Effect of Parathyroid Hormone on Calcium and Other Plasma Constituents of Dairy Cattle near Parturition1

EFFECT

OF PARATHYROID PLASMA

HORMONE

CONSTITUENTS

ON C A L C I U M A N D

OF DAIRY

OTHER

CATTL~

NEAR PARTURITION 1 It. D. JACKSON, A. R. PAPPENHAGEN, 2 G. D. GOETSCH, AND C. H. NOLLER Departments of Biochemistry, Veterinary Physiology and Pharmacology, and Animal Sciences Purdue University, Lafayette, Indiana SU~:SiARY

The administration of parathyroid extract within 2 hr postpartum to first calf heifers, normal mature cows, and mature cows with a past history of nfilk fever did not appreciably alter the plasma levels of calcium, magnesium, or alkaline phosphatase. Plasma inorganic phosphorus was increased moderately in most of the animals by the action of parathyroid extract. At the time of parturition mature animals showed a greater decrease in calcium and inorganic phosphorus and a greater increase in magnesium than did the younger animals. Alkaline phosphatase remained relatively constant in all animals. Thirty-one parturitions were studied and six cases of milk fever observed. Data suggest that milk fever can result from either a low level of calcium or a low level of both phosphorus and calcium. Cases of milk fever which relapsed were of the latter type. Parturient cows were not responsive to parathyroid extract and its administration just after calving had no apparent effect on the occurrence or severity of milk fever.

The parathyroid deficiency theory of par~urient paresis or milk fever has had wide acceptance since it was first proposed by Dryerre and Greig in 1925 (3). However, there is little experimental evidence supporting this theory, other than the observation of hypocalcenfia and the successful use of calcium to relieve the symptmns of this disorder. Although parathyroid extract ( P T E ) , when administered intramuscularly, has been shown to elevate the level of blood calcium in most species of animals (20), it has not been tested adequately in the prevention of bovine milk fever. Little and Mattick (9) administered to two cows 450 or 600 Hanson Units 3 of parathyroid activity, Received for publication April 11, 1962. 1Journal Paper No. 1900, Purdue University Agricultural Experiment Station. Taken in part from a thesis presented by A. R. Pappenhagen to the Graduate School in partial fulfillment of the Ph.D. degree. Present address: Presbyterian Hospital, Chicago, Illinois. ~One Hanson unit is 0.01 of the amount of parathyroid activity required to increase serum calcium by 1.0 mg % in parathyroldectomlzed dogs within 6 hr (20). One U.S.P. parathyroid unit is 0.01 of the amount of parathyroid activity required to increase serum calcium by 1.0 mg % in normal dogs within 16-18 hr (21).

intramuscularly, daily for four days prepartum to one day postpartum. Data showed no definite effect of the Paroidin injections over the controls for that year, but the treated animals showed lower calcium levels at the next parturition a year later, when no parathyroid extract was given and when one of the cows developed milk fever. These workers concluded that the parathyroid extract may have been beneficial to some extent, possibly by preventing milk fever. Seekles, Sjollema, and Van Der Kaay (17) were able to relieve the symptoms of milk fever in one cow by the intravenous adnfinistration of 100 USP units of parathyroid hormone, tIibbs, Pounden, and Krauss (8) have given a brief report that parathyroid extract (3,000-5,000 USP units/animal) was of no benefit to three cows with early milk fever symptoms but was of benefit to a similar cow when injected in conjunction with oral vitamin D. Stott and Smith (19) thyroparathyroideetomized seven cows, 3-6 yr of age, approximately one month prepartum without the subsequent occurrence of symptoms of milk milk fever. The parathyroid deficiency theory of milk fever has been reviewed by Hibbs (7) and the role of the parathyroids in regulating the level of blood calcium has been reviewed by Boda and Cole (2). Preliminary experiments have indicated that 897

898

H.D.

JACKSON ET AL

parathyroid extract administered intranluscularly to dailT cattle elevated plasma calcium to a maximum level in 8 to 12 hr and had a duration of action of more than 30 hr (12). Since milk fever is most likely to occur within 12 to 24 hr postpartum, the administration of parathyroid extract within 2 hr postpartmn could be expected to increase plasma calcium during this critical pmiod. This experiment was designed to study the effects of the postpartum administration of parathyroid hormone on the occurrence of milk fever, and to determine whether normal cows and cows with a past history of milk fever have a different response of plasma calcium, phosphorus, magnesium, and alkaline phosphatase to the administration of parathyroid hormone at parturition. E X P E R I M E N T A L PROCEDURES

Twenty-seven dairy animals of Holstein, Guernsey, or Jersey breeding were studied in 31 parturitions during this experiment (Table 1). First calf heifers (Group I) and the norTABLE 1 Distribution of animals used to test the effect of parathyroid extract at parturition b

a (with (no PTE) PTE ")

Group I II

II[

First calf heifers

4

Normal mature cows in their fourth or higher lactation with no milk fever history. 5

of the P T E used in the experiment was of the same batch. Since the experiment required more titan a year, the potency of the P T E was tested near the end of the experiment by its administration to two dry cows, to determine if it had lost its ability to elevate plasnm ealeimn. Blood samples were obtained in heparinized syringes from the jugular vein and were centrifuged to remove red cells. Plasma was analyzed for inorganic phosphorus by adding one volume of plasma dropwise to four volmnes of 10% trichloroacetic, filtering, and determining the phosphorus in an aliquot of the filtrate by the A.O.A.C. eolorimetric method (1). Phosphorus and alkaline phosphatase (18) were determined within 12 hr after collection of the blood. Magnesium was determined by the method of Grette (4). Calcimu levels were initially determined by Grette's method (4), which involves oxalate precipitation and titration of the calcimn with ethylenediaminetetraaeetic acid ( E D T A ) , but most of the calcium levels were determined by a new nmthod involving the dir e c t titration of the plasma calcium with EDTA, using Cal-Red as the indicator (13). Daily plasma levels of these constituents were determined in most eases from seven days prepartum to seven days postpartum; for 48 hr postpartum blood samples were drawn every 2 hr for the first 12 h~,, event 4 hr for the next 12 hr, and every 6 hr for the next 24 hr.

4 R E S U L T S AND DISCUSSIOIg

4

Mature animals with milk fever histories in at least the last two parturitions. S

6 ......... " 400 U.S.P. units/100 lb (3,000-6,000 units/ animal). real nmture cows (Group I I ) were from the Purdue University Dairy Herd. Animals with a history of milk fever in their last two lactations (Group I I I ) were obtained from Indiana farmers and maintained at the School of Veterinary Science and ?,fcdicine. All of the animals were fed and managed by the practices used in the University Dairy Herd. Animals which received parathyroid extract were given 400 U S P units/1O0 lb (3,000-6,000 units/head) intramuscularly in the cervical region of the neck within 2 hr postpartum. All

The mature cows in this study showed a wide variation between animals in their plasma levels of calcium, inorganic phosphorus, and magnesimn. In contrast, the plasma levels of these constituents in the first calf heifers were much more uniform. Alkaline phosphatase values were quite consistent in all groups at approximately 2 to 3 m ~ units (18) and these wtlues did not change at the time of parturition, with tile age of the animal, or with the administration of parathyroid hormone. The first calf heifers all calved normally and none developed milk fever. The plasma levels of calcium and inorganic phosphorus were mildly depressed at parturition, but magnesium showed no change. Administration of parathyroid extract within 2 hr after parturition had no appreciable effect in these young aniurals (Figure 1, Groups I a attd b). Average calcium levels decreased 1.4 mg % just after parturition in control heifers and 1.3 mg % hi heifers which received PTE. The lowest level of plasma calcium was observed at the tinm of

EFFECT

OF P A R A T H Y R O I D

HORMONE

parturition, followed by a slow postpartal increase and a second decline in six of the eight heifers 16 to 24 hr postpartum. TILe normal mature cows did not have a history of previous milk fever and, like the first calf heifers, did not show symptoms of nfilk fever during their current lactation (Figure 1, Groups I I a and b). Furthermore, eight of the 14 cows with a past history of milk fever were unaffected during the current parturition (Figure 1, Groups I I I a and b). Four of these eight cows had received P T E at parturition and four had not. I f milk fever did not occur, the mature animals in the several categories (with and without previous milk fever and with and without P T E ) could not be distinguished from mature cows in the other categories by their post-parturient changes of plasma calcium or magnesium, even though there was variation between individual animals within a group. These cows appeared to have a modest hlcrease in plasma inorganic phosphorus due to PTE. Together, these mature animals without nfilk fever showed an average decline of 2.5 mg % of plasma calcium at parturition and this decline was not substantially affected by P T E administration (Figure 1; Groups I I a and b, and Groups I I I a and b). Other scientists (11, 15) have also observed a greater decrease in plasma calcium and in inorganic phosphorus at parturition in mature cows than in younger cows. Since the prepartal decline ill the level of plasma calcium has been associated with milk secretion (15), higher-producing mature cows could be expected to show a greater decline in plasma calcium than firstcalf heifers. Preliminary experiments with E D T A infusions (12), and the experiments of Hansard, Comar, and Plumlee (6) show that young cattle metabolize calcium more efficiently than do mature cattle. I n the younger animals of the current experiment the lowest level of plasma calcium was observed 1 to 2 hr after parturition and it rose rapidly. I n the mature cows not showing milk fever the lowest level of plasma calcium was not observed until 6 to 12 hr postpartum and it rose more slowly. By 48 hr postpartum, the plasma calcium in both age groups had returned to a normal level of about 10 mg %. I n the mature animals there was a gradual increase in plasma magnesium which reached its nmximum average level (3.0 mg %) about 4 hr postpartum, then slowly returned to a normal level of approximately 2.0 mg %. Of the 14 cows with a previous history of

ON P L A S M A C O N S T I T U E N T S

899

milk fever six developed milk fever during the experiment and two of these relapsed (Figure 2). The data suggest that two types of milk fever occur and these result from a low level of plasma calcium, or a low level of both phosphorus and calcium. Three cases of the low calcium type were observed and one of the animals received P T E at the time of parturition. When the symptoms of milk fever were observed in these cows plasma calcium levels were 4.7 to 6.4 mg %, inorganic phosphorus levels were 2.0 to 5.4 mg %, and magnesium levels were 3.2 to 4.0 mg % (Figure 2: Cows A-5, A-6, aud B-4). These cases of low calcium milk fever all showed good recovm}- following a single treatment with a commercial calciumphosphorus preparation. Milk fever of the low-calcium low-phosphorus type was observed in three cows, of which one had received P T E at parturition (Figure 2: Cows A-2, A-4, and B-3). When treatment with a commercial calcium-phosphorus preparation was initiated, plasma calcium levels were 3.0 to 4.8 mg %, phosphorus levels were 0.4 to 0.8 mg %, and plasma magnesium levels were 3.1 to 4.8 mg %. Magnesium frequently varied inversely with calcium. Two of these cases relapsed and required multiple infusions of calcium-phosphorus solution. At the time of the third treatment, P T E was injected intramuscularly (400 U.S.P. units/100 lb). After this dual treatment, both inorganic phosphorus and calcium were elevated and remained at an adequate level so that milk fever symptoms did not reoccur. I t is believed that P T E was beneficial in the treatment of these two eases of low-calcium, low-phosphorus nfilk fever. I n the third low-calcium, low-phosphorus ease the cow had been injected with P T E following parturition and calcium therapy was delayed to allow the horlnone to exert its effect. Within 2 hr after nlilk fever symptoms were first observed the animal rolled on to her side and bloated. Following calcium-phosphorus therapy milk fever symptoms were alleviated and plasma levels of calcium and inorganic phosphorus increased (Figure 2, B-3). However, the animal died 4 hr after calcium-phosphorus therapy, due to inhalation of rumen ingesta as revealed by post-mortcm examination. The observations on low-calcium, low-phosphorus milk fever are similar to those reported by ~¢[arr, ~[oodie, and Robertson (10) and Robertson et al. (14), who found that milk fever animals which relapse or show a poor response to calcium therapy often have a very low plasma level of both phosphorus and calcium. They

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FIG. 1. Average plasma ]eve]s of ealelum, inorg~nle phosphorus, and magnesium in cows which calved normally (the number of animals in each subgroup is show~ in parentheses).

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F[o. 2. P l a s m a levels of calcium, i n o r g a n i c p h o s p h o r u s , a n d m a g n e s i u m in cows w h i c h developed p a r t u r i e n t paresis. T h e a r r o w s (1") i n d i c a t e t h e t i m e of t r e a t m e n t with p a r a t h y r o i d e x t r a c t a n d / o r c a l c i u m - p h o s p h o r u s solutions.

902

H+ D. JACKSON ET AL

observed that the changes in magnesium in milk f e v e r cows are no g r e a t e r than those in normal parturitions and also indicated that udder inflation was beneficial in severe cases of milk fever, as it p r e v e n t e d milk secretion and the additional drain of calcium and phosphorus f r o m the body. H a l l g r e n (5) related that downers which did not respond to calcium t h e r a p y could be treated by the f u r t h e r injection of phosphorus alone. These reports help emphasize the role of blood phosphorus in p a r turient paresis. E x p e r i m e n t s of Robinson, tIuffman, and B u r t (16) with dairy calves have shown that P T E causes a marked increase in the level of plasma calcium and an Luerease in the excretion of u r i n a r y phosphorus. E x p e r i m e n t s in this laboratory using E D T A infusions to depress plasma calcium (12) have shown t h a t yearling heifers have a greater response to exogenous P T E than do mature cattle. F o r these reasons it is especially difficult to explain why the first calf heifers were not responsive to the administration of P T E at parturition. N e a r the end of the experiment a portion of the P T E was administered to two dry mature cows and their plasma calcium was increased 1.7-1.9 mg % a f t e r 16 h r and the maximum levels were maintained through 32 hr f r o m the tinie of injection ( F i g u r e 3). Thus, the P T E had remained

marked increase in plasma calcium in response to P T E , and since p a r t u r i e n t cows did not, the experiments show that exogenous parathyroid extract is ineffective in p a r t u r i e n t cows. Either an inactivation of the hormone (8) or a tempor a r y nonresponsiveness of the cow at p a r t u r i tion could explain the a p p a r e n t ineffectiveness of p a r a t h y r o i d hormone at calving time. ACKNOWLEDGMENT

The authors are indebted to Eli Lilly Company, Indianapolis, Indiana, for their generous contribution of parathyroid extract, and to Professor A. R. Allen, Medical Illustrations Unit, School of Veterinary Science and Medicine, for preparation of the figures. REFERENCES (1) ASSOCIATION OP

(2)

(3)

(4) -COW S-go COW W-13

IZ E

_8

(5)

I0

OFFICIAL

AGRICULTURAL

CHB~ISTS. Official Methods of Analysis. 8th ed., p. 115. Association of Official Agricultural Chemists, Washington, D. C. 1955. BONA, ft. M., AND CObE, H. H. Calcium Metabolism with Special Reference to Parturient Paresis (Milk Fever) in Dairy Cattle: A Review. J. Dairy Sci., 39: 1027. 1956. D~Y~, H., .(ND Gm~IG, J. R. Milk Fever: Its Possible Association with Derangements in the Internal Secretions. Vet. Record, 5: 225. 1925. Gm~r~, K. Micromethod for the Determination of Calcium and Magnesium in Serum. Scand. J. Clin. & Lab. Invest., 5: 151. 19'53. H A L L G ~ , W. Studies on Parturient Paresis in Dairy Cows. Nord. Veterinaermed., 7: 43'3. 1955.

(8) HANSARD, S. L., CO:~IAI~, C. L., AND PLUML]~,

i

2

Me

TIME PROM ADMINISTRATION OF PARATHYROID EXTRACT, HOURS

FIG. 3. Response of two dry mature cows to parathyroid extract. potent throughout the experiment. P l a s m a phosphorus was increased modestly in one cow and decreased modestly in the other due to the p a r a t h y r o i d injecti•n, but there was no change in the level of plasma magnesium. These data extend the observation of ttibbs et al. (8), that P T E will elevate plasma calcium in nonp a r t u r i e n t cows. Since dry cows showed a

M . P . The Effects of Age upon Calcium Utilization and Maintenance Requirements in the Bovine. J. Animal Sci., 13: 25. 1954. (7) HIB~S, J. W. Milk Fever (Parturient Paresis) in Dairy Cows--A Review. J. Dairy Sci., 33: 758. 1950. (8) tIm~s, J. W., Pou~vl)z¢~, W. D., ANI) Kr~uss, W. E. Further Studies on the Effect of Vitamin D and of Parathyroid Extract, Paroidin, on the Blood Changes of Normal and Milk Fever Cows at Parturition. J. Dairy Sci., 30: 564~. 1947. (9) LITTLe, W. L., ANn MATTICK, E. C. V. The

Calcium Content of Cow's Blood. II. (a) Calcium in the Blood of Parturient Heifers. (b) Effect of Injections of Parathyroid Extract, Irradiation, etc.; (e) Some Csse Reports of Milk Fever. Vet. Record, 13: 1091. 1933. (10) MAI~R,,A., Moo~)~, E. W., AND ROBElCTSON,A. Some Biochemical and Clinical Aspects of Milk Fever. J. Comp. Path. Therap., 65" 347. 1955.

EFFECT OF PARATHYROID HORMONE ON PLASMA CONSTITUENTS (11) MOODII~,E. W., I~¢IARR,A., AN]) ROBEI~TSOI~, A. Serum Calcium and Magnesium and Plasma Phosphate Levels in Normal Parturient Cows. J. Comp. Path. Therap., 65: 20. 1955. (12) PAPP]~IxrI-IAG]~N~A. R. Parathyroid Hormone and Parturient Paresis in Dairy Cattle. Ph.D. thesis, Purdue University. June, 1959. (13) PAPP]~NHAGEN, A. R., AND JACKSON, H. D. Modified Method for the Determination of Serum Calcium in the Presence of Magnesium Using Ca]-Red Indicator. Clin. Chem., 6: 582. 1960. (14.) ROB]~RTSOIT, A., BUF~GI~]SS, J. W., MAR~, A., AIqD MILNE, BF.TSY J. Some Observations on Milk Fever. Vet. Record, 60: 505. 1948. (15) ROB]~RTS0hr,A., MAKR, A., AN]) MOOI)I~, E. W. Milk Fever. Vet. Record, 68: 173. 1956. (16) ROBINSON, C. S., HUFFMAN, C. F., AND BU~T, K. L. The Effect of the Administration of Parathyroid Extract on Normal Calves. J. Biol. Chem., 74: 477. 1927.

903

(17) SEEKL~S, L., SJOLLF.]~IA~ B., AND VAI~ DER KAAY, F. C. Zur Pathogenese der Paresis Puerpcralis. Ein Beitrag uber den Zusammenhang Zwlschen Calciumstoffwechsel und Muskeltonisierung. Acta Brevia Neerl., 2: 200. 1932. (18) S0~M~.~, A. 5. The Determination of Acid and Alkaline Phosphatase Using p-Nitrophenyl Phosphate as Substrate. Med. Bull. St. Louis University, 4: 165. 1952. (19) STO~T, G. •., AN]) SMITH, V. R. Parturient Paresis. V I I I . Results of Parathyroidectomy of Cows. J. Dairy Sei., 40: 897. 1957. (20) TUaNF~, C. D. General Endocrinology. 3rd ed., chap. 4. W. B. Saunders Co., Philadelphia. 1960. (21) U.S.P. The Pharmacopeia of the United States of America, 16th rev., United States Pharmacopeial Convention, Inc., Washington, D. C. 1960.