Molecular analysis of the AGXT gene in Italian patients with primary hyperoxaluria type 1 (PH1).

Specimens were collected from 22 Italian patients with primary hyperoxaluria type 1 (PH1). Ten of them had already been analyzed by molecular biology. To clarify the molecular characteristics of the AGXT gene disease responsible for PH1, DNA samples were examined for known mutations by hybridisation of PCR products with Sequence Specific Oligonucleotides (PCR-SSO). We planned to identify new mutations of the AGXT gene by heteroduplex analysis followed by direct sequencing. We had already standardized a) the conditions for the amplification of the 11 exons of AGXT, b) the PCR-SSO technique and c) the heteroduplex analysis of amplified products. Preliminary results demonstrated that the AGXT mutations described in previous studies were found only in 40% of the examined Italian patients with PH1. The remaining 60% of mutations should be characterised in future studies.

Clinical and biochemical patterns of presentation in monolateral and bilateral calcium nephrolithiasis.

To investigate patterns of monolateral and bilateral nephrolithiasis, we enrolled 196 patients with idiopathic calcium stone disease (ICaSD) and 36 with proven primary hyperparathyroidism (PHP). Monolateral disease occurred in 45 subjects with ICaSD and 3 with PHP. All had had three or more stone events. They were studied for a number of clinical and biochemical parameters. The expected prevalence of monolateral stone disease was calculated according to the binomial distribution of random events. Whereas the observed and expected prevalence of monolateral nephrolithiasis did not differ in PHP, the distribution did not follow a chance pattern in ICaSD, since monolateral disease was still frequent among patient with more than 6 episodes. To find out whether monolateral and bilateral ICaSD had distinct pathogenic mechanisms the two groups were compared for clinical and biochemical patterns: no differences emerged concerning metabolic derangements, urine saturation and diet-related biochemistries. Bilateral stone-formers had a higher recurrence rate, but a similar number of stone-operations or ESWL. In 81 of 151 bilateral idiopathic stone-formers in which we were able to assess the exact number of stone events in left and right kidney, the distribution of stones between kidneys did not differ from the binomial distribution. In conclusion, while PHP-associated nephrolithiasis presents predictable patterns, ICaSD comprises a subset in which the disease occurs monolaterally. These forms cannot be distinguished from bilateral forms with common clinical features or routine biochemistries.

Increased serum pancreatic enzymes in uremia: relation with treatment modality and pancreatic involvement.

Increased levels of pancreatic enzymes have been reported in patients with renal insufficiency and ascribed either to impaired urinary excretion or, in a few studies, to the presence of pancreatic damage. In the present study serum total amylase, pancreatic amylase, and lipase were evaluated in 63 patients with chronic renal insufficiency (CRF), in 98 patients on hemodialysis (HD), in 28 patients on continuous ambulatory peritoneal dialysis (CAPD), in 23 patients with renal transplantation (RT), and in 34 normal volunteers (C). Serum parathyroid hormone and triglyceride levels were also measured in the majority of patients. Ultrasound examination of the pancreas was performed in a select number of cases. Mean values of pancreatic enzymes were significantly higher in all the study groups in comparison with controls, but values exceeding three times the upper normal limit were detected only in HD patients, who also showed amylase and lipase levels significantly highly than those of CAPD and CRF subjects. Negligible amounts of pancreatic enzymes were detected in peritoneal fluid of CAPD patients. Significant correlations were found with serum creatinine in CRF, with parathyroid hormone in HD and CAPD, and with duration of treatment in HD. No pancreatic abnormalities were detected by ultrasound. In conclusion, very high levels of pancreatic enzymes are seen mainly in HD patients and might be related more to the metabolic derangement of long-term dialysis treatment than to the occurrence of acute pancreatic damage.

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.

[Bartter's syndrome in children and adults. Study of 6 cases]. / La sindrome di Bartter nel bambino e nell'adulto. Studio di 6 casi.

Six patients (3 children and 3 adults) with the clinical and biochemical features of Bartter's syndrome are presented. Pediatric cases included a more severe form, in one patient, with physical and mental retardation, hypercalciuria and nephrocalcinosis, and a less severe one, including two patients, with milder clinical features, low calcium and high magnesium excretion and hypomagnesiemia. Adult patients were affected by either the mild congenital form (case n. 4) or the acquired variety (cases n.5 and 6). Tubular function was investigated in the 3 adults by assessing clearance measurements during maximal diuresis. There was a defective fractional distal solute reabsorption (FDR) ranging between 0.52 and 0.60. This was well below the results obtained in one patient with psychogenous vomiting (FDR 0.94) and comparable to those in two patients with interstitial nephropathies caused by vesico-ureteral reflux (FDR 0.63 and 0.67 respectively). We concluded that: 1) the etiopathogenetic spectrum of Bartter's syndrome corresponds to different clinical presentation (mild, heavy, congenital or acquired varieties), and alterations in mineral and electrolyte renal handling; 2) reduction in FDR is a feature neither essential nor exclusive of this syndrome.

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.

[Renal damage in rheumatoid arthritis]. / Il danno renale in corso di artrite reumatoide.

Renal damage is not uncommon in rheumatoid arthritis, but the causative role of the disease per se is not well defined yet. In this paper the updated literature data are reported and our own experience as well. In particular, we describe renal syndromes associated with non-steroidal antiinflammatory drugs, remission-inducing agents (gold and penicillamine) and cytotoxic drugs, secondary amyloidosis, systemic rheumatoid vasculitis, glomerular and tubulo-interstitial nephritis not related to drug therapy.

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.

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.

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.

High-performance liquid chromatographic assay for L-glyceric acid in body fluids. Application in primary hyperoxaluria type 2.

We describe a liquid chromatographic technique to determine L-glycerate in body fluids. The method is based on the derivatisation of the L-glycerate by incubation with lactate dehydrogenase and nicotinamide-adenine dinucleotide in the presence of phenylhydrazine. Oxidation of L-glycerate forms beta-hydroxypyruvate which is converted in turn into the related phenylhydrazone. The UV-absorbing derivative is determined using reversed-phase high performance liquid chromatography. The sensitivity was 5 mumol/l and 50 microliters of sample were required. The imprecision relative standard deviation was 4.5% and the recovery was 96.5 +/- 6.8% for L-glycerate in plasma. L-Glycerate concentrations in urine and plasma were less than 5 mumol/l in both normal individuals and patients with glycolic aciduria. In a patient with systemic oxalosis and normal plasma glycolate, plasma L-glyceric acid was 887 mumol/l.

Plasma and urine glycolate assays for differentiating the hyperoxaluria syndromes.

To differentiate hyperoxaluria syndromes we measured plasma and urine glycolate by a novel high performance liquid chromatographic procedure. Mean glycolate level was 7.9 +/- 2.4 mumol./l. in plasma and 422 +/- 137 mumol./24 hours in urine from 19 control subjects. Renal clearance was about 50% the glomerular filtration rate irrespective of the underlying disease. There was close correlation between glycolate and oxalate in plasma. Plasma glycolate was normal in all but 8 patients who had primary hyperoxaluria 1. Plasma assay detected the disease more efficiently than urine assay. Pyridoxine decreased oxalate biosynthesis in 2 of the 4 patients treated with it and glycolate assay confirmed this behavior. Glycolate excretion was significantly high in 3 of 8 patients of primary hyperoxaluria 1 patients. Idiopathic stone formers had mild increases in glycolate excretion but this was not related with oxalate excretion. Glycolate levels were normal in 5 patients with enteric hyperoxaluria. We conclude that glycolate assay is essential for identifying patients with primary hyperoxaluria 1 and may represent a valuable tool for differentiating hyperoxaluria.

High-performance liquid chromatographic microassay for L-alanine:glyoxylate aminotransferase activity in human liver.

We examine the suitability of a rapid and sensitive liquid chromatographic technique to determine L-alanine:glyoxylate aminotransferase (AGT) activity in human liver. Homogenised tissue was incubated for 30 min in the presence of substrates and the generated pyruvate was converted into the corresponding phenylhydrazone which was determined using reversed-phase high-performance liquid chromatography (HPLC). The procedure allowed the detection of the enzyme activity expressed by 10 micrograms of liver protein and was rapid enough resulting more sensitive and less time-consuming than the previous colorimetric one. We found that AGT activity in two hyperoxaluria type 1 patients was reduced as compared with controls. Also, cirrhotic patients had very low enzyme activities, even in the absence of detectable disorders of oxalate metabolism and this was ascribed to abnormal liver morphology. This may represent a misleading drawback if diagnosis of type 1 primary hyperoxaluria (PH1) uniquely relies on AGT assay.

Oxalate balance studies in patients on hemodialysis for type I primary hyperoxaluria.

Primary hyperoxaluria type I (PH1) always leads to end-stage renal failure (ESRF) due to deposition of calcium oxalate in the kidney. Regular dialysis therapy (RDT) can not overcome the excess production of oxalate, hence, systemic oxalate deposition occurs. The extent of tissue deposition and the rate at which oxalate accumulates influence the quality of life and survival of the patients. Therefore, an estimate of the oxalate balance needs to be made for patients on RDT. In this study, we suggest a simple model by which some of the main parameters of oxalate turnover can be assessed without using radioactive materials. Levels of oxalate, glycolate, and urea, and degrees of calcium oxalate saturation, were assessed on plasma ultrafiltrates from two patients with PH1, sampled before, at the end of a dialysis session, and over the entire interdialytic interval. In patients with PH1, oxalate increased linearly during the early phases and then the curve flattened at a concentration corresponding to approximately threefold saturation. The initial phase of the relationship was used to estimate generation rate of oxalate. The delayed phase was ascribed to the deposition of newly generated oxalate out of its miscible pool. Conversely, the relationship for glycolate and urea remained linear. This was also different from the values obtained in four patients with oxalosis-unrelated ESRF, whose oxalate levels increased linearly over the entire interdialytic interval. In the two patients with PH1, the overall oxalate generation was assessed at 4 to 7 mmol/d. The difference between generation and dialysis removal indicated that tissue deposition was greater than 50 mumol/kg body weight/d.(ABSTRACT TRUNCATED AT 250 WORDS)