Chemistry: analytical and immunological testing – Nuclear magnetic resonance – electron spin resonance or other...
Reexamination Certificate
1998-07-16
2001-12-11
Soderquist, Arlen (Department: 1743)
Chemistry: analytical and immunological testing
Nuclear magnetic resonance, electron spin resonance or other...
C424S009300, C424S009350, C435S004000, C435S029000, C435S030000, C435S035000, C436S056000, C436S063000, C436S094000, C436S095000, C436S127000, C436S128000, C436S129000
Reexamination Certificate
active
06329208
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to convenient and efficient methods of assay and diagnosis of metabolic states, particularly gluconeogenesis, pyruvate flux and anapleurosis.
2. Description of Related Art
Current methods for the measurement of gluconeogenesis in humans require exposure to radioactive materials. Unfortunately, this feature precludes almost any quantitative studies of these pathways in patients. The development of
13
C NMR for clinical applications is very attractive because of its convenience and the dramatic improvement in metabolic detail which it can provide. Although direct in vivo NMR spectroscopy is the most exciting application for
13
C tracer studies, it is unlikely that appropriate whole-body systems will be widely available any time soon for clinical research. On the other hand, analytical NMR spectrometers suitable for
13
C NMR studies of human blood or urine are already in place in every medical school in this country.
Measurement of hepatic gluconeogenesis using both
13
C and
14
C-labeled glucose is by now a well-developed approach and had been successfully applied in both animals and humans. However, measurements of absolute anaplerotic fluxes have been largely restricted to perfused organs and tissues and the methods cannot be performed in vivo. Since only gluconeogenesis can currently be measured noninvasively, a necessary simplification is to assume that gluconeogenesis is equal to anapleurosis. The penalty for this assumption is that the total anaplerotic flux is underestimated to an unknown degree, and the allocation of anaplerotic carbons between gluconeogenesis and the other biosynthetic pathways is not known.
There is widespread interest in measurement of flux through gluconeogenesis in animals and humans with the goal of a better understanding of dietary and hormonal regulation of fluxes through all associated pathways. Numerous techniques have been used for such measurements, including GC-mass spectrometry (Katz et al., 1993; Katz, 1985; Tayek and Katz, 1996) NMR (Cohen, 1981; Cohen, 1987; Cohen, et al. 1981), and radiotracers (Strisower et al., 1952; Landau et al., 1993). One key control point that connects these two pathways is the interconversion of PEP and pyruvate, catalyzed in the forward direction by pyruvate kinase and in the reverse direction by the combined action of pyruvate carboxylase and phosphoenolpyruvate carboxykinase. Many early studies of gluconeogenesis detected excess cycling of three carbon units through this metabolic intersection. This process, often referred to as pyruvate recycling on an excess substrate cycle, has been detected in studies of isolated hepatocytes (Clark et al., 1973; Jones and Titheradge, 1996), perfused livers (Friedman et al., 1971), and in rats and humans (Magnusson et al., 1990; Petersen et al., 1994).
Most early metabolic models have relied on measurements of isotope enrichment in two or more sites of various product molecules. In prior metabolic studies of pathways associated with the Krebs citric acid cycle, the detection of
13
C-
13
C spin-spin NMR couplings in product molecules has been demonstrated. This approach, which is referred to as
13
C isotopomer analysis, can in some cases provide more information about isotope labeling patterns than other tracer methods. Recently, the analysis of propionate metabolism in the isolated heart under conditions where the pathway succinyl-CoA→pyruvate→acetyl-CoA was significant (Jeffrey et al., 1996) was reported. In contrast to entry of labeled acetyl-CoA and scrambling of this label in cycle reactions, this metabolic condition yields a series of nonlinear equations, which in general are difficult to solve analytically. Although the Newton-Raphson procedure is well known (Press et al., 1988), the complexity of the equations in the comprehensive model obscures some simple and helpful relations between the
13
C NMR spectrum and metabolic state. One such condition occurs during hepatic metabolism of [1,2,3-
13
C]propionate.
SUMMARY
The present invention seeks to overcome the aforesaid and other drawbacks by providing a method allowing gluconeogenesis and total anapleurosis to be measured independently, but simultaneously, in a non-invasive manner. This method is unique because it does not require radioactive tracers and can be easily transferred to the clinical environment for studies of abnormalities in glucose metabolism and hepatic synthetic function. The method allows routine and convenient analysis of gluconeogenesis and other pathways intersecting the citric acid cycle of the liver under almost any clinical circumstance. The basic approach is applicable to animal models and to humans.
Simple interpretations of the
13
C NMR spectra of glucose, glutamate, glucuronide and phenylacetylglutamine are presented in terms of gluconeogenesis and pyruvate recycling. The methods are demonstrated in isolated perfused rat livers, in samples collected from rats after intragastric versus intravenous administration of propionate, and in humans after oral ingestion of propionate, phenylacetate, and acetaminophen. The data demonstrate that gluconeogenesis and pyruvate recycling can be measured in a single
13
C NMR spectrum of glucose obtained from blood samples or of glucuronide or phenylacetylglutamine obtained from urine samples after oral administration of [1,2,3-
13
C]propionate, phenylacetate, and acetaminophen. The simple processing protocols for both blood and urine are well within the capability of a standard medical analytical laboratory. The disclosed methods will be particularly useful in studying and assessing liver damage, such as caused by diabetes, sepsis, cirrhosis and other diseases that affect liver function. Additionally, the methods may be useful in assessing the effect of liver damage caused by trauma.
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Cottam G. Larry
Jeffrey F. M. H.
Jones John G.
Malloy Craig. R.
Sherry A. Dean
Board of Regents , The University of Texas System
Fulbright & Jaworski
Soderquist Arlen
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