Utah State University DigitalCommons@USU All Graduate Theses and Dissertations Graduate Studies 5-1966 Cholesterol Levels in Serum and 5β – Pregnane – 3α,20α - diol in Urine of University Students Gertrude Kuei-Shu Chiang Utah State University Follow this and additional works at: https://digitalcommons.usu.edu/etd Part of the Food Science Commons Recommended Citation Chiang, Gertrude Kuei-Shu, "Cholesterol Levels in Serum and 5β – Pregnane – 3α,20α - diol in Urine of University Students" (1966). All Graduate Theses and Dissertations. 4820. https://digitalcommons.usu.edu/etd/4820 This Thesis is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. CHOLESTEROL LEVELS IN SERUM AND 5� -PREGNANE-3D( ,20o(.-DIOL IN URINE OF UNIVERSITY STUDENTS by Gertrude Kuei-Shu Chiang A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in Nutrition and Biochemistry UTAH STATE UNIVERSITY� Logan, Utah 1966 TABLE OF CONTENTS INTRODUCTION 1 REVIEW OF LITERATURE 4 Physiological effects of progesterone 4 Source and metabolism 4 Function in the body and its excretion 6 Analytical method of urinary pregnanediol The relationships of serum cholesterol and coronary atherosclerosis 8 The influence of the sex hormones on the circulating lipids . 13 Serum cholesterol levels and eating frequency 14 Analytical method of serum cholesterol 16 METHOD AND PROCEDURE 18 Experimental design 18 Collection and storage of urine specimens 20 Collection 20 Storage . 22 Analysis of ur inary Sft-pregnane -~, 2~-diol 22 Method 22 Equipment 22 Reagents 23 Procedure 23 Hydrolysis 24 Extraction 24 llage Washing and drying 24 Preparing the glass plates 25 Chromatography 26 Spectrophotometry 28 Calculation 29 Recovery 29 Preparation of serum samples 29 Analysis of serum cholesterol 31 Method 32 Equipment 32 Reagents 33 Procedure 34 Extraction and sampling 34 Precipitation of total cholesterol 34 Precipitation of free cholesterol . 35 Washing of the cholesterol digitonide precipitates 35 Color development 36 Calculation 37 Recovery 37 RESULTS AND DISCUSSION 39 Urinary pregnanediol levels 39 Effect of menstrual cycle 43 Effect of two meals versus three meals per day 43 Serum cholesterol levels 55 Effect of menstrual cycle 63 Page Effect of two meals versus three meals per day 63 Effect of dietary factors 66 Relationships between serum choles terol and degra­ dation products of androgens and progesterone 72 COMMENTS AND CONCLUSIONS 76 SUMMARY 78 LITERATURE CITED 80 APPENDIX 84 LIST OF TABLES Table Page 1. Experimental design 19 2. Mean age, weight, and height of subjects 21 3. Recovery test of urinary pregnanediol 30 4. Recovery test of serum cholesterol 38 5. Summary of mean squares obtained by analysis of variance of data on urinary pregnanediol 40 6. Mean values of pregnanediol in urine of subjects consuming two meals and three meals per day 41 7. Urinary pregnanediol values of normal humans as reported in the literature . 42 8. Summary of mean squares obtained by analysis of variance of data on serum cholesterol of male and female subjects 56 9. Summary of mean squares obtained by analysis of variance of data on serum cholesterol of female subjects 57 10. Percentage distribution of nine subjects within various ranges of serum total cholesterol values 58 11. Mean serum cholesterol concentrat ion by sex and meals 12. Mean values of total and free cholesterol in serum of subjects consuming two and three meals per day 61 13. The range of serum total cholesterol values for individual subjects by number of meals . 62 14. The effect of menstrual cycle on the concentration of the serum cholesterol 15. Mean food intake of the subjects 70 16. The relationship between serum cholesterol and urinary neutral 17-ketosteroid, and pregnanediol by sex 75 Table Page 17. Urinary 5$ -pregnane3oL,20ol-diol values for individual subjects consuming two meals and three meals per day 18. Urinary pregnanediol excretion values for the individual subjects consuming two meals and three meals per day by day of menstrual cycle 19. Urinary pregnanediol excretion values for the individual subjects by day of menstrual cycle 87 20. Serum total cholesterol values for individual subjects eating two meals and three meals per day 88 21. Serum free cholesterol values for individual subjects consuming two meals and three meals per day LIST OF FIGURES Figure Page 1. Biosynthesis and metabolism of progesterone 2. The effect of day of menstrual cycle on excretion values of pregnanediol and the levels of serum cholesterol 44 3. The effect of day of menstrual cycle on mean excretion values of pregnanediol of 5 subjects 44A 4. Urinary pregnanediol excretion values and serum cholesterol levels by day of menstrual cycle for subject S.R. 45 5. Urinary pregnanediol excretion values and serum cholesterol levels by day of menstrual cycle for subject T.D. 46 6. Urinary pregnanediol excretion values and serum cholesterol levels by day of menstrual cycle for subject J .A. 47 7. Urinary pregnanediol excretion values and serum cholesterol levels by day of menstrual cycle for subject B.N. 48 8. Urinary pregnanediol excretion values and serum cholesterol levels by day of menstrual cycle for subject M.J. 49 9. The effect of number of meals on mean excretion values of urinary pregnanediol for male subjects 50 10. The effect of number of meals by day of test period on pregnanediol excretion values for male subjects 51 11. The effect of number of meals on mean excretion values of urinary pregnanediol for female subjects 53 12. The effect of number of meals by day of menstrual cycle on pregnanediol excretion values 54 13. Mean total cholesterol values for men with or with- out lunch by days of study . 65 Figure Page 14. Mean total cholesterol values for women with or with­ out lunch by days of menstrual cycle 67 15. The effect of number of meals by days of study on total serum cholesterol values fo r men 68 16. The effect of number of meals by days of menstrual cycle on total serum cholesterol values 69 17. Mean serum cholesterol and urinary neutral 17- ketosteroid and pregnanediol values by days of study for men 73 18. Mean serum cholesterol and urinary neutral 17- ketos teroid and pregnanediol values by days of menstrual cycle for women ACKNOWLEDGEMENTS The author wishes to express her gratitude to Dr. Ethelwyn B. Wilcox for her untiring efforts in initiating this problem and in making suggestions and counseling throughout this study. Appreciation is also expressed to Dr. Joseph C. Street, Mrs. Grace J. Smith, and Dr. Elveda Smith for their special assistance and suggestions. Thanks are given to Dr. Donald V. Sisson for his help with the statistical analysis. This study was part of the Western Regional Research Project on Biological Interrelationships in Lipid Metabolism of Importance to Man. It was supported in part by W-44 funds obtained under the Research and Marketing Act. Acknowledgement is given to all others who gave their assistance and encouragement and thanks to those persons who acted as experimental subjects. INTRODUCTION Since there is a high incidence of coronary heart disease in humans of middle-age and older in the United States, and the mortality of this disease is still increasing, a great deal of research has been done in this field. Although many theories have been formulated con­ cerning the cause of atherosclerosis, it is a very complex disease, and appears to be influenced by many factors. Disturbed lipid meta­ bolism is widely believed to be involved in the development of the vascular lesions found in arteriosclerosis, although the nature and origin of the disturbance is unknown. The mechanism for the elevation of serum cholesterol and triglyceride levels is not known; but sex hormones and dietary constituents are believed to be among the factors involved. The concept that atherosclerosis is a metabolic disorder involving lipids and lipoproteins has stimulated extensive investigation of the endocrine influences on circulating lipids and on lipid metabolism. The results have consistently indicated that estrogens decrease cir­ culating cholesterol and prevent cholesterol induced coronary atheros­ clerosis, while androgen administration tends to increase circulating cholesterol . There are relative few studies of the effect of other sex hormones, such as progesterone. There is only limited information on the relationship between the level of progesterone and the concentra­ tion of serum cholesterol in healthy adults while living under ordinary living conditions. Further work with more subjects is desirable. Since the discovery by Marrian and co-workers (1929) of S~preg­ nane-3ol,200l-diol in urine of pregnant woman and the perfection of a quantitative method for its determination by Venning (1937), this compound has received wide attention because of its close relation to the meta- holism of the corpus luteum hormone, progesterone. Pregnanediol is the major end product of progesterone catabolism and its estimation provides a useful index of luteal function. Progesterone has been isolated from three mammalian tissue sources, the corpus luteum, the adrenal, and the placenta. The function of progesterone i s to promote the prolifer­ ation of uterine mucosa and thus to prepare this tissue to receive the fertilized ovum. Cholesterol, literally meaning bile solid-alcohol, derives its name from the fact that it was first isolated from human gallstones, of which it is generally the chief component. The amount of chole­ sterol in animal tissues varies widely. It is particularly abundant in brain and nerve tissue, adrenal glands , and egg yolk. It had been shown that the administration of deuterium-labeled cholesterol to a pregnant woman gave rise t o labeled pregnanediol in the urine.