Plasma cyclic AMP response to calcitonin: A potential clinical marker of bone turnover

Bone, 6, 285-290 (1985) Printed in the USA. All rights reserved

Copyright

8756-3282185 $3.00 -t 00 @ 1985 Pergamon Press Ltd.

Plasma Cyclic AMP Response to Calcitonin: A Potential Clinical Marker of Bone Turnover J.R. MINKOFF,

B.F. GRANT, and R. MARCUS

Gerontology Diwsion, Department of Medione, Stanford University, and the Aging Study Unit, Geriatrics Research fducabon Center, VA Medical Center, Palo Alto, CA, USA. Address

for correspondence

and Chcal

and repnnts: Dr. R. Marcus, GREGG 182-B, VAMC. Palo Alto, CA 94304, USA

1982). Although this technique allows accurate determrnation of bone remodeling in relatively small groups of patients, the need to evaluate initial status and response to therapy in routine clinical practice makes it desirable to develop noninvasive approaches to assess bone turnover. Brown et al. (1984) recently found that serum levels of the gammacarboxyglutamic acid-containing peptide, bone Gla protein (BGP or osteocalcin), correlate well with histomorphometric measures of bone formation. To date, however, no reliable index of bone resorption has been forthcoming. Blanc et al. (1977) reported that the degree of hypocalcemia induced by an injection of calcitonin mrght provrde a clinically useful index of bone resorption. They showed that this calcemic response was greater in patients with hyperparathyroidism and Paget’s disease than in normal subjects and that control of these diseases was associated with reduction in the magnitude of response to calcrtonin. However, this test, as originally described, has not proved to be generally useful. The change in serum calcium was often little greater than the error of the analytical method, and the duration of response required frequent sampling over a 24-h period. The physiological actions of calcitonln appear to be mediated by an initial interaction of the hormone with specific cell surface receptors in its target tissues, leading to activation of the adenylate cyclase-CAMP system (Heersche et al., 1974). Reflecting this fact, administration of calcitonin in vivo results in a rapid increase in plasma concentrations of CAMP (Ardaillou et al., 1976; Caniggia et al., 1980; Gennari et al., 1981). Although this response could be due to an action of the hormone on either kidney or bone, evidence has been presented that it arises primarily from bone (Ardaillou et al., 1976; Caniggia et al., 1980). We have conducted experiments to characterize the acute plasma CAMP response to calcitonrn. These include studies in rats that address the relative contribution of bone and kidney to this response and whether the intensity of response may reflect the degree of bone turnover. In addition, we have administered calcitonln to normal men and women and to subjects with disorders of mineral metabolism to determine whether the CAMP response might provrde a clinically useful index of bone turnover in humans. These studies form the basis of this reoort.

Abstract A noninvasive marker of bone turnover would be useful in predicting which patients are at risk of rapid bone loss and in monitoring response to therapy. Calcitonin (CT)-induced hypocalcemia correlates with bone turnover but has not been a clinically useful test because changes in serum calcium are small, and test duration is long. CT rapidly increases plasma CAMP levels. We conducted studies in rats and in man to characterize the origin of this rise, and to determine its suitability as a potential clinical marker of bone resorption. Administration of CT to rats resulted in a prompt and sustained rise in plasma CAMP. This effect was not blunted by nephrectomy and was greater in calciumdeprived rats with increased bone resorption. Thus, it appears to reflect CT action on bone An intramuscular injection of 100 units salmon CT elevated plasma CAMP in 18 normal men and women. This effect was observed by 20 min after injection and persisted over 2 h. Although basal levels of plasma CAMP were similar in healthy postmenopausal women and men, the response to CT was greater in women. The response of women with hyperparathyroidism was greater than that of normal women, and the response of pagetic men was greater than that of normal men. The rise in plasma CAMP following CT is rapid and easily measured and appears to correlate with the state of bone remodeling. Additional studies will be required in osteoporotic subjects with high and low remodeling activity before the clinical utility of this test can be determined.

Key Words: Bone Turnover-Calcitonin-Cyclic AMP-Hyperparathyroidism-Paget’s Dtsease-Osteoporosis.

Introduction Osteoporosis is a heterogeneous disorder, and quantitative histomorphometry of trabecular bone from iliac crest confirms that some patients have little active remodeling, whereas others show increased prevalence of resorption surfaces and osteoclasts (Meunrer, 1980; Whyte et al., 205

286

J.R. Mlnkoff, B.F. Grant, and R. Marcus: Plasma CAMP response to calcitonln and bone turnover

Materials and Methods Synthetic salmon calcltonln (sCT) was obtalned as a lyophilized powder and was found to have a potency of 4300. u&s/mg (Schwartz et al., 1981). For studies In rats sCT was dissolved. lmo/ ml, in 0.005 N acetic acid and stored at -20°C in small allquo&. For clinical studies, sCT(Calcimar) was purchased from the United States Vitamin Corp. Specialized diets were purchased from the Nutritional Biochemical Corp, Cleveland, OH and their calcium contents were determined in our laboratory by titration against EGTA (Corning Calcium Analyzer, Medfield MA) The fortified control diet was found to contain 4.7% calcium, and the calcium-deficient diet contained 0.01% calcium Sprague-Dawley male rats were purchased from Simonsen Labs (Gilroy, CA) and were maintained under usual conditions of housing and diet unless indicated otherwise. Materials for the radioimmunoassay of CAMP were purchased from the Becton Dickinson Corp. (Orangeburg, NY)

Sfudfes in rats Rats, 150-250 g, were administered

varying doses of sCT by intraperitoneal injection They were decapitated at the appropriate intervals, and blood was funneled into glass centrifuge tubes containing 0.5 ml 15% Na,EDTA. The tubes were immediately agitated and centrifuged at 1000 g for 10 min. Plasma was deproteinized with equal volumes of 10% trichioroacetic acid (TCA) Following neutralization with solid CaCo, (Tihon et al., 1977), CAMP was measured by radioimmunoassay. In one experiment 30 rats, 80-1009, were anesthetized with Metafane by inhalation. Fifteen ammals underwent bilateral nephrectomy via flank incisions, and the remainder underwent sham operations that involved mobllizlng the kidneys to the extenor and then replacing them in the renal bed. One and two days later 5 rats from each group were Injected with 50 ~1 Isotonic saline, and 10 rats were given sCT, 50 U/100 g body weight. Rats were killed, and blood was taken for CAMP 30 mln later as described. In another experiment, 15 rats were fed a control diet containing 4.7% calcium, and 15 rats were fed a diet containing 0.01% calcium. Between 45 and 48 days after Instituting the diets, they were injected either with saline or sCT, 50 U/100 g body weight. After 30 mln, rats were killed, and blood, renal cortex, and calvarla were taken for measurement of CAMP Calvaria were rapldly fixed by mlcrowave irradiation, and renal cortices were immersed directly into 6% TCA as described previously (Marcus et al., 1980) Studies in man Nine men (age 46-76) and nine postmenopausal women (age 5469) served as controls. They were free of medical conditions known to affect calcium or skeletal homeostasis. No sublect had clinically apparent osteoporosis All denied loss of height. and spine radlographs revealed no instance In which compresslon or loss of 10% of anterior or middle-vertebral height had been sustained. In addltion, we studied five women with mild hyperparathyroidlsm dlagnosed by multlple smultaneous elevations In the serum concentrations of calcium and lmmunoreactlve PTH (Madvig et al., 1984) Creatlnine clearances of the hyperparathyrold women (56$-8 ml/ mln) were IndIstinguishable from those of the normal women In this study (65f 10) or from those of age-matched controls (Marcus et al., 1984) Three men with Paget’s disease of bone were also studled This diagnosis was based on typlcal bone radiographs and scans and elevated serum alkaline phosphatase activity. Serum alkaline phosphatase activity was 288-2690 IU/L (upper limtts of normal, 155 IU/L). They had not received treatment witti either calcltonin or diphosphonate within the previous 3 months. Except for one of these men, no subject was receiving medication known to Influence calcium homeostasis, e.g , thiazide dluretlcs. lithium carbonate, diphenylhydantoln, or glucocorticolds. One pagetlc man was receiving chronic therapy with llthlum carbonate. None of the women took estrogen repalcement therapy No subject was recelving methylxanthines or other drugs known to affect CAMP generatlon or metabolism The protocol was approved by the Human Subjects Committee of Stanford University, and each subject pcbvlded written consent

Subjects were admltted to the Aging Study Unit, a cllnlcal Investigation ward. After an overnight fast an indwelling catheter was placed in a forearm vein. Thirty minutes later a blood sample was obtalned, and sCT. 100 units, was administered by intramuscular injection. Additional blood samples were obtained 10, 20. 40, 60, 90, 120, and 180 min later. Subjects remained recumbent in a quiet room throughout the study period. Five men received injectIons of isotonic saline on a separate day. Blood samples were collected Into tubes containing EDTA. Following centrifugation at 1000 g for 10 min, plasma was stored at -20%. Plasma was deproteinized and assayed for CAMP by radioimmunoassay (Becton-Dickinson, Orangeburg, NY) as above. Data analysis Plasma CAMP is expressed as picomoles/ml. Results are given as the mean + standard error of the mean. Statlstlcal analyses were performed at the central computer facility of Stanford University. Data were analyzed by analysis of variance with Neuman-Keuls analysis and t-tests where appropriate. Areas under the curve were determined with a microcomputer Incremental areas under the curve were calculated by subtracting the area under the baseline value from the area under the entlre curve.

Results Studies in rats The basal level of CAMP in rat plasma was 33.0 pmol/ml. Injection of saline caused no change in CAMP concentration. sCT led to a rapid and prolonged rise in plasma CAMP (Fig. 1A). This rise was detectable by 10 min and persisted over the subsequent 3 h. A significant rise in plasma CAMP was produced increase

by 5 U/100 g body weight, with a dose-related to 50 units/100 g body weight (Fig. 1B). Bilateral nephrectomy led to a rise in basal levels of CAMP within 24 h compared to levels in sham-operated control

rats (75 + 8 vs 55 & 7 pmol/ml, (P
50 TIME (mm1

sCT (units/100 $Jbody welght)

Fig. 1. Effect of salmon calcitonin on plasma CAMP In rats. Each point represents the mean f SE M. of five animals. A. Time course of response to 50 U/lOOg body weight sCT. A significant rise was observed from lo-180 min., P~O.01.8. Dose-response, PcO.05 for 5 U/lOOg body weight or more.

J.R. Mtnkoff. B.F. Grant, and R. Marcus: Plasma CAMP response to calcitonin and bone turnover

NEPHRECTOMY

SHAM =

200

-5 E 160 EL 9

120

i

80

2

40 0

287

rats (4.6 + 0.3 pmol/mg protein) was significantly higher than that of the control rats (2.6 + 0.4 pmol/mg, PcO.05, Fig. 3C). A significant rise in renal cortical CAMP occurred in control rats (3.8 f 0.4 pmol/mg) in response to sCT, but this increase did not differ from that which occured in calcium-deficient rats (4.8 f 0.3 pmol/mg). Thus, the exaggerated rise in plasma CAMP following sCT in calcium-deprived rats is not attributable to the kidney and most likely represents an increased responsiveness of bone. Studies in man

Saline (5)

sCT (101

Saline (5)

sCT 110)

Fig. 2. Effect of nephrectomy on the plasma CAMP response to sCT. Animals underwent nephrectomy or sham operation 24-48 h before sCT injection. Numbers in parentheses represent number of animals in each group. ‘Differs from sham-operated rats receiving saline, P
CAMP level in saline-treated calcium deficient animals (46 f 8 pmol/ml) was significantly higher than that of the controls (28 + 7 pmol/ml, P ~0.05, Fig. 3A). In response to calcitonin, the peak level of plasma CAMP in calcium-deficient rats (134 & 10 pmol/ml) was also significantly higher than that of the controls (46 & 9 pmol/ml, P
PLASMA 160

++

Normal subjects. Baseline plasma CAMP levels were 9.6 + 0.7 pmol/ml in men and 11.8 + 1.0 pmol/ml in the healthy postmenopausal women (P> 0.05, Table I). Injection of isotonic saline led to no significant change in CAMP concentration in the five men tested (Fig. 4). Injection of sCT caused a rise in plasma CAMP in all subjects. This rise was detectable as early as 20 min, with a peak value between 40 and 120 min. Men and healthy postmenopausal women had different responses to sCT. Baseline CAMP levels were similar (see above), but the response of these women was significantly greater than that of men at 60, 90, and 120 min (P=zO.O25) (Fig. 4). In addition, the area under the response curve was greater in the healthy women, 3378 + 321 vs 2550 f 63, PcO.025 (Table I, Fig. 5, Left). This difference was marginally significant when expressed as incremental areas under the curve: women, 1262 + 180; men, 816 + 139, P=O.O6. Neither the maximum Increment (max6) of CAMP over baseline nor the percentage increase (not shown) differed between men and women (Table I, Fig. 5,

Right). Patients with d/sorders of skeletal remodeling. The baseline plasma level of CAMP did not differ significantly from that of normal postmenopausal women in five women with hyperparathyroidism (18.2 + 1.8 vs 11.8 & 1.O). In response to sCT, plasma CAMP was significantly greater than normal at 90, 120, and 180 min (Fig. 4). The area under the curve was greater in the hyperparathyroid women, either when expressed as total area (5768 IL- 667 vs 3378 f 321,

BONE

,,-

KIDNEY 1

CALCIUM DIET cl LOW I NORMAL I

-

,

-

,

Saline

sCT

Saline

sCT

m

Fig. 3. Effect of dietary calcium restriction on the plasma CAMP response to sCT in kidney, bone, and plasma. Rats were divided Into two groups. Control animals received a 4.7% calcium diet, and restricted animals consumed 0.01% calcium. For each dtetary group, saline was administered to 5 animals and sCT, 50 U/lOOg, was given to 10 animals. A. Plasma; B. Calvaria; C. Renal cortex. ‘PC 0.05, l*P
288

J.R Mlnkoff, B.F. Grant, and R. Marcus: Plasma CAMP response to calcltonin and bone turnover

fable I. The effect of salmon calcltonln on plasma CAMP In healthy adults. Plasma CAMP (pmoles/ml)

Group

N

Normal Women Normal Men ‘Differs

Basellne

Age

9

63.6k1.6

9

65.6$-3

11.8+1 5

90 Min

10

9.6&O 7

Maxlmum

A Max

Area Under Curve (AUC)

Incremental Area Under Curve

23.2_t2.8’

27 Of3.2’

15 7+2.6

3378+321’

1262+180

18.0+ 1.6

19.7k1.7

10.2i1.8

2550+

816,139

163

from normal men, p < ,025.

PcO.01) or as the incremental area (2488 f 444 vs 1262 + 180, PcO.01, Fig. 5, Table II). However, the maximum concentrations and percentage increases were not different between these two groups (Table II, Fig. 5, Right). The three men with Paget’s disease had significantly different responses from those of the normal male controls. Baseline plasma CAMP (17.5 + 5.8 pmol/ml) was higher than that of normals (9.6 + 0.7, P < ,025) (Table II, Fig. 5, Right). Plasma CAMP was higher at 90 min (63.3 + 26.1 vs 18.0 ?I 1.6) and at its maximal value (64.6 + 25.9 vs 19.7 k 1.7) in the men with Paget’s disease (Table II, Fig. 5B). Pagetlc men also had significantly greater areas under the curve, incremental area under the curve, and percent increase in plasma CAMP after sCT injection (Table II, Fig. 5). The number of cases studied to date does not allow meaningful correlation between symptoms or biochemical parameters and response to calcitonin. However, the hyperparathyroid woman with the greatest elevation In serum calcium had the most exuberant plasma CAMP response to calcitonin. The elevation in serum alkaline phosphatase activity appeared to correlate closely with CAMP response in the three pagetic men. The plasma CAMP value 90 min after Injection correlated well with the Incremental area under the curve (r=0.94, P
5 A”

’

01020

’

I

I

I

I

40

60

90

120

TIME

1 f/

180

(mln)

Fig. 4. Effect of salmon calcltonln, 100 U IM. In healthy men (Cl. n=9), healthy women (S, n=9), and women with hyperparathyroldlsm (0, n=5), or saline placebo in healthy men (a n=5) Mean + SEM ‘P < 0 025, compared lo normal women “P < 0 01, compared to normal women

Discussion We have presented data showing a consistent, rapid, and sustained rise in plasma CAMP concentration in response to sCT administration in both rat and humans, This effect was seen in normal animals and humans and was greater in states of increased bone turnover. Kaminsky et al. (1970) first described this phenomenon after intravenous infusion of CT in humans, and it has subsequently been confirmed by Ardaillou et al. (1976) and by Caniggia et al. (1980). The latter investigators administered salmon, porcine, and human CT intravenously to normals and to patients with Paget’s disease. Although plasma CAMP rose more quickly in that study, their results also indicate an increased response in patients with Paget’s disease. The rise in plasma CAMP is not due to renal effects of CT. Indeed, nephrectomized rats had a greater response to sCT than did rats that had undergone sham operation. The primary target cell in bone for CT is presumed to be the osteoclast. Although direct stimulation of CAMP formation by CT has not yet been shown directly for osteoclasts, the hormone fails to elevate CAMP levels in osteoblasts, whereas it does promote CAMP formation in cells with osteoclastlike characteristics (Wong et al., 1977). The most reasonable Interpretation of our results is that the rise in plasma CAMP reflects activation of the adenylate cyclaseCAMP complex In bone. We have shown that CT-dependent accumulation of CAMP is greatly increased In bone and plasma from rats with dietary calcium deficiency. Thus, the response to CT is related to the rate of bone turnover. However, the data do not permit us to exclude the possibility that organs other than bone contribute to the calcitonininduced rise in plasma CAMP of control rats or of animals given a high calcium diet. It IS also possible that the response to CT in humans may be confounded by diseases affecting organs other than bone. For example, renal failure could affect the clearance of both CT and CAMP, as well as end-organ responsiveness to hormone stimulation. However, at least one study (Sztics and Horvath, 1980) showed no difference in plasma CAMP response to calcitonin between normal and uremic patients. It is of interest that healthy postmenopausal women had a marginally greater reponse to sCT than had men. Several studies have shown lower CT levels and CT reserve In women than In men (Heath and Sizemore, 1977; Hillyard et al., 1978, Deftos et al., 1980; Body and Heath, 1983), and it IS tempting to hypothesize that receptors in women are upregulated. Alternatively, one may propose that remodeling is greater in postmenopausal women than in men. The dose of sCT we administered is so large that it would be difficult to invoke higher per kilogram dosage as a cause for the greater CAMP response In women.

289

J.R. Minkoff, B.F. Grant, and R. Marcus: Plasma CAMP response lo calcitonin and bone turnover

go t >

5

s

6000

2 -

5000

>” 5 0

4000

K :

3000

z 1 2

2000

2

1000

f a zi

30 20 10

0

TOTAL

0

INCREMENTAL

BASELINE

90 min MAXIMUM

MAX A

Fig. 5. Effect of sCT, 100 U IM, on plasma CAMP in healthy men (0, n=9), women (8, n=9), women with hyperparathyroidism (a, n=5), and men with Paget’s disease (B, r-1=3). Left. Total area under the response curve from 0 to 180 min and incremental area under the curve (TOTAL-BASELINE). Right. Plasma CAMP (pmol/ml) at baseline at 90 min, at the peak of the response (MAXIMUM), and the difference between maximal response and baseline compared lo normal women.

(MAXA). +P<0.025

compared

Hyperparathyroid postmenopausal women had a greater plasma CAMP response to CT than had healthy postmenopausal women, and the response of men with Paget’s disease was substantially larger than that of normal men. These results support the conclusion that the rise in plasma CAMP reflects the degree of bone remodeling. Previous studies using CT challenge as a marker of bone remodeling have focused on changes in serum calcium (Blanc et al., 1977;

Table

II. The effect of salmon calcitonln

to normal men. lP
compared

to normal men “P ~0.01

Bataille and Sany, 1982). Patients with disease states manifested by increased remodeling showed a greater decrease in serum calcium than did normal controls, and in one study (Chapuy et al., 1975), the decrease in calcium concentration correlated with both alkaline phosphatase activity and urinary hydroxyproline excretion. However, the time required to conduct this test and the small changes in serum calcium produced have limited its practical applicability. The CAMP

on plasma CAMP patients with disorders

of mineral metabolism.

Plasma CAMP (pmoles/ml) Group

N

Hyperparathyroidism: EK Cla LP VW FH Mean, _tSEM 5 Pager’s disease: ALb CJ IS Mean, *SEM 3

Age

Baseline

60 73 64 71 54 64.4k3.5

24 4 15.2 20.0 15.5 16.0 18.2f

75 67 78 73.3k3.3

6.7 8.0 27.9 17.5+5.8**

1.8

90 Mln

A Max

59.0 36.9 40.8 18.4 31.2 37.3k6.6’

59.0 39.5 41.7 27.9 31.2 39.955.4’

34.6 24.3 21 7 12.4 15.2 21.6k3.9

7940 6166 5920 4021 4793 5768+667’

113.4 25.4 51.1 63.3+26.1”

113.4 25.4 55 0 64.6+25.9”

106.7 17.4 27.1 50.4i28.3”

10140 3321 8245 7235+2032”

a Secondary hyperparathyroidlsm b On other medtcations. LICQ and diphenylhydantoin * Differs from normal women, p < .05 **Differs from normal men, p < ,025 l

Maximum

Area Under Curve (AUC)

Incremental Area Under Curve

3548 3430 2320 1231 1913 2488+44’ 8934 1881 3223 4679+2162”

290

J.R. Minkoff, B.F. Grant, and R Marcus: Plasma CAMP response to calcltonin and bone turnover

response to CT offers the advantages of a shorter time course and a greater change in the measured variable and might therefore allow better separation of high-remodeling from low-remodeling states. The correlation between 90 min and incremental area under the curve is so strong that paired baseline and 90 min CAMP values probably provide as accurate a reflection of response to CT as does more prolonged monitoring. Clearly, there would be no practical utility in a CT challenge test if it were useful only to distinguish individuals with Paget’s disease or hyperparathyroidism from normal. The potential for such a test lies in the possibility that it might distinguish individuals with osteoporosis on the basis of remodeling activity and in providing a noninvasive means to follow the effects of therapy on bone resorption. To validate such a use will require correlating the response to CT with other biochemical markers of bone turnover, to histomorphometric indices of resorption, and to sequential changes in bone mass. Acknowledgements: We thank Mrs. Marilyn Kazeml and the staff of the Aging Study Unit for their enthusiastic and expert support. The manuscript was prepared by Ms. Susan Singh and Ms. Anna Boberg. This work was supported by the Research Services, Veterans Administration. Synthetic sCT was the generous gift of Dr. Joseph Orlowski, Armour Pharmaceuticals, Kankakee, IL.

References Ardalllou R.. Isaac R.. Nlvez M.P.. Kuhn J M Cazor J L.. and Flllastre J.P Effect of salmon calcltonln on renal excretion of adenosme X-monophosphate Horm Mefab Res 8 136-140, 1976 Bataille R and Sany J.. Cllnlcal evaluation of myeloma osteoclastlc bone lesions Il. Induced hypocalcemla test usmg salmon calcltomn Mefab Bone DIS Rel Res 439-42, 1982. Blanc D , Chapuy M.C., and Meunler P Evaluation de I’actlvlte osteoclastlque par le test d’hypocalcemle provoquee par la calcltonlne de saumon Nov Presse Med 6.2489-2494, 1977 Body J -J. and Heath H /Il. Estimates of clrculatmg monomeric calcitonln PhysIologIcal studies In normal and thyroldectomized man J Clan. Endocrinol. Mefab 57 897-903, 1983 Brown J.P., Malaval L.. Chapuy M.C., Delmas P D Edouard C and Meunler P.J Serum bone Gla-protem. A speclftc marker for bone formatlon In postmenopausal osteoporosis. Lance1 1(8386),1091-1093. 1984 Canlggla A., Gennari C., Lore F Nut1 R and Gall1 M Effects of parathyrold hormone and calcitonln on plasma and nephrogenous cyclic adenoslne~ 3’,5’-monophosphate In normal subjects and in pathological condltlons Europ. J C/m invesl 10.99-105. 1980 Chapuy M C.. Meunler P Terner M , David L and Ugnon G Effects b!ologlques a court terme de la calcltonine synthetlque de saumon dans la

maladle de Paget Influence de la posologle Pathol 010 23 349.359, 1975 Deftos L J , Welsman M.H.. Wllllams G W.. Karpf D.B., Frumar A M.. Davldson E J , Parthemore J G.. and Judd, H L Influence of age and sex on plasma calcltonln In human beings N Engi J Med 302 1351.1353. 1980. Gennari C., Chienchettl S.M., Vibrelil C., FrancIn G., Maloll E., and Gonnelll S: Acute effects of salmon, human, and porcme calcitonm In plasma CAMP levels In man. Curr. Ther. Res. 30:1024-1032, 1981. Heath H Ill and SIzemore G.W.: Plasma calcitomn in normal man. Dlfierences between men and women. d. Ckn Invest. 60:1135-l 140, 1977 Heersche J.N.M.. Marcus R., and Aurbach G.D.: Calcltonin and the formatlon of 3’,5’-AMP in bone and kidney. Endocrinology 94:241-247, 1974 Hillyard C J., Stevenson J.C. and Maclntyre I.: Relative deficiency of plasma calcitonin In normal women. Lancef 1:961-962, 1978. Kamlnsky N.I., Ball J.H., Broadus A.E.. Hardman J.G., Sutherland E.W. and Liddle G.W : Hormonal effects on extracellular cyclic nucleotldes In man Trans. Assoc. Am. Phys. 83:235-244, 1970. Madvig P.. Young G and Marucs R: Assessment of adenosine 3’,5’-monophosphate excretion and an oral calcium tolerance test In the diagnosis of mild primary hyperparathyroldlsm. J. C/in. Endocnnol. Melab. 58.480407, 1984 Marcus R.. Madvig P. and Young G: Age-related changes In parathyroid hormone and parathyroid hormone action in normal humans. J C/m. Endocrrnol Mefab. 58:223-230, 1984. Marcus R., Orner F.B. and Bnckman A.S.: Effects of in VIVO vitamin D metabolites and 17-estradlol on parathyrold hormone-dependent formation of adenoslne 3’,5’-monophosphate in rat bone. Endocnnology 107.1593. 1599, 1980. Meunler P.J Bone biopsy in diagnosis of metabolic bone disease, In: Hormonal Control of Calcium Mefabolism. Excerpta Medica, AmsterdamOxford-PrInceton InternatIonal Conference on Calcium Regulattng Hormones, InternatIonal Congress Series, 115. 1980. Schwartz K.E., Orlowskl R.C. and Marcus R.: des-Ser 2 salmon calcltonln. A biologically potent synthetic analog. Endocrinology 108:831-835, 1981 Stock J.L and Coderre J.A Calcitonln and PTH mhiblt accumulation of CAMP In stimulated human mononuclear cells Blochem BIophys Res. Commun. 109 (3):935-942, 1982 Szucs J. and Horvath T.. Effect of calcltonin on plasma CAMP in uremic man Harm. Mefab. Res. 12:44-45, 1980 Tihon C., Goren M.P., Spitz E. and Rlckenberg H.V Convenient ellminatlon of tnchloroacetlc acid prior to radiolmmunoassay of cyclic nucleoldes. Anal. Blochem 80, 652-655, 1977. Whyte M P.. Bergfeld M.A., Murphy W.A , AVIOII L.V. and Teitelbaum S L.. Postmenopausal osteoporosis: A heterogeneous disorder as assessed by hlstomorphometrlc analysis of iliac crest bone from untreated patients Am. J Med. 72 193-202, 1982 Wong G L., Luben R.A and Cohn D.V 1.25.Dlhydroxycholecalciferol and parathyroid hormone. Effects on Isolated osteoclast-llke and osteoblastllke cells Soence 19?:663-665, 1977

Recerved November 13, 1984 Revrsed March 4. 1985 Accepted Aprrl 3. 1985