TY - JOUR
T1 - In Vitro Monitoring of Total Choline Levels in a Bioartificial Pancreas
T2 - 1H NMR Spectroscopic Studies of the Effects of Oxygen Level
AU - Long, Robert C.
AU - Papas, Klearchos K.
AU - Sambanis, Athanassios
AU - Constantinidis, Ioannis
N1 - Funding Information:
The authors gratefully acknowledge support in part from the NIH (DK47858), the NSF (BES9410703), and the ERC Program of the National Science Foundation under Award EEC9731643. We also acknowledge the excellent skilled technical assistance of Mrs. Inge Rask.
PY - 2000/9
Y1 - 2000/9
N2 - This investigation implements specifically designed solvent-suppressed adiabatic pulses whose properties make possible the long-term monitoring of 1H NMR detectable metabolites from alginate/poly-L-lysine/alginate (APA)-encapsulated βTC3 cells. Our encapsulated preparations were maintained in a perfusion bioreactor for periods exceeding 30 days. During this prolonged cultivation period, the cells were exposed to repetitive hypoxic episodes of 4 and 24 h. The ratio of the total choline signal (3.20 ppm) to the reference signal (observed at 0.94 ppm assigned to isoleucine, leucine, and valine) decreased by 8-10% for the 4-h and by 20-32% for the 24-h episodes and returned to its prehypoxic level upon reoxygenation. The decrease in the mean value of total choline to reference signal ratio for three 4-h and two 24-h episodes in two different cultures was highly significant (P < 0.01). The rate of recovery by this ratio was slower than the rates of recovery by oxygen consumption, lactate production, or glucose consumption. A step-up in oxygen level led to a new, higher value for the total choline to reference ratio. From spectra of extracts at 400 MHz, it was determined that 63.6% of the total choline signal is due to intracellular phosphorylcholine. Therefore, it is inferred that the observed changes in total choline signal are linked to an oxygen level dependence of the intracellular phosphorylcholine. Several possible mechanisms in which oxygen may influence phosphorylcholine metabolism are suggested. In addition, the implications of these findings to the development of a noninvasive monitoring method for tissue-engineered constructs composed of encapsulated cells are discussed.
AB - This investigation implements specifically designed solvent-suppressed adiabatic pulses whose properties make possible the long-term monitoring of 1H NMR detectable metabolites from alginate/poly-L-lysine/alginate (APA)-encapsulated βTC3 cells. Our encapsulated preparations were maintained in a perfusion bioreactor for periods exceeding 30 days. During this prolonged cultivation period, the cells were exposed to repetitive hypoxic episodes of 4 and 24 h. The ratio of the total choline signal (3.20 ppm) to the reference signal (observed at 0.94 ppm assigned to isoleucine, leucine, and valine) decreased by 8-10% for the 4-h and by 20-32% for the 24-h episodes and returned to its prehypoxic level upon reoxygenation. The decrease in the mean value of total choline to reference signal ratio for three 4-h and two 24-h episodes in two different cultures was highly significant (P < 0.01). The rate of recovery by this ratio was slower than the rates of recovery by oxygen consumption, lactate production, or glucose consumption. A step-up in oxygen level led to a new, higher value for the total choline to reference ratio. From spectra of extracts at 400 MHz, it was determined that 63.6% of the total choline signal is due to intracellular phosphorylcholine. Therefore, it is inferred that the observed changes in total choline signal are linked to an oxygen level dependence of the intracellular phosphorylcholine. Several possible mechanisms in which oxygen may influence phosphorylcholine metabolism are suggested. In addition, the implications of these findings to the development of a noninvasive monitoring method for tissue-engineered constructs composed of encapsulated cells are discussed.
KW - H NMR
KW - Hypoxia
KW - Total choline
KW - βTC3 insulinoma cells
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U2 - 10.1006/jmre.2000.2112
DO - 10.1006/jmre.2000.2112
M3 - Article
C2 - 10968957
AN - SCOPUS:0034266119
SN - 1090-7807
VL - 146
SP - 49
EP - 57
JO - Journal of Magnetic Resonance
JF - Journal of Magnetic Resonance
IS - 1
ER -