CUN. CHEM. 21/3, 420-424(1975)
Mechanized Enzymatic Determination of Triglycerides in Serum
Giovanni Bucolo,” Juanlto Yabut, and Ting Yen Chang
A procedure for enzymatic determination of serum triglycerides [CNn. Chem. 19, 476 (1973)] has been
adapted foruse incontinuous-flow
analysis(Technicon
AutoAnalyzer). A very simple manifold is used; serum is
incubatedat 37 #{176}C
with the lipase and cr-chymotrypsin
inpotassium phosphate buffer (0.1 mol/liter, pH 7, Containing 1.50 g of bovine serum albumin per liter). The liberated glycerol Is dialyzed againstthecompleteglycerol
reagent. The change in absorbance
at 340 nm resulting
from oxidation of NADH isproportional
to the dialyzed
gly#{243}erol.
The same manifold can be used to determine
preformed glycerol if the hydrolyzing enzymes are omitted. The lipase used need not be extensively purified because the dlalyzer removes coloring material and contaminating enzymes that would interfere with the components of the glycerol reagent in the manual procedure. The hydrolysis Is complete, as shown by the use
of equivalent glycerol standards. No prior treatment of
the samples is necessary. Assays are run at 60 per
hour in the AutoAnalyzer I, 80 per hour in the AutoAnalyzer II. Results with both instruments for 150 samples
correlated well with those obtained by the same enzymatic manual method and by the AutoAnalyzer fluorometric procedure.
Addftlonal K.yphraes:
AutoAnalyzer
#{149}glycerol
the assay to be done under mild conditions and with a very
simple manifold. The complexity of the enzymatic assay is
only apparent, because all the reactions proceed smoothly
and without interfering with each other.
The method for the AutoAnalyzer
I and II described
here is based on the reaction sequence shown in Figure 1.
The glycerol liberated by enzymatic hydrolysis of triglycerides is dialyzed and measured as already described (3).
Materials
Apparatus
Spectrophotometric
measurements
associated
with
TRIGLYCERIDESenzymatic
GLYCEROL+ FFA
hydrolysis
determi-
nation
GLYCEROL
Knowledge
of serum triglyceride
concentrations
has
great significance
in the recognition and management
of
hyperlipidemia
(1). Consequently,
many sera are routinely
screened. To handle the resulting volume of assays, automated techniques
are required and were developed (2).
However, the automated procedures developed for the AutoAnalyzer
require a manual extraction into a suitable solvent and a preliminary purification of the extract, before it
is introduced into the instrument. Thus, the procedures are
only partly mechanized. Additionally,
the assay requires a
relatively high temperature
for the alkaline hydrolysis of
glycerides. Because of the harsh chemicals and solvents
used, it is necessary
to use special and expensive tubing,
which must be changed frequently. The assay is complex
and as a result the manifolds used are in most cases rather
ADP
+
+
PYRUVATE
Fig.
1.
GK
PEP
+
a-GP
ATP
NADH
Triglycerides
dialysis
ATP
LDH
assay.
+
+
PYRUVATE
LACTATE
Reaction
ADP
sequence,
+
NAD
indicating
step
complicated.
By comparison, a method base4 on the specific and complete enzymatic hydrolysis described by us (3) would allow
Biomedical Research Laboratories, Calbiochem, 10933 N. Torrey Pines Rd., La Jolla, Calif. 92037.
1 Present
address: Technicon
Instruments
Corp., Tarrytown,
N. Y. 10591.
Received June 24, 1974; accepted Dec. 24, 1974.
4Q
CLINICAL CHEMISTRY, Vol. 21, No.3. 1975
this
work were carried out with a Model 25 kinetic assay system
(Beckman
Instruments
Co., Fullerton, Calif. 92634) and a
Model 240 spectrophotometer
(Gilford Instruments,
Inc.,
Oberlin, Ohio 44704). A single-channel
AutoAnalyzer Sampler II system (Technicon Instruments
Corp., Tarrytown,
Fig.
2.
tion
in
Schematic
the
AutoAnalyzer
flow
diagram
I
for
the
triglyceride
determina-
the
“at,,”
N. Y. 10591)with a Model 111 fluorometer(G. K. Turner
Assoc.,Palo Alto, Calif.94303)was used forthe automated
fluorometric
assays
(2). A 405-nm
narrow-pass
filter
(Turner cat.No. 110-812)was used as the primary and a
485-nm sharp cut (Turnercat.No. 110-817)was used asthe
secondary filter.
The same AutoAnalyzer
Sampler II Sys-
tem, supplemented
with a Model 300 N spectrophotometer
4015 recorder interface (both from Gilford Instruments, Inc.) was used for the enzymatic assay as adapted to the AutoAnalyzer
I, according to the schematic diagram shown in Figure 2.
The AutoAnalyzer II System (Figure 3) was assembled
from standardTechnicon parts.A Pump III,a cartridgekit
assembly, type A, and a single-channel
colorimeterforthe
AutoAnalyzer II were used. A 153-cm (60-inch) dialyzer
was used forthe dialysis.
Membranes used were Type C
(cat. No. 170-0472-02)or Type H (cat. No. 196-0146-A).
All
pH measurements were made with a Model 4 Radiometer
pH meter (The London Co.,Westlake,Ohio 44091).
The SonifierCell Disruptor, Model W185D, was a product of Heat System-UltraSonics,
Inc., Plainview, L.I., N. Y.
and a Model
11803.
Fig. 3. Schematic flow diagram for the triglycerides determination in the AutoAnalyzer II
Double manifold for blank correction
of lactatedehydrogenase,
170 U of glycerolkinase,and 42
mg of NADH.
The above reagentsare available
alreadypreparedinstable dry form from Calbiochem,
as “Triglycerides
MaxPack” (cat. No. 869862).
Glycerol
Chemicals
stock
standard,
containing
about
10 mg/mI.
Weight 1.1g of reagent-gradeglycerol95% into a 100-ml
Calbiochem
productsused were:a-chymotrypsin
(peptivolumetricflask.Diluteto volume with distilled water and
dyl-peptidehydrolase;EC 3.4.4.5),
glycerolkinasefrom E.
mix well.Determine the exact concentrationof this stock
coli (ATP:glycerol phosphotransferase;
BC 2.7.1.30), lac- standard by assayinga suitabledilution(e.g.,
1 ml diluted
tate dehydrogenase (L-lactate:NAD
oxidoreductase;
BC
to 250 ml with distilled
water)with Calbiochem’s
Triglyc1.1.1.27),
lipasefrom R. delemar (cat. No. 437611) (glycerol
erides Stat-Pack. The value obtained is expressed in mg/dl,
ester hydrolase;
EC 3.1.1.3), pyruvate kinase (ATP:pyras triolein.
#{149}
uvate phosphotransferase;
BC 2.7.1.40), bovineserum albuWorking glycerol standards.
Prepare dilutions of the
min, adenosine-5-triphosphate
(ATP), phosphoenolpyrustock in 6 g/dl bovine serum albumin solution to contain
vate (PEP), reduced nicotinarnide
adenine dinucleotide
100, 200, 300, and 400 mg/dl, expressed
as tniolein. The
(NADH), and triolein.
same dilutions
can be made in water without any apparent
Gum acacia was from Hathaway Allied Products (Harbor
difference.
#{149}
City, Calif. 90710). Commercial triglycerides
controls used
Triolein
stock standard.
Add 2.00 g of pure tniolein to
were “ElevatedLipid Control”(Lederle Diagnostics,
Pearl
100 ml of a 3.5 g/dl solution of gum acacia.Immerse the
River,N. Y. 10965),“Tn-El” (A.R. Smith Laboratories, container in an ice bath and emulsify the mixture with the
Los Angeles, Calif. 90017), and Calbiochem’ “Calipitrol.”
sonifier cell disruptor
setat 100 W for 5 mm. The emulsion
Glycerol and Triglycerides
“Stat Packs” (Calbiochem)
contains1958 mg/dl oftriolein.
Itisstableforseveraldays
were used for the manual assaysof glyceroland triglycer- at room temperature,but itmust be mixed wellby shaking
ides. Serum samples were obtained from hospital and combeforeuse.
mercial laboratories and from volunteers.
Working triolein standards.
Make working triolein stanThe wetting agents used (Table 2) were Technicon proddards
by appropriate
dilutions of the well-mixed stock
ucts: Aerosol 22 (T21-0300), Brij-35, 300 gfliter (T21-0110),
standard with the 6 g/dlbovineserum albumin, to contain
Tween 20 (T21-0309), Levor IV (T21-0110), FC 134, 2 g/
a final concentration
of 100, 200, 300, and 400 mg/dl oftnliter(T01-0384),Wetting Agent A (Ultrawet60 L TOlolein. These standards can be used foratleast24 h.
0214). In our evaluation, 0.5 ml ofeach wettingagent,as reWorking triglyceride standards.
Make working triglycceived, was added to 1 liter of potassium phosphate buffer.
eride standards
by appropriate
dilutions of commercial
control sera or of pooled patients’ sera with bovine serum
Reagents and Standards
albumin solution, 60 g/liten.
Potassium
phosphate
buffer (0.1 mol/liter,
pH 7.0) (Reagent A). Dissolve 10.7g of anhydrous
K2HPO4 and 5.3g of
KH2PO4
in distilled
water and dilute
to about
950 ml.
Check the pH and adjustit to 7 with either potassium
hydroxide or phosphoric
acid, 1 mol/liter.
Add 830 mg of bovine serum albumin and let dissolve. Add 0.5 ml of Wetting
Agent A (Ultrawet60L),diluteto a finalvolume of 1 liter,
and check thepH again.
Hydrolyzing
enzymes solution (Reagent B). To 100 ml
ofthe potassiumphosphate bufferdescribed above add 100
U of a-chymotrypsin
amount
determined
and
27000
U
(or any
to be needed for complete
equivalent
triglycerides
hydrolysis)
of lipase.
Glycerol
reagent
(Reagent
C). To 100 ml of the phosphate
buffer described
above add 33 mg of magnesium
chloride hexahydrate,
20 mg of ATP disodium salt, 15 mg
of phosphoenolpyruvate,
500 U of pyruvate
kinase, 170 U
Procedure
Serum or plasma (ethylenediaminetetraacetic
acid anticoagulant) samples were stored frozen until assay. Care was
taken to ensure that the samples were homogeneous, by adequate mixing after thawing and during storage and assay.
Calbiochem’s
Triglycerides
and Glycerol Stat-Packs
were used formanual assays(3).
All reagents were stored refrigerated
and also during
assay.The refrigerated
reagentsarestable for about 48 h.
For the AA J2 operationthe recorderis adjusted by use
of the Gilford 300 N recorder “Scale Adjust” dial and the
Model 4015 recorder interface, while pumping through the
2
Nonstandard
AutoAnalyzer
abbreviations
used:
AA I, AutoAnalyzer
I; AA II,
II.
CLINICAL CHEMISTRY. Vol. 21, No.3, 1975
421
Table 1. Day-to-Day Precision, for 20 Sera, of
the Automated Enzymatic Method for
Triglycerides in the AA II, Double Manifolda
Mean
SD
CV, %
mg/dI
129
.88
81
83
76
5.6
5.1
5.7
7.3
9.0
2.5
3.0
7.6
6.6
3.3
5.8
8.2
7.0
3.1
6.3
2.0
11.7
123
210
75
120
79
213
87
139
8.6
153
7,5
175
73
6.3
2.7
7.7
8.0
9.7
151
101
82
absorbance-concentration
curves
a
Day-to-day precision was evaluated by daily duplicate
assays of 20 sera for 22 days (Table 1).
The excellentcorrelation
between results by the manual
enzymatic method and the present automated procedure in
the AA II,double manifold,is shown in Figure 4. Results
=
=
=
=
=
=
5.4
=
DIscussion
5.8
6.1
4.9
3.6
3.6
5.5
5.2
9.6
4.8
4.0
Linear
were obtained
in both instruments,up to and including
triglyceride
concentration
ofatleast400 mg/dl.
from 150 seraassayedby the two methods produced a correlation coefficient (r) of 0.99 1 and a linear regression
of y
O.980x + 2.8. The same samples were assayed in the AA I
and by the automated
fluorometric
procedure of Kessler
and Lederer (2). The followingcorrelations were obtained:
manual (x) vs. AA I: r 0.992and y
O.986x + 12,fluorometric (x) vs. AA I: r 0.993and y
0.952x + 10,fluorometric (x) vs. AA II: r
0.992and y
0.997x+ 4.6.Allassayswere correctedforfreeglycerol.
4.1
5.2
2.5
5.1
140
-
7.3
concentrations.
‘Each sample was run in duplicate for each of the 22 days, a
total of 44 assays per sample.
instrument either water containing 0.5 ml of Wetting Agent
A per liter
or the reagents.
For operationof the AA II,with the reversingswitch in
the “I” position, the baseline dial set at midpoint and the
standard
calibration
set at “1” and while the reagents are
through the instrument, the recorder is adjusted
toO by using the A and B apertures. The instrument is calibrated with the Standard Calibration, to read 40 while a
pumped
containing
200 mg/dl of triglycerides
is assayed.
The resultsobtained in the AA I and the AA II (with a
singlemanifold) must be corrected
by a blank assay in
which reagentA (phosphatebuffer)replacesreagentB (hydrolyzingenzymes).
This separaterun isnot needed in the AA II manifold
shown
in Figure
3,sincethe blank correctionis appliedau-
standard
tomatically using the double beam colorimeter.
Glycerol standards cannot be used in the AutoAnalyzer
II with double manifold. They are useful, however, for setting up calibration curves in the AA I and in the AA II with
a single manifold. Their main value lies in establishing a
base forthe degreeof hydrolysis of triglycerides in the samples,tniolein
standards,or controlseraof known triglycerides content. Serum or plasma (ethylenediaminetetraacetic
acid anticoagulant)
can both be used forthe assay. Samples
The present procedure offers several distinct advantages
over the same assay as conducted manually. Aside from
considerations
of speed of analysis,
the aitomated procedure separates the two main steps of the assay: enzymatic
hydrolysis and determination
of glycerol. These two phases
of the assay are combined in the AutoAnalyzer
at the dialyzerstep.At thispoint coloredmaterials and interfering
enzymes are effectively separated from the more sensitive
part of the assay devoted to the determination
of glycerol.
Colored materialsthat may pass through the membranes
resultina constantinterference
and thusdo not change the
baseline.
Hydrolysis
of phosphate
compounds
by contaminating enzymes
in the lipase preparation
with a resulting
troublesome
change in absorbance,
such as seen in the coupled manual assay, is sharply diminished.
In the automated
procedure,
this interference
would cause a shift in the baseline. The efficiency
of the dialysis
allowed
us to employ
cruder lipases than those used in the manual
assay. Even
possible contaminating
enzymes
in the sample (more trou-
blesome because they would
the components of the glycerol
the only blank found in the
deriving from contaminating
produce variable changes in
reagent) are removed. Thus,
automated
procedure is that
enzymes present in pyruvate
350
300
250
V
200
-J
4
z
4
150
that tend to separate on standing must be thoroughly
100
mixed before assay.
The two manifolds
in the AA H arephased by substitut(0.1 mol/liter)
for the sample and a solution con-
ing NaOH
taining 5 ml of phenolphthalein
luted to 1 liter with distilled
in ethanol
(100 mg/dl),
50
di-
water instead of Reagent C.
100
Results
150
AUTOANALVZER
200
II
-
250
mg/dV
In the AA I and AA IIwith singlemanifoldor with double manifold but with water used in the blank side, the results obtained with working serum standards exactly dupli-
between results for triglycerides obtained
for 150 samples by the manual and by the AutoAnalyzer II enzymatic assay
cated thoseobtainedwith glycerol standards
Double manifold
422
CLINICAL
CHEMISTRY,
Vol. 21, No.3. 1975
of equivalent
Fig. 4. Correlation
Table 2. Effect of Various Wettin9 Agents on
Results of the Manual Triglycerides Assay
R.cov.ry
Time6
Control5
Brij 35
V
0
z
0
“I
D
-J
4
>
of added
%
triglycerides,
0 time
2h
100%
100%
58%
66%
80%
78%
54%
Aerosol 22
Tween 20
Levor IV
FC 134
96%
103%
Ultrawet-60 L
101%
65%
80%
100%
105%
‘Time elapsed from preparation of the reagent.
Reagentwithout additions.
VALUES OF STANDARDS
(6)
mgldl
Fig. 5. Recoveries of triglycerides obtained at various acthiity
of lipase from R. delemar
300
Assay supplemented wIth 10 U of a-chymotrypsln per ml In AutoAnalyzer I.
1, 2, 3, 4, and 5 represent assays containing, respectIvely, 45, 90, 135. 180.
and 250 U of Ilpase per ml of hycolyzing enzymes reagent. Control Is “Tngiyce.ldes Max Pack”
250
P5200
kinase,lactatedehydrogenase, and glycerolkinase. All
theseenzymes arenow available
ina highdegreeofpurity.
When running the assay inthe AA I or inthe AA IIwith
a singlemanifold,the resultsincludefreeglycerol,
glycerol
liberated
from glyceridesand other compounds that may
be present in serum, such as pyruvate. For this reason itis
important
to run the assay by the same procedure, but eliminating the use of lipase, and to subtract these results
from the total assay. This blank correction is accomplished
automatically in the AA IIwith double manifold.
At a lipase activity below that needed to obtain complete
hydrolysis of triglycerides in the sample, the liberated glycerol appears to be proportional to the concentration of the
triglycerides. Results obtained with R. delemar lipase supplemented by a-chymotrypsin are shown in Figure 5, in
which the activity of lipase is correlated to triglyceride recovery.
Thus it would appear that complete hydrolysis
is
not a requisiteforthe assay,but this isnot necessarily so.
The hydrolysis of triglycerides
depends not only on the
particular activity of the lipase employed, but also on the
physical form of the lipids in the sample. At an activity of
lipase
below the optimum needed forcomplete hydrolysis,
the resultswould be highly variableand depend verymuch
on any loss ofthe enzyme duringthe assay. This would also
diminish the upper limitofdetectionand decreasesensitivity. Furthermore, any free glycerol present would represent
a much higher percentage of the total glycerol.
The optimum lipase activity needed for complete triglycerides hydrolysis is very close to that needed in the manual
assay, under the same conditions of temperature, time of
hydrolysis, and ratio of sample-to-reagent-volume.
Because
the ability of lipase to hydrolyze triglycerides varies from
batch to batch and is only poorly correlated
with results of
assay of the enzyme by titration of fatty acids released
from a substrate of olive oil, it becomes necessary to determine the optimum amount
for every new lot of lipase,
which is done by a stepwise increase in lipase activity until
the glycerol liberated from triolein standards
and from
control sera of known triglycerides concentration is found
to be the same as that recovered from glycerol standards
of
0
z
0
150
Cd,
UI
l00
(5)
>
(4)
50
(3)
-1II
-
50
100
150
200
VALUES OF STANDARDS - m/dI
250
-
300
350
Fig. 6. Effect of a-chymotrypsln on the automated triglycerides assay. Lipase Is from R. delemar
1, 25 U lipase per ml. No chymotrypsln; 2, 25 U lipase and 10 U a.chymotrypsin per ml; 3,75 U lipase per ml. No chymotrypsln; 4,75 U lipase and 10
U a-chymotrypsln per ml; 5,250 U lipase per ml. No chymotrypsln; 6,250 U
lipase and lOU a-chymotrypsln per ml
equivalent
concentration. Under the conditions of our
lipase activity established for each lot,
assay and with the
the hydrolysis
must
be completed
by the time
the mixture
leaves the heating bath and before it enters the dialyzer.
This is indeed the case, as can be shown by collecting
the
reactionmixture before itentersthe dialyzer, immersing
the containerin boilingwater,and assaying the solution.
Under the conditions of assay used in our manifolds, no tri-
glycerides can be measured at this point, all the glycerol
being assayed and accounted for as free glycerol.
In our earlyexperiments we used type “C” dialyzing
membranes for both AA I and AA II.The sensitivity
achieved with the AA I was satisfactory,
whilethe concentration
of glycerol dialyzed
in the dialyzer of the AA II was
very
low. By collecting samples
every
2
mm at the exit of
the dialyzerduring continuousassay of triglycerides (250
mg/dl) and measuring the residualabsorbanceof the glyc-
erol reagent, the concentration of dialyzed glycerol could be
derived. If the glycerol reagent was replaced by potassium
phosphate buffer(0.1mol/liter,
pH 7),then the collected
CLINICAL CHEMISTRY, Vol. 21, No.3, 1975
423
could be assayed for glyceroL In the first assay,
the ratio of the average decrease in absorbance between AA
I and AA II was 2.7. The average assay for glycerol in the
second case yielded a ratio of 2.8. These figures correlate
well with the ratio of the length of the two dialyzers (refractions
spectively,
221 cm and 61 cm), which is3.6.The sensitivity
of the assay in the AA II was increased when we replaced
the type “C” with the new type “H” membranes (Technicon).
One problem inherent
but for
which a correction can easily be applied in the manual
assay, is the blank caused by contaminating
activities or
substratesin the enzymes used. Unless removed, theseconin the
automated
assay,
taminantsproduce a shiftinthe baseline that isdifficult to
compensate.
The dialysis
step,as already mentioned, very
effectively
removes contaminants
present in the lipase of
a-chymotrypsin.Of the otherenzymes, the one found to be
In the presence of some wetting agents commonly used
in the AutoAnalyzer,
recoveries of added triglycerides are
lower than expected. We determined
the effect of several of
these compounds
on the extent of hydrolysis (Table 2). Of
the compounds
tested, Ultrawet
60 L and FC 134 had no
effecton the assay. The reagent used was the same as that
used in the manual
procedure. The temperature
of the
assay was changed from 30 to 37 #{176}C.
This has no effect on
the overallhydrolysisin the AA I,because the incubation
time issufficiently
long(16 mm); however, in the AA II the
time isconsiderablyshorter(approximately5 mm) but an
increasein the temperature of hydrolysis compensates for
this. Changing
the molarityof the bufferfrom 50 to 150
mmol/liter
and increasingthe pH to 7.5 had no effect on
the assay. The effect of a-chymotrypsin,
when R. delemar
lipase isused,isjustas dramatic as the one found during
the development
of the manual
assay.
Figure
6 shows
the
the major contributor to the blank is glycerol kinase. This
enzyme is now available in purified form with a much re-
increaseinhydrolysispromoted by the additionof the protease to the assay.
content of contaminants.
The effectof these contaminants can be further decreasedifthe enzyme is kept
separated
from the remainder of theglycerol
reagent and if
References
duced
the two reagents
are mixed on stream. This procedure
re-
ofthe contaminantswith the substrates
(phosphoenolpyruvate,
ATP, NADH)
in the reagent, and as a result, the blank is also decreased. An
duces
the time of contact
added advantage
is the longer stability of glycerol kinase in
the more concentrated solution. As the cost of this enzyme
accounts
for about half of the total cost of the combined reagent, this is an important
consideration.
CIJN.
Cl-EM. 21/3,
1. Fredrickson, D. S., Levy, R. I., and Lees, R. S., Fat transport in
lipoproteins-An
integrated approach to mechanisms and disorders. New Engi. J. Med. 276,32(1967).
2. Kessler, G., and Lederer, H., Fluorometric measurement
of triglycerides in automation. In Automation
in Analytical Chemistry,
Technicon Symposium
1965; L. T. Skeggs, Jr. et al. Eds. Mediad,
New York, N. Y., 1966, p 341.
3. Bucolo, G., and David, H., Quantitative determination of serum
triglycerides by use of enzymes. Clin. Chem. 19,476 (1973).
424-426(1975)
Lipase-Triggered Kinetic Assay of Serum Triglycerides
GiovannI
Bucolo,1
Ralph McCroskey, and Nadine Whittaker
We describe a kinetic method for assay of serum or
plasma triglycerides, by use of an enzymatic hydrolysis
and reaction sequence already described [Clin. Chem.
19, 476 (1973)]. The reaction is triggered by addition of
lipase, at a time when free glycerol, or pyruvate (or
both) are no longer present. In this method, therefore,
there is no need for a blank glycerol assay. In the procedure, reagents are used that are available commercially in the form of stable, dry powders; the method for
the preparation of the reagents has been changed to
achieve improved stability and performance. Stability
and recovery of added triglycerides are satisfactory.
when applied to automated instruments,because
tion time is considerably shorter
than
correspondingendpoint method and
an enzymatic assay is measured in which the rate-limiting
component is the substrate to be determined. Assay procedures based on this principle are particularly attractive
Biochemical
Research Laboratories,
Calbiochem,
10933 North
Torrey Pines Rd., La Jolla, Calif.
92037.
I Present address:
TechniconInstruments
Corp., 511 Benedict
Ave., Tarrytown, N. Y. 10591.
Received June 25, 1974; accepted Dec. 24, 1974.
424
CLINICAL
CHEMISTRY,
Vol. 21, No. 3. 1975
required
observaby the
thus the analysisis
speeded.
The most obvious kineticadaptationof the enzymatic
triglycerides
assay we have described
(3) would be to
trigger the series of reactions described below by adding
glycerol kinase2 to the total assay mixture at a time when
all the triglycerides have been completely hydrolyzed by
the action of lipase:
glycerol
Glycerol
+
kinase
ATP
glycerol-
Kinetic assay for several substrates has been described in
a number of papers (e.g., 1,2). In these methods the rate of
that
pyruvate
ADP
+
P-enolypyruvate
+
NADH
+
+
ADP
kinase
ATP
4
lactate
Pyruvate
1-phosphate
+
pyruvate
dehydrogenase
H
lactate+ NAD
2Glycerol
kinase
(ATP:glycerol
phosphotransferase;
BC
2.7.1.30); lactate dehydrogenase (L-lactate:NAD
oxidoreductase;
BC 1.1.1.27); lipase (glycerol ester hydrolase; EC 3.1.1.3); pyruvate
kinase (ATP:pyruvate
phosphotransferase;
BC 2.7.1.40).