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,2
* Departments of Biochemistry & Molecular Biology and
Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR 72205;
** General Clinical Research Center, University of Arkansas for Medical Sciences, Little Rock, AR 72205; and
Clinical Research Center, University of Iowa College of Medicine, Iowa City, IA
2To whom correspondence should be addressed. E-mail: MockDonaldM{at}uams.edu.
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ABSTRACT |
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 TOP ABSTRACT SUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • organic acid • biotin deficiency • human • 3-hydroxypropionic acid • methylcitric acid
Although safe and adequate intakes for the water-soluble vitamin biotin have been recommended (1,2), the human requirement for biotin at various ages and in special situations, such as pregnancy, remains uncertain. This surprising gap in modern nutritional knowledge arises at least in part from the absence of studies validating the indices of biotin status during progressive experimental biotin deficiency. Recent clinical studies have provided evidence that marginal biotin deficiency can develop in such disparate clinical circumstances as pregnancy (3,4), protein energy malnutrition (5,6), and long-term therapy with certain anticonvulsants (7–10). Marginal, asymptomatic biotin deficiency is a common occurrence in normal human pregnancy as judged by increased 3-hydroxyisovaleric acid (3HIA)3 excretion, and this increased excretion can be reversed by biotin supplementation (11). To assess the severity of biotin deficiency in these clinical situations and to understand whether marginal biotin deficiency can cause human birth defects, there is a need to develop and validate indicators of marginal biotin deficiency (12). To this end, we induced progressive, but asymptomatic biotin deficiency in normal adults and tested the validity of four additional organic acids in detecting marginal biotin deficiency.
Biotin is a covalently bound prosthetic group for five mammalian carboxylases. Two of these, propionyl-CoA carboxylase (PCC) and methylcrotonyl-CoA carboxylase (MCC), catalyze essential steps in the intermediary metabolism of propionate and leucine, respectively. Decreased activity of PCC shunts the substrate propionyl CoA into alternative metabolic pathways producing 3-hydroxypropionic acid (3HPA) and methylcitric acid (MCA), which are then excreted in increased quantities in urine. Decreased activity of MCC shunts the substrate 3-methylcrotonyl CoA to alternate metabolic pathways, producing 3HIA, 3-methylcrotonylglycine (3MCG), and isovalerylglycine (IVG). In severe biotin deficiency caused by egg-white feeding (13) or by biotin-free parenteral nutrition (14), the urinary excretion of 3HPA, MCA, 3HIA, 3MCG, and IVG are often 10- to 100-fold above normal.
In two previous studies (15,16), we observed that increased urinary excretion of 3HIA was an early and sensitive indicator of biotin deficiency. In this study, we investigated the diagnostic utility of 3HPA, MCA, 3MCG, and IVG in the same subjects (15,16) in whom biotin deficiency was induced by egg-white feeding.
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SUBJECTS AND METHODS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Induction of marginal biotin deficiency
Study 1. The details of the diet and sample and diet collection for this study were published previously (15). Briefly, 10 subjects (3 women) completed a 3-wk inpatient study at the University of Iowa Clinical Research Center; subjects consumed a low-biotin, 30 g egg white/100 g dry weight diet that produced substantial decreases in the urinary excretion of biotin and substantial increases in the urinary excretion of 3HIA. Sufficiency of other vitamins and minerals was ensured by provision of a multivitamin and multimineral supplement containing 100% of the 1989 RDA for a variety of vitamins and minerals. At the end of the egg-white feeding period, subjects were given 300 µg (1.2 µmol) biotin/d for 10 d to restore biotin status. Samples were stored at -70°C. Initial analysis of samples was completed within 1 y.
Study 2. Subjects were fed a similar diet high in native egg white. Details of this study were published previously (16). In brief, 11 subjects (8 women) completed the study. Study 2 differed importantly from Study 1 because Study 2 included biotin-loading (300 µg biotin on Study Day -14 to -8) and washout phases (zero biotin on Study Day -7 to Study Day 0) before the start of the egg-white diet to ensure biotin adequacy of all subjects. In addition, subjects received a daily vitamin and mineral supplement not containing biotin.
At weekly intervals, 24-h urine collections were made; completeness of each collection was evaluated by determining the total creatinine excretion/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 was established in our laboratory and was similar to published normal ranges.
Analytic methods
Before GC/MS analysis, the creatinine concentrations were measured for all urine samples by the picric acid method using a Beckman Creatinine Analyzer 2 (Beckman Instruments).
Internal standards. Deuterated (D8) 3-hydroxyisovaleric acid (3HIA) was synthesized as described previously (17) and was used as the internal standard for the quantitation of 3-hydroxypropionic acid (3HPA). Because the structures of these two organic acids are similar and because the retention times for GC/MS are within 2 min of each other, 3HIA is an appropriate internal standard for 3HPA.
Deuterated 3HIA was also used as the internal standard for relative quantitation of isovalerylglycine (IVG) and 3-methylcrotonylglycine (3MCG). Neither IVG nor 3MCG is commercially available. For this study, the excretion rates of IVG and 3MCG are expressed relative to deuterated 3HIA.
Deuterated (D4) citric acid was used as the internal standard for the quantitation of MCA. The structures of these two organic acids are similar, and the retention times for GC/MS are within 2 min of each other.
Extraction.
Before extraction, all urine samples were diluted to a creatinine concentration of 4.4 mmol/L. For samples with original creatinine concentrations of <4.4 mmol/L, no dilution was carried out. To each diluted urine were added 10 µL of 1.59 mmol/L deuterated 3HIA and 10 µL of 2.91 mmol/L deuterated citric acid. The concentration of deuterated 3HIA added to each sample produced an area under the curve (AUC) for the quantitation ion (m/z 137) of the di-trimethylsilane (di-TMS) derivative that was similar in magnitude to the AUC of the quantitation ion for 3HPA-di-TMS (m/z 219). The AUC for the 3HIA quantitation ion (m/z 137) was also similar to the AUC of the quantitation ion for 3-methylcrotonylglycine-di-TMS (m/z 102). The concentration of deuterated citric acid added to each sample was chosen to produce an AUC for the quantitation ion (m/z 276) of the di-TMS derivative that was similar in magnitude to the AUC of the quantitation ion for methylcitric acid-TMS-4 (m/z 287). After the addition of 0.5 mL of 0.15 mol/L Ba(OH)2 (Sigma-Aldrich), the sample was centrifuged at 
1000 x g for 10 min. An aliquot of the supernatant (0.750 mL) was acidified with 6 mol/L HCl to a pH of 1.0, saturated with NaCl, and extracted by the discontinuous organic solvent method twice using 2 mL of ethyl acetate (Aldrich). The two extracts were pooled. A 1-mL aliquot of the extract was placed in a conical-bottomed centrifuge tube and dried completely using a Meyer analytical evaporator. Under these conditions, there is no detectable loss of 3HPA.
TMS derivation. To the dried sample, 10 µL of pyridine (Burdick and Jackson) was added and the sample was mixed on a vortex. BSTFA (40 µL) with 1% TMCS (Pierce, Rockford, IL) was added. The sample was capped, mixed again on a vortex, and incubated at 70°C for 20 min.
GC and MS. GC/MS analyses were performed as described previously (17). For quantitation, a characteristic mass fragment of the unknown (m/z 219 for 3HPA-di-TMS and m/z 287 for methylcitric acid-TMS-4) is normalized by the AUC of a characteristic mass fragment of the internal standard (m/z 137 for deuterated 3HIA-di-TMS and m/z 276 for citric acid-TMS-4). Relative quantitation of IVG and 3MCG is expressed as the AUC of the characteristic mass fragment (m/z 288 for isovalerylglycine-di-TMS and m/z 102 for 3-methylcrotonylglycine-di-TMS) and is normalized by the AUC of a characteristic mass fragment of the internal standard (m/z 137 for deuterated 3-hydroxyisovaleric acid-di-TMS).
External standards. Standard curves were generated with each GC/MS analytical run from samples containing varying amounts of 3HPA and a constant amount of deuterated 3HIA. Standard curves were linear over the concentration range of the samples analyzed in this study; a typical correlation coefficient was 0.99. Aqueous hydrolysis of ß-propionyl lactone (Sigma-Aldrich) was used to prepare 3HPA. The identity and purity of the synthesized 3HPA was confirmed by GC/MS of the di-TMS derivatives. Standardization of 3HPA was by volumetric titration with sodium hydroxide solution (Sigma-Aldrich).
Because MCA is not commercially available or easily synthesized, the standard curve for quantitating MCA was constructed by using a human urine sample containing a high concentration of MCA. The concentration of that reference urine was standardized against deuterated (D4) citric acid. Standard curves generated from standards with varying content of MCA and a constant content of deuterated citric acid were linear over the concentration range of the samples analyzed in this study; a typical correlation coefficient was 0.99.
Normal ranges. Normal ranges for urinary 3HIA excretion were determined in our laboratory from minimum and maximum excretion rates from 17 normal adults (9 women) (15,16,18). Normal ranges for 3HPA, MCA, 3MCG, and IVG were determined from minimum and maximum excretion rates for the 11 adults at Study Day 0 from Study 1.
Statistical methods
StatView 5.01 (SAS Institute) was used for all analyses.
Comparison of Study 1 and Study 2 at Study Day 0. The significance of differences between Study 1 and Study 2 at Study Day 0 was tested using Student’s unpaired, two-tailed t test with significance set at P < 0.05.
Testing of differences between Study 1 and Study 2 during progressive biotin deficiency. The volunteer populations in Study 1 and Study 2 were similar; the essential study designs including the egg-white feeding were similar; the method of quantitating the organic acid 3HIA was similar; and the analysis of the various organic acids were performed at the same time. Therefore, the results of the two studies could be directly compared. For the three organic acids quantitated in both Study 1 and Study 2, the significance of differences between studies and with time in subjects consuming the egg-white diet were tested by 2-way ANOVA with repeated measures. When significant differences between studies were detected, post-hoc testing was performed using Fisher’s test (19); data from Study 1 and Study 2 were not pooled. To determine the point at which in the progression of biotin deficiency the excretion of a particular organic acid had increased significantly from Study Day 0, 1-way ANOVA with repeated measures was performed as described below.
Testing of trends in individual studies with duration of egg-white feeding. For organic acids quantitated at only 2 time points (e.g., Study Day 0 and Study Day 21), the significance of differences was tested using the Mann Whitney U test. For organic acids quantitated at more than 2 time points, the significance of trends was tested by one-way ANOVA with repeated measures. If the trend was significant at P < 0.05, Fisher’s post-hoc test (19) was used to identify the points at which the group mean was significantly different from the baseline value. Values in the text are means ± SD.
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RESULTS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Mean 3-hydroxypropionic acid (3HPA) excretion rates in both Study 1 and Study 2 remained within the normal range during biotin depletion. In Study 1, excretion of 3HPA was greater than normal for only 1 of 10 subjects by Study Day 21. In Study 2, excretion of 3HPA was greater than normal for only 4 of 11 subjects by Study Day 28.
In Study 1, excretion of methylcitric acid (MCA) increased (P < 0.0001) with time in subjects consuming the egg-white diet (Fig. 3). Excretion of MCA differed from Study Day 0 by Study Day 7 (P < 0.0003) and was greater than normal for all 10 subjects by Study Day 21. In striking contrast, mean MCA excretion in Study 2 did not increase significantly at any point; MCA excretion was normal for all subjects at Study Day 28.
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DISCUSSION |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Excretion of 3HPA was consistently normal and exhibited a consistently poor sensitivity in detecting marginal biotin deficiency in both studies. Urinary excretion of MCA increased early in Study 1 and remained elevated. However, this promising diagnostic utility was not confirmed in Study 2. The loading and washout periods that preceded the second study with the attendant increase in biotin status may have caused the different results in the two studies.
Differences in analytical techniques did not contribute to the observed differences. Samples from the first and second studies were reanalyzed on the same day with identical internal standards. This reanalysis confirmed the values from the original analyses and provided no evidence for an analytical difference. We investigated whether the differences in protein or amino acid content of the study diets might have contributed to the difference. Protein and amino acid composition and intakes were similar, as were the amino acid profiles of the dietary protein.
We contend that the most likely explanation for the failure to observe an increase in MCA in the second study is that PCC did not become rate limiting in the intermediary metabolism of propionyl CoA.
The findings of this study should not be interpreted as negating the validity of urinary 3HPA, MCA, 3MCG, or IVG in the diagnosis of severe biotin deficiency, multiple carboxylase deficiency, or isolated deficiencies of the relevant carboxylases. Our interpretation is that marginal biotin deficiency does not reliably increase the excretion of any of these organic acids with the exception of 3HIA, but severe deficiency will increase the excretion of all of them.
The observations from this study are consistent with the hypothesis that substantial reductions of the activities of biotin-dependent enzymes are required to produce the substantial increases in organic acid excretion reported in moderate and severe biotin deficiency.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: AUC, area under the curve; 3HIA, 3-hydroxyisovaleric acid; 3HPA, 3-hydroxypropionic acid; IVG, isovalerylglycine; MCA, methylcitric acid; MCC, methylcrotonyl-CoA carboxylase; 3MCG, 3-methylcrotonylglycine; PCC, propionyl-CoA carboxylase; TMS, trimethylsilane.
Manuscript received 26 August 2003. Initial review completed 23 September 2003. Revision accepted 4 November 2003.
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LITERATURE CITED |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Mock, D. M. & Said, H. (1998) Biotin. 1998 http://www.nutrition.org/nutinfo/ ed. 2001. The American Society for Nutritional Sciences web site [homepage on the Internet]. Accessed on 12/2/03.
2. Mock, D. M. (2001) Biotin. Rucker, B. Suttie, J. McCormick, D. Machlin, L. eds. Handbook of Vitamins 2001 Marcel Dekker New York, NY. .
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. Mock, D. M., Stadler, D., Stratton, S. & Mock, N. I. (1997) Biotin status assessed longitudinally in pregnant women. J. Nutr. 127:710-716.
5. Velazquez, A., Martin-del-Campo, C., Baez, A., Zamudio, S., Quiterio, M., Aguilar, J. L., Perez-Ortiz, B., Sanchez-Ardines, M., Guzman-Hernandez, & Casanueva, E. (1988) Biotin deficiency in protein-energy malnutrition. Eur. J. Clin. Nutr. 43:169-173.
6. 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.
7. Krause, K.-H., Berlit, P. & Bonjour, J.-P. (1982) Impaired biotin status in anticonvulsant therapy. Ann. Neurol. 12:485-486.[Medline]
8. 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]
9. Krause, K.-H., Kochen, W., Berlit, P. & Bonjour, J.-P. (1984) Excretion of organic acids associated with biotin deficiency in chronic anticonvulsant therapy. Int. J. Vitam. Nutr. Res. 54:217-222.[Medline]
10. 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]
11. Mock, D. M., Quirk, J. G. & Mock, N. I. (2002) Marginal biotin deficiency during normal pregnancy. Am. J. Clin. Nutr. 75:295-299.
12. Said, H. M. (1999) Biotin bioavailability and estimated average requirement: why bother?. Am. J. Clin. Nutr. 69:352-353.
13. Sweetman, L., Surh, L., Baker, H., Peterson, R. M. & Nyhan, W. L. (1981) Clinical and metabolic abnormalities in a boy with dietary deficiency of biotin. Pediatrics 68:553-558.
14. Mock, D. M. (1996) Biotin. Ziegler, E. E. Filer, L. J., Jr eds. Present Knowledge in Nutrition 7th ed. 1996 International Life Sciences Institutes, Nutrition Foundation Washington, DC. .
15. Mock, N. I., Malik, M. I., Stumbo, P. J., Bishop, W. P. & Mock, D. M. (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.
16. Mock, D. M., Henrich, C. L., Carnell, N. & Mock, N. I. (2002) Indicators of marginal biotin deficiency and repletion in humans: validation of 3-hydroxyisovaleric acid excretion and a leucine challenge. Am. J. Clin. Nutr. 76:1061-1068.
17. 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]
18. Mock, D. M., Lankford, G. L. & Cazin, J., Jr (1993) Biotin and biotin analogs in human urine: biotin accounts for only half of the total. J. Nutr. 123:1844-1851.
19. Zar, J. H. (1974) Biostatistical Analysis 1974 Prentice-Hall Englewood Cliffs, NJ.
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