This assay of plasma lipoperoxides involves hydrolysis in dilute H3PO4 at 100 degrees C; complexation of malondialdehyde (MDA), a hydrolysis product, with thiobarbituric acid (TBA); methanol precipitation of plasma proteins; fractionation of the protein-free extract on a C18 column; and spectrophotometric quantification of the MDA-TBA adduct at 532 nm. The detection limit was 0.15 mumol of MDA per liter of plasma. Run-to-run precision (CV) averaged 8 to 13%. Analytical recovery of MDA after addition of tetraethoxypropane standards to 21 specimens of human or rat plasma averaged 98% (SD 7%). Lipoperoxide concentrations (as MDA) averaged 0.60 (SD 0.13) mumol/L in plasma specimens from 41 healthy persons and 1.4 (SD 0.3) mumol/L in plasma specimens from 12 control rats. Mean lipoperoxide concentrations were 1.5 to 2.3 times as great in plasma sampled from rats one to three days after subcutaneous administration of NiCl2 at dosages (250 to 750 mumol per kilogram body wt) previously shown to induce lipid peroxidation in lung, liver, and kidney.
Optimum reaction conditions were evaluated for assay of serum ceruloplasmin by measurement of its p-phenylenediamine oxidase activity. The pH optima of p-phenylenediamine oxidase activities of human and rat ceruloplasmins were 5.45 and 5.2, respectively. The p-phenylenediamine oxidase activity of rat ceruloplasmin was markedly inhibited by phosphate (0.1 mol/liter), that of human ceruloplasmin only slightly. These reaction conditions were judged to be optimum for the ceruloplasmin assay: (a) substrate: p-phenylenediamine dihydrochloride, 8.9 mmol/liter; (b) buffer: acetate, 0.1 mol/liter; (c) chloride concentration: 21 mmol/liter; (d) serum dilution: 31-fold; (e) incubation: 37°C for 30 min; (f) spectrophotometry: 530 nm (vs. a "serum blank" containing NaN3). Using these reaction conditions, we devised an improved technique for measuring serum ceruloplasmin. The coefficients of variation of replicate analyses by this technique were 1.25% (for "within-run" precision) and 2.8% (for "day-to-day" precision). The mean concentration of ceruloplasmin in sera from 29 healthy men was 0.315 g/liter (SD = ± 0.049, range = 0.233 to 0.402).
Thirty-two workers in an electroplating plant accidently drank water contaminated with nickel sulfate and chloride (1.63 g Ni/liter). Twenty workers promptly developed symptoms (e.g., nausea, vomiting, abdominal discomfort, diarrhea, giddiness, lassitude, headache, cough, shortness of breath) that typically lasted a few hours but persisted 1-2 days in 7 cases. The Ni doses in workers with symptoms were estimated to range from 0.5 to 2.5 g. In 15 exposed workers who were tested on day 1 postexposure, serum Ni concentrations ranged from 13 to 1,340 micrograms/liter and urine Ni concentrations ranged from 0.15 to 12 mg/g creatinine. Ten subjects (with initial urine Ni concentrations greater than 0.8 mg/g creatinine) were hospitalized and treated for 3 days with intravenous fluids to induce diuresis, resulting in a mean elimination half-time (T1/2) for serum Ni of 27 hours (SD +/- 7 hour), which was significantly shorter (p less than .001) than the mean T1/2 of 60 hours (SD +/- 11 hours) in 11 subjects who did not receive intravenous fluids. Laboratory tests showed transiently elevated levels of blood reticulocytes (N = 7), urine albumin (N = 3), and serum bilirubin (N = 2). All subjects recovered rapidly, without evident sequellae, and returned to work by the eighth day after exposure.
Helicobacter pylori is a human gastrointestinal pathogen involved in gastritis, duodenal ulcers, and gastric neoplasia. This microorganism produces large amounts of a urease which, like all known ureases, has nickel in the active site. We have identified a protein in clinical isolates of H. pylori and an identical protein in the ferret pathogen Helicobacter mustelae that strongly binds Ni 2؉ and Zn 2؉. This protein has been named Hpn to emphasize its origins in H. pylori and its affinity for nickel. The encoding hpn gene, cloned and expressed in Escherichia coli ER1793, has an open reading frame (180 bp) that specifies a protein with a calculated molecular mass of 7,077 Da and with the same amino-terminal sequence as that of wild-type Hpn. The deduced sequence of Hpn consists of 60 amino acids, of which 28 (47%) are histidines. The hpn gene does not map with the urease gene cluster on the H. pylori chromosome. An Hpn-negative, isogenic H. pylori strain, generated by hpn gene deletion and grown on blood agar, had the same urease activity that wild-type cells did. Thus, the role of Hpn in helicobacters is unknown.
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