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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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.

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 (β = 0.012, SE = 0.002, P = 2.98 × 10 -12 ) and the nucleotide uridine (β = 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 (β = 0.050, SE = 0.015, P = 7.53 × 10 -4 ) and validated in the urine of 236 UK twins (β = 0.091, SE = 0.032, P = 0.004). Two metabolites from the targeted platform, hydroxysphingomyelin C14:1 (β = 0.034, SE = 0.005, P = 9.75 × 10 -14 ) and diacylphosphatidylcholine C28:1 (β = 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.

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.

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.

Background: Alcohol-dependence (AD) is a ravaging public health and social problem. AD diagnosis depends on questionnaires and some biomarkers, which lack specificity and sensitivity, however, often leading to less precise diagnosis, as well as delaying treatment. This represents a great burden, not only on AD individuals but also on their families. Metabolomics using nuclear magnetic resonance spectroscopy (NMR) can provide novel techniques for the identification of novel biomarkers of AD. These putative biomarkers can facilitate early diagnosis of AD., Objectives: To identify novel biomarkers able to discriminate between alcohol-dependent, non-AD alcohol drinkers and controls using metabolomics., Method: Urine samples were collected from 30 alcohol-dependent persons who did not yet start AD treatment, 54 social drinkers and 60 controls, who were then analysed using NMR. Data analysis was done using multivariate analysis including principal component analysis (PCA) and orthogonal partial least square-discriminate analysis (OPLS-DA), followed by univariate and multivariate logistic regression to develop the discriminatory model. The reproducibility was done using intraclass correlation coefficient (ICC)., Results: The OPLS-DA revealed significant discrimination between AD and other groups with sensitivity 86.21%, specificity 97.25% and accuracy 94.93%. Six biomarkers were significantly associated with AD in the multivariate logistic regression model. These biomarkers were cis-aconitic acid, citric acid, alanine, lactic acid, 1,2-propanediol and 2-hydroxyisovaleric acid. The reproducibility of all biomarkers was excellent (0.81-1.0)., Conclusion: This study revealed that metabolomics analysis of urine using NMR identified AD novel biomarkers which can discriminate AD from social drinkers and controls with high accuracy., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

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 β-hydroxy-β-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.

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.

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., (© 2014 American Society for Nutrition.)

Gamma-valerolactone (GVL) is reported to be a substance that can be used as a legal substitute for gamma-hydroxybutyric acid (GHB), which is currently a controlled substance in several countries. Unlike gamma-butyrolactone and 1,4-butanediol, GVL is not metabolized to GHB, which causes the effects after uptake of these two chemicals. In the case of GVL, the lactone ring is split to gamma-hydroxyvaleric acid (GHV or 4-methyl-GHB) by a lactonase. Because of its affinity for the GHB receptor, GHV reveals similar effects to GHB, although it is less potent. Intoxications with GVL, or its use as a date rape drug, are conceivable. Despite these facts, there are no publications in the literature regarding detections of GHV in human samples. This study reports three cases, including five urine samples, in which GHV could be detected in concentrations between 3 and 5.8 mg/L. In one of these cases, a drug-facilitated sexual assault (DFSA) was assumed; four of these samples were from two people suspected of abusing GHB. The results indicate that GVL is used as an alternative to GHB and its precursors and should be taken seriously. GVL or GHV should be included in toxicological analysis, particularly in DFSA cases. More information is needed regarding the pharmacokinetics of GVL/GHV for the meaningful interpretation of positive or negative results.

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.

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.

The leucine metabolite, β-hydroxy-β-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.

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.

Patients with SUCLA2 gene defects characteristically develop the trias of early hypotonia, progressive dystonia and sensori-neural deafness. We describe the clinical course and biochemical phenotype in 16 children from the Faroe Islands with a homozygous SUCLA2 splice site mutation. Elevated urinary 3-hydroxyisovaleric acid is a novel biochemical feature in patients. Progressive hearing loss, in combination with a characteristic metabolite profile (increased lactate, methylmalonic acid, C4-dicarboxylic carnitine, 3-hydroxyisovaleric acid) should lead the clinician to the correct diagnosis even in patients with only intermittent lactic acidemia. Direct SUCLA2 sequence analysis is suggested instead of an invasive muscle biopsy to obtain the diagnosis. Nutritional intervention may be considered in SUCLA2 patients.

A family with 3-methylcrotonyl-CoA carboxylase deficiency with different clinical features is described. A 15-month-old boy, who was the index patient, was admitted to the hospital with atonic seizure. His brother had delayed language development and their uncle had been followed with diagnosis of epilepsy for the last 5 years. Urinary organic acid analysis displayed elevated 3-hydroxyisovaleric acid and 3-methylcrotonylglycine, analysis of acylcarnitines showed elevated 3-hydroxyisovalerylcarnitine and decreased free carnitine levels in both the patients and their uncle. Methylcrotonyl-CoA carboxylase activity in cultured fibroblasts displayed a low residual activity of 2.2% of the median control value while propionyl-CoA carboxylase activity was normal in the index patient. Mutation analysis revealed a large homozygous deletion of 2264 bp (c.873+4524&#95;6787de12264) in the MCCA gene, which has not been described to date. Adult-onset afebrile seizures have not been reported in the literature. Our cases are an example of this wide phenotypic variability within a single family.

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.

2-Methylbutyryl-CoA dehydrogenase (MBD; coded by the ACADSB gene) catalyzes the step in isoleucine metabolism that corresponds to the isovaleryl-CoA dehydrogenase reaction in the degradation of leucine. Deficiencies of both enzymes may be detected by expanded neonatal screening with tandem-mass spectrometry due to elevated pentanoylcarnitine (C5 acylcarnitine) in blood, but little information is available on the clinical relevance of MBD deficiency. We biochemically and genetically characterize six individuals with MBD deficiency from four families of different ethnic backgrounds. None of the six individuals showed clinical symptoms attributable to MBD deficiency although the defect in isoleucine catabolism was demonstrated both in vivo and in vitro. Several mutations in the ACADSB gene were identified, including a novel one. MBD deficiency may be a harmless metabolic variant although significant impairment of valproic acid metabolism cannot be excluded and further study is required to assess the long-term outcome of individuals with this condition. The relatively high prevalence of ACADSB gene mutations in control subjects suggests that MBD deficiency may be more common than previously thought but is not detected because of its usually benign nature.

Pyruvate carboxylase (PC) is a biotin-dependent enzyme that plays a crucial role in gluconeogenesis, lipogenesis, Krebs cycle anaplerosis and amino acid catabolism. Biotin deficiency reduces its mass besides its activity. Enzyme mass is the result of its cellular turnover, i.e., its rates of synthesis and degradation. We have now investigated, by a pulse and chase approach in cultured primary hepatocytes, the effects of biotin deficiency on these rates. Wistar rats were fed a biotin-deficient diet and the controls were fed the same diet supplemented with biotin; their biotin status was monitored measuring lymphocytes propionyl-CoA carboxylase activity and urinary 3-hydroxyisovaleric acid. After 6-7 weeks primary hepatocytes were cultured in biotin-deficient or complete DMEM. PC activity was determined by measuring the incorporation of (14)C-bicarbonate into acid-non-volatile products, and its mass by streptavidin Western blots. Its synthesis rate was estimated from [(35)S] methionine incorporation into anti-PC antibody immunoprecipitate. Its degradation rate was calculated from the loss of radioactivity from previously labeled hepatocytes, in a medium containing an excess of non-radioactive methionine. PC synthesis rate in biotin-deficient hepatocytes was approximately 4.5-fold lower than in the controls, and its degradation rate was 5.1-fold higher. Therefore, the decrement of PC mass during biotin deficiency results both from a decrease in its synthesis and an increase in its degradation rates. To our knowledge, this is the first instance where a mammalian enzyme cofactor is necessary to sustain both processes.

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.

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.

In this paper, we describe a distinct clinical subtype of 3-methylglutaconic aciduria. 3-Methylglutaconic aciduria is a group of different metabolic disorders biochemically characterized by increased urinary excretion of 3-methylglutaconic acid. We performed biochemical and genetic investigations, including urine organic acid analysis, NMR spectroscopy, measurement of 3-methylglutaconyl-CoA hydratase activity, cardiolipin levels, OPA3 gene analysis and measurement of the oxidative phosphorylation in four female patients with 3-methylglutaconic aciduria. 3-Methylglutaconic aciduria type I, Barth syndrome, and Costeff syndrome were excluded as the activity of 3-methylglutaconyl-CoA hydratase, the cardiolipin levels, and molecular analysis of the OPA3 gene, respectively, showed no abnormalities. The children presented with characteristic association of hearing loss and the neuro-radiological evidence of Leigh disease. They also had neonatal hypotonia, recurrent lactic acidemia, episodes with hypoglycemia and severe recurrent infections, feeding difficulties, failure to thrive, developmental delay, and progressive spasticity with extrapyramidal symptoms. Our patients were further biochemically characterized by a mitochondrial dysfunction and persistent urinary excretion of 3-methylglutaconic acid.

A diagnosis of 3-methylglutaconic aciduria type I (OMIM: 250950) based on elevated urinary excretion of 3-methylglutaconic acid (3MGA), 3-methylglutaric acid (3MG) and 3-hydroxyisovaleric acid (3HIVA) was made in a 61-year-old female patient presenting with leukoencephalopathy slowly progressing over more than 30 years. The diagnosis was confirmed at the enzymatic and molecular level. In vivo brain MR spectroscopic imaging (MRSI) was performed at 3.0 T, and one-dimensional and two-dimensional in vitro NMR spectroscopy of body fluids of the patient was performed at 11.7 T. Additionally, we measured 1D (1)H-NMR spectra of urine of seven patients with a total of four different inborn errors of leucine metabolism. Increased concentrations of 3HIVA, 3MGA (cis and trans) and 3MG were observed in the NMR spectra of the patient's urine. In the cerebrospinal fluid, the 3HIVA concentration was 10 times higher than in the plasma of the patient and only the cis isomer of 3MGA was observed. In vivo brain MRSI showed an abnormal resonance at 1.28 ppm that may be caused by 3HIVA. Comparison of (1)H-NMR spectra of urine samples from all eight patients studied, representing five different inborn errors of leucine metabolism, showed that each disease has typical NMR characteristics. Our leukoencephalopathy patient suffers from a late-onset form of 3-methylglutaconic aciduria type I. In the literature, only very few adult patients with this conditions have been described, and 3HIVA accumulation in white matter in the brain has not been presented before in these patients. Our data demonstrate that (1)H-NMR spectroscopy of urine can easily discriminate between the known inborn errors of leucine metabolism and provide the correct diagnosis.

We report a 3-year-old boy with isolated 3-methylcrotonyl-coenzyme A deficiency with unexpectedly severe presentation, seizures and history of cerebral ischae-mic episode.

Background: Isolated excretion of 2-methylbutyrylglycine (2-MBG) is the hallmark of short/branched-chain acyl-CoA dehydrogenase deficiency (SBCADD), a recently identified defect in the proximal pathway of L-isoleucine oxidation. SBCADD might be underdiagnosed because detection and recognition of urine acylglycines is problematic. Excretion of 2-ethylhydracrylic acid (2-EHA), an intermediate formed in the normally minor R-pathway of L-isoleucine oxidation, has not previously been described in SBCADD., Methods: Samples from four patients with 2-MBG excretion were analyzed by gas chromatography-mass spectrometry for urine organic acids, quantification of 2-MBG, and chiral determination of 2-methylbutyric acid. Blood-spot acylcarnitines were measured by electrospray-tandem mass spectrometry. Mutations in the ACADSB gene encoding SBCAD were identified by direct sequencing., Results: SBCADD was confirmed in each patient by demonstration of different ACADSB gene mutations. In multiple urine samples, organic acid analysis revealed a prominent 2-EHA peak usually exceeding the size of the 2-MBG peak. Approximately 40-46% of total 2-methylbutyric acid conjugates were in the form of the R-isomer, indicating significant metabolism via the R-pathway., Conclusions: If, as generally believed, SBCAD is responsible for R-2-MBG dehydrogenation in the R-pathway, 2-EHA would not be produced in SBCADD. Our observation of 2-ethylhydracrylic aciduria in SBCADD implies that a different or alternative enzyme serves this function. Increased flux through the R-pathway may act as a safety valve for overflow of accumulating S-pathway metabolites and thereby mitigate the severity of SBCADD. Awareness of 2-ethylhydracrylic aciduria as a diagnostic marker could lead to increased detection of SBCADD and improved definition of its clinical phenotype.

We developed an assay for measuring urinary 3-hydroxyisovaleric acid (3-HIA) using HPLC after derivatization with 2-nitrophenylhydrazine hydrochloride (2-NPH . HCl). The derivatized 3-HIA was extracted into n-hexane and separated isocratically on a C8 reversed-phase column for fatty acids (YMC-Pack FA). We used this method to measure 3-HIA in urine extracts from mice fed a biotin-deficient diet for >4 wk and in an infant who was fed a special Japanese formula and was suspected of being biotin deficient. Urinary 3-HIA could be assayed within the range of 0.42-8.5 mmol/L with high accuracy by this method, as an indicator of biotin deficiency. Therefore, the HPLC method for 3-HIA described here may be a useful tool clinically as well as in the research laboratory.

A patient with a severe neonatal variant of 3-methylcrotonyl-CoA carboxylase (MCC) deficiency is reported. The first child of healthy consanguineous Turkish parents presented on the second day of life with dehydration, cyanosis, no sucking, generalized muscular hypotonia, encephalopathy, respiratory depression requiring mechanic ventilation, macrocephaly, severe acidosis and hypoglycaemia. Elevated C5-OH-carnitine in dried blood spot by tandem MS and elevated urinary excretion of 3-hydroxyisovaleric acid and 3-methylcrotonylglycine suggested MCC deficiency, confirmed by enzyme analysis in cultured fibroblasts. Cerebral ultrasonography and cranial CT findings revealed progressive changes such as disseminated encephalomalacia, cystic changes, ventricular dilatation and cerebral atrophy. Treatment with high-dose biotin and protein-restricted diet was ineffective and the patient died at the age of 33 days with progressive neurological deterioration. Mutation analysis revealed a homozygous mutation in the splice acceptor site of intron 15 in the MCC beta-subunit. Early-onset severe necrotizing encephalopathy should be included in the differential diagnosis of isolated MCC deficiency.

Deficiency of 3-methylcrotonyl-CoA carboxylase (MCC) results in elevated excretion of 3-methylcrotonylglycine (3-MCG) and 3-hydroxyisovaleric acid (3-HIVA). MCC is a heteromeric mitochondrial enzyme comprising biotin-containing alpha subunits and smaller beta subunits, encoded by MCCA and MCCB, respectively. Mutations in these genes cause isolated MCC deficiency, an autosomal recessive disorder with a variable phenotype that ranges from severe neonatal to asymptomatic adult forms. No reported patients have responded to biotin therapy. Here, we describe two patients with a biochemical and, in one case, clinical phenotype of MCC deficiency, both of whom were responsive to biotin. The first patient presented at 3 months with seizures and progressive psychomotor retardation. Metabolic investigation at 2 years revealed elevated excretion of 3-MCG and 3-HIVA, suggesting MCC deficiency. High-dose biotin therapy was associated with a dramatic reduction in seizures, normalization of the electroencephalogram, and correction of the organic aciduria, within 4 weeks. MCC activity in fibroblasts was 25% of normal levels. The second patient, a newborn detected by tandem-mass-spectrometry newborn screening, displayed the same biochemical phenotype and remained asymptomatic with biotin up to the age of 18 months. In both patients, sequence analysis of the complete open reading frames of MCCA and MCCB revealed heterozygosity for MCCA-R385S and for the known polymorphic variant MCCA-P464H but revealed no other coding alterations. MCCA-R385S is unusual, in that it has a normal amount of MCC alpha protein but confers no MCC activity. We show that MCCA-R385S, but not other MCCA missense alleles, reduces the MCC activity of cotransfected MCCA-wild-type allele. Our results suggest that MCCA-R385S is a dominant negative allele and is biotin responsive in vivo.

Background: Smoking accelerates the degradation of many nutrients, including lipids, antioxidants, and certain B vitamins. Accelerated biotin catabolism is of concern in women because marginal biotin deficiency is teratogenic in mammals., Objective: The objective was to assess the effect of smoking on the biotin status of women., Design: A preliminary study of 7 women and 3 men examined the urinary concentrations of biotin and its metabolites biotin sulfoxide and bisnorbiotin in smokers. The interpretation of the results of this study was limited by the lack of a contemporaneous control group; consequently, we conducted a cohort-controlled study. Smoking women (n = 8) and nonsmoking control subjects (n = 15) provided 24-h urine samples; excretion rates of biotin, the biotin metabolites, and 3-hydroxyisovaleric acid were determined. Increased urinary excretion of 3-hydroxyisovaleric acid, which reflects a reduced activity of the biotin-dependent enzyme 3-methylcrotonyl-Co A carboxylase, is a sensitive indicator of biotin depletion at the tissue level., Results: Compared with control subjects from previous studies, the smoking women in the preliminary study excreted significantly less urinary biotin (P = 0.02). Moreover, the ratio of urinary biotin sulfoxide to biotin increased (P = 0.04) in these women. In the cohort-controlled study, the urinary excretion of biotin decreased by 30% (P = 0.04), and the ratios of urinary bisnorbiotin and biotin sulfoxide to biotin increased significantly, which indicated accelerated catabolism in smokers. Moreover, the urinary excretion of 3-hydroxyisovaleric acid was greater in the smokers than in the control subjects (P = 0.04), which indicated biotin depletion in the smokers at the tissue level., Conclusion: These data provide evidence of accelerated biotin metabolism in smoking women, which results in marginal biotin deficiency.

3-Methylglutaconic-aciduria type I (MGA1, OMIM 250950) resulting from 3-Methylglutaconyl-coenzyme A hydratase deficiency is a rare inherited metabolic disorder of l-leucine catabolism. We diagnosed this condition in a 4-year-old German male with generalized fever-associated seizures from the age of 12 months and normal psychomotor development. First he was considered to suffer from uncomplicated febrile seizures. After his eighth seizure, laboratory investigations were performed to exclude inborn errors of metabolism. Analysis of organic acids in urine indicated highly elevated concentrations of 3-methylglutaconic and 3-hydroxyisovaleric acids. 3-Methylglutaconyl-coenzyme A hydratase activity was markedly decreased in skin fibroblasts. Mutation analysis in the AUH gene revealed homozygosity for a novel splice site mutation IVS9-2A>G. We conclude that MGA1 may be associated with fever-associated seizures even in children without delayed psychomotor development.

We report the clinical course and biochemical findings of a 10-year-old, mentally retarded girl with late-onset holocarboxylase synthetase (HCS, gene symbol HLCS) deficiency and only partial response to biotin. On treatment, even with an unusually high dose of 200mg/day, activities of the biotin-dependent mitochondrial carboxylases in lymphocytes remained below 50% of the mean control values. Not only urinary 3-hydroxyisovaleric acid excretion has been persistently elevated, but also plasma and, with even higher concentrations, cerebrospinal fluid 3-hydroxyisovaleric acid have not normalized. The unusual and insufficient response of this patient to biotin treatment can be explained by the effect of the combination of the common HLCS allele IVS10 +5 g>a on one chromosome and a truncating mutation on the other. This case illustrates mechanisms involved in the genotype-phenotype correlation that unequivocally exists in HCS deficiency.

Background: The results of clinical studies have provided evidence that marginal biotin deficiency is more common than was previously thought. A previous study of 10 subjects showed that the urinary excretion of biotin and 3-hydroxyisovaleric acid (3HIA) are early and sensitive indicators of marginal biotin deficiency., Objective: Marginal biotin deficiency was experimentally induced and corrected to assess the utility of 3 indicators of biotin status: urinary excretion of biotin and 3HIA and the increase in 3HIA excretion after leucine loading., Design: Eleven healthy adults consumed an egg white diet for 28 d. Blood and 24-h urine samples were collected before the start of the diet and twice weekly thereafter. In 5 subjects, an oral leucine challenge was performed weekly for 4 wk. After depletion, biotin status was restored with a general diet with or without a supplement containing 80 micro g biotin. Urinary excretion of biotin, bisnorbiotin, and biotin sulfoxides was determined by avidin-binding assay after HPLC. Excretion of 3HIA, an indicator of reduced activity of the biotin-dependent enzyme methylcrotonyl-CoA carboxylase (EC 6.4.1.4), was measured by gas chromatography-mass spectrometry., Results: 3HIA excretion increased significantly with time on the egg white diet (P < 0.0001), as did 3HIA excretion in response to the leucine challenge (P < 0.002); the excretion of both biotin and bisnorbiotin decreased significantly with time (P < 0.0001). In most subjects, biotin status returned to normal after 1 wk of a general diet., Conclusions: Excretion of 3HIA and of biotin are early and sensitive indicators of biotin deficiency. 3HIA excretion after a leucine challenge is at least as sensitive.

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.

Background: Biotin deficiency is teratogenic in several mammalian species. Approximately 50% of pregnant women have an abnormally increased urinary excretion of 3-hydroxyisovaleric acid (3-HIA), which probably reflects decreased activity of the biotin-dependent enzyme methylcrotonyl-CoA carboxylase. However, increased 3-HIA excretion could result from pregnancy per se (eg, from an effect of pregnancy on renal handling of organic acids)., Objective: We tested the hypothesis that biotin supplementation significantly decreases 3-HIA excretion in pregnant women with abnormally increased 3-HIA excretion., Design: Twenty-six pregnant women with increased 3-HIA excretion were studied in a randomized, placebo-controlled trial; 10 women were studied during early pregnancy (6-17 wk gestation) and 16 women during late pregnancy (21-37 wk gestation). Urine samples were collected before and after 14 d of supplementation with 300 microg (1.2 micromol) biotin/d or placebo., Results: In the early-pregnancy group, 3-HIA excretion decreased (P < 0.006) by 11.7 +/- 3.6 mmol/mol creatinine (mean +/- SEM) in the 5 women who received biotin supplements, whereas 3-HIA excretion increased by 1.6 +/- 0.6 mmol/mol creatinine in the 5 women who received placebo. In the late-pregnancy group, 3-HIA excretion decreased (P < 0.002) by 7.1 +/- 1.2 mmol/mol creatinine in the 8 women who received biotin supplements, whereas 3-HIA excretion increased by 0.9 +/- 1.8 mmol/mol creatinine in the 8 women who received placebo., Conclusions: This study provides evidence that the increased excretion of 3-HIA seen frequently in normal pregnancy reflects reduced biotin status. The conclusion that marginal biotin deficiency occurs frequently in the first trimester further raises concern about potential human teratogenicity.

To assess the effects of marginal biotin deficiency on immune function and thereby evaluate immune function as a potential marker for impaired biotin status, we investigated immune function in a rat model during progression from sufficiency to moderate biotin deficiency. As immune function indicators, we assessed the IgG response to a vaccine and the cytokine responses and relative proportions of lymphocyte subpopulations in the immunocytes in blood, spleen and thymus. Neither phenotype nor organ redistribution of lymphocytes differed between biotin-deficient and biotin-sufficient rats. Assessment of immune function by mitogen T cell proliferation, mitogen-induced interferon-gamma and interleukin-4 levels, IgG antibody responses and natural killer cell activity were not significantly different in mild to moderately biotin-deficient rats compared with biotin-sufficient controls. The absence of effects on immune function was not attributable to failure to induce biotin deficiency; the rats exhibited unequivocal evidence of biotin deficiency, including reduced hepatic biotin and impaired leucine metabolism resulting from deficiency of the biotin-dependent enzyme methylcrotonyl-CoA carboxylase. We conclude that the immune markers examined are not promising candidates as indicators of mild to moderate deficiency in humans.

Objectives: The effect of administration of the antiepileptic drug valproate (VPA), on the composition of the plasma acylcarnitine profile (including free carnitine) was investigated., Design and Methods: Plasma samples were obtained from 18 individuals (13 males:5 females; 15-65 y) on long-term treatment with VPA (resulting in plasma levels of 14.6-135.0 mg/L; therapeutic conc.: 40-100 mg/L). Acylcarnitines (AC) in plasma were quantified by electrospray tandem mass spectrometry (ESI-MS/MS)., Results: VPA was found to increase the levels (mean +/- SD, microM) of 3-hydroxy-isovalerylcarnitine (0.10 +/- 0.04; controls: 0.02-0.06), C14:2 acylcarnitine (0.11 +/- 0.05; controls: 0.02-0.08), propylglutarylcarnitine (0.06 +/- 0.05; controls: 0.00-0.04), and C18-0H-acylcarnitine (0.09 +/- 0.05; controls: 0.00-0.04). The free carnitine (C) (42.2 +/- 9.0; controls: 22.3-54.9) and the total carnitine (52.3 +/- 10.1; controls: 26.5-73.6) were not significantly altered by VPA. Other AC (C2-C18, monounsaturated and hydroxylated) were all within the control range and especially no increase of C8 (valproyl) carnitine was observed. A positive correlation was found between the ratios [AC] / [C] (p < 0.05) or [long-chain AC (C10-C18)] / [C] (p < 0.09) with the plasma VPA concentration., Conclusions: The unequivocal increase in 3-hydroxy-isovalerylcarnitine is consistent with the increase of 3-hydroxy-isovaleric acid observed in urine of VPA treated patients. This finding suggests an interaction mechanism of VPA with specific enzymes, namely involved in leucine metabolism. Adult patients under VPA monotherapy do not suffer from carnitine deficiency; the effect of the accumulating acylcarnitines is ill-defined.

The results of previous studies in the baboon have suggested that HPTP, the tetrahydropyridinyl analog of haloperidol causes a urinary biochemical marker profile similar to those seen in humans suffering from inborn errors of mitochondrial respiration. In order to identify a possible relationship between compromised cellular energy production and neuronal damage we now have compared the urinary profiles of rats treated with the pro-neurotoxin, MPTP as well as with HPTP. Significantly increased urinary excretion of lactic acid and 2-ethylhydracrylic acid in MPTP and HPTP treated rats was observed, indicating that both MPTP and HPTP and/or their respective metabolites cause mitochondrial inhibition in the rat.

Although estimated average requirements for biotin have been proposed, the human requirements for biotin in specific populations and at various ages remain uncertain, in part because indicators of biotin status have not been validated. With the use of improved methods for measuring biotin and metabolites, a recent study indicated that decreased urinary excretion of biotin and bisnorbiotin is an early and sensitive indicator of biotin deficiency, but decreased serum concentration of biotin is not. Increased urinary excretion of 3-hydroxyisovaleric acid (3-HIA), a leucine metabolite that is excreted in increased quantities with deficiency of the biotin-dependent enzyme beta-methylcrotonyl-CoA carboxylase, is also an early and sensitive indicator of biotin deficiency. When these indicators were assessed longitudinally in 13 pregnant women, biotin excretion was not significantly decreased early in pregnancy but did decrease significantly from early to late pregnancy. Excretion of 3-HIA was abnormally increased in about three-fourths of the women studied in both early and late pregnancy. Thus, each indicator detected biotin deficiency late in pregnancy, but assessment of biotin status for the two indicators conflicted early in pregnancy. Preliminary results from a trial assessing response of 3-HIA excretion to biotin treatment indicate that biotin status is indeed impaired both early and late in pregnancy.

Background: In subjects undergoing long-term therapy with carbamazepine and/or phenytoin, reduced plasma concentrations of biotin have been reported. However, the diagnostic value of plasma biotin is unclear, in part because of the presence of significant plasma concentrations of biotin metabolites. Pathologic organic aciduria has also been reported with long-term anticonvulsant therapy, suggesting biotin deficiency, but no mechanism leading to deficiency has yet been determined., Methods: In the current study, we sought to determine whether biotin catabolism was accelerated in children receiving long-term treatment with certain anticonvulsants and to assess biotin status as judged by urinary excretion of biotin and 3-hydroxyisovaleric acid, an organic acid that is an indicator of deficiency of a biotin-dependent enzyme. Seven children treated with carbamazepine and/or phenytoin and six treated with phenobarbital provided untimed urine samples. Sixteen healthy children receiving no anticonvulsants served as controls. Biotin and biotin metabolites were determined by high-performance liquid chromatography/avidin-binding assay. Urinary excretion of 3-hydroxyisovaleric acid was determined using gas chromatography/mass spectrometry., Results: Bisnorbiotin excretion was increased significantly in the carbamazepine/phenytoin group and in the phenobarbital group. Biotin sulfoxide excretion was significantly increased in the carbamazepine/phenytoin group but not in the phenobarbital group. 3-Hydroxyisovaleric acid excretion was increased significantly in the carbamazepine/phenytoin group. However, only one child (carbamazepine/phenytoin group) had a decreased urinary excretion of biotin., Conclusion: These data provide evidence that long-term administration of some anticonvulsants can accelerate biotin catabolism, but the indicators of biotin status conflict.

Using serum biotin concentration as the indicator, a previous study reported biotin deficiency resulting from long-term anticonvulsant therapy. However, serum biotin may not be a good indicator of tissue biotin status. Using better indicators of biotin status in anticonvulsant-treated subjects, we found increased urinary excretion of biotin catabolites and 3-hydroxyisovaleric acid, an organic acid produced in greater quantities secondary to reduced activity of a biotin-dependent carboxylase. We conclude that anticonvulsant treatment led to increased biotin catabolism and probably to reduced biotin status.

Objective: To assess biotin nutritional status during normal human gestation., Methods: Urine samples were obtained in a cross-sectional design from 16 women in early pregnancy (17 +/- 1 weeks, mean +/- 1 SD) and from 13 women in late pregnancy (36 +/- 1 weeks). The urinary excretion of biotin, two metabolites bisnorbiotin (BNB) and biotin sulfoxide (BSO), and the organic acid 3-hydroxyisovaleric acid (3-HIA) were measured by HPLC/avidin-binding assay and GC/MS, respectively. Excretion rates were expressed as concentration ratios to urinary creatinine., Results: In both early and late pregnancy, 3-HIA excretion was increased compared to controls (p < 0.0001), suggesting decreased activity of a biotin-dependent enzyme caused by tissue biotin depletion. In early pregnancy, urinary excretion of biotin was normal; in late pregnancy, excretion was increased (p < 0.0002), suggesting biotin status was not decreased. In late pregnancy, urinary excretion of BNB and BSO were increased (p < 0.009)., Conclusion: The apparent conflict in the indices of biotin status is not explained by this study but could be resolved by two alternate explanations: 1) Pregnancy caused an impairment of renal reclamation of biotin, BNB, and BSO leading to a paradoxical increase in biotin excretion 2) Pregnancy caused metabolic or renal effects that increased 3-HIA excretion nonspecifically; hence, the increased 3-HIA excretion did not reflect biotin deficiency. We speculate that some of the women studied were marginally biotin deficient and that renal wasting and accelerated breakdown of biotin contributed to the deficiency.

This study assessed biotin nutritional status longitudinally during pregnancy as judged by urinary excretion of biotin and biotin metabolites and by serum concentration of biotin. 3-Hydroxyisovaleric acid excretion was also assessed because increased excretion of that acid reflects decreased tissue activity of the biotin-dependent enzyme, methylcrotonyl-CoA carboxylase. Thirteen women provided untimed urine samples during both early and late pregnancy. Twelve nonpregnant women served as controls. Biotin and metabolites were determined by a combined HPLC/avidin-binding assay. 3-Hydroxyisovaleric acid was determined by gas chromatography/mass spectrophotometry. Significance of changes from early to late pregnancy was tested by paired t test; to compare nonpregnant controls with early and late pregnancy, ANOVA was used. During early pregnancy, biotin excretion was not significantly different than controls; however, 3-hydroxyisovaleric acid excretion was significantly increased relative to controls (P < 0.0001) and was greater than the upper limit of normal in 9 of 13 women. From early to late pregnancy, biotin excretion decreased in 10 of 13 women (P < 0.01); by late pregnancy, biotin excretion was less than normal in six women. During late pregnancy, 3-hydroxyisovaleric acid remained significantly increased relative to controls (P < 0.0001). Serum concentrations of biotin were significantly greater than those of controls during early pregnancy (P < 0.0001) and decreased in each woman from early to late pregnancy (P < 0.0001). These data provide evidence that biotin status decreases during pregnancy.

To assess the utility of various indicators of biotin status, marginal biotin deficiency was induced experimentally in normal adults. Ten subjects consumed a diet that contained enough avidin to bind seven times more biotin than that in the diet. Blood and 24-h urine samples were collected before the diet began and twice weekly thereafter for 20 d. The urinary excretion and serum concentration of biotin and its two principal inactive metabolites bisnorbiotin and biotin sulfoxide were determined after HPLC separation with an avidin-binding assay. The urinary concentration of 3-hydroxyisovaleric acid, an indicator of reduced activity of a biotin-dependent enzyme, was quantitated by gas chromatography-mass spectrometry. The urinary excretion of 3-hydroxyisovaleric acid increased significantly (P < 0.0001). For all subjects, the urinary excretion of both biotin and bisnorbiotin decreased significantly (P < 0.0001 for each). In contrast, the mean serum concentration of biotin did not decrease significantly (P = 0.06). These data provide evidence that the urinary excretion of 3-hydroxyisovaleric acid and the urinary excretion of biotin are early and sensitive indicators of biotin deficiency and that the serum concentration of biotin is not.