CUN. CHEM. 20/10. 1282-1286 (1974)
Evaluation of an Enzymatic Procedure for Determination of
Serum Cholesterol with the Abbott ABA- 100
David 1. Wife, David A. Barrett II, and Delane A. Wycoff
A previously reported direct enzymatic method for cholesterol [C/in. Chem. 20, 470 (1974)] was critically
evaluated in the clinical laboratory. Other methods compared to this enzymatic procedure were: (a) the manual
Abell-Kendall procedure, giving a regression slope (proportional sensitivity) of 0.998 and a negative bias of 9.7
mg/dI in the enzymatic procedure; (b) Lipid Research
Clinic procedure, giving a regression slope of 1.005 and
a negative bias of 8.8 mg/dl in the enzymatic procedure; and (C) the SMA 12/60 (Technicon) method, giving
a regression slope of 0.881 and a negative bias of 20.8
in the enzymatic procedure. Replicate analyses of
pooled serum by the enzymatic procedure demonstrated the following inter-run precision: mean = 178
mg/dI, SD = 4 mg/dl, CV = 2.2%. Bilirubin caused a
slight positive interference (1 mg of bilirubin per deciliter
appears as 1-2 mg of cholesterol). Hemolysis, lipemia,
and a series of steroid drugs caused no interference. A
group of 465 normal subjects was tested by the enzymatic procedure. This population had a mean cholesterol of 203 mg/dl. The 2.5 percentile was 130 mg/dl, and
the 97.5 percentile was 316 mg/dl.
stable and corrosive
reagents,
and a host of interfering substances
(most notably
bilirubin,
hemoglobin,
and bromide).
Recently
the federal
government,
through
establishment
of Lipid Research
Clinics, has
sought to standardize
the determination
of cholesterol by the Liebermann-Burchard
reaction
and special
serum calibrators.
This is the currently
recognized
standard
of performance
and is directly related to the
manual Abell-Kendall
determination
(12).
Even more recently,
the specificity
provided
by enzyme reagents
has been introduced
into cholesterol
determinations
(8-10).
This paper presents
a critical
evaluation
of the enzymatic
cholesterol
measuring
system described
by Allain et al. (10), in which three
enzymes
are used: cholesterol
ester hydrolase
(EC
3.1.1.13), cholesterol
oxidase
(EC 1.1.3.6), and horse
radish peroxidase
(EC 1.11.1.7).
The latter
enzyme
couples the hydrogen
peroxide
formed by cholesterol
oxidase to 4-amino-antipyrine
and phenol, to form a
colored compound
that absorbs at 500 nm.
AddItIonal Keyphrases: normal
Methods and Materials
comparison
values
and
chromogenicily
#{149}
enzymatic
inter-method
reactivity
of
various steroids
Because of clinical importance
and analytical difficulties, the determination
of serum cholesterol has
been the subject of numerous investigations.
Classically, the methodology
has revolved around the dehydration and dimerization
reactions of cholesterol
in
the presence of metal catalysts and strong acids. This
literature has been reviewed by Tonks (1). Since this
review appeared,
two new but similar colorimetric
methods for cholesterol have been described: that of
Parekh and Jung (2) in which uranyl acetate isused,
and that of Wybenga et al. (3) in which ferric perchlorate is used. These procedures
have been subsequently
modified
and automated
(4-6). More recently, the initial period of these colorimetric
reactions
has been followed kinetically
in an effort to increase
specificity
and accuracy
(7).
The colorimetric
reactions
have been plagued with
a long list of difficulties:
lack of specificity,
difficulty
in standardization,
variable
reactivity
of esters, un-
Department
of Pathology,
College
Iowa, Iowa City, Iowa 52242.
Received June 13, 1974; accepted
1282
CLINICAL
CHEMISTRY,
of
Medicine,
July 25, 1974.
Vol. 20, No. 10, 1974
University
of
Standards.
Solutions
of National
Bureau
of Standards SRM 911 cholesterol
in isopropanol
were used
as standards
for the ABA-100
(Abbott
Bichromatic
Analyzer;
Abbott
Labs., Diagnostics
Division,
Pasadena, Calif. 91030) and manual Abell-Kendall
determinations.
The extinction
coefficient
of this material
was determined
before use by adding
3 ml of a 100
mg/dl solution
of the SRM 911 in glacial acetic acid
to 10 ml of Liebermann-Burchard
reagent
prepared
as specified
by the National
Bureau
of Standards
(11). The average
observed
molar extinction
coefficient at 535 nm as measured
on a Gilford 240 spectrophotometer
was 582 mol
cm
with a range of 570594 mol
cm’
for the eight measurements.
The
NBS reported
the extinction
coefficient
as 590 mol’
cm.
Manual
A bell-Kendall
determinations
were done
as described
in the literature
(12), with use of onehalf the volumes stated.
Enzymatic
cholesterol
determinations
were done
as described
in the ABA procedures
manual with reagent supplied
by Abbott Diagnostics.
The automated procedure
requires
5
of serum and 500 il of reagent and is a single-step
assay with a 10-mm incubation time at 37 #{176}C.
The exact methodology
has been
previously
described
(10).
The SMA 12/60 determinations
were done by unmodified
Technicon
procedures.
SMA
reference
serum supplied
by Harleco
(lot 64288) was used to
standardize
the SMA 12/60.
Lipid Research Clinic (LRC) values were generated in the University
of Iowa Lipid Research
Clinic
according
to their standard
analytical
procedure,
in
which are used a zeolite and isopropanol
extract
and
a Technicon
AutoAnalyzer
II standardized
with
serum calibratorsprovided by the Center for Disease
Control (22).
Drug preparations.
All drugs used in this study
were injectablepreparations obtained
from the hospital pharmacy. These drugs were: (a) Merck’s Hydrocortone
phosphate
(hydrocortisone),
(b) Upjohn’s
Depo Medrol (methyl prednisolone), (c) Organon’s
Hexadrol Phosphate Injection (dexamethasone),
(d)
Squibb’s Delestrogen (estradiol valerate),
and (e)
Upjohn’s
cortisone
acetate,
USP.
Normal
subjects.
These were volunteers
from the
production
department
of a local electronics
firm. All
individuals
completed
a questionnaire
giving age, sex,
height,
weight,
drug
history,
statement
of their
health,
fasting status, and history of recent physician
visits.
Linear regressions.
Except where indicated, the
regression
lines were calculated
by using the geometric fit method of Wycoff.1 This method minimizes
the
perpendicular distances from the points to the lineof
best fit.
Table 1. Regression Parameters for
Methods Comparisons
Method x
Slope
y-intercept
ABA
Method y
LRC
1.005
-8.8
SMA12/60
Manual A-K
Manual A.K
LRC
LRC
ABA
0.899
+9.8
+0.5
SMA12/60
ABA
1.004
0.998
0.881
+9.7
+20.8
Table 2. Inter-run Precision Data
Method
SMA 12/60
LRCpoolA
LRC pool B
ABA
Manual A-K
CV, %
x ± SD, mg/dI
192 ± 9
163.0±2.6
4.7%
1.6%
260.2
1.2%
±
3.1
178±4
2.2%
200 ± 18
9Q%a
Estimated from differences between duplicates.
Results
Methods
comparisons.
Forty-eight patients’sera,
two lyophilized sera, and one frozen pool of human
serum were analyzed as described above, by the four
procedures. The most pertinent statistical
results
in
method comparisons are those generated by linear
statistical
fitting(13). These data are summarized
in
Table 1 and presented
in Figures 1-5. Those symbolized by + are individual
patient’s
samples;
other
symbols are pooled quality-control
materials.
Linearity.
Linearity
for each method
was verified
with solutions
of cholesterol
in isopropanol
for values
up to 500 mg/dl with the Abell-Kendall
procedure
and up to 800 mg/dl with the ABA. Linearity
wtih
patient
samples
was verified up to 430 mg/dl for the
ABA. No attempt
to find the limits of linearity
by
using higher samples was undertaken.
Precision.
Precision
data
for the methods
described are given in Table 2.
Interferences.
Possible
interfering
substances
were
investigated
by the usual addition
technique,
with
use of pooled
human
serum and of cholesterol-free
lyophilized
serum. Each sample
contained
identical
volumes
of serum
and added solvent,
and only the
The geometric fit takes into account
from random variations in the reference
test
method.
which ignores
scatter.
This
is in contrast
the contribution
with
the data scatter resulting
method, as well as in the
the
least-squares
of the reference
method
method,
to
data
Fig. 1. Comparison of ABA (y-axis) and Lipid Research Clinic
(x-axis) methods: slope = 1.005. y.intercept = -8.8 mg/dl.
geometric
fit
Fig.2.Comparison of SMA 12/60 (y.axis) and Upid Research
Clinic (x-axis) methods: slope = 0.899. y4ntercept = +9.8
mg/dl, geometricfit
amount of added interferentwas varied (15). Bilirubin interferencewith the cholesterolmeasurement on
the ABA 100 ispresented in Table 3.
Similar additions of erythrocyte
lysate and water
to pooled serum demonstrated that hemoglobin did
not interfere(seeTable 4).
CLINICAL
CHEMISTRY.
Vol. 20. No. 10. 1974
1283
-
Table 3. Effect of Bilirubin Added to
Cholesterol-Free Serum
BIIIrubln
added
PBELL-KENOPLL
LrPw:EsRc>//
Apparent cholesterol
mg/dl
0
2.5
3.8
00
06
07
09
16
25
6.3
12.5
18.8
C1’CL(STCL
Table 4. Effect of Erythrocyte Lysate Added
to a Pool of Human Serum
Hemoglobin0 added
(flC/.I
Fig.3. Comparison of manual Abell-Kendall
(y-axis) and Lipid
Research Clinic (x-axls) methods: slope = 1.004. y-intercept
= +.5 mg/dl, standard least-squares fit
Apparent 89
cholesterol
mg/dl
26
53
90
89
105
90
263
89
Table 5. Effect of Drugs Added to a Pool of
Human Serum and to Cholesterol-Free Serum
Apparent
cholesterol
Drug
my drug/mi
No drug
Cortisone acetate
Estradiol valerate
Methyl prednisolone
Hydrocortisone
Dexamethasone
6.3
2.5
10.p
13.0
1.0
All values
cholesterol-free
were
zero for
a
Normal serums
89
85
of manual Abell-Kendall
(y-axis) and ABA
(x.exls) methods: slope = 0.998. y-intercept +9.7 mg/dl,
standardleast-squares fit
Fig. 4. Comparison
84
85
85
86
corresponding
SMP
12/60
VS flOP
examination of
//
300
serum.
0
A seriesof drugs with structuressimilarto cholesterol was added to serum pools. The resultsare summarized
in Table 5.
Lipemia.
A seriesof 26 lipemicsera with absorbances at 600 nm ranging from 0.5to above 2.0and
with triglyceride
values up to 3600 m/dl were split
and analyzed by the enzymatic method and by the
Lipid Research Clinicmethod. The best fittedline
for this data had a slope of 1.02and the interceptindicated
dure.
a -12.5
Normal
of whom
values. A total of 465 subjects were tested,
280 were taking no medications. The
ranged
in age from 18 to 65 years.The nor-
subjects
mal ranges
mg/dl
bias
were determined
in the
enzymatic
by ranking
the values
CLINICAL CHEMISTRY, Vol. 20, No. 10. 1974
+
200
+
+
I
#{149}100
100
ri#i
proce-
200
rcTrw’u
300
iMrir
,
Fig. 5. Comparison of SMA 12/60 (y.exis) and ABA (x.exis)
methods: slope = 0.881. y-lntercept = +20.8 mg/dl. geometric fit
in
ascending order and determining the appropriate
nonparametric
percentileswithout rejectionof outliers (16, 17). The normal ranges obtained
are shown
in Table 6.
Figure6 shows the frequencydistribution
ofvalues
for the populationof 465 subjects.A detailedstudy
on age-and sex-relateddifferences
as wellas specific
drug effectsisinprogressforthe same population.
1284
E
Discussion
The purpose ofthisstudy was to critically
evaluate
an enzymatic procedure for serum cholesterolin a
clinical
laboratorysetting.
The colorimetric
determination of serum cholesterol
has been difficult owing
to poor specificity,
unstableand acidicreagents,and
variability
in coloryield.The enzymatic procedure
(4-
Table 6. Values Observed for a
Normal Population
12(_.)
Percentile
Population
All
All, no drugs
Men, no drugs
Women,
no drugs
2.5
97.5
No. subjects
316
316
465
133
203
200
280
133
203
318
140
133
196
295
140
130
50
to-
Li
D
(fl
>
64.
2-
SO
Ii
I
(
(00
ISO
I
I
I
I
I
200 2S0 300 360 400 460 600
CHOLESTEROL
(MC/OL)
Fig. 6. Frequency distribution of values obtained for 465
subjects
discussed
here offers an alternative
that is more specific, simple, and accurate.
To evaluate
accuracy,
we assayed
each sample by
four different
methods,
one of which (12) is the most
widely accepted
“reference”
method,
and another
of
which, the Technicon
SMA 12/60, is probably
the
most commonly
used laboratory
screening
method.
Figure 4 demonstrates
the correlation
between
results of the enzymatic
procedure
and the manual
Abell-Kendall
procedure
performed
in our laboratory. The determinations
were both standardized
with
the same isopropanol
solutions
of National
Bureau of
Standards
SRM 911 cholesterol.
The average 10 mgI
dl negative
bias in the enzymatic
procedure
has been
previously
reported
(10) and is caused by the differing reactivities
of naturally
occurring
noncholesterol
serum sterols.
These sterols have differing
chromogenicities
with Liebermann-Burchard
reagent
and
also differing
reactivities
with the cholesterol
oxidase
and cholesterol
ester hydrolase.
The normal
serum
content
of these sterols caused an average
20 mg/dl
error in the enzymatic
procedure
and an average
38
mg/dl error in the Abell-Kendall
procedure
(10). Figure 1 shows the correlation
of the enzymatic
procedure with the University
of Iowa Lipid
Research
Clinic analyses.
Again, a similar negative
9 mg/dl bias
is evident,
because
the Lipid Research
Clinic determination
is directly
related
to the Abell-Kendall
(22). The standard
error of the estimate
is considerably smaller
when the two automated
methods
are
compared
than in Figure 4, where a manual and automated method
were compared.
Figure 2 and Figure 5
show the comparisons
of the Technicon
SMA 12/60
to the enzymatic
procedure
and the Lipid Research
Clinic,
respectively.
It has been previously
shown
that the SMA 12/60, a direct colorimetric
reaction,
is
a nonspecific
method
and therefore
tends to give erroneously
high results. Standardization
by use of reference sera values determined
by methods
other than
the SMA 12/60 method
partially
alleviate
this problem. Our SMA 12/60 reference
material
has a set
point
determined
by the Abell-Kendall
method,
which helps explain the correlation
observed
between
the SMA 12/60 and the “reference”
methods.
This
method
of standardization
has been discussed
by Seligson (17), who states that it is analogous
to assuming that all simple
sera have the same amount
of
“CRUD”
(Chemicals Reacting Unfortunatelyas the
Desired
one). As Figures
2 and 5 show, on the average this approach
seems to work fairly well, because
these plots show good correlation.
However,
on any
given sample, one is never sure the CRUD is the same
as that in the standard.
This will be particularly
true
for samples
with high bilirubin
content.
The fittedline data reported
in Table 1 show that the SMA 12/
60 sensitivity
to the substance
measured
by the ABA
was 88.1%. Compared
to the ABA, the SMA 12/60
had a positive
constant
error of 20.8 mg/dl. The result is that the SMA 12/60 and the ABA produced
similar
values
in the center
of the normal
range.
However,
at lower values, the SMA 12/60 values were
higher values than those for the ABA, and at higher
values the SMA 12/60 values were lower than those
for the ABA.
The preceding
discussion
demonstrates
the accuracy and correlation
of the enzymatic
procedure
to
other methods.
The procedure
also showed good precision. The inter-run
precision
for a quality
control
pool (Table 2) indicates
a CV of 2.2% at the concentration
of 178 mg/dl. The values used to calculate
the
inter-run
precision
were taken from samples
placed
at both the start and finish of the run.
For patients’
samples,
the enzymatic
procedure
is
linear to a cholesterol
concentration
of at least 430
mg/dl, a significant
advantage,
because
many procedures are linear to only 300 mg/dl.
Many substances
interfere
with colorimetric
methods for serum cholesterol
(18, 19). Because
the enzymatic approach
involves completely
different
chemical reactions,
interferents
with the colorimetric
procedure
would
not necessarily
interfere
with this
method.
Bilirubin
caused slight positive
interference,
1 mg
of bilirubin
per deciliter
being measured
as 1-2 mg of
cholesterol
(Table 3). However,
this is much less than
that reported
for many colorimetric
methods
(20).
With the ABA-100
optical
system,
the absorbance
difference
(Ad) which is used to quantitate
cholesterol, is measured
by subtracting
A600 (the absorbance
at 600 nm) from A500 (the absorbance
at 500 nm).
Because
bilirubin
absorbs
more strongly
at 500 than
CLINICAL
CHEMISTRY.
Vol. 20, No. 10, 1974
1285
at 600 nm, it is not surprising
positive interference.
that
it causes a slight
The enzymatic procedurewas not subjectto hemoglobin interference
at concentrations
that may reasonably be encountered
in routine laboratory
specimens (Table 4). Lipemic sera with absorbances
greater than 2.0 and triglyceride concentrations
up to 3600
mg/dl also showed no interference
with the enzymatic procedure, as indicated by the similarity of the regression parameters
obtained for normal and lipemic
sera.
Because cholesterol oxidase has a particular specificity for cholesterol and similar compounds,
causing
a negative bias relative to the Abell-Kendall
procedure, it seemed possible that some of the commonly
used steroid drugs may interfere with the enzymatic
procedure. These drugs could conceivably interfere in
two ways: by acting as substrates,
and so cause a positive interference,
or by acting as inhibitors,
and so
cause a negative interference.
The former seemed unlikely (10), because most of the drugs were already
3-ketones. The data shown in Table 5 indicate that
even in concentrations
exceeding
the therapeutic
range, no positive or negative interference
was seen.
The bias noted between the manual Abell-Kendall
and the enzymatic procedures is accounted for by different reactivities
of naturally occurring steroids, as
discussed by Allain et al. (10).
The enzymatic procedure
couples to the 4-amino
antipyrine
(phenazone)
peroxidase-hydrogen
peroxide measuring system, which is the same system as
that used by Trinder for measuring glucose oxidase
(20).
Both Trinder (20) and Pennock et al. (21) have
demonstrated
no interference
by uric acid, glutathione, or ascorbic acid. Allain et al. (10) also reported no ascorbic acid interference
in the enzymatic
holesterol
method.
We acknowledge the technical assistance of Dr. Donald Wiebe,
Director of the University of Iowa Lipid Research Clinic, and Mrs.
CLINICAL
CHEMISTRY,
during
tient
Vol. 20, No. 10, 1974
the normal
technical
value study.
assistance
Finally,
of Polly
we acknowledge
the pa-
Fassler.
References
1. Tonks,
cation and
D. B., The estimation
of cholesterol
in serum; a classificritical review of methods.
Clin. Biochem.
1, 12 (1967).
Parekh, A. C., and Jung, D. H., Cholesterol determination
with
ferric acetate-uranium
acetate and sulfuric acid-ferrous sulfate reagents.
Anal. Chem. 42, 1423 (1970).
3. Wybenga, D. R., Pileggi, V. J., Dirstine, P. H., and Di Giorgio,
2.
J., Direct determination
of serum total cholesterol
stable reagent. Clin. Chem. 16, 980 (1970).
with
a single
4. Holub, W. R., and Galli, R. A., Automated
direct method for
measurement of serum cholesterol, with use of primary standards
and a stable reagent. Clin. Chem. 18, 239 (1972).
5. Witte, D. L., Bienhoff, R. L., and Routh, J. F., Direct determination of serum
cholesterol with ferric perchlorate-sulfuric
acidethyl acetate reagent, comparison with the SMA and N24a methods. Clin. Chem. 19, 669 (1973). Abstract.
6. Dow Diagnotest, Dow Chemical Co., Indianapolis, md.
7. Slickers, K. A., Edwards,
L., Daly, J., and Ertingshausen,
G.,
Automated reaction-rate method for the direct determination
total serum cholesterol by use of the “CentrifiChem”
parallel
of
fast
analyzer. Clin. Chern. 19, 937 (1973).
8. Flegg, H. M., An investigation
of the determination
of serum
cholesterol by an enzymatic method. Ann. Clin. Biochern. 10, 79
(1973).
9. Richmond, W., Preparation
and properties of a cholesterol oxidase from Nocardia
sp. and its application to the enzymatic assay
of total cholesterol in serum. Clin. Chem. 19. 1350 (1973).
10. Allain, C. C., Poon, L. S., Chan, C. S. G., Richmond, W., and
Fu, P. C., Enzymatic
determination
of total serum cholesterol.
Clin.
Chem.
11. National
20, 470 (1974).
Bureau
of Standards,
Standard
Reference
Material
911 Cholesterol Bulletin.
12. Abel!, L. L., Levy, B.
lesterol in serum. Stand.
13. Westgard, J. 0., and
common
Chem.
Finally, several practical points should be considered in the routine use of the enzymatic cholesterol
procedure
on the ABA 100. Quality-control
sera at
two concentrations
should be used to ascertain proper linearity in each run, means and standard deviations of the Ad’s observed for the standards
and the
reagent blanks should be recorded to help in troubleshooting problems, and finally, because evaporation
of samples can be a problem that is greatly magnified
with the isopropanol
standard
solutions, standards
should be aliquoted immediately
before starting the
run. “Sera Seal,” a commercially
available silicone
compound used to layer over serum to prevent evaporation, cannot be used with the isopropanol
standards because Sera Seal is miscible with isopropanol
and causes relative dilution of the standards.
In conclusion, we think that the enzymatic procedure for serum cholesterol offers a good alternative
to
the classical colorimetric
methods. This method offers ease of operation, acceptable accuracy and precision, and greater specificity.
1286
Sandy Streeter. We also thank Abbott Scientific for providing material support and the people of Collins Radio for their cooperation
statistical
NBS, Washington, D.C.
B., Brodie, B. B. and Kendall, F. E., ChoMethods Clin. Chem. 2, 26 (1958).
Hunt, M. R., Use and interpretation
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tests
in method-comparison
studies. Clin.
19, 49 (1973).
14. Laessig, R. H., Schwartz, T. H., and Paskey, T. A., Determination of SMA-Mark X set points for bilirubin by standard addition
technique. Clin. Chem.
18, 48 (1972).
15. Reed, A. H., Hewy, R. J., and Mason, W. B., Influence
tistical
method
used on the resulting
estimate
of normal
Gun. Chem. 17, 275 (1971).
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16. Herrera,
L., The precision of percentiles in establishing normal
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17. Seligson, D., Effect of computers on the precision and accuracy
limits
in medicine.
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results. In Automation
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cal Laboratory,
G. E. Westlake
and J. L. Bennington,
in the Clini-
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18. Zak. B., Weiner, L. M., and Welsh, B., Spectrophotometric
study of bilirubin interference
in the Hiang reaction for cholesterol. Clin. Chim. Acta 30,697 (1970).
19. Young, D. S., Thomas, D. W., Friedman, R. B., and Pestaner,
L. D., Bibliography:
Drug interferences
with clinical laboratory
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20. Trinder,
P., Determination
of glucose in blood using glucose
oxidase with an alternative
oxygen receptor.
Ann. Clin. Biochem.
6,24 (1969).
21. Pennock, C. A., Murphy, D., Sellers, J., and Longdon, K. J., A
comparison of AutoAnalyzer methods for the estimation of glucose
in blood. Clin. Chim. Acta 48, 193 (1973).
22. Rush, R. L., Leon, L., and Turrell, J., Automated
simultaneous
cholesterol
and triglyceride
determination
on the AutoAnalyzer
II
instrument.
In Advances
in Automated
Analysis,
Tech nicon International
Congress
1970, I, Clinical
Analysis;
C. E. Barton et
al., Eds. Thurman
Associates,
Miami, Fla. 33132, 1971, p 503.