Page 278 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioTechnology

P1: GPB/GRB P2: GLQ Final pages
Encyclopedia of Physical Science and Technology EN016J-783 August 1, 2001 10:58

Tissue Engineering 829

pentose phosphate, urea cycle, tricarboxylic acid cycle, since evolved into metabolic flux balance models that
fatty acid synthesis and oxidation) are interrelated through can predict intracellular fluxes in complex metabolic net-
common precursors and metabolic intermediates. Thus, works. This methodology utilizes a stoichiometric model
an enhanced perspective of metabolism and cellular that describes the major intracellular reactions at steady
function can be obtained by considering a framework state. Extracellular fluxes, which correspond to rates of
that incorporates all the major participating reactions, consumption/production of extracellular metabolites, are
rather than a few isolated ones. Two methodologies experimentally determined, and intracellular fluxes are
for the characterization and analysis of cell metabo- calculated based on the stoichiometric constraints of the
lism that are especially useful for the analyses of metab- intracellular reaction network. This approach has been
olic abnormalities in human disease are metabolic flux used extensively to study and improve strains of microor-
analysis and metabolic control analysis. ganisms (bacteria and yeasts) of significance in biotech-
Metabolic flux can be defined as the net rate of conver- nology. As of now, applications of metabolic flux balance
sion of one metabolic precursor to a product. Metabolic models to mammalian cell systems have been more lim-
flux analysis refers to the calculation of fluxes through ited but are gaining in popularity.
metabolic pathways. Two techniques are primarily used The starting point in this analysis is the construction of a
for flux determination: (1) mass isotopomer analysis, and list of steady-state material balance equations to describe
(2) extracellular metabolite balance models. Mass iso- the conversion of substrates to metabolic products for the
topomer analysis has been used extensively to quantitate biochemical system of interest. For example, if one con-
fluxes in mammalian cells and tissues including brain, siders a simplified scheme of amino acid metabolism in
heart, and liver. In this approach, the body is fed, or the iso- liver, one can write a set of steady-state material balance
lated tissue is perfused with, substrates labeled with stable equations that represent the flow of metabolites through
13
isotopes (e.g., C). A different labeling pattern of metabo- the network (Fig. 9). The equations contain measurable
lites in the blood, perfusate, and/or tissue extract arises de- quantities(thesearemarkedwithanasterisk)whicharethe
pending on the pathways utilizing these substrates. The la- rates of consumption/production of extracellular metabo-
beling patterns are experimentally determined by nuclear lites. The concentrations of strictly intracellular metabo-
magnetic resonance or mass spectroscopy. These labeling lites (e.g., argininosuccinate) are assumed to be constant.
patterns are analyzed in conjunction with a mathematical In this particular case, we have eight fluxes to be deter-
∗
∗
∗
∗
model to calculate the fluxes through the various pathways mined, five of which are measurable (F ,F ,F ,F ,F ).
∗
1 2 4 7 8
which best account for the observed labeling patterns. Al- The five equations listed here, which relate these fluxes
though isotopomer analysis is a powerful and generally to each other, can be reduced to four independent equa-
noninvasive method, stably labeled compounds and the tions. Thus, the system can be solved to yield the three
instruments required to determine the isotopomer distri- unknown intracellular fluxes (F 1 ,F 2 ,F 4 ). Because the sys-
butions of key metabolites are relatively expensive. tem is overdetermined, it provides an internal check for
Material balances of whole-body macronutrients have consistency of the data with each other and the assumed
been used since the 19th century for evaluating bulk ma- biochemistry.
terial processing with the body (e.g., to study the con- While this method is very useful, there is a limit to
version of carbohydrates to fat). Material balances have the extent to which complex metabolic networks can be

FIGURE 9 System of mass balance equations describing the flow of metabolites through the urea cycle of hepato-
cytes. Measurable fluxes are labeled with a star.