Biotin eksikliği doğuştan olabildiği gibi yetersiz beslenen kişilerde de görülebilen bir durumdur.Tırnak (tırnakta kırılma,incelme,yumuşamama,uzamama gibi sorunlar) ve saç sorunları (kalite kaybı,dökülme,kırılma,sönükleşme gibi sorunlar) ile karşımıza çıkar.
Am J Clin Nutr 2002;76:1061–8. Printed in USA. © 2002 American Society for Clinical Nutrition 1061
Indicators of marginal biotin deficiency and repletion in humans:
validation of 3-hydroxyisovaleric acid excretion and a leucine
challenge1–3
Donald M Mock, Cindy L Henrich, Nadine Carnell, and Nell I Mock
ABSTRACT
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 _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. Am J Clin Nutr
2002;76:1061–8.
KEY WORDS Biotin, leucine, metabolism, 3-hydroxyisovaleric
acid, bisnorbiotin, egg whites
INTRODUCTION
The results of clinical studies conducted by our group and others
have provided evidence that marginal biotin deficiency in such
disparate clinical circumstances as pregnancy (1, 2), proteinenergy
malnutrition (3), and long-term therapy with certain anticonvulsants
(4–8) is not rare. As noted in an editorial in this journal,
there is a need to develop valid indicators of marginal biotin
deficiency (9).
1 From the Departments of Biochemistry and Molecular Biology and of
Pediatrics (DMM, CLH, and NIM) and the General Clinical Research Center
(NC), University of Arkansas for Medical Sciences, Little Rock.
2 Supported by DDK 36823 from the National Institutes of Health and by
the NIH General Clinical Research Program of the National Center for
Research Resources: M01RR14288 (University of Arkansas for Medical Sciences)
and RR 00059 (University of Iowa).
3 Address reprint requests to DM Mock, Department of Biochemistry and
Molecular Biology, University of Arkansas for Medical Sciences, 4301 West
Markham Street, Slot 516, Little Rock, AR 72205. E-mail: mockdonaldm@
uams.edu.
Received June 26, 2001.
Accepted for publication November 27, 2001.
In 1997 we reported the results of the first experimental study to
successfully induce biotin deficiency in human subjects since the
1940s. In that study, we evaluated 3 indexes of biotin status that
depended on the previous development of 2 analytic methods: 1) a
sensitive, chemically specific assay for biotin that combines HPLC
with an avidin-binding assay and 2) a gas chromatography–mass
spectrometry method for measuring 3-hydroxyisovaleric acid
(3HIA) in urine (10).
Biotin is a covalently bound prosthetic group for 4 mammalian
carboxylases; one of these, methylcrotonyl-CoA carboxylase
(EC 6.4.1.4), catalyzes an essential step in the intermediary
metabolism of the branched-chain amino acid leucine. Decreased
activity of methylcrotonyl-CoA carboxylase shunts the substrate
3-methylcrotonyl CoA to an alternate metabolic pathway,
producing 3HIA, which is then excreted in urine.
In that first study, we observed that decreased urinary excretion
of biotin was an early and sensitive indicator of biotin deficiency;
biotin deficiency was identified before the onset of symptoms and
was detected in almost all subjects. However, a decrease in the
plasma concentration of biotin was detected in less than one-half
of the subjects. Increased urinary excretion of 3HIA was also an
early and sensitive indicator of biotin deficiency.
Although that study was a reasonable first effort, the conclusions
were limited in the following ways. 1) Homogeneity of
biotin status was not established with a biotin loading-washout
phase. 2) Only 10 subjects were studied (7 men, 3 women). 3)
Recovery from deficiency was not studied in the repletion phase.
We addressed these limitations in the study reported here and
expanded the scope of the study to include evaluation of an oral
leucine challenge as an index of biotin status.
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1062 MOCK ET AL
TABLE 1
Calculated nutrient composition of the 2 diets1
Nutrient Menu 1 Menu 2
Protein (g) 129 186
Leucine (g) 9 13
Fat (g) 68 99
Carbohydrate (g) 273 395
Dietary fiber (g) 22 31
Cholesterol (mg) 74 107
Biotin (_g) 23 13.4
1 Amount per 10 MJ.
SUBJECTS AND METHODS
Study subjects
All human studies were approved by the University of Arkansas
for Medical Sciences’ Human Research Advisory Committee;
informed consent was obtained at enrollment. Initially, 15 healthy
adult volunteers (10 women) resided in a general clinical research
center (GCRC) for 28 d. The subjects were allowed to go to work
on weekdays and took the noon meal and urine collection vessels
with them; otherwise, the subjects resided fulltime at the GCRC.
Individuals who were supplementing their diets deliberately or
inadvertently with dietary biotin were excluded from enrolling.
Examples of supplements that substantially increase biotin intake
include many breakfast cereals, weight-gain supplements, weightloss
supplements, and multivitamin supplements. These foods
contain amounts of biotin that are substantial compared with the
estimated daily dietary intake of 35–70 _g/d (143–287 nmol/d)
(11–15). Because biotin is often present in foods covalently bound
to protein and thus is difficult to measure, and because most assays
do not discriminate between biotin and its various metabolites, the
estimates of biotin content are not precise. Thus, potential volunteers
taking any type of supplement were excluded unless negligible
biotin content was confirmed by assay.
Eleven subjects (8 women) completed the study. The 4 subjects
who did not complete the study withdrew during the biotin depletion
phase of the study due to difficulties in complying with the
diet and housing protocols.
Study design
Loading and washout
On study day _14, the subjects began the loading phase by
receiving a daily supplement containing 300 _g biotin (1.2 _mol).
This supplement is 10 times the recommended adequate intake of
30 _g (16). In addition, a daily vitamin supplement not containing
biotin was given. This supplement contained vitamin A
(5000 IU), vitamin C (60 mg, or 340 _mol), vitamin D (400 IU),
vitamin E (15 IU), thiamine (1.5 mg, or 5 _mol), riboflavin (1.7 mg,
or 5 _mol), niacin (20 mg, or 163 _mol), vitamin B-6 (2 mg, or
10 _mol), folate (400 _g, or 1 _mol), vitamin B-12 (6 _g, or
4 nmol), and iron (18 mg, or 322 _mol).
On study day _7, the biotin supplement was discontinued to
begin the washout period. The multivitamin supplement without
biotin was continued through study day 28. The amount of biotin
in the multivitamin supplement without biotin was measured by
direct assay with the avidin-binding assay (17). The mean biotin
content was 0.01 _g (48 pmol); the range was between 0.004 and
0.03 _g (18–120 pmol; n = 4). This amount is < 0.09% of the
estimated daily dietary intake of 35–70 _g/d (143–287 nmol/d)
(11, 12) and 0.3% of the study dietary intake of 10.2 _g (42 nmol)
for women and 13.8 _g (56 nmol) for men.
Biotin depletion
On study day _1, the subjects were admitted to the GCRC.
They provided a 24-h urine collection, which served a test run for
proper collection technique. A urine chorionic gonadotropin pregnancy
test was performed on the samples from the women and all
test results were negative. On study day _1, the subjects received
a self-selected, general diet. At 0730 of study day 0, the subjects
completed a second 24-h urine collection.
Feeding of the research diet (referred to hereafter as the egg
white diet) commenced on study day 0. The egg white was provided
as dried albumen (Wakefield Brand; M.G. Waldbaum,Wakefield,
NE) in a blended beverage containing 16 g dried egg
white/MJ dietary energy; the egg white accounted for 22–32% of
the diet’s dry weight, depending on consumption of the alternate
diets described below. This egg white content successfully produced
biotin deficiency in the previous study (10). The egg white
content was chosen to contain sufficient avidin to bind _78 times
the dietary biotin intake, which was calculated from estimates of
biotin in food sources (11, 18, 19) and was measured directly in
our laboratory as described below. The calculation used a published
value for the avidin content of egg white (20, 21), and the estimate
of binding capacity used an avidin-to-biotin binding ratio of 1:4.
The egg white beverage was consumed at breakfast (25%),
lunch (33%), and dinner (42%). Energy expenditure for each volunteer
was calculated by using the Harris Benedict equation (22)
to provide a euenergetic menu. The typical daily energy intake was
9700 kJ for women and 14 000 kJ for men: 23% from protein, 50%
from carbohydrate, and 27% from fat; nutrient content was estimated
by using NUTRITIONIST 5, version 1.6 (First Data Bank,
San Bruno, CA). The typical American diet provides _12–15%
protein, 45–50% carbohydrate, and 35–50% fat. The egg white
beverage was provided in proportion to the energy content of each
meal; because high-biotin foods were avoided in the protocol
meals, energy content roughly paralleled biotin content. Nutrient
concentrations were as shown in Table 1 as menu 1 and menu 2.
Subjects received a 2-d rotating menu cycle. To increase variety,
the foods in each menu were rearranged to produce 2 additional
menu cycles. For each subject, food amounts were factored
proportionately to provide equal daily energy intake. Foods were
weighed to the nearest gram. Subjects were allowed up to 12 oz
(360 mL) coffee, tea, or diet soda throughout the day. Dietary
compliance was judged by observing meals eaten, by inquiring
daily about other foods consumed, by daily morning weigh-ins,
and by subjects completing daily journals kept in the GCRC. Subjects
were required to consume the entire egg beverage with each
meal. Although subjects were strongly encouraged to consume
each meal entirely, they were allowed to refuse portions of foods.
Uneaten food was recorded; data for actual food consumption
were used to calculate average nutrient intake.
Repletion phase
On study day 28, the egg white diet was discontinued, and all
subjects consumed a self-selected, mixed general diet from study
day 28 to 49. For the first week of repletion (study day 28 to 35),
5 subjects received a daily multivitamin and multimineral supplement
with biotin. For these subjects, the intent was to provide a
supplement that contained an amount of biotin (30 _g) equal to
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BIOTIN INDICATORS IN DEFICIENCY AND REPLETION 1063
the adequate intake. The labeled content was 30 _g biotin
(123 nmol), and our previous measurements have indicated that
labeled biotin content in commercial vitamins is typically accurate
within 10% (17). However, analysis of the multivitamin after
the subjects began consuming the supplement showed that the
biotin content was greater than the labeled amount: 80 ｱ13 _g
(328 ｱ53 nmol; ｱSD; n = 5). The supplement contained the
same vitamin content as the biotin-free supplement with the following
exceptions: vitamin K (25 _g, or 0.1 _mol), vitamin B-6
(2.9 mg, or 14 _mol), and pantothenic acid (10 mg, or 46 _mol).
Six subjects continued to receive the daily multivitamin supplement
without biotin through study day 35. Thereafter, all subjects
received the 80-_g biotin supplement.
Leucine dose selection
In support of the main study described here, we empirically
established a suitable dose of leucine for the leucine challenge
tests by conducting a small leucine-dose-ranging study. In this
study, the biotin status of 3 healthy adults was altered as follows:
lowered biotin status (induced by avidin feeding), normal biotin
status (mixed general diet), and augmented biotin status (produced
by supplementation with 300 _g biotin/d). Individuals were studied
at 2 leucine doses. After providing a baseline void, subjects
drank the leucine dose suspended in water; subsequent voids were
collected at 2-h intervals for 6 h.
Leucine challenge
In the depletion phase of the main study, 5 subjects underwent
a leucine challenge on study days 1, 7, 14, and 21. These 5 subjects
consumed 70 mg leucine/kg (534 _mol/kg) dissolved in
orange juice after collecting the last void of the previous 24-h collection.
All voids were collected for 5 h after ingestion and pooled.
No other food was consumed during this 5-h period. The full egg
white diet for the day, including the egg white beverage, was consumed
later in the day.
Sample collection and handling for biotin and
3-hydroxyisovaleric acid excretion
In addition to the collection on study day 0 as noted above, 24-h
urine samples were collected on study days 3, 7, 10, 14, 17, 21, 24,
28, 35, and 49. During 24-h urine collections, individual voids were
pooled and refrigerated immediately; no preservative was added.
Before storage, urine samples were warmed to 50 _C for 30 min,
centrifuged (10 min at 1200 _ g and room temperature) to remove
any particulates, and portioned for storage at_20 _C until analyzed.
The completeness of each urine collection was evaluated by
determining the total creatinine excretion per 24 h. Creatinine
excretion was not less than the lower limit of the normal range for
any collection. The normal range for men and women established
in our laboratory was similar to published normal ranges.
Analytic methods
The presence of substantial amounts of biotin metabolites in
human urine and plasma in subjects who are biotin deficient, biotin
adequate, and biotin supplemented (10, 23–27) requires the use of a
chemically specific assay. To specifically measure concentrations of
biotin and biotin metabolites, we used a 2-step approach. First, biotin
and the biotin metabolites were separated by C18 reversed-phase
chromatography (28); next, each compound was quantitated against
the authentic biotin analogue. Authentic standards were required
because the various biotin analogues differ in avidin-binding
affinity (21). For this study, HPLC retention times were determined
by using radiolabeled biotin, bisnorbiotin, and biotin sulfoxides;
the appropriate HPLC fractions were collected and analyzed
against authentic standards as described previously (24, 29). The
term sulfoxides is used because the d and l isomers are not resolved
by this HPLC method.
The urinary excretion of 3HIA was determined by gas
chromatography–mass spectrometry (10). This method uses
authentic unlabeled and deuterated 3HIA as the external and internal
standards, respectively. Creatinine was measured in our laboratory
by the picric acid method (30, 31) with a Beckman Creatinine
Analyzer 2 (Beckman Instruments, Inc, Brea, CA).
Statistics
Normal ranges
Over the past decade, for each study of biotin status within a subject
population, our laboratory has recruited individuals to serve as
control subjects. As our understanding of biotin nutriture has grown,
we have reanalyzed our stored samples with the use of improved
techniques and have identified factors that disqualify some individuals
from participating as control subjects, eg, the use of birth control
medication. Taken together, these results provide a reasonable
normal range with which to compare the results from the current
study. Despite the exclusion of individuals who acknowledged
biotin supplementation, our measurements of the biotin excretion
of free-living subjects showed a bimodal distribution; we speculate
that the small subgroup that excreted larger amounts of biotin inadvertently
or deliberately supplemented their biotin intake. Because
the distribution was not normal, the normal range for biotin excretion
(19–62 nmol/d) was chosen as the 10th percentile to the 90th
percentile of 19 subjects (11 women). A similar approach was used
to establish the normal ranges for bisnorbiotin (12–54 nmol/d) and
biotin sulfoxides (6–15 nmol/d) by using values from the same subjects.
The normal range for the excretion of 3HIA (39–150 _mol/d)
was determined from 17 subjects (9 women); the distribution was
monomodal and roughly normal.
Comparison of subjects at study day 0 with control subjects
(ie, not subjected to prior biotin load and washout)
For nutritional status indicators with normal distributions,
the significance of differences between subjects was tested by
using Student’s unpaired, two-tailed t test with significance set
at P < 0.05. For indicators with nonnormal distributions, the significance
of differences was tested by using the Mann Whitney U test
with significance set at P < 0.05.
Studies of 3-hydroxyisovaleric acid excretion for baseline and
leucine challenge
The significance of differences in baseline 3HIA excretion
among the individuals with different biotin status was tested by
one-way analysis of variance (ANOVA). The significance of differences
in challenge 3HIA excretion among the individuals with
different biotin status was also tested by one-way ANOVA. If overall
differences were significant at P < 0.05, Fisher’s post hoc test
with Bonferroni correction was used to define pairwise differences.
Testing of trends with duration of egg white feeding and
repletion
The significance of trends with duration of egg white feeding
was tested by one-way ANOVA with repeated measures.
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1064 MOCK ET AL
FIGURE 1. Depletion phase. Mean (ｱSD) urinary excretion of 3-hydroxyisovaleric acid (3HIA), biotin, bisnorbiotin (BNB), and biotin sulfoxides
(BSOs) in 11 subjects from study day 0 to study day 28. Subjects consumed an egg white beverage with each meal to produce marginal biotin deficiency.
NR, normal range. *Significantly different from study day 0 at the level indicated in each panel by post hoc test after ANOVA.
If overall trends were significant at P < 0.05, Fisher’s post
hoc test with Bonferroni correction (32) was used to define
the time points that were significantly different from study
day 0. The P values given in the figures are the least significant
value between any 2 time points and are not less than
the overall significance of the ANOVA itself. The significance
of trends with duration of repletion was tested by twoway
ANOVA with repeated measures; the within factor was
time and the between factor was supplement group (immediate
versus late supplement). If trends with time were significant
(P < 0.05), Fisher’s post hoc test with Bonferroni correction
was used to define the time points that were
significantly different from study day 28. If the interaction
of group with time was significant, treatment subgroups were
analyzed separately. If the interaction was not significant,
immediate and late groups were pooled before post hoc testing
of the time points. STATVIEW 5.01 (SAS Institute, Cary,
NC) was used for the analyses.
RESULTS
Effects of loading and washout
Neither the mean urinary excretion of biotin (50 ｱ27 nmol/d)
nor the mean urinary excretion of 3HIA (98 ｱ31 _mol/d) on
study day 0 was significantly different from that of a group
studied previously without a load and washout (49 ｱ31 nmol/d
and 113 ｱ34 _mol/d, respectively). Thus, these indicators suggest
that initial biotin status was not altered by loading and
washout.
Signs and symptoms of biotin deficiency
There was a significant (P = 0.026 by paired means comparison)
but biologically unimportant weight loss; the mean (ｱSD)
body weight difference for study day 28 compared with study
day 0 was _0.69 ｱ0.88 kg. Each subject was closely monitored
by the GCRC staff for characteristic signs of biotin deficiency
such as hair loss and skin rash. No subject developed such symptoms.
One subject reported an evanescent red rash on study day 28;
the rash was not apparent to an examiner at discharge (study
day 28) and did not recur. About one-half of the subjects noticed
an unusual body odor or were reported by roommates or staff to
have an unusual body odor; to our knowledge, body odor has not
been reported previously as a consequence of biotin deficiency.
Biotin depletion
The mean urinary excretion rates of 3HIA, biotin, bisnorbiotin,
and biotin sulfoxides for the 11 subjects over the 28 d of egg white
feeding are shown in Figure 1. Excretion of 3HIA increased steadily
and significantly (P < 0.0001). Compared with study day 0, the mean
increase was significant by study day 17. Excretion of 3HIA was
greater than the upper limit of normal for 3 subjects by study day 7,
for 8 subjects by study day 14, and for 9 subjects by study day 28.
Biotin excretion decreased rapidly and significantly during
depletion (P < 0.0001). Compared with study day 0, the mean
decrease was significant by study day 7. For all subjects, the mean
biotin excretion on study day 28 was 22% of the value on study
day 0. On study day 14, biotin excretion was less than normal for
8 of the 11 subjects; on study day 28, biotin excretion was less
than the lower limit of normal for 9 of the 11 subjects.
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BIOTIN INDICATORS IN DEFICIENCY AND REPLETION 1065
FIGURE 2. 3-Hydroxyisovaleric acid (3HIA) response to a leucine challenge: dose selection study. _, baseline 3HIA excretion; _, challenge 3HIA
excretion; Supp, supplemented. Error bars denote ｱ1 SD of analytic replicates (n ≥3 replicates). A: Dose used was 35 mg leucine/kg body wt;
n = 1 subject per biotin status category. Challenge 3HIA excretion increased with decreasing biotin status. Baseline 3HIA excretion also increased with
decreasing biotin status. *Significantly different from supplemented and normal, P < 0.0003. **Significantly different from supplemented and deficient,
P < 0.0003. ***Significantly different from supplemented, P < 0.0003. B: Dose used was 70 mg leucine/kg body wt. Challenge 3HIA excretion was related
to biotin status, such that supplemented < normal < deficient. Baseline excretion also increased with decreasing biotin status.*Significantly different from
supplemented and normal, P < 0.0003. **Significantly different from supplemented and deficient, P < 0.001. ***Significantly different from supplemented,
P < 0.0001.
Before egg white feeding, the bisnorbiotin excretion rates of
the subjects were not significantly different from the group used
to determine the normal range. No subject excreted less bisnorbiotin
than the lower limit of normal. However, 4 subjects excreted
more bisnorbiotin than the upper limit of normal. Bisnorbiotin
excretion decreased rapidly and significantly during depletion
(P < 0.0001). Compared with study day 0, the mean decrease was
significant by study day 3. For all subjects, the mean bisnorbiotin
excretion on study day 28 was 9% of the value at study day 0. On
study day 14, bisnorbiotin excretion was less than normal for 7 of
the 11 subjects; on study day 28, bisnorbiotin excretion was less
than the lower limit of normal for 10 of the 11 subjects.
Before egg white feeding, the mean excretion rate of biotin sulfoxides
of the study subjects was not significantly different from
normal. One subject excreted less biotin sulfoxides than the lower
limit of normal, and 6 subjects excreted more biotin sulfoxides than
the upper limit of normal. Biotin sulfoxide excretion decreased
significantly with depletion (P < 0.0001); excretion of biotin sulfoxides
decreased by _50% during the first week of depletion and
changed slowly thereafter. Compared with study day 0, the decrease
was significant by study day 3. For all subjects, the mean biotin sulfoxide
excretion on study day 28 was 9%of the value at study day 0.
On study day 14, biotin sulfoxide excretion was less than normal
for 6 of the 11 subjects; on study day 28, biotin sulfoxide excretion
was less than the lower limit of normal for 7 of the 11 subjects.
We sought to determine whether either the urinary excretion of
3HIA or the urinary excretion of biotin differed between the sexes.
We concluded that pooling of the data from the current study (n = 3
men and 8 women) and the study reported in 1997 (n = 8 men and
2 women; 10) was justified despite differences in study design
such as the loading and washout period in the current study
because initial biotin status was not significantly different between
the 2 studies. Before egg white feeding, 3HIA excretion was not
significantly different between men (x– ｱSD: 114 ｱ36 _mol/d;
n = 10) and women (98 ｱ30 _mol/d; n = 11). Nor was biotin
excretion significantly different between men (58 ｱ36 nmol/d)
and women (42 ｱ18 nmol/d).
We also sought to determine whether susceptibility to biotin
depletion differed between the sexes.We used the pooled data on the
urinary excretion of 3HIA and biotin on study day 21 from the 1997
study and from the current study. Mean 3HIA excretion was not
significantly different between men (412 ｱ197 _mol/d) and women
(282 ｱ212_mol/d). Likewise, biotin excretion did not differ significantly
between the sexes (17 ｱ11 for men and 13 ｱ5 nmol/d for women).
Preliminary leucine dose study
The dose used for the leucine challenge in the current study was
determined in a preliminary dose-ranging study that included 2
doses of leucine. We initially evaluated 35 mg leucine/kg body wt
because this dose is one-half of the average daily intake. We
observed that the highest rate of 3HIA excretion varied among the
individuals (data not shown); the increased excretion over baseline
was largely complete by 6 h after ingestion of the leucine. On
the basis of the excretion rates observed in the 2-h collections, a
collection interval of 5 h was selected for the standard leucine
challenge; all voids during this interval were pooled. Results were
expressed as mmol 3HIA excreted/mol creatinine in the pooled
urine collection. As shown in Figure 2, both baseline 3HIA
excretion and challenge 3HIA excretion increased significantly
(P < 0.0001 by one-way ANOVA). Single individuals were tested
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1066 MOCK ET AL
FIGURE 3. Leucine challenge. Urinary excretion of 3-hydroxyisovaleric
acid (3HIA) in 5 subjects (2 women) who underwent an oral leucine
challenge on study days 0, 7, 14, and 21. Bars representing analytic variability
were approximately the size of the symbols and are not shown. The
leucine dose of 70 mg/kg body wt was taken in the morning; all urine was
collected for the next 5 h. The clear box represents the range of excretion
at study day 0.
at each dose and biotin status; thus, we could not infer whether
differences were attributable to biotin status or individual differences.
For the purposes of designing the leucine challenge, we
assumed that the differences arose from biotin status rather than
individual differences.
We next evaluated 70 mg/kg (Figure 2). Greater challenge
3HIA excretion rates were observed with the larger dose, and challenge
3HIA excretion rates increased significantly as biotin status
decreased (P < 0.0001); no adverse symptoms were elicited.
This dose was chosen for the current study.
Leucine challenge during depletion
The final 5 subjects participating in the main depletion and
repletion study underwent a 70-mg/kg leucine challenge on study
days 0, 7, 14, and 21. The mean challenge excretion rate for the
group of 5 subjects increased significantly with time on the egg
white diet (P < 0.002 by one-way ANOVA). By study day 7, the
challenge 3HIA excretion rates for 4 of the 5 subjects were
greater than the greatest challenge 3HIA excretion rate on day 0
(Figure 3). On study days 14 and 21, the values for all 5 subjects
were greater than the baseline range.
Repletion phase
The mean urinary excretion rates of 3HIA, biotin, bisnorbiotin,
and biotin sulfoxides during 21 d of biotin repletion are shown in
Figure 4. 3HIA excretion decreased dramatically and significantly
(P < 0.0001). Excretion of 3HIA was not significantly different
between those who started an 80 _g biotin supplement on day 28
(immediate supplement group; n = 5) and those who consumed
only a mixed general diet until day 35 (late supplement group;
n = 6). The interaction between time and supplementation group
was also not significant. For the pool of all subjects, excretion on
both day 35 and day 49 was significantly less than that on day 28
(P < 0.0002). By study day 49, the 3HIA excretion rates of all 11
subjects were normal.
For biotin excretion, two-wayANOVAwith repeated measureswas
significant for time (P < 0.0001), supplement group (P = 0.02), and for
the interaction (P = 0.03). For the immediate supplement group, biotin
excretion increased dramatically and significantly (P < 0.003) from
study day 28 to 35. A further modest increase from study day 35 to
study day 49was observed, but the increasewas not significant. For the
late supplement group, biotin excretion did not increase significantly
from study day 28 to 35.Asubstantial and significant increase occurred
in response to the later initiation of biotin supplementation (P < 0.003
for study day 49 compared with study day 0). In the immediate supplement
group, biotin excretion returned to normal for 5 of 5 subjects
by study day 35. Biotin excretion of one subject (no. 9) in this group had
returned to normal by study day 35, then fell to belowthe normal range
by study day 49 despite 14 d of supplementation, raising the question
of compliance with the biotin supplementation regimen as an outpatient.
In the late supplement group, biotin excretion for 1 of 6 subjects
remained abnormal at study day 35, but was normal by study day 49.
In both groups, the response of bisnorbiotin excretion to repletion
was similar to that of biotin and was significant (P < 0.0001).
Neither the difference between supplement groups nor the interaction
was significant. In the pooled group of 11 subjects, the
increases from study day 0 to study day 35 and to study day 49
were both significant (P < 0.0001). Subject no. 9 also excreted
abnormally low bisnorbiotin at study day 49.
The response of biotin sulfoxide excretion to repletion was similar
to that of biotin and was significant (P < 0.0002). Neither the
difference between supplement groups nor the interaction was
significant. In the pooled group of 11 subjects, the increases from
study day 0 to study day 35 and to study day 49 were significant
(P < 0.0004). The biotin sulfoxide excretion of subject no. 9 also
decreased dramatically from study day 35 to study day 49.
DISCUSSION
The results of clinical studies have provided evidence that marginal
biotin deficiency is more common than previously thought.
For example, our recent studies provide evidence that marginal,
asymptomatic biotin deficiency is common in normal human
pregnancy (1, 2). To cite another example, Velazquez (3) reported
laboratory evidence of biotin deficiency and biotin-responsive
rashes in children with severe protein-energy malnutrition. He
speculated that biotin deficiency might be rate limiting in the
nutritional rehabilitation of these patients. To cite a third example
in both children and adults, marginal biotin deficiency
appears to be a frequent consequence of long-term therapy with
certain anticonvulsants (4–8).
Properly diagnosing marginal biotin deficiency is important
because marginal deficiency may have deleterious health effects.
For example, marginal biotin deficiency may be teratogenic (33).
In all those studies, assertions concerning the presence of biotin
deficiency depended on the validity of indicators of biotin status.
The findings of this study provide confirmation that the urinary
excretion of biotin and the urinary excretion of 3HIA are early
and sensitive indicators of biotin deficiency. These studies also
provide evidence that resumption of a mixed general diet
repletes biotin status within 1 wk in most individuals as judged
both by urinary biotin excretion and 3HIA excretion. Moreover,
the rate of repletion is accelerated by supplementation with 80 _g
(328 nmol) biotin/d.
In this study, the urinary excretion rates of biotin and bisnorbiotin
were abnormally decreased in one individual despite 7 d of a
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BIOTIN INDICATORS IN DEFICIENCY AND REPLETION 1067
FIGURE 4. Repletion phase. Mean (ｱSD) urinary excretion of 3-hydroxyisovaleric acid (3HIA), biotin, bisnorbiotin (BNB), and biotin sulfoxides
(BSOs) for the immediate supplement (filled symbols; n = 5) and late supplement (open symbols; n = 6) groups at study days 28, 35, and 49. Time points
have been offset slightly for graphic clarity. NR, normal range. Because neither supplement group nor the interaction of supplement group with time was
significantly different for 3HIA, BNB, or BSO, for these indicators of biotin status, testing of trends with time was performed on the pooled subjects
(n = 11); P values are provided on bars. For biotin, both the effect of supplement group and the interaction of supplement group with time were significant
(P = 0.02 and P = 0.03, respectively); statistical comparisons are for supplement groups considered separately. *Significantly different from study
day 28, P < 0.001 (post hoc test after ANOVA).
mixed general diet followed by 14 d of biotin supplementation.
This subject’s 3HIA excretion returned to normal after 1 wk on a
general diet and remained normal during biotin supplementation.
This subject was 1 of the 2 individuals whose biotin excretion on
day 0 was less than the lower limit of normal, although 3HIA
excretion was normal. This may represent population variation,
although failure to take the biotin supplement is also a possibility.
Thus, these 2 indicators conflicted concerning this subject’s biotin
status. The urinary 3HIA excretion of a different individual
remained well within the normal range for the first 24 d of egg
white feeding, rising to twice the upper limit of normal on the last
day of the biotin depletion. Yet, her excretion of biotin and bisnorbiotin
was abnormally low after study day 14. We speculate
that this individual was indeed becoming biotin deficient and that
she represents a variant of normal in which the byproducts of the
buildup of methylcrotonyl CoA are not shunted primarily into
3HIA. Further studies of this individual are underway. Results
from these 2 subjects exemplify our growing impression that neither
of these 2 indicators used alone is sufficient to identify every
individual who is marginally deficient.
Both of these validated indicators depend on renal function.
Development of a valid indicator that does not depend on renal
function would likely be useful. Unfortunately, the plasma concentration
of biotin is not particularly useful in detecting marginal
biotin deficiency. Moreover, the plasma concentration of biotin is
quite elevated in the first trimester of pregnancy for unknown reasons
(2); the increase is not attributable to increased protein binding
(2). Likewise, the concentration of biotin in erythrocytes is
similar to the plasma concentration for the same blood sample
(unpublished data); this equivalency likely depends on time for
biotin to equilibrate across the erythrocyte membrane, either in
vivo or in vitro. Studies of propionyl-CoA carboxylase (EC
4.4.4.41) in lymphocytes of biotin deficient patients receiving parenteral
nutrition (34) suggest this enzyme activity may be a more
promising indicator for future studies.
One approach to comparing the sensitivity of several indicators
in detecting marginal biotin deficiency is to compare the proportion
of individuals who are identified as deficient at various points
during progressive deficiency. For example, by study day 14, biotin
excretion was less than the lower limit of normal for 8 of the 11
subjects and bisnorbiotin excretion was less than the lower limit
of normal for 7. At this same point in the progression of biotin deficiency,
the urinary excretion of 3HIA was increased in 8 subjects.
Thus, it appears that biotin, bisnorbiotin, and 3HIA have approximately
equal efficacy in detecting marginal biotin deficiency.
If the normal range is defined as the smallest and largest challenge
3HIA excretion rates for the 5 subjects on study day 0, then
4 of the 5 would be identified as biotin deficient by study day 7
and all 5 thereafter. For comparison, biotin excretion was less than
the lower limit of normal in 4 and bisnorbiotin was less than the
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1068 MOCK ET AL
lower limit of normal in 2 of the 11 subjects on study day 7; 3HIA
excretion was greater than the upper limit of normal in 3 of the 11
subjects on study day 7. By these criteria, the urinary excretion of
3HIA in response to a leucine challenge is a potentially useful
index of marginal biotin deficiency. The strength of this conclusion
is currently limited by the small number of subjects studied.
A variety of lines of evidence indicate that increased urinary
excretion of 3HIA is the result of accumulation of methylcrotonyl
CoA (the substrate for methylcrotonyl-CoA carboxylase) or its
immediate precursor isovaleryl CoA. The observed progressive
increases in 3HIA excretion after a leucine challenge during progressive
biotin deficiency are consistent with this mechanism.
The blunted responses to leucine challenge observed in biotinsupplemented
subjects compared with that in subjects with normal
biotin status suggest that methylcrotonyl-CoA carboxylase is
close to rate limiting in the leucine degradation pathway, even in
persons with normal biotin status. Thus, the observation that 24-h
excretion is an early indicator of biotin depletion is consistent with
the leucine challenge results.
To assess whether supplementation at the adequate intake
would have a significant effect on accelerating repletion, we provided
a commercially available biotin supplement with a labeled
content of 30 _g during the repletion phase. Although our previous
measurement of the biotin content of a broad range of commercially
available vitamins compounded from pure sources indicated
that the content was accurate (17), the assayed content of
the chosen supplement was 80 ｱ13 _g. Thus, the results of this
study indicate that supplementation at roughly 3 times the adequate
intake (16) augments the urinary excretion of biotin to
above the normal range in most patients, but the data from this
study do not address the effect of the supplement at the adequate
intake.
We acknowledge the assistance of Kim Stuckey and Jeanne Poppelreiter in
recruitment, patient contact, and sample transportation. Melain Raguseo and
Mike Ruckle performed biotin and analogue analyses, and Teresa Evans measured
organic acids.
REFERENCES
1. Mock DM, Stadler DD. Conflicting indicators of biotin status from
a cross-sectional study of normal pregnancy. J Am Coll Nutr 1997;
16:252–7.
2. Mock DM, Stadler D, Stratton S, Mock NI. Biotin status assessed
longitudinally in pregnant women. J Nutr 1997;127:710–6.
3. Velazquez A, Martin-del-Campo C, Baez A, et al. Biotin deficiency
in protein-energy malnutrition. Eur J Clin Nutr 1988;43:169–73.
4. Krause K-H, Berlit P, Bonjour J-P. Impaired biotin status in anticonvulsant
therapy. Ann Neurol 1982;12:485–6.
5. Krause K-H, Berlit P, Bonjour J-P. Vitamin status in patients on
chronic anticonvulsant therapy. Int J Vitam Nutr Res 1982;52:375–85.
6. Krause K-H, Kochen W, Berlit P, Bonjour J-P. Excretion of organic
acids associated with biotin deficiency in chronic anticonvulsant therapy.
Int J Vitam Nutr Res 1984;54:217–22.
7. Mock DM, Dyken ME. Biotin catabolism is accelerated in adults
receiving long-term therapy with anticonvulsants. Neurology 1997;
49:1444–7.
8. Mock DM, Mock NI, Lombard KA, Nelson RP. Disturbances in
biotin metabolism in children undergoing long-term anticonvulsant
therapy. J Pediatr Gastroenterol Nutr 1998;26:245–50.
9. Said HM. Biotin bioavailability and estimated average requirement:
why bother? Am J Clin Nutr 1999;69:352–3 (editorial).
10. Mock N, Malik M, Stumbo P, Bishop W, Mock D. Increased urinary
excretion of 3-hydroxyisovaleric acid and decreased urinary excretion
of biotin are sensitive early indicators of decreased status in
experimental biotin deficiency. Am J Clin Nutr 1997;65:951–8.
11. Hardinge MG, Crooks H. Lesser known vitamins in food. J Am Diet
Assoc 1961;38:240–5.
12. Hoppner K, Lampi B, Smith DC. An appraisal of the daily intakes of
vitamin B12, pantothenic acid and biotin from a composite Canadian
diet. Can Inst Food Sci Technol J 1978;11:71–4.
13. Bull NL, Buss DH. Biotin, pantothenic acid and vitamin E in the
British household food supply. Hum Nutr Appl Nutr 1982;36A:
125–9.
14. Lewis J, Buss DH. Trace nutrients: minerals and vitamins in the
British household food supply. Br J Nutr 1988;60:413–24.
15. Zempleni J, Mock DM. Biotin biochemistry and human requirements.
J Nutr Biochem 1999;10:128–38.
16. National Research Council. Dietary reference intakes for thiamin,
riboflavin, niacin, vitamin B-6, folate, vitamin B-12, pantothenic
acid, biotin, and choline. In: Food and Nutrition Board, Institute of
Medicine, ed. Recommended dietary allowances. 11th ed. Washington,
DC: National Academy Press, 1998:415.
17. Tolaymat N, Mock DM. Biotin analysis of commercial vitamin and
other nutritional supplements. J Nutr 1989;119:1357–60.
18. Pennington JAT. Biotin. Bowes and Church’s food values of portions
commonly used. 15th ed. Philadelphia: JP Lippincott Co, 1989.
19. Pennington JAT, Church HN. Biotin. Bowes and Church’s food values
of portions commonly used. 14th ed. NewYork: Harper & Row,
1985.
20. Green NM, Toms EJ. Purification and crystallization of avidin.
Biochem J 1970;118:67–70.
21. Green NM. Avidin. Adv Protein Chem 1975;29:85–133.
22. Zeman FJ. Clinical nutrition and dietetics. 2nd ed. New York:
Macmillan Publishing, 1991.
23. Mock DM, Malik MI. Distribution of biotin in human plasma: most
of the biotin is not bound to protein. Am J Clin Nutr 1992;56:427–32.
24. Mock DM, Lankford GL, Cazin Jr J. Biotin and biotin analogs in
human urine: biotin accounts for only half of the total. J Nutr 1993;
123:1844–51.
25. Zempleni J, McCormick DB, Mock DM. Identification of biotin sulfone,
bisnorbiotin methyl ketone, and tetranorbiotin-l-sulfoxide in
human urine. Am J Clin Nutr 1997;65:508–11.
26. Mock DM, Mock NI. Serum concentrations of bisnorbiotin and biotin
sulfoxide increase during both acute and chronic biotin supplementation.
J Lab Clin Med 1997;129:384–8.
27. Mock DM, Heird GM. Urinary biotin analogs increase in humans
during chronic supplementation: the analogs are biotin metabolites.
Am J Physiol Endocrinol Metab 1997;272:83–7.
28. Mock DM. Determinations of biotin in biological fluids. In:
McCormick DB, Suttie JW,Wagner C, eds. Methods in enzymology.
New York: Academic Press, 1997:265–75.
29. Mock DM, Lankford GL, Mock NI. Biotin accounts for only half of the
total avidin-binding substances in human serum. J Nutr 1995;125:941–6.
30. Heingard D, Tiderstrom G. Determination of serum creatinine by a
direct calorimetric method. Clin Chim Acta 1973;43:305–10.
31. Rose DB. Clinical physiology of acid-base and electrolyte disorders.
In: Jeffers J, Navrozov M, eds. Renal physiology. New York:
McGraw-Hill, Inc, 1994:44–65.
32. Zar JH. Biostatistical analysis. Englewood Cliffs, NJ: Prentice-Hall,
Inc, 1974.
33. Zempleni J, Mock DM. Marginal biotin deficiency is teratogenic.
Proc Soc Exp Biol Med 2000;223:14–21.
34. Velazquez A, Zamudio S, Baez A, Murguia-Corral R, Rangel-Peniche B,
Carrasco A. Indicators of biotin status: a study of patients on prolonged
total parenteral nutrition. Eur J Clin Nutr 1990;44:11–6.
Downloaded from ajcn.nutrition.org by guest on October 29, 2012
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