ABSTRACT: Continuous monitoring of lactate production from cardiomyocytes is of great physiological and pathological importance since the level of lactate in extracellular fluid is closely associated with myocardial energy metabolism with implication in the diagnosis and therapeutics of myocardial hypoxia and ischemia. This study demonstrates an electrochemical approach to continuous monitoring of lactate production from neonatal rat cardiomyocytes following myocardial hypoxia with a dehydrogenase-based electrochemical biosensor and a negative pressure driven culture sampling. To eliminate the effect of pH variation occurring following the cardiomyocyte hypoxia on the biosensor response and to supply nicotinamide adenine dinucleotide (NAD(+)) cofactor necessary for the enzymatic reaction of lactate dehydrogenase (LDH), artificial cerebrospinal fluid (aCSF) containing NAD(+) cofactor is externally perfused and mixed online with cell culture before the culture goes to the detector. The method exhibits a high selectivity against the electrochemically active species endogenously existing in the extracellular culture of cardiomyocytes and a high tolerance against the variation of pH following cardiomyocyte hypoxia. The dynamic linear range for lactate detection is from 0.20 to 10 mM (I (nA) = 25.6 C(Lactate) (mM) + 20.1, γ = 0.996) with a detection limit of 0.16 mM (S/N = 3). The physiological level of the extracellular lactate of neonatal rat cardiomyocytes is determined to be 1.1 ± 0.1 mM (n = 3) with the cell density of about 0.5 × 10(3) cells/mm(2). When the cardiomyocytes are subject to hypoxia induced with anoxic reagents, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), the extracellular lactate increases to 255 ± 30.3% (n = 3), relative to the physiological level, following 20 min of the hypoxia. This study essentially offers a new and effective electrochemical platform for investigating energy metabolism during cardiac physiological and pathological processes.
Analytical Chemistry 05/2012; 84(12):5285-91. · 5.86 Impact Factor