In Vitro Analysis of Apoptotic Cell Death Using Proton Nuclear Magnetic Resonance Spectroscopy

Shin, Dong Gun; Kim, Sang Kyung; Kim, Jong Ki

Abstract

PURPOSES: Cells undergoing apoptosis display profound morphologic and biochemical changes in the nucleus and cytoplasm, loss of membrane phospholipid asymmetry, resulting in the exposure of phosphatidylserine (PS) at the surface of the cell, membrane blebbing, and decreased membrane microviscosity. Proton nuclear magnetic resonance spectroscopy ('H NMR spectroscopy) is able to detect the mobile fraction of lipids contained in the cell, and thus is sensitive to membrane fluidity modifications related to lipid composition changes. We have used 'H NMR spectroscopy in HL-60 cell line to detect and characterize the changes in plasma membrane lipid associated with apoptotic cell death.
MATERIALS AND METHODS
We performed annexin-FITC and propidium iodide dual fluorescence flow cytometry, DNA gel electrophoresis, and obtained 200 MHz 'H NMR spectra of the HL-60 cell cultures before and at 6, 12, 18, 24, 36 and 48 hours after the addition of doxorubicin (100 ng/mL).
RESULTS
The onset of apoptosis is accompanied by a greater than four fold increase in signal intensity ratio of the membrane lipid methylene (-CH2) resonance (at 1.2 ppm) to the methyl (-CH3) resonance (at 0.9 ppm). The quantitative relationship between apoptosis and the H NMR signal intensity was determined by fluorescein-annexin V flow cytometry, and showed that increases in the CH2/CH3 resonance signal intensity ratio paralleled the surface expression of PS as an early marker of apoptosis ( y =0.80, N 18 samples). The gradual decrease in the ratio of choline resonance (at 3.2 ppm) to CH3 signal intensity after 12 hours in the time course' experiment is directly proportional to the percentage of apoptotic cells ( y =0.96, N=18 samples).
CONCLUSIONS
Monitoring of the CH2 and choline resonance signal intensity may therefore be useful in detecting apoptosis. Further studies using various stimuli to induce apoptotic cell death will be necessary to better determine the capabilities of 'H NMR spectroscopy for the detection and estimation of apoptosis in vitro.