A Rapid, Accurate Method for the Determination of Total Cholesterol in Serum SIDXEY PEARSOS, SIDNEY STERN, AND THORIAS H. McGAVACK The New York Medical College, illetropolitan Hospital Research Unit, Y e w York, N . Y Absorption Curve and Stability of Color. Over the range 550 mp to 650 mp, the absorption curve of serum cholesterol parallels that of free cholesterol and cholesteryl acetate (Figure 2). Although the observed absorption was highest a t 650 mp, the color density varies continually with time a t that wave length A4t550 mp, the optical density remains constant from 10 t o 15 minutes after the 20 minute reaction period. Therefore, optical density is measured a t 550 mG.
for the determination of total serum cholesterol are tedious and subject to possible errors as a result of incomplete extraction of cholesterol, and of manipulative processes required by the methods (2, 3 ) . These sources of error have been eliminated or grentll- reduced in a new, rapid, accurate procedure which requires only the addition of reagents to serum and the nirnaurement of the density of the resultant color. This method gives results which agree with those obtained by the Schoenheinier-Sperry procedure ( e ) . The determination is completed i n less than 30 minutes. ETHODS
2
7
5
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3IATERIALS A N D REAGENTS
250.
All C.P. reagents n-ere obtained from Fisher Scientific Co. Acetic anhydride, C.P. p-Toluenesulfonic acid solution. Dissolve 12 grams of C.P. p-toluenesulfonic acid in 100 mi. of C . P . glacial acetic acid. Cholesterol, c.P., m.p. not less than 1 4 i " C. Recrystallize from ethylene chloride when the m.p. drops belox7 1 4 i o C. Cholesteryl aretate, obtained from Eastman Iiodak Co., Rochester, S . T. Sulfuric acid, C.P. Spectrophotometer, Coleman junior, Model 6A.
0 0
s 200.
looi/
PROCEDURE
To 0.1 nil. of serum in a test tube add, in the following order, 0.1 nil. of glacial acetic acid, 0.5 ml. of p-toluenesulfonic acid solution, and 1.5 nil. of acetic anhydride. Alllow the solution to stand, without mixing, a t room temperature until the material cools: and then add 0.2 ml. of concentrated sulfuric acid. Mix thoroughly, immediately after the addition of the acid, until the precipitate is completely dissolved. After the solution has stood a t room temperature for 20 minutes, measure the optical density a t 550 mp. Prepare a standard of 0.1 ml. of a glacial acetic acid solution of cholesterol (200 mg. per 100 ml.) in the same way, but add 0.1 nil. of water to the cholesterol solution instead of the 0.1 ml. of g1:tcial acetic acid that, 17-as added to the serum. Prepare the solution with which the zero point of the spectrophotometer is set according to the procedure used for the sample, using 0.1 ml. of water. Prepare blanks to correct for interfering substances in serum that absorb a t 550 mp by the addition of 2.3,ml."ofp-toluenesulfonic acid solution to 0.1 ml. of serum. Use the p-toluenesulfonic acid solution to set the zero point, before measurement of the optical tlt,nsit,y of the blanks. RESULTS
Beer's Law. Figure 1 illustrates t h e relationship between optical density and the amount of cholesterol, cholesteryl acetate, and serum assayed. Cholesterol and cholesteryl acetate have equal optical densities, mol. for mol.
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, M L . X 4 SERUM , .IO .20 .30 .40 0 .05 ,IO I5 .20 .25 .30 .35 .40 5
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.
MG. CHOL. Figure 1. Relationship between Optical Density at 550 mp and the Amount of Cholesterol, Cholesteryl Acetate, or Serum A , serum cholesterol; X, cholesteryl acetate; and 0 , free cholesterol
Reproducibility. Five different serums were analyzed for cholesterol; the analyses v-ere repeated seven times on each serum a t intervals of 1 hour or more over a 24-hour period. The absolute extinction values of the color developed Table I. Reproducibility of Results of Determinations of Cholesterol i n Serum by a given serum varied somewhat from Optical Density X 1000 analysis t o analysis, and, therefore, a Analysis S o . standard cholesterol solution is included Specimen 1 2 3 4 5 6 7 8 in each series of serum analyses. The Serum 1 190 198 206 186 190 191 180 179 calculated cholesterol content of each Serum 2 400 411 421 400 395 398 375 373 186 172 170 Serum 3 181 188 I90 175 181 serum was reproducible. See Table I. Serum 4 160 166 172 163 160 167 151 154 Average deviations for the eight deterSerum 5 160 163 160 158 160 160 153 152 Standard cholesterol minations on serums 1 to 5 , respecsoln. 150 155 161 146 147 148 140 138 tively, were 0.4%, 0.6%, 1.30J0, 2.3%, Cholesterol Content of Serum, Mg./100 1\11. and 2.4%. Analysis KO, Serum 1 2 3 4 5 6 7 8 Recovery. Various amounts of choles256 255 259 253 256 256 257 256 terol and cholesteryl acetate (0.05 mg. 522 544 538 534 535 532 630 532 236 240 241 243 246 250 245 213 to 0.25 mg.) were added to different 213 214 218 214 223 224 216 220 210 199 216 218 serums, and in ten determinations re213 215 218 217 coveries averaged 99.2 & 3.60/,.
813
ANALYTICAL CHEMISTRY
814
7
Table 11. Comparison of the New and the SchoenheimerSperry Methods hlg. Cholesterol per 100 111.
S e w methoda 253 195 22: 25a 222 221 267 216 257 251 a
S.-S. method 258 193 229 260 204 220 250 220 25s 243
Kew methoda 224 227 218 162 130 193 308 256 262 194
S.-S. method
223 241 229 153 133 190
282 245 242 198
-x8 500 0
Average of two determinations
Comparison with Accepted Method for Determination of Total Cholesterol. Tn-enty different serum specimens were analyzed in duplicate, and the results were compared with analyses of samples of the same serums by the Schoenheimer Sperry method ( 2 ) . Results are shown in Table 11. With the new method, values were obtained M hich had an average deviation of &3.5y0 from the values found with the SchoenheimerSperry procedure. CALCULATIONS
The milligrams of cholesterol in 100 ml. of serum is calculated as follons: Optical density of unknown - optical density of blank X Optical density of standard mg. of cholesterol in standard X lo3 = mg. of cholesterol in 100 nil. of serum
All serums and standards are run in duplicate, and the average optical densities of the duplicates are used to calculate the final results. Duplicates have been found to show an average deviation of less than 5%.
01
400
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450
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500
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550
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600
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650
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700
A ,MU Figure 2. Absorption Curves of the Reaction Products of Cholesterol, Cholesteryl Acetate, or Serum A, serum; X, cholesteryl acetate; and 0 , free cholesterol Schoenheimer-Sperry method shows that the same holds true for cholesterol and cholesteryl esters in serum. ACKNOWLEDGMENT
The authors wish to thank L. Le& ;ibell of Columbia University for the analyses by the Schoenheimer-Sperry method.
DISCUSSION
I t has been stated that methods based upon direct colorimetry without saponification are inaccurate because cholesteryl esters give higher color densities than cholesterol (1). I n the new method, equimolar quantities of cholesterol and cholesteryl acetate give equal color densities, and the agreement between results obtained by the new method and thope obtained by the
LITERATURE CITED
(1) Soyons, E. C., Biochem. Z . , 303, 415 ( 1 9 3 8 ) . (2) Schoenheimer, R., and Sperry, W. M.,J . B i d . Chem., 106, 745 (1934). (3) Zuckerman, J. L., and Natelson, S., J . Lab. Clin. .\led., 33, 1322 (1948). RECEIVED for review August 6, 1952. Accepted December 13, 1952.
Colorimetric Estimation of lsonicotinic Acid Hydrazide E. L. PRATT, Winthrop-Stearns Inc., Rensselaer, N . Y . application of isonicotinic acid hydrazide in the treatRE""" ment of tuberculosis has brought forth the need of a specific T
analytical procedure for control testing this compound. The method dcscribed herein is based on the absorbancy measurement of the chromophore resulting \Then isonicotinic acid hydrazide is reacted with sodium P-naphthoquinone-4-sulfonate and 2 N sodium hydroxide. All steps are carried out in a complete aqueous system a t room temperature. Absorbancy maximum, 480 mfi. Subsequent to the utilization of isonicotinic acid hydrazide in the treatment of tuberculosis analytical procedures for the estimation of this compound appeared. One of the early methods was based on the titration of the hydrazide substituent with nitrous acid (9). An iodometric procedure was proposed by Canback ( I ) , and a similar method, carried out in acid solution, was described by Alicino (1). A second method in the publication of Alicino (1) was based on the titration of the basic ring nitrogen and end amino nitrogen of the hydrazide m-ith perchloric acid in nonaqueous media. The substituted pyridine ring has been
characterized by ultraviolet spectrophotometry (9). and the effect of various solvents and aqueous acidic p H levels in producing both bathochroniic and hyperchromic spectrum shifts has been observed by Auerbach ( 2 ) . A colorimetric procedure has recently been reported by Ballard and Scott ( 3 ) based on the chromophore produced when the T acid, l-chloro-2,4-dinitrobenzene, combines with the basic nitrogen of r-picoline and isonicotinic acid hydrazide. The procedure described herein is based on the early work of Ehrlich and Herter ( 5 ) and the later writings of Feigl (6). I n Feigl's text the identification of reactive CH2 and NH, groups by reaction with alkaline sodium ~-naphthoquinone-4-sulfonate is discussed. I n this paper the use of the aforementioned reagent in estimating the hydrazide substituent of isonicotinic acid hydrazide in microgram quantities is considered. Subsequent to the preparation of this article, Scott (10) has described the qualitative reaction of 1,2-naphthoquinone-4-sulfonate with isonicotinic acid hydrazide.
