A mutation creating an out-of-frame alternative translation initiation site in the GRHPR 5'UTR causing primary hyperoxaluria type II.

Primary hyperoxaluria type II is a recessive genetic disorder caused by mutations in the GRHPR gene. Although several dozen mutations have been described, all affect coding or transcript splicing. A man suspected of having primary hyperoxaluria type II was heterozygous for a novel single-nucleotide deletion (c.694delC) in GRHPR affecting Gln(232) , which introduced a pre-mature termination (p.Gln232Argfs*3). Two 5'untranslated region (UTR) variants of unknown significance were also noted. We show that these two variants occur in cis, on the opposite allele, and introduce - immediately upstream of the canonical translation initiation site - a novel out-of-frame translational start site. In vitro studies using the GRHPR 5'UTR fused to a luciferase reporter show that the variant start site pre-empted initiation at the canonical translational start site, and this was corroborated within the broader context of 1.3 kb of the GRHPR proximal promoter. This latter mechanism may be underappreciated in general; reports of clinically significant functional variation of this type are extremely rare.

Metabolism of fructose to oxalate and glycolate.

Much attention has been recently directed at fructose consumption because of its association with obesity and subsequent development of chronic diseases. It was recently reported that an increased fructose intake increases the risk of forming kidney stones. It was postulated that fructose consumption may increase urinary oxalate, a risk factor for calcium oxalate kidney stone disease. However, conflicting results have been obtained in human studies examining the relationship between fructose metabolism and oxalate synthesis. To test whether fructose intake influences urinary excretions impacting kidney stone risk, healthy subjects consumed diets controlled in their contents of fructose, oxalate, calcium, and other nutrients. Subjects consumed diets containing 4, 13, and 21% of calories as fructose in a randomized order. No changes in the excretions of oxalate, calcium, and uric acid were observed. In vitro investigations with cultured liver cells incubated with (13)C-labeled sugars indicated that neither fructose nor glucose was converted to oxalate by these cells. Fructose metabolism accounted for 12.4 ± 1.6% of the glycolate detected in the culture medium and glucose 6.4 ± 0.9%. Our results suggest that mechanisms for stone risk associated with fructose intake may lie in factors other than those affecting the major stone risk parameters in urine.

The absence of glyoxylate cycle enzymes in rodent and embryonic chick liver.

There have been several reports over the past decade of the presence of the glyoxylate cycle enzymes, isocitrate lyase and malate synthase, in animal tissues. Reaction products in these assays have been measured principally by chromatographic separation of isotopes or by colorimetric procedures. In this report more sensitive and accurate HPLC and HPCE analyses were used to detect enzymatic activity. Reversed phase HPLC revealed the absence of detectable isocitrate lyase activity in guinea pig, rat and chick embryonic liver. The formation of several other alpha-keto acids was detected and this may account for the previously reported activities. Using HPCE to monitor malate formation malate synthase activity was not detected in these tissues. These results indicate that when assaying enzyme activities in crude tissue homogenates specific methods for the identification of end products are required.

The isolation of coupled mitochondria from Physarum polycephalum and their response to Ca2+.

A method for the isolation of coupled mitochondria from the acellular slime mould Physarum polycephalum is described. The mitochondria oxidize respiratory substrates at rates comparable to those of mitochondria from other microorganisms and show similar responses to respiratory inhibitors. ADP/O values approach similar values to those obtained with mitochondria from higher organisms: 3 with NAD-linked substrates, 2 with succinate, and 1 with ascorbate-TMPD. Mitochondria actively take up low concentrations of Ca2+ with stimulation of their respiration. With succinate or pyruvate-malate as substrates respiratory responses are depressed by Ca2+ concentrations in excess of 200 micron in the presence or absence of phosphate. Exogenous NADH is unique in supporting the uptake of large amounts of Ca2+ in the presence of phosphate and in showing an unusual 'uncoupled' response in the absence of phosphate. A sigmoidal relationship occurs between initial velocity of Ca2+ uptake and Ca2+ concentration with a maximum velocity of approx. 15 nmol/s per mg protein and half maximum velocity occurring at approx. 50 micron Ca2+.

25-Hydroxysterols increase the permeability of liposomes to Ca2+ and other cations.

25-Hydroxycholesterol and 25-hydroxy vitamin D-3 increased the permeability of liposomes to Ca2+ measured by the arsenazo III encapsulation technique. This effect was sensitive to the lipid composition of the membrane, with changes that decreased the motional freedom of phospholipid acyl chains decreasing Ca2+ permeability. The greatest permeability was observed with the zwitter-ionic phospholipids, phosphatidylcholine and phosphatidylethanolamine, whereas the acidic phospholipids, phosphatidylinositol and phosphatidylserine, depressed Ca2+ permeability. The effect was not specific for Ca2+. Other divalent cations were translocated in the order Mn2+ greater than Mg2+ = Ca2+ much greater than Sr2+ = Ba2+. The permeability of liposomes to the monovalent cation, Na+, was also substantially increased. The effect did not appear to be due to ionophoretic properties of the sterols, and it is suggested that perturbation of the membranes by the polar 25-hydroxyl group may play a role in increasing membrane permeability.

The saturation and isomerization of dietary fatty acids and the respiratory properties of rat heart mitochondria.

Weanling rats were fed semi-purified diets containing 15% by weight of either corn oil, a high oleic acid safflower oil, lard or hydrogenated soybean oil. Significant changes in the fatty acid composition of heart mitochondrial preparations were induced by these dietary fats. Despite these changes in membrane composition, no effects on the respiratory properties of the mitochondria were observed. These results suggest that mitochondrial membranes adapt to changes in dietary fatty acids in a way which prevents changes in their functional properties.

The effect of the saturation and isomerization of dietary fatty acids on the osmotic fragility and water diffusional permeability of rat erythrocytes.

Weanling rats were fed semi-purified diets containing 15% by weight of either corn oil, a high oleic acid safflower oil, lard or hydrogenated soybean oil. Significant changes in the fatty acid composition of erythrocytes were induced by these dietary fats. The compositional changes did not effect water diffusional permeability, but did affect their osmotic fragility. An increased fragility appeared to be associated with an increased octadecenoate content of the membranes.

Failure of leukotriene B4 to translocate calcium across phosphatidylcholine membranes.

It has been reported that leukotriene B4 can translocate calcium across model membranes (Serhan et. al., (1982) J. Biol. Chem., 257: 4746). Such ionophoretic behavior could account for its biological effects. We have examined the effect of chromatographically pure leukotriene B4 on Ca2+ permeability when added exogenously at 3 microM to phosphatidylcholine liposomes and when incorporated at 5 mole % in the lipid mixture used to prepare liposomes. No effect was observed with either procedure. An oxidized preparation of leukotriene B4 stimulated calcium permeability, however, suggesting that oxidation may account for the previously reported ionophoretic behavior of leukotriene B4.

The influence of dietary isomeric and saturated fatty acids on atherosclerosis and eicosanoid synthesis in swine.

Weanling swine were fed for 6 months high fat diets containing as fat source, a high oleic acid safflower oil, lard, or a partially hydrogenated soybean oil blended with soybean oil. The extent of atherosclerosis in left coronary arteries and the ability of vascular components to synthesize eicosanoids important for blood clotting were determined. There was no significant difference (p greater than 0.05) in the extent of atherosclerosis or the synthesis of thromboxane A2. Significant effects were observed on serum cholesterol, which was elevated in the lard fed group, serum triacylglycerol, which was highest in the safflower oil group, and prostacyclin synthesis, which was depressed in both the lard and hydrogenated soybean oil diets compared to the safflower oil diet. No unique effect on the development of heart disease appears to be attributable to hydrogenated fats. The hydrogenated fat was similar to lard in decreasing prostacyclin synthesis, suggesting that the saturation of dietary fatty acids may be a contributory factor in the development of heart disease, through its effect on thrombotic processes.

Severe oligohydramnios induced by cyclooxygenase-2 inhibitor nimesulide.

BACKGROUND: Cyclooxygenase-2 inhibitors might have fewer adverse fetal effects than conventional nonsteroidal anti-inflammatory drugs that inhibit both isoforms of the enzyme. Although cyclooxygenase-2 is expressed in fetal kidneys, there are no reports of adverse effects in human pregnancy. CASE: A 27-year-old woman, gravida 2, para 0, with a twin pregnancy at 24 weeks' gestation had placement of a cervical cerclage. Nimesulide was prescribed for postoperative preterm labor prophylaxis. Three weeks later, severe oligohydramnios was identified in both sacs, despite normal growth, renal anatomy, and umbilical artery and renal artery Doppler flow velocimetry. After stopping the drug, amniotic fluid volumes returned to normal over 2 weeks. There were no adverse neonatal renal effects. CONCLUSION: Selective cyclooxygenase-2 inhibition might cause severe oligohydramnios. If it is used, we advise close fetal surveillance.

Role of diet in the therapy of urolithiasis.

The data reviewed in this paper indicate that there is compelling direct and indirect evidence that certain dietary modifications can limit the risk for stone formation. Fluid therapy should be a front-line approach for all stone formers, because it is safe, cheap, and effective. Restricting sodium and animal-protein consumption produces changes in the urinary environment that should benefit the majority of stone formers, including a decrease in calcium and increase in citrate excretion. Minimizing the intake of processed goods limits sodium gluttony. These dietary modifications also reduce cardiovascular risks. Indiscriminant calcium restriction should be avoided, because it could accelerate stone formation and violate skeletal integrity. Oxalate restriction should be considered for calcium oxalate stone formers, especially those with hyperoxaluria. Specific recommendations for modifying the consumption of other nutrients cannot be made at this time because of the limited available information about the resultant effects. The aforementioned goals can be achieved within the context of a nutritionally balanced diet providing adequate sources of fruits and vegetables. There is a definite need for better designed studies of the nutritional effects on stone disease. This would promote a better understanding of the interplay between the genetic and environmental components of this disorder.

The taurine and hypotaurine content of human semen.

Taurine and hypotaurine levels were measured in human sperm and seminal fluid. Sperm taurine ranged from 17 nmol/mg DNA to 348 nmol/mg DNA, and hypotaurine from 0 nmol/mg DNA to 251 nmol/mg DNA. Seminal fluid contained 319 mumol/L to 1590 mumol/L of taurine, but no detectable hypotaurine. The coefficient of variation in multiple ejaculates from a single man for these components ranged from 12% for hypotaurine to 24% for seminal fluid taurine, indicating a relative constancy in their concentrations. Sperm hypotaurine content was significantly correlated with sperm morphology, sperm relative forward progression, the percentage of motile sperm, and the total number of sperm in the ejaculate. By contrast, sperm taurine content was negatively correlated with these parameters. The mean hypotaurine content of sperm from 8 fertile men was 149 +/- 92 nmol/mg DNA, four times higher than that of sperm from 9 infertile men, which was 35 +/- 19 nmol/mg DNA (P = 0.011). In contrast, the mean sperm taurine content of the fertile men was lower than that of the infertile men (83 +/- 33 nmol/mg DNA versus 168 +/- 119 nmol/mg DNA, respectively; P = 0.07). Seminal fluid taurine concentrations, however, were similar for both groups. Hypotaurine, an antioxidant, may play an important role in protecting sperm from reactive oxygen species. Higher concentrations of taurine in the sperm of infertile men suggest that accelerated oxidation of hypotaurine to taurine may accompany the observed decline in other sperm parameters.

A lack of coordination in the release of urinary lysosomal and brush border enzymes following renovascular surgery.

The urinary secretion of two lysosomal enzymes, N-acetyl-D-glucosaminidase (NAG, EC 3.2.1.30) and beta-glucuronidase (GLR, EC 3.2.1.31), and two brush border enzymes, alanine aminopeptidase (AAP, EC 3.4.11.2) and gamma-glutamyltransferase (GGT, EC 2.3.2.2), was examined in apparently healthy individuals and in patients before and after renovascular surgery for treatment of hypertension. Eight out of nine patients had elevated levels of at least one enzyme before surgery. The ranking in their frequency of elevation was NAG greater than AAP greater than GLR greater than GGT. In comparing the release of any two enzymes in apparently healthy individuals, the release was coordinated except for GGT and GLR. In individual patients following surgery the excretion of the lysosomal enzymes was highly coordinated whereas the release of the brush border enzymes was less coordinated. Comparisons of lysosomal to brush border enzyme activities revealed dissimilar release patterns between these two classes of enzymes. Analysis of variance over the entire hospitalization period showed that NAG/GLR (p = 0.42) and AAP/GGT (p = 0.12) did not vary significantly whereas all comparisons of lysosomal to brush border enzymes varied significantly (p less than or equal to 0.03). These results indicate that enzymes derived from different subcellular organelles, lysosomes or brush borders, have similar release patterns. However, the lack of a significant correlation between lysosomal and brush border enzyme excretion implies that the two processes are not interdependent. These studies further suggest that the transient pathophysiological changes that occur within renal cells following renovascular surgery affect these cellular components in different ways.

Pharmacological approaches in the treatment of primary hyperoxaluria.

Drug therapy receives scant attention as a treatment mode for primary hyperoxaluria (PH). Currently, pyridoxine is the only drug in the arsenal and only a minority of PH1 patients respond to it. In this report a pathway describing the synthesis of glyoxylate, the major precursor of oxalate, is proposed and potential drugs that may be effective in inhibiting hepatic oxalate synthesis are discussed. One of these, (L)-oxothiazolidine-4-carboxylate (OTZ), is currently undergoing evaluation in a Phase II clinical trial. It is suggested that an ideal drug may be an antisense oligonucleotide that blocks the expression of glycolate oxidase, a key enzyme in hepatic oxalate synthesis.

Maternal insulin-like growth factor binding protein-1, body mass index, and fetal growth.

AIM: To examine the hypothesis that the maternal insulin-like growth factor system may constrain fetal growth. METHODS: A prospective observational study of maternal serum insulin-like growth factor binding protein-1 (IGFBP-1) and fetal growth was undertaken in neonates with birthweights below the 5th centile. They had been classified either as having fetal growth restriction (FGR) due to placental dysfunction (increased umbilical artery Doppler pulsatility index (PI); n = 25) or as being small for gestational age (SGA; normal umbilical artery PI, growth velocity and amniotic fluid; n = 27). Eighty nine controls had normal birthweights (5th-95th centile), umbilical artery PI, growth velocity, and amniotic fluid. IGFBP-1 was measured by radioimmunoassay. RESULTS: Among the controls, there was no significant correlation between IGFBP-1 and birthweight after allowing for body mass index (BMI). Maternal BMI was high in FGR and after adjusting for this, IGFBP-1 was increased (109 ng/ml) compared with SGA babies (69 ng/ml) and controls (57 ng/ml) and correlated with the umbilical artery PI. CONCLUSIONS: Maternal IGFBP-1 is probably not part of normal placental function. Its increase in FGR could be the cause or consequence of impaired placental perfusion, but high IGFBP-1 concentrations might further reduce the availability of maternal IGF-I to the placenta. This could worsen placental function and so adversely affect fetal growth.

Urinary oxalate and citrate.

The amounts of oxalate and citrate excreted in urine, and their urinary concentrations are important risk factors for the development of calcium oxalate kidney stones (1). The most widely used procedures to estimate these analytes are enzyme-based procedures using commercially available reagent kits (2,3). Components in the urine matrix may interfere with these assays, and some sample cleanup is required to remove them for oxalate analysis. Ion chromatography, although well suited to these determinations (4,5), is less widely used presumably because of long assay times, the need for expensive equipment, and the maintenance costs associated with the procedure. Capillary electrophoresis enables the rapid determination of oxalate and citrate in the same run, as well as the simultaneous measurement of chloride and sulfate. Estimation of these anions is useful for the calculation of relative supersaturations of urine with calcium oxalate and calcium phosphate. The method developed here utilizes indirect absorption to detect anions (6), and relies on the change in absorption observed when oxalate and citrate migrate through the detection window, and displace chromate in the electrolyte. It is important that the chromophoric electrolyte chosen has a migration time similar to that of the anions of interest. Pyromellitic acid is another suitable chromophore that can be used as the electrolyte (7).

Guaifenesin- and ephedrine-induced stones.

PURPOSE: We report a new type of drug-induced stone that is caused by overconsumption of preparations containing guaifenesin and ephedrine. MATERIALS AND METHODS: Clinical and stone analysis data from the Molecular Structure Laboratory at the Veterans Affairs Medical Center in Milwaukee, Wisconsin, were reviewed. Stone analysis was performed by Fourier transform infrared spectroscopy, high-resolution X-ray crystallographic powder diffraction, or both. The urine and stone material from one of the subjects were analyzed with high-performance liquid chromatography. RESULTS: Stone analysis from seven patients demonstrated metabolites of guaifenesin. High-performance liquid chromatography revealed that the stone and urine from one subject had a high content of guaifenesin metabolites and a small amount of ephedrine. Demographic data were available on five patients. Three had a history of alcohol or drug dependency. All were consuming over-the-counter preparations containing ephedrine and guaifenesin. Four admitted to taking excessive quantities of these agents, mainly as a stimulant. Hypocitraturia was identified in two individuals subjected to urinary metabolic testing. These stones are radiolucent on standard X-ray imaging but can be demonstrated on unenhanced CT. Shockwave lithotripsy was performed in two patients, and the calculi fragmented easily. CONCLUSIONS: Individuals consuming large quantities of preparations containing ephedrine and guaifenesin may be at risk to develop stones derived mainly from metabolites of guaifenesin and small quantities of ephedrine. These patients may be prone to drug or alcohol dependency.