Postmenopausal osteoporosis

Postmenopausal Osteoporosis A Heterogeneous Disorder as Assessed by Histomorphometric Analysis of Iliac Crest Bone from Untreated Patients

MICHAEL P. WHYTE, M.D. MICHELE A. BERGFELD, B.A. WILLIAM A. MURPHY, M.D. LOUIS V. AVIOLI, M.D. STEVEN L. TEITELBAUM, M.D. St. Louis. Missouri

From the Divisionof Bone and Mineral Metabolism,Dep&ment of Medicine(Mpw, LVA),and the Deperbnentof PathologyandLaboratoryMedicine (MAB, SLT). Jewish Hospital of St. Louis; and Section of Musculoskeletal Radiology, Malljnckrodt Institute of Radiology (WAM); Washington LtniversltySchool of Medicine, St. Louis, Missouri. This work was supported in part by NIH Training Grant A&07033, NIH Grant AMl4570, CRC &ant RR-00036 and a grant-in-aid from the Shriners Hospitals for Crippled Children (St. Louis Unit). Presented in part at the American Federation for Clinical Research, Southem Section Meeting, New Orleans, Louisiana, January 16, 1979 (Clin Res 1976; 26: 776A.L and the 1st Annual Meeting, American Society for Bone and Mineral Research, Anaheim, California, June 12, 1979 (Calcif Tissue Int 1979; 28: 161.). Reprint requests should be addressed to Dr. Michael P. Whyte, Division of Bone and Mineral Metabolism, The Jewish Hospital of St. Louis, 216 South Kingshighway, St. Louis, Missouri 63110. Manuscript accepted September 15. 1981.

Twenty-six women with untreated postmenopausal osteoporosis underwent iliac crest biopsy following tetracycline-labeling and mineral metabolism studies. Hkkmorphomebk assessment of their bone remodeling rates, including formation determined by the tetracycline-labeling technkfue, revealed considerable variatkn. Efght women had no evidence of active bone formation (inactive remodeling osteoporosis), whereas the others showed a spectrum of bone formation rates (active remodeling osteoporosis). Clinical and biochemical studies failed to predict the histomorphometric findings. Postmenopausal osteoporosis is a histologically heterogeneous disorder with morphologic expression in bone that cannot be predicted by single or combined routine clinical and laboratory parameters. Bone bkpsy, necessary to identify the histokgk leston and assess skeletal dynamics, may prove to be important for optimal therapy of osteoporosk, as a varkty of agents-with dtfferent effects on bone remodellng-are available. Osteoporosis is generally regarded as a disorder characterized by decreased skeletal mass without abnormality in either the composition or proportion of the mineral and organic phases of bone [ 1,2]. However, as bone is a living tissue that is constantly undergoing formation and resorption (remodeling or ‘turnover”) [3], assessment of the skeletal dynamics in postmenopausal osteoporosis would enhance our understanding of the pathophysiology of this common and important metabolic bone disease. During the past two decades, recognition that tetracycline markers would enable measurement of the rate of bone formation [3], development of relatively atraumatic techniques to sample bone tissue by closed iliac crest bone biopsy [4], and progress with methods to process thin undecalcified sections for histomorphometric analysis [5] have made possible the assessment of skeletal dynamics in a variety of metabolic bone diseases. Using these techniques in 26 untreated women with postmenopausal osteoporosis, we have found that this is a histologically heterogeneous disorder with morphologic expression in bone that cannot be predicted by routine clinical or biochemical studies. PATIENTS AND METHODS Patients. To be eligible for the protocol study, women referred to us by their physicians for osteopenia had to be both untreated for at least eight months

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(i.e., had not received hormones, fluoride, calcium supplements, vitamin D or diphosphonate) and postmenopausal. Subjects were ineligible if they had a history of hepatic or renal disease; gastrointestinal surgery; malignancy; significant immobilization; symptoms of gastrointestinal malabsorption; or anticonvulsant, antacid, glucocorticoid or heparin therapy. Protocol. Oxytetracycline (250 mg orally every 6 hours for three days) was administered to subjects two weeks before they were studied as in-patients at the Clinical Research Center of Barnes Hospital. The patients recorded the dates that the tetracycline was taken. After informed consent and medical history were obtained and physical examination was carried out, they received a second identical course of oxytetracycline and were fed a hydroxyproline-free diet during the first three days of the five-day study. Thereafter, they ate their usual diets. All were encouraged to ambulate as accustomed. Blood specimens were obtained in the morning after patients had fasted from midnight. During the third day, 24-hour urine collections for hydroxyproline assay were obtained. Tubular reabsorption of phosphate (TRP), tubular maximum of phosphate per glomerular filtration rate (TmP04/GFR-the phosphate threshold) [6] and the fasting urine calcium-to-creatinine ratio [7] were determined with two P-hour urine collections obtained between 8 and 10 AM and 10 and 12 AM after subjects had fasted from midnight (each patient drank 500 cc water at 6:30 AM and then 250 cc hourly during the study; blood was collected at 9 and 11 AM).

To exclude disorders other than postmenopausal osteoporosis associated with osteopenia, results of the following studies were also obtained with the protocol studies: radiographs of the chest, thoracic and lumbar spine, hands, hips and pelvis; technetium 99m pyrophosphate bone scan; serum and urine protein electrophoresis; routine urinalysis; complete blood cell count with leukocyte differential; serum SMA-18 analysis (Technicon Instruments Corp., Tarrytown, New York); Tq; Ts; carotene: and assay of immunoreactive parathyroid hormone [ 81. Bone Mass Measurements. Cortical bone mass was assessed by metacarpal cortical width determination as described by Gam [9] and by photon absorption at the mid-radial cortex using a Norland-Cameron Bone Mineral Analyzer (Norland Instruments, Fort Atkinson, Wisconsin) [ 10,i 11. Cancellous bone mass was assessed by evaluation of the trabecular pattern of the femoral neck and calculation of the “Singh index” [ 121. Both the metacarpal width and Singh index were determined by a musculoskeletal radiologist (WAM). Biochemical Assays. Circulating immunoreactive parathyroid hormone (IPTH) was assayed by the method of Hruska et al. [8] using CH-9 antiserum which binds to the carboxyterminal of the PTH molecule; serum immunoreactive calcitonin (ET) by the method of Rojanasathit and Haddad [ 131; and 25hydroxyvitamin D (25-OHD) by the competitive protein-binding method of Haddad and Chyu [ 141. Ionized calcium was measured by an Orion flow-through electrode. Serum follicle-stimulating and luteinizing hormone levels and urine total hydroxyproline were assayed by Bioscience (St. Louis Branch).

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Iliac Crest Bone Biopsies. Trans-iliac bone biopsies were performed using local anesthesia with a trocar having a 0.5 cm internal diameter according to the method of Bordier et al. [4]. Specimens, which included both cortices and intervening trabeculae, were fixed in neutral buffered formalin, embedded without prior decalcification in methylmethacrylate, and then cut into 5- and 1O-pm sections on a Jung Model-K Sledge microtome (Jung Instruments, Heidelberg, Germany). Histomorphometric measurements with a MerzSchenk Integrating Eyepiece or a linear micrometer [ 151, performed by one observer (MAB), were made by routine light microscopy at a magnification of 200 on the 5-pm sections stained by a modification of the Masson trichrome stain [ 161. Unstained lO+m sections were examined, as just described, with fluorescent microscopy for tetracycline-based histomorphometry. The following parameters were evaluated in trabecular bone: l Total bone volume-percentage of bone medullary space occupied by trabecular bone tissue (mineralized and nonmineralized). l Relative osteoid volume-percentage of trabecular bone tissue that was unmineralized, i.e., osteoid. l Total osteoid surface-percentage of trabecular bone surface covered by osteoid. l Osteoblastic osteoid surface--percentage at trabecular bone surface covered by osteoid that is lined by typical, cuboidal (“active”) osteoblasts. l Mean osteoid seam width-the average width of the osteoid seams on the trabecular surface Mm) (a derived value obtained by dividing the relative osteoid volume by the absolute linear extent of osteoid over the trabecular surface). l Osteoclasts per square millimeter of medullary space. l Osteoclasts per millimeter of trabecular perimeternumber of osteoclasts per millimeter of trabecular bone surface. l Total resorptive surface-percentage of trabecular bone surface with “resorption bays” (Howship’s lacunae). l Osteoclastic resorptive surface-percentage of trabecular bone surface with Howship’s lacunae containing osteoclasts juxtaposed to bone. l Fibrotic surface-percentage of trabecular bone surface covered by fibrous tissue. l Cellular rate bf mineralization-the mean distance between the two (“double”) fluorescent tetracycline labels divided by the duration between the administration of the two courses of tetracycline @m/day). Normal values for each of the “static” (non-tetracyclinebased) parameters were obtained previously from identically processed iliac crest specimens from 12 Caucasian women-mean age 48.8 f 18.5 (SD) years-who had died suddenly. Values for the cellular rate of mineralization have been published for 29 normal women (mean age 29 years; range 19-53) by Melsen and Mosekilde [ 171. For both cortices of the iliac crest specimens, the number of double tetracycline labels were counted and the following histomorphometric parameters were assessed: l Cortical wisth-calculated width of the cortical bone (mm).

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TABLE I

Results of Biochemical Studies in 26 Women with Untreated Postmenopausal Dsteowrosis Patient Mean f 2 SD

Parameter Blood Total calcium (mg/dl) Ionized calcium (mg/dl) Inorganic phosphate (mg/dl) lmmunoreactive parathyroid hormone

9.5 4.5 3.9 6.2

(CLIeq/mt) Alkaline phosphatase (IU/liter) lmmunoreactive calcitonin (pg/mt) 25-OHD (ng/ml) Magnesium (meqjliter) Creatinine (mg/dl) Carotene (pg/dl) Estradiol (pg/ml) Thyroxine (pgldl) Urine+ Calcium (mg) Phosphorus (mg) Hydroxyproline (mg) Free cortisol (gg) 17-Hydroxysteroids (mg) Fasting calciumlcreatinine ratio Renal Function Creatinine clearance (ml/min) TRP (%) TmPOd/GFR (mg/dl)

f 1.0 f 0.4 f 0.8 f 6.0

(26)

217 992 23 55 6.8 0.187

f f f f f f

35-95 8 [ 141 1.5-2.4 0.6-1.1 70-250 15-40 5.0-l 1.0

164 560 18 48 4.8 0.138

(22) (21) (21) (24) (20) (20)

100-300 500-1500 15-43 20-90 2-13 0.01-0.15 [7]

88 f 50 86f 3.8 f

9.0-10.3 4.5-5.0 3.0-5.0