asking; furthermore, recovery of magnesium added to the sample often differed markedly from 100%. RECOMMENDED METHOD
Table VII.
Specimen S O .
Determination of Calcium. Using a stock solution of lanthanum chloride
containing 50,000 p.p.m. of La, prepare standard solutions of 0, 5, 10, 15, and 20 p.p.m. of Ca containing also 10.000 p.p.m. of La. Prepare a solution of each urine specimen such t h a t its calcium concentration lies in the 5- t o 20-p.p.m. range, adding enough lanthanum stock solution t o give a final concentration of 10,000 p.p.m. of La, and clearing any cloudiness by adding a drop or two of hydrochloric acid. (Since the calcium content of urine is so variable it may be advisable to determine the degree of dilution required by roughly measuring the absorption of 0.5 ml. of urine and 2 ml. of stock La solution diluted to 10 ml.) Measure the absorptions of the solutions in the following order: standards, samples, standards, samples, standards. Each reading takes about 7 seconds and rcxquires approximately 0.3 ml. of solution. Average the absorption values for each solution, plot the calibration curve
1
6 9 9
14
14 16
Results of Recovery Experiments on Determination of Magnesium in Urine
Concentration in solution 0..55 0.74 0.74 0.71
1.02 1.02 0.96
Added 0.29 0.31 0.29 0.29 0.50 1.00 0.80
and read off the concentration of the sample solutions. The absorbance-concentration curve is almost a straight line. Determination of Magnesium. Prepare standards containing 0.5, 1.0, 1.5. and 2.0 p.p.m. of Mg and dilute the urine specimens so t h a t the magnesium concentration lies between 0.5 and 2 p.p.m. Measure standards and samples in the same way as for calcium.
Total
Recovered
0.84
0.87
i.05
1.03 1.00 1.52 2.02 1.76
1.05 1.04
0.99 1.52 2.04 1.77
Recovery % 103.5 100 101 99 100 101 100.5 Av. 100.7
and calcium contents by methods.
chemical
LITERATURE CITED
(1) Allan, J. E., Analyst 83, 466 (1958). (2) Box, G. F., Walsh, A., Spectrochim. Acta 16, 255 (1960). (3) Fletcher, R. F., Henly, A. A., Sammons. H. G.. Sauire. J. R.. Lancet D.
. .
ACKNOWLEDGMEN'I
The writer thanks Roger Melick, who supplied the samples of pathological urine and measured their phosphorus
(1955). (6) Willis, J. B., Zbid., 16, 259 (1960). ( 7 ) Ibid., p. 273 (1960). RECEIVED for review October 19, 1960. Accepted December 27, 1960.
Extens io n O f the Porter-Si1ber Reaction to 17-Deoxy-aIpha- keto1ic Steroids MARVIN
L. LEWBART
and VERNON R. MATTOX
Mayo Clinic and Mayo Foundation, Rochester, Minn.
b A method for the quantitative microdetermination of 17-deoxy-aketolic steroids i s described. The method consists of a preliminary cupric acetate oxidation of such compounds to the corresponding glyoxals followed b y treatment with the PorterSilber reagent. For all a-ketolic steroids tested a strict adherence to Beer's law was obtained.
Porter-Silber reaction to include such compounds. This paper presents a method for the microestimation of pure ketols employing cupric acetate oxidation followed by treatment with the Porter-Silber reagent. I n addition, a procedure for converting 17-OHketols and ketols to glyoxals on paper chromatograms is described. MATERIALS
I
1950 Porter and Silber (7) described a color reaction for 17,21dihydroxy-20-ketosteroids (17-OH-ketols) rvhich has since proved to be of great valur in the quantitative determination of these substances. *kltliough 21-hydroxy-20-ketosteroids (ketols) give no color a i t h the reagent, their corresponding 21-aldehydes or glyoxals react even more rapidly than 17-OH-kptols (8). Since ketols can be oxidized readily in high yield to glyoxals by reaction with cupric acetate (3,9), it has been possible to extend the N
llethanol. Distilled from anhydrous potassium carbonate. Phenylhydrazine Hydrochloride. Recrystallized three to four times from 95% ethyl alcohol. Porter-Silber Reagent A. For quantitative analysis. This was prepared by dissolving 50 mg. of phenylhydrazine hydrochloride in 100 ml. of an 8:2 mixture of concentrated HQS04 and HzO. For color development, 2 nil. of this solution freshly prepared and mixed with 1 ml. of methanol containing the dissolved steroid. Porter-Silber Reagent B. For use on paper chromatograms. This was
prepared by mixing t n o parts of 7 : 3 H2SOa-H20 containing 0.5 mg. per ml. of phenylhydrazine hydrochloride and one part of 95% ethyl alcohol. 0.005M Cupric Acetate Solution. One milligram of C U ( O A C )H~2 0 (llallinckrodt) per milliliter of methanol. Solutions retain their full oxidizing activity for a t least 2 15-eeks despite some color change and precipitation. Abbreviations Used. The following abbreviations are employed: 'I'HA4 = 3 ~ ~ 2- 1dihydroxypregnane - 11,20dione; T H B = 3 a , l l B,21-trihydroxypregnan-20-one; THQ = 3cq21dihydrosypregnan-20-one; T H E = 3a,17,21- trihydroxypregnane - 11,20-dione; T H F = 3n,llp,l7,21-tetrahydroxypregnan-20-one; THS = 3~~,17,21-trihydroxypregnan-20-one; AlG-THA = 3a,21-dihydroxy-l B-pregnene - 11,2O-dione ; compound H = 3p,21-dihydroxyallopregnane-11,20-dione; substance R = 3p711p,21-trihydroxyallopregnan-20one; THA glyoxal hydrate = 3ahydroxy-11,2O-dioxopregnan-21-a1 hyVOL. 33, NO. 4, APRIL 1961
559
drate; 4l6-THA glyoxal hydrate = 3 a - hydroxy - 11,20 - dioxo - 16 - pregnen-21-a1 hydrate; T H Q glyoxal hydrate = 3a-hydroxy-20-oxopregnan21-a1 hydrate. METHODS AND RESULTS
Quantitative Estimation of Ketolic Steroids. I n 1.5 by 15 cm. glassstoppered, tapered reaction tubes 4t o 60-pg. amounts of steroids were dissolved in 100 pl. of methanol. T o these solutions were added 100 pl. of cupric acetate solution. After 30 minutes in air at room temperature, the solvent was evaporated in vacuo over anhydrous calcium chloride. Kithin a period of not more than 1 hour after removal of the solvent the residues were treated with 1 ml. of methanol and 2 ml. of Porter-Silber reagent A. After 30 minutes a t room temperature, readings were made a t the absorption maxima against appropriate blanks in a Beckman Model DU spectrophotometer equipped for microcells (6). For estimation of 0.4 t o 8.0 pg. of aldosterone the procedure was modified as follow: The oxidation was carried out in 50 pl. each of methanol and cupric acetate solution for 50 minutes. The residues were treated with 100 p!. of methanol and 200 pl. of Porter-Silber reagent containing phenylhydrazine in a concentration twice that in reagent A. The absorbances were measured a t 400 mp after 45 minutes at room temperature. Several 17-OH-ketols were subjected t o cupric acetate oxidation follom-ed by treatment with the Porter-Silber reagent and the values were compared with those obtained on the unoxidized compounds. The Porter-Silber procedure
Table I. Chrornogenicity of Oxidized a-Ketols and Crystalline Glyoxals Molar Absorptivity Steroid x 10-8 THA 23.5 24.6 THA glyoxal hydrate 12.1 THB 12.6 THB glyoxal hydrate 15.1 THQ 16.1 THQ glyoxal hydrate 30r,lla,21-Trihydroxypregnan-20-one 22.7 3a,21-Dihydroxy-9( 11)pregnen-20-one 12.9 Corticosterone 20.6 21.8 Deoxycorticosterone Aldosterone 18.5 (19.5)" 11-Epicorticosterone 23.1 13.4 Substance R Compound H 23.6 3a,21-Dihydroxy-l60r,l7a-
epoxypregnane-l1,20dioneb 23.6 Ale-THA 6.9 AIG-THA glyoxal hydrate 7.7 Value for modified procedure (total volume of 0.3 ml.). b Unoxidized compound is Porter-Silber negative. 0
560
ANALYTICAL CHEMISTRY
Figure 1. Relative chrornogenicities of oxidized and unoxidized a-ketols with Porter-Silber reagent
used for oxidized 17-OH-ketols mas the same as for oxidized ketols. For unoxidized 17-OH-ketols, color development proceeded for 18 hours a t room temperature. Reaction mixtures from unoxidized and oxidized 17-OH-ketols were read a t their respective maxima. Unoxidized 17-OH-ketols and oxidized ketols had maximal absorption at 410 to 415 mp in the Porter-Silber reaction. The only exception was aldosterone, which had peak absorption a t about 400 mp. The cupric acetate oxidation products of both 17-OH-ketols and A%-ketolic steroids had peak absorption a t 425 to 430 mp. A11 tests showed a linear relationship between the amount of ketols oxidized and color produced in the Porter-Silber reaction. The molar absorptivities of the Porter-Silber products of the oxidized ketols tested are listed in Table I. The values obtained on 17-OHketols with and without cupric acetate oxidation are shown in Figure 1. Of the six 17-OH-ketols and two A16a-ketols tested, only cortisone and THE remained equally chromogenic after oxidation; the other six compounds gave considerably less color. The glyoxals from THA, THB, THQ, and 4l8-TH-4 were prepared by cupric acetate oxidation of millimolar quantities of a-ketols. The yields of the crystalline glyoxal hydrates were consistently 90% or better (9). Their molar absorptivities as obtained in the Porter-Silber reaction are included in the table. The values obtained from the crystalline glyoxals and from the corresponding ketols after micro-oxidation with cupric acetate are in close agreement. With both 17-OH-ketols and oxidized ketols the relative concentration of sulfuric acid and methanol necessary for maximal color development was variable. Porter-Silber reagent A, which gave maximal color with oxidized TK4 and aldosterone, was used throughout the study even though the amount of sulfuric acid present was higher than optimal for many of the other compounds tested. When, for example, the molar absorptivities of the glyoxal hydrates derived from THA, THB, and T H Q were determined with the less
acidic reagent of Silber and Porter (8), the values obtained after 3 hours' incubation a t room temperature were 24,400, 23,500, and 20,800, respectively. After an additional 16 hours, the absorbances had decreased by approximately 10%. Oxidation of 17-OH-Ketols and Ketols on Paper Chromatograms. I n duplicate and on separate strips of paper, 20 pg, amounts of steroid were chromatographed in appropriate systems. Cortisone, cortisol, T H E , THF, 3p,17a.21 - trihydroxy - 5 - pregnen - 20one,6/3-h~-droxy-ll-deoxycortisol,11epicorticosterone, and T H B were chromatographed in system C of Bush ( 2 ) . 11-Deoxycortisol, THS, 17,21-dihydroxy-prepnane-3,11,20-trione,deoxycorticosterone, THQ, THA, THB, substance R, and compound H were chromatographed in toluene-iso-octanemethanol-water (275:225:400: 100) (4). The finished chromatograms of one series were dipped in the 0.005X methanolic cupric acetate, dried briefly and suspended for 1 hour a t room temperature in a methanol atmosphere. The strips of both the oxidized and the unoxidized series were then dipped in Porter-Silber reagent B, placed between glass plates and left a t room temperature. All unoxidized 17-OH-ketols gave typical yellow spots which developed slowly, reaching maximal intensity in 2 to 3 hours. Both 17-OH-ketols and ketols, after treatment with cupric acetate, gave yellow colors almost immediately. As little as 2 pg. of THA could be detected by this method.
DISCUSSION
Our interest in the copper-catalyzed oxidation of a-ketols arose from a n attempt to determine the cause of loss of microgram quantities of cortisone and related steroids during paper chromatography. It was found that traces of copper in the glassware were the cause of destruction (5) and that, of the several artifacts formed, one was the corresponding 21-aldehyde. Further study showed that of various cupric salts tested, cupric acetate gave by far the most rapid and complete oxidation of a-ketols t o glyoxals. At the time this work was done, we were unaware that Merck and Company had received several patents dealing with the preparation of steroidal glyoxals from cyketols using cupric acetate as the oxidant in both catalytic and stoichiometric amounts ( 3 ) . We have found that steroidal glyoxals are not stable in the presence of cupric acetate and slowly undergo rearrangement to a-hydroxy acids. The interval of time between completion of oxidation of a-ketols to glyoxals and treatment with the Porter-Silber reagent should, therefore,
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)’; \
I
Weiljard, J., Ibid., 2,773,078. (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
