Vancomycin serum concentrations in patients with renal dysfunction: a comparison of fluorescence polarization immunoassay and the enzyme-multiplied immunoassay technique.

A study was conducted to determine whether assay-specific quantitative differences exist in the determination of vancomycin serum concentrations obtained from patients with renal dysfunction. Vancomycin serum concentrations were obtained during the first week of therapy for each of three time intervals: 48-96 h, 96-144 h, and 144-192 h after administration of the first dose of vancomycin. Vancomycin serum concentrations were measured using the enzyme-multiplied immunoassay technique (EMIT) and fluorescence polarization immunoassay (FPIA). Twenty patients with an estimated creatinine clearance < 40 ml/min who were receiving intravenous vancomycin were evaluated. Hemodialysis was required in 16 of 20 patients. Fifty samples were included in the data analysis. The mean (+/-SD) serum concentrations obtained with EMIT and FPIA were 10.9 mg/L (+/-5.3) and 12.6 mg/L (+/-5.7), respectively (p = 0.13), and were not statistically different. A linear relationship was observed between EMIT and FPIA (EMIT = 0.89 x FPIA - 0.24; r2 = 0.93). No statistically significant differences were observed in the calculated pharmacokinetic parameters between methods. FPIA and EMIT are comparable methods in determining vancomycin serum concentrations within the first week of vancomycin therapy in patients with moderate to severe renal dysfunction.

Gene therapy for hemophilia B: host immunosuppression prolongs the therapeutic effect of adenovirus-mediated factor IX expression.

Hemophilia B is caused by a deficiency of blood clotting factor IX (FIX). Previous studies have shown that the delivery of a recombinant adenoviral vector expressing canine FIX (cFIX) resulted in a complete correction of hemophilia B in FIX-deficient dogs, but that cFIX expression decreased to only about 1-2% of normal levels 3 weeks after treatment. In the present study, therapeutic levels of cFIX expression capable of producing a partial correction of hemophilia B were maintained for at least 6 months after the coadministration of the cFIX-expressing adenovirus and the immunosuppressive agent cyclosporin A (CsA). These findings support a recent report (Yang et al., 1994) that host T-cell-mediated immunity against virally transduced cells is a major contributing factor to the transient nature of adenovirus-mediated gene expression in immunocompetent animals. Although a second administration of the cFIX-expressing adenovirus 6 months after the first infusion had only a minimal effect on plasma FIX levels in a dog that had been continuously treated with CsA, the prolonged expression of the transgene indicates that immunosuppression may be applicable in attaining long-term treatment of clinically relevant disorders.

Transient expression mediated by the Trypanosoma cruzi rRNA promoter.

Plasmid constructs containing a putative Trypanosoma cruzi rRNA promoter and transcription start point upstream from the bacterial chloramphenicol acetyltransferase (CAT) reporter gene were transfected into cultured T. cruzi epimastigotes to verify the presence of a promoter activity. Constructs bearing the putative promoter and a 3' trans-splicing acceptor site in the proper orientation yielded approx. two orders of magnitude greater CAT expression than that previously observed with the T. cruzi spliced leader (SL) gene promoter. In contrast, similar constructs lacking the known 3' splice site yielded reduced but readily measurable expression suggesting that sequences near the promoter may function as cryptic 3' splice sites. A repeated sequence upstream from the putative basal rRNA promoter in a position analogous to rRNA gene enhancer elements in other eukaryotes did not enhance expression from the T. cruzi rRNA promoter. Finally, these constructs were functional in some but not all T. cruzi isolates, and were inactive in other kinetoplastid species, suggesting that the T. cruzi rRNA promoter may have a limited host range.

Determination of N-desmethylmethsuximide serum concentrations using enzyme-multiplied and fluorescence polarization immunoassays.

N-Desmethylmethsuximide (NDM), the active metabolite of the antiepileptic agent methsuximide, has been analyzed by gas-liquid chromatography and high-performance liquid chromatography (HPLC) in the past. This study compares methods using two commercially available immunoassays for ethosuximide, the enzyme multiplied immunoassay technique (EMIT) and fluorescence polarization immunoassay (FPIA), with an HPLC method for the measurement of NDM concentrations in serum. Within-day precision studies, utilizing low therapeutic (15.0 mg/L) and toxic (45.0 mg/L) NDM concentrations (n = 20), resulted in coefficients of variation (CVs) of 4.6 and 4.2%, respectively, for EMIT and 5.4 and 3.2%, respectively, for FPIA. Day-to-day precision studies (n = 10) resulted in CVs of 7.6 and 5.5%, respectively, for EMIT and 3.5 and 2.4%, respectively, for FPIA. No interference was observed from toxic concentrations of acetaminophen, caffeine, carbamazepine, methsuximide, phenobarbital, phensuximide, phenytoin, primidone, salicylate, and valproic acid in the EMIT and FPIA procedures. There was good linear correlation between EMIT and HPLC NDM determinations of 50 patient samples (r = 0.970; y = 0.96 x + 0.03), and a similar correlation between FPIA and HPLC NDM determinations in 48 patient samples (r = 0.975; y = 0.91 x + 1.24). Using ethosuximide reagents, both EMIT and FPIA systems can be adapted to reliably measure NDM serum concentrations.

Measurement of methsuximide and N-desmethylmethsuximide using solid-phase extraction and wide-bore capillary gas chromatography.

A method for the determination of the anti-epileptic drug methsuximide (MSM) and its active metabolite N-desmethylmethsuximide (NDM) is presented. 5-Methyl-5-phenylhydantoin is used as the internal standard. A simple solid-phase extraction procedure utilizing disposable reversed-phase C18 columns is described. Samples are analyzed by gas chromatography with flame ionization detection using a wide-bore capillary column with a permanently bonded, non-polar stationary phase. The MSM assay possesses linearity to 6.0 micrograms/mL, sensitivity to 0.5 microgram/mL, recovery ranging from 93 to 110%, and precision reflected by a SD of +/- 0.37 microgram/mL. The NDM assay displays linearity up to 80.0 micrograms/mL, sensitivity to 5.0 micrograms/mL, recovery of 90 to 100%, and precision reflected by a SD +/- 0.90 microgram/dL. Lack interference is documented for 6 commonly prescribed anti-epileptic drugs and 4 drugs with similar retention times on this stationary phase; only guaifenesin was found to potentially interfere with the determination of methsuximide. We conclude that the method reported here is ideally suited for monitoring therapeutic and toxic levels of this anti-epileptic drug.

Measurement of trazodone using solid-phase extraction and wide-bore capillary gas chromatography with nitrogen-selective detection.

A method for the determination of the antidepressant drug trazodone is presented. 8-Hydroxyloxapine is used as the internal standard. A simple solid-phase extraction procedure utilizing disposable reversed-phase C18 columns is described. Samples are analyzed by gas chromatography with nitrogen-selective detection using a wide-bore capillary column with a permanently bonded, nonpolar stationary phase. The assay possesses linearity to 3.0 micrograms/mL, sensitivity to at least 0.25 microgram/mL, recovery averaging 96%, and between-run precision reflected by a CV of 5.6%. We conclude that the method reported here is ideally suited for monitoring therapeutic and toxic levels of trazodone.

Measurement of antidepressants using solid-phase extraction and wide-bore capillary gas chromatography with nitrogen-selective detection.

A comprehensive method is presented for the determination of nine antidepressant drugs and metabolites in serum: (1) amitriptyline, (2) nortriptyline, (3) imipramine, (4) desipramine, (5) maprotiline, (6) doxepin, (7) desmethyldoxepin, (8) protriptyline, and (9) trimipramine. Chlorimipramine is used as the internal standard. A simple solid-phase extraction procedure utilizing disposable reversed-phase C18 columns is described. Samples are analyzed by gas chromatography with nitrogen-selective detection using a wide-bore capillary column with a permanently bonded, non-polar stationary phase. The assay possesses linearity to 800 ng/mL for maprotiline and 500 ng/mL for the other antidepressants, sensitivity to at least 25 ng/mL, recovery ranging from 96 to 107%, and between-run precision reflected by CVs of 4.4 to 8.1%. Lack of interference is documented for over 27 commonly prescribed drugs. We conclude that the method reported here is ideally suited for monitoring therapeutic and toxic levels of antidepressant drugs.

Community hospital to trauma center.

The Committee on Trauma of the American College of Surgeons published a report in 1976 charging hospitals to provide care for seriously injured patients. Implementing an effective emergency care/trauma system in a not-for-profit community hospital was a task that demanded leadership, substantial time, and commitment. The building process could not have begun without a strong commitment from the hospital's board, administration, medical staff, and nursing service. Initially, the operating rooms, radiology, intensive care units, and emergency departments were renovated or replaced. General surgeons and surgical subspecialists committed to trauma care were recruited. Emergency department (ED) physicians were upgraded and resident rotations in the ED were begun. A ground and helicopter transport system was initiated; dispatch was centered in the ED. Educational programs in prehospital critical care and stabilization for flight nurses and EMT's were developed. The operating rooms began 24-hour service with in-house anesthesia coverage. Radiology provided 24-hour coverage of specialty services. Physicians began in-house coverage of the critical care units. The department of surgery developed a trauma section to encompass all the general surgeons and subspecialty physicians in emergency care. Monthly in-service programs were begun for the intensive care unit (ICU) and ED nurses. In each of the past 3 years, a 2-day trauma update program has been provided to the regional Emergency Medical Services (EMS) and medical community. The dedication and commitment of many people during the past 5 years has resulted in a sound system of emergency/trauma care in a community hospital.

Chromium deficiency after long-term total parenteral nutrition.

A 63-year-old female developed unexplained hyperglycemia and glycosuria during administration of a total parenteral nutrition regimen on which she had been stable for several months. Because the patient had no history of diabetes or evidence of an infection, chromium deficiency was considered. Plasma chromium level was 0.1 microgram/dl (laboratory reference interval: 1.8-3.8 micrograms/dl). Fourteen days of supplemental intravenous chromium chloride (200 micrograms/day) allowed complete withdrawal of exogenous insulin with no further hyperglycemia or glycosuria. Correction of unexplained glucose intolerance following vigorous chromium supplementation indicates that the patient had chromium deficiency. Subsequent plasma chromium levels remained unchanged, possibly reflecting the sensitivity limits of the assay that was used, the uncertainty that exists regarding appropriate reference intervals for this element, and the fact that plasma levels do not always correlate with total body stores. The patient did not manifest peripheral neuropathy, which was present in one of the two previously reported cases, nor encephalopathy, which was reported in the other. We conclude that this patient developed chromium deficiency as a result of inadequate administration of chromium in the parenteral formula (6 micrograms/day) plus excessive enteric losses, and she presented with glucose intolerance as the only clinical manifestation of the deficiency. Caution should be exercised when interpreting plasma chromium in patients with suspected deficiency.

A single-dose pharmacokinetic study of the antisickling agent cetiedil.

Cetiedil citrate is an antisickling agent shown to be effective in reducing the severity and duration of acute sickle cell crisis. With the use of a sensitive GC/MS assay, the pharmacokinetic profile of cetiedil was studied in normal men and in men with sickle cell anemia who were not in crisis at the time of study. A peak cetiedil concentration of 70 to 200 ng/ml was found immediately after a 30-minute drug infusion. The plasma level then gradually declined to approximately 10 ng/ml during a 3-hour distributive phase. Computer analysis of the data was most consistent with a three-compartment model. No pharmacokinetic differences were found between the normal men and the subjects with sickle cell. Because the cetiedil plasma levels achieved during this in vivo study are well below concentrations that exhibit antisickling activity in vitro, additional clinical studies will be necessary before an optimal dosing regimen can be established.

DNA relationships among some tox-bearing corynebacteriophages.

The DNA genomes of a number of tox-bearing, temperate corynebacteriophages isolated from strains of Corynebacterium diphtheriae and Corynebacterium ulcerans were compared. With one exception, these phages displayed similarities in their restriction enzyme digest profiles and extensive homology with prototypic beta converting phage. The exception, phage delta, had a unique restriction profile and exhibited homology with beta over a limited portion of its genome. DNAs of phages from each host contained cohesive ends and integrated as prophage by a mechanism analogous to that employed by coliphage lambda. It is proposed that these tox-bearing phages belong to a common family, the beta family. The role of the beta family in the movement of the tox gene between strains of C. diphtheriae and C. ulcerans is discussed.

Inhibition of theophylline clearance by cimetidine but not ranitidine.

This study was designed to compare the effects of equivalent therapeutic doses of two H2 antagonists, cimetidine and ranitidine, on theophylline pharmacokinetics and to determine whether the previously described cimetidine-theophylline interaction is dose dependent. Twelve healthy adult men were given a 6-mg/kg intravenous aminophylline dose on four occasions. Subjects were randomly assigned four treatments: no treatment (control); cimetidine, 1,200 mg/day; cimetidine, 2,400 mg/day; and ranitidine, 300 mg/day. Cimetidine, 1,200 mg/day, significantly decreased theophylline clearance by 36% (range, 22% to 49%) and increased the mean elimination half-life from 5.7 hours (control) to 9.2 hours. A significant difference was not found between the two cimetidine dosages, indicating dose independence of the interaction over the dosage range studied. Ranitidine did not significantly alter theophylline pharmacokinetics. Theophylline plasma protein binding was not affected by any treatment. The relative effects of cimetidine and ranitidine on the elimination of cytochrome P-450 metabolized drugs such as theophylline indicate a useful property of ranitidine as compared with cimetidine.

Effect of ethanol on lactic acidosis in experimental hemorrhagic shock.

Many trauma victims who have hemorrhagic shock are also intoxicated. Ethanol could worsen the severity of shock and decrease the amount of blood loss necessary to reach or maintain the shock state, perhaps by increasing lactic acidosis. We examined the effect of ethanol on lactic acidosis in a group of rats that were intoxicated, then put in a state of hemorrhagic shock (MAP = 40 mm Hg). These animals were compared to a control group that were in a similar state of hemorrhagic shock but not intoxicated. The volumes of blood necessary to reach and maintain the predetermined model state of shock for two hours in each group were also measured. The animals were paralyzed and placed on controlled ventilation. The ethanol produced an expected baseline lactic acidosis, and it took significantly less blood volume loss to keep the intoxicated group in shock. However, during shock there was no significant difference in the state of lactic acidosis. These results suggest that acute ethanol intoxication made the animals more sensitive to hemorrhage. This effect was not mediated by an increase in lactic acidosis in our model.

Evaluation of the IFCC-recommended procedure for serum aspartate aminotransferase as modified for use with the centrifugal analyzer.

The IFCC-recommended procedure for aspartate aminotransferase (EC 2.6.1.1) was adapted to the centrifugal analyzer and evaluated during five years. The main hindrance to widespread use of the recommended method is the need for pre-incubation with pyridoxal phosphate. The present method includes a 10-min pre-incubation with pyridoxal phosphate. Extensive evaluation of the method with and without this pre-incubation confirms earlier reports that it eliminates some significant errors, especially for samples from patients with cardiac disease. However, the pre-incubation can be briefer than the recommended 10 min and still provide acceptable results.