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PubMed2000

Chemiluminescence-HPLC for the assay of guanidino compounds.

A chemiluminescence-HPLC system for the assay of serum guanidino compounds was established. In this study, we found that lucigenin added to the reaction mixture containing guanidino compounds and alkaline-ninhydrin reagent resulted in strong chemiluminescence. The lucigenin chemiluminescence was not derived from fluorescent derivatives of guanidino compounds or ninhydrin. The interaction of guanidino compounds and ninhydrin under alkaline conditions produced reactive oxygen species. We applied this phenomenon to the microanalysis of guanidino compounds.

Acridines↗

PubMed1980

Automated simultaneous determination of ammonia and total amino acids in ruminal fluid and in vitro media.

Catalyzed phenol-hypochlorite and ninhydrin colorimetric procedures were adapted to the Technicon AutoAnalyzer for simultaneous determination of ammonia and total amino acids in ruminal fluid or ruminal in vitro media. The manifold developed was compatible with a sampling rate of 40/h without significant sample-to-sample carryover. With proper storage, reagents for both the phenol-hypochlorite and the air-stable ninhydrin systems were stable for 8 mo or more. Response of individual amino acids in the phenol-hypochlorite system were generally 1% or less than equimolar amounts of ammonia. Certain amino acids inhibited ammonia color yield 10 to 15% when with equimolar amounts of ammonia; however, the inhibitory effect of casein amino acids was only 2 to 3%. Although ninhydrin response, relative to leucine, of individual alpha-amino acids ranged from 62 (valine) to 151% (histidine), recoveries of casein amino acids from ruminal fluid had coefficients of variation of 1% or less. Coefficients of variation for ammonia recoveries from ruminal fluid by the phenol-hypochlorite procedure were about half of those for the Conway microdiffusion technique. Intraclass correlations for the adapted procedures indicated high degrees of accuracy and precision for both ammonia and amino acid analyses.

Amino Acids↗

PubMed2002

Effects of cytotoxic agents on functional integrity and antioxidant enzymes in clonal beta-cells.

Effects of cytotoxic agents and hydrogen peroxide were examined using pancreatic BRIN-BD11 cells and the parental insulinoma RINm5F cell line. Cell viability was determined using the MTT colorimetric assay and the TUNEL assay was used to assess apoptosis and acridine orange assay was used to determine levels of apoptosis versus necrosis. RT-PCR studies were employed to investigate the effects of the toxins on the expression of antioxidative enzymes, superoxide dismutase (SOD), glutathionine peroxidase (GPX) and catalase (CAT). Streptozotocin, hydrogen peroxide, alloxan and ninhydrin exerted time- and concentration-dependent toxic effects on BRIN-BD11 and RINm5F cells. RT-PCR showed that 90 minutes exposure of BRIN-BD11 cells or RINm5F cells to 5 mM ninhydrin down regulates SOD, GPX and CAT antioxidative enzymes. Glutathionine peroxidase gene expression was also down regulated in both types of cell by hydrogen peroxide. There were no significant differences in antioxidant gene expression after exposure to the other toxins under the conditions employed. TUNEL assay revealed that streptozotocin (8 mM) and hydrogen peroxide (125 microM) had no significant effect on the number of cells undergoing apoptosis. However after exposure to ninhydrin (5 mM) almost 100% of the non-viable BRIN-BD11 cells and around 50% of the RINm5F cells were dying by apoptosis. With the BRIN-BD11 cells there was around a 30% increase in the number of apoptotic cells compared with 50% in the RINm5F cells after exposure to alloxan (16 mM). The results indicate multiple effects of cytotoxic agents on functional integrity and antioxidant enzyme gene expression in clonal beta-cells.

Alloxan↗

PubMed1979

[Quantitative determination of the activity of acid peptidases of industrial origin].

Reagent ninhydrine-Cd++, reacts with free alpha and epsilon amino groups of proteins. Horse-heart apomyoglobin was subjected to exhaustive succinylation, rendering the product non reactive to ninhydrine. The succinylglobin was submitted to enzyme digestion at pH 2.0, 4.0, 4.7 and 6.0. The commercially available enzymes contain mainly pepsin-like and chymosin-like enzymes. The enzymatic digests of succinyl-globin contain new free alpha-amino groups reacting with ninhydrin. Enzymatic digestion was performed under various condition (ratio E/S, pH). The results were compared to those obtained with synthetic substrate: PRO-HIS-LEU-SER-PHE(NO2)-NLEU-ALA-LEU-OME. The price of the synthetic substrate used, was more than 100 times the cost of succinyl-globin, thus the use of this substrate is a valuable tool for the quantitative estimation of peptidase activity in commercially available (pepsin, chymosin-like) enzymes.

Apoproteins↗

PubMed1978

Evaluation of lysinoalanine determinations in food proteins.

A comparison is made between lysinoalanine (LAL) determinations both with an automatic amino acid analyzer (AAA) and with thin layer chromatography-densitometry (TLC) in different types of food and food ingredients, taken from the Dutch market. Generally there is a reasonable agreement between the LAL content obtained by both methods. However, some results indicate that a single technique is not always conclusive about the real identity of the ninhydrin-positive compound at the same position as LAL on the chromatogram. By TLC for instance, in yeast a content of about 800 mg of LAL/kg in protein is found, but according to the AAA method no LAL is present. In heated milk and milk products the LAL content determined by the TLC method is also higher than that found by the AAA method. This is caused by a preceding unknown ninhydrin-positive compound in TLC, occurring in all heated milk products and practically coinciding with LAL. In the AAA technique similar interferences of unknown ninhydrin-positive compounds could be avoided by choosing a suitable elution temperature; however, application of this temperature modification to foaming agents gave no satisfactory results.

Chromatography, Thin Layer↗

PubMed1987

Protein precipitation assay for quantitation of tannins: determination of protein in tannin-protein complex.

A protein precipitation method for the determination of tannins has been developed. The protein in the tannin-protein complexes was measured using the ninhydrin assay of amino acids released by alkaline hydrolysis of the complex. Standard protein and the complex were hydrolyzed with 13.5 N NaOH at 120 degrees C for 20 min and the amino acids released were measured with ninhydrin. Tannins did not interfere in the determination of protein by ninhydrin assay. The bovine serum albumin (BSA) precipitated (y; mg) increased linearly with increase in tannic acid (x) from 0.2 to 0.9 mg (y = 2.598x - 0.258). The protein precipitation capacities (mg BSA precipitated/g dry wt) measured by the method for young and mature leaves of oaks were Quercus incana (young, 42.21; mature, 79.51), Q. ilex (young, 1.86; mature, 1.86), and Q. semecarpifolia (young, 733.54; mature, 304.32). The method can provide valuable information on the mechanisms of protein-tannin interactions and nutritional and physiological significances of tannins.

Animals↗

PubMed1981

Decrease of a peptide in the cat spinal cord after upper cervical transection.

The effects of a transection on the peptide contents in the cat cervical spinal cord have been investigated. Seven days following the hemisection at the C1 level, the C5-8 levels of the spinal cord were used for the biochemical analyses. A crude peptide extract was fractionated on a Sephadex G-15 column. The third major peak which contained smaller molecular weight peptides was collected and separated with a peptide map on the cellulose TLC plate. Three ninhydrin-positive and two ninhydrin-negative peptides were found on the TLC plate. When we compared between the operated side and the control side, a ninhydrin-negative peptide, whose acid hydrolysate was composed of glutamic acid and aspartic acid in a ratio of 1:2, was decreased by approximately 30% in the operated side of the spinal cord.

Animals↗

PubMed1977

Comparative studies on the nonprotein but nondialysable sugar-peptide fraction isolated from the plasma of various classes of vertebrates.

1. The nonprotein but nondialysable ninhydrin-positive substances isolated from plasma of some species belonging to four classes of vertebrates (fishes, amphibians, birds and mammals) were studied. 2. In all species studied (Cyprinus carpio, Salmo gairdneri, Rana esculenta, Rana temporaria, Gallus domestica and Rattus rattus) a similar group of nondialysable ninhydrin-positive substances may be isolated from the plasma by Sephadex G-25 filtration followed by dialysis. 3. Thin-layer Sephadex G-25 electrophoresis disclosed in birds (Gallus domestica) the lack of one ninhydrin-positive band contrary to the species of other classes of vertebrates. 4. Electrochromatography showed in all species studied an alteration in the composition of the basic peptide fractions and in birds the lack of one neutral peptide.

Animals↗

PubMed2005

A further study to investigate the detection and enhancement of latent fingerprints using visible absorption and luminescence chemical imaging.

This study investigated the application of chemical imaging to the detection of latent fingerprints using the Condor macroscopic chemical imaging system (ChemImage Corp., Pittsburgh, USA). Methods were developed and optimised for the visualisation of untreated latent fingerprints and fingerprints processed with DFO, ninhydrin, cyanoacrylate, and cyanoacrylate plus rhodamine 6G stain. The results obtained with chemical imaging were compared to the detection achieved using conventional imaging techniques. The Condor significantly improved the detection of many prints, especially those that might be considered poor quality or borderline prints. Prints on newspaper treated with ninhydrin and DFO, and prints on white and yellow paper treated with ninhydrin, benefited the most from chemical imaging detection. In many cases, fingerprints undetectable using conventional imaging techniques could be visualised with chemical imaging. Ridge detail from untreated prints on yellow paper was also detected using the Condor. When prints of high quality were examined, both detection techniques produced quality results. The results of this project demonstrate that chemical imaging offers advantages over conventional visualisation techniques when examining latent fingerprints, especially those that would be considered difficult, such as weak prints or prints on surfaces that produce highly luminescent backgrounds. Standard testing procedures for the detection and enhancement of fingerprints by chemical imaging are presented and discussed.

Dermatoglyphics↗

PubMed1978

Comparison of thin-layer chromatographic detection methods for histamine from food extracts.

The specificity and sensitivity of four visualization reagents, ninhydrin, o-phthalaldehyde (OPT), fluorescamine and o-diacetylbenzene (DAB), for the detection of histamine on thin-layer chromatograms developed in several optimal solvent systems were compared. While the ninhydrin reagent is the most sensitive with a detection limit of 0.4 nmoles of histamine, the DAB and fluorescamine reagents offer the most potential as histamine-specific visualization reagents. The OPT reagent was unsatisfactory because the fluorescent sports developed with this reagent were unstable. Of 19 amines tested in addition to histamine, ninhydrine reacted with 17, fluorescamine with 8 and DAB with 15. Since only histidyl-leucine, octopamine, serotonin and tryptophan interfere in the resolution of histamine in any of the solvent systems used, DAB provides some advantages by not reacting with histidyl-leucine. In addition, with the acetone-ammonia (95:5) solvent system, which adequately separates histamine from all other tested amines, no background interference problems were noted when using DAB as the visualization reagent. Background interference problems with the acetone-ammonia system were evident with the other visualization reagents. The sensitivity of the DAB and fluorescamine methods (4 nmoles of histamine) allows both reagents to act as semi-quantitative screening agents for potentially toxic levels of histamine in food extracts. With a variety of food samples, DAB visualization following development in acetone-ammonia (95:5) was the optimal thin-layer chromatographic method.

Chromatography, Thin Layer↗

PubMedDate not supplied

Determination of protein loading in biodegradable polymer microspheres containing tetanus toxoid.

Various methods to determine loading of vaccine in biodegradable polymer microspheres encapsulating tetanus toxoid were evaluated. The microspheres were composed of poly (D-lactic acid) (PLA) and poly (DL-lactic-co-glycolic acid) (PLGA). Dissolution of microspheres in organic solvents such as methylene chloride, chloroform, or dimethyl sulfoxide and extraction of vaccine antigen or total protein with phosphate buffered saline gave variable results which depended upon the characteristics of the microspheres, such as type of polymer, excipients used in the microspheres and formulation conditions. Microspheres made from low molecular weight PLGA polymer and showing a large burst release exhibited up to 25% extraction of antigen whereas microspheres made from PLA microspheres with low burst release showed < 1% extraction. Extraction of total protein with 0.1 N NaOH and 5% sodium dodecyl sulfate showed results similar to those obtained with organic solvent extraction method. Partial digestion of microspheres with 6 N HCl at 60 degrees C for 20 h resulted in approximately 30% loss in TT protein by micro-bicinchoninic acid (BCA) assay. The major problem with this method was strong reactions in the micro-BCA assay of stabilizers, particularly sugars (glucose, sucrose) used in the microsphere formulations. Complete digestion of microspheres with 6 N HCl at 110 degrees C for 20 h or with 13.5 N NaOH at 121 degrees C for 1 h and quantitation of amino acids by a modified ninhydrin assay showed reproducible results on the protein loading in the microspheres. However, this method was affected by the presence of stabilizers, such as gelatin, which contain amino acids. Further, sucrose concentrations higher than 10% caused interference in the ninhydrin assay on samples hydrolyzed with 6 N HCl. In contrast, hydrolysis with 13.5 N NaOH did not show any interference by sucrose. Stabilizers used outside the microspheres for lyophilization purposes may be removed by washing the microspheres before loading determination or by dialysis but stabilizers used inside the microspheres would still cause interference. For reliable determination of total protein in the microspheres containing vaccines, we suggest complete digestion of microspheres with acid or base followed by amino acid analysis by colorimeteric assays such as ninhydrin method or using amino acid analyzers. The method needs to be optimized for each type of formulation to eliminate interference by the excipients. Alternatively, total protein nitrogen in the microspheres may be determined by the Kjel-dahl method if no amino acids or other nitrogen containing stabilizer is used inside the microspheres.

Antigens, Bacterial↗

PubMed1986

Profiling of amino acids in body fluids and tissues by means of liquid chromatography.

The needs of urgent diagnoses and the needs emerging from acute forms of diseases have directed progress in amino acid profiling to modern, rapid, automated analyses that can be done at reasonable cost. The first step in this direction was the short programmes of classical ion-exchange chromatography. At the beginning of this review we attempted to survey methods of sample preparation and sample treatment, as these are frequently neglected stages where artefacts or erroneous results may arise. There are basically the following approaches in amino acid profiling by liquid chromatographic techniques. For preliminary screening of a large number of samples in clinical routine planar procedures are the methods of choice, as they allow large numbers of samples to be handled with minimum effort and at very reasonable cost. For more precise profiling, particularly where quantitative data are essential, one can choose between some of the modern procedures for separating underivatized amino acids using modern equipment for cation-exchange chromatography, by making use of a stepped series of lithium citrate buffers with ninhydrin, o-phthalaldehyde or 4-fluoro-7-nitrobenzo-2,1,3-oxadiazole detection. Ninhydrin detection is preferred in those situations where the demands on sensitivity are not high. Where, however, only small amounts of samples are available or high sensitivity is required, one of the latter two methods is preferred. The o-phthalaldehyde procedure is not suitable for the detection of secondary amines and, if these are of interest, then diazole derivatization is to be preferred. At present, however, the ninhydrin and o-phthalaldehyde detection procedures are the most popular. The other choice is to use one of the sophisticated HPLC systems equipped with fluorescence detection and to separate amino acids as derivatives. Here o-phthalaldehyde and 4-fluoro-7-nitrobenzo-2,1,3-oxadiazole derivatives offer the most versatile possibilities. Automation and computerization have penetrated both categories of liquid column separation and are applied to automated sample delivery, automated and computerized gradient formation and quantitation of the data obtained. The tables of metabolic disorders of amino acids and the roles of different amino acids in these disorders should provide preliminary information for clinical chemists.

Amino Acids↗

PubMed1990

Serum amino acid analysis with pre-column derivatization: comparison of the o-phthaldialdehyde and N,N-diethyl-2,4-dinitro-5-fluoroaniline methods.

We compared two pre-column derivatization methods, o-phthaldialdehyde (OPA) and N,N-diethyl-2,4-dinitro-5-fluoroaniline (FDNDEA), for analysis of serum amino acids by reversed-phase high-performance liquid chromatography. Separations took 102 and 106 min for FDNDEA and OPA (reconditioning included), respectively, allowing a very good resolution of 30 amino acids by the former process and 38 by the latter. Linearity, within- and between-day variability and advantages in terms of accuracy and speed were studied for both methods. Twenty serum samples from healthy volunteers were assayed with OPA, FDNDEA and with the reference method of ion-exchange and post-column ninhydrin reaction (amino acid analyser), which took 170 min. The correlation between OPA and ninhydrin was good for all the amino acids (r = 0.959) except for the last-eluting lysine. Good agreement was found for FDNDEA (r = 0.987), which appeared in general to be a highly reproducible technique. Both pre-column methods were more sensitive than the post-column ninhydrin method.

Adult↗

PubMed1965

Associations of amino acids and related compounds in the seeds of forty-seven species of Vicia: their taxonomic and nutritional significance.

1. The genus Vicia may be subdivided into groups of species characterized by associations of ninhydrin-positive compounds occurring in high concentrations in their seeds. Despite these subdivisions the overall distribution pattern emphasizes the unity of the genus and the possible value of such studies in establishing degrees of relationship between species within the genus. 2. Canavanine is a major constituent in the seeds of 17 species. 3. gamma-Hydroxyarginine, an amino acid that has not been found in other plant genera, occurs in 18 species. 4. gamma-Hydroxyornithine, a new natural amino acid, is found in two species. 5. A new naturally occurring ureido amino acid tentatively identified as ;gamma-hydroxycitrulline' is found in two species. 6. High concentrations of alphabeta-diaminopropionic acid are found in seed of V. baicalensis and of alphagamma-diaminobutyric acid in seed of V. aurantica. 7. The neurotoxic amino acid beta-cyanoalanine and its gamma-glutamyl peptide are found together in high concentrations in the seeds of 16 species of this agriculturally important genus. 8. Seven unidentified ninhydrin-positive compounds occur in high concentration (about 1% of the dry weight or more) in the seed of various species. Details of their R(F) values, ionic mobilities and colour reactions are given. 9. The total concentration of extractable ninhydrin-positive compounds varies little between seeds of different species and these compounds may, as has been suggested for those in Lathyrus, constitute a readily available form of storage nitrogen. 10. The nature and distribution, as opposed to the total concentration, of the amino acids and related compounds accumulated in the seeds of Vicia are different from those accumulated in the seeds of the related genus, Lathyrus. One particularly interesting difference is the accumulation of C(6) guanidino amino acids (arginine and gamma-hydroxyarginine) by Vicia and the accumulation of C(7) guanidino amino acids (homoarginine, gamma-hydroxyhomoarginine and the related amino acid lathyrine) by Lathyrus. Such differences afford a method for distinguishing between species of these genera.

Journal Article↗

PubMed1966

The determination of the substitution achieved at the alpha-amino, epsilon-amino and imidazole groups of proteins with special reference to derivatives of gelatin.

1. The diminution of the quantity of pigment formed in the ninhydrin colorimetric reaction or the titre in two types of formol titration of a protein after substitution is used to determine the degree of substitution achieved at the various amino groups of a protein. 2. The determination of the substitution achieved at alpha-amino plus in-amino groups by the ninhydrin colorimetric method as described by Cobbett, Gibbs & Leach (1964) has been modified to correct for the hydrolysis of the peptide chain which takes place during colour development. 3. The degree of substitution at the in-amino groups was determined by the formol titration carried out at pH9, essentially as described by the above authors. 4. Substitution at the alpha-amino plus in-amino plus imidazole groups was determined from the diminution of the titre of a modified formol titration. This titration was carried out by adjusting the protein solution to pH6.5 followed by the addition of formaldehyde and then titrating to pH9.0. 5. The three methods have been applied to carbamoylated and benzenesulphonylated gelatin derivatives. The values for the degrees of substitution obtained by the ninhydrin (alpha-amino plus in-amino) and the formol (pH9.0, in-amino) methods were shown to be almost identical. The values from the formol (pH6.5-9.0, alpha-amino plus in-amino plus imidazole) titration were consistently lower than the values obtained by the other methods and corresponded to no substitution having taken place at the imidazole groups by the preparative methods employed. 6. The application of the methods to other protein systems is discussed.

Amino Acids↗

PubMed1970

Chemical constitution of the host-specific toxin of Helminthosporium carbonum.

The host-specific toxin of Helminthosporium carbonum Ullstrup has a molecular formula approximating C(32)H(50)N(6)O(10). The compound has been crystallized and a crystalline hydrochloride derivative has been produced. The molecular weight, as determined by chromatography on Sephadex G-10, is slightly less than 700. The toxin appears to be a cyclic peptide, since, although it does not react with ninhydrin or dinitrofluorobenzene, it yields, on hydrolysis, compounds which react to these reagents. It is unstable in dilute acids, yielding ninhydrin-reacting products. Complete acid hydrolysis yields alanine, proline, and three other ninhydrin-reacting components. The infrared spectrum of the toxin reveals an ester band in addition to amide absorption. Its ultraviolet spectrum reveals the presence of unsaturation in the molecule. The toxin is relatively unstable and loses its specific toxicity. This loss of activity appears to be associated with loss of nitrogen and with decreased solubility in water.

Crystallization↗

PubMed1980

Purification of GABA on small columns of Dowex 50W; Combination with a method for separation of biogenic amines.

Elution of GABA has been included in the method for separation of biogenic amines on small Dowex 50W columns described by Atack & Magnusson (1978). After extraction with perchloric acid and neutralization to pH 3.0, the sample is passed through the column. 5-HIAA might thereafter be eluted as described by Lindqvist (1971). After washes with water and 8 ml of a 0.025 M sodium citrate buffer pH 4.5, GABA is eluted in 3 ml of a 0.05 M sodium citrate buffer pH 5.3-5.4. The elution of tryptophane and the above mentioned amines can thereafter be performed. The recovery of GABA from the column is 100%. Silica thin-layer chromatography of the GABA eluate gives only one ninhydrine positive spot with a Rf value identical to GABA standard. When the GABA eluate from a whole brain and spinal cord is run on an automatic amino acid analyser, only one peak is detectable, eluted in the same position as GABA standard. Fluorescence analysis of the GABA eluate gives similar values if a( ninhydrine, b) acetylacetone plus formaldehyde or c) fluorescamine is used to form the flurophore. The GABA values are identical whether the samples reacted with ninhydrine are diluted with water or copper tartrate. The GABA concentrations found in some parts of the rat brain are of the same order of magnitude as reported by other authors.

5-Hydroxytryptophan↗

PubMed1978

Comparison of the toxicities of patulin and patulin adducts formed with cysteine.

The toxicities of patulin and of the patulin adducts formed with cysteine were compared using the mutation-sensitive strain Escherichia coli W3110 thy polA1 and its polA1+ revertant. The acute toxicities of patulin and of the adduct mixture were also compared using NMRI mice. The adduct mixture was shown by thin-layer chromatography to consist of one ninhydrin-positive, one ninhydrin- and MBTH (3-methyl-2-benzothiazolinone hydrazone)-positive, three MBTH-positive, and two ninhydrin- and MBTH-negative components. The results showed that patulin was over 100 times more toxic to E. coli than the adduct complex. Neither patulin nor the adduct mixture was found to induce the repair effect in E. coli. In the mouse feeding tests, the oral 50% lethal dose for patulin was 29 mg/kg, while that of the adduct mixture was greater than 2,370 mg/kg.

Animals↗