CLIN. CHEM.

19/5, 476-482 (1973)

Quantitative Determination of Serum Triglycerides

by the Use of Enzymes
Giovanni Bucolo and Harold David

We describe a novel method for determining

serum
triglycerides,
in which an enzymatic
hydrolysis
replaces the more commonly
used saponification
procedure. Under the conditions
of the assay, the enzymatic hydrolysis
can be completed
in less than 10
mm by the combined action of a microbial lipase and
a protease. We have been able to demonstrate
complete hydrolysis of triglycerides
by thin-layer chromatography
of the reaction
products,
by recovery
of
glycerol
from sera of known triglycerides
content,
and by comparison
of triglyceride
assays on a number of sera assayed by our method vs. the AutoAnalyzer procedure.
The hydrolysis is directly coupled to
the enzymatic
determination
of glycerol,
and is followed through absorbance
changes at 340 nm. The
assay is simple, rapid, and requires only 50 tl or
less of sample.
Because the enzymes used do not
release glycerol from other compounds
in serum, the
hydrolysis
can be considered
specific for triglycerides.
Additional

trysin
reaction

Keyphrases:
R. delemar Iipase #{149}a-chymodetermination of glycerol by coupled enzymatic
#{149}
thin-layer chromatography
#{149}
AutoAnalyzer

#{149}

The most widely

used methods
for determining
serum triglycerides
require
at least a chemical
hydrolysis
(1-7), usually
carried
out on a solvent
extract of the serum lipids. Compounds
that may interfere with the final determination
of the hydrolysis
products
must, in most procedures,
be removed
by
appropriate
purification
procedures.
All these methods
yield precise
and accurate
results,
but they are cumbersome,
time consuming,
and require considerable
skill.
On the other hand,
hydrolysis
of triglycerides
by
one or more enzymes
under
mild conditions
would
eliminate
many of the deficiencies
and complexities
From
the Biochemical
Research
Laboratories,
10933 North Torrey Pines Road, Calif. 92037.
Received May 23, 1972; accepted
Feb. 13, 1973.

476

CLINICAL CHEMISTRY, Vol. 19, No.5, 1973

Calbiochem,

of the chemical
procedures.
It would be simple and
specific.
Errors
or losses possible
in more complex
techniques
would be minimized
if prior treatment
of
the sample was not required
and if the complete
determination
was performed
in a single reaction
vessel.
We have developed
a simple,
precise,
and reproducible
procedure
that links the enzymatic
hydrolysis of serum triglycerides
to the simultaneous
enzymatic determination
of glycerol.
In our opinion,
the
self-evident
advantages
of the new technique
render
it the method
of choice for the assay of serum triglycerides.

Materials
Apparatus
A Model DBG Spectrophotometer
(Beckman
Instruments,
Inc., Fullerton,
Calif. 92634),
a Model
2000 Spectrophotometer
(Gilford
Instruments,
Inc.,
Oberlin,
Ohio 44704) and a Model 330 Spectrophotometer
(G. K. Turner
Assoc.,
Palo Alto,
Calif.
94303) were used for the spectrophotometric
assays.
A single-channel
AutoAnalyzer
Sampler
II System
(Technicon
Instrument
Corp.,
Tarrytown,
N. Y.
10591) with a Model 111 Fluorometer
(G. K. Turner
Assoc.) was used for the automated
determination
of
serum triglycerides.
Matched
Pyrex cuvets (Calbiochem, La Jolla, Calif. 92037) were used for all spectrophotometric
readings
at 340 nm. Oxford Samplers
(Calbiochem)
were used in all assays.
Chemicals
Calbiochem
products
used were: a-Chymotrypsin
(peptidylpeptide
hydrolase;
EC 3.4.4.5),
elastase
(pancreato-peptidase
E; EC 3.4.4.7),
glycerol kinase
(ATP: glycerol
phosphotransferase;
EC
2.7.1.30),
lactate
dehydrogenase
(L-lactate : NAD oxidoreductase; EC 1.1.1.27),
lipase (glycerol
ester hydrolase;
EC 3.1.1.3) from hog pancreas
(Pronase),
pyruvate

kinase
(ATP: pyruvate
phosphotransferase;
EC
2.7.1.40),
trypsin
( peptidylpeptide
hydrolase;
EC
3.4.4.4),
adenosine-5-diphosphate
(ADP),
adenosine-5-triphosphate
(ATP),
bovine serum albumin,
L-a-glycerophOSphate,
lecithin,
linolenic
acid, lysolecithin,
methyl
linoleate,
methyl
linolenate,
methyl
oleate,
reduced
nicotinamide
adenine
dinucleotide
(NADH),
oleic acid, P-enolpyruvate
(PEP),
and tnolein. 1,3-Diolein
was purchased
from Applied
Sciences Laboratory,
Inc., State College, Pa. 16801. Lipases (glycerol
ester hydrolase;
EC 3.1.1.3) from R.
delemar,
A. niger and G. candidum
were products
of
Miles Laboratories,
Inc., Kankakee,
Ill. 60901. All
other chemicals
were either
Analytical
Reagent
or
the otherwise
best available
grade.
Samples
of serum were obtained
from hospital
and
commercial
laboratories
and from donors.
All samples and pooled sera were assayed,
subdivided,
and
maintained
frozen or lyophilized
until needed. In our
experience
(to be reported
in a future
communication) triglycerides
in serum
are stable under these
conditions.

Reagents and Procedures

Triglycerides
were assayed
with the AutoAnalyzer
according
to the procedure
of Kessler
and Lederer
(4).
Triglycerides
were hydrolyzed
to glycerol and fatty
acid either enzymatically
with a mixture
of R. delemar lipase and a-chymotrypsin
or by saponification
with potassium
hydroxide
in ethanol.
The resulting
free glycerol
was then determined
enzymatically
by
the following series of reactions:

Glycerol
ADP

glycerol

kinase

pyruvate

kinaoe

ATP

glycerol-i-P

#{247}
PEP

-.

lactate

Pyruvate

+ NADH

ATP

ADP

#{247}
pyruvate

dehydrogenaae
-

Lactate

NAD

The decrease
in absorbance
at 340 nm is proportional to the concentration
of glycerol.
The glycerol assay reagent contains,
in a final volume of 3 ml: 5 mol
of magnesium,
0.9 mol
of ATP,
0.9 zmol of PEP, 6 U of pyruvate
kinase, 2 U of lactate dehydrogenase,
NADH to a final absorbance
at
340 nm of 0.9-1.0
(approx.
0.4 mg) in potassium
phosphate
buffer (pH 7, 0.1 mol/liter).
The triglyceride
reagent
has the same composition
as the glycerol reagent;
per assay, it contains
additionally
400 U of R. delemar
lipase, 30 U of a-chymotrypsin,
and 5 mg of bovine serum albumin.
A glycerol kinase solution
containing
2 U in 50 zl
is prepared
by diluting
the enzyme
in potassium
phosphate
buffer (pH 7, 0.1 mol/liter)
containing
0.1
g of bovine serum albumin
per 100 ml.
The glycerol
and the triglycerides
reagents
have
proven to be stable for at least one year in dry form
in the refrigerator
(Triglycerides
and Glycerol
StatPack,
Calbiochem,
La Jolla, Calif. 92037). After re-

constitution
with water and storage at 4#{176}C,
these reagents can be used for at least 24 h.
The same glycerol reagent is used for the enzymatic determination
of glycerol after alkaline
hydrolysis,
but the final volume of the reagent
is 2.5 ml instead
of 3 ml. The following additional
reagents
are needed
for the alkaline hydrolysis:
Potassium
hydroxide,
approx.
0.7 mol/liter.
Dissolve 4 g of potassium
hydroxide
in 10 ml distilled
water. When cool, dilute to 100 ml with ethanol.
Magnesium
sulfate,
approx.
0.18 mol/liter.
Dissolve 2.17 g of anhydrous
magnesium
sulfate
in
water and dilute to 100 ml.
Free GyceroI

in Serum

1. To 3 ml of glycerol
reagent
in a cuvet with a
10-mm light path, add 0.2 ml of serum.
Mix. Incubate for about 5 mm at 30#{176}C,
to consume
endogenous substrates.
2. Read the absorbance
(A0) at 340 nm, with distilled water as a blank. Add 50 l of the glycerol kinase solution,
mix, and incubate
at 30#{176}C.
3. Read the absorbance
of the sample 10 mm (A10)
after the addition
of glycerol kinase.
Obtain iXA by subtracting
A10 from Ao.
Calculations:
M x 23.7 = mg glycerol in 100 ml
of serum;
LA x 228 = mg glycerol
in 100 ml of
serum, expressed
as triolein.
Triglycerides

in Serum

A. Enzymatic
hydrolysis.
1. To 3 ml of triglycerides reagent
in a cuvet with a 10-mm light path, add
50 cl of serum,
mix, and incubate
at 30#{176}C
for about
10mm.
2. Read the absorbance
(A0) of the sample
at 340
nm, with distilled
water as a blank.
Add 50 l of
glycerol kinase, mix, and incubate
at 30#{176}C.
3. Read the absorbance
at 340 nm 10 mm (A10)
after the addition
of glycerol kinase.
4. Obtain &4 by subtracting
A10 from A0.
5. Calculations:
.A
X 883 = mg triglycerides
in
100 ml of serum, expressed
as triolein.
B. Alkaline
hydrolysis.
1. Into a screw-capped
culture tube, pipet 0.2 ml of serum and 0.5 ml of potassium hydroxide
solution,
mix, cap the tube and place
it in a water bath at 60#{176}-70#{176}C
for 30 mm. Cool to
room temperature.
2. Add 1 ml of magnesium
sulfate
solution,
mix,
and centrifuge.
Use the clear supernatant
fluid in the
following assay.
3. To 2.5 ml of the glycerol reagent in a cuvet with
a 10-mm light path, add 0.5 ml of supernatant
fluid
(from step 2), incubate
for 5 mm at 30#{176}C,
and read
the absorbance
(A0) of the sample
at 340 nm, with
distilled water as a blank.
4. Add 50 tl of glycerol kinase solution.
Mix and
incubate
at 30#{176}C.
Read the absorbance
10 mm (A10)
after the addition
of glycerol kinase.
5. Obtain A
by subtractingA1o
from Ao.
6. Calculations:
Corrected
M x 738 = mg triglycerides in 100 ml of serum, as triolein.
CLINICAL CHEMISTRY, Vol. 19. No. 5, 1973

477

For the methods

described,
the following considerations should be noted:
1. A blank determination,
in which a volume
of
water
is used that is equivalent
to the volume
of
sample,
must be performed
at least once for every
batch
of reagent
to compensate
for contaminants
(substrates
or enzymes)
in the components
of the
assay.
2. No correction
has been made in the calculations
to account
for the dilution
made when 50 l of glycerol kinase is added to the reaction
mixture.
The decrease in initial absorbance
(Ao) resulting
from this
dilution
can be obtained
by multiplying
A0 by the
factors
0.985 for glycerol
or 0.984 for triglycerides,
after enzymatic
or alkaline hydrolysis.
3. Preformed
free glycerol in the sample
must be
determined
and the results
(expressed
as tniolein)
subtracted
from the total triglycerides
value.
4. The factors used in the calculation
are based on
a molar absorptivity
for NADH of 6.22 X 10.
Thin-Layer

Chromatography

Chloroform:methanol
(2:1 by vol) was used to
extract
the lipids from incubation
mixtures
(8). The
solvent was evaporated
at low temperature
(less than
40#{176}C)
under a stream
of nitrogen.
The residue
was
dissolved
in a volume of chloroform:methanol
(2:1)
equivalent
to one-tenth
the original
volume
of the
serum.
Standard
solutions
were prepared
to contain
10 g of triglycerides
(or equivalent
weight of other
compounds)
per liter. A 5-l sample of each solution
was applied
to an Eastman
Kodak
Silica Gel No.
6060 Chromagram,
1 cm above the bottom edge. The
ascending
chromatogram
was developed
(9, 10) in
diethyl
ether: benzene: ethanol: acetic
acid (40:50: 2:0.2) until the solvent front had reached
a height of
4 cm above the point of sample application.
The chromatogram
was then
dried
and developed
in the
same direction
in hexane:diethyl
ether:acetic
acid
(180:30:2).
The spots were made visible by exposure
to iodine vapors in a closed tank.
Evaluation

of Lipases

Lipases
were evaluated
by one or more of the following procedures:
1. Titration
of fatty acids liberated
from an emulsion of olive oil (11). Units of lipase are expressed
as
microequivalents
of fatty acid liberated
per minute
by this procedure
at 30#{176}C.
2. Determination
of the glycerol liberated
by various amounts
of lipase from the same olive oil emulsion (11). The reaction
was stopped
at selected
times
by adding
an equal volume of trichloroacetic
acid, 5
g in 100 ml of water. The acid and the unreacted
olive oil were removed
by extracting
twice with four
volumes
of chloroform
and once with four volumes of
diethyl
ether. The last traces of ether were removed
by bubbling
nitrogen
through
the solution.
This
treatment
left a clear aqueous
solution
containing
glycerol in amounts
proportional
to the extent of hydrolysis.
Glycerol
was determined
in a suitable
frac478

CLINICAL CHEMISTRY, Vol. 19, No.5, 1973

tion of this solution by the procedure

described.
3. The final and most important
consideration
in
the selection
of a lipase was, of course, its ability to
completely
hydrolyze
triglycerides
within the parameters of our assay. Crude enzyme
preparations,
not
suitable
for direct
spectrophotometnic
assay,
were
evaluated
before purification
by a two-step
procedure. In this case, serum was incubated
with the iipase in the presence
of a-chymotrypsin
for 15 mm at
30#{176}C.
Proteins
were precipitated
with perchloric
acid
and the assay mixture
was centrifuged.
The clear supernatant
fluid was neutralized
with solid potassium
carbonate.
The potassium
perchlorate
was removed
by centrifugation
and the glycerol in the supernatant
fluid was assayed as described.

Results and Discussion

The most important
considerations
in the selection
of a lipase for our spectrophotometnic
assay were the
extent
of hydrolysis
of triglycerides,
the specificity
for these compounds,
and the period
required
for
maximum
activity.
However,
none of the lipases
from different
sources (e.g., from A. niger, G. candidum, hog pancreas,
or R. delemar)
catalyzed
complete hydrolysis
of serum triglycerides
by itself. We
assumed
for these lipases a mode of action similar to
that reported
for pancreatic
lipase. This enzyme has
been shown to preferentially
hydrolyze
the esters at
the primary
hydroxyl
groups of glycerol,
while hydrolysis at the secondary
hydroxyl
group is very slow
and limited
(12-15).
On the basis of this model, it
was conceivable
that a nonspecific
esterase
could
complete
the hydrolysis
of the ester in the beta position. a-Chymotrypsin
was chosen
as the auxiliary
enzyme
because
of its well-known
esterase
activity
(16); moreover,
the enzyme is available
in highly purified form, free of contaminants
having an adverse
effect on the other constituents
of the glycerol
reagent, and it is readily soluble.
To test the validity
of this assumption,
various
amounts
of a-chymotrypsin
were added to the glycerol reagent
and the lipases were evaluated
in the
triglycerides
assay. Among the various
lipases,
only
the one from R. delemar
(17)
completely
hydrolyzed
triglycerides
in the reagent
supplemented
with achymotrypsmn.
The optimum
concentration
of the
latter
enzyme
was found to be 30 U/3 ml of assay
mixture.
a-Chymotrypsin
exerts
a similar
action
when
added to lipases from other sources.
For example,
if
30 U of .chymotrypsin
is added to 800 U of partially purified
porcine
pancreas
lipase, recovery
of free
glycerol from serum triglycerides
increases
from 18%
to 65%.
Figure 1 demonstrates
the increase
in glycerol production
by the R. delemar
lipase in the presence
of
a-chymotrypsin
from an olive oil emulsion
as compared with glycerol liberated
by lipase alone by the
procedure
described.
An increase
in glycerol liberated
by R. delemar
IIpase from serum triglycerides
was also found when

Table 1. Effect of Proteases on the Hydrolysis

of Serum Triglycerides by
A. delemar Lipase (400 U)
Protease added

GLYCEROL
(AS
IRIOLEIN)

mq

No addition
a-Chymotrypsin
Elastase
Pronase
Trypsin
S

IS

IS

25

25
UNITS

Fig. 1. Production
by R. delemar lipase,

of glycerol

30

35

40

45

Percent
hydrolysis

Amount

30-40%
100%
90%
98%
73%

30 U
15 U
90 P.U.K. units
5000 N.F. units

54

OF LIPASEPERASSAY

from an olive oil emulsion

with and without

s-chymotrypsin

other
proteases-such
as elastase
(15 U), trypsin
(5000 N.F.
units)
(18), and Pronase
(90 PUK
units)
(19)-were
substituted
for a-chymotrypsin
(Table
1). Because
of the common
behavior
of these
enzymes,
we believe
that
esterase
activity
is not
their function.
The mechanism
of action of the proteases in our assay is currently
under investigation.
No lipase activity
has been found in any of the
proteolytic
enzymes
investigated,
as glycerol
is not
released
from triglycerides
by these enzymes.
Thinlayer chromatography
(Figure 3) similarly
shows the
lack of activity
toward triglycerides
by a-chymotrypsin in the absence of lipase.
One of our major concerns
was the possibility
that
the
lipase-protease
combination
could
hydrolyze
phospholipids.
Under the conditions
of our assay, no
free glycerol
was formed from lecithin,
lysolecithin,
or indeed
from L-a-glycerol
phosphate.
We did not
investigate
whether
fatty acids are liberated
by our
hydrolyzing
enzymes
from these
compounds.
This
would be an important
consideration
if triglycerides
were determined
by assaying
free fatty acids produced, rather than glycerol.
Thin-layer
chromatography
was used to separate
and identify
the components
formed by the following
incubation
mixture:
serum
(0.5 ml) was incubated
for 30 mm at 30#{176}C
with 50 U of a-chymotrypsin
or
1300 U of lipase, or both enzymes
in 2 ml of potassium phosphate
buffer (0.1 mol/liter,
pH 7), or with
the complete
triglycerides
reagent.
The lipids were
extracted
from these
mixtures
and separated
by
thin-layer
chromatography
as described
(Figures
2-4). Esters of cholesterol
and of fatty acids are not
hydrolyzed
by the reagent
(Figure
4). Free fatty
acids were evident
after a very short period of interaction of the reagent
with serum
(Figure
4, C). At
this time the triglycerides
spot was very faint. After
10 mm, this spot had completely
disappeared.
Glycerol monooleate
and triglycerides
are not hydrolyzed
by chymotrypsin
alone,
but are rapidly
attacked
when lipase is added.
Hydrolysis
of triglycerides
is
complete
when both enzymes
are present;
the other
components
of the glycerol
reagent
are not needed
for the hydrolysis,
as shown in Figure 3.

S.:

S.

#{149}

I
S

#{149}

A
Fig.
phy

2. Standards

A, Cholesteryl

oleate;

separated
B. Methyl

E
F
by thin-layer

linolenate;

G
chromatogra-

C, Triolein;

D, Mixture

of all

E, Linolenic acid; F, Diolein

standards;

II.

0
Fig. 3. Thin-layer
from serum lipids
A, Untreated

treated
without
pase

serum;

____

______

-.--

chromatography

of hydrolysis

,-&
4;#{149}r1

products

B, Standards
without methyl linolenate;
C, Serum
D, Serum treated with lipase; E. Standards
F, Serum treated with o-chymotrypsin
and Ii-

with a-chymotrypsin;
cholesteryl
oleate;

CLINICAL

CHEMISTRY,

Vol. 19. No.5,

1973

479

:t

#{248}.

S.

ii

#{149}0
I.

J_

.,.

,_.

A
-$t
#{149}-#{176}i
f
Fig. 4. Thin-layer chromatography
of hydrolysis products
from serum lipids after treatment with the total triglycerides reagent
.

6
T.

10 12 54 $6 1$ 20
1. MipuNs

Fig. 5. Influence of the components

mixture on the triglycerides reaction.

6
Ti..

S 10 12 14 16 1$ 20
I. MI..tss

of the hydrolysis

Curve A, Albumin; Curve B, t-Chymotrypsin; Curve C, Albumin + lipase;

Curve D, Albumin + s-chymotrypsin; Curve E, Lipase; Curve F, Lipase
+ t-chymotrypsin;
Curve G, Albumin + lipase + -chymotrypsin.
Glycerol kinase added after 10 minute incubation period at 30#{176}C

A, Untreated serum; B, Standards without cholesteryl oleate; C, Serum

with reagent-30
seconds incubation; D. Standards without methyl 1mblenate; E, Serum with reagent after 10 minutes incubation; F. Reagent

We found that there was generally

a poor relationship between
the specific activity
of lipases as measured by titration
of fatty acids, by the measurement
of glycerol
(Figure
1) produced
from a
olive oil
emulsion,
and by the concentration
of enzyme
required for complete
hydrolysis
of triglycerides.
These
parameters
would be more directly
related in a highly purified
enzyme preparation;
such a preparation
is
not required
in our assay and would be very costly.
For this reason,
the amount
of lipase
needed
per
assay must be optimized
in every new preparation;
for R. delemar
lipase,
this amount
varied between
100 and 400 U.
The glycerol reagent,
even though based on a published
procedure
(3), was modified
to perform
in
combination
with the hydrolyzing
enzymes.
The
course of events that guided us during this development is represented
in Figure 5, which shows patterns of absorbance
changes
at 340 nm during
the
assay. When a-chymotrypsin
is added to the assay
with and without
lipase (Figure
5, B, D, F, G) the
serum clears,
as indicated
by a rapid initial fall in
absorbance,
which is barely visible at the beginning
of the charts.
This is a common
phenomenon,
observed to varying
degrees
with many samples,
but
more evident
with lipemic
samples.
In one experiment, 200 l of lipemic serum was added to the glycerol reagent
supplemented
with 30 U of a-chymotrypsin.
.A,
at 340 nm, before addition
of glycerol
kinase was 0.5; the change in the absence
of a-chymotrypsin
was negligible.
The same
clarification
takes place with lipase alone (Figure 5, E), but after
an initial lag period. In the absence
of bovine serum
albumin,
turbidity
develops
in the system after the
initial phase of clearing
(Figure 5, F), as indicated
by
a slow increase
in absorbance
before the glycerol kinase is added.
The absence
of turbidity
when albu480

CLINICAL CHEMISTRY, Vol. 19, No. 5, 1973

mm is present,
as demonstrated
by the constant
rate
of decrease
in absorbance
of the system
before the
addition
of glycerol kinase (Figure 5, G), may be the
result
of the binding
of fatty acids to this protein
and thus to their solubilization
and removal from the
reactants.
This was the principal
reason for adding
this protein to the reagent.
The blank rate generated
in the assay is due to
phosphatases
acting
on PEP and ATP. The major
sources of these contaminants
are lipase, glycerol kinase, and possibly the sample itself.
The rate owing to contaminating
enzymes
or loss
in absorbance
caused by contaminating
substrates
in
the reagent
can be determined
for every batch by
performing
a blank assay with water in the place of
the sample.
Phosphate
ions are known, to inhibit
phosphatase
activity
(20), and their effect in reducing the activity
of the contaminants
in the reagent is
shown in Table 2. The pH chosen was 7, even though
the Table
indicates
that the phosphatase
activity
from the reagent
is somewhat
higher at this pH. On
the other hand,
it is important
to inhibit
alkaline
phosphatases
from serum,
which may have higher
activities
than those contaminating
the enzymes
of
the reagent.
Under these conditions,
and with this
buffer, increase
in blank rate caused by phosphatases in the sample
has not been observed
by us. Even
when purified
human intestinal
or placental
alkaline
phosphatase
was added to serum (2000 U per liter),
the increase
in blank rate of the system is negligible.
The average
increase
noted at this concentration
of
enzyme was less than 0.015 for 10 mm, equivalent
to
13 mg of triglycerides
per 100 ml under the conditions of our assay. However,
our experience
in the
application
of this method has not yet been so extensive as to rule out increase
in rate or inhibitors
from
sera in some pathological
conditions
or as a result of
a particular
medication.
In these cases, individual

Table 2. Effect of Various Buffers (0.1

mol/liter) and pH on the Phosphatase and

ATPase Activity of Contaminants in the
Triglycerides

Reagent at 30#{176}C
inhibition,

Buffer

pH

Av A/mIn

Tris-maleate
Tris-succinate
Tris-succinate
Potassium phosphate

7
7.6
8.0
8.0

0.089
0.0098
0.0016
0.0006

0
90
98
99

Potassium
Potassium

7.6
7.0

0.0015
0.002

98
97

phosphate
phosphate

AUTO
ANALYZER

100

200

300

400

500

600

700

PRESENT METHOD

correction
could be applied by taking a third absorbance reading 20 mm after addition
of glycerol kinase,
and subtracting
the xA between
10 and 20 mm from
that between
0 and 10 mm. Should
the blank rate
thus determined
be lower than that found with the
reagent
alone,
it would
indicate
a depletion
in
NADH or inhibition
of some of the enzymes
in the
system.
NADH may be depleted
at high concentrations of triglycerides
or as a result of high pyruvate
content
in the sample.
This last compound
may be
present in high concentration
in some control sera.
The triglycerides
reagent
was modified
according
to these considerations
and to the optimum
concentration
of the components
as determined
under the
conditions
of our assay. The reagent
as finally developed contained:
magnesium,
5 zmol; ATP, 0.9 zmol;
PEP, 0.9 mol;
bovine serum albumin,
5 mg; pyruvate kinase,
6 U; lactate
dehydrogenase,
2 U; R.
delemar
lipase,
400 U; a-chymotrypsin,
30 U;
NADH,
0.4 mg, all in a final volume of 3 ml in potassium
phosphate
buffer (pH 7, 0.1 mol/liter).
Standard
solutions
of glycerol can be used to provide a control for the reagent.
A solution
containing
1 g of triolein
in 100 ml of 2-propanol
can also be
used. Aliquots
of 5, 10, and 15 l of this solution
will
give a decrease
in absorbance
equivalent
to that obtained
by using 50 l of serum
containing,
respectively, 100, 200, and 300 mg triglycerides
in 100 ml.
This will serve as a control
for the hydrolyzing
enzymes as well as for the glycerol reagent.
We prefer
to use a serum control standardized
by assay of glycerol after alkaline
hydrolysis
or by the AutoAnalyzer
procedure.
Results
for 38 sera by the present
method
vs. the
AutoAnalyzer
procedure
(4) produced
a correlation
coefficient
(r) of 0.993 and a linear regression
of y =
1.048 x - 4 (Figure 6).
Three
serum
pools were assayed
on 10 different
days by the present
method.
The results
appear
in
Table 3.
Other methods
for determining
glycerol
liberated
in the hydrolysis
of triglycerides
are currently
under
investigation.
In general,
they are not as convenient
as the one presented
here because
in most cases additional
steps are required,
and they do not lend
themselves
as readily to the use of a single reagent.

Fig. 6. Triglycerides
assays of 38 serum
AutoAnalyzer and by present method
Figures

are in mg/100

samples

by

ml

Table 3. Results of 30 Assays on

Each of Three Serum Pools
Pool
1

104
97
95
102
101

151
153
156
151
155

276
274
273
273
278

91

155

273

100

162

282

95

160

278

94

154

274

97
100
103

158
157
158

278
271
271

106
104
103
102
104

159
155
159
155
157

271
275
265
273
273

95

151

290

95
101
101

151
153
151

285
275
276

103

155
153
156
160

274
271
273
261

102
98
102

Mean

98

159

275

108

160

273

104
102
100

151
156
154

290
280
278

100

156

275

SD

3.86

3.22

7.00

CV %

3.86

2.06

2.54

We thank
tions, sound

Drs. W. Drell and J. K. Pollard,

advice, and constant
support.
CLINICAL

CHEMISTRY.

Vol.

Jr. for their

19. No.5,

1973

sugges-

481

References
1. Carlson,
L. A., and Wadstrom,
L. B., Determination
ides in blood serum. Clin. Chim. Acta 4, 197 (1959).
2. Henry,
R. J., Clinical
Chemistry:
Principles
and
Hoeber, New York, N. Y., 1964, p 864.

of glycerTechnics,

3. Eggstein,
M., and Kreutz,
F. H., Eine neue Bestimmung
der
Neutralfette
in Blutserum
and Gewebe.
KIm. Wochenschr. 44,
262(1966).
4. Kessler,
G., and Lederer,
H., Fluorometric
measurement
of triglycerides.
In Automation
in Analytical
Chemistry,
Technicon
Symposia.
1965; L. T. Skeggs,
et al, Eds. Mediad,
New York,
N.Y., 1966,p341.
5. Rice, E. W., Triglycerides
(neutral
fat)
in serum.
Stand.
Methods Clin.Chem. 6,215(1970).
6. Soloni,
F. G., Simplified
manual
micromethod
tion of serum triglycerides.
Clin.Chem. 17,529(1971).

for determina-

7. Chin, H. P., Abd El-Meguid,

S. S., and Blankenhorn,
D. H.,
An improved
method
for determination
of serum and plasma triglycerides.
Clin. Chim. Acta 31, 381 (1971).
8. Folch,
J., Lees, M., and Sloane-Stanley,
G. H., A simple
method
for the isolation
and purification
of total lipids from animal tissue. J. Biol.Chem. 226, 497 (1957).
9. Freeman,
C. P., and West, D., Complete
separation
of lipid
classes on a single thin-layer
plate. J. Lipid Res. 7,324(1966).
10. Louis-Ferdinand,
R. T., Therriault,
D. G., Blatt, W. F., and
Mager,
M., Application
of thin-layer
chromatography
to the

482

CLINICAL

CHEMISTRY,

Vol.

19, No.5,

1973

quantitation
of plasma
neutral
lipids and free fatty acids. Clin.
Chem. 13,773(1967).
11. Laboureur,
P., and Labrousse,
M., Lipase de Rhizopus arrhizus. Obtention,
purification,
et proprietes
de Ia lipase de Rhizopus arrhizus var. delemar.
Bull. Soc. Chim. Biol. 48, 747 (1966).
12. Willis, E. D., Lipases. Advan.
Lipid Res. 3, 197 (1965).
13. Mattson,
triglycerides

F. H., and
by pancreatic

Beck, L. W., The digestion

in vitro
lipase. J. Biol. Chem. 214, 115 (1955).

of

14. Mattson,
F. H., and Beck, L. W., The specificity
of pancreatic lipase for the primary
hydroxyl
groups
of glycerides.
J. Biol.
Chem. 219, 735(1956).
15. Mattson,
F. H., and Volpenheim,
R. A., Hydrolysis
of primary and secondary
esters
of glycerol
by pancreatic
juice. J.
Lipid Res. 9,79(1968).
16. Desnuelle,
P., Chymotrypsin.
In The Enzymes,
2nd ed., 4, P.
D. Boyer, H. Lardy, and K. Myrback,
Eds. Academic
Press, New
York, N. Y., 1960, pp 111 and 112.
17. Fukumoto,
J., Lwai, M., and Tsujisaka,
Y., Studies
on lipase.
IV. Purification
and properties
of a lipase secreted
by Rhizopus
delemar. J. Gen. Appl. Microbiol.
10, 257 (1964).
18. National
Formularv,
13th ed., American
Pharmaceutical
Association,
Washington,
D. C. 20037, p 747.
19. Brochure
on Pronase
by Kaken
Chemical
Co., Ltd.,
Japan. Distributed
by Calbiochem,
La Jolla, Calif. 92037.

Tokyo,

20. tadtman,
T. C., Alkaline
phosphatases.
In The Enzymes,
2nd ed., 5, P. D. Boyer, H. Lardy,
and K. Myrback,
Eds. Academic Press, New York, N. Y., 1960, p55.