@Copyright 1992 by The Humana Press, Inc. All rights of any nature, whatsoever, reserved. 0163-4984/92/3502~)185 $03.00
Tissues and Organs as Indicators of Intestinal Absorption of Minerals and Trace Elements, Evaluated in Rats TORBEN LARSEN *'~ AND BRI'I-FMARIE SANDSTROM 2
1National Institute of Animal Science, Animal Physiology and Biochemistry, Foulum, P.O. Box 39, 8830 Tjele, Denmark; and 2Research Department of Human Nutrition, Royal Veterinary and Agricultural University, Rolighedsvej 25, DK-1958 Frederiksberg C, Denmark Received January 23, 1992; Accepted February 17, 1992
ABSTRACT Tissue and organ deposition and blood parameters were evaluated as indices of mineral and trace element absorption in rats. The absorption of elements was quantified in relation to nitrogen retention, i.e., considering the weight gain and new tissue synthesis. A rapeseed meal diet was supplied with three levels of calcium, two levels of zinc, and two levels of copper in a factorial design. In general, an increase in dietary mineral content increased the relative absorption, which in turn, increased the tissue deposition progressively. Striated muscle, however, did not respond to either an increased calcium or zinc supply. Furthermore, an increased calcium absorption caused a depression of the fractional phosphorus and magnesium content of femur bones. The copper content of the kidneys and the heart muscle was directly proportional to the amount of absorbed zinc and iron, respectively. The iron content of tissues was, in general, inversely proportional to zinc absorption and showed a tendency to be directly proportional to copper absorption. The zinc level in tissues was, in a similar way, inversely correlated to measured calcium absorption. In conclusion, interactions between ele*Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
1 85
Vol. 35, 1992
186
Larsen and Sandstr6m
ments do not only affect the intestinal element absorption, but also the distribution of already absorbed elements in tissues and organs. Index Entries: Minerals; trace elements; tissue deposition; organ storage; translocation; mineral interaction.
INTRODUCTION Several organs and tissues as well as blood parameters have been proposed as indicators of mineral and trace element availability and absorption. Most studies considering these aspects evaluate tissue deposition in relation to dietary intake, not considering the possibility of mineral interactions or mutual antagonism between elements after the absorptive phase. Reports on the deposition of elements in tissue as a consequence of the actual absorbed amounts of the elements are less common. The absorption/retention of nitrogen is considered a positive event in animal production, since it represents a deposition and an increment in lean body mass. Calcium and phosphorus retention is usually regarded similarly, since it creates materials for a stronger bone structure. The deposition of other elements, such as zinc in bone, copper in liver, or iron in kidney, is, beyond a certain limit, not necessarily a positive event, but reflects absorption of an available element. The present article is part of a more comprehensive study with the intention of: 1. Elucidating the bioavailability of the intrinsic trace elements and minerals in defatted rapeseeds; 2. Observing the effect of an increasing calcium supplementation to an intrinsic, high-phytate level in diets; and 3. Studying the interactive effect of dietary addition of inorganic calcium, copper, and zinc on intestinal element absorption, and the consecutive internal distribution and deposition in organs and tissues. In a previous paper (1), the interactions between elements in the absorptive phase were examined in relation to the dietary content of minerals. It was found that dietary addition of calcium and zinc markedly affected the feed intake, the body growth, and the feed conversion of the weight gain. The fractional nitrogen absorption and retention was influenced. Similarly, the calcium, magnesium, and phosphorus absorption/ retention was affected by dietary supply of calcium and zinc, and the iron and zinc absorption was affected, both considered in proportion to the nitrogen retention (growth) and as fractions of ingested material. In general, interactions between elements were obvious in the absorptive phase, but the effect continued beyond absorption per se. The purpose of the present study was to follow the deposition and distribution of the minerals and trace elements as a function of the actually absorbed amounts of elements. This article reports on the appearance of selected minerals and trace elements in the bone, liver, kidney, spleen, heart muscle, striated muscle, and intestinal tissue of rats. Analyses of wholeBiological Trace Element Research
I1"ol.35, 1992
Tissue and Organ Deposition of Trace Elements
187
blood hemoglobin, plasma zinc, plasma alkaline phosphatase, and plasma ceruloplasmin activity are also reported.
EXPERIMENTAL
Diets and Dietary Composition The experimental diet was composed of rapeseed meal and potato starch with minor additions of other ingredients; the composition is presented in Table 1. Sodium chloride was added to the diet in moderate amounts, as the only basic mineral. The basic (intrinsic) mineral content is presented in Table 1. Origin of minerals was predominantly from the rapeseed meal, since the starch used was extremely low in minerals. Phytate and degradation products from it also originated from the rapeseeds.
Experimental Design The experiment was planned and carried out as a complete, balanced, factorial design. Dietary modifications of calcium, copper, and zinc constituted the differences in experimental treatments. Calcium (as carbonate) was added to the diets at three levels (g/kg DM): 0, 3.7, and 7.4. Copper was supplied as sulfate at two levels: 0 and 20 mg/kg DM; zinc was correspondingly supplied as sulfate at two levels: 0 and 40 mg/ kg DM. The dietary design is outlined in Table 2, where the analyzed levels of calcium, copper, and zinc are given. The 12 experimental diets (3*2*2) were randomized on 12 rats; this procedure was repeated six times, separated by time. The entire experiment consequently involved 72 animals.
Animals, Feeding and Dissection Experimental animals were male, Wistar rats. Initial body wt was on average 70.0 g. The feeding period extended over 28 d. The rats were fed ad libitum and had free access to redistilled drinking water. Housing was individual in Plexiglas TM cages, with mesh bottoms of stainless-steel; environmental conditions (temperature, humidity, and light-dark periods) were kept constant. On day 28 of the experiment, the animals were anesthetized in an atmosphere of carbon dioxide and killed by a subsequent puncture of the heart with a stainless-steel needle. Blood was drawn from the heart and stored in heparinized tubes, placed in icecooled medium. Heart muscle, kidneys, liver, and spleen were liberated from loose fat and connective tissue, and rinsed in ice-cooled, saline water. The thigh muscles from both legs were cut loose, and both femur bones were liberated from tissue and cartilage. The entire small intestine was dissectBiological Trace Element Research
Vol. 35, 1992
188
Larsen a n d S a n d s t r 6 m
Table 1 Basic Composition, g/kg Dry Matter (Left) and Chemical Constituents (Right) of the Unsupplemented Experimental Diet, per kg Dry Matter
Rapeseed meal Saccharose Cellulose powder Soya oil Potato starch a Vitamin mixture b Sodium chloride~
400.0 52.0 30.3 30.3 470.4 16.0 1.0
Calcium, g Phosphorus, g Magnesium, g Iron, mg Zinc, mg Manganese, mg Copper, mg IP6_3, mmol ~
3.29 6.31 1.91 99 25 23 3.4 22.7
aAutoclaved. bAccording to reference (32). CAnalytical grade, Merck, Darmstadt, Germany. aInositol phosphates containing three to six phosphates per inositol residue.
Table 2" Diet code 1 2 3 4 5 6 7 8 9 10 11 12
Dietary inclusion I' Ca0 . " . Cal . " . Ca2 . " .
(3290) . .
Cu0 (3.4) . Cul (23)
. . (6400) . .
.
. . (10100) . .
.
.
.
Cu0 (3.4) . Cul (23) Cu0 (3.4) . Cul (23)
.
Zn;~ (25) Znl (63) Zn0 (25) Znl (63) Zn0 (25) Znl (63) Zn0 (25) Znj (63) Zn0 (25) Znl (63) Zn0 (25) Znl (63)
"The experimental design was balanced and factorial (3*2*2)regarding dietary supply of calcium, copper, and zinc. Ca~, Cu~ and Zn~ designates the native, intrinsic level of minerals. Ca1 and Ca, indicate calcium supply of 3.7 and 7.4 g (as carbonate), respectively, and Cu~ and Zn 1supply of 20 mg copper and 40 mg of zinc (as sulfate) per kg diet DM, respectively. Figures in parenthesis are the actual measured amounts of mineral, mg/kg DM feed I'Supplied minerals were analytical grade, Merck, Darmstadt, Germany. ed a n d f l u s h e d w i t h 10 m L of a cold saline solution. All collected o r g a n s a n d tissues w e r e f r o z e n a n d lyophilized before f u r t h e r analyses. Materials in direct contact w i t h the animals a n d diets, a n d tools u s e d in dissection w e r e w a s h e d in dilute nitric acid a n d t h o r o u g h l y r i n s e d w i t h distilled w a t e r before use.
Analyb'cal Methods The collected o r g a n s a n d tissues w e r e d r y a s h e d in crucibles of silica (525~ for 6 h). A s h e s w e r e dissolved in h o t dilute hydrochloric/nitric Biological Trace Element Research
Vol. 35, 1992
Tissue and Organ Deposition of Trace Elements
189
acid (proanalysis/Suprapur). Measurements of calcium, magnesium, zinc, iron, copper, and manganese were analyzed by atomic absorption spectrophotometry (AAS) (PU 9400 X, Philips Scientific, Cambridge, UK). Phosphorus was quantified colorimetrically by the vanadomolybdate procedure (2). Blood hemoglobin was determined according to the cyanmethemoglobin method. Zinc content in plasma was measured directly after dilution by AAS. Activity of alkaline phosphatase in plasma was quantified using p-nitrophenyl phosphate as substrate (3). Ceruloplasmin activity was assessed as p-phenylenediamine oxidase (4). Inositol phosphates (phytate) were determined by an HPLC method (5).
Presentation of Results The results are presented as probabilities that absorbed minerals/ trace elements have affected the element content of the organs/tissues, either positively or negatively. Since the animals were fed ad libitum, the absorption of iron, zinc, copper and calcium was furthermore expressed in relation to the nitrogen retention. The justification for this expression is that nitrogen retention is correlated to the feed intake and growth, and that the mineral demand is proportional to lean body increase. Statistical Methods The experiment was performed as a complete, balanced, randomized, factorial trial, carried out in block sections. The material was subjected to statistical analysis of multiple regression using the following model (6): Y = [3o + ~lXl -}- ~2X2 q- ~3X3 -}- ~4X4 q- e where the f3s are the unknown parameters, the es are statistical errors, Y is the response, and X1, X2, X3, and X4 are the predictors, i.e., absorbed iron, copper, zinc, and calcium, respectively. In statistical analysis where a factor did not appear significant, the factor was eventually excluded from the statistical model, the effect was accordingly included in the error (root MSE) of the statistical test. Mean values of responses presented are from all animals in trial, irrespective of dietary treatment; the standard deviations are accordingly presented as a measure of variation within all animals. Regression analyses were performed on individual figures. All statistical analyses were performed with the SAS system (7).
RESULTS Calcium, Phosphorus, and Magnesium The fractional amount of inorganic material of femur bones was on average 60.1% of bone DM. The amount of absorbed minerals did not Biological Trace Element Research
Vol. 35, 1992
190
Larsen a n d S a n d s t r 6 m 12
185
SE = 2.0
O) "~" E9 180
p
Tissues and Organs as Indicators of Intestinal Absorption of Minerals and Trace Elements, Evaluated in Rats TORBEN LARSEN *'~ AND BRI'I-FMARIE SANDSTROM 2
1National Institute of Animal Science, Animal Physiology and Biochemistry, Foulum, P.O. Box 39, 8830 Tjele, Denmark; and 2Research Department of Human Nutrition, Royal Veterinary and Agricultural University, Rolighedsvej 25, DK-1958 Frederiksberg C, Denmark Received January 23, 1992; Accepted February 17, 1992
ABSTRACT Tissue and organ deposition and blood parameters were evaluated as indices of mineral and trace element absorption in rats. The absorption of elements was quantified in relation to nitrogen retention, i.e., considering the weight gain and new tissue synthesis. A rapeseed meal diet was supplied with three levels of calcium, two levels of zinc, and two levels of copper in a factorial design. In general, an increase in dietary mineral content increased the relative absorption, which in turn, increased the tissue deposition progressively. Striated muscle, however, did not respond to either an increased calcium or zinc supply. Furthermore, an increased calcium absorption caused a depression of the fractional phosphorus and magnesium content of femur bones. The copper content of the kidneys and the heart muscle was directly proportional to the amount of absorbed zinc and iron, respectively. The iron content of tissues was, in general, inversely proportional to zinc absorption and showed a tendency to be directly proportional to copper absorption. The zinc level in tissues was, in a similar way, inversely correlated to measured calcium absorption. In conclusion, interactions between ele*Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
1 85
Vol. 35, 1992
186
Larsen and Sandstr6m
ments do not only affect the intestinal element absorption, but also the distribution of already absorbed elements in tissues and organs. Index Entries: Minerals; trace elements; tissue deposition; organ storage; translocation; mineral interaction.
INTRODUCTION Several organs and tissues as well as blood parameters have been proposed as indicators of mineral and trace element availability and absorption. Most studies considering these aspects evaluate tissue deposition in relation to dietary intake, not considering the possibility of mineral interactions or mutual antagonism between elements after the absorptive phase. Reports on the deposition of elements in tissue as a consequence of the actual absorbed amounts of the elements are less common. The absorption/retention of nitrogen is considered a positive event in animal production, since it represents a deposition and an increment in lean body mass. Calcium and phosphorus retention is usually regarded similarly, since it creates materials for a stronger bone structure. The deposition of other elements, such as zinc in bone, copper in liver, or iron in kidney, is, beyond a certain limit, not necessarily a positive event, but reflects absorption of an available element. The present article is part of a more comprehensive study with the intention of: 1. Elucidating the bioavailability of the intrinsic trace elements and minerals in defatted rapeseeds; 2. Observing the effect of an increasing calcium supplementation to an intrinsic, high-phytate level in diets; and 3. Studying the interactive effect of dietary addition of inorganic calcium, copper, and zinc on intestinal element absorption, and the consecutive internal distribution and deposition in organs and tissues. In a previous paper (1), the interactions between elements in the absorptive phase were examined in relation to the dietary content of minerals. It was found that dietary addition of calcium and zinc markedly affected the feed intake, the body growth, and the feed conversion of the weight gain. The fractional nitrogen absorption and retention was influenced. Similarly, the calcium, magnesium, and phosphorus absorption/ retention was affected by dietary supply of calcium and zinc, and the iron and zinc absorption was affected, both considered in proportion to the nitrogen retention (growth) and as fractions of ingested material. In general, interactions between elements were obvious in the absorptive phase, but the effect continued beyond absorption per se. The purpose of the present study was to follow the deposition and distribution of the minerals and trace elements as a function of the actually absorbed amounts of elements. This article reports on the appearance of selected minerals and trace elements in the bone, liver, kidney, spleen, heart muscle, striated muscle, and intestinal tissue of rats. Analyses of wholeBiological Trace Element Research
I1"ol.35, 1992
Tissue and Organ Deposition of Trace Elements
187
blood hemoglobin, plasma zinc, plasma alkaline phosphatase, and plasma ceruloplasmin activity are also reported.
EXPERIMENTAL
Diets and Dietary Composition The experimental diet was composed of rapeseed meal and potato starch with minor additions of other ingredients; the composition is presented in Table 1. Sodium chloride was added to the diet in moderate amounts, as the only basic mineral. The basic (intrinsic) mineral content is presented in Table 1. Origin of minerals was predominantly from the rapeseed meal, since the starch used was extremely low in minerals. Phytate and degradation products from it also originated from the rapeseeds.
Experimental Design The experiment was planned and carried out as a complete, balanced, factorial design. Dietary modifications of calcium, copper, and zinc constituted the differences in experimental treatments. Calcium (as carbonate) was added to the diets at three levels (g/kg DM): 0, 3.7, and 7.4. Copper was supplied as sulfate at two levels: 0 and 20 mg/kg DM; zinc was correspondingly supplied as sulfate at two levels: 0 and 40 mg/ kg DM. The dietary design is outlined in Table 2, where the analyzed levels of calcium, copper, and zinc are given. The 12 experimental diets (3*2*2) were randomized on 12 rats; this procedure was repeated six times, separated by time. The entire experiment consequently involved 72 animals.
Animals, Feeding and Dissection Experimental animals were male, Wistar rats. Initial body wt was on average 70.0 g. The feeding period extended over 28 d. The rats were fed ad libitum and had free access to redistilled drinking water. Housing was individual in Plexiglas TM cages, with mesh bottoms of stainless-steel; environmental conditions (temperature, humidity, and light-dark periods) were kept constant. On day 28 of the experiment, the animals were anesthetized in an atmosphere of carbon dioxide and killed by a subsequent puncture of the heart with a stainless-steel needle. Blood was drawn from the heart and stored in heparinized tubes, placed in icecooled medium. Heart muscle, kidneys, liver, and spleen were liberated from loose fat and connective tissue, and rinsed in ice-cooled, saline water. The thigh muscles from both legs were cut loose, and both femur bones were liberated from tissue and cartilage. The entire small intestine was dissectBiological Trace Element Research
Vol. 35, 1992
188
Larsen a n d S a n d s t r 6 m
Table 1 Basic Composition, g/kg Dry Matter (Left) and Chemical Constituents (Right) of the Unsupplemented Experimental Diet, per kg Dry Matter
Rapeseed meal Saccharose Cellulose powder Soya oil Potato starch a Vitamin mixture b Sodium chloride~
400.0 52.0 30.3 30.3 470.4 16.0 1.0
Calcium, g Phosphorus, g Magnesium, g Iron, mg Zinc, mg Manganese, mg Copper, mg IP6_3, mmol ~
3.29 6.31 1.91 99 25 23 3.4 22.7
aAutoclaved. bAccording to reference (32). CAnalytical grade, Merck, Darmstadt, Germany. aInositol phosphates containing three to six phosphates per inositol residue.
Table 2" Diet code 1 2 3 4 5 6 7 8 9 10 11 12
Dietary inclusion I' Ca0 . " . Cal . " . Ca2 . " .
(3290) . .
Cu0 (3.4) . Cul (23)
. . (6400) . .
.
. . (10100) . .
.
.
.
Cu0 (3.4) . Cul (23) Cu0 (3.4) . Cul (23)
.
Zn;~ (25) Znl (63) Zn0 (25) Znl (63) Zn0 (25) Znl (63) Zn0 (25) Znj (63) Zn0 (25) Znl (63) Zn0 (25) Znl (63)
"The experimental design was balanced and factorial (3*2*2)regarding dietary supply of calcium, copper, and zinc. Ca~, Cu~ and Zn~ designates the native, intrinsic level of minerals. Ca1 and Ca, indicate calcium supply of 3.7 and 7.4 g (as carbonate), respectively, and Cu~ and Zn 1supply of 20 mg copper and 40 mg of zinc (as sulfate) per kg diet DM, respectively. Figures in parenthesis are the actual measured amounts of mineral, mg/kg DM feed I'Supplied minerals were analytical grade, Merck, Darmstadt, Germany. ed a n d f l u s h e d w i t h 10 m L of a cold saline solution. All collected o r g a n s a n d tissues w e r e f r o z e n a n d lyophilized before f u r t h e r analyses. Materials in direct contact w i t h the animals a n d diets, a n d tools u s e d in dissection w e r e w a s h e d in dilute nitric acid a n d t h o r o u g h l y r i n s e d w i t h distilled w a t e r before use.
Analyb'cal Methods The collected o r g a n s a n d tissues w e r e d r y a s h e d in crucibles of silica (525~ for 6 h). A s h e s w e r e dissolved in h o t dilute hydrochloric/nitric Biological Trace Element Research
Vol. 35, 1992
Tissue and Organ Deposition of Trace Elements
189
acid (proanalysis/Suprapur). Measurements of calcium, magnesium, zinc, iron, copper, and manganese were analyzed by atomic absorption spectrophotometry (AAS) (PU 9400 X, Philips Scientific, Cambridge, UK). Phosphorus was quantified colorimetrically by the vanadomolybdate procedure (2). Blood hemoglobin was determined according to the cyanmethemoglobin method. Zinc content in plasma was measured directly after dilution by AAS. Activity of alkaline phosphatase in plasma was quantified using p-nitrophenyl phosphate as substrate (3). Ceruloplasmin activity was assessed as p-phenylenediamine oxidase (4). Inositol phosphates (phytate) were determined by an HPLC method (5).
Presentation of Results The results are presented as probabilities that absorbed minerals/ trace elements have affected the element content of the organs/tissues, either positively or negatively. Since the animals were fed ad libitum, the absorption of iron, zinc, copper and calcium was furthermore expressed in relation to the nitrogen retention. The justification for this expression is that nitrogen retention is correlated to the feed intake and growth, and that the mineral demand is proportional to lean body increase. Statistical Methods The experiment was performed as a complete, balanced, randomized, factorial trial, carried out in block sections. The material was subjected to statistical analysis of multiple regression using the following model (6): Y = [3o + ~lXl -}- ~2X2 q- ~3X3 -}- ~4X4 q- e where the f3s are the unknown parameters, the es are statistical errors, Y is the response, and X1, X2, X3, and X4 are the predictors, i.e., absorbed iron, copper, zinc, and calcium, respectively. In statistical analysis where a factor did not appear significant, the factor was eventually excluded from the statistical model, the effect was accordingly included in the error (root MSE) of the statistical test. Mean values of responses presented are from all animals in trial, irrespective of dietary treatment; the standard deviations are accordingly presented as a measure of variation within all animals. Regression analyses were performed on individual figures. All statistical analyses were performed with the SAS system (7).
RESULTS Calcium, Phosphorus, and Magnesium The fractional amount of inorganic material of femur bones was on average 60.1% of bone DM. The amount of absorbed minerals did not Biological Trace Element Research
Vol. 35, 1992
190
Larsen a n d S a n d s t r 6 m 12
185
SE = 2.0
O) "~" E9 180
p
