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The effect of age on calcium absorption and accumulation of 1,25-hydroxyvitamin D3 in intestinal mucosa of rats
Metab.
Bone
Dis.
Metabolic Bone Disease 8 Related Research
& Ref. Res. 1, 29-33 /I9781
@ by S.N.P.M.D. (Paris 1978)
The effect of age on calcium absorption of l,Z!i-diil oxyvitamin 03 in intestinal R.L. HORST, H.F. DELUCA, Departments of Dairy Science Madison, Madison, Wisconsin
and accumulation mucosa ‘of rats
AND N.A. JORGENSEN and Biochimistry. 53706 U.S.A.
College
of
: Dr H. F. DeLuca. Address for correspondance and reprints of Wisconsin-Madison, Madison, WI 53706, U.S.A.
Agricultural Department
and of
Life
Sciences,
Ekochemistry,
420
University
Henry
Fordtran, 1973) and sheep (Braithwaite 1971) but not in aged subjects.
Abstract Calcium transport in the proximal duodenum was measured in male rats of various ages by the everted intestinal sac method. Calcium transport markedly and progressively diminished from 54 days to 384 days of age despite adequate intakes of vitamin D3 (IO IU/ day). Additionally, feeding low phosphorus and/or low calcium diets for 24 days did not result in an enhanced calcium transport in old [I year) rats while showing the expected enhancement in The administration of 325 young rats (24 days]. pmoles of either 1.25.dihydroxyvitamin D3 or la-hy droxyvitamin D3 daily increased intestinal calcium transport in both young and old rats. This response was accompanied by increased serum calcium in young rats and increased serum phosphorus in old rats. Administration of vitamin [1,23H]D3 daily (IO IUI for 6 days orally and chromatography of the extracts of serum and intestine permitted the demonstration that the diminished calcium transport with age is positively correlated with diminished levels of 1,25dihydroxy-[1GZ3H1 D, in serum and intestinal mucosa. It appears that the decreased intestinal absorption with age is a result of a diminished formation of 1.2% dihydroxyvitmain DL The inability of aged rates to alter this condition under dietary depletion of calcium and/or phosphorus may play a significant role in the development of bone disease in the aged.
of Mall,
WisconsinUniversity
and Riaduddin,
demonstraRecent advances in vitamin D3 metabolism te that it is converted in the liver to 25-hydroxyvitamin D3 (25OH-D3) which is transported to the kidney, and under the influence of parathyroid hormone (PTH) and/or low serum phosphorus is converted to f ,25dihydroxyvitamin D3 (f,25-(OHlzD31, the active form of vitamin D3 (DeLuca, 1974). Furthermore, the stimulation of intestinal absorption of Ca by PTH has been shown to be mediated by its effect on f ,25-IOHIzD3 synthesis (Ribovich and DeLuca, 1975; Garabedian et al., 19741. The objectives of the present study were to determine: the effects of advancing age on the intestinal transport of calcium-45 (45Cal; the effects of age on the ability of the rat to alter intestinal 45Ca transport under conditions of acute dietary deprivation of Ca and phosphorus IPI or a dietary excess of Ca; and the relationship between intestinal 45Ca transport and the acumulation of 1,25-(OHIzD3 in the mucosa and serum of young and aged rats.
Methods
-
Experiment ‘1. Male albino rats were purchased at the ages of 30, 60, 120, and 360 days from Sprague-Dawley Co. (Madison. Wis.). They were housed in individual hanging wire cages and fed for 24 days one of the following vitamin D-deficient diets containing: (A) 2.0% Ca, 0.1% P; (B) 0.47% Ca, 0.3% P: (Cl 0.1% Ca, 0.1% P. Each rat was given orally 10 IU of vitamin 03 each day. The animals were starved for 24 hr prior to decapitation. Intestinal 45Ca transport studies were carried out according to the method of Martin and DeLuca (1969) and results were expressed as a ratio of serosal (S) 45Ca/mucosal (M) 45Ca.
in intestinal calcium (Ca) age and diet have been in animal studies. Young growing rats absorb more Ca than mature rats, measured both in vitro and in vivo (Schacter et al., 1960; Zornitzer and Bronner, 1970). Feeding a low Ca diet also stimulated Ca conservation by the digestive system in young humans (Ireland and
Experiment II. Male albino rats [Holtzman). 30 days of age (young) and retired breeders (360 days), were fed one of five vitamin D-deficient diets (Suds et al., 19711 for 24 days (Table II. Each rat was given 10 IU of vitamin 03 orally each day. The rats within an age group were randomly placed into one of four subgroups: the groups were given (A) 0.05 ml of 95% ethanol intravenously [IV) 7 to 9 hr or 24 hr prior to sacrifice; (B) 0.125 pg of ICXhydroxyvitamin D3 [la-OH-D31 24 hr prior to sacrifices;
Key words: Calcium I ,25_dihydroxyvitamin
- Transport
- Duodenum
- Age
D”.
Introduction Physiological variations absorption related to repeatedly demonstrated
30
R.L. Horst
Table I. Intestinal ment I).
calcium
transport
response
of
rats
in dietary
Age
and
1,25-Dihydroxyvitamin
calcium
(Cal
and
phosphorus
DS
(P) (Experi-
Diet’
Age
B
A
(days1
54 84 144 384
to changes
et al.:
4.22 2.52 1.90 1.04
I+ f -t *
0.24 0.38 0.20 0.02
(lO)a (51 (2) (5)
2.63 1.83 1.85 1.10
t r ?z +
0.18 0.23 0.19 0.20
C S/M (9)b (41 (4) (5)
45Ca ratio & SE2 3.40 * 0.33 2.18 -+ 0.19 1.94 i 0.18 1.05 -c 0.03
Age Average
(9)’ (5) (5) (41
“Diets fed were: (A) Ca, 2%: P. 0.1%; (B) Ca, 0.47%; P. 0.3%; (C) Ca, 0.1%; P, 0.1%. 2Serosal to mucosal concentration ratio. The number of rats used in the calculation of the mean a.bGcMeans with different superscripts are differeni (P < 0.05). d.e.fsgMeans with different superscripts are different (P < 0.05).
(Cl 0.125 pg of 1.25-(OH)zD3 7 to 9 hr prior to sacrifices. and (D) 10 IU of vitamin D3 daily for 17 day followed by IO IU vitamin [l.23H] DS (731 dpm/pmole) daily for 6 days. A dose of 10 IU of vitamin 03 daily was selected as a maintenance dose for all these expetiments (Lindquist, 1952) so storage would not occur. This then permitted replacing the unlabeled vitamin D3 with 3H vitamin 03 for 6 consecutive days. This procedure permitted 90-100% replacement of the unlabeled vitamin D compounds. The ;ats in this subgroup were killed 24 hr after the last oral dose of vitamin [l,23H]D3. The number of rats per diet per age group in subgroups A through D were 12, 6, 6 and 6. To confirm the findings rats of similar age and similarly treated were bled and 1,25-(OH)zD3 levels were measured by a modification of the Eisman et al (1976) in parentheses technique. The values obtained shown in Table III illustrate the agreement between the direct measurement of 1,25-(OHlzD3 and the levels estimated by the radioactive measurements following 6 days of 3H vitamin 03. The rats in subgroups A through C were starved for 24 hr and killed by decapitation. Blood was collected and the serum harvested immediately for analysis of Ca and P. The intestine was removed and 45Ca transport measurements were conducted as in Experiment I. Subgroup D rats were sedated with ether and the blood was collected by heart puncture. The serum and intestinal mucosa were harvested and pooled according to diet and age. The separation of the vitamin D3 metabolites was achieved by the method of Holick and DeLuca (1971). Recoveries of the radioactivity from Sephadex LH-20 chromatography ranged from 80 to 94% in the serum and 84 to 104% in the intestine. The identity of metabolites was confirmed by co-chromatography with authentic compound on celite (Suda et al., 1970) or high pressure liquid chromatography (Jones and DeLuca, 19751. Radioactivity was counted in a Packard Tri-Carb Scintillation Count&. Serum Ca was measured on an atomic absorotion spectroohotometer (Perkin-Elmer Core.. 19651 and P’ was measured by the method of Chen et a’l. (19563. Statistics were calculated according to Duncan as described by Steel and Torrie (1960).
Results of Ca and/or P Experiment 1. Dietary restriction (diets A and C) resulted in increased (P<.O5) intestinal 45Ca transport in 54 day old rats and a nonsignificant increase in 84 day old rats (Table I) when compared to rats fed diet B (normal in Ca and P). The restriction of these two elements had no effect on intestinal 45Ca transport in 144 and 384 day old rats (Table I). Comparison of age group means (Table I) showed a
decline
ficantly
3.45 2.20 1.90 1.04
+ 0.19 f 0.17 & 0.10 z!I0.07
is in
(28)d (14)e (lllf (1419
parentheses.
in 45Ca transport with all means being signidifferent (P<.O5).
Experiment
II. Intestinal 45Ca transport was stimulated (P<.O5) by low dietary levels of P (diets 1 and 2) and Ca (diet 3) in young rats when compared to diet 5, normal Ca and P (Table II). Feeding excess Ca to young rats depressed (Pc.05) transport of 45Ca when coupled with normal P levels (diet 4). Aged rats did not show significant alterations in 45Ca transport within the five dietary groups. However, there was a slight, but nonsignificant, increased transport provoked by diets 1 through 3, and a slight depression by diet 4 [Table II).
The stimulatory effects of exogenous la-OH-D3 and 1,25-(OH)zD3 on transport of 45Ca were also studied. Comparisons of la-OH-D3 and 1,25(OH)zD3. treated rats to control rats showed a significant overall effect (including all diets) of each treatment in both age groups (Table II). The apparent inability of 1,25-(OH)zD3 and la-OH-D3 to stimulate 45Catransport in the young animals fed low Ca was of interest. This result is unexplained but may demonstrate that the intestinal receptors are already saturated with 1,25-(OH)zD3, and exogenous vitamin has no further effect. The concentration of 1,25-(OH)zD3 in the pooled serum (Table Ill) and intestinal mucosa (Table IV) lipid extracts exhibited a high correlation (P<.Ol) with the mean intestinal 45Ca transport values in Table II (r = .954, r = .937). This simple regression equation of Y (S/M ratio) vs. X [mucosal 1,25-(OHlzD3 (pmoles/g], diagrammatically shown in Fig. 1, was Y = .979 + 5.86 (X). Serum Ca was significantly lower (7.1 mg/lOO ml1 in the young rats on the low Ca diet than on the normal diet (IO.2 mg/lOO ml Pc.05). The highest levels of Ca in the young group were observed in those rats fed the low P and high Ca-normal P diets (11.5-12.5 mg/lOO ml). Injections of la-OH-D3 and 1,25-(OH)zD3 caused an increase in serum Ca in the young rats (average of each treatment). There was, however, no effect of either of the diets or these treatments on serum Ca in aged rats. Contrary to the effects observed on serum Ca, I.V. injections of la-OH-D3 and 1,25-(0H)zDs did cause a significant increase in serum P in the young (8.2 mg/lOO ml to 9.7 mg/lOOO ml) and aged rats (6.9 mg/lOO ml to 9.8 mg/lOO mll. The 1,25-(OH1zD3
R.L. Horst
Table
et al.:
II. Effects
Age
and
1,25Dihydroxyvitamin
of age, diet
and
31
D3
I-hydroxylated
vitamin
D treatment
on intestinal
calcium
transport,
mean
+
SE.
Treatment Diet’
1,25-[OH]zD3
1x-OH-Da
Control
2 2.00 0.1 3 0.02 0.3 4 2.00 0.3 5 0.47 0.3 Age Average
4.18 3.85 3.81 2.54 2.91 3.49
* 0.32c -fi 0.38’ * 0.33’ I!Z 0.33d r+- 0.26d f 0.17a
Young 4.78 f 0.38 4.40 i 0 34 3.95 i 0.46 3.20 i 0.22 4.66 -t 0.26 4.41 t 0.18”
5.04 6.98 3.46 3.96 4.04 4.70
i i& -+ * f
0.26 1.20 0.30 0.49 0.32 0.36”
1 2 3 4 5 Age
1.13 1.20 1.39 0.95 1.12 1.16
X!z 0.12 z? 0.11 * 0.12 i- 0.04 t 0.10 & 0.05”
1.87 2.20 2.01 1.19 1.64 1.75
Aged i 0.11 i- 0.20 -I- 0.20 + 0.08 ? 0.10 t 0.10”
2.08 1.88 1.99 1.99 1.47 1.88
c & +* f t
0.29 0.23 0.10 0.10 0.11 O.lOh
Ca
P
1 0.47
0.1
0.47 0.1 2.00 0.1 0.02 0.3 2.00 0.3 0.47 0.3 Average
‘Refer to Table 1 for diet key and Ca and P levels are given a,bMeans with different superscripts are different (P < 0.05). c.dMeans with different superscripts are different (P < 0.05).
Table III. Serum
concentrations
of vitamin
[1.2YH]Ds
and its
in “0.
metabolites
as affected
by age diet,
pmoles/ml
Diet Sterol 1
2
D3 25-OH-D3 24,25-(OH]zD3 1.25-(OHlzD3
0.55 7.02 1.09 0.54
3.73 10.04 1.54 0.66
D3 25-OH-D3 24,25-(OHlzD3 1.257(OHlzD3
1.30 7.31 1.17 0.16
0.84 6.08 1.11 0.08
Diet 1: 0.47% Ca, 0.1% P Diet Diet 5: 0.47% Ca, 0.3% P ‘represent Numbers in parentheses Eisman et al. technique (1976).
Table IV. Intestinal
mucosa
2: 2.00%
Ca. 0.1%
actual
1,25-(OHIzD3
concentration
of vitamin
3
4
Young 0.38 2.68 0.28 0.77 (0.80)
1.52 11.05 2.68 0.34 (0.331
1.22 8.00 2.04 0.44 (0.50)
0.67 6.32 0.90 0.04 (0.03)
0.97 6.88 0.89 0.03 (0.02]
Aged 0.75 6.38 1.19 0.11
P -
Diet
3: 0.02%
determined
[1.23H]D3
on
Ca. 0.3%
rats
and its metabolites
similarly
5
P -
Diet treated,
as affected
4: 2.00% but
Ca, 9.3%, P -
measured
by age and diet,
by
the
pmoles/g.
Diet Sterol 1
3
2
4
5
0.69 1.21 0.17 0.20
0.52 0.83 0.13 0.19
0.70 0.32 0.45 0.04
0.58 0.38 0.06 0.06
Young ::-OH-D3 24,25-(OH]zD3 1.25-(OHlzD3
0.61 1.20 0.18 0.48
0.51 1.51 0.14 0.45
D3 25-OH-D3 24,25-(OH)zD3 1,25-(O!-i)zD3
0.74 0.37 0.09 0.08
0.60 0.37 0.08 0.08
Diet Diet
1: 0.47% 5: 0.47%
Ca, 0.1% P Ca. 0.3% P.
Diet
2: 2.00%
Ca, 0.1%
0.31 1.10 0.09 0.61 Aoed 023 0.35 0.08 0.07
P -
Diet
3: 0.02%
Ca. 0.3%
P -
Diet
4: 2.1~0% Ca, 0,301~ _
32
R.L. Horst
/ A
A
z
2
-
3. L
A
/
A
/
! j.< ,,,,,,,
)
.I MUCOSAL
.2 l.25-(0H)z
.3
.4 Da
(PMOLES
5
.6
.7
et al.:
Age
and
1.25-Dihydroxyvitamin
Da
skeletal calcium for support of serum calcium concentration which would then result in a loss of bone mass over a long period of time. A complete lack of 1,25-(OHlzD3 would be expected to give osteomalacia but an inadequate amount would preclude adequate calcium absorption to meet needs but would permit utilization of skeletal stores of calcium contributing to osteoporosis. Osteomalacia and osteoporosis are both found in aging man Furthermore a (Nordin, 1971; Aaron et al. 1974). decrease in calcium absorption is known to occur in man with advancing age as is the inability to increase calcium absorption when low Ca diets are fed (Ireland and Fordtran, 1973; Aaron et al. 1974).
/G)
Fig. 1. The relationship
between mucosal 1,25-dihydroxyvitamin D3 levels and calcium-45 transport. Y = 0.979 + 5,86(x], r = 0.937. Note the intercept of the Y axis is 0.979. A value of 1 would be indicative of complete lack of active calcium transport in the in vitro media.
serum P than caused a significantly higher (P<.O5) the IGOH-Ds in the aged rats, whereas these steroids had indistinguishable hyperphosphatemic activity in the young rats.
Discussion The sterol 1,25-(OHlzD3 has been shown to be the regulator of intestinal Ca transport (DeLuca, 1974). Therefore, the hypothesis that the decline in Ca transport with age is related to a decrease of this hormone in intestinal mucosa, was tested. Our data clearly demonstrate a positive relationship between the declining level of radioactivity isolated as 1,25-(OH)zD3 in the mucosa (Table IV] and the decline in 45Ca transport with advancing age (Table III. Our data likewise provide additional exidence that the ability of young rats to increase calcium transport when fed a low calcium or low P diet is related to the amount of 1,25-(OH)zD3 in the intestine. Such an adaptation by the intestine was not apparent in the aged rat, nor was an increased accumulation of 1,25-(OH)zD, in mucosa of rats fed low dietary calcium observed. An apparent reason for the lack of intestinal accumulation of 1,25-(OH)zD3 in the mucosa is possib\y a declining rate of synthesis in the kidney as reflected [Table IV), rather by serum levels of this metabolite than a lack of ability to accumulate 1,25-(OHIzD3 in Further evidence of this point is the mucosa. reflected in the demonstrable acceleration in Ca induced by exogenous 1c(-OH-De and transport 1,25-(OHlzD3 in the aged rats (Table II). Nicolaysen et a/. (1953) suggested there is a close relationship between the skeletal needs for Ca and the rate Ca transport, this relationship being mediated by an .endogenous factorn that required the presence this of vitamin D. Boyle et al. (1971) suggested mendogenous factorm was 1,25-(OH)zD3. Therefore, the decrease in 1,25-(OHIzD3 production with age may be related to advanced skeletal maturation and This phenomenon could also explain calcification. the enhanced production of 1,25-(OHIzD3 and the rapid adaptation to low Ca and P diets in the young rats. On the other hand, the inability to synthesize sufficient amounts of 1,25-(OH)zD3 to meet the needs for calcium may be a primary event in the aging process. This lack could result in a reliance upon
The synthesis of 1,25-(OHlzD3 is under the control of both serum Ca (Boyle et al. 19711 and P (Tanaka and DeLuca, 1973). These observations were confirmed by the measurements of serum 1,25-(OHIzD3 levels by Hughes et al. (1975). Boyle et al. (19711 showed that hypocalcemia led to an increase in the amount of 1,25-(OHIzD3 in the serum. A similar result was observed in our experiment, where young rats fed a Ca-deficient diet had a resulting hypocalcemia and a marked increase in serum 1,25-(OHIzD3 (Table Ill). The proposed mechanism whereby 1,25-(OH@ is increased was recently discussed (Deiuca, 1974). Hypocalcemia leads to a rise in circulating PTH which acts as a stimulating factor in the conversion of 25-OH-D3 to 1,25-(OHIzD3. Hypophosphatemia is also attended by increased serum 1,25-(OH)zD3 levels (Table Ill), a result similar to that of Tanaka and DeLuca (1973). Of interest was the observation that a similar elevation of serum Ca and depression of serum P resulted in young rats fed diet 1 (0.45 % Ca, 0.1 % P) and diet 4 (2.0 % Ca, 0.3 % PI. However, regardless of similarity in serum concentration of these two elements, the level of 1,25-(OH)zD3 was 56 % higher in the rats fed diet 1. An increased flow of Ca and/or P though the kidney (a possible consequence from the offering of diet 4) may be responsible for this effect. However, no data exists which correlates the flow of Ca and/or P. through the kidney and 1,25-(OH)zD3 synthesis. Blood Ca was significantly increased (P<.O5) by injections of exogenous 1a-OH-D3 and 1,25-(OHIzD3 in the young rat, but not in aged rats (Table VI. An explanation for the lack of hypercalcemia might be the lowered response of aged bone and intestine to Ca mobilizing stimuli (Messer et a/. 19741. Unlike serum Ca, P levels in both age groups were significantly (P c.051 elevated by injections of la-OH-D3 and 1,25-(OH)zD3. This rise has been shown to take place even with a diet almost devoid of P (Messer et a/. 1974). Bone mobilization, stimulated by 1,25-(OH)zD3, is likely to be the cause. Although a response of intestinal calcium transport to exogenous la-OH-D3 or f,25-(OHlzD3 was observed in aged rats, it did not approach that found in young In addition to an inability to synthesize rats. sufficient amounts of 1,25-(OH)zD3 it seems likely that the intestinal calcium transport system is less responsive in the aged rats. These results thus indicate that aging produces both a less responsive renal 25-OH-D-I-hydroxylase system and a less responsive intestinal calcium transport
system. These factors therefore to contribute to the development loss.
might be expected of senescent bone
A.L. Horst et al.: Age and i,25Dihydroxyvitamin
33
D3
Research supported by the College of Agricultural and Life Sciences, University of WisconsinMadison, Hatch Project 5051, U.S. ERDA EY-76-S-02-1668,a grant from the Procter and Gamble Company and the Harry Steenbock Research Fund.
Aknowledgement:
References Aaron, J.E., Gallagher, J.C., Anderson, J., Stasiak. L., Longton, E.B. and Nordin, B.E.C.: Frequency of osteomalacia and osteoporosis in fractures of the proximal femur. Lancet i: 229-233, 1974. R.W. and DeLuca, H.F.: Regulations by calcium of in vivo synthesis of 1.25-dihydroxychole. Proc. Nat. calciferol and 21,25-dihydroxycholecalciferol.
Boyle,
I.T., Gray,
Acad.
Sci. 66: 2131-2734,
1971.
Braithwaite, G.D. and Riaduddin. S.H.: The effect of age and level of dietary calcium intake on calcium metabolism in sheep. &. J. Notr. 26: 215-225, 1971. Chen, P.S., Jr., Toribara T.Y. and Warner H.: Microdetermination of phosphorus. Anal. Chem. 28: 1756-1758, 1956.
~ Martin,
D.L. and DeLuca HF.: Influence of sodium on calcium transport by rat small intestine. Am, J. Physio/. 216: 1351-1359, 1969.
Messer, H.H., Yuen, S.Y. and Copp. D.H.: Effect of age on bone calcium and phosphate metabolism in organ culture. Calc. Tiss. Res. i6: 89-92, 1974. Nicolaysen. R., Eeg-Larsen, N. and Malm O.J.: Physiology of calcium metabolism. Physiol. Rev. 33: 424-444, 1953. Nordin, B.E.C.: The clinical significance and pathogenesis of osteoporosis. Brit. Med. J. 1: 571-576, 1971. Perkin-Elmer, Corporation. Analytical Absorption Spectrophotometry. Norwalk, CT, 1965.
Ribovich, M.L. and DeLuca H.F.: The influence of dietary calcium and phosphorus on intestinal calcium transArch. Bioport in rats given vitamin D Metabolites. them. Schacter, port Am. Steel,
DeLuca, H.F.: Vitamin D: the vitamin Fed. Proc. 33: 2211-2219, 1974.
or the hormone.
Methods of Atomic Perkin-Elmer Corp.
Biophys.
170:
529-535,
1975.
D., Dowdle, E.B. and Schenker H.: Active transof calcium by the small intestine of the rat. J. Physiol. 198: 263-268, 1960.
R.G.D.
and
of Statistics.
Torrie
J.H.:
McGraw-Hill
Principles and Procedures Book Co., Inc., New York,
1960.
Eisman, J.A., Hamstra, A.J., Kream, B.E. and DeLuca H.F.: A sensitive, precise and convenient method for determination of 1,25-dihydroxyvitamin D in human plasma. Arch. Biochem. Biophys. 177: 225-243, 1976.
Suda, T., DeLuca, H.F. and Tanaka Y.: Biological activity of 25-hvdroxvcholecalciferol in rats. J. Notr. 100: 1049-105-2,1970.
Garabedian, M., Tanaka, Y., Holick, M.F. and DeLuca H.F.: Response of intestinal calcium transport and bone calcium mobilization to 1,25-dihydroxyvitamin D3 in
Suda, T., DeLuca, H.F., Schnoes, H.K., Tanaka, Y. and Holick, M.F.: 25-26-Dihydroxyergocalciferol, a metabolite of vitamin D3 with intestinal calcium transport activity. Biochemistry. 9: 4776-4780, 1970.
-the thyroparathyroidectomized
rats. Endocrinology.
94:
1022-1027. 1974.
Tanaka, Y. and DeLuca. H.F.: The control
Holick, M.F. and DeLuca H.F.: A new chromatographic system for vitamin D3 and its metabolites: resolution of a new vitamin 03 metabolite. J. Lipid. Res. 12: 460-465, 1971. Ireland,
P. and Fordtran
and age on jejunal studied by intestinal 2672-2681,
J.S.: Effect of dietary calcium calcium absorption in humans perfusion.
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Invest.
52:
1973.
of 25-hydroxyvitamin D metabolism by inorganic phosphorus. Arch. Biochem. Biophys. 154: 566-574. 1973.
Tanaka, Y. and DeLuca, H.F.: Role of 1,25-dihydroxyvitamin 03
in rickets.
maintaining serum phosphorus and curing hoc. Nat. Acad. Sci. 71: 1040-1044, 1974.
Zornitzer, A.E. and Bronner. F.: In situ studies of calcium absorption in rats. Cal&. Tissue Res. 4 (suppI.): 161, 1970.
H.F.: High-pressure liquid chromatography: separation of the metabolites of vitamins Dz and D3 on small-particle silica columns. J. Lipid
Jones,
G. and DeLuca
Res. 16: 448-453, 1975. Lindquist, B.: Effect of vitamin D on the metabolism of radiocalcium in rachitic rats. Acta Paediat. 41: Suppl. 86, 1952.
Received: January 16, 1978 Revised: April 17, 1978 Accepted: April 25, 1978
Bone
Dis.
Metabolic Bone Disease 8 Related Research
& Ref. Res. 1, 29-33 /I9781
@ by S.N.P.M.D. (Paris 1978)
The effect of age on calcium absorption of l,Z!i-diil oxyvitamin 03 in intestinal R.L. HORST, H.F. DELUCA, Departments of Dairy Science Madison, Madison, Wisconsin
and accumulation mucosa ‘of rats
AND N.A. JORGENSEN and Biochimistry. 53706 U.S.A.
College
of
: Dr H. F. DeLuca. Address for correspondance and reprints of Wisconsin-Madison, Madison, WI 53706, U.S.A.
Agricultural Department
and of
Life
Sciences,
Ekochemistry,
420
University
Henry
Fordtran, 1973) and sheep (Braithwaite 1971) but not in aged subjects.
Abstract Calcium transport in the proximal duodenum was measured in male rats of various ages by the everted intestinal sac method. Calcium transport markedly and progressively diminished from 54 days to 384 days of age despite adequate intakes of vitamin D3 (IO IU/ day). Additionally, feeding low phosphorus and/or low calcium diets for 24 days did not result in an enhanced calcium transport in old [I year) rats while showing the expected enhancement in The administration of 325 young rats (24 days]. pmoles of either 1.25.dihydroxyvitamin D3 or la-hy droxyvitamin D3 daily increased intestinal calcium transport in both young and old rats. This response was accompanied by increased serum calcium in young rats and increased serum phosphorus in old rats. Administration of vitamin [1,23H]D3 daily (IO IUI for 6 days orally and chromatography of the extracts of serum and intestine permitted the demonstration that the diminished calcium transport with age is positively correlated with diminished levels of 1,25dihydroxy-[1GZ3H1 D, in serum and intestinal mucosa. It appears that the decreased intestinal absorption with age is a result of a diminished formation of 1.2% dihydroxyvitmain DL The inability of aged rates to alter this condition under dietary depletion of calcium and/or phosphorus may play a significant role in the development of bone disease in the aged.
of Mall,
WisconsinUniversity
and Riaduddin,
demonstraRecent advances in vitamin D3 metabolism te that it is converted in the liver to 25-hydroxyvitamin D3 (25OH-D3) which is transported to the kidney, and under the influence of parathyroid hormone (PTH) and/or low serum phosphorus is converted to f ,25dihydroxyvitamin D3 (f,25-(OHlzD31, the active form of vitamin D3 (DeLuca, 1974). Furthermore, the stimulation of intestinal absorption of Ca by PTH has been shown to be mediated by its effect on f ,25-IOHIzD3 synthesis (Ribovich and DeLuca, 1975; Garabedian et al., 19741. The objectives of the present study were to determine: the effects of advancing age on the intestinal transport of calcium-45 (45Cal; the effects of age on the ability of the rat to alter intestinal 45Ca transport under conditions of acute dietary deprivation of Ca and phosphorus IPI or a dietary excess of Ca; and the relationship between intestinal 45Ca transport and the acumulation of 1,25-(OHIzD3 in the mucosa and serum of young and aged rats.
Methods
-
Experiment ‘1. Male albino rats were purchased at the ages of 30, 60, 120, and 360 days from Sprague-Dawley Co. (Madison. Wis.). They were housed in individual hanging wire cages and fed for 24 days one of the following vitamin D-deficient diets containing: (A) 2.0% Ca, 0.1% P; (B) 0.47% Ca, 0.3% P: (Cl 0.1% Ca, 0.1% P. Each rat was given orally 10 IU of vitamin 03 each day. The animals were starved for 24 hr prior to decapitation. Intestinal 45Ca transport studies were carried out according to the method of Martin and DeLuca (1969) and results were expressed as a ratio of serosal (S) 45Ca/mucosal (M) 45Ca.
in intestinal calcium (Ca) age and diet have been in animal studies. Young growing rats absorb more Ca than mature rats, measured both in vitro and in vivo (Schacter et al., 1960; Zornitzer and Bronner, 1970). Feeding a low Ca diet also stimulated Ca conservation by the digestive system in young humans (Ireland and
Experiment II. Male albino rats [Holtzman). 30 days of age (young) and retired breeders (360 days), were fed one of five vitamin D-deficient diets (Suds et al., 19711 for 24 days (Table II. Each rat was given 10 IU of vitamin 03 orally each day. The rats within an age group were randomly placed into one of four subgroups: the groups were given (A) 0.05 ml of 95% ethanol intravenously [IV) 7 to 9 hr or 24 hr prior to sacrifice; (B) 0.125 pg of ICXhydroxyvitamin D3 [la-OH-D31 24 hr prior to sacrifices;
Key words: Calcium I ,25_dihydroxyvitamin
- Transport
- Duodenum
- Age
D”.
Introduction Physiological variations absorption related to repeatedly demonstrated
30
R.L. Horst
Table I. Intestinal ment I).
calcium
transport
response
of
rats
in dietary
Age
and
1,25-Dihydroxyvitamin
calcium
(Cal
and
phosphorus
DS
(P) (Experi-
Diet’
Age
B
A
(days1
54 84 144 384
to changes
et al.:
4.22 2.52 1.90 1.04
I+ f -t *
0.24 0.38 0.20 0.02
(lO)a (51 (2) (5)
2.63 1.83 1.85 1.10
t r ?z +
0.18 0.23 0.19 0.20
C S/M (9)b (41 (4) (5)
45Ca ratio & SE2 3.40 * 0.33 2.18 -+ 0.19 1.94 i 0.18 1.05 -c 0.03
Age Average
(9)’ (5) (5) (41
“Diets fed were: (A) Ca, 2%: P. 0.1%; (B) Ca, 0.47%; P. 0.3%; (C) Ca, 0.1%; P, 0.1%. 2Serosal to mucosal concentration ratio. The number of rats used in the calculation of the mean a.bGcMeans with different superscripts are differeni (P < 0.05). d.e.fsgMeans with different superscripts are different (P < 0.05).
(Cl 0.125 pg of 1.25-(OH)zD3 7 to 9 hr prior to sacrifices. and (D) 10 IU of vitamin D3 daily for 17 day followed by IO IU vitamin [l.23H] DS (731 dpm/pmole) daily for 6 days. A dose of 10 IU of vitamin 03 daily was selected as a maintenance dose for all these expetiments (Lindquist, 1952) so storage would not occur. This then permitted replacing the unlabeled vitamin D3 with 3H vitamin 03 for 6 consecutive days. This procedure permitted 90-100% replacement of the unlabeled vitamin D compounds. The ;ats in this subgroup were killed 24 hr after the last oral dose of vitamin [l,23H]D3. The number of rats per diet per age group in subgroups A through D were 12, 6, 6 and 6. To confirm the findings rats of similar age and similarly treated were bled and 1,25-(OH)zD3 levels were measured by a modification of the Eisman et al (1976) in parentheses technique. The values obtained shown in Table III illustrate the agreement between the direct measurement of 1,25-(OHlzD3 and the levels estimated by the radioactive measurements following 6 days of 3H vitamin 03. The rats in subgroups A through C were starved for 24 hr and killed by decapitation. Blood was collected and the serum harvested immediately for analysis of Ca and P. The intestine was removed and 45Ca transport measurements were conducted as in Experiment I. Subgroup D rats were sedated with ether and the blood was collected by heart puncture. The serum and intestinal mucosa were harvested and pooled according to diet and age. The separation of the vitamin D3 metabolites was achieved by the method of Holick and DeLuca (1971). Recoveries of the radioactivity from Sephadex LH-20 chromatography ranged from 80 to 94% in the serum and 84 to 104% in the intestine. The identity of metabolites was confirmed by co-chromatography with authentic compound on celite (Suda et al., 1970) or high pressure liquid chromatography (Jones and DeLuca, 19751. Radioactivity was counted in a Packard Tri-Carb Scintillation Count&. Serum Ca was measured on an atomic absorotion spectroohotometer (Perkin-Elmer Core.. 19651 and P’ was measured by the method of Chen et a’l. (19563. Statistics were calculated according to Duncan as described by Steel and Torrie (1960).
Results of Ca and/or P Experiment 1. Dietary restriction (diets A and C) resulted in increased (P<.O5) intestinal 45Ca transport in 54 day old rats and a nonsignificant increase in 84 day old rats (Table I) when compared to rats fed diet B (normal in Ca and P). The restriction of these two elements had no effect on intestinal 45Ca transport in 144 and 384 day old rats (Table I). Comparison of age group means (Table I) showed a
decline
ficantly
3.45 2.20 1.90 1.04
+ 0.19 f 0.17 & 0.10 z!I0.07
is in
(28)d (14)e (lllf (1419
parentheses.
in 45Ca transport with all means being signidifferent (P<.O5).
Experiment
II. Intestinal 45Ca transport was stimulated (P<.O5) by low dietary levels of P (diets 1 and 2) and Ca (diet 3) in young rats when compared to diet 5, normal Ca and P (Table II). Feeding excess Ca to young rats depressed (Pc.05) transport of 45Ca when coupled with normal P levels (diet 4). Aged rats did not show significant alterations in 45Ca transport within the five dietary groups. However, there was a slight, but nonsignificant, increased transport provoked by diets 1 through 3, and a slight depression by diet 4 [Table II).
The stimulatory effects of exogenous la-OH-D3 and 1,25-(OH)zD3 on transport of 45Ca were also studied. Comparisons of la-OH-D3 and 1,25(OH)zD3. treated rats to control rats showed a significant overall effect (including all diets) of each treatment in both age groups (Table II). The apparent inability of 1,25-(OH)zD3 and la-OH-D3 to stimulate 45Catransport in the young animals fed low Ca was of interest. This result is unexplained but may demonstrate that the intestinal receptors are already saturated with 1,25-(OH)zD3, and exogenous vitamin has no further effect. The concentration of 1,25-(OH)zD3 in the pooled serum (Table Ill) and intestinal mucosa (Table IV) lipid extracts exhibited a high correlation (P<.Ol) with the mean intestinal 45Ca transport values in Table II (r = .954, r = .937). This simple regression equation of Y (S/M ratio) vs. X [mucosal 1,25-(OHlzD3 (pmoles/g], diagrammatically shown in Fig. 1, was Y = .979 + 5.86 (X). Serum Ca was significantly lower (7.1 mg/lOO ml1 in the young rats on the low Ca diet than on the normal diet (IO.2 mg/lOO ml Pc.05). The highest levels of Ca in the young group were observed in those rats fed the low P and high Ca-normal P diets (11.5-12.5 mg/lOO ml). Injections of la-OH-D3 and 1,25-(OH)zD3 caused an increase in serum Ca in the young rats (average of each treatment). There was, however, no effect of either of the diets or these treatments on serum Ca in aged rats. Contrary to the effects observed on serum Ca, I.V. injections of la-OH-D3 and 1,25-(0H)zDs did cause a significant increase in serum P in the young (8.2 mg/lOO ml to 9.7 mg/lOOO ml) and aged rats (6.9 mg/lOO ml to 9.8 mg/lOO mll. The 1,25-(OH1zD3
R.L. Horst
Table
et al.:
II. Effects
Age
and
1,25Dihydroxyvitamin
of age, diet
and
31
D3
I-hydroxylated
vitamin
D treatment
on intestinal
calcium
transport,
mean
+
SE.
Treatment Diet’
1,25-[OH]zD3
1x-OH-Da
Control
2 2.00 0.1 3 0.02 0.3 4 2.00 0.3 5 0.47 0.3 Age Average
4.18 3.85 3.81 2.54 2.91 3.49
* 0.32c -fi 0.38’ * 0.33’ I!Z 0.33d r+- 0.26d f 0.17a
Young 4.78 f 0.38 4.40 i 0 34 3.95 i 0.46 3.20 i 0.22 4.66 -t 0.26 4.41 t 0.18”
5.04 6.98 3.46 3.96 4.04 4.70
i i& -+ * f
0.26 1.20 0.30 0.49 0.32 0.36”
1 2 3 4 5 Age
1.13 1.20 1.39 0.95 1.12 1.16
X!z 0.12 z? 0.11 * 0.12 i- 0.04 t 0.10 & 0.05”
1.87 2.20 2.01 1.19 1.64 1.75
Aged i 0.11 i- 0.20 -I- 0.20 + 0.08 ? 0.10 t 0.10”
2.08 1.88 1.99 1.99 1.47 1.88
c & +* f t
0.29 0.23 0.10 0.10 0.11 O.lOh
Ca
P
1 0.47
0.1
0.47 0.1 2.00 0.1 0.02 0.3 2.00 0.3 0.47 0.3 Average
‘Refer to Table 1 for diet key and Ca and P levels are given a,bMeans with different superscripts are different (P < 0.05). c.dMeans with different superscripts are different (P < 0.05).
Table III. Serum
concentrations
of vitamin
[1.2YH]Ds
and its
in “0.
metabolites
as affected
by age diet,
pmoles/ml
Diet Sterol 1
2
D3 25-OH-D3 24,25-(OH]zD3 1.25-(OHlzD3
0.55 7.02 1.09 0.54
3.73 10.04 1.54 0.66
D3 25-OH-D3 24,25-(OHlzD3 1.257(OHlzD3
1.30 7.31 1.17 0.16
0.84 6.08 1.11 0.08
Diet 1: 0.47% Ca, 0.1% P Diet Diet 5: 0.47% Ca, 0.3% P ‘represent Numbers in parentheses Eisman et al. technique (1976).
Table IV. Intestinal
mucosa
2: 2.00%
Ca. 0.1%
actual
1,25-(OHIzD3
concentration
of vitamin
3
4
Young 0.38 2.68 0.28 0.77 (0.80)
1.52 11.05 2.68 0.34 (0.331
1.22 8.00 2.04 0.44 (0.50)
0.67 6.32 0.90 0.04 (0.03)
0.97 6.88 0.89 0.03 (0.02]
Aged 0.75 6.38 1.19 0.11
P -
Diet
3: 0.02%
determined
[1.23H]D3
on
Ca. 0.3%
rats
and its metabolites
similarly
5
P -
Diet treated,
as affected
4: 2.00% but
Ca, 9.3%, P -
measured
by age and diet,
by
the
pmoles/g.
Diet Sterol 1
3
2
4
5
0.69 1.21 0.17 0.20
0.52 0.83 0.13 0.19
0.70 0.32 0.45 0.04
0.58 0.38 0.06 0.06
Young ::-OH-D3 24,25-(OH]zD3 1.25-(OHlzD3
0.61 1.20 0.18 0.48
0.51 1.51 0.14 0.45
D3 25-OH-D3 24,25-(OH)zD3 1,25-(O!-i)zD3
0.74 0.37 0.09 0.08
0.60 0.37 0.08 0.08
Diet Diet
1: 0.47% 5: 0.47%
Ca, 0.1% P Ca. 0.3% P.
Diet
2: 2.00%
Ca, 0.1%
0.31 1.10 0.09 0.61 Aoed 023 0.35 0.08 0.07
P -
Diet
3: 0.02%
Ca. 0.3%
P -
Diet
4: 2.1~0% Ca, 0,301~ _
32
R.L. Horst
/ A
A
z
2
-
3. L
A
/
A
/
! j.< ,,,,,,,
)
.I MUCOSAL
.2 l.25-(0H)z
.3
.4 Da
(PMOLES
5
.6
.7
et al.:
Age
and
1.25-Dihydroxyvitamin
Da
skeletal calcium for support of serum calcium concentration which would then result in a loss of bone mass over a long period of time. A complete lack of 1,25-(OHlzD3 would be expected to give osteomalacia but an inadequate amount would preclude adequate calcium absorption to meet needs but would permit utilization of skeletal stores of calcium contributing to osteoporosis. Osteomalacia and osteoporosis are both found in aging man Furthermore a (Nordin, 1971; Aaron et al. 1974). decrease in calcium absorption is known to occur in man with advancing age as is the inability to increase calcium absorption when low Ca diets are fed (Ireland and Fordtran, 1973; Aaron et al. 1974).
/G)
Fig. 1. The relationship
between mucosal 1,25-dihydroxyvitamin D3 levels and calcium-45 transport. Y = 0.979 + 5,86(x], r = 0.937. Note the intercept of the Y axis is 0.979. A value of 1 would be indicative of complete lack of active calcium transport in the in vitro media.
serum P than caused a significantly higher (P<.O5) the IGOH-Ds in the aged rats, whereas these steroids had indistinguishable hyperphosphatemic activity in the young rats.
Discussion The sterol 1,25-(OHlzD3 has been shown to be the regulator of intestinal Ca transport (DeLuca, 1974). Therefore, the hypothesis that the decline in Ca transport with age is related to a decrease of this hormone in intestinal mucosa, was tested. Our data clearly demonstrate a positive relationship between the declining level of radioactivity isolated as 1,25-(OH)zD3 in the mucosa (Table IV] and the decline in 45Ca transport with advancing age (Table III. Our data likewise provide additional exidence that the ability of young rats to increase calcium transport when fed a low calcium or low P diet is related to the amount of 1,25-(OH)zD3 in the intestine. Such an adaptation by the intestine was not apparent in the aged rat, nor was an increased accumulation of 1,25-(OH)zD, in mucosa of rats fed low dietary calcium observed. An apparent reason for the lack of intestinal accumulation of 1,25-(OH)zD3 in the mucosa is possib\y a declining rate of synthesis in the kidney as reflected [Table IV), rather by serum levels of this metabolite than a lack of ability to accumulate 1,25-(OHIzD3 in Further evidence of this point is the mucosa. reflected in the demonstrable acceleration in Ca induced by exogenous 1c(-OH-De and transport 1,25-(OHlzD3 in the aged rats (Table II). Nicolaysen et a/. (1953) suggested there is a close relationship between the skeletal needs for Ca and the rate Ca transport, this relationship being mediated by an .endogenous factorn that required the presence this of vitamin D. Boyle et al. (1971) suggested mendogenous factorm was 1,25-(OH)zD3. Therefore, the decrease in 1,25-(OHIzD3 production with age may be related to advanced skeletal maturation and This phenomenon could also explain calcification. the enhanced production of 1,25-(OHIzD3 and the rapid adaptation to low Ca and P diets in the young rats. On the other hand, the inability to synthesize sufficient amounts of 1,25-(OH)zD3 to meet the needs for calcium may be a primary event in the aging process. This lack could result in a reliance upon
The synthesis of 1,25-(OHlzD3 is under the control of both serum Ca (Boyle et al. 19711 and P (Tanaka and DeLuca, 1973). These observations were confirmed by the measurements of serum 1,25-(OHIzD3 levels by Hughes et al. (1975). Boyle et al. (19711 showed that hypocalcemia led to an increase in the amount of 1,25-(OHIzD3 in the serum. A similar result was observed in our experiment, where young rats fed a Ca-deficient diet had a resulting hypocalcemia and a marked increase in serum 1,25-(OHIzD3 (Table Ill). The proposed mechanism whereby 1,25-(OH@ is increased was recently discussed (Deiuca, 1974). Hypocalcemia leads to a rise in circulating PTH which acts as a stimulating factor in the conversion of 25-OH-D3 to 1,25-(OHIzD3. Hypophosphatemia is also attended by increased serum 1,25-(OH)zD3 levels (Table Ill), a result similar to that of Tanaka and DeLuca (1973). Of interest was the observation that a similar elevation of serum Ca and depression of serum P resulted in young rats fed diet 1 (0.45 % Ca, 0.1 % P) and diet 4 (2.0 % Ca, 0.3 % PI. However, regardless of similarity in serum concentration of these two elements, the level of 1,25-(OH)zD3 was 56 % higher in the rats fed diet 1. An increased flow of Ca and/or P though the kidney (a possible consequence from the offering of diet 4) may be responsible for this effect. However, no data exists which correlates the flow of Ca and/or P. through the kidney and 1,25-(OH)zD3 synthesis. Blood Ca was significantly increased (P<.O5) by injections of exogenous 1a-OH-D3 and 1,25-(OHIzD3 in the young rat, but not in aged rats (Table VI. An explanation for the lack of hypercalcemia might be the lowered response of aged bone and intestine to Ca mobilizing stimuli (Messer et a/. 19741. Unlike serum Ca, P levels in both age groups were significantly (P c.051 elevated by injections of la-OH-D3 and 1,25-(OH)zD3. This rise has been shown to take place even with a diet almost devoid of P (Messer et a/. 1974). Bone mobilization, stimulated by 1,25-(OH)zD3, is likely to be the cause. Although a response of intestinal calcium transport to exogenous la-OH-D3 or f,25-(OHlzD3 was observed in aged rats, it did not approach that found in young In addition to an inability to synthesize rats. sufficient amounts of 1,25-(OH)zD3 it seems likely that the intestinal calcium transport system is less responsive in the aged rats. These results thus indicate that aging produces both a less responsive renal 25-OH-D-I-hydroxylase system and a less responsive intestinal calcium transport
system. These factors therefore to contribute to the development loss.
might be expected of senescent bone
A.L. Horst et al.: Age and i,25Dihydroxyvitamin
33
D3
Research supported by the College of Agricultural and Life Sciences, University of WisconsinMadison, Hatch Project 5051, U.S. ERDA EY-76-S-02-1668,a grant from the Procter and Gamble Company and the Harry Steenbock Research Fund.
Aknowledgement:
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Received: January 16, 1978 Revised: April 17, 1978 Accepted: April 25, 1978