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 analyses

Cat no

Quantity

Reagents

KR1321

1000 mlx1

Mobile Phase

KR1322

100 mlx1

İnternal Standart

KR1323

200 mlx1

Hemolysis Solution

KR1324

20mlx1

Reducing Solution

KR1325

0.5 mlx2

Derivatization Solution

KR1326

5 mlx1

Deproteinization Solution

KR1327

25mlx1

Buffer Solution

KR13280

0.5 mlx5

Calibrator

 

ACCESSORIES

KR1320

1 adet

HPLC column for the analysis of Glutathione in Whole Blood.

KR1329

100 adet

Autosampler vial with teflon caps

 

CONTROLS

KR13281

0.5 ml x5

Whole Blood Control, Level I

KR13282

0.5 ml x5

Whole Blood Control, Level II

 

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 Buffer Solution is added.

50 μL Reducing Solution is added.

50 μL 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 temprature

50 μL 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.

© KREON MEDİKAL SİSTEMLER SAN.VE TİC.A.Ş. 2017