Biotin Depletion Research Articles

A-041 Measurement of Plasma 3-Hydroxyisovalerylcarnitine by LC-MS/MS to Predict Marginal Biotin Deficiency in Inflammatory Bowel Disease

Abstract Background Biotin’s role in the immune response have emergently revealed that biotin is related to inflammation and that its deficiency leads to an increase in the level of proinflammatory cytokines. A biotin-deficient diet was recently shown to induce a colitis-like phenotype in mice, alleviated by biotin substitution. Mice with dextran sulphate sodium (DSS)-induced colitis showed biotin deficiency and significantly reduced biotin absorption. Oral biotin substitution reversed DSS colitis and induced remission. Diagnosis of biotin deficiency is rather challenging since based solely on measurement of circulating biotin levels has been shown to be insufficiently sensitive for clinical purposes. Increased urinary excretion of biotin metabolites, is suggested as a sensitive indicator of biotin deficiency. However, 24 h urine collection is time consuming, expensive, and the test relies on correct collection of samples by the patient, thus if this is not done properly the results may be inaccurate. 3HIVc increases in the case of biotin deficiency as a response to the decreased activity of biotin dependent enzyme methyl crotonyl-coenzyme. Analysis of circulating 3HIVc levels by LC-MS/MS has been found to be one of the most sensitive markers of biotin depletion. Methods Study cohort conducted with 100 inflammatory bowel disease (IBD) patients (20–60 years, 40 females) whom diagnosed according to standard clinical, radiological and pathological criteria and 100 healthy controls (20–60 years, 40 females). Of the patients 58/100 had Crohn’s Disease (CD), 42/100 had ulcerative colitis (UC) and 29/100 were in inflammatory state (serum high sensitivity C reactive protein (hsCRP) concentrations >5 mg/L). Standard serum and citrate-plasma samples were withdrawn between 8:00–10:00 h in the clinic after overnight fasting and stored at −20 °C until the analysis. Complete blood count (CBC), high sensitivity C reactive protein (hsCRP), fecal calprotectin (fCal) were determined with standard methods in serum matrix. 3-Hydroxyisovalerylcarntine (3HIVc) levels were determined by a commerical LC-MS/MS Kit in citrate plasma (KM3200, Immundiagnostik AG, Germany). Results Patients with IBD were found to have significantly higher 3HIVc levels than controls (6.1 ± 2.4 ng/mL vs 5.1 ± 2.1 ng/mL respectively, P = 0.004). 3HIVc concentrations were compared according to different disease characteristics within the patient group: circulating 3HIVc levels were found to be similar in patients with CD and UC (5.2 ± 2.1 ng/mL vs 4.9 ± 2.1, P = 0.451). Patients were also found to have similar circulating 3HIVc concentrations irrespective of whether or not inflammatory activity was present (5.1 ± 2.0 ng/mL in inflammatory vs 5.7 ± 2.4 ng/mL in noninflammatory conditions, P = 0.131). Conclusion The clinical findings of this study are in line with the existing preclinical evidence indicating that biotin deficiency is more common in patients with IBD in comparison to healthy controls in a clinical setting. However, surprisingly, the biotin status of patients with IBD was not found to differ according to inflammatory status. Therefore, there is still a room for more comprehensive look into the relationship between IBD related inflammation and biotin deficiency. Moreover, LC-MS/MS analysis of plasma 3HIVc has the potential to help clinicians to define biotin repletion and help to clearify the latter relation.

Depletion and Repletion of Biotinyl Enzymes in Liver of Biotin-Deficient Rats: Evidence of a Biotin Storage System

The quantities of biotinyl proteins in liver of young rats were compared with age-matched controls at intervals during depletion and repletion of biotin. Growth rate and the concentrations of biotinyl proteins previously proposed as mitochondrial storage forms of acetyl CoA carboxylase rapidly decreased in response to biotin deprivation, whereas neither the concentration nor activity of cytosolic acetyl CoA carboxylase was affected. Concentrations of carboxylases active within mitochondria (pyruvate carboxylase, propionyl CoA carboxylase and 3-methyl crotonyl CoA carboxylase) decreased only after d 28. When biotin was injected into biotin-deficient rats, concentrations of the carboxylases active within mitochondria were restored to control levels within 3 h, whereas the concentrations of putative mitochondrial storage forms of acetyl CoA carboxylase reached normal levels only after 9 h, indicating that the injected biotin was preferentially used for the synthesis of the carboxylases active within mitochondria rather than acetyl CoA carboxylase. Mitochondrial acetyl CoA carboxylase may serve as a reservoir to maintain a normal concentration of cytosolic acetyl CoA carboxylase in liver of rats deprived of biotin and provide biotin, indirectly, to maintain essentially normal concentrations of the biotinyl enzymes active within mitochondria for several weeks after rats were fed a biotin-deficient diet.

Regulatory Landscape of the Pseudomonas aeruginosa Phosphoethanolamine Transferase Gene eptA in the Context of Colistin Resistance

Pseudomonas aeruginosa has the genetic potential to acquire colistin resistance through the modification of lipopolysaccharide by the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) or phosphoethanolamine (PEtN), mediated by the arn operon or the eptA gene, respectively. However, in vitro evolution experiments and genetic analysis of clinical isolates indicate that lipopolysaccharide modification with L-Ara4N is invariably preferred over PEtN addition as the colistin resistance mechanism in this bacterium. Since little is known about eptA regulation in P. aeruginosa, we generated luminescent derivatives of the reference strain P. aeruginosa PAO1 to monitor arn and eptA promoter activity. We performed transposon mutagenesis assays to compare the likelihood of acquiring mutations leading to arn or eptA induction and to identify eptA regulators. The analysis revealed that eptA was slightly induced under certain stress conditions, such as arginine or biotin depletion and accumulation of the signal molecule diadenosine tetraphosphate, but the induction did not confer colistin resistance. Moreover, we demonstrated that spontaneous mutations leading to colistin resistance invariably triggered arn rather than eptA expression, and that eptA was not induced in resistant mutants upon colistin exposure. Overall, these results suggest that the contribution of eptA to colistin resistance in P. aeruginosa may be limited by regulatory restraints.

Differential growth inhibition, cell cycle arrest and apoptosis of MCF-7 and MDA-MB-231 cells to holocarboxylase synthetase suppression

Holocarboxylase synthetase (HLCS) catalyzes the covalent attachment of biotin onto the biotin-dependent carboxylases. Recent studies have shown that HLCS is over-expressed in breast cancer patients. Here we investigated the functional roles of free biotin and HLCS in supporting growth and migration of breast cancer cell lines. Depletion of biotin from culture medium markedly reduced biotinylation of the two most abundant biotin-carboxylases, acetyl-CoA carboxylase and pyruvate carboxylase. This was accompanied by a marked decrease in cell growth. Suppression of HLCS expression in the low invasive breast cancer cell line MCF-7 resulted in an 80% reduction of biotinylated ACC, but not PC. HLCS knockdown MCF-7 cell lines showed 40-50% reduction of proliferation and 35% reduction of migration, accompanied by G1 cell cycle-arrest-induced apoptosis. In contrast, knockdown of HLCS expression in the highly invasive cell line MDA-MB-231 resulted in only marginal reduction of biotinylation of both ACC and PC, accompanied by 30% reduction of proliferation and 30% reduction of migration. Our studies provide new insights to use HLCS as a novel anti-cancer drug target.

Mitigation of Biotin Interference in Manual and Automated Immunoassays by Preconjugating Biotinylated Antibodies to the Streptavidin Surface as an Alternative to Biotin Depletion.

Streptavidin-to-biotin binding is one of the strongest noncovalent interactions in nature and incorporated into many immunoassays. Biotin-streptavidin coupling assays are susceptible to interference from free biotin in patient specimens, which may falsely decrease or increase results. To prevent biotin interference, we evaluated a method to preconjugate biotinylated antibodies to the assay's streptavidin solid surface before adding patient specimen and compared this technique to a biotin depletion protocol. Biotin interference in 3 manual ELISAs and 2 automated immunoassays was established. Mitigation of biotin interference by preincubation was evaluated in each assay by adding biotinylated antibody to the streptavidin-coated surface before adding biotin- or PBS-spiked serum. Lastly, the preincubation method was compared to a biotin-depletion protocol to compare the effectiveness of mitigating biotin interference. In the presence of 400 µg/L biotin, analyte detection was reduced to 10% to 15% of total in the ELISA assays and to 15.2% in the automated sandwich (thyroglobulin) immunoassay. In the automated competitive (free thyroxine) immunoassay, biotin caused an increased detection of 551.6%. Preconjugation of the biotinylated capture antibody to the streptavidin surface in the ELISA assays resulted in 84% to 99% activity recovery, compared to 84% to 97% by a biotin depletion protocol. Similarly, automated sandwich and competitive immunoassays obtained 97.1% and 116.5% recovery by preconjugation, compared to 95.6% and 100.3% by the depletion method, respectively. This study demonstrates how assay redesign to include preconjugation of biotinylated capture antibody to streptavidin is an effective alternative to biotin-depletion methods to mitigate biotin interference.

Validation of a Commercial Reagent for the Depletion of Biotin from Serum/Plasma: A Rapid and Simple Tool to Detect Biotin Interference with Immunoassay Testing

It is important for clinical laboratories to have protocols for investigating suspected biotin interference in patient samples. VeraPrep Biotin™ is a commercial product used to rapidly deplete biotin from serum/plasma samples. The objectives of this study were to verify that VeraPrep Biotin™: (a) does not impact immunoassay analyte recovery in control samples and (b) can effectively deplete biotin from samples (both biotin-spiked and samples from donors who ingested biotin supplements). De-identified residual waste serum/plasma samples were combined to create 9 pools for each immunoassay. Plasma/serum samples (n = 23) were obtained from 6 healthy donors at varying times following ingestion of biotin (20 mg, 100 mg, or 200 mg). Nine Elecsys immunoassays were evaluated using the e 602 (Roche Diagnostics Inc.). Control, biotin-spiked (n = 10, ∼400 ng/mL), and donor samples were assayed pre- and post-VeraPrep treatment. Percentage analyte recovery [(posttreatment/pretreatment) × 100] was calculated for control samples. A laboratory-developed LC-MS/MS method was used to quantify biotin. In control samples (n = 81), 90-110% analyte recovery was observed post-VeraPrep treatment in over 95% of samples (77/81). The pre- and post-VeraPrep treatment biotin concentration [mean ± standard deviation (SD)] for specimens spiked with up to 500 ng/mL biotin was 357 ± 47 ng/mL and 1.0 ± 0.6 ng/mL, respectively. The mean (range) biotin concentration for the donor samples pre- and post-treatment was 166 (15-1029) ng/mL and 0.2 (<0.1-3) ng/mL, respectively (P = 0.004). These data demonstrate that treatment with VeraPrep Biotin™ does not affect analyte recovery in biotin-negative samples and effectively depletes both spiked and endogenous biotin in serum/plasma.

Biotin rescues mitochondrial dysfunction and neurotoxicity in a tauopathy model

Mitochondrial and metabolic dysfunction are often implicated in neurological disease, but effective mechanism-based therapies remain elusive. We performed a genome-scale forward genetic screen in a Drosophila model of tauopathy, a class of neurodegenerative disorders characterized by the accumulation of the protein tau, and identified manipulation of the B-vitamin biotin as a potential therapeutic approach in tauopathy. We show that tau transgenic flies have an innate biotin deficiency due to tau-mediated relaxation of chromatin and consequent aberrant expression of multiple biotin-related genes, disrupting both carboxylase and mitochondrial function. Biotin depletion alone causes mitochondrial pathology and neurodegeneration in both flies and human neurons, implicating mitochondrial dysfunction as a mechanism in biotin deficiency. Finally, carboxylase biotin levels are reduced in mammalian tauopathies, including brains of human Alzheimer's disease patients. These results provide insight into pathogenic mechanisms of human biotin deficiency, the resulting effects on neuronal health, and a potential therapeutic pathway in the treatment of tau-mediated neurotoxicity.

Fungal biotin homeostasis is essential for immune evasion after macrophage phagocytosis and virulence.

Biotin is an important cofactor for multiple enzymes in central metabolic processes. While many bacteria and most fungi are able to synthesise biotin de novo, Candida spp. are auxotrophic for this vitamin and thus require efficient uptake systems to facilitate biotin acquisition during infection. Here we show that Candida glabrata and Candida albicans use a largely conserved system for biotin uptake and regulation, consisting of the high-affinity biotin transporter Vht1 and the transcription factor Vhr1. Both species induce expression of biotin-metabolic genes upon in vitro biotin depletion and following phagocytosis by macrophages, indicating low biotin levels in the Candida-containing phagosome. In line with this, we observed reduced intracellular proliferation of both Candida cells pre-starved of biotin and deletion mutants lacking VHR1 or VHT1 genes. VHT1 was essential for the full virulence of C. albicans during systemic mouse infections, and the lack of VHT1 led to reduced fungal burden in C. glabrata-infected brains and C. albicans-infected brains and kidneys. Together, our data suggest a critical role of Vht1-mediated biotin acquisition for C. glabrata and C. albicans during intracellular growth in macrophages and systemic infections.

Interference From High-Dose Biotin Intake in Immunoassays for Potentially Time-Critical Analytes by Roche.

Immunoassays using the interaction between streptavidin and biotin are used for clinical chemical analytes on platforms by many different manufacturers. The design can be susceptible to interference from high-dose biotin intake in patients, which remains an often-overlooked confounder despite recently increased awareness. To evaluate an easily implementable method of in vitro biotin depletion for the removal of biotin interference in immunoassays for potentially time-critical analytes. A biotin stock solution was made and de-identified patient samples were spiked to reach a biotin concentration of 1.126 × 106 pg/mL, the maximum reported biotin concentration 1 to 2 hours after a single oral dose of 300 mg biotin. Then, the resulting interference in Elecsys immunoassays for cortisol, cyclosporine A, tacrolimus, digitoxin, thyroid-stimulating hormone, free triiodothyronine, free thyroxine, C-peptide, insulin, N-terminal pro-B-type natriuretic peptide, troponin T high sensitive, human immunodeficiency virus, procalcitonin, β human chorionic gonadotropin, toxoplasma immunoglobulin M, and toxoplasma immunoglobulin G was evaluated before and after biotin depletion using streptavidin particles. All tested immunoassays, with the exception of toxoplasma immunoglobulin M and toxoplasma immunoglobulin G, suffered from significant biotin interference. The depletion protocol removed assay interference due to biotin and produced results that were close or identical to initial prespike measurements. Despite an increase in turnaround times, biotin adsorption is a feasible countermeasure for biotin interference in Elecsys immunoassays. Until test kits with an increased resistance to the interference from high-dose biotin intake are distributed, the evaluated protocol can provide results properly reflecting the patient's clinical condition.

Host biotin is required for liver stage development in malaria parasites

Acetyl-CoA carboxylase (ACC) is a biotin-dependent enzyme that is the target of several classes of herbicides. Malaria parasites contain a plant-like ACC, and this is the only protein predicted to be biotinylated in the parasite. We found that ACC is expressed in the apicoplast organelle in liver- and blood-stage malaria parasites; however, it is activated through biotinylation only in the liver stages. Consistent with this observation, deletion of the biotin ligase responsible for ACC biotinylation does not impede blood-stage growth, but results in late liver-stage developmental defects. Biotin depletion increases the severity of the developmental defects, demonstrating that parasite and host biotin metabolism are required for normal liver-stage progression. This finding may link the development of liver-stage malaria parasites to the nutritional status of the host, as neither the parasite nor the human host can synthesize biotin.

Depletion of biotin using streptavidin-coated microparticles: a validated solution to the problem of biotin interference in streptavidin-biotin immunoassays.

Background Biotin interference in streptavidin-based immunoassays causes widespread analytical distortions that may lead to clinical confusion, inappropriate patient management and, ultimately, adverse events. Its prevalence has increased recently due to the increased use of high-dose biotin therapy in specific patient groups (notably multiple sclerosis) and possibly the general community. Methods We have developed a method to deplete biotin from samples using the streptavidin-coated magnetic microparticles that are a component of most susceptible assays. Results We show that high concentrations of spiked biotin can be adequately depleted from serum using this approach, and that gross biochemical derangements can be restored to normality. We also show that biotin in samples derived from multiple sclerosis patients receiving 300 mg biotin daily can be adequately depleted to remove associated analytical interference and restore normal results. The method is applicable to competitive and sandwich immunoassays and importantly, because it does not change the volume of the sample, suitable for the measurement of free thyroid hormone assays. Application of the method does not significantly change the precision of measurement, and for the majority of analytes, the accuracy is not substantially altered. Conclusions Adopting this method enables laboratories to confirm biotin interference in the appropriate clinical setting. Moreover, it enables laboratories to remove the interference and report accurate and reliable results, without the need for patients to withhold beneficial therapy prior to blood tests. Until the biotin tolerance of susceptible assays is improved, our method gives laboratories a safe alternative for reporting results using streptavidin-based methods.

Biotin-independent strains of Escherichia coli for enhanced streptavidin production

Biotin is an archetypal vitamin used as cofactor for carboxylation reactions found in all forms of life. However, biotin biosynthesis is an elaborate multi-enzymatic process and metabolically costly. Moreover, many industrially relevant organisms are incapable of biotin synthesis resulting in the requirement to supplement defined media. Here we describe the creation of biotin-independent strains of Escherichia coli and Corynebacterium glutamicum through installation of an optimized malonyl-CoA bypass, which re-routes natural fatty acid synthesis, rendering the previously essential vitamin completely obsolete. We utilize biotin-independent E. coli for the production of the high-value protein streptavidin which was hitherto restricted because of toxic effects due to biotin depletion. The engineered strain revealed significantly improved streptavidin production resulting in the highest titers and productivities reported for this protein to date.

Selective accumulation of biotin in arterial chemoreceptors: requirement for carotid body exocytotic dopamine secretion

Biotin, a vitamin whose main role is as a coenzyme for carboxylases, accumulates at unusually large amounts within cells of the carotid body (CB). In biotin-deficient rats biotin rapidly disappears from the blood; however, it remains at relatively high levels in CB glomus cells. The CB contains high levels of mRNA for SLC5a6, a biotin transporter, and SLC19a3, a thiamine transporter regulated by biotin. Animals with biotin deficiency exhibit pronounced metabolic lactic acidosis. Remarkably, glomus cells from these animals have normal electrical and neurochemical properties. However, they show a marked decrease in the size of quantal dopaminergic secretory events. Inhibitors of the vesicular monoamine transporter 2 (VMAT2) mimic the effect of biotin deficiency. In biotin-deficient animals, VMAT2 protein expression decreases in parallel with biotin depletion in CB cells. These data suggest that dopamine transport and/or storage in small secretory granules in glomus cells depend on biotin. Biotin is a water-soluble vitamin required for the function of carboxylases as well as for the regulation of gene expression. Here, we report that biotin accumulates in unusually large amounts in cells of arterial chemoreceptors, carotid body (CB) and adrenal medulla (AM). We show in a biotin-deficient rat model that the vitamin rapidly disappears from the blood and other tissues (including the AM), while remaining at relatively high levels in the CB. We have also observed that, in comparison with other peripheral neural tissues, CB cells contain high levels of SLC5a6, a biotin transporter, and SLC19a3, a thiamine transporter regulated by biotin. Biotin-deficient rats show a syndrome characterized by marked weight loss, metabolic lactic acidosis, aciduria and accelerated breathing with normal responsiveness to hypoxia. Remarkably, CB cells from biotin-deficient animals have normal electrophysiological and neurochemical (ATP levels and catecholamine synthesis) properties; however, they exhibit a marked decrease in the size of quantal catecholaminergic secretory events, which is not seen in AM cells. A similar differential secretory dysfunction is observed in CB cells treated with tetrabenazine, a selective inhibitor of the vesicular monoamine transporter 2 (VMAT2). VMAT2 is highly expressed in glomus cells (in comparison with VMAT1), and in biotin-deficient animals VMAT2 protein expression decreases in parallel with the decrease of biotin accumulated in CB cells. These data suggest that biotin has an essential role in the homeostasis of dopaminergic transmission modulating the transport and/or storage of transmitters within small secretory granules in glomus cells.

Use of Nutritional Requirements for Brettanomyces bruxellensis to Limit Infections in Wine

Specific vitamin requirements of the wine spoilage yeast, Brettanomyces bruxellensis, were evaluated. Previous studies had not taken into influences of ethanol or nutrient carry-over between sequential transfers into vitamin-omitted media. Knowing nutritional needs, limiting growth of the yeast in wine by selective removal of important vitamins was investigated. Six strains of B. bruxellensis were grown and sequentially transferred into single vitamin-omitted media. None of the strains required p-aminobenzoic acid, folic acid, nicotinic acid, myo-inositol, pantothenic acid, or riboflavin. While some needed thiamin depending on the absence/presence of ethanol, growth of all strains was greatly affected by biotin. Here, concentrations ≥0.2 µg/L were required to achieve yeast populations &gt;106 cfu/mL for high (104 cfu/mL) or low (102 cfu/mL) initial inoculums. At concentrations &lt;0.2 µg/L, culturabilities either remained unchanged or increased less than two logs after 40 days. Since the protein avidin binds irreversibly to biotin, egg whites containing avidin or the purified protein were added to a wine to diminish bioavailability of the vitamin. While biotin concentrations were reduced, populations of B. bruxellensis achieved were decreased by one to three logs, thereby supporting further development of biotin depletion strategies in winemaking.

Microarray analysis of pancreatic gene expression during biotin repletion in biotin-deficient rats.

Biotin is a B vitamin involved in multiple metabolic pathways. In humans, biotin deficiency is relatively rare but can cause dermatitis, alopecia, and perosis. Low biotin levels occur in individuals with type-2 diabetes, and supplementation with biotin plus chromium may improve blood sugar control. The acute effect on pancreatic gene expression of biotin repletion following chronic deficiency is unclear, therefore we induced biotin deficiency in adult male rats by feeding them a 20% raw egg white diet for 6 weeks. Animals were then randomized into 2 groups: one group received a single biotin supplement and returned to normal chow lacking egg white, while the second group remained on the depletion diet. After 1 week, pancreata were removed from biotin-deficient (BD) and biotin-repleted (BR) animals and RNA was isolated for microarray analysis. Biotin depletion altered gene expression in a manner indicative of inflammation, fibrosis, and defective pancreatic function. Conversely, biotin repletion activated numerous repair and anti-inflammatory pathways, reduced fibrotic gene expression, and induced multiple genes involved in pancreatic endocrine and exocrine function. A subset of the results was confirmed by quantitative real-time PCR analysis, as well as by treatment of pancreatic AR42J cells with biotin. The results indicate that biotin repletion, even after lengthy deficiency, results in the rapid induction of repair processes in the pancreas.

Biotinylation of the c‐myc promoter binding protein MBP‐1 decreases c‐myc expression in mammary carcinoma MCF‐7 cells

Biotin feeding studies in mice predisposed to developing breast cancer suggest that the tumor load increases by ~100% in biotin‐deficient mice compared with controls, and that the increase is associated with metastasis rather than new tumors. The oncogene c‐myc is implicated in cancer metastasis, and its expression is repressed by binding of MBP‐1 to the c‐myc promoter. Discovery‐based studies for identifying novel biotin‐containing proteins suggest that MBP‐1 contains covalently bound biotin, judged by mass spectrometry. We hypothesized that biotinylation of MBP‐1 increases the binding of MBP‐1 to the c‐myc promoter, thereby decreasing c‐myc expression and metastasis. MCF‐7 cells were cultured in biotin‐defined media (deficient, sufficient, supplemented) and transfected with a reporter gene construct for the c‐myc promoter. Reporter activity was ~100% greater in biotin‐deficient cells compared with sufficient and supplemented controls. Likewise, c‐myc mRNA levels were 10% higher in deficient compared with sufficient cells, and 10% high in sufficient compared with supplemented cells. Biotinylation of MBP‐1 is an enzymatic process, judged by streptavidin blots. These observations are consistent with our hypothesis and preliminary studies, suggesting that biotin depletion causes an increased expression of the oncogene c‐myc, thereby increasing cancer risk.Grant Funding Source: ARD Hatch, NIFA, and NIH

Epigenetic synergies between methyl donors and biotin in gene repression are mediated by holocarboxylase synthetase (HLCS)

HLCS catalyzes the binding of biotin to proteins in humans. Previously, we have shown (1) that HLCS interacts physically with the methyl transferase EHMT‐1, which creates histone H3 K9 methylation (H3K9me) gene repression marks, (2) that biotinylation of EHMT‐1 strengthens interactions with HLCS, (3) that biotin depletion and HLCS knockdown cause a loss of H3K9me marks leading to de‐repression of long terminal repeats (LTRs) and to chromosomal abnormalities, and (4) that erasure of DNA methylation impairs HLCS‐dependent biotinylation events. Here, we tested the novel hypothesis that methyl donors and biotin synergize in mediating gene repression through epigenetic marks, and that HLCS contributes toward mediating these synergies. Co‐immunoprecipitation (Co‐IP) and limited proteolysis assays (LPA) confirmed that HLCS interacts physically with the DNA methyl transferase DNMT1 and methyl CpG binding protein MeCP2. Overexpression of HLCS in HEK293 cells caused a 200% increase of H3K9me marks in LTRs (Kruskal‐Wallis, P&lt;0.001), judged by ChIP, and an 84% decrease in LTR mRNA (t‐test, P&lt;0.05), judged by qRT‐PCR, compared with controls; erasure of DNA methylation by azacytidine abrogated effects of HLCS. We conclude that interactions between HLCS and chromatin proteins create epigenetic synergies between biotin and methyl donors in gene repression, thereby maintaining genome stability.Grant Funding Source: ARD Hatch, NIFA, NIH

The Biotinidase Gene Variants Registry: A Paradigm Public Database

The BTD gene codes for production of biotinidase, the enzyme responsible for helping the body reuse and recycle the biotin found in foods. Biotinidase deficiency is an autosomal recessively inherited disorder resulting in the inability to recycle the vitamin biotin and affects approximately 1 in 60,000 newborns. If untreated, the depletion of intracellular biotin leads to impaired activities of the biotin-dependent carboxylases and can result in cutaneous and neurological abnormalities in individuals with the disorder. Mutations in the biotinidase gene (BTD) alter enzymatic function. To date, more than 165 mutations in BTD have been reported. Our group has developed a database that characterizes the known mutations and sequence variants in BTD. (http://arup.utah.edu/database/BTD/BTD_welcome.php). All sequence variants have been verified for their positions within the BTD gene and designated according to standard nomenclature suggested by Human Genome Variation Society (HGVS). In addition, we describe the change in the protein, indicate whether the variant is a known or likely mutation vs. a benign polymorphism, and include the reference that first described the alteration. We also indicate whether the alteration is known to be clinically pathological based on an observation of a known symptomatic individual or predicted to be pathological based on enzymatic activity or putative disruption of the protein structure. We incorporated the published phenotype to help establish genotype-phenotype correlations and facilitate this process for those performing mutation analysis and/or interpreting results. Other features of this database include disease information, relevant links about biotinidase deficiency, reference sequences, ability to query by various criteria, and the process for submitting novel variations. This database is free to the public and will be updated quarterly. This database is a paradigm for formulating databases for other inherited metabolic disorders.

Differential effect of biotin on carboxylase activity and mice skeletal muscle metabolism

In mammalian skeletal muscle there are four carboxylases involved in several biochemical processes like gluconeogenesis, tricarboxylic acid cycle anaplerosis, metabolism of fatty acids and metabolism of various amino acids. It has been shown that biotin deficiency reduces body weight at the expense of muscular mass. When necessary, the liver uses skeletal muscle protein to provide glucose and amino acids to organs in need of such compounds. In this paper we analyzed carboxylase specific activities in hind limb skeletal muscle of 3 weeks old BALB/c male mice, at 0, 1, 4, 7, and 14 days of a specific diet with different biotin concentrations. Biotin was used at 0.0, 1.8 or 98.2 mg per kg of food; and was referred to as biotin deficient, sufficient and supplemented, respectively. Water and food supply and consumption by the three groups of mice were the same. Therefore, the observed effects were directly related to biotin ingestion. The body weight of biotin supplemented mice was the same as the body weight of mice in the biotin sufficient group, while biotin deficiency caused body weight reduction after 7 days of biotin depletion. We found that the total protein concentration in the vastus lateralis muscle is associated with the biotin content in the diet. After 7 days, the muscle total protein content was lower in mice of the biotin deficient group while it was higher in the mice from the biotin supplemented group (P

Temporal development of genetic and metabolic effects of biotin deprivation. A search for the optimum time to study a vitamin deficiency

Biotin deficiency (Bt-D) is usually studied at the point at which the animal model exhibits the signs of full-blown deficiency symptoms; in rats, this typically occurs at 6–8weeks of feeding a deficient diet. To differentiate specific deficiency effects from those of undernutrition, biotin sufficient and deficient rats were studied at 2, 3, 4, and 5weeks on the deficiency diet, before the onset of weight loss and deficiency signs. The deficiency state was confirmed by biochemical and molecular analyses. Blood and liver metabolites were determined and western blots of signaling proteins, and qRT-PCR gene expression studies. The main effects of Bt-D were already well established by the fourth week on the diet; thus, we consider the fourth week as the optimum time to study the consequences of biotin depletion. Early effects, which were already apparent at week 2, included cellular energy deficit (as assessed by increased AMP/ATP ratio), activation of the AMPK energy sensor, and changes of carbon metabolism gene transcripts (e.g., phosphoenolpyruvate carboxykinase, carnitine palmitoyl transferase 1, liver glucokinase and fatty acid synthetase). Reduced post-prandial blood concentrations of glucose were also observed early; we speculate that these are attributable to augmented sensitivity to insulin and increased glucose utilization, a likely effect of AMPK induction of translocation of glucose transporter GLUT4 to the cell membranes and increased hexokinase expression. Other late-onset changes (week 4) included increased serum concentrations of lactate and free fatty acids and decreased liver glycogen and serum concentrations of triglycerides and total cholesterol. The identification of the early specific molecular and metabolic disturbances of biotin deficiency might be useful in identifying individuals with marginal deficiency of this vitamin, which appears to be common in normal human pregnancy. The study of time-course of other vitamin deficiencies, such as this one, might help to better understand and cope with their effects.