The relationship between hypotonically-induced taurine and K fluxes in trout red blood cells.

Hypotonic swelling of teleost erythrocytes activates multiple transport systems leading to the regulatory decrease of cell volume. We have examined using pharmacological manipulation the swelling-induced taurine flux pathway in red blood cells of the rainbow trout and its relationship to swelling-induced K flux pathways. We show that the activation and deactivation of taurine flux is rapid and that the flux is a sigmoidal function of cell volume. N-ethylmaleimide (NEM) and the non-specific protein kinase inhibitor, staurosporine, both inactivated the hypotonically-induced taurine flux with concentrations eliciting half-maximal inhibition (IC50s) of 212 and 17 micromol(-1), respectively. The low taurine fluxes under isotonic conditions were unaffected. By contrast, the tyrosine kinase inhibitor, genistein, partially inhibited taurine flux under both isotonic and hypotonic conditions. The specific phosphatase inhibitor, calyculin A, had no inhibitory or stimulatory effect under either condition whilst the less-specific phosphatase inhibitor, ortho-vanadate, reduced taurine flux only under hypotonic conditions. In these respects the regulatory control of the taurine pathway differs from the Cl-dependent K flux. However, NEM and staurosporine also inhibited the Cl-independent K flux, both with similar IC50s to those observed for taurine fluxes. This supports the idea of the hypotonically-induced taurine flux and the Cl-independent K flux sharing the same transport pathway.

A comparison in normal individuals and sickle cell patients of reduced glutathione precursors and their transport between plasma and red cells.

INTRODUCTION: Reduced glutathione is an important antioxidant in red cells whose depletion may contribute to the pathophysiology of sickle cell disease. The current study was designed to examine the availability of reduced glutathione precursors (glutamate, cysteine, glycine and possibly glutamine) together with the activity of the main transport pathways for their uptake (system ASC for cysteine and glycine; system gly for glycine). MATERIALS AND METHODS: Blood samples were obtained from normal (HbAA, HbA cells) and sickle cell disease patients (HbSS, HbS cells); amino acids were measured by HPLC; and transporter activity was measured by radioactive tracer fluxes (using serine and glycine for activity of system ASC; and glycine for that of system gly). RESULTS: Plasma concentrations of cysteine and glycine were increased and concentrations of all amino acids were elevated in HbS cells. The activity of system ASC was increased in HbS cells (both transport capacity and affinity were elevated for serine transport; transport capacity only for glycine). Activity of system gly was also increased (twofold increase in V(max) for glycine flux), though not significantly. Oxygenation also increased the activity of both transporters in normal and HbS cells. CO prevented deoxy-inhibition of glycine transport. Staurosporine (5 microM) inhibited O(2)-stimulated glycine transport through system ASC. It also inhibited the absolute magnitude of transport through system gly, but the O(2)-dependent flux was unaffected. CONCLUSION: Low reduced glutathione levels in HbS cells were not due to decreased substrate availability and O(2) stimulated transport of reduced glutathione precursors in both normal and HbS cells, through a mechanism that is likely to involve Hb and possibly protein phosphorylation.

Characterization of the K+ (Na+)/H+ monovalent cation exchanger in the human red blood cell membrane: effects of transport inhibitors.

The (ouabain + bumetanide + EGTA)-insensitive K+ influx (defined as residual K+ influx) in the human erythrocyte was investigated with respect to the characterization of the recently identified K+(Na+)/H+ exchanger (Richter et al. 1997). In particular, the effects of selected ion transport inhibitors on this flux in physiological ionic strength (high ionic strength, HIS) as well as low ionic strength (LIS) solutions were qstudied. The stimulation of the K+ influx observed in LIS medium was further enhanced when DIDS, phloretin, eosin-5-maleimide, furosemide, DIOA, NPPB, or DCDPC was present at a concentration of 0.1 mmol/l. This paradoxical, inhibitor-induced increase of the K+ influx was more pronounced in LIS media where chloride (7.5 mmol/l) was replaced by nitrate. For DNDS, niflumic acid, and MK-196 (0.1 mmol/l) an enhanced K+ transport could only be observed in nitrate-containing LIS solution. Bumetanide and purine riboside, at a concentration of 0.1 mmol/l, did not cause significant changes of the K+ influx in either chloride- or nitrate-containing LIS media. Dipyridamole and ruthenium red (0.1 mmol/l), which are positively charged, significantly reduced the K+ influx in both chloride- and nitrate-containing LIS media. In nitrate-containing HIS solution only dipyridamole inhibited the K+ influx. The residual K+ influx in LIS solution was significantly increased by removing internal [Mg2+], and decreased by quinacrine (1 mmol/l). In HIS solution, no effect of altering intracellular Mg2+ occurred but a stimulation of the flux by quinacrine was observed. The results are discussed in terms of a more general surface charge effect of the used inhibitors on the K+(Na+)/H+ exchanger.

Responses of the Na+/H+ exchanger of European flounder red blood cells to hypertonic, beta-adrenergic and acidotic stimuli.

The transport pathways mediating regulatory volume increase (RVI) and beta-adrenergic responses in red cells of the European flounder Platichthys flesus have been investigated. Hypertonic treatment under a low-PO2 atmosphere led to a complete RVI and to a three- to fourfold increase in Na+ influx. The RVI and the activated Na+ influx were blocked by the transport inhibitors amiloride and 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), both at a concentration of 10(-4 )mol l-1, and the RVI was abolished in a Na+-free saline, indicating the involvement of a hypertonically induced Na+/H+ exchanger and an accompanying Cl-/HCO3- exchanger. Both the hypertonically induced Na+ influx and the RVI were blocked by oxygenation of shrunk cells. The beta-adrenergic agonist isoproterenol also strongly activated a Na+ influx and caused cell swelling. This response was also inhibited by amiloride and DIDS but was unaffected by oxygenation. Simultaneous application of isoproterenol and hypertonic shrinkage did not lead to additive Na+ influxes, suggesting that both responses were mediated by the same pool of exchangers. Mild cell acidification activated a Na+ influx under iso-osmotic conditions; amiloride caused partial inhibition of this influx, but oxygenation had no effect. Acid-induced and isoproterenol-induced Na+ fluxes were again non-additive. Thus, the Na+/H+ exchanger of flounder red cells is strongly activated by three physiological stimuli: hypertonic shrinkage, beta-adrenergic hormones and cell acidification. Of these responses, only the first is affected by oxygenation, indicating some differentiation of their respective transduction mechanisms. These characteristics contrast with those of the corresponding exchangers from rainbow trout and eel red cells.

An examination of the role of intracellular ATP in the activation of store-operated Ca2+ influx and Ca2+-dependent capacitance increases in rat basophilic leukaemia cells.

The role of ATP in both the activation of store-operated Ca2+ current ICRAC and in Ca2+-dependent vesicular fusion was examined in a study of rat basophilic leukaemia (RBL) cells using the whole-cell patch-clamp technique. Fusion was monitored via changes in plasma membrane capacitance. Following a decrease in the levels of intracellular ATP, achieved using the mitochondrial poison antimycin and the ATP synthase inhibitor oligomycin, as well as a reduction of glycolysis by removal of external glucose, ICRAC activated in a manner similar to control cells when stores are depleted by dialysis with a pipette solution containing either inositol 1,4, 5-trisphosphate (InsP3) or ionomycin together with a high concentration of EGTA. Dialysis of cells for 150 s with the non-hydrolysable ATP analogue 5'-adenylylimidodiphosphate (AMP-PNP) (2 mM) in addition to the mitochondrial inhibitors also failed to prevent activation of ICRAC following external application of ionomycin and thapsigargin, when compared with control recordings obtained with 2 mM ATP instead. Ca2+-dependent vesicular fusion was triggered by dialysing cells with 10 microM Ca2+ and guanosine-5'-O-(3-thiotriphosphate (GTP[gamma-S]). The capacitance increase was unaffected by inhibition of glycolysis, mitochondrial inhibitors or dialysis with either AMP-PNP or adenosine 5'-O-(3-thiotriphosphate) (ATP[gamma-S]) instead of ATP. We conclude that ATP hydrolysis does not seem to be necessary for the activation of ICRAC or for the capacitance increases elicited by high concentrations of intracellular Ca2+.

Transport of L-arginine and the nitric oxide inhibitor NG-monomethyl-L-arginine in human erythrocytes in chronic renal failure.

1. Transport of L-arginine and the nitric oxide synthase inhibitors NG-monomethyl-L-arginine and NG-nitro-L-arginine was investigated in human erythrocytes from healthy donors and uraemic patients on haemodialysis. 2. Although K(m) values for total L-arginine influx were not significantly different in erythrocytes freshly isolated from controls or uraemic patients, uraemia was associated with an increase in the Vmax for transport (826 compared with 1176 mumol h-1 l-1 of cells) which was reduced to control values after dialysis. 3. Saturable influx of L-arginine was mediated by the classical cationic amino acid transport system y+ and system y+L, known to transport cationic and neutral amino acids with higher affinity. 4. Under zero-trans conditions, the Vmax for L-arginine transport via system y+increased from 271 to 700 mumol h-1 l-1 of cells in uraemia, while K(m) values increased from 44 to 94 mumol/l. Dialysis had no significant effect on the kinetic parameters altered by uraemia. 5. Under zero-trans conditions, and with system y+ inhibited by N-ethylmaleimide (0.2 mmol/l), transport of L-arginine via system y+L was unaffected by uraemia. 6. Saturable influx of NG-monomethyl-L-arginine was also mediated by systems y+ (K(m) = 56 mumol/l, Vmax = 353 mumol h-1 l-1 of cells) and y+L (K(m) = 17 mumol/l, Vmax = 51.3 mumol h-1 l-1 of cells) and, as with L-arginine, uraemia increased the transport capacity for NG-monomethyl-L-arginine. 7. Influx of the neutral nitric oxide synthase inhibitor NG-nitro-L-arginine was not readily saturable. 8. Intracellular concentrations of L-arginine and NG-monomethyl-L-arginine were significantly increased in erythrocytes from uraemic patients when compared with controls, consistent with an increased transport capacity for L-arginine and NG-monomethyl-L-arginine. 9. The present study provides evidence that system y+ mediates the increased transport of L-arginine and NG-monomethyl-L-arginine in human erythrocytes from patients with chronic renal failure. Our findings may have implications for the activity of the L-arginine-nitric oxide signalling pathway in vascular endothelial and smooth-muscle cells in uraemia.

Spawning induces a shift in energy metabolism from glucose to lipid in rainbow trout white muscle.

Enzymatic changes that occur in the white somatic muscle of rainbow trout (Oncorhynchus mykiss) in response to spawning were investigated, and the evenness of their distribution across the ventral-dorsal plane of this muscle was assessed. Four enzymes that are involved in energy metabolism were measured (phosphofructokinase: glycolytic capacity, 3-hydroxyacyl-CoA dehydrogenase: ß-oxidation, citrate synthase: citric acid cycle, cytochrome oxidase: oxidative capacity). The enzyme activities were followed in different parts of the white muscle of non-spawning female rainbow trout from May, four months after their first spawning, until December, at second spawning. Samples were taken from white epaxial muscle along the lateral line, on the dorsum, and in between. Samples were also taken from red muscle of non-spawning fish. The isoforms of myosin heavy chains (MyHC) were electrophoretically identified on 6% SDS-PAGE gel to study possible changes in contractile properties of the muscle.Transformation from the non-spawning to spawning phase was associated with dramatic changes in the activity of the enzymes studied in white muscle: glycolytic capacity decreased to less than half, whereas oxidative metabolism increased about two- to four-fold in all areas. Significant quantitative differences in enzyme activities were found between the three epaxial muscle areas: in the non-spawning fish lateral line samples differed from those taken in the other two areas, whereas in spawning fish the dorsal sample difered from the other two. No difference in the expression of MyHC-isoforms was found between spawning and non-spawning fish. Co-expression of both slow and fast isoforms was found in single fibres isolated from red muscle.The results show that the energy metabolism in white muscle of domestic rainbow trout is altered during spawning; i.e., the metabolism becomes increasingly aerobic, with an increased capacity for fatty acid utilization, concomitant with phenotypic changes associated with sexual maturation. These changes are especially pronounced in ventral, superficially located fibres.

Mycotoxins in Swedish grains and mixed feeds.

Approximately 68 samples of grains and mixed feeds associated with disturbances in animal breeding from 1977 and 1978 were analyzed for ochratoxin A, zearalenone, aflatoxin, and 9 trichothecenes. Six contained ochratoxin A (40-1690 ppb, average of 750 ppb) and 2 contained zearalenone (1200 and less than 100 ppb). Aflatoxin was detected in a sample from formic acid-treated oats with a level of 2600 ppb at the surface of the stored grain. Deoxynivalenol was detected in two samples. One contained approximately 2 ppm and was confirmed by gas-liquid chromatography mass spectrometry (GLC/MS).

On the bioactivation and genotoxic action of fluoranthene.

Fluoranthene (FA) was studied with respect to possible mechanisms of its high mutagenicity but low carcinogenicity, in comparison with the corresponding properties of benzo[a]pyrene (BaP), and with regard to the synergism of these two compounds shown by van Duuren and Goldschmidt (J Natl Cancer Inst 56, 1976, 1237). FA and BaP activated by S9 from Aroclor 1254 (PCB)-treated rats induce HPRT mutations in CHO cells with about equal effectiveness at the same exposure doses, which also lead to the same frequencies of repairable DNA adducts, enzyme-induced strand breaks being used as an indirect measure of adducts to DNA. FA was also shown to be an efficient inducer of SCE in human peripheral lymphocytes cocultivated with PCB-treated HepG2 cells or with liver cells from PCB-pretreated rats. For the induction of SCE, FA and BaP were shown to act additively. From metabolic studies with liver microsomes from C57Bl/6 mice it is concluded that, whereas BaP induces the metabolism of BaP to the mutagenic epoxide, neither BaP nor FA is able to induce the metabolism of FA. In mutation experiments with V79 cells (XEM2) constitutive for P450 IA1 activity, BaP 7,8-diol but not FA 2,3-diol provokes a high frequency of HPRT mutations. In cells constitutive for P450 IA2 enzymatic activity FA and BaP are but weakly mutagenic and practically nonmutagenic, respectively. Due to the additivity of the genotoxic effects of FA and BaP, induction of an error-prone condition by the latter compound seems to be excluded.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased frequency of acetaldehyde-induced sister-chromatid exchanges in human lymphocytes treated with an aldehyde dehydrogenase inhibitor.

Acetaldehyde, the first metabolite of ethanol oxidation, in concentrations ranging from 100 microM to 400 microM caused a dose-dependent linear increase in the frequency of sister-chromatid exchanges (SCE) in cultured human peripheral lymphocytes. The SCE frequency was on an average 2-fold higher when the cells were exposed to the acetaldehyde after 24 h incubation instead of at the time of mitogen stimulation (0 h). When acetaldehyde was added together with the potent aldehyde dehydrogenase inhibitor 1-aminocyclopropanol (0.1 mM), the SCE response was significantly (p less than 0.05) increased. The present results indicate that acetaldehyde is metabolized within human lymphocytes, and, moreover, that alcohol consumption during treatment with drugs that inactivate aldehyde dehydrogenase may cause a further increased incidence of acetaldehyde-induced SCE and concomitant lesions.

Genotoxic effects of ethylene oxide and propylene oxide: a comparative study.

The two alkylating agents ethylene oxide (EO) and propylene oxide (PO) were compared for genotoxic effectiveness in various test systems. The study was undertaken partly to shed light on the difference between the compounds found after chronic exposure of monkeys (Lynch et al., 1984) where EO but not PO was able to induce SCE and chromosomal aberrations. In the present study EO was found to be 5-10 times more effective than PO with respect to gene conversion and reverse mutation in Saccharomyces cerevisiae D7 and sister-chromatid conversion in S. cerevisiae RS112. In contrast, the abilities of the two compounds to induce point mutation in S. typhimurium strains and SCE in human lymphocytes were approximately equal. One possible cause of EO being more effective than PO in certain respects, discussed on the basis of inference from earlier studies, is an expected difference in ability to cause strand breaks via alkylation of DNA-phosphate groups.

Induction of sister-chromatid exchanges by direct and indirect mutagens in human lymphocytes, co-cultured with intact rat liver cells. Effect of enzyme induction and preservation of the liver cells by freezing in liquid nitrogen.

An in vitro assay system using intact rat hepatocytes and human peripheral lymphocytes is described which has been developed with the aim of bringing test conditions closer to in vivo conditions, thereby broadening the available battery of simple in vitro assays. A culture vessel, which contains an inner chamber with a semipermeable bottom, has been designed to allow easy removal of the hepatocytes. Determination of sister-chromatid exchange rate was used as the experimental end point. For validation, a series of chemicals were used which have been tested previously in a large interlaboratory investigation of short-term test methods. Our study supplies complementary information to this investigation in as much as some chemicals could be correctly assigned as positive or negative, in contrast to what was found in the earlier tests. Furthermore, we show that the metabolic capacity of both normal and induced liver cells can be preserved in liquid nitrogen for long periods.

Liquid chromatographic determination of the estrogens daidzein, formononetin, coumestrol, and equol in bovine blood plasma and urine.

A liquid chromatographic (LC) method is described for the determination of the plant estrogens diadzein, formononetin, and coumestrol and the estrogenically active metabolite equol in bovine blood plasma and urine. The blood and urine samples are incubated overnight with and without beta-glucuronidase/sulfatase for analysis of both free and conjugated forms of estrogens. Samples are applied to Extrelut columns, extracted with ethyl acetate, and evaporated to dryness. Residues from urine samples are dissolved in methanol, diluted with water, acidified with HCl, and purified by injection through a Sep-Pak C18 cartridge. This eluate is used for LC analysis. Residues from blood samples are dissolved in benzene-petroleum ether (1 + 1), extracted with ammonium hydroxide, acidified with glacial acetic acid, and extracted with ethyl acetate. The ethyl acetate extract is evaporated, dissolved in 80% methanol, injected onto a LC reverse-phase column, and separated in a linear gradient system between 40 and 80% methanol in phosphate buffer. Quantitation is performed by means of UV and fluorescence responses. The method was sensitive enough to determine 0.4 ng/mL of daidzein and formononetin and 0.1 and 13 ng/mL of coumestrol and equol, respectively, in blood, and 130, 80, and 7 ng/mL of daidzein, formononetin, and coumestrol, respectively, and 4 micrograms/mL of equol in urine. The applicability of the method was checked by the determination of total and free plant estrogens in blood samples from a dairy cow fed a normal diet.

Activation of human T cells by neuraminidase-galactose oxidase-treated erythrocytes involving CD2 (T11) and its complementary structure.

Human T lymphocyte proliferation induced by neuraminidase-galactose oxidase (NAGO)-treated autologous erythrocytes (HENAGO) plus polyethylene glycol (PEG) has previously been shown to be independent of accessory cells. Here, we show that the response to HENAGO + PEG was accompanied by interleukin 2 (IL-2) release and was inhibited by anti-IL-2 and anti-IL-2 receptor antibodies. HENAGO alone initiated DNA synthesis together with phorbol ester (12-O-tetradecanoyl-phorbol-13-acetate; TPA). To elucidate the nature of the stimulatory signals NAGO-treated sheep erythrocytes (SENAGO) were used in additional experiments. In parallel to the superior rosetting capacity of SE compared to HE. SENAGO were by themselves stimulatory, and the response was further enhanced by PEG or TPA. Antibody L180/1, specific for the T11 (CD2) target structure (T11TS) on SE, homologous to the human CD2 ligand LFA-3, abolished the response to SENAGO alone or when combined with PEG or TPA. The results suggest that ENAGO induce T-cell response through CD2-LFA-3-T11TS interaction, and via other surface antigens bound by the oxidatively induced aldehyde groups on ENAGO.

Accessory cell-independent activation of T cells by oxidized red blood cells plus polyethylene glycol.

We have previously shown that the combination of neuraminidase-galactose oxidase (NAGO)-treated autologous erythrocytes (EOX) and polyethylene glycol (PEG) is highly mitogenic for human peripheral blood lymphocytes (PBL). In this report, we show that EOX plus PEG-induced T lymphocyte proliferation is independent of HLA-DR and Leu M3-positive accessory cells (AC). Purified T (pT) cells and PBL were equally stimulated by EOX + PEG, while pT cells were unresponsive to the mitogens phytohemagglutinin (PHA), NAGO, and the anti-CD3 antibody UCHT1, even in the presence of PEG. These findings indicate that specific signals from AC may be replaced by unspecific stimuli in T cell activation.

Nicotinamide deficiency and benzamide-induced sister chromatid exchanges.

Benzamides induce sister chromatid exchanges (SCE) in L1210 cells. This induction is strongly potentiated when the cells are grown in nicotinamide-free medium. There is no dependence on the concentration of bromodeoxyuridine (BrdUrd) except at toxic doses, high enough to inhibit BrdUrd incorporation into the DNA. Nicotinamide starvation by itself does not increase the frequency of SCE markedly. These observations are consistent with the notion that BrdUrd and benzamides induce SCE by different mechanisms. The mode of action of benzamides in inducing SCE is still unclear.

Metabolism of zearalenone by sow intestinal mucosa in vitro.

Homogenized intestinal mucosa samples from sows were incubated with zearalenone in the presence of NADPH or UDPGA. In addition, UDPglucuronosyltransferase activity in the microsomal fraction of mucosa was determined using 1-naphthol as substrate. In the presence of NADPH, zearalenone was reduced to both alpha- and beta-zearalenol (0.37 +/- 0.18 and 0.29 +/- 0.11 nmol/mg protein/hr in the duodenum and jejunum, respectively). The beta-isomer was the predominant metabolite. Glucuronide conjugation of zearalenone was very high compared with the level of reduction occurring (11.3 +/- 6.1 and 9.4 +/- 5.8 nmol conjugated/mg protein/hr in the duodenum and jejunum, respectively). There was no correlation between the rates of glucuronide conjugation of zearalenone and 1-naphthol, indicating that they depend upon two different isoenzymes of UDPglucuronosyltransferase.

Effect of T2 toxin on the spontaneous antibody-secreting cells and other non-lymphoid cells in the murine spleen.

The Fusarium secondary metabolite, T2 toxin was investigated for its effects upon spontaneous antibody-producing cells in the murine spleen. Changes in the number of non-lymphoid cells in the spleen were also studied. A statistically significant increase in the number of spontaneous antibody-secreting cells, as detected by the protein A plaque assay, was observed. A dose-dependent increase was found after chronic exposure to T2 toxin for 3 wk. An increased number of erythroblast cells was also found in the spleens of these animals. At all the T2 toxin concentrations studied, the mice showed a dose-dependent reduction in blood levels of haemoglobin. Reduction in the suppression of B cell growth and stimulation of B cells caused by erythropoiesis or activated macrophages could be responsible for the increase in antibody production.

Changes in neck muscles in Swedish reindeer bucks during rutting season.

Three neck muscles in Swedish reindeer bucks have been studied before and during the rutting season. These were M. splenius, M. sternocephalicus and M. brachiocephalicus. For comparison, M. longissimus dorsi was chosen. Fibre composition and fibre size were studied in the four muscles as also was the metabolic potential of three enzymes, representing respiratory chain (cytochrome oxidase), beta-oxidation of fatty acids (3-hydroxyacyl-CoA dehydrogenase) and anaerobic glycolysis (lactate dehydrogenase). The extreme increase in size of certain muscles in the neck in connection with the rutting season (e.g. sternocephalicus, which increases from 250 g to 1,500 g) was to a great extent due to an increase in fibre size. In splenius, all three fibre types studied increased (I, IIA, IIB); in brachiocephalicus, mainly IIA and IIB; and in sternocephalicus, only the IIB. No corresponding fibre increase could be found in longissimus dorsi. In splenius and sternocephalicus from bucks older than 54 months, 60-70% of the fibres were of type I, and in brachiocephalicus, only about 40%. In all muscles but one, oxidative capacity (cytochrome oxidase) and beta-oxidation of fatty acids (3-hydroxyacyl-CoA dehydrogenase) decreased significantly during the rutting season. This indicates that purposes other than the enhancement of energy production by fatty acid oxidation must account for the enlargement of the neck muscles.

Quantitative liquid chromatographic method using fluorescence detection for determining zearalenone and its metabolites in blood plasma and urine.

The liquid chromatographic (LC) method described, suitable for use with both blood plasma and urine, is applicable for determination of zearalenone and alpha-zearalenol at levels as low as 0.5 ng/mL plasma and 5 ng/mL urine. The sample is incubated overnight with beta-glucuronidase to analyze for both conjugated and unconjugated forms of zearalenone. The next day, the sample is acidified with H3PO4, extracted with chloroform, and evaporated to dryness. The residue is dissolved in toluene and loaded onto a silica gel cartridge which is washed with toluene and eluted with toluene-acetone (88 + 12). The eluate is evaporated, and the residue is dissolved in chloroform, extracted with 0.18M NaOH, neutralized with H3PO4, and re-extracted with chloroform. The chloroform extract is evaporated, dissolved in mobile phase for LC, and injected onto a normal phase column under the following chromatographic conditions: mobile phase of water-saturated dichloromethane containing 2% 1-propanol, and fluorescence detector, excitation wave-length 236 nm, and 418 nm cut-off emission filter. Recoveries of zearalenone and its metabolites from blood plasma and urine are 80-89% in the range 2.0-10 ng standard/mL plasma, and 81-90% in the range 10-30 ng standard/mL urine. This method was used to analyze blood and urine samples from a pig fed zearalenone-contaminated feed (5 mg/kg), corresponding to 80 micrograms/kg body weight. Zearalenone was rapidly metabolized to alpha-zearalenol, which appeared in the blood only 30 min after feeding. Almost all zearalenone and alpha-zearalenol was found conjugated with glucuronic acid in both blood plasma and urine.