Discrimination Between Drug Abuse and Medical Therapy: Case report of a tranylcypromine overdose-related fatality.

Tranylcypromine is an effective antidepressant from the class of monoamine oxidase inhibitors and is structurally related to amphetamine. However, reports differ regarding the potential metabolism of tranylcypromine to amphetamine and methamphetamine within the human body. We report a 25-year-old woman with severe depression who died due to a fatal tranylcypromine overdose in 2016. She had been prescribed tranylcypromine one day previously and had no history of previous suicide attempts or substance abuse. The body was transferred to a forensic medicine department in Tehran, Iran for the autopsy. A urine sample was positive for tranylcypromine, amphetamine and methamphetamine using gas chromatography/mass spectrometry after derivatisation with heptafluorobutyric acid. As amphetamines were present in the urine sample, it was assumed that the tranylcypromine had been converted to amphetamines metabolically. As such, it is possible that the legitimate use of certain prescription drugs may complicate the interpretation of test results for illegal drugs.

Raised endogenous monoamine oxidase inhibitor output in postwithdrawal alcoholics: effects of L-dopa and ethanol.

Urinary output of endogenous monoamine oxidase inhibitor was significantly greater in a group of postwithdrawal alcoholics than in controls. An oral dose of 0.5 g of L-dopa reduced output to control values in the alcoholics, but in the controls themselves output was unaffected. A similar excretion pattern to unextracted samples was observed in ethyl acetate extracts of these urine samples, acidified to pH 1. In a second group of postwithdrawal alcoholics, where the L-dopa effect was confirmed, ethanol administration brought about a small but not significant reduction in inhibitor output.

Daidzein conjugates are more bioavailable than genistein conjugates in rats.

This study compared the bioavailability of conjugates of the soy isoflavones genistein and daidzein in rats. Rats were given a single oral dose of a soy extract that provided 74 micromol genistein and 77 micromol daidzein/kg body wt (as conjugates). Plasma samples were obtained from treated and untreated rats; urine and fecal samples were obtained before and after treatment. Isoflavones, equol (the main end product of bacterial degradation of daidzein), and 4-ethyl phenol (the main end product from genistein) were measured by HPLC. The plasma daidzein concentration was maximal at 2 h (9.5 +/- 0.71 micromol/L) and was almost double that of genistein (P = 0.009). Between 2 and 15 h, the plasma daidzein concentration declined by 32%, but the concentration of genistein changed little. At 15 h, the concentrations of daidzein and genistein were not significantly different. Urinary excretion of daidzein over the 48-h postdose period was 17.4 +/- 1.2% of the dose, but only 11.9 +/- 1.1% of the genistein dose was excreted in urine. Equol excretion was 5.0 +/- 1.5% of the daidzein dose, but 41.9 +/- 5.0% of the genistein dose was excreted as 4-ethyl phenol. Fecal daidzein accounted for 2.3 +/- 0.5% and fecal genistein for 3.4 +/- 0.4% of the respective doses. It is concluded that conjugates of daidzein are more bioavailable than those of genistein, probably because of the greater resistance of the former to degradation by gut bacteria.

Plasma and urinary kinetics of the isoflavones daidzein and genistein after a single soy meal in humans.

The aim of this study was to determine the pharmacokinetics and urinary excretion patterns of the soy isoflavones daidzein and genistein in humans. Six healthy men with a mean age of 37 y and a mean body mass index (in kg/m2) of 24 consumed a soybean flour-based meal on two occasions approximately 6 d apart. Blood samples and total urine were collected at intervals for the measurement of daidzein and genistein with HPLC. Isoflavone concentrations rose slowly and reached maximum values of 3.14 +/- 0.36 micromol/L at 7.42 +/- 0.74 h for daidzein and 4.09 +/- 0.94 micromol/L at 8.42 +/- 0.69 h for genistein. Elimination half-lives were 4.7 +/- 1.1 and 5.7 +/- 1.3 h for daidzein and genistein, respectively. The slow increase in plasma concentrations is consistent with the facilitation of absorption by hydrolysis in the small and large intestines of the glycosidic forms of the isoflavones present in soybean-containing foods to their corresponding aglycones. The rate of urinary excretion of daidzein was greater than that of genistein throughout the postmeal period, with mean recoveries of 62 +/- 6% and 22 +/- 4% (P < 0.001) for daidzein and genistein, respectively. However, the ratio of the areas under the plasma concentration versus time curves for genistein and daidzein was equal to the ratio of the concentrations of the respective isoflavones in the soy meal. It is concluded that the bioavailabilities of daidzein and genistein are similar, not withstanding the difference in urinary excretion.

Urinary excretion of isoflavonoids and the risk of breast cancer.

Isoflavonoids are a group of biologically active phytochemicals that humans are exposed to mainly through soy food intake. Because of the similar chemical structure of these compounds and estradiol, it has been hypothesized that isoflavonoids may be related to the risk of breast cancer. Overnight urine samples from 60 incident breast cancer cases and their individually matched controls were assayed for urinary excretion rates of five major isoflavonoids (daidzein, genistein, glycitein, equol, and O-desmethylangolensin) and total phenols. These subjects were from a large population-based case-control study conducted in Shanghai, and urine samples from breast cancer cases were collected before any cancer therapy to minimize the potential influence of the disease and its sequelae on study results. Urinary excretion of total phenols and all individual isoflavonoids, particularly glycitein, was substantially lower in breast cancer cases than controls. For total isoflavonoids, the mean excretion was 13.95 nmol/mg creatinine (SD, 20.76 nmol/mg creatinine) for cases and 19.52 nmol/mg creatinine (SD, 25.36 nmol/mg creatinine) for controls (P for difference = 0.04). The case-control difference was more evident when median levels of these compounds were compared, with the median excretion of all major isoflavonoids being 50-65% lower in cases than in controls. Individuals in the highest tertile of daidzein, glycitein, and total isoflavonoids had about half the cancer risk of those in the lowest tertile. The adjusted odds ratio for breast cancer was 0.14 (95% confidence interval, 0.02-0.88) for women whose urinary excretion of both phenol and total isoflavonoids was in the upper 50% compared with those in the lower 50%. The results from this study support the hypothesis that a high intake of soy foods may reduce the risk of breast cancer.

Increased urinary tribulin output in generalised anxiety disorder.

Urinary output of an endogenous monoamine oxidase inhibitor and benzodiazepine receptor binding inhibitor (tribulin) was raised in a group of patients with generalised anxiety disorder compared with controls. Tribulin levels remained relatively constant in individual patients over the 6-week period of observation, mean values remaining high even after reduction of anxiety following non-drug behaviour therapy.

Regional and molecular separation of the four bioactivities of 'tribulin'.

The endocoid tribulin has four known bioactivities: monoamine oxidase A and B inhibitory activities (MAO-AI and MAO-BI) and peripheral and central benzodiazepine receptor binding inhibitory activities (PBR-I and CBR-I). Analysis of the four bioactivities in rat tissue reveals regional dissociation. Most notably liver was particularly rich in PBR-I yet contained no detectable MAO-AI. In addition we have succeeded in separating the four activities from human urine. MAO-AI and MAO-BI have greater retention on charcoal than PBR-I and CBR-I when eluted step-wise with aqueous methanol. MAO-AI can be separated from MAO-BI and in addition PBR-I can be separated from CBR-I by step aqueous methanol elution from Amberlite XAD-4. Hence we present two lines of evidence that, contrary to the original view, tribulin is composed of a number of distinct molecular components.

Urinary output of endogenous monoamine oxidase inhibitory activity is related to everyday stress.

The MAO A and B inhibitory components of urinary tribulin were investigated in normal individuals (11 males and 24 females, mean age +/- SD; 27.1 +/- 4.5 years) in relation to everyday stress. Volunteers collected a urine sample at the same time (late evening) on five days over a single week. On each occasion subjects also completed a mood adjective checklist which measured perceived stress levels over the day in question. For each subject all daily measures were aggregated. Mean individual urinary MAO A and B inhibitory activity was found to be positively correlated with stress scores both before (r = 0.38, p < 0.05 and r = 0.37, p < 0.05 respectively, n = 35) and after (r = 0.35, p < 0.05 and r = 0.33, p < 0.05 respectively, n = 35) correction for the effects of urinary volume. These results suggest that in normal healthy individuals high endogenous MAO inhibitory activity in the urine is indicative of a relatively enduring state of everyday stress.

New endogenous benzodiazepine receptor ligand in human urine: identity with endogenous monoamine oxidase inhibitor?

Normal human urine contains both monoamine oxidase-inhibiting and benzodiazepine receptor-binding material. Each was extracted into ethyl acetate at pH 1 and subjected to high performance liquid chromatography: they ran similarly, showing three major peaks. The correlation coefficient between the pattern of MAO inhibition and inhibition of 3H-flunitrazepam binding to benzodiazepine receptors in the second half of the elution process was 0.78 (p less than 0.001): most UV-absorbing material present was eluted earlier in the run. These results are compatible with, although they do not prove, the hypothesis that the endogenous MAO inhibitor, previously shown to be increased in stress, is also an endogenous inhibitor of 3H-flunitrazepam binding to the benzodiazepine receptor. This material is different from other putative endogenous ligands: it migrates more rapidly than the potent but artefactual beta-carboline-3-carboxylic acid ethyl ester previously isolated from human urine; nor can the effect we have identified derive from harmane, inosine, hypoxanthine or nicotinamide which fail to extract into ethyl acetate at pH 1.

Urinary excretion of deprenyl metabolites.

(+)-Deprenyl metabolites in rat's urine, such as nordeprenyl. methamphetamine amphetamine and p-hydroxy. methamphetamine were identified by HPLC-MS. After oral administration of 10 mg of pure (-)- and (+)-deprenyl to human volunteers, their urine was analyzed by gas chromatography. The concentration of methamphetamine was found to be overwhelming in the case of the (-)-isomer, while amphetamine and methamphetamine were excreted in equal amounts when (+)-deprenyl was administered. The metabolic processes of deprenyl resulted in metabolites possessing different lipophilicity, as it has been shown by planar displacement chromatography.

Quantitative liquid chromatography-mass spectrometry determination of isatin in urine using automated on-line extraction.

Here we describe a simple, fast and sensitive liquid chromatography/mass spectrometry method with automated on-line extraction to quantify isatin, an endogenous monoamine oxidase, and atrial natriuretic peptide inhibitor, in urine. After derivatisation of isatin to isatinoxime with hydroxylamine hydrochloride and zinc sulfate precipitation, samples were loaded on the extraction column, washed and, after activation of the column-switching valve, backflushed onto the analytical column. Using electrospray ionisation, [M+H]+ ions could be detected in the selected ion monitoring mode. The assay was linear from 5 to 5000 ng/ml (r2>0.99) and analytical recovery was >80%. Inter-assay precision for the quality control samples was less than 3% and inter-assay accuracy was within +/- 5%.

Urinary MAO inhibitor in psychiatric illness.

Normal human urine contains an endogenous monoamine oxidase inhibitor. We have now investigated its activity in urine samples from psychiatric patients in various diagnostic categories. Significantly higher values were observed in alcoholics recently withdrawn from ethanol, compared with controls. Inhibitory activity was not specifically related to primary affective disorders. Inhibitor output may be positively related to certain symptom clusters rather than to disease entities (i.e. alcohol withdrawal, agitation, and hyperkinesis). Significantly lower inhibitor output was also found in a small group of patients with chronic schizophrenia.

Psychiatric morbidity, platelet monoamine oxidase and tribulin output in headache.

A significantly higher proportion of patients with headache showed scores in the psychopathological range of the General Health Questionnaire (GHQ) compared with controls, with ratings particularly high on the anxiety and depression subscales. Across the whole group, there was a significant negative correlation between platelet monoamine oxidase (MAO) activity and GHQ score overall, and with the anxiety and depression subscales. There was a significant positive correlation between platelet MAO activity and urinary output of the endogenous MAO inhibitor, tribulin. Within the migraine group, there was a significant negative correlation between tribulin output and GHQ score. These findings suggest that the biochemical nature of the anxiety associated with migraine may differ from that in other conditions such as generalized anxiety disorder where high platelet MAO activity and high tribulin output have been reported.

Gas-chromatographic study on the stereoselectivity of deprenyl metabolism.

The metabolism and urinary elimination of both (-)-deprenyl and (+)-deprenyl have been studied. Gas-chromatographic analysis with mass specific detection indicated that the metabolism of (-)-deprenyl results in a large excess of methamphetamine compared to amphetamine, while the metabolism of (+)-deprenyl gave nearly equal amounts of amphetamine and methamphetamine. A novel deprenyl metabolite, phenylacetone, was also identified in our studies.

Determination of enantiomeric metabolites of l-deprenyl, d-methamphetamine, and racemic methamphetamine in urine by capillary electrophoresis: comparison of deprenyl use and methamphetamine use.

The enantiomeric analysis of urine collected from rats administered l-deprenyl, d-methamphetamine (MA), or dl-MA and from healthy male volunteers who ingested l-deprenyl by capillary electrophoresis (CE) using carboxy methylated-beta-cyclodextrin (CMCD) as a chiral selector was investigated to compare the metabolic pattern of l-deprenyl with the metabolism of d- or dl-MA. Urine from illegal drug abusers was also analyzed for the comparison of therapeutic drug (l-deprenyl) use with illicit drug (d-MA) use. MA enantiomers (l-, d-), amphetamine (AM) enantiomers (l-, d-), l-deprenyl, and desmethylselegiline (DMS) enantiomers (l-, d-) were simultaneously separated and detected with clear resolution. L-deprenyl and its metabolites, l-MA, l-AM, and l-DMS, were detected in rat urine sample collected up to 24 h after oral administration of l-deprenyl (10 mg/kg), and the urinary l-AM/l-MA ratio was 2.45 +/- 0.55. This AM/MA ratio was significantly higher than the ratios obtained from rats administered with d-MA (5 mg/kg) and dl-MA (10 mg/kg). The d-AM/d-MA ratio was 0.98 +/- 0.25 for the d-MA treatment, and the d-AM/d-MA and l-AM/l-MA ratios were 0.72 +/- 0.24 and 0.71 +/- 0.21, respectively, for the dl-MA treatment. Analysis of human urine revealed that, unlike in rat urine, the MA content was much greater than the AM content, resulting in the AM/MA ratios being far lower in cases of healthy adult men treated with l-deprenyl (10 mg) and MA abusers. The AM/MA ratio from l-deprenyl users (0.33 +/- 0.03) was significantly higher than the ratio from MA abusers (0.20 +/- 0.12). Results indicate that although metabolic patterns of the drugs in rat and humans may be different, the AM/MA ratio from l-deprenyl use is significantly higher than the ratio from MA use in both rat and human urine. This ratio, however, cannot give conclusive proof of deprenyl or MA use in humans. The simultaneous chiral separation for all the metabolites of l-deprenyl and MA by CE analysis used in this study could provide rapid and simple discrimination between therapeutic drug use and illegal drug abuse.

Detection, quantification, metabolism, and behavioral effects of selegiline in horses.

Selegiline ([R]-[-]N,alpha-dimethyl-N-2- propynylphenethylamine or l-deprenyl), an irreversible inhibitor of monoamine oxidase, is a classic antidyskinetic and antiparkinsonian agent widely used in human medicine both as monotherapy and as an adjunct to levodopa therapy. Selegiline is classified by the Association of Racing Commissioners International (ARCI) as a class 2 agent, and is considered to have high abuse potential in racing horses. A highly sensitive LC/MS/MS quantitative analytical method has been developed for selegiline and its potential metabolites amphetamine and methamphetamine using commercially available deuterated analogs of these compounds as internal standards. After administering 40 mg of selegiline orally to two horses, relatively low (<60 ng/ml) concentrations of parent selegiline, amphetamine, and methamphetamine were recovered in urine samples. However, relatively high urinary concentrations of another selegiline metabolite were found, tentatively identified as N- desmethylselegiline. This metabolite was synthesized and found to be indistinguishable from the new metabolite recovered from horse urine, thereby confirming the chemical identity of the equine metabolite. Additionally, analysis of urine samples from four horses dosed with 50 mg of selegiline confirmed that N-desmethylselegiline is the major urinary metabolite of selegiline in horses. In related behavior studies, p.o. and i.v. administration of 30 mg of selegiline produced no significant changes in either locomotor activities or heart rates.

Quantification of antidepressants using gas chromatography-mass spectrometry.

Antidepressants are of great interest to clinical and forensic toxicologists as they are frequently used in suicidal gestures; they can be the source of drug interactions and some have narrow therapeutic indices making the potential for toxicity more likely. There are five categories of antidepressants based on function and/or structure. These are monoamine oxidase inhibitors (MAOI), cyclic antidepressants including tricyclic and tetracyclic compounds (TCA), selective serotonin reuptake inhibitors (SSRI), serotonin-norepinephrine reuptake inhibitors (SNRI), and atypical compounds. This method is designed to detect the presence of antidepressant drugs in blood/serum, urine, and tissue specimens using gas chromatography/mass spectrometry (GC/MS) following liquid-liquid extraction (LLE) and identified by relative retention times and mass spectra.