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The metabolic fate and mode of excretion of cyclohexylphenyl 4-[35S]sulphate were studied in the guinea pig. Up to 54.8% of the dose appeared in the bile, the majority as unchanged ester. Substantial amounts of hydroxylated cyclohexylphenyl 4-[35S]sulphate were also excreted in the bile together with minor amounts of the corresponding glucuronic acid conjugate. When isolated guinea-pig livers were perfused with cyclohexylphenyl 4-[35S]sulphate the biliary components were the same as those in the intact animal, although the relative concentration of the hydroxylated derivative was significantly greater. When the hydroxylated derivative was re-injected into guinea pigs it was excreted almost entirely unchanged in the bile. However, in the rat, it was excreted in the bile as a glucuronic acid conjugate. These findings are discussed in relation to studies carried out in the rat [Hearse, Powell, Olavesen & Dodgson (1969) Biochem. Pharmacol. 18, 181--195] and to differences in enzyme activities in rat and guinea-pig liver. The results are also discussed in terms of the molecular-weight threshold for the excretion of anions in guinea-pig bile.

1. The biliary excretion of phenolphthalein di[35S]sulphate was studied in rats. 2. The conjugate was administered by continuous infusion at rates of 3, 4.5, 6, 9 and 12 mug/min, and kinetic analysis of the rate of biliary excretion was consistent with a two-compartment open-model system. 3. The results obtained after single injections of the ester were also consistent with the model. 4. An essential feature of the model is the presence of a compartment into which the ester may pass as an alternative to direct excretion via the bile. 5. It is suggested that such a compartment may be located within the liver.

1H n.m.r. spectra in [2H6]dimethyl sulphoxide of dodecyltrimethylammonium salts of chondroitin sulphates and hyaluronate, or sodium salts of oligomers from hyaluronate, showed unambiguous NH signals. The acetamido NH occurs in two different environments: environment I ('normal') in simple sugars, and environment II (hydrogen-bonded NH) appearing in tri- or tetrasaccharides, indicating a secondary structure in hyaluronate (and some chondroitin sulphates) involving a hydrogen-bonded acetamido NH.

The metabolic fates and modes of excretion of diethylstilboestrol mono[35S]sulphate and diethylstilboestrol di[35S]sulphate were studied in the guinea pig. Comparative studies were also made with [G-3H]diethylstilboestrol and phenolphthalein di[35S]sulphate. Diethylstiboesterol di[35S]sulphate was extensively eliminated in the bile unchanged. After administration of diethylstilboestrol mono[35S]sulphate, extensive biliary elimination of radioactivity was also recorded. Radioactive components were identified as diethylstilboestrol disulphate, diethylstilboestrol monosulphate monoglucuronide and unchanged diethylstilboestrol monosulphate. When [G-3H]diethylstilboestrol was administered, 3H-labelled diethylstilboestrol monoglucuronide, diethylstilboestrol monosulphate monoglucuronide and diethylstilboestrol disulphate appeared in the bile. Phenolphthalein di[35S]sulphate was excreted unchanged in bile. These findings are discussed in relation to studies carried out in the rat [Barford, Olavesen, Curtis & Powell (1977) Biochem. J. 164, 423--430] and species differences are related to differences in enzyme activities in rat and guinea-pig liver.

The metabolic fates and modes of excretion of diethylstilboestrol mono[35S]sulphate and diethylstilboestrol di[35S]sulphate were studied in the rat. Both of the esters were desulphated to some extent in vivo. In addition, significant amounts of radioactivity appeared in the bile as diethylstilboestrol mono[35S]sulphate monoglucuronide. The percentage of the dose appearing in bile as the diconjugate was substantially greater in experiments with diethylstilboestrol mono[35S]sulphate than with diethylstilboestrol di[35S]sulphate. Whole-body radioautography and studies with isolated perfused liver confirmed the liver as the major metabolic organ for both esters. When the metabolite diethylstilboestrol mono[35S]sulphate monoglucuronide isolated from the bile was reinjected, it was excreted in the bile unchanged. Studies in vitro demonstrated that both esters were substrates for arylsulphatase C with Km values in the range 52-76 micrometer. The metabolic fates and modes of excretion of the esters are discussed in relation to the enzyme complement of rat liver.

The metabolic fates of the synthetic surfactants, sodium [1-(14)C]undecyltriethoxy sulphate and sodium [1-(14)C]dodecyltriethoxy sulphate were studied in the rat. Both compounds were extensively metabolized regardless of the route of administration, oral, intraperitoneal or intravenous. Short-chain radioactive products were eliminated in the urine: the major metabolite of the dodecyl homologue in the urine was identified as (-)O(2)C(14)CH(2)- (OC(2)H(4))(3)OSO(3) (-) by n.m.r. and g.l.c.-mass spectrometry, whereas the major metabolite of the undecyl homologue in the urine was tentatively identified as (-)O(2)CCH(2) (14)CH(2)- (OC(2)H(4))(3)OSO(3) (-). In contrast with experiments with the dodecyl derivative, when [1-(14)C]undecyltriethoxy sulphate was administered to rats, appreciable amounts of radioactivity were recovered as (14)CO(2) in expired air. Whole-body radioautography implicated the liver as the major site of metabolism of both surfactants. The nature of the metabolic products establishes that both compounds are degraded by omega,beta-oxidation. Cleavage of the ether linkage proximal to the sulphate moiety may account for the small amounts of (14)CO(2) recovered in expired air after the administration of [1-(14)C]dodecyltriethoxy sulphate. It is suggested the substantial amounts of (14)CO(2) recovered after [1-(14)C]-undecyltriethoxy sulphate administration originate from (-)O(2) (14)C(OC(2)H(4))(3) OSO(3) (-), an unstable product of omega,beta-oxidation. An n.m.r. spectrum of the metabolite identified as 2-(triethoxy sulphate)acetic acid and a mass spectrum of the trimethylsilyl derivative of the parent alcohol of that metabolite have been deposited as Supplementary Publication SUP50086 (5 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.

The growth of the mould Trichoderma viride on a defined medium containing either potassium d-glucose 6-O-sulphate or potassium d-galactose 6-O-sulphate as sole sources of both carbon and sulphur is marked by the production of an enzyme system capable of liberating inorganic SO(4) (2-) ions from either of the sulphate esters. The enzyme is not produced when the organism is grown with glucose (or galactose) and potassium sulphate or with glucose and methionine as sole sources of carbon and sulphur. Experimental conditions are described whereby inorganic SO(4) (2-) ions liberated from potassium glucose 6-O-sulphate by the growing mould appear in the culture medium after a constant lag period of 21-24hr. The enzyme has been shown to be a simple glycosulphatase that is active towards the 6-O-sulphate esters of d-glucose and d-galactose but not towards potassium glucose 3-O-sulphate. The properties of the crude glycosulphatase show the enzyme to be appreciably different from analogous molluscan enzymes that can degrade monosaccharide sulphate esters.

1. The metabolism of sodium cortisone 21-[(35)S]sulphate was investigated in rats. 2. Quantitative and qualitative experiments showed that substantial amounts of (35)SO(4) (2-) appeared in the urine of free-ranging rats receiving the ester. 3. Whole-body radioautograms indicated considerable biliary elimination of (35)S and also pointed to the liver as the site of metabolism. 4. When female rats with bile-duct cannulae received sodium cortisone 21-[(35)S]sulphate approx. 70% of the dose appeared in the bile as a doubly conjugated steroid (metabolite I). This metabolite was identified as 3alpha-(beta-d-glucopyranuronosido)- 17alpha-hydroxy-21-[(35)S]sulpho-oxy-5alpha-pregnane-11,20-dione. 5. When metabolite I was administered to a rat with a bile-duct cannula 90% of the dose appeared in the bile unchanged. After the administration (intraperitoneally or orally) of metabolite I to free-ranging rats considerable amounts of (35)SO(4) (2-) appeared in the urine. 6. The route by which (35)SO(4) (2-) might be produced from cortisone [(35)S]sulphate in free-ranging animals is discussed.

The metabolic fate of potassium dodecyl [(35)S]sulphate was studied in rats. Intraperitoneal and oral administration of the ester into free-ranging animals were followed by the excretion of the bulk of the radioactivity in the urine within 12hr., approximately 17% being eliminated as inorganic [(35)S]sulphate. Similar results were obtained in experiments in which potassium dodecyl [(35)S]sulphate was injected intravenously into anaesthetized rats with bile-duct and ureter cannulae. Analysis of urinary radioactivity revealed the presence of a new ester sulphate (metabolite A). This metabolite was isolated, purified and subsequently identified as the sulphate ester of 4-hydroxybutyric acid by paper, thin-layer and gas chromatography, by paper electrophoresis and by comparison of its properties with those of authentic butyric acid 4-sulphate. The identity of the metabolite was confirmed by isotope-dilution experiments. When either purified metabolite A or authentic potassium butyric acid 4[(35)S]-sulphate was administered to free-ranging rats the bulk of the radioactivity was eliminated unchanged in the urine within 12hr., approx. 20% of the dose appearing as inorganic [(35)S]sulphate. Whole-body radioautography and isolated-liver-perfusion experiments implicated the liver as the major site of metabolism of potassium dodecyl [(35)S]sulphate. It is suggested that butyric acid 4-sulphate probably arises by omega-oxidation of dodecyl sulphate to a fatty acid-like compound, which is then degraded by beta-oxidation.

The most recently published method for the assay of testicular hyaluronidase preparations was based on the premise that the enzyme also exhibited carboxylesterase activity towards indoxyl acetate. Studies on the relative enzyme activities of various hyaluronidase preparations towards hyaluronate and indoxyl acetate, the relative stabilities towards pH, temperature and mechanical shaking and the behaviour towards a variety of inhibitors, showed that the activities towards the two substrates reflected the presence of at least two different enzyme systems in the preparations. Gel chromatography and polyacrylamide-gel-electrophoresis experiments confirmed these conclusions and the collective findings clearly establish that methods based on the use of indoxyl acetate cannot be employed to measure testicular hyaluronidase activity.