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Studies of alcaptonuriainfra-red spectra of deuterated homogentisic acid solutions.

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BRIEF NOTE
Studies of Alcaptonuria : Infra-Red Spectra of Deuterated
Homogentisic Acid Solutions*
By ROBERTAUSTINMILCH
P
REVIOUS HEPORTS HAVE demonstrated that the autoxidized quinoneform polymers of homogentisic acid ( HGA-OP ) , but not the corresponding hydroquinoneform monomers,l are capable of binding to and acting as
interchain crosslinking agents for collagen structures.":' These reactions presumably account for the later clinical development in alcaptonuric individuals
of ochronosis and of ochronotic arthritis and arterio~clerosis.~
Little is known directly, however, of the additional properties or structure
of the autoxidized compounds, save for the fact that they readily form phenylhydrazone derivatives. The presently reported investigation was thus undertaken specifically ( a ) to determine and compare the infra-red spectra of
monomeric and polymeric homogentisic acid solutions and ( b ) to determine
the effects of oxidizing agents on the probable structure of the homogentisic
acid monomer.
Approximately 20 per cent solutions of a commercially available ( Nutritional
Biochemicals Corp., Cleveland) homogentisic acid sample were prepared in
99.5' per cent deuterium oxide (Volk Radiochemical Corp., Skokie, Ill.) and
in 1 per cent D 2 0 solutions of K:{Fe(CN),;. Fresh samples were immediately
placed in a thermoregulated Irtran-2 cell and spectra obtained as outlined
previously"*7using a Beckman IR-5A recording spectrophotometer. Autoxidized
samples were permitted to stand in open vescels at room temperature
(25 -t 3 ° C ) for 21-23 days before being analyzed. Rims were obtained at
room temperature and with the sample cell maintained at approximately 50°C.
Commercially available samples of hydroquinone and phenylacetic acid
were also run under identical experimental conditions, as were the difference
spectra of monomeric and polymeric homogentisic acid against phenyIacetic
acid and against hydroquinone.
Representative spectra are presented in figures 1-3. Probable assignments
Aided b y grants from the Nationul Institute of Arthritis und Metabolic Diseases
(AM-02642 M e t ) and the American Cancer Society (IN-11F).
ROnEnr AUSTIN MILCH, M.D., F.A.C.S.: Associate Professor of Orthopaedic Surgery and
Director, Orthopueldic Research Laboratory, T h e Johns Hopkins University School of
Medicine; Orthopaedic Surgeon, Children's Metlical and Surgical Center, ?'he Johns
Elopkins Hospital, Baltimore, M d .
'Dedicated to the memory of Dr. Hdnry Milch of New York, a distinguished and brilliantly imaginative orthopaedic surgeon, who pioneered in developing inany of the
mathematical principles of modern bone snrgery. He first intercsted the author in the
problems presented by the alcaptonuria syndrome and provided much of the stimulus
and enthusiasm for these and later stndics, which prophetically, have been basically designed to idmtify the tissue processes which ultimately caused his sridden and most untimely death.
1002
ARTHRITISAND
1~IiEUMATISlrl,VOL.
8, NO. 5
(OCTOBER),
1965
1003
STUDIES OF ALCAPTONURIA
0
2
6
4
12
8
10
WAVELENGTH I N M I C R O N S
I4
16
Fig. 1.-Spectra of monomeric (freshly prepared) honiogentisic acid in deuteriuin oxide at 23°C (solid line) and at .50°C (dashed line).
o
~
>
,
,
,
,
WAa
V E L E N G ,T H I N M
, I C R O N,S
,
,
,
, I4
,
,
,
Fig. S.-Spectra of autoxidized (solid line) and ferricyanide-oxidized, (dashed
line) homogentisic acid at 25°C.
I-----
100-
z
9
80-
e
l
.
..
I
60-
c
IL
c 40Y
B
.
20-
0
2
4
6
8
10
WAVELENGTH I N M I C R O N S
I2
14
16
Fig. 3.-Spectra of hydroquinone (solid line) at 23°C and phenylacetic acid
(dashed line) at 60°C.
of the various vibrations are summarized in table 1in terms of data published
earlier by M e ~ k e ,Blout,*
~
S h r e ~ b u r y ,Shimanouchi,
~
Tsuboi, Takenishi and
Iwata,lo Brownll and Bellamy et al.lOJ3
The data indicate ( a ) that there is no similarity between the present homogentisic acid spectra and those reported for different melanins16 and ( b )
that there is no detectable infra-red spectral difference between colorless
monomeric and pigmented polymeric homogentisic acid solutions. Furthermore, they suggest that, in contrast to phenylacetic acid (which probably
exists in D 2 0 solution in the cyclic dimeric form) (fig. 3) both hydroquinone
10()4
ROBERT AUSTIN MILCH
Table 1.-Band
Assignments of Homogentisic Acid Solutions
Frequency
(microns)
Probable Assignment
3.0
0 - D and 0-H stretches (Coupled with weak C-D
stretch at 3 . 3 , ~ )
Coupled C-D and 0-D stretches
C = 0 stretch
Ring vibration
-CO-OD
Coupled C-C. C - 0 and OD stretches
( ? -CH,-COOD, see ref. 12)
Coupled C-Dand in-plane 0 - D stretches
Coupled ring C-D and out-of-plane OD bends
4.6, 4.9, 5.2
5.9
6.7
7.0,7.3, 7.7
8 .O-8.5
9.5-10.3
12.3
and the monomeric and polymeric homogentisic acid compounds possess
broad, heat-labile bands in the 0-D stretching region and hence presumably
occur mainly in a deuterium-bonded open lattice form (fig. 4 ) . Carbonyl
groups of the quinone nucleus, unlike the carbonyl functions of collagenreactive aldehydes,6 are not deuterated to the corresponding aldol or gemdihydroxy forms, as evidenced by the lack of broad vibrations in the 9-11
micron regions. Binding of homogentisic acid oxidation polymers to collagen
structures and the failure of the unoxidized monomers to so bind can, therefore, be attributed primarily to steric factors. These in turn allow for the
secondary occurence of covalent bonding, presumably of the Sciff base type,
between ring carbonyl groups on the one hand and the amino and/or amide
groups of collagen network polymers on the other.
0=
o="
A
O------
OD
t
1
0
II
I
0
II
I
I
oc"<)-
/
DO
II
0
0
o\c<A)
/
0II
90
I
II
Fig. 4.-Postulated
um oxide solution,
II
0
structure of oxidized homogentisic acid molecules in deuteri-
1005
STUDIES OF ALCAPTOSUHIA
ACKNOWLEDGMENT
The writer wishes to acknowledge the excellent technical assistance of Angelo Russo
and Richard Murray and to express his gratitude to Dr. Lionel J. Bellamy, The British
Ministry of Aviation, Essex, England, for critically reviewing this manuscript.
REFERENCES
Milch, R. A., Titus, E. U., and Loo,
T. L.: Atmospheric oxidation of
homogentisic acid sohitions: spectrophotometric studies. Science 126:
209, 1957; ( b ) Milch, R. A. and
Magnns, I. C . : Kinetics of homogentisic acid autoxidation, Arch.
Biochem. Biophys. 7:513> 1958; ( c )
Milch, R. A. and Schirnier, H. K. A.:
Studies of alcaptonuria: Kinetics of
homogentisic acid autoxidation. Arch.
Biochem. Biophys. 89:27, 1960.
2. ( a ) Milch, R. A,, and Titus, E. D.:
Studies of alcaptonuria : Absorption
spectra of homogentisic acid-chondroitin sulfate solutions. Arth. &
Rheumat. L:366, 1958; ( b ) Milch,
R. A,, and Robinson. R. A , : Content and density of the water and
solid phases of ochronotic cartilage.
J. Chron. Dis. 12:409, 1960; ( c )
Milch, R. A , : Studies of alcaptonnria: Mechanisms of swelling of
honiogentisic acid-collagen preparations. Arth. & Rheumat. 4:253, 1961;
( d ) Milch, R. A,, and Murray, R. A.:
Studies of alcaptonnria: Absorption
of homogentisic acid solutions on
collagen chromatographic colnmns.
Arth. I% Rheumat. 4:268, 1961; ( e )
Stndies of alcaptonuria: Binding of
homogentisic acid solntions to hide
powder collagen. Proc. Soc. Exp. Biol.
bled. 106:68, 1961; ( f ) Milch, R. A.:
Studies of alcaptonuria: Collagenase
degradation of hornogentisic tanned
hide powder collagen. Proc. Soc. Exp.
Biol. Med. 107:183, 1961.
3. ( a ) La Du. B. N.. O’Brien. W. M.. and
Zannoni, V. G.: Stndies on Ochronosis. I. The distribution of homogentisic acid in guinea pigs. Arth. it
Rheumat. 5:81, 1962; ( b ) Zannoni,
V. G.. Malawista, S. E. and La Du,
B. N.: Studies on ochronosis. 11.
Studies on benzoquinoneacetic acid,
a probable interinediatc in the con-
1.
(a)
nective tissue pigmentation of aicaptonuria. Arth. & Rheumat. 5:547,
1962.
4. vii]&. R. A. : ~ i ~ ~ studies
h ~ on ~
the pathogenesis of collagen tissue
changes in alcaptonuria. Clin. Orthopaedics 24:213, 1962.
.!j.-: Infra-red
spectra of cleuterotecl
6.
-:
gelatin sols. Nature 202:84, 1965.
Aqueous solution infra-red spectra
of collagen-reactive aldehydes. Biochim. Biophys. Acta 93:45, 1964;
( b ) Milch, R. A., and Murray, R. A.:
Infra-red spectra of aqueous dialdehyde starch solutions. J. Am. Leather
Chems. Assn. 59:310, 1964.
7 . Mecke, R.: Infra-red spectra of hydroxylic compounds. Disc. Faraday
SOC. 9:161, 1950.
8. Blout, E. R.: Aqueous solution infrarcd
spectroscopy of biochemical polymers. Ann. N. Y. Acad. Sc. 69:84,
19.57.
9. Schrewsbnry, D. D.: The infra-red
spectra of nlkyl phenols. Spectrochim. Acta 16:1294, 1960.
10. Shimanouchi, T., Tsuboi, M., Takenishi,
T., and Iwata, N.:A strong band
at 1200 cm-1, characteristic of the
-CH,-COOH
group. Spectrochim.
Acta 16:1328, 1960.
11. Brown, T. L.: The infra-red carbonyl
absorption in some p-quinones and
related substances. Spectrochim. Acta
18:1065, 1962.
12. Bellamy, L. J., and Pace, R. J.: Hydrogen bonding in carboxylic acids.
I. Oxalic acids. Spectrochim. Acta
19:435, 1963.
13. -, Lake, R. F., and Pace, R. J.: I-lydrogen bonding in carboxylic acids.
11. Monocarboxylic acids. Spectrochim. Acta 19:443, 1963.
14. Bonner, T. G., and Duncan, A.: Infrared spectra of some melanins. Naturc
194: 1078, 1962,
~
i
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