Calcium Metabolism and Its Control — A Review1

Calcium Metabolism and Its Control — A Review1 j . H. SOARES, JR. Department of Poultry Science, University of Maryland, College Park, Maryland 20742 (Received for publication November 27, 1983)

1984 Poultry Science 63:2075-2083 INTRODUCTION

DISCUSSION

F e w e l e m e n t s , if a n y , can be implicated in so m a n y metabolic reactions of t h e intact organism as calcium. It is i m p o r t a n t t h e n t h a t t h e r e is critical control within a very n a r r o w range of circulating calcium in t h e n o r m a l animal. H o r m o n a l control of this process appears t o be a d o m i n a n t factor influencing major calcium reservoirs such as t h e skeleton and t h e b l o o d plasma. N o less t h a n four e n d o c r i n e systems (calcitonin, p a r a t h y r o i d h o r m o n e , 1,25-dihyd r o x y v i t a m i n D [ l , 2 5 ( O H ) 2 D ] , and estrogen) c o n t r o l these processes.

Metabolism of Vitamin D and Calcium Homeostasis. It has been clearly s h o w n t h a t birds can efficiently metabolize only t h e cholecalciferol ( D 3 ) form of vitamin D . Plant sources of this vitamin (ergocalciferols o r D 2 ) are essentially nonfunctional (Valinietse and Bauman, 1981), having only a b o u t 5% of t h e activity of D 3 . A l t h o u g h t h e exact reason for this is n o t clear, t h e r e is s o m e indication t h a t this is due t o a rapid t u r n o v e r rate of D 2 forms, because t h e r e is less efficient binding t o plasma t r a n s p o r t proteins (Belsey et al., 1 9 7 4 ) . It is well k n o w n t h a t t h e precursor for D 3 (i.e., 7-dehydrocholesterol) is synthesized in t h e liver during n o r m a l steroid synthesis. This comp o u n d is t r a n s p o r t e d via t h e circulatory system o n a t r a n s p o r t p r o t e i n to t h e skin where irradiation with ultraviolet (UV) light results in t h e synthesis of D 3 (Figure 1). A binding p r o t e i n carries D 3 t o t h e liver w h e r e further m e t a b o lism or storage occurs. L u n d and D e L u c a ( 1 9 6 6 ) first d e m o n s t r a t e d t h a t f u r t h e r m e t a b o lism of D 3 to 2 5 - h y d r o x y v i t a m i n D 3 ( 2 5 - O H D 3 ) was necessary for n o r m a l activity of this vitamin. It is n o w clear t h a t the site of synthesis of this m e t a b o l i t e is t h e liver, although in t h e chicken t h e intestine and k i d n e y are capable of synthesis of 2 5 - O H D 3 t o some degree ( T u c k e r

During t h e last 15 years or so there has been an explosive d e v e l o p m e n t of o u r k n o w l e d g e of t h e metabolism of vitamin D and of calcium homeostasis in general. No a t t e m p t is m a d e in this paper t o show t h e sum total of this massive a m o u n t of literature. A n effort will b e m a d e , however, t o outline t h e role of vitamin D and a n u m b e r of t h e factors t h a t modify its function in t h e control of calcium m e t a b o l i s m in t h e avian.

'Scientific Article No. A-3776, Contribution No. 6753 of the Maryland Agriculture Experiment Station (Department of Poultry Science).

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ABSTRACT It is now established that avians can only utilize the cholecalciferol form of vitamin D, which must be converted to the hormone 1,25-dihydroxyvitamin D 3 [l,25-(OH) 2 D 3 ] to perform normal calcium metabolism. Although l,25(OH) 2 D 3 is the final active form of vitamin D, hens fed only this form of vitamin D do not have normal hatchability of eggs. The problem appears to be caused by abnormal calcification and development of the embryonic beak. This appears to be caused by inadequate transport of l,25(OH) 2 D 3 into the egg. Although l,25(OH) 2 D 3 is not incorporated into the egg adequately, its precursor, 25-hydroxyvitamin D 3 (25-OH-D 3 ), is. The developing embryo however, can utilize l,25(OH) 2 D and does so at least as early as Day 10 of incubation. During periods of maximal shell calcification and high circulating estradiol levels, the hen produces high levels of l,25(OH) 2 D 3 . The kidney hydroxylase responsible for the final hydroxylation of the vitamin D hormone can be further stimulated by in vivo or in vitro administration of estradiol and, to a lesser extent, prolactin and parathyroid hormone. When eggs are not produced, as in the senescent or prepubertal stages of life, plasma l,25(OH) 2 D 3 concentrations are less than half that occurring during periods of active lay. Hens selected for their ability to produce thin or thick shells have l,25(OH) 2 D 3 concentrations in plasma that are positively correlated to their ability to produce egg shell. (Key words: calcium, vitamin D, homeostasis, 1,25-dihydroxyvitamin D 3 , avians)

SOARES, JR.

OH

UV Light Skin

J

*

Liver

f "

/

Kidney

> i CHg Microsomes

/S^CH

—> 2

i

Mitochondria

/^^CH

9

HO

HO 7 - Dehydrocholesterol

,25 (0H1 2 D 3 Kidney

/ OH

( 2 4 R ) - 1,24,25 (OH) 3 D 3

FIG. 1. Activation of vitamin D 3 and conversion to the hormone 1,25 dihydroxyvitamin D 3 [l,25(OH) 2 D 3 ].

et al, 1973). Within the microsomal fraction of the liver, the enzyme vitamin D3-25-hydroxylase is capable of hydroxylating D3 at the 25 position in the presence of nicotinamide adenine dinucleotide phosphate, reduced (NADPH), molecular oxygen, and cytochrome P-450 (DeLuca, 1976). There appears to be only limited control of the production of this metabolite and the supplementation of D3 from low to high doses results in an almost linear increase in circulating 25-OHD 3 (Clark and Potts, 1977). Measurement of 25-OHD 3 , therefore, would appear to be a useful means of assessing D3 status. Further metabolism of 25-OHD 3 is known to occur in the kidney and was first shown in the chicken by Haussler et al. (1968) and Lawson et al. (1969). Gray et al. (1972) demonstrated that 25-OHD-la-hydroxylase was a mitochondrial enzyme of the kidney and also a mixed function oxidase. It apparently also requires the presence of the reductase ferredoxin (Pedersen et al., 1976) for normal activity. Several other metabolites of D3 have been isolated, and considerable speculation exists as to their function. The most studied of these is probably 24,25-dihydroxyvitamin D 3 [24,25 ( O H ) 2 D 3 ] . This metabolite is also produced in the renal mitochondria by action of a 25OHD 3 -24-hydroxylase (Knutson and DeLuca,

1974). There is some evidence that this metabolite is needed for normal hatchability of chicks (Henry and Norman, 1978). This point will be discussed further later in this paper. Apparently this same enzyme system can hydroxylate the 24 position on l,25(OH) 2 D 3 to form 1,24,25(OH) 2 D 3 , which has about 60% of the activity of l,25(OH) 2 D 3 . The rate limiting step for activation of the vitamin D endocrine system is catalyzed by the 25-OHD-la-hydroxylase (DeLuca, 1974, 1976; Haussler, 1974). Figure 2 illustrates a number of important aspects of the calcium homeostatic system. It is apparent that there is both direct and indirect control of the D3 endocrine system by plasma calcium and possibly plasma phosphorus as well. Hypocalcemia is known to stimulate the activity of the la-hydroxylase, thereby increasing the production of 1,25(OH) 2 D 3 (DeLuca, 1980). In addition, parathyroid hormone (PTH) from the parathyroid gland is secreted in response to low plasma calcium. Parathyroid hormone is believed to stimulate the renal la-hydroxylase system in addition to its other target tissues. Interestingly, plasma l,25(OH) 2 D3 has been shown to bind to receptors in the parathyroids and stimulate PTH release itself (Wecksler et al., 1977). In the hypocalcemic state, l,25(OH) 2 D 3 is known to stimulate increased active and passive absorption of calcium and phosphorus along

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( 2 4 R ) - 24,25 (0H) 2 D 3

H

SYMPOSIUM: METABOLISM AND GROWTH IN POULTRY the entire intestinal tract. Although there are some differences of opinion, it is thought that passive or facilitative mechanisms for calcium absorption predominate when dietary calcium is adequate. Under deficient or low calcium conditions, the active calcium absorption mechanism(s) are believed to be more important (Spencer et al, 1978; Rasmussen et al, 1979). Therefore l,25(OH) 2 D controls both passive and active mechanisms for calcium absorption. In addition to the intestine, l,25(OH) 2 D3 and PTH are thought to act on the osteoclasts

2077

of the skeletal system to release calcium and phosphorus into circulation (Garabedian et al., 1974). Upon the return of normocalcemia, it is generally thought that calcitonin is released from the thyroid to inhibit the action of D3 and PTH on bone. Relatively little is known about the mechanism of action of calcitonin. Coincidental with normocalcemia or hypercalcemia the activity of the renal la-hydroxylase declines and the 24-hydroxylase system is stimulated. The result is an increase in circulating 24,25(OH) 2 D 3 and a decrease in 1,25-

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Shut down • Start up

FIG. 2. Vitamin D metabolism and calcium homeostasis.

»

2078

SOARES, JR.

TABLE 1. Percent abnormal embryos of eggs from hens fed various vitamin D sources (from Sunde and DeLuca, 1978)

Treatment

% Abnormal embryos

D-Deficient Vitamin D, 12 mg/kg l,25(OH) 2 D 3 ,2mg/kg l,25(OH) 2 D 3 ,4mg/kg

50 15 78 80

Calcium Metabolism and the Ovulatory Cycle. The average table egg contains 2 g of

TABLE 2. Reproductive performance of hens fed vitamin D 3 (Dz) or la-hydroxyvitamin D 3 (la-OHD3) (from Soares et al., 1979)

Number of hens Eggs laid' Average production (H/d) % Fertility % Hatchability 2 % Abnormalities 4 1

D3

la-OHDj

19 86

18 80

.65 67 ± 3.3 58 + 3.3 0

.64 62 ± 6.2 45 ± 5.23 33.0

Total for the 21st week.

2

Mean ± SEM, based on production of hens laying at least one fertile egg during the 21st week of the experiment. 3 Significantly different from the control group (P<.05). 4

Based on total number of dead embryos.

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(OH) 2 D 3 (DeLuca, 1976, 1980). Considerable controversy remains as to the actual metabolic role of this metabolite. Vitamin D Endocrine System in Embryonic Development. Henry and Norman (1978) concluded that feeding 24,25(OH) 2 D 3 was necessary to obtain normal hatchability of chickens. When hens had access to only 1,25(OH) 2 D 3 as a vitamin D3 source for 30 weeks, they exhibited poor hatchability. Although Henry and Norman's conclusion has not been definitely proven, Sunde and DeLuca (1978) also showed that hatchability of chicken eggs was abnormally low and there was a high incidence of abnormal embryos after 28 weeks of feeding a D 3 -deficient diet supplemented with crystalline l,25(OH) 2 D 3 . Egg production, shell calcification, and egg size were not affected by this treatment, but upper mandible and pipping tooth development appeared to be retarded (Table 1). Similarly, Soares et al. (1979) showed that egg production and fertility were normal after 21 weeks of feeding la-OHD 3 (a synthetic analogue of l,25(OH) 2 D 3 ), but hatchability was decreased and the incidence of embryonic abnormalities was significantly increased (Table 2). Similar findings have also been reported by Abdulrahim et al. (1979). Although the embryos die before pipping, no other gross anatomical defects have been reported. Normal hatchability can be restored by supplementation of the hen's diet with D 3 or 25-OH-D3. Interestingly, Sunde and DeLuca (1978) showed that hatchability was greatly improved when eggs from the l,25(OH) 2 D3 fed hens were injected with l,25(OH) 2 D 3 . This indicates that the chick embryo can effectively use l,25(OH) 2 D 3 . It appears that there is some aspect of the transport of l,25(OH) 2 D 3 and possibly the la-OH-D 3 analogue that does not allow this metabolite to enter the egg in sufficient quantity to support normal embryonic development.

Because the chick embryonic skeletal system contains over 120 mg of calcium and the yolk has only about 20 mg calcium, a large proportion of the calcium needed by the embryonic skeleton must come from other sources, such as the shell of the egg. According to Tuan and Scott (1977), onset of calcium transport by the chorioallantoic membrane occurs about Days 10 to 12 of embryonic development. Maximal calcium transport activity occurs at Day 19 and then declines. Kubota et al. (1981) reported that renal 25-OH-D 3 -la-hydroxylase activity was significantly enhanced as early as Day 8. These workers showed that 19-day-old embryos had plasma concentrations of 2.3 ng/ml 25OH-D 3> .6 ng/ml 24,25(OH) 2 D 3 , and 300 pg/ml l,25(OH) 2 D 3 . Interestingly, they report that 24,25(OH) 2 D 3 production in the 21-dayold embryo is six times higher than that of l,25(OH) 2 D 3 . In contrast, it has been shown (Seino et al., 1982) that serum l,25(OH) 2 D and 24,25(OH) 2 D concentrations reach normal levels (Table 3) between 15 and 19 days of embryonic development and are generally lower than indicated by Kubota and coworkers (1981). Therefore, essentially normal plasma levels of l,25(OH) 2 D are attained as early as the 18th day of embryonic development. By 15 days of incubation, the embryo also produces a detectable duodenal l,25(OH) 2 D 3 receptor protein. The activity of this receptor is maximal at hatching.

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SYMPOSIUM: METABOLISM AND GROWTH IN POULTRY TABLE 3. Vitamin D metabolites in embryonic and posthatching chick serum'

l,25(OH) 2 D 4

24,25(OH) 2 D 3

25-OHD2

Age

(pg/ml)

(days) Embryonic 15 18 19 20

8.0 10.8 11.1 10.0

2.2 2.4 2.7 2.8

44.9 49.3 60.7 62.7

Posthatch 1 2 118

9.0 9.5 15.5

2.8 2.8 2.9

57.2 57.7 54.7

From Seino et al. (1982). 25-Hydroxyvitamin D.

3 4

24,25-Dihydroxyvitamin D.

1,25-Dihydroxyvitamin D.

calcium, and the typical body weight of the laying hens is about 2 kg. The hen's skeleton contains a total of approximately 20 g of calcium. Therefore, each egg contains about 10% of the total body calcium. Considering the ovulatory cycle of the laying hen to be about 25—26 hr, we can estimate that almost 1000 mg Ca/kg body weight per day is needed by the regularly laying hen for egg shell formation alone. The need for calcium by the reproductively active avian female is enormous, and efficient transport of calcium into the uterus is of utmost importance. It is known that the

active laying hens has plasma calcium concentrations of 20 to 35 mg/dl while nonlayers have concentrations about one-half this. Estrogens apparently play a major role in these changes and injections of estrogens can stimulate high blood calcium in nonlayers as well as in males (Taylor et al, 1971; Mueller, 1976). With adequate dietary calcium, most of the elevated calcium demand is met by increased intestinal absorption and secondarily from increased bone turnover. Simkiss and Taylor (1971) estimated that the shell gland has a maximal transport of calcium of 100 to 150 mg/h. At this rate the

TABLE 4. Hormonal control of renal vitamin D hydroxylases Treatment

— (pmol g Females Estradiol Progesterone Control Males Testosterone Progesterone Estradiol PTH 3 PTH and estradiol Control

24,25(OH) 2 D 3 2

l,25(OH),D 3

4.5 1.0 .5 9 20 134 17 103 8.5

1

1,25-Dihydroxyvitamin D 3 .

2

24,25-Dihydroxyvitamin D 3 .

3

PTH = Parathyroid hormone.

min

Reference

) 3 3.5 4.0 88 104 19 44 37 139

Baksi and Kenny (1977) Baksi and Kenny (1977) Baksi and Kenny (1977) Castillo Castillo Castillo Castillo Castillo Castillo

et et et et et et

al. al. al. al. al. al.

(1977) (1977) (1977) (1977) (1977) (1977)

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1 2

2080

SOARES, JR. TABLE 5. Hormonal control of renal hydroxylases in mature quail hens

Treatment

l,25(OH),D3>

Estradiol Estradiol Control

141 181 31

— (pmol g

min

24,25(OH)2D3

Reference

) ND ND ND

Baksi and Kenny (1980) Baksi and Kenny (1980) Baksi and Kenny (1980)

(pg/ml) 190 90

Estradiol Control

Kaetzel and Soares (1981) Kaetzel and Soares (1981)

1

1,25-Dihydroxyvitamin D 3 . 24,25-Dihydroxyvitamin D 3 . 3 ND = Not detectable.

2

i

300

250 230 Q J? I

2

210

150

in —

\ from: * Abe et al. (1979)

w E - ~- 130

< °£ no < °-

from: Castillo et al. (1979)

90 70 50

0 2 4 6 8 10 12 14 16 18 2022 24 26 HOURS

AFTER

OVULATION

FIG. 3. Circadian rhythm of 1,25-dihydroxyvitamin D, [l,25(OH)2D3] in hens.

(1976). He demonstrated that l,25(OH) 2 D 3 production is enhanced during the 24-hr period following ovulation. Elevation of l,25(OH) 2 D 3 took place within the first 6 to 7 hr following ovipositioning. If ovipositioning is followed by a pause in ovulation, l,25(OH) 2 D 3 production declines. Other work (Baksi and Kenny, 1977; Castillo et al., 1977) has shown that 17/3estradiol has a strong effect on stimulating the renal la-hydroxylase system needed for synthesis of l,25(OH) 2 D 3 (Table 4). Simultaneously, the 24-hydroxylase system is suppressed. Other hormones (progesterone, testosterone, and PTH) appear to have some but lesser degrees of la-hydroxylase stimulating activity than estrogens in the immature male and female as well as mature male quail. In addition, Spanos et al. (1976) were able to show that prolactin injection (100 /Jg/day) in immature chicks caused increased plasma concentrations of l,25(OH) 2 D 3 . Not only was this study one of the first in vivo demonstrations of exogenous hormonal stimulation of 1,25(OH) 2 D 3 synthesis, but is also showed that prolactin is a very strong stimulator of renal la-hydroxylase activity. Mature female quail also respond to injected estradiol by exhibiting significantly increased renal la-hydroxylase activity in tissue homogenates (Baksi and Kenny, 1980; Table 5). Maximal response to estradiol injection occurred at .03 mg 17/3-estradiol/kg, a dose more nearly compatible to normal physiologic conditions than had been previously tested. It is noteworthy that the 24-hydroxylase system is very inactive in mature hens. This would further indicate the relatively minor role

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blood would be depleted in less than 30 min, if increased intestinal absorption and bone turnover did not occur. Eastin and Spaziani (1978) have shown that net calcium secretion in the shell gland ranges from an initially low rate of 13 to 28 jdm h"1 g _1 dry weight of uterus before arrival of an egg to a maximal rate of 150 /xm I f ' g " 1 at 14 to 18 hr postovipositioning and then declines to basal levels 2 hr before the next ovipositioning. If one assumes that there is about 20 g of dry weight in the average uterus, then these data agree with those of Simkiss and Taylor (1971). The first to report a strong regulatory control by exogenous estrogen or actual ovulation on vitamin D metabolism was Kenny

SYMPOSIUM: METABOLISM AND GROWTH IN POULTRY TABLE 6. Effect of age on plasma dibydroxyvitamin D3 [l,25(OH)lDz]in female quail and Leghorn chickens Reference

Quail Kaetzel(1981)

Chickens Soares « a/. (1983)

Age

l,25(OH) 2 D 3

(wk)

(pg/ml)

8 20 120

330 270 85*

8 16 60

60 46 260*

24,25 (OH) 2 D 3 has in stimulating egg shell calcification. These studies demonstrated that the la-hydroxylase system can be further stimulated even over the high rate observed in the reproductively active hen. We have been able to show (Kaetzel and Soares, 1981) that plasma l,25(OH) 2 D 3 is elevated when mature nonlaying quail hens were implanted with 170-estradiol (Table 5). If exogenous estradiol can stimulate 1,25(OH) 2 D 3 production in the kidney, will physiologic fluctuations in circulating estrogen stimulate a circadian rhythm of plasma l,25(OH) 2 D 3 ? Several laboratories have recently addressed this question with variable results (Castillo et al, 1979; Abe et al, 1979; Soares et al, 1980). Both renal la-hydroxylase and circulating l,25(OH) 2 D3 were measured in the studies of Castillo et al. (1979). The la-hydroxylase activities reached maximal rates at about 22 to 24 weeks, an age when pullets should begin egg production. These data are shown in Figure 3 and are corrected for an apparent error in estimating the time of ovulation after ovipositioning (.5 vs. 3 hr). When this is done the data are more similar to that of Abe and coworkers (1979), who showed a circadian rhythm for plasma l,25(OH) 2 D 3 as well as renal lahydroxylase activity. Their data indicate that laying hens show peak l,25(OH) 2 D 3 production around 15 hrs postovulation when maximal egg shell calcification is taking place in the uterus. They also observed that 24-hydroxylase activity remained low and did not change throughout the cycle. However, 25OH-D 3 tended to rise slightly around 20 hr

postovulation. Both groups stated that the hens in these studies were fed adequate dietary calcium (~3.5%). We have measured circulating l,25(OH) 2 D 3 in laying quail hens fed diets higher than adequate (3.5%) or marginal (1.7%) in calcium (Kaetzel and Soares, 1984). Marginal calcium diets enhanced the circulating levels of 1,25(OH) 2 D 3 (approximately 25%), while no affect was seen on 25-OH-D 3 . In agreement with Abe and coworkers (1979), our data show that plasma l,25(OH) 2 D 3 had a tendency to rise to maximal concentrations around 15 hr postovulation. Because this trend only occurred when quail were fed marginal levels of dietary calcium, it may be that the fluctuations in plasma l,25(OH) 2 D 3 are magnified in calcium stress and secondarily from circulating estrogens. It is also possible that the successive estrogenic pulses culminating around the 15th to 18th hr postovulation are needed to stimulate the renal la-hydroxylase system to produce l,25(OH) 2 D 3 . Additional investigations are needed to develop a clear mechanism for these responses. Because gonadal function has a marked effect on the metabolism of vitamin D, it would seem logical that those periods of low or quiescent gonadal activity, particularly in females, would result in relatively low concentrations of peripheral circulating l,25(OH) 2 D 3 . Studies conducted in our laboratory indicate that the prepubertal and senescent states of life are periods of low plasma l,25(OH) 2 D 3 in the avian female (Table 6). Similar declines in plasma l,25(OH) 2 D 3 are known to occur in nonlaying mature females as well (Kaetzel, 1981). It may be asked what applied benefit a knowledge of the vitamin D endocrine system might have. We (Soares et al., 1980) have attempted to relate our findings to the problem of egg shell quality. In a collaborative study with the Pennsylvania State University Department of Poultry Science, blood was drawn from hens that had been selected over a period of years for markedly different abilities to calcify egg shells. For reference purposes these lines are called "thick" and "thin" as a description of the type of egg shells they produced. The data showed that these two lines had significantly different averages of 10.3 and 8.8% shell for thick and thin respectively. Plasma total calcium was not significantly different when determined at 5 and 18 hr postovipositioning.

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•Significantly different from other group values (P<.05).

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SOARES, JR. REFERENCES Abdulrahim, S. M., M. B. Patel, and J. McGinnis, 1979. Effects of vitamin D 3 and D 3 metabolites on production parameters and hatchability of eggs. Poultry Sci. 58:858-863. Abe, E., R. Tanabe, T. Suda, and S. Yoshiki, 1979. Circadian rhythm of 1,25 dihydroxyvitamin D 3 production in egg-laying hens. Biochem. Biophys. Res. Comm. 88:500-507. Baksi, S. N., and A. D. Kenny, 1977. Vitamin D 3 metabolism in immature Japanese quail: Effects of ovarian hormones. Endocrinology 101:1216— 1220. Baksi, S. N., and A. D. Kenny, 1980. Estradiol-induced stimulations of 25-hydroxyvitamin D-la-hydroxylase in vitamin D-deficient Japanese quail. Pharmacology 20:298-303. Belsey, R. F., H. F. DeLuca, and J. T. Potts, 1974. Selective binding properties of vitamin D transport protein in chick plasma in vitro. Nature 247:208-209. Castillo, L., T. Tanaka, and H. F. DeLuca, 1977. The stimulation of 25-hydroxyvitamin D-la-hydroxylase by estrogen. Arch. Biochem. Biophys. 179:211-217. Castillo, L., Y. Tanaka, M. J. Wineland, J. O. Jousey, and H. F. DeLuca, 1979. Production of 1,25dihydroxyvitamin D 3 and formation of medullary bone in the egg-laying hen. Endocrinology 104:1598-1601. Clark, M. B., and J. T. Potts, Jr., 1977. 25-Hydroxyvitamin D 3 regulation. Calcif. Tissue Res. 22 (Suppl):29-34. DeLuca, H. F., 1974. Vitamin D: The vitamin and the hormone. Fed. Proc. 33:2211-2219. DeLuca, H. F., 1976. Metabolism of vitamin D: Current status. Am. J. Clin. Nutr. 29:1258-1270. DeLuca, H. F., 1980. Some new concepts emanating from a study of the metabolism and function of vitamin D. Nutr. Rev. 38:169-182. Eastin, W. C , Jr., and E. Spanziani, 1978. On the control of calcium secretion in the avian shell gland (uterus). Biol. Reprod. 19:493-504. Garabedian, M., Y. Tanaka, M. F. Holick, and H. F. DeLuca, 1974. Response of intestinal calcium transport and bone calcium mobilization of 1,25-dihydroxyvitamin D in thyroparathyroidectomized rats. Endocrinology 94:1022-1027. Gray, R. M., J. L. Omdahl, J. G. Ghazarian, and H. F. DeLuca, 1972. 25-hydroxycholecalciferol-la-hydroxylase. Subcellular location and properties. J. Biol. Chem. 247:7528-7532. Grunder, A. A., K. G. Hollands, and C.P.W. Tsang, 1983. Plasma estrogen, calcium and shell quality in two strains of White Leghorns. Poultry Sci. 62:1294-1296. Haussler, M. R., 1974. Vitamin D: Mode of action and biomedical applications. Nutr. Rev. 32:257-266. Haussler, M. R., J. F. Myrtle, and A. W. Norman, 1968. The association of a metabolite of vitamin D 3 with intestinal mucosa chromatin in vivo. J. Biol. Chem. 243:4055-4064. Henry, H. L., and W. W. Norman, 1978. Vitamin D: Two dihydroxylated metabolites are required for normal chicken egg hatchability. Science 201:

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Plasma l,25(OH) 2 D 3 was determined at 7 and 12 months of age and at early and late stages of the ovulatory cycle. The l,25(OH)2D concentrations were significantly higher in the thick line (231 ± 15 vs. 313 ± 18). Interestingly, Grunder et al. (1983) recently reported on a similar study in which plasma estradiol concentrations were somewhat higher in hens that lay eggs with high egg shell quality than in those with low shell quality. It is clear that vitamin D is further metabolized to the hormone l,25(OH) 2 D 3 , which has a major role in regulating calcium homeostasis by stimulating intestinal absorption as well as bone mobilization of calcium and phosphorus. Several reports now clearly show that l,25(OH) 2 D 3 as the only source of dietary vitamin D is not adequately transported into the egg to support the normal development of the chick embryo. If, however, D 3 or 25-OH-D is supplied in the diet, adequate supplies of these precursors of l,25(OH) 2 D 3 are transported into the egg and normal embryonic development and hatchability will occur. The embryonic chick can utilize l,25(OH) 2 D3 if it is injected into the egg and, in fact, produces its own l,25(OH) 2 D 3 as early as the 10th day of incubation. Estrogenic influence of l,25(OH) 2 D 3 metabolism appears to be surpassed only by the influence of low calcium stress. Exogenous estrogen, or normal release during the ovulatory cycle, stimulates the la-hydroxylase system in the kidney to produce l,25(OH) 2 D3 in immature and mature birds of both sexes. Testosterone (in males) and prolactin also stimulate this system but to a lesser extent than estradiol. Consequently, it can be seen that l,25(OH) 2 D 3 reaches maximal circulating levels when an egg is undergoing rapid calcification in the uterus. Similarly, during periods of low gonadal activity in the female, such as prepuberty and senescence, plasma l,25(OH) 2 D 3 concentrations decline to values between 40 to 100 pg/ml. When the mature female ceases egg production temporarily, such as during a molt, plasma values also fall to these basal levels. The male will consistently have levels of 1,25(OH) 2 D 3 similar to the nonlaying female throughout its life. Recent studies have shown that hens with markedly low egg shell quality tend to have lower circulating levels of l,25(OH) 2 D 3 than similar hens that produce well-calcified eggs.

SYMPOSIUM: METABOLISM AND GROWTH IN POULTRY

Avioli, 1982. Biochemical characterization of l,25(OH) 2 D 3 receptors in chick embryonal duodenal cytosol. Calcif. Tiss. Int. 34:265-269. Simkiss, K., and T. G. Taylor, 1971. Shell formation. Pages 1331- 1334 in Physiology and Biochemistry of the Domestic Fowl. Vol. 3. Academic Press, New York, NY. Soares, J. H., Jr., M. A. Ottinger, E. G. Buss, and D. M. Kaetzel, Jr., 1980. Plasma concentration of l,25(OH) 2 D 3 , estradiol and calcium in laying hens selected for differential egg shell quality. Poultry Sci. 59:1662. (Abstr.) Soares, J. H., Jr., M. R. Swerdel, and M. A. Ottinger, 1979. The effectiveness of the vitamin D analog la-OH-D 3 in promoting fertility and hatchability in the laying hen. Poultry Sci. 58:1004-1006. Spanos, E., J. W. Pike, M. R. Haussler, K. W. Coston, I.M.A. Evans, A. M. Goldner, T. A. McCain, and I. Maclntyre, 1976. Circulating la-25-dihydroxyvitamin D in the chicken: Enhancement by injection of prolactin and during egg laying. Life Sci. 19:1751-1756. Spencer, R., M. Charman, P. W. Wilson, and D.E.M. Lawson, 1978. The relationship between vitamin D stimulated calcium transport and intestinal calcium-binding protein in the chicken. Biochem. J. 170:93-101. Sunde, M. L., and H. F. DeLuca, 1978. The essentiality of vitamin D metabolites for embryonic chick development. Science 200:1067—1069. Taylor, T. G., K. Simkiss, and D. A. Stringer, 1971. The skeleton: Its structure and metabolism. Pages 621—640 in Physiology and Biochemistry of the Domestic Fowl. Vol. 2. Academic Press, New York, NY. Tuan, R. S., and W. A. Scott, 1977. Calcium-binding protein of chorioallantoic membrane: Identification and developmental expression. Proc. Natl. Acad. Sci. 74:1946-1949. Tucker, G., R. E. Gagnor, and M. R. Haussler, 1973. Vitamin D 3 -25-hydroxylase: Tissue occurrence and apparent lack of regulation. Arch. Biochem. Biophys. 155:47-57. Valinietse, M. Yu., and V. K. Bauman, 1981. Comparative antirachitic activity of vitamin D 2 and D 3 in chicks. Appl. Biochem. Microb. 17:531—537. Wecksler, W. R., H. L. Henry, and A. W. Norman, 1977. Studies on the mode of action of calciferol. Arch. Biochem. Biophys. 183:168-175.

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