Errors Associated with the Direct Measurement of Human Serum Cholesterol Using the FeCI3 Color Reagent

Transcription

1 Errors Associated with the Direct Measurement of Human Serum Cholesterol Using the FeCI3 Color Reagent Robert V. Moore and Edwin Boyle, Jr. Evidence is presented concerning errors in the direct determination of human serum cholesterol with a ferric-chioride color reagent. It is shown that in physiologic concentrations the vitamins A, D, E, K, and niotinic acid cause no error, but that hemoglobin, anticoagulants, and impurities in acetic acid can causeerrors. The control of these errors is discussed. Errors caused by steroids, added salts, and high bilirubin content of serumare discussed. THERE ARE reports (1, 2) criticizing the direct-cholesterol method of Zlatkis et al. (3), which utilizes a ferric-chioride color reagent, for giving high values for the cholesterol concentrations in human sera. Zlatkis et al. warned that glyoxyiic acid, which occurs in commercial acetic acid, might interfere with the test. (Glyoxylic acid reacts with tryptophan, from the protein in serum, in the presence of concentrated 112S04, to give a color which is measurable at the wavelength used in the cholesterol procedure.) When acetic acid is refluxed with Cr03, then distilled, as described by Maclntyre and Ralston (4), all impurities which affect this test are removed and the cholesterol values obtained using the Fe Cl3 color reagent are comparable to values obtained using extraction followed by the Liebermann-Burchard color reagent. Henly (5) and Morris (2) using similarly distilled acetic acid confirmed this finding. These three papers, however, did not show why Cr03 was necessary. Henly (5) also pointed out that the original color reagent of Zlatkis was unstable and that it was water sensitive. These From the Lipid Metabolism Laboratory, Department of Medicine, Medical College of South Carolina, Charleston, S. 0. This work was supported in part by the American Heart Association and the South Carolina Heart Association. Received for publication Nov. 2,

2 Vol. 9, No. 2, 1963 SERUM CHOLESTEROL 157 objections are overcome by the use of the FeC13 color reagent modifications of Rosenthal et at. (6). The sensitivity of the FeC13 color reagent, which is greater than the Liebermann-Burchard reagent, is pointed out by Henly (5) and Smit and Weymeyer (7). This sensitivity, which magnifies small ei rors in technic and measurement, and errors caused by changes in initial temperatures are pointed out by Furst and Lange (8). Kinley and Krause (9) have reported that vitamin A seriously affects direct human serum cholesterol values. fbilinger et at. (10), without giving any data, claim that the FeCl3 reagent of Zlatkis... reacted with so many agents in blood serum as to preclude specificity for cholesterol. It is the purpose of this paper to present data relative to the aforementioned criticisms as applied to human serum-cholesterol measurements using the Rosenthal color reagent with the Zlatkis procedure. The reasons for distilling and the method of distillation of acetic acid will be given. Also included are data on the effects of hemolysis, vitamins other than A, and errors caused by anticoagulants used in preparing plasma. Materials and Methods All reagents and all solvents were reagent grade except the following: cholesterol, which was recrystallized until the final product had a melting point of #{176} ; vitamins A, D, E, and K, which were commercial forms; and nicotinic acid powder U.S.P. The cholesterol procedure used was that described by Zlatkis et at (3) using the color reagent modification of Rosenthal et al (6). Essentially it consists in pipetting ml. of serum into a suitable container* adding 3 ml. of redistilled acetic acid, then adding 2 ml. of fernc chloride color reagent down the side of the container ill such a way as to layer it below the acetic acid. The container is capped and the mixture is abruptly and thoroughly mixed. After 10 miii. the absorbance is read at 560 m in a spectrophotometer. The color is completely stable for 10 mm. A standard cholesterol in acetic acid is measured daily. Cholesterol values of lipoprotein fractions obtained by preparative ultracentrifugation are measured as previously described (11). Distillation of acetic acid was carried out in an all-glass apparatus using a Claisen distilling head. Distillation was started with 2 L. of acid and stopped after it was shown that the acetic acid obtained gave This laboratory has found the Plastainer vial (Owens-Illinois Glass Co.), 7 dram, to be a very satisfactory container as it is resistant to chemical reaction and breakage.

3 158 MOORE & BOYLE Chnical Ch.mhtry reproducible values, which occurred after about 1 L. of acid had beeii distilled. The first 10-mi. aliquot from each 50 ml. distilled was tested for purity by its effect on a standard cholesterol solution, human serum, and a protein and high-density lipoprotein fraction of human serum (11)-the infranate of serum separated at gm. density in a preparative ultracentrifuge. Acetic acids from Baker and Adamson,.J. A. Baker Chemical Company, Nallinckrodt Chemical Works, E. I. du Pont, and Merck and Company, Inc., have all been found to need distillation, but the quantity of impurity has been found to vary from batch to batch within each company s product. In the distillation experiment reported only one batch of acid was used. After proper distillation the acids from all sources are equivalent. Results and Discussion The error in serum cholesterol values caused by the reaction of glyoxylic acid in undistilled acetic acid with tryptophan from human serum was investigated by following the course of acetic-acid distillations. A standard cholesterol solution, human serum, and the protein and high-density lipoprotein component (hereafter called the protein solution) of human serum were used in the test. The protein solution was selected for use because it contains all the serum proteins and the high-density lipoproteins, which contain only a small percentage of the total serum cholesterol. This would enhance any error caused by the reaction of the tryptophan from the protein with glyoxylic acid when enough of the material was used to insure a measurable amount of cholesterol. Five different types of distillations were made: (1) plain, with no modifications; (2) under an N9 atmosphere; (3) from FeSO4, 1 gm. per liter, under an N0 atmosphere; (4) after 3 hr. refiuxing in the presence of zinc granules under N2; and (3) from a K0Cr907 (0.1 gm. per liter) solution. Distillation 5 was from an oxidizing solution as first suggested by Maclntyre and Ralston (4) and confirmed by Morris (2) arid Henly (5). The use of K0Cr907 in an acid solution is equivtlent to the use of Cr03 reported by these investigators. Distillation Types 1-4 gave similar results, varying only a little in the time to reach plateau values. Figure 1 shows the typical result. l)istillation Type 5 differed. The plateau values shown in Fig. 1 were reached as SOOn as the boiling point of acetic acid was reached, and these values continued until the concentration of K0Cr907 became so high (due to removal of acetic acid) that secondary oxidation began

4 Vol. 9, No. 2, 1963 SERUM CHOLESTEROL 159 to take place as showim by a rapid change of color and a rise in tile temperature. Refluxing the acid prior to distillation is not necessary. All acid used for subsequent experiments was distilled from K9Cr907. The most striking finding from this experiment is that there are at >- U z a:i standard serum protein solution 0 ci VOLUME (ml. acetic acid X 50) Fig. 1. Distillation of acetic acid from FeSO, under N, is typical of all distillations. Effect of impurities in commercial acetic acid on intensity of color developed in reaction of cholesterol with FeCL, color reagent. least two different impurities in commercial acetic acid. This is shown by the rising values of the standard indicating one impurity, and by the decreasing values of the pi otein solution and the serum curves indicating an overriding second impurity. This second impurity could be the glyoxylic acid. The first impurity is hitherto unreported. It may be peracetic acid which could oxidize the cholesterol to cholestenone (12), thus reducing the amount of color formed by the color reagent, as cholestenone does not affect the FeC13 reagent (13). (This impurity has been found in most but not all acetic acid batches tested.) The presence of this impurity and the reducible substance glyoxylic acid led to the use of reducing agents prior to or during distillation. Reducing agents appear to improve distillation results slightly but not in a manner which is conclusive. Errors caused by the two impurities are large. The immediate removal of these impurities from acetic acid when distilled from K2Cr907 make this the method of choice. The elimination of these two impurities make the direct serum cholesterol values much more comparable to values obtained through extraction

5 160 MOORE & BOYLE Clinical Chemistry procedures. Specifically, when compared to the procedure of Abeil et at. (14) using 21 consecutive human sera, the mean values were 316 ± mg./100 ml. for the FeCl. procedure and 310 ± 3.3 mg./100 ml. for the Abell procedure. Furst and Lange (8) found that changes in the initial temperatures of reagents caused changes in the amount of color developed by the Zlatkis color reagent. There is an upward trend to absorbancies read with respect to an increase in temperatures of the Rosenthal reagents. Alterations in absorbance over a range of temperatures from 2#{176} to 47#{176} are well within the range of ± 3%, which is the range of reproducibility. Certainly the comparatively small variations in room temperature which would occur during the course of an analysis would cause errors so small as to be negligible, especially when compared to a standard done at the same temperature. If the procedure as described is followed carefully, heats of reaction will be uniform and error from this source negligible. Furst and Lange also noted a day-to-day difference in color development. This is also true of the Rosenthal color reagent. This is demonstrated by a daily decrease in the amount of color developed by the cholesterol standard. The ferric chloride reagent itself becomes noticeably less yellow. This necessitates measuring a standard daily. In order to have a control, a single serum was pipetted fresh into suitable containers and frozen until used. Measurements were made on this colitrol serum each time a new standard or a new color reagent solution was made up. Values obtained over a period of a few months from any control serum so used were reproducible within experimental error. An effort was made to confirm the results of Kinley and Krause (9) concerning the error in cholesterol values caused by vitamin A. The study was expanded to include the oil-soluble vitamins D, E, and K, and also to include nicotinic acid. The results are shown in Table 1. The vitamins measured had to be in milligram or gram quantities to obtain the small errors noted. If these concentrations are extrapolated to physiologic values, the error becomes immeasurably small. The report of Kinley and Krause stated that chloroform was used to dissolve the vitamin A added to the serum they measured. We have found that ml. of chloroform added to a standard caused an increase of 11 mg./100 ml. in the cholesterol standard. It is also notable that in their report Kinley and Krause could not correct for physiologic vitamin A, but only for added vitamin A.

6 Vol. 9, No. 2, 1963 SERUM CHOLESTEROL 161 Table 1. ERROR IN CHOLESTEROL VALUES CAUSED BY ADDED VITAMINs Added Cholesterol Error caused vitamins valve by vitamins Physiologic p er 100 ml. per 100 ml. per 100 ml. serum Solution (my.) (my.) (my.) concentrations Cholesterol standard Vitamin A pg. (16) Vitamin D pg. (17) Vitamin E mg. (18,19) Vitamin K Not known Nicotinic acid pg. (19) The error in cholesterol values due to hemolysis of the serum was investigated. A standardization solution of hemoglobin, made from hemolyzed washed red cells, was prepared, and portions of this solution were added to serum for measurement. The error is significant, but it is not proportional to the concentration (Table 2). Sera, not plasma, should always be used for cholesterol determinations. Plasma from blood containing the anticoagulants oxalate, citrate, and fluoride gives values about mg./100 ml. lower than Sera. Plasma containing heparin or EDTA also give low values, hut in the range 5-15 mg./100 ml. The use of any of these anticoagulants, in concentrations found in plasma, with a pure cholesterol standard does not cause an appreciable error. Values obtained on plasma and sera using the direct method were duplicated using the method of Abel et at. (14). The results indicate that serum should be used in that extraction procedure also. The anticoagulant effect is probably due to increased osmotic pressure which dilutes plasma with cell water. A high concentration of chloride ion will also cause an error in cholesterol values. This error becomes appreciable in the high concen- Table 2. Eaoa IN SERUM CHOLESTEROL VALUES CAVsED BY HEMOLYSIS Hemolysis Solution Hemoplobin (mg./100 ml.) Cholesterol (mg./100 ml.) error (mg./100 ml.) Solution coloir Yellow Yellow Amber Pink Decidedly pink Red Very red

7 162 MOORE & BOYLE Clinical Chemistry trations used in ultracentrifugation. This error is discussed in full by Boyle et at. (11). Bischoff and Turner (is) tested a number of steroids with the ferric chloride reagent and reported that most have no effect. They also noted that lathosterol, a cholesterol precursor, would cause some error with the ferric chloride reagent, but would cause a much greater error with the Liebermann-Burchard reagent. Desmosterol in patients treated with MER/29* appears to affect Liebermann-Burchard and ferric chloride color reagents similarly. Cholesterol values obtained in this laboratory by the two color reagents on persons treated with MER/29 are similar. This is in agreement with Frantz et at. (15). Bilirubin will cause error, but that error in the great majority of sera would be negligible because of the low physiologic concentrations. In those sera which have a high bilirubin value, a correction can be applied by substracting 1.4 mg. of cholesterol per 100 ml. of serum for 1 mg. of bihirubin per 100 ml. of serum (4). These values have been confirmed in this laboratory. References 1. Best, M., Van Loon, E. J., Watheni, J., and Seger, A. J., An,. J. Med. 16, 601 (1954). 2. Morris, T. G., J. Clin. Path. 12,518 (1959). 3. Zlatkis, A., Zak, B., and Boyle, A. 3., J. Lab. Gun. Med. 41, 486 (1953). 4. Maclntyre, I., and Ralston, M., Biocheni. J. 5-6, xliii(1954). 5. Henly, A. A., Anal yst 82,286 (1957). 6. Rosenthal, H. L., Pfluke, M. L., and Buscaglia, S.,J. Lab. GUn. Med. 50, 318 (1957). 7. Smit, Z. M., and Weymeyer, A. S.,South African J. Lab. GUn Med. 3,321 (1957). 8. Furst, V., Jr., and Lange, R., Scand. J. GUn.. Lab. Invest. 6, 60 (1954). 9. Kinley, L. J., and Krause, R. F., Proc. Soc. Exptt. Biol. Med. 99, 244 (1958). 10. Hollinger, N. F., Austin, E., Chandler, P., and Lansing, R. K., Clin. Chem. 5, 458 (1959). 11. Boyle, B., Wilson, J., and Moore, R. V., J. Lipid Res. 2, 191 (1961). 12. Deuel, H. J., Jr., The Lipids, vol.1, Interscience Publishers, New York, 1951, p Bischoff, P., and Turner, J. G., Jr., GUn. Chem. 4, 300 (1958). 14. Abell, L. L., Levy, B. B., Brodie, B. B., and Kendall, P. E., J. Biol. CIte,,,. 195, 357 (1952). 15. Frantz, I. D., Jr., Mobberley, M. L., amid Sehroepter, G..1., Jr., Progress in Gardiera.ucular Diseases 2, 511 (1960). 16. Week, E. F., and Sevigne, F. J., J. Nutrition 40, 563 (1950). 17. Warkany, J., Biochein. Z. 293, 415 (1937). 18. Quaife, M. L., Swanson, W. J., Dju, M. Y., and Harris, P. L., An-n. N. Y. Acad. Sci. 52, 300 (1949). 19. Sunderman, F. W., and Boerner, F., Normal Values in Clinical Medicine. Saunders Company, Philadelphia, 1949, p The Wm. S. Merrell Co., Cincinnati, Ohio.