Page 42 - Read Online

Bennett. J Transl Genet Genom 2020;4:36-49 I https://doi.org/10.20517/jtgg.2020.17 Page 41

In contrast, studies in the present author’s laboratory indicate that tissue samples frozen between 30 s and
3 min of being harvested from freshly sacrificed animals were rich in signals from MRC redox centers, and
the spectra showed no time-dependent changes over that time span. Samples taken from different parts of
muscle, liver and lung from the same wild-type (w/t) rat exhibited astounding reproducibility, with signal
intensities within 5% for all signals. Between animals, the reproducibility was within 10% (except for the
intensities of signals due to blood components, which did vary significantly). Liver samples have been found
to be completely stable for at least 6 months at -80 °C. A sample of diced brain that had been stored for
1 year at -80 °C and had experienced substantial sampling handling (during which time it was likely that the
temperature rose significantly above -80 °C, though it had never been thawed) showed significant change
(+ 10%) in the aconitase region of the signal, indicative of oxidative conversion of the labile [4Fe4S] to
[3Fe4S]. Otherwise, only a very minor change (< 5%) in the composite FeS resonance at g = 1.92 was
eff
seen. Liver MRC signals were surprisingly tolerant of freeze-thawing, with only very small changes (< 5%)
observed in the aconitase/S3/UQ region of the spectrum. Muscle was much less tolerant of freeze-thaw
2
cycles, with large increases in the signals from ferriheme, and aconitase and/or S3, indicating both structural
damage and oxidation. Small but significant changes in other signals included an increase in Cu , a decrease
A
in the Complex I/II composite FeS signal at g = 1.92, and specifically a larger decrease in N3 compared
eff
to N4 or the g = 1.93 feature, suggesting an increase in the overall redox potential. It appears then that
eff
medium term storage at -80 °C preserves the EPR signals and thus the redox status of the mitochondria, but
sample handling must be carried out carefully to avoid warming, and thawing must be avoided.

Samples are most readily prepared by transfer of fresh tissue into an EPR tube followed by freezing in dry-
ice/methanol, liquid nitrogen-chilled isopentane, or liquid nitrogen; the latter is a slower freezing method but
is more convenient and safer in a clinical environment due to the lack of flammability. The sample is typically
prepared by rapid extrusion of intact tissue from a syringe into an EPR tube that is blown from a length of
quartz to leave a small hole in the bottom to prevent air spring resistance. Ideally, samples should completely
fill the volume of the tube that occupies the active length of the EPR resonator for reproducible quantitation.
Smaller samples can be mounted in the center of the active region of the resonator by first freezing a platform
of a water-glycerol mixture at the bottom of the tube, adding the tissue, and adding more water-glycerol
to the desired height before freezing. The latter step maintains a more-or-less uniform dielectric constant
along the active region of the resonator, which maintains consistency of B , the oscillating field due to the
1
microwave, across the sample and between samples for a given microwave power and resonator.

QUANTITATIVE ANALYSIS OF EPR SPECTRA
There are a number of challenges to data analysis. One complicating factor is that each of the contributory
signals exhibits different dependencies on temperature and microwave power. Therefore, an empirical scaling
factor needs to be determined for each signal for any given set of conditions. Phenomena that can reduce
the observed intensity of the EPR signal include rapid-passage effects at too-low temperatures, relaxation-
broadening at too-high temperatures, and power saturation at too-high microwave power. The most efficient
way to arrive at scaling factors is to first determine the temperature dependence of each signal in order to (1)
identify a temperature (or a small number of temperatures) at which most or all of the signals are observable;
and (2) provide a temperature coefficient to account for any deficiency in signal intensity at the temperatures
to be used for routine measurement compared to the maximum intensity that the temperature-dependence
experiments predict. Then, a power-dependence at the preferred temperatures is carried out. Modern
commercial instruments allow for two-dimensional experiments that can be programmed and run overnight
to rapidly facilitate this process.

A second challenge is that many of the signals overlap, sometimes extensively. From the 24 MRC redox
centers distributed among Complexes I-IV, 18 discrete EPR signals have been observed from tissues and
•
mammalian cells under various conditions (FeS N1b, N2, N3 & N4 from Complex I; ferriheme, [UQ ]
2