Biomedical subjects

Ross P Holmes

Publications and source records attributed to Ross P Holmes.

7 recordsLinked to original sources

PubMed2006

Glyoxylate reductase activity in blood mononuclear cells and the diagnosis of primary hyperoxaluria type 2.

BACKGROUND: Primary hyperoxaluria type 2 (PH2) is a rare monogenic disorder characterized by an elevated urinary excretion of oxalate. Increased oxalate excretion in PH2 patients can cause nephrolithiasis and nephrocalcinosis, and can, in some cases, result in renal failure and systemic oxalate deposition. The disease is due to a deficiency of glyoxylate reductase/hydroxypyruvate reductase (GRHPR) activity. A definitive diagnosis of PH2 is currently made by the analysis of GR activity in a liver biopsy. GRHPR is expressed in virtually every tissue in the body, suggesting that utilization of more readily available cells could be used to determine GRHPR deficiency. In this study, we have evaluated the potential of determining GR and d-glycerate dehydrogenase (DGDH) activity in blood mononuclear cells (BMC) as a diagnostic indicator of PH2. METHODS: Blood samples were obtained from 10 male and 10 female normal subjects, median age 31, range 21-63, at the Wake Forest University Medical Center and from primary hyperoxaluria patients at the Mayo Clinic. The BMC were isolated and GR and DGDH activities measured in cell lysates. RESULTS: An assay of 20 normal individuals indicated that BMC contained a DGDH and GR activity of 0.97+/-0.20 (range 0.62-1.45), and 10.6+/-3.3 (range 8.3-16.6) nmol/min/mg protein, respectively. The intra-assay coefficient of variation for DGDH and GR activity was 8.2 and 11.5%, respectively. The BMC lysates from normal adult subjects and patients with PH1 showed similar GR and DGDH activities. This was confirmed by the presence of immunoreactive GRHPR protein by western blot analysis. In contrast, PH2 BMC lysates did not exhibit DGDH or GR activity, and showed no immunoreactive GRHPR by western blot analysis. CONCLUSION: These results suggest that the assay of DGDH or GR activity in BMC could be used as a minimally invasive diagnostic test for PH2.

Adult↗

PubMed2005

Mitochondrial hydroxyproline metabolism: implications for primary hyperoxaluria.

BACKGROUND/AIMS: Primary hyperoxaluria results from an alteration in enzymes that metabolize glyoxylate. The metabolism that leads to glyoxylate synthesis is not well defined. The aim of this study was to investigate the production of glyoxylate in liver mitochondria when they metabolize hydroxyproline. METHODS: Mitochondria were isolated from mouse liver using Percoll gradient centrifugation. The metabolism of hydroxyproline was examined by a combination of HPLC and ion chromatography/mass spectrometry techniques. RESULTS: Glyoxylate production was substantially greater when mitochondria were incubated with hydroxyproline in comparison with proline. Inclusion of malate and glutamate with hydroxyproline resulted in a drop in glyoxylate and an increase in glycolate in the incubation mixture. This suggests an increased NAD(P)+ reduction which occurred with the inclusion of glutamate/malate and that the NAD(P)H production was required to stimulate the glyoxylate reductase-catalyzed conversion of glyoxylate to glycolate. The presence of glyoxylate reductase in these mitochondria was confirmed by measuring enzymatic activity and by Western blotting. CONCLUSION: These results indicate that studies on isolated mitochondria have the potential to help unravel the metabolism associated with glyoxylate and oxalate production and understand the metabolic function of glyoxylate reductase.

Animals↗

PubMed2005

Genetic and dietary factors in urinary citrate excretion.

BACKGROUND AND PURPOSE: Hypocitraturia, an important risk factor for calcium oxalate nephrolithiasis, is the result of numerous factors. We studied citrate excretion by patients with and without stones consuming normal and controlled formula diets. SUBJECTS AND METHODS: Subjects with and without a history of calcium oxalate stones (N = 101 per group) provided two or three 24-hour urine specimens during consumption of self-selected diets. Data also were collected on subsets of subjects consuming formula (Ensure) diets. Citrate was determined using the citrate lyase method of Petrarulo and associates, and values for multiple specimens were averaged. The data were adjusted for creatinine excretion and examined on a per-day basis. RESULTS: The mean citrate excretion of the non-stone formers was slightly but not significantly higher than that of the stone formers (442 +/- 217 versus 378 +/- 153 mg/g of creatinine). All statistical analyses revealed highly significant differences between, but not within, individuals, a result compatible with a genetic influence. In the normal population, 5% of subjects had a citrate excretion <200 mg/g of creatinine, whereas this result was seen in 34% of the stone-forming subjects. When the subjects consumed a formula diet, women in both groups had much higher citrate excretion than when on a self-selected diet, but little difference was seen in the men. The patterns of citrate recovery suggest low, intermediate, and high excretors. In the normal population, 15% of subjects excreted <340 mg/g of creatinine, whereas this was true of 43% of the stone-forming subjects. Analysis of six families suggested three excretor phenotypes, with a codominant pattern of inheritance. CONCLUSION: These findings imply a genetic influence on citrate excretion, as has already been demonstrated for calcium excretion. Further studies of genetic influences on calcium oxalate stone formation are warranted.

Adult↗

PubMed2005

Dietary oxalate loads and renal oxalate handling.

PURPOSE: Dietary oxalate makes a significant contribution to urinary oxalate excretion and, thus, may have a role in calcium oxalate kidney stone formation. Studies have indicated that the ingestion of oxalate rich foods results in transient increases in plasma oxalate concentrations and urinary oxalate excretion. We examined changes in plasma and urinary oxalate following oral crystalline oxalate loading under controlled dietary conditions to further define the renal handling of oxalate by normal adults. MATERIALS AND METHODS: Six normal adult subjects consumed controlled diets of known oxalate content for 1 week before ingesting loads of 0, 2, 4 and 8 mmol of oxalate. Urinary and plasma changes were measured to assess renal oxalate handling. Urinary excretion of proximal tubule derived enzymes and isoprostanes was monitored to assess for renal injury and oxidative stress. RESULTS: Time and dose dependent changes in plasma oxalate, urinary oxalate and in the clearance ratio of oxalate-to-creatinine were observed. A significant correlation (r=0.43, p <0.001) between the oxalate-to-creatinine clearance ratio and plasma oxalate levels was identified. No changes in urinary markers of oxidative stress or renal injury were observed following the 8 mmol oxalate load. CONCLUSIONS: Oxalate is rapidly absorbed and cleared by the kidney by filtration and secretion following an oral oxalate load. Renal oxalate secretion has a significant role in the renal handling of an oral oxalate load. There is no evidence of acute renal injury or oxidative stress with oral oxalate loads in these experimental conditions.

Adult↗

PubMed2004

Glycolate and glyoxylate metabolism in HepG2 cells.

Oxalate synthesis in human hepatocytes is not well defined despite the clinical significance of its overproduction in diseases such as the primary hyperoxalurias. To further define these steps, the metabolism to oxalate of the oxalate precursors glycolate and glyoxylate and the possible pathways involved were examined in HepG2 cells. These cells were found to contain oxalate, glyoxylate, and glycolate as intracellular metabolites and to excrete oxalate and glycolate into the medium. Glycolate was taken up more effectively by cells than glyoxylate, but glyoxylate was more efficiently converted to oxalate. Oxalate was formed from exogenous glycolate only when cells were exposed to high concentrations. Peroxisomes in HepG2 cells, in contrast to those in human hepatocytes, were not involved in glycolate metabolism. Incubations with purified lactate dehydrogenase suggested that this enzyme was responsible for the metabolism of glycolate to oxalate in HepG2 cells. The formation of 14C-labeled glycine from 14C-labeled glycolate was observed only when cell membranes were permeabilized with Triton X-100. These results imply that peroxisome permeability to glycolate is restricted in these cells. Mitochondria, which produce glyoxylate from hydroxyproline metabolism, contained both alanine:glyoxylate aminotransferase (AGT)2 and glyoxylate reductase activities, which can convert glyoxylate to glycine and glycolate, respectively. Expression of AGT2 mRNA in HepG2 cells was confirmed by RT-PCR. These results indicate that HepG2 cells will be useful in clarifying the nonperoxisomal metabolism associated with oxalate synthesis in human hepatocytes.

Alcohol Oxidoreductases↗

PubMed2004

The impact of dietary oxalate on kidney stone formation.

The role of dietary oxalate in calcium oxalate kidney stone formation remains unclear. However, due to the risk for stone disease that is associated with a low calcium intake, dietary oxalate is believed to be an important contributing factor. In this review, we have examined the available evidence related to the ingestion of dietary oxalate, its intestinal absorption, and its handling by the kidney. The only difference identified to date between normal individuals and those who form stones is in the intestinal absorption of oxalate. Differences in dietary oxalate intake and in renal oxalate excretion are two other parameters that are likely to receive close scrutiny in the near future, because the research tools required for these investigations are now available. Such research, together with more extensive examinations of intestinal oxalate absorption, should help clarify the role of dietary oxalate in stone formation.

Animals↗

PubMed2002

Oxalobacter formigenes and its potential role in human health.

Oxalate degradation by the anaerobic bacterium Oxalobacter formigenes is important for human health, helping to prevent hyperoxaluria and disorders such as the development of kidney stones. Oxalate-degrading activity cannot be detected in the gut flora of some individuals, possibly because Oxalobacter is susceptible to commonly used antimicrobials. Here, clarithromycin, doxycycline, and some other antibiotics inhibited oxalate degradation by two human strains of O. formigenes. These strains varied in their response to gut environmental factors, including exposure to gastric acidity and bile salts. O. formigenes strains established oxalate breakdown in fermentors which were preinoculated with fecal bacteria from individuals lacking oxalate-degrading activity. Reducing the concentration of oxalate in the medium reduced the numbers of O. formigenes bacteria. Oxalate degradation was established and maintained at dilution rates comparable to colonic transit times in healthy individuals. A single oral ingestion of O. formigenes by adult volunteers was, for the first time, shown to result in (i) reduced urinary oxalate excretion following administration of an oxalate load, (ii) the recovery of oxalate-degrading activity in feces, and (iii) prolonged retention of colonization.

Anaerobiosis↗