Methodology is Philosophy - Systems Biology: Philosophical Foundations - page 77

signals from in vivo metabolites, e.g., glucose, ATP, glutamate, and glutamine
that are usually, but not always, small molecules. Measurements of the proton
or natural abundance 13 C signals of such metabolites can be used to determine
their concentrations. These concentrations can be localized using the NMR
methods that support the parallel method of magnetic resonance imaging (MRI)
which images the internal body. After having identified the metabolites by their
characteristic NMR spectrum their concentrations are localized to particular body
regions by MRI methods.
The theoretical component of the methodology based on the MRS experimen-
tal results is supplied by metabolic control analysis (MCA). An excellent review
of the concepts of MCA can be found in Fell (1997). MCA, also described
elsewhere in this topic, provides definitions of experimental parameters and a
logical analysis of the results so as to evaluate these parameters. The parameters
describe the control of in vivo fluxes in terms of the in vitro properties of the
constituent enzymes. They also describe the effects of these enzymes and the
in vivo fluxes on concentrations of metabolites. Therefore, MCA provides a
framework for relating the measured properties of the constituent enzymes to
the in vivo properties of metabolic pathways. It allows one to go up one level
of complexity, from molecules to metabolism.
Metabolic fluxes are measured by labeling a molecule, usually at the beginning
of a pathway, and following the label by MRS. The most useful label has been
13 C, which is only 1.1% naturally abundant. The weak natural abundance allows
signals to be obtained from unlabeled metabolites only when they are more
concentrated. One particularly useful signal, even in the absence of labeling,
has been from the large glycogen molecule, which was early shown to give a
well-resolved MRS signal, acting in the NMR spectrum like a small molecule
because of its mobile molecular structure. Infusing 99% labeled 13 C glucose into
the human or animal blood stream allows the flow of glucose into glycogen to be
measured even more quantitatively by 13 C MRS in the human skeletal muscle,
heart, or liver (Fig. 1). This particularly quantitative measurement of fluxes
has allowed our experiments to illustrate the inseparability of methodology and
philosophy.
Our experiments have explored the pathway of glycogen synthesis, figura-
tively shown in Fig. 2. (For a review see Shulman & Rothman, 2001.) Plasma
glucose is taken up by the muscle via passive glucose transporters. For the ranges
of plasma glucose concentrations studied, the transporters operate in a linear
range. They can be recruited by insulin, thereby increasing their velocity, but
in the experiments described insulin levels were maintained constant. The flux
from glucose to glycogen was measured by clamping plasma glucose at two dif-
ferent levels and measuring the rate of incorporation of the glucose into muscle
glycogen by 13 C MRS. To improve the signal strength, plasma glucose was
enriched by infusing 13 C-enriched glucose. The plasma glucose concentrations