Increased Aspartate Aminotransferase Activity of Serum after

in Vitro Supplementation with Pyridoxal Phosphate

Robert Rej, Charles F. Fasce, Jr.,1 and Raymond E. Vanderlinde

We examined the effect of pyridoxal phosphate sup- cal assay for determining the AST activity of serum.
plementation on the apparent aspartate aminotrans- Reports differ on its effect on serum AST activity.
ferase (EC 2.6.1.1.) activity of human serum. Sup- Karmen et al. (1) were unable to show any measur-
plementation by 25 mol/liter effected an average able effect of pyridoxal phosphate, or of boiled rat
increase of 16% in the results for kinetic assay. The liver extract, on serum aminotransferase activity.
increase was not the result of increased enzymatic Similar conclusions have been reported by others
or nonenzymatic blanks, and, within a small range,
(9-11). Hamfelt (12), however, was able to demon-
sample dilution had no significant effect. Part of the
strate increased serum AST activity after supple-
increase was attributableto the enzyme being pro-
tected against the loss of activity that occurs during mentation with pyridoxal phosphate. The increases
preincubation with L-aspartate. A similar increase he reported varied with health and age of subjects
was not demonstrated in a two-point colorimetric but were not limited to cases of Vitamin B6 deficien-
method, perhaps because of the short reaction time, cy, as has been previously suggested (10).
without preincubation, and the initialpresence of Currently, standardization of optimized reference
both substrates in the assay. We attempted to Corre- enzyme methods is being attempted, so that results
late such stimulation of aminotransferase activity will be repeatable and comparable (13-15). The
and the patient’s diagnosis or treatment. Pyridoxal Deutsche Gesellschaft f#{252}r
Klinische Chemie (14) has
phosphate should be included in the reaction mixture recently recommended measurement of enzyme ac-
when aspartate aminotransferase activity is being tivities under optimal or optimized concentrations of
measured clinically. substrate, cofactors, activators, and buffers. In the
same publication this society proposed optimized
Additional Keyphrases: differences in results by kinetic
and colorimetric methods - serum enzyme activity
methods for determination of the activity of several
enzymes found in serum. The method cited for mea-
The diagnostic use of measurements of serum suring AST activity does not include pyridoxal phos-
AST2 activity is well known. In 1955 Karmen et al. phate supplementation.
(1) demonstrated increased aminotransferase activity Reference methods of lasting importance and use-
in sera from patients with various clinical diagnoses; fulness can be achieved only by thorough investiga-
and AST activity of serum is now known to be in- tion of each factor influencing the activity of the en-
creased in a variety of physiological conditions (2, 3). zyme examined. Toward this end the present study
The requirement of pyridoxal phosphate for amino- was initiated.
transferase activity is also well documented. 0 ‘Kane
Materials and Methods
and Gunsalus (4) in 1947 and Meister et al. (5) in
1954 were able to activate AST in porcine heart Kinetic AST assays were performed by the method
preparations by incubating them with pyridoxal or of Henry et al. (16), with slight modifications. Sam-
pyridoxamine phosphate. Pyridoxal phosphate is ple volume was either 0.2 or 0.5 ml, as specified.
now firmly established as the cofactor for AST and Total assay volume was 3.0 ml in all cases. The final
other aminotransferases (6-8). control substrate mixture contained the following,
Despite this knowledge, pyridoxal phosphate has per liter: 90 mmol of phosphate buffer, pH 7.5; 125
not been incorporated into any commonly used clini- mmol of L-aspartate; 6.7 mmol of 2-oxoglutarate;
0.18 mmol of NADH; 867 U of malate dehydrogenase
From the New York State Health Department, Division of Lab- (2.6 U per test); 6.9 mmol of sodium azide; 225
oratories & Research, Albany, N.Y. 12201.
mmol of glycerol; and 0.2 mmol of (NH4)2S04. Pyri-
1 Present address: City of Kingston Laboratory, Kingston, N.Y.
12401. doxal-phosphate-supplemented substrate was the
2 Nonstandard abbreviations used, and enzyme identification: same except for the additional presence of 25 zmol of
AST, aspartate aminotransferase, EC 2.6.1.1, L-aspartate : 2-oxo-
pyridoxal phosphate per liter. Pyridoxal phosphate
glutarate aminotransferase [formerly known as glutamic-oxalo-
acetic transaminase (GOT)J; malate dehydrogenase, EC 1.1.1.37, and NADH solutions were prepared daily.
L-malate:NAD oxidoreductase;GD, glutamate dehydrogenase, All reagents except malate dehydrogenase, which
EC 1.4.1.3,t-glutamate:NAD (P) oxidoreductase (deaminating); was prepared as a glycerol-water (equal volumes)
and lactatedehydrogenase,EC 1.1.1.27,
L-lactate:NADoxidore-
ductase. suspension, were filtered through filters of 0.22-tm
Received Oct. 18, 1972; accepted Nov. 10, 1972. average pore diameter (Millipore Corp., Bedford,

92 CLINICAL CHEMISTRY, Vol.19,No.1,1973

Mass. 01730) and contained 0.5 g of sodium azide phosphate buffer, pH 7.5; 134 mmol of L-aspartate;
per liter as an antimicrobial agent. Sodium azide, at 0.19 mmol of NADFI; 2.7 U of malate dehydrogenase
this concentration, has no detectable effect on AST and, when supplemented, 26.8 ltmol of pynidoxal
activity. Reaction rates were measured at 340 nm phosphate.
with either “Kintrac VII” (Beckman Instruments, When incubation was complete, 2-oxoglutarate
Fullerton, Calif. 92634) or Model 2000 (Gilford In- was added and AST activity was measured at 30#{176}C.
strumentation Labs. Inc., Oberlin, Ohio 44074) spec- Each serum specimen was assayed in duplicate with
trophotometric systems. All kinetic assays were per- each substrate preparation. We observed a mean in-
formed at 30#{176}C, at which temperature the units (U) crease of 16% in AST activity with the supplemented
are micromoles per minute, unless otherwise stated. substrate for 125 sera (P < 0.001). “Normal” serum
The normal range that we find by this procedure is 6 specimens were also obtained from 16 healthy labo-
to 22 U/liter of serum sample. ratory personnel, and assays were begun within 1 h. A
A diazonium salt assay was used to determine the 16% increase was also observed for pynidoxal phos-
effect of pyridoxal phosphate in a two-point colon- phate-supplemented sera in this population (P <
metric method. The procedure and reagent concen- 0.001).
trations were identical to those described by Babson A scatter diagram for the results obtained by these
et al. in their original method (17), except that 10 g two methods for all sera tested with initial activities
of polyvinylpyrrolidone per liter was included in the less than 52 U/liter (135 specimens) is shown in Fig-
control buffer-the concentration specified for the ure 1. The ratio of AST activity measured using pyr-
substrate medium. Assay temperature was 37#{176}C. Ox- idoxal-phosphate-supplemented substrate, compared
aloacetic acid standards were prepared in 0.01 molar to control, for four groups of specimens is shown in
HC1, dispensed in 2.0-ml aliquots, lyophilized, and Table 1. While a slight decrease in this ratio is ob-
stored at -40#{176}C.
Concentration of oxalacetate for served for sera with elevated AST activity (>40 U/
each lot was determined by the enzymatic method of liter), no statistical difference can be demonstrated
Hohorst and Reim (18). Standards were reconstitut- between the ratios of any two groups (P > 0.25 in
ed with 0.01 molar HC1 immediately before use. Ab- each case).
sorbance at 530 nm was recorded in a Model DB-G
spectrophotometer (Beckman), and a serum blank Two-Point Colorimetric Assay
was prepared for each serum assayed. The AST activities of 39 human sera were estimat-
L-Aspartic acid; 2-oxoglutanic acid; pynidoxal ed by the assay method of Babson et al. (17), with
phosphate; cis-oxalacetic acid (grade I); /3-nicotin- and without pynidoxal phosphate supplementation
amide adenine dinucleotide, reduced form [/3-NADA (25 smol/liter during incubation). The results are
(Grade III)]; 6-benzamido-4-methoxy-m-toluidine di-
azonium chloride [Fast Violet B salt (grade I)]; and
crystalline glutamate dehydrogenase [bovine liver,
60
glycerol suspension (Type II)] were all obtained from
Sigma Chemical Co., St. Louis, Mo. 63178. Malate
dehydrogenase (derived from porcine heart) was ob-
50
tained from Boehringer Mannheim Corp., New York, 0

N.Y. 10017; polyvinylpyrrolidone (PVP; av mol wt, w

z
30,000) from Technicon Instruments, Tarrytown, w
N.Y. 10591; and sodium azide, purified, from Fisher w4c
a- ,
Scientific Co., Pittsburgh, Pa. 15219. All other a- /
:2
(I) - /
chemicals were of reagent grade. Distilled, de-ionized -J
water (>15 Mci/cm) was used throughout. Human 3C
0
erythrocyte AST was prepared by the method of Rej 0

etal. (19). 2-
a-
Statistical equality was analyzed by use of the t- t 2C
Ui
distribution (20). I-
-J -

.#.- 1

Results 10

Kinetic Assay
Human sera, chosen without conscious bias from
specimens collected for a hospital clinical laboratory, 0 ‘0 20 30 40 50
U/LITER
were kept at 4#{176}C
and assayed within 24 h from the
Fig. 1. Correlation between AST activitydetermined with
time of drawing. Aliquots of 0.2 ml of each specimen and without pyridoxal phosphate supplementation in a
were added to control and to pyridoxal phosphate- kinetic assay
supplemented substrates lacking 2-oxoglutarate, and Each point represents the mean of duplicate analyses by each method.
Activities are reported at 30#{176}C:
sample size was 0.2 ml. Pyridoxal con-
were incubated for 1 h at 30#{176}
C. The composition of the centration was 25 mol/liter; ( ) represents a slope of 1.0; (-)
preincubation mixture was, per liter: 89 mmol of represents the regression fit of y = 1.16 x - 0.26. r = 0.987

CLINICAL CHEMISTRY, Vol.19,No. 1,1973 93

Table 1. Increase Effected in Aspartate 0
Aminotransferase (AST) Activity of Human Ui
I-
Serum by Added Pyridoxal Phosphate z 60
Lii
Pyridoxal-
Lii
AST activIty, No. supplemented/ -J
Population U/liter specimens controi#{176} a-
0
Normal 9-22 16 1.16±0.02
U)
Hospital 4-24 97 1.18±0.03 40
-J
(normalserum AST)
2<
Hospital 25-40 16 1.15± 0.02 0
0
(moderately elevated
serum AST) >-
20
Hospital >40 12 1.12±0.03
Ui
(elevated serum AST)
I-
#{176}Mean
± std. error.
-J

0 20 40 60
shown in Figure 2. The best linear model to describe U/LITER
the relationship between control (X) and supple- Fig. 2. Correlation between AST activity determined with
mented .(Y) substrates is Y = X + 0.7. This regres- and without pyridoxal phosphate supplementation in a
sion fit, with a slope of 1.0 and an intercept of 0.7, is colorimetric method
Activities are reported in units described by Babson et al. (17) at 37#{176}c.
consistent with increased blank because of addition Where present, pyridoxal phosphate concentration was 26 tmol/liter.
of pynidoxal phosphate without stimulation of en- Regressionlinedescribesy = x + 0.7:r = 0.985

zyme activity. Although the magnitude of the in-

creased blank is small (<1 U/liter), a statistically
significant difference can be demonstrated between lyzed by GD, is a source of blank activity in the ki-
results by the two conditions (P <0.05). netic assay of AST (24):
Factors Influencing the Effect of Pyridoxal 2-oxoglutarate + NH3 + NADH
Phosphate on AST Activity L-glutamate + NAD+ H20
Blank. Pyridoxal phosphate can artifactually in- Pyridoxal phosphate might stimulate serum GD ac-
crease measured aminotransferase by stimulation of tivity in the kinetic assay, thereby increasing the ap-
apo-AST, which may be present as a contaminant in parent aminotransferase activity. But this possibility
the malate dehydrogenase suspension (21). In addi- is unlikely, since (a) GD is barely present in normal
tion, the pyridoxal phosphate moiety is able to du- serum (25) and (b) the Km of the human serum en-
plicate AST catalytic activity in the absence of apo- zyme for ammonium ion is about 24 mmol/liter (26),
enzyme (22). while the final concentration of ammonium ion in
The blank activity of the kinetic AST assay was the described assay is only 0.4 mmol/liter. Even with
measured after 60-mm incubation with and without elevated serum GD activity and ammonium ion con-
pynidoxal phosphate (25 zmol/liter). Control sub- centrations, an increase in GD blank activity with
strate exhibited a 0.5 ± 0.1 U/liter blank activity; increased pynidoxal phosphate supplementation is
for the supplemented substrate the activity was 0.6 unlikely.
± 0.2 U/liter. Measurements also include effects on GD blank activity was measured using conditions
spontaneous breakdown
of NADH (23). identical to those in the kinetic AST assay omitting
Preincubation conditions. One-hour preincuba- L-aspartate. Five human sera were selected in which
tions at 30#{176}C
were used in these experiments. Previ- pynidoxal phosphate supplementation produced an
ous data (5, 12) indicate that a 60-mm incubation is increase of greater than 25% AST activity over con-
sufficient for complete saturation of aminotransfer- trol. Samples were incubated for 1 h at 30#{176}C,both
ase at pyridoxal phosphate concentrations in the with and without supplementation. No increase
range of 25 zmol/liter. Increasing incubation times could be demonstrated in GD blank activity as the
to 120 and 180 mm did not observably increase the result of increased pyridoxal phosphate concentra-
enhancement of AST activity measured after 60-mm tion. Blank activity was <1 U/liter for all sera.
incubation. Decreasing incubation time to 30 mm The effect of high amounts of GD activity on the
lowered the stimulation observed to a mean of 10% kinetic AST assay was also measured. A glycerol
for 12 human sera investigated. suspension of bovine-liver GD was prepared to give
Increasing temperature to 37#{176}C
for both 1-h prein- an activity of 400 U/liter when measured at optimal
cubation and assay gave a mean increase of 15%, not pH and ammonium ion concentration (27). This
statistically different from the increase observed with preparation gave a blank activity of 100 ± 7 U/liter
30#{176}C
incubation and assay. in the control assay and 103 ± 8 U/liter in the pyri-
Glutamate dehydrogenase activity. Oxidation of doxal-phosphate-supplemented assay. While there
NADH by 2-oxoglutarate and ammonium ion, cata- was no difference between the two blank activities,

94 CLINICAL CHEMISTRY, Vol. 19, No.1,1973

the magnitude of the blank activity at such low am-
monium ion concentrations was unexpected. This 60
may be explained by a lower Km (NH4) for the bo-
vine enzyme (28) than for the human serum.
Sample size. The sample size, and consequently
40
serum dilution, varies considerably in clinically used Ui
AST assays: 60-fold dilution in the Eskalab method a.
(29), 2.5-fold dilution in the Technicon SMA proce-
.3C
dure (30, 31). For spectrophotometric assays, Henry
U)
et al. (16) use 0.2 ml of specimen in a total volume of d
3.0 ml, while Bergmeyer and Bernt (25) recommend Ui
F- 20
0.5 ml of sample in the same total volume. We com- -i
pared the AST activity of 67 human sera at both 0.5
ml per assay (sixfold dilution) and 0.2 ml per assay
(15-fold dilution), with substrate concentrations and 10
assay conditions identical to those described in “Ma-
terials and Methods,” and with no exogenous pyni-
doxal phosphate. Specimens were equilibrated and
0 0 20 30 40 50
assayed at 30#{176}C. The ratio between the assays was U/LITER (0.2ml SAMPLE)
1.0, and no significant difference could be demon- Fig.3. Comparison of AST activity determined by kinetic
strated between the results of these two procedures assay, with 0.5 ml and 0.2 ml serum in a total assay
(Figure 3). volume of 3.0 ml
Activities are reported at 30#{176}C.
LInear regression fit is y = x - 0.06;r =
We also investigated the effects of pyridoxal phos-
0.997
phate supplementation on the measurement of AST
activity, by use of a sample size of 0.5 ml. Samples
of 69 human sera were added to control and supple-
mented substrates, incubated for 1 h at 30#{176}C,and
assayed after addition of 2-oxoglutarate. AST activi-
ty increased 15% over control with the supplemented.
substrate (P < 0.001). Figure 4 compares results by
these two procedures.
AST stability. We have previously shown (19) that
AST activity found in serum and erythrocytes is la-
bile when incubated at 45#{176}C
in the presence of as-
partate. Our experiments and the procedure of
Henry et al. (16) call for incubation of AST in the
presence of aspartate. Pyridoxal phosphate thus may
exert its effect by preserving rather than enhancing
AST activity. To examine this role of pyridoxal
phosphate, we preincubated two aliquots from each of
17 human sera in control substrate at 30#{176}C
for 6 mm
and 60 mm, respectively. A 6-mm preincubation was
required both for temperature equilibration and for
completion of the side reaction (oxidation of NADH
20 30
by pyruvate, catalyzed by lactate dehydrogenase). U/LITER
Aminotransferase activity measured after the 60- Fig.4. Correlationbetween AST activitywith and without
mm incubation exhibited a decrease of 4% (4 ± 1%, pyridoxalphosphate supplementationina kineticassay
mean ± standard error), which is slight but statisti- Sample sizewas 0.5 ml in a totalassay volume of 3.0 ml. Activities
are
reported at 30#{176}C:
pyridoxal phosphate concentration was 25 !imoI/Ilter.
cally significant (P < 0.005), from that measured ) representsa slopeof 1.0,(-) describes the regression
equa-
after 6 mm. However, this average decrease was not tiony = 1.15x -0.41: r = 0.984
sufficient to explain the 16% increase in activity ob-
served when pynidoxal phosphate was added to the
preincubation mixture. each sample was removed from the 45#{176}Cbath and
To further elucidate the effect of pyridoxal phos- cooled to 30#{176}C,
2-oxoglutarate was added, and the
phate, we added human cytoplasmic AST prepared mixture was assayed for AST activity. The results
from erythrocytes (19) to pooled human serum. Of (Figure 5) show a dramatic decrease in AST activity
this highly active serum (95 U of AST activity per with use of the control substrate, consistent with our
liter at 30#{176}C),
0.2-ml samples were added to both previous findings (19), and a 13% increase over the
control and pyridoxal-supplemented substrates first 20 mm in the supplemented substrate. AST ac-
(without 2-oxoglutarate). The samples were then in- tivity in the supplemented substrate remained near
cubated for various lengths of time at 45#{176}C. After this elevated level throughout the experiment. Pyri-

CLINICAL CHEMISTRY, Vol.19,No. 1, 1973 95

 AST is protected from denaturation by incubation
with 2-oxoglutarate (32). Here, pyridoxal phosphate
was able to effect a 14 ± 2% increase on a sample
that showed 17 ± 1% increase when incubated in a
medium containing aspartate. As Figure 5 also dem-
onstrates, this protective action of pyridoxal phos-
phate is increasingly important at higher incubation
temperatures.
Bowers (33) has suggested that the effect of the
00 220 ratio of the sample volume to total reaction volume
MINUTES in kinetic assays must be examined before the advis-
Fig.5. Effectsof pyridoxalphosphate and L-aSpartateon ahility of using pyridoxal phosphate in a reference
preincubation
Serum-based human cytoplasmic AST was incubated with 134 mmol L-
method for AST can be decided. Arbitrary variations
aspartate per liter, assay preincubation concentration, at 45#{176}c,.X = con- of 6- to 15-fold serum dilution are now used in such
trol. Supplemented medium (0) also contained 26.8 zmol/liter pyridoxal
phosphate. At times shown, samples were withdrawn from heating bath,
assays. It is tempting to theorize that these differing
immediately cooled and assayed for AST activity at 30#{176}C dilutions might explain the discrepancies previously
reported (1, 9-12) in the degree of pyridoxal phos-
phate stimulation of AST. Our results, however,
doxal phosphate thus not only enhances AST activi- showed that pyridoxal phosphate stimulates AST
ty but protects it from inactivation in the presence of activity to about the same degree with use of either
aspartate as well. serum volume (Figures 1 and 4). Furthermore, in the
nonsupplemented assay, there is no significant dif-
Discussion ference when either 0.2 or 0.5 ml of serum is used in
An added 25 mol of pyridoxal phosphate per liter the 3.0-ml kinetic assay (Figure 3).
increases the activity of AST in serum as determined The stimulation of AST activity observed with
in a kinetic assay (Table 1, Figures 1 and 3). The re- pyridoxal phosphate supplementation indicates that
sults obtained show an average increase of 16% in this enzyme is not fully saturated with coenzyme in
measured AST activity for 141 human sera. This in- the kinetic assay medium. For a normal serum, with
crease is attributable neither to pyridoxal phosphate a pyridoxal phosphate concentration of 24 zg/liter
stimulation of contaminant apo-AST nor to nonpro- (34), final concentrations of this coenzyme in a 3.0-
tein catalysis by the coenzyme moiety alone. Pyri- ml total assay volume are 6 X 10 mol/liter with
doxal phosphate stimulation of GD blank activity 0.2 ml of serum and 1.5 X 10-8 mol/liter with 0.5 ml
can also be dismissed as the cause of the observed of serum. Turano et al. (35) have shown that with
increase. Since identical mean increases of 16% were modified preparations of porcine AST, pyridoxamine
observed in sera assayed within 24 h and within 1 h#{149} phosphate dissociates from the holoenzyme at low
of sample collection, pyridoxal phosphate stimula- concentrations in the presence of the amino acid
tion is not the result of coenzyme lability in speci- substrate. In addition, while the binding of coen-
mens during the <24-h storage at 4#{176}C. zyme to the aminotransferase is the result of many
As this study demonstrates, pyridoxal phosphate, factors, the contribution of the phosphate group in
at a concentration of 25 zmol/liter, protects serum the binding is important. Inorganic phosphate buff-
cytoplasmic AST from loss in activity when incubat- er, the buffer most frequently used in AST assays,
ed with L-aspartate (Figure 5). For us to examine the has been shown to inhibit competitively the recom-
true stimulation of AST by pyridoxal phosphate, it bination of coenzyme with apoenzyme (36). Albu-
was necessary to investigate this protective effect. min, present in serum at a molar concentration
Our experiments included 1-h preincubations at about 7 X 10 times that of AST, has also been
30#{176}C
for control and supplemented substrates. While shown to have an affinity for pyridoxal phosphate
we have here and previously (19) shown a significant (37). Dissociation of the coenzyme, perhaps in the
decrease in measured AST activity when sera are in- more easily resolved pyridoxamine form (38), from
cubated at 45#{176}C
in the presence of L-aspartate, we the holoenzyme and inhibition of its recombination
did not expect large decreases with 30#{176}C incuba- with apoenzyme is thus a likely possibility in the
tions. This was borne out when sera incubated at AST reaction mixture. Our data also suggest that
30#{176}C
for 60 mm exhibited 96% of the a&ivity ob- the enzyme present in serum is not fully saturated
served with 6-mm incubations. While slight, this de- with coenzyme.
crease was statistically significant, but it can ac- In our experiments, no comparable stimulation of
count for only a portion of the 16% increase in AST serum AST could be observed when the assay meth-
activity observed with pyridoxal phosphate supple- od of Babson et al. (17) was used. Recombination of
mentation. This was confirmed by altering the order pyridoxal phosphate with apo-AST has been shown
of substrate addition, allowing sera to incubate in the to be retarded by the presence of either 2-oxoglutar-
presence of 2-oxoglutarate and initiating the reaction ate or (less effectively) L-aspartate (4). In the Babson
by addition of L-aspartate. It has been shown that assay method (unlike the kinetic method), both

96 CLINICAL CHEMISTRY, Vol. 19, No. 1, 1973

these substrates are present in the assay mixture protects against a loss of activity during preincuba-
when serum is added. In addition, only 20 mm is re- tion that becomes increasingly important as preincu-
quired for enzyme catalytic activity, without prein- bation temperature is increased; (b) increases mea-
cubation. These factors may explain the lack of ob- sured activity by about 16%; and (c) cancels the ef-
servable effect of pyridoxal phosphate in this meth- fects of age, nutritional state, and similar conditions
od, and may also account for the lack of effect ob- that artifactually influence AST activity by varying
served by other authors (9-11) who used similar the concentration of vitamin B6 in the plasma. Thus
methods. pyridoxal phosphate supplementation seems to be
The reported lack of AST stimulation by pyridoxal desirable in clinical and reference methods for the
phosphate may be due in some cases to oversup- estimation of true aspartate aminotransferase activi-
plementation of coenzyme. Holzer and Schreiber ty in human serum.
(39) have shown that higher concentrations of pyri-
doxal phosphate (150 zmol/liter) inhibit the recom-
bination of the coenzyme with the apoenzyme ob- We gratefully acknowledge theexcellent technical assistance of
tained from yeast. This is also consistent with our Ms. Catherine Nelli.

observations regarding AST of human cytoplasmic References

origin. Gonnard and Nguyen-Philippon (9), who re-
1. Karmen, A., Wr#{244}blewski,F., and LaDue, J. S., Transaminase
ported no pyridoxal phosphate stimulation of serum activity in human blood. J. Clin. Invest. 34, 126(1955).
AST, used it in concentrations as high as 320 tmol/ 2. Schwartz, M. K., Clinical aspects of aspartate and alanine
liter. aminotransferases. Methods Enzymol. 27 B, 866(1971).
Pyridoxal phosphate addition also elicited a 15% 3. Zimmerman, H. J., and Henry, J. B., Serum enzyme determi-
nationsas an aid to diagnosis: Transaminases.In Todd-Sanford:
mean increase when the preincubation and assay Clinical Diagnosis by Laboratory Methods, 14th ed.,I. David-
were done at 37#{176}C. Thus the lack of effect in the sohn, and J. B. Henry, Eds. W. B. SaundersCo.,Philadelphia,
Babson colorimetric assay is not due to the higher Pa.,1969, pp 721-723.
assay temperature. 4. O’Kane, D. E.,and Gunsalus,I.C.,The resolution and purifi-
cation of glutamic-aspartic transaminase. J. Biol. Chem. 170, 425
We also attempted to correlate the diagnoses of the (1947).
patients with large increases in AST activity pro- 5. Meister, A., Sober, H. A., and Peterson, E. A., Studies on the
duced by added pyridoxal phosphate. Four of 141 coenzyme activation of glutamic-aspartic apotransaminase. J.
sera examined (Table 1, Figure 1) demonstrated in- Biol. Chem. 206,89(1954).
creases greater than 45% in aminotransferase activi- 6. Fasella, P., and Turano, C., Structure and catalytic role of the
functional groups of aspartate aminotransferase.Vitam. Horm.
ty when they were supplemented with pyridoxal (New York) 28, 157 (1970).
phosphate. One specimen, the activity of which in- 7. Braunstein,A. E.,Some topochemicalaspectsofthe structure
creased by 46%, was obtained from a 41-year-old and functionof aspartatetransaminase,FEBS(Fed. Ear. Bio-
chem. Soc.) Syrnp. 18,101(1970).
male subject. The day before the sample was ob-
8. Jenkins,W. T., and Sizer,I.W., Glutamic aspartic transami-
tained the patient had undergone 12 h of single-pass, nase. IV. The mechanism of transamination.J. Biol. Chern. 235,
warm hemodialysis, with regional heparmnization. A 620(1959).
recent note by Wolf et al. (40) discusses low serum 9. Gonnard, P., and Nguyen-Philippon,C., Dosage manom#{233}tri-
AST activity in patients uhdergoing hemodialysis. que de Iatransaminaseglutamique-aspartique skrique. Ann. Biol.
Clin. (Paris) 17,206(1959).
These authors suggest that hemodialysis may result 10.Mohun, A. F., and Cook,I.J. Y., Simple
methods for mea-
in depletion of plasma pyridoxal and pyridoxammne suring serum levelsof the glutamic-oxaloacetic and glutamic-py-
phosphate and consequent lowered AST activity. ruvic transaminases in routine laboratories.J. Clin. Pathol. 10,
394(1957).
The large stimulation in AST activity we observed
11. Sall, T., Richards, H. K., Harrison, E.,and Myerson, R. M.,
supports this hypothesis. A colorimetricprocedure for the determinationof serum glu-
A specimen drawn from another patient (female, tamic-oxaloacetictransaminase.J. Lab. Clin. Med. 50, 291
62 years old), diagnosed as having squamous cell (1957).
12. Hamfelt,A.,The effectof pyridoxalphosphateon the amino-
carcinoma of the liver, exhibited a 56% increase in
transferase assay in blood. Scand. J. Clin. Lab. Invest 18 (Suppl.
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98 CLINICALCHEMISTRY,Vol. 19, No.1,1973