Analysis of plasma thiols by high-performance liquid chromatography with ultraviolet detection

Bald, Edward; Chwatko, Grażyna; Głowacki, Rafał; Kuśmierek, Krzysztof

doi:10.1016/j.chroma.2003.11.030

Cited by 130 publications

(103 citation statements)

References 12 publications

(13 reference statements)

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“…The classical technique HPLC has been used to analysis of Hcys or HTL in human plasma. The HPLC analysis was performed with a Hewlett-Packard 1100 Series system according to Głowacki et al (2001) and Bald et al (2004). Human plasma was incubated with the reduced form of L-Hcys (at final doses of 10-100 µmol/l) or with L-Hcys HTL (0.1-1 µmol/l) for following times: 5, 30 and 60 min, at 37°C.…”

Section: Isolation Of Plasmamentioning

confidence: 99%

May modifications of human plasma proteins stimulated by homocysteine and its thiolactone induce changes of hemostatic function of plasma in vitro?

Olas¹,

Kołodziejczyk²,

Malinowska³

2010

gpb

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Abstract. Homocysteine (Hcys) may be implicated in different diseases, especially in cardiovascular illnesses. The most reactive form of Hcys is its cyclic thioester -homocysteine thiolactone (HTL), which is formed in plasma and represents up to 0.29% of plasma total Hcys. Recently, it has been observed that Hcys and HTL may modify plasma proteins, including albumin, hemoglobin or fibrinogen, but the role of this process is not yet well known. The aim of our study in vitro was to investigate the modifications of human plasma total proteins after incubation with the reduced form of Hcys in concentrations 10-100 µmol/l, and HTL in concentrations 1-0.1 µmol/l, which correspond to levels found in human plasma during hyperhomocysteinemia in vivo. The aim of our study was also to explain the effects of Hcys and HTL on coagulation activity of human plasma. We showed that in model system in vitro Hcys and HTL change the level of thiol, amino and carbonyl groups in plasma total proteins. Moreover, our studies reported that not only Hcys (10-100 µmol/l), but also HTL (at lower concentrations than Hcys) modulates the coagulation properties of human plasma.

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Section: Isolation Of Plasmamentioning

confidence: 99%

May modifications of human plasma proteins stimulated by homocysteine and its thiolactone induce changes of hemostatic function of plasma in vitro?

Olas¹,

Kołodziejczyk²,

Malinowska³

2010

gpb

View full text Add to dashboard Cite

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“…However, it has poor concentration sensitivity because of the small optical path length (25-75 µm) limited by the inner diameter of the capillary and small sample injection volumes (~ nL). To improve the sensitivity of UV detection, the analytes can be chemically derivatized by UV labeling agents with strong UV absorption [13]. EC detection is more sensitive, but the integration of an EC detection cell with an electrophoretic separation system can be problematic because the separation current has to be separated effectively from the detection circuit [14,15].…”

Section: Introductionmentioning

confidence: 99%

On-column preconcentration of glutathione and glutathione disulfide using pH-mediated base stacking for the analysis of microdialysis samples by capillary electrophoresis

Hoque

Arnett

Lunte

2005

Journal of Chromatography B

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Capillary electrophoresis (CE) has become a useful analytical tool for the analysis of microdialysis samples. However, CE with UV detection (CE-UV) is not sensitive enough to quantify glutathione (GSH) and glutathione disulfide (GSSG) in high ionic strength sample matrices such as microdialysates, because of the small optical path length in the capillary. To overcome this limitation, an on-column preconcentration technique, pH-mediated base stacking, was used in this study to improve the sensitivity of CE-UV. In pH-mediated base stacking, sample injection is followed by the injection of sodium hydroxide to achieve on-column preconcentration of analytes. This stacking technique allowed large volumes of high ionic strength samples to be injected without deterioration of the separation efficiency and resolution. The limit of detection for GSH and GSSG was found to be 0.75 µM (S/N = 6) and 0.25 µM (S/N = 6), respectively. A 26-fold increase in sensitivity was achieved for both GSH and GSSG using the pH-mediated base stacking, relative to normal injection without stacking. The developed method was used to analyze GSH and GSSG in liver microdialysates of anesthetized Sprague Dawley male rats. The basal concentrations of GSH and GSSG in the liver microdialysates of male rats were found to be 4.73 ± 2.08 µM (n = 7) and 5.52 ± 3.66 µM (n = 7), respectively.

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“…To control the real Hcy concentration under our experimental conditions, High Performance Liquid Chromatography (HPLC) with ultraviolet detection was used (HP 110 Series System, Hewlett-Packard, Waldbronn, Germany) [15]. The level of Hcy was calculated using calibration standards.…”

Section: Hcy Analysismentioning

confidence: 99%

Endothelial microparticle formation in moderate concentrations of homocysteine and methionine in vitro

Sekuła

Janawa

Stankiewicz

et al. 2011

Cellular and Molecular Biology Letters

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Microparticles (MPs) are small membrane vesicles released by stimulated or apoptotic cells, including the endothelium. Hyperhomocysteinemia (HHcy) is a blood disorder characterized by an increase in the plasma concentrations of total homocysteine (Hcy). The plasma Hcy level is determined by environmental factors (dietary habits, i.e. the intake of folic acid, FA) and genetic factors (N 5,N 10-methylenetetrahydro-folate reductase, MTHFR, polymorphism 677C>T). To evaluate whether moderate Hcy concentrations induce endothelial MP formation, the role of FA supplementation and the influence of MTHFR polymorphism were analysed. Human umbilical vein endothelial cells (HUVEC) were treated in vitro with 50 μM of Hcy and methionine (Met). The MP number and apoptotic phenotype were analyzed using flow cytometry. Increasing doses of FA (5, 15 and 50 μM) were used to reduce the HHcy effect. The MTHFR 677C>T polymorphism was determined. HUVEC stimulated by Hcy produced significantly more MPs than HUVEC under the control conditions: 3,551 ± 620 vs 2,270 ± 657 kMP (p = 0.02). Supplementation with FA at concentrations of 5, 15 and 50 μM reduced the MP count in the cell culture supernatant to 345 ± 332, 873 ± 329, and 688 ± 453 kMP, respectively (p = 0.03). MTHFR 677C>T heterozygosity was associated with a significant increase in MP formation after stimulation with Hcy compared to the control conditions: 3,617 ± 152 vs 1,518 ± 343 kMP (p = 0.02). Furthermore, the MTHFR genotype altered MP formation after Met loading. On average, 24% of the entire MP population was apoptotic (annexin V-positive). Endothelial function impairment due to HHcy is related to MP shedding, which may involve platelets and other blood and vascular cells. MP shedding is a physiological response to moderate HHcy.

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Analysis of plasma thiols by high-performance liquid chromatography with ultraviolet detection

Cited by 130 publications

References 12 publications

May modifications of human plasma proteins stimulated by homocysteine and its thiolactone induce changes of hemostatic function of plasma in vitro?

May modifications of human plasma proteins stimulated by homocysteine and its thiolactone induce changes of hemostatic function of plasma in vitro?

On-column preconcentration of glutathione and glutathione disulfide using pH-mediated base stacking for the analysis of microdialysis samples by capillary electrophoresis

Endothelial microparticle formation in moderate concentrations of homocysteine and methionine in vitro

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