UVM Theses and Dissertations
Format:
Print
Author:
Kaur, Pushwinder
Dept./Program:
Chemistry
Year:
2013
Degree:
MS
Abstract:
Glutathione ([gamma]-glutamyl-cysteinyl-glycine, GSH) is an antioxidant that protects cells from reactive oxygen species. During this process, GSH is oxidized to glumthione disulfide (GSSG). During high oxidative stress, the balance between GSH and GSSG is disturbed. The reason may be a decrease in synthesis rate of GSH or the increase in its oxidation. Stable isotopes of amino acids have been used to determine the synthesis rate of GSH in red blood cells. The stable amino acid tracers are infused into the subjects and from their rate of incorporation into the newly synthesized GSH, the rate of GSH synthesis can be calculated. Studies have shown that the rate of GSH synthesis declines during different disease conditions. The methods reported in literature have been used to measure isotopic enrichments of GSH in whole blood, but they are not sensitive enough to measure the isotopic enrichments in plasma.
This is because plasma has three orders of magnitude lower concentration of GSH than whole blood ([mu]M vs mM). We have developed two liquid chromatography-mass spectrometry (LCMS) methods to determine both the concentration and isotopic enrichments of GSH in human blood and plasma. For quantification of GSH, homoglutathione (hGSH) was used as an internal standard. The first method was developed using an ion trap mass spectrometer in pseudo selected ion monitoring mode (pSIM), and requires only 150 [mu]1 of whole blood (=150 nmol GSH). A more sensitive method was developed using a triple sector quadrupole mass spectrometer and selected reaction monitoring (SRM) that needs only 100 [mu]1 of plasma (=200 pmol). It is believed that primary source of plasma GSH is liver, and plasma GSH can represent status of liver GSH.
Therefore, by measuring the GSH tracer enrichment in plasma, the synthesis rate of GSH in liver can be calculated. Thus, this method may lead to a better understanding of liver GSH without the need of a biopsy. In both methods, thiol groups of GSH and hGSH were derivatized with 5, 5'-dithiobis-2-nitrobenzoic acid (DTNB). The internal standard hGSH was validated for blood and plasma samples using standard addition. Both methods involve easy sample preparation and are quick (need only a 7 minute isocratic run). GSSG can also be quantified along with GSH measurements by altering the LC conditions. The limit of detection for quantification of GSH and GSSG is 26 nM and 40 nM, respectively, for the SRM method. The isotopic enrichments for GSH are measured as tracer tracee ratio (TTR), and the limit of detection is 0.04% TTR, i.e. 4 labeled GSH molecules out of 10,000 GSH molecules. can be identified.
This is because plasma has three orders of magnitude lower concentration of GSH than whole blood ([mu]M vs mM). We have developed two liquid chromatography-mass spectrometry (LCMS) methods to determine both the concentration and isotopic enrichments of GSH in human blood and plasma. For quantification of GSH, homoglutathione (hGSH) was used as an internal standard. The first method was developed using an ion trap mass spectrometer in pseudo selected ion monitoring mode (pSIM), and requires only 150 [mu]1 of whole blood (=150 nmol GSH). A more sensitive method was developed using a triple sector quadrupole mass spectrometer and selected reaction monitoring (SRM) that needs only 100 [mu]1 of plasma (=200 pmol). It is believed that primary source of plasma GSH is liver, and plasma GSH can represent status of liver GSH.
Therefore, by measuring the GSH tracer enrichment in plasma, the synthesis rate of GSH in liver can be calculated. Thus, this method may lead to a better understanding of liver GSH without the need of a biopsy. In both methods, thiol groups of GSH and hGSH were derivatized with 5, 5'-dithiobis-2-nitrobenzoic acid (DTNB). The internal standard hGSH was validated for blood and plasma samples using standard addition. Both methods involve easy sample preparation and are quick (need only a 7 minute isocratic run). GSSG can also be quantified along with GSH measurements by altering the LC conditions. The limit of detection for quantification of GSH and GSSG is 26 nM and 40 nM, respectively, for the SRM method. The isotopic enrichments for GSH are measured as tracer tracee ratio (TTR), and the limit of detection is 0.04% TTR, i.e. 4 labeled GSH molecules out of 10,000 GSH molecules. can be identified.