71-18,082
SINGH, Ram Awadh, 1942^ EFFECT OF CERTAIN COMPOUNDS ON STEROL METABOLISM OF LAYING HENS.
The Ohio State University, Ph.D., 1970 Biochemistry
University Microfilms. A XEROX Company , Ann Arbor, Michigan
THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED EFFECT OF CERTAIN COMPOUNDS ON STEROL METABOLISM OF LAYING HENS
DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By Ram Awadh Singh, M.Sc,(Ag.)
******
The Ohio State University 1970
Approved by
Adviser Department of Poultry Science PLEASE NOTE:
Some pages have snail and indistinct type. Filmed as received.
University Microfilms ACKNOWLEDGMENT
I am gTateful to my adviser, Professor E. C. Naber, whose help and guidance made this work possible. The guidance and encouragement of all faculty members are greatly appreciated, as is also the patience of my fellow colleagues. Thanks are due Dr. J. B. Allred for his advice dur ing all phases of my dissertation. I am also thankful to Dr. J. F. Weiss and Mr, J. Elliot for theiT help in analy sis of samples. I am greatly indebted to the Agency for Internation al Development for financial assistance, as well as the government of India and the University of Udaipur for sponsoring me to avail this opportunity. I am very grateful to my wife for her unusual pa tience and understanding during the period of my study in U.S.A.
ii VITA
December 30, 1942 . . , Born, Utter Pradesh, India
1960, a ■ a • ■ • a * a B.Sc.(Ag.), Agra University, Agra, India
1962* * * * * * • a • • M,Sc,(Ag,), Agra University, Agra, India 1962-1963 ...... Research Assistant, Nutrition, Balawant Rajput College, Agra, India 1963-1967 ...... Lecturer, University of Udaipur, Rajasthan, India 1967...... Graduate Student, Department of Poultry Science, The Ohio State University, Columbus, Ohio
FIELDS OF STUDY Major Field: Poultry Science Studies in Nutrition, Professor E. C. Naber Studies in Physiological Chemistry. Professor D. G* Cornwell Studies in Physiology, Professor H, S. Weiss
iii TABLE OF CONTENTS
Page ACKNOWLEDGMENT...... ii VITA...... iii LIST OF TABLES...... vii LIST OF FIGURES...... ix INTRODUCTION...... 1 REVIEW...... 5 Azacholesterol ...... 13 D-Thyroxine...... , ...... IS Nicotinic Acid . . . , ...... 18 Estrogen...... 21 Vanadium ...... 22 Saponin...... 23 Ion Exchange Resins...... 24 Benzmalacene ...... 25 Inositol ...... 26 Sterol Extraction and Determination...... 27 Extraction and Isolation of Lipid ...... 27 Color Development ...... 27 Determination of Cholesterol and Desmosterol in Mixture...... 29 MATERIALS AND METHODS ...... 30 Cholesterol Analysis ...... 30 1. Collection of Blood and Separation of Plasma ...... 30 2. Extraction of Egg Yolk...... 33 3. Extraction of Blood Plasma...... 33 4. Cholesterol Determination ...... 34 A. Pearson's Method...... 34 B. Modified Zlatkis Method ...... 35 C. Modified Lavine and Zak Method, . . 36
iv TABLE OF CONTENTS (continued) Page Thin Layer Chromatography...... 36 Gas-Liquid Chromatography...... 38 Statistical Analysis ...... 38 RESULTS...... 39 DISCUSSION...... 74 Nicotinic A c i d ...... 74 Vanadium ...... 75 Saponin...... 76 Ion Exchange Resins...... 77 Estrogen ...... 78 D-Thyroxine, . • 79 Benzmalacene ...... 80 Inositol...... 80 U-22,593A, U-11.607A and Azacholesterol...... 81 U- 11,10 0A...... 94 SUMMARY AND CONCLUSIONS ...... 9 6 APPENDIX A. Sources of the Drugs Tested in this Study, . 10 0 B. Effect of Selected Drugs on Blood and Egg Cholesterol, Egg Production and Egg Weight - Experiment 1...... 101 C. Effect of Selected Drugs on Blood and Egg Cholesterol, Egg Production and Egg Weight - Experiment 2 ...... ,,102 D. Effect of Selected Drugs on Blood and Egg Cholesterol - Experiment 3 ...... 103 E. Effect of U-11,100A and Azacholesterol on Egg Cholesterol Measured at 550 and 625 mu Wavelength - Experiment 5...... 10^ F. Effect of U-11,100A and Azacholesterol on Egg Weight and Egg Yolk Weight - Experiment 5,,.., ...... 105
v TABLE OF CONTENTS (continued) Page G. Effect of U-11,10OA and Azacholesterol on Egg Production - Experiment 5...... 106 BIBLIOGRAPHY...... 107
vi LIST OF TABLES
Table Page 1, Compounds Tested, , , , ...... • *.«.. 31 2, Content of Basal Ration...... 32 3, Comparison of the Modified Zlatkis and Pearson Methods for Cholesterol Determination ...... 40 4, Effect of Selected Drugs on Changes in Plasma and Egg Cholesterol, Egg Production and Egg Weight - Experiment 1 ...... 46 5, Comparison of Control Group with Drug Treated Groups for Selected Variables - Experiment 1* , 47 6, Effect of Selected Drugs on Changes in Plasma and Egg Cholesterol, Egg Production and Egg Weight - Experiment 2, ...... 48 7, Comparison of Drug Treated Groups with Control for Different Variables Observed - Experiment 2,,.,, ...... 49 8, Effect of Selected Drugs on Changes in Plasma and Egg Cholesterol, Egg Production and Egg Weight - Experiment 3 , , , S5 9, Effect of U-22,593A on Egg Weight - Experiment 4...... 58 10, Effect of U-22,593A on Egg Production - Experiment 4 . , , ...... 59 11, Effect of U-22,S93A on Changes in Absorbance Related to Egg Sterol Content - Experiment 4, , 61 12, Least Square Means of Changes in Absorbance Related to Drug Level of U-22,593A - Experiment 4,,.., ...... 62
vii LIST OF TABLES (continued) Table Page 13. Least Square Means of Changes in Absorbance Related to Time Following Feeding of U-22,593A - Experiment 4...... 64 14. Effect of Azacholesterol and U-11,100A on Changes in Blood and Egg "Cholesterol", Egg Production and Egg Yolk Weight - Experiment 5 . 6S 15. The Rf Values of Sterols in the Reversed Phase Thin Layer Chromatogram , ...... 67 16. Measurement of Different Sterols in Blood and Egg Extracts by Gas-Liquid Chromatograph, . . . 69 17. Effect of Different Levels of Azacholesterol on Concentrations of Cholesterol and Desmosterol, Cholesterol + Desmosterol and Ratio of Cholesterol/Desmosterol in Eggs .and Blood of Laying Hens - Experiment 6 ...... 71 18. Effect of Different Levels of Azacholesterol on Change in Egg Weight and Egg Yolk Weight - Experiment 6...... 72 19. Effect of Different Levels of Azacholesterol on Egg Production - Experiment 6, , ...... 73
viii LIST OF FIGURES
Figure Page 1. The Cholesterol Balance in Laying Hens...... 6 2. Outline of the Pathway of Cholesterol Biosynthesis...... 8 a. The Reactions Between Acetate and Mevalonate b. The reactions etween Mevalonate and Squalene c. The Reactions Between Squalene and Cholesterol 3. Absorption Spectrum of Different Sterols with the p-Toluenesulfonic Acid Reagent Used in Pearson's Method for Cholesterol Determination. 42 4. Change with Time in Absorbance of Sterols Developed Using Pearson's Method of Cholesterol Determination at 550 and 625 ...... 44 5. Effect of p-Toluenesulfonic Acid on Color Development in Pearson's Method of Cholesterol Determination When Absorbance is Measured at Different Wavelengths ...... 45 6. Effect of Different Levels of Azacholesterol on Blood and Egg Cholesterol of Laying Hens . . 83 7. Absorption Spectrum of Different Sterols with p-Toluenesulfonic Acid Reagent Used in Pear son's Method for Cholesterol Determination. . . 84 8. Effect of Different Levels of Azacholesterol on Change in Egg Concentration of Cholesterol and Desmosterol...... 86 9. Effect of Duration of Treatment of Azacholes- terol on Egg Cholesterol and Egg Weight .... 89
ix LIST OF FIGURES (continued) Figure Page 10. Effect of Different Levels of Azacholesterol on Egg S t e r o l ...... 90 11. Change in Absorbance of Egg Extract with Time as Affected by U-22,593ATreatment ...... 92 12. Effect of Different Levels of U-22,593A on Absorbance of Egg Extract...... 93 13. Effect of Different Levels of U-11(100A on Egg Cholesterol ...... 95
x INTRODUCTION
Many years ago, when atherosclerosis was first linked to feeding cholesterol in rabbits, it seemed that dietary restriction of cholesterol might solve the problem of atherosclerosis in humans. However, subsequent study by Wilkinson et al.* using 83 persons from a kindred in which essential familial hypercholesterolemia was present and using a diet of choice reported that the absolute amount of cholesterol in the diet had no demonstrable re lation to the level of total serum cholesterol. This re port was supported by Gould,^ using labeled dietary cholesterol in rats and mice. He demonstrated that a very large portion of labeled was found in respiratory car bon dioxide and in feces possibly as bile acids and neu tral sterols. Of the labeled cholesterol present in the body, a very small fraction was present in the blood in dicating that serum cholesterol is virtually independent of its intake in rats. This probably cannot be general ized. On the other hand vegetarians with low cholesterol intakes tend to have lower serum cholesterol levels than non-vegetarians.^ Later on, Connor ££ an<* Messin- ger et al,® questioned the above findings and demonstrated a significant increase in serum cholesterol when egg yolk was included in the diet of men. Withdrawal of egg yolk from the diet decreased the blood cholesterol level. Yet, chemically pure cholesterol is not very effective when fed without other lipids in altering the serum cholesterol in men, 4 A great deal of interest has been shown in the rela tionship among cholesterol metabolism, atherosclerosis, and the dietary intake of saturated vs, the unsaturated essential fatty acids. It is now fairly conclusively established that dietary intake of polyunsaturated essen tial fatty acids lowers the plasma cholesterol level in animals as well as in man where it was first reported by Tuttle, It seems probable that unsaturated fatty acids are concerned with both oxidation and excertion of choles terol. Gordon et_ al,,® Lewis,® and Intengan*® reported increased oxidation of cholesterol to bile acids when polyunsaturated essential fatty acids were incorporated into the diet, Heilman et_ jal. ** in man and Wilson^ in the rat, using -^C-cholesterol, showed that linoleate markedly enhanced the reduction of cholesterol to copro- stanol in the large intestine where it is excreted whereas oleate and palmitate depressed reduction and excretion. These observations and others led the American Heart Association and other organizations to advocate the use of "modified fat diets", particularly the use of unsaturated fats in place of "hard fats" and consumption of a low cholesterol diet to reduce blood serum cholesterol lev els. This led to the modification of egg yolk fat in favor of unsaturated fatty acids^^but modified eggs had no advantage over the ordinary eggs in their ability to lower blood cholesterol. An equally plausible approach to lower blood choles terol is to lower the level of dietary cholesterol and/or modify the nature of dietary cholesterol so that it is not absorbed and utilized by the body. Since cholesterol is absorbed from the intestine like the long chain fatty acids, in the form of micelles, a change in structure of cholesterol or an agent inhibiting incorporation of cho lesterol into the micelles in the intestinal lumen would serve to reduce blood cholesterol concentration. This hypothesis is substantiated by dietary inclusion of plant sterols which have been shown to lower serum cholesterol by 15 per cent.22 These sterols probably block mucosal esterification and acceptor sites, and also form insoluble sitosterol-cholesterol crystals which are not absorbed.2^ There is evidence that egg cholesterol can be manipulated. Weiss et al. 2 A reported increased cholesterol content of eggs when hens were fed diets containing large amounts of unsaturated fat. Edwards et al,25 showed that feeding of 0.1 per cent of lithocholic acid significantly increased total lipids and egg cholesterol. Attempts have not been reported to produce low cholesterol eggs and/or modify the structure of sterols in the egg either by manipulating the diet of chickens or by drug treatment. The present study was an attempt to alter the level and/or nature of egg sterols with the use of chemothera peutic agents. If a chemotherapeutic agent interfered with cholesterol biosynthesis and/or increased intestinal excretion of sterols and bile acids, blood levels of cho lesterol would be reduced, transport to the ovary would be reduced and if cholesterol synthesis in the ovary were not increased, egg yolk levels of cholesterol might be re duced without adverse effects on egg production. If how ever the agent was extremely effective in inhibiting cho lesterol biosynthesis and prevented sterol reutilization, cholesterol would not be available for estrogen and adre nal hormone synthesis and as a result reproductive per formance would decline, reflecting itself in reduced egg output and egg size. REVIEW
There are several ways drugs might be involved in the cholesterol metabolism. Control of a cholesterol pool size within the animal body leads to the consideration of its destruction and excretion, absorption and utilization, endogenous synthesis and transport and transfer between different body pools. A brief discussion of cholesterol balance in the body and its biosynthesis would be in order to review the roles of selected drugs related to cholesterol metabolism. A simplified summary of cholesterol balance in the body is shown in Figure 1. Although most body cells can synthesize cholesterol, the liver is the main site of cho lesterol biosynthesis. Most of the sterol present in the body is of endogenous origin except for a small but vari able quantity coming from the diet. Once the cholesterol has been synthesized from acetyl coenzyme A, it can be in corporated by liver into lipoprotein molecules, either as the free alcohol, or esterified with fatty acids and re leased into the circulation. For practical purposes, cholesterol present in the body can be considered as a part of several body pools-- 6
FIG. I
THE CHOLESTEROL BALANCE IN LAYIN G HEN.
EWTEROHEPATIC CIRCULATION ' FXOGENOUS ENDOGENOUS Cholesterol Dietary Acetate or absorption B ile acids
BLOOD OVARY
esterified eggs! Birds I
INTESTINAL TRACT______"Bile acids free or conjugated
Steroid hormones free or conjugated
OVARY AND ADRENALS TESTES Glucocorticoids. Estrogen, Progesterone [Mineralocorticoids Testestorone. etc. for example, blood cholesterol pool, liver cholesterol pool, enterohepatic cholesterol pool, brain cholesterol pool, ovary pool, etc. These pools exchange with each other through the blood pool at varying rates depending upon the characteristics of barriers between body pools. Cholesterol is readily synthesized and the only im portant pathway of cholesterol excretion is via the bili ary system into the intestinal tract except in bird and reptile females where very large quantities are excreted through the eggs. Liver excretes some of the cholesterol unchanged but largely in the form of bile acids. Once the cholesterol and bile acids are excreted in the intestine they do not necessarily leave the body. Some of them are recirculated through enterohepatic system. The concept of a series of cholesterol pools in the body is particularly useful when one considers the mechan ism of action of the various agents used to manipulate cholesterol level. For example, one may well question the ultimate clinical value of an agent which lowers serum cholesterol by transferring cholesterol from the blood to other body pools. This review is concerned with inhibi tors of cholesterol biosynthesis; therefore, mention of the metabolic pathways involved is desirable. The syn thetic pathway is shown in Figures 2a, 2b, and 2c. The first step involves the activation of acetate to acetyl 8
FIG. 2
OUTLINE OF THE PATHWAY OF CHOLESTEROL BIOSYNTHESIS,
CHrrCooH
A TP CoA 1 C itrate ♦ CO, * CH,— CO—SCoA MOOC-CH,—CO-SCoA Krebs cycle Acetyl CoA Malonyl CoA ATP
+C H ,— CO—SCoA Patty acid a Synthesis Spiral CH,—CO—CH,—CO -SCoA 9y r Acetoaeetyl CoA Fatty acid + CH,— CO—SCoA
S-OH-Ae,l CoA OK CH,— CO— SCoA CH.— C—1 CH.— CO—SCoA j Triglyceride CK, CHt—CO— CH p - COOH i phospholipid COOH Aectoacetic add p-Hjrdroay*P-ineihylglutaryl CoA
CH,— C— CH,— CH,— OH yh* COOH Mevalonic acid a. The reactions between acetate and mevalonate. 9
n a - i, - v OHI COOH COOH ■•(•bak kM b n t n k pki|lwli r it i V-f -CHj-CHj-O- P - 0 - P - OH ATP Hti«H4ltN) - CO j p 2 OH 1>H -H }0 COOH Imhili rfrtpkti^lHi* 5 kmnu ■V-jj-Clj-CHj-O-f-O.f.• * It -P.I OH -OH HjC-^CH-CKj-O-P-C I CM] OH OH CH, OH OK
rlk irtrb lljl ifittk u k M
NjC>C»CM-CK?-CM,-C-CM-CM,-0 - 5P- 0-P fi - OH CM)I CK)I OHI o I k (mill »TT»fk«l»i*U 7 US HjC -C *CH - CMj -CHj -C i CK - CHj -CH2-C = CH - C H j-0 -P-O-P-OH Atf, CH) OH CM Fmtfl ITPMH 8 [K)Cr -CtCH-CHj-CM,-C *CH I’ - CKj- CHj-C = CK -CM, -1 I c m, Am , c m , J, VoIni b. The reactions between mevalonate and squalene. S^athw ItnMltral 2 I (-COj)
i t i a a l l i n
H 4,4 PI—ri>|ltUm Ulni«-B,2 HO H -4- af«-l,24-4i»*- The reactions between squalene and cholesterol, 11 coenzyme A, and a third molecule condenses with it, in a reaction analogous to citrate formation, to form a branched-chain acid, B-hydroxy-8-methylglutaryl CoA, This compound is then reduced by hydroxymethylglutar>1 CoA re ductase to form mevalonic acid (Figure 2a). Mevalonate is phosphorylated in two successive steps to form the pyro phosphate derivative. This derivative is then decarboxy- lated to form isopentyl pyrophosphate in a rather unique reaction that appears to involve a transient intermediate carrying a third phosphate derived from ATP. Isopentyl pyrophosphate is in equilibrium with dimethylallyl pyro phosphate, Two moles of the 5 carbon intermediates con dense to form Cjq intermediate, geranyl pyrophosphate, and then a third molecule of Cj condenses with geranyl pyro phosphate to form a farnesyl pyrophosphate-15 carbon in termediate. Two moles of farnesyl pyrophosphate condense to form squalene (Figure 2b). The conversion of squalene to lanosterol is an oxygen-requiring reaction, as is the subsequent removal of three methyl groups of lanosterol. Finally, there is a shift of the nuclear double bond from A 8 to A 5 (which actually involves several transitions and 24 requires oxygen at one step) and reduction of the A double bond in the side chain, an anaerobic reaction which results in cholesterol formation (Figure 2c). Availabil ity of a number of excellent reviews makes it unnecessary 12 to detail here the experimental evidence for the normal pathway.26.27.28.29.30 The ideal site of regulating cholesterol synthesis is between g-hydroxy-B-methylglutaric acid and mevalonic acid as suggested by Gould,since a block at this point would not interfere with other metabolic processes; for example, acetyl CoA is oxidized via the Krebs cycle, or is converted to malonyl CoA as indicated by reaction 5 of Figure 2a, and subsequently to fatty acids, triglycerides, and phospholipids. It would seem undesirable to attempt to interfere with the pathway from acetate to $-hydroxy-8_ methylglutaryl CoA. The use of this site is further sup ported by several workers as reviewed by Migicovsky,32 ^ second point for control lies between mevalonic acid and squalene. The liver can dispose of polyprenalpyrophos- phate, a precursor of squalene, preventing an accumulation of this intermediate in the presence of inhibitors. 33 Biosynthetic steps between squalene and cholesterol can be modified with the consequent production of sterol inter mediates that may not move from ovary to eggs or from in testine to blood. Little evidence is available in this regard, and careful examination of this aspect in produc ing modified foods low in cholesterol is needed. Excellent reviews covering effects of drugs on lipid metabolism are available.34*35*36•37 The following review 13 pertains only to drugs used in this study and their rela tionship with sterol metabolism. Azacholesterol Three classes of nitrogen containing steroids have been studied in the past several years, namely 4-azaster- oids with several tertiary derivations, e.g., SKF 12621, SKF 12622, SKF 23476; side chain azasteroids, e.g., SC11952, SC12937, 25-azacholes terol, etc.; and 3- (fj-dial- kylaminoethoxy)-substituted steroids. Some of the 4-azasteroids markedly decrease the con centration of both plasma and liver cholesterol in the TO mouse. Gas liquid chromatographic analysis of the non* saponifiable fraction of liver and blood plasma of treated mice and rats has shown that there is an accumulation of desmosterol.^ Conversion of one of the 4-azasteroids (SKF 12622) to a tertiary amine, namely 3-0-benzyl-4 ,4- dimethyl- 4-aza-5 ct-cholestone chlorid (SKF 14531-A) re- TO suited in a hypolipidemia when given to dogs, ° This com pound reduced cholesterol and triglycerides in plasma. However liver concentration of triglyceride was increased although liver cholesterol was reduced. There was no in dication of accumulation of any other sterol. It is sug gested that SKF 14531-A interferes at a very early stage of acetate incorporation. Another group of azasteroids in which carbon atoms 14 of the side chain have been isosterically replaced by nitrogen have been used. The first compound in this group was 20 a-(2-dimethylaminoethyl) amino-5 a-pregnan-3 8-ol dihydrochloride or SC-11952 or 22,25-diazacholesterol which was studied by Ranney and Counsell. They sug gested that this compound blocks cholesterol synthesis in vivo at the hydroxymethylglutaryl-CoA reductase step. The same site is involved in the homeostatic mechanism for regulating cholesterol synthesis in the liver.40 »4* How- ever, Sachs and Wolfman42 reported progressive accumula tion of desmosterol with fall in blood cholesterol in 14 hypercholesterolemic patients when they received 75 mg daily of 22,25-diazacholesterol. The results of Sachs and Wolfman were further supported by Dvornik4^ who found that 22.25-diazacholesterol markedly lowered cholesterol in the serum, liver, and adrenals of rats. The effectiveness of 22.25-diazacholesterol (SC-11952) in reducing serum cho lesterol levels in experimental animals and in man led Counsell et al.44 to investigate additional analogues of this compound. A series of N-methyl-N (dialkylaminc) al kyl derivates of 17-B-amino-androst-Sen-3B-ol sterically similar to cholesterol were synthesized. Of these 20,25- diazacholes terol (SC-12937) proved to be most potent de rivative based upon the reduction of acetate-1-14C incor poration into hepatic cholesterol. Thompson et al.45 have 15 shown that 20,25-diazacholcsterol fed to rats at 10 mg per kg body weight per day caused accumulation of large amounts of 24-dehydrocholesterol in serum, liver, and car cass. Ahrens et al.*** and Fumagalli and Nilmiro^ inves tigated the possible origin of desmosterol in brain sterol of 20,25-diazacholesterol fed rats, and postulated that desmosterol was synthesized within the brain and not transferred from blood based upon the incorporation of i^c-acetate and glucose in brain and liver of control and 20,25-diazacholesterol fed rats. Counsell et re ported that 25-azacholesterol caused a dose related accu mulation of desmosterol and a total reduction of sterol in turn suggesting a second site of action prior to the cy- clization of squalene. Gordon et_ al. ^ and Phillips et_ al. ^ reported 3-fi- B-dimethylaminoethoxyl) androst-5-en-17-one as a hycholes- terolemic agent, which lowers cholesterol by inhibiting the biosynthesis of cholesterol at the desmosterol stage. D-Thyroxine Results of acetate-1 - incorporation by hens fed high and low fat basal diets have indicated that D-thyrox- ine injection into laying chickens fed these diets, de creased plasma cholesterol and increased egg cholesterol M CO levels. It appears that the ovary works as an excre tory organ for cholesterol in c h i c k e n s because 16 Stamler a^. showed that the greater resistance of hens to dietary induced hypercholesterolemia, compared to cocks, was abolished by oviduct ligation. In myxedema, there are considerable changes in blood lipids, particularly an increase in plasma cholesterol. This increase occurs equally in the free and esterified fractions. The ratio of free to esterified cholesterol usually remains within the range of 0,24-0,32,54 Accord ing to Malmros and Swahn,55 the increase in cholesterol is mainly in the portion associated with 6-lipoprotein. There is a high degree of species variation in the rise of cholesterol level in thyroid deficiency. In humans and dogs the relative rise is much greater than in rats. Turner and Khayat^® have failed to find this rise after thyroidectomy in rabbits. The blood cholesterol of ani mals receiving thyroid treatment is influenced by the diet. As mentioned by Hurxthal,^ the plasma cholesterol level can be reduced in myxedema by giving the patient a diet with a low fat content, and the degree of change de pends upon species. If cholesterol is added to the diet of thyroid-deficient dogs, plasma cholesterol level rises,In D-thyroxine-treated birds, there is not much effect from high-fat diets on blood and egg cholesterol when compared to chickens on a normal diet,^^ From a clinical viewpoint, use of thyroxine in low ering cholesterol level in blood without raising basal 17 metabolic rate has been unsuccessful. However, several reports suggest the usefulness of the D-isomer of Tj and in lowering cholesterol level without much effect on metabolic r a t e . ^ * ^ ' ^ Karp and Stetten®^ showed greater incorporation of deuterium from body water into glycogen, fatty acids, and sterols of liver and carcass in thyroxine- treated rats than in normal controls. This indicates an increased rate of sterol synthesis. Rosenman et al,®* re ported that thyroid treatment causes a greater excretion of bile acids in canulated animals than is true of non treated animals. Treatment with thiouracil decreased the excretion of bile acids.®* Myant and Eder®® showed in their jin vitro rat liver slices study that cholesterol synthesis in liver is increased two to three-fold when bile acids are drained through a canula. A similar con clusion may be drawn from the work of other research ers.®®*®^ Eriksen®® has studied the influence of thyroid activity upon excretion of bile acids in bile duct fisu- lated rats. In the hypothyroid state, formation of cholic acid and chenodeoxycholic acid is diminished, but in the hyperthyroid state there is a slight increase in excretion of bile acids with different composition. Since most of the plasma cholesterol is converted to bile acids, any in crease in plasma cholesterol is expected to increase bile acids excretion. These facts prompted the belief that 18 during thyroid treatment, the rate of cholesterol turnover is greater than the increased synthesis in liver, result ing in an overall decrease in blood plasma cholesterol level. The rise in synthesis may be due to several factors and one of them may be the faster rate of turnover of cho lesterol in the thyroxine treated subject since cholester ol synthesis in liver is inversely related with its con centration in blood. However, there is evidence against the above explanation. Karp and Stetten®^ reported that treatment with thyroid hormone either had no significant effect on concentration of total cholesterol in the liver or led to a slight increase.6^*70 These observations sug gest that under the influence of thyroid hormone the in verse relationship between synthesis and concentration of cholesterol does not exist, though the possibility of fall of free cholesterol in liver was not excluded. Nicotinic Acid Since the discovery of nicotinic acid in 1937 as a pellagra preventive factor, a number of functions have been ascribed to this compound.7* Altshul et al.7^ have reported this compound to be hypocholesteremic in man. As a result, a number of investigators have studied nicotinic acid and related compounds as potential hypocholesteremic agents in man and other species. 19 Friedman and Byers 71 did not observe a change in the level of blood cholesterol due to nicotinic acid in rab bits fed a high cholesterol-fat diet* This observation is contrary to that of Duncan and Best7** who reported that nicotinic acid has a small but significant effect on low ering blood cholesterol in rats. However, their subse quent research contradicted their earlier results.In dogs, Comesana al.^ reported that nicotinic acid had a more rapid and marked effect on reducing blood cholesterol than did the phenylethylacetic acid amide. Treatment with the combination of nicotinic acid and the phenylethylace- tic amide had a more pronounced effect than either one of the substances ingested separately, Norcia et al.7** were unable to confirm these studies. Many researchers have verified the original findings of Altschul et al.72 in man.77*78*75 Gaylor et al.80 reported that blood choles terol level of chicks fed a cholesterol containing diet (0.5%) was depressed by nicotinic acid and slightly de pressed by nicotinamide. However, liver pyridine nucleo tide levels were markedly elevated by both nicotinic acid and nicotinamide while isonicotinic acid and benzoic acid produced no change. Different derivates of nicotinic acid have been in vestigated for their effect on lipid metabolism. On an equimolar basis, nicotinic acid is more potent than its 20 amide,?2,75 Miller et al. ®* tested nicotinuric acid (the major excretory product of nicotinic acid) and its ana logues in order to overcome the absorption problem of nicotinuric acid, and concluded that nicotinuric acid is converted to nicotinic acid in the body before producing its action. According to Mill e r , ®^ quinolinic acid did not lower serum lipids. Hardy et al.®® reported isonico- tinic acid to be more potent than nicotinic acid in reduc ing blood cholesterol in rats and chicks. Salts and es ters of nicotinic acid have been studied in an effort to avoid the side effects of nicotinic acid--gastrointestinal disturbances probably due to acidity of the compound and vasodilation resulting in the flushing of skin--v/ithout much success. It may be seen that the reports on nicotin ic acid as a hypocholesteremic agent are not consistent. Gaylor®® reported increased synthesis of cholesterol in rats and chicks when fed nicotinic acid whereas Parson®^ reported decreased synthesis of cholesterol from acetate in man. Other evidence®®*®6 suggests that nicotinic acid causes increased ^n vitro production of CO2 from acetate in rat liver homogenates indicating the inhibition prior to the formation of mevalonic acid in cholesterol biosyn thesis. This inhibition appears to be at the reduction of 0-hydroxy-0-methylglutaryl CoA to mevalonic acid. 21 Estrogen Clearcut evidence to support the existence of a narked sex difference in blood cholesterol level in the human appears only during the age of 30 to 50 years. Jones ejt al. , Adlersberg et_ al. , and Lawry e£ al. ®^ reported that men have higher levels of blood cholesterol from 30 to 50 years of age than do women of the same age, but levels tend to equalize during the menopause period of women. Keys et al. Block £t al. and Barr et al.®^ failed to support the existence of sex differences of serum cholesterol levels in humans prior to the age of 40 years. However, there are strong indications of signifi cant differences in serum lipoprotein patterns of pre menopausal women and adult men and post-menopausal women. Ultracentrifugal studies revealed that prior to the age of 40 years, men exhibit higher concentrations of low density lipoprotein CL.D.L.)®^*® ^ a n d correspondingly lower serum a-lipoprotein®^than women of comparable age. Lorenz e£ al^reported that the sexually mature female bird has a greater concentration of neutral fat, phospholipids, and cholesterol in blood serum than the male bird. Wood et al.®^ confirmed the above report and added that the immature female bird has a higher serum cholesterol content than the male of the same age and that increases take place in both sexes as they mature. 22 q q Lorenz et al., using estrogenic material extracted from pregnant mare urine administered to immature chicks of both sexes, reported an increase in neutral lipids, phospholipids, and cholesterol in blood serum due to estrogen treatment. Several studies followed thereafter using different kinds of natural and synthetic estrogenic materials in male and female, in young and mature chicks, confirming that estrogen induced elevation of serum lipids.^ *101* It was also noted that the blood serum cholesterol increase was not simply a transfer from other body pools, since a similar increase was also noted in liver, gut, aorta, heart, lung, kidney, etc. Caldwell et also revealed a decrease in cholesterol/ phospholipids ratio in estrogen treated birds. This de crease was due to an increase in phospholipids. Vanadium Curran105 showed that manganese in the presence of an optimum dose of magnesium ions causes an increased in corporation of 14C-acetate into cholesterol. This led him to investigate the effect of other closely related ele ments on cholesterol synthesis. Vanadium and iron de creased incorporation of labeled acetate into cholesterol, and chromium and manganese increased the incorporation. Curran and Costello100 and Mountain et al.10^ further re ported that hepatic cholesterol synthesis in rabbits, 23 measured by l^C-acetate incorporation , was inhibited by the addition of 0.05% vanadylsulfate to the diet. Mountain et_ al^. 10 7 demonstrated a decrease in serum cholesterol as a result of vanadium treatment. They pro posed that this decrease might be due to increased catab olism of cholesterol. This hypothesis was not supported 1 Afi by Whitehouse et^ al_. #AWO based upon an in vitro system prepared from both rat and mouse liver, capable of oxidiz ing up to 10 per cent of terminal methyl groups of choles terol to carbon dioxide. Vanadates were found to have no effect on oxidation of cholesterol in this system. Curran et al.**^ reported the accumulation of B-hydroxy-B-methyl- glutaric acid in rat liver slices incubated with ^C- acetate in the presence of vanadium, suggesting inhibition of cholesterol biosynthesis at the mevalonic acid step. Azarnaf et aKreported another site of action between mevalonate and squalene, most likely at the squalene syn thetase step. Several other mechanisms of inhibition have been reported, suggesting a lack of ATP to phosphorylated mevalonate or a decrease of coenzyme A and Q in liver.111-112 Saponin Incorporation of saponin in the diets of hypercho- lesterolemic chickens decreased blood plasma cholesterol level. It has been suggested that complexing of 24 saponin with cholesterol secreted in the intestinal lumen makes less cholesterol available for reabsorption from the intestinal tract• Newman et al.114 observed reduc tion of cholesterol level in the blood and liver of cholesterol-fed chickens when treated with saponin. Nahm**^ noticed the inhibition of cholesterol absorption with ginseng powder CPanax schinseng) and thus speculated that the presence of saponin in the material produced this effect. Ion Exchange Resins Cholestyramine, a quarternary ammonium styrenedi- vinylbenzene copolymer, has been used as a bile acid bind ing resin. Evidence indicates that the rate of oxidation of cholesterol is regulated by the need for bile acids in the enterohepatic cycle. If this is correct, removal of bile acids from the cycle by introducing a bile acid se- questrant into the intestine should increase the oxidation of cholesterol. Tennent ejt al.fed high molecular weight polymeric quarternary ammonium salts to cholesterol fed cockerels. At the one per cent level, the rise in blood cholesterol was inhibited by 50 per cent. In a longer experiment, plaque formation was reduced by feeding the resin. Similar results were also obtained in dogs without side effects. The compound used in the above ex periment was MK-135, which has also been used in human 25 experiments by Bergen et al, 117 * These workers reported that total blood cholesterol levels were lowered more than 10 per cent in 23 out of 26 patients. Average decrease in serum cholesterol for all subjects was 20 per cent. In further experiments, Tennent et al.**** tested two bile acid binding polymeric organic bases, MK-325 and MK-135, on cholesterol-fed and normocholesterolemic cockerels and dogs, and reported that these bases inhibited the choles terol rise and aortic plaque formation in cholesterol-fed cockerels; lowered plasma cholesterol in normocholesterol emic cockerels and dogs. In an experiment lasting for one year, the bases did not produce any visible toxic effect. When cholestyramine was fed with inhibitors of cholesterol synthesis, their effects were additive. Benzmalacene N-(1-methyl-2,di-p-chlorophenylpropyl) maleamic acid (Benzmalacene) is reported to be a non-competitive inhibi tor of incorporation of mavalonic acid into cholesterol in rat liver homogenates.*20 When it was fed orally as a calcium salt to rats it decreased blood cholesterol by 22 to 30 per cent within ten days following treatment. The acid form was less effective than the calcium salt, but had no detrimental effect on growth of rats. Holmes and DiTullio^l reported indirect evidence that benzmalacene blocked the in vitro synthesis of cholesterol at the 26 polyprenol pyrophosphate stage; however, the exact site is unknown. This compound has also been investigated clinic- 122 12 3 ally on normo- and hypercholesterolemic patients. * It does decrease the cholesterol level in blood, but toxic side effects rule out the possibility of its use as a hypocholesterolemic agent in human patients. Inositol The effect of inositol as a lipotropic agent with or without choline is known, but limited effort has been made to study it as a hypocholesterolemic compound, Herrmann*^* reported that the feeding of 0.5 gm of inositol daily to old hens from high production stock fed a high fat basal diet reduced blood sterol by 11 and 13 per cent in choles terol and cholesterol ester, respectively, after a 25-day treatment. After S5 to 68 days of inositol feeding, the average blood cholesterol dropped to 22 per cent, the 12 5 ester, 17 per cent. On the other hand, Stamler et al. found no effect on blood cholesterol level induced by feeding lipotropic agents, namely choline and inositol, when cholesterol was fed to produce a hypercholesterolemia in cockerels. Instead, these agents tend to aggravate the hypercholesterolemia. Dotti et al.*2^ reported that inositol inhibits the expected rise in cholesterol and phospholipid levels in serum of rabbits on high choles terol diets. 27 Review of Sterol Extraction and Determination Extraction and Isolation of Lipid A mixture of ethanol-ether (3:1) was introduced by Bloor, 12 7 and was used for many years to extract lipid from tissues containing largely neutral lipids like adi pose tissue, A better solvent, chloroform-methanol (2:1) was tried by Folch e£ al, , to extract tissues contain ing phospholipids, cholesterol, and neutral lipids. These solvent systems are not suited for quantitative extraction of highly polar phospholipids, bile acids, and urinary or fecal steroids.>130,131 Lipids which may be found in these extracts are hydrocarbons, cholesterol, esters of cholesterol and other sterols, mono-, di-, triglycerides, wax esters, free-fatty acids, ceramides, most phospholi pids, fat-soluble vitamins, and pigments,Digitonin is used for isolation of sterols. In 1933, Schoenheimer and DamA1 3 3 published a method for the formation and cleavage of the digitonide. Later in 1934, an improved method of determination of free and esterified cholesterol was pub- lished. i ^id This method is not specific for cholesterol. Several compounds similar to cholesterol in structure are also precipitated by digitonin. Color Development Since 188S, the Lieberman-Burchard*33 reaction for 28 determination of cholesterol has been used. In 1910( Grigaut136 developed a quantitative method using the Lieberman-Burchard reaction and it has been modified sev eral times since then, most extensively by Abell.Sev eral other workers have attempted to find a more specific and consistent method. Zlatkis and others published sev eral papers on a method in which they used ferric chloride and sulfuric acid as a color reagent. This is a direct method involving no hydrolysis prior to color de velopment. However, Weiss et al.51 have shown that un saturated fatty acids of egg interfere with Zlatkis rea gent to give color that interferes with the determination. Unsaturated carotenoids also contribute to the color de velopment. Hanel and Dam*^* simplified a method for use with blood. In this method Tschugaeff reagent (acetyl- chloride and zinc chloride) is used. Pearson et al,^^ determined cholesterol directly from serum without inter vening the phases of extraction and hydrolysis, and color was developed with p-toluenesulfonic acid. This method was evaluated for reaction time, reaction temperature, intensity and speed of color development due to free and cholesterol ester, loss of color with time by Leppanen1^3 and was used in present work after further evaluation and modification. 29 Determination of Cholesterol and Desmosterol 1 n~~ :Ti xture Several analytical approaches have been used to de termine the relative concentrations of desmosterol and cholesterol. Avigan et a K noted that desmosterol ab sorbs only 61 per cent of total light that was absorbed by cholesterol at 635 my in the Liebermann-Burchard reaction, FTantz e£ a^. observed desmosterol to give half the color yield of cholesterol with Liebermann-Burchard reac tion, but both gave equal color yields with Zlatkis rea gent. Based on these observations, Frantz et^ al. ana lyzed two aliquots of the same sample, one by Zlatkis method and another by Abell. From two determinations the separate value of each sterol can be calculated by solving two simultaneous equations for two unknowns, Hollander ej^al^.l^ followed a similar approach using the Abell and Anthrone precipitating method. In our laboratory, desmosterol and cholesterol were measured at 550 and 625 my, respectively, which are ab sorption maxima when Pearson*s method of color development is used. Since the colors are measured at wavelengths of maximum absorption, this technique may be more sensitive than others. MATERIALS AND METHODS A series of six experiments was conducted in this investigation. The first two experiments were designed to evaluate the twelve compounds listed in Table 1. In all experiments, egg production type chickens were used. They were housed in individual cages and given 14 hours of light each day. Feed and water was supplied every day on an ad libitum basis. Drugs were administered by incorporation into the basal ration which was a high energy, all mash, chicken layer and breeder ration (Table 2). Feeds were kept isocaloric, using sucrose as carrier for mixing the drugs. One kilogram of basal ration was mixed with 50 grams of sucrose or sucrose premix to pro duce the experimental diets. More details pertinent to the individual experiments will precede the results of each experiment in the next section. Cholesterol Analysis 1. Collection of Blood and Separation of Plasma Blood samples were collected either by heart punc ture using a syringe with an 18G, 2" needle or by 30 31 TABLE 1 COMPOUNDS TESTED Experiments 1 2 3 4 5 6 Compound Levels in Feed Nicotinic Acid 1.0 UmAfO 10.0 20.0 Vanadium 10.0 (mg/kg) 25.0 from V 02O5 50.0 Saponin 3.0 (gm/kg) 6.0 12.0 U-11.100A 10.0 25.0 2.5 (mg/kg) 25.0 50.0 5.0 so.o 100.0 200.0 U-11.607A 10.0 2.0 (»g/*g) 25.0 5.0 50.0 10.0 25.0 U -22,593A 10.0 2.0 0.5 0.1 (■g/ke) 25.0 5.0 1.0 0.25 50.0 10.0 2.0 0.5 5.0 1.0 Atacholesterol 1.0 5.0 0.5 2.0 0.5 (mg/kg) 2.0 10.0 1.0 5.0 1.0 5.0 20.0 2.0 2.5 5.0 5.0 Ion Exchange 5.0 Resin*- 10.0 Cholestyramine 20.0 (»g/kg) D-Thyroxine 10.0 (*b A e ) 20.0 30.0 Estrogen 10.0 (■g/kg) 40.0 •0.0 Benzmalacene 0.0 (gm/kg) 0.5 1.0 Inositol 2.0 Cg»/kg) 5.0 10.0 32 TABLE 2 CONTENT OF BASAL RATION Ingredients gm/kg of Feed Ground Yellow Corn 672.9 441 Soybean Meal 155,5 50t Meat and Bone Scrap 25,0 60t Dried Fish Solubles 15,0 17% Alfalfa Meal 25.0 16% Dried Whey Product 25.0 Dicalcium Phosphate (16% Ca; 21% P) 12.0 Feeding Limestone (35% Ca) 65.0 Iodized Salt 4.8 Micronutrients Added rcg/kg of Feed Riboflavin 1.77 Pantothenic Acid 3.85 Vitamin B]? 0,06 DL-Methionine 489,00 Antioxidant (BUT) 124.00 Zinc (Zinc Oxide) 137.50 Manganese (Manganous Oxide) 116,50 I.U./kg of Feed Vitamin A 2,200 Vitamin Dj 1,056 33 puncturing the brachial vein using a 23 G, 1” needle. The blood was transferred immediately to heparinized test tubes. Plasma was separated by centrifuging the blood samples at S90XG for ten minutes* Plasma was stored in a refrigerator at 4°C for further analysis. 2, Extraction of Egg Yolk Egg yolks were separated and weighed in aluminum foil dishes. After thorough mixing, 10 gm yolk samples were weighed, and 30 ml of chloroform-methanol (2:1 v/v) solvent was added with mixing. Mixtures were allowed to stand for two hours before they were filtered through sin tered glass funnels using a vacuum pump. An additional 40 ml of solvent was added and the extraction was re peated, Extracts were diluted to 100 ml with the same solvent in volumetric flasks. One ml of extract was diluted with 9 ml of isopro panol when the autoanalyzer was used for cholesterol anal ysis, 3, Extraction of Blood Plasma Extracts of blood plasma were used. When the Tech- nicon autoanalyzer was employed for analysis, (9.5 ml. of 99 per cent solution) Isopropanol was added in series of test tubes with an automatic burette, followed by 0.5 ml of plasma from a volumetric pipette. Contents of test 34 tubes were mixed thoroughly and centrifuged in a refriger ated centrifuge to reduce loss of solvent by evaporation, A suitable aliquot of supernatant liquid was then intro duced in Technicon autoanalyzer for cholesterol determina tion. 4. Cholesterol Determination A. Pearson's Method142 In this method blood plasma was analyzed directly without any extraction. Solutions were pipetted into test tubes in the following order: 0.2 ml of blood plasma or egg yolk extract, 0.2 ml of glacial acetic acid, 1.0 ml of p-toluenesulfonic acid (13.25 gm p-toluenesulfonic acid dissolved in 100 ml of glacial acetic acid) and 3,0 ml of acetic anhydride. Tubes containing plasma samples were mixed thoroughly to completely disperse the protein pre cipitate. The mixture was cooled to room temperature. Previously cooled 0,4 ml of concentrated sulfuric acid was added. Tubes were mixed and allowed to stand for 20 min utes for color development. The absorbance of the colored solution was measured at 625 mp wavelength using a Coleman Autoset Spectrophotometer or a Bausch and Lomb Spectronic 20 Spectrophotometer. A standard curve was prepared by using known quantities of cholesterol standard solution (200 mg recrystallized cholesterol per 100 ml glacial acetic acid) in place of the plasma or egg yolk extract. 35 The color was developed in the manner described for plasma or egg extract. B. Modified Zlatkis Method^ The lipids contained in 0.5 ml,of egg yolk extract or plasma were saponified with 5 ml alcoholic potassium hydroxide (6 ml of 33 per cent potassium hydroxide in 94 ml of 95 per cent ethanol) for one hour at 65®C. Five ml of a cholesterol standard solution (0,4 mg recrystallized cholesterol/ml absolute ethanol) was saponified with 0,3 ml of a 33 per cent alcoholic potassium hydroxide under the same conditions as the samples. Ten ml of petroleum ether (B,P. 65-85°C) was added to saponification mixture and the tubes were shaken. Five ml of water was added washing the stopper and sides of test tube. Two aliquots of the petroleum ether extract (2 ml) were taken for the analysis of each sample and standard. Petroleum ether fTom the extracts was evaporated under nitrogen at 6S®C before the addition of any color reagents. Three-tenths ml of glacial acetic acid was added to the sample residues followed by 2 ml of freshly prepared Zlatkis reagent (99 ml of concentrated sulfuric acid added to 1 ml of a solu tion of 1 gm ferric chloride in 10 ml glacial acetic acid) by means of a pipette. Care was taken to avoid the imme diate mixing of acetic acid and Zlatkis reagent. After the reagent was added to all the samples, the bottoms of 36 tubes were struck sharply to effect immediate mixing and even heat distribution. The color mixture was transferred to cuvettes after 20 minutes and optical densities were measured at the 560 mu wavelength. Cholesterol values were determined by dividing the absorbance of the samples by average absorbance of cholesterol obtained from the standards. C. Modified Levine and Zak Method^*® This method has been modified by Block e£ al. for automated analysis using a Technicon autoanalyzer. An anhydrous reagent containing ferric chloride dissolved in a mixture of acetic acid and sulfuric acid is used. This reagent is preheated to 95°C in a long glass coil. After leaving the heating bath the stream is segmented with air and the plasma extract is introduced. The reaction takes place in vacuum jacketed mixing coils. Color is developed and read at the 550 mu wavelength in a tubular flow cell with a 15 mm light path. Thin Layer Chromatography Reversed phase thin layer chromatography described by Souza and Nes**® was used to separate different ster ols, This method employs the use of paraffin oil as the stationary phase and aqueous acetone saturated with paraf fin as the mobile phase. Anisaldehyde-sulfuric acid was used as detecting reagent. The materials for analysis 37 were obtained from commercial sources.* About 500 ml of acetone-water 4:1 was shaken in a separatory funnel with 50 ml paraffin oil. The mixture was allowed to stand several hours at room temperature. The paraffin oil layer was separated and diluted with petroleum ether (B.P. SO-bO^C) to a five per cent solution of paraffin oil. This solution was used to impregnate the JCieselguhr-G coated thin layer plate which had been previ ously activated at 110°C for one hour. The acetone-water mixture was introduced in chromatographic development tank and was used as solvent. The sterols (in chloroform- methanol solution) were applied on the plates and the chromatoplate was developed. After the solvent front had ascended to 15 cm, the plates were dried in air and sprayed with the reagent containing 1.5 gm of p-anisalde- hyde and 1.5 ml of concentrated sulfuric acid in 27 ml of 90 per cent ethanol, and heated for 5-10 minutes at 110°C. The sterols spots were blue on a pale pink background. *Kieselguhr-G chromatographic plates - Brinkmann Instru ment Inc., Westbury. Paraffin Oil, Light (Mineral Oil) - Will Scientific, Inc. p-Anisaldehyde - J. T, Baker Chemical Company, New Jersey. Cholesterol - Applied Science Laboratories, Pennsylvania. Desmosterol - Applied Science Laboratories, Pennsylvania. Lanosterol - Steraloids, Inc., Queens, New York. A'-Cholestene-38-ol - Calbiochem. Cholestane - Applied Science Laboratories, Pennsylvania, Cholesterol Acetate - Hormel Foundation. 7-Dehydrocholesterol - Steraloids, Inc., New York. 5a-Cholestan-38,5a,60-Triol - Steraloids, Inc, Sitosterols - Applied Science Laboratories, Pennsylvania. 38 Gas-Liquid Chromatography Aliquots of chloroform-methanol extract were evap orated to a small volume, and were saponified with 2 per cent potassium hydroxide in absolute ethanol for 90 min utes at 60°C. Five ml of water was added and the mixture was extracted with 7 ml of hexane. The extract was evap orated to dryness under nitrogen. Trimethylsilyl ether derivatives were prepared by adding 0.1 ml of a mixture of pyridine:hexamethyldisilazane:trimethylchlorosilane (9:3:1), The mixture was allowed to stand at room temper ature for at least 15 minutes, then it was evaporated under nitrogen and vacuum. The residue was dissolved in a small amount of carbon disulfide and was injected in a gas-liquid 4 foot glass column containing 1 per cent Hi- Eff-3BP (Applied Science) at 200°C. Inlet and detector (hydrogen flame) were at 220°C. Flow of nitrogen was kept at 40 cc/minute, air at 400 cc/minute, and hydrogen at 25 cc/minute. Statistical Analysis Statistical analyses were done using a generalized least square analysis program described by W, R. Harvey. 149 RESULTS Evaluation of Pearson's Method of Cholesterol Determination Pearson's method of cholesterol determination was compared to the well-known method described by Zlatkis and modified by Weiss at a^, Blood plasma and chloroform- methanol extracts of egg yolk were studied. The results obtained are presented in Table 3, It is clear from the data that Pearson's method consistently produced lower values for plasma cholesterol when compared to the Zlatkis method. In the case of the egg extract, Pearson's method resulted in an average content of 1606 mg/100 gm yolk whereas the Zlatkis method gave 1587 mg/100 gm. However, this difference is negligible. It is possible that plasma contains some compound which interferes with color devel opment in Pearson's method and is removed when plasma is extracted with a fat solvent. This hypothesis is further supported by the finding that egg yolk extracts yield sim ilar values with both methods,and the difference in plasma cholesterol concentration determined by the two methods became negligible when Pearson's method was corrected for recovery (Table 3). 39 40 TABLE 3 COMPARISON OF THE MODIFIED ZLATKIS AND PEARSON FETHODS FOR CHOLESTEROL DETERMINATION Pearson * s Corrected for Pearson * s Recovery Zlatkisa Source mg/100 ml Plasma 1 101.9 131.3 131.0 2 106.0 136.6 131.5 3 12S.0 161.0 142.4 4 109.5 141.1 138.3 Average 110.6 142.5 135.8 mg/100 gm yolk Egg Extract 1 1530 1440 2 1619 1548 3 1620 1553 4 1654 1805 Average 1606 1587 aZlatkis method involves the extraction of blood plasma. 41 Pearson's method is a c-iveci o:. * and does not in volve the saponification and xt:vacti -n of blood plasma. It is much more rapid than tht* i'uJ.K.-' method, and suited to experiments where large nurlers of analyses are in volved. Pearson's method was also subjected to internal hecking for the recovery of pure cholesterol, change in ibsorbance with time following color development, use of j-toluenosu1fonic acid as the oxi ABSORPTION SPECTRUM OF DIFFERENT STEROLS WITH THE P TOLUENESULFONIC ACID REAGENT USED IN PEARSONS METHOD FOR CHOLESTEROL DETERMINATION LAN STEROL...... CHOLESTEROL---" to< Egg Ext.-AZACHOLESTEROL-...... CONTROL EGG EXTr DESMO STEROL------ I o. 4 * A UJ 10 U2 < □o0£ loO oo< j 0- I 0 * >00 no 4 00 t 00 too WAVELENGTH mu 43 550 my, as indicated in Figure 4, However, if the optical density was measured within 15 to 20 minutes after the color was developed, the discrepancy due to the loss of color was avoided. Since the absorption at the 625 my wavelength was several times greater than at 550 mp, meas urement of cholesterol was much more sensitive at 625 my. The use of p-toluenesulfonic acid as an oxidizing agent resulted in greater color development and as a result greater absorbance at 625 my, as indicated in Figure 5. Hence the use of p-toluenesulfonic acid in addition to sulfuric acid in the color reaction increased the sensi tivity of the assay. Screening of Hypocholesterolemic Drugs The first two experiments were conducted to screen several drugs for their effects on blood and egg choles terol, egg production, and egg weight. The results ob tained in these experiments are presented in Tables 4 and 6 as least square means of the changes before and after the drug treatment. Plasma cholesterol, egg cholesterol, egg production, and egg weight changes are shown. The original observations are presented in Appendices B and C. The analysis of variance of the dose response of the drugs are also reported in Tables 4 and 6, Only significant effects are mentioned. Tables 5 and 7 contain the t-values for drug treated groups compared to control groups where ABSORBANCE X 1000 4 3 00 300 1 00 CHANGE W IT H T IM E IN A B S O R B A N C E OF STEROL COLOR COLOR STEROL OF E C N A B R O S B A IN E IM T H IT W CHANGE DEVELOPED U S IN G PEARSONS METHOD OF CHOLESTEROL CHOLESTEROL OF METHOD PEARSONS G IN S U DEVELOPED 40 ERMI I T 5 AND 65 u m 625 D N A 550 AT N TIO A IN M R TE E D »0 ME I NUT S TE U IN M IN E IM T 3 10300 100 130 L AT 5 AND 65 u m 625 D N A 550 T A K N BLA HLSEO AT 50 _ u m 550 T A CHOLESTEROL CHOLESTEROL A T 625 625 T A CHOLESTEROL 4 . G I F EUM AT 65 . . - - - u m 625 T A M SERU ...... 44 EFFECT OF P-TOLUENESULFONIC ACID ON COLOR DEVELOPMENT IN PEARSON'S METHOD OF CHOLESTEROL DETERMINATION WHEN ABSORBANCE MEASURED AT DIFFERENT WAVELENGTH CHOLESTEROL WITH P-TOLUENESULFONIC AC ID - CHOLESTEROL WITHOUT P-TOLUENESULFONIC ACID \ SERUM WITH P-TOLUENESULFONIC ACID’ SERUM WITHOUT P-TOLUENESULFONIC ACID- # \# \ \0 # \0 \# \ \0 ___ 500 550 600 625 700 WAVELENGTH- mu 4 6 T A B L E * a E F F E C T O F SELECTED DRUGS ON CHANCES TN PL ASMA AND EGG CHOLESTEROL, EGG PRODUCTION, AND EGG A E I G l i T P l a s m a t r s E c 3 EE* £ g g £fig Cholesterol Choles terol Product ion P r o d u c t i o n L'eii;ht b’e l e h t L e v e l s £a - b ) f A - B ) (D-B) (A-B) (D-B) (A-B) D ru g s I n F e e d m g / J O O m l rip/lOO yolk I 1 S" g m Experiment 1 N i c o t i n i c 1 g m / k g - 56 - 1 1 1 - 1 • 4 0 * 3 A c i d 1 0 g m / k g -16 -36 -3 • 11 -2 ♦ 3 2 0 g m / k g - 2 1 - 1 6 0 - 4 - 2 + 1 + 6 V a n a d i u m 1 0 » ( t / k * - 1 2 • 14 - S - 1 3 ♦ 1 + 4 2 5 m g / k g ♦ 2 - 2 9 9 ♦ 2 + 6 + 6 + 6 S O m g / k g - 1 7 • 5 1 4 • 4 • 3 • 3 + 6 S a p o n in 3 gm/kg -24 - 391*L ♦17**L + 1S*L *I»*Q ♦ 3 0 g m / k g -go - 4 4 0 ♦ 3 - 1 - 4 •1 1 2 g m / k g - 3 S • 9 - S + 6 •» ♦ * U - 1 1 . 1 0 0 A 10 mg/kg -SO •60 -4 • B ♦ 4 + 3 2 S m g / k g • 4 6 + 64 - 9 - 6 ♦ 1 + 1 S O m g / k g - 6 5 - 3 4 5 - 1 • S ♦ 1 + 2 U - 2 2 , S 9 3 A 1 0 m g / k g - S*L - 4 1 T * * Q - S 2 “ Q - 4 4 “ L - 1 ♦ 6 m g / k g + 6 1 - 1 S 9 • 3 9 • 4 6 - 6 + 1 S O m g / k g + 71 - 6 2 2 - 5 6 - 7 6 - 4 - 3 0 - l l , 6 O T A 1 0 m g / k g + S S - 2 2 6 • 49 - S2 • 6 * 1 2 S m g / k g + 107 - 6 3 3 - $ 4 - 6 3 - 3 • 2 S O n g / k g + 7 J • 3 6 3 - 4 S - 6 0 -3 + 1 Aaacholetterol I m g / k g - S*Q - g a » Q - 1 0 - 4 2 0 • 1 2 m g / k g + 6 1 - 1 7 4 - 1 0 - 3 9 +1 -3 5 m g / k g + 1 4 • 6 4 1 • IS • 4 0 • 2 - 2 C o n t r o l ♦ 1 • 1 0 4 - 4 - 2 0 ♦ 2 + 3 •cholesterol was determined by Pearson's method *t 6ZS mu wavelength. Following abbreviations have also been used in other experiment*; (A-B) sterds for change; increase ( + ) or decrease (-) ftora pro-experimental to post- expcriir.ental period. £P-B) stands for change; increase (+) or decrease (-) from pro-experimental to during experimental period. *1 - Linear effect at SI level of probability, +Q - Quadratic effect at SI level of probability. **L - Linear affect at It level of probability. ■*Q - Quadratic effect at 11 level of probability. 47 TABLE 5 COMPARISON OF CONTROL GROUP WITH DRUG TREATED GROUPS FOR SELECTED VARIABLES Experiment 1 Drugs Variables Calculated t-value U-22 ,593A Blood Cholesterol (A- B) 1. 58 Egg Cholesterol (A-B) 1.56 Egg Production (D-B) 10.00 Egg Production (A-B) 9.46 Egg Weight (D-B) 2.24 U-11,100A Blood Cholesterol (A-B) 1.97 U-11.607A Egg Production (D-B) 3.80 Azacholesterol Egg Production (A-B) 6.70 Saponin Blood Cholesterol (A-B) 1.67 Egg Cholesterol (A-B) 1.50 Tabular t-value Required t-value for 1,22 d.f. 10% probability 1.717 5% probability 2.074 1% probability 2.819 TABLE 6 EFFECT OF SELECTED DRUGS ON CHANGES IN PLASMA AND EGG CHOLESTEROL,* EGG PRODUCTION, AND EGG WEIGHT Experiment 2 Plasma Egg Egg Egg Egg Egg Cholesterol Cholesterol Production Production Weight Weight Levels (A-B) (A-B) (D-B) (A-B) (D-B) (A-B) Drug* In Feed mg/100 ml mg/100 gm yolk \ \ gm «* Aiecholesterol 5 mg/kg + 49*L -194*L -16 -42**L -6 0 10 mg/kg ♦ 148 -142 -39 -63 -9 0 20 mg/kg ♦166 * 52 -49 -85 -10 0 U-22,S93A 2 mg/kg ♦ 69 * 100*L -42 -35 •10 0 S mg/kg ♦171 -1SS -52 •44 -S 0 10 mg/kg ♦ 169 *321 -43 -40 *6 0 Choleityraaine 0,5 gm/kg ♦ 7S*L -1SS + 4 -2 ♦ 3 0 1 gm/kg +22 -214 + 7 + 2 ♦ 4 0 2 gm/kg -26 -180 -8 -10 ♦ 1 0 D- Thyroxine 10 mg/kg -13*Q +39**Q •S 0 ♦ 3 0 20 mg/kg -141 ♦ 165 -12 -8 + 1 0 30 mg/kg -40 ♦417 -29 -21 -1 0 Eitrogen 10 mg/kg -41 -78 +17*1 + 14*L ♦ 1 0 40 mg/kg + 13 -104 ♦S ♦1 ♦ 3 0 80 mg/kg +25 • 132 -8 -9 ♦S 0 Benin*licen* 0.S gm/kg ♦ 38 ♦ 127 -13 -16 0 0 1.0 gm/kg ♦46 ♦39 0 -3 0 0 Inoiltol 2 gm/kg ♦61 -87 ♦ 20 ♦18 ♦ 2 0 5 g»/kg ♦ 34 -187 + 1 -1 ♦ 1 0 10 gm/kg ♦36 -289 ♦ 5 ♦6 ♦ 2 0 Control ♦ 7 •48 ■1 ♦5 ♦2 ♦1 "Cholesterol wo* determined by Petrion'* method *t 62S mu wovelength. 49 TABLE 7 COMPARISON OF DRUG TREATED GROUPS WITH CONTROL FOR FOR DIFFERENT VARIABLES OBSERVED / Experiment 2 Drugs Variable Calculated t-value Azacholesterol Plasma Cholesteral (A-B) 2. SO Egg Production (D-B) 3.46 Egg Production (A-B) 7.65 Egg Weight (D-B) 4.02 U- 22,59 3A Plasma Cholesterol (A-B) 2.89 Egg Production (D-B) 4.60 Egg Production (A-B) 4.90 Egg Weight (D-B) 3.23 D-Thyroxine Plasma Cholesterol (A-B) 2.12 Egg Cholesterol (A-B) 3.68 Estrogen Egg Weight (A-B) 2.20 Bcnzmalacene Egg Production (D-B) 3,71 Egg Production (A-B) 5.30 Tabular t-value Required t-value for 1,22 d.f. at 101 probability 1.717 at 5% probability 2.074 at It probability 2 .819 50 statistically significant differences due to drug treat ment were found. Six birds were used at each level of treatment in the first two experiments. The birds on treatment level were divided into two sub-groups of three birds each, and blood and egg cholesterol were analyzed from pooled sam ples of the sub-groups. Total cholesterol content of blood plasma and egg yolk was determined before the drug treatment and three weeks after the drug treatment. Pear son^ method of cholesterol analysis was used and samples were measured at the 625 mu wavelength. Statistical anal ysis was done on the difference of observations before and after treatment. It appears that nicotinic acid, vanadium, saponin, U-11,100A decreased the blood "cholesterol" level after three weeks of treatment, whereas U-22,593A, U-11,607A and azacholesterol increased the blood cholesterol level over three weeks of treatment (Table ^). None of these changes were significantly different from the control at 5 per cent level of probability. However, the dose effect of U-22,593A at 10, 20, and 50 mg per kg of feed shows that egg "cholesterol" was reduced after three weeks of feeding the drugs but none of these changes were significantly differ ent from the control at the 5 per cent level of probability. In the control group, egg cholesterol decreased by 10*f mg/100 6® egg yolk, which is a reduction of 8.9 per cent from 51 before the treatment. This decline may be due to in creased size which was about 5,3 per cent, Slight decrease in egg production nay also be a contributing factor in the decline of egg "cholesterol" level. Saponin, fJ-22 , 593A and azacholesterol produced significant dose effects on change in egg cholesterol at the 5 per cent of probability. The drugs U-22,593A and azacholesterol produced highly signifi cant negative quadratic dose effects but in the case of the saponin effect, egg cholesterol decreased as the treat ment dose of saponin increased. Egg production decreased very significantly during the treatment, particularly in the cases of U-22,593A, U-11,607A and azacholesterol, These reductions were significantly different from the control. There was a linear negative dose effect of sap onin on egg production and quadratic effect of U-22,593A on egg production. The dose effect of U-11,607A on change in egg cholesterol and egg production was similar to the effects of U-22,593A and azacholesterol, Based upon these results, it was decided to re-test U-22,593A at lower levels and azacholesterol at higher levels because U-22,593A at 10, 25, and 50 mg/kg of feed severely hampered the egg production and azacholesterol at 1, 2, and 5 gm/kg feed resulted in a linear decline in egg "Cholesterol" implies apparent cholesterol and may also include other sterols. 52 cholesterol. The second experiment was similar to the first experiment except that five new drugs were tested. The birds in this experiment were four months older than the first experiment. Results are presented in Tables 6 and 7. Azacholesterol at 5, 10, and 20 mg/kg of diet had a significant linear effect on changes in plasma and egg cholesterol. The increase in blood cholesterol and de crease in egg cholesterol was significantly different from the control at the 5 and 1 per cent levels of probability respectively (Table 7), Significant reductions in egg production and egg weight also were evident, U-22,S93A at 2, 5, and 10 mg/kg had a significant linear effect on re duction of egg cholesterol. When compared with the con trol by the t-test, plasma cholesterol was increased and egg production and egg weight were decreased. All these changes were significant at 1 per cent level of probabil ity, D-thyroxine at 10, 20, and 30 mg/kg decreased blood cholesterol and increased egg cholesterol. These changes were quadratic and significant at 5 per cent level of c 7 probability and confirm the findings of Weiss et al. * No significant changes in egg production or egg weight oc curred due to D-thyroxine treatment. Feeding of estrogen at 10, 40, and 80 mg/kg decreased egg production signifi cantly at the 5 per cent level of probability. 53 Benzmalacene at 0*5 gm and 1.0 gm/kg of ration in creased the blood and egg cholesterol, and the effect was more severe at 0.5 gm level than 1,0 gm/kg of feed. There was significant reduction in egg production when compared to control group. Inositol appeared to decrease egg cholesterol when fed at 2, 5, and 10 gm/kg of feed but blood cholesterol appeared to increase. However, these changes were not statistically different from the control. There were no noticeable effects of Inositol on egg production or egg weight. Cholestyramine seemed to increase blood choles terol at lower levels and decreased it at higher levels. Egg cholesterol decreased at all doses but there was no obvious trend. Three of the twelve compounds studied in the above experiments met partially our criteria of selecting drugs which reduce egg cholesterol without severely reducing egg production and egg quality. Therefore, it was decided to study U-22,593A, azacholesterol, and U-11,100A in more detail. Three compounds from the Upjohn Company (U-22,593A, U-11,100A, and U-11,607A) and azacholesterol from the G. D. Searle Company were studied in the third experiment. All drugs were fed at four levels and eight egg production type hens were fed at each treatment level. No control 54 group was maintained. Each group served as its own con trol for processing of the data. Egg and blood choles terol were determined on individual samples before and three weeks after the drug feeding. The modified Levine and Zak method designed for the Technicon autoanalyzer with spectrophotometric examination at 550 mp was used for cholesterol measurement. Results obtained are presented in Table 8. The general effect of all the compounds studied in this experiment was the same as reported in the first two experiments. U-22,S93A at 0,1, 1, 2, and 5 mg/kg of feed resulted in a quadratic effect on the increase in blood cholesterol in which a maximum increase was noted at 2 mg/kg of feed. This is not consistent with the observa tions of the first two experiments, where a linear change was found up to 50 mg/kg of feed. A similar inconsistency was observed in the decrease in egg cholesterol. In this experiment the reduction in egg cholesterol was smaller as the treatment level of drug increased, which was just the opposite of results reported from the first two experi ments, However the range of drug levels employed was somewhat different. Egg production decreased due to treatment and the results were similar to those reported earlier. Effect on reduction in egg weight was much more pronounced than in the previous experiments. TABLE 8 EFFECT OF SELECTED DRUGS ON CHANGES IN PLASMA AND EGG CHOLESTEROL,3 EGG PRODUCTION AND EGG WEIGHT Experiment 3 Plasma Egg Egg Egg Egg Egg Level of Cholesterol Cholesterol Production Production Weight Weight Feed (A-B) (A-B) (D-B) (A-B) (D-B) (A-B) Drugs mg/kg mg/100 ml mg/100 gm yolk t t gm gm U-22,593 0.5 + 47 -274 -li -21 -4 + 2 1 + 105 -184 -24 -24 -7 0 2 +179 -173 -42 -17 -6 + 1 5 ♦ 116 -46 -37 -32 -11 -5 Azacholesterol 0.5 -6 -205 -43 -26 ♦ 1 + 1 1 -56 -169 -21 -20 -2 -2 2 ♦ 58 -121 -25 -34 -5 -2 5 + 141 -114 -37 -39 -6 -4 U-11,100A 10 -64 -176 -40 -IS 0 + 3 20 -39 -199 -18 -9 0 + 2 25 -89 - 203 -26 -16 0 0 SO -3 -105 -32 -39 -2 + 2 U-11,607A 10 + 33 -70 -47 -41 -9 -1 25 + 125 - 290 -52 -51 -6 -1 50 + 34 -130 -56 -58 - 8 -5 100 + 186 -49 -48 -63 -13 -3 *Cholesterol was determined by Levine and Zak method (1964) at 550 mu wavelength. inVi 56 The effect of azacholesterol on blood and egg choles terol, egg production, and egg weight was very similar to that of U-22,593A. The drug U-11,607A also had an effect similar to that of U-22,593A and azacholesterol, with a greater range of variation. The compound U-11,100A de creased blood and egg cholesterol and egg production at 10, 20, 2 5 , and 50 mg/kg of feed without any significant trend due to dose. The effect of feeding U-11,100A in this experiment was similar to that observed in experiment 1 although the effects observed in experiment 1 were not statistically significant. In all three of the previously reported experiments the effect of the drugs was determined after three weeks of treatment which was considered to be enough time for the drugs to reflect their effect on egg composition. It usually takes 8 to 10 days of rapid growth for ova to reach the stage of maturation and the effects of metabolic changes are accumulating over this period of time. There fore, after three weeks of treatment all the eggs produced would reflect the effect of drug tested. In all the above experiments only the net effect of drug could be assessed. In order to determine the long-term effect of the drugs on the pattern of egg sterol change during the experimental period an experiment was conducted in which twelve egg production type laying hens were fed 0.1, 0.25, 0.5, and 57 1.0 mg of U-22,593A per kg of feed for nine weeks. Eggs were collected weekly, weighed, and analyzed for total cholesterol using Pearson's method at the 550 mu wave length, No other strains were imposed on the birds during the experiment since no blood samples were collected. The weekly averages of egg weight and egg production during the experiment are presented in Tables 9 and 10, respect ively. Decrease in egg weight was dose related (Table g). At the two highest levels of drug treatment an effect on egg weight was apparent as early as the third week of the experiment. At 1 mg of U-22,593A per kg of feed, the average egg weight decreased by 12,9 per cent when com pared to average egg weight of the controls. Egg weight was also negatively correlated with concentration of egg sterols, and regression of sterol concentration on egg weight was significant at 1 per cent level of probability (F-value 14.1) indicating that as egg weight decreased, egg sterol concentration increased and vice versa. Table 10 shows that at the 1 mg/kg level, U-22,593A decreased egg production by 37 per cent. From the data presented it is clear that the highest level of drug affected egg pro duction during the first week of the study, while the ef fect of the 0.5 mg level was not apparent until the sixth week of the study. At lower levels of the drug there was TABLE 9 EFFECT OF U-22.S93A ON EGG WEIGHT (GM) Experiment 4 Levels Weeks mg/kg % Feed 1 2 3 4 5 6 7 8 9 Ave. Decrease Control 52.8 54.0 54.0 55.1 54.3 57.4 56.1 56.3 S5.4 55.0 0 0.1 53.3 55.0 54.6 55.4 S4.9 55.3 54.3 55.1 53.8 54.6 0.7 0.25 53,4 54.4 54,0 53.7 54.7 52.5 52.9 52.9 51.9 53.3 3.1 0.5 52,7 52.7 50,7 49.7 50.0 50.4 48.9 48.7 51.1 50.5 8.2 1.0 54,9 53.5 48.4 46.8 44,4 44.7 47.1 46.6 46.3 47.9 12.9 Coi/i TABLE 10 EFFECT OF U-22,593A ON EGG PRODUCTION* Experiment 4 Levels Weeks % Change in Feed from (mg/kg) 1 2 3 4 5 6 7 8 9 Ave. Control Control 70 68 69 77 73 75 69 73 60 70 0 0.1 78 77 73 84 70 67 73 68 73 74 +6 0.25 79 75 70 78 73 67 52 73 59 69 -1 0.5 74 68 70 78 66 49 49 52 47 61 -13 1.0 60 57 52 44 37 33 43 37 37 44 -37 aEgg production is presented on a hen-day basis as per cent. 60 no clear effect on egg production during the entire nine week experiment. Egg sterol concentrations in this experiment are presented as absorbance at 550 mu because at this wave length, absorbance appears to measure total sterols rather than cholesterol alone (Figure 3). Hence the data are not interpreted as concentration figures for sterols but left as absorbance. Table 11 shows the effect of different levels of U-22,593A and duration of treatment on absorbance of egg sterols measured at 550 mp. It is evident from the table that absorbance increased as the level of drug increased or with duration of treatment. However, the effect of interaction of level and duration of treatment on absorb ance of egg sterol was statistically non-significant. Since the interaction of level and duration of treatment was not significant, it was desirable to examine separate ly the effect of levels of treatment and duration of treatment on the absorbance of egg sterol. Statistical analysis indicated that there was a linear increase in absorbance of the egg extract with in crease in drug level. The average absorbance as affected by dose is presented in Table 12 with F-value obtained from analysis of variance. There was also a linear in crease in absorbance with increased time on the drug TABLE 11 EFFECT OF U-22,593A ON CHANGES IN ABSORBANCE* RELATED TO EGG STEROL CONTENT Experiment 4 Levels Weeks in Feed (mg/kg) 1 2 3 4 5 6 7 8 9 Ave, Control 0.104 0,097 0.107 0.099 0.115 0.112 0,108 0.109 0.105 0,106 0.1 0.107 0.096 0.103 0,105 0.121 0,114 0,103 0.115 0,115 0.109 0.25 0.108 0.104 0.109 0.114 0,122 0.121 0,116 0.128 0.124 0,116 0.5 0.107 0.103 0.113 0.113 0.125 0.108 0.129 0.135 0.127 0.118 1.0 0.112 0,116 0,117 0,128 0.135 0.119 0,130 0.133 0.136 0,125 aColor was developed using Pearson's method of cholesterol determination at 550 mu wavelength. 62 TABLE 12 LEAST SQUARE MEANS OF CHANGES IN ABSORBANCE RELATED TO DRUG LEVEL OF U-22,593A Experiment 4 Levels Absorbance x 1000 Control 106.0 0.1 108.9 0.25 116.0 0.50 117.7 1.00 125.1 Tabular F-Value for 1132 d.f, at Calculated F-Value S% P 1% P Linear 75.26 4.15 7.5 Quadratic 5.20 63 treatment but in this case there were also significant higher order effects indicating that the changes did not follow a smooth polynomial function. Table 13 shows the least square means of absorbance related to time. Dura tion of the drug treatment has highly significant linear effect on absorbance. Maximum effect of the azacholes terol was reached after 8 or 9 weeks of treatment. A similar long-term experiment was also conducted with U-11,100A and azacholesterol to measure changes in the egg sterol pattern related to dose. Results obtained are presented in Table 14. In this experiment blood and egg cholesterol were determined using the modified Pear son^ method at both the 550 and 625 wavelength. Azacholesterol at 2.5 and 5 mg/kg increased the blood cholesterol and decreased the egg cholesterol when absorbance was measured at 625 my but when the absorbance was measured at 550 mv, egg cholesterol appeared to in crease with these treatments. Egg production decreased by 14 and 42 per cent from control period with 2.5 and 5 mg/kg, respectively. Egg yolk weight has decreased by 4.6 and 8.1 gm with azacholesterol at 2.5 and 5 mg/kg of feed. At 550 ini', U-11,100A produced a very slight increase in egg cholesterol concentration when compared to the control group but gave a lower figure when total sterol per egg was con sidered. Decrease in egg sterol due to U-11,100A was 64 TABLE 13 LEAST SQUARE MEANS OF CHANGES IN ABSORBANCE RELATED TO TIME FOLLOWING FEEDING OF U-22.593A Experiment 4 Weeks Absorbance x 1000 1 107.3 2 103.3 3 109.7 4 111.6 5 123. 7 6 114.7 7 117.1 8 123.8 9 121.6 Tabular F-Value for 1,32 d.f. at Calculated F-Value St P It P Linear 90.39 4.15 7.5 TABLE 14 EFFECT OF AZACHOLESTEROL AND U-11,100A ON CHANGES IN BLOOD AND EGG "CHOLESTEROL"ta EGG PRODUCTION, AND EGG YOLK WEIGHT Experiment 5 Changes in Egg "Cholesterol" Compared to Change in Blood Control Group after Seven Weeks Treatment Egg Egg Yolk "Cholesterol" — ■ ■ ■ — ...... Production Weight Level from Control 550 mp wavelength 625 mp wavelength from , from mg/kg 625 mp wavelength (mg/100 Pot Egg (mg/100 Per Egg Control Control Drugs of Feed (mg/100 ml plasma) gm) (mg) gm) (mg) (I) (gm) Azacholesterol 2.5 +10 ♦ 360 -1 -518 -157 -14 -4.6 5.0 ♦ 123 ♦444 -34 -524 -164 -42 -8.1 U-11.100A 2,5 -13 +64 -15 -163 -46 -3 -0.4 5.0 -40 + 42 -26 -310 -89 + 3 -1.4 ‘Cholesterol was determined by Pearson's method both at 625 and 550 mp wavelength. ^Egg production of control group was also decreased by 4.5 per cent. 66 considerable when considered at the 625 n*j wavelength and lower blood cholesterol was also noted. There was no sig nificant effect of U-ll(100A on egg production and egg weight at the lower levels used in experiment 5- The differences in egg cholesterol concentration when measured at the 550 and 625 nm wavelength led us to believe that compounds other than cholesterol were accumu lating in eggs as a result of azacholesterol treatment and that these compounds were giving different absorption spectra when reacted with the p-toluenesulfonic acid rea gent. Since previous work showed that sterols other than cholesterol may accumulate in rats given diazacholesterol, thin layer chromatography was used to separate and identi fy the sterols accumulating in blood and egg yolk. The Rf. values of the sterols chromatographed are presented in Table 15. Only one spot was observed in the case of aza cholesterol treated plasma and egg extract which had an Rf. value comparable to desmosterol. It appears that aza- cholesterol-treated plasma and egg extract contains mainly desmosterol and that the extract from the control group contains mainly cholesterol. Evidence of accumulation of desmosterol in azacho lesterol -treated blood plasma samples and egg yolks led us to quantitate the different sterols present in blood and egg samples from control, azacholesterol, U-11,100A. 67 TABLE 15 THE Rf VALUES OF STEROLS IN THE REVERSED PHASE THIN LAYER CHROMATOGRAM: (Paraffin Oil)/ (Acetone-Water 4:1) Sterols Rf Standards S-Cholester-33-ol (Cholesterol) 0.46 Cholesterol Acetate 0.06 5,24-Cholestadien-33-ol (Desmosterol) 0. 53 8124-Lanostadien-33-ol (Lanosterol) 0.32 Experimental Samoles Azacholesterol-treated plasma and egg extract 0. 52 Control - plasma and egg extract 0.46 U-22,593A-treated hens using gas-liquid chromatography. Results are shown in Table 1 6 . It is evident from the table that blood and egg extracts from control birds con tain exclusively cholesterol whereas samples from birds fed at 5 mg of azacholesterol per kg feed contain 79.1 and 7^,6 per cent desmosterol in egg and blood extracts, respectively. The remaining sterol was mostly cholesterol. Very similar results were obtained from egg extract from U-22,593A-treated birds. U-ll,100A seemed to be differ ent. Blood extract at the 5 mg level contained mainly cholesterol and about 3.6 per cent of total sterol had the same retention time as desmosterol and 7-dehydrocholesterol. Thin layer chromatography failed to demonstrate the presence of desmosterol in the blood or egg extract. About 6.2 per cent of other sterols which were present were not Identified. The sixth experiment was conducted to determine whether or not the decrease in egg sterol observed when azacholesterol is fed is due to a reduction of total ster ol or to a change in the ratio of cholesterol and des mosterol. Observations on liver and blood sterols were also made. An experiment was conducted in which twelve birds were placed at 0, 0.5» 1.0, 2.5f and 5.0 mg/kg levels of azacholesterol for three weeks. Cholesterol and desmosterol were determined by calculation from the ab sorbance obtained at 550 and 625 mu wavelength using TABLE 16 MEASUREMENT OF DIFFERENT STEROLS IN BLOOD AND EGG EXTRACTS BY GAS-LIQUID CHROMATOGRAPH Cholesterol Desmosterol Others Blood Extract - Control > 99% 0.0 trace Blood Extract • Azacholesterol 5.0 mg/kg feed 25.4 74.6 trace Blood Extract - U-11P100A 5 mg/kg feed 90,2 3.6 6.2 Egg Extract - Control > 99% 0.0 trace Egg Extract - Azacholesterol 2,5 mg/kg feed 38.5 61.5 trace 5.0 mg/kg feed 20,9 79,1 trace Egg Extract - U-11(100A 5 mg/kg feed > 991 0.0 trace Egg Extract - U-22f593A 5.0 mg/kg feed 26,5 73.5 trace 70 Pearson's method. Results obtained were statistically analyzed for the regression of cholesterol and desmosterol on dose. Table 17 shows the least square means of sterol concentration expressed in terms of optical density. Rec ords were also kept for egg production, egg weight, and yolk weight, and are presented in Tables 18 and 19, From Table 17 it is clear that as the cholesterol level in egg extract went down due to the treatment level of drug, desmosterol level increased and the ratio between cholesterol and desmosterol decreased from 15.9 to 1,2. This indicates that presence of desmosterol in egg extract is mostly at the expense of cholesterol. This is also true in blood extract of treated birds. Egg production, egg weight, and egg yolk weight decreased due to drug treatment and decrease was dose related as shown in Tables 18 and 19. Reduced egg yolk weight was relatively greater than reduction in total egg weight at all levels of drug feeding. There was very little effect of azacho lesterol at 0.5 and 1,0 mg/kg of feed on egg weight, egg yolk weight and egg production, but reduction became very severe at 2.5 mg/kg of feed and higher. TABLE 17 EFFECT OF DIFFERENT LEVELS OF AZACHOLESTEROL ON THE CONCENTRATION3 OF CHOLESTEROL, DESMOSTEROL, CHOLESTEROL PLUS DESMOSTEROL, AND RATIO OF CHOLESTEROL/DESMOSTEROL IN ECCS AND BLOOD OF LAYING HENS Experiment 6 Optical Demity in Err Optical Density in Blood Level* of Cholesterol Desmosterol Cholesterol Cholesterol/ Cholesterol ^Desmosterol Cholesterol Cholesterol/ Axacholesterol plus Desmosterol plus Desmosterol (■g/k| feed) Desmosterol Ratio Desmosterol Ratio Control 0.606 0,041 0.729 15.9 0.361 0.0 0.361 -- 0.5 0.64S 0.119 0.764 5.4 0,477 0,315 0.792 1,51 1.0 0.560 0.105 0.745 3,0 0,401 0.236 0.637 1.69 3.5 ■ 0.495 0.117 0.012 1,5 0.686 0.267 0. 63 2.56 5.0 0.471 0,319 0.060 1.2 0. 384 0.637 1.021 0,60 Analysis of Variance Calculated F*value for Tabular F1•value at Calculated F-value for Tabular F- ’Value at 1.140 d.f. St P It P 1.S3 d.f. 51 P It P Linear 64.4 127.2 3.04 6.63 0.037 8.09 4.0 7.08 Quadratic 15.3 13,1 5.9 0,041 VVHhVMA(«BAUM9 Lit hILIM VI p-toluenesulfonic acid reagent, ^Adjusted for blank value. 72 TABLE 18 EFFECT OF DIFFERENT LEVELS OF AZACHOLESTEROL ON CHANGE IN EGG WEIGHT AND EGG YOLK WEIGHT Experiment 6 Egg Weight in Grams Average Levels % Chan; in Feed 1st 2nd 3rd 4 th 5th from tl (mg/kg) Week Week Week Week Week Average ContTO Control 48.1 50. 4 50. 3 50. 4 50. 7 50.0 0.5 48.0 50. 7 50,4 48. 5 51.0 49. 7 -0.6 1.0 50.4 51.0 48.3 47. 3 48.6 49.1 -1.8 2.5 49.8 50.6 46.3 44.1 45. 5 47. 3 -5.4 S.O 50.9 48.9 42.7 37. 4 38. 7 43.7 -12,6 Err Yolk Weight in Grams Control 13.2 14.3 13.4 14. 4 16.0 14. 3 0.5 12.6 13. 7 13.1 12.9 15.4 13.5 -5.5 1.0 13,1 13.9 12.6 12. 5 14.4 13.3 -6.9 2.5 13.0 13.3 11.1 11. 3 13.9 12.5 -12.5 5.0 13.9 13.0 9.9 9,4 11.6 11.6 -18.9 73 TABLE 19 EFFECT OF DIFFERENT LEVELS OF AZACHOLESTEROL ON EGG PRODUCTION3 Experiment 6 ^During Pre- cDuring Levels in Feed Experimental Experimental (rog/kg) Period (%) Period (3) % Change Control 84 82 -2 0. 5 87 78 -9 1.0 83 86 + 3 2, 5 80 67 -13 5.0 82 44 -38 aEgg production is calculated on a hen day basis and is presented as I. ^Based on 3 weeks of egg production data before the drug feeding, cBased on 9 weeks of egg production data when drug was fed. DISCUSSION Nicotinic Acid There are conflicting reports regarding the hypocho- lesterolemic property of nicotinic acid depending upon species of experimental animals, derivatives and levels of nicotinic acid used in the studies. In the present study, nicotinic acid fed in a practical ration at 1, 10 and 20 gm per kg of feed appeared to decrease plasma cholesterol, egg cholesterol, and egg production, and may have in creased egg weight slightly. None of these effects were statistically significant at the 5 per cent level of prob ability. The levels of added nicotinic acid used are 200 to 4000 times the requirement for the vitamin and thus are greatly beyond the nutritional range. The only report available on the effect of feeding a high level of nico tinic acid (0*1 per cent) to laying hens shows that there is no effect on blood or egg cholesterol.Gaylor et £1.80 reported depressed cholesterol levels in young chicks fed a 0,5 per cent cholesterol containing diet when treated with nicotinic acid. There are reports showing increased blood cholesterol levels in r a b b i t s , ^3 and de creased levels in rats,^* dogs^ and men,^ The small 74 75 increase in egg weight observed in the present experiment may be attributed to the maturation of pullets. No toxic effect of feeding high levels of nicotinic acid to the hens was observed. The previous reports suggested that the nicotinic acid inhibited reduction of 6-hydroxy-8- methylglutaryl CoA to mevalonic acid. Within the range of nicotinic acid levels used in the present study and levels employed by Weiss et al. 5* there was no significant effect observed on blood or egg cholesterol. The failure to ob tain any effect may be due to the levels of nicotinic acid used in these studies or may be due to a failure of nico tinic acid to significantly inhibit mevalonic acid synthe sis in the hen. Vanadium No report is available on vanadium treatment of lay ing hens but Curran and Costello106 and Mountain ejt al.107 showed decreased blood cholesterol levels in rats as a re sult of vanadylsulfate treatment. The present study of vanadium pentoxide showed no definite trend for blood cho lesterol but there was a non-significant decline in egg cholesterol. There was little effect on egg production. Egg weight increase may have been due to the maturation of experimental pullets. None of these effects was statis tically significant. The apparent reduction in egg cho lesterol may have been related to increased egg size. It 76 is possible that the levels of vanadium used in the pres ent study were too low to affect cholesterol metabolism or that the form of vanadium used was less active than forms used by others. Saponin Newman et_ al..*** Griminger and Fisher**3 have sug gested that saponin may complex with cholesterol in the intestinal lumen and as a consequence less cholesterol may be absorbed resulting in increased excretion. Newman e£ al.**^ have reported reduction in blood cholesterol of cholesterol fed young chicks due to saponin, which sup ports the observations from the present study. In our ex periment, feeding of saponin significantly reduced egg cholesterol but did not significantly reduce blood choles terol. Reduction in egg cholesterol was maximum at the 6 gm/kg level of treatment. There was a significant nega tive linear dose effect on egg production. At the lowest level of feeding saponin egg production was increased by 16 per cent. Egg weight was reduced at 6.0 gm/kg, but in creased at 12 gm/kg of diet. This compound needs further study in the range of 3.0 to 6.0 gm/kg of feed because it decreased egg cholesterol with no adverse effect on egg production. It is clear that the reduction in egg choles terol cannot be due to changes in egg production or egg weight since the intermediate level of saponin resulted in 77 reduced egg cholesterol with a significant increase in egg production. Ion Exchange Resins Tennent et have studied the effect of choles tyramine resin on blood cholesterol level and the forma tion of aortic plaques in normocholesterolemic and hyper- cholesterolemic cockerels. Other workers have studied its effect on other species including human patients. There is no information available on laying hens. In our exper iment, levels of 5 and 10 gm of Cuemid (containing 83.25$ cholestyramine resin and 16,75% inert material) per kg of feed increased blood cholesterol significantly in laying hens, but there was a reduction in blood cholesterol at 20 gm per kg of feed. It may be that this compound is ef fective in sequestering the bile acids at levels of 20 gm/ *8 or more while it is ineffective at lower levels in the laying hen. Our finding does not support the observation of Tennent*^ who reported a reduction of blood cholester ol in cockerels by 50 per cent at the 10 gm per kg feed. This difference in observations may be partially due to the use of Cuemid which contains only 83,25% resin. Therefore our 10 gm level corresponds to 8,32 gm choles tyramine resin/kg feed. The difference in results may al so be due to variations in rate of cholesterol synthesis which are high in the laying hen, low in the cockerel fed 78 a normal diet and virtually nonexistent in the cockerel fed cholesterol. Hence greater rates of synthesis are associated with a larger entrohepatic turnover of sterols and bile acids which might be attacked by a sequesterant. Estrogen Estrogen had no significant effect on blood plasma or e2g cholesterol. At lower levels, egg production was significantly increased by estrogen treatment. An im provement in egg weight was also observed during the ex periment. No report is available on the effect of estro- • gen on sterol metabolism in laying hens. However, there are reports9** »97 »98 that estrogenic treatment in day old chicks to 7 and 10 weeks of age increased blood cholester ol along with other lipid fractions. Estrogen supplementa tion seems to have the lipogenic property in young chicks when the level of endogenous estrogen is very low, but failed to exhibit an additional lipogenic property in the case of laying hens , where the endogenous estrogen level was already very high. Estrogen might be a limiting fac tor during egg production because dietary supplementation improved the egg production and egg weight. The increase in blood cholesterol in the young chicks may also be due to simply handling of the birds every day for injection of estrogen or mode of administration may be responsible for the discrepancy. 79 D-Thyroxine Deficiency of thyroxine increased plasma cholesterol and treatment \*ith thyroxine normalized the level in most species. The effectiveness of D-thyroxine treatment in reducing plasma cholesterol varies with species; for ex ample , dogs and men are more sensitive than are rats, mice and rabbits to this drug. Laying hens probably fall in the latter category. In the present study feeding of D- thyroxine at 10, 20, and 30 mg/kg of feed significantly decreased plasma cholesterol and significantly increased egg cholesterol at the 1 per cent level of probability. Egg production was depressed 29 per cent in the three weeks experimental period but birds seemed to recover quickly when the drug was withdrawn. Practically no ef fect was observed on egg weight. In previous reports from our laboratory, Weiss £££l. ,51*52 reported that the in creased plasma cholesterol and decreased egg cholesterol were observed when they subcutaneously injected laying hens with 40 and 80 ug/100 g body weight/day. This obser vation directly supports our findings. Reports®*have presented direct or indirect evi dence for elevated synthesis and excretion of bile acids during thyroid treatment and decreased synthesis or excre tion in the case of thyroidectomy or thiouracil treatment as well as in the case of myxedema in human patients. 80 These reports prompted the belief that both catabolism and anabolism of sterols are increased during thyroid treat ment but that the rate of catabolism is higher than ana bolism, resulting in an overall decrease in blood plasma cholesterol level. Elevated cholesterol level in the egg due to D-thyroxine treatment may represent increased ex cretion which further supports the above hypothesis. Benzmalacene There are reports that benzmalacene decreases blood cholesterol in rats and human patients. However, in our experiment this drug appeared to increase blood and egg cholesterol when it was fed at 0.5 and 1.0 gm per kg of feed. No consistent effect on egg production and egg weight was observed. In the experiments of others higher levels of benzmalacene have been used. Limitations on the amount of drug available prohibited feeding at higher levels, and as a result the levels used may have been in adequate to influence cholesterol metabolism. Inos itol Inositol was fed in the present experiment at levels of 2, 5 and 10 gm/kg of feed, which is comparable to the levels of inositol fed by Stamler e^t al. assuming that the birds consumed 100 gm feed/day/bird. Inositol failed to significantly increase blood cholesterol in cholesterol 81 fed cockerels, Herrmann124 reported decreased blood cho lesterol level in old laying hens. In this study inositol failed to decrease egg and blood cholesterol. Variable effects on egg production and egg weight were observed. This might possibly be due to the presence of a high level of endogenous estrogen in laying hens, a strong lipogenic agent which counteracted the effect of inositol, a lipo tropic agent; resulting in no significant change in blood and egg cholesterol and variable effect on egg production and egg weight. 20.25-Diazacholesterol CAzacholesterol)» ij-^z. 593A ana" V- n,bi)/A The above compounds have been studied in greater de tail in these experiments, and are discussed here together because of the similarity of their effects on the hen. Evidence has been presented in the results section for the accumulation of desmosterol in egg and blood plas ma of laying hens when fed diazacholesterol. This evi dence is based upon spectrophotometric absorption of the sterols reacted with p-toluenesulfonic acid at the 550 and 62S mp, thin layer chromatographic study and gas-liquid chromatographic analysis. This finding is consistent with the observations of Renney and Counsell^S based upon a gas-liquid chromatographic study that 4-azasteroid treated rats and mice accumulated desmosterol in blood. The same 82 researchers have also reported that 20,25-diazacholesterol effects cholesterol synthesis by inhibiting 6-hydroxy-8“ methylglutaryl CoA reductase, thus blocking conversion of g-hydroxy-6-methylglutaryl CoA to mevalonic acid. How ever, Thompson £t al.^ reported that 20,25-diazacholes- terol caused accumulation of desmosterol in the serum, liver and carcass of rats, and suggested the probable in hibition of desmosterol reductase, thus blocking the con version of desmosterol to cholesterol. On the basis of desmosterol accumulation in egg due to the azacholesterol treatment, it is possible to explain the increased egg cholesterol found when measurements were made at the 550 my wavelength and the decrease found when measurements were made at 625 my when the same egg extract obtained from azacholesterol treated birds was examined. This effect is shown in Figure 6. From Figure 7 it is ob vious that equimolar amounts of desmosterol absorbed only 28 per cent of the light absorbed by cholesterol at a wavelength of 625 my. At 550 my, desmosterol absorbed al most the same amount of light as it did on 625 my but cho lesterol absorption is reduced by 46 per cent. Therefore, any accumulation of desmosterol at expense of cholesterol will reflect itself in a large decrease in total absorp tion at the 625 my wavelength. At the same time, accumu lation of desmosterol would decrease the absorption at c h a im o b mgoio s t e r o l FETO DFEETLVL O ZCOETRL N LO N G CHOLESTEROL EGG AND BLOOD ON AZACHOLESTEROL OF LEVELS DIFFERENT EFFECTOF ' 0 0 + 5 (Pearson's method at 550 and 625 mu wavelength) mu625 550 method and at (Pearson's FIGB OFLAYING G COETRL a 50 mu) 550 (at CHOLESTEROL EGG 44 LO COETRL a 65 mu) 625 (at CHOLESTEROL BLOOD EGG CHOLESTEROL mu) 625 (at HENS mg/kg 83 8* FIQ 7 ABSORPTION SPECTRUM OF DIFFERENT STEROLS WITH THE P TOLUENESULfONlC ACID REAGENT USED IN PEARSONS METHOD FOR CHOLESTEROL DETERMINATION LAN STEROL CHOLESTEROL Egg Ext. -AZACHOLESTEROL"-...... CONTROL EGG EXT: desmosterol ------ 300 * *0 * 00 7 00 ■ 00 W AVELENGTH — mu 85 550 mu slightly because at this wavelength cholesterol absorption is reduced while desmosterol absorption is similar. In order to answer the question of whether or not the reduction in egg sterol measured at 625 mp is due to a change in total sterol concentration or is simply due to a change in the ratio of cholesterol to desmosterol, the data obtained in experiment 6, in which concentrations of cholesterol and desmosterol were measured, was examined by the method of multicomponent spectral analysis. The rela tionships are presented in figure 8. It is evident from the figure that desmosterol accumulates in egg at the ex pense of cholesterol resulting in a change of the choles terol to desmosterol ratio from 15:9 to 1:2. This is a major factor in reduction of sterol level at 625 my in treated birds, because desmosterol absorbs only 28 per cent of the light absorbed by cholesterol at the 625 my. The accumulation of desmosterol at the expense of choles terol amounted to 79,1 per cent of the total sterol as re ported in Table 16 after a three weeks period of treat ment. There is also indication of a 17,5 per cent (Table 17) increase in total sterol of egg extract from azacho lesterol treated birds at the 5 mp/kg level when compared to the untreated controls. This increase in total sterol may be accounted for by a decrease in egg or yolk size 86 FIO 8 EFFECT OF DIFFERENT LEVELS OF AZACHOLESTEROL ON CHANGE IN EGG CONCENTRATION OF CHOLESTEROL AND DESMOSTEROL (Spectrum analysis using Pearsons method) 9 * 90 IS s o 75 \ 70 * 5 60 IS so x CHOLESTEROL 4 5- 4 » ,« DESMOSTEROL 3 S 3 0 3 S 2 0 1 S- 1 0 5 ■ 1 ■ I ■ | l I I 1 ■ 1 1—— * S 10 IS 20 2$ SO 35 40 45 50 55 6 0 ™ J '« | LEVEL Of TREATMENT 87 which was about 19 per cent. Egg weight is negatively correlated with egg sterol concentration. Therefore, the apparent reduction in egg sterol when measured at 625 my in the azacholesterol treated group is the net result of the accumulation of desmosterol at the cost of cholesterol and increased total sterol concentration due to decreased egg weight. However, accumulation of desmosterol in place of cholesterol is the major contributing factor. Since the increase of total sterol in egg is com pletely accounted for by a decrease in egg size, it is possible to assume that the azacholesterol treatment did not interfere with the rate of cholesterol synthesis, ex cept in the last step, and that desmosterol is transferred to the egg as efficiently as cholesterol. The inhibition of cholesterol synthesis is probably due therefore to the inhibition of desmosterol reductase thus blocking the con version of desmosterol to cholesterol. Desmosterol is probably transported from blood across the ovarian membrane to the egg in a manner like that for cholesterol since the ratios of the two sterols in blood and egg yolk are similar (Table 16), There is however an increase in total blood sterol associated with azacholesterol treatment not reflected in egg yolk sterol accumulation when corrected for egg size. This may mean that sterol oxidation to bile acids is reduced or that 88 bile acids re-utilization is increased, Azacholesterol severely depressed egg production and egg weight. This effect may have been due to a toxic ef fect of the drug or to its effect in inhibition of choles terol biosynthesis at the last step. Limited cholesterol production might inhibit the production of sex hormones and as a result limit egg production and egg weight. This hypothesis might be easily tested by administering the sex hormones along with the azacholesterol. There is no evi dence at present to support the above hypothesis and it needs careful study. The effect of azacholesterol seemed to be cumulative with time of drug feeding. In order to examine the cumu lative effect of this drug on egg cholesterol, egg weight and egg production, data from experiment is displayed graphically in Figure 9. It is obvious that the feeding of azacholesterol for seven weeks continuously increased the sterol level when measured at 550 mp while egg weights decreased for the first five weeks and then tended to level off. Therefore measurement of effects of azacholes terol in short term feeding experiments might produce mis leading results. From Figure 10 it can be seen that egg sterol level increased when measured at 550 mu as the dose of azacholesterol increased, and it seems that a maximum effect was obtained at a level of 2.5 mg/kg of diet. EGG CHOLESTEROL mg 1100 gm EGG YOLK I | 1 »»* I I** FET F ON O TETET F ZCOLSEO O EG CHOLESTEROL EGG ON LESTEROL AZACHO OF TREATMENT OF N IO T A R U D OF EFFECT 1 Pear s met 50 wavel h ) th g n le e v a w u m 550 t a d o th e m 's n o rs a e (P G WEGHT H EIG W EGG 3 UAIN F RAMN (Weeks! TREATMENT OF DURATION ND EG I T H EIG W EGG D AN 4 S * G COETRL * * CHOLESTEROL EGG / T 9 1 *4 B 4 3 4 ; >4 -ss SO EGG WEIGHT IN Gm EGG CHOLESTEROL mg / 100 gm EGG YOLK I 140- 1**0 EFFECT OF DIFFERENT LEVELS OF AZACHOLESTEROL ON EGG STEROL. STEROL. EGG ON AZACHOLESTEROL OF LEVELS DIFFERENT OF EFFECT L i nd s mehd t 5 mu wavel h th g n le e v a w u m 550 at ethod m 's k a Z d an e vin La < EE O TREATMENT OF LEVEL O l Q I P ) 90 91 There are no public reports on the Upjohn Company's compound, U-22,593A. However this drug behaves exactly like azacholesterol on every parameter examined in this study. It appears that this drug is an azacholesterol- like compound. The compound U-22.59 3A seems to have cumulative ef fect on egg sterol as is obvious from Figure 11, where the change in absorbance as affected by this drug is plotted against the duration of treatment. There was a highly significant positive linear effect of duration of treat ment on change in absorbance, indicating that hens should be treated for nine weeks or more with this drug in order to obtain complete expression of the effect on eggs. Sim ilar data is presented in Figure 12, where the change in absorbance is plotted against the levels of treatment and shows that absorption increased as the dose of the drug was increased to 1 mg/kg of feed; and this linear effect of dose on change in absorbance was observed up to 10 mg/ kg of feed in other experiments. The compound U-11,607A, in general, increased blood cholesterol, and decreased egg cholesterol and egg produc tion. Its action also seems to be similar to U-22,593A and azacholesterol, but a limited supply of this drug made it impossible to study its effect in detail. a b s o r b a n c e X 1000 t *► I 14^ I 1 •> CHANGE IN A B S O R B A N C E OF EGG EXTRACT W IT H T IM E AS AFFECTED AFFECTED AS E IM T H IT W EXTRACT EGG OF E C N A B R O S B A IN CHANGE Pear mehd t 5 mu engt ! th g n le e v a w u m 550 at ethod m s n o rs a e (P B Y U ' 22 .593 * A TREATMENT TREATMENT A * .593 22 ' U Y B I. 11 FIG. 92 ABSORBANCE X 1 0 00 tot 104 . tio in. It* lie m FET F IFRN LVL O U2.9- O ABSORBANCE ON U-22.593-A OF LEVELS DIFFERENT OF EFFECT Pasns ehd t 5 m wavelength) mu 550 at method (Pearson's -40 F G EXTRACT EGGOF 7 00 *70 I. 12 FIG. 91 U-11.100A U-11,100A was markedly different from other U-series compounds studied. At lower levels, it decreased blood and egg yolk cholesterol without significant accumulation of any other sterols. There was no significant effect on egg production when the compound was fed at levels of 2.5 and 5.0 mg/kg. The effects of U-11,100A on egg sterol measured at 550 mP showed no consistent pattern of change with time. It seems that reduction in egg cholesterol is dose related at lower levels of treatment (Fig. 13) when measured at 625 mP wavelength. The compound U-llt100A seemed to be ideal in an effort to lower egg sterol because it lowered the level of egg cholesterol without the accumulation of any other sterols (Table 16), In addition, it did not reduce egg production and egg weight at lower levels and no toxic effect was observed. Further study of this compound was not possible because of a limited supply. Additional study is needed. EGG CHOLESTEROL mg 1100 gm EGG YOLK 1100 1 13 10S0 40 00 0 - FET F IFRN LVL O INI, IO o EG CHOLESTEROL EGG on IOOA , I N I OF LEVELS DIFFERENT OF EFFECT Pasns ehd t 2 m wavelength) mu 625 at method (Pearson's FIG mg/kg 13 95 SUMMARY AND CONCLUSIONS The present study was directed toward the manipula tion of the quantity and nature of egg sterols with the use of drugs. The first two experiments were conducted to screen twelve selected compounds in laying hens for their effect on blood and egg cholesterol concentration. The remaining experiments \*ere devoted to a detailed study of U-22,593A, U-11,100A and 20,25-diazacholesterol. Based upon the results obtained, the following conclusions were drawn: 1. U-22,593A and 20,25-diazacholesterol appeared to have similar effects on every parameter studied in these experiments. The chemical nature of U-22,593A is not known, but it is presumed to he an azacholesterol-like compound, 2. U-22,S93A and azacholesterol were found to in crease egg cholesterol concentration in treated birds when measured at 550 my and to reduce con centration when measured at 625 my, Bgg produc tion and egg weight were considerably reduced by the treatment with these two drugs. 3. Evidence has been presented for the accumulation 96 of desmosterol at the expense of cholesterol in egg and blood of azacholesterol and U-22P523A treated birds. The difference in the pattern of egg sterol change in treated birds when measured at the 5S0 and 62 5 mv wavelengths was mainly due to the ac cumulation of desmosterol and due in small part to the increase in total sterol concentration. The increase in total sterol concentration of eggs from azacholesterol treated birds when ex pressed as mg4 of egg yolk was accounted for by the decreased egg size observed. Concentration of egg sterols was found to be negatively corre lated to egg weight. It is speculated that U-22P593A and azacholes terol treatment reduced the rate of cholesterol biosynthesis by inhibiting desmosterol reductase which blocks the conversion of desmosterol to cholesterol as suggested by Thompson e£ al^in rats. Desmosterol appears to be transported across the ovarian membrane as efficiently as cholesterol because the concentration ratios of the two sterols in blood and egg yolk are simi lar. Elevations in total blood sterol concentration suggest that desmosterol is not converted to 'bile acids or to different steroid hormones as easily as cholesterol. 8 . Limited production of cholesterol in the adre nals and ovaries may limit the production of steroid hormones and as a result egg production was severely affected, 9. The accumulation of desmosterol in egg and blood of U-22,593A and azacholesterol treated birds is a linear function of dose and time. Sterol con centrations appear to be changing after seven or more weeks of treatment, 10, The effect of azacholesterol and U-22I593A as a very potent inhibitor of cholesterol biosynthesis supports our hypothesis that a very low level of cholesterol in the bodies of laying hens would result in lower production of steroid hormone. As a result, reproductive performance would de cline, reflecting itself in reduced egg produc tion and egg weight, 11. The compound U-11,100A is unlike U-22,593A and azacholesterol. It decreased egg and blood cho lesterol concentration without affecting egg pro duction and egg weight significantly at lower levels of treatment. The drug treatment did not result in an accumulation of significant amounts of other sterols in the eggs and blood of treated birds, The reduction in egg cholesterol seemed to be dose related up to 5 mg/kg of feed, Lim ited availability of this drug made it impossible to study it further. The effects of nicotinic acid, vanadium, saponin, ion exchange resin, D-thyroxine, estrogen, benz- malacene and inositol on blood and egg choles terol, egg production and egg weight in laying hens did not produce consistent results or ef fects that required further investigation based on the objectives of this study, A brief discus sion has been presented about these compounds in the discussion section. APPENDIX A SOURCES OF THE COMPOUNDS TESTED IN THIS STUDY Cuenid (Cholestyramine Resin) Merck Sharp £ Dohme West Point, Pennsylvania Vanadium Pentoxide J, T, Baker Chemical Company Phillipsburg, New Jersey Saponin (Technical Powder) Matheson Colenan G Bell Norwood (Cincinnati) , Ohio Inositol (Meso) Nutritional Biochemicals Corporation Cleveland, Ohio Benzmalacene Merck Sharp G Dohme Research Lab Rahtvay, New Jersey Nicotinic Acid (Niacin) Nutritional Biochemicals Corporation Cleveland, Ohio U-22 ,593A Research Laboratories of the U-ll, 100A Upjohn Company U-11,607A Kalamazoo, Michigan Azacholesterol G. D. Searle 5 Company Chicago, Illinois Sodium D-Thyroxine Flint, Eaton G Company Morton Grove, Illinois 100 APPENDIX . I Effect of Selected P m ki on tlooJ and I tf Cheleiterel, Eft Productinn and Egg weight Eiperlaent 1 StOOd M l ■Ee E Production Ejg Weight Chelnterol Choir* terol Cholesterol Chsle'ierol Level Befnre A fte r Orfnre A fte r Sefsre During Afte r Pefore Purine A fte r T reitnen t Treatment Treatment T re if-e n t T r u t - e n t p * r ;» Treatment Treatment T re iire rtt T r e it 'e n t T r e itr e n t Drug f«4d or 3 BE 5 r r 5 f t 1 t 1 I (Of) (,-n) {;p) C entral 116,7 117.7 1171.7 1033.5 *7.3 82.5 *7.3 55.5 57.1 57.4 111.6 120.S 115-', 9 10 *2.6 *7 .) *4.1 8 5.3 SO. 5 57.3 59.0 tflcetlnic Acid 0.1 I" 159,1 103.1 1209.0 1127.5 14.1 80.9 80.9 57.0 56.6 59.3 1973.1 0.1 *" 123,4 68.0 1223.3 14.1 *5,7 70.1 47, 5 50.0 5 * .l 1 I" 151,0 152.1 1223.5 1280.0 S2.5 70.3 68.2 55.5 56.2 58.0 1 0* 15S.I 126.1 i: : s . 2 ID"1). 4 79,3 76,1 71.4 55.5 51.V 59.5 I 1" 125,0 92.6 l.’ OO.I 1060,4 76,1 71.4 7b. 1 53,7 56.1 69, 2 2 *■ 121,0 OS.4 1294.1 10 70.1 82,5 7’’, 3 77, 7 53.7 5t.,6 55,8 Yenidlua 10 n 135. 1 109,4 12:*.2 1467,4 SC.9 74.6 69, 8 57,0 57. 1 59.* 10 WE 154.9 116,3 16’9.4 1.112,2 *3.3 79 . 3 68.2 54.0 56.1 60.0 25 B* 167,0 139,7 145*.I 11S5.6 12,3 14.1 *5. 7 49.6 54.7 S t.7 23 Bg 111. * 145,4 1341.1 1015.6 77.7 79.3 *5.7 50.5 58.1 59.1 50 f t 14.',1 137.1 130). 5 1058,1 85.7 77.7 79.3 52,3 55.1 S l.l SO me 146.7 128.4 1411.7 1029.0 76.1 76.1 7'«.1 57.5 57.1 63,1 9 70,9 Sepeain 3 t " 104.0 101.S 1492.1 Ob.6 90.4 *4.1 54.0 54.0 54.3 3 1:0.2 85. I 1117.6 961.9 73.0 *2.5 80.9 53.0 56,0 59,1 6 t=> 223.1 105.2 1270.5 *27.7 (9.1 76.1 68.2 62 . 0 36,9 61,1 6 m 1 14,0 71.9 1176.4 -I'.: 74.6 74.6 71.6 61.0 58.1 5 \ 2 12 *B 1S1.0 167.7 1176.4 1275.1 *6.0 *0,9 83.3 45,0 55.0 54.5 1)72,2 12 n 127,1 79.2 1341.1 *2,5 71.4 77.7 52.5 55.5 55,7 0*11,100A 10 »t 1)4.1 51.1 1121,4 1396,7 95.1 90.4 *7.3 52.4 57.2 54.5 10 lrv-1.7 1)7.3 1223.5 11*5.6 15.7 82. 5 77,7 51.3 58.2 58.2 1073.1 25 "» 1)5.5 31.6 1091.7 18.5 80.3 12.5 59.0 5*.7 61.5 25 BE 145.2 105.1 m s .2 1221.4 18.0 7 'M 82.5 49.5 31.8 52.0 50 "E 1ST. 3 72.4 1 159.S 10 '6.7 * 9.9 *3.1 10,9 55.5 56,5 56.5 So BP 136.5 92, 7 1251.7 1051,4 84. 1 2 '.3 74,6 56.0 5f.,6 51.4 11*22,593A 10 n( 150.* 9 4 .S m i . 7 9)1.5 8 *, 1 36. 5 34.9 59.0 59.1 tu.O 10 131.0 206, 7 1251.6 891.1 *4.1 33.1 5.1,0 S3. S 51.5 56.5 25 ng 91.4 1)7.3 n : i .0 1)51.2 *5.7 42.1 4 2,* 51.0 41.0 49.5 25 BE 167,(7 246.9 1294,1 l ) 1* . 2 *1.3 47,6 30. 4 51.0 41.7 55.0 50 "I 1)5.5 2 35.6 1299.4 4 ’2.1 *5.7 25.3 19.0 55.5 52.1 44.7 50 ■E 11S.S 197.7 1175.4 618.7 87, 3 36. 5 1.5 51.5 18.0 58.0 0*11,607 10 ng 111. 4 201,3 1105.1 7'. 9.6 79.3 34.9 31.7 54,5 48.3 56.5 10 BE 129.2 130. 3 1162.3 I ' M . 4 93.4 36.5 33.3 51.6 46.3 52.5 25 B* 256,6 421,4 i n s ,2 811.) 80,9 37.9 33.5 49.0 58.0 25 B* 165.6 715.6 1 V- 4. T 715.1 90. 4 33,1 22.2 52,0 49.6 52,0 SO f‘K 14’ . t 196,0 1520.4 1297.5 *2.5 26.9 19,0 59,5 50,5 56.5 50 BE 14*.* 241,6 1121.1 926,1 14,1 50.7 26,9 53.3 46.3 49.0 A ia d t * l* lt « r « ] 1 n( 145.i 1ST.4 1170.4 1341,2 7b.1 6S.0 13.6 54,0 52.1 57.0 1 r ( 135.5 112.7 1256.4 1071,1 | i. 7 70,1 Jg,0 54,5 5J.1 55,1 1 ■! 132,5 206,7 1341,1 1310,9 14.1 76,1 13.1 52,5 51,1 46.0 101 I * | 111,9 211.1 1200,0 (72.4 I I . I 76,1 61.9 50,9 51,6 31,1 5 H 134,1 144,0 1270.3 641.7 90.4 10,9 47,6 50.1 46,6 47,0 1 ■! 115.1 114,1 1129.4 419.7 92.0 71.5 55.S 46.0 45.1 44.1 unmt * c Effect «f hip m IlMf n< Ell ChtlMttrel, E|| an* Ef| waltfit Eipcrintnt I 1 *■a » < * • ► © f.9 w t b ^ b V £ V b N © hi v P * — «e « * * & C I- — bC u M • CE~ v *- * ►^ T PI- L . i m ( r c 4 c v v * n - i [ ^ r»^ ^ ^ *4 Pt h p*f>4 p*f>4 h O ►'lO V ^ M A P K iai k n / i * krt e ' V V f l i/| l f V V ' e krt * i « e » s a * ■ •*, 1 ^ ■a 4 4 p * h r N f g N V 4 4 * S » » . » C * — V K f t S C E r * * * ■ < Pi * t> « A t t n fl . f fcl l i n i i tf) i 0 H « «» • N « Vi *» *if Ntnfi^ v*lr*i B ^ p*i aiNhaii ^ k/> -« viMftaeo P k ^ Pj a f h ^ h-Oi p . ^ Pk«t c ft ^ M ) V * / ) V I i l l m m ^ m i i a ^ ^ 9 p ^ k / k ^ p p P t * r n » r VfECPPPfi^ t f l v i V i V I v > V i a ^ ^ IN| O O c Pe C M P i P p O P I r > »• s n e r e c ! r * ■ i w ■ p - « j* » i * f ■ - f » l ,f* * - f t V i V - V * ^ V *n4 0 4 6 C C a f t F f t f C f t A C M l f k S i I / I M « c > P I c . v v p - i t r t i r t ■ f * i C C p i M Ml *4 4 a C * ) ^ c h> ^ ““■ i“ ^ ^ P 0 1/1 >0 0 7 4 Ml. ' i r« Mi C> pk V ^ O ^ I W 0 4 ^ NP «4 0 4 m pi r^ n P 4 1 ia pw p.« a M M O P ■ rv h |k pj a K/k. r^p sooeed CkP/inM« 0 fp Pt • PP N/ 0 Mltf MIVMP V i /JO v* iCP V i v i V i v < ~ t P * - V i ^ | M n £ A o e,n > M l **> > M l *M P*P M> lA SPJ *4 — ■ ^ O > * > ^ • * M l i / % l A l A M l l / l <10 A fi 0 4 HNttKKU t n « # ■3 4 * ^ M CfH C p c ■* 0 P« pi pi « « a » « 0 a 0 4 4 p« ^ p> a V> M ■ B «P 4 0 «4 6 ~ 4 ^ r K» » «K *- v: vpi«n e * e 4 0 »4 0 .0 *0 • p* *4 0 0 7 0 |H 0 4 1 i * * * 4/1 0 4/1 V 0 —4 P 4 4 3 o o « t t 4 a t r i q o o o d ^ ' •*» O s © O ^ — vi a «3npi ■AlAtrt kArtu M t 4 P t n * ^ > 1 * 4 o f f l P t 4 ) E E ft S C « > - i n * » '> t */l P Ml i iP o ’Pi * V I k / 1 V I ^ I *4 k o» o n n. O O VI O ►-. W% 4 VI li « « ■ n *>N K * p ^ « « e e un ik k/1 i/k n u p X ^ ^ 4 /o 1 « P ^ pfr i Ifl £ 4 f7 n — j p * p i j — p^i *A I p"% V w pO p Pv ^ C 1 . n s ir :fi*t b ^ ^ ^ pp 4/1 ^ V n o n v p re ( *r D e> 4 « p)tn vlvi M C>k ^| I f p p - P < £ » K v n a l 102 j» * * j» *^ i APPENDIX - D Effect of Selected Drugs on Blood and Egg Cholesterol Experiment 3 Blood Cholesterol Egg Cholesterol Level Before After Before After per Kg Treatment Treatment Treatment Treatment Drug Feed mg % mg mg I mg % U-22.593A 0.5 mg 167 213 1433 1169 1.0 171 276 1510 1326 2,0 162 340 1473 1349 5.0 155 287 1480 1531 Azacholesterol 0.5 mg 147 142 1433 1228 1.0 149 94 1461 1293 2.5 153 207 1459 1334 5.0 155 274 1430 1338 U-ll,100A 2,0 mg 174 140 1420 1256 5.0 144 131 1437 1336 10,0 131 67 1515 1339 25.0 172 102 1410 1206 U-11.607A 10.0 mg 149 203 1395 1315 25.0 108 241 1454 1134 50.0 139 189 1509 1395 100.0 174 365 1450 1401 APPENDIX - E Effect of U-ll^lOOA and Azacholesterol on Egg Cholesterol Measured at 550 and 625 mp Wavelength Using Pearson's Method Experiment 5 At 550 mp Wavelength At 625 mu Wavelength Level Weeks of Treatment Weeks of Treatment per Kg Drug Feed 1 2 3 4 5 6 7 6 7 Control 1325 1419 1464 1219 1394 1351 1366 1514 1414 U-11,100A 2.5 mg 1252 1371 1307 1425 1451 1315 1409 1352 1315 5.0 mg 1301 1495 1177 1204 1292 1342 1318 1204 1098 Azacholesterol 2.5 mg 1384 1432 1429 1332 1806 1451 1725 997 1001 5.0 mg 1347 1264 1343 1580 2075 1721 1810 990 1224 APPENDIX - F Effect of U-lljlOOA and Azacholesterol on Egg Weight and Egg Yolk Weight Experiment 5 Egg Weight in Gm Egg Yolk in Gm Level Weeks of Treatment Weeks of Treatment per Kg DrugFeed 1 2 3 4 S 6 7 3 4 5 6 7 Control 59.8 58.9 57.5 59.6 59.0 -- 60.0 17,9 19.1 19.0 19.3 20.0 U-11.100A 2.5 mg 59.2 59,1 60.3 60.8 61.7 -- 60.7 18,7 19.0 19,5 18,8 19,6 5.0 mg 59.4 59.4 57.3 57.9 58.0 -- 58.5 18.2 18.3 18.0 17.7 18.7 Azacholesterol 2.5 mg 57.7 56.8 53.9 51.8 49.8 -- 50.0 16.2 15.2 15.2 14.4 15.4 5.0 mg 58,0 57.5 52,3 48.5 46.0 -- 44.3 15.4 14.3 13.2 13.9 11,9 M a APPENDIX - G Effect of U-11,100A and Azacholesterol on Egg Production Experiment 5 Level Egg Production Egg Production per Kg Before Treatment After Treatment Drug Feed * i Control 75.0 70.4 U-11.100A 2.5 mg 75,8 73.1 5.0 mg 75,5 78,2 Azacholesterol 2.5 mg 72,4 58.5 5.0 mg 77.3 36,0 106 BIBLIOGRAPHY 1. 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