Diagnostic dilemma of patients with methylmalonic aciduria: Experience from a tertiary care centre in Pakistan.

OBJECTIVE: To determine the frequency of disorders leading to methylmalonic acidurias. METHODS: This cross-sectional study was conducted from January 2013 to April 2016 at the Aga Khan University Hospital, Karachi, and comprised patients diagnosed with methylmalonic acidurias based on urine organic acid analysis. Clinical history and biochemical data was collected from the biochemical genetics laboratory requisition forms. Organic acid chromatograms of all the subjects were critically reviewed by a biochemical pathologist and a metabolic physician. For assessing the clinical outcome, medical charts of the patients were reviewed. SPSS 19 was used for data analysis. RESULTS: Of the 1,778 patients 50(2.81%) were detected with methylmalonic acidurias. After excluding patients with non-significant peaks of methylmalonic acidemia, 41(2.31%) were included in the final analysis. Of these, 20(48.7%) were females, while the overall median age was 11.5 months (interquartile range: 6-41.5). On stratification by type of disorders leading to methylmalonic acidurias, 9(22%) had methylmalonic acidemia, 12(29%) had Cobalamin-related remethylation disorders, nonspecific methylmalonic acidurias in 16(39%), while 2(5%) each had succinyl coenzyme A synthetase and Vitamin B12 deficiency. respectively. CONCLUSIONS: Screening tests, including urine organic acid, provided valuable clues to the aetiology of methylmalonic acidurias.

Severe child form of primary hyperoxaluria type 2 - a case report revealing consequence of GRHPR deficiency on metabolism.

BACKGROUND: Primary hyperoxaluria type 2 is a rare monogenic disorder inherited in an autosomal recessive pattern. It results from the absence of the enzyme glyoxylate reductase/hydroxypyruvate reductase (GRHPR). As a consequence of deficient enzyme activity, excessive amounts of oxalate and L-glycerate are excreted in the urine, and are a source for the formation of calcium oxalate stones that result in recurrent nephrolithiasis and less frequently nephrocalcinosis. CASE PRESENTATION: We report a case of a 10-month-old patient diagnosed with urolithiasis. Screening of inborn errors of metabolism, including the performance of GC/MS urine organic acid profiling and HPLC amino acid profiling, showed abnormalities, which suggested deficiency of GRHPR enzyme. Additional metabolic disturbances observed in the patient led us to seek other genetic determinants and the elucidation of these findings. Besides the elevated excretion of 3-OH-butyrate, adipic acid, which are typical marks of ketosis, other metabolites such as 3-aminoisobutyric acid, 3-hydroxyisobutyric acid, 3-hydroxypropionic acid and 2-ethyl-3-hydroxypropionic acids were observed in increased amounts in the urine. Direct sequencing of the GRHPR gene revealed novel mutation, described for the first time in this article c.454dup (p.Thr152Asnfs*39) in homozygous form. The frequent nucleotide variants were found in AGXT2 gene. CONCLUSIONS: The study presents metabolomic and molecular-genetic findings in a patient with PH2. Mutation analysis broadens the allelic spectrum of the GRHPR gene to include a novel c.454dup mutation that causes the truncation of the GRHPR protein and loss of its two functional domains. We also evaluated whether nucleotide variants in the AGXT2 gene could influence the biochemical profile in PH2 and the overproduction of metabolites, especially in ketosis. We suppose that some metabolomic changes might be explained by the inhibition of the MMSADH enzyme by metabolites that increase as a consequence of GRHPR and AGXT2 enzyme deficiency. Several facts support an assumption that catabolic conditions in our patient could worsen the degree of hyperoxaluria and glyceric aciduria as a consequence of the elevated production of free amino acids and their intermediary products.

Metabolites of milk intake: a metabolomic approach in UK twins with findings replicated in two European cohorts.

PURPOSE: Milk provides a significant source of calcium, protein, vitamins and other minerals to Western populations throughout life. Due to its widespread use, the metabolic and health impact of milk consumption warrants further investigation and biomarkers would aid epidemiological studies. METHODS: Milk intake assessed by a validated food frequency questionnaire was analyzed against fasting blood metabolomic profiles from two metabolomic platforms in females from the TwinsUK cohort (n = 3559). The top metabolites were then replicated in two independent populations (EGCUT, n = 1109 and KORA, n = 1593), and the results from all cohorts were meta-analyzed. RESULTS: Four metabolites were significantly associated with milk intake in the TwinsUK cohort after adjustment for multiple testing (P < 8.08 × 10-5) and covariates (BMI, age, batch effects, family relatedness and dietary covariates) and replicated in the independent cohorts. Among the metabolites identified, the carnitine metabolite trimethyl-N-aminovalerate (ß = 0.012, SE = 0.002, P = 2.98 × 10-12) and the nucleotide uridine (ß = 0.004, SE = 0.001, P = 9.86 × 10-6) were the strongest novel predictive biomarkers from the non-targeted platform. Notably, the association between trimethyl-N-aminovalerate and milk intake was significant in a group of MZ twins discordant for milk intake (ß = 0.050, SE = 0.015, P = 7.53 × 10-4) and validated in the urine of 236 UK twins (ß = 0.091, SE = 0.032, P = 0.004). Two metabolites from the targeted platform, hydroxysphingomyelin C14:1 (ß = 0.034, SE = 0.005, P = 9.75 × 10-14) and diacylphosphatidylcholine C28:1 (ß = 0.034, SE = 0.004, P = 4.53 × 10-16), were also replicated. CONCLUSIONS: We identified and replicated in independent populations four novel biomarkers of milk intake: trimethyl-N-aminovalerate, uridine, hydroxysphingomyelin C14:1 and diacylphosphatidylcholine C28:1. Together, these metabolites have potential to objectively examine and refine milk-disease associations.

A (14)C-leucine absorption, distribution, metabolism and excretion (ADME) study in adult Sprague-Dawley rat reveals ß-hydroxy-ß-methylbutyrate as a metabolite.

Leucine is an essential branched-chain amino acid that acts as a substrate for protein synthesis and as a signaling molecule. Leucine not incorporated into muscle protein is ultimately oxidized through intermediates such as ß-hydroxy-ß-methylbutyrate (HMB) which itself is reported to enhance muscle mass and function in rats and humans. HMB has been reported in the plasma following oral leucine administration in sheep and pigs but not in Sprague-Dawley rats, the standard preclinical model. Therefore, we conducted radiolabeled absorption, distribution, metabolism and excretion (ADME) studies in rats using a low (3 mg/kg) or high dose (1,000 mg/kg) of (14)C-leucine. Blood, tissue, and urine samples were analyzed for (14)C-leucine and its metabolites by HPLC-MS. Our results show for the first time that (14)C-HMB appears in plasma and urine of rats following an oral dose of (14)C-leucine. (14)C-leucine appears in plasma as (14)C-α-ketoisocaproic acid (KIC) with a slower time course than (14)C-HMB, a putative product of KIC. Further, two novel metabolites of leucine were detected in urine, N-acetyl leucine and glycyl leucine. Mass balance studies demonstrate that excretory routes accounted for no more than 0.9 % of the radiolabel and approximately 61 % of the dose was recovered in the carcass. Approximately 65 % of the dose was recovered in total, suggesting that approximately one-third of the leucine dose is oxidized to CO2. In conclusion, this study demonstrates endogenous production of HMB from leucine in adult rats, a standard preclinical model used to guide design of clinical trials in nutrition.

Biotin-deficient diet induces chromosome misalignment and spindle defects in mouse oocytes.

Increased abnormal oocytes due to meiotic chromosome misalignment and spindle defects lead to elevated rates of infertility, miscarriage, and trisomic conceptions. Here, we investigated the effect of biotin deficiency on oocyte quality. Three-week-old female ICR mice were fed a biotin-deficient or control diet (0, 0.004 g biotin/kg diet) for 21 days. On day 22, these mouse oocytes were analyzed by immunofluorescence. Due to biotin, undernutrition increased the frequency of abnormal oocytes (the biotin deficient vs. control: 40 vs. 16%). Next, the remaining mice in the biotin-deficient group were fed a control or biotin-deficient diet from day 22 to 42. Although biotin nutritional status in the recovery group was restored, the frequency of abnormal oocytes in the recovery group was still higher than that in the control group (48 vs. 18%). Our results indicate that steady, sufficient biotin intake is required for the production of high-quality oocytes in mice.

Pregnancy and lactation alter biomarkers of biotin metabolism in women consuming a controlled diet.

BACKGROUND: Biotin functions as a cofactor for several carboxylase enzymes with key roles in metabolism. At present, the dietary requirement for biotin is unknown and intake recommendations are provided as Adequate Intakes (AIs). The biotin AI for adults and pregnant women is 30 µg/d, whereas 35 µg/d is recommended for lactating women. However, pregnant and lactating women may require more biotin to meet the demands of these reproductive states. OBJECTIVE: The current study sought to quantify the impact of reproductive state on biotin status response to a known dietary intake of biotin. METHODS: To achieve this aim, we measured a panel of biotin biomarkers among pregnant (gestational week 27 at study entry; n = 26), lactating (postnatal week 5 at study entry; n = 28), and control (n = 21) women who participated in a 10- to 12-wk feeding study providing 57 µg of dietary biotin/d as part of a mixed diet. RESULTS: Over the course of the study, pregnant women excreted 69% more (vs. control; P < 0.001) 3-hydroxyisovaleric acid (3-HIA), a metabolite that accumulates during the catabolism of leucine when the activity of biotin-dependent methylcrotonyl-coenzyme A carboxylase is impaired. Interestingly, urinary excretion of 3-hydroxyisovaleryl-carnitine (3-HIA-carnitine), a downstream metabolite of 3-HIA, was 27% lower (P = 0.05) among pregnant (vs. control) women, a finding that may arise from carnitine inadequacy during gestation. No differences (P > 0.05) were detected in plasma biotin, urinary biotin, or urinary bisnorbiotin between pregnant and control women. Lactating women excreted 76% more (vs. control; P = 0.001) of the biotin catabolite bisnorbiotin, indicating that lactation accelerates biotin turnover and loss. Notably, with respect to control women, lactating women excreted 23% less (P = 0.04) urinary 3-HIA and 26% less (P = 0.05) urinary 3-HIA-carnitine, suggesting that lactation reduces leucine catabolism and that these metabolites may not be useful indicators of biotin status during lactation. CONCLUSIONS: Overall, these data demonstrate significant alterations in markers of biotin metabolism during pregnancy and lactation and suggest that biotin intakes exceeding current recommendations are needed to meet the demands of these reproductive states. This trial was registered at clinicaltrials.gov as NCT01127022.

Urinary excretion of 3-hydroxyisovaleric acid and 3-hydroxyisovaleryl carnitine increases in response to a leucine challenge in marginally biotin-deficient humans.

Experimentally increasing metabolic flux in a pathway in which an essential step is catalyzed by a vitamin-dependent enzyme (a challenge test) has been used in assessing functional vitamin status and elucidating common and alternate metabolic pathways. Conversion of 3-methylcrotonyl CoA to 3-methylglutaconyl CoA in the leucine catabolic pathway is catalyzed by the biotin-dependent enzyme methylcrotonyl-CoA carboxylase (MCC). Marginal biotin deficiency reduces MCC activity and increases urinary excretion of 3-hydroxyisovaleric acid (3HIA) and 3-hydroxyisovaleryl carnitine (3HIA-carnitine) measured in 24-h urine collections. We assessed urinary excretion of 3HIA and 3HIA-carnitine in response to a leucine challenge in humans made progressively biotin deficient by egg white consumption. In 2 cohorts of healthy adults (Study 1: n = 5; Study 2: n = 7) rendered biotin deficient over 28 d, urinary excretion of 3HIA and 3HIA-carnitine in response to a leucine challenge was quantitated weekly for 3 or 4 wk, respectively. In both studies, mean urinary excretion of both 3HIA and 3HIA-carnitine increased >2-fold by d 14 (P < 0.002 for both indicators for both studies). Diagnostically, both indicators were highly sensitive, but diagnostic sensitivities were not superior to those of 24-h excretion of 3HIA and 3HIA-carnitine. These studies provide evidence that urinary excretions of 3HIA and 3HIA-carnitine in response to an oral leucine challenge are early and sensitive indicators of marginal biotin deficiency in humans. The variability of the proportion of leucine catabolites excreted as 3HIA suggests substantial population heterogeneity in the metabolic capacity of the 3HIA-carnitine detoxification pathway.

Free acid gel form of ß-hydroxy-ß-methylbutyrate (HMB) improves HMB clearance from plasma in human subjects compared with the calcium HMB salt.

The leucine metabolite, ß-hydroxy-ß-methylbutyrate (HMB), is a nutritional supplement that increases lean muscle and strength with exercise and in disease states. HMB is presently available as the Ca salt (CaHMB). The present study was designed to examine whether HMB in free acid gel form will improve HMB availability to tissues. Two studies were conducted and in each study four males and four females were given three treatments in a randomised, cross-over design. Treatments were CaHMB (gelatin capsule, 1 g), equivalent HMB free acid gel swallowed (FASW) and free acid gel held sublingual for 15 s then swallowed (FASL). Plasma HMB was measured for 3 h following treatment in study 1 and 24 h with urine collection in study 2. In both the studies, the times to peak plasma HMB were 128 (sem 11), 38 (sem 4) and 38 (sem 1) min (P < 0·0001) for CaHMB, FASW and FASL, respectively. The peak concentrations were 131 (sem 6), 249 (sem 14) and 239 (sem 14) µmol/l (P < 0·0001) for CaHMB, FASW and FASL, respectively. The areas under the curve were almost double for FASW and FASL (P < 0·0001). Daily urinary HMB excretion was not significantly increased resulting in more HMB retained (P < 0·003) with FASW and FASL. Half-lives were 3·17 (sem 0·22), 2·50 (sem 0·13) and 2·51 (sem 0·14) h for CaHMB, FASW and FASL, respectively (P < 0·004). Free acid gel resulted in quicker and greater plasma concentrations (+185%) and improved clearance (+25%) of HMB from plasma. In conclusion, HMB free acid gel could improve HMB availability and efficacy to tissues in health and disease.

Measurement of 3-hydroxyisovaleric acid in urine from marginally biotin-deficient humans by UPLC-MS/MS.

Measurement of 3-hydroxyisovaleric acid (3HIA) in human urine has been shown to be a useful indicator of biotin status for a variety of clinical situations, including pregnancy. The work described herein presents a novel UPLC-MS/MS method for accurate and precise quantitation of urinary 3HIA. This method utilizes sample preparation prior to quantitation that has been simplified compared to the previous GC-MS method. To demonstrate the suitability of the UPLC-MS/MS method for human bio-monitoring, this method was used to measure 3-HIA in 64 human urine samples from eight healthy adults in whom marginal biotin deficiency had been induced experimentally by egg white feeding. 3HIA was detected in all specimens; the mean concentration [±standard deviation (SD)] was 80.6 ± 51 µM prior to inducing biotin deficiency. Mean excretion rate for 3HIA (expressed per mol urinary creatinine) before beginning the biotin-deficient diet was 8.5 ± 3.2 mmol 3HIA per mol creatinine and the mean increased threefold with deficiency. These specimens had been previously analyzed by GC-MS; the two data sets showed strong linear relationship with a correlation coefficient of 0.97. These results provide evidence that this method is suitable for bio-monitoring of biotin status in larger populations.

Isovaleric and -methylbutyric acidemias induced by hypoglycin A: mechanism of Jamaican vomiting sickness.

Hypoglycin A, the causative agent of the Jamaican vomiting sickness, produced a marked increase in concentration of isovaleric acid in the plasma of rats, when administered in a single dose. alpha-Methylbutyric acid, a position isomer, also accumulated. The use of hypoglycin A reproduced some features of human isovaleric acidemia. Accumulation of these branched pentanoic acids may be another factor contributing to the pathogenesis of the Jamaican vomiting sickness.

Positive newborn screen in the biochemically normal infant of a mother with treated holocarboxylase synthetase deficiency.

Expanded programmes of newborn screening permit early diagnosis in time to prevent serious complications. These programmes have begun to detect patients who might otherwise remain asymptomatic. An additional confounding variable is the positive screen that results from maternal rather than neonatal disease. This was the case in an infant in whom elevated hydroxyisovalerylcarnitine (C(5)OH) in his newborn screen was the result of placental transfer from his mother, whose holocarboxylase synthetase deficiency was being successfully treated with biotin. The mother had been diagnosed and treated with biotin prenatally. She had no phenotypic feature of holocarboxylase synthetase deficiency, most importantly no episodes ever of acute metabolic acidosis. In the infant a repeat screen was also positive. On day 28 the infant's plasma C(5)OH carnitine was 0.05 mumol/L (normal) and urinary organic acids on day 39 were normal. The mother's excretion of 3-hydroxyisovaleric acid was 109 mmol/mol creatinine. These observations indicate that holocarboxylase synthetase deficiency is one more maternal metabolic disease which may lead to a positive screen in her unaffected newborn infant. They also make the point that holocarboxylase synthetase deficiency in an infant should be detectable in programmes of neonatal screening, which was not clear previously.

Effects of biotin deficiency on embryonic development in mice.

OBJECTIVE: The purpose of this investigation was to determine the effects of biotin deficiency on maternal metabolism and embryonic development in pregnant mouse dams. METHODS: The pregnant mice were randomly assigned to one of three dietary groups and given a biotin-deficient diet, biotin-supplemented diet, or biotin-control diet during gestation. On days of gestation (dgs) 0, 4, 8, 12, and 16, organic acids including 3-hydroxyisovaleric acid in urine were discovered by high-performance liquid chromatography, and the biotin level in the serum and urine was determined by a bioassay. On dg 18, fetuses were examined for morphologic development. RESULTS: In the biotin-deficient group, biotin excretion in urine decreased on dg 4 and was subsequently below the lower limit, whereas the urinary concentration of 3-hydroxyisovaleric acid increased after dg 12. In contrast, the biotin concentration in urine significantly increased on dgs 4, 8 and 12 in the biotin-supplemented group, but decreased on dg 16 in the biotin-supplemented and biotin-control groups. The urinary excretion of pyruvic acid in the biotin-deficient group was significantly higher than that in the biotin-supplemented group throughout the entire gestation. These concentrations in urine significantly increased on dg 16 compared with dg 0. The inhibition of embryonic development and external malformations such as cleft palate (100%), micrognathia (100%), and micromelia (91.4%) were also detected in biotin-deficient fetuses. CONCLUSION: These findings indicated that, as the requirement of biotin increases during gestation and/or embryonic development, a large amount of biotin is necessary for maintaining normal reproductive performance during the late stage of gestation.

The urinary organic acids profile in single large-scale mitochondrial DNA deletion disorders.

Single large-scale mitochondrial DNA deletions disorders are classified into three main phenotypes with frequent clinical overlap: Pearson marrow-pancreas syndrome (PMS), Kearns-Sayre syndrome (KSS) and chronic progressive external ophtalmoplegia (PEO). So far, only few anecdotal studies have reported on the urinary organic acids profile in this disease class. In this single-center retrospective study, we performed quantitative evaluation of urinary organic acids in a series of 15 pediatric patients, 7 with PMS and 8 with KSS. PMS patients showed an organic acids profile almost constantly altered, whereas KSS patients frequently presented with normal profiles. Lactate, 3-hydroxybutyrate, 3-hydroxyisobutyrate, fumarate, pyruvate, 2-hydroxybutyrate, 2-ethyl-3-hydroxypropionate, and 3-methylglutaconate represented the most frequent metabolites observed in PMS urine. We also found novel metabolites, 3-methylglutarate, tiglylglycine and 2-methyl-2,3-dihydroxybutyrate, so far never reported in this disease. Interestingly, patients with a disease onset as PMS evolving overtime into KSS phenotype, presented persistent and more pronounced alterations of organic acid signature than in patients with a pure KSS phenotype. Our study shows that the quantitative analysis of urinary organic acid profile represents a helpful tool for the diagnosis of PMS and for the differential diagnosis with other inherited diseases causing abnormal organic acidurias.

Biotin dependency due to a defect in biotin transport.

We describe a 3-year-old boy with biotin dependency not caused by biotinidase, holocarboxylase synthetase, or nutritional biotin deficiency. We sought to define the mechanism of his biotin dependency. The child became acutely encephalopathic at age 18 months. Urinary organic acids indicated deficiency of several biotin-dependent carboxylases. Symptoms improved rapidly following biotin supplementation. Serum biotinidase activity and Biotinidase gene sequence were normal. Activities of biotin-dependent carboxylases in PBMCs and cultured skin fibroblasts were normal, excluding biotin holocarboxylase synthetase deficiency. Despite extracellular biotin sufficiency, biotin withdrawal caused recurrent abnormal organic aciduria, indicating intracellular biotin deficiency. Biotin uptake rates into fresh PBMCs from the child and into his PBMCs transformed with Epstein Barr virus were about 10% of normal fresh and transformed control cells, respectively. For fresh and transformed PBMCs from his parents, biotin uptake rates were consistent with heterozygosity for an autosomal recessive genetic defect. Increased biotin breakdown was ruled out, as were artifacts of biotin supplementation and generalized defects in membrane permeability for biotin. These results provide evidence for a novel genetic defect in biotin transport. This child is the first known with this defect, which should now be included in the identified causes of biotin dependency.

Changes in measures of biotin status do not reflect milk yield responses when dairy cows are fed supplemental biotin.

A sensitive indicator of biotin status for lactating dairy cows is necessary to understand factors that affect milk yield responses to biotin supplementation. 3-Hydroxyisovaleric acid (3HIA) is an alternative metabolite in the pathway of Leu catabolism when the biotin-dependent enzyme methylcrotonyl-coenzyme A carboxylase is limiting. We evaluated urinary excretion of 3HIA as a determinant of biotin status in lactating dairy cows. We hypothesized that high-producing cows would have a greater biotin requirement and excrete more 3HIA than low-producing cows and that biotin supplementation would decrease 3HIA excretion. Twenty high-producing and 20 low-producing Holstein cows (43 +/- 5 and 23 +/- 4 kg/d of milk, respectively) were fed diets that contained either 0 or 0.96 mg/kg of supplemental biotin. On d 16 cows were given an intraruminal infusion of 1.4 mol of isovaleric acid and urine was sampled. Biotin supplementation did not affect basal urinary excretion of 3HIA. The infusion of isovaleric acid increased urinary excretion of 3HIA (maximum at 8 h after infusion), but biotin supplementation did not attenuate this increase. The increase in urinary 3HIA excretion was less for low-producing cows than for high-producing cows. Biotin increased yields of milk and milk components in high-producing cows but had no effect in low-producing cows. However, potential measures of biotin status (concentrations of avidin-binding substances in the plasma, milk, and urine, and urinary 3HIA excretion) responded similarly to biotin supplementation for both high- and low-producing cows. A sensitive indicator of biotin status for lactating dairy cows is still needed.

2-methyl butyramide, a previously identified urine biomarker for Ascaris lumbricoides, is not present in infected Indonesian individuals.

ᅟ: Previous reports suggest that the 2-methyl butyramide and 2-methyl valeramide metabolites of Ascaris lumbricoides in urine of infected individuals could be considered as urinary biomarkers for active infection. We have developed an LC-MS method with a detection limit of 10 ng/mL using synthetic chemicals as reference material. Urine samples (n = 21) of infected individuals were analyzed for the presence of these metabolites, but they were not detected in any of the samples. Furthermore, the recorded 1H-NMR spectrum for reference 2-methyl butyramide did not match with the spectrum that was described for the Ascaris metabolite. Based on these two observations, we concluded that the urinary biomarkers that were detected for A. lumbricoides infection are not 2-methyl butyramide nor 2-methylvaleramide. New discovery efforts will be required to identify the structure of these metabolite biomarkers in urine of infected individuals. TRIAL REGISTRATION: Urine samples used in this study were collected as part of a clinical trial with trial number ISRCTN75636394 (12 November 2013).

Metabolomic profiles investigation on athletes' urine 35 minutes after an 800-meter race.

BACKGROUND: The aim of this study was to identify possible biological pathways of the metabolite profile changes in athletes' urine samples before and after 800-m runs. METHODS: We used an NMR-based metabolomics analysis to evaluate the metabolite profile changes in 19 young male athletes' urine samples after 800-m runs and provide an overall picture of its impact. Various multivariate data analysis methods, including principal component analysis (PCA), partial least squares-discrimination analysis (PLS-DA), and orthogonal projection of latent-structure-discrimination analysis (OPLS-DA) were applied to analyze the NMR data and thus identify possible correlations between the metabolite profile changes and the alterations in biological pathways. RESULTS: The potential biological mechanism of an 800-m race was finally elucidated based on the multivariate statistical analysis results. The levels of blood lactate (Lac), 2-hydroxyisovalerate (2HIV), leucine, 2-hydroxyisobutyrate (2HIB), alanine, N-acetyl-glucoprotein, pyruvate, creatinine, fumarate, inosine (Ino) and hypoxanthine (Hyx) were up-regulated in the post samples, whereas the levels of certain metabolites, including 3-hydroxyisovalerate, citrate, taurine, glycine and formate were down-regulated in the postsamples. CONCLUSIONS: Our study provides novel insights into the 800-m race metabolic characteristic. Separation of pre- from postexercise samples was related to the Krebs cycle, Cori cycle, Cahill cycle, HIFs and ROS. Besides the Lac change, the increased concentrations of Ino, 2HIV concentrations in the postexercise urine samples represent potential indices which indicate the high percent of glycolysis during the 800-m run. The increase of concentrations of Hyx, 2HIB may indicated oxidative stress with concomitant ROS generation in the athletes' bodies during the 800-m race.

Lymphocyte propionyl-CoA carboxylase and its activation by biotin are sensitive indicators of marginal biotin deficiency in humans.

BACKGROUND: Marginal biotin deficiency may be a human teratogen. A biotin status indicator that is not dependent on renal function may be useful in studies of biotin status during pregnancy. A previous study of experimental biotin deficiency suggested that propionyl-coenzyme A carboxylase (PCC) activity in peripheral blood lymphocytes (PBLs) is a sensitive indicator of biotin status. OBJECTIVE: We examined the utility of measuring PCC activity and the activation of PCC by biotin in detecting marginal biotin deficiency. DESIGN: Marginal biotin deficiency was induced in 7 adults (3 women) by egg-white feeding for 28 d. Blood and urine were obtained on days 0, 14, and 28 (depletion phase) and 44 and 65 (repletion phase). PBLs were incubated with (activated) or without (control) biotin before PCC assay. The activation coefficient of PCC is the ratio of PCC activity in activated PBLs to that in control PBLs. The significance of differences for all measurements was tested by repeated-measures analysis of variance with Fisher's post hoc test and Bonferroni correction. RESULTS: Changes in the urinary excretion of biotin and of 3-hydroxyisovaleric acid confirmed that marginal biotin deficiency was successfully induced. By day 14, PCC activity had decreased (P < 0.0001) to below the lower limit of normal in all subjects. By day 28, the activation coefficient of PCC had increased significantly (P = 0.003) and was above the upper limit of normal in 6 of 7 subjects. CONCLUSION: PCC activity is the most sensitive indicator of biotin status tested to date. In future pregnancy studies, the use of lymphocyte PCC activity data should prove valuable in the assessment of biotin status.