In a third set, PRP was incubated for 72 hours with lactic acid (30% in water) (Sigma Aldrich) or metformin (16,600 mg/L) plus sodium mainly bicarbonate. Lactic acid was added to PRP every 24 hours so as to reach the same lactate level as the samples incubated with metformin (16,600 mg/L). Sodium bicarbonate was added every 24 hours to PRP already treated with metformin (16,600 mg/L) to maintain bicarbonate at the same level as the samples incubated with saline. Plasma pH, lactate levels and platelet oxygen consumption were measured at 72 hours.Finally, we incubated human red blood cells, instead of platelets, with saline or metformin (16,600 mg/L) and measured pH and lactate levels every 24 hours, up to 72 hours.ex vivo experimentWe enrolled ten consecutive patients admitted since 2008 to one Hospital in Milan (Italy) with lactic acidosis (arterial pH < 7.

30 and lactate concentration > 5 mmol/L), serum metformin concentration > 10 mg/L (therapeutic level is < 4 mg/L) and no other primary explanation for lactic acidosis (such as, for instance, overt respiratory, heart or liver failure). Exclusion criteria were pre-existing mitochondrial disease and hemoglobin < 8 g/dl (< 10 g/dl in the case of ischemic cardiomyopathy). Platelet mitochondrial function was studied within 48 hours of diagnosis. Blood was anticoagulated with ethylenediamine tetraacetic acid (EDTA) (30 ml) (for measuring platelet mitochondrial respiratory chain complex activities, always done) or citrate (20 ml) (for measuring platelet mitochondrial membrane potential, only performed since the beginning of 2010).

It was then sedimented and centrifuged (2,500 g for 10 min) and PRP collected for further analysis (see below). Ten healthy volunteers (similar in sex and age to intoxicated patients) acted as controls.Mitochondrial respiratory chain complex enzyme activitiesPRP (either from in vitro or ex vivo experiments) was washed with distilled water, centrifuged at 5,000 g for 10 min (14,500 g from the second cycle on) and then GSK-3 washed again with PBS until a clear platelet pellet could be stored at -80��C (two or three cycles were usually required). At the time of analysis, the platelet pellet was diluted in buffer (KCl 120 mM, HEPES 20 mM, MgCl2 5 mM and EGTA 1 mM; pH 7.2, 300 to 400 ��l), sonicated (two cycles at 60 W for 10 seconds) and centrifuged (750 g for 10 min) while kept at 4��C. Supernatant was then analyzed using spectrophotometry (at 30��C). We measured the activity of respiratory chain NADH-ubiquinone 1 reductase (complex I), succinate-cytochrome c reductase (complex II+III) and cytochrome c oxidase (complex IV) and expressed it relative to that of citrate synthase (a marker of mitochondrial density) [26]. Proteins were measured using Lowry’s method.