Detection of novel visible-light region absorbance peaks in the urine after alkalization in patients with alkaptonuria.
BACKGROUND
Alkaptonuria, caused by a deficiency of homogentisate 1,2-dioxygenase, results in the accumulation of homogentisic acid (2,5-dihydroxyphenylacetic acid, HGA) in the urine. Alkaptonuria is suspected when the urine changes color after it is left to stand at room temperature for several hours to days; oxidation of homogentisic acid to benzoquinone acetic acid underlies this color change, which is accelerated by the addition of alkali. In an attempt to develop a facile screening test for alkaptonuria, we added alkali to urine samples obtained from patients with alkaptonuria and measured the absorbance spectra in the visible light region.


METHODS
We evaluated the characteristics of the absorption spectra of urine samples obtained from patients with alkaptonuria (n = 2) and compared them with those of urine specimens obtained from healthy volunteers (n = 5) and patients with phenylketonuria (n = 3), and also of synthetic homogentisic acid solution after alkalization. Alkalization of the urine samples and HGA solution was carried out by the addition of NaOH, KOH or NH4OH. The sample solutions were incubated at room temperature for 1 min, followed by measurement of the absorption spectra.


RESULTS
Addition of alkali to alkaptonuric urine yielded characteristic absorption peaks at 406 nm and 430 nm; an identical result was obtained from HGA solution after alkalization. The absorbance values at both 406 nm and 430 nm increased in a time-dependent manner. In addition, the absorbance values at these peaks were greater in strongly alkaline samples (NaOH- KOH-added) as compared with those in weakly alkaline samples (NH4OH-added). In addition, the peaks disappeared following the addition of ascorbic acid to the samples.


CONCLUSIONS
We found two characteristic peaks at 406 nm and 430 nm in both alkaptonuric urine and HGA solution after alkalization. This new quick and easy method may pave the way for the development of an easy method for the diagnosis of alkaptonuria.
Department of Clinical Laboratory, The University of Tokyo Hospital, Tokyo, Japan ; The Group of Neurobiology, Division of Health Sciences, Graduate School of Medicine, Osaka University, Osaka, Japan.
Yasunori
Y
Tokuhara
Department of Clinical Laboratory, The University of Tokyo Hospital, Tokyo, Japan.
Kenichi
K
Shukuya
Department of Clinical Laboratory, The University of Tokyo Hospital, Tokyo, Japan.
Masami
M
Tanaka
Department of Clinical Laboratory, The University of Tokyo Hospital, Tokyo, Japan.
Mariko
M
Mouri
Department of Clinical Laboratory, The University of Tokyo Hospital, Tokyo, Japan.
Ryunosuke
R
Ohkawa
Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
Midori
M
Fujishiro
Department of Pediatrics, Shimane University Faculty of Medicine, Izumo, Japan.
Tomoo
T
Takahashi
Department of Clinical Laboratory, The University of Tokyo Hospital, Tokyo, Japan.
Shigeo
S
Okubo
Department of Clinical Laboratory, The University of Tokyo Hospital, Tokyo, Japan.
Hiromitsu
H
Yokota
Department of Clinical Laboratory, The University of Tokyo Hospital, Tokyo, Japan ; Department of Clinical Laboratory Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
Makoto
M
Kurano
Department of Clinical Laboratory, The University of Tokyo Hospital, Tokyo, Japan ; Department of Clinical Laboratory Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
Hitoshi
H
Ikeda
Department of Pediatrics, Shimane University Faculty of Medicine, Izumo, Japan.
Seiji
S
Yamaguchi
The Group of Neurobiology, Division of Health Sciences, Graduate School of Medicine, Osaka University, Osaka, Japan.
Shinobu
S
Inagaki
Department of Pediatrics and Child Health, Nihon University School of Medicine, Tokyo, Japan.
Mika
M
Ishige-Wada
Department of Pediatrics and Child Health, Nihon University School of Medicine, Tokyo, Japan.
Hiromi
H
Usui
Department of Clinical Laboratory, The University of Tokyo Hospital, Tokyo, Japan ; Department of Clinical Laboratory Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
Yutaka
Y
Yatomi
Department of Clinical Laboratory, The University of Tokyo Hospital, Tokyo, Japan ; Department of Clinical Laboratory Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
Tatsuo
T
Shimosawa
D000328:Adult; D000474:Alkaptonuria / Q000175:diagnosis / Q000652:urine*; D016022:Case-Control Studies; D005260:Female; D064368:Healthy Volunteers; D006713:Homogentisic Acid / Q000652:urine*; D006801:Humans; D006878:Hydroxides / Q000494:pharmacology*; D008027:Light; D008297:Male; D008875:Middle Aged; D010084:Oxidation-Reduction; D010661:Phenylketonurias / Q000175:diagnosis / Q000652:urine*; D017680:Potassium Compounds / Q000494:pharmacology*; D012972:Sodium Hydroxide / Q000494:pharmacology*; D013053:Spectrophotometry; D055815:Young Adult
D006878:Hydroxides; D017680:Potassium Compounds; D012972:Sodium Hydroxide; D006713:Homogentisic Acid; C029943:potassium hydroxide