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At least 199 records · Page 11Linked to original sources
PubMed2005

MALDI mass spectrometry analysis of high molecular weight proteins from whole bacterial cells: pretreatment of samples with surfactants.

The use of surfactants as additives in conjunction with on-probe whole cell bacterial protein analysis employing MALDI-TOF-MS is described. Nonionic and zwitterionic surfactants were used to enhance the detection of high molecular weight proteins. Three nonionic, N-octyl-B-D-glactopyranoside, N-decyl-B-D-maltopyranoside, and N-dodecyl-B-D-maltoside, and two zwitterionic surfactants, N,N-dimethyldodecylamine-N-oxide and zwittergent 3-12 were evaluated with five different MALDI matrix systems. New peaks in the mass range of 2 to 80 kDa were produced with all of the various combinations of matrix and surfactant from both whole cell gram-positive and gram-negative bacteria. Ferulic acid used in conjunction with a 1.0 mM solution of N-octyl-B-D-glactopyranoside produced the highest quality spectra with high signal to noise ratios and peaks up to 140 kDa.

Bacteria↗
PubMed2005

Selective quantitative bioanalysis of proteins in biological fluids by on-line immunoaffinity chromatography-protein digestion-liquid chromatography-mass spectrometry.

A quantitative method for the determination of proteins in complex biological matrices has been developed based on the selectivity of antibodies for sample purification followed by proteolytic digestion and quantitative mass spectrometry. An immunosorbent of polyclonal anti-bovine serum albumin (BSA) antibodies immobilized on CNBR agarose is used in the on-line mode for selective sample pretreatment. Next, the purified sample is trypsin digested to obtain protein specific peptide markers. Subsequent analysis of the peptide mixture using a desalination procedure and a separation step coupled, on-line to an ion-trap mass spectrometer, reveals that this method enables selective determination of proteins in biological matrices like diluted human plasma. This approach enhances substantially the selectivity compared to common quantitative analysis executed with immunoassays and colorimetry, fluorimetry or luminescence detection. Hyphenation of the immunoaffinity chromatography with on-line digestion and chromatography-mass spectrometry is performed and a completely on-line quantification of the model protein BSA in bovine and human urine was established. A detection limit of 170 nmol/l and a quantification limit of 280 nmol/l is obtained using 50 microl of either standard or spiked biological matrix. The model system allows fully automated absolute quantitative mass spectrometric analysis of intact proteins in biological matrices without time-consuming labeling procedures.

Animals↗
PubMed2006

19F NMR as a powerful technique for the assay of anti-psychotic drug haloperidol in human serum and pharmaceutical formulations.

19F nuclear magnetic resonance was used as a suitable analytical tool for the identification and selective determination of haloperidol in human serum and pharmaceutical preparations. The method is based on the integration of appropriate signals of haloperidol and trifluoroacetic acid as an internal standard. The proposed method is a rapid and facile, while without any sample pretreatment, manipulation of large samples and lengthy instrument time. The regression equation for haloperidol in human serum showed a good linearity in the range of 60-600 microg ml(-1) with a detection limit of 1.4 microg ml(-1). The mean recovery results on human serum samples ranged from about 96-103%, with relative standard deviations <8%. The method was also applied successfully to the determination of haloperidol in real pharmaceutical samples, and compared with the results obtained by a reference method. The drug's degradation was studied by the proposed method in hydrochloric acid media and main products were identified.

Antipsychotic Agents↗
PubMed2002

Determination of reboxetine, a recent antidepressant drug, in human plasma by means of two high-performance liquid chromatography methods.

Reboxetine is a new norepinephrine reuptake inhibitor (NRI) drug recently introduced in the therapy for depressed patients. It is effective in the treatment of severe depression and safer to use than traditional tricyclic antidepressants. In this paper an original high-performance liquid chromatography (HPLC) method with ultraviolet detection for the determination of reboxetine in human plasma is described. It uses a C8 reversed-phase column and a mobile phase composed of acetonitrile and aqueous tetramethylammonium perchlorate. For the analysis of plasma samples containing very low levels of reboxetine, another HPLC method with fluorimetric detection was developed (limit of quantitation, LOQ=11 ng ml(-1); limit of detection, LOD=4 ng ml(-1)). The fluorimetric method is based on precolumn derivatisation of reboxetine with 9-fluorenylmethyl chloroformate. An accurate sample pretreatment of human plasma samples has been implemented by means of solid-phase extraction (SPE) on Oasis HLB (hydrophilic-lipophilic balance) cartridges with very high extraction yields (>95%). Both methods were applied to the analysis of plasma samples from depressed patients undergoing therapy with reboxetine and gave satisfactory results in terms of precision (RSD<4.5%) and accuracy (mean recovery>94%).

Antidepressive Agents↗
PubMed2001

Prevalence of the K76T mutation in the putative Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene and its relation to chloroquine resistance in Mozambique.

K76T, a mutation in the Plasmodium falciparum chloroquine (CQ) resistance transporter protein, has been implicated in resistance to CQ. A modified 14-day in vivo test to estimate the CQ resistance level was done in southern Mozambique: 21 (42%) of 50 subjects who completed the follow-up were CQ susceptible. Use of msa2-restriction fragment length polymorphism (RFLP) genotyping to differentiate new from recrudescent infections made little difference in the estimated prevalence of resistance. The K76T mutation prevalence was estimated by RFLP-polymerase chain reaction and sequencing, and its relation to parasitological CQ resistance was explored on day 0 samples: 51 of 56 pretreatment samples presented the T76 codon, and it was present in 100% of children with parasitological resistance. T76 also was present in 18 of 23 subjects in whom the infection resolved after CQ treatment. These findings show a high prevalence of the K76T mutation among wild isolates but also suggest additional factors responsible for CQ resistance.

Animals↗
PubMed1990

The bio/immuno ratio of serum luteinizing hormone increases after orchiectomy in prostatic cancer patients and is associated with decreased molecular weight and appearance of isohormones with alkaline pI values.

Serum concentrations of bioactive (B) and immunoreactive (I) luteinizing hormone (LH) were determined in six patients with prostatic cancer before castration and at frequent intervals after the operation up to 6 mo. B-LH increased in 6 mo from 11 +/- 1 to 90 +/- 9 (mean +/- SEM, n = 6) IU/liter (p less than 0.01), and I-LH from 9 +/- 1 to 37 +/- 5 IU/liter (p less than 0.01). Accordingly, a significant increase in the B/I ratio of LH occurred at the same time, from 1.3 +/- 0.1 to 2.4 +/- 0.2 (p less than 0.01). To elucidate the molecular basis of the B/I ratio change, serum samples obtained before and 2-6 mo after orchiectomy were fractionated by gel filtration and chromatofocusing, and the eluted fractions were analyzed for B-LH and I-LH. In gel filtration, the fractions with the highest B-LH and I-LH contents were eluted later in the post-castration samples than in the pretreatment samples (mean Ve/Vo 1.31-1.32 vs. 1.26-1.28; p less than 0.02-0.01), indicating a small reduction in the average Mr of the circulating LH after castration. In chromatofocusing, a single major peak of immunoreactivity with a pI value of 7.4 was identified before castration, but in post-castration samples, a significantly large proportion of the immunoreactivity was eluted in the alkaline pI range 7.4-9 (22.2 +/- 2.4% before, 56.5 +/- 5.2% after castration, p less than 0.05). These findings indicate that after castration, the increased B/I ratio of serum LH is explained by a preferential increase in isohormones with slightly reduced molecular weights and alkaline pI values.

Aged↗
PubMed2002

Enzymatic amplification by substrate recycling for alpha-ketoglutarate in conjunction with capillary electrophoresis.

The application of substrate cycling for alpha-ketoglutarate (AKG) through enzymatic amplification has been demonstrated in conjunction with capillary electrophoresis. AKG is determined using an off-line assay, which involves the coupled enzymatic reactions catalyzed by glutamate dehydrogenase and glutamic oxalacetic transaminase. Varying concentrations of the substrate and set concentrations of cofactors and enzyme are incubated together outside the CE instrument. The accumulated product, nicotinamide adenine dinucleotide, reduced form (NADH), was detected at 340 nm using CE. Amplification of a factor of 10 over just the reversed glutamate dehydrogenase reactions permitted an AKG detection limit of 10 microM. Throughput for each assay is about 5 min (including wash and equilibrium steps). After simple dilution as the only sample pretreatment, spiked AKG serum samples were determined with an average recovery of 96% with a 3% RSD.

Animals↗
PubMedDate not supplied

Enzymatic hydrolysis of biological and environmental samples as pretreatment for analysis.

Four commercially available proteases were tested, in conjunction with a lipase, for efficacy in hydrolyzing 3 tissue substrates: cod fillet, chicken egg, and bovine liver. Enzymatic hydrolysis of tissues minimizes the formation of emulsions during liquid-liquid extraction and does not accelerate the decomposition of acid- or base-labile analytes. Recovery of hexane and benzene phases from the hydrolysates was also evaluated. Protease from Streptomyces griseus combined with lipase from Candida cylindracea (available commercially) produced the highest percent hydrolysis (relative to classical acid hydrolysis) in all 3 tested tissues (60-95%) and the greatest recovery of hexane (100%) and benzene (92-100%) solvent phases.

Animals↗
PubMed1986

Rapid enzymatic pretreatment of samples before determining chromium in serum or plasma.

A simplified method for preparing blood serum and plasma for Zeeman electrothermal atomic absorption spectrometry is described and applied to the measurement of chromium in human serum and plasma. This enzymatic degradation with bacterial protease (EC 3.4.21.3) requires little laboratory apparatus, decreases the work and time of sample preparation, and obviates some potential sources of contamination. We used bovine reference serum (USDA No. 7292) to validate Cr concentration. There was less Cr in serum than in plasma, whether sodium heparin or sodium citrate was used as anticoagulant. For six human subjects, Cr in serum averaged 0.15 (SD 0.02) micrograms/L, 0.26 (SD 0.03) micrograms/L in heparinized plasma, and 0.28 (SD 0.02) micrograms/L in citrated plasma. We postulate that the Cr concentration is lower in serum because some of the Cr binds to proteins complexed with the clot in the coagulation process.

Adult↗
PubMed1993

Ion-chromatographic determination of plasma oxalate reexamined.

This new procedure for determining oxalic acid in plasma is based on sample deproteinization with hydrochloric acid and acetonitrile and subsequent ion-chromatographic assay of the neutralized supernate. Sample pretreatment produces very clean samples, which ensures long column life. Mean analytical recovery of oxalate (5.0-10.0 mumol/L) added to plasma samples averaged 98.6 +/- 6.2%; imprecision (CV) was 5.2% (at 2.2 mumol/L) and the detection limit was 0.5 mumol/L at a signal-to-noise ratio of 5:1. Ascorbate to oxalate conversion was < 0.2%, indicating that the procedure is free from ascorbate interference. Plasma oxalate concentrations, measured in samples from 31 healthy persons, ranged from 0.8 to 3.4 mumol/L (mean 1.89, SD 0.75 mumol/L), which agrees with results from indirect radioisotopic dilution methods.

Adult↗
PubMed1993

Binding of a new monoclonal antibody against N-terminal heptapeptide of fibrin alpha-chain to fibrin polymerization site 'A': effects of fibrinogen and fibrinogen derivatives, and pretreatment of samples with NaSCN.

A novel murine monoclonal antibody against the fibrin alpha-chain N-terminus is presented, which reacts with desAA- and desAABB-fibrin. In immunoblot procedures, the antibody reacted with fibrin degradation products X and Y of non-crosslinked fibrin, and fragment E. No binding was observed to the fibrin fragment D-dimer, and fibrinogen fragments D and E. Minor binding to fibrinogen fragments X, and Y, and desBB-fibrin were presumably due to minor contamination with (desAA)-fibrin. A prerequisite for binding was release of fibrinopeptides A (FpA), the binding site being a fibrin-specific neo-epitope. No binding was observed to fibrinogen or to thrombin-treated dysfibrinogen MANNHEIM III (A alpha 16 Arg-->Cys) molecules, which do not release FpA. The antibody bound to abnormal fibrin molecules prepared from dysfibrinogen MANNHEIM I (A alpha 19 Arg-->Gly), albeit to a lesser extent than to normal fibrin. Binding of the antibody to the fibrin epitope was greatly enhanced by denaturation, e.g. by heat, or by treatment with chaotropic ions. Soluble fibrin in clinical samples is generally found as a complex with fibrinogen, since polymerization sites 'A' exposed by release of FpA react with complementary binding sites 'a' on the D-domains of other fibrin and fibrinogen molecules. Treatment of samples with NaSCN caused dissociation of fibrin monomer complexes. Reassociation was prevented by denaturation of both polymerization sites 'A' and 'a'. The antibody in combination with NaSCN-treatment of samples was useful for specific detection of fibrin monomer in plasma samples. Measurement was not influenced by fibrinogen degradation products, whereas fibrin degradation products at very high concentration caused some underestimation of fibrin monomer concentration.

Animals↗
PubMed1988

Immunoturbidimetric method for routine determinations of apolipoproteins A-I, A-II, and B in normo- and hyperlipemic sera compared with immunonephelometry.

We describe a method for routine immunoturbidimetry of apolipoproteins (apo) A-I, A-II, and B in both normo- and hyperlipemic sera. A special antiserum reagent, consisting of a highly concentrated mixture of nonionic and anionic detergents (final concentration in the assay, 36 g/L), rapidly removes intrinsic turbidities of even strongly lipemic sera without interfering with the antigen-antibody precipitation reaction. The method has good precision, and obviates the need for special sample pretreatment, extended incubation periods, and measurment of sample blanks. A comparison with established immunoephelometric assays generally showed close agreement for analytical recoveries of the three apolipoproteins. However, in samples containing greater than or equal to 18 g of triglycerides per liter, the nephelometric assays yielded about two- to threefold higher values for apo A-II and B than did the turbidimetric procedure. To elucidate this discrepancy, we used the turbidimetric methods to assay sera with and without enzymatic lipolytic pretreatment. Even for samples with triglyceride concentrations up to 60 g/L, complete enzymatic lipolysis (as evidenced by thin-layer chromatography) did not significantly alter the recoveries of apo A-II and B from those obtained with the untreated specimens. Thus the immunoturbidimetric methods yield reliable results for apo A-I, A-II, and B, not only in normo- but also in hyperlipemic sera.

Apolipoprotein A-I↗