Changes in Brain Energy Metabolism and Protein Synthesis Following Transient Bilateral Ischemia in the Gerbil

The time course of the reduction in brain protein synthesis following transient bilateral ischemia in the gerbil was characterized and compared with changes in a number of metabolites related to brain energy metabolism. The recovery of brain protein synthesis was similar following ischemic periods of 5, 10, or 20 min; in vitro incorporation activity of brain supernatants was reduced to approximately 10% of control at 10 or 30 min recirculation, showed slight recovery at 60 min, and returned to 60% of control activity by 4 h. Protein synthesis activity was indistinguishable from control at 24 h. One minute of ischemia produced no detectable effect on protein synthesis measured after 30 min reperfusion; longer periods of ischemia resulted in progressive inhibition, with 5 min producing the maximal effect. Pentobarbital (50 mg/kg) increased by 1-2 min the threshold ischemic duration required to produce a given effect. Whereas most metabolites recovered quickly following 5 min ischemia, glycogen showed a delayed recovery comparable to that seen for protein synthesis. These results are discussed in relation to possible mechanisms for the coordinate regulation of brain energy metabolism and protein synthesis. An improved method for the fluorimetric measurement of guanine nucleotides is described.     

Functional, Metabolic, and Circulatory Changes Associated with Seizure Activity in the Postischemic Brain

Abstract: The present study was undertaken to explore how transient ischemia in rats alters cerebral metabolic capacity and how postischemic metabolism and blood flow are coupled during intense activation. After 6 h of recovery following transient forebrain ischemia 15 min in duration, bicuculline seizures were induced, and brains were frozen in situ after 0.5 or 5 min of seizure discharge. At these times, levels of labile tissue metabolites were measured, whereas the cerebral metabolic rate for oxygen (CMRO₂) and cerebral blood flow (CBF) were measured after 5 min of seizure activity. After 6 h of recovery, and before seizures, animals had a 40–50% reduction in CMRO₂, and CBF. However, because CMRO₂ rose threefold and CBF fivefold during seizures, CMRO₂ and CBF during seizures were similar in control and postischemic rats. Changes in labile metabolites due to the preceding ischemia encompassed an increased phosphocreatine/ creatine ratio, as well as raised glucose and glycogen concentrations. Seizures gave rise to minimal metabolic perturbation, essentially comprising reduced glucose and glycogen contents and raised lactate concentrations. It is concluded that although transient ischemia leads to metabolic depression and a fall in CBF, the metabolic capacity of the tissue is retained, and drug-induced seizures lead to a coupled rise in metabolic rate and blood flow.     

CEREBRAL METABOLIC CHANGES DURING PROLONGED EPILEPTIC SEIZURES IN RATS

Abstract— Sustained epileptic seizures were induced in paralysed, artificially ventilated and anaesthetized (70% N₂O) rats by means of intravenous bicuculline (1.2mgkg^?1), and cerebral cortical tissue was frozen in situ after periods varying between 10 s and 2 h for analyses of labile phosphates, glycolytic metabolites, citric acid cycle intermediates, and associated amino acids and ammonia, using enzymic fluorometric techniques. Body temperature was kept at 37°C, and arterial hypotension, arterial hypoxaemia and hypoglycaemia were prevented. Cortical glycogen concentrations fell progressively (to 23% of control levels) between 1 and 20 min after seizure onset but returned to control concentrations after 120 min of seizure activity. Cortical glucose concentration fell to 30% of control after 1 min of seizure activity, remained close to 50% of control for 1 h, and fell again to 30% after 2 h of seizure activity. Cortical lactate concentration was doubled in brains frozen 10 s after bicuculline injection. It rose over the following 20 min, reaching a steady concentration of about 10μmolg^?1 wet wt. The changes in lactate and glucose concentration indicated a 34-fold increase in the rate of glycolysis during the first minute of seizure. Phosphocreatine concentration was reduced by nearly 50% after 10 and 30 s of seizure activity, and subsequently stabilized at a concentration 2/3 of normal. ATP concentration was maximally reduced (by 7%) after 30 s and remained close to normal thereafter. Larger, initial reductions occurred in ATP/ADP and ATP/ AMP ratios, as well as in the adenylate energy charge. All these parameters remained significantly reduced for the rest of the 2 h seizure period. However, the changes were moderate since the energy charge was maintained within 2% of control. Changes in citric acid cycle intermediates included initial reductions in α-ketoglutarate and oxaloacetate (calculated) and progressive increases in fumarate, malate and citrate. After long periods of seizures all citric acid cycle intermediates except oxaloacetate were increased in concentration. Ammonia increased during the first min to reach steady state values of 200% of control. Alanine increased progressively during the first 20 min, to stabilize at 200% of control thereafter. GABA increased at 5 min and subsequently rose to almost twice the control value (120 min). At 20 min and onwards there were progressive decreases in glutamate and aspartate, and a progressive increase in glutamine. The sum of amino acids measured increased significantly and the sum of ammonia equivalents rose substantially. Intracellular pH calculated from the creatine kinase equilibrium decreased by 0.25 units during the first minute. However, since the pH calculated from P_co, and cellular buffer base changes remained close to normal during this period, it is concluded that the components of the creatine kinase reaction were not in equilibrium, and the pH values calculated from this equilibrium were incorrect. Tentative calculations of NADH/NAD⁺ ratios indicated that redox changes of opposite direction occurred in cytoplasm (reduction) and mitochondria (oxidation).     

Comparison of Rates of Local Cerebral Glucose Utilization Determined with Deoxy[l-14C]glucose and Deoxy[6-14C]glucose

The activity of the pentose phosphate shunt pathway in brain is thought to be linked to neurotransmitter metabolism, glutathione reduction, and synthetic pathways requiring NADPH. There is currently no method available to assess flux of glucose through the pentose phosphate pathway in localized regions of the brain of conscious animals in vivo. Because metabolites of deoxy[1-14C]glucose are lost from brain when the experimental period of the deoxy[14C]glucose method exceeds 45 min, the possibility was considered that the loss reflected activity of this shunt pathway and that this hexose might be used to assay regional pentose phosphate shunt pathway activity in brain. Decarboxylation of deoxy[1-14C]glucose by brain extracts was detected in vitro, and small quantities of 14C were recovered in the 6-phosphodeoxygluconate fraction when deoxy[14C]glucose metabolites were isolated from freeze-blown brains and separated by HPLC. Local rates of glucose utilization determined with deoxy[1-14C]glucose and deoxy[6-14C]glucose were, however, similar in 20 brain structures at 45, 60, 90, and 120 min after the pulse, indicating that the rate of loss of 14CO2 from deoxy[1-14C]glucose-6-phosphate in normal adult rat brain is too low to permit assay pentose phosphate shunt activity in vivo. Further metabolism of deoxy[1-14]glucose-6-phosphate via this pathway does not interfere during routine use of the deoxyglucose method or explain the progressive decrease in calculated metabolic rate when the experimental period exceeds 45 min.     

Visualization of spatiotemporal energy dynamics of hippocampal neurons by mass spectrometry during a kainate-induced seizure

We report the use of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry combined with capillary electrophoresis (CE) mass spectrometry to visualize energy metabolism in the mouse hippocampus by imaging energy-related metabolites. We show the distribution patterns of ATP, ADP, and AMP in the hippocampus as well as changes in their amounts and distribution patterns in a murine model of limbic, kainate-induced seizure. As an acute response to kainate administration, we found massive and moderate reductions in ATP and ADP levels, respectively, but no significant changes in AMP levels--especially in cells of the CA3 layer. The results suggest the existence of CA3 neuron-selective energy metabolism at the anhydride bonds of ATP and ADP in the hippocampal neurons during seizure. In addition, metabolome analysis of energy synthesis pathways indicates accelerated glycolysis and possibly TCA cycle activity during seizure, presumably due to the depletion of ATP. Consistent with this result, the observed energy depletion significantly recovered up to 180 min after kainate administration. However, the recovery rate was remarkably low in part of the data-pixel population in the CA3 cell layer region, which likely reflects acute and CA3-selective neural death. Taken together, the present approach successfully revealed the spatiotemporal energy metabolism of the mouse hippocampus at a cellular resolution--both quantitatively and qualitatively. We aim to further elucidate various metabolic processes in the neural system.     

N-Substituted amino acid N'-benzylamides: synthesis, anticonvulsant, and metabolic activities

Amino acid amides (AAA) were prepared and evaluated in seizure models. The AAA displayed moderate-to-excellent activity in the maximal electroshock seizure (MES) test and were devoid of activity in the subcutaneous Metrazol-induced (scMet) seizure test. The AAA anticonvulsant activity was neither strongly influenced by the C(2) substituent nor by the degree of terminal amine substitution. An in vitro metabolism study suggested that the structure-activity relationship pattern was due, in part, to metabolic processes that occurred at the N-terminal amine unit.     

Identification of the in vivo metabolites of the antimalarial arteether by thermospray high-performance liquid chromatography/mass spectrometry.

The thermospray mass spectra of arteether and 16 of its potential metabolites all showed strong [M + NH4]+ ions and with only a few exceptions these compounds also showed spectral peaks corresponding to [M + NH4 - HOR]+ and [M + H - HOR]+, where OR represents the alkoxy or hydroxy group at the 12-position. A method for quantifying the metabolites was developed in which the plasma was spiked with an internal standard (the propyl ether analog of arteether), extracted using a C-18 solid-phase cartridge, then subjected to thermospray high-performance liquid chromatographic/mass spectrometric analysis using selected ion monitoring and a C-18 reversed-phase analytical column. Following the intravenous administration of arteether (11.6 mg kg-1), the plasma was found to contain 12 metabolites of arteether in the 10-1000 ng ml-1 range 15 min post-injection, and within 60 min two of these metabolites attained higher concentrations than that of the parent compound, while several other of the metabolites attained concentrations similar to the parent compound. The pseudo-first-order half-life of arteether was found to be 10.0 +/- 0.6 min, while the apparent half-lives of most of the metabolites were in the 15-30 min range. Nine of these metabolites were identified by comparison to authentic reference standards and the structures of three remaining metabolites were tentatively assigned from their spectral and chromatographic properties. The metabolic pathways leading to these 12 metabolites was a rather complex, multiple-step process, but most of the metabolites arose from an enzymatic oxidation at one of three sites; 3 alpha, 9 alpha, or the CH2 of the side-chain. Conversion of the endoperoxide group to an cyclic ether was not a major pathway. The in vitro antimalarial activity of reference standards of several of the metabolites was determined and all of those tested were found to be active in the low nanogram per milliliter range.     

Dopamine and Serotonin Metabolism in Brain Sites Ipsi- and Contralateral to Direction of Conditioned Turning in Rats

Concentrations of dopamine, serotonin, and some of their metabolites were analyzed by means of HPLC in brain samples obtained from rats operantly conditioned to turn in circles to obtain water reinforcement. In experiment 1 using Wistar rats, no differences in the levels of transmitters or metabolites were detected between brain samples (frontal cortex, ventral striatum, dorsal striatum, septum, amygdala, substantia nigra) from the hemispheres located ipsi- and contralateral to the direction of turning. A higher dopamine metabolism (indicated by higher metabolite/transmitter ratios) in ventral striatum, dorsal striatum, and amygdala was found after 15 min than after 5 min of turning in both hemispheres. A higher dopamine metabolism was found in water-deprived rats compared to nondeprived rats independently of whether or not deprived rats were trained to turn for water reinforcement. In two additional experiments, no differences in dopamine metabolism were found between the ipsi- and contralateral striatum of Wistar rats after 25 min and Sprague-Dawley rats after 10 min of operantly conditioned turning. The present results confirm that dopamine metabolism can change with different behavioral or physiological states; they do not support the hypothesis that conditioned turning is correlated with asymmetrical changes in the metabolism of dopamine or serotonin in the brain.     

The Influence of Bicuculline-Induced Seizures on Free Fatty Acid Concentrations in Cerebral Cortex, Hippocampus, and Cerebellum

Abstract: Using ventilated rats maintained on N₂O-O₂ (70:30, vol/vol) we induced continuous seizures with i.v. bicuculline and analysed free fatty acids (FFA) in cerebral cortex, hippocampus, and cerebellum after seizure durations of 1–120 min. In the cerebral cortex, peak FFA concentrations were observed after 5 min, with a threefold increase in total FFA content. The values then remained unchanged for the next 15-20 min, but decreased thereafter. At 60 and 120 min, total FFA contents were only moderately increased above control. In the initial period, arachidonic acid increased about 10-fold and stearic acid 2- to 3-fold, with little change in palmitic acid and linoleic acid concentrations. At all times, the docosahexenoic acid concentration was markedly increased. Following its massive accumulation at 1 min, arachidonic acid gradually decreased in concentration. Pretreatment of animals with indomethacin did not alter this behaviour. After 20 and 120 min of seizure activity, changes in total and individual FFA concentrations in the hippocampus were similar to those observed in the cerebral cortex. The cerebellum behaved differently. Thus, at 20 min the only significant change was a 5- to 10-fold increase in arachidonic acid concentration and, after 120 min, total and individual FFA concentrations were similar to control values. Furthermore, since the control values for arachidonic acid were much lower in the cerebellum, the 20-min values were only about 20% of those observed in the cerebral cortex and the hippocampus.     

In vitro metabolism of polychlorinated biphenyl congeners by beluga whale (Delphinapterus leucas) and pilot whale (Globicephala melas) and relationship to cytochrome P450 expression

We measured rates of oxidative metabolism of two tetrachlorobiphenyl (TCB) congeners by hepatic microsomes of two marine mammal species, beluga whale and pilot whale, as related to content of selected cytochrome P450 (CYP) forms. Beluga liver microsomes oxidized 3,3',4,4'-TCB at rates averaging 21 and 5 pmol/min per mg for males and females, respectively, while pilot whale samples oxidized this congener at 0.3 pmol/min per mg or less. However, rates of 3,3',4,4'-TCB metabolism correlated with immunodetected CYP1A1 protein content in liver microsomes of both species. The CYP1A inhibitor alpha-naphthoflavone inhibited 3,3',4,4'-TCB metabolism by 40% in beluga, supporting a role for a cetacean CYP1A as a catalyst of this activity. Major metabolites of 3,3',4,4'-TCB generated by beluga liver microsomes were 4-OH-3,3',4',5-TCB and 5-OH-3,3',4,4'-TCB (98% of total), similar to metabolites formed by other species CYP1A1, and suggesting a 4,5-epoxide-TCB intermediate. Liver microsomes of both species metabolized 2,2',5,5'-TCB at rates of 0.2-1.5 pmol/min per mg. Both species also expressed microsomal proteins cross-reactive with antibodies raised against some mammalian CYP2Bs (rabbit; dog), but not others (rat; scup). Whether CYP2B homologues occur and function in cetaceans is uncertain. This study demonstrates that PCBs are metabolized to aqueous-soluble products by cetacean liver enzymes, and that in beluga, rates of metabolism of 3,3',4,4'-TCB are substantially greater than those of 2,2',5,5'-TCB. These directly measured rates generally support the view that PCB metabolism plays a role in shaping the distribution patterns of PCB residues found in cetacean tissue.     

Pregnenolone metabolites in rat testis: endogenous concentrations,and intracellular distribution in whole testes during incubation in vitro

The intracellular movements of pregnenolone in rat testes were investigated. Whole testes were incubated in the presence or absence of pregnenolone (2.5 mM) in the medium for 120 min (in some studies 30, 60, and 90 min). The testes were homogenised, subcellular fractions prepared and analysed in quadruplicate for steroid content by gas chromatography–mass spectrometry with selected ion monitoring. Quantification of pregnenolone and 11 of its metabolites, obtained from non-incubated whole testes, provided values for endogenous amounts. Pregnenolone was the only steroid of quantitative importance found initially in the mitochondrial fraction but was subsequently found in the microsomal fraction, where metabolism occurred. Identification and quantification of metabolites indicated that both classical pathways for testosterone production were operating, with the 4-en-3-oxosteroid pathway predominating. By 120 min, virtually all pregnenolone metabolites, including pregnenolone itself, were found in the cytosol, consistent with an overall movement from mitochondria to endoplasmic reticulum to cytosol.     

Effects of Handling or Immobilization on Plasma Levels of 3,4-Dihydroxyphenylalanine, Catecholamines, and Metabolites in Rats

In conscious animals, handling and immobilization increase plasma levels of the catecholamines norepinephrine (NE) and epinephrine (EPI). This study examined plasma concentrations of endogenous compounds related to catecholamine synthesis and metabolism during and after exposure to these stressors in conscious rats. Plasma levels of 3,4-dihydroxyphenylalanine (DOPA), NE, EPI, and dopamine (DA), the deaminated catechol metabolites 3,4-dihydroxyphenylglycol (DHPG), and 3,4-dihydroxyphenylacetic acid (DOPAC), and their O-methylated derivatives methoxyhydroxyphenylglycol (MHPG) and homovanillic acid (HVA) were measured using liquid chromatography with electrochemical detection at 1, 3, 5, 20, 60, and 120 min of immobilization. By 1 min of immobilization, plasma NE and EPI levels had already reached peak values, and plasma levels of DOPA, DHPG, DOPAC, and MHPG were increased significantly from baseline, whereas plasma DA and HVA levels were unchanged. During the remainder of the immobilization period, the increased levels of DOPA, NE, and EPI were maintained, whereas levels of the metabolites progressively increased. In animals immobilized briefly (5 min), elevated concentrations of the metabolites persisted after release from the restraint, whereas DOPA and catecholamine levels returned to baseline. Gentle handling for 1 min also significantly increased plasma levels of DOPA, NE, EPI, and the NE metabolites DHPG and MHPG, without increasing levels of DA or HVA. The results show that in conscious rats, immobilization or even gentle handling rapidly increases plasma levels of catecholamines, the catecholamine precursor DOPA, and metabolites of NE and DA, indicating rapid increases in the synthesis, release, reuptake, and metabolism of catecholamines.     

Tissue Distribution, Metabolism, Anticonvulsant Efficacy, and Effect on Brain Amino Acid Levels of the Glia-Selective γ-Aminobutyric Acid Transport Inhibitor 4,5,6,7-Tetrahydroisoxazolo[4,5-c]pyridin-3-ol in Mice and Chicks

Using tritium-labelled 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO) its tissue distribution and metabolism were investigated in adult mice and 4-day-old chicks after systemic administration of the drug. It was found not to be significantly metabolized in the brain since metabolites of THPO corresponding to only approximately 8% of the parent compound could be detected 30 min after administration of the drug intramuscularly in mice. In the liver, however, THPO was found to be metabolized to a considerable extent. In chicks THPO metabolites were found in the brain but they accounted for less than 35% of the radioactivity. The brain concentration of THPO in mice and chicks corresponded to respectively 10 and 50% of the dose injected intramuscularly and the tissue level was essentially constant for at least 3 h after injection. Following systemic administration of THPO to mice and chicks the contents of aspartate, glutamate, glutamine, and gamma-aminobutyric acid (GABA) in whole brain and in synaptosomes was determined. It was found that only GABA contents were affected being increased in synaptosomes from mice and decreased in whole brain in chicks. Doses of THPO, which in chicks but not in mice led to brain levels that were sufficient to inhibit glial GABA uptake, were found to protect chicks but not mice against isonicotinic acid hydrazide-induced seizures. The findings are compatible with the notion that THPO exerts its anticonvulsant activity by inhibition of astrocytic GABA uptake.     

Increased dopamine and serotonin metabolites in CSF during severe insulin-induced hypoglycemia in freely moving rats

The effect of insulin on dopamine (DA) and serotonin (5-HT) metabolites was determined in the cerebrospinal fluid (CSF) of the rat and compared with glucose levels in blood and CSF. CSF was continuously withdrawn from the third ventricle of freely moving rats at a constant rate of 1 μl/min. Liquid chromatography with electrochemical detection was used for the direct assay of DA and 5-HT metabolites in the CSF. The metabolites were stable during the first hour after insulin injection (6IU/Kg). A progressive increase occurred thereafter in animals which had no access to food during the time of the experiment. The maximal effect was observed 2.5 h after insulin, with respective mean increases of 80% for dihydroxyphenylacetic acid, 47% for homovanillic acid and 33% for 5-hydroxyindolacetic acid. These increases in monoamine metabolites were not observed when rats received glucose (5g/Kg ip) 45 min after insulin or when food was made available. The period for insulin-induced increase in DA and 5-HT metabolites corresponded to a maximal fall of glucose levels both in blood and CSF although the CSF glucose decrease was delayed when compared to the fall of blood glucose. The role of brain glucose and brain insulin in the control of central DA and 5-HT metabolism is discussed.     

Comparative metabolism of 7H-dibenzo[c,g]carbazole and dibenz[a,j]acridine by mouse and rat liver microsomes.

The comparative metabolism of the carcinogenic pollutants 7H-dibenzo[c,g]-carbazole (DBC) and dibenz[a,j]acridine (DBA) was investigated in vitro using 3-methylcholanthrene (3MC) induced Sprague-Dawley rat and Hsd:ICR(Br) mouse liver microsomal preparations with benzo[a]pyrene (BaP) as the positive control. Metabolites were isolated and separated by HPLC and identified by spectroscopic and co-chromatographic techniques using synthetic standards. The major metabolites of DBC were the phenols: the 5-OH-DBC, 3-OH-DBC, and 2-OH-DBC. Traces of 1-OH-DBC were also found yet no dihydrodiols were identified. The major metabolites of DBA were the 3,4-diol-DBA and 5,6-diol-DBA, 1,2-diol-DBA, DBA-5,6-oxide and 4-OH-DBA. Treatment of both mice and rats with 3MC resulted in significant (P less than or equal to 0.05) increases relative to control in the microsomal metabolism of DBA to dihydrodiol and phenol metabolites, similar to that observed for BaP. 3MC-induced rat liver microsomes significantly (P less than or equal to 0.05) increased DBC metabolism relative to control microsomes whereas DBC metabolism was not increased with 3MC-induced mouse liver microsomes. These data indicate that different enzymatic pathways are involved in the metabolic activation of DBC in the Hsd:ICR(Br) mouse and Sprague-Dawley rat.     

The regulation of arachidonic acid metabolism in human first trimester trophoblast by cyclic AMP

Human trophoblast cells are known to release a range of arachidonic acid metabolites into culture medium, including cyclo-oxygenase, lipoxygenase and epoxygenase products. In this study we investigated the effects of dibutyryl cyclic AMP (db cAMP) on arachidonic acid metabolism in human first trimester trophoblast cells, and also determined the distribution of metabolites between intracellular and extracellular compartments. db cAMP increased intracellular levels of radioactivity within 2 min, and extracellular levels of radioactivity were increased after 30 min. These changes were reflected in increased levels of arachidonic acid metabolites in both compartments, indicating that arachidonic acid was metabolised. db cAMP increased intracellular levels of 5,6-epoxyeicosatrienoic acid (5,6-EpETrE) within 2 min of addition to cultured cells. No changes were detected after 5-10 min, but substantial changes were found 30 min after the addition of db cAMP. The dihydroxyeicosatrienoic acid (DiHETrE) breakdown products also increased with similar kinetics. In contrast, levels of 14,15-EpETrE increased after 5-10 min.     

Progesterone metabolism in the gastric mucosa microsomes of guinea pig

The microsomes from guinea pig gastric mucosa were found to convert [4-14C]progesterone to two major metabolites in the presence of NADPH. The gastric metabolizing activity was the highest among the gastrointestinal tissues of guinea pig. 5 alpha-Pregnane-3,20-dione and 3 beta-hydroxy-5 alpha-pregnan-20-one were identified as the major metabolites by thin-layer chromatography and crystallization to constant specific activity, suggesting the presence of steroid 5 alpha-reductase and 3 beta-hydroxysteroid dehydrogenase activities in the gastric mucosa microsomes. Furthermore, time course of progesterone metabolism and analysis of 5 alpha-pregnane-3,20-dione metabolites suggest that the gastric progesterone metabolism is initiated by 5 alpha-reductase and followed by 3 beta-hydroxysteroid dehydrogenase. The progesterone-metabolizing activity was strongly inhibited by SKF 525-A and disulfiram. The activity was also inhibited by methyrapone to a somewhat lesser extent than the above inhibitors. From gastric mucosa microsomes, the progesterone-metabolizing activity was successfully solubilized with 2% digitonin using 0.1 M potassium chloride and 1 mM dithiothreitol, 0.4 mM NADPH and 20% glycerol as stabilizers for the solubilized activity. Among these stabilizers, glycerol was found to be most effective for stabilizing the activity of the solubilized microsomes.     

Cryopreserved rat liver slices: a critical evaluation of cell viability, histological integrity, and drug-metabolizing enzymes

The effects of a cryopreservation procedure on the biochemical, morphological and functional integrity of rat liver slices just after thawing and after 24 h culture were evaluated. Freshly prepared slices were incubated in modified University of Wisconsin solution containing 50% fetal calf serum and 10% dimethyl sulfoxide for 20 min on ice prior to a rapid cooling in liquid nitrogen. After 10-40 days, slices were thawed rapidly at 42 degrees C. Total protein content and (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) (MTT) reduction were well preserved at thawing, whereas ATP content was markedly decreased relative to freshly prepared slices (-83%). The major microscopic findings in sections of just-thawed liver slices consisted of hepatocellular dissociation and minimal apoptosis. The qualitative profile of antipyrine (AP) metabolism was well preserved in cryopreserved slices, but the amounts of phase I and phase II AP metabolites produced over a 3-h incubation period were markedly reduced relative to fresh slices (-58 to -71%). When cryopreserved slices were cultured for 24 h after thawing, the viability was markedly reduced, as reflected by the almost complete absence of MTT reduction and the loss of ATP content. Histological examinations showed extensive cellular necrosis. The amount of AP metabolites produced by cryopreserved slices was similar after a 3- or a 24-h culture period, indicating that AP metabolism capacities were lost at 24 h culture. In conclusion, our results suggest that cryopreserved rat liver slices may be a useful model for short-term in vitro determination of drug metabolism pathways. Further work is required to extend their use for toxicological studies.     

Monitoring the specific activities of dopamine and its metabolites in striatum and olfactory tubercle after intravenous administration of l-[3H]tyrosine: Complex relations,indicating more than two compartments

It is still a matter of debate whether in dopaminergic nerve endings dopamine (DA) is present in different functional and/or metabolic compartments. To investigate this, DA metabolism was studied in vivo by measuring the specific activity of DA and its metabolites after intravenous administration of l-[3,5-³H]tyrosine (200 μCi/rat) to freely moving animals. The incorporation of ³H into DA and metabolites was determined in striatum and olfactory tubercle at 5, 10, 20, 40, 60 and 80 min after [³H]tyrosine administration. In both structures the level of [³H]tyrosine initially declined monoexponentially, but deviated from that pattern later on. The curves representing the formation in time of [³H]DA and [³H]metabolites were very similar in both structures, although as a whole, the levels in the olfactory tubercle were higher. The relative patterns of the specific activities of DA and those of its metabolites, a possible clue to DA compartmentation, neither indicated a clearcut metabolic one-compartment, nor a two-compartment system. The flow of radioactivity through DA metabolism could in fact only be explained by assuming more complex metabolic relations.     

Analysis of the compartmentation of glycolytic intermediates, nucleotides, sugars, organic acids, amino acids, and sugar alcohols in potato tubers using a nonaqueous fractionation method

The compartmentation of metabolism in heterotrophic plant tissues is poorly understood due to the lack of data on metabolite distributions and fluxes between subcellular organelles. The main reason for this is the lack of suitable experimental methods with which intracellular metabolism can be measured. Here, we describe a nonaqueous fractionation method that allows the subcellular distributions of metabolites in developing potato (Solanum tuberosum L. cv Desiree) tubers to be calculated. In addition, we have coupled this fractionation method to a recently described gas chromatography-mass spectrometry procedure that allows the measurement of a wide range of small metabolites. To calculate the subcellular metabolite concentrations, we have analyzed organelle volumes in growing potato tubers using electron microscopy. The relative volume distributions in tubers are very similar to the ones for source leaves. More than 60% of most sugars, sugar alcohols, organic acids, and amino acids were found in the vacuole, although the concentrations of these metabolites is often higher in the cytosol. Significant amounts of the substrates for starch biosynthesis, hexose phosphates, and ATP were found in the plastid. However, pyrophosphate was located almost exclusively in the cytosol. Calculation of the mass action ratios of sucrose synthase, UDP-glucose pyrophosphorylase, phosphoglucosisomerase, and phosphoglucomutase indicate that these enzymes are close to equilibrium in developing potato tubers. However, due to the low plastidic pyrophosphate concentration, the reaction catalyzed by ADP-glucose pyrophosphorylase was estimated to be far removed from equilibrium.