ELEVATED XANTHINE OXIDASE IN VITAMIN E DEFICIENCY lmmunochemical evidence is presented that the increased xanthine oxidase activity that occurs in the livers of rabbits deficient in vitamin E is due to an increased accumulation of the enzyme protein.
Key Words: vitamin E, xanthine oxidase
An observation that vitamin E deficiency in rabbits and monkeys led to a marked increase in allantoin excretion prompted J. S. Dinning' to investigate liver xanthine oxidase and uricase levels in this deficiency. A marked increase in liver xanthine oxidase was found to occur while uricase activity was normal. The increased activity o f xanthine oxidase was not affected by the in vitro addition o f either a-tocopherol phosphate or normal liver homogenates. Further studies by G. L. Catignani and J. S. Dinning2 tended to rule out t h e possibility that an activation of preformed xanthine oxidase was occurring in vitamin E deficiency and these authors suggested that vitamin E regulates the synthesis of the enzyme. Evidence has now been presented by G. L Catignani and his co-workers3 that an accelerated de novo synthesis probably occurs in vitamin E deficiency in rabbits. These recent studies of Catignani e t al. were made possible by the ability of the authors to make a preparation of xanthine oxidase from the livers of rabbits deficient in vitamin E which showed homogeneity on acrylamide gel electrophoresis (unpublished results). Antibodies to the enzyme were obtained by injecting the enzyme into a sheep. lmmunoelectrophoresis of the antibody preparation showed a single precipitin arc in the presence of the purified xanthine oxidase. Livers from normal and vitamin E deficient rabbits were homogenized and centrifuged a t 105,000 x-g. Increasing amounts of both supernatants
(cytosol) were then incubated in the presence of a fixed amount of either control or immune serum, and the xanthine oxidase activity measured. A plot of activity versus the amount of cytosol yielded an equivalence point, the amount of cytosol necessary to furnish sufficient xanthine oxidase to react with the fixed amount of antibody that was employed. A series of such experiments of immunochemical titration revealed that the cytosol prepared from livers of vitamin E deficient rabbits had four to nine times the capacity to neutralize the enzyme antibody as that from normal animals. The enzyme activity of the cytosol prepared from the experimental animals was also four to nine times that obtained from the normal animals. A graph is presented by the authors in which the activity of original cytosol is plotted against the activity found in the presence of the fixed amount of antibody. Such a graph reveals identical lines for both normal and experimental cytosol and demonstrates that the xanthine oxidase which accumulates during vitamin E deficiency is immunologically indistinguishable from that in the normal animal. A second approach to the problem is made by measuring the incorporation of radioactive leucine into xanthine oxidase. Control and vitamin E deficient rabbits were fasted overnight and 14C-leucine was injected. Six hours later the animals were sacrificed and liver cytosols prepared. From these cytosols the following fractions were made and analyzed for radioactivity: immunoprecipitable protein, total protein NUTRITION REVIEWS/VOL 33. NO. ZIFEBRUARY 1975
59
as precipitated by trichloracetic acid, and
leucine isolated from the trichloracetic acid supernatants. The radioactivity of the immunoprecipitable protein obtained from the deficient animals was 357 and 821 percent higher than that of the control animals. In these experiments the enzyme activity of the cytosol from the deficient animals was 670 and 890 percent of the control value. No significant difference was observed between experimental and control animals in the radioactivity of the protein precipitated from the cytosol by trichloracetic acid. The specific activity of the leucine isolated from the trichloracetic acid supernatant of the cytosol from the deficient rabbits was 40 percent lower than that from the control animal. This latter finding of an apparent "decreased availability'' of labeled leucine in the deficient animals is interpreted by the authors to substantiate "the concept that an elevated rate o f synthesis of the enzyme occurs during vitamin E deficiency". It is puzzling that this decrease in the pool o f labeled leucine i s not reflected in the radioactivity of the protein precipitated by trichloracetic acid from the liver cytosol o f the deficient an imal s. The authors make the reasonable conclusion that their results show that the mechanism of increase of liver xanthine oxidase in vitamin E deficient rabbits involves the accumulation of the enzyme protein rather than an activation o f the enzyme. They favor the interpretation that the accumulation of xanthine oxidase reflects an accelerated de novo synthesis of the enzyme. They are careful, however, t o
60
NUTRITION REVIEWSIVOL. 33,
NO. ZIFEBRUARY
1975
point out that a part of the accumulation of the enzyme may be due to inhibition o f i t s degradation. In any case their results seem t o indicate clearly the direction that future investigations must follow in elucidating the mechanism responsible for this change. One possibility suggested by the authors is that in vitamin E deficiency there is an alteration of the transcription of the messenger RNA for xanthine oxidase. Earlier workers' ,2 suggested that vitamin E somehow regulates the synthesis of the enzyme. The possibility must not be overlooked that the increase in xanthine oxidase may be secondary to a more primary aberration in nucleic acid metabolism as suggested by Dinning.' Thus the increase in xanthine oxidase might be a reflection of an increase in the flux of i t s subtrates and resemble the marked changes in liver glucose-6-phosphate dehydrogenase activity that occurs with alterations in dietary carbohydrate intake. The relationship between xanthine oxidase activity and vitamin E levels certainly seems to be worthy of more intensive study. 0
1. J. S. Dinning: An Elevated Xanthine Oxidase in Livers of Vitamin E Deficient Rabbits. J. Biol. Chem. 202: 213-215, 1953 2. G. L. Catignani, Jr. and J. S. Dinning: Role of Vitamin E in the Regulation of Rabbit Liver Xanthine Oxidase Dehydrogenase Activity. J. Nu irition 101 : 1327- 1330, 197 1 3. G. L. Catignani, F. Chytil, and W. J. Darby: Vitamin E Deficiency: lmmunochemical Evidence for Increased Accumulation of Liver Xanthine Oxidase. Proc. Nat. Acad. Sci. USA 7 1 1966-1968,1974
Key Words: vitamin E, xanthine oxidase
An observation that vitamin E deficiency in rabbits and monkeys led to a marked increase in allantoin excretion prompted J. S. Dinning' to investigate liver xanthine oxidase and uricase levels in this deficiency. A marked increase in liver xanthine oxidase was found to occur while uricase activity was normal. The increased activity o f xanthine oxidase was not affected by the in vitro addition o f either a-tocopherol phosphate or normal liver homogenates. Further studies by G. L. Catignani and J. S. Dinning2 tended to rule out t h e possibility that an activation of preformed xanthine oxidase was occurring in vitamin E deficiency and these authors suggested that vitamin E regulates the synthesis of the enzyme. Evidence has now been presented by G. L Catignani and his co-workers3 that an accelerated de novo synthesis probably occurs in vitamin E deficiency in rabbits. These recent studies of Catignani e t al. were made possible by the ability of the authors to make a preparation of xanthine oxidase from the livers of rabbits deficient in vitamin E which showed homogeneity on acrylamide gel electrophoresis (unpublished results). Antibodies to the enzyme were obtained by injecting the enzyme into a sheep. lmmunoelectrophoresis of the antibody preparation showed a single precipitin arc in the presence of the purified xanthine oxidase. Livers from normal and vitamin E deficient rabbits were homogenized and centrifuged a t 105,000 x-g. Increasing amounts of both supernatants
(cytosol) were then incubated in the presence of a fixed amount of either control or immune serum, and the xanthine oxidase activity measured. A plot of activity versus the amount of cytosol yielded an equivalence point, the amount of cytosol necessary to furnish sufficient xanthine oxidase to react with the fixed amount of antibody that was employed. A series of such experiments of immunochemical titration revealed that the cytosol prepared from livers of vitamin E deficient rabbits had four to nine times the capacity to neutralize the enzyme antibody as that from normal animals. The enzyme activity of the cytosol prepared from the experimental animals was also four to nine times that obtained from the normal animals. A graph is presented by the authors in which the activity of original cytosol is plotted against the activity found in the presence of the fixed amount of antibody. Such a graph reveals identical lines for both normal and experimental cytosol and demonstrates that the xanthine oxidase which accumulates during vitamin E deficiency is immunologically indistinguishable from that in the normal animal. A second approach to the problem is made by measuring the incorporation of radioactive leucine into xanthine oxidase. Control and vitamin E deficient rabbits were fasted overnight and 14C-leucine was injected. Six hours later the animals were sacrificed and liver cytosols prepared. From these cytosols the following fractions were made and analyzed for radioactivity: immunoprecipitable protein, total protein NUTRITION REVIEWS/VOL 33. NO. ZIFEBRUARY 1975
59
as precipitated by trichloracetic acid, and
leucine isolated from the trichloracetic acid supernatants. The radioactivity of the immunoprecipitable protein obtained from the deficient animals was 357 and 821 percent higher than that of the control animals. In these experiments the enzyme activity of the cytosol from the deficient animals was 670 and 890 percent of the control value. No significant difference was observed between experimental and control animals in the radioactivity of the protein precipitated from the cytosol by trichloracetic acid. The specific activity of the leucine isolated from the trichloracetic acid supernatant of the cytosol from the deficient rabbits was 40 percent lower than that from the control animal. This latter finding of an apparent "decreased availability'' of labeled leucine in the deficient animals is interpreted by the authors to substantiate "the concept that an elevated rate o f synthesis of the enzyme occurs during vitamin E deficiency". It is puzzling that this decrease in the pool o f labeled leucine i s not reflected in the radioactivity of the protein precipitated by trichloracetic acid from the liver cytosol o f the deficient an imal s. The authors make the reasonable conclusion that their results show that the mechanism of increase of liver xanthine oxidase in vitamin E deficient rabbits involves the accumulation of the enzyme protein rather than an activation o f the enzyme. They favor the interpretation that the accumulation of xanthine oxidase reflects an accelerated de novo synthesis of the enzyme. They are careful, however, t o
60
NUTRITION REVIEWSIVOL. 33,
NO. ZIFEBRUARY
1975
point out that a part of the accumulation of the enzyme may be due to inhibition o f i t s degradation. In any case their results seem t o indicate clearly the direction that future investigations must follow in elucidating the mechanism responsible for this change. One possibility suggested by the authors is that in vitamin E deficiency there is an alteration of the transcription of the messenger RNA for xanthine oxidase. Earlier workers' ,2 suggested that vitamin E somehow regulates the synthesis of the enzyme. The possibility must not be overlooked that the increase in xanthine oxidase may be secondary to a more primary aberration in nucleic acid metabolism as suggested by Dinning.' Thus the increase in xanthine oxidase might be a reflection of an increase in the flux of i t s subtrates and resemble the marked changes in liver glucose-6-phosphate dehydrogenase activity that occurs with alterations in dietary carbohydrate intake. The relationship between xanthine oxidase activity and vitamin E levels certainly seems to be worthy of more intensive study. 0
1. J. S. Dinning: An Elevated Xanthine Oxidase in Livers of Vitamin E Deficient Rabbits. J. Biol. Chem. 202: 213-215, 1953 2. G. L. Catignani, Jr. and J. S. Dinning: Role of Vitamin E in the Regulation of Rabbit Liver Xanthine Oxidase Dehydrogenase Activity. J. Nu irition 101 : 1327- 1330, 197 1 3. G. L. Catignani, F. Chytil, and W. J. Darby: Vitamin E Deficiency: lmmunochemical Evidence for Increased Accumulation of Liver Xanthine Oxidase. Proc. Nat. Acad. Sci. USA 7 1 1966-1968,1974
