Red cell volume can be accurately determined in sheep using a nonradioactive biotin label.

The sheep has served as an informative animal model for investigation of human fetal and newborn erythropoiesis and red blood cell (RBC) kinetics. We previously validated the permanent label (14C)cyanate for measuring red cell volume (RCV) in sheep. Here, we validate biotin labeling of RBCs as a nonradioactive method for measuring RCV in sheep with the anticipation that it can be applied in studies of human infants. The RCV was determined simultaneously using two techniques for quantitation of the biotin label. The first one quantified total blood concentration of biotin label on biotin-labeled RBCs using (125I)streptavidin. The second one enumerated biotin-labeled RBCs by flow cytometry after incubation with fluorescein-conjugated avidin. RCV measurements made using the two biotin quantitation techniques were validated against both (14C)cyanate and 51Cr as reference methods. Both biotin techniques produced RCV values that agreed well with the reference methods and with each other, producing correlation coefficients averaging >or =0.93. Sequential repetitive measurements in the same animal also agreed with the (14C)cyanate method and each other (average difference <10%). These results establish biotin-labeled RBCs as an accurate method for performing RCV measurements in sheep. This biotin method can be applied in studies that model neonatal erythropoiesis.

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

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

Indicators of marginal biotin deficiency and repletion in humans: validation of 3-hydroxyisovaleric acid excretion and a leucine challenge.

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.

Marginal biotin deficiency during normal pregnancy.

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.

A biotin-protein bond with stability in plasma.

A nonradioactive label for peptide hormones would be useful for pharmacokinetic studies in infants, children, and pregnant women. Because the binding affinity between biotin and avidin is large (Ka=10(15) M(-1)), biotin could also serve as a covalent label for subsequent detection using a variety of avidin conjugates. However, biotin labels produced by most commercially available biotinylating reagents are rapidly cleaved from protein in plasma. We sought to synthesize a stable biotin label for protein. With the use of immunoglobulin G (IgG) as a model protein, biotin was conjugated through a cysteine residue; a carboxylate group was positioned alpha to the biotinamide bond. Stability of the bond in the presence of plasma and buffer control was assessed by release of biotin. Released biotin was separated from biotinylated IgG by ultrafiltration and was quantitated by an avidin-binding assay. In plasma, less than 0.6% of bound biotin was released. This release rate is not significantly different from buffer and is less than 7% of the release rate for IgG biotinylated by N-hydroxysuccinimide-LC-biotin. We conclude that this biotin-protein bond is stable in plasma. We speculate that many uses of avidin-biotin technology could be improved by using this method for protein labeling.

Biotin deficiency reduces expression of SLC19A3, a potential biotin transporter, in leukocytes from human blood.

In evaluating potential indicators of biotin status, we quantitated the expression of biotin-related genes in leukocytes from human blood of normal subjects before and after inducing marginal biotin deficiency. Biotin deficiency was induced experimentally by feeding an egg-white diet for 28 d. Gene expression was quantitated for the following biotin-related proteins: methylcrotonyl-CoA carboxylase chains A (MCCA) and B (MCCB); propionyl-CoA carboxylase chains A (PCCA) and B (PCCB); pyruvate carboxylase (PC); acetyl-CoA carboxylase isoforms A (ACCA) and B (ACCB); holocarboxylase synthetase (HCS); biotinidase; and 2 potential biotin transporters: sodium-dependent multivitamin transporter (SMVT) and solute carrier family 19 member 3 (SLC19A3). For 7 subjects who successfully completed the study, the abundance of the specific mRNAs was determined by quantitative real-time RT-PCR at d 0 and 28. At d 28, SLC19A3 expression had decreased to 33% of d 0 (P < 0.02 by two-tailed, paired t test). Expression of MCCA, PCCA, PC, ACCA, ACCB, HCS, biotinidase, and SMVT decreased to approximately 80% of d 0 (P < 0.05). Expression of the MCCB and PCCB chains that do not carry the biotin-binding motif did not change significantly; we speculate that expression of the biotin-binding chains of biotin-dependent carboxylases is more responsive to biotin status changes. These data provide evidence that expression of SLC19A3 is a relatively sensitive indicator of marginal biotin deficiency.

Lymphocyte propionyl-CoA carboxylase is an early and sensitive indicator of biotin deficiency in rats, but urinary excretion of 3-hydroxypropionic acid is not.

Recent clinical studies have provided evidence that marginal biotin deficiency is more common than previously thought. The validity of that conclusion rests on two indicators of biotin status that depend on renal function. Our goal was to develop and assess the usefulness of two additional indicators in detecting marginal biotin status in a rat model, i.e., 1) activity of the biotin-dependent enzyme propionyl-CoA carboxylase in lymphocytes; and 2) urinary excretion of 3-hydroxypropionic acid, an organic acid that reflects decreased activity of propionyl-CoA carboxylase. Marginal-to-moderate biotin deficiency was induced experimentally by an egg-white diet (deficient rats); the biotin-supplemented rats were fed the egg-white diet plus supplemental biotin. Propionyl-CoA carboxylase activity was determined by an optimized H(14)CO(3)(-) incorporation assay. Urinary organic acids were determined by gas chromatography/mass spectrometry. Lymphocyte propionyl-CoA carboxylase activity decreased dramatically and in parallel with hepatic propionyl-CoA carboxylase activity. By d 7, lymphocyte propionyl-CoA carboxylase activity in each rat in the deficient group had decreased to less than the lowest value of any rat on d 0. By two-way ANOVA, the effects of diet (P < 0.0001), time (P < 0.005) and their interaction (P < 0.0001) were all significant. The urinary excretion of 3-hydroxypropionic acid did not differ between the two groups. Lymphocyte propionyl-CoA carboxylase activity is an early and sensitive indicator of marginal biotin deficiency, whereas the urinary excretion of 3-hydroxypropionic acid is not.

Instability of the biotin-protein bond in human plasma.

Labeling proteins with biotin offers an alternative to labeling with radioisotopes for pharmacokinetic studies in humans. However, stability of the biotin-protein bond is a critical tacit assumption. Using release of biotin from immunoglobulin G as the outcome, we individually evaluated stability of the biotin label produced by six biotinylation agents: biotin PEO-amine, 5-(biotinamido)-pentylamine, iodoacetyl-LC-biotin, NHS-LC-biotin, sulfo-NHS-LC-biotin, and biotin-LC-hydrazide. Each of the six biotinylated proteins was incubated at room temperature for 4h in human plasma or in phosphate-buffered saline (control). Free biotin was separated from the biotinylated protein by ultrafiltration and quantitated by avidin-binding assay. For each biotinylation reagent, biotin release was significantly increased by plasma (p < 0.0001 vs control by unpaired t test). Moreover, the hydrazide bond was also unstable in buffer. Biotin remaining on the protein was quantitated directly using capture of europium-streptavidin by the immobilized biotinylated immunoglobulin G. Consistent with biotin release data, streptavidin capture was reduced by plasma to 8% of control. We conclude that all of the biotinylating agents produce biotin-protein bonds that are susceptible to hydrolysis by factors present in human plasma; five of six are stable in buffer.

Release of biotin from biotinylated proteins occurs enzymatically and nonenzymatically in human plasma.

Studies in our laboratory and others indicate that biotin is released from biotinylated proteins in vivo and in vitro in human plasma. Using immunoglobulin G (IgG) as the model protein and four different biotinylating reagents, we investigated the mechanism of release. All of the biotin bonds shared an amide link to the carboxyl group of biotin but differed in the chemical links (amide, thioether, and hydrazone) between spacer arm and the various functional groups on IgG. Biotinylated IgG was incubated with phosphate-buffered saline, plasma, or plasma treated to either inactivate enzymes or remove all macromolecules. Released biotin was separated from bound biotin by ultrafiltration and quantitated by avidin-binding assay. As judged by high-performance liquid chromatography, greater than 95% of the released avidin-binding activity was biotin. We infer that the amide bond between the biotin and the spacer arm rather than the bond attaching the spacer to the protein was cleaved. Sodium dodecyl sulfate gel electrophoresis detected no proteolytic degradation of biotinylated IgG. Neither heat inactivation of plasma nor ultrafiltration of plasma to remove macromolecules completely eliminated biotin cleavage. We conclude that cleavage of biotin from protein occurs by both enzymatic and nonenzymatic mechanisms.

3-Hydroxypropionic acid and methylcitric acid are not reliable indicators of marginal biotin deficiency in humans.

In two studies comprising 10 and 11 subjects, respectively, marginal biotin deficiency was induced experimentally by an egg-white diet in healthy men and women. The following urinary organic acids were assessed for their usefulness in detecting marginal biotin status: 1) 3-hydroxypropionic acid and methylcitric acid, organic acids that reflect decreased activity of the biotin-dependent enzyme propionyl-CoA carboxylase and 2) methylcrotonylglycine and isovalerylglycine, organic acids that reflect decreased activity of methylcrotonyl-CoA carboxylase. Mean 3-hydroxypropionic acid excretion rates remained normal during biotin depletion in both studies. By the end of the depletion period, 3-hydroxypropionic acid excretion identified only 5 of 21 marginally deficient subjects. Mean methylcitric acid excretion increased (P < 0.0001) in the first study but not in the second. Mean methylcrotonylglycine excretion increased in each study (P < 0.004 and P < 0.05, respectively); methylcrotonylglycine excretion identified 13 of 21 marginally deficient subjects. Mean isovalerylglycine excretion increased only in the first study (P = 0.006) and identified only 6 of 21 deficient subjects. We conclude that none of these organic acids is as sensitive an indicator of marginal biotin deficiency as 3-hydroxyisovaleric acid, which reflects decreased methylcrotonyl-CoA carboxylase.

Marginal biotin deficiency is teratogenic in ICR mice.

The incidence of marginal biotin deficiency in normal human gestation is approximately one in three. In ICR mice, maternal biotin deficiency results in cleft palate, micrognathia, microglossia and limb hypoplasia. However, the relationships among the severity of maternal biotin deficiency, fetal biotin status and malformations have not been reported. This study utilized validated indices of biotin status to investigate the relationships among maternal biotin status, fetal biotin status and the rate of fetal malformations in ICR mice. Biotin status was controlled by feeding diets with varying egg white concentration. In dams and fetuses, biotin status was assessed by hepatic biotin content and hepatic activity of the biotin-dependent enzyme propionyl-CoA carboxylase; in dams, status was also assessed by urinary excretion of biotin and 3-hydroxyisovaleric acid. Malformations were assessed morphologically. Biotin was measured by HPLC/avidin-binding assay. Propionyl-CoA carboxylase (PCC) activity was determined by H(14)CO(3) incorporation. 3-Hydroxyisovaleric acid concentration was determined by GC/MS. Although no overt signs of deficiency appeared, metabolic disturbances caused by biotin deficiency were detectable in dams and fetuses. These disturbances increased with increasing egg white. Fetal biotin status correlated significantly with maternal biotin status (fetal vs. dam hepatic biotin, r = 0.671; fetal vs. dam PCC activity, r = 0.70). The incidences of malformations were strikingly dependent on egg white concentration. We conclude that in ICR mice, marginal maternal biotin deficiency causes fetal biotin deficiency. We speculate that the fetal malformations are primarily the consequence of fetal biotin deficiency. Because murine malformations appeared at degrees of biotin deficiency that are similar to those in human gestation, we speculate that some human fetal malformations may be caused by biotin deficiency.

Circulating RBC volume, measured with biotinylated RBCs, is superior to the Hct to document the hematologic effects of delayed versus immediate umbilical cord clamping in preterm neonates.

BACKGROUND: One problem assessing the hematologic physiology of preterm infants after delivery and/or the efficacy and toxicity of therapeutic interventions affecting RBC measurements is the inability of blood Hct values to accurately reflect circulating RBC volume-owing to changes in plasma volume that influence Hct (i.e., a fall in plasma volume concentrates RBCs to increase Hct; a rise in plasma volume dilutes RBCs to decrease Hct). STUDY DESIGN AND METHODS: As part of a randomized, clinical trial testing the hypothesis that delayed clamping of the umbilical cord at delivery expands neonatal circulating RBC volume, blood Hct was compared to circulating RBC volume results measured directly with autologous, biotinylated RBCs or estimated mathematically with neonatal body weight and Hct values in neonates after immediate or delayed (60 sec) cord clamping. RESULTS: Circulating RBC volume measured directly with biotinylated RBCs significantly increased (p=0.04) in neonates after delayed (42.1 +/- 7.8 mL/kg) versus immediate (36.8 +/- 6.3 mL/kg) cord clamping-a difference not detected indirectly by measuring Hct or estimating circulating RBC volume mathematically. CONCLUSIONS: Because true hematologic effects of delayed versus immediate cord clamping may not be apparent or may be misinterpreted, when based on indirect measurements of Hct or calculations of circulating RBC volume, it is important to measure circulating RBC volume directly-as done with autologous, biotinylated RBCs-to document whether delayed cord clamping truly results in a transfer of significant quantities of RBCs from placenta to neonate. The clinical benefits and potential toxicities of increased RBC transfer to neonates require further studies.