Thesis Approved by Major Advisor

Thesis Approved b y Major Advisor A NEW SPECTROPHOTOMETRIC METtilGD FOR THE ESTIMATION OF 3-BETA-HYDROXY- STEROL S BY BENJAMIN 3AHAGIAN A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE IN BIOCHEMISTRY at the CREIGHTON UNIVERSITY OMAHA, 1959 ACKNOWLEDGEMENT I wish to express my sincere gratitude and appreciation to Dr. Victor E. Levine under whose direction and guidance this work was acconplished. ' 3.0 3 0001 00061 0990 TABLE OF CONTENTS “ o' List of Tables V List of Figures v i I. Introduction 1 lie Experimental Methods 7 A. Qualitative ProcProcedures for the Detection of Sugars 7 1*1, Reaction of Sugars with Resorcinol 7 2#2* Reaction of Xylose, Galactose, Digitonin and DigitonideDigitox with Various Phenols 8 3. Blank Tests 11 B. Quantitative Procedures for the Determination of Sugars 11 1. Precipitations of Cholesterol with Digitonin 11 2e Washing of the Cholesteryl Digitonide 12 3. A New Technique for Washing and Centrifuging the Precipitate is Introduced lit lie Development of the Color-Coup lex of Cholesteryl Digitonide with Resorcinol and with Phloro- glucinol 16 5. The Determination of Wavelength of Maximum Absorbance for Resorcinol Color-Coup lex 17 6« The Conformity of Resorcinol Method to the Beer-Lambert Law 17 7 # The Determination of Wavelength of Maximum Absorbance for the Phloroglucinol Color-Conplex 20 8. The Conformity of the Phloroglucinol Color- Conplex with the Beer-Lambert Law 20 9» Extraction of Cholesterol and Cholesterol Esters from Blood Serum 27 10. Hydrolysis of Esters and the Precipitation of Total Cholesterol 30 C. Standardization Procedures and Corqparative Data Presented by Three Different Methods 31 1. The Standardization of the Schoenheimer- Sperry Method 31 2. The Standardization of the Rosenthal Modifi cation of the Zlatkis and Zak Method 33 iii Page 3, Comparative data on Cholesterol and Cholesterol Esters Obtained by the Three Different Methods 33 lu P.ecovery of Cholesterol Added to Serum by the Phloroglucinol Method 35 III. Results and Discussion 38 A, Importance of Cholesterol and Cholesterol Ester Determinations 38 B, Evaluation of Methods Available for Cholesterol Determinations 38 C, Development of the Phloroglucinol Method ill IV. Summary U7 V. References U8 iv LIST OF TABLES Page Table I. Description of colors resulting from the reaction of resorcinol, sugars and concen trated mineral acids 9 Table II. Description of colors resulting from reaction of various phenols with sugars and 70 per cent sulfuric acid 10 Table III. Cooperative data of free cholesterol and total cholesterol values by the three different methods 36 Table IV. Recoveries of cholesterol added to pooled serum by the phloroglucinol method 37 v i LIST OF FIGURES Page Figure 1 . Formulae of cholesterol, digitonin, resorcinol and related compounds k Figure 2 . Special apparatus devised for washing the precipitate of cholesteryl digitonide 15 Figure 3 . The absorption spectrum of the color-complex of cholesteryl digitonide with resorcinol 18 Figure lu The absorption spectrum of the color-complex of cholesteryl digitonide with resorcinol 19 Figure 5 . Conformity to Beer-Lambert Law using the resorcinol cholesteryl digitonide color- coirplex 21 Figure 6 . The absolution spectrum of the color-complex of pure xylose and phloroglucinol 22 Figure 7 . The absorption spectrum of the color-complex of pure galactose with phloroglucinol 23 Figure 8 . The absorption spectrum of the color-coirplex of xylose and galactose with phloroglucinol 2h Figure 9 . The absorption spectrum of the color-complex with digitonin and phloroglucinol 25 Figure 1 0 . The absorption spectrum of the color-complex with cholesteryl digitonide and phloroglucinol 26 Figure 1 1 . Standard curve for the phloroglucinol method 28 Figure 1 2 . Standard curve for the phloroglucinol method 29 Figure 1 3 . Standard curve for the Schoenheimer-Sperry method 32 Figure lit. Standard curve for the Zlatkis-Zak method 3h INTRODUCTION In recent years a great deal of attention has been focused ip on lipid metabolism and in particular ip on cholesterol metabolism. Therefore the estimation of cholesterol and its esters in body fluids has assumed prime importance. There are at least two reasons for this. One is that cholesterol and its esters bear a close rela tionship to the other steroids in the body, and the other is that cholesterol is believed to be involved m some way in certain de generative changes which occur in the walls of arteries, thus bring ing about the condition known as atherosclerosis. Accumulations of cholesterol are not necessarily dependent upon excess dietary intake of cholesterol since the body is capable of synthesizing its own cholesterol from small molecular units such as acetate (1, 2). Similarly, emotional stress situations can increase the cholesterol concentration of body fluids regardless of dietary restrictions (3 ). Although cholesterol concentrations per se do not explain in toto the derangement of lipid metabolism or indicate the onset of atherosclerosis, a very complex disease which develops insidiously over a period of many years, nevertheless a great deal of emphasis has been brought to bear ip on the importance of determining cho lesterol and correlating cholesterol values with other findings (h, 5, 6). Hence the need for an accurate and sirrple method for the estimation of cholesterol in body fluids has been ever present. 2 Cholesterol is the principal 3-beta-hydroxysterol of the animal body« Only animals have cholesterol; plants have other sterols. Cholesterol is of wide distribution in practical3.y all cells of the b o d y and e¡specially in nervous tissue. Blood serum contains both the free form of cholesterol and the ester form. Erythrocytes con tain only the free form of cholesterol. The total blood cholesterol range in the normal individual is approximately 150-250 mg p e r 100 ml of serum, and of this amount about 20-i|0 per cent is present as free cholesterol. Structurally, cholesterol is a sterol which has its nucleus designated as cyclopentanoperhydrophenanthrene in common with other steroids and a saturated side chain attached to Oyj of the cyclopen tane ring. Cholesterol is 3-beta-hydroxy-5,6-cholestene. The 3-beta- hydroxy group which is trans to the angular methyl group at C^Q is the functionally active part of the molecule and occurs at C^ of ring A. The double bond occurs between C^ and C position in ring B. 6 Esterification takes place with the C^ beta-hydroxyl group. The non- hydroxylic part of the molecule is strictly hydrocarbon in nature and accounts for the solubility characteristics of cholesterol which are like those of true lipids. Cholesterol and other 3-beta-hydroxy- steroias are readily precipitated from an alcohol-acetone or alcohol- ether solution by means of digitonin (7, 8). Digitonin, gitonin and tigonin are members of the digitalis saponins which occur with the cardiac glycosides of the digitalis group. The digitalis saponins form solid molecular compounds with 3 the higher alcohols, the phenols and the thiophenols (9 , 10). The digitalis saponins also contain the cyclop entanop erhydrophenan- threne nucleus. Most of the other saponins are built up on some other ring system. The addition conpounds formed with digitonin have been studied more thoroughly than those formed with the other saponins, and in all cases the ratio of the saponin to alcohol or phenol is 1 : 1 (9, 10). These saponins are tetrosides and pento- sides which on hydrolysis yield a number of sugars such as galactose, xylose, glucose, and the aglycones or sapogenins— digitogenin, gitogenin and tigogenin. Digitonin xpon hydrolysis yields the aglycone digitogenin which is a 2-alpha, 3-beta, 15- beta-triol and the pentasaccharide moiety consisting of four mole cules of galactose and one molecule of xylose (11). The digitonides of the beta-sterols and beta-hyoroxysteroias are hydrophilic but those of the alpha-sterols are hydrophobic. The addition conpounds are insoluble in water but are usually soluble in alcohol. However, tne addition conpounds formed with the 3-beta-hytiroxysteroids are insoluble in alcohol. Digitonin is prepared and used as a 1% solu tion in 50% ethanol. Cholesterol esters are not precipitated by digitonin so that a separation of the free cholesterol from that of the ester variety can be readily effected (Fig. 1). The object of this investigation has been to find an accurate and sinple procedure for the determination of 3-beta-hydroxysterols and, in particular, of free cholesterol and cholesterol esters in blood serum. The method developed was one of extraction of FIGURE 1 Formulae of cholesterol, digitonin, resorcinol and related compounds li 19 / n X l X / T X (u n /i /<• / l \ / < ? \ ja VO 7J X « / X 6/ Steroid nucleus Cholesteryl arachidonate Resorcinol Phloroglucinol Orcinol ose i o ¿ J a c t ó s e 0 6 cholesterol and cholesterol esters from blood serum* (11 , 12, 13) by means of alcohol-ether mixture (3 : 1 ) and precipitation of cholesterol with digitonin and corrplete and quantitative separa tion of the cholesteryl digitonide from all traces of excess digitonin. This was acconplished by the use of a mixture of sodium sulfate solution and isopropyl alcohol as wash solution. The cho lesteryl digitonide was treated with sulfuric acid and phloroglucinol to develop a color-conplex with the pentasaccharide moiety of the digitonide. The carbohydrate moiety may be determined by a number of color reactions involving strong sulfuric acid or hydrochloric acid and a suitable compound such as: alpha-naphthol, resorcinol, orcinol, phloroglucinol, anthrone or carbazole (15# 1 6, 17), The action of strong acids on the carbohydrate moiety probably results in the formation of furfural and hydroxymethylfurfural and these con dense with the phenols introduced into the mixture, Phloroglucinol gives a violet-red color with pentoses and uronic acids in the presence of hydrochloric acid.

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