Analysis of thiamine and its phosphate esters by high-performance liquid chromatography.

High-performance liquid chromatography was used to separate thiamine and its phosphate esters after conversion to corresponding highly fluorescent thiochrome derivatives by alkaline oxidation. These compounds were absorbed on LiChrosorb-NH₂, eluted with acetonitrile-90 mm potassium phosphate buffer (pH 8.4), and determined spectrofluorometrically. A complete, rapid, and quantitative separation of thiochrome and its phosphate derivatives was made and the minimum amount detected was 1 pmol for each of these compounds.     

Thiamine inhibits formation of dityrosine,a specific marker of oxidative injury,in reactions catalyzed by oxoferryl forms of hemoglobin

Effects of thiamine and its derivatives on inhibition of dityrosine formation were studied in reactions catalyzed by oxoferryl forms of hemoglobin. At high thiamine concentrations, a complete inhibition of dityrosine formation was observed due to interaction of tyrosyl radicals with thiamine tricyclic and thiol forms. In neutral and alkaline media, tyrosyl radicals oxidized thiamine to thiochrome, oxodihydrothiochrome, and thiamine disulfide. In the absence of tyrosine, oxoferryl forms of hemoglobin manifested peroxidase activity towards thiamine and its phosphate esters by inducing their oxidation to disulfide compounds, thiochrome, oxodihydrothiochrome, and their phosphate esters, respectively, in neutral media. Thiamine and its phosphate esters were oxidized by both oxoferryl forms of hemoglobin, viz., +*Hb(IV=O) (compound I with an additional radical on the globin) and Hb(IV=O) (compound II). Putative mechanisms of thiamine conversions under oxidative stress and the protective role of hydrophobic thiamine metabolites are discussed.     

Analysis of abscisic acid by high-performance liquid chromatography.

Methodology for the ready analysis of abscisic acid (ABA) in plant tissues based upon application of high-performance liquid chromatography (hplc) has been developed. The method involves isolation of the acid fraction, preparation of the methyl esters with diazomethane, and hplc using a combination procedure of two columns: (1) reversed-phase C₁₈, and (2) porous silica in the absorption mode. Only isocratic elution is required so the method is readily adaptable to laboratories having limited hplc capability. Measured recoveries are 70% and the use of an internal standard allows quantification of ABA levels to 1 ng/g of tissue with minimum absolute detectable levels of ABA of 20 ng. The method is illustrated by analysis of ABA concentration in potato tubers at various times postcutting.     

Analysis of (3-phenyl,2-thio)hydantoin amino acids by high-performance liquid chromatography: Comparison of three programs with particular reference to the glutamic and aspartic derivatives

(3-Phenyl,2-thio)hydantoin-amino acids are readily separated and quantitated by high-performance liquid chromatography (hplc). Three reversed-phase column procedures were investigated. We found the aspartic and glutamic acid derivatives difficult to separate and quantitate by any of the three until we determined the effects on hplc of several injection solvents and the pH of the elutrient buffers. We found 90% acetic acid or acetonitrile: 90% formic acid (8:2, v:v) satisfactory as injection solvents. Each of the three hplc programs also required pH changes for optimal separation of the aspartic and glutamic acid derivatives from the other (3-phenyl,2-thio)hydantoin-amino acids; none of these had retention times that varied with pH in the ranges investigated. Using the three modified systems, the retention times of 19 commonly found (3-phenyl,2-thio)hydantoin-amino acids were compared. In addition, the N-terminal residues of two model human plasma proteins were chromatographed, fibrinogen and plasminogen. In all of these, the aspartic and glutamic acid derivatives were clearly separable without diminishing resolution of the other amino acids.     

Separation and detection of uv-absorbing derivatives of fecal bile acid metabolites by high-performance liquid chromatography

Major fecal bile acid metabolites related to lithocholic acid were resolved by high-performance liquid chromatography (hplc). The uv-absorbing p-nitrobenzyl ester derivatives of lithocholic, isolithocholic, 3-keto-5β-cholanic, and 5β-cholanic acids were prepared using the reagent o,p-nitrobenzyl-N,N′-diisopropylisourea. Separation was achieved in less than 20 min on a microparticulate silica column using isocratic elution with 2% isopropanol in isooctane as the mobile phase. The p-chlorobenzoyl esters of methylated lithocholic and isolithocholic acids were also prepared but required purification by thin-layer chromatography before separation by hplc. These derivatives were eluted from a Porasil T column using 5% diisopropyl ether in isooctane as the mobile phase. Lithocholic and isolithocholic acids produced by microbial metabolism of [¹⁴COOH]taurolithocholic acid were separated and identified by preparing p-nitrobenzyl derivatives and monitoring the column effluent for both uv and radioactivity. This technique is a rapid and sensitive method for isolating bile acid metabolites.     

Regional alterations of thiamine phosphate esters and of thiamine diphosphate-dependent enzymes in relation to function in experimental Wernicke's encephalopathy

Thiamine phosphate esters (thiamine monophosphate-TMP; thiamine diphosphate-TDP and thiamine triphosphate-TTP) were measured as their thiochrome derivatives by High Performance Liquid Chromatography in the brains of pyrithiamine-treated rats at various stages during the development of thiamine deficiency encephalopathy. Severe encephalopathy was accompanied by significant reductions of all three thiamine phosphate esters in brain. Neurological symptoms of thiamine deficiency appeared when brain levels of TMP and TDP fell below 15% of normal values. Activities of the TDP-dependent enzyme -ketoglutarate dehydrogenase were more severely reduced in thalamus compared to cerebral cortex, a less vulnerable brain structure. On the other hand, reductions of TTP, the non-cofactor form of thiamine, occurred to a greater extent in cerebral cortex than thalamus. Early reductions of TDP-dependent enzymes and the ensuing metabolic pertubations such as lactic acidosis impaired brain energy metabolism, and NMDA-receptor mediated excitotoxicity offer rational explanations for the selective vulnerability of brain structures such as thalamus to the deleterious effects of thiamine deficiency.     

Hexose-, inositol-, and nucleoside phosphate esters in germinating seeds of crested wheatgrass

Net synthesis of phosphate esters was measured in crested wheatgrass seeds [Agropyron desertorum (Fisch.) Schult.] to learn what phosphorylating reactions occur in early stages of germination. Phosphate esters were separated on ion exchange columns and identified by cochromatography and chemical analyses. Synthesis of adenosine triphosphate and UDP-hexose was detected 6 hours after the beginning of water absorption. This synthesis occurred during the period of rapid increase in water potential. Synthesis of hexose phosphate and uridine triphosphate was detected 12 hours after the beginning of water absorption. The concentrations of these esters increased during 48 hours. Increases in concentration of inositol di-, tri-, and tetraphosphate suggested that hydrolysis of inositol hexaphosphate began after 12 hours.     

Determination of pyridoxal 5′-phosphate as the semicarbazone derivative using high-performance liquid chromatography

A high-performance liquid chromatographic (hplc) procedure is described for the determination of pyridoxal 5′-phosphate (PLP) in animal tissues. The procedure is based on extraction with perchloric acid and treatment with semicarbazide to form PLP-semicarbazone. This derivative is quantitatively determined using hplc with an octylsilica column, an acidic phosphate mobile phase buffer, and fluorometric detection. The validity of the method was confirmed by inspection of the fluorescence spectra of the PLP-semicarbazone hplc peaks of semicarbazide-treated standards and tissue extracts. Preparative chromatography for extract purification is not required because of the hplc efficiency and detection specificity. This method provides a simple technique for the rapid, direct assay of PLP in animal tissues.     

Fluorescent derivatives of the pyruvate dehydrogenase component of the Escherichia coli pyruvate dehydrogenase complex.

One sulfhydryl group per polypeptide chain of the pyruvate dehydrogenase component of the pyruvate dehydrogenase multienzyme complex from Escherichia coli was selectively labeled with N-[P-(2-benzoxazoyl)phenyl]-maleimide (NBM), 4-dimethylamino-4-magnitude of-maleimidostilbene (NSM), and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) in 0.05 M potassium phosphate (pH 7). Modification of the sulfhydryl group did not alter the enzymatic activity or the binding of 8-anilino-1-naphthalenesulfonate (ANS) or thiochrome diphosphate to the enzyme. The fluorescence of the NBM or NSM coupled to the sulfhydryl group on the enzyme was quenched by binding to the enzyme of the substrate pyruvate the coenzyme thiamine diphosphate, the coenzyme analogue thiochrome diphosphate, the regulatory ligands acetyl-CoA, GTP, and phosphoenolpyruvate, and the acetyl-CoA analogue, ANS. Fluorescence energy transfer measurements were carried out for the enzyme-bound donor-acceptor pairs NBM-ANS, NBM-thiochrome diphosphate ANS-DDPM, and thiochrome diphosphate-DDM. The results indicate that the modified sulfhydryl group is more than 40 A from the active site and approximately 49 A from the acetyl-CoA regulatory site. Thus, a conformational change must accompany the binding of ligands to the regulatory and catalytic sites. Anisotropy depolarization measurements with ANS bound on the isolated pyruvate dehydrogenase in 0.05 M potassium phosphate (pH 7.0) suggest that under these conditions the enzyme is dimeric.     

Succinate transport in Bacillus subtilis. Dependence on inorganic anions

Cations were generally ineffective in stimulating succinate transport in a succinate dehydrogenase mutant of Bacillus subtilis unless accompanied by polyvalent anions; phosphate and sulfate being particularly active. The K_m values for the phosphate or sulfate requirement were approx. 3 mM.Biphasic kinetics were characteristic of both the succinate (K_m values 0.1 and 1 mM), and inorganic phosphate (K_m values 0.1 and 3 mM) transport system(s). The phosphate transport system(s) was repressed by high inorganic phosphate and a coordinate increase in the transport of phosphate, arsenate, and phosphate-stimulated succinate transport accompanied growth in low phosphate media.A class of arsenate resistant mutants were simultaneously defective in the transport of arsenate, phosphate and succinate when cells were repressed for phosphate transport, however, the transport of these ions was regained in these mutants when grown in low phosphate media. Organic phosphate esters did not stimulate succinate transport in arsenate resistant mutants but were effective after growth in low phosphate media. Growth under phosphate limitation permitted the simultaneous regain of both phosphate and sulfate dependent succinate transport activities whereas sulfate limitation alone was ineffective.Succinate was not transported by an anion exchange diffusion mechanism since phosphate efflux was low or absent during succinate transport.The transport of C₄-dicarboxylates in B. subtilis is strongly stimulated by intracellular polyvalent anions. The absence of an anion permeability mechanism precludes succinate transport but partial escape from this restriction is mediated by the derepression of a phosphate transport system.     

Synthetic and biosynthetic studies of porphyrins: III. Structures of the intermediates between uroporphyrinogen-III and coproporphyrinogen-III: Synthesis of fourteen heptacarboxylic,hexacarboxylic, and pentacarboxylic porphyrins related to uroporphyrin-III

Four heptacarboxylic, six hexacarboxylic, and four pentacarboxylic porphyrins related to uroporphyrin-III by decarboxylation of one, two, or three of the acetic acid side chains have been synthesised as their methyl esters by application of the MacDonald or b-oxobilane methods, as appropriate. Comparison (mixed mp, “mixed” nmr spectra, and hplc) of the synthetic materials with the methyl esters of hepta-, hexa-, and pentacarboxylic porphyrins isolated from natural sources showed that the structures of the latter corresponded to the D-ring methyl, the DA-dimethyl, and the DAB-trimethyl analogs of uroporphyrin-III. Because the naturally occurring porphyrins arise by oxidation of intermediate porphyrinogens, we conclude that the enzymic decarboxylation of uroporphyrinogen-III to coproporphyrinogen-III takes place in a preferred sequential clockwise fashion (both in normal and abnormal metabolism) starting with the acetic acid moiety on the D-ring and followed by those on the A, B, and C rings.     

Determination of three different pools of reduced one-carbon-substituted folates. III. Reversed-phase high-performance liquid chromatography of the azo dye derivatives of p-aminobenzoylpoly-gamma-glutamates and its application to the study of unlabeled endogenous pteroylpolyglutamates of rat liver

A new reversed-phase high-performance liquid chromatographic (hplc) method is described for the separation and quantitation of picomole amounts of the azo dye derivatives of p-aminobenzoylpoly-γ-glutamates. In conjunction with our previously described procedures for the differential cleavage of one-carbon-substituted, reduced folates, this hplc method provides a rapid, sensitive, and reproducible approach to the quantitation and chain-length determination of three pools of unlabeled endogenous pteroylpolyglutamates. Analysis of rat liver (n = 9) yielded the following results ($\text{x}{}^1\text{±}\text{SE}$): total folates 14.5 ± 1.0 nmol/g; folates of pool 1 (5,10-methylenetetrahydro- and unsubstituted tetra- and dihydrofolates) 2.65 ± 0.74 nmol/g; folates of pool 2 (5-methyltetrahydrofolates) 5.30 ± 0.36 nmol/g; and folates of pool 3 (5,10-methenyltetrahydro-, 10-formyltetrahydro-, 5-formyltetrahydro-, and 5-formiminotetrahydrofolates) 6.40 ± 1.60 nmol/g. Most of the folates of rat liver occur as penta- (7.60 ± 0.69 nmol/g) and hexaglutamates (6.00 ± 0.29 nmol/g). In pool 3 the hexaglutamates predominate. We also report experiments showing that folate patterns based on the amount of radioactive label incorporated after a pulse dose of [³H]folic acid differ at all times from the true steady-state pattern of unlabeled endogenous folates.     

Hydrolytic enzyme substrates. 3. Phosphomonoesterase substrates

This report documents the use of a new and sensitive colorimetric method for measuring phosphomonoesterase activity. The substrates are the phosphate esters of 4-(p-nitrophenoxy)-1,2-butanediol (PNB), 4-(2,4-dinitrophenoxy)-1,2-butanediol (DNB) and 3-(p-nitrophenoxy)-1,2-propanediol (PNG). The key intermediate in the assay is the nitrophenoxy diol which is obtained by enzyme hydrolysis of its phosphate ester. Periodate oxidation of this substance in solution containing methylamine quantitatively yields its nitrophenolate ion whose concentration is determined colorimetrically. The amount of nitrophenolate ion is thus equivalent to the amount of nitrophenoxy diol whose concentration is a function of the phosphomonoesterase activity in the assay sample. The unhydrolyzed phosphomonoester is completely stable to periodate and the hydrolytic conditions used in the assay. The enzymes used to test the substrates were E. coli alkaline phosphomonoesterase and wheat germ phosphomonoesterase. These new esters were all better substrates than the glycerol phosphate esters. Their Michaelis-Menten constants were determined for E. coli phosphomonoesterase.     

Simultaneous analysis of disopyramide and quinidine in plasma by high-performance liquid chromatography

A new reversed-phase high-performance liquid chromatographic method allowing simultaneous measurement of plasma concentrations of disopyramide and quinidine is described. Disopyramide and quinidine were separated on a reversed-phase column using 0.05 M phosphate buffer (pH 3.0)—acetonitrile (73:27, v/v), as mobile phase and the peaks were monitored by UV absorbance at the wavelengths of 254 and 325 nm. The drugs were extracted from alkaline plasma with chloroform containing the internal standard. The organic phase was evaporated to dryness and the residue was redissolved in a small volume of the mobile phase before analysis by high-performance liquid chromatography. The method is convenient and reliable in routine monitoring of both drugs.     

Effect of Sodium Nitrite on the Destruction of Thiamine

The effect of sodium nitrite on the destruction of thiamine was investigated. When sodium nitrite-containing thiamine solution was treated by the condition of heating at 75°C for 60 min, elemental sulfur and 4-methyl-5-(β-hydroxyethyl) thiazole were identified, and thiochrome was estimated. When sodium nitrite-free thiamine solution was heated at 75°C for 60 min, 4-methyl-5-(β-hydroxyethyl) thiazole was a main product, and elemental sulfur and thiochrome were not produced. From these results, it showed that elemental sulfur and thiochrome were produced from thiamine by the effect of sodium nitrite.     

Isolation and characterization of 3,3′,4,6-tetra-O-acylated sucrose esters from the type B glandular trichomes of Solanum berthaultii Hawkes (PI 265857)

A series of polar, high-molecular-weight compounds that comprise the major portion of nonvolatile constituents in the exudate from type B glandular trichomes of S. berthaultii Hawkes (PI 265857) were isolated by silica gel t.l.c. and identified by spectroscopic and chemical means as a complex of 3,3′,4,6-tetra-O-acylated sucrose esters. Two of the principal sucrose esters were resolved by reversed-phase t.l.c. and characterized as 6-O-capryl-3,3′,4-tri-O-isobutyrylsucrose and 6-O-capryl-3′-O-isobutyryl-3,4-di-O-(2-methylbutyryl)sucrose.     

Determination of thiamin diphosphate in whole blood samples by high-performance liquid chromatography—A method suitable for pediatric diagnostics

An improved and easy to use method for the determination of thiamin diphosphate (TDP) in 100 μl of whole blood was developed. The small sample volume makes it possible to assess the nutritional status of vitamin B₁ in infants and even in preterm infants. Sample preparation comprises the extraction of TDP from whole blood by hemolysis, protein precipitation with trichloroacetic acid, and subsequent centrifugation. Potassium ferricyanide is used for pre-column derivatization of TDP to its fluorescent thiochrome derivative. Chromatographic separation was carried out using a reversed-phase column and an isocratic elution which consisted of a phosphate buffer and acetonitrile. TDP was detected fluorimetrically and quantified by external standardization. Method validation showed a high precision, almost complete recovery, and a high sensitivity. The lower limit of detection and the lower limit of quantification were 0.2 ng/ml and 4 ng/ml, respectively. Linearity was demonstrated over the expected concentration range of 4–400 ng/ml. In conclusion, we present a convenient HPLC method for the determination of TDP which is precise, sensitive and suitable for pediatric diagnostics.     

Improved assay method for the determination of pyronaridine in plasma and whole blood by high-performance liquid chromatography for application to clinical pharmacokinetic studies

An improved high-performance liquid chromatography method using a diisopropyl-C₁₄ reversed-phase column (Zorbax Bonus-RP column) and a liquid–liquid extraction technique with UV detection is presented for the analysis of pyronaridine in human whole blood and plasma. Tribasic phosphate buffer (50 mM, pH 10.3) and diethyl ether were used for liquid–liquid extraction. The mobile phase consists of acetonitrile–0.08 M potassium dihydrogen phosphate buffer (13:87, v/v) with the pH 2.8 adjusted by orthophosphoric acid. Amodiaquine was found to be a suitable internal standard for the method. The quantification limit with UV detection at 275 nm was 3 ng on-column for both plasma and blood samples. The method was applied to plasma and blood specimens from a rabbit after a single intramuscular dose of pyronaridine tetraphosphate (20 mg/kg as base). From this in vivo study, evidence was found that pyronaridine is concentrated in blood cells, with a blood:plasma ratio ranging from 4.9 to 17.8. We conclude that blood is the preferred matrix for clinical pharmacokinetic studies.     

Use of reversed-phase high-performance liquid chromatography for simultaneous determination of glutamine synthetase and glutamic acid decarboxylase in crude extracts

Glutamine and γ-aminobutyric acid (GABA), formed from glutamic acid in crude tissue extracts by glutamine synthetase and glutamic acid decarboxylase respectively, were separated by derivatization with dansyl chloride followed by reversed-phase high-performance liquid chromatography on an Altex Ultrasphere ODS-5 column. The mobile phase was a gradient of 100 mM potassium dihydrogen phosphate (pH 2.1) with 0–40% acetonitrile. The amounts of glutamine and GABA formed from glutamic acid were determined under different reaction conditions.     

Effect of phosphorus deficiency on levels of phosphorus compounds in spirodela

When Spirodela plants are transferred to a phosphate-deficient medium, growth slows down immediately, and ceases after 14 days. During this time, inorganic phosphate content falls from 30 to 0.7 μmoles/g fresh weight of tissue, phosphate ester content from 3.5 to 0.6 μmoles/g, phospholipid content from 3.5 to 1.2 μmoles/g, and residual phosphate (mainly RNA) content from 7.5 to 2.0 μmoles/g. Relative proportions of the various phosphate esters, and relative proportions of the various phospholipids, are not markedly affected by phosphate deficiency. Turnover rates of phosphate esters are somewhat higher in phosphate-deficient tissue. In control tissue, inorganic phosphate is present in 2 pools; a metabolic (12%) and a non-metabolic pool (88%). In phosphate-deficient tissues, most of the inorganic phosphate (>90%) is in the metabolic pool. Non-metabolic phosphate is presumably stored in the vacuole, and is not readily accessible to the tissue, so that growth normally occurs at the expense of external phosphate. During deficiency, growth is limited by the rate at which phosphate can be transported through the tonoplast and tissue to the growing point. Growth ceases when the supply of non-metabolic phosphate is exhausted. Metabolic phosphate is presumably located in the cytoplasm: it can not be used for growth. Nor can the plant respond to deficiency by making some phosphorus compounds at the expense of others. In this respect, phosphorus deficiency and nitrogen deficiency are dissimilar.