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2
Departments of
*
Biochemistry & Molecular Biology and
Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR 72205
2To whom correspondence should be addressed. E-mail: MockDonaldM{at}uams.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • lymphocyte propionyl-CoA carboxylase • biotin • deficiency • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–3
) and in individuals treated for extended periods with certain anticonvulsants (4
–9
). In rats (10
) and humans (11
), reduced urinary excretion of biotin and increased urinary excretion of 3-hydroxyisovaleric acid (3HIA), 3
are early and sensitive indicators of impaired biotin deficiency. Both of these validated indicators depend on renal function. Increased 3HIA excretion reflects decreased activity of the biotin-dependent enzyme methylcrotonyl-CoA carboxylase. Development of a valid indicator of biotin status 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 (11
). The concentration of biotin in erythrocytes is similar to the plasma concentration in the same blood sample (unpublished data); thus is not likely to be useful for detecting marginal biotin deficiency.
Propionyl-CoA carboxylase (PCC) is a biotin-dependent enzyme found in a variety of tissues including liver and lymphocytes. Studies of biotin-deficient patients receiving parenteral nutrition (12
) or suffering from protein-energy malnutrition (13
) suggest that lymphocyte PCC activity reflects biotin status in moderate-to-severe biotin deficiency. Using egg-white–fed rats, a well-established model of biotin deficiency, we sought to determine whether the activity of the PCC in lymphocytes is useful in detecting marginal biotin deficiency. We also evaluated whether urinary excretion of 3-hydroxypropionic acid (3HPA), an organic acid that reflects decreased activity of PCC, is useful in detecting marginal biotin deficiency in this rat model.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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On d 1 of the study, rats were randomly assigned to the biotin-deficient group or the biotin-supplemented group. The deficient group was fed a diet4
containing 30 g of egg-white solids per 100 g diet (TD 87400, Harlan Teklad, Madison, WI). This quantity of egg white has been shown to reliably produce biotin deficiency in rats (14
,15
). The biotin-supplemented group received a diet containing 30 g of egg-white solids/100 g diet to which 16.4 µmol biotin/kg diet had been added (TD 97197, Harlan). This is sufficient biotin to occupy all of the biotin-binding sites on avidin as well as enough excess biotin to meet the metabolic requirement even if the bioavailability of the added biotin may be significantly <100% (15
,16
). The amount of food fed to the biotin-supplemented rats each day was adjusted to track the consumption of the deficient group. In previous studies (17
,18
), we used pair-feeding on the basis of weight of diet consumed by the deficient rat in the pair, but significant weight differences developed despite pair-feeding. For this study, to maintain similar weights in the two diet groups, we adjusted daily the amount of food available to the supplemented group. The approach was successful in producing similar weight gains in the two experimental groups. However, in the last week of the experiment, the body weights diverged significantly (see Results).
For 24 h before killing on d 1, 7, 14, 28 and 40, urine was collected in metabolic cages. During the collection, rats were deprived of food to prevent contamination of urine with the avidin-containing diet. Under intraperitoneal sodium pentobarbital anesthesia (150 mg/kg weight), rats were exsanguinated by vena cava puncture and livers were removed. To obtain sufficient material for lymphocyte carboxylase measurements, blood samples from several rats were pooled to make five pools for each diet group at each of the first three time points. Specifically, blood samples from four rats were pooled for d 1, three for d 7 and two for d 14. Blood from only one rat per diet group was sufficient for d 28 and 40. After processing, all samples were stored at -70°C until analysis.
On d 14, two biotin-supplemented rats were noted to have generalized hemorrhage, decreased movement and decreased body temperature. The rats were killed; autopsies revealed that one rat had only one kidney. Otherwise, the autopsies were not revealing. Neither rat’s tissues were used for the study. The cause of the illness was suspected to be a virus. No other rats displayed these findings.
Analytical methods.
Urinary excretion of biotin was measured by the horse radish peroxidase (HRP)-avidin-binding assay after separation of biotin from biotin analogs with HPLC (19
). Total hepatic biotin was measured by the HRP-avidin assay after acid hydrolysis to release all protein-bound biotin followed by HPLC separation of the biotin from the other hydrolysis products (20
,21
).
Activity of PCC in liver was measured using a14C-bicarbonate incorporation assay described previously (22
) with some modifications. Briefly, a portion of liver was homogenized in 250 mmol/L sucrose, 50 mmol/L Tris buffer, pH 7.9, 5 mmol/L reduced glutathione and 1 mmol/L EDTA at a ratio of 1 g liver to 5 mL buffer. The crude homogenate was sonicated on ice four times for a duration of 10 s each. After centrifugation at 105,000 x g, the supernatant was retained for enzymatic and protein assay. The assay was conducted in a 96-well U-bottomed plate at 30°C for 15 min. The reaction was stopped by the addition of 25 µL of 800 mmol/L perchloric acid; 40 µL of reaction mixture was transferred to a well in a 96-well plate containing solid scintillant (LumaPlates, Packard Instruments, Downers Grove, IL). The plates were dried in a hood to remove the unreacted NaH14CO3. The disintegration rate for the 14C-labeled product was determined using a quench curve for 14C in a scintillation counter (Topcount, Packard Instruments). Activity was expressed per milligram protein (BAC Protein Assay kit, Pierce, Rockford, IL) or per gram liver. The normalized values reported in the Results were expressed per gram liver. This assay was linear with time for at least 30 min. Within-run precision (CV) was typically <5%; between-run precision was typically 
10%.
Lymphocyte PCC activity was measured using the optimized 14C-bicarbonate incorporation assay described above. Blood drawn from the inferior vena cava was pooled as described above to yield 6 mL. Lymphocytes were isolated by density gradient separation as follows. Blood was overlaid onto Histopaque (Sigma-Aldrich, St; Louis, MO) and centrifuged at 400 x g for 30 min. The buffy coat layer containing the mononuclear cells was removed, resuspended in two volumes of PBS and sedimented at 200 x g for 10 min at room temperature. When mononuclear cells are isolated by this gradient centrifugation procedure, the cell preparation contains almost entirely lymphocytes (23
). In blood, lymphocytes and monocytes are quantitatively the most important mononuclear cells; eosinophils and basophils account for 
3% of total blood mononuclear cells (23
). The monocytes are removed during the process of cell isolation because of their selective binding to plastic surfaces. In this paper, the term lymphocytes will be used.
The lymphocyte pellet was resuspended in 1 mL of PBS for every 1.5 mL of starting volume of blood. This suspension was divided into aliquots, placed in 2-mL microfuge tubes for subsequent assays and lymphocytes were pelleted at 3800 x g for 5 min. The supernatant was aspirated and the pellet containing 0.2 mg of protein was resuspended in 90 µL of the buffer described above with an additional 25 µL of 0.5% Triton X-100. The lymphocyte pellets were stored at -70°C for assay of all time points at once. The pellets were vortexed vigorously to resuspend the lymphocytes.
The PCC assay method for lymphocytes was the same as for liver PCC with the following differences. To obtain enough lymphocyte protein for all analyses, blood was pooled from several rats at the early time points (d 0, n = 4; d 7, n = 3; d 14, n = 2; and d 28 and 40, n = 1). There were five discrete pools for each diet group at each time point. Because of the limited protein available in each of the five pools, the PCC activity was determined once from each pool using 50 µL of suspension (500 mg/L protein) in a single replicate incubated at 30°C for 30 min with correction. Activity of PCC was normalized by protein.
Organic acid concentrations in urine were determined by gas chromatography/mass spectrometry (GC/MS) of the trimethylsilyl (TMS) derivatives as follows. Before GC/MS analysis, creatinine concentration of each sample was measured by the picric acid method (24
–26
) in our laboratory using the Beckman Creatinine Analyzer 2 (Beckman Instruments, Brea, CA).
Quantitation of 3HIA.
Excretion of 3HIA was quantitated using GC/MS detection as previously described (10
). This method employs authentic unlabeled and deuterated 3HIA as external and internal standards, respectively. The depicted normal range was established from the maximum and minimum values excreted by all rats on d 0 and the biotin-supplemented rats at all subsequent time points.
3-Hydroxypropionic acid (3HPA) and 3-methylcrotonylglycine (3MCG)
Internal standards.
Deuterated (D8) 3HIA, synthesized as described previously (27
), was used as the internal standard for the quantitation of 3HPA. The structures of these two organic acids are similar, and the retention times on GC/MS are within 2 min of each other, making 3HIA a reasonable internal standard. Only relative quantitation was possible for 3MCG. Deuterated 3HIA was also used as the internal standard for quantitation of 3MCG.
Extraction.
Before extraction, all urine samples were diluted with distilled water to a concentration of 4.4 mmol/L creatinine. For urine samples with original creatinine concentrations of <4.4 mmol/L creatinine, no dilution was made. Within experimental error, the same result is obtained with any creatinine concentrations > 0.88 mmol/L creatinine (unpublished data). To 0.5 mL of the diluted urine, we added 10 µL of 1.59 mmol/L deuterated 3HIA followed by vortexing and addition of 0.5 mL of 0.15 mol/L Ba(OH)2 (Sigma Diagnostics, St. Louis, MO). The concentration of deuterated 3HIA added to each sample produced an area under the curve for the quantitation ion (m/z 137) of the di-TMS derivative that was similar in magnitude to the area under the curve of the quantitation ion for 3HPA-di-TMS (m/z 219). This area was also similar to the area under the curve of the quantitation ion for 3MCG-di-TMS (m/z 102). Sample extraction and derivatization with N,O-bis(trimethylsilyl)-triflouroacetamide with 1% trimethylchlorosilane (Pierce) were as previously described (10
).
Absolute and relative quantitation of these organic acids was performed by selected ion monitoring. A characteristic mass fragment of the unknown (m/z 219 for 3HPA-di-TMS) was normalized by the area under the curve of a characteristic mass fragment of the internal standard (m/z137 for deuterated 3HIA-di-TMS). Relative quantitation of 3MCG is expressed as the area under the curve of the characteristic mass fragment (m/z 102 3-methylcrotonylglycine-di-TMS) and is normalized by the area under the curve of a characteristic mass fragment of the internal standard (m/z 137 for deuterated 3HIA-di-TMS). Because the internal standard is added at a constant ratio to creatinine in the sample, the area under the curve ratios are proportional to the absolute excretion rates normalized by creatinine.
External standard.
Standard curves generated from samples containing varying amounts of 3HPA and a constant amount of deuterated 3HIA were linear over the concentration range of the samples analyzed in this study; the correlation coefficient was typically 
0.99. Preparation of 3HPA was by the hydrolysis of ß-propionyl lactone (Sigma/Aldrich) in aqueous solution. The identity and purity of the synthesized 3HPA were confirmed by GC/MS of the di-TMS derivatives. The 3HPA stock solution was standardized by volumetric titration with a standardized NaOH solution (Sigma/Aldrich).
Excretion rates for all organic acids were expressed per mole creatinine. Results expressed per 24 h were somewhat less well grouped, but the statistical significance and conclusions did not change. We infer that this results from incomplete urine collections in the metabolic cages.
Statistical methods.
Differences in hepatic biotin from d 0 to 28 and between diet groups at d 28 were tested for significance using the unpaired t test with Bonferroni’s correction for multiple t tests. Weight gain, urinary excretion of biotin, urinary excretion of 3HIA, 3HPA, and 3MCG, and liver and lymphocyte PCC activity were evaluated by a two-way ANOVA (time x treatment) using StatView 5.0 (28
). If the two-way ANOVA indicated a significant difference for time, the individual treatment groups were analyzed by one-way ANOVA with Fisher’s post-hoc testing using correction for multiple comparisons to determine the time point at which the changes were significant relative to d 1.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Neither body weight (Fig. 1
) nor liver weights differed between the biotin-deficient and biotin-supplemented rats. Physical signs of biotin deficiency were first noted in the biotin-deficient rats during wk 4; hair was rough and moderately sparse in 15 of 23 rats. By wk 6, partial alopecia was noted in eight of 13; kangaroo gait was noted in two of 13 and irritability was present in all. Appearance and behavior of supplemented rats was normal except as discussed in Materials and Methods.
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Biotin deficiency was successfully induced by the egg-white diet without added biotin as judged by several established indicators. Urinary biotin excretion decreased rapidly (Fig. 2
). By d 7, urinary biotin excretion for each rat in the deficient group had decreased to less than the lowest value of any rat on d 0; the range on d 0 was 19–125 nmol/mol creatinine. Urinary biotin excretion for the supplemented rats increased 20-fold, providing evidence of biotin sufficiency. By two-way ANOVA, the effects of diet (P < 0.0001), time (P = 0.0001) and their interaction were significant (P = 0.0001).
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), likely reflecting decreased tissue (especially hepatic) activity of methylcrotonyl-CoA carboxylase. By d 7, urinary 3HIA excretion for each deficient rat was greater than the largest excretion of 3HIA for any rat on d 0. At all subsequent time points, the 3HIA excretion of each deficient rat was at least twice the greatest value at d 0. In the biotin-supplemented rats, the urinary 3HIA excretion remained within the d 0 range (30–88 µmol/mol creatinine). The change in 3HIA excretion was affected by diet (P < 0.0001), time (P < 0.0001), and the interaction of diet with time (P < 0.0001).
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Hepatic PCC activity decreased rapidly (Fig. 4
). By d7, hepatic PCC activity in each rat in the deficient group had decreased to less than the lowest activity of any rat on d 0. The hepatic PCC activity in the biotin-supplemented rats increased 60%, providing evidence of biotin sufficiency. The effects of diet and time and their interaction were significant (P < 0.0001).
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Lymphocyte PCC activity decreased dramatically with time of consuming the egg-white diet in the deficient rats (Fig. 4)
. By d 7, lymphocyte PCC activity in each rat in the deficient group had decreased to less than the lowest value of any rat on d 0. The course of the decrease in lymphocyte PCC activity was strikingly similar to that of hepatic PCC activity. For the supplemented rats, lymphocyte PCC activity increased to more than two times that of the control value but decreased from maximum values after d 14. The effects of diet and time and their interaction were significant (P < 0.0001).
Diagnostic utility of urinary 3-hydroxypropionic acid (3HPA) and 3 methylcrotonyl glycine (3MCG).
Urinary excretion of 3HPA decreased in both deficient and supplemented rats (Fig. 5
). Although the deficient group excreted more 3HPA on d 14 and 28, the difference was not significant (P = 0.52 by diet) and the excretion rates were nearly identical on d 40.
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). On d 7, the 3MCG excretion rates for 10 of 15 deficient rats were greater than the largest value on d 0. By d 14, four of five were abnormal. At subsequent time points, the 3MCG excretion of each deficient rat was at least four times the greatest value at d 0. In the group consuming the diet with added biotin, the urinary 3MCG excretion remained within the d 0 range. The change in 3MCG excretion was affected (P < 0.0001) by diet, time and the interaction of diet with time.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,3
). In both children and adults, marginal biotin deficiency appears to be a frequent consequence of long-term therapy with certain anticonvulsants (4
,5
,8
,9
,29
). In children suffering from severe protein-energy malnutrition, Velazquez reported biotin-responsive rashes and evidence of biotin deficiency (13
,30
). Velazquez and co-workers speculated that biotin might be rate-limiting in the nutritional rehabilitation of these patients.
Properly diagnosing marginal biotin deficiency is important because marginal deficiency may have deleterious health effects. For example, marginal biotin deficiency may be teratogenic (31
). In most studies, assertions concerning the presence of biotin deficiency depended on indicators of biotin status that also reflect renal function. For instance, as biotin intake decreases, the renal tubule might increase biotin reclamation, producing a decrease in urinary biotin excretion that precedes actual tissue biotin deficiency. However, Velazquez reported that the PCC activity of lymphocytes in children with severe protein-energy malnutrition was decreased on average and that lymphocyte PCC activity increased significantly during nutritional rehabilitation with biotin compared with a control group who were rehabilitated without biotin. The three children with biotin-responsive rashes had the lowest PCC activities and the greatest activation index. A similar decrease of lymphocyte PCC and restoration by biotin repletion has been reported in patients receiving long-term parenteral nutrition without biotin (12
). These findings suggest that PCC might be a useful indicator of marginal biotin status.
The findings from this study provide evidence that the decrease in lymphocyte PCC activity closely parallels the decrease in hepatic PCC activity in biotin-deficient rats. Lymphocyte PCC can be used to detect marginal biotin deficiency as early as d 7 of egg-white feeding. On the basis of the ability to identify unequivocally all of the deficient rats, lymphocyte PCC activity is as sensitive and specific as urinary biotin excretion and urinary excretion of 3HIA, at least in this rat model. Renal excretion of biotin likely reflects both biotin depletion and the homeostatic attempt of the kidney to retain biotin as the total body pool of biotin becomes progressively depleted. Because lymphocyte PCC does not depend upon renal function, this indicator of biotin status could potentially serve an important role in assessing biotin status if a similar diagnostic sensitivity applies to humans.
In contrast, urinary excretion of 3HPA is not an early or sensitive indicator of marginal biotin deficiency in this rat model. On the basis of the decline of hepatic PCC activity, an increase in 3HPA might have been predicted in analogy to the increase in 3HIA that results from decreased activity of methylcrotonyl-CoA carboxylase. Indeed, Rodriguez-Melendez and co-workers (32
) recently reported that mRNA levels encoding holocarboxylase synthetase decreased significantly in liver, kidney, muscle and brain in biotin-deficient rats. In contrast, mRNA for pyruvate-carboxylase and propionyl-CoA carboxylase did not decrease significantly despite the expected decreases in activity and protein mass for these two biotin-dependent carboxylases. The complexity of the response to biotin depletion is further highlighted by the observation that urinary excretion of 3HPA does not increase in marginal biotin deficiency in this rat model. We speculate that PCC may not become rate-limiting in this pathway as quickly as methylcrotonyl-CoA carboxylase becomes limiting in the metabolism of leucine. Although this increase in 3MCG excretion is delayed compared with 3HIA, the presence of the increase in 3MCG is consistent with this interpretation. Theoretically, an increase in holocarboxylase synthetase activity in response to biotin supplementation might explain the observed increase in hepatic and lymphocyte PCC activity in the biotin-supplemented group. The observed increases in PCC activity in liver and lymphocytes and in urinary excretion of biotin in the control group guided our choice of the term "biotin supplemented" rather than biotin sufficient.
The findings of this study should not be interpreted as negating the validity of urinary 3HPA or 3MCG in the diagnosis of severe biotin deficiency, multiple carboxylase deficiency or isolated deficiencies of the relevant carboxylases. In severe biotin deficiency caused by egg-white feeding (33
) and in biotin deficiency during biotin-free parenteral nutrition (34
), the urinary excretion of 3HPA and 3MCG are typically 10- to 100-fold above normal; the magnitudes of elevation for these organic acids (and for 3HIA and methylcitric acid) vary from patient to patient.
The results of this study contrast, but do not conflict, with the results of Liu et al. (35
). These investigators induced severe biotin deficiency in rats with >7 wk of egg-white feeding. In that study, all rats exhibited skin rash, alopecia, conjunctivitis and central nervous system dysfunction; weight decreased to 103 ± 9 g. An ad libitum consumption group weighed an average of 328 ± 30 g. In these severely biotin-deficient rats, Liu et al. (35
) detected > 100-fold elevations of 3HPA and 3MCG excretion and > 50-fold increase in excretion of 3HIA and methylcitric acid. In comparison, in this study and in a previous study in rats (36
), the biotin deficiency was not as severe. The signs of biotin deficiency were just emerging after 4 wk of consuming the egg-white diet; reduced rates of weight gain appeared only at 40 d, the final time point in this study. We interpret this difference as evidence that mild-to-moderate biotin deficiency does not increase the excretion of 3HPA. However, increased 3HPA excretion likely would have ensued had we allowed the rats to progress to severe deficiency.
The failure to detect increased excretion of 3HPA is not easily attributable to technical errors. Urine from a biotin-deficient individual was used as a reference standard for increased excretion of 3HPA. Moreover, the same analytical method was successful in detecting normal and abnormal 3HPA excretion during biotin withdrawal in a child with a biotin-dependent inborn error of metabolism (unpublished data).
The validity of the conclusions of this paper rests on two assumptions inherent in the experimental design. The first is that the differences between the two experimental groups were caused primarily by biotin deficiency rather than any biotin excess in the biotin-supplemented group. The observations concerning increased urinary excretion of 3-HIA and decreased lymphocyte PCC activity compared with d 0 are both consistent with this assumption. The second assumption is that the changes observed were attributable only to changes in biotin status, rather than to a transition from the nonpurified diet used during the stabilization phase to the semipurified diets used during the experimental phase of the study. This assumption seems reasonable in light of the specific role of biotin as the covalently bound prosthetic group for the carboxylases that catalyze the step that becomes rate-limiting, giving rise to increased excretion of the organic acids examined in this study. However, the possibility remains that a portion of the changes reported here could be the result of changes in some other component of the diet.
In summary, this optimized technique for measuring carboxylase activity in a 96-well format is satisfactory for measuring PCC activity using volumes of blood that can easily be obtained from humans, including term neonates. Application of the technique to a rat model of biotin deficiency revealed that lymphocyte PCC activity is an early and sensitive indicator of marginal deficiency. We speculate that lymphocyte PCC may be a useful indicator of biotin status in humans. In rats, urinary excretion of 3-MCG was not useful in detecting marginal biotin deficiency, but was useful in detecting moderate biotin deficiency; however, urinary excretion of 3-HPA was not useful in detecting either marginal or moderate biotin deficiency.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: GC/MS, gas chromatography/mass spectrometry; 3HIA, 3-hydroxyisovaleric acid; 3HPA, 3-hydroxypropionic acid; HRP, horse radish peroxidase; 3MCG, 3-methylcrotonylglycine; PCC, propionyl-CoA carboxylase; TMS, trimethylsilyl.
4 The egg-white diet contained the following: (g/kg) egg white solids, spray-dried, 300.0; casein, "vitamin-free" test, 44.0; dextrose, monohydrate, 798.06; corn oil, 100.0; cellulose, 20.0; mineral mix, Ca-P deficient (TD 79055), 13.37; calcium phosphate, dibasic CaHPO4, 19.3; calcium carbonate CaCO3, 1.15; biotin (omitted in the deficient diet), 0.004; vitamin B-12 (0.1% in mannitol), 0.03; calcium pantothenate, 0.066; choline dihydrogen citrate, 3.497; folic acid, 0.002; menadione, 0.05; niacin, 0.099; pyridoxine HCl, 0.022; riboflavin, 0.022; thiamine HCl, 0.022; dry vitamin A palmitate (500,000 U/g), 0.04; dry cholecalciferol (500,000 U/g), 0.004; dry vitamin E acetate (500 U/g), 0.242; ethoxyquin (antioxidant), 0.02.
Manuscript received 11 October 2001. Initial review completed 21 January 2001. Revision accepted 26 March 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Mock, D. M., Stadler, D., Stratton, S. & Mock, N. I. (1997) Biotin status assessed longitudinally in pregnant women. J. Nutr. 127:710-716.
2. Mock, D. M., Mock, N. I., Stratton, S. L., Heird, G. M. & Stadler, D. D. (1996) Increased excretion of 3-hydroxyisovaleric acid provides evidence of biotin deficiency during pregnancy. FASEB J. 10:A464(abs.).
3. Mock, D. M. & Stadler, D. D. (1997) Conflicting indicators of biotin status from a cross-sectional study of normal pregnancy. J. Am. Coll. Nutr. 16:252-257.[Abstract]
4. Krause, K.-H., Berlit, P. & Bonjour, J.-P. (1982) Impaired biotin status in anticonvulsant therapy. Ann. Neurol. 12:485-486.[Medline]
5. Krause, K.-H., Berlit, P. & Bonjour, J.-P. (1982) Vitamin status in patients on chronic anticonvulsant therapy. Int. J. Vitam. Nutr. Res. 52:375-385.[Medline]
6. Krause, K.-H., Bonjour, J.-P., Berlit, P., Kynast, G., Schmidt-Gayk, H. & Schellenberg, B. (1988) Effect of long-term treatment with antiepileptic drugs on vitamin status. Drug Nutr. Interact. 5:317-343.[Medline]
7. Mock, D. M. & Dyken, M. E. (1995) Biotin deficiency results from long-term therapy with anticonvulsants. Gastroenterology 108:A740(abs.).
8. Mock, D. M. & Dyken, M. E. (1997) Biotin catabolism is accelerated in adults receiving long-term therapy with anticonvulsants. Neurology 49:1444-1447.
9. Mock, D. M., Mock, N. I., Lombard, K. A. & Nelson, R. P. (1998) Disturbances in biotin metabolism in children undergoing long-term anticonvulsant therapy. J. Pediatr. Gastroenterol. Nutr. 26:245-250.[Medline]
10. Mock, N. I. & Mock, D. M. (1989) Use of an improved method for determination of urinary 3-hydroxyisovaleric acid allows earlier detection of biotin deficiency in the rat. Clin. Res. 37:955A(abs.).
11. Mock, N., Malik, M., Stumbo, P., Bishop, W. & Mock, D. (1997) 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. 65:951-958.
12. Velazquez, A., Zamudio, S., Baez, A., Murguia-Corral, R., Rangel-Peniche, B. & Carrasco, A. (1990) Indicators of biotin status: a study of patients on prolonged total parenteral nutrition. Eur. J. Clin. Nutr. 44:11-16.[Medline]
13. Velazquez, A., Teran, M., Baez, A., Gutierrez, J. & Rodriguez, R. (1995) Biotin supplementation affects lymphocyte carboxylases and plasma biotin in severe protein-energy malnutrition. Am. J. Clin. Nutr. 61:385-391.
14. Mock, D. M. (1988) Evidence for a pathogenetic role of fatty acid (FA) abnormalities in the cutaneous manifestations of biotin deficiency. FASEB J. 2:A1204(abs.).
15. Kramer, T. R., Briske-Anderson, M., Johnson, S. B. & Holman, R. T. (1984) Effects of biotin deficiency on polyunsaturated fatty acid metabolism in rats. J. Nutr. 114:2047-2052.
16. Klevay, L. M. (1976) The biotin requirement of rats fed 20% egg white. J. Nutr 106:1643-1646.
17. Mock, D. M. (1990) Evidence for a pathogenic role of 
6 polyunsaturated fatty acid in the cutaneous manifestations of biotin deficiency. J. Pediatr. Gastroenterol. Nutr. 10:222-229.[Medline]
18. Mock, D. M., Mock, N. I., Johnson, S. B. & Holman, R. T. (1988) Effects of biotin deficiency on plasma and tissue fatty acid composition: evidence for abnormalities in rats. Pediatr. Res. 24:396-403.[Medline]
19. Mock, D. M. (1997) Determinations of biotin in biological fluids. McCormick, D. B. Suttie, J. W. Wagner, C. eds. Methods in Enzymology 1997:265-275 Academic Press New York, NY. .
20. Mock, D. M. & Malik, M. I. (1992) Distribution of biotin in human plasma: most of the biotin is not bound to protein. Am. J. Clin. Nutr. 56:427-432.
21. Mock, D. M., Mock, N. I. & Langbehn, S. E. (1992) Biotin in human milk: methods, location, and chemical form. J. Nutr. 122:535-545.
22. Zempleni, J., Trusty, T. A. & Mock, D. M. (1997) Lipoic acid reduces the activities of biotin-dependent carboxylases in rat liver. J. Nutr. 127:1776-1781.
23. Ratnoff, O. D. (1988) Blood. Berne, R. M. Levy, M. N. eds. Physiology 2nd ed. 1988:357-370 C. V. Mosby Company St. Louis, MO. .
24. O’Brien, D., Ibbott, F. A. & Rodgerson, D. O. (1968) Laboratory Manual of Pediatric Micro-Biochemical Techniques 1968 Hoeber Medical Division, Harper & Row New York, NY. .
25. Heingard, D. & Tiderstrom, G. (1973) Determination of serum creatinine by a direct calorimetric method. Clin. Chim. Acta 43:305-310.[Medline]
26. Rose, D. B. (1994) Clinical Physiology of Acid-Base and Electrolyte Disorders. Jeffers, J. Navrozov, M. eds. Renal Physiology 1994:44-65 McGraw-Hill New York, NY. .
27. Mock, D. M., Jackson, H., Lankford, G. L., Mock, N. I. & Weintraub, S. T. (1989) Quantitation of urinary 3-hydroxyisovaleric acid using deuterated 3-hydroxyisovaleric acid as internal standard. Biomed. Environ. Mass Spectrom. 18:652-656.[Medline]
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P < 0.02; *P < 0.003.
