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859
SHORT REPORT
Specificity of the commonly used enzymatic assay for
plasma cholesterol determination
M H Moghadasian, J J Frohlich, C H Scudamore
.............................................................................................................................
J Clin Pathol 2002;55:859861
Aim: To assess the specificity and sensitivity of the
commonly used enzymatic colorimetric test for plasma
cholesterol determination.
Methods: Interference with an enzymatic method for cholesterol measurement by several non-cholesterol sterols (
sitosterol, campesterol, stigmasterol, stigmastanol, desmosterol, and lathosterol) was assessed. Some of these
compounds are present in plasma at higher than normal
concentrations either in rare genetic disorders, such as
phytosterolaemia, or after the consumption of phytosterol
enriched foods.
Results: The non-cholesterol sterols were detected by the
assay in a linear manner. There was no competitive interference in the presence of cholesterol.
Conclusions: This crossreactivity may affect the diagnosis
and treatment of non-cholesterol dyslipidaemias, including
phytosterolaemia and cerebrotendinous xanthomatosis.
Similarly, changes in plasma lipid compositions after the
consumption of phytosterol enriched foods cannot be specifically determined by this enzymatic assay. Until a more
specific enzymatic assay is developed, alternative methods such as gas chromatography should be used to differentiate between cholesterol and non-cholesterol sterols.
ncreased plasma cholesterol concentrations are a causal
factor in the development of coronary heart disease (CHD).
Both American and Canadian guidelines recommend
annual plasma cholesterol/lipid measurements in patients
with more than two major risk factors for CHD.1 2 Patients
with fewer risk factors may only need to check their plasma
cholesterol values every five years or less.1 2 In addition, CHD
develops in rare non-hypercholesterolaemic recessive genetic
disorders, such as phytosterolaemia3 and cerebrotendinous
xanthomatosis (CTX)4; in particular, CHD related deaths are
frequently reported in young men with phytosterolaemia.5
Higher absorption and lower excretion of plant sterols play a
causal role in the development of phytosterolaemia.5 The
accumulation of cholestanol in the plasma and tissues of
patients with CTX may account for multiple coronary artery
aneurysms and myocardial ischaemia.4
Patients with hypercholesterolaemia respond to treatment
with cholesterol lowering agents such as statins, fibrates, or bile
acid resins. However, some of these agents are not of value in
phytosterolaemia and CTX. In fact, bile acid resins, a treatment
of choice for sitosterolaemia, are contraindicated in CTX.
Another strategy for reducing plasma cholesterol concentrations in individuals with hypercholesterolaemia is the consumption of phytosterol enriched foods.3 These may cause an
increase in plasma concentrations of plant sterols, particularly
sitosterol and campesterol.6 7 Similarly, both human and animal
studies showed that the consumption of plant sterols is associated with increases or the appearance of desmosterol and
lathosterol in plasma.810 Although the longterm importance of
this small increase in the concentrations of non-cholesterol
sterols in human plasma is unknown, both patients and physicians should be able to detect such changes.
Higher absorption and lower excretion of plant sterols
play a causal role in the development of phytosterolaemia
The commonly used enzymatic assay for cholesterol
measurement lacks the specificity to discriminate plasma
cholesterol from non-cholesterol sterols. The aim of our
present study was to determine the extent of specificity of the
enzymatic cholesterol assay for several phytosterols and other
non-cholesterol sterols that may appear in plasma as a result
of changes in cholesterol metabolism during the management
of hypercholesterolaemia, or in certain rare genetic disorders
of lipoprotein metabolism.
MATERIALS AND METHODS
All chemicals including cholesterol (catalogue number, C 8667;
purity, > 99%), sitosterol (catalogue number, S 9889; purity,
> 97%), campesterol (catalogue number, C 5157; purity, 65%),
stigmasterol (catalogue number, S 2424; purity, 95%), stigmastanol (catalogue number, S 4297; purity, > 95%), desmosterol
(catalogue number, D 6513; purity, > 85%), and lathosterol
(catalogue number, C 3652) were purchased from Sigma
(Oakville, Ontario, Canada). The stock solutions were prepared
by dissolving the chemicals in cholesterol diluent (Wako
Chemicals GmbH, Neuss, Germany) using a hot plate and stirring. Five different concentrations were made from each stock
solution. The concentrations of the chemicals were determined
by the enzymatic colorimetric test for cholesterol determination
currently used in the clinical laboratory, St Pauls Hospital, Vancouver, Canada. Briefly, Hitachi analyser 911 (Boehringer Mannheim, Indianapolis, Indiana, USA) and cholesterol reagent
(catalogue number, 1489232; Boehringer Mannheim) were
used. This method is based on the determination of 4
cholestenone after enzymatic cleavage of the cholesterol ester
by cholesterol esterase, conversion of cholesterol by cholesterol
oxidase, and the consequent formation of a red dyestuff after
the reaction of 4-aminophenazone with phenol under the catalytic action of peroxidase.11 The colour intensity is directly
proportional to the concentration of cholesterol and is
determined photometrically. The false positive results (cholesterol reading) were plotted against the real concentrations of
each specific compound. A free cholesterol standard solution
(catalogue number, 27447109; Wako Chemicals GmbH)
containing 2.6 mmol/litre cholesterol was also used to determine the extent of competitive interference in the presence of
native cholesterol. There was no need to obtain ethics approval
.............................................................
Abbreviations: CHD, coronary heart disease; CTX, cerebrotendinous
xanthomatosis
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860
Short report
Figure 1 False determination of cholesterol by various non-cholesterol sterols including sitosterol, campesterol, stigmasterol, stigmastanol,
desmosterol, and lathosterol using a common enzymatic assay.
for our study because of the lack of use of animals, humans, or
their tissues.
RESULTS
Figure 1 shows values for non-cholesterol sterols as determined by the routinely used enzymatic assay for cholesterol.
www.jclinpath.com
All compounds tested were detected by the cholesterol assay
in a linear manner. The assay was sensitive for all these compounds; the lowest concentration of sterols tested and
detected was > 0.2 mmol/litre. The assay crossreacted with
sitosterol and campesterol (two plant sterols abundant in
nature with relatively high absorption rates and plasma
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Short report
concentrations). For example, the assay read equivalent to 3.5
and 2.6 mmol/litre cholesterol for 5.2 mmol/litre of sitosterol
and campesterol, respectively, whereas it read 1.7 mmol/litre
for 2.6 mmol/litre of both compounds. The reading was comparable to the actual concentrations at lower values
(< 2.6 mmol/litre) for both compounds. In contrast, the assay
consistently showed higher (by 3070%) concentrations of
desmosterol. This was more pronounced at lower actual
concentrations (1.4 mmol/litre for 0.8 mmol/litre, a 70%
increase in reading) of desmosterol as compared with higher
actual concentrations (16.7 mmol/litre for 13 mmol/litre, a
28% increase in reading). The results were comparable to the
actual concentrations for other compounds including cholesterol, lathosterol, stigmastanol, and stigmasterol. When
various known concentrations of non-cholesterol sterol were
added to a cholesterol containing solution (2.6 mmol/litre) the
assay read the concentrations of both sterols (cholesterol and
non-cholesterol) as cholesterol in an additive manner. This
indicates a lack of competitive interference in the presence of
native cholesterol.
DISCUSSION
Plant sterols have chemical structures similar to that of cholesterol. Therefore, they interfere with cholesterol absorption
and thus can be used for dietary management of mild hypercholesterolaemia. For plant sterols, normally only up to 5% of
the dose ingested is absorbed, whereas the absorption rate for
cholesterol is up to 50%. The consumption of plant sterol
enriched foods results in higher plasma concentrations of
some phytosterols, such as sitosterol and campesterol, and
endogenous sterols such as desmosterol and lathosterol.69 For
example, the ratio of plasma campesterol to cholesterol
concentrations increased more than 50% (from 3.39 to 5.18)
in men with hypercholesterolaemia during a 30 day period of
consumption of a phytosterol enriched diet.7 This reflects significant decreases in the plasma cholesterol concentration and
increases in plasma campesterol concentrations. An intake of
2 g of plant sterols each day may double plasma campesterol
and sitosterol values.
In patients with sitosterolaemia (phytosterolaemia), a rare
genetic disorder, plant sterols accumulate in both the plasma
and the tissues. Plasma plant sterols are increased from
approximately 0.05 mmol/litre in healthy individuals to 1.2
mmol/litre in patients with sitosterolaemia.3 Although plasma
cholesterol concentrations may be in the normal range or
raised, the clinical symptoms of the disease resemble those of
hypercholesterolaemia, including coronary artery disease and
xanthomatosis.3 5
Our data provide evidence for the possibility of a low detection rate for sitosterolaemia world wide. In general, such
patients may be misdiagnosed as having hypercholesterolaemia based on the results of plasma cholesterol determination.
Thus, their treatment may not be appropriate. Moreover, the
use of phytosterol enriched food products as one of the current
strategies in the management of mild hypercholesterolaemia1
may result in the accumulation of plant sterols6 7 and other
endogenous non-cholesterol sterols (desmosterol and
lathosterol)8 9 in plasma, which interfere with plasma cholesterol determination.
Our data provide evidence for the possibility of a low
detection rate for sitosterolaemia world wide
It is well documented that unlike cholesterol, relatively
small increases in plasma concentrations of non-cholesterol
sterols, such as plant sterols (in phytosterolaemia) or
cholestanol (in CTX), can result in clinical events, including
861
Take home messages
Non-cholesterol sterols were detected by the commonly
used enzymatic colorimetric test assay in a linear manner and there was no competitive interference in the
presence of cholesterol
This crossreactivity may affect the diagnosis and
treatment of non-cholesterol dyslipidaemias, including
phytosterolaemia and cerebrotendinous xanthomatosis
In addition, changes in plasma lipid compositions after
the consumption of phytosterol enriched foods cannot
be specifically determined by this enzymatic assay
Until a more specific enzymatic assay is developed,
alternative methods such as gas chromatography should
be used to differentiate between cholesterol and
non-cholesterol sterols
myocardial infarction. Therefore, it is important to develop an
enzymatic assay with a higher specificity for cholesterol. In the
meantime, the use of other methods, including gas chromatography, is recommended in patients who show no observable cholesterol lowering in response to hypocholesterolaemic
agents, those with symptoms of CHD in the absence of high
plasma cholesterol concentrations, subjects with a family history of sitosterolaemia or CTX, or people who are consuming
phytosterol enriched diets for a prolonged period.
.....................
Authors affiliations
M H Moghadasian, J J Frohlich, C H Scudamore, Department of
Pathology and Laboratory Medicine, The University of British Columbia,
Vancouver, Canada
Correspondence to: Dr M Moghadasian, Healthy Heart Program, St
Pauls Hospital, 1081 Burrard Street, Vancouver, BC, Canada V6Z 1Y6;
[email protected]
Accepted for publication 15 May 2002
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Specificity of the commonly used enzymatic
assay for plasma cholesterol determination
M H Moghadasian, J J Frohlich and C H Scudamore
J Clin Pathol 2002 55: 859-861
doi: 10.1136/jcp.55.11.859
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