Nicotinamida adenina dinucleótido (NADH) en pacientes con síndrome de fatiga crónica / Nicotinamide adenine dinucleotide (NADH) in patients with chronic fatigue syndrome

AntecedentesLa nicotinamida adenina dinucleótido (NADH) podría estar deplecionada en el síndrome de fatiga crónica. El objetivo del estudio fue evaluar la eficacia de la suplementación con NADH en estos pacientes.Material y métodosSe realizó un ensayo clínico, doble ciego, controlado con placebo de 3 meses de duración. Los pacientes fueron randomizados a NADH oral 20mg o placebo durante los 2 primeros meses. Se evaluó la intensidad de la fatiga, estado funcional, estado de ánimo, impacto funcional de la fatiga, calidad de vida, calidad del sueño, capacidad al ejercicio y reserva funcional, así como la opinión del investigador y pacientes sobre la eficacia de la intervención, antes y a los 30, 60 y 90 días del inicio del tratamiento. En la visita basal y a los 60 días (último día de tratamiento doble ciego) se realizó una prueba de esfuerzo.ResultadosSe incluyeron 86 enfermos de los cuales 77 concluyeron el estudio (edad media, 47 años; 72 mujeres). No se hallaron diferencias significativas en la mayoría de las variables estudiadas al finalizar el estudio. La administración de NADH se asoció a una disminución de estado de ansiedad de −1,0 puntos (p<0,05) y de −0,2 puntos (p=NS) en el grupo asignado a placebo. La frecuencia cardiaca máxima tras la prueba de esfuerzo disminuyó una media de −8,1l/min (p<0,05) en el grupo NADH y ascendió +1,7l/min en el grupo placebo (p=0,73). No se hallaron diferencias en la percepción de eficacia con NADH y placebo, por parte del investigador y pacientes.ConclusionesLa administración de NADH oral se asoció a una disminución de la ansiedad y de la frecuencia cardiaca máxima, tras una prueba de esfuerzo, en los pacientes con síndrome de fatiga crónica. Por el contrario, este tratamiento no modificó otras variables clínicas y el estado funcional global(AU)

BackgroundNicotinamide adenine dinucleotide (NADH) may be depleted in chronic fatigue syndrome (SFC). The purpose of the study was to evaluate the efficacy of supplementation with NADH in these patients.ResultsA total of 86 patients, 77 of whom completed the study (mean age, 47 years, 72 women) were enrolled. No significant differences were found in most of the variable studied at the end of the study. Administration of NADH was associated to a decrease in anxiety condition of −1.0 points (p<0.05) and of −0.2 points (p=NS) in the placebo assigned group. Maximum heart rate after the stress test decreased a mean of −8.1l/min (p<0.05) in the NADH group and increased by +1.7l/min in the placebo group (p=0.73). No differences were found in the perception of efficacy with NADH and placebo, by the investigator and patients.ConclusionsAdministration of oral NADH was associated to a decrease in anxiety and maximum heart rate, after a stress test in patients with CFS. On the contrary, this treatment did not modify other clinical variables and the global functional performance(AU)

Pharmacokinetic aspects of reduced nicotinamide adenine dinucleotide (NADH) in rats.

Nicotinamide adenine dinucleotide (NADH) plays a major role in cellular metabolism and mitochondrial dysfunction and is thought that NAD+/NADH decrease neuronal degeneration and improve behavioral deficits. This potential use of NAD+ or NADH as neuroprotective drugs requires an insight on the pharmacokinetic properties of these compounds. For this reason, we assessed the absorption of NADH in the small intestine in vitro using the everted gut sac technique. We show an enteral absorption of the intact NADH molecule. In the gut sac, NADH had a concentration-independent absorption rate of about 5 percent and the in vivo laser-induced fluorescence spectroscopy revealed a relatively quick absorption of NADH starting after a few minute reaching a plateau (about 5 percent ) after 20-30 minutes. Theses results show that, should NADH be protected against the acidic conditions of the stomach, NADH is absorbed principally in the small intestine.

Transformation products of extracellular NAD(+) in the rat liver: kinetics of formation and metabolic action.

The perfused rat liver responds in several ways to NAD(+) infusion (20-100 microM). Increases in portal perfusion pressure and glycogenolysis and transient inhibition of oxygen consumption and gluconeogenesis are some of the effects that were observed. Extracellular NAD(+) is also extensively transformed in the liver. The purpose of the present work was to determine the main products of extracellular NAD(+) transformation under various conditions and to investigate the possible contribution of these products for the metabolic effects of the parent compound. The experiments were done with the isolated perfused rat liver. The NAD(+) transformation was monitored by HPLC. Confirming previous findings, the single-pass transformation of 100 microM NAD(+) ranged between 75% at 1.5 min after starting infusion to 95% at 8 min. The most important products of single-pass NAD(+) transformation appearing in the outflowing perfusate were nicotinamide, ADP-ribose, uric acid, and inosine. The relative proportions of these products presented some variations with the time after initiation of NAD(+) infusion and the perfusion conditions, but ADP-ribose was always more abundant than uric acid and inosine. Cyclic ADP-ribose (cADP-ribose) as well as adenosine were not detected in the outflowing perfusate. The metabolic effects of ADP-ribose were essentially those already described for NAD(+). These effects were sensitive to suramin (P2(XY) purinergic receptor antagonist) and insensitive to 3,7-dimethyl-1-(2-propargyl)-xanthine (A2 purinergic receptor antagonist). Inosine, a known purinergic A3 agonist, was also active on metabolism, but uric acid and nicotinamide were inactive. It was concluded that the metabolic and hemodynamic effects of extracellular NAD(+) are caused mainly by interactions with purinergic receptors with a highly significant participation of its main transformation product ADP-ribose.

P2X7 receptors mediate NADH transport across the plasma membranes of astrocytes.

NADH plays critical roles in mitochondrial functions and energy metabolism. There has been no study demonstrating that NADH can be transported across the plasma membranes of cells. In this study we tested our hypothesis that NADH can be transported across the plasma membranes of astrocytes by a P2X7 receptor (P2X7R)-mediated mechanism. We found that treatment of astrocytes with NADH led to increases in both intracellular NADH and NAD+. Three lines of studies suggest that P2X7R mediates the NADH transport into astrocytes: the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) blocked the NADH transport; RNAi knockdown of P2X7R led to decreased NADH transport; and transfection of HEK293 cells with mouse P2X7R cDNA led to increased NADH transport. Collectively, our study provides the first direct evidence demonstrating that NADH can be transported across the plasma membranes of astrocytes by a P2X7R-mediated mechanism. Our study also suggests a novel approach for manipulating intracellular NADH and NAD+ levels.

Comparison of metabolic fates of nicotinamide, NAD+ and NADH administered orally and intraperitoneally; characterization of oral NADH.

Since NADH has been implicated in medication for some symptoms and as a possible supplement for health, we characterized the metabolic fate of NADH orally given to mice by comparing with those of nicotinamide (Nam), NAD+ and NADH intraperitoneally or orally administered. Mice were individually housed in metabolic cages, and divided into two sets of four groups. Within each set, one group was intraperitoneally or orally administered saline and the other three groups received intraperitoneal or oral administration of a pharmacological dose of Nam, NAD+ or NADH (5 micromol/mouse). Twenty-four hour urine samples for the day before and days 1 to 4 after administration were collected and analyzed for Nam and its metabolites. When mice were administered saline alone, urinary excretion of Nam and its metabolites, such as nicotinamide N-oxide (Nam N-oxide), N1-methylnicotinamide (MNA), N1-methyl-2-pyridone-5-carboxamide (2-Py), and N1-methyl-4-pyridone-3-carboxamide (4-Py), was unchanged from day 0 to day 4. Intraperitoneal injection of Nam, NAD+ and NADH produced significant increases in urinary excretion of Nam and its metabolites. Similar results were obtained when Nam and NAD+ were given orally. On the other hand, oral administration of NADH did not bring about an increase in urinary excretion of Nam and its metabolites, suggesting that NADH in digestive organs has been decomposed to a compound(s) that cannot yield Nam. In fact, incubation of NADH at acidic pH to mimic the stomach resulted in rapid conversion of NADH to an unknown compound. Better understanding of the fate of oral NADH is needed for its therapeutic and supplemental use.

Isatin interaction with glyceraldehyde-3-phosphate dehydrogenase, a putative target of neuroprotective drugs: partial agonism with deprenyl.

There is evidence that the binding of deprenyl, a monoamine oxidase (MAO) B inhibitor, and other propargylamines to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is primarily responsible for their neuroprotective and antiapoptotic effects. Thus, GAPDH may be a target for other neuroprotective drugs. Using two independent approaches, radioligand analysis and an optical biosensor technique, we demonstrate here that GAPDH also interacts with the endogenous, reversible MAO B inhibitor, isatin. Deprenyl inhibited both [3H]isatin binding to GAPDH, and the binding of this enzyme to an isatin analogue, 5-aminoisatin, immobilized on to an optical biosensor cell. Another MAO inhibitor, tranylcypromine, was ineffective. Both deprenyl and isatin inhibited GAPDH-mediated cleavage of E. coli tRNA, and their effects were not additive. We suggest that isatin may be an endogenous partial functional agonist of deprenyl in its effect on GAPDH and GAPDH-mediated RNA cleavage. Changes in level of endogenous isatin may influence the neuroprotective effect of deprenyl in vivo.

The effects of organic solvents on poly(ADP-ribose) polymerase-1 activity: implications for neurotoxicity.

Poly(ADP-ribose) polymerase-1 (PARP-1; EC 2.4.2.30), also termed as poly(ADP-ribose) synthetase, is a key enzyme in the recognition and repair of damaged DNA. Several conditions (e.g., ischemia-reperfusion or chemical-induced injury) have been shown to overactivate PARP-1, causing neurodegeneration and necrotic or apoptotic cell death from NAD+ and ATP depletion. In contrast, inhibitors of PARP-1 have been shown to have a neuroprotective effect by ameliorating this response. The purpose of this study was to determine the effects of three routinely used organic solvents (ethanol, methanol, and dimethyl sulfoxide (DMSO)) on the activity of purified PARP-1. A dose-response was examined with each of these solvents. A 112% and 82% increase in PARP-1 activity was observed with 15% ethanol and 20% methanol, respectively. In contrast, a near 20% decrease in the activity was observed with 4% DMSO. Kinetic analysis revealed that the maximal velocity remained unchanged with increasing concentrations of DMSO up to 20%, indicating that DMSO is a competitive inhibitor of PARP-1. Thus, PARP-1 inhibition by DMSO depends on NAD+ concentration and in some pathological processes might be significant even at low DMSO concentrations. Our findings suggest that the interpretation of data from dose-response studies obtained when using common organic solvents may be dramatically skewed, either exaggerating the inherent toxicity of the compound or masking its potential for damage.

On the safety of reduced nicotinamide adenine dinucleotide (NADH).

The objective of the study was to determine both the toxicity of the stabilized orally absorbable form of nicotinamide adenine dinucleotide (NADH) (ENADA) and the maximum tolerated intravenous dose (MTD) of betaNADH (the reduced form of NADH) in beagle dogs. The administration of the stabilized orally absorbable form of NADH to beagle dogs at dose levels of 20, 100, and 150 mg/kg for 14 days elicited no signs of a toxicological effect. A transitory change in stool formation was observed with the intermediate and high dose in males. There were also apparent increases in adrenal, heart, kidney, liver, brain, and thyroid weights, particularly in males, but none of these changes were considered to be toxicologically significant. In addition, four dogs (two of each sex) received intravenous infusions of 100 mg NADH/kg/day for 4 days, followed by 200 mg NADH/kg/day for 3 days, followed by 500 mg NADH/kg/day for 4 days, and 1000 mg NADH/kg/day on the final day. At the end of the MTD phase, the control animals that had received saline solution in the MTD phase were used to evaluate the potential toxicity of the established MTD. These animals received 500 mg NADH/kg/day for 14 days (fixed dose phase). There were no deaths. At dose levels between 100 and 1000 mg/kg/day, effects on the cardiovascular system and also some evidence of an effect on the central nervous system and on the adrenals were observed. At doses of 500 mg/kg/day and above, food consumption and body weight were reduced. On the basis of the observed changes, the maximum intravenous dose of NADH tolerated by beagle dogs was considered to be 500 mg/kg/day. There were no gross histological findings indicative of toxicity in the organs of tissues examined. Based on these findings, the stabilized orally absorbable form of NADH (ENADA) can be regarded as safe.

NADH supplementation decreases pinacidil-primed I K ATP in ventricular cardiomyocytes by increasing intracellular ATP.

1 The aim of this study was to investigate the effect of nicotinamide-adenine dinucleotide (NADH) supplementation on the metabolic condition of isolated guinea-pig ventricular cardiomyocytes. The pinacidil-primed ATP-dependent potassium current I(K(ATP)) was used as an indicator of subsarcolemmal ATP concentration and intracellular adenine nucleotide contents were measured. 2 Membrane currents were studied using the patch-clamp technique in the whole-cell recording mode at 36-37 degrees C. Adenine nucleotides were determined by HPLC. 3 Under physiological conditions (4.3 mM ATP in the pipette solution, ATP(i)) I(K(ATP)) did not contribute to basal electrical activity. 4 The ATP-dependent potassium (K((ATP))) channel opener pinacidil activated I(K(ATP)) dependent on [ATP](i) showing a significantly more pronounced activation at lower (1 mM) [ATP](i). 5 Supplementation of cardiomyocytes with 300 micro g ml(-1) NADH (4-6 h) resulted in a significantly reduced I(K(ATP)) activation by pinacidil compared to control cells. The current density was 13.8+/-3.78 (n=6) versus 28.9+/-3.38 pA pF(-1) (n=19; P<0.05). 6 Equimolar amounts of the related compounds nicotinamide and NAD(+) did not achieve a similar effect like NADH. 7 Measurement of adenine nucleotides by HPLC revealed a significant increase in intracellular ATP (NADH supplementation: 45.6+/-1.88 nmol mg(-1) protein versus control: 35.4+/-2.57 nmol mg(-1) protein, P<0.000005). 8 These data show that supplementation of guinea-pig ventricular cardiomyocytes with NADH results in a decreased activation of I(K(ATP)) by pinacidil compared to control myocytes, indicating a higher subsarcolemmal ATP concentration. 9 Analysis of intracellular adenine nucleotides by HPLC confirmed the significant increase in ATP.

Skeletal muscle sarcoplasmic reticulum contains a NADH-dependent oxidase that generates superoxide.

Skeletal muscle sarcoplasmic reticulum (SR) is shown to contain an NADH-dependent oxidase (NOX) that reduces molecular oxygen to generate superoxide. Its activity is coupled to an activation of the Ca2+ release mechanism, as evident by stimulation in the rate of high-affinity ryanodine binding. NOX activity, coupled to the production of superoxide, is not derived from the mitochondria but is SR in origin. The SR preparation also contains a significant NADH oxidase activity, which is not coupled to the production of superoxide and appears to be mitochondrial in origin. This mitochondrial component is preferentially associated with the terminal cisternae region of the SR. Its activity is inhibited by diphenylene iodonium (10 microM), antimycin A (200 nM), and rotenone (40 nM) but is not coupled to the generation of superoxide or the stimulation of the ryanodine receptor. The rate of superoxide production per milligram of protein is larger in SR than in mitochondria. This NOX may be a major source of oxidative stress in muscle.

Selective recovery of lactate dehydrogenase using affinity foam.

Selective isolation of lactate dehydrogenase (LDH) from porcine muscle extract was studied using foam generated from the vigorous stirring of a non-ionic surfactant, Triton X-114 derivatized with Cibacron blue. The cloud point of the surfactant-dye conjugate was higher than that of the native Triton X-114, and also the foam prepared from the affinity surfactant was more rigid taking a longer time to collapse. The equilibrium dissociation constant between pure LDH and surfactant-dye conjugate was 5.0 microM as compared to the value of 2.2 microM for the enzyme and free dye as measured by differential spectroscopy. The isolation procedure involved mixing of the porcine muscle extract with the affinity foam, separating and collapsing the foam, and warming the solution formed to 37 degrees C to yield the surfactant-dye phase and an aqueous phase containing the enzyme. The effect of surfactant concentration and protein load on enzyme recovery and purification was investigated. Under optimal conditions, LDH was quantitatively recovered with high purification factor in a very short time. Both recovery and purification were higher when foam prepared from an equivalent mixture of surfactant-dye conjugate and unmodified surfactant was used. The selectivity of interaction between LDH and detergent-dye conjugate was confirmed by lowered recovery when NADH was included during the binding step.

Bioavailability of reduced nicotinamide-adenine-dinucleotide (NADH) in the central nervous system of the anaesthetized rat measured by laser-induced fluorescence spectroscopy.

Drugs intended to increase wellness or quality of life ("lifestyle drugs") have gained popularity and/or importance over recent years. Biogenic substances like nicotinamide adenine dinucleotide (NADH) are supposed to increase the physical and intellectual performance without side-effects. NADH is an energy-delivering co-substrate in the respiratory chain. Clinical studies showed positive effects of peripherally given NADH in Morbus Parkinson and major depression. NADH can be measured by its fluorescence. In this study a pulsed N2-laser combined with a fibre-optic probe and photomultipliers was used to induce and measure NADH fluorescence in the rat cortex. The aims of the study were to assess the suitability of the laser-induced spectroscopy for in vivo and on-line measurement of NADH in neuroscience and the assessment of the central availability of NADH after peripheral administration. NADH (50 mg/kg) but not the precursor nicotinamide caused a significant rise of the NADH fluorescence intensity indicating an increase of the NADH concentration in the rat cortex. In conclusion, the results suggest that NADH given orally or intraperitoneally increases the amounts of NADH in the brain. The results may thus help to explain the clinical effects reported.

Elevation of blood NAD level after moderate exercise in young women and mice.

We previously reported that the blood NAD levels are decreased by severe exercise, and administration of nicotinamide, a precursor of NAD, improves the endurance capacity of mice. In the present study, we determined whether moderate exercise changes the blood NAD levels in humans and mice. College female students exercised moderately with bike-ergometers. The blood NAD levels elevated after moderate exercise. Mice were forced to swim in a running water pool for 5 min as a moderate exercise, 15 min as a strong exercise, and until exhaustion as a severe exercise (average swimming time was 28.7 min). A 5 min swim gave a result similar to that of moderate exercise by human subjects. However, the blood NAD levels decreased after all-out exercise. The changes in whole blood tryptophan (a precursor of pyridine nucleotides) levels were similar to that in NAD. The glucose levels in whole blood and the non-esterified fatty acid levels in serum decreased according to exercising time. These data are the first demonstration of moderate exercise raising the blood NAD levels in human and mice. Elevation of the blood NAD levels may reflect changes in niacin metabolism that occur in response to exercise.

Identification of the enzymatic active site of CD38 by site-directed mutagenesis.

CD38 is a ubiquitous protein originally identified as a lymphocyte antigen and recently also found to be a multifunctional enzyme participating in the synthesis and metabolism of two Ca(2+) messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate. It is homologous to Aplysia ADP-ribosyl cyclase, where the crystal structure has been determined. Residues of CD38 corresponding to those at the active site of the Aplysia cyclase were mutagenized. Changing Glu-226, which corresponded to the catalytic residue of the cyclase, to Asp, Asn, Gln, Leu, or Gly eliminated essentially all enzymatic activities of CD38, indicating it is most likely the catalytic residue. Photoaffinity labeling showed that E226G, nevertheless, retained substantial NAD binding activity. The secondary structures of these inactive mutants as measured by circular dichroism were essentially unperturbed as compared with the wild type. Other nearby residues were also investigated. The mutants D147V and E146L showed 7- and 19-fold reduction in NADase activity, respectively. The cADPR hydrolase activity of the two mutants was similarly reduced. Asp-155, on the other hand, was crucial for the GDP-ribosyl cyclase activity since its substitution with either Glu, Asn, or Gln stimulated the activity 3-15-fold, whereas other activities remained essentially unchanged. In addition to these acidic residues, two tryptophans were also important, since all enzyme activities of W125F, W125Y, W189G and W189Y were substantially reduced. This is consistent with the two tryptophans serving a substrate positioning function. A good correlation was observed when the NADase activity of all the mutants was plotted against the cADPR hydrolase activity. Homology modeling revealed all these critical residues are clustered in a pocket near the center of the CD38 molecule. The results indicate a strong structural homology between the active sites of CD38 and the Aplysia cyclase.

Mapping of capillary flow, cellular redox state, and resting membrane potential in hypoperfused rat myocardium.

The influence on myocyte viability of ischemia-induced changes in capillary perfusion was studied in the hearts of anesthetized rats subjected to partial occlusion of the left coronary artery for 45 min. Timed plasma labeling was applied to determine perfusion patterns. Changes in the fluorescence of preloaded potential-sensitive dyes [tetramethylrhodamine methyl ester (TMRM) and bis-oxonol], of trypan blue, and of endogeneous NADH were utilized in characterizing myocyte viability in histological sections of the heart. Within the hypoperfused zone, localized areas appeared vascularly nonlabeled for periods of at least 10 min. Within these areas a reduction in TMRM fluorescence occurred in 82. 5% of the tissue, signaling a reduced resting membrane potential. In the same areas 37.7% of the myocytes revealed an NADH fluorescence lower than that regularly found in anoxic tissues. This correlated with an especially low level of TMRM, with increased fluorescence bis-oxonol and with an accumulation of trypan blue. In conclusion, in localized hypoperfusion-induced zones lacking capillary flow, an inhomogeneous pattern of reductions in myocyte viability develops, which appears to be relevant in ischemia-induced arrhythmias.

Characterization of the active site of ADP-ribosyl cyclase.

ADP-ribosyl cyclase synthesizes two Ca(2+) messengers by cyclizing NAD to produce cyclic ADP-ribose and exchanging nicotinic acid with the nicotinamide group of NADP to produce nicotinic acid adenine dinucleotide phosphate. Recombinant Aplysia cyclase was expressed in yeast and co-crystallized with a substrate, nicotinamide. x-ray crystallography showed that the nicotinamide was bound in a pocket formed in part by a conserved segment and was near the central cleft of the cyclase. Glu(98), Asn(107) and Trp(140) were within 3.5 A of the bound nicotinamide and appeared to coordinate it. Substituting Glu(98) with either Gln, Gly, Leu, or Asn reduced the cyclase activity by 16-222-fold, depending on the substitution. The mutant N107G exhibited only a 2-fold decrease in activity, while the activity of W140G was essentially eliminated. The base exchange activity of all mutants followed a similar pattern of reduction, suggesting that both reactions occur at the same active site. In addition to NAD, the wild-type cyclase also cyclizes nicotinamide guanine dinucleotide to cyclic GDP-ribose. All mutant enzymes had at least half of the GDP-ribosyl cyclase activity of the wild type, some even 2-3-fold higher, indicating that the three coordinating amino acids are responsible for positioning of the substrate but not absolutely critical for catalysis. To search for the catalytic residues, other amino acids in the binding pocket were mutagenized. E179G was totally devoid of GDP-ribosyl cyclase activity, and both its ADP-ribosyl cyclase and the base exchange activities were reduced by 10,000- and 18,000-fold, respectively. Substituting Glu(179) with either Asn, Leu, Asp, or Gln produced similar inactive enzymes, and so was the conversion of Trp(77) to Gly. However, both E179G and the double mutant E179G/W77G retained NAD-binding ability as shown by photoaffinity labeling with [(32)P]8-azido-NAD. These results indicate that both Glu(179) and Trp(77) are crucial for catalysis and that Glu(179) may indeed be the catalytic residue.

Murine pharmacokinetics of 6-aminonicotinamide (NSC 21206), a novel biochemical modulating agent.

The pyridine nucleotide 6-aminonicotinamide (6AN) was shown recently to sensitize a number of human tumor cell lines to cisplatin in vitro. The present studies were undertaken to compare the drug concentrations and length of exposure required for this sensitization in vitro with the drug exposure that could be achieved in mice in vivo. Human K562 leukemia cells and A549 lung cancer cells were incubated with 6AN for various lengths of time, exposed to cisplatin for 1-2 hr, and assayed for Pt-DNA adducts as well as the ability to form colonies. K562 cells displayed progressive increases in Pt-DNA adducts and cisplatin sensitivity during the first 10 hr of 6AN exposure. An 18-hr 6AN exposure was likewise more effective than a 6-hr 6AN exposure in sensitizing A549 cells to cisplatin. HPLC analysis of 6AN and its metabolite, 6-amino-NAD+, permitted assessment of exposures achieved in vivo after i.v. administration of 10 mg/kg of 6AN to CD2F1 mice. 6AN reached peak serum concentrations of 80-90 microM and was cleared rapidly, with T1/2alpha and T1/2beta values of 7.4 and 31.3 min, respectively. Bioavailability was 80-100% with identical plasma pharmacokinetics after i.p. administration. At least 25% of the 6AN was excreted unchanged in the urine. The metabolite 6-amino-NAD+ was detected in perchloric acid extracts of brain, liver, kidney, and spleen, but not in serum. Efforts to prolong systemic 6AN exposure by administering multiple i.p. doses or using osmotic pumps resulted in lethal toxicity. These results demonstrated that 6AN exposures required to sensitize tumor cells to cisplatin in vitro are difficult to achieve in vivo.

BiP maintains the permeability barrier of the ER membrane by sealing the lumenal end of the translocon pore before and early in translocation.

Secretory proteins are cotranslationally translocated across the mammalian ER membrane through an aqueous pore in the translocon while the permeability barrier is maintained by a tight ribosome-membrane junction. The lumenal end of the pore is also blocked early in translocation. Extraction of soluble lumenal proteins from microsomes and reconstitution with purified proteins demonstrate, by fluorescence collisional quenching, that BiP seals the lumenal end of this pore. BiP also seals translocons that are assembled but are not engaged in translocation. These ribosome-free translocons have smaller pores (9-15 A diameter versus 40-60 A in functioning translocons) and are generated when ribosomes dissociate from functioning translocons with large pores. BiP therefore maintains the permeability barrier by sealing both nontranslocating and newly targeted translocons.

Hepatic mitochondrial respiration and transport of reducing equivalents in rats fed an energy dense diet.

OBJECTIVE: To measure hepatic mitochondrial respiration as well as the ability of hepatic mitochondria to transport reducing equivalents by shuttle systems in rats fed an energy dense diet. DESIGN: Rats were fed a control (CD) or energy dense (ED) diet for 15 days. MEASUREMENTS: State 3 and State 4 oxygen consumption were measured in isolated mitochondria using glutamate+malate or pyruvate+malate as substrate. We also measured malate-aspartate shuttle activity and mitochondrial alpha-glycerophosphate dehydrogenase activity. RESULTS: ED rats, in comparison with CD rats, showed a significantly greater energy intake without a corresponding greater body weight gain and carcass lipid content. Compared to CD rats, ED rats also showed a significant increase in resting metabolic rate, which was abolished by propranolol. Hepatic mitochondrial state 3 respiration using glutamate+malate or pyruvate+malate as substrate as well as malate-aspartate shuttle activity significantly decreased, while mitochondrial alpha-glycerophosphate dehydrogenase significantly increased in ED rats compared to CD rats. CONCLUSIONS: Mitochondrial NADH oxidation is reduced and a greater fraction of cytoplasmic NADH can be transported to the mitochondria through the alpha-glycerophosphate shuttle and oxidized through the respiratory chain from complex II onwards. This mechanism could lead to a decrease in hepatic metabolic efficiency which is in line with the increased energy expenditure occurring in rats fed an energy dense diet.

Virtual cofactors for an Escherichia coli nitroreductase enzyme: relevance to reductively activated prodrugs in antibody directed enzyme prodrug therapy (ADEPT).

A nitroreductase enzyme has been isolated from Escherichia coli that has the unusual property of being equally capable of using either NADH or NADPH as a cofactor for the reduction of its substrates which include menadione as well as 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954). This property is shared with the mammalian enzyme, DT diaphorase. The nitroreductase can, like DT diaphorase, also use simple reduced pyridinium compounds as virtual cofactors. The intact NAD(P)H molecule is not required and the simplest quaternary (and therefore reducible) derivative of nicotinamide, 1-methylnicotinamide (reduced), is as effective as NAD(P)H in its ability to act as an electron donor for the nitroreductase. The structure-activity relationship is not identical to that of DT diaphorase and nicotinic acid riboside (reduced) is selective, being active only for the nitroreductase. Irrespective of the virtual cofactor used, the nitroreductase formed the same reduction products of CB 1954 (the 2- and 4-hydroxylamino derivatives in equal proportions). Nicotinic acid riboside (reduced), unlike NADH, was stable to metabolism by serum enzymes and had a plasma half-life of seven minutes in the mouse after an i.v. bolus administration. NADH had an unmeasurably short half-life. Nicotinic acid riboside (reduced) could also be produced in vivo by administration of nicotinic acid 5'-O-benzoyl riboside (reduced). These results demonstrate that the requirement for a cofactor need not be a limitation in the use of reductive enzymes in antibody directed enzyme prodrug therapy (ADEPT). It is proposed that the E. coli nitroreductase would be a suitable enzyme for ADEPT in combination with CB 1954 and a synthetic, enzyme-selective, virtual cofactor such as nicotinic acid riboside (reduced).