CLIN.CHEM. 23/7. 1201-1214(1977)
CholesterolMethodologies:A Review
Bennie Zak
Jntroductlon
The introduction
of enzymes as reagents into the
methodologies
of cholesterol determination
(1-6) has
drastically altered the limited strong-acid approaches
taken by most analysts of the past 90 years (7-19).
There has been a considerably
heightened interest in
determining
the compound because of new diagnostic
approaches such as hyperlipoprotein
phenotyping
(13)
with the implied relationship of high cholesterol values
as an underlying cause of cardiovascular disease (14,15),
and abnormal cholesterol values in several other disease
states (16, 17).
From a historical point of view, modern cholesterol
determinations
had their beginnings’ in the late nineteenth century when Salkowski described a color reaction (18) for this analyte, which was isolated from gallstones about a century earlier (19). Three studies then
gave impetus to what might be described as a great analytical chemistry movement for the determination
of
cholesterol, and these were the development of a practicable color reaction (20), its modification (21), and the
discovery
that digitonin
precipitates
cholesterol
quantitatively
and relatively easily (22). The latter was
accomplished by Windaus, even though as late as 1928,
in his Nobel Prize address, he described the cholesterol
structure incorrectly, showing an ethyl group at carbon
10(23,24). When it was discovered that oxidation with
selenium resulted in chrysene (25), and on the basis of
x-ray measurements
on the mycosterol, ergosterol, it
was then believed that the ring nucleus of cholesterol
was perhydrochrysene.
Later, several investigators
(26-28) concluded that the correct nucleus was cyclopentanoperhydrophenanthrene,
a structure
that
seemed in harmony with data derived from earlier experimentations
(24).
The combination of a color reaction for quantification
and digitonide precipitation
for purification led to the
first colorimetric procedure for serum (29), which was
based on the gravimetric procedure of Windaus (22). By
that time he had introduced saponification,
a step that
showed that some idea of the presence of free cholesterol
Department
of Pathology, Wayne State University School of
Medicine; and Detroit General Hospital, Detroit, Mich. 48201.
Received and accepted April 1, 1977.
and the nature of ester cholesterol in blood was already
known to these early analysts (22,30). It is interesting
that the first acceptable reference procedure for the
determination
of cholesterol
was this gravimetric
method of Windaus (29). It was later used by Schoenheimer and Sperry (10) to prove the accuracy of their
own elegant procedure, which then became the reference method used to prove the Abel! et al. procedure (7),
a significant chain of events. Both have now been accepted as reference procedures,
which indicates that
present day accuracy is easier to accomplish but perhaps
no better than that obtained by the gravimetric means
of the past.
Between the colorimetric,
fluorometric,
and electrochemical
enzyme-reagent
techniques
of the
present-some
manual, some mechanized-and
the
beginning of cholesterol
methodologies
of the late
nineteenth
century, all of which were manual, many
suggestions were set forth for procedures to determine
this interesting
and important
compound
(31-41).
Literally hundreds of papers, many little more than
miniscule modifications of others, have been proposed.
Oddly, early studies showed the necessity for purifica
tion of the analyte before resorting to an endpoint reaction. But as time went on and mechanization
of
methodology
appeared,
the direct reactions became
more popular because mechanical devices were limited
in the sorts and numbers of reaction steps they could
perform. It was then suggested that a reference procedure such as the Schoenheimer-Sperry
and a direct
Liebermann-Burchard
procedure produced results that
were indistinguishable
from one another, a complete
turnabout from the past (42). More will be said of this
later under interferences
and standardization.
Before attempting, to discuss the various methods
available for the determination
of cholesterol, it might
be useful to classify them in order that one may peruse
and choose according to the application
and need to
which the measurement
is to be applied. Such a chemical categorization
could simplify the quandary most
users feel when faced with variety and a number of
choices. Because of the advent of newer methodologies
involving completely enzymatic systems (43,62) as well
as the procedures leading to them (1-6), it appears that
some revisions in classification
may be necessary that
CUNICAL CHEMISTRY. Vol. 23, No. 7, 1977
1201
would include the newer approaches, even though it is
apparent that the new systems should fit into the older
classification
(40).
Several methodological
classifications
have previously been proposed (36, 38-40). Among these, one
was described in which methods were grouped according
to the procedures of handling (40) rather than by the
color reaction used as the endpoint device at the equilibrium of the last step in the procedural outline (39).
This seemed and still seems quite suitable as a classification plan if it were modified somewhat to include
enzyme reagents and other kinds of endpoint reactions
other than spectrophotometric
ones. It demonstrates
in brief fashion the techniques involved, and enables the
selector to make a decision that is based on the limitations of the procedure selected for the kind of data required in terms of the accuracy and precision that is
adequate for the needs of the user. One should also
consider the medium in which the determination
was
to be done. It would be of critical importance,
for example, if the medium of interest contained insoluble
particulate
matter, which might preclude the use of a
simple, direct reaction that is better suited to a simpler
sample that presents
no such solubility
problems.
However, it might be possible in the future to avoid
problems engendered by particulate matter if the endpoint reaction, for example an electrochemical
one
rather than an optical one, could react with an analyte
generated in that procedure.
of Cholesterol Methods
I. Direct Reactions for the Determination of
Cholesterol
Classification
A. No separation
or partial separation
of cholesterol
is carried out. The reagents are mixed with sample and
the endpoint
is determined
by spectrophotometry
(9,
electrochemical
measurement
of a product
of reaction (49,56), or any other kind of endpoint device
deemed suitable to that purpose. A direct reaction
would undoubtedly
have to be considered to be one in
which the reagents are added to the sample and in which
no fraction of the sample such as the proteins are removed before a measurable reaction is allowed to take
place; that is, there is no phase separation in direct reactions. The Wybenga procedure with an Fe(III) reaction (63), the procedures of Pearson et al. (9) or Huang
(64), each based on the Liebermann-Burchard
reaction,
and the Allain procedure (5) in which a coupled enzymatic system is used are examples of direct reactions.
The Parekh-Jung
modification of an Fe(III) procedure
is improperly
described as a direct reaction (84-88).
They themselves stated that the reagent precipitates
out the proteins of serum and extracts the cholesterol
from its binding sites (88). Like the similar Zak (89),
Henley (90), Chiamori and Henry (91), or Bowman and
Wolf procedures (92, 93) that are the basis on which the
Parekh-Jung
procedure is founded, it would have to be
considered an extraction
procedure. Any claims to a
direct reaction for a material such as serum (85) or
12, 63-83),
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CLINICAL CHEMISTRY,
Vol. 23. No. 7, 1977
adrenal tissue (84) should be viewed as an error. The
belief is also incorrect that a certain kind of organic
solvent or mixture is required to extract cholesterol from
serum, as perhaps a liquid-liquid
system. Any solvent
that can precipitate proteins and at the same time solubilize cholesterol from its binding site is an extraction
solvent.
B. Blanking
1. Only a reagent blank is used (5,68,64,94-101).
The
assumption is made that no irrelevant absorption due
to color or turbidity could be present in the sample,
perhaps because of high dilution, and therefore no
serum blank is required, or it is assumed that the serum
blank effect is small enough to be ignored, or that a fast
kinetic rate measurement
obviates the blank.
2. Reagent blank and a serum blank are both subtracted. A serum blank of several kinds has been suggested. A change in the solvent matrix is made such that
cholesterol cannot react, so the background color of the
serum itself is subtracted (40,66, 69), or the reagents are
changed so that cholesterol does not react but the interfering compound (or compounds) can react, allowing
a blank correction for a dynamic interference along with
any residual irrelevant absorption caused by other static
colors or turbidities in the serum itself (75).
C. Advantages
and (or) limitations.
The primary
advantage of a direct procedure lies in its simplicity. If
a procedure yields reasonable
values so easily, it is
readily amenable to manual and simplified automated
systems. In the latter methodologies,
automated
liquid-solid or liquid-liquid
extractions
are rather uncommon, although each has been reported (102, 103).
Obviously such systems can be subject to problems involving static and (or) dynamic errors of either an absolute or relative type (104). In the case of the latter, the
situation can be improved by use of the principle of internal standardization
(105), a process that will be
discussed later.
II. Partial Purification of Cholesterol with Organic
Solvents
A. Partial isolation
can be achieved
or liquid-liquid
extractions
(106-120).
by liquid-solid
In the case of
liquid-solid
extractions, the extracting fluid is soluble
in water and serves to remove the cholesterols from their
protein-binding
sites into the liquid phase, where the
measurement
can be made either directly (121) or on
a later solubilized residue of an evaporated
extract
(122). Alternatively,
the serum is dried into one of several solid materials, from which it can be eluted into a
separating
liquid (123-125).
Biphasic liquid-liquid
extractions
with (7) or without (8) saponification
of
cholesterol esters before the extraction have been used.
In some procedures, saponification
is required because
the extracting fluid will not completely separate both
free and esterified cholesterol
(7). In the automated
mode, there has been at least one report in an enzymatic
procedure (47) of dialysis of product, peroxide, where
the latter could be measured
in a diffusate,
which
seemed free of interferences. Any potential competition
of bilirubin with the color reaction was avoided by a
preliminary treatment of the sample with alkali to oxidize it when total cholesterol was determined, because
peroxide was generated on the same side of the membrane where the bilirubin was present. However, the
reaction of bilirubin with peroxide is minimized if no
peroxidase is present to catalyze it.
B. Blanking. Irrelevant absorbing compounds of the
static type (i.e., absorption does not change during the
reaction) can be isolated from cholesterol (126) by the
extraction process. In addition, known dynamic interferences can also be separated (126). Therefore, procedures involving extraction
systems do not usually
describe the use of serum blanks, and rely primarily on
reagent blanks.
C. Advantages
and limitations.
Partial purification
and elimination of most interferences
should result in
a more nearly pure end-point determination
and more
nearly accurate values. However, the more steps that
there are in the reaction process, the more likely the
possibility that errors can occur. In addition, the potential for automation
decreases as the complexity of
the process increases. Because the determination
of
cholesterol is frequently requested,
it intensifies the
problem of handling many samples on a daily basis.
Ill. Complete Isolation of Cholesterol
A. Free cholesterol has been isolated, after extraction
and saponification, by precipitation with one of a variety
of precipitating
agents (30, 114, 127-133) and this purified derivative has then been subjected to endpoint
analyses (10, 30, 117, 118). The precipitation
has
sometimes been hastened with the aid of a gathering
step for which either aluminum
hydroxide (128) or
aluminum chloride (127) is used.
B. Cholesterol or its esters, or both, have been separated by means of different chromatographic
processes
after a preliminary
partial purification
by extraction
into an organic solvent. These have included gas chromatography (133-141), liquid column chromatography
(142, 143), and paper chromatography
or thin-layer
chromatography
(144-149).
C. Advantages
and limitations.
The advantage of
purification of an analyte before it is determined is that
it can then serve as a reference procedure for the more
common procedures that are used routinely. The disadvantages of complexities of processes and equipment
along with the requirement
of greater skills for the analyst are outweighed by the need for reference procedures (150). In the case of purification by precipitation
with digitonin, some compounds that are structurally
similar to cholesterol
and present
in measurable
amounts appear to be present in serum (137, 141), but
the extent of their interference is not clearly defined as
yet. In addition, if they are not esterified, they would
contaminate
free cholesterol
disproportionately
as
compared to total cholesterol. An advantage of the digitonide, however, is that it provides an additional analytical grouping for measurement
because of the carbohydrate moiety on digitonin (151-156). Another ad-
vantage of isolation and purification by digitonide formation is that this treatment would exclude potential
interfering sterols whose structures preclude reaction
with digitonin.
IV. Miscellaneous Procedures
A. Cholesterol and its esters have been extracted from
the protein zones known to contain them after electrophoresis of serum proteins was carried out (157-160).
B. Lipoproteins
containing
cholesterol
have been
selectively precipitated out with dextran sulfate before
colorimetric determination
by either the LiebermannBurchard or Fe(III) reactions (161, 162).
C. A selective partition procedure has been described
for isolating free cholesterol (163).
D. Any procedure devised in the future that does not
fit into I, II, or III would undoubtedly
fit here in IV.
V. Screening, Definitive and Reference Procedures
No established
definitive procedure for the determination of serum cholesterol has been described as yet.
However, at least three procedures have proven worthy
as (as yet unofficial) reference methods (7, 10, 22), while
all of the rest have been applied to the routine circumstances. The techniques that have provided most of the
diagnostic data in recent years might best be termed the
screening procedures (9, 42, 63, 64), whose function it
is to find abnormal values quickly, although perhaps not
providing nearly the accuracy of the reference procedures when the latter are carefully carried out (40). The
advent of such simple screening procedures as a part of
automated multiphasic instrumentation
(42), or highspeed discrete sampling analyzers (5) where they can
be dedicated for screening in hyperlipidemia,
has generated an era in which all can easily obtain the advantage of relatively inexpensive
cholesterol assay. The
introduction
of enzymes as reagents into the more recent methods for the determination
of serum cholesterol
appears to serve the ideal: a screening procedure that
is as accurate as the reference procedures
for most
samples (40). Because the enzymatic procedures
are
very simple in terms of handling steps as compared to
the considerably
more complicated
reference procedures (7, 10) and because the reagents are easier to
handle, the precision of routine determinations
is superior to what it was in the recent past, and may very
well be better than that of the reference procedures
unless the latter are carried out with an extreme care not
usually practiced in the routine area. We appear to be
approaching
that analytical circumstance
where some
techniques
for the determination
of cholesterol
are
simple, accurate, and rapid, making them ideal for automation and still offering the promise of narrower
“gray” areas for perhaps better diagnostic definition.
It seems obvious even at this early period in the development of the enzymatic methods, that a cholesterol
oxidase approach to a substitute analyte for cholesterol,
namely hydrogen peroxide, when coupled to a preliminary purification process as in the Abell procedure (7)
should lead to a more nearly ideal reference procedure
than those in which the strong-acid endpoint reactions
CLINICAL CHEMISTRY,
Vol. 23, No. 7, 1977
1203
are used, with all of their known attendant
problems.
However, in considering reference procedures, one must
determine
how a reference
method is established
by a
contemporary
group of scientists.
In recent years one
has seen the introduction
of the concept of a Reference
Method
for calcium (164), alleged to be the first and
only Reference Method in all of clinical chemistry.
From
it a reference
procedure
was established
and later confirmed as acceptable
(165). It behooves us to question
whether
other reference
procedures,
such as those described for the determination
of cholesterol,
have been
established
by similar rigorous investigations
or simply
accepted
by common
usage. This question
has been
raised-and
it should be answered-as
to who establishes reference
procedures
(166).
Other Analytical Considerations
Interferences
This is one of the most important
areas of any analytical procedure.
Innovators
of analytical
procedures
cannot always predict what the future of any analytical
system will hold when testing
it in a heterogeneous
sample subject to wide-swinging
changes in concentration of its constituents
owing to abnormal
physiological
states and often characterized
by the presence of compounds
(drugs) perhaps
ingested
by an individual
or
administered
to a monitored
individual
in an attempt
to return a patient
back to the normal state. Even a
clean-looking
biological sample such as normal serum
contains
many compounds
that are not always suspected as interferences
because
they do not become
troublesome
until they are present in rather high concentrations
in the tested fluid. Samples that are jaundiced, turbid,
lipemic, or hemolyzed
may present
an
important
problem, especially if a direct determination
is to be made. The most important
of these problem
samples
is caused by jaundice,
because
bilirubin
is a
reactive compound,
differing
in the way it acts in the
several endpoint
systems
that are in common
use in
present
cholesterol
methodologies.
In the Liebermann-Burchard
and iron reactions,
if bilirubin
is not
removed, it reacts to form stable biliverdin,
which gives
a positive absolute interference.
The magnitude
of that
interference
is greater for the Liebermann-Burchard
reaction
because bilirubin
forms more measurable
absorbance
by conversion
to biliverdin
than does cholesterol in generating
a similarly shaped spectral absorbance curve in the region of the measurement
wavelength. This easy conversion
of bilirubin
to biliverdin
exemplifies
an oxidative
action of the LiebermannBurchard
reagent.
In methods
in which enzymes
are
used as reagents,
authors
of three similar systems described how bilirubin
results in a small positive error
(43), a small negative error (5), or no error at all (167).
If one considers that the effect of interference
could be
determined
by testing bilirubin in albumin without the
possibility
of peroxide/peroxidase
side reactions,
the
correct interpretation
would be for a positive error. If
1204
CLINICAL CHEMISTRY,
Vol. 23, No. 7, 1977
cholesterol
and bilirubin
were present simultaneously,
then the error could be negative. However, it is difficult
to rationalize
no error at all when a very high bilirubin
concentration
is present
and a competitive
substrate
reaction
should take place to destroy a portion of the
peroxide generated. In other words, bilirubin
can interfere with enzymatic procedures
for the determination
of cholesterol
positively
in the static mode and negatively in the dynamic mode for colorimetric
endpoints.
It would seem that it could only interfere
negatively
in
fluorescent
methods
and possibly
negatively
in electrochemical
measurements.
Investigators
of fluorescent
or electrochemical
methods
may believe that there
should be no error because bilirubin
does not fluoresce
under these conditions
since it requires an acid medium
and albumin
(168) or that it does not react with a selective-ion electrode. The latter technique
should suffer
minimally
from bilirubin
because
no peroxidase
is
present.
However,
the potential
reaction
of bilirubin
with peroxide
may be critical to all endpoint
measurements. The more peroxide
side-reacts
under the conditions specified for a particular
endpoint measurement,
the lower will be the final result and the greater
the
deviation
from the true indirectly
obtained
value for
cholesterol.
Enzymes
used as chemical
reagents
for
producing
peroxide,
where the latter becomes the derived or substitute
analyte for cholesterol,
have some
selective
specificity
in generating
peroxide,
but from
that point in the sequence of reactions
the enzymes are
no longer involved. Since the peroxide
is capable of a
side reaction with a competitive
substrate
such as bilirubin in preference
to those of the anticipated
endpoint
reaction,
if aminoantipyrene-phenol
is the example,
then bilirubin
can interfere
by lowering the concentration
of final compound,
hydrogen
peroxide,
which
is supposed
to reflect cholesterol
content.
In addition,
the measurement
wavelength
becomes
important
for
the final result because bilirubin
absorbs somewhat
at
the endpoint
peak maximum
of 500 nm, and any residual bilirubin
will act compensatorily
for the substitute colored complex that was not formed (177). If the
wavelength
is shifted toward the infrared to get away
from bilirubin absorbance,
as has been proposed
(167),
then it would seem reasonable
to assume
that the
compensating
irrelevant
absorption
of bilirubin
is
eliminated
and the final apparent
concentration
of
cholesterol
should be lower.
Interferences
in Fe(III)
reactions
can cause either
absolute or relative errors, which depend in part on how
the sample is treated before the colorimetric
step. If one
embarked
on isolation
processes
such as those used in
the procedure
of Abell et al. (7) or SchoenheimerSperry (10), and then substituted
an Fe(III) reaction
for the Liebermann-Burchard
reaction,
the results
should be no different
than those of the LiebermannBurchard
reaction on the finally treated residue, unless
rather
high concentrations
of reactive
compounds,
perhaps
other i5-sterols,
were extracted
along with
cholesterol
during the isolation.
With the Fe(III) reaction, the final color obtained
would be more stable and
considerably more sensitive. It is for the direct mode or
for the partly isolated system that some interferences
have been reported. Bromide causes relative errors of
enhancement (169), although it is not often encountered
and maybe easily removed (91, 126, 169). Its enhancing
effect is more suitably obviated by the method of internal standardization
(105) rather then by isolation
from the interference.
Thiouracil is now known to be
unnecessary
for the now-defunct
test for butanol-extractable iodine, azide is not a highly regarded preservative anymore
because of its potential
explosive
qualities, while the amount of nitrate required for notable inhibition,
presumably
that contaminating
the
sulfuric acid, is much greater than the tolerable amount
in that reagent (174). Bilirubin is quickly oxidized to
biliverdin by any of the Fe(III) reactions, buL the extent
of its interference is less than that of most other colorimetric procedures,
including the Liebermann-Burchard reaction and the Hantzsch and Trinder reactions
of the enzymatic procedures (176, 177).
Many substances were previously reported as interferences in the Liebermann-Burchard
reaction for
cholesterol
(39, 40, 178-182).
The actual chemical
documentation
is fragmentary,
mostly stemming from
the early days involving visual bicolorimetric
measurements. The compound that causes the most such
trouble is bilirubin (39, 40). The reported extent of this
interference
has ranged from questionable
(70), 3
mg/mg (42), and 10-12 mg/mg (183) to erratically high
and variable (66, 76). However, it can easily be shown
that, in the most common modification of the Liebermann-Burchard
procedure
for cholesterol
(64), the
reaction with bilirubin is linear when allowed to go to
completion, the fmal color is stable (far more stable than
that for cholesterol itself), the color formed is linearly
related to concentration
of bilirubin, the reaction is
independent
of the presence of cholesterol, and the
product formed absorbs more strongly than does that
of cholesterol. Several of these points are also identical
to those found with all Fe(III) reactions for cholesterol,
in all of which bilirubin is easily converted to biliverdin.
However, if the procedure for cholesterol based on the
Liebermann-Burchard
reaction is carried out at low
temperature
as suggested (68), which would slow up the
bilirubin oxidation, and if the wavelength of measurement is 540 nm as also suggested (68), then the bilirubin
interference
owing to unstable intermediates
formed
in its oxidative conversion might be too formidable to
overcome by blanking.
Interferences
in which enzyme reagents are used for
the subsequent
colorimetric determination
of cholesterol through its oxidation to form peroxide have been
evaluated to some extent (5, 6), but it is perhaps really
the peroxide itself that needs the most study. If one
considers the entire analytical scheme, peroxide seems
the most vulnerable to side reactions, and the problems
associated are quite similar to those with other oxidase
systems in which peroxide reflects the concentration
of
the analyte. If peroxide is also the compound actually
being measured in the fluorometric procedures (51), it
would appear that it should result in a similar problem,
if a side reaction could compete with the fluorescence
reaction for the measured compound. Compounds already tested as interferants
both for cholesterol (3, 5)
and in other oxidase procedures
for other analytes
(184-186) include steroids, bilirubin, hemoglobin, drugs,
uric acid, urea, creatinine, glucose, bromide, ascorbic
acid, glutathione,
L-dopa, and epinephrine.
Bilirubin
has already been shown to be a competing substrate for
peroxide, and a mechanism
in proof was described
(176). Cholesterol was allowed to react to completion
to deplete the peroxide formed before bilirubin in a
solution of albumin was added and the resulting spectrum scanned. This combined
spectrum
was then
compared to exactly the same amount of bilirubin plus
cholesterol reacted simultaneously,
where the bilirubin
had an opportunity to compete for the peroxide with the
intended color reagent. The latter combined spectrum
subtracted
from the first combined spectrum should
have resulted in a straight line of 0.0 A along the absorbance abscissa if no bilirubin interference occurred,
but instead the second spectrum was considerably lower
than the first spectrum, proving bilirubin interaction.
In addition, when a spectrum for pure cholesterol representing exactly the same concentration
of cholesterol
was subtracted
from the simultaneous
reaction spectrum, then the remaining absorption did not account
for all the bilirubin. Conversely, when the spectrum for
a pure bilirubin solution was subtracted
from the simultaneous
reaction spectrum,
then something
less
than the true cholesterol spectrum resulted (177).
The enzymatic procedure in which the oxygen consumed in the cholesterol oxidase reaction with cholesterol is measured
electrochemically
and where the
peroxide is blocked (by the addition of azide) from any
reaction with contaminating
catalase in the glucose
oxidase to form oxygen is less likely to be interfered with
by bilirubin (56). The pH at which the oxidase action
takes place and the short time of reaction, including the
hydrolase action to free the esterfied forms, allows more
specificity than when the peroxide is coupled sequentially to catalase or peroxidase reactions as final spectrophotometric
endpoint reactions.
To get around any problems engendered by the peroxide-peroxidase
reaction with interferences,
several
alternatives
present themselves
for the enzymatic
procedures in which peroxide is normally the measured
compound. Bilirubin can be destroyed by preliminary
alkaline action, which also hydrolyzes the esters before
cholesterol oxidase action (47); peroxide can be dialyzed
before it reacts with peroxidase and color reagents, a
process in which the reaction between bilirubin and
peroxide is minimized by the absence of the enzyme to
catalyze it (45); or cholesterol can be removed from the
serum in which the bilirubin is present before peroxide
is generated (4). In addition, the reacting compound,
oxygen, of the cholesterol oxidase step in polarographic
systems can be measured instead of measuring reaction
products of enzyme action while the peroxide generated
is protected from catalase action by the addition of soCLINICAL CHEMISTRY,
Vol. 23, No. 7, 1977
1205
dium azide (56). In the event that cholestenone
rather
than peroxide is measured,
it is a strong ultraviolet
absorber (240 nm) or its dinitrophenylhydrazone
can
be measured in the near-visible range (1, 4).
Other Sterols
From time to time reports have appeared in which
sterols other than cholesterol are said to be present in
serum (137, 141, 187). In procedures in which cross reactions are possible, a positive interference
results,
whereas if a separatory
step is incorporated
into the
procedure before the detection and determination
step,
then a more nearly true value is obtained, as is the case
with extraction followed by gas chromatography-mass
spectrometry for identification and quantification
(138).
Procedures in which cholesterol, either free or total, is
isolated by precipitation
with saponins (10) should be
more free of interference
by other reactive sterols, for
only those sterols of the alleged contaminants
with the
correct spatial configuration
should precipitate.
In
addition, digitonides of these interfering sterols may be
unreactive in a color reaction or have much lower molar
absorptivities
than does cholesterol. This area of the
other reactive sterols for the several colorimetric procedures needs more clarifying investigation
than we
have seen, because results which are the basis of our
present suppositions are based on quite poor analytical
techniques (187).
Blanking
The simplest approach to the determination
of any
of serum would be its direct measurement
without treatment
except by means of the measuring
device itself. For the moment that is not an available
alternative for cholesterol measurement.
Therefore, an
alternative way to determine cholesterol in serum would
be to react the analyte of the sample directly with reagent, a reaction that ideally would involve only the
desired constituent,
with all other components
of the
sample inert or at least subliminal to the chosen measuring device. Primarily,
such treatment
of sample
would result in a reaction product that most suitably
would be monitored spectrophotometrically.
More recently, electrochemical
detectors have been suggested
with which suitable products of enzyme reagent action
could be adequately
determined
(49, 56). In several
procedures,
blanking-either
of reagent, sample, or
both-has
been described. Blanking remains one of the
question mark areas of cholesterol methodology.
One
sees proposals for reagent blanks and serum blanks for
direct reactions in which the serum blanks may be static
(9, 188) or dynamic (66, 75, 76), and in at least one case
(189) a blank in which it was claimed that the medium
was inhibitory to the reaction of cholesterol, allowing
only the interferences to react in that particular matrix.
The static approach can only correct for the background
color of serum, and since this must involve a change in
solvent character that obviates the reaction of cholesterol, it must be assumed that there are no changes in
the irrelevant absorption measured at the peak maxianalyte
1206
CLINICAL CHEMISTRY,
Vol. 23, No. 7, 1977
mum in spite of the matrix differences.
Obviously,
this
would fail with either the Liebermann-Burchard
or
Fe(III) reactions if the interference
was reactive; for
example, bilirubin would be easily and quickly oxidized
to biliverdin. The biliverdin spectrum has a minimum
where the Fe(III) reaction has a maximum, but its peak
absorbance is at about the same wavelength as that for
the Liebermann-Burchard
reaction for cholesterol.
Therefore, there is a near 10-fold difference between the
two reactions in the extent of its interference
and interference is greater with the Liebermann-Burchard
reaction-in
some procedures (76) the latter amounts
to about 6:1 on a weight basis and therefore 9:1 on an
equivalent basis.
The previous discussion on blanking was primarily
concerned with interferences
yielding absolute errors,
but it can easily be shown that in Fe(III) reactions relative errors can occur that are corrected for by neither
static nor dynamic blanking. It then becomes necessary
either to remove the interference or separate the analyte
from the interfering matrix, a task which few would
relish. Blanking serves no purpose in this case because
the interferant
itself does not react with the reagents,
but interacts only when the analyte is present (105). An
alternative proposal would be to put standard into the
sample, allow it to come under the inhibiting or enhancing influence of the sample matrix, and then calculate from the additive absorbances of the analyte and
its standard when both are subjected to the same influences (105).
Another novel form of blanking in direct reactions
was by bichromatic
measurement
wherein it was assumed that bilirubin would constitute the only interference (82). Two wavelengths were chosen, the measurement one at the peak for cholesterol and the corrective one toward the red end of the spectrum at which
the absorbance reading for biliverdin was the same as
at the peak, thus nulling out the effect of the bilirubin
conversion.
Dynamic blanking by means of a change in reagent
matrix to create an analytical circumstance
in which
cholesterol is not reactive but bilirubin or perhaps other
interferences
are reactive (189) has been shown to be
inaccurate for at least one such version of an Liebermann-Burchard
reaction
(190) because
the green
spectrum formed with the Liebermann-Burchard
reaction for bilirubin is displaced bathochromically
in the
blanking version, causing an underestimation
of the
interference
effect at the measurement
wavelength.
Although described as a reaction in which the cholesterol reaction with the Liebermann-Burchard
reagent
is inhibited,
it is really
not an Liebermann-Burchard
reagent when a compound
is substituted
for a large
such as dimethylformamide
portion
of the Liebermann-
Burchard reagent. Other organic compounds used in
place of dimethylformamide
were shown to have a
similar spectral effect (190).
In studying interference
effects for blanking, it is
perhaps important to see if the interference reacts with
the reagent or is only interactive when both reagent and
analyte are present with the interfering compound. For
example, in the case of the Liebermann-Burchard
and
Fe(ffl) reactions with bilirubin, the reaction takes place
independently
from the reaction with cholesterol and
the error is additive and absolute. In the case of the
enzyme reagents sequencing to the peroxide/peroxidase
reaction with the Trinder reagent or the catalase/
methanol/peroxide
of the Hantzsch reaction, there is
no reaction with bilirubin without cholesterol because
no peroxide has formed. But when cholesterol is present
to generate peroxide by the cholesterol oxidase reaction,
then bilirubin in the presence of peroxide can and does
act as a competing substrate by destroying peroxide and
causing a lowered Trinder or Hantzsch reaction (176).
However, if one looks at the spectrum of bilirubin one
can see that the infrared side of the curve gives a substantial reading at 500 nm, the peak of the Trinder reaction, while the ultraviolet side gives an even more
substantial one at 410 nm, the measurement wavelength
of the Hantzsch reaction. Therefore, the user faces a
dilemma in blanking for bilirubin in either reaction. If
a serum blank in the Trinder reaction is subtracted
where part of the bilirubin has been converted by the
action of peroxide, the full concentration
of bilirubin
absorbance will be subtracted, whereas somewhat less
than that concentration
is actually residual in the final
measurement,
an overcorrective effect (176). Similarly,
in the Hantzsch reaction, bilirubin in the sample will be
partly or completely converted and the Hantzsch reaction yield is low, whereas a serum blank will contain
all of the bilirubin, again resulting in oversubtraction
of blank absorbance (176). Different versions of what
appear to be identical reaction systems may show
somewhat smaller or larger interferences, depending on
reagent makeup, especially with enzyme reagents, because, for example, reaction rates in the sequence of
reactions can be quite different, allowing more time for
the bilirubin and peroxide to react. In any sequence of
reactions in which conditions are favorable for peroxide/bilirubin
reaction or peroxide-competing
substrate
reaction, the endpoint reaction will underestimate
the
cholesterol because the substitute
analyte for cholesterol, having itself been converted by the competing
substrate, is no longer available for the necessary endpoint reaction, and the overall effect is a decreased apparent cholesterol concentration.
It would appear that
this should be true if peroxide is measured fluorometrically, colorimetrically,
or sequenced to yield a coenzyme-coupled ultraviolet measurement
(5, 44, 51, 191).
This blanking problem would not be encountered if the
cholestenone
could be determined,
a difficult measurement in a direct reaction, or if the oxygen used in
the oxidation of cholesterol to cholestenone is measured
while the peroxide generated is hindered or removed by
side reactions, since it would not be the endpoint device
in this kind of a procedure (56). If a colorimetric measurement
is made based on a peroxide/peroxidase
coupled reaction, and any bilirubin remains as an interfering color to contribute
absorbance at the measurement wavelengths, then that reading will serve as
a compensating
to aggravate
error unless a full serum blank is used
the measurement.
What Should Constitute a Sample
Although there may be secondary
objectives for
cholesterol determinations,
the usual sample in which
it is to be measured is blood. If it seems reasonable to
assume that the most useful portion of that sample is
serum, it might still be worthwhile to recapitulate a bit
on why this should be so. To begin with, one must consider how cholesterol is distributed
in blood, in what
forms it exists, where those forms are found, and in what
concentrations they could be measured. One often reads
statements
claiming that cholesterol
esters are the
predominant
form in plasma whereas free cholesterol
predominates in the erythrocytes (187). However, there
is no cholesterol inside the erythrocyte;
it is all in the
membrane, and apparently none of it is esterified-that
was an artifact of previous analytical error (192). The
cholesterol in the membrane is dynamic and has been
described as existing in equilibrium with the serum pool
of free cholesterol (192). There is also the huffy coat,
which contains cholesterol and which varies in volume
in certain diseases. Because the erythrocytes
also vary
in volume and thus act as a diluent of unknown proportions, analysis of whole-blood suffers from serious
deficiencies that are alleviated by the use of serum. The
use of plasma rather than serum also can be the source
of certain problems owing to the anticoagulants,
which
could interfere with some reactions by interaction or by
water transfer between compartments
of blood (40).
Standardization
The process of standardization
of cholesterol for the
different kinds of methodologies
has not been a simple
one in several colorimetric procedures. In fact, it is one
of the most complex and confusing aspects of cholesterol
determinations.
Because the cholesterol
in serum is
present mostly in the esterified form and the esters have
been suspected of reacting differently than does free
cholesterol
in some procedural
versions, then the
problem of the form measured must be considered in
those cases (10, 37, 40, 194). Different approaches have
been taken to circumvent the problem. These have included saponification
to convert all esters to the free
form (10, 115), mixing ester and free cholesterol in a
calibration standard to approximate
the normal value
(109), use of a factor to correct the ester value to the free
cholesterol value (112), or changing the solvent character of the medium to make free and ester cholesterol
react equivalently
(9, 188).
Simple, direct colorimetric
procedures
of the acid
type, such as those involving the still dominant Liebermann-Burchard
reaction, have not been simple to
standardize,
especially if one wished to adapt them to
automation,
and even more so in the automated
multiphasic systems where detergent-solubilized
or denyatized cholesterol would not be helpful because a serum
control was required for calibrating
the other serum
constituents
measured
in the multidetermination
CLINICAL CHEMISTRY,
Vol. 23, No. 7, 1977
1207
package offered. Originally, when the Huang procedure
(64), which had the advantage of a more stable reagent
than those devised for earlier methods (7, 10), was
compared in an automated
version (42) to the Schoeheimer-Sperry
method (10) by using nonicteric serum
standards as calibrators whose values had been determined by their own reference extraction procedure (42),
it was proven that the results were reasonably similar,
indicating that the direct automated method was very
accurate
indeed. However, after the Framingham
studies were reported (195), the values of the serum
standard for cholesterol for multiphasic
systems were
determined
by the Abell et al. procedure (7, 196), a
second reference procedure, and these values were then
assigned to the lock-in controls. This value represented
only some 85% of the color generated, which meant that
an arbitrary subtraction of about 15% of the absorbance
of the standard would then be applied to all patients’
samples. This standardization
treatment
leads to several interesting
points to consider. Fifteen percent
subtracted
from any samples, could only roughly approximate the background of irrelevant absorption for
all of them because pathological
samples, unlike controls, are apt to be quite different one from the other in
background absorbance characteristics.
The major interferant
in direct Liebermann-Burchard
reactions,
bilirubin, gives an interference
equivalent
to about
50-60 mg of serum cholesterol per liter for each 10 mg
of serum bilirubin per liter (177). Therefore, 200mg of
bilirubin per liter should cause an absolute error of at
least 1000 mg of serum cholesterol pen liter. But that too
would be corrected partly by the standardization
step
in which an absolute percentage is subtracted from all
samples. As a consequence,
one must use a suggested
(197) corrective equation (bilirubin
10 mg/liter) X 4
= cholesterol
to be subtracted,
in mg/liter. For 200 mg
of bilirubin per liter, one would then subtract 760
mg/liter rather than 1000 mg/liter, and the error would
appear smaller. To compound the confusion, investigators who developed new procedures and compared
their values to those of the automated procedures might
find that results of the compared procedures did not
appear to be very far apart (5,48) even though they were
actually much further apart than they appeared to be
because of the serum correction factor. Uncorrected
jaundiced specimens, if compared, would obviously be
worse if every 100 mg of bilirubin per liter appeared to
be about 500 to 600 mg of cholesterol per liter in the
automated
procedures. This deliberate devaluation of
a standard value in an effort to make results conform
to those believed to be more nearly correct is confusing.
It may be questionable,
in fact, to assign a true value to
a standard in which interfering and variable background
colors, either static or dynamic in nature, are arbitrarily
subtracted from that standard. There is an implied assumption here that all sera, normal or pathological,
must have the same characteristics
as the control with
respect to irrelevant absorbances.
Assuming that the value for control serum can be
established by the Abell et al. procedure, perhaps its
-
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CLINICAL CHEMISTRY,
Vol. 23, No. 7, 1977
true cholesterol value, it was also rather surprising to
find values for a single multiphasic control serum stated
to be 2100 mg/liter for the Liebermann-Burchard
direct
reaction and 1450 mg/liter for the enzymatic procedure,
a difference of about 30%. If the Liebermann-Burchard
reaction uses a value derived from the Abell et al. procedure and the enzymatic procedure gives the same
values as the Abell et al. procedure (Technicon Lot No.
B5J810) it is difficult to understand how the two stated
values could be 30% apart.
Reaction Mechanisms
Undoubtedly,
a main reason why the enzymatic
procedures for cholesterol are more easily accepted than
other techniques is that they are the best understood
in terms of their reaction-sequence
mechanism than are
those methods that are based on either the Fe(III) or
Liebermann-Burchard
reactions.
In the enzymatic
procedures, every step in the entire sequence not only
is clearly understood but the products of each sequence,
including that of the quantifying equilibrium reaction,
have been identified (1-6). Relatively few studies describe the mechanisms
for the Liebermann-Burchard
reactions (198-204) or the Fe(III) reactions (200, 205,
206). This is partly because it is difficult to define
products that are relatively unstable and that defy easy
isolation for characterization.
It is also not clearly understood whether those products that represent the
endpoint
reactions
of Liebermann-Burchard
and
Fe(III) procedures can exist in stable form when isolated
from the milieu in which they are generated.
In the
Liebermann-Burchard
reaction, if one waits until final
color stability is achieved, the peak maximum of the
final product spectrum is at 430 nm. From this finding,
one might infer that the green product most frequently
measured, which peaks at 625 nm, is an unstable intermediate whose structure might be postulated from
that of the compound which it theoretically
precedes
as a final product.
Some attempts have been made to characterize
the
structures
of the final products of the LiebermannBurchard (201, 204) and Fe(III) reactions (200). The
product of an L-B reaction has been isolated and reported (204). A recent quite elegant study on both
mechanisms has been described, in which it was determined that the product of the Fe(III) reaction that absorbs maximally at 563 nm is a cholestatetraenylic
cation and the Liebermann-Burchard
product which
exhibits a peak maximum at 620 nm is a pentaenylic
cation (200). In addition these investigators believe that
both reactions involve similar oxidative mechanisms,
which result in a series of cholestapolyenes.
They also
present evidence that the two colored species are “the
corresponding
enylic carbonium ions of the respective
conjugated polyenes” (200).
Phases of Procedure
Even though the tendency of routine laboratories
is
toward direct determination
of cholesterol as classified
in IA above, mostly avoiding a serum blank, there are
still many reports in which the isolation procedures of
the other analytical classifications
are described. Indeed, two of the reference procedures (7, 10) include
alkaline saponification and extraction while one of them
(10) also uses digitonin for more nearly complete purification before color development.
It is unlikely that all
investigators
will rely on direct systems or that every
sample will necessarily be a uniform aqueous liquid such
as serum, or even that sera are uniform in composition,
including lipid concentrations
and in vivo or in vitro
interferences.
Thus it would seem worthwhile first to
discuss these preliminary phases of the analysis before
discussing the means of final determination,
the endpoints.
Extraction.
The primary reason for extraction is the
partial purification of cholesterol before it is reacted in
the extract (70) or in a residue of the extract (7). In other
procedures this might be the first step in a multi-step
purification scheme. For example, extraction might be
followed by isolation as a precipitated
derivative (10,
114) or by thin-layer
chromatography
(207, 208), column chromatography
(209), or gas chromatography
(210).
Saponification.
Hydrolysis of the cholesterol esters
to convert them to the free form has been resorted to for
several reasons. It would be necessary to saponify the
esters before extraction if the solvent of choice was not
capable of quantitatively
extracting esterified cholesterol (7). Moreover, free cholesterol and its esters may
react at different rates or result in unequal molar absorptivities in the Liebermann-Burchard
reaction (10,
194). If precipitation
by digitonin (10) or tomatine (114,
211) is required for a purification step, or if the turbidity
(or nephelometry)
of the precipitate
is to be what is finally measured (212,213), or if ultraviolet measurement
is the determinative
device (214), then it might be
necessary to saponify the esters of cholesterol.
Endpoint Devices
Most cholesterol procedures result in visible colors
which have useful quantitative
properties in that they
obey Beer’s law over a wide analytical range. In the
modern era, two reactions (12, 20, 21) and the many
modifications of modifications of them have been a part
of the profuse offerings during a relatively short time.
Reviews describing the strengths and shortcomings
of
both color reactions are available (36-40). Other spectrophotometric
procedures
have also been suggested
(11, 31, 215-218)
along with physicochemical
measurements (153, 219-221) that are resourcefully devised
but have yet to achieve popularity.
Of the latter, the
electrometric
approach involving enzymes as reagents
appears quite promising and it will certainly be an
available system to be offered in a compact instrument
for measurement
of peroxide generated by cholesterol
oxidase in sequence with hydrolysis by cholesterol esterase. It will be unnecessary
to add catalase, peroxidase, and color reagents coupled to those enzymes because the procedure can end with either the determi-
nation of the peroxide itself or the rate of oxygen uptake
in the cholesterol oxidase step.
Liebermann-Burchard
reaction.
The LiebermannBurchard reaction has the longest history of common
use of all of the endpoint devices now available (20,21).
Reagent modifications have been suggested which alter
reaction rates or color intensity achieved, by changing
the ratio of acetic anhydride to sulfuric acid or the organic solvent in which the reaction takes place (109,211,
222). In recent years, the stability of the reagent has
been increased from hours (10) to weeks (64) by a
change in reagent makeup. The inclusion of a high
concentration
of Na2SO4 or p -toluenesulfonic
acid (9)
resulted in reagents with longer shelf lives. The stabilities of the several similar reagents proposed popularized them as one-step direct procedures for use with
mechanized instrumentations
(42). However, because
interferences
were difficult to overcome with this type
of method (66, 76, 82) and because of the confidence
engendered by the advent of enzymatic approaches, use
of this reaction is beginning to decline and it soon will
probably disappear entirely. At present, its single advantage over enzymes is cost per test.
Iron reactions.
Modifications
of the original Fe(III)
reagents (12), all of which resulted in more sensitive
color reactions than the Liebermann-Burchard
reactions and with color that was far more stable, enhanced
the use of this reaction. Its sensitivity made it useful at
very low concentration
of cholesterol extracted from
small amounts of tissue (84,223) or with micro amounts
of sample (68), and its fluorescent properties made it
amenable to the determination
of cholesterol in cerebrospinal fluid (224). Although, it is reported to be
markedly affected by bilirubin, the error resulting from
this interference
is small when compared to this error
with the Liebermann-Burchard
reaction (76) and in
fact is smaller than the error from bilirubin in some
cholesterol
oxidase procedures
(176). However, the
strong acids used, the viscosity of the final mix, reported
errors from interferences-some
valid (169, 170, 174)
some not (225, 226)-and
the advent and immediate
acceptance of the enzymatic techniques undoubtedly
will lead to disuse of this reaction in routine laboratories.
Like the Liebermann-Burchard
reaction, its major
advantage is low materials cost, which could keep acid
systems in use for screening procedures.
Enzyme
reagents.
When cholesterol
reacts with
cholesterol
oxidase to generate a cholestenone
and
peroxide it provides several potential reaction possibilities, most of which have been exploited to some extent. The original procedure was quite clumsy and time
consuming
(227) and involved measurement
of the
disappearance
of cholesterol
by use of the Liebermann-Burchard
reaction. Alternatively,
the cholestenone generated by the action of a cholesterol dehydrogenase from a Mycobacterium
was measured at 240
nm, or the 2,4-dinitrophenylhydrazone
of this ketone
was measured at 390 nm (1, 4). Later, Flegg (4) also
measured cholestenone
in the ultraviolet at the wavelength of the peak maximum or alternatively,
the 2,4CLINICAL CHEMISTRY,
Vol. 23. No. 7, 1977
1209
dinitrophenylhydrazone
was formed and measured. It
was in this paper that the author suggested other ways
of measurement
if the reaction by the oxidase proved
to be a peroxide generator. Once it was determined that
peroxide did form, the way was open to treat the procedure in a manner similar to other analytes such as
glucose, uric acid, and galactose, whose oxidase enzymes
also generate peroxide during the reaction with their
analytes. Thus colorimetry, fluorometry,
and electrochemical detection became available for cholesterol,
already having been established for the determination
of the peroxide generated
in other procedures.
The
primary thrust from the inception of available procedures has been to modify from one endpoint device to
another, to subject those procedures
to mechanized
handling, and to study the interferences, many of which
had already been investigated
in the other techniques
involving peroxide generating analytes.
Miscellaneous
other endpoint
reactions.
There has
been a great deal of interest in method development
with respect to cholesterol and this has resulted in reactions for and modifications
of numerous endpoint
devices for the final step of cholesterol
procedures.
Aside from the Fe(III) and Liebermann-Burchard
reactions, which some believe to be variations of the same
reaction (200, 201), there has been a continuous stream
beginning with gravimetric determination
of the digitonide (30) and terminating
for the moment in the enzymatic reagents that have already resulted in a myriad
of variations for determination.
In between, one can find
the Tschugaeff-Bernoulli
reaction (11, 31, 228), turbidimetric or nephelometric
measurements
(212, 213),
carbohydrate
determinations
on the pentasaccharide
chain of saponin derivatives (151, 229), metal reactions
other than iron (126,230,231),
an erythrocyte
hemolysis
technique (232), titrimetry
(233), gasometry (234), oscilloscopic polarography
(229), colorimetry
by a differential thermal effect (125, 235), mass spectrometry
or flame ionization following gas chromatography
(136,
138), unesterified
cholesterol by hemolysis inhibition
by lucensomycin
(236), radioactivity
(153), oxidation
with perchloric
acid-acetyl
chloride in ethylene dichloride (217), ultraviolet
spectrophotometry
(214),
reaction with o-phthalaldehyde
(216, 237, 238), and
oxidative charring (152). This list is by no means complete (39, 40) and other miscellaneous
techniques for
cholesterol exist. However, their use in hospital or research laboratories
is negligible compared to Liebermann-Burchard,
Fe(III), and enzymatic procedures.
Supported in part by a Grant-in
Hospital Research Corporation.
Aid from the Detroit
General
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