IV. Determination of aromatic L-amino acid decarboxylase in serum of various animals by high-performance liquid chromatography with electrochemical detection

This paper describes the distribution of aromatic L-amino acid decarboxylase (AADC), using both L-DOPA and L-5-hydroxytryptophan (L-5-HTP) as substrates, in serum of various animals. The ratio of the activities of the enzyme towards both substrates was also determined in the same serum. AADC activity was discovered in serum using our new and highly sensitive assay for AADC activity by high-performance liquid chromatography (HPLC) with electrochemical detection (ED) with L-DOPA and L-5-HTP as substrates. It was found that among the species used, guinea pig serum had the highest activity, and we made a systematic study on guinea pig serum AADC.     

Assay of short-chain acyl coenzyme A intermediates in tissue extracts by high-pressure liquid chromatography

A high-pressure liquid chromatographic method for the measurement of short- and medium-chain-length acyl-CoA compounds is described. Compounds are separated on a reverse-phase μBondapak C₁₈ column with the order of elution based on differences in lipophilicity. The mobile phase consisted of variable mixtures of methanol and 50 mm KH₂PO₄, pH 5.3. Conditions are described that allow isocratic separation of groups of compounds of similar lipophilicity. With increasing methanol concentration, the more lipophilic compounds are eluted earlier. This has the effect of sharpening the peaks and improving quantitation. Detection of acyl-CoA intermediates is achieved using a uv detector and is based on the high absorbance of CoA-containing compounds at 254 nm. Neutralized perchloric acid extracts of tissues can thus be analyzed directly without further purification or derivatization. A mobile phase consisting of a 9:1 phosphate buffer-to-methanol mixture is used to separate CoASH, methylmalonyl-CoA, succinyl-CoA, β-hydroxy-β-methylglutaryl-CoA and acetyl-CoA. Increasing the methanol concentration to a 4:1 mixture allows separation of acetyl-CoA, propionyl-CoA, and isobutyryl-CoA, while with a 7:3 mixture of phosphate buffer to methanol, β-methylcrotonyl-CoA and isovaleryl-CoA are readily separated. Examples of results obtained using extracts from isolated hepatocytes, rat liver mitochondria, and perfused rat hearts incubated with α-ketoisocaproate, α-ketoisovalerate, or propionate are presented. In addition, methods and optimal conditions are presented for the analysis of malonyl-CoA, glutathione-CoA, dephospho-CoA, and oxidized CoA in tissue extracts.     

The analysis of the betaine homarine by high pressure liquid chromatography

A procedure is presented which is suitable for the qualitative and quantitative analysis of the betaine homarine in aqueous tissue extracts. After preliminary purification of the extract by gel permeation chromatography on Sephadex G-25, quantitative analysis of the homarine content is performed by high pressure liquid chromatography on a 1-m column of Corasil II.     

The analysis of nanogram levels of free amino acids by reverse-phase high-pressure liquid chromatography.

A simple method and apparatus are described for the efficient recovery of proteins from sodium dodecyl sulfate-polyacrylamide gel systems after electrophoretic resolution. This procedure provides for high yields of proteins which are free of sodium dodecyl sulfate and in certain cases, exhibit significant levels of biological activity.     

Separation of hemoglobin types by cation-exchange high-performance liquid chromatography

The use of a recently developed cation-exchange HPLC packing material for the separation of hemoglobin types in human blood has been investigated. Adult and newborn hemolysates from normal individuals and from subjects with hemoglobin disorders were analyzed using a weak cation carboxymethyl-bonded phase on 5-micron-particle-size silica. Elution was accomplished using a Bistris (2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1, 3-propanediol) gradient. Seven well-resolved HbA1 fractions eluted before the major HbA peak. Hbs A1a, A1b, A1c and an HbA1 fraction that increased with aging of the hemolysates were separately eluted. HbF when present or when added to the hemolysates eluted as a distinct peak. HbA was followed by Hbs A2, S, and C when present. An early-eluting peak corresponding to Hb Bart's was identified in newborn hemolysates. It is concluded that cation-exchange HPLC provides a new tool for the reliable separation of minor hemoglobin components.     

Assay for aliphatic amino acid decarboxylases by high-performance liquid chromatography

A sensitive and rapid assay for aliphatic amino acid decarboxylases based on separation of the product from the substrate by ion-pairing reversed-phase high-performance liquid chromatography and subsequent fluorometric detection has been developed. The resolution of substrates and products of seven amino acid decarboxylases, namely, arginine, aspartate, 2,6-diaminopimelate, histidine, glutamate, lysine, and ornithine decarboxylase, is complete within 15 to 35 min of isocratic elution. The limit of detection for the product is 40 pmol. The applicability of the procedure was assessed with glutamate decarboxylase. The formation of the product 4-aminobutyrate proved to be linear with time and protein concentration. The method allows the time course of the reaction to be followed in a single assay and works well with crude extracts of bacteria or tissues.     

The analysis of aflatoxins by high-performance liquid chromatography.

The potential of high-performance liquid chromatography as a technique for separating aflatoxins B₁ B₂, G₁, G₂, B_2a, Q₁, M₁, P₁, aflatoxicol, and a degradation product of aflatoxin B₁, 2,3-dihydrodiol, has been assessed. A microparticulate silica adsorption column used with a 1:1 chloroform -dichloromethane eluant provided good resolution of aflatoxins B₁, B₂, G₁, and G₂ but the addition of 1% propan-2-ol was necessary for the elution of aflatoxins M₁ and Q₁. By selecting appropriate solvent mixtures, good resolution of all of the aflatoxins studied was obtained using columns containing an octadecyl (C₁₈) reversed-phase bonded to a microparticulate support. Details are given for resolving: (1) aflatoxins B₁, B₂, G₁, and G₂ using a 5% tetrahydrofuran-15% dimethylformamide in water eluant and (2) aflatoxins B₁ B_2a, Q₁ M₁ P₁ aflatoxicol, and a product of aflatoxin B₁ 2,3-dihydrodiol treated with Tris-buffer, using either 15% dimethylformamide in water or 10% tetrahydrofuran in water as eluant.     

Advances in the analysis of amino acid phenylthiohydantoins by high performance liquid chromatography

All the organic soluble amino acid phenylthiohydantoins are eluted (but not totally resolved) in under 40 min on either a Zorbax ODS or Permaphase ETH column. The superior resolution of the Zorbax ODS column and the complimentary characteristic of the Permaphase ETH column allow the isocratic analysis of 15 phenylthiohydantoins in less than 20 min when the columns are operated simultaneously. The water soluble PTH-His and -Arg are analyzed separately in under 10 min with the Zorbax ODS column. Five picomoles of a derivative may be detected with a sample to noise ratio of 6, HPLC is the most sensitive, practical method for PTH analysis.     

Assay of bovine fibrinopeptides by high performance liquid chromatography.

A method for separating small amounts (<10^?5 mol) of bovine fibrinopeptides A and B employing high performance liquid chromatography has been developed. The limit of detectability of this method is about 10^?10 mol of fibrinopeptide. The separation was achieved within 20 min under reversed phase conditions using isocratic elution with aqueous buffer-acetonitrile solvent systems.     

Assay of tryptophan hydroxylase and aromatic L-amino acid decarboxylase based on rapid separation of the reaction product by high performance liquid chromatography

A rapid separation of 5-hydroxytryptophan by high performance liquid chromatography (HPLC) was achieved for the assay of tryptophan hydroxylase. "Bulk separation" of the product from all other components in the reaction mixture by HPLC was achieved by 1) the choice of a suitable column-solvent system so as to elute the reaction product ahead of other components in the sample mixture, 2) the use of a monitor selective for the reaction product, 3) minimization of the column length so as to achieve rapid separation of the product from the substrate. The method finally employed a reversed phase column of 5 cm length, relatively rapid elution at 2 ml/min and fluorescence detection at 350 nm with an excitation at 302 nm. The assay is convenient and as sensitive as the radioisotope method. The advantages of the method are 1) almost no pretreatment of samples, 2) repeatability every 2 min, 3) wide latitude of product determination from picomole to nanomole amounts per assay. The method was extended to the assay of 5-hydroxytryptophan decarboxylase by essentially the same procedures.     

Separation of substituted pteroyl monoglutamates and pteroyl oligo-gamma-L-glutamates by high pressure liquid chromatography.

Rapid analysis of substituted and unsubstituted pteroyl-oligo-γ-l-glutamates at the nanomole level is carried out by high performance liquid chromatography. The use of a siliceous microparticulate anion-exchanger column and gradient elution at pH 6.5 with increasing salt concentration facilitates the separation of the species containing up to seven glutamyl residues without decomposition in 30 min. The column effluent is monitored with a uv detector at 254 nm, and the peaks are conveniently identified by their retention.     

Analysis of porphyrin carboxylic acids in biological fluids by high-performance liquid chromatography

A method for the rapid and sensitive fluorometric analysis of porphyrin carboxylic acids by reverse-phase high-performance liquid chromatography is described. Separation of free porphyrin carboxylic acids was carried out with a microparticulate octadecylsilane column with elution by a gradient of methanol in phosphate buffer containing tetrabutylammonium hydroxide. Separation and quantitation of di-, tri-, tetra-, penta-, hexa-, hepta-, and octacar-boxylic porphyrins was achieved within 25 min at picomolar concentrations. The method is also capable of separating the type I and type III isomers of tetracarboxylic through hexacarboxylic porphyrins. By using a stopped flow technique, one can record fluorescence excitation and emission spectra of porphyrin carboxylic acids. This method is directly applicable to biological fluids such as urine, plasma, red cell lysates, or medium or extracts from cell culture.     

Analysis of the amino acid indospicine in biological samples by high performance liquid chromatography

Indospicine is a hepatotoxic amino acid that accumulates in the meat of horses that consume the legume Indigofera linnaei. A method to determine indospicine concentration in biological samples using an amino acid analyser has been reported, but the analysis time is long and therefore not suited to the analysis of large numbers of samples. A rapid and reliable method was developed for the analysis of indospicine in horsemeat and serum using High Performance Liquid Chromatography. Horsemeat and serum were extracted with either water or 0.01 N hydrochloric acid, respectively, and deproteinized by ultrafiltration. Precolumn derivatization of samples with phenylisothiocyanate was followed by separation of indospicine from other amino acids on a Pico-Tag C 18 column and UV detection at 254 nm. The calibration curves for indospicine in horsemeat extract were linear over the concentration range 0.4 microg ml(-1) to 20 microg ml(-1), while for indospicine in serum, the linear range was from 0.17 microg ml(-1) to 16.67 microg ml(-1). The mean recovery of indospicine in horsemeat extract was 87.2 +/- 6.8% and in serum was 97.3 +/- 9.9%. Analysis time for indospicine in horsemeat samples was 31 min and in serum samples was 36 min.     

Separation of estrogen conjugates by high pressure liquid chromatography.

High pressure liquid chromatography using a prepacked commercial strong anion exchanger column (mu Partisil 10 SAX, 25 cm x 4.6 mm) was used to separate a mixture of eight estrogen conjugates. Chromatographic conditions using a 0.01 M potassium phosphate or 0.1 M NaCl as solvent in the isocratic mode are described for the separation of estrone glucosiduronate, 17beta-estradiol-3-glucosiduronate, 17beta-estradiol-17-glucosiduronate, estriol-3-glucosiduronate, estriol-16alpha-glucosiduronate, estriol-17-glucosiduronate, estrone sulfate and 17beta-estradiol-3-sulfate. This system gives high resolution of the estrogen conjugates in small eluent volumes in less than 30 min. The advantages of this high pressure liquid chromatographic system over other methods of separation are discussed.     

A high-performance liquid chromatography method for the assay of cytidine monophosphate-sialic acid synthetase

An HPLC method has been developed for the assay of cytidine monophosphate-sialic acid synthetase (EC 2.7.7.43) using ion-pair chromatography and gradient elution. This procedure permits the assay of alternative substrates and inhibitors of the enzyme and is not subject to the limitation of the colorimetric method. The newly synthesized N-acetyl-9-deoxy-9-fluoro-D-neuraminic acid was found to be a good substrate of the enzyme with a Km of 6.35 mM as compared to 1.84 mM for N-acetylneuraminic acid.     

High-pressure liquid chromatography of neurotoxic phenylphosphonothioate esters and related compounds

It has been suggested that perfluorooctanoic acid occurs in human plasma; however, no method of analysis for this compound in biological samples has been published to date. A method is presented for the analysis of perfluorooctanoic acid in plasma, urine, and liver tissue based on conversion of the acid to its methyl ester followed by separation and quantitation by gas chromatography.     

Determination of methylputrescine oxidase by high performance liquid chromatography

N-Methyl-Δ¹-pyrrolinium chloride, the product of the title enzyme, was synthesized by methylation of aminobutyraldehyde diethylacetal followed by acidic cleavage. After purification to homogeneity, it was characterized by NMR and UV spectroscopy. The compound had an absorption maximum at 210 nm; previous data indicating a maximum at 267 nm were shown to arise from an impurity. An HPLC method for the assay of N-methylputrescine oxidase from plant material was developed based on the separation of N-methyl-Δ¹-pyrrolinium chloride on a cation exchange column and direct detection at 210 nm. The enzyme activity was measured in the protein fraction extracted from plant roots and treated by gel filtration on disposable PD 10 columns. A K_m value of 1.9 mM was determined for methylputrescine and the enzyme from tobacco roots. The enzyme activities from N. tabacum and Datura stramonium were compared.     

Characterization of proteins and peptides by high-performance liquid chromatography and fluorescence monitoring of their tryptic digests.

Proteins and peptides can be characterized and compared at the subnanomole level by treatment with trypsin followed by high-performance liquid chromatography on reverse-phase partition columns. A fluorescamine monitoring system automatically analyzes a portion of the column effluent while the remainder can be collected for further studies. The method has already been used for characterization of rat β-endorphin and a protein which cross-reacts with antiserum prepared against prolyl hydroxylase.     

Determination of 3-methoxy-4-hydroxyphenylglycol in urine by high-performance liquid chromatography with amperometric detection

A reversed-phase high-performance liquid chromatographic method has been used for the quantitative determination of 3-methoxy-4-hydroxyphenylglycol (MHPG) in urine. After incubation with glusulase, free MHPG is extracted into ethyl acetate and further isolated by a combination of thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The addition of amperometric detection provides increased sensitivity to a highly specific assay.