Determination of Total Cholesterol in Blood Serum with Perchloric

Perchloric Acid-Phosphoric Acid—Ferric Chloride Reagent. Sir: The current methods for the determination of total cholesterol in blood serum are base...
0 downloads 0 Views 424KB Size
obtained with the soft-glass capillaries might be low because of the effect of alkali-except t h a t t k e melting points of the glucoses as obtained with the softglass capillaries agree with those melting points obtained on the Dennis melting point bar where there are no glass surfaces. Furthermore, the mixture of powdered borosilicate glass and the sugar still gave higher melting points in t h e soft-glass capillary and the differential thermal anilysis apparatus. Perhaps boron from the borosilicate has interacted with the sugars (boron does complex with the hydroxyl group) or has catalyzed some molccular rearrangement. The cause oi the phenomena should be further inve,itigated.

Finally, we believe that as glass capillaries are used extensively there is some need for further testing borosilicate melting point capillaries for suitability, particularly in ccnnection with those compounds t h a t are capable of mutarotation, bear hydroxyl groups, or have aldehydic or ketonic groups capable of undergoing keto-enol transformations. ACKNOWLEDGMENT

‘The authors are indebted t o C. 11. Conrad for the differential thermal analyses made on the samples. LITERATURE CITED

( I ) Dieckmann, IT., Chem. Rei. 49, 2203, 2213 (1916).

( 2 ) Jones, H. A.; IND.ENG.CHEM.,ASAL. ED. 13, 819 (1941). (3) Jones. H. A.. Wood, J. W.. J . Am. Chem SOC.63, 1760 (1941).

(4) Morton, -4.A., “Laboratory Technique in Organic Chemistry,” pp. 22-23, McGraw-Hill, New York, 1938. LEONSEGAL DAVIDJ. STANONIS Plant Fibers Pioneering Research Laboratory Southern Utilization Research and Development Division U. S. Department of Agriculture Kew Orleans, La. Use of a company and/or product named b r t h e Department does not imply approval or recommendation of the product to the exclusion of others which may also be suitable.

Determinatiton of Total Cholesterol in Blood Serum with Perc hIoric Acid-P hos ph o ric Acid-Fe rric ChIoride Rea g e nt SIR: The current methods for the determination of tot a1 cholesterol in blood serum are based on either the Liebermann-Burchard reaction (4) or the reaction with ferric chloride-sulfuric acid reagent (6). Homver, the former reaction requires manv strict conditions of performance to obtain a reproducible color developrient ; while in the latter reaction, small amounts of impurities in sulfuric acid frequently give higher chole-terol values ( I , 2, 5 ) . The method presenied here is very simple to use with a large number of samples, and gives :holesterol values analogous t o those determined by Schoenheimer and ,Sperry’s method, which is generally accepted as a standard method for the determination. EXPERlMENTAl

Reagents. Color reagent. Dissolve 8.0 grams of l ~ e C l ~ . 6 H 2(rea0 gent grade) in a mixture of 200 ml. of perchloric acid (70%, reagent grade) and 600 ml. of phosphoric acid (85%, reagent grade). This reagent is very stable and can be stored at room temperature for a long time. Ferric chloride, 0.1 %, glacial acetic acid solution. Ferric chloride, 0.2’%, aqueous acetic acid solution. Dissolve 1.18 grams of FeC13.6H20 in a mixture of 590 ml. of glacial acetic acid and 20.0 ml. of water. Standard cholester 31 stock solution, 0.2 mg. per ml. Dissolve 50 mg. of pure cholesterol (3)in 250 ml. of glacial acetic acid. Working standard cholesterol solutions. Dilute 10.0, 20.0, 30.0, 40.0, 50.0, and 60.0 ml. of the standard stock qolution with glacial ztcetic acid to give a total volume of 61.0 ml. in each case. The working standard solutions thus obtained have equivalent concentrations

of total serum cholesterol of 100, 200, 300, 400, 500, and 600 mg. per 100 ml., respectively. Procedure. To 6.00 ml. of O.lY0 FeC13 solution in a centrifuge tube is added 0.100 ml. of serum. The mixture is stirred thoroughly with a stirring rod t o complete coagulation of proteins. The rod is removed and t h e tube is ceiitrifuged a t about 3000 r.p.m. for 10 minutes. T o 4.00 ml. of t h e supernatant solution in a test tube is added 2.00 ml. of color reagent. A blank tube with a mixture of 2.00 nil. of glacial acetic acid and 2.00 ml. of 0.2% FeCI3 aqueous acetic acid solution is also prepared. I n the case of calculating unknown cholesterol values from the absorbance of a single working standard solution, another tube with a mixture of 2.00 nil. of a single working standard solution, 2.00 ml. of 0.2% FeCL aqueous acetic acid solution, and 2.00 ml. of color reagent is also prepared. The contents of all tubes are

mixed well by shaking. Then the tube.; are heated in a boiling water bath for 20 minutes in such a way that the surfaces of the contents are kept under the surface of boiling water. The tubes are cooled in running water. When the contents of the tubes are thoroughly chilled, the absorbance of the sample (and of a single working standard solution) is read at 450 mp on a spectrophotometer with a glass cell of 10 mm. sample path length against the reagent blank. Calculation. The cholesterol values of t h e samples are calculated from t h e calibration curve which is described below or conveniently from t h e ratio of absorbance of unknown t o t h a t of a single working standard solution employed. Calibration Curve. Three aliquots of 2.00 ml. of each working standard cholesterol solution are pipuetted into test tubes, and 2.00 ml. of 0.2% FeClr aqueous acetic acid solution and 2.00

Table I. Influence of Composition of Color Developing Agents o n Coloration Concentration of FeC13 Ratio of HC1O4 Color of (%, w./v.) to &Po4 (v./v.) blank tube Amax (mp) E460 mlr 0 1:3 colorless 455 0.350 2: 1 yellow 0.600 1:1 faint yellow 0.560 0.5 1:2 colorless 450 0.500 1:3 colorless 454 0,450 4.53 0.400 1:4 rolorless ‘>. -.1 yellow 379 0.800 1: 1 yellow 0.520 1.0 1:2 Taint yellow 452 0.500 1:3 faint vellow 452 0.440 1:4 colorlkss 453 0.440 1:2 yellow 376 0.450 2.0 1:s faint yellow 0.420 1:s faint yellow 383 0.390 3.0 1:4 faint yellow 363 0.340

VOL 35, NO. 11, OCTOBER 1963

1751

Table II.

Influence

Ratio of HClOd to Hap04 1:2 1:3c

of Concentration of Color Developing Agent on Coloration"

Volume of color developing agent (ml.) 1.00 1.50 2.00 1.00 2.00 3.00

Volume of glacial acetic acid (ml.) 1.00 0.50 0 1.00

0 0

Volume of final mixture (ml.) 6.00

6.00 6.00 6.00 6.00 7.00

EWmlr'

0.130 0.184 0.214 0. 185 0.423 0.386

a In each case 2.00 ml. of cholesterol solution and 2.00 ml. of 0.2% FeC13aqueous acetic acid solution were used. Av. of three tubes. Cholesterol solution used was different from that used in 1:2 series.

Table 111. Influence of Ratio of Perchloric Acid to Phosphoric Acid in Color Developing Agent on Cholesterol Value in Serum

Cholesterol value (mg./100 ml.) Schoenheimer and Ratio of HClO, Sperry'a to HaPo, Serum method 1:2 1:3 1:4 1 223 230 223 217 2 187 194 184 178 3 176 198 186 175 4 99 99 91 85 5 182 185 184 171 G 192 208 204 193 7 173 187 182 176 ~8 i96 zio 202 i8S 9 244 246 242 223 Mean 186 195 189 178

Table IV. Comparison of Present Method to Schoenheimer and Sperry's and to Zak's Methods

Cholesterol value (mg./100 ml.)

BY Schoenheimer and BY By Sperry's present Zak s Serum method method method 10 358 350 358 11 178 186 197 12 312 318 319 13 237 235 250 14 190 199 212 229 224 15 228 16 176 166 161 17 205 204 195 168 174 184 18 172 183 19 188 20 190 187 198 21 178 181 181 22 158 160 172 152 159 163 24 178 193 23 190 25 127 130 140 26 176 181 179 162 163 I66 28 27 129 128 136 Mean 194 194 202 340 189 6 Hyland Normal Clinical Chemistry Control Serum which contained 185 mg./100 ml. of cholesterol. Hyland Laboratories, LOBAngelea, Calif., U. S. A.

1752

ANALYTICAL CHEMISTRY

ml. of color reagent are successively added. A blank tube with 2.00 ml. of glacial acetic acid and 2.00 ml. each of both reagents is also prepared. The subsequent treatment is identical with the one described under procedure. The absorbances are read a t 450 mp against the reagent blank. The calibration curve thus drawn up is a straight line. RESULTS AND DISCUSSION

The deproteinization of serum was successfully carried out with 0.1% FeCla in glacial acetic acid (1). Since a larger amount of FeCla was given to the deproteinized solution by adding the color reagent, the diminution of FeC13by deproteinization had no influence upon the coloration of the final liquid. From our preliminary experiments it became clear that cholesterol developed a color by heating with perchloric acid (70%) in acetic acid and the addition of FeC4 intensified the color. A yellow color of ferric chloride in the mixture was lightened by the addition of phosphoric acid (85y0). Therefore, a mixture of perchloric acid, FeCla, and phosphoric acid was used as a color developing agent. To determine the composition of the color developing agent, 20 kinds of mixtures of three chemicals were prepared. To 2.00 ml. of the same cholesterol solution in glacial acetic acid were added 2.00 ml. of each mixture and 2.00 ml. of 0.2% FeC13 aqueous acetic acid solution. The resulting 20 mixtures were heated in a boiling water bath for 20 minutes. Their visible-light absorption spectra were measured by a Beckman DK-2 ratio recording spectrophotometer with their reagent blanks and summarized in Table I, except five mixtures which gave yellow blank tests. This table clearly shows the following points: yellow blank tests were given when the concentration of FeCL exceeded l.Oyo or when the ratios of perchloric acid to phosphoric acid were 2: 1 and 1:1. 4 less intense color was developed when a mixture mas lacking in FeC13. The preferable concentration of FeClo was thought to be 1.0% to

avoid the influence on the coloration which might be caused by the diminution of FeCls in the deproteinization. Next, the influence of concentration of color developing agent in the final mixture on the coloration was examined. Here the final mixture means a mixture which contains color developing agent, 0.2% FeCb aqueous acetic acid solution, and cholesterol solution. The final mixtures prepared were heated in a boiling water bath for 20 minutes and their absorbances were read a t 450 mp against the respective blanks. The result shown in Table I1 reveals that the most intense color is given in the presence of 2.00 ml. of color developing agent in 6.00 ml. of the final mixture. It is important to note that the ratio of perchloric acid to phosphoric acid in the color developing agent affects the cholesterol value in serum even when the concentration of FeC18 is fixed as 1.0%. To select the best condition, three reagents were prepared which contained perchloric acid in the ratios of 1 :2, 1:3, and 1:4 to phosphoric acid, respectively. These reagents were applied to the same serum by the method described under procedure, and the cholesterol values were read from each calibration curve. The resulting data are shown in Table I11 with those of parallel tests by Schoenheimer and Sperry's method. The cholesterol values increase with increasing concentration of perchloric acid, and almost agree with those of Schoenheimer and Sperry's method when the ratio of perchloric acid to phosphoric acid is 1:3. The necessary time of heating to reach a maximum absorbance was between 15 and 25 minutes, and 20 minutes' heating was thought to be preferable in routine work. Both visible-light absorption curves of developed color with a standard cholesterol solution and serum had practica,lly the same shape. They had a tendency of gradual increasing intensity toward shorter wavelength region with a shoulder a t 452 mp.

Table V.

Recovery Tests of Method

Cholesterol value (mg./100 ml.) After addition of cholesterol (equivalent to 100 Remg./100 ml. covery, Serum Initial in serum) % 99 327 29 228 102 318 30 216 291 100 191 31 101 266 165 32 263 98 165 33 102 291 34" 189 See footnote of Table IV. 0

The color was s t a b k and its aliiorbance ivas constant for about 4 hours not only in the dark but also in the light. It followed Bc'er's lam- when a concentration of chole3terol in a standard solution lay betw3en zero and 0.2 nig. per ml. Therefore, when serum has a higher cholesterol value than 600 nig. per 100 ml. it should he diluted with water before determination. Table 1V presents a result of comparison of the present method to Zak's and to Schoenheimcbr and Sperry's methods. Recovery tests of t'his method gave 100 i 2 7 , recovery as ihown in Table I-. Cholesteryl acetate and oleate alio developed colors, botE of xhich had an identical visible-light ab;.orption spectrum with that of c:holesterol. And 100 =t 1% and 97 =t1%; recoveries of equivalent cholesterol v:rlues were given by cholest'eryl acetat,. and oleate! respect'ively (Table TI). The relative standard deviation ~i this method a t a level of 225 mg. 1)er 100 nil. cholesterol in seri1.n n.a. O.itiyL:,.

Table VI.

Acetate Equivalent cholesterol values (mg./100 ml.) Added Recovered 181 180 180 161

Recovery Tests of Cholesteryl Esters

Recovery, r /O

101

100 101

Oleate Equivalent cholesterol values (mg./100 d . 1 Added Recovered 121

Recovery,

118

116 117

%

98 96 97

271

ACKNOWLEDGMENT

The authors express their gratitude to Junji Nagai, Central Clinical Laboratory of the University Hospital, for the supply of sera. LITERATURE CITED

Ishii, T., Rinsho Byori, Special Issue S o . 10, p. 152 (1959). 12) Kitamura, hI., Arimatsu, Y., -Yisshin Igaku 48,456 (1961). 1 3 ) "Organic Synthesis,!' Vol. 35, p. 43, Wiley, Sew York, 1 9 ~ 0 . 14) Schoenheimer, R., Sperry, IV. X, (1)

J . Bid. Chem. 106,745 (1934); "Standard Methods of Clinical Chemistry," 5'01. 1, p. 43-54, Academic Press, New Y-ork, 1953. (5) Yoshikawa, H. et al., Igaku no Ayumi, 33, 375 (1960). (6) Zak, B., Ani. J . Clin. Path. 27, 583 (1967). TSUTOMU R~OMOSE Yo VEDA KYOKO YAMAMOTO TOSHIKO MASUMURA KIYOKO OHT.~ Institute of Pharmaceutical Sciences Faculty of Medicine, Kyushu University Fukuoka, Japan

Methyl Orange Spectrophotometric Method for Schiff Bases amineq by deposition on the glass surSIR:I n n rcJccnt issue of .IXALYTICAL

CHEMISTRY,Silverstfin (3) de-crihed the devc1ol)meiit of a methyl orange method for measurem1:nt of high molecular weight' amines in aqueous solution. It \vas I m c d on reaction of the ainiiics with methyl orange s t pH 3 tcJ 4 to form a yellon- coniljex soluble in an organic liquid. The color intensity o i the organic phase Jva:j used as a direct measure of tot'al amine concentration. This procedure was made specific for the detcct'ion and measurement of primary, secondary, arid tertiary amines. The basic methyl orange colorimetric method for arnincb >vas introduced by 13rodie and Udenfriend (1: in 1945 for the determination of cinchona alkaloids arid other organic bases in plasma and urinc.. Later. the writclr and Ellenboge.1 (wj applied this technique to t>he (.stirnation of damphetamine and dr'-amphetamine in blood and urine. I n the interim period this method ha, been u5ed by t'ht vvrit'er and,'or colle igues for many analyses involving det'ection and measurement of amines. Several Txriations hare been used compared to t'he method reported by Silverstein ( 3 ) . The methyl oracge technique ii made riiorc' reliable, especially a t the low coilcentration levels) by coating the glass equipment with a dicone film such as Desicote, which works very well. This prevents the loas of

faces and makes i t possible to obtain reproducible results under a variety of experimental conditions. The sensitivity of the methyl orange procedure iq increased for measurement of total amines by extracting the yellow methyl orange complex from the organic phase into aqueous acid solution. The abborbance of the resulting red solution at 508 m p is significantly higher than for the organic phase a t 430 mp. Other method variations mist including the use of benzene as a solvent instead of ethylene dichloride and the use of a different buffer system. This report describes a further application of the methyl orange procedure a j used in this laboratory for meaburernent of 100 p.p.m. of Schiff base3 in S o . 2 fuel oils. I n general, ,Schiff babes represent the class of compound3 derived by chemical reaction (condensation) of aldehydes or ketones with primary amines. The general formula for the lveakly basic Schiff The Schiff base is RR'-C=S-R". base for this work, I, is represented by the formula tert-Cl,-lsH2,-31-T=CH2, hich is a mixture of highly branched primary amines ha1 ing a tertiary alkyl structure (nitrogen attached t o the tertiary carbon atom). The oil sample containing Schiff base I is dissolved in benzene and given a n alkaline wash. The benzene is shaken

u-ith methyl orange reagent at p H 5 and thcl excess methyl orange is removed. The methyl orange which dissolveb in the benzene through complex formatioii with Schiff baqe I is returned to an acid solution and measured spectrophotometrically. EXPERIMENTAL

Apparatus. Coat t h e inside of glass stoppered bottles, 100-ml. capacity, with a silicone product such as Desicott., produced by Becknian Instrument Co. A Cary Model 11 spectrophotometer with 2 em. fused quartz cells was used for measurement i n t h e visible range. Reagents. Benzene Containing l.?iyOIsoamyl Alcohol. A technical grade of benzene is purified by successive washings with 1N sodium hydroyide, 1 S hydrochloric acid, a n d distilled water. .4 reagent grade of isoamyl alcohol is mashed with 0.2 volunie of 1-1- hydrochloric acid followed by several washes of distilled water. The absorbance of t h e reagent blank should not exceed o.ol"0. Methyl Orange Reagent. Dissolve 500 mg. of methyl orange crystals in 100 ml. of warm water. Immediately before use. mix eaual volumes of the methyl orange solition and saturated boric acid solution. Standard Solution of Schiff Base I. To prepare a stock solution, weigh VOL. 35, NO. 1 1, OCTOBER 1963

1753