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Studies of alcaptonuriaAbsorption spectra of homogentisic acid-chondroitin sulfate solutions.

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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.
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acid, solutions, sulfate, chondroitin, alcaptonuriaabsorption, homogentisic, studies, spectral
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