Biomedical subjects
M Petrarulo
Publications and source records attributed to M Petrarulo.
Low levels of urinary inorganic pyrophosphate indicating systemic pyrophosphate deficiency in a boy with idiopathic infantile arterial calcification.
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Liquid chromatographic determination of ethyl alcohol in body fluids.
A high-performance liquid chromatographic technique for ethyl alcohol determination in body fluids is proposed. Ethyl alcohol is quantitatively converted into acetaldehyde-phenylhydrazone by oxidation in the presence of alcohol dehydrogenase, nicotinamide-adenine dinucleotide and phenylhydrazine. The derivative is suitable for reversed-phase liquid chromatography and ultraviolet detection at 276 nm. The limits of linearity, detection and quantification as well as accuracy and reproducibility were investigated in water, serum and whole blood. Analytical responses were linear within the 0.008 to 5 g/l range, and the limit of quantification was 0.02 g/l both in aqueous standard and in biological matrix assays. Mean analytical recovery of ethyl alcohol in blood serum averaged 98.2+/-4.2%, imprecision (CV%) at 0.80 g/l was 2.2%, and the limit of quantification was 0.02 g/l. Serum concentrations of persons that avoided alcoholic beverages for a week were less than the limit of quantification. Ethyl alcohol concentrations in serum and whole blood compared well with those obtained by headspace gas chromatography. This simple and reliable procedure, which was also used for a urine assay, could be suitable for validation of the screening procedures used to monitor ethanol abuse.
Effects of mineral composition of drinking water on risk for stone formation and bone metabolism in idiopathic calcium nephrolithiasis.
1. To assess whether the mineral content of drinking water influences both risk of stone formation and bone metabolism in idiopathic calcium nephrolithiasis, 21 patients were switched from their usual home diets to a 10 mmol calcium, low-oxalate, protein-controlled diet, supplemented with 21 of three different types of mineral water. Drinking water added 1, 6 and 20 mmol of calcium and 0.5, 10 and 50 mmol of bicarbonate respectively to the controlled diet. 2. The three controlled study periods lasted 1 month each and were separated by a 20 day washout interval. Blood and urine chemistries, including intact parathyroid hormone, calcitriol and two markers of bone resorption, were performed at the end of each study period. The stone-forming risk was assessed by calculating urine saturation with calcium oxalate (beta CaOx), calcium phosphate (beta bsh) and uric acid (beta UA). 3. The addition of any mineral water produced the expected increase in urine output and was associated with similar decreases in beta CaOx and beta UA, whereas beta bsh varied marginally. These equal decreases in beta CaOx, however, resulted from peculiar changes in calcium, oxalate and citrate excretion during each study period. The increase in overall calcium intake due to different drinking water induced modest increases in calcium excretion, whereas oxalate excretion tended to decrease. The changes in oxalate excretion during any one study period compared with another were significantly related to those in calcium intake. Citrate excretion was significantly higher with the high-calcium, alkaline water. 4. Parathyroid hormone, calcitriol and markers of bone resorption increased when patients were changed from the high-calcium, alkaline to the low-calcium drinking water. 5. We suggest that overall calcium intake may be tailored by supplying calcium in drinking water. Adverse effects on bone turnover with low-calcium diets can be prevented by giving high-calcium, alkaline drinking water, and the stone-forming risk can be decreased as effectively as with low-calcium drinking water.
Bony content of oxalate in patients with primary hyperoxaluria or oxalosis-unrelated renal failure.
Oxalate retention occurs in end-stage renal failure. Regular dialysis treatment does not prevent progressive accumulation of oxalate in cases of ESRF due to primary hyperoxaluria (PH), whereas such accumulation seldom seems to occur in oxalosis-unrelated ESRF. To elucidate this issue we have measured the bony content of oxalate on biopsies of the iliac crest taken from 32 uremic patients, 7 of them with ESRF associated with PH1 (6 cases) or PH2 (1 case). Ten subjects with normal renal function and no evidence of metabolic bone disease were taken as controls. Only trace amounts levels of oxalate were detected in normal subjects and oxalate to phosphate ratio was below 3:10,000. Non-PH dialyzed patients exhibited fivefold increases in oxalate levels, which rose to 5.1 +/- 3.6 mumol/g bony tissue. Calcium oxalate was estimated to represent 0.18% of the hydroxyapatite content of bone. Oxalate amounts were neither related to pre-dialysis plasma levels of oxalate, nor with duration of dialysis treatment, suggesting that accumulation was not progressive disorder. Oxalate levels were slightly higher in patients with a low turnover osteodystrophy compared to those with a high turnover pattern. Dialyzed patients with PH had remarkable increases in oxalate levels, which ranged between 14.8 and 907 mumol/g bony tissue. Oxalate deposition appeared to be progressive in that oxalate levels were significantly related to time on dialysis. In three patients calcium oxalate was a significant fraction of the mineralized bone. The occurrence of calcium oxalate crystals affected the histomorphometric patterns, that were featured by an increase in resorptive areas and a decrease in bone formation rate.(ABSTRACT TRUNCATED AT 250 WORDS)
Erythrocyte transmembrane flux and renal clearance of oxalate in idiopathic calcium nephrolithiasis.
An anomaly in erythrocyte oxalate transport has been reported in patients with idiopathic calcium oxalate nephrolithiasis. Even if clinical and experimental evidence suggests a causal role of this cellular anomaly in calcium nephrolithiasis, a definitive answer to this fundamental question is still lacking. We approached this problem by searching for a possible relationship between the erythrocyte oxalate self-exchange anomaly and the renal clearance of this anion in stone formers. In 10 idiopathic calcium-oxalate renal stone formers, and 10 healthy subjects we evaluated the erythrocyte oxalate flux rate, and the renal fractional clearance of oxalate by a recently described enzymathic procedures for plasma oxalate determination. With respect to controls, stone formers had higher oxalate flux rate in erythrocytes, and higher oxalate renal fractional clearance with a significant direct correlation between the two parameters. These data are compatible with a membrane transport abnormality within the kidney of these stone formers, and the existence of a common defect of the oxalate transport shared by both erythrocytes and tubular renal cells. The latter may be crucial in the pathogenesis of calcium oxalate nephrolithiasis, by modifying the renal handling of oxalate.
The clinical significance of assessment of serum calcium oxalate saturation in the hyperoxaluria syndromes.
Estimating calcium oxalate saturation (beta CaOx) in body fluids is proposed as a simple and reproducible procedure to assess the risk of systemic oxalosis in several clinical conditions associated with oxalate retention. beta CaOx was computerized from the measured concentrations of main serum ions. Accurate assay of serum oxalate was crucial for reliability of beta CaOx estimates. However, beta CaOx also depended upon changes of calcium and magnesium concentrations. Patients with end-stage renal failure (ESRF) due to primary or enteric hyperoxaluria had beta CaOx greater than saturation, whereas this happened in only 10 of 25 and two of 24 of those with oxalosis-unrelated ESRF. Bony content of oxalate measured in some of these patients was consistent with these results. In patients with maintained renal function beta CaOx was inversely related to glomerular filtration rate, but the slope was steeper in patients with than in those without hyperoxaluria and beta CaOx reached saturation at earlier stages of renal insufficiency.
Citrate in urine determined with a new citrate lyase method.
An enzyme-spectrophotometric method to determine citrate in biological fluids is proposed, based on citrate lyase-catalyzed and phenylhydrazine reactions. The enzyme converts citrate into oxaloacetate, which, in the presence of phenylhydrazine, is transformed into the corresponding phenylhydrazone. The ultraviolet-absorbing product is determined by absorbance measurement at 330 nm. The method is more precise and twice as sensitive as the traditional citrate lyase method and, because it does not require the use of additional enzymes and coenzymes, is cheaper and simpler. Mean analytical recovery of citrate averaged 100.7% +/- 2.2%, imprecision (CV) of the assay for citrate at 0.96 mmol/L (urine) was 2.0%, and the lower limit of quantification was 0.08 mmol/L. Results correlated well with those by both ion-chromatographic and traditional citrate lyase methods.
Detection of primary hyperoxaluria type 2 (L-glyceric aciduria) in patients with maintained renal function or end-stage renal failure.
Primary hyperoxaluria (PH) type 1 and type 2 are autosomal recessive defects of oxalate metabolism resulting from glyoxylate accumulation which occurs by two distinct pathways. PH1 is associated to glycolic aciduria; PH2 to L-glyceric aciduria. Because hyperoxaluria leads to nephrolithiasis or nephrocalcinosis in both, they can be differentiated only through detection of the associated acidurias. However, glycolate and L-glycerate assays are not widely available and, in the setting of ESRF, diagnosis is hampered by a number of misleading events. At any stage of the disease diagnosis is crucial because there are differences between the two forms in clinical behaviour, long-term prognosis, and treatment. In this paper we outline diagnostic criteria for identification of PH2 in two patients, one with maintained renal function and one with ESRF on CPD, based on the use of a novel HPLC assay of L-glycerate in different body fluids. With the routine application of this procedure PH2 has been identified in two of 23 patients fulfilling criteria for diagnosis of PH. This suggests that the type 2 variant of PH may occur more frequently than so far suspected, and should be tested for even in the setting of ESRF.
End-stage renal failure in primary hyperoxaluria type 2.
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Solubility and structure of N-carboxymethylchitosan.
N-Carboxymethylchitosan from crab and shrimp chitosans was obtained in water-soluble form by proper selection of the reactant ratio, i.e. using equimolar quantities of glyoxylic acid and amino groups. HPLC determinations of glyoxylic and glycolic acids, in conjunction with NMR analysis, permitted identification of the structure of the product, which is partly N-mono-carboxymethylated (0.3), N,N-dicarboxymethylated (0.3) and N-acetylated depending on the level of deacetylation of the starting chitosan (0.08-0.15). The preparation can be made successfully even in the presence of large concentrations of glycolic acid. The use of enzymes exerting hydrolysing activity on the high-molecular-weight fractions helps to avoid gel formation during storage and precipitate formation on addition of anti-microbial agents.
Assay of plasma oxalate with soluble oxalate oxidase.
We use oxalate oxidase from barley seedlings for the colorimetric determination of oxalate in plasma. The oxalate is converted to hydrogen peroxide, which, in the presence of peroxidase, is detected by a Trinder-like chromogenic system. Optimization of the assay, including deproteinization and elimination of interferences from reducing substrates, is described. Ascorbate additions (200 mumol/L) did not affect oxalate concentration in plasma, even after long frozen storage. Mean analytical recovery of oxalate averaged 102% +/- 6.9%, imprecision (CV) at 2.0 mumol/L was 7.2%, and the lower limit of quantification (CV = 20%) was 0.6 mumol/L. Results correlated well with those by chromatography (r = 0.999, Sy/x = 0.29 mumol/L, n = 32, range for x, y = 0-140 mumol/L). Plasma oxalate concentrations measured in 32 healthy subjects ranged from 0.6 to 2.9 mumol/L (mean 1.28, SD 0.71 mumol/L), which agrees with those measurable by using indirect radioisotopic dilution methods. Patients with primary hyperoxaluria and chronic renal failure exhibited markedly greater plasma concentrations of oxalate.
High-performance liquid chromatographic microassay for L-glutamate:glyoxylate aminotransferase activity in human liver. Application in primary hyperoxaluria type 1.
A rapid and sensitive liquid chromatographic technique to determine L-glutamate:glyoxylate and aminotransferase (EC 2.6.1.4) activity in human liver is described. Homogenised tissue was incubated for 60 min in the presence of substrates and the 2-oxoglutarate generated was converted into the corresponding phenylhydrazone which was determined using reversed-phase high-performance liquid chromatography. The procedure allowed the detection of the enzyme activity expressed by 7.5 micrograms of liver protein, it was more sensitive and less time-consuming than the spectrophotometric procedure previously used. No significant differences were found between normal controls and patients with primary hyperoxaluria. In an 8-month-infant with primary hyperoxaluria type 1, the enzyme activity was reduced to 16% of the average control values.
Determinants of oxalate balance in patients on chronic peritoneal dialysis.
We assessed plasma levels and removal rates of oxalate in 24 patients on chronic peritoneal dialysis (CPD) for oxalosis-unrelated renal failure. The ion-chromatographic (IC) measurements of oxalate in plasma, dialysate, and urine (in seven patients with residual renal function) were used to calculate peritoneal and renal clearances of oxalate. The serum state of saturation with calcium oxalate was calculated by means of a computer-based model system. Patient data were compared with those from 19 healthy individuals. Peritoneal clearance of oxalate was 6.3 +/- 4.7 mL/min, ie, 8% of the normal renal clearance. As a result, both plasma oxalate and calcium oxalate saturation were higher than in controls and did not overlap. Plasma was supersaturated with calcium oxalate in only two of 24 patients (8%). Removal of oxalate by dialysis was related to the amount of fluid infused. Overall removal of oxalate (dialysate plus urine) was similar to 24-hour excretion of normal subjects and was taken as a measure of its generation. Oxalate generation rate was dependent on protein (whole and animal) intake, but not on caloric intake or pyridoxine status. Pyridoxine supplementation, 75 and 300 mg daily for 1 months, was not effective in reducing plasma levels or generation rates of oxalate. Residual renal function had a minor influence on oxalate patterns. We conclude that current programs are adequate to maintain oxalate balance in patients on CPD under basic conditions.
Effects of oral and intravenous calcitriol on serum calcium oxalate saturation in dialysis patients.
1. To determine whether the multiple changes in the blood chemistry profile induced by calcitriol may be conducive to secondary systemic oxalosis we have studied nine patients on regular dialysis treatment under three different regimens: (1) oral calcitriol, 0.25 microgram/daily for at least 6 months. (2) off calcitriol, a 1-month withdrawal of the drug, taken as the baseline study period; (3) intravenous calcitriol, 1 microgram three times weekly at the end of dialysis, with tests performed at 1 and 3 months from initiation. 2. Serum concentrations were measured pre- and post-dialysis at the end of each study period. The whole dialysate was used for the determination of the overall calcium and oxalate removal by dialysis. The degree of saturation with calcium oxalate monohydrate was estimated by a computer program. Serum calcitriol concentrations were also assessed. 3. Total and ionized serum calcium did not change on average, although mild hypercalcaemia developed in some patients on intravenous calcitriol. There was an increase in plasma level of oxalate during both oral and intravenous calcitriol treatment, but this was less pronounced during intravenous therapy. Removal of oxalate by dialysis was also greater in patients on oral calcitriol. 4. These increases were probably originated from intestinal absorption and secondary to hyperabsorption of dietary calcium. Consequently, the degree of saturation with calcium oxalate before dialysis rose during calcitriol treatment, irrespective of the route of administration. 5. These results emphasize that, in addition to soft tissue calcification due to calcium phosphates, ectopic calcium oxalate crystallization must also be viewed as a potential risk associated with long-term administration of calcitriol.
Long-term survival on renal replacement therapy for primary hyperoxaluria type I.
We describe the case of a patient in end-stage renal failure due to primary hyperoxaluria type I (PH1) who started hemodialysis in 1977 and is still alive and active. The diagnosis of PH1 was first suspected after a bone biopsy performed in 1981 to investigate hyperparathyroidism. Oxalosis recurred as early as 3 months after transplantation in a cadaver kidney grafted in 1987; nevertheless, graft function remained good enough to make possible the discontinuation of dialysis treatment for 5 months and thereafter to have only 1 dialysis a week for 17 months. The diagnosis of PH1 has been recently confirmed despite the patient being already anuric by means of the determination of plasma oxalate and glycolate levels as well as by determining hepatic alanine:glyoxylate amino-transferase.
Care of the adult patients with cystic fibrosis.
Although no therapeutic breakthrough has taken place, the life expectancy of patients with cystic fibrosis (CF) has dramatically increased in the last 20 yrs. Nowadays, more than 80% of the patients outlive 18 yrs of age, with a mean survival age approaching 28 yrs. These favourable results are due mainly to early diagnosis and continuous treatment in specialized centres. The main therapeutic items are: prevention and early treatment of lung infections, mainly due to Pseudomonas aeruginosa, drainage of bronchial secretions (postural drainage, forced expiratory techniques, positive-expiratory-pressure mask, autogenic drainage,...), substitutive therapy with pancreatic enzymes and hypercaloric diet. Thus cystic fibrosis is increasingly a disease which involves adults, with many related problems, which span from the difficulties of prescribing an effective antibiotic therapy in cases with multiple resistances, to combining the need for a long daily physiotherapy schedule with school or a working life. Sexual problems and related psychological troubles are an important issue in the management of cystic fibrosis in adult patients, and are accordingly treated.
Thresholds of serum calcium oxalate supersaturation in relation to renal function in patients with or without primary hyperoxaluria.
Systemic oxalosis is a constant feature in patients with primary hyperoxaluria type 1 (PH1) and chronic renal failure (CRF) and is not prevented by regular dialysis (RDT), because removal cannot keep up with retention and overproduction of oxalate. These patients are candidates to kidney and/or liver transplantation, which should be ideally planned prior to the development of oxalosis. However, methods to detect the presence and extent of oxalosis are invasive and poorly reproducible, and only indirect approaches are feasible. Because supersaturation of body fluids is an essential condition for oxalotic deposits to form, we have assessed serum calcium oxalate saturation (beta CaOx) in 12 patients with PH1 and 26 with PH1-unrelated renal diseases and varying degrees of CRF. Nineteen healthy individuals were taken as controls. beta CaOx was closely dependent on oxalate serum levels. Serum oxalate and beta CaOx were increased in patients with CRF as compared to controls, and were inversely related to GFR, assessed as creatinine clearance. However, at any level of GFR, both were always greater in PH1 patients. From the slopes of the regression of beta CaOx over ClCr, saturation was predicted to be obtained at ClCr ranging 24-34 and 8-11 ml/min/1.73 m2 in PH1 and non-PH1 patients respectively. Based on the dependence of beta CaOx on oxalate, saturation was associated with serum oxalate between 44 and 46 mumol/l, irrespective of either the prevailing GFR or the underlying disease. These simple procedures represent a valuable non-invasive tool to define the risk of systemic oxalosis and may assist in timing of transplantation.