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Bone Vol. 4, No. 6 June 1996:629-632

Salmon Calcitonin in the Prevention of Bone Loss at Perimenopause I. A R N A L A , 1 J. SAASTAMOINEN, 2 and E. M. A L H A V A 1

i Department of Surgery, Kuopio University Hospital and A. I. Virtanen Institute, University of Kuopio, Kuopio, Finland 2 Lasaretti Hospital, Kuopio, Finland

The objective of this study was to determine whether intra- nasal salmon calcitonin prevents physiological bone loss at perimenopause. A double-blind study of 120 perimenopausal women without present or past disease or medication that could affect bone metabolism were studied. The subjects were randomized in two groups and provided with nasal spray bottles containing either placebo (excipient only) or active compound (excipient plus 50 international units (IU) salmon calcitonin per dose). Subjects took one puff from the nasal spray in each nostril every morning. All subjects took one soluble tablet of calcium (1000 mg) per day. Serum bio- chemistry, dual-energy X-ray absorptiometry of lumbar spine and proximal femur, quantitative computed tomogra- phy of lumbar spine, and single photon attenuation of fore- arm were used to evaluate bone mineral density (BMD). There were no differences in demographic characteristics or hormone status at entry. No fractures were recorded during the study period. Serum calcium increased and serum dihy- droxyvitamin D and osteocalcin decreased in both groups. There was no difference in biochemical parameters between the groups. The BMD of upper femur did not change during the study, but it was decreased in the lumbar spine in both groups. The mineral content of distal radius increased in both groups. In conclusion, nasal salmon calcitonin, 100 IU daily, has no protective effect on bone mass and does not modify bone metabolism at perimenopause. (Bone 18:629- 632; 1996)

Key Words: Bone loss; Bone mineral density; Calcitonin; Menopause; Osteoporosis; Perimenopause.

Introduction

Calcitonin has been widely used in the treatment of postmeno- pausal osteoporosis. There are many publications on the inhibi- tory effect of injectable 2'1° and nasal 13'~7 salmon calcitonin on bone loss. Also, it has been shown that calcitonin protects against hip fractures in postmenopausal women. 8 To date there have been no publications on the effect of calcitonin on bone in peri- menopausal women. It is well known that bone loss accelerates after menopause. Prevention of osteoporosis in women depends on maximizing bone mass through physical activity, good nutri-

Address for correspondence and reprints: I lkka A m a l a , M.D., Depart-

ment of Surgery, Kuopio University Hospital, FIN-70210 Kuopio, Fin- land.

tion, and especially, treatment to prevent bone loss after meno- pause. Hormone replacement therapy (HRT) is the most success- ful treatment currently available, and it is relatively inexpensive. The long-term use of HRT, however, is restricted by the incon- venience of continued monthly bleeding in elderly women and by concerns about the safety of such extended treatment. In addition, women at risk for breast cancer are unable to receive HRT. The purpose of this study was to examine the possible protective effect of calcitonin on bone loss in perimenopause using biochemical analysis and bone mineral density (BMD) measurements in the evaluation of bone turnover.

Materials and Methods

Subjects

A total of 120 women were enrolled in the study. The subjects were perimenopausal (menstrual cycles had become shorter, longer, or more irregular than usual) women over the age of 40 years, who had had a menstrual period within the previous 6 months. They were otherwise healthy, without evidence of pre- sent or past disease, and were not receiving medication influenc- ing bone metabolism.

The protocol was approved by the Ethics Committee of Kuo- pio University and Kuopio University Central Hospital. All sub- jects were informed by the investigator of the nature and purpose of the study and of the withdrawal of possible benefits of HRT during the study. The subjects gave written informed consent prior to the start of the study and were computer randomized in the study into groups of four.

Study Protocol

The subjects were provided nasal spray boules containing either placebo (excipient alone) or active compound [excipient plus 50 international units (IU) salmon calcitonin per dose], in a double- blinded manner. One puff of nasal spray was taken in each nostril every morning. To ensure adequate calcium intake during the study, all subjects took one soluble tablet of calcium (1000 mg) per day. This regimen was followed for 36 months. After the main study period a volunteer subgroup of subjects continued the experiment up to 60 months. After 3 years all subjects were given salmon calcitonin at the same dose (100 IU/day) and 1000 mg of calcium daily. Only BMDs of lumbar spine, proximal femur, and distal radius were recorded during the extension of the study.

© 1996 by Elsevier Science Inc. 629 8756-3282/96/$15.00 All rights reserved. Pn $8756-3282(96)00084-1

630 I. Arnala et al. Bone Vol. 4, No. 6 Calcitonin and bone loss at perimenopause June 1996:629-632

Biochemistry

Serum levels of calcium (S-Ca), phosphate (S-Pi), alkaline phos- phatase (S-ALP), serum albumine (S-AIb), creatinine (S-crea) c-terminal peptide of parathyroid hormone (S-PTH), luteinizing hormone (S-LH), and follicle-stimulating hormone (S-FSH), and estradiol and urine analysis were determined using routine labo- ratory methods (Progress Clinical Chemical Analyzer; Kone, Es- poo, Finland). Serum osteocalcin (S-BGP) was measured using commercial ly available methods (lncstar, Stillwater, MN). The 1,25 (OH)2D 3 metabolite of vitamin D was analyzed (Medix, Helsinki, Finland).

Dual-Energy X-ray Absorptiometo' (DEXA )

Bone mineral density of lumbar spine (L2-4) and proximal fe- mur (femoral neck) was measured using DEXA (Lunar DPX; Lunar Radiation, Madison, WI). D E X A analyses were available at 12 months.

Quantitative Computed Tomography (QCT)

The BMD of the lumbar spine was also analyzed by QCT. The first four lumbar vertebrae were measured by an X-ray computed tomography scanner (Somatom 2, Siemens, Erlangen, Germany) from 4 mm consecutive slices with an energy of 96 kV and 300 mA, and 720 projections. A calibration phantom of water and air was scanned together with the subjects. Each slice was manually framed as the region of interest and the density was recorded using the Hounsfield scale. The final result is given as the mean of 11-13 slices. The coefficient of variation in the spine was 2.8%. ~'

Single Photon Attenuation (SPA)

The forearm BMD was measured by the gamma-ray attenuation method. The source of radiation was 1665 MBq (45 mCi) of Am-241 radionuclide with an energy of 60 keV. A straight col- limator (diameter of 3.5 mm) was connected coaxially with the

radiation source and detector. The apparatus was calibrated be- fore and after the subject 's measurement. The forearm to be measured was fixed in a water bath between the collimated source and detector. Two sets of the measurements were ob- tained. The first was taken at the site approximately 1.5 cm from the dorsal end of the distal radius. The mineral density of the distal radius (grams per cubic centimeter) was calculated as an average of the three or four scans as previously described. ~,9 The values obtained were corrected to eliminate the effects of fat. The reproducibility of BMD in the distal radius was within 1.9%. Measurements of cortical bone were done at the site between the middle and distal thirds of the radius and ulna. In the region of compact bone the mineral content is given as grams per square centimeter. The results were recorded as the mean of three or four scans. The reproducibility of the bone mineral content was within 5% in cortical bone. 9

Statistical Methods

The statistical analyses were performed using the SPSSPC 5.0 statistical package. The Mann-Whi tney U-test was used to ana- lyze the differences between the groups, and Wilcoxon matched- pairs s igned-ranks test was used to evaluate the differences within the groups from baseline.

Results

The women were recruited into the trial and randomized to the treatment groups so that there were 60 women in the group given 100 IU salmon calcitonin/day and 60 in the group given placebo. In the treatment group 50 women, and in the placebo group 46 women, completed the 3-year study. Ten patients in the treatment group prematurely withdrew from the study: four quit because of severe climacteric symptoms, three were uncooperative, two were treated for gastrointestinal cancer, and one cited personal reasons. Six women in the placebo group discontinued the study because of severe climacteric symptoms, six for personal rea- sons, and two for protocol violations. Eight subjects in the treat- ment group and five in the placebo group were menopausal after

Table 1. Serum biochemistry

Time (months)

Parameter 0 In = (t)/(p) 60/60] 12 [n = (t)/(p) 55/54] 24 In (t)/(p) 53/50] 36 In (t)/(p) 50/46]

S-Ca (t) 2.21 + 0.13 2.30 + 0.11 a 2.33 _+ 0.11 d 2.31 _+ 0.08 d S-Ca (p) 2.24 + 0.11 2.28 _+ 0.08 2.31 _+ 0.10 ~ 2.31 -+ 0.07 a S-Pi (t) 1.09 + 0.11 1.08 + 0.25 1.14 _+ 0 . 2 3 a'b 1.12 -+ 0.21 S-Pi (p) I.//9 _+ 0.21 1.05 -+ 0.18 1.06 + 0.14 1.(/8 -+ 0.14 S-Alp (t) 119.2 _+ 27.9 109.8 + 30.7 118.6 _+ 40.8 117.6 -+ 43.8 S-Alp (p) 120.1 _+ 30.6 104.8 + 24.5 c 112.3 + 29.4 111.6 -+ 28.5 S-BGP (t) 3.33 _+ 0.98 2.04 _+ 0.91 a 2.31 _+ ().82 ~ 2.91 -+- 1 .04 b

S-BGP (p) 3.44 + 1.20 2.29 _+ 0.97 a 2.35 _+ 1.01 a 2.65 -+ 1.1 I a S-1,25 D 3 115.4 _+ 26.1 90.3 -+ 26.3 a 76.4 _+ 22.8 a 82.3 -+ 19.4 d S-1,25 D~ 110.0_+ 28.3 83.3 _+ 29.6 d 76.2 + 18.9 d 80.4_+ 17.1 a S-PTH (t) 48.9_+ 22.0 51.3 + 18.5 55.1 + 23.4 48.1 + 17.7 S-PTH (p) 49.3 _+ 18.3 49.4 _+ 18.2 57.3 + 18.9 46.7 -+ 16.5 S-Crea (t) 76.0 _+ 12.9 ~' 74.9 -+ 9.6 78.6 + 9.8 75.3 -+ 9.5 S-Crea (p) 69.6 + 13.6 72.2 _+ 12.9 76.6_+ 12.6 d 73.7-+ I I.0 h S-AIb (t) 42.5 _+ 4.8 42.9 _+ 3.8 42.2 _+ 5.9 4(/.2 -+ 2.6 ~ S-AIb (p) 41.0 + 4.5 41.9 _+ 3.4 40.4 _+ 3.9 40.8 -+ 3.5

(t) = treatment group; (p) - placebo group; S-Ca = serum calcium (mmol/L); S-Pi - serum phosphate (mmol/L); S-Alp = serum alkaline phosphatase (U/L); S-BGP = serum osteocalcine (btg/L); S-1,25 D 3 = serum 1,25 metabolite of vitamin D (pmol/L); S-PTH = serum parathyroid hormone (pmol/L); S-Crea - serum creatinine (~xmol/L); S-Alb = serum albumine (g/L). Difference between groups: ap < 0.05. Difference from baseline: ~'p < 0.05; ~p < 0.01; ap < 0.001.

Bone Vol. 4, No. 6 I. Aruala et al. 631 June 1996:629-632 Calcitonin and bone loss at perimenopause

Table 2. Serum sex hormone levels and urine analysis

Time (months)

Parameter 0 [n = (t)/(p) 60/60] 12 [n = (t)/(p) 55/54] 24 [n = (t)/(p) 53/50] 36 [n = (t)/(p) 50/46]

S-Estra (t) 0.30 _+ 0.28 0.31 + 0.18 0.30 _+ 0.20 0.22 _+ 0.14 b S-Estra (p) 0.29 + 0.31 0.30 _+ 0.20 0.31 _+ 0.19 0.26 _+ 0.38 S-LH (t) 7.56 + 4.65 8.78 + 6.53 12.65 __. 14.63 8.37 _+ 7.39 S-LH (p) 7.52 + 3.47 11.86 + 17.63 10.23 _+ 16.69 8.72 _+ 14.52 S-FSH (t) 9.56 -+ 4.14 13.62 _+ 9.32 ~ 21.54 + 25.63 c 16.33 _+ 17.58 b S-FSH (p) 9.75 _+ 6.07 18.51 __ 29.76 c 14.07 _+ 14.89 b 15.00 _+ 17.34 b U-Ca/Crea (t) 542 + 259 521 -+ 234 584 _+ 232 527 -+ 188 U-Ca/Crea (p) 434 + 170 a 549 + 205 d 535 __ 241 d 474 + 203 U-HP/Crea (t) 16.6 _+_ 10.4 20.6 _+ 10.3 c 17.8 _+ 7.0 19.1 + 6.9 c U-HP/Crea (p) 15.3 +_ 9.1 19.0 __ 7.4 c 18.6 + 9.3 20.4 _ 9.5 a

(t) = treatment group; (p) = placebo group; S-Estra = serum estradiol (nmol/L); S-LH = serum luteinizing hormone (U/L); S-FSH = serum follicle stimulating hormone (U/L); U-Ca/Crea = urine calcium/creatinine ratio; U-HP/Crea = urine hydroxyproline/creatinine ratio. Difference between groups: ap < 0.05; difference from baseline: ap < 0.05; bp < 0.01; Cp < 0.001.

36 mon ths . There were no d i f fe rences in the demograph ic char- acterist ics or h o r m o n e status at entry. No fractures were recorded dur ing the s tudy period.

Biochemistry

At the beg inn ing o f the s tudy s e rum creat inine was s l ight ly h igher in the t rea tment g roup than in the placebo group (p < 0.05). There were no d i f fe rences in any other paramete rs at base- line. Se rum ca l c ium increased in both groups (p < 0.01). There was no di f ference in s e rum ca lc ium be tween the g roups at any poin t o f evaluat ion. Se rum d ihyd roxyv i t amin D decreased sig- n i f icant ly in both groups (p < 0.001). S e r u m os teocalc in de- c reased in both groups . In the evalua t ion o f sex h o r m o n e s an ex tens ive var ia t ion in individual resul ts was seen. In the calci- tonin group s e rum estradiol levels decreased s igni f icant ly by the end o f the s tudy (p < 0.01). S -FSH increased in both g roups after 12 m o n t h s (p < 0.01). A t 36 m o n t h s there were no s igni f icant d i f ferences be tween the groups in any o f the parameters . The resul ts are s u m m a r i z e d in T a b l e s 1 a n d 2.

BMD

M e a s u r e m e n t o f B M D us ing Q C T revealed no di f ference be- tween the groups . Wi th this m e t h o d B M D decreased in the treat- m e n t g roup at 36 m o n t h s (p < 0.05) and in both groups at 60 m o n t h s (p < 0.001). The minera l content o f the distal radius increased in bo th groups (p < 0.01) dur ing the 36 m o n t h s tudy period. B M D of the upper f emur did not change , bu t it was

decreased in the l umba r spine in both groups dur ing the exten- s ion o f the s tudy ( T a b l e s 3 a n d 4). Regard less o f the me th o d used to m e a s u r e bone m a s s there was no correlat ion be tween B M D and S-FSH or estradiol concentrat ion.

Discussion

Our me thods to s tudy B M D are val id and were used in previ- ous ly pub l i shed papers , In this s tudy, bone loss was not seen with D E X A dur ing the first 36 mon ths , bu t it was ev ident in the l umba r sp ine in both g roups after 48 months . Also, Q C T revealed s ignif icant (p < 0.05) loss o f bone in the l umba r region dur ing the ex tens ion o f the study. In the p rox imal f e m u r no bone loss was seen. This could be expla ined by the fact that the upper f em u r ( femoral co l lum) conta ins more cortical bone than the vertebrae. There is no clear-cut explana t ion for why B M D increased sig- nif icant ly (p < 0.01) in the distal radius in both groups. Perhaps this f ind ing resul ted f rom the rather h igh dose o f ca lc ium supple- menta t ion g iven to all subjects and the u n e v e n dis t r ibut ion o f ca lc ium in the skeleton. Prev ious ly it was shown that ca lc ium intake > 1000 m g / d a y is related to bone m a s s in p remenopause . 14 In another study, 5 h igh dietary ca lc ium intake was associa ted with increased bone m a s s at menopause . Subst i tu t ion o f 1000 m g ca lc ium dai ly in our s tudy for both groups m a y have hampe red smal l changes in B M D induced by calci tonin. The se rum levels o f ca lc ium increased s ignif icant ly in both the calci tonin and pla- cebo groups . Die tary ca l c ium supp l emen ta t i on in p o s t m e n o - pausa l w o m e n has been s h o w n to reduce bone loss. 15 B iochemi - cal da ta r evea led no acce le ra t ion in bone resorpt ion . 1,25-

Table 3. Bone mineral density of lumbar spine and forearm (QCT and SPA)

Method 0 months [n = (t)/(p) 60/60] 24 months [n = (t)/(p) 53/50] 36 months [n = (t)/(p) 50/46] 60 months [n = (t)/(p) 30/28] a

QCT (t) 230.0 -+ 42.3 229,2 _+ 43.4 225.7 _+ 45.4 b 220.9 _ 51.4 a QCT (p) 232.6 + 35.9 231.0 _+ 37.4 231.9 _ 36.7 219.3 _ 30.3 a SPADR (t) 257.9 + 18.0 266.2 _ 21.0 a 266.3 + 22.8 c 263.6 _ 48.6 a SPADR (p) 260.6 _ 25.9 264.0 _ 24.7 266.6 _+ 23.2 c 267.4 _+ 19.4 d SPARC (t) 0.88 _ 0.07 0.90 _ 0.07 0.89 _+ 0.06 0.87 _+ 0.05 SPARC (p) 0.89 - 0.08 0.91 +_ 0.07 0.89 -+ 0.07 0.88 _+ 0.05 SPAUC (t) 0.95 _ 0.09 0.95 __. 0.10 0.94 _ 0.09 0.89 + 0.06 a SPAUC (p) 0.94 _+ 0.08 0.94 _+ 0.08 0.92 _ 0.08 0.89 _ 0.08 a

(t) = treatment group; (p) = placebo group; QCT = BMD of lumbar spine (HU); SPADR = SPA distal radius (mg/cm3); SPAR = SPA cortical radius (g/cm2); SPAUC = SPA cortical ulna (g/cm2). aCalcitonin treatment after 36 months. Difference from baseline: bp < 0.05; Cp < 0.01; ap < 0.001.

632 I. A m a l a et al. Bone Vol. 4, No. 6 Calci tonin and bone loss at pe r imenopause June 1996 :629-632

Table 4. Bone mineral density of lumbar spine and upper femur (DEXA)

12 months 24 months 36 months 48 months 60 months Site [n = (t)/(p) 53/50] [n = (t)/(p) 46/45] In = (t)/(p) 36/37] [n = (t)/(p) 32/29] [n = (t)/(p) 26/20] ~

F e m u r (t) 0.94 ± 0.11 0.93 + 0.10 0.92 _+ 0.11 0.92 _+ 0.11 0.92 ± 0.11 Femur (p) 0.94 ___ 0.09 0.96 ___ 0.09 0.95 ___ 0.09 0.94 ± 0.09 0.92 _ 0.11 Spine (t) 1.18 ± 0.16 1.18 ± 0.15 1.15 _+ 0.15 1.15 ± 0.16 b 1.14 ± 0.17 c Spine (p) 1.20 ± 0 .14 1.19 ± 0.15 1.19 ± 0.14 1.15 ± 0.14 c 1.12 _+ 0.17 c

(t) = treatment group; (p) = placebo group; Femur = BMD of femoral col lum (g/cm2); Spine = BMD of lumbar vertebra L2-4 (g/cm2). ~Calcitonin treatment after 36 months. Difference from baseline: bp < 0.05; Cp < 0.01.

dihydroxycholecalciferol decreased significantly (p < 0.001) in both groups beginning at 12 months. The levels of circulating active metabolites of vitamin D are known to decrease after menopause. 3 This may at least partially explain the low vitamin D concentrations in this study after 1 year.

Calcitonin has been used effectively in the treatment of es- tablished osteoporosis.~l'12 To date, there have been no publica- tions of the effect of calcitonin on bone metabolism at perimeno- pause. It would be ideal to protect bone from accelerated resorp- tion at the point when bone loss begins especially if the peak bone mass is already low. When treatment should start is diffi- cult, because in some patients irregularities in menstruation and hormonal imbalance can exist for an extended period of time. In this study, the levels of sex hormones varied to a large extent among subjects, although there was no difference in hormonal status between the groups at enrollment or during the study. The estradiol levels decreased significantly in the calcitonin group toward the end of the study. The negative result of the study can be explained by too early an intervention for some of the women, because of the ongoing physiologically effective production of estrogen. Calcitonin may not modify bone metabolism under conditions when sex hormone production is normal. According to a previous publication, the daily dose of calcitonin should have been sufficiently high. ~6 The development of secondary resis- tance to calcitonin may play a role in continuous treatment, and perhaps this effect could have been avoided in this study by using cyclical administration. 4 This could have been an inhibitory fac- tor in the effect of calcitonin toward the end of the study, when bone loss was accelerating. A previous study 17 showed that women in the early postmenopausal state responded well to in- termittent treatment with doses as low as 50 IU. In a prospective study ~8 it was demonstrated that intermittent treatment with salmon calcitonin 100 IU/day for 10 days per month increased total body bone mineral content.

Exercise is beneficial in preserving bone mass and restoring muscle strength. 7 In our study, the subjects' level of physical activity was normal, and their lifestyle was active. There was no difference in exercise habits between the groups. During the study period no fractures were recorded in the groups.

In conclusion, nasal salmon calcitonin 100 IU daily has no protective effect on bone mass and does not modify bone me- tabolism at perimenopause.

Acknowledgment: This s tudy was f inancial ly supported by Sandoz, Hel- sinki, Finland.

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4. Gennari, C., Agnusdei, D., and Camporeale, A. Long-term treatment with calcitonin in osteoporosis. Horm Metab Res 25:484-485; 1993.

5. Hansen, M. A., Overgaard, K., Riis, B. J., and Cristiansen, C. Potential risk factors for development of postmenopansal osteoporosis---examined over 12- year period. Osteopor Int 1:95-102; 1991.

6. H~m~i, M., Karjalalnen, P., Hoikka, V., and Alhava, E. Bone density in women with spinal and hip fractures. Acta Orthop Scand 56:380-385; 1985.

7. Jangson, B., Ringsberg, K., Josefsson, P. O., Johnell, O., and Birch-Jensen, M. Effects of physical activity on bone mineral content and muscle strength in women: A cross-sectional study. Bone 13:191-195; 1992.

8. Kanis, J. A., Johnell, O., Gullberg, B., Allander, E., Dilsen, G., Genoari, C., Lopes-Vaz, A. A., Lyritis, G. P., Mazzuoli, G., and Miravet, L., et al. Evidence for efficacy of drugs affecting bone metabolism in preventing hip fractures. BMJ 305:t124-1128; 1992.

9. Karjalalnen, P. A method for determination of the mineral content and mineral density of the distal radius using gamma ray attenuation. Ann Clin Res 5:231- 237; 1973.

10. Mazzuoli, G. F., Passeri, M., Gennari, C., Minisola, S., Antonelli, R., Valtorte, C., Palumeri, E., Cervellin, G. F., and Francinin, G. Effects of salmon calci- tonin in postmenopausal osteoporosis: A controlled double-blind study. Calcif Tissue Int 38:3-8; 1986.

11. Mclntyre, I., Whitehead, M. I., Banks, L. M., Stevenson, J. C., Wimalawansa, S. A., and Healy, M. J. R. Calcitonin for prevention of postmenopausal bone loss. Lancet i:900-901; 1988.

12. Overgaard, K., Hansen, M. A., Jensen, S. B., and Christiansen, C. Effect of salcatonin given intranasally on bone mass and fracture rates in established osteoporosis: A dose-response study. BMJ 305:556-561; 1992.

13. Overgaard, K., Riis, B. J., Christiansen, C., and Hansen, M. A. Effect of sal- catonin given intranasally on early postmenopausal bone toss. BMJ 299:477- 479; 1989.

14. Picard, D., Ste-Marie, L. G., Coutu, D., Carrier, L., Chartrand, R., Lepage, R., Fugere, P., and D'Amour, P. Premenopausal bone mineral content relates to height, weight and calcium intake during early adulthood. Bone Miner 4:299- 309; 1988.

t5. Prince, R. The calcium controversy revisited: Implications of new data. Med J Aust 159:404-407; 1993.

16. Reginster, J. Y. Calcitonin for prevention and treatment of osteoporosis. Am J Med 95:44S-47S; 1993.

17. Reginster, J. Y., Meurmans, L., Deroisy, L., Jupsin, I., Biquet, 1., Albert, A., and Franchimont, P. A 5-year controlled randomized study of prevention of postmenopausal trabecular bone loss with nasal salmon catcitonin and calcium. Eur J Clin Invest 24:565-569; 1994.

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Date Received: November 8, 1995 Date Revised: February 6, 1996 Date Accepted: March 5, 1996