N,O-diacylated-N-hydroxyarylsulfonamides: nitroxyl precursors with potent smooth muscle relaxant properties.

N,O-Diacylated-N-hydroxyarylsulfonamides are capable of slowly releasing nitroxyl (HNO) by simple, non-enzymatic hydrolysis in Krebs solution at 37 degrees C. Release of nitric oxide (NO) was not seen. These compounds were also found to elicit vasorelaxation in rabbit thoracic aorta in vitro, presumably as a result of their ability to release HNO. This effect was enhanced by the addition of superoxide dismutase (SOD). Thus, these results are consistent with previous work indicating that HNO is a potent vasorelaxant.     

Xanthine oxidase converts nitric oxide to nitroxyl that inactivates the enzyme

Xanthine oxidase (XO) was found to convert nitric oxide (NO* ) released from spermine-NONOate to nitroxyl (HNO), the one-electron reduction product of NO*, in the presence of its substrate hypoxanthine under anaerobic conditions. Under these conditions, XO lost its activity. Upon aerobic incubation of XO with its substrate, neither conversion of NO* to HNO nor inactivation of the enzyme was observed. Angeli's salt (an HNO generator) or synthetic peroxynitrite inactivated XO at low concentrations, whereas high concentrations of diethylamine-NONOate (an NO* donor) and SIN-1 (which generates peroxynitrite by releasing both NO* and superoxide) were required to inactivate XO. These results suggest that HNO generated by XO under anaerobic conditions inactivates XO. As both XO and NO* synthase are activated and/or induced in ischemia-reperfusion injury, HNO formed by XO may contribute to pathogenesis by exerting its potent oxidation activity against a variety of biological compounds.     

Generation of Nitric Oxide and Possibly Nitroxyl by Nitrosation of Sulfohydroxamic Acids and Hydroxamic Acids

Diazeniumdiolates (NONOates) and sulfohydroxamic acids are chemical entities that spontaneously generate nitric oxide (NO) and nitroxyl (HNO), respectively, at physiological pH and temperature. By combining the functional aspects of the NONOates with the hydroxamic acids and sulfohydroxamic acids, hybrid NONOate-type compounds that could theoretically generate nitroxyl or nitric oxide can be rationalized. Although the instability of these compounds, viz., the N-nitrosohydroxamic acids and the N-nitrososulfohydroxamic acids, precluded their chemical characterization by actual isolation, their transient existence was deduced by identification of the products of their decomposition. Thus, treatment of benzohydroxamic acid (BHA) with limiting or excess nitrous acid (from NaNO(2) and H(3)PO(4)) gave rise to quantitative generation of N(2)O, possibly via HNO, based on the limiting reactant. Nitrosation of N-t-butyloxycarbonyl hydroxylamine gave similar results. The organic acid produced from BHA was identified as benzoic acid. No nitric oxide was detected from these reactions. In contrast, treatment of Piloty's acid (benzenesulfohydroxamic acid) and methanesulfohydroxamic acid (MSHA) with nitrous acid under the same conditions as above gave 36% of the theoretical quantity of NO from Piloty's acid and 47% of NO from MSHA, although finite quantities of HNO (measured as N(2)O) were also formed. The organic acid produced from Piloty's acid was identified by reverse-phase HPLC as the redox product, benzenesulfinic acid.     

Fully habituated sugarbeet callus: Under permanent stress?

Summary A fully habituated nonorganogenic (HNO) sugarbeet callus line, compared to a normal (hormone dependent) one originated from the same plant exhibits many characteristics of a vitrified tissue and several traits common to animal cancer cells. Four types of biochemical or metabolic characteristics of HNO callus [deficiency of tetrapyrrole-containing compounds; lipid (per)oxidation and malondialdehyde formation; high activity of enzyme protective systems; proline, glutamate, and polyamine accumulation] may be interpreted as responses to stress. The deficiency of tetrapyrrole-containing compounds can be considered an indirect protection against activated forms of oxygen as well as the higher activity of the antioxidant defense mechanisms. This fits with suggestions in the literature that the autonomy associated with plant cancer tissue is explained on the basis of antioxidants as stimulators of cell division and corresponding inhibitors of cell differentiation. Such changes occurring in HNO cells may in turn be responsible for a greater absorption and sensitivity to ammonium ions. An altered nitrogen metabolism leads to proline, glutamate, and polyamine accumulation. Lipid peroxidation and malondialdehyde accumulation rapidly occur in this very sensitive HNO callus in a prolonged culture cycle, which might be related to the appearance of necrosis bands. Thus there are arguments allowing us to consider HNO cells as mutants adapted to some stresses, but resulting changes in their structure might have rendered them still more sensitive to other factors.     

Darkness improves growth and delays necrosis in a nonchlorophyllous habituated sugarbeet callus: Biochemical changes

The transfer of light-cultured green normal (N) and white habituated (HNO) sugarbeet callus to darkness reduced the growth of N callus and improved growth and delayed necrosis in the HNO callus. The decrease of dry matter of N callus under darkness was accompanied by a reduced content of carotenoids and by decreased CO₂ fixation, which was compensated by an increased dependency on externally supplied sucrose. The levels of some organic nitrogen compounds such as glutamate, proline, and free polyamines were not affected by transfer to darkness of N or HNO callus. Darkness decreased ethylene emissions in both callus types. In the HNO callus, the sucrose growth dependency and the CO₂ fixation were unaffected by darkness. Chlorophylls were absent both in light and darkness, whereas some carotenoids were accumulated in the HNO callus only in dark conditions. In another connection, a significant increase of peroxidase activity, which did not occur in the N callus, was induced by darkness in the HNO callus. A decreased content of thio-barbituric acid (TBA)-reactive substances was measured in the HNO callus transferred to darkness, whereas an increase was noticed in the N callus placed in the same conditions. These metabolic changes and the reduction of cellular damage in darkness revealed light-induced stress reactions leading to necrosis and to reduced growth of HNO callus. It appeared that darkness allowed the HNO callus to avoid the photooxidation stress. Therefore, the favorable effect of darkness on HNO growth might be explained by the suppression of photooxidative damage due to the absence of carotenoids. The higher peroxidase activity in the HNO callus maintained in darkness raised the problem of heme synthesis in this heterotrophic callus.     

Effect of nitroxyl on the hamster retinal nitridergic pathway

There is a growing body of evidence on the role of nitric oxide (NO) in retinal physiology. Recently, interest has developed in the functional role of an alternative redox form of NO, namely nitroxyl (HNO/NO(-)), because it is formed by a number of diverse biochemical reactions. The aim of the present report was to comparatively analyze the effect of HNO and NO on the retinal nitridergic pathway in the golden hamster. For this purpose, sodium trioxodinitrate (Angeli's salt) and diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO) were used as HNO and NO releasers, respectively. Angeli's salt and DEA/NO significantly decreased nitric oxide synthase activity. In addition, Angeli's salt (but not DEA/NO) significantly decreased l-arginine uptake. DEA/NO significantly increased cGMP accumulation at low micromolar concentrations, while Angeli's salt affected this parameter with a threshold concentration of 200muM. Although Angeli's salt and DEA/NO significantly diminished reduced glutathione and protein thiol levels in a similar way, DEA/NO was significantly more effective than AS in increasing S-nitrosothiol levels. None of these compounds increased retinal lipid peroxidation. These results suggest that HNO could regulate the hamster retinal nitridergic pathway by acting through a mechanism that only partly overlaps with that involved in NO response.     

Acyloxy Nitroso Compounds Inhibit LIF Signaling in Endothelial Cells and Cardiac Myocytes: Evidence That STAT3 Signaling Is Redox-Sensitive

We previously showed that oxidative stress inhibits leukemia inhibitory factor (LIF) signaling by targeting JAK1, and the catalytic domains of JAK 1 and 2 have a cysteine-based redox switch. Thus, we postulated that the NO sibling and thiophylic compound, nitroxyl (HNO), would inhibit LIF-induced JAK-STAT3 activation. Pretreatment of human microvascular endothelial cells (HMEC-1) or neonatal rat cardiomyocytes with the HNO donors Angeli's salt or nitrosocyclohexyl acetate (NCA) inhibited LIF-induced STAT3 activation. NCA pretreatment also blocked the induction of downstream inflammatory genes (e.g. intercellular adhesion molecule 1, CCAAT/enhancer binding protein delta). The related 1-nitrosocyclohexyl pivalate (NCP; not a nitroxyl donor) was equally effective in inhibiting STAT3 activation, suggesting that these compounds act as thiolate targeting electrophiles. The JAK1 redox switch is likely not a target of acyloxy nitroso compounds, as NCA had no effect on JAK1 catalytic activity and only modestly affected JAK1-induced phosphorylation of the LIF receptor. However, pretreatment of recombinant human STAT3 with NCA or NCP reduced labeling of free sulfhydryl residues. We show that NCP in the presence of diamide enhanced STAT3 glutathionylation and dimerization in adult mouse cardiac myocytes and altered STAT3 under non-reducing conditions. Finally, we show that monomeric STAT3 levels are decreased in the Gαq model of heart failure in a redox-sensitive manner. Altogether, our evidence indicates that STAT3 has redox-sensitive cysteines that regulate its activation and are targeted by HNO donors and acyloxy nitroso compounds. These findings raise the possibility of new therapeutic strategies to target STAT3 signaling via a redox-dependent manner, particularly in the context of cardiac and non-cardiac diseases with prominent pro-inflammatory signaling.     

Human carbonyl reductase catalyzes reduction of 4-oxonon-2-enal

4-Oxonon-2-enal (4ONE) was demonstrated to be a product of lipid peroxidation, and previous studies found that it was highly reactive toward DNA and protein. The present study sought to determine whether carbonyl reductase (CR) catalyzes reduction of 4ONE, representing a potential pathway for metabolism of the lipid peroxidation product. Recombinant CR was cloned from a human liver cDNA library, expressed in Escherichia coli, and purified by metal chelate chromatography. Both 4ONE and its glutathione conjugate were found to be substrates for CR, and kinetic parameters were calculated. TLC analysis of reaction products revealed the presence of three compounds, two of which were identified as 4-hydroxynon-2-enal (4HNE) and 1-hydroxynon-2-en-4-one (1HNO). GC/MS analysis confirmed 4HNE and 1HNO and identified the unknown reaction product as 4-oxononanal (4ONA). Analysis of oxime derivatives of the reaction products via LC/MS confirmed the unknown as 4ONA. The time course for CR-mediated, NADPH-dependent 4ONE reduction and appearance of 4HNE and 1HNO was determined using HPLC, demonstrating 4HNE to be a major product and 1HNO and 4ONA to be minor products. Simulated structures of 4ONE in the active site of CR/NADPH calculated via docking experiments predict the ketone positioned as primary hydride acceptor. Results of the present study demonstrate that 4ONE is a substrate for CR/NADPH and the enzyme may represent a pathway for biotransformation of the lipid. Furthermore, these findings reveal that CR catalyzes hydride transfer selectively to the ketone but also to the aldehyde and C=C of 4ONE, resulting in 4HNE, 1HNO, and 4ONA, respectively.     

Morphological and proteomic analyses of sugar beet cultures and identifying putative markers for cell differentiation

Normal (N), habituated nonorganogenic (HNO), and tumour (T) sugar beet cell lines were analysed in order to establish specific patterns of extracellular proteins and identify protein markers that might explain the distinct phenotypical characteristics. Electron microscopy showed that the ultrastructure of N cells corresponds to that of parenchyma cells, and that these cells contain plastids with large starch grains. HNO and T cells had enlarged, lobed nuclei with an increased number of nucleoli; the number of nuclei in HNO cells was greater than in T cells. The T plastids were elongated, with reduced thylakoids and abundant phytoferritin deposits, while HNO plastids were small and vacuolated, with an irregular, underdeveloped thylakoid system. The extracellular proteome of the cells was separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Greater differences in protein expression were observed between the HNO and N lines than between the T and N lines. Sixteen of the most prominent bands differentially expressed among the cell lines were cut out from the gel and analyzed by mass spectrometry. Cell wall-modifying enzymes were identified, including a peroxidase whose expression was twofold higher in N and T tissue than in HNO tissue; pectinesterase, which was expressed at a level threefold lower in the T line than in the other cell lines; and xyloglucan endotransglucosylase, which was expressed at a level sixfold higher in HNO and T tissue. Three proteins belonged to the chitinase gene family and their expression was higher in HNO and T tissue than in N tissue. The differential expression of these proteins suggests that these play a role in cell line-specific cell wall composition and cell-to-cell adhesion.     

Chemotherapeutic potential of diazeniumdiolate-based aspirin prodrugs in breast cancer

Diazeniumdiolate-based aspirin prodrugs have previously been shown to retain the anti-inflammatory properties of aspirin while protecting against the common side effect of stomach ulceration. Initial analysis of two new prodrugs of aspirin that also release either nitroxyl (HNO) or nitric oxide (NO) demonstrated increased cytotoxicity toward human lung carcinoma cells compared to either aspirin or the parent nitrogen oxide donor. In addition, cytotoxicity was significantly lower in endothelial cells, suggesting cancer-specific sensitivity. To assess the chemotherapeutic potential of these new prodrugs in treatment of breast cancer, we studied their effect both in cultured cells and in a nude mouse model. Both prodrugs reduced growth of breast adenocarcinoma cells more effectively than the parent compounds while not being appreciably cytotoxic in a related nontumorigenic cell line (MCF-10A). The HNO donor also was more cytotoxic than the related NO donor. The basis for the observed specificity was investigated in terms of impact on metabolism, DNA damage and repair, apoptosis, angiogenesis and metastasis. The results suggest a significant pharmacological potential for treatment of breast cancer.     

Orthogonal properties of the redox siblings nitroxyl and nitric oxide in the cardiovascular system: a novel redox paradigm

Endogenous formation of nitric oxide (NO) and related nitrogen oxides in the vascular system is critical to regulation of multiple physiological functions. An imbalance in the production or availability of these species can result in progression of disease. Nitrogen oxide research in the cardiovascular system has primarily focused on the effects of NO and higher oxidation products. However, nitroxyl (HNO), the one-electron-reduction product of NO, has recently been shown to have unique and potentially beneficial pharmacological properties. HNO and NO often induce discrete biological responses, providing an interesting redox system. This article discusses the emerging aspects of HNO chemistry and attempts to provide a framework for the distinct effects of NO and HNO in vivo.     

Shemin pathway and peroxidase deficiency in a fully habituated and fully heterotrophic non-organogenic sugarbeet callus: an adaptative strategy or the consequence of modified hormonal balances and sensitivities in these cancerous cells? A review and reassessment

There are many arguments for considering a specific fully habituated (auxin and cytokinin-independent) and fully heterotrophic non-organogenic (HNO) sugarbeet callus cell line as terminating a neoplastic progression, and thus to be made of cancerous cells. The similarities with animal tumour and cancer cells are recalled. All types of habituated tissues examined in the literature share at least three common biochemical characteristics: low apparent peroxidase activity, high content of polyamines (PAs) and low production of ethylene. However, results concerning their auxin and cytokinin levels are not consistent. Peroxidase synthesis in the achlorophyllous HNO callus appears to arise from aminolevulinic acid (ALA) synthesis through the Shemin pathway, commonly used by animals and fungi. This pathway is limited by disturbed nitrogen metabolism that diverts glutamate (directly used for ALA synthesis in green higher plants) from the Kreb's cycle into PA synthesis. There is no argument to suggest that the low ethylene production is caused by a competition with PAs for their common precursor, S-adenosylmethionine. The results we report here indicate modified anabolic and catabolic pathways of auxins and cytokinins but also the possibilities of unusual compounds playing similar roles (dehydrodiconiferyl alcohol glucosides, for instance). A higher turnover of PAs is shown in the HNO callus, which could suggest a role for H2O2 and gamma-aminobutyric acid, products or intermediates in the PA catabolic pathway, as secondary messengers. The habituated cells retain some sensitivity towards exogenous auxins and cytokinins. Their increased sensitivity to PAs and ethylene suggests modified hormonal balances for the control of these actively dividing cells.     

Hydroxylamine acutely activates glucose uptake in L929 fibroblast cells

Nitroxyl (HNO) has a unique, but varied, set of biological properties including beneficial effects on cardiac contractility and stimulation of glucose uptake by GLUT1. These biological effects are largely initiated by HNO's reaction with cysteine residues of key proteins. The intracellular production of HNO has not yet been demonstrated, but the small molecule, hydroxylamine (HA), has been suggested as possible intracellular source. We examined the effects of this molecule on glucose uptake in L929 fibroblast cells. HA activates glucose uptake from 2 to 5-fold within two minutes. Prior treatment with thiol-active compounds, such as iodoacetamide (IA), cinnamaldehyde (CA), or phenylarsine oxide (PAO) blocks HA-activation of glucose uptake. Incubation of HA with the peroxidase inhibitor, sodium azide, also blocks the stimulatory effects of HA. This suggests that HA is oxidized to HNO by L929 fibroblast cells, which then reacts with cysteine residues to exert its stimulatory effects. The data suggest that GLUT1 is acutely activated in L929 cells by modification of cysteine residues, possibly the formation of a disulfide bond within GLUT1 itself.     

Comparison of the reactivity of nitric oxide and nitroxyl with heme proteins. A chemical discussion of the differential biological effects of these redox related products of NOS

Investigations on the biological effects of nitric oxide (NO) derived from nitric oxide synthase (NOS) have led to an explosion in biomedical research over the last decade. The chemistry of this diatomic radical is key to its biological effects. Recently, nitroxyl (HNO/NO(-)) has been proposed to be another important constituent of NO biology. However, these redox siblings often exhibit orthogonal behavior in physiological and cellular responses. We therefore explored the chemistry of NO and HNO with heme proteins in different redox states and observed that HNO favors reaction with ferric heme while NO favors ferrous, consistent with previous reports. Further results show that HNO and NO were equally effective in inhibiting cytochrome P450 activity, which involves ferric and ferrous complexes. The differential chemical behavior of NO and HNO toward heme proteins provides insight into mechanisms of activity that not only helps explain some of the opposing effects observed in NOS-mediated events, but offers a unique control mechanism for the biological action of NO.     

The emergence of nitroxyl (HNO) as a pharmacological agent

Once a virtually unknown nitrogen oxide, nitroxyl (HNO) has emerged as a potential pharmacological agent. Recent advances in the understanding of the chemistry of HNO has led to the an understanding of HNO biochemistry which is vastly different from the known chemistry and biochemistry of nitric oxide (NO), the one-electron oxidation product of HNO. The cardiovascular roles of NO have been extensively studied, as NO is a key modulator of vascular tone and is involved in a number of vascular related pathologies. HNO displays unique cardiovascular properties and has been shown to have positive lusitropic and ionotropic effects in failing hearts without a chronotropic effect. Additionally, HNO causes a release of CGRP and modulates calcium channels such as ryanodine receptors. HNO has shown beneficial effects in ischemia reperfusion injury, as HNO treatment before ischemia-reperfusion reduces infarct size. In addition to the cardiovascular effects observed, HNO has shown initial promise in the realm of cancer therapy. HNO has been demonstrated to inhibit GAPDH, a key glycolytic enzyme. Due to the Warburg effect, inhibiting glycolysis is an attractive target for inhibiting tumor proliferation. Indeed, HNO has recently been shown to inhibit tumor proliferation in mouse xenografts. Additionally, HNO inhibits tumor angiogenesis and induces cancer cell apoptosis. The effects seen with HNO donors are quite different from NO donors and in some cases are opposite. The chemical nature of HNO explains how HNO and NO, although closely chemically related, act so differently in biochemical systems. This also gives insight into the potential molecular motifs that may be reactive towards HNO and opens up a novel field of pharmacological development.     

Nitroxyl (HNO) acutely activates the glucose uptake activity of GLUT1

Nitroxyl (HNO) is a molecule of significant interest due to its unique pharmacological properties, particularly within the cardiovascular system. A large portion of HNO biological effects can be attributed to its reactivity with protein thiols, where it can generate disulfide bonds. Evidence from studies in erythrocytes suggests that the activity of GLUT1 is enhanced by the formation of an internal disulfide bond. However, there are no reports that document the effects of HNO on glucose uptake. Therefore, we examined the acute effects of Angeli’s salt (AS), a HNO donor, on glucose uptake activity of GLUT1 in L929 fibroblast cells. We report that AS stimulates glucose uptake with a maximum effective concentration of 5.0 mM. An initial 7.2-fold increase occurs within 2 min, which decreases and plateaus to a 4.0-fold activation after 10 min. About 60% of the 4.0-fold activation recovers within 10 min, and 40% remains after an hour. The activation is blocked by the pretreatment of cells with thiol-reactive compounds, iodoacetamide (0.75 mM), cinnamaldehyde (2.0 mM), and phenylarsine oxide (10 μM). The effects of AS are not additive to the stimulatory effects of other acute activators of glucose uptake in L929 cells, such as azide (5 mM), berberine (50 μM), or glucose deprivation. These data suggest that GLUT1 is acutely activated in L929 cells by the formation of a disulfide bond, likely within GLUT1 itself.     

Erythromycin as a tool to investigate the tetrapyrrole biosynthetic pathways in habituated and normal sugarbeet calli

Erythromycin (ERT) has been shown to reduce the 5-aminolevulinic acid (ALA) synthesizing capacity of a normal (N) chlorophyllous sugarbeet callus, grown under light, in contrast to a habituated achlorophyllous non-organogenic (HNO) callus of the same species. Similar effects were obtained on total hemes and on catalase which is a hemoprotein used as marker. The effect of ERT, which is an inhibitor of plastid differentiation and of chlorophyll synthesis, was reversed in the N callus by a supply of glycine and succinate. The compounds are the precursors of ALA synthesized through 5-aminolevulinic acid synthase (ALAS) which is implied in the Shemin pathway. The involvement of ALAS appeared to be favoured when plastids were undifferentiated (HNO callus) or when plastids were inefficient (N callus under darkness or under light after ERT treatment).     

HNO induces DNA deletions in the yeast S. cerevisiae

HNO is genotoxic but its mechanism is not well understood. There are many possible mechanisms by which HNO can attack DNA. Since HNO is electrophilic, it may react with exocyclic amine groups on DNA bases and through a series of subsequent reactions form a deaminated product. Alternatively, HNO may induce radical chemistry through O(2)-dependent (or possibly O(2)-independent) chemistry. In cell free systems, experiments have shown that HNO does react with DNA, resulting in base oxidation and strand cleavage. In this study, we used a whole-cell system in the yeast Saccharomyces cerevisiae to study the mechanism of HNO induced DNA damage with Angeli's salt as HNO donor. The yeast DEL assay provided a measure of intrachromosomal recombination leading to DNA deletions. We also examined interchromosomal recombination leading to genomic rearrangements and used the canavanine (CAN) assay to study induction of forward point mutations. HNO was a potent inducer of DNA deletions and recombination but it was negative for induction of point mutations. This suggests that HNO causes DNA strand breaks rather than base damage. Genotoxicity was observed under aerobic and anaerobic conditions and NAC protected against HNO induced DNA deletions. Since HNO is genotoxic under anaerobic conditions, NAC probably protected against radicals generated by HNO independent of oxygen.     

Inhibition of yeast glycolysis by nitroxyl (HNO): mechanism of HNO toxicity and implications to HNO biology

Nitroxyl (HNO) was found to inhibit glycolysis in the yeast Saccharomyces cerevisiae. The toxicity of HNO in yeast positively correlated with the dependence of yeast on glycolysis for cellular energy. HNO was found to potently inhibit the crucial glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH), an effect which is likely to be responsible for the observed inhibition of glycolysis in whole cell preparations. It is proposed that GAPDH inhibition occurs through reaction of HNO with the active site thiolate residue of GAPDH. Significantly, levels of HNO that inhibit GAPDH do not alter the levels or redox status of intracellular glutathione (GSH), indicating that HNO has thiol selectivity. The ability of HNO to inhibit GAPDH in an intracellular environment that contains relatively large concentrations of GSH is an important aspect of HNO pharmacology and possibly, physiology.     

High-performance liquid chromatographic method for the detection and quantitation of haloperidol and seven of its metabolites in microsomal preparations

An isocratic high-performance liquid chromatographic (HPLC) system was developed to analyze haloperidol and its potential metabolites. These compounds included 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), haloperidol N-oxide (HNO), reduced haloperidol (RHAL), the 1,2,3,6-tetrahydropyridine analogue and its N-oxide, and the pyridinium ion from haloperidol (HP⁺). The HPLC system comprised a Hypersil CPS5 column with a mobile phase of acetonitrile (67%) and ammonium acetate (final concentration 10 mM) which was adjusted to pH 5.4 by acetic acid. The solvent was delivered at 1 ml/min. RHAL and CPHP were determined by an ultraviolet detector at 220 nm with a detection limit of 1 nmol/ml. All other compounds were determined at 245 nm and had a detection limit of 0.3 nmol/ml. This system was used to analyze a microsomal metabolic mixture of haloperidol. It was found that all above compounds except HNO were metabolites of haloperidol. In addition, two other metabolites were also well separated in this HPLC system which are proposed to be oxygenated haloperidol and the pyridone analogue of haloperidol. The HPLC system was used to carry out quantitative metabolic studies of haloperidol. It was found that the metabolism of haloperidol exhibits large inter-species differences. The apparent enzyme kinetic parameters were also determined using mice microsomes.