Alkaline phosphatases in the complex chronic kidney disease-mineral and bone disorders

Alkaline phosphatases (APs) remove the phosphate (dephosphorylation) needed in multiple metabolic processes (from many molecules such as proteins, nucleotides, or pyrophosphate). Therefore, APs are important for bone mineralization but paradoxically they can also be deleterious for other processes, such as vascular calcification and the increasingly known cross-talk between bone and vessels. A proper balance between beneficial and harmful activities is further complicated in the context of chronic kidney disease (CKD). In this narrative review, we will briefly update the complexity of the enzyme, including its different isoforms such as the bone-specific alkaline phosphatase or the most recently discovered B1x. We will also analyze the correlations and potential discrepancies with parathyroid hormone and bone turnover and, most importantly, the valuable recent associations of AP's with cardiovascular disease and/or vascular calcification, and survival. Finally, a basic knowledge of the synthetic and degradation pathways of APs promises to open new therapeutic strategies for the treatment of the CKD-Mineral and Bone Disorder (CKD-MBD) in the near future, as well as for other processes such as sepsis, acute kidney injury, inflammation, endothelial dysfunction, metabolic syndrome or, in diabetes, cardiovascular complications. However, no studies have been done using APs as a primary therapeutic target for clinical outcomes, and therefore, AP's levels cannot yet be used alone as an isolated primary target in the treatment of CKD-MBD. Nonetheless, its diagnostic and prognostic potential should be underlined

Glycan analysis of the chicken synaptic plasma membrane glycoproteins - a major synaptic N-glycan carries the LewisX determinant

The majority of plasma membrane components are glycosylated. It is now widely accepted that this post-translational modification is crucial during the establishment, maintenance and function of the nervous system. Despite its significance, structural information about the glycosylation of nervous system specific glycoproteins is very limited. In the present study the major glycan structure of the chicken synaptic plasma membrane (SPM) associated glycoprotein glycans were determined. N-glycans were released by hydozinnolysis, labelled with 2-aminobenzam,ide, treated with neuraminidase and subsequently fractionated by size exclusion chromatography. Individual fractions were characterized by combination of high-pressure liquid chromatography, exoglicosidase treatment or reagent array analysis method (RAAM). In addition to oligomannose-type glycans, core-fucosylated complex glycans with biantennary bisecting glycans carrying the LewisX epitope were most abundant. The overall chicken glycan profile was strikingly similar to the rat brain glycan profile. The presence of the LewisX determinant in relatively large proportions suggests a tissue-specific function for these glycans

Inositol specific phospholipase D in health and disease a GPI-anchor cleaving enzyme

In recent years a novel mechanism by which proteins are anchored to cell membranes has been elucidated. In excess of 100 molecules of differing structure and function are known to be linked to the cell surface by these glycan phosphatidyl inositol anchors (GPI-anchors). These include cell adhesion molecules, complement regulatory factors, lymphocyte differentiation antigens and ectoenzymes. The relatively recently discovered circulating enzyme; inositol specific phospholipase D (PIPLD) is now known to be capable of cleaving GPI anchors. The complete elucidation of the mechanisms of action and physiological function of PIPLD will therefore have important scientific and clinical implications. In this work; the activity of PIPLD has been investigated. In addition to established methods of analysis, novel analytical systems called aqueous polymer phase systems were developed and adapted for this use. Proteins, membrane particles and cells partition in these phases on the basis of their relative hydrophobicity. Alkaline phosphatase (ALP) was used as a model substrate representative of GPI-anchored molecules to investigate the activity of PIPLD in health and disease. The mechanism of action of PIPLD was studied by conducting in vitro experiments aimed at investigating the effect of PIPLD contained in serum on cultured cells, on prepared membrane fractions and on solubilised GPI-anchor containing molecules. The serum ALP in hepatobiliary and bone disease is measured only as a raised activity however evidence shows that it is present in a range of molecular forms probably related to the specific disease and to mechanisms of production and release. The various isoforms and the mechanisms underlying their formation were investigated. The enzyme was characterised and assay systems developed to examine its physiological function and its activity in disease. Patients with cholestasis and infections were investigated. The range of PIPLD activity was measured in sera from apparently healthy subjects and shown to vary considerably with age and disease. Evidence has been provided which indicates that its principal site of synthesis is the liver and it has been shown to be of probable use as a marker of liver synthetic reserve. In addition PIPLD activity was shown to act as an acute phase reactant. These studies further emphasise the important role PIPLD plays in the metabolism of the groups of molecules linked to the cell surface by GPI anchors

Altered alkaline phosphatase activity in obese Zucker rats liver respect to lean Zucker and Wistar rats discussed in terms of all putative roles ascribed to the enzyme

Biliary complications often lead to acute and chronic liver injury after orthotopic liver transplantation (OLT). Bile composition and secretion depend on the integrated action of all the components of the biliary tree, starting from hepatocytes. Fatty livers are often discarded as grafts for OLT, since they are extremely vulnerable to conventional cold storage (CS). However, the insufficiency of donors has stimulated research to improve the usage of such marginal organs as well as grafts. Our group has recently developed a machine perfusion system at subnormothermic temperature (20°C; MP20) that allows a marked improvement in preservation of fatty and even of normal rat livers as compared with CS. We sought to evaluate the response of the biliary tree of fatty liver to MP20, and a suitable marker was essential to this purpose. Alkaline phosphatase (AlkP, EC 3.1.3.1), frequently used as marker of membrane transport in hepatocytes and bile ducts, was our first choice. Since no histochemical data were available on AlkP distribution and activity in fatty liver, we have first settled to investigate AlkP activity in the steatotic liver of fatty Zucker rats (fa/fa), using as controls lean Zucker (fa/+) and normal Wistar rats. The AlkP reaction in Wistar rats was in accordance with the existing data and, in particular, was present in bile canaliculi of hepatocytes in the periportal region and midzone, in the canals of Hering and in small bile ducts but not in large bile ducts. In lean ZR liver the AlkP reaction in Hering canals and small bile ducts was similar to Wistar rat liver but hepatocytes had lower canalicular activity and besides presented moderate basolateral reaction. The difference between lean Zucker and Wistar rats, both phenotypically normal animals, could be related to the fact that lean Zucker rats are genotypically heterozygous for a recessive mutated allele. In fatty liver, the activity in ductules and small bile ducts was unchanged, but most hepatocytes were devoid of AlkP activity with the exception of clusters of macrosteatotic hepatocytes in the mid-zone, where the reaction was intense in basolateral domains and in distorted canaliculi, a typical pattern of cholestasis. The interpretation of these data was hindered by the fact that the physiological role of AlkP is still under debate. In the present study, the various functions proposed for the role of the enzyme in bile canaliculi and in cholangiocytes are reviewed. Independently of the AlkP role, our data suggest that AlkP does not seem to be a reliable marker to study the initial step of bile production during OLT of fatty livers, but may still be used to investigate the behaviour of bile ductules and small bile ducts

Plasma membrane associated enzymes of mammary tumours as the biochemical indicators of metastasizing capacity. Analyses of enriched plasma membrane preparations.

Plasma membranes from 6 spontaneously metastasizing and 4 non-metastasizing rat mammary carcinomata were isolated by discontinuous sucrose density gradient centrifugation of microsomal pellets. The starting microsomal fraction contained 40-50% plasma membranes as determined by the levels of 5'-nucleotidase activity, with a negligible amount of nuclear (1%), mitochondrial (5%) and lysomal (7%) contamination. Five distinct fractions (F1-F5) were banded at densities 1 X 09, 1 X 13, 1 X 15, 1 X 17 and 1 X 21 at 25 degrees C, in addition to a pellet (F6) obtained by centrifuging at 76,000 g for 17 h. The fractions F1 through F5, all contained various concentrations of membranous structures, while the pellet (F6) contained only amorphous materials as evidenced by electron microscopy. The F3 fraction at the gradient 1 X 15 had the highest specific as well as total activity of the plasma membrane marker enzyme, with aggregates of the least contaminated plasma membranes in vesicular forms. This fraction also had the lowest specific activity for glucose-6-phosphatase (smooth ER marker) and for beta-D-glucuronidase (lysomal marker), and therefore was considered to be the "cleanest" plasma membrane fraction. When the activity of 4 additional plasma membrane marker enzymes, i.e., alkaline phosphatase, phosphodiesterase I, nucleotide pyrophosphatase and alkaline ribonuclease was determined in the same F3 fraction, their levels were significantly lower in every metastasizing tumour than in the non-metastasizing ones, with the enzyme activity decreasing in direct proportion to the metastasizing capacity. On the other hand, the marker enzymes were high in all non-metastasizing tumours, with the activity seemingly increasing with the immunogenicity of tumour cells. There was no significant difference between the 2 groups of mammary tumours in the levels of sialic acid, hexosamine, phospholipid or cholesterol in the plasma membranes. Thus, the level of plasma membrane marker enzymes is considered an accurate indicator for metastasizing capacity in the rat mammary tumour system

Alkaline phosphatase activity as a biochemical biomarker in aqua-toxicological studies

Alkaline phosphatase (ALP) is a glycoprotein with a metallophosphatase structure that catalyzes the hydrolysis of monophosphate esters of biomolecule esters at alkaline pH. ALP activity is a useful bioindicator to assess the physiological health of cellular membranes, cell growth, apoptosis and cell migration, cellular metabolic status, hepatocyte function, and detoxification activity in hepatocytes. ALP activity is detected in a colorimetric method using the para-nitrophenyl phosphate substrate (p-NPP) at a wavelength of 405 nm in biological samples. Cell hemolysis, especially erythrocytes; increased levels of sex hormones and corticosteroids, biological infections, and poor nutrition can adversely affect ALP activity

Subcellular localization of monoglyceride acyltransferase, xanthine oxidation, NADP: isocitrate dehydrogenase and alkaline phosphatase in the mucosa of the guinea-pig small intestine.

1. Rate dependent and isopycnic banding in a zonal rotor were used to analyse the subcellular sites of enzymes in homogenates of guinea-pig small intestinal mucosa. 2, The results demonstrate the following localizations: monoglyceride acyltransferase-microsomal; xanthine oxidase and dehydrogenase-soluble phase, and NADP: isocitrate dehydrogenase-soluble phase and mitochondrial. 3, Alkaline phosphatase is confined to brush borders and is absent from the basolateral plasma membrane. A variable proportion of the activity, up to 40%, is on brush borders which during homogenization break up into particles of reduced density and slow sedimentation rate