T. F. Gallagher

ISOTOPIC STUDIES OF PLASMA CHOLESTEROL OF ENDOGENOUS AND EXOGENOUS ORIGINS

This study was undertaken in order to compare the metabolic behavior of plasma cholesterol derived from the diet with that of cholesterol synthesized in vAvo from acetate (1). Cholesterol labeled with either isotopic carbon or hydrogen was administered orally to human subjects and the incorporation into plasma cholesterol was followed over an extended period. In certain instances, both endogenous and exogenous cholesterol metabolism were studied simultaneously by the use of appropriately labeled acetate and cholesterol. These techniques were also applied to an examination of the behavior of plasma cholesterol in four patients with hypercholesterolemia.

Cortisol Metabolism in Cirrhosis

The production and peripheral metabolism of cortisol have been studied in 10 cirrhotics and 11 controls after i.v. tracers of cortisol-(14)C. The findings were as follows: (a) Total urinary excretion of radioactivity was normal (81% of the dose) but a decreased fraction was present as glucosiduronates: 18-47% of the dose (average 34%) compared to a normal average of 54%. (b) There was a distinctively abnormal pattern of cortisol metabolites, not previously observed in other illnesses: tetrahydrocortisone was decreased to 14% of the enzyme hydrolysate (normal 26%); cortolones were increased to 34% (normal 19%), owing entirely to an increase in cortolone (20alpha) formation, since beta-cortolone (20beta) was not significantly increased; Reichsteins substances U and epi-U were increased, averaging 2.6% for the former and 4.7% for the latter; tetrahydrocortisol, allotetrahydrocortisol and cortols were normal. This pattern was independent of the degree of decreased glucosiduronate formation and also independent of the presence or absence of a portacaval shunt. (c) Cortisol production, determined by isotope dilution, was normal in each of six cirrhotic patients. From these data, taken in conjunction with our previously reported findings concerning the influence of norethandrolone on cortisol metabolism, the following conclusions were drawn: (a) Cirrhotic patients have decreased A-ring reduction of cortisone to tetrahydrocortisone and correspondingly increased 20-ketone reduction of cortisone to Reichsteins substances U and epi-U and then to the cortolones. (b) Intrahepatic cholestasis, a regular pathophysiological feature of cirrhosis, may be responsible for the observed abnormal cortisol metabolite pattern in this disease. (c) The slowed metabolic turnover rate of cortisol in cirrhosis may be due to decreased transport and/or binding of cortisol to its intracellular metabolic sites rather than to abnormalities of any specific metabolizing enzymes.

Psychoendocrine aspects of cancer of the breast.

&NA; Thirty women in the hospital awaiting biopsy of a breast tumor were interviewed in order to assess the adequacy of their ego defenses in this presumably threatening situation. The criteria for this psychological evaluation were the patients affective state, functional disruption, and defensive reserve impairment. Concurrent with these assessments, daily production rates of hydrocortisone and urinary excretion levels of the principal hydrocortisone and androgen metabolites were measured. A rank order correlation significant at the .02 level was found between rating scores for extent of defensive failing and hydrocortisone production rates, and at the .05 level between this psychological variable and the principal hydrocortisone metabolites; however, no correlation existed with the androgen metabolites. Since other studies have indicated a correlation between prognosis in breast cancer and a numerical ratio calculated on the basis of the patients endogenous corticoid to androgenic steroids, this study raises questions about the determination and nature of such a ratio and about the possible role of psychological phenomena in the course of this neoplasm.

Metabolism of estriol-17α-3H in man

Abstract Estriol stereospecifically labelled with tritium at the 17 α position was prepared and administered together with 14 C labelled estriol to three subjects including one cirrhotic. No significant tritium was present in the plasma or urine water and hence no oxidation at C-17 had occurred. Excretion of radioactivity was very rapid particularly in the cirrhotic subjects. Analysis of the enzyme and acid hydrolysates showed that they consisted of only three compounds. The majority of the excreted radioactivity consisted of estriol which did not lose any of the tritium at 17 α . The only other compounds were 16-ketoestradiol (0.8%) and 16-epiestriol (0.2%). Both metabolites retained the major part of the tritium at C-17 α and hence could not have arisen via 17-keto intermediates.

Analysis of estrogen metabolites by isotope derivative and by fluorimetry

Abstract Following intravenous administration of a trace quantity of estradiol-3H the urinary metabolites were obtained after β-glucuronidase hydrolysis. These were separated by partition chromatography and estrone and estriol were further purified by thin layer chromatography on silica gel. These were acetylated with 14C labeled reagent and the acetates after carrier addition were chromatographed on thin layer silica gel. Additional carrier was added and the compounds were recrystallized until constant isotope ratio was achieved. The procedure was checked by addition of unlabeled estrone and estriol to the crude extract and repetition of the purification and isolation. The estrone fraction from the partition column was reduced to estradiol, purified and analyzed as the 14C diacetate. The specific activities so obtained were in agreement for the respective metabolites. No extraneous radioactivity was detected by gas chromatography and radioactive monitoring on the purified acetates at constant specific activity. An attempt was made to employ fluorimetry for measurement of the mass of estrone and estriol prior to carrier addition and recrystallization. This was unsuccessful and it was evident that more extensive purification would be required to use this technique.

Tetrahydrocortisol metabolism in man

Abstract Tetrahydrocortisol (THF) metabolism was studied by I.V. administration of trace amounts of 3H-THF to 3 normal men and 4 patients with miscellaneous illnesses. The principal urinary steroid was THF. This constituted about half of the “glucosiduronates” in the normal subjects, but significantly more (61–79%) in 3 of the 4 patients. Reduction of the 20-ketone of THF was quantitatively less prominent than the corresponding reaction for THE, but similarly stereoselective. Total cortols constituted 2–12% of the “glucosiduronates”, and the 111β-epimer predominated by about 15:1. The major conversion product of THF was 11β-hydroxyetiocholanolone, 12–25% of the “glucosiduronates” in normal subjects. Three of the four patients showed significantly diminished formation of this metabolite (3–5% of the “gluco-siduronate”). This suggests that diminished side-chain removal of THF may occur non-specifically in ill subjects. In one of these 3 patients, the low 11β-hydroxy-etiocholanolone formation was associated with extremely high β-cortol formation (32% of the “glucosiduronate” suggesting that β-cortol serves as one of the immediate precursors of 11β-hydroxyetiocholanolone.

Reproducibility of a double isotope derivative analysis for cortisol metabolites

Abstract Replicate measurements of the specific activity of urinary tetra-hydrocortisol and tetra hydrocortisone after injection of 14C-cortisol in human subjects, were made by a double isotope derivative technique employing carrier addition and recrystallization to constant isotope ratio. Twenty subjects were studied and reproducibility was found to be excellent.

Cortisol metabolism in the morning and evening; relation to cortisol secretion rate measurements

Abstract The question was studied whether the discrepancies observed in cortisol secretion rate determinations from several metabolites were due to changes in metabolic pathway of cortisol during the course of the day. Cortisol-4- 14 C was administered to two subjects in the morning at about 0800 hr. The studies were repeated in the same subjects twice in the evening at about 1800 hr and one subject received cortisol-4- 14 C at 1415 hr as well. The specific activities of 6 metabolites were determined and the apparent cortisol secretion rates were calculated. The production rates from the various metabolites were not in precise agreement at any of the times of day studied; the disagreement was apparently greatest when the tracer was given in the evening. That the time of day at which labeled cortisol is injected is not the factor responsible for the different estimates was shown in an adrenalectomized subject maintained on dexamethasone. Tracer cortisol-4- 14 C was given to this patient at 1750 hr and 36 hours later another tracer of cortisol 1,2- 3 H was given at 0900 hr. Complete urine collections were obtained from the time of administration of the first tracer until 36 hours after the second. The urine was processed as a single collection. The 3 H/ 14 C ratios of all the metabolites were identical demonstrating that the labeled cortisol followed identical metabolic pathway in the morning and in the evening. It thus appears that a diurnal variation in cortisol metabolizing enzymes is not responsible for the demonstrated discrepancies between metabolites when secretion rate is measured by the isotope dilution method from urinary metabolites.

Metabolism of adrenosterone

Abstract The metabolism of simultaneously administered adrenosterone-1,2-3H and 11β-hydroxy-Δ4-androstene-3,17-dione-4-14C has been studied in a normal and a hypothyroid subject. Conversion to the metabolites from either hormone was essentially alike demonstrating ready interconversion of the 11β-hydroxy and 11-keto groups of the administered hormones. Following a large dose of adrenosterone to a hypothyroid subject 3α-hydroxy-Δ4-and-rostene-11, 17-dione and its reaction product with urea, 3α-ureido-Δ4-and-rostene-11, 17-dione were isolated and characterized.

Metabolism of pregnane-3α,17,20β-triol in man

Abstract The metabolism of pregnane-3α,17,20β-triol has been studied in man. Evidence was obtained for oxidation at C-20 and epimerization to pregnane-3α,17,20α-triol. The principal urinary product was the administered steroid. It was concluded that pregnane-3α,17,20β-triol is not on the major pathway to pregnane-3α,17,20α-triol production.