Fraction Of Rat Liver Research Articles

Comparative metabolism of warfarin in rats and chickens

Warfarin, a coumarin rodenticide, is commonly used worldwide for rodent control, and is often reported as the cause for poisoning accidents in nontarget animals, in particular bird species. However, the metabolism of warfarin in birds is still unclear. In a previous study, we found an unknown warfarin metabolite in chicken cytosolic fractions. In the present study, we aimed to clarify the cytosolic warfarin metabolites in chickens compared with those in rats. The cytosol fractions of both chicken and rat livers showed the metabolic activity of 2 diastereomers and 2 enantiomers of warfarin alcohol. In chicken cytosol, we found that the production level of (S)-warfarin-(S)-alcohol was markedly higher (32-fold) than that in rat cytosol. From the results of the inhibition assay, we finally suggest that aldehyde oxidase may mainly contribute to the warfarin alcohol products in chicken cytosol.

Kinetics of ethylene and ethylene oxide in subcellular fractions of lungs and livers of male B6C3F1 mice and male fischer 344 rats and of human livers.

Ethylene (ET) is metabolized in mammals to the carcinogenic ethylene oxide (EO). Although both gases are of high industrial relevance, only limited data exist on the toxicokinetics of ET in mice and of EO in humans. Metabolism of ET is related to cytochrome P450-dependent mono-oxygenase (CYP) and of EO to epoxide hydrolase (EH) and glutathione S-transferase (GST). Kinetics of ET metabolism to EO and of elimination of EO were investigated in headspace vessels containing incubations of subcellular fractions of mouse, rat, or human liver or of mouse or rat lung. CYP-associated metabolism of ET and GST-related metabolism of EO were found in microsomes and cytosol, respectively, of each species. EH-related metabolism of EO was not detectable in hepatic microsomes of rats and mice but obeyed saturation kinetics in hepatic microsomes of humans. In ET-exposed liver microsomes, metabolism of ET to EO followed Michaelis-Menten-like kinetics. Mean values of Vmax [nmol/(min·mg protein)] and of the apparent Michaelis constant (Km [mmol/l ET in microsomal suspension]) were 0.567 and 0.0093 (mouse), 0.401 and 0.031 (rat), and 0.219 and 0.013 (human). In lung microsomes, Vmax values were 0.073 (mouse) and 0.055 (rat). During ET exposure, the rate of EO production decreased rapidly. By modeling a suicide inhibition mechanism, rate constants for CYP-mediated catalysis and CYP inactivation were estimated. In liver cytosol, mean GST activities to EO expressed as Vmax/Km [μl/(min·mg protein)] were 27.90 (mouse), 5.30 (rat), and 1.14 (human). The parameters are most relevant for reducing uncertainties in the risk assessment of ET and EO.

Biodegradation of tetrabromobisphenol A by oxidases in basidiomycetous fungi and estrogenic activity of the biotransformation products

Tetrabromobisphenol A (TBBPA) degradation was investigated using white rot fungi and their oxidative enzymes. Strains of the Trametes, Pleurotus, Bjerkandera and Dichomitus genera eliminated almost 1mM TBBPA within 4days. Laccase, whose role in TBBPA degradation was demonstrated in fungal cultures, was applied to TBBPA degradation alone and in combination with cellobiose dehydrogenase from Sclerotium rolfsii. Purified laccase from Trametes versicolor degraded approximately 2mM TBBPA within 5h, while the addition of cellobiose dehydrogenase increased the degradation rate to almost 2.5mM within 3h. Laccase was used to prepare TBBPA metabolites 2,6-dibromo-4-(2-hydroxypropane-2-yl) phenol (1), 2,6-dibromo-4-(2-methoxypropane-2-yl) phenol (2) and 1-(3,5-dibromo-4-hydroxyphen-1-yl)-2,2′,6,6′-tetrabromo-4,4′-isopropylidene diphenol (3). As compounds 1 and 3 were identical to the TBBPA metabolites prepared by using rat and human liver fractions (Zalko et al., 2006), laccase can provide a simple means of preparing these metabolites for toxicity studies. Products 1 and 2 exhibited estrogenic effects, unlike TBBPA, but lower cell toxicity.

Combination of Micellar Electrokinetic and High-Performance Liquid Chromatographies to Assess Age-Related Changes in the In Vitro Metabolism of Fischer 344 Rat Liver

The metabolism of doxorubicin, a widely used anticancer drug, is different in young adult and old cancer patients. In this study, we demonstrate that micellar electrokinetic chromatography with laser-induced fluorescence detection is highly suited to monitor the metabolism of doxorubicin in subcellular fractions isolated from young adult (11 months, 100% survival rate) and old (26 months, ~25% survival rate) Fischer 344 rat livers. The relative amounts of doxorubicin metabolized in both mitochondria-enriched and postmitochondria fractions of young adult were larger than the respective fractions of old rat liver. 7-Deoxydoxorubicinolone and 7-deoxydoxorubicinone were identified using internal standard addition and structural elucidation by high-performance liquid chromatography with combined laser-induced fluorescence and mass spectrometry detection. Although high-performance liquid chromatography with combined laser-induced fluorescence and mass spectrometry detection is more useful in the identification of compounds, micellar electrokinetic chromatography with laser-induced fluorescence detection has low-sample requirements, simplified sample processing procedures, short analysis times and low limit of detection. Therefore, the combination of these two techniques provides a powerful approach to investigate metabolism of fluorescent drugs in aging studies.

Design, Synthesis, ADME Properties, and Pharmacological Activities of β‐Alanyl‐D‐histidine (D‐Carnosine) Prodrugs with Improved Bioavailability

β-Alanyl-D-histidine (D-CAR, the enantiomer of the natural dipeptide carnosine) is a selective and potent sequestering agent of reactive carbonyl species (RCS) that is stable against carnosinase, but is poorly absorbed in the gastrointestinal tract. Herein we report a drug discovery approach aimed at increasing the oral bioavailability of D-CAR. In our study we designed, synthesized, and evaluated a series of novel lipophilic D-CAR prodrugs. The considered prodrugs can be divided into two categories: 1) derivatives with both terminal groups modified, in which the carboxyl terminus is always esterified while the amino terminus is protected by an amidic (N-acetyl derivatives) or a carbamate (ethyloxy or benzyloxy derivatives) function; 2) derivatives with only one terminus modified, which can be alkyl esters as well as amidic or carbamate derivatives. The prodrugs were designed considering their expected lipophilicity and their hydrolysis predicted by docking simulations on the most important human carboxylesterase (hCES1). The stability and metabolic profile of the prodrugs were studied by incubating them with rat and human serum and liver fractions. The octyl ester of D-CAR (compound 13) was chosen as a candidate for further pharmacological studies due to its rapid hydrolysis to the bioactive metabolite in vitro. Pharmacokinetic studies in rats confirmed the in vitro data and demonstrated that the oral bioavailability of D-CAR is increased 2.6-fold if given as an octyl ester relative to D-CAR. Compound 13 was then found to dose-dependently (at daily doses of 3 and 30 mg kg(-1) equivalent of D-CAR) decrease the development of hypertension and dyslipidemia, to restore renal functions of Zucker fa/fa obese rats, and to inhibit the carbonylation process (AGEs and pentosidine) as well as oxidative stress (urinary 8-epi-prostaglandin F2α and nitrotyrosine). A plausible mechanism underlying the protective effects of 13 is RCS sequestration, as evidenced by the significant increase in the level of adduct between CAR and 4-hydroxy-trans-2-nonenal (HNE, the main RCS generated by lipid oxidation) in the urine of treated animals.

Effect of diazepam and chlordiazepoxide on the metabolism of other drugs

Abstract Diazepam and chlordiazepoxide, even at high single doses, do not inhibit the metabolic transformation of p-nitroanisol, aniline and aminopyrine by the 9,000 g fraction of rat liver. Although diazepam and chlordiazepoxide increase the sleeping time induced by pentobarbitone, this effect is not explained by an increase in the brain level of pentobarbitone. Repeated administrations of the two benzodiazepines increase the metabolism of p-nitroanisol, aniline, amidopyrine and shorten the sleeping time induced by pentobarbitone in rats. There was a concomitant reduction of the concentration of brain pentobarbitone.

Some aspects of the metabolism of morphine-N-oxide.

Abstract After administration of morphine-N-oxide (MNO) to rats the opiates appearing in the urine were morphine (61%) and MNO (39%). After administration of morphine, the urinary opiates were morphine (80%) and normorphine (20%). When tacrine was given with morphine the urine also contained MNO (46% of total urinary opiates) and the amount of normorphine was much decreased (to 1%), the remainder being morphine (53%). Tacrine and amiphenazole inhibited demethylation of morphine and codeine by a rat liver fraction in vitro. MNO had weak inhibitory activity. Neither MNO nor codeine-N-oxide were demethylated in vitro.

Butobarbitone metabolism in man: identification of 3î-ketobutobarbitone

Abstract After single 200 mg oral doses of butobarbitone (I) in man, 5·4% was excreted in urine unchanged and 28·2% as the 3î-hydroxy metabolite (II) in six days. A new metabolite, the ketone (III), was detected in urine following oral administration both of I and of II. Oxidation of the hydroxy to the ketone metabolite was demonstrated in vitro with the 9000g fraction of rat liver. Use was made of selective ion monitoring g.c.-m.s. methods in metabolite detection.

Human Multiple Inositol Polyphosphate Phosphatase as Apoptosis Biomarker

The multiple inositol polyphosphate phosphatase (MINPP) which regulates cellular levels of inositol phosphates, is located on chromosome 10q23 in human cells and is a member of histidine phosphatases family. In this study, we have determined the antigenicity of MINPP. We proposed different possible peptides and designed the experimental methodology to produce MINPP polyclonal antibodies. To further elucidate the physiological role of MINPP we have characterized the human MINPP antibodies using direct western immunoblotting of bacteria (E. coli), yeast (Saccharomyces cerevisae) expressing MINPP. SDS‐PAGE identified 58 KDa protein band using mammalian pancreatic cancer cell lysates (Panc1) and homogenate from rat liver and microsomal fraction of rat liver and kidney after immunoprecipitations with MINPP antibodies. The MINPP enzyme activity was determined by two methods. First, inositol phosphates separation by column chromatography using the microsomal fraction of rat liver and kidney, total cell lysates from rat liver in a scintillation counter using tritiated‐inositol (3H‐inositol). Second, a customized colorimetric method was developed without any radio‐active 3H ‐inositol using 96 wells plate reader. Using different apoptosis inducers, we have seen that MINPP expression increased in programmed cell death in a dose dependent manner. Supported by University of Arkansas at Little Rock.

A Highly Sensitive RP-HPLC-Fluorescence Method to Study Aldehyde Oxidase Activity

Aldehyde oxidase is a widely distributed enzyme that is involved in the metabolism of an extensive range of aldehydes and N-heterocyclic compounds with physiological, pharmacological, and toxicological relevance. In the present study, a highly sensitive RP-HPLC-fluorescence method based on the oxidation of phenanthridine to phenanthridinone has been developed and validated to assay aldehyde oxidase activity in biological samples. Determination of phenanthridinone was achieved on a C18 column using 10 mmol/L phosphate buffer (pH 5.0) containing 0.1 mmol/L EDTA-acetonitrile (40 + 60, v/v) as the mobile phase. The fluorescence intensity of phenanthridinone was measured at 364 nm with excitation at 236 nm. The proposed method was precise, accurate, specific and rapid (analysis time, approximately 8 min) with a mean RSD of 2.54%. Peak responses were linear from 0.5 to 100 nmol/L, with an LOD of 0.125 nmol/L. The applicability of the method was demonstrated by measurement of aldehyde oxidase activity in rat liver, kidney, ovary, and heart fractions.

Analysis of Enoyl-Coenzyme A Hydratase Activity and Its Stereospecificity Using High-Performance Liquid Chromatography Equipped with Chiral Separation Column

Enoyl-coenzyme A (CoA) hydratase catalyzes the hydration of trans-2-enoyl-CoA to yield 3-hydroxyacyl-CoA during fatty acid degradation (β-oxidation). Although much research has focused on the stereospecificities of 2-enoyl-CoA hydratases, a direct quantification of the production of 3(R)- and 3(S)-hydroxyacyl-CoA has not yet been established. Therefore, we developed a method of concurrently quantifying 3(R)- and 3(S)-hydroxyacyl-CoA using high-performance liquid chromatography (HPLC) equipped with a chiral separation column. The optimized conditions for the separation of 3(R)-, 3(S)-hydroxyhexadecanoyl-CoA and trans-2-hexadecenoyl-CoA, were determined to be as follows: mobile phase of 35/65 (v/v) of 50 mM phosphate buffer (pH 5.0)/methanol; flow rate of 0.5 mL/min; detection at 260 nm; and column temperature of 25°C. This method was applied to subcellular fractions of rat liver; the results directly confirmed that 3(S)-hydroxyhexadecanoyl-CoA is the dominant product obtained from the heat-stable enoyl-CoA hydratase-catalyzed reaction of trans-2-hexadecenoyl-CoA. Finally, the stereospecificities of L-bifunctional protein (L-BP) and D-bifunctional protein (D-BP) were reinvestigated using this method, and it was confirmed that L- and D-BP yielded 3(S)- and 3(R)-hydroxyhexadecanoyl-CoA were yielded from trans-2-hexadecenoyl-CoA, respectively. 3(R)-Hydroxyacyl-CoA is a peroxisomal β-oxidation-specific intermediate. Therefore, this method is potentially useful not only studies regarding the stereochemistry of enoyl-CoA hydratase but also for the diagnosis of diseases caused by defects of peroxisomal enoyl-CoA hydratase.

In vitro biotransformation and investigation of metabolic enzymes possibly responsible for the metabolism of bisdesoxyolaquindox in the liver fractions of rats, chicken, and pigs

Bisdesoxyolaquindox is a reduced metabolite of olaquindox which is used as a medicinal feed additive in veterinary medicine. The relevant metabolism studies of bisdesoxyolaquindox have been carried out for the first time in rat, chicken, and pig liver subcellular fractions in order to understand the metabolic enzymes that are possibly responsible for the metabolism of olaquindox. The metabolites were characterized by high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry. The major metabolic pathways of bisdesoxyolaquindox in the three species were the oxidation of hydroxyl to bisdesoxyolaquindox-2′-carboxyl acid (O10) and the N-dealkylation of the side chain to 3-methylquinoxaline 2-carboxamide (O12). Other metabolic pathways were also proposed which involved the direct methyl oxidation and N-oxide on the quinoxaline ring in the three species as well as N-hydroxylation only in rat. The intrinsic clearance values in the liver microsomes for O10 and O12 were ranked in the order of chicken > pig ≫ rat and rat > pig ≫ chicken, respectively. Inhibition studies indicated that 8-methoxypsoralen, 4-methylpyrazole and α-naphthoflavone could inhibit the formations of O10 and O12 in all species. Quinidine, troleandomycin, diethyldithiocarbamate, and disulfiram showed an interspecies difference in the inhibition of the formation of two metabolites. In rat and pig liver cytosol, 4-methylpyrazole, menadione and chlorpromazine strongly inhibited the formation of O10. Both diethyldithiocarbamate and disulfiram were found to inhibit O10 formation in rat cytosol but not in pig cytosol. These results indicated the following: In rat liver microsomes, CYP2A might be involved in the formation of O10, and CYP1A, CYP2A and CYP2E would be involved in the O12 formation. In pig liver microsomes, CYP1A and CYP2E might catalyze the formations of O10 and O12. In rat cytosol, alcohol dehydrogenase, aldehyde oxidase and aldehyde dehydrogenase should catalyze the O10 formation. In pig cytosol, alcohol dehydrogenase and aldehyde oxidase might be involved in the formation of O10. In chicken, it was found that various CYP isoenzymes were capable of catalyzing the two reactions; none of the inhibitors of cytosol enzymes inhibited O10 formation in chicken cytosol.

Mitochondrion is a novel site of biotransformation of dichloroacetate by glutathione transferase zeta

Dichloroacetate (DCA) is an environmental contaminant as well as a potential therapeutic agent through its ability to stimulate the pyruvate dehydrogenase complex. Repeated doses of DCA result in reduced drug clearance, probably through inhibition of glutathione transferase zeta 1 (GSTz1), a cytosolic enzyme that converts DCA to glyoxylate. Because DCA is known to be taken up into mitochondria, its principal site of action, it is of interest to explore the role of mitochondria in DCA biotransformation. Incubation of human and rat liver mitochondria with 14C‐DCA resulted in biotransformation only when glutathione was added, and glyoxylate was identified as the major metabolite. Protein expression of GSTz was detected in human and rat liver mitochondria by Western Blotting against human GSTz1 antibody. In rats, the expression of GSTz per gram of liver is 14 times as much in cytosol as in mitochondria. Similar to cytosolic GSTz, the expression and activity of mitochondrial GSTz was markedly reduced in rats treated with 500 mg DCA/kg for 8 weeks. Studies of sub‐mitochondrial fractions of rat liver showed that GSTz is localized in the mitochondrial matrix. We conclude that the mitochondrion is a site of DCA biotransformation catalyzed by GSTz, an enzyme shown for the first time to be co‐localized in cytosol and mitochondrial matrix. Supported in part by the US Public Health Service, ES 07355, ES 014617, and 1UL1 RR029890.

Cysteine S-conjugate β-lyases: important roles in the metabolism of naturally occurring sulfur and selenium-containing compounds, xenobiotics and anticancer agents

Cysteine S-conjugate β-lyases are pyridoxal 5'-phosphate-containing enzymes that catalyze β-elimination reactions with cysteine S-conjugates that possess a good leaving group in the β-position. The end products are aminoacrylate and a sulfur-containing fragment. The aminoacrylate tautomerizes and hydrolyzes to pyruvate and ammonia. The mammalian cysteine S-conjugate β-lyases thus far identified are enzymes involved in amino acid metabolism that catalyze β-lyase reactions as non-physiological side reactions. Most are aminotransferases. In some cases the lyase is inactivated by reaction products. The cysteine S-conjugate β-lyases are of much interest to toxicologists because they play an important key role in the bioactivation (toxication) of halogenated alkenes, some of which are produced on an industrial scale and are environmental contaminants. The cysteine S-conjugate β-lyases have been reviewed in this journal previously (Cooper and Pinto in Amino Acids 30:1-15, 2006). Here, we focus on more recent findings regarding: (1) the identification of enzymes associated with high-M(r) cysteine S-conjugate β-lyases in the cytosolic and mitochondrial fractions of rat liver and kidney; (2) the mechanism of syncatalytic inactivation of rat liver mitochondrial aspartate aminotransferase by the nephrotoxic β-lyase substrate S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (the cysteine S-conjugate of tetrafluoroethylene); (3) toxicant channeling of reactive fragments from the active site of mitochondrial aspartate aminotransferase to susceptible proteins in the mitochondria; (4) the involvement of cysteine S-conjugate β-lyases in the metabolism/bioactivation of drugs and natural products; and (5) the role of cysteine S-conjugate β-lyases in the metabolism of selenocysteine Se-conjugates. This review emphasizes the fact that the cysteine S-conjugate β-lyases are biologically more important than hitherto appreciated.

Identification of nevadensin as an important herb-based constituent inhibiting estragole bioactivation and physiology-based biokinetic modeling of its possible in vivo effect

Estragole is a natural constituent of several herbs and spices including sweet basil. In rodent bioassays, estragole induces hepatomas, an effect ascribed to estragole bioactivation to 1′-sulfooxyestragole resulting in DNA adduct formation. The present paper identifies nevadensin as a basil constituent able to inhibit DNA adduct formation in rat hepatocytes exposed to the proximate carcinogen 1′-hydroxyestragole and nevadensin. This inhibition occurs at the level of sulfotransferase (SULT)-mediated bioactivation of 1′-hydroxyestragole. The Ki for SULT inhibition by nevadensin was 4 nM in male rat and human liver fractions. Furthermore, nevadensin up to 20 μM did not inhibit 1′-hydroxyestragole detoxification by glucuronidation and oxidation. The inhibition of SULT by nevadensin was incorporated into the recently developed physiologically based biokinetic (PBBK) rat and human models for estragole bioactivation and detoxification. The results predict that co-administration of estragole at a level inducing hepatic tumors in vivo (50 mg/kg bw) with nevadensin at a molar ratio of 0.06, representing the ratio of their occurrence in basil, results in almost 100% inhibition of the ultimate carcinogen 1′-sulfooxyestragole when assuming 100% uptake of nevadensin. Assuming 1% uptake, inhibition would still amount to more than 83%. Altogether these data point at a nevadensin-mediated inhibition of the formation of the ultimate carcinogenic metabolite of estragole, without reducing the capacity to detoxify 1′-hydroxyestragole via glucuronidation or oxidation. These data also point at a potential reduction of the cancer risk when estragole exposure occurs within a food matrix containing SULT inhibitors compared to what is observed upon exposure to pure estragole.

The riddle of mitochondrial caspase-3 from liver

Caspase-3 is one of the main executors of apoptosis. Its zymogen procaspase-3 was localized to cytosol, mitochondria and nuclei. The subcellular location of procaspase-3 in liver was reported by several studies to be either cytosolic or cytosolic and mitochondrial. Our aim was to investigate these separate procaspase-3 pools to differentiate the pathways of their activation. By cell fractionation, immunocytochemistry, and confocal microscopy we report that there is a single procaspase-3 pool located to the cytosol in primary hepatocytes and in fractions of rat liver. In contrast, it depends on the isolation purity whether procaspase-3 is located in mitochondria of non-parenchymal liver cells, or not. All preparations with mitochondrial procaspase-3 fractions contain traces of haemoglobin, indicating the presence of some erythrocytes, which are the source of mitochondrial procaspase-3. Since erythrocytes migrate with mitochondria in subcellular fractionations, it is important to check for haemoglobin, before localizing the protein to mitochondria.

Sulfation of N-acyl dopamines in rat tissues

Sulfation of N-acyl dopamines has been shown for the first time in cytosolic fractions of rat liver and nervous system. Sulfation of dopamine amides of docosahexaenoic and oleic acids occurred in all tissues studied, N-arachidonoyl dopamine was sulfated in the liver and spinal cord, and N-stearoyl dopamine was sulfated only in the liver. Depending on the substrate and tissue, the sulfation activity varied from 0.5 to 3.5 nmol/min per mg total protein. Kinetic parameters of N-docosahexaenoyl dopamine sulfation in the brain were determined. The findings characterize the sulfation system as the most productive metabolic pathway of N-acyl dopamines, but the role of this system in the body is unclear because of high K(m) value.

STUDIES IN IRON METABOLISM

An enzyme system incorporating inorganic iron into protoporphyrin has been demonstrated in human and rat liver, kidney, spleen and in human bone marrow and measured, using radioiron as a tracer. Studies on the enzyme content of the intracellular fractions of human and rat liver showed that while there was some activity in all fractions, the mitochondrial fraction was the most active. Dialysis of tissue homogenates led to a loss of enzymic activity which could be restored by the addition of ascorbic acid, GSH, cysteine or DPNH. The effect of these substances was not entirely due to an effect of reduction of the ferric iron since potentiation of activity resulted when these substances were added to a system containing iron in the ferrous form. Observations have also been made on the in vitro biosynthesis of haem from protoporphyrin and iron bound to siderophilin or to rat liver homogenate. Rat liver homogenate or a preparation from it of mitochondria served as the source of iron-incorporating enzyme. The reducing substances namely ascorbic acid, GSH, cysteine and DPNH, which were effective in increasing the incorporation into protoporphyrin of inorganic ferric iron also substantially increased the transfer of protein bound iron from haem biosynthesis. ATP enhanced the transfer of iron bound to siderophilin for incorporation into protophyrin but had no effect on the transfer of iron bound to rat liver protein. From this it is clear that physiological reducing substances play an important role in the transfer of iron from its protein-bound form for incorporation into protoporphyrin in the process of haem biosynthesis. A technique has been established for the measurement of iron incorporating enzyme activity in haemolysates of rabbit bone marrow containing a known number of red cell precursors. The method has been applied to marrows of normal and bled rabbits. In the bled animal, the enzymic activity of a unit number of cells was greatly decreased as compared to normal but was increased by the addition of reducing substances. This would indicate that the marrow responds to blood loss by an increase in the production of cells rather than an increased capacity of each cell to form haemoglobin. The enzymic activity of liver homogenates was also measured in normal and bled rabbits. In this tissue there was no difference in the enzymic activity per gram of liver between the normal and the bled animals. There was, however, a marked decrease in iron incorporation by liver cells from animals which had been injected with 300 mgs. Imferon. In two rabbits made iron deficient and anaemic there was an increase in iron incorporation as compared to normal. From this it is clear that gross changes in the size of the iron pool will vitiate the correct interpretation of results on the measurement of haem synthesis using radio-iron as a tracer. Nakao et al (1960) have suggested that plasma samples from a variety of haematological disorders possess a factor (or factors) stimulating haem synthesis as compared to normal plasma. The 'haem-stimulating activity' was measured by an in vitro technique in which radio-iron incorporation was used as a measure of haem synthesis. This work has been repeated using the same technique and results show that any marked increase in haem formation, measured in this way, could be explained by the diminution of the residual iron-binding capacity of the plasma sample. It is concluded that no definite evidence has yet been obtained for the presence of a specific haem-stimulating factor in human plasma. A statistically significant difference has been shown between the absorption of radio-iron given along with a standard meal, between patients with acid present in their gastric juice and patients with a histamine-fast achlorhydria. Both of these groups of patients were anaemic and there was an absence of stainable iron in the bone marrow of both groups. The mean absorption from the meal of the patients with acid in their gastric juice was 57.5 per cent while the mean absorption of those patients with histamine-fast achlorhyrdria was 18.5 per cent.

Chemical synthesis of bile acid acyl-adenylates and formation by a rat liver microsomal fraction

In mammals, unconjugated bile acids formed in the intestine by bacterial deconjugation are reconjugated ( N-acylamidated) with taurine or glycine during hepatocyte transport. Activation of the carboxyl group of bile acids to form acyl-adenylates is a likely key intermediate step in bile acid N-acylamidation. To gain more insight into the process of bile acid adenylate formation, we first synthesized the adenylates of five common, natural bile acids (cholic, deoxycholic, chenodeoxycholic, ursodeoxycholic, and lithocholic acid), and confirmed their structure by proton NMR. We then investigated adenylate formation by subcellular fractions of rat liver (microsomes, mitochondria, cytosol) using a newly developed LC method for quantifying adenylate formation. The highest activity was observed in the microsomal fraction. The reaction required Mg 2+ and its optimum pH was about pH 7.0. In term of maximum velocity ( V max) and the Michaelis constant ( K m), the catalytic efficiency of the enzyme under the conditions used was highest with cholic acid of the bile acids tested. The formation of cholyl-adenylate was strongly inhibited by lithocholic and deoxycholic acid, as well as by palmitic acid; ibuprofen and valproic acid were weak inhibitors. In cholestatic disease, such adenylate formation might lead to subsequent bile acid conjugation with glutathione or proteins.

Characterization of Escherichia coli nitroreductase NfsB in the metabolism of nitrobenzodiazepines

Nitrobenzodiazepine (NBDZ) is a sedative-hypnotic drug used in the treatment of anxiety and sleep problems. Overdose of NBDZ may cause severe neurological effects, especially for people in drug abuse or addiction. In the present study, we investigated NBDZ nitroreduction in rat enteric contents and characterized the role of enterobacterial nitroreductase in the reductive pathway. Nitroreduction of flunitrazepam (FZ) was studied in the microsomal membrane fractions of rat liver, jejunum and jejunal microflora using HPLC analysis. In the jejunal microflora, FZ was demonstrated to be significantly reduced to its amino derivative under anaerobic condition. Escherichia coli type I nitroreductase NfsB (EC 1.5.1.34) was found in rat jejunal microflora via PCR technique and Western blotting. The participation of NfsB in FZ nitroreduction was demonstrated from inhibition studies. Kinetic study of the purified recombinant NfsB indicated that nitroreduction of FZ, nitrazepam (NZ) and clonazepam (CZ) are mediated by NfsB, where CZ shows lower k cat/ K M ratio than that of the other two. Finally, two other nitroreductases E. cloacae NR (EC 1.6.99.7) and S. typhimurium Cnr were also found to be responsible for FZ nitroreduction. These results provide that the reduction of NBDZ in normal flora is catalyzed by type I nitroreductase NfsB.