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© 2005 The American Society for Nutritional Sciences J. Nutr. 135:615-618, March 2005

Nutritional Methodology

Measurement of 3-Hydroxyisovaleric Acid in Urine of Biotin-Deficient Infants and Mice by HPLC1

Toshiaki Watanabe2, Ken-Ichi Oguchi, Shuhei Ebara and Toru Fukui*

School of Human Science and Environment, Himeji Institute of Technology, University of Hyogo, Himeji, 6700092, Japan and * Byotai Seiri Laboratory, Tokyo, 1730025, Japan

2To whom correspondence should be addressed. E-mail: watanabe{at}shse.u-hyogo.ac.jp.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
We developed an assay for measuring urinary 3-hydroxyisovaleric acid (3-HIA) using HPLC after derivatization with 2-nitrophenylhydrazine hydrochloride (2-NPH · HCl). The derivatized 3-HIA was extracted into n-hexane and separated isocratically on a C8 reversed-phase column for fatty acids (YMC-Pack FA). We used this method to measure 3-HIA in urine extracts from mice fed a biotin-deficient diet for >4 wk and in an infant who was fed a special Japanese formula and was suspected of being biotin deficient. Urinary 3-HIA could be assayed within the range of 0.42–8.5 mmol/L with high accuracy by this method, as an indicator of biotin deficiency. Therefore, the HPLC method for 3-HIA described here may be a useful tool clinically as well as in the research laboratory.

KEY WORDS: • 3-hydroxyisovaleric acid • HPLC • biotin deficiency • methylcrotonyl CoA carboxylase • urine

Biotin is a cofactor for several carboxylases used in fatty acid synthesis, gluconeogenesis, and BCAA metabolism. The major biotin-containing enzymes are ß-methylcrotonyl-CoA carboxylase (MCC),3 propionyl-CoA carboxylase, pyruvate carboxylase, and acetyl CoA carboxylase. MCC catalyzes an essential step in the degradation of leucine, which converts ß-methylcrotonyl-CoA to 3-methylglutaconyl-CoA. The reduced activity of MCC leads to an elevated excretion of 3-methylcrotonic acid, the product of its hydration (3-hydroxyisovaleric acid: 3-HIA), and 3-methylcrotonylglycine, formed by conjugation with glycine. The increased urinary excretion of these abnormal metabolites reflects reduced activity of MCC or is due to dietary biotin depletion in genetically normal individuals.

Conditions that cause biotin deficiency include the following: long-term consumption of undenatured egg white; infant diets that do not include biotin; deficiency of one or more of the biotin-dependent carboxylases in inherited metabolic disorders; biotinidase deficiency and holocarboxylase synthetase deficiency; and biotin transporter deficiency (1,2). One of the most important criteria for the diagnosis of biotin deficiency is the detection of organic acids in urine, such as 3-HIA and 3-hydroxypropionic acid, which are elevated in biotin deficiency (3–5).

In general, 3-HIA in the urine and/or serum of biotin-deficient patients and experimental animals has been assayed by GC/MS, which is specific for assaying 3-HIA in screening tests for biotin deficiency. However, the procedure is complex and difficult. Therefore, we devised an assay for urinary 3-HIA by HPLC, using equipment that is available in most laboratories. We also discuss the usefulness of this method for assaying 3-HIA in the urine of biotin-deficient and biotin-supplemented mice and human infants.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Animal care and urine collection. Male mice (ICR/Jcl) were obtained from CLEA Japan at 8 wk of age. The biotin-deficient diet was purchased from Oriental Yeast in pelleted form. The components of the biotin-deficient diet (g/kg) were as follows: egg white, 245; cornstarch, 465; sucrose, 100; nonnutritive cellulose, 50; corn oil, 60; mineral mix, 70; and vitamin mix, 10. The control diet was made by supplementing the biotin-deficient diet with biotin (5.0 mg of biotin/kg diet). Mice were kept in an animal room maintained at constant temperature (23 ± 2°C) with a 12-h light:dark cycle (0700–1900 h).

The mice consumed a biotin-deficient or biotin-supplemented diet ad libitum and had free access to distilled water for 6 wk. The body weights of the 14-wk-old mice were 27.9 ± 2.5 and 28.2 ± 2.0 g in the biotin-deficient and biotin-supplemented groups, respectively. Consumption of the diet was also confirmed to be approximately the same in both diet groups, as shown in our previous study (6). Urine was collected from 3 mice fed the biotin-deficient or biotin-supplemented diet every week for 6 wk. The urine of the mice in the individual metabolic cages was collected for 24 h and stored at –40°C until use. All procedures were performed in accordance with the standards related to the care and management of experimental animals of the Japanese Prime Minister’s Office (7).

    Urine collection in an infant. Because biotin is not yet registered in Japan as a food additive, except in some foods such as dietary supplements, it cannot be added to infant formulas. We demonstrated previously that the biotin concentration of special formulas for medical treatment and prevention of disease in Japan was less than a fifth of the level in American products (8). Therefore, it has often been reported that biotin deficiency develops in infants with food allergies or inborn errors of metabolism who have been fed special Japanese formulas (9).

In the present case, the infant studied was a 5-mo-old Japanese male. At 4 wks of age, he was diagnosed as having dyspepsia and started to receive maternal milk and/or special formula called Elemental Formula (Meiji Milk Products). Afterwards, prominent erythematous skin lesions developed on his eyelids, perioral region, and neck. Dietary biotin deficiency was strongly suspected and oral treatment with 1 mg/d biotin was started. After 2 wk, the skin lesions disappeared rapidly, and the infant recovered and remained well. Urine was collected 1 wk before and after the biotin treatment. This study was performed in accordance with the ethical principles for medical research involving human subjects (Declaration of Helsinki, World Medical Association). Informed consent was obtained from the parents at enrollment.

    Reagents. For the HPLC analysis of standards, 3-HIA was obtained from Tokyo Kasei Kogyo. A kit (X8RFAR 01) for the analysis of short- and long-chain FFA by HPLC (YMC) was used for the pretreatment of the 3-HIA standard solution and urine samples. This kit contains a derivatized reagent that converts the carboxyl moiety of FFA into 2-nitrophenylhydrazide (2-NPH). Sensitive detection in the UV-visible range thus becomes possible after a simple derivatization procedure. Analytical reagent-grade acetonitrile was obtained from Wako Chemical Industries. The control urine "AUTION CHECK II," which is prepared from human urine and is used to monitor the precision of urinalysis, was obtained from Arkray.

    Derivatization procedures. The derivatization procedure for fatty acids, including 3-HIA, was a modification of a previously reported technique (10,11) and was as follows:

  1. Sample and standard solutions of 3-HIA (100 µL), 200 µL of 2-NPH · HCl (20 mmol/L) solution, and 200 µL of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (250 mmol/L) solution were added in turn to a 15-mL tube. The mixture was heated at 60°C for 20 min.
  2. After the addition of 200 µL of 15% KOH solution (in 20% methanol), the mixture was heated at 60°C for 15 min and then cooled to room temperature.
  3. To the resulting mixture, 4 mL of 0.033 mmol/L phosphate buffer (pH 6.4)-0.5 mol/L HCl (3.8:0.4 v:v) and 5 mL of n-hexane were added and mixed on a vortex mixer.
  4. The n-hexane layer, separated after centrifugation at 2800 x g for 5 min, was taken to remove the long-chain fatty acids. This step was repeated.
  5. Diethyl ether (5 mL) was added and mixed on a vortex mixer. After centrifugation at 2800 x g for 5 min, the diethyl ether layer was collected in a new 15-mL tube.
  6. Milli-Q water (3 mL) was added to the diethyl ether layer, mixed on a vortex mixer, and centrifuged at 2800 x g for 5 min. The diethyl ether layer was collected in a fresh tube.
  7. The diethyl ether layer was collected and dried under decompression at room temperature. The residue was dissolved in 1 mL of methyl alcohol, and an aliquot (10 µL) was used as a sample for measurement.

    Chromatographic analyses and conditions. Chromatographic separations were performed using a HITACHI HPLC system (Hitachi). The HPLC column was a C8 reversed-phase column for fatty acids (YMC-Pack FA), which was packed with Si 60 (particle size 5 µm, 250 x 4.6 mm) only for carboxylic acid.

The solvent system for elution from the YMC-Pack FA column consisted of acetonitrile:methyl alcohol:Milli-Q water (30:16:54, by vol). Solvents were adjusted to pH 4.5 with 0.01 mol/L HCl; they were filtered through a 0.45-µm membrane filter and degassed under decompression with ULVAC (Sinku Kiko). Separations were made at a flow rate of 1.0 mL/min. The column temperature was kept constant at 50°C. The elution patterns were monitored at 230 nm to detect the derivatized carbonyl moiety of 3-HIA. The retention time (RT, in min) on a YMC-Pack FA column ranged from 5 to 11 min.

    Biochemical analyses. The biotin concentrations in the urine were quantified using a microtiter plate adaptation of the microbiological assay with Lactobacillus plantarum ATCC 8014. The microbioassay lacks specificity compared with biotin analysis using an HPLC/streptavidin assay. Urinary specimens were filtered through 0.45-µm membranes and assayed without hydrolysis. The creatinine concentrations were measured in all urine samples with the picric acid method using a Creatinine Test Wako kit (Wako Chemical Industries). The concentrations of urinary biotin and 3-HIA were expressed as µmol/mol creatinine and mmol/mol creatinine, respectively.

    Statistical analyses. For statistical evaluation of the data in mice, repeated-measures ANOVA and Student’s t test were used. Differences of P < 0.05 were considered significant. StatView 5.01 (SAS Institute) software was used for all statistical analyses. Values in the text are means ± SD.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
In the chromatograms of the 3-HIA standard solution and of the urine analyzed by HPLC, the peak of authentic 3-HIA was identified as the position with an RT of 8.32 min (Fig. 1a). In the chromatogram of the extract of the urine of mice fed a biotin-deficient diet for 6 wks, a high peak was detected at 8.33 min and low unknown peaks appeared (Fig. 1b). 3-HIA was separated completely from other fatty acids in each sample. A peak was detected at 8.72 min in the urine of the biotin-supplemented mice at a position markedly different from the peak of 3-HIA (Fig. 1c). No peak was present at 8.33 min.



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  		FIGURE 1 Chromatograms (HPLC) of derivatized 3-HIA in biotin-deficient and -supplemented mice and in an infant suspected of being biotin deficient. (a) Standard 3-HIA; (b) Urinary extract of biotin-deficient mice; (c) Urinary extract of biotin-supplemented mice. A new peak appeared at 8.72 min at a position markedly different from that of the 3-HIA peak; (d) Extract of the infant’s urine collected before biotin treatment; (e) Extract of the infant’s urine collected after biotin treatment. Large arrows and figures indicate the peak of 3-HIA.

 
Standard 3-HIA concentrations ranging from 0.42 to 8.5 mmol/L and the peak area were correlated (r = 0.999, P < 0.01) (Fig. 2a). In mice fed the biotin-deficient diet for 6 wk, the concentration of urinary 3-HIA was 114.2 ± 69.6 mmol/mol creatinine, which was higher than the concentration of 35.2 ± 23.8 mmol/mol creatinine in mice fed the biotin-deficient diet for 4 wk (P < 0.046; Table 1). However, no profound clinical signs of biotin deficiency, such as loss of hair or dermatitis, were observed in these biotin-deficient mice during the experimental period.



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  		FIGURE 2 Development of the standard curve (panel a) and assessment of the relation between urinary biotin and 3-HIA concentrations in biotin-deficient mice (panel b, r = –0.581, P = 0.021).

 

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  		TABLE 1 Effects of biotin deficiency on the excretion of 3-HIA in urine of mice1

 
3-HIA was detected by HPLC in the urine of biotin-deficient mice (3-HIA-positive mice) (Table 1). The incidence of 3-HIA-positive mice increased with the length of time the biotin-deficient diet was fed. There were no 3-HIA-positive mice within 3 wk of feeding, but all biotin-deficient mice excreted 3-HIA in their urine after 4 wk of feeding. Urinary biotin and 3-HIA concentrations in biotin-deficient mice were inversely correlated (r = –0.58, P = 0.021; Fig. 2b). When the biotin concentration in the urine was <15 µmol/mol creatinine, 3-HIA was detected in the urine of 6 of 7 mice. On the other hand, no 3-HIA was detected in the urine of 8 biotin-deficient mice that had a urinary biotin level > 15 µmol/mol creatinine. Moreover, no 3-HIA was detected in the urine of the biotin-supplemented mice (Table 1).

The chromatogram of urine obtained from the infant fed a special formula manufactured in Japan and suspected of being biotin deficient had its highest peak at 8.27 min(Fig. 1d). Another peak was detected at 8.77 min. However, these peaks were not detected in the urine after biotin treatment (Fig. 1e).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
GLC was used previously to detect 3-HIA and ß-methylcrotonylglycine after hydrogenation of urinary extracts from humans with MCC deficiency (12). Subsequently, the first application of GC/MS for the quantitative detection of 3-HIA in urine was reported by Mock et al. (13). Derivatized 3-HIA was detected in urine extracts of patients with biotin deficiency. However, in addition to the high cost of the necessary equipment, after repeated extraction and the separation of the organic acids in the urine, a derivative for separation by GC must also be made (14). In addition, measurement of urinary 3-HIA is a time-consuming process.

Several HPLC methods were developed for the analysis of fatty acids in serum and urine; these employ precolumn derivatization techniques to increase the sensitivity and specificity of detection (10,11,15,16). In the present study, the preprocessing, including labeling, was simplified by the use of the derivatization technique, and a method for 3-HIA measurement was established using a standard HPLC system. The lower limit of detection of urinary 3-HIA was 0.042 mmol/L. This sensitivity was sufficient to measure 3-HIA in urine samples from an infant suspected of being biotin deficient. A relatively higher peak of 3-HIA was obtained before biotin treatment compared with other fatty acids present in the urine. These findings suggest that this HPLC method is sufficiently sensitive for assaying urinary 3-HIA in screening tests for biotin deficiency in humans.

The recovery of 3-HIA was in the range of 90.1–108.8%. This indicates that the present method does not have completely satisfactory precision for analyzing the 3-HIA concentration in urine because the method used here has several extraction and separation steps, which may affect the recovery of 3-HIA. The normal range of urinary 3-HIA in humans was reported to be from 5.1 to 10.7 mmol/mol creatinine as assessed by GC/MS, and it is increased several-fold by biotin deficiency (17). In the present study, the urinary 3-HIA concentration was 78.6 mmol/mol creatinine in an infant fed a special Japanese formula. However, no 3-HIA was detected after biotin treatment of this infant.

The concentrations of biotin and organic acids, and the activity of carboxylase in the serum and urine are generally used as indicators of biotin status (18,19). Mock et al. (3–5) demonstrated that decreased urinary biotin and increased urinary 3-HIA are sensitive indicators of early biotin deficiency, but methylcrotonylglycine and isovalerylglycine, which are also produced due to the decreased activity of MCC, are not. 3-HIA is detected in urine before the appearance of clinical signs of biotin deficiency; thus, it is expected to be a useful indicator of early biotin deficiency. The measurement of 3-HIA in urine may thus be useful for the diagnosis of biotin deficiency.

3-HIA is also a sensitive indicator of biotin deficiency in mice. 3-HIA was detected within a short time after the beginning of the feeding of a biotin-deficient diet, along with a decrease of biotin in the urine. Thus, the HPLC method for 3-HIA described here can be a useful tool clinically as well as in the research laboratory.


   ACKNOWLEDGMENTS
 
The authors thank Ms. Ayumi Taniguchi for excellent technical support with the biotin assay.


   FOOTNOTES
 
1 Supported by a grant from the University of Hyogo 2004. Back

3 Abbreviations used: 3-HIA, 3-hydroxyisovaleric acid; MCC, methylcrotonyl CoA carboxylase; 2-NPH, 2-nitrophenylhydrazide; RT, retention time. Back

Manuscript received 19 October 2004. Initial review completed 4 November 2004. Revision accepted 17 December 2004.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

1. Mock, D. M., DeLorimer, A. A., Liebman, W. M., Sweetman, L. & Baker, H. (1981) Biotin deficiency: an unusual complication of parenteral alimentation. N. Engl. J. Med. 304:820-823.[Medline]

2. Wolf, B. & Feldman, G. L. (1982) The biotin-dependent carboxylase deficiencies. Am. J. Hum. Genet. 34:699-716.[Medline]

3. 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 biotin status in experimental biotin deficiency. Am. J. Clin. Nutr. 65:951-958.[Abstract/Free Full Text]

4. Mock, D. M., Henrich-Shell, C. L., Carnell, N., Stumbo, P. & Mock, N. I. (2004) 3-Hydroxypropionic acid and methylcitric acid are not reliable indicators of marginal biotin deficiency in humans. J. Nutr. 134:317-320.[Abstract/Free Full Text]

5. 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.[Abstract/Free Full Text]

6. Watanabe, T. (1983) Teratogenic effects of biotin deficiency in mice. J. Nutr. 113:574-581.

7. Japanese Prime Minister’s Office (1980) The Standards Related to the Care and Management of Experimental Animals. Notification No. 6, March 27 1980.

8. Watanabe, T. & Fukui, T. (1998) Low biotin content of infant formulas made in Japan. Food Add. Contam. 15:619-625.

9. Higuchi, R., Noda, E., Koyama, Y., Shirai, T., Horino, A., Juri, T. & Koike, M. (1996) Biotin deficiency in an infant fed with amino acid formula and hypoallergenic rice. Acta Paediatr. 85:872-874.[Medline]

10. Miwa, H., Yamamoto, M., Nishida, T., Nunoi, K. & Kikuchi, M. (1987) High-performance liquid chromatographic analysis of serum long-chain fatty acids by direct derivatization method. J. Chromatogr. 416:237-245.[Medline]

11. Miwa, H. & Yamamoto, M. (1987) High-performance liquid chromatographic analysis of serum short-chain fatty acids by direct derivatization. J. Chromatogr. 421:33-41.[Medline]

12. Stokke, O., Eldjarn, L., Jellum, E., Pande, H. & Waaler, P. E. (1972) Beta-methylcrotonyl-CoA carboxylase deficiency: a new metabolic error in leucine degradation. Pediatrics 49:726-735.[Abstract/Free Full Text]

13. Mock, D. M., Jackson, H., Lankford, G. L., Mock, N. I. & Weintraub, S. T. (1989) Quantification of urinary 3-hydroxyisovaleric acid using deuterated 3-hydroxyisovaleric acid as internal standard. Biomed. Environ. Mass Spectrom. 18:652-656.[Medline]

14. Teran-Garcia, M., Ibarra, I. & Velazquez, A. (1998) Urinary organic acids in infant malnutrition. Pediatr. Res. 44:386-391.[Medline]

15. D’Amboise, M. & Gendreau, M. (1979) Isocratic separation of human blood plasma long chain free fatty acid derivatives by reversed phase liquid chromatography. Anal. Lett. 12:381-395.

16. Yamaguchi, M., Matsunaga, R., Hara, S., Nakamura, M. & Ohkura, Y. (1986) Highly sensitive determination of free fatty acids in human serum by high-performance liquid chromatography with fluorescence detection. J. Chromatogr. 375:27-35.[Medline]

17. Mock, D. M., Quirk, J. G. & Mock, N. I. (2002) Marginal biotin deficiency during normal pregnancy. Am. J. Clin. Nutr. 75:295-299.[Abstract/Free Full Text]

18. Chastain, J. L., Bowers-Komro, D. M. & McCormick, D. B. (1985) High-performance liquid chromatography of biotin and analogues. J. Chromatogr. 330:153-158.[Medline]

19. Zempleni, J., McCormick, D. B. & Mock, D. M. (1997) Identification of biotin sulfone, bisnorbiotin methyl ketone, and tetranorbiotin-l-sulfoxide in human urine. Am. J. Clin. Nutr. 65:508-511.[Abstract/Free Full Text]





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