Liquid chromatographic determination of atropine in nerve gas antidotes and other dosage forms.

A simple and specific liquid chromatographic method was developed for the determination of atropine in nerve gas antidotes and several other dosage forms. The method is also used simultaneously to quantitate phenol, an antimicrobial agent present in nerve gas antidotes, and to monitor the level of tropic acid, a principal degradation product of atropine. The system uses a Spherisorb CN column and a mobile phase of acetonitrile-0.05M sodium phosphate monobasic (10 + 90), pH 4.0. The detection wavelength is 220 nm. The method was validated by testing for accuracy, linearity, reproducibility and precision. In addition, the proposed method was applied to 8 commercial preparations of atropine, including injectables, ophthalmic solutions, and ointments, and was found to be satisfactory and free from interferences from preservatives, such as benzyl alcohol, methylparaben, benzalkonium chloride and chlorobutanol, that are present in these formulations.

Assessment of the possible role of iron and copper in cisplatin-induced nephrotoxicity in the rat.

Nephrotoxic lesions induced by cisplatin in rats are characterized by acute tubular necrosis in the outer stripe of the medulla. The purpose of this study was to examine the potential role of changes in metal binding proteins, and iron and copper content in urine and renal tissue in cisplatin-induced nephrotoxicity. Cisplatin was administered intravenously to groups of 20 rats at single doses of 0, 1, 2.5, and 5 mg/kg and rats were sacrificed at 1, 2, 3 and 6 days after treatment. Increased serum BUN and creatinine were observed at a dose of 5 mg/kg cisplatin on day 2 through day 6. Increased urinary copper excretion coincided with necrosis and increased BUN and creatinine on day 3 in the high-dose group. Evidence of renal injury was apparent histologically as karyomegaly at all dose levels as early as 48 hours after injection of cisplatin, prior to increases in urinary copper levels. No change in the distribution of metal binding proteins (transferrin, ferritin, ceruloplasmin, and metallothionein) evaluated by immunohistochemical staining, was seen. Based upon these results, it is unlikely that changes in metal excretion play a primary role in cisplatin-induced nephrotoxicity however, changes in nuclear function indicated by karyomegaly may be involved in early renal injury.

Extraction of physostigmine from biologic fluids and analysis by liquid chromatography with electrochemical detection.

A rapid and simple method is described for the extraction of physostigmine (Phy) and its hydrolysis product, eseroline, from plasma, whole blood, and cerebrospinal fluid (CSF) and their subsequent quantitation by high-performance liquid chromatography (HPLC) with dual electrode electrochemical detection. Phy and eseroline were extracted from biologic fluids with cyano-phase columns eluted with 0.1 M citrate buffer, pH 4 containing 20% acetonitrile. Phy recovery from citrate buffer and CSF was nearly 100%. Phy recovery from plasma was 82% when methanol was used to precipitate proteins and 62% when HClO4 was used to precipitate proteins. Phy recovery from whole blood was only 17%. These results are discussed in the context of attempting to measure Phy in fluids of patients receiving this drug in clinical trials for the treatment of Alzheimer's disease.

Studies on new, centrally active and reversible acetylcholinesterase inhibitors.

We have synthesized the tertiary amines of pyridostigmine and neostigmine, 3-pyridinol dimethylcarbamate (norpyridostigmine) and 3-dimethylaminophenol dimethylcarbamate (norneostigmine) respectively, and we have tested their abilities to cross the blood-brain barrier and inhibit mouse brain AChE activity. The in vivo inhibition of AChE activity by norpyridostigmine reaches 72% at 10 minutes which is comparable to that seen with physostigmine (73% at 10 minutes). Inhibition by norneostigmine is less effective (50% at 10 minutes) and approaches that obtained with tetrahydroaminoacridine (57% at 10 minutes). These data show that both norpyridostigmine and norneostigmine cross the blood-brain barrier and that they are effective inhibitors of mouse brain AChE activity. These drugs could be useful in the treatment of memory impairment associated with Alzheimer's disease, and other memory disorders.

Thyrotoxic periodic paralysis.

Periodic flaccid paralysis of skeletal muscle occurring in a thyrotoxic patient, an unusual and dramatic event, is seen predominantly in Oriental men. It is important to remember the clinical association between periodic paralysis and thyrotoxicosis.

In vivo production of ethylene from 2-keto-4-methylthiobutyrate in mice.

The use of 2-keto-4-methylthiobutyric acid (KMB), the alpha-keto analog of methionine, was studied as a potential means of detecting free radical generation in vivo. KMB-dependent ethylene production (presumably from free radical interception), and ethane production from in vivo lipid peroxidation, were monitored simultaneously by measuring the rate of exhalation of these hydrocarbons by mice. Injection of KMB (1 g/kg) into mice resulted in an 8-fold increase in ethylene production above endogenous levels seen in saline-injected controls (1.47 +/- 0.35 vs 0.17 +/- 0.02 nmoles/100 g/hr respectively). Administration of CCl4 (3.0 g/kg) to initiate hepatic lipid peroxidation, 20 min prior to KMB injection, augmented the production of ethylene (2.37 +/- 0.10 nmoles/100 g/hr). Lipid peroxidation following injection of CCl4 was monitored via the increased exhalation of ethane. Pretreating the mice with vitamin E (100 mg/kg daily for 3 days), an inhibitor of lipid peroxidation, did not result in a significant change in ethylene production from KMB by itself or after prior injection of CCl4. However, vitamin E did suppress ethane production initiated by CCl4. Similar results were obtained with mouse liver slices studied in vitro. Metyrapone (150 mg/kg), an inhibitor of hepatic mixed function oxidase activity, also suppressed significantly the CCl4-stimulated production of ethane, but not the CCl4-stimulated production of ethylene from KMB. It appears that ethylene production from KMB does not derive from free radicals generated during in vivo lipid peroxidation since suppression of lipid peroxidation by vitamin E or metyrapone did not suppress ethylene production.

Euthyroid hyperthyroxinemia and thyroxine-binding prealbumin excess in islet cell carcinoma.

This manuscript describes euthyroid hyperthyroxinemia secondary to elevated serum T4-binding prealbumin (TBPA) concentrations in a patient with islet cell carcinoma and reports serum TBPA measurements in other patients with islet cell carcinoma. A 73-yr-old man with a 17-yr history of metastatic islet cell carcinoma was found to have hyperthyroxinemia. His total serum T4 concentration was 18.5 micrograms/dl (normal, 5.5-11.5). Eight years previously, his serum T4 concentration was normal. His free T4 concentration, as determined by equilibrium dialysis, was 1.3 ng/dl (normal, 0.9-2.1). Serum T3, TSH, and T4-binding globulin (TBG) concentrations were normal, as was the TSH response to TRH administration. Polyacrylamide gel electrophoresis of the patient's serum in the presence of tracer amounts of [125I]T4 revealed that, compared to normal sera, [125I]T4 binding to TBPA was increased from 30.0 +/- 6.0% (mean +/- SD) to 52.0%. The distribution of [125I]T3 amont albumin, TBG, and TBPA was normal in this electrophoretic procedure. The concentration of TBPA in the patient's serum was 189 mg/dl. In contrast, the mean serum TBPA concentration in normal men was 40 +/- 4 (mean +/- SD) mg/dl, and that in normal women was 18 +/- 4 mg/dl. Sera from the patient's daughter, his brother, 2 sisters, and 2 male paternal cousins contained normal amounts of TBPA, ranging from 34-47 mg/dl in his male relatives and from 26-30 mg/dl in his female relatives. Serum free T4 index determinations in his relatives also were normal. Serum TBPA concentrations were determined in 14 additional patients with islet cell carcinoma. In 1 of these patients, a man with an insulinoma, serum TBPA was elevated (66 mg/dl). This patient's serum T4 level was 7.3 micrograms/dl, and his free T4 index was 7.0. These data and another study suggest that islet cell carcinoma may rarely produce a TBPA-like protein resulting in an elevated serum TBPA concentration. Markedly elevated serum TBPA is associated with euthyroid hyperthyroxinemia.

Male pseudohermaphrodism with persistent müllerian and wolffian structures complicated by intra-abdominal seminoma.

A fifty-year-old male patient with bilateral cryptorchidism and abdominal mass due to a large seminoma was found to have persistent müllerian duct derivatives: uterus and fallopian tubes. The patient underwent total hysterosalpingectomy and bilateral intra-abdominal orchidectomy of a seminoma and testes. He was given maintenance testosterone and pelvic irradiation.

Metastatic insulinoma with long survival and glucagonoma syndrome.

In 1966, during cholecystectomy for cholecystolithiasis, a 56-year-old man was found to have islet-cell carcinoma metastatic to the liver; his fasting serum glucose level was normal. In 1971, he developed peptic ulcer disease and symptoms of fasting hypoglycemia; inappropriate secretion of insulin was shown. His primary pancreatic tumor was removed in 1973. During the next 9 years, his liver metastases continued to grow and his fasting serum glucose level was maintained at 35 to 116 mg/dL with diazoxide and hydrochlorothiazide therapy. In 1982, he developed clinical evidence of the glucagonoma syndrome, with glucagon levels between 4000 and 11 000 pg/mL. Since then, his fasting serum glucose level has been maintained at 58 to 119 mg/dL without medication. This patient has survived 17 years with a malignant insulinoma and without islet-cell chemotherapy. His course shows that malignant insulinomas may secrete other peptide hormones that can induce various clinical syndromes.

Metastatic glucagonoma. improvement after surgical debulking.

A patient with the classic manifestations of the glucagonoma syndrome and hepatic metastases is presented. Clinical improvement occurred after removal of the primary tumor by distal pancreatectomy and splenectomy. No further antitumor therapy has been needed over a 15 month postoperative follow-up despite metastatic disease.

Potentiometric titrations and oxidation-reduction potentials of manganese and copper-zinc superoxide dismutases.

Bovine erythrocyte superoxide dismutase and two manganese-containing superoxide dismutases have been reduced by the indirect coulometric titration method with methylviologen as the mediator-titrant. On the basis of the titration data the manganese-containing superoxide dismutases contain 1 g-atom of metal per mol of enzyme (dimer). E0' = +0.31 V for the enzyme from Escherichia coli which exhibits a complicated pH dependence above neutral pH. The Bacillus stearothermophilus manganese-containing enzyme has an E0' = +0.26 V and delta Em/pH is 50 mV. Bovine erythrocyte superoxide dismutase exhibits anomalous behavior in the coulometric titration curves, which is indicative of two nonequivalent copper centers in the enzyme. Addition K3Fe(CN)6 or K2IrCl6 to the enzyme solution, prior to coulometric titration, indicates that these anions bind preferentially to one of the copper centers.

Model studies for molybdenum enzymes. Reduction of cytochrome c complexes by mu-oxo-bis[oxodihydroxo(L-cysteinato)molybdate(V)].

The reduction by mu-oxo-bis[oxodihydroxo(L-cysteinato)molybdate(V)] (I) of cytochrome c complexes in which the methionine-80 ligand has been replaced by N3-, CN-, or imidazole has been investigated. The N3- and CN-substituted species are reduced by I in a first-order reaction that appears to proceed via a slow dissociation of N3- or CN-, followed by rapid reduction of native cytochrome c. At low concentrations of I, reduction of the imidazole-cytochrome complex occurs by the same mechanism, while, at higher concentrations of I, direct reduction by I and by a monomeric Mo(V) complex in equilibrium with I appears to occur, although at much lower rates than with native cytochrome c. Potentiometric measurements of E degrees for the cytochrome c complexes with N3- and imidazole indicate the lack of reducibility, or reduction in rate relative to native cytochrome c, is not due to thermodynamic reasons. In the case of the CN- complex, E degrees may be too low for direct reduction by I. The effects on the reduction rates are attributed to a conformational change accompanying the replacement of the methionine-80 ligand, which makes the exposed heme edge less available to attack by outer sphere reductants.

Model studies for molybdenum enzymes. The reduction of cytochrome c by molybdenum(V)-cysteine complexes.

The reduction of ferricytochrome c by two molybdenum(V)-cysteine complexes has been investigated as a model for electron transfer in the molybdenum enzymes sulfite oxidase and nitrate reductase. The reduction by the dioxo-bridged Mo(V)-cysteine complex, di-mu-oxo-bis-[oxo(L-cysteinato)molybdate(V)] (I), is relatively slow and its rate is first order in cyt cIII and zero order in I (k = (1.09 +/- 0.10) times 10(-3) sec minus 1, pH 7.5, 20 degrees). The reduction by the monoxo-bridged complex, mu-oxo-bis[oxodihydroxo(L-cysteinato)molybdate(V)] (II), is extremely rapid and its rate is first order in both reactants (k = (2.6 +/- 0.7) times 10(7) M minus 1 sec minus 1, pH 7.0, 25 degrees). Above pH 7.5, the reduction by II follows biphasic kinetics due to the fast reduction of a low pH form of cyt cIII and a slower reduction of a high pH form (at pH 10.0, 25 degrees, k = 2.9 times 10(6) M minus 1 sec minus 1 for the low pH form and k = 7.2 times 10(4) M minus 1 sec minus 1 for the high pH form). Reaction mechanisms for reductions by both I and II are proposed and the biological implications of the results, both for sulfite oxidase and mechanisms of electron transfer to cytochrome c, are discussed.