Sensitive and selective determination of glutathione in probiotic bacteria by capillary electrophoresis–laser induced fluorescence

Abstract

Glutathione (GSH) is a thiol with an important function in protecting tissue against the oxidative stress which has been related to carcinogenesis in the colon. For this reason the development of probiotic species producing glutathione could be of great interest. To determine the glutathione content of some probiotic bacteria of the Bifidobacterium and Lactococcus genera, a very sensitive and selective analytical method based on capillary electrophoresis coupled to laser-induced fluorescence detection has been developed. Pretreatment of cell-lysate samples is very simple--precipitation of protein with acetonitrile in 1:2 volume ratio. The fluorophore 5-iodoacetamidofluorescein (5-IAF) was chosen for glutathione derivatisation; it reacts with thiols at pH 12.5, forming a fluorescent adduct which is excited by a laser at 488 nm for detection. The reaction conditions optimised were temperature, time, and 5-IAF/GSH molar ratio. Electrophoresis was performed with a carbonate buffer (25 mmol L(-1), pH 9.8) as background electrolyte and a voltage of 30 kV; an electrophoretic run was complete in less than 7 min. There was a good linear relationship between concentration and response in the range 2.5-500 ng mL(-1) and the LOD was 0.5 ng mL(-1). The glutathione content of probiotic cells was determined by using the standard additions method to reduce matrix effects. The method was fully validated and shown to be of suitable sensitivity and selectivity for determination of GSH in probiotic cell lysates.

Simultaneous Determination of Glutathione and Glutathione Disulfide in an Acid Extract of Plant Shoot and Root by Capillary Electrophoresis

“…Limit of detection values are comparable to those reported by Herrero et al 5 using CE with a diode array detector or those reported by Carru et al 12 using photometric detection. Values are also similar to those found through chromatographic techniques 8 , although not as low as those found through CE coupled to the use of more sensitive detectors such as laser-induced fluorescence 18 and mass spectrometry 19 . EC methods have been described based on other modalities such as sample stacking 32 or MECK 13 with lower detection limits than those found in the present study, but they have not been successfully applied to highly acid matrices such as those formed by MPA.…”

“…If we go through proposed methods based on capillary zone electrophoresis (CZE), a similarity is observed in the process of analyte separation, where the background analyte is usually constituted by borate in the 0.05-0.3 M concentration range, with pH values near its pK a 16,17 , BGE concentration and pH, together with capillary length and voltage applied having the strongest effect on the time required for analyte separation. On the other hand, detection using CE has been carried out with different systems, the most sensitive of which are based on laser-induced fluorescence 18 , mass spectrometry 19 , and, less sensitive even though more widely used, systems based on photometric detection 11 .…”

Effect of Daily Consumption of Probiotic Yogurt on Oxidative Stress in Pregnant Women: A Randomized Controlled Clinical Trial

“…GSH is an important regulator of intracellular redox homeostasis and it appears that probiotics can increase plasma GSH levels in several ways. It has been reported that certain probiotics including L. fermentum, Lactobacillus reuteri, Bifidobacterium and Lactococcus genera can release GSH from other tissues [25,26,44]. Probiotics can also cause GSH synthesis through enhanced glutamate-cysteine ligase (GCL) activity, increased mRNA expression of both of the GCL subunits, increased local synthesis and systemic GSH levels [42,43].…”

Recent developments in CE and CEC of peptides

“…The sensitivity of the method was sufficient for determination of GSH and GSSG in human plasma and tobacco leaves. The other CE-LIF techniques were applied to simultaneous and rapid determination of GSH and reactive oxygen species in individual red blod cells [202,203], in apoptotic leukemia cells [204], and in probiotic bacteria [205].…”