Joseph E. Zerwekh

The Effects of Twelve Weeks of Bed Rest on Bone Histology, Biochemical Markers of Bone Turnover, and Calcium Homeostasis in Eleven Normal Subjects

This study was undertaken to examine the effects of 12 weeks of skeletal unloading on parameters of calcium homeostasis, calcitropic hormones, bone histology, and biochemical markers of bone turnover in 11 normal subjects (9 men, 2 women; 34 ± 11 years of age). Following an ambulatory control evaluation, all subjects underwent 12 weeks of bed rest. An additional metabolic evaluation was performed after 12 days of reambulation. Bone mineral density declined at the spine (−2.9%, p = 0.092) and at the hip (−3.8%, p = 0.002 for the trochanter). Bed rest prompted a rapid, sustained, significant increase in urinary calcium and phosphorus as well as a significant increase in serum calcium. Urinary calcium increased from a pre‐bed rest value of 5.3 mmol/day to values as high as 7.3 mmol/day during bed rest. Immunoreactive parathyroid hormone and serum 1,25‐dihydroxyvitamin D declined significantly during bed rest, although the mean values remained within normal limits. Significant changes in bone histology included a suppression of osteoblastic surface for cancellous bone (3.1 ± 1.3% to 1.9 ± 1.5%, p = 0.0142) and increased bone resorption for both cancellous and cortical bone. Cortical eroded surface increased from 3.5 ± 1.1% to 7.3 ± 4.0% (p = 0.018) as did active osteoclastic surface (0.2 ± 0.3% to 0.7 ± 0.7%, p = 0.021). Cancellous eroded surface increased from 2.1 ± 1.1% to 4.7 ± 2.2% (p = 0.002), while mean active osteoclastic surface doubled (0.2 ± 0.2% to 0.4 ± 0.3%, p = 0.020). Serum biochemical markers of bone formation (osteocalcin, bone‐specific alkaline phosphatase, and type I procollagen extension peptide) did not change significantly during bed rest. Urinary biochemical markers of bone resorption (hydroxyproline, deoxypyridinoline, and N‐telopeptide of type I collagen) as well as a serum marker of bone resorption (type I collagen carboxytelopeptide) all demonstrated significant increases during bed rest which declined toward normal during reambulation. Thus, under the conditions of this study, the human skeleton appears to respond to unloading by a rapid and sustained increase in bone resorption and a more subtle decrease in bone formation.

Disruption of Cholesterol 7α-Hydroxylase Gene in Mice II. BILE ACID DEFICIENCY IS OVERCOME BY INDUCTION OF OXYSTEROL 7α-HYDROXYLASE

Past experiments and current paradigms of cholesterol homeostasis suggest that cholesterol 7α-hydroxylase plays a crucial role in sterol metabolism by controlling the conversion of cholesterol into bile acids. Consistent with this conclusion, we show in the accompanying paper that mice deficient in cholesterol 7α-hydroxylase (Cyp7−/− mice) exhibit a complex phenotype consisting of abnormal lipid excretion, skin pathologies, and behavioral irregularities (Ishibashi, S., Schwarz, M., Frykman, P. K., Herz, J., and Russell, D. W. (1996) J. Biol. Chem. 261, 18017-18023). Aspects of lipid metabolism in the Cyp7−/− mice are characterized here to deduce the physiological basis of this phenotype. Serum lipid, cholesterol, and lipoprotein contents are indistinguishable between wild-type and Cyp7−/− mice. Vitamin D3 and E levels are low to undetectable in knockout animals. Stool fat content is significantly elevated in newborn Cyp7−/− mice and gradually declines to wild-type levels at 28 days of age. Several species of 7α-hydroxylated bile acids are detected in the bile and stool of adult Cyp7−/− animals. A hepatic oxysterol 7α-hydroxylase enzyme activity that may account for the 7α-hydroxylated bile acids is induced between days 21 and 30 in both wild-type and deficient mice. An anomalous oily coat in the Cyp7−/− animals is due to the presence of excess monoglyceride esters in the fur. These data show that 7α-hydroxylase and the pathway of bile acid synthesis initiated by this enzyme are essential for proper absorption of dietary lipids and fat-soluble vitamins in newborn mice, but not for the maintenance of serum cholesterol and lipid levels. In older animals, an alternate pathway of bile acid synthesis involving an inducible oxysterol 7α-hydroxylase plays a crucial role in lipid and bile acid metabolism.

Hypercalcemia Associated with Increased Serum Calcitriol Levels in Three Patients with Lymphoma

A radioreceptor assay for serum 1,25-dihydroxyvitamin D (calcitriol) was used to screen patients with hypercalcemia of malignancy. Three patients with non-Hodgkins lymphoma and hypercalcemia (serum Ca, 12.0, 13.4, and 13.0 mg/dL, respectively) had increased serum calcitriol levels (56, 72, and 77 pg/mL, respectively; normal, less than 50 pg/mL). Elevated levels of calcitriol, an active vitamin D metabolite, occurred in the presence of significant renal impairment (creatinine clearance, 8 to 19 mL/min) and relative parathyroid suppression (serum immunoreactive parathyroid hormone, 17 to 39 microL-eq/mL; mean value in end-stage renal disease, 182 +/- 39 microL-eq/mL). Hypercalcemia and excessive serum calcitriol levels responded to glucocorticosteroid therapy. In two patients, the hypercalcemia and increased serum calcitriol level were related to a tumor, but not to the serum immunoreactive parathyroid hormone level. Fractional intestinal 47Ca absorption, measured in one patient, was increased (0.94; normal, less than 0.61) and varied directly with the serum calcitriol level. No patient had evidence of sarcoidosis. Hypercalcemia associated with certain lymphomas may be caused by the increased synthesis of calcitriol by lymphoma cells.

Bone Has a Sexually Dimorphic Response to Aromatase Deficiency

Aromatase synthesizes estrogen from androgen precursors. To better understand the role of estrogen in skeletal metabolism and growth, we have assessed long bone growth and histomorphometry in aromatase‐deficient (ArKO) mice. The age range for the animals was 5–7 months. At this age mice have already achieved peak bone density but continue slow bone growth. Femur length, an index of long bone growth, showed decreased growth in ArKO males compared with wild‐type (wt) littermates but no significant difference in females. Radiographically, compared with age‐ and sex‐ matched littermates both ArKO males and females showed osteopenia in the lumbar spine. Histologically, both ArKO males and females showed an osteoporotic‐type picture, characterized by significant decreases in trabecular bone volume and trabecular thickness. However, compared with wt littermates female ArKO animals showed a bone remodeling picture consistent with increased bone turnover, much like early postmenopausal osteoporosis in humans. On the other hand, male ArKO animals showed decreases in both osteoblastic and osteoclastic surfaces compared with wt littermates, similar to age‐related osteopenia. These findings suggest that osteoporosis seen in aromatase‐deficient mice may arise from different bone remodeling activities between males and females. These results also show that the ArKO model exhibits the expected results of estrogen deficiency and may be a good model for investigating sex‐specific responses to estrogen deficiency. Furthermore, they imply that estrogen is important for attaining peak bone mass in male as well as in female mice.

Unusual mid‐shaft fractures during long‐term bisphosphonate therapy

Background  Bisphosphonates are the most commonly prescribed medications for the treatment of osteoporosis. Although existing evidence supports a good safety profile, there is concern that chronic administration of these agents could result in severe suppression of bone turnover with increased risk of nonvertebral fractures.

Management of Cystine Nephrolithiasis with Alpha-Mercaptopropionylglycine

The effect of long-term treatment with alpha-mercaptopropionylglycine was examined in 66 patients with cystinuria. Of the patients 49 took D-penicillamine before therapy, whereas 17 did not. Over-all side effects to alpha-mercaptopropionylglycine were common, and occurred in 75.5 per cent of the patients with and 64.7 per cent without a history of D-penicillamine treatment, compared to 83.7 per cent who suffered toxicity to D-penicillamine. Serious adverse reactions requiring cessation of therapy were less common with alpha-mercaptopropionylglycine. Among the patients who took both drugs 30.6 per cent had to stop taking alpha-mercaptopropionylglycine, whereas 69.4 per cent could not tolerate D-penicillamine. Of the latter group with toxicity to D-penicillamine before therapy, whereas 17 did therapy only 5.9 per cent had side effects to alpha-mercaptopropionylglycine of sufficient severity to require withdrawal. Alpha-mercaptopropionylglycine was equally as effective as D-penicillamine in reducing cystine excretion. During long-term treatment with alpha-mercaptopropionylglycine (average dose 1,193 mg. per day) urinary cystine levels were maintained at 350 to 560 mg. per day and urinary cystine was kept at undersaturated levels. Commensurate with these changes, alpha-mercaptopropionylglycine produced remission of stone formation in 63 to 71 per cent of the patients and reduced individual stone formation rate in 81 to 94 per cent. Thus, alpha-mercaptopropionylglycine has a definite therapeutic role in cystinuric patients with toxicity to D-penicillamine.

The measurement of vitamin D: analytical aspects.

In the past quarter of a century, our understanding of the metabolism and mechanism of action of vitamin D has been elucidated. During this period, many metabolites of vitamin D have been identified and a small proportion of these assayed in blood. The ability to assay these vitamin D metabolites has led to a better appreciation of the pathological role that altered vitamin D metabolism plays in the development of diseases of calcium homeostasis. However, for many physicians it is not clear which vitamin D metabolites should be quantitated and what the information gained tells us. Of the four major circulating vitamin D metabolites in blood, only two, namely 25-hydroxyvitamin D (25OHD) and 1,25-dihydroxyvitamin D [1,25(OH)2D], have warranted measurement. Of these, the need for assessing serum 1,25(OH)2D is actually quite limited and should therefore not be considered as part of the standard vitamin D testing regimen. 25OHD, on the other hand, provides us with the single best assessment of vitamin D nutritional status and should be the only vitamin D assay typically ordered for this reason. Which of the many methods that are available should a laboratory use for quantitating either of these vitamin D metabolites? Early methods required large volumes of blood, organic solvent extractions, and extensive purification of the vitamin D metabolites prior to assay. Today, these time-consuming and costly methods have given way to a range of radioimmunoassays and enzyme-linked immunosorbent assays that can accurately and conveniently provide important information concerning an individuals vitamin D status. This review will consider when vitamin D measurements should be undertaken and how best to perform such assays.

Bone phenotype of the aromatase deficient mouse

Estrogens are important for normal bone growth and metabolism. The mechanisms are incompletely understood. Thus, we have undertaken characterization of the skeletal phenotype of aromatase (ArKO) deficient mice. No abnormalities have been noted in skeletal patterning in newborns. Adult ArKO mice show decreased femur length and decreased peak Bone Mineral Density (BMD) with accelerated bone loss by 7 months of age in females. Magnetic resonance microscopy (MR) and microCT (microCT) imaging disclosed decreased cancellous connectivity and reduced cancellous bone volume in ArKO females. Bone formation rate (BFR) is increased in ArKO females and decreased in ArKO males. Estradiol therapy reverses these changes. This anabolic effect of estradiol in the male skeleton is supported by 18-F- Positron Emission Tomography (PET) imaging, which clearly demonstrates decreased spinal uptake, but marked increase after estradiol therapy. Serum IGF-1 levels are high in young female ArKO mice but low in young ArKO males. The reduced BMD in ArKO females, despite the presence of elevated serum IGF 1, suggests that other mechanism(s) are operative. There is increased B-cell lymphopoiesis in adult female ArKO bone marrow cells. These results show that ArKO mice show the effects of estrogen deficiency on bone growth, mass, metabolism, microarchitecture and the hematopoietic microenvironment.

Impaired bone formation in male idiopathic osteoporosis: Further reduction in the presence of concomitant hypercalciuria

We present iliac bone histomorphometric data and related biochemical data from 16 nonalcoholic men (50±11 (SD) years) referred for evaluation of spontaneous skeletal and/or appendicular fractures and reduced spinal bone density. All men were eugonadal and had no known underlying disorder associated with osteopenia. For the group, mean serum chemistry values were within normal limits including immunoreactive parathyroid hormone, osteocalcin and serum 1,25-dihydroxyvitamin D [1,25(OH)2D]. Nine men demonstrated hypercalciuria (⩾0.1 mmol/kg per day) while on a constant metabolic diet of 20 mmol/day Ca. Their 24-hour urinary calcium was significantly greater than that for the remaining 7 men (7.4±1.6 vs. 5.0±0.8 mmol/day,p=0.003), as was their calciuric response to a 1 g oral calcium load (0.23±0.06 vs. 0.15±0.05 Ca/creatinine,p=0.042). Serum parameters (including parathyroid hormone and 1,25(OH)2D) of hypercal-ciuric and normocalciuric men were not significantly different. Histomorphometric indices for cancellous bone demonstrated significant differences between the entire group of osteoporotic men and age-adjusted normal values for bone volume (11.4±4.0% vs. 23.2±4.4%), osteoid surface (5.6±3.9% vs. 12.1±4.6%), osteoblastic surface (2.0±2.3% vs. 3.9±1.9%), and mineralizing surface (1.9±2.4% vs. 5.1±2.7%);there were also significant differences in bone formation rate (total surface referent) (0.004±0.001 vs. 0.011±0.006 mm3/mm2 per year). Compared with the normocalciuric group the 9 hypercalciuric men had significantly lower osteoblastic surfaces (1.6±1.9% vs. 2.5±2.6%) and mineralizing surfaces (1.4±1.5% vs. 2.7±3.2%). Cortical bone indices demonstrated a similar trend in formation parameters although these differences did not reach significance. These results suggest that idiopathic osteoporosis in men is characterized by suppressed bone formation due to reduced osteoblast proliferation and that this defect is exaggerated in hypercalciuric men as opposed to normocalciuric men. The cause for suppressed bone formation and increased intestinal absorption of calcium in some men is not known but may be the result of 1,25(OH)2D or some previously unrecognized factor(s).

Biochemical changes associated with the symptomatic human intervertebral disk

Significant changes in disk biochemistry were evident in the nuclei pulposi of 25 symptomatic disks that demonstrated abnormal diskography. Compared with the results from ten normal disk from young subjects (mean age, ten years) and from ten asymptomatic disks from adult patients (mean age, 33 years), the symptomatic disks demonstrated significantly greater collagen content than the young and asymptomatic adult subjects, respectively. Significantly fewer total glycosaminoglycans (GAGs) and significantly less water content were also evident for the diseased disks. The mean pH of the nucleus pulposus from 23 patients was also significantly reduced for the symptomatic disks as compared with the mean pH measured for four asymptomatic adult subjects. Although significantly fewer total GAGs were observed for the symptomatic disks, there were no significant differences in the percent composition of hyaluronic acid (HA), chondroitin sulfate (CS), or keratan sulfate (KS) among the normal and symptomatic disks. Significant changes in disk biochemistry are associated with the symptomatic disk, and these changes could contribute to the development of the painful disk syndrome.