A new color reaction for cholesterol assay

CLINICA

CHIMICA

ACTA

A NEW COLOR REACTION

D.

H.

JUNG

73

FOR CHOLESTEROL

AND A. C. PAREKH*

Clinical Laboratory, Indiana University Medical Center, IIOO Indiana 46202 (U.S.A.) and Division of Clinical Pathology, 750 E. Adams Street, Syracuse, N.Y. 13210 (U.S.A.) (Received

ASSAY

W. Michigan Street, Indianapolis, S.U.N.Y. Upstate Medical Center,

March 2, 1971)

SUMMARY

A new iron-cholesterol reaction, based on the Parekh-Jung method of determination of cholesterol, is described. The reagents consist of ferric acetate-uranium acetate and sulfuric acid-ferrous sulfate. Unlike the commonly employed LiebemlannBurchard and ferric chloride-sulfuric acid reactions, the color obtained is stable and is unaffected by instability of reaction temperature or moisture content of the sulfuric acid. The color reaction obeys Beer’s law over a wide clinical range of the serum cholesterol. The reaction can be applied to the assay of cholesterol either as part of the direct method of determination or to the cholesterol pre-extracted by employing other techniques. A close correlation with simultaneous measurements by the method of Abel1 et al. was obtained. A study of the importance of the use of ferrous sulfate in the reagent system is described. The analytical data are discussed with reference to the reported significance of the chromogenicity of the free and ester forms of cholesterol. No effect of the presence of digitonin on the color reaction was noted. The proposed iron-cholesterol reaction is presented as the color reaction of choice for cholesterol assays.

INTRODUCTION

The author+ recently proposed a new direct method for determination of serum cholesterol that employed ferric acetate-uranium acetate and sulfuric acid-ferrous sulfate reagents. In this direct method, the reagent ferric acetate-uranium acetate serves as a unique precipitating agent that removes bilirubin (as biliverdin) with proteins, clears the serum of the lipids and serves to extract total cholesterol without the need of solvents. The acetate reagent extract when mixed with the sulfate reagent

* Reprint requests should be moiled to A.C. Parekh; present address: (Dept Clin. Pathol., tram Administration Hospital, San Francisco, Calif. 94121 (U.S.A.) C&z. Chim. Acta

Ve-

35 (1971) 73-78

yields a purple color with an absorption

maximum

at 500 nm. The maximum

develops within 15 min and is stable for at least I 11. In the foilowing text, the authors describe their

experience

color

with the use of

ParekhJung reagents primarily in obtaining a color reaction since occasionally one may find it expedient to employ other technics to extract cholesterol from tire sample. Also a further study on the advantage

of employing

ferrous sulfate with concentrated

sulfuric acid is presented. The merits of the use of ferric acetate in place of ferric chloride are considered and the chromogenicity of free zleysus esterified cholesterol is discussed with regard to the proposed color reaction. Further, the effect of digitonin on the color reaction as well as on the assay of cholesterol were studied. METHODS AND MATERIALS Cholesterol determinations were carried out by the Pareklr-Jung1 well as by the method of Abel1 et aLz and the reagents were prepared

method as accordingly.

The cholesterol standards were prepared from Standard Reference Material No. 911 obtained from the National Bureau of Standards consisting of 99.4 *0.37$ pure cholesterol.

Serachol,

a commercial

reference

preparation

(available

Chilcott Co., Morris Plains, New Jersey) was also used. Digitonin was obtained from the Fisher Scientific Company.

from Warner

(certified

reagent)

Determinations were also carried out by applying the proposed color reaction extracted from the serum according to the Abel1 procedure. The

to the cholesterol

E G 0.8: v) 0.7: z < ; 0

:

<

0.60.50.4-

0

III 0

100

III 200

300

400

CONCENTRATION Fig.

I. Calibration

CZin. Chim.

Acta,

curve. 35

(1971)

73-78

500

600

IIll 700 800

(mg/lOO

900

ml)

1000

CHOLESTEROL

7.5

ASSAY

extract was evaporated under nitrogen followed by addition of 3 ml of ferric acetateuranium acetate reagent and z ml sulfuric acid-ferrous sulfate reagent to obtain the color. The absorbance was measured in a Coleman Jr. II spectrophotometer employing the reaction cuvettes described by Parekh and Jung’. Each point displayed in the figures and each datum shown in Table I referred to in the following text represents a mean of three analyses. RESULTS

Various concentrations of cholesterol standard as well as of Serachol were analysed by the Parekh-Jung1 method and the data obtained are presented in Fig. I. It illustrates the linearity displayed by the proposed color reaction throughout the usually encountered clinical range of the serum cholesterol. The absorbance values thus obtained are conveniently measurable with the spectrophotometer. TABLE

I

COMPARISON ACETATE THOSE

OF

CHOLESTEROL

IN ACETIC OBTAINED

ACID

BYTHE

(mg/IOO

ml)

REAGENTTREATEDWITH PROCEDURES

OFABELL

Abel1 et al. procedure

H,SO,-F&O,

Cone. H,SO,

1. 2. 3. 4. 5. 6.

375 305 170 128 I73

400 326 176 132 180

I29

‘35

375 307 168 I27 I72 126

DETERMINATION

H,SO,-FeSO,

WITH

FERRIC

ACETATE--URANIUM

REAGENT AND

H,SO,ALONE

TO

etal.

Table I consists of data obtained on simultaneous analyses of a group of random sera by the method of Abel1 et aL2 and by the method of Parekh and Jung’ with and without the presence of ferrous sulfate in the concentrated sulfuric acid. The results obtained demonstrate a very close agreement between the two methods when ferrous sulfate was part of the reagent system in the latter. On the omission of the ferrous sulfate, falsely elevated values were obtained which were inconsistent with the results obtained simultaneously by employing the method of Abel1 et aL2. Fig. z displays a scattergram obtained on cholesterol determination in IOO different sera carried out as follows. Each sample was analysed for its total cholesterol employing the method of Abel1 et a1.2. Also, each sample was assayed for the total cholesterol by subjecting it to the extraction procedure of Abel1 et al. followed by application of the proposed color reaction as described in the preceding section. The results obtained demonstrate a close correlation emphasizing the role of the ParekhJung’ reagents in providing a reliable color reaction with cholesterol. Choles .erol standards (zoo, 400 and 600 ,ug) were taken with and without the addition of 0.6 mg digitonin. These samples were then subjected to color development by the proposed reaction as described in the preceding section. Neither a change in the shade or the color was observed nor any significant difference in absorbance measured.

Clin. Chim. Acta,

35 (1971) 73-78

I

I

0

200

lb0

I

300

I

I

I

460

500

600

TOTALCHOLESTEROL(~~/I~~~)BYABELLEXTRACTI~N WITH NEW Fe REAGENT Fig.

2. Scattergram

of results obtained

on analysts

of 100 random

sera.

DISCUSSION

It is evident

from the foregoing

and sulfuric acid-ferrous

sulfate reagents

data that the ferric acetate-uranium can, when necessary,

be successfully

acetate applied

as color developing reagents only. As shown in the original method, the proposed reaction is very stable and the timed measurements are not necessary. This color stability has earlier been attributed by the author+ to the unusual temperature stability of the proposed color reaction. The need for stability of temperature in case of ferric chloride-sulfuric acid reaction3 has been demonstrated by Webste+, Chiamory and Henry5 and RadzioG. These authors have recognized the need for either a constant temperature room or a constant temperature bath in order to control the temperature of the exothermic reaction. Largan et aL7 have suggested the use of a 50” waterbath. Parekh and Jung8 have earlier demonstrated the partial evolution of hydrochloric acid in case of the ferric chloride-sulfuric acid reaction leading to the unavoidable instability of the reaction temperature. This serious shortcoming of the ferric chloride-sulfuric acid reaction has been overcome by the authors by employing ferric acetate in place of ferric chloride. As a result, the temperature of the proposed color reaction is maintained within a narrow range of 48-50”. Furst and Lange9 have criticized the inconsistency of results obtained on minor alterations of the quantities of sulfuric acid or ferric chloride or both. Chiamory and Henry5 and Zak et al.1° pointed to significant color suppression obtained in presence C&n.

Chim.

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CHOLESTEROL

ASSAY

77

of waterin concentratedsulfuric acid. Badzioe has similarly documented non-specificity of the ferric chloride-sulfuric acid reaction in presence of moisture. The authors’ data (Table I) strongly point to a significant role played by the anhydrous ferrous sulfate added to the concentrated sulfuric acid. However, the amount of ferrous sulfate (IOO mg/l) in the reagent is so small that the author9 have felt that it might be conferring the stability to the color by some mechanism, as yet unknown. There have been many reports in the literature emphasizing the different chromogenicity of the free and ester forms of cholesterol. Brownll has found all cholesterol methods to agree only after a non-saponifiable extract was first obtained. This then points to another form of discrepancy usually added to the commonly employed direct methods of measurement of cholesterol whether employing Liebermann-Burchard reaction12 or the ferric chloride-sulfuric acid reaction13p14. In general, such direct methods tend to yield a value 7-15% higher than the true value in case of normal sera. On examining the data in Table I and Fig. z, one can conclude that indeed the proposed color reaction, whether employed as part of the direct Parekh-Jung1 method or in conjunction with saponification and extraction by the technique of Abel1 et al. yields total cholesterol values in very close agreement with those obtained by simultaneous application of the method of Abel1 et al. 2. It is thereby implied that in case of the proposed color reaction, a difference in the chromogenicity of free or esterified cholesterol is not observed. Digitonin itself reacts in many of the color reactions for cholesterol. Yasudals has shown that a positive error of measurement of cholesterol may occur unless digitonin is removed prior to color formation in case of the Liebermann-Burchard reaction. Rosenthal and Jud16 have shown that inclusion of digitonin in the final reaction yields an orange-red instead of a purple color in case of a modification of the reaction of Zlatkis et aL3. As described in the experiment with varying concentrations of cholesterol and digitonin, it is apparent that neither the shade of the color alters nor is the measured cholesterol value affected in any way in the proposed reaction. In this regard, our experience is similar to that of Schoenheimer and Sperry” who did not observe any effect of digitonin on the color. These authors have commented on possible enhancement of color by digitonin obtained from the different sources. The accuracy of a method of determination of cholesterol thus depends not only on the extraction and purification steps but also on the color reaction used. The shortcomings of the ferric chloride-sulfuric acid color reaction are already mentioned. The Liebermann-Burchard reaction is a very complex color reaction which is affected by most of the variables described above in addition to being light sensitive. The authors therefore propose the use of Parekh-Jung iron-cholesterol reaction as the color reaction of choice. ACKNOWLEDGEMENT

Authors appreciate the technical assistance rendered by Mr. R. Creno. This investigation was partially supported by the Research Foundation No. 11--8520-A of the State University of New York.

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Acta,

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35 (1971) 73-78

RBFBRBNCES I A1.C. PAREKH AXU D. H. Jrxc;, Amzl. Chum., 42 (1970) 14~3. 2 L. L. ABELL, B. 13. LEVY, 13. 15.I~R~DIE ASD 1'.E. KENDALL, ,I.Viol. C/WHI., 19.j (I~l,iZ) .‘,.jT. 3 A. ZLATKIS, H. ZAK ANI) .A.J. 13oYLE, ./.Lab. Clip?. ilfrd, 41 (19j3) J8h .+ I>.WEBSTER, CZzx. Chim. Acta, S (1963) 731. 5 N. CHIAMORY AND Ii.J. HENRY, ilmw. J. Cl&. Puthol.. 31 (1959) 3O.j. 6 T. BADZIO, Clzn. Chim. ,-1cta, II (1965) j3. 7 T. A. I,ARGAN, E. 1~. D~RRKJM AKU W. P. JENCKS, J.Clzn. Invrst., 34 (1955) 11~7. 8 X. C. PAREKH AXU D. H. JUNG, Abstracts - 7th I&vwational Gmgzg~r~ssof Clinical f’atholog_v, Montreal, Canada. 1909, 1’. j6. 0 (1954) 60. 9 V. FURST, JR. AKD 1~. LAXGE, Scmzd. J. Clie. Lab. Inwst., 10 1~. ZAK, R. L. DICKENMAX, I<. (;. WHITE, H. HURXET ASD P. J. CE~IEKSEY,Anrr. ,/. CZzn. P&hot., 21 (1954) 1307. IT W. D. BROWN, A ustv. ,I. Ikp. Biol. ;Zied. Sci.. 39 (1961) 209. 12 P. VANNIXI AND -4.I)EL \'~ccHIo, Arch. “E. ~~~avagliam” Pathol. Cli~z., I L (1956) 793. 13 P. \'.FERRO ASD .\. B. HAM, Amrv. J, Clin. Pathol.. 33 (1960) 545. 14 T. C. HUANG, C. I’. CHES, \‘. \VEPLER AKL) A. RAPTERY, Axal. Chcm., 33 (1961) ‘1o.j. 1.5 31. Y~suua, J. B!iochrn/., LJ (1936) 429. 16 H. L. ROSEKTHAL AND 1~. JuL?, /. Lab. C/in. Ilf?d.. j1 (Igj8) 11.3. 17 R. SCHOENHEIMER ANII \I:.31. SPERR‘I.,.I.Biol. Chrnz., 106 (1934) 74j.

Clit~. Chiwz. .4cta, 3.5 (1971) 73 7S