A fast and sensitive ¹H NMR method to measure the turnover of the H2 hydrogen of lactate

Abstract

A fast and sensitive procedure to determine the turnover of the H2 hydrogen of lactate and quantify its 2 H-enrichment by 1 H NMR is illustrated using C6 cells metabolizing (3-13 C) lactate in 50% 2 H 2 O (vol/vol). 2 H substitution of the lactate H2 hydrogen resulted in two easily detectable transformations of the vicinal H3 doublet resonance: 1) the formation of an H3 singlet due to the disappearance of the homonuclear coupling to H2 ( 3 J ␤H-␣H ‫؍‬ 7.0 Hz), and 2) an upfield isotopic shift derived from the vicinal 2 H2 substitution (⌬ 3 ‫؍‬ -0.007 ppm). Only those lactate molecules that have passed through the cell cytosol experience these effects, since H2 deuteration involves lactate dehydrogenase activity and NAD( 2 H). Thus, analysis of the observed shifted and unshifted H3 lactate resonances from the incubation medium allows the discrimination of the perprotonated (3-13 C) lactate added as substrate, and the (3-13 C, 2-2 H) lactate recycled to the incubation medium after passage through the cytosol. Magn Reson Med 54:1014 -1019, 2005.

In vivo 13C saturation transfer effect of the lactate dehydrogenase reaction

“…Finally, the fast exchange rate between lactate and pyruvate determined in this study indicates that LDH is not the rate-limiting step for the much slower cytosolic deuteration of lactate H2 which involves several enzymes (40).…”

Increased Oxidative Metabolism and Neurotransmitter Cycling in the Brain of Mice Lacking the Thyroid Hormone Transporter Slc16a2 (Mct8)

“…This was possible since myo -inositol had a low turnover and did not become enriched after a 30 min (1- 13 C) glucose infusion independently of the thyroid status [38], providing a robust internal reference from which all 13 C enrichments can be derived. The absolute amount of 13 C incorporation in the different carbons and the fractional enrichment was determined as previously described [41], [42].…”

Kinetic properties of the redox switch/redox coupling mechanism as determined in primary cultures of cortical neurons and astrocytes from rat brain

“…In the original ANLS hypothesis, a flux of lactate was proposed to operate from astrocyte to neuron. The reversibility of the lactate dehydrogenase isozymes and the monocarboxylate transporters of neurons and astrocytes allows these neural cells to produce and consume lactate or pyruvate, resulting in recycling of carbon and reducing equivalents through the corresponding plasma membranes (Rodrigues and Cerdan, 2005b; Rodrigues et al, 2005). Moreover, because both lactate and pyruvate can exchange reversibly between the cytosol of each cell and the extracellular space, it follows that these metabolites can also be exchanged transcellularly between both cells in a lactate/pyruvate shuttle, an aspect previously not envisioned.…”