In a previous publication (1) the isolation of a green pigment from the urine of pyridoxine-deficient rats was described. The green pigment was shown to be the product of a reaction between ferric ammonium sulfate or other ferric salts and a compound whose nature was unknown. This compound has now been isolated in crystalline form. It is a yellow pigment and has been identified as xanthurenic acid

Haagen-Smit, A. J.

Lepkovsky, Samuel

Roboz, Elizabeth

English

Caltech Authors - Main

XANTHURENIC ACID AND ITS ROLE IN THE TRYPTOPHANE 
METABOLISM OF PYRIDOXINE-DEFICIENT RATS* 
BY SAMUEL LEPKOVSKY, ELISABETH ROBOZ, AND A. J. HAAGEN-SMIT 
(From the Division of Poultry Husbandry, College of Agriculture, University of 
California, Berkeley, and the William G. Kerckhof Laboratories of the 
Biological Sciences, California Institute of Technology, Pasadena) 
(Received for publication, April 2, 1943) 
In a previous publication (1) the isolation of a green pigment from the 
urine of pyridoxine-deficient rats was described. The green pigment was 
shown to be the product of a reaction between ferric ammonium sulfate or 
other ferric salts and a compound whose nature was unknown. This com- 
pound has now been isolated in crystalline form. It is a yellow pigment and 
has been identified as xanthurenic acid. 
Isolation of Xanthurenic Acid-The xanthurenic acid was isolated from 
the urine of pyridoxine-deficient rats with the help of the paper-packed 
chromatographic column described in a previous publication (1). Cotton 
pulp has been used successfully and can replace the paper pulp. The yel- 
low pigment was lightly adsorbed from rat urine’ which had been saturated 
with sodium chloride. The column was washed with saturated sodium 
chloride solution, which carried through most of the urinary constituents 
before the yellow pigment came through. This process was repeated until 
the yellow pigment was obtained in a fair degree of purity. The sodium 
chloride-saturated solution containing the yellow pigment was then con- 
centrated to about one-tenth of its volume and the precipitated sodium 
chloride removed by filtration. At this point, two different procedures 
were employed. 
The solution was further concentrated until, along with sodium chloride, 
the yellow pigment crystallized out in needles which aggregated to form 
rosettes. The crystal form of the yellow pigment is illustrated in Fig. 1. 
The yellow pigment could be separated from the sodium chloride by re- 
crystallization. 
The second procedure consisted of eliminating most of the salt from the 
solution with alcohol and acetone. 2 to 4 volumes of alcohol were added to 
the salt-saturated solution and to this alcoholic solution was added an equal 
volume of acetone. The precipitate was discarded and the filtrate was 
* This work was supported in part by a grant from the American Cyanamid 
Company. 
1 Much of the urine from pyridoxine-deficient rats was kindly given to us by the 
Vitab research laboratory, Emeryville, California. 
195 
196 PYRIDOXINE AND TRYPTOPHANE METABOLISM 
evaporated in a vacuum until the yellow pigment crystallized out. It was 
recrystallized from solutions containing small amounts of sodium chloride. 
Attempts to recrystallize the yellow pigment from distilled water failed. 
The yellow pigment differs in many ways from its green iron complex. 
The green pigment is more readily adsorbed on the paper-packed column 
than the yellow pigment, and so far it has not been obtained in the form of 
well defined crystals. The yellow pigment, like the green iron complex, is 
soluble in strong alcoholic solutions and in alcohol-acetone solutions. These 
solubility characteristics have been taken advantage of in the isolation of 
the yellow pigment, as described above. 
Identification of Yellow Pigment As Xanthurenic Acid-The elementary 
analysis of the yellow pigment showed a considerable amount of ash, owing 
to the presence of sodium. To obtain the free acid, the pigment was dis- 
solved in 50 per cent ethyl alcohol and the acid precipitated with 1 N 
FIG. 1. Crystals of the Na salt of xanthurenic acid 
HzS04. It was purified further by dissolving in warm 50 per cent alcohol, 
adding excess 0.1 N NaOH to form the sodium salt, and filtering off the 
impurities. The acid was reprecipitated with 0.1 N HCl. After two pre- 
cipitations the melting point, 288”, of the substance could not be raised by 
further precipitation. The acid, which is a light yellow pigment, changes 
to yellowish brown in alkaline solution. When an excess of HCl is added, 
the free acid which is precipitated is redissolved, forming a light yellow- 
green solution. If the acid solution is left standing or is warmed, it turns 
nearly colorless. With methyl red it is monobasic.; with phenolphthalein, 
dibasic. 
The compound is nearly insoluble in water, slightly soluble in 50 per cent 
alcohol and in dilute HCI, and easily soluble in alkali hydroxides, carbonates, 
and hot dilute HCl. 
The microanalysis gave the following results. 
GoH7NOa. Calculated. C 58.53, H 3.42, N 6.83 
Found. “ 58.27, “ 3.70, “ 7.12 
LEPKOVSKY, ROBOZ, AND HAAGEN-SMIT 197 
The equivalent weight was found by titration to be 103.3. Calculated for 
a dibasic acid of the formula C&H7N04 = equivalent weight 102.5. 
The analytical data are best explained by assuming that the yellow pig- 
ment is the sodium salt of a monohydroxyquinolinecarboxylic acid with 1 
molecule of water of crystallization or that of a dihydroxyquinolinecarbox- 
ylic acid. 
A search of the literature revealed that recently a similar acid had been 
found by Musajo (2) in urine. This acid is a 4,8-dihydroxyquinoline-2- 
carboxylic acid and was called xanthurenic acid. The properties of this 
xanthurenic acid resemble closely those of our yellow pigment. 
Since it was not possible to obtain a sample of Musajo’s acid and the 
melting points of kynurenic and xanthurenic acids are close together, and 
since other properties are also very similar, it was desirable to carry out 
specific color reactions and prepare the methyl ester for more reliable iden- 
tification. The methyl ester of kynurenic acid melts at 224”, whereas the 
methyl ester of xanthurenic acid melts at 262”. 
Preparation of Methyl Ester of Xanthurenic Acid-The free acid was sus- 
pended in absolute methyl alcohol (3) (25 mg. in 2 cc.). It was heated in a 
water bath at 60” and dry HCl bubbled through it for 3 hours. When the 
esterification was completed, the reaction mixture was cooled for 2 hours in 
ice-salt mixture, centrifuged, and washed twice with cold methyl alcohol. 
The precipitate was taken up in 25 cc. of boiling water and after being 
cooled in ice was neutralized with NaHC03. It was stirred again and al- 
lowed to stand overnight under refrigeration. The precipitate was re- 
crystallized from hot 50 per cent ethyl alcohol. After three recrystalliza- 
tions, 12.4 mg. of methyl ester were obtained with an approximate yield of 
50 per cent. It crystallized as light yellow, silky needles which melted at 
260-261”. It was dried at 110” and analyzed. 
C,lHgNOa. Calculated. C 60.25, H 4.14, N 6.40 
Found. “ 60.32, “ 4.25, “ 6.522 
The following color reactions were carried out with the free acid: (1) Millon’s 
reagent, orange-red color; (2) acetic acid-acetic anhydride and benzene, 
light brown-violet color; (3) freshly synthesized diazobenzosulfonic acid, 
intense red color. 
In order to distinguish between the kynurenic acid and xanthurenic acid, 
reactions were carried out which gave different colors with these two quino- 
linecarboxylic acids (4). (1) FeC4 added in the presence of NaHCOs gave 
an intense green color. This is characteristic of xanthurenic acid, whereas 
kynurenic acid gives no color reaction. (2) Diazobenzosulfonic acid in the 
presence of NaHC03 gave the red color of xanthurenic acid. Again kynu- 
2 The authors wish to express their thanks to Dr. G. Oppenheimer for carrying 
out the microanalyses. 
198 PYRIDOXINE AND TRYPTOPHANE METABOLISM 
renic acid did not give this color reaction. (3) Xanthurenic acid evaporated 
with concentrated HCI and KC103 turns brown when moistened with r\TH3. 
Kynurenic acid similarly treated gave a green color. (4) The reaction of 
Kotake and Shichiri (5); the substance was boiled in alcoholic solution 
with phenylhydrazine hydrochloride and sodium carbonate and extracted, 
after being cooled, with ether. On addition of concentrated H$04, after 
removal of the ether, no color reaction was observed, whereas kynureninc 
gives a blue color. 
The yellow compound isolated from the urine of pyridoxine-deficient rats 
must therefore be identical with xanthurenic acid, the formula of which is 
H 
0 
COOH 
0 N 
H 
Relation of Pyridozine Dejiciency to Protein (Tryptophane) Metabolism-- 
Since xanthurenic acid occurs in the urine of pyridoxine-deficient rats and 
immediately disappears from the urine on addition of pyridoxine to the diet, 
it would seem that xanthurenic acid is the result of some sort of metabolic 
derangement brought about in the rat by inadequate pyridoxine intake. 
That this metabolic derangement might involve tryptophane metabolism 
follows from the work of Musajo (6) who found that on high protein diets 
rats and rabbits excrete both kynurenic acid and xanthurenic acid in the 
urine. He suggested that these acids found their origin in tryptophane as 
follows : 
kynurenic acid 
,J” 
Tryptophane -----+ kynurenine 
is 
xanthurenic acid 
If, in pyridoxine-deficient rat,s, xanthurenic acid originated from dietary 
tryptophane, the feeding of a tryptophane-deficient diet should result in the 
disappearance of xanthurenic acid from the urine of pyridoxine-deficient 
rat,s and the addition of tryptophane should cause t.he reappearance of xan- 
thurenic acid in the urine of such rats. This has in fact been found experi- 
mentally to be the case. 
Pyridoxine-deficient rats were put in metabolism cages” and the presence 
of xanthurenic acid in their urine was established by the production of the 
3 The authors wish to express their thanks t.o Mrs. Della Parsons for the cart 
of t,he animals used in this work. 
LEPKOVSKY, ROBOZ, AND IIAAGEN-SMIT 199 
green color with ferric ammonium sulfate. The casein was then removed 
from their diets and replaced with zein and gelatin, a protein mixture very 
low in tryptophane. The xanthurenic acid disappeared from the rat urine 
in from 6 to 12 hours. Each rat was then fed 20 mg. daily of Z-tryptophane 
and within 6 to 12 hours xanthurenic acid reappeared in their urine. Addi- 
tion of 20 y of pyridoxine4 daily to the diet of each rat immediately caused 
the disappearance of xanthurenic acid from the rat urine. 
This experiment was repeated with acid-hydrolyzed caaein replacing the 
casein in the pyridoxine-deficient diet. The absence of tryptophane in the 
acid-hydrolyzed casein was established by qualitative tests. The results 
obtained with this tryptophane-deficient diet were identical with those ob- 
tained with the use of zein and gelatin as the tryptophane-deficient proteins. 
This leaves little doubt that the function of pyridoxine is relat,ed t’o trypto- 
phane metabolism. 
These results are in harmony with those of Voris and hloore (7) who 
showed that pyridoxine is related to protein metabolism. They used 
paired rats, one rat of each pair receiving pyridoxine. The pyridoxine-fed 
rat gained more weight than its paired pyridoxine-deficient mate, and the 
gains in weight were exclusively protein, whereas in similar studies with 
thiamine and riboflavin the gains were represented by fat. 
Foy and Cerecedo (8) studied pyridoxine deficiency in rats on diets con- 
taining 15, 30, and 45 per cent casein. At the low level of protein intake, 
little dermatitis developed in 70 days, while at the intermediate level the 
rats developed dermatitis after 30 days. At the highest level of protein 
intake (45 per cent casein) severe dermatitis developed in 26 days followed 
shortly after by death. Apparently high protein (tryptophane?) intake 
seems to increase the severity of the nutritional disorder in rats due to in- 
adequate intake of pyridoxine. Perhaps the excess protein destroys pyri- 
doxinc in the rat in some unknown way. Such a phenomenon would 
explain the early appearance of xanthurenic acid in the urine of rats and 
rabbits (6) fed high protein diets. 
Species Dij’erences--While pyridoxine-deficient rats excrete an abun- 
dance of xanthurenic acid in their urines, pyridoxine-deficient dogs excrete 
very little (9), even when they have become very anemic as a result of the 
deficiency. So far, no xanthurenic acid has been demonstrated in the 
droppings of chicks.5 Wintrobe et al. (10) have reported the occurrence of 
a compound in the urine of pyridoxine-deficient pigs which gives a green 
color with the addition of ferric ammonium sulfate, indicating the presence 
of xanthurenic acid. 
4 The pyridoxine was generously supplied by Merck and Company, Inc., Rahway, 
New Jersey. 
6 Iiratzer, I?. II., unpublished results. 
200 PYRIDOXINE AND TRYPTOPHANE METABOLISM 
DISCUSSION 
In the past, three related compounds, namely kynurenic acid, kynurenine, 
and xanthurenic acid, have been isolated from urine as products of protein 
metabolism. Kynurenic acid was first isolated by Liebig (11) in 1853, and 
Ellinger and Matsuoka (12) found it after feeding tryptophane. Kotake 
and Iwao (13) isolated kynurenine as an intermediary product in 
tryptophane metabolism and reported that kynurenine is excreted as a po- 
tassium salt, whereas kynurenic acid is found as a sodium salt. Musajo (2) 
found a compound similar to kynurenic acid in connection with feeding high 
protein diets and named it xanthurenic acid. 
The excretion of kynurenic acid, kynurenine, and xanthurenic acid seems 
to be connected with vitamin deficiencies. Rabbits on a diet of polished 
rice, which is deficient in the vitamin B complex, excrete not only kynurenic 
acid but also kynurenine (13). In this paper it is shown that the urine of 
rats on a diet deficient in pyridoxine contains xanthurenic acid. Whether 
other members of the vitamin B complex are also related to tryptophane 
metabolism remains to be shown. Thus far all that is known is that panto- 
thenic acid-deficient rats do not excrete any xanthurenic acid in their urine. 
The species differences, especially as found with the dog and rat, are of 
interest. Pyridoxine-deficient rats excrete an abundance of xanthurenic 
acid in their urine, but they do not become so severely anemic as pyridoxine- 
deficient dogs (9) ; yet such dogs excrete but very little xanthurenic acid (9). 
It must follow, therefore, that the metabolism of tryptophane does not 
follow the same pathway in both the rat and the dog. 
SUMMARY 
1. A yellow compound was isolated from the urine of pyridoxine- 
deficient rats. 
2. This yellow compound is shown to be identical with Musajo’s xan- 
thurenic acid, a 4,8-dihydroxyquinoline-2-carboxylic acid. 
3. Xanthurenic acid was shown to originate in dietary tryptophane. 
4. The relation of pyridoxine to tryptophane and protein metabolism has 
been discussed. 
BIBLIOGRAPHY 
1. Lepkovsky, S., and Nielsen, E., J. Biol. Chem., 144, 135 (1942). 
2. Musajo, L., Chew Abst., 29, 7347 (1935); Atti accad. Lincei, 21, 368 (1935). 
3. Gordon, W. G., Kaufman, R. E., and Jackson, R. W., J. Biol. Chem., 113, 125 
(1936). 
4. Musajo, L., Chem. Abst., 31,706O (1937); Gazz. chim. ital., 67, 165 (1937). 
5. Kotakc, Y., and Shichiri, G., Z. physiol. Chem., 196, 158 (1931). 
6. Musajo, L., Chem. Abst., 29,6292 (1935); Boll. Sot. ital. biol. sper., 10,290 (1935). 
7. Voris, L., and Moore, 1%. I?., J. Nutrition, 26,7 (1943). 
LEPKOVSKY, ROBOZ, AND HAAGEN-SMIT 201 
8. Foy, J. R., and Cerecedo, L. R., Abstracts, Division of Biological Chemistry, 
American Chemical Society, Atlantic City, Sept. 8 (1941). 
9. Fouts, P. J., and Lepkovsky, S., Proc. Sot. Exp. Biol. and Med., 60, 221 (1942). 
10. Wintrobe, M. M., Follis, R. H., Jr., Miller, M. H., Stein, H. J., Alcayaga, R., 
Humphreys, S., Suksta, A., and Cartwright, G. E., Bull. Johns Hopkins Hosp.. 
72, 1 (1943). 
11. Robson, W., J. Biol. Chem., 62, 495 (1924-25). 
12. Ellinger, A., and Matsuoka, Z., %. physiol. Chem., 109, 259 (1920). 
13. Kotake, Y., and Iwao, J., Z. physiol. Chem., 196, 139 (1931). 


Xanthurenic acid and its rôle in the trytophane metabolism of pyroxidine-deficient rats

Johns Hopkins Hosp..

https://authors.library.caltech.edu/10329/1/LEPjbc43.pdf

Xanthurenic acid and its rôle in the trytophane metabolism of pyroxidine-deficient rats

Abstract

Similar works

Full text

Available Versions

Caltech Authors - Main