Natl. Acad. Sci. 8: 231396.
21. Carpenter, K. J. (1994) The life and times of W. O. Atwater (1844
1907). J. Nutr. 124: 1707S1714S.
22. Benedict, F. G. & Milner, R. D. (1907) Experiments on the metabolism
of matter and energy, 19031904. USDA Office of Experiment Stations, Bulletin
175. Government Printing Office, Washington, DC.
23. Bunge, G. (1885) Ueber die Assimilation des Eisens. Hoppe-Seyler Z.
Physiol. Chem. 9: 49 59.
24. Carpenter, K. J. (1990) The history of a controversy over the role of
inorganic iron in the treatment of anemia. J. Nutr. 120: 141147.
25. Hamburger, E. W. (1878 9) U ber die Aufnahme und Ausscheidung
des Eisens. Z. Physiol. Chem. 2: 191205.
26. Stockman, R. (1893) The treatment of chlorosis by iron and some
other drugs. Br. Med. J. i: 881 885, 942944.
27. Boussingault, J. B. (1872) Du fer contenu dans le sang et dans les
aliments. C. R. Acad. Sci. Paris 74: 13531359.
28. Stockman, R. (1895) On the amount of iron in ordinary dietaries and in
some articles of food. J. Physiol. 21: 5557.
29. Carpenter, K. J. (2000) Beriberi, White Rice and Vitamin B: A Disease,
A Cause and a Cure, pp. 2 4, 10. University of California Press, Berkeley, CA.
30. Takaki, K. (1885) On the cause and prevention of kakke. Sei-I-Kwai
Med. J. 4 (suppl. 4): 29 37.
31. Takaki, K. (1887) Special report of the kakke patients in the Imperial
Japanese Navy from 1878 to 1886. Sei-I-Kwai Med. J. 6: 7374.
32. Pekelharing, C. A. & Winkler, C. (1888) Recherches sur la Nature et la
Cause du Be ribe ri et sur les Moyens de le Combattre. Kemink & Fils, Utrecht, the
Netherlands.
33. Eijkman, C. (1990) Polyneuritis in Chickens, or the Origin of Vitamin
Research, pp. 41 43. Hoffman-La Roche, Basel, Switzerland. [An English translation of his papers originally published in Dutch, 1890 96.]
34. Eijkman, C. (1929) Nobel lecture: antineuritic vitamin and beriberi. In:
Nobel Lectures: Physiology or Medicine, 19221941. Elsevier, Amsterdam, 1965.
35. Eijkman (1990) See cit. no. 33, p. 74.
36. Jansen, B.C.P. (1950) C. Eijkman. J. Nutr. 42: 3 8.
37. Carpenter, K. J. (2000) See cit. no. 29, p. 52.
38. Vorderman, A. G. (1897) Onderzoek naar het gevangenissen op Java
en Madoera het voorkomen van beri-beri onder de genterneerden. Jav. Boekh. &
Drukkerij, Batavia, Indonesia.
39. Carpenter, K. J. (2000) See cit. no. 29, pp. 46 51.
40. Grijns, G. (1935) Researches on Vitamins, 1900 1911. J. Noorduyn en
Zoon, p. 38. Gorinchem, the Netherlands (English translation of papers published
in Dutch, 19011909).
41. Hulshoff Pol (1902) Katjang idjo, un nouveau medicament contre le

be ri-be ri. Janus 7: 524- 534, 570 581.
42. Carpenter, K. J. (2000) See cit. no. 29, pp. 88 89.
43. Braddon, W. L. (1907) The Cause and Prevention of Beri-beri. Rebman, London, UK.
44. Fraser, H. & Stanton, A. T. (1909) The etiology of beri-beri. Studies
from the Institute for Medical Research, Federated Malay States, no. 11. F.M.S.
Govt. Print. Off., Kuala Lumpur, Malaysia.
45. Carpenter, K. J. (2000) See cit. no. 29, pp 8190.
46. Chamberlain, W. P. & Vedder, E. B. (1912) The cure of infantile beriberi
by the administration to the infant of an extract of rice polishings. Bull. Manila
Med. Soc. 4: 26 29.
47. Schaumann, H. (1911) Further contributions to the etiology of beriberi.
Trans. Soc. Trop. Med. Hyg. 5: 59 75.
48. Funk, C. (1911) On the chemical nature of the substance which cures
polyneuritis in birds, induced by a diet of polished rice. J. Physiol. 43: 395 400.
49. Suzuki, U., Shimamura, T. & Odake, S. (1912) U ber Oryzanin, ein
Bestandteil der Reisklei und seine physiologische Bedeutung. Biochem. Z. 43:
89 153.
50. Cheadle, W. B. (1888) A discussion on rickets. Br. Med. J. ii: 1145
1148.
51. Palm, T. A. (1890) The geographical distribution and aetiology of
rickets. Practitioner 45: 270 279, 321342.
52. Carpenter, K. J. (1986) The History of Scurvy and Vitamin C, pp.
158 172. Cambridge University Press, New York, NY.
53. Griffith, J.P.C., Jennings, C. G. & Morse, J. L. zems(1898) The American
Pediatric Societys collective investigation on infantile scurvy in North America.
Arch. Pediatr. 15: 481508.
54. Carpenter, K. J. (1986) See cit. no. 52, 146 147.
55. Jackson, F. G. (1899) A Thousand Days in the Arctic. 2 vol. Harper
Bros., London, vol. 2, pp. 387388.
56. Jackson, F. G. & Harley, V. (1900) An experimental enquiry into scurvy.
Proc. R. Soc. (Lond.) 66: 250 266.
57. Koettlitz, R. (1902) The British Antarctic Expedition. Precautions
against scurvy in the victualling of the Discovery. Br. Med. J. i: 342343.
58. Carpenter, K. H. (1986) See cit. no. 52, pp. 152153.
59. Holst, A. & Fro lich, T. (1907) Experimental studies relating to ship
beri-beri and scurvy. II. On the etiology of scurvy. J. Hyg. (Cambridge) 7:
634 671.
60. Wolf, G. 1998 M. Moris definitive recognition of vitamin A deficiency
and its cure in children. Nutrition 14: 481 484.
61. Guggeneim, K. Y. (1981) Nutrition and Nutritional Diseases: the Evolution of Concepts, p. 271. D. C. Heath, Lexington, MA.
62. Ibid p. 283.
63. Hart, E. B., McCollum E. V., Steenbock, H. & Humphrey, G. C. (1911)
Physiological effect on growth and reproduction of rations balanced from restricted sources. University of Wisconsin Agricultural Experiment Station Bull. 17:
131205.
64. Harper, A. E. (1997) Liebigs concept of nutritional adequacy challenged. J. Nutr. 127: 1027S1028S.
65. Funk, C. (1922) The Vitamines, 2nd ed. (H. E. Dubin translator) Williams & Wilkins, Baltimore, MD.
nearly 1,000 papers had been published on vitamins with over
300 on vitamin D alone in just 12 months (6).
It is sometimes asked, Who first had the idea of vitamins?
One can find tantalizing early quotations, but they were not
followed up at the time. In 1804 Thomas Christie, a physician
working in Sri Lanka, wrote, The chief cause of beriberi is
certainly a want of stimulating and nourishing diet . . . .
However, giving acid fruits, which I find of great value in
scurvy has no effect in beriberi . . . . I can suppose the
difference to depend on some nice chemical combination
(7,8). In 1830 John Elliotson lecturing at a London teaching
hospital said that, scurvy is a purely chemical disease . . . each
part of the system is ready to perform all its functions, but one
of the external things necessary for its doing so is taken away
. . . the remedy for this state is fresh food (9). In 1842 George
Budd also lecturing in London added, Scurvy is only one of a
number of diseases due to specific dietary deficiencies, another
is rickets and a third is characterized by a peculiar ulceration
of the cornea (10,11). Gerrit Grijns pronouncement already
quoted in A Short History of Nutritional Science: Part 2
(18851912), (2) was probably the first clear statement, based
on his own work, of the existence of an organic nutrient
required only in small amounts.
In the early years of the 20th century, the number of

researchers engaged in nutritional work expanded enormously.
Space does not permit more than an introduction to the
proliferation of literature on any specific topic and it has
unfortunately not been possible to fully distribute the credit to
several groups of researchers working on the same problem.
Also I have probably not given a due share to those publishing
in languages other than English. When a development has
been presented in a series of papers, I have tried to cite a
relatively recent one because it is easier for readers to work
their way back through the literature than try to go forward.
The vitamin era
In 1912 Casimir Funk wrote that the antiberiberi factor,
because of the different conditions under which it could be
extracted from rice polishings and then precipitated, must be
an organic base and therefore contain an amine group. He
went on to suggest that pellagra, scurvy and rickets were
caused by deficiencies of other yet to be identified factors. This
was not original, but he then went on to hypothesize that
these factors would all be found to have the same basic
character, i.e., to be vital amines, for which he coined the
name vitamines (3). It seems that he felt intuitively that
they would be chemically similar in a manner analogous to the
components of protein having different properties even
though they were all amino acids. This was a leap of faith and
not a correct prophecy, but some 10 years later he would claim
that the name contributed in no small measure to the dissemination of these ideas (4). Another group wrote in agreement, the name was a stroke of genius . . . such a captivating
word . . . necessary to focus attention upon the possibilities of
the field (5).
Certainly vitamins (with the e omitted after it was
realized that they could not all be amines) formed a new vein
of knowledge waiting to be mined and were to be the major
topic of nutritional research for the next 30 years. Looking for
example, at the first two volumes of The Journal of Nutrition,
published from 1928 1930, we see that more papers (some
40%) dealt with this general topic than any other. In 1933
Leslie Harris, who had been given the responsibility of reviewing the previous years work in the field, pointed out that
Rats and mice fed purified diets

Previously most work started with a diet that had been
found in practice to be associated with a particular disease, and
this type of work was to continue with important results that
we shall consider shortly. However, from this point on, much
of the productive work was to start from the bottom, so to
speak, with purified diets, meaning mixtures prepared from
only the major nutrients (protein, carbohydrates and fat) in
forms as pure as possible, as well as minerals. These diets would
be deficient in all of the hypothetical vitamins, and the task
would be to find what combinations of supplements would
restore them to promoting good performance. McCollum explained, The older practice of experimenting with combinations of natural foodstuffs is not searching enough in character
to reveal any of the fundamental principles of nutrition, or to
. . . lay the foundation of a system of feeding based upon
scientific principles (12).
The work of E. V. McCollum
To begin with I will follow the progress in the field through
the work of McCollum and his volunteer assistant Marguerite
Davis at the Wisconsin Agricultural Experiment Station attached to the University of Wisconsin; they were certainly
1
This is the third of four invited papers on the history of nutritional science.
The first and second papers in the series were published in the March and April
2003 issues of The Journal of Nutrition (1,2).
2
Manuscript received 29 May 2003.
3023
3024
CARPENTER
leading the pack in the early years (13). It is interesting in

view of the importance of their discoveries that McCollums
young colleagues at Yale told him that he was foolish to accept
a position in which he would be working in nutrition since,
the subject was already worked out, with nothing remaining
to be discovered! (14). He had been recruited from Yale, as
has been previously related (2), to work on the single-grain
experiment with cattle that had been producing dramatic and
inexplicable results.
Searching the literature
McCollum had already mastered German and began with a
careful reading of the earlier European work summarized in
Malys Jahresbericht uber die Fortschritte der Tier-Chemie, having
bought the 37 volumes describing work published between
1870 and 1907. He found there descriptions of 13 experiments
in which efforts to maintain small animals on purified diets
had all failed. The influential professor Von Bunge believed
that the elements iron and phosphorus needed to be part of
organic complexes in order to be absorbed, and he attributed
the failures of these diets to the complexes having been decomposed by the purification procedures rather than to lack of
any unknown nutrient (1517). In any case the studies had
not been followed up.
Cornelis Pekelharing, whose work as the leader of the
Beriberi Commission in Indonesia has been previously described (2), also reported that mice would not thrive on a
simplified diet of casein, egg albumin, rice flour and minerals.
He buried his brief announcement in a 1905 paper on a
different subject, but made it clear that the mice were deficient
in something that he could supply to them in whey (i.e., milk
with the fat and casein removed). He added that he would say
no more about it because he could not identify the missing
factor. Indeed his work remained generally unknown until
translated into English 20 years later (18) and McCollum was
not aware of it. Strangely, Gowland Hopkins, Professor of
Biochemistry at Cambridge University, said something very
similar in the following year. Toward the end of an obscure
published lecture we find, No animal can live upon a mixture
of pure protein, fat and carbohydrate, and even when the
necessary inorganic material is carefully supplied the animal
still cannot flourish. . . . In diseases such as rickets, and
particularly in scurvy, we have had for long years knowledge of
a dietetic factor; but, though we know how to benefit these
conditions empirically, the real errors in the diet are to this day
quite obscure. They are, however, certainly of the kind . . . that
I am considering. Many years later in his Nobel Prize speech,
he said that he had based his remarks on studies he had made
with mice in 1906 1907, but like Pekelharing had thought
that they would not be taken seriously until he had identified
the missing factor (19,20). He was probably the first to associate the deficiencies of a purified diet with human disease
conditions.
It is interesting that McCollum had also failed to find any
reference to the important work of Eijkman and Grijns in
studying the disease produced by feeding chickens white rice
and the latters conclusion that it was caused by deficiency of
a relatively unstable water-soluble organic compound (2).
In 1909 a Swiss ophthalmologist, who had seen in another
trial the mention of eye lesions appearing in rats fed a purified
diet, repeated the experiment and identified the xerophthalmia and keratomalacia that he saw with conditions found in
human subjects that had proved responsive to cod liver oil
(21,22).
McCollums first trials

It had been speculated at this time that animals needed to
be supplied with phosphorus in the form of nucleic acids.
McCollum set out to test this by using vegetable proteins
prepared without any significant phosphorus compounds attached. He fed three rats a mixture of the plant proteins
edestin (from hemp seed) and zein (from corn) at levels
providing 1218% protein in powdered diets that also included 5% butter fat and 8% minerals as well as starch and
cane sugar, all mixed with a little finely divided cellulose and
water, and dried at 38C. The rats, initially weighing 100 170
g, all lost weight and showed little appetite after one week. He
tried adding different flavorings, e.g., banana, bacon etc.,
which restimulated the appetite for a day or two, and concluded that the lack of palatability was the limiting factor with
such diets. However, from phosphorus balance data he concluded that rats could manufacture their own nucleic acid
without the need for either organic phosphorus or purines
(16). In Connecticut Osborne and Mendel confirmed this last
point in 1911 (23). These workers were primarily interested in
being able to compare the nutritional values of a variety of
isolated proteins tested in diets with no other protein source
present. However, in order to do that they needed a basal
protein-free mixture that would meet all the other needs of
young rats, and that brought them willy-nilly into the vitamin
field.
Factors A and B
McCollum could now use the phosphoprotein casein as his
purified protein source without worrying about its phosphorus
content and he experimented to improve his mineral mix. In
1913 he reported with Marguerite Davis that rats fed diets
containing 1218% casein, 20% lard, 20% lactose, 6% minerals and starch, would grow well for 8 to14 weeks but then
stop and in some cases lose weight. However, growth would
restart if the rats were given 1 g of ether extract of egg on
alternate days. In the following year they reported that butterfat, but neither olive oil or cottonseed oil, would also
stimulate rats to grow again. They also reported that if butterfat were saponified and the emulsion shaken with olive oil, the
olive oil was then active in restarting rat growth, which
suggested that the active factor was nonsaponifiable and remained fat soluble (24). Again the need for a fat-soluble
substance was confirmed by the Connecticut group who also
reported the high potency of cod liver oil (25).
By this time, Hopkins had argued that the two American
groups had only been able to obtain growth with their diets
because the casein and lactose were insufficiently purified from a
water-soluble growth factor (26,27). McCollum and his colleagues looked into this and agreed, concluding that rats needed
both a fat-soluble Factor A and a water-soluble Factor B that
was identical to the antiberiberi factor deficiency which developed in chickens and pigeons fed white rice (28).
Here we see the beginning of the scheme for identifying
vitamins by letters before their chemistry had been worked
out. They then reported that leaves showed Factor A activity even though their ether extracts and also the plant oils that
they had tested did not do so. It was also noticed that the
deficiency of Factor A resulted in severe ophthalmia. This of
course linked up with the clinical work previously described
dealing with night blindness leading to xerophthalmia and its
prevention with cod liver oil (2). We will return to the slow
and complex progress in understanding Factor A in a later
section.
3025
Rickets and vitamin D

In 1917 McCollum moved to the newly established and
well-funded School of Public Health at Johns Hopkins University in Baltimore. There, in further rat experiments, he
discovered that he had produced an experimental model of
another human disease, rickets.
As previously described, rickets had become a serious problem among young children in the large industrial cities of
Western Europe and the northern United States (Fig. 1) (2).
It was particularly serious in Glasgow, Scotlands largest city,
whose medical school had an active group concerned with the
problem. They reported in 1908 that puppies fed bread or
oatmeal with whole milk would develop rickets if kept indoors,
but not if taken for outdoor walks (29). From a study conducted in the citys slums, it was concluded that inadequate
fresh air and exercise were potent factors in determining the
onset of rickets, and in a further trial with puppies it appeared
that having an outside run was more important than the
intake of milk fat (30).
In contrast Edward Mellanby in England, who had worked
under Gowland Hopkins and was familiar with the subject of
accessory food factors, reported in 1921 that he had produced
rickets in puppies kept indoors by limiting their milk intake to
200 mL/d. However, he could prevent its occurrence with
supplements of a variety of foods including butter and cod liver
oil without allowing the dogs outdoors or giving them exercise
(Fig. 2) (31). He and the Scots were therefore understandably
skeptical of each others conclusions, and it seemed to be an
additional insult when Mellanby reported at a meeting held in
FIGURE 1 A sister and brother (aged 5 and 3 y, respectively)

showing the effects of earlier rickets, Rachford, 1912 (126).
FIGURE 2 A puppy with rickets as a result of being reared indoors

on a rachitic diet, Mellanby, 1921 (31).
Glasgow that oatmeal had a rachitic effect on his puppies. This

was the traditional cereal food of the Highlanders who prided
themselves on their physique. In reaction a Scottish newspaper published a cartoon of an addle-brained professor saying
that porridge was useless because it did not agree with his dog,
and then being offered some bones to see how he liked them
(32,33).
The controversy was soon resolved. Exposing an infant to
sunlight had already been one of the traditional folk treatments for rickets in Northern Europe, and in 1919 exposure to
ultraviolet lamps had also been reported to be effective. At
this time, in the aftermath of World War I, there had been
great food shortages in Central Europe, and a group of scientists financed by the Society of Friends and led by Harriette
Chick of Londons Lister Institute went to Vienna where the
well-equipped Childrens Hospital had many cases of rickets.
There they were able to demonstrate, with the aid of X-ray
photographs, that either the use of cod liver oil or irradiation
with ultraviolet light would cure the condition, which otherwise developed even under the most hygienic conditions (34).
By this time, McCollum and his medical colleagues at Johns
Hopkins had, as already mentioned, found that the more
convenient rat could also be used to provide a model for
rickets if its diet were severely imbalanced in ratio of calcium
to phosphorus. They then found that cod liver oil would
prevent the disease even after it had been aerated in a way that
destroyed its antiophthalmic (factor A) value (35).
The next extraordinary finding, back in Wisconsin in 1924,
was that not only did irradiation of rachitic rats with ultraviolet light have a curative effect, but so did irradiation of the
diet from which they had developed the disease (36). Many
groups tried to determine what factor might be activated in
this way. It was quickly traced to lipid, then to the sterol
fraction and finally to ergosterol and, in 1931, the activated
material itself now named vitamin D was crystallized (37).
CARPENTER
3026
(The letter C had already been allocated to the antiscorbutic

vitamin.)
With this new knowledge rickets ceased to be an intractable public health problem. It was later realized that there was
more than one form of active vitamin, and that their relative
activities depended on the species under consideration.
Experimental scurvy
Surprisingly, after the discovery in 1907 that guinea pigs
could provide an animal model for scurvy, very little use was
made of them for several years (2). McCollum became interested because his rats clearly did not need antiscorbutics to
survive, and if species could differ drastically in their requirements it would be a challenge to the general significance of
results obtained with rats. He and a colleague fed guinea pigs
a combination of oats milk. Some survived, but others
died and were found to have the cecum distended with
putrefying feces that they assumed to be causing autointoxication. In 1917 on the basis of this one finding, they wrote,
The significance of this interpretation is far reaching: it
removes from the list one of the syndromes [scurvy] which has
been generally accepted as due to dietary deficiency (38).
Harriette Chick and E. M. Hume, the first independent
women scientists to be mentioned in this history, were soon
able to demonstrate that cows milk had only a low antiscorbutic activity; guinea pigs receiving an oats milk diet
needed some 50 mL per day to remain healthy and if the milk
went at all sour they would not touch it. It was essential
therefore to monitor individual consumption in order to interpret the results. Scurvy consistently appeared with autoclaved milk that had apparently lost most of its vitamin C
activity (39).
Because of suspicions that commercial lime juice was not an
effective antiscorbutic, the same group now used guinea pigs to
test this in what may have been the first bioassay for vitamin
activity. They found that commercial lime juices had less than
one tenth the activity of freshly squeezed lemon juice (Table
1) (40). The processing almost certainly included pumping
through copper pipes and probably some form of sterilization.
This of course confirmed that the Victorians had been correct
to doubt that lime juice as they knew it was effective in
preventing outbreaks of scurvy on long expeditions.
Other workers now attempted to isolate vitamin C from
lemons, which proved difficult because of its instability.
Strangely, it was first achieved in 1928 by Albert SzentGyo rgi, who was not seeking a vitamin but a factor involved in
the catalysis of oxidation-reduction reactions in mammalian

metabolism. He used adrenal cortex tissues from slaughterhouses and rapid in vitro assays (41). Only four years later after
its isolation from lemon juice by another group was it realized
that Szent-Gyo rgi had already isolated the vitamin (42).
Then, in a remarkably short time, chemists were able to
determine its molecular structure, synthesize it and confirm
the products biological activity (43). Vitamins could no
longer be described by skeptics as hypothetical entities.
In 1939 a surgeon at Harvard Medical School fed himself a
diet containing no vitamin C, but supplements of all other
vitamins. After 26 weeks he developed hemorrhages on his
legs, a wound inflicted on his back failed to heal and he rapidly
became exhausted. Upon dosing with ascorbic acid these problems quickly disappeared (44).
Beriberi and vitamin B
We have seen already that McCollum and others had
produced signs of polyneuritis in rats using purified diets with
a source of vitamin A and had characterized the antiberiberi
factor as vitamin B. It was then realized that autoclaved yeast,
although it had lost its anti-neuritis activity, still promoted
growth in rats so that it must contain a second factor; these
were then called B1 and B2, respectively.
The isolation of B1 was achieved in 1926 by Dutch scientists in Java using small rice birds fed on washed white rice
supplemented with cod liver oil for their assays. Starting with
nearly 700 pounds of rice polishings, they obtained 100 mg of
crystals so potent that only 10 g was needed to cure a
deficient pigeon (45). The next problem was to determine the
structure of the crystals of the chloride salt that had an
empirical formula of C12H18Cl2N4OS. This was finally
achieved and a biologically active compound synthesized in
1936 (46,47). It was named Thiamin(e) as the vitamin containing sulfur (thios in, Greek).
Meanwhile Rudolph Peters at Oxford was investigating the
function of the vitamin. It was known that deficient subjects
maintained unusually high levels of pyruvic or lactic acid in
their blood after exercise, and his group obtained evidence
that thiamin pyrophosphate served as a cofactor for the enzyme pyruvate decarboxylase. He introduced the term biochemical lesion to describe the effect of its deficiency (48).
This was the first of a long series of findings that B-vitamins
generally served as part of coenzymes concerned with different
aspects of metabolism.
Pellagra in the United States
TABLE 1
A selection of the results obtained from feeding guinea pigs a
scorbutic diet supplemented with lemon juice or lime juice
from different sources (38)
Material tested
Lemon juice, fresh

Lemon juice, fresh
Lime juice, fresh
Lime juice, fresh
Lime juice, navy issue (stored)
Lime juice, navy issue (stored)
Lime juice, 6 commercial samples
Daily dose
Degree of
protection1
ml
1.5
2.5
2.5
5
5
10
5
0
0
0
1 Full protection, ; partial, ; slight, ; none, 0.
This is a disease characterized by dermatitis of areas exposed

to sunshine, and gastrointestinal and mental disturbances (Fig.
3). There probably were a few earlier cases of pellagra in the
Southern States, but from 1905 it became common. At one
mental hospital in 1906 there were 88 sufferers of whom 57
died (49). By 1909 pellagra had spread to many parts of the
South, and nearly 350 physicians attended the first national
conference held in South Carolina. There were many ideas as
to the cause of the disease. One from Italy, where pellagra had
long been a problem, was that corn that had been damaged or
inadequately dried became moldy with pathogenic fungi (50).
Another, because it occurred mostly in the poorer areas, was
that flies made their way into inadequately screened outdoor
privies and then carried infected excreta onto food being
prepared in kitchens (51).
Joseph Goldberger, put in charge of the Public Health
Services pellagra program in 1914, began systematic investi-
3027
extracts. Early work with monkeys and rats failed to elicit

anything resembling the human disease. McCollum too had
been finding that rats failed to show anything like pellagra
when fed diets resembling those used in Goldbergers prison
experiment and referring to the doubts as to whether pellagra
had been produced there, he concluded that probably . . .
pellagra is caused by an infectious agent (55). So the man
whose name is linked to the discovery of vitamins wanted at
one point to erase both pellagra and scurvy from the list of
deficiency diseases.
Goldbergers group persevered with searching for an animal
model. Dogs given mixtures with mostly cornmeal and no
meat or milk powder developed a condition called blacktongue, showing red lips with patches of necrosis, drooling
and loss of appetite. The group considered this to be a model
for pellagra after the dogs responded rapidly to yeast, and this
proved equally valuable for pellagrins (57).
Dogs were then used to assay fractions obtained from yeast and
liver. Finally in 1937 after nicotinic acid had been found to be a
bacterial growth factor, both it and nicotinic amide were found in
Wisconsin to be extremely potent in curing blacktongue, and also
pellagrous patients in Alabama (58,59). This was the only example of a vitamin, an already familiar chemical, now being given
the blander name of niacin. This is not the end of the story; it
will be continued in Part 4 of this series.
Riboflavin
FIGURE 3 A patient with pellagra showing dermatitis on the parts

of her body that had been exposed to strong sunlight, H.F. Harris, 1916
(127).
gations. Because nurses or physicians treating the sick never

developed the condition, he believed that pellagra was not
infectious and was willing to put his life on the line for it. He
received subcutaneous injections of blood from patients, then
had skin eruptions rubbed into his nasal mucosa, and finally
ate some of their excreta; then some of his fellow physicians
followed (52). It was a tradition of the Service that officers
would expose themselves to the same degree of risk as those in
the Armed Services. In this case none was made ill.
Goldberger thought that an unbalanced diet was responsible and persuaded authorities in Mississippi to allow 12 prisoners to volunteer to eat for six months an experimental diet
that might induce pellagra. In return the prisonerswould be
released at the end of the periodif still alive! The diet had
abundant corn and other cereals but no meat or dairy products.
After five months, six of the men had developed dermatitis on
the scrotum and in a few cases, on the back of their hands
(53). Goldberger was satisfied that this was pellagra, but the
volunteers immediately fled after obtaining their release and
he could not demonstrate their condition to physicians who
doubted whether he truly had produced the disease (54).
In another study in South Carolina mill villages, Goldbergers
group found that pellagrous families had purchased a very similar
pattern of foods to that of healthy families, but the latter nearly
all kept a cow and were obtaining abundant milk (55).
They now tried to find an animal model with which they
could assay the antipellagrous activity of different foods and
It was soon realized that the B2 in autoclaved yeast was a

complex of factors. The first to be investigated, and originally
called vitamin G, was tentatively identified with the greenfluorescent pigment of whey (60). It was isolated and then
synthesized in 1935, and named riboflavin (61). It was then
discovered that some dogs on a blacktongue diet that had
collapsed with a prior condition of yellow liver, could be
saved by giving them riboflavin. It was also found that pellagrins who still showed cheilosis (lesions about the mouth) after
being treated with niacin responded rapidly to riboflavin (62).
It appeared that most patients with signs of pellagra were also
at least marginally deficient in riboflavin in addition to niacin.
Folic acid
The trail to the discovery of this vitamin began in India.
Lucy Wills went to Bombay in 1928 to investigate the macrocytic (large cell) anemia of pregnancy most commonly seen
in Mohammedan women. After failing to associate the condition with infection or deficiency of vitamins A or C, she
found that yeast and its proprietary extract Marmite were
highly effective in curing the condition (63). Back in England
she and colleagues reported in 1937 that feeding a poor Bombay diet to rhesus monkeys also induced macrocytic anemia
and leucopenia; this responded to both Marmite and to crude
liver extracts, but more refined liver extracts given parenterally, and effective in patients with pernicious anemia, had no
effect (64). Others confirmed that the condition in monkeys
did not respond to any of the known vitamins and called the
deficient factor vitamin M (for monkey) (65).
In the same period workers interested in poultry nutrition
had found that chicks fed purified diets containing all of the
then known vitamins still grew slowly and developed a macrocytic anemia, and in 1944, that this could be prevented by
giving them crystalline vitamin Bc isolated as a growth factor
for certain bacteria (66). The same or a closely related compound had also been obtained from spinach and named folic
acid because it had come from foliage. These materials were
CARPENTER
3028
also active in treating the experimental monkey anemia and it

was hoped that they would be clinically effective in India
when supplies became available (65).
The chemical identification and syntheses of these active
compounds will be considered in Part 4.
Other B vitamins
Once it was clear that vitamin B-2 was a combination of
different factors, the race was on to identify them. Space does
not permit describing this demanding work but in principle, it
was of the same type that had been used to separate and
identify the first vitamins discovered using baby chicks, yeast
and lactobacilli, as well as rats (68,69). By 1937 pantothenic
acid, B6 [the pyridoxine (PN) group] and biotin were added
to the list of water-soluble vitamins (Table 2). Choline also
was recognized to be essential for poultry in addition to having
a lipotropic value for mammals under some conditions (70).
The existence of other factors, including B4, B10 and B11 was
claimed and then quietly abandoned (71).
The fat-soluble vitamins
Vitamin A and carotene. In the first section of this history
Part 3, we reviewed early work that led to the concept of
vitamin A as a fat-soluble vitamin needed by young rats to
support growth and to prevent the development of xerophthalmia. In Denmark during World War I, when fats were in
short supply, an unintended experiment occurred in a childrens home. In one group of 16 children, 8 developed xerophthalmia, while no cases developed in a second group. The only
difference in the diets of the two groups was that the second
had received whole cream milk in the previous six months.
Carl Bloch, the pediatrician in charge, then began to give cod
liver oil to the affected group; their eye problems cleared up in
eight days and they began to grow faster (72,73). Clearly, the
work with rats had some practical relevance.
The problem now was to identify the vitamin, which appeared to exist in at least two forms: a highly colored form in
leaves and carrots, and a colorless form in animal fat. Crystals
of -carotene, a polyunsaturated hydrocarbon, were obtained
from carrots and found to be active. The colorless factor was
more difficult to obtain but the activity of extracts correlated
with a characteristic color formation with antimony trichloTABLE 2
Timelines for the discovery of the vitamins up to 1944.
Modified, with permission, from Combs, 1992 (128)
Vitamin
Thiamin
Vitamin C
Vitamin A
Vitamin D
Vitamin E
Niacin
Biotin
Vitamin K
Pantothenic acid
Folate
Riboflavin
Vitamin B6
Year
proposed
Isolated
Structure
determined
Synthesis
achieved
1901
1907
1915
1919
1922
1926
1926
1929
1931
1931
1933
1934
1926
1926
1939
1931
1936
1937
1939
1939
1939
1939
1933
1936
1936
1932
1942
1932
1938
1937
1942
1939
1939
1934
1938
1936
1933
1932
1938
18671
1943
1940
1940
1935
1939
1 This compound, nicotinic acid, was known long before its vitamin
activity had been discovered.
ride that could be differentiated from the color obtained with

carotene. It was then found that giving carotene to rats depleted of vitamin A resulted in the reappearance of the color
reaction of the animal factor in extracts from their livers
(74). Thus carotene appeared to be a precursor of the final
vitamin, and this was confirmed when the actual vitamin was
finally isolated from fish liver oils in 1939 using centrifugal
molecular stills, and its structure identified (75,76).
Synthesis proved particularly difficult. A major contributor
wrote, After so many years, victory has come and the romance of high hopes and bitter disappointment will in a few
years simply be recorded in textbooks of organic chemistry in
a few terse sentences (76). Sad but realistic. The vitamin,
now named retinol, was an alcohol attached to a long
unsaturated carbon chain linked in turn to a -ionone ring,
and -carotene could be considered as two retinol molecules
condensed through their alcohol groups.
Vitamins E and K. In 1922 H. M. Evans and Katharine
Bishop working at Berkeley found that a purified diet with
vitamin supplements that supported good growth in female
rats nevertheless failed to support normal reproduction; the
embryos were being resorbed before the end of pregnancy (77).
Lettuce was the first food found to prevent this problem, but
then wheat and in particular, wheat germ oil. Cod liver oil
seemed unexpectedly to increase the problem. The active
factor was named vitamin E and following further investigations by many groups, it was isolated in 1935 and named
tocopherol (from Greek terms signifying the childbirthproducing alcohol). Three years later the Swiss chemist Paul
Karrer synthesized it by condensing phytyl bromide with trimethyl hydroquinone (78). He too received a Nobel Prize for
his work on the chemistry of several vitamins.
During this period there was a change in thinking about the
effects of a deficiency of vitamin E. In rats, males showed
testicular degeneration, but it was realized that in pregnancy it
was the fetus that failed to develop rather than the dam being
at fault. It was also found that vitamin E deficiency in lambs
and rabbits resulted in muscular degeneration rather than
infertility; and in chicks it resulted in exudative diathesis
and/or encephalomalacia, both related to disturbances in the
vascular system (79).
Vitamin E became a popular treatment, backed by reports
of early successes, for a number of clinical conditions including
abortions, impotence and various forms of muscular dystrophy,
but with more controlled testing, few if any of these claims
could be confirmed (80).
Hemorrhaging in chicks, which responded to dosing with
cabbage, was another disease that at one time was thought to
possibly be caused by a deficiency of vitamin E. The Danish
worker Henrik Dam reported in 1935 that it was the deficiency
of a new fat-soluble vitamin, which he named vitamin K in
recognition of its essential role in blood coagulation (Koagulation in Danish and German) (81). It was discovered to
occur naturally in modified forms in many plants and to be
produced by bacterial growth in stored animal products
(82,83). The vitamin also cured the hemorrhaging of patients
with obstructive jaundice who lacked bile to aid absorption of
the vitamin and of cattle that had been eating sweet clover
hay that contained an anti-vitamin (84).
Herman Almquist and a colleague at Berkeley could have
published the discovery of vitamin K before Dam, but had to
delay publication until a controversy on campus about the
cause of the chick disease had been resolved and so missed a
Nobel prize. By this period there were so many people at work
on nutritional problems that such things were almost inevitable. Many years later Almquist was to write philosophically,
More often than not, a discovery is really born of a converging culmination of scientific leads . . . to all of which many
have contributed . . . a stage has been set . . . there are many
who can do the job if they happen to be on the scene (85).
Essential fatty acids
In 1929 George and Mildred Burr, who had moved from
working with H. M. Evans and were now at the University of
Minnesota, went to great trouble to prepare diets that were
completely free from fat using sucrose instead of corn starch
(because the latter contains 0.7% lipid unextractable with
ether) and supplying vitamins A and D by saponifying cod
liver oil and adding only the nonsaponifiable fraction. They
found that rats fed such diets for many months failed to reach
their normal mature weight; they also lost fur and their tails
became inflamed and scaly. In the following year they found
that giving the animals small quantities of methyl linoleate,
but not butterfat, prevented the condition (86). Much more
would be discovered later about the functions of the polyunsaturated acids.
Proteins and amino acids
We now return to the beginning of the period. One aim of
the early work with rats was to investigate the relative nutritional value of different proteins and this became possible with
diets that included protein-free milk (skimmed milk acidified,
heated and filtered). Osborne and Mendel had already found
that gliadin, isolated from wheat, supported far slower growth
than was obtainable with casein (Fig. 4). Now they found that
with 18% zein, the corn protein that lacked tryptophan and
lysine, rats failed to grow unless the diet was supplemented
with both these amino acids (87,88). It appeared therefore
that certain amino acids were essential, meaning that they
could not be synthesized by the body and needed to be provided in the diet.
Further work showed that mixing proteins deficient in
different amino acids could result in mutual complementation
(89). However, proving that the value of proteins was deter-
FIGURE 4 Two female rats from the same litter at 140 d of age
after receiving: (A) 18% casein as the sole protein source, and (B)
gliadin (from wheat); other diet components being identical, Osborne &
Mendel, 1911 (23, Plate 1).
3029
mined only by their digestibility and amino acid balance took

considerably longer.
Chemical methods of amino acid analysis were slow and
subject to interference (90). However, microbiological methods taking advantage of the complex requirements of lactobacilli were developed in the 1940s and allowed many more
assays to be completed (91). From these it was possible to
determine the chemical score of food proteins calculated as
the lowest percentage value when the level of each essential
amino acid was compared with that found in whole egg (taken
as a provisional standard). These scores were then found in
most cases to correlate well with the relative values of the
same foods as determined in protein quality tests with young
rats, based either on weight gain or nitrogen balance (92).
In 1930 William Rose, who had earlier been a graduate
student with Mendel and attended Chittendens lectures, was
a professor at the University of Illinois, Urbana campus. He
and his group set out to develop amino acid mixtures that
would support good growth in rats. They began with 19 known
amino acids, but the animals failed to grow. With hindsight it
is surprising that their list did not include methionine, discovered at Harvard in the 1920s as being required for the growth
of certain bacteria (93). However, it was largely ignored by
nutritionists for another decade, perhaps because they knew
that rats receiving casein as their sole protein would grow
faster if cystine were added to the diet, so that cystine appeared
to be the sulfur-containing essential amino acid. Even with the
inclusion of methionine, rats still failed to grow at Urbana
unless they received in addition the mix of amino acids obtained from an acid-hydrolysate of casein.
Attempts to isolate an active factor from the hydrolysate
proved very frustrating until it was realized that it was supplying two additional materials. With further careful fractionation, Madelyn Womack working with Rose, discovered that
one was isoleucine (94). At that time there was no method for
determining isoleucine separately from leucine. It was included in the original mix, but at a level well below what
turned out to be its requirement.
The second factor was a previously unknown amino acid,
which they identified as 2-amino-3-hydroxybutanoic acid and
named threonine (95). When it was included in the amino
acid mix the rats grew well so that this was a most important
finding. Reviewers have referred to the very high level of
chemical competence and skill with which these studies were
conducted and, for example, to fivefold crystallizations of
tryptophan and histidine, even after analyses had shown
them to be essentially pure (96).
Starting in 1942, Rose set out to extend the study to human
adults. This involved preparing much larger quantities of
amino acids, and then careful control of the energy intakes of
volunteers and measurement of their nitrogen balance. All of
this took time and the findings will be considered in Part 4.
Meanwhile, the availability of isotopes had allowed a new
approach to studying the fate and distribution of nutrients in
the body. In 1939 Rudolf Schoenheimer and his colleagues at
Columbia University reported results from feeding rats for
three days a physiological dose of l-leucine, doubly labeled
with N15 and deuterium (replacing hydrogen in the side
chain). They found that less than one third of the N15 had
appeared in the urine, but that 57% was incorporated into
body proteins, much of it in other amino acids with the
exception of lysine (97). It was assumed that this was the
consequence of transamination reactions.
Schoenheimer thought at this time of protein molecules
opening to release one amino acid molecule at a time into the
blood stream before reattaching a replacement. In any case, it
3030
CARPENTER
appeared that most synthesis of body proteins must be coming

from recycled amino acids rather than from newly digested
dietary protein. As he was to write, If the starting materials
are available, all chemical reactions which the animal is capable of are carried out continually and The synthesis of
amino acids, like that of fatty acids . . . proceeds even when
there is no obvious need for it (98).
Mineral elements
The first workers with purified diets felt that they could meet
their animals mineral requirements by giving them the residual
ash from combustion of an apparently satisfactory food such as
milk or dog biscuits. Then when the problems of supplying
vitamins and amino acids had been solved they could begin to
study individual mineral requirements. One early finding, already
quoted, was that rats would become rachitic if either the calcium
or phosphorus content of the diet was greatly increased in the
absence of vitamin D or irradiation.
The development by 1929 of analytical procedures using
emission spectroscopy allowed the detection of trace elements
in foods. Thus, it was found that cows milk contained strontium and vanadium in addition to the previously detected
iron, copper, zinc and manganese, as well as larger quantities of
calcium, magnesium, potassium, sodium and phosphorus (97).
Chlorine and iodine had been found using other procedures.
There was no doubt in the minds of workers that the more
abundant elements were essential, and studies now began on
possible requirements for the trace elements (100).
At Wisconsin copper had already been found to be required
for the production of hemoglobin in rats fed purified diets
(101). Then in 1931 a deficiency of copper was found to be
responsible for a characteristic sickness occurring in cattle in
parts of Florida (102). In the same year McCollums group
reported results with young rats fed a purified diet designed to
be as low as possible in manganese content. They grew normally, but the males were sterile with testicular degeneration,
and the females would breed when mated with normal males,
but were unable to suckle their young (103). Again, a few years
later manganese deficiency was recognized at Cornell as being
a practical problem, this time in intensive poultry production
and responsible for perosis (a crippling deformity of the leg
bones) in young birds (104).
Magnesium was known to be present in both bones and soft
tissues of animals but it proved difficult to obtain a deficiency
condition in rats. Finally, by feeding rats a diet that after
careful purification contained only 1.8 g/g of the element,
McCollums group produced a characteristic condition of tetany (105). In contrast, grass-fed cattle would sometimes develop tetany that responded to dosing with magnesium salts
even when the level in their blood appeared normal (106).
It also proved possible to produce zinc deficiency with a
highly purified diet on which rats showed slow growth and loss
of hair (107). However, no evidence of zinc deficiency being a
practical problem was seen in the period under review. In
contrast, serious problems affecting cattle and sheep in parts of
Australia were recognized in 1937 as being due to cobalt
deficiency without its having first been produced experimentally in laboratory animals (108). The special function of
cobalt in ruminant nutrition would only be worked out in a
later period.
The importance of iron in the prevention of microcytic
anemia in human subjects was previously discussed (2). Interestingly, iron deficiency had been much less of a problem in
traditional animal husbandry, in large part because animals
had access to soil which is rich in iron. However, when sows
began to be brought indoors in the 1920s for farrowing, the

piglets had a higher death rate. This was found to be the result
of anemia and workers in Scotland prevented it by adding iron
salts to the sows feed (109). It was thought that the piglets
obtained additional iron from contamination with her feed
and feces rather than from any additional iron appearing in the
sows milk and this was confirmed in Wisconsin (110).
In 1937 Robert McCance and Elsie Widdowson, working in
London at that time, published a classic paper arguing that
contrary to current opinion, humans had little or no ability to
excrete iron, and that there must therefore be a mechanism
that regulated its absorption according to need (111). This was
confirmed by work with dogs using radioactive iron when it
became available from the Berkeley cyclotron. It was interesting that absorption did not increase immediately after dogs
had been bled to induce anemia, but only seven days later
when body iron stores had been exhausted as a consequence of
increased synthesis of red cells (112).
Iodine
As previously stated, the old idea that goiter was caused by
deficiency of iodine became discredited in the 1800s as a result
of toxic overdosing (1). However, it gradually became recognized that iodine was concentrated in the thyroid gland and
that the enlarged thyroid in goiter had a low iodine content
(113). The first thyroid hormone, thyroxine was also found to
contain iodine in its molecule (114).
Old observations that the incidence of goiter did not seem
to parallel the deficiency of iodine in local water and food
supplies had made many enquirers believe that other factors
must also be at work. In 1928 workers at Johns Hopkins
discovered that rabbits fed mainly cabbage and being used for
a study of infections had developed goiters (115). Deliberate
studies with rats then showed that most Brassica plants and
also unprocessed soybeans had goitrogenic activity that responded to higher intakes of iodide (114,115). However, feeding Brassica seeds resulted in goiters that were reversed with
thyroxine but not with iodine (116).
In 1917 David Marine organized a large-scale trial of iodine
supplementation of schoolgirls in an area of Ohio where the
disease was endemic (119). The results of reexamination of
these subjects six months after their first treatment are summarized in Table 3. Longer periods of treatment produced
more complete freedom from the problem with no evidence of
harm. After this iodized salt began to be produced in many
parts of the world with government encouragement.
Fluorine
Fluoride ions were recognized in the 1930s as being toxic
for livestock in areas where fluoride-rich ash was scattered as a
TABLE 3
Results from the reexamination of the thyroids of schoolgirls
in Akron, Ohio 6 mo after dosing with 2.0 g
sodium iodide (119)
Control groups
Number initially normal
Worsened after 6 months, %
Number initially enlarged
Worsened after 6 mo, %
Unchanged after 6 mo, %
Improved after 6 mo, %
1257
Treated groups
908
27.6
1048
0.2
1282
14.1
72.0
14.0
0.2
39.5
60.3
result of mining or smoke from the manufacture of aluminum,

with animals becoming stiff and lame and without appetite
(120). In the same period it was realized that mottling of the
enamel of human teeth in particular areas was associated with
a relatively high fluorine content of the local water supply
(121). It was then appreciated that at the other extreme
children growing up where the water was low in fluoride had
more dental caries. This was accepted only with great reluctance, and the fluoridation of water supplies low in fluoride was
delayed for several years (122).
Diet restriction and life span
Much of the research in this period was concerned with
discovering what was needed to obtain maximum growth rates
in young animals, presumably with an underlying assumption
that this was a good thing. However, in 1917 the Connecticut group reported that stunting female rats for their first year
of life by restricting their food supply actually led to their
living and remaining fertile, with vigorous young, to a greater
age (123).
Clive McCay had learned of this work while with Lafayette
Mendel on a postdoctoral fellowship. Mendel had told him
that a younger man was needed to pursue such long-term work.
McCay, who had moved to a faculty position at Cornell in
1927, decided to take it on. He and his colleagues confirmed
that, indeed, the life of rats, particularly males, could be greatly
extended by restricting their diet for an initial year or even two
years, though he commented that, it seems little short of
heresy to present data [supporting] the ancient theory that
slow growth favors longevity (124,125). However, they also
had to report that when the heating failed in the animal room
it was only the skinny ones that succumbed!
In addition to the advances in scientific knowledge in this
period, there were important advances in its practical application. Distributing iodized salt, as already mentioned, significantly reduced the incidence of goiter. But there can be
difficulties; for example, fortifying foods with iron salts may
result in rancidity and destruction of vitamin A. Solving such
technical problems can be as demanding as the original discoveries of nutrients, but is outside the scope of these articles
as is the work of developing standards for the level of individual nutrients in adequate diets and also the evaluation of
nutritional status in different population groups.
Those who had begun their careers in the 1930s looked
back on it as the golden age of nutrition, with its rapid
discoveries of one nutrient after another. We are certainly
indebted to them for their findings, but did they leave anything still to be discovered? That is what will have to be
considered in Part 4.
LITERATURE CITED
(17851885). J. Nutr. 133: 638 645.
(18851912). J. Nutr. 133: 975984.
3. Funk, C. (1912) The etiology of the deficiency diseases. J. State Med.
20: 341368.
4. Funk, C. (1922) The Vitamines, 2nd ed. Williams & Wilkins, Baltimore,
MD.
5. Williams, R. R. & Spies, T. D. (1938) Vitamin B1 (Thiamin) and its Use
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3031
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3032
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65. Jukes, T. H. & Stokstad, E.L.R. (1948) Pteroylglutamic acid and
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67. Day, P. L., Mims, V., Totter, J. R., Stokstad, E.L.R., Hutchings, B. L. &
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76. Heilbron, I. M. (1948) Recent developments in the vitamin A field.
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77. Evans, H. M. & Bishop, K. S. (1922) On the existence of a hitherto
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78. Evans, H. M. (1962) The pioneer history of vitamin E. Vit. Horm. 20:
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79. Mason, K. E. (1944) Physiological action of vitamin E and its homologues. Vit. Horm. 2: 107153.
80. Anonymous (1941) Vitamin E falls from grace. Lancet. ii: 219 220.
81. Dam, H. (1935) The antihaemorrhagic vitamin of the chick. Nature,
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82. Almquist, H. J. & Close, A. A. (1939) The anti-hemorrhagic activity of
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83. Butt, H. R. & Snell, A. M. (1941) Vitamin K. Saunders, Philadelphia,
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86. Burr, G. O. & Burr, M. M. (1930) On the nature and role of the fatty
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87. Osborne, T. & Mendel, L. B. (1916) The amino-acid minimum for
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89. Mitchell, H. H. & Carman, C. G. (1926) The biological value of mixtures of patent white flour and animal foods. J. Biol. Chem. 68: 183215.
90. Block, R. J. & Bolling, D. (1945) The Amino Acid Composition of
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92. Block, R. J. & Mitchell, H. H. (1946) The correlation of the amino-acid
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93. Mueller, J. H. (1923) A new sulfur-containing amino acid isolated
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97. Schoenheimer, R., Ratner, S. & Rittenberg, D. (1939) The metabolic
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99. Wright, N. C. & Papish, J. (1929) The inorganic constituents of milk.
Science 69: 78.
100. Underwood, E. J. (1971) Trace Elements in Human and Animal
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101. Hart, E. B., Steenbock, H., Waddell, J. & Elvehjem, C. A. (1928)
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77: 797 812.
102. Neal, W. M., Becker, R. B. & Shealy, A. L. (1931) A natural copper
deficiency in cattle rations. Science 74: 418 419.
103. Orent, E. S. & McCollum, E. V. (1931) Effects of deprivation of
manganese in the rat. J. Biol. Chem. 92: 651 678.
104. Wilgus, H. S., Norris, L. C. & Heuser, G. F. (1937) The role of
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105. Kruse, H. D., Schmidt, M. M. & McCollum, E. V. (1934) Changes in
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106. Duckworth, J. (1939) Magnesium in animal nutrition. Nutr. Abstr. Rev.
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107. Todd, W. R., Elvehjem, C. A. & Hart, E. B. (1934) Zinc in the nutrition
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118. Purves, H. D. (1943) The effect of di-iodotyrosine and thyroxine on
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121. Ibid, pp. 395397.
122. Hodge, H. C. & Smith, F. A. (1965) Effects of fluoride on bones and
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294 295.
124. McCay, C. M., Maynard, L. A., Sperling, G. & Barnes, L. L. (1939)
Retarded growth, life span, ultimate body size and age changes in the albino rat
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125. Swan, P. B. (1997) To live longer, eat less! (McCay, 1934 1939). J.
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126. Rachford, B. K. (1912) Diseases of Children. Appleton, New York.
127. Harris, H. F. (1916) Pellagra. MacMillan, New York.
128. Combs, G. F., Jr. (1992) The Vitamins: Fundamental Aspects in
Nutrition and Health. Academic Press, San Diego, CA.
Vitamins
Folic acid. As described in Part 3, it had been found by
1944 that the material named folic acid was a vitamin
required by monkeys and chickens to prevent macrocytic
anemia and was also a growth factor for some bacteria. It was
then discovered that other bacteria could synthesize the factor,
and this was made use of in the preparation of larger quantities
for the study of its chemistry. In 1946 it was identified by
1
This is the last of four invited papers on the history of nutritional science. The
previous papers (13) were published in the March, April and October 2003 issues
of The Journal of Nutrition.
2
3
Abbreviations used: EFA, essential fatty acids; FPC, fish protein concentrate; IHD, ischemic heart disease; MeTH4F, methyltetrahydrofolic acid; TH4F,
tetrahydrofolic acid.
Manuscript received 25 July 2003.
3331
Robert Stokstad and colleagues at the Lederle Laboratories in

the U.S. as N-[(6-pteridinyl)-methyl]-p-aminobenzoic acid
conjugated with one or more L-glutamic acid residues (4). This
nomenclature was simplified to pteroyl mono (or poly) glutamate. After absorption it was reduced to tetrahydrofolic acid
(TH4F)3 and also methylated (MeTH4F). Humans and animals can use all these forms, with intestinal enzymes removing
excess glutamates before absorption, but some microorganisms
require the monoglutamate specifically (5). This work involved many people, including an active group at the Parke
Davis Company.
Folic acid, in relatively large doses and given either by
mouth or injection, was also found to be active in stimulating
red cell production in pernicious anemia patients, although it
was realized that it could not be the active factor in the liver
extracts being used for the same purpose. However, by 1948, it
had also been found that patients treated in this way for
several months typically began to show neurological disturbances that responded to liver extracts (6). We will return to
this puzzle in the following section.
In general, young rats would thrive without any source of
folic acid in their diet, but, when a relatively insoluble sulfonamide drug was added to their diet, they rapidly developed
leucopenia (a shortage of white cells in the blood) and this
condition did respond to folic acid. It was determined that the
sulfonamide structure selectively inhibited the introduction of
the rather similar p-aminobenzoic acid into the molecules of
folic acid that were normally being synthesized by bacteria in
the large intestine, so that the rat no longer obtained its
requirement of the vitamin by coprophagy (feces eating) (7).
Making further use of the concept of the antagonism between similar molecules, analogues of folic acid were synthesized and several, but particularly methotrexate (4-amino-10methyl folic acid), by 1950 were found to antagonize the
functions of folic acid in animal tissues, and to be useful in the
chemotherapy of cancer in which the rapid growth of tumors
needed to be inhibited (7).
Another line of interest in folic acid came from observations in the UK indicating an unusually large proportion of
mothers whose babies were born with neural tube defects
(spina bifida, etc.) seemed to come from lower income groups.
Obviously, this could have a multitude of causes. One of the
first suggestions receiving follow-up was that a high level of
potato consumption might increase the risk (8). Another was
Because of the huge number of papers published from 1945

to 1985 (250,000 were abstracted in Nutrition Abstracts and
Reviews), I have restricted coverage in the first section of this
paper to those dealing most directly with the discovery of new
nutrients, the effects of deficiency and the interaction of other
factors affecting their availability. This omits biochemical
mechanisms of absorption and function except where they
directly affect nutritional requirements. This is an important
restriction because so many interesting mechanisms were
worked out during this period, for example, the conversion of
vitamin D to an active hormone and the role of vitamin A in
the visual cycle. But space is a limiting factor here.
Less than one in a thousand of the papers published can be
listed here, with the selection inevitably influenced (and
therefore biased) by the authors greater exposure to some
areas than to others. Some senior scientists may therefore see
that their work in this period has not received the attention
that it deserves. For this I can only apologize. I have also cited
reviews as well as original papers, especially where several
groups have contributed to a particular development.
So much was discovered in the period from 1912 to 1944,
covered in Part 3 (3), that it was not surprising that some
people thought that the subject had been exhausted. Oxford
University closed its nutrition group soon after World War II
with that justification. In fact, important findings were still to
come from the lines of research that were already in progress,
which we can classify roughly as the discovery of new nutrients, to be reviewed first. But there was also a whole new
approach to nutritional questions that could be described as
A critique of the affluent diet and more not always being
better, and this proved to be of great practical importance for
the adult population as well as in stimulating new lines of
research.
CARPENTER
3332
TABLE 1
The recurrence of neural tube defects (NTD) in the babies/
embryos of women having previously experienced NTD
in their next pregnancy when taking a multivitamin
supplement compared with an unsupplemented group 1
Unsupplemented
women2
Multivitamin
supplement users
NTD/total pregnancies (%)

Southeast
England3
Northern
Ireland
Combined data
9/250 (3.6)
3/196 (1.5)
14/293 (4.8)
23/543 (4.2)
2/225 (0.9)
5/421 (1.2)
1 Modified from Seller & Nevin (10).

2 These were not true controls. It had been forbidden, as unethical,
for women who had volunteered for the study to be allocated to a

placebo without their knowledge. They were therefore women who
lived in the same general areas, became pregnant at the same times,
and were considered to have similar risks of recurrence.
3 The two areas were chosen because the first had the lowest, and
the second the highest recorded incidences of NTD in the UK.
assay for the activity of liver extracts. So every fraction had to

be tested by measuring the response in the blood picture of a
number of pernicious anemia patients. However, in 1948 a
group at the Merck Laboratories in New York State announced the successful isolation of the liver factor. This had
been aided by assays for a microbial growth factor that they
had correctly guessed would turn out to be the same thing, as
well as by the color of the more potent extracts. They named
their crystals vitamin B-12 (12). Another group in the UK
followed them a few weeks later, having also guessed that the
increasingly pink color of their more potent extracts could
serve as a guide (13). Analysis of the crystals gave a sensational
resultthe presence of the trace metal cobalt! The vitamin
then received the alternative name cobalamin. It also
proved active in extraordinarily small quantities. Even 5 g
was found to give a response in patients and it was realized that
some of the injectable liver concentrates in use had contained
only one part of the vitamin in a million.
Microbiological assays indicated that no plants synthesized
the vitamin, only certain microorganisms (14,15). This in
itself was something entirely new. Up until that time, it
appeared that the grand scheme of nature on Earth was that
the Animal Kingdom as a whole lived on the Plant Kingdom,
that could utilize solar irradiation as the energy source for its
syntheses. The only known role for microorganisms in the
grand system was that they broke down dead tissues from both
kingdoms and prepared them for reuse by plants. But here, for
the first time, animals were found to require something that
they could only obtain from microbial synthesis.
As described in Part 3, cobalt had already been found to be
required by cattle, but it had not been proven possible to
obtain a response to cobalt in the usual small animal species,
rats or chicks. A lot of things now began to come together.
The organisms living in the rumen (or forestomach) of cattle
and of other ruminants such as sheep were determined to be
particularly active. This explained why these animals benefited from a supply of inorganic cobalt salts; the fermentation
occurred ahead of the ordinary system for digestion and absorption in the true stomach and small intestine, and this
made the microbial cobalamin available for transfer to the
tissues of the host animal.
In poultry, on the other hand, it appeared that the cobalamin produced by fermentation in the large intestine was not
absorbed into the blood stream. It had already been found that
intensively farmed chickens would not thrive indefinitely on a
purely plant diet, and there had been studies of their need for
an animal protein factor known to be supplied by fish meal,
but also by dried cow manure. However, chicks hatched from
eggs laid by a hen well provided with cobalamin could survive
on stores for a long period, and it was only the next generation
that showed severe deficiency. Poultry, and also pigs, kept
outdoors appeared to obtain sufficient cobalamin from soil
organisms and worms (16).
It appeared that rats too were unable to absorb the cobalamin produced by organisms in their large intestine. However,
they managed to indulge in coprophagy even when housed on
raised wire screens and they could obtain some vitamin in this
way, though not always enough for maximum growth (17,18).
Monogastric herbivores, like the horse or elephant, have hindgut fermentation that adds to the energy that they can extract
from feed. It appeared that significant quantities of cobalamin
were produced by this fermentation, and it was found that
labeled cobalamin injected into a horses cecum was absorbed
into the blood stream (19,20). Humans also have some cobalamin production in the cecum but it is unclear as to whether
or not any significant quantity is absorbed. Vegans, who obtain
that only a small proportion of the mothers had been taking

vitamin supplements before or during their pregnancy.
In view of the finding of malformations in the embryos of
rats deficient in folic acid, there was a study of the folic acid
status of mothers who had been pregnant with malformed
embryos, compared with that of mothers who had delivered
healthy babies. Of 35 mothers who had infants with malformations of the central nervous system, 24 were judged to be
folate deficient, whereas of the same number of otherwise
similar mothers with healthy babies, only 6 were judged deficient by the FIGLU test, an important difference (9). Studies
were then set up with women who had already had a malformed baby, because they were known to have a higher risk of
its recurrence. Women who volunteered to join the study were
given a multivitamin supplement that included folic acid
before their next conception. The timing was important because neural tube defects occur within the first 21 d of conception, before a woman would normally know that she is
pregnant. The results from the trial (Table 1) were that the
incidence of malformed embryos was 3.6 times greater among
women who had not taken the multivitamin supplement than
in those who had, i.e., 4.2% compared with 1.2% (10).
This was the subject of serious concern by 1985 and proposals had already been made in several countries for the
enrichment of bread flour with folic acid. These proposals were
reinforced by another possible effect of higher folic acid intakes on the health of adultsreducing the level of homocysteine circulating in the bloodstream. This will be discussed in
the following section.
Vitamin B-12. As stated previously, it was realized that
folic acid, although it produced a response in pernicious anemia patients, was not the active factor in the increasingly
concentrated liver extract preparations being used for this
condition. It had also been known for many years that sufferers
from the disease lacked an intrinsic factor secreted into the
stomach that normally in some way activated an extrinsic
factor present in meat and particularly in liver. This latter
factor was still effective if given to patients by injection, but
only to a very slight extent if given by mouth (11).
No animal model had been discovered that could be used to
deficiency specifically in men failed (32). There is also the

question of why pellagra has not been a traditional problem in
Mexico where corn has been the staple, with little in the way
of animal foods for the poorest section of the population.
Evidence, mainly with experimental animals, has indicated
that niacin is released from a nutritionally unavailable bound
form during the alkaline treatment of corn during the traditional preparation of tortillas, and this contributes at least
some additional niacin to the diet (3335).
Minerals
Factor 3. Several lines of work had been leading to the
idea that there might be one more vitamin to be discovered. It
had been realized that alcoholics suffering from cirrhosis of the
liver typically had very imbalanced diets of low protein content and were greatly helped by having their diet improved.
This led to studies with rats of the effect of such diets on their
livers even in the absence of alcohol, and by 1944 it had been
found that supplementation with methionine was effective
against the liver necrosis produced with such diets (36,37). At
the same time, Klaus Schwarz at Heidelberg University was
hoping to find a new vitamin by working with such rat diets.
His protein source was casein purified by boiling in mild alkali
and reprecipitating with acid. He found that liver necrosis was
produced only with casein pretreated in this way, and that it
could be prevented with wheat germ and then with vitamin E
specifically (38). It is surprising to think of Schwarz being able
to carry on this basic research in the final years of World War
II when Germany was being so heavily bombed, but Heidelberg was spared by the Allied Air Forces, as were Oxford and
Cambridge by the Luftwaffe.
After the war, Schwarz moved to the U.S. and continued
his work at NIH. There he found that diets based on yeast as
the protein source failed to induce liver necrosis, although
they had done so in Germany. In 1951 he reported that this
was explained, not by the varieties of yeast in use, but by the
medium on which they were grown. Corn steep liquor consumed in the U.S. seemed to be supplying a protective factor
not present in the sulfite liquors consumed in Europe (39). In
his next paper he went back to his earlier experience in which
treatment with alkali had caused casein to lose its protective
factor that he now called Factor 3, because it could not have
been either vitamin E or methionine, and he described how he
had been able to make concentrates from casein fractions (40).
I myself learned of this puzzle through the frustrations of an
older friend and colleague at the Rowett Institute in Scotland.
He had been studying environmental conditions that affected
whether or not rats would actually develop necrosis on a
supposedly necrogenic casein-based diet, but suddenly he
could no longer produce the condition. One possible explanation was that he had to change his source of casein, because
the original company was no longer marketing a vitamin-free
grade. After many trials this was found to be the factor,
independent of the degree of purification of the casein (41).
All he knew was that the first company had obtained its crude
casein from New Zealand and the second from Europe. He
seemed to be getting nowhere, and this very experienced
pathologist abandoned his research career and moved abroad
to a routine clinical position. With the benefit of hindsight, we
can see that if his friend had been a soil scientist, New
Zealand might have rung a bell and led to his making the
decisive breakthrough.
In fact, the breakthrough came three years later from
Schwarz himself and from the Lederle research group, both of
whom discovered in 1957 that Factor 3 contained the element
none from their food, have low levels of the vitamin in their
blood, but some have remained healthy for a decade or more,
despite declaring that they have remained on a strictly vegan
diet (21).
There are enough cobalamin-synthesizing microorganisms
in the sea to account for the presence of this vitamin in the
zoo-systems living in water, with the larger fish living on
smaller organisms right down to the microscopic level (22).
Pernicious anemia. Now we return to the puzzle of why
two such different molecules as folate and cobalamin should
both reverse pernicious anemia. It was soon realized that only
cobalamin prevents the slower development of neurological
complications with degeneration of the spinal cord in pernicious anemia patients and most vegans (6). Because of this,
folic acid supplements were seen as a possible danger in that
they hid the existence of a vitamin B-12 deficiency.
It was reported in 1962 that in vitamin B-12-deficient
patients, folic acid accumulated in the blood in the methylated
form, MeTH4F (23). Further biochemical studies led to the
concept that cobalamin had an essential role in allowing
MeTH4F to transfer its methyl group to a second homocysteine-methionine cycle, so that its deficiency led essentially to
a functional folic acid deficiency unless larger quantities of
folic acid were supplied in the diet (24). The transferred
methyl groups were normally used through a succession of
cycles for the synthesis of DNA components needed for the
continual production of blood cells.
Another effect of the deficiency of either vitamin is a rise in
the concentration of homocysteine in the blood. This has
been another matter of concern because people with very high
levels of this compound in the blood as result of a genetic
effect are particularly subject to early onset arteriosclerosis
(25). This has become an active field of research, but almost
entirely with publications after 1985.
Pellagra and niacin. By 1945 there were already reasons
for thinking that pellagra might not be the result of a straightforward deficiency of niacin. Using a newly developed method
of analysis for niacin, it had been found that poor rice diets
from India (where pellagra was not a problem) contained less
niacin than Rumanian corn-based diets, where it was a problem (26,27). In addition, rats fed a standard purified diet with
15% casein as the protein source thrived without any source of
the vitamin and actually excreted niacin and a methylated
derivative in their urine.
It was then found that rats would become niacin deficient
if their purified diet was diluted with 40% corn meal, the food
that had traditionally been associated with the disease in
Europe. Furthermore, growth could be restored if the diet was
supplemented with 0.05% of the amino acid tryptophan that is
present at a lower level in corn than in other grains (28). It
was then shown that tryptophan could also be used to treat
humans afflicted with pellagra, with some 3% of the amino
acids being converted to niacin under the conditions of one
trial (29). Cats showed no conversion, and dogs much less
than rats because they would become niacin deficient on diets
containing relatively well balanced protein (30).
At first it was thought that the additional tryptophan might
be stimulating microbial synthesis of niacin in the small intestine, but later it was found by isotopic labeling that there
was actually an enzymic route for the conversion of a portion
of the tryptophan molecules to niacin (31).
There are still problems in understanding pellagra. No
animal model of niacin deficiency has shown the sun-induced
areas of dermatitis seen in pellagrins. It is now realized that
Goldbergers own trial with convicts had actually produced
riboflavin deficiency and a later attempt to induce niacin
3333
3334
CARPENTER
FIGURE 1 Pigs with parakeratosis before supplementation (3),

and 8 wk after their rations had been supplemented with 0.02% zinc
carbonate (4,56). (By permission of the Society for Experimental Biology and Medicine)
rus, and Boyd ODell reviewed his own and others evidence
that phytic acid can greatly reduce the availability of zinc as
well as of other minerals (58).
In the 1960s, Anasta Prasad and colleagues, under a grant
to Vanderbilt University, began the investigation of dwarfing
and hypogonadism among teenage boys in Egypt and found
that, in addition to their anemia being corrected with iron
salts, they had low levels of plasma zinc (59). They then found
that supplementation with zinc salts stimulated growth and
maturity (60). Two further trials failed to give clear-cut results,
but positive findings were observed from a controlled study of
a comparable group in Iran using a higher level (40 mg/d) of
supplemental zinc and with both control and supplemented
groups receiving a range of other trace minerals (Table 2)
(61).
The daily intake of zinc by boys in this region was not
particularly low, but their staple food, together with beans, was
whole wheat bread that had not been leavened with yeast and
still contained its intact phytic acid. Evidence indicated that
this, and also the geophagia (soil-eating) of these subjects,
resulted in the formation of indigestible zinc complexes in the
digestive tract (58,62). There were similar findings in Turkish
villages where growth depression and delayed puberty were
studied in girls as well as boys. Gains in height increased
selenium and that its activity could be replaced by sodium

selenite (42,43).
Selenium. Up to this time, selenium was known only as a
toxic element that made it impossible to graze animals on areas
where the soil, and thus the plants growing on it, were particularly rich in it (44). It was also suspected of being a
carcinogen, so that it was illegal to add it to animal feed; but
it now appeared likely that this element was a hitherto unrecognized essential trace element. It had been found to prevent
liver necrosis in rats and exudative diathesis in chicks, both of
which were already known to be preventable with vitamin E,
but in the form of sodium selenite, it was 500 times as active
as the vitamin (42,43). This was reminiscent of the puzzle over
rickets being prevented by such different treatments as irradiation and dosing with cod liver oil.
Although the disorders in rat and chicks had been induced
with special diets designed to be deficient, it was reported from
New Zealand, where volcanic pumice soils are particularly low
in selenium, that specific diseases in sheep, cattle, pigs, horses
and poultry on farms were all responding to supplementation
with selenite (45). White muscle disease in lambs and
hepatosis dietetica in pigs, which were practical problems in
many parts of Scandinavia and the U.S., also responded to
supplementation of their rations with 0.1 g/g of sodium
selenite (46). Most of these conditions also responded to
dosing with vitamin E, but usually to a lesser extent (47,48).
That both might be having an antioxidant role seemed more
likely when selenium was found to be an essential component
of the enzyme glutathione peroxidase (49,50)
A group at Cornell carried out the first chick bioassays of
the relative potency of selenium in different forms and foods,
but results were found to differ according to the procedure used
(51).
The next question was whether humans were also at risk
from selenium deficiency. This was first studied in New Zealand, but no clear evidence of a problem was obtained (52).
However, in the Keshan area of China, where selenium levels
are equally low, a characteristic cardiac myopathy (Keshan
disease) was seen that appeared to respond to selenium supplements, but was not a simple cause and effect. To examine
this fascinating problem, limited to the work done by 1985,
could occupy this whole article, but good reviews are available
(52,53).
Chromium. During their study of Factor 3, Klaus Schwarz
and Walter Mertz discovered that rats fed some of their experimental diets showed an impaired glucose tolerance that
was not reversed with concentrates of Factor 3 (54). Eventually the deficiency was found to be reversed with trivalent
chromium that appeared to act as a cofactor with insulin (55).
Hexavalent chromium was inactive.
Zinc. In the period covered in Part 3 it had been difficult
to produce an experimental zinc deficiency. Rats and mice
appeared to need only about 1 g/g of zinc in their purified
diets. However, in the 1950s it was discovered that parakeratosis, a fairly common problem in growing pigs characterized
by dermatitis, diarrhea and anorexia, was the result of a zinc
deficiency (Fig. 1) (56). This was despite their diets containing some 40 g/g of the element. It appeared to occur when
plant protein concentrates were being used in the rations and
particularly when high levels of either bone meal or calcium
carbonate were also included; but when the level of zinc was
doubled, growth and health were excellent (57).
Other groups then reported that growth depression, reversible with supplements of zinc salts, could also occur in young
chickens whose diets included high levels of soy protein concentrate. This material included 0.5% phytic acid phospho-
3335
TABLE 2
TABLE 3
Summarized findings from a study in which 13-y-old boys

in rural Iran were divided into two groups of 20 receiving
supplement with or without additional zinc1
Roses findings of the amino acids needed by the growing

rat and the quantities needed for nitrogen balance
in young men (68,69)1
Mean gain
Mean gain
Mean gain
Proportion
showing
in height, cm
in weight, kg
in bone age, mo
of boys examined
genital maturation
Gp B (Zn)
Gp C (Zn)
P value
14.6
4.4
13.1
23.6
5.5
22.9
0.05
0.05
0.01
4/13
7/13
NS
1 One group (B) received on school days (6/wk) a liquid supplement

of protein, corn oil, vitamins and minerals other than zinc. The other
group (C) received the same supplement with the addition of 40 mg of
zinc as carbonate. The trial continued for 20 months, but without
supplementation during a 4-mo school vacation in the middle of the trial
(61).
Protein
Amino acid patterns. I have not tried to cover work designed to measure the quantitative requirements for individual
nutrients; however, protein is not in this category. No two
proteins are identical, nor is the mix of proteins from one food
identical to that from another. Therefore, the question remained as to how closely the amino acid pattern of our food
needed to match that of our body proteins, which in practice
related to the extent to which either animal protein or synthetic amino acids were needed to balance vegetable proteins
for a diet to be ideal.
Workers hoped to overcome this problem by stating requirements in terms of individual amino acids, but in 1945 it
had not been demonstrated that mixtures of amino acids could
completely replace protein in the human diet. William Rose
and his colleagues at the University of Illinois had been
working to resolve this since 1942 and they reported their
findings from 1948 to 1955 (Table 3) (68 70), with a final
discussion in 1957 (71).
They found that young men would remain in nitrogen
balance with surprisingly low levels of amino acids (equivalent
to only 24 g crude protein) but only with energy intakes higher
than were required with equivalent quantities of intact protein
(Table 4). This was disturbing because it was known that
increased energy intakes had an effect on the retention of
Lysine
Tryptophan
Histidine
Phenylalanine
Leucine
Isoleucine
Threonine
Methionine
Valine
Arginine2
Total of the upper levels of
essential amino acid needs
Daily need of
human adults
Subjects
tested
0.40.8
0.150.25
Not needed
0.81.1
0.51.1
0.650.7
0.30.5
0.81.1
0.40.8
Not needed
27
31
22
8
8
19
13
23
6.35
1 Additional glycine and urea were added to raise the total nitrogen
intake of the men to 10 g/day, equivalent to 62.5 g crude protein (167).
In further trials with double the upper level of each essential amino acid,
it was found that nitrogen balance could be maintained if urea was
eliminated and the glycine reduced to 6.5 g/d, so that the total nitrogen
content of the diet was only 3.85 g, equivalent to 24 g of crude protein
(70).
2 Arginine can be synthesized by the rat, but not at a sufficiently
rapid rate to meet the demands for maximum growth. Its classification,
therefore, as essential or non-essential is purely a matter of definition
(68).
nitrogen (72). Even though the quantitative requirements had

a question mark attached to them, Rose made the point that
the list of essential amino acids required by human adults must
now be complete because, in the absence of even one, nitrogen
balance would not be obtained at any level of intake (71).
Elsie Widdowson and Robert McCance took advantage of
the unusual situation in Germany after World War II when
food rations were severely limited, so that it was ethical to
compare the performance of orphanage children receiving
different kinds of special supplements. One group of 47 children was provided with all the bread (85% extraction, i.e.,
brown but not whole wheat) that they could eat, with calcium
and vitamin supplements and their small ration of milk that
contributed only 8.8 g protein. Another matched group received the same with extra milk providing three times as much
animal protein. These treatments continued for 6 mo under
TABLE 4
The nitrogen balance (g/d) of an experimental subject
receiving 10 g nitrogen/d and either 35 or 45 kcal/d
of total energy intake (71)
Nitrogen source
Whole casein
Acid-hydrolyzed casein
tryptophan
Enzymically hydrolyzed casein
8 essential amino acids
glycine urea
1 Duration of test period (d).
35 kcal/d1
45 kcal/d
0.14 (7)1; 0.46 (5)
0.63 (5)
0.29 (8)
0.09 (6)
0.50 (6)
0.91 (6)
0.33 (5)
significantly in response to 6 mo of receiving 27 mg zinc per

day as the sulfate (63).
Bioavailability. It had become clear with zinc, as in the
previous sections dealing with niacin and selenium, that simple analysis for the total concentration of a nutrient in the diet
was not a sufficient measure of its adequacy, and that one had
to face the more difficult question of its bioavailability (64).
Evidence had mounted that increasing the intake of one
mineral could result in impaired absorption of others (65).
Also, for selenium, chromium and niacin (as well as other Bvitamins), availability was influenced by the chemical forms or
combinations in which the nutrient was present.
It was found that copper-deficient animals failed to synthesize normal elastin, with the result that their arterial walls were
greatly weakened (66). It has been suggested that many human
diets, as a consequence of their high zinc:copper ratios, have a
relative copper deficiency and that this could be a factor
contributing to the high incidence of ischemic heart disease
(67).
Amino acids found essential

for the growing rat
CARPENTER
3336
TABLE 5
The performance of orphanage children receiving unrestricted
bread and different rations of milk for 6 mo (73)
Protein intake, g/d

85% extr. wheat bread
Other vegetable foods
Animal foods
Total
Total energy intake, kcal/(kg d)
Nitrogen intake, mg/(kg d)
Weight gain over 6 mo, kg
FAO/WHO calculations
N gain, mg/(kg d)
Obligatory N loss, mg/(kg d)
Theoretical N need, mg/(kg d)
Efficiency required of dietary N, %
Low milk
Higher milk
41.0
11.5
8.8
61.4
66.6
322
2.5
34.6
11.5
26.5
72.6
67.0
381
2.5
13.9
73
87
27
13.9
73
87
23
FIGURE 2 Kwashiorkor characterized by edema of the face (left),

and by skin lesions on the back (right). (Courtesy of Dr. R. G. Whitehead)
careful supervision (Table 5) (73). The children, averaging

age 9 10 y, grew equally well on both diets, even though in
one 12% of their energy came from protein and of this only
14% was animal protein (or first class according to some
writers). It is unfortunate that this important study was published in a series of monographs not available in many academic libraries, although some of the results have been summarized and discussed elsewhere (74).
These orphanage children were growing both in height and
weight at a rate 25% above the average for their ages,
perhaps as a catch up phenomenon. It is notable that this
was possible on the low protein diet, in which mixed proteins
themselves contained only 3.7% lysine, about half the level
in our own body proteins. Human growth is extremely slow
and the children were estimated to have gained on average
only 14 mg N/(kg body wtd) with an intake of 322 mg/(kgd).
A group at the Massachusetts Institute of Technology suggested that, although people on relatively low protein intakes
were in nitrogen balance, their equilibrium might be at the
expense of lower rates of protein turnover and of potential
synthesis of antibodies when exposed to infection (75). This
was an important question and by 1985 procedures were being
developed for its measurement using turnover and oxidation
studies with isotope-labeled amino acids (76). However, no
definitive answer had been obtained and another worker recommended caution in justifying the need for increased protein
on the basis of such measurements (77).
It was also demonstrated that the requirement of growing
chicks for the limiting essential amino acid, lysine, was increased from 0.85% of the diet to 1.1% when the total level
of protein in the diet was raised from 20% to 30% (78). Alfred
Harper and colleagues at Wisconsin then demonstrated that
adding a single amino acid at a fairly high level, for example
2% L-histidine to a diet containing 12% casein (plus methionine) that supported rapid weight gains (56 g in 9 d) in young
rats, could inhibit their appetite and performance (in this
example to 45 g) (79). Attempts have been made to divide
effects of this general kind into toxicities, antagonisms and
imbalances (80). However, there was no evidence that they
were likely to occur in practice with humans. One possible
concern was that high protein Western diets might be causing
an acidosis that resulted in a compensating loss of calcium, and
thus of bone (81).
The world protein problem. The period had begun therefore with studies indicating that the supply of protein, at least
for diets based on cereals, was not a problem (82). Nevertheless, in 1960, a senior nutritionist said, We have moved from
the era of vitamin research to protein research, and the head
of the Nutrition Division of FAO (the Food and Agriculture
Organization of the United Nations) wrote that, deficiency of
protein in the diet is the most serious and widespread problem
in the world (83 85).
This idea grew from the finding that a serious disease, called
kwashiorkor in West Africa and recognized by flaky dermatitis, hair changes, edema and apathy, was also common
among 1 4-y-old children in other parts of the developing
world (Fig. 2) (86). It was found to respond to concentrated
relatively high protein nutritional supplements such as skim
milk powder, and the previous idea that it was an infantile
form of pellagra was abandoned because it did not respond to
nicotinic acid or other B-vitamins (87).
Kwashiorkor was also characterized by liver damage and,
because cirrhosis of the liver was common among adults in
Africa, it was initially suspected at FAO that the African diet
remained protein-deficient throughout life, and that the same
might be true throughout the developing world. Milk and milk
powder were expensive and in short supply, and it was urged
that substitutes needed to be developed (88).
Much work was carried out in areas where the problem
existed, for example at the Institute for Nutrition in Central
America and Panama, to develop and test cheaper alternatives
to milk powder based on locally available cereals and oilseed
flours. These could prevent the condition from developing and
also cure it, although not quite as quickly as with milk powder
(89). Individual babies could also be deficient in electrolytes
and vitamins as well as in protein and energy (90). Others
suggested that essential fatty acids might also be deficient
(91,92).
In 1968 the United Nations published a paper entitled
International Action to Avert the Impending Protein Crisis (93).
By then, several projects had been set up, with substantial
funding (some from governments and foundations), to develop
processes and machinery in advanced countries for the preparation of stable, solvent-extracted high protein powders from
fish [fish protein concentrate (FPC)] and other materials. This
was encouraged by enthusiastic international conferences,
even though the original idea had been to devise new crops or
Essential fatty acids

Linoleic acid. As described in Part 3 (3) it was already
known that rats would show scabby tails, have excessive water
loss and fail to breed successfully if reared fed a diet lacking the
polyunsaturated linoleic acid 18:2(n-6). The symbol n, or
in the earlier literature, refers to the number of carbon atoms,
counting from the hydrocarbon end of the molecule, before an
unsaturated bond is reached.
The next obvious question was whether humans had a
similar requirement. In 1963, as the result of a large study of
over 400 infants fed formulas for 6 mo differing in fat content,
it was concluded that children receiving 0.1% of their energy as linoleic acid developed dry thickened skin and in many
cases showed unsatisfactory growth. When more linoleic acid
was provided, the problems promptly disappeared (106). The
same signs of deficiency were seen after quite short periods in
infants that had to be fed parenterally and were not receiving
lipid (Fig. 3) (107). In one case, rubbing the skin with oil rich
in linoleic acid proved sufficient to correct the condition
(108).
FIGURE 3 The foot of an infant suffering from a deficiency of

linoleic acid as a consequence of fat-free intravenous alimentation
(107). (By permission of the American Journal of Clinical Nutrition)
Work with rats originally had also shown that a considerable proportion of absorbed linoleic acid was metabolized to
gamma-linolenic acid 18:3(n-6) and then to arachidonic acid
20:4(n-6). Gamma-linolenic acid, which could be obtained
from evening primrose oil and a few other plants, was found to
be helpful in treating some skin disorders, perhaps because of
subjects limited ability to synthesize arachidonic acid from
linoleic acid (109).
When animals were limited in the supply of linoleic acid
there was a correspondingly larger synthesis of eicosotrienoic
acid 20:3(n-9) from the nonessential oleic acid 18:1(n-9)
(110,111). The eicosotrienoic:arachidonic ratio in the blood
was found to be an indicator of marginal linoleic acid deficiency, even before the appearance of clinical symptoms, in
rats and pigs, and then in humans (106,112,113).
Cats gave quite different results. They were found to require
arachidonic acid in their diet and to lack the enzymes needed
to lengthen and desaturate linoleic acid (114). This was analogous to their inability to use carotene as a source of retinol.
As carnivores they can, of course, obtain both preformed
retinol and arachidonic acids from the animal tissues that they
consume and do not have to produce the molecules required
by the Animal Kingdom from a precursor found in plants. This
seemed to suggest that linoleic acid served for other species
only as a precursor for arachidonic acid. However, later work
demonstrated that the waterproofing effect of linoleic acid on
rat skin was caused by epidermal sphingolipids incorporating
linoleic acid as such (115).
Alpha-linolenic acid. Because the gamma molecule 18:
3(n-6) is also considered a linolenic acid, one should perhaps
always use the prefix alpha for the 18:3(n-3) form, but in
practice it is assumed that is what is meant by linolenic acid.
In the Burrs pioneering work with rats it appeared that linolenic acid could fully replace linoleic acid, but later workers,
perhaps using purer preparations, did not find this to be the
case (116,117). In a critical trial lasting three generations, rats
grew well and appeared to behave normally with linoleic, but
without linolenic acid (118). However, it was noticed that the
simple methods of food processing that could be adopted in

underdeveloped villages. In addition, even more sophisticated
processes were being planned to produce single cell protein
(SCP) from yeasts, fungi and bacteria, grown on media ranging
from molasses waste to petroleum (94). Doris Calloway drew
attention to the poor tolerance and even toxicity of some SCP
materials, and that their high content of nucleic acids was also
a problem for humans, who metabolize purines only to the
relatively insoluble uric acid, so that they were more suited for
animal species that do not have this problem (95).
Hi-tech projects, which were supposed to be aiding relatively primitive communities, received particularly bitter criticism from the faculty at the London School of Hygiene and
Tropical Medicine who referred to a continuing process of
justifying scientific enthusiasms by the drawing of facile and
tenuous links between research which is intellectually exciting
to the investigator and problems which are of sufficient public
concern to make it politically attractive to devote funds to
them (96). Even in the U.S., where scientists are usually less
willing to risk giving offense, the leader of the governmentfinanced FPC project was to say later (in a book worth reading) that, Much of the motivation for FPC development had
little or nothing to do with the ostensible and well-publicized
humanitarian goal (97).
This is an episode in our history that nutritional scientists
would probably like to forget, but one use of history is to learn
from our mistakes and to not repeat them. It was brought to an
end by the realization that most kwashiorkor victims had been
receiving diets that were as deficient in energy as they were in
protein, and too bulky for the youngsters to take in sufficient
amounts (98). The general need was to provide more concentrated foods and correct electrolyte deficiencies rather than
concentrate just on protein (99 101). It was especially difficult to improve diets based on roots such as cassava that were
very bulky as well as low in protein (102). The United Nations
Organization, which had previously emphasized its concerns
about a world protein problem, made no mention of it at its
1974 World Food Conference (103).
The synthetic production of essential amino acids likely to
be first-limiting in Third World diets was also stimulated in
the 1960s (104). Although results with human trials were
generally disappointing, these compounds have found practical uses in intensive pig and poultry feeding (105).
3337
3338
CARPENTER
Shortcomings of the affluent diet

Was more always better? The following is a quotation
from 1957: For practically all of the first half of this century
. . . problems of deficiency disease and undernutrition have
been emphasized. Public nutrition programs have been dedicated largely toward increased consumption of milk, meat, eggs
. . . and practically everything in the usual diet. . . . The term
allowance for safety is predicated upon the idea that excess is
preferable to limitation . . . . It is worthwhile considering how
much responsibility the nutritionist has for the incidence of
obesity in this country. . . . The evidence that good diets, by
past definition, are an important causative factor in atherosclerosis, diabetes and other diseases that are largely of the
upper income group, is so strong as to be nearly conclusive.
Those who have known it will recognize the clear and independent voice of Mark Hegsted here (126).
After World War II it was realized that in some ways the
most affluent countries did not have the best health records,
and as stated above, their dietary patterns were actually conducive to chronic, noninfective diseases in middle age. The
evidence for long-term effects in adults had, of course, to come
from epidemiological studies. One surprise was that during the
war, when supplies of food, and particularly of animal foods in
European countries were severely restricted, the incidence of
some diseases was generally reduced (127129).
The first aspects of the affluent diet to be under suspicion
were the high levels of saturated fat and the low levels of fiber
(coupled, in the minds of some workers, with the high intakes
of sugars). We will consider first the studies related to dietary
lipids.
Dietary fat and cholesterol. One important cause of death

in Western countries was ischemic heart disease (or IHD).
This is caused by a diminution in blood supply to a portion of
the heart muscle that may result in necrosis or myocardial
infarction. It was known that autopsies of IHD cases typically
showed a narrowing of coronary blood vessels by atherosclerosis, i.e., deposition of plaques rich in cholesterol on the
walls. This had long been a subject of interest among pathologists and in 1934 one wrote, The literature contains a
multitude of reports correlating atherosclerosis with diet,
blood pressure, race etc. and later: . . . where the neutral fat
intake is low atherosclerosis is not prevalent (130), but it does
not seem to have caught the attention of nutritionists prior to
WWII.
A characteristic of most Western diets was that a high
proportion of energy came from fat, much of it animal. The
first thought therefore was that the IHD problem came from
ingesting too much cholesterol found only in animal foods.
Feeding cholesterol to guinea pigs, and to other herbivores
that did not receive it in their normal diet, had been found to
result in increased levels of serum cholesterol and in the
formation of plaques. However, in human studies, the feeding
of different types of fat did not affect serum cholesterol levels
in proportion to the contribution of cholesterol, although the
level of dietary cholesterol did have some effect (131133).
From 1950 on, Ancel Keys, working with numerous collaborators in different countries, studied many environmental
aspects of the IHD epidemic in the U.S. and different parts of
Europe. He began work in Naples where the percentage of
dietary energy coming from fat was only half of that in the
U.S., i.e., 20% vs. 40%, and the death rate of men aged 30 50
y from degenerative heart disease was only one third or less
(134). Classification of cause of death can differ from country
to country, but all other major causes seemed to be recorded at
similar rates in Italy and the U.S., so that the lower total death
rate in Italy could be mainly attributed to degenerative heart
disease (135). Serum cholesterol levels did not rise in middle
age in Naples to the same extent as in the U.S. or England,
and it seemed that total fat intake was the determining factor
and much more important than either physical activity or
degree of obesity (136).
In 1954 it was reported from the Rockefeller Foundation in
New York that a mix of plant fats supported a considerably
lower level of serum cholesterol than did the same diet with fat
from animal sources (1.55 vs. 1.96 g/L) (137). The group then
found, after feeding different plant and land animal fats, that
the resulting cholesterol levels were lowest with fats of higher
iodine value (i.e., more unsaturated), regardless of which kingdom they came from (138).
Contrasting national diets. It was reported in 1959, that
when published national IHD death rates were compared with
different dietary characteristics, the closest correlation was
with the percentage of energy coming from saturated fat
(139). Keys too, in his Seven Countries Study of 16 different
communities, found the closest correlation (r 0.84) between
deaths from IHD and this measure (134). His data, further
compressed into single groups of the same nationality, are
summarized in Table 6. We see again that deaths in communities in the U.S. and other countries with about the same
high intakes of saturated fat were similar, and roughly three
times as great as in the Mediterranean countries which had
much lower intakes. This was exemplified to me in 1956 when
a friend told me that his Greek-American, businessman father
had just died in Boston of a heart attack, while his grandfather
was still living in good health on his farm back in Greece.
rats strongly retained docosohexanoic acid 22:6(n-3) that was

formed from linolenic acid, and others found it to be particularly concentrated in the outer segments of retina rods (119).
In contrast, there was no doubt that fish required n-3 acids for
normal growth (120).
In 1982 it was reported that a 6-y-old girl, who had for
several years been receiving only parenteral feeding with a
formula containing very little linolenic acid, began to show
neurological abnormalities including blurred vision. These disappeared when the formula was changed to include more
linolenic acid (121). When rhesus monkeys were fed mixes
free of linolenic acid and their babies received the same mix,
it was observed that at 12 wk of age they responded poorly in
a visual acuity test of preferential looking compared with
controls (122). It seemed reasonable to conclude therefore
that linolenic acid, or longer chain n-3 acids, were required in
the diet.
Prostaglandins It had long been known that extracts from
male accessory glands of animals had a vasodepressor effect,
and in 1963 Sune Bergstro m and colleagues in Stockholm
identified some of these active compounds, which had been
called prostaglandins, as derivatives of C20-cyclopentanoic
acid (123). Two years later they showed that they could be
synthesized in the body from arachidonic acid, and suggested
that at least some of the effects of essential fatty acid (EFA)
deficiency could be because of inadequate synthesis of prostaglandin hormones (124). Later it was shown that increasing
the linoleate content of a diet increased prostaglandin synthesis (125). We will return to this subject in a later section
headed Polyunsaturated fats where their importance had
reappeared as an end point of quite a different sphere of
investigation.
TABLE 6
The 10-y death rates from IHD of 40 59-y-old men from
communities grouped by countries with the estimated
percentage of dietary energy received from saturated fat 1
Percentage
of energy
from
saturated fat
U.S. (N.W.)
Finland
Netherlands
Italy
Yugoslavia
Greece
Japan
Northern
Mediterranean
18
20.5
19
10
12
7.5
3
19
10
IHD deaths
per 10,000
men
425
475
320
200
150
75
60
407
142
1 Data recalculated from Keys, 1980 (134).
diet and nutrition on cancer reported in 1982 that, of all the

dietary components studied, the combined epidemiological
and experimental evidence is most suggestive for a causal
relationship between fat intake and the occurrence of cancer,
. . . particularly breast and colon cancer. . . . Experimental data
on cholesterol and cancer risk are too limited to permit any
inferences to be drawn (148).
Polyunsaturated fats. As discussed in an earlier section,
PUFA, after lengthening to C-20 forms, had been shown to be
the source from which a variety of prostaglandins with very
varied hormonal actions were synthesized. In particular, some
stimulated the aggregation of platelets, but the major action
was inhibition (149). This could explain the reduced formation of arterial plaques in subjects consuming more polyunsaturated fats.
It was also suggested that the observed effect of polyunsaturated acids in reducing blood pressure was mediated by the
synthesis of a favorable balance of prostaglandins (150). However, in experiments in which prostaglandins were injected
into rats, only short-term effects were observed, presumably
because they are unstable and perhaps need to be synthesized
within the tissues in which they are active (125).
When unsaturated vegetable fats, in contrast to more saturated animal fats, were first found to reduce serum cholesterol
levels in both humans and rats, it was assumed to be due to
their higher content of the recognized EFA, i.e., linoleic and
perhaps linolenic acids. Since fish oils had little of these two
acids, although they were highly unsaturated, it had not been
expected that they too would reduce serum cholesterol levels.
In fact, they were found to be equally or even more effective
than vegetable oils in both humans and rats (151153).
It was then suggested that fish oils might have an additional
beneficial effect on the incidence of IHD, in addition to
reducing serum lipids. It was found that eicosapentaenoic acid,
the C20:5(n-3) polyunsaturated acid in fish oils, is metabolized
to prostaglandins that antagonize platelet aggregations to a
greater extent than those derived from the C20:4(n-6) derivative of the linoleic acid in vegetable oils (154). The value of
fish oils was confirmed in further work, but it was also observed
that bleeding times were somewhat increased (155).
At the very end of this period, results were reported from
the Netherlands of a follow-up study of over 800 middle-aged
men whose diet varied greatly in fish consumption, from none
to 45 g/d. In the 20 y of the study 78 died from IHD with the
risk rate for the high-consumers being only 42% that of the
abstainers, and with a significant trend for decreasing risk with
higher consumption (156).
Dietary fiber. Two medical men, with long experience in
different parts of Africa, had each become impressed by seeing
there so little of some of the noninfective diseases most common in industrialized countries. In 1969 Denis Burkitt pointed
out that cancer of the colon and rectum among 35 64-y-old
men was 10 times as frequent in Connecticut as it was in East
Africa, and intermediate in Puerto Rico and most Asian
countries. He commented, Bowel cancer and other noninfective diseases of the bowel are rare in every community examined which exists on high-residue diet, and common in every
country where a low-residue diet has been adopted. It seems
likely that carcinogens produced by the action of an abnormal
bowel flora, when held for a prolonged period in contact with
the bowel mucosa, may account for the high incidence of these
diseases . . . (157). In 1972 Hugh Trowell suggested that it was
a higher intake of dietary fiber that also helped to protect
people in less developed countries from IHD (158). Following
these and other papers, there was increased interest in the
There were many further epidemiological studies, some of

them comparing the health records of emigrants with those of
people who had not emigrated in order to eliminate possible
racial differences in susceptibility (140). In one study it was
found that Japanese men who had migrated to California and
had at least partially Americanized their diet, had approximately three times the very low incidence of IHD of a corresponding population group in Japan (141).
No one suggested that Americans could be persuaded to
adopt a Japanese diet, and the Mediterranean diet began to
be considered as a more practicable ideal, but what did it
consist of in the 1950s and 60s? There were some big differences. In one study area in Yugoslavia 96 g/d of fish was eaten,
and in another none, but the IHD rates were similar. Only 35
g/d of meat were eaten in the Greek communities, but over
200 g/d in one Yugoslav community where the IHD rate was
again similar. The Greek and Italian communities all had a
high consumption of fruit and vegetables, but the Yugoslavs
had no more than the Dutch and American groups (142,143).
There remains the question posed by Philip James and
colleagues as to whether the Mediterranean diets are protective or simply nontoxic (144). Were the more affluent diets
elsewhere worsened by the presence of trans fatty acids from
hydrogenated oils or a lack of antioxidants from leafy vegetables? In 1985 all these points remained to be investigated
further.
Lipoproteins. Cholesterol is carried in the blood in combinations with proteins and as early as 1950, a Berkeley group
that had devised a method of separating different classes of
serum lipoproteins suggested that high levels of the low density
or -fraction might indicate an increased risk of atherosclerosis (145). In 1959 Robert Olson presented additional evidence
for the importance of this fraction (146). Then in 1977 it was
reported from the NIHs large Framingham study that the
analysis of 79 coronary heart disease deaths, among subjects
whose lipoproteins had been studied, showed that the greatest
risk was among those with the lowest levels of cholesterol in
HDL or -lipoproteins. Of those with 44 mg/100 mL or less
the incidence was 105/1,000, and among those with more it
was only 48/1,000 (147). In other words, HDL was apparently
a good form of cholesterol. However, cholesterol in this combination made up only a small proportion of the total, so that
earlier studies in which just total cholesterol was measured still
had value.
A committee set up by the NRC to study the influence of
3339
CARPENTER
3340
Epilogue
If it seemed to some people that nutritional science was
virtually complete in 1945, it was certainly not the case 40
years later.
The incidence of ischemic heart disease was falling in the
affluent countries, presumably in part as a result of people
taking nutritional advice, but also from smoking less and the
availability of improved drugs. However, the problem of obesity with its attendant diabetes was still growing, and nutritional science had not been able to come up with an easily
adopted solution for people with a sedentary lifestyle. By 1985,
as technology advanced but the human machine remained the
same, a few people were reversing the traditional work-rest
cycle, i.e., doing their work (and associated travel) sitting
down, but spending their breaks on a treadmill.
We can look back with respect on the labors of our predecessors over a period of 200 y and on their remarkable accomplishments, while recognizing that there remained, and still
remains, much more to be done in terms of the particular
individual as well as of the statistically average member of the
population. Only the surface has been scratched in investigating the effects of the nonnutrient chemicals in foods on
resistance to disease.
ACKNOWLEDGMENTS
I thank the anonymous reviewers who have commented on the
drafts in this series (particularly in relation to the topic of lipids in
Part 4) and Patricia Swan, whose editing throughout the series
balanced criticism and encouragement.
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A Short History of Nutritional Sciences, Carpenter K.J. (2003)

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History of Nutrition

A Short History of Nutritional Science: Part 1 (17851885)

This is the first of four invited articles planned to provide a

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82.25%, respectively (1). This was impressive work and one

HISTORY OF NUTRITIONAL SCIENCE (17851885)

FIGURE 1 Schematic drawing by

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and horses, whose common feeds had the reputation of being

Synthesis only by plants

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Apparent daily balance

criticism, Boussingault explained, I am far from regarding

HISTORY OF NUTRITIONAL SCIENCE (17851885)

The composition of animal substance

because there was reason to believe that legume crops had a

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every atom is identical. Chemical combination occurs when

FIGURE 2 Picture of the treadwheel in a London prison of the type

The next stage in this story involves another line of basic

law in England, was developing a technique for measuring

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The conservation of energy

HISTORY OF NUTRITIONAL SCIENCE (17851885)

States. However, two names are remembered from this time.

nourishment. . . . These disorders are, no doubt, frequently

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mislead explorers for a considerable time, and to provide an

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detergent action] that helped to free perspiration pores in

HISTORY OF NUTRITIONAL SCIENCE (17851885)

bution to reducing human suffering but, at least for a period,

24. Wo hler, F. (1832) Researches respecting the radical of benzoic acid.

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1. Berthollet, C. L. (1785) Analyse de lalkali volatil. Me m. Acad. Sci. Paris

Before 1885, nearly all of the nutritional studies had been

Protein digestion and interconversion

that proteins ingested in foods were absorbed almost intact and

substitutes for dietary protein. Most found that meat proteins

FIGURE 1 A photograph used by Chittenden to demonstrate that

HISTORY OF NUTRITIONAL SCIENCE (18851912)

daughter having to make up accounts of what it was still being

FIGURE 2 A diagram used by Atwater to illustrate the kind of equipment

1 The calculated net balance was not included in the original

nutrient would depend on the level ingested or on the prior

Because of this, bread had been considered to be as rich in iron

HISTORY OF NUTRITIONAL SCIENCE (18851912)

FIGURE 3 Victims of beriberi, with

brown rice. He therefore concentrated on what factor in the

HISTORY OF NUTRITIONAL SCIENCE (18851912)

200,000) and many were brought back to temporary hospitals

FIGURE 5 A pigeon showing characteristic head retraction after it

an organic base, and 50 mg was sufficient to cure a deficient

HISTORY OF NUTRITIONAL SCIENCE (18851912)

Night blindness and xerophthalmia

Mean weight gain

dying before the end of the trial. In contrast, the corn-fed

41. Hulshoff Pol (1902) Katjang idjo, un nouveau medicament contre le

In the early years of the 20th century, the number of

Rats and mice fed purified diets

leading the pack in the early years (13). It is interesting in

McCollums first trials

HISTORY OF NUTRITIONAL SCIENCE (19121944)

Rickets and vitamin D