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Patients rely on their primary care physician to manage multiple, often chronic medical conditions that require prescription medications. Balancing the risk to benefit of treatments can be challenging and requires that the primary care physician stay abreast of new information regarding risks and benefits. The number of medications with reported adverse effects on skeletal health is expanding. This review focuses on medications recently added to the list of "bad to the bone" drugs and on recent advances in management of glucocorticoid-induced osteoporosis. A practical approach to assessing and managing the skeletal risks is outlined. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of this study is to test the ability of risedronate and estradiol, alone or in combination, to prevent bone loss associated with androgen deprivation therapy in men with prostate cancer.Materials and Methods: This is a randomized placebo-controlled trial of risedronate and estradiol, alone or in combination, in men with prostate cancer receiving androgen deprivation therapy. The primary outcome was change in hip bone mineral density at 1 year.Results: No statistical difference was found among the groups for bone mineral density changes. The only side effects of note were increased gynecomastia and breast tenderness associated with estrogen therapy. The study was limited by poor accrual and subsequent lack of statistical power.Conclusions: Men receiving androgen deprivation therapy for prostate cancer are at risk for bone loss and should receive appropriate bone density monitoring and preventive advice about calcium, vitamin D, exercise, and fall prevention. Prescription drugs proven in this patient population should be used when the risk of fracture is high. [ABSTRACT FROM AUTHOR]

Acquired osteosclerosis is a rare disorder of bone formation but an important consideration in adults with sclerotic bones or elevated bone density results. In such patients, malignancy, hepatitis C, and fluorosis should all be considered when making a diagnosis. We describe 4 patients evaluated at our Metabolic Bone Disease Clinic from May 1, 1997, to July 1, 2006, whose bone disorders resulted from chronic fluoride exposure due to excessive tea intake. Three of these patients had toxic serum fluoride levels (> 15 micromol/L). Although the clinical presentation of the patients varied, all 4 had an unexpectedly elevated spine bone mineral density that was proportionately higher than the bone mineral density at the hip. Other clinical features included gastrointestinal symptoms such as nausea, vomiting, and weight loss; lower extremity pain sometimes associated with stress fractures of the lower extremities; renal insufficiency; and elevated alkaline phosphatase levels. Readily available, tea often contains high levels of fluoride. Obsessive-compulsive drinking behaviors and renal insufficiency may predispose to excessive fluoride consumption and accumulation. The current cases show that fluoride-related bone disease is an important clinical consideration in patients with dense bones or gastrointestinal symptoms and a history of excessive tea consumption. Furthermore, fluoride excess should be considered in all patients with a history of excessive tea consumption, especially due to its insidious nature and nonspecific clinical presentation. [ABSTRACT FROM AUTHOR]

Megestrol acetate is a progestational agent for treatment of metastatic breast cancer and endometrial cancer. Megestrol has also been used as an appetite stimulant for patients with human immunodeficiency virus and malignancy who experience cachexia and wasting; also, megestrol can be beneficial in relieving hot flashes in women and men. Megestrol has been shown to have a glucocorticoidlike effect and has been associated with substantial suppression of plasma estradiol levels. We describe 2 patients who recently presented to our Metabolic Bone Disease Clinic with severe osteoporosis complicated by multiple vertebral fractures experienced while the patients were receiving high-dose megestrol therapy. The patients had evidence of adrenal axis suppression but recovered fully after megestrol was discontinued. We speculate that megestrol was an important factor in the development of osteoporosis and subsequent fractures. Further study is warranted to clarify the relationship between megestrol and its potential for adversely affecting the skeleton. [ABSTRACT FROM AUTHOR]

Background: Most periprosthetic fractures following total hip arthroplasty (THA) are fragility fractures that qualify patients for osteoporosis diagnoses. However, it remains unknown how many patients were diagnosed who had osteoporosis before injury or received the proper evaluation, diagnosis, and treatment after injury., Methods: We identified 171 Vancouver B2 (109) and B3 (62) periprosthetic femur fractures treated with a modular fluted tapered stem from 2000 to 2018 at 1 institution. The mean patient age was 75 years (range, 35 to 94), 50% were women, and the mean body mass index was 29 (range, 17 to 60). We identified patients who had osteoporosis or osteopenia diagnoses, a fracture risk assessment tool (FRAX), bone mineral density (BMD) testing, an endocrinology consult, and osteoporosis medications. Age-appropriate BMD testing was defined as no later than 1 year after the recommended ages of 65 (women) or 70 years (men). The mean follow-up was 11 years (range, 4 to 21)., Results: Falls from standing height caused 94% of fractures and thus, by definition, qualified as osteoporosis-defining events. The prevalence of osteoporosis diagnosis increased from 20% before periprosthetic fracture to 39% after (P < .001). The prevalence of osteopenia diagnosis increased from 13% before the fracture to 24% after (P < .001). The prevalence of either diagnosis increased from 24% before fracture to 44% after (P < .001). No patients had documented FRAX scores before fracture, and only 2% had scores after. The prevalence of BMD testing was 21% before fracture and 22% after (P = .88). By the end of the final follow-up, only 16% had received age-appropriate BMD testing. The proportion of patients who had endocrinology consults increased from 6% before the fracture to 25% after (P < .001). The proportion on bisphosphonate therapy was 19% before fracture and 25% after (P = .08)., Conclusions: Although most periprosthetic fractures following THA are fragility fractures that qualify patients for osteoporosis diagnoses, there remain major gaps in diagnosis, screening, endocrinology follow-up, and treatment. Like nonarthroplasty fragility fractures, a systematic approach is needed after periprosthetic fractures., Level of Evidence: Level III, retrospective cohort study., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Osteoporotic fractures among long-term care residents have substantial economic and human costs. After a fracture, many older adults do not receive an osteoporosis diagnosis or evidence-based treatment, which leads to increased risk of recurrent fractures. Optimal processes are well defined for transitioning medical care after a hip or vertebral fracture for osteoporosis evaluation, but the handoff process from the specialist back to a primary care practitioner (PCP) or to a rehabilitative setting is not well defined. Our interdisciplinary quality improvement team developed and evaluated a program for transitioning care from a hospital-based fracture liaison clinic (FLC) to PCPs caring for older adults across the care continuum. To understand the current process of postfracture care transitions, we analyzed the postfracture patient experience. We surveyed PCPs to assess barriers to osteoporosis treatment, and retrospectively conducted a baseline analysis of 87 patients who had sustained an osteoporotic fracture in 2020. This preliminary work showed several opportunities for practice improvement and helped us develop a practical multicomponent intervention aimed at improving care transitions from the FLC to PCPs. The intervention (June-September 2021) comprised a standardized documentation template in the electronic health record (EHR) for FLC clinicians, a structured handoff process, and an engagement tool for patients outlining the roles and responsibilities of each care team member. We compared care transition measures before and after intervention. EHR documentation of an osteoporosis diagnosis increased from 56% (49 of 87 patients) before intervention to 92% (48 of 52) after intervention (P < .001). Additionally, increases were observed in documentation of treatment recommendations, associated risk factors, and PCP discussions with patients regarding osteoporosis and related treatment. This practical, commonsense intervention established clear roles for each care team member. The intervention addressed systemwide barriers in facilitating a safe transition from a subspecialty care team to PCPs providing care to older adults with osteoporosis., (Copyright © 2023 AMDA – The Society for Post-Acute and Long-Term Care Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: Osteoporosis is a common condition that can be caused or exacerbated by estrogen deficiency., Methods: This narrative review will discuss optimizing bone health in the setting of adjuvant endocrine treatments for hormone receptor-positive breast cancer and the current use of antiresorptive agents as adjuvant therapy and as bone modifying agents., Results: Adjuvant endocrine treatments for hormone receptor-positive breast cancer (tamoxifen and aromatase inhibitors) affect bone health. The exact effect depends on the agent used and the menopausal state of the woman. Antiresorptive medications for osteoporosis, bisphosphonates and denosumab, lower the risk of bone loss from aromatase inhibitors. Use of bisphosphonates as adjuvant treatment in breast cancer, regardless of hormone receptor status, is increasing because of benefits seen to cancer relapse and survival., Conclusion: Optimizing bone health in women with breast cancer during and after cancer treatment is informed by an understanding of breast cancer treatment and its skeletal effect., (Copyright © 2022 AACE. Published by Elsevier Inc. All rights reserved.)

Introduction: Both osteoporosis and cardiovascular disease (CVD) increase in women after menopause. Estrogen deficiency is thought to be an underlying mechanism for both these conditions., Methods: Healthy menopausal women (n = 374, age 42-58 years) underwent cardiac CT scans over four years as participants in the Kronos Early Estrogen Prevention Study (KEEPS), a randomized, controlled trial to Women randomized to either oral conjugated equine estrogens (o-CEE, n = 104), transdermal 17β-estradiol (t-E2, n = 119) or placebo (n-115). CAC (Agatston units, AU), and BMD (mg/cm 3 ) were measured from thoracic vertebrae at baseline and at the 4 years of the study using validated software. ANOVA and multiple linear regression analyzed the association between incident CAC or progression of CAC and BMD among the treatment groups., Results: At baseline 374 women, 40 participants with CAC >0 had greater decrements in BMD than the 334 participants with CAC = 0 at baseline, The average change in BMD in o-CEE group with CAC was -9.6 ± 13.3 versus -3.1 ± 19.5 in those with zero CAC, p = 0.0018. With t-E2, BMD changed by -11.7 ± 26.2 in those with CAC versus +5.7 ± 26.2 in the zero CAC group, p ≤ 0. 0001. Similarly in the 66 participants that showed progression of CAC >1, had more BMD loss, than those with stable CAC regardless of the treatment., Conclusion: Progression of bone loss is reduced among women treated with o-CEE or t-E2. Progression of CAC is associated with greater BMD loss, a relationship that is differentially modified by t-E2 and o-CEE., Competing Interests: Declaration of competing interest Matthew J. Budoff, M.D., FACC discloses work for the National Institutes of Health and General Electric Healthcare; All other authors have no conflicts of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Context: Survival in patients with primary hyperparathyroidism (PHPT) remains uncertain., Objective: To update survival in patients with PHPT in a United States community population., Design: Retrospective cohort study., Setting: Community population in Rochester, Minnesota., Participants: Residents who met criteria for PHPT from 1965 to 2010., Interventions: Survival was estimated using the Kaplan Meier product-limit method. The Cox proportional hazards model was used to determine associations, as relative hazards (RR) with 95% confidence intervals (CI), of various risk factors with time to death., Main Outcome Measure: The overall age and gender-adjusted survival compared to white Minnesota residents., Results: We identified 1139 PHPT individuals, 76% female, with a median age of 58 years. Most were observed without parathyroidectomy (69%). The relative risk of death among the entire cohort was 0.996 (95% CI: 0.91-1.09, P = 0.935) which was not different compared to Minnesota residents. Those with maximum serum calcium level ≥ 10.8 mg/dL (0.7 mg/dL above the reference range) had an increase in mortality (RR 1.32, 95% CI: 1.10-1.58, P = 0.002). Survival among all PHPT individuals after parathyroidectomy was no different from expected (RR = 1.06, 95% CI 0.89-1.28; P = 0.508). Mortality was significantly decreased after parathyroidectomy in those with serum calcium levels ≥10.8 mg/dL (HR 0.47, 95% CI: 0.36-0.61, P < 0.001)., Conclusions: Mortality in the entire cohort was not different from expected. PHPT patients with a maximum serum calcium level ≥ 10.8 mg/dL had increased mortality. Survival was improved after parathyroidectomy in those with this degree of hypercalcemia., (Copyright © 2021. Published by Elsevier Inc.)

Objective: Orthotopic liver transplant recipients are at high risk of fragility fractures both in pre-liver transplant (pre-LT) and in the immediate posttransplant (post-LT) period. The aims of this study were to identify risk factors associated with post-LT fracture and identify factors that contribute to changes in bone mineral density (BMD) in post-LT as they relate to the risk of fracture in the immediate post-LT period., Methods: We conducted a retrospective cohort study of first-time LT recipients who had BMD testing within 2-year pre-LT and 1-year post-LT. We assessed factors associated with immediate post-LT fracture using logistic regression models and linear regression models., Results: New fractures occurred in 41/286 (14.3%) of LT recipients during the first year following LT. In multivariate analysis, we noted an increased odds of fracture for patients with prior history of fracture (P < .001), patients who were older (P = .03), patients with higher end-stage liver disease score (P = .03), and patients with lower BMD. After adjustment for multiple testing, only a history of prior fracture was statistically significant., Conclusion: Our study demonstrated that prior fracture at any site was associated with developing a new fracture in the first year post-LT., (Copyright © 2021 AACE. Published by Elsevier Inc. All rights reserved.)

Background: Cardiac fat is emerging as an important parameter for cardiovascular risk stratification. Accurate and reproducible volumetric measurements can facilitate in the serial assessment of cardiac fat by computed tomography (CT). We assessed the intra- and inter-observer variability of cardiac fat volumetric measurements using a semi-automated CT software., Methods: We used non-contrast coronary calcium CT scans to quantify epicardial and intra-thoracic fat volumes. Two expert readers analyzed baseline and follow up CT scans of 45 subjects by using a semi-automated CT software (QFAT 2.0, Cedars Sinai-Medical Center). Correlation and Bland-Altman analysis was performed for both intra- and inter-observer comparisons for each cardiac fat type., Results: The intra-observer correlation coefficients ranged between 0.86 to 0.99 and 0.87 to 0.99 for epicardial (median fat per reader (cm 3 ) 20.9 to 25.7) and intra-thoracic (median fat per reader (cm 3 ) 27.1 to 31.6) fat volumes respectively, with no significant differences between individual data points (all p > 0.38). The inter-observer correlation coefficient was 0.99 (p < 0.0001 for correlation) for both epicardial and intra-thoracic fat. By Bland-Altman analysis for epicardial fat measurements, mean difference of intra-observer was 0.90 cm 3 with 95% confidence intervals (0.22,1.7) and -1.8 cm 3 for inter-observer, with 95% CI (-2.9, -0.69). Bland-Altman plots for intra-thoracic fat measurements were similarly impressive for both inter- and intra-observer reads., Conclusions: Our data showed that measuring epicardial and intra-thoracic fat volumes by CT using a semi-automated software has excellent intra-observer and inter-observer reliability. Cardiac fat volumes can be obtained easily and reproducibly from routine calcium scoring scans and may help in assessing cardiovascular risk., Clinical Trial Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT00154180; Keywords: Epicardial fat volume; intra-thoracic fat volume; computed tomography; intra-observer; inter-observer., (Copyright © 2020 Society of Cardiovascular Computed Tomography. Published by Elsevier Inc. All rights reserved.)

Objective: Several studies indicate that patients with primary hyperparathyroidism (PHPT) undergoing parathyroid surgery have improvement in mood and neuropsychological functioning. The current analysis aims to examine the relationship between biochemical and clinical variables and the improvement in depression scores and in specific symptoms, after parathyroidectomy., Design: A prospective observational case-control study at a referral centre., Patients: Patients with PHPT undergoing parathyroidectomy (n = 88) or thyroid surgery (n = 85)., Measurements: The Patient Health Questionnaire-9 (PHQ-9) was utilized to obtain depression scores at enrolment and 12 months after surgery. The changes in PHQ-9 were analysed and correlated with baseline clinical and biochemical parameters., Results: At enrolment, there was no difference between the groups in the number with a depression diagnosis (PHPT 34.1%, thyroid surgery, 35.5%, P = 0.86). However, baseline PHQ-9 scores were significantly higher in PHPT (median 7.5, range 0-27) than thyroid surgery patients (median 3.0, range 0-18, P < 0.0001). Following surgery, all PHQ-9 scores, total and symptom group (cognitive, somatic) improved and were no longer different between PHPT (total PHQ-9 median 2, range 0-16) and thyroid (median 1, range 0-14, P = 0.31) groups. Baseline parathyroid hormone level, but not calcium, had a weak relationship with change in PHQ-9 score after parathyroid surgery (P = 0.003). Baseline PHQ-9 score was correlated with change in PHQ-9 score at 12 months after parathyroid surgery (P < 0.001)., Conclusions: Depression scores improve in both somatic and cognitive domains after parathyroidectomy for PHPT and baseline severity of depression predicts the response., (© 2019 John Wiley & Sons Ltd.)

Background Heart fats (epicardial and paracardial adipose tissue [PAT]) are greater after menopause. Endogenous estrogen may regulate these fat depots. We evaluated the differential effects of hormone therapy formulations on heart fat accumulations and their associations with coronary artery calcification (CAC) progression in recently menopausal women from KEEPS (Kronos Early Estrogen Prevention Study). Methods and Results KEEPS was a multicenter, randomized, placebo-controlled trial of the effects of 0.45 mg/d oral conjugated equine estrogens and 50 µg/d transdermal 17β-estradiol, compared with placebo, on 48-month progression of subclinical atherosclerosis among 727 early menopausal women. CAC progression was defined if baseline CAC score was 0 and 48-month CAC score was >0 or if baseline CAC score was >0 and <100 and annualized change in CAC score was ≥10. Of 727 KEEPS participants, 474 (mean age: 52.7 [SD: 2.6]; 78.1% white) had computed tomography-based heart fat and CAC measures at both baseline and 48 months. Compared with women on placebo, women on oral conjugated equine estrogens were less likely to have any increase in epicardial adipose tissue (odds ratio for oral conjugated equine estrogens versus placebo: 0.62 [95% CI, 0.40-0.97]; P =0.03). PAT did not change in any group. Changes in epicardial adipose tissue and PAT did not differ by treatment group. CAC increased in 14% of participants. The assigned treatment modified the association between PAT changes and CAC progression ( P =0.02) such that PAT increases were associated with CAC increases only in the transdermal 17β-estradiol group. Conclusions In recently menopausal women, oral conjugated equine estrogens may slow epicardial adipose tissue accumulation, whereas transdermal 17β-estradiol may increase progression of CAC associated with PAT accumulation. Clinical Trial Registration URL: http://www.clinicaltrials.gov. Unique identifier: NCT00154180.

Objective: This study examined relationships, by pregnancy histories, between bone mineral density (BMD) and coronary artery calcification (CAC) in postmenopausal women., Methods: Forty women identified from their medical record as having pre-eclampsia (PE) were age/parity-matched with 40 women having a normotensive pregnancy (NP). Vertebral (T4-9) BMD and CAC were assessed by quantitative computed tomography in 73 (37 with PE and 36 with NP) of the 80 women. Analyses included linear regression using generalized estimating equations., Results: Women averaged 59 years of age and 35 years from the index pregnancy. There were no significant differences in cortical, trabecular or central BMD between groups. CAC was significantly greater in the PE group (p = 0.026). In multivariable analysis, CAC was positively associated with cortical BMD (p = 0.001) and negatively associated with central BMD (p = 0.036). There was a borderline difference in the association between CAC and central BMD by pregnancy history (interaction, p = 0.057)., Conclusions: Although CAC was greater in women with a history of PE, vertebral BMD did not differ between groups. However, both cortical and central BMD were associated with CAC. The central BMD association was marginally different by pregnancy history, suggesting perhaps differences in underlying mechanisms of soft tissue calcification.

Context: Thiazide diuretics, the antihypertensive agent prescribed most frequently worldwide, are commonly associated with hypercalcemia. However, the epidemiology and clinical features are poorly understood., Objective: To update the incidence of thiazide-associated hypercalcemia and clarify its clinical features., Patients and Methods: In a population-based descriptive study, Olmsted County, Minnesota, residents with thiazide-associated hypercalcemia were identified through the Rochester Epidemiology Project and the Mayo Clinic Laboratory Information System from 2002-2010 and were added to the historical cohort beginning in 1992., Main Outcome: Incidence rates were adjusted to the 2010 United States white population., Results: Overall, 221 Olmsted County residents were identified with thiazide-associated hypercalcemia an average of 5.2 years after initiation of treatment. Subjects were older (mean age, 67 years) and primarily women (86.4%). The incidence of thiazide-associated hypercalcemia increased after 1997 and peaked in 2006 with an annual incidence of 20 per 100,000, compared to an overall rate of 12 per 100,000 in 1992-2010. Severe hypercalcemia was not observed in the cohort despite continuation of thiazide treatment in 62.4%. Of patients discontinuing thiazides, 71% continued to have hypercalcemia. Primary hyperparathyroidism was diagnosed in 53 patients (24%), including five patients who underwent parathyroidectomy without thiazide discontinuation., Conclusions: Many patients with thiazide-associated hypercalcemia have underlying primary hyperparathyroidism. Additionally, a sharp rise in thiazide-associated hypercalcemia incidence began in 1998, paralleling the increase observed in primary hyperparathyroidism in this community. Case ascertainment bias from targeted osteoporosis screening is the most likely explanation.

Objective: To determine which residency characteristics are associated with performance during endocrinology fellowship training as measured by competency-based faculty evaluation scores and faculty global ratings of trainee performance., Methods: We performed a retrospective review of interview applications from endocrinology fellows who graduated from a single academic institution between 2006 and 2013. Performance measures included competency-based faculty evaluation scores and faculty global ratings. The association between applicant characteristics and measures of performance during fellowship was examined by linear regression., Results: The presence of a laudatory comparative statement in the residency program director's letter of recommendation (LoR) or experience as a chief resident was significantly associated with competency-based faculty evaluation scores (β = 0.22, P = .001; and β = 0.24, P = .009, respectively) and faculty global ratings (β = 0.85, P = .006; and β = 0.96, P = .015, respectively)., Conclusion: The presence of a laudatory comparative statement in the residency program director's LoR or experience as a chief resident were significantly associated with overall performance during subspecialty fellowship training. Future studies are needed in other cohorts to determine the broader implications of these findings in the application and selection process.

Background: Postmenopausal women with breast cancer receiving aromatase inhibitors are at an increased risk of bone loss. The current study was undertaken to determine whether upfront versus delayed treatment with zoledronic acid (ZA) impacted bone loss. This report described the 5-year follow-up results., Methods: A total of 551 postmenopausal women with breast cancer who completed tamoxifen treatment and were undergoing daily letrozole treatment were randomized to either upfront (274 patients) or delayed (277 patients) ZA at a dose of 4 mg intravenously every 6 months. In the patients on the delayed treatment arm, ZA was initiated for a postbaseline bone mineral density T-score of <-2.0 or fracture., Results: The incidence of a 5% decrease in the total lumbar spine bone mineral density at 5 years was 10.2% in the upfront treatment arm versus 41.2% in the delayed treatment arm (P<.0001). A total of 41 patients in the delayed treatment arm were eventually started on ZA. With the exception of increased NCI Common Toxicity Criteria (CTC) grade 1/2 elevated creatinine and fever in the patients treated on the upfront arm and cerebrovascular ischemia among those in the delayed treatment arm, there were no significant differences observed between arms with respect to the most common adverse events of arthralgia and back pain. Osteoporosis occurred less frequently in the upfront treatment arm (2 vs 8 cumulative cases), although this difference was not found to be statistically significant. Bone fractures occurred in 24 patients in the upfront treatment arm versus 25 patients in the delayed treatment arm., Conclusions: Immediate treatment with ZA prevented bone loss compared with delayed treatment in postmenopausal women receiving letrozole and these differences were maintained at 5 years. The incidence of osteoporosis or fractures was not found to be significantly different between treatment arms., (© 2015 American Cancer Society.)

Introduction of automated serum calcium measurements in the 1970s resulted in a sharp rise in primary hyperparathyroidism (PHPT) incidence. However, recent investigations suggest a significant rise in PHPT incidence for unclear reasons. Our objective was to update our population-based secular trends in PHPT incidence, to determine if there has been a significant rise in PHPT incidence as suggested by others, and, if possible, to identify changes in clinical practice that might be responsible. Rochester, Minnesota, residents who met the criteria for PHPT from 2002 through 2010 were identified through the medical records-linkage system of the Rochester Epidemiology Project and added to the historical cohort beginning in 1965. Incidence rates were adjusted to the 2010 US white population. Altogether, 1142 Rochester residents have been diagnosed with PHPT since 1965, including 341 in 2002-2010. Over time, two periods of increased PHPT incidence occurred, one beginning in 1974 (121.7 per 100,000 person-years) and a second peak (86.2 per 100,000 person-years) starting in 1998. The median age of PHPT subjects has increased significantly from 55 years in 1985-1997 to 60 years of age in 1998-2010 and more patients (36%) had a parathyroidectomy in 1998-2010. Although serum calcium measurement has declined since 1996, there was a progressive increase in parathyroid hormone testing between 1994 and 2008. There was also a rise in orders for bone mineral density measurements in women since 1998, which peaked in 2003-2004. A second sharp rise in PHPT incidence occurred in our community in 1998, simultaneously with the introduction of national osteoporosis screening guidelines, Medicare coverage for bone density measurement, and new medications for the treatment of osteoporosis. Case ascertainment bias from targeted PHPT screening in patients being evaluated for osteoporosis is the most likely explanation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Purpose: To assess the quality of an initiative to improve the diagnosis and management of osteoporosis in patients over 50 years of age with distal radius fractures (DRF)., Methods: A retrospective review was conducted to determine the baseline percentage of individuals undergoing osteoporosis screening after DRF. Thereafter, a study was implemented in which DRF patients who were not being treated for osteoporosis or had not recently undergone screening were offered a dual-energy x-ray absorptiometry scan and referral to endocrinology at the initial hand surgery clinic visit. Patients who declined participation were contacted by a patient educator to discuss the benefits of screening and address their concerns. Those who then wanted to receive an osteoporosis evaluation were scheduled for bone scanning and endocrinology consultation., Results: During the baseline period, 7 patients (15%) were screened, and 41 (85%) were not screened. During the active phase of the initiative, 82 patients over 50 years of age were treated for a DRF at our institution. A total of 44 patients were identified for potential osteoporosis screening, and 35 patients met inclusion criteria. Of these, 19 (54%) agreed to screening after the initial orthopedic evaluation, and 16 declined. After speaking to a patient educator, 9 of these 16 patients agreed to screening. Of the remaining 7 patients, 4 again declined screening and 3 were unavailable by telephone. Overall, 80% of patients who were identified in the initiative agreed to osteoporosis screening after the combination of recommendation during hand surgery clinic visit and patient education by telephone, and 64% were diagnosed with osteoporosis/osteopenia as a result of completing screening., Conclusions: An integrated model of care among orthopedic surgeons, patient educators, and endocrinologists substantially increased screening for osteoporosis after DRF., Type of Study/level of Evidence: Prognostic II., (Copyright © 2014 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.)

Context: In women, cortical bone mass decreases significantly at menopause. By contrast, loss of trabecular bone begins in the third decade and accelerates after menopause., Objective: The aim of the study was to investigate the effects of estrogen on cortical and trabecular bone., Design: The Kronos Early Estrogen Prevention Study is a double-blind, randomized, placebo-controlled trial of menopausal hormone treatment (MHT) in women, enrolled within 6-36 months of their final menstrual period., Setting: The study was conducted at the Mayo Clinic, Rochester, Minnesota., Intervention: Subjects were treated with placebo (n = 31), or .45 mg/d conjugated equine estrogens (n = 20), or transdermal 50 μg/d 17β-estradiol (n = 25) with pulsed micronized progesterone., Main Outcome Measures: Cortical and trabecular microarchitecture at the distal radius was assessed by high-resolution peripheral quantitative computed tomography., Results: At the distal radius, cortical volumetric bone mineral density (vBMD) decreased, and cortical porosity increased in the placebo group; MHT prevented these changes. By contrast, MHT did not prevent decreases in trabecular microarchitecture at the radius. However, MHT prevented decreases in trabecular vBMD at the thoracic spine (assessed in a subset of subjects; n = 51). These results indicate that MHT prevents deterioration in radial cortical vBMD and porosity in recently menopausal women., Conclusion: The maintenance of cortical bone in response to estrogen likely has important clinical implications because cortical bone morphology plays an important role in bone strength. However, effects of MHT on trabecular bone at the radius differ from those at the thoracic spine. Underlying mechanisms for these site-specific effects of MHT on cortical vs trabecular bone require further investigation.

Background: Patients with primary hyperparathyroidism often lack classic symptoms but can have reductions in bone mineral density and increased fracture risk. We sought to determine bone mineral density improvement after successful surgery and associated factors., Methods: A review of patients with osteopenia or osteoporosis with curative parathyroidectomy and both pre- and postoperative dual-energy X-ray absorptiometry bone mineral density scans was conducted. We compared patients with declining (<0%), moderate improvement (0.1-5%), and significant improvement (>5%) on dual-energy X-ray absorptiometry bone mineral density scans., Results: We identified 420 patients who underwent a dual-energy X-ray absorptiometry bone mineral density scan preoperatively and within 36 months postoperatively. At the most affected site, 38% had significant improvement, 31% moderate improvement, and 31% declining bone mineral density. Patients who significantly improved were younger (P = .01), had lesser preoperative dual-energy X-ray absorptiometry (P = .001), and had greater preoperative levels of parathyroid hormone (P = .04), serum calcium (P = .03), and preoperative urinary calcium. There was no difference in outcomes between sex and with preoperative bisphosphonate use. Average hip and spine bone mineral density had similar responses to surgery., Conclusion: Bone mineral density improves in up to 75% of patients after curative parathyroidectomy for primary hyperparathyroidism. The hip and lumbar spine responded similarly. Younger patients and those with severe primary hyperparathyroidism may derive the most skeletal benefits from parathyroidectomy, but the uniform positive response supports parathyroidectomy in patients with osteoporosis and possibly osteopenia., (Copyright © 2012 Mosby, Inc. All rights reserved.)

Objectives: Coronary artery disease and osteoporosis increase in women after menopause. Computed tomography (CT) scans of the heart used to evaluate coronary arterial calcification include images of the thoracic vertebrae. The utility of using these images to assess bone health in women remains to be defined. Analyses of thoracic spine volumetric bone mineral density (vBMD) from CT scans of the heart were performed to determine how specific calibration affects the ability to assess vBMD in recently menopausal women and to evaluate how vBMD relates to areal bone mineral density (aBMD) using dual-energy X-ray absorptiometry (DEXA)., Methods: Women (n = 111) enrolled in the Kronos Early Estrogen Prevention Study (KEEPS) at Mayo Clinic underwent a CT scan of the heart that included calibration phantoms and a DEXA of the lumbar spine. The Spine Cancer Assessment program was used to determine vBMD of thoracic vertebrae with and without the calibration correction., Results: Trabecular bone vBMD at T8 averaged 163.57±28.58 and 157.94±27.55 mg/cc (mean±standard deviation, SD) for calibrated and uncalibrated values, respectively. The relationship between calibrated and uncalibrated measures approached unity (R = 0.98). Lumbar spine (L2-4) aBMD was 1.19±0.16 g/cm(2) (mean±SD). Both calibrated and uncalibrated thoracic vBMD correlated positively and significantly with lumbar aBMD, but the relationship was less than unity (R = 0.63)., Conclusion: Uncalibrated measures of thoracic spine vBMD obtained from CT scans of the heart may provide clinically relevant information about bone health and osteoporosis/osteopenia risk in recently menopausal women.

Context: Patients with primary hyperparathyroidism (PHP) often report nonspecific symptoms including mood disturbances., Objective: The objective of the study was to determine the frequency of depression in PHP and assess its response to parathyroidectomy., Methods: A case-control study at a referral center in Rochester, MN, performed Patient Health Questionnaire-9 (PHQ-9) assessments in observed (n = 81) and surgical (n = 88) PHP and benign nontoxic surgical thyroid disease (n = 85) at baseline and 1, 3, 6, and 12 months after surgery or the initial questionnaire in observed PHP. Baseline PHQ-9 scores and their response to surgery were evaluated., Results: The groups were similar in gender and depression history, but PHP patients were older. Baseline PHQ-9 scores were 1.71 points higher in PHP than controls after adjusting for age and gender (P = .004). Clinically significant PHQ-9 scores (≥10) were twice as common in PHP (31.4%) compared with thyroid subjects (15.3%). Parathyroidectomy resulted in significant and sustained reductions in PHQ-9 scores, which were greater than observed PHP at all time points (P < .001). PHP patients with clinically significant PHQ-9 scores dropped to 7.4% (P < .001) and 7.6% (P < .001) at 1 month and 1 yr after parathyroidectomy. There were greater declines in PHQ-9 scores after parathyroidectomy at 1, 3, and 6 months (P < .001) and 1 yr (P = .061) compared with thyroid surgery., Conclusions: Depression is common in patients with PHP. Parathyroidectomy results in greater improvement in PHQ-9 scores compared with thyroid surgery or observation of PHP.

Objectives: Cardiovascular disease and osteoporosis increase in women after menopause. While aortic calcification is associated with bone loss in women, a similar relationship for coronary arterial calcification (CAC), a risk factor for coronary artery disease in women, is less clear. This study was designed to examine the relationship between CAC and volumetric bone mineral density (vBMD) in women (n=137) who were within a median of 18 months past their last menses at screening for the Kronos Early Estrogen Prevention Study (KEEPS)., Methods: CAC was measured using 64-slice computed tomography; vBMD was measured from these images using the Spine Cancer Assessment program. Concentrations of osteocalcin, bone alkaline phosphatase, tartrate-resident acid phosphatase-5b and osteopontin as bone matrix protein in serum and plasma were evaluated by ELISA., Results: CAC scores ranged from 0 to 327.6 Agatston Units (AU); 113 women had a score of 0 AU, 20 had a CAC score between 0 and 50 AU, and four had a CAC score>50 AU. Although not statistically significant, there was a trend toward decreasing central density of thoracic T9 with increasing CAC. On average, levels of markers of bone turnover were within the normal range but did not correlate with age or with months past menopause., Conclusions: Clinically significant CAC and spine vBMD are quantifiable from the same scans within the first 3 years of menopause. Additional work is needed to determine how these measurements change with increasing age or with estrogenic treatments.

Unlabelled: The decline in hip fracture incidence is now accompanied by a further reduction in the likelihood of a recurrent hip fracture among survivors of the first fracture., Introduction: Hip fracture incidence is declining in North America, but trends in hip fracture recurrence have not been described., Methods: All hip fracture events among Olmsted County, Minnesota residents in 1980-2006 were identified. Secular trends were assessed using Poisson regression, and predictors of recurrence were evaluated with Andersen-Gill time-to-fracture regression models., Results: Altogether, 2,752 hip fractures (median age, 83 years; 76% female) were observed, including 311 recurrences. Between 1980 and 2006, the incidence of a first-ever hip fracture declined by 1.37%/year for women (p < 0.001) and 0.06%/year for men (p = 0.917). Among 2,434 residents with a first-ever hip fracture, the cumulative incidence of a second hip fracture after 10 years was 11% in women and 6% in men with death treated as a competing risk. Age and calendar year of fracture were independently associated with hip fracture recurrence. Accounting for the reduction in first-ever hip fracture rates over time, hip fracture recurrence appeared to decline after 1997., Conclusion: A recent reduction in hip fracture recurrence is somewhat greater than expected from the declining incidence of hip fractures generally. Additional research is needed to determine the extent to which this can be attributed to improved patient management.

Osteoporosis is a common cause of vertebral compression fractures. Although vertebroplasty is used to treat the pain, the risk of additional compression fractures is very high in these patients. Adequate evaluation and management of the underlying osteoporosis is critical to reducing the risk of subsequent fractures. Such an evaluation involves understanding the underlying physiology of osteoporosis and the role of calcium, vitamin D, prescription medication, and lifestyle changes. This brief review is intended to familiarize neuroradiologists with these aspects so they can advise patients about optimizing fracture risk reduction.

To better define the relationship between vascular calcification and bone mass/structure, we assessed abdominal aortic calcification (AAC), BMD, and bone microstructure in an age-stratified, random sample of 693 Rochester, MN, residents. Participants underwent QCT of the spine and hip and high-resolution pQCT (HRpQCT) of the radius to define volumetric BMD (vBMD) and microstructural parameters. AAC was quantified with the Agatston scoring method. In men, AAC correlated with lower vertebral trabecular and femoral neck vBMD (p < 0.001), but not after age or multivariable (age, body mass index, smoking status) adjustment. Separation into <50 and >or=50 yr showed this pattern only in the older men. BV/TV and Tb.Th inversely correlated with AAC in all men (p < 0.001), and Tb.Th remained significantly correlated after age adjustment (p < 0.05). Tb.N positively correlated with AAC in younger men (p < 0.001) but negatively correlated in older men (p < 0.001). The opposite was true with Tb.Sp (p = 0.01 and p < 0.001, respectively). Lower Tb.N and higher Tb.Sp correlated with AAC in older men even after multivariable adjustment. Among all women and postmenopausal women, AAC correlated with lower vertebral and femoral neck vBMD (p < 0.001) but not after adjustment. Lower BV/TV and Tb.Th correlated with AAC (p = 0.03 and p = 0.04, respectively) in women, but not after adjustment. Our findings support an age-dependent association between AAC and vBMD. We also found that AAC correlates with specific bone microstructural parameters in older men, suggesting a possible common pathogenesis for vascular calcification and deterioration in bone structure. However, sex-specific differences exist.

Osteoclasts and osteoblasts dictate skeletal mass, structure, and strength via their respective roles in resorbing and forming bone. Bone remodeling is a spatially coordinated lifelong process whereby old bone is removed by osteoclasts and replaced by bone-forming osteoblasts. The refilling of resorption cavities is incomplete in many pathological states, which leads to a net loss of bone mass with each remodeling cycle. Postmenopausal osteoporosis and other conditions are associated with an increased rate of bone remodeling, which leads to accelerated bone loss and increased risk of fracture. Bone resorption is dependent on a cytokine known as RANKL (receptor activator of nuclear factor kappaB ligand), a TNF family member that is essential for osteoclast formation, activity, and survival in normal and pathological states of bone remodeling. The catabolic effects of RANKL are prevented by osteoprotegerin (OPG), a TNF receptor family member that binds RANKL and thereby prevents activation of its single cognate receptor called RANK. Osteoclast activity is likely to depend, at least in part, on the relative balance of RANKL and OPG. Studies in numerous animal models of bone disease show that RANKL inhibition leads to marked suppression of bone resorption and increases in cortical and cancellous bone volume, density, and strength. RANKL inhibitors also prevent focal bone loss that occurs in animal models of rheumatoid arthritis and bone metastasis. Clinical trials are exploring the effects of denosumab, a fully human anti-RANKL antibody, on bone loss in patients with osteoporosis, bone metastasis, myeloma, and rheumatoid arthritis.

Purpose: The study determines the incidence of thiazide-associated hypercalcemia and clarifies its clinical features and natural history., Methods: In a population-based descriptive study, Olmsted County, Minn, residents with thiazide-associated hypercalcemia were identified through the Rochester Epidemiology Project and the Mayo Clinic Laboratory Information System. Changes in incidence rates were evaluated by Poisson regression., Results: Seventy-two Olmsted County residents (68 women and 4 men; mean age, 64 years) with thiazide-associated hypercalcemia first recognized in 1992 to 2001 were identified. The overall annual age- and sex-adjusted (to 2000 US whites) incidence was 7.7 (95% confidence interval [CI], 5.9-9.5) per 100,000. There was an increase in incidence after 1996, peaking at 16.3 (95% CI, 8.3-24.3) per 100,000 in 1998. The highest rate was 55.3 per 100,000 in 70- to 79-year-old women. Hypercalcemia was identified a mean of 6+/-7 years after thiazide initiation, and the average highest serum calcium was 10.7+/-0.3 mg/dL with serum parathyroid hormone (obtained in 53 patients) of 4.8+/-2.7 pmol/L. Of 33 patients who discontinued the thiazide, 21 (64%) had persistent hypercalcemia. Patients subsequently diagnosed with primary hyperparathyroidism had the highest average serum calcium and parathyroid hormone levels of 11.0+/-0.3 mg/dL and 6.3+/-2.4 pmol/L, respectively., Conclusion: The persistence of hypercalcemia in patients discontinuing thiazides, and similarities in the clinical spectrum, suggest that underlying primary hyperparathyroidism is common in patients who develop hypercalcemia while taking thiazide diuretics.

Primary hyperparathyroidism remains the most common cause of hypercalcemia encountered in outpatient practice. Management of the asymptomatic patient with mild disease remains controversial. Surgery is the only curative therapy, and minimally invasive procedures are now possible.

Sex steroid hormones are essential to normal skeletal growth and maintenance throughout life in both men and women. The importance of estrogens to bone health in women becomes obvious at menopause when estrogen deficiency occurs and results in accelerated bone loss. After menopause, estrogen deficiency results in drastic changes in the androgen-estrogen ratio. Thus, the relative importance of androgens after menopause may increase. Androgens also appear to be important for bone health in pre-menopausal women. Evidence from human, animal, and laboratory studies is leading to a better understanding of the effects of androgens on bone in women.

Steroid receptor coactivator (SRC)-1 is an important nuclear receptor coactivator that enhances estrogen (E) action in many tissues, but its role in mediating E effects on bone is unknown. Thus, we assessed the skeletal response to ovariectomy (ovx) and E replacement in SRC-1 knockout (KO) mice compared with wild-type (WT) littermates. Bone mineral density was measured by dual-energy x-ray absorptiometry and peripheral quantitative computed tomography at baseline and after 2 months of sham surgery, ovx, or ovx plus E replacement. Microcomputed tomography and bone histomorphometry were also performed at the end of the study. Both WT and SRC-1 KO mice lost bone at multiple sites after ovx; however, although an estradiol (E(2)) dose of 10 microg/kg.d completely prevented loss of cancellous bone (at the lumbar spine and tibial metaphysis) in the WT mice, it was entirely ineffective in preventing cancellous bone loss at these sites in the SRC-1 KO mice. This E(2) dose was, however, equally effective on cortical bone in the tibia in the SRC-1 KO and WT mice. Moreover, a 4-fold higher dose of E(2) was able to overcome the deficit in E action in cancellous bone in the SRC-1 KO mice. These findings establish that, in mice, loss of SRC-1 leads to skeletal resistance to E predominantly in cancellous bone.

Hypercalcemia is frequently encountered in healthy outpatients. Reliable measurements of the mediators of hypercalcemia have improved diagnostic certainty about the etiology in most patients. Hyperparathyroidism is overwhelmingly the most common cause. Medical evaluation of the patient with hyperparathyroidism requires an understanding of the complications of the disorder and the associated syndromes. At present decreased bone mineral density and nephrolithiasis are the major sequelae of hyperparathyroidism. Most cases of primary hyperparathyroidism are sporadic; however, hereditary forms can occur in patients with the multiple endocrine neoplasia syndromes. Surgery is the only curative therapy. Results are excellent when an experienced endocrine surgeon performs parathyroid surgery.

The bone morphogenic proteins (BMPs) play a key role in skeletal development and patterning. Using the technique of differential display polymerase chain reaction (ddPCR), we have identified a novel gene whose expression is increased during BMP-2-induced differentiation of the prechondroblastic cell line, MLB13MYC clone 17, to an osteoblastic phenotype. The 6.5-kilobase mRNA recognized by this ddPCR product is increased 10-fold by BMP-2 treatment of the MLB13MYC clone 17 cells. The mRNA recognized by this ddPCR product is also increased as MC3T3-E1 cells recapitulate the program of osteoblast differentiation during prolonged culture. The full-length transcript corresponding to this ddPCR product was cloned from a MLB13MYC clone 17 cell cDNA library. Analysis of the deduced amino acid sequence demonstrated that this gene encodes a novel 126-kDa putative serine/threonine protein kinase containing a nuclear localization signal. The kinase domain, expressed in Escherichia coli, is capable of autophosphorylation as well as phosphorylation of myelin basic protein. The gene was, therefore, named BIKe (BMP-2-Inducible Kinase). The BIKe nuclear localization signal is able to direct green fluorescent protein to the nucleus in transfected COS-7 cells. When stably expressed in MC3T3-E1 cells, BIKe significantly decreases alkaline phosphatase activity and osteocalcin mRNA levels and retards mineral deposition relative to vector control. This novel kinase, therefore, is likely to play an important regulatory role in attenuating the program of osteoblast differentiation.

Objective: To compare the prolactogenic effects of risperidone, clozapine, and typical antipsychotic agents in an outpatient community-based psychiatric population., Methods: Prolactin and thyroid-stimulating hormone (TSH) concentrations were measured in 68 outpatients with schizophrenia who were receiving antipsychotic medications and were recruited from a community mental health clinic., Results: The percentage of women with increased prolactin concentrations was significantly greater in the risperidone group (100%, 12 of 12 patients) than in the clozapine group (25%, 1 of 4) (P = 0.0071) but not in comparison with the typical antipsychotic agent group (83%, 5 of 6) (P = 0.333). The percentage of men with increased prolactin concentrations was significantly greater in the risperidone group (94%, 17 of 18) than in the clozapine group (18%, 3 of 17) (P<0.0001) and in comparison with the typical antipsychotic agent group (27%, 3 of 11) (P = 0.0003). The mean prolactin concentration (all ng/mL +/- standard deviation) was also significantly higher in patients taking risperidone (women, 125.0 +/- 56.6; men, 37.3 +/- 23.9) than clozapine (women, 22.0 +/- 25.9; men, 13.3 +/- 22.4) (female patients, P = 0.0004; male patients, P<0.0001) or typical antipsychotic agents (women, 69.0 +/- 59.8; men, 13. 3 +/- 9.1) (female patients, P = 0.036; male patients, P = 0.0003). In the risperidone group, gender affected prolactin level, with women having higher concentrations than men, but the duration of therapy did not. In this group, prolactin was inversely dependent on age. No difference was noted in TSH concentrations between medication groups., Conclusion: Risperidone is a potent inducer of hyperprolactinemia in outpatients with schizophrenia in a community population. The higher and more frequently increased prolactin concentrations caused by risperidone could adversely affect patient health and compliance.

Activins are members of the transforming growth factor beta (TGF-beta) superfamily and have been shown to be multifunctional regulators of development and cell differentiation. Increasing evidence suggests activin betaA is involved in skeletal development. Using differential display PCR we have identified activin betaA as a gene associated with recombinant human bone morphogenetic protein-2 (rhBMP-2) induced differentiation of a mouse limb bud cell line, MLB13MYC clone 17, from a prechondroblastic to an osteoblastic phenotype. The expression of activin betaA peaks at 24 h of rhBMP-2 treatment, before detection of osteocalcin mRNA expression. Cycloheximide treatment inhibits induction of activin betaA, indicating a requirement for new protein synthesis. The induction of the mRNA encoding follistatin, an activin binding protein, was also examined. Follistatin mRNA increases within 18 h of rhBMP-2 treatment, as activin betaA mRNA increases but before it peaks. Treatment of MLB13MYC clone 17 cells with purified activin betaA concomitant with rhBMP-2 does not affect markers of chondrocyte or osteoblast differentiation, nor does treatment with purified activin betaA alone. This suggests that activin betaA exerts its effect via a paracrine mechanism. In situ hybridization analysis demonstrates that activin betaA expression is localized to cells in the developing interphalangeal joints of embryonic mouse limbs. This is consistent with in vivo induction by BMP-2 which is also expressed in the developing joints. Activin betaA, therefore, is downstream from BMP-2 in the cascade of events that result in skeletal development., (Copyright 2000 Wiley-Liss, Inc.)

The regulation of osteocalcin gene transcription is complex, involving multiple positive and negative regulators. Previous studies have demonstrated that an intronic sequence, TTTCTTT (+118 to +124) is capable of mediating transcriptional repression of osteocalcin-CAT fusion genes in cells of the osteoblast lineage, by interacting with a specific nuclear protein. Further analyses of intronic sequences have identified a second silencer motif in this region. Two copies of a CCTCCT motif are present within the first intron of the rat osteocalcin gene (+106 to +111 and +135 to +140) and are capable of mediating transcriptional repression of osteocalcin-CAT fusion genes in rat osteosarcoma cells. Transient gene expression assays of wild-type and mutant osteocalcin-CAT fusion genes into ROS 17/2.8 cells demonstrate that mutagenesis of either of these CCTCCT motifs in isolation results in a 1.6-fold increase in CAT activity relative to the parent fusion gene. Moreover, a 5-fold increase in reporter gene activity is observed when both motifs are mutated together. These sequences are also capable of suppressing osteocalcin promoter activity when placed upstream to the osteocalcin promoter. Gel retardation and southwestern analyses demonstrate that the CCTCCT motifs interact with specific proteins present in nuclear extracts from ROS 17/2.8 and UMR 106 osteosarcoma cells but not COS-7 kidney cells. Mutations that abolish suppressor function of this motif markedly impair interactions with this specific nuclear protein. These data demonstrate that at least two different silencer motifs (TTTCTTT and CCTCCT) in the first intron of the rat osteocalcin gene contribute to its transcriptional repression.

The subcellular site of xylosylation, the first carbohydrate modification of the core protein that initiates glycosaminoglycan chain synthesis, was characterized in situ. Methods were developed to combine electron microscopic (EM) autoradiography and the radiolabeling of semi-intact chondrocytes. In the accompanying paper, Kearns et al. (Kearns, A. E., Vertel, B. M., and Schwartz, N. B. (1993) J. Biol. Chem. 268, 11097-11104) presented biochemical and subcellular fractionation studies that utilized semi-intact chondrocytes and radiolabeled UDP sugars to overcome obstacles to the direct analysis of xylosylation. The results suggested that xylosylation begins in the endoplasmic reticulum (ER) and continues in the Golgi. The site of xylosylation was not specified further due to the limitations of subcellular fractionation techniques. The studies described in this report were undertaken to localize these modifications directly in situ. Semi-intact cell preparations were optimized for ultrastructural preservation by modifications of permeabilization methods utilizing nitrocellulose filter overlays. Biochemical analysis demonstrated the exclusive incorporation of UDP-xylose into the cartilage chondroitin sulfate proteoglycan (aggrecan) core protein and 3'-phosphoadenosine 5'-phosphosulfate (PAPS) into the highly modified proteoglycan monomer. Immunolocalization studies showed the equivalence of cytoplasmic subcompartments in normal and semi-intact chondrocytes at the levels of light and electron microscopy. Once the biochemical and morphological equivalence of intact and semi-intact cells was established, EM autoradiographic studies were pursued using UDP-[3H]xylose and [35S]PAPS. Based on both qualitative and quantitative data, silver grains resulting from incorporated sulfate were concentrated in the perinuclear Golgi, while those resulting from incorporated xylose were found at the cis or forming face of the Golgi and in vesicular regions of the peripheral cytoplasm associated with the late ER. These data support the view that xylose addition begins in a late ER compartment and continues in intermediate compartments, perhaps including the cis-Golgi.

In order to define the location and organization of the numerous reactions involved in polysaccharide assembly during synthesis of proteoglycans and glycoproteins, the topography of some of the glycosylation reactions in chondroitin sulfate synthesis was examined using a relatively new technique for generating permeable cells. Permeable chondrocytes were shown to directly take up nucleotide sugar precursors and incorporate them into chondroitin sulfate proteoglycan (CSPG), allowing specific labeling at each step in chondroitin sulfate synthesis. Subcellular fractionation following labeling with UDP-[14C]xylose, UDP-[14C]galactose, UDP-[14C]glucuronic acid, or [35S]PAPS localized the labeled CSPG to the compartment where each glycosylation reaction occurred. From these experiments it appears that xylose addition begins in the endoplasmic reticulum and continues in the Golgi apparatus where galactose, glucuronic acid, and sulfate are added. This conclusion was confirmed by direct visualization of xylose incorporation using electron microscopic autoradiography (Vertel, B. M., Walters, L. M., Flay, N., Kearns, A. E., and Schwartz, N. B. (1993) J. Biol. Chem. 268, 11105-11112). Further examination of xylose addition showed that permeable chondrocytes can utilize both exogenous UDP-xylose transported into the lumen and UDP-xylose generated from UDP-glucuronic acid within the lumen. The enzyme responsible for this reaction, UDP-glucuronate carboxy-lyase, co-localized with xylosyltransferase activity in subcellular fractions. Orientation toward the lumen in subcellular compartments was determined by trypsin sensitivity in the permeable chondrocytes. Therefore, we conclude that UDP-xylose can be produced in the lumen of the compartment where it is utilized in CSPG synthesis, obviating the need for a direct transport mechanism for this nucleotide sugar and providing close regulation of UDP-xylose and UDP-glucuronic acid levels.

3'-Phosphoadenosine 5'-phosphosulfate (PAPS) functions as the high-energy sulfate donor for sulfate ester synthesis in all higher organisms. This activated sulfate, like its adenosine 5'-phosphosulfate precursor, is both chemically labile and vulnerable to sulfohydrolase degradation. These obstacles have limited the utility of the native PAPS in the purification and mechanistic description of the numerous PAPS-utilizing enzymes. This paper describes the synthesis of the 2'- and 3'-isomers of a nonhydrolysable, and thus stable, PAPS analog, beta-methylene-PAPS, from the previously described beta-methylene-APS (L. Callahan et al., Anal. Biochem. 177, 67-71, 1989). The method involves phosphorylation of beta-methylene-APS with trimetaphosphate and separation of the resulting mixed 2'(3')-isomers by ion-pair reverse-phase HPLC. The utilization of this analog as an inhibitor of APS kinase and PAPS translocase, two of the numerous PAPS-utilizing activities, as well as an affinity ligand for purification of APS kinase, is described.

The nature of the primary signals important for the addition of xylose to serines on the core protein of the cartilage chondroitin sulfate proteoglycan has been investigated. The importance of consensus sequence elements (Acidic-Acidic-Xxx-Ser-Gly-Xxx-Gly) in the natural acceptor was shown by the significant decrease in acceptor capability of peptide fragments derived by digestion of deglycosylated core protein with Staphylococcus aureus V8 protease, which cleaves within the consensus sequence, compared to the similar reactivity of trypsin-derived peptide fragments, in which consensus sequences remain intact. A comparison of the acceptor efficiencies (Vmax/Km) of synthetic peptides containing the proposed xylosylation consensus sequence and the natural acceptor (deglycosylated core protein) was then made by use of the in vitro xylosyltransferase assay. The two types of substrates were found to have nearly equivalent acceptor efficiencies and to be competitive inhibitors of each other's acceptor capability, with Km = Kiapparent. These results suggest that the artificial peptides containing the consensus sequence are analogues of individual substitution sites on the core protein and allowed the kinetic mechanism of the xylosyltransferase reaction to be investigated, with one of the artificial peptides as a model substrate. The most probable kinetic mechanism for the xylosyltransferase reaction was found to be an ordered single displacement with UDP-xylose as the leading substrate and the xylosylated peptide as the first product released. This represents the first reported formal kinetic mechanism for this glycosyltransferase and the only one reported for a nucleotide sugar:protein transferase.