Insight into cell metabolism will help interpret PET, MRI scans
13 July 2005
By discovering a crucial piece of submicroscopic information about how
the brain converts fuel into energy for neurons, Cornell University
biophysicists have gleaned new insights into brain cell metabolism that will
allow neurologists to better interpret data from such diagnostic tests as
PET scans and blood oxygen level dependent functional magnetic resonance
imaging (BOLD-fMRI) scans.
The discovery uncovers a key piece of information that's been missing for
years about cell metabolism — how the compound beta —nicotinamide adenine
dinucleotide (NADH) interacts in the mitochondria. The researchers
discovered that some molecules of NADH are bound to other molecules in the
mitochondria, while some are free in two different conformations. Whether
NADH is bound or free affects how much it fluoresces in diagnostic tests —
and not knowing this has led scientists in the past to misjudge the amount
of activity in neural cells.
The findings, published as the paper of the week in the July 1 issue of
the Journal of Biological Chemistry (Vol. 280), are based on research in the
biophysics lab directed by Watt W. Webb, the S.B. Eckert Professor in
Engineering at Cornell. The journal's cover illustration was designed by
Webb with images from his biophysics lab by Karl Kasischke, Harshad
Vishwasrao, and Dan Dombeck.
Vishwasrao, the lead author of the paper and a former graduate student of
Webb's, was able to differentiate between bound and the two forms of free
states of NADH molecules based on the rate that molecules rotate, or don't
rotate, over nanoseconds of time. He used a technique developed by Ahmed
Heikal (now of Pennsylvania State University) in Webb's lab.
NADH concentration has been used as an indicator for cell metabolism for
some 50 years, but harmful levels of ultraviolet radiation were required to
induce the fluorescence needed for the measurements. Webb and his
colleagues, however, devised a technique several years ago that uses short,
intense laser pulses of harmless infrared instead of ultraviolet radiation.
The technique, called multi-photon laser scanning microscopy (MPLSM),
allowed the Vishwasrao team to measure NADH levels in cells with controlled
levels of oxygen saturation without damaging the cells. And unlike other
methods, MPLSM can simultaneously show how the orientation of NADH molecules
changes (by measuring their anisotropy) within fractions of a nanosecond.
The results, said Vishwasrao, now a postdoctoral fellow at Columbia
University, indicate that the unbound NADH molecules rotate much more
quickly — and therefore lose their fluorescence more quickly — than bound
"One bound NADH molecule is about as bright as 10 free ones," said
Vishwasrao. "When we first got evidence that there was free NADH, we thought
we made a big mistake. We thought we were crazy. We went back, and the more
we talked about it, and the more experiments we did, it became clear. Other
groups were seeing the same thing."
When the team used the data to calculate the proportion of bound-to-free
NADH molecules in a section of tissue, they found that their calculations
resolved inconsistencies that had troubled researchers for years. "The
effect is large enough to account for the frequently seen problems," said
NADH is a good indicator of cell activity for several reasons. First, the
molecule is ubiquitous in the mitochondria, where oxidative metabolism takes
place. It also naturally fluoresces, which means it can be detected without
adding artificial tracers or dyes. And because NADH is converted in the
metabolic process to non-fluorescent NAD+, researchers can gauge how much
oxidation is occurring in a cell based on its fluorescence.
With this new information, Vishwasrao said, scientists and physicians who
study the effects of stroke, Alzheimer's disease and other brain injuries
and pathologies will be better equipped to interpret quantitative data from
diagnostic techniques they've been using—without fully understanding—for
"The role of multi-photon imaging and spectroscopy of NADH is not to
replace other imaging techniques," he said, "but rather to provide a more
detailed microscopic framework of brain metabolic dynamics within which
macroscopic techniques, such as such as PET, and BOLD-fMRI, and optical
scanning, can interpret their respective detected signals."