R. E. Silman

TYPING SCHEME FOR CLOSTRIDIUM DIFFICILE: ITS APPLICATION IN CLINICAL AND EPIDEMIOLOGICAL STUDIES

Epidemiological studies of Clostridium difficile diarrhoeal disease have been hindered by the lack of a typing scheme for this organism. A typing method based on the incorporation of sulphur-35-labelled methionine into cellular proteins and their separation by sodium dodecylsulphate/polyacrylamide gel electrophoresis showed clear pattern differences between strains, and nine distinct groups within the C difficile species were established. 98% of 250 clinical strains derived from four hospitals were typable. Group X was the commonest group and was associated with outbreaks of pseudomembranous colitis and antibiotic-associated colitis in two hospitals. Groups A-D were isolated predominantly from mothers and newborn infants. In outbreaks of antibiotic-associated colitis in oncology and orthopaedic wards the same strains, group X and group E, respectively, were isolated from patients and their environment, providing strong evidence of cross-infection between patients and of hospital acquisition of C difficile.

The isolation, purification, and characterisation of the principal urinary metabolites of melatonin

Melatonin is metabolised by hydroxylation to form 6‐hydroxy‐melatonin by demethylation to form N‐acetyl‐serotonin, which are excreted as sulphate glucuronide conjugates. We required these metabolites as pure powders therefore undertook their isolation characterisation. Three volunteers ingested 1 g each of melatonin, their urine was collected pooled. For the sulphate conjugates, a Lichoprep column was used to concentrate the metabolites to remove most of the urea. The sulphate conjugates were separated from the glucuronides on a Florisil column further purified on a fractogel column. They were separated by high‐performance liquid chromatography (HPLC) resulting in white powders of 6‐hydroxy‐melatonin sulphate (SaMT) N‐acetyl‐serotonin sulphate (SNAS). For the glucuronide conjugates, an aliquot of the pooled urine was taken to dryness, the residue was dissolved in methanol, the solution was filtered. The methanol filtrate was taken to dryness, the residue was applied to a Florisil column. The isolated glucuronide conjugates were recrystallized prior to separation by HPLC, which gave pure white powders of N‐acetyl‐serotonin glucuronide (GNAS) 6‐hydroxy‐melatonin glucuronide (GaMT). Characterisation was achieved by using infrared ultraviolet spectroscopy, thin‐layer chromatography (TLC), gas chromatography‐mass spectrometry (GCMS). These techniques unambiguously confirmed the assigned structures for SaMT SNAS fully supported the assigned structures for GNAS GaMT. Three TLC solvent systems were used, in each case the individual conjugated metabolite appeared as a discreet spot. Purity, as assessed by GCMS, was shown to be greater than 95% for SNAS, SaMT, GaMT to be 88% for GNAS.

An enzyme immunoassay for 6-sulphatoxy-melatonin in human urine

Abstract: We describe a newly developed enzyme immunoassay (EIA) for the determination of 6‐sulphatoxy‐melatonin (aMT6s) in human urine, using a aMT6s‐bovine serum albumin‐horseradish peroxidase (aMT6s‐BSA‐HRP) conjugate as the enzyme label. The assay incorporates a highly specific antibody raised in rabbits. The EIA has a sensitivity of 2 pg/well (40 pg/ml) with intraassay coefficients of variation of 2.3–6.1% in the range of the assay. The material with the highest level of cross‐reactivity was N‐acetyl serotonin sulphate, with a relative potency of 0.000078%. One hundred thirty‐four urine samples from children and adults at different time points were assayed and the results compared with those from an established radioimmunoassay (RIA) and with a newly developed RIA using the same antibody as the EIA. The correlation coefficient, r, comparing the two RIAs was 0.9869, and the regression equation was log (kit) = 0.9340 log (new) + 0.1213. The correlation coefficient, r, comparing the EIA with the newly developed RIA, was 0.9686, and regression equation log (new) = 0.9674 log (EIA) + 0.0600. The EIA for the measurement of aMT6s in urine represents a new approach in the investigation of pineal function.

Activity rhythms of hamsters in a single cage compared to a simulated burrow system

A method for measuring activity of hamsters using a stabilimeter at a 1 second sampling rate with data computer recorded as 5 minutes integrated values was developed. In a single cage without a running wheel a consistent pattern for activity was observed, consisting of (a) low levels of daytime activity until one or two hours before lights off when activity increased significantly; and (b) a peak of nocturnal activity in the first hour of the dark cycle. The inclusion of a running wheel increased and altered significantly the pattern of nocturnal activity. In further experiments animals were housed in two linked cages, one acting as light-proof burrow and the other exposed to light. Measurements were recorded from each cage independently and from two position detectors in the interconnecting tunnel. The results showed: (a) total activity, i.e., the summation of activity in both cages, was not different from activity in a single cage system; (b) low daytime activity was composed of prolonged periods of rest in the burrow plus short periods of activity in the exposed cage; the increased activity one hour before lights off was localised to the light-proof burrow; and (c) after lights off, the animals began to spend increasing periods of time in the exposed cage reaching a maximum after one hour. Replacing artificial with natural light did not change the principal features of behaviour.

The pineal and extra-pineal origins of 5-sulphatoxy N-acetyl-serotonin in humans

Abstract: In humans 6‐sulphatoxy melatonin (SaMT) is the principal metabolite of endogenous and exogenous melatonin. 5‐sulphatoxy N‐acetyl‐serotonin (SNAS) is a minor metabolite of exogenous melatonin, but it has not been established whether the levels of endogenous SNAS in plasma derives principally from endogenous melatonin. We have developed the first radioimmunoassay (RIA) for SNAS and used it (together with RIAs for melatonin and SaMT) to determine whether endogenous SNAS derives from endogenous melatonin or from platelet serotonin. Our results show a) the values of endogenous SNAS, unlike endogenous SaMT, increased with blood collection procedures that increased the values of serotonin, b) the values of endogenous SNAS in urine or in platelet‐poor plasma were approximately the same as those of endogenous SaMT, but, unlike SaMT, did not show a diurnal rhythm, and c) we confirmed that SNAS was a minor metabolite of orally ingested melatonin. Thus, our conclusion is that SNAS is a minor metabolite of exogenous melatonin, but is not a significant metabolite of endogenous melatonin. In all probability, endogenous SNAS is principally the metabolite of platelet serotonin.

The purification and characterization of biological 6‐sulphatoxymelatonin and comparison with synthetic 6‐sulphatoxymelatonin

Abstract: We have purified the major metabolite of melatonin, 6‐sulphatoxymelatonin, from urine and compared it to its synthetic counterpart. For preparation of the biological material, oral melatonin was administered to human volunteers and their urine extracted onto Amberlite XAD‐2 resin to remove urea; the glucuronide metabolites of melatonin were removed by silica chromatography; and 6‐sulphatoxymelatonin was separated from N‐acetyl serotonin sulphate, the other sulphate metabolite of melatonin, by preparative thin‐layer chromatography. Synthetic 6‐sulphatoxymelatonin was produced by reacting 6‐hydroxymelatonin with chlorosulphonic acid in dimethylformamide; the reaction mixture was purified on Florisil and preparative thin‐layer chromatography was used to remove indolic by‐products of the reaction. Elemental and X‐ray microanalysis of the biological and synthetic products showed that classical methods used for their purification introduced inorganic impurities, such as silicon‐ and chlorine‐ containing compounds, which were not detectable by thin‐layer chromatography, infrared spectroscopy, nuclear magnetic resonance spectroscopy, or gas chromatography‐mass spectrometry. We introduced further purification steps to remove these inorganic impurities, monitoring the process using elemental and X‐ray microanalysis. Extensive characterization of the resulting purified products showed that the biological and synthetic compounds were identical.

An investigation of demethylation in the metabolism of methoxytryptamine and methoxytryptophol.

Though melatonin is primarily metabolised to 6‐hydroxy‐melatonin, we have recently shown that it can also be demethylated to form N‐acetyl‐serotonin. The question therefore arises as to whether demethylation is a general metabolic pathway that can apply to other pineal methoxyindoles. To investigate this possibility we administered deuterated methoxy‐tryptophol (dML) and deuterated methoxy‐tryptamine (dMT) to rats and analysed the urine for the presence of deuterated methoxyindole acetic acid (dMIAA) and deuterated hydroxyindole acetic acid (dHIAA). The method of analysis was gas chromatography mass spectrometry (GCMS), where the relevant molecular ion and fragment ions were monitored. The results showed that the major metabolite in all cases was dMIAA. There was no evidence to suggest that the compounds had been demethylated to form dHIAA. The study therefore indicates that the demethylation of melatonin is a specific metabolic pathway that does not apply to other methoxyindoles.