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Monitoring of ATP Levels in Red Blood Cells and T Cells of Healthy and Ill Subjects and the Effects of Age on Mitochondrial Potential

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Monitoring of ATP Levels in Red Blood Cells and T Cells of Healthy and Ill Subjects and the Effects of Age on Mitochondrial Potential Monitoring of ATP Levels in Red Blood Cells and T Cells of Healthy and Ill Subjects and the Effects of Age on Mitochondrial Potential Nina Mikirova, Ph.D.;1 Hugh D. Riordan, M.D.;1 R.K. Kirby, M.D.;1 A. Klykov, B.S.;1 James A. Jackson, Ph.D.1 Introduction tent is correlated with red cell viability, Energy metabolism plays a role in hereditary metabolic abnormalities, and health and aging as well as disease. Energy functional diseases with increased or de- must be available for the work of synthesiz- creased ATP. Measurements of the level of ing new cellular material, maintaining mem- ATP in red blood cells may be used for branes and organelles, and to fuel movement evaluation of the pathobiochemical shift in and active transport. Determination of the energy production and for evaluation of concentration of the energy carrier mol- the therapeutic effect of treatments. ecule, adenosine-5'-triphosphate (ATP), may Measurements of the level of bio- assess the energy state in cells. Cellular ATP energetics of T cells are important in the is an important determinant of cell death assessment of disease management and for by apoptosis or necrosis.1 Cells stay alive as estimation of the level of cellular immune long as a certain level of ATP is maintained. functioning. ATP measurements can pro- When ATP falls below this level, apoptosis vide an easy and fast method for estima- is activated.2 A severe drop in cellular ATP tion of the T cell bioenergy and response will result in cellular necrosis. of cell activation to a variety of stimuli. The Metabolic conditions such as, trauma method can be used for monitoring the or stress may increase requirements for infectious disease and response to treat- ATP or reduce the regeneration of ATP, ments. It may be useful in determining the therefore decreasing the overall ATP avail- response to nutritional supplements or the able to the body. Several pathological con- effect of aging on cellular metabolism. ditions may also decrease ATP production Measurements of mitochondrial po- in cells. In addition, age and age-related tential are important since mitochondria diseases may be due to a fall in energy play the primary role in energy production metabolism in the mitochondria. For exam- and ATP generation. Mitochondria are the ple, patients with chronic fatigue syndrome energy furnaces of the body. When mito- (CFS) have relatively low intracellular ATP chondria make insufficient ATP, there is concentrations after exercise and a lower inadequate energy for the body. This study ATP synthesis rate during recovery.3,4 In sought to evaluate the energy state of mi- type II diabetes, alterations in the ATP syn- tochondria and dependence of the level of thesis may contribute to the pathogenesis energy production on age. of this disease.5 There are several techniques for meas- Lower ATP levels, number of hemoglobin uring ATP in cells, including enzymatic, and RBC deficiencies are common in cancer fluorometric, and chromatographic meth- patients. The cellular level of ATP in subjects ods.7-11 The more widely used is the with solid tumors may be 30 % lower than that bioluminescent method.12-18 This method of normal adults.6 uses the firefly luciferase assay and has a In RBCs, the adenine nucleotide con- high level of sensitivity and specificity. 1. The Bio-Communications Research Institute, Inc., 3100 In previous methods, cells were lysed N. Hillside Avenue, Wichita KS, 67219 with cold trichloroacetic acid then 50 ATP Levels in RBCs and T Cells and the Effects of Age on Mitochondrial Potential nucleotides were determined in the lysate were removed by aspiration. Packed cells after dilution with buffer. This step resulted were diluted by PBS with 10 mM glucose in a significant loss of ATP in the and 1mg/ml BSA (bovine serum albumin) supernatant. to preserve the normal shape of RBCs. The RBCs were then washed two times by PBS Materials and methods and centrifuged at 200 g for 2-3 min. The The method that we used for ATP final dilution of cells by PBS was greater measurements complements other ATP than 1:3000. Before ATP measurements, assay methods. In our measurements, di- cells were counted by flow-cytometer. luted cells were counted by flow-cytom- eter. Therefore, an analysis of the level of T cells separation ATP in a known number of cells was per- T cells were separated by “PosetteSep” formed without incubation or troublesome procedure from Stem Cell Technology.19 In extraction. For ATP determination, we this procedure, 50 uL of RosetteSep cock- used a kit from Promega Company, tail was added to each mL of blood and “CellTiter-Glo Luminescent Cell Viability mixed well. Cells were incubated 20 min at Assay”. room temperature. After incubation, cells Hemoglobin interferes with the were diluted with an equal volume of PBS luciferease-luminescence assay. The reac- containing 2% FBS (fetal bovine serum), tion produces a flash of yellow-green light mixed well and layered on the top of Ficoll- with peak emission at 560 nm. The major Paque (Amersham Bioscience). After 20 peak of hemoglobin absorption occurs at min of centrifugation at 1200 g, enriched 408 nm. As hemoglobin exists in several cells were separated, washed in PBS with forms, each of which has different absorb- FBS, counted, and used for analysis. ance spectra, there are additional absorb- To remove residual red cells, the solution ance at the peak values of 575 nm and was lysed with ammonium chloride. Enriched 540nm. These forms of hemoglobin are re- cells were washed one more time after lysis by sponsible for the interference with the PBS with 1 mM EDTA. Part of the cell suspen- luciferase spectrum. sion was counted by flow cytometer. Other We analyzed the effect of hemoglobin portions of cells were used for ATP analysis presented in supernatant at the measured and mitochondrial potential analysis. ATP level in cells. The hemoglobin pre- sented in samples was determined by light ATP measurements absorption at wavelength 408 nm and 560 The level of ATP in T cells and RBCs nm. Our results demonstrated an effect was determined by The CellTiter- of hemoglobin on the luminescence inten- GloLuminescent cell Viability Assay kit sity in samples with concentrations of cells (Promega). The assay generates a glow higher than 10-15 million cells per mL. type signal produced by luciferase reac- In addition, the level of absorption did tion, which is proportional to the amount not demonstrate a detectable level of of ATP present in cells. In this assay, the hemolysed cells in solution after cells were signal half-life was greater than one hour. washed 2-3 times with PBS (phosphate For photon count, 50 uL of reaction mix- buffered saline). ture was mixed with 50 uL of cells and the count was measured by luminometer (BD Red cell separation Biosciences). RBCs were separated from other cells During the development of the pro- and plasma by centrifugation at 500 g for cedure, the relationship between lumines- 10 min. The supernatant and “buffy coat” cence output and the number of T cells 51 Journal of Orthomolecular Medicine Vol. 20, No. 1, 2004 and RBCs in culture was determined. The of reproducibility was demonstrated with experiments demonstrated significant triplicate measurements of the samples. correlations between the number of cells and Mitochondrial Potential Measurements luminescent emission (Figure 1, below). To measure mitochondrial potential, There was a linear relationship be- cells were harvested from experimental 6 tween the cell number in the range of 0.1- samples at concentrations of 0.5*10 cells 1.5 million cells and the level of output sig- per mL. Cells were stained in PBS with nal (coefficient of correlation R=0.99). 2.5ug/ml JC-1 and kept in a dark place at The level of ATP was determined room temperature for 15-20 min. After ex- from the standard curve. For determina- posure, cells were washed twice in PBS by tion of the standard curve, ATP (Sigma) centrifuging at 500 g for 5min. For detec- was dissolved in PBS to make concentra- tion of emission by fluorometer, cells were tions of 50-500 nM. The solution was lysed in a solution of PBS with 0.2% Triton calibrated to an absorbance extinction X-100 containing 0.5mM dithiothreitol coefficient equal to 15.4/mM-1cm. The (DTT) (Sigma). standard was prepared for each day of Mitochondrial potential was assessed measurements. The standard curve was by using the fluorescent potentiometric linear in the range of ATP at 0-1000 nM. dye, JC-1 (5,52,6,62,-tetrachloro-1,12,3,32 A linear relationship was observed be- tetraethylbenzimidazolylcarbo-cyanine io- tween the number of cells and the dide).20 JC-1 is able to selectively enter the amount of ATP in the cells. A high level mitochondria of intact cells and form ag- Figure 1. Correlation of cell number with luminescent output. 3.5*105 y= -18915 + 2.4229e + 05x R= 0.99582 3.0*105 2.5*105 2.0*105 1.5*105 3.5*105 Intensity of Luminescence Intensity 1.0*105 0.5*105 0 0 0.51 1.5 Number of Cells (M) 52 ATP Levels in RBCs and T Cells and the Effects of Age on Mitochondrial Potential gregates that emit at 585 nm (orange-red). manner. This indicates a loss of mitochon- If the mitochondrial potential is reduced, drial potential. In addition, the microscopic JC-1 disaggregates to monomers that fluo- observation of cell stained by this dye resce at 527 nm (green). The ratio between showed that the dye was indeed taken into the red and the green signals is indicative the mitochondrial membrane.
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