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PubMed1983

Liquid-chromatographic determination of indole-3-acetic acid and 5-hydroxyindole-3-acetic acid in human plasma.

We describe a "high-performance" reversed-phase liquid-chromatographic method for determination of indole-3-acetic acid (I) and 5-hydroxyindole-3-acetic acid (II) in human plasma. I is eluted at 1.0 mL/min with a mixture of 1-pentanesulfonic acid (pH 3.1), methanol, and water. It is detected by fluorometry. A mixture of citric acid/sodium phosphate solution (pH 4.8) and methanol, at 1.5 mL/min, is used to elute II, which is detected with an electrochemical cell. Platelet-poor plasma samples were pretreated with HCl, perchloric acid, and trichloroacetic acid for protein precipitation. Best results were obtained with the last (protein precipitation is incomplete with HCl, while recoveries of I are concentration dependent with perchloric acid). Analytical recoveries were 58% (SD 3.1%, CV 5.3%, n = 12) and 79% (SD 3.3%, CV 5.3%, n = 9) for I and II, respectively. Concentrations of I and II in plasma ranged from 0.61 to 3.32 (mean 1.54, SD 0.59, n = 15) mumol/L and from 33.0 to 102.6 (mean 51.8, SD 20.1, n = 16) nmol/L, respectively.

Adult↗
PubMed1999

Drug uptake and cellular targets of hydroxymethylacylfulvene (HMAF).

Hydroxymethylacylfulvene (HMAF, MGI 114) is a novel antitumor drug and a potent pro-apoptotic agent that has the potential to alkylate cellular nucleophiles. The objective of these studies was to characterize drug uptake and cellular targets for drug binding in human leukemia CEM cells. The uptake of [14C]HMAF had two components: a rapid phase (0-10 min) and a slow phase. At 10 microM drug (37 degrees), the rapid and slower phase amounted to 0.86 and 0.13 pmol/min/10(6)cells, respectively. HMAF uptake was inhibited 82% by low temperature (4 degrees) at 4 hr. Cell-associated HMAF localized to nuclear (50%), cytoplasmic (37%), and membrane fractions (10%). Continued drug uptake appeared to be driven by covalent binding to cellular macromolecules. Approximately 1/4 and 2/3 of cell-associated HMAF formed covalent adducts after 10 min and 4 hr, respectively, as found by perchloric acid precipitation. Drug adducts were not readily reversible; 77% of the covalently bound radiolabel was retained by the cells 20 hr after drug treatment. Combinations of DNase, RNase, and proteinase K with perchloric acid precipitation showed that approximately 60, 30, and 10% of the covalently bound drug was associated with the protein, DNA, and RNA fractions, respectively. Incubation of 100 microM [14C]HMAF (24 hr) with purified DNA, serum albumin, thioredoxin, and thioredoxin reductase resulted in 6, 22, 14, and 11 pmol [14C]HMAF/microg DNA or protein, respectively. Results indicate that multiple targets for HMAF binding may contribute to the pro-apoptotic and antiproliferative action of the drug.

Antineoplastic Agents↗
PubMedDate not supplied

[The isolation of anti-carcinoembryonic antigen (CEA) immunoglobulins and their use in immunocytochemical and immunoenzyme (ELISA) technics].

Carcinoembryonic anti-antigen (CEA) polyclonal antibodies were obtained in ram and rabbit using as antigen source the extracts with perchloric acid from the human colon adenocarcinomas. CEA was purified by gel filtration on Sepharose 4BCL and ion-exchange chromatography (DEAE-Sephadex A50). The total antiserum was absorbed with human serum and perchloric acid extract from the normal colon. IgG anti-CEA was purified by chromatography of the monospecific antiserum, then tested for bonding specificity, at cryostat on sections of colon adenocarcinoma, by indirect immunoperoxidase. The specific reaction was compared with that obtained by the same technique, using two monoclonal antibodies specific to the CEA molecule (MAb-26/3/13 and MAb-26/5/1 respectively). IgG anti-CEA was also used for obtaining some IgG-peroxidase conjugates, with an immunoenzymatic system, ELISA type, with two antibodies according to the model of the ELISA kits produced by the Cantacuzino Institute (ELISA-AFP). The ELISA-CEA kit was standardized using an international CEA standard. CEA was quantitatively determined with this immunoenzymatic system (ELISA-CEA) on a group of 15 healthy subjects (average: 4.8 +/- 0.12 ng/ml) and on 30 patients with colorectal tumours (average: 26.6 +/- 0.15 ng/ml). ELISA-CEA kit, sensitive and reproducible, allow the usual quantitative determination of CEA, a useful marker in diagnosing and monitoring tumour evolution.

Antibody Specificity↗
PubMed1992

Monitoring propofol serum levels by rapid and sensitive reversed-phase high-performance liquid chromatography during prolonged sedation in ICU patients.

A quick and sensitive reversed-phase high-performance liquid chromatography (HPLC) method has been developed in order to determine the concentration of Propofol (2,6 diisopropylphenol) in human serum. Propofol can be isolated from serum by adding 0.5 mL precipitating solution. This consists of an acetonitrile and perchloric acid (67:33, v/v) mixture, which also contains dibutylphthalate (2 mg/100 mL) as internal standard. The sample is then mixed for 1 min on a vortex-mixer. The endogenous serum substances precipitated by acetonitrile and perchloric acid are further separated by centrifugation. The supernatant is directly injected into the HPLC system. A 250- x 4.6-mm column, packed with 10-microns Spherisorb reversed-phase octadecylsilane particles (C18), is used for chromatographic separation. The mobile phase consists of an acetonitrile-water mixture (67:33 ratio) with 0.4 mL acetic acid (pH 4). Propofol is monitored by a UV-visible detector at 270 nm and 0.1-0.002 absorbance units full scale (AUFS). The detection limit of Propofol (in human serum) is 0.1 mg/L for a 20-microL injection volume. The time of the assay is less than 20 min, including sample preparation.

Acetonitriles↗
PubMed1975

Simplified direct radioimmunoassay method for measuring carcinoembryonic antigen.

The radioimmunoassay for carcinoembryonic antigen as usually carried out is an involved, time-consuming procedure complicated by the use of a dialysis step to remove perchloric acid from an extract of the sample. A simplified direct assay for the antigen in serum or plasma is reported here. It is evaluated in comparison with our earlier method which was done using perchloric acid extraction of the samples. The results of the simplified method compare favorably with those of the indirect method.

Animals↗
PubMed2006

Ion-pair association and acid-base equilibria in nonaqueous capillary electrophoresis of weakly basic compounds.

CE in nonaqueous media was used to study the migrating behavior of two weakly basic s-triazine pesticides and one of their metabolites. The target pesticides were selected to be representative for each of the two main groups: propazine and deethylatrazine for the chloro-s-triazines group and ametryn for the methylthio-s-triazines group. To elucidate the phenomena involved, systematic studies were carried out in the different organic media studied. Absolute mobilities were determined in 50% v/v methanol (MeOH)/ACN by extrapolation of the effective mobilities to zero ionic strength in the presence of different concentrations of perchloric acid. Conductivity measurements performed in MeOH and 50 and 20% v/v methanol/ACN permitted the evaluation of the associations of the components of the BGE. The effects of ionic strength on the actual mobilities of the compounds were determined in the presence of perchloric acid and SDS in different organic media. Two different ion-pair equilibria were considered: one due to the presence of perchlorate anions present in the BGE and second that from the added dodecyl sulfate anions. Bearing in mind that these weakly basic compounds can exhibit ion-pair and acid-base equilibria, the acid-base and ion-pair parasite reaction coefficients were determined. Finally, the effects of ionic strength, ion-pair interactions and acid-base properties on the effective electrophoretic mobilities of the analytes are discussed.

Acetonitriles↗
PubMed1996

Detection of methylglyoxal as a degradation product of DNA and nucleic acid components treated with strong acid.

The 1,2-diaminobenzene derivation assay for methylglyoxal in biological systems involves the use of perchloric acid, both as a deproteinizing agent and to prevent the spontaneous formation of methylglyoxal from glycolytic pathway intermediates. However, while using a modification of the standard literature assay to measure methylglyoxal in Chinese hamster ovary cells, we found that oxidation of nucleic acids and related compounds by perchloric or trichloroacetic acid results in the formation of methylglyoxal. Compounds containing 2-deoxyribose gave higher levels of methylglyoxal than those containing ribose; purine nucleotides and deoxynucleotides gave more methylglyoxal than did the pyrimidines. Nucleic acids were the most susceptible to degradation, with 12-fold more methylglyoxal being formed from DNA than RNA. Oxidation of nucleic acids increased with higher temperatures and with decreasing nucleic acid fragment size. Another product of nucleic acid oxidation was 2,3-butanedione, the 1,2-diaminobenzene derivative of which is sometimes used as an internal standard during methylglyoxal measurement. Unless accounted for during the assay procedure, the generation of methylglyoxal and 2,3-butanedione due to the oxidation of nucleic acids may lead to substantial errors in the determination of methylglyoxal concentrations in biological systems.

Animals↗
PubMed1996

Detection of different metabolites in the rabbit lens by high resolution 1H NMR spectroscopy.

PURPOSE: To investigate the metabolic profile of the rabbit lens using high resolution 1H NMR spectroscopy including two-dimensional shift correlated (COSY) technique. METHODS: Perchloric acid extracts of the rabbit lens were analysed with a Bruker AM-500 spectrometer and the metabolites were assigned in the spectra. Some of these were also quantified. RESULTS: More than 20 metabolites were detected in the perchloric acid extract of a single lens, including amino acids, nucleotides and other related compounds. Of particular importance is the ability to detect and identify glutathione, myoinositol, scyllo-inositol and taurine. CONCLUSIONS: The study demonstrated the potential of 1H NMR spectroscopy for monitoring the metabolic profile of the lens in normal and pathologic conditions.

Amino Acids↗
PubMed1979

Determination of the ADP concentration available to participate in energy metabolism in an actin-rich cell, the platelet.

Almost all cells contain actin, which in its polymerized form, F-actin, binds 1 molecule of ADP/monomer. Little is known about the availability to metabolism of this bound ADP. A comparison was therefore made between perchloric acid and EDTA/ethanol extracts of human blood platelets. When the cells were extracted under conditions where the ATPase activity was negligible, the ethanol extracts had a 75% higher ATP/ADP ratio and a higher adenylate energy charge than perchloric acid extracts. The methods differed in that a considerable portion of protein-bound ADP was not extracted by ethanol. This bound ADP behaved as though it were unavailable to energy metabolism and should thus be considered as a compartment separate from the bulk metabolic pool of extragranular platelet adenine nucleotides. These results suggest that the level of ADP obtained with the common acid extraction overestimates the level available to participation in metabolism.

Actins↗
PubMed1996

Determination of serum and urinary aluminum by HPLC with fluorometric detection of Al-lumogallion complex.

We describe a simple and sensitive HPLC method for quantifying aluminum (Al) in biological fluids by measuring the fluorescence of the Al-lumogallion complex (excitation wavelength 500 nm, emission wavelength 575 nm). Serum samples are deproteinized with 0.83 mol/L perchloric acid and centrifuged; the supernates are mixed with lumogallion reagent. Urine samples are pretreated with sodium hydroxide (2 mol/L) and methanol, kept for 1 h at -20 degrees C, and then centrifuged; the precipitate is resuspended in perchloric acid and mixed with lumogallion reagent, as for serum. The maximal fluorescence complex is formed after 1 h at pH 5 +/- 0.5. The HPLC mobile phase consists of (per liter) 100 mL, of 0.2 mol/L potassium hydrogen phthalate, 220 mL of acetonitrile, and distilled deionized water. The flow rate is 1 mL/min, and the injection volume is 5 microliters. The major aluminum species is eluted at 3.5 min, the lowest detection limit being 0.45 pg. We validated the method with samples collected from normal subjects and from workers occupationally exposed to aluminum. Comparing the results with those by traditional atomic absorption spectrometry of urinary aluminum suggests that the proposed method is reliable.

Adult↗
PubMed1984

An automated micro-method for the enzymatic determination of D-B-hydroxybutyrate in ruminant plasma.

An enzymatic 'reaction rate' micro-method for the rapid routine estimation of D-B-hydroxybutyrate (D-B- HOB ) in ruminant plasma, using an I.L. Multistat III centrifugal analyzer, is described. Reaction conditions were optimized to give a linear response for plasma D-B- HOB concentrations between 100 and 2500 mumoles per litre, at 30 degrees C and pH 9.0. For the standardized method the within-run and between-run coefficients of variation for deproteinised ovine plasma were consistently less than 3.5%. There was good agreement between plasma concentrations obtained by the present method and both original U.V. end-point technique (r = 0. 927b = 0.950) and a colorimetric end-point procedure (r = 0.937. b = 0.879). Untreated ovine and bovine plasma consistently exhibited high 'blank' activity and this was directly correlated with plasma lactate dehydrogenase (LDH) activity in both species (r = 0.971; p less than 0.001 and r = 0.949; p less than 0.001 respectively). The distribution of LDH activity in man was similar to sheep but, contrastingly , non-specific interference was extremely low in human plasma and unrelated to LDH. Horse, chicken and rat had negligible 'blank' activity and comparatively low LDH levels. In both cattle and sheep non-specific interference was abolished by perchloric acid precipitation. In the sheep subtraction of 'blank' activity gave D-B- HOB concentrations for untreated plasma comparable to those in deproteinised samples. However, in the bovine, D-B- HOB levels remained significantly (t = 6.44; p less than 0.001) higher even after 'blank' correction. In contrast to man and other non-ruminants, perchloric acid precipitation is essential in ruminants to avoid false overestimation of plasma D-B- HOB levels. Plasma with EDTA as anticoagulant and serum gave concentrations of D-B- HOB approximately 60% lower, than samples containing heparin or oxalate/fluoride. However, heparin was associated with much higher (up to 50%) non-specific NAD reduction than oxalate/fluoride. High levels of acetoacetate (400-1000 mumoles per litre) reduced the recovery of D-B- HOB from ovine plasma by less than 10%. This effect was negated by the inclusion of hydrazine hydrate in the reaction mixture. Perchlorate ion concentrations above 25 mumoles per litre per test dramatically inhibited the assay in ovine plasma, and therefore precipitation conditions must be carefully controlled. Plasma with oxalate/fluoride as anticoagulant showed the greatest stability in storage; 24 hours at room temperature, one week at +4 degrees C and at least one month at -20 degrees C.

3-Hydroxybutyric Acid↗
PubMedDate not supplied

Comparative 31P and 1H NMR studies on rat astrocytes and C6 glioma cells in culture.

Rat astroglial cells in primary culture (95% enrichment) and C6 glioma cells were adapted to grow on microcarrier beads. In vivo 31P NMR spectra were collected from cell-covered beads perfused in the NMR tube. The NMR-visible phosphorylated metabolite contents of both cell types were determined using saturation factors calculated from the values of longitudinal relaxation times determined for C6 cells using progressive saturation experiments. On the other hand, the amounts of phosphorylated metabolites in cells were determined from proton decoupled 31P NMR spectra of cell perchloric acid extracts. The results indicate that the NTP and Pi contents of the normal and tumoral cells were similar, whereas the PCr level was higher in C6 cells and the NDP and phosphomonoester levels higher in astrocytes. The comparison of 1H NMR spectra of cell perchloric acid extracts evidenced larger inositol and alanine contents in C6 cells, whereas larger taurine and choline (and choline derivatives) contents were found in astrocytes. The Glu/Gln ratio was very different, 3.5 and 1 in C6 cells and astrocytes, respectively. In both cases, the more intense resonance in the 1H NMR spectrum was assigned to glycine. Based on the comparison of the metabolite content of a tumoral and a normal cell of glial origin, this work emphasizes the usefulness of a multinuclear NMR study in characterizing intrinsic differences between normal and tumoral cells.

Animals↗
PubMed1985

Phosphorus-31 and proton NMR analysis of reproductive organs of male rats.

Phorphorus NMR spectra of the whole reproductive organs of male rats and their perchloric acid extracts indicate the presence of phosphocholine and phosphoethanolamine in the testis, glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE) in the epididymis, and creatine phosphate, GPC, and GPE in the seminal vesicles. High amounts of carnitine and inositol were observed by the proton NMR of perchloric acid extract of the corpus and cauda epididymis. Smaller amounts of these compounds were observed in the caput epididymis and vas deferens; they were totally absent in the testis. Creatine is present in high concentrations in the testis, vas deferens, and seminal vesicles. It is almost absent in all parts of the epididymis.

Animals↗
PubMed1993

High-performance liquid chromatographic determination of thiopurine metabolites of azathioprine in biological fluids.

A selective and sensitive reversed-phase liquid chromatographic method for the analysis of thiopurine bases, nucleosides and nucleotides in biological samples was developed. A simple and rapid sample treatment procedure using perchloric acid deproteinization with dithiothreitol for the analysis of thiopurine bases and nucleosides is presented. The addition of dithiothreitol during sample collection and treatment improves recoveries. This procedure also allows the determination of thiopurine nucleotides by hydrolysis to their free bases after heating of the perchloric acid extract. The method was applied to the analysis of thiopurine metabolites in plasma and erythrocytes from lung-transplant patients under azathioprine therapy.

Azathioprine↗
PubMed2004

Concomitant isolation of protein C inhibitor and unnicked beta2-glycoprotein I.

beta2-Glycoprotein I (beta2GPI) is the major target molecule for so-called anticardiolipin antibodies. We evaluated the isolation procedure of beta2GPI from human plasma with special emphasis on the time of precipitation, composition of different isolated fractions and their antigenic properties. The isolation was initiated by perchloric acid precipitation for either 3, 18 or 50 min, followed by heparin affinity and cationic exchange chromatography. The properties of isolated proteins were tested by rocket electrophoresis, enzyme-linked immunosorbent assay, polyacrylamide gel electrophoresis, immunoblotting and N-terminal sequencing. Each isolation procedure, regardless of the perchloric acid precipitation duration, resulted in three distinct protein peaks, differing in protein composition qualitatively. Comparing sequential peaks between the isolations of different precipitation times, we found that all the three first peaks (set of peaks No. 1), all the three second peaks (set No. 2) as well as the three third peaks (set No. 3) consisted of identical proteins but in different quantities. Set No. 1 was composed of immunoglobulins and a lesser amount of beta2GPI. In set No. 2 only unnicked beta2GPI was detected. Protein C inhibitor was found in addition to smaller amounts of unnicked betaGPI in set No. 3. Oxidation or degradation of beta2GPI during the isolation procedure did not result in a mixture of different forms of beta2GPI but rather in a lower yield of wild-type beta2GPI. The co-existence of beta2GPI and protein C inhibitor in the isolated fractions may suggest their protein-protein interactions in vivo.

Autoantibodies↗
PubMed2005

Liquid chromatography-tandem mass spectrometry analysis of erythrocyte thiopurine nucleotides and effect of thiopurine methyltransferase gene variants on these metabolites in patients receiving azathioprine/6-mercaptopurine therapy.

BACKGROUND: Polymorphic thiopurine S-methyltransferase (TPMT) is a major determinant of thiopurine toxicity. METHODS: We extracted 6-thioguanine nucleotides (6-TGNs) and 6-methylmercaptopurine nucleotides (6-MMPNs) from erythrocytes with perchloric acid and converted them to 6-thioguanine (6-TG) and a 6-methylmercaptopurine (6-MMP) derivative during a 60-min acid hydrolysis step. The liquid chromatography system consisted of a C(18) column with an ammonium acetate-formic acid-acetonitrile buffer. 8-Bromoadenine was the internal standard. Analytes were measured with positive ionization and multiple reaction monitoring mode. With PCR-restriction fragment length polymorphism analysis and TaqMan allelic discrimination, common TPMT alleles (*1, *2, *3A, *3B, *3C) were determined in 31 792 individuals. We used perchloric acid extraction, acid hydrolysis, and HPLC with ultraviolet detection to measure erythrocyte 6-TG and 6-MMP nucleotide concentrations in 6189 patients with inflammatory bowel disease receiving azathioprine/6-mercaptopurine therapy. RESULTS: Intra- and interday imprecision were <10% at low and high analyte concentrations. The conversion of 6-TG and 6-MMP nucleoside mono-, di-, and triphosphates was complete after hydrolysis. Allelic frequency for TPMT variant alleles ranged from 0.0063% (*3B) to 3.61% (*3A). Compared with wild types, TPMT heterozygotes had an 8.3-fold higher risk for 6-TGNs >450 pmol/8 x 10(8) erythrocytes (concentration associated with increased risk for leukopenia), but an 8.2-fold lower risk for 6-MMPNs >5700 pmol/8 x 10(8) erythrocytes (concentration associated with increased risk for hepatotoxicity). CONCLUSIONS: The liquid chromatography-tandem mass spectrometry method can be applied to the routine monitoring of thiopurine therapy. The association between TPMT genotype and metabolite concentrations illustrates the utility of pharmacogenetics in the management of patients undergoing treatment with thiopurines.

Adult↗