Determination of alkaline phosphatase activity in human sera by mid-FTIR spectroscopy

Fourier transform infrared spectroscopy is applied to the determination of alkaline phosphatase (ALP) activity in human sera. 4-nitrophenylphosphate was found to be an excellent ALP substrate for FT-IR spectroscopic detection. The developed method is based on the acquisition of two FT-IR spectra: one recorded immediately after mixing the sample and assay solution and the other after an incubation time of 10 min. Spectral changes in the mid IR range due to the enzymatic reaction could be correlated to the ALP activity in the sample. Experimental conditions were established such that the clinically relevant range for determination of ALP activity in human sera (50 to 900 U/L) was covered. The method was applied to the analysis of ALP activities in standard solutions as well as in human sera yielding results which agreed well with those obtained by a standard reference method. Received: 20 June 1997 / Accepted: 16 September 1997     

2-Carboxy-1-naphthyl phosphate as a substrate for the fluorimetric determination of alkaline phosphatase

A new substrate, 2-carboxy-1-naphthyl phosphate (CNP), was developed for the fluorimetric determination of alkaline phosphatase (ALP) activity. The product of the enzyme reaction is 1-hydroxy-2-naphthoic acid (HNA), which is a strong fluorescent product. The amount of HNA generated is proportional to ALP activity. Optimal conditions for the determination of ALP were investigated. The linear range and detection limit for the determination of ALP are 0.01-4.8 U/L and 7.44 mU/L, respectively. This method is simple, practical and can be successfully applied to assess ALP in human serum with good accuracy and precision. The results were evaluated by comparison with a standard colorimetric assay using p-nitrophenyl phosphate as ALP substrate.     

Electrochemical Determination of Alkaline Phosphatase in Human Serum by Differential Pulse Voltammetry

Differential pulse voltammetry(DPV) was applied to the determination of alkaline phosphatase(ALP) activity in human serum with phenyl phosphate as the substrate. Phenyl phosphate can enzymatically be hydrolyzed to produce phenol which is quantified by DPV at 660 mV(vs. Ag/AgCl) in the concentration rangeof 2.0--100 μmol/L. The standard curve for ALP is linear over the range from 0.06 to 1000U/L with a relative standard deviation of 3.0%. The conditions for the enzymatic reaction and voltammetric detection were optimized and the kinetic constants were also examined. The human serum samples were tested by this method and the results were in good agreement with those obtained by the routine p-nitrophenyl phosphate spectrophotometric method.     

荧光光度法测定血清中碱性磷酸酶

合成了一种新的底物水杨酸磷酸酯 (SP),用于荧光光度法测定血清中碱性磷酸酶(ALP)活力.在37.0 ℃的Tris-HCl缓冲液(pH 9.0)条件下, 碱性磷酸酶作用于荧光底物SP,水解生成强荧光产物水杨酸(SA),生成的SA的量与参与反应的ALP 的活力成正比.据此建立了荧光光度法测定血清中碱性磷酸酶活力的新方法.测定碱性磷酸酶的线性范围是0.03～6.00 U/L,检测限为7.04 mU/L.本方法适用于血清中碱性磷酸酶的测定.测定结果与临床上常用的以对硝基苯磷酸酯为底物的分光光度法相比,无显著性差异.     

Sensitive flow-injection amperometric detection of iodide using Mn3+ and As3+.

A rapid, selective, and sensitive kinetic flow-injection method for iodide content determination with amperometric detection on a platinum electrode was developed. The method is based on the catalytic effect of iodide on the Mn3+ reaction with As3+ in the presence of sulfuric acid. The calibration curve was linear in the concentration range from 5.0 x 10(-7) to 1.0 x 10(-4) mol/L iodide. The limit of detection (LOD) was found to be 5.0 x 10(-9) mol/L iodide. The relative standard deviations (RSD) were 1.68% and 3.03% for 1.0 x 10(-3) mol/L standard and 1.0 x 10(-6) mol/L iodide solution (n = 6), respectively. The method has been successfully applied for determination of iodide in waters, table salts, fodder, organic substances and human blood sera. The results were compared with those obtained by a standard AOAC (Association of Official Analytical Chemists) method, as well as with those obtained by a kinetic spectrophotometric procedure for determination of iodide.     

Determination of fipronil by solid-phase microextraction and gas chromatography-mass spectrometry.

A method for the determination of trace amounts of the insecticide fipronil was developed using solid-phase microextraction-gas chromatography-mass spectrometry and selected ion monitoring. Fipronil was extracted with a fused-silica fiber coated with 85 microm polyacrylate. The effects of pH, ionic strength, sample volume, extraction and desorption times as well as the extraction temperature were studied. Lindane was used as an internal standard. The linear concentration range of application was 0.3-100 ng ml(-1) of fipronil, with a relative standard deviation of 9.5% (for a level of 50 ng ml(-1)) and a detection limit of 0.08 ng ml(-1). The method was applied to check the eventual existence of fipronil above this limit in water and soil samples from Granada (Spain) as well as in human urine samples. The method validation was completed with spiked matrix samples. The method can be applied as a monitoring tool for water, soil and urine, in the investigation of environmental and occupational exposure to fipronil.     

Potentiometric flow-injection system for determination of alkaline phosphatase in human serum

A flow-injection system for detection of alkaline phosphatase (ALP) activity in human serum samples has been developed. As a specific and inexpensive ALP substrate for this kinetic assay monofluorophosphate (MFP) was applied. For detection of fluoride ions, generated in the course of the biocatalytic hydrolysis of MFP, conventional fluoride ion-selective electrode based on LaF₃-crystalline membrane was applied. After optimization the system allows analysis of human serum with high selectivity and relatively short time of analysis (5–6 samples h⁻¹). Volume of serum required for analysis is 0.05 mL. The system is useful for determination of the enzyme activity in human serum samples at physiological and pathological levels as well as for detection of isoenzymatic forms of ALP.     

Determination of air-loop volume and radon partition coefficient for measuring radon in water sample

A simple method for the direct determination of the air-loop volume in a RAD7 system as well as the radon partition coefficient was developed allowing for an accurate measurement of the radon activity in any type of water. The air-loop volume may be measured directly using an external radon source and an empty bottle with a precisely measured volume. The partition coefficient and activity of radon in the water sample may then be determined via the RAD7 using the determined air-loop volume. Activity ratios instead of absolute activities were used to measure the air-loop volume and the radon partition coefficient. In order to verify this approach, we measured the radon partition coefficient in deionized water in the temperature range of 10–30 °C and compared the values to those calculated from the well-known Weigel equation. The results were within 5 % variance throughout the temperature range. We also applied the approach for measurement of the radon partition coefficient in synthetic saline water (0–75 ppt salinity) as well as tap water. The radon activity of the tap water sample was determined by this method as well as the standard RAD-H₂O and BigBottle RAD-H₂O. The results have shown good agreement between this method and the standard methods.     

Biparametric multicommutated flow analysis system for determination of human serum phosphoesterase activity

A multicommutated flow analysis (MCFA) system constructed of microsolenoid valves and pumps offering simultaneous determination of activity of acid phosphatase (ACP) and alkaline phosphatase (ALP) in human serum samples has been developed. The MCFA system is based on optoelectronic flow-through detector made of two light emitting diodes and operating according to paired emitter detector diode (PEDD) principle. This photometric PEDD device has been dedicated for detection of p-nitrophenol (NP) generated in the course of enzymatic hydrolysis of p-nitrophenyl phosphate and optimized for the determination of NP in human serum samples. The developed PEDD-based MCFA system allows independent optimization of conditions for reaction and detection steps of photometric ACP and ALP bioassays. Moreover, it allows elimination of photometric interferences from serum matrix components according to two-points kinetic mode of measurement. The single measurement cycle takes 12 min, consists of four measurements (two for each phosphoesterase) and enables determination of serum ACP and ALP activities at physiological and pathological levels. The real analytical utility of the developed MCFA system has been confirmed by analysis of control sera as well as real human serum samples from healthy persons and oncological patients.     

Improved spectrophotometric vitellogenin determination via alkali-labile phosphate in fish plasma–a cost effective approach for assessment of endocrine disruption

Vitellogenin (VTG) is a well known protein biomarker for exposure to environmental estrogens and possible endocrine disruption in fish. VTG is very dominant in plasma after the onset of vitellogenesis and the protein is heavily phosphorylated. This enables indirect quantification through measurement of alkali-labile protein bound phosphate (ALP) as an alternative to the more expensive enzyme linked immunosorbent assay. Good correlation has previously been shown between ALP and actual VTG levels but little effort has been made to investigate the method in an analytical way e.g., to assure the origin of the measured phosphate. During this method development care has been taken to rule out non-VTG sources of phosphate such as phospholipids and free phosphate in the blood plasma. Sample preparation has been simplified and unnecessary steps have been omitted. The common spectrophotometric measurement for ALP involves measurement at two wavelengths and calculation of corrected absorbance values. With a quick phase separation step the spectrophotometric phosphate determination using molybdic acid and ascorbic acid has been improved and all matrix interference has been eliminated. The final ALP method presented here has a detection limit of 3.2 µg PO₄ ^3?/ml plasma which is six times lower than similar methods and it also has less variability. A high sample throughput in comparison to previous ALP methods is possible after scaling down sample and reagent volumes to fit in a 96 well microtiter plate. The cost for buying all chemicals and plastic consumer goods for setting up the indirect protocol for the analysis of 1000 samples is only circa 350 euro. This is only 1% of the material cost for buying commercially available test kit for direct quantification of VTG in the same number of samples. The ALP method should thus be of interest also for applied scientists outside advanced research laboratories.     

Quantification of Carcinoembryonic Antigen by Capillary Electrophoresis–Chemiluminescence Detection using Internal Standard Method

Abstract

Sensitive determination of tumor marker carcinoembryonic antigen (CEA) by capillary electrophoresis–chemiluminescence detection (CE-CL) has been developed using an internal standard method. Parameters affecting the CE separation and CL detection were systematically optimized. Under the optimal conditions, the free horseradish peroxidase (HRP)–labeled antibody (Ab?), the bound Ab?-antigen complex (Ab?-Ag), and HRP used as internal standard were well separated within 5 min. The proposed method was successfully applied for the quantification of CEA in human sera obtained from both healthy persons and patients.     

Improved capillary electrophoresis method for the determination of carbohydrate-deficient transferrin in patient sera

Capillary zone electrophoresis (CZE) with a dynamic double coating formed by charged polymeric reagents represents an effective tool for the separation of iron-saturated transferrin (Tf) isoforms and thus the determination of carbohydrate-deficient transferrin (CDT, sum of asialo-, monosialo- and disialo-Tf in relation to total Tf) in human serum. Using the CEofix-CDT reagents, a 50 microm inner diameter (ID) capillary of 60 cm total length and the P/ACE MDQ under optimized instrumental conditions (20 kV and 30 degrees C) is demonstrated to provide outstanding assay precision for the determination of CDT in human serum. For CDT levels of 1.0% and 4.5%, precision relative standard deviation (RSD) values (n = 8) were determined to be < 3.0% and < 1.5%, respectively. During the first year of operation under routine conditions, more than 600 patient samples were analyzed in a total of 62 sets of runs. Except for selected samples of patients with severe liver diseases, interference-free Tf patterns were detected. Asialo-Tf was not detected in control sera and in patient sera with a CDT level < 1.70%, but became detectable in 89.6% of sera with > 2.3% disialo-Tf. Monosialo-Tf was only detected in two sera containing > 13.3% CDT. The optimized CZE assay was applied to confirm positive CDT results produced by an immunoassay during long-term monitoring of a patient which led to the determination of the elimination kinetics of asialo-Tf, disialo-Tf, and CDT after an episode of high alcohol consumption (estimated apparent half lifes of 4.86, 7.24, and 6.74 days, respectively). The optimized CZE assay with an upper reference limit for CDT of 1.70% represents an attractive alternative to high-performance liquid chromatography (HPLC). It features simpler sample preparation, faster analysis time, and higher isoform resolution compared to the most recent HPLC approach and can thus be regarded as a new candidate of a reference method for CDT.     

Spectrofluorometric determination of kynurenic acid with horseradish peroxidase in the presence of hydrogen peroxide.

The catalytic activity of horseradish peroxidase (HRP) in the presence of hydrogen peroxide has been investigated for the fluorescent derivatization of kynurenic acid under conditions with no exposure to light. Non-fluorescent kynurenic acid was converted into a fluorescent compound (Ex: 367 nm, Em: 470 nm) with HRP in the presence of hydrogen peroxide, and the optimum conditions of this fluorescent derivatization were investigated. Moreover, this fluorescent derivatization was developed for a spectrofluorometric determination of trace amounts of kynurenic acid by measuring the fluorescence intensity of the fluorescent compound. The calibration curve obtained was linear from 1.0 to 10.0 nmol of kynurenic acid in a 1.0 mL sample solution. The relative standard deviation at 5.0 nmol of kynurenic acid was 5.71% (n=5). By adjusting the bandwidths for both the excitation and emission to 15 nm, the calibration curve was also linear in the range between 0.1 to 1.0 nmol of kynurenic acid in a 1.0 mL sample solution. This method was applied to the fluorometric determination of trace amounts of kynurenic acid in the control sera.     

Fixed-time kinetic enthalpimetry: improved sensitivity for enthalpimetric enzyme activity determinations in homogeneous and heterogeneous systems

A fixed-time (integral) method is described for the enthalpimetric determination of enzyme activity. The method involves the determination of residual unreacted substrate after a fixed incubation time with the sample. Results are presented for the determination of cholinesterase in aqueous solution and in 0.1cm³ samples of reference sera using 30-min incubation periods. Results are correlated with a spectrophotometric procedure. A precision of 1.8% relative standard deviation is reported for serum assays. Preliminary data are also presented for the enthalpimetric determination of cholinesterase activity after immobilization onto non-porous glass beads by carbodiimide and glutaraldehyde coupling procedures.     

Development and validation of RP-HPLC,HPTLC and UV-visible spectrophotometric methods for simultaneous estimation of alprazolam and propranolol hydrochloride in their combined dosage form

Three accurate, sensitive and reproducible methods are described for the quantitative determination of alprazolam (ALP) and propranolol hydrochloride (PNL) in their combined dosage form. The first method involves an RP-HPLC separation on the C₁₈ column using acetonitrile-25 mM ammonium acetate buffer and 0.2% triethylamine (pH of buffer adjusted to 4 with glacial acetic acid) in the ratio of 35: 65 (v/v) as mobile phase. Symmetrical peaks with good separation, ALP at 9.3 min and PNL at 3.5 min, were achieved. Quantification was done with photo diode array detection at 255 nm over the concentration ranges of 0.5–50 and 10–250 μg/mL for ALP and PNL, respectively. The second method is based on the separation of drugs by HPTLC using chloroform-methanol-ammonia 7: 0.8: 0.1 (v/v/v) as mobile phase. Quantification was achieved using UV detection at 248 nm over the concentration range of 100–600 ng/spot and 5–30 μg/spot for ALP and PNL, respectively. The third method involves dual wavelength UV-visible spectrophotometric method. It is based on the determination of PNL at 319.4 nm using its absorptivity value and ALP at 258.2 nm after deduction of absorbance due to PNL. Quantification was achieved over the concentration range of 1–40 and 80–200 μg/mL for ALP and PNL, respectively. All methods were validated according to ICH guidelines and successively applied to marketed pharmaceutical formulation, and the results of all three methods were compared statistically as well. No interference from the tablet excipients was found.     

基于微孔板化学发光法检测碱性磷酸酶的研究

CDP-Star能被ALP迅速水解,且水解产物能够持续发光.本研究对化学发光法检测ALP的反应条件进行了优化.优化后的反应条件为:125 μtmol/L的CDP- Star在pH 9.5缓冲液中,37℃下与ALP反应10min.在此条件下,△RLU与ALP的活力呈线性关系.因此,建立了一种基于微孔板化学发光法检测ALP的实验方法.结果表明,本方法检测ALP的检测线性范围是0.05～10 U/L和10～1000 U/L,相对于光吸收法具有速度快、灵敏度高、重复性好等优点.因此,本方法可以被开发成试剂盒,应用于高通量全自动生化分析仪的测试分析.     

基于可控相转变温度热敏高分子的人IgG酶联荧光免疫分析

以温度敏感高分子聚N－异丙基丙烯酰胺－丙烯酰胺[P(NIP-AA)]作为载体,建立了酶联荧光免疫分析人IgG的新方法。AA摩尔含量为8％的高分子P(NIP-AA)其临界溶解温度为37 °C。竞争型免疫测定中,被固定的IgG和标准溶液（或样品）在33 °C均相条件下竞争性地与辣根过氧化物酶标记抗体反应,升高温度分离出高分子免疫复合物,沉淀重新溶解后通过偶联过氧化氢与对羟基苯乙酸的荧光反应进行定量,线性范围为100-1000 ng/mL,检测限为2.0 ng/mL。方法灵敏、快速操作简便,且提高了免疫反应效率。此外,灵敏度与用传统微孔板做载体相似,但测定时间更快（从100-120分钟减少到30分钟）。该法用于测定人血清中IgG的含量,结果满意。     

Amperometric determination of total cholesterol in serum with use of immobilized cholesterol esterase and cholesterol oxidase

Cholesterol esterase and cholesterol oxidase were immobilized on octyl-agarose gel, activated with cyanogen bromide and placed in a reactor. The sensor system for total cholesterol was assembled with the immobilized enzyme reactor, a hydrogen peroxide electrode and a peristaltic pump. Characteristics of the sensor system were investigated by using cholesterol palmitate as a standard substrate. A linear relationship was obtained between peak current and cholesterol palmitate concentration below 1000 mg dl^-1 (10.3 mM). A 10-μl sample could be assayed in 5 min. Total cholesterol in human serum was determined in the range 100–400 mg dl^-1. The standard deviation for the determination of 50 samples of 300 mg dl^-1 was 6 mg dl^-1 (2%). The system was used for 300 assays without loss of enzymatic activity. The correlation coefficient was 0.94 for 27 samples of human sera analyzed by the system proposed and by the conventional chemical method.     

顺序注射在线稀释测定人体液中尿酸

基于酸性条件下甲醛对尿酸-KMnO4发光反应的增敏作用,建立了在线稀释顺序注射化学发光联用技术测定人体液中尿酸的新方法。在选定的实验条件下的7个浓度梯度范围内,尿酸浓度在5.0×10-6mol/L~1.0×10-3mol/L范围内与发光强度呈良好线性关系,相关系数0.9942~0.9998,RSD在2.0%~3.5%之间,回收率为98.0%~103.0%。每小时可分析80个样品,在线稀释测定结果与手工稀释法一致。     

Polydopamine nanodots are viable probes for fluorometric determination of the activity of alkaline phosphatase via the in situ regulation of a redox reaction triggered by the enzyme

The authors describe an environmentally friendly and fast (~14 min) method for the synthesis of homogeneously distributed fluorescent polydopamine nanodots (PDA-NDs) using KMnO₄ as the oxidant. Alkaline phosphatase (ALP) catalyzes the hydrolysis of ascorbic acid 2-phosphate to release free ascorbic acid which undergoes an in-situ redox reaction with KMnO₄. Depending on the activity of ALP, more or less KMnO₄ is consumed, and this affects the formation of the PDA-NDs. Based on this finding, a sensitive method was worked out to quantify the activity of ALP via real-time formation of fluorescent PDA-NDs. The fluorometric signal (best measured at excitation/emission peaks of 390/500 nm) is linear in the 1 to 50 mU·mL^?1 ALP activity range, and the limit of the detection is as low as 0.94 mU·mL^?1 (based on 3 σ/m). The method was successfully applied to the determination of ALP activity in spiked human serum and in MCF-7 cell lysates. It was also applied in a method to screen for inhibitors of ALP.

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Graphical abstract Schematic of a fluorometric method for the determination of alkaline phosphatase (ALP) activity. The method is based on the in-situ regulation of the formation of fluorescent polydopamine nanodots (PDA-NDs) through the competition between the KMnO₄-induced polymerization of dopamine and ALP-directed ascorbic acid 2-phosphate (Asc-2P) hydrolysis. AA: Ascorbic acid.