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The effect of vitamin D on urinary citrate in relation to calcium, phosphorus and urinary pH
The Effect of Vitamin D on Urinary Citrate in Relation to Calcium, Phosphorus and Urinary pH1 Stuart A. Bellin, David C. Herting, John W. Cramer, and Harry Steenbock
Vincent J. Pileggi
From the Department of Biochemistry, College of Agriculture, of Wisconsin, Madison, Wisconsin Received
May
University
29, 1953
With the observation that the increase in urinary citrate induced by the administration of vitamin D can be correlated with an increase in blood citrate (l), it became increasingly important to determine what dietary relations affect this increase. Admittedly, this is hard to establish because, as judged by the increments in citrate excretion following the ingestion of base (2-7), and the great capacity of the animal to catabolize orally ingested citrate (8-12), the range within which an equilibrium can be established must be very wide. So far, we know that in rachitic rats, while the citrate response to vitamin D is accompanied by an increase in urinary pH, it apparently is not dependent on this increase because it is elicited even when the urines are already strongly alkaline as the result of the inclusion of generous amounts of NaHC03 in the ration. It remains to be ascertained if vitamin D will have the same effect in the absence of a demonstrable increase in urinary pH. In addition, we have sought to determine the effect of Ca, P, and other minerals. Our interest in Ca stemmed from the fact that citratemia has been associated with hypercalcemia (11) and that Ca is well known as forming soluble non-ionized complexes with citrate. These complexes might be excreted readily during a hypercalcemia induced by vitamin D (13). Phosphorus has already been shown (7) to affect the intensity of citraturia in that citrate excretion was observed to be at 1 Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. We are indebted to the Wisconsin Alumni Research Foundation for the funds which made this research possible.
18
VITAMIN
D EFFECT ON URINARY
CITRATE
19
the highest level on low-P rations but it was increased the most, by vitamin D, with P-containing rations. In some of our presently reported experiments the Ca and P contents of the rations were reduced to very low levels (approximately 0.02 %). In others, all mineral additions to the organic basal ration, except NaCl, were dispensed with. NaHC03 was added in one series to increase the urinary pH. The above experiments were undertaken in full realization of the limitations in the value of urinary excretion as an index of citrate metabolism. Not only could increased synthesis in one tissue or organ be counterbalanced by increased catabolism in another, but changes in blood citrate might not be reflected by comparable changes in excretion (6). Nevertheless, we felt that a demonstration of the generality of a vitamin D effect might emphasize the desirability of more definitive research with tissues and organs. EXPERIMENTAL
Methods Young rats varying from 60 to 166 g. in weight in different series, but within a range of 10 g. for individual experiments, were fed a basal synthetic ration of glucose, cooked egg white, roughage, cottonseed oil, salts, and vitamins as previously described (1, 7). The salts in the basal ration were limited to a Ca-free, P-free mixture; the vitamins to a vitamin D-free mixture. The various omissions from, and supplements to, the basal constituents are presented, with the results, in the various tables. When required by the experimental plan, Ca was added as CaCOl , unless otherwise indicated; P was added as a neutral equimolar mixture of KHzPO, and KIHPOd , and vitamin D as a cottonseed oil solution of calciferol. The latter supplement, equivalent to 75 I.U., was given orally to each rat every 3 days. Records were kept of food consumed and, in some cases, as noted, consumption was equalized between D and non-D groups. The urines, preserved with toluene, were collected in 3-day periods. They were analyzed for citrate essentially by the use of the Pucher, Sherman, and Vickery technique (14), and for Ca and P, respectively, by KMnO, titration of the oxalate, and by the Fiske-SubbaRow calorimetric method (15) after perchloric-nitric acid digestion. The pH of the urine was determined at the end of each 3-day period with a glass electrode on fresh samples of urine collected in a few hours in clean, dry metabolism cages.
Results A gross inspection of the results in Table I reveals significantly that modifications of the low-P, low-Ca basal ration by addition of either P or Ca, singly or in multiple, did not have as much effect on citrate excretion as an increase in base intake in the form of NaHC03 alone, or re-
20
BELLIN,
The Eflect
of Vitamin
HERTING,
CRAMER,
PILEGGI
TABLE
=
Series
D on Urinary
Citrate
Keight ktial
l-l% III IV
42,Sl
4&i%
V II(a) II(b) I
Low ca Low P Low Ca Low P NaHCOa Low Ca Low P NaCl No other Normal Normal NaCl No other Low Ca High P Medium Normal Medium Normal High Ca Low P NaHC03
minerals Ca P
STEENBOCK
I in Relation
Vita& D
Ration
AND
to Calcium of rats
Urinary
Final
Ca ____
and PhosDhorusa ex~;tbp
P I
(daily/100
Citric
acid
!
g.
g.
g.
0.02 0.02 0.02 0.02 10.00 0.01 0.03 0.40
0 + 0 +
96 95 89 96
89 86 87 87
107 107 102 110
3.57 6.45 3.44 6.28
0.12 4.25 0.12 8.63 0.1821.5 0.2433.4
0 +
103 116
103 106
124 141
2.98 8.10
0.11 0.13
0.40 0.32 0.40
0 +
90 125
102 110
119 148
0.48 0.68
0.02 0.62 0.20 0.30 0.21 0.33 1.26 0.02 6.60
0 + 0 + 0 + 0 +
42 102 69 98 60 106 61 64
75 100 59 59 90 92 62 60
68 127 70 98 98 135 68 73
0.5424.7 1.70 0.4037.7 7.80 0.5710.2 2.30 0.61 8.5612.50 0.69 0.91 0.58 2.54 1.6 0.2021.3 8.2 0.1736.6
103 56
2.36 4.38 85
6.86 6.19
2.22 8.90
300
minerals
Ca P Cab P
359 445 179 72
a All groups contained six rats except series II(b) and 42,& in which there were three rats. All rats were males except those in series 42 which were females. All data are for the last four collections for 3 days each and therefore represent averages for the collections from the 9th to the 21st day except for series II(a); in this way they represent the averages for the 6th to the 18th day. The food consumed and body weights, initial and final, are likewise for these la-day periods. To each rat was given 75 I.U. of vitamin D every 3 days when indicated. A basal mineral mixture “free” from Ca and P was included in all rations except those fed in series 42,Sd and 42,Sh. b Ca was given as the sulfate; in all other series it was given as the carbonate.
placed, in part, by an equivalent amount of CaC03. Furthermore, even when citrate excretion was raised to a high level, supplementation with vitamin D elicited a further increase which, expressed in milligrams, was as large as that obtained with any of the other rations. Obviously, even
VITAMIN
D
EFFECT
ON
URINARY
21
CITRATE
the lowest levels of Ca and P did not prevent the excretion of citrate or the vitamin D effect. Citrate was always excreted in the urine and vitamin D always increased its excretion. This response, though limited to the area covered by our experiments, is expected to be very general, except possibly under more acidic conditions than those studied by us. The role of an increase in urinary pH or systemic pH is not so easily assessed. We have reported (12) that vitamin D will increase urinary pH when given to rats on rachitogenic rations, that is, on rations low 0; physiologically low, in assimilable P, but relatively high in Ca. On other rations, e.g., those nonrachitogenic in character, this effect was not obtained (Table II), yet citrate excretion was always increased even in those instances in which a reduction in urinary pH occurred. With urines from the bicarbonate series we were unable to obtain measurement of pH to a sufficient degree of accuracy; they were too heavily charged with CO* . If it is accepted that an increase in urinary citrate is not necessarily accompanied by an increase in urinary pH, it does not necessarily follow that the vitamin D effect and pH are not interrelated; an increase in hydroxyl ions may nevertheless be induced at the loci of citrate formation. Folis (16) has reported his inability to demonstrate any deviation in pH from the normal physiological range in bone and cartilage by the TABLE Vitamin
D in
Relation
Ration Expt.
+o. No. (
II
= I
to Urinary
Characteristic
Citrate
= I
and
Citrate
mg./lOO
g. rat/day
Before
D”
After
D*
pH PR
Before
D”
After
Db
-9
4
11A
26
12
11c
SP
6
1lD
0.20% calcium &s carbonate 0.30% phosphorus Wesson salti4 4.0% No calcium 0.30% phosphorus
5.30 (4.75-5.81) 3.57 (0.99-4.59) 1.31 (0.83-2.30) Without
42 42
6 3
S4 SS
No salts except 0.40% NsCl No salts except 0.40% NaCI. 1.0% CSCOI , and 0.32% P
16.92 (9.39-20.70) 10.60 (3.66-24.60) 6.16 (3.64-9.21) DC
With
3.51 6.70
-
7.27 (6.53-7.75) 6.29 (5.49-7.61) 7.32 (6.2C8.04)
DC
Without
6.37 11.56
-
7.29 7.60
5.61 (5.14-6.04) 6.08 (5.39-7.70) 6.18 (6.32-7.23) DC
With
-I
DC
7.29 7.60
-
a Collection after 6th~9th day on ration. * Collection after 9th-12th day on ration; 525 I.U. vitamin D was given over the a-day period. c Average values obtained in 21 days from rata started on rations with and without vitamin D when received from the breeders. The vitamin D supplemented group received 75 I.U. each day, every 3 days. d Wesson, L. G., Science Xi, 339 (1932).
22
BELLIN,
HERTING,
CRAMER,
PILEGGI
AND
STEENBOCK
application of a series of indicators. It remains to be seen if such a test is adequate. The skeleton has been considered of minor importance in citrate production (17). Our data do not provide the answer as to whether the increase in citrate elimination was due to an increase in citrate synthesis or to a decrease in its destruction. Tentatively we favor the former because the very high level of citrate excretion induced by 5 y0 NaHCOs , as previously reported (l), was increased only 8 y0 by raising the dietary bicarbonate from the 5 y0 to the 10 y0 level, while vitamin D increased it 34%. In accordance with the neutralization theory of &tberg (3), the excessive intake of bicarbonate should have elicited the excretion of much more citrate if it had been possible for the rats to synthesize more in the absence of vitamin D. While P is a potent factor in determining citrate equilibrium, our efforts to duplicate the effect of vitamin D by the addition of P were unsuccessful. In fact, in harmony with the low level of citrate excretion obtained with rations which contained P but no vitamin D, supplementing a rachitogenic ration with neutral phosphates reduced the citrate output. Later, when vitamin D was given, a rise in urinary citrate followed promptly. The urinary pH was not increased (Table III). Further evidence of a possible P effect was obtained with rats on TABLE III Effect of Phosphate on Urinary Citrate The basal ration was 23A, a low-P (0.02%), medium-Ca (0.21% Ca as CaCOr) ration. The P supplement was given gradually in a few days to avoid tetany. In series S-l, 136 mg. P was given per rat as a solution of KzHPOI and KHzPOa . In series W-l, P was incorporated in the ration, equivalent to 0.30yo P. The vitamin D dose was 525 I.U. given every 3 days. Before Collection
P
With
P
After
D
days 4-6
i Mg. citrate,
1
7-9
x-12
1
13-15
/ 8.0 )
7.2
i 100 g. rat, daily
11618
19-21
1 22-24
Series W-l (8 rats) Series S-l (12 rats) Urinary i 7.6 /
7.2
pH ) 7.0 1
]
VITAMIN
D EFFECT ON URINARY
CITRATE
23
Ration 2965 (18). As is well known, vitamin D increases the availability of P from this cereal ration. With four rats made rachitic to a degree comparable to that produced on our synthetic rations and then given vitamin D, citrate excretion was reduced to a level 42 y0 below that found in a comparable 6-day period immediately preceding the administration of the vitamin. It was not until the lapse of an additional 3 days that the excretion of urinary citrate rose to a level of 35 % above the pre-vitamin D period. This effect is quite at variance with the increases of 35-37 y0 obtained with our low P (0.02 ~~0)synthetic rations in the first 3 days. We were unable to find any correlation between the excretions of citrate and Ca or P. SUMMARY
Vitamin D increased the amount of urinary citrate excreted by young rats kept on rations varying widely in mineral content. These rations ranged in composition from those providing an adequate mineral intake to those made grossly deficient in Ca, P, and other minerals. The biggest percentage increase in citrate was obtained with P-containing rations, but supplements of P, given in the form of neutral solutions of K phosphates to a low-P ration, reduced citrate elimination. An increase in urinary pH induced by vitamin D with rats on low-P rachitogenie rations was not obtained with rations adequate in P, yet an increase in citrate elimination resulted in all instances. Inasmuch as a supplement of vitamin D increased urinary citrate far more than did an additional large intake of NaHC03, it appears that the effect of vitamin D is due to an increase in citrate synthesis rather than to a decrease in its destruction. REFERENCES 1. STEENBOCK, H., AND BELLIN, S. A., J. Biol. Chem. 206,985 (1953). 2. FASOLD, H., 2. Kinderheilk. 49, 709 (1930). 3. E)STBERG, O., Stand. Arch. Physiol. 62, 81 (1931). 4. SULLMAN, H., AND SHEARER, E., Schweiz. med. Wochschr. 13, 619 (1932). 5. SCHUCK, C., J. Nutrition 7, 679 (1934). 6. SHERMAN, C. C., MENDEL, L. B., AND SMITH, A. H., 3. Biol. Chem. 113, 247, 265 (1936). 7. BELLIN, S. A., AND STEENBOCK, H., J. Biol. Chem. 194, 311 (1952). 8. GONCE, J. E., AND TENPLETON, H. L., Am. J. Diseases ChiZdren 39,265 (1930). 9. KUYPER, A. C., AND MATTILL, H. A., J. Biol. Chem. 103, 51 (1933).
24
BELLIN,
10. KUETHER,
HERTING,
C. A., MEYER,
CRAMER,
C. E.,
PILEGGI
AND
SMITH,
AND
A.
H.,
STEENBOCK
Proc. Sot. Exptl.
Biol.
Med. 44, 224 (1940). 11. SMITH, A. H., FREEMAN, S., AND CHANG, T. S., Am. J. Physiol. 160,341 (1950). 12. STEENBOCK, H., BELLIN, S. A., AND WIEST, W. G., J. Biol. Chem. 193, 843 (1951). 13. JONES, J. H., J. Nutrition 28, 7 (1944). 14. PUCHER, G. W., SHERMAN, C. C., AND VICKERY, H. B., J. Biol. Chem. 113, 235 (1936). 15. FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem. 66,375 (1925). 16. FOLIS, R. H., Transactions First Conference Metabolic Interrelations I, 30 (1949) Josiah Macy, Jr. Foundation, New York. 17. CLASS, R. N., AND SMITH, A. H., J. Biol. Chem. 161,363 (1943). 18. STEENBOCK, H., AND BLACK, A., J. Biol. Chem. 84, 263 (1925).
Vincent J. Pileggi
From the Department of Biochemistry, College of Agriculture, of Wisconsin, Madison, Wisconsin Received
May
University
29, 1953
With the observation that the increase in urinary citrate induced by the administration of vitamin D can be correlated with an increase in blood citrate (l), it became increasingly important to determine what dietary relations affect this increase. Admittedly, this is hard to establish because, as judged by the increments in citrate excretion following the ingestion of base (2-7), and the great capacity of the animal to catabolize orally ingested citrate (8-12), the range within which an equilibrium can be established must be very wide. So far, we know that in rachitic rats, while the citrate response to vitamin D is accompanied by an increase in urinary pH, it apparently is not dependent on this increase because it is elicited even when the urines are already strongly alkaline as the result of the inclusion of generous amounts of NaHC03 in the ration. It remains to be ascertained if vitamin D will have the same effect in the absence of a demonstrable increase in urinary pH. In addition, we have sought to determine the effect of Ca, P, and other minerals. Our interest in Ca stemmed from the fact that citratemia has been associated with hypercalcemia (11) and that Ca is well known as forming soluble non-ionized complexes with citrate. These complexes might be excreted readily during a hypercalcemia induced by vitamin D (13). Phosphorus has already been shown (7) to affect the intensity of citraturia in that citrate excretion was observed to be at 1 Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. We are indebted to the Wisconsin Alumni Research Foundation for the funds which made this research possible.
18
VITAMIN
D EFFECT ON URINARY
CITRATE
19
the highest level on low-P rations but it was increased the most, by vitamin D, with P-containing rations. In some of our presently reported experiments the Ca and P contents of the rations were reduced to very low levels (approximately 0.02 %). In others, all mineral additions to the organic basal ration, except NaCl, were dispensed with. NaHC03 was added in one series to increase the urinary pH. The above experiments were undertaken in full realization of the limitations in the value of urinary excretion as an index of citrate metabolism. Not only could increased synthesis in one tissue or organ be counterbalanced by increased catabolism in another, but changes in blood citrate might not be reflected by comparable changes in excretion (6). Nevertheless, we felt that a demonstration of the generality of a vitamin D effect might emphasize the desirability of more definitive research with tissues and organs. EXPERIMENTAL
Methods Young rats varying from 60 to 166 g. in weight in different series, but within a range of 10 g. for individual experiments, were fed a basal synthetic ration of glucose, cooked egg white, roughage, cottonseed oil, salts, and vitamins as previously described (1, 7). The salts in the basal ration were limited to a Ca-free, P-free mixture; the vitamins to a vitamin D-free mixture. The various omissions from, and supplements to, the basal constituents are presented, with the results, in the various tables. When required by the experimental plan, Ca was added as CaCOl , unless otherwise indicated; P was added as a neutral equimolar mixture of KHzPO, and KIHPOd , and vitamin D as a cottonseed oil solution of calciferol. The latter supplement, equivalent to 75 I.U., was given orally to each rat every 3 days. Records were kept of food consumed and, in some cases, as noted, consumption was equalized between D and non-D groups. The urines, preserved with toluene, were collected in 3-day periods. They were analyzed for citrate essentially by the use of the Pucher, Sherman, and Vickery technique (14), and for Ca and P, respectively, by KMnO, titration of the oxalate, and by the Fiske-SubbaRow calorimetric method (15) after perchloric-nitric acid digestion. The pH of the urine was determined at the end of each 3-day period with a glass electrode on fresh samples of urine collected in a few hours in clean, dry metabolism cages.
Results A gross inspection of the results in Table I reveals significantly that modifications of the low-P, low-Ca basal ration by addition of either P or Ca, singly or in multiple, did not have as much effect on citrate excretion as an increase in base intake in the form of NaHC03 alone, or re-
20
BELLIN,
The Eflect
of Vitamin
HERTING,
CRAMER,
PILEGGI
TABLE
=
Series
D on Urinary
Citrate
Keight ktial
l-l% III IV
42,Sl
4&i%
V II(a) II(b) I
Low ca Low P Low Ca Low P NaHCOa Low Ca Low P NaCl No other Normal Normal NaCl No other Low Ca High P Medium Normal Medium Normal High Ca Low P NaHC03
minerals Ca P
STEENBOCK
I in Relation
Vita& D
Ration
AND
to Calcium of rats
Urinary
Final
Ca ____
and PhosDhorusa ex~;tbp
P I
(daily/100
Citric
acid
!
g.
g.
g.
0.02 0.02 0.02 0.02 10.00 0.01 0.03 0.40
0 + 0 +
96 95 89 96
89 86 87 87
107 107 102 110
3.57 6.45 3.44 6.28
0.12 4.25 0.12 8.63 0.1821.5 0.2433.4
0 +
103 116
103 106
124 141
2.98 8.10
0.11 0.13
0.40 0.32 0.40
0 +
90 125
102 110
119 148
0.48 0.68
0.02 0.62 0.20 0.30 0.21 0.33 1.26 0.02 6.60
0 + 0 + 0 + 0 +
42 102 69 98 60 106 61 64
75 100 59 59 90 92 62 60
68 127 70 98 98 135 68 73
0.5424.7 1.70 0.4037.7 7.80 0.5710.2 2.30 0.61 8.5612.50 0.69 0.91 0.58 2.54 1.6 0.2021.3 8.2 0.1736.6
103 56
2.36 4.38 85
6.86 6.19
2.22 8.90
300
minerals
Ca P Cab P
359 445 179 72
a All groups contained six rats except series II(b) and 42,& in which there were three rats. All rats were males except those in series 42 which were females. All data are for the last four collections for 3 days each and therefore represent averages for the collections from the 9th to the 21st day except for series II(a); in this way they represent the averages for the 6th to the 18th day. The food consumed and body weights, initial and final, are likewise for these la-day periods. To each rat was given 75 I.U. of vitamin D every 3 days when indicated. A basal mineral mixture “free” from Ca and P was included in all rations except those fed in series 42,Sd and 42,Sh. b Ca was given as the sulfate; in all other series it was given as the carbonate.
placed, in part, by an equivalent amount of CaC03. Furthermore, even when citrate excretion was raised to a high level, supplementation with vitamin D elicited a further increase which, expressed in milligrams, was as large as that obtained with any of the other rations. Obviously, even
VITAMIN
D
EFFECT
ON
URINARY
21
CITRATE
the lowest levels of Ca and P did not prevent the excretion of citrate or the vitamin D effect. Citrate was always excreted in the urine and vitamin D always increased its excretion. This response, though limited to the area covered by our experiments, is expected to be very general, except possibly under more acidic conditions than those studied by us. The role of an increase in urinary pH or systemic pH is not so easily assessed. We have reported (12) that vitamin D will increase urinary pH when given to rats on rachitogenic rations, that is, on rations low 0; physiologically low, in assimilable P, but relatively high in Ca. On other rations, e.g., those nonrachitogenic in character, this effect was not obtained (Table II), yet citrate excretion was always increased even in those instances in which a reduction in urinary pH occurred. With urines from the bicarbonate series we were unable to obtain measurement of pH to a sufficient degree of accuracy; they were too heavily charged with CO* . If it is accepted that an increase in urinary citrate is not necessarily accompanied by an increase in urinary pH, it does not necessarily follow that the vitamin D effect and pH are not interrelated; an increase in hydroxyl ions may nevertheless be induced at the loci of citrate formation. Folis (16) has reported his inability to demonstrate any deviation in pH from the normal physiological range in bone and cartilage by the TABLE Vitamin
D in
Relation
Ration Expt.
+o. No. (
II
= I
to Urinary
Characteristic
Citrate
= I
and
Citrate
mg./lOO
g. rat/day
Before
D”
After
D*
pH PR
Before
D”
After
Db
-9
4
11A
26
12
11c
SP
6
1lD
0.20% calcium &s carbonate 0.30% phosphorus Wesson salti4 4.0% No calcium 0.30% phosphorus
5.30 (4.75-5.81) 3.57 (0.99-4.59) 1.31 (0.83-2.30) Without
42 42
6 3
S4 SS
No salts except 0.40% NsCl No salts except 0.40% NaCI. 1.0% CSCOI , and 0.32% P
16.92 (9.39-20.70) 10.60 (3.66-24.60) 6.16 (3.64-9.21) DC
With
3.51 6.70
-
7.27 (6.53-7.75) 6.29 (5.49-7.61) 7.32 (6.2C8.04)
DC
Without
6.37 11.56
-
7.29 7.60
5.61 (5.14-6.04) 6.08 (5.39-7.70) 6.18 (6.32-7.23) DC
With
-I
DC
7.29 7.60
-
a Collection after 6th~9th day on ration. * Collection after 9th-12th day on ration; 525 I.U. vitamin D was given over the a-day period. c Average values obtained in 21 days from rata started on rations with and without vitamin D when received from the breeders. The vitamin D supplemented group received 75 I.U. each day, every 3 days. d Wesson, L. G., Science Xi, 339 (1932).
22
BELLIN,
HERTING,
CRAMER,
PILEGGI
AND
STEENBOCK
application of a series of indicators. It remains to be seen if such a test is adequate. The skeleton has been considered of minor importance in citrate production (17). Our data do not provide the answer as to whether the increase in citrate elimination was due to an increase in citrate synthesis or to a decrease in its destruction. Tentatively we favor the former because the very high level of citrate excretion induced by 5 y0 NaHCOs , as previously reported (l), was increased only 8 y0 by raising the dietary bicarbonate from the 5 y0 to the 10 y0 level, while vitamin D increased it 34%. In accordance with the neutralization theory of &tberg (3), the excessive intake of bicarbonate should have elicited the excretion of much more citrate if it had been possible for the rats to synthesize more in the absence of vitamin D. While P is a potent factor in determining citrate equilibrium, our efforts to duplicate the effect of vitamin D by the addition of P were unsuccessful. In fact, in harmony with the low level of citrate excretion obtained with rations which contained P but no vitamin D, supplementing a rachitogenic ration with neutral phosphates reduced the citrate output. Later, when vitamin D was given, a rise in urinary citrate followed promptly. The urinary pH was not increased (Table III). Further evidence of a possible P effect was obtained with rats on TABLE III Effect of Phosphate on Urinary Citrate The basal ration was 23A, a low-P (0.02%), medium-Ca (0.21% Ca as CaCOr) ration. The P supplement was given gradually in a few days to avoid tetany. In series S-l, 136 mg. P was given per rat as a solution of KzHPOI and KHzPOa . In series W-l, P was incorporated in the ration, equivalent to 0.30yo P. The vitamin D dose was 525 I.U. given every 3 days. Before Collection
P
With
P
After
D
days 4-6
i Mg. citrate,
1
7-9
x-12
1
13-15
/ 8.0 )
7.2
i 100 g. rat, daily
11618
19-21
1 22-24
Series W-l (8 rats) Series S-l (12 rats) Urinary i 7.6 /
7.2
pH ) 7.0 1
]
VITAMIN
D EFFECT ON URINARY
CITRATE
23
Ration 2965 (18). As is well known, vitamin D increases the availability of P from this cereal ration. With four rats made rachitic to a degree comparable to that produced on our synthetic rations and then given vitamin D, citrate excretion was reduced to a level 42 y0 below that found in a comparable 6-day period immediately preceding the administration of the vitamin. It was not until the lapse of an additional 3 days that the excretion of urinary citrate rose to a level of 35 % above the pre-vitamin D period. This effect is quite at variance with the increases of 35-37 y0 obtained with our low P (0.02 ~~0)synthetic rations in the first 3 days. We were unable to find any correlation between the excretions of citrate and Ca or P. SUMMARY
Vitamin D increased the amount of urinary citrate excreted by young rats kept on rations varying widely in mineral content. These rations ranged in composition from those providing an adequate mineral intake to those made grossly deficient in Ca, P, and other minerals. The biggest percentage increase in citrate was obtained with P-containing rations, but supplements of P, given in the form of neutral solutions of K phosphates to a low-P ration, reduced citrate elimination. An increase in urinary pH induced by vitamin D with rats on low-P rachitogenie rations was not obtained with rations adequate in P, yet an increase in citrate elimination resulted in all instances. Inasmuch as a supplement of vitamin D increased urinary citrate far more than did an additional large intake of NaHC03, it appears that the effect of vitamin D is due to an increase in citrate synthesis rather than to a decrease in its destruction. REFERENCES 1. STEENBOCK, H., AND BELLIN, S. A., J. Biol. Chem. 206,985 (1953). 2. FASOLD, H., 2. Kinderheilk. 49, 709 (1930). 3. E)STBERG, O., Stand. Arch. Physiol. 62, 81 (1931). 4. SULLMAN, H., AND SHEARER, E., Schweiz. med. Wochschr. 13, 619 (1932). 5. SCHUCK, C., J. Nutrition 7, 679 (1934). 6. SHERMAN, C. C., MENDEL, L. B., AND SMITH, A. H., 3. Biol. Chem. 113, 247, 265 (1936). 7. BELLIN, S. A., AND STEENBOCK, H., J. Biol. Chem. 194, 311 (1952). 8. GONCE, J. E., AND TENPLETON, H. L., Am. J. Diseases ChiZdren 39,265 (1930). 9. KUYPER, A. C., AND MATTILL, H. A., J. Biol. Chem. 103, 51 (1933).
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