David A. Ontjes

Increased Rate of Fractures and Severe Kyphosis: Sequelae of Living into Adulthood with Cystic Fibrosis

Cystic fibrosis is the most common fatal autosomal recessive genetic disease in white persons; it affects approximately 30 000 Americans and a similar number of Europeans [1]. Cystic fibrosis mutations occur in approximately 1 of every 2500 live births in the white population and lead to death or lung transplantation in more than 500 persons annually in the United States alone [2]. Although respiratory disease is the greatest cause of illness and death in patients with cystic fibrosis, improved therapy for chronic pulmonary infection has markedly extended life expectancy and has led to the discovery of myriad other problems that afflict these patients [3]. Osteoporosis, in particular, increases pain and debilitation in patients with cystic fibrosis as they live into adulthood. Patients with cystic fibrosis have increased risk for osteoporosis as a result of multiple factors [4]. Poor nutrition, pancreatic insufficiency, reduced absorption of calcium and vitamin D, reduced physical activity, delayed and reduced production of sex steroids, use of corticosteroids, and increased circulating concentrations of osteoclast-activating factors (such as tumor necrosis factor- and interleukin-1) may all cause reduced bone mineral density in patients with cystic fibrosis. Young patients with cystic fibrosis invariably fail to reach peak bone mass [5-10], and this contributes to low bone mineral density in adulthood. Unbalanced bone formation and resorption [11], caused by accelerated bone loss, may lead to further bone demineralization in early adulthood. Both increased bone resorption and decreased bone formation probably play a role in osteoporosis in cystic fibrosis [8, 12, 13]. Osteoporosis in patients with cystic fibrosis is well documented [5-10, 12-16]. Hahn and colleagues [5] were the first to show low bone mineral density (mean reduction, 15%) in the distal radii of these patients. In the past decade, reductions in bone mineral density for the femur (mean decrease, 11.1%), lumbar spine (mean decreases, 12.5% to 35%), and total body (mean decrease, 10%) [6, 12, 14] have been reported in adults with cystic fibrosis. Henderson and Madsen [7] recently found that patients with cystic fibrosis had low bone mineral density in childhood and that it worsened with age. The average z-scores for the lumbar spine were 0.39 for children aged 5 to 8 years, 0.99 for children aged 8 to 12 years, and 1.69 for children aged 12 to 18 years. Taken together, these results show that osteopenia may occur as early as the first decade of life in patients with cystic fibrosis and that bone loss accelerates during adolescence and early adulthood. Although low bone mineral density in cystic fibrosis has attracted considerable attention, data on the complications of osteoporosis, including fractures and kyphosis, are limited. Despite low bone mineral density and anecdotal reports of increased fracture rates [9, 14, 15, 17-20], no reports have documented significant increases in fracture rates in adults with cystic fibrosis. It has been suggested that the decreased activity level that accompanies progressive cystic fibrosis offers a protective influence with respect to the occurrence of fractures. Our main goal was to quantitate the clinical sequelae of osteoporosis, namely, fractures and kyphosis, in a large group of adults with cystic fibrosis to test the hypothesis that fracture rates and kyphosis angles are greater in patients with cystic fibrosis than in the general population. A second goal was to investigate the role of potentially important clinical variables in the pathogenesis of osteoporosis in cystic fibrosis. Methods Patients The study sample consisted of 70 adults (older than 18 years of age) with advanced cystic fibrosis who were referred for lung transplantation at the University of North Carolina between January 1994 and December 1996. The Committee on Human Research (IRB #96-Med-336) approved this retrospective cohort study and required verbal consent from participating patients. Cystic fibrosis was diagnosed by elevated sweat chloride concentrations and an appropriate clinical picture. All patients had end-stage lung disease and an anticipated survival of less than 2 to 3 years [21]. Fracture History The history, date, and mechanism of fracture were determined by personal interviews done using methods similar to those of the National Health Interview Study [22]. Fractures were required to have patient report of radiographic confirmation at the time of the fracture, but we did not review these radiographs. Confirmation of long-bone fractures required patient report of treatment with casting. Fractures occurring between birth and 6 years of age were not assessed because most patients were unable to provide accurate histories for those years. The number of years that each patient was at risk for fracture was summed by age interval to determine the total number of patient-years for this cohort. All patients were asked to give their date of puberty; to give a detailed history of corticosteroid use (expressed as cumulative dose of prednisone in grams); to state whether they had received therapy for osteoporosis; and, if they were female, to state whether and when they had had oligomenorrhea. Bone Densitometry Bone mineral density was measured in all patients by a single, registered radiologic technologist using dual-energy x-ray absorptiometry (Hologic QDR 1000/W, Waltham, Massachusetts) [23]. Lumbar spine (L1-L4), nondominant femoral neck, and total-body bone mineral densities were measured; measurements were expressed in grams of bone mineral per cm2 of bone. Quality control was maintained by daily scanning of an anthropomorphic spine phantom. The coefficient of variation for our Hologic QDR 1000/W densitometer is 0.3%, and the reference limits for variation are 1.5%. Results were expressed as T-scores, which are the number of SDs that the bone mineral density measurement is above (positive value) or below (negative value) expected peak bone mass. The age at which peak bone mass is achieved differs slightly for each site but is usually between 20 and 30 years. Using World Health Organization guidelines, we defined osteopenia as a T-score greater than 2.5 and 1.0 or less. Osteoporosis was defined as a T-score of 2.5 or less [24]. Normal bone mineral density was defined as a T-score of 0 1. Laboratory Measurements Serum calcium, phosphorus, alkaline phosphatase, and creatinine concentrations were measured with an automatic analyzer (Hitachi 911, Boehringer Mannheim, Indianapolis, Indiana). Vitamin D metabolites were extracted from serum specimens (obtained while patients were fasting) with column chromatography and were measured with radiobinding assays (Nichols Institute, Raleigh, North Carolina). The 1,25-dihydroxyvitamin D and 25-hydroxyvitamin D assays had sensitivities of 5 pg/mL and 5 ng/mL, respectively. Testosterone levels were measured by using a competitive radioimmunoassay (Diagnostics Product Corp., Los Angeles, California). Genotype analysis for the 15 most common CFTR mutations was done by using published methods [25]. Radiographic Analysis Screening of the most recent posteroanterior and lateral chest radiographs for the extent of thoracic kyphosis as well as rib and vertebral body fractures was done by a single musculoskeletal radiologist on 65 patients (5 patients did not undergo radiography at the University of North Carolina). Thoracic kyphosis was measured from the second or third to the twelfth thoracic vertebral body by using a modification of the method of Cobb [26]. The number of vertebral compression fractures was also determined from the lateral radiograph by measuring anterior and posterior vertebral body height and expressing the difference as a percentage [27]. The posteroanterior chest radiograph was examined for the presence of rib fractures. Nonacute fractures appeared as focal deformities in the rib contour with varying degrees of reparative bone formation. Statistical Analysis Analyses were done with SAS software (version 6.12, SAS Institute, Cary, North Carolina) [28], and significance was based on two-tailed tests with a P value less than 0.05. Discrete variables are summarized as percentages, and continuous variables are summarized as means SD. Nonparametric methods were used when the distribution of the continuous variable was skewed. Student t-tests were used to compare differences in all clinical and anthropomorphic variables except fracture rates [29]. Relationships between spine and femur T-scores and seven continuous variables-age, age at puberty, cumulative steroid dose, body mass index, FEV1, FVC, and vitamin D levels-were assessed with either the Pearson or Kendall correlation coefficients [30]. Backward stepwise regression analysis was used to determine the multivariate relation between spine and femur T-scores and the aforementioned predictors. Clinical predictors and T-scores were compared, using the Kruskal-Wallis test, by dividing the patients into three groups: those without fractures, those with one fracture, and those with more than one fracture. This was done to determine whether differences existed between groups. Using spine or femur z-scores (the number of SDs that a bone mineral density measurement was above or below that of age- and sex-matched normal controls) rather than T-scores gave similar results (data not shown) because most patients were in the age range at which peak bone mass is expected. For the analysis of fractures and kyphosis angles, patients were grouped by age to conform with published databases (normal databases are based on age) [22, 31]. Two separate fracture rate analyses were done: In one, all fractures were used as the numerator; in the other, all persons with any fracture were used as the numerator. The latter analysis was done to eliminate the possibility that fracture clustering had affected the results. We used person-years as the denominator fo

Thyrocalcitonin: Stimulation of Secretion by Pentagastrin

Administration of a small dose of pentagastrin, a synthetic pentapeptide containing the biologically active portion of the native hormone gastrin, results in a marked, rapid, transitory increase in thyrocalcitonin secretion in the pig. Gastrin or a related gastrointestinal peptide may be important in the physiological secretion of thyrocalcitonin, such as that which occurs when calcium salts are introduced into the gastrointestinal tract.

Abnormal bone turnover in cystic fibrosis adults.

Abstract: Cystic fibrosis (CF) patients often have low bone mineral density (BMD) and may suffer from fractures and kyphosis. The pathogenesis of low BMD in CF is multifactorial. To study bone metabolism, we collected fasting serum and urine from 50 clinically stable CF adults (mean age 28 years) and 53 matched controls to measure markers of bone formation and bone resorption. The CF subjects had moderate lung disease (FEV1: 46.1 ± 18.6% predicted) and malnutrition (BMI: 20.0 ± 3.3 kg/m2). Only 3 subjects had normal BMD. CF subjects had higher urinary N-telopeptides of type I collagen (81.0 ± 60.0 vs 49.0 ± 24.2 nm BCE/mmol creatinine, p= 0.0006) and free deoxypyridinoline (7.3 ± 5.0 vs 5.3 ± 1.9 nM/mM, p= 0.004) levels than controls. Serum osteocalcin levels were similar in the two groups, a result confirmed by two immunoassays that recognize different epitopes on osteocalcin. Serum bone-specific alkaline phosphatase levels were elevated in CF patients (32.0 ± 11.3 vs 21.8 ± 7.0 U/l, p<0.0001), but were much more closely associated with serum total alkaline phosphatase levels (r = 0.51, p = 0.001) than with age or gender. Parathyroid hormone levels were elevated (p= 0.007) and 25-hydroxyvitamin D levels were depressed (p= 0.0002) in the CF patients in comparison with controls. These results indicate that adults with CF have increased bone resorption with little change in bone formation. Medications that decrease bone resorption or improve calcium homeostasis may be effective therapies for CF bone disease.

Altered calcium homeostasis in adults with cystic fibrosis.

Abstract: Bone mineral density (BMD) in cystic fibrosis (CF) patients falls progressively below normal with advancing age, in part due to steroid administration, low levels of sex hormones, chronic inflammatory disease, physical inactivity, and chronic malabsorption of calcium and/or vitamin D. The purpose of this study was to compare the fractional absorption of 45Ca and urinary excretion of calcium in CF subjects and normal controls following a high-calcium breakfast containing 45Ca. Seven young men and 5 young women with CF with pancreatic insufficiency were studied on two separate occasions, with and without administration of pancreatic enzymes. Eleven healthy young adults with normal BMD measurements served as controls. Mean T-scores at the lumbar spine and femur were significantly lower in the CF subjects (p<0.002). Following baseline, fasting collections, timed serum and urine samples were obtained for 5 h after the meal. Fractional absorption (FA) of 45Ca was estimated by the method of Marshall and Nordin. At baseline, CF subjects had lower mean serum 25-hydroxyvitamin D, calcium and albumin values (p<0.03 for each), slightly, but not significantly (p= 0.12), lower albumin-corrected calcium values, equivalent serum 1,25-dihydroxyvitamin D values and a trend toward a higher mean serum parathyroid hormone (PTH) value (p= 0.10). Without pancreatic enzymes, CF subjects showed significantly impaired calcium absorption (5 h FA: 11.8 ± 0.5 for controls vs 8.9 ± 0.2 for CF subjects, p= 0.02) and excretion (4 h excretion: 0.20 ± 0.08 mg Ca/mg creatinine for controls vs 0.16 ± 0.09 mg Ca/mg for CF subjects, p= 0.025). Addition of pancreatic enzymes did not fully compensate for this deficiency. In addition, CF patients had higher serum PTH values after a high-calcium meal (p= 0.03), suggesting mild secondary hyperparathyroidism. Altered calcium homeostasis is likely to be a factor in the development of bone disease in CF patients.

Treatment of bone disease in cystic fibrosis

Purpose of review As individuals with cystic fibrosis (CF) have experienced marked improvements in longevity over the last three decades, bone disease has emerged as a new problem. Bone disease in CF has not been previously reviewed in this journal. Therefore, this review will give a brief overview of bone disease in CF and then concentrate on treatment options. Recent findings In some series, as many as three fourths of adults with CF have low bone density. Decreased absorption of fat-soluble vitamins due to pancreatic insufficiency, altered sex hormone production, chronic inflammation, physical inactivity, and glucocorticoid treatment are some of the factors that contribute to this problem. Vitamin D depletion most likely contributes to bone disease, but identifying the safest and most efficacious vitamin D supplementation has yet to be resolved. Calcium and vitamin K supplementations are important if the diet contains less than the recommended amounts. Treatment of delayed puberty and adult hypogonadism with hormone replacement is recommended to achieve peak bone mass and maintain bone density. Bisphosphonates, including pamidronate and alendronate, are beneficial in improving bone mineral density before and after transplantation in CF adults. Bisphosphonates have not been studied in CF children. Summary Although much progress has been made in our understanding of the pathogenesis, natural history, and clinical manifestations of bone disease in CF, treatment options are still evolving. More attention to nutrition, in terms of the maintenance of lean body mass and vitamin D and calcium supplementation, is likely to decrease bone complications. Bisphosphonates can be of value in CF adults with low bone density. Several clinical trials are under way to help optimize the treatment of CF bone disease.

Tests of anterior pituitary function

T IS THE PURPOSE OF THIS PAPER to review the different tests that provide an index of the production of the anterior pituitary hormones. In many instances these tests involve direct measurement of pituitary hormone concentrations in blood or hormone excretion in urine. Such measurements have been greatly advanced in recent years by the development of sensitive radioimmunoassay techniques. In spite of the remarkable progress made in the direct measurement of the hormones, the assessment of the effects of the hormones on their target tissues still plays an important diagnostic role. Most pituitary function tests begin with the determination of hormone production in the basal state. Where pituitary hypofunction is suspected, provocative tests that indicate the reserve capacity of the pituitary for increased hormone production above basal levels often allow the detection of more subtle limitations in function. Conversely where pituitary hyperfunction is suspected, tests that determine suppressibility of hormone production may aid in revealing functional abnormalities. Tests that directly or indirectly assess thyrotropin, growth hormone, adrenocorticotropin, luteinizing hormone, and follicle stimulating hormone levels will be reviewed. It should be noted that a sensitive assay for the beta melanocyte stimulating hormone has been developed. l Alpha melanocyte stimulating hormone has not been specifically measurable in human biological fluids. Recent evidence suggests that the human pituitary, like that of other species, produces a prolactin that is a separate molecular entity from growth hormone.2 Application of prolactin assays to human biological fluids will undoubtedly prove useful in the characterization of disorders of lactation.3

Radiochemical assay for renin utilizing a synthetic insoluble substrate

In order to provide a convenient in vitro assay for renin activity, a radiolabeled renin substrate analog, N-acetyl-Asn-Arg-Val-Tyr-Ile-His-Pro-Phe-His-[3H]-Leu-Leu-Val-Tyr-Ser-Gly-Lys-Pro-OH, was prepared by solid-phase synthesis. The substrate peptide was bound covalently to agarose through the ϵ-amino group of its lysine residue. Incubation of this insoluble complex with partially purified hog renin resulted in the release of biologically active tritiated peptide into the soluble phase of the incubation mixture, at a rate proportional to the quantity of renin added. The optimum pH for cleavage was 6.5. The apparent Km of the substrate was 1 × 10−4M, and the Vmax was 83 pmoles tritiated peptide released/min/mg renin preparation added. The minimum amount of renin detectable by the assay was 2 μg, a quantity that would be expected to generate 1.0 pmole angiotensin per minute from the natural plasma substrate. Chymotrypsin, trypsin, papain, and pseudorenin, were also effective in cleaving labeled peptides from the insoluble substrate, but leucine aminopeptidase did not appear to release soluble radioactivity. The assay, as described, is useful for the measurement of large numbers of renin samples because of the speed and ease with which it may be performed. It is not yet sufficiently sensitive nor specific to measure the low levels of renin found in plasma.

Affinity chromatography of renin using inhibitory renin substrate analogs.

Summary Several short peptide analogs of renin substrate were tested for their ability to inhibit renin in solution and to bind renin in affinity chromatography. The peptides Leu-Leu-Val-Tyr-OCH3, Leu-Leu-Val-Phe-OCH3 and Leu-Val-Phe-OCH3 had in vitro inhibitory activity against both hog and human renin. Kinetic studies of the prototype inhibitor Leu-Leu-Val-Phe-OCH3 demonstrated classical competitive inhibition, with a K i of 180 μM. When impure hog renin was eluted from succinylated aminoethyl agarose columns containing co-valently bound Leu-Leu-Val-Phe-OCH3 or Leu-Val-Phe-OCH3, the elution of renin activity was delayed in comparison with the elution of inactive contaminating proteins. The binding of human renin to agarose bound inhibitors could also be demonstrated if a longer connecting arm was interposed between the peptide ligand and the supporting polysaccharide.

The pharmacologic control of adrenal steroidogenesis

Abstract The control of cortisol secretion by ACTH and of aldosterone secretion by angiotensin is exerted upon separate cell populations in the adrenal cortex. Cells of the zona faciculata and the zona glomerulosa, while sharing common steroidogenic pathways, are affected differently by hormones and drugs. Fasciculata cells demonstrate increased cAMP formation and cortisol output primarily in response to ACTH. ACTH receptors, when occupied by hormone, transmit an activating signal to membrane-bound adenylate cyclase by a mechanism that may require the translocation of Ca2+. Although the precise way in which increased intracellular cAMP leads to increased steroidogenesis is unknown, protein phosphorylation and new protein synthesis are probably involved. Glomerulosa cells also respond to ACTH, but are uniquely responsive to physiological concentrations of angiotensin II and K+. The responsiveness of these cells to angiotensin may be governed by alterations in receptor number. Whether occupied angiotensin receptors activate steroidogenesis via cAMP is uncertain, but alterations in Ca2+ distribution within the cell may again be involved. Dopamine probably exerts a tonic inhibitory effect on glomerulosa cell function. Competitive inhibitory analogs for both ACTH and angiotensin II are available, but thus far all inhibitors have retained weak agonist properties. Because the regulatory processes for both cortisol and aldosterone are complex, a wide variety of drugs can affect rates of steroidogenesis in vivo .

Divergent Effects of Forskolin on 3',5' Cyclic Adenosine Monophosphate Production and Parathyroid Hormone Secretion

SummaryForskolin, a diterpene which directly stimulates adenylate cyclase, markedly stimulated cAMP production in intact rat parathyroid glands and dispersed cells from hyperplastic and adenomatous human parathyroid tissues. Stimulation of cAMP production in human parathyroid adenomas occurred as early as 2 min and continued for at least 2 h; furthermore, a dose-response relationship was observed, with a maximal 80-fold cAMP response occurring at 100 µM forskolin. When PTH secretion by rat or human parathyroid tissues was studied at low (0.5 mM) and high (2.5 mM) extracellular Ca2+ in either the presence or absence of forskolin, no significant stimulation by forskolin was observed at 15 min, 1 h, and 2 h. When 10 human parathyroid specimens were studied with varying concentrations of forskolin at 1 mM Ca2+, 6 failed to show stimulation of PTH secretion and 4 showed modest but detectable increases in PTH that did not appear dose-related. We conclude that (1) at low and high Ca2+ levels, marked stimulation of cAMP production by forskolin can occur without a corresponding increase in PTH secretion; (2) inhibition of PTH secretion by high extracellular Ca2+ levels continues unchanged despite stimulation of cAMP production by forskolin; and (3) at intermediate Ca2+ levels (1.0 mM), PTH secretion is affected either minimally or not at all by forskolin in human hyperparathyroid tissue preparations. The marked stimulation of parathyroid adenylate cyclase by forskolin without concomitant increases in PTH secretion in the majority of tissues suggests that the level of cAMP production is not a primary or sufficient determinant of hormone secretion.