GLUTATHIONE (WHOLE BLOOD) HPLC

Glutathione (Whole Blood) For HPLC

INTENDED USE

KR132 Reagent kit for the HPLC determination of Glutathione in Whole Blood.

KIT SPECIFICATION

Contents for 100 analyzes

CONTROLS

INTRODUCTION

One of the samples of tripeptide is Glutathione (GSH). It includes an unusual linkage between the amine group of cysteine and the carboxyl group of the glutamate side chain. Glutathione is an antioxidant substance and it protects cells from toxins like free radicals. Thanks to reducing form, Glutathion can be obtained. Glutathione reductase is active and inducible upon oxidative stress. In fact, the ratio of reduced to oxidized glutathione within cells is often used such as a measure of cellular toxicity.
Glutathione can be synthesized from the amino acids L-cysteine, L-glutamate and glycine. Glutamate cysteine ligase (GCL) is a heterodimeric enzyme involved of a catalytic (GCLC) and modulatory (GCLM) subunit. All the enzymatic activity is formed by GCLC, while GCLM increases the catalytic efficiency of GCLC. In healthy tissue, more than 90% of the total glutathione pool is in the reduced form and less than 10% exists in the disulfide form (GSSG). An increment of GSSG/GSH ratio is considered indicative of oxidative stress.

EQUIPMENT AND INSTRUMENTAL PARAMETERS

The analysis of Glutathione requires a HPLC system including isocratic pump, autosampler and FLD detector.
The use of a solvent degasser prevents air bubbles and ensure a stable baseline.

Sampler: Injection volume 50 μl, analytical run time 5 min..
HPLC pump: Flow rate 1.3 ml/min is recommended
Column temperature: Ambient (~25 °C)
FLD detector: EX 385 nm, EM 515 nm

COLLECTION AND STORAGE OF PATIENT SAMPLES

Pipette 3 ml of whole blood in a tube with anticoagulant citrate.
Stable 3 weeks at 2–8 °C or 4 months at – 20 °C.

SAMPLE PREPARATION PROCEDURE

50 μl sample is Dilueted with 1950 μl Hemolysis Solution in vial.

50 μL diluted sample is added to a reaction vial.

200 µL of Buffer Solution is added.

50 μL of Reducing Solution is added.

50 µL of internal standard is added.

30 μL Derivatization Solution is added.

Vortex the mix for 10s.

Incubate at 55 °C for 15 min. Then cool to room temperature

50 µL of Deproteinization Solution is added.

Vortex the mix for 10s.

Centrifuge at 5 000 rpm for 5 min.

Samples prepared as above can be stored at ambient temperature for about 24 hours, at +2 to +8 °C up to 36 h, frozen below -18 °C up to 3 months.