4 research outputs found
Rapid and reproducible characterization of sickling during automated deoxygenation in sickle cell disease patients
In sickle cell disease (SCD), sickle hemoglobin (HbS) polymerizes upon deoxygenation,
resulting in sickling of red blood cells (RBCs). These sickled RBCs have strongly reduced
deformability, leading to vaso-occlusive crises and chronic hemolytic anemia. To date,
there are no reliable laboratory parameters or assays capable of predicting disease severity
or monitoring treatment effects. We here report on the oxygenscan, a newly developed
method to measure RBC deformability (expressed as Elongation Index - EI) as a function of
pO2. Upon a standardized, 22 minute, automated cycle of deoxygenation (pO2 median
16 mmHg ± 0.17) and reoxygenation, a number of clinically relevant parameters are produced in a highly reproducible manner (coefficients of variation <5%). In particular, physiological modulators of oxygen affinity, such as, pH and 2,3-diphosphoglycerate showed a
significant correlation (respectively R = ‑0.993 and R = 0.980) with Point of Sickling
(PoS5%), which is defined as the pO2 where a 5% decrease in EI is observed during deoxygenation. Furthermore, in vitro treatment with antisickling agents, including GBT440,
which alter the oxygen affinity of hemoglobin, caused a reproducible left-shift of the PoS,
indicating improved deformability at lower oxygen tensions. When RBCs from 21 SCD
patients were analyzed, we observed a significantly higher PoS in untreated homozygous
SCD patients compared to treated patients and other genotypes. We conclude that the
oxygenscan is a state-of-the-art technique that allows for rapid analysis of sickling behavior in SCD patients. The method is promising for personalized treatment, development of
new treatment strategies and could have potential in prediction of complications
Structural and functional studies on human and Escherichia coli pyridoxal kinases
Pyridoxal kinase is an ATP dependent enzyme that phosphorylates pyridoxal, pyridoxine, and pyridoxamine forming their
respective 5-phosphorylated esters. The kinase is a part of the salvage pathway for re-utilizing pyridoxal 5-phosphate, which serves
as a coenzyme for dozens of enzymes involved in amino acid and sugar metabolism. Clones of two pyridoxal kinases from Esche-
richia coli and one from human were inserted into a pET 22b plasmid and expressed in E. coli. All three enzymes were puriWed to
near homogeneity and kinetic constants were determined for the three vitamin substrates. Previous studies had suggested that
ZnATP was the preferred trinucleotide substrate, but our studies show that under physiological conditions MgATP is the preferred
substrate. One of the two E. coli kinases has very low activity for pyridoxal, pyridoxine, and pyridoxamine. We conclude that in vivo
this kinase may have an alternate substrate involved in another metabolic pathway and that pyridoxal has only a poor secondary
activity for this kinase