AMERICAN JOURNAL OF CLINICAL PATHOLOGY Vol. 31, No. 4, April, 1959, pp. 305-309 Printed in U.S.A. STUDY OF THE FERRIC CHLORIDE METHOD FOR DETERMINATION OF TOTAL CHOLESTEROL AND CHOLESTEROL ESTERS NEIL CHIAMORI, B.S., AND RICHARD J. HENRY, M.D. Bio-Science Research Foundation, Los Angeles, California Several of the newer technics that have been introduced for the determination of serum cholesterol so far have been applied only to the total cholesterol. For routine tests in a clinical laboratory, it is desirable to have a cholesterol method that can be performed not only rapidly and precisely in testing a large number of samples, but that can also be applied to the determination of cholesterol esters when requested. The present authors, therefore, attempted application of some of these methods to analysis for the esters. Trials with the p-toluenesulfonic acid method of Pearson and his associates 10 failed because of conspicuous interference from even a trace of digitonide. The direct ferric chloride method of Zlatkis and co-workers, 17 in which the reaction is performed without precipitation of proteins, was deemed unsuitable because of the interference from bilirubin in icteric serums when total cholesterol is determined. Zak and associates, 16 Crawford, 4 and Ferrand and RiefTel 5 circumvented this difficulty by applying the ferric chloride reaction to organic solvent extracts of serum. Henly 6 and Zak, 15 in a different approach, precipitated the proteins with a ferric chloride-acetic acid reagent, bilirubin being effectively removed by coprecipitation. This article includes a study of the method of Henly 6 for the determination of total cholesterol with adaptation for the determination of cholesterol esters. These technics are then evaluated by comparison with the methods of Schoenheimer and Sperry 13 and Bloor. 2 METHODS Reagents 1. Ferric chloride-acetic acid reagent. FeCIs'GHaO, 0.05 per cent (w/v) in glacial Received, October 13, 1958; accepted for publication December 2. Mr. Chiamori is Research Chemist, and Dr. Henry is Director. Their address is 12318 Santa Monica Boulevard, Los Angeles 25, California. 305 acetic acid. Store at room temperature in polyethylene bottle. Stable at least 1 month. 2. Concentrated H 2 S0 4. 3. Elhanol-ether solvent mixture. 3 vols, of 95 per cent ethanol + 1 vol. of ether. Ethanol denatured with methanol is satisfactory if 5 per cent water is added. 4. Digitonin solution. Digitonin solution, 0.5 per cent, in 95 per cent ethanol. 5. Petroleum ether. B. P. 30 to 60 C. 6. Stock cholesterol standard, in chloroform, 2.00 mg. per ml. 7. Working cholesterol standard, 0.2 mg. per ml. Dilute the stock standard 1:10 with chloroform. Procedure for Total Cholesterol Using a T. C. pipet, rinse 0.1 ml. serum or plasma into 9.9 ml. of the FeCU-acetic acid reagent contained in a test tube. Mix the contents well and set aside for 10 to 15 min. Heat the tubes in a 60 C. water-bath for 2 min. with occasional shaking. Allow to cool to room temperature and centrifuge. Set up the following in large test tubes (25 by 150 mm.) : Blank. Five milliliters FeCl3-acetic acid reagent. Standard. Place 1.0 ml. working cholesterol standard in test tube, bring to dryness at 60 C, then add 5 ml. FeCU-acetic acid reagent. Evaporation of the CHCI3 can be hastened by a forced stream of air or N 2. Unknown. Five milliliters of supernatant fluid. Add 3.0 ml. concentrated H2SO4 to each tube from a buret and mix immediately. After 10 min., read the absorbance (A) at 560 m/x or with a filter with nominal wavelength in this region (a Klett No. 54 is satisfactory) against a water blank. Calculation: = mg. of cholesterol per 100 ml.
306 CHIAMORI AND HENRY Vol. 81 Procedure for Cholesterol Esters Transfer 0.4 ml. serum or plasma to a dry, 10-ml. volumetric flask. Add 5 ml. ethanolether solvent mixture and bring to boiling point for a few seconds by immersion in a 60 C. water-bath. Let flask come to room temperature, add solvent mixture to volume, and mix. Filter through a dry Whatman No. 1 filter paper. Transfer 2.5 ml. of filtrate to a large test tube (25 by 150 mm.), add 0.5 ml. of digitonin solution, and mix. Evaporate to dryness in a water-bath at 60 to 80 C. This evaporation can be hastened by a stream of air or N 2. Extract the cholesterol esters from the residue by adding approximately 5 ml. of petroleum ether, bring to boil in a 60 C. water-bath, allow to cool, stopper, centrifuge, and decant supernatant fluid into a large test tube. Repeat the extraction and combine extracts. Evaporate to dryness under air or nitrogen at 60 to 80 C. Proceed as for total cholesterol, setting up a blank and standard as for total cholesterol. Calculation: A, A B X 200 A H - A B = mg. of cholesterol esters, as free cholesterol, per 100 ml. INVESTIGATION OF METHOD Characteristics of color produced. The color reached a maximum in 5 min. and was stable for at least 1 hr. The absorption curves for free cholesterol and cholesterol esters (palmitate, oleate, and stearate) were identical, peak absorbance occurring at 560 nut. The color developed in accordance with Beer's law in a Beckman model DU spectrophotometer and in Klett photometer with a No. 54 filter. Addition of the concentrated sulfuric acid to the ferric chloride-acetic acid reagent results in evolution of considerable heat, which hastens the attainment of peak color intensity and increases the degree of color formed. Thus, in one typical experiment, the Klett readings obtained at 30 min. for 200 Mg. standards, mixed and immediately placed in water-baths at 18, 25, and 37 C, were 299, 306, and 322, respectively. Another standard kept in air at room temperature (25 C.) read 330. In laboratories where the room temperature is subject to wide fluctuation it would be best to control temperature by use of a constant temperature water-bath. The reaction is performed in the absence of water, and great care must be taken that there is no more than trace water contamination. For example, a bottle of concentrated sulfuric acid can absorb sufficient water from the air to result in decreased color. Thus, the presence of 0.2 ml. or more of water in the final color causes significant color suppression. This effect of water has been previously reported. 16 Relative absorptivilies of free cholesterol and its esters. The ratio of color intensity formed with cholesterol esters to that with free cholesterol in equimolar concentration is given in Table 1 for both the Liebermann- Burchard reaction and the ferric chloridesulfuric acid reaction. The predominant TABLE 1 ABSORPTIVITIES OF CHOLESTEROL ESTERS RELATIVE TO FREE CHOLESTEROL Cholesterol Cholesterol palmitate Cholesterol oleate Cholesterol stearate Source and Number of Lot Reagent Chemicals, 190* K & K Laboratories, 83011 K & K Laboratories, 5812f K & K Laboratories, 5809t Temperature of Liquefaction Found * Standard Scientific Supply Corp., New York 12, New York, t K & K Laboratories, Inc., Long Island City 1, New York. Reported by Cook! degrees C. degrees C. 144-146 149.5-150 72-74 75 42-43 35 74-76 71 Absorptivity Relative to Free Cholesterol Liebermann- Burchard 1.17 1.18 1.24 I'erric cliloridesulfuric acid 0.95 0.95 0.86
April 1959 TOTAL CHOLESTEROL AND CHOLESTEROL ESTERS 307 cholesterol esters of human blood serum are reported to be the palmitate, oleate, and linoleate. 7 ' 9 ' 14 It is revealed in Table 1 that the palmitate and oleate manifested approximately 17 to 18 per cent more color than free cholesterol in the Liebermann- Burchard reaction, and approximately 5 per cent less color in the ferric chloride-sulfuric acid reaction. There were somewhat greater discrepancies with the stearate, but the amount of this ester in human blood seems to be relatively small. 7 ' u The authors were unable to locate any cholesterol linoleate for this study. Study of interferences. Severe icterus and lipemia did not interfere with the ferric chloride-sulfuric acid method. The following substances in the stated concentrations, however, did cause approximately a 10 per cent positive error: 1 per cent hemoglobin, 10 mg. bromide as NaBr per 100 ml., and 10 mg. iodide per 100 ml. The interference by hemoglobin makes the method inapplicable to whole blood, but slight hemolysis in serum can be tolerated. Iodide interference can be ignored, inasmuch as such high levels would not be encountered in serum. Interference by bromide does constitute a problem, inasmuch as levels as high as 300 mg. per 100 ml. may occasionally be encountered. The use of silver iodate to precipitate the bromide, used by Rice and Lukasiewicz 11 in the method of Zak and associates, 16 where cholesterol is extracted by organic solvent, proved to be unsatisfactory, inasmuch as excess silver iodate interfered with the subsequent color reaction. Removal of the bromide, however, was successfully accomplished by batchwise use of an ion exchange resin. Approximately 1 ml. of serum and 200 mg. Dowex 2 (Cl~ form, 200 to 400 mesh) are placed in a test tube and continuously mixed on a shaking machine for 10 min. The sample for analysis is then removed from the supernatant fluid. The results of 3 typical experiments are given in Table 2. Fortunately, the presence of interfering quantities of bromide is easily detectable early in an analysis as a distinct yellow color appearing in the supernatant fluid and the protein precipitate after addition of the ferric chloride-acetic acid reagent, in contrast to the usual straw color. TABLE 2 REMOVAL OF INTERFERENCE BY BROMIDE Total Cholesterol (mg. per WO ml.) Serum 1 Serum 2 Serum 3 Serum Serum + 320 mg. of NaBr per 100 nil. Serum + resin Serum + 320 mg. of NaBr per 100 ml. + resin 24S 307 255 25S 220 279 230 231 201 249 20S 212 TABLE 3 COMPARISON OF RESULTS FROM THREE METHODS Cholesterol Total, expressed as free Esters, expressed as free Free * See text. Schoen- heimer- Sperry 220 161 59 FeCls- Sulfuric Acid 225 164 61 ISS 63 Bloor Uncorrected 251 Corrected* 219 156 Completeness of petroleum ether extraction of cholesterol esters. Six serums were studied in duplicate, the cholesterol esters determined in 4 successive extractions of the digitonide precipitate. The first extraction removed 90 to 97 per cent of the esters (x 95 per cent), and the first 2 extractions removed 97 to 100 per cent (x 99 per cent). Comparison of results with the methods of Bloor and Schoenheimer-Sperry. A comparison of total cholesterol, cholesterol esters, and free cholesterol, as determined by the 3 methods, is given in Table 3. The results represent the means of quadruplicate determinations on 6 different serums. Results obtained by the Schoenheimer-Sperry method and the ferric chloride-sulfuric acid reaction are in excellent agreement. Bloor's method, however, yielded the expected higher results for total cholesterol and cholesterol esters, attributable to the higher relative absorptivity of the esters in the Liebermann-Burchard reaction. In the last column of Table 3 the results are corrected
308 CHIAMORI AND HENRY Vol. 31 for this difference in relative absorptivity, by subtracting 17 per cent from the values for the esters. This correction succeeded in bringing the results in close agreement with the other 2 methods. Precision of the ferric chloride-sulfuric acid method. The precision (2 standard deviations) for the determination of total and ester cholesterol determined from 25 duplicate routine analyses were ± 6.5 and ± 10.1 mg. per 100 ml., respectively. The 2 standard deviation limits for total cholesterol from a quality control chart on a pooled serum were =fc 12 mg. per 100 ml. These are more realistic limits, inasmuch as they include the variation between days and analysts. DISCUSS [ON Since its introduction in 1916 until relatively recently, the method of Bloor 2 was probably the most widely used method for the routine determination of cholesterol in the clinical laboratory. The method of Schoenheimer and Sperry 13 and subsequent modifications, in which cholesterol esters are saponified to free cholesterol, have been generally recognized as the methods of reference, but the technics have been too time consuming for routine use. It has been known for some time that any method, such as Bloor's, that applies the Liebermann- Burchard color reaction without prior saponification of the cholesterol esters, is inaccurate because of the higher relative absorptivities of the esters in the color reaction compared to free cholesterol. 8 Kingsley and Schaffert 8 decreased this error by applying a standard correction based on an average ratio of esters to free cholesterol. Without correction, however, results are spuriously high by approximately 16 per cent. 1 For more than 2 decades, therefore, this has constituted a major source of discrepancy of results reported from different laboratories, and is a partial explanation for such interlaboratory disagreement as noted by Rivin and associates. 12 The ferric chloride-sulfuric acid reaction, as used in the technic described herein, or in a similar method, possesses definite advantages either for routine or research use over most methods previously suggested: (1) it is relatively simple and is adaptable to determinations in large numbers, (2) the reaction is very sensitive, permitting routine analysis at micro or ultra-micro levels, (3) reproducibility is quite satisfactory and better than many previous technics, probably because of fewer manipulations, (4) the method is accurate, as judged by agreement with the method of Schoenheimer and Sperry, and (5) it can be applied to the determination of cholesterol esters. SUMMARY A study has been made of the determination of total cholesterol and cholesterol esters in serum by precipitation of protein with ferric chloride-acetic acid reagent and then developing color with sulfuric acid. Interference by any bromide present can be avoided by removal with an ion exchange resin. Evidence of the accuracy of the technic was obtained by a study of various pure cholesterol esters and a comparison of results obtained on serums with the Schoenheimer- Sperry method. SUMMARIO IN INTERLINGUA Esseva effectuate un studio del methodo de determinar cholesterol total e esteres de cholesterol in sero per precipitar le proteina con un reagente de chloruro ferric e acido acetic, sequite per le disveloppamento de color per medio de acido sulfuric. Disturbationes resultante del presentia possibile de bromuro pote esser evitate per eliminar le bromuro per medio de un resina de excambio ionic. Un prova del accuratia del technica esseva obtenite per un studio de varie esteres cholesterolic pur e un comparation del resultatos obtenite in seros per medio del methodo de Schoenheimer-Sperry. REFERENCES 1. ANDERSON, J. T., AND KEYS, A.: Cholesterol in serum and lipoprotein fractions. Clin. Chem., 2: 145-159, 1956. 2. BLOOR, W. R.: The determination of cholesterol in blood. J. Biol. Chem., 24: 227-231, 1916. 3. COOK, R. P.: Cholesterol. New York: Academic Press, Inc., 1958, pp. 24, 89. 4. CRAWFORD, N.: An improved method for the determination of free and total cholesterol using the ferric chloride reaction. Clin. Chim. Acta., 3: 357-367, 1958. 5. FERRAND, P., AND RIEFFEL, C: Etude de la methode de Zak pour le dosage du cholesterol serique par la reaction de Zlatkis
April 1959 TOTAL CHOLESTEROL AND CHOLESTEROL ESTERS 309 et Zak. Ann. de Biol. Clinique, 16: 299-307, 1958. 6. HENLY, A. A.: The determination of serum cholesterol. Analyst, 82: 286-287, 1957. 7. JAMBS, A. T., LOVELOCK, J. E., WEBB, J., AND TROTTER, W. R.: The fatty acids of the blood in coronary-artery disease. Lancet, 1: 705-708, 1957. S. KINCSLEY, G. R., AND SCHAFPBBT, R. R.: Determination of free and total cholesterol by direct chloroform extraction. J. Biol. Chem., 180: 315-328, 1949. 9. MICHALEC, C: Nature of cholesterol esters in human blood serum. Biochim. et Biophys. Acta, 19: 187-188, 1956. 10. PEARSON, S., STERN, S., AND MCGAVACK, T. H.: A rapid, accurate method for the determination of total cholesterol in serum. Anal. Chem., 25: 813-814, 1953. 11. RICE, E. W., AND LUKASIEWICZ, D. B.: Interference of bromide in the Zak ferric chloridesulfuric acid cholesterol method, and means of eliminating this interference. Clin. Chem., 3: 160-162, 1957. 12. RIVIN, A. V., YOSHINO, J., SHICKMAN, M., AND SCHJBIDE, 0. A.: Serum cholesterol measurement hazards in clinical interpretation. J. A. M. A., 166: 210S-2111, 195S. 13. SCHOENHEIMBR, R., AND SPBRRY, W. M.: A micromethod for the determination of free and combined cholesterol. J. Biol. Chem., 106: 745-760, 1934. 14. TUNA, N., RECKERS, L., AND FRANTZ, I. D., JR.: The fatty acids of total lipids and cholesterol esters from normal plasma and atheromatous plaques. J. Clin. Invest., 37: 1153-1165, 195S. 15. ZAK, B.: Simple rapid microtechnic for serum total cholesterol. Am. J. Clin. Path., 27: 583-58S, 1957. 16. ZAK, B., DICKENMAN, R. L., WHITE, E. G., BURNET, H., AND CHERNEY, P. J.: Rapid estimation of free and total cholesterol. Am. J. Clin. Path., 24: 1307-1315, 1954. 17. ZLATKIS, A., ZAK, B., AND BOYLE, A. J.: A new method for the direct determination of serum cholesterol. J. Lab. & Clin. Med., 41: 4S6-492, 1953.