Life Sciences Vol . 19, pp . 887-892, 1976 . Printed in the U .S .A .
Pergamon Presa
RANTHINE ORIDASE ACTIVITY IN RAT SERUM AFTER ADMINISTRATION OF HOMOGENIZED BOVINE CREAM PREPARATION A . J . Clark, D . E . Pratt, Department of Foods and Nutrition and J . V . Chambers, Department of Animal Sciences Purdue University, West Lafayette, IN 47907 U .S .A . (Received in final form August 3, 1976) SUMMARY Rate were given a single dose of saline, saline supplemented with aanthine oxidaee (RO), half cream and half milk (H/H) and H/H supplemented with RO . RO was determined by a apectrophotometric method at 297 ~ in serum at 0, 2, 4 and 6 hours after administration . The method ie rapid, reliable and compares favorably with reported assays . No significant difference was obtained between the two saline treatments . The RO activity in serum of animals receiving the H/H increased significantly at 2 hours and then decreased . The H/H supplemented with RO demonstrated a maaimum activity in serum at 4 hours and then declined to a value similar to that of the H/H treatment and below the RO level at 0 time . The initial increase in %0 activity in serum of rate receiving the H/H treatments may indicate that RO is absorbed in the gaetrointnetinal tract or that the H/H materials stimulated endogenous RO activity . Recent reports by Oeter and his associates (1,2,3) have focused attention on the possible role of aanthine oxidaee (RO) in the atharosclerotic process of blood vessels . Previous reports (4,5) have characterized RO as the metallo flavoprotein which can enzymatically oaidiza hypoxanthine, xanthine, pyrimidinee, pterine, aldehydes, and reduced diphosphopyridine . Concurrent with these oaidative reactions would be the hydrogen transfer to such acceptors as molecular orygen, cytochrome c, ferricyanide, ferritin and several redox dyes . RO has been demonstrated in the tissue of the liver, the small intestinal mucosa (6), rat blood serum (7) and bovine milk (8,9) . Mazur et al . (10) suggest that a biological function of RO is to aid the release of iron from the liver stores (stored as ferritin-iron complexes) and the transport of dietary iron across the intestinal mucoea into the blood stream . Dietary molybdenum is reported by Seelig (11) to stimulate rat intestinal RO activity rates whereas, iron deficient conditions in the rat's body stimulate the mobilization of iron from the liver stores by increasing liver RO activity (12) . In contrast to the findings of Mazur et al . (10) and Seelig (11), Ostar suggested that because bovine milk is a major source of dietary RO that this enzyme can pace into the blood stream via the lymphatic system and become ectopically located in the blood vessels thereby initiating the atherosclerotic process . Since the initial controversy focuses upon the ability of the catalytically functional enzyme to pass through the intestinal mucoea, it was the purpose of this study to eaamina the dietary influnnca of commercially avail1 Journal paper No . 6232 .
Agricultural Experiment Station, Purdue University . 887
88 8
Xanthine Oxidase Activity in Serum
Vol . 19, No . 6
able half and half (H/H) (commonly used ae a coffee creamer) on %0 activity in rat serum. Ia the process of investigating this phenomenon, a modified spectrophotometric method for datermiaiag RO activity in rat serum was developed . It was ascertained that several %0 assay methods were available but those methods warn either too involved, required elaborate analytical equipment and reagents or have employed uncommon substrates and end products . The current method being reported was found to be convenient sad reliable for determination of serum RO activity . Methode Mal~ rats (3lrague-Dawley strain) weighing 140-180 g were used in this study. Rats were allowed Purina chow ad libitum sad they were fasted for 12 hours prior to giving the treatments . The rate were gives a single doss consisting of 6 ml of test materials via a plastic stomach tube . The treatments consisted of : a) saline (0 .85X), b) saline supplemented with 0 .16 unite per ml of %0 (EC 1.2 .3 .2), (Sigma Chemical Co .), c) H/H and d) H/H supplemented with 0 .16 units of %0 per ml . The mutates contained 0 .5 ml of Tween 20 per 66 ml of test material sad were The H/H was a paeteurizad and homogenized mixed for 2 min in a blaring blandor. commercial preparation consisting of approximately 12X butter fat . The H/H The ezperiment consisted of 9 contained approximately 0.04 unite of RO per ml . rate/dose-time treatment for a total of 144 rats . One unit of anzyma activity is defined as the amount of enzyme required to oxidize 1 .0 pmole of hypoxanthine to uric acid per minute at 25°C . Rats were aaesthetizad with ether and blood was removed from the heart with a syringe and needle at 0, 2, 4 and 6 hours after intubation of the traatBlood was immersed in an ice bath, allowed to clot and centrifuged at meats . 4°C for 25 minutes at 5000 z g. The serum was removed and analyzed for xanthine ozidase activity is duplicate. Samples ware stored at 4°C sad analyzed Assays revealed that RO activity was not altered during the within 3 days . storage period although activity decrnasad for samples etorad longer than 4-5 The measurement of %0 activity in serum was a modification of the method days . A double beam racordiag spectrophotometer described by Avis et al . (8,9) . The reference cuvette contained 0 .4 ml (Beckman, Model 25) at 297 nm was used . Tha test of serum and 1 .6 mT of 0.05 M sodium pyrophosphate buffer, pH 8.3 . solution contained 0.4 ml of serum, 1.0 ml pf pyrophosphate buffer and 0.6 ml Thn assay medium was mixed and abeorbaaca readings of 150 pM hypozanthina . takes immediately. At 15 and 30 miauteB after addition of hypozanthine The difference between absorbance abeorbance measurements ware recorded . measuremante at 30 and 15 minutes (pA) was used to calculate %0 activity . The formula used for calculating %0 activity in serum was : %0 activity (unit/m~ serum protein) ~ A z 1000/15 min a 1.22 z 10 x 14 mg protein/ml serum. The molar axtiaction coafficieat of uric acid was 1 .22 z 104 . The protein content of rat serum ~!as determined by Lowery method (13) to have a mean coaThe reported method was compared with the ceatratioa of 14 mg/ml of serum. :auorometric assay tschniqua described by Hainiag sad Legaa (14) using 2-amino4-hydroayptnridine (AHP) as substrate . Reeulta FIG . 1 shows the rate of oxidation of hypozaathine to uric acid catalyzed by R0 . The reaction rata was considered linear from fifteen to thirty minutes.
Vol . 19, No . 6
Ranthiae Ozidasa Activity is Serum
88 9
a.~
w
xo
w
MINUTES
w
so
FIG . 1 Ozidation of hypozaathine catalysed by %0 and measured by absorbaace as a function of time . The incubation mixture contained 0 .6 ml of 150~u?f hypoaaathine, and 1 .4 ml of pyrophosphate buffer, pH 8 .3 . The reaction miztura contained 6 .4 a 10 - unite of %0 par ml . FIG . 2 shove the absorbance spectra for uric acid with absorption mazimum at 289 nm . In serum, the absorption maxima was altered shaving a bathochromic shift of 8 ~ . The addition of uric acid to the serum samples did not change the absorbance ~n=i,m ~. The curves demonstrate that the rata of uric acid formation ie proportional to %0 activity .
FIG . 2 Abaorbaace spectra for uric acid ( 0 .04 pmole uric acid/ml sad - - - 0 .02,umole uric acid/ml) ; for ( . . . . .) uric acid produced in reaction medium ; and for (---) 0 .0125~umole uric acid/ml added to reaction medium (0 .6 ml 150 pM hypozaathine ; 1 .0 ml 0 .05 M pyrophosphate buffer, pH 8 .3 ; sad 0 .4 ml serum) . FIG . 3 shows the time-treatmeat response of %0 activity in serum after giving rats a dose of the test materials . The data for saline sad saline plus %0 were pooled because aeseatially no differaacee were dieceraed between these traatmaats . The %0 activity for the saline treatments shaved a slight elevation from 0 time to 6 hours after administration . The RO activity in serum of animals receiving the H/H increased markedly at 2 hours sad then decreased aignificaatly . The H/H supplemented with %0 demonstrated a TMs=i+m++~ %0 activity in serum at 4 hours and then declined to a value similar to that of the H/H .
89 0
Vol . 19, No . 6
Ranthina Oxidase Activity in Serum
FIG . 3 Effect of H/H, H/H + RO and saline on the activity of RO in rat serum . Saline (D---o), H/H (~-+d) and H/H + RO ( o o ) . The composition of the reaction miztures is given under Methods . The use of analysis of variance demonstrated that a significant differeaca in RO activity (P
Pergamon Presa
RANTHINE ORIDASE ACTIVITY IN RAT SERUM AFTER ADMINISTRATION OF HOMOGENIZED BOVINE CREAM PREPARATION A . J . Clark, D . E . Pratt, Department of Foods and Nutrition and J . V . Chambers, Department of Animal Sciences Purdue University, West Lafayette, IN 47907 U .S .A . (Received in final form August 3, 1976) SUMMARY Rate were given a single dose of saline, saline supplemented with aanthine oxidaee (RO), half cream and half milk (H/H) and H/H supplemented with RO . RO was determined by a apectrophotometric method at 297 ~ in serum at 0, 2, 4 and 6 hours after administration . The method ie rapid, reliable and compares favorably with reported assays . No significant difference was obtained between the two saline treatments . The RO activity in serum of animals receiving the H/H increased significantly at 2 hours and then decreased . The H/H supplemented with RO demonstrated a maaimum activity in serum at 4 hours and then declined to a value similar to that of the H/H treatment and below the RO level at 0 time . The initial increase in %0 activity in serum of rate receiving the H/H treatments may indicate that RO is absorbed in the gaetrointnetinal tract or that the H/H materials stimulated endogenous RO activity . Recent reports by Oeter and his associates (1,2,3) have focused attention on the possible role of aanthine oxidaee (RO) in the atharosclerotic process of blood vessels . Previous reports (4,5) have characterized RO as the metallo flavoprotein which can enzymatically oaidiza hypoxanthine, xanthine, pyrimidinee, pterine, aldehydes, and reduced diphosphopyridine . Concurrent with these oaidative reactions would be the hydrogen transfer to such acceptors as molecular orygen, cytochrome c, ferricyanide, ferritin and several redox dyes . RO has been demonstrated in the tissue of the liver, the small intestinal mucosa (6), rat blood serum (7) and bovine milk (8,9) . Mazur et al . (10) suggest that a biological function of RO is to aid the release of iron from the liver stores (stored as ferritin-iron complexes) and the transport of dietary iron across the intestinal mucoea into the blood stream . Dietary molybdenum is reported by Seelig (11) to stimulate rat intestinal RO activity rates whereas, iron deficient conditions in the rat's body stimulate the mobilization of iron from the liver stores by increasing liver RO activity (12) . In contrast to the findings of Mazur et al . (10) and Seelig (11), Ostar suggested that because bovine milk is a major source of dietary RO that this enzyme can pace into the blood stream via the lymphatic system and become ectopically located in the blood vessels thereby initiating the atherosclerotic process . Since the initial controversy focuses upon the ability of the catalytically functional enzyme to pass through the intestinal mucoea, it was the purpose of this study to eaamina the dietary influnnca of commercially avail1 Journal paper No . 6232 .
Agricultural Experiment Station, Purdue University . 887
88 8
Xanthine Oxidase Activity in Serum
Vol . 19, No . 6
able half and half (H/H) (commonly used ae a coffee creamer) on %0 activity in rat serum. Ia the process of investigating this phenomenon, a modified spectrophotometric method for datermiaiag RO activity in rat serum was developed . It was ascertained that several %0 assay methods were available but those methods warn either too involved, required elaborate analytical equipment and reagents or have employed uncommon substrates and end products . The current method being reported was found to be convenient sad reliable for determination of serum RO activity . Methode Mal~ rats (3lrague-Dawley strain) weighing 140-180 g were used in this study. Rats were allowed Purina chow ad libitum sad they were fasted for 12 hours prior to giving the treatments . The rate were gives a single doss consisting of 6 ml of test materials via a plastic stomach tube . The treatments consisted of : a) saline (0 .85X), b) saline supplemented with 0 .16 unite per ml of %0 (EC 1.2 .3 .2), (Sigma Chemical Co .), c) H/H and d) H/H supplemented with 0 .16 units of %0 per ml . The mutates contained 0 .5 ml of Tween 20 per 66 ml of test material sad were The H/H was a paeteurizad and homogenized mixed for 2 min in a blaring blandor. commercial preparation consisting of approximately 12X butter fat . The H/H The ezperiment consisted of 9 contained approximately 0.04 unite of RO per ml . rate/dose-time treatment for a total of 144 rats . One unit of anzyma activity is defined as the amount of enzyme required to oxidize 1 .0 pmole of hypoxanthine to uric acid per minute at 25°C . Rats were aaesthetizad with ether and blood was removed from the heart with a syringe and needle at 0, 2, 4 and 6 hours after intubation of the traatBlood was immersed in an ice bath, allowed to clot and centrifuged at meats . 4°C for 25 minutes at 5000 z g. The serum was removed and analyzed for xanthine ozidase activity is duplicate. Samples ware stored at 4°C sad analyzed Assays revealed that RO activity was not altered during the within 3 days . storage period although activity decrnasad for samples etorad longer than 4-5 The measurement of %0 activity in serum was a modification of the method days . A double beam racordiag spectrophotometer described by Avis et al . (8,9) . The reference cuvette contained 0 .4 ml (Beckman, Model 25) at 297 nm was used . Tha test of serum and 1 .6 mT of 0.05 M sodium pyrophosphate buffer, pH 8.3 . solution contained 0.4 ml of serum, 1.0 ml pf pyrophosphate buffer and 0.6 ml Thn assay medium was mixed and abeorbaaca readings of 150 pM hypozanthina . takes immediately. At 15 and 30 miauteB after addition of hypozanthine The difference between absorbance abeorbance measurements ware recorded . measuremante at 30 and 15 minutes (pA) was used to calculate %0 activity . The formula used for calculating %0 activity in serum was : %0 activity (unit/m~ serum protein) ~ A z 1000/15 min a 1.22 z 10 x 14 mg protein/ml serum. The molar axtiaction coafficieat of uric acid was 1 .22 z 104 . The protein content of rat serum ~!as determined by Lowery method (13) to have a mean coaThe reported method was compared with the ceatratioa of 14 mg/ml of serum. :auorometric assay tschniqua described by Hainiag sad Legaa (14) using 2-amino4-hydroayptnridine (AHP) as substrate . Reeulta FIG . 1 shows the rate of oxidation of hypozaathine to uric acid catalyzed by R0 . The reaction rata was considered linear from fifteen to thirty minutes.
Vol . 19, No . 6
Ranthiae Ozidasa Activity is Serum
88 9
a.~
w
xo
w
MINUTES
w
so
FIG . 1 Ozidation of hypozaathine catalysed by %0 and measured by absorbaace as a function of time . The incubation mixture contained 0 .6 ml of 150~u?f hypoaaathine, and 1 .4 ml of pyrophosphate buffer, pH 8 .3 . The reaction miztura contained 6 .4 a 10 - unite of %0 par ml . FIG . 2 shove the absorbance spectra for uric acid with absorption mazimum at 289 nm . In serum, the absorption maxima was altered shaving a bathochromic shift of 8 ~ . The addition of uric acid to the serum samples did not change the absorbance ~n=i,m ~. The curves demonstrate that the rata of uric acid formation ie proportional to %0 activity .
FIG . 2 Abaorbaace spectra for uric acid ( 0 .04 pmole uric acid/ml sad - - - 0 .02,umole uric acid/ml) ; for ( . . . . .) uric acid produced in reaction medium ; and for (---) 0 .0125~umole uric acid/ml added to reaction medium (0 .6 ml 150 pM hypozaathine ; 1 .0 ml 0 .05 M pyrophosphate buffer, pH 8 .3 ; sad 0 .4 ml serum) . FIG . 3 shows the time-treatmeat response of %0 activity in serum after giving rats a dose of the test materials . The data for saline sad saline plus %0 were pooled because aeseatially no differaacee were dieceraed between these traatmaats . The %0 activity for the saline treatments shaved a slight elevation from 0 time to 6 hours after administration . The RO activity in serum of animals receiving the H/H increased markedly at 2 hours sad then decreased aignificaatly . The H/H supplemented with %0 demonstrated a TMs=i+m++~ %0 activity in serum at 4 hours and then declined to a value similar to that of the H/H .
89 0
Vol . 19, No . 6
Ranthina Oxidase Activity in Serum
FIG . 3 Effect of H/H, H/H + RO and saline on the activity of RO in rat serum . Saline (D---o), H/H (~-+d) and H/H + RO ( o o ) . The composition of the reaction miztures is given under Methods . The use of analysis of variance demonstrated that a significant differeaca in RO activity (P
