In vivo, continuous and automatic monitoring of extracellular ascorbic acid by microdialysis and on-line liquid chromatography

A system for in vivo, automatic, continuous monitoring of organ extracellular ascorbic acid in anesthetized rat is described. This system involves microdialysis perfusion and a LC system equipped with an electrochemical detector. Microdialysate, eluted from a microdialysis probe implanted in the brain cortex or in the left ventricular myocardium of anesthetized rats was collected in the sample loop of an on-line injector for direct injection onto the LC system. This automated method provides a shortened sample processing time. This system was utilized to investigate the effect of cerebral ischemia on cortex extracellular ascorbic acid and the effect of myocardial ischemia on left ventricular myocardium extracellular ascorbic acid in anesthetized rats. Basal ascorbic acid concentrations in the cortex and left ventricular myocardium ranged from 9.7 to 15.4 μM (mean±S.D. 12.7±2.5 μM from the results of eight rats) and from 9.3 to 36.0 μM (mean±S.D., 24.3±8.9 μM from the results of twelve rats), respectively. Cerebral ischemia significantly elevated ascorbic acid levels in the cortex extracellular space, while myocardial ischemia did not significantly alter ascorbic acid levels in the left ventricular myocardium extracellular space.     

Rapid mass spectrometric analysis for ascorbate and related organic acids in small volumes of plasma for use in pediatric subjects

A gas chromatographic–mass spectrometric isotope dilution method was developed for analysis of ascorbate on 10 μl samples of plasma. This assay was reproducible (standard deviation of less than 4%) and gave values for plasma ascorbate content within 8% of our previously published gas chromatographic–mass spectrometric method. Non-specific sample preparation allowed other analytes to be determined on the same sample by adjusting data acquisition parameters and adding the appropriate internal standard. Analysis on 28 subjects fell within the expected range for plasma ascorbate 68±29 μm (11.9±5.0 μg/ml) and established a normal range for plasma threonate of 28.1±2.4 μm (3.8±0.4 μg/ml).     

Simultaneous high-performance liquid chromatographic determination of ascorbic acid and dehydroascorbic acid in biological samples

The ascorbic acid (AA)—dehydroascorbic acid redox couple is an important component of many biological systems, and various physiological roles have been described for this vitamin. Simultaneous measurement of both AA and dehydroascorbate using high-performance liquid chromatography (HPLC) has proven difficult owing to detection problems. A simple, single-step HPLC assay for the simultaneous detection of both AA and dehydroascorbate was developed without the burden of derivatization of either compounds. This has proven to be a reliable technique and should be applicable to a wide variety of biological samples.     

Liquid chromatographic determination of hydroxyproline in tissue samples

We describe a reversed-phase assay of hydroxyproline in rat lung tissue using sarcosine for the internal standard and pre-injection reaction with both o-phthalaldehyde (OPA) and 9-fluorenylmethylchloroformate (FMOC). Intra-assay variability in the concentration range of 25-500 microM hydroxyproline was less than 1%. Normal rat (left) lung was found to have a hydroxyproline content of 1.08+/-0.18 mg/lung. This ability to measure minute amounts of hydroxyproline is being applied to the measure of collagen and pathological fibrosis.     

Enzymatic measurement of ethanol or NAD in acid extracts of biological samples

An enzymatic method for the measurement of ethanol has been developed to permit analyses with unneutralized acid extracts of blood, liver, cell suspensions, or other biological materials. Components of the assay mixture include NAD, yeast alcohol dehydrogenase, tris(hydroxymethyl)aminomethane (Tris), and lysine. Tris is a trapping agent for the reaction product, acetaldehyde. Lysine is used to maintain the pH at 9.7 where oxidation of ethanol is quantitative and most rapid, even when as much as 0.2 ml of 0.5 n HClO₄ is added. Lysine also causes the reaction to be 2 to 4 times faster than it is when either glycine or 2-amino-2-methyl-1-propanol is used as the buffer. The assay is linear up to an ethanol concentration of 0.125 mm in the reaction mixture and is complete by 4 min. By substituting ethanol for NAD in the reagents, the assay performs equally well in measuring NAD.     

A method for the estimation of amino acid radioactivity in biological samples

A method for quantitative estimation of total radioactivity present in the free amino acid fraction of tissue samples has been described. Samples deproteinized with cold acetone were extracted, in acidic medium, with ethyl (peroxide free); after centrifugation, the aqueous phase was used for amino acid derivatization at 40°C for 15 h with 1-flouro-2,4-dinitrobenzene in bicarbonate-buffered medium. Aliquots of the derivatized samples were acidified and extracted twice again with ethyl ether. The combined organic phases were placed in glass scintilation vials, dried, and used for the determination of its radiactivity, corresponding to the radioactivity present in the free amino acid fraction of the sample. Deproteinized samples of rat blood plasma, as well as hen egg white and yolk were tested after addition of known quantities of ¹⁴C-labelled amino acids or glucose, for validation of the method. No glucose radioactivity was found in any of the extracted samples. All radioactivity added to the samples in the form of ¹⁴C-labelled alanine, glutamic acid, leucine and phenylalanine was quantitatively recovered in the derivatized fraction; only a fraction of arginine radioactivity was recovered.     

structure of ascorbic acid and its biological function

Ascorbic acid/isoascorbic acid are present as radicals at physiological pH with the unpaired electron located in the C(4) region. Since a distinction can be made between both types of radicals, the electron spin resonance technique can be used for discrimination between the epimers of vitamin C. The radical has a cyclic side-chain structure which is formed by the hydrogen bond C(3)-O^– ... HO-C(6) ( 2.7 kJ) and which engulfs Na⁺ or K⁺ in the case of the ascorbyl or the isoascorbyl radical, respectively. The radicals Na-ASC and K-Iso-ASC are electroneutral. Red. glutathione affects both types of radicals by restoring the original electronic configuration at C(4) without changing the electroneutral bicyclic structure. In this way, the mobile carriers Na-ASC and K-Iso-ASC can transport Na⁺ and K⁺ across membranes. Its highest efficiency is around 37 C and pH 7, that is, at physiological values. The biological importance of the side chain of vitamin C is outlined and a possible transport mechanism proposed.     

Different efficacy of iodoacetic acid and N-ethylmaleimide in high-performance liquid chromatographic measurement of liver glutathione

The widely used high-performance liquid chromatography (HPLC) procedure to determine glutathione in biological samples utilizing iodoacetic acid as thiol quenching agent and 1-fluoro-2,4-dinitrobenzene for derivatization has been modified regarding tissue sample processing and storage of the working solutions. The modified procedure compared with the original method reduces artifactual oxidation in rat liver glutathione measurement (1.47±0.8% vs. 2.84±0.69%, respectively). In both HPLC procedures, an increase in artifactual oxidation was found in both standard glutathione solutions and hepatic samples when N-ethylmaleimide instead of iodoacetic acid was used for thiol trapping.     

Fluorometric measurement of tin-protoporphyrin in biological samples

Sn-protoporphyrin is a potent competitive inhibitor of heme oxygenase, can suppress neonatal and other forms of hyperbilirubinemia in laboratory animals, and represents a potential new approach to the treatment of neonatal jaundice in humans. In order to study the disposition of Sn-protoporphyrin in vivo we have developed a sensitive fluorometric method for the quantitation of this metalloporphyrin in biological samples. The method is sensitive to concentrations as low as 0.01 nmol/ml, and is specific for Sn-protoporphyrin even in the presence of other porphyrins such as protoporphyrin.     

Liquid chromatography for iothalamate in biological samples

We have previously reported an iothalamate assay for the assessment of the glomerular filtration rate (GFR) that required a long column equilibration time and 22 min run time per sample. We now report a simpler assay that requires a run time of only 5.5 min and is more precise and accurate than the earlier technique. The mobile phase consisted of methanol-acetonitrile-50 mM sodium monobasic phosphate (10:5:85, v/v) at pH 4.4, pumped at a rate of 1.5 ml/min on a C(18) reversed-phase column. Samples of plasma and urine were deproteinized with 1 volume of 4% perchloric acid or 9 volumes of 2% perchloric acid, respectively. No internal standard was used. The diode array detection system collected absorbance at 240 nm and the peak height areas of iothalamate were determined. The iothalamate peak appeared at 3.5 min. Detector response was linear over the range tested (10-2000 microg/ml). Within-run precision was <3% for both plasma and urine and accuracy was 96-102%. Between-day precision for plasma and urine analyses were <7%. The recovery of iothalamate in urine and plasma were 102% and 91%, respectively. There was excellent thermal and pH stability of iothalamate. No interference was found with para-amino hippuric acid (PAH) or N-acetyl PAH, which can be simultaneously assayed, if desired.     

Solid-phase extraction and liquid chromatographic quantitation of quinfamide in biological samples

This paper describes a high-performance liquid chromatographic method for the assay of quinfamide and its main metabolite, 1-(dichloroacetyl)-1,2,3,4,-tetrahydro-6-quinolinol, in plasma, urine and feces. It requires 1 ml of biological fluid, an extraction using Sep-Pack cartridges and acetonitrile for drug elution. Analysis was performed on a CN column (5 μm) using water–acetonitrile–methanol (40:50:10) as a mobile phase at 269 nm. Results showed that the assay was linear in the range between 0.08 and 2.0 μg/ml. The limit of quantitation was 0.08 μg/ml. Maximum assay coefficient of variation was 14%. Recovery obtained in plasma, urine and feces ranged from 82% to 98%.     

Rapid,fluorimetric-liquid chromatographic determination of malondialdehyde in biological samples

Capillary zone electrophoresis was employed for the determination of lactate using end-column amperometric detection at a carbon fiber bundle microdisk electrode. The optimum conditions of separation and detection are 3.6 x 10(-3) mol/l Na(2)HPO(4)-1.4 x 10(-3) mol/l NaH(2)PO (pH 7.2) for the buffer solution, 18 kV for the separation voltage and 1.60 V versus the saturated calomel electrode for the detection potential. The limit of detection is 7.6 x 10(-7) mol/l or 1.7 fmol (S/N=3) and the linear range is 1.7 x 10(-6)-8.2 x 10(-4) mol/l for the injection voltage of 6 kV and injection time of 5 s. The RSD is 1.8% for the migration time and 3.3% for the electrophoretic peak current. The method was applied to the determination of lactate in human saliva. The recovery of the method is between 95 and 109%.     

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.     

High-performance liquid chromatographic measurement of urocanic acid isomers and their ratios in naturally light-exposed skin and naturally shielded skin

We have developed methods for sampling and extraction of trans-urocanic acid and cis-urocanic acid from human skin, and subsequent high-performance liquid chromatographic measurement of these isomers. Sampling involves applying cellophane adhesive tape to the skin for 10 s. Urocanic acid isomers were completely extracted by immersing the tape in KOH solution. The HPLC column was a Tosoh ODS 80_TS (250×4.6 mm I.D., 7 μm average particle size) eluted with 20 mM potassium dihydrogenphosphate containing 1 g/l sodium heptanesulphonate (pH 3.7)–acetonitrile (93:7, v/v) at a flow-rate of 1.0 ml/min. The isomers were detected by UV absorbance at 264 nm. This technique was used to analyze the ratio of trans-urocanic acid/cis-urocanic acid on human skin at various sites on the body. It was found that the ratio was low in naturally light-exposed skin and high in naturally shielded skin.     

Reaction of thymidine and ascorbic acid induced by UV in the presence of salicylic acid

When a neutral solution of thymidine and ascorbic acid was irradiated with UV light of wavelength longer than 300 nm in the presence of salicylic acid as a photosensitizer, six product peaks appeared in an HPLC chromatogram in addition to small amounts of thymidine dimers. The six products were identified as three pairs of diastereomers of 5-(2-deoxy-2-l-ascorbyl)-5,6-dihydrothymidine, 5-(2-l-ascorbyl)-5,6-dihydrothymidine, and 5,6-dihydrothymidine. These results suggest that novel DNA damage may be generated by ascorbic acid with salicylic acid induced by sunlight.     

Methods for chromatographic determination of amanitins and related toxins in biological samples

Methods for the chromatographic determination of amanitins, toxins of Amanita phalloides (Fr.), Link mushrooms and related toxins are reviewed; particular emphasis is given to high-performance liquid chromatographic methods. The main chemical and toxicological aspects are discussed, but the focus of the present review is on the analytical problems arising in a laboratory charged with the setting up of a procedure which can direct the appropriate clinical management of an intoxicated patient or solve a forensic case.     

High-performance liquid chromatographic analysis of cytosine arabinoside and metabolites in biological samples

A rapid, non-radioactive method to quantitate therapeutically realistic levels of 1-β- -arabinofuranosylcytosine (Ara-C) and its metabolites would be useful both in the clinic, for monitoring drug levels, and in the laboratory for correlating drug levels with cellular and molecular perturbations. Liquid chromatographic analysis of arabinose-nucleoside analogs in biological samples is complicated by the presence of interfering nucleosides and nucleotides. We report the development of two analytic procedures to measure Ara-C and metabolite levels in biological samples. One method uses a quaternary ammonium type anion-exchange resin to achieve isocratic separation in less than one hour. The second method utilizes a boronate-derivatized polyacrylamide column which binds cis-diols to selectively retain cytosine and uridine, while arabinose compounds are eluted with recovery approaching 100%. The eluted compounds are then easily quantitated on a reversed-phase C₁₅ column. The sensitivity of both procedures was sufficient to obtain pharmacokinetic data on Ara-C and uracil-arabinose levels in serum and urine and on Ara-C triphosphate levels in tumor cells.     

Ascorbic acid conversion to erythroascorbic acid, mediated by ubiquitin

We recently identified a microbial conversion of l-ascorbic acid (AsA) to l-erythroascorbic acid (eAsA), a five-carbon analog of AsA. In this paper, we show that ubiquitin plays a crucial role in this process. Based on an assay that determined AsA decomposition, we purified proteins that had N-terminal amino acid sequences identical to that of yeast ubiquitin. Purified ubiquitin facilitated decompositions of AsA and dehydro-AsA, accompanying a partial conversion to eAsA through C1-elimination. Acetylation or limited hydrolysis of ubiquitin abolished its activity. A mutant ubiquitin, with Lys⁶ replaced by Arg, completely lost activity, whereas a mutant, with six other Lys residues (positions at 11, 27, 29, 33, 48 and 63) substituted by Arg, retained activity. Thus, Lys⁶, which locates in close proximity to His⁶⁸, is crucial for ubiquitin activity in the AsA conversion to eAsA.