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*
Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, CA 95616, and
Waltham Centre for Pet Nutrition, Waltham-on-the-Wolds, Melton Mowbray, LE14 4RT, United Kingdom
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • cholecalciferol • 25-hydroxyvitamin D • kitten • cats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Hazewinkel et al. (1987)
reported that dogs exposed to
UV light were not able to synthesize adequate vitamin D to prevent
clinical signs of vitamin D deficiency. Morris (1999)
also found that plasma concentration of 25-hydroxyvitamin D (25-OHD) of
kittens given a vitamin D-free diet and exposed to direct summer sun in
California for 15 h/wk declined at the same rate as kittens kept
indoors, and they developed clinical signs of vitamin D deficiency.
Similarly, no synthesis was observed under laboratory conditions where
cats were exposed for 3 h/d to UV lights, and these cats also developed
clinical signs of vitamin D deficiency.
How et al. (1994)
did not find pre-vitamin D in isolated
cat skin exposed to UVB light and attributed the lack of synthesis to a
low concentration of 7-dehydrocholesterol (the precursor of pre-vitamin
D) in the skin of cats. Morris (1999)
demonstrated that
vitamin D synthesis could be induced in vitamin D-deficient cats by
administering an inhibitor of
7-dehydrocholesterol-
7-reductase. The inhibitor produced
a fivefold increase in skin concentration of 7-dehydrocholesterol, and
the cats had increasing levels of 25-OHD in their plasma with time of
exposure. Vitamin D-deficient cats exposed to UV light and not given
the inhibitor had no increase in the concentration of 25-OHD in plasma.
Vitamin D deficiency in growing kittens was investigated by
Gershoff et al. (1957)
, who gave three- to six-mo-old
kittens a purified vitamin D-free diet based on casein, corn oil and
hydrogenated fat as sources of protein and fat. The diet was
supplemented with most vitamins, but did not contain supplemental
sources of taurine, vitamin E or arachidonate (or its precursor). These
authors reported that the clinical signs of vitamin D deficiency could
be prevented by oral administration of 6.25 µg (250 IU) of
cholecalciferol twice weekly. A high incidence of deaths occurred in
kittens given the diet with added vitamin D, so the requirement is
questionable. Rivers et al. (1979)
observed no signs of
vitamin D deficiency in adult cats fed a vitamin D-free diet for over a
year and suggested that the requirement of vitamin D for adult cats may
be very low. Based on the experiment of Gershoff et al. (1957)
, the National Research Council (1986)
proposed a minimal vitamin D requirement for growing kittens of 12.5
µg cholecalciferol (500 IU)/kg diet dry matter.
The objective of this study was to determine the dietary concentrations of cholecalciferol that maintained plasma 25-OHD at a concentration normal for other mammals. An additional aim of the study was to avoid overt clinical signs of vitamin D deficiency and the possible animal discomfort associated with it.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Kittens used in the study were produced from British domestic shorthair
queens given an expanded (dry) diet containing only the vitamin D
derived from the natural ingredients. The diet was given to the queens
before mating, during gestation (approximately 64 d) and
lactation. This procedure was followed to reduce maternal transfer of
vitamin D, so vitamin D status of the kittens entering the study would
not be high. Kittens were given a purified diet without supplemented
vitamin D at 3 wk of age to accustom them to a purified diet. Weaning
began at 7 wk of age, and the kittens continued to receive the vitamin
D-free diet until 9 wk of age when they were randomly assigned to the
experimental diets containing the graded levels of vitamin D. After
weaning, kittens were housed in individual enclosures (1.15 x
0.60 x 0.55 m) with food and water available at all times.
The temperature and light/dark cycle in the room was 21 ± 2°C
and a 12/12 h, respectively. The experimental protocol adhered to the
Guide for the Care and Use of Laboratory Animals
(National Research Council 1985
).
Diet.
A purified vitamin D-free diet was prepared from the ingredients given
in Table 1.
Because the diet was devoid of animal fat and because the synthesis of
arachidonate by cats is limited, evening primrose oil was added as a
source of 
-linoleic acid. The basal diet was formulated to provide
all essential nutrients (other than vitamin D) at levels in excess of
those recommended by the National Research Council (1986)
and was confirmed by analysis. Concentration of calcium
and phosphorus on analysis was 12 and 8 g/kg diet dry matter,
respectively. The basal diet was analyzed for vitamin D by a method
adapted to pet foods (Morris 1999
). To the basal diet,
0.0, 3.125, 6.25, 12.5, 18.75 or 25 µg/kg of cholecalciferol (Sigma
Reference Standard; Sigma Chemical Co., St. Louis, MO) in sunflower oil
was added. The added cholecalciferol was equivalent to 0, 125, 250,
500, 750 or 1000 IU of cholecalciferol/kg.
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As kittens were weaned, they were assigned to the dietary treatments on
the basis of sex, such that each treatment contained four female and
three male kittens. All kittens received the diets containing various
levels of vitamin D until they were 22 wk of age. The two groups of
kittens assigned to the 0.0 and 3.125 µg/kg diets continued these
dietary treatments until 34 wk of age. Samples of blood were taken in
heparinized syringes from unanesthetized kittens at 7, 9, 10, 14, 18
and 22 wk of age, and also from the kittens in the 0.0 and 3.125 µg
groups at 26, 30 and 34 wk of age. The following measurements were made
on the samples collected at wk 9 and 20: total calcium and phosphorus
(Coulter CPA Analyzer; Coulter Electronics, Luton, Beds) and ionized
potassium and ionized calcium by a Ciba-Corning 228 Analyzer
(Ciba-Corning Medfield, MA); at 8 and 20 wk of age: hemoglobin,
packed-cell volume, red and white cell numbers (Serono-Baker
Diagnostics System Analyzer 9000; Allentown, PA), total plasma protein,
albumin, urea and cholesterol concentrations, and alanine and aspartic
aminotransferase activity (Coulter CPA Analyzer). At wk 12 and 20,
plasma taurine concentration was measured by an amino acid analyzer.
The concentration of 25-OHD in the plasma of kittens was measured by a
protein-binding assay (Chen et al. 1990
) when the
kittens were 7, 10, 14, 18 and 22 wk of age and at 34 wk of age for
kittens in 0.0 and 3.125 µg treatments. Body weight was measured
weekly and food intake recorded daily.
Radiographs were taken of the forelimbs of all kittens at 22 wk of age and again at 34 wk of age for the kittens in the 0.0 and 3.125 µg/kg treatment groups and were read by a Board Certified Veterinary radiologist who was not aware of the treatments.
Observations were subjected to a three-way analysis of variance using a general linear model, and P < 0.05 was taken as significant. Pair-wise multiple comparisons were made by the Student-Newman-Keuls method. Values are means ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Neither plasma total nor ionizable calcium was significantly affected by dietary treatments, sex or age. Plasma total and ionizable calcium at 9 and at 20 wk of age were 2.82 ± 0.23 and 2.79 ± 0.04 mmol/L and 1.33 ± 0.012 and 1.30 ± 0.012 mmol/L, respectively. Plasma inorganic phosphorus concentration was not significantly affected by treatments, or sex at 9 and 20 wk of age (2.09 ± 0.10 mmol/L). Similarly, ionized potassium, hemoglobin, packed-cell volume, red and white cell numbers, plasma total protein, albumin, urea and taurine were not affected by treatments. Alkaline phosphatase was not significantly different due to treatments, sex or age (8 and 20 wk values were 174 ± 8.9 and 266 ± 12.0 units/L, respectively), and the activities of alanine and aspartic aminotransferases were also not significantly affected by these variables. Radiographs of the kittens at 22 wk of age, examined by a radiologist, did not reveal any treatment differences in width of the growth plate.
Concentration of 25-OHD in the plasma of kittens at 7 wk of age was
20 ± 1.9 nmol/L, indicating that their reserves were depleted.
There was no significant difference in plasma concentration of 25-OHD
before dietary treatments were applied (P > 0.05).
After consumption of the diet for only 1 wk (10 wk old, Fig. 1
),there was a significant increase in plasma concentration of 25-OHD in
kittens receiving diets containing more than 6.250 µg of
cholecalciferol/kg. A significant rectilinear relationship
(y = a + bx) between group
mean plasma concentration of 25-OHD (y) and vitamin D
concentration of the diet (x) occurred at 10, 14, 18 and 22
wk of age. The slope (b) increased with time the diets were
fed from 10 to 14 wk and 14 to 18 wk of age. However, the b
value for the regression at 22 wk of age was lower, but not
significantly different from the 18-wk value, indicating that the
concentrations in plasma had stabilized. At 22 wk of age the
r2 value of the regression equation was 0.99
(P < 0.001). At 22 wk of age, analysis of variance
indicated a significant difference due to treatments (P < 0.001), and all pairwise multiple comparisons were significant
(P < 0.05). The mean concentrations of 25-OHD in the
plasma of kittens in the 0.0 µg/kg group at 22 and 34 wk were less
than the 7 wk mean value (P = 0.006). In contrast, all
kittens but two receiving the diet containing 3.125 µg/kg had higher
25-OHD concentrations at 22 and 34 wk than at 7 wk. These two kittens
had 25-OHD concentrations at 7 wk of age of 52 and 25 nmol/L and at 34
wk of age, 34 and 20 nmol/L, respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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demonstrated that kittens require vitamin D in the diet,
and in an independent experiment, one of the authors (J.G.M.), using a
diet similar to the purified diet used in these experiments (but
containing 7 g calcium and 6 g phosphorus/kg diet), produced
vitamin D deficiency in kittens (Morris et al. 1994
).
Analysis of the basal diet used gave undetectable levels of vitamin D,
and as the queens were given a diet with vitamin D supplied only by the
natural ingredients, the kittens' reserves of the vitamin were low as
evidenced by the 7-wk 25-OHD values. In some species, such as rats,
vitamin D deficiency is only evident when the diet contains a low
concentration of calcium and/or a high concentration of phosphorus.
Extensive non-vitamin D-mediated absorption of calcium occurs in the
ileum of neonatal and pregnant rats, which exceeds the total calcium
flux across the duodenum and jejunum (Marcus and Lengemann 1962
). Also in rats, the adaptation of the ileum to calcium
restriction results in a fourfold increase in net absorption, whereas
only a twofold increase occurs in the duodenum (Petith and Schedl 1976
). Similarly, in adult humans the distal intestine
equals or exceeds the contribution of the duodenum to calcium
absorption (Birge et al. 1969
). Therefore, a
high-calcium diet masks the effects of vitamin D deficiency, which
functions through calcitriol induction of calbindin to promote
absorption of calcium primarily in the duodenum. The kinetics of
calcium absorption by the intestine are best described by a saturable,
energy-dependent transcellular process that is subject to hormonal
regulation and a nonsaturable voltage-dependent diffusional component
(Bronner et al. 1986
, Bringhurst 1989
).
It is only in the active process of calcium uptake that vitamin D,
primarily as calcitriol, is involved in the enterocytes (Holick 1989
). The concentrations of calcium and phosphorus in the diet
that we used were in considerable excess of the minimal requirements
proposed by the National Research Council for growing kittens (8.0 g
calcium and 6.0 g phosphorus/kg diet). We (Morris and Earle,
unpublished) have subsequently demonstrated that the growing kitten's
requirement for calcium is considerably less than that proposed by the
National Research Council (1986)
.
The calcium-to-phosphorus ratio of the diet also affects the severity
of vitamin D deficiency in cats. Gershoff et al. (1957)
reported that "rickets produced (in the kittens) on the 1:1 ratio
(Ca/P) was more severe than that on the 2:0.65 ratio," and all of the
six kittens given the vitamin D-deficient diet with the 2.06 Ca/1P
ratio survived to 21 mo, whereas only one of seven kittens given the
1:1 ratio diet was alive at 21 mo.
The rectilinear relationship we observed between plasma 25-OHD, and
dietary intake of vitamin D was of the same form as reported by
Holick and Clark (1978)
for vitamin D-deficient rats
given various oral doses of cholecalciferol. The response of 25-OHD in
rats to oral cholecalciferol extends well into the pharmacological
range (Holick 1989
). Therefore, there is no plateau
value of 25-OHD that can be used to assess adequacy of this vitamin.
But, because the relationship in cats between the concentration of
vitamin D in the diet and the 25-OHD concentration in plasma is very
close, circulating levels of 25-OHD could be used as a bioassay for the
available vitamin D in foods. It appears that cats, like other animals,
have a large reserve activity of vitamin D-25-hydroxylase in liver.
The lack of an elevation in alkaline phosphatase in kittens given the
0.0 µg cholecalciferol/kg diet is consistent with observations of
kittens exhibiting frank clinical signs of vitamin D deficiency
(Morris, unpublished data). Alkaline phosphatase in kittens does not
appear to be a sensitive indicator of vitamin D deficiency, especially
in the early stages of the disease when the diet is well supplied with
calcium. In humans, alkaline phosphatase increases in active rickets,
and Gershoff et al. (1957)
suggested on limited data
that alkaline phosphatase may be the most sensitive chemical means
(then available) for evaluating the state of rickets in cats.
Definition of a requirement for an essential nutrient requires that
some physiological function be selected which is optimized or
maximized, or some metabolic variable, such as circulating
concentration of a nutrient, be maintained at a certain level
(Morris and Rogers 1994
). For the vitamin D requirements
of kittens, we suggest that the circulating level of 25-OHD could be
used to estimate a requirement. The concentration of 25-OHD in plasma
is more stable than the concentration of either vitamin D or
calcitriol. The half-life of vitamin D in the plasma of humans is about
24 h, that of 25-OHD about 3 wk and that of calcitriol 4 to 6 h (Holick 1990
). Because the concentration of 25-OHD
increased in the group given the 3.125 µg/kg diet and significantly
decreased in the group given 0.0 µg cholecalciferol/kg diet, it could
be inferred that 3.125 µg cholecalciferol/kg diet may be adequate for
kittens. However, the concentration in the plasma of three kittens in
the 3.125 µg/kg group was less than 10 ng/mL (25 nmol/L) which has
been suggested by Holick (1990)
as indicating impending
or frank vitamin D deficiency in humans. In contrast, the concentration
of 25-OHD in the plasma of all kittens in the group receiving the diet
containing 6.25 µg cholecalciferol/kg at 22 wk of age was in excess
of 20 ng/mL (50 nmol/L) which is regarded as normal for humans.
Therefore a conservative estimate of requirement including a margin of
safety is 6.25 µg of cholecalciferol/kg diet with a metabolizable
energy value of ~20 kJ/g. This value is half that of the
National Research Council (1986)
recommendation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Abbreviations used: 25-OHD, 25-hydroxyvitamin D;
UV, ultraviolet.
Manuscript received October 28, 1998. Revision accepted December 22, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1. Birge S. J., Peck W. A., Burman M., Wheldon G. D. Study of calcium absorption in man: A kinetic analysis and a physiologic model. J. Clin. Invest. 1969;48:1705-1713
2. Bringhurst F. R. Calcium and phosphate distribution, turnover, and metabolic actions. De Groot L. J. eds. Endocrinology 1989;vol. 2:805-843 W. B. Saunders Co Philadelphia Chap. 52
3. Bronner F., Pansu D., Stein W. D. An analysis of the intestinal calcium transport across the rat intestine. Am. J. Physiol. (Gastrointest. Liver Physiol.) 1986;13:G561-G569
4. Chen T. C., Turner A. K., Holick M. F. Method for the determination of the circulating concentration of 25-hydroxyvitamin D. J. Nutr. Biochem. 1990;1:315-319[Medline]
5. Gershoff S. N., Legg M. A., O'Connor F. J., Hegsted D. M. The effect of vitamin D-deficient diets containing various Ca:P ratios on cats. J. Nutr. 1957;63:79-93[Medline]
6. Hazewinkel, H.A.W., How, K. L., Bosch, R., Goedegebuure, S. A. & Voorhout, G.(1987)Inadequate photosynthesis of vitamin D in dogs. In: Nutrition, Malnutrition, and Dietetics in the Dog and Cat. Proceedings of the International Symposium held at Hanover, September 3 to 4, 1987 (Edney, A.T.B., ed.) British Veterinary Association in collaboration with the Waltham Centre for Pet Nutrition.
7. Holick M. F. Vitamin D biosynthesis, metabolism, and mode of action. DeGroot L. J. eds. Endocrinology 1989:902-926 W. B. Saunders Co Philadelphia.
8. Holick M. F. The use and interpretation of assays for vitamin D and its metabolites. J. Nutr. 1990;120:1464-1469
9. Holick M. F. Vitamin D. Shils M. E. Olson J. A. Shike M. eds. Modern Nutrition in Health and Disease 8th edn. 1994:308-325 Lea & Febiger Philadelphia.
10. Holick M. F., Clark M. B. The photobiogenesis and metabolism of vitamin D. Fed. Proc. 1978;37:2567-2574[Medline]
11. How K. L., Hazewinkel H.A.W., Mol J. A. Dietary vitamin D dependence of cat and dog due to inadequate cutaneous synthesis of vitamin D. Gen. Comp. Endocrin. 1994;96:12-18[Medline]
12. Marcus C. S., Lengemann F. W. Use of radioyttrium to study food movement in the small intestine of the rat. J. Nutr. 1962;76:179-182
13.
Morris J. G. Ineffective vitamin D synthesis in cats is reversed by an inhibitor of 7-dehydrocholestrol-7-reductase. J. Nutr. 1999;129:903-908
14. Morris J. G., Kirk C. A., Burek K. A. Vitamin D deficiency in kittens exposed to ultraviolet light or sunlight. FASEB J 1994;8:A190abs 1099
15. Morris J. G., Rogers Q. R. Assessment of the nutritional adequacy of pet foods through the life cycle. J. Nutr. 1994;124:2520S-2534S
16. National Research Council(1985)Guide for the Care and Use of Laboratory Animals, Publication no. 85-23 (rev.), National Institutes of Health, Bethesda, MD.
17. National Research Council Nutrient Requirements of Cats 1986 National Academy of Sciences Press Washington, D.C.
18.
Petith M. M., Schedl H. P. Duodenal and ileal adaptation to dietary calcium restriction: in vivo studies in the rat. Am. J. Physiol. 1976;231:865-871
19. Rivers J.P.W., Frankel T. C., Juttla S., Hay A.W.M. Vitamin D in the nutrition of the cat. Proc. Nutr. Soc. 1979;38:36A[Medline]
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