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Biomedical subjects

P G Werness

Publications and source records attributed to P G Werness.

At least 19 recordsLinked to original sources
PubMed1986

Inhibition of hydroxyapatite crystal growth by bone-specific and other calcium-binding proteins.

Mineralization of bone matrix may be influenced by the presence of specific, noncollagenous bone proteins. The quantitative influence of two bone-specific proteins--bone gamma-carboxyglutamic acid (Gla) protein and osteonectin--and other proteins that decreased the rate of crystal growth was measured by adding seed crystals of hydroxyapatite to a solution of CaCl2 and KH2PO4, pH 7.4 at 37 degrees C. The molar concentrations of proteins needed to inhibit the rate of crystal growth by 50% were as follows: osteonectin, 0.15 microM; bone Gla protein, 0.8 microM; prothrombin, 0.9 microM; prothrombin fragment 1, 1.0 microM; soybean trypsin inhibitor, 3 microM; prethrombin 1, 9 microM; cytochrome c, 30 microM. Calmodulin and parvalbumin were found to be less active than prothrombin fragment 1 and had no activity in the micromolar range. The combination of two inhibitors resulted in a mixture with an inhibitory activity that was the sum of the two inhibitors. Decarboxylation of bone Gla protein significantly reduced its inhibitory activity. These results indicate that the inhibitory activity of a protein does not correlate with Ca2+-binding affinity under these conditions, that the mixture of inhibitors has an additive effect, and that gamma-carboxyglutamic acid residues enhance the ability of a protein to inhibit hydroxyapatite-seeded crystal growth.

Animals↗
PubMed1986

Ascorbic acid levels in the aqueous humor of nocturnal and diurnal mammals.

Ascorbic acid concentration is known to be very high in the aqueous humor of humans and most animals. The role it might play in ocular function is a subject of conjecture. Some have proposed that it might protect the eye against light-induced damage. We examined the aqueous humor from 22 species of mammals to determine the range of levels and to see if there was a correlation with behavior. A wide range of ascorbic acid levels in the aqueous humor was found. Most of the animals considered to be diurnal had higher ascorbic acid levels than the nocturnal animals. This would suggest that ascorbic acid in the aqueous humor may play a protective role in those animals who are most exposed to light. Regardless, any theory proposing a role for ascorbic acid in the eye in mammals must take the wide range of ascorbic acid levels into account.

Animals↗
PubMed1985

Isolation and characterization of native adult osteonectin.

Noncollagenous bovine bone proteins were obtained from EDTA-solubilized extracts of adult bovine bone in the absence of denaturants. Native osteonectin was isolated from the noncollagenous bone proteins by ion-exchange chromatography using DEAE-Sephadex A-50 and DEAE-Sephadex A-25, followed by gel filtration on Sephadex G-100. Comparison of the physical and chemical properties (i.e. electrophoric mobility, amino acid composition and pI) of this protein with those reported by Termine et al. (Termine, J.D., Belcourt, A.B., Conn, K.M., and Kleinman, H.K. (1981) J. Biol. Chem. 256, 10403-10408) indicate that this protein is osteonectin. Sedimentation equilibrium analyses in the presence of 6 M guandinium chloride, 10 mM Ca2+, 10 mM EDTA, or 0.15 M NaCl all yielded a molecular weight of 29,100 +/- 900. 125I-Osteonectin underwent saturable and exchangeable binding to hydroxyapatite and calf skin collagen. Eleven mg of 125I-osteonectin bound to 1.0 g of hydroxyapatite with a Kd of 8 X 10(-8) M. The intrinsic fluorescence of bovine osteonectin was partially quenched when micromolar Ca2+ was added, indicating a high affinity Ca2+ interaction. Native osteonectin was found to reduce the rate of hydroxyapatite crystal seeded growth by 50% (1 IU) at a concentration of 1.6 X 10(-7) M at pH 7.4, 37 degrees C in 0.15 M NaCl. This makes osteonectin one of the most potent inhibitors of hydroxyapatite formation presently known and more than 5 times as effective as bone Gla protein (1 IU = 8 X 10(-7) M).

Amino Acids↗
PubMed1985

EQUIL2: a BASIC computer program for the calculation of urinary saturation.

A BASIC computer program for the calculation of urinary supersaturation with respect to the common kidney stone components is described. In vitro and in vivo tests show that the program described accurately calculates supersaturation. The application of this computer program to urolithiasis research is discussed.

Computers↗
PubMed1985

Inhibitors of crystal growth of hydroxyapatite: a constant composition approach.

Pyrophosphate, citrate and magnesium, inhibitors of hydroxyapatite crystal growth, were studied using a seeded crystal growth system of constant composition at pH 5.80, 6.60 and 7.40. With this technique, crystal growth was studied at constant supersaturation at different pH values without the induction of other calcium phosphate phases. One inhibitor unit (that concentration of material that results in a reduction of 50 per cent in the growth rate from control) was calculated using the Langmuir adsorption isotherm. Pyrophosphate and citrate increased inhibitor activity with decreasing pH, whereas magnesium increased inhibitor activity with increasing pH. These data suggest that, at the urinary concentrations of these inhibitors, pyrophosphate is the most potent inhibitor, citrate less, and magnesium least. Pooled urine collections were studied using the same system and were found to have decreased inhibitor activity as pH decreased. This suggests that other modulators of hydroxyapatite, either promoters or inhibitors, are active in this system at the pH values studied.

Citrates↗
PubMed1985

Sulfate and methyldopa metabolism: metabolite patterns and platelet phenol sulfotransferase activity.

Sulfate conjugation catalyzed by phenol sulfotransferase (PST) is the major metabolic pathway for methyldopa. Methyldopa is also O-methylated in a reaction catalyzed by catechol-O-methyltransferase (COMT). Our studies were performed to determine whether sodium sulfate alters methyldopa metabolism. Methyldopa powder, 3.5 mg/kg, was taken with and without sodium sulfate, 13.25 mg/kg, by 24 subjects in a randomized, crossover design. Compared with results obtained when only methyldopa was taken, sodium sulfate taken with methyldopa increased the proportion of drug excreted as methyldopa sulfate expressed as the percentage of all urinary metabolites (66.0% +/- 5.3% and 50.1% +/- 7.5%; means +/- SD). The percentage of free methyldopa excreted also decreased (17.1% +/- 3.7% and 27.3% +/- 5.5%). Platelet PST and red blood cell COMT activities were measured in blood samples from these subjects. When sodium sulfate was taken with methyldopa, there was a significant correlation between platelet PST activities and percentages of metabolites excreted as methyldopa sulfate (r = 0.545; P less than 0.01). This correlation was not significant when methyldopa was taken alone (r = -0.340; P greater than 0.10). There was a significant correlation between red blood cell COMT activities and the proportion of urinary metabolites excreted as 3-O-methyl-alpha-methyldopa when methyldopa was taken alone (r = 0.532; P less than 0.01) but not when it was taken with sodium sulfate (r = 0.153; P greater than 0.20). Our data support the conclusion that variation in sulfate availability may be one factor responsible for individual differences in the metabolism of clinically used doses of methyldopa.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗
PubMed1985

Bladder secretion of inhibitors of calcium oxalate crystal growth.

Differences in calcium oxalate crystal growth inhibition were studied in normally voided urine (bladder urine) and in urine collected directly from the kidney (kidney urine) in nine dogs. Urine samples were collected before and 10 days after bilateral ureterostomies. Calcium oxalate crystal growth inhibition was measured in a standard seeded crystal growth system. The alcian blue-precipitable material of the urine samples was determined. Significantly lower values were observed in kidney urine than in bladder urine for calcium oxalate crystal inhibition (mean difference, 0.07 +/- 0.02 inhibitor units/mg creatinine; P less than 0.01) and for the alcian blue-precipitable material (mean difference, 0.07 +/- 0.02 mg/mg creatinine; P less than 0.01). We conclude that the bladder adds calcium oxalate crystal growth inhibition to urine. Glycosaminoglycans from the bladder mucosa may be responsible; however, other acidic polymers such as RNA fragments or glycopeptides have been shown to be a constituent of the alcian blue-precipitable material. These are potent inhibitors of calcium oxalate crystal growth, and their participation in the increase of inhibition observed in bladder urine cannot be excluded. Total calcium oxalate crystal growth inhibition present in normally voided urine may be an overestimation of the actual inhibition present at the level of the kidney, where calculi usually form.

Alcian Blue↗
PubMed1984

Pentosan polysulfate as an inhibitor of calcium oxalate crystal growth.

Pentosan polysulfate was studied as a calcium oxalate crystal growth inhibitor. The inhibition from pentosan polysulfate at concentrations ranging from 3.5 to 110 mg./l. was measured in simple inorganic calcium oxalate solutions at pH 5, 6 and 7. Pentosan polysulfate also was mixed with urine at concentrations from 10 to 350 mg./l. and inhibition determined. Measurement of calcium oxalate crystal growth inhibition was performed in a seeded crystal growth system. One inhibitory unit (concentration of pentosan polysulfate necessary to give a 50 per cent reduction in the rate of crystal growth) was 5.7 +/- 2 mg./l. at pH 5, 7.2 +/- 1.1 mg./l. at pH 6 and 6.0 +/- 2.1 mg./l. at pH 7. Urine-pentosan polysulfate mixtures showed more inhibitory activity than the predicted inhibition present. Addition of pentosan polysulfate to urine at a concentration of 10 mg./l. increased the inhibitory activity from 45 +/- 3 to 59 +/- 3 IU/l. Pentosan polysulfate is a potent calcium oxalate crystal growth inhibitor in urine at physiologic pH levels. If the urinary concentration of pentosan polysulfate after oral administration reaches 10 mg./l., the increase in inhibitory activity in urine that may occur might be important in the treatment of patients who form calcium oxalate calculi within the urinary tract.

Calcium Oxalate↗
PubMed1984

Calcium oxalate dihydrate formation in urine.

Factors that promote the formation of calcium oxalate dihydrate (COD) in urine were investigated. Crystals resulting from the incubation of 25-ml aliquots of the solution to be tested and 1 ml of 0.05 M ammonium oxalate were examined by infrared spectrophotometry. With reference spectra of known mixtures, the fractional content of COD could be estimated. At pH 6.5, only COD formed in human urine. In a supersaturated inorganic solution of calcium oxalate, the percentage of COD was 7.5 +/- 1.4. Pyrophosphate (1 to 8 X 10(-5) M), citrate (10(-4) to 2 X 10(-3) M), RNA from yeast (5 X 10(-9) to 0.5 X 10(-7) M), or heparin (2 X 10(-9) to 2 X 10(-7) M) added to a supersaturated solution of calcium oxalate increased the percentage of COD proportional to the concentration of the additives. Chondroitin sulfate and magnesium had no effect. An increase in pH increased the formation of COD in inorganic solutions containing citrate, pyrophosphate, and heparin and in undiluted urine. RNA-citrates and citrate-pyrophosphate mixtures showed additivity. Urine showed an effect that was inversely proportional to its dilution. Substances that promote COD formation in this system had their phase-stabilizing effects at concentrations normally found in urine. Further, these same substances are known inhibitors of calcium oxalate crystal formation. With both inhibition and phase stabilization, there is additivity of effects, and changes in pH alter the response.

Adult↗
PubMed1984

Oxalate absorption and postprandial urine supersaturation in an experimental human model of absorptive hypercalciuria.

The effect of 1.25-dihydroxyvitamin D [1,25-(OH)2D] on dietary oxalate absorption and postprandial urine supersaturation with calcium oxalate was determined in 11 normal subjects. 1,25-(OH)2D increased the urinary excretion of orally administered [14C]oxalate in the 8 h period after a liquid meal containing 1.875 mmol of calcium and 0.83 mmol of oxalate (P less than 0.01), and during a 48 h period when the subjects ingested a diet containing 25 mmol of calcium and 3.3 mmol of oxalate/day (P less than 0.01); however, 1,25-(OH)2D administration had no effect on [14C]oxalate excretion when calcium was removed from the liquid meal. 1.25-(OH)2D increased 24 h urinary oxalate excretion from 28.7 +/- 2.1 mmol/mol of creatinine to 36.8 +/- 2.6 mmol/mol of creatinine (P less than 0.05) on the 10 mmol/day calcium diet and from 26.4 +/- 2.9 to 33.2 +/- 2.2 mmol/mol of creatinine (P less than 0.1) on the 25 mmol/day calcium diet. A linear correlation (r = 0.72) was found between plasma 1,25-(OH)2D levels and urinary [14C]oxalate excretion after the liquid meal. 1,25-(OH)2D administration produced postprandial supersaturation of urine with calcium oxalate and calcium oxalate crystalluria. These studies suggest that 1,25-(OH)2D increases oxalate absorption (and urinary excretion) by increasing calcium absorption, which results in less binding of calcium to oxalate in the intestine; therefore more oxalate is available for absorption. The combined effect of increased calcium and oxalate absorption results in postprandial supersaturation of urine with calcium oxalate, with resultant crystalluria.

Adult↗
PubMed1983

Binding of nicotinamide adenine dinucleotide by the renal brush border membrane from rat kidney cortex.

The characteristics of nicotinamide adenine dinucleotide (NAD) binding on brush border membranes prepared from rat renal cortex were investigated with the use of radioactively labelled NAD, [adenine-2,8-3H]NAD+, as a ligand. (1) We found that NAD binds on brush border membrane and that the extent of NAD binding is linearly proportional to the brush border membrane protein, and progressively increases with concentration of NAD in the medium. (2) The rate of NAD binding was dependent on temperature. At 20 degrees C, the equilibrium binding was obtained at 15 min, while NAD binding at 0 degree C was slower, but the final level of binding reached at 120 min was similar to that plateau of binding observed at 20 degrees C. Brush border membrane inactivated by heating at 95 degrees C for 3 min did not bind NAD. Binding of NAD on brush border membranes was reversed by simple dilution or by the addition of unlabelled NAD. Both alpha-NAD and beta-NAD stereoisomers displaced bound [3H]NAD. Reduced NAD (NADH) caused less displacement of bound NAD than oxidized NAD+. Adenine, nicotinamide, pyrophosphate, of 5'-AMP did not displace bound NAD. (3) The NAD binding to brush border membranes was nearly saturable, approximating saturation at 10(-4) M NAD. Kinetic analysis by Scatchard plot indicates two sets of NAD binding sites in brush border membranes: a high-affinity binding site (Kd = 1.9 . 10(-5) M) and a low-affinity binding site (Kd = 2.2 . 10(-3) M). (4) Unlike concentrative uptake of D-[14C]glucose by brush border membrane vesicles, binding of NAD was not dependent on the presence of an outside-in sodium gradient [Na+0 greater than Na+i], nor was it abolished by repeated freezing and thawing of brush border membranes. Unlike D-[14C]glucose uptake, NAD binding by brush border membranes did not change upon decrease of intravesicular volume in hypertonic media. These observations indicate that NAD association with brush border membranes is true binding rather than intravesicular uptake of this compound. (5) The presence of specific binding sites in renal brush border membrane capable of binding of NAD with a high degree of affinity suggests that such sites may be involved in previously observed (Kempson, S.A., Colon-Otero, G., Ou, S.L., Turner, S.T. and Dousa, T.P. (1981) J. Clin. Invest. 67, 1347) modulatory effect of NAD on sodium-gradient-dependent uptake of phosphate across luminal brush border membrane of proximal tubules.

Animals↗
PubMed1983

Sulphate conjugation of p-hydroxytriamterene by platelet phenol sulphotransferase: assay conditions and correlation with metabolism in man.

1 Sulphate conjugation catalyzed by phenol sulphotransferase (PST) is an important pathway in the metabolism of many drugs including triamterene. Variations in PST activity in an easily obtained tissue such as the platelet might reflect individual differences in the sulphate conjugation in other organs and tissues. Human platelets contain at least two forms of PST, a thermolabile (TL) form for which dopamine is a substrate and a thermostable (TS) form for which low concentrations of p-nitrophenol serve as a substrate. 2 p-OH-triamterene, the major metabolite of triamterene, is conjugated with sulphate in vivo. p-OH-triamterene was a substrate for platelet PST with an apparent Michaelis-Menten value of 26 microM. Thermal stability studies indicated that p-OH-triamterene was a substrate for only the TS form of platelet PST. 3 When platelet homogenates from 29 individual subjects were tested, there was a significant correlation between PST activities measured with 4 microM p-nitrophenol and with p-OH-triamterene (r = 0.985, P less than 0.0001) but not between activities measured with dopamine and with p-OH-triamterene (r = 0.023, P greater than 0.2). These results confirmed that p-OH-triamterene was a substrate for only the TS form of human platelet PST. 4 The same 29 subjects were treated with 1 mg/kg of triamterene orally. 24-h urinary excretions of triamterene, p-OH-triamterene and p-OH-triamterene sulphate averaged 15.3%, 6.3% and 78.4%, respectively, of the total of triamterene plus measured metabolites excreted. The excretion of triamterene plus the two metabolites averaged 43.1 +/- 2.6% (mean +/- s.e. mean) of the ingested dose. There was not a significant correlation between the proportion of p-OH-triamterene excreted as sulphate conjugate and the activities of either the TS or TL forms of platelet PST activity.

Adult↗
PubMed1982

Triamterene urolithiasis: solubility, pk, effect on crystal formation, and matrix binding of triamterene and its metabolites.

The commonly used diuretic Tri and its metabolites have been identified recently as major components of some kidney stones. We have carried out basic physical chemical studies to determine the mechanism of Tri incorporation into kidney stones. The solubility and pK of Tri and its major metabolites, Tri-OH and the Tri-So4, have been determined at 37 C in 0.15 M NaCl. The effect of Tri and its metabolites on crystal formation has been measured in the calcium oxalate monohydrate, hydroxyapatite, and uric acid crystal systems. Tri and its metabolites do not promote crystal nucleation, growth, or aggregation in any of these crystal systems and are not incorporated as these crystals form. We can demonstrate binding of Tri and its metabolites to the protein matrix isolated from kidney stones. These data suggest that Tri and its metabolites to the protein matrix isolated from kidney stones. These data suggest that Tri and its metabolites do not promote the formation of kidney stones but rather become incorporated into existing stones or stone nidi by binding to the protein matrix found in all kidney stones.

Calcium Oxalate↗
PubMed1981

Urinary crystal growth: effect of inhibitor mixtures.

1. The crystal growth inhibitory activity of mixtures of known inhibitors and of mixtures of known inhibitors with normal urine was determined in calcium oxalate monohydrate and hydroxyapatite seeded crystal growth systems. 2. The inhibitory activity of the mixtures was compared with the measured activity of the individual components of the mixtures. All mixtures had inhibitory activity equal to the sum of the activities of their components, with the exception of RNA/urine mixtures in the calcium oxalate monohydrate system. 3. RNA/urine mixtures had inhibitory activity toward calcium oxalate monohydrate crystal growth which was less than would be predicted from the activity of the RNA and of the urine which were added. This reduced inhibitory activity was shown to be due probably to hydrolysis of RNA by the ribonuclease activity normally present in urine. 4. The results of these experiments make it possible to determine quantitatively the contribution of various naturally occurring urinary crystal growth inhibitors to the total measured inhibition observed in urine.

Calcium Oxalate↗
PubMed1980

Renal brush border membrane adaptation to phosphorus deprivation: effects of fasting versus low-phosphorus diet.

Alimentary phosphorus deprivation due to a low-phosphorus diet (LPD) elicits a profound antiphosphaturia and an increase in sodium-dependent inorganic phosphate (Pi) uptake by renal cortical brush border membrane (BBM) vesicles. But, in alimentary phosphorus deprivation due to total fasting, high urinary excretion of Pi persists. In the present study, we determined whether low tubular reabsorption of Pi in fasting is due to a diminished capacity of the specific Pi transport system with the renal cortical luminal BBM or whether it is due to a reduced transepithelial reabsorption of Pi because of metabolic conditions occurring in proximal tubule cells during fasting. Sodium-dependent Pi transport in compared with fasted rats or rats fed a normal phosphorus diet. Sodium-dependent uptake of D-glucose was significantly lower in LPD rats, compared with fast animals or animals fed a normal diet. Thus, in contrast to LPD, fasting does nt elicit an increase in Pi transport and a decrease in D-glucose transport across the isolated renal BBM. The same differences in BBM transport of Pi were present also in thyroparathyroidectomized rats. Further experiments demonstrated that the adaptation of renal function and the renal BBM transport to LPD are overridden by a subsequent period of total fasting. Results of the present study show that fasting both prevents and reverses the renal response of rats to alimentary phosphorus deprivation. The differences in Pi excretion between fasted rats, LPD rats, and LPD rats subsequently fasted are attributed, at least in part, to specific adaptive changes in sodium-dependent Pi transport across the luminal BBM, rather than to alterations in other cellular (metabolic) components of transepithelial Pi reabsorption in the proximal tubule.

Animals↗