Analytical sensitivity and specificity of the metabolic activity lung cancer test (MA-LC)
The Metabolic Activity test measures the change in acid concentration over time in reaction to exposure to a stimulant. We use 8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS)—a highly water-soluble, membrane-impermeant pH indicator (pH 6.6 to 8.0) that is added to each plate’s well. HPTS exhibits a pH-dependent adsorption shift that allows the performance of ratiometric pH measurements by using the excitation ratio of 403/455 nm (d) that correlates with acidity. Analytical sensitivity of the newly developed metabolic activity test represents the smallest amount of change over time of acidity in a sample that can be accurately measured by the MA test.To calculate the analytical sensitivity, we first determined the limit of blank (LOB). Our blank is PBMCs without a stimulant. We measure the highest result that is likely to be observed with a blank with certainty of 90% (z-score of 1.645 times the standard deviation of the repeats). Next, we’ve added to the LOB the lowest measurement result that is likely to be observed with PBMCs with a stimulant with certainty of 90%. The calculated lower limit of quantification (LLOQ) for metabolism activity based on the above calculations is 0.000119 (d/minute) in change of acidity over time. This points out the high sensitivity of the MA test to detect tiny changes in acid concentration over time.The MA test measures time-dependent changes in acid concentration of the extracellular fluid in a reaction that relates to the glycolysis metabolic cycle. We verified that the test specifically quantifies glycolysis by measuring the acidity (d) change over time. We use 2-deoxy-D-glucose (2-DG), a glucose analogue able to suppress glycolysis by competitively inhibiting hexokinase 2 (HK2). Adding a general stimulant (mitogen PHA, stimulates metabolic activity in a nonspecific manner) to PBMCs causes detectable extracellular acidification reaction resulting from the secretion of lactic acid, a product of the glycolysis pathway into the extracellular fluid (blue line—Fig. Acidity signal is related to glycolysis metabolic pathway (2DG suppresses acidification/glycolysis). The blue line shows acidity (d) changes over time of PBMCs together with a general stimulant (mitogen PHA). The orange line shows acidity (d) changes over time of PBMCs together with a general stimulant (mitogen PHA) and 2DG (10 mM/well)Oligomycin (OMC) is an inhibitor of the oxidative phosphorylation metabolic cycle. It is an Adenosine Triphosphate (ATP) synthase inhibitor that prevents phosphorylation of Adenosine Diphosphate (ADP) to ATP. Inhibition of ATP synthase via the oxidative phosphorylation metabolic cycle stimulates the increase of the glycolysis cycle in order to meet the energy production need of the cell. We verified that adding OMC (4 μM/well) together with PHA to PBMCs increased the glycolysis rate (orange line—Fig. Inhibition of oxidative phosphorylation metabolic pathway by OMC increases glycolysis. LThe orange line shows acidity (d) changes over time of PBMCs together with a general stimulant (mitogen PHA) and OMC. The blue line shows acidity (d) changes over time of PBMCs together with a general stimulant (mitogen PHA)These two experiments show that acidity changes over time in PBMCs extracellular fluid correlate with changes of the glycolysis metabolic cycle.