A comparison of fluoride bioavailability from a sustained-release NaF preparation (Neosten) and other fluoride preparations.

Twelve normal subjects completed a crossover study with sustained-release sodium fluoride (Neosten, 11.3 mg F), monofluorophosphate (MFP, 10 mg F), and plain sodium fluoride (P-NaF, 11.3 mg F). After each preparation was given with 400 mg calcium, serum fluoride (Fser) was measured for 24 hours, and pharmacokinetic data were calculated. Fluoride absorption in the Neosten group, as measured by change in the area under the curve (delta AUC) of Fser, was less than 33% of that in the MFP and P-NaF treated groups. Both peak Fser (Cmax) and peak-basal variation in the Neosten group were 25% that found in the other groups. t1/2 was nearly twofold greater after Neosten. MFP and P-NaF showed greater bioavailability than Neosten and much higher Cmax that exceeded the toxic threshold of Fser (190 ng/ml). These findings could explain the ineffectiveness of MFP and P-NaF observed in recent clinical trials.

Mapping a gene defect in absorptive hypercalciuria to chromosome 1q23.3-q24.

Absorptive hypercalciuria (AH), a common cause of kidney stones, is due to intestinal hyperabsorption of calcium. The presence of a family history of nephrolithiasis, in about half of the affected individuals studied indicates that an inherited genetic defect is one likely cause of AH. Although it is known that intestinal calcium absorption is regulated by a number of factors, the molecular biological basis for the increased calcium absorption in AH is unknown. This study was designed to determine the chromosomal locus of the gene defect linked to the AH phenotype in three families with a severe form of AH. Three kindreds were evaluated in a systematic autosomal genome-wide linkage analysis study. The AH phenotype, characterized by hyperabsorption of calcium and hypercalciuria, was linked to only one chromosomal locus, 1q23.3-q24. A 2-point logarithm of odds score of 3.3 was obtained with markers D1S318 and D1S196 at a recombination frequency of theta = 0. Nonparametric multipoint linkage analysis yielded a peak nonparametric linkage Z(all)-score of 12.7, P = 6 x 10(-6) Analysis of key recombinants within the families studied localized the gene to a 4.3-megabase region between markers D1S2681 (centromere) and D1S2815. A trait associated with intestinal hyperabsorption of calcium in a severe form of absorptive hypercalciuria has been mapped to chromosome 1q23.3-q24.

Mutations in the genomic deoxyribonucleic acid for SLC3A1 in patients with cystinuria.

Cystinuria is an inherited transport disorder characterized by defective renal resorption of cystine and other dibasic amino acids. We have studied the occurrence of mutations in the SLC3A1 gene, which codes for a dibasic amino acid transporter-like protein, in 33 unrelated cystinurics. We found mutations in 34 of the 66 chromosomes studied. There were 14 different mutations in our study population, 8 of which had not been previously described. Of these new mutations, 4 were missense mutations: G1934C, C1259G, T1607G, and G1373A. The other 4 mutations consisted of a single base insertion mutation (2022 ins T), a single base deletion mutation (163 del C), a 23-base deletion mutation (del 782A-804A), and a complex mutation that consisted of a 36-base deletion (del C431-3 to T463) and a duplication insertion of 468 T to 474 A after nucleotide 474.

Recent advances in the biochemical and molecular biological basis of cystinuria.

PURPOSE: We reviewed the literature describing recent advances in the understanding of the nature of the transport proteins involved in the renal transport of cystine, properties of the solute carrier family 3, member 1 (SLC3A1) gene, which is involved in renal cystine transport, and the mutations reported in this gene, which have been shown to be the causative factor in approximately half of the cases of type I cystinuria studied. MATERIALS AND METHODS: The MEDLINE data base from 1966 to date and the internet online mendelian inheritance in man were searched using cystinuria, cystine crossed with biological transporters and cystine transporter as key words. Selected citations within these references were also reviewed. RESULTS: The SLC3A1 gene has been shown to code for a protein that, when expressed in Xenopus oocytes, confers on these cells the ability to transport cystine, arginine, lysine and ornithine. To date 21 different mutations and 9 polymorphisms have been reported in the SLC3A1 gene isolated from cystinuric patients. CONCLUSIONS: Type I cystinuria appears to be due to mutations in the SLC3A1 gene, while the molecular genetic determinants of types II and III cystinuria remain to be delineated.

In vivo effects of lipopolysaccharide on hepatic free-NAD(P)(+)-linked redox states and cytosolic phosphorylation potential in 48-hour-fasted rats.

This study was performed to determine the magnitude and time of onset of in vivo changes in hepatic bioenergetics in response to a sublethal dose of lipopolysaccharide (LPS), a bacterial endotoxin. Male rats (48-hour-fasted) were administered an intraperitoneal injection of LPS (5 mg/kg body weight) or vehicle alone, and the livers were freeze-clamped 5, 30, or 180 minutes or 24 hours later. Liver tissue was extracted with perchloric acid, and the metabolites necessary to calculate NAD(+)- and NADP(+)-linked redox states and the cytosolic phosphorylation potential were measured. There was no significant difference in hepatic cytosolic phosphorylation potential between LPS and control groups at any of the times investigated. This indicated that the ability of the liver to synthesize adenosine triphosphate (ATP) was not compromised under the conditions of the study. No changes in hepatic redox states were observed 5 or 30 minutes after LPS treatment. Three hours after LPS treatment, hepatic cytosolic and mitochondrial free-[NAD+]/[NADH] redox states and the cytosolic free-[NADP+]/[NADPH] redox state were more oxidized. By 24 hours, only NAD(+)-linked redox states were more oxidized than the time-matched controls. Hepatic urea content was elevated at both 3 and 24 hours, compatible with an increased rate of urea synthesis as a consequence of increased amino acid metabolism, whereas hepatic beta-hydroxybutyrate and total ketone bodies were decreased 24 hours after LPS treatment, indicating decreased hepatic ketogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of chloride potential in perfused rat hearts by nuclear magnetic resonance spectroscopy.

Isolated beating rat hearts were perfused with trifluoroacetamide (TFM) and trifluoroacetate (TFA) and monitored by 19F-nuclear magnetic resonance (NMR). The average membrane TFA potential in spontaneously beating rat hearts, calculated according to standard principles assuming that TFA is distributed in its anionic form, was found to be -36.2 +/- 3.2 mV (n = 9) under normoxic conditions. In separate experiments, the chloride and potassium potentials were determined to be -38.5 +/- 3.6 mV (n = 7) and -85.3 +/- 3.3 mV (n = 7), respectively, from freeze-clamped heart tissue. In the presence of the anion-exchange inhibitor, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), TFA uptake into heart was significantly reduced, suggesting that TFA uptake occurs partly via the Cl(-)-HCO3- exchanger. Based on these results and the results of R. E. London and S. A. Gabel (Biochemistry 28: 2378-2382, 1989), we conclude that the distribution of TFA in hearts reflects the chloride potential (ECl) and not the membrane potential. A time-dependent change in the ECl occurs during global ischemia, and changes in ECl were also observed when the hearts were perfused with high concentrations of KCl. These results demonstrate that 19F-NMR may be utilized to monitor the ECl of perfused hearts under a variety of conditions.

Phosphate free perfusion prevents washout of tissue creatine in Langendorff perfused rabbit heart.

Rabbit hearts were perfused with Krebs-Henseleit bicarbonate buffer supplemented with 15 mM glucose and 10 mU/ml of insulin +/- Pi. At the end of 60 min the hearts were freeze-clamped and the content of ATP, creatine phosphate, creatine, lactate, pyruvate, DHAP and 3-P glycerate were determined enzymatically in neutralized perchloric acid tissue extracts. The free cytosolic ADP and Pi and the cytosolic NAD+ redox and phosphorylation potentials were calculated from the measured metabolite concentrations. Pi free perfusion resulted in increased creatine, free cytosolic ADP and cytosolic phosphorylation potential, decreased calculated free Pi and no change in cardiac ATP and creatine phosphate content. The increase in the cytosolic phosphorylation potential was due to the lowering of cytosolic free Pi. The increase in ADP was due to the increase in creatine. The increase in creatine appeared to be due to an inhibition of creatine efflux from the heart during Pi free perfusion which was mediated by an enhanced Na+ electrochemical gradient.

Thermodynamics of hydrolysis of sugar phosphates.

Thermodynamics of the enzyme-catalyzed (alkaline phosphatase, EC 3.1.3.1) hydrolysis of glucose 6-phosphate, mannose 6-phosphate, fructose 6-phosphate, ribose 5-phosphate, and ribulose 5-phosphate have been investigated using microcalorimetry and, for the hydrolysis of fructose 6-phosphate, chemical equilibrium measurements. Results of these measurements for the processes sugar phosphate2- (aqueous) + H2O (liquid) = sugar (aqueous) + HPO2++-(4) (aqueous) at 25 degrees C follow: delta Ho = 0.91 +/- 0.35 kJ.mol-1 and delta Cop = -48 +/- 18 J.mol-1.K-1 for glucose 6-phosphate; delta Ho = 1.40 +/- 0.31 kJ.mol-1 and delta Cop = -46 +/- 11 J.mol-1.dK-1 for mannose 6-phosphate; delta Go = -13.70 +/- 0.28 kJ.mol-1, delta Ho = -7.61 +/- 0.68 kJ.mol-1, and delta Cop = -28 +/- 42 J.mol-1.K-1 for fructose 6-phosphate; delta Ho = -5.69 +/- 0.52 kJ.mol-1 and delta Cop = -63 +/- 37 J.mol-1.K-1 for ribose 5-phosphate; and delta Ho = -12.43 +/- 0.45 kJ.mol-1 and delta Cop = -84 +/- 30 J.mol-1.K-1 for the hydrolysis of ribulose 5-phosphate. The standard state is the hypothetical ideal solution of unit molality. Estimates are made for the equilibrium constants for the hydrolysis of ribose and ribulose 5-phosphates. The effects of pH, magnesium ion concentration, and ionic strength on the thermodynamics of these reactions are considered.

The medical and metabolic consequences of administration of sodium acetate.

1. The standard total parenteral nutrition, peritoneal dialysis, hemodialysis and many surgical fluids in use today contain 36 to 45 mM D,L-lactate or 2 to 140 mM acetate whereas the normal blood level of D-lactate is 0.02 mM L-lactate 0.5 to 5 mM and acetate 0.1 nM. The reasons for the continued use in patients of such unphysiological concentrations of these anions appear to be historic. 2. Administration of similar concentrations of these anions to the rat causes widespread metabolic disturbances which mimic many of the untoward complications associated with current parenteral and dialysis therapy. Understanding of the mechanisms attendant upon the metabolism of these anions may serve as a guide for designing improved parenteral fluids for human patients. 3. Elevation of blood D-lactate to 5 mM is associated with cerebral dysfunction in human patients. 4. Acetate stimulates the release of the inflammatory leukokine, interleukin-1 from human monocytes. Use of 35 to 45 mM acetate in peritoneal dialysis fluids led to peritoneal fibrosis. Patients exposed to acetate containing hemodialysis fluids have 12-fold elevation in their plasma interleukin-1 levels. 5. Administration of 20 mM sodium acetate to rats leads to a number of metabolic disturbances similar to those seen in human dialysis patients: (a) Acetate elevates blood glucose in the rat and may contribute to the exacerbation of the carbohydrate intolerance seen in uremic patients. (b) Acetate increases the levels of hepatic malonyl CoA, the rate controlling substrate of fatty acid synthesis and may exacerbate the hypertriglyceridemia characteristic of dialysis patients. (c) Acetate administration in the rat leads to a decrease in the cytosolic phosphorylation potential, reduction of the redox state of the free cytosolic NAD couple and paradoxical oxidation of the mitochondrial NAD couple in a pattern analogous to that produced by uncouplers of oxidative phosphorylation and may account in part for the elevation of temperature reported in patients undergoing hemodialysis with acetate. (d) Acetate administration in the rat leads to an increase in intracellular phosphorylated intermediates, adenine nucleotides, inorganic phosphate, inorganic pyrophosphate, calcium and magnesium. On cessation of acetate metabolism, the inorganic phosphate and calcium accumulated intracellularly leave the intracellular space. In patients undergoing hemodialysis, the blood phosphate returns to predialysis levels, within 6 hr after the completion of treatment, leaving significant numbers of patients with chronic hyperphosphatemia and the multiple complications attendant to that state.(ABSTRACT TRUNCATED AT 400 WORDS)

Early metabolic effects of platelet-derived growth factor and transforming growth factor-beta in rat liver in vivo.

The short term metabolic effects of the in vivo administration of platelet-derived growth factor have been examined in the liver of the rat. Meal-fed male Wistar rats weighing between 150-180 g received an intraperitoneal injection of platelet-derived growth factor (17 units/100 g weight), transforming growth factor-beta (185 ng/100 g weight), or saline. At 5 min after injection, the livers were freeze-clamped. Samples of the tissue were subsequently assayed for metabolite content and enzyme activities. Platelet-derived growth factor injection caused an elevation in the liver content of pyruvate from 0.14 +/- 0.012 to 0.19 +/- 0.009 mumol/g wet weight liver (p less than or equal to 0.01) and an increase in the cytosolic phosphorylation potential [sigma ATP]/[sigma ADP][sigma Pi] from 6670 +/- 540 to 8970 +/- 750 (p less than or equal to 0.01). In addition an increase in the hepatic content of the hexose monophosphate pathway metabolites, 6-phosphogluconate (0.027 +/- 0.004 to 0.037 +/- 0.005 mumol/g wet weight) (p less than or equal to 0.05), ribulose 5-phosphate (0.013 +/- 0.001 to 0.017 +/- 0.001 mumol/g wet weight) (p less than or equal to 0.05) and combined sedoheptulose 7-phosphate and ribose 5-phosphate (0.052 +/- 0.007 to 0.062 +/- 0.004 mumol/g wet weight) (p less than or equal to 0.05) was observed. The elevation in the hexose monophosphate pathway metabolites resulted from a 1.3-fold elevation in the activity of glucose-6-phosphate dehydrogenase [EC 1.1.1.49] when measured in a crude homogenate. Kinetic analysis performed on partially purified glucose-6-phosphate dehydrogenase demonstrated no significant change in the Km of the enzyme for either NADP+ or glucose 6-phosphate, while a 2.4-fold increase in the Vmax was observed. In view of the rapidity of the change in total measured enzyme activity and increase in the Vmax of glucose-6-phosphate dehydrogenase, it is postulated that platelet-derived growth factor causes a covalent modification of the existing enzyme. Transforming growth factor-beta caused no change in the hepatic metabolite content in the treated animals when compared to saline treated controls.

The accumulation of pyrophosphate by rat hepatocytes.

Hepatocytes that were isolated from 48 hr starved rats and incubated in Krebs-Henseleit bicarbonate buffer containing 10 microM A23187, 10mM l-lactate, 1mM pyruvate and 2mM l-lysine were found to contain 0.064 mumol of inorganic pyrophosphate/g wet wgt cells. Addition of either 20mM acetate or butyrate, which caused the formation of pyrophosphate in both the cytosol and the mitochondrial matrix or only the matrix, resulted in an increase of 0.915 and 1.91 mumol pyrophosphate/g wet wgt cells, respectively. The accumulation of pyrophosphate was shown to be non-linear with time and dependent on the calcium concentration of the incubation media. In contrast, incubations containing a combination of 10 mM NH4Cl and 5 mM ornithine, which resulted in the formation of pyrophosphate only in the cytosol, had a pyrophosphate content of 0.032 mumol/g wgt cells. When isolated hepatocytes that had been incubated with acetate or butyrate were subjected to digitonin fractionation, all of the recoverable pyrophosphate was present in the particulate fraction. It is concluded that pyrophosphate accumulates in isolated rat hepatocytes only in the presence of calcium and a calcium ionophore, only within the mitochondrial matrix and only when pyrophosphate is formed within the mitochondrial matrix.

Purification and kinetic properties of ox brain histamine N-methyltransferase.

Histamine N-methyltransferase (EC 2.1.1.8) was purified 1100-fold from ox brain. The native enzyme has an Mr of 34800 +/- 2400 as measured by gel filtration on Sephadex G-100. The enzyme is highly specific for histamine. It does not methylate noradrenaline, adrenaline, DL-3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, 3-hydroxytyramine or imidazole-4-acetic acid. Unlike the enzyme from rat and mouse brain, ox brain histamine N-methyltransferase did not exhibit substrate inhibition by histamine. Initial rate and product inhibition studies were consistent with an ordered steady-state mechanism with S-adenosylmethionine being the first substrate to bind to the enzyme and N-methylhistamine being the first product to dissociate.

The effect of short chain fatty acid administration on hepatic glucose, phosphate, magnesium and calcium metabolism.

Intra peritoneal administration of the short chain fatty acids, acetate, propionate and butyrate, in amounts calculated to reach 20 mM in total body water were given to fed and 48 hour starved male Wistar rats. One half hour after administration, the livers were freeze-clamped and the hepatic contents of various intermediary metabolites were measured. The liver content of total glycolytic intermediates was elevated by short chain fatty acids. In fed animals, the portion of glycolysis from fructose 1,6-bisphosphate (FBP) to PEP was elevated 2 to 4 fold. In 48 hour starved animals, where gluconeogenesis is active, the portion of the gluconeogenetic pathway from FBP to glucose was elevated 1.5 to 3.5 fold with the exception of the butyrate treated animals where blood glucose was not elevated. The metabolites of the hexose-monophosphate pathway that were measured, namely 6-phosphogluconate, ribulose 5-phosphate and xylose 5-phosphate were increased in both fed and starved animals. The free cytoplasmic [NAD+]/[NADH], [NADP+]/[NADPH], and [epsilon ATP]/[epsilon ADP] X [epsilon Pi] ratios were all decreased in both fed and starved animals after short chain fatty acid administration. The liver content of calcium increased 1.2 to 2 fold in fed animals and 2 to 3 fold in starved animals while total liver magnesium was either unchanged or increased only 1.2 times. The liver pyrophosphate (PPi) content increased a minimum of 10 fold in fed animals and over 100 fold in starved animals. In all cases no PPi could be detected in vivo by 31P NMR even though in the starved rats the PPi levels approached those of ATP. The liver content of inorganic Pi increased 1.3 to 1.5 fold in fed animals and 1.5 to 2 fold in starved animals. The total "rapidly metabolizing" Pi pool, that includes adenine and guanine nucleotides, glycolytic and shunt intermediates, Pi and PPi increased 1.3 times in fed animals (from 13.8 mumole/g fresh weight) and 1.5 to 1.7 fold in starved animals (from 15.7 mumol/g fresh weight). The total phosphate taken up from blood and entering the rapidly turning over pool of liver phosphate ranged between 4 and 12 mumols/g of liver.(ABSTRACT TRUNCATED AT 400 WORDS)

Determination of Km and V of an enzyme for an unstable substrate and its application to the oxidation of N tau-methylimidazol-3-ylacetaldehyde by aldehyde dehydrogenase.

The method of Storer & Cornish-Bowden [(1974) Biochem. J. 141, 205-209] for determining the lag time in coupled enzyme assays was adapted to enable the kinetic parameters of the second (coupling) enzyme for the intermediate to be calculated. The validity and accuracy of this method of progress-curve analysis was established by comparing the Km value of glucose 6-phosphate dehydrogenase for glucose 6-phosphate generated in situ by the action of glucose phosphate isomerase on fructose 6-phosphate with that determined from initial-rate measurements. The method was applied to the determination of the Km value of ox liver cytoplasmic aldehyde dehydrogenase for N tau-methylimidazol-3-ylacetaldehyde that was generated in situ by the action of plasma amine oxidase on N tau-methylhistamine.

The role of cytoplasmic aldehyde dehydrogenase in the metabolism of N-tele-methylhistamine.

The subcellular distributions of aldehyde dehydrogenase activities towards acetaldehyde have been compared with those toward N-tele-methylimidazole acetaldehyde, the aldehyde derived from the oxidation of N-tele-methylhistamine. At high concentrations of acetaldehyde (3.0 mM), significant aldehyde dehydrogenase activity can be found in the mitochondrial, light mitochondrial, microsomal and cytoplasmic fractions whereas, when the activity is determined with 15 microM acetaldehyde, the enzyme activity is enriched only in the mitochondrial fraction suggesting that this organelle will be the dominant site for the metabolism of acetaldehyde derived from ingested ethanol. The activity towards N-tele-methylimidazole acetaldehyde was determined by generating this compound in the assay by the oxidation of N-tele-methylhistamine in the presence of beef plasma amine oxidase. At the low steady-state aldehyde concentrations that will be present in such an assay, only the cytoplasmic form of aldehyde dehydrogenase showed activity towards this substrate.