UDP-glucuronosyltransferases.

Glucuronidation represents a major pathway which enhances the elimination of many lipophilic xenobiotics and endobiotics to more water-soluble compounds. The UDP-glucuronosyltransferase (UGT) family catalyzes the glucuronidation of the glycosyl group of a nucleotide sugar to an acceptor compound (aglycone) at a nucleophilic functional group of oxygen (eg, hydroxyl or carboxylic acid groups), nitrogen (eg, amines), sulfur (eg, thiols), and carbon, with the formation of a beta-D-glucuronide product. At this time, over 35 different UGT gene products have been described from several different species. UGTs have been divided into two distinct subfamilies based on sequence identities, UGT1 and UGT2. The UGT1 gene subfamily consists of a number of UGTs that result from alternate splicing of multiple first exons and share common exons 2-5. The substrate specificities of the various isoforms have been examined in cultured cell experiments, and include bilirubin, amines, and planar and bulky phenol. The UGT2 gene family is different in that the UGT2 mRNAs are transcribed from individual genes. The UGT2 subfamily consists of numerous enzymes which catalyze the glucuronidation of a diverse chemical base including steroids, bile acids, and opioids. Until recently, the liver has been the major focus for studying the metabolism of xenobiotics and endobiotics. Several groups have identified extrahepatic tissues that express UGT isoforms including the kidney, gastrointestinal tract and brain. This review discusses the two UGT gene families, substrate specificities, and the recent discoveries of UGTs in extrahepatic tissues.

Expression of UDP-glucuronosyltransferases (UGTs) 2B7 and 1A6 in the human brain and identification of 5-hydroxytryptamine as a substrate.

The extrahepatic expression of UDP-glucuronosyltransferases (UGTs) is important in the detoxification of a number of endogenous and exogenous compounds, including 5-hydroxytryptamine and morphine. Studies were designed to investigate the extrahepatic expression of human UGTs using RT-PCR techniques and to determine the UGTs involved in the glucuronidation of 5-hydroxytryptamine. Human UGT2B7 expression was found in the human liver, kidney, pancreas, and brain, while UGT1A6 expression is found in the liver, kidney, and brain. This is the first observation of UGTs present in the human central nervous system. Using glucuronidation assays, a significant amount of 5-hydroxytryptamine glucuronide was found to be catalyzed by UGT1A6. These studies suggest that UGT2B7 may play an important role in the overall contribution of morphine analgesia by serving to generate the potent morphine-6-O-glucuronide in situ. UGT1A6 could play an important role in the glucuronidation of 5-hydroxytryptamine in vivo, therefore terminating the actions of the neurotransmitter.

Folate and folate-dependent enzymes associated with rat CNS development.

Folic acid and its derivatives are important mediators in growth-related cellular processes. The concentration of folate and two folate-dependent enzymes, 10-formyltetrahydrofolate synthetase (10-FTHFS) and 10-formyltetrahydrofolate dehydrogenase (10-FTHFDH), was determined in brain regions over the early period of rat development. Folate concentrations determined at birth were high in all brain regions studied. During the first 2 weeks, folate concentrations declined steadily, followed by a period of significant increase. High and invariant activity of 10-FTHFS was found throughout the period of study. Low amounts of 10-FTHFDH were seen for the first 2 weeks, but increased significantly from postnatal days 14 to 28. These changes correlated with changes determined in the concentration of folate, supporting the idea that this protein is involved with folate uptake and/or storage. Furthermore, immunohistochemical expression of 10-FTHFDH in different rat brain regions revealed glial cells as a preferential cellular location for this folate-binding protein.

Glucuronidation of catechol estrogens by expressed human UDP-glucuronosyltransferases (UGTs) 1A1, 1A3, and 2B7.

Catechol estrogens are major estrogen metabolites in mammals and are the most potent naturally occurring inhibitors of catecholamine metabolism. These estrogen compounds have been implicated in carcinogenic activity and the 4/2-hydroxyestradiol concentration has been shown to be elevated in neoplastic human mammary tissue compared to normal human breast tissue. Three human liver UDP-glucuronosyltransferases, UGT2B7, UGT1A1, and UGT1A3, have been shown to catalyze the glucuronidation of catechol estrogens and lead to their enhanced elimination via urine or bile. The present study was designed to study the kinetic interaction of expressed human UGT2B7(Y) or (H), UGT1A1, and UGT1A3 toward 2- and 4-hydroxycatechol estrogens. cDNAs encoding UGT2B7(Y) or (H), UGT1A1, and UGT1A3 were expressed in HK293 cells, and cell homogenates or membrane preparations were used to determine their glucuronidation ability. UGT2B7(Y) reacted with higher efficiency toward 4-hydroxyestrogenic catechols, whereas UGT1A1 and UGT1A3 showed higher activities toward 2-hydroxyestrogens. UGT2B7(H) catalyzed estrogen catechol glucuronidation with efficiencies similar to UGT2B7(Y). Flunitrazepam (FNZ), a competitive inhibitor of morphine glucuronidation in hepatic microsomes, competitively inhibited catechol estrogen glucuronidation catalyzed by UGT2B7(Y), UGT1A1, and UGT1A3. Buprenorphine, an opioid substrate that reacts at high efficiency with each of these UGTs, was also studied. FNZ competitively inhibited buprenorphine glucuronidation with UGT1A1 and UGT2B7 but had no inhibitory activity toward UGT1A3. This suggests that buprenorphine and 2-hydroxycatechol estrogens react with separate active sites of UGT1A3. A catecholamine, norepinephrine, did not inhibit UGT2B7(Y)-, UGT1A1-, and UGT1A3-catalyzed glucuronidation of catechol estrogens. These results also suggest that drug-endobiotic interactions are possible in humans and may have implication in carcinogenesis.

Immunohistochemical localization of UDP-glucuronosyltransferases in rat brain during early development.

Extrahepatic glucuronidation, such as that in the central nervous system (CNS), may play a very important role in xenobiotic disposition and may serve to protect the CNS from potentially toxic xenobiotics. UDP-glucuronosyltransferase (UGT) 1A6 is an important catalyst for phenol and polycyclic aromatic hydrocarbon glucuronidation. Studies were designed to determine the immunohistochemical localization of UGT1A6 and the steroid-reactive UGTs 2B2 and 2B3 in brain regions throughout the rat development. Neuronal cells, such as pyramidal cells, in sections from cerebral cortex and hippocampus displayed intensive UGT1A6-specific staining. UGT1A6-specific staining was also found in neuronal cells throughout the cerebral cortex, as well as in the cerebellar white matter. Glial cells revealed no apparent staining. In addition, staining for UGT1A6 was seen in choroid plexus at a later developmental stage. Although UGT1A6 staining was evident, brain sections analyzed for UGT2B2 and UGT2B3 immunoreactivity showed no significant staining. These results provide the first definitive evidence for the presence and cellular localization of UGT1A6, in neurons of developing rat brain, whereas UGT2B2 and UGT2B3 were not detected.

The glucuronidation of opioids, other xenobiotics, and androgens by human UGT2B7Y(268) and UGT2B7H(268).

UGT2B7 has been cloned and expressed previously in COS cells and HK293 cells. Two forms have been identified: one with a tyrosine and one with a histidine at position 268. UGT2B7 has been shown to catalyze NSAIDs, catechol estrogens, and morphine-3- and -6-glucuronidation. cDNAs for UGT2B7Y268 and H268 were cloned and stably expressed in HK 293 cells. Studies were designed to test each form for reactivity toward a number of opioid compounds, xenobiotics such as menthol, oxazepam, and propranolol, and androgens such as androsterone and testosterone using membrane preparations derived from HK 293 cells. Both UGT2B7Y and UGT2B7H are highly reactive with many opioids, menthol, androsterone, and (R)- and (S)-propranolol, and similar kinetic values were observed. UGT2B7Y and UGT2B7H react poorly with oxazepam and no difference in (R)- or (S)-glucuronidation rate ratios was found. Thus, UGT2B7Y and H cannot account for the variability in the plasma or urine concentrations of these glucuronides in human populations. Our data suggest that UGT2B7 is a major isoform responsible for the glucuronidation of androsterone. Neither UGT2B7Y nor H catalyzes the glucuronidation of testosterone although each catalyzes the glucuronidation of epitestosterone. UGT2B7 seems to be a major human isoform responsible for the glucuronidation of opioids of the morphinan and oripavine class and is capable of catalyzing the glucuronidation of both the 3- and 6-hydroxyl moieties on these molecules. Thus, UGT2B7 plays a major role in the conversion of morphine to morphine-6-glucuronide, the potent analgesic metabolite of morphine.

Glucuronidation of retinoids by rat recombinant UDP: glucuronosyltransferase 1.1 (bilirubin UGT).

Rat liver recombinant BR1UGT1.1 was found to have significant activity toward retinoid substrates. UGT1.1 glucuronidation activity was 91 +/- 18 pmol/mg x min for atRA and 113 +/- 19 pmol/mg x min for 5,6-epoxy-atRA. The apparent K(M) and V(max) of atRA acid glucuronidation by UGT1.1 were 59.1 +/- 5.4 microM and 158 +/- 43 pmol/mg x min, respectively. SDS-PAGE and Western blot analysis of UGT1.1-transfected HK293 membrane proteins photolabeled with [11,12-3H]atRA revealed a protein of approximately 56 kDa that was labeled by [3H]atRA, detected by anti-pNP UGT antibody and not present in membranes from nontransfected HK293 cells. Liver microsomes from Gunn rats, which lack UGT1.1, had significant activity toward atRA (111 +/- 28 pmol/mg x min).

Outreach services: issues and challenges.

Tompkins-McCaw Library of Virginia Commonwealth University has planned and implemented four one-year outreach service projects during the past two years. These projects were funded by the National Library of Medicine and the National Network of Libraries of Medicine Southeastern/Atlantic Region. The projects focus on information access for public health nurses, HIV/AIDS information access, and circuit librarian services in rural Southern Virginia. This article documents issues and challenges which have been identified from these projects and suggests ways to resolve them.

Comparison of stably expressed rat UGT1.1 and UGT2B1 in the glucuronidation of opioid compounds.

Opioids are important drugs used as analgesics, antitussives, antidiarrheals, and in the therapy of myocardial infarctions, and as antagonists of opioid intoxication. The glucuronidation of these compounds, catalyzed by UDP-glucuronosyltransferases (UGTs), is well known to be a primary step in their metabolism to hydrophilic products and in their ultimate excretion. The present study was designed to compare the reactivity and relative glucuronidation efficiencies of opioid agonists, antagonists, and partial agonists with two rat UGT isoforms; UGT1.1, which is generally considered the "bilirubin UGT," and UGT2B1, which has previously been shown to catalyze the glucuronidation of testosterone, chloramphenicol, and (-)-morphine. Rat UGT2B1, stably expressed in HK293 cells, exhibited high glucuronidation rates and catalytic efficiencies for many opioids, although values for (-)-morphine and nalorphine were the highest. In contrast, these compounds were very poor substrates for expressed rat UGT1.1. Comparably high glucuronidation rates and efficiencies were found for buprenorphine and diprenorphine with both UGT isoforms. These results suggest that opioids with morphinan-based chemical structures similar to (-)-morphine interact with UGTs differently than those with oripavine-based chemical structures similar to buprenorphine. To investigate the contribution of rat UGT1.1 and UGT2B1 in the overall rate of glucuronidation of buprenorphine in the rat liver, hepatic microsomes from Gunn rats (where UGT1.1 activity is absent) and Wistar rats (where UGT1.1 activity is present) were studied. Buprenorphine glucuronidation activity in Gunn rat liver microsomes exhibit approximately 25% of rates observed in Wistar rat liver microsomes, whereas (-)-morphine, naloxone, and naltrexone glucuronidation rates were not significantly different in microsomal preparations from Gunn and Wistar rats. These data suggest that UGT2B1 is the major hepatic enzyme involved in the glucuronidation of (-)-morphine and naloxone in livers from untreated rats, whereas buprenorphine glucuronidation is preferentially catalyzed by rat UGT1.1.

Human UGT2B7 catalyzes morphine glucuronidation.

A human UDP-glucuronosyltransferase (UGT) catalyzing the glucuronidation of morphine has been identified. A full length cDNA was isolated from a human liver cDNA library and found to be identical to the UGT2B7 form having a tyrosine at position 288. This cDNA was transfected into HK 293 cells, and stable expression was achieved. Cell homogenates and membrane preparations from HK 293 cells expressing UGT2B7 catalyzed the glucuronidation of morphine and other clinically significant opioid agonists, antagonists, and partial agonists. UGT2B7 catalyzed morphine glucuronidation at the 3- and 6-hydroxy positions and also mediated the formation of codeine-6-glucuronide from codeine. This represents the first demonstration of a UGT capable of catalyzing the glucuronidation of both the 3- and 6-positions of opioids. Since humans excrete morphine-3-glucuronide and morphine-6-glucuronide after morphine administration, it is likely that UGT2B7 is a major isoform in humans responsible for the metabolism of this important drug and its surrogates.

The glucuronidation of exogenous and endogenous compounds by stably expressed rat and human UDP-glucuronosyltransferase 1.1.

Rat and human UDP-glucuronosyltransferase (UGT) 1.1 share > 70% identity in their deduced primary amino acid sequences. We have previously shown that rat UGT1.1, stably expressed in human embryonic kidney 293 cells, catalyzes the glucuronidation of bilirubin and the mixed opioid agonist/antagonist buprenorphine with high efficiency. The present study was designed to characterize the reactivity of expressed human UGT1.1 with opioid compounds and compare its substrate specificity for opioids to that of the expressed rat enzyme. The results show that both rat and human UGT1.1 catalyze the glucuronidation of opioids with a relative reactivity of buprenorphine > > nalorphine approximately naltrexone. Comparison of glucuronidation activities in livers from Crigler-Najjar type 1 patients and normal patients indicates that UGT1.1 catalyzes at least 75% of buprenorphine conjugation in normal human liver. In separate studies, the reactivity of expressed rat UGT1.1 was characterized toward various xeno-and endobiotics of various compound classes. It was found that both rat and human UGT1.1 exhibited comparable substrate specificities and efficiencies (Vmax/Km) of glucuronide formation for anthraquinones, coumarins, estrogens, flavonoids, and phenolic compounds. Neither rat nor human UGT1.1 catalyzed the glucuronidation of amines, monoterpenoid alcohols, androgens, or progestins. In general, these data indicate that rat and human UGT1.1 are functionally identical and can be considered orthologous enzymes.

Purification and properties of two rat liver phenobarbital-inducible UDP-glucuronosyltransferases that catalyze the glucuronidation of opioids.

Glucuronidation of xenobiotics and endobiotics is catalyzed by a group of intrinsic membrane proteins of the endoplasmic reticulum of cells: the UDP-glucuronosyltransferases. Two isoforms with glucuronidation activity toward opioids have been purified and characterized from liver microsomes obtained from phenobarbital-treated Wistar rats. The proteins have been identified as the gene products of UGT2B1 and UGT1.1r. The purified proteins exhibited the same apparent KM values for morphine glucuronidation (2-3 mM). However, the purified UGT1.1r enzyme exhibited glucuronidation activity toward buprenorphine and bilirubin with high efficiency, but the UGT2B1 protein did not react with these compounds. Both purified enzymes glucuronidated chloramphenicol, 4-hydroxybiphenyl, chrysin, and ibuprofen. Flunitrazepam photoaffinity labeling was demonstrated for both enzymes, and naloxone, the opioid antagonist, antagonized the photoaffinity labeling reactions.

Effects of formula temperature on postprandial thermogenesis and body temperature of premature infants.

To study the effect of formula temperature on the thermogenic response to gavage feeding, we fed formula at room temperature (mean 24.0 degrees C, SD 1.1) and at body temperature (mean 36.9 degrees C, SD 1.7) to premature infants in a crossover design while monitoring their metabolic heat production and gastric, rectal, and skin temperatures. After feeding with room temperature formula, stomach temperature fell by 6.9 degrees C, rectal temperature by 0.2 degree C, and mean skin temperature by 0.6 degree C, and metabolic rate increased by 16% in the first postprandial hour. After body temperature feedings, mean skin temperature fell by 0.2 degree C, but stomach and rectal temperatures did not change appreciably. The metabolic rate rose by 12% in the first hour, which was not significantly less than the rise after room temperature feeding. The heat required to warm the formula to body temperature did not result in a detectably greater rise in metabolic rate after cool feeding than after warm feeding. The effects of feed temperatures below room temperature were not studied, but it remains possible that cooler feedings might produce even greater body cooling and a greater thermogenic response.

Estimation of 24-hour energy expenditure from shorter measurement periods in premature infants.

We performed continuous indirect calorimetry for 24 h on nine occasions in small premature infants. Oxygen consumption, carbon dioxide production, respiratory quotient, and energy expenditure were calculated for each 2-h period. The mean energy expenditure during the first 6 h was within 6.5% of the mean for the whole 24-h period in all but one case. The mean error in estimating total daily energy expenditure from 6-h measurements was 0.9%. Because positive and negative errors tend to offset each other, we also calculated the mean absolute error, which was 5.6%. The mean coefficient of variation in energy expenditure among the 2-h periods was 11.0%. The mean coefficients of variation in oxygen consumption, carbon dioxide production, and respiratory quotient were 12.8, 9.9, and 14.1%, respectively. Total daily energy expenditure of small premature infants can be estimated from measurements as short as 6 h with sufficient accuracy for most purposes.

Effects of environmental warming on blood components dispensed in syringes for neonatal transfusions.

We examined the quality of blood components dispensed in syringes for transfusion into neonates, including the effects on quality of environmental conditions to which blood is exposed when it is transfused into neonates nursed in warm-air incubators or under radiant warmers. Syringes of blood placed in incubators rapidly warmed to 36 degrees C. Blood placed under radiant warmers operated at full power was heated rapidly to temperatures approaching 45 degrees C, and all blood components exhibited evidence of falling pH and cellular damage after 6 hours exposure to radiant energy. Erythrocyte damage was suggested by an increase in plasma hemoglobin, potassium, and lactic dehydrogenase. Platelets lost the ability to recover from hypotonic shock. Granulocytes exhibited a marked defect in the ability to produce superoxide anion after stimulation. The excessive warming and functional abnormalities exhibited by all blood components exposed to infrared energy were abrogated when syringes were shielded by covering them with aluminum foil. The clinical importance of these experimental findings remains to be established. Meanwhile, it would seem prudent either to shield syringes of blood placed under radiant warmers by covering them with aluminum foil or to limit the volume of a single transfusion to a quantity that can be infused within a relatively brief period.

Tympanic membrane temperature of term and preterm neonates.

Deep body temperatures of 70 term and 24 preterm newborn infants were measured at two sites: deep rectum (5 cm beyond the anus) and tympanic membrane. A significant correlation was found between deep rectal and tympanic membrane temperatures in both term and preterm infants. Mean deep rectal and tympanic membrane temperatures in term infants were 37.01 degrees C and 36.83 degrees C, respectively. Mean deep rectal and tympanic membrane temperatures in preterm infants were both 36.69 degrees C.

Temperature measurement in term and preterm neonates.

Body temperatures of 99 term and 44 preterm infants were measured at four sites: core (5 cm beyond the anus, with an electronic telethermometer), rectum (2 cm, with a mercury-in-glass thermometer), axilla, and between the skin and mattress. Temperatures measured at the four sites agreed closely in this group of largely normothermic infants. However, five of seven term infants with abnormal core temperature (greater than 1.5 SD below or above the mean) would have been judged to be normothermic by each of the three other measurements. The temperatures in preterm infants were lower and varied less with the site of measurement, indicating a smaller core-surface temperature gradient because of their relative lack of thermal insulation by body fat. Axillary temperature was as reliable as rectal temperature measured in the usual way with a mercury-in-glass thermometer. Measurement of the temperature between the skin and mattress was nearly as accurate as the other more frequently used methods. Ninety percent of temperatures were within 0.1 degree C of their final stabilization readings by 5 minutes for each type of thermometer and measurement site.

Air versus skin temperature servocontrol of infant incubators.

Air temperature servocontrol was compared with skin temperature servocontrol and manual control as methods for regulating the heat output of a single-walled incubator (Air-Shields C-86) (1) when optimally used in the laboratory and (2) when operated by staff nurses in the nursery. The subjects were eight premature infants with birth weights between 1.07 and 1.54 kg. When the three methods were used to produce neutral air and skin temperatures during 2-hour measurement periods in the laboratory, there were no differences in mean air, skin, or rectal temperature, metabolic heat production, or body heat loss. There were also no differences among the three methods in mean air, skin, or rectal temperature when used by the nurses in the nursery for periods of 24 hours. When incubator wall temperature is stable, air temperature servocontrol can be used as effectively as skin temperature servocontrol to operate infant incubators.

Performance characteristics of two double-walled infant incubators.

Two double-walled incubators, the Air Shields C-100 and the Ohio IC, were evaluated for performance characteristics. The Ohio incubator heated more rapidly from room temperature to 36 degrees C, but overshot the preset air temperature and produced greater fluctuation in air temperature due to the operation of the servocontrol system at equilibrium. Neither incubator produced excessive air currents, sound levels, or carbon dioxide accumulation.

A double-walled incubator alters the partition of body heat loss of premature infants.

Partitional calorimetry was used to assess the influence of a double-walled incubator (Air-Shields C-86 Isolette) on the body heat loss of eight premature newborn infants (birth weights 1.44-1.89 kg, ages 6-19 days). Each infant was studied in the same incubator with and without the inner wall. Incubator heater output was regulated by servocontrol to maintain the abdominal skin temperature at 36.5 degrees C. Operative environmental temperature was the same (mean 33.0 degrees C) in both incubators. There were no differences in body temperatures, oxygen consumption, carbon dioxide production, respiratory quotient, or evaporative water and heat losses. The double-walled incubator reduced radiant heat loss but increased convective heat loss, so that the total rate of body heat loss was unchanged.