Naphthyl Phosphate Method

CoMPAKisoN OP Acid Phosphatase Activities Determined by Different Methods 

It is apparent from the preceding discussion that the rate of action of the acid phosphatase present in normal serum varies with the par- 

As the preceding considerations illustrate and as was noted at the beginning of this section (2.1), comparison of acid phosphatase activities obtained in different studies must take into account the method employed. Some workers have attempted to do this by using the terms 8-glycerophosphatase, phenylphosphatase, etc. to designate the substrate employed (B6, T6). However, such usage may imply that different acid phosphatases are responsible for these actions, and we shall therefore attempt to avoid this usage in the present review. 

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In practice it is often more convenient to measure the release of a phenol from an aryl phosphomonoester. Standard serum phosphatase methods employ phenyl phosphate (188), p-nitrophenyl phosphate (189), phenolphthalein monophosphate (140), or thymolphthalein monophosphate (141) where the phenol released can be determined spectrophoto-metrically [only the Bodansky method (13) uses a Pi determination]. A number of fluorogenic substrates have been used for phosphatase studies, e.g., jS-naphthyl phosphate (30, 148), 4-methylumbelliferyl phosphate (143), and 3-O-methylfluorescein phosphate (144) The main advantage here is the much greater sensitivity of fluorescence as compared with spectrophotometric assays as little as 1 pmole of 4-methyl-umbelliferone can be detected in continuous assay. 

A spectrofluorometric method for the estimation of acid phosphatase has been devised. It uses a-naphthyl phosphate as substrate thus, it is somewhat more specific for prostatic acid phosphatase than most (37). 

Most of the substrates that have been used in measuring alkaline phosphatase activity have also been used to measure acid phosphatase, e.g. p-nitrophenylphosphate, phenylphosphate and a-naphthyl phosphate. In most cases of acid phosphatase estimation, it is the level of the prostatic phosphatase which needs to be known, and so specific inhibitors are included in the reaction mixtures. The prostatic isoenzyme is strongly inhibited by tartrate and so in many methods it is tartrate-labile acid phosphatase which is measured. On the pther hand, the red cell enzyme which contributes significantly to the total serum activity is inhibited by formaldehyde and cupric ions. Many laboratories therefore measure formaldehyde-stable acid phosphatase as an indication of prostatic acid phosphatase. 

Stabilisers are usually determined by a time-consuming extraction from the polymer, followed by an IR or UV spectrophotometric measurement on the extract. Most stabilisers are complex aromatic compounds which exhibit intense UV absorption and therefore should show luminescence in many cases. The fluorescence emission spectra of Irgafos 168 and its phosphate degradation product, recorded in hexane at an excitation wavelength of 270 nm, are not spectrally distinct. However, the fluorescence quantum yield of the phosphate greatly exceeds that of the phosphite and this difference may enable quantitation of the phosphate concentration [150]. The application of emission spectroscopy to additive analysis was illustrated for Nonox Cl (/V./V -di-/i-naphthyl-p-phcnylene-diamine) [149] with fluorescence ex/em peaks at 392/490 nm and phosphorescence ex/em at 382/516 nm. Parker and Barnes [151] have reported the use of fluorescence for the determination of V-phenyl-l-naphthylamine and N-phenyl-2-naphthylamine in extracted vulcanised rubber. While pine tar and other additives in the rubber seriously interfered with the absorption spectrophotometric method this was not the case with the fluoromet-ric method. 

Most of the older methods of fluorimetric analysis of pesticides involved hydrolysis to form fluorescent anions. Co-ral (coumaphos) [147] was hydrolyzed in alkali to the hydroxybenzopyran, which was subsequently determined by means of its fluorescence. Guthion (azinphosmethyl) was hydrolyzed to anthranilic acid for fluorimetric analysis [148,149]. A method was developed [150] for Maretin (N-hydroxynaphthalimide diethyl phosphate) in fat and meat which involved hydrolysis in 0.5 M methanolic sodium hydroxide followed by determination of the fluorescence of the liberated naphthalimide moiety. Carbaryl (1-naphthyl N-methylcarbamate) and its metabolites have been determined by a number of workers using base hydrolysis and the fluorescence of the resulting naphtholate anion [151-153]. Nanogram quantities of the naphtholate anion could be detected. Zectran (4-dimethylamino-3,5-xylyl N-methylcarbamate) has been determined by the fluorescence of its hydrolysis product [154]. The fluorescence behaviour of other carbamate insecticides in neutral and basic media has been reported [155]. Gibberellin spray used on cherries has been determined fluorimetrically after treatment with strong acid [156]. Benomyl (methyl N-[l-(butylcarbamoyl)-2-benzimidazolyl]carbamate) has been analyzed by fluorimetry after hydrolysis to 2-aminobenzimidazole [157]. 

Free fatty acids in human serum were derivatized with 1-naphthylamine after being converted to acyl chlorides [109], Serum (0.5 ml) was mixed with 0.1 ml of methanol containing an internal standard and 1.4 ml of 1/15 M phosphate buffer and poured into a column packed with Ig of Extrelut. The adsorbed fatty acid was recovered by elution with 10 ml of chloroform. After removal of solvent, the residue was dissolved in 0.6 ml of benzene. A solution of oxalyl chloride in benzene (2%) was added to the fatty acids and the mixture was allowed to react at 70 °C for 30 min. The solvent was removed at reduced pressure. Then naphthylamine solution (0.1ml) and triethylamine solution (0.01ml) were added. The reaction was carried out at 30 °C for 15 min, and 2 fi of the mixture was injected onto a pBondapak Cjg column at 40 °C. The mobile phase was methanol/water (81 19 v/v) and detection was at 280 nm Each fatty acid was quantitatively converted into its acid chloride and the overall recovery of naphthyl amides by this method was 94-106%. The main free fatty acids in human serum (14 0,16 0,16.1,18 0,18 1,18 2) and the internal standard (17 0) were separated in 30 min. 

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