AmI. clin.

Biochem, 10 (1')73) 79

AMES AWARD LECTURE 1972

An Investigation of the Determination of Serum Cholesterol by an Enzymatic Method

HEATHER M. FLEGG
Wolfson Research Laboratories, Qu<'C'n Elizabeth Medical Crntrr Birmingham BI5 2TH

The enzymatic determination of total serum cholesterol is described. The source of the enzyme
cholesterol dehydrogenase is the bacterium Nocardia erythropolis. A comparison is made between
the enzymatic assay and an automated Liebermann-Burchard serum cholesterol determination.

Serum cholesterol is frequently determined in

clinical biochemistry, but the existing colorimetric
methods, although relatively simple, convenient to
carry out and useful in comparative studies, are
nevertheless non-specific. Since cholesterol is a
relevant biochemical investigation, it is considered
that the development of more reliable methods is
worthwhile.
Past and present methods for cholesterol deter-
mination utilise several physical and chemical
techniques. The majority of cholesterol assays
involve colorimetric determinations preceded by
extraction into many organic solvent combinations
and concentrated acids, occasionally including
digitonin precipitation. Often extraction and/or
saponification are omitted before colour develop-
ment. It appears that very little attempt has been
made to alter the concept of analysis, with workers
merely making slight modifications to the general
principles of cholesterol analysis. The lack of
specificity with acidic, colorimetric methods may be
replaced by the specificity provided by enzymatic
assays.
The use of enzymes as reagents has developed due
to modern techniques for enzyme isolation and
induction of bacteria. Absolute accuracy can be
more readily approached when an enzyme is used as a
reagent to determine the concentration of a serum
component. Advantages of enzyme reagent systems
reside in sensitivity and greater specificity. Dis-
advantages include the presence of possible enzyme
inhibitors in the serum and greater attention must
be paid to pH, temperature and molarity of the
reaction mixture. The principles of enzymatic
reagent analysis may be based on the determination
of reaction rate or a reading at reaction equilibrium.
Ideally a pure enzyme should be used.
With the knowledge that some soil bacteria are
capable of metabolising cholesterol, the possibility
of an enzymatic method for determining serum
cholesterol was considered. The enzyme chosen to
be used for the subsequent assay of serum cholesterol
was cholesterol dehydrogenase (Fig. I). The product
79
140'
~{llesterol
Fig. I-Enzyme reaction.
..1 4 cholestenone has an absorption maximum at
240 nm and is capable of reacting with 2,4 dini-
trophenylhydrazine.
A suitable bacterium was sought by investigating
the ability of a number of soil organisms to
metabolise cholesterol by determining cholesterol
disappearance and ..1 4 cholestenone appearance in an
agar containing cholesterol. The analytical scheme
was modified and developed from a study of soil
bacteria active on cholesterol (Zanin, 1968).
As the agar growth was sparse, a more appro-
priate mode for the large scale preparation of
bacterial cells capable of cholesterol metabolism was
thought to be a liquid medium with cholesterol as the
sole carbon source. Nocardia erythropolis (Turfitt,
1948), the most active organism tested in a choles-
terol broth, was grown in an ultrasonicated choles-
terol-rnineral medium, providing cells for the
preparation of cholesterol dehydrogenase (Fig. 2).
Cholesterol dehydrogenase was separated from
many of the other cell enzymes, notably the enzyme
activity on ..1~ cholestenone, by ammonium sulphate
fractionation of the cell free supernatant. Several
assays for the determination of enzyme activity were
devised, all employing the measurement of the
reaction product ..1~ cholestenone. With the aid of
these techniques the enzyme properties needed to
derive optimal conditions for the future serum
80

4 Phosphate buffers, 0·1 rnol/l, pH 5·8 and 7·2.

pH .JJg 15. cholestenone mg cholester I Prepared as described by Dawson, Elliott, and
1100ml 1100ml 50 Jones (1969).
Bovine serum albumin, 7 g/ I DO ml. This concen-
8.0 40 tration is obtained by dilution of a 'Sigma' pre-
'0 paration of 35 gil DO ml bovine serum albumin with
0·1 mol/I phosphate buffers (pH 5·8 for the test
'. 30 reaction and pH 7·2 for the blank reaction).
,, Determination of .14 cholestenone
7.0
--,-
,, - 5 -. ptL. 20
, Isopropanol.
, ~4chol
--. ------.
,, Calibration
,, 10
,,
Cholesterol standards, 6-19 f-Lg, in 40 f-Ll ethanol:
dioxane, 0·9:1·1 tvlv).
6.0 \ 0 _. .c_h.!.JL 0
'Serachol' standards. 'Serachol ' is a commercial
-0-'- 2 3 4 5 6 7 control serum in which known quantities of free and
days esterified cholesterol are added to serum. This mode
Fig. 2-Nocardia erythropolis incubated in a sonicated of calibration overcame the need for recovery
cholesterol medium at 25"C, pH 7'0-7,2. ..1' choles- corrections.
tenone is produced and further metabolised by the cell
enzymes.
METHODS

Preparation of cholesterol dehydrogenase

cholesterol assay were determined since the enzyme
As cholesterol dehydrogenase is an inducible
had not been sufficiently characterised in the previous
intracellular enzyme, it is prepared from bacterial
literature (Stadtrnan et al., 1954).
cells grown on cholesterol. After cell disruption by
The development of an enzymatic serum choles-
sound waves, residual cholesterol which does not
terol assay included a serum extraction technique
sediment on centrifuging, is separated by serial
utilising solvents non-inhibitory to cholesterol
filtration. As the cell contains other enzymes in
dehydrogenase, a reaction mixture, saponification
addition to cholesterol dehydrogenase, especially
conditions and a solvent for the final .14 choles-
enzymes acting on Lt4 cholestenone, partial puri-
tenone extraction. An ultra-violet assay, involving
fication was undertaken with ammonium sulphate.
the increase in extinction at 240 nm in isopropanol
The yield from 30 g ammonium sulphate/IDO ml
extracts, was developed to provide an e~zymatic
cell extract is 50~;'; but the method is fast and
determination of serum cholesterol, which was
effective in the removal of the second enzyme of the
subsequently assessed by comparison with an
bacterial catabolic pathway of cholesterol.
automated Liebermann-Burchard serum cholesterol
Serum was extracted with ethanol :dioxane,
method. Two modes of calibration were tested, using
0·9: 1·1 (v/v) and after centrifugation the extract was
cholesterol standards in ethanol :dioxane, 0·9: 1·1
saponified with KOH. Saponified serum extract,
(v/v) and cholesterol standards involving the dilution
50 f-LI, was added to a phosphate buffer pH 5·8 con-
of a commercial cholesterol control serum, 'Serachol'.
taining 3·5 g/IDO ml bovine serum albumin: the fi~al
'Serachol' calibration provided the better correlation
pH is 7·2. The partially purified enzyme preparation
with Liebermann-Burchard methodology.
was then introduced and the incubation performed
at 37"C. A
MATERIALS The reaction proceeded to equilibrium, the £.14
cholestenone being estimated at 240 nm after an
The chemicals and solvents used are BDH
extraction with 3 ml isopropanol. Since the reaction
•Analar' analytical grade unless otherwise stated.
is an end point determination with the final estima-
tions in the UV, reaction blanks are required.
Enzyme reaction system
Calibration of serum cholesterol assay
Solvent system for serum cholesterol extraction:
ethanol:dioxane, 0·9:1·1 (v/v). Initially, calibration consisted of the enzymatic
KOH, 56g/IDO ml. conversion of cholesterol standards in ethanol:
 81

dioxane, 0·9: 1·1 (v/v) to ..1 4 cholestenone, from Table 1. Substrate specificiry of cholesterol dehydro-
which the serum cholesterol values were obtained. genase.
To overcome the need for recovery corrections by
this method a commercial control serum 'Serachcl'
Rate of .14 cholestenone production
was used for calibration purposes. Dilutions of 309 as a percentage of enzymatic rate with
mgjl 00 ml serum cholesterol produced standards at cholesterol
206 and 103 mg/IOO ml cholesterol, Fig. 3. Both Sterol"
methods of calibration were used to correlate with the Dinitrophenylhydrazine 240nm assay
automated Liebermann-Burchard method for serum assay
cholesterol.
Cell free Salt Salt
supernatant fraction fraction

Cholestanol 24·0 18·7 0

1/ 7-dehydro-

/~
cholesterol 54·6 69·0 31'5
Ergosterol 11·4 11-4 15·2
/ 20«-hydroxy- > 100 >100 > 100
/ cholesterol
/
./ / .'
// "The first two sterols occur in serum; the others do not.

/~
E
c::
C
o::t
mg/ I00 ml. The standard deviation of the difference

-
N

nl
"/ in extinction between the test and blank results is
0·010 which gives a coefficient of variation of 5 %.
W
<J

/
/ There was an improved correlation when 'Serachol'
calibration was used, as seen from the correlation
coefficients which were significantly different, Table
2. Also the serum cholesterol results from the two
assays were significantly different. The mean dif-
4 -r8---12: 16 :20 2~g cho!. ference in cholesterol between the two methods does
not greatly alter between the two modes of calibra-
~100 ..200 ",300 mg%chol. tion. With 'Serachol' calibration, the intercepts
Fig. 3.-Enzymic conversion of I. Cholesterol standards in calculated from the regression analysis gave values
ethanol-dioxane, 2. 'Serachol' serum standards, to .1' greater than the difference between the mean
cholestenone. cholesterol results, Table 3 and Fig. 4. The regression
lines are removed from A (the theoretical line at 45"
for two identical procedures) towards the Lieber-
RESULTS mann-Burchard axis.

Enzyme substrate specificity studies were per- DISCUSSION

formed with sterols related to cholesterol, especially
the compounds present in normal serum, except for
..1; cholestenol which was not commercially
available. As the enzymatic rate with cholestanol and
7-dehydro-cholesterol is less than the rate with
cholesterol, an enzymatic serum cholesterol assay
should possess a greater specificity than established
colorimetric assays (Henry, 1964) provided the
slower reactions with related sterols do not reach
completion. The comparison of enzymatic rates on
these compounds assumes that all the ketone deriva-
tives exhibit the same extinction (Table I).
Fifteen test and blank cholesterol determinations
by the enzymatic assay were carried out on a serum
specimen with a cholesterol concentration of 310
Since sterols other than cholesterol exist in serum,
specificity of cholesterol for its substrate was
investigated, as lack of cholesterol specificity is
one of the drawbacks in conventional cholesterol
assays. A list of typical serum concentrations of
'non-cholesterol' sterols is given by Henry (1964).
Although complete specificity for cholesterol is not
exhibited by cholesterol dehydrogenase, the enzy-
matic technique appears to be more specific than
colorimetric cholesterol assays.
Determination of the enzyme product ..1 4
cholestenone was chosen for the assay of serum
cholesterol. As there is a stable accumulation of
product the analysis is made at reaction equilibrium.
82

Table 2. Statistical analysis of the correlation of enzymatic and Liebermann-Burchard serum cholesterol assays using
two modes of calibration,

Calibration Mean (S.D.) cholesterol No. of Correlation Difference Paired Level of

method (mgjl(}() ml) readings coefficient between student significance
------- ------ means t test (P)
L-B Enzymic (mgjl ()() ml) t value
------- ------ ------ --~--- - - - - - - - - - - - - - - - - - - - --------.
Pure choles- 205·35 173-41 31 0·8065 31'94! 9·7 6·54 ooo I
terol organic (37'30) (46'(}()
standards
'Serachol' 215-60 188·30 38 0'9437 27·30 \ 9·\ 5·83 ooo 1
standards (69'70) (75-60)

By Fisher analysis Z 2·2 therefore the correlation coefficients are significantly different.

Table 3. Regression analysis oj" the correlation oj"

Enzymatic and Liebermann-Burchard serum cholesterol 400
assays using two modes of calibration.
"CI
y = a\ bx a = intercept b = regression coefficient Q
.c

Calibration x y b a
~3oo
a:I
method
----------_._-- - - -.-.-- - _ ....._ - _ . _ - - -------- I
~
Cholesterol
15-200
...
standards in
ethanol:
dioxane.
H

0·9: 1'1, (v/v) b

'Serachol' a
L-B

Enzyme
L-B
L-B
Enzyme
Enzyme 0·9997

0·6506
1·0145
-31,9

-! 92·5
-29,9
-CI:l
I II
CI:l
15
-5100
standards b I
Enzyme L-B 0·8466 +55'7 E
2
CI:l

The incubation time is long (up to 2 h), since the
quantity of enzyme activity capable of being pro-
duced was low, and was limited by the size of
bacterial cultures. With a higher enzyme activity
some other aspect of the reaction could be exploited.
If, after elucidation of the reaction mechanism, the
enzyme is found to be an oxidase, the hydrogen
peroxide produced could be linked, via the enzyme
peroxidase, to a dye. However, for this feature to be
demonstrated and used with cholesterol dehydro-
drogenase from this bacterium, high enzyme activi-
ties are required to complete the reaction within
several minutes, so that the labile hydrogen peroxide
can be quickly converted and coupled to an 'oxygen-
acceptor' dye.
Technical problems with the enzyme preparation
and cholesterol assay has been accentuated by the
relative insolubility of cholesterol in aqueous media
~
en 0
E
o 100 200 300 400
mg%serum cholesterol, enzyme method
Fig. 4.-Correlation of the enzymic method with the
Liebermann-Burchard method of analysis for cholesterol.
The 45° line is marked A. The correlation coefficient
0,94, mean difference 27·3 mg/IOO mi.
needed to be overcome in the cholesterol agar, the
aqueous cholesterol growth medium and the
bacterial cell free supernatant. Also removal of
enzyme activity on Ll4 cholestenone is needed for an
assay involving the measurement of this substance.
The enzyme assay requires serum cholesterol
extraction and saponification. The solvent system
has to satisfy several criteria and the maintenance of
cholesterol in a 0,) /Lmol/l phosphate medium is
necessary for the enzyme reaction to take place. The

saponified serum extract needs to be well buffered
and the high blanks at 240 nm are reduced by
extractions with isopropanol.
The correlation graphs show that a relationship
exists between the enzymatic and the Liebermann-
Burchard determination of serum cholesterol
although most of the experimental observations do
not coincide with the line A (Fig. 4). This would
imply complete identity between the two methods
whereas their specificities and interferences (both
positive and negative) differ. This is exemplified by
the results from the correlation coefficients which
indicate a trend but not a perfect correlation. Also,
the majority of the observations fall to one side of the
45" line, providing lower serum cholesterol values for
the enzymatic method.
The area of experimental points on the correlation
graphs are an indication of the combined error in the
two methods. The automated Liebermann-Burchard
assay has a c.y. of 5'y" at 200 mg/lOO rnl ; for the
enzyme method the c.y. is 5% at 300 mgflOO ml.
The precision of the enzymatic assay in its present
form, is disappointing, but inevitable when there is
a need for blank determinations together with small
differences in extinction between test and blank
values. The sensitivity of the reaction is limited by the
amount of cholesterol that can be added to the
reaction medium, which is a function of the serum:
solvent ratio of the initial extraction.
Table 3 presents the results from two regression
analyses. Section a refers to the regression of
enzyme on Liebermann-Burchard and section b to
Liebermann-Burchard on enzyme.
Extrapolation can be hazardous because the
regression applies within the range of observed data
an~ derivation of a value for the intercept is only
valid when the relationship is linear over the whole
range. In order to obtain data from the two equations
quoted for 'Serachol ' calibration, Table 3, the
objective must be defined. If the serum cholesterol
concentration that would be given by the enzyme
assay is required from the Liebermann-Burchard
cholesterol value, equation 2a is used. To obtain the
value on the Liebermann-Burchard axis when the
enzyme axis equals 0 mgflOO ml serum cholesterol
2b is used.
The intercepts quoted in Table 3 and the mean
differences obtained between the two cholesterol
assays. Table 2. may be compared with an average
serum concentration of 28 mgflOO ml for circulating
sterols other than cholesterol. The experimental
results may be a reflection of the true serum situation
or the inhibition of cholesterol dehydrogenase by
compounds present in sera.
Further research will no doubt provide more
answers and explanations.
83
CONCLUSION
The assay system described is not easily
automated. It is possible that with more enzyme
activity, hydrogen peroxide production may be
demonstrated and used. There is ample scope for
future research and development. Further enzyme
characterisation such as molecular weight, reaction
mechanism and other properties need to be studied.
One of the drawbacks of an enzymatic cholesterol
assay is the need to saponify serum cholesterol
esters. In the literature the ability of Nocardia
erythropolis to metabolise cholesterol palmitate as the
sole carbon source has been described (Schatz et al.,
1949). Also mammalian liver features a cholesterol
esterase (Deykin et al., 1962) with a high affinity
for the series of cholesterol esters present in normal
sera.
The enzymatic assay of serum cholesterol.
monitored by the production of Ll' choiestenone has
been shown to have more specificity than present
colorimetric methods, with comparable precision.
The enzymatic assay is more sensitive, since micro-
gram quantities of cholesterol may be detected by
dehydrogenation to Ll4cholestenone, but, at present,
the analytical time is longer than for conventional
cholesterol methods.
The routine use of such an approach depends on
the production of higher enzyme activity. to complete
the reaction in a shorter incubation and to utilise
some other aspect of the reaction when the mechanism
has been elucidated. Also it would be advantageous
to saponify cholesterol esters enzymically.
Whenever an enzyme is used which has the
minimum of described properties, the research
worker is faced with many different aspects to
investigate. Since the object of this piece of work was
to use the enzyme as a reagent for serum cholesterol
it was considered that a balance had to be found
between research on enzyme characterisation and its
use as a reagent. Therefore the properties and mode
of purification which were studied were those which
seemed to be necessary for the development of a
satisfactory assay method with the amount of
enzyme which was capable of being produced.
'
REFERI'NCES
Dawson, R. M. C, Elliott, D. C, Jones, K. M. Data for
Biochemical Research, 2nd edn., 1969, Oxford University
Press, 489.
Deykin, D., Goodman, D. S. The hydrolysis of long chain
fatty acid esters of cholesterol with rat liver enzymes.
J. biol. Chem. 237 (1962) 3649.
Henry, R. J. Clinical Chemistry, Principles and Technics,
Harper Row, New York, 1964,844 and 860.
84
Schatz, A., Savard, K., Pintner, I. J. The ability of soil
microorganisms to decompose steroids. J. Bact. S8
(1949)117.
Stadtman, T. C, Cherkes, A., Anfinsen, C B. Studies on
the microbiological degradation of cholesterol. J. bioi.
Chern. 206 (1954) 511.
Turfitt, G. E. The microbiological degradation of
steroids. 4. Fission of the steroid molecule. Biochem. J.
42 (1948) 376.
Zanin, V. A. Selection of Actinomycetes decomposing
cholesterol. Mikro. 37 (1968) 919.