Enzymology of oxidation of tropic acid to phenylacetic acid in metabolism of atropine by Pseudomonas sp. strain AT3.

Pseudomonas sp. strain AT3 grew with dl-tropic acid, the aromatic component of the alkaloid atropine, as the sole source of carbon and energy. Tropic acid-grown cells rapidly oxidized the growth substrate, phenylacetaldehyde, and phenylacetic acid. Crude cell extracts, prepared from dl-tropic acid-grown cells, contained two NAD+-linked dehydrogenases which were separated by ion-exchange chromatography and shown to be specific for their respective substrates, dl-tropic acid and phenylacetaldehyde. Phenylacetaldehyde dehydrogenase was relatively unstable. The stable tropic acid dehydrogenase was purified to homogeneity by a combination of ion-exchange, molecular-sieve, and affinity chromatography. It had a pH optimum of 9.5 and was equally active with both enantiomers of tropic acid, and at this pH, phenylacetaldehyde was the only detectable product of tropic acid oxidation. The formation of phenylacetaldehyde from tropic acid requires, in addition to dehydrogenation, a decarboxylation step. By analogy with NAD+-specific isocitrate and malate dehydrogenases, phenylmalonic semialdehyde, a 3-oxoacid, would be expected to be the precursor of phenylacetaldehyde. Other workers have established that isocitrate and malate dehydrogenases catalyze the decarboxylation of enzyme-bound or added 3-oxoacid intermediates, a reaction that requires Mn2+ or Mg2+ ions. Studies with tropic acid dehydrogenase were hampered by lack of availability of phenylmalonic semialdehyde, but in the absence of added divalent metal ions, both enantiomers of tropic acid were completely oxidized and we have not, by a number of approaches, found any evidence for the transient accumulation of phenylmalonic semialdehyde.

Atropine Metabolism by Pseudomonas sp. Strain AT3: Evidence for Nortropine as an Intermediate in Tropine Breakdown and Reactions Leading to Succinate.

Pseudomonas strain AT3, isolated by elective culture with atropine, hydrolyzed atropine and grew diauxically, first on the tropic acid and then on the tropine. Tropine was also used as a sole carbon and energy source. The methyl group of tropine was eliminated as formaldehyde, and the nortropine thus formed was a precursor of 6-hydroxycyclohepta-1,4-dione. Ammonia was detected as a product of nitrogen elimination. 6-Hydroxycyclohepta-1,4-dione was oxidized to cyclohepta-1,3,5-trione by an induced NAD(sup+)-specific dehydrogenase. Although cyclohepta-1,3,5-trione is a (beta)-diketone with two potential hydrolytic cleavage sites, an induced hydrolase was specific for one of these sites, with 4,6-dioxoheptanoate as the only hydrolysis product. Unlike the alternative cleavage product (3,6-dioxoheptanoate), this compound is also a (beta)-diketone, and a second hydrolytic cleavage formed succinate and acetone. Although Pseudomonas strain AT3 was not capable of growth with acetone, the compound was not detected in the culture medium and may have been lost to the atmosphere. Exhaustive experimentation with a wide range of conditions did not result in detection of the enzymes required for cleavage of the carbon-nitrogen bonds leading to the formation of nortropine and 6-hydroxycyclohepta-1,4-dione.

Tropine dehydrogenase: purification, some properties and an evaluation of its role in the bacterial metabolism of tropine.

Tropine dehydrogenase was induced by growth of Pseudomonas AT3 on atropine, tropine or tropinone. It was NADP(+)-dependent and gave no activity with NAD+. The enzyme was very unstable but a rapid purification procedure using affinity chromatography that gave highly purified enzyme was developed. The enzyme gave a single band on isoelectric focusing with an isoelectric point at approximately pH 4. The native enzyme had an M(r) of 58,000 by gel filtration and 28,000 by SDS/PAGE and therefore consists of two subunits of equal size. The enzyme displayed a narrow range of specificity and was active with tropine and nortropine but not with pseudotropine, pseudonortropine, or a number of related compounds. The apparent Kms were 6.06 microM for tropine and 73.4 microM for nortropine with the specificity constant (Vmax/Km) for tropine 7.8 times that for pseudotropine. The apparent Km for NADP+ was 48 microM. The deuterium of [3-2H]tropine and [3-2H]pseudotropine was retained when these compounds were converted into 6-hydroxycyclohepta-1,4-dione, an intermediate in tropine catabolism, showing that the tropine dehydrogenase, although induced by growth on tropine, is not involved in the catabolic pathway for this compound. 6-Hydroxycyclohepta-1,4-dione was also implicated as an intermediate in the pathways for pseudotropine and tropinone catabolism.

The isolation and identification of 6-hydroxycyclohepta-1,4-dione as a novel intermediate in the bacterial degradation of atropine.

Growth of Pseudomonas AT3 on the alkaloid atropine as its sole source of carbon and nitrogen is nitrogen-limited and proceeds by degradation of the tropic acid part of the molecule, with the metabolism of the tropine being limited to the point of release of its nitrogen. A nitrogen-free compound accumulated in the growth medium and was isolated and identified as 6-hydroxycyclohepta-1,4-dione. This novel compound is proposed as an intermediate in tropine metabolism. It served as a growth substrate for the organism and was also the substrate for an NAD(+)-linked dehydrogenase present in cell extracts. The enzyme was induced during the tropine phase of diauxic growth on atropine or during growth on tropine alone.

Toxin production by strains of Aeromonas hydrophila grown in laboratory media and prawn purée.

Toxin production by four strains of Aeromonas hydrophila grown at 30 and 37 degrees C in two laboratory media and prawn purée was studied. Three different cell lines were used to test for cytotoxic activity, haemolytic activity was tested against rabbit and guinea pig erythrocytes, proteolytic activity was assayed with azo-casein and enterotoxic activity using the suckling mouse assay. Results showed reduced cytotoxic and haemolytic activities in prawn purée compared with the two media, but in most cases increased proteolytic activity. No enterotoxic activity was observed in prawn purée although it was occasionally detectable in both laboratory media.

Evaluation of ELISA, RPLA, and Vero cell assays for detecting Clostridium perfringens enterotoxin in faecal specimens.

Three hundred and ninety two faecal specimens from 70 separate outbreaks of suspected Clostridium perfringens food poisoning were examined by enzyme linked immunosorbent assay (ELISA), reversed passive latex agglutination (RPLA), and Vero cell assays for the presence of enterotoxin. Although the most time consuming method, ELISA was the most specific and reproducible. RPLA was slightly more sensitive than ELISA, but it showed some non-specific reactions. The Vero cell assay was the least sensitive and least reproducible method, being affected by some non-specific cytotoxic and cytotonic reactions. Normal rabbit serum should be included in the Vero cell assay as a control for the neutralisation of cytotoxic effects.

Scombrotoxic fish poisoning in Britain: features of over 250 suspected incidents from 1976 to 1986.

Between 1976 and 1986, 258 incidents of suspected scombrotoxic fish poisoning were reported in Britain. Histamine analysis was carried out on 240 fish samples from these incidents, and 101 were found to contain greater than 5 mg histamine/100 g fish. The symptoms most consistently reported were rash, diarrhoea, flushing and headache. In recent years there has been a decrease in the number of confirmed scombrotoxic outbreaks and a trend towards more sporadic incidents. Of fish samples with greater than 20 mg histamine/100 g, 94% were from incidents in which scombrotoxic symptoms were characteristic, but where fish had 5-20 mg/100 g only 38% of incidents were clinically distinctive. Guidelines are presented based on the interpretation of quantitative histamine analysis of fish samples from scombrotoxic poisoning incidents.

Assessment of an enzyme immunoassay for the detection of salmonellas in foods and animal feeding stuffs.

The Salmonella Bio-EnzaBead Screening Kit, in its modified form with both the MOPC 467 and the 6H4 antibodies, was used for the detection of salmonellas in naturally contaminated foods and animal feeding stuffs in parallel with a traditional cultural procedure. Initial results showed an 82% agreement between the enzyme immunoassay (EIA) and cultural methods when using the criterion recommended by the manufacturer as a cut-off for all types of foods. By adjusting the cut-off for each type of food, the number of EIA positive, culture negative samples was reduced although the number of EIA negative, culture positive samples increased. The EIA may be more sensitive than the cultural methods as in many cases the EIA positive, culture negative results could be real positives which were not detected by the cultural methods. The screening kit provides a simple and convenient method for the detection of salmonella in foods and feeds and a presumptive positive result can be reported within 48 h. The advantages and disadvantages of the method are discussed.

Alpha-mannosidase activity in synovial fluid.

A microassay technique for the evaluation of synovial fluid alpha-mannosidase activity is presented with the evaluation of 184 synovial fluid samples from a variety of arthropathic conditions. The results indicate a close correlation of enzyme activity with synovial fluid cell count and the degree of inflammation associated with the various types of joint disease. It is suggested that the evaluation of this and other lysosomal glycosidases offer a quantitative method for the evaluation of inflammatory joint disease.

Development and application of an enzyme linked immunosorbent assay for Clostridium perfringens type A enterotoxin.

An enzyme linked immunosorbent assay (ELISA) has been developed to quantitate faecal Clostridium perfringens enterotoxin in the investigation of C perfringens food poisoning. The sandwich ELISA could be carried out in 24 h and was sensitive enough to detect as little as 5 ng/g of enterotoxin in faeces. Specificity of the assay was shown by comparing results with those obtained from other standard toxin assays, such as double gel diffusion and counterimmunoelectrophoresis, and by the assay of faecal material from control groups. By means of the ELISA method, 515 faecal samples from 50 separate outbreaks of C perfringens food poisoning were examined, together with 21 food samples from 12 of the outbreaks. A clear distinction was noted between faecal samples collected on the first two days of an outbreak, where 77% were enterotoxin positive, and those specimens collected later than the second day, when only 33% had detectable enterotoxin. The ELISA is recommended as a valuable tool in the investigation of C perfringens foodborne illness.

SS-A (Ro) antibody in random mother-infant pairs.

In a study of the occurrence of detectable antibodies to SS-A and SS-B in 300 randomly selected mother-infant pairs, three (1%) mother-infant pairs were positive for precipitating antibodies to SS-A. No matched pairs were positive for SS-B. Review of the clinical history of the mother-infant pairs with SS-A antibodies failed to reveal evidence of connective tissue disease or the neonatal lupus syndrome. Follow up of two of the three SS-A positive mother-infant pairs two months after delivery also showed no evidence of disease. While the SS-A antibody may be closely associated with the development of the neonatal lupus syndrome, our study does not support the proposed aetiological nature of the antibody. Random maternal screening for possible SS-A antibody positivity and potential neonatal lupus syndrome does not appear to be warranted.

Pernicious anaemia, intragastric bacterial overgrowth, and possible consequences.

Intragastric bacterial colonization is well known in pernicious anaemia (PA), but its consequences have rarely been investigated. We have studied the clinical history, blood samples, and endoscopic biopsies from the stomach and duodenum of 80 patients with PA. In a random subgroup of 22 patients gastric juice was collected for aerobic culture and for estimation of nitrate, nitrate-reducing bacteria, nitrite, and N-nitrosamines; duodenal juice was studied in parallel in eight of these subjects. Gastric and duodenal juice had high bacterial counts; faecal organisms were found in 14 patients. The mean count of nitrate-reducing bacteria was significantly higher than in a control group of patients with peptic ulcer disease (p less than 0.001), as was the nitrite concentration (p less than 0.001). Thirty-three of the 80 patients had gastric dysplasias; 1 early gastric carcinoma was also found. Duodenitis was present in 39 out of 80 cases, in 6 associated with partial villous atrophy. A history of malabsorption and/or chronic intermittent diarrhoea was obtained significantly more often from patients with duodenitis. Four patients developed acute gastroenteritis shortly before or during the time of the study, two having a salmonella infection. Bacterial overgrowth in PA may be facilitated by altered immunological conditions, since low serum levels of IgA and IgG were found in this patient group.

Enterotoxigenic Clostridium perfringens: a possible cause of antibiotic-associated diarrhoea.

Free Clostridium perfringens enterotoxin was detected in the stools of 11 patients with diarrhoea. All had high faecal counts of enterotoxigenic strains of C perfringens, mostly of serotypes not commonly associated with food poisoning. 10 of these 11 patients had severe or prolonged diarrhoea which had developed after antibiotic treatment. Enterotoxigenic C perfringens appears to be one of the causes of antibiotic-associated diarrhoea.

Bacterial colonization and nitrite concentration in the achlorhydric stomachs of patients with primary hypogammaglobulinaemia or classical pernicious anaemia.

The degree and type of bacterial colonization was similar in achlorhydric patients with either severe primary hypogammaglobulinaemia or classical pernicious anaemia. This suggests that antibodies do not play a significant part in controlling the gastric flora in achlorhydric patients. The nitrite concentration in gastric juice was higher in the hypogammaglobulinaemia patients, raising the possibility that these patients may have very high levels of nitrite-derived mutagenic compounds in their gastric juice. This may account for the high incidence of gastric cancer in these patients.

Intragastric nitrites, nitrosamines, and bacterial overgrowth during cimetidine treatment.

A six week course of cimetidine (1 g/day) healed peptic ulcers in 20 of 23 patients (14 with duodenal ulcer, nine with gastric ulcer). Reduction of basal acid output by 73% and peak acid output by 36% led to a rise in concentrations of intragastric aerobic bacteria and nitrate-reducing bacteria. While the mean intragastric concentration of nitrate was unchanged by treatment, there were statistically significant rises in nitrite and N-nitrosamine concentrations. The conversion from nitrates to nitrites was closely related to the occurrence of nitrate-reducing bacteria. In three patients the intragastric milieu had returned to normal two months after cimetidine treatment had been discontinued. Mean nitrite and N-nitrosamine concentrations did not return to pre-treatment levels in the group of eight patients who remained on maintenance cimetidine (0.4 g at night-time) for three months after the full dose treatment. This study shows that cimetidine treatment can create an intragastric milieu resembling that of atrophic gastritis. Large scale and long-term studies are necessary to establish whether these findings have any clinical significance.

Effect of cimetidine on the gastric bacterial flora.

Bacteriological studies were carried out on samples of gastric juice obtained from 31 patients with peptic ulcer before and immediately after a month's course of cimetidine. After cimetidine there were major increases in both total bacterial counts and counts of nitrate-reducing organisms and qualitative changes suggesting the establishment of a flora which included large numbers of "faecal" organisms.

Gastric bacteria, nitrate, nitrite and nitrosamines in patients with pernicious anaemia and in patients treated with cimetidine.

Patients with pernicious anaemia have a high risk of gastric cancer and we are investigating the role of N-nitroso compounds in their gastric carcinogenesis. We have carried out bacteriological studies and analyses of nitrite and N-nitroso compounds. Patients with duodenal or gastric ulcer are often treated with H2-receptor inhibitors such as cimetidine. This treatment induces hypochlorhydria similar to that in pernicious anaemia patients and it has been suggested that this might put these patients at risk of developing gastric cancer later. Analyses similar to those for p.a. patients have therefore been carried out in patients treated with cimetidine. We have set up an animal model of achlorhydria, using piglets, that will allow us to test in vivo the efficacy of compounds known to inhibit N-nitrosation in vitro. This is clearly necessary, in view of the growing size of the population now thought to be at risk.

Cyclophosphamide therapy for rheumatoid arthritis.

Cyclophosphamide in high doses was given for six months to 19 patients with rheumatoid arthritis. A second group of patients with rheumatoid arthritis whose conditions were stable on low-dose prednisone received in addition either cyclophosphamide or placebo for six months. Measurements of joint function and joint inflammation were used to estimate disease activity. Joint inflammation progressively decreased and joint function improved in the high-dose group. The low-dose cyclophosphamide-plus-prednisone group had a similar response that was different from the prednisone-plus-placebo group. Cyclophosphamide toxicity was common in the high-dose group and minimal in the low-dose-plus-prednisone group. Cyclophosphamide therapy improved the arthritis of these patients. The results were almost as good in the low-dose-plus-prednisone group, and the toxicity was much less.