- Email: [email protected]
Silicon facilitation of copper utilization in the rat
Silicon facilitation of copper utilization in the rat Royce J. Emerick and Henry Kayongo-Male Departments of Chemistry and Biology, South Dakota State University, Brookings, SD, USA
An eight-week, 2 × 4 factorial rat experiment using two levels o f dietary copper and four levels o f dietary silicon was conducted to further delineate a previously observed silicon-copper interaction in which silicon appears to mimic copper in its effect on the composition o f the aorta. Dietary copper concentrations were 1.4 (deficient) and 5.4 (adequate) mg/kg diet, and silicon concentrations were 5, 135, 270, and 540 mg/kg diet. Compared with the lowest level o f silicon and copper, weight gains were 15.5% higher f o r rats f e d 540 mg silicon/kg diet and 14.3% higher f o r those f e d 5.4 mg eopper/kg diet. The growth-promoting effects o f silicon and copper were additive. Evidence that silicon elevated the copper status o f copper-deficient rats includes an increase in packed-cell volume by 540 mg silicon/kg diet in the otherwise packed-cell volume-depressed, copper-deficient rats, accompanied by a trend toward higher hemoglobin values and lower relative heart weights. In the copper-adequate rats, evidence that 540 mg silicon/kg diet elevated their copper status includes a two-fold increase in the blood-plasma copper concentration, a three-fold increase in ceruloplasmin activity, and an increase in cardiac, renal, and hepatic copper concentrations. In addition, 540 mg silicon/kg diet resulted in higher aortic dry mass and aortic elastin content in both copper-deficient and copper-adequate rats. While dietary silicon concentrations o f 135, 270, and 540 mg/kg diet were all effective in increasing aortic elastin in the copper-adequate rats, only 540 mg silicon/kg diet increased aortic elastin in the copperdeficient rats. These data indicate that some o f the metabolic" effects attributed to silicon may be manifested through a silicon-facilitated increase in copper utilization.
Keywords: silicon; copper; connective tissue; aorta; elastin; collagen Introduction Essential functions of silicon identified in animals appear to be largely associated with formation of connective tissue with specific i n v o l v e m e n t in collagen synthesis. This subject has been reviewed by Carlisle. 1.2 In addition to silicon, c o p p e r is also involved in connective tissue synthesis. C o p p e r is a c o m p o n e n t of lysyl oxidase, the e n z y m e active in the formation of cross-linkages b e t w e e n polypeptide chains of the connective tissue proteins, collagen and elastin. 3 Aortic a n e u r y s m s and ruptures occurring in several species 4-6 often a p p e a r to be a c o n s e q u e n c e of defective elastin
Address reprint requests to Dr. R.J. Emerick, Olson Biochemistry Laboratories, Animal Science Bldg., South Dakota State University, Brookings, SD 57007, USA. Published with approval of the Director of the South Dakota Agricultural Experiment Station as publication number 2486 of the journal series. Received February 16, 1990; accepted April 11, 1990. ©
1990 Butterworth-Heinemann
synthesis. Recently, E m e r i c k and K a y o n g o - M a l e 7 reported that 270 mg silicon per kg of diet appeared to mimic the gross effects of c o p p e r in promoting higher aortic elastin levels in copper-deficient rats. This study was conducted to further delineate the silicon-copper interrelationship, and data are presented in support of the hypothesis that silicon facilitates c o p p e r utilization in the rat.
Materials and methods One-hundred-twenty male S p r a g u e - D a w l e y rats (SASCO, O m a h a , N E , USA) were used in a 2 × 4 factorial experiment involving diets providing two concentrations of copper, 1.4 and 5.4 mg per kg of diet, and four concentrations of silicon, 5, 135, 270, and 540 mg per kg of diet. The rats initially averaged 54 _+ 5.6 g in weight and were randomly allotted across the eight treatments providing 15 rats per treatment. T h e y were housed individually in hanging stainlesssteel cages with wire m e s h floors in a r o o m maintained at 23 to 25°C. A 12-hour light-to-dark cycle was main-
J. Nutr. Biochem., 1990, vol. 1, September
487
Research Communications Table 1
Basal diet composition
Ingredient Dextrose, anhydrous Egg albumin, spray dried a Corn oil Salt mixtureb Vitamin mixturec
Amount (%) 69.0 20.0 5.0 40 20
a Spray dried egg albumin was autoclaved at 120°C for 1 hour and dried at 120°C for 1 hour for denaturation. b The percent composition of salt mixture (salt mixture P-H with a reduced amount of CuSO4 and an increased amount of ZnCl2, ICN Nutritional Biochemicals, Cleveland, OH, USA); dipotassium phosphate, 32.2; calcium carbonate, 30.0; sodium chloride, 16.7; magnesium sulfate (hydrate), 10.2; manganese sulfate, 0.51; dibasic calcium phosphate dihydrate, 7.5; ferric citrate, 2.75; potassium iodide, 0.08; copper sulfate pentahydrate, 0.0098; zinc chloride, 0.0624; cobalt chloride, 0.005. c Vitamin mixture composition in grams/kilogram triturated in dextrose (vitamin diet fortification mixture, ICN Nutritional Biochemicals): vitamin A concentrate (500,000 IU/g), 1.8; vitamin D concentrate (850,000 IU/g), 0.125; alpha-tocopherol (250 IU/g), 22.0; ascorbic acid, 45; inositol, 5.0; choline chloride, 75; menadione, 2.25; p-aminobenzoic acid, 5.0; niacin, 4.25; riboflavin, 1.0; pyridoxine hydrochloride, 1.0; thiamine hydrochloride, 1.0; calcium pantothenate, 3.0; biotin, 0.020; folic acid, 0.090; and vitamin B-12, 0.00135
tained. Attempts were made to minimize environmental silica with a thorough initial cleaning of the room and related equipment followed by a weekly flushing of the painted concrete floor. The diets were provided ad libitum in glass jars, and distilled water was provided in polyethylene bottles fitted with stainless-steel drinking-tips. The basal diet is shown in Table 1. Before use, the spray-dried egg albumin was autoclaved at 120°C for 1 hour and dried at 120°C for 1 hour to prevent avidin interference with biotin absorption. Additional copper, above the 0.4 mg/kg diet inherent in the non-mineral diet ingredients, was provided as CuSO4"5H2O. The additional silicon was provided by tetraethylorthosilicate (J. T. Baker Chemical Co., Phillipsburg, N J, USA). Tetraethylorthosilicate is rapidly hydrolyzed in HCI equivalent to that of the gastric stomach, and it has been used extensively in a rat model at concentrations providing up to 2,700 mg silicon/kg diet for the study of silica metabolism and silica urolithiasis. 7d2 Tha rats were weighed weekly during the 8-week experiment. Blood samples were obtained at termination by cardiac puncture following anesthetization with Halothane (Abbott Laboratories, Chicago, IL, USA). Blood was placed in heparinized tubes. Following the immediate determination of hemoglobin, as described below, and packed cell volume (PCV) (Autocrit Centrifuge, Clay Adams, Inc., New York, NY, USA), plasma was separated and stored at - 25°C until analyzed. While continuing under anesthetization, the rats were sacrificed by decapitation and the liver, kidneys, heart, femurs, the femoral-tibial (F-T)joints including the respective condyles, the patella and associated articular cartilage, and a sample of aorta were
488
J. Nutr. Biochem., 1990, vol. 1, September
removed, weighed and stored at - 25°C until analyzed. The sample of aorta represented - 3 cm of the descending thoracic aorta beginning immediately below the aortic arch. Concentrations of calcium and magnesium in plasma, and copper in nitric acid digests of all soft tissues were determined by flame atomic absorption spectrophotometry (Perkin-Elmer model 5000, Norwalk, CT, USA). Copper was determined in plasma by graphite furnace atomic absorption (Perkin-Elmer model 5000 equipped with model 500 heated graphite atomizer and autosampler) using diluted plasma containing 0.2% of concentrated nitric acid (Ultrex, J. T. Baker Chemical Co., Phillipsburg, N J, USA). Recovery of copper from spiked samples averaged 103% by this method. Plasma inorganic phosphorus was determined using the Fiske and Subbarow phosphomolybdate method. ~3 Silicon content of the basal diet was determined by graphite furnace atomic absorption spectrophotometry in an aqueous suspension prepared with a tissue homogenizer (Tekmar Co., Cincinnati, OH, USA). Atomic absorption conditions were the same as those described herein for plasma. Total hemoglobin was determined by the cyanomethemoglobin method (test kit: hemoglobin 525, Sigma Chemical Co., St. Louis, MO, USA). Ceruloplasmin activity was determined using o-dianisidine dihydrochloride substrate as described by others. 14 Silicon in blood plasma was determined by graphite furnace atomic absorption (Perkin-Elmer model 5000 equipped with model 500 heated graphite atomizer and autosampler) using nickel nitrate matrix modification as described by Emerick and Kayongo-Male.v The aortas were extracted for 20 minutes with chloroform/methanol (2:1 vol/vol), washed with ethanol and ether, and allowed to air dry. Aortic elastin was determined as the amount of the aorta remaining insoluble following treatment with 0.1 N NaOH at 98°C for 1 hour.~5 Collagen content of the aortas was calculated from hydroxyproline contained in an aliquot of the 0.1 N NaOH supernatant solution. Hydroxyproline was determined colorimetrically, 16 and was assumed to comprise 10% of the weight of collagen. 17 F-T joints were crushed and extracted with 2:1 vol/ vol chloroform/methanol. Elastin and collagen were determined as described for aortas except that elastin was corrected for bone ash following ashing at 500°C overnight. Elastin and collagen were calculated as a percentage of the dry, fat-free organic portion of the F-T joint. Femurs were extracted for 12 hours with ethanol followed by a 12-hour extraction with diethylether. After air-drying, they were ashed overnight at 500°C, and ash was calculated as percent of the dry, fat-free bone. Calcium, magnesium, and phosphorus were determined on the acid-soluble bone ash by the methods described for plasma. Statistical analyses were made by analysis of variance using a model consisting of the copper and silicon main effects and the two-way interaction. J8 Significant
Silicon-copper interaction: Emerick and Kayongo-Male differences between appropriate means were identified by the method of least significant differences protected by a significant F value.
Results
Body weights Body weights at 0, 4, and 8 weeks of the experiment are presented in Table 2. Weight differences related to treatment main effects were first observed at 4 weeks, and they increased in magnitude during the final 4 weeks of the 8-week study. Compared with the lowest treatment level of silicon and copper, weight gains at 8 weeks were 15.5% higher (P < 0.01) for rats fed 540 mg silicon/kg diet and 14.3% higher (P < 0.01) for those fed 5.4 mg copper/kg diet. The growthpromoting effects of 540 mg silicon and 5.4 mg copper/ kg diet were additive.
Blood and blood plasma Packed-cell volume and hemoglobin data are shown in Table 2, and the plasma data are shown in Table 3. Copper-deficient rats fed 540 mg silicon/kg diet had a higher average PCV (42.7 vs. 39.3%, P < 0.05) than those fed the low-silicon basal diet. Hemoglobin values followed the same trend as PCV, but the differences attributable to silicon were not significant (P > 0.05). Copper-adequate rats fed 540 mg silicon/kg diet had average plasma copper concentration that was twofold higher (0.51 vs. 0.28 mg copper/L; P < 0.01) than the value for rats fed the basal diet inherently containing only 5 mg silicon/kg diet. Plasma ceruloplasmin
activity was likewise three-fold higher (38.9 vs. 12.2 U/ L; P < 0.01) for copper-adequate rats fed 540 vs. 5 mg silicon/kg diet. Lower dietary silicon concentrations of 135 or 270 mg/kg had no apparent effect on plasma copper concentrations or ceruloplasmin activity. Furthermore, none of the dietary silicon additions had an effect on these components in the copper-deficient rats where average plasma copper levels were only 0.07 mg/L. Compared to rats fed no supplemental dietary silicon, plasma silicon concentrations were increased in copper-deficient rats fed 270 and 540 mg silicon/kg diet, and in copper-adequate rats fed 540 mg silicon/kg diet. Increases (P < 0.01) in plasma concentrations of calcium, magnesium, and phosphorus were associated with an increase in dietary copper. An increase (P < 0.05) in plasma calcium was also associated with the 540 mg silicon/kg diet in the copper-deficient rats and with 135 mg silicon/kg diet in the copper-adequate rats. Plasma phosphorus concentration was lowered by 540 mg silicon/kg diet in the copper-adequate rats. Dietary silicon had no effect (P > 0.05) on plasma magnesium.
Aorta Aorta data are shown in Table 4. Rats fed 540 mg silicon/kg diet, with either of the two levels of dietary copper, had higher (P < 0.01) average aortic mass (4.9 and 5.0 mg vs. 4.0 mg dry mass/cm length) when compared with those fed the corresponding low-silicon basal diet. Elastin content of the aorta was also increased (P < 0.01) by silicon. While dietary silicon concentrations of 135,270, and 540 mg/kg diet were all effective in increasing aortic elastin in the copper-
Table 2 Body weights, packed cell volume, and hemoglobin values of rats fed 4 levels of silicon with 2 levels of copper in the diet for 8 weeks a'b Dietary copper and silicon (mg/kg diet) Copper, 1.4 Silicon, 5 Silicon, 135 Silicon, 270 Silicon, 540 Copper, 5.4 Silicon, 5 Silicon, 135 Silicon, 270 Silicon, 540 SEM c ANOVA d Copper Silicon Interaction
Initial (g)
Body weights 4 weeks (g)
8 weeks (g)
Packed cell volume (%)
Hemoglobin (g/d0
56.9 56.9 57.3 57.1
208 210 209 220
267 a 280 a'b 263 a 300 b
39.3 a 36.7 a 38.3 a 42.7 b
12.9 12.0 12.4 13.7
56.1 58.3 56.8 57.9 1.5
213 a 210 a 215 a 232 b 4.7
296 a 304 a 304 a 333 b 7.2
44.6 44.7 45.5 44.3 1.2
14.7 14.6 14.9 14.6 0.36
NS NS NS
NS 0.002 NS
0.0001 0.0001 NS
0.0001 NS 0.04
0.0001 NS NS
a N - 15/treatment. b Mean values within a column and within the same copper treatment not sharing a common superscript symbol differ by the method of least significant differences protected by a significant F value (P < 0.05). ° Standard error of the mean calculated from the error mean square. d A 2 x 4 analysis of variance; numerical values are levels of probability for main effects and interaction, NS = not significant (P > 0.05).
J. Nutr. Biochem, 1990, vol. 1, September
489
Research Communications Table 3
Blood plasma concentrations of selected elements for rats fed 4 levels of silicon with 2 levels of copper in the diet for 8 weeks a'b
Dietary copper and silicon (mg/kg diet) Copper, 1.4 Silicon, 5 Silicon, 135 Silicon, 270 Silicon, 540 Copper, 5.4 Silicon, 5 Silicon, 135 Silicon, 270 Silicon, 540 SEM c ANOVA d Copper Silicon Interaction
Copper (mg/L)
Ceruloplasmin (U/L)
Silicon (mg/L)
Calcium (mg/L)
Magnesium (mg/L)
Phosphorus (mg/L)
0.09 0.06 0.06 0.07
0.7 1.0 0.8 0.8
0.27 a 0.27 a 0.36 b 0.39 b
103 a 105 ab 102 a 107 b
19.0 19.5 19.1 20.0
68 73 75 72
12.2 a 3.8 a 14.7 a 38.9 b 4.9
0.28 a 0.28 a 0.32 a,b 0.42 b 0.02
10P 112 b 110 ~'b 109 ~'b 1.4
21.0 22.0 21.8 20.3 0.45
81 a 78 a'b 84 ~ 73 b 2.5
NS 0.0001 NS
0.0001 0.02 NS
0.0001 NS NS
0.28 a 0.17 a 0.22 a 0.51 b 0.046 0.0001 0.001 0.004
0.0001 0.005 0.006
0.0001 0.04 NS
a N = 15/treatment. b Mean values within a column and within the same copper treatment not sharing a common superscript symbol differ by the method of least significant differences protected by a significant F value (P < 0.05). c Standard error of the mean calculated from the error mean square. d A 2 x 4 analysis of variance; numerical values are levels of probability for main effects and interaction, NS = not significant (P > 0.05).
Table 4.
Aortic and femoral-tibial joint characteristics of rats fed 4 levels of silicon with 2 levels of copper in the diet for 8 weeks a'b Aorta
Dietary copper and silicon (mg/kg diet) Copper, 1.4 Silicon, 5 Silicon, 135 Silicon, 270 Silicon, 540 Copper, 5.4 Silicon, 5 Silicon, 135 Silicon, 270 Silicon, 540 SEM c ANQVA ~ Copper Silicon Interaction
Mass (mg/cm)
Femoral-tibial joint
Elastin
Collagen
Elastin
Collagen (percent) (Dry, fat-free basis)
(percent) (Dry, fat-free basis)
4.0 a 4.2 ~ 4.2 a 4.9 b
40.8 a 42.2 ~b 42.4 a'b 44.1 b
39.9 38.9 39.2 38.3
15.7 15.0 14.9 14.2
40.4 39.1 41.0 39.8
4.0 a 4.2 a 4.3 a 5.0 b 0.20
42.6 a 46.5 b 46.9 b 46.2 b 1.0
40.4 40.4 41.0 39.5 095
16.5 16.8 17.5 17.0 0.73
36.0 38.2 36.3 37.0 1.0
NS 0.0001 NS
0.0001 0.005 NS
NS NS NS
0.0006 NS NS
0.0001 NS NS
a N = 15/treatment. b Mean values within a column and within the same copper treatment not sharing a common superscript symbol differ by the method of least significant differences protected by a significant F value (P < 0.05). c Standard error of the mean calculated from the error mean square. A 2 x 4 analysis of variance; numerical values are levels of probability for main effects and interaction, NS = not significant (P > 0.05).
adequate rats, only 540 mg silicon/kg diet increased aortic elastin in the copper-deficient rats. In these instances, aortic elastin values of the siliconsupplemented rats were 109% of those fed no added silicon. Aortic collagen percentages tended to be increased by copper (P = 0.08) but were unaffected by silicon (P > . 10).
490
J. Nutr. Biochem., 1990, vol. 1, September
Femur diaphysis, and F-T joint Average of the treatment means + SEM for femurdiaphysis ash (data not shown) was 61.0 _ 0.3% of the dry, fat-free bone, and the bone ash contained 35.4 _+ 0.2% calcium, 17.3 _+ 0.1% phosphorus, and 0.64 _+ 0.01% magnesium with no treatment differences.
Silicon-copper interaction: Emerick and Kayongo-Male Table 5 Heart weights, and heart, liver and kidney copper contents for rats fed 4 levels of silicon with 2 levels of copper in the diet for 8 weeks a'b
Dietary copper and silicon (mg/kg diet) Copper, 1.4 Silicon, 5 Silicon, 135 Silicon, 270 Silicon, 540 Copper, 5 4 Silicon, 5 Silicon, 135 Silicon, 270 Silicon, 540 SEM c ANOVA d Copper Silicon Interaction
Relative heart weights (g/lO0 g body wt)
Absolute heart weights (g)
0.43 a 0.44 a 0.43 a 0.39 b
1.13 1.21 1.11 1.14
0.36 a'b 0.36 a'b 0.37 a 0.33 b 0.12
1.06 1.09 1.14 1.10 0.033
13.1 a 14.5 ~'b 12.8 a 16.6 b 0.84
0.0001 0.003 NS
NS NS NS
0.0001 0.02 NS
Heart copper
7.9 6.9 7.9 8.6
Liver copper (mg/kg, dry basis)
Kidney copper
30 3.7 3.3 40
13.7 13.6 13.3 14.5
8.0 76 7.6 9.4 0.59
18.3 a 18.0 a 18.6 a 21.4 b O71
0.0001 NS NS
0.0001 0.008 NS
a N = 15/treatment b Mean values within a column and within the same copper treatment not sharing a common superscript symbol differ by the method of least significant differences protected by a significant F value (P < 0.05). c Standard error of the mean calculated from the error mean square. d A 2 x 4 analysis of variance; numerical values are levels of probability for main effects and interaction, NS = not significant (P > 0.05).
Elastin and collagen contents of the dry, fat-free, organic portion of the F-T joint are shown in Table 4. Compared to the copper-deficient treatment, adequate copper increased (P < 0.01) elastin and lowered (P 0.01) collagen concentrations in the fat-free organic portion of the F-T joint. The average percentage of elastin in the F-T joint for copper-adequate rats was 113.4% and collagen was 92.0% of the respective values for copper-deficient rats. Silicon had no effect on elastin or collagen contents of the F-T joint. Collagen was negatively correlated with elastin (r = -0.36, P < 0.01).
Heart, liver, and kidneys Heart, liver, and kidney data are presented in Table 5. Absolute heart weights (in grams) did not differ among treatments (P > 0.05). However, the average relative heart weight (g/100 g body weight) of all copperdeficient rats was 119% of the average of all copperadequate rats (P < 0.01). The highest level of silicon (540 mg/kg diet) appeared to prevent this cardiac hypertrophy relative to body weight in copper-deficient rats (P < 0.05) and tended (P = 0.10) to further reduce relative heart weights in copper-adequate rats. In the copper-adequate rats, the highest dietary silicon level, compared to no supplemental silicon, increased copper concentrations (dry basis) in the heart (16.6 vs. 13.1 mg copper/kg, P < 0.05), kidney (21.4 vs. 18.3 mg copper/kg, P < 0.01), and liver (9.4 vs. 8.0 mg copper/kg, P = 0.09).
Discussion The inclusion of 1.0 mg copper (from CuSO4 • 5HzO)/ kg diet to raise the copper concentration of the basal
diet to 1.4 mg/kg was done to avoid deaths that may have occurred with a more severe copper deficiency. On the other hand, 5.4 mg copper/kg diet used as the higher level is normally considered to be adequate. 19 Data obtained here indicate that 540 mg silicon/kg diet elevated the copper status of rats whether they were fed the copper-deficient basal diet (1.4 mg copper/kg diet) or the copper-adequate diet (5.4 mg copper/kg diet). Evidence that silicon raised the copper status of copper-deficient rats includes an increase in PCV to near normal in the otherwise PCV-depressed, copperdeficient rats, accompanied by a trend toward higher hemoglobin values. Furthermore, a lower average relative heart weight for the high-silicon rats is consistent with the physiologic effect of adequate copper. 9 In the copper-adequate rats, evidence that 540 mg silicon/kg diet elevated their copper status was more precise, and included a two-fold increase in the blood-plasma copper concentration, a three-fold increase in ceruloplasrain activity, and in increase in cardiac, renal, and hepatic copper concentrations. Independent increases in weight gain in response to silicon or copper supplementation, and their appearance of being additive, made it difficult to estimate how much, if any, of this function of silicon may have been manifested through an elevation of copper status. The effectiveness of 540 mg silicon/kg diet in promoting weight gain, and the ineffectiveness of lower levels for this purpose, is in accord with the earlier findings of Schwarz and Milne 2° that 500, but not 250, mg silicon/kg diet gave a growth response in rats. Another apparent independent effect of silicon is the increase in aortic dry mass (weight/length) associated with 540 mg silicon/kg of diet. That the greater aortic mass may be a component of the heavier body
J. Nutr. Biochem., 1990, vol. 1, September
491
Research Communications weights o f rats in this t r e a t m e n t c a n n o t be ruled out at this time. H o w e v e r , a n o t h e r aortic benefit attributable to increases in d i e t a r y silicon, the m a g n i t u d e o f w h i c h a p p e a r e d to be partially d e p e n d e n t u p o n the p r e s e n c e o f a potentially a d e q u a t e level o f dietary c o p p e r , inc l u d e d an i n c r e a s e in aortic elastin. While all dietary additions o f silicon (135, 270, and 540 mg silicon/kg diet) effected increases in aortic elastin in the c o p p e r a d e q u a t e rats, o n l y the highest level (540 mg/kg diet) o f silicon p r o d u c e d this r e s p o n s e in the copper-deficient rats. In a p r e v i o u s s t u d y b y E m e r i c k and K a y o n g o Male, 7 270 m g silicon/kg diet i n c r e a s e d aortic elastin in c o p p e r - d e f i c i e n t rats. In c o n t r a s t to the effect o f silicon and c o p p e r on aortic elastin, o n l y c o p p e r e f f e c t e d an increase in the p e r c e n t a g e elastin in the fat-free organic fraction o f the F - T joint. T h e p a r a d o x o f a c o p p e r - i n d u c e d increase in F - T joint elastin that did not r e s p o n d to the elevation in c o p p e r status i n d u c e d b y silicon s u p p l e m e n t a t i o n r e m a i n s u n e x p l a i n e d at this time. A c o n c o m i t a n t d e c r e a s e in collagen, a c c o m p a n y i n g the c o p p e r - i n d u c e d i n c r e a s e in elastin, resulted in a relatively c o n s t a n t p e r c e n t a g e for the c o m b i n a t i o n o f elastin and collagen in the fat-free organic fraction o f the F - T joint. T h e F - T joint included the f e m o r a l and tibial c o n d y l e s and the patella in addition to the associated articular cartilage. T h u s , the dry, fat-free F-T joint samples c o n t a i n e d - 62% b o n e as calculated f r o m the p e r c e n t a g e ash, and the fat-free organic fraction o f the joint c o n t a i n e d b o n e matrix as well as articular cartilage a n d a s s o c i a t e d ligaments. T h e a b s e n c e o f a silicon effect on p e r c e n t a g e o f collagen in the fatfree organic m a t t e r o f the F - T joint and in the aorta a p p e a r s to be c o n t r a r y to r e p o r t s b y others 21-23 implicating silicon in collagen biosynthesis. F u r t h e r m o r e , Carlisle 24 r e p o r t e d that 250 mg silicon/kg diet h a s t e n e d the rate o f b o n e mineralization. N o effect o f silicon o n b o n e ash or b o n e c a l c i u m - p h o s p h o r u s ratio was f o u n d in this or in the p r e v i o u s s t u d y b y E m e r i c k and K a y o n g o - M a l e , 7 and effects o f silicon o n p l a s m a c o n c e n t r a t i o n s o f c a l c i u m and p h o s p h o r u s a p p e a r e d to be t o o small to be o f p h y s i o l o g i c i m p o r t a n c e . T h e s e d a t a are in a g r e e m e n t with the p r e v i o u s findings o f E m e r i c k and K a y o n g o - M a l e 7 w h o concluded that the aortic effects o f silicon m i m i c k e d the effects o f c o p p e r . H o w e v e r , rats fed 540 mg silicon/kg diet here exhibited m o r e o b v i o u s indications o f an elev a t e d c o p p e r status t h a n w e r e o b s e r v e d for rats fed 270 m g silicon/kg diet in the p r e v i o u s study. 7 It is c o n c l u d e d that s o m e o f the metabolic effects attributed to silicon m a y be m a n i f e s t e d t h r o u g h a silicon-facilitated increase in c o p p e r utilization.
References 1 2 3 4
5 6 7 8 9
10 11 12 13 14
15 16 17
18 19 20 21 22
Acknowledgments
23
T h e a u t h o r s gratefully a c k n o w l e d g e the technical assistance o f R e n a t a W n u k .
24
492
J. Nutr. Biochem., 1990, vol. 1, September
Carlisle, E.M. (1986). Silicon as an essential trace element in animal nutrition. Ciba Foundation Symposium 121, 123-139 Carlisle, E.M. (1988). Silicon as a trace nutrient. Sci. Total Environ. 73, 95-106 Hill, C.H., Starcher, B., and Kim, C. (1967). Role of copper in the formation of elastin. Fed. Proc. 26, 129-133 Shields, G.S., Coulson, W.F., Kimball, D.A., Carnes, W.H., Cartwright, G.E., and Wintrobe, M.M. (1962). Studies on copper metabolism. XXXII. Cardiovascular lesions in copperdeficient swine. Am. J. Pathol. 41,603-621 Guenthner, E., Carlson, C.W., and Emerick, R.J. (1978). Copper salts for growth stimulation and alleviation of aortic rupture losses in turkeys. Poult. Sci. 57, 1313-1324 Fields, M., Ferretti, R.J., Smith, J.C., and Reiser, S. (1983). Effect of copper deficiency on metabolism and mortality in rats fed sucrose or starch diets. J. Nutr. 113, 1335-1345 Emerick, R.J. and Kayongo-Male, H. (1990). Interactive effects of dietary silicon, copper, and zinc in the rat. J. Nutr. Biochem. 1, 35-40 Emerick, R.J. (1984). Chloride and phosphate as impediments to silica urinary calculi in rats fed tetraethylorthosilicate. J. Nutr. 114, 733-738 Schreier, C.J. and Emerick, R.J. (1986). Diet calcium, phosphorus and acidifying and alkalizing salts as factors influencing silica urolithiasis in rats fed tetraethylorthosilicate. J. Nutr. 116, 823-830 Emerick, R.J. (1986). Animal age and duration of exposure to a urolithic diet as factors influencing silica urolithiasis incidence in an animal model. Nutr. Rep. Int. 34, 907-913 Emerick, R.J. and Lu, D. (1987). A possible synergism of dietary phosphate and urine acidifying salts in preventing silica urolithiasis in a rat model. J. Nutr. 117, 1603-1608 Emerick, R.J. (1987). Phosphate inhibition of proteinpolysilicic acid complex formation as a factor in preventing silica urolithiasis. J. Nutr. 117, 1924-1928 Oser, B.L. (1965). Hawk's Physiological Chemistry, 14th ed., McGraw-Hill, New York, pp. 1113-1114 Lehmann, H.P., Schosinsky, K.H., and Beeler, M.F. (1974). Standardization of serum ceruloplasmin concentrations in international enzyme units with o-dianisidine dihydrochloride as substrate. Clin. Chem. 20, 1564-1567 Starcher, B., Hill, C.H., and Matrone, G. (1964). Importance of dietary copper in the formation of aortic elastin. J. Nutr. 82, 318-322 Woessner, J.F., Jr. (1961). The determination of hydroxyproline in tissue and protein samples containing small proportions of this amino acid. Arch. Biochem. Biophys. 93, 440-447 Berg, R.A. (1982). Determination of 3- and 4-hydroxyproline. In Methods In Enzymology (L.W. Cunningham and D.W. Frederiksen, eds.), 82, pp. 372-398, Academic Press, Inc., New York SAS. (1985). SAS/STAT Guide for Personal Computers. Version 6 ed. SAS Institute Inc., Cary, NC National Research Council (1987). Nutrient requirements of Laboratory Animals, 3rd Ed., National Academy of Science, Washington, DC Schwarz, K. and Milne, D.B. (1972). Growth-promoting effects of silicon in rats. Nature 239, 333-334 Carlisle, E.M., Berger, J.W., and Alpenfels, W.F. (1981). A silicon requirement for prolyl hydroxylase activity. Fed. Proc. 40:866 (abstr) Carlisle, E.M. and Garvey, D.L. (1982). The effect of silicon on formation of extracellular matrix components by chondrocytes in culture. Fed. Proc. 41:461 (abstr) Carlisle, E.M. and Alpenfels, W.F. (1984). The role of silicon in proline synthesis. Fed. Proc. 43:680 (abstr) Carlisle, E.M. (1974). Silicon as an essential element. Fed. Proc. 33, 1758-1766
An eight-week, 2 × 4 factorial rat experiment using two levels o f dietary copper and four levels o f dietary silicon was conducted to further delineate a previously observed silicon-copper interaction in which silicon appears to mimic copper in its effect on the composition o f the aorta. Dietary copper concentrations were 1.4 (deficient) and 5.4 (adequate) mg/kg diet, and silicon concentrations were 5, 135, 270, and 540 mg/kg diet. Compared with the lowest level o f silicon and copper, weight gains were 15.5% higher f o r rats f e d 540 mg silicon/kg diet and 14.3% higher f o r those f e d 5.4 mg eopper/kg diet. The growth-promoting effects o f silicon and copper were additive. Evidence that silicon elevated the copper status o f copper-deficient rats includes an increase in packed-cell volume by 540 mg silicon/kg diet in the otherwise packed-cell volume-depressed, copper-deficient rats, accompanied by a trend toward higher hemoglobin values and lower relative heart weights. In the copper-adequate rats, evidence that 540 mg silicon/kg diet elevated their copper status includes a two-fold increase in the blood-plasma copper concentration, a three-fold increase in ceruloplasmin activity, and an increase in cardiac, renal, and hepatic copper concentrations. In addition, 540 mg silicon/kg diet resulted in higher aortic dry mass and aortic elastin content in both copper-deficient and copper-adequate rats. While dietary silicon concentrations o f 135, 270, and 540 mg/kg diet were all effective in increasing aortic elastin in the copper-adequate rats, only 540 mg silicon/kg diet increased aortic elastin in the copperdeficient rats. These data indicate that some o f the metabolic" effects attributed to silicon may be manifested through a silicon-facilitated increase in copper utilization.
Keywords: silicon; copper; connective tissue; aorta; elastin; collagen Introduction Essential functions of silicon identified in animals appear to be largely associated with formation of connective tissue with specific i n v o l v e m e n t in collagen synthesis. This subject has been reviewed by Carlisle. 1.2 In addition to silicon, c o p p e r is also involved in connective tissue synthesis. C o p p e r is a c o m p o n e n t of lysyl oxidase, the e n z y m e active in the formation of cross-linkages b e t w e e n polypeptide chains of the connective tissue proteins, collagen and elastin. 3 Aortic a n e u r y s m s and ruptures occurring in several species 4-6 often a p p e a r to be a c o n s e q u e n c e of defective elastin
Address reprint requests to Dr. R.J. Emerick, Olson Biochemistry Laboratories, Animal Science Bldg., South Dakota State University, Brookings, SD 57007, USA. Published with approval of the Director of the South Dakota Agricultural Experiment Station as publication number 2486 of the journal series. Received February 16, 1990; accepted April 11, 1990. ©
1990 Butterworth-Heinemann
synthesis. Recently, E m e r i c k and K a y o n g o - M a l e 7 reported that 270 mg silicon per kg of diet appeared to mimic the gross effects of c o p p e r in promoting higher aortic elastin levels in copper-deficient rats. This study was conducted to further delineate the silicon-copper interrelationship, and data are presented in support of the hypothesis that silicon facilitates c o p p e r utilization in the rat.
Materials and methods One-hundred-twenty male S p r a g u e - D a w l e y rats (SASCO, O m a h a , N E , USA) were used in a 2 × 4 factorial experiment involving diets providing two concentrations of copper, 1.4 and 5.4 mg per kg of diet, and four concentrations of silicon, 5, 135, 270, and 540 mg per kg of diet. The rats initially averaged 54 _+ 5.6 g in weight and were randomly allotted across the eight treatments providing 15 rats per treatment. T h e y were housed individually in hanging stainlesssteel cages with wire m e s h floors in a r o o m maintained at 23 to 25°C. A 12-hour light-to-dark cycle was main-
J. Nutr. Biochem., 1990, vol. 1, September
487
Research Communications Table 1
Basal diet composition
Ingredient Dextrose, anhydrous Egg albumin, spray dried a Corn oil Salt mixtureb Vitamin mixturec
Amount (%) 69.0 20.0 5.0 40 20
a Spray dried egg albumin was autoclaved at 120°C for 1 hour and dried at 120°C for 1 hour for denaturation. b The percent composition of salt mixture (salt mixture P-H with a reduced amount of CuSO4 and an increased amount of ZnCl2, ICN Nutritional Biochemicals, Cleveland, OH, USA); dipotassium phosphate, 32.2; calcium carbonate, 30.0; sodium chloride, 16.7; magnesium sulfate (hydrate), 10.2; manganese sulfate, 0.51; dibasic calcium phosphate dihydrate, 7.5; ferric citrate, 2.75; potassium iodide, 0.08; copper sulfate pentahydrate, 0.0098; zinc chloride, 0.0624; cobalt chloride, 0.005. c Vitamin mixture composition in grams/kilogram triturated in dextrose (vitamin diet fortification mixture, ICN Nutritional Biochemicals): vitamin A concentrate (500,000 IU/g), 1.8; vitamin D concentrate (850,000 IU/g), 0.125; alpha-tocopherol (250 IU/g), 22.0; ascorbic acid, 45; inositol, 5.0; choline chloride, 75; menadione, 2.25; p-aminobenzoic acid, 5.0; niacin, 4.25; riboflavin, 1.0; pyridoxine hydrochloride, 1.0; thiamine hydrochloride, 1.0; calcium pantothenate, 3.0; biotin, 0.020; folic acid, 0.090; and vitamin B-12, 0.00135
tained. Attempts were made to minimize environmental silica with a thorough initial cleaning of the room and related equipment followed by a weekly flushing of the painted concrete floor. The diets were provided ad libitum in glass jars, and distilled water was provided in polyethylene bottles fitted with stainless-steel drinking-tips. The basal diet is shown in Table 1. Before use, the spray-dried egg albumin was autoclaved at 120°C for 1 hour and dried at 120°C for 1 hour to prevent avidin interference with biotin absorption. Additional copper, above the 0.4 mg/kg diet inherent in the non-mineral diet ingredients, was provided as CuSO4"5H2O. The additional silicon was provided by tetraethylorthosilicate (J. T. Baker Chemical Co., Phillipsburg, N J, USA). Tetraethylorthosilicate is rapidly hydrolyzed in HCI equivalent to that of the gastric stomach, and it has been used extensively in a rat model at concentrations providing up to 2,700 mg silicon/kg diet for the study of silica metabolism and silica urolithiasis. 7d2 Tha rats were weighed weekly during the 8-week experiment. Blood samples were obtained at termination by cardiac puncture following anesthetization with Halothane (Abbott Laboratories, Chicago, IL, USA). Blood was placed in heparinized tubes. Following the immediate determination of hemoglobin, as described below, and packed cell volume (PCV) (Autocrit Centrifuge, Clay Adams, Inc., New York, NY, USA), plasma was separated and stored at - 25°C until analyzed. While continuing under anesthetization, the rats were sacrificed by decapitation and the liver, kidneys, heart, femurs, the femoral-tibial (F-T)joints including the respective condyles, the patella and associated articular cartilage, and a sample of aorta were
488
J. Nutr. Biochem., 1990, vol. 1, September
removed, weighed and stored at - 25°C until analyzed. The sample of aorta represented - 3 cm of the descending thoracic aorta beginning immediately below the aortic arch. Concentrations of calcium and magnesium in plasma, and copper in nitric acid digests of all soft tissues were determined by flame atomic absorption spectrophotometry (Perkin-Elmer model 5000, Norwalk, CT, USA). Copper was determined in plasma by graphite furnace atomic absorption (Perkin-Elmer model 5000 equipped with model 500 heated graphite atomizer and autosampler) using diluted plasma containing 0.2% of concentrated nitric acid (Ultrex, J. T. Baker Chemical Co., Phillipsburg, N J, USA). Recovery of copper from spiked samples averaged 103% by this method. Plasma inorganic phosphorus was determined using the Fiske and Subbarow phosphomolybdate method. ~3 Silicon content of the basal diet was determined by graphite furnace atomic absorption spectrophotometry in an aqueous suspension prepared with a tissue homogenizer (Tekmar Co., Cincinnati, OH, USA). Atomic absorption conditions were the same as those described herein for plasma. Total hemoglobin was determined by the cyanomethemoglobin method (test kit: hemoglobin 525, Sigma Chemical Co., St. Louis, MO, USA). Ceruloplasmin activity was determined using o-dianisidine dihydrochloride substrate as described by others. 14 Silicon in blood plasma was determined by graphite furnace atomic absorption (Perkin-Elmer model 5000 equipped with model 500 heated graphite atomizer and autosampler) using nickel nitrate matrix modification as described by Emerick and Kayongo-Male.v The aortas were extracted for 20 minutes with chloroform/methanol (2:1 vol/vol), washed with ethanol and ether, and allowed to air dry. Aortic elastin was determined as the amount of the aorta remaining insoluble following treatment with 0.1 N NaOH at 98°C for 1 hour.~5 Collagen content of the aortas was calculated from hydroxyproline contained in an aliquot of the 0.1 N NaOH supernatant solution. Hydroxyproline was determined colorimetrically, 16 and was assumed to comprise 10% of the weight of collagen. 17 F-T joints were crushed and extracted with 2:1 vol/ vol chloroform/methanol. Elastin and collagen were determined as described for aortas except that elastin was corrected for bone ash following ashing at 500°C overnight. Elastin and collagen were calculated as a percentage of the dry, fat-free organic portion of the F-T joint. Femurs were extracted for 12 hours with ethanol followed by a 12-hour extraction with diethylether. After air-drying, they were ashed overnight at 500°C, and ash was calculated as percent of the dry, fat-free bone. Calcium, magnesium, and phosphorus were determined on the acid-soluble bone ash by the methods described for plasma. Statistical analyses were made by analysis of variance using a model consisting of the copper and silicon main effects and the two-way interaction. J8 Significant
Silicon-copper interaction: Emerick and Kayongo-Male differences between appropriate means were identified by the method of least significant differences protected by a significant F value.
Results
Body weights Body weights at 0, 4, and 8 weeks of the experiment are presented in Table 2. Weight differences related to treatment main effects were first observed at 4 weeks, and they increased in magnitude during the final 4 weeks of the 8-week study. Compared with the lowest treatment level of silicon and copper, weight gains at 8 weeks were 15.5% higher (P < 0.01) for rats fed 540 mg silicon/kg diet and 14.3% higher (P < 0.01) for those fed 5.4 mg copper/kg diet. The growthpromoting effects of 540 mg silicon and 5.4 mg copper/ kg diet were additive.
Blood and blood plasma Packed-cell volume and hemoglobin data are shown in Table 2, and the plasma data are shown in Table 3. Copper-deficient rats fed 540 mg silicon/kg diet had a higher average PCV (42.7 vs. 39.3%, P < 0.05) than those fed the low-silicon basal diet. Hemoglobin values followed the same trend as PCV, but the differences attributable to silicon were not significant (P > 0.05). Copper-adequate rats fed 540 mg silicon/kg diet had average plasma copper concentration that was twofold higher (0.51 vs. 0.28 mg copper/L; P < 0.01) than the value for rats fed the basal diet inherently containing only 5 mg silicon/kg diet. Plasma ceruloplasmin
activity was likewise three-fold higher (38.9 vs. 12.2 U/ L; P < 0.01) for copper-adequate rats fed 540 vs. 5 mg silicon/kg diet. Lower dietary silicon concentrations of 135 or 270 mg/kg had no apparent effect on plasma copper concentrations or ceruloplasmin activity. Furthermore, none of the dietary silicon additions had an effect on these components in the copper-deficient rats where average plasma copper levels were only 0.07 mg/L. Compared to rats fed no supplemental dietary silicon, plasma silicon concentrations were increased in copper-deficient rats fed 270 and 540 mg silicon/kg diet, and in copper-adequate rats fed 540 mg silicon/kg diet. Increases (P < 0.01) in plasma concentrations of calcium, magnesium, and phosphorus were associated with an increase in dietary copper. An increase (P < 0.05) in plasma calcium was also associated with the 540 mg silicon/kg diet in the copper-deficient rats and with 135 mg silicon/kg diet in the copper-adequate rats. Plasma phosphorus concentration was lowered by 540 mg silicon/kg diet in the copper-adequate rats. Dietary silicon had no effect (P > 0.05) on plasma magnesium.
Aorta Aorta data are shown in Table 4. Rats fed 540 mg silicon/kg diet, with either of the two levels of dietary copper, had higher (P < 0.01) average aortic mass (4.9 and 5.0 mg vs. 4.0 mg dry mass/cm length) when compared with those fed the corresponding low-silicon basal diet. Elastin content of the aorta was also increased (P < 0.01) by silicon. While dietary silicon concentrations of 135,270, and 540 mg/kg diet were all effective in increasing aortic elastin in the copper-
Table 2 Body weights, packed cell volume, and hemoglobin values of rats fed 4 levels of silicon with 2 levels of copper in the diet for 8 weeks a'b Dietary copper and silicon (mg/kg diet) Copper, 1.4 Silicon, 5 Silicon, 135 Silicon, 270 Silicon, 540 Copper, 5.4 Silicon, 5 Silicon, 135 Silicon, 270 Silicon, 540 SEM c ANOVA d Copper Silicon Interaction
Initial (g)
Body weights 4 weeks (g)
8 weeks (g)
Packed cell volume (%)
Hemoglobin (g/d0
56.9 56.9 57.3 57.1
208 210 209 220
267 a 280 a'b 263 a 300 b
39.3 a 36.7 a 38.3 a 42.7 b
12.9 12.0 12.4 13.7
56.1 58.3 56.8 57.9 1.5
213 a 210 a 215 a 232 b 4.7
296 a 304 a 304 a 333 b 7.2
44.6 44.7 45.5 44.3 1.2
14.7 14.6 14.9 14.6 0.36
NS NS NS
NS 0.002 NS
0.0001 0.0001 NS
0.0001 NS 0.04
0.0001 NS NS
a N - 15/treatment. b Mean values within a column and within the same copper treatment not sharing a common superscript symbol differ by the method of least significant differences protected by a significant F value (P < 0.05). ° Standard error of the mean calculated from the error mean square. d A 2 x 4 analysis of variance; numerical values are levels of probability for main effects and interaction, NS = not significant (P > 0.05).
J. Nutr. Biochem, 1990, vol. 1, September
489
Research Communications Table 3
Blood plasma concentrations of selected elements for rats fed 4 levels of silicon with 2 levels of copper in the diet for 8 weeks a'b
Dietary copper and silicon (mg/kg diet) Copper, 1.4 Silicon, 5 Silicon, 135 Silicon, 270 Silicon, 540 Copper, 5.4 Silicon, 5 Silicon, 135 Silicon, 270 Silicon, 540 SEM c ANOVA d Copper Silicon Interaction
Copper (mg/L)
Ceruloplasmin (U/L)
Silicon (mg/L)
Calcium (mg/L)
Magnesium (mg/L)
Phosphorus (mg/L)
0.09 0.06 0.06 0.07
0.7 1.0 0.8 0.8
0.27 a 0.27 a 0.36 b 0.39 b
103 a 105 ab 102 a 107 b
19.0 19.5 19.1 20.0
68 73 75 72
12.2 a 3.8 a 14.7 a 38.9 b 4.9
0.28 a 0.28 a 0.32 a,b 0.42 b 0.02
10P 112 b 110 ~'b 109 ~'b 1.4
21.0 22.0 21.8 20.3 0.45
81 a 78 a'b 84 ~ 73 b 2.5
NS 0.0001 NS
0.0001 0.02 NS
0.0001 NS NS
0.28 a 0.17 a 0.22 a 0.51 b 0.046 0.0001 0.001 0.004
0.0001 0.005 0.006
0.0001 0.04 NS
a N = 15/treatment. b Mean values within a column and within the same copper treatment not sharing a common superscript symbol differ by the method of least significant differences protected by a significant F value (P < 0.05). c Standard error of the mean calculated from the error mean square. d A 2 x 4 analysis of variance; numerical values are levels of probability for main effects and interaction, NS = not significant (P > 0.05).
Table 4.
Aortic and femoral-tibial joint characteristics of rats fed 4 levels of silicon with 2 levels of copper in the diet for 8 weeks a'b Aorta
Dietary copper and silicon (mg/kg diet) Copper, 1.4 Silicon, 5 Silicon, 135 Silicon, 270 Silicon, 540 Copper, 5.4 Silicon, 5 Silicon, 135 Silicon, 270 Silicon, 540 SEM c ANQVA ~ Copper Silicon Interaction
Mass (mg/cm)
Femoral-tibial joint
Elastin
Collagen
Elastin
Collagen (percent) (Dry, fat-free basis)
(percent) (Dry, fat-free basis)
4.0 a 4.2 ~ 4.2 a 4.9 b
40.8 a 42.2 ~b 42.4 a'b 44.1 b
39.9 38.9 39.2 38.3
15.7 15.0 14.9 14.2
40.4 39.1 41.0 39.8
4.0 a 4.2 a 4.3 a 5.0 b 0.20
42.6 a 46.5 b 46.9 b 46.2 b 1.0
40.4 40.4 41.0 39.5 095
16.5 16.8 17.5 17.0 0.73
36.0 38.2 36.3 37.0 1.0
NS 0.0001 NS
0.0001 0.005 NS
NS NS NS
0.0006 NS NS
0.0001 NS NS
a N = 15/treatment. b Mean values within a column and within the same copper treatment not sharing a common superscript symbol differ by the method of least significant differences protected by a significant F value (P < 0.05). c Standard error of the mean calculated from the error mean square. A 2 x 4 analysis of variance; numerical values are levels of probability for main effects and interaction, NS = not significant (P > 0.05).
adequate rats, only 540 mg silicon/kg diet increased aortic elastin in the copper-deficient rats. In these instances, aortic elastin values of the siliconsupplemented rats were 109% of those fed no added silicon. Aortic collagen percentages tended to be increased by copper (P = 0.08) but were unaffected by silicon (P > . 10).
490
J. Nutr. Biochem., 1990, vol. 1, September
Femur diaphysis, and F-T joint Average of the treatment means + SEM for femurdiaphysis ash (data not shown) was 61.0 _ 0.3% of the dry, fat-free bone, and the bone ash contained 35.4 _+ 0.2% calcium, 17.3 _+ 0.1% phosphorus, and 0.64 _+ 0.01% magnesium with no treatment differences.
Silicon-copper interaction: Emerick and Kayongo-Male Table 5 Heart weights, and heart, liver and kidney copper contents for rats fed 4 levels of silicon with 2 levels of copper in the diet for 8 weeks a'b
Dietary copper and silicon (mg/kg diet) Copper, 1.4 Silicon, 5 Silicon, 135 Silicon, 270 Silicon, 540 Copper, 5 4 Silicon, 5 Silicon, 135 Silicon, 270 Silicon, 540 SEM c ANOVA d Copper Silicon Interaction
Relative heart weights (g/lO0 g body wt)
Absolute heart weights (g)
0.43 a 0.44 a 0.43 a 0.39 b
1.13 1.21 1.11 1.14
0.36 a'b 0.36 a'b 0.37 a 0.33 b 0.12
1.06 1.09 1.14 1.10 0.033
13.1 a 14.5 ~'b 12.8 a 16.6 b 0.84
0.0001 0.003 NS
NS NS NS
0.0001 0.02 NS
Heart copper
7.9 6.9 7.9 8.6
Liver copper (mg/kg, dry basis)
Kidney copper
30 3.7 3.3 40
13.7 13.6 13.3 14.5
8.0 76 7.6 9.4 0.59
18.3 a 18.0 a 18.6 a 21.4 b O71
0.0001 NS NS
0.0001 0.008 NS
a N = 15/treatment b Mean values within a column and within the same copper treatment not sharing a common superscript symbol differ by the method of least significant differences protected by a significant F value (P < 0.05). c Standard error of the mean calculated from the error mean square. d A 2 x 4 analysis of variance; numerical values are levels of probability for main effects and interaction, NS = not significant (P > 0.05).
Elastin and collagen contents of the dry, fat-free, organic portion of the F-T joint are shown in Table 4. Compared to the copper-deficient treatment, adequate copper increased (P < 0.01) elastin and lowered (P 0.01) collagen concentrations in the fat-free organic portion of the F-T joint. The average percentage of elastin in the F-T joint for copper-adequate rats was 113.4% and collagen was 92.0% of the respective values for copper-deficient rats. Silicon had no effect on elastin or collagen contents of the F-T joint. Collagen was negatively correlated with elastin (r = -0.36, P < 0.01).
Heart, liver, and kidneys Heart, liver, and kidney data are presented in Table 5. Absolute heart weights (in grams) did not differ among treatments (P > 0.05). However, the average relative heart weight (g/100 g body weight) of all copperdeficient rats was 119% of the average of all copperadequate rats (P < 0.01). The highest level of silicon (540 mg/kg diet) appeared to prevent this cardiac hypertrophy relative to body weight in copper-deficient rats (P < 0.05) and tended (P = 0.10) to further reduce relative heart weights in copper-adequate rats. In the copper-adequate rats, the highest dietary silicon level, compared to no supplemental silicon, increased copper concentrations (dry basis) in the heart (16.6 vs. 13.1 mg copper/kg, P < 0.05), kidney (21.4 vs. 18.3 mg copper/kg, P < 0.01), and liver (9.4 vs. 8.0 mg copper/kg, P = 0.09).
Discussion The inclusion of 1.0 mg copper (from CuSO4 • 5HzO)/ kg diet to raise the copper concentration of the basal
diet to 1.4 mg/kg was done to avoid deaths that may have occurred with a more severe copper deficiency. On the other hand, 5.4 mg copper/kg diet used as the higher level is normally considered to be adequate. 19 Data obtained here indicate that 540 mg silicon/kg diet elevated the copper status of rats whether they were fed the copper-deficient basal diet (1.4 mg copper/kg diet) or the copper-adequate diet (5.4 mg copper/kg diet). Evidence that silicon raised the copper status of copper-deficient rats includes an increase in PCV to near normal in the otherwise PCV-depressed, copperdeficient rats, accompanied by a trend toward higher hemoglobin values. Furthermore, a lower average relative heart weight for the high-silicon rats is consistent with the physiologic effect of adequate copper. 9 In the copper-adequate rats, evidence that 540 mg silicon/kg diet elevated their copper status was more precise, and included a two-fold increase in the blood-plasma copper concentration, a three-fold increase in ceruloplasrain activity, and in increase in cardiac, renal, and hepatic copper concentrations. Independent increases in weight gain in response to silicon or copper supplementation, and their appearance of being additive, made it difficult to estimate how much, if any, of this function of silicon may have been manifested through an elevation of copper status. The effectiveness of 540 mg silicon/kg diet in promoting weight gain, and the ineffectiveness of lower levels for this purpose, is in accord with the earlier findings of Schwarz and Milne 2° that 500, but not 250, mg silicon/kg diet gave a growth response in rats. Another apparent independent effect of silicon is the increase in aortic dry mass (weight/length) associated with 540 mg silicon/kg of diet. That the greater aortic mass may be a component of the heavier body
J. Nutr. Biochem., 1990, vol. 1, September
491
Research Communications weights o f rats in this t r e a t m e n t c a n n o t be ruled out at this time. H o w e v e r , a n o t h e r aortic benefit attributable to increases in d i e t a r y silicon, the m a g n i t u d e o f w h i c h a p p e a r e d to be partially d e p e n d e n t u p o n the p r e s e n c e o f a potentially a d e q u a t e level o f dietary c o p p e r , inc l u d e d an i n c r e a s e in aortic elastin. While all dietary additions o f silicon (135, 270, and 540 mg silicon/kg diet) effected increases in aortic elastin in the c o p p e r a d e q u a t e rats, o n l y the highest level (540 mg/kg diet) o f silicon p r o d u c e d this r e s p o n s e in the copper-deficient rats. In a p r e v i o u s s t u d y b y E m e r i c k and K a y o n g o Male, 7 270 m g silicon/kg diet i n c r e a s e d aortic elastin in c o p p e r - d e f i c i e n t rats. In c o n t r a s t to the effect o f silicon and c o p p e r on aortic elastin, o n l y c o p p e r e f f e c t e d an increase in the p e r c e n t a g e elastin in the fat-free organic fraction o f the F - T joint. T h e p a r a d o x o f a c o p p e r - i n d u c e d increase in F - T joint elastin that did not r e s p o n d to the elevation in c o p p e r status i n d u c e d b y silicon s u p p l e m e n t a t i o n r e m a i n s u n e x p l a i n e d at this time. A c o n c o m i t a n t d e c r e a s e in collagen, a c c o m p a n y i n g the c o p p e r - i n d u c e d i n c r e a s e in elastin, resulted in a relatively c o n s t a n t p e r c e n t a g e for the c o m b i n a t i o n o f elastin and collagen in the fat-free organic fraction o f the F - T joint. T h e F - T joint included the f e m o r a l and tibial c o n d y l e s and the patella in addition to the associated articular cartilage. T h u s , the dry, fat-free F-T joint samples c o n t a i n e d - 62% b o n e as calculated f r o m the p e r c e n t a g e ash, and the fat-free organic fraction o f the joint c o n t a i n e d b o n e matrix as well as articular cartilage a n d a s s o c i a t e d ligaments. T h e a b s e n c e o f a silicon effect on p e r c e n t a g e o f collagen in the fatfree organic m a t t e r o f the F - T joint and in the aorta a p p e a r s to be c o n t r a r y to r e p o r t s b y others 21-23 implicating silicon in collagen biosynthesis. F u r t h e r m o r e , Carlisle 24 r e p o r t e d that 250 mg silicon/kg diet h a s t e n e d the rate o f b o n e mineralization. N o effect o f silicon o n b o n e ash or b o n e c a l c i u m - p h o s p h o r u s ratio was f o u n d in this or in the p r e v i o u s s t u d y b y E m e r i c k and K a y o n g o - M a l e , 7 and effects o f silicon o n p l a s m a c o n c e n t r a t i o n s o f c a l c i u m and p h o s p h o r u s a p p e a r e d to be t o o small to be o f p h y s i o l o g i c i m p o r t a n c e . T h e s e d a t a are in a g r e e m e n t with the p r e v i o u s findings o f E m e r i c k and K a y o n g o - M a l e 7 w h o concluded that the aortic effects o f silicon m i m i c k e d the effects o f c o p p e r . H o w e v e r , rats fed 540 mg silicon/kg diet here exhibited m o r e o b v i o u s indications o f an elev a t e d c o p p e r status t h a n w e r e o b s e r v e d for rats fed 270 m g silicon/kg diet in the p r e v i o u s study. 7 It is c o n c l u d e d that s o m e o f the metabolic effects attributed to silicon m a y be m a n i f e s t e d t h r o u g h a silicon-facilitated increase in c o p p e r utilization.
References 1 2 3 4
5 6 7 8 9
10 11 12 13 14
15 16 17
18 19 20 21 22
Acknowledgments
23
T h e a u t h o r s gratefully a c k n o w l e d g e the technical assistance o f R e n a t a W n u k .
24
492
J. Nutr. Biochem., 1990, vol. 1, September
Carlisle, E.M. (1986). Silicon as an essential trace element in animal nutrition. Ciba Foundation Symposium 121, 123-139 Carlisle, E.M. (1988). Silicon as a trace nutrient. Sci. Total Environ. 73, 95-106 Hill, C.H., Starcher, B., and Kim, C. (1967). Role of copper in the formation of elastin. Fed. Proc. 26, 129-133 Shields, G.S., Coulson, W.F., Kimball, D.A., Carnes, W.H., Cartwright, G.E., and Wintrobe, M.M. (1962). Studies on copper metabolism. XXXII. Cardiovascular lesions in copperdeficient swine. Am. J. Pathol. 41,603-621 Guenthner, E., Carlson, C.W., and Emerick, R.J. (1978). Copper salts for growth stimulation and alleviation of aortic rupture losses in turkeys. Poult. Sci. 57, 1313-1324 Fields, M., Ferretti, R.J., Smith, J.C., and Reiser, S. (1983). Effect of copper deficiency on metabolism and mortality in rats fed sucrose or starch diets. J. Nutr. 113, 1335-1345 Emerick, R.J. and Kayongo-Male, H. (1990). Interactive effects of dietary silicon, copper, and zinc in the rat. J. Nutr. Biochem. 1, 35-40 Emerick, R.J. (1984). Chloride and phosphate as impediments to silica urinary calculi in rats fed tetraethylorthosilicate. J. Nutr. 114, 733-738 Schreier, C.J. and Emerick, R.J. (1986). Diet calcium, phosphorus and acidifying and alkalizing salts as factors influencing silica urolithiasis in rats fed tetraethylorthosilicate. J. Nutr. 116, 823-830 Emerick, R.J. (1986). Animal age and duration of exposure to a urolithic diet as factors influencing silica urolithiasis incidence in an animal model. Nutr. Rep. Int. 34, 907-913 Emerick, R.J. and Lu, D. (1987). A possible synergism of dietary phosphate and urine acidifying salts in preventing silica urolithiasis in a rat model. J. Nutr. 117, 1603-1608 Emerick, R.J. (1987). Phosphate inhibition of proteinpolysilicic acid complex formation as a factor in preventing silica urolithiasis. J. Nutr. 117, 1924-1928 Oser, B.L. (1965). Hawk's Physiological Chemistry, 14th ed., McGraw-Hill, New York, pp. 1113-1114 Lehmann, H.P., Schosinsky, K.H., and Beeler, M.F. (1974). Standardization of serum ceruloplasmin concentrations in international enzyme units with o-dianisidine dihydrochloride as substrate. Clin. Chem. 20, 1564-1567 Starcher, B., Hill, C.H., and Matrone, G. (1964). Importance of dietary copper in the formation of aortic elastin. J. Nutr. 82, 318-322 Woessner, J.F., Jr. (1961). The determination of hydroxyproline in tissue and protein samples containing small proportions of this amino acid. Arch. Biochem. Biophys. 93, 440-447 Berg, R.A. (1982). Determination of 3- and 4-hydroxyproline. In Methods In Enzymology (L.W. Cunningham and D.W. Frederiksen, eds.), 82, pp. 372-398, Academic Press, Inc., New York SAS. (1985). SAS/STAT Guide for Personal Computers. Version 6 ed. SAS Institute Inc., Cary, NC National Research Council (1987). Nutrient requirements of Laboratory Animals, 3rd Ed., National Academy of Science, Washington, DC Schwarz, K. and Milne, D.B. (1972). Growth-promoting effects of silicon in rats. Nature 239, 333-334 Carlisle, E.M., Berger, J.W., and Alpenfels, W.F. (1981). A silicon requirement for prolyl hydroxylase activity. Fed. Proc. 40:866 (abstr) Carlisle, E.M. and Garvey, D.L. (1982). The effect of silicon on formation of extracellular matrix components by chondrocytes in culture. Fed. Proc. 41:461 (abstr) Carlisle, E.M. and Alpenfels, W.F. (1984). The role of silicon in proline synthesis. Fed. Proc. 43:680 (abstr) Carlisle, E.M. (1974). Silicon as an essential element. Fed. Proc. 33, 1758-1766