The problem of 4,5-dioxovaleric acid as a precursor of 5-aminolevulinic acid and chlorophyll in green algae

During chlorophyll biosynthesis, 4,5-dioxovaleric acid is supposed to be an intermediate between 2-oxoglutarate and 5-aminolevulinic acid. Although the occurrence of 4,5-dioxovaleric acid in cells and culture filtrates of unicellular green algae treated with levulinic acid has been previously reported, improvement of the analytical method now shows that 4,5-dioxovaleric acid does not occur in these organisms but arises due to interference of 5-aminolevulinic acid in 4,5-dioxovaleric acid analysis. The significance of 4,5-dioxovaleric acid as a free metabolite in 5-aminolevulinic acid biosynthesis must therefore be reconsidered.     

Chemical synthesis of 4,5-dioxovaleric acid and its nonenzymatic transamination to 5-aminolevulinic acid

4,5-Dioxovaleric acid (DOVA) was synthesized from 5-bromolevulinic acid via formation of the pyridinium bromide of 5-bromolevulinic acid, followed by nitrone formation with p-nitrosodimethylaniline, and hydrolysis of the nitrone to yield DOVA. Partial purification of DOVA was obtained by passage of the reaction mixture through a cation exchange column. DOVA was identified by paper electrophoresis and by a specific fluorometric assay. DOVA was nonenzymatically transaminated to 5-aminolevulinic acid (ALA) with glycine serving as the amino donor. Other compounds tested were less effective amino donors. Glyoxylic acid was identified as a reaction product by paper electrophoresis and a specific calorimetric test. ALA was identified by paper electrophoresis, paper chromatography of a pyrrole derivative, reaction with Ehrlich reagent, and by its enzymatic conversion by a barley extract to porphobilinogen and uroporphyrin. The nonenzymatic transamination was inhibited by Tris and was stimulated by high pH. The existence of this nonenzymatic activity is discussed in relation to previous reports of dova transaminase activity in cell extracts.     

Determination of 5-aminolevulinic acid in blood plasma, tissues and cell cultures by high-performance liquid chromatography with electrochemical detection

A simple and sensitive method for determining 5-aminolevulinic acid (ALA) in biological samples is described. ALA is derivatized with o-phthaldehyde to give a compound with favorable properties for high-performance liquid chromatography with electrochemical detection. The method does not require extensive pretreatment of the samples and its detection limit is in the range of 1 pmol/20 μl injection. This method was applied to the determination of plasma ALA from normal and lead-exposed subjects, where 0.26±0.08 μM (n=30) and 2.6±0.75 μM (n=30), respectively were found. We also determined ALA in rat tissues, namely liver and brain, and the uptake of ALA by cultured fibroblasts and hepatocytes to illustrate the diversified applicability of the method.     

The role of 4,5-dioxovaleric acid in porphyrin and vitamin B12 formation by clostridia

4,5-Dioxovalerate, which has been proposed as an intermediate in the newly discovered so-called C₅ pathway that leads from L-glutamate to δ-aminolevulinate, strongly inhibits uroporphyrin formation from δ-aminolevulinate in cells of $\text{Clostridium}\text{tetanomorphum}$ and in cell-free extracts of this organism, in spite of the presence of L-alanine: 4,5-dioxovalerate aminotransferase (aminolevulinate aminotransferase, EC 2.6.1.43). The interference by 4,5-dioxovalerate with porphyrin formation is due to strong inhibition of δ-aminolevulinate dehydratase (EC 4.2.1.24). Since 4,5-dioxovalerate hence effectively prevents the operation of the reaction sequence from L-glutamate to porphyrin, it is concluded that 4,5-dioxovalerate does not function as a physiological δ-aminolevulinate precursor.     

Quantification of urinary 5-aminolevulinic acid by gas chromatography-mass spectrometry

In this report, we describe a method for the specific quantification of urinary 5-aminolevulinic acid. It is based on gas chromatographic mass spectrometric measurement of the trimethylsilyl ester of the ethylacetoacetate pyrrole derivative of 5-aminolevulinic acid. Selected ion monitoring (SIM) was used for quantification using 3-hydroxymyristic acid as an internal standard. The detection limit of this method was 0.1 nmol/ml. This method was applied to the determination of urinary 5-aminolevulinic acid from a tyrosinemia type I patient and normal subjects, and 21.4 mmol/mol creatinine and 0.54+/- 0.49 mmol/mol creatinine (n = 7), respectively, were detected. Less than 0.2 ml urine was sufficient for the determination of 5-aminolevulinic acid in healthy subjects.     

Bio-analysis of vinorelbine by high-performance liquid chromatography with fluorescence detection

A simple and selective procedure for the determination of vinorelbine, a new semi-synthetic vinca alkaloid, is presented. The method is based on ion-exchange high-performance liquid chromatography on normal-phase silica with fluorescence detection, combined with liquid—liquid extraction using diethyl ether for sample clean-up. The absence of endogenous interferences and the excellent chromatographic behaviour of vinca alkaloids provides accurate results even at low concentrations. The limit of determination in plasma is 1.5 μg/l (500-μl sample). Reproducible recoveries in urine were obtained if 10–50 μl of sample were processed supplemented with 500 μl of blank plasma.     

Analysis of perillic acid in plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A simple and sensitive isocratic high-performance liquid chromatographic (HPLC) method with UV detection for the quantitation of perillic acid, a major circulating metabolite of perillyl alcohol and d-limonene, in plasma is described. Sample preparation involved protein precipitation and subsequent transfer and dilution with 10 mM NaHCO₃. The mobile phase consisted of acetonitrile (36%) and 0.05 M ammonium acetate buffer pH 5.0 (64%). Separations were achieved on a C₁₈ column and the effluent monitored for UV absorption at the analytes' respective UV_max. Separation was excellent with no interference from endogenous plasma constituents. This method was found suitable for quantifying drug concentrations in the range of 0.25 to 200.0 μg/ml using a 0.05-ml plasma sample, and was used to study the plasma pharmacokinetics of perillic acid in mice.     

Measurement of hydroxyl radical-generated methane sulfinic acid by high-performance liquid chromatography and electrochemical detection

A liquid chromatographic (HPLC) method has been developed for direct quantitative determination of methane sulfinic acid (MSA) produced by hydroxyl radical oxidation of dimethyl sulfoxide. This method measures MSA directly by HPLC separation and electrochemical oxidation following rapid extraction from intact cells. MSA can be measured in tissue extracts at 0.04 nmol (equivalent to 2 microM). Using this technique, MSA production in paraquat-treated bean leaves is demonstrated. When compared with the widely used dye-binding technique, this method simplifies the preparation of the extract by eliminating two steps required in the dye-binding method: removal of interfering lipophilic compounds and the derivitization (color reaction) of the MSA.     

Determination of uric acid in human saliva by high-performance liquid chromatography with amperometric electrochemical detection

The aim of the present study is to establish a highly sensitive method for the determination of uric acid (UA) in human saliva. The monitoring of UA levels in less invasive biological samples such as saliva is suggested for the diagnosis and therapy of gout, hyperuricemia, and the Lesch-Nyhan syndrome, and for detecting such conditions as alcohol dependence, obesity, diabetes, high cholesterol, high blood pressure, kidney disease, and heart disease. Reversed-phase high-performance liquid chromatography with electrochemical detection (HPLC-ED) was employed for the determination of UA obtained by solid-phase extraction from saliva. To quantify UA, we compared the ED efficiencies of an amperometric ED (Ampero-ED) with a single electrode and a coulometric ED (Coulo-ED) with a multiple electrode array. The results showed that the detection limits (S/N=3) were 3 nM for Ampero-ED and 6 nM for Coulo-ED, and the linearity of the calibration curves of 60-6000 nM had correlation coefficients exceeding 0.999. In addition, the total analytical time was 10 min. In the sample preparation of UA in saliva, an Oasis MAX solid-phase cartridge was used. The recoveries of UA spiked at 0.6 and 3 microM in saliva were above 95% with a relative standard deviation (RSD) of less than 15%. Therefore, the present method may be used in the routine and diagnostic determination of UA in human saliva.     

Simultaneous determination of lysophospholipids by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatography (HPLC) procedure for the separation of choline lysophospholids including 1-acyl-lysophosphatidylcholines and 1-O-alkyl-lysophosphatidyl-cholines, like the lysoform of the platelet activating factor (2-lysoPAF), is described. The lysophospholipids are derivatized at the sn-2 position of the hydroxyl group by 7-diethylaminocoumarin-3-carbonylazide, which converts them into the corresponding carbamoyl derivatives. The derivatized compounds were well separated by reversed-phase HPLC and quantified by fluorimetric detection. This method shows a high sensitivity and allows the separation and quantification of mixtures of lysophospholipids at picomolar level. The method was applied to assay enzyme activities, like phospholipase A₂ and PAF-acetylhydrolase, on single phospholipids or their mixtures.     

Determination of plasma homocysteine by high-performance liquid chromatography with fluorescence detection

Severe homocystinemia is frequently associated with vascular disease while the pathological consequences of moderate or slightly elevated plasma homocysteine are unknown. Cobalamin and folate deficiencies may result in an elevation of plasma homocysteine. A sensitive and reproducible assay for total plasma homocysteine has been developed. The essential steps in the assay include (i) conversion of homocysteine disulfides to free homocysteine with borohydride reduction; (ii) conjugation of homocysteine with monobromobimane; (iii) separation of homocysteine-bimane from other plasma thiol-bimane adducts by reverse-phase high-performance liquid chromatography; and (iv) detection and quantitation of homocysteine-bimane by fluorometry. The method has a sensitivity of 4.4 pmol of homocysteine and is highly reproducible (intra- and interassay coefficients of variation = 4.97 and 4.53%, respectively). The mean concentration of total plasma homocysteine in nonfasting adult males (n = 12) and females (n = 12) was 15.8 (range, 7.0-23.7) and 16.5 nmol/ml (range, 8.6-20.7), respectively. Markedly elevated levels of homocysteine were found in patients with cobalamin and folate deficiency. Total plasma homocysteine represents approximately 4% of borohydride-generated thiol reactivity in the plasma of normal individuals.     

Simultaneous determination of tryptophan and its metabolites in mouse brain by high-performance liquid chromatography with fluorometric detection

A new method for the determination of tryptophan and its metabolites in a single mouse brain using high-performance liquid chromatography (HPLC) with fluorometric detection is described. Tryptophan, serotonin, 5-hydroxyindoleacetic acid, indoleacetic acid, and tryptophol were clearly separated by a C8 reverse-phase column. Tissue preparation is performed only to centrifuge homogenates of brain prior to the injection to HPLC. The sensitivity is in the range from 10 to 15 pg.     

Determination of physostigmine in plasma by high-performance liquid chromatography and fluorescence detection

Physostigmine (PHY) is an anticholinergic drug used in the treatment of neuromuscular disorders and organophosphate poisoning. We described a sensitive, accurate, and reproducible method for PHY determination in biological materials. The method utilized a liquid/liquid, ion pair extraction, normal phase HPLC separation, and fluorometric quantitation at 240 nm excitation and 360 nm emission wavelength. We used neostigmine as a stabilizing agent to protect PHY from degradation and dimethylphysostigmine as an internal standard. The peak-height ratio vs concentration was linear over a working range from 0.50 to 25.0 ng/ml of PHY in plasma. Sensitivity of the method was 100 pg/ml of plasma which was the limit of quantitative detection under the experimental conditions used. Precision of the method was evaluated using plasma spiked with two concentrations of PHY: 1.0 and 10.0 ng/ml. Intra-day coefficient of variation (CV) ranged from 3.8 to 5.3%, and inter-day CV ranged from 1.8 to 3.6% for the two levels. The average recovery was 92%. We applied the method to examine the stability of PHY in plasma stored at -15 and -80 degrees C. The data indicated that PHY can be stored at either temperature for 9 weeks without undergoing significant alterations.     

Simultaneous determination of alpha-lipoic acid and its reduced form by high-performance liquid chromatography with fluorescence detection

The simultaneous determination of alpha-lipoic acid (LA) and DHLA (reduced form of LA) was carried out by HPLC with fluorescence detection. DHLA in the sample was first labeled with ABD-F at room temperature for 10 min and then the LA was labeled with SBD-F at 50 degrees C for 1 h after conversion to DHLA using the reducing agent, TCEP. The resulting fluorophores, ABD-DHLA and SBD-DHLA, were separated by reversed-phase chromatography and detected at 510 nm (excitation at 380 nm). Both fluorophors were completely separated without any interference of endogenous thiols and disulfides in the sample and sensitively detected by fluorimetry. The proposed method was applied to the assay of the LA supplement and the determination in human plasma after the oral administration of LA tablets. The concentration (%) of LA in the tablet was reasonable to the stated amount. Furthermore, the result of a time course study in the plasma after the administration of LA did not differ from a previous report. Thus, the present method seems to be applicable to the simultaneous determination of LA and DHLA in various biological specimens.     

高效液相荧光法测定大鼠脑内氨基酸类神经递质方法的改良

目的 改良测定大鼠脑组织氨基酸类神经递质的反相高效液相色谱荧光法.方法 改良使用磷酸盐-甲醇-乙腈作为流动相,反相高效液相色谱洗脱,高丝氨酸作为内标,邻苯二甲醛柱前衍生和荧光检测器,检测大鼠大脑皮质、海马、纹状体、中脑、小脑和下丘脑6个脑区中天冬氨酸(Asp)、谷氨酸(Glu)、谷氨酰胺(G1n)、甘氨酸(Gly)、γ-氨基丁酸(GABA)和牛磺酸(Tau)6种氨基酸类神经递质含量.结果 6种氨基酸在20 min内洗脱完全,分离效果良好;在6.25～ 400 μmol/L浓度范围有较好的线性关系,其相关系数不低于0.99;6种氨基酸日内试验精密度范围为1.38％ ～7.59％;日间试验精密度为2.7％～8.68％;6种氨基酸回收率不低于80％.结论 改良后的反相高效液相色谱荧光法灵敏度较高、重复性好,能有效分离检测大鼠脑组织分区中氨基酸类神经递质含量.     

Determination of acetylsalicylic acid and salicylic acid in skin and plasma by high-performance liquid chromatography

This study describes a HPLC method to determine the concentrations of acetylsalicylic acid (ASA) and salicylic acid (SA) in human stratum corneum and in plasma. The stratum corneum layers for ASA/SA analysis were removed from three patients with postherpetic hyperalgesia treated with topical and oral aspirin. Blood samples were also collected from the same patients. Tape strippings were placed in acetonitrile and sonicated for 15 min. After centrifuging, aliquots of the supernatant were injected into the chromatograph. ASA and SA from plasma samples were extracted on Isolute C₈ columns. Due to interfering peaks in the tape samples, HPLC conditions were slightly different for tape and plasma samples. ASA and SA were separated on a LiChrospher 100 RP-18 column at 1 ml/min using a water–phosphate buffer (pH 2.5)–acetonitrile mobile phase (35:40:25, v/v/v). A linear response to quantities of ASA from 0.1 to 100 μg/cm² and of SA from 0.1 to 5 μg/cm² in tape and to quantities of ASA 0.1 to 2 μg/ml and 1 to 50 μg/ml was obtained and the recovery from tape and plasma samples was over 98%. The method is sensitive (0.1 μg/cm²) and specific enough to allow the determination of the drugs in the skin not only after topical but also after oral administration. A good sensitivity was also obtained in plasma (0.1 μg/ml) allowing study of the kinetics of ASA and SA in plasma after oral administration. Concentrations of ASA after topical administration were 100–200 times higher than after oral administration. Plasma levels of ASA and SA after oral administration were similar to those previously found. No ASA or SA were detected in plasma after topical ASA administration.     

Pharmacokinetics of ciprofloxacin in eels by high-performance liquid chromatography with fluorescence detection

The plasma kinetics of ciprofloxacin (CF) were investigated in the eels after administration by oral gavage and bath treatment. Plasma concentrations of CF were determined by high-performance liquid chromatography with fluorescence detection. The mean concentration time data after oral gavage of a single dose (10.0 mg/kg CF) and after bath treatment by exposure (10 microg/ml CF) to medicated water for 48 h were both best fitted by a one-compartment model. After oral gavage in eels, the half-time of absorption (T1/2Ka) was 0.10 h, the half-time of elimination (T1/2Ke) was 51.87 h, and the maximum plasma concentration (Cmax) was 0.4552 microg/ml at Tmax 0.88 h. After bath treatment, the (T1/2Ka) was 0.02 h, the (T1/2Ke) was 15.46 h, and the Cmax was 0.1175 microg/mL at Tmax 0.22 h.     

Rapid determination of nitrite by reversed-phase high-performance liquid chromatography with fluorescence detection

Measurement of nitrite and nitrate, the stable oxidation products of nitric oxide (NO), provides a useful tool to study NO synthesis in vivo and in cell cultures. A simple and rapid fluorometric HPLC method was developed for determination of nitrite through its derivatization with 2,3-diaminonaphthalene (DAN). Nitrite, in standard solution, cell culture medium, or biological samples, readily reacted with DAN under acidic conditions to yield the highly fluorescent 2,3-naphthotriazole (NAT). For analysis of nitrate, it was converted to nitrite by nitrate reductase, followed by the derivatization of nitrite with DAN to form NAT. NAT was separated on a 5-μm reversed-phase C₈ column (150×4.6 mm, I.D.) guarded by a 40-μm reversed-phase C₁₈ column (50×4.6 mm, I.D.), and eluted with 15 mM sodium phosphate buffer (pH 7.5) containing 50% methanol (flow-rate, 1.3 ml/min). Fluorescence was monitored with excitation at 375 nm and emission at 415 nm. Mean retention time for NAT was 4.4 min. The fluorescence intensity of NAT was linear with nitrite or nitrate concentrations ranging from 12.5 to 2000 nM in water, cell culture media, plasma and urine. The detection limit for nitrite and nitrate was 10 pmol/ml. Because NAT is well separated from DAN and other fluorescent components present in biological samples, our HPLC method offers the advantages of high sensitivity and specificity as well as easy automation for quantifying picomole levels of nitrite and nitrate in cell culture medium and biological samples.     

Quantitation of monohydroxy fatty acids by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic (HPLC) procedure for the separation of hydroxyeicosatetraenoic acids (HETEs) and hydroxyoctadecanoic acids (HODEs) after derivatization of the hydroxy group with 1-anthroylnitrile is described. Anthroyl esters of HETEs were separated from those of HODEs by reversed-phase HPLC. The positional isomers of the HETEs and HODEs were well separated by normal-phase HPLC. The fluorimetric HPLC method has a high sensitivity and naturally occurring HETEs can be quantitatively analyzed at the picomolar level. The amount of 5-HETE in A23187-stimulated polymorphonuclear leukocytes (PMNLs) was determined by the present method. PMNLs produced approximately 150 ng of 5-HETE per 10⁷ cells at 5 min stimulation. The amount of 5-HETE determined by fluorimetric detection was consistent with that determined by ultraviolet detection (235 nm).