High Concentrations Of Biotin Research Articles

ESC 0/1h-algorithm using the updated, biotin-resistant high-sensitivity cardiac troponin T assay

Abstract Background For rapid and safe triage of patients with suspected non-ST-elevation myocardial infarction (NSTEMI), the European Society of Cardiology (ESC) recommends the use of a 0/1h-algorithm based on high-sensitivity cardiac troponin (hs-cTn) T or I. Recently, multiple widely used immuno-assays including hs-cTnT have been reported to interfere with high biotin concentrations, potentially leading to false-negative results. To address this major concern, an updated, biotin-resistant hs-cTnT assays has been developed by the manufacturer to replace its previous version. Purpose We aimed to assess and compare the clinical performance of the updated, biotin-resistant hs-cTnT Elecsys assay when using the original ESC hs-cTnT 0/1h-algorithm. Methods In a prospective international diagnostic study enrolling patients presenting with suspected NSTEMI to the emergency department (ED), hs-cTnT was serially measured at ED presentation and after one hour with the original, biotin-sensitive (hs-cTnT-original) and the updated, biotin-resistant hs-cTnT (hs-cTnT-updated) assay. Patients presenting with STEMI were excluded. The final diagnosis was centrally adjudicated by two independent cardiologists using all available data including coronary angiography, echocardiography, follow-up data, and serial measurements of hs-cTn. We assessed the overall efficacy, rule-out safety and rule-in accuracy of the ESC hs-cTnT 0/1h-algorithm when using hs-cTnT-updated and compared it with the use of hs-cTnT-original. Results NSTEMI was the final diagnosis in 162 of 865 (19%) patients. The ESC 0/1h-algorithm using hs-cTnT-updated triaged 58% of patients towards "rule-out", 21% towards "rule-in", and 21% towards "observe" compared to 56%, 21% and 23% using hs-cTnT-original, respectively (Figure 1, p=ns). Only 6 patients were reclassified using hs-cTnT-updated. Safety of rule-out was very high using hs-cTnT-updated with a negative predictive value for NSTEMI of 100% (95% confidence interval (CI), 99.2-100%) and a sensitivity of 100% (95%CI, 97.7-100%) and was highly comparable to hs-cTnT-original (NPV 100% [95%CI 99.2-100], sensitivity 100% [95%CI, 97.7-100%], all p-values for comparison not significant). Of note, no NSTEMI-patient was missed by the ESC 0/1h-algorithm neither using hs-cTnT-updated nor original. Similarly, accuracy of rule-in was very high (positive predictive value 73.7% [95%CI, 66.8-79.6%], specificity 93.3% [95%CI, 91.2-94.9%]) and highly comparable to hs-cTnT-original (PPV 73.9% [95%CI 67.1-79.7%], specificity 93.2% [95%CI, 91.1-94.8%], all p-values for comparison not significant). Conclusions The safety and efficacy of the ESC 0/1h-algorithm using the novel updated, biotin-resistant hs-cTnT-assay is excellent and similar to the use of the original assay without the need to adjust the cutoffs to the new assay.Figure 1

Effects of High-Biotin Sample Interference on Antibody Concentrations in Sandwich Immunoassays.

The use of antimicrobial growth promoters (AGPs) is banned because of problems associated with drug residues in animal products and increased bacterial resistance. The immunization of chickens with specific antigens is a promising strategy for generating specific antibodies that can target a wide range of antibiotic-resistant bacteria and can be used as an alternative to antibiotics. Immunoglobulin Y (IgY) antibodies in a polyclonal antibody (pAb) format, when administered orally, modulate the ruminal microbiome and maintain animal health and performance; however, there are concerns pertaining to protein impurities and biotin concentrations in the samples. Signal amplification strategies involving the noncovalent interaction of biotin with streptavidin is extensively used in diagnosis and scientific research, particularly in enzyme-linked immunosorbent assays (ELISAs). However, the high concentrations of biotin in samples, especially in those derived from rich sources such as egg yolk, can pose challenges and potentially harm the accuracy of diagnostic tests and protein concentration measurements. This study aimed to evaluate the influence of biotin on the measurement of IgY in freeze-dried egg yolk samples obtained from immunized laying hens using immunoassays with biotin-avidin/streptavidin. The detection of IgY in yolk samples using ELISA with streptavidin-biotin binding could lead to misdiagnosis due to biotin interference; the level of interference varies with the specific assay conditions and the concentration of biotin in the yolk samples. An ELISA without streptavidin-biotin binding is advisable to avoid interactions between biotin and target proteins, prevent biotin interference with the results, and achieve more reliable and accurate results.

Biotin interference in routine IDS-iSYS immunoassays for aldosterone, renin, insulin-like growth factor 1, growth hormone and bone alkaline phosphatase

Biotin is increasingly used as dietary supplement. As many immunoassays rely on a binding between biotin and streptavidin, intake of biotin may interfere with laboratory tests, leading to spurious test results. We examined the extent to which levels of aldosterone, renin, insulin-like growth factor 1 (IGF-1), growth hormone (GH) and bone alkaline phosphatase (BAP) were affected by biotin. In an experimental study performed at Aarhus University Hospital, Denmark, patient samples (plasma or serum) were pooled and spiked with biotin in increasing concentrations (0, 20, 50, 100 and 500 ng/mL). All biomarkers were analyzed using Immunodiagnostic Systems (IDS-iSYS) Multi-Discipline Automated System assays. The average bias (%) was calculated, as the difference in concentrations between the sample without biotin (reference) and the samples with increasing concentrations of biotin. Both aldosterone and renin assays showed substantial biotin interference in a dose-dependent manner, with biases up to +3484% for aldosterone and −98% for renin in the highest concentrations of biotin (100–500 ng/mL). IGF-1, GH and BAP results were generally less affected by added biotin and significant bias (>10%) was observed only when the biotin concentration was 100 ng/mL (IGF-1 and GH) or 500 ng/mL (BAP). In conclusion, biotin interfered with the IDS-iSYS immunoassays, particularly for aldosterone and renin. The assays for GH, IGF-1 and BAP were less sensitive and only with high concentrations of biotin.

Serum biotin interference: A troublemaker in hormone immunoassays

ObjectivesBiotin therapy can affect the results of many immunoassay procedures. The present study investigates biotin’s interference on 25-hydroxy vitamin D (25-OHD), parathyroidhormone (PTH) and thyroid-stimulating hormone (TSH) tests using four different assay systems and biotin neutralization. Design and methodsEnrolled in the study were 50 children diagnosed with biotinidase deficiency (BTD) undergoing treatment with biotin (5–20 mg/day) who were subjected to a series of analyses involving 25-OHD (Roche Diagnostics assays, Beckman Coulter assays, HPLC, LC/MS-MS), TSH, PTH (Roche Diagnostics assays, Beckman Coulter assays) and biotin (LC/MS-MS), before and after biotin neutralization with Streptavidin-coated magnetic particles (SMP). ResultsThe median biotin concentration was found to be 175.2 [94.0–307.1] μg/L. There was no significant difference in the 25-OHD results before and after neutralization with the Beckman Coulter, HPLC and LC-MS/MS assays. In contrast, the median 25-OHD level was seen to decrease from 90.2 [35.9–105.3] ng/mL to 29.1 [22.6–37.6] ng/mL after neutralization with the Roche assay (p < 0.0001). While there was no statistically significant difference in the values recorded before and after neutralization in PTH analysis using Beckman assay, the median PTH levels increased from 7.8 [1.6–21.6] pg/mL to 28.2 [22.5–41.9] pg/mL after neutralization with the Roche assay (p < 0.0001). The cut-off values at which serum biotin interfered in the Roche assay PTH test, with 25-OHD levels determined as 51.4 μg/L and 62.9 μg/L, respectively. A significant increase was detected in the TSH levels analyzed with a Roche assay after neutralization (from 2.36 [1.85–3.00] mIU/L to 2.74 [1.93–3.70] mIU/L, p < 0.0001). ConclusionsThe PTH, 25-OHD and TSH results were found to be affected by high biotin concentrations in Roche assays, leading to a risk of misdiagnosis, although SMP neutralization can suppress any such interference efficiently.

Biotin Interference in Point of Care HIV Immunoassay.

The use of high concentrations of biotin as a dietary supplement to improve hair, skin, and nail quality has increased in the United States over the past few years. High concentrations of biotin have been shown to interfere with some diagnostic assays that use streptavidin–biotin interactions as one of the steps in the assay. The objective of this report is to evaluate potential biotin interference on the analytical and clinical sensitivity of a point of care (POC) antigen–antibody combo HIV-1 assay. We spiked biotin at concentrations ranging from 12.5 to 400 ng/mL into serum and plasma containing HIV-1 subtype B p24 antigen derived from culture supernatant. The p24 antigen was present in the matrices at 30 pg/mL. Fifty microliters of each sample was applied to Alere Determine HIV-1/2 Ag/Ab combo assay strips in duplicate and results were read by eye after 20 to 30 min. Biotin interfered with detection of HIV-1 p24 in serum and plasma. HIV-1 p24 was not detected at 30 pg/mL p24 when biotin was present at 200 ng/mL concentration. Our study demonstrated that elevated levels of biotin in samples may interfere with POC assays. It is important to consider biotin supplements as potential sources of falsely increased or decreased test results, especially in cases wherein supplementation cannot be ruled out.

Dietary Biotin Supplementation Impairs Testis Morphology and Sperm Quality.

Supplements containing pharmacological concentrations of biotin are commercially available over the counter. Classical toxicity studies have considered biotin administration as harmless; however, recent investigations have shown that biotin supplementation modifies tissue morphology without changes in toxicity markers, raising concerns about the consequences of morphological changes on tissues' functions and the safety of pharmacological concentrations of the vitamin. Testes are very sensitive to toxicants, and testicular histology is a reliable method to study its function. In this work, we investigated the effects of dietary biotin supplementation on testis morphology and spermatogenesis function using an experimental model, in which we have not observed unfavorable effects on other tissue functions or toxicity markers. Male BALB/cAnNHsd mice were fed a control or a biotin-supplemented diet (1.76 or 97.7 mg biotin/kg diet) for 8 weeks. Compared to the control group, the biotin-supplemented mice presented remarkable testis morphology changes, including increased spermatogonia layers; the cellular mechanism involved is related to increased proliferation. Sperm count and serum testosterone levels were not affected, but spermatozoa motility and morphology were significantly impaired in the biotin-supplemented mice. These results caution against the use of supplements with high concentrations of biotin and indicate that biotin's pharmacological effects on morphology need to be considered in toxicological studies.

In vitro BioID: mapping the CENP-A microenvironment with high temporal and spatial resolution.

The centromere is located at the primary constriction of condensed chromosomes where it acts as a platform regulating chromosome segregation. The histone H3 variant CENP-A is the foundation for kinetochore formation. CENP-A directs the formation of a highly dynamic molecular neighborhood whose temporal characterization during mitosis remains a challenge due to limitations in available techniques. BioID is a method that exploits a “promiscuous” biotin ligase (BirA118R or BirA*) to identify proteins within close proximity to a fusion protein of interest. As originally described, cells expressing BirA* fusions were exposed to high biotin concentrations for 24 h during which the ligase transferred activated biotin (BioAmp) to other proteins within the immediate vicinity. The protein neighborhood could then be characterized by streptavidin-based purification and mass spectrometry. Here we describe a further development to this technique, allowing CENP-A interactors to be characterized within only a few minutes, in an in vitro reaction in lysed cells whose physiological progression is “frozen.” This approach, termed in vitro BioID (ivBioID), has the potential to study the molecular neighborhood of any structural protein whose interactions change either during the cell cycle or in response to other changes in cell physiology.

Biotin and high-sensitivity cardiac troponin T assay

IntroductionThe high-sensitivity cardiac troponin T assay of Roche Diagnostics is known to have interference with high concentrations of biotin as this assay uses biotin-streptavidin binding as detection method. As studies so far have not shown if different biotin concentrations could have diverse influence on various troponin concentrations and whether interference could be removed by available protocol within corresponding turnaround time we aimed to investigate it.Materials and methodsPlasma samples were spiked with different concentration of biotin solution. Troponin T concentrations were tested on a Roche Cobas® 8000 module 602 analyser. Final concentrations of biotin and troponin T were 50, 100, 500 and 1000 μg/L and 18, 59, 201 and 6423 ng/L, respectively. Impact of different incubation times following biotin neutralization protocol described by Piketty et al. was also tested.ResultsWe observed a mean of negative biases of 24, 56, 97, and 98% of the troponin T expected value at biotin concentrations of 50, 100, 500, 1000 μg/L. Neutralization protocol was applied on the sample with initial concentration of TnT of 59 ng/L at a biotin concentration of 1000 μg/L. Same results across different incubation times from 60 to 0 minutes were obtained (mean value 56.8 ng/L, coefficient of variation of 1.31%). We demonstrated that neutralization process had a dilution effect of the troponin concentration (loss of 4.5% to 9.6% of initial troponin value).ConclusionsBiotin interference is not dependent of initial troponin value. Interference could be successfully neutralized within a time frame compatible with emergency but results still should be carefully interpreted due to possible dilution effect.

Screening of Proximal and Interacting Proteins in Rice Protoplasts by Proximity-Dependent Biotinylation.

Proximity-dependent biotin identification (BioID), which detects physiologically relevant proteins based on the proximity-dependent biotinylation process, has been successfully used in different organisms. In this report, we established the BioID system in rice protoplasts. Biotin ligase BirAG was obtained by removing a cryptic intron site in the BirA∗ gene when expressed in rice protoplasts. We found that protein biotinylation in rice protoplasts increased with increased expression levels of BirAG. The biotinylation effects can also be achieved by exogenous supplementation of high concentrations of biotin and long incubation time with protoplasts. By using this system, multiple proteins were identified that associated with and/or were proximate to OsFD2 in vivo. Our results suggest that BioID is a useful and generally applicable method to screen for both interacting and neighboring proteins in their native cellular environment in plant cell.

High-dose biotin therapy leading to false biochemical endocrine profiles: validation of a simple method to overcome biotin interference.

High-dose biotin therapy is beneficial in progressive multiple sclerosis (MS) and is expected to be adopted by a large number of patients. Biotin therapy leads to analytical interference in many immunoassays that utilize streptavidin-biotin capture techniques, yielding skewed results that can mimic various endocrine disorders. We aimed at exploring this interference, to be able to remove biotin and avoid misleading results. We measured free triiodothyronine (fT3), free thyroxine (fT4), thyroid-stimulating hormone (TSH), parathyroid homrone (PTH), 25-hydroxyvitamin D (25OHD), follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin, C-peptide, cortisol (Roche Diagnostics assays), biotin and its main metabolites (liquid chromatography tandem mass spectrometry) in 23 plasmas from MS patients and healthy volunteers receiving high-dose biotin, and in 39 biotin-unsupplemented patients, before and after a simple procedure (designated N5) designed to remove biotin by means of streptavidin-coated microparticles. We also assayed fT4, TSH and PTH in the 23 high-biotin plasmas using assays not employing streptavidin-biotin binding. The biotin concentration ranged from 31.7 to 1160 µg/L in the 23 high-biotin plasmas samples. After the N5 protocol, the biotin concentration was below the detection limit in all but two samples (8.3 and 27.6 μg/L). Most hormones results were abnormal, but normalized after N5. All results with the alternative methods were normal except two slight PTH elevations. In the 39 biotin-unsupplemented patients, the N5 protocol did not affect the results for any of the hormones, apart from an 8.4% decrease in PTH. We confirm that most streptavidin-biotin hormone immunoassays are affected by high biotin concentrations, leading to a risk of misdiagnosis. Our simple neutralization method efficiently suppresses biotin interference.

A versatile Escherichia coli strain for identification of biotin transporters and for biotin quantification

Biotin is an essential cofactor of carboxylase enzymes in all kingdoms of life. The vitamin is produced by many prokaryotes, certain fungi, and plants. Animals depend on biotin uptake from their diet and in humans lack of the vitamin is associated with serious disorders. Many aspects of biotin metabolism, uptake, and intracellular transport remain to be elucidated. In order to characterize the activity of novel biotin transporters by a sensitive assay, an Escherichia coli strain lacking both biotin synthesis and its endogenous high-affinity biotin importer was constructed. This strain requires artificially high biotin concentrations for growth. When only trace levels of biotin are available, it is viable only if equipped with a heterologous high-affinity biotin transporter. This feature was used to ascribe transport activity to members of the BioY protein family in previous work. Here we show that this strain together with its parent is also useful as a diagnostic tool for wide-concentration-range bioassays.

Identification of biotin‐ and holocarboxylase synthetase‐dependent microRNAs in human fibroblasts

MicroRNAs (miRs) regulate their target genes by binding to the 3′‐UTR. Previously we reported that the expression of miR‐539 depends on biotin, thereby regulating the expression of holocarboxylase synthetase (HCS) in human kidney cells. HCS appears to be part of a multiprotein gene repression complex in human chromatin. We hypothesized that both biotin and HCS play important roles in regulating miRs. IMR‐90 primary human fibroblasts were cultured in media containing deficient, normal, and high biotin concentrations; in addition, HCS mutant fibroblast were cultured in high biotin medium to mimic the scenario in HCS‐deficient patients receiving biotin supplements. miRs were sequenced using the SOLiD platform. Expression analyses were performed using edgeR, R software. The expression of miR‐378 and miR‐34a* was greater in biotin‐deficient and ‐supplemented IMR‐90 cells, respectively, compared with biotin‐normal controls (p &lt; 0.01). The expression of miR‐2110 and miR‐150 was lower, in biotin‐deficient and supplemented cells, respectively, compared with controls (p &lt; 0.01). The expression of 58 miRs increased or decreased in HCS‐mutant fibroblasts compared to IMR‐90 cells in biotin‐normal medium (p &lt; 0.01). We conclude that HCS activity is of greater importance than biotin supply in regulating miR expression. (SSW and SAM contributed equally.)(UNL ARD Hatch Act, NIH DK063945, DK077816, and DK082476)

A biosensor based on photonic crystal surface waves with an independent registration of the liquid refractive index

A high-precision optical biosensor technique capable of independently determining the refractive index (RI) of liquids is presented. Photonic crystal surface waves were used to detect surface binding events, while an independent registration of the critical angle was used for accurate determination of the liquid RI. This technique was tested using binding of biotin molecules to a streptavidin monolayer at low and high biotin concentrations. The attained baseline noise is 5 × 1 0 − 13 m/Hz 1/2 for adlayer thickness changes and 9 × 1 0 − 8 RIU/Hz 1/2 for RI changes.

Homeostasis of biotin in human lymphoma and liver cells

Biotin is covalently attached to carboxylases and histones in a reaction catalysed by holocarboxylase synthetase (HCS). Here we determined whether biotin concentrations in culture media affect the expression of genes maintaining biotin homeostasis in human lymphoid (Jurkat) cells. As a secondary objective we determined whether mechanisms of biotin homeostasis are distinct in cells with great metabolic activities, i.e., human hepatocarcinoma (HepG2) cells. Jurkat cells were cultured in media containing deficient, physiological, or high concentrations of biotin for 36 d. While the abundances of biotinylated carboxylases responded rapidly (&lt;3 d) to altered biotin supply, changes in histone biotinylation were not apparent until day 36 when biotin supplementation caused increased biotinylation of histones in nuclear extracts. The expression of genes linked with biotin homeostasis depended on biotin in Jurkat cells. The expression of biotin transporters SMVT and MCT1 increased in biotin‐deficient cells, whereas the expression of HCS increased in biotin‐supplemented cells. In ongoing studies we investigate the effects of biotin supply on histone biotinylation at distinct genomic loci by ChIP assays, and we compare biotin homeostasis in Jurkat cells to HepG2 liver cells. (Supported by UNL ARD Hatch Act funds, NIH DK063945, DK077816 and ES015206, USDA CSREES 2006‐35200‐17138, and NSF EPSCoR EPS‐0701892.)

Proliferation and differentiation of cultured human follicular keratinocytes are not influenced by biotin.

In humans and in animals, biotin deficiency causes pathological changes in the skin and its appendages. High doses of biotin may also have beneficial effects on skin, hair and fingernails in humans and animals with normal biotin status. Therefore, we investigated the effects of low and high concentrations of biotin on proliferation and differentiation of cultured outer root sheath cells from human hair follicles as an in vitro model for skin. The activities of biotin-dependent carboxylases were measured to evaluate the biotin status of the cells. In monolayer cultures of outer root sheath cells, proliferation and expression of the differentiation-specific keratins K1 and K10 were not influenced by extremely low concentrations of biotin (<2 x 10(-10) mol/l) or by pharmacological doses of biotin (10(-5) mol/l). Biotin deficiency of the cells was confirmed under the former condition by demonstrating decreased activities of the mitochondrial carboxylases. In organotypic cocultures of outer root sheath cells and dermal fibroblasts, in which stratified epithelia resembling epidermis were developed, the biotin concentration had no effect on the expression of all tested epidermal differentiation markers, including the suprabasal keratins K1 and K10, the hyperproliferation-associated keratin K16, involucrin and filaggrin.

Flow cytometric detection of receptors for interleukin-6 on bone marrow and peripheral blood cells of humans and rhesus monkeys

The expression of receptors for interleukin-6 (IL-6) on human and rhesus monkey peripheral blood and bone marrow (BM) cells was examined by multiparameter flow cytometry after staining with biologically active, biotin-labeled human IL-6 and phycoerythrin-conjugated streptavidin. Consistent with the multiple biologic effects of IL-6 in stimulating immune functions and hematopoiesis, IL-6 receptors were detectable on a wide variety of cell types. In peripheral blood, IL-6 receptors were detectable on monocytes, granulocytes, and on CD4+ T lymphocytes but not on resting, CD19+ B lymphocytes and CD56+ natural killer (NK) cells. CD8+ T lymphocytes also expressed IL-6 receptors but at lower levels than CD4+ cells. The IL-6 receptors on granulocytes were only detectable after staining with high concentrations of biotin- IL-6, suggesting that most IL-6 receptors on these cells represent low- affinity sites. In contrast, IL-6 receptors on both CD4+ and CD8+ T lymphocytes were detectable at biotin-IL-6 concentrations as low as 10 pmol/L, indicating that these cells bind IL-6 with high affinity. IL-6 receptor expression patterns on rhesus monkey and human blood cells were very similar except that receptor levels on granulocytes were lower in humans than in rhesus monkeys. Similar differences in expression levels were observed for IL-6 receptors that were detectable on most granulocyte precursors in the mononuclear fraction of rhesus monkey and human bone marrow. In addition to these relatively mature cell types, IL-6 receptors were detectable on a large fraction of human and rhesus monkey BM blast cells that express the CD34 antigen. The presence of IL-6 receptors on CD34+ BM blast cells, which are the precursor cells of most, if not all, BM-derived blood cells, is consistent with the ability of IL-6, in conjunction with other cytokines, to stimulate immature hemopoietic cells in vitro and to promote blood cell production when administered in vivo.

Taenia taeniaeformis: Early Inflammatory Response around Developing Metacestodes in the Liver of Resistant and Susceptible Mice I. Identification of Leukocyte Response with Monoclonal Antibodies

Female BALB/cJ (resistant), C3H/HeJ (intermediate resistant), and C3H/HeDub (susceptible) inbred mice, 4-5 wk old, were infected with Taenia taeniaeformis. Liver sections were stained by an immunoperoxidase technique (avidin-biotin complex, ABC) for the differentiation antigens Lyt-1, Lyt-2, Mac-1, Mac-2, Mac-3, and B220. Binding of ABC to the cytoplasm of hepatocytes around the developing parasite was observed at 4 days postinfection (PI) in all 3 strains of mice, persisting in BALB/cJ and C3H/HeJ liver sections at 5 and 6 days PI, suggesting the presence of high concentrations of biotin, a fatty acid synthesis mediator. Two cell populations were labeled with B220 monoclonal antibodies: lymphocytes and polymorphonuclear (PMN) cells. At 4 days PI the number of labeled PMN cells peaked in infected C3H/HeJ and BALB/cJ mice; however a low number of PMN cells were labeled in infected C3H/HeDub mice. Few lymphocytes bound the B220 antibody in either BALB/cJ, C3H/HeJ, or C3H/HeDub infected mice. The number of Mac-1+ cells detected in infected C3H/HeJ and BALB/cJ liver sections were similar whereas fewer Mac-1+ cells were present in infected C3H/HeDub mice. Mac-2+ cells appeared in high numbers around the growing parasite at 5 and 6 days PI in the liver of C3H/HeDub mice, but not in the liver of BALB/cJ mice. Mac-3+ cells followed a similar pattern to that of the cell population defined by Mac-2. Few Lyt-1+ and Lyt-2+ cells were detected around the parasite site in the 3 strains of mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of biotin responsiveness in cultured fibroblasts from patients with propionic acidemia: absence of response by structurally altered carboxylases.

We have demonstrated that, although propionyl CoA carboxylase (PCC) activity is deficient in fibroblast extracts from PCC-deficient patients belonging to the two major and two minor genetic complementation groups, the activity of another biotin-dependent carboxylase, beta-methylcrotonyl CoA carboxylase (betaMCC), is normal. Moreover, betaMCC activity is stimulated when the fibroblasts are cultured in high concentrations of biotin, in the same way that it is in normal fibroblasts, whereas the depressed PCC activity remains essentially unchanged. Because these results are parallel with the in vivo failure of high-dose biotin to stimulate PCC activity in peripheral blood leukocytes, we conclude that the biotin responsiveness of PCC in cultured fibroblasts from patients with PCC deficiency may be used to predict or confirm biotin responsiveness in vivo.

A mutant of Escherichia coli that requires high concentrations of biotin.

A recessive mutation (bir) of E. coli, at 77 min on the standard map, destroys ability of the bacteria to take up biotin or to grow except at high concentrations of biotin. In a biotin prototroph, the bir mutation causes overproduction and excretion of biotin, and derepression of at least one biosynthetic gene (bioD). Growth of the bir mutant is inhibited by avidin or high concentrations of dethiobiotin. Both effects are reversed by biotin.

Irreversible inhibition of biotin transport in yeast by biotinyl-p-nitrophenyl ester.

Biotinyl-p-nitrophenyl ester (BNP), an active-ester derivative of biotin, irreversibly inactivates biotin transport in the yeast Saccharomyces cerevisiae. Transport inactivation is progressive with time and occurs at concentrations of the ester as low as 10(-7) M. In the presence of sodium azide, a reagent known to block biotin accumulation in yeast, the derivative is still effective. The specificity of inactivation by the ester is revealed by the following findings: (a) Biotinyl-p-nitroanilide and acetyl-p-nitrophenyl ester do not affect biotin transport; (b) the nitrophenyl ester does not affect the transport of lysine and aspartic acid, or that of L-sorbose; (c) inactivation of biotin transport by the ester is partially prevented when the cells are incubated with it in the presence of relatively high concentrations of biotin.