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The urinary excretion of calcium, hydroxyproline and uronic acids in the laying fowl after the administration of parathyoid extract
Comp. Biochem. Physiol., 1970, Vol. 32, pp. 703 to 707. Pergamon Press. Printed in Great Britain
T H E URINARY EXCRETION OF CALCIUM, HYDROXYPROLINE AND URONIC ACIDS IN THE LAYING FOWL AFTER THE A D M I N I S T R A T I O N OF PARATHYROID EXTRACT J O H N K. C A N D L I S H A.R.C. Poultry Research Centre, King's Buildings, West Mains Road, Edinburgh 9 (Received 24 J u l y 1969)
Intravenous and subcutaneous injections of parathyroid extract were given to laying hens with the object of detecting any possible phasing of the urinary excretion of the major bone metabolites calcium, hydroxyproline and uronic acids. 2. The excretion of hydroxyproline tended to be more prolonged than that of calcium and uronic acids. 3. The results are consistent with the theory that parathyroid hormone actively stimulates lysis of all the major structural constituents of bone.
Abstract--1.
INTRODUCTION THE BLOOD level of calcium in the laying fowl (Gallus domesticus) is elevated by 5 mg/100 ml 1.5 to 2 hr after injection of 200 U.S.P. units parathyroid extract ( P T E ) (Hertelendy, 1962), p r e s u m a b l y due to the breakdown of bone mineral. T h e r e is a conflict of views as to the importance of parathyroid h o r m o n e in the control of skeletal resorption during the egg cycle. Some consider that it plays a major role (Taylor, 1969), while others deny its importance (Urist, 1967). I n the present work P T E was employed as a stimulus for bone resorptiort in laying fowls to allow examination of the phasing of the excretion of certain bone materials, namely, calcium (derived f r o m the hydroxyapatite crystals), hydroxyproline (from the fibrillar collagenous matrix) and uronic acids (from the chondroitin sulphates in the a m o r p h o u s fraction of the matrix). T h e aim was to follow the dynamics of elimination of these breakdown products of the major structural components of bone, and, in particular, to assess whether in the fowl the excretion of matrixderived hydroxyproline and uronic acids is obligatory upon mineral release. MATERIALS AND M E T H O D S A population of medium hybrid hens, derived from Thomber 404 stock aged 12-18 months, was maintained on a normal laying diet. Individuals were selected immediately after laying and a catheter (for the provision of which I am indebted to Dr. L. Martindale of this Centre) was inserted in the cloaca. This allows faeces to be passi~d to the exterior while urine is collected in the bell-shaped cup of the catheter from wher~ ~*/isdrained away into a collecting tube. The birds were then restrained and allowed to t ~ t for 3 hr, after which subcutaneous (Expt. 1) or intravenous (Expt. 2) injections of 100 or 250 U.S.P. units P T E 703
704
JOHN K. CANDLISH
(Parathormone, Lilly) were administered. I n each series, control birds were treated in the same way except that they were not injected. In the case of the subcutaneous (s.c.) injections, the urine collections were made at hourly intervals from the time the catheter was first inserted. With intravenous (i.v.) injections the collection period was 12 rain since it is known that the response to i.v. is more rapid than to s.c. injection (Hertelendy, 1962), and no collections were made during the initial 3-hr resting period while the birds became accustomed to the presence of the catheter. The results were expressed as total weights of materials excreted per collection period. It was not possible to determine concentrations because of the need to wash out the catheter at frequent intervals to prevent blockage by urates and mucoid. The urine was centrifuged and the supernatant was taken for analysis. Calcium was analysed on the Technicon AutoAnalyser (Kessler & Wolfman, 1964). After hydrolysis with 6 N HC1 overnight at 105°C, aliquots were estimated for hydroxyproline using an AutoAnalyser (Grant, 1964); uronic acids were determined by an orcinol method (Davidson, 1966).
20-
Calcium
~-
T I/
]-
-'- \
;/t
/t\
l\I
0
o.
/U: Uronicacids
15
1
J 0.5 ~ L
2
3
4
5
6
7
8
FIG. 1. Mean urirmry excretion of calcium, hydroxyproline and uronic acids in eight hens after '~ ) U.S.P. units P T E (s.c. injection) ( , ) and in four controls ( 0 ) (mg/l'a'). V e ~ c a l bars show S.E.M. P T E was administered at end of the third period. Horizontal axis--periods of 1 hr; vertical--mg.
PARATHYROID
ADMINISTRATION
IN FOWLS
705
To eliminate variations due to differences in endogenous bone resorption, urines collected from birds which did not lay the day after the experiment were not assayed. R E S U L T S AND D I S C U S S I O N W h e n collections were taken at h o u r l y intervals after insertion of the catheter it was immediately evident that there was great variation in response f r o m bird to bird b o t h before and after P T E injection. Results are given in Fig. 1. Excretion of uronic acids was similar in control and injected birds, but a definite response to the h o r m o n e occurred in the case of calcium and hydroxyproline. T h e effect on calcium
0oz-ooc0
/vii
l/
0
Hydroxyproline 1.4-i
,]
.L
0
0.6--
Uronic acids
J
0.5--
o, t ~=~ ~ ~ ~ ~ .~t~-~ ~-~ ~-~--t I
Z
5
4
5
6
7
S
9
10
I1
12
fB
14
15
16
Fxo. 2. Mean urinary excretion of calcium, hydroxyproline and uronic acids in four hens after 250 U.S.P. units PTE (i.v. injection) (ll) and in four controls (0) (mg/12 rain). Vertical bars show S.E.M. PTE was administered at end of the fourth period. Horizontal axis--periods of 12 min; vertical axis--mg.
JOHN K. CANDLISH
706
was marked but transient, lasting no more than 2 hr. T h e hydroxyproline elimination was more prolonged and even after 5 hr significantly more hydroxyproline was excreted in the treated than in the control birds. Figure 2 shows the results of Expt. 2, using 250 U.S.P. units P T E injected i.v. T h e r e were significant initial rises in the excretion of all three components, but with 100 units there were no marked responses and these data are not shown. One of the most striking effects of the i.v. injections of P T E was a marked diuresis, copious quantities of dilute urine appearing almost instantaneously. This effect has also been observed in mammals (Charbon et al., 1968), in which it is accompanied by a diuresis of sodium, potassium, calcium and phosphate. T h e main problem in the interpretation of the present results is to distinguish between the responses due to a direct effect of the P T E on the kidney and those effecting the bone and expressed subsequently as urinary responses. T h e effect of P T E on water diuresis was almost immediate, and it seems probable therefore that its direct effect on the urinary excretion of calcium, hydroxyproline and uronic acids was also rapid and responsible for the rises in excretion taking place in the first halfhour. When the data from Expt. 2 were expressed as calcium ratios (Fig. 3), it was clear that the maximum values for the calcium : hydroxyproline and calcium: uronic acid ratios were substantially greater than the basal values or the values 1 hr after the injections; it appears likely, therefore, that calcium was being actively
6--
5 Q.
44 o
3
g 2 12E
~..1
f
I
I
1
t
I
r
!
]
5T M 6
7
8
9
I0
II
12
13
14
15
Sampling
t6
periods of 12min
FIC. 3. Ratios of calcium : hydroxyproline (©) and of calcium : uronic acids (0) in the urine of hens after i.v. injection of PTE. Further explanation in text. Vertical axis--ratios of excretory products; horizontal axis--sampling periods of 12 rain.
PARATHYROID ADMINISTRATION IN FOWLS
707
released from the bone at this time and that the very large increase in its secretion during periods 6 to 8 was a combined effect on bone and kidney. T h e current controversy over the mechanism of action of parathyroid hormone (Klein & Talmage, 1968) centres on whether it affects one only of the major structural components of bone (hydroxyapatite, collagen or mucopolysaccharide) in a fashion which destabilizes the others, rendering their resorption a secondary effect, or whether it so stimulates osteolytic activity that removal of all components occurs concomitantly. T h e results obtained here are consistent with the view that on P T E administration the mineral fraction of bone is resorbed first, followed by a slower elimination of matrix. This is most readily observed in Fig. 3. T h e calcium : hydroxyproline and calcium : uronic acids ratios rise up to 1 hr, when the urine is being progressively enriched with calcium relative to the organic endproducts of bone metabolism, but after that there is a reversal from the basal conditions (sampling time 4 on the graph) and the urine becomes enriched with the organic constituents relative to calcium. This latter trend is reversed after about 2 hr (periods 15 and 16), with signs of a return to the basal state. It is easy to conceive a mechanism of bone resorption by which destabilization of the organic matrix results ill hydroxyapatite release. It is mechanistically difficult, however, to imagine the initial removal of mineral observed in this work causing obligatory destabilization of collagen. For this reason, the results support the view that parathyroid hormone actively causes resorption of both the mineral and organic phases of bone, but not necessarily simultaneously. Furthermore, the response to the hormone seems to be sufficiently rapid for it to have a possible role ill the provision of calcium for egg shell formation.
REFERENCES CHARBON G. A., BRUMMERF. & RENEMAN R. S. (1968) Diuretic and vascular action of parathyroid extracts in animals and man. Archsint. Pharmacol. 171, 1-11. DAVIDSON E. A. (1966) Analysis of sugars found in mucopolysaccharides. In Methods in Enzymology, Vol. VIII: Complex Carbohydrates (Edited by NEUFIELDE. F. • GINSBORG V.). Academic Press, New York. GRANT R. A. (1964) Estimation of hydroxyproline by the Auto Analyser..7. clin. Path. 17, 685-686. HERTELENDYF. (1962) Biochemical studies on the tissues of the domestic fowl in relation to reproduction. Ph.D. thesis, University of Reading. KESSLERG. & WOLFMANM. (1964) An automated method for the simultaneous determination of calcium and phosphorus. Clin. Chem. 10, 686-703. KLEIN n . C. ~ TALMAGE R. V. (1968) Thyrocalcitonin suppression of hydroxyproline release from bone. Proc. Soc. exp. Biol. Med. 127, 95-99. TAYLOR T. G. (1969) The role of the skeleton in eggshell formation. Annls Biol. anita. Biochim. Biophys. In press. URIST M. R. (1967) Avian parathyroid physiology: including a special comment on calcitonin. Am. Zoologist 7, 883-895. Key Word Index--Fowl; parathyroid extract; urinary calcium; urinary hydroxyproline;
urinary uronic acids.
T H E URINARY EXCRETION OF CALCIUM, HYDROXYPROLINE AND URONIC ACIDS IN THE LAYING FOWL AFTER THE A D M I N I S T R A T I O N OF PARATHYROID EXTRACT J O H N K. C A N D L I S H A.R.C. Poultry Research Centre, King's Buildings, West Mains Road, Edinburgh 9 (Received 24 J u l y 1969)
Intravenous and subcutaneous injections of parathyroid extract were given to laying hens with the object of detecting any possible phasing of the urinary excretion of the major bone metabolites calcium, hydroxyproline and uronic acids. 2. The excretion of hydroxyproline tended to be more prolonged than that of calcium and uronic acids. 3. The results are consistent with the theory that parathyroid hormone actively stimulates lysis of all the major structural constituents of bone.
Abstract--1.
INTRODUCTION THE BLOOD level of calcium in the laying fowl (Gallus domesticus) is elevated by 5 mg/100 ml 1.5 to 2 hr after injection of 200 U.S.P. units parathyroid extract ( P T E ) (Hertelendy, 1962), p r e s u m a b l y due to the breakdown of bone mineral. T h e r e is a conflict of views as to the importance of parathyroid h o r m o n e in the control of skeletal resorption during the egg cycle. Some consider that it plays a major role (Taylor, 1969), while others deny its importance (Urist, 1967). I n the present work P T E was employed as a stimulus for bone resorptiort in laying fowls to allow examination of the phasing of the excretion of certain bone materials, namely, calcium (derived f r o m the hydroxyapatite crystals), hydroxyproline (from the fibrillar collagenous matrix) and uronic acids (from the chondroitin sulphates in the a m o r p h o u s fraction of the matrix). T h e aim was to follow the dynamics of elimination of these breakdown products of the major structural components of bone, and, in particular, to assess whether in the fowl the excretion of matrixderived hydroxyproline and uronic acids is obligatory upon mineral release. MATERIALS AND M E T H O D S A population of medium hybrid hens, derived from Thomber 404 stock aged 12-18 months, was maintained on a normal laying diet. Individuals were selected immediately after laying and a catheter (for the provision of which I am indebted to Dr. L. Martindale of this Centre) was inserted in the cloaca. This allows faeces to be passi~d to the exterior while urine is collected in the bell-shaped cup of the catheter from wher~ ~*/isdrained away into a collecting tube. The birds were then restrained and allowed to t ~ t for 3 hr, after which subcutaneous (Expt. 1) or intravenous (Expt. 2) injections of 100 or 250 U.S.P. units P T E 703
704
JOHN K. CANDLISH
(Parathormone, Lilly) were administered. I n each series, control birds were treated in the same way except that they were not injected. In the case of the subcutaneous (s.c.) injections, the urine collections were made at hourly intervals from the time the catheter was first inserted. With intravenous (i.v.) injections the collection period was 12 rain since it is known that the response to i.v. is more rapid than to s.c. injection (Hertelendy, 1962), and no collections were made during the initial 3-hr resting period while the birds became accustomed to the presence of the catheter. The results were expressed as total weights of materials excreted per collection period. It was not possible to determine concentrations because of the need to wash out the catheter at frequent intervals to prevent blockage by urates and mucoid. The urine was centrifuged and the supernatant was taken for analysis. Calcium was analysed on the Technicon AutoAnalyser (Kessler & Wolfman, 1964). After hydrolysis with 6 N HC1 overnight at 105°C, aliquots were estimated for hydroxyproline using an AutoAnalyser (Grant, 1964); uronic acids were determined by an orcinol method (Davidson, 1966).
20-
Calcium
~-
T I/
]-
-'- \
;/t
/t\
l\I
0
o.
/U: Uronicacids
15
1
J 0.5 ~ L
2
3
4
5
6
7
8
FIG. 1. Mean urirmry excretion of calcium, hydroxyproline and uronic acids in eight hens after '~ ) U.S.P. units P T E (s.c. injection) ( , ) and in four controls ( 0 ) (mg/l'a'). V e ~ c a l bars show S.E.M. P T E was administered at end of the third period. Horizontal axis--periods of 1 hr; vertical--mg.
PARATHYROID
ADMINISTRATION
IN FOWLS
705
To eliminate variations due to differences in endogenous bone resorption, urines collected from birds which did not lay the day after the experiment were not assayed. R E S U L T S AND D I S C U S S I O N W h e n collections were taken at h o u r l y intervals after insertion of the catheter it was immediately evident that there was great variation in response f r o m bird to bird b o t h before and after P T E injection. Results are given in Fig. 1. Excretion of uronic acids was similar in control and injected birds, but a definite response to the h o r m o n e occurred in the case of calcium and hydroxyproline. T h e effect on calcium
0oz-ooc0
/vii
l/
0
Hydroxyproline 1.4-i
,]
.L
0
0.6--
Uronic acids
J
0.5--
o, t ~=~ ~ ~ ~ ~ .~t~-~ ~-~ ~-~--t I
Z
5
4
5
6
7
S
9
10
I1
12
fB
14
15
16
Fxo. 2. Mean urinary excretion of calcium, hydroxyproline and uronic acids in four hens after 250 U.S.P. units PTE (i.v. injection) (ll) and in four controls (0) (mg/12 rain). Vertical bars show S.E.M. PTE was administered at end of the fourth period. Horizontal axis--periods of 12 min; vertical axis--mg.
JOHN K. CANDLISH
706
was marked but transient, lasting no more than 2 hr. T h e hydroxyproline elimination was more prolonged and even after 5 hr significantly more hydroxyproline was excreted in the treated than in the control birds. Figure 2 shows the results of Expt. 2, using 250 U.S.P. units P T E injected i.v. T h e r e were significant initial rises in the excretion of all three components, but with 100 units there were no marked responses and these data are not shown. One of the most striking effects of the i.v. injections of P T E was a marked diuresis, copious quantities of dilute urine appearing almost instantaneously. This effect has also been observed in mammals (Charbon et al., 1968), in which it is accompanied by a diuresis of sodium, potassium, calcium and phosphate. T h e main problem in the interpretation of the present results is to distinguish between the responses due to a direct effect of the P T E on the kidney and those effecting the bone and expressed subsequently as urinary responses. T h e effect of P T E on water diuresis was almost immediate, and it seems probable therefore that its direct effect on the urinary excretion of calcium, hydroxyproline and uronic acids was also rapid and responsible for the rises in excretion taking place in the first halfhour. When the data from Expt. 2 were expressed as calcium ratios (Fig. 3), it was clear that the maximum values for the calcium : hydroxyproline and calcium: uronic acid ratios were substantially greater than the basal values or the values 1 hr after the injections; it appears likely, therefore, that calcium was being actively
6--
5 Q.
44 o
3
g 2 12E
~..1
f
I
I
1
t
I
r
!
]
5T M 6
7
8
9
I0
II
12
13
14
15
Sampling
t6
periods of 12min
FIC. 3. Ratios of calcium : hydroxyproline (©) and of calcium : uronic acids (0) in the urine of hens after i.v. injection of PTE. Further explanation in text. Vertical axis--ratios of excretory products; horizontal axis--sampling periods of 12 rain.
PARATHYROID ADMINISTRATION IN FOWLS
707
released from the bone at this time and that the very large increase in its secretion during periods 6 to 8 was a combined effect on bone and kidney. T h e current controversy over the mechanism of action of parathyroid hormone (Klein & Talmage, 1968) centres on whether it affects one only of the major structural components of bone (hydroxyapatite, collagen or mucopolysaccharide) in a fashion which destabilizes the others, rendering their resorption a secondary effect, or whether it so stimulates osteolytic activity that removal of all components occurs concomitantly. T h e results obtained here are consistent with the view that on P T E administration the mineral fraction of bone is resorbed first, followed by a slower elimination of matrix. This is most readily observed in Fig. 3. T h e calcium : hydroxyproline and calcium : uronic acids ratios rise up to 1 hr, when the urine is being progressively enriched with calcium relative to the organic endproducts of bone metabolism, but after that there is a reversal from the basal conditions (sampling time 4 on the graph) and the urine becomes enriched with the organic constituents relative to calcium. This latter trend is reversed after about 2 hr (periods 15 and 16), with signs of a return to the basal state. It is easy to conceive a mechanism of bone resorption by which destabilization of the organic matrix results ill hydroxyapatite release. It is mechanistically difficult, however, to imagine the initial removal of mineral observed in this work causing obligatory destabilization of collagen. For this reason, the results support the view that parathyroid hormone actively causes resorption of both the mineral and organic phases of bone, but not necessarily simultaneously. Furthermore, the response to the hormone seems to be sufficiently rapid for it to have a possible role ill the provision of calcium for egg shell formation.
REFERENCES CHARBON G. A., BRUMMERF. & RENEMAN R. S. (1968) Diuretic and vascular action of parathyroid extracts in animals and man. Archsint. Pharmacol. 171, 1-11. DAVIDSON E. A. (1966) Analysis of sugars found in mucopolysaccharides. In Methods in Enzymology, Vol. VIII: Complex Carbohydrates (Edited by NEUFIELDE. F. • GINSBORG V.). Academic Press, New York. GRANT R. A. (1964) Estimation of hydroxyproline by the Auto Analyser..7. clin. Path. 17, 685-686. HERTELENDYF. (1962) Biochemical studies on the tissues of the domestic fowl in relation to reproduction. Ph.D. thesis, University of Reading. KESSLERG. & WOLFMANM. (1964) An automated method for the simultaneous determination of calcium and phosphorus. Clin. Chem. 10, 686-703. KLEIN n . C. ~ TALMAGE R. V. (1968) Thyrocalcitonin suppression of hydroxyproline release from bone. Proc. Soc. exp. Biol. Med. 127, 95-99. TAYLOR T. G. (1969) The role of the skeleton in eggshell formation. Annls Biol. anita. Biochim. Biophys. In press. URIST M. R. (1967) Avian parathyroid physiology: including a special comment on calcitonin. Am. Zoologist 7, 883-895. Key Word Index--Fowl; parathyroid extract; urinary calcium; urinary hydroxyproline;
urinary uronic acids.