Does deteriorating oxidative defence and impaired γ-glutamyl cycle drive pathophysiology in individuals with sickle cell disease?

Published: 14 May 2024| Version 1 | DOI: 10.17632/k89yd4jrbj.1
Contributor:
Shruti Bhatt

Description

ickle cell disease (SCD) is a global health crisis, affecting two-thirds of the children born in Africa and India. Oxidative stress, pivotal in SCD pathophysiology, induces endothelial dysfunction and inflammation. This study reassesses oxidative stress in SCD via intricate interplay of metabolites, antioxidant enzymes, and ion profiles. We assessed the plasma elemental levels of SCD patients, trait and healthy controls (n=10) via ICP-MS. Additionally, comparative erythrocyte metabolomics of SCD and healthy (n=5) was carried out using LC-MS mass-spectrometry. Followed by assessment of antioxidant defence (AD) enzymes such as glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT) in erythrocytes and plasma of SCD patients (n=31) compared to trait (n=8) and healthy (n=9). In principal component analysis, 442 metabolic features clustered separately with 135 exhibiting differential expression in the SCD vs HC. In SCD, upregulation of N6,N6,N6-Trimethyl-L-lysine, Pyroglutamate and 2-Aminoisobutyric while downregulation of Glutathione, Aminolevulinate and D-Glutamine was observed. SCD suffered dysregulations in D-Glutamine/Glutamate, Sphingolipid, Arginine biosynthesis, Gly/SerThr, β-Alanine, and Glutathione pathway. In SCD, plasma ion profiling yielded an elevated 24Mg, 44Ca, 66Zn,208Pb,39K and a decreased 57Fe,77Se, 85Rb indicating hemolysis, ion-channelopathy and anemia. SCD exhibited repressed activities of GR, SOD, and CAT in erythrocytes and plasma. SCD-RBCs displayed negligible GR and CAT activity changes, while trait and HC exhibited significant AD activity increase under hypoxia. Our findings highlight metabolic deregulations in SS-RBCs, a compromised antioxidant defence, reduced heme synthesis, ion-channel dysfunction, a shift to ATP consuming aberrant γ-glutamyl cycle leading to clinical manifestations such as oxidative stress, anemia, dehydration and hemolysis.

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Untargeted metabolite profiling of sickle RBCs The 0.5 ml aliquots of human donor erythrocytes (Sri Sri University, Cuttack, Odisha, India) that had been pre-treated with heparin anti-coagulant were placed in 2.0 ml microcentrifuge tubes (MCT). After adding internal standards, they were centrifuged at 1000×g and 4 °C for 2 min and then placed on ice while the supernatant was aspirated. The RBCs were washed twice with their resuspension in 1×phosphate-buffered saline (PBS) through centrifugation, and finally, the supernatant was aspirated to obtain an RBC pellet. These wash cycles remove non-erythrocytic metabolites and other compounds that may still be present outside the cells; however, it also delays quenching, and might leave residual traces of phosphate salts. Metabolite extraction To the RBC pellet of each tube, 0.15 mL of ice-cold, ultrapure water (Milli-Q Millipore, Mississauga, Canada) was added to resuspend the erythrocytes. The tubes were first plunged into the dry ice for 30 sec followed by 20 sec of incubation in the water bath at 37 °C to quench metabolism and lyse the cells. After quenching with dry ice, 0.6 ml of methanol at 20 °C temperature was added, and the tubes were then vortexed to ensure complete mixing. The tubes were then plunged again into dry ice where 0.45 ml of chloroform was added to each tube. These tubes were vortexed briefly every 5 min for 30 min, and between each brief vortexing interval, they were placed in a cold bath. After 6 brief vortexes, the tubes were transferred to room temperature and 0.15 ml of ice-cold, ultrapure water (Milli-Q Millipore, Mississauga, Canada) was added to drive the phase separation between methanol and chloroform. The tubes were centrifuged at 1,000 ×g for 2 min at 4 °C so that a clear separation of the two phases could be observed above and below the compact disk of erythrocytes. After centrifugation, the tubes were transferred to a –20 °C freezer for an overnight incubation to allow residual chloroform to precipitate out of the aqueous methanol phase. The two liquid phases in each tube were transferred to separate 1.5 mL microcentrifuge tubes without disturbing the compact disk of erythrocytes or transferring any erythrocytes to the new tubes. The final volumes translated to a methanol/water/chloroform ratio of 4:2:3 for extraction and phase separation. The samples were then dried with speed-vac and resuspended in 0.2 mL LC mobile phase (97.9% ultra-pure water / 2% acetonitrile / 0.1% formic acid)

Institutions

University of Delhi - South Campus

Categories

Mass Spectrometry, Erythrocyte, Patient with Sickle Cell Disease, Untargeted Metabolomics

Funding

University of Delhi

IOE/FRP/LS/2020/27

Birla Institute of Technology and Science, Pilani

BPGC/RIG/2022-23/09-2022/01; GOA/ACG/2022-2023/Oct/10

Indian Council of Medical Research

EM/SG/Dev.Res/122/3151-2023; ISRM: IIRP-2023-3151

Department of Biotechnology

DBT/2019/UDSC/1179

International Centre for Genetic Engineering and Biotechnology

Core grant

Licence