Growth and Skeletal Development
Calcium Metabolism in Great Dane Dogs Fed Diets with Various Calcium and Phosphorus Levels1 HERMAN A. W. HAZEWINKEL,*2 WALTER E. VAN DEN BROM* ARIE TH. VAN T KLOOSTERj GEORGE VOORHOUTÃŽ AND ANK VAN WEES* 'Department of Clinical Sciences of Companion Animals, ^Department of Husbandry and nutrition, and ^Department of Veterinary Radiology, Faculty of Veterinary Medicine, Uniuersity of Utrecht, The Netherlands in a calcium absorption and retention not differing from that in control dogs, the latter despite the occurrance of severe osteoporosis. J. Nutr. 121 : S99-S106,1991.
ABSTRACT The influence of dietary calcium on cal cium metabolism was investigated in growing dogs that reach an adult body weight similar to that of humans. Seven groups of dogs (n > 5) were raised on a diet with a composition meeting the National Research Council (NRC) requirements (1974), but differing in calcium content, with or without a constant ratio to phosphorus. Control dogs fed 1.1% calcium and 0.9% phosphorus (all on a dry matter basis) were fed ad libitum (n = 10) or in restricted amounts (n = 6); dogs fed high calcium (3.3%) diets received either 0.9% phosphorus (n = 6) or 3.0% phosphorus (n = 6); dogs fed low calcium (0.55%) diets received either 0.9% phosphorus (n = 5 + 6) or 0.5% phosphorus (n = 8). Food intake, circulating total calcium and inorganic phosphorus concentrations and calcium metabolism, with 45Ca kinetics, were studied at 8, 14, 20 and 26 wk of age. Except for the difference in food intake in two groups at 14 wk (i.e., 0.55% calcium-0.9% phos phorus higher and 3.3% calcium-0.9% phosphorus lower, respectively, than the controls) no differences were noticed during the rest of the study. The mean plasma calcium concentrations did not differ between groups during the studies, whereas that of inorganic phosphorus revealed temporal aberrations in two groups. An absorption coefficient a of 45-66% was found for the control group. High and low calcium diets gave rise to values of 23-43% and 70-97%, respec tively, for a irrespective of the phosphorus content of the diet. The amount of calcium absorbed was nev ertheless considerably higher in the dogs fed the high calcium diet than in the control dogs. The dogs fed the high calcium diets had also a higher calcium retention than found in the low calcium group, which was not different from the control group. The conclusions were young dogs fed a diet with a calcium content of 3.3% (with either 0.9 or 3.0% phosphorus) have a signifi cantly elevated absorption and retention of calcium and seem unable to protect themselves against chronic ex cessive calcium intake, and young dogs of this large breed elevate the absorption of calcium to >90% of the ingested amount when raised on a diet with 0.55% calcium (with either 0.5 or 0.9% phosphorus), resulting
0022-3166/91
INDEXING KEY WORDS:
•symposium •dogs •dietary calcium •dietary phosphate •skeletal mineralization •45Cakinetics
Dogs, especially those with an adult body weight similar to that of humans (i.e., 60-70 kg), are of in terest for studies of calcium metabolism for clinical as well as comparative nutritional knowledge. More than any other laboratory mammalian species, dogs have a metabolic activity remarkably similar to that of humans (1). Net absorption and retention of calcium can be measured with radioactive or stable calcium isotopes (2-5). It has been shown in the young dog that any gain or loss of calcium to the body is a gain or loss to the skeleton and is therefore considered to be a good estimate of bone mineralization (6). Because dogs (7), like humans (3), are able to adapt to lower or higher levels of calcium intake, a diet of constant calcium content should be fed to allow for conclusions from a long-term study of calcium me tabolism. Since the phosphorus and calcium content of the diet may reciprocally influence absorption and accretion of both nutrients, the phosphorus content of the diet should be taken into account (8). To gain 1 Presented as part of the Waltham International Symposium on Nutrition of Small Companion Animals, at University of California, Davis, CA 95616, on September 4-8, 1990. Guest editors for the symposium were James G. Morris, D'Ann C. Finley and Quinton R. Rogers. 1 To whom correspondence should be addressed: Department of Clinical Sciences of Companion Animals, University Utrecht, P.O. Box 80.154, 3508 TD Utrecht, The Netherlands.
$3.00 ©1991 American Institute of Nutrition.
$99 Downloaded from https://academic.oup.com/jn/article-abstract/121/suppl_11/S99/4744095 by Washington University School of Medicine Library user on 06 June 2018
HAZEWINKEL
SlOO
more insight into the effects of intakes of calcium higher and lower than the requirement (9), with or without constant ratios to phosphorus, calcium me tabolism was studied in seven groups of Great Dane dogs during growth from weaning until one-half of adult weight. Because a variety of nutritional factors, including the ratio of calcium to phosphorus, the presence of phytin and fat content may influence cal cium availability, a dry dog food differing only in its calcium and/or phosphorus content was used and at tempts were made to standardize all other factors.
MATERIALS AMD METHODS Animais. Forty-seven Great Dane dogs, from eight different litters, 5-10 wk old, were studied in several groups of five to six dogs each, in which males and females were divided at random. Dogs were individ ually housed in cages with access to an outside run; during the kinetic studies the animals were housed individually in metabolism cages. Water was available ad libitum. Each group of dogs was fed one diet for the entire period of the study, ranging from 11 to 26 wk. Diets. The diet differed among groups only in cal cium and/or phosphorus content, as given in Table 1, and otherwise met the U.S. National Research Coun cil's Nutrient Requirements of Dogs, 1974 (9). The composition of the diet used in all studies is given in Table 2. The food intake of the HCHP3 group was expected to be low, based on previous experiences with
TABLE 1
Dietary calcium and phosphorus and number of Great Dane dogs per experimental group1 Calcium
Phosphorus
g/ÃŽOO g dry matter Normal dietsNCNP calcium lib.NCNP-rHigh ad dietsHCNPHCHPLow calcium
ET AL. TABLE 2
Composition of the dry feed used in the different experiments1
Ingredient Bloodmeal (spray dehydrated) Casein (dehydrated) Soybean flour Corn gluten meal Corn/potato starch mixture Tallow Soybean oil Sugar DL-Methionine Cellulose Vitamin/Mineral supplement1
are used to indicate the Ca and P
content of the diets (given as percentage of the dry matter basis of the diets) as fed to the experimental dogs: LCLP, 0.55% Ca-0.5% P; LCNP-1 and LCNP-2,0.55% Ca-0.9%P; NCNP and NCNP-r, 1.1% Ca-0.9% P; HCNP, 3.3% Ca-0.9% P; HCHP, 3.3% Ca-3.0% P (-1 and -2 refer to successive experiments, -r to restricted feeding as indicated in the text).
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5.00-381 5.01-162 5.04-593 5.02-900 4.02-889/4.07-850 4.08-128 4.07-983
g/kg diet
62 57 100 62 491 68 20 50 2 20 60
1 Proximate analysis (in g/100 g): Crude protein, 21.0; Ether ex tract, 9.9; N-Free extract, 49.5; Crude Fiber, 3.0; Ash, 6.6; Moisture, 10.0. 2 Additives: retinyl acetate, 5000 IE/kg; cholecalciferol, 1000 IE/ kg; dl-a-tocopheryl acetate, 50 IE/kg; B.H.T., 1 g/kg; Monocalcium phosphate, Potassium phosphate, Calcium carbonate.
feeding the HCNP3 diet. For that reason a separate control group (NCNP-r)3 was fed isoenergetically re stricted to the intake of the HCHP dogs. All other dogs were fed ad libitum. LCNP3 diet (calcium 0.55%, phosphorus 0.9%) was used in two separate experi ments and the results are given separately (i.e., LCNPl3 and LCNP-23). Blood chemistry. Blood collection and investi gation for total plasma calcium and plasma inorganic phosphorus was performed as described previousCalcium kinetics. Calcium kinetic studies were performed with 45Ca (Amersham International, Amersham, UK; specific activity 12 mBq/mg) admin istered intravenously (185 kBq) and, 4-7 d later, orally (370-540 kBq) at the age of 8, 14, 20 and 26 wk. Urine and feces were collected for 3 and 4 days after 45Ca administration, respectively. Feces were mixed and a sample was dried, ashed and diluted with HC1. An
dietsLCNP,LCNPZLCLP1.11.13.33.30.550.550.550.90.90.93.00.90.90.510666568 calcium
1The following abbreviations
International Feed Number
3 Abbreviations: HC, high calcium = 3.3% calcium on dry matter basis; HP, high phosphorus = 3.0% phosphorus on dry matter basis; LC, low calcium = 0.55% calcium on dry matter basis; LP, low phos phorus = 0.5% phosphorus on dry matter basis; NC, normal calcium = 1.1% calcium on dry matter basis (according to 1974 NRC); NP, normal phosphorus = 0.9% phosphorus on dry matter basis (ac cording to 1974 NRC); NP-1 and 2, two separate experiments with diet containing 0.9% phosphorus on dry matter basis; NP-r, diet containing 0.9% phosphorus, fed isoenergetic restricted to the food intake of dogs fed a diet with 3.3% calcium and 3.0% phosphorus; NP-ad lib., diet containing 0.9% phosphorus, fed ad libitum.
S101
MINERAL EFFECTS ON CALCIUM KINETICS
aliquot was used for 45Ca counting with 10 mL scintillator (Instagel, Packard, Downers Grove, IL) in a liquid scintillation counter (Packard) using low potas sium glass vials. The collected urine of each dog was mixed and aliquots were processed for 45Cacounting. The daily calcium intake (mmol/kg BW) Vj was cal culated as the averaged intake of four consecutive days of the metabolic study. By means of a computerized nonlinear least-squares fitting procedure a bi-exponential function c(t) = Ae~at + Be~btwas fitted to the plasma 45Ca concentration. Employing the plasma concentration of stable calcium, c(t) was expressed in counts/mmol. The turnover T of the plasma equivalent pool (in terpreted as the exchangeable calcium pool) was cal culated as follows: T =D where D denotes the dose (injected amount of activity). T is expressed in mmol Ca-kg body weight"'-d"1: mmol • kg BW"1• d"1. The daily amount of calcium ab sorption (Va)was calculated from Va = "v^- (VF- Vf), where VFis the total daily fecal calcium excretion, and Vf is the daily endogenous fecal calcium excretion. Vf is given by R4//o c(t) • dt, where R4 is the part of the injected dose of 45Caexcreted in the feces during 4 d after intravenous 45Caadministration. The daily renal clearance of calcium (Vu,mmol/kg BW)was calculated with Vu = RS//O c(t) • dt, where R3 is the activity ex creted into the urine during 3 d after intravenous 45Ca administration. Intestinal absorption coefficient a was defined as a = Va/V, X 100% and calculated from (Dor- RF)/Dor X 100%, where Doris the orally administered activity and RFis the activity recovered from the feces. It was assumed that the 45Ca originating from endogenous fecal excretion after oral 45Ca administration was of negligible magnitude. Assuming that during the time course of the study the exchangeable calcium pool was in steady state, turnover = input = output and thus T = Va + V0- = Vu + Vf + V0+, where V„-= bone ré sorption and V0+ = calcium accretion, and thus V0+ = T - (Vu+ Vf) and V0~= T - Va. The calcium reten tion, defined as the difference between calcium accre tion and résorption,is given by V0+- V0~= Va - (Vu + Vf), which can be calculated. Statistics. Results of the kinetic studies were an alyzed for each experiment using nonparametric anal ysis of variances (Kruskal-Wallis test), followed by multiple comparisons (Dunn's procedure) in which the HC and LC groups were compared with the control groups (NCNP and NCNP-r). RESULTS The food intake in groups raised on LCNP diet dif fered: LCNP-1 (but not LCNP-2) groups ate more than
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their controls (NCNP). This was only statistically sig nificant at 14 wk of age (Fig. 1). The HCHP group appeared to have an ample food intake at an early age. As a consequence the food intake did not differ sig nificantly between the dogs fed the NCNP diet re stricted or ad libitum. Pooled values of the first study of blood chemistry and calcium metabolism at the age of 8 wk revealed the following mean (±SEM,n = 17) values: total plasma calcium and inorganic phosphorus concentra tions were 2.79 ±0.02 and 2.66 ±0.09 mmol/L, re spectively; Vj was 16.2 ±0.5 mmol-kg BW^-d"1, with an absorption coefficient (a) of 70 ±2.4%, VFwas 1.25 ±0.05 mmol-kg BW^-d"1, calcium accretion was 36.6 ±0.7 mmol-kg BW"1-d"1, calcium résorp tion was 27.3 ±0.8 mmol • kg BW"1• d"1, and calcium retention 9.8 ±0.5 mmol-kg BW"1-d"1. Vf was ~1 mmol-kg BW"1-d"1 at 8 wk of age. Vu became sig nificantly higher in the HC dogs [i.e., 0.4 mmol-kg BW^-d"1] than in the NC dogs [0.2 mmol-kg BW^-d-1] and LC dogs [80% of the calcium intake. This was especially impressive in the dogs fed the LCLP diet. Low calcium intake will be detected quickly by the parathyroid glands, which respond with an in creased parathyroid hormone (PTH) secretion (14). Under prolonged exposure to high levels of PTH, renal production of calcitriol will rise (15), resulting in an enhanced intestinal absorption of calcium and phos phorus to keep circulating calcium concentration within narrow physiological ranges. The high a for calcium found in the dogs fed the LC diets resulted in daily calcium absorption values comparable to those in the dogs fed the NC diet. This was especially notable in one group (LCNP-1 ), raised on a LC diet with a high food intake, at the age of 14 wk. The significantly high plasma phosphorus concentration in the LCLP group may reflect an elevated phosphorus absorption as well as an enhanced bone résorption,both mediated by cal citriol activity (15). Dogs raised on a diet with three times the recom mended amount of calcium but with a normal calcium: phosphorus ratio (i.e., HCHP diet), had indeed a sig nificantly lower a at 14 wk of age than the control dogs fed the NCNP diet restricted to be isoenergetic with the intake of the HCHP dogs. Although the ab sorption percentage in the HCHP group increased during the study, it stayed significantly behind that of the control dogs during the entire study. However, the value of a in dogs raised on the HCNP diet did not differ significantly from that in control dogs, ex cept at the age of 20 wk. As a consequence, the dogs fed the HCNP diet had a high calcium absorption dur ing the entire study, contrary to what was to be ex pected. The following model for calcium homeostasis in dogs raised on a diet with a high calcium content can be proposed tentatively: high calcium intake reduces PTH secretion and consequently calcitriol production (14). However, in the dogs raised on the diet containing 3.3% calcium and 0.9% phosphorus (i.e., HCNP diet), a tendency to hypophosphatemia developed at 14 wk of age, which may have compensated for the influence of the decreased PTH level on calcitriol synthesis (16, 17). As a result, this hypophosphatemia may have pre vented a decrease in active calcium absorption in the HCNP group, whereas in the dogs raised on the HCHP diet, the decrease in a was more effective. In this study, it appears that at least some dogs fed the HCHP diet eventually developed an adaptation mechanism to de crease calcium absorption to normal levels. At 20 wk of age, a was still lower in both groups fed the HC diet than in the control dogs, but at the end of the study the values of a in dogs raised on NC and HC diets reached the same magnitude (Fig. 6). The expla nation for this could be that calcium absorption is the sum of carrier-mediated active transport and passive
MINERAL EFFECTS ON CALCIUM KINETICS
diffusion, the latter predominating at a young age and in the presence of a high intestinal calcium concen tration (18). Thus, in the dogs raised on the HC diets, the amount of calcium absorbed became directly re lated to the daily calcium intake, suggesting that young dogs chronically fed a diet with high calcium content might lack an effective mechanism to protect them selves against excessive calcium absorption. A decrease in calcium accretion at 14 and 26 wk and in calcium résorptionduring the whole study in the dogs fed the HCNP diet, as well as the more pos itive values for calcium retention in both groups of growing dogs fed an HC diet, were apparent in the calcium kinetic studies. These findings are in accor dance with previously reported findings in the HCNP dogs, in which -y-densitometry (10) and bone histomorphometry (19) revealed the skeleton to be more mineralized than that of control dogs raised on the NCNP diet. On the contrary, no differences were found in values for accretion, résorption,and consequently, retention of calcium in dogs fed the LC diet, in spite of the fact that these dogs developed severe clinical and histomorphometrical signs of osteoporosis, including pathological fractures (20). A possible explanation for this contrast is that these calcium kinetic studies do not give a reliable insight into the degree of skeletal mineralization when bone turnover is rapid. One cause could be a return of tracer-containing calcium to the labile pool by way of bone résorptionbefore equilib rium has been achieved within that pool. This has been suggested to occur in humans with Paget's disease (2). In addition, these calcium kinetic parameters will not reflect a reuse of calcium within the stabile pool with out calcium entering the labile pool, which may play a significant role during fast modeling of the rapidly growing skeleton when calcium moves inside the bone fluid compartment (14). Thus, identical values for ac cretion, résorptionand retention do not imply iden tical remodeling rates. Great Danes fed a restricted amount of food rich in protein, energy and calcium had less severe skeletal abnormalities than those fed ad libitum (21). This led Hedhammar et al. (21) to the conclusion that overnutrition of growing dogs should be avoided. In the present study, however, the food intake of the NCNPr and NCNP-ad lib.3 dogs did not differ; hence, our findings do not allow for any conclusions in this re gard. In contrast, the HCNP dogs with a lower food intake than the NCNP dogs at the age of 14 wk had severe skeletal abnormalities. Whether osteochondrosis is the consequence of hypercalcitoninemia due to a high calcium absorption (22-24) or is a direct ef fect of calcium on cartilage (25) needs to be elucidated. From the findings of this study it may be concluded that the minimum requirements for calcium as given in the NRC Nutrient Requirements of Dogs 1985 (12) Downloaded from https://academic.oup.com/jn/article-abstract/121/suppl_11/S99/4744095 by Washington University School of Medicine Library user on 06 June 2018
S105
(i.e., 0.59% calcium on dry matter basis) is most prob ably too low for rapidly growing dogs of a large breed, especially when it is taken into account that the avail ability of calcium from the diet of our study (Table 2) is high in comparison with dry dog food with a higher content of phytates. In summary, it is clear from these studies that in dogs on a low calcium intake, absorp tion can increase to S;80% and that in dogs raised on a diet with an excessive calcium content calcium re tention increases proportionally. Within the limits of the calciumrphosphorus ratios of this study, it can be concluded that the calcium:phosphorus ratio is not as important a long-term influence on calcium absorption and retention as is the calcium content per se in the diet in young rapidly growing dogs. LITERATURE
CITED
1. URIST,M. R. (1989) Bone morphogenetic protein, bone re generation, heterotropic ossification and the bone-bone marrow consortium. In: Bone and Mineral Research (Peck, W. A., ed.), vol. 6, pp. 57-112, Elsevier, Amsterdam, The Netherlands. 2. HARRIS,W. H. & HEANEY,R. P. (1969) Skeletal renewal and metabolic bone disease. New Engl. /. Med. 280: 193202. 3. PATERSON, C. R. (1974) Metabolic Disorders of Bone. Blackwell Scientific Publications, Oxford, United Kingdom. 4. HILLMAN, L. S.,TACK,E., COVELL, D. G., VIEIRA,N. E. & YERGEY, A. H. (1988) Measurement of true calcium absorption in pre mature infants using intravenous "Ca and oral "Ca. Pediatr. Res. 23(6): 589-594. 5. HAZEWINKEL, H. A. W., HACKENG,W. H. L., BOSCH,R., GOEDEGEBUURE, S. A., VOORHOUT,
G., VAN DEN BROM, W. E. &
BEVERS, M. M. (1987) Influences of different calcium intakes on calciotropic hormones and skeletal developments in young growing dogs. Front. Norm. Res. 17: 221-232. 6. LEE,W. R., MARSHALL,J. H. & SISSONS,H. A. (1965) Calcium accretion and bone formation in dogs: an experimental com parison between the results of Ca45kinetic analysis and tetracycline labelling. /. Bone Joint Surg. [ßrj 47B: 157-180. 7. GERSHOFF,S. N., LEGG,M. A. & HEGSTEDT,D. M. (1958) Adaptation to different calcium intakes in dogs. /. Nutr. 64: 303-312. 8. LEWIS,L. D., MORRIS,M. L. & HAND,M. S. (1987) Small An imal Clinical Nutrition 111,Mark Morris Associates, Topeka, KS, pp. 1-1-13-18. 9. NATIONALRESEARCH COUNCIL(1974) Nutrient Requirements of Dogs. National Academy Press, Washington DC. 10. HAZEWINKEL, H. A. W., GOEDEGEBUURE, S. A., POULOS, P. W. & WOLVEKAMP, W. TH. C. (1985) Influences of chronic calcium excess on the skeletal development of growing Great Danes. Am. Anim. Hasp. Assoc. 21: 377-391. 11. KIRK,R. W. (1966) Growth curves for fifteen breeds of dogs. In: Current Veterinary Therapy 111(Kirk, R. W., ed.) p. 716, W. B. Saunders, Philadelphia, PA. 12. NATIONAL RESEARCH COUNCIL(1985) Nutrient Requirements of Dogs. National Academy Press, Washington DC. 13. SHEFFY, B. E. (1989) The 1985 revision of the National Research Council nutrient requirements of dogs and its impact on the pet food industry. In: Nutrient of the Dog and Cat, Waltham Symposium number 7 (Burger, I. H. & Rivers, J. P. W., eds.), pp. 11-26, Cambridge University Press, Cambridge, United Kingdom.
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14. WONG,K. M. & KLEIN,L. (1981) Fluctuation in bone and di etary contribution to blood calcium, in normal, calcium deficient and thyroparathyroidectomized dogs. Calci/. Tissue Int. 33: 301 (abs.) 15. MARCUS,R. (1988) Endocrine control of bone and mineral me tabolism. In: Metabolic Bone and Mineral Disorders (Manolagas, S. C. &.Olefsky, J. M., eds.), pp. 13-32, Churchill Livingstone, New York, NY. le. DELUCA,H. F. &. SCHNOES,H. K. (1983) Vitamin D: recent advances. Ann. Rev. Biochem. 52: 411-439. 17. PERAULT-STAUB, A. M., STAUB,J. F. & MILHAUD,G. (1990) Extracellular calcium homeostasis. In: Bone and Mineral Re search 7 (Heersche, N. M. & Kanis, J. A., eds.), pp. 1-102, Elsevier, Amsterdam, The Netherlands. 18. ALLEN,L. H. (1982) Calcium bioavailability and absorption: a review. Am. /. Clin. Nutr. 35: 783-808. 19. GOEDEGEBUURE,
S.
A.
&
HAZEWINKEL,
H.
A.
W.
(1986)
Morphological findings in young dogs chronically fed a diet containing excess calcium. Vet. Pathol. 23: 594-605.
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ET AL. 20. VOORHOUT,G. & HAZEWINKEL, H. A. W. (1987) Radiographie studies on the skeletal development in Great Danes on different calcium intakes. Vet. Radial. 28: 152-157. 21. HEDHAMMER, A., Wu, F. M., KROOK,L., SCHRYVER, H. F., DE LAHUNTA,A., WHALEN,J. P., KALLFELZ, F. A., NUNEZ,E. A., HINTZ,H. F., SHEFFY, B. E. & RYAN,G. D. (1974) Overnutrition and skeletal disease, an experimental study in growing Great Dane dogs. Cornell Vet. 64(Suppl. 5): 11-160. 22. TALMAGE, R. V., COOPER,C.W. & TOVERUD,S.U. (1983) The physiological significance of calcitonin. In: Bone and Mineral Research Annual 1 (Peck, W. A., éd.),pp. 74-143, Excerpta Medica, Amsterdam, The Netherlands. 23. AZRIA,M. (1989) The Calcitonins, Physiology and Pharma cology, Karger, New York, NY, pp. 1-152. 24. CORBELLINI, C. N., KROOK,L., NATHANIELSZ, P. W. & KALLFELZ, F. A. (1991) Osteochondrosis in fetuses of ewes overfed cal cium. Calcif. Tissue Int. 48: 37-45. 25. HOWELL,D. S., PITA,J. C. & ALVAREZ, J. (1976) Possible role of extracellular matrix vesicles in initial calcification of healing rachitic cartilage. Fed. Proc. 35: 122-126.
Calcium Metabolism in Great Dane Dogs Fed Diets with Various Calcium and Phosphorus Levels1 HERMAN A. W. HAZEWINKEL,*2 WALTER E. VAN DEN BROM* ARIE TH. VAN T KLOOSTERj GEORGE VOORHOUTÃŽ AND ANK VAN WEES* 'Department of Clinical Sciences of Companion Animals, ^Department of Husbandry and nutrition, and ^Department of Veterinary Radiology, Faculty of Veterinary Medicine, Uniuersity of Utrecht, The Netherlands in a calcium absorption and retention not differing from that in control dogs, the latter despite the occurrance of severe osteoporosis. J. Nutr. 121 : S99-S106,1991.
ABSTRACT The influence of dietary calcium on cal cium metabolism was investigated in growing dogs that reach an adult body weight similar to that of humans. Seven groups of dogs (n > 5) were raised on a diet with a composition meeting the National Research Council (NRC) requirements (1974), but differing in calcium content, with or without a constant ratio to phosphorus. Control dogs fed 1.1% calcium and 0.9% phosphorus (all on a dry matter basis) were fed ad libitum (n = 10) or in restricted amounts (n = 6); dogs fed high calcium (3.3%) diets received either 0.9% phosphorus (n = 6) or 3.0% phosphorus (n = 6); dogs fed low calcium (0.55%) diets received either 0.9% phosphorus (n = 5 + 6) or 0.5% phosphorus (n = 8). Food intake, circulating total calcium and inorganic phosphorus concentrations and calcium metabolism, with 45Ca kinetics, were studied at 8, 14, 20 and 26 wk of age. Except for the difference in food intake in two groups at 14 wk (i.e., 0.55% calcium-0.9% phos phorus higher and 3.3% calcium-0.9% phosphorus lower, respectively, than the controls) no differences were noticed during the rest of the study. The mean plasma calcium concentrations did not differ between groups during the studies, whereas that of inorganic phosphorus revealed temporal aberrations in two groups. An absorption coefficient a of 45-66% was found for the control group. High and low calcium diets gave rise to values of 23-43% and 70-97%, respec tively, for a irrespective of the phosphorus content of the diet. The amount of calcium absorbed was nev ertheless considerably higher in the dogs fed the high calcium diet than in the control dogs. The dogs fed the high calcium diets had also a higher calcium retention than found in the low calcium group, which was not different from the control group. The conclusions were young dogs fed a diet with a calcium content of 3.3% (with either 0.9 or 3.0% phosphorus) have a signifi cantly elevated absorption and retention of calcium and seem unable to protect themselves against chronic ex cessive calcium intake, and young dogs of this large breed elevate the absorption of calcium to >90% of the ingested amount when raised on a diet with 0.55% calcium (with either 0.5 or 0.9% phosphorus), resulting
0022-3166/91
INDEXING KEY WORDS:
•symposium •dogs •dietary calcium •dietary phosphate •skeletal mineralization •45Cakinetics
Dogs, especially those with an adult body weight similar to that of humans (i.e., 60-70 kg), are of in terest for studies of calcium metabolism for clinical as well as comparative nutritional knowledge. More than any other laboratory mammalian species, dogs have a metabolic activity remarkably similar to that of humans (1). Net absorption and retention of calcium can be measured with radioactive or stable calcium isotopes (2-5). It has been shown in the young dog that any gain or loss of calcium to the body is a gain or loss to the skeleton and is therefore considered to be a good estimate of bone mineralization (6). Because dogs (7), like humans (3), are able to adapt to lower or higher levels of calcium intake, a diet of constant calcium content should be fed to allow for conclusions from a long-term study of calcium me tabolism. Since the phosphorus and calcium content of the diet may reciprocally influence absorption and accretion of both nutrients, the phosphorus content of the diet should be taken into account (8). To gain 1 Presented as part of the Waltham International Symposium on Nutrition of Small Companion Animals, at University of California, Davis, CA 95616, on September 4-8, 1990. Guest editors for the symposium were James G. Morris, D'Ann C. Finley and Quinton R. Rogers. 1 To whom correspondence should be addressed: Department of Clinical Sciences of Companion Animals, University Utrecht, P.O. Box 80.154, 3508 TD Utrecht, The Netherlands.
$3.00 ©1991 American Institute of Nutrition.
$99 Downloaded from https://academic.oup.com/jn/article-abstract/121/suppl_11/S99/4744095 by Washington University School of Medicine Library user on 06 June 2018
HAZEWINKEL
SlOO
more insight into the effects of intakes of calcium higher and lower than the requirement (9), with or without constant ratios to phosphorus, calcium me tabolism was studied in seven groups of Great Dane dogs during growth from weaning until one-half of adult weight. Because a variety of nutritional factors, including the ratio of calcium to phosphorus, the presence of phytin and fat content may influence cal cium availability, a dry dog food differing only in its calcium and/or phosphorus content was used and at tempts were made to standardize all other factors.
MATERIALS AMD METHODS Animais. Forty-seven Great Dane dogs, from eight different litters, 5-10 wk old, were studied in several groups of five to six dogs each, in which males and females were divided at random. Dogs were individ ually housed in cages with access to an outside run; during the kinetic studies the animals were housed individually in metabolism cages. Water was available ad libitum. Each group of dogs was fed one diet for the entire period of the study, ranging from 11 to 26 wk. Diets. The diet differed among groups only in cal cium and/or phosphorus content, as given in Table 1, and otherwise met the U.S. National Research Coun cil's Nutrient Requirements of Dogs, 1974 (9). The composition of the diet used in all studies is given in Table 2. The food intake of the HCHP3 group was expected to be low, based on previous experiences with
TABLE 1
Dietary calcium and phosphorus and number of Great Dane dogs per experimental group1 Calcium
Phosphorus
g/ÃŽOO g dry matter Normal dietsNCNP calcium lib.NCNP-rHigh ad dietsHCNPHCHPLow calcium
ET AL. TABLE 2
Composition of the dry feed used in the different experiments1
Ingredient Bloodmeal (spray dehydrated) Casein (dehydrated) Soybean flour Corn gluten meal Corn/potato starch mixture Tallow Soybean oil Sugar DL-Methionine Cellulose Vitamin/Mineral supplement1
are used to indicate the Ca and P
content of the diets (given as percentage of the dry matter basis of the diets) as fed to the experimental dogs: LCLP, 0.55% Ca-0.5% P; LCNP-1 and LCNP-2,0.55% Ca-0.9%P; NCNP and NCNP-r, 1.1% Ca-0.9% P; HCNP, 3.3% Ca-0.9% P; HCHP, 3.3% Ca-3.0% P (-1 and -2 refer to successive experiments, -r to restricted feeding as indicated in the text).
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5.00-381 5.01-162 5.04-593 5.02-900 4.02-889/4.07-850 4.08-128 4.07-983
g/kg diet
62 57 100 62 491 68 20 50 2 20 60
1 Proximate analysis (in g/100 g): Crude protein, 21.0; Ether ex tract, 9.9; N-Free extract, 49.5; Crude Fiber, 3.0; Ash, 6.6; Moisture, 10.0. 2 Additives: retinyl acetate, 5000 IE/kg; cholecalciferol, 1000 IE/ kg; dl-a-tocopheryl acetate, 50 IE/kg; B.H.T., 1 g/kg; Monocalcium phosphate, Potassium phosphate, Calcium carbonate.
feeding the HCNP3 diet. For that reason a separate control group (NCNP-r)3 was fed isoenergetically re stricted to the intake of the HCHP dogs. All other dogs were fed ad libitum. LCNP3 diet (calcium 0.55%, phosphorus 0.9%) was used in two separate experi ments and the results are given separately (i.e., LCNPl3 and LCNP-23). Blood chemistry. Blood collection and investi gation for total plasma calcium and plasma inorganic phosphorus was performed as described previousCalcium kinetics. Calcium kinetic studies were performed with 45Ca (Amersham International, Amersham, UK; specific activity 12 mBq/mg) admin istered intravenously (185 kBq) and, 4-7 d later, orally (370-540 kBq) at the age of 8, 14, 20 and 26 wk. Urine and feces were collected for 3 and 4 days after 45Ca administration, respectively. Feces were mixed and a sample was dried, ashed and diluted with HC1. An
dietsLCNP,LCNPZLCLP1.11.13.33.30.550.550.550.90.90.93.00.90.90.510666568 calcium
1The following abbreviations
International Feed Number
3 Abbreviations: HC, high calcium = 3.3% calcium on dry matter basis; HP, high phosphorus = 3.0% phosphorus on dry matter basis; LC, low calcium = 0.55% calcium on dry matter basis; LP, low phos phorus = 0.5% phosphorus on dry matter basis; NC, normal calcium = 1.1% calcium on dry matter basis (according to 1974 NRC); NP, normal phosphorus = 0.9% phosphorus on dry matter basis (ac cording to 1974 NRC); NP-1 and 2, two separate experiments with diet containing 0.9% phosphorus on dry matter basis; NP-r, diet containing 0.9% phosphorus, fed isoenergetic restricted to the food intake of dogs fed a diet with 3.3% calcium and 3.0% phosphorus; NP-ad lib., diet containing 0.9% phosphorus, fed ad libitum.
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MINERAL EFFECTS ON CALCIUM KINETICS
aliquot was used for 45Ca counting with 10 mL scintillator (Instagel, Packard, Downers Grove, IL) in a liquid scintillation counter (Packard) using low potas sium glass vials. The collected urine of each dog was mixed and aliquots were processed for 45Cacounting. The daily calcium intake (mmol/kg BW) Vj was cal culated as the averaged intake of four consecutive days of the metabolic study. By means of a computerized nonlinear least-squares fitting procedure a bi-exponential function c(t) = Ae~at + Be~btwas fitted to the plasma 45Ca concentration. Employing the plasma concentration of stable calcium, c(t) was expressed in counts/mmol. The turnover T of the plasma equivalent pool (in terpreted as the exchangeable calcium pool) was cal culated as follows: T =D where D denotes the dose (injected amount of activity). T is expressed in mmol Ca-kg body weight"'-d"1: mmol • kg BW"1• d"1. The daily amount of calcium ab sorption (Va)was calculated from Va = "v^- (VF- Vf), where VFis the total daily fecal calcium excretion, and Vf is the daily endogenous fecal calcium excretion. Vf is given by R4//o c(t) • dt, where R4 is the part of the injected dose of 45Caexcreted in the feces during 4 d after intravenous 45Caadministration. The daily renal clearance of calcium (Vu,mmol/kg BW)was calculated with Vu = RS//O c(t) • dt, where R3 is the activity ex creted into the urine during 3 d after intravenous 45Ca administration. Intestinal absorption coefficient a was defined as a = Va/V, X 100% and calculated from (Dor- RF)/Dor X 100%, where Doris the orally administered activity and RFis the activity recovered from the feces. It was assumed that the 45Ca originating from endogenous fecal excretion after oral 45Ca administration was of negligible magnitude. Assuming that during the time course of the study the exchangeable calcium pool was in steady state, turnover = input = output and thus T = Va + V0- = Vu + Vf + V0+, where V„-= bone ré sorption and V0+ = calcium accretion, and thus V0+ = T - (Vu+ Vf) and V0~= T - Va. The calcium reten tion, defined as the difference between calcium accre tion and résorption,is given by V0+- V0~= Va - (Vu + Vf), which can be calculated. Statistics. Results of the kinetic studies were an alyzed for each experiment using nonparametric anal ysis of variances (Kruskal-Wallis test), followed by multiple comparisons (Dunn's procedure) in which the HC and LC groups were compared with the control groups (NCNP and NCNP-r). RESULTS The food intake in groups raised on LCNP diet dif fered: LCNP-1 (but not LCNP-2) groups ate more than
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their controls (NCNP). This was only statistically sig nificant at 14 wk of age (Fig. 1). The HCHP group appeared to have an ample food intake at an early age. As a consequence the food intake did not differ sig nificantly between the dogs fed the NCNP diet re stricted or ad libitum. Pooled values of the first study of blood chemistry and calcium metabolism at the age of 8 wk revealed the following mean (±SEM,n = 17) values: total plasma calcium and inorganic phosphorus concentra tions were 2.79 ±0.02 and 2.66 ±0.09 mmol/L, re spectively; Vj was 16.2 ±0.5 mmol-kg BW^-d"1, with an absorption coefficient (a) of 70 ±2.4%, VFwas 1.25 ±0.05 mmol-kg BW^-d"1, calcium accretion was 36.6 ±0.7 mmol-kg BW"1-d"1, calcium résorp tion was 27.3 ±0.8 mmol • kg BW"1• d"1, and calcium retention 9.8 ±0.5 mmol-kg BW"1-d"1. Vf was ~1 mmol-kg BW"1-d"1 at 8 wk of age. Vu became sig nificantly higher in the HC dogs [i.e., 0.4 mmol-kg BW^-d"1] than in the NC dogs [0.2 mmol-kg BW^-d-1] and LC dogs [80% of the calcium intake. This was especially impressive in the dogs fed the LCLP diet. Low calcium intake will be detected quickly by the parathyroid glands, which respond with an in creased parathyroid hormone (PTH) secretion (14). Under prolonged exposure to high levels of PTH, renal production of calcitriol will rise (15), resulting in an enhanced intestinal absorption of calcium and phos phorus to keep circulating calcium concentration within narrow physiological ranges. The high a for calcium found in the dogs fed the LC diets resulted in daily calcium absorption values comparable to those in the dogs fed the NC diet. This was especially notable in one group (LCNP-1 ), raised on a LC diet with a high food intake, at the age of 14 wk. The significantly high plasma phosphorus concentration in the LCLP group may reflect an elevated phosphorus absorption as well as an enhanced bone résorption,both mediated by cal citriol activity (15). Dogs raised on a diet with three times the recom mended amount of calcium but with a normal calcium: phosphorus ratio (i.e., HCHP diet), had indeed a sig nificantly lower a at 14 wk of age than the control dogs fed the NCNP diet restricted to be isoenergetic with the intake of the HCHP dogs. Although the ab sorption percentage in the HCHP group increased during the study, it stayed significantly behind that of the control dogs during the entire study. However, the value of a in dogs raised on the HCNP diet did not differ significantly from that in control dogs, ex cept at the age of 20 wk. As a consequence, the dogs fed the HCNP diet had a high calcium absorption dur ing the entire study, contrary to what was to be ex pected. The following model for calcium homeostasis in dogs raised on a diet with a high calcium content can be proposed tentatively: high calcium intake reduces PTH secretion and consequently calcitriol production (14). However, in the dogs raised on the diet containing 3.3% calcium and 0.9% phosphorus (i.e., HCNP diet), a tendency to hypophosphatemia developed at 14 wk of age, which may have compensated for the influence of the decreased PTH level on calcitriol synthesis (16, 17). As a result, this hypophosphatemia may have pre vented a decrease in active calcium absorption in the HCNP group, whereas in the dogs raised on the HCHP diet, the decrease in a was more effective. In this study, it appears that at least some dogs fed the HCHP diet eventually developed an adaptation mechanism to de crease calcium absorption to normal levels. At 20 wk of age, a was still lower in both groups fed the HC diet than in the control dogs, but at the end of the study the values of a in dogs raised on NC and HC diets reached the same magnitude (Fig. 6). The expla nation for this could be that calcium absorption is the sum of carrier-mediated active transport and passive
MINERAL EFFECTS ON CALCIUM KINETICS
diffusion, the latter predominating at a young age and in the presence of a high intestinal calcium concen tration (18). Thus, in the dogs raised on the HC diets, the amount of calcium absorbed became directly re lated to the daily calcium intake, suggesting that young dogs chronically fed a diet with high calcium content might lack an effective mechanism to protect them selves against excessive calcium absorption. A decrease in calcium accretion at 14 and 26 wk and in calcium résorptionduring the whole study in the dogs fed the HCNP diet, as well as the more pos itive values for calcium retention in both groups of growing dogs fed an HC diet, were apparent in the calcium kinetic studies. These findings are in accor dance with previously reported findings in the HCNP dogs, in which -y-densitometry (10) and bone histomorphometry (19) revealed the skeleton to be more mineralized than that of control dogs raised on the NCNP diet. On the contrary, no differences were found in values for accretion, résorption,and consequently, retention of calcium in dogs fed the LC diet, in spite of the fact that these dogs developed severe clinical and histomorphometrical signs of osteoporosis, including pathological fractures (20). A possible explanation for this contrast is that these calcium kinetic studies do not give a reliable insight into the degree of skeletal mineralization when bone turnover is rapid. One cause could be a return of tracer-containing calcium to the labile pool by way of bone résorptionbefore equilib rium has been achieved within that pool. This has been suggested to occur in humans with Paget's disease (2). In addition, these calcium kinetic parameters will not reflect a reuse of calcium within the stabile pool with out calcium entering the labile pool, which may play a significant role during fast modeling of the rapidly growing skeleton when calcium moves inside the bone fluid compartment (14). Thus, identical values for ac cretion, résorptionand retention do not imply iden tical remodeling rates. Great Danes fed a restricted amount of food rich in protein, energy and calcium had less severe skeletal abnormalities than those fed ad libitum (21). This led Hedhammar et al. (21) to the conclusion that overnutrition of growing dogs should be avoided. In the present study, however, the food intake of the NCNPr and NCNP-ad lib.3 dogs did not differ; hence, our findings do not allow for any conclusions in this re gard. In contrast, the HCNP dogs with a lower food intake than the NCNP dogs at the age of 14 wk had severe skeletal abnormalities. Whether osteochondrosis is the consequence of hypercalcitoninemia due to a high calcium absorption (22-24) or is a direct ef fect of calcium on cartilage (25) needs to be elucidated. From the findings of this study it may be concluded that the minimum requirements for calcium as given in the NRC Nutrient Requirements of Dogs 1985 (12) Downloaded from https://academic.oup.com/jn/article-abstract/121/suppl_11/S99/4744095 by Washington University School of Medicine Library user on 06 June 2018
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(i.e., 0.59% calcium on dry matter basis) is most prob ably too low for rapidly growing dogs of a large breed, especially when it is taken into account that the avail ability of calcium from the diet of our study (Table 2) is high in comparison with dry dog food with a higher content of phytates. In summary, it is clear from these studies that in dogs on a low calcium intake, absorp tion can increase to S;80% and that in dogs raised on a diet with an excessive calcium content calcium re tention increases proportionally. Within the limits of the calciumrphosphorus ratios of this study, it can be concluded that the calcium:phosphorus ratio is not as important a long-term influence on calcium absorption and retention as is the calcium content per se in the diet in young rapidly growing dogs. LITERATURE
CITED
1. URIST,M. R. (1989) Bone morphogenetic protein, bone re generation, heterotropic ossification and the bone-bone marrow consortium. In: Bone and Mineral Research (Peck, W. A., ed.), vol. 6, pp. 57-112, Elsevier, Amsterdam, The Netherlands. 2. HARRIS,W. H. & HEANEY,R. P. (1969) Skeletal renewal and metabolic bone disease. New Engl. /. Med. 280: 193202. 3. PATERSON, C. R. (1974) Metabolic Disorders of Bone. Blackwell Scientific Publications, Oxford, United Kingdom. 4. HILLMAN, L. S.,TACK,E., COVELL, D. G., VIEIRA,N. E. & YERGEY, A. H. (1988) Measurement of true calcium absorption in pre mature infants using intravenous "Ca and oral "Ca. Pediatr. Res. 23(6): 589-594. 5. HAZEWINKEL, H. A. W., HACKENG,W. H. L., BOSCH,R., GOEDEGEBUURE, S. A., VOORHOUT,
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BEVERS, M. M. (1987) Influences of different calcium intakes on calciotropic hormones and skeletal developments in young growing dogs. Front. Norm. Res. 17: 221-232. 6. LEE,W. R., MARSHALL,J. H. & SISSONS,H. A. (1965) Calcium accretion and bone formation in dogs: an experimental com parison between the results of Ca45kinetic analysis and tetracycline labelling. /. Bone Joint Surg. [ßrj 47B: 157-180. 7. GERSHOFF,S. N., LEGG,M. A. & HEGSTEDT,D. M. (1958) Adaptation to different calcium intakes in dogs. /. Nutr. 64: 303-312. 8. LEWIS,L. D., MORRIS,M. L. & HAND,M. S. (1987) Small An imal Clinical Nutrition 111,Mark Morris Associates, Topeka, KS, pp. 1-1-13-18. 9. NATIONALRESEARCH COUNCIL(1974) Nutrient Requirements of Dogs. National Academy Press, Washington DC. 10. HAZEWINKEL, H. A. W., GOEDEGEBUURE, S. A., POULOS, P. W. & WOLVEKAMP, W. TH. C. (1985) Influences of chronic calcium excess on the skeletal development of growing Great Danes. Am. Anim. Hasp. Assoc. 21: 377-391. 11. KIRK,R. W. (1966) Growth curves for fifteen breeds of dogs. In: Current Veterinary Therapy 111(Kirk, R. W., ed.) p. 716, W. B. Saunders, Philadelphia, PA. 12. NATIONAL RESEARCH COUNCIL(1985) Nutrient Requirements of Dogs. National Academy Press, Washington DC. 13. SHEFFY, B. E. (1989) The 1985 revision of the National Research Council nutrient requirements of dogs and its impact on the pet food industry. In: Nutrient of the Dog and Cat, Waltham Symposium number 7 (Burger, I. H. & Rivers, J. P. W., eds.), pp. 11-26, Cambridge University Press, Cambridge, United Kingdom.
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14. WONG,K. M. & KLEIN,L. (1981) Fluctuation in bone and di etary contribution to blood calcium, in normal, calcium deficient and thyroparathyroidectomized dogs. Calci/. Tissue Int. 33: 301 (abs.) 15. MARCUS,R. (1988) Endocrine control of bone and mineral me tabolism. In: Metabolic Bone and Mineral Disorders (Manolagas, S. C. &.Olefsky, J. M., eds.), pp. 13-32, Churchill Livingstone, New York, NY. le. DELUCA,H. F. &. SCHNOES,H. K. (1983) Vitamin D: recent advances. Ann. Rev. Biochem. 52: 411-439. 17. PERAULT-STAUB, A. M., STAUB,J. F. & MILHAUD,G. (1990) Extracellular calcium homeostasis. In: Bone and Mineral Re search 7 (Heersche, N. M. & Kanis, J. A., eds.), pp. 1-102, Elsevier, Amsterdam, The Netherlands. 18. ALLEN,L. H. (1982) Calcium bioavailability and absorption: a review. Am. /. Clin. Nutr. 35: 783-808. 19. GOEDEGEBUURE,
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ET AL. 20. VOORHOUT,G. & HAZEWINKEL, H. A. W. (1987) Radiographie studies on the skeletal development in Great Danes on different calcium intakes. Vet. Radial. 28: 152-157. 21. HEDHAMMER, A., Wu, F. M., KROOK,L., SCHRYVER, H. F., DE LAHUNTA,A., WHALEN,J. P., KALLFELZ, F. A., NUNEZ,E. A., HINTZ,H. F., SHEFFY, B. E. & RYAN,G. D. (1974) Overnutrition and skeletal disease, an experimental study in growing Great Dane dogs. Cornell Vet. 64(Suppl. 5): 11-160. 22. TALMAGE, R. V., COOPER,C.W. & TOVERUD,S.U. (1983) The physiological significance of calcitonin. In: Bone and Mineral Research Annual 1 (Peck, W. A., éd.),pp. 74-143, Excerpta Medica, Amsterdam, The Netherlands. 23. AZRIA,M. (1989) The Calcitonins, Physiology and Pharma cology, Karger, New York, NY, pp. 1-152. 24. CORBELLINI, C. N., KROOK,L., NATHANIELSZ, P. W. & KALLFELZ, F. A. (1991) Osteochondrosis in fetuses of ewes overfed cal cium. Calcif. Tissue Int. 48: 37-45. 25. HOWELL,D. S., PITA,J. C. & ALVAREZ, J. (1976) Possible role of extracellular matrix vesicles in initial calcification of healing rachitic cartilage. Fed. Proc. 35: 122-126.
