Some normal biochemical parameters of pigs in Pupua New Guinea

The normal values of serum glutamic oxaloacetic transminase, serum glutamic pyruvic transaminase, serum lactic dehydrogenase and serum alkaline phosphatase, total protein, urea, creatine, cholesterol, glucose, magnesium, calcium and inorganic phosphorus were measured monthly over a 12-month period from 10 "pure" Native and 10 Native X British Crossbred pigs. Except for cholesterol, no significant difference was found between the two groups. Similar estimations were made for 5-month and 11-month Village pigs in which the serum alkaline phosphatase, inorganic phosphorus, total protein, urea, creatinine and calcium were significantly lower when compared with the corresponding age group of the pure Native pigs and Crossbred pigs. These lower values are thought to be due to the effects of the malnutrition-parasite complex of Village pigs.     

Alkaline phosphatase (EC 3.1.3.1) in serum is inhibited by physiological concentrations of inorganic phosphate

Natural and artificial manipulation of tissue nonspecific alkaline phosphatase activity indicates that pyrophosphate, phosphoethanolamine, and pyridoxal 5'-phosphate are among the natural substrates for this enzyme. Although inorganic phosphate has been recognized as a competitive inhibitor of this enzyme for many years, the influence of phosphate on alkaline phosphatase activity in serum under physiological conditions has not been previously reported. We examined the kinetics of tissue nonspecific alkaline phosphatase from bovine kidney and sera from 49 patients with a wide range of endogenous phosphate concentrations using pyridoxine 5'-phosphate as a substrate at pH 7.4. For the bovine kidney enzyme, the Km was 0.42 +/- 0.04 micromol/L, and the Ki for phosphate was 2.4 +/- 0.2 micromol/L. Analysis of the kinetics using pyridoxine 5'-phosphate in undiluted serum from 10 subjects with phosphorus ranging from 0.5-2.1 mmol/L and alkaline phosphatase activity ranging from 41-165 nmol/min x mL gave estimates for the Km of 56 +/- 11 micromol/L and for the Ki of 540 +/- 82 micromol/L for phosphate. This indicates that under physiological conditions alkaline phosphatase activity toward pyridoxine 5'-phosphate is reduced approximately 50% by the normal phosphate concentration and that it will increase or decrease significantly in response to changes in phosphate concentration within the ranges observed clinically.     

Epidemiological study of osteoporosis

An epidemiological investigation of osteoporosis was carried out in a community in which fishing is the primary industry, and correlations were investigated between the bone mass and various risk factors for osteoporosis and biochemical findings. In 852, who could be examined directly, of the 3541 males and females aged 40 years or above living in Nansei Town, Watarai District, Mie Prefecture, the age, sex, height, body weight, years after menopause, married or unmarried, area of residence, daily activity, and intakes of milk, fish, and alcohol were studied by direct oral inquiry, and the serum calcium, inorganic phosphorus, alkaline phosphatase, and total protein were measured. The subjects were divided into a decreased bone mass group who showed grade I or more advanced loss in bone mass and a normal bone mass group who showed no or only slight loss in bone mass on the basis of microdensitometry (MD). Eighty-two subjects (9.6%) were classified as the decreased bone mass group. Items that showed a close correlation with sigma GS/D, which is an index of bone density, were age, years after menopause, serum alkaline phosphatase level, height, and body weight in females, age and serum alkaline phosphatase level in males. In the decreased bone mass group, 79 of the 82 subjects were females. Significant differences were observed between the decreased bone mass group and the normal bone mass group in age, years after menopause, serum alkaline phosphatase level, height, and body weight. From these results, a high age, being a female, low height, and a low body weight were found to be factors correlated with osteoporosis. Also, the bone mass was correlated with the serum alkaline phosphatase level and years after menopause.     

25-hydroxycholecalciferol serum levels in patients with Crohn's disease

In 37 patients with Crohn's disease the 25-hydroxycholecalciferol (25-HCC) serum level, serum concentration of calcium and inorganic phosphate, and the enzyme activity of alkaline phosphatase were measured. Furthermore the activity index of Crohn's disease was determined in every patient. There was no statistically significant difference of 25-HCC serum levels in these patients compared to a healthy control group. Correspondingly most patients showed normal alkaline phosphatase enzyme activity and normal serum concentration of calcium and inorganic phosphate. No correlation between 25-HCC concentration and site of inflammation or activity index was found.     

Induction of the blood/brain-barrier-associated enzyme alkaline phosphatase in endothelial cells from cerebral capillaries is mediated via cAMP

The mammalian blood/brain barrier is located at the endothelial cells of the cerebral capillaries. Alkaline phosphatase is associated to a very large extent with these cells and has been established as a marker enzyme for a differentiated blood/brain barrier phenotype in vivo and in vitro. Nevertheless cultured brain capillary endothelial cells (BCEC) lose this marker enzyme because of a cessation of de novo synthesis. Since astrocytes have been shown to possess the capability to re-induce the enzymic activity of alkaline phosphatase in BCEC in vitro we were interested in the second messengers involved in the signal-transduction mechanism of this induction in BCEC. For this reason we treated cultured porcine BCEC with a water-soluble and membrane-permeable analogue of cAMP, 8-(4-chlorophenylthio)-cAMP (C1PhS-cAMP) in the absence of astrocytes. By means of enzymic activity assays we were able to show that within three days the activity of alkaline phosphatase increased up to sixfold compared with the controls. The total activity of alkaline phosphatase in C1PhS-cAMP-treated BCEC was comparable to that of freshly isolated cells. Addition of cycloheximide inhibited the alkaline phosphatase activity increase. We conclude that cAMP is one of the second messengers involved in the induction of alkaline phosphatase activity in BCEC in vitro.     

Values of the serum components in Japanese black beef steers at farms with high productivity and low frequencies of disease and death in Miyazaki Prefecture

Few reference values for use in metabolic profile tests for the maintenance of high productivity and the prevention of production diseases have been reported in Japanese Black beef cattle. To obtain basic data, 101 healthy steers at farms with high productivity and low frequencies of disease and death in Miyazaki Prefecture, Japan, were examined for the values of their serum components in this preliminary study. At the later fattening stage (5 to 20 months after introduction), statistically significant increases were observed in the mean serum activities of lactic dehydrogenase, glutamic-oxalacetic transaminase, gamma-glutamyl transpeptidase, and creatine phosphokinase, the mean serum contents of triglyceride, total cholesterol, albumin (Alb), total protein, blood urea nitrogen, magnesium, and vitamin E, and the mean serum calcium (Ca)/inorganic phosphorus (IP) ratio, and statistically significant decreases were seen in the mean serum alkaline phosphatase activity and the mean serum contents of glucose, IP, and vitamin A. The mean serum Alb/globulin ratio and the mean serum Ca and nonesterified fatty acids contents demonstrated no statistically significant changes.     

Phosphaturia in thalassemia

Thirteen phosphorus balances were performed in four thalassemic children aged 6 to 10 years. No correlation was found between phosphorus intake and serum level or between phosphorus intake and net absorption. There was a positive correlation among daily phosphorus intake, net absorption, and 24-hour urinary excretion. The 24-hour urinary excretion level was higher than net absorption, indicating that these children have normal phosphorus absorption but abnormally high renal phosphaturia, which leads to a deficiency of phosphorus. A strongly positive correlation was found between values for hemoglobin and serum alkaline phosphatase. In the thalassemic patients with hemoglobin levels larger than or equal to 7.5 gm/100 ml, the serum alkaline phosphatase values were larger than or equal to 15 King-Armstrong units, suggesting normal osteoblast function.     

Skeletal scintigraphic findings in endemic skeletal fluorosis

Endemic skeletal fluorosis is characterized by bone, joint and muscle pain, progressive ankylosis of various joints and crippling deformities. Whole body skeletal scintigraphy with 99Tcm-methylene diphosphonate was performed for 17 symptomatic subjects suffering from this disorder. The fluoride content of drinking water ranged from 4.1 to 12.9 mg l-1 (normal < 1 mg l-1). Urinary and serum fluoride levels were markedly elevated. Serum calcium (total and ionized), inorganic phosphorus, creatinine and albumin were essentially normal while serum alkaline phosphatase was elevated in six subjects (mean +/- S.D. 206 +/- 106; range 22-1072 IU l-1). Skeletal radiology revealed a wide spectrum of bony abnormalities. Skeletal scintigraphy revealed a picture similar to metabolic 'superscan' in all subjects, i.e. increased tracer uptake in axial and appendicular skeleton, reduced soft tissue uptake, poor or absent renal images, prominent costochondral junction and 'tie' sign in sternum. Increased uptake was present in all subjects irrespective of age, water fluoride content, serum alkaline phosphatase level and radiological abnormalities. Our findings suggest the presence of a high bone turnover state in endemic skeletal fluorosis irrespective of other variables.     

Postcholestatic alkaline phosphatase activity after relief of bile duct obstruction in the rat

The effects of obstructive cholestasis on the activity of alkaline phosphatase have been extensively studied in serum and liver tissue. However, very little is known about the activity of this enzyme in the postcholestatic condition after relief of the biliary obstruction. The purpose of this study has been to characterize alkaline phosphatase activity in serum, liver and bile in the postcholestatic period and to relate it to changes in bile acid secretory rate. Serum activity and biliary secretory rates of alkaline phosphatase were markedly increased in rats subjected to a reversible obstructive cholestasis for 24 hr or 48 hr and progressively declined along the postcholestatic period to values not significantly different from those of control rats within 48 hr. A significant direct linear relationship between the biliary secretory rates of enzyme activity and bile salts was apparent both in cholestatic groups and in the control groups. The slope of the regression line (units of alkaline phosphatase secreted per micromole of bile salts) was 1.5-fold to 3-fold higher in cholestatic animals. Remarkably, a positive y-intercept of regression lines suggested that a significant fraction of the enzyme was secreted independently of bile salts; this fraction was 18-fold and 34-fold greater in 24-hr and 48 hr cholestatic rats, respectively, compared with that in controls. Sodium taurocholate administered intravenously, either as a bolus or as an infusion at increasing submaximal rates, resulted in parallel increases of bile salt and alkaline phosphatase secretory rates into bile. The enzyme activity secreted per micromole of taurocholate was significantly greater in cholestatic than in control rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gallium nitrate inhibits alkaline phosphatase activity in a differentiating mesenchymal cell culture

The effect of gallium nitrate on alkaline phosphatase activity in a differentiating chick limb-bud mesenchymal cell culture was monitored in order to gain insight into the observation that rachitic rats treated with gallium nitrate failed to show the expected increase in serum alkaline phosphatase activity. Cultures maintained in media containing 15 microM gallium nitrate showed drastically decreased alkaline phosphatase activities in the absence of significant alterations in total protein synthesis and DNA content. However, addition of 15 microM gallium nitrate to cultures 18 h before assay for alkaline phosphatase activity had little effect. At the light microscopic and electron microscopic level, gallium-treated cultures differed morphologically from gallium-free cultures: with gallium present, there were fewer hypertrophic chondrocytes and cartilage nodules were flatter and further apart. Because of altered morphology, staining with an antibody against chick cartilage alkaline phosphatase appeared less extensive; however, all nodules stained equivalently relative to gallium-free controls. Histochemical staining for alkaline phosphatase activity was negative in gallium-treated cultures, demonstrating that the alkaline phosphatase protein present was not active. The defective alkaline phosphatase activity in cultures maintained in the presence of gallium was also evidenced when cultures were supplemented with the alkaline phosphatase substrate, beta-glycerophosphate (beta GP). The data presented suggest that gallium inhibits alkaline phosphatase activity in this culture system and that gallium causes alterations in the differentiation of mesenchymal cells into hypertrophic chondrocytes.     

Subcellular localization of enterokinase (enteropeptidase EC 3.4.21.9) in rat small intestine

The subcellular localization of enterokinase is controversial. In this study, enterokinase was extracted from a soluble fraction and a brush border fraction of rat small intestine by differential centrifugation. The soluble fraction contained 41% of the initial enterokinase activity while the brush border fraction contained only 4.6% of the initial activity. In contrast, alkaline phosphatase monitored as a brush border marker, yielded 26.3% in the brush border fraction and only 6% in the soluble fraction. Further separation of the soluble fraction on a Sepharose 4B column revealed three peaks of enterokinase activity. One small peak (3%) of a bound enzyme (Mr, 2 - 10(6)) and two larger peaks of free enzyme (Mr, 3 - 10(5) and 9 -10). In contrast, alkaline phosphatase major fraction was in a high molecular weight peak of bound enzyme. When the brush border fraction was chromatographed only a single peak of bound enterokinase and alkaline phosphatase were found. In the lower part of the small intestine, no brush border-bound enterokinase was found, while the peak of alkaline phosphatase was the same as in the upper intestine. These data suggest that enterokinase activity in the rat intestine is mainly in a free form localized in the mucin and soluble fraction and to a negligible extent in the brush border.     

Course of blood levels of calcium, inorganic phosphate, alkaline phosphatase, parathyroid hormone and calcidiol (25-OH-D3) in one and two year old thoroughbred horses

The present study was aimed to determine the contents of calcium, inorganic phosphate, parathormon, 25-OH-D3 and the activity of alkaline phosphatase in the plasma of one- and two-years-old thoroughbred horses. Data were obtained monthly from 44 one-year-old thoroughbred of 4 different studs from May during grazing-season and from October during stable-, resp. training-season up to april of the following year. Calcium, inorganic phosphate and the activity of alkaline phosphatase were measured with a photometric method and the concentration of PTH and 25-OH-D3 were determined with a radioimmunoassay. The following results were obtained: Calcium: The concentration of calcium in the plasma of one-year-old thoroughbred horses was 3.03 +/- 0.23 mmol/l during grazing-season and 3.14 +/- 0.14 mmol/l during the following stable-, resp. training-season. Inorganic phosphate: The concentration of inorganic phosphate was significantly affected by the age. The average was 1.7 +/- 0.19 mmol/l during grazing-season and 1.3 +/- 0.19 mmol/l during the following stable- and training-season. Activity of alkaline phosphatase: The activity of alkaline phosphatase was also significantly affected by the age. The average of the activity was 403 +/- 86 U/l during grazing-season and 308 +/- 65 U/l during the following winter period. Parathormon: There were big differences between the averages of parathormon during grazing-season (1.27 +/- 0.45 ng/ml) and the following winter-season (0.9 +/- 39 ng/ml). Besides from that there were big individual differences. 25-OH-D3: The concentration of 25-OH-D3 during grazing-season (10.38 +/- 3.08 ng/ml) was lower than during the winter period (13.03 +/- 2.86 ng/ml). The significance of the obtained results is discussed in relation to the corresponding literature.     

State of protein and mineral metabolism following thyrocalcitonin administration during experimental osteogenesis

Prolonged administration of thyrocalcitonine (TC) into rats (at a dose 5 un per animal) led to some alterations in protein and mineral metabolism: decrease in content of calcium and phosphorus in blood, activation of alkaline phosphatase in blood, decrease in excretion of hydroxyproline, increase in excretion of aminonitrogen with urine. Distinct correlation was observed between the metabolic activity and definite steps in reparative osteogenesis. Exogenous TC activated the cellular structures, which participated in the regenerative processes, promoted the rapid turnover of bone generation and decreased the periods of tracture healing.     

Phosphatase-negative mutants of Legionella pneumophila and their behavior in mammalian cell infection

Microbial phosphatases are known or suspected to play a role in the pathogenesis of several intracellular pathogens, including Legionella micdadei. Legionella pneumophila also possess phosphatase activities, but their possible roles in cellular infection are unknown. We generated mutants of a serogroup 1 isolate of L. pneumophila that lack the major phosphatase. Isolation of a Pho- mutant after random mutagenesis with transposon MudII4041 allowed us to dissociate the major alkaline phosphatase (pH optimum approximately 8) from a minor acid phosphatase activity. Both activities were concentrated in the bacterial periplasm. The gene encoding the major alkaline phosphatase (pho) was cloned by expression in E. coli and used to generate a site directed mutation in two L. pneumophila strains. Each parent-mutant pair was compared in a U937 cell tissue culture assay for capacity to infect, lyse, and grow within mammalian cells. Although the parental stains differed in their U937 cell cytopathicity, neither was significantly more infective than its Pho- derivative, suggesting that the alkaline phosphatase activity is not essential for cellular infection. Because they are not attenuated, Pho- mutants can be used to generate gene fusions with E. coli alkaline phosphatase to study and secretion and cellular infectivity in L. pneumophila.     

Gay men who engage repeatedly in unprotected anal intercourse with casual partners: the Sydney Men and Sexual Health Study

A probable outbreak of oak (Quercus calliprinos) toxicosis in a herd of beef cattle--heifers and first-calving cows--grazing in the Judean foothills of Israel is described. Toxicosis probably occurred because of the consumption of oak leaves and buds during a period of pasture scarcity without any feed supplementation. A progressive syndrome of wasting, dullness, anorexia, polyuria, nephrosis, constipation and recumbency, culminating in death, was seen. A high mortality rate of 83% (38/46 animals) was noted. The clinical-pathological findings revealed increases in blood urea, creatinine, aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), creatine kinase (CK), lactate dehydrogenase (LDH) and inorganic phosphorus. Decreases were found in alkaline phosphatase (ALP), total serum protein, albumin (ALB), triglyceride (TG), calcium (Ca), magnesium (Mg), sodium (Na) and chloride (CI). The main pathological findings were severe nephrosis, chronic interstitial nephritis, and occasional intestinal ulceration. On the basis of epidemiology, clinical signs, clinical-pathological and pathological findings and renal histology, a tentative diagnosis of oak toxicosis was made.     

The integration host factor (IHF) affects the expression of the phosphate-binding protein and of alkaline phosphatase in Escherichia coli

The genes encoding alkaline phosphatase (phoA) and the inducible inorganic phosphate transport system Pst (pstS,C,A,B,U) belong to the PHO regulon. Mutants of Escherichia coli lacking the global regulatory protein integration host factor (IHF) show an increased level of alkaline phosphatase and a decreased level of Pst. IHF binds weakly but specifically to a DNA fragment containing the promoter region of the pst operon but does not bind to a fragment that includes the promoter region of phoA. It is proposed that IHF is a positive regulator of the pst operon and as such controls indirectly the expression of phoA.     

Plasma bone-specific alkaline phosphatase as an indicator of osteoblastic activity

The total plasma alkaline phosphatase level has long been recognised as an indicator of osteoblastic activity, but lack of specificity makes it an insensitive index of the progress of disease and the response to treatment. Selective precipitation by wheatgerm lectin allows measurement of the plasma bone-specific alkaline phosphatase. We measured the plasma levels of this isoenzyme in 170 normal Chinese adolescents and adults, in 49 adults with fractures of a long bone, in 15 patients with osteosarcoma and in 38 patients with osteolytic metastases. The enzyme activity was also determined in 39 patients with liver disease. Of the patients with fractures, 94% had increased plasma activity during the healing process. The level was also increased in those with osteosarcoma but not in those with osteolytic bone metastases. There was no significant increase in activity in the patients with liver disease. We conclude that the plasma bone-specific alkaline phosphatase activity is a sensitive and reliable measure of osteoblastic activity.     

Dephosphorylation activates the purified plant plasma membrane H+-ATPase--possible function of phosphothreonine residues in a mechanism not involving the regulatory C-terminal domain of the enzyme

The plasma membrane H+-ATPase was purified from tobacco cells (line BY-2). After solubilization by lysophosphatidylcholine followed by separation on a glycerol gradient, a fraction with a high specific activity of 9 micromol ATP x min(-1) x mg protein(-1) was obtained, in which the H+-ATPase polypeptide represented at least 80% of the protein. The incubation of this fraction in the presence of alkaline phosphatase increased H+-ATPase activity by 40%, in a manner consistent with dephosphorylation of the enzyme itself. The hydrolytic activity of the solubilized enzyme and its proton translocating activity, after reconstitution into proteoliposomes, were stimulated to the same extent. Alkaline phosphatase treatment was also accompanied by a 92% decrease in the H+-ATPase phosphothreonine content, whereas the phosphoserine residues were almost unaffected. The dephosphorylation induced a slight decrease of the affinity of the enzyme towards ATP. The purified enzyme was not activated by lysophosphatidylcholine addition nor by trypsin-mediated proteolysis, two treatments reported to release the inhibitory control by the C-terminal domain of the H+-ATPase and to increase the affinity of the enzyme towards ATP. Based on these results, the regulatory phosphorylation evoked by alkaline phosphatase most likely differs from the autoinhibitory control of the H+-ATPase by its C-terminal domain.     

Spatial physiological heterogeneity in Pseudomonas aeruginosa biofilm is determined by oxygen availability

The role of oxygen availability in determining the local physiological activity of Pseudomonas aeruginosa growing in biofilms was investigated. Biofilms grown in an ambient-air environment expressed approximately 1/15th the alkaline phosphatase specific activity of planktonic bacteria subjected to the same phosphate limitation treatment. Biofilms grown in a gaseous environment of pure oxygen exhibited 1.9 times the amount of alkaline phosphatase specific activity of air-grown biofilms, whereas biofilms grown in an environment in which the air was replaced with pure nitrogen prior to the inducing treatment did not develop alkaline phosphatase activity. Frozen cross sections of biofilms stained for alkaline phosphatase activity with a fluorogenic stain demonstrated that alkaline phosphatase activity was concentrated in distinct bands adjacent to the gaseous interfaces. These bands were approximately 30 micron thick with biofilms grown in air, 2 micron thick with biofilms grown in pure nitrogen, and 46 micron thick with biofilms grown in pure oxygen. Overall biofilm thickness ranged from approximately 117 to approximately 151 micron. Measurements with an oxygen microelectrode indicated that oxygen was depleted locally within the biofilm and that the oxygen-replete zone was of a dimension similar to that of the biologically active zone, as indicated by alkaline phosphatase induction. These experiments revealed marked spatial physiological heterogeneity within P. aeruginosa biofilms in which active protein synthesis was restricted by oxygen availability to the upper 30 micron of the biofilm. Such physiological heterogeneity has implications for microbial ecology and for understanding the reduced susceptibilities of biofilms to antimicrobial agents.     

1.25(OH)2 cholecalciferol pulse therapy and the effects of different dialysis membranes on serum PTH levels of haemodialysis patients

Either oral, intravenous or subcutaneous 1.25(OH)2 cholecalciferol is used in the therapy of hyperparathyroidism, which is a serious complication in patients on haemodialysis. We studied a total of 30 patients (10 women and 20 men) and divided them into two groups depending on the different types of dialysis membranes used. In the polysulfone group, mean age was 43.7 +/- 0.97 years and the average dialysis period lasted 29.9 +/- 1.23 months. For the 15 cases in which we used cuprophane membrane the mean age was 40.2 +/- 1.31 years and the average dialysis period lasted 16.2 +/- 0.86 months. The calcium level of the dialysate in both groups was 1.5 mmol/l. According to the study protocol, the determined oral calcitriol dose was 0.07 mg/kg and it was administered intermittently. After one month on high dose calcitriol therapy, treatment was continued with a maintenance dose of 0.03 mg/kg for a further six months. As a phosphate binding agent, daily 3 g calcium carbonate was administered. Before starting this treatment protocol, patients went on a 1 mg/day calcitriol therapy, although the mean PTH level was 424.63 pg/ml and the mean serum alkaline phosphatase level was 290.2 U/l. During the pretreatment period, levels of PTH, alkaline phosphatase, ionized calcium, and total calcium remained significantly within normal limits as a result of the new therapy protocol applied. PTH and phosphorus clearance rates were compared in the patient groups in which different dialysis membranes had been used. PTH and phosphorus clearances were 15.2 +/- 3 ml/min and 239.1 +/- 19.2 ml/min, respectively, in the polysulfone membrane group, and 1.1 +/- 0.3 ml/min and 112.8 +/- 9.88 ml/min, respectively, in the cuprophane membrane group (p < 0.05).