Clinical meaning of increased alkaline phosphatase

Interleukin-1 (IL-1) is secreted by endothelial cells (ECs) and smooth-muscle cells (SMCs), which are two major component cells of vessels and detected in atherosclerotic lesions. To evaluate the effect of hyperglycemia on the secretion of IL-1 beta in endothelial cells in diabetic patients, we investigated the effects of high glucose and hyperosmolar conditions on the secretion of IL-1 beta from cultured human aortic endothelial cells (HAECs). HAECs were treated with high concentration of glucose or hyperosmolar condition for 3 days. IL-1 beta in the supernatant was measured by high sensitive enzyme-linked immunosorbent assay (ELISA). Under high concentration of glucose (16.6 mmol/L) and hyperosmolar condition (glucose 5.5 mmol/L + mannitol 11.1 mmol/L), the secretion of IL-1 beta was significantly increased (41.0 +/- 2.8 and 26.3 +/- 5.9% increase, respectively, compared with that of 5.5 mmol/L glucose). In conclusion, high glucose and hyperosmolar condition increase the secretion of IL-1 beta in HAECs. The results suggest that diabetic macroangiopathies might be accelerated partly through the increase of IL-1 beta secretion in HAECs.     

Inhibition of alkaline phosphatase by several diuretics

Acetazolamide, furosemide, ethacrynic acid and chlorothiazide, diuretics of considerable structural diversity, inhibit alkaline phosphatase. The inhibition is reversible and the mechanism is of the mixed type, having both competitive and non-competitive characteristics. Ki is calculated to be 8.4, 7.0, 2.8 and 0.1 mmol/l for acetazolamide, furosemide, ethacrynic acid and chlorothiazide, respectively. Chlorothiazide is a much more potent inhibitor of alkaline phosphatase than the other three diuretics. The combination of ethacrynic acid and cysteine, itself an alkaline phosphatase inhibitor, is less inhibitory than ethacrynic acid alone. Rat and human kidney alkaline phosphatase are equally sensitive to chlorothiazide, ethacrynic acid and furosemide.     

Is skewed X inactivation responsible for symptoms in female carriers for adrenoleucodystrophy?

A study of X inactivation in 12 female carriers for adrenoleucodystrophy showed no evidence that skewed patterns are related to clinical manifestation. Other possible mechanisms to explain manifestation in females are considered.     

Kinetics of inactivation of green crab (Scylla Serrata) alkaline phosphatase during removal of zinc ions by ethylenediaminetetraacetic acid disodium

Green crab (Scylla Serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, the each active site in which contains a tight cluster of two zinc ions and one magnesium ion. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou has been applied to a study on the kinetics of the course of inactivation of the enzyme by ethylenediaminetetraacetic acid disodium (EDTA). The kinetics of the substrate reaction with different concentrations of the substrate p-nitrophenyl phosphate (PNPP) and inactivator EDTA suggested a complexing mechanism for inactivation by, and substrate competition with, EDTA at the active site. The inactivation kinetics are single phasic, showing the initial formation of an enzyme-EDTA complex is a relatively rapid reaction, followed a slow inactivation step that probably involves a conformational change of the enzyme. Zinc ions are finally removed from the enzyme. The presence of metal ions apparently stabilizes an active-site conformation required for enzyme activity.     

Catalysis of the hydrolysis of phosphorylated pyridines by alkaline phosphatase has little or no dependence on the pKa of the leaving group

Bacterial alkaline phosphatase is an active catalyst for the hydrolysis of N-phosphorylated pyridines, with values of the second-order rate constant kcat/Km in the range 0.4-1.2 x 10(6) M-1 s-1 at pH 8.0, 25 degrees C. There is little or no dependence of the rate on the pKa of the leaving group; the value of beta 1g is 0 +/- 0.05, which may be compared with beta 1g = -1.0 for the nonenzymic reaction. Phosphorylated pyridines do not have a free electron pair available for protonation or coordination of the leaving group. Therefore, this result means that the similar, small dependence on leaving group structure for the enzyme-catalyzed hydrolysis of phosphate esters [Hall, A. D., & Williams, A. (1986) Biochemistry 25, 4784-4790) does not provide evidence for general acid catalysis or electrophilic assistance of leaving group expulsion. The results are consistent with the hypothesis that productive binding of the substrate, which may involve a conformational change, is largely rate limiting for turnover of the enzyme at low substrate concentrations.     

Evidence for a glycosylinositolphospholipid-anchored alkaline phosphatase in the aquatic plant Spirodela oligorrhiza

Several acutely acting antimigraine drugs, including sumatriptan and other second generation 5-HT1D receptor agonists, have the ability to constrict porcine carotid arteriovenous anastomoses as well as the human isolated coronary artery. These two experimental models seem to serve as indicators, respectively, for the therapeutic and coronary side-effect potential of the compounds. Using these two models, we have now investigated the effects of GMC2021 (3-[2-(dimethylanimo)ethyl]-5-[(trifluoromethyl)sulfonyl]oxy][1 H]indole oxalate, a close analogue of sumatriptan. GMC2021 (30, 100, 300 and 1000 micrograms.kg-1, i.v.) decreased the total carotid blood flow by exclusively decreasing arteriovenous anastomotic blood flow; capillary blood flow to the skin and ears was moderately increased. The mean +/- S.E.M. dose of GMC2021 eliciting a 50% decrease (ED50) in the porcine carotid arteriovenous anastomotic blood flow was found to be 1.1 +/- 0.3 mumol.kg-1 and the highest dose (1000 micrograms.kg-1) produced a 67 +/- 4% reduction. The carotid haemodynamic effects of GMC2021 were reduced by the selective 5-HT1D receptor antagonist, GR127935 (N-[methoxy-3-(4-methyl-1- piperazinyl)phenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)[1 , 1-biphenyl]-4-carboxamide hydrochloride), which completely antagonizes porcine carotid haemodynamic responses to sumatriptan (ED50: 0.16 mumol.kg-1, i.v.). Compared to sumatriptan (pD2: 6.12 +/- 0.15; Emax: 31.3 +/- 12.3% of contractions to 100 mM K+), GMC2021 was less potent in constricting the human isolated coronary artery (pD2: 5.45 +/- 0.2; Emax: 21.0 +/- 4.8% of contractions to 100 mM K+). The above results suggest that GMC2021 constricts carotid arteriovenous anastomoses partly by a 5-HT1D receptor and partly by another, probably novel, receptor and that GMC2021 should be able to abort migraine headaches in patients, with perhaps a less propensity for coronary side effects.     

Oxidative inactivation of glutamine synthetase from the cyanobacterium Anabaena variabilis

In crude extracts of the cyanobacterium Anabaena variabilis, glutamine synthetase (GS) could be effectively inactivated by the addition of NADH. GS inactivation was completed within 30 min. Both the inactivated GS and the active enzyme were isolated. No difference between the two enzyme forms was seen in sodium dodecyl sulfate-gels, and only minor differences were detectable by UV spectra, which excludes modification by a nucleotide. Mass spectrometry revealed that the molecular masses of active and inactive GS are equal. While the Km values of the substrates were unchanged, the Vmax values of the inactive GS were lower, reflecting the inactivation factor in the crude extract. This result indicates that the active site was affected. From the crude extract, a fraction mediating GS inactivation could be enriched by ammonium sulfate precipitation and gel filtration. GS inactivation by this fraction required the presence of NAD(P)H, Fe3+, and oxygen. In the absence of the GS-inactivating fraction, GS could be inactivated by Fe2+ and H2O2. The GS-inactivating fraction produced Fe2+ and H2O2, using NADPH, Fe3+, and oxygen. Accordingly, the inactivating fraction was inhibited by catalase and EDTA. This GS-inactivating system of Anabaena is similar to that described for oxidative GS inactivation in Escherichia coli. We conclude that GS inactivation by NAD(P)H is caused by irreversible oxidative damage and is not due to a regulatory mechanism of nitrogen assimilation.     

Isolation and properties of extracellular alkaline phosphatase from Bacillus intermedius

Patients with chronic disease may be excluded from capitated managed care plans due to higher than average expected costs. In an attempt to remedy this inequity, one type of risk adjustment technique proposes to set separate capitation rates for certain chronic illnesses, including coronary artery disease (CAD). Cardiologists, who increasingly are requested to accept capitation, will benefit from understanding the impact of using clinical factors as opposed to using demographic factors to set capitation rates. Using a 5% national random sample of the 1992 Medicare population, we determined mean annual expenditures and variation in expenditures of individuals with CAD. We compared the use of 2 demographic factors currently used for capitation rate adjustment (age and gender) with 2 factors not currently used--3-digit International Classification of Disease (ICD-9) code (a measure for severity) and Charlson index (a measure for comorbidity). Mean annual expenditures for individuals with CAD were more than double mean annual expenditures for the general Medicare population ($6,944 vs $3,247). Among individuals with CAD, mean expenditures of subgroups defined by both age and gender ranged from $6,205 to $7,724. In comparison, stratifying by measures of severity and comorbidity identified subgroups with lower and higher mean expenditures, producing a range of $1,702 to $19,959. Substantial variation of expenditures for individuals within subgroups defined by severity and comorbidity remained, with few patients having substantially higher expenditures than the rest. When capitation rates are set with the use of demographic factors alone, patients may be subjected to risk selection and physicians to financial loss. Using clinical measures may decrease the incentive for patient risk selection, but substantial financial risk to physicians would remain, because of a relatively few patients with high expenditures (or costs).     

Partial purification and some properties of human liver alkaline phosphatase

1. Alkaline phosphatase (EC 3.1.3.1) from human liver was solubilized from the homogenate using 0.2% Triton X-100 containing 0.2 M lithium 3,5-diiodosalicylate, and the pellet obtained was resolubilized with 20% n-butanol. The procedure resulting in 3842-fold purification included acetone fractionation, ammonium sulfate precipitation, DEAE-cellulose chromatography, Sephadex G-200 gel filtration, hydroxyapatite gel chromatography and further concanavalin A/Sepharose 4B affinity chromatography. 2. The highly purified enzyme showed one major protein band on acrylamide gel electrophoresis at pH 8.6, and exhibited one-seventh of the alkaline p-nitrophenylphosphatase activity in the hepatic enzyme preparation contains of the alkaline pyrophosphatase activity. 3. The highly purified enzyme was a sialic-acid containing glycoprotein. 4. Sialidase-treated hepatic enzyme clearly presented the phenomenon of delayed mobility, and the delayed enzyme fraction stained more strongly than that of non-treated hepatic alkaline phosphatase. 5. In order to investigate the role of the carbohydrate region(s) of the hepatic alkaline phosphatase molecule on substrate binding, the effect of sialidase treatment on the rate of substrate inhibition of alkaline phosphatase was studied. In the case of hepatic enzyme without sialidase, substrate inhibition of alkaline phosphatase activity was clearly shown, while in the case of the hepatic enzyme with sialidase, there was hardly any substrate inhibition in the range of 1-8 mM p-nitrophenylphosphate.     

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.     

Assessment of corticosteroid-induced alkaline phosphatase isoenzyme as a screening test for hyperadrenocorticism in dogs

Quantitative determination of the corticosteroid-induced isoenzyme of alkaline phosphatase (CAP) was evaluated as a screening test for hyperadrenocorticism (HAC) in dogs. A series of 40 dogs with HAC (CAP range, 96 to 14,872 U/L), 30 clinically normal dogs (CAP range, 0 to 38 U/L), and 80 dogs with various diseases (non-HAC) and without history of exogenous glucocorticoid exposure for a minimum of 60 days (CAP range, 0 to 1163 U/L) were used to evaluate the test. Sensitivity and specificity of CAP was calculated at various cutoff points for absolute CAP activity and for CAP activity expressed as a percentage of total alkaline phosphatase activity. A cutoff point of 90 U/L was selected as optimal for use of this assay as a screening test for HAC. A prevalence survey then was done of all canine serum samples submitted to our diagnostic laboratory over a 3-month period, to calculate the predictive values of a positive and a negative test result in a clinical population and to determine the relative frequency and magnitude of CAP activity in dogs that had received glucocorticoids. The predictive values of a positive and a negative test result at the 90 U/L cutoff value were 21.43% (95% confidence limits, 8.3 to 40.95%) and 100% (95% confidence limit > 96%), respectively. It was concluded that CAP isoenzyme activity, determined by routine biochemical analysis by an automated levamisole-inhibition assay, could function as a screening test for HAC; however, the predictive value of a positive test result was too low to recommend the assay as a diagnostic test.(ABSTRACT TRUNCATED AT 250 WORDS)     

Qualitative and quantitative detection of alkaline phosphatase coupled to an oligonucleotide probe for somatostatin mRNA after in situ hybridization using unfixed rat brain tissue

In situ hybridization (ISH) of somatostatin (SOM) mRNA was carried out on sections of rat brain using an alkaline phosphatase (AP) coupled oligonucleotide probe. Different hybridization and AP development conditions were tested for qualitative and quantitative detection of target mRNA on sections of unfixed tissue. Hybridization signal intensities after 24 h of hybridization were high. Comparison with adjacent formaldehyde-fixed tissue sections and hybridization for various lengths of time (2-42 h) indicated that in unfixed tissue retention of SOM mRNA was at least as high as after fixation, and that the mRNA was not degraded during hybridization. The use of tetranitroblue instead of nitroblue tetrazolium chloride in the AP detection medium provided a superior signal-to-noise ratio, and medium stability was improved for quantitative studies on unfixed sections by adding 10% polyvinyl alcohol at pH 8.5. Microphotometric measurements of mean optical densities (MOD) of the formazan reaction product in a defined area within individual neurons of the lateral central amygdaloid nucleus showed a linear increase over the first 23 h of AP reaction time. The mean MOD values per neuron were comparably high in various equally thick sections of the nucleus and increased with section thickness in a linear manner. The findings indicate that the ISH and detection reagents penetrate the entire section and that there is a linear relationship between the amount of AP reaction product measured and the amount of mRNA present in the measured area. Thus, ISH using an AP-coupled oligonucleotide on sections of unfixed tissue appears suitable for quantitative mRNA detection.     

Activity of alkaline phosphatase in mouse brain at various periods of extrauterine life and following ethylnirosourea administration during fetal life]

It has been possible by seeing twenty cases of cholestasis in pregnancy to define certain characteristics of the condition. This pathology of pregnancy is frequently associated with twin pregnancy. The prognosis for the fetus is especially affected by prematurity. There is no evidence of small-for-dates babies, but the neonate gains weight slowly. Anaemia and jaundice in the neonates are common. The treatment of threatened premature labour using beta-mimetic drug is useful and of no danger to the mother.