Nedosiran Dramatically Reduces Serum Oxalate in Dialysis-Dependent Primary Hyperoxaluria 1: A Compassionate Use Case Report.

Primary hyperoxaluria 1 (PH1) is a devastating condition involving recurrent urolithiasis, early end-stage renal disease and multisystemic deposition of calcium oxalate crystals. Treatment options for PH1 are limited, inevitably requiring transplantation, usually combined kidney and liver transplant. Here we report successful compassionate use of Nedosiran, an RNA interference targeting lactate dehydrogenase, in an index patient. Monthly Nedosiran injections led to dramatically decreased plasma oxalate levels, decreased frequency of weekly hemodialysis sessions from 6 to 3, and deferral of combined kidney and liver transplant. Nedosiran represents a novel and impactful potential therapeutic for PH1 patients with end-stage renal disease.

Oxalosis Associated With High-Dose Vitamin C Ingestion in a Peritoneal Dialysis Patient.

We report a case of systemic oxalosis involving the eyes and joints due to long-term use of high-dose vitamin C in a patient receiving maintenance peritoneal dialysis (PD). This 76-year-old woman with autosomal dominant polycystic kidney disease underwent living unrelated kidney transplantation 10 years earlier. The transplant failed 6 months before presentation, and she initiated hemodialysis therapy before transitioning to PD therapy 4 months later. During the month before presentation, the patient noted worsening arthralgias and decreased vision. Ophthalmologic examination revealed proliferative retinopathy and calcium oxalate crystals. Plasma oxalate level was markedly elevated at 187 (reference range, <1.7) µmol/L, and urine oxalate-creatinine ratio was high (0.18mg/mg). The patient reported taking up to 4g of vitamin C per day for several years. Workup for causes of primary and secondary hyperoxaluria was otherwise negative. Vitamin C use was discontinued, and the patient transitioned to daily hemodialysis for 2 weeks. Plasma oxalate level before the dialysis session decreased but remained higher (30-53µmol/L) than typical for dialysis patients. Upon discharge, the patient remained on thrice-weekly hemodialysis therapy with stabilized vision and improved joint symptoms. This case highlights the risk of high-dose vitamin C use in patients with advanced chronic kidney disease, especially when maintained on PD therapy.

Design and Characterization of Apocynin Loaded PLGA Nanoparticles and their In vivo Efficacy in Hyperoxaluric Rats.

BACKGROUND: Apocynin has become a drug of choice in NADPH oxidase induced pathological conditions. Hyperoxaluria is one such pathological condition where NADPH oxidase is involved in eliciting renal injury. OBJECTIVE: Recently apocynin has shown to reverse the transcriptome profile of the NADPH oxidaseassociated genes and reduced oxidative burden in hyperoxaluric animals. The poor solubility of this drug creates certain apprehensions about its bioavailability. PLGA (Poly Lactic co-Glycolic Acid) encapsulation of drug nanoparticles have showed to induce sustain release and henceforth enhance the efficiency and bioavailability of drugs. Therefore, the present study is aimed to envisage a novel approach of synthesizing apocynin doped PLGA nanoparticles. METHODS: The PLGA nanoparticles (both unloaded and loaded) were prepared using solvent extraction method and analyzed for size and stability by Dynamic Light Scattering (DLS), TEM (transmission electron microscopy) and zeta potential. Furthermore, the drug release and encapsulation efficiency of the drug was calculated in vitro. RESULTS: The nanoencapsulation formed was stable with desired size (217-259 nm) and posses a controlled drug release of 20%. Further this nanoencapsulation was explored for its potential to reduce hyperoxaluric manifestations in rats given ethylene glycol with ammonium chloride for 9 days. CONCLUSION: In comparison to free apocynin, it was found that nanoparticles containing apocynin showed moderately better results in vivo by maintaining serum urea and createnine levels. These nanoparticles can be used in diseases where a sustained release of apocynin is required.

Metabolic syndrome contributes to renal injury mediated by hyperoxaluria in a murine model of nephrolithiasis.

Metabolic syndrome (MS) individuals have a higher risk of developing chronic kidney disease through unclear pathogenic mechanisms. MS has been also related with higher nephrolithiasis prevalence. To establish the influence of MS on renal function, we designed a murine model of combined metabolic syndrome and hyperoxaluria. Four groups of male Sprague-Dawley rats were established: (1) control group (n = 10) fed with standard chow; (2) stone former group (SF) (n = 10) fed with standard chow plus 0.75% ethylene glycol administered in the drinking water; (3) metabolic syndrome group (MS) (n = 10), fed with 60% fructose diet; (4) metabolic syndrome + stone former group (MS + SF) (n = 10), 60% fructose diet and 0.75% EG in the drinking water. MS group showed a significant injury to renal function when hyperoxaluria was induced. It was demonstrated by a significant decrease of creatinine clearance (p < 0.001), with higher tubular damage (34.3%, CI 95% 23.9-44.7, p < 0.001), produced by deposition of crystals, and increased tubular synthesis of osteopontin as a response to tubular damage. Induction of hyperoxaluria in rats with MS causes severe morphological alterations with a significant impairment of renal function. This impairment is not produced in rats without MS. Therefore, this model can be useful for the study of the influence of MS in stone formation.

Different effects of γ-linolenic acid (GLA) supplementation on plasma and red blood cell phospholipid fatty acid composition and calcium oxalate kidney stone risk factors in healthy subjects from two race groups with different risk profiles pose questions about the GLA-arachidonic acid-oxaluria metabolic pathway: pilot study.

Fatty acid (FA) composition of phospholipids in plasma and red blood cells (RBC) can influence calciuria, oxaluria and renal stone formation. In this regard, the ratio of arachidonic acid (AA) and its precursor linoleic acid (LA) appears to be important. Administration of γ-linolenic acid (GLA) has been shown to increase the concentration of dihomo-gamma linoleic acid (DGLA) relative to AA indicating that it may attenuate biosynthesis of the latter. Such effects have not been investigated in race groups having difference stone occurrence rates. Black (B) and white (W) healthy males ingested capsules containing linoleic acid (LA) and GLA, for 30 days. Plasma and RBC total phospholipid (TPL) FA profiles, serum and 24 h urine biomarkers of hypercalciuria and urinary stone risk factors were determined on days 0 and 30. Data were tested for statistical significance using GraphPadInstat version 3.02. Concentration and percentage content of DGLA in plasma TPL increased in W but not in B. Arachidonic acid (AA) did not change in either group. There was no change in calcium excretion in either group but oxalate and citrate excretion increased in W. We suggest that elongation of GLA to DGLA may occur more rapidly than desaturation of DGLA to AA in W and that depressed activity of the enzyme elongase may occur in B. Calciuric and citraturic effects may be dependent on the quantity of LA or on the mass ratio of LA/GLA in the FA supplement. Questions about the mooted DGLA-AA-oxaluria pathway arise. We speculate that there exists a potential for using GLA as a conservative treatment for hypocitraturia. The observation of different responses in B and W indicates that such differences may play a role in stone formation and prevention.

Bilateral native nephrectomy reduces systemic oxalate level after combined liver-kidney transplant: A case report.

Primary hyperoxaluria type 1 (PH1) is a rare liver enzymatic defect that causes overproduction of plasma oxalate. Accumulation of oxalate in the kidney and subsequent renal failure are fatal to PH1 patients often in pediatric age. Combined liver and kidney transplantation is the therapy of choice for end-stage renal disease due to PH1. Levels of plasma oxalate remain elevated for several months after liver transplantation, as the residual body oxalate is slowly excreted. Patients with persistent hyperoxaluria after transplant often require hemodialysis, and accumulation of residual oxalate in the kidney can induce graft dysfunction. As the native kidneys are the main target of calcium oxalate accumulation, we postulated that removal of native kidneys could drastically decrease total body oxalate levels after transplantation. Here, we report a case of bilateral nephrectomy at the time of combined liver-kidney transplantation in a pediatric PH1 patient. Bilateral nephrectomy induced a rapid decrease in plasma oxalate to normal levels in less than 20 days, compared to the several months reported in the literature. Our results suggest that removal of native kidneys could be an effective strategy to decrease the need for hemodialysis and the risk of renal dysfunction after combined liver-kidney transplantation in patients with PH1.

Polyacrylic acid attenuates ethylene glycol induced hyperoxaluric damage and prevents crystal aggregation in vitro and in vivo.

The study explores calcium oxalate crystal inhibiting characteristic of polyacrylic acid (pAA), an anionic polymer in in vitro and in vivo. Animals were divided into 5 groups where group 1 served as control, group 2 were made hyperoxaluric by supplementing with Ethylene glycol (EG) 0.75% (v/v) for 30 days. Group 3, 4 & 5 were also given with EG and treated simultaneously with 2.5, 5 & 10 mg of pAA/kg of body weight, respectively. Urine, serum and tissue analyses along with histological studies were performed at the end of the 30 days study. In vitro crystallization was significantly inhibited by pAA and further it was supported by particle size analyses, XRD and FT-IR studies. Toxicological analyses showed that pAA was safe to use in animals at concentrations below 100 mg/kg BW. In vivo anti-urolithic study showed significant improvement in urinary lithogenic factors (calcium, oxalate, phosphate, citrate & magnesium) and renal function parameters (creatinine, urea and protein). Tissue analyses on anti-oxidant enzyme activity and lipid peroxides showed maintenance of tissue antioxidant status in the pAA supplemented rats and histological studies demonstrated the nephroprotection offered by pAA and were concurrent to the biochemical analyses. Supplementation of pAA not only reduces the crystal aggregation but also regulates the expression and localization of crystal inhibiting proteins and gene expression of inflammatory cytokines in experimental animals. In summary, pAA is a potent anti-urolithic agent in rats and we can propose that 10 mg/kg body weight is the effective dosage of pAA and this concentration can be used for further studies.

Reduction of plasma oxalate levels by oral application of Oxalobacter formigenes in 2 patients with infantile oxalosis.

The spectrum of primary hyperoxaluria type I is extremely heterogeneous, ranging from singular to recurrent urolithiasis and early end-stage renal disease (ESRD). In infantile oxalosis, the most devastating form, ESRD occurs as early as within the first weeks of life. No kidney replacement therapy sufficiently removes endogenously overproduced oxalate. However, curative combined liver-kidney transplant often is impracticable in small infants. Oxalobacter formigenes (O formigenes), an anaerobic oxalate-degrading bacterium, is a colonizer of the healthy human colon. Oral administration of O formigenes has been shown to significantly decrease urine and plasma oxalate levels in patients with primary hyperoxaluria. We report compassionate use of O formigenes in two 11-month-old girls with infantile oxalosis and ESRD. They received O formigenes twice a day for 4 weeks (or until transplant). Dialysis regimens were unchanged. Plasma oxalate levels decreased from >110 µmol/L before to 71.53 µmol/L under treatment in patient 1 and from >90 to 68.56 µmol/L (first treatment period) and 50.05 µmol/L (second treatment period) in patient 2. O formigenes was well tolerated. No serious side effects were reported. Extremely increased plasma oxalate levels in patients with infantile oxalosis may enable intestinal elimination of endogenous oxalate in the presence of O formigenes. Therefore, O formigenes therapy may be helpful as a bridging procedure until transplant in such patients.

Ethylene glycol induced hyperoxaluria increases plasma and renal tissue asymmetrical dimethylarginine in rats: a new pathogenetic link in hyperoxaluria induced disorders.

PURPOSE: The pathogenesis of kidney stones remains elusive. There is some evidence that hyperoxaluria may effect vascular endothelium and many studies link renal stones to atherosclerosis. Also, renal vascular endothelial cells regulate proximal tubular epithelial cell function. We determined the effect of hyperoxaluria on plasma and tissue levels of asymmetrical dimethylarginine. The secondary aim was to determine the effect of verapamil on asymmetrical dimethylarginine. MATERIALS AND METHODS: A total of 42 Sprague-Dawley rats were included in the study. In groups 1A, 1B and 1C hyperoxaluria was induced with ethylene glycol for 2 weeks. Groups 2A, 2B and 2C received ethylene glycol for 14 days and verapamil for 28 days. Control group 3 received no specific medication but distilled water. Blood samples were obtained at 24 hours and at study end, and kidney samples were obtained at 24 hours, and 7 and 28 days for histopathological evaluation. RESULTS: Plasma asymmetrical dimethylarginine increased early in the hyperoxaluric group (p = 0.0002). The effect was retained at the end of the study period (p = 0.01). There was no increase in asymmetrical dimethylarginine in the verapamil group on short-term and long-term followup. Hyperoxaluria induced a significantly dense staining pattern in renal tissue asymmetrical dimethylarginine vs controls (p = 0.01). Asymmetrical dimethylarginine staining did not differ in the control and verapamil groups. CONCLUSIONS: Increased systemic and local tissue asymmetrical dimethylarginine may help explain the pathogenetic mechanisms of hyperoxaluria induced disorders such as nephrolithiasis and atherosclerosis.

[Effects of mangesium salts and their combinations with vitamin B6 on oxalates crystalluria in rats fed with pyridoxine-deficient diet].

We studied the effects of oral magnesium (Mg) salts either alone or in combination with pyridoxine hydrochloride in rats on pyridoxine-deficient diet. Fifty-four male rats were randomized into two groups and were fed either a standard diet or a pyridoxine-deficient diet for 3 weeks. A significant rise of the EGOT index ( > 1.5), oxaluria (from 74.8 +/- 5.2 to 117.9 +/- 12.3 mcM/l, p = 0.035), and crystalluria in rats fed with pyridoxine deficient diet were revealed. Oral Mg chloride, Mg L-aspartate either alone or in combination with pyridoxine in comparison with magnesium sulfate, magne B6 (Mg lactate with pyridoxine) and pyridoxine alone were administered (50 mg of magnesium and/or 5 mg of pyridoxine per kg body weight). Magnesium salts in combination with pyridoxine lowered an oxalate level and crystalluria whereas magnesium salts alone reduced only crystalluria. Antilithis effects of Mg L-aspartate and Mg chloride in combination with pyridoxine were comparable with those observed in magne B6 or pyridoxine treatment and were significantly higher than in magnesium sulfate treatment.

Plasma oxalate level in pediatric calcium stone formers with or without secondary hyperoxaluria.

Plasma oxalate (POx) concentration is significantly elevated in primary hyperoxaluria, severe renal failure or ethylene glycol poisoning. In these conditions, the degree of hyperoxalemia correlates with the severity of systemic calcium oxalate (CaOx) deposition and should be therefore carefully monitored. Although secondary hyperoxaluria (secHyOx) is a common finding in pediatric patients with kidney stone disease, very little is known about POx in this condition. We therefore evaluated POx level in 59 children and adolescence with calcium urolithiasis (34 confirmed by CaOx stone analysis and 25 children with a strong clinical suspicion of this type of urolithiasis), with or without "mild" secHyOx. A control group consisted of 41 healthy sex- and age-matched children. We found that POx was significantly increased in children with calcium urolithiasis and secHyOx compared to healthy children (9.16 +/- 3.60 vs. 6.42 +/- 2.53 micromol/l), but that was not the case in children with calcium urolithiasis but with normal urinary oxalate excretion (7.12 +/- 3.33 micromol/l). We conclude that POx may be slightly increased in some pediatric calcium stone formers with secHyOx, probably related to intestinal oxalate hyperabsorption.

Effect of sulphated polysaccharides on erythrocyte changes due to oxidative and nitrosative stress in experimental hyperoxaluria.

Kidney stones are known to haunt humanity for centuries and increase in oxalate is a predominant risk factor for stone formation. The present study was initiated with a notion to study the oxidative and nitrosative stress on erythrocytes under oxalate stress and the putative role of sulphated polysaccharides. Hyperoxaluria was induced in two groups by the administration of 0.75% ethylene glycol in drinking water for 28 days and one of them was treated with sulphated polysaccharides from Fucus vesiculosus from the 8th day to the end of the experimental period of 28 days at a dose of 5 mg/kg body weight subcutaneously. Control and drug control (sulphated polysaccharides alone) were also included in the study. Glycolic and glyoxylic acid levels of urine were analyzed as an index of hyperoxaluria. The plasma enzymic markers of cellular integrity, redox status of red blood cells, osmotic fragility, and (14)C-oxalate binding were investigated. Urine and plasma nitric oxide metabolites, expression of inducible nitric oxide synthase protein, and mRNA were assessed in kidney to evaluate the nitrosative stress. Increased levels of glycolic and glyoxylic acid in urine indicated the prevalence of hyperoxaluria in ethylene glycol-administered groups. Plasma aspartate and alanine transaminase were not altered, but alkaline phosphatase and lactate dehydrogenase of hyperoxaluric group were increased indicating tissue damage. Activities of antioxidant enzymes were decreased, whereas erythrocyte membrane lipid peroxidation was increased in hyperoxaluric rats. Moreover, an altered fragility with an increase in oxalate binding activity was observed in hyperoxaluric group. Increase in nitric oxide metabolites levels in urine and plasma along with an increase in expression of inducible nitric oxide synthase protein and mRNA in kidney were observed in hyperoxaluric rats. Administration of sulphated polysaccharides to hyperoxaluric rats averted the abnormal increase in urinary glycolic and glyoxylic acid levels and enzyme activities, decreased lipid peroxidation, and increased the activities of antioxidant enzymes. Furthermore, increased nitrosative stress accompanying hyperoxaluria was also normalized on sulphated polysaccharides treatment. To conclude, sulphated polysaccharide administration was able to maintain the integrity of erythrocyte membrane and decrease the damage to erythrocytes in hyperoxaluria.

Vitamin B6 metabolites in idiopathic calcium stone formers: no evidence for a link to hyperoxaluria.

Vitamin B6 metabolites and their potential correlates to urinary oxalate excretion in idiopathic calcium stone formers (ICSF) compared with healthy subjects were investigated. This clinical study was performed in a population of male ICSF with (Hyperoxalurics, n=55) or without hyperoxaluria (Normooxalurics, n=57) as well as in 100 healthy male control subjects. Pyridoxal 5'-phosphate serum concentration (S-pyridoxal 5'P) and 24-h urinary excretion of 4-pyridoxic acid (U-4pyridoxic acid) were measured using HPLC; 24-h urinary excretion of oxalate (U-oxalate) was measured concurrently. A subgroup of subjects (40 Hyperoxalurics, 15 Normooxalurics and 50 controls) underwent the same measurements before and after 7-day pyridoxine loading per os (pyridoxine hydrochloride, 300 mg/d). Under usual conditions, U-4pyridoxic acid was similar in the three groups, whereas mean S-pyridoxal 5'P was significantly lower ( p<0.0001) in the Hyperoxalurics (59.6+/-21.2 nmol/L) and in the Normooxalurics (64.9+/-19.7 nmol/L) than in the controls (86.0+/-31.0 nmol/L). No correlation could be found between U-oxalate and U-4pyridoxic acid or S-pyridoxal 5'P. After B6 loading, S-pyridoxal 5'P was still significantly lower in the Hyperoxalurics (415+/-180 nmol/L, p<0.001) and in the Normooxalurics (429+/-115 nmol/L, p=0.036) than in the controls (546+/-180 nmol/L), although there was no difference between groups for U-4pyridoxic acid. No correlation in any group could be found between changes in U-oxalate and changes in U-4pyridoxic acid or S-pyridoxal 5'P. Although there is no vitamin B6 deficiency in ICSF with or without hyperoxaluria, these patients, on average, have lower levels of S-pyridoxal 5'P than healthy subjects. However, this slight decrease does not seem to account for idiopathic hyperoxaluria.

High-calcium intake abolishes hyperoxaluria and reduces urinary crystallization during a 20-fold normal oxalate load in humans.

BACKGROUND: The aim of the study was to test whether increasing dietary calcium intake lowers intestinal oxalate absorption and thereby prevents hyperoxaluria and urinary crystallization during a 20-fold normal oxalate load in healthy subjects. METHODS: Fourteen healthy male volunteers (age 23-44 years, BMI 21.5-27.7 kg/m2) collected 24-h urines while on free-choice diet as well as on two standardized diets. The latter contained 2545 kcal, 2500 ml of mineral water, 102 g of protein, 13.6 g of sodium chloride and 2220 mg of oxalate (approximately 20-fold content of an average diet). Subjects were studied twice while on the standardized diet, once while eating a normal amount of calcium (1211 mg/day, oxalate-rich diet), and once while eating 3858 mg of calcium/day (calcium and oxalate-rich diet). RESULTS: Compared with the free-choice diet (322+/-36 micromol/d), UOx x V increased to 780+/-72 micromol/d on the oxalate-rich diet (P=0.001) and fell again to 326+/-31 micromol/d on calcium and oxalate-rich diet (P=0.001 vs oxalate-rich diet). Urinary glycolate (a metabolic precursor of Ox) always remained below the upper limit of the normal range and did not change between different diets, indicating that changes in UOX x V reflect respective variations in intestinal absorption of Ox. Uca x V was 4.60+/-0.45 mmol/d on the free-choice diet and 3.20+/-0.32 mmol/d on the oxalate-rich diet (P=0.011 vs free-choice diet); it increased to 7.28+/-0.74 mmol/d on the calcium- and oxalate-rich diet (P=0.001 vs free-choice and oxalate-rich diets). As indicated by the AP (CaOx) index (Tiselius), urinary supersaturation did not vary significantly between the three diets. In freshly voided morning urines (studied in 8/14 subjects) on the oxalate-rich diet, CaOx crystals or crystal aggregates of up to 80 microm diameter were found in 5/8 urines, whereas this never occurred on the free-choice diet and only t once on the calcium- and oxalate-rich diet. CONCLUSION: . Increasing calcium intake while eating Ox-rich food prevents dietary hyperoxaluria and reduces CaOx crystallization in healthy subjects. This further illustrates that dietary counseling to idiopathic calcium-stone formers should ensure sufficient calcium intake, especially during oxalate-rich meals.

Plasma calcium-oxalate saturation in children with renal insufficiency and in children with primary hyperoxaluria.

BACKGROUND: Calcium-oxalate (CaOx) deposition and systemic oxalosis are uncommon in children with chronic renal failure (CRI), but frequent in children with primary hyperoxaluria type I (PH-1). We hypothesized a difference in plasma CaOx saturation (betaCaOx) and its determining factors would explain this discrepancy. METHODS: Therefore, in addition to common biochemical measurements, plasma-oxalate (POx), citrate (PCit) and sulfate (PSulf) (plasma anions) were measured and betaCaOx was calculated in 17 PH-1 patients with normal renal function receiving pyridoxine and citrate therapy, in 54 children with CRI (SCr 0.9 to 5.9 mg/dl), and in 50 healthy children (NL). Plasma anions were analyzed by ion-chromatography and betaCaOx was calculated using a PC-based program for solution equilibria. RESULTS: Compared to NL, all plasma anion levels and betaCaOx were higher in PH-1 and CRI; POx, PCit and betaCaOx were higher in PH-1 than in CRI (P < 0.05), but PSulf was higher in CRI (P < 0.01). BetaCaOx and POx were correlated in all groups (r = 0.63 to 0.95, P < 10(-4)). POx and betaCaOx were both inversely correlated to a decrease in GFR in CRI patients. PCit and PSulf did not influence betaCaOx. Although supersaturation (betaCaOx > 1) was found in 7 CRI and in 4 PH-1 patients, eye examinations were suspicious for CaOx depositions only in the PH-1 patients, while systemic oxalosis was confirmed in one PH patient because of oxalate osteopathy. CONCLUSIONS: In PH-1, POx and betaCaOx are elevated even with normal renal function, which increases the likelihood of CaOx crystal deposition. Therefore, more effective therapy to decrease betaCaOx is crucial to reduce the risk of systemic oxalosis. In children with CRI unknown, but presumably protective substances, help prevent the risk of systemic oxalosis, despite increased POx and betaCaOx levels, often to supersaturation levels.

Primary hyperoxaluria in an adult with renal failure, livedo reticularis, retinopathy, and peripheral neuropathy.

We present the case of a young woman who developed renal failure of unknown cause, and after 2 months of maintenance hemodialysis developed livedo reticularis, retinopathy, and peripheral sensory neuropathy. The patient was subsequently shown to have primary oxalosis type I, a rare autosomal recessive error of metabolism characterized by accumulation of calcium oxalate crystals in the kidneys, eyes, skin, and other organs. Intravascular obstruction, caused by deposition of calcium oxalate crystals in cutaneous arterioles, is thought to be responsible for the ischemic livedo reticularis lesions observed in this patient. A method is described for measuring serum glycolate by isotope dilution gas chromatography-mass spectrometry (GC-MS). An approach to the diagnosis and management is also briefly mentioned.

Vitamin B6 deficiency and galactose induced alterations in morphology and osmotic fragility of rat erythrocytes.

Recently, red blood cells have been investigated mainly for alterations in ion transporting capacity, membrane bound enzymes or modifications in the structure of its individual constituents in clinical and experimental urolithiasis. However, the implication of such modifications on the physical state or morphology of cells has not been investigated. Scanning electron microscopic studies performed in vitamin B6 deficient and/or galactose fed rat (established hyperoxaluric models) erythrocytes, showed the presence of large number of stomatocytes, spherocytes and other variously deformed cells as compared to discocytic cells seen in normal control group. These changes in shape were in concurrence with red cell osmotic fragility, which decreased both in vitamin B6 deficient and vitamin B6 deficient + galactose fed group (19 % and 33 % hemolysis at 4 g/l NaCl, respectively) while it increased in galactose control group (73 % hemolysis at 4 g/l NaCl) as compared to normal control group (55 % hemolysis at 4 g/l NaCl). These morphological and physical state alterations could be correlated with red blood cells' membrane cholesterol and phospholipid sub-class distribution. These findings suggest that some structural membrane changes occur due to vitamin B6 deficiency and/or galactose feeding, which may be responsible for the altered membrane functions known to be associated with pathogenesis of urolithiasis.

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.

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.