Structural and analytical comparison of gallbladder stones collected from a single patient: studies of five cases

We observed the gross and fine structure of gallbladder stones collected from five adult patients (cases I–V) by optical photography, radiography, scanning electron microscopy, and backscattered electron microscopy, and then measured the components by energy-dispersive X-ray microanalysis and infrared spectroscopy. From the stones, calcium (Ca) phosphate, Ca bilirubinate, and Ca palmitate or fatty acid Ca were identified. The 3 cholesterol stones (case I) and the 2 brown pigment stones (case II) showed macroscopic homogeneity, respectively. In addition, their fine structure and components were also similar to each other. The black pigment stones (case III) showed macroscopic homogeneity, but they were divided into radiopaque (30 stones) and radiolucent types (60 stones). The former had Ca phosphate in the center surrounded with Ca bilirubinate, and the latter was dotted with minute deposits of Ca bilirubinate. The 6 cholesterol stones (case IV) were divided into two types in size. The 5 large stones, of macroscopic homogeneity, had a core region of Ca palmitate and clear concentric rings of Ca phosphate, whereas the smaller stone was almost filled with Ca phosphate deposits in the center. From the different distributions of Ca phosphate, the smaller stone may have been formed later than the 5 large stones. Case V contained 4 stones. The 3 large cholesterol stones, of more or less macroscopic homogeneity, had a core region and concentric rings of Ca phosphate, but 1 smaller stone was dotted with minute deposits mainly containing iron (Fe) and/or silicon elements (rare type). Therefore, the stones of cases III, IV, and V showed considerable heterogeneity, respectively. In many stones, the initial precipitation of Ca salts will have become the nidus, and the concentric rings and dotted deposits of Ca salts may have accelerated cholesterol stone growth. In addition, the dotted deposits of Ca bilirubinate in the black pigment stones and the dotted deposits containing Fe in the rare stone may have become also the nidi.     

A combined qualitative and quantitative procedure for the chemical analysis of urinary calculi

A better understanding of the physico-chemical principles underlying the formation of calculus has led to a need for more precise information on the chemical composition of stones. A combined qualitative and quantitative procedure for the chemical analysis of urinary calculi which is suitable for routine use is presented. The procedure involves five simple qualitative tests followed by the quantitative determination of calcium, magnesium, inorganic phosphate, and oxalate. These data are used to calculate the composition of the stone in terms of calcium oxalate, apatite, and magnesium ammonium phosphate. Analytical results and derived values for five representative types of calculi are presented.     

X-ray microanalysis of spontaneously formed urinary calculi in a jaundiced strain of rat

A high incidence of spontaneously formed urinary stone was found in the females of a jaundiced strain of rat developed from a cross between Gunn's rat and Wistar-Imamichi rat. In this colony, 42.3% of the females had urinary calculi. Elemental analyses of these urinary calculi were carried out with an analytical electron microscope, a high-resolution transmission electron microscope fitted with an energy dispersive type X-ray microanalyzer and a scanning device. In the surface and middle areas of the stone, the main components were recognized as magnesium (Mg) and phosphorus (P). In the central region of the stone, calcium (Ca) and phosphorus (P) were found as the main elements with trace amounts of sodium (Na), chlorine (Cl), and potassium (K). The analyses indicated that the spontaneous urinary stone consisted of phosphate salts with calcium or magnesium. In addition, the mass ratio of the $\text{Mg}\text{P}$ or $\text{Ca}\text{P}$ in the stones was calculated from X-ray pulse intensity ratios and compared with the mass ratio of a standard sample. The results suggested that the magnesium and phosphorus in the urinary stones existed as ammonium magnesium phosphate, MgNH₄PO₄, and the calcium and phosphorus as tribasic calcium phosphate, Ca₃(PO₄)₂.     

Electron microscopic findings of the liver of a patient with Dubin–Johnson syndrome complicated by chronic hepatitis C and iron overload

A patient was first found to have Dubin–Johnson syndrome and chronic hepatitis at the age of 25 years. Two years later, he underwent gastrectomy because of massive bleeding from duodenal ulcer, followed by posttransfusion self-limited biochemical liver damage. Twenty-five years later, his jaundice worsened to a serum bilirubin level of 10 mg/dl. The test for circulating hepatitis C virus RNA was highly positive, and liver histology showed cirrhosis with brown pigment granules in small numbers of hepatocytes. Some pigment granules were positive for histochemical iron stain. Ultrastructural study on the liver showed (1) the presence of iron-specific X-ray-positive pigment granules, and (2) large numbers of myelin-like bodies throughout the hepatocyte cytoplasm. Histologically advanced hepatic lesions of hepatitis C virus infection and posttransfu-sion iron overload may exacerbate bilirubin transport dysfunction of the syndrome.     

Binding of calcium and phosphate ions to dentin phosphophoryn

The concomitant binding of calcium and inorganic phosphate ions by the highly phosphorylated rat dentin phosphophoryn (HP) was measured in the pH range of 7.4-8.5 using an ultrafiltration procedure. HP binds almost exclusively the triply charged PO4(3-)ion, and for each PO4(3-)ion bound, the protein binds about 1.5 additional Ca2+ ions. The protein-mineral ion complex can be described as a protein with two different ligands, Ca2+ ions and calcium phosphate clusters having a stoichiometry of about Ca1.5PO4. The stoichiometry of the bound clusters is similar to amorphous calcium phosphate, indicating that the protein does not sequester crystal embryos of octacalcium phosphate or hydroxyapatite. The protein-mineral ion complex is amorphous by electron diffraction analysis and does not catalyze the formation of a crystalline phase when aged in contact with its solution.     

尿结石患者体重指数对血尿生化及结石成分的影响

目的 分析尿路结石患者体重指数（BMI）对24h尿液、血液生化指标及结石成分的影响。方法 对2017年1月~2018年7月我院收治的尿路结石患者150例进行血尿生化分析,按照BMI分为体重正常组45例、超重组65例,肥胖组40例,检测24h尿钙、磷、尿酸、血钙、血磷、血尿酸及尿pH等指标,同时收集患者结石标本进行红外光谱结石成分分析。比较组间各指标及结石成分的差异性;根据结石成分不同分为四组（草酸钙组、磷酸钙组、尿酸组和磷酸镁铵组）,比较组间各指标的差异性。结果 超重组和肥胖组尿液pH均低于正常组（P＜0.05）;体重正常组患者24h尿钙、尿磷、尿酸和血磷、尿酸明显低于超重或肥胖组（P＜0.05）。磷酸镁铵组患者尿液pH高于其它组（P＜0.05）。尿酸组和磷酸钙组24h尿磷高于草酸钙组和磷酸镁铵组（P＜0.05）。尿酸组24h尿酸高于其它组,磷酸镁铵组24h尿酸低于其它组（P＜0.05）。尿酸组血尿酸高于其它组,磷酸镁铵组血尿酸低于磷酸钙组和尿酸组（P＜0.05）。磷酸镁铵组BMI低于其它组（P＜0.05）。肥胖组、超重组草酸钙、尿酸结石比例均高于正常组（P＜0.05）,而磷酸镁铵比例则低于正常组（P＜0.05）。BMI与尿液pH的相关性分析表明两者高度相关（r=-0.725,P＜0.05）。结论 肥胖及超重对血尿生化、血生化、尿pH及结石成分比例有一定影响,且存在性别差异,建议肥胖或超重的尿结石患者行血尿生化、结石成分等评估,注意饮食控制、减重等措施。     

新疆哈密地区汉族泌尿系结石患者尿路结石成分分析

目的 研究新疆哈密地区汉族泌尿系结石患者的尿路结石成分及其分布特征。方法 收集选取2013年4月~2016年12月我院汉族泌尿系结石患者557例,分析其结石成分,并对其结石构成进行检测。结果 结石患者男女比为2.92:1,上、下尿路结石比为6.96:1。检出率最高的为草酸钙469例（84.22%）,其次是碳酸磷灰石216例（38.79%）,尿酸85例（15.27%）、磷酸镁铵62例（11.13%）、胱氨酸3例（0.54%）等成分结石占比较低。患者的发病率随年龄逐渐升高,40~60岁时达高峰。尿路结石主要分布于肾脏,占60.32%。各部位的尿路结石均以草酸钙、碳酸磷灰石为主,尿酸等成分较少。结论 新疆哈密地区汉族泌尿系结石患者的结石成分以草酸钙为主,结石发病率较高与饮食结构及生活习惯关系密切。     

Influence of ATP on sarcoplasmic reticulum function of vascular smooth muscle

A vesicular fraction isolated from bovine aorta and enriched in fragmented sarcoplasmic reticulum (FSR) exhibited active calcium transport and ATPase activity. By use of a hypotonic NaHCO3 extraction solution, an active preparation was isolated that retained activity for up to 4 days. A small but significant (P less than 0.05) Ca2+-stimulated, Mg2+-dependent ATPase associated with calcium transport was demonstrated with a specific activity of 0.33 mumol inorganic phosphate (Pi).mg-1.min-1. The basal Mg2+ ATPase demonstrated Michaelis-Menten kinetics [Km(Mg2+-ATP) = 0.44 +/- 0.01 X 10(-3) M; Vmax = 2.22 +/- 0.01 mumolPi.mg-1.min-1]. The Ca2+-stimulated, Mg2+-ATPase demonstrated apparent substrate inhibition (Ks approximately 10 mM) with no evidence for end-product (ADP) or excess added Ca2+ contributing to this inhibition. Oxalate-supported active calcium uptake velocities also exhibited quantitatively similar substrate inhibition. These results suggest that FSR from vascular smooth muscle contains either two enzymes or one enzyme with two isomeric forms, one of which is associated with the calcium uptake activity of this structure and the other of unknown function.     

Relation of plasma calcium to total protein and albumin in African grey (Psittacus erithacus) and Amazon (Amazona spp.) parrots

A significant correlation was found between total calcium and albumin concentration in the plasma of 70 African grey parrots (r=0.37; P<0.05). A correlation formula for plasma calcium concentration in the African grey parrot was derived on the basis of the concentration of albumin: Adjusted Ca (mmol/1) = Ca (mmol/1) - 0.015 Albumin (g/1) + 0.4. About 14% of the variability in calcium was attributable to the change in the concentration of plasma albumin concentration (R2=0.137). The correlation between calcium and total protein in African greys and between calcium and albumin and calcium and total protein in Amazons was not significant.     

Serum inorganic phosphate and serum calcium in middle-aged men. II. Relation to glucose and insulin parameters in glucose tolerance tests

Fasting serum inorganic phosphate and fasting serum calcium were measured in 752 49-year-old Malm? males, in whom an oral or i.v. glucose tolerance test, in 189 cases including plasma insulin measurements, was performed as part of an internal medical screening examination. The serum inorganic phosphate and calcium levels were analysed statistically in relation to various glucose and insulin response parameters. A significant, positive correlation of serum inorganic phosphate with the early insulin response in the i.v. glucose tolerance tests was found but not with any of the other glucose or insulin response parameters included in the study. Serum calcium showed no significant correlations. These findings are discussed in relation to the pertinent literature.     

尿路结石成分分析及临床应用研究

目的探讨本地区尿路结石成分构成情况,并根据尿路结石成分构成指导尿路结石的治疗和预防。方法收集江门市中心医院住院和门诊上、下尿路结石标本120例,采用化学显色法对尿路结石中磷酸盐、尿酸盐、草酸盐、碳酸盐、钙、铵、胱氨酸等化学成分进行定性分析。结果本组尿路结石样本中含钙结石最多,占80.8%,含磷酸盐结石次之,占67.5%。单纯性结石占35%,其中草酸钙结石为最多,占69%。感染性结石中以磷酸盐结石最多,占68.7%,含铵结石占61.2%。复发结石含磷酸盐占88.2%,其比例显著高于初发结石。上尿路结石中含钙和磷酸盐结石显著高于下尿路结石,下尿路结石中含尿酸盐结石显著高于上尿路。结论根据结石分析得到的结石化学成分特点,可有针对性地指导临床治疗,并可指导患者从饮食、生活习惯等方面预防尿路结石的发生和复发。     

Relationship of plasma total protein and albumin to total calcium in peregrine falcons (Falco peregrinus)

A significant correlation was found between total calcium and total protein concentration in 124 plasma samples of captive peregrine falcons (r = 0.65; P<0.01). About 42% of the variability in calcium was attributable to the change in the plasma total protein concentration (R2 = 0.417). The correlation between calcium and albumin was significant (r = 0.33; P<0.01), but significantly smaller than the correlation between calcium and total protein (P<0.01). Only 11% of the plasma calcium concentration was attributable to difference in concentration of albumin (R2 = 0.108). An adjustment formula for plasma calcium concentration in the peregrine falcon was derived on the basis of the total protein concentration: Adj.Ca (mmol/1) = Ca (mmol/1) -0.02 Total protein (g/1) + 0.67.     

Calcium fluxes in isolated pancreatic acini: effects of secretagogues

45Ca2+ exchange and total calcium content were measured in isolated mouse pancreatic acini. 45Ca2+ uptake could be described as the sum of a constant and a single exponential kinetic component; about 60% of total acinar calcium was exchangeable. Stimulation by bethanechol increased 45Ca2+ uptake, but the time course of uptake could be fit only by the addition of a more rapid kinetic component without any change in the total exchangeable Ca2+. 45Ca2+ washout after 1-h loading could be fit as the sum of two exponential components. Stimulation increased the rate of 45Ca2+ washout with the appearance of a third and more rapid kinetic component. There was not, however, a good correspondence between the exponential constants measured in uptake and washout protocols in unstimulated acini. Exponential constants were also affected by the concentration of calcium in the medium, further indicating the presence of nonlinearities in 45Ca2+ exchange. The dose-response relationships were similar for bethanechol stimulation of 45Ca2+ uptake and amylase release, whereas stimulation of 45Ca2+ washout reached a maximum at a higher concentration of bethanechol. Both 45Ca2+ uptake and analytical measurement of total Ca2+ showed a rapid drop in acinar Ca2+ content followed by a gradual reuptake on stimulation by bethanechol. It is concluded that the initial primary effect of secretagogues is to increase Ca2+ efflux, which is interpreted to be the result of release of sequestered calcium into the cytosol.     

Scanning Electron Microscope Studies of Human Gallstones After Plasma Etching

The morphological relationship between cholesterol and the calcium carbonate polymorphs and other inorganic constituents in intact human gallstones was investigated with scanning electron microscopy (SEM) after plasma etching the specimen instead of chemical dissolution of the organic constituents. The technique allowed magnification of more than 22,000× without structural damage or distortion. A framework of inorganic material that also contained crystals of calcium carbonate, vaterite, aragonite, calcite, and apatite remained after plasma etching. All cholesterol monohydrate and bile pigment compounds had been removed. The plasma etching technique allowed SEM studies of intact gallstones. An inorganic framework matrix was found in all stones. Its possible role in their formation is discussed.     

Scanning Electron Microscope Studies of Human Gallstones After Plasma Etching

The morphological relationship between cholesterol and the calcium carbonate polymorphs and other inorganic constituents in intact human gallstones was investigated with scanning electron microscopy (SEM) after plasma etching the specimen instead of chemical dissolution of the organic constituents. The technique allowed magnification of more than 22,000× without structural damage or distortion. A framework of inorganic material that also contained crystals of calcium carbonate, vaterite, aragonite, calcite, and apatite remained after plasma etching. All cholesterol monohydrate and bile pigment compounds had been removed. The plasma etching technique allowed SEM studies of intact gallstones. An inorganic framework matrix was found in all stones. Its possible role in their formation is discussed.     

Calcium phosphate formation at the surface of bioactive glass in vitro

The calcium phosphate formation at the surface of bioactive glass was studied in vitro. Glass rods and grains were immersed in different aqueous solutions and studied by means of scanning electron microscopy and energy dispersive x-ray analysis. Surface morphological changes and weight loss of corroded grains were monitored. In-depth compositional profiles were determined for rods immersed in the different solutions. The solutions used were tris-buffer (tris-hydroxymethylaminomethane + HCl), tris-buffer prepared using citric acid (tris-hydroxymethylaminomethane + C6H8O7.H2O), and a simulated body fluid, SBF, containing inorganic ions close in concentration to those in human blood plasma. It was found that the calcium phosphate formation at the surface of bioactive glass in vitro proceeds in two stages. When immersing the glass in tris or in SBF a Ca,P-rich surface layer forms. This accumulation takes place within the silica structure. Later, apatite crystals forming spherulites appear on the surface. The Ca/P-ratio of initially formed calcium phosphate was found to be about unity. It is proposed that this is due to bonding of phosphate to a silica gel. The surface is stabilized, i.e., leaching is retarded, by the rapid Ca,P-accumulation within the silica structure before apatite crystals are observed on the surface. It is proposed that the initially formed calcium phosphate is initiated within the silica gel. The crystallizing surface provides nucleation sites for extensive apatite formation on the glass surface. In the presence of citrate no Ca,P-accumulation occur at the glass surface, but soluble Ca-citrate complexes form. By comparing the weight loss during corrosion in tris with that in the calcium and phosphate containing SBF, it is possible to establish whether the glass can induce apatite formation at its surface or not.     

Effect of serum fibroblast growth factor receptor 2 and CAPS proteins on calcium status in β-thalassaemia major patients who are free from overt inflammation

Bone disorders and disturbed calcium (Ca) homeostasis are common disorders in β-thalassaemia major (β-TM). In the present study, two bone related markers are studied in β-TM patients with negative C-reactive protein for the first time; fibroblast growth factor receptor 2 (FGFR2) and CAPS protein. Another goal is to estimate the correlation between the recent parameters and bone biomaterials as a function of iron status parameters in β-TM patients. The results revealed that, in patients with β-TM serum FGFR2, CAPS, alkaline phosphatase (ALP) and Mg significantly increased while serum Ca levels were low as compared with controls. Ca status is correlated with iron overload in β-TM. A significant correlation was present between CAPS and FGFR2. In conclusion, FGFR2 and CAPS associated with Ca status and subsequent bone disturbances in β-TM patients. Their level can be predicted from the equation: CAPS =0.001ALP +0.48FGFR2-1.26Ca – 3.95Pi +12.76 with acceptable applicability.     

Muscle fatigue: the role of intracellular calcium stores.

Force declines when muscles are used repeatedly and intensively and a variety of intracellular mechanisms appear to contribute to this muscle fatigue. Intracellular calcium release declines during fatigue and has been shown to contribute to the reduction in force. Three new approaches have helped to define the role of calcium stores to this decline in calcium release. Skinned fibre experiments show that when intracellular phosphate is increased the amount of Ca2+ released from the sarcoplasmic reticulum (SR) declines. Intact fibre experiments show that the size of the calcium store declines during fatigue and recovers on rest. Intact muscles which lack the enzyme creatine kinase, do not exhibit the usual rise of phosphate during fatigue and, under these conditions, the decline of Ca2+ release is absent or delayed. These results can be explained by the "calcium phosphate precipitation" hypothesis. This proposes that if phosphate in the myoplasm rises, it enters the SR and binds to Ca2+ as Ca2+ phosphate. The resultant reduction in free Ca2+ within the SR contributes to the reduced Ca2+ release during fatigue.     

Microhardness measurements of renal calculi: regional differences and effects of microstructure.

Microhardnesses of five types of renal calculi: calcium apatite (82.5%)/magnesium ammonium phosphate hydrogen (10%)/calcium oxalate monohydrate (7.5%); calcium apatite (95%)/calcium oxalate monohydrate (5%); magnesium ammonium phosphate hydrogen (90%)/calcium apatite (10%); calcium oxalate monohydrate (85%)/calcium apatite (15%); and cystine (100%) were measured. Using Knoop and Vickers indenters the effects of chemical composition and microstructure on the microhardness measurement were assessed. Calcium oxalate monohydrate, magnesium ammonium phosphate hydrogen, and cystine stones, without apparent structure pattern, showed neither regional nor directional differences in their microhardness. In contrast, calcium apatite stones, with distinctly concentric laminae structure, showed regional variations which were correlated with the chemical composition of stone constituents. Scanning electron microscopy of the indenter impressions were taken to help in interpreting the directional dependence in Knoop hardness measurements with respect to the microstructure of the calculi. Vickers measurements showed the crystalline stones were isotropic within a layer. Combined results of Knoop and Vickers measurements indicate that the anisotropic Knoop hardness readings seen in the laminated regions were structural but not material-based. Implications of the results for the fragmentation of renal calculi in extracorporeal shock wave lithotripsy are discussed.     

Excretion of magnesium,calcium, and inorganic phosphate by the cat submandibular gland

Summary The excretion of magnesium, calcium, and inorganic phosphate by the submandibular gland of normal cats was studied during electrical stimulation of the parasympathetic nerve fibers to the gland.The concentration of magnesium and calcium decreased rapidly after the beginning of constant stimulation. Thereafter a constant level was reached. The changes of phosphate concentrations were less marked.Steady state magnesium and calcium concentrations were independent of the salivary flow when this was above 100 l/min×g gland weight. The concentration of both magnesium and calcium increased at flows below this value. Steady state concentration of inorganic phosphate was independent of salivary flow when this was higher than 150–200 l/min×g gland weight and increased at lower secretory rates. The concentrations of all the three elements were always lower than the serum concentrations.Acute thyroparathyroidectomy did not alter the secretory pattern. In acutely thyroparathyroidectomized animals an increase in serum magnesium concentration from 2–5 meq/l did not change steady state salivary magnesium concentrations. This was only moderately augmented by still higher concentrations. A rise in serum calcium concentration to 15 meq/l did not affect the steady state salivary calcium concentration.The technical assistance of Mrs. K. Nielsen and Miss K. Videbæk is gratefully acknowledged.