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Lack of changes in histomorphometric, bone mass and biochemical parameters in ovariohysterectomized dogs
8756-3282192 $5.00 + .OO Copyright 0 1992 Pergamon Press Ltd.
Bone, 13, 311-316, (1992) Printed in the USA. All rights reserved.
Lack of Changes in Histomorphometric, Bone Mass, and Biochemical Parameters in Ovariohysterectomized Dogs V. SHEN, D. W. DEMPSTER,‘,’ R. LINDSAY’.’
R. BIRCHMAN,’
R. W. E. MELLISH,’
E. CHURCH,3
D. KOHN3 and
’ Regional Bone Center, Helen Hayes Hospital, New York Stare Department of Health, W. Haverstraw, NY, U.S.A. ’ Departments of Medicine and Pathology and 3 Institute of Comparative Medicine, Columbia University, New York, NY, U.S.A. Address for correspondence and reprints: V. Shen, Ph.D., U.S.A.
Regional Bone Center, Helen Hayes Hospital,
Abstract
Rt. 9W, W. Haverstraw,
NY 10993,
of this process. An animal model can be employed to study the cellular mechanisms of bone loss, as well as to evaluate the potential therapeutic value of pharmaceutical agents where it is inappropriate or impractical to do so in humans. Only a few primate species experience a natural menopause comparable to that in humans (Martin et al. 1987). However, their experimental use is generally cost prohibitive. In other species, one may surgically create ovarian dysfunction. Ovariectomized rats have been proposed as a model (Wronski et al. 1985), but rat bones continue to grow during most of the animal’s life, and they do not exhibit the same remodeling process as humans. Dogs undergo a bone remodeling process resembling that in humans in situations of age-related (Jee et al. 1970; Martin et al. 1981; Williams et al. 1982) or disuse-related (Uhthoff & Jaworski 1978; Jaworski et al. 1980) bone loss. Several studies have evaluated the effects of ovariectomy on histomorphometric variables in cancellous and cortical bone in the dog. The results appear to be heterogeneous (Snow et al. 1984; Malluche et al. 1986; Dannucci et al. 1987; Martin et al. 1987; Boyce et al. 1990). There are no data on the effects of altering the PTH/vitamin D axis in ovariohysterectornized dogs, and basic biochemical data are also limited. In this study, we have combined several techniques to ascertain whether the dog can be useful for the study of ovariandysfunction induced osteopenia.
A predictable animal model with skeletal remodeling characteristics similar to those of humans is needed to facilitate the understanding of the mechanism of postmenopausal osteoporosis. We have utilized the ovariohysterectomized (Ovh) dog to examine cellular and biochemical responses to estrogen depletion and PTH stimulation. Histomorphometric measurements of bone biopsies taken prior to (first biopsy) and five months after the operation (second biopsy) showed no significant differences in static and dynamic parameters. Bone mineral density of the excised vertebrae displayed the same values between the two groups six months after surgery. Between the second biopsy and sacrifice, two infusion studies were performed. A two-hour infusion of EDTA followed by a two-hour recovery period elicited a rapid response in PTH production, highly correlated to the changes in ionized calcium, but no significant difference in response was observed between Sham and Ovh groups. A short-term (24-h) infusion of l-34 hPTH increased circulating ionized calcium and 1,25-(OH),-D levels to a similar extent in both groups. The levels of alkaline phosphatase were constant and both groups showed a small but nonsignificant increase in osteocalcin. The lack of sizable responses in histomorphometric, bone mass, and biochemical parameters may limit the utility of dogs for the study of cancellous bone loss in ovarian-dysfunction osteoporosis.
Materials and Methods Animal husbandry
Key Words:
Ovariohysterectomy-Osteopenia-PTH-Bone histomorphometry .
and surgical procedures
Eight four-year-old female breeder beagle dogs were purchased from Laboratory Research Enterprises, Kalamazoo, MI. They were housed and cared for at the AAALAC accredited animal facility of the Institute of Comparative Medicine, Columbia University. Animal protocols used in this study were approved by the IACUCs of Columbia University and Helen Hayes Hospital. The beagles were penned in pairs and were given Purina Dog Chow and tap water ad libitum. Four beagles were Shamoperated and four beagles were subjected to ovariohysterectomy. The dogs were grouped by matching initial body weight. Prior to this procedure and the bone biopsies, the dogs were premedicated with 0.05 mg/kg of atropine and 0.02 mg/kg of ace. promazme l.m., with anesthesia induced by 17 mglkg of thiopental administered iv., and maintained with 1.5% isofloranel 0,. At the same time as the surgery was performed, the first
Introduction
The changes in skeletal remodeling that result from ovarian dysfunction in women after menopause or oophorectomy are well studied, but are far from being completely understood. Histomorphometry (Parfitt et al. 1980, 1983; Steiniche et al. 1989), biochemistry (Christiansen et al. 1982), calcium kinetics (Reeker et al. 1977) and dynamic testing of skeletal, and parathyroid gland and renal function (Duda et al. 1987; Tsai et al. 1989; Sorensen et al. 1982) have been used to facilitate the understanding of the underlying mechanisms of bone loss in humans. However, a predictable and reproducible animal model of ovariandysfunction bone loss would greatly enhance our understanding 311
312
bone biopsies were obtained from a predetermined position of the dorsal surface of the iliac crest shown to present minimal structural variation in the dog (Hardt & Jee 1982). Two left and two right biopsies were obtained from each of the two groups (Sham and Ovh). The second bone biopsy was taken five months later from the side opposite the first biopsy, at a position on the iliac crest comparable to that of the first biopsy. The biopsies were taken with a Bordier trephine (7 mm diameter). The selection of the position was aided by the use of a fluoroscope. Double tetracycline labels for the first biopsy were administered by i.v. injections of 20 mg/kg of declomycin with a schedule of I day on, 13 days off, 1 day on, 11 days off. prior to the biopsy. Prior to the second biopsy, the animals were labeled by i.v. administration of achromycin (20 mg/kg) with a labeling schedule of 1 day on, 14 days off, I day on, 14 days off. Histomorphometty The biopsies were immediately fixed in 70% alcohol and processed, undecalcified, following the standard dehydration and embedding protocols used in our laboratory (Parisien et al. 1990). Seven-pm-thick sections were cut with a Jung model K microtome (Reichert-Jung, Heidelberg, Germany) and stained with Goldner’s trichrome for analysis of static parameters. Sections were obtained from four different planes of the biopsy. Each plane was separated from the other by approximately 350 pm. One section from each of the four planes was used for the measurements. The average number of fields of measurement on each section was 7.83 (8.11 mm’). To avoid subcortical bone, the distance at the base of the thickes: trabecula emanating from the cortex on each side was measured. Subcortical bone with a distance equal to twice this value was excluded. Measurements of static variables were performed by the point counting method using a Merz grid at a total magnification of X 125, with the exception of cancellous bone volume, which was made using a Zeiss Integrations II grid at the same magnification. The coefficient of intra- and interobserver variations for cancellous bone volume were 2.4% and 3.6%, respectively. Ten-pm sections were mounted unstained for assessment of dynamic parameters by fluorescence microscopy (Meunier et al. 1980). The distance between the two labels was measured using a semi-automated image analyzer (Optomax VIDS IV, Optomax Inc., Hollis, NH). The mineral apposition rate (MAR) was calculated by dividing the distance between double labels by the number of days between the two labels and the values expressed in micrometers per day. Mineralizing surfaces were defined as the proportion of trabecular surface exhibiting single and double tetracycline labels; these labels were measured separately and expressed as single-labeled surfaces (sLS/BS), double-labeled surfaces (dLS/ BS), and double-plus single-labeled surfaces (TLS/BS). TLS/BS was used for the calculation of the bone formation rate at the tissue level (BFRIBS) and the adjusted appositional rate (Aj.AR); these values were defined as the volume of mineralized bone formed per unit bone surface and per unit osteoid surface/ day, respectively. EDTA and PTH infusion studies Two weeks after the second biopsy, each beagle received 25 mg/kg of Na, . EDTA in 125 ml of saline with 5% dextrose plus 5 ml of lignocaine solution over a two-hour period by i.v. infusion. The beagles were individually caged without restraint. Infusion was stopped after two hours. Blood samples were taken at 0,60, 120, 180, and 240 min from the beginning of the infusion. The procedure is similar to the one previously used by Torrance
V. Shen et al.: Effects of ovariohysterectomy on bone metabolism in dogs and Nachreiner (1989) on normal dogs. Ionized calcium and intact PTH were measured in the serum samples. Two weeks after the EDTA infusion, a PTH infusion was performed, with all the beagles in individual cages without restraint. The procedure is similar to that used in human studies in our laboratory (Cosman et al. 1990). Briefly, the beagles received 0.55 U/kg of human l-34 PTH fragment dissolved in 120 ml of saline over a 24-hour period by i.v. infusion via a syringe pump. Blood samples were collected at 0, 4, 8, 12, 16, 20, and 24 hours after the initiation of the PTH infusion. Measurements included ionized calcium, phosphorus, osteocalcin, N-terminal PTH, intact PTH, 25-(OH)-D, and l,25-(OH),-D. Sacriffce and bone mineral density measurements Two weeks after the PTH infusion studies, the animals were euthanized by administration of pentobarbital (60 mglkg) i.v., and the pelvis and lower vertebrae were dissected out and preserved in 70% alcohol. The bone mineral density of the L,-L, lumbar vertebrae was determined using a dual energy X-ray absorptiometer (QDR-1000, Hologic, Waltham, MA) under 50 cm of 70% alcohol in a glass container. Measurements
of biochemical parameters
Serum calcium, phosphorus, creatinine, and alkaline phosphatase were measured by routine calorimetric methods. Serum ionized calcium was analyzed with a Nova 8 ionized calcium analyzer (Nova Biomedical, Waltham, MA). Serum osteocalcin was measured by radioimmunoassay specific for canine osteocalcin. The purified canine osteocalcin and diluted antibody were generously provided to us by Dr. Barbara Miller of Norwich Eaton Pharmaceutical Co. Briefly, canine osteocalcin was purified from canine femurs, labeled by the standard chloramine T method, and the labeled ligand separated by G-25 column chromatography in assay buffer, 0.01 M Tris, 0.14 M NaCl 25 mM EDTA, 0.25% RIA grade bovine serum albumin, and 0.1% Tween 20, pH 7.4. After incubating the standards or canine serum with anti-canine osteocalcin antisera, raised in rabbit, for 22 hours at 4” C, the antigen-antibody complex was precipitated with goat anti-rabbit gamma globulin. The concentration of osteocalcin in the serum was determined by comparison to the standard curve. The nonspecific binding was determined by using non-immune rabbit serum. Intact PTH in the serum was determined by a radioimmunometric assay of intact human PTH (Nichols Institute, San Juan Capistrano, CA). The feasibility of using this human assay kit for dogs has been previously documented (Torrance & Nachreiner 1989). Radioimmunoassay of N-terminal PTH molecules was performed using antibody, standards, and procedure kindly provided to us by Dr. G. Segre, Massachusetts General Hospital, Boston, MA (Segre 1983). Vitamin D metabolites were separated after organic solvent extraction of serum and sequential chromatography on Cl8 Sep-Pak cartridges, and further purified on silica Sep-Pak cartridges, as outlined previously (Reinhardt et al. 1984). 25-(OH)-D was assayed by a competitive protein binding method (Preece et al. 1974) using a highly diluted vitamin D depleted rat serum. 1,25(OH),-D was measured by a radioreceptor assay that uses a 1,25-(OH),-D receptor protein isolated from calf thymus (Incstar Corp., Stillwater, MN), by the procedure of Reinhardt et al. (1984). Statistical analysis Data are expressed as mean 2 standard error. Comparison between means was performed using Student’s t test. Power anal-
V. Shen et al.: Effects of ovariohysterectomy on bone metabolism in dogs
313 PTH infusion
ysis of the differences in cancellous bone volume measurements before and after the operation on the same animal in Ovh groups was performed using the formula in Duncan et al. (1983). The estimation of the power was obtained by altering one variable at a time in the calculation, either an assumed 20% decrement from the mean in the pre-operation measurements in cancellous bone volume or a doubling of the sample size. The variance was assumed to be constant in all calculations.
A 24-hour infusion of hF’TH (l-34), at a dose of 0.55 U/kg/h, which is identical to the dose we administer in our human studies (Cosman et al. 1990), was performed on each of the animals (Table III). Although there were differences between Sham and Ovh groups in some of the variables measured in the basal state, the differences were not statistically significant. Ionized calcium showed a steady increase throughout the infusion period from 1.33to 1.81 mMintheshamgroupand 1.3Oto 1.76mMinthe Ovh group. After 8 hours, the level of ionized calcium was significantly higher than the basal level for both groups. N-terminal PTH, which measures both endogenous N-terminal F’TH fragments and exogenously infused l-34 hPTH, showed a lo- to 20-fold increase over basal levels at the first sampling point (4 h) in both groups. Intact FTH, which measures endogenous intact FTH only, decreased rapidly in both groups to below detectable limits after 8 hours. Samples collected from both groups after 16 hours showed a small increase in osteocalcin levels (11 vs. 1315 ng/ml), although the differences were not significantly different from the basal level. Alkaline phosphatase activity, phosphorus, and levels of 25-(OH)-D remained constant throughout the infusion period. 1,25-(OH&-D increased significantly after 12 hours from 44 to 57 pg/mI (p < .05) in Sham and 43 to 63 pg/ml @ < .05) in Ovh groups. There were very few differences between Sham and Ovh groups at any of the time points, except 1,25-(OH),-D at 16 hours (56 vs 69 pg/mI in Sham and Ovh, p < .02) and phosphorus at 24 hours (5.6 mg% in Sham and 4.3 mg% in Ovh, p < .02).
Results Histomorphometric
parameters
and body weight of Sham and
Ovh beagles
Body weight was monitored in each animal prior to the biopsy. Ovh animals increased their body weight significantly @ < .Ol) over the course of the study, while the Sham group demonstrated a nonsignificant increase in body weight (Table I). Measurements of cancellous bone volume, osteoid surface, single-, double-, and total-labeled surface, bone formation rate, and adjusted apposition rate in Sham and Ovh animals showed no differences before or after the operation, either between or within groups (Table I). There was a significant increase @ < .05) in mineral apposition rate in Ovh animals compared with Sham animals five months after the operation. However, this difference may be the result of an inherently higher mineralization rate in the Ovh animals as their preoperative values were also higher when compared to Sham animals. EDTA infusion
Bone mineral density measurements
Each animal was infused with 25 mglkg of Na, . EDTA over a two-hour time period and then allowed to recover. Ionized calcium and intact PTH levels were measured at 0, 1,2,3,4, hours after beginning the infusion (Table II). EDTA infusion decreased the levels of ionized calcium by approximately 10% within the first hour. Endogenous intact F’TH responded vigorously to the lowered ionized calcium level with an approximately four-fold increase. Ionized calcium recovered rapidly to preinfusion levels two hours after the termination of EDTA infusion. Intact FTH levels decreased when the EDTA infusion was terminated, but were not reduced to basal levels. There was a somewhat greater response in PTH production in the Ovh group than the Sham group, but the difference did not reach statistical significance.
Excised lumbar vertebrae were subjected to bone mineral density measurements after termination of the experiment. There were no statistically significant differences in bone mineral density between Sham and Ovh animals (Table IV). The mean values in the Ovh group were 4%) 4%) 1% , and - 3% different from those in the Sham group in lumbar vertebrae L,, L,, L4, and L,, respectively. Discussion There have been few studies examining the effects of ovarian dysfunction on bone and calcium metabolism in dogs. In the present study we have compared serum biochemistries, both
Table I. Histomorphometric parameters and body weight of Sham-operated and ovariohysterectomized beagles
Ovh
Sham Pre BV/TV (a) OSlBS (%) SLS (%,) DLS (%) TLS (%) MAR (*m/d) BFR (~m3/~m*ld) (tissue level surface ref.) BFR (p,m?pm’/d) (tissue level volume ref.) Aj.AR (pm3/pm2/d) Body wt (kg)
23.96 23.49 16.98 7.21 24.25 0.6975
? 2 2 2 + +
Post 2.59 8.69 3.46 2.41 5.30 0.0347
22.42 23.70 16.04 10.54 26.58 0.6926
2 2 ? ? 2 2
Pre 2.46 4.96 1.67 3.02 2.58 0.0242
23.18 26.08 15.63 9.12 24.74 0.8250
2 ? 2 2 -c *
Post 3.38 7.68 3.37 2.83 6.34 0.0933
24.06 24.80 15.46 8.89 24.35 0.8400
‘2 -c ” ” ?a
2.55 2.85 1.98 1.15 2.36 0.0536
0.1706 2 0.0448
0.1681 + 0.0222
0.2184 t 0.0795
0.2033 + 0.0188
1.3274 ? 0.3601 0.8934 2 0.1577 12.45 4 0.59
1.3228 2 0.1833 0.7618 -+ 0.0923 14.95 ” 1.32
1.8070 f 0.6934 0.8679 ? 0.2030 12.55 2 0.33
1.5644 h 0.2074 0.8515 -c 0.1157 17.45 t 0.78b
Results are expressed as mean ‘r SEM. “different from Sham, post-op; p < .05. bdifferent from Ovh, pre-op; p < .OI.
V Shen et al.: Effects of ovariohysterectomy
314 Table II. Biochemical
parameters
Sham Ovh
A EDTA Ih
1.44 2 0.03 1.36 2 0.02
Sham Ovh
18.9 12.4
+ 4.4 i 3.8
in dogs
in EDTA Infusion experiment Sampling
Oh
on bone metabolism
1.32 2 I.28 + 69.7 54.9
time
I
- EDTA
2h
3h
Ionized Calcium (mM) 0.07 1.25 2 0.07” 0.03” I. I8 t 0.02”
t 19.6” 2 11.5”
4h
1.35 t 1.36 2
Intact PTH (pg/ml) 74.9 t 24.2 144.0 + 27.8”
74.9 172.2
0.03 0.02
1.41 t 1.35 +
2 18.7” ? 74.1
36.5 72.8
0.07 0.01
t 17.3 t 37.3
Results are expressed as mean t SEM. “Different from 0 hr sample, p < ,005.
static and kinetic responses to PTH and EDTA, bone histomorphometry, and bone mass measurements in Sham-operated and Ovh dogs. Our results support the premise that the dog may have limited utility for the study of cancellous bone loss in ovariandysfunction osteoporosis. The effects of ovariectomy or ovariohysterectomy on cancellous bone in the beagle dog are controversial. Cancellous bone volume was shown to be either decreased (Faugere et al. 1990: Malluche et al. 1986; Martin et al. 1987) or unchanged (Danucci et al. 1987; Snow et al. 1986; Boyce et al. 1990; our findings). Changes in biochemical and other histomorphometric data also differ in each study. Using sequential biopsies during the first
Table III.
Biochemical
Sampling time
parameters
Sham Ovh
in FTH infusion experiment
4h
Oh
1.33 ” 0.03 1.30 ir 0.03
Sham Ovh
26 II
18 -i-5
Sham Ovh
31 18
29 +
Sham Ovh
11.0 11.0
Sham Ovh
1.40 t 1.30 ? 209 210
I2 h
8h
0.03 0.03
f 41‘ _t 40
1.54 t 1.42 +
1I5 122
I6 h
Ionized calcium (mM) 0.04’ 1.60 ? 0.03” 0.03d I.53 * 0.04
f 46 ? 64
N-terminal PTH (pgiml) IS5 !Z 56 I29 2 34*
145 I43
+ 5
1” Ih
Intact PTH (pgiml) 3 r I” 4 t Ob
4 5
2.1 3.8
II.8 8.7
+ 2
1.6 2.3
Osteocalcin (ng!ml) 12.5 i- 2.0 10.3 -+ 1.7
13.8 13.0
3.5 8.3
16.1 21.1
Alkaline phosphatase (IU!L) t 6.2 17.9 + 4.9 ? II.3 20.6 ? IO.6
i3 i-6
36 47
-+6 + IO
~3 t II
50 53
Z ?
2 7
+ *
0.3 0.3
r7 t3d
2 2.0 t 1.7
10.3 10.3
? ?
17.5 21.1
* 3.1 ‘- 4.4
16.7 20.0
? 2
Sham Ovh
44 48
-t5 _t7
36 43
Sham Ovh
44 43
*4 56
42 43
25 (0H)D (@ml) 36 -t 7 49 r 15
20 h
1.73 + 0.03” 1.61 _f 0.06d
4 3
15 7
1
four months after surgery, two reports (Faugere et al. 1990; Malluche et al. 1986) showed a small but rapid loss of cancellous bone volume. They found no change in bone resorption indices at any dme. but the tetracycline-labeled surface and bone formation rate were reduced. They deduced that the loss of bone must have resulted from an early hyper-resorption within the first month, followed by maintenance of low bone mass ascribable to osteoblast insufficiency. Another report showed a significant 15% trabecular bone loss in the spine and an associated high turnover phenomenon, accompanied by increased serum osteocalcin (Martin et al. 1987). Danucci’s observations of increased osteoid surface, resorption surface, bone formation rate, and
? 62 ? 4@
1.78 * 1.69 ? 207 I85
24 h
0.03” O.Ogd
2 43’ + 65d
1.81 t 1.76 +-
0.03” 0.11”
I85 250
? -t
32‘ 10”
3 4
‘t
I” Ih
5 4
2 +
2’1 Ih
* I.5 IT 4.0
13.0 15.0
2 5
I.0 6.0
15.0 14.7
t -+
I.8 5.9
16.6 20.6
? 4.1 + 12.2
17.3 21.1
t 4.5 + 12.0
17.5 20.6
* i
5.1 II.4
36 43
t8 t 15
42 46
25 * 10
38 49
t +
3 10
56 69
t I?
I Id.’
63 66
_t 5
4d 9
65 68
I 2
5d 5d
i i
0.3 0.5
t t
0.3 0.2
-t +
0.2 0.2’
f t
Id I’
I ,2XOH),D (pg/ml)
Sham Ovh
4.9 4.3
All results are “different from bdifferent from ‘different from ddifferent from ‘different from
? 0.2 ? 0.2
4.9 5.3
expressed as mean time 0 hr sample, time 0 hr sample, time 0 hr sample, time 0 hr sample, control sample, p
2 p p p p <
? 2
0.3 0.5
SEM. < .0001 < .OOI. < .Ol < .OS. .02.
4.8 4.3
57 63
? i
2d 1”
Phosphorus (mg %) 4.9 * 0.2 4.5 i_ 0.2
4.6 4.9
3
4.9 4.2
5.6 4.3
V. Shen et al.: Effects of ovariohysterectomy
on bone metabolism
in dogs
Table IV. Bone mineral density measurements
315 in ovariohysterectomized
dogs (gm/cm2)
Lumbar vertebrae
Sham Ovh
L,
L,
L,
L,
0.587 2 0.014 0.613 * 0.005
0.578 ” 0.020 0.601 ? 0.006
0.578 + 0.017 0.585 + 0.010
0.549 r 0.021 0.531 2 0.013
Results are expressed as mean 2 SEM. Excised lumbar vertebrae were used for the measurements.
labeled surface are in agreement with high turnover. However, they failed to observe a significant decrease in cancellous bone volume six months after surgery (Danucci et al. 1987). A recent report (Boyce et al. 1990) made a more detailed examination of the histomorphometric changes at I, 3, 6, and 10 months after surgery. At one to three months post-operation, they observed a transient increase in mineralizing surface and bone formation rate; at six months post-op the values were depressed below the pre-surgery levels; and at 10 months post-op the levels returned to pre-surgery values. No change in cancellous bone volume was detected at any of the time points, perhaps because of the brief nature of the changes in bone remodeling. It is noteworthy that our second biopsy falls exactly between the increased bone formation rate at three months and decreased bone formation rate at six months, as seen previously (Boyce et al. 1990), which may explain why we did not observe any changes in remodeling parameters. In contrast to many metabolic diseases, the mean values for biochemical parameters may deviate slightly, but generally remain within the normal range in osteoporosis. There is a large overlap of individual values for all indices (Meunier et al. 1980; Parfitt et al. 1980; Whyte et al. 1982). perhaps because of the large age range of the study populations or the heterogeneous temporal variation in the disease process. Biochemical indices in early and late stages of estrogen deficiency do differ. A stimulation test for skeletal and renal products might provide greater discrimination between normal and osteoporotic subjects than is possible with static measurements of circulating levels (Tsai et al. 1989). We examined the response of the parathyroid gland to fluctuations of serum calcium using a short-term EDTA infusion. The PTH concentration was highly correlated with the level of circulating ionized calcium (r = 53, p < .Ol, data not shown), but there was no difference in responsivity between Ovh and Sham-operated animals. It has been suggested that osteoporotic women and ovariohysterectomized rats have an increased sensitivity to PTH (Heaney 1965; Jasani et al. 1965; Orimo 1972). In our PTH stimulation experiment, the levels of N-PTH increased rapidly during the infusion (lo- to 20-fold in the first four hours), and although suppression of endogenous PTH production by exogenously infused l-34 hPTH was highly significant, few differences were observed between the two groups. PTH infusion, as expected, stimulated renal production of 1,25-(OH),-D, but again there was no difference between the two groups except for a minor difference at one of the time points, in contrast to our experience in humans (Cosman et al. 1990). Alkaline phosphatase levels were not altered, but osteocalcin, a more specific bone formation marker, showed a small but nonsignificant increase at the later time points. This increase could be the result of increased synthesis of 1,25-(OH),-D in the kidney, which is known to stimulate osteocalcin synthesis (Price et al. 1980; Gundberg et al. 1983). Although a hormonal challenge approach did not provide us with additional information, more sensitive and discriminating challenges may not yield useful results in the future.
There are several reasons why we may not have detected any differences between Sham-operated dogs and those that underwent ovariohysterectomy: (a) estrogen may not play an important role in skeletal metabolism because of the relatively low levels of estrogen and the semiannual estrus cycle of the dog (Fox & Laird 1970); (b) the small sample size and limited diameter of the pelvis in the dog may not have provided enough statistical power to estimate the differences, in light of the highly varied individual values in these animals. However, based on present data, we should have been able to detect a 20% difference in cancellous bone volume with 75% power. An increase of sample size to twice that used in the experiment would only yield a 10% increase in power, and (c) it is possible that current methods for determining acme changes of bone turnover are not sensitive enough to detect small differences in biochemical and histomorphometric parameters. Using dual-energy X-radiography, a method with better reproducibility than histomorphometry, a recent report showed a significant change in bone density between oophorectomized and control dogs (Drezner & Nesbitt 1990). However, using the same techniques (with a coefficient of variation of 1% with the ex vivo bone samples), we could not detect any differences in bone density between the two groups six months after operation. There are several ways in which one could improve the chances of detecting bone loss due to ovarian dysfunction in dogs. One may perform the ovariohysterectomy at a specific time point in the estrus cycle, and limit calcium intake and weight gain. Extra adipose tissue could increase conversion of androstenedione to estrone (Casey et al. 1983) to compensate for the small loss in estradiol and estrone. A vigorous weight control program for Ovh animals may be needed, although it has been shown that weight gain only partially prevents bone loss in ovarectomized rats and that ovariectomy causes marked bone loss regardless of body weight (Wronski et al. 1987). High calcium intake in the routine laboratory diet (1.1%) may mask the small changes in skeletal metabolism. Although our results and others (Dannucci et al. 1987; Snow et al. 1986; Boyce et al. 1990) suggest limited use of ovariectomized dogs for the study of cancellous bone loss in estrogen-depleted dogs, a study of cortical bone remodeling may yet provide useful information, as suggested by the recent comprehensive review of all the available oophorectomized dog data (Kimmel 1992).
The study was supported by NIH grants AR39191 and AR35647. The authors acknowledge the outstanding technical assistance of Wendy Herbert and Michele Schnitzer.
Acknowledgments:
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Bone, 13, 311-316, (1992) Printed in the USA. All rights reserved.
Lack of Changes in Histomorphometric, Bone Mass, and Biochemical Parameters in Ovariohysterectomized Dogs V. SHEN, D. W. DEMPSTER,‘,’ R. LINDSAY’.’
R. BIRCHMAN,’
R. W. E. MELLISH,’
E. CHURCH,3
D. KOHN3 and
’ Regional Bone Center, Helen Hayes Hospital, New York Stare Department of Health, W. Haverstraw, NY, U.S.A. ’ Departments of Medicine and Pathology and 3 Institute of Comparative Medicine, Columbia University, New York, NY, U.S.A. Address for correspondence and reprints: V. Shen, Ph.D., U.S.A.
Regional Bone Center, Helen Hayes Hospital,
Abstract
Rt. 9W, W. Haverstraw,
NY 10993,
of this process. An animal model can be employed to study the cellular mechanisms of bone loss, as well as to evaluate the potential therapeutic value of pharmaceutical agents where it is inappropriate or impractical to do so in humans. Only a few primate species experience a natural menopause comparable to that in humans (Martin et al. 1987). However, their experimental use is generally cost prohibitive. In other species, one may surgically create ovarian dysfunction. Ovariectomized rats have been proposed as a model (Wronski et al. 1985), but rat bones continue to grow during most of the animal’s life, and they do not exhibit the same remodeling process as humans. Dogs undergo a bone remodeling process resembling that in humans in situations of age-related (Jee et al. 1970; Martin et al. 1981; Williams et al. 1982) or disuse-related (Uhthoff & Jaworski 1978; Jaworski et al. 1980) bone loss. Several studies have evaluated the effects of ovariectomy on histomorphometric variables in cancellous and cortical bone in the dog. The results appear to be heterogeneous (Snow et al. 1984; Malluche et al. 1986; Dannucci et al. 1987; Martin et al. 1987; Boyce et al. 1990). There are no data on the effects of altering the PTH/vitamin D axis in ovariohysterectornized dogs, and basic biochemical data are also limited. In this study, we have combined several techniques to ascertain whether the dog can be useful for the study of ovariandysfunction induced osteopenia.
A predictable animal model with skeletal remodeling characteristics similar to those of humans is needed to facilitate the understanding of the mechanism of postmenopausal osteoporosis. We have utilized the ovariohysterectomized (Ovh) dog to examine cellular and biochemical responses to estrogen depletion and PTH stimulation. Histomorphometric measurements of bone biopsies taken prior to (first biopsy) and five months after the operation (second biopsy) showed no significant differences in static and dynamic parameters. Bone mineral density of the excised vertebrae displayed the same values between the two groups six months after surgery. Between the second biopsy and sacrifice, two infusion studies were performed. A two-hour infusion of EDTA followed by a two-hour recovery period elicited a rapid response in PTH production, highly correlated to the changes in ionized calcium, but no significant difference in response was observed between Sham and Ovh groups. A short-term (24-h) infusion of l-34 hPTH increased circulating ionized calcium and 1,25-(OH),-D levels to a similar extent in both groups. The levels of alkaline phosphatase were constant and both groups showed a small but nonsignificant increase in osteocalcin. The lack of sizable responses in histomorphometric, bone mass, and biochemical parameters may limit the utility of dogs for the study of cancellous bone loss in ovarian-dysfunction osteoporosis.
Materials and Methods Animal husbandry
Key Words:
Ovariohysterectomy-Osteopenia-PTH-Bone histomorphometry .
and surgical procedures
Eight four-year-old female breeder beagle dogs were purchased from Laboratory Research Enterprises, Kalamazoo, MI. They were housed and cared for at the AAALAC accredited animal facility of the Institute of Comparative Medicine, Columbia University. Animal protocols used in this study were approved by the IACUCs of Columbia University and Helen Hayes Hospital. The beagles were penned in pairs and were given Purina Dog Chow and tap water ad libitum. Four beagles were Shamoperated and four beagles were subjected to ovariohysterectomy. The dogs were grouped by matching initial body weight. Prior to this procedure and the bone biopsies, the dogs were premedicated with 0.05 mg/kg of atropine and 0.02 mg/kg of ace. promazme l.m., with anesthesia induced by 17 mglkg of thiopental administered iv., and maintained with 1.5% isofloranel 0,. At the same time as the surgery was performed, the first
Introduction
The changes in skeletal remodeling that result from ovarian dysfunction in women after menopause or oophorectomy are well studied, but are far from being completely understood. Histomorphometry (Parfitt et al. 1980, 1983; Steiniche et al. 1989), biochemistry (Christiansen et al. 1982), calcium kinetics (Reeker et al. 1977) and dynamic testing of skeletal, and parathyroid gland and renal function (Duda et al. 1987; Tsai et al. 1989; Sorensen et al. 1982) have been used to facilitate the understanding of the underlying mechanisms of bone loss in humans. However, a predictable and reproducible animal model of ovariandysfunction bone loss would greatly enhance our understanding 311
312
bone biopsies were obtained from a predetermined position of the dorsal surface of the iliac crest shown to present minimal structural variation in the dog (Hardt & Jee 1982). Two left and two right biopsies were obtained from each of the two groups (Sham and Ovh). The second bone biopsy was taken five months later from the side opposite the first biopsy, at a position on the iliac crest comparable to that of the first biopsy. The biopsies were taken with a Bordier trephine (7 mm diameter). The selection of the position was aided by the use of a fluoroscope. Double tetracycline labels for the first biopsy were administered by i.v. injections of 20 mg/kg of declomycin with a schedule of I day on, 13 days off, 1 day on, 11 days off. prior to the biopsy. Prior to the second biopsy, the animals were labeled by i.v. administration of achromycin (20 mg/kg) with a labeling schedule of 1 day on, 14 days off, I day on, 14 days off. Histomorphometty The biopsies were immediately fixed in 70% alcohol and processed, undecalcified, following the standard dehydration and embedding protocols used in our laboratory (Parisien et al. 1990). Seven-pm-thick sections were cut with a Jung model K microtome (Reichert-Jung, Heidelberg, Germany) and stained with Goldner’s trichrome for analysis of static parameters. Sections were obtained from four different planes of the biopsy. Each plane was separated from the other by approximately 350 pm. One section from each of the four planes was used for the measurements. The average number of fields of measurement on each section was 7.83 (8.11 mm’). To avoid subcortical bone, the distance at the base of the thickes: trabecula emanating from the cortex on each side was measured. Subcortical bone with a distance equal to twice this value was excluded. Measurements of static variables were performed by the point counting method using a Merz grid at a total magnification of X 125, with the exception of cancellous bone volume, which was made using a Zeiss Integrations II grid at the same magnification. The coefficient of intra- and interobserver variations for cancellous bone volume were 2.4% and 3.6%, respectively. Ten-pm sections were mounted unstained for assessment of dynamic parameters by fluorescence microscopy (Meunier et al. 1980). The distance between the two labels was measured using a semi-automated image analyzer (Optomax VIDS IV, Optomax Inc., Hollis, NH). The mineral apposition rate (MAR) was calculated by dividing the distance between double labels by the number of days between the two labels and the values expressed in micrometers per day. Mineralizing surfaces were defined as the proportion of trabecular surface exhibiting single and double tetracycline labels; these labels were measured separately and expressed as single-labeled surfaces (sLS/BS), double-labeled surfaces (dLS/ BS), and double-plus single-labeled surfaces (TLS/BS). TLS/BS was used for the calculation of the bone formation rate at the tissue level (BFRIBS) and the adjusted appositional rate (Aj.AR); these values were defined as the volume of mineralized bone formed per unit bone surface and per unit osteoid surface/ day, respectively. EDTA and PTH infusion studies Two weeks after the second biopsy, each beagle received 25 mg/kg of Na, . EDTA in 125 ml of saline with 5% dextrose plus 5 ml of lignocaine solution over a two-hour period by i.v. infusion. The beagles were individually caged without restraint. Infusion was stopped after two hours. Blood samples were taken at 0,60, 120, 180, and 240 min from the beginning of the infusion. The procedure is similar to the one previously used by Torrance
V. Shen et al.: Effects of ovariohysterectomy on bone metabolism in dogs and Nachreiner (1989) on normal dogs. Ionized calcium and intact PTH were measured in the serum samples. Two weeks after the EDTA infusion, a PTH infusion was performed, with all the beagles in individual cages without restraint. The procedure is similar to that used in human studies in our laboratory (Cosman et al. 1990). Briefly, the beagles received 0.55 U/kg of human l-34 PTH fragment dissolved in 120 ml of saline over a 24-hour period by i.v. infusion via a syringe pump. Blood samples were collected at 0, 4, 8, 12, 16, 20, and 24 hours after the initiation of the PTH infusion. Measurements included ionized calcium, phosphorus, osteocalcin, N-terminal PTH, intact PTH, 25-(OH)-D, and l,25-(OH),-D. Sacriffce and bone mineral density measurements Two weeks after the PTH infusion studies, the animals were euthanized by administration of pentobarbital (60 mglkg) i.v., and the pelvis and lower vertebrae were dissected out and preserved in 70% alcohol. The bone mineral density of the L,-L, lumbar vertebrae was determined using a dual energy X-ray absorptiometer (QDR-1000, Hologic, Waltham, MA) under 50 cm of 70% alcohol in a glass container. Measurements
of biochemical parameters
Serum calcium, phosphorus, creatinine, and alkaline phosphatase were measured by routine calorimetric methods. Serum ionized calcium was analyzed with a Nova 8 ionized calcium analyzer (Nova Biomedical, Waltham, MA). Serum osteocalcin was measured by radioimmunoassay specific for canine osteocalcin. The purified canine osteocalcin and diluted antibody were generously provided to us by Dr. Barbara Miller of Norwich Eaton Pharmaceutical Co. Briefly, canine osteocalcin was purified from canine femurs, labeled by the standard chloramine T method, and the labeled ligand separated by G-25 column chromatography in assay buffer, 0.01 M Tris, 0.14 M NaCl 25 mM EDTA, 0.25% RIA grade bovine serum albumin, and 0.1% Tween 20, pH 7.4. After incubating the standards or canine serum with anti-canine osteocalcin antisera, raised in rabbit, for 22 hours at 4” C, the antigen-antibody complex was precipitated with goat anti-rabbit gamma globulin. The concentration of osteocalcin in the serum was determined by comparison to the standard curve. The nonspecific binding was determined by using non-immune rabbit serum. Intact PTH in the serum was determined by a radioimmunometric assay of intact human PTH (Nichols Institute, San Juan Capistrano, CA). The feasibility of using this human assay kit for dogs has been previously documented (Torrance & Nachreiner 1989). Radioimmunoassay of N-terminal PTH molecules was performed using antibody, standards, and procedure kindly provided to us by Dr. G. Segre, Massachusetts General Hospital, Boston, MA (Segre 1983). Vitamin D metabolites were separated after organic solvent extraction of serum and sequential chromatography on Cl8 Sep-Pak cartridges, and further purified on silica Sep-Pak cartridges, as outlined previously (Reinhardt et al. 1984). 25-(OH)-D was assayed by a competitive protein binding method (Preece et al. 1974) using a highly diluted vitamin D depleted rat serum. 1,25(OH),-D was measured by a radioreceptor assay that uses a 1,25-(OH),-D receptor protein isolated from calf thymus (Incstar Corp., Stillwater, MN), by the procedure of Reinhardt et al. (1984). Statistical analysis Data are expressed as mean 2 standard error. Comparison between means was performed using Student’s t test. Power anal-
V. Shen et al.: Effects of ovariohysterectomy on bone metabolism in dogs
313 PTH infusion
ysis of the differences in cancellous bone volume measurements before and after the operation on the same animal in Ovh groups was performed using the formula in Duncan et al. (1983). The estimation of the power was obtained by altering one variable at a time in the calculation, either an assumed 20% decrement from the mean in the pre-operation measurements in cancellous bone volume or a doubling of the sample size. The variance was assumed to be constant in all calculations.
A 24-hour infusion of hF’TH (l-34), at a dose of 0.55 U/kg/h, which is identical to the dose we administer in our human studies (Cosman et al. 1990), was performed on each of the animals (Table III). Although there were differences between Sham and Ovh groups in some of the variables measured in the basal state, the differences were not statistically significant. Ionized calcium showed a steady increase throughout the infusion period from 1.33to 1.81 mMintheshamgroupand 1.3Oto 1.76mMinthe Ovh group. After 8 hours, the level of ionized calcium was significantly higher than the basal level for both groups. N-terminal PTH, which measures both endogenous N-terminal F’TH fragments and exogenously infused l-34 hPTH, showed a lo- to 20-fold increase over basal levels at the first sampling point (4 h) in both groups. Intact FTH, which measures endogenous intact FTH only, decreased rapidly in both groups to below detectable limits after 8 hours. Samples collected from both groups after 16 hours showed a small increase in osteocalcin levels (11 vs. 1315 ng/ml), although the differences were not significantly different from the basal level. Alkaline phosphatase activity, phosphorus, and levels of 25-(OH)-D remained constant throughout the infusion period. 1,25-(OH&-D increased significantly after 12 hours from 44 to 57 pg/mI (p < .05) in Sham and 43 to 63 pg/ml @ < .05) in Ovh groups. There were very few differences between Sham and Ovh groups at any of the time points, except 1,25-(OH),-D at 16 hours (56 vs 69 pg/mI in Sham and Ovh, p < .02) and phosphorus at 24 hours (5.6 mg% in Sham and 4.3 mg% in Ovh, p < .02).
Results Histomorphometric
parameters
and body weight of Sham and
Ovh beagles
Body weight was monitored in each animal prior to the biopsy. Ovh animals increased their body weight significantly @ < .Ol) over the course of the study, while the Sham group demonstrated a nonsignificant increase in body weight (Table I). Measurements of cancellous bone volume, osteoid surface, single-, double-, and total-labeled surface, bone formation rate, and adjusted apposition rate in Sham and Ovh animals showed no differences before or after the operation, either between or within groups (Table I). There was a significant increase @ < .05) in mineral apposition rate in Ovh animals compared with Sham animals five months after the operation. However, this difference may be the result of an inherently higher mineralization rate in the Ovh animals as their preoperative values were also higher when compared to Sham animals. EDTA infusion
Bone mineral density measurements
Each animal was infused with 25 mglkg of Na, . EDTA over a two-hour time period and then allowed to recover. Ionized calcium and intact PTH levels were measured at 0, 1,2,3,4, hours after beginning the infusion (Table II). EDTA infusion decreased the levels of ionized calcium by approximately 10% within the first hour. Endogenous intact F’TH responded vigorously to the lowered ionized calcium level with an approximately four-fold increase. Ionized calcium recovered rapidly to preinfusion levels two hours after the termination of EDTA infusion. Intact FTH levels decreased when the EDTA infusion was terminated, but were not reduced to basal levels. There was a somewhat greater response in PTH production in the Ovh group than the Sham group, but the difference did not reach statistical significance.
Excised lumbar vertebrae were subjected to bone mineral density measurements after termination of the experiment. There were no statistically significant differences in bone mineral density between Sham and Ovh animals (Table IV). The mean values in the Ovh group were 4%) 4%) 1% , and - 3% different from those in the Sham group in lumbar vertebrae L,, L,, L4, and L,, respectively. Discussion There have been few studies examining the effects of ovarian dysfunction on bone and calcium metabolism in dogs. In the present study we have compared serum biochemistries, both
Table I. Histomorphometric parameters and body weight of Sham-operated and ovariohysterectomized beagles
Ovh
Sham Pre BV/TV (a) OSlBS (%) SLS (%,) DLS (%) TLS (%) MAR (*m/d) BFR (~m3/~m*ld) (tissue level surface ref.) BFR (p,m?pm’/d) (tissue level volume ref.) Aj.AR (pm3/pm2/d) Body wt (kg)
23.96 23.49 16.98 7.21 24.25 0.6975
? 2 2 2 + +
Post 2.59 8.69 3.46 2.41 5.30 0.0347
22.42 23.70 16.04 10.54 26.58 0.6926
2 2 ? ? 2 2
Pre 2.46 4.96 1.67 3.02 2.58 0.0242
23.18 26.08 15.63 9.12 24.74 0.8250
2 ? 2 2 -c *
Post 3.38 7.68 3.37 2.83 6.34 0.0933
24.06 24.80 15.46 8.89 24.35 0.8400
‘2 -c ” ” ?a
2.55 2.85 1.98 1.15 2.36 0.0536
0.1706 2 0.0448
0.1681 + 0.0222
0.2184 t 0.0795
0.2033 + 0.0188
1.3274 ? 0.3601 0.8934 2 0.1577 12.45 4 0.59
1.3228 2 0.1833 0.7618 -+ 0.0923 14.95 ” 1.32
1.8070 f 0.6934 0.8679 ? 0.2030 12.55 2 0.33
1.5644 h 0.2074 0.8515 -c 0.1157 17.45 t 0.78b
Results are expressed as mean ‘r SEM. “different from Sham, post-op; p < .05. bdifferent from Ovh, pre-op; p < .OI.
V Shen et al.: Effects of ovariohysterectomy
314 Table II. Biochemical
parameters
Sham Ovh
A EDTA Ih
1.44 2 0.03 1.36 2 0.02
Sham Ovh
18.9 12.4
+ 4.4 i 3.8
in dogs
in EDTA Infusion experiment Sampling
Oh
on bone metabolism
1.32 2 I.28 + 69.7 54.9
time
I
- EDTA
2h
3h
Ionized Calcium (mM) 0.07 1.25 2 0.07” 0.03” I. I8 t 0.02”
t 19.6” 2 11.5”
4h
1.35 t 1.36 2
Intact PTH (pg/ml) 74.9 t 24.2 144.0 + 27.8”
74.9 172.2
0.03 0.02
1.41 t 1.35 +
2 18.7” ? 74.1
36.5 72.8
0.07 0.01
t 17.3 t 37.3
Results are expressed as mean t SEM. “Different from 0 hr sample, p < ,005.
static and kinetic responses to PTH and EDTA, bone histomorphometry, and bone mass measurements in Sham-operated and Ovh dogs. Our results support the premise that the dog may have limited utility for the study of cancellous bone loss in ovariandysfunction osteoporosis. The effects of ovariectomy or ovariohysterectomy on cancellous bone in the beagle dog are controversial. Cancellous bone volume was shown to be either decreased (Faugere et al. 1990: Malluche et al. 1986; Martin et al. 1987) or unchanged (Danucci et al. 1987; Snow et al. 1986; Boyce et al. 1990; our findings). Changes in biochemical and other histomorphometric data also differ in each study. Using sequential biopsies during the first
Table III.
Biochemical
Sampling time
parameters
Sham Ovh
in FTH infusion experiment
4h
Oh
1.33 ” 0.03 1.30 ir 0.03
Sham Ovh
26 II
18 -i-5
Sham Ovh
31 18
29 +
Sham Ovh
11.0 11.0
Sham Ovh
1.40 t 1.30 ? 209 210
I2 h
8h
0.03 0.03
f 41‘ _t 40
1.54 t 1.42 +
1I5 122
I6 h
Ionized calcium (mM) 0.04’ 1.60 ? 0.03” 0.03d I.53 * 0.04
f 46 ? 64
N-terminal PTH (pgiml) IS5 !Z 56 I29 2 34*
145 I43
+ 5
1” Ih
Intact PTH (pgiml) 3 r I” 4 t Ob
4 5
2.1 3.8
II.8 8.7
+ 2
1.6 2.3
Osteocalcin (ng!ml) 12.5 i- 2.0 10.3 -+ 1.7
13.8 13.0
3.5 8.3
16.1 21.1
Alkaline phosphatase (IU!L) t 6.2 17.9 + 4.9 ? II.3 20.6 ? IO.6
i3 i-6
36 47
-+6 + IO
~3 t II
50 53
Z ?
2 7
+ *
0.3 0.3
r7 t3d
2 2.0 t 1.7
10.3 10.3
? ?
17.5 21.1
* 3.1 ‘- 4.4
16.7 20.0
? 2
Sham Ovh
44 48
-t5 _t7
36 43
Sham Ovh
44 43
*4 56
42 43
25 (0H)D (@ml) 36 -t 7 49 r 15
20 h
1.73 + 0.03” 1.61 _f 0.06d
4 3
15 7
1
four months after surgery, two reports (Faugere et al. 1990; Malluche et al. 1986) showed a small but rapid loss of cancellous bone volume. They found no change in bone resorption indices at any dme. but the tetracycline-labeled surface and bone formation rate were reduced. They deduced that the loss of bone must have resulted from an early hyper-resorption within the first month, followed by maintenance of low bone mass ascribable to osteoblast insufficiency. Another report showed a significant 15% trabecular bone loss in the spine and an associated high turnover phenomenon, accompanied by increased serum osteocalcin (Martin et al. 1987). Danucci’s observations of increased osteoid surface, resorption surface, bone formation rate, and
? 62 ? 4@
1.78 * 1.69 ? 207 I85
24 h
0.03” O.Ogd
2 43’ + 65d
1.81 t 1.76 +-
0.03” 0.11”
I85 250
? -t
32‘ 10”
3 4
‘t
I” Ih
5 4
2 +
2’1 Ih
* I.5 IT 4.0
13.0 15.0
2 5
I.0 6.0
15.0 14.7
t -+
I.8 5.9
16.6 20.6
? 4.1 + 12.2
17.3 21.1
t 4.5 + 12.0
17.5 20.6
* i
5.1 II.4
36 43
t8 t 15
42 46
25 * 10
38 49
t +
3 10
56 69
t I?
I Id.’
63 66
_t 5
4d 9
65 68
I 2
5d 5d
i i
0.3 0.5
t t
0.3 0.2
-t +
0.2 0.2’
f t
Id I’
I ,2XOH),D (pg/ml)
Sham Ovh
4.9 4.3
All results are “different from bdifferent from ‘different from ddifferent from ‘different from
? 0.2 ? 0.2
4.9 5.3
expressed as mean time 0 hr sample, time 0 hr sample, time 0 hr sample, time 0 hr sample, control sample, p
2 p p p p <
? 2
0.3 0.5
SEM. < .0001 < .OOI. < .Ol < .OS. .02.
4.8 4.3
57 63
? i
2d 1”
Phosphorus (mg %) 4.9 * 0.2 4.5 i_ 0.2
4.6 4.9
3
4.9 4.2
5.6 4.3
V. Shen et al.: Effects of ovariohysterectomy
on bone metabolism
in dogs
Table IV. Bone mineral density measurements
315 in ovariohysterectomized
dogs (gm/cm2)
Lumbar vertebrae
Sham Ovh
L,
L,
L,
L,
0.587 2 0.014 0.613 * 0.005
0.578 ” 0.020 0.601 ? 0.006
0.578 + 0.017 0.585 + 0.010
0.549 r 0.021 0.531 2 0.013
Results are expressed as mean 2 SEM. Excised lumbar vertebrae were used for the measurements.
labeled surface are in agreement with high turnover. However, they failed to observe a significant decrease in cancellous bone volume six months after surgery (Danucci et al. 1987). A recent report (Boyce et al. 1990) made a more detailed examination of the histomorphometric changes at I, 3, 6, and 10 months after surgery. At one to three months post-operation, they observed a transient increase in mineralizing surface and bone formation rate; at six months post-op the values were depressed below the pre-surgery levels; and at 10 months post-op the levels returned to pre-surgery values. No change in cancellous bone volume was detected at any of the time points, perhaps because of the brief nature of the changes in bone remodeling. It is noteworthy that our second biopsy falls exactly between the increased bone formation rate at three months and decreased bone formation rate at six months, as seen previously (Boyce et al. 1990), which may explain why we did not observe any changes in remodeling parameters. In contrast to many metabolic diseases, the mean values for biochemical parameters may deviate slightly, but generally remain within the normal range in osteoporosis. There is a large overlap of individual values for all indices (Meunier et al. 1980; Parfitt et al. 1980; Whyte et al. 1982). perhaps because of the large age range of the study populations or the heterogeneous temporal variation in the disease process. Biochemical indices in early and late stages of estrogen deficiency do differ. A stimulation test for skeletal and renal products might provide greater discrimination between normal and osteoporotic subjects than is possible with static measurements of circulating levels (Tsai et al. 1989). We examined the response of the parathyroid gland to fluctuations of serum calcium using a short-term EDTA infusion. The PTH concentration was highly correlated with the level of circulating ionized calcium (r = 53, p < .Ol, data not shown), but there was no difference in responsivity between Ovh and Sham-operated animals. It has been suggested that osteoporotic women and ovariohysterectomized rats have an increased sensitivity to PTH (Heaney 1965; Jasani et al. 1965; Orimo 1972). In our PTH stimulation experiment, the levels of N-PTH increased rapidly during the infusion (lo- to 20-fold in the first four hours), and although suppression of endogenous PTH production by exogenously infused l-34 hPTH was highly significant, few differences were observed between the two groups. PTH infusion, as expected, stimulated renal production of 1,25-(OH),-D, but again there was no difference between the two groups except for a minor difference at one of the time points, in contrast to our experience in humans (Cosman et al. 1990). Alkaline phosphatase levels were not altered, but osteocalcin, a more specific bone formation marker, showed a small but nonsignificant increase at the later time points. This increase could be the result of increased synthesis of 1,25-(OH),-D in the kidney, which is known to stimulate osteocalcin synthesis (Price et al. 1980; Gundberg et al. 1983). Although a hormonal challenge approach did not provide us with additional information, more sensitive and discriminating challenges may not yield useful results in the future.
There are several reasons why we may not have detected any differences between Sham-operated dogs and those that underwent ovariohysterectomy: (a) estrogen may not play an important role in skeletal metabolism because of the relatively low levels of estrogen and the semiannual estrus cycle of the dog (Fox & Laird 1970); (b) the small sample size and limited diameter of the pelvis in the dog may not have provided enough statistical power to estimate the differences, in light of the highly varied individual values in these animals. However, based on present data, we should have been able to detect a 20% difference in cancellous bone volume with 75% power. An increase of sample size to twice that used in the experiment would only yield a 10% increase in power, and (c) it is possible that current methods for determining acme changes of bone turnover are not sensitive enough to detect small differences in biochemical and histomorphometric parameters. Using dual-energy X-radiography, a method with better reproducibility than histomorphometry, a recent report showed a significant change in bone density between oophorectomized and control dogs (Drezner & Nesbitt 1990). However, using the same techniques (with a coefficient of variation of 1% with the ex vivo bone samples), we could not detect any differences in bone density between the two groups six months after operation. There are several ways in which one could improve the chances of detecting bone loss due to ovarian dysfunction in dogs. One may perform the ovariohysterectomy at a specific time point in the estrus cycle, and limit calcium intake and weight gain. Extra adipose tissue could increase conversion of androstenedione to estrone (Casey et al. 1983) to compensate for the small loss in estradiol and estrone. A vigorous weight control program for Ovh animals may be needed, although it has been shown that weight gain only partially prevents bone loss in ovarectomized rats and that ovariectomy causes marked bone loss regardless of body weight (Wronski et al. 1987). High calcium intake in the routine laboratory diet (1.1%) may mask the small changes in skeletal metabolism. Although our results and others (Dannucci et al. 1987; Snow et al. 1986; Boyce et al. 1990) suggest limited use of ovariectomized dogs for the study of cancellous bone loss in estrogen-depleted dogs, a study of cortical bone remodeling may yet provide useful information, as suggested by the recent comprehensive review of all the available oophorectomized dog data (Kimmel 1992).
The study was supported by NIH grants AR39191 and AR35647. The authors acknowledge the outstanding technical assistance of Wendy Herbert and Michele Schnitzer.
Acknowledgments:
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