Studies of alcaptonuriaAbsorption spectra of homogentisic acid-chondroitin sulfate solutions.код для вставкиСкачать
PROGRESSEREPORT Studies of Alcaptonuria: Absorption Spectra of Homogentisic Acid-Chondroitin Sulfate Solutions By ROBERTAUSTIN~ ~ I L C IAND I EDWARD D. TITUS D EPOSITION of pigmentary material in the mesodermal tissues of the skeletal and cardiovascular systems of patients with alcaptonuric ochronosis has been repeatedly demonstrated both clinically and morphologically. The nature of the pigment, however, is not well demonstrated (other than the suggestion that it is probably quite unlike certain of the naturally occurring melanins’ ), and the intimate mechanisms of its biologic production remain almost entirely undiscovered. Furthermore, despite the observed localization of pigment in tissues which tend to be characterized by high chondroitin sulfate contents and observations that a number of anionic polyelectrolytes, including oxidation polymers of both gentisic and homogentisic acids, are capable of inhibiting hyaluronidase in vitro, virtually no recorded attention appears to have been directed toward possible interactions between homogentisic acid, its oxidation products and various connective tissue components. The present study was undertaken, therefore, as a preliminary investigation of the interactions between homogentisic acid solutions and various connective tissue components. MATER~ALS AND METHODS Synthetic, commercially available samples of homogentisic acid ( HGA ), homogentidc acid isolated and recrystallized from the urines of patients with alcaptonuric ochronosis and commercially available chondroitin sulfate (CSA) preparations were used. Each of the HGA preparations behaved identically and, thus, a single standardized sample was used; this had a m.p. equal to 146 to 148”C., with A,n,, = 290mp. and loge = 3.58. The CSA preparations were obtained from bovine nasal speta, had an optical rotation (a),,m = -Soand on elementary analysis, the following composition: C, 30.06; H, 5.36; N, 4.12; S, 1.75; ash, 20.84. Solutions of HGA, CSA and HGA + CSA in HGA:CSA mole ratios from 1:l to 1:8 were prepared in phosphate buffers over the pH range 6.8 to 7.6. Atmospheric oxidation was facilitated by placing the solutions in Erlenmeyer flasks a t room temperature (25°C.) and by constantly bubbling tank 0, through the solutions for periods from one minute to 120 hours. Ultraviolet absorption spectra, against internally compensated standards, were obtained of all preparations a t the end of each run, using a Cary llPM recording spectrophotometer. RESULTSAND DISCUSSION Absorption spectra of HGA solutions in every instance showed an initial peak at 290 mp. prior to autoxidation; subsequently, an additional peak at 250 mp. developed, as has been previously reported. CSA solutions showed only negligible absorption, without any absorption maxima, over the entire ultraviolet spectral range. CSA did not alter the absorption maximum of HGA solutions in any of the From the National Cancer Imtitute, National Institutes of Health, Bethesdu, Md. 566 567 STUDIES OF ALCAPTONUAIA 0. 0.1 I --- H.G.A.+ -H.G.A. C.S.A. 0.’ 0: 0. 0. 0 Od FIG. 1.-Ultraviolet absorption spectra of HGA and HGA+CSA (mole ratio = 1:4, HGA = 80 y/ml.). The ordinate is optical density, and the abscissa is wave length in millimicra. It is apparent that there is no spectrophotometric evidence of association between HGA and CSA and that slight inhibition of HGA autoxidation occurred in the HGA+CSA solution. studies, irrespective of pH or time of autoxidation. No spectrophotometric evidence of binding of HGA to CSA was found under the described experimental conditions. At pH’s greater than 7.2, however, slight inhibition of HGA autoxidation was observed in solutions having HGA:CSA mole ratios exceeding 1:4 (fig. 1). The present data would tend to suggest, therefore, that HGA does not bind to CSA in vitro at physiologic pH’s and that, in the system studied, CSA is in itself capable of inhibiting the atmospheric oxidation of HGA. Direct interaction of the salt type between the two moieties, both of which exist as anions in solutions, would not be expected, nor would it be likely that any negative ion, including homogentisate, would appreciably influence the high negative potential of CSA solutions.2 Other types of association states could occur theoretically. That this is probably not the case, however, is suggested in the present studies both by the lack of any detectable bathochromic shift in the absorption maximum of the HGA molecule and by the apparent 568 PROGRESS REPORT stabilization of the HGA molecule, at least as regards loss of electrons, by the addition of CSA to aqueous solutions fully saturated with respect to molecular oxygen. The data would tend to imply, therefore, that the mechanism( s ) of pigment formation in alcaptonuric ochronosis in all likelihood do not directly involve interaction between HGA, CSA and molecular 0 2 . Also, owing to the known low PO, of cartilage and the observation that HGA autoxidation does not proceed at detectable rates at low pO;s, it would seem likely that if molecular O2 be at all involved in HGA autoxidation in vivo, it is presumably involved via the agency of an enzyme system present locally. REFERENCES 1. Milch, R. A., Titus, E. D. and Loo, T. L.: 2. Mathews, M. B.: Condrotinsulfuric acidAtmospheric oxidation of homogentisic A linear polyelectrolyte. Arch. Biochem. Biophys. 43~151,1953. acid: Spectrophotonietric studies. Science 136:209-210, 1957. Robert Austin Milch, M.D.,Assistant Resident Orthopaedic Surgeon, The Johns Hopkins Hospital and Assistant in Orthopaedic Surgey, The Johns Hopkins University School of Medicine, Baltimore, Md.; formerly, Clinical Associate in Surgery, National Cancer Institute, National Institutes of Health, Bethesclu, Md. Student, School of Medicine, Stanford Edward D. Titus, M.S., Uniuersity, San Francisco, Calif .; formerly Research Associute, Clinical Phurmucology and Experimental Therapeutics Section, National Cancer Institute, Bethesda, Md.