Biomedical subjects

T R Tephly

Publications and source records attributed to T R Tephly.

At least 55 records · Page 3Linked to original sources

PubMed1995

Expressed human UGT1.4 protein catalyzes the formation of quaternary ammonium-linked glucuronides.

In humans, the metabolism of a number of tertiary amine-containing pharmacological agents to quaternary ammonium-linked glucuronides, catalyzed by UDP-glucuronosyltransferase (UGT), represents a unique and important metabolic pathway for these compounds. A full-length cDNA-encoding human UGT1.4 (the so-called "minor" human bilirubin UGT) was inserted into the expression vector pREP9 and transfected into human embryonic kidney 293 cells, and stable transfectants were obtained after geneticin selection. As expected, the expressed protein had low catalytic activity toward bilirubin. However, expressed human UGT1.4 protein exhibited glucuronidation activity toward tertiary amine substrates, such as imipramine, cyproheptadine, tripelennamine, and chlorpromazine, which form quaternary ammonium-linked glucuronides. Carcinogenic primary amines (beta-naphthylamine, benzidine, and 4-aminobiphenyl) also reacted with the expressed UGT1.4 protein at rates approximately 10-fold higher than the rates for quaternary ammonium glucuronide formation. Although a number of other UGT gene products are capable of catalyzing the glucuronidation of primary amine substrates, expressed human UGT1.4 protein is the only UGT isoform that has been shown to conjugate tertiary amine substrates, forming quaternary ammonium-linked glucuronides.

Carbohydrate Conformation↗

PubMed1994

Detection and quantification of 10-formyltetrahydrofolate dehydrogenase (10-FTHFDH) in rat retina, optic nerve, and brain.

Methanol poisoning is characterized by the accumulation of formic acid, a metabolite of methanol, which can lead to metabolic acidosis and ocular toxicity. Formate metabolism to CO2 is governed by tissue H4folate and 10-FTHFDH levels. Presumably, rats are not normally susceptible to formate toxicity because they possess high hepatic H4folate and 10-FTHFDH levels. However, the ability of target tissues to metabolize formate is not known. Therefore, studies were performed to determine whether 10-FTHFDH was present in rat retina, optic nerve, and brain. 10-FTHFDH levels were determined using Western blot analysis of mitochondrial and postmitochondrial preparations from these tissues. Hepatic mitochondrial and postmitochondrial levels of 10-FTHFDH were 13 and 12 ng/micrograms protein, respectively. Postmitochondrial levels of 10-FTHFDH in rat retina, optic nerve and whole brain were 0.2, 1.3, and 2.1 ng/micrograms protein; mitochondrial values in retina and brain were 0.2 and 1.5 ng/micrograms protein, respectively. Postmitochondrial values obtained for rat brain regions were similar to those found for whole brain. These results suggest that, in rats, target tissues possess the capacity to metabolize formate to CO2 and may be protected from formate toxicity through this folate-dependent system.

Animals↗

PubMed1993

Cloning and characterization of rabbit liver UDP-glucuronosyltransferase cDNAs. Developmental and inducible expression of 4-hydroxybiphenyl UGT2B13.

A polyclonal antibody generated against rabbit liver p-nitrophenol UDP-glucuronosyltransferase (UGT) was used to screen a rabbit liver cDNA expression library constructed in lambda gt11. A 500-base pair cDNA clone, termed pPNP, generated a fusion protein that was antigenic with the antibody. Clone pPNP encoded the 3' region of a UGT. To identify larger recombinants, clone pPNP was used as a probe to screen a second cDNA library constructed in lambda ZAP. Two different cDNA clones were identified by DNA sequence analysis. Based upon their predicted amino acid sequence analysis, the clones encode transferases belonging to the UGT2 subfamily, and have been identified as UGT2B13 and UGT2B14. The predicted N-terminal sequence of UGT2B13 is identical to that determined for the purified rabbit liver estrone UGT. However, expression of the UGT2B13 cDNA in COS-1 cells displayed no activity in the presence of estrone but efficiently conjugated 4-hydroxybiphenyl. Results of Southern blot analysis using the 5' divergent region of the UGT2B13 cDNA that encodes exon 1 demonstrates that multiple genes share sequence homology to UGT2B13, an observation which indicates that the estrone UGT and UGT2B13 genes are encoded by separate alleles. When the 5' variable regions of the cDNAs where used in Northern blot analysis, the expression of UGT2B13 and UGT2B14 were shown to be expressed primarily in adult rabbits. However, when neonatal rabbits were treated with either dexamethasone or rifampicin, UGT2B13 mRNA levels were induced. The neonatal induction of UGT2B13 mRNA corresponded with similar increases in 4-hydroxybiphenyl UGT activity. The expression and induction of UGT2B13 paralleled that of the developmentally regulated rabbit liver progesterone 6 beta-hydroxylase P4503A6.

Amino Acid Sequence↗

PubMed1992

Formate metabolism in micropigs.

The toxicity of methanol is directly related to the accumulation of formate which, in turn, is related to the adequacy of the folate-dependent metabolism of formate to carbon dioxide. Thus, humans who possess low hepatic folates and low 10-CHO H4folate dehydrogenase activity metabolize formate poorly and are sensitive to methanol. Conversely, most laboratory species do not exhibit methanol toxicity because they metabolize formate at high rates. Studies reported here show that the Yucatan micropig has the lowest hepatic folates of any animal species studied. Formate oxidation rates in micropigs were 23% of rates reported for rats. The half-life of formate disappearances from the blood was 74 min, a value twice that reported for rats. In addition, 10-CHO H4folate dehydrogenase activity and amount in micropig liver is markedly reduced. Micropigs may prove useful in studies of methanol poisoning due to their low capacity for formate oxidation and their reasonable size and ease in handling.

Animals↗

PubMed1991

The toxicity of methanol.

Methanol toxicity in humans and monkeys is characterized by a latent period of many hours followed by a metabolic acidosis and ocular toxicity. This is not observed in most lower animals. The metabolic acidosis and blindness is apparently due to formic acid accumulation in humans and monkeys, a feature not seen in lower animals. The accumulation of formate is due to a deficiency in formate metabolism which is, in turn, related, in part, to low hepatic tetrahydrofolate (H4 folate). An excellent correlation between hepatic H4 folate and formate oxidation rates has been shown within and across species. Thus, humans and monkeys possess low hepatic H4 folate levels, low rates of formate oxidation and accumulation of formate after methanol. Formate, itself, produces blindness in monkeys in the absence of metabolic acidosis. In addition to low hepatic H4 folate concentrations, monkeys and humans also have low hepatic 10-formyl H4 folate dehydrogenase levels, the enzyme which is the ultimate catalyst for conversion of formate to carbon dioxide. This review presents the basis for the role of folic acid-dependent reactions in the regulation of methanol toxicity.

Animals↗

PubMed1990

Formate metabolism in young swine.

Formate generated from methanol metabolism in vivo is the chemical entity responsible for the development of the methanol toxicity syndrome in the monkey. Compared to rats, monkeys are in a state of folate deficiency. This leads to a decreased ability to dispose of formate generated leading to its accumulation and the subsequent development of the classic symptoms of methanol toxicity. Rats possess a more efficient folate system; therefore, they metabolize formate very readily and do not exhibit methanol toxicity symptoms. In this report, the hepatic folate content and the ability to handle a formate "load" were evaluated in another animal species, the pig. The results obtained indicate that the pig, compared to all other species studied, has extremely low levels of folates and very low levels of a key enzyme in the folate pathway, namely 10-formyl H4folate dehydrogenase. Also the pig's ability to dispose of formate was extremely limited and slower than that observed in rats or monkeys. These results suggest that the pig may be a suitable animal model for studying formate metabolism and possibly methanol toxicity.

Animals↗

PubMed1990

Characterization and primary sequence of a human hepatic microsomal estriol UDPglucuronosyltransferase.

A human liver microsomal UDP glucuronosyltransferase (UDPGT) that demonstrates reactivity with estriol (pI 7.4 UDPGT) has been purified to homogeneity and characterized further. No activity toward morphine, 4-hydroxybiphenyl, bilirubin, or tripelennamine was observed. The estriol UDPGT shows immunoreactivity with antibodies raised against rat hepatic microsomal 3 alpha- and 17 beta-hydroxysteroid UDPGTs but not with antibodies raised against rat hepatic microsomal p-nitrophenol UDPGT. The NH2-terminal sequence of the purified protein was determined and found to correspond to an identical sequence in the deduced amino acid sequence of a cDNA obtained from a human liver library in lambda gt11 (HLUG4). Sequence analysis revealed that HLUG4 is 2094 bp in length and encodes a protein of 523 amino acids which has a 16 amino acid leader sequence, followed by an untranslated 3' region of 525 bp. Three potential N-glycosylation sites were identified in the predicted sequence. The deduced amino acid sequence of estriol UDPGT showed 82% identity with the deduced amino acid sequence of another human hepatic cDNA (HLUG25), which has been expressed as a UDPGT capable of 6 alpha-hydroxyglucuronidation of hyodeoxycholic acid, strongly suggesting that these proteins are members of the same gene subfamily.

Amino Acid Sequence↗

PubMed1990

Repeated ingestion of aspartame-sweetened beverages: further observations in individuals heterozygous for phenylketonuria.

Six adults heterozygous for phenylketonuria (PKU) ingested eight successive servings of unsweetened and aspartame (APM)-sweetened beverage at 1-hour intervals in a randomized, balanced, crossover design. In one part, the eight beverage servings were not sweetened. In the other, each of the eight beverage servings provided 600 mg of APM, a dose equivalent to the amount provided by 36 oz of an APM-sweetened diet beverage. Plasma aspartate concentration was not significantly increased after ingestion of unsweetened or APM-sweetened beverage. Similarly, ingestion of the unsweetened beverage had no significant effect on plasma phenylalanine concentration. However, ingestion of APM-sweetened beverage significantly increased plasma phenylalanine concentrations 2.35 to 4.03 mumol/dL above baseline 30 minutes after ingestion. Plasma phenylalanine values reached a steady-state after administration of five servings of APM-sweetened beverage and were slightly, but significantly higher than usual postprandial values for adults heterozygous for PKU. Similarly, the ratio of the plasma phenylalanine concentration to the sum of the concentration of the large neutral amino acids was significantly higher than usual postprandial values. Blood methanol and formate concentrations remained within normal limits. These data indicate that a fasting adult heterozygous for PKU could consume the equivalent of 24 12-oz servings of APM-sweetened beverage over an 8-hour period and only increase plasma phenylalanine concentration to a modest degree.

Adult↗

PubMed1990

UDP-glucuronosyltransferases: a family of detoxifying enzymes.

Glucuronidation is an important process in the metabolism of xenobiotic and endogenous substances leading to enhancement of excretion of these compounds from the body. A multigene family encodes a number of UDP-glucuronosyltransferase enzymes which catalyse this route of metabolism. Recent advances in biochemical and molecular biological approaches, reviewed here by Thomas Tephly and Brian Burchell, have given new insight into the function and structure of UDP-glucuronosyltransferases. These proteins have surprising similarities and yet appear to be capable of conjugating a remarkable number of different chemicals.

Glucuronosyltransferase↗

PubMed1990

Photoaffinity labeling of rat liver microsomal morphine UDP-glucuronosyltransferase by [3H]flunitrazepam.

Benzodiazepines have been shown to competitively inhibit morphine glucuronidation in rat and human hepatic microsomes. Flunitrazepam exerted a potent competitive inhibition of rat hepatic morphine UDP-glucuronosyltransferase (UDPGT) activity (Ki = 130 microM). It has no effect on the activity of p-nitrophenol, 17 beta-hydroxysteroid, 3 alpha-hydroxysteroid, or 4-hydroxybiphenyl UDPGTs. Because flunitrazepam is an effective photoaffinity label for benzodiazepine receptors, studied were performed in solubilized rat hepatic microsomes and with partially purified preparations of morphine UDPGT to determine the enhancement of flunitrazepam inhibition and binding to morphine UDPGT promoted by exposure to UV light. Under UV light, flunitrazepam inhibition was markedly enhanced. UV light exposure also led to a marked increase in binding of [3H]flunitrazepam to microsomal protein, which was protected substantially by preincubation with morphine. Testosterone, androsterone, and UDP-glucuronic acid did not protect against UV-enhanced flunitrazepam binding, and morphine did not reverse flunitrazepam binding once binding had occurred. As morphine UDPGT was purified, a good correlation was found between the increases in specific activity of morphine UDPGT and flunitrazepam binding to protein. Chromatofocusing chromatography showed that flunitrazepam bound only to fractions containing active morphine UDPGT, and no binding to 4-hydroxybiphenyl UDPGT was observed. Fluorography of a sodium dodecyl sulfate-polyacrylamide electrophoresis gel of solubilized hepatic microsomes that had been treated with [3H] flunitrazepam under UV light revealed a band with a monomeric molecular weight between 54,000 and 58,000. This monomeric molecular weight compares favorably with the reported monomeric molecular weight of homogeneous morphine UDPGT (56,000). These studies suggest that flunitrazepam binds rather selectively to the morphine binding site of morphine UDPGT and may prove to be a useful probe for this enzyme.

Affinity Labels↗

PubMed1989

N-glycosylation of purified rat and rabbit hepatic UDP-glucuronosyltransferases.

Five UDP-glucuronosyltransferases (UDPGTs) have been isolated to apparent homogeneity from rat and rabbit liver and have been characterized for their glycoprotein nature by reacting these proteins with commercially available endo- and exoglycosidases. The enzymes studied were rat hepatic p-nitrophenol, 17 beta-hydroxysteroid, and 3 alpha-hydroxysteroid UDPGTs and rabbit hepatic p-nitrophenol and estrone UDPGTs. Hydrolysis of oligosaccharide moieties was evidenced by an increase in the mobility (decreased apparent molecular weight) of the protein subunits after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified rabbit hepatic estrone and p-nitrophenol UDPGTs were hydrolyzed by almond glycopeptidase A and endo-beta-N-acetylglucosaminidase H from Streptomyces plicatus (endo H), but not by endo-beta-N-acetylglucosaminidase D from Diplococus pneumoniae (endo D) suggesting that these transferases are glycoproteins of the high mannose type and not of the complex type. Likewise, purified rat hepatic 3 alpha-hydroxysteroid and p-nitrophenol UDPGTs were substrates for glycopeptidase A and endo H but not for endo D. One enzyme, 17 beta-hydroxysteroid UDPGT, was not glycosylated since it was not hydrolyzed by any of the three endoglycosidases. All four glycosylated UDPGTs could serve as substrates for jack bean alpha-mannosidase, confirming the high mannose nature of the oligosaccharide. Deglycosylation of the purified UDPGTs by endo H did not have an effect on the catalytic activities of these proteins.

Animals↗

PubMed1989

Effect of repeated ingestion of aspartame-sweetened beverage on plasma amino acid, blood methanol, and blood formate concentrations in normal adults.

Aspartame (APM) is a widely used dipeptide sweetener (L-aspartyl-L-phenylalanine methyl ester). It has been suggested that excessive use of APM might elevate plasma aspartate, phenylalanine, and/or methanol concentrations to levels that are potentially harmful. Six normal young adults ingested eight successive servings of unsweetened and APM-sweetened beverage at one-hour intervals in a balanced crossover design. In one part, the beverage was not sweetened. In the other, each serving of beverage provided 600 mg APM, a dose equivalent to the amount provided by 36 oz of APM-sweetened diet beverage. Plasma aspartate concentration was not significantly increased after ingestion of unsweetened or APM-sweetened beverage. Similarly, ingestion of the unsweetened beverage had no significant effect on plasma phenylalanine concentration. However, ingestion of APM-sweetened beverage significantly increased plasma phenylalanine levels 1.41 to 2.35 mumol/dL above baseline 30 minutes after ingestion. Plasma phenylalanine values reached a steady state after administration of four to five servings and did not exceed normal postprandial values at any time. Blood methanol and formate concentrations remained within normal limits. The data indicate ready metabolism of APM when administered at levels that may be ingested by normal individuals who are heavy users of diet beverages.

Adult↗

PubMed1989

UDP-glucuronosyltransferases in the metabolic disposition of xenobiotics.

UDPGT isoenzymes are products of multiple gene families as demonstrated by sequence analysis of purified proteins and by molecular cloning experiments. These isoenzymes are relatively specific for endogenous substrates but have broad substrate specificities for xenobiotic substrates. They are important metabolic enzymes capable of converting exogenous and endogenous substances to more hydrophilic metabolites. Each species has its own pattern of UDPGTs and it is not possible at this time to extrapolate information directly from one species to another.

Animals↗

PubMed1989

Studies on the mechanism of methanol poisoning: purification and comparison of rat and human liver 10-formyltetrahydrofolate dehydrogenase.

Methanol poisoning in primates and humans is due to formate accumulation as a result of low rates of formate oxidation. This toxicity is not seen in rats, where formate oxidation rates are high. Formate oxidation in vivo is dependent on hepatic tetrahydrofolate levels and on the activity of the enzyme 10-formyl-tetrahydrofolate (10-formyl-H4folate) dehydrogenase (EC 1.5.1.6). Because hepatic 10-formyl-H4folate dehydrogenase activity is lower in human liver than in rat liver, studies were performed investigating the properties of this enzyme in rat and human liver. 10-Formyl-H4folate dehydrogenase was purified to homogeneity from rat and human liver and was found to possess similar subunit molecular weights on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (96,000). N-Terminal amino acid analysis of the pure proteins showed an identical sequence for the first 16 amino acids. Antibodies raised in rabbits against the rat liver enzyme were inhibitory toward the activity of both rat and human liver enzymes and appeared to recognize only the 10-formyl-H4folate dehydrogenase in cytosolic preparations of rat and human liver. Immunoblots of pure rat and human liver 10-formyl-H4folate dehydrogenase showed similar staining intensity. It is concluded that rat and human liver 10-formyl-H4folate dehydrogenase possess very similar properties and that the activity of the enzyme in human liver is lower than that of rat liver, due to a reduced amount of enzyme protein in human liver. This may be an important factor in regulating formate oxidation in humans and may explain, in part, the accumulation of formate and the mechanism of toxicity of methanol in humans.

Amino Acid Sequence↗

PubMed1989

Safety of long-term large doses of aspartame.

Safety of long-term administration of 75 mg/kg of aspartame per day was evaluated with the use of a randomized, double-blind, placebo-controlled, parallel-group design in 108 male and female volunteers aged 18 to 62 years. Subjects received either aspartame or placebo in capsule form three times daily for 24 weeks. No persistent changes over time were noted in either group in vital signs; body weight; results of standard laboratory tests; fasting blood levels of aspartame's constituent amino acids (aspartic acid and phenylalanine), other amino acids, and methanol; or blood formate levels and 24-hour urinary excretion of formate. There also were no statistically significant differences between groups in the number of subjects experiencing symptoms or in the number of symptoms per subject. These results further document the safety of the long-term consumption of aspartame at doses equivalent to the amount of aspartame in approximately 10 L of beverage per day.

Adolescent↗

PubMed1988

Properties of a 3-methylcholanthrene-inducible phenol UDP-glucuronosyltransferase from rat liver.

Functional and molecular probes are described which are useful to identify a 3-methylcholanthrene-inducible phenol UDP-glucuronosyltransferase (GTMC) from rat liver. Two different procedures for isolation of GTMC were compared, method 1 utilizing DEAE-Sepharose chromatography or method 2, chromatofocusing. Method 2 appeared to be superior in separating different isoenzymes. Subsequently the enzyme was purified by affinity chromatography on UDP-hexanolamine Sepharose. With both methods a protein was purified with a subunit Mr of 55,000, catalyzing glucuronidation of a variety of planar phenols and, in particular, of benzo(a)pyrene-3,6-quinol to its mono- and diglucuronide. Antibodies to GTMC recognized a polypeptide with a subunit Mr of 55,000 as the major 3-methylcholanthrene-inducible isoenzyme in rat liver microsomes. The described functional and molecular probes may help to differentiate GTMC from similar isoenzymes conjugating planar phenols and to elucidate its regulation and biological function.

Animals↗

PubMed1988

Characterization of antibodies to a rabbit hepatic UDP-glucuronosyltransferase and the identification of an immunologically similar enzyme in human liver.

An antibody to a UDP-glucuronosyltransferase (UDPGT) isoenzyme which catalyzes the glucuronidation of p-nitrophenol (PNP) in rabbit liver was raised in sheep and used to identify immunologically similar UDPGTs in rabbit and human livers. Immunoblotting experiments showed that the antisera specifically recognized PNP UDPGT but not estrone UDPGT purified from rabbit liver. Sheep anti-rabbit liver PNP UDPGT IgG immunoprecipitated PNP, 1-naphthol, and 4-methylumbelliferone glucuronidation activities in rabbit and human liver microsomal preparations. In rabbit liver microsomes the antibody did not immunoprecipitate estrone or estradiol glucuronidation activities. In human liver microsomes, 4-aminobiphenyl but not estriol glucuronidation activities were immunoprecipitated, suggesting that the antibody recognizes a specific UDPGT (pI 6.2) in human liver microsomes.

Animals↗

PubMed1988

Immunohistochemical demonstration of isozyme- and strain-specific differences in the intralobular localizations and distributions of UDP-glucuronosyltransferases in livers of untreated rats.

Antibodies directed against three isozymes of rat hepatic microsomal UDP-glucuronosyltransferase (EC 2.4.1.17), p-nitrophenol, 3 alpha-hydroxysteroid, and 17 beta-hydroxysteroid UDP-glucuronosyltransferases were used to localize these enzymes at the light microscopic level in livers of untreated Sprague-Dawley and Wistar rats. Avidin-biotin-peroxidase staining revealed the presence of each isozyme within parenchymal cells throughout the liver lobule in rats of both strains. However, although antibodies to the 3 alpha- and 17 beta-hydroxysteroid UDP-glucuronosyltransferases appeared to stain hepatocytes across the liver lobule quite uniformly, centrilobular hepatocytes were stained much more intensely for p-nitrophenol UDP-glucuronosyltransferase than were midzonal and periportal cells. Additionally, appreciable immunohistochemical staining for p-nitrophenol UDP-glucuronosyltransferase, but not for the two hydroxysteroid UDP-glucuronosyltransferases, was detected within the epithelium of the hepatic bile duct and the endothelium of the hepatic artery and portal vein. Another difference was noted in livers of Wistar rats: hepatocytes of rats possessing low 3 alpha-hydroxysteroid (i.e., androsterone) UDP-glucuronosyltransferase activity were stained much less intensely for the 3 alpha-hydroxysteroid UDP-glucuronosyltransferase than were those of rats exhibiting high rates of androsterone glucuronidation, whereas differences in immunoperoxidase staining for p-nitrophenol and 17 beta-hydroxysteroid UDP-glucuronosyltransferases were not apparent between the two subclasses of Wistar rats. These immunohistochemical findings demonstrate that different UDP-glucuronosyltransferase isozymes are distributed across the liver lobule in significantly different manners and, furthermore, suggest that xenobiotics may be glucuronidated within epithelial cells of the hepatic bile duct and endothelial cells of the hepatic artery and portal vein, as well as within hepatocytes. The results of this study also provide evidence that differences in the content of 3 alpha-hydroxysteroid UDP-glucuronosyltransferase within hepatocytes account for genetically determined variations in the rates at which androsterone and certain other xenobiotics are glucuronidated in livers of Wistar rats.

Animals↗