PMC

2,3-DPG reduces progeny number. Merozoite count within late-stage schizonts growing in presence or absence of 2,3-DPG 8mM. n=132 schizonts; Presence (light dots, median = 14.00, mean 14.51, SD 3.075) or absence (dark dots, median = 20.00, mean 20.65, SD 5.169) of 2,3-DPG. ****p < 0.0001.

Excess adenosine acts through A_2BRs to induce 2,3-DPG and subsequent sickling in SCD transgenic mice. (a) Concentration of 2,3-DPG in RBCs of wild-type and SCD transgenic mice with or without chronic PEG-ADA enzyme therapy. (b) In vivo measurement of oxygen saturation of hemoglobin in wild-type and SCD transgenic mice with or without PEG-ADA treatment for 8 weeks. (c) 2,3-DPG concentrations in isolated RBCs treated with NECA in the presence or absence of theophylline. (d) 2,3-DPG concentrations in RBCs isolated from wild-type or mice deficient in each of the four types of adenosine receptors cultured in the presence or absence of NECA. (e) Immunostaining of A_2BRs in RBCs from wild-type and A_2BR-deficient mice. (f) 2,3-DPG concentrations in RBCs isolated from wild-type and adenosine receptor–deficient mice under hypoxic conditions. (g) cAMP concentrations in RBCs isolated from wild-type and A_2BR-deficient mice treated with or without NECA. (h) 2,3-DPG concentrations in RBCs isolated from wild-type mice treated with NECA in the presence or absence of the PKA-specific inhibitor H-89. (i) 2,3-DPG concentrations in RBCs of wild-type and SCD transgenic mice treated with or without PSB1115. (j) Lifespan of RBCs in SCD transgenic mice treated with or without PSB1115. Mean ± s.e.m.; *P < 0.05 relative to untreated controls, **P < 0.05 relative to treated or positive samples; Each experiment was repeated five to seven times. n = 5–8 mice per group.

After correcting for Hb, 2,3-DPG concentrations were higher in SCD patients than in controls (p<0.001) and Z-scores for 2,3-DPG, as assessed by DIHRMS, were similar in patients and controls. The Z-scores positively correlated with 2,3-DPG concentrations (Fig.A, 0.353, p=0.004). Because of the anaemia in SCD, RBCs and plasma make up lower and higher volumes in the DBS, respectively, compared to healthy controls. DIHRMS detects a wide range of RBC and plasma metabolites, whereas the quantitative measurement is restricted to measuring 2,3-DPG of RBCs. To correct for those differences between methods, we applied a correction factor to the DIHRMS data using the formula (1/Ht)*(1-Ht/1), correcting for the RBC volume (1/Ht) and the plasma volume (1-Ht/1). This resulted in a trend towards higher Z-scores in patients than controls (p=0.0597). Moreover, the positive correlation with 2,3-DPG concentrations increased to 0.526 (Fig B, p<0.001). As 2,3-DPG affects the oxygen affinity of Hb, all measurements were correlated to p50. Expectedly, 2,3-DPG concentrations positively correlated with p50 (0.842, p<0.001). After applying the correction factor to the DIHRMS data, p50 correlations increased from 0.361 (p=0.003) to 0.529 (p<0.001).

Phagocytosis of apoptotic thymocytes by P388D1 macrophages. (a) Effect of apoptotic thymocytes on the phospholipase D (PLD) activity of P388D1 macrophages. P388D1 cells were labelled with [³H]palmitic acid and incubated with or without apoptotic thymocytes for 1 hr, as described in the Materials and methods. For control data, the PLD activity of P388D1 cells in the freshly prepared thymocytes was also determined. PLD activity was presented as percentage formation of [³H]phosphaytidylbutanol (PBt) from total labelled lipids. Data represent mean values ± standard deviation (SD) of triplicate determinations. (b) The effect of PLD action blockers, 2,3-diphosphoglycerate (2,3-DPG) and 1-butanol (1-BuOH), on the phagocytotic capability of P388D1 macrophages. P388D1 cells were incubated with apoptotic thymocytes for 1 hr in the presence or absence of 2,3-DPG (10 mm), 1- and 2-butanol (0·3%, vol/vol), or phospho-l-serine (PLS, 1 mm), as described in the Materials and methods. Data are presented as numbers of phagocytosed thymocytes per 100 P388D1 cells. Data represent mean values ± standard deviation (SD) of three determinations from random fields. (Inset) Effect of 2,3-DPG on the PLD activity of P388D1 macrophages. [³H]-Labelled P388D1 cells were incubated for 30 min in the presence or absence of 2,3-DPG (10 mm). The 2,3-DPG effect in the presence of apoptotic thymocytes (2,3-DPG + apoptotic thy) was also measured. PLD activity was presented as percentage formation of [³H]PBt from total labelled lipids. Data represent mean values ± standard deviation (SD) of triplicate determinations.