C. Y. HO, R. T. CRANE 3 ANDA. J. CLIFFORD Department of Nutrition, University of California, Davis, California 95616 ABSTRACT Three experiments were conducted to evaluate the follow ing: the absorption of mükxanthine oxidase (XO) via the lymphatic sys tem; the effect of oral fat on serum XO concentration; and the possibility that milk XO might be a source of riboflavin. Lymph was obtained from cannulated thoracic ducts of rats after stomach intubation of a mixture of half cream and half milk ( H/H ) fortified with XO. The presence of XO in lymph could not be demonstrated by the microcomplement fixation tech nique, and consequently, it was concluded that the enzyme was not ab sorbed by this route. Orally administered corn oil alone increased serum XO level demonstrating that the increased serum XO obtained with orally administered H/H, reported earlier, was probably due to the fat rather than the XO in the H/H. The possibility that milk XO might serve as a dietary source of riboflavin was also studied in a growth trial with chicks which were fed a riboflavin-free diet for 2 weeks, and were then divided into treatment groups and given dietary supplements of riboflavin (2 & 4 mg/kg ) or oral or intraperitoneal supplements of XO ( 15 mg/day ) for the following 15 days. Dietary supplements of XO produced only small incre ments in growth rate which was equivalent to that which would be ob tained with 0.5 mg riboflavin/kg of diet. The data demonstrate that orally administered fat increased serum XO, that bovine milk XO was not ab sorbed in the lymph of the rat, and that the enzyme was not a good source of riboflavin for the chick. J. Nutr. 108: 55-60, 1978. xanthine oxidase •lymphatic absorption INDEXING KEY WORDS •function Xanthine oxidase (XO) (EC 1.2.3.2), a metalloflavoprotein enzyme which occurs in relatively high concentrations in bovine milk, was recently postulated to be ab sorbed intact from the intestine and to be associated in a causative way with the de velopment of coronary disease (1-8). The postulated hypothesis involved the deple tion of arterial plasmalogens by homogen ized milk XO absorbed from the gut via the lymphatic system. One recent investi gation (9) was unable to deplete arterial plasmalogen in rabbits with large oral or intravenous doses of the enzyme given over

an extended time period. Another study (10) was unable to confirm significant ab sorption of the enzyme. Although a mix ture of half cream and half milk (H/H) fortified with XO was shown to increase serum XO (11), it was not apparent which component of the H/H was responsible Received for publication June 2, 1977. 1 Supported by National Dairy Council, Chicago, Illinois. 2 Address reprint requests to A. J. Clifford, Depart ment of Nutrition, University of California, Davis, Calif. 95616. 3 Department of Internal Medicine, Section of Car diology, School of Medicine, University of California, Davis, Calif. 95616. 55

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Studies on Lymphatic Absorption of and the Availability of Riboflavin from Bovine Milk Xanthine Oxidase1'2

56

HO, CRANE AND CLIFFORD

A homogenized and pasteurized mixture of half milk and half cream (H/H) was purchased at a local supermarket and for tified with 0.015 volumes of purified XO,4 0.0075 volumes of detergent5 and 1.0 vol umes of corn oil,6 and thoroughly mixed. One-half of the mixture was placed in a boiling water bath for 30 minutes to in

' Grade IV. Activity was 0.22 units/ml. Purchased from Sigma Chemical Co.. St. Louis, Missouri. "Triton X-100, purchased from Sigma Chemical Co., St. Louis, Missouri. • Mazóla. Corn Products Co.. New York. 7 Ho, C. Y., Barr, L. G. & Clifford, A. J. Immunologlcal relatedness of mammalian xanthine oxidases (manuscript submitted). 8 Simonson, Sunyvale, Calif. 9 Cary 118C Spectrophotometer, VarÃ-an Instrument Division, Cary Techtron Products, 611 Hansen Way, Palo Alto, Calif.

IO

5 (O

4l

4«

1512

ANTIGEN

61035

DILUTION

Fig. 1 Microcomplement fixation assay for xanthine oxidase (XO) in milk (A A), a mixture of half milk and half cream (H/H) forti fied with XO (• •),and boiled XO forti fied H/H, or lymph from rats intubated with XO fortified H/H or boiled XO fortified H/H

for the increased serum XO. We evaluated the lymphatic absorption of XO with di rect measurements for the enzyme in lymph from rats given XO fortified H/H. We also measured the effect of orally administered fat on serum XO activity. The physiological function of XO in bo vine milk is unknown. Since each mole of XO contains 2 moles of flavin adenine dinucleotide (FAD) (12), the extent to which this enzyme might serve as a source of riboflavin was determined. This report shows that under the conditions of the present experiments XO was not absorbed via the lymphatic system, orally adminis tered fat increased serum XO and the en zyme did not serve as a good source of riboflavin.

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MATERIALS AND METHODS

activate the enzyme. The boiled and nonboiled mixtures were tested for XO by microcomplement fixation (13) using serum from rabbits immunized with XO.7 To test the lymphatic absorption of XO, adult male Sprague-Dawley rats8 (200 g) were given 6 ml of either the boiled or nonboiled mixture by stomach tube and 2 hours later were fitted with thoracic duct cannulas (14). Installation of the cannulas was accomplished in less than 30 minutes. Lymph was collected for the next 4.5 hours in tubes standing in ice since an earlier report (11) has shown that serum XO was highest 4 hours after H/H fortified with XO was given. Aliquots of the collected lymph were tested for the presence of XO by microcomplement fixation (13). To determine the effect of exogenous fat on serum XO levels, adult male Sprague Dawley rats 8 (180 g) were intubated with 1 ml of corn oil or 1 ml of 154 mM saline, and blood was collected 3 and 6 hours later. The corn oil and saline were fortified with 0.0625 volumes of detergent.5 The serum was isolated by centrifugation and assayed spectrophotometrically9 for XO (11). To evaluate XO as a source of riboflavin, 64 3-day-old New Hampshire cockerels were wing banded and weighed. Ten chicks were randomly selected and fed a complete purified diet (15) while the re maining chicks were fed the same diet but without riboflavin during the next 14 days. These 14 days (period 1) represented a riboflavin depletion period intended to condition the birds for maximum growth response to supplements of riboflavin or XO. During the following 14 days (period 2), the 10 chicks receiving the complete diet (in period 1) continued to be fed this diet in period 2 and served as positive controls. Forty of the 54 chicks fed the riboflavin-free diet were assigned (16) during period 2 of the following treat-

100

MILK XANTHINE

OXIDASE ABSORPTION

RESULTS The effect of XO fortified H/H on lym phatic XO is shown in figure 1. All the lymph samples and the boiled XO fortified H/H failed to show micro-complement fix ation. Consequently, it was concluded that XO was not absorbed via the lymphatic system in the rat. The data also show that heat inactivation of XO rendered the en zyme antigenically inactive. Corn oil, administered orally to rats, in creased serum XO levels compared with saline (table 1) 3 hours after administra tion, but the difference was no longer sig nificant at 6 hours after administration. Data from the chick growth experiment are summarized in table 2. At the end of

TABLE 1 Effect of corn oil on serum xanthine oxidase activity1 Saline

Treatment No. of rats Time after intubation 3 hours 6 hours

Corn oil 10

5.642±1.01° 7.97 ±2.39