J. Biol. Chem., Vol. 265, Issue 36, 22097-22100, 12, 1990
Rate of ATP synthesis in the perfused rat liver by 31P cryo-NMR
I Ikai, M Okuda, N Doliba and B Chance
Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia 19104.
ATP concentrations in the perfused rat liver during normoxic perfusion,
transient ischemia, and recovery from transient ischemia were measured
using the modified 31P cryo-NMR method (Chance, B., Nakase, Y., Bond, M.,
Leigh, J. S., Jr., and McDonald, G. (1978) Proc. Natl. Acad. Sci. U.S.A.
75, 4925-4929). Transient ischemia was induced in the perfused livers of
starved rats, and multiple freeze-trapped tissue samples were taken from
each liver at short intervals (15-30 s) during ischemia or following
reperfusion. The freeze-trapped tissue was pulverized together with an
antifreezing agent and high energy metabolites were measured by 31P NMR at
243 K after thawing. By using the cryo-NMR technique, a biochemical time
resolution of 2 s could be achieved. Absolute metabolite concentrations
were calculated by comparing the peak areas with internal standards mixed
into the samples. Good time resolution and reliable concentration
measurements provided by the cryo- NMR method enable us to estimate the ATP
synthesis rate in the perfused liver during reperfusion following transient
ischemia. The rate of ATP synthesis in the normoxic perfusion was 1.95
mumol/min/g wet weight; the maximal ATP synthesis rate during the recovery
phase from ischemia was 5.75 mumol/min/g wet weight.
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