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Methods are described to extract and measure 2,3-bis(3-hydroxybenzyl)-?-butyrolactone (enterolactone) and 2,3-bis(3-hydroxybenzyl)butane-1,4-diol (enterodiol) from physiological fluids. They are based on the use of stable labelled analogues of each compound as internal standards with end point assay being by gas chromatography mass spectrometry. The methods were developed to quantify enterolactone and enterodiol in experiments designed to investigate the significance and source of these compounds in man and animals. Examples of studies on human ovarian blood and investigations of their excretion in urine following dietary manipulation are described.

[4-14C] Cortisol was administered intramuscularly to one adult female macaque monkey, MF3 (Macaca fascicularis). To adult female macaque monkey, MF4, [4-14C]corticosterone was administered intramuscularly. Urine samples were collected and the metabolites excreted identified using gas chromatography, radio-gas chromatography and gas chromatography-mass spectrometry.The principal metabolites of cortisol were identified as glucuronide conjugates of 11-oxygenated-17-oxosteroids. The excretion of tetrahydrocortisol and tetrahydrocortisone relative to the other corticosteroid metabolites was low compared with that of man. Two compounds, 3β-cortol and 3β-cortolone not normally present in human urine were identified in the urine from this species.The principal metabolites of corticosterone were glucuronide conjugates of hexahydroCompound A and hexahydrocorticosterone. Two unidentified radioactive compounds were also present.

The definitive identification of the first lignans to be found in humans and animals is described. Gas chromatography-mass spectrometry, n.m.r. spectroscopy, i.r. spectroscopy and chemical techniques were employed to establish the structures of two lignans as trans-2,3-bis(3-hydroxybenzyl)-gamma-butyrolactone and 2,3-bis(3-hydroxybenzyl)butane-1,4-diol. Both compounds are essetially racemic. Evidence was also found for several methoxy analogues of these lignans in the vervet monkey.

1. Pruritus was assessed in 19 patients by measurement of nocturnal limb movement. 2. Serum (nine pruritic, ten non-pruritic) and interstitial fluid (five pruritic, three non-pruritic) bile acids were fractionated according to their mode of conjugation by using DEAP-Sephadex LH-20 and measured by gas chromatography—mass spectrometry. 3. No correlation was found between serum or interstitial fluid total bile acid or individual bile acid concentrations and pruritus. Bile acid profiles in the two groups of patients were similar and there was no correlation between pruritus and the conjugation pattern. 4. The bile acid profile of interstitial fluid reflected that of serum and a linear relationship was found between serum and interstitial fluid bile acid concentrations (r = 0·95, P < 0·001). 5. The proportion of bile acid sulphate in interstitial fluid was significantly smaller than that in serum (P < 0·025), where sulphates accounted for up to 46% of the total bile acids. 6. In three patients, a decrease in serum bile acid concentrations achieved by percutaneous transhepatic biliary drainage had little or no effect on pruritus. 7. These findings suggest that bile acids do not have a causative role in the pruritus of cholestatic liver disease.

The half-life of plasma cortisol in the rhesus monkey (M. mulatta), determined by two methods, was about 130 min and longer than that in man; it was unaffected by administration of dexamethasone. Dexamethasone (5 mg, i.v.) immediately inhibited the secretion of ACTH from the monkey pituitary. The plasma half-life of NH2-terminal immunoreactive ACTH was found to be about 55 min which was much longer than the biological half-life. The adrenal synthesis of cortisol was inhibited by metyrapone which caused a prompt increase in the plasma concentration of ACTH and 11-deoxycortisol. The hypothalamicpituitary-adrenal system of the rhesus monkey sedated with phencyclidine hydrochloride responded rapidly to alteration in the level of steroids in the circulation.

Computerised gas chromatography-mass spectrometry was employed in the identification of polar corticosteroid metabolites excreted in the urine from the macaque monkey (Macaca fascicularis) and the baboon (Papio hamadryas). The following steroids were identified in significant amounts in the urine from both species: 3α,17α,20α,21-tetrahydroxy-5β-pregnan-11-one; 3α,17α,20β,21-tetrahydroxy-5β-pregnan-11-one; 5β-pregnane-3α,11β,17α,20α,21-pentol; 5β-pregnane-3α,11β,17α,20β-pentol; 5α-pregnane-3β, 11β, 17α,20β,21 -pentol.11β,17α,21-Trihydroxy-4-pregnene-3,20-dione (cortisol), 11β,17α,20β,21-tetrahydroxy-4-pregnen-3-one and 11β, 17α,20β,21 -tetrahydroxy-5ξ-pregnan-3-one were identified in macaque monkey urine. Two steroids, 17α,20β,21-trihydroxy-4-pregnane-3,11-dione and 17α,20α,21-trihydroxy-4-pregnene-3,11-dione were excreted as major C21metabolites in the baboon but were not identified in the urine from the macaque monkey. 3β-Hydroxy-5α-pregnane metabolites were identified in the urine from both species. All these steroids were excreted conjugated to glucuronic acid, evidenced by their recovery after hydrolysis with β-glucuronidase enzyme.An efficient 20β-reduction of corticosteroids in both species is apparent, and the excretion pattern of polar steroid metabolites in the two species was shown to be similar.

The dietary origin of the weak oestrogen equol (7-hydroxy-3-(4′-hydroxyphenyl)-chroman) present in human urine has been investigated using gas chromatography–mass spectrometry. Feeding experiments with different food constituents and monitoring the urinary excretion of equol revealed that soya food yields more than 0·1 mg urinary equol/g flour ingested. From this source the glucoside of daidzein (4′,7-dihydroxyisoflavone) has been isolated and identified as a precursor of equol. Both equol and daidzein were characterized as monoglucuronide conjugates in human urine and the concentration of urinary equol exceeded the concentrations of the classical oestrogens by 100- to 1000-fold after ingestion of a single meal containing soya protein. The potential biological significance of this result is discussed.J. Endocr.(1984) 102, 49–56

The physiological regulation of the plasma corticosteroid concentration, measured by competitive protein-binding, was studied in female rhesus monkeys (M. mulatta) sedated with phencyclidine hydrochloride. Morning basal levels of plasma corticosteroids were found to be in the range 8·0–25·2 μg/100 ml, which is lower than that previously reported in this species. A circadian rhythm in plasma cortisol concentration was demonstrated. Prolonged sedation with phencyclidine was associated with a gradual increase in the plasma cortisol concentration. Synthetic α1–24adrenocorticotrophic hormone given intravenously caused a rapid rise in plasma cortisol, the minimum effective dose was between 1 and 10 ng/kg body weight and the response was maximal after 1000 ng/kg. The administration of lysinevasopressin and the induction of hypoglycaemia by insulin were both followed by an increase in the plasma corticosteroid concentration. Metyrapone caused a decline in plasma 11-hydroxycorticosteroids and a concomitant increase in total corticosteroids measured by competitive protein-binding. It is concluded that the hypothalamic-pituitary-adrenal system in the rhesus monkey functions in a manner which is qualitatively and quantitatively similar to that of man.

Past experiments and current paradigms of cholesterol homeostasis suggest that cholesterol 7alpha-hydroxylase plays a crucial role in sterol metabolism by controlling the conversion of cholesterol into bile acids. Consistent with this conclusion, we show in the accompanying paper that mice deficient in cholesterol 7alpha-hydroxylase (Cyp7-/- mice) exhibit a complex phenotype consisting of abnormal lipid excretion, skin pathologies, and behavioral irregularities (Ishibashi, S., Schwarz, M., Frykman, P. K. , Herz, J., and Russell, D. W.(1996) J. Biol. Chem. 261, 18017-18023). Aspects of lipid metabolism in the Cyp7-/- mice are characterized here to deduce the physiological basis of this phenotype. Serum lipid, cholesterol, and lipoprotein contents are indistinguishable between wild-type and Cyp7-/- mice. Vitamin D3 and E levels are low to undetectable in knockout animals. Stool fat content is significantly elevated in newborn Cyp7-/- mice and gradually declines to wild-type levels at 28 days of age. Several species of 7alpha-hydroxylated bile acids are detected in the bile and stool of adult Cyp7-/- animals. A hepatic oxysterol 7alpha-hydroxylase enzyme activity that may account for the 7alpha-hydroxylated bile acids is induced between days 21 and 30 in both wild-type and deficient mice. An anomalous oily coat in the Cyp7-/- animals is due to the presence of excess monoglyceride esters in the fur. These data show that 7alpha-hydroxylase and the pathway of bile acid synthesis initiated by this enzyme are essential for proper absorption of dietary lipids and fat-soluble vitamins in newborn mice, but not for the maintenance of serum cholesterol and lipid levels. In older animals, an alternate pathway of bile acid synthesis involving an inducible oxysterol 7alpha-hydroxylase plays a crucial role in lipid and bile acid metabolism.

Urinary steroids and steroid conjugates were measured in the squirrel monkey (Saimuri sciureus). The principal steroids excreted were cortisol, 11β,17α,20β,21-tetrahydroxy-4pregnen-3-one (20β-dihydrocortisol), 11β,17α,20α,21-tetrahydroxy-4-pregnen-3-one (20α-dihydrocortisol), α- and β-cortol and α- and β-cortolone. The majority of the steroids were excreted unconjugated and a conspicuous feature of the pattern was the large amount of urinary free cortisol. Unlike man there was an insignificant excretion of 3α,17α,21-trihydroxy-5β-pregnane-11,20-dione (tetrahydrocortisone) and 3α,11β,17α,21-tetrahydroxy-5β-pregnan-20-one (tetrahydrocortisol). A steroid not previously identified in urine from any species was one of the major glucuronide conjugates; it was characterized as having the structure 3β,17α,20ξ,21-tetrahydroxy-5β-pregnan-11-one. Administration of dexamethasone resulted in complete suppression of steroid output, whilst the response to adrenocorticotrophic hormone was inconstant.

A detailed study of the qualitative and quantitative composition of bile acids in human fetal gallbladder bile is described. Bile was collected during early gestation (weeks 16-19) and analyzed by gas chromatography and mass spectrometry, fast atom bombardment ionization mass spectrometry, and high performance liquid chromatography. Bile acids were separated into different conjugate groups by chromatography on the lipophilic anion exchange gel, diethylaminohydroxypropyl Sephadex LH-20. Quantitatively more than 80% of the bile acids were secreted into bile conjugated to taurine. Unconjugated bile acids and glycine conjugates accounted for 5-10% of the total biliary bile acids. Bile acid sulfates were present only in trace amounts indicating that quantitatively sulfation is not an important pathway in bile acid metabolism during development. Total biliary bile acid concentrations were low (0.1-0.4 mM) when compared to reported values for adult bile (greater than 10 mM). Chenodeoxycholic acid was the major biliary bile acid and exceeded cholic acid concentrations by 1.43-fold indicating either a relative immaturity in 12 alpha-hydroxylase activity during early life or a dominance of alternative pathways for chenodeoxycholic acid synthesis. A relatively large proportion of the biliary bile acids comprised metabolites not found in adult bile. The presence of relatively high proportions of hyocholic acid (often greater than cholic acid) and several 1 beta-hydroxycholanoic acid isomers indicates that C-1 and C-6 hydroxylation are important pathways in bile acid synthesis during development. We describe, for the first time, evidence for the existence of a C-4 hydroxylation pathway in the metabolism of bile acids, which may be unique to early human development. Mass spectrometry was used to confirm the identification of 3 alpha,4 beta,7 alpha-trihydroxy-5 beta-cholanoic and 3 alpha,4 beta-dihydroxy-5 beta-cholanoic acids. Quantitatively, these C-4 hydroxylated bile acids accounted for 5-15% of the total biliary bile acids of the fetus, suggesting that C-4 hydroxylation is quantitatively an important pathway in the bile acid metabolism during early life.

The assays of testosterone and corticosteroids in plasma from adult male rhesus monkeys using competitive protein-binding and radioimmunoassay techniques are described. The radioimmunoassay for testosterone was conducted without chromatography and, therefore, additionally estimated 17β-hydroxy-5α-androstan-3-one (dihydrotestosterone). Levels of testosterone in the peripheral plasma of 14 intact male rhesus monkeys showed marked fluctuations over a period of 24 h. Concentrations of testosterone at 22.00 h (1776 ± 814 ( ± s.d.) ng/100 ml) were approximately double those at 08.00 h (858 ± 407 ng/100 ml), 12.00 h (898 ± 316 ng/100 ml) and 16.00 h (784 ± 530 ng/100 ml). Castration resulted in low plasma testosterone levels (85 ± 29 ng/100 ml), and the increases at 22.00 h were no longer observed. In intact males, the 'basal' plasma corticosteroidlevel(08.00 h) was 22·4 ± 6·0 μg/100 ml. Administration of synthetic corticotrophin raised plasma corticosteroid levels without changing plasma testosterone concentration. Because plasma testosterone levels were not related to changes in adrenocortical activity, the noctural rises appear to be due to changes in testicular secretion.

The identification (by gas chromatography–mass spectrometry and n.m.r.) for the first time of the weak oestrogen equol [7-hydroxy-3-(4′-hydroxyphenyl)chroman] in human urine is described. Preliminary results of its quantitative excretion in urine are reported and the potential significance of the occurrence of this compound is discussed.