1. No category

CLIN.CHEM. 25/3.439-442(1979)
AutomatedMeasurementof Total Cholesteroland Triglycerides,in
“Tandem,” on the DiscreteSample Analyzer,GilfordSystem 3500
Lynn Smith, Diane Lucas, and Gary Lehnus
We have developed an automated procedure
on a discrete
sample analyzer, Gilford#{174}
System 3500, which alternately
measures both total serum cholesterol and triglyceride
concentrations as a “tandem” procedure. We used Dow
Diagnostic’s fully enzymatic, colorimetric reagents and
aqueous standards to calculate unknowns ratiometrically.
Cholesteroland then triglyceride
reagent aredispensed
into alternatecups; the produced color at 500 nm is
measured after an ambient temperature incubation of 20
mm. Reagent and sample carryover is less than 1.6%.
Correlation coefficients of 0.997 for comparison of both
automated tests with the manual methods at 30 #{176}C
and a
typical CV of less than 2.0% show this “tandem” procedure to be reliable and accurate.
Cholesterol
determinations
have become
they have been associated with
various lipid disorders such as hyperlipidemia,
diabetes
mellitus, hypothyroidism,
liver disease, atherosclerosis,
and
other metabolic derangements
(1,2). The quantitation
of both
exceedingly
Method and Materials
Serum samples are loaded on the instrument in duplicate.
Serum and either cholesterol or triglyceride reagent are separately dispensed into alternate cups. After a 20-mm incubation at ambient temperature,
the reaction mixtures are
sequentially
aspirated and the amount of color formed is
measured at 500 nm. The concentrations
of total cholesterol
or triglycerides in the samples are calculated ratiometrically,
according to the respective standard’s absorbance.
In the cholesterol reaction, samples are incubated with
cholesterol esterase and cholesterol oxidase, which convert
cholesterol esters to cholest-4-en-3-one
and hydrogen peroxide. Peroxidase converts 4-aminoantipyrine,
in the presence
of hydrogen peroxide and phenol, to a quinone imine chromogen.
and triglyceride
important
because
lipid constituents
is helpful in classifying
familial hyperlipoproteinemias
and, under appropriate
circumstances,
may be
sufficient for diagnosis (3). Early clinical procedures
required
strong saponification
chemicals
and mineral acids in multistep processes
(4-8).
Modern procedures
use microbial
and
animal
enzymes
to rapidly
hydrolyze
cholesterol
and triglycerides,
making them more adaptable
to modern instrumentation
(9-13).
Although the Gilford System 3500 (Gilford Instrument
Laboratories,
Inc., Oberlin, OH 44074) is primarily a discrete
sample analyzer, its programmable
versatility
allows it to be
used as a dual-channel
instrument.
Prerequisites
in the development
of this tandem procedure
include identical wavelengths, timing, temperature,
and sample volumes; reagent
compatibility; and the ability to under-reconstitute
reagents
for both tests.
The tandem procedure
uses test parameters
similar to the
reagent
manufacturer’s
manual
methods.
The differences
between the two methods are the reduced sample and reagent
volumes, incubation
at ambient temperatures,
and elimination
of the addition
of acid in the triglyceride
test immediately
after sample hydrolysis to stabilize the final color. The addition of the acid reagent is unnecessary
in the tandem procedure because the tests are processed in an exact time se-
cholesterol
esterase
Cholesterol esters
cholesterol
(EC
3.1.1.13)
+ fatty
cholesterol oxidase
Cholesterol
+ 02
cholest-4-en-3-one
(EC
1.1.3.6)
+ H2O2
2H2O2 + 4-aminoantipyrine
peroxidase
+ phenol
quinone
Inc.,
132
lipases
Triglycerides
glycerol
accepted
glycerol
Glycerol
kinase
+ ATP
glycerol
(EC
Glycerol 1-phosphate
1-phosphate
+ ADP
2.7.1.30)
+ NAB
glycerol.
1-phosphate
dehydrogenase
(EC 1.1.1.8)
Artino Street, Oberlin,
Dec. 19, 1978.
+ fatty acids
(EC 3.1.1:1)
phosphate
+ NADH
diaphorase
NADH + INT
1978;
chromogen
The triglyceride
reaction involves two microbial lipases that
completely
hydrolyze
triglycerides
to glycerol. Glycerol
is
converted
to glycerol-1-phosphate
by glycerol kinase, which
then reacts with NAD in the presence of glycerol - 1-phosphate
dehydrogenase
to yield dihydroxyacetone
phosphate
and
NADH. The reduced NADH in the presence of diaphorase
reacts
with INT [2- (p -iodophenyl) -3-p -nitrophenyl-5phenyltetrazolium
chloride] to yield a colored formazan.
dihydroxyacetone
Laboratories,
imine
(EC 1.11.1.7)
quence.
Gilford Instrument
OH 44074.
Received July 26,
acids
formazan
+ NAD
(EC 1.6.4.3)
CLINICALCHEMISTRY,Vol. 25, No. 3, 1979 439
Table 1. Correlation of Tandem Cholesterol
Determination with Two Other Methods
Manual
method
Correlation coefficient
Slope
Intercept
No. samples
0.997
1.002
0.03
55
Table 3. Within-Run Precision of Tandem
Determination
3500 BMC
procedure
0.993
1.014
-0.10
79
Table 2. Correlation of Tandem Triglyceride
Determination with Two Other Methods
Manual
method
Correlation
coefficient
Slope
Intercept
0.997
0.958
0.03
No. samples
46
3500
BMC
procedure
0.983
1.035
-0.19
67
Instrumentation
The Gilford System 3500 discrete sample analyzer was used
for the tandem automated
procedure
with program card and
procedure
(part no. 17831X82,
Gilford).
The reference
instrument for the manual methods was a Model 250 Spectro(Gilford) with a temperature-controlled
cuvet
maintained at 25 ± 0.1 #{176}C.
A water bath was used at 37 ± 0.5
#{176}C
forincubation,
and a Pipetter/Diluter
(Gilford) was used
to dispense samples.
photometer
Reagents
Reagents supplied by Dow Diagnostics
(Dow Chemical Co.,
P.O. Box 68511, Indianapolis,
IN 46268)
were used throughout. Each reagent
was reconstituted
to two-thirds
regular
volume to allow for the addition of water to dispense samples
and to maintain
final concentrations
similar to those in the
manual methods.
Each vial of cholesterol
reagent (cat. no.
46650) was reconstituted
with 10.0 mL of the supplied buffer
containing phenol, and the triglyceride
reagent (cat. no. 46676)
was reconstituted
with 7.0 mL of the supplied tris(hydroxymethyl)methylamine
buffer.
The following
reconstituted
reagent concentrations
(per liter) give concentrations
similar
to those used by the manufacturer
when additional
water is
added:
Cholesterol
analysis.
15.5 mmol sodium cholate, 3.4 mmol
4-aminoantipyrine,
32 mmol phenol, 102 U cholesterol
esterase, 186 U cholesterol
oxidase, and 71 300 U peroxidase;
buffer, pH 6.7.
Triglycerides
analysis. 79000 U lipases, 236 mg magnesium
chloride,
1.4 g ATP, 5.7 g NAD, 190 mg INT, 44 000 U diaphorase,
720 U glycerol kinase, and 10 800 U glycerol-iphosphate
dehydrogenase;
buffer, pH 7.4.
Reagents for the manual methods were prepared
according
to the package inserts.
Cholesterol,
TriglycerIdes,
20
20
20
Procedures
Manual
methods:
Reconstitute
the cholesterol
440 CLINICALCHEMISTRY,Vol.25,No.3,1979
reagent
with
g/L
20
3.230 0.763 1.555 2.608 3.865
0.030 0.006 0.007 0.037 0.029
0.93 0.76 0.44 1.41 0.76
Make calculations as described in the package inserts for
both tests to obtain results in grams per liter.
Automated
procedure:
Reconstitute cholesterol and triglyceride reagents with 10.0 mL and 7.0 mL of reconstituting
buffer, respectively, and allow to equilibrate
to ambient
temperature for 10-15 mm. Program the instrument with the
special program card. Dispenser A delivers 5 tL of sample and
0.25 mL of distilled water into each reaction cup. Dispenser
B delivers 0.5 mL of cholesterol
reagent into the first cup of
each sample pair, and Dispenser
C delivers 0.5 mL of triglyceride reagent into the second cup of each sample pair.
In determining the cholesterol and triglyceride factors, the
instrument divides the concentration
of the standard by the
difference
between
absorbance
blank [standard
conc/(Astendard
absorbance
values are converted
of the standard
-
and of the
factor]. Sample
to grams per liter by the
A blank) =
following formula:
in g/L. The blank
(Asample
Ablk)
X factor = sample value
and standard
absorbances
and the factors
are printed as a check on the instrument/reagent
system.
Load the reaction strips over the sample cups and set the
instrument
controls as instructed
by the tape printout;
start
the program by depressing the RUN push button. The samples are automatically
processed
in sequence, alternating
cholesterol
and triglyceride
reagent addition. The instrument
times the 20-mm hydrolysis period and aspirates the mixtures
into the thermal cuvet, which is regulated at 25 ± 0.1 #{176}C.
After
a 5-s equilibration time, the instrument
and prints the results.
reads the absorbances
Results
Correlation
samples,
less than one week old and stored tightly
available lyophilized controls were used to evaluate the tandem cholesterol!
triglyceride
procedure.
Comparisons
with manual methods
and BMC’s System 3500 procedures
(Tables 1 and 2) were
made. The BMC triglyceride
procedure
uses a serum blank
for each sample and calculates
results from the difference
in
absorbance
between blank and sample. The tandem cholesterol procedure
correlates
to the Liebermann-Burchard
method (4,5) with a slope of 1.010 and a correlation
coefficient
of 0.98 1. The tandem triglyceride
procedure
correlates
to the
capped at 4 #{176}C
until use, and commercially
Aqueous cholesterol
and triglyceride
standards
were provided by the manufacturer
in the reagent kits. The cholesterol
standard
contained
2 g of cholesterol
per liter in ethylene
glycol monomethyl
ether, and the triglyceride
standard
contained 2.08 g of glycerol per liter, equivalent
to a triolein
concentration
of 2 g/L.
20
15.5 mL of buffer as described
in the package
insert and
preincubate
at 37 #{176}C
for 7-45 mm. Pipet 10 L of blank,
standard,
and samples into disposable
glass test tubes containing 1.5 mL of pre-warmed
reagent, mix by inversion,
and
incubate
at 37 #{176}C
for 10 mm. Read absorbances
at 500 nm
within 15 mm after hydrolysis,
to avoid color deterioration.
Reconstitute
the triglyceride
manual method
reagent
as
described
with 5.5 mL of buffer solution and preincubate
at
37 #{176}C
for 5-10 mm. Pipet 20 sL of blank, standard,
and
samples into disposable
glass test tubes containing
1.0 mL of
pre-warmed
reagent, mix by inversion,
and incubate exactly
10 mm at 37 #{176}C;
then immediately
pipet 2.0 mL of acid reagent into each tube. Mix by inversion,
then record absorbances at 500 nm.
Patients’
Standards
g/L
No.
20
20
20
assays
Mean 1.024 1.746 2.014
SD
0.008 0.028 0.014
CV, %0.83
1.63 0.67
Table 5. Results of Reagent Sample Carryover
Tests
Table 4. Day-to-Day Precision of Tandem
Determination
Cholesterol, g/L
No.
24
assays
24
24
TrIglycerIdes,
24
24
24
% carryover
g/L
24
24
Mean 1.453 2.080 2.314 1.995 0.830 1.460 1.817 3.079
SD
0.037 0.042 0.038 0.040 0.028 0.048 0.064 0.098
CV, %2.53 2.02 1.62 1.35 3.33 3.30 3.49 3.19
Worthington
Diagnostic saponification
method
of 1.046 and a correlation coefficient of 0.994.
with a slope
.
High Cholesterol to low triglycerides
Low cholesterol to high triglycerides
High triglycerides to low cholesterol
Low triglycerides to high cholesterol
1.55
0.81
1.35
1.58
pies. Each set of four results was averaged to establish mean
values for separate and tandem runs and used in a formula
proposed by Broughton (14) in a scheme for instrument
evaluation. Overall carryover was calculated to be less than
Precision
1.6%(Table 5).
Precision data are listed in Tables 3 and 4 for the tandem
cholesterol/triglyceride procedure. Within-run precision was
evaluated on replicate assays of four pooled sera having cholesterol values ranging from 1.02 to 3.23 g/L and triglyceride
values ranging from 0.76 to 3.86 g/L. Results showed coeffi-
Linearity
cients of variation of less than 1.7% for cholesterol and 1.5%
for triglycerides
(Table 3). Day-to-day precision was accumulated over a three-day period on four pooled sera with
concentrations
ranging from 1.45 to 3.00 g/L for cholesterol
and 0.83 to 3.08 g/L for triglycerides. Freshly reconstituted
reagent and standards
were used for each assay. Results
showed coefficients of variation of less than 2.6% for cholesterol and 3.5% for triglycerides (Table 4).
or both. Each pool was diluted with human sera with low
amounts of analyte to give appropriate concentrations.
Observed results, y, are plotted vs. expected results, x (Figures
1 and 2).
Acceptable linearities were obtained up to 6 g/L for cholesterol and to 5 g/L for triglycerides. Values greater than
these should be diluted with low-concentration
sera and
reassayed.
Carryover
Discussion
Carryover studies were performed
to determine
the cross-
contamination
effects between
the alternately
aspirated
cholesterol
and triglyceride
sample/reagent
mixtures. Human
samples were selected that contained abnormally
high and low
cholesterol
and triglyceride
concentrations.
Each high or low
sample was separately
assayed in quadruplicate,
then reassayed with the tandem
procedure,
alternating
various con-
centration
combinations
of cholesterol
and triglyceride
Linearities of the automated tandem procedure were determined by assaying dilutions of pools of human sera with
abnormally high concentrations
of cholesterol, triglyceride,
Most discrete sample analyzers
cannot be used as a dual-
channel system to measure two serum constituents
within the
course of one analysis; the Gilford System 3500, however, can
be used to routinely assay both total cholesterol and triglyceride serum concentrations
as a tandem procedure.
Assays for total cholesterol
and triglyceride
concentrations
are frequent
tests on patients
exhibiting
various lipid disor-
sam-
7
N
6
5
C)
C,
a.,
4..
0)
0
a,
>
a)
a)
.0
4
C)
>
C,
Cl)
.0
3
0
0
2
1
Expected
Fig. 1. Linear
range of tandem
(g/liter)
cholesterol
dilutions
ofabnormalpatients’
samples
procedure
from
Expected (g/liter)
Fig. 2. Linear range of tandem triglyceride procedure from
dilutions
ofabnormalpatients’
samples
CLINICAL CHEMISTRY,
Vol. 25, No. 3, 1979
441
This unique dual-measuring procedure offers considerable savings over discrete determinations routinely used by
ders.
batch analyzers.
Productivity
of the tandem cholesterol/triglycerides
procedure
is 46 patient pairs/h.
We have shown that this procedure
is comparable
to the
manual methods, System 3500 BMC procedures,
and to Lie-
bermann-Burchard
and saponification
methods.
It offers
excellent
precision
reagents,minimum
and repeatability
with economical
use of
use of personnel,
and readily available
commercial
reagents.
Other reagents cannot be interchanged
for this procedure.
We thank
Dr. Andris Indriksons
and Robert Mack (Dow Diag-
5. Burchard, H., Beitrage zur Kenntnis des Cholesterins. Chem.
Zentralbl. 61, 25 (1890).
6. Griguat, A., Proc#{233}d#{233}
colorim#{233}trique
de dosage de Ia cholest#{233}rine
dans l’organisme. C. R. Soc. Biol. 68, 791 (1910).
7. Abell, L. L., Levy, B. B., Brodie, B. B., and Kendall, F. E., Simplified method for the estimation of total cholesterol in serum and
demonstration
of its specificity. J. Biol. Chem. 195, 357 (1952).
8. Wybenga, D. R., Pileggi, V. J., Dirstine, P. H., and DiGiorgio, J.,
Direct manual determination of serum total cholesterol with a single
stable reagent. Clin. Chem. 12, 980 (1970).
9. Bucolo, G., and David, H., Quantitative determinations
of serum
triglycerides by the use of enzymes. Clin. Chem. 19, 476 (1973).
10. Flegg, H. M., Investigation of the determination
of serum cholesterol by an enzymatic method. Ann. Clin. Biochem.
10, 79
(1973).
nostics) for their help with this procedure.
References
1. Fredrickson, D. S., Physician’s guide to hyperlipidemia.
Mod.
Concepts Cardiovasc. Dis. 41,31(1972).
2. Tietz, N. W., Fundamentals of Clinical Chemistry, W.B. Saunders
Co., Philadelphia, PA, 1970, p 323, 329.
3. Davidsohn, I., and Henry, J. B., Clinical Diagnosis by Laboratory
Methods, W.B, Saunders Co., Philadelphia, PA, 1974, p 626.
4. Liebermann, C., Ueberges Oxychinoterpen.
Ben. Dtsch. Chem.
Ges. 18, 1803 (1855).
442
CLINICALCHEMISTRY.
Vol. 25,No.3, 1979
11. Richmond, W., Preparation and properties of a cholesterol oxidase
from Nocardia sp. and its applications to the enzymatic assay to total
cholesterol in serum. Clin. Chem. 19, 1350 (1973).
12 Allain, C. C., Poon, L. S., Chan, C. S. C., et al., Enzymatic determinations of total serum cholesterol. Clin. Chem. 20,470 (1974).
13. Roeschlau, P., Bernt, E., and Gruber, W., Enzymatic determination of total cholesterol in serum. J. Clin. Chem. Clin. Biochem.
12,226
(1974).
14. Broughton, P. M. G., Gowenlock, A. H., McCormack, J.J.,and
Neill, D. W., Revised scheme for the evaluation of automatic instruments for use in clinical chemistry. Ann. Clin. Biochem. 11, 207
(1974).