Moshides: Kinetic enzymatic detennination of HOL cholesterol 583

J. Clin. Chem. Clin. Biochem.Vol. 25, 1987, pp. 583-587© 1987 Walter de Gruyter & Co.

Berlin · New York

Kinetic Enzymatic Method for Automated Determinationof HDL Cholesterol in Plasma

j By /. S. Moshides

\ Department of Clinical Chemistry, Prince of Wales Hospital, Randwick, Sydney, Australia

(Received March 2/June l, 1987)

Summary: A sensitive fixed-time kinetic enzymatic method for the measurement of high density lipoproteincholesterol is described, äs adapted for the Cobas Bio centrifugal analyser (Hoffmann LaRoche). TheBoehringer Mannheim cholesterol esterase/cholesterol oxidase/peroxidase/3,4-dichlorophenol kinetic reagentwas modified by the inclusion of 2,4,6-tribromo-3-hydroxybenzoic acid which reacts with hydrogen peroxideand 4-aminophenazone to produce a quinone-imine dye with a greater molar absorptivity than that producedwith phenol. The method has been developed for the detennination of HDL fractions isolated with polyethyl-ene glycol 6000, for which a reagent of high sensitivity is required.

The method is linear to at least 3.88 mmol/1 of HDL cholesterol and the coefficients of Variation for within-run and day-to-day precision were less than 3.0%. Correlation of the kinetic HDL cholesterol method withan equilibrium method was good (R = 0.9980).

The ässay is rapid, inexpensive and large numbers of specimens can be processed conveniently.

Introduction

The analysis of high density lipoprotein cholesterolhas, in epidemiological and clinical studies (1^6),shown that a low HDL cholesterol level üsually indi-cates a risk for coronary heart disease. The increaseddemand for HDL cholesterol assays had led to theneed for a method capable of handling large numbersof assays generated frona clinical and epidemiologicalworkf The Boehringer Männheim cholesterol oxidase4-aminophenazone (CHOD-PAP) reagent for cho-lesterol has been adapted for use with a centrifugalanälyser to measure HDL cholesterol "äs an equili^brium method (7). The incorpQration of 3,4-dichloro-phenol in the Boehringer Mannheim CHOD-PAPreagent has enabled the assay of total serum choles-terol üsing automated fixed-time kinetic methodology(8). The kinetic cholesterol method has coüsiderablyreduced the analytical time require4 therefore allow-ing more efficient use of automated instruments, andhas the advantage of being less sensitive to interfer-ences than are equilibrium methods.

However, the adaptation of the kinetic method forthe analysis of HDL cholesterol would involve themeasurement of significäntly smaller absorbance dif-ferences ( ). Since a relatively high photometricerror would be incurred in the measurement of suchlow absorbance values (9), the assay was modified byadding i^o^tribromo-S-hydroxybenzoic acid to theBoehringer Mannheim kinetic cholesterol reagent.The tribromohydroxybenzoic acid reacts with hydro-gen peroxide and the 4-aminophenazone/peroxidaseSystem to produce quantitative amounts of quinone-imine dye. Details of the modified assay are reported.

Materials and MethodsApparatusCobas Bio centrifugal analyser (F. Hoflfman-La Röche and Co.,Limited Company, Basle, Switzerland) and related accessories.Hettich Universal K2S refrigerated centrifuge (Andreas Het-tich, 1\ittiingen, W. Germany).

J. Clin. Chem. Clin. Biochem. / Vol. 25,1987 / No. 9

584 Moshides: Kinetic enzymatic deterrnination of HDL cholesterol

ReagentsBuffercd polyethylene glycol 6000: Polyethylene glycol, averagerelative molecular mass Mr = 6000 (British Drug Houses) 200g/l in glycine/NaOH buffer, 0.2 mol/1, pH 10.

2,4,6-Tribromo-3-hydroxybenzoic acid: Synthesised by bromi-nation of Analar grade 3-hydroxybenzoic acid (10).

Cholesterol reagenl: Boehringer Mannheim "CHOD-PAP" ki-netic colorimetric test (catalogue number 692905).

Cholesterol Standards: Preciset® High Performance Standards(Boehringer Mannheim, GmbH catalogue number 709905).

High Density Lipoprotein Cholesterol Controi: BoehringerMannheim Precinorm®L (lyophilised) catalogue number781827, lot number 154747.

Procedures

Isolation of HDL

Buffered polyethylene glycol 6000 solution (0.2 ml) was mixedwith plasma (0.2 ml). After 10 min at 20 — 25 °C, the mixturewas centrifuged at 2000 g for 20 min at 4 °C. The supernatantwas aspirated for cholesterol analysis. Positive displacementpipettes were used for all quantitative pipetting procedures.

Cobas Bio reagent boat

Equal quantities of 9 g/l NaCl solution and polyethylene glycol6000 solution were mixed and placed in Standard well l. Cho-lesterol Standards were mixed with equal quantities of polyetr>ylene glycol 6000 solution. Boehringer Mannheim Preciset Stan-dard 1.29 mmol/1 was placed in Standard well 2, and Standard2.59 mmol/1 in well 3. For 70 assays, the reconstituted CHOD-PAP reagent (25 ml) was combined with 2,4,6-tribromo-3-hydroxybenzoic acid and placed in the main well. This reagentwas discarded after use. Various concentrations of 2,4,6^tri-bromo-3-hydroxybenzoic acid ranging from 0—20 mmol/1 wereevaluated to determine the most suitable strength.

Cobas Bio procedure

Various plasma/reagent ratios were tried in a series of prelimi-nary experiments and the most suitable was found to be l to55. This ratio and the Optimum 2,4,6-tribromo-3-hydroxyben-zoic acid concentration was a compromise to obtain the desiredsensitivity required for a rapid kinetic method. The maximumabsorbance of the quinone-imine dye, which represents the finalproduct of the reaction, was at 515 nm. Absorbances measuredin this region are less likely to be affected by colorimetricinterference due to haemolysed or icteric plasmas. The instru-ment was operated according to the settings listed in table I.

Results

Concentration of 2,4,6-tribromo-3-hy-droxybenzoic acid

The concentration of 10 mmol/1 2,4,6-tribromo-3-hydroxybenzoic acid in the Boehringer Mannheimreagent produced the largest absorbance differences(ΔΑ) for a total reaction time of 50 seconds. Theseabsorbance differences obtained using 2,4,6-tribro-mo-3-hydroxybenzoic acid were three times greater'

Tab. 1. Parameter listing for determination of high densitylipoprotein cholesterol on the Cobas-Bio centrifugalanalyser by the fixed-time kinetic method.

Units mmol/1

Calculation factorStandard l conc.Standard 2 conc.Standard 3 conc.Limit (mmol/1)Temperature °CType of analysisWavelength (nm)Sample Volume (ul)Diluent Volume (μΐ)Reagent Volume (ul)Incubation time (s)Start reagent volume (μΐ)Time of First Reading (s)Time intervai (s)Number of ReadingsBlank mode

0.0 (Polyethylene glycol blank)1.29 , ,2.594.0

305

5151530

350300

10106l (reagent blank)

than those produced with phenol alone in the un-modified CHOD-PAP kinetic cholesterol reagent (fig.1). Subsequent work was carried out using 10 mmol/1.2,4,6-tribromo-34iydroxybenzoic acid.

Course of the reaction

The feasibility of an assay protocol based on first-order kinetics was examined by following the progiressc rve of four aqueous cholesterol Standards (0.65,1.29, 2.59 and 3.88 mmol/1) and two patient plasmas(0.80 and 1.95 mmol/1 HDL eholesterol), all treatedwith polyethylene glycol. The semilogarithmic plot ofabsorbance at completion of reaction minus absorb-ance at time t versus t indicates that the reactionfollows first-order kinetics since the plot is linearfrom 10 seconds to at least four min tes (fig. 2). Theinitial lag phase is terminated within the first 10seconds of the reaction. A total reaction time of60 seconds provides sufficient linearity and a shortanalysis time. Therefore, the subsequent kinetic pro-tocol was based on the preineubation of the 2,4,6-tribromo-3-hydroxybenzoic acid modified reagent at30 °C for 30 seconds, the absorbance difference toeingcalculated from the difference between the absorb-ances at 60 seconds and 10 seconds after plasma andreagent mixing (ΔΑ/50 seconds).

Linearityl..;

Linearity was determined with the Preciset® choles-terol Standards and their diltitions (fig. 3) and serialdilutions of a patient plasma with a HDL cholesterolvalue of 2.45 mmol/1 (fig. 4). The linear r nge for the

J. Clin. Chem. Clin. Biochem. / Vol. 25,1987 / No. 9

Moshides: Kinetic enzymatic determination of HDL cholesterol 585

Standards was from at least 0.25 to 3.88 mmol/1. TheΔΑ values for the dilutions of the patient plasmawere proportional within the dilution r nge tested(l: l to l: 16). The linear r nge of the assay thereforecovers the majority of the population values likely tobe encountered (0.5 to 2.5 mmol/1) (7).

Accuracy and precision

Comparison of 48 patient HDL cholesterol plasmavalues obtained for a fixed-time kinetic method (x)and an equilibrium method (y) (7) showed goodcorrespondence of results (r = 9980, y = 1.01 Ix+ 0.016) (fig. 5).

11.0

0.8

10.6

<c<30.4

0.2

HD L-cholesterol[mmol/U

\ >

0 5 10 15 202,4,6-Tribromo-3-hydroxybenzoic acid [mmol/l]

Fig. 1. Absorbance plots of HDL cholesterol Standards utilis-mg varying concentrations of 2,4,6-tribroimo-3-hy-droxybenzoic acid in the enzymic cholesterol reagent.

3.0

2.0

i v,

0 1 2 3 4t Lmin]

Fig. 2. Urne course of reaction.At, absorbance at time t; A^, absorbance after compte?tion of reaction; rate constant, k = — ΔΙηζΑ^ — AJ/At (slope of curves). Specimens: l, 3, 5, 6, PrecisetCholesterol'high performance (0.65, 1.29, 2.59, 3.88mmol/1, respectively); 2, 4, human plasma (0.85, 1.99mmol/1 HDL cholesterol, respectively).

1.0

0.8

I 0.6

0.4

Q.2

1:0 2.0 3.0HDL-cholesterol [mmol/1]

4.0

Fig. 3. Linearity of the fixed-time kinetic HDL cholesterol as-say utilising 10 mmol/1 2,4,6-tribromo-3-hydroxyben-zoic acid in the enzymic cholesterol reagent (duplicateassay s).

0.5

0.4

0.3

<3

0.2

0.1

1:16-1:8 1:4 1:2Sample dilution

1:1(undiluted)

Fig. 4. Linearity of the fixed-time kinetic HDL cholesterol as-say on serial dilutions of a human plasma containing2.45 mmol/1 HDL cholesterol (duplicate assay s).

3.0

2.0

S 1.0

31 0 1.0 2.0 3.0HOL-cholesterol (fixed-time kinetic method)

[mmol/l]

Fig. 5. Comparison of an equilibrium method (y) and the fixed-time kinetic HDL cholesterol method (x) in 48 humanplasmas (slope = 1.011, axis interccpt = 0.016 mmol/l,correlation coefilcient - 0.9980).

J. Clin. Chem. Cltaf Biochem. / Vol. 25,1987 / No. 9

586 Moshides: Kinetic enzymatic determination of HOL cholesterol

Multiple analysis of the precinorm® L control serumproduced mean values close to the manufacturer'svalue of 1.14 mmol/1 (ränge = 0.96 to 1.32 mmol/1).The within-run precision using Precinorm L wasrepresented by a coefficient of Variation of 2.0%(x = 1.202 mmol/1, n = 20). The between-run preci-sion was 2.6% (x = 1.180, n = 20). Analysis of aplasma pool with a low concentration of HDL cho-lesterol produced within-run and between-run coeffi-cients of Variation of 1.4% (x = 0.6763, n = 20) and2.9% (x = 0.6902, n = 20) respectively. The within-run and between run precision of a plasma pool witha high concentration of HDL cholesterol were 1.1%(x = 1.6988, n = 20) and 1.6% (x = 1.7234, n = 20)respectively.

Interferences

The results of interferences by drugs, plasma metab-olites or coloured constituents of blood are repre-sented in table 2. Concentrations of the substancesexamined Were added to a pooled specimen of humanplasma. This plasma pool had a HDL cholesterolvalue of 1.24 mmol/1 (mean of 10 assays) in theabsence of any added substance.

Tab. 2. Interference of added substances (plasma concentra-tions of drugs usually found after administration oftherapeutic doses in brackets).

Substances added, final concentration Percentage ofHDL cholesterolaccounted for

Urea, 200 mg/1Creatinine, 100 mg/1Uric acid, 200 mg/1Glucose, 4000 mg/1Bilirubin, 50 mg/1Bilirubin, 100 mg/1Haemoglobin, 1000 mg/1Haemoglobin, 2000 mg/1Salicylate, 1000 mg/1 (200 mg/1)Gentisic acid, 30 mg/1 (l mg/1)Gentisic acid, 50 mg/1L-Dopa, 5 mg/1 (l mg/1)L-Dopa, 10 mg/1a-Methyldopa, 30 mg/1 (l mg/1)a-Methyldopa, 50 mg/1Ascorbic acid, 20 mg/1 after 0 hAscorbic acid, 50 mg/1 after 0 hAscorbic acid, 20 mg/1 after l hAscorbic acid, 50 mg/1 after l hAscorbic acid, 20 mg/1 after 2 hAscorbic acid, 50 mg/1 after 2 hAscorbic acid, 20 mg/1 after 3 hAscorbic acid, 50 mg/1 after 3 h

at 20 °Cat 20 °Cat 20 °Cat 20 °Cat 20 °Cat 20 °Cat 20 °Cat 20 °C

9999999899989897

1019490908679718463938195909997

Discussion

The kinetic fixed-time techniqüe for enzymatic ana-lysis of total serum cholesterol was made possible bythe use of cholesterol oxidase from a Streptomycesspecies instead of the enzyme from Nocardia erythro-polis (8). The Michaelis constant (Km) with respect tocholesterol of the Streptomycti enzyme can appar-ently be increased, by adding the competitive iiihibi-tor, 3,4-dichlorophenol, to such an extent that thecholesterol oxidase reaction would follow first-orderkinetics over a large ränge of cholesterol concentra-tions. By exploiting this Inhibition, together with thevery efficient cholesterol esterase, a kinetic cholesterolreagent was developed. In this kinetic System, thecholesterol oxidase reaction is the rate limiting step,so that the indicator reaction follows first-order kine-tics (Boehringer Mannheim catalogue riumber692 905 and 725 242).

Addition of 2,4,6-tribromo-3-hydroxybenzoic acid tothis reagent to enhance the indicator reaction pro*duces an increase in the sensitivity with respect tocholesterol so that a kinetic protocol mäy also beused to measure HDL cholesterol. Compared withthe unmodified reagent, the method described in thisreport enables a three-fold increase in the absorbancedifferences ( ) of HDL cholesterol supernatantsprepared with buffered polyethylene glycol 6000, aprocedure whieh involves a two-fold dilution ofplasma. The use of buffered polyethylene glycol 6000to isolate HDL enables the Clearing of most lipaemicplasmas which would otherwise cause an over-estiina-tion of HDL cholesterol (11). The polyethylene glycol6000 procedure is also äcourate and efficient (12).Preliminary results also indicate that HDL süperna*tants prepared by the phosphotungstic acid/MgCk(13) or dextran sulphate 500/MgCl2 (14) ß-lipoproteinprecipitation methods may be assayed with the kineticmethod on the Cobas-Bio analyser provided that itis programiiied with a sample volume of 7 , insteadof 15 1.

The first-order kinetics of the HDL cholesterol ki-netic method show a parallelism among the aqueousStandards and the patient plasmas (flg. 2), indicatingthat the protocol described is suitable for routineestimation of patient HDL cholesterol values. Corre-lation between the kinetic and equilibrium methodswas excellent and there was good agreement betweenthe value obtained for the Precinorm® L control aiidthe manufacturer's value. Linearity and precision arecomparable to equilibrium methods (7,11).

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Moshides: Kinetic enzymatic determination of HDL cholesterol 587

In contrast to the kinetic method for total serumcholesterol (8), in vitro addition of large amounts ofsubstituted phenols such äs L-dopa, methyldopa andgentisic acid to the plasma pool caused significantinterference. However, the concentrations that thesedrugs attain in vivo would interfere negligibly (15).Interference from added ascorbic acid was minimisedby leaving the HDL supernatant at room temperaturefor three hours before analysis. Ascorbic acid in solü-tion rapidly oxidises in the presence of oxygen, es-pecially at pH > 7.0 (16). In any case, a normalplasma ascorbic acid level of less than 20 mg/1 shouldnot interfere significantly and any higher levels dueto, excessive administration of vitamin C can be over-come by extending to 3 hours the interval betweenblood-taking and HDL cholesterol determination.

In conclusion, the present method involving 2,4,6-tribromo-3-hydroxybenzoic acid addition to theBoehringer Mannheim kinetic cholesterol reagent af-fords a faster, convenient analysis of HDL cholesterolthan the corresponding equilibrium methods. Hence,the method is suitable for routine use in the auto-mated laboratory. The method is also relatively cheapand has the potential of being used to measure thecholesterol in LDL and HDL subfraction isolates(HDL2 and HDL3).

Acknowledgements

This study was supported by laboratory funds and facilitiesmade available by Dr. Maithew Meerkin, Chairman of theDepartment of Clinical Chemistry, Prince of Wales Hospital(Sydney).Dr. Garry Graham, senior lecturer in the Faculty of Medicineof the University of N. S. W., made helpful comments.

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Mr. James S. MoshidesDepartment of Clinical ChemistryPrince of Wales HospitalRandwick, N.S.W. 2031Australia

J. Clin. Chem. Clin. Biochem. / Vol. 25,1987 / No. 9