[Hallucinogen-induced psychological disorders]. / Halluzinogen-induzierte psychische Störungen.

OBJECTIVE: The purpose of this article is to provide an overview of the current research on hallucinogen induced psychiatric disorders. In addition to LSD and psilocybin hallucinogens of biologic origin are increasingly used by adolescents and young adults. METHODS: Relevant literature and related articles were identified by means of a computerized MEDLINE search including the years 1997 - 2007. As keywords "hallucinogen induced psychosis", "hallucinogen induced flashback", "hallucinogen persisting perception disorder (HPPD)" were used. Finally, 64 journal articles and books out of 103 were included in the review. RESULTS: Acute psychotic syndromes in adolescents are rarely due to intoxications with hallucinogenic drugs. However, clinical relevance of flashback phenomena as post-hallucinogenic psychiatric disorder has to be disputed. Because of the high popularity of biogenic hallucinogens and LSD knowledge of intoxications and resulting psychiatric disorders as well as medical complications and therapeutical approaches are clinically important. Especially intoxications with drugs of herbal origin like tropanalcaloids play an important role in emergency situations.

Synthetic approaches and bio-distribution studies of [11C]methyl-phenidate.

PURPOSE: The purpose of this study was a) to present a facilitated method for the preparation and workup of [11C]d-threo-methylphenidate ([11C]d-threo-MP) (a ligand that was shown to bind selectively to the presynaptic dopaminergic transporters) from [11C]methyliodide ([11C]CH3I), b) to demonstrate that the ligand can as well be produced by an alternative labeling method employing [11C]diazomethane as the labeling agent and c) to present biodistribution data for this tracer obtained in rats. METHODS: 11C-labeling with [11C]CH3I was performed using either [d-threo-1-(2-nitrophenylsulfanyl)piperidin-2-yl]phenyl-acetic acid (d-threo-N-NPS-ritalinic acid) under addition of sodium hydroxide as base or the previously prepared sodium salt of d-threo-N-NPS-ritalinic acid. The two approaches were compared with regard to radiochemical yield and purification procedures needed in order to obtain a sufficiently pure tracer solution for human use. For the alternative reaction pathway using [11C]diazo-methane as the labeling agent the reaction was performed with d-threo-N-NPS-ritalinic acid. The biodistribution of [11C]d-threo-MP was determined in rats at 5, 10 and 30 min post injection of the tracer. RESULTS: The application of the sodium salt of d-threo-N-NPS-ritalinic acid as precursor resulted in higher radiochemical yields than the use of the free acid under basic conditions, the yields were 20 +/- 8% and 6 +/- 3%, respectively for the final isolated product (based on [11C]CH3I starting activity). The alternative labeling approach by means of [11C]diazomethane as the labeling agent was demonstrated to give radiochemical yields of 76 +/- 8% (based on [11C]diazomethane starting activity, determined by HPLC analysis of the crude reaction mixture before final work-up) within shorter process times. Based on [11C]methane starting activity both approaches result in similar yields (17% and 15%, respectively) Biodistribution studies in rats revealed a low blood activity (0.09% injected dose/g (% ID/g)) at 5 min post injection (p.i.), as well as a relatively high liver uptake (15.9% ID at 30 min) compared to a lower kidney uptake (3.2% ID at 30 min). Brain uptake was 0.9% ID/g already 5 and 10 min p.i.. CONCLUSIONS: The application of the sodium salt of d-threo-N-NPS-ritalinic acid as precursor for the radiosynthesis of [11C]d-threo-MP reduces the amount of [11C]methanol formed from the reaction of [11C]CH3I with sodium hydroxide, that is added to generate the carboxylic anion of d-threo-N-NPS-ritalinic acid needed for labeling with [11C]CH3I. The purification process could be simplified (omission of one solid phase extraction step), resulting in an easily automated process for the production of the tracer. The preparation of [11C]d-threo-MP by means of [11C]diazomethane as the labeling agent appears to be an interesting alternative to the [11C]CH3I methods because of shorter overall process times and high labeling yields. Biodistribution data show a rapid extraction of the tracer from the blood pool. Tracer excretion seems to take place predominantly via the hepatic pathway since liver uptake at 30 min was considerably higher than kidney uptake. [11C]d-threo-MP exhibits a rapid and sufficiently high brain uptake in rats.

Prepulse inhibition of the startle reflex and its attentional modulation in the human S-ketamine and N,N-dimethyltryptamine (DMT) models of psychosis.

Patients with schizophrenia exhibit diminished prepulse inhibition (PPI) of the acoustic startle reflex and deficits in the attentional modulation of PPI. Pharmacological challenges with hallucinogens are used as models for psychosis in both humans and animals. Remarkably, in contrast to the findings in schizophrenic patients and in animal hallucinogen models of psychosis, previous studies with healthy volunteers demonstrated increased levels of PPI after administration of low to moderate doses of either the antiglutamatergic hallucinogen ketamine or the serotonergic hallucinogen psilocybin. The aim of the present study was to investigate the influence of moderate and high doses of the serotonergic hallucinogen N,N-dimethyltryptamine (DMT) and the N-methyl-D-aspartate antagonist S-ketamine on PPI and its attentional modulation in humans. Fifteen healthy volunteers were included in a double-blind cross-over study with two doses of DMT and S-ketamine. Effects on PPI and its attentional modulation were investigated. Nine subjects completed both experimental days with the two doses of both drugs. S-ketamine increased PPI in both dosages, whereas DMT had no significant effects on PPI. S-ketamine decreased and DMT tended to decrease startle magnitude. There were no significant effects of either drug on the attentional modulation of PPI. In human experimental hallucinogen psychoses, and even with high, clearly psychotogenic doses of DMT or S-ketamine, healthy subjects failed to exhibit the predicted attenuation of PPI. In contrast, PPI was augmented and the startle magnitude was decreased after S-ketamine. These data point to important differences between human hallucinogen models and both animal hallucinogen models of psychosis and naturally occurring schizophrenia.

Psychological effects of (S)-ketamine and N,N-dimethyltryptamine (DMT): a double-blind, cross-over study in healthy volunteers.

INTRODUCTION: Pharmacological challenges with hallucinogens are used as models for psychosis in experimental research. The state induced by glutamate antagonists such as phencyclidine (PCP) is often considered as a more appropriate model of psychosis than the state induced by serotonergic hallucinogens such as lysergic acid diethylamide (LSD), psilocybin and N,N-dimethyltryptamine (DMT). However, so far, the psychological profiles of the two types of hallucinogenic drugs have never been studied directly in an experimental within-subject design. METHODS: Fifteen healthy volunteers were included in a double-blind, cross-over study with two doses of the serotonin 5-HT2A agonist DMT and the glutamate N-methyl-D-aspartate (NMDA) antagonist (S)-ketamine. RESULTS: Data are reported for nine subjects who completed both experimental days with both doses of the two drugs. The intensity of global psychological effects was similar for DMT and (S)-ketamine. However, phenomena resembling positive symptoms of schizophrenia, particularly positive formal thought disorder and inappropriate affect, were stronger after DMT. Phenomena resembling negative symptoms of schizophrenia, attention deficits, body perception disturbances and catatonia-like motor phenomena were stronger after (S)-ketamine. DISCUSSION: The present study suggests that the NMDA antagonist model of psychosis is not overall superior to the serotonin 5-HT2A agonist model. Rather, the two classes of drugs tend to model different aspects or types of schizophrenia. The NMDA antagonist state may be an appropriate model for psychoses with prominent negative and possibly also catatonic features, while the 5-HT2A agonist state may be a better model for psychoses of the paranoid type.

Analysis of plant extracts by NIRS: simultaneous determination of kavapyrones and water in dry extracts of Piper methysticum Forst.

A near-infrared reflection spectroscopy (NIRS) method was developed to determine the total content of kavapyrones, kavain and water in dry extracts of Piper methysticum Forst. (kava kava, Piperaceae). Based on the recorded spectra and the reference data, performed by HPLC and Karl Fischer titration, a chemometrical analysis was calculated using PLS 2 algorithm. In general, good calibration statistics are obtained for the prediction of the different contents presenting high correlation coefficients (r(2) > 0.9913) and low root mean square errors of prediction (RMSEP < 0.094%). Usually the main water bands are "cut out" of the spectra to improve the model, however this is associated with the loss of relevant spectroscopic information. Thus, the entire spectrum including the OH bands is used, as these are not only found in water but also in the kavapyrones. The use of this new strategy succeeds in overcoming the difficulties in NIRS and establishes NIRS as a valid alternative in the routine quality control of plant extracts.

High-performance liquid chromatography-coordination ion spray-mass spectrometry (HPLC-CIS/MS): a new tool for the analysis of non-polar compound classes in plant extracts using the example of Piper methysticum Forst.

An efficient method to characterise complex plant extracts is described using the example of Piper methysticum Forst. (kava; Piperaceae). The method is based on the on-line coupling of high-performance liquid chromatography to a new detection technique: coordination ion spray-mass spectrometry (CIS/MS). CIS/MS is a universal, novel ionisation technique improving selectivity as well as sensitivity. Charged complexes were formed through addition of central complexing ions such as sodium, silver and cobalt. The advantages of CIS/MS detection compared with the electrospray ionisation detection are discussed. The experimental set-up and the application of this simple and robust technique is described to show the its various fields of application in the analysis of plant extracts.

Is the Ecstasy-induced ipsilateral rotation in 6-hydroxydopamine unilaterally lesioned rats dopamine independent?

3,4-Methylenedioxymethamphetamine (MDMA) has recently been hypothesized to be effective against the symptoms of Parkinson's disease. Therefore we tested the effects of MDMA-derivatives in the rotational behavioural model. Male Sprague Dawley rats were lesioned unilaterally with 6-hydroxydopamine at the medial forebrain bundle. MDMA was administered at doses of 2.5, 5.0 and 10.0 mg/kg, its derivatives N-Methyl-1-(1,3-benzodioxol-5-yl)-2-butananamine (MBDB), 3,4-Methylenedioxy-N-ethylamphetamine (MDE) and 3,4-Methylenedioxyamphetamine (MDA) at 5.0 mg/kg respectively. All substances induced ipsilateral rotations, MDA being the most effective. MDMA induced rotations were attenuated by the selective serotonin reuptake inhibitor Citalopram but were only slightly reduced by pre-treatment with the selective serotonin synthesis inhibitor PCPA (para-chlorophenylalanine). The effects of MDMA can therefore not fully be explained by serotonin release or by dopaminergic activity of the drugs.

Effects of the hallucinogen psilocybin on covert orienting of visual attention in humans.

Hallucinogenic drug-induced states are considered as models for acute schizophrenic disorders (experimental psychoses). In a double-blind study with healthy volunteers we investigated the influence of the serotonergic hallucinogen psilocybin, the ecstasy-like drug 3,4-methylenedioxyethylamphetamine (MDE), the stimulant d- methamphetamine and placebo on covert orienting of spatial attention (n = 8 in each group). Reaction times were prolonged after ingestion of psilocybin > MDE, but not after d-methamphetamine. In addition, subjects on psilocybin exhibited particularly slow reaction times in invalid trials at short cue target intervals and failure of response inhibition in valid trials at long cue target intervals for right visual field targets. Despite some methodological limitations, these results are in line with both bilateral impairment of disengagement of attention and a lateralized impairment of inhibition of return (IOR) in productive psychotic states. Additional investigations with larger samples, different hallucinogenic substances (serotonergic agonists vs. NMDA antagonists) and different dose regimens are needed in order to further explore the suggested relationship between visuospatial attentional dysfunction and acute psychotic conditions.

Rapid quantification of constituents in St. John's wort extracts by NIR spectroscopy.

A quantitative near-infrared reflectance spectroscopy (NIRS) method was established for the determination of two major constituents (hyperforin and I3,II8-biapigenin) in St. John's wort extracts. Hyperforin was chosen due to the fact that it is found in a concentration range from 1 to 5%, a common one for NIRS determinations. I3,II8-Biapigenin on the other hand was selected as a constituent with very low concentrations (0.1-0.7%) but an extensive chromophore that allows very precise measurements in the ultraviolet (UV) and thus exact reference values that are vital for proper NIRS calibrations. Reference measurements were performed by reversed-phase high performance liquid chromatography (HPLC), determining the constituents' content in 35 pharmaceutical dry extracts of different origins. The reference method was validated according to the ICH guideline Q2B. Using partial-least squares (PLS) regression a multivariate calibration was done for the two ingredients each (PLS1). Satisfactory calibration statistics were obtained for hyperforin with a root mean square error of calibration (RMSEC) of 0.17 and a root mean square error of prediction (RMSEP) of 0.22 at a concentration range from 1 to 6% in the dry extracts. Due to the very low concentrations of I3,II8-biapigenin the accuracy of prediction is somewhat lower. However, it is possible to obtain very good results and reliable prediction by dividing the concentration range at 0.35%. The study emphasizes the potential of NIRS as a rapid and highly effective alternative method to conventional quantitative analysis of plant extracts.

Quantitative determination of phospholipids in a pharmaceutical drug by scanning and video densitometry.

This paper describes a convenient and practice method for quantitation of surfactant phospholipids (1,2-dipalmitoyl-3-sn-phosphatidyl choline [DPPC] and 1-palmitayl-2-oleyl-3-sn-phosphatidyl glycerol [POPG]) in a recombinant surfactant lyophile (Venticute) by high-performance, thin-layer chromatography (HPTLC) with video densitometry. DPPC and POPG were extracted from Venticute-lyophile using methanol. Separation from the other active ingredients and excipients was accomplished by HPTLC on silica gel F254 plates with a mixture of chloroform, methanol, glacial acetic acid, and water as development solvent. Postchromatographic derivatization by dipping in copper sulphate/phosphoric acid reagent and subsequent heating shows grey-brown bands on a light blue background. These were detected with the video densitometer in the VIS range, and with scanning densitometry at 365 nm. Linear calibration in a working range of 0.7-1.3 microg DPPC and 0.35-0.65 microg POPG was demonstrated by integrating the area under the peaks. Good results were obtained with recovery experiments. When compared to classical slit scanning densitometry, video densitometry represents a fast alternative to quantitate thin-layer chromatograms in surfactant phospholipid analysis.

Enantio-selective cognitive and brain activation effects of N-ethyl-3,4-methylenedioxyamphetamine in humans.

In a randomised double-blind trial the subjective, neuropsychological and brain activation effects of the two enantiomers of the MDMA (ecstasy-) like drug N-ethyl-3,4-methylenedioxyamphetamine (MDE) were studied in five normal subjects using functional magnetic resonance imaging (fMRI). (S)-MDE produced elevated mood, impairments in conceptually driven cognition and marked right frontal activation. In contrast, (R)-MDE produced increased depression, enhanced visual feature processing, and activation of visual cortical and left frontal areas. Plasma concentrations were higher for the (R)-enantiomer. The so-called entactogenic effects of MDE are likely to be caused by the (S)-enantiomer, whereas (R)-MDE appears to be responsible for neurotoxic effects.

Changes in serotonin, dopamine and noradrenaline levels in striatum and nucleus accumbens after repeated administration of the abused drug MDMA in rats.

The selective neurotoxic action of the abused drug 3,4-methylenedioxymethamphetamine (MDMA) on the serotonergic axons ascending from the dorsal raphe nucleus (DRN) is well known. The present study examined the long-term effects of subchronic MDMA treatment on rat brain tissue contents of catecholaminergic neurotransmitters. Two and four weeks after cessation of repeated MDMA treatment (ten consecutive days, 20 mg/kg/day), the tissue neurotransmitter concentrations were measured by means of electrochemical detected HPLC in several forebrain areas and DRN. We found reduced serotonin levels in the whole forebrain at both instants of time. In nucleus accumbens (NAC), the noradrenaline levels were also decreased, whereas dopamine levels were increased 4 weeks after treatment. It is concluded that MDMA causes changes of monoamine transmitter levels outlasting cessation of drug intake for at least 4 weeks. Decreased noradrenaline and/or serotonin may subsequently cause the augmentation of dopamine in NAC, a structure crucially involved in motivation circuits. With exception of transmitter alterations in the NAC, the post drug effects are opposite to the acute effects of MDMA and may underlie the psychiatric changes after MDMA intake in humans.

Stereospecific analysis of ecstasy-like N-ethyl-3,4-methylenedioxyamphetamine and its metabolites in humans.

A chiral HPLC method has been developed for the ecstasy analogue (R,S)-N-ethyl-3,4-methylenedioxyamphetamine (MDE) and its metabolites o-glucuronyl-(R,S)-N-ethyl-4-hydroxy-3-methoxyamphetamine (HME) and (R,S)-3,4-methylenedioxyamphetamine (MDA) in human plasma. The chiral discrimination of the compounds was carried out with an enantioselective HPLC method using beta-cyclodextrin in the mobile phase for MDE and MDA and a chiral protein phase (chiral-CBH) for HME. MDE and MDA were detected fluorimetrically at 322 nm, while the major metabolite HME was selectively determined by electrochemical detection at +600 mV. After hydrolysis of the conjugates using beta-glucuronidase/arylsulfatase and solid-phase extraction with a cation-exchange phase for sample preparation high recovery rates of more than 95% were yielded. The limit of quantitation for the enantiomers of MDE and its metabolites in plasma were between 1.2 (MDA) and 16 ng/ml (HME) and the relative method standard deviations (V(xO), Table 1) were less than 3%. The methods described have been used successfully in the enantioselective quantitation of the compounds in plasma samples obtained from six healthy volunteers in a clinical study after oral administration of 140 mg racemic MDE hydrochloride. Significant differences were found in the plasma concentrations of the examined stereoisomers. Whereas the R-enantiomer of the parent substance, MDE, was predominant in the plasma samples investigated, higher plasma concentrations of the S-enantiomers of the metabolites MDA and HME were measured.

New sorbent for HPTLC--FTIR in situ determination of impurities in flurazepam.

An efficient HPTLC--UV/FTIR coupling procedure is presented for the separation and rapid identification of flurazepam hydrochloride and its related substances in bulk powder and capsules. An optimized mobile phase was developed for the separation on specialized plates for HPTLC--FTIR in situ measurement containing a proportion of 50% magnesium tungstate. The proposed procedure shows several advantages to the related compound test of the pharmacopoeia, e.g. baseline separation of the known impurities and detection of the substances as peaks in the UV, Gram-Schmidt or window chromatograms. Furthermore, unambiguous identification is obtained by postrun extraction of the DRIFT spectra and comparison with reference spectra in the library. Quantification of the related compounds was carried out densitometrically. This method shows that the current resurgence of interest in modern instrumental TLC is rightful based on the flexibility and efficiency of this analytical method.

Flupirtine and retigabine prevent L-glutamate toxicity in rat pheochromocytoma PC 12 cells.

Flupirtine is an analgesic drug thought to have NMDA receptor antagonistic and antiapoptotic effects. We investigated the effects of Ethyl-2-amino-6-(4-(4-fluorbenzyl)amino)-pyridine-3-carbamamic+ ++ acid, maleate (flupirtine) and the related compound N-(2-amino-4-(4-fluorobenzylamino)-phenyl)-carbamic acid, ethyl ester) (retigabine) (Desaza-flupirtine) on the toxicity of L-glutamate and L-3,4-dihydroxyphenylalanine (L-DOPA) in rat pheochromocytoma PC 12 cells in vitro. Both drugs (10 microM) markedly decreased nonreceptor-mediated necrotic cell death in PC 12 cultures treated with L-glutamate (10 mM) for 72 h. In contrast, apoptosis induced by L-DOPA (250 microM) after 48 h was not affected by either substance. While L-DOPA elicited massive generation of reactive oxygen intermediates, L-glutamate-induced cell death was accompanied by only slightly increased levels of reactive oxygen intermediates. Flupirtine and retigabine exerted anti-oxidative effects in PC 12 cultures independent of their ability to prevent cell death. Further examination of the protective action of flupirtine and retigabine against L-glutamate toxicity showed that it had no influence on monoamine oxidase (monoamine: oxygen oxidoreductase (deaminating), EC 1.4.3.4., MAO) activity. Thus, flupirtine and retigabine provided protection against cystine deprivation and L-glutamate toxicity but did not protect against L-glutamate under cystine-free conditions indicating that both compounds are sufficiently effective to compensate the oxidative stress elicited by cystine deprivation but not excessive activity of monoamine oxidase after L-glutamate treatment.

Development of a 5-hydroxytryptamine(2A) receptor binding assay for high throughput screening using 96-well microfilter plates.

A high throughput screening method for the analysis of 5-hydroxytryptamine(2A) (5-HT(2A)) receptor binding parameters has been developed, using 96-well filter plates of the Millipore MultiScreen system in combination with a MicroBeta PLUS microplate scintillation counter. MAFB filter plates (GF/B filter over a Durapore membrane) were used because of the lower nonspecific binding of the radioligand to GF/B filter material than to GF/C filters. Comparing different scintillation cocktails, highest counting efficiency and shortest equilibration time were detected with Betaplatescint, after drying the plates at 50 degrees C for 2 h. Measuring the plates without the plastic underdrain increased the counting efficiency by about 39% as compared with counting the plate with the underdrain intact. Presoaking the wells with 0.5% polyethyleneimine for 2 h reduced the nonspecific binding to the filter material by about 50%. A linear relationship of protein concentration and radioligand binding was established up to a protein concentration of 165 microg of protein/well. In the assays, 70 microg of protein/well was generally used, which has turned out to be favorable with respect to the number of counts obtained. When a higher concentration of protein was used, the period of time needed to aspirate the plate was too long because of obstruction of the filter material. Receptor-radioligand equilibration was reached after about 20 min at concentrations less than 0.05 nM [(3)H]ketanserin-HCl; at higher concentrations it was reached after about 10 min. Saturation analysis of [(3)H]ketanserin-HCl resulted in a mean B(max) of 393 fmol/mg protein and a K(D) of 2.0 nM using rat frontal cortex as a receptor source. Competition experiments with known 5-HT(2A) receptor ligands-DOB-HCl (K(i) = 59 nM), DOET-HCl (K(i) = 137 nM), DOM-HCl (K(i) = 533 nM), DMT (K(i) = 1,985 nM), and TMA-HCl (K(i) = 22,340 nM)-were in accordance with literature values.

Effect of 1-methyl-4-phenylpyridinium on glutathione in rat pheochromocytoma PC 12 cells.

We investigated the effect of the selective dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+) on glutathione redox status and the generation of reactive oxygen intermediates (ROI) in rat pheochromocytoma PC 12 cells in vitro. Treatment with MPP+ (250 microM) led to a 63% increase of reduced glutathione (GSH) after 24 h, while a 10-fold higher concentration of MPP+ (2.5 mM) depleted cellular GSH to 12.5% of control levels within that time. Similarly, the complex I-inhibitor rotenone induced a time-dependent loss of GSH at 1 and 10 microM, whereas treatment with lower concentrations of rotenone (0.1, 0.01 microM) increased cellular GSH. Both MPP+ and rotenone increased cellular levels of oxidised glutathione (GSSG) and the higher concentrations of both compounds led to an elevated ratio of oxidised glutathione (GSSG) vs total glutathione (GSH + GSSG) indicating a shift in cellular redox balance. MPP+ or rotenone did not induce the generation of ROI or significant elevation of intracellular levels of thiobabituric acid reactive substances (TBARS) for up to 48 h. Our data suggest that MPP+ has differential effects on glutathione homeostasis depending on the degree of complex I-inhibition and that inhibition of complex I is not sufficient to generate ROI in this paradigm.

On-line coupling of automated solid-phase extraction with high-performance liquid chromatography and electrochemical detection. Quantitation of oxidizable drugs of abuse and their metabolites in plasma and urine.

The concentration effect of automated on-line solid-phase extraction (SPE) in combination with HPLC and very sensitive electrochemical detection was employed for the determination of N-ethyl-4-hydroxy-3-methoxy-amphetamine (HMEA, the main metabolite of the ecstasy analogue MDE), delta 9-tetrahydrocannabinol (THC) and 11-nor-delta 9-tetrahydrocannabinol-carboxylic acid (THC-COOH) in plasma and urine in comparison to a previously published psilocin assay. For the SPE either CBA (functional group: carboxypropyl)- or CH (functional group: cyclohexyl)-sorbent was used. The following separation was carried out on a reversed-phase column (LiChroCart, Superspher 60 RP select B from Merck). Depending on the hydrodynamic voltammogram of the analyzed substance the oxidation potential varied from 920 mV up to 1.2 V. In spite of using high potentials, precision and accuracy were always within the accepted statistical requirements. The limits of quantitation were between 5 ng/ml (THC, THC-COOH in plasma) and 20 ng/ml (HMEA in plasma). Advantages of on-line SPE in comparison with off-line methods were less manual effort, evidently smaller volumes (< or = 400 microliters) of plasma or urine and almost always higher recovery rates (> 93%). The assays have been successfully proven with real biological samples and found suitable for use in routine analysis.

Differential effects of L-buthionine sulfoximine and ethacrynic acid on glutathione levels and mitochondrial function in PC12 cells.

We investigated the effect of glutathione (GSH) depletion on mitochondrial function and generation of reactive oxygen intermediates (ROI) in PC12 cells in vitro. Direct depletion of cellular GSH using ethacrynic acid (EA, 500 mM) resulted in a concentration-dependent generation of ROI and cell death within 24 h. Treatment with 500 microM L-buthionine sulfoximine (BSO), which inhibits GSH synthesis, reduced cellular GSH but did not lead to generation of ROI. Furthermore, cells remained viable up to 72 h. Analysis of subcellular fractions revealed complete loss of cytosolic and mitochondrial GSH within 4 h of EA treatment. In contrast, BSO-treated cells still maintained 100% GSH in the mitochondrial fraction for 4 h and 6% for 48 h. Mitochondrial complex II/IIi and IV activities were not significantly decreased up to 48 h of BSO treatment while EA treatment resulted in a complete loss of complex II/III activity and a 70% reduction of complex IV activity within 4 h. These findings suggest that mitochondrial GSH is critical for the maintenance of mitochondrial function and cellular viability.