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 DOI: 10.1177/000456329202900601

1992 29: 577Ann Clin BiochemW Richmond

Analytical Reviews in Clinical Biochemistry: The Quantitative Analysis of Cholesterol  

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Review Article Ann CUn Biochem 1992; 29: 577-597

Analytical reviews in clinical biochemistry: the quantitativeanalysis of cholesterolW RichmondFrom the Department ofChemical Pathology, St Mary's Hospital, Praed Street, London W21PG, UK

Studies on the occurrence and chemical nature ofcholesterol can be traced'v to the observation,circa 1733, of the partial solubility of gallstones inalcohol! and the subsequent crystallization" of anunsaponifiable substance' from this source. It wasnamed cholesterine by Chevreul" in 1816accordingto its origin [Greek: chole, bile, steros, solid] andfirst identified in human blood in 1838by Lecanu.?Its discovery in atheromatous arteries by Vogelin 18438 was an early indication of the patho­logical significance of this important compound.

Introduction of the name cholesterol andidentification of cholesterol esters in plasmafollowed Berthelot's work (1859)showing 'choles­terine' to be an alcohol.9 The exact empiricalformulae was established by Reinitzer in 1888.10

Elucidation of the chemically reactive portions ofthe molecule was completed by 1904 with theidentification of a double bond!' and demon­stration that the alcohol group was secondary. 12

The colour reactions of cholesterol with strongacids were first noted by Salkowski (1872).13Many variants of this type of reaction werereported and applied empirically to quantitativeanalysis over the next century as will be discussedin detail later.

The immense task of elucidating the structuralformula of cholesterol began circa 1900and took30 years work by the most notable chemists tounravel. Weiland and Windaus received the Nobelprize in 1928 for their brilliant work in this field.X-ray crystallographic studies by Bernal inCambridge (1932)14 indicated however that theWeiland structure gave a molecule of the wrongdimensions. Rosenheim and King working at MillHill used this information and additionalchemical evidence produced by Diels andGadke" to deduce the correct cyclopenta­nophenanthrene structure in 1932.16•17

This review was commissioned by the Analytical MethodsWorking Party of the Scientific Committee of the Associationof Clinical Biochemists. The views expressed are those of theauthor and are not necessarily those of the ScientificCommittee.

Cholesterol was first demonstrated in plasmalipoprotein complexes by Macheboeuf (1929).18Developments in electrophoresis (Tiselius,1941),19 analytical ultracentrifugation (Gofmanet ai, 1949),20 and preparative ultracentri­fugation (Havel et al, 1955)21 demonstrated theexistence of different lipoprotein fractions andenabled their classification according to electro­phoretic mobility and density. Low density lipo­proteins and high density lipoproteins were shownto be the major cholesterol transporting fractions.

By 1950a great deal of evidence had accrued tosupport the hypothesis that serum cholesterol con­centrations tended to be higher in individuals pre­senting with coronary heart disease than in normalsmatched for age, sex, weight, etc.22,23 Furthermore,it had becomeevidentthat atherosclerosis was promi­nent in disease states, notably diabetes" and nep­hrosis,2s accompanied by hypercholesterolaemia.

The relationship between low density lipo­proteins and atherosclerosis had been demon­strated by Gofman using the ultracentrifugationtechniques developed by his group." He claimedthat the correlation between these measurementsand atherosclerosis was much closer than thatbetween blood cholesterol concentrations and thisdisease. The majority opinion of the United StatesNational Heart Advisory Council, following alarge cooperative study completed in 1956, wasthat lipoprotein measurements presented noadvantage over cholesterol analysis in identifyingindividuals prone to develop coronary heartdisease." Cholesterol measurements were thusestablished as an important aspect of aetiologicaland epidemiological studies while lipoproteinstudies would make an important contribution tounderstanding the complexities of cholesteroltransport and lipoprotein metabolism.

THE MEASUREMENT OF TOTAL ANDFREE CHOLESTEROL IN SERUM

The chemical approachThe reaction of cholesterol with strong acid underdehydrating conditions to give coloured products

577

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578 Richmond

has been widely used for the quantitativedetermination of cholesterol. Of the manyvariants of this reaction reported, the Liebermann­Burchard and Zak reactions have found thewidest application in clinical analysis. Theformer was described initially by Liebermanrr?in 1885, and applied to cholesterol analysis shortlyafter by Burchard.P Cholesterol was allowed toreact with sulphuric acid and acetic anhydride inchloroform solvent in the original studies, but theLiebermann-Burchard reaction is now carried outin an acetic acid-sulphuric acid-acetic anhydridemedium. The Zak reaction was first applied tocholesterol analysis by Zlatkis et al. in 195329

and is carried out in an acetic acid-sulphuric acidmedium. In this reaction, however, ferric ionsmust be present to obtain the required colouredspecies as explained below.

Early mechanistic studies on colour productionby the action of acids on cholesterol have beenreviewed by Kritchevesky.f It was suggested thatthe mechanism of the sulphuric acid-initiatedcolour reactions of cholesterol involves an initialdehydration to cholestadiene followed by theformation of coloured sulphonated dimers whichcan undergo further polymerization. Subse­quent studies suggested that a more probablemechanism involved the oxidation of cholesterolto a pentadiene.P More recently, Burke et al.31

have presented evidence for the mechanisms ofthe Zak and Liebermann-Burchard colourreactions proposed in Fig. 1.

According to these proposals, which arebased on the correlation of spectral and massspectrophotometric data with S02 and Fe2+measurements, both reactions have a commoninitial step, i.e. protonation of the 3{j-hydroxygroup in cholesterol and subsequent loss of waterto give the carbonium ion of 3,5-cholestadiene.Serial oxidation of this carbonium ion, by S03in the Liebermann-Burchard reaction and Fe3+

in the Zak reaction, yields the cholestapolyenecarbonium ions shown. Thus it was suggested thatthe red product (>.. max 536 nm) typically mea­sured in the Zak reaction is predominantly thecholestatetraenylic species, and the blue-greenproduct in the Liebermann-Burchard reaction(>.. max 620 nm) is predominantly the pentaenylicspecies. These studies indicate that the colourreactions of cholesterol produce a number ofproducts with varying absorptivities. This is anundesirable situation in quantitative analysis, andindicates the need to use these reactions understrictly controlled conditions. Furthermore, thesereactions are very non-specific and require some

Dienylic ccticn Pentcenylic cctionA mcx- 412 nm A mcx- 620 nm

~••• +FeT2~!..... +S0 2

., + ~ I.- •• SO OH "'" ~

Trienylic cction 2 Cholestchexcene sulphcnicAmox-47Bnm ccid A mcx-410nm

!

,~~Tetrceny lic cctionAmcx- 563nm

FIGURE 1. Proposed mechanisms of the Zak andLiebermann-Burchard reaction. JJ

degree of purification of the analyte before theycan be applied successfully to the quantitativemeasurement of cholesterol in biological tissuesor fluids.

Despite these limitations methods have beenproposed for the determination of cholesterol inserum based on the colour reactions describedabove. In some, colourimetric reactions have beenapplied directly to serum29,32-34 while in otherscholesterol is isolated from serum and purifiedto various degrees prior to colourlmetric measure­ment. In many procedures cholesterol is separatedfrom protein by solvent extraction prior tocolourimetric determination.P<" Alternatively,serum may be subjected to alkaline saponi­fication and the nonsaponifiable material ex­tracted with chloroform or petroleum spirit. 39-41This procedure allows a more complete andspecific extraction of cholesterol, a colourreaction with cholesterol which has all beenconverted to the free form and, in some

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methods, further purification by precipitating theextracted cholesterol with digitonin. 4O,42,43

The method of Abell et al.39 which hasbecome widely accepted as the method ofreference for total cholesterol, involves alkalinesaponification of cholesterol esters, extractionwith petroleum ether, evaporation of a portionof the extract, and determination of cholesterolby the Liebermann-Burchard reaction. Theextraction is based on the partition of cholesterolbetween aqueous alcohol and petroleum spirit andthis reference procedure owes its success to therelatively specific and quantitative recovery ofcholesterol in the non-aqueous phase.

The current reference procedure developed bythe Lipid Standardisation Laboratory of theCenters for Disease Control (CDC) is a modi­fication of the Abell procedure in which hexaneis substituted for petroleum spirit in the sol­vent extraction system.v' It has been demons­trated that, under very rigidly controlled condi­tions, this manual procedure was transferablebetween reference Iaboratories." By comparisonwith the OC/MS definitive method a mean bias,for all 14 laboratories, of less than 31170 wasobtained with a mean within-laboratory CV ofless than 1'51170. The between-laboratory CV wasless than 31170.

In the procedure used in the Lipid ResearchClinic Studies (from which current clinical actionlimits for total cholesterol have been derived)46.47the Liebermann-Burchard reaction was applied,on an AutoAnalyser II system calibrated withfree cholesterol standards, to isopropanol ex­tracts of plasma which had been purified with azeolite mixture.f In the absence of a hydrolysisstep cholesterol esters give a greater chromo­genic response than free cholesterol with theLiebermann-Burchard reagent; consequently,falsely high plasma values are recorded with thisprocedure. It was therefore necessary to apply acorrection factor which was determined byassaying serum control materials with valuesassigned by the CDC reference procedure."Measurement of free cholesterol requires theextraction and separation of free-from-esterifiedcholesterol by precipitatiorr'" or chromato­graphic techniques." In the method of Sperryand Webb,43 long regarded as the referencemethod, free cholesterol is precipitated as thedigitonide from an acetone-ethanol extract ofserum and measured by the Liebermann­Burchard reaction.

The limitations of these chemical methods arewell documented in several major reviews,4O,51-55

Analytical reviews in clinical biochemistry 579

and can largely be attributed to the non-specifityof the reactions employed, their susceptibility tointerferences, the reactivity of their reagents, andtheir critical dependence on a number of variablessuch as temperature, water content and time.These assays are consequently difficult to controland interlaboratory quality control surveys haveindicated poor performance in the field. 54,55 Thedirect methods for total cholesterol are parti­cularly prone to interference from haemoglobinand bilirubin and in many cases give differentcolour yields for cholesterol and cholesterolesters. Saponification and extraction steps aretherefore essential if good accuracy is to beachieved. These factors, together with thehazardous nature of the reagents employed, createdifficulties in the automation of these assays, andconsequently make them unsuitable for use in thegeneral clinical laboratory.

GC, HPLC AND MASS SPECTROMETRY

Gas liquid chromatographyGas liquid chromatography (OLC) has becomea most powerful analytical tool in sterol analysis.Although accurate, specific and capable ofautomation the technique requires preliminaryextraction of analyte into a suitable solvent. It hasnot, therefore, been generally adopted as aroutine procedure for cholesterol measurement.It has been proposed as a reference procedurehowever" and forms the basis of definitiveisotope dilution, mass spectrometric (ID/MS)methods.

Quantitative OLC procedures involve: theaddition of an internal standard to serum whichhas a retention time fairly close to that ofcholesterol; hydrolysis of cholesterol esters andextraction of the total cholesterol; preparation ofa chemical derivative to increase the thermalstability and volatility of the molecule; chroma­tography to resolve cholesterol from othersterols and flame ionization detection. Thederivatization step has been omitted in someprocedures in order to simplify the method.57.58

There are a number of internal standardswhich have been successfully employed. Theseinclude epicoprostanol," ll-ketoprogester­one,59 stigmasterol.W" 5-cholestane61.62 and n­octasane."

The saponification step for hydrolysis ofcholesterol esters has been carried out using eitheralcoholic KOH56-58,63 or tetramethylammoniumhydroxide (TMH).61.62 TMH does not give the

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adverse effect on the columns' that is obtainedwith KOH57 if either is inadvertently injected.

The two most commonly used chemical deri­vatives for the GLC of cholesterol are trifluoro­acetyl esters60,65 and trimethylsilyl ethers. 56,66

The methods of Derks et al. presented as acandidate reference method's minimizes thepossibility of interference from cholestane,reported by Gambert;" by use of efficientcapillary columns and a novel peak integrationtechnique. Rigorous use of volumetric andgravimetric procedures and bracketing standardstechniques" ensure a very precise and accurateprocedure" and a candidate definitive GC/MSprocedure."

Gas chromatography/mass spectrometryThe first isotope dilution gas chromatographymass spectrometry (GC/MS) method for thedetermination of total cholesterol in serum wasdeveloped by Bjorkhem et al.(f) at the KarolinskaInstitutet. Several methods have since beendescribed67,68.70-74 all of which are basically verysimilar analytically. They involve the followingsteps:

(a) The precise addition of isotopically labelledcholesterol, as internal standard, to anaccurately measured sample of the specimen.

(b) Chemical hydrolysis of cholesterol esters.(c) Solvent extraction of the liberated free

cholesterol.(d) Preparation of a volatile derivative of

cholesterol.(e) Isolation by gas chromatography.(f) Mass spectrometric measurement of the ratio

of cholesterol to internal standard using theintensities of an equivalent ion in the spec­trum of each compound.

(g) Computation of the concentration of choles­terol in the sample from calibration curvederived from measurements of the ratios ofthe same ions in standard mixtures ofdifferent amounts of pure cholesterol in afixed amount of internal standard.

Only two of these procedures'<" have met theprecision requirements advocated for a definitivemethod: a coefficient of variation of <0·5OJo.Both these methods use similar bracketingstandard and peak integration techniques. Theydiffer in the type of mass spectrometer used(quadrupole vs magnetic sector); the GC column(fused silica vs packed stainless steel); the internalstandard (l3C vs 2H cholesterol); and the isotope

dilution technique (automatic pipetting vsweighing each aliquot of sample, standard, andinternal standard).

High-performance liquid chromatographyThe introduction of high performance spectro­photometers with low volume flow cells whichcan operate at wavelengths as low as 200 nm,has enabled high performance liquid chromato­graphy (HPLC) to be applied to the deter­mination of total and free cholesterol. A pro­cedure has been described in which reversedphase chromatography on a /LBondapack C18,10 /Lm column is used with detection at 200 nm."The sample for total cholesterol estimation issubjected to alkaline hydrolysis according to theAbell-Kendal procedure." An aliquot of ahexane extract of the hydrolysate is evaporatedto dryness and redissolved in isopropanol beforechromatographic analysis. For free cholesterol anisopropanol extract of serum is used directly.The mobile phase for total cholesterol wasisopropanol-acetonitrile (l: 1, by volume); forfree cholesterol isopropanol-acetonitrile-water(60:30: 10) was used. In this procedure potentiallyinterfering sterols .,17-cholestenol and 7-dehydro­cholesterol co-eluted with cholesterol. Choles­tanol, lacking a double bond does not absorbat 200 nm and consequently is not detected.

HPLC/MSIn a recent report a similar HPLC system has beenapplied to the measurement of total serumcholesterol by isotope dilution/mass spectro­merry." In this procedure 3,4-l3C choles­terol is added to serum prior to the alkalinehydrolysis of cholesterol esters and hexaneextraction. The hexane extract is evaporatedunder a stream of nitrogen and the residuedissolved in methanol. Aliquots of the methanolicsolution are chromatographed on a Finepack SILC18-5 column using acetonitrile-isopropanol (3:1)mobile phase. The cholesterol fraction detectedat 200 nm is collected, evaporated under nitrogenand the residue dissolved in ethanol. For massspectrometric measurement this solution isintroduced directly into the ion source, in agraphite sample tube. Isotope ratios are measuredby multiple ion detection at mass to charge ratios(m/z) of 386 and 388. The CV of this procedurewas less than 10J0. It was considered that thismight be improved with on-line coupling ofHPLC to MS.

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THE ENZYMIC APPROACH

Tbe occurrence and properties ofcbolesterol oxidaseThe ability of soil microorganisms to oxidizecholesterol was first observed in 1913 bySohngen.?" Some 30 years later Tak78 demon­strated that such organisms could be isolatedby the enrichment culture technique. Turfitt''?isolated cholest-4-en-3-one (~4-cholestenone)

from cultures of Nocardia erythropolis grownon cholesterol as carbon source and con­cluded that the initial stage of bacterial attackwas a dehydrogenase catalysed oxidation ofthe 3(3 hydroxyl group accompanied by arearrangement of the double bond from the~5 to the 4 position. Subsequent reports indicatedthat although different pathways are evident innumerous organisms with the ability to degradecholesterol8o-86 the oxidation of cholesterol to~4-cholestenone is a common initial step.

Stadtrnan'" demonstrated that when a Myco­bacterium'" was grown on glycerol as solecarbon source, the cells were able to effectthis conversion quantitatively without furtherdegradation of ~4-cholestenone.The 'dehydro­genase' catalyzing this reaction was extractedafter grinding the cells with solid carbondioxide or alumina, and partially purified byammonium sulphate precipitation. The low yieldsof soluble enzyme obtained were attributed to theresistant nature of the cell wall. Conversion ofcholesterol to ~4-cholestenone by these pre­parations was quantitated by (a) measure­ment of cholesterol dissappearance with theLiebermann-Burchard reaction, or (b) ~-4

cholestenone production using its UV absorptionat 240 nm or its 2,4-dinitrophenolhydrazonederivative." Though the mechanism of thereaction was not elucidated, a minimal loss ofactivity on dialysis which could not be restoredby the addition of NAD+ or NADP effectivelyruled out the involvement of loosely boundnicotinamide coenzymes.

Flegg,88 using a similar approach, isolated andpartially purified a similar enzyme from Nocardiaerythropolis (grown on cholesterol) by ultrasonicdisruption of the cells and ammonium sulphatefractionation of the cell free extract. Thispreparation was applied to the estimation of totalcholesterol in serum. The procedure involvedalkaline hydrolysis of cholesterol esters in anethanol:dioxane extract of serum, enzymicoxidation of a portion of the extract inbuffered aqueous medium, measurement of the

Analytical reviews in clinical biochemistry 581

Cholesterol Cholest-4- en- 3 one

FIGURE 2. The reaction catalysed by cholesterol oxi­dase,

~4-cholestenone produced by its UV absorptionin a further isopropanol extract. Blanks werenecessary and incubation times of up to 2 h wererequired.

Richmond89-91 demonstrated that the choles­terol oxidizing enzyme from another Nocardiaspecies (NCIB 10554, National Collection ofIndustrial Bacteria, Torry Research Station,Aberdeen, Scotland) used oxygen as hydrogenacceptor producing one mole of hydrogenperoxide for each mole of cholesterol oxidized(Fig. 2). Furthermore the enzyme was shownto be situated on the surface membrane ofthe organism and could be selectively removedby extraction with detergent. No lysis of thebacterium occurred during this process thusproviding an enzyme of high specific activitywhich could be adequately purified in a single stepby an ion exchange chromatography. This novelprocess enabled the economic large scaleproduction of enzyme. 90 ,92 The enzyme wasstable, active over a wide pH range, exhibitedfavourable kinetics and specificity. Thus for thefirst time practicable options for the enzymicmeasurement of serum cholesterol based onhydrogen peroxide detection were available.

Cholesterol oxidases were also demonstrated inStreptomyces violascens'" and Brevibacteriumsterolicum.?' These enzymes were secreted intothe culture medium. After extensive purificationthey were also shown to be oxygen; oxido­reductases producing hydrogen peroxide.95,96

The Streptomyces enzyme, like those fromNocardia species, does not appear to requireexternal electron acceptors other than molecularoxygen. The cholesterol oxidase from Brevi­bacterium, in contrast, is a flavoprotein. Thisenzyme has also been used in serum cholesterolassay reagents. 97,98

The enzymes described are active over a widepH range (3' 5-9' 0) with maximal activity atpH 7·0 in phosphate buffer. The optimumtemperature is 50°C for the Streptomyces,Brevibacterium and Nocardia (NCIB 10554)91,99

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enzymes and 32 DC for the Nocardia erythropolisenzyme.99,HJO All exhibit considerable thermalstability. The reported Michaelis constants (Km)for cholesterol are 1 X 10-6, 7 X 10-6 and14 X 10-6 the Nocardia enzymes 90,10l,102 and4·5-6·7x 10- 4 for the Streptomyces enzyme."

The specificities of cholesterol oxidases havebeen extensively reviewed. 99,I03 In summary, theNocardia and Brevibacterium enzymes are similarin their relative affinities for sterols and specificfor the 3{:J orientation of the hydroxyl group: 3 X

sterols are not oxidized. Saturated sterols and{:J sitosterol, the plant sterol, are oxidized atreduced rates relative to cholesterol. Activitydecreases as the length of the side chain on carbon17 of the D ring decreases.

In contrast the Streptomyces enzyme is notinfluenced to the same extent by side chain length.Unlike other 3a hydroxy compounds, androst­4-ene-3a, 17{:J diol was oxidised by the enzyme.This reactivity was attributed to the uniquesteric configuration of this molecule.

A number of 3{:J sterols have been demonstratedin normal serum.'?' These include 5a-cholestan­3{:J-ol (cholestanol), 5a-cholest-7-ene-3{:J-ol (t:.7­cholestenol), and cholesta-5,7-dien-3{:J-ol (7­dehydrocholesterol). Although the latter hasbeen reported to be present in normal sera atconcentrations of up to 1'0 mmol/Lv'P' thisestimate has been shown to be artifactual andthe available evidence suggests that these non­cholesterol sterols amount to less than 10J0 of thetotal sterols in human plasma. 106 Although upto 250 mg of plant sterols, predominantly {:Jsitosterol, may be consumed daily in the typicaldiet'P? absorption is poor and plasma concen­trations are typically less than 25 /Lmol/L,108except in very rare cases of {:J sistosterolaemia.l'"It can therefore be concluded that the con­centrations of potentially interfering sterols areinsignificant and the specificity of cholesteroloxidase is adequate for the analysis of choles­terol in serum.

The occurrence and properties ofcholesterol esteraseCholesterol esterase occurs in many mamma­lian tissues!'? and has been isolated and wellcharacterized from porcine pancreas. I11 The en­zyme has also been isolated from a numberof microorganisms including Nocardia restric­tus,1l2 Pseudomonas fluorescens.!'! Fusariumoxysporurn.!" and Streptomyces lavendulae!'!and an unspecified species.!" The microbial andpancreatic enzymes exhibit interesting differences

in stability, bile salt activation and substratespecificity which are of importance in theiranalytical application.

The pancreatic enzyme is labile to heat,proteolysis and pH. The microbial enzymeis active over a wide pH range and morethermostable.!" The pancreatic enzyme requiresthe presence of trihydroxy bile salts to form afully active hexamer. llO,III,117,118 Polymerizationalso confers protection against proteolytic, pHand sulphydryl inactivation.I'Z''!'? Bile saltconcentration can affect the direction of thereaction: a high concentration favours hydrolysiswhile a lower concentration favours esteri­fication.P"

Microbial esterases have no absolute require­ment for bile salt and are active in the pre­sence of non-ionic detergents. I16,121 However,the Pseudomonas enzyme is stimulated by thefurther addition of bile salt!" and sodiumcholate is included in the effective reagentformulation for a microbial enzyme of unspeci­fied origin. 122

With the pancreatic esterase, rates of hydrolysisof saturated esters of cholesterol decrease withincreasing chain length. 123 The reverse is true ofmicrobial esterases. They consequently show littleactivity against cholesterol acetate.

The activity of pancreatic esterase on un­saturated esters of a given chain length in­creases with the degree of unsaturation.F' Thesituation is less clear with microbial esterases.Although some data indicate a direct relationshipbetween activity and degree of unsaturatlon,'!'it is evident that this varies between organisms andwith reaction conditions.PrP" Of particularnote is the inability of certain preparationsto achieve rapid or complete hydrolysis ofcholesterol arachidonate.P'-!"

Analytical applications of cholesteroloxidase and esteraseThe introduction of enzymes as reagents intocholesterol methodology was timely in view of theincreasing awareness of the importance of serumcholesterol as a risk factor for coronary heartdisease and the difficulties in automating thechemical methods hitherto available. Manylaboratory benches and instruments bo~e the scarsof these highly corrosive and dangerous reagents.The availability of cholesterol oxidase imme­diately placed the experience gained with otheroxidases, notably glucose oxidase, 127 at theanalyst's disposal. A multiplicity of reagentsevolved based on hydrogen peroxide measurement.

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Additionally some investigators pursued themeasurement of d4-cholestenone and others theconsumption of oxygen during the reaction.

In the earliest applications of cholesteroloxidase in the measurement of serum totalcholesterol Richmond employed an alkalineethanolic hydrolysing reagent to release choles­terol from lipo-proteins and hydrolyse its estersto the free form. The liberated cholesterolwas held in aqueous micellar solution withsurfactant for enzymic oxidation. 90,128 Theintroduction of cholesterol esterases quicklyfollowed and the first totally enzymic reagentsystemincorporating the hydrolytic, oxidative andindicator reactions in a single reagent wasdeveloped by Allain et al.129 A number ofproblems were encountered with both chemicaland enzymic hydrolysis and these are nowdiscussed.

Problems associated with alkaline hydrolysisA wide range of conditions have been employedwhich achieve complete hydrolysis of cholesterolesters. In the method of Abell39 one part ofserum was treated with 10 parts of ethanolicpotassium hydroxide (0'35 mol/L) at 37-40 °Cfor 55 min. In order to reduce this reactiontime to 5 min, Richmond used more severeconditions.!" One part of serum was treatedwith five parts of ethanolic potassium hydroxide(1'0 mol/L) at 75°C. Under these conditions theproduction of interfering peroxides was avoidedand proteins were sufficiently denatured toremain in solution on aqueous neutralizationof the hydrolysate. Reducing substances, sub­sequently confirmed to be thiol cornpounds.P!were generated however. and removed by reactionwith mercuric ions90 or iodoacetamide.P" In acareful study comparing a similar chemical systemwith enzymic hydrolysis, it was demonstrated thatremoval of interfering thiols by a variety ofalkylating and oxidizing reagents, includingiodoacetamide, was incomplete. 123 This resultedin low recoveries of cholesterol when hydrogenperoxide was measured with the Trinder chromo­gen.P? Chemical hydrolysis did have the advan­tage however of eliminating interference byascorbic acid and reducing that of bilirubin to anegligible extent.

Problems associated with enzymic hydrolysisDeacon and Dawson.F' using a human serumpool, established the optimum concentration ofsodium cholate (8'Ommol/L) and Triton X-loo(6'0 giL) for pancreatic cholesterol esterase

Analytical reviews in clinical biochemistry 583

activity. When these conditions were applied tothe analysis of individual sera, some samplesproduced spuriously low results. These could notbe raised by increasing the reaction time or byincreasing the amount of cholesterol esterase usedin the assay. A detailed investigation of one ofthese atypical sera revealed that a 30070 increasein recovery could be obtained at sodium cholate(6'Ommol/L) and Triton X-loo (15'Og/L)concentrations that were not optimal for choles­terol esterase activity. It was concluded thatthese concentrations of surfactant were necessaryto achieve complete disruption of lipoproteins andrelease of cholesterol and its esters into micellarsolution. The authors suggested that this was themost critical factor in the enzymic hydrolysissystem.

Enzyme specificity studies with substratessolubilized in detergent require cautious inter­pretation because of the uncertain accessibilityof the enzyme to such substrates. None the less,the available evidence suggests that, in con­trast to the pancreatic enzyme, esterases ofmicrobial origin have low activity with cholesterolacetate and cholesterol arachidonate. Althoughcholesterol acetate does not occur in human serumit is sometimes incorporated into control sera.Cholesterol arachidonate occurs to a small butsignificant extent in human serum and is the mostcommon ester in rat serum. It has been suggestedthat this apparent difference in substratespecificity may account for the negative bias givenby certain reagent systems containing microbialcholesterol esterases.P'!" Alternatively it isquite possible that substrate accessibility isa limiting factor for complete hydrolysis ofcholesterol esters in these formulations. Amicrobial esterase (origin not specified) has beendemonstrated to achieve complete hydrolysis ofserum cholesterol esters in a reagent containingother lipolytic enzymes and 3,4-dichloro­phenol. 122 The latter ingredients may assist inthe rupture of lipoproteins and, together withoptimal concentrations of effective surfactants,buffers and activating cations,12.126 enablecomplete hydrolysis of cholesterol esters.

In summary, chemical hydrolysis of cholesterolesters is complete but necessitates additionalprocedural steps for neutralization of the hydro­lysate and removal of interfering thiols ifhydrogen peroxide detection is to be used as anend point. Measurement of total cholesterol withenzymic hydrolysis can be achieved, on the otherhand, in a single combined reagent. The sourceof the esterase and the reagent formulation are

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critical however, for complete hydrolysis of estersoccurring in plasma and control sera.

ANALYTICAL OPTIONS OF THECHOLESTEROL OXIDASE REACTION

Direct measurement of the extent of this reac­tion can be achieved either by polarographicmeasurement of oxygen consumption or spectro­photometric measurement (at 240 nm) of .:14­cholestenone production. Alternatively coupledindicator reactions can be used for the colouri­metric determination of hydrogen peroxide pro­duction.

Measurement of oxygenNoma and Nakayama!" were among the firstto use an oxygen electrode to measure freecholesterol with a cholesterol oxidase/surfactantreagent. Total cholesterol was measured by theinclusion of a microbial cholesterol esterase in thereagent system. Sodium azide was included toprevent interference from catalase contaminationof the reagents. Total oxygen consumption wasmeasured preferentially as rate measurementswere influenced by variations in the oxygencontent of the reagents, and required moreenzyme in the reagent. The assay took 2 min persample was linear to 15' 5 mmol/L and insensitiveto bilirubin and ascorbate interference.

UV measurement of .:14-cholestenoneThis procedure was first used by Flegg88 andRoschlau et al. m to measure serum cholesterolas described earlier. Trinder 'P refined the pro­cedure with the incorporation of a quantitativeextraction procedure and the use of surfactantsthat had low absorbances at the measurementwavelength (232 nm). The assay is a single tubeprocedure and involves hydrolysis of cholesterolester in 20 JLL plasma with 200 JLL ethanolic KOHfor 2 min at 100 "C, The mixture is cooled to50 0 e and incubated at this temperature for15 min with 2·0 mL buffered enzyme reagentcontaining cholesterol oxidase and surfactant.Ethanolic KOH (1. 8 mL) is added followedby 5' 0 mL iso-octane. The .:14-cholestenoneproduced is then extracted into the iso-octane andmeasured by its absorbance at 232 nm. The yieldof .:14-cholestenone is quantitative. Plasma blanks(prepared by omitting cholesterol oxidase fromthe enzyme reagent) are negligible.

This approach has the advantage of specificity.Sterols which are oxidized by cholesterol oxidasebut which do not give a conjugated UV absorbing

product (e.g. cholestanol) will not interfere.Similarly, interference, from sterols with oxi­dative products with lower molar absorptivitythan .:14-cholestenone (.:15-cholestenol, 7-de­hydrocholesterol), will be reduced commen­surately.

This method is also free from interference fromhaemoglobin, bilirubin and ascorbic acid. It is notamenable to automation but merits considerationas an elegant and simple reference procedure.There is also potential for further refinementof this procedure with measurement of .:14­cholestenone in the iso-octane extracts byHPLC.

Measurement of hydrogen peroxideNon-enzymic methodsRichmond'? measured hydrogen peroxide bychelation with Ti(lV) and xylenol orange. 133, 134

This indicator reaction is specific and free ofspectral or chemical interference from bilirubin.It is limited, however, by the low pH of thereaction which precluded its combination with thecholesterol oxidase component of the reagentsystem.

Papastathopoulos and Rechnitz!" appliednon-selective electrode technology using themolybdenum (VI) catalysed reaction of peroxideand iodide:

This procedure is also limited because of the needto acidify the indicator reaction. It was performedon a continuous flow analyser but throughputwas very low (20 samples/h). This reaction hasalso been applied colourimetrically.!"

ENZYMIC METHODS

The Hantzsch reaction (shown below) was appliedto cholesterol analysis by Roschlau et al. 1I 6

HP2 + Methanol--+CATALASE....Formaldehyde + H20

Formaldehyde + Acetylacetone + Ammonia....Dihydro­lutidine Derivative +3H20

The dihydrolutidine derivative has both absorp­tion and fluorescence characteristics, How­ever, the non-enzymic condensation reactionis slow and serum blanking is recommended. Theprocedure is reported to be very insensitive tointerference'J' and has been automated in thecolourimetric!" and fluorimetric'P modes. Afaster kinetic version of the colourimetric procedure

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for a centrifugal analyser has been described. 140

The method was free from interference frombilirubin and haemolysis but was subject topositive interference from ascorbic acid andnegative interference in lipaemic samples.

Haekel and Perlick!" described a similarreaction in which ethanol is oxidized in a reac­tion with hydrogen peroxide and catalase. Theacetaldehyde serves as substrate for the aldehydedehydrogenase indicator reaction in which theconversion of NAD(P)+ to NAD(P)H is moni­tored at 340 nm. Calculations of serum choles­terol concentrations were based on the molarabsorptivity of the reduced pyridine nucleotide.The reaction was faster than the Hantzschreaction but required serum blanks.

Huang et al.142 described a procedure in whichhydrogen peroxide is measured fluorimetricallyin the peroxidase catalysed oxidation of homo­vanillic acid to a highly fluorescent product.

The peroxidase catalysed oxidative couplingof 4-aminophenazone and phenol to form aquinonemine, the Trinder reaction.P? was usedin the first totally enzymic cholesterol assay129and has emerged as the most popular chromogen:

2H202 + 4-aminophenazone + phenol-sPEROXIDASE-+4-(p-benzoquinone-mono-imino)­

phenazone +4H20

The reaction is fast, does not require serum blanksand compares favourably with other chromogensin terms of sensitivity, solubility and non­inhibition of the cholesterol oxidase and esterasereactions.

In developing a kinetic procedure using theTrinder chromogen Deeg and Ziegenhorn143found the Km of cholesterol oxidase fromNocardia erythropolis to be too low to achievefirst order kinetics over an acceptable choles­terol concentration range. Competitve inhibitors,which could have been used to elevate theapparent Km of the enzyme, could not be found.A cholesterol oxidase from a Streptomyces specieswas identified however which exhibited suitablekinetics on inhibition with 3,4-dichlorophenol.Fortuitously this substituted phenol was alreadyincorporated, at a suitable concentration, as acomponent of the detergent system in a totallyenzymic cholesterol end point assay reagent. 122This system incorporated the cholesterol oxidasefrom Nocardia, a microbial esterase, and hadbeen optimized for complete hydrolysis ofcholesterol esters in human serum. Substitutionof the cholesterol oxidase from Nocardia in

Analytical reviews in clinical biochemistry 585

this reagent system with the Streptomyces enzymewas therefore all that was required to satisfy theconditions for the kinetic assay: The cholesteroloxidase reaction became rate limiting and theindicator reaction followed pseudo first orderkinetics with respect to cholesterol. A newprimary cholesterol standard (Preciset cholesterolhigh performance) was developed which had thesame reaction rate constant as serum andtherefore could be used as calibrator for thekinetic assay. The procedure was reported tobe free from interference from bilirubin,haemoglobin and lipaemia and less subject todrug and ascorbate interference than the parentendpoint assay. Serum samples containing ascor­bic acid at I· 0 giL gave results 260/0 lowerin the end point method.l"

The Trinder reaction has found a furtherapplication in the radiative energy attenuation(REA) technology developed by Abbot Diag­nostics for use on their microprocessor con­trolled automated fluorimeters (TDx analysers).In this system" the absorbance of the quino­neimine dye produced in the Trinder reaction(A max 505 nm) overlaps both the excitationand emission spectrum of a fluorescent indi­cator added to the reaction. The fluorescenceintensity measured in the system consequentlydecreases logarithmically with the increasingconcentration of quinoneimine dye and thus bearsan inverse relationship to the cholesterolconcentration of the sample. The system isfree from interference from lipaemia. Bilirubininterference has been demonstrated and methyl­dopa interference can be anticipated.P'

DRY CHEMISTRY SYSTEMS

Dry chemistry systems have been developed for'desk top' analysers intended for use in extra­laboratory situations. The principles of thesesystems have been reviewed14S.146 and their per­formance with respect to cholesterol measure­ment evaluated. The available systems are basedon totally enzymic cholesterol reagents withperoxidase linked chromogens. The reagentcomponents are partitioned in dry form inspecially designed matrices. Reactions are carriedout within this matrix in the aqueous medium ofthe applied sample. The colour developed ismeasured at end point by reflectance photometry.

The Boehringer Reflotron incorporates a cellseparation step, requires 30/olL capillary bloodand uses a Trinder-type chromogen the precisechemical nature of which is not disclosed.

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The Kodak Ektachem DT60 requires 10 p.Lserum/plasma and uses the peroxidase catalysedoxidation of a triarylimidazole leuco dye forcolour development. The Ames Seralyzer requires20 p.L of ninefold diluted serum or plasma. Theindicator reaction in this case is the oxidativecoupling of 3-methyl-2-benzothiazolinone andprimaquine diphosphate.

Reports on the performance of these sys­temsl47-1s0 have favoured the convenience ofworking with whole blood on the Reflotron butquestion the accuracy of this instrument, anegative bias being evident at cholesterolconcentrations in excess of 7· 5 mmollL. It wasconcluded that this instrument could satisfactorilybe used to identify individuals with cholesterolvalues greater than 7· 0 mmollL for referral fortreatment and further monitoring by laboratoryanalyses.

An evaluation of the Seralyzer for cholesterolassay':" has demonstrated a positive bias at6· 5 mmollL and suggested that the precisionattainable by junior medical staff with thisinstrument was not adequate. A subsequent studyfound that acceptable accuracy and precisioncould be obtained by trained laboratory staff."?

Acceptable bias and precision has been demon­strated into two evaluations of the EktachemDT60. 149,ls0 These instruments can be recali­brated using correlation data to give resultsidentical to those obtained with the appliedreference method.

ANALYTICAL CONSIDERATIONS

Totally enzymic peroxidase-linked assays are nowused universally for routine measurement ofcholesterol. A large number of reagent kits areavailable and are applied to a wide range ofautomated analysers which place differentconstraints on the methodology. It is impor­tant therefore to consider factors which cancompromise the accuracy of these systems.

Completion of cholesterol ester hydrolysisAs discussed above the source of esterase and thenature and concentration of the detergent systemare critical for the complete hydrolysis ofcholesterol esters. It is necessary therefore to usethe reagent at the concentration indicated by themanufacturer.

Interference from sample turbidityComplete disruption of lipoproteins is a pre­requisite for the quantitative analysis of their

cholesterol content. This is accomplished duringthe course of the reaction by the action ofsurfactant and lipolytic enzymes as discussedabove. Consequently, turbidity conferred bytriglyceride-rich lipoproteins at the initiation ofthe reaction will have been completely abolishedat the end point. If then, as in some analysers,an initial sample blank reading is taken imme­diately after mixing sample and reagent andsubtracted from the final absorbance after colourdevelopment, the absorbance change will beunderestimated by the turbidity component of theinitial blank reading.

No such interference will occur if a reagentblank is used unless, of course, turbidity is notcompletely dispersed by the reagent system duringthe course of the reaction. In this case positiveinterference would result. Similar errors will occurin kinetic procedures unless the turbidity iscompletely dispersed before the reaction rate ismeasured.

Detergent interactionsInterference has been reported with the useof Carbowax (polyethylene glycol 6000) assurfactant in one reagent formulation due topartial precipitation of apoB containing lipo­proteins. 121,126 The resulting turbidity causesoverestimation of cholesterol values. Similardevelopment of turbidity resulting in a positivebias has been noted in other forrnulations.!"

Bilirubin interfaceThe absorbance spectrum of bilirubin is such that350/0 of its maximal absorbance (at 475 nm) ispresent at 500 nm-the absorbance maximum ofmost Trinder chromogens. The extent anddirection of spectral interference by bilirubin atthis wavelength is determined, however, by theeffects of its chemical interference and theblanking mode employed.

Bilirubin is thought to react chemically with anintermediate in the peroxidase catalysed reactionthereby diminishing the amount of chromophoreformed. In addition bilirubin is oxidized tobiliverdinIS1 during the reaction with a corres­ponding reduction in absorbance at the mea­suring wavelength. Consequently, when reagentblanking is employed spectral interference isreduced as the bilirubin destroyed during thereaction does not contribute to the final absor­bance. Furthermore, the absorbance of any resi­dual bilirubin will commensurately offset thereduced chromophore formation resulting fromchemical interference.

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If sample blanking is used, the bilirubincontribution to the blank absorbance, which hasbeen removed during this reaction, will beerroneously subtracted from the final absorbance.The resulting negative spectral interference willtherefore exaggerate the negative chemical inter­ference from bilirubin. 152

Spectral interference can be reduced by mea­surement off peak at slightly higher wavelengths(520nm) where bilirubin absorbance is minimaland adequate chromogen sensitivity remains.Thus reagent blanking and absorbance measure­ment at 520 nm are recommended.

Chemical interference from bilirubin can bereduced by stabilizing the reacting intermediatewith ferrocyanide.Pl-P' Alternatively, the samplecan be pretreated with bilirubin oxidase to removebilirubin!" and so eliminate both chemical andspectral interference. It is then necessary, how­ever, to incorporate azide in the reagent to inhibitthe subsequent 'chromogen oxidase' activity ofbilirubin oxidase on the peroxidase chromogensystem. ISS, l S6 .

Although bilirubin reacts chemically with mostperoxidase linked chromogens1S1,154. IS7 the KodakEktachem triarylimidazole reagent and the DupontACA system in which an aniline derivative, ratherthan phenol, is oxidatively coupled with amino­phenazone appear to be exceptions to this rule.P'

HAEMOGLOBIN INTERFERENCE

The oxyhaemoglobin spectrum has the majorsoret peak at 410 nm and dual peaks at 547 and575 nm. A trough occurs at 500-520 nm andspectral interference is acceptably low if reagentblanking is employed and measurements are madeat 520nm.

Bichromatic and polychromatic analysisBichromatic analysis, while correcting for varia­tions in the optical characteristics of reactioncuvettes and detector sensitivity, has limitationswhen applied to the correction of spectralinterference or turbidity in the sample. It assumes,for example:

(a) The absorbance caused by each interferent isthe same at blanking (XI) and measuring (X:z)wavelengths.

(b) The relative absorbance between Al and A2 isthe same for calibrants and samples withoutinterferents.

(c) Any absorbance of the chromogen at Alfollows the Beer-Lambert Law.

Analytical reviews in clinical biochemistry 587

Blank correction by complex algorithm followingpolychromatic measurement on a diluted sampleis invalid if the interferent changes during thecourse of the reaction as described above in thecase of lipaemic and icteric sera.

CALmRATION OF ENZYMICPROCEDURES

A potential source of inaccuracy in cholesterolmeasurement is the common practise of usingcommercial control sera as calibrants. Some ofthese materials are very turbid while others ofanimal origin can be highly pigmented withcarotene. They are in some cases 'spiked' withcholesterol esters and stabilizers not found inhuman serum. Target values assigned to thesematerials are often 'consensus' values derivedfrom analyses performed in different laboratories.In view of the analytical considerations discussedearlier it is not surprising to find wide 'acceptableranges' quoted which reflect the wide responsegiven by these materials in different analyti­cal systems used to determine the 'targetvalue' .

Occasionally these materials are assigned valuesby reference or definitive methods. It is evident,however, that matrix changes introduced duringthe manufacture of lyophilized or frozen refer­ence sera can modify their response to varyingdegrees in different enzymic reagent systems. ISS

Consequently, they cannot be used with com­plete reliability to assess or eliminate bias.

This problem has recently been acknowledgedby the Laboratory Standardisation Panel of theUnited States National Cholesterol EducationProgramme as a potential obstacle to improvingaccuracy with the use of their certified serumbased reference materials.!" It is evident that theonly reliable means of assessing the accuracy ofcalibration of an enzymic system is by directcomparison of results on fresh patients' serawith that obtained by the reference method.Manufacturers have consequently been recom­mended to assign values to their calibratingmaterials by this route for the analytical systemsto which they are to be applied."?

A national reference laboratory network hasbeen established in the United Statesl S9 to pro­vide manufacturers and clinical laboratorieswith the resources to establish the accuracy oftheir analytical systems in this manner. No similarreference laboratory network is available inthe UK and reliance is placed on the attainmentof acceptable performance in external quality

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assurance schemes. Although it has beendemonstrated that the all-method means obtainedfor serum pools used in these schemes are likelyto be within 11170 of the true value,l60,161 thesematerials are also subject to matrix effects, andattainment of the correct value on a given pooldoes not guarantee that results obtained on freshsera with a particular system will be entirelywithout bias.

Reference sera prepared by procedures deve­loped to minimize the production of matrixeffects 162 are currently the most reliable methodsavailable for checking accuracy in the absence ofaccess to reference methods for comparisons onfresh sera. They can be obtained from the Collegeof American Pathologists, 5202 Old OrchardRoad, Skokie, IL 60077, USA.

A number of free cholesterol standards pre­pared from NBS certified cholesterol dissolvedin aqueous detergent solutions are availablecommercially. Although it is attractive to considerthese as primary standards in automated systems,care must be taken that they have the samesampling characteristics as serum and are com­patible with the reagent formulations to whichthey are applied. The author has found BoehringerHigh Performance aqueous cholesterol standard­ised (Catalogue No. 125504) to give veryacceptable results when used with BoehringerCHaD-PAP reagent on an olympus AU 5022ANALYSER: Bias < 1'5% on College ofAmerican Pathologists survey pools and meanbiases of - I . 3% and + O·23% with respect tothe all-method mean on the UKEQAS andWellcome QC schemes, respectively. When suchmaterials can be shown to give accurate cali­bration with a particular reagent/instrumentcombination they have great advantages ofstability, batch to batch consistancy, andeconomy. They are not universally applicable,however, and accuracy checks should be made,ideally by comparing results on fresh sera withthose obtained by a reference method or byanother enzymic system whose accuracy has beenvalidated.

Free cholesterol calibrants have a furtheradvantage in that when used together with aserum-based control material the chromogenicresponse of the former can be monitored tocheck the patency of the cholesterol oxidaseand indicator reactions while the relativeresponse of the control provides an index ofthe efficiency of the hydrolytic reaction andrecovery of cholesterol from lipoprotein com­plexes.

ANALYTICAL GOALS

AccuracyA number of epidemiological studies163-165 haveconfirmed that plasma cholesterol concentrationsare significantly correlated with the prevalence ofcoronary heart disease (CHD). Two majorprospective studies-the Multiple Risk FactorIntervention Triall66,167 in the United States andthe 10 year follow up of the Whitehall Study168in London have demonstrated a continuousand curvilinear relationship between plasmacholesterol concentrations and CHD mortalityover the entire plasma cholesterol distribution. Amodest increase in risk occurs from the lowestconcentrations up to 5·2 mmollL. Risk isdoubled at 6· 5 mmollL and quadrupled at7' 8 mmollL. Intervention studies have revealedthat cholesterol lowering by a combination ofdrug and diet reduce the incidence of CHDproportionately's-I'" and can retard developmentof atheroma and indeed initiate regression in somecases. 170

Attention has focused on these statistics andconsensus policy statements have been publishedby American,'?' European'P and British'P studygroups to provide strategies for the prevention ofCHD. These guidelines define cholesterol levelsto assign risk, initiate appropriate therapy, andset therapeutic goals. With this approach accuracyof cholesterol measurement has become ofincreasing importance. The recommended actionlimits . are based on Lipid Research Clinicmeasurements'" the accuracy of which is trace­able to the CDC reference method." It followsthe cholesterol measurements should comparewell with the reference procedure.

It is recommended that method bias should notexceed ± 5% and ultimately a national goal of< 3% should be achieved. 159,174 In order toachieve these goals the development, and uni­versal application, of stable calibration materialswith acceptable matrix specifications is urgentlyrequired.

PRECISION

The interlaboratory precision attained in ex­ternal quality control schemes has improvedconsiderably over the last 15 years as theproportion of laboratories using enzymic methodshas increased. In the College of AmericanPathologists'I" and United Kingdom ExternalQuality Assurance Schemes!" schemes aninterlaboratory precision of approximately 6% is

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Analytical reviews in clinical biochemistry 589

CVAnalytial CV TOIaI SSD (OJ.)

0 5'50 15'4I 5'58 15'52 5'85 16'43 6'26 17'54 6·80 19·05 7'47 20·7

be able to detect a clinically significant change of5OJo in response to therapy. The above dataillustrate, however, that the magnitude ofbiological variation is such that cholesterol statusor response to therapy cannot be determinedaccurately on a single measurement even at thelowest levels of analytical imprecision. Therecommendation for CVA to be one half CVB isconsequently a reasonable analytical goal.

TABLE I. C V,Ol'" and SSD calculated at different levelsof analytical imprecision and assuming a biologicalvariation of 5.5'"

8+20

~ +10 i!

7 c

~

I0 j

6 ti-10 2

!-20

I 2 3 4 !> ev.!>-6 !>9 63 68 7-4 tv,

Coeffoclent01\IOnotlOll ("Yo)

FIGl'RI' 3. The relative contributions of analyticalICV,) and total ICV,) imprecision to the range ofresults 1+1 SD) that would beexpected from sequentialmeasurements ofcholesterol in an individual with a truemean value of 5·5 mmol/L, Biological variation of5·5% is assumed. ~ = Total imprecision l±lSD);Ell = analytical imprecision I:!: 1 SD).

currently achieved. A mean intralaboratory CVof 3· 5OJo has been recorded l14 ,175 and it has beendemonstrated that an analytical CV of < IOJois attainable in carefully controlled specialistlaboratories.!" A target analytical CV of ~ 3OJohas been recommended for routine labora­tories.!" This is in accord with proposals thatthe analytical CV should be ~ to one halfof the intraindividual biological CVI77-I80 whichhas been found to be approximately 5· 5OJo fortotal cholesterol in human subjects.I80.ISI

Figure 3 shows the range of results, within 95OJoconfidence limits, that would be expected fromsequential estimations at monthly intervals!" ina patient with a true mean cholesterol value of6' 5 mmol/L. The relative contributions of ana­lytical imprecision (CVA)' from 0-5OJo, andtotal imprecision (CVT) to the overall distri­bution of results have been calculated assumingintraindividual biological variation (CVB ) of5·5OJo.

Similarly, the smallest significant difference(SSO) between two sequential measurernents'Pcan be calculated as follows:

cvr=J(CVA)l + (CVB)l

SSD (OJ.)= ±2'8XCVr

The SSOs at varying degrees of analyticalimprecision and assuming intraindividual bio­logical variation of 5, 5OJo are listed in Table I.

Broughton and Buckley!" have suggested atarget of IOJo analytical imprecision in order to

CHOLESTEROL MEASUREMENT INLIPOPROTEIN FRACTIONS

The predictive value of total cholesterol measure­ments is limited by the fact that while LOLis atherogenic HOL, especially HOL2, appearsto be cardioprotective. Attention has there­fore focused on the use of total/HOL andLOL/HOL cholesterol ratios for risk assess­ment l83 and has stimulated interest in the deve­lopment of simple procedures for the measure­ment of cholesterol associated with these lipo­proteins that do not involve ultracentrifugation.A number of selective precipitation procedureshave been developed which enable total HOLcholesterol to be measured in a supernatefollowing precipitation of apo B containinglipoproteins (LOL & VLOL). These methods havebeen extensively reviewed. 1u - l89

The most commonly used precipitants areheparin-Mn- +, dextran sulphate-Mg! +, Phos­photungstate-Mg!", and polyethylene glycol.HOL3 cholesterol can be measured in thesesupernates following precipitation of HOL2allowing HOL2 cholesterol to be calculated bysubtraction. 190-193

LOL cholesterol can be calculated frommeasurements of total cholesterol, HOL choles­terol and an estimate of VLOL cholesterol(plasma triglyceride/z- 19) according to theFriedwald'P' formula:

LDL cholesterol = total cholesterol­(HDL +TG/2'19)mmoIlL

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The estimate of VLDL cholesterol is not validat triglyceride concentrations > 4· 6 mmol/L, intype III hyperlipidaemia, or when chylomicronsare present in the sample. Alternatively, LDLcholesterol can be calculated from the differencebetween measurements of cholesterol before andafter selective precipitation of LDL.19S-198

LDL cholesterol, derived from three measure­ments by the Friedwald formula or by twomeasurements in direct precipitation proce­dures, is a rather imprecise measurement andthere has been much debate about the validityof the Friedwald formula. 199-202 None the less, itis a bonus when calculated from three use­ful measurements and has found acceptancein assigning risk and making decisions ontherapy. 171

Total HDL cholesterol can be measured withacceptable precision.P! Accuracy is difficult todefine, however, because of heterogeneity HDLand the qualitative differences between fractionsobtained by selective precipitation and ultra­centrifugation. For example Lp(a) , an apoB­containing lipoprotein, is isolated in the HDLfraction prepared by ultracentrifugation butis removed together with LDL and VLDL inprecipitation procedures.P' Apo E containingHDL is precipitated by dextran sulphate-Mgt"but not by the heparin-Mn-:' reagent.P'

Considerable matrix effects are evident incontrol materials (author's unpublished studies)and significant differences are obtained onpatients' samples with different precipitationprocedures although the results are highly cor­related. 184.18S.187.20S Rate zonapos and densitygradientl06 ultracentrifugation studies on HDLsub fractions prepared by precipitation indi­cate that although the ultracentrifugation andprecipitation techniques give similar results fortotal HDL cholesterol, they assigned differentdistributions to HDL subfraction cholesterol.

No interferences from precipitating reagentshave been reported in the reference cholesterolmethod. Manganese ions can cause turbidity todevelop in certain enzymic reagent systems.P?This can be readily overcome by incorporationof EDTA in the reagent. Dextran sulphate of highmolecular weight (500 (00) has been shown to givelow results in another enzymic system!" possiblyby inhibition of pancreatic cholesterol ester­ase. 208 No other interferences by the precipi­tating reagents in cholesterol assays have beenreported. It has been demonstrated, however,that increased plasma concentrations of EDTAresulting from incomplete filling of blood

collection tubes cause serious overestimations ofHDL3 cholesterol due to chelation of divalentcations in the precipitating reagents and con­sequently incomplete precipitation of HDL2.209

Despite these analytical limitations a low totalHDL cholesterol remains an important riskfactor for coronary heart disease''" particu­larly in women"? and individuals with elevatedplasma triglyceride concentrations.i!'

HDL2 cholesterol, being derived from thedifference between two measurements at relativelylow concentration, is an imprecise measurementand is not likely to be accepted as a diagnosticor prognostic test until the physiological functionsof HDL subfractions are more clearly under­stood.

Factors to be considered in the selection ofprecipitation procedures for HDL cholesterolmeasurement include:

(a) The quality and availability of precipitationreagents

(b) Freedom from interference in cholesterolreagent systems

(c) The ability to give stable precipitates at hightriglyceride concentrations

(d) The degree of dilution of sample by theprecipitating reagent

Polyethylene glycol (PEG) and phosphotungstate­Mg2+ reagents are readily available in 'kit' form.These systems have no reported interferences withcholesterol reagents and are tolerant of highsample triglyceride concentrations. The sample isdiluted threefold by these precipitants, how­ever, which necessitates measuring cholesterolat low concentrations. The viscosity of thePEG solutions is a potential source of impre­cision and/or inaccuracy with some pipettingsystems.

An advantage of the heparin-Mn-" andDextran Sulphate-Mg-" systems is their lowdilution of sample (10%). However, they are lesstolerant of high triglyceride concentrations.Mn2+ ions can interfere with some cholesterolreagents and the source of heparin of definedactivity is critical. Similarly, the molecular weightof dextran sulphate is important.

The author has found the procedure describedby Warnick et a/.186 using dextran sulphate of50 ()()() molecular weight [Dextralip 50, fromSochibo, Villacoublay, France] to be a mostrobust procedure providing an economic methodwith minimal sample dilution.

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THE BLOOD SAMPLE FOR LIPIDAND LIPOPROTEIN STUDIES

The selection, collection, and storage of bloodsamples for lipoprotein analyses has beenextensively reviewed by Bachorik.i'f A briefsummary is given here. Patients should befollowing their normal eating habits and shouldnot be investigated during an acute illness orpregnancy. For the assessment of cholesterolstatus alone a fasting sample is not essential. A12h fast is mandatory for a profile includingtriglyceride measurements or lipoprotein studies.Venipuncture should be carried out on patientswho have been in the sitting position for at least5 min. The tourniquet should be used for asbrief a period as possible and released beforewithdrawing the sample. Either plasma orserum can be used but it should be notedthat the osmotic effects of low molecularweight anticoagulants shift water from ery­throcytes to plasma causing a reduction inlipoprotein concentrations.i" Samples anti­coagulated with EDTA at a final concetltra­tion of 1· 5 mg/mL give results 3070 lowerthan simultaneously obtained serum samples.Blood bottles should be filled to the correctvolume to minimize variation caused by thiseffect. Variations in sample EDTA concen­trations can also seriously affect selective lipo­protein precipitating procedures involving diva­lent cations.P? EDTA does have the advantagehowever of chelating heavy metals such asCu2 + that promote auto-oxidation of unsatur­ated fatty acids and cholesterol. EDTA is alsoan inhibitor of phospholipase C that can arisefrom bacterial contamination. EDTA is there­fore the preferred anticoagulant if sampleshave to be stored prior to lipoprotein analysis andcan be added to serum samples for this purpose.Samples should be allowed to clot in glass tubesat room temperature for 45 min. Serum or plasmashould be separated from cells within 3 h. Plasmacan be stored at 4 DC for total cholesterol andtriglyceride analyses or frozen at - 20 DC or belowfor longer periods. There is a great deal ofconflicting evidence, however, on the stability ofHDL during frozen storage.!" Lipoproteinfractionation should be done as soon as possibleon fresh samples.

CONCLUSION

The complex structure and heterogeneity oflipoproteins present the major difficulty in theformulation of enzymic reagent systems which are

Analytical reviews in clinical biochemistry 591

required to disrupt these complexes, achievecomplete hydrolysis of cholesterol esters, andensure the quantitative oxidation of cholesterolby cholesterol oxidase. Matrix effects introducedby freezing, lyophilization or the addition of non­human materials make it difficult to producecalibration and control sera which do notintroduce varying degrees of bias in differentreagent systems. Consequently, although ex­cellent precision can be achieved with the manyenzymic reagent kits now available, accuracyand inter-laboratory consensus are more elu­sive. Laboratories should therefore be care­ful to select calibrators and reagent systemswith proven performance from reputable manu­facturers and apply these on sound analyticalprinciples to minimize interference. Accuracyshould be assessed with certified referencematerials and by comparison of results onfresh patient sera with those obtained by areference procedure or a procedure with provenaccuracy.

After a century of use in clinical analysisthe application of the Liebermann-Burchardreaction, with all its limitations, in referenceprocedures should also be reviewed. Accuratespectrophotometric measurement of ~4 choles­tenone following chemical hydrolysis of choles­terol esters, extraction and enzymic oxidationof cholesterol as proposed by Trinder'P' deservesserious consideration as a practicable alternative.This procedure is within the scope of everylaboratory with an adequate spectrophotometerand could be refined by the application of HPLCto the measurement of ~4 cholestenone. It isalso evident that excellent accuracy and precisionare attainable by gas chromatography and it islikely that this technique will find increasingapplication in reference laboratories.

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2 Kritchevsky D. In: Cholesterol. New York: Wiley1958 •

3 Va1lsnieri A. Opere Fisco-mediche, Stampate andManoscritte, Vol. 3. Venice: Coleti, 1733; 594

4 De Fourcroy AF. De la substance feuillettee etcrystalline contenue dans les calculs biliares, et de lanatures des concentrations cystiques crista1isees.AnnChim 1789; 3: 242-52

5 Chevreul ME. Researches chimiques sur plusieurscorps gras, et particulierement sur leurs combinationsavec les alcalis. Cinquieme Memoire. Ann Chim 1815;95: 5-50

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6 Chevreul ME. Sixieme Memoire, Examen des graissed'homrne, de mouton, de boeuf', de jaguar et d'oie.Ann Chim et Phys 1816; 2: 339-72

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9 Berthelot M. Sur plusiers alcools nouveaux. AnnChim Phys 1859; 56: 51-98

10 Reinitzer F. Beitrage Zur Kenntniss des Cholesterins.Sitzber Akad Wiss Wein, Math-Naturw, KI Abt i1888; 97; 167-87

II Wislicenus J, Moldenhauer W. Ueber das cho­lesterindibromur. Ann Chim 1868; 146: 175-80

12 Diels 0, Aberhalden E. Zur Kenntniss des Cho­lesterins. Ber 1904; 37: 3092-103

13 Salkowski E. Kleinere Mittheilungen physiologisch­chemischen Inhalts (II). Pfluger's Arch ges Phsiol1872; 6: 207-22

14 Bernal JD. Crystal structures of vitamin D andrelated compounds. Nature 1932; 129: 277-8

15 Diels 0, Gadke W. Uber die Bildung von Chrysenbei der Dehydrierung des Cholesterins. Ber 1927; 60:140-7

16 Rosenheim 0, King H. The ring system of sterolsand bile acids. Nature 1932; 130: 315

17 Rosenheim 0, King H. The ring system of sterolsand bile acids. J Soc Chem Ind (Land) 1932; 51:464-6

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Accepted for publication 18 February 1992

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