New insight into cellular metabolism will help neurologists better interpret dia

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New insight into cellular metabolism will help neurologists better

interpret diagnostic tests

12 Jul 2005 Medical News Today

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 positron emission tomography (PET) scans and

a specialized magnetic resonance imaging (MRI) test.

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 a 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, such as PET and

blood oxygen level dependent functional magnetic resonance imaging

(BOLD-fMRI), 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 NADH molecules.

" 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 Webb.

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 years.

" 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 BOLD-fMRI , PET and

optical scanning, can interpret their respective detected signals. "

Webb's lab, the Developmental Resource for Biophysical Imaging Opto-

electronics (DRBIO), has yielded other significant advances recently.

Last year, Kasischke, formerly of Cornell and now a resident in

neurology in Germany, used MPLSM to tease apart the metabolic

functions of neurons (nerve cells) and astrocytes (star-shaped cells

that provide neurons with fuel). Kasischke's paper filled an

important piece of the metabolic puzzle by showing that brain cells

have distinct roles in the metabolic process. His team (which

included Vishwasrao and Webb) found that neurons use oxygen to

convert carbohydrate to energy (a process called oxidation) and that

astrocytes kick in subsequently to produce lactate fuel (a process

called glycolysis). This confirmed a controversial function based on

the hypothesis known as the astrocyte-neuron lactate shuttle, and the

finding helped researchers to better understand how the metabolic

process works on a microscopic level.

Vishwasrao's current paper is co-authored by Kasischke, Webb and

Heikal.

http://www.news.cornell.edu