Digestion and Absorption of Bovine Milk Xanthine Oxidase and Its Role as an Aldehyde Oxidase1
ABSTRACT The effects of acidic and intestinal proteolytic environ ments on bovine milk xanthine oxidase (XO) activity were determined in order to evaluate the extent to which this enzyme was absorbed in biologi cally active form. The inhibition of XO by folie acid and the relative affinities of XO for the oxidation of palmitaldehyde, stearaldehyde, and xanthine were compared. The effects of acid and gastric juice on XO activity were measured by incubating purified enzyme, and non-purified enzyme (milk), in buffers ranging in pH from 2 to 9. Fresh gastric juice was also incubated with milk. Increasing amounts of the enzyme were inactivated as the pH of the incubation mixture was reduced below pH 6.5. Below pH 3.5, the enzyme was completely inactivated. Gastric juice, pH 2.2, also reduced the enzyme activity in proportion to the amount of gastric juice incubated with milk. Milk XO activity was reduced 36% when milk was incubated with an equal volume of gastric juice. Homogenized milk had 59% less XO activity compared with raw milk. Fresh raw milk XO, homogenized milk XO, and purified XO were equally susceptible to inactivation by acid or gastric juice. After incubation of milk with gastric juice, or gastric juice followed by pancreatin, XO activity was associated with a macromolecule of 300,000 daltons molecular weight and subunits containing activity were not found. It was estimated that 0.00008% of the XO in the intestine was absorbed. Both folie acid and allopurinol inhibited XO activity in vitro. Allopurinol was 3.5 times more potent an inhibitor than folie acid. A large excess of dietary folie acid did not reduce rat liver or intestinal XO activity in vivo. XO had a much greater affinity for xanthine than for palmitaldehyde or stearaldehyde substrates. It was esti mated that of 100 mg of XO in fresh raw milk, 41 mg remained after homogenization, 27 mg entered the intestine and only 20 ng were ab sorbed as intact enzyme. J. Nutr. 106; 1600-1609, 1976. INDEXING KEY WORDS xanthine oxidase •absorption •function Xanthine oxidase (EC 1.2.3.2) is a metallo-flavoprotein containing flavin adenine dinucleotide, molybdenum, iron and having a molecular weight of 300,000 daltons. The enzyme has been found in the milk of cows, sheep, goats, and rabbits but not in the milk of sows, mares, or humans.2 Milk xanthine oxidase was re cently reported to be absorbed intact and to be associated in a causative way with the development of coronary disease (1-8).
from homogenized milk was absorbed from the gut in biological active form and oxidized phospholipid plasmalogen of arterial and myocardial tissues, leading to scar formation, local deposition of cholesteryl esters and ultimately atherosclerotic lesions. It was further postulated that the
It
oxidase
was
postulated
that
xanthine
Oxidase
Receivedfor publicationApril 20, 1976. miSSST^ by NaU°nal Da'ry C°unc"'CT"cag°! ¿&&&SFÕÕ ^ fAf^'Al
1600
In milk. Proc. Nutr.
Soc. 18, I (abstract).
Downloaded from https://academic.oup.com/jn/article-abstract/106/11/1600/4768772 by guest on 07 November 2018
CHARLES Y. HO ANDANDREW J. CLIFFORD Department of Nutrition, University of California, Davis, California 95616
MILK XANTHINE OXIDASE ABSORPTION AND FUNCTION
1601
TABLE 1 Experimental protocols for evaluating the effect of digestion on milk xanthine oxidase activity
.Experiment 1Purified (nil)Buffer, XO, (ml)pH mixtureExperiment of
3Haw
(ml)IICl/ Milk, (normality)pH mixtureExperiment of 4Haw
Downloaded from https://academic.oup.com/jn/article-abstract/106/11/1600/4768772 by guest on 07 November 2018
2 (ml)Buffer Haw Milk (ml)pH mixtureExperiment of
(ml)Gastric Milk (nil)pH Juice,* of mixture0.10.6"2.250.10.6"2.80.10.22.530.00.62.20.10.6°3.50.10.6"3.350.10.152.870.10.53.560.10.6"4.50.10.6°3.850.10.133.050.2 " 0.05 M citrate phosphate buffer. ò0.05 M phosphate buffer. c0.05 M glycine-KOH buffer. "*Volume used = 0.3 ml. ' Freshly aspirated human gastric juice was pH 2.2 and 0.084 N with respect to a 1,000,000) was 5 times less than that of horse spleen ferritin (molecular weight 650,000). From these data (20, 21), it appears that as the molecular size was doubled, the extent of absorption into the intestinal cell was re duced 5-fold. If a linear relationship be tween molecular size and transepithelial migration is assumed, it is possible to cal culate that as the molecular weight in creased from 40,000 (horseradish perox idase) to 300,000 (xanthine oxidase), the percent absorption would decrease from 0.01% to 0.00008%. Application of these calculations to the present study indicate that of the 27% residual units of xanthine oxidase activity in the small intestine, only 2 X 10-" units would be absorbed. The size of the xanthine oxidase pool in liver can be estimated to be 53 mg (22). Thus the
1607
1608
CHARLES Y. HO AND ANDREW J. CLIFFORD
ACKNOWLEDGMENTS
This research was funded by a grant from the National Dairy Council, Chicago, Illinois. The authors are grateful to Pro fessor W. L. Dunkley for use of his oxygraph to measure xanthine oxidase activity and to Professor C. H. Halsted, and Dr. C. R. Fleming and R. S. Newton for pro viding gastric iuice. Technical help from Randy H. Shaffer is appreciated. LITERATURE CITED 1. Oster, K. A. (1968) Treatment of angina pectoris according to a new theory of its origin. Cardiol. Digest. 3, 29-34. 2. Oster, K. A. (1971) Plasmalogen diseases: a new concept of the etiology of the athero sclerotic process. Am. J. Clin. Res. 2, 30-35. 3. Oster, K. A. (1972) Predisposition to atherosclerosis. J. Am. Med. Assoc. 222, 704. 4. Oster, K. A. (1973) Is an enzyme in homogenized milk the culprit in dietaryinduced atherosclerosis? Med. Counterpoint 5 (November), 26-36. 5. Oster, K. A. (1974a) Plasmalogen disease and the rarity of atherosclerosis in the Masai. In: Myocardiology in Africa, pp. 209-214, E. A. Community (CPS) Printer, Nairobi.
6. Oster, K. A. (1974b) Bovine milk xanthine oxidase as one of the dietary causes of early atherosclerosis. Med. Counterpoint 6 (Novem ber), 39-42. 7. Oster, K. A. & Hope-Ross, P. (1966) Plas mai reaction in a case of recent myocardial infarction. Am. J. Cardiol. 17, 83-85. 8. Oster, K. A., Oster, J. B. & Ross, D. J. (1974) Immune response to bovine xanthine oxidase in atherosclerotic patients. Am. Lab. (August) 41-47. 9. Kalchar, H. M. (1947) Differential spectrophotometry of purine compounds by means of specific enzymes. I. Determination of hydroxypurine compounds. J. Biol. Chem 167 429-443. 10. Clark, L. C., Jr. (1956) Monitor and con trol of blood and tissue oxygen tension. Trans. Am. Soc. Artificial. Internal. Organs. 2, 4145. 11. Briley, M. S. & Eisenthal, R. (1974) Asso ciation of xanthine oxidase with the bovine milk-fat globule membrane. Catalytic prop erties of the free and membrane-bound en zyme. Biochem. J. 143, 149-157. 12. Dixon, M. (1953) The determination of enzyme inhibitor constants. Biochem. I. 55, 170-171. 13. Food and Nutrition Board, National Research Council (1974) Recommended Dietary Allowances, ed. 8, National Academy of Sciences, Washington, D.C. 14. Lineweaver, H. & Burk, D. (1934) The determination of enzyme dissociation con stants. J. Am. Chem. Soc. 56, 658-666. 15. Smith, I. (1968) Acrylamide gel disc electrophoresis. In: Chromatographie and electrophoretic techniques. Vol. 2, 365-418. J. Wiley, New York. 16. Dittmer, J. C. & Wells, M. A. (1969) Quantitative and qualitative analysis of lipids and lipid components. In: Methods in Enzymology XIV, 482-530. 17. Menlitz, A., Gierschner, K. U. & Minas, T. ( 1963) Dunnschichtchromatographische Trennung von 2,4-Dinitrophenylhydrazonen. Chemiker-Zeitung 87, 573-576. 18. Bray, R. C., Palmer, G. & Bennert, H. ( 1964) Direct studies on the electron ¿ansfer sequence in xanthine oxidase by electron paramagnetic resonance spectroscopy. II. Kinetic studies employing rapid freezing. Î. Biol. Chem. 239, 2667-2676. 19. Greenbank, G. R. & Pallansch, M. J. (1962) Inactivation and reactivation of xanthine oxi dase in dairy products. J. Diary Sci. 45, 958961. 20. Walker, W. A. & Isselbacher, K. J. (1971) Uptake and transport of macromolecules by the intestine. Possible role in clinical dis orders. Gastroenterology 67, 531-550. 21. Worthington, B. S. & Syrotuck, J. (1976) Intestinal permeability to large panticles in normal and protein-deficient adult rats. J. Nutr. 106, 20-32. 22. Krenitsky, T. A. (1974) Xanthine oxidase and aldehyde oxidase. Adv. Exp. Med. Biol. 41A, 57-64.
Downloaded from https://academic.oup.com/jn/article-abstract/106/11/1600/4768772 by guest on 07 November 2018
ize rapidly under physiological conditions (28) and consequently their availability as substrates for xanthine oxidase would be reduced even more. These results support the concept that xanthine oxidase was pri marily concerned with purine metabolism and appeared to be minimally involved in fatty aldehyde oxidation. Although xanthine oxidase occurs in rather high levels in bovine milk prior to homogenization, its biological role is not clear. Homogenization and exposure to gastric juice markedly reduced the activ ity of this enzyme. Subsequent exposure to pancreatin completed to release of the enzyme from the milk fat-droplet mem brane to yield free enzyme with a molec ular weight of 300,000 daltons. The extent to which this enzyme was absorbed was calculated to be practically zero. Our data indicates that xanthine oxidase absorption was minimal; the enzyme was not inhib ited in vivo by dietary folate; and the enzyme was unlikely to oxidase plasmai in vivo. Consequently the data would not support the hypothesis (1-8) of xanthine oxidase absorption and plasmalogen de pletion.
MILK XANTHINE
OXIDASE ABSORPTION
1609
26. Clifford, A. J. & Story, D. L. (1976) Levels of purines in foods and their metabolic effects in rats. J. Nutr. 106, 435-442. 27. Wittenberg, J. B., Korey, S. R. & Swenson, F. H. (1956) The determination of higher fatty aldehydes in tissues. J. Biol. Chem. 219, 39-47. 28. Gray, G. M. (1969) The preparation and assay of long-chain fatty aldehydes. In: Methods in Enzymology (Colowick, S. P. & Kaplan, N. O., eds.), Vol. XIV, pp. 678-684, Academic Press, New York.
Downloaded from https://academic.oup.com/jn/article-abstract/106/11/1600/4768772 by guest on 07 November 2018
23. Tamura, T. & Stockstad, E. L. R. (1973) The availability of food folate in man. Br. J. Haematol. 25, 513-532. 24. De Renzo, E. C. (1956) Chemistry and biochemistry of xanthine oxidase. Adv. Enzymol. 17, 293-328. 25. Herbert, V. (1975) Folie acid (pteroylglutamic acid): Transport, distribution, stor age, fate, and excretion. In: The Pharma cological Basis of Therapeutics (Goodman, L. S. & Oilman, A., eds.), pp. 1341-1342, 5th edition, MacMillan Publishing Co., Inc., New York, N.Y.
AND FUNCTION
ABSTRACT The effects of acidic and intestinal proteolytic environ ments on bovine milk xanthine oxidase (XO) activity were determined in order to evaluate the extent to which this enzyme was absorbed in biologi cally active form. The inhibition of XO by folie acid and the relative affinities of XO for the oxidation of palmitaldehyde, stearaldehyde, and xanthine were compared. The effects of acid and gastric juice on XO activity were measured by incubating purified enzyme, and non-purified enzyme (milk), in buffers ranging in pH from 2 to 9. Fresh gastric juice was also incubated with milk. Increasing amounts of the enzyme were inactivated as the pH of the incubation mixture was reduced below pH 6.5. Below pH 3.5, the enzyme was completely inactivated. Gastric juice, pH 2.2, also reduced the enzyme activity in proportion to the amount of gastric juice incubated with milk. Milk XO activity was reduced 36% when milk was incubated with an equal volume of gastric juice. Homogenized milk had 59% less XO activity compared with raw milk. Fresh raw milk XO, homogenized milk XO, and purified XO were equally susceptible to inactivation by acid or gastric juice. After incubation of milk with gastric juice, or gastric juice followed by pancreatin, XO activity was associated with a macromolecule of 300,000 daltons molecular weight and subunits containing activity were not found. It was estimated that 0.00008% of the XO in the intestine was absorbed. Both folie acid and allopurinol inhibited XO activity in vitro. Allopurinol was 3.5 times more potent an inhibitor than folie acid. A large excess of dietary folie acid did not reduce rat liver or intestinal XO activity in vivo. XO had a much greater affinity for xanthine than for palmitaldehyde or stearaldehyde substrates. It was esti mated that of 100 mg of XO in fresh raw milk, 41 mg remained after homogenization, 27 mg entered the intestine and only 20 ng were ab sorbed as intact enzyme. J. Nutr. 106; 1600-1609, 1976. INDEXING KEY WORDS xanthine oxidase •absorption •function Xanthine oxidase (EC 1.2.3.2) is a metallo-flavoprotein containing flavin adenine dinucleotide, molybdenum, iron and having a molecular weight of 300,000 daltons. The enzyme has been found in the milk of cows, sheep, goats, and rabbits but not in the milk of sows, mares, or humans.2 Milk xanthine oxidase was re cently reported to be absorbed intact and to be associated in a causative way with the development of coronary disease (1-8).
from homogenized milk was absorbed from the gut in biological active form and oxidized phospholipid plasmalogen of arterial and myocardial tissues, leading to scar formation, local deposition of cholesteryl esters and ultimately atherosclerotic lesions. It was further postulated that the
It
oxidase
was
postulated
that
xanthine
Oxidase
Receivedfor publicationApril 20, 1976. miSSST^ by NaU°nal Da'ry C°unc"'CT"cag°! ¿&&&SFÕÕ ^ fAf^'Al
1600
In milk. Proc. Nutr.
Soc. 18, I (abstract).
Downloaded from https://academic.oup.com/jn/article-abstract/106/11/1600/4768772 by guest on 07 November 2018
CHARLES Y. HO ANDANDREW J. CLIFFORD Department of Nutrition, University of California, Davis, California 95616
MILK XANTHINE OXIDASE ABSORPTION AND FUNCTION
1601
TABLE 1 Experimental protocols for evaluating the effect of digestion on milk xanthine oxidase activity
.Experiment 1Purified (nil)Buffer, XO, (ml)pH mixtureExperiment of
3Haw
(ml)IICl/ Milk, (normality)pH mixtureExperiment of 4Haw
Downloaded from https://academic.oup.com/jn/article-abstract/106/11/1600/4768772 by guest on 07 November 2018
2 (ml)Buffer Haw Milk (ml)pH mixtureExperiment of
(ml)Gastric Milk (nil)pH Juice,* of mixture0.10.6"2.250.10.6"2.80.10.22.530.00.62.20.10.6°3.50.10.6"3.350.10.152.870.10.53.560.10.6"4.50.10.6°3.850.10.133.050.2 " 0.05 M citrate phosphate buffer. ò0.05 M phosphate buffer. c0.05 M glycine-KOH buffer. "*Volume used = 0.3 ml. ' Freshly aspirated human gastric juice was pH 2.2 and 0.084 N with respect to a 1,000,000) was 5 times less than that of horse spleen ferritin (molecular weight 650,000). From these data (20, 21), it appears that as the molecular size was doubled, the extent of absorption into the intestinal cell was re duced 5-fold. If a linear relationship be tween molecular size and transepithelial migration is assumed, it is possible to cal culate that as the molecular weight in creased from 40,000 (horseradish perox idase) to 300,000 (xanthine oxidase), the percent absorption would decrease from 0.01% to 0.00008%. Application of these calculations to the present study indicate that of the 27% residual units of xanthine oxidase activity in the small intestine, only 2 X 10-" units would be absorbed. The size of the xanthine oxidase pool in liver can be estimated to be 53 mg (22). Thus the
1607
1608
CHARLES Y. HO AND ANDREW J. CLIFFORD
ACKNOWLEDGMENTS
This research was funded by a grant from the National Dairy Council, Chicago, Illinois. The authors are grateful to Pro fessor W. L. Dunkley for use of his oxygraph to measure xanthine oxidase activity and to Professor C. H. Halsted, and Dr. C. R. Fleming and R. S. Newton for pro viding gastric iuice. Technical help from Randy H. Shaffer is appreciated. LITERATURE CITED 1. Oster, K. A. (1968) Treatment of angina pectoris according to a new theory of its origin. Cardiol. Digest. 3, 29-34. 2. Oster, K. A. (1971) Plasmalogen diseases: a new concept of the etiology of the athero sclerotic process. Am. J. Clin. Res. 2, 30-35. 3. Oster, K. A. (1972) Predisposition to atherosclerosis. J. Am. Med. Assoc. 222, 704. 4. Oster, K. A. (1973) Is an enzyme in homogenized milk the culprit in dietaryinduced atherosclerosis? Med. Counterpoint 5 (November), 26-36. 5. Oster, K. A. (1974a) Plasmalogen disease and the rarity of atherosclerosis in the Masai. In: Myocardiology in Africa, pp. 209-214, E. A. Community (CPS) Printer, Nairobi.
6. Oster, K. A. (1974b) Bovine milk xanthine oxidase as one of the dietary causes of early atherosclerosis. Med. Counterpoint 6 (Novem ber), 39-42. 7. Oster, K. A. & Hope-Ross, P. (1966) Plas mai reaction in a case of recent myocardial infarction. Am. J. Cardiol. 17, 83-85. 8. Oster, K. A., Oster, J. B. & Ross, D. J. (1974) Immune response to bovine xanthine oxidase in atherosclerotic patients. Am. Lab. (August) 41-47. 9. Kalchar, H. M. (1947) Differential spectrophotometry of purine compounds by means of specific enzymes. I. Determination of hydroxypurine compounds. J. Biol. Chem 167 429-443. 10. Clark, L. C., Jr. (1956) Monitor and con trol of blood and tissue oxygen tension. Trans. Am. Soc. Artificial. Internal. Organs. 2, 4145. 11. Briley, M. S. & Eisenthal, R. (1974) Asso ciation of xanthine oxidase with the bovine milk-fat globule membrane. Catalytic prop erties of the free and membrane-bound en zyme. Biochem. J. 143, 149-157. 12. Dixon, M. (1953) The determination of enzyme inhibitor constants. Biochem. I. 55, 170-171. 13. Food and Nutrition Board, National Research Council (1974) Recommended Dietary Allowances, ed. 8, National Academy of Sciences, Washington, D.C. 14. Lineweaver, H. & Burk, D. (1934) The determination of enzyme dissociation con stants. J. Am. Chem. Soc. 56, 658-666. 15. Smith, I. (1968) Acrylamide gel disc electrophoresis. In: Chromatographie and electrophoretic techniques. Vol. 2, 365-418. J. Wiley, New York. 16. Dittmer, J. C. & Wells, M. A. (1969) Quantitative and qualitative analysis of lipids and lipid components. In: Methods in Enzymology XIV, 482-530. 17. Menlitz, A., Gierschner, K. U. & Minas, T. ( 1963) Dunnschichtchromatographische Trennung von 2,4-Dinitrophenylhydrazonen. Chemiker-Zeitung 87, 573-576. 18. Bray, R. C., Palmer, G. & Bennert, H. ( 1964) Direct studies on the electron ¿ansfer sequence in xanthine oxidase by electron paramagnetic resonance spectroscopy. II. Kinetic studies employing rapid freezing. Î. Biol. Chem. 239, 2667-2676. 19. Greenbank, G. R. & Pallansch, M. J. (1962) Inactivation and reactivation of xanthine oxi dase in dairy products. J. Diary Sci. 45, 958961. 20. Walker, W. A. & Isselbacher, K. J. (1971) Uptake and transport of macromolecules by the intestine. Possible role in clinical dis orders. Gastroenterology 67, 531-550. 21. Worthington, B. S. & Syrotuck, J. (1976) Intestinal permeability to large panticles in normal and protein-deficient adult rats. J. Nutr. 106, 20-32. 22. Krenitsky, T. A. (1974) Xanthine oxidase and aldehyde oxidase. Adv. Exp. Med. Biol. 41A, 57-64.
Downloaded from https://academic.oup.com/jn/article-abstract/106/11/1600/4768772 by guest on 07 November 2018
ize rapidly under physiological conditions (28) and consequently their availability as substrates for xanthine oxidase would be reduced even more. These results support the concept that xanthine oxidase was pri marily concerned with purine metabolism and appeared to be minimally involved in fatty aldehyde oxidation. Although xanthine oxidase occurs in rather high levels in bovine milk prior to homogenization, its biological role is not clear. Homogenization and exposure to gastric juice markedly reduced the activ ity of this enzyme. Subsequent exposure to pancreatin completed to release of the enzyme from the milk fat-droplet mem brane to yield free enzyme with a molec ular weight of 300,000 daltons. The extent to which this enzyme was absorbed was calculated to be practically zero. Our data indicates that xanthine oxidase absorption was minimal; the enzyme was not inhib ited in vivo by dietary folate; and the enzyme was unlikely to oxidase plasmai in vivo. Consequently the data would not support the hypothesis (1-8) of xanthine oxidase absorption and plasmalogen de pletion.
MILK XANTHINE
OXIDASE ABSORPTION
1609
26. Clifford, A. J. & Story, D. L. (1976) Levels of purines in foods and their metabolic effects in rats. J. Nutr. 106, 435-442. 27. Wittenberg, J. B., Korey, S. R. & Swenson, F. H. (1956) The determination of higher fatty aldehydes in tissues. J. Biol. Chem. 219, 39-47. 28. Gray, G. M. (1969) The preparation and assay of long-chain fatty aldehydes. In: Methods in Enzymology (Colowick, S. P. & Kaplan, N. O., eds.), Vol. XIV, pp. 678-684, Academic Press, New York.
Downloaded from https://academic.oup.com/jn/article-abstract/106/11/1600/4768772 by guest on 07 November 2018
23. Tamura, T. & Stockstad, E. L. R. (1973) The availability of food folate in man. Br. J. Haematol. 25, 513-532. 24. De Renzo, E. C. (1956) Chemistry and biochemistry of xanthine oxidase. Adv. Enzymol. 17, 293-328. 25. Herbert, V. (1975) Folie acid (pteroylglutamic acid): Transport, distribution, stor age, fate, and excretion. In: The Pharma cological Basis of Therapeutics (Goodman, L. S. & Oilman, A., eds.), pp. 1341-1342, 5th edition, MacMillan Publishing Co., Inc., New York, N.Y.
AND FUNCTION
