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Effects of ascorbic acid and inflammation on ceruloplasmin activity levels in guinea pigs
NUTRITION RESEARCH, Vol. 6, pp. 1009-1012, 1986 0271-5317/86 $3.00 + .00 Printed in the USA. Copyright (c) 1986 Pergamon Journals Ltd. All rights reserved.
EFFECTS OF ASCORBIC ACID AND INFLAMMATION ON CERULOPLASMIN ACTIVITY LEVELS IN GUINEA PIGS Robert A. DiSilvestro, Ph.D. Department of Foods and Nutrition, Purdue University West Lafayette, IN 47907
ABST~CT
The possibility that ascorblc acid exerts a postabsorptlve effect on copper metabolism in guinea pigs was examined. A pair of ascorbic acid injections (20 mg/kg, ip, 18 h apart) caused a small but statistically significant increase in oxldase activity levels of ceruloplasmin, a serum copper protein. In contrast, dietary ascorbic acid deprivation for 14 days did not prevent increases in ceruloplasmin activity levels caused by turpentlne-lnduced inflammation. The ascorbic acid deprivation did reduce weight gain and adrenal ascorblc acid contents but serum ascorbic acid concentrations were not significantly lowered. In summary, ascorblc acid can produce postabsorptive changes in copper metabolism but adequate intake of the vitamin is not necessary for Inflammation-lnduced elevations of ceruloplasmin activity levels. Keywords:
Ceruloplasmin, Ascorblc Acid, Inflammation I~TRODUCTION
Ceruloplasmln (Cp) has been proposed to transport serum copper as well as function enzymatlcally (I). Activity levels of this protein rise during a variety of pathological and nonpathologlcal stress states in humans and experimental animals ( i ) . The mechanisms behind these changes remain largely uncharacterlzed but increased Cp protein synthesis is suspected. The copper needed for the increased Cp could originate from copper storage proteins such as metallothioneln which is found in several tissues (i). A decrease in the amount of copper bound to renal metallothlonein precedes an increase in rat serum Cp levels following injection with Interleukln-l, a hormone released by phagocytes in response to stress (2,3). Some evidence suggests that ascorbic acid (AA) promotes release of copper from metallothloneln resulting in increased copper incorporation into Cp. For example, copper release from metallothioneln in vitro is enhanced by ascorblc acid (4). In addition, AA injections in chicks produce a strong rise in Cp activity levels (5). Furthermore, AA administration raises Cp levels in a child with Menke's Syndrome, a disease characterized by low amounts of copper bound to Cp but high amounts to metallothionein (6). Thus, copper redistribution in vivo could involve some action of AA in certain tissues. On the other hand, AA does not seem to be required for maintaining normal Cp levels. AA deficiency in guinea pigs actually increases Cp activity concentrations (7). Presumably, this occurs because dietary AA can antagonize
1009
1010
R.A. DiSILVESTRO
copper absorption, at least in rats (8). The present studies examine the possibility that AA deficiency in guinea pigs reduces inflammatlon-induced increases in Cp activity levels. NETIIODS
Female, Hartley guinea pigs weighing 250-299 g were obtained frem Harlan Sprague Dawley, Inc. The AA deficient diet was purchased from U.S. Biochemicals with or without supplementation with I ppm AA. This diet contains 20% skim milk, 40% rolled oats, 15% bran, 10% vitamin-free casein, 8% alfalfa, 5% vegetable oil, 1% Ca carbonate, 0.5% magnesium sulfate, and 0.5% NaCI. Animals were fed the diet ad libltum for 14 days. Inflammation was induced by injecting 0.I ml of turpentine Im in the leg on day 12. AA in~ections (20 mg/kg in saline) were given ip 18 h apart on days 13 and 14 with the animals sacrificed 7 h after the second injection. All guinea pigs were killed in mid afternoon to avoid possible circadian variations in Cp activity levels. The animals were anesthetized with ketamine (30 mg/kg, im), bled by heart puncture and sacrificed by severing the diaphram. Cp activity was measured by oxidation of p-phenylenedlamine (PPD) as described by Rice (9). AA was measured in adrenals and serum by the method of Roe and Kuether (i0). Adrenals were analyzed as 3 pools of tissues from 3 animals per pool. Serum copper was evaluated by atomic absorbance spectrometry on serum diluted i:i with deionlzed water. ~SULTS AA injectlons in guinea pigs on AA supplemented diets produced a statistically significant 30% mean increase in Cp oxidase activity levels compared to noninjected animals (TABLE i). Total serum copper was elevated, but not significantly. AA deficient guinea pigs, compared to the AA supplemented group, showed low body weight and adrenal AA contents but no significant difference in serum AA (TABLE 2). In agreement with previous work (7) AA deficiency significantly increased serum copper and Cp oxidase activity concentrations (TABLE 3). Turpentine-induced inflammation raised both these parameters to about the same levels in AA deficient and AA adequate guinea pigs (TABLE 3). TABLE 1
Serum Copper and Cp Activity i n AA Injected Guinea Pigs. Serum Copper g/dl
Cp Activity units*/dl
AAAdequate - AA Injections
77 + 16
63 + 19
+AA
86 + 18
82 + 13"*
Injection
All values are means + SD (n=9) *Units represent AA ~'0 per 15 mln due to oxidation of PPD **P <.05 (Student t T e s ~ nm AA injected twice 18 h apart (20 mg AA/kg) animals killed 7 h after second injection
ASCORBIC ACID AND CERULOPLASMIN
T~LE
1011
2
Influence of Diet on AA Status Assessors. Body Weight g
Serum AA g/ml
Adrenal AA g/g
AA Adequate
349 + 21
5.2 + 0.6
i073 + 91
AA Deficlent
308 + 27*
4.5 + 0.7
205 +
8**
All values represent the means obtained from 9 animals + SD. *p ~05 compared to AA adequate. **p ~001 compared to AA adequate. TABLE 3
Effect of AA Status and Inflammation on Serum Copper and Cp Oxldase
Serum Copper ug/dl
Cp Oxldase Unlts/dl
AA Adequate + Turpentine
84 + 19 173 ~ 44**
67 + 19 210 ~ 42**
AA Deflclent + Turpentine
105 + 9* 196 ~ 25**
91 + 15" 216 ~ 52**
All values are means + SD. Animals were injected 48 h before sacrifice. *P <.05 compared to AAAdequate, no injection **P ~001 compared to AAAdequate, no injection DISCUSSIOH
This study demonstrates that AA injections can raise Cp activity levels in guinea pigs but that inflammatlon-induced increases in Cp activities can occur despite dietary AA restriction. The percent increase in Cp activity concentrations caused by AA injections (TABLE I) was much lower than that produced in chicks (5). This difference might arise simply because chicks have very low Cp activity levels before injection (5). Alternatively, AA injections might raise tissue AA contents to higher values in chicks than in guinea pigs since the former synthesizes AA endogenously. Both the AA and turpentine injections raised Cp oxldase activities to a greater extent than they did serum copper (TABLES I and 3), A similar pattern was obtained by Wirth et al when comparing colon cancer patients to healthy controls (Ii). This pattern could result from changes in nonCp serum copper disproportionate to changes in Cp bound copper. The amount of serum copper accounted for by the nonCp fraction is unknown in guinea pigs but could be close to 40% of the total copper in human serum (II). It is also possible that the specific oxidase activity per Cp bound copper atom rises above normal in certain circumstances (ii).
1012
R.A. DiSILVESTRO
Body AA contents can be depleted beyond that found here (TABLE 2) by a longer feeding of a different AA deficient diet (7). However, this approach also produces increases in serum copper and Cp activity that are more than I0 fold greater than the increases found in the present study (7). Such high serum copper and Cp values before inflammation would make it difficult to evaluate any effects of AA deficiency on inflammation-induced elevation in Cp levels. The present study does not rule out the possibility that AA deficiency reduces Cp elevations caused by situations other than inflammation. ACKNOWLEDGEMENTS
The author wishes to recognize the fine technical assistance of Deanna Case, Mary Frahm, Renee Massad, Lynn Yoder and Laura Hess. This work was supported in part by the Showalter Endowment Fund. REFERENCES
i.
Cousins RJ. Absorption, transport and hepatic metabolism of copper and zinc - special reference to metallothionein and ceruloplasmin. Physiol. Rev. 1985; 65:238-309.
2.
Pekareck R, Wannemacher R, Powanda M, Abeles F, Mosher R, Dinterman R, Beisel, W. Further evidence that leukocytic endogenous mediator (LEM) is not endotoxin. Life Sci. 1974; 14:1765-1776.
3.
DiSilvestro RA, Cousins RJ. Mediation of endotoxin-induced changes in zinc metabolism in rats. Am. J. Physiol. 1984; 247:E436-E441.
4.
Evans GW, Majors PF, Cornatzer WE. Ascorbic acid interactions with metallothionein. Biochem. Biophys. Res. Comm. 1970; 41:1244-1247.
5.
DiSilvestro RA, Harris ED. A postabsorptive effect of L-ascorbic acid on copper metabolism in chicks. J. Nutr. 1981; 111:1964-1968.
6.
Ueki Y, Narazakl O, Hanai T. Menkes disease: Is vitamin C treatment effective? Brain Dev. 1985; 7:519-522.
7.
Hitierp Y. Repercussions de la carence en acide ascorbique sur la ceruloplasmine et le cuivre tissulaire. Int. J. Vit. Nutr. Res. 1975; 46:48-57.
8.
Campen DV, Gross E. Influence of ascorbic acid on the absorption of copper by rats. J. Nutr. 1968; 95:617-622.
9.
Rice, E.W. Standardization of ceruloplasmin activity in terms of international enzyme units. Anal. Biochem. 1962; 3:452-456.
I0.
Roe, J.H., Kuether, C.A. The determination of ascorbic acid in whole blood and urine through the 2,4-dinitrophenylhydrazine derivative of ascorbic acid. J. Biol. Chem. 1943; 147:399-402.
ii.
Wirth PL, Linder MC. Distribution of copper among components of human serum. JNCI. 1985; 75:277-284.
Accepted
for p u b l i c a t i o n
July
12,
1986
EFFECTS OF ASCORBIC ACID AND INFLAMMATION ON CERULOPLASMIN ACTIVITY LEVELS IN GUINEA PIGS Robert A. DiSilvestro, Ph.D. Department of Foods and Nutrition, Purdue University West Lafayette, IN 47907
ABST~CT
The possibility that ascorblc acid exerts a postabsorptlve effect on copper metabolism in guinea pigs was examined. A pair of ascorbic acid injections (20 mg/kg, ip, 18 h apart) caused a small but statistically significant increase in oxldase activity levels of ceruloplasmin, a serum copper protein. In contrast, dietary ascorbic acid deprivation for 14 days did not prevent increases in ceruloplasmin activity levels caused by turpentlne-lnduced inflammation. The ascorbic acid deprivation did reduce weight gain and adrenal ascorblc acid contents but serum ascorbic acid concentrations were not significantly lowered. In summary, ascorblc acid can produce postabsorptive changes in copper metabolism but adequate intake of the vitamin is not necessary for Inflammation-lnduced elevations of ceruloplasmin activity levels. Keywords:
Ceruloplasmin, Ascorblc Acid, Inflammation I~TRODUCTION
Ceruloplasmln (Cp) has been proposed to transport serum copper as well as function enzymatlcally (I). Activity levels of this protein rise during a variety of pathological and nonpathologlcal stress states in humans and experimental animals ( i ) . The mechanisms behind these changes remain largely uncharacterlzed but increased Cp protein synthesis is suspected. The copper needed for the increased Cp could originate from copper storage proteins such as metallothioneln which is found in several tissues (i). A decrease in the amount of copper bound to renal metallothlonein precedes an increase in rat serum Cp levels following injection with Interleukln-l, a hormone released by phagocytes in response to stress (2,3). Some evidence suggests that ascorbic acid (AA) promotes release of copper from metallothloneln resulting in increased copper incorporation into Cp. For example, copper release from metallothioneln in vitro is enhanced by ascorblc acid (4). In addition, AA injections in chicks produce a strong rise in Cp activity levels (5). Furthermore, AA administration raises Cp levels in a child with Menke's Syndrome, a disease characterized by low amounts of copper bound to Cp but high amounts to metallothionein (6). Thus, copper redistribution in vivo could involve some action of AA in certain tissues. On the other hand, AA does not seem to be required for maintaining normal Cp levels. AA deficiency in guinea pigs actually increases Cp activity concentrations (7). Presumably, this occurs because dietary AA can antagonize
1009
1010
R.A. DiSILVESTRO
copper absorption, at least in rats (8). The present studies examine the possibility that AA deficiency in guinea pigs reduces inflammatlon-induced increases in Cp activity levels. NETIIODS
Female, Hartley guinea pigs weighing 250-299 g were obtained frem Harlan Sprague Dawley, Inc. The AA deficient diet was purchased from U.S. Biochemicals with or without supplementation with I ppm AA. This diet contains 20% skim milk, 40% rolled oats, 15% bran, 10% vitamin-free casein, 8% alfalfa, 5% vegetable oil, 1% Ca carbonate, 0.5% magnesium sulfate, and 0.5% NaCI. Animals were fed the diet ad libltum for 14 days. Inflammation was induced by injecting 0.I ml of turpentine Im in the leg on day 12. AA in~ections (20 mg/kg in saline) were given ip 18 h apart on days 13 and 14 with the animals sacrificed 7 h after the second injection. All guinea pigs were killed in mid afternoon to avoid possible circadian variations in Cp activity levels. The animals were anesthetized with ketamine (30 mg/kg, im), bled by heart puncture and sacrificed by severing the diaphram. Cp activity was measured by oxidation of p-phenylenedlamine (PPD) as described by Rice (9). AA was measured in adrenals and serum by the method of Roe and Kuether (i0). Adrenals were analyzed as 3 pools of tissues from 3 animals per pool. Serum copper was evaluated by atomic absorbance spectrometry on serum diluted i:i with deionlzed water. ~SULTS AA injectlons in guinea pigs on AA supplemented diets produced a statistically significant 30% mean increase in Cp oxidase activity levels compared to noninjected animals (TABLE i). Total serum copper was elevated, but not significantly. AA deficient guinea pigs, compared to the AA supplemented group, showed low body weight and adrenal AA contents but no significant difference in serum AA (TABLE 2). In agreement with previous work (7) AA deficiency significantly increased serum copper and Cp oxidase activity concentrations (TABLE 3). Turpentine-induced inflammation raised both these parameters to about the same levels in AA deficient and AA adequate guinea pigs (TABLE 3). TABLE 1
Serum Copper and Cp Activity i n AA Injected Guinea Pigs. Serum Copper g/dl
Cp Activity units*/dl
AAAdequate - AA Injections
77 + 16
63 + 19
+AA
86 + 18
82 + 13"*
Injection
All values are means + SD (n=9) *Units represent AA ~'0 per 15 mln due to oxidation of PPD **P <.05 (Student t T e s ~ nm AA injected twice 18 h apart (20 mg AA/kg) animals killed 7 h after second injection
ASCORBIC ACID AND CERULOPLASMIN
T~LE
1011
2
Influence of Diet on AA Status Assessors. Body Weight g
Serum AA g/ml
Adrenal AA g/g
AA Adequate
349 + 21
5.2 + 0.6
i073 + 91
AA Deficlent
308 + 27*
4.5 + 0.7
205 +
8**
All values represent the means obtained from 9 animals + SD. *p ~05 compared to AA adequate. **p ~001 compared to AA adequate. TABLE 3
Effect of AA Status and Inflammation on Serum Copper and Cp Oxldase
Serum Copper ug/dl
Cp Oxldase Unlts/dl
AA Adequate + Turpentine
84 + 19 173 ~ 44**
67 + 19 210 ~ 42**
AA Deflclent + Turpentine
105 + 9* 196 ~ 25**
91 + 15" 216 ~ 52**
All values are means + SD. Animals were injected 48 h before sacrifice. *P <.05 compared to AAAdequate, no injection **P ~001 compared to AAAdequate, no injection DISCUSSIOH
This study demonstrates that AA injections can raise Cp activity levels in guinea pigs but that inflammatlon-induced increases in Cp activities can occur despite dietary AA restriction. The percent increase in Cp activity concentrations caused by AA injections (TABLE I) was much lower than that produced in chicks (5). This difference might arise simply because chicks have very low Cp activity levels before injection (5). Alternatively, AA injections might raise tissue AA contents to higher values in chicks than in guinea pigs since the former synthesizes AA endogenously. Both the AA and turpentine injections raised Cp oxldase activities to a greater extent than they did serum copper (TABLES I and 3), A similar pattern was obtained by Wirth et al when comparing colon cancer patients to healthy controls (Ii). This pattern could result from changes in nonCp serum copper disproportionate to changes in Cp bound copper. The amount of serum copper accounted for by the nonCp fraction is unknown in guinea pigs but could be close to 40% of the total copper in human serum (II). It is also possible that the specific oxidase activity per Cp bound copper atom rises above normal in certain circumstances (ii).
1012
R.A. DiSILVESTRO
Body AA contents can be depleted beyond that found here (TABLE 2) by a longer feeding of a different AA deficient diet (7). However, this approach also produces increases in serum copper and Cp activity that are more than I0 fold greater than the increases found in the present study (7). Such high serum copper and Cp values before inflammation would make it difficult to evaluate any effects of AA deficiency on inflammation-induced elevation in Cp levels. The present study does not rule out the possibility that AA deficiency reduces Cp elevations caused by situations other than inflammation. ACKNOWLEDGEMENTS
The author wishes to recognize the fine technical assistance of Deanna Case, Mary Frahm, Renee Massad, Lynn Yoder and Laura Hess. This work was supported in part by the Showalter Endowment Fund. REFERENCES
i.
Cousins RJ. Absorption, transport and hepatic metabolism of copper and zinc - special reference to metallothionein and ceruloplasmin. Physiol. Rev. 1985; 65:238-309.
2.
Pekareck R, Wannemacher R, Powanda M, Abeles F, Mosher R, Dinterman R, Beisel, W. Further evidence that leukocytic endogenous mediator (LEM) is not endotoxin. Life Sci. 1974; 14:1765-1776.
3.
DiSilvestro RA, Cousins RJ. Mediation of endotoxin-induced changes in zinc metabolism in rats. Am. J. Physiol. 1984; 247:E436-E441.
4.
Evans GW, Majors PF, Cornatzer WE. Ascorbic acid interactions with metallothionein. Biochem. Biophys. Res. Comm. 1970; 41:1244-1247.
5.
DiSilvestro RA, Harris ED. A postabsorptive effect of L-ascorbic acid on copper metabolism in chicks. J. Nutr. 1981; 111:1964-1968.
6.
Ueki Y, Narazakl O, Hanai T. Menkes disease: Is vitamin C treatment effective? Brain Dev. 1985; 7:519-522.
7.
Hitierp Y. Repercussions de la carence en acide ascorbique sur la ceruloplasmine et le cuivre tissulaire. Int. J. Vit. Nutr. Res. 1975; 46:48-57.
8.
Campen DV, Gross E. Influence of ascorbic acid on the absorption of copper by rats. J. Nutr. 1968; 95:617-622.
9.
Rice, E.W. Standardization of ceruloplasmin activity in terms of international enzyme units. Anal. Biochem. 1962; 3:452-456.
I0.
Roe, J.H., Kuether, C.A. The determination of ascorbic acid in whole blood and urine through the 2,4-dinitrophenylhydrazine derivative of ascorbic acid. J. Biol. Chem. 1943; 147:399-402.
ii.
Wirth PL, Linder MC. Distribution of copper among components of human serum. JNCI. 1985; 75:277-284.
Accepted
for p u b l i c a t i o n
July
12,
1986