be kept to a minimum. A detailed study of this rearrangement is currently being conducted in this laboratory. I t has been suggested that both 17OH-ketols and AIE-ketols rearrange to 17-deoxy glyoxals in the course of the Porter-Silber reaction (8). This would explain the rapid reaction of the cupric acetate oxidation products, thereby eliminating the necessity for heating or prolonged reaction a t room temperature. Despite a strict adherence to Beer’s law, the a-ketolic steroids tested in the cupric acetate-Porter-Silber method showed considerable difference in chromogenicity. Since the crystalline glyoxal hydrates derived from THA, THB, ’I”&, and A16-THA were only slightly more chromogenic in the Porter-Silber reaction than the corresponding ketols following micro-oxidation (Table I), it is evident that for these compounds conversion of keto1 to glyoxal was almost quantitative. The differences among THA, THQ, and T H B are in agreement with those of the corresponding 17-hydroxy analogs in that T H E is more chromogenic than THS or THF. From these results and others obtained with the Porter-Silber reaction under our conditions, we have concluded that among the 17-deoxy glyoxals differing by substitution a t C-11, the order of
decreasing chromogenicity is 11-keto > llcu-hydroxy > 11-deoxy > 1lphydroxy. The exkreme variability in the relative chromogenicity of the 17-OHketols and their corresponding oxidation products with cupric acetate as shown in Figure 1 makes it unlikely that one could apply cupric acetate oxidation to mixtures of 17-OH-ketols and ketols to determine independently the two classes of compounds. The foregoing method should find use in the determination of individual ketols following isolation by paper chromatography. The value of the method lies in its simplicity and relative specificity, and in that blank values on eluates from paper are appreciably lower than those obtained from methods for estimating ketols, such as blue tetrazolium reduction. For example, the corrected absorbance determined on a residue obtaincd from a 5 by 7 cm. area of unwashed Whatman No. 1 paper by elution with water was 0.007 a t 410 mp, equivalent to 0.3 pg, of THA. Use of the Porter-Silber reagent to detect both 17-OH-ketols and glyoxals on paper chromatograms has been reported recently by Birmingham (1). It was found that 17-OH-ketols gave maximal color after 1 to 2 hours, whereas
glyoxals produced yellow spots immediately. We have extended this application of the Porter-Silber reagent by oxidizing both 17-OH-ketols and ketols to the corresponding glyoxals on paper chromatograms. This technique enables one to use the Porter-Silber reaction to detect ketols, which without oxidation give no color with the reagent. The 17-OH-ketols are converted to 17-OH-glyoxals which also give yellow spots immediately with the PorterSilber reagent. LITERATURE CITED
(1) Birmingham, Marion K., Nature 184,
67 (1959).
(2) B y h , I. E., Biochem. J. 50, 370 ( 1932).
(31 Conbere. J. P. (to Merck & Co.. Inc.) U. S. Patknt 2,773,077 (Dec. 4, i956)’; Weiljard, J., Ibid., 2,773,078. \
I
(4) Eberlein, W. R., Bongiovanni, A. M., J . Clin. Endocrinol. 18, 300 (1958). (5) TLewbart, &I. L., Mattox, V. R., h ature 183, 820 (1959). (6) Lowry, 0. H., Bessey, 0. A,, J . Biol. Chem. 16, 633 (1946). (7) Porter, C. C., Silber, R. H., Ibid., 185, 201 (1950). (8) Silber, R. H., Porter, C. C., “Methods of Biochemistry Analysis,” Vol. 4, p. 139. Interscience. Yew York. 1957. (9) Uhpublished observations. ’ RECEIVED for review September 28, 1960. Accepted Pu’ovember 9, 1960.
Turbidimetric Determination of Total Serum Cholesterol GEORGE R. KINGSLEY and OZlE ROBNETT Clinical Biochemistry Laboratory, Veterans Administration Center, and Deparfment of Physiological Chemisfry, School of Medicine, University of California, los Angeles 24, Calif.
b Total serum cholesterol can b e quantitatively determined b y absorbance measurement of the turbidity produced upon the addition of sodium alcoholate to serum. The relationship of turbidity produced to cholesterol concentration appears to b e linear within the limits described b y the procedure. The results obtained by this simple turbidimetric technique for the rapid determination of serum cholesterol are in good agreement with those b y established methods.
V
and Velu ( 6 , 6 ) have described a simple sodium alcoholate turbidimetric method for the determination of serum cholesterol. ELU
The only reagent used, sodium alcoholate, is prepared by diluting 1.25 ml. of 36’ Baume sodium hydroxide (3Oy0) to 100 ml. with ethyl alcohol to obtain a 50% alcohol concentration in the final reaction mixture. In their analytical procedure 0.5 ml. of serum is added to 1 ml. of saline; 4.5 ml. of sodium ethylate is then added and
mixed and the mixture is incubated a t 55’ C. for 30 minutes. A blank is prepared by substituting saline for serum. After incubation, the turbidity developed by the serum is measured photometrically against the blank with a red filter, Wratten KO.70. Since the authors were unable to obtain reproducible results with this method, all of the optimum conditions for its use, such as time, temperature, concentration of reagents, etc., were determined. The method finally adopted differed from that of Velu and Velu in the use of a lower incubation time (10 minutes) and temperature (20’ to 25’ C.) and higher concentration of ethyl alcohol (56%). This investigation resulted in the development of an unusually simple, accurate, and reproducible method for determining cholesterol. EXPERIMENTAL
REAGENT. 56y0 Sodium Ethylate Reagent (make fresh before use). Mix
35 ml. of H20, 45 ml. of absolute ethyl alcohol, and 1 ml. of 30% NaOH, carbonate free. This reagent will give 56y0 sodium ethylate in the final reaction mixture. PROCEDURE. Mix 0.2 ml. (if cholesterol concentration is in the range of 100 to 300 mg. per 100 ml.) of serum with 1.0 ml. of saline in a photometer cuvette. Use 0.4 ml. of serum below 100 mg. % cholesterol and 0.1 ml. or less above 300 mg. per 100 ml. Prepare a blank with 6.0 ml. of saline solution (0.9% NaC1) and 0.2 ml. of serum. The difference in absorbance of sodium ethylate and saline is not significant. Add 5 ml. of sodium ethylate reagent to serum specimen and mix. Keep the reaction mixture a t 20’ to 25’ C. for 10 minutes. Read the serum sample against the blank a t 550-mp light transmittance. The transmittance readings are stable for 10 to 30 minutes after start of reaction. STANDARDIZATION. Run three pooled serums containing 100, 200, and 300 mg. yo of cholesterol as determined by a colorimetric cholesterol method (3) through the procedure as described for serum, and set up standardization. VOL. 33, NO. 4, APRIL 1961
0
561
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"'.-..* .......,
*
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0.30-
6 Z
--,
i
E
0.20-
si 7 0.10I
,
I
30
40
I
I
50
60
70 % ALCOHOL
I
1
I
80
90
100
Figure 1. Determination of optimum concentration of organic solvents to use in turbidimetric determination of cholesterol 1.
.2....Ethanol Stable from 10 to 30 minutes a t 56% 2-Propanol
and 25' C.
- Stable 10 to 30 minutes at 50%.- and 2 5 ' -3. - Acetone Stable 30 to 40 minutes a t 50% and 25'
C. C.
4.
Methanol Stable 10 to 30 minuter a t 75% and 2 5 ' Points represent triplicate determinations
---.
Absorbance Ratio for Colorimetric cholesterol = Method = 300 mg. absorbance of 0.39 (with Coleman, Jr., spectrophotometer KO. 6 4 , Barium
Sulfate
Standardization.
REAGEKTS. Bas04 Standard. Add 3 ml. of 0.0962N BaClz to a 100-ml. volumetric flask and dilute to volume with cold (10' C.) 0.2N HzS04. Adjust temperature of mixture t o 20" C. before diluting to volume. This standard is equivalent to 300 mg. of cholesterol per 100 ml. Bas04 standard and ethylate reagents may be obtained from K.M.W. Science Search, Inc., 10866 La Grange Ave., Los Angeles 25, Calif. STANDARDIZATION PROCEDURE. Place 0, 2, 4, 5, 8, and 10 ml. of undiluted standard in colorimeter cuvettes and dilute each to 10 ml. a t 10' C. with 0.2N H2S04. Let stand a t 20" to 25" C. for 30 minutes. Mix well before reading. Set zero standard a t 100% T with 550-mp light transmittance and read standards; 10 ml. of standard is equivalent to 300 mg. of cholesterol per 100 ml. (Coleman, Jr., No. 6 spectrophotometer), Calculate K in the formula C/lOO ml. = K ( 2 - log % T ) from concentration of standards and transmittance data, and prepare a table for conversion of per cent transmittance to milligrams of cholesterol per 100 ml. of serum DETERMINATION OF APPROPRIATE SOLVENT AND OPTIMUM CONCENTRATION
Water-soluble organic solvents ethanol, 2-propanol, methanol, and acetone were studied to determine which would produce the greatest absorbance over the widest range of solvent concentrations for measurement of turbidity production in the quantitative measurement of cholesterol concentration (Figure 1). The data presented in
562
ANALYTICAL CHEMISTRY
C.
Figure 1 indicate that ethanol in concentrations of 56 to 63% gave maximum density for cholesterol turbidimetric measurement. The concentration of NaOH used in the preparation of sodium ethylate was not critical and could be varied by the addition of 1 ml. of 20% to 1 ml. of 70% NaOH without any apparent change in absorbance or stability in the time interval of 5 to 30 minutes. A range of temperature from 5" t o 60" C. had little effect on stability of turbidity from 10 to 30 minutes, but lower absorbances were obtained a t higher temperatures. Maximum absorbance was obtained in a range of 5"
Table 1. Comparison of Colorimetric and Turbidimetric Cholesterol Methods a t Optimum Working Conditions
~ ~ l ~ Turbidimetric ~ j Methods, metric Mg. % Na htethod Na Na (S), M g . ethyl- methyl- isopro% ate ate pylate 250 250 250 255 143 140 145 130 191 195 200 200 68 70 68 70 615 600 600 610 68 70 75 7.5 ~. 160 155 150 150 210 195 198 200 250 250 250 260 224 220 223 217 192 180 185 185 150 160 160 150 255 250 240 245 130 135 125 140 125 115 125 115 225 225 223 215 140 130 140 130 165 165 160 175 150 160 160 165 163 150 155 150
to 25" C. ,4 considerable decrease in absorbance was obtained at 55" C., a temperature recommended by Velu and Velu (5, 6). I n order to determine the linearity obtained n ith different alcoholates in the turbidimetric measurement of cholesterol, standardization curves were prepared with serial dilutions of a pooled serum of known cholesterol content over a range of 50 t o 250 mg. % (Figure 2). If optimum working conditions for the use of ethanol, methanol, and 2-propanol were used in the preparation and use of their alcoholates, good agreement was obtained between these turbidimetric methods and the colorimetric cholesterol method of Kingsley and Schaffert (3). The data in Table I represent 20 comparative examples taken from over 1000 serum analyses. The reproducibility of duplicate determinations made by the turbidimetric alcoholate method was within one standard deviation and those made by the colorimetric method of Kingsley and Schaffert (3)were within approximately two standard deviations. The reproducibility of the turbidimetric methods was greater because of greater simplicity. STANDARDIZATION O F METHOD WITH BARIUM SULFATE STANDARDS
Pure cholesterol standards cannot be used for standardizing the turbidimetric cholesterol method, and lyophilized commercial serum standards are unsatisfactory because the natural lipide-calcium-protein complex in these standards is altered, especially in its solubility. The use of barium sulfate as a turbidimetric reference standard was investigated because of the lack of other suitable standards. Barium sulfate has been used as a satisfactory turbidity standard for the thymol turbidity method (9). The standard and method of preparation as described in
*
I 50
100
50
mg c n o L : s - E a o -
200
25C
30 m i
Figure 2. Linearity of turbidimetric cholesterol standardization with different solvents Serial dilutions of serum containing 300 mg. of cholesterol per 100 ml. Points represent single determinations X 56% sodium ethylate, K = 6 2 5 0 50% sodium isopropylate, K = 730 0 75% sodium methylate, K = 730 K. Constant for Coleman, Jr., spectrophotometer
,
