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PubMed1973

Partial reactions of D-glucose 6-phosphate-1L-myoinositiol 1-phosphate cyclase.

After removal of tightly bound NAD(+) by using charcoal, a preparation of d-glucose 6-phosphate-1 l-myoinositol 1-phosphate cyclase catalysed the reduction of 5-keto-d-glucitol 6-phosphate and 5-keto-d-glucose 6-phosphate by [4-(3)H]NADH to give [5-(3)H]-glucitol 6-phosphate and [5-(3)H]glucose 6-phosphate respectively. The position of the tritium atom in the latter was shown by degradation. Both enzyme-catalysed reductions were strongly inhibited by 2-deoxy-d-glucose 6-phosphate, a powerful competitive inhibitor of inositol cyclase. The charcoal-treated enzyme preparation also converted 5-keto-d-glucose 6-phosphate into [(3)H]myoinositol 1-phosphate in the presence of [4-(3)H]NADH, but less effectively. These partial reactions of inositol cyclase are interpreted as providing strong evidence for the formation of 5-keto-d-glucose 6-phosphate as an enzyme-bound intermediate in the conversion of d-glucose 6-phosphate into 1 l-myoinositol 1-phosphate. The enzyme was partially inactivated by NaBH(4) in the presence of NAD(+). Glucose 6-phosphate did not increase the inactivation, and there was no inactivation in the absence of NAD(+). There was no evidence for Schiff base formation during the cyclization. d-Glucitol 6-phosphate (l-sorbitol 1-phosphate) was a good inhibitor of the overall reaction. It did not inactivate the enzyme. The apparent molecular weight of inositol cyclase as determined by Sephadex chromatography was 2.15x10(5).

Animals↗
PubMed1974

Transition of metabolisms in living popular bark from growing to wintering stages and vice versa: changes in glucose 6-phosphate and 6-phosphogluconate dehydrogenase activities and in the levels of sugar phosphates.

Activities of glucose 6-phosphate, 6-phosphogluconate, and isocitrate dehydrogenases, together with intermediate levels of the glycolytic pathway and the pentose phosphate cycle, were measured throughout a year in the living bark of poplar (Populus gelrica). Shoots, immediately after budding (early May), contained very high levels of the three enzyme activities, which fell gradually by early or mid-July to a level, roughly equivalent to budding (May) or growing (July) 2-year-old twigs. In September, the former two dehydrogenase activities of the new shoots and 2-year-old twigs began to rise, while the latter activity started to decrease. The rise of the two dehydrogenase activities continued until late November (or early December). The high level of the two dehydrogenase activities lasted until early in April of the following year and then the decrease in the activities began prior to the onset of budding, reaching a low, basal level in early May. The profile of changes in the two dehydrogenase activities appeared to coincide with the increase and decrease of soluble proteins.Normal concentrations of total hexose phosphates in the glycolytic pathway plus 6-phosphogluconate were found to be 288 to 895 mumoles/kilogram dry weight. During the metabolism transition (September and April), a transient and striking increase of 6-phosphogluconate was observed. In September, 6-phosphogluconate reached a level on the order of 10(-4)m and was 4 times that of fructose 6-phosphate. The increase in 6-phosphogluconate coincided with the increase in the glucose 6-phosphate dehydrogenase activity. Coincidentally, with the change of 6-phosphogluconate level, a large deviation of the in vivo ratio of fructose 6-phosphate to glucose 6-phosphate from the known equilibrium constant was observed, showing the relation of pentose phosphate cycle enzyme activity to the control of glycolysis. The ratio of glucose 6-phosphate to glucose 1-phosphate deviated from that predicted. These ratios fluctuated throughout the year and were affected by the growth phases. The levels of pentose phosphate cycle metabolites, except for 6-phosphogluconate, in the bark were extremely low.The level of inorganic phosphate in the living bark throughout the year was dependent upon the growth phases, suggesting the presence of a regulatory mechanism to maintain inorganic phosphate at a given level as the growth phase changed.Sugar phosphate levels of popular twigs or potato tubers (Solanum tuberosum) remained constant after they were stored for 2 weeks at low temperatures, whereas in sweet potato roots (Ipomoea batatas), the level rose to about 9-fold of the control, indicating the presence of a strict regulatory system for the synthesis and catabolism of sugar phosphate in the former two.

Journal Article↗
PubMed2006

Unchanged expression of the sodium-dependent phosphate cotransporter NaPi-IIa despite diurnal changes in renal phosphate excretion.

Renal phosphate excretion is subjected to circadian rhythmicity. The bulk of filtered inorganic phosphate (P(i)) is reabsorbed by the sodium-dependent phosphate cotransporter NaPi-IIa. The regulation of proximal tubular phosphate reabsorptive capacity is largely attributed to the altered abundance of NaPi-IIa residing in the brush border membrane (BBM) of proximal tubular cells. Therefore, we examined if the diurnal rise in renal phosphate excretion is accompanied by a corresponding change in NaPi-IIa expression. Renal phosphate excretion, creatinine clearance, and serum phosphate were determined at consecutive time points in rats, starting from 8 a.m. until 5 p.m. During this period, renal phosphate excretion (fractional P(i) excretion) increased more than eightfold until 5 p.m. compared to the morning values at 8 a.m. In addition, serum phosphate and creatinine clearance as well as the calculated tubular phosphate threshold increased. Neither immunoblot analysis of BBMs nor immunohistochemical staining for NaPi-IIa yielded evidence for a lower abundance of NaPi-IIa in kidneys collected in the afternoon compared to those in the morning. However, kidneys sampled in the afternoon showed a small decrease (14%) in (32)P uptakes into BBM vesicles (BBMVs). Thus, the diurnal rise in renal phosphate excretion was associated with a mild reduction in the sodium-dependent phosphate transport rate in proximal tubular BBMs. There was no apparent downregulation of NaPi-IIa abundance and only a small reduction in Na(+)-dependent Pi-transport activity. Thus, the diurnal changes in urinary phosphate excretion appear to be mainly related to changes in serum phosphate and tubular threshold but not to NaPi-IIa expression.

Algorithms↗
PubMed1989

A kinetic study of the effects of phosphate and organic phosphates on the activity of phosphoenolpyruvate carboxylase from Crassula argentea.

The effects of phosphate and several phosphate-containing compounds on the activity of purified phosphoenolpyruvate carboxylase (PEPC) from the crassulacean acid metabolism plant, Crassula argentea, were investigated. When assayed at subsaturating phosphoenolpyruvate (PEP) concentrations, low concentrations of most of the compounds tested were found to stimulate PEPC activity. This activation, variable in extent, was found in all cases to be competitive with glucose 6-phosphate (Glc-6-P) stimulation, suggesting that these effectors bind to the Glc-6-P site. At higher concentrations, depending upon the effector molecule studied, deactivation, inhibition, or no response was observed. More detailed studies were performed with Glc-6-P, AMP, phosphoglycolate, and phosphate. AMP had previously been shown to be a specific ligand for the Glc-6-P site. The main effect of Glc-6-P and AMP on the kinetic parameters was to decrease the apparent Km and increase Vmax/Km. AMP also caused a decrease in the Vmax of the reaction. In contrast, phosphoglycolate acted essentially as a competitive inhibitor increasing the apparent Km for PEP and decreasing Vmax/Km. Inorganic phosphate had a biphasic effect on the kinetic parameters, resulting in a transient decrease in Km followed by an increase of the apparent Km for PEP with increasing concentration of phosphate. The Vmax also was decreased with increasing phosphate concentrations. Further, the enzyme appeared to respond to the complex of phosphate with magnesium. In the presence of a saturating concentration of AMP, no activation but rather inhibition was observed with increasing phosphate concentration. This is consistent with the binding of phosphate to two separate sites--the Glc-6-P activation site and an inhibitory site, a phenomenon that may be occurring with other phosphate containing compounds. High concentrations of phosphate with magnesium were found to protect enzyme activity when PEPC, previously shown to contain an essential arginine at the active site, was incubated with the specific arginyl reagent 2,3-butanedione, consistent with the binding of phosphate at the active site. Data were successfully fitted to a rapid equilibrium model allowing for binding of the phosphate-magnesium complex to both the activation site and the active site which accounts for the activation/deactivation observed at low substrate concentrations. Effects on the Vmax of the reaction are also addressed. Factors controlling the differential affinity of various effectors to the active site or activation site appear to include charge distribution, size, and other steric factors.

Adenosine Monophosphate↗
PubMed1987

[Formation of a pentose phosphate cycle metabolite, erythrose-4-phosphate, from initial compounds of glycolysis by transketolase from the rat liver].

Using ion-exchange chromatography of sucrose phosphates on Dowex-1, it was demonstrated that the highly purified rat liver transketolase (specific activity 1.7 mumol/min.mg protein) is capable of catalyzing the synthesis of erythrose-4-phosphate, a metabolite of the pentose phosphate pathway non-oxidizing step, from the initial participants of glycolysis, i. e., glucose-6-phosphate and fructose-6-phosphate. As can be evidenced from the reaction course, the second product of this synthesis is octulose-8-phosphate. The reaction was assayed by accumulation of erythrose-4-phosphate. The soluble fraction from rat liver catalyzes under identical conditions the synthesis of heptulose-7-phosphate (but not erythrose-4-phosphate), which points to the utilization of the erythrose-4-phosphate formed in the course of the transketolase reaction by transaldolase which is also present in the soluble fraction. The role of the transketolase reaction reversal from the synthesis of pentose phosphate derivatives to glycolytic products is discussed. The transketolase reaction provides for the relationship between glycolysis and the anaerobic step of the pentose phosphate pathway which share common metabolites, i. e. glucose-6-phosphate and fructose-6-phosphate.

Animals↗
PubMed1981

Divalent metal ion, inorganic phosphate, and inorganic phosphate analogue binding to yeast inorganic pyrophosphatase.

Four different techniques, equilibrium dialysis, protection of enzymatic activity against chemical inactivation, 31P relaxation rats, and water proton relaxation rates, are used to study divalent metal ion, inorganic phosphate, and inorganic phosphate analogue binding to yeast inorganic pyrophosphatase, EC 3.6.1.1. A major new finding is that the binding of a third divalent metal ion per subunit, which has elsewhere been implicated as being necessary for enzymatic activity [Springs, B., Welsh, K. M., & Cooperman, B. S. (1981) Biochemistry (in press)], only becomes evident in the presence of added inorganic phosphate and that, reciprocally, inorganic phosphate binding to both its high- and low-affinity sites on the enzyme is markedly enhanced in the presence of divalent metal ions, with Mn2+ causing an especially large increase in affinity. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and inorganic phosphate or of Co2+ and inorganic phosphate to the enzyme and show that the high-affinity site has greater specificity for inorganic phosphate than the low-affinity site. In addition, they provide. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and inorganic phosphate or of Co2+ and inorganic phosphate to the enzyme and show that the high-affinity site has greater specificity for inorganic phosphate than the low-affinity site. In addition, they provide. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and inorganic phosphate or of Co2+ and inorganic phosphate to the enzyme and show that the high-affinity site has greater specificity for inorganic phosphate than the low-affinity site. In addition, they provide evidence against divalent metal ion inner sphere binding to phosphate for enzyme subunits having one or two divalent metal ions bound per subunit and evidence for a conformational change restricting active-site accessibility to solvent on the binding of a third divalent metal ion per subunit.

Binding Sites↗
PubMed1980

Effects of phosphate loading and parathyroid hormone on starling renal phosphate excretion.

The effects of phosphate loading were examined in intact, parathyroidectomized (PTX), and PTX starlings infused with parathyroid extract (PTX +/- PTE). In intact starlings, there was no correlation between the filtered phosphate load and the quantity of phosphate excreted. Net secretion predominated at lower filtered loads (2.5-20 mu mol . kg-1 . min-1) but net reabsorption occurred at filtered loads between 20 and 35 mu mol . kg-1 . min-1. Phosphate-loaded PTX starlings reabsorb phosphate over a wide range of filtered loads (5-45 mu mol . kg-1 . min-1). Excretion was significantly correlated with the filtered load in PTX birds (r = 0.943). Phosphate loaded PTX + PTE starlings secreted phosphate when filtered loads ranged from 2.5 to 30 mu mol . kg-1 . min-1. Excretion also was significantly correlated with the filtered load in this group (r = 0.972). The results of these experiments indicate that a) variations in endogenous PTH release in intact starlings result in wide variations in the renal pattern of phosphate excretion during phosphate loading; b) net phosphate secretion rarely occurs during phosphate loading in PTX starlings, but always occurs in PTX + PTE starlings; and c) both phosphate secretion and phosphate reabsorption are limited by maximal tubular transport capacities. Additional evidence indicates that PTH inhibits tubular phosphate reabsorption in starlings.

Animals↗
PubMed2001

Dietary phosphate-dependent growth is not mediated by changes in plasma phosphate concentration.

The present study was undertaken in order to assess the role of dietary phosphate in growth. A diet deficient in phosphate led to a suppression of growth in juvenile rats. The phenomenon is similar to that described for diets deficient in other essential single components such as Mg, Zn or K. However, unlike the other constituents, dietary phosphate restriction affected the growth rate much more than it altered the serum phosphate concentration; addition of phosphate to the drinking water of rats fed a low-phosphate diet restored the growth rate without a concomitant change in serum phosphate concentration. The suppression of growth rate by the deletion of phosphate was associated with a delayed decrease in food intake. This finding implies that the variation in appetite was secondary to the change in growth. The increase in body weight following phosphate supplementation was associated with a concomitant increment in food intake. The phosphate-dependent growth was, however, evident also in rats that were pair-fed with those that were not supplied with phosphate. It is concluded that dietary phosphate-dependent growth is not mediated by changes in phosphate concentrations in the extracellular fluid. It is plausible that signals arising from receptors for phosphate in the digestive system constitute part of the growth control apparatus in rats.

Animals↗
PubMed2005

Quantification of sugar phosphate intermediates of the pentose phosphate pathway by LC-MS/MS: application to two new inherited defects of metabolism.

We describe a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to quantify pentose phosphate pathway intermediates (triose-3-phosphates, tetrose-4-phosphate, pentose-5-phosphate, pentulose-5-phosphates, hexose-6-phosphates and sedoheptulose-7-phosphate (sed-7P)) in bloodspots, fibroblasts and lymphoblasts. Liquid chromatography was performed using an ion pair loaded C(18) HPLC column and detection of the sugar phosphates was carried out by tandem mass spectrometry using an electron ion spray source operating in the negative mode and multiple reaction monitoring. Reference values for the pentose phosphate pathway intermediates in blood spots, fibroblasts and lymphoblasts were established. The method was applied to cells from patients affected with a deficiency of transaldolase. The transaldolase-deficient cells showed an increased concentration of sedoheptulose-7-phosphate. (Bloodspots: 5.19 and 5.43 micromol/L [0.49-3.33 micromol/L]; fibroblasts 7.43 and 26.46 micromol/mg protein [0.31-1.14 micromol/mg protein]; lymphoblasts 16.03 micromol/mg protein [0.61-2.09 micromol/mg protein].) The method was also applied to study enzymes of the pentose phosphate pathway by incubating fibroblasts or lymphoblasts homogenates with ribose-5-phosphate or 6-phosphogluconate and the subsequent analysis of the formed sugar phosphates.

Cells, Cultured↗
PubMed1984

Allosteric interactions of glycogen phosphorylase b. A crystallographic study of glucose 6-phosphate and inorganic phosphate binding to di-imidate-cross-linked phosphorylase b.

The binding to glycogen phosphorylase b of glucose 6-phosphate and inorganic phosphate (respectively allosteric inhibitor and substrate/activator of the enzyme) were studied in the crystal at 0.3 nm (3A) resolution. Glucose 6-phosphate binds in the alpha-configuration at a site that is close to the AMP allosteric effector site at the subunit-subunit interface and promotes several conformational changes. The phosphate-binding site of the enzyme for glucose 6-phosphate involves contacts to two cationic residues, Arg-309 and Lys-247. This site is also occupied in the inorganic-phosphate-binding studies and is therefore identified as a high-affinity phosphate-binding site. It is distinct from the weaker phosphate-binding site of the enzyme for AMP, which is 0.27 nm (2.7A) away. The glucose moiety of glucose 6-phosphate and the adenosine moiety of AMP do not overlap. The results provide a structural explanation for the kinetic observations that glucose 6-phosphate inhibition of AMP activation of phosphorylase b is partially competitive and highly co-operative. The results suggest that the transmission of allosteric conformational changes involves an increase in affinity at phosphate-binding sites and relative movements of alpha-helices. In order to study glucose 6-phosphate and phosphate binding it was necessary to cross-link the crystals. The use of dimethyl malondi-imidate as a new cross-linking reagent in protein crystallography is discussed.

Adenosine Monophosphate↗
PubMed1991

Possible involvement of Lys603 from Escherichia coli glucosamine-6-phosphate synthase in the binding of its substrate fructose 6-phosphate.

Pyridoxal 5'-phosphate is a competitive inhibitor of glucosamine-6-phosphate synthase with respect to the substrate fructose 6-phosphate. Irreversible inactivation of pyridoxal-5'-phosphate-treated enzyme with [14C]-cyanide resulted in covalent incorporation of close to 1 mol pyridoxal 5'-phosphate/mol enzyme subunit. The enzyme-pyridoxal-5'-phosphate complex could also be inactivated by reduction with NaBH3CN. Sequence analysis of the unique radioactively labelled tryptic peptide, resulting from inactivation with [3H]NaBH3CN, identified the C-terminal nonapeptide encompassing the modified Lys603. The presence of fructose 6-phosphate protected this residue from pyridoxylation. Direct evidence that a lysine residue is involved in the binding of the substrate as a Schiff base came from the isolation at 4 degrees C of a enzyme-fructose-6-phosphate complex in a 1:1 molar ratio. Treatment of the enzyme-[14C]fructose-6-phosphate complex with NaBH3CN revealed one site of modification in the tryptic peptide map. In contrast, trapping the same complex with potassium cyanide resulted in the isolation of several radiolabelled peptides containing lysines which could potentially bind fructose 6-phosphate. However, since the radioactivity was not specifically associated with the lysine residues, it is suggested that these 14C-labelled peptides resulted from the decomposition of an unstable alpha,alpha'-dihydroxyaminonitrile adduct rather than from a lack of specificity of fructose 6-phosphate fixation. Lys603 is then the candidate of choice for fructose 6-phosphate binding since it lies at or near the active site as demonstrated by the trapping experiments with pyridoxal 5'-phosphate described above, and among the lysines which belong to the sugar-binding domain this is the only one conserved between the three members of the purF, glutamine-dependent, amidotransferase subfamily which include the glucosamine-6-phosphate synthase from Escherichia coli, Saccharomyces cerevisiae and the Rhizobium nodulation protein NodM.

Amino Acid Sequence↗
PubMed1994

A unique protonated phosphate group in bone mineral not present in synthetic calcium phosphates. Identification by phosphorus-31 solid state NMR spectroscopy.

The detailed chemical composition and microstructure of freshly deposited bone mineral, and how these properties change with maturation of the mineral, have been studied intensively and still remain controversial. For example, current analytical technology is inadequate for the unambiguous characterization of the monohydrogen phosphate ions in bone mineral. Using a differential cross polarization/magic angle spinning solid state nuclear magnetic resonance spectroscopy technique, we suppress the dominant orthophosphate (PO4-3) signal to reveal the spectra of the minor phosphate constituents. This method depends upon differences in the cross polarization time constants for phosphorus-31 nuclei in protonated and non-protonated phosphate ions. It is now possible for the first time to directly measure both the proportion of acid phosphate (HPO4-2) as well as the parameters which characterize its isotropic and anisotropic chemical shift. In bone from three species at several developmental stages, we have found a single type of acid phosphate species, identical in all of the specimens examined. The phosphorus-31 isotropic chemical shift of this acid phosphate group in bone mineral corresponds precisely with that of acid phosphate in octacalcium phosphate, and not with that of brushite. In contrast, the bone acid phosphate anisotropic chemical shift parameters are close to those of brushite, and differ significantly from those of octacalcium phosphate. The orthophosphate resonances of bone mineral, synthetic hydroxyapatite and synthetic octacalcium phosphate share identical chemical isotropic shifts, and similar chemical shift anisotropies. The implication of these results is that the intimate structure of the acid phosphate group in bone mineral is unique, and that none of the common synthetic calcium phosphates accounts well for all of the observed solid state phosphorus-31 NMR properties of bone mineral.

Animals↗
PubMed2001

X-ray structure of Escherichia coli pyridoxine 5'-phosphate oxidase complexed with pyridoxal 5'-phosphate at 2.0 A resolution.

Escherichia coli pyridoxine 5'-phosphate oxidase catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate by the FMN oxidation of pyridoxine 5'-phosphate forming FMNH(2) and H(2)O(2). Recent studies have shown that in addition to the active site, pyridoxine 5'-phosphate oxidase contains a non-catalytic site that binds pyridoxal 5'-phosphate tightly. The crystal structure of pyridoxine 5'-phosphate oxidase from E. coli with one or two molecules of pyridoxal 5'-phosphate bound to each monomer has been determined to 2.0 A resolution. One of the pyridoxal 5'-phosphate molecules is clearly bound at the active site with the aldehyde at C4' of pyridoxal 5'-phosphate near N5 of the bound FMN. A protein conformational change has occurred that partially closes the active site. The orientation of the bound pyridoxal 5'-phosphate suggests that the enzyme catalyzes a hydride ion transfer between C4' of pyridoxal 5'-phosphate and N5 of FMN. When the crystals are soaked with excess pyridoxal 5'-phosphate an additional molecule of this cofactor is also bound about 11 A from the active site. A possible tunnel exists between the two sites so that pyridoxal 5'-phosphate formed at the active site may transfer to the non-catalytic site without passing though the solvent.

Binding Sites↗
PubMed2003

Mechanism of flavin mononucleotide cofactor binding to the Desulfovibrio vulgaris flavodoxin. 1. Kinetic evidence for cooperative effects associated with the binding of inorganic phosphate and the 5'-phosphate moiety of the cofactor.

The pathway(s) by which the flavin cofactor binds to the apoflavoprotein is the subject of some debate. The crystal and NMR structures of several different flavodoxins have provided some insight, although there is disagreement about the location of the initial interaction between the flavin mononucleotide (FMN) and the apoflavodoxin and the degree of protein conformational change associated with cofactor binding [Genzor, C. G., Perales-Alcon, A., Sancho, J., and Romero, A. (1996) Nat. Struct. Biol. 3, 329-332; Steensma, E., and van Mierlo, C. P. M. (1998) J. Mol. Biol. 282, 653-666]. Binding kinetics using stopped-flow spectrofluorimetry and phosphate competition studies were used to develop a model for flavin binding to the flavodoxin from Desulfovibrio vulgaris. In the presence of phosphate, the time course of fluorescence quenching associated with FMN binding to apoflavodoxin was biphasic, whereas riboflavin, which lacks the 5'-phosphate group of FMN, displayed monophasic binding kinetics. When the concentration of phosphate in solution was increased, the FMN binding rates of the two phases behaved differently; the rate of one phase decreased, while the rate of the other increased. A similar increase in the single phase associated with riboflavin binding was also observed. This has led to the following model. The binding of the flavin isoalloxazine ring to its subsite is dependent on the presence of a phosphate group in the phosphate-binding subsite. When phosphate is in the buffer solution, FMN can bind in either of two ways: by the initial insertion of the 5'-phosphate group followed by ring binding or, when inorganic phosphate from solution is bound, the insertion of the isoalloxazine ring first. Riboflavin, which lacks the phosphate moiety of FMN, binds only in the presence of inorganic phosphate, presumably due to the binding of this group in the phosphate-binding subsite. These results suggest that cooperative interactions exist between the phosphate subsite and the ring-binding region in the D. vulgaris flavodoxin that are necessary for isoalloxazine ring binding.

Alanine↗
PubMed1998

Control of serum phosphate without any phosphate binders in patients treated with nocturnal hemodialysis.

We compared the efficacy and the long-term effects of nocturnal hemodialysis (NHD) versus conventional hemodialysis (CHD) in controlling serum phosphate levels in patients with end-stage renal disease (ESRD). Patients underwent thrice weekly CHD and were subsequently switched to NHD six nights weekly. In the "acute" study serum and dialysate phosphate were measured during and after dialysis, and the total dialysate was collected to calculate mass solute removal. Although pre-dialysis (1.7 +/- 0.6 vs. 1.5 +/- 0.8 mM) serum phosphate levels were similar in CHD and NHD, respectively, post-dialysis levels were slightly lower with CHD (0.7 +/- 0.2 vs. 0.8 +/- 0.2 mM, P < 0.05). The measured phosphate removed per session of CHD or NHD was comparable, 25.3 +/- 7.5 versus 26.9 +/- 9.8 mumol/session, respectively. On the other hand, the cumulative weekly phosphate removal was significantly higher with NHD as compared to CHD, 75.8 +/- 22.5 versus 161.6 +/- 59.0 mumol/week (P < 0.01). In the "chronic" study serum phosphate levels were measured monthly for five months on CHD and for five months after the patients were switched to NHD. Dietary phosphate intake and the dosage of phosphate binders were tabulated. Serum phosphate levels fell during NHD: 2.1 +/- 0.5 mM at the beginning of the study and 1.3 +/- 0.2 mM five months after being switched to NHD (P < 0.001). At the same time dietary phosphate intake increased by 50%. By the fourth month of NHD therapy none of the patients was taking any phosphate binders. In conclusion, NHD is more effective in controlling serum phosphate levels than CHD, allowing patients to discontinue their phosphate binders completely and to ingest a more liberal diet.

Cross-Over Studies↗
PubMed1985

Phosphate excretion in uremic rats: effects of parathyroidectomy and phosphate restriction.

As progressive renal failure develops, phosphate excretion per functioning nephron increases, thus preserving homeostasis. To test whether dietary phosphate supply might contribute to the regulation of renal phosphate excretion in the uremic setting, groups of male Sprague-Dawley rats that were either parathyroidectomized (PTX) or sham PTX (S-PTX) and either five-sixths nephrectomized (Nx) or sham Nx (S-Nx) were studied following a 4-wk dietary regimen consisting of 0.1 or 0.7% phosphate. For Nx rats fed the 0.7% phosphate diet the fractional excretion of phosphate (FEPi) was enhanced (47 +/- 6 vs. 21 +/- 3%) and the maximum tubular reabsorption of phosphate per milliliter GFR (TmPi/GFR) was suppressed (1.65 +/- 0.19 vs. 2.33 +/- 0.19 mumol/ml). FEPi was unchanged by PTX in these Nx animals (42 +/- 6 vs. 47 +/- 6%). TmPi/GFR remained suppressed in PTX, NX animals when compared with S-Nx, PTX controls (3.38 +/- 0.33 vs. 5.07 +/- 0.41 mumol/ml). For rats fed the 0.1% phosphate diet Nx did not affect TmPi/GFR in either S-PTX (5.40 +/- 0.43 vs. 4.97 +/- 0.34 mumol/ml) or PTX (7.03 +/- 0.23 vs. 6.98 +/- 0.21 mumol/ml) animals. For both S-Nx and Nx animals the effects of PTX and dietary phosphate restriction on TmPi/GFR were independent and additive. In all groups of animals, tubular reabsorption of phosphate per milliliter GFR (TRPi/GFR) dropped acutely with continued infusion of phosphate once TmPi/GFR was achieved. Thus, a resetting of TRPi/GFR occurs among Nx rats in response to both chronic dietary phosphate deprivation and acute intravenous phosphate loading.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗
PubMed1988

Effect of cAMP analogue infusion on phosphate reabsorption in phosphate-deprived rats.

The present study was performed to compare the effects of 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (cAMP analogue) and parathyroid hormone (PTH) infusion on segmental phosphate reabsorption in phosphate-deprived rats. Micropunctures of the late proximal and the early distal tubules were performed in acutely thyroparathyroidectomized (TPTX) rats fed either a normal (NPD) or low phosphate diet (LPD), and the phosphaturic response to infusion of PTH and cAMP analogue was evaluated. In NPD rats, PTH (n = 10) and the cAMP analogues (n = 11) markedly increased urinary phosphate excretion, due to inhibition of phosphate reabsorption along the proximal convoluted tubule and pars recta. In phosphate-deprived rats, PTH (n = 10) or the cAMP analogue (n = 11) did not increase urinary phosphate excretion. However, PTH and the cAMP analogue inhibited phosphate reabsorption along the proximal convoluted tubule but not in the pars recta in phosphate-deprived rats. We conclude that cAMP analogue infusion mimics the effect of PTH infusion on phosphate reabsorption along the proximal convoluted and proximal straight tubule in normal and phosphate-deprived rats. The resistance to the phosphaturic effect of PTH and cAMP infusions is a result of a blunted inhibition of phosphate reabsorption by the proximal convoluted tubule and also an increased phosphate reabsorption by the proximal straight tubule.

Animals↗
PubMed1995

Reversible structural changes of octacalcium phosphate and labile acid phosphate.

Acid phosphate is one of the major impurities incorporated into bioapatites, and its quantity and environment in forming mineral have been used as diagnostic probes to pursue acidic precursor(s). Currently, little is known about the structural feature of nonstoichiometric octacalcium phosphate (OCP), which has been advocated to be, most plausibly, mineral salt initially formed during amelogenesis. In the present report, we attempt to define the state of acid phosphate in OCP crystals which were Ca-deficient and contained 40% total phosphate as acid phosphate. We assessed fractions of acid phosphate in discrete environments by extracting the crystals in either deionized water, 10 mmol/L NaOH solution (initial pH 11), or 150 mmol/L Tris buffer at pH 7.4. Solid samples before and after the treatments were examined by chemical analyses and x-ray diffraction. The results indicated that successive extractions with use of the alkaline solution brought about a reversible change (not hydrolysis) in the interior structure of OCP, which accompanied a marked decrease in acid phosphate. A substantial part of the lost acid phosphate was restored during subsequent treatments at neutral pH, and, intriguingly, this restoration accompanied a re-ordering of OCP structure. The data suggested that the acid phosphate in OCP is separated into three pools: (a) a stable pool corresponding to roughly 50 to 60% of the total acid phosphate, (b) a reversibly exchangeable pool corresponding to 25 to 30% of the acid phosphate which may exist either in the water layer or on crystal surfaces, and (c) an unstable (or irreversibly lost) pool corresponding to 15 to 20% of the acid phosphate, a part of which might be explained by the presence of excess hydrogen in OCP. The present work supports the concept that protons and, to a lesser magnitude, phosphate species can diffuse into and out of the OCP lattice prior to initiation of its hydrolytic transition into apatite.

Apatites↗