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Diagnosis of Alkaptonuria by NMR Urinalysis : Rapid Qualitative and Quantitative Analysis of Homogentisic Acid

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Diagnosis of Alkaptonuria by NMR Urinalysis : Rapid Qualitative and Quantitative Analysis of Homogentisic Acid Tohoku J. exp. Med., 1986, 150, 227-228 Short Report Diagnosis of Alkaptonuria by NMR Urinalysis : Rapid Qualitative and Quantitative Analysis of Homogentisic Acid SHUICHIYAMAGUCHI, NAOYA KODA and TOYA OHASHI* .Division of Endocrinology and Metabolism and *Division of Neurology, Saitama Children's Medical Center,Iwatsuki 339 YAMAGUCHI,S., KODA,N. and OHASHI,T. Diagnosis of Alkaptonuria by NMR Urinalysis. Rapid Qualitative and Quantitative Analysis of Homogentisic Acid. Tohoku J. exp. Med., 1986, 150 (2), 227-228 Urinalysis of alkaptonur- ia using NMR spectroscopy revealed the abnormal amount of homogentisic acid. Qualitative and quantitative determination was performed simultaneously with untreated urine sample and within 15 min. NMR ; alkaptonuria ; homogentisic acid Alkaptonuria is an inborn error of metabolism characterized by ochronosis as dark deposits in the sclerae, early and severe osteoarthritis, and high incidence of heart disease. They excrete dark urine because of homogentisic aciduria that results from homogentisic acid oxidase deficiency. Biochemically, homogentisic acid has been identified by paper chromatography (Knox and LeMay-Knox 1951) or thin layer chromatography (Sankoff and Sourkes 1963). Quantitative analysis is only performed by enzymatic method, which needs a cumbersome procedure (Seegmiller et al. 1961). Currently proton nuclear magnetic resonance spectroscopy ('H-NMR) is reported as a useful method for the detection of metabolites in urine, especially in case of inborn error of metabolism (Yamaguchi et al. 1984, 1985; Iles et al. 1985). 'H-NMR urinalysis has several advantages ; 1) the quick procedure with untreated urine samples, 2) simultaneous analy- sis of metabolites such as amino acids, organic acids, carbohydrates and others. Qualitative and quantitative analysis of homogentisic acid with NMR urinalysis was studied in the patients with alkaptonuria. The diagnosis of alkaptonuria using this technique is easier than the methods ever established. METHODAND MATERIALS Control urine specimens were collected from children without any known metabolic or neurologic disorders. Urine samples from two patients with alkaptonuria were provided by Dr. William L. Nyhan, University of California and Dr. Hatae Maesaka, Kanagawa Chil- dren's Medical Center. Aliquots of untreated urine, 0.45 mL, were placed with 0.05 mL of 10 mM trimethylsilylpropionate-2, 2, 3, 3, -d4 (TSP-d4, MSD Isotopes, Montreal, Quebec, Canada) in D20 as an internal standard in 5 mm i.d. NMR tubes. 'H-NMR was recorded at 89.55 MHz with JEOL FX-90Q as previously described (Yamaguchi et al. 1984). Received July 8, 1986 ; accepted for publication August 14, 1986. Present address : Department of Pediatrics, Mount Sinai School of Medicine, New York, NY 10029, USA. 227 228 S. Yamaguchi et al. Fig 1. 1H-NMR spectra of urine from normal control (A) and patients with alakptonuria (B). Chemical shifts (ppm) were referred to TSP-d4. Authentic standard of homogentisic acid was dissolved in 10 mM phosphate buffered saline, pH 7.4. RESULTSAND DISCUSSION 1H-NMR spectra in the urine of normal control subjects (Fig. la) showed signals for water (3.5 to 5.3 ppm) and creative plus creatinine (3.0 ppm). Since the signals of water not necessary for our purposes, they were not recorded. 1H-NMR spectra of authentic homogentisic acid were shown as two separated signals, 6.76 ppm and 6.78 ppm. In the urine of a patient with alkaptonuria, two signals of homogentisic acid were observed as chemical shift 6.76 ppm and 6.78 ppm (Fig. lb). The dependency of homogentisic acid to the intensity of the signals, 6.76 ppm, was studied. Linearity was observed between 5 mM to 125 mM of homogentisic acid and when applied to the signal of 6.78 ppm, a similar linearity was obtained. The amount of homogentisic acid in calculated from the division of the intensities of signals of homogentisic acid and TSP-d4. Relative or absolute concen- tration in calculated with creatinine or with daily urine volume. This procedure needed only 15 minutes, which is far rapider than that of the enzymatic ones. It is advantages to require very small amount of urine, not to need pretreatment and to be able to make a qualitative and quantitative determination simultaneously. Acknowledgments We thank Director Dr. Takeshi Mori, Prof. Dr. Kihei Maekawa, Dr. Kikumaro Aoki and Dr. Yoshikatsu Eto for encouragement, Prof. Dr. Machiko Tozawa, Dr. Kazuhiro Matsushita for technical suggestions and Mr. Hideaki Yamamoto for preparation of the manuscript. References 1) Iles, R.A., Hind, A.J. & Chalmers, R.A. (1985) Use of proton magnetic resonance spectroscopy in detection and study of organic acidurias. Clin. Chem., 31,1795-1801. 2) Knox, W.E. & LeMay-Knox, M. (1951) The oxidation in liver of L-tyrosine to acetoacetate through p-hydroxyphenylpyruvate and homogentisic acid. Biochem. J., 49, 686-693. 3) Sankoff, I. & Sourkes, T.L. (1963) Determination by thin-layer chromatography of urinary homovanillic acid in normal and disease states. Canad. J. Biochem. Physiol., 41, 1381-1388. 4) Seegmiller, J.E., Zannoni, V.G., Laster, L. & La Du, B.N. (1961) An enzymatic spectrophotometric method for the determination of homogentisic acid in plasma and urine. J. biol. Chem., 236, 774-777. 5) Yamaguchi, S., Koda, N., Eto, Y. & Aoki, K. (1984) Rapid screening of metabolic disease by proton NMR urinalysis. Lancet, 2, 284. 6) Yamaguchi, S., Koda, N., Eto, Y. & Aoki, K. (1985) Quick screening and diagnosis of organic acidemia by NMR urinalysis. J. Pediat., 106, 620-622..
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