Phosphates inorganic, determination

Daly JA, Ertingshausen G. Direct method for determining inorganic phosphate in serum with Centri-fichem. Clin Chem 1972 18 263-5. 

Shukla, S.S. Kyers, J.K. Armstrong, D.E. Arsenic Interference in the Determination of Inorganic Phosphate in Lake Sediments. J. Environ. Quality, 1972, 1, 292-295. 

Enzyme Assay. Na , K -ATPase, and sarcoplasmic reticulum Ca - ATPase were prepared from rat hearts (22) and dog hearts (23), respectively. Bovine heart cyclic AMP phosphodiesterase was purchased from Sigma. The enzyme reaction was carried out after 5-min pretreatment with the drug, and the amount of inorganic phosphate liberated during the reaction period was determined. 

Although it is widely accepted that ischaemia/ reperfusion-induced oxidant stress is associated with a reduction of Na/K ATPase activity, it is difficult to determine which features of this process are responsible for this effect. A classical approach to this type of problem has been to determine the effect of the application of selected metabolites or agents on the activity of the enzyme of interest, an approach that has been exploited for the sarcolemmal Na/K ATPase and glutathione (Haddock et al., 1990). The application of GSH (O.l-l.OmM) induces a concentration-dependent increase in the activity of a bovine isolated ventricular Na/K ATPase preparation (determined by the ouabain-sensitive hydrolysis of ATP to release inorganic phosphate). In the presence of 1 mM GSH there was a 38% stimulation of activity compared to untreated control... 

Figure 4.9 Effect of reduced glutathione (GSH) (0.25-1.0 ihm) and oxidized glutathione (GSSG) (0.25-1.0 mM) on ouabain-sensitive Na/K ATPase activity. An isolated Na/K ATPase preparation was prepared from fresh bovine ventricular tissue. Na/K ATPase activity was determined and quantified by the ouabain-sensitive hydrolysis of ATP to yield Inorganic phosphate. The rate of inorganic phosphate production was compared prior to and following the addition of either GSH or GSSG to the Incubation mixture. The data are presented as...

The pH of a medium also impacts the formation of metal-phosphate precipitates. For example, divalent ionic cadmium (Cd2+) concentrations rapidly decline as both phosphate concentration and pH increase. Sandrin and Hoffman121 determined that when no phosphate is present in a commonly used mineral salts medium, the concentration of divalent ionic cadmium remains relatively constant until an abrupt decline above pH 8. When 15 mM inorganic phosphate is added to the medium, divalent cadmium ion concentrations rapidly decline at pH values above only 6. 

Airey et al. [8] have described a method for removing sulfide prior to the determination of these anions in anoxic estuarine waters. Mercury(II) chloride was used to precipitate free sulfide from samples of anoxic water. The sulfide-free supernatant solution was used to estimate sulfide by measuring the concentration of unreacted mercury(II), as well as to determine sulfate, inorganic phosphate, and nitrate by spectrophotometric methods, in which sulfide interferes. Sulfide concentrations in the range 0.5-180 000 ig/l sulfur could be measured, while the lower limits for sulfate, ammonia, nitrite, and inorganic phosphate were 0.024, -1.0 and 1 xg/l, respectively. 

Because the preceding chromogenic assay rely on choline quantitation, the hydrolysis of substrates with headgroups other than choline cannot be followed. To circumvent this problem, another useful protocol was devised whereby the phosphorylated headgroup produced by the PLCBc hydrolysis is treated with APase, and the inorganic phosphate (Pi) that is thus generated is quantitated by the formation of a blue complex with ammonium molybdate/ascorbic acid 5 nmol of phosphate may be easily detected. This assay, which may also be performed in a 96-well format, has been utilized to determine the kinetic parameters for the hydrolysis of a number of substrates by PLCBc [37,38]. 

Phytic acid solutions were prepared by titrating sodium phytate (Sigma Chemical Company) with HCl the concentration was determined by analyzing for inorganic phosphate after wet ashing with HoSOu-HNOo (3 2) for 45 minutes. The concentration of CaCl stock solutions was measured by atomic absorption spectrometry. 

The coefficient of variation obtained for the determination of total phosphorus in sediment at the 1400ppm level was 2.5%. Some 98-100% recovery of inorganic phosphate was obtained in spiking experiments carried out on sediments. 

Shulka et al. [75] investigated interference by arsenic in the spectrophotometric determination of inorganic phosphate in lake sediments. 

Paper Chromatography.—A ketonic solvent system has been recommended for the rapid determination of nucleotide-inorganic phosphate mixtures,221 and a procedure for the simultaneous determination of metabolites of various chemical classes has been developed.222... 

Similarly, specific catalysts called enzymes are important factors in determining what reactions occur at an appreciable rate in biological systems. For example, adenosine triphosphate is thermodynamically unstable in aqueous solution with respect to hydrolysis to adenosine diphosphate and inorganic phosphate. Yet this reaction proceeds very slowly in the absence of the specific enzyme adenosine triphosphatase. This combination of thermodynamic control of direction and enzyme control of rate makes possible the finely balanced system that is a hving cell. 

Procedure (determination of inorganic phosphate (a) in the acetic acid extract). The 8-hydroxyquinoline forms a precipitate in acidic ammonium molybdate solution, which will interfere unless the aliquot is <5 ml. It should therefore be removed by ignition as follows. Transfer 10 ml acetic acid extract to a 45-ml silica basin, add 0.5 ml 1 M magnesium acetate and evaporate to dryness on a water-bath. (Note do not use magnesium nitrate, which reacts adversely on heating with 8-hydroxyquinoline.)... 

Procedure (determination of inorganic phosphate (b) in the alkaline extract). Pipette 25 ml of the alkaline extract into a 50-ml volumetric flask followed by 3.5 ml 1 M HCI from a dispenser. This will neutralize the sodium hydroxide and leave 1 ml excess of acid, causing precipitation of organic matter. Make up to 50 ml and mix. Filter through a dry Whatman No. 44, 9 cm, filter paper... 

Determine the inorganic phosphate in a 0.5 ml aliquot of each sample (procedure see below). The specific activity of the oubain-sensitive Na,K-ATPase as well as the total ATPase activity is calculated from inorganic phosphate formed in the presence of ouabain... 

The Permeable Cell Assay is a simple method to measure the cellular ATP synthetic activity. ATP should be synthesized from domestic ADP and externally added inorganic phosphate mainly by the glycolytic pathway with consumption of glucose. High concentration of inorganic phosphate inhibits luciferase. The used concentration of inorganic phosphate (1.5 mM) was determined that it does not inhibit the activity of luciferase too much. 

The release of inorganic phosphate may be assayed but the other product is usually determined. Phenol is colourless but forms a coloured complex on reaction with one of several reagents, e.g. 2,6-dichloroquinonechloroimide, with which it forms a blue complex. p-Nitrophenol is yellow while phenolphthalein is red at the alkaline pH of the assay (10) and hence the concentration of either of these may be determined easily. 

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