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Biomedical subjects

R L Veech

Publications and source records attributed to R L Veech.

At least 19 recordsLinked to original sources
PubMed1992

Pre-translational induction of pentoxyresorufin O-depentylase by pyridine.

Pentoxyresorufin O-depentylase activity, mainly associated with phenobarbital-inducible cytochrome P450IIB1 (designated CYP2B1), was increased after a single treatment of pyridine (250 mg/kg, i.p.), and further increased by repeated treatments for 5 days. The catalytic activity and immunoreactive protein of CYP2B recognized by polyclonal antibodies were significantly induced by a relatively high dose of pyridine (250 mg/kg, i.p.) while ethanol-inducible cytochrome P450IIE1 (CYP2E1) could be induced by a low dosage (25 mg/kg, i.p.). Unlike CYP2E1 induction without changing its mRNA level, the induction of CYP2B by pyridine was accompanied by an elevation of its mRNA, indicating a pre-translational activation of this enzyme. These results indicate that pyridine induces various isozymes of cytochromes P450 by different induction mechanisms.

Aniline Hydroxylase↗
PubMed1992

Regulation of the purine salvage pathway in rat liver.

The regulation of purine metabolism in rat liver has been examined under conditions that alter the flux through the pathway. Rats were given intraperitoneal injections of ethanol, sodium acetate, or sodium phosphate to attain body water concentrations of approximately 70, 20, and 10 mM, respectively. The livers were freeze-clamped after 30 min, and extracts were made for the analysis of metabolites, cofactors, purine bases, and nucleosides; homogenates were made for the measurement of the activities and kinetic parameters of seven enzymes that participate in purine salvage. The values of the equilibrium constants of nine reactions were determined in vitro and compared with the ratios of the reactants measured in liver. The changes in phosphoribosylpyrophosphate (PRPP), a key intermediate in both the de novo and salvage pathways of purine metabolism, were directly correlated with the changes in ribose 5-phosphate (ribose-5-P); ([PRPP] = 1.7[ribose-5-P] - 7.4 mumol/kg). Ribose-5-P concentrations in turn could be predicted from the liver content of fructose 6-phosphate and glyceraldehyde 3-phosphate by calculation from the known equilibria. The maximum velocities in the tissue of the seven enzymes measured were calculated from the measured substrate values in the liver and with consideration of other effectors of enzyme activity. PRPP synthetase was the least active of the enzymes measured, indicating a possible rate-limiting step. The delta G of the enzyme steps differed from equilibrium values by factors ranging from 4 (nucleoside phosphorylase) to 10(5) (PRPP synthetase and purine transferase reactions). The regulation of purine salvage appeared to depend on the levels of PRPP and ribose-5-P.

Acetates↗
PubMed1992

Substrate regulation of mitochondrial oxidative phosphorylation in hypercapnic rabbit muscle.

Endurance muscle performance is highly dependent on ATP production from mitochondrial oxidative phosphorylation. To study the role of the mitochondrial oxidative enzymes in muscle fatigue, we analyzed the relationship between the concentrations of substrates associated with ATP synthesis and the muscle performance of electrically stimulated rabbit muscle under CO2-induced acidosis. Two different conditions of pacing-induced muscle performance were produced in the gastrocnemius and soleus muscle groups in anesthetized rabbits by stimulating the sciatic nerve submaximally at two frequencies. Phosphorus nuclear magnetic resonance was used to measure ATP, phosphocreatine, and Pi and to provide data for a calculation of intracellular pH and free ADP. To induce acidosis, the animal was ventilated with 20% CO2. The administration of CO2 effectively reduced the intracellular pH from 6.9 to 6.7 and reduced the isometric tension-time integral (TTI) to below half the value measured in normocapnia at the low pacing frequency. A twofold increase in the pacing frequency resulted in a doubling of the TTI in normocapnia and a tripling of TTI in hypercapnia. The increases in TTI corresponded with increases in free ADP and Pi concentrations. Under the various conditions, all free ADP values were near the in vitro Michaelis-Menten constant (Km) of ADP. The Michaelis-Menten relationship of the oxidative phosphorylative enzymes was applied to the change in substrate concentrations with respect to TTI. From this relationship we observed that the in vivo Km of free ADP was 26 microM, which is close to the in nitro Km, and that Km and maximal reaction velocity did not change under hypercapnia and increased pacing frequency.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis↗
PubMed1992

Pretranslational activation of cytochrome P450IIE during ketosis induced by a high fat diet.

Ethanol-inducible cytochrome P450 (P450IIE) is reported to be induced by ketosis. In the present study, the effects of a high fat diet on P450IIE induction and the relationship between ketone body concentration and P450IIE induction were studied by the following: 1) measurement of the activity of aniline hydroxylase, 2) immunoblot analysis for P450IIE protein, and 3) Northern blot analysis for P450IIE mRNA. The enzyme activities (aniline hydroxylase) in hepatic and renal microsomes were elevated about 2-3-fold by feeding with a high fat diet for 3 days. The increases in enzyme activities were also accompanied by 3-fold increases in immunoreactive P450IIE protein and its mRNA. In contrast, no differences were observed for the catalytic activities of N-alkoxyresorufin dealkylases or the amounts of immunoreactive P450IA and P450IIC, indicating a specific induction of P450IIE by high fat feeding. Furthermore, the increases in the levels of P450IIE mRNA correlated positively (r = 0.73) with plasma concentrations of acetoacetate and beta-hydroxybutyrate but not with that of acetone, which induces P450IIE without changing its mRNA level. Our data thus indicated that P450IIE induction during the ketosis of a high fat feeding appears to be due to pretranslational activation and that is similar to the induction mechanism of fasted and diabetic animals.

Aniline Hydroxylase↗
PubMed1991

Post-translational reduction of cytochrome P450IIE by CCl4, its substrate.

The molecular mechanism of cytochrome P450IIE reduction by CCl4 was reexamined by measuring its enzyme activity, immunoreactive protein contents, and mRNA levels. Aniline hydroxylase and the amounts of immunoreactive P450IIE were rapidly decreased in a time-dependent manner after a single dose of CCl4. No changes were observed in the amounts of immunoreactive P450IIC and P450IA despite significant decreases decrease in their catalytic activities. However, the decreases in P450IIE enzyme activity and immunoreactive protein by CCl4 were not accompanied by a decline in its mRNA level. The data thus suggested a post-translational reduction of P450IIE by CCl4, probably due to specific destruction of the P450IIE protein by its own substrate rather than heme moiety.

Aniline Hydroxylase↗
PubMed1991

The metabolism of lactate.

Lactate contents of tissues can vary during normal physiological conditions in mammals from 0.5 to over 5 mM, and higher in pathological states, suitable for measurement by NMR. Lactate itself is a dead-end metabolite, metabolized only by lactate dehydrogenase. The extent and direction of that reaction is determined by the free [NAD+]/[NADH][H+] ratio of cytoplasm with which lactate and pyruvate are in near-equilibrium. Pyruvate is a crossroads of most of the major degradative and synthetic pathways, but present in about one-tenth the amount of lactate. Information on the content of both lactate and its redox partner pyruvate is likely to provide more information on the metabolic state of tissue than are measurements of lactate alone.

Biological Transport↗
PubMed1991

Enzymatic determination of total CO2 in freeze-clamped animal tissues and plasma.

An enzymatic method for measuring total carbon dioxide content in freeze-clamped animal tissues is described. Total carbon dioxide content [TCO2] was defined as the sum of the dissolved CO2, the bicarbonate concentration, and the carbonate concentration. Tissue was extracted in 80% methanol, 20 mM 2-amino-2-methyl-1-propanol, pH 9.5 at 25 degrees C and homogenized in a 1.5-ml Sardstat screw-top test tube containing 0.5-mm glass beads and a minibead beater. Total CO2 was determined as bicarbonate/carbonate by monitoring the oxidation of NADH at 340 nm using the coupled assay of phosphoenolpyruvate carboxylase (EC 4.1.1.31) and malate dehydrogenase (EC 1.1.1.37). In the coupled assay system, 1 mumol of bicarbonate/carbonate consumed is equivalent to the oxidation of 1 mumol NADH at 340 nm. The assay medium comprised 50 mM 2-amino-2-methyl-1-propanol, pH 9.0 at 25 degrees C, 5 mM phosphoenolpyruvate (PEP), 0.25 mM NADH, 5 mM MgCl2, 5 mM mercaptoethanol, 0.02% bovine serum albumin, 10 mM oxamate, PEP carboxylase (0.5 units/ml), and malate dehydrogenase (0.5 units/ml). The total CO2 content measured in freeze-clamped rat heart, liver, brain, and skeletal muscle was 20.53 +/- 0.64, 17.34 +/- 0.67, 17.00 +/- 0.48, 16.06 +/- 0.53 mumol/g wet wt tissue, respectively (n = 5). The total CO2 in the crusher muscle of the lobster was found to be 5.0 +/- 0.33 mumol/g wet wt. Total CO2 was also enzymatically measured in arterial plasma from four chronically cannulated male wistar rats and was 24.65 +/- 1.81 mumol/ml plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Age dependence of steady state mitochondrial oxidative metabolism in the in vivo hypoxic dog brain.

Mitochondrial bioenergetics were investigated in newborn, neonatal and adult dog brains during normoxia and hypoxia. The ratio of the rate of ATP synthesis to the maximum synthesis rate (V/Vmax), phosphorylation potential, [ADP] and PCr/Pi, were used to evaluate age related mitochondrial hypoxic tolerance. These indicators were calculated from the phosphorus compounds measured by in vivo 31P MRS quantitatively using ATP as an internal reference. Indicators and substrates of mitochondrial function, V/Vmax, ADP, and Pi reached a peak value during the neonatal (3-21 days) period of development, suggesting that the oxidative metabolism of the neonate is more vulnerable to stress when compared to newborns and adults. Distinction among newborns and neonates became apparent during hypoxia. Newborns (0-2 days old) showed substantial tolerance by maintaining V/Vmax until exposure to severe hypoxia. Older neonates (3-21 days old) showed increases in V/Vmax, [Pi] and [ADP] under less than severe conditions of hypoxia. Adults exhibited low V/Vmax values even during exposure to severe hypoxia, further indicating that mitochondrial oxidative processes are more stable in adults than in newborns and neonates. This study provides evidence that newborns and adults are more capable of maintaining mitochondrial function under conditions of minimal to moderate hypoxia than 3-21 day old neonates.

Adenosine Triphosphate↗
PubMed1991

Altered expression of HLA antigens and CD16 Fc receptors on leukocytes of alcoholic subjects and uremic patients.

The possible influences of ethanol and its metabolic product acetate on the surface expression of HLA class I and class II antigens and CD16 Fc receptors were examined. Fluorescent-labeled monoclonal antibodies and flow cytometry were used to measure these antigens on leukocytes from reference controls, subjects admitted for alcohol detoxification, uremic patients undergoing hemodialysis using Cu-prophan dialyzers and fluids containing 4 to 37 mM acetate, and uremic patients that were not hemodialyzed. In comparison to the controls, the mean intensity of staining for class I antigens was not changed significantly on lymphocytes or monocytes from alcoholics but was depressed on cells from eight of 12 uremic patients. Interferon-gamma above 5 units/ml was detected in less than 15% of plasma samples from controls, uremic patients or alcoholics on admission but was detected in four of eight samples from alcoholics at discharge (2-4 days after admission). The intensity of staining for class II antigens was depressed by more than 50% on lymphocytes from alcoholics and uremic patients. The expression of HLA class I and class II antigens was depressed whether uremic patients were hemodialyzed or not. The percentage of lymphocytes expressing CD16 was depressed in three of seven alcoholics and five of seven hemodialyzed patients. In contrast, the percentage of monocytes expressing CD16 was increased in six of seven hemodialyzed patients and three of five uremic patients not undergoing hemodialysis suggesting activation of monocytes in these patients. Plasma levels of beta 2-microglobulin were elevated by 61% in alcoholics, 50-fold in hemodialyzed patients, and 26-fold in nonhemodialyzed uremic patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Microsomal ethanol oxidizing system: transcriptional and posttranscriptional regulation of cytochrome P450, CYP2E1.

CYP2E1 is solely responsible for microsomal P450-mediated ethanol oxidation activity. This enzyme is also involved in a pathway leading to gluconeogenesis from ketone bodies and in metabolic activation of numerous foreign compounds to intermediates that can be toxic to cells. Metabolic activation of certain procarcinogens by CYP2E1 may also lead to cell transformation. Regulation of CYP2E1 is especially intriguing. The CYP2E1 gene is transcriptionally activated in rat liver from a dormant state within a few hours after birth. This activation is due in part to the participation of a transcription factor designated hepatocyte nuclear factor 1 or HNF-1. In adult animals, constitutive expression of the enzyme is controlled to some degree by growth hormone. CYP2E1 is also regulated by many of its substrates through a substrate-induced stabilization of the enzyme. Under extreme conditions of fasting and uncontrolled diabetes CYP2E1 mRNA is stabilized.

Animals↗
PubMed1990

The Gibbs-Donnan near-equilibrium system of heart.

The gradients of the major inorganic ions across the plasma membrane of heart were examined to determine the factors controlling the extent and direction of the changes induced during injury, certain diseases, and electrolyte disturbances. The ionic environment was altered by changing only the concentration of inorganic phosphate, [sigma Pi]o, from 0 to 1.2 to 5 mM in the Krebs-Henseleit buffer perfusing working rat hearts. Raising [sigma Pi]o from 1.2 to 5 mM resulted in a decrease in total Mg2+ content and calculated free cytosolic [Mg2+] from 0.44 to 0.04 mM, conversion of 4 mmol of MgATP2- to ATP4- and a decrease in measured intracellular [Cl-]i from 41 to 16 mM. At all levels of [sigma Pi]o, both the [Na+]i and [K+]i were invariant at about 3 mM and 130 mM, respectively, as was the energy of hydrolysis of the terminal phosphate bond of sigma ATP, delta GATP Hydr, of -13.2 kcal/mol. The relationship maintained between the ions on both sides of the plasma membrane by the 3Na+/2K(+)transporting ATPase (EC 3.6.1.37) and an open K+ channel was: (formula; see text) The energy of the gradients of the other inorganic ions across the plasma membrane, delta G[ion]o/i, exhibited three distinct quanta of energy derived from the prime quantum of delta GATP Hydr of -13.2 kcal/mol. The second quantum was about one-third of delta GATP Hydr or +/- 4.4 kcal/mol and comprised the delta G[Na+]o/i, delta G[Mg2+]o/i, and delta G[HPO42-]o/i. These results indicated near-equilibrium was achieved by the reactants of the 3Na+/2K(+)-ATPase, the K+ channel, the Na(+)-Pi co-transporter, and a postulated net Mg2+/H2PO4- exchanger. The third quantum was one-third of delta G[Na+]o/i or about +/- 1.5 kcal/mol and comprised delta G[H+]o/i, delta G[HCO3-]o/i, and delta G[Cl-]o/i. The delta G[K+]o/i was 0, indicating near-equilibrium between the chemical energy of [K+]o/i and the E across the plasma membrane of -83 mV. It is concluded that the gradients of the major inorganic ions across the plasma membrane and the potential across that membrane constitute a Gibbs-Donnan equilibrium system catalyzed by transport enzymes sharing common substrates. The chemical and electrical energies of those gradients are equal in magnitude and opposite in sign to the chemical energy of ATP hydrolysis.

Adenosine Diphosphate↗
PubMed1990

In vivo portal-hepatic venous gradients of glycogenic precursors and incorporation of D-[3-3H]glucose into liver glycogen in the awake rat.

Male Wistar fed rats were chronically cannulated and fed ground chow for 2 h for 6 days. On the 7th post-operative day, blood was simultaneously drawn from the portal and hepatic veins over a 2-h feeding period. The position of the hepatic vein cannula was verified using a tritiated water washout technique. In separate experiments, 200 microCi of [3-3H]glucose was added to the food in order to determine the relative contribution of D-glucose and 3-C precursors to newly synthesized glycogen. The 22-h fasting plasma portal vein concentrations of D-glucose, L-lactate, and L-alanine were 4.8 +/- 0.03, 0.81 +/- 0.06, and 0.20 +/- 0.03 mM, respectively (n = 5). The fasting hepatic vein plasma concentrations were 5.1 +/- 0.2, 0.70 +/- 0.15 and 0.19 +/- 0.03 mM, respectively. The portal-hepatic vein gradients after 22 h were -0.24, +0.16, and +0.01 mM for D-glucose, L-lactate, and L-alanine, respectively. At 20 min after beginning the meal, the respective gradients were +2.2, +0.53, and +0.44 mM, indicating hepatic uptake of all glycogen precursors. Of the total carbon from the three major precursors entering the liver as C-6, D-glucose contributed 82%, while alanine and lactate contributed 18% at 20 min. As portal vein D-glucose and L-alanine levels exceeded 6.65 +/- 0.69 and 0.32 +/- 0.07 mM, respectively, the portal-hepatic venous gradient became positive and increased linearly with portal concentrations. The glycogen concentration in the liver increased from a 22-h fast value of 5 mumol of glucosyl units/g wet weight to 101 +/- 7 mumol/g 2 h after the meal. The mean specific activity of portal vein plasma of [3-3H]glucose was 11,490 +/- 1,180 dpm/mumol (+/- S.E.) and that in the glycogen isolated from liver was 8,175 +/- 785 dpm/mumol of glycosyl units 2 h after the meal. The specific activity of liver [3H]glycogen relative to glucose after the meal was 0.73 +/- 0.08. It was concluded that a minimum of 73% of the newly synthesized glycogen was formed from the uptake and direct phosphorylation of portal blood D-glucose by the liver without prior conversion of glucose to 3-C units.

Alanine↗
PubMed1990

A new transient activator of phosphofructokinase during initiation of rapid glycolysis in brain.

The tissue contents of previously known allosteric effectors of brain phosphofructokinase (EC 2.7.1.11) (PFK) and the kinetic behavior of isolated PFK were investigated during the initiation of rapid glycolytic flux in freeze-blown rat brain. Comparing 0- with 5-s brains revealed that there was a 4-fold drop in total tissue content of Fru-6-P and a 5.6-fold increase in Fru-1,6-P2 consistent with activation of PFK. Additionally, analysis of brain content showed a 15-fold increase in AMP, a 3-fold decrease in ATP, a 3-fold decrease in Pi, and a 1.6-fold increase in NH4+. There was no change in Fru-2,6-P2, H+, citrate, or Glc-1,6-P2 or the kinetic profiles of isolated PFK for ATP inhibition or Fru-2,6-P2 activation. We concluded that the observed change in PFK activity could be accounted for only partially by changes in the concentrations of adenine nucleotides and other known effectors. High performance liquid chromatography fractions of extracts obtained from 5-s brains showed the activator with a mobility identical to ribose 1,5-P2 and gave 2 nmol/g (wet weight) at 0 s, 10 nmol/g at 5 s, and 2 nmol/g at 20 s. Assay of PFK in the presence of effectors determined to be in tissue at 5 s showed that addition of 10 nmol/ml ribose 1,5-P2 gave a 4-fold activation of PFK. Based on the rapidity of its formation, its potency of activation, and its similarity in chemical properties to authentic ribose 1,5-P2, we conclude that ribose 1,5-P2 served as the initial activator of PFK in brain.

Adenosine Monophosphate↗
PubMed1990

Radiometric measurement of phosphoribosylpyrophosphate and ribose 5-phosphate by enzymatic procedures.

Methods for the measurement of phosphoribosylpyrophosphate (PRPP) and ribose 5-phosphate (R-5-P) in tissues have been developed. The lability of these compounds during tissue extraction and the recovery of standards from tissue preparations have been examined. Enzymatic conversion of phosphoribosylpyrophosphate to [14C]AMP in the presence of labeled adenine or formation of [14C]GMP ([14C]IMP) in the presence of labeled guanine or hypoxanthine was accomplished in the first step. In the second step, the labeled product was separated from the substrate. For the measurement of R-5-P, the first step included phosphoribosylpyrophosphate synthetase, as well as the appropriate substrate and effector (ATP and Pi), in combination with adenine phosphoribosyl transferase. The product [14C]AMP was measured in three ways: (1) HPLC separation with an on-line radioisotope detector; (2) butanol extraction of the labeled base, and measurement of an aliquot of the aqueous phase in a scintillation counter; (3) filtration of the incubation mixture with chromatographic filter paper disks, which were then counted in a scintillation counter. When [14C]guanine was the substrate, HPLC separation was used because the butanol or paper separation was not adequate. Measurement of 5-125 pmol of PRPP or R-5-P gave a linear response.

Adenine Phosphoribosyltransferase↗
PubMed1990

Short chain diol metabolism in human disease states.

Recent clinical studies have shown the presence of two short chain diols, meso-2,3-butanediol and D/L-2,3-butanediol, and in most cases 1,2-propanediol in either serum or urine collected from humans in several apparently unrelated disease states: congenital propionic and methylmalonic acidemia, premature infants, and alcoholics both in the presence and absence of ethanol. In addition 1,2-propanediol has been shown in patients during prolonged starvation, and in patients with diabetic keto-acidosis. No common defect is known to exist in these metabolic states. Understanding how these compounds are produced in clinically well-defined diseases such as methyl malonic and propionic aciduria, however, may help explain how and why these compounds are produced in alcoholics.

Acetoin↗
PubMed1990

Plasma tumor necrosis factor alpha predicts decreased long-term survival in severe alcoholic hepatitis.

Plasma tumor necrosis factor alpha (TNF alpha), interleukin 1 alpha (IL-1 alpha), and interleukin 1 beta (IL-1 beta) were measured in plasma samples obtained from 23 patients with severe alcoholic hepatitis on admission and after 30 days of hospitalization. Over a 2-year follow-up period, 14 patients died at a mean time of 8 months following discharge. The presence of elevated plasma TNF alpha either at admission or discharge from the hospital was associated with death in 82% (14/17) of patients. By contrast absence of elevated plasma TNF alpha was associated with survival in 100% (6/6). The difference in survival with and without detectable plasma TNF alpha was significant at p = 0.0022. Plasma TNF alpha was not elevated in alcoholic patients without clinically apparent liver disease, with alcoholic cirrhosis, or in nonalcoholic healthy controls. Plasma IL-1 alpha was also significantly increased in alcoholic hepatitis whereas IL-1 beta was not. Neither IL-1 alpha nor beta was correlated with outcome in the alcoholic hepatitis group. It is concluded that the presence of elevated plasma TNF alpha is a significant predictor of decreased long-term survival in patients with severe alcoholic hepatitis.

Adult↗
PubMed1990

Cytochrome P450IIE1 is elevated in lymphocytes from poorly controlled insulin-dependent diabetics.

Cytochrome P450IIE1, a member of the cytochrome P450 supergene family, was measured in peripheral lymphocytes of 14 patients with insulin-dependent diabetes mellitus who were in poor metabolic control, as evidenced by elevated hemoglobin A1 levels (mean, 11.9 +/- 2.8%; normal, less than 7.8). Only one major form (mol wt, 48,000 daltons) of cytochrome P450IIE1 was detected with a specific polyclonal antibody against P450IIE1. Levels of cytochrome P450IIE1 were very low to undetectable in human lymphocytes from seven normal subjects. However, levels of P450IIE1 were elevated in lymphocytes from patients with insulin-dependent diabetes mellitus. Elevated levels of cytochrome P450IIE1 determined by immunoblot analysis correlate positively with the levels of hemoglobin A1 (r = 0.8), a metabolic indicator in diabetic subjects. In one study subject in whom diabetic control was improved, the drop in hemoglobin A1C levels was accompanied by normalization of P450IIE1 levels.

Adolescent↗