The
Oxidation of L-tryptophan-carboxyl-14C And DL-tryptophan (Pyrrole-2-14C)
to 14CO2 In Vitamin B6-Deficient Rats
Ngo Manh
Tran and Marcel Laplante
Département de Medicine Nucléaire et de Radiobiologie,
Centre Hospitalier Universitaire, Sherbrooke, Québec, Canada
Published in International Journal of Biochemistry,
2: 307-311, 1971
Despite the availability of considerable biochemical information concerning the function of pyridoxine, the effective early diagnosis of Vitamin B6 deficiency awaits the development of simple, rapid techniques for screening populations for the metabolic defects attendant on this vitamin B6 deficiency state. The present work is directed towards development of such techniques similar to the development of a method for early diagnosis of beri-beri which has been demonstrated recently (Ngo Tran, Winchell, Williams, Finley, and Lawrence, 1969).
The apparent dependency of the oxidation of the 1-carbon
of L-tryptophan to CO2 upon the presence of vitamin B6 suggested that
appearance of 14CO2 in the breath following administration of L-tryptophan-carboxyl-14C
might be a measure of vitamin B6 deficiency, and thus such measurements
might be useful in early diagnosis of pyridoxine deficiency in man.
Inbred rats (Rattus rattus) (Canadian Breeding Laboratories,
St-Constant Co., Laprairie, Québec) were divided into control and vitamin
B6-deficient groups. Control and vitamin B6-deficient diets were obtained
from Nutritional Biochemicals Corporation, Cleveland, Ohio. The diet of
the control rats had the following composition, expressed as percentages:
alphacel 2.0; Oil 2.0; hydrogenated cotton-seed oil 6.0; salt mixture
hawk over 4.0; corn starch 28.0; sucrose 29; vitamin-free casein 30.0;
vitamin diet fortification mixture. The deficient diet contained the same
formula as above except that it was deficient in vitamin B6. The experiments
were conducted from 4 to 6 weeks after initiation of these diets. In these
experiments, the control rats weighed an average of 274.2 g. at the beginning
and 410.2 g. at the end of the experimental period, while vitamin B6 deficient
rats weighed an average of 278.4 g at the beginning and 301.8 g. at the
end of the experimental period.
In control rats, approximately 2.9 per cent of the 14C administered as L-tryptophan-carboxyl-14C and 0.8 percent of the 14C administered as DL-tryptophan (pyrrole-2-14C) appear in the breath as 14CO2 within the first 180-minute period. A significant increase in CO2 production from the oxidation of the labeled-carboxyl L-tryptophan is noted as compared to that obtained from the 2-carbon of the pyrrole ring of DL-tryptophan (P < 0.01). Such a significant difference may be due to the fact that the 2-carbon of the pyrrole ring of DL-tryptophan would be oxidized to CO2 only by way of α-amino-β-carboxyl muconic semialdehyde, while the 1-carbon of the labeled-carboxyl L-tryptophan could arise by directly or after its oxidation of L-hydroxytryptophan, by the metabolism of the side chain as alanine, and by the oxidative catabolism of indopyruvic acid (Happold and Hoyle, 1935; Wood and others, 1947; Gholson and others, 1960; Madras and Sourkes, 1968; Sourkes and Missala, 1969). In addition, since D-tryptophan is oxidized by a specific pyrrolase, a smaller amount of 14C obtained from DL-tryptophan (pyrrol-2-14C) confirms the evidence that this metabolic pathway would not contribute significantly to the 14CO2 excretion in the breath of control rats (Madras and Sourkes, 1968).
In Vitamin B6-deficient rats, there is a delay in the
oxidation of L-tryptophan-carboxyl-14C to 14CO2. This result may be due
to a dependency of the oxidation of L-tryptophan, upon the presence of
vitamin B6, on both tryptophanase and kyrurenase. The result obtained
suggests further that the most extensive conversion of this carbon to
CO2 arises through the oxidative catabolism of both indolpyruvic acid
and aniline rather than through the decarboxylation of 5-hydroxytryptophan
to serotonin. 14CO2 production is, however, unchanged in vitamin B6-deficient
rats after administration of DL-tryptophan (pyrrole-2-14C). This result
suggests that vitamin B6 deficiency does not influence tryptophan pyrrolase
activity.
The fact that alterations in the metabolism of carboxyl-labelled
tryptophan in the presence of vitamin B6 deficiency can be detected in
the intact animal by measuring 14CO2 excretion in the breath suggests
the possible application of this approach to the early diagnosis of pyridoxine
deficiency in man.
The fact that the tryptophan pyrrolase enzyme can be reconstituted
with either haem or haematin (Soukes and Missala, 1969) suggests the possible
application of this reliable method with DL-tryptophan (pyrrole-2-14C)
to the metabolic studies of various types of anaemias in man.
This work was supported by the University
of Sherbrooke Medical School Fund.
Marcel Laplante was a recipient of the Medical Research Council
of Canada. He was also a Ph.D. candidate in Radiobiology and Nuclear Medicine
of the University of Sherbrooke Medical School, Québec, Canada
