Elemental microanalysis of organelles in proximal tubules. I. Alterations in transport and metabolism

Oxygen deprivation to the kidney causes a multifactorial series of morphological, physiological, and biochemical alterations that occur as a function of time. One of the earliest events involves significant changes in the cellular contents of the physiologically important elements (ions) Na and K. Controversy exists as to the nature of changes in the content of the regulatory ion Ca, in either its free or bound form, and much less is known regarding in situ distribution and amounts of other elements such as Mg, P, S, and Cl during physiological or pathophysiological states. The objective of these studies was to evaluate element compartmentation in proximal renal tubules by using quantitative electron probe x-ray microanalysis, during specific conditions which are at least partially manifested during oxygen deprivation. Cells from control proximal tubule suspensions were compared with those exposed to (1) ouabain, to inhibit (Na+, K+)-ATPase; (2) mitochondrial uncouplers, to rapidly deplete ATP content; or (3) calcium ionophores, to cause a rapid elevation in cytoplasmic free calcium. In parallel with electron probe x-ray microanalysis imaging of subcellular elemental content, total cell potassium and ATP contents, enzyme release, oxygen consumption, cytoplasmic free calcium levels, and ultrastructural alterations were assessed. Results indicated that ATP depletion was, in the short term, more deleterious to renal proximal tubules than any of the tested ionic alterations. Intracellular organelles including mitochondria and nuclei appeared to be readily permeable to Na, K, and Cl, altering their concentrations of these ions in parallel with cytoplasmic concentrations. Lysosomes exhibited evidence of Cl accumulation, consistent with an inwardly directed proton ATPase with accompanying Cl transport. Whereas in the cytoplasm Na, K and Cl appeared to be mostly free, a large fraction of these ions within intracellular organelles seemed bound.     

Unusual elements in renal calculi

A group of 54 renal calculi were spontaneously passed renal stone after a nephritic colic. Two groups of calculi were found: papillary and non-papillary calculi. All calculi were analyzed by infrared spectroscopy and electronic microscopy scan (EMS) and EDAX. When the stones were analyzed with EDAX, elements such as C, N, O, Na, S, Mg, Al, Si, Cl, K, Ca, Mn, Fe, Ni, Zn were detected. The possible origin of these elements is discussed in this work.     

Evaluation of mineral content of enamel prepared by erbium, chromium:yttrium–scandium–gallium–garnet laser

The aim of this study was to evaluate the mineral content of enamel etched at two different power settings with an erbium, chromium:yttrium–scandium–gallium–garnet (Er,Cr:YSGG) laser. Buccal, lingual and mesial or distal surfaces of five premolar teeth were cut, and three enamel slabs were obtained from each tooth. Fifteen enamel specimens were divided into three groups (1 W, 2 W and control) of five specimens each and subjected to Er,Cr:YSGG laser. The mean percentage weights of the five elements [calcium (Ca), potassium (K), magnesium (Mg), sodium (Na) and phosphorus (P)] in each slab were measured by inductively coupled plasma-atomic emission spectrometry (ICP-AES). One way analysis of variance (ANOVA) was used to analyze differences among the groups (1 W, 2 W and control). There were no significant differences among the groups (1 W, 2 W and control) for Ca, K, Mg, Na, or P, or for the Ca/P ratio (P > 0.05). Scanning electron microscopy (SEM) photographs indicated that the surface irregularities increased with increased power setting. Laser treatment did not affect the mean percentage weights of Ca, K, Mg, Na, and P, or the Ca/P ratio, in any group.     

Ablation of Cathepsin K Activity in the Young Mouse Causes Hypermineralization of Long Bone and Growth Plates

Cathepsin K deficiency in humans causes pycnodysostosis, which is characterized by dwarfism and osteosclerosis. Earlier studies of 10-week-old male cathepsin K-deficient (knockout, KO) mice showed their bones were mechanically more brittle, while histomorphometry showed that both osteoclasts and osteoblasts had impaired activity relative to the wild type (WT). Here, we report detailed mineral and matrix analyses of the tibia of these animals based on Fourier transform infrared microspectroscopy and imaging. At 10 weeks, there was significant hypercalcification of the calcified cartilage and cortices in the KO. Carbonate content was elevated in the KO calcified cartilage as well as cortical and cancellous bone areas. These data suggest that cathepsin K does not affect mineral deposition but has a significant effect on mineralized tissue remodeling. Since growth plate abnormalities were extensive despite reported low levels of cathepsin K expression in the calcified cartilage, we used a differentiating chick limb-bud mesenchymal cell system that mimics endochondral ossification but does not contain osteoclasts, to show that cathepsin K inhibition during initial stages of mineral deposition retards the mineralization process while general inhibition of cathepsins can increase mineralization. These data suggest that the hypercalcification of the cathepsin K-deficient growth plate is due to persistence of calcified cartilage and point to a role of cathepsin K in bone tissue development as well as skeletal remodeling.     

Electrolytes of isolated epiphyseal chondrocytes,matrix vesicles,and extracellular fluid

Summary Chondrocyte, matrix vesicle, and membrane fractions, as well as interstitial fluid samples from the proliferating and hypertrophic zones of chicken epiphyseal cartilage were analyzed for electrolyte content. Intracellular Ca levels were 1.4–2.1 mM, over 90% of which was nondiffusible. Isolated hypertrophic chondrocytes had higher intracellular Na and lower K than proliferating cells. Matrix vesicles contained 25 to 50 times higher concentrations of Ca than the adjacent cells. Vesicles from the zone of hypertrophy contained twice as much Ca as did those from the proliferating area. Ca/P₁ molar ratios of matrix vesicles were much higher than those of cells or of later mineral deposits. These findings indicate that Ca is concentrated in matrix vesicles during formation, but acuumulation of Ca and P₁ must continue in the matrix. X-ray diffraction of freeze-dried vesicle and membrane fractions failed to detect crystalline apatite, suggesting that crystals seen in electron micrographs of matrix vesicles may be artifacts. Interstitial fluid expressed from epiphyseal cartilage was higher in K, P_i, Mg and nucleotides, and lower in Na and Cl, than blood plasma. Fluid from the hypertrophic zone was higher in K and nucleotides, but not P_i or Mg, than that from the proliferating layer. These data suggest that selective leakage or extrusion of these constituents, which are normally intracellular, must occur, especially in the hypertrophic zone. More of the Ca and Mg, and less of the P_i, was protein-bound in cartilage fluid than in blood plasma. There was more binding of the divalent cations in fluid from proliferating than from hypertrophic cartilage. The presence of greater amounts of ultrafilterable peptides in fluid from hypertrophic than from proliferating cartilage or blood plasma, suggests that proteolytic activity may release bound divalent cations during mineralization.     

Mass spectrometric trace element analysis of calcium oxalate uroliths

Mass spectrometric trace element analysis of whewellite and weddellite uroliths revealed 55 elements, from which 20 could be well assessed quantitatively. The total trace element content in weddellite increased by 77.5% with respect to whewellite, the main portion being made up from Na, P, Mg, and Cl. In physiological conditions weddellite is stabilized by trace elements. The importance of trace element analysis of Si, Al, Sr, Rb, S, F, Cl, and Br in uroliths is indicated.     

Distribution of elements and water in peripheral nerve of streptozocin-induced diabetic rats

Accumulating evidence suggests that alterations in Na, Ca, K, and other biologically relevant elements play a role in the mechanism of cell injury. The pathogenesis of experimental diabetic neuropathy is unknown but might include changes in the distribution of these elements in morphological compartments. In this study, this possibility was examined via electron-probe X-ray microanalysis to measure both concentrations of elements (millimoles of element per kilogram dry or wet weight) and cell water content (percent water) in frozen, unfixed, unstained sections of peripheral nerve from control and streptozocin-induced diabetic rats. Our results indicate that after 20 wk of experimental diabetes, mitochondria and axoplasm from myelinated axons of proximal sciatic nerve displayed diminished K and Cl content, whereas in tibial nerve, the intraaxonal levels of these elements increased. In distal sciatic nerve, mitochondrial and axoplasmic levels of Ca were increased, whereas other elemental alterations were not observed. These regional changes resulted in a reversal of the decreasing proximodistal concentration gradients for K and Cl, which exist in nondiabetic rat sciatic nerve. Our results cannot be explained on the basis of altered water. Highly distinctive changes in elemental distribution observed might be a critical component of the neurotoxic mechanism underlying diabetic neuropathy.     

应用电子探针能谱分析法测定低温对家兔睾丸元素含量的影响

本文报道用电子探针能谱分析法,直接测定了低温后各实验组兔睾丸局部组织的元素含量,3、6、10天组与正常对照组相比,Mg、K元素WT％含量明显降低,而Na、Cl元素又明显升高。P、S元素含量各实验组与正常对照组间无明显差异。     

Evaluation of mineral content of dentin prepared by erbium, chromium:yttrium scandium gallium garnet laser

Laser etching has an effect on the mineral content of dentin. The aim of this study was to evaluate the mineral content of dentin prepared at three different power settings with an erbium, chromium:yttrium scandium gallium garnet (Er,Cr:YSGG) laser. The enamel of five, lower, wisdom, molar teeth was removed to expose the dentin surface. Four dentin slabs were obtained, then each tooth was randomly divided into four portions (groups 1 W, 2 W, 3 W and control) so that we could evaluate the effect of laser treatment. The Er,Cr:YSGG laser used for the study had a pulse duration of 140 μs, a pulse repetition rate of 20 Hz and a power output range of 0 W to 6 W. Laser energy was delivered through a fiberoptic system to a sapphire tip terminal 6 mm long and 600 μm in diameter, using a non-contact mode. The levels of five elements: magnesium (Mg), phosphorus (P), calcium (Ca), potassium (K), and sodium (Na), in each slab were measured by inductively coupled plasma–atomic emission spectrometry (ICP-AES). There were significant differences between the groups (1 W, 2 W, 3 W and control) for Ca, Mg, Na, P and Ca/P ratio (P < 0.05); however, there were no significant differences for K (P = 0.43). Laser treatment at 1 W significantly affected the mean percentage weight of all element groups except K. Scanning electron microscopy (SEM) photographs indicated that the surface irregularities increased with increasing power setting.     

Effects of laser and acid etching and air abrasion on mineral content of dentin

The aim of this study was to evaluate the mineral content of dentin prepared using an Er,Cr:YSGG laser at four different power settings, acid etching, and air abrasion. The study teeth comprised 35 molars which were randomly divided into seven equal groups. The occlusal third of the crowns were cut with a slow-speed diamond saw. The groups were as follows: group A, control group; group B, dentin etched with 35% buffered phosphoric acid for 30 s; group C, dentin abraded at 60 psi with 50-μm aluminium oxide for 1 s; groups D–G, dentin irradiated with the Er,Cr:YSGG laser at 1.50 W (group D), 2.25 W (group E), 3.00 W (group F), and 3.50 W (group G). The levels of Mg, P, Ca, K and Na in each dentin slab were measured by inductively coupled plasma-atomic emission spectrometry (ICP-AES). Data were analysed by one way analysis of variance and Tukey HSD tests. There were no significant differences between the groups in the levels of Ca, P and Na, and the Ca/P ratio (p>0.05); however, there were significant differences in the levels of K (p<0.001) and Mg (p=0.13). In addition, the levels of Mg in the air abrasion group were higher than in the other groups (p<0.01). Etching with the Er,Cr:YSGG laser system, air abrasion and acid etching did not affect the levels of Ca, P and Na, or the Ca/P ratio, in the dentin surface.     

High concentration of sweat calcium, magnesium and phosphate in chronic renal failure

Sweat collected from the forearm of chronic renal failure and control patients, after iontophoretic stimulation with pilocarpine, was analyzed for Na, K, Cl, Mg, phosphate and urea. Concentrations of Ca, Mg and phosphate in sweat from chronic renal failure patients were significantly elevated (p less than 0.05) as compared to controls, while the concentrations of Na, K, and Cl were normal. The mean sweat rates and apparent skin conductivities were lower in uremics, but not significantly different from controls (0.05 less than p less than 0.10). Tentatively it is concluded that the increase of Ca, Mg and phosphate in uremic sweat is due to an increase in the secretion of these electrolytes in the secretory portion of the sweat gland, while the reabsorptive duct is normal.     

实验性骨关节炎关节软骨P、K、Ca、S元素的变化

目的　研究骨关节炎发病过程中关节软骨不同层次的元素含量的变化 ,探讨元素变化与生物力学的关系。方法　采用成年 Wistar大鼠复制实验性骨关节炎模型 ,于造模后 1个月和 3个月对关节软骨不同层次磷 ( P)、钾 ( K)、钙 ( Ca)、硫 ( S)等元素的变化进行观测。结果与结论　 ( 1 )软骨表层拉伸度降低与 K、Ca的吸附增多有关 ;( 2 )压力增加时 ,其关节软骨表面出现硫酸软骨素的破坏 ,随即开始重建以适应高压状态 ,而较深层主要以重建方式适应高压状态 ,再深层其硫酸软骨素还将保持相对稳定状态 ;( 3 )软骨钙化随着病情发展而加重。     

Urinary mineral excretion in healthy Iranian children

The purpose of this study was to determine normal reference values for urinary calcium/creatinine (Ca/Cr), phosphate/creatinine (P/Cr), magnesium/creatinine (Mg/Cr), sodium/creatinine (Na/Cr), potassium/creatinine (K/Cr), sodium/potassium (Na/K), calcium/sodium (Ca/Na), and uric acid/creatinine ratios in healthy Iranian children. Nine hundred and ninety children (515 boys, 475 girls) aged 1 month to 14 years were studied. Two non-fasting random urine specimens (1 week apart) from each subject and 24-h urine collections from 114 children were analyzed for Ca, P, Mg, uric acid, Na, K, and Cr. Urinary Ca/Cr, P/Cr, Mg/Cr, Na/Cr, K/Cr, Na/K, Ca/Na, and uric acid/creatinine ratios were determined from each sample. Non-fasting Ca/Cr, P/Cr, Na/Cr, K/Cr, Na/K, Ca/Na, and uric acid/creatinine ratios were not significantly different between the sexes (P>0.05). Urinary Mg/Cr ratios were higher in girls than boys (P<0.001). No significant relationships were found between urinary Ca/Cr and urinary Na/Cr, urinary Ca/Cr and urinary Na/K, and urinary Ca/Cr and urinary Ca/Na (P>0.05). The P/Cr values showed a gradual decrease with age (from mean+/-SD 0.962+/-0.172 mg/mg at 1 month of life to 0.318+/-0.124 mg/mg at 14 years) (P<0.05). The Ca/Cr ratio was highest between 6 months and 3 years (mean+/-SD=0.047+/-0.041 mg/mg). Following a moderate decrease it stabilized by the age of 7 years (mean+/-SD=0.038+/-0.044 mg/mg). Urinary ratios of Mg/Cr were significantly higher in children under 10 years (mean+/-SD=0.042+/-0.015 mg/mg) compared with the 11- to 14-year age group (mean+/-SD=0.031+/-0.001 mg/mg) (P<0.05). The uric acid/creatinine ratios decreased from 0.060+/-0.077 mg/mg in those less than 7 years to 0.041+/-0.033 in the 11- to 14-year group. Urinary Na/Cr ratios were significantly lower in younger age groups compared with the older age group (P<0.05). Urinary K/Cr ratio was highest in younger children, and then steadily decreased with age. There was no correlation between 24-h urinary Ca and Na excretion. The mean Ca/Na ratios significantly decreased with advancing age (P<0.05). The solute/creatinine ratios in the non-fasting urine samples correlated well with the 24-h solute excretion. We provide reference values for urinary Ca/Cr, P/Cr, Mg/Cr, Na/K, K/Cr, and uric acid/creatinine ratios in normal Iranian children. A child's age and ethnicity should be taken into consideration when assessing the urinary solute/creatinine ratios.     

兔脊髓损伤后钠钾钙镁含量变化及临床意义

目的研究脊髓损伤前后血清及脊髓内钠、钾、钙、镁含量的变化,为临床治疗脊髓损伤提供依据。方法３０只家兔用改良Ａｌｅｎ氏法造成脊髓损伤,６只家兔暴露脊髓不造成损伤。在伤前和伤后６ｈ、２４ｈ、４８ｈ、７２ｈ及６ｄ,用离子选择电极法和原子吸收光谱法测定血清和脊髓组织中离子钠、钾、钙、镁和总钠、钾、钙、镁含量。结果脊髓损伤后,血清中离子钙含量升高,总镁含量下降;脊髓组织中总钙、总钠含量升高,总镁、总钾含量下降。结论脊髓损伤后体内微量元素发生了不同程度的变化。     

The inorganic components of the testicle,epididymis, seminal vesicle,prostate and ejaculate of young men

The concentration of ashes, Ca, Mg, P, K, Na, Fe, Zn and Cu in testicle, epididymis, seminal vesicle, prostate and ejaculates from 21 healthy human males between 16 and 37 was analysed and compared, and the values discussed. With the exception of K (referred to dry weight) the ejaculate contained the highest quantum and tracer element concentration in comparison with those organs producing and storing the ejaculate liquid. The quantities of various elements found in various organs made conclusions possible about organs, through which such elements are put into the ejaculate liquid.     

槟榔牙表面着色沉积物的XRF及红外分析

目的通过对槟榔牙表面着色沉积物的成分分析,探究槟榔牙着色的可能原因。方法利用X射线荧光光谱(XRF)分析槟榔牙表面着色沉积物的元素组成;借助傅里叶变换红外光谱仪(FTIR)分析着色沉积物中可能的有机成分。结果着色区域含有O、C、Ca、P、S、Cl、Fe、Mn、Na等元素。着色区域有机成分中可能含有蛋白质、磷酸根、磷酸一氢根、碳酸根。结论槟榔牙无机着色与Fe、Mn、S元素相关,有机着色可能与蛋白质氧化有关。     

Body weight, early growth and antler size influence antler bone mineral composition of Iberian red deer (Cervus elaphus hispanicus)

Researchers have devoted little attention to the possibility that the chemical composition of bone might be variable under normal nutrition conditions. This study assessed antler bone composition of 25 one-year old deer (spikes). Antler content of ash, Ca, P, K, Na, Mg, Fe and Zn was assessed in base and tine, and the mean composition or the difference in composition between tine and base was used to explain variability in antler length, weight and perimeter. In turn, mean composition and difference in concentration of each mineral were related to body measures at 1 year of age, weight at birth, weight at 1 year of age and weight gains during lactation, or between weaning and year of age. Chemical composition differed between base and tine in ash, Ca, P, K, Zn and Fe, but not in Na or Mg. Composition explained a mean variability of 77% in antler length and weight. Body weight and size, in turn, influenced mineral composition. The greatest body effect was that of gains during lactation on principal components analysis factor related to Ca, P and other major minerals such as Na, K or Mg. Antler bone composition is variable in normal conditions and such variability may play a role in biomechanical properties of the antler, but it is also likely to show the nutritional status or physiological effort to grow antlers. Assessing bone composition may emerge as a new useful tool to obtain information regarding bone biology and its bearer in other species including ours.     

A zonal analysis of inorganic and organic constituents of the epiphysis during endochondral calcification

A dissection procedure has been devised to permit zonal analysis of the epiphyseal plate of fetal calf leg bones. Samples of whole and washed tissue from the various zones were analyzed for their content of electrolyte and organic constituents, as well as for density, ash and moisture. Results showed that as ash content and density increased, water content decreased. Ash levels in calcifying cartilage zones were unexpectedly high. When expressed as a percentage of dry weight, washed calcified cartilage had the highest content of any zone. In the early stages of the mineralization of cartilage, Na content (mmoles/l of fresh tissue) decreased as Ca and inorganic P increased. Magnesium levels increased as calcification proceeded, but only at a fraction of the rate of Ca and P. Ratios of Ca/inorganic P were highest in resting cartilage (non-differentiated hyaline cartilage), suggesting an initial binding of Ca to chondromucoproteins. However, at the onset of calcification in proliferating cartilage, Ca/P ratios were much lower (ca. 1.50), but gradually increased with advancing mineralization. Marked changes occurred in the composition of the organic phase during endochondral calcification. As determined by hydroxyproline analysis, collagen content progressively decreased during cartilaginous calcification, but increased rapidly during bone formation. As determined by hexosamine and sulfur analysis, chondromucoproteins were at highest levels in proliferating cartilage and decreased steadily as calcification increased. However, although calcification was already well advanced in hypertrophic cartilage, large amounts of mucopolysaccharide still were present. Sulfur/hexosamine ratios showed a slight decline during the early stages of calcification, but increased markedly with further mineralization. Sialic acid levels were elevated in epiphyseal cartilage over those in resting cartilage or bone. Lipids increased rapidly during cartilaginous calcification, but were greatly reduced in fully-formed bone. The significance of these findings is discussed.     

广西胆结石成分的19年变化(1981-1999)

目的：为了进一步研究广西胆石病的特点，我们在总结胆石病临床调查的同时对胆石的结构成分进行两个时期（1981-1990年，1991-1999年）的比较研究，在微观上探讨广西胆石病的特点。方法：对这两个时期以来不同部位（胆囊、胆总管、肝内胆管）的胆石进行铁、锌、钙、铜、镁、钾、钠、磷等8种微量元素以及胆固醇、磷脂、胆红素等的检测。结果：两个时期的不同部位结石均为以胆红素为主的色素性结石，所测微量元素中钙的含量始终最高。两时期比较无明显改变。结论：19年来广西胆结石的成分和种类基本没有改变。     

Magnesium Deficiency: Effect on Bone and Mineral Metabolism in the Mouse

Insufficient dietary magnesium (Mg) intake has been associated in humans with low bone mass. Mg deficiency in the rat has suggested bone loss is due to increased bone resorption and/or inadequate bone formation during remodeling. The purpose of this study was to assess the effect of a low Mg diet on bone and mineral metabolism in the young and mature BALB/c mouse and explore the hypothesis that inflammatory cytokines may contribute to Mg deficiency-induced osteoporosis. Using an artificial diet, we induced targeted Mg depletion (0.002% Mg) with all other nutrients maintained at the normal level. In all Mg-depleted mice, hypomagnesemia developed and skeletal Mg content fell significantly. The serum Ca in Mg-deficient mice was higher than in control mice; however, serum PTH levels were not significantly different. Osteoprotegerin (OPG) in dosages that inhibit osteoclastic bone resorption did not prevent hypercalcemia in Mg-deficient animals. No significant difference in serum Ca was observed between groups when dietary Ca was reduced by 50%, suggesting that a compensatory increase in intestinal absorption might account for the hypercalcemia. Growth plate width decreased 33% in young Mg-deficient animals and chondrocyte columns decreased in number and length, suggesting that Mg deficiency reduced bone growth. Trabecular bone volume in the metaphysis of the tibia in these animals was decreased and osteoclast number was increased by 135%. Osteoblast number was significantly reduced. Immunohistochemistry revealed that substance P increased 230% and 200% in megakaryocytes and lymphocytes, respectively, after 1 day of Mg depletion. IL-1 increased by 140% in osteoclasts by day 3 and TNF alpha increased in osteoclasts by 120% and 500% in megakaryocytes on day 12. This study demonstrates a profound effect of Mg depletion on bone characterized by impaired bone growth, decreased osteoblast number, increased osteoclast number in young animals, and loss of trabecular bone with stimulation of cytokine activity in bone.