Fluorogenic Product Research Articles

Hantzsch reaction approach for determination of teicoplanin and vancomycin in real human plasma: Application to pharmaceutical preparations and to synthetic mixture with rifampicin for drug-resistant strain of Staphylococcus aureus

Teicoplanin (TEIC) and vancomycin hydrochloride (VAN) are glycopeptide antimicrobial drugs against methicillin-resistant Staphylococcus aureus and Gram-positive organisms. So, simple, quick, cost-effective and ultra-sensitive spectrofluorimetric method was developed and bio-analytically validated for assay of TEIC and VAN. The proposed method is based on the reaction of acetylacetone with primary amine moiety in the presence of formaldehyde producing highly fluorogenic product measured at 485 nm (λex at 420 nm). The calibration range was constructed from (50–400 ng mL−1) and (70–800 ng mL−1) for TEIC and VAN, respectively. The presented method exhibited low LOD (7.04 and 3.55 ng mL−1) and LOQ (21.33 and 10.77 ng mL−1) values for TEIC and VAN, respectively. The presented method was effectively applied to determine TEIC and VAN in real human plasma with percentage of recovery ranged from 95.57 ± 2.06% to 97.43 ± 1.66% for the studied drugs. Also, the developed method was applied for the first time to study synthetic mixtures of the studied drugs with rifampicin (RIF), as a combination therapy to treat drug-resistant strains of Staphylococcus aureus without interferences from RIF. The synthetic mixtures of TEIC or VAN with RIF were prepared in three different ratios in order to measure the stability of the studied drugs TEIC or VAN in presence of RIF in pure forms and human plasma.

Donor Substrate Specificity of 4- -Glucanotransferase of Porcine Liver Glycogen Debranching Enzyme and Complementary Action to Glycogen Phosphorylase on Debranching

Glycogen debranching enzyme (GDE) has both 4-alpha-glucanotransferase and amylo-alpha-1,6-glucosidase activities. Here, we examined 4-alpha-glucanotransferase action of porcine liver GDE on four 6(4)-O-alpha-maltooligosyl-pyridylamino(PA)-maltooctaoses, in the presence or absence of an acceptor, maltohexaose. HPLC analysis of digested fluorogenic branched dextrins revealed that in the presence or absence of acceptor, 6(4)-O-alpha-glucosyl-PA-maltooctaose (B4/81) was liberated from 6(4)-O-alpha-maltopentaosyl-PA-maltooctaose (B4/85), 6(4)-O-alpha-maltotetraosyl-PA-maltooctaose (B4/84) and 6(4)-O-alpha-maltotriosyl-PA-maltooctaose (B4/83), whereas 6(4)-O-alpha-maltosyl-PA-maltooctaose (B4/82) was resistant to the enzyme. The fluorogenic product was further hydrolyzed by amylo-alpha-1,6-glucosidase to PA-maltooctaose (G8PA) and glucose. The ratio of the rates of 4-alpha-glucanotransferase actions on B4/85, B4/84 and B4/83 in the absence of the acceptor was 0.15, 0.42 and 1.00, respectively. The rates increased with increasing amounts of acceptor, changing the ratio of the rates to 0.09, 1.00 and 0.60 (with 0.5 mM maltohexaose) and 0.10, 1.00 and 0.58 (with 1.0 mM maltohexaose), respectively. Donor substrate specificity of GDE 4-alpha-glucanotransferase suggests complementary action of GDE and glycogen phosphorylase on glycogen degradation in the porcine liver. Glycogen phosphorylase degrades the maltooligosaccharide branches of glycogen by phosphorolysis to form maltotetraosyl branches, and phosphorolysis does not proceed further. GDE 4-alpha-glucanotransferase removes a maltotriosyl residue from the maltotetraosyl branch such that the alpha-1,6-linked glucosyl residue is retained.

Evaluation of unfolded-partial least-squares coupled to residual trilinearization for four-way calibration of folic acid and methotrexate in human serum samples

The combination of unfolded-partial least-squares (U-PLS) with a recently proposed separate procedure, known as residual trilinearization (RTL), has been successfully employed for four-way data calibration. The chemometric method employs the evolution of excitation–emission matrices (EEMs) with time, for the resolution of folic acid–methotrexate mixtures, in human serum samples. The fluorogenic products monitored correspond to the oxidation of the studied analytes with potassium permanganate, in slightly acidic medium. The reaction is developed in 7 min and followed using a fast-scanning spectrofluorimeter, capable of recording each complete EEM in 12 s. This allows the acquisition of 10 successive EEMs, at different reaction times, during the development of the oxidation reaction, given rise to the four-way data set employed. The procedure, which had been previously reported for urine determination, is extended to serum analysis in this work. The combination of U-PLS/RTL is providing enhanced predictive results in comparison with standard methods as PARAFAC and N-PLS, in the presence of human serum, where significant unexpected components and or inner filter effects may occur.

Quantitative measurement of caspase-3 activity in a living starfish egg

If not fertilized, synchronous apoptosis is induced in starfish eggs at ∼11 h after stimulation with the hormone, 1-methyladenine. In this study, a membrane-impermeant substrate of caspase-3, acetyl-Asp-Glu-Val-Asp-coumarylamido-4-methanesulfonic acid (Ac-DEVD-CAMS), was synthesized and microinjected into a starfish egg. Caspase-3 activity in unfertilized egg was detected ∼30 min before blebbing by quantifying the accumulation rate of a membrane-impermeant, fluorogenic product, 7-aminocoumarin-4-methanesulfonic acid (ACMS), using a photomultiplier mounted on a fluorescence microscope. When active recombinant human caspase-3 was microinjected into an egg at 3 h after 1-methyladenine treatment, the injected caspase-3 activity decreased and disappeared within 2 h. This decrease is probably due to proteasome-dependent degradation of the enzyme, since the injected caspase-3 was degraded and a proteasome inhibitor blocked its degradation. In contrast, in aged eggs at ∼10 h after 1-methyladenine treatment, no degradation of the injected caspase-3 was observed, suggesting that endogenous caspase-3 may stabilize at this point, therefore, inducing apoptosis.

Activation of 4-α-Glucanotransferase Activity of Porcine Liver Glycogen Debranching Enzyme with Cyclodextrins

Glycogen debranching enzyme (GDE) is a single polypeptide chain containing distinct active sites for 4-alpha-glucanotransferase and amylo-alpha-1,6-glucosidase activities. Debranching of phosphorylase limit dextrin from glycogen is carried out by cooperation of the two activities. We examined the effects of cyclodextrins (CDs) on debranching activity of porcine liver GDE using a fluorogenic branched dextrin, Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B5/84), as a substrate. B5/84 was hydrolyzed by the hydrolytic action of 4-alpha-glucanotransferase to B5/81 and maltotriose. The fluorogenic product was further hydrolyzed by the amylo-alpha-1,6-glucosidase activity to the debranched product, Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (G8PA), and glucose. alpha-, beta- and gamma-CDs accelerated the liberation of B5/81 from B5/84, indicating that the 4-alpha-glucanotransferase activity was activated by CDs to remove the maltotriosyl residue from the maltotetraosyl branch. This led to acceleration of B5/84 debranching. The extent of 4-alpha-glucanotransferase activation increased with CD concentration before reaching a constant value. This suggests that there is an activator binding site and that the binding of CDs stimulates 4-alpha-glucanotransferase activity. In the porcine liver, glycogen degradation may be partially stimulated by the binding of a glycogen branch to this activator binding site.

Conversion of γ-hydroxybutyric acid to a fluorescent derivative: a method for screening

Conversion of γ-hydroxybutyric acid (GHB) to a fluorescent derivative using 3-bromomethyl-6,7-dimethoxy-1-methyl-1,2-dihydroquinoxaline-2-one (Br-DMEQ), and its application to drug screening were studied. Br-DMEQ reacted with the carboxyl group of sodium GHB in the presence of a potassium salt and crown ether to produce a fluorescent derivative, which could be easily detected by thin-layer chromatography (TLC). An electrospray ionization mass spectrum of the fluorogenic product supported the expected structure. The Br-DMEQ-derivatized GHB gave an Rf value of 0.49 on TLC, which was easily distinguished from 19 other carboxylic acids; all of the latter had Rf values over 0.61. Various sodium carboxylates including sodium GHB reacted with Br-DMEQ in the presence of KCl, but the free forms of the carboxylic acids did not react under these conditions. When aqueous solution containing GHB was pretreated with sulfuric acid, GHB was converted to γ-butyrolactone, resulting in removal of its reactivity with Br-DMEQ. By such analysis following the derivatization, with and without the above pre-treatment with sulfuric acid, GHB added to human urine could be specifically detected, although the limit of detection was about 100μg/ml, which was 20 times higher than the endogenous blood GHB level. Therefore, the present method seems useful for screening GHB present in a solid state such as powder and tablets, and also in urine samples obtained in overdose cases.

Fluorogenic substrates of glycogen debranching enzyme for assaying debranching activity

Glycogen debranching enzyme (GDE) degrades glycogen in concert with glycogen phosphorylase. GDE has two distinct active sites for maltooligosaccharide transferase and amylo-1,6-glucosidase activities. Phosphorylase limit dextrin from glycogen is debranched by cooperation of the two activities. Fluorogenic branched dextrins were prepared as substrates of GDE from pyridylaminated maltooctaose (PA-maltooctaose) and maltotetraose, taking advantage of the synthetic action of Klebsiella pneumoniae pullulanase. Their structures were as follows: Glcα1-4Glcα1-4Glcα1-4Glcα1-4Glcα1-4(Glcα1-4Glcα1-4Glcα1-4Glcα1-6)Glcα1-4Glcα1-4GlcPA (B3), Glcα1-4Glcα1-4Glcα1-4Glcα1-4(Glcα1-4Glcα1-4Glcα1-4Glcα1-6)Glcα1-4Glcα1-4Glcα1-4GlcPA (B4), Glcα1-4Glcα1-4Glcα1-4(Glcα1-4Glcα1-4Glcα1-4Glcα1-6)Glcα1-4Glcα1-4Glcα1-4Glcα1-4GlcPA (B5), Glcα1-4Glcα1-4(Glcα1-4Glcα1-4Glcα1-4Glcα1-6)Glcα1-4Glcα1-4Glcα1-4Glcα1-4Glcα1-4GlcPA (B6), Glcα1-4(Glcα1-4Glcα1-4Glcα1-4Glcα1-6)Glcα1-4Glcα1-4Glcα1-4Glcα1-4Glcα1-4Glcα1-4GlcPA (B7), and Glcα1-4Glcα1-4Glcα1-4Glcα1-6Glcα1-4Glcα1-4Glcα1-4Glcα1-4Glcα1-4Glcα1-4Glcα1-4GlcPA (B8). These dextrins were incubated with porcine skeletal muscle GDE. No fluorogenic product was found in the digest of B8. The fluorogenic products from B3, B4, and B5 were PA-maltooctaose only. PA-maltooctaose, PA-maltoundecaose, and 6 7- O-α-glucosyl-PA-maltooctaose were from B7. PA-maltooctaose and 6 6- O-α-glucosyl-PA-maltooctaose were from B6. These results indicate that the maltooligosaccharide transferase removed the maltotriosyl residues from the maltotetraosyl branches by hydrolysis or intramolecular transglycosylation to expose 6- O-α-glucosyl residues, and then the amylo-1,6-glucosidase hydrolyzed the α-1,6-glycosidic linkages of the products rapidly. Probably, 6- O-α-glucosyl-PA-maltooctaoses from B7 and B6 were less susceptible to the amylo-1,6-glucosidase than were those from B3, B4, and B5. Taking this into account, B3, B4, and B5 are suitable substrates for GDE assay.

Sex-specific biotransformation and detoxification after xenobiotic exposure of primary cultured hepatocytes of European flounder ( Platichthys flesus L .)

Sex-specific effects of sublethal concentrations of known effective pro-oxidants such as 100, 200 and 400 μM benzo[ a]pyrene (B[ a]p), 50 μM nitrofurantoin (NF) and 100 μM hydrogen peroxide (H 2O 2) on biotransformation pathways were studied in isolated hepatocytes of immature female and male European flounder ( Platichthys flesus L.). Cell responses were assessed at the level of: (1) stress induction as measured by formation of reactive oxygen species (ROS), mainly superoxide radicals, and induction of cytochrome P450 (CYP450) biotransformation activity; (2) cellular antioxidant defences, both non-enzymatic (reduced glutathione) and enzymatic (DT-diaphorase (DTD) or quinone oxidoreductase, EC 1.6.99.2); (3) detoxification (aldehyde dehydrogenase (ALDH), EC 1.2.1.3); and (4) cellular damage as measured by reduced lysosomal membrane stability and cell death. As there is increasing evidence that 17-β-estradiol interferes with certain pathways of xenobiotic biotransformation, we additionally tested the effects of different concentrations of 17-β-estradiol (0.2–10 μM) alone and 17-β-estradiol (1 μM) in combination with 100 μM B[ a]p. Parameters were monitored after 1 and 9 days of exposure by quantitative image analysis of chromogenic or fluorogenic reaction products. Our study revealed sex-dependent differences in cellular stress responses. In hepatocytes of female flounder, biotransformation was slower and the capacity of non-enzymatic antioxidant defences and detoxification of toxic aldehydes was lower than in males. Additional administration of 17-β-estradiol enlarged these differences between the sexes with respect to biotransformation activity and antioxidant defence in xenobiotic-induced injury. These findings may explain the higher susceptibility of female flounder to toxic and carcinogenic compounds in the marine environment.

A fluorogenic assay using pressure-driven flow on a microchip.

A fluorogenic assay for human T-cell phosphatase (TCPTP) was conducted on an etched glass microchip using pressure driven flow. The TCPTP enzyme catalyzes the removal of a phosphate group from 6,8-difluoro-4-methylumbelliferyl/phosphate (DiFMUP) to produce the fluorogenic product 6,8-difluoro-4-methylumbelliferone (DiFMU). Enzyme assays with real-time on-chip dilution were performed in both low-viscosity (1 cP) buffer and an enzyme solution containing 50% glycerol (6 cP). Single side channels connect a series of reagent wells to a main channel where the fluorescent product of the enzyme reaction passes the detector region. Flow regulation of mixed viscosity fluids requires a pressure control on each arm of the chip contributing to the overall flow. An 8-channel pressure controller was built to regulate the air pressure above all wells feeding channels of the chip, thereby controlling the dilution ratios of buffer, substrate and enzyme. Well pressures maintained a constant concentration of enzyme in the detector channel while adjusting the flow contribution of substrate and buffer. The substrate concentration was stepped over two orders of magnitude while verifying fluid dilutions using marker dyes. The kinetic parameters, Km, Vmax, and Kcat, showed good agreement with the values determined using a standard well plate and fluorometer.

Why Does Threonine, and Not Serine, Function as the Active Site Nucleophile in Proteasomes?

Proteasomes belong to the N-terminal nucleophile group of amidases and function through a novel proteolytic mechanism, in which the hydroxyl group of the N-terminal threonines is the catalytic nucleophile. However, it is unclear why threonine has been conserved in all proteasomal active sites, because its replacement by a serine in proteasomes from the archaeon Thermoplasma acidophilum (T1S mutant) does not alter the rates of hydrolysis of Suc-LLVY-amc (Seemüller, E., Lupas, A., Stock, D., Lowe, J., Huber, R., and Baumeister, W. (1995) Science 268, 579-582) and other standard peptide amide substrates. However, we found that true peptide bonds in decapeptide libraries were cleaved by the T1S mutant 10-fold slower than by wild type (wt) proteasomes. In degrading proteins, the T1S proteasome was 3.5- to 6-fold slower than the wt, and this difference increased when proteolysis was stimulated using the proteasome-activating nucleotidase (PAN) ATPase complex. With mutant proteasomes, peptide bond cleavage appeared to be rate-limiting in protein breakdown, unlike with wt. Surprisingly, a peptide ester was hydrolyzed by both particles much faster than the corresponding amide, and the T1S mutant cleaved it faster than the wt. Moreover, the T1S mutant was inactivated by the ester inhibitor clasto-lactacystin-beta-lactone severalfold faster than the wt, but reacted with nonester irreversible inhibitors at similar rates. T1A and T1C mutants were completely inactive in all these assays. Thus, proteasomes lack additional active sites, and the N-terminal threonine evolved because it allows more efficient protein breakdown than serine.

Inhibition and Labeling of Enzymes and Abzymes by Phosphonate Diesters

Reactive phosphonate diesters were designed and prepared as inhibitors of serine proteases and esterases. Inactivation of trypsin, chymotrypsin, and butyrylcholinesterase was determined by residual enzymatic activity as well as by the release of a chromogenic or fluorogenic product of the inhibition reaction. Second-order rate constants were determined from rates of nitrophenol formation. Application of the reaction for active-site titration of enzyme preparations is demonstrated. A basic functional group present in the nitrophenyl tropane phosphonate diester was shown to confer selectivity for inactivation of trypsin and chymotrypsin. Biotinylated derivatives of the phosphonate diesters were prepared to permit analysis of proteins modified in the inhibition reaction. Labeled polypeptides were resolved by SDS-PAGE, electroblotted, and detected by streptavidin-peroxidase staining. A detection limit of less than 4 ng, corresponding to 20 nM of trypsin, was demonstrated. Pretreatment of enzymes with DFP or nonbiotinylated phosphonates specifically blocks the labeling. This technique permits identification of serine proteases in complex mixtures with good sensitivity and specificity.

An assay method for glycogen debranching enzyme using new fluorogenic substrates and its application to detection of the enzyme in mouse brain.

An assay method for glycogen debranching enzyme involving fluorogenic dextrins as substrates was developed. Two dextrins were prepared from 6-O-alpha-D-glucosyl-alpha-cyclodextrin and glucose by taking advantage of the action of Bacillus macerans cyclodextrin glucanotransferase, and converted by pyridylamination to fluorogenic derivatives. Structural analysis of the fluorogenic dextrins by FAB-MS, partial acid hydrolysis, and glucoamylase digestion revealed that they were Glcalpha1-4(Glcalpha1-6)Glcalpha1-4Glcalpha1- 4Glcalpha1-4Glc-PA (FD6) and Glcalpha1-4Glcalpha1-4(Glcalpha1-6)Glcalpha1- 4Glcalpha1-4Glcalpha1-4G lc-PA (FD7). Using the glycogen debranching enzyme from rabbit muscle, FD6 and FD7 were, respectively, hydrolyzed to PA-maltopentaose and PA-maltohexaose, in addition to glucose, showing that these two fluorogenic dextrins are suitable substrates for assaying the glycogen debranching enzyme. An assay method involving the separation and quantification by HPLC of the characteristic fluorogenic products was successfully applied to determination of the distribution of the enzyme activity in mouse cerebrum.

High Performance Liquid Chromatography with Peroxyoxalate Chemiluminescence Detection of Synthetic Peptide, Ebiratide

Ebiratide [L-methionyl-L-glutamyl-L-histidyl-L-phenyl alanyl-D-lysyl- N-(8-aminooctyl)- L- phenylalanin -amide-S, S-dioxide] is one of the synthetic analogues of ACTH4-9 for Alzheimer disease. High performance liquid chromatography with peroxyoxalate chemiluminescence detection (HPLC-PO-CL) has been examined for the analysis of this compound. The fluorogenic reaction product of ebiratide with DBD-F [4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole] was detected by the chemiluminescence generated with bis(2,4,6-trichlorophenyl)oxalate (TCPO) or bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl]oxalate (TDPO) in the presence of hydrogen peroxide. TDPO gave about five times higher CL intensity than TCPO using 100mM hydrogen peroxide. The detection limit of DBD derivative of ebiratide was 25 fmol on column with TDPO. The HPLC-PO-CL was applied to the determination of ebiratide extracted from rat plasma by the solid phase extraction with Bond Elute C18. The method gave a good linearity over the range from 0.25 to 10 pmol on column with the recovery of 85% for the spiked rat plasma and was able to estimate the time course of ebiratide at a sub-pmol/mL level.

Presence of UDP-D-xylose: beta-D-glucoside alpha-1,3-D-xylosyltransferase involved in the biosynthesis of the Xyl alpha 1-3Glc beta-Ser structure of glycoproteins in the human hepatoma cell line HepG2.

HepG2 cells were examined for the presence of UDP-D-xylose: beta-D-glucoside alpha-1,3-xylosyltransferase activity by using 2-[(2-pyridyl)amino]ethyl beta-D-glucopyranoside (Glc beta-R) as a fluorogenic acceptor. UDP-D-xylose and the acceptor were incubated with the HepG2 microsomal fraction, and the enzymatic reaction mixture was analyzed by HPLC. Structural analysis of the fluorogenic product by alpha-D-xylosidase digestion, FAB-MS, and Smith degradation revealed that it was Xyl alpha 1-3Glc beta-R, thus demonstrating the existence of beta-D-glucoside alpha-1,3-xylosyltransferase. A solubilization study of the enzyme from the microsomal fraction indicated that it differed from UDP-D-xylose: alpha-D-xylodie alpha-1,3-xylosyltransferase of the HepG2 microsomal fraction producing Xyl alpha 1-3Xyl alpha 1-3Glc-R' [R', (2-pyridyl)amino-] from UDP-D-xylose and Xyl alpha 1-3Glc-R' [Minamida, S., Aoki, K., Natsuka, S., Omichi, K., Fukase, K., Kusumoto, S. & Hase, S. (1996) J. Biochem. (Tokyo) 120, 1002-1006]. The newly detected enzyme appears to be involved in the biosynthesis of the Xyl alpha 1-3Glc beta-Ser structure of glycoproteins such as human blood coagulation factors VII and IX.

Anion-Exchange High-Performance Liquid Chromatographic Assay of Plasma Lipoproteins

A fast, accurate, and precise high-performance liquid chromatographic (HPLC) assay method for human plasma lipoproteins has been developed. The method includes complete separation of high-, low-, and very-low-density lipoproteins within 20 min using a DEAE–glucomannan gel with stepwise elution, enzymatic oxidation of cholesterol (following ester hydrolysis) followed by a two-step reaction to form a fluorogenic product, and fluorometric detection as the total cholesterol (TC) level of each lipoprotein. The calibration range is 5–1000 μg/ml for the TC level of 20-μl plasma samples and the relative standard deviation (n= 5) of the precision at 5 μg/ml is less than 10%. Each lipoprotein level determined by this optimized HPLC method agrees well with levels determined by ultracentrifugation or precipitation methods. This new method was successfully applied to assays of plasma lipoproteins in various forms of hyperlipidemia.

Linkage Position Analysis of Pyridylamino-Disaccharides by HPLC of Fluorogenic Smith Degradation Products1

Oligosaccharides are often converted to fluorogenic pyridylamino-oligosaccharides (PA-oligosaccharides) to be analyzed sensitively. A method for determining the glycosidic linkage position to the PA-reducing-end residue was developed with PA-disaccharides as model compounds. Periodate oxidation of PA-disaccharides was carried out at 0 degrees C for 15 min or at 4 degrees C for 40 h, and the reaction mixtures were reduced with borohydride. The fluorogenic products obtained at 4 degrees C for 40 h were purified by reversed phase HPLC, and the fraction collected were hydrolyzed with acid. The hydrolysates were analyzed by reversed phase HPLC. PA-glyceraldehyde was formed from 2-substituted PA-disaccharides with PA-hexose, PA-threose (or PA-erythrose) from 3-substituted ones, and PA-glycolaldehyde from 4- or 6-substituted ones. HPLC analysis of the products obtained at 0 degrees C for 15 min revealed a difference between 4- and 6-substituted ones. PA-glyceraldehyde was formed from 6-substituted ones, but not from 4-substituted ones. The linkage position, therefore, can be determined by analyzing fluorogenic product(s). As for PA-disaccharides with PA-N-acetylglucosamine, the linkage position can be simply determined by analysis of 40-h oxidation-reduction mixtures. 2-Acetamido-2-deoxy derivatives of PA-threose, PA-xylose, and PA-glyceraldehyde were formed from 3-, 4-, and 6-substituted ones, respectively. The linkage position analysis was successfully applied to determination of the structures of two Fuc-Man-PAs produced through the transglycosylation action of bovine kidney alpha-L-fucosidase.

Renin assay using a fluorogenic substrate and high performance liquid chromatography

Measurement of renin activity in human fluids using a fluorogenic substrate and high performance liquid chromatography (HPLC) is described. A nine amino acid peptide containing the fluorogenic residue, N-(2-pyridyl) glycine (Pg) is used as a substrate. The peptide sequence is homologous with the cleavage site of human angiotensinogen. This substrate is hydrolyzed by renin to generate fluorogenic and non-fluorogenic products. The amount of fluorogenic product is directly measured by reversed phase HPLC. Optimization of assay conditions and measurement of human serum renin levels are described. Assay results correlated well with those from radioimmunoassay. The method is simple, convenient, highly sensitive and can be used for routine clinical renin assays.

Enzyme immunoassay techniques an overview

In spite of the great variety of enzyme immunoassays (EIA) they can be classified into two groups ‘analyte-observed’ and ‘ reagent-observed’ assays, depending on their reaction principle. The latter are favored by use of monoclonal antibodies and are characterized by a greater sensitivity, a larger measuring range, a lower susceptibility to disturbing influences. They can be used only for detection of macromolecules. For heterogeneous EIAs to be used on laboratory scale, simple adsorption of antigens and antibodies is still recommendable though affinity constants decrease by at least one order of magnitude and antibody density at the solid phase and analyte binding capacity are not parallel due to increasing steric hindrance. For this reason, the antibody with the higher affinity constant should therefore always be used as solid-phase antibody. Microparticles used as solid phase for heterogeneous assays, due to their very high binding capacity for the analyte and extremely short diffusion distances, guarantee ‘one step’ assays of only a few minutes. Of the limited number of enzymes suitable as markers in immunoassays, horseradish peroxidase is the enzyme of choice followed by alkaline phosphatase. Although enzyme and enzyme-labelled reagents are detectable by fluorogenic product measuring with a sensitivity, which is 10–1000 times higher than using chromogenic substrates, the sensitivity of the assays can be increased only by factor 2–10. Labelling enzymes cannot only be covalently bound to the antibody, but also via anti-enzyme antibodies. Pros and cons of the different methods of coupling the enzyme/anti-enzymes complex to analyte-containing immune complexes are discussed. Different EIA variants to detect specific antibodies are reviewed. Among them only capture EIAs permit precise isotype analysis of antibodies of a distinct idiotype. Homogeneous EIAs are widely spread for hapten determination but even variants based on proximal linkage are no alternatives to heterogeneous EIAs for determination of macromolecules. Different parameters are defined which permit to assess the quality of an immunoassay and which should be used in routine assays as internal controls in the laboratory.

Membrane filtration differentiation of E. coli from coliforms in the examination of water.

A membrane filter-Endo agar method for enumerating Escherichia coli as distinct from other coliforms in drinking water was developed. Membranes containing coliform colonies are transferred to nutrient agar containing 4-methyl umbelliferyl-beta-D-glucuronide (MUG) and incubated at 35 degrees C for 4 h. The MUG is hydrolyzed by the glucuronidase of E. coli and the fluorogenic product is visualized. The method recovered 98% of E. coli without false positives and is proposed as an additional test in routine water examination for the detection of pollution.

Detection of Mold in Processed Foods by High Performance Liquid Chromatography

A high-performance liquid chromatographic (HPLC) method was developed to analyze for the degree of mold contamination in processed fruit and vegetable products. The method is based on detection of glucosamine, a breakdown product of chitin which is one of the major constituents of fungal cell walls Food samples were hydrolyzed at 121°C for 2 h to release glucosamine from fungal chitin. The hydrolysates were partially purified by passing them through a Dowex-50 cation exchanger. Effluents were derivatized by o-phthalaldehyde and the fluorogenic reaction products were separated by reversed-phase HPLC and detected by a spectroflurometer. Six species of mold, Alternaria alternata, Alternaria solani, Colletotrichum coccodes, Fusarium oxysporum, Geotrichum candidum and Rhizopus stolonifer, which are commonly associated with fruit and vegetable products, were used in this study and different levels (0.1 to 2.5 mg/g of sample) of mold mycelium were added to rot-free tomato products. A linear relationship between concentration and glucosamine was observed. However, different species of mold gave different amounts of glucosamine. The effect of insect contamination on the assay was negligible unless high levels were present. Results obtained by the HPLC method were compared with those obtained with an amino acid analyzer and reasonable correlation between the two methods was evident.