In Vivo Assessment of Cellular Metabolism in Humans

The tricarboxylic (TCA) or Krebs cycle is the "central hub of cellular metabolism" that takes place within the mitochondria. It is a series of sequential chemical reactions that generate cellular energy in the form of ATP. In addition, the cycle provides intermediate metabolites that are utilized in the biosynthesis of amino acids and fatty acids as well as reducing agents such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2) that are used in numerous biochemical reactions. The dysfunction of the TCA cycle is recognized for its association in neurodegenerative and cardiovascular diseases, metabolic syndromes, tumorigenesis and aging. Hence, being able to measure the activity or flux of the TCA cycle either in vitro or in vivo holds significant clinical significance. Almost all studies are based on in vitro approaches except NMRS based studies that involve multiple non-validated assumptions.

Various stable isotope labeling studies have been used to estimate the TCA cycle flux by measuring one or more labelled intermediate metabolites within the cycle. Unfortunately, these labelled intermediates are often present through only partial segments of the cycle due to exchange, anaplerosis (entrance into the cycle), cataplerosis (export out of the cycle) or incomplete cycling. Though these previous isotope labeling studies of the TCA cycle flux were qualitatively informative, many were quantitatively inaccurate due to unexpected dilutions of the TCA cycle intermediates arising from unlabeled precursors.

This is a pilot study to establish a novel methodology using mass-isotopomer flux analysis after infusions of 2-13C-Acetate, 2-15N-Glutamine and D5-phenylalanine to measure the in vivo TCA cycle flux in tissues of human beings. This study will simultaneously determine the validity of measuring the TCA cycle flux in tissue indirectly through dynamic differences in enrichment of labelled TCA cycle intermediates between arterial and venous blood supplies of that particular tissue bed (i.e. arteriovenous model or A-V balance technique). We propose to measure the rates of the different metabolic reactions within the TCA cycle by tracing the position-specific 13C and 15N transfer between the intermediate metabolites in order to characterize the oxidative, anaplerotic, cataplerotic and exchange rates across the TCA cycle. The use of 2-15N-Glutamine will specifically allow us to determine the rate of glutamine entry into the cycle via its conversion to glutamate, thus providing a more accurate quantification of the TCA flux.

This methodology will be validated in the setting of controlled physiologic perturbations in human study participants such as low endogenous insulin levels alone or in combination with high glucagon levels.

Finally, correlative studies evaluating the mitochondrial activity in the skeletal muscle tissue, the oxygen consumption in the skeletal and splanchnic tissue beds, the role of circulating exosomes derived from the arteriovenous circulation of the skeletal and splanchnic tissue beds and the changes in the whole body metabolome will also be performed:

• First, mitochondrial respiration will be measured by high resolution respirometry (Oxygraph, Oroboros Instruments, Innsbruck, Austria) using a stepwise protocol to evaluate various components of the electron transport system. Protein content of the mitochondrial suspension will be measured using a colorimetric assay (Pierce 660-nm Protein Assay). Oxygen flux rates will be expressed per tissue-wet weight and per milligram of mitochondrial protein.
• Secondly, reactive oxygen species (ROS) emissions will also be evaluated on all skeletal muscle tissue samples. Briefly, a Fluorolog 3 (Horiba Jobin Yvon) spectrofluorometer with temperature control and continuous stirring will be used to monitor Amplex Red (Invitrogen) oxidation in freshly isolated mitochondrial suspensions obtained from the skeletal muscle biopsies. Amplex Red oxidation will be measured in the presence of glutamate (10 mmol/L), malate (2 mmol/L), and succinate (10 mmol/L). The fluorescent signal will be corrected for background auto-oxidation and calibrated to a standard curve. The H2O2 production rates will be expressed relative to mitochondrial protein.
• Third, simultaneous assessments of the oxygen consumption and carbon dioxide production will be determined through blood gas measurements from the arteriovenous samples obtained from the splanchnic and skeletal muscle tissue beds. These assessments will be performed at all three time points of blood sample assessments and correlated with the measured TCA cycle flux in their respective tissue beds.
• Circulating exosomes will also be derived from the arteriovenous samples of the splanchnic and skeletal muscle tissue beds to determine its intra-exosome proteome and metabolome and its relationship with the TCA cycle flux in their respective tissue beds. Incorporation of D5-phenylalanine will help trace the protein formation in the exosomes.
• Finally, changes in the whole body metabolome and proteome determined via the arteriovenous samples obtained from the splanchnic and skeletal muscle tissue beds will also be performed and correlated with the TCA cycle flux in their respective tissue beds.