9 199l by The Humana Press, Inc. All rights of any nature, whatsoever, reserved. 0163-4984/91/2903--0193 $02.00
Inhibition of Copper Absorption by Zinc Effect of Histidine RAUL A. WAPNIR* AND CHERYL BALKMAN
Department of Pediatrics, North S h o r e University Hospitai~Comell University Medical College,/Vlanhasset, NY 11030 Received May 22, 1990; Accepted August 4, 1990
ABSTRACT Copper and zinc interact at the intestinal mucosal level, affecting copper absorption. Amino acids, such as histidine, may affect the absorption of these two elements by chelating these cations. The two mechanisms could have additive potential. This possibility was investigated using a duodenal-jejunal single-pass perfusion procedure in anesthetized rats. Copper absorption and tissue retention from solutions containing 0.1 mM copper were determined in the presence of either no zinc or equimolar zinc, or at a zinc/copper ratio of 10/1, either without histidine or with histidine at a 10/1 or 20/1 ratio to copper. Copper removal from the intestinal lumen was decreased by zinc, and further reduced by increasing concentrations of histidine. There was a greater accumulation of copper in the small intestine, reaching a maximum with a 10-fold excess of histidine. With zinc at a 10/1 ratio to copper, the addition of a 10- or 20-fold molar excess of histidine further decreased the net uptake of copper from the perfusate while greater copper accumulation in the tissue occurred. Histidine thus enhances the inhibitory effects of zinc on copper absorption, suggesting the application of convergent mechanisms for diminishing copper uptake. This could be relevant for the treatment of Wilson's disease. Index Entries: Copper absorption; zinc; histidine; intestinal absorption; amino acids. *Author to whom all correspondence and reprint requests should be addressed.
Biological Trace Element Research
] 93
VoL 29, 1991
194
Wapnir and Balkman
INTRODUCTION It has been well established that copper (Cu) and zinc (Zn) interact during intestinal absorption, resulting in a decreased uptake of Cu (1,2). This antagonism has also been d e m o n s t r a t e d in foods (3), although they can be viewed as one of the several interactions between elements of similar chemical characteristics reflected in physiological and nutritional manifestations (4). It is now accepted that C u - Z n interplay is mediated through intracellular metallothioneins (MT), which are inducible by certain divalent cations, for w h o m they have the greatest affinity (5,6). The competition between Zn and Cu may also take place at the brush border during the luminal phase of absorption, but the effects at this site have not been well characterized. The interaction between these two elements has therapeutic implications, since it has been demonstrated that the oral administration of Zn salts can reduce Cu accumulation in the liver in patients with Wilson's disease (8-11). Certain amino acids, such as histidine (His), exhibit high affinity for tile transition elements Cu and Zn. They form complexes with both metals and have very low dissociation constants (12). Free amino acids may be present in the intestinal lumen during the absorptive process, as they become released from dietary protein, or from proteolysis of mucosal cells shed in the course of normal tissue breakdown. Previous studies have indicated that His may regulate tile serum concentration of Zn and Cu (13), and also decrease the rate of Cu uptake from the intestinal lumen (14). The objective of this study was to compare Zn and Cu interactions during the luminal phase of Cu absorption, in terms of its rate of removal from the intestinal lumen and its mucosal retention, at two Zn/Cu ratios, 1/1 and 10/1, as well as the effects of His on Cu absorption and retention in the presence of Zn at various His/Zn and His/Cu ratios. We hypothesized that the reduction of Cu absorption attributable to Z n - C u interaction could be modified by the amino acid and result in a further decrease in Cu absorption.
MATERIALS AND METHODS The intestinal absorption of Cu was assessed by a perfusion procedure, in situ, on male rats. The animals weighed 125-150 g (Crl:[WI]BR, Charles River Laboratories, Kingston, NY). Rats were fed a standard laboratory feed (Purina Lab Chow, Ralston Purina Company, St. Louis, MO). They were tested after at least 48 h of acclimatization in the institutional animal-housing facility. Following an overnight fast, anesthesia was induced with u r e t h a n e (1.3 g/kg ip). A 20 to 30-cm segment, distal to the ligament of Treitz, was cannulated. The solutions to be tested w e r e p u m p e d through at 0.18-0.20 mL/min (Harvard peristaltic Biological Trace Element Research
Vol. 29, 1991
Inhibition of Copper Absorption by Zinc
195
pumps, model 1203, Boston, MA), for a 1-h equilibration period and, subsequently, for 2 h, during which fractions were collected at 15-min intervals and individually analyzed. Cu, as the nitrate, was perfused at 0.1 mM in solutions containing 140 mM NaCl, buffered at pH 6.8 with 5 mM PIPES-KOH. P1PES was selected as a buffer because of its lack of chelating properties toward Cu and Zn (15). Zn, as the sulfate, and His, as the base (Sigma Chemical Company, St. Louis, MO), were added, as indicated, prior to pH adjustment. At the end of the procedure, the perfused s e g m e n t was excised; slit open; rinsed in isotonic, ice-cold NaC1; placed in a test tube; and treated with 2 mL of 15.5M nitric acid. The covered tubes were occasionally shaken and were left at room temperature until total dissolution of the tissue, which took 7-10 d. Preliminary experiments s h o w e d that washing with isotonic NaCI sufficed to remove adsorbed Cu from the perfusates. Treatment with a chelating agent, such as EDTA, did not alter tissue metal levels, as determined in separate test perfusions with or without Cu. Cu concentration was determined by atomic-absorption spectroscopy (Varian SpectrAA 10), using as reference certified external standards (Fisher Scientific Co., Fair Lawn, NJ). The rates of Cu extraction from the perfusate were corrected for fluid exchanges using the nonabsorbable marker phenol red (20 mg/L). The dye was assayed spectrophotometrically (16), and the values obtained also served to calculate net water absorption. The unidirectional lumen-to-serosa water influx was d e t e r m i n e d by addition of 5 I~Ci 3H~O and gravimetric m e a s u r e m e n t of water disappearance from the perfused segment. The methodology has been previously described in detail (17,18). Differences between means were assessed by one-way analysis of variance and subsequent Dunnett's test for the determination of significance by comparison with the reference solution, which contained Cu only (19).
RESULTS Cu Absorption and Retention in the Presence of Isomolar Concentrations of Zn Plus Variable Amounts of His We d e t e r m i n e d the rates of Cu absorption by the rat small intestine in the presence or absence of isomolar (0.1 mM) Zn, either with no a d d e d His or with 0.1, 1.0, or 2.0 mM of the amino acid (Fig. 1). The addition of equimolar Zn reduced Cu uptake rates compared to the absorption rates in the absence of the putative inhibitor. The introduction of His in the perfusate further decreased Cu removal from the lumen: w h e n 2.0 mM His and 0.1 m M Zn were present, Cu absorption was one-third that observed in the absence of the competitors. Biological Trace Element Research
Vol. 29, 1991
Wapnir and Balkman
196
Fig. l. Cu absorption rates obtained in tile absence and in tile presence of equimolar concentrations of Zn, with or without the addition of His. (N) = N u m b e r of rats in each group. The significance of tile differences was assessed against perfusions with no Zn or His (open bar). Tile Dunnett's test was applied for all statistical analyses 1791.
Fig. 2. Cu retention by the perfused segment of rat intestine prepared as indicated in the text. The asterisks indicate significant differences against the results obtained in the absence of Zn and His. (N) = N u m b e r of rats in each group.
Biological Trace Element Research
VoL 29, ! 991
Inhibition of Copper Absorption by Zinc
197
Table l Net Water Absorption and Lumen-to-Serosa (lm~) Fluid Inflow in Intestinal Segments Exposed to Equimolar Concentrations of Copper and Zinc, With or Without I-lisddine Additions Cu
Zn
Net Water Absorption His
ImM]
0.1
0
~L/min x g
[~L/min x cm]
2.890 • 1.040 (21) {0.174 + 0.0571
0.I
0,I
0
0.i
0.i
2.816 • 1.471 (20)
0.I
1.0
0.407 • 1.616 (12)
0.I
2.0
-3.190 • 1.673 (i0)"
Data are means + SEM.
72.128 ! 5.930 [3.957 !0.163]"
-2.370 ! 2.747 (8) [0.116 t 0.157]
72.730 • 2.140 {3.868 i 0.061]"
[-0.180 t 0.090]"
0.i
81.935 • 3.220 I4.124 t 0.083]
[0.036 t 0.089}
0.I
82.492 i 3.240 [4.517 i 0.152l
[0.174 + 0.075]
0.I
Inflow
~L/min x g
[~L/min x cm]
0
Lumen-to-Muqosa
(8)
70.897 ~ 3.874 4.026 i 0.131
(N) = Number of rats.
"P
Inhibition of Copper Absorption by Zinc Effect of Histidine RAUL A. WAPNIR* AND CHERYL BALKMAN
Department of Pediatrics, North S h o r e University Hospitai~Comell University Medical College,/Vlanhasset, NY 11030 Received May 22, 1990; Accepted August 4, 1990
ABSTRACT Copper and zinc interact at the intestinal mucosal level, affecting copper absorption. Amino acids, such as histidine, may affect the absorption of these two elements by chelating these cations. The two mechanisms could have additive potential. This possibility was investigated using a duodenal-jejunal single-pass perfusion procedure in anesthetized rats. Copper absorption and tissue retention from solutions containing 0.1 mM copper were determined in the presence of either no zinc or equimolar zinc, or at a zinc/copper ratio of 10/1, either without histidine or with histidine at a 10/1 or 20/1 ratio to copper. Copper removal from the intestinal lumen was decreased by zinc, and further reduced by increasing concentrations of histidine. There was a greater accumulation of copper in the small intestine, reaching a maximum with a 10-fold excess of histidine. With zinc at a 10/1 ratio to copper, the addition of a 10- or 20-fold molar excess of histidine further decreased the net uptake of copper from the perfusate while greater copper accumulation in the tissue occurred. Histidine thus enhances the inhibitory effects of zinc on copper absorption, suggesting the application of convergent mechanisms for diminishing copper uptake. This could be relevant for the treatment of Wilson's disease. Index Entries: Copper absorption; zinc; histidine; intestinal absorption; amino acids. *Author to whom all correspondence and reprint requests should be addressed.
Biological Trace Element Research
] 93
VoL 29, 1991
194
Wapnir and Balkman
INTRODUCTION It has been well established that copper (Cu) and zinc (Zn) interact during intestinal absorption, resulting in a decreased uptake of Cu (1,2). This antagonism has also been d e m o n s t r a t e d in foods (3), although they can be viewed as one of the several interactions between elements of similar chemical characteristics reflected in physiological and nutritional manifestations (4). It is now accepted that C u - Z n interplay is mediated through intracellular metallothioneins (MT), which are inducible by certain divalent cations, for w h o m they have the greatest affinity (5,6). The competition between Zn and Cu may also take place at the brush border during the luminal phase of absorption, but the effects at this site have not been well characterized. The interaction between these two elements has therapeutic implications, since it has been demonstrated that the oral administration of Zn salts can reduce Cu accumulation in the liver in patients with Wilson's disease (8-11). Certain amino acids, such as histidine (His), exhibit high affinity for tile transition elements Cu and Zn. They form complexes with both metals and have very low dissociation constants (12). Free amino acids may be present in the intestinal lumen during the absorptive process, as they become released from dietary protein, or from proteolysis of mucosal cells shed in the course of normal tissue breakdown. Previous studies have indicated that His may regulate tile serum concentration of Zn and Cu (13), and also decrease the rate of Cu uptake from the intestinal lumen (14). The objective of this study was to compare Zn and Cu interactions during the luminal phase of Cu absorption, in terms of its rate of removal from the intestinal lumen and its mucosal retention, at two Zn/Cu ratios, 1/1 and 10/1, as well as the effects of His on Cu absorption and retention in the presence of Zn at various His/Zn and His/Cu ratios. We hypothesized that the reduction of Cu absorption attributable to Z n - C u interaction could be modified by the amino acid and result in a further decrease in Cu absorption.
MATERIALS AND METHODS The intestinal absorption of Cu was assessed by a perfusion procedure, in situ, on male rats. The animals weighed 125-150 g (Crl:[WI]BR, Charles River Laboratories, Kingston, NY). Rats were fed a standard laboratory feed (Purina Lab Chow, Ralston Purina Company, St. Louis, MO). They were tested after at least 48 h of acclimatization in the institutional animal-housing facility. Following an overnight fast, anesthesia was induced with u r e t h a n e (1.3 g/kg ip). A 20 to 30-cm segment, distal to the ligament of Treitz, was cannulated. The solutions to be tested w e r e p u m p e d through at 0.18-0.20 mL/min (Harvard peristaltic Biological Trace Element Research
Vol. 29, 1991
Inhibition of Copper Absorption by Zinc
195
pumps, model 1203, Boston, MA), for a 1-h equilibration period and, subsequently, for 2 h, during which fractions were collected at 15-min intervals and individually analyzed. Cu, as the nitrate, was perfused at 0.1 mM in solutions containing 140 mM NaCl, buffered at pH 6.8 with 5 mM PIPES-KOH. P1PES was selected as a buffer because of its lack of chelating properties toward Cu and Zn (15). Zn, as the sulfate, and His, as the base (Sigma Chemical Company, St. Louis, MO), were added, as indicated, prior to pH adjustment. At the end of the procedure, the perfused s e g m e n t was excised; slit open; rinsed in isotonic, ice-cold NaC1; placed in a test tube; and treated with 2 mL of 15.5M nitric acid. The covered tubes were occasionally shaken and were left at room temperature until total dissolution of the tissue, which took 7-10 d. Preliminary experiments s h o w e d that washing with isotonic NaCI sufficed to remove adsorbed Cu from the perfusates. Treatment with a chelating agent, such as EDTA, did not alter tissue metal levels, as determined in separate test perfusions with or without Cu. Cu concentration was determined by atomic-absorption spectroscopy (Varian SpectrAA 10), using as reference certified external standards (Fisher Scientific Co., Fair Lawn, NJ). The rates of Cu extraction from the perfusate were corrected for fluid exchanges using the nonabsorbable marker phenol red (20 mg/L). The dye was assayed spectrophotometrically (16), and the values obtained also served to calculate net water absorption. The unidirectional lumen-to-serosa water influx was d e t e r m i n e d by addition of 5 I~Ci 3H~O and gravimetric m e a s u r e m e n t of water disappearance from the perfused segment. The methodology has been previously described in detail (17,18). Differences between means were assessed by one-way analysis of variance and subsequent Dunnett's test for the determination of significance by comparison with the reference solution, which contained Cu only (19).
RESULTS Cu Absorption and Retention in the Presence of Isomolar Concentrations of Zn Plus Variable Amounts of His We d e t e r m i n e d the rates of Cu absorption by the rat small intestine in the presence or absence of isomolar (0.1 mM) Zn, either with no a d d e d His or with 0.1, 1.0, or 2.0 mM of the amino acid (Fig. 1). The addition of equimolar Zn reduced Cu uptake rates compared to the absorption rates in the absence of the putative inhibitor. The introduction of His in the perfusate further decreased Cu removal from the lumen: w h e n 2.0 mM His and 0.1 m M Zn were present, Cu absorption was one-third that observed in the absence of the competitors. Biological Trace Element Research
Vol. 29, 1991
Wapnir and Balkman
196
Fig. l. Cu absorption rates obtained in tile absence and in tile presence of equimolar concentrations of Zn, with or without the addition of His. (N) = N u m b e r of rats in each group. The significance of tile differences was assessed against perfusions with no Zn or His (open bar). Tile Dunnett's test was applied for all statistical analyses 1791.
Fig. 2. Cu retention by the perfused segment of rat intestine prepared as indicated in the text. The asterisks indicate significant differences against the results obtained in the absence of Zn and His. (N) = N u m b e r of rats in each group.
Biological Trace Element Research
VoL 29, ! 991
Inhibition of Copper Absorption by Zinc
197
Table l Net Water Absorption and Lumen-to-Serosa (lm~) Fluid Inflow in Intestinal Segments Exposed to Equimolar Concentrations of Copper and Zinc, With or Without I-lisddine Additions Cu
Zn
Net Water Absorption His
ImM]
0.1
0
~L/min x g
[~L/min x cm]
2.890 • 1.040 (21) {0.174 + 0.0571
0.I
0,I
0
0.i
0.i
2.816 • 1.471 (20)
0.I
1.0
0.407 • 1.616 (12)
0.I
2.0
-3.190 • 1.673 (i0)"
Data are means + SEM.
72.128 ! 5.930 [3.957 !0.163]"
-2.370 ! 2.747 (8) [0.116 t 0.157]
72.730 • 2.140 {3.868 i 0.061]"
[-0.180 t 0.090]"
0.i
81.935 • 3.220 I4.124 t 0.083]
[0.036 t 0.089}
0.I
82.492 i 3.240 [4.517 i 0.152l
[0.174 + 0.075]
0.I
Inflow
~L/min x g
[~L/min x cm]
0
Lumen-to-Muqosa
(8)
70.897 ~ 3.874 4.026 i 0.131
(N) = Number of rats.
"P
