Disulfiram use in patients with abnormal liver function test results.

BACKGROUND: Concern about the precipitation of severe hepatitis by disulfiram often causes clinicians to avoid using this effective treatment in patients who have elevated baseline transaminase levels, even though no empirical evidence has so far shown severe hepatotoxicity to be related to such laboratory abnormalities. This study examines the effects of disulfiram in alcohol-dependent patients with elevated liver function test results and/or serologic evidence of hepatitis C virus (HCV) infection. METHOD: Hepatitis serologies and baseline transaminase levels were obtained for 57 male alcoholics starting treatment with disulfiram. Sequential liver function test results were obtained for up to 12 weeks while subjects took disulfiram. RESULTS: Although subjects with elevated baseline transaminase levels and serologic evidence of HCV infection were the most likely to evidence marked elevations in transaminase levels while taking disulfiram, most subjects took disulfiram without other adverse consequences. In only 1 subject did elevations appear directly related to disulfiram. CONCLUSION: Monitoring of liver function test results is warranted for patients taking disulfiram and permits most patients with moderately elevated transaminase levels to take it safely.

Comparison of three commercial PSA assays: results of restandardization of the Ciba Corning method.

BACKGROUND: Consistency in prostate-specific antigen (PSA) quantitation by different PSA test manufacturers would minimize potential clinical confusion. The Ciba Corning ACS PSA2 calibration has been adjusted for alignment with a proposed international standard and clinical concordance with the Hybritech Tandem R assay. Herein we evaluate the clinical effectiveness of this recalibrated PSA test by comparing it with the IMx (Abbott Laboratories) and Tandem R (Hybritech) assays. METHODS: Archival serum was used that had been stored at -70 degrees C from men who underwent ultrasound-guided prostate needle biopsy. Assays were run according to each manufacturer's specifications in singlicate on a single thaw. RESULTS: The study included sera of 191 patients; 44 of the patients had carcinoma. There were 151 men with PSA (Tandem R) in the range of 0-10.0 ng/ml, 28 of whom had cancer. The correlation coefficients for Tandem R versus ACS, Tandem R versus IMx, and ACS versus IMx were 0.958, 0.955, 0.979 for benign patients and 0.960, 0.954, and 0.985 for those with cancer, respectively. The corresponding slopes were 1.029, 0.855, and 0.824 for men without and 1.044, 0.830, and 0.790, respectively, for those with malignancy. CONCLUSIONS: These data demonstrate substantial equivalence of the restandardized ACS assay and of the Hybritech method. Significant bias exists between these methods and the IMx assay with lower results being identified with the latter. These findings have significant implication, particularly in screening when results of an IMx assay are compared to other assays.

Analysis of urinary benzodiazepines using solid-phase extraction and gas chromatography-mass spectrometry.

A solid-phase extraction and GC-MS confirmation method was developed for certain urinary diazolo- and triazolobenzodiazepines, including the metabolites of lorazepam, clonazepam, alprazolam, and triazolam. The latter two do not form benzophenones, and the others are not readily confirmed by conventional thin-layer chromatography or GC-MS techniques. Samples were hydrolyzed with glucuronidase at 37 degrees C, adjusted to pH 4.5, extracted with Bond Elut Certify columns, dried, and derivatized using BSTFA with 1% TMCS. Sample preparation time averaged 4 hours. A GC-MS selected-ion-monitoring acquisition method targeting retention time, molecular ion abundances, and qualifier ion ratios was used to determine positive results. The recovery of 7-NH2-clonazepam was 95%, and recoveries of alpha-hydroxyalprazolam, alpha-hydroxytriazolam, and lorazepam were greater than 66%. Linearity was demonstrated from 0.1 to 1.0 microgram/mL for each drug. Within-run CVs were less than 11%, and between-run CVs were less than 16%. Using this technique, we have been able to confirm suspected cases of abuse that had not been confirmed by previous techniques.

Semiquantitation of cannabinoid immunoassays? A reexamination of the EMIT 20-ng/mL assay.

Concerns regarding the ability to semiquantitate drugs-of-abuse urine immunoassay results, particularly THC-COOH, prompted us to reexamine EMIT results. The Syva EMIT d.a.u. cannabinoid 20-ng/mL assay was performed on the Cobas Bio centrifugal analyzer, and positive samples were confirmed by Toxi-Lab or GC/MS. Of 39 specimens tested, 17 were confirmed positive. However, four specimens were not accurately semiquantitated by EMIT. These four had very high levels (greater than 100 ng/mL) of the primary metabolite but yielded EMIT results between the low and medium calibrator (20-75 ng/mL). Linearly studies confirmed that the absorbance changes with the EMIT immunoassay plateau near the medium calibrator. In addition, we obtained false positive EMIT results due to sample carryover when samples containing 500 ng/mL (or greater) of THC-COOH preceded a negative specimen. Significant carryover was not observed when concentrations up to 1,300 ng/mL were used with the 100-ng/mL immunoassay, and this effect may be explained by the higher cutoff used. Because EMIT cannabinoid results on the Cobas Bio analyzer can be affected by carryover and a hook effect, they should not be reported even semiquantitatively.

A nationwide survey of urinalysis practices of methadone maintenance clinics. Utilization of laboratory services.

The urinalysis practices of 324 methadone maintenance clinics were surveyed by using a brief self-report questionnaire. Results indicated that there was a wide variability in collection practices and utilization of laboratory techniques. The most frequent collection schedules were monthly (40%) and weekly (32%). The most frequent laboratory techniques used for initial screening were thin-layer chromatography and enzyme immunoassay. Confirmatory testing by a second technique was used by 69% of the laboratories. The most popular confirmatory techniques used were thin-layer chromatography, enzyme immunoassay, and gas chromatography-mass spectrometry. The geographic differences in urinalysis practices also were noted. The implications for drug abuse treatment were determined.

Four fluorescence polarization immunoassays for therapeutic drug monitoring evaluated.

We evaluated four fluorescence polarization immunoassays--those for phenytoin, procainamide, N-acetylprocainamide (NAPA), and quinidine--from Roche Diagnostic Systems, done in the Cobas Bio FP centrifugal analyzer. The assays for phenytoin, NAPA, and quinidine demonstrated a linear response over the expected ranges of concentrations, and analytical recovery of test drug added to drug-free sera was greater than 90%. However, recovery in the procainamide assay was poor (69-82%) for samples containing greater than 8 mg/L, owing to nonlinearity. Results of method-comparison studies of the four assays paralleled the recovery studies, although the quinidine assay demonstrated a bias (1.0 mg/L higher) when compared with EMIT (Syva). The precision of the phenytoin assay was acceptable at all concentrations tested (total CV less than 7.0%). Imprecision of the other assays was significant at certain concentrations: total CV greater than 10.0% at subtherapeutic values for NAPA and quinidine, and greater than 9.0% for low (2.4 mg/L) and moderate concentrations (9.6 mg/L) of procainamide. Interferences were not significant for hemolyzed, icteric, or lipemic specimens or for specimens with added drug metabolites. The calibration curves for all four assays had good stability (greater than 60 days).

Clinical evaluation and use of urine screening for drug abuse.

Urine drug screening is indicated to evaluate patients who show mental status or behavioral changes and to monitor the abstinence of drug abusers. The appropriate timing for collecting urine specimens may vary depending on the suspected drug of abuse and on laboratory factors. Laboratories use a variety of techniques to do urine screens, and these must be understood by clinicians ordering the screens to interpret results correctly. In treating drug-abusing patients, clinicians must apply structured reinforcement in conjunction with urine screen results to aid patients in achieving abstinence.

Differentiation of renal from non-renal hematuria by microscopic examination of erythrocytes in urine.

Recent studies indicate that hematuria of renal parenchymal origin can be differentiated from hematuria of other origin by the presence of dysmorphic urinary erythrocytes (cells exhibiting irregular membranes or small surface blebs). We investigated the utility of this simple screening assay in a routine clinical laboratory. Dysmorphic erythrocytes in urine from 69 patients (18 with renal-parenchymal disease) were quantified on unstained slides by medical technologists using phase-contrast microscopes. Samples stored at 4 degrees C or 23 degrees C for up to 5 h had no significant changes in percentages of dysmorphic erythrocytes (PDE). PDE was also not modified by urea nitrogen concentration, osmolality, or pH over the physiological ranges of these variables. Receiver-operating characteristic (ROC) curves indicated an optimal sensitivity of 88% and specifity of 94% at a decision level of 14% dysmorphic erythrocytes per high-power field. Thus, the presence of fewer than 14% dysmorphic cells is suggestive of extra-renal disease; more than 14% is suggestive of intra-renal disease.

Clinical and laboratory evaluation of a radioimmunoassay kit for measuring the mid-molecule fraction of parathyrin.

We report the results of a laboratory and clinical evaluation of a commercial kit procedure (Immuno Nuclear Corp.) for measuring the mid-region of the parathyrin molecule. The estimated dose at 50% binding averaged 130 pmol/L, and the minimum detectable concentration was 14 pmol/L. The within-assay CV was less than or equal to 6.6%, the between-assay CV less than or equal to 12.5%. Relative analytical recoveries of various parathyrin fragments averaged 93% (intact, amino acid residues 1-84), 100% (midregion, 44-68), less than 0.01% (N-terminal, 1-34), and less than 0.01% (C-terminal, 69-84). Correlation of results obtained for 59 patients' samples with a radioimmunoassay for midregion/C-terminal parathyrin performed by the Mayo Medical Laboratory yielded the equation INC = 1.16 (Mayo)-1.94 pmol/L (r = 0.971). Clinical evaluation indicates that the INC results for parathyrin correlate with the diagnoses of patients at least as well as the results obtained with the Mayo assay; in some cases, the INC procedure better distinguishes hyperfunction from normal parathyroid function. The INC procedure can be easily performed in hospital laboratories, with a 4-h turnaround time for results.

Simplified high performance liquid chromatographic method for the determination of clonazepam and other benzodiazepines in serum.

A convenient and sensitive high performance liquid chromatographic method was developed for determination of clonazepam in serum using a C-18 reverse-phase column, and mobile phase consisting of a 50:35:15 mixture by volume of pH 6.0 phosphate buffer:methanol:acetonitrile. Quantitation was performed at 313 nm with flunitrazepam as the internal standard. Using 1 ml of serum for extraction, the assay is linear for clonazepam concentrations between 10 and 250 ng/ml. The relative recovery averaged 100.3%, and the coefficient of variation for between-day and within-day assays was less than 7%. A simple modification permits analysis of 200 microliter of serum, with little loss of precision and with a detection limit of 20 ng/ml. Only three drugs tested (nitrazepam, methaqualone, and norchlordiazepoxide) interfered with the assay, and none are likely to be used therapeutically with clonazepam. Importantly, carbamazepine and carbamazepine-10,11-epoxide do not interfere under the conditions of the assay. The method is equally suitable for the determination of nitrazepam. By adjusting the mobile phase so that volume ratios of phosphate buffer, methanol, and acetonitrile are 45:35:20, and using nitrazepam as the internal standard, seven other benzodiazepines (demoxepam, oxazepam, chlordiazepoxide, norchlordiazepoxide, temazepam, diazepam, and nordiazepam) can be resolved at 254 nm.

Effect of protein concentration on the determination of digoxin in serum by fluorescence polarization immunoassay.

Determination of digoxin by fluorescence polarization immunoassay (FPIA) with the Abbott "TDx" is significantly influenced by the concentration of total serum protein. Each 10 g/L increase in serum protein results in an 8% decrease in measured digoxin. Studies with [3H]digoxin confirmed that digoxin binds to the protein pellet during the trichloroacetic acid precipitation step before the immunoassay. Serum protein, or equal concentrations of albumin or gamma-globulin, exert an equivalent effect on the apparent digoxin value. Because the total protein concentration of the assay calibrators is low (50 g/L) compared with its reference interval in serum (60-80 g/L), results by FPIA may be expected to be low by an average of 16% (range, 8-24%). Digoxin results by FPIA will be most nearly accurate when the calibrators include a total protein concentration of about 70 g/L. Patients' specimens with abnormally high or low protein content will give falsely high or low results for digoxin.

Characterization of the thrombin-induced contraction of vascular smooth muscle.

Purified human alpha-thrombin induced a sustained contraction of isolated rabbit aorta and dog coronary arteries. These vascular tissues also exhibited a refractoriness towards a second thrombin exposure. The extent of tachyphylaxis exhibited by the aorta correlated with the initial concentration of thrombin and the length of time the tissue was exposed to thrombin. The thrombin-induced contraction in the aorta was not blocked by phospholipase or cyclooxygenase inhibitors, but it was inhibited in the presence of hirudin, heparin, nitroglycerin, and nitroprusside. Nitroglycerin, nitroprusside, and hirudin also inhibited the contraction in the dog coronary artery. Ca++ channel blockers did not inhibit the thrombin-induced contraction in the coronary artery, although a small inhibition was observed in Ca++-free media. In both tissues, equivalent contractile responses were obtained using equimolar quantities of beta-, tetranitromethane-, and alpha-thrombin, even though the latter's coagulant activity was 30-40 times that of the modified thrombins. However, if the catalytic activity of thrombin was inhibited by modification with Tos-Lys-CH2Cl, hirudin, or heparin/antithrombin III, the vasoconstrictor activity was also lost. These studies suggest that alterations of the fibrinogen-binding site do not affect the contractile activity of thrombin. The contraction may be the result of a proteolytic interaction of the active site of the enzyme with vascular smooth muscle.

Generation of a vasoactive substance in human plasma during coagulation. Evidence of thrombin-induced contraction of rabbit aorta and dog coronary artery.

A stimulant of vascular smooth muscle contraction was generated in fresh, citrated human plasma during activation of the clotting system. Plasma, exposed briefly to thromboplastin and Ca++, induced a contraction of isolated rabbit aorta and dog coronary arteries that was slow in development and persisted after washout. The contractile activity was not blocked by phenoxybenzamine, atropine, or angiotensin inhibitor, but was blocked when heparin or hirudin was incubated with the plasma. The contractile stimulant produced in the plasma was short-lived (less than 3 min) and paralleled the appearance of thrombin in plasma. Purified human alpha-thrombin also induced a sustained contraction in these blood vessels that was not inhibited by phenoxybenzamine, atropine, or angiotensin inhibitor, but was blocked by hirudin. Partial relaxation of the thrombin-treated blood vessel was achieved by the addition of heparin. These results suggest that this vasoactive component of thromboplastin-activated human plasma is alpha-thrombin. Because of its potent and persistent effects, thrombin-induced vasospasm may be an important mechanism in the etiology of ischemic heart disease.

Functional fractionation of platelets: aggregation kinetics and glycoprotein labeling of differing platelet population.

Platelet heterogeneity has been studied with a technique called functional fractionation which employs gentle centrifugation to yield subpopulations ("reactive" and "less-reactive" platelets) after exposure to small doses of aggregating agent. Aggregation kinetics of the different platelet populations were investigated by quenched-flow aggregometry. The larger, "reactive" platelets were more sensitive to ADP (Ka = 1.74 microM) than the smaller "less-reactive" platelets (Ka = 4.08 microM). However, their maximal rate of aggregation (Vmax, % of the platelets aggregating per sec) of 23.3 was significantly lower than the "less-reactive" platelets (Vmax = 34.7). The "reactive" platelets had a 2.2 fold higher level of cyclic AMP. Platelet glycoproteins were labeled using the neuraminidase-galactose oxidase--[H3]-NaBH4 technique. When platelets were labeled after reversible aggregation, the "reactive" platelets showed a two-fold decrease in labeling efficiency (versus control platelets). However, examination of whole cells or membrane preparations from reversibly aggregated platelets revealed no significant difference in Coomassie or PAS (Schiff) staining. These results suggest that the large, "reactive" platelets are more sensitive to ADP but are not hyperaggregable in a kinetic sense. Reversible aggregation may cause a re-orientation of membrane glycoproteins that is apparently not characterized by a major loss of glycoprotein material.

Functional fractionation of platelets.

Studies of platelet populations suggest that they are heterogeneous in size, age, and metabolic parameters. In an attempt to correlate these parameters with efficiency of aggregation, a new technique, functional fractionation, was developed. Platelet populations are separated by their differential reactivity to aggregating agents. For example, low doses of ADP (0.1 to 0.7 microM) are added to stirred PRP, after which gentle centrifugation is used to remove aggregates from single unreacted platelets. The loose aggregates can be readily dispersed for comparison of the physical or biochemical properties of the reacted versus unreacted platelets. It was found that reactive platelets were larger (6.5 micrometer3) than unreacted platelets (5.51 micrometer3). No significant difference in density existed between the two populations, and no release of [14C]serotonin from prelabeled platelets occurred during functional fractionation. Scanning and transmission electron microscopy confirmed the size difference and revealed that in both populations platelets were structurally intact with a normal discoid shape and no significant difference in organelle content. Human platelets most reactive to ADP were also enriched in glycogen (3.6-fold), ATP (1.6-fold), and ADP (twofold), compared with less reactive cells. These "reactive" cells took up more 51[Cr] and contained 1.9 times more surface sialic acid. In an in vivo aging experiment, rats were injected with 75[Se]methionine. Shortly after labeling (1 day), the most reactive platelets possessed the highest amount of 75[Se]. These results reveal that functionally active platelets, which are also larger, are more active metabolically than less reactive platelets, possess a higher negative surface charge, and may be a younger population.