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In an attempt to test null hypothesis (Ho): that prenatal lead exposure does not increase the risk of prematurity and the delivery of SGA infants, a case-control study was performed in four hospitals of Southern Poland (Kraków, Rabka, Limanowa, Zakopane). Lead content was determined in maternal and cord blood as well as in head and pubic hair by the GF AAS (Perkin Elmer). A significant interregional variation of lead content in maternal blood was observed. Lead concentration in maternal and cord blood was significantly higher in the group of mothers of SGA newborns when compared to the controls. This was not the case with respect to the mothers of preterm infants. Also, the comparison of lead concentration in head and pubic hair revealed no statistically significant case-control differences. For a combined population of cases and controls, a significant gradient of lead concentration between maternal and cord blood was demonstrated. The correlation between lead content in different body compartments was observed. Conclusions. Different blood lead levels observed in mothers from four hospitals suggest different exposure. Higher lead concentration in maternal blood was associated with an increased risk of the delivery of SGA infant.

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Proximal tubular ammoniagenesis is amplified under conditions of acute and chronic metabolic acidosis. Current hypotheses postulate that alterations in intracellular pH (pHi) or in the pH gradient across the inner mitochondrial membrane (delta pHm) influence mitochondrial glutamine metabolism. Enhanced glutamine transport across the inner mitochondrial membrane might constitute a key regulatory factor in acidosis. To examine changes in delta pHm, a technique was used to determine pHi and intramitochondrial pH (pHm) simultaneously. Regulation of the enzyme alpha ketoglutarate dehydrogenase (alpha KGDH) was assessed by evaluating enzyme activity at varied levels of medium pH, Ca++, and adenosine diphosphate (ADP). The results indicate that pHi decreased with an acid external pH. A fall in pHi correlated to increase activity of alpha KGDH associated with increased affinity for the substrate, alpha KG. Increments in either buffer Ca++ or ADP concentration increased enzyme affinity for alpha KG at pH 7.6 but not at pH 6.8. These results, compatible with previous reports, indicate that pH, Ca++, and ADP are effectors of the enzyme alpha KGDH. Alterations in pH across the inner mitochondrial membrane might augment flux through alpha KG by accelerating glutamine metabolism. Increased alpha KG oxidation over the range of 10 to 500 mumol/L Ca++ concentration is compatable with data for Ca++ regulation reported for the solubilized enzyme. These studies provide evidence that the above factors, through enhancing alpha KGDH activity, participate in regulation of ammoniagenesis during states of acidosis.

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The effects of oxalate on kidney mitochondria were evaluated in vitro to test whether oxalate exposure leads to derangement(s) in mitochondrial function that could in turn promote the formation of kidney stones. Our previous studies demonstrated that oxalate is transported across the mitochondrial membrane via the dicarboxylate carrier. The present studies indicated that oxalate competitively inhibits the uptake and oxidation of exogenous malate and succinate in isolated mitochondria but has no effect on mitochondrial respiration in the presence of a mixture of glutamate plus malate or glutamate plus pyruvate. Oxalate attenuates the increase in mitochondrial respiration produced by the uncoupler CCCP or by the Ca2+ ionophore A23187, and the latter effect is more pronounced in kidney than in liver mitochondria. The apparent Ki of oxalate for the response to Ca2+ ionophore is 1.9 +/- 0.3 mM in kidney and 6.1 +/- 0.2 mM in liver mitochondria. Similarly, the ability of oxalate to attenuate calcium-induced swelling of mitochondria is more dramatic in kidney than in liver mitochondria (apparent KiS of 1.7 +/- 0.1 and 18.2 +/- 0.7 mM, respectively). Oxalate has no effect on the rate of calcium uptake by energized mitochondria or on the rate of ruthenium red-insensitive calcium efflux from mitochondria in either tissue. The above findings indicate that oxalate interacts with the inner mitochondrial membrane or with processes controlling membrane integrity to a greater extent in kidney than liver mitochondria. The effects of oxalate on membrane permeability or integrity may be more important than its effects on mitochondrial energy production or calcium sequestration in the pathogenesis of calcium oxalate microlith formation in the kidney.

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This study was performed to evaluate whether cyclosporine penetrates kidney mitochondria and impairs mitochondrial functions, causing nephrotoxicity. Exposure of rat kidney cortical mitochondria in vitro to cyclosporine had little effect on the oxidation of glutamate plus malate. Oxidation of succinate was markedly inhibited by a toxic level of cyclosporine (25 to 50 nmol/mg protein) under resting (State 4) and ADP-stimulated (State 3) conditions. Under uncoupling conditions, induced by the proton ionophore, CCCP or by the calcium ionophore, A23187 plus calcium, mitochondrial respiration was unchanged by cyclosporine. In mitochondria isolated from rats treated with an immunosuppressive dose of cyclosporine (25 mg/kg/day, p.o.), respiration was not significantly impaired. The respiration stimulated by ADP was only diminished in mitochondria from rats treated with 75 mg/kg. The rate of calcium uptake was unchanged by cyclosporine under in vitro and in vivo conditions. Kidney mitochondria of untreated rats maintained in a medium containing respiratory substrates and phosphate released spontaneously accumulated calcium that was accompanied by large amplitude swelling and enhanced respiration. Cyclosporine in vitro inhibited the process of spontaneous calcium discharge at the concentration range of 0.1 to 0.5 nmol/mg protein. Swelling and respiration induced by accumulated calcium was significantly diminished in kidney mitochondria isolated from cyclosporine-treated rats given doses of 25 or 75 mg/kg. The data obtained indicate that cyclosporine interacts with the membrane of kidney mitochondria in virtually the same way under in vitro and in vivo conditions. Cyclosporine at an immunosuppressive level impairs calcium-induced membrane permeability and at a toxic level, the rate of ADP phosphorylation.

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Data from a number of laboratories suggest that the exchange of glutamate for aspartate across the mitochondrial inner membrane is stimulated by glucagon and by Ca2+-mobilizing hormones. The purpose of this study was to determine the site of action of these hormones. Two possibilities were considered and tested. The first hypothesis is that the mitochondrial membrane electrical potential gradient (delta psi m) in the cells is increased by the hormones; and that the putative increase in delta psi m stimulates aspartate efflux. The second possibility is that Ca2+ mediates decreases in cellular levels of alpha-ketoglutarate, secondary to stimulation of alpha-ketoglutarate dehydrogenase, and that the decrease in alpha-ketoglutarate stimulates aspartate production by mitochondria. The effect of glucagon on delta psi m was estimated in intact hepatocytes using the lipophilic cation tetraphenyl phosphonium. No increase in delta psi m was observed due to hormone treatment. On the other hand, alpha-ketoglutarate was found to be an effective competitive inhibitor of aspartate formation via glutamate transamination by isolated liver mitochondria (Ki = 0.55 mM).

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Oxalate, a metabolic end product, forms calcium oxalate deposits in the tissues under a variety of pathological conditions. In order to determine whether oxalate is able to penetrate the mitochondrial matrix, the uptake of oxalate by rat liver and kidney cortical mitochondria was characterized. Mitochondria did not swell in an iso-osmotic medium of ammonium oxalate unless a small amount of phosphate was provided. This phosphate-induced swelling was prevented by N-ethylmaleimide. The uptake of [14C]oxalate by liver and kidney mitochondria followed first order kinetics and was inhibited by mersalyl an inhibitor of the phosphate and dicarboxylate carriers. Accumulation of [14C]oxalate at equilibrium was significantly higher by mitochondria energized with succinate than by rotenone-inhibited mitochondria due to higher matrix pH as determined by the [14C]5,5'-dimethyloxazolidine-2, 4-dione distribution ratio. The velocity of oxalate accumulation by mitochondria was temperature dependent. The activation energy was 81.5 and 86.5 J/mol for liver and kidney mitochondria, respectively. In both types of mitochondria, the rate of oxalate uptake was hyperbolic with respect to the concentration of oxalate. The apparent Km was 28.8 +/- 0.6 and 13.4 +/- 1.2 mM and the Vmax 87.1 +/- 1.1 and 66.1 +/- 3.1 nmol X mg-1 X min-1 at 12 degrees C for liver and kidney mitochondria, respectively. Phenylsuccinate exhibited mixed inhibition of the rate of oxalate uptake. Oxalate exhibited also a mixed inhibition of the uptake and oxidation of malate by mitochondria. The data obtained provide evidence that oxalate is transported across the mitochondrial membrane by a phosphate-linked, carrier-mediated system similar to or identical to the dicarboxylate transporter.

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Presumptive evidence suggests that the brown fat mitochondrial uncoupling protein, thermogenin, is involved in the mechanism of stimulation of respiration by norepinephrine in the intact tissue. Conflicting data have been reported which suggest involvement of either adenine nucleotides, or fatty acids, or long chain acyl-CoA, or protons in the physiological regulation. We measured the electrical potential gradient across the mitochondrial membrane (delta psi m) in control cells and in cells stimulated with norepinephrine, using the accumulation of lipophilic cation, tetraphenylphosphonium, as an indicator of the potential gradient. The value of delta psi m in the cells in the control state is 116 mV, and in the hormonally stimulated state it is 56.6 mV. This supports the view that the protein is involved in the mechanism of hormone action. Other studies were designed to distinguish between the effects of fatty acids and ATP levels on the uncoupling protein in isolated mitochondria and in the adipocytes. ATP levels and fatty acid levels inside intact cells were independently varied using oligomycin or external fatty acids. Their effect on thermogenin was monitored as the capacity of the cells for reverse electron transport from durohydroquinone. The results suggest that ATP modulates the activity of thermogenin, while fatty acids can alter the relationship between ATP and thermogenin activity such that the protein appears to be activated at a higher cellular ATP level in the presence of fatty acids than in their absence.

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The inner mitochondrial membrane of rat kidney mitochondria was altered by 0.03% Triton X-100 treatment in such a way as to render it permeable to NAD and CoA molecules without release of phosphate-dependent glutaminase. A break of linearity in the Arrhenius plot of the enzyme activity was characteristic for a conformational change of a membrane-bound enzyme. The activity of phosphate-dependent glutaminase immobilized in the inner mitochondrial membrane, as studied in 0.03% Triton X-100-treated mitochondria, and solubilized, as in the supernatant of sonicated mitochondria, was hyperbolic with respect to glutamine concentration. Under optimal conditions (pH 8.6 and 100 mM phosphate) the Vmax and Km were 216 +/- 12 nmol/mg per min and 2.7 +/- 0.4 mM, respectively, for Triton X-100-treated mitochondria, and 121 +/- 8 nmol/mg per min and 15.9 +/- 1.8 mM for sonicated mitochondria. Under near physiological conditions (pH 7.8 and 20 mM phosphate), distinct differences in phosphate-dependent glutaminase kinetics were observed. The Vmax as 29.8 +/- 0.4 and 2.6 /- 0.3 nmol/mg per min and the apparent Km 1.55 +/- 0.06 and 24.5 +/- 6.6 mM for Triton X-100 and sonicated mitochondria, respectively. The sigmoidal activation by phosphate at pH 7.8 was significantly shifted to the left in Triton X-100-treated as compared to sonicated mitochondria. As opposed to the data obtained in sonicated mitochondria, the kinetics of phosphate-dependent glutaminase in 0.03% Triton X-100-treated mitochondria agreed quite well with those obtained in intact, rotenone-inhibited and metabolically active mitochondria. These results suggest that an attachment of phosphate-dependent glutaminase to the inner membrane of kidney mitochondria has a profound effect on its kinetics, particularly under near physiological conditions.

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Data from numerous laboratories show that mitochondria isolated from livers treated acutely with glucagon have higher rates of state 3 respiration than control mitochondria. The purpose of the present study was to learn whether this phenomenon is an isolation artifact resulting from a stabilization of the mitochondrial membrane or whether it represents a real increase in the maximal respiratory capacity of liver cells due to glucagon treatment. Electron transport was measured through different spans of the electron transport chain by using ferricyanide as an alternate electron acceptor to O2. With isolated intact liver mitochondria, pretreatment with glucagon was found to cause an increase in electron flow, through both Complex I and Complex III, suggesting that the effect of glucagon was not specific for a single site in the electron transport chain. Using intact isolated hepatocytes, different results are obtained. Respiration was measured in isolated hepatocytes after quantitation of the hepatocyte mitochondrial content by assay of citrate synthase. Hepatocyte respiration could therefore be reported per mg of mitochondrial protein. By providing durohydroquinone to the cells, it was possible to measure electron flow from coenzyme Q to O2 in the absence of the physiological regulation of substrate supply. Likewise, the addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone released the in situ mitochondria from control by the cytosolic ATP/ADP ratio and it was possible to measure maximal electron flow rates through Complex III. In the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, electron flow was higher in mitochondria in the cell than in isolated mitochondria. Glucagon caused no increase in mitochondrial respiration in situ either in the presence of the physiological substrates or in the presence of durohydroquinone. The data obtained do not support a role for the electron transport chain as a target of glucagon action in hepatocytes.

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Liver mitochondria isolated from rats treated acutely with glucagon exhibit higher respiration-dependent H+ ion gradients across the mitochondrial inner membrane than mitochondria from control rats. It has been suggested that similar increases in mitochondrial delta pH in situ could stimulate gluconeogenesis, chiefly because the transport of pyruvate into mitochondria would increase in response to the increase in mitochondrial matrix pH. In order to determine whether the increased delta pH observed in vitro in isolated mitochondria also occurs in situ, the effect of glucagon on the pH in the cytosol and mitochondria matrix spaces of isolated hepatocytes was determined. For qualitative results, the spectral responses of intracellularly trapped 6-carboxyfluorescein was used to monitor cytosol pH, while fluorescein-loaded hepatocytes were used to monitor the mitochondrial pH. Hepatocytes were incubated with the diacetate ester derivatives of these dyes. The esters are permeable to the cell membranes, but are rapidly hydrolyzed in the cells. The free unesterified dyes are relatively impermeable to the cell membranes. After being trapped in the cell, 6-carboxyfluorescein remains localized in the cell cytosol, whereas fluorescein is taken up by the mitochondria as a function of the mitochondrial delta pH. In order to quantitate the actual pH in these compartments, the spectral responses (490-465 nm) of 6-carboxyfluorescein-loaded hepatocytes were used to determine the cytosolic pH. Calibration of these responses was obtained within the cell by determination of the dye's differential absorption coefficient (epsilon 490-465 nm) in various high K+ buffers after equilibration of the internal and external pH with valinomycin and the uncoupler 1799. All absorbance values were corrected for dye leakage. Equal hematocrits of unloaded cells were used to correct for absorbance contributions from cellular constituents. The mitochondrial pH was determined by a combination of the indicator dye and [14C]5,5'-demethyloxazolidine-2, 4-dione (DMO) distribution ratio methods. The weak acid DMO freely distributes across the plasma membrane and mitochondrial membrane in whole cells according to the pH gradient across each membrane. Knowledge of the cytoplasmic pH from the 6-carboxyfluorescein data allows the expected distribution of DMO across the plasma membrane to be calculated. The excess accumulation of DMO in intact hepatocytes over that predicted from the plasma membrane pH gradient alone was then used to calculate the pH gradient across the mitochondrial inner membrane. The effects of valinomycin, uncouplers, and hormones on the pH in cytosolic and mitochondrial compartm

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To test the significance of the purine nucleotide cycle in renal ammoniagenesis, studies were conducted with rat kidney cortical slices using glutamate or glutamine labelled in the alpha-amino group with 15N. Glucose production by normal kidney slices with 2 mM-glutamine was equal to that with 3 mM-glutamate. With L-[15N]glutamate as sole substrate, one-third of the total ammonia produced by kidney slices was labelled, indicating significant deamination of glutamate or other amino acids from the cellular pool. Ammonia produced from the amino group of L-[alpha-15N]glutamine was 4-fold higher than from glutamate at similar glucose production rates. Glucose and ammonia formation from glutamine by kidney slices obtained from rats with chronic metabolic acidosis was found to be 70% higher than by normal kidney slices. The contribution of the amino group of glutamine to total ammonia production was similar in both types of kidneys. No 15N was found in the amino group of adenine nucleotides after incubation of kidney slices from normal or chronically acidotic rats with labelled glutamine. Addition of Pi, a strong inhibitor of AMP deaminase, had no effect on ammonia formation from glutamine. Likewise, fructose, which may induce a decrease in endogenous Pi, had no effect on ammonia formation. The data obtained suggest that the contribution of the purine nucleotide cycle to ammonia formation from glutamine in rat renal tissue is insignificant.

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Effects of maleate on the content of CoA derivatives in isolated mitochondria and in the tissues of maleate-intoxicated rats have been studied. The addition of maleate to kidney mitochondria incubated with 2-oxo-glutarate decreased CoA-SH and acid-soluble acyl-CoA concentrations while acid insoluble acyl-CoA content remained unchanged. As a result, a substantial loss (depletion) of the total CoA occurred. Similar changes in CoA content were found in vivo in the kidneys of maleate-treated rats. Neither in the isolated liver mitochondria nor in the liver of intoxicated animals have such changes been observed before. Acetoacetate, the substrate for CoA transferase, added to kidney mitochondria before maleate, abolished its inhibitory effect on oxidation of 2-oxoglutarate and prevented the decrease of CoA content. The data are in accord with the previous findings indicating that maleate can bind and sequester CoA in the form of a stable and metabolically inert compound.

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