The coulometric determination of cholesterol in serum

CLINICA

THE

CHIMICA

ACT.4

COULOMETRIC

15.5

DETERMINATION

OF

CHOLESTEROL

IN

SERCM*

SUMMART

Numerous rapid and precise methods have been developed for the routine analysis of cholesterol in serum. However, these methods and many of the current reference techniques are not amenable to the analysis of biological samples which may contain either major or minor concentrations of other steroids. In the present method accurate cholesterol values are obtained by a thin-layer separation and coulometric quantitation procedure. This procedure may be of considerable value in those samples where other steroids exist.

One of the first sensitive methods for the detection of cholesterol was proposed by Salkowskil in 1872. This was based on the color formed from the reaction of cholesterol in chloroform with sulfuric acid. Liebermann2 and Burchard3 studied this reaction in greater detail in the presence of acetic anhydride and many of the subsequent analytical methods are based on these original studies. Similar color reactions, based on the reaction of cholesterol with Lewis acids, are the source of most quantitative methods for the estimation of cholesterol in clinical laboratories, However, they do require that one must adhere to a rigorous set of conditions with respect to temperature, reagent concentrations, and the presence of light. Most of these Lewis acids will react with steroids other than cholesterol, which can cause a limitation with respect to selectivity. The first step in most methods for the determination of cholesterol involves the extraction of cholesterol into an organic solvent. This generally results in a gross extraction of lipids, i.e. cholesterol, cholesterol esters, phospholipids, triglycerides, and fatty acids. In addition, certain other organic compounds, such as urea or glucose, may be extracted. Most methods claim to achieve specificity on the color reagent used to estimate cholesterol in this crude extraction. One of the earliest methods used to isolate cholesterol was proposed by Windaus” and is based an the selective precipitation of 3/?-hydroxy steroids with digitonin; this method does provide some selectivity in the isolation step. Numerous paper, thin-layer, and column chro-

.__ * Taken in part from the Ph.D. Dissertation of Dr. Robert J. Troy, ** Present address: Jackson Laboratory, E.I. du Pont de Nemours Del. 19899.

University of Maryland, 1969. & Co., Box 525, Wilmington,

C/in. Chilpz. Acta,

26 (1969)

Isj-163

nlatvgraphic techniques have provided good separation rnethocls classes of lipids present in l~lood but in general, special techniques effect a separation of the similar isumers of chc~lestcr01.

for the \2rious are requited to

Since cholesterol is reported to be 7 51;, csterified in serum, man\: methods for tlw determination of total cholestel-ol require the hydrolysis of these estws. Tlris is usuall~~ acc~tIl~~~islle~1by hydrolysis lvitlt alcoltctlic potassium hydroside. Although this is a convenient tuetl~oct, there is a danger of oxidation of cholesterol in the preseimy of 2ir. Few titrimetric wethods have hccu applied to the analysis of c~irolcsterol. (&-don” 1~s reported the ml\, Illicro nlethod for the determination of cholcsttw~l. This method is ~XMY~on the reaction of an csccss of bromine wit11 c~hc~lcsterol; tIlta residual bromine is allowed to react with a dye, and the t1ccrea.w in ahsorhanw of this dye is used to measure tile amcwnt of cholesterol present. Although I;() one 1~1s titrated cholesterol directly with bromine, Leiscy and C;rutsch@ have titrated c,~rclohexene with couloi~~etricall~~ generated hron~inr. 12 the piweut paper, (.o~lloil~~tri(. titrations are applied tn the dete~-~~~i~~~~tio~~ of cll(~lestert~l in serum. This ill~esti~~~tioll is part of a continuing stud!, on the application of coulometric titrations to chid chemistry’.

R&Xg:cdS

further

X11 chemicais were reagent grade whenever pmsible purificati~)ll. Diosane, ether, hesane, and cyrlohexenc

and were used without were redistilled

prior to

use. Primary grade cholesterol was prepared according by Kadin*. This consisted of a reaction of the cholesterol

to the procedure descrilmd with bromine in ether, col-

lectiou of the dibrornide, a wash with acetic acid, removal of the bromide wit11 zinc and acetic acid, collection of the purified cholesterol, and recrystallization with ethanol five times. The product was characterized by the melting point (found 148.4 149.4O, reported 149.3-151.3~) and (,~l~l~~arison with the standard infrared spectruul:‘. It was checked for the presence of impurities with the chr~~~lat~)gra~hic system dtscrihed in the section on thin-layer chromatograph>~. Lathosterol was prepared according to a procedure described by Fieser and of 7-dt.h~drocholesterol wit11 Herzl”. This procedure consisted of the reduction Raney nickel aud recrystallization with acetonemmwater. Thin-layer cllro~llatogra~)l~~~ indicated that on&, two-thirds of the materjal was converted to lathosterol. Lathostcrol was isolated on a preparative thin-layer plate for further studies. It was eluted from the plate with cl~lorof~rt~l and recrystallized with ethanol. Tl~is material n-as tllarac.reported 126’) and comparison wit11 terized ht- melting point {found 125.3-16.3”, its standard infrared spectrunlll. I. Potassium hydroxide, 33Yr, (w/v) 2. Absolute ethanol 3. Alcoholic potassium hydroxide. This consisted

of 6 ml of 33”.

potasslunl

hydroxide and 94 ml of absolute ethanol. 4, Arsenious acid, o.og880 X 5. Generating reagent. This consisted of a mixture of IZO ml nlethan~~~ Z&I ml acetic acid, z4 ml 5 M sodium bromide, and 8 g mercuric acetate. Bromine was added

COULOMETRY

OF SERUM

CHOLESTEROL

I57

in excess dropwise 6. 7.

to this mixture and stirred 24 11.The excess bromine was consumed by adding a saturated solution of arsenious acid in methanol. Hexane Chloroform-methanol (3 : I) 8. Chloroform-ether (90: z) 9. Silver nitrate, 100 mg;; (w/v) IO. Sulfuric acid II. Liebermann-Burchard reagent. This was prepared from a mixture of acetic anhydride, sulfuric acid, and acetic acid as described by HenryI”. 12. Cholesterol standard, 0.4 mg()b in absolute alcohol 13. Cholesterol standard, 2.0 mgq, in chloroform 14. Ferric chloride reagent, 0.059; (w/v) FeC1,.6H,O in glacial acetic acid.

Coulometric measurements were made with a ChrisFeld Microcoulometric Quantalyzer, Model 6. This instrument was calibrated by measurement of the potential drop across a roe-ohm, 0.10/o precision resistor with a Leeds and Northrup 8687 Volt Potentiometer. Bromine was generated coulometrically from a platinum anode (1.0 cm2). The platinum cathode (2.4 cm2) was isolated from the test solution with a tube constructed of “thirsty glass” (Corning Glass Works, Corning, N.Y.) sealed onto the end of a glass tube with a Manolok Teflon sleeve (Manostat Corp., New York). A IO?,; lithium chloride solution served as the catholyte. The titration assembly consisted of a ro-ml titration cell and a cover in which the two generating and two indicating electrodes were located. The indicating electrodes were two platinum wire electrodes (0.025 cm2) with 150mV impressed between them. The impressed potential was supplied by a Model XV Sargent Polarograph. All biamperometric measurements were made by recording the change in current on the polarograph. The end point was determined by recording the current until an arbitrary current reading was attained above the base line for both the blank and test solutions. Visible absorbance was recorded on a Beckman DB spectrophotometer. Thin-layer chromatographic studies were made using the ascending technique with MN-Polygram silica gel plates (Brinkmann Instruments Inc., Westbury, N.Y.) and ChromAR Sheet IOOO (Mallinckrodt Chemical Works, St. Louis, MO.). The latter material is composed of 70% SilicAR-TLC 75 (silicic acid) with 30% glass fiber binder and is I mm thick. Detection reagents were applied using the spray technique with an aerosol detection kit (NBC Co., Cleveland, Ohio).

Thin-layer

chromatography

Test compounds were generally prepared at a concentration of IO mg/ml in chloroform. Solutions of standards were applied with a 5-,LI~pipet. The thin-layer plates were impregnated with silver nitrate by placing them in a tank containing IOO mg”/o silver nitrate and allowing the solvent to migrate over the entire plate. The plates were air dried in the dark, placed in a 100~ oven for 30 mm, and stored in a desiccator which was protected from direct light. Two solutions, a chloroform and a chloroform-ether (90 : 2) solution which was saturated with water, were used to develop the chromatograms. Clzn. Chim. Acta, 26 (1~60) 155-163

158

TROY,

PL’RJ)Y

Test compounds were detected by spraying the plates with 5010 sulfuric acid and heating them on a hot plate. Materials were quantitatively eluted from Mallinckrodt ChromAK Sheet \vith the Osdova elution tecllnique13. This consisted of cutting the separated material from the strip in the form of a “benzene ring,” applying the solvent at one point, and collecting it off the opposite point. A chloroforn~methanol (3: I) solution was the eluting solvent and was applied with a syringe and a zz-gauge needle. COULOMETRIC

ANALYSIS

OF

TOTAL

CHOLESTEROL

1Z-G SERL~JI

Cholesterol esters were converted to free cholesterol by incubating o.5 ml of serum in 5.0 ml of alcoholic potassium hydroxide at 37’ for 55 min. The mixture lyas then cooled to room temperature. Ten ml of hexane was added and mixed. Five ml of water was added and the mixture was shaken vigorously for I min. The phases were allowed to separate. A z-ml aliquot of the hexane phase was placed in a rz-ml centrifuge tube with a conical bottom and the hexane was evaporated at 60” under nitrogen. The residue was dissolved in IOO 1’1 of chloroform and the tube was washed with two additional IOO-,~l portions of chloroform. This extract was transferred to a Mallinckrodt ChromAK Sheet impregnated with silver nitrate. It had the dimensions of 2.5 x 20 cm, and the solution was applied in the form of a streak 2 cm from the edge of the sheet. This was chromatographed for 30 min in a solvent of chloroforn-ether (90 : 2). The location of the cholesterol was determined by comparison with a cholesterol standard chromatographed in the same manner. The material was eluted with IO ml of chloroforni-methanol (3: I) into the titration cell. The solvent was evaporated at 60” under nitrogen and cooled to room temperature. The isolated cholesterol fraction was dissolved in IO ml of generating reagent, and 3 Ciequiv of bromine was generated coulometrically. The bromine was allowed to react for 5 min at room temperature and then 25 1’1 of standard arsenious acid was added. The residual arsenic (III) was determined with coulometrically generated bromine and the end point was determined biamperometrically. The bromine was generated at a current of 0.965 mA which generated IO nequiv/sec. The end point was taken as the current reading o.1 ,LJA above the base line. The blank was determined bv treating 0.5 ml of water in an identical manner except that z pequiv of bromine was generated in excess. 7‘ is defined as the total bromine consumed by the sample and B is the total bromine consumed by the blank, both expressed in terms of seconds. Calculations Titer (serum chOlesterO1, 0.01

/leqUiV/SW

I.933 =

nig”,) X 0.1933

= (?‘--H) lllg/,LEqUiV

X

1.933 X 100

0.1 ml

Comparison methods The method of Abel1 et aLI is generally accepted as the reference method in clinical analysis. This method is described in detail by HenryIs. It consists of a saponification of the cholesterol esters with alcoholic potassium hydroxide, extraction into hexane, evaporation of the solvent, and measurement of the cholesterol with the Liebermann-Burchard color reaction. A commonly used routine method for the Clin. Chim.

Acta.

26

(1969)

15.y16.3

COULOMETRY

OF SERUM CHOLESTEROL

159

analysis is based on the color reaction of cholesterol with ferric chloride and sulfuric acid; the details of the procedure are described by Henry12. Briefly, the procedure consists of a precipitation of the proteins with the ferric chloride-acetic acid reagent, and the addition of sulfuric acid to an aliquot of the supernatant for calorimetric comparison. RESULTS AND DISCUSSIOW

of cho2esterol In developing a workable coulometric cholesterol, three conditions must be fulfilled:

Brominatio9t

TABLE

procedure for the determination of cholesterol must be soluble in the

(I)

I

TITXATIOK

EFFICIENCY

AT

VARIOUS

CURRENT

DENSITIES

Cuwent density (pA/cwS)

Compound

Arsmious acid Arscnious acid Arsenious acid Cyclohexene Cyciohcsene Cyclohexene

Titration e@cicncy (96)

..-

96.5

92.0

2x9.5 965 96.5 289.5 965

97.3 99-g 92.9 96.3 99.9

._ ._.~

solvent medium, (2) cholesterol should react rapidly with bromine in this medium, and (3) bromine should be generated with IOO~/~ titration efficiency in this medium. Various systems of sodium bromide in dioxane-water, pyridine sulfate-acetic acid, and methanol-acetic acid were evaluated for ease of bromination with coulometritally generated bromine. A system which was proposed by Leisey and Grutsche for the coulometric titration of cyclohexene yielded the most promising results. The biamperometric end point did not correspond to the stoichiometric end point. This titration was studied as a function of temperature and it was noted that the titration gave a sharp biamperometric break at a temperature of 4~~. This end point indicated that more than one mole of bromine was consumed by cholesterol, which probably means that secondary substitution reactions were also proceeding. At temperatures less than 45”, the reaction proceeded at a slower rate and it was difficult to get reproducible end points. This system was evaluated for titration efficiency with cyclohexene and arsenite at various current densities. Table I shows the results of these studies. Results indicated that a current of 0.965 mA gave more reproducible values and approached IOO 0; titration efficiency. TABLE

II

REACTION

OF

T:;, Excess

bromine

rn.0

CHOLESTEROL

5

mill

pg Tlatwy 250.6 250.6 250.6 ‘25.3

0.2 2.4

125.3

0.8

“25.3

4.8

250.0

256.6 125.3 124.3 13X.3

-

FOR

215.5

100.0

LOO.0

RROMINE

pg Cafe.

200.0 IO.0 100.0

WITH

‘J;>rxffcveltcc 2.0

0.0

-_--

160

Preliminary results demonstrated that reproducible results would be difficult to obtain in direct titrations of cholesterol. Residual titrations were investigated; excess bromine was generated and allowed to react with cholesterol for given periods of time. Results in Table II show the izg of cholesterol calculated from residual titrationswhen different levels of bromine were allowed to react for 5 min at room temperature (24”). Table III shows a similar study in which bromine was allowed to react for IO min. In general, the increased time tended to increase the danger of substitution; therefore a 5-min reaction time was used in subsequent studies. Isolation techbqlccs Since the bromination

of organic

compounds

is a very common

reaction,

it is

necessary to isolate cholesterol in a highly purified form before any meaningful quantitative results can be obtained in the analysis of biological materials. Cholesterol is present as the free alcohol and esterified with fatty acids in most biological samples.

RECOVERY

OF CHOLESTEROL

FROM

Before cholesterol is reacted alcohol form and separate it et al.‘* employed a procedure the isolation step. It consists and extraction of the steroid

HEXANE-EXTRACI’ABLE

A HEX.\T\;E

EXTRACT

with bromine, it is necessary to convert it to the free from other materials which react with bromine. Abel1 which does provide an effective extraction technique for of a saponification with alcoholic potassium hydroxide into hexane. Table TV shows the per cent recovery of

JIATERIALS

IX

NORMAL

SERUM

. Comnfxmd

il’owml

Cholesterol r7-Ketosteroids \‘itamin A Carotene Tocophcrol Calciferot A ‘-Steroids Cholestanol _ ._

‘97

CEiul.Chim. Acta,

stwtm

0.17’

0.058 0.220

o.qso 0.00275 1.1 5.0 26 (r969) 155-10.3

value ._

(wtg~~]

COULOMETRY

il.

OF SERVM

161

CHOLESTEROL

COULOMETRIC ASSAYOF VARIOUSExTRACTSOF COXTROL

-.

Watev wash

HCL wash

Digitonin

(%%)

(wObl

(%?Ol

(%?61

Hexane

.Sampk

\‘ersatol .I

extract

Hyland

20x.0

102.2

87.4 184.X

H?;la~ltl A

200.2 652.7

182.7 628.0

Serachol b.

SERUN

COMPARISON

COLORIMETRIC Assay-I15

85.’

187.8 182.4 636.9

isolatim

X3.8

170.0 156.3 307.9

TECHSIQUES (mxg:o)

so.0 168.0

Assay-212

(mg%)

go.0 166.3 161.6

‘57.5 344~7

319.6

cholesterol when it was carried through such a procedure; the isolated cholesterol was analyzed coulometrically. Table V shows the composition of steroids and some other hexane-extractable materials which are known to be normally present in serum. Prom this table it would appear that IT-ketosteroids, tocopherol, carotene, and the .1’steroids would contribute to the greater error in the analysis of cholesterol through bromination. The residual bromination technique was used on untreated hexane extracts for total cholesterol. These extracts were subsequently washed with an additional s-ml portion of water, and then with an additional 5-ml portion of 0.1 Ai HCl. Finally, they were isolated through a technique described by Kelsey 20 in which cholesterol is isolated as the digitonide. Cholesterol can be regenerated from the digitonide by treating the precipitate with boiling benzene. Table VI shows the progression of results after these various treatments when compared to two standard calorimetric assays of cholesterol. It may be noted that the results are about zoy/, high in the untreated hexane extracts, with the exception of the Serachol samples, when compared to the calorimetric procedures. Serachol does contain a material which improves the solubility of cholesterol, and does not interfere with the calorimetric techniques. The digitonin isolation procedure does isolate cholesterol from many interfering substances, but does not necessarily isolate it from other steroids which may be present in pathological or pharmacologically induced states. Thin-layer chromatography was used to study the separation of certain test TABLE

VII

RELATIVE

MOBILITY OF SELECTED STEROIDS

Comfmunds

Rdntiue

Cholesterol* 7-Dehydrocholesterol Desmosterol Dihydrocholesterol Lanosterol Lathosterol *

Cholesterol

mobility

1.00 0.38 0.78 1.27 I.88 1.18

RF 0. ii. Clin. Chim.

Acta,

16 (1969) 755-163

TROY, PUKD\r

162

steroids which are reported to be present in serum or are precursors in the synthesis of cholesterol. Table VII shows the relative mobility of such compounds in a system of water-saturated chloroform-ether (go : 2) in which a silver nitrate impregnated ChromAR sheet was used. Preliminary studies indicated that similar results were obtained with chloroform, but subsequent studies confirmed that the mobilities were less reproducible. This system did effectively separate many of the steroids and was chosen for use in further investigations on the basis of its ability to separate cholesterol from its f17-isomer. The ChromAR sheet was chosen for chromatographic studies for two reasons: (I) it has the advantage of allowing the separation to occur in a relatively short period of time (30 min) and (2) due to its method of construction,

samples

may be easily eluted.

Many of the elution

techniques

described

for paper

chromatography may be used without the attendant losses of adsorbent that are frequently a problem with TLC. Table VIII shows the recoveries of a IOO-pg sample of cholesterol from ChromAR sheets when the Osdova elution techniqueI is used. Analysis of seyurn samples Since the TLC technique seemed capable of yielding good recoveries of cholesterol, it was used to analyze control serum and IO serum samples chosen at random. An aliquot of the hexane extract was transferred to the ChromAR sheet, chromatographed, eluted, and titrated. A small amount of silver nitrate was eluted with the solvent, but this did not interfere with the titration. The amount of excess bromine TABLE

IX

Versatol A Hyland Hpland

80.0 168.0

80.0 168.0 I 60.0 349.9 184.4 228.9 14I.I 165.~ 163.2 164.9 186.9 212.2 161.8 I42.2 X.6

157.5

161.6

Serachol I

344.7 183.9

319.6 1X6.2

2 3 4 5 6 7 s 9

242.1 140.1 167.6 163.5 rho.2 IXI.6 217.X 173.5

233.9 I42.2 166.2 171.6

IO Std. Dev.

“47.5 2.5

754.9 4.0

Clin.

A

90.0 166.3

Chim.

Acta,

26 (1969)

‘77.4 187.6 220.1

178.2

155-163

I02.2 20x.0 200.2 “52.7 212.7 228.7 163.H 198.0 187.8 196.6 2IS.3 26j.0 204.0 IX1.j 8.8

COULOMETRY

OF SERCM

CHOLESTEROL

163

was arbitrarily chosen. It was based on the assumption that there is a o.5-,Llequiv blank and most serum samples have a concentration range for cholesterol between IOO and 400 rng%. The excess bromine in such an experimental design will range from 50 to ZOOMS.The results of these titrations are shown in Table IX. The t-test at the 75O;, probability level indicated that there was no significant difference between the TLC-coulometric procedure and the Assay I procedure (Abel1 et ~1.~~).Since the TLCcoulometric method is an entirely different approach to cholesterol analysis, the results in Table IX seem to substantiate the specificity claimed by the Abel1 calorimetric procedure in the analysis of routine serum samples. Direct coulometric titrations of hexane extracts on IO normal sera also tend to give high results when compared to the Abel1 calorimetric method. It is difficult to determine all the materials which contribute to this procedure’s positive error. Tocopherol, carotene, 7-ketosteroids, and the d7-steroids could contribute to only half of this quantity. Some other sources may be the porphyrin metabolites of hemoglobin and bilirubin. Although the results of the TLC-coulometric method do not yield the same precision as the calorimetric methods, this former method may value in accurate determinations in those cases where an abnormal other closely related steroids in serum.

be of considerable state may produce

ACKNOWLEDGEMENT

The authors are indebted to the Office of the Surgeon General of the U.S. Army for partial support of this work under Contract No. DADA17-67~C7161. REFERENCES I

2 3 4 5 6 7 8 9 10 II

12 13 14 15 16 17 18 19 20

E. SALKOWSKI, Pflugev’s Arch. Ges. Physiol., 6 (1872) 207. C. LIEBERMANN, Ber., 18 (1885) 1893. H. BURCHARD, Ph.D. Dissertation, Restock, Germany, 1889. A. WINDAUS, Rer., 49 (1916) 1724. H. T. GORDON, Anal. Chem., 23 (1951) 1853. F. A. LEISEY AND J. F. GRUTSCH, Anal. Chem., 28 (1956) 1553. W. C. PURDY, 2. Anal. Chem., 243 (1968) 17. N. RADIN, in S. MEITES (Ed.), StandardMethods of Clinical Chemistry, Vol. T’, Academic Press, New York, 1965, p. 91. K. DOBRIXER, E. R. KATZENELLENBOGEN AND R. N. JOSES, Infrared Absorption ofSteroids, 1~01. I, Interscience Publishers, Inc., New York, 1953. p. 41. L. F. FIESER AND J. E. HERZ, J. Am. Chem. Sot., 75 (1953) 121. K. DOBRINER, E. R. KATZENLLENROGEN AND R. N. JONES, op. cit., p. 44. R. J. HENRY, Clinical Chemistry, Harper & Row Publishers, New York, 1964, p. 855. 0. V. DOMINQUEZ, in H. CARSTENSEX (Ed.), Standard Hormone Analysis, 1’01. I, Marcel Dekker, New York, 1967, p. 302. L. I,. ABELL, B. B. LEVY B. B. BRODIE AND F. E. KENDALL, ,I, Biol. Chem., 195 (1932) 357. R. J. HER.RY, op. cit., p. 852. D. L. ALTMAN, in D. S. DITTMER (Ed.), Blood and Other Body Fluids, Fed. of Am. Sot. Exptl. Biol., Washington, D.C., 1961. K. NAKANISHI, B. K. BHATTACHARRY~ AND L. F. FIESER, J. Am. Chem. Sot., 75 (1953) 4415. XI. GREIG AND R. P. COOK, B&hem. J., 77 (1960) 16P. R. SCHOENHEIMER, R. H. BOHR~NG AND R. HUMMEL, Z. Physiol. Chem., 192 (1930) 93. F. E. KELSEY, J, Biol. Chem., 127 (1939) 15.

C/in. Chim.

Acta,

26 (1969)

155-163