Marginal Biotin Deficiency Can Be Induced Experimentally in Humans Using a Cost-Effective Outpatient Design

Abstract

To date, marginal, asymptomatic biotin deficiency has been successfully induced experimentally by the use of labor-intensive inpatient designs requiring rigorous dietary control. We sought to determine if marginal biotin deficiency could be induced in humans in a less expensive outpatient design incorporating a self-selected, mixed general diet. We sought to examine the efficacy of three outpatient study designs: two based on oral avidin dosing and one based on a diet high in undenatured egg white for a period of 28 d. In study design 1, participants (n = 4; 3 women) received avidin in capsules with a biotin binding capacity of 7 times the estimated dietary biotin intake of a typical self-selected diet. In study design 2, participants (n = 2; 2 women) received double the amount of avidin capsules (14 times the estimated dietary biotin intake). In study design 3, participants (n = 5; 3 women) consumed egg-white beverages containing avidin with a biotin binding capacity of 7 times the estimated dietary biotin intake. Established indices of biotin status [lymphocyte propionyl-CoA carboxylase activity; urinary excretion of 3-hydroxyisovaleric acid, 3-hydroxyisovaleryl carnitine (3HIA-carnitine), and biotin; and plasma concentration of 3HIA-carnitine] indicated that study designs 1 and 2 were not effective in inducing marginal biotin deficiency, but study design 3 was as effective as previous inpatient study designs that induced deficiency by egg-white beverage. Marginal biotin deficiency can be induced experimentally by using a cost-effective outpatient design by avidin delivery in egg-white beverages. This design should be useful to the broader nutritional research community.

Introduction

Biotin is a water-soluble vitamin that serves as a required cofactor for five carboxylases that catalyze essential steps in human intermediary metabolism (1–3). Studies indicate that marginal, asymptomatic biotin deficiency is teratogenic in a number of animal species, including mammals (4–8). In view of recent studies indicating that marginal biotin deficiency develops frequently during normal human gestation, there has been increased investigation of the potential mechanisms of teratogenesis in mammalian and nonmammalian models as well as increased efforts to identify valid human indicators of biotin status (9, 10).

Often, studies inducing vitamin deficiency were conducted in an inpatient setting. For reasons of cost and participant acceptance, more recent studies have tended to be conducted in an outpatient setting with strong dietary support (11). Studies that induce biotin deficiency experimentally in humans have uniformly been conducted in an inpatient setting, such as a CRC⁸. These studies have characteristically utilized a diet high in undenatured egg white delivered as a beverage consumed with meals, and the depletion periods have ranges from 3 to 12 wk (12–15). Egg white contains avidin, a tetrameric glycoprotein that binds 4 mol biotin/mol protein with very high affinity.

Although successful in inducing marginal asymptomatic biotin deficiency, past studies conducted by our laboratory have been labor intensive and expensive. An important contribution to the expense was the inpatient study design. Each study employed daily interaction with CRC dietitians, nurses, and staff. Meals for participants designed to be low in biotin were carefully planned, prepared, and served by CRC dietary staff. Additionally, participants were housed in the CRC for 28 inpatient days.

We sought to determine if marginal biotin deficiency could be induced in humans in a less expensive outpatient design incorporating a self-selected, mixed general diet. In addition, we examined the relative sensitivity of several validated indicators of biotin status during increasing severity of deficiency.

Participants and Methods

Determination of biotin binding capacity of source avidin.

To empirically determine the biotin binding capacity of the avidin (Belovo) used in study designs 1 and 2, biotin binding stoichiometry was assessed using an optical dye displacement (HABA) assay as previously described (16). The HABA assay showed the avidin to bind a mean of 3.5 mol biotin/mol avidin.

Mouse study.

Inadvertent denaturation could affect the ability of dietary avidin to induce biotin deficiency even though the avidin exhibited adequate biotin binding stoichiometry. To assess the ability of dietary avidin from the commercial source to induce biotin deficiency, a preliminary study was performed in 3 mice. This study was approved by the University of Arkansas for Medical Sciences Animal Care and Use Committee. The dietary biotin content per gram of a standard mouse diet (Harlan Teklad 22/5 Rodent diet no. 8640) was analyzed by the avidin-binding assay as previously reported (7, 17). Then an avidin-containing diet was prepared by grinding diet pellets to powder using a manual mortar and pestle. The powder was thoroughly mixed with a gravimetrically determined mass of avidin that had the capacity to bind 7 times the biotin content per gram of diet powder. The diet powder was then moistened and reformed into pellets. Mice were housed in hanging wire metabolic cages and allowed free access to water and the avidin-containing diet. Weight and food intake were measured daily. On the day prior to initiating the avidin-containing diet and again on d 7, all food was removed from the cages and 24-h urine samples were collected. Mice were feed deprived during the collection to prevent contamination of the urine by the avidin-containing diet. Urinary excretion of biotin (total avidin-binding substances) was determined in each of these collections.

Human studies.

This research was approved by the Institutional Review Board of the University of Arkansas for Medical Sciences. Written informed consent was obtained from each participant as part of the ongoing consent process. Inclusion criteria included good health without a history of renal disease. Exclusion criteria included current history of smoking (18), consumption of dietary supplements known to contain biotin, and a recent history of therapy with certain medications known to accelerate biotin catabolism, such as certain anticonvulsants (19–21). Eight participants (5 women; mean age 36 y; range 23–56 y) successfully completed the study. One participant (woman) completed study design 1 and 2. Another participant (woman) completed study designs 1, 2 and 3.

Biotin sufficiency on d 0 was ensured in each of the 3 study designs by biotin loading and washout prior the start of the depletion phase of the studies as previously described (15). To ensure sufficiency of other vitamins, all participants consumed a multivitamin supplement without biotin daily during the depletion phase of the 3 study designs; the content of the multivitamin supplement was previously described (15).

After completing the washout regimen, participants were seen as outpatients in the CRC on d 0 and began the biotin depletion regimen. The biotin depletion regimen consisted of a self-selected, mixed general diet with avoidance of high-biotin foods, including organ meats, eggs, Swiss chard, and nuts. Biotin depletion was induced by consumption of purified avidin in capsule form or an egg-white beverage with meals and snacks (summarized in Supplemental Table 1). Participants continued the depletion regimen through d 27. Biotin intakes were monitored bi-weekly using 3-d diet histories.

In study designs 1 and 2, participants consumed commercially compounded capsules (Custom Compounding Pharmacy) containing purified avidin before each meal and snack. A sufficient number of capsules were consumed each day to provide ~14 mg (207 nmol) avidin (study design 1). This mass of avidin will bind 7 times the mean estimated biotin intake of study participants based on 3-d diet history and analysis by Nutritionist Pro, version 2.4 (First DataBank) and by reference to tables of biotin content of foods determined by Dr. Mock’s laboratory, where no published biotin content was found by A.M.D. (CRC Research Dietitian). In study design 2, ~28 mg (414 nmol) avidin was consumed per day (14-fold the estimated dietary biotin intake). On the basis of dietary record analysis, the intake of biotin was roughly proportional to energy consumption. Consequently, participants were given pillboxes with a 1-wk avidin supply divided into doses individually tailored to each person’s typical energy consumption for 3 meals and 1 snack/d.

In study design 3, avidin was consumed in egg-white (Michael Foods) beverages provided with 3 meals and 1 snack/d as previously described (13–15). The egg white beverage provided ~35% of the daily energy intake and 7 times the mean estimated biotin intake of study participants; the relationship to energy intake and biotin intake is similar to previous successful inpatient studies. The egg-white beverages were apportioned among meals and snacks according to the individual participant’s energy intake from a representative 3-d diet history as described above for avidin capsule distribution. The beverages were made in batch, refrigerated, and distributed to participants every 4 d. Participants were instructed to refrigerate beverages and consume one-half of the beverage just prior to the meal and the remainder of the beverage with the meal.

Participants in the 3 study designs repleted biotin status after the biotin depletion phase by consuming a self-selected, unrestricted diet and a daily multivitamin. The multivitamin had the same formulation as the multivitamin used in the depletion phase except that the supplement also contained 30 μg of biotin. The repletion regimen was initiated on d 29 and was continued for 2 wk. Urinary biotin excretion levels were monitored in all participants for up to an additional 30 d to ensure that urinary biotin of all participants reached normal range before discharge from the study.

Blood and urine sample collection.

Peripheral venous blood was collected from fasting participants in a heparinized syringe on d 0, 14, and 28 of the depletion phase and on d 48 of the repletion phase as previously described (22). Peripheral blood mononuclear cells and plasma were obtained from the blood by density gradient centrifugation and were stored at −70°C as previously described (22).

Complete urine collections were obtained for the 24-h period ending at 0800 on d 0, 7, 14, 21, and 28 during depletion and on d 48 during repletion. Five-hour urine collections were obtained on d 0, 7, 14, 21, and 28 after a leucine challenge test was administered as previously described (23). Samples were processed and stored as previously described (14).

Determination of blood and urinary indicators of biotin status.

Lymphocyte PCC activities were measured by ¹⁴CO₂ incorporation as previously described (22). Activities were normalized by lymphocyte protein content. Plasma 3HIA-carnitine was extracted and quantitated by liquid chromatography-tandem MS as previously described (24, 25). Urinary biotin was quantitated by HPLC and avidin-binding assay as previously described (26). Urinary 3HIA was quantitated by ultra-performance liquid chromatography-tandem MS as previously described (27). Urinary 3HIA-carnitine was quantitated in diluted urine without prior extraction by liquid chromatography-tandem MS as previously described (28). Excretion of all urinary indicators was normalized by urinary concentration of creatinine. Urinary creatinine was determined by the picric acid method using an automated creatinine analyzer (Beckman Life Sciences) as previously described (29).

Statistical analyses.

For the 3 study designs, the differences in the various group mean indicators during depletion were tested for significance by 1-way ANOVA with repeated measures. When significant (P < 0.05), Dunnett’s post hoc test was used to test for significance of differences between depletion phase study days and d 0. To compare the degree of biotin deficiency induced in study design 3 to a previous study, the various biotin indicators were tested by Wilcoxon-Mann-Whitney rank sum test. All tests were performed using KaleidaGraph (version 4.1.1; Synergy Software).

Normal ranges.

Normal ranges for lymphocyte PCC activities and urinary excretion of biotin were determined as previously described in 19 individuals who were biotin sufficient (established by loading and washout) in 2 previous studies (14, 30) plus data from d 0 of the current study (8 participants). For each indicator, values of the subgroups from the different sources did not differ by Wilcoxon-Mann-Whitney rank sum test. Accordingly, values for the subgroups were combined (n = 27) and the 10th and 90th percentiles were calculated to form the basis of the normal range for biotin sufficiency.

Conversion of 3-methylcrotonyl CoA to 3-methylglutaconyl CoA in the leucine catabolic pathway is catalyzed by the biotin-dependent enzyme MCC. Marginal biotin deficiency reduces MCC activity and increases urinary excretion of 3HIA and 3HIA-carnitine in 24-h urine collections (23, 31) as well as immediately after an oral leucine challenge (23). The normal ranges for urinary excretion of 3HIA and 3HIA-carnitine were determined from 82 individuals as previously described (23). The 10th and 90th percentiles were used as range limits. Normal ranges for urinary excretion of 3HIA and 3HIA-carnitine after leucine challenge on d 0 were chosen as the full range of values measured in 5-h urine collections before beginning the egg-white diet when the participants were biotin sufficient.

Normal ranges for plasma 3HIA-carnitine concentrations were established from measurements in 63 individuals. Of these 63 individuals, 27 had been rendered biotin sufficient by loading and washout (including 8 participants from d 0 of the current study). The other 36 individuals were free-living individuals who had not undergone loading and washout and who, by history, had not supplemented their diets with biotin above the RDA. Mean fasted plasma 3HIA-carnitine concentrations did not differ for these 2 subgroups (0.011 and 0.012 μmol/L, respectively). Values for the subgroups were pooled and 10th and 90th percentiles were used as range limits.

Results and Discussion

Pilot mouse study.

Mean urinary biotin excretion decreased 25-fold (from 8.7 ± 4.9 μmol/mol creatinine to 0.35 ± 0.23 μmol/mol creatinine) over 7 d of consuming the avidin-containing diet. Ratios of the d 7 to d 0 excretion rates varied in individual mice from 6-fold to a 100-fold decrease. These values are consistent with reductions in urinary biotin previously reported in rodent experiments that were successful in inducing overt biotin deficiency (7).

Dietary analysis for the three study designs.

For the three study designs, participants maintained body weight throughout the depletion phase of the study. Mean weight change for all participants was −0.0125 kg. No observed differences in weight loss were found for the different study designs. Biotin intakes were similar among the 3 study designs and were 18.2 ± 13 μg/d (range 2.3–60.8 μg/d).

Human study designs 1 and 2.

Neither study design 1 nor study design 2 was uniformly effective in inducing biotin deficiency in the participants. Study design 1 decreased lymphocyte PCC activity to less than the lower limit of normal in 3 of the 4 participants (Supplemental Fig. 1A). Moreover, urinary 3HIA, plasma 3HIA-carnitine, and urinary 3HIA-carnitine did not increase to greater than the upper limit of the normal range in 3 of the 4 participants (Supplemental Fig. 1B–D). Urinary biotin did not decrease to less than the upper limit of the normal range in 3 of the 4 participants (Supplemental Fig. 1E).

On the basis of the results of study design 1, we speculated that the avidin dose might be effectively insufficient, despite a 7-fold excess of biotin binding capacity. We doubled the total avidin supplement (study design 2) and studied 2 participants. Study design 2 did reduce PCC activity and urinary biotin below the lower limit of normal in both participants (Supplemental Fig. 1A,E). However, urinary 3HIA, plasma 3HIA-carnitine, and urinary 3HIA-carnitine did not increase to greater than the upper limit of the normal range in 1 of the 2 participants (Supplemental Fig. 1B–D). At this point, we speculated that inadequate biotin binding capacity might not be the root cause of inconsistent efficacy in inducing biotin deficiency.

Human study design 3.

Consequently, we conducted a larger third study (study design 3) that utilized egg white feeding in an outpatient design. For these 5 participants, the preponderance of the results indicated that marginal biotin deficiency was induced to the same degree as in past inpatient studies (Table 1). In all 5 participants, lymphocyte PCC activity decreased to less than the lower limit of normal (Fig. 1A). Urinary 3HIA and 3HIA-carnitine increased to greater than the upper limit of normal (Fig. 1B,D). Urinary biotin decreased below normal in all 5 participants as well (Fig. 1E). Of note, urinary 3HIA-carnitine was abnormal in all 5 participants by d 7, whereas urinary 3HIA and biotin were abnormal in only 3 of 5 participants. The plasma concentration of 3HIA-carnitine increased to greater than the normal range in 4 of the 5 participants (Fig. 1C).

TABLE 1.

Degree of marginal biotin deficiency induced by d 28 in outpatient study design 3 is similar to previous inpatient study¹

Indicator	Normal range	Previous inpatient study d 282	Diagnostic sensitivity3, %	Current outpatient study d 282	Diagnostic sensitivity3, %	P value
Lymphocyte PCC, pmol · min · mg protein−1	300–680	112 ± 43	100	168 ± 57	100	>0.05
Urinary 3HIA, mmol/mol creatinine	3.3–11.5	21.6 ± 11.9	86	31.2 ± 12.3	100	>0.05
Plasma 3HIA-carnitine, μmol/L	0.004–0.018	0.051 ± 0.016	100	0.028 ± 0.01	80	0.02
Urinary 3HIA-carnitine, mmol/mol creatinine	0.06–0.16	0.42 ± 0.14	100	0.45 ± 0.12	100	>0.05
Urinary biotin, μmol/mol creatinine	1.8–7.7	0.81 ± 0.27	100	0.97 ± 0.27	100	>0.05

¹From (15). 3HIA, 3-hydroxyisovaleric acid; 3HIA-carnitine, 3-hydroxyisovaleryl carnitine; PCC, propionyl-CoA carboxylase.

²Values are mean ± SD, = 5 (outpatient) or 7 (inpatient).

³Defined as percentage of participants whose values were above or below the established normal range at d 28.

FIGURE 1.

Indicators of biotin status [lymphocyte PCC activity (A), urinary excretion of 3HIA (B), plasma 3HIA-carnitine concentration (C), urinary excretion of 3HIA-carnitine (D), and urinary excretion of biotin (E)] for participants in study design 3 (n = 5) showed avidin delivery by egg-white shake form was effective in inducing marginal biotin deficiency. The gray rectangle denotes the normal range. Asterisks indicate different from d 0: *P < 0.05, **P < 0.003, *** P ≤ 0.0001. 3HIA, 3-hydroxyisovaleric acid; 3HIA-carnitine, 3-hydroxyisovaleryl carnitine; PCC, propionyl-CoA carboxylase.

Leucine challenge.

We recently investigated a new indicator of biotin status: the increase in urinary 3HIA and 3HIA-carnitine excretion in the 5 h after oral administration of 70 mg leucine/kg body weight. This indicator is not as well characterized as the 5 validated indicators depicted in Figure 1; hence, results from leucine challenges in study designs 1, 2, and 3 are presented and discussed separately.

For study design 1, urinary excretion of 3HIA and 3HIA-carnitine in response to a leucine challenge increased earlier in the course of avidin feeding (Supplemental Fig. 2A,B) than the 24-h urinary excretion of 3HIA and 3HIA-carnitine (Supplemental Fig. 1B,D). By the end of study design 1, urinary excretion of 3HIA after leucine challenge increased to greater than the upper limit of normal in all 4 of the study participants and urinary 3HIA-carnitine after leucine challenge increased in 3 of 4 participants. By the end of study design 2, urinary excretion of 3HIA and 3HIA-carnitine after leucine challenge increased in both of the participants (Supplemental Fig. 2A,B). These observations suggest that a subtle degree of biotin deficiency was indeed induced by study designs 1 and 2. Further, we speculate that this subtle degree of biotin deficiency and the accompanying modest MCC deficiency were unmasked when metabolic flux down the leucine catabolic pathway became limiting at the MCC step under a leucine load, as indicated by the increase in urinary excretion of 3HIA-carnitine.

In study design 3, the diagnostic sensitivity of urinary excretion of 3HIA and 3HIA-carnitine excretion in response to the leucine challenge was similar to that observed for the steady-state, 24-h urinary excretion of 3HIA and 3HIA-carnitine (Fig. 2). Increased urinary excretion of 3HIA and 3HIA-carnitine in response to leucine challenge was observed in all 5 participants after just 2 wk of the egg-white diet. The group means tended to continue to increase further at d 21 and 28.

FIGURE 2.

Urinary excretion of 3HIA (A) and 3HIA-carnitine (B) in response to leucine challenges for participants in study design 3 (n = 5) showed that participants were clearly marginally biotin deficient. The gray rectangle denotes the normal range. Asterisks indicate different from d 0: *P < 0.02, **P < 0.002, ***P < 0.0001. 3HIA, 3-hydroxyisovaleric acid; 3HIA-carnitine, 3-hydroxyisovaleryl carnitine.

Although the factors limiting effectiveness of the avidin capsule administration are not known, we speculate that factors involving solubility, source, and timing in relationship to meals may have been responsible for the lower effectiveness of avidin in capsules. Avidin may not have readily dissolved in gastric contents when administered in gelatin capsules. Avidin might have been sufficiently structurally altered during purification, storage, or shipping to be susceptible to further denaturing on exposure to the pH of 1–2 found in the fasting stomach. Timing of avidin capsule consumption may have rendered the avidin unavailable to bind biotin at the exact time free biotin was released into the aqueous phase of intestinal contents. In view of the mouse study results, we favor prior denaturation and ineffective timing relative to biotin release as the most likely mechanisms for failure of study designs 1 and 2.

In summary, the outpatient study designs investigated here incorporated fewer CRC visits, mixed general diets that were self-selected and self-provided, and administration of avidin in either purified capsule form or in an egg-white beverage. We conclude that avidin in capsule form was not consistently effective in inducing marginal biotin deficiency in an outpatient setting, but administration of an egg-white beverage with meals and snacks was as effective as previous inpatient studies. Given the resources required for inpatient studies, the outpatient design described here is likely to provide a more cost-effective and participant-acceptable design for studying marginal biotin deficiency.