Drug screening with EMIT reagents: a quantitative approach to quality control.

We investigated the precision, linearity, accuracy, and stability of quantitative results for five drugs of abuse [amphetamines, benzoylecgonine, opiates, phencyclidine, and the cannabinoid-tetrahydrocannabinol (THC)-9-acid metabolite], analyzed in control specimens by using EMIT d.a.u. reagents (Syva Co.) with a Monarch 2000 analyzer with a nonlinear interpolation curve-fitting algorithm. The within-day and between-days coefficients of variation (CVs) were less than 5% for all drugs except THC-9-acid, which had a CV between 10% and 20%. The drift of control values during a 30-day stability study was less than 10% from target values for three weeks after a single calibration, except for THC-9-acid control values, which were stable for only two to three days. Daily calibration reduced the drift away from target values during the 30-day stability study and produced optimum precision of all drug assays. Mean control values near the National Institute on Drug Abuse cutoff limits were within 10% of their target values.

Thyroid function in a healthy elderly population: implications for clinical evaluation.

Evaluation of thyroid function in elderly people is complex and has generated some controversy about what is normal. This study analyzed thyroid function assays in an identified healthy elderly population of 216 subjects. Thyroxine, free thyroxine, triiodothyronine, T3 uptake, "supersensitive" thyrotropin, and thyroid antibody titers were performed. Histories of treatment for thyroid conditions were present in 13.9% (n = 30) of the population, and test results for an additional 4.3% (n = 8) revealed some hypothyroidism. These subjects were excluded from statistical analysis. Test results revealed significant differences from younger controls as well as skewed distributions for T4, FT4, and TSH. There were no significant correlations with increasing age or gender within the elderly population. 11.8% (n = 21) of the population exhibited elevated TSF levels with normal T4 values, and 23.0% (n = 41) exhibited a titer of one or both thyroid antibodies. Current reference ranges for thyroid tests are broad enough to include the range of values seen in the healthy elderly, but some cautions are discussed.

Failure of single dose methotrexate followed by citrovorum factor in nonmetastatic gestational trophoblastic neoplasia.

With increasing cost of medical care, newer methods of administering chemotherapy on an outpatient basis are being sought to reduce the need for hospitalization and protracted losses of valuable time for patients with nonmetastatic gestational trophoblastic neoplasia (NMGTN). To achieve this end, two NMGTN patients were treated with a single course of one dose of methotrexate (MTX) followed by multidose Citrovorum Factor (CF) without observing the expected response. Failure to respond appeared to be due to the schedule of administration. Although the dose and plasma concentrations of MTX were considered to be adequate for cell kill, fractionation--as established by conventional schedules of MTX administration--appeared necessary for response by exposing the maximum number of trophoblastic cells to inhibitory levels of MTX during the S-phase of the cell cycle.

Depletion of guanine nucleotides with mycophenolic acid suppresses IgE receptor-mediated degranulation in rat basophilic leukemia cells.

In RBL-2H3 rat basophilic leukemia cells, Ag that crosslink IgE-receptor complexes stimulate the turnover of inositol phospholipids, the mobilization of Ca2+ from intra- and extracellular sources, the release of serotonin and other substances from granules and the transformation of the cell surface from a microvillous to a lamellar architecture. This study explores the role of GTP-binding proteins (G proteins) in the control of these biochemical and functional responses. We report that incubating RBL-2H3 cells for 4 h with 10 microM mycophenolic acid (MPA), an inhibitor of de novo GTP synthesis, reduces GTP levels by over 60% and causes an average reduction of 50% in Ag-stimulated serotonin release. This inhibition of secretion is associated with a 50% decrease in the rate of 45Ca2+ influx in MPA-treated cells. In contrast, Ag-stimulated inositol trisphosphate production is only slightly reduced, indicating that the phosphatidylinositol-specific phospholipase C can be activated by Ag in GTP-depleted cells. The membrane responses to IgE receptor cross-linking are unaffected by incubating cells with MPA. Exogenous guanine or guanosine protects the GTP pools in MPA-treated cells and permits normal ion transport and secretory responses to Ag; adenine does not. These results implicate a guanine nucleotide-binding protein in the control of IgE receptor-dependent signal transduction in RBL-2H3 cells. This protein may particularly control the Ca2+ influx pathway that is essential for secretion.

Serum methotrexate and human chorionic gonadotropin concentrations during five-day chemotherapy for low-risk metastatic gestational trophoblastic neoplasia.

A recent report of serum methotrexate (MTX) levels measured during treatment of gestational trophoblastic neoplasia (GTN) led us to determine MTX and human chorionic gonadotropin (beta-hCG) levels in a patient with low-risk metastatic GTN with a pulmonary metastasis. Peak MTX concentrations exceeded 10(-6) mol/l considered by many investigators to be within the therapeutic range against many human tumors. Serum beta-hCG levels did not decline during MTX administration; however, after 5 days of MTX a steep dose-response curve was observed which continued during 5 courses of chemotherapy.

Fatal intoxication by tocainide.

A 26-year-old woman committed suicide by ingestion of a large quantity of tocainide, a recently developed oral antiarrhythmic agent with chemical similarities to lidocaine. Blood and bodily fluid analysis by thin-layer chromatography, high pressure liquid chromatography, and mass spectroscopy confirmed the presence of tocainide, with a serum level of 68 mg/L, nearly 7 times the upper recommended therapeutic level for this drug. Tocainide was also detected at significant levels in vitreous fluid and bile. Although the mechanism of death from tocainide intoxication in animal studies is related to central nervous system toxicity, the presentation of ventricular tachyarrhythmias with coma in this patient suggests that tocainide at high levels may have primary myocardiotoxicity in humans.

Partial characterization of a mitomycin-C resistant T cell.

A cell resistant to concentrations of mitomycin-C associated with the inactivation of many in vitro immune responses is described. This cell is responsive to concanavalin A (Con A), poorly adherent to nylon wool, present in relatively greater numbers in lymph node than other lymphoid tissues, and is relatively radiosensitive. In part, the resistance of this T cell appears to relate to a reduced activity of a microsomal reductase responsible for converting mitomycin-C to its more active form. Discrepancies in reductase activity may represent a way to differentiate among subsets of lymphocytes.

Cisplatin neuropathy. Clinical, electrophysiologic, morphologic, and toxicologic studies.

Ten of 11 patients with ovarian cancer receiving cisplatin developed a distal sensory neuropathy, manifested early by decreased vibratory sensibility in toes and depressed ankle jerks and later by uncomfortable paresthesias. Eleven patients receiving cisplatin, 50 mg/m2 monthly (mean total, 580 mg/m2) were studied prospectively with monthly neurologic examinations and conduction velocity determinations of median, peroneal, and sural nerves. Early signs were decreased vibratory sensibility in toes (mean dose, 417 +/- 132 mg/m2 [SD]) and loss of ankle jerks (mean dose, 455 +/- 86 mg/m2). With continued therapy, four developed paresthesias. Strength was unaffected. Sural nerve responses abruptly disappeared in six of peripheral nerves from four patients showed axonal degeneration and secondary myelin breakdown. Platinum concentrations in three patients were similar in tumor (3.3 micrograms/g), sural nerves (3.5 micrograms/g), and spinal ganglia (3.8 micrograms/g), but lower in brain (0.17 microgram/g). This may explain the cisplatin toxicity of peripheral nerves with relative sparing of the central nervous system.

Phase II and pharmacokinetic study of high-dose methotrexate in the treatment of advanced gynecologic malignancy. A Southwest Oncology Group Trial.

Fifteen patients with advanced or recurrent gynecologic malignancy were treated with high-dose methotrexate (HDMTX) (1-8 g/M2) and citrovorum factor rescue (10-100 mg/M2). One complete response (13%) and two improved responses occurred in eight patients (25%) with squamous cell carcinoma and one of seven patients (14%) with nonsquamous nontrophoblastic carcinoma had stable disease for 7 months. The median duration of survival in the squamous group was 9 months and in the nonsquamous groups 6.5 months. Mean serum MTX concentrations were proportional to the doses administered and typical two compartment plasma disappearance curves were seen. Adverse toxic reactions were not observed at serum MTX levels less than 7.8 X 10(-7) M at 24 hr and 1 X 10(-7) M at 48 hr post-MTX. Hematopoietic toxicity occurred most frequently with leukopenia observed in 19.5% of courses. Hepatic, renal, gastrointestinal, and dermatologic toxicities were observed infrequently. Drug-induced nephrotoxicity occurred in one patient and possibly related leukoencephalopathy occurred in another patient. On the basis of the relatively low response rate observed in this trial and the high expense of HDMTX therapy, the value of such therapy may be limited in advanced nontrophoblastic gynecologic cancer.

A comparison of irradiation and mitomycin as blocking agents in the mixed lymphocyte reaction.

In comparison with administration of mitomycin, lethal irradiation (2,000 rad) of the stimulator cells in a one-way mixed leukocyte culture results in a reduced response due at least in part to the release of inhibitory materials by the irradiated cells. These inhibitory molecules may be partially removed by washing and possess differential reactivity with respect to phytohemagglutinin, concanavalin A, lipopolysaccharide, and pokeweed mitogen.

Trophoblastic cell sensitivity to 8-day chemotherapy in nonmetastatic gestational trophoblastic neoplasia.

Serial radioimmunoassay determinations of serum beta hCG and methotrexate were compared in two patients with nonmetastatic gestational trophoblastic neoplasia (NMGTN) treated with Goldstein's modification of Bagshawe's intermediate-dose methotrexate-citrovorum factor rescue-treatment program. Pretreatment beta hCG levels (mIU/ml) ranged within the outer limits of the 10(3) log level. Following intravenous methotrexate, sharp serum peaks between 10(-6) and 10(-5) M were observed. Plasma disappearance was rapid with a 3 log drop noted within 24 hr to levels incapable of inhibiting DNA synthesis. beta hCG levels manifested a 1 to 1.5 log drop over the 8 days of chemotherapy and complete remission was noted within 5 to 6 weeks of the first dose of methotrexate. No significant clinical or laboratory toxicity was observed. Although cell culture studies show that 100% of cell death can be achieved with serum levels of 10(-5) M in methotrexate-resistant choriocarcinoma, similar data do not exist for previously untreated trophoblastic neoplastic cells. These preliminary observations suggest that serum methotrexate levels are important for establishing sensitivity levels in a heterogeneous population of trophoblastic cells in NMGTN and that the total dose of methotrexate may be safely preselected on the basis of the pretreatment beta hCG.

Effects of irradiation on the interaction of fluorescent probes with lymphocytes.

Low doses of radiation reduce the rate and magnitude of fluorescent probe interaction with spleen cells. The uptake of 8-anilino-naphthalene sulfonic acid (ANS), but not diphenyl-hexatriene (DPH), is reduced in a dose-dependent fashion in cells that receive 25-100 rads. The effects of irradiation are most evident in a "medium" fluorescent subpopulation with no effect observed for a "dim" subpopulation. Splenic lymphocytes enriched for T cells show lower uptake of ANS, compared with a B-cell-enriched population. In addition, the normal decrease in ANS fluorescence polarization that occurs during the initial 10 minutes of probe interaction is attenuated in irradiated cells. These findings are consistent with the notion that low doses of radiation limit the penetration of ANS, but not DPH, into a less restrictive microenvironment within the plasma membranes of T, but not B, cells.

Evaluation of a colorimetric method for determination of glycosylated hemoglobin.

We evaluated a colorimetric assay for glycosylated hemoglobin to determine the effects of several variables --oxalic acid concentration, extraneous glucose, hemoglobin concentration, hydrolysis interval, and 5-hydroxymethylfurfural destruction--and the precision. The interference seen when the blood glucose concentration exceeds 2.0 g/L (11 mmol/L) can be eliminated by washing the erythrocytes with 9 g/L saline. The accuracy of this assay is not influenced by hemoglobin concentrations from 80 to 150 g/L. The background nonspecific color, although substantial, is similar from sample to sample. After a 5-h hydrolysis at 100 degrees C, about 85% of the hexose is released, and the analytical recovery of 5-hydroxymethylfurfural is constant over a wide range of glycosylated hemoglobin concentrations. The 5th to 95th percentile reference interval for a population of 65 nondiabetic individuals was 4.6 to 6.1 mol per 100 mol of total hemoglobin. The range of values for a population of 85 diabetic patients was 6.9 to 20.4 mol per 100 mol.

Acute thallium poisoning: toxicological and morphological studies of the nervous system.

Nine days following ingestion of 5 to 10 gm of thallium nitrate, a young man died with severe cranial and peripheral neuropathy, anuria, and heart failure. Ultrastructural examination of nerves obtained on days 7 and 9 demonstrated axonal degeneration with secondary myelin loss. Axons were swollen and contained distended mitochondria and vacuoles. Thallium levels in more than twenty organs and body fluids ranged from below 1.0 to 178 microgram/gm; concentrations in twenty areas of the nervous system ranged from 29 to 140 microgram/gm. The highest brain levels of thallium were found in gray matter. In the thalamus, 87% of the thallium was present in cell sap. Tissue concentrations of thallium did not parallel those reported for potassium, suggesting that thallium distribution differs from potassium distribution in human beings.

Five-day programmed intraperitoneal insulin delivery in insulin-dependent diabetic man.

This study examined the feasibility of continuous programmed intraperitoneal insulin infusion to maintain glycemic control in insulin-dependent, "C"-peptide-negative diabetic man over a 5-day period. The insulin was delivered via a transcutaneous ip catheter from a portable, programmable insulin delivery pump. All 10 diabetic subjects received ip insulin during the day and night, and plasma glucose, free insulin, and C-peptide concentrations were evaluated at 16 intervals throughout each 24-h period. Standard American Diabetes Association recommended diets were provided, and the insulin dosage was adjusted for both premeal glycemia and the quantity of calories ingested. All subjects maintained normal daily activities including attendance at work or school but slept in the Clinical Research Center at night. Our results demonstrate that continuous programmed ip insulin infusion can maintain glycemic control in insulin-dependent diabetic man for 5 days. Furthermore, normalization of plasma free insulin profiles can be achieved, with sharp peaks of insulin coincident with the rise in glucose at each meal. We conclude that the peritoneum may be an appropriate insulin delivery site for C-peptide-negative diabetic man.

"Normal" insulin secretion: the goal of artificial insulin delivery systems?

Prehepatic insulin production can be determined from analysis of connecting-peptide behavior in the plasma. In the present study, we have determined prehepatic insulin production in six normal men throughout a day that included three typical 750-cal meals. Total insulin secretion for the 24 h was 45.4 U, secreted as 10.6 U with breakfast, 13.4 U with lunch, and 13.8 U with dinner. The remaining 7.6 U was secreted during the 9 h night at a rate of 0.85 U/h. At least 50% of the newly secreted insulin is known to be extracted by the liver during the initial transhepatic passage, so that total peripheral delivery can be estimated as approximately 22 U/day. Consequently, portal vein insulin levels are in excess of those seen in peripheral blood by at least 20 +/- 8 microU/ml in the fasted state, and by as much as 115 +/- 15 microU/ml in the 2-h postabsorptive state. The data suggest that insulinization of the liver, without peripheral hyperinsulinemia, may be a goal of artificial insulin delivery.

Death associated with nitrite ingestion: report of a case.

The tissue concentrations of nitrite and nitrate found at autopsy of a case of intentional ingestion of nitrite salts have been reported. The percentage of methemoglobin and the serum nitrite concentrations are consistent with those reported for acute overdoses. We conclude that noth nitrite and nitrate salts may be identified in tissues from persons ingesting only nitrite salts and that a significant conversion to nitrate may result from oxidation of nitrite during the conversion of heme iron to Fe3+.

Solid-phase enzyme immunoassay for serum ferritin.

We describe a solid-phase enzyme immunoassay for ferritin in human serum, with use of a horseradish peroxidase-labeled antibody and a highly sensitive chromogen, 2,2'-azino-di(3-ethyl-benzthiazoline-6-sulfonate). The assay requires only 10 microL of serum per assay, relatively less time and labor than other assys for ferritin, and as little as 10 pg of ferritin can be measured. We believe this assay offers a reliable alternative to radioimmunoassay for serum ferritin.