Discordance between bone turnover and bone loss: effects of aging and ovariectomy in the rat.

Mechanical strain maintains bone architecture even under conditions of increased bone turnover such as occurs with ovarian hormone deficiency. The rat distal femur contains two sites that apparently experience different levels of mechanical strain and therefore the rat is a suitable model for investigating such effects. The femoral epiphysis experiences higher strain energy compared with the metaphysis and we report the effects of aging between 7 and 12 months and the postovariectomy effects over the same time period on cancellous bone variables measured at these two sites. Age-related bone loss in sham-operated (Sham) animals occurred in both regions, with a greater fall in the metaphysis than in the epiphysis (trabecular bone volume [BV/TV, %] Mean [SEM] Metaphysis: day 0, 25. 9 [2.4]; day 150, 8.8 [1.3]: Epiphysis: day 0, 44.8 [1.7]; day 150, 36.7 [1.4] [p < 0.0001]). With ovariectomy (OVX) there was a 73% reduction in cancellous bone at the metaphysis compared with no specific loss at the epiphysis (BV/TV [%] OVX: Metaphysis: day 150, 2.4 [0.4] [p < 0.01 compared with Sham]: Epiphysis: day 150 29.3 [2. 7] [NS]). Osteoblast cell activity and osteoclast surface were increased after ovariectomy in both regions. The mineral apposition rate decreased at 9.5 months of age in both regions (p < 0.0001), independent of ovariectomy, and was coincident with a reduction in trabecular number in the epiphyses of both operative groups and in the metaphysis of the ovary-intact group. These data suggest that local mechanical strain governs bone balance with aging and that architectural changes resulting from age-related bone loss may mirror those following estrogen deficiency but occur via a different cellular mechanism.

Comparison of three methods for estimation of bone resorption following ovariectomy in the distal femur and the proximal tibia of the rat.

The study of estrogen-deficient bone loss requires accurate assessment of bone resorption; however, there has been considerable variance in the values reported for this variable. We compared three techniques for estimating bone resorption in the adult rat at four anatomical sites, the metaphysis and epiphysis of the distal femur and the proximal tibia. The techniques include an osteoclast-morphology-based method (VK-Oc), a bone-surface-based method (Pit-Oc), and an enzymatic method (AP-Oc). Thirty 6-month-old rats were either ovariectomized (ovx) or sham operated (sham) and killed at 0, 9, or 18 days postoperation. Each method was analyzed for variance in the ovary-intact groups and for the ability to detect the increase in osteoclast surface known to occur following ovariectomy. A 50-fold variation for the estimation of extent of osteoclast surface was entirely accounted for by these three methods for osteoclast estimation. The VK-Oc method was the most consistent for discriminating levels of osteoclast surface between ovary-intact and ovariectomized rats, detecting an increase at three of the four sites. There was no difference between the methods in their ability to produce consistent values for ovary-intact groups. The Pit-Oc method produced the largest numerical difference between the two operation groups with a 2.5-3-fold increase compared with 1.25-1.5-fold for the other methods (p < 0.001). However, the greater variance associated with this method limited the ability to detect the increase in osteoclast surface following ovariectomy. The AP-Oc method lacked the sensitivity of the other two methods. The oophorectomy-induced increase of osteoclast surface in the metaphysis of distal femur was larger and more consistently demonstrated than increases at the other sites.

Variation in the short-term changes in bone cell activity in three regions of the distal femur immediately following ovariectomy.

The effect of ovariectomy (OVX) on cancellous bone in the rat is not uniform at all sites of the skeleton. We report variation in the short-term effects of adult OVX in three regions of the distal femur: the diaphysis (DIA), the metaphysis (META), and the epiphysis (EPI). Cancellous bone parameters were estimated in the three separate zones of the femora and compared with changes in bone cell activity, as estimated by osteoclast surface (Oc.S) and bone formation rate (BFR). Changes were studied for 30 days in a series of rats either sham-operated (Sham) or ovariectomized (OVX) at 7 months of age. Oc.S and BFR were elevated following OVX in all regions. The time course for the OVX-induced changes differed between regions: DIA, both Oc.S and BFR were elevated at day 9; META, Oc.S was also elevated at day 9, while the rise in BFR was delayed until day 21; EPI, Oc.S remained stable but increased relative to ovary-intact rats by day 18 due to reduced levels in the latter, but BFR did not rise until day 28. These changes in bone cell activity following OVX produced a 71% reduction of cancellous bone in the DIA and a 35% reduction in the META. In contrast, no OVX-induced bone loss was observed in the EPI. This study shows that bone cell activity increases in each region of the distal femur within the first 30 days following OVX, independent of bone loss. However, the time course of increased bone cell activity is not uniform. These data highlight the role of local factors in the response to ovarian hormone deficiency.

Prepubertal oophorectomy limits the accumulation of cancellous bone in the femur of growing rats with long-term effects on metaphyseal bone architecture.

Bone loss after oophorectomy of adult rats is more rapid and complete in the metaphysis than in the epiphysis of the femur, particularly in the proximal region of the metaphysis distant from the growth plate. This study was undertaken to determine the effects of prepubertal oophorectomy, on femoral cancellous bone acquisition during growth. Rats were oophorectomized (OVX) or sham operated at 3 weeks of age and killed at intervals up to 78 weeks for scanning electron microscopy and histomorphometry of the distal femur. Differences in cancellous bone architecture between the two groups was evident after 6 weeks of age. Relatively minor differences were found in the part of the metaphysis near the growth plate and in the epiphysis, with less trabeculae in the primary spongiosa and 1 to 2 less trabeculae/mm in the secondary spongiosa. However, as metaphyseal growth proceeded, trabeculae were present for a greater distance up the femoral shaft in controls than in OVX rats, with mean BV/TV in the proximal part of the metaphysis increasing from 1.4% at 6 weeks to 13.4% at 20 weeks in controls, with no increase in the OVX rats. We find that the lack of ovarian hormones increases the rate of destruction of trabeculae near the metaphyseal-diaphyseal junction.

Estradiol treatment transiently increases trabecular bone volume in ovariectomized rats.

The effect of short-term estradiol treatment, administered from the time of ovariectomy, on increased bone turnover and subsequent bone loss was studied in the rat. Adult female Sprague-Dawley rats were ovariectomized and administered daily subcutaneous (s.c.) injections of 17 beta-estradiol at 8 micrograms/ kg per day (Low) and 20 micrograms/kg per day (High) or vehicle alone (Veh). Femoral trabecular bone volume (BV/TV) and trabecular number (Tb.N) in the distal femur were transiently increased at 6 days postoperation in a dose-dependent manner following estradiol administration [mean +/- SEM: BV/TV (%), day 0, 6.6 +/- 0.2; day 6, Veh 7.8 +/- 0.4, Low 10.2 +/- 2.2, High 12.8 +/- 1.7 (p < 0.05); Tb.N (/mm), day 0, 2.30 +/- 0.24; day 6, Veh 2.89 +/- 0.33, Low 3.4 +/- 0.7, High 4.39 +/- 0.34 (p < 0.05)]. Estradiol prevented the ovariectomy-induced decrease in BV/TV and Tb.N between 9 and 15 days observed in Veh rats. Both serum alkaline phosphatase and urine hydroxyproline excretion were maintained at preoperative levels or lower from day 6 postoperation with high dose estradiol. Serum osteocalcin, however, rose above preoperative levels with estradiol at days 6 and 9, but returned to these values on days 15 and 21 postoperation. These results suggest that estradiol, administered from the time of ovariectomy, immediately suppressed markers associated with osteoblast proliferation/matrix synthesis and bone resorption. Mineralization does not appear to be so rapidly suppressed by estradiol with relatively high levels immediately following administration, resulting in a transient increase in trabecular bone volume and trabecular number.

Increased bone resorption precedes increased bone formation in the ovariectomized rat.

This study describes an increase in biochemical and histomorphometric markers of bone resorption prior to increased bone formation and trabecular bone loss in the ovariectomized rat. Six-month-old, female Sprague Dawley rats were either sham operated or ovariectomized (Ovx) and killed at 0, 6, 9, 15, 18, 21, and 42 days postoperation when femora were collected and trabecular bone volume (BV/TV) was determined from von Kossa silver-stained sections using the Quantimet 520 image analysis system in the distal region. A number of these sections were also examined unstained for fluorochrome labels, and stained for acid phosphatase to detect osteoclast-like cells (ACP surface). At 18 days postoperation, lumbar vertebrae were examined. Blood and urine specimens were analyzed for bone-related biochemical variables. ACP surface was significantly greater in Ovx rats compared with sham at 6 days postoperation (mean ACP surface (%TS) +/- SEM: sham 36.4 +/- 1.9; Ovx 40.3 +/- 1.2, P < 0.05) as was urinary hydroxyproline excretion. Serum osteocalcin and alkaline phosphatase activity were not elevated in Ovx rats compared with Sham until 9 days postoperation. Mineral apposition rate (MAR) was increased at 12 days after ovariectomy (mean MAR (microm/day) +/- SEM: sham 0.85 +/- 0.06; Ovx 1.23 +/- 0.06, P < 0.05). Trabecular bone volume (BV/TV) at a specific site in the metaphyseal-diaphyseal core area was significantly lower at 15 days postoperation (mean (%) +/- SEM: Sham 7.40 +/- 1.23, Ovx 4.25 0 0.65, P < 0.05). There was no difference in lumbar vertebral BV/TV between the two groups at 18 days postoperation, however, ACP surface was elevated in the Ovx rats (P < 0.05). A systemic increase in bone resorption at 6 days postovariectomy precedes increased formation whereas the length of time required for the dissolution of trabeculae postoperation is determined locally.

Parathyroidectomy does not prevent bone loss in the oophorectomized rat.

The evidence for a role of parathyroid hormone in the bone loss after the menopause remains controversial. This study examines the effect of parathyroidectomy on femoral trabecular bone volume, thickness, and spacing and biochemical markers of bone turnover in the oophorectomized rat. Female Sprague-Dawley rats 3 months old were double sham operated (sham), oophorectomized (OPX), parathyroidectomized (PTX), or oophorectomized and parathyroidectomized (O/P) under halothane anesthesia. At 9 weeks postoperation, femoral trabecular bone volume (BV/TV) was lower in OPX and O/P rats compared with sham or PTX animals (BV/TV, %, mean +/- SEM): sham 25.9 +/- 0.5, OPX 15.1 +/- 0.9, PTX 24.1 +/- 0.9, O/P 17.3 +/- 0.5; p < 0.001). Urinary hydroxyproline excretion, serum osteocalcin, and alkaline phosphatase activity were higher in OPX and O/P rats compared with control animals at 3 weeks postoperation (OHPE microM GF, mean +/- SEM: sham 1.37 +/- 0.16, OPX 2.16 +/- 0.26, PTX 0.95 +/- 0.21, O/P 1.92 +/- 0.22, p < 0.005; osteocalcin, microgram/liter, sham 31.8 +/- 1.8, OPX 33.7 +/- 2.7, PTX 24.5 +/- 2.1, O/P 34.3 +/- 2.1, p < 0.025; alkaline phosphatase, U/liter, sham 90 +/- 3, OPX 125 +/- 9, PTX 87 +/- 9, O/P 116 +/- 11, p < 0.005). These data indicate postoophorectomy bone loss is not prevented by parathyroidectomy.

Anabolic steroids in postmenopausal osteoporosis.

The anabolic steroids were first developed in the 1950's to provide the anabolic advantages of androgens with less androgenic action. They are widely used in the treatment of osteoporosis, although their effectiveness has only recently been demonstrated with the advent of bone densitometry. It is now established that their administration leads to a significant increase in bone mass in osteoporotic men and women associated with an apparent conversion of fat into muscle tissue. The bone gain is of the order of 3% per annum but the maximal effect is obtained in the first few months. The bone gain is probably due to stimulation of bone formation and is associated with elevation of serum albumin and fat-free skinfold thickness. The most serious side-effect with parenteral use is lowering of voice pitch but there is some evidence that oral anabolic steroids may adversely affect liver function. The optimum dose and duration of therapy has not yet been established.

Effects of oophorectomy on biochemical and bone variables in the rat.

The metabolic effects of oophorectomy (Oophx) were studied in 6-month-old rats maintained on a normal chow diet. Nine weeks following operation, Oophx animals had a significantly lower femoral trabecular bone volume (BV/TV) than sham-operated animals; mean (SD) Oophx 8.5 (3.8)%; Sham 13.4 (2.5)%; P = 0.013). They also had a higher urine hydroxyproline (P less than 0.001), serum alkaline phosphatase activity (P less than 0.001), serum phosphate (P less than 0.001) and lower serum albumin (P less than 0.001) than the controls. Serum osteocalcin was inversely related to the BV/TV in the Oophx animals at 9 weeks post operation (r = -0.85, P = 0.007, n = 8). A fall in serum ionized calcium from 3 to 9 weeks post operation correlated with a fall in urinary hydroxyproline in the Oophx animals (r = 0.57, P = 0.002, n = 27). The data are consistent with a model of ovarian hormones acting directly to modulate bone cell activity as well as exerting an effect on the renal handling of phosphate.

Trabecular spacing in post-menopausal Australian women with and without vertebral fractures.

Histomorphometric measurements were made from iliac crest biopsies of 32 women with vertebral fractures and 37 women without fracture. All were post-menopausal Australian women who had presented with back pain to a hospital out-patient endocrinology clinic. Bone from the fracture cases was characterised by loss of individual trabecular elements, with the remaining trabeculae being spaced further apart than those in the non-fracture women (p less than 0.0001). This resulted in a significant decrease in trabecular bone volume (p less than 0.01). In addition osteoid surface was reduced (p less than 0.01). Dynamic parameters of bone turnover were not significantly different between the two groups. These data should be useful for the assessment of iliac bone histomorphometry in Australian post-menopausal women suspected of having osteoporosis.

Progressive cancellous bone loss in rats after adrenalectomy and oophorectomy.

Female Sprague-Dawley rats (n = 72), 6 months old, underwent either sham operation, oophorectomy, adrenalectomy, or combined oophorectomy and adrenalectomy (O&A). They were all maintained on normal saline ad libitum and 20 g/day 1.1% calcium chow. Nine weeks after operation, the trabecular bone volume of the distal femoral shaft was significantly lower (P less than 0.001) in the adrenalectomized (11.1%), oophorectomized (7.0%), and O&A (8.3%) animals than in sham-operated animals (19.8%). Eighteen weeks after operation, the trabecular bone volume in O&A animals had fallen to a mean of 3.8% (sham 17.0%), and the length of the femur had increased to 38.8 mm after O&A (sham 36.8 mm, P less than 0.01). O&A animals treated with 0.35 mg/kg/week nandrolone decanoate from 9 weeks postoperatively onward, had twice the femoral trabecular bone volume of untreated animals at 18 weeks (P less than 0.05). By contrast, no significant differences were found in vertebral body trabecular bone between any groups, including groups receiving treatment with androgens. We have found that, by 9 weeks after operation, adrenalectomy alone causes significant loss of metaphyseal trabecular bone, similar to the progressive loss seen after oophorectomy.

Use of Chernoff faces to follow trends in laboratory data.

Graphical representation of laboratory data speeds up recognition of abnormalities. The Chernoff face is an excellent tool to represent patient data; the human eye and mind are exquisitely sensitive to subtle changes in facial appearances and expression. One of the best uses of the faces is in illustrating trends in the data, particularly in acutely ill patients where many laboratory studies are performed. The faces are especially useful if comparisons to a former or normal state are made. A very brief examination of adjacent faces suffices to identify changes in laboratory data.

Variation in histomorphometric estimates across different sites of the iliac crest.

Bone specimens were obtained from four different sites in the ilium of necropsy cases using a Jamshidi 8G trephine needle. Undecalcified histological sections were prepared and two structural histomorphometric parameters were estimated. Estimates of the percentage of trabecular bone volume (%TBV) deviated systematically, with variation of the biopsy site around the usual location, increasing in magnitude from the anterior to the posterior ilium. Such differences highlight the importance of understanding the degree of variation which exists in histomorphometric data. The histomorphometry of a vertical biopsy specimen of the iliac crest did not differ significantly from that of a transilial biopsy specimen of similar diameter taken from an adjacent site. When taken at the standard site, and when providing a long core of cancellous bone, with minimal discomfort to the patient, the vertical biopsy specimen compares well with the transiliac biopsy specimen for use for investigating metabolic bone disorders.

Comparison of two bone trephine instruments used for quantitative histomorphometry.

Transilial bone samples were obtained at necropsy using trephine instruments with internal diameters of 2 mm and 7 mm. Undecalcified histological sections were prepared and two structural histomorphometric variables (percentage of trabecular bone volume and surface density of bone) were estimated by a Quantimet 720 image analyser. The differences between the variables measured from the two adjacent cores were due to differences in biopsy site rather than the size of the sample. The smaller diameter needle is therefore recommended for use as the biopsy procedure is simpler and tissue sections can be prepared sooner than is the case with the larger needle.

Clinical responsiveness to laboratory results.

Digoxin prescriptions written by physicians after obtaining digoxin assay results were reviewed for 100 patients in order to study aspects of experts' interpretation of test results; also 77 pathologists' interpretations of serum digoxin assay results and glucose tolerance curves were studied. There was agreement on the interpretation of only some extreme test result values. Borderline values evoked a mixture of opinions. Responsiveness to result values was measured in terms of the extent of agreement among interpreters and was found to depend on the amount of deviation of the result value from referent values. This is consistent with the idea that the attention given to tests varies with the result value according to its perceived contribution toward reducing clinical error rates. Thus, there is no requirement for unusual analytic precision near referent values, as clinicians respond to results with more skill than anticipated.

Applying descriptive discriminant analysis as a visual aid for physicians interpreting biochemical test results.

Descriptive discriminant analysis displays for classes of common hepatic and biliary disorders and other mimicking conditions using 11 biochemical measurements, were demonstrated to physicians as an aid to interpretation. Displayed on a video, biochemical distinctions among disorders were made apparent to viewers. Physician users could easily see where their patient's results fell in relation to other results from patients with relevant diseases. Users had difficulty specifying which diseases they wanted included in displays. This technique would be useful for verifying experts' test result interpretations, when the differential diagnosis can be explicitly stated.

Effects of the introduction of the dexamethasone suppression test on diagnosis and treatment of depression by psychiatrists.

A review of case notes before and after the introduction of the DST into clinical psychiatric practice revealed considerable changes in diagnosis and management. Specifically there were increases in the diagnosis of biological depression and treatment with somatic antidepressant therapy. There was no association between DST results and particular management plans. There was a strong association between requesting the DST and management with antidepressants. It is suggested that the introduction of laboratory tests for psychiatric disorders may firm the belief of psychiatrists in the biological basis of some forms of depression and thus alter their diagnostic and treatment practice.

Clinical acceptance of a multi-test reference region for biochemical-panel results.

Results of a panel of six biochemical tests on a patient's specimen were mathematically combined into a "six-test signal strength" (STSS) value. This value indicated the overall extent of change from physiological results, and it was calculated in a way that ensured that a STSS value less than or equal to 1 occurred in 95% of apparently healthy people. STSS was reported with the test results for hospital inpatients during a four-month trial period. Doctors requested a repeat of the panel less often when a low STSS was reported, even if some test results were outside their separate reference intervals. Clinicians expressed differing opinions about its usefulness, some finding that a high STSS value had saved them from overlooking abnormal results, others not finding the value to be any practical advantage. Using a multi-test normal region resolves a statistical dilemma, while compounding the problem of knowing what results really mean.

The diagnostic utility index.

A method for quantifying the value placed on diagnostic accuracy is described. Named the Diagnostic Utility Index (DUI), it was completed by fifty subjects (including pathologists, physicians and medical students). Analysis of the responses reveals that attitudes to diagnostic perfectionism differ from one disease to another. It is suggested that the DUI may be useful in the clarifying of subjective factors which influence medical attitudes to the diagnosis and investigation of disease.