Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving glucose or galactose
Reexamination Certificate
2000-11-06
2003-11-25
Witz, Jean C. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving glucose or galactose
C435S004000
Reexamination Certificate
active
06653090
ABSTRACT:
TECHNICAL FIELD
This invention relates to medicine and physiology. In particular, the invention provides methods for measuring the metabolism of the heart. The invention also provides methods for screening for compounds that can effect the metabolism of the heart under normal and abnormal, such as stressed, e.g., ischemic, conditions. These screening methods can be used to identify therapeutic drugs.
BACKGROUND
Glycogen is an important source of glucose for energy substrate metabolism and ATP generation in the heart. The contribution of glycogen as an endogenous source of glucose depends on both energy substrate availability as well as the metabolic status of the heart. The regulation of glycogen synthesis and degradation (turnover) has been extensively studied in the liver. Although the heart has a great potential for the synthesis and storage of glycogen, myocardial glycogen turnover is less well understood, particularly under ischemic or reperfused conditions.
When cardiac muscle is adequately perfused, exogenous glucose is transported into myocytes, where it primarily either enters the glycolytic pathway or is stored as glycogen. This glycogen can also be subsequently mobilized to provide a source of endogenous glucose for glycolysis. During ischemia, when the supply of O
2
and exogenous substrates is impaired, fatty acid and glucose oxidation are inhibited and ATP generation from anaerobic glycolysis increases. Although glycolysis produces ATP in the absence of O
2
, excessive rates of glycolysis may be deleterious during and after severe ischemia due to the production of protons from the hydrolysis of glycolytically derived ATP. During reperfusion of ischemic hearts, glycolytic rates continue to exceed glucose oxidation rates. This uncoupling of glycolysis from glucose oxidation continues to be an important source of protons and leads to intracellular acidosis, Na
+
accumulation, and Ca
2+
overload. Glucose released from glycogen under conditions of glycogenolysis may be preferentially oxidized. If so, then endogenous glucose may be contributing less to proton production than exogenous glucose. Thus, the preferential utilization of endogenous glucose, rather than exogenous glucose, may result in lower rates of proton production and Ca
2+
overload.
However, the relationships between various parameters of metabolism, including endogenous and exogenous glucose metabolism, proton production, fatty acid utilization and the like, are not completely understood.
Current methods of detecting energy metabolism rely on indirect approaches and thus have significant drawbacks. These include: (1) they only indirectly assess energy metabolism in the heart; (2) they cannot quantitatively determine flux through the individual energy producing pathways; (3) they cannot directly assess either the contribution of glucose from glycogen or fatty acid from triacyiglycerol as a source of energy; (4) they cannot measure energy metabolism in the presence of physiological workloads or in the presence of myocardial ischemia, and (5) they cannot directly compare energy metabolism to oxygen consumption in the heart.
SUMMARY
The invention provides novel methods for measuring overall endogenous and exogenous fatty acid utilization and glucose metabolism, complete energy metabolism and exogenous energy metabolism by a working heart under “normal” and ischemic conditions. Using these novel methods, the invention provides methods for screening for compounds that can effect the metabolism of the heart under normal and abnormal, such as stressed, e.g., ischemic, conditions. These screening methods can be used to identify therapeutic drugs.
The invention provides methods for measuring overall endogenous and exogenous fatty acid utilization by a working heart comprising the following steps: (a) providing an isolated working heart; (b) measuring fatty acid oxidation and lactate oxidation in the working heart simultaneously; and, (c) measuring triacyiglycerol turnover in the working heart, wherein step (c) can be performed during or after step (b), thereby measuring overall endogenous and exogenous fatty acid utilization in the working heart.
In this method, as in all methods of the invention, the heart can be derived from any source; in one embodiment, the isolated working heart is a mammalian heart, such as a monkey or ape heart, a rat heart, a rabbit heart, or a mouse heart, The heart can be isolated by any methodology. In this and all methods of the invention the isolated working heart can be subjected to a trauma or a drug before a measurement or sample is taken; for example, the trauma can be an induced ischemia. Alternatively, the ischemia can be induced during or after initiation of any of the measurements. The measuring can be done or the sample taken before, during and/or after the ischemic event. In one embodiment, the isolated working heart is reperfused before measuring or sampling. The reperfusion can be designed to induce and/or prolong a state of ischemia. The reperfusion can be designed to allow slow or fast recovery from an ischemic event.
In one embodiment, the isolated working heart is reperfused to induce a low-flow ischemia. The ischemia can be induced by stopping perfusate flow through a left atrial inflow line and limiting flow throughout an aortic outflow line, thereby generating any range of aortic perfusion rates and any degree of ischemia. Severe ischemia can be induced by generating aortic perfusion rates ranging from about 0.5 ml/min to about 2 ml/min. Moderate ischemia can be induced by generating aortic perfusion rates from about 2 ml/min to about 5 ml/min. Mild ischemia can be induced by generating aortic perfusion rates from about 5 ml/min to about 15 ml/min.
In the methods of the invention, fatty acid oxidation can be measured by any known methodology or protocol. In one exemplary embodiment, fatty acid oxidation of the isolated heart is measured by quantitative collection of
14
CO
2
from [
14
C]palmitate, as described, e.g., by Saddik (1991) J. Biol. Chem. 266:8162-8170; Saddik (1992) J. Biol. Chem. 267:3825-3831; Lopaschuk (1997) Mol. Cell. Biochem. 172:137-147; or, Barr (1997) J. Pharmacol. Methods 38:11-17; or, combinations thereof or variations thereof.
In another exemplary embodiment, fatty acid oxidation of the isolated heart is measured by quantitative collection of
3
H
2
O production from [
3
H]palmitate, as described, e.g., by Saddik (1991) supra; Saddik (1992) supra; or, Lopaschuk (1997) supra; or, Barr (1997) supra; or, combinations thereof or variations thereof.
In the methods of the invention, lactate oxidation can be measured by any known methodology or protocol; for example, it can be measured by quantitative collection of
14
CO
2
from [
14
C]lactate, as described, e.g., by Liu (1996) Circ. Res. 79:940-948; or, Lopaschuk (1997) supra; or, Barr (1997) supra; or combinations thereof or variations thereof.
In another exemplary embodiment, lactate oxidation is measured by quantitative collection of
14
CO
2
from [
14
C]lactate, while simultaneously measuring palmitate oxidation, as described by Liu (1996) supra, or variations thereof.
In the methods of the invention, triacylglycerol turnover can be measured by any known methodology or protocol; for example, it can be measured using a pulse-chase procedure in which quantitative collection of
3
H
2
O production from exogenous [
3
H]palmitate and quantitative collection of
14
CO
2
from endogenous [
14
C]palmitate labeled triacylglycerol is measured simultaneously, as described, e.g., by Saddik (1991) supra, Saddik (1992) supra; or variations thereof.
In another exemplary embodiment, triacylglycerol turnover is measured by quantitative collection of
14
CO
2
from exogenous [
14
C]palmitate, and quantitative collection of
3
H
2
O production from endogenous [
3
H]palmitate labeled triacylglycerol, as described, e.g., by Saddik (1991) supra; Saddik (1992) supra; or variations thereof.
The invention also provides
University of Alberta
Wetherell, Jr. John R.
Witz Jean C.
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