Study on measurement of δ-aminolevulinic acid in plasma by high-performance liquid chromatography

A method for the determination of δ-aminolevulinic acid in plasma of lead-exposed workers by high-performance liquid chromatography with fluorescence detection of a fluorescent δ-aminolevulinic acid derivative (2-methylidineamino-3,5-diacetyl-4,6-dimethylpropionic acid) was established. The detection limit of δ-aminolevulinic acid in plasma was 0.01 μg/ml at a signal-to-noise ratio of 5:1. A linear correlation was obtained between the amounts of δ-aminolevulinic acid injected from 0.01 to 0.5 μg/ml (r = 0.999). The recovery of 0.05 and 0.1 μg/ml of δ-aminolevulinic acid added to plasma with various concentrations of δ-aminolevulinic acid in plasma ranged from 80.0 to 100.8%. This method, combined with the use of an automatic sampler, should facilitate the routine measurement of δ-aminolevulinic acid in plasma.     

Determination of R- and S-3-methyl-2-oxopentanoate enantiomers in human plasma: suitable method for label enrichment analysis

A sensitive method for the determination of S- and R-3-methyl-2-oxopentanoate enantiomers (KMV, (α-keto-β-methylval-erate) in physiological fluids suitable for isotope enrichment analysis is described: after extraction with acid, 2-oxo acids are separated from interfering amino acids by cation-exchange chromatography. Reductive amination of the branched-chain 2-oxo acids by use of - leucine dehydrogenase yields the corresponding -amino acids. -Isoleucine and -alloisoleucine which are formed from S- and R-3-methyl-2-oxopentanoate, respectively, are then quantified by amino acid analysis. The method was used for determination of the R-/S-3-methyl-2-oxopentanoate ratio in plasma of healthy subjects and patients with diabetes mellitus and maple syrup urine disease. Applicability for gas chromatographic-mass spectrometric analysis of ¹³C-label enrichment in plasma S-3-methyl-2-oxopentanoate is demonstrated.     

Rapid and sensitive step gradient assays of glutamate, glycine, taurine and γ-aminobutyric acid by high-performance liquid chromatography–fluorescence detection with o-phthalaldehyde–mercaptoethanol derivatization with an emphasis on microdialysis samples

We developed a rapid step-gradient HPLC method for determination of glutamate, glycine and taurine, and a separate method for determination of γ-aminobutyric acid (GABA) in striatal microdialysates. The amino acids were pre-column derivatized with o-phthalaldehyde–2-mercaptoethanol by using an automated refrigerated autoinjector. Separation of the amino acids was established with a non-porous ODS-II HPLC column, late-eluting substances were washed out with a one-step low-pressure gradient. Concentrations of the amino acids were determined with a fixed-wavelength fluorescence detector. The detection limit for GABA was 80 fmol in a 15 μl sample, detection limits for glutamate, glycine and taurine were not determined because their concentrations in striatal perfusates were far above their detection limits. Total analysis time was less than 12 min, including the wash-out step. The methods described are relatively simple, sensitive, inexpensive, and fast enough to keep up with the microdialysis sampling.     

Simple high-performance liquid chromatographic method for the determination of tocotrienols in human plasma

A simple high-performance liquid chromatographic method using fluorescence detection was developed for the determination of vitamin E especially δ-, γ- and α-tocotrienols in human plasma. The method entailed direct injection of plasma sample after deproteinization using a 3:2 mixture of acetonitrile–tetrahydrofuran. The mobile phase comprised 0.5% (v/v) of distilled water in methanol. Analyses were run at a flow-rate of 1.5 ml/min with the detector operating at an excitation wavelength of 296 nm and emission wavelength of 330 nm. This method is specific and sensitive, with a quantification limit of approximately 40, 34 and 16 ng/ml for α-, γ- and δ-tocotrienol, respectively. The mean absolute recovery values were about 98% while the within-day and between-day relative standard deviation and percent error values of the assay method were all less than 12.0% for α-, γ- and δ-tocotrienol. The calibration curve was linear over a concentration range of 40–2500, 30–4000 and 16–1000 ng/ml for α-, γ- and δ-tocotrienol, respectively. Application of the method in a bioavailability study for determination of the above compounds was also demonstrated.     

Simultaneous determination of mevalonate and 7α-hydroxycholesterol in human plasma by gas chromatography—mass spectrometry as indices of cholesterol and bile acid biosynthesis

A very sensitive and specific method for the simultaneous determination of mevalonate and 7α-hydroxycholesterol in human plasma is described. The assay is based on isotope dilution mass spectrometry: the extracts from plasma were treated with benzylamine followed by dimethylethylsilylimidazole, then the resulting dimethylethylsilyl ether derivatives of mevalonylbenzylamide and 7α-hydroxycholesterol were determined by gas chromatography—mass spectrometry using high-resolution selected-ion monitoring. Simple regression analysis revealed significant correlations between the plasma level of mevalonate and the hepatic activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (EC 1.1.1.34) (r = 0.83, P < 0.01) and between the plasma level of free 7α-hydroxycholesterol and the hepatic activity of cholesterol 7α-hydroxylase (EC 1.14.13.7) (r = 0.76, P < 0.05).     

Determination of estramustine and its 17-keto metabolite in plasma by high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of estramustine and its 17-keto metabolite in plasma. The assay involves extraction of the compounds into hexane from plasma buffered to pH 9.0, the residue obtained by evaporation of the hexane extract is dissolved in the mobile phase hexane—ethanol (92.5:7.5) with HPLC analysis performed on a 5-μm silica gel column using a fluorescence detector with excitation at 195 nm and emission at wavelengths greater than 250 nm. The overall recoveries and limits of sensitivity for estramustine and the 17-keto metabolite are 74.7% and 40 ng/ml of plasma and 85.1% and 50 ng/ml of plasma, respectively. The method was used to obtain plasma concentration—time profiles in three subjects with prostatic cancer following oral administration of a single 7 mg/kg dose of estramustine phosphate.     

Fatty Acid Production from Amino Acids and α-Keto Acids by Brevibacterium linens BL2

Low concentrations of branched-chain fatty acids, such as isobutyric and isovaleric acids, develop during the ripening of hard cheeses and contribute to the beneficial flavor profile. Catabolism of amino acids, such as branched-chain amino acids, by bacteria via aminotransferase reactions and α-keto acids is one mechanism to generate these flavorful compounds; however, metabolism of α-keto acids to flavor-associated compounds is controversial. The objective of this study was to determine the ability of Brevibacterium linens BL2 to produce fatty acids from amino acids and α-keto acids and determine the occurrence of the likely genes in the draft genome sequence. BL2 catabolized amino acids to fatty acids only under carbohydrate starvation conditions. The primary fatty acid end products from leucine were isovaleric acid, acetic acid, and propionic acid. In contrast, logarithmic-phase cells of BL2 produced fatty acids from α-keto acids only. BL2 also converted α-keto acids to branched-chain fatty acids after carbohydrate starvation was achieved. At least 100 genes are potentially involved in five different metabolic pathways. The genome of B. linens ATCC 9174 contained these genes for production and degradation of fatty acids. These data indicate that brevibacteria have the ability to produce fatty acids from amino and α-keto acids and that carbon metabolism is important in regulating this event.     

Efficient assay for the determination of atenolol in human plasma and urine by high-performance liquid chromatography with fluorescence detection

An efficient method for the determination of atenolol in human plasma and urine was developed and validated. α-Hydroxymetoprolol, a compound with a similar polarity to atenolol, was used as the internal standard in the present high-performance liquid chromatographic analysis with fluorescence detection. The assay was validated for the concentration range of 2 to 5000 ng/ml in plasma and 1 to 20 μg.ml in urine. For both plasma and urine, the lower limit of detection was 1 ng/ml. The intra-day and inter-day variabilities for plasma samples at 40 and 900 ng/ml, and urine samples at 9.5 μg/ml were <3% (n=5).     

Ability of Thermophilic Lactic Acid Bacteria To Produce Aroma Compounds from Amino Acids

Although a large number of key odorants of Swiss-type cheese result from amino acid catabolism, the amino acid catabolic pathways in the bacteria present in these cheeses are not well known. In this study, we compared the in vitro abilities of Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, and Streptococcus thermophilus to produce aroma compounds from three amino acids, leucine, phenylalanine, and methionine, under mid-pH conditions of cheese ripening (pH 5.5), and we investigated the catabolic pathways used by these bacteria. In the three lactic acid bacterial species, amino acid catabolism was initiated by a transamination step, which requires the presence of an α-keto acid such as α-ketoglutarate (α-KG) as the amino group acceptor, and produced α-keto acids. Only S. thermophilus exhibited glutamate dehydrogenase activity, which produces α-KG from glutamate, and consequently only S. thermophilus was capable of catabolizing amino acids in the reaction medium without α-KG addition. In the presence of α-KG, lactobacilli produced much more varied aroma compounds such as acids, aldehydes, and alcohols than S. thermophilus, which mainly produced α-keto acids and a small amount of hydroxy acids and acids. L. helveticus mainly produced acids from phenylalanine and leucine, while L. delbrueckii subsp. lactis produced larger amounts of alcohols and/or aldehydes. Formation of aldehydes, alcohols, and acids from α-keto acids by L. delbrueckii subsp. lactis mainly results from the action of an α-keto acid decarboxylase, which produces aldehydes that are then oxidized or reduced to acids or alcohols. In contrast, the enzyme involved in the α-keto acid conversion to acids in L. helveticus and S. thermophilus is an α-keto acid dehydrogenase that produces acyl coenzymes A.     

High-performance liquid chromatographic determination of α-keto acids in human urine and plasma

A high-performance liquid chromatographic method has been developed for the determination of α-keto acids in human urine and plasma. These acids were prepurified using a column of hydrazide gel and derivatized with o-phenylenediamine into 2-quinoxalinol derivatives, which were extracted into ethyl acetate. The 2-quinoxialinol derivatives were separated by reversed-phase paired-ion chromatography using a 250 × 4 mm-i.d. column packed with LiChrosorb RP-8 (5 μm). This method is sensitive, selective, and reproducible. The α-keto acids in urine and plasma from normal individuals were determined.     

Role of Aminotransferase IlvE in Production of Branched-Chain Fatty Acids by Lactococcus lactis subsp. lactis

The objective of this study was to determine the role of a lactococcal branched-chain amino acid aminotransferase gene, ilvE, in the production of branched-chain fatty acids. Lactococcus lactis subsp. lactis LM0230 and an ilvE deletion mutant, JLS450, produced branched-chain fatty acids from amino and α-keto acids at levels above α-keto acid spontaneous degradation and the fatty acids' flavor thresholds. The deletion mutant produced the same amounts of branched-chain fatty acids from precursor amino acids as did the parent. This was not the case, however, for the production of branched-chain fatty acids from the corresponding precursor α-keto acids. The deletion mutant produced a set of fatty acids different from that produced by the parent. We concluded from these observations that ilvE plays a role in the specific type of fatty acids produced but has little influence on the total amount of fatty acids produced by lactococci.     

An enzyme-linked immunosorbent assay for 6-keto PGF

An enzyme-linked immunosorbent assay for 6-keto prostaglandin F_1α, a stable metabolite of prostacyclin, has been developed. The assay allows quantitation of 6-keto PGF_1α in the range 1–200 pg/0.1 ml and shows very low cross reactivity to nine other prostaglandins. Dose dependent stimulation by thrombin of 6-keto PGF_1α formation in human endothelial cells in culture has been used to verify the assay. Quantitation by the enzyme linked immunosorbent assay agrees closely with determination by radioimmunoassay.     

Validation of the determination of amino acids in plasma by high-performance liquid chromatography using automated pre-column derivatization with o-phthaldialdehyde

A sensitive and reproducible fully automated method for the determination of amino acids in plasma based on reversed-phase high-performance liquid chromatography and o-phthaldialdehyde pre-column derivatization is described. A 5-μm Spherisorb ODS 2 column (125 × 3 mm I.D.) was selected for routine determination. Over 40 physiological amino acids could be determined within 49 min (injection to injection) and 48 samples could be processed unattended. The coefficients of variation for most amino acids in plasma were below 4%. We were also able to measure trace amounts of amino acids in plasma normally not detected in a routine analysis. The results obtained with the method described compared favourably with those of conventional amino acid analysis (r = 0.997) and were in excellent agreement with those of other laboratories (r = 0.999).     

Electrophysiological evidence for detection and discrimination of pheromonal bile acids by the olfactory epithelium of female sea lampreys (<Emphasis Type="Italic">Petromyzon marinus</Emphasis>)

Electro-olfactograms were used to determine sensitivity and specificity of olfactory organs of female sea lampreys (Petromyzon marinus) to four bile acids: 3-keto petromyzonol sulfate and 3-keto allocholic acid from spermiating males and petromyzonol sulfate and allocholic acid from larvae. Spermiating male bile acids are thought to function as a mating pheromone and larval bile acids as a migratory pheromone. The response threshold was 10^–12 mol l^–1 for 3-keto petromyzonol sulfate and 10^–10 mol l^–1 for the other bile acids. At concentrations above 10^–9 mol l^–1, the sulfated bile acids showed almost identical potency, as did the non-sulfated bile acids. The two sulfated bile acids were more potent than the two non-sulfated ones. In addition, 3-keto petromyzonol sulfate and water conditioned with spermiating males induced similar concentration-response curves and response thresholds. Cross-adaptation experiments demonstrated that the sulfated and non-sulfated bile acids represent different odors to the olfactory epithelium of females. Further exploration revealed that 3-keto petromyzonol sulfate represents a different odor than petromyzonol sulfate, while 3-keto allocholic acid and allocholic acid represent the same odor. Results indicate that male-specific bile acids are potent and specific stimulants to the female olfactory organ, supporting the previous hypothesis that these bile acids function as a pheromone.Abbreviations 3kACA 3-keto allocholic acid - 3kPZS 3-keto petromyzonol sulfate - ACA allocholic acid - ANOVA analysis of variance - ELISA enzyme-linked immunosorbent assay - EOG electro-olfactogram - PIR percent initial response - PZS petromyzonol sulfate - SMW spermiating male washings     

Simultaneous determination of serum retinol and α- and γ-tocopherol levels in type II diabetic patients using high-performance liquid chromatography with fluorescence detection

This paper presents a simple reversed-phase high-performance liquid chromatographic method for the simultaneous determination of retinol, and α- and γ-tocopherols in human serum using a fluorescence detector. For chromatographic separation a binary gradient was used: phase A; acetonitrile–butanol (95:5); phase B; water, at a flow-rate of 1.5 ml/min. Serum retinol, and α- and γ-tocopherol levels were measured in patients with non-insulin-dependent diabetes mellitus. Small sample requirement, good reproducibility and sensitivity make this method useful for the determination of the serum levels of these compounds in patients with diabetes mellitus.     

High-performance liquid chromatographic assay for a new fasciolicide agent, αBIOF10, in biological fluids

This paper describes a high-performance liquid chromatographic method with ultraviolet absorbance detection at 304 nm for the determination of 6-chloro-5-(1-naphthyloxy)-2-methylthio benzimidazole (αBIOF10) — a new fasciolicide agent — and its sulphoxide (SOαBIOF10), in plasma and urine. It requires 2 ml of biological fluid, an extraction using Sep-Pak cartridges, and methanol for drug elution. Analysis is performed on a μBondapak C₁₈ (10 μm) column, using methanol–acetonitrile–water (40:30:30, v/v) as the mobile phase. Results showed that the assay is sensitive: 12 ng/ml for αBIOF10 and SOαBIOF10 in plasma and 3.6 ng/ml for both compounds in urine. The response was linear between 0.195 and 12.5 μg/ml. Maximum intra-day coefficient of variation was 5.3%. Recovery obtained was 97.8% for both αBIOF10 and SOαBIOF10. In urine, recovery was 99.6% and 93.1% for αBIOF10 and SOαBIOF10 respectively. The method was used to perform a preliminary pharmacokinetic study in two sheep and was found to be satisfactory.     

Simultaneous determination of norethisterone and six metabolites in human plasma by capillary gas chromatography with mass-selective detection

A method for the simultaneous determination of norethisterone (NET) and six metabolites in human plasma by capillary gas chromatography-mass-selective detection (GC-MS) is described. The compounds are determined in plasma after enzymatic hydrolysis. After addition of norgestrel as the internal standard, the compounds are extracted from plasma at pH 5 using an Extrelut column and elution with dichloromethane. After evaporation, the compounds are converted into bistrimethylsilyl derivatives which are determined by gas chromatography using a mass-selective detector at m/z 429 for the two dihydro-NET (5β-NET and 5α-NET), m/z 431 for the four tetrahydro-NET (3α,5α-NET, 3α,5β-NET, 3β,5β-NET and 3β,5α-NET), m/z 442 for NET and m/z 456 for the internal standard. The reproducibility and accuracy of the method were found suitable over the range of concentrations between 0.50 and 8 ng/ml for NET, and metabolites except for 5α-dihydro-NET (between 1 and 8 ng/ml). The method was applied to clinical samples.     

Improved high-performance liquid chromatographic determination with amperometric detection of α-amanitin in human plasma based on its voltammetric study

A sensitive and simple method is described for the selective determination in human plasma of α-amanitin, the most poisonous and prevalent toxin in the lethal fungi of species Amanita, using high-performance liquid chromatography with amperometric detection. After an extraction of plasma with disposable C₁₈ silica cartridges, the extracts were separated by isocratic reversed-phase chromatography using a macroporous poly(styrene—divinylbenzene) column and a mobile phase of 0.05 M phosphate buffer—acetonitrile (91:9) at the apparent pH of 9.5. Amperometric detection was performed by applying an oxidation potential as low as +350 mV (vs. Ag/AgCl) to a glassy carbon electrode, in a thin-layer flow-cell. The linear range for α-amanitin was 3–200 ng/ml, and the relative limit of detection in plasma was 2 ng/ml at a signal-to-noise ratio of 2. The intra-assay precision was evaluated at levels of 10 and 200 ng/ml; the coefficients of variation were 4.5 and 2.6% (n=5), respectively. Inter-assay coefficients of variation were 6.5 and 4.2% (n=5) for the same concentrations of toxin. These analytical conditions have been chosen on the basis of a preliminary in batch cyclic voltammetric investigation of α-, β- and γ-amanitins, which has allowed their oxidation process to be clarified and the pH dependence of their oxidation potentials to be determined. All three amanitins are oxidized at the same potential values, and adsorption onto the electrode surface of both reactant and products was found in all cases. This adsorption did not affect the signal recorded for α- and γ-amanitins at the amperometric detector, and for β-amanitin a stronger adsorption for the anodic product was found, which leads to a marked positive shift of the potential required for the oxidation of this isomer in the amperometric detector cell.     

Uptake of α-Ketoglutarate by Citrate Transporter CitP Drives Transamination in Lactococcus lactis

Transamination is the first step in the conversion of amino acids into aroma compounds by lactic acid bacteria (LAB) used in food fermentations. The process is limited by the availability of α-ketoglutarate, which is the best α-keto donor for transaminases in LAB. Here, uptake of α-ketoglutarate by the citrate transporter CitP is reported. Cells of Lactococcus lactis IL1403 expressing CitP showed significant levels of transamination activity in the presence of α-ketoglutarate and one of the amino acids Ile, Leu, Val, Phe, or Met, while the same cells lacking CitP showed transamination activity only after permeabilization of the cell membrane. Moreover, the transamination activity of the cells followed the levels of CitP in a controlled expression system. The involvement of CitP in the uptake of the α-keto donor was further demonstrated by the increased consumption rate in the presence of l-lactate, which drives CitP in the fast exchange mode of transport. Transamination is the only active pathway for the conversion of α-ketoglutarate in IL1403; a stoichiometric conversion to glutamate and the corresponding α-keto acid from the amino acids was observed. The transamination activity by both the cells and the cytoplasmic fraction showed a remarkably flat pH profile over the range from pH 5 to pH 8, especially with the branched-chain amino acids. Further metabolism of the produced α-keto acids into α-hydroxy acids and other flavor compounds required the coupling of transamination to glycolysis. The results suggest a much broader role of the citrate transporter CitP in LAB than citrate uptake in the citrate fermentation pathway alone.     

Minimal CK2 activity required for yeast growth

Protein kinase CK2 is essential for the growth of Saccharomyces cerevisiae. Yeast cells that lack the functional genes coding for both the catalytic subunits of protein kinase CK2 can grow only if they are complemented by exogenous cDNAs coding for this subunit. A series of deletion mutants of CK2α from Xenopus laevis was constructed. These mutants that have carboxyl end deletions yield a CK2α product that varies over four orders of magnitude in its capacity to phosphorylate casein in vitro. Complementation of yeast RPG41-1a, a mutant defective in CKA1 and CKA2 genes, with wild-type X. laevis CK2α and with cDNAs coding for truncated CK2α having amino acids 1–328 and 1–327 resulted in cells that grew in gal-minimal media at 30 ^∘C as well as the cells harboring the yeast CKA2 gene. However, the growth was significantly diminished when cells were complemented with X. laevis CK2α containing 1–326 amido acids. This mutant has 0.6% of the catalytic activity of the wild-type enzyme. Yeast cells that expressed CK2α 1–324 and 1–323 which have 10-and 100-fold less activity, respectively, were not able to grow. The growth of cells containing the CK2α 1–326 mutant was very sensitive to temperature, and minimal growth was observed at 37 ^∘C. This mutant was also more sensitive to UV radiation but was not significantly affected by 0.4 M NaCl.Both authors contributed equally to this work