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Digestion and Absorption of Bovine Milk Xanthine Oxidase and Its Role as an Aldehyde Oxidase1

CHARLES Y. HO ANDANDREW J. CLIFFORD Department of Nutrition, University of California, Davis, California 95616 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).
It was postulated that xanthine Oxidase

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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 Received publicationApril 20, 1976. for miSSST^ by NaUnal Da'ry Cunc"' CT"cag! &&&SF ^ fAf^'Al
oxidase In milk. Proc. Nutr. Soc. 18, I (abstract).

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MILK XANTHINE OXIDASE ABSORPTION AND FUNCTION TABLE 1


Experimental protocols for evaluating the effect of digestion on milk xanthine oxidase activity

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.Experiment 1Purified (nil)Buffer, XO, (ml)pH


mixtureExperiment of

2 (ml)Buffer Haw Milk (ml)pH mixtureExperiment of

(ml)IICl/ Milk, (normality)pH mixtureExperiment of


4Haw

3Haw

(ml)Gastric Milk (nil)pH Juice,* of mixture0.10.6"2.250.10.6"2.80.10.22.530.00.62.20.10.63.50.10.6"3.350.10.152.870.10.53.560.10.6"4.50.10.63.850.10.133.050.2 " 0.05 M citrate phosphate buffer. 0.05 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<;id.

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administration of folie acid, a xanthine oxidase inhibitor, reduced xanthine oxi dase activity in vivo, reduced the inci dence of coronary disease and slowed the development of coronary disease already underway. The significance of xanthine oxidase in the etiology of atherosclerosis has recently been reviewed.3 Since little is known concerning the stability of xanthine oxidase during passage through the stom ach and intestine of man, this report describes a series of experiments designed to evaluate the extent to which xanthine oxidase escapes degradation in the acid environment of the stomach and the proteolytic environment in the human intes tine. We have also measured the inhibition of xanthine oxidase by folie acid to evalu ate the extent of inhibition at physiologi cal concentrations of folate.

activity (experiment 3); the effects of adding human gastric juice to milk on its xanthine oxidase activity (experiment 4); and the effect of human gastric juice fol lowed by pancreatin on fresh raw milk xanthine oxidase activity (experiment 5). In all experiments, the purified xanthine oxidase,4 fresh raw milk5 and homogenized milk6 were incubated (37) at different pH (table 1) for varying periods of time up to 240, 20, 60 and 30 minutes for ex periments 1, 2, 3, and 4, respectively. At the end of the incubation period the mix tures containing purified enzyme were ad justed to pH 8.1 with Tris-HCl buffer (0.136 M) and the residual xanthine oxi dase activity measured spectrophotometrically (9). Incubation mixtures containing raw milk and homogenized milk were ad justed to pH 7.4 with sodium pyrophosphate buffer (0.2 M) and the residual MATERIALS AND METHODS xanthine oxidase activity measured polaroTo evaluate the extent to which milk graphically (10). Xanthine was used as xanthine oxidase was inactivated under substrate in all experiments, and in addi conditions similar to those found in the tion, NADH was used as substrate in ex stomach and intestine of healthy humans, periment 5 since the ratio of activities with a series of five experiments were con xanthine and NADH was reported to reducted. The experiments (table 1) mea sured the effect of pH on purified xanthine 3 Carr, C. J., Talbot, J. M. & Fisher, K. D. (1975) A review oxidase activity using buffers ranging in oxidase in of the significance of bovine milk xanthine the etiology of atherosclerosis. Report pre pH from 2.25 to 9.5 (experiment 1); the pared for Division of Nutrition, Bureau of Foods, Food and Drug Administration, Washington, effect of pH on fresh raw milk xanthine pp. 1-65. Life Science Office, Fed. Am. Soc. D.C., Exp. Maryland. oxidase activity using buffers ranging in Biol.. Bethesda, from Sigma Chemical Company, St. 1 Purchased pH from 2.8 to 7.2 (experiment 2); the Louis, Missouri. 6 from pooled effect of adding increasing levels of HC1 sity Samples were obtained dairy herd. milk, Univer of California, Davis, 6 Purchased at local supermarket. to fresh raw milk on its xanthine oxidase

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CHARLES Y. HO AND ANDREW J. CLIFFORD

fleet the relative amounts of membranebound and free enzyme present (11). Purified xanthine oxidase, fresh raw milk, and homogenized milk incubated with 0.2 M sodium pyrophosphate buffer, pH 7.4, under identical conditions served as controls in each experiment. In experiment 5, 0.3 ml of raw milk and 0.3 ml of human gastric juice7 were incu bated (37) for 30 minutes and then 0.6 ml of 2% pancreatin * in sodium pyrophosphate buffer (pH 7.4) was added, and the incubation continued for a further 80 minutes. The pH of the milk and gastric juice mixture was 5.36 which increased to 7.4 when the pancreatin was added. Mea surements of xanthine oxidase activity with xanthine and NADH as substrates were made after 10, 20, 30, 40, 50, 60, 70 and 80 minutes of incubation with pancreatin. The effect of gastric juice, and gastric juice followed by pancreatin on the molec ular size of xanthine oxidase in milk was evaluated in experiment 6. Aliquots of fresh raw milk (20 ml) were incubated (30 minutes at 37) with equal volumes of phosphate buffer (0.1 N sodium pyrophosphate, pH 7.45, containing 0.27 HIM EDTA), fresh gastric juice, or fresh gastric juice followed by neutralization to pH 7.4; all samples were further incubated (30 minutes at 37 with pancreatin in which ) the final concentration was \%. Xanthine oxidase activity was isolated on Sepharose 2B 8 columns ( 5 X 40 cm ) previously equilibrated with phosphate buffer (0.1 N sodium pyrophosphate, pH 7.45, con taining 0.27 mM EDTA). The columns were eluted with the same phosphate buf fer (15 ml/hour) and the effluent col lected in 5 ml fractions. To further confirm the homogeneity of the peaks of xanthine oxidase activity which were absorbed onto and eluted from the sepharose columns, the fractions con taining activity from each column were pooled and concentrated ( PM 30 filters 9 at 0 to 5. Since the filtrate did not have ) enzyme activity, it was discarded. The concentrated solutions were re-chromatographed on Sephadex G-2008 columns (3.3 X 49 cm) equilibrated with phos phate buffer (0.1 M sodium pyrophosphate, pH 7.45 containing 0.27 mM EDTA) and eluted with the same phosphate buffer

(6 ml/hour) and the effluent collected in 2 ml fractions. Since marked symptomatic improve ment of atherosclerotic patients was re ported with folie acid treatment (5, 6), the inhibition of xanthine oxidase by folie acid was evaluated in vitro and in vivo (experiment 7). Xanthine oxidase activity was measured in the presence of varying concentrations of folie acid10 and the in hibition constant (Ki) determined from inverse plots of activity and inhibitor con centration ( 12 ). Allopurinol4 [4-hydroxypyraxol-(3,4-d)-pyrimidine] was also used as an inhibitor of xanthine oxidase for com parative purposes. The in vivo inhibition of xanthine oxidase by folie acid was evaluated by measuring xanthine oxidase activity in liver and intestine of young adult male Sprague-Dawley rats fed a stock diet (control) " or the same diet supplemented with folie acid [l g/100 g diet i.e. 1.5 X IO4 times the human Recom mended Daily Allowance (13)] for 15 days. In order to determine the extent to which xanthine oxidase might function as an aldehyde oxidase, Michealis constants (K,n) and maximum velocities (Vmax) for the enzyme were measured (14) using xanthine,4 palmitaldehyde 12 and stearaldehyde 12 as substrates (experiment 8). The expression Km/Vnwx was then calculated to describe the affinity of xanthine oxidase for each of the substrates. Xanthine oxidase purity was confirmed on polyacrylamide gels (15). Palmitalde hyde and stearaldehyde purity was checked by thin layer chromatography (16, 17). The purity of xanthine oxidase and the aldehydes were in excess of 90 and 98%, respectively. RESULTS The effect of increasing acidity on xan thine oxidase activity is shown in figure 1.
'Obtained from healthy young adults undergoing gastric analysis by C. H. Halsted, M.D. and C. R. Fleming, M.D., Department of Internal Medicine, University of California. Sacramento Medical Center. Purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. irr,]. Corporation, Am Lexington, Massachusetts. 10Purchased from ICN Pharmaceuticals, Inc., Cleveland, Ohio. 11Purina rat chow, Ralston Purina Company, St. Louis, Missouri. a Purchased from nalabs Inc., North Haven, Con necticut.

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MILK XANTHINE OXIDASE ABSORPTION AND FUNCTION


100

1(503

RM TIME ZERO

<f*j!

50 u. O 25

3 pH

Fig. 1 Influence of pH on bovine milk xanthine oxidase activity. Purified xanthine oxi dase ( PE ) and raw milk ( RM ) were incubated at pH values shown for 30 minutes at 37.The pi I was then adjusted to 7.4 for milk samples and to 8.1 for purified enzyme and the xanthine oxidase activity measured. PE zero time X X and PE 30 minutes X X represent activity measurements after 0 and 30 minutes incubation, respectively. RM zero O O and RM 30 min utes O O represent activity measurements after 0 and 30 minutes, respectively.

Two peaks of xanthine oxidase activity were observed when fresh raw milk was diluted with phosphate buffer and applied to the Sepharose 2B columns (fig. 4). The first peak of activity was eluted in the void volume of the column (295 ml) and accounted for 18% of the applied activity. The second peak of activity was recov ered in fractions 90 to 190 (450-950 ml) and accounted for 73 % of the applied ac tivity. When the milk was incubated with gastric juice and also when this incubation was followed by further incubation with pancreatin, xanthine oxidase activity was eluted in a single peak with Q5% recovery of the applied activity in fractions 90 to 190 (450-950 ml). There was no enzyme activity recovered in the void volume of the column when the milk was incubated with gastric juice or with gastric juice fol-

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As the pH was reduced from 7.0, increas ing amounts of the enzyme were inacti vated; when the pH was reduced below 3.5, the purified enzyme as well as that in milk was completely inactivated. The puri fied xanthine oxidase and the enzyme in raw and homogenized milks were equally susceptible to inactivation by incubation in acid media. The effects of incubating raw and ho mogenized milk with varying amounts of human gastric juice is shown in figure 2. The inhibition of xanthine oxidase activ ity was proportional to the relative amounts of milk and gastric juice in the incubation mixture. Homogenized milk had lower enzyme activity compared with raw milk. When milk was incubated first with gastric juice and the mixture further in cubated with pancreatin (fig. 3), no fur ther loss in enzyme activity was observed with xanthine as substrate. However, a 4-fold reduction in enzyme activity oc curred when NADH was the substrate used to assay the enzyme. When the changes in activity were expressed as that activity with xanthine as substrate di vided by the corresponding activity with NADH as substrate, the activity ratio (X/N) increased 4.5-fold.

HM + SJ

.1 .5

.2 .4

.3 .3

.5 .1

.6 0 6.8

MILK

Im

GASTRIC JUICE (mil pH

Fig. 2 Influence of human gastric juice on bovine milk xanthine oxidase activity. Fresh raw milk (RM) and homogenized milk (HM) were incubated with sodium pyro phosphate buffer (B) (pH 7.4) O O, O O or fresh human gastric juice (GJ) X X, X X, respec tively, in the ratios shown for 30 minutes at 37. The pH of all samples after incubation were ad justed to 7.4 and the remaining xanthine oxidase activity measured. The pH values shown in the figure represent the pH of the mixture of milk and gastric juice.

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CHARLES Y. HO AND ANDREW J. CLIFFORD

Supplementing rat diets with folie acid did not reduce liver or intestinal xanthine oxidase activity in vivo (table 2). Inhibition constants (Ki) for folie acid and allopurinol were calculated to be 3.26 tM 0.91 /M, and respectively (fig. 6), thus demonstrating that allopurinol was 3.5 times more potent an inhibitor of xanthine oxidase than was folie acid. The enzyme activity plots (fig. 6) also show that both folie acid and allopurinol were competi tive inhibitors. The Km and VmKI alues determined with v xanthine, palmitaldehyde, and stearaldehyde (table 3) show the Km/Vmai was much greater (130 times) for xanthine compared with the aldehydes. These data demonstrate that xanthine (purine) rather than aldehyde was the much preferred substrate for xanthine oxidase. DISCUSSION In developing the hypothesis of a causal relationship between milk xanthine oxidase and coronary Disease" was coined based "Plasmalogen heart disease, a new term on the concept of a deficiency of plasmalogens in arterial intima and myocardial cells (1-8). It was postulated that xan thine oxidase from bovine milk destroyed the aldehydes liberated from cell mem brane-based plasmalogens, thus damaging the arterial intima and myocardial cell membranes. The resulting histochemical injury was reported to evoke repair pro cesses manifested by cell proliferation, scar formation, local deposition of cholesteryl esters, and ultimate development of typical atherosclerotic lesions in the ar teries and affected myocardium. According to this hypothesis, xanthine oxidase from milk, especially homogenized milk, not only survived passage through the human stomach, but also was presum ably absorbed, enzymatically intact, and in sufficient quantity, from the gastrointes tinal tract. Because homogenization of milk reduced the size of the fat droplet with which xanthine oxidase activity is associated, it was assumed that homogeni zation increased the biological availability of the enzyme. Thus, it was concluded that consumption of milk is a predisposing fac tor in the atherosclerotic process (1-8). Since xanthine oxidase, a unimolecule,

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IO 20

30 40 50 TIME (min)

60 70

80

Fig. 3 Influence of pancreatin on xanthine oxidase activity. Raw milk was incubated with an equal volume of gastric juice for 30 minutes at 37.Pancreatin was added and the incubation continued for a further 80 minutes at 37.Aliquots were removed at the time intervals shown and xanthine oxidase activity measured using xanthine or NADH as substrates.

lowed by further incubation with pan creatin. Rechromatography on Sephadex G-200 of the Sepharose 2B xanthine oxi dase activity peaks from milk treated with gastric juice or gastric juice followed by pancreatin yielded a single peak of activ ity which was eluted in fractions 30 to 90 (60-180 ml) with 95% of the applied activity recovered (fig. 5). These data demonstrate that xanthine oxidase activity in fresh raw milk was associated with par ticles of 3 X IO5 and > 3 X IO7 Daltons molecular weight. When fresh raw milk was incubated with gastric juice or gastric juice followed by further incubation with pancreatin, the xanthine oxidase activity of molecular weight > 3 X IO7 was trapped in the curd, and when the curd was ex tracted and chromatographed on Sepha rose 2B, the xanthine oxidase activity was recovered in fractions 90 to 190.

MILK XANTHINE OXIDASE ABSORPTION AND FUNCTION

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loo

i o FRACTION NUNSEM

PIIACTION KIMtlt

100 1*0 FRACTION NUMBER

Fig. 4 Influence of gastric juice and pancreatiti on the molecular size of bovine milk xanthine oxidase. Milk was incubated with an equal volume of buffer (sodium pyrophosphate pH 7.4), gastric juice (pH 2.2) or gastric juice followed by a further incubation with pancreatin. Each incubating was for 30 minutes at 37.At the end of the incubation period the mixture was centrifugation ( 3,000 X g, 10 minutes, at 5 and the supernatant applied ) to Sepharose 2B columns. The columns were eluted with sodium pyrophosphate buffer (0.1 M) containing 0.27 HIM EDTA, pH 7.45 and xanthine oxidase activity ( ) in the effluent was measured. Protein in the column effluent ( ) was also measured. Fractions 0 to 90 represent the void volume. Milk incubated with buffer contained a peak of enzyme activity in the void volume. On treatment with gastric juice the enzyme activity in the void volume disappeared.

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multicomponent enzyme (18) of about 300,000 daltons molecular weight appeared likely to lose much activity during gastric digestion (low pH) and by virtue of its molecular weight, seemed likely to be ab sorbed by the intestine only to a very lim ited extent; we evaluated the effects of a variety of pH environments, of gastric juice, and of pancreatin on the activity of this enzyme. Below pH 3.5, both the puri fied enzyme and the enzyme in milk were

completely inactivated. The presence of milk did not have a protective effect on the enzyme. The effect of pH in reducing xanthine oxidase activity was independent of the buffers, the HC1, or the gastric juice used. With a 1:1 ratio of milk to gastric juice, enzyme activity was reduced 36%. At lower ratios of milk to gastric juice, considerably greater quantities of enzyme activity were inactivated. Homogenized milk had 59% less xan-

MILK ASTRIC JUICE

oo

too

ieo

60

100

ISO

80 FRACTION

100 NUMI

ISO

FRACTIOk NUN

FIUCTIO NUNKR

Fig. 5 Rechromatography of xanthine oxidase activity peaks (from fig. 4) on Sephadex G-200. Fractions of 100-190 (fig. 4) were combined, concentrated by ultrafiltration and applied to Sephadex G-200. Sodium pyrophosphate buffer (pH 7.4 containing 0.27 mM EDTA) was used to elute the enzyme. Xanthine oxidase activity ( ) and protein ( ) were measured in the column effluent. Xanthine oxidase was eluted as a single peak, and rechromatographed with purified xanthine oxidase (molecular weight '300,000).

1GOG

CHARLES Y. HO AND ANDREW J. CLIFFORD TABLE 2

Effect of dietary folie acid on rat liver and intestine xanthine oxidase
Control No. of rata Liver xanthine oxidase Intestine xanthine oxidase 6 1.16 0.23 0.690.18 Control + folie acid 6 1.37 0.44 0.790.12

Adult male Sprague-Dawley rate were fed a stock diet (control) and the same diet supplemented with folie acid (0.2 u KHIg diet) for 14 days. Values are pinoles uric acid formed/min/ tissue protein at 25. alues are means SD. V

thine oxidase activity than fresh raw milk. This lower activity in homogenized milk agreed well with previously published data (19). The inhibitory effect of gastric juice on xanthine oxidase activity was the same for raw and homogenized milks. The lower ( 59% ) xanthine oxidase activity in homogenized milk coupled with the fur ther reduction (36%) due to gastric juice, left only 27% of the initial xanthine oxi-

4.28 XIO'^M

1.4 2.t 4.2 5.6 FOLIC ACID CONC (fjM]

dase activity of raw milk. The 27% re sidual activity probably represented a maximum value because it was based on a 30-minute incubation period with gastric juice, a time chosen because it approxi mates the transit time in the stomach when milk alone is consumed. This time of incubation with gastric juice was also chosen because milk, in the presence of gastric juice, forms a curd in which about 30% of the xanthine oxidase activity be comes entrapped, and therefore has a con siderably longer transit time in the stom ach. Since milk is often consumed at meal time along with other foods (cereals, etc.), the transit time in the stomach would probably increase and result in a greater inactivation of xanthine oxidase. Pancreatin, a mixture of hydrolytic en zymes, acts upon that xanthine oxidase which escapes digestion in the stomach. Since the relative activities of xanthine oxidase with xanthine and NADH (X/N) as substrates was reported to reflect the free or bound state of the enzyme (11), the X/N activity ratios of milk which was incubated with gastric juice for 30 min utes and further incubated with pancreatin for 30 minutes were measured. This effect of pancreatin was interpreted to mean that xanthine oxidase may enter the intestine partly as a membrane bound enzyme and there be converted to free enzyme. Although xanthine oxidase was previously reported to be absorbed into the lymphatic system (8) by virtue of being membrane-bound (fat globule mem brane), our in vitro data suggest that in the intestine the enzyme may be entirely free rather than membrane-bound. TABLE 3
Effect of substrate1 on bovine milk xanthine oxidase activity

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SubstrateXanthinePalmi
0.16 0.72 1.08 1.44

ALLOPURINCL CONC. (pM)

10-2.5 X 107 X 10-3.5 X X taldehydeStearaldehydeMichealisconstant1(Km)3.5 10*8 X 10-Maximumvelocity*(V.,)35.718.227.7Vm.,K.10 X 10 1Purified xanthine oxidase (0.025 unita) was incubated with the substrates. The XO activity was determined polarographically at 37. s Values are molar. *Values were determined from the slope (Oiconsumed/1.8 ml reaction mixture per 5 min).

Fig. 6 Inhibition of bovine milk xanthine oxidase by folie acid and allopurinol. Activity of purified enzyme (0.02 units) was measured in the presence of the inhibitor and substrate concen trations shown.

MILK XANTHINE OXIDASE ABSORPTION AND FUNCTION

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Results from experiment 6 showed 2 peaks of xanthine oxidase activity in fresh raw milk and only 1 peak after incubation with gastric or gastric juice followed by pancreatin. The first eluted peak for fresh raw milk had a molecular weight in ex cess of 3 X IO7 Daltons because it was eluted in the void volume of the Sepharose 2B columns. Incubation with gastric juice or gastric juice followed by pancreatin re sulted in the formation of a curd which trapped about 30% of the enzyme activity. This activity was washed from the curd, and when applied to a Sephadex G-200 column, had an activity peak of 3 X IO5 Daltons molecular weight. This peak of activity corresponded well to that obtained when purified xanthine oxidase was chromatographed as the reference standard on Sephadex G-200. Experiments 5 and 6 in dicate that the treatment of milk with gastric juice and pancreatin under condi tion designed to simulate those in the human intestine suggested that in the in testine the milk xanthine oxidase existed as free enzyme with a molecular weight of approximately 300,000 daltons. With respect to the intestinal absorp tion of macromolecules, Walker and Isselbacher (20) recently reported that 0.01 % of a single dose of horseradish peroxidase (molecular weight 40,000) was absorbed in 4 hours. More recently, Worthington and Syrotuck (21) have shown that the transepithelial migration of adenovirus particles (molecular weight > 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

amount of xanthine oxidase absorbed would appear to be very small relative to the total pool of xanthine oxidase present in liver. Since intestinal tissue also con tains xanthine oxidase, the amount of xanthine oxidase absorbed relative to the total body pool of this enzyme would be still smaller. Folie acid did not inhibit liver or in testinal xanthine oxidase in the rat when given in very large doses, i.e. 1.5 X IO4 times the human Recommended Daily Al lowance (13) or 1.2x10 times the human nutritional requirement (23) even though folie acid is a well known inhibitor of xanthine oxidase in vitro (24). The apparent discrepancy can be accounted for by the fact that human tissue satura tion with folates can be maintained with an intake of 2 mg every other day after an initial loading dose (23) and intakes above this amount are presumably ex creted in urine (23, 25). Folie acid con centration in liver tissue is approximately 30 (.M (23), and an intake of 2 mg every other day can be calculated to increase the liver concentration to approximately 33 fj.M; an increase, which would not detectibly inhibit xanthine oxidase activity. Thus, the beneficial effects ascribed to folie acid (5, 6) seem very unlikely due to its inhibition of xanthine oxidase. Previous reports have shown that liver purine (xanthine and hypoxanthine) levels were in the range of 2 to 10 X IO"3M (26) and liver fatty aldehyde concentrations were approximately 1 to 5 X IO-3 M (27). Comparing these values with the Km values for xanthine oxidase obtained in the present study, it is apparent that the liver purine concentrations were 1,000 times greater than the Km value with purine substrate while the liver fatty aldehyde concentrations were only 10 times greater than the Km value with aldehyde substrate. Furthermore, liver purine concentrations were 2 times greater than fattv aldehyde concentrations. The greater affinity (130fold) of xanthine oxidase for xanthine coupled with the greater purine (2-fold) concentrations demonstrated that purines would be 260 times more available than fatty aldehydes as substrates for xanthine oxidase. In addition, fatty aldehydes are unstable compounds that tend to polymer-

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CHARLES Y. HO AND ANDREW J. CLIFFORD 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. . 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.

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.
ACKNOWLEDGMENTS

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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.
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