Matrix vesicles in chicken epiphyseal cartilage. Separation from lysosomes and the distribution of inorganic pyrophosphatase activity

Summary The extracellular matrix vesicles from epiphyseal cartilage of chickens were isolated by differential centrifugation. The matrix vesicles obtained showed considerable activity of lysosomal enzymes. This appears to have been due to lysosomal contamination because when we used a new density gradient medium (Percoll?), the lysosomal enzyme activities and the activity of alkaline phosphatase could be totally separated. Electron microscopy of the alkaline phosphatase-rich fraction showed matrix vesicle-like structures. Phosphatase activities of the cells and matrix vesicles were further studied by Sephadex G-200 gel filtration. Specific magnesium-activated inorganic pyrophosphatase, distinct from nonspecific alkaline phosphatase, could be demonstrated in the cellular fraction. No such separate activity could be demonstrated in the matrix vesicle fraction, and it is supposed that the pyrophosphatase activity in the matrix vesicles originates from the alkaline phosphatase.     

Vitamin A deficiency stimulated phosphatase activity in epiphyseal chick cartilage

This paper reports studies of the epiphyseal cartilage in normal and vitamin A deficient chicks. The organic composition and the phosphatase activity in the resting cartilage, ossifying cartilage and new bone were measured. The ossifying cartilage and new bone had a higher content of inorganic material, phosphate and collagen than the resting cartilage. Vitamin A deficiency caused increase in the phospholipid content of all three tissues. The resting cartilage from vitamin A deficient tissue had, after homogenisation and centrifugation, a supernatant with an activity of alkaline phosphatase and glycerophosphatase higher than that in control samples. It is considered that effects of vitamin A deficiency on enzymes are related to defects of the lysosomal membrane with release of phosphatases, and that normal mineralisation also involves phosphatases activity.     

Organization and cellular biology of the perichondrial ossification groove of ranvier: a morphological study in rabbits.

The perichondrial ossification groove of Ranvier, a circumferential groove in the periphery of the epiphyseal cartilage, was studied in rabbits whose ages ranged from one week to eight months using light and electron microscopy, autoradiography after labeling with 3H-thymidine, 3H-proline, and 3H-glucosamine, and histochemical staining for proteoglycans and alkaline phosphatase. By these methods, three groups of cells were identified within the groove: 1. A group of densely packed cells deep in the groove, which are the progenitor cells for the osteoblasts that form the bone bark, a cuff of bone surrounding the epiphyseal growth-plate region and the adjacent part of the metaphysis. 2. A group of more widely dispersed, relatively undifferentiated mesenchymal cells and fibroblasts, some of which are chondroblast precursors that probably contribute to appositional chondrogenesis and growth in width of the epiphyseal cartilage. 3. Fibroblasts and fibrocytes among sheets of highly oriented and organized collagen fibers which form a fibrous layer that is continuous with the outer fibrous layer of the periosteum and with the perichondrium. This layer also sends fibers into the epiphyseal cartilage and anchors the periosteum firmly to the epiphyses as bone growth proceeds.     

Bone composition and phosphatase activity in magnesium deficiency in rats.

The effect of magnesium deficiency on phosphatase activity and bone composition was determined in the femora of young rats. In the right distal metaphysis the acid and alkaline phosphatase activities were decreased in magnesium-deficient rats, and the activity of bone alkaline phosphatase in the incubation mixture after adding magnesium was significantly greater in the magnesium-deficient than in the control rats. In the left distal metaphysis the water content was significantly lower in the magnesium-deficient rats at the fifth week but not at the third week. Conversely, the ash content of metaphyseal bone was significantly increased in magnesium-deficient rats at the fifth week, but not at the third week. The magnesium and phosphorus contents were abnormally low in the deficient bone at both periods. The calcium content was increased in the deficient bone at the third week, but not at the fifth week.     

Effect of phosphate supplementation on the expression of the mutant phenotype in murine X-linked hypophosphatemic rickets

The X-linked mouse, a murine homologue of X-linked hypophosphatemia in humans, is characterized by rachitic bone disease, hypophosphatemia, impaired renal brushborder membrane Na⁺-phosphate cotransport and abnormal regulation of renal vitamin D metabolism. We demonstrated that short-term phosphate supplementation decreases renal 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D) catabolism and increases serum 1,25-(OH)₂D levels in mice (Tenenhouse & Jones 1990). In the present study, we compared several other parameters in normal and mice fed control (1%) and high (1.6%) phosphate diets for 4 days. Phosphate supplementation significantly raised serum phosphate levels and decreased renal brush-border membrane Na⁺-phosphate, but not Na⁺-glucose, cotransport in both genotypes (67% of control diet, p < 0.05). However, under both dietary conditions, the phosphate/glucose transport ratio was significantly reduced in mice (58% of normal littermates, p < 0.05). Renal PTH-stimulated cAMP accumulation, which was significantly blunted in mice compared to normal mice under control dietary conditions (p < 0.05), was not altered by phosphate supplementation in either genotype. Serum alkaline phosphatase activity was significantly higher than normal in mice on the control diet and was further increased in mutants but not in normals fed the high phosphate diet (p < 0.05). Measurements of serum bilirubin and electrophoresis of serum alkaline phosphatase suggested that the elevation in serum alkaline phosphatase activity in phosphate-supplemented mice represents the bone-derived isozyme. The present study demonstrates that, in contrast to the abnormality in vitamin D metabolism, the abnormalities in renal brush-border membrane Na⁺-phosphate cotransport, PTH-stimulated cAMP accumulation and serum alkaline phosphatase activity are not corrected by phosphate supplementation of mice. We suggest that the increase in serum alkaline phosphatase activity in phosphatesupplemented mice represents a stimulation of osteoblastic activity in response to the increase in available phosphate and elevated serum 1,25-(OH)₂D levels.     

Gestational changes of thyroid hormone action in the developing fetal bovine epiphysis

Summary Thyroid hormones are essential for cartilage growth and maturation. In order to assess their actions during different periods of skeletal development, [¹²⁵I]-triiodothyronine (T₃) binding capacity in epiphyseal cartilage and triiodothyronine concentrations in serum were quantitated in bovine fetuses of the second and third trimesters of gestation (equivalent to fetal sizes of 10–90 cm crownrump (CR) length), and related to the alkaline phosphatase activities in the same cartilaginous tissues. Nuclear T₃ binding levels, which were initially low during 10–30 cm CR, rose to a peak value (1.7 pmol/mg DNA) at the end of the second trimester (40–50 cm CR). Then, following a sharp decline at 50–60 cm CR, T₃ binding rose to a moderate level in the later gestational period (60–90 cm CR). Serum total triiodothyronine rose transiently in fetuses of 30–50 cm CR to a peak level (34 ng/100 ml), and subsequently increased continuously in the later period (60–90 cm CR). Alkaline phosphatase activities measured in epiphyseal chondrocytes rose significantly in fetuses of 60–90 cm CR. The coincident rise of cartilage triiodothyronine binding capacity and serum circulating triiodothyronine levels in the late second trimester suggests that this thyroid hormone induces its own binding sites in bovine epiphyseal cartilage; the dramatic increase in the level of serum triiodothyronine during the third trimester of gestation is temporally related to the increase in alkaline phosphatase activity of chondrocytes, as well as other recognized developmental changes in the fetal bovine skeletal tissues.     

慢性肾脏病血清镁离子、钙离子水平与周围神经功能的相关性研究

目的研究慢性肾脏病(chronic kidney disease,CKD)患者血清Ca²⁺和Mg²⁺水平及其与周围神经功能的相关性。方法选取2011年10月至2019年10月中国人民解放军联勤保障部队第961医院收治的CKD患者300例为样本进行横断面研究,根据肾小球滤过率分为1~5期,比较各期血清Ca²⁺和Mg²⁺水平,同时测量正中神经和腓总神经的感觉及运动传导潜伏期、传导速度和波幅,比较不同血钙和血镁水平患者的神经传导速度、潜伏期和波幅等神经电生理指标的差异,并分析各指标与血清Ca²⁺、Mg²⁺水平的相关性。结果CKD 1~3期患者血清Ca²⁺水平明显高于4~5期(P<0.05),血清Mg²⁺水平明显低于4~5期(P<0.05),CKD 1~3期低钙血症和高镁血症发生率明显低于4~5期患者(P<0.05)。正常血钙组和高钙血症组正中神经和腓总神经的感觉及运动传导潜伏期低于低钙血症组,神经传导速度及波幅均高于低钙血症组,且高钙血症组正中神经和腓总神经的运动传导波幅高于正常血钙组,差异均有统计学意义(P<0.05);正常血镁组和高镁血症组正中神经和腓总神经的感觉及运动传导潜伏期低于低镁血症组,神经传导速度及波幅高于低镁血症组,且高镁血症组腓总神经的运动传导波幅高于正常血镁组,差异均有统计学意义(P<0.05)。CKD患者正中神经和腓总神经的感觉及运动传导潜伏期与血清Ca²⁺和Mg²⁺水平均具有明显负相关性(P<0.05),神经传导速度和波幅均与血清Ca²⁺和Mg²⁺水平具有明显正相关性(P<0.05)。结论CKD患者常合并Ca²⁺和Mg²⁺代谢紊乱,并可能对周围神经功能造成不利影响。     

Comparison of the effect of gluconate, lactose, and xylitol on bone recalcification in calcium-deficient rats

The therapeutic value of three calcium absorption promoting carbohydrates, lactose, gluconate and xylitol, in bone calcification was evaluated in 7-week-old male rats which were fed on a semisynthetic Ca-deficient diet for 3 weeks. Lactose + CaCO₃, xylitol + CaCO₃, Ca-gluconate, or CaCO₃ alone were administered to the Ca-deficient rats for 2 weeks; the carbohydrate and Ca contents of the diets were 5% and 0.5%, respectively. The Ca-deficient rats showed a decrease in serum total Ca and ionized Ca²⁺ and in tibial Ca, Mg, P and density, with a concomitant increase in bone hydroxyproline concentration. Bone and serum tartrateresistant acid phosphatase activities were increased 2-fold and the serum 1,25(OH)₂D₃ level 5-fold. Smaller increases were found in serum calcitonin, PTH, alkaline phosphatase and osteocalcin levels. These changes (except calcitonin) were reversed by the administration of Ca and the carbohydrates. It was observed that all three agents improved the recalcification of bones compared with the effect of CaCO₃ alone. The effect of lactose and xylitol was superior to that of gluconate. These results suggest advantages in the use of xylitol in Ca-supplements.     

Deficiency of insulin receptor substrate-1 impairs skeletal growth through early closure of epiphyseal cartilage.

Morphological analyses in and around the epiphyseal cartilage of mice deficient in insulin receptor substrate-1 (IRS-1) showed IRS-1 signaling to be important for skeletal growth by preventing early closure of the epiphyseal cartilage and maintaining the subsequent bone turnover at the primary spongiosa. Introduction: IRS-1 is an essential molecule for intracellular signaling by IGF-I and insulin, both of which are potent anabolic regulators of cartilage and bone metabolism. To clarify the role of IRS-1 signaling in the skeletal growth, morphological analyses were performed in and around the epiphyseal cartilage of mice deficient in IRS-1 (IRS-1(-/-)), whose limbs and trunk were 20-30% shorter than wildtype (WT) mice. MATERIALS AND METHODS: The epiphyseal cartilage and the primary spongiosa at proximal tibias of homozygous IRS-1(-/-) and WT male littermates were compared using histological, immunohistochemical, enzyme cytohistochemical, ultrastructural, and bone histomorphometrical analyses. RESULTS: In and around the WT epiphyseal cartilage, IRS-1 and insulin-like growth factor (IGF)-1 receptors were widely expressed, whereas IRS-2 was weakly localized in bone cells. Chronological observation revealed that height of the proliferative zone and the size of hypertrophic chondrocytes were decreased in WT mice as a function of age, and these decreases were accelerated in the IRS-1 (-/-) cartilage, whose findings at 12 weeks were similar to those of WT at 24 weeks. In the IRS-1(-/-) cartilage, proliferating chondrocytes with positive proliferating cell nuclear antigen (PCNA) or parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor immunostaining had almost disappeared by 12 weeks. Contrarily, TUNEL+ apoptotic cells were increased in the hypertrophic zone, at the bottom of which most of the chondrocytes were surrounded by the calcified matrix, suggesting the closure of the cartilage. In the primary spongiosa, bone volume, alkaline phosphatase (ALP)+ osteoblasts, TRACP+ osteoclasts, and the osteopontin-positive cement line were markedly decreased. Bone histomorphometrical parameters for both bone formation and resorption were significantly lower in IRS-1(-/-) mice, indicating the suppression of bone turnover. CONCLUSION: The IRS-1(-/-) epiphyseal cartilage exhibited insufficient proliferation of chondrocytes, calcification of hypertrophic chondrocytes, acceleration of apoptosis, and early closure of the growth plate. Thus, the data strongly suggest that IRS-1 signaling is important for the skeletal growth by preventing early closure of the epiphyseal cartilage and by maintaining the subsequent bone turnover at the primary spongiosa.     

Hypocalcemia associated with estrogen therapy for metastatic adenocarcinoma of the prostate

We report 2 cases of true hypocalcemia (not caused by decreased binding proteins) associated with metastatic prostate cancer and review previously reported cases. Hypocalcemia is a common but frequently unrecognized complication of prostatic cancer. Estrogen therapy often is associated with the hypocalcemia, which may be asymptomatic. The hypocalcemia is always associated with osteoblastic metastases and usually it is associated with increased serum alkaline phosphatase activity, acid phosphatase activity and serum parathyroid hormone concentration. Serum concentrations of magnesium, phosphorus and vitamin D frequently are decreased. Patients are in a positive calcium balance. The osteoblastic metastases seem to act as a calcium sink, creating a "hungry tumor phenomenon". The role of estrogens may be to stop the resorption of normal bone resulting in lower serum calcium concentrations.