1H-Observe/13C-decouple spectroscopic measurements of lactate and glutamate in the rat brain in vivo.

Rothman, Douglas L.; Behar, Kevin L.; Hetherington, Hoby P.; Hollander, Jan A. den; Bendall, M.Robin; Petroff, Ognen A. C.; Shulman, Robert G.

doi:10.1073/pnas.82.6.1633

Cited by 219 publications

(116 citation statements)

References 19 publications

(20 reference statements)

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“…These results are in good agreement with a previous estimate of 15-20 min determined for rat brain by Rothman et al (1985); however, the period of ob servation in that study was only =20 min and the data did not permit assessment of animal-to-animal variation. The improved sensitivity (and time reso lution) in the present study resulted in a well characterized time course for each animal.…”

Section: Discussionsupporting

confidence: 88%

The Flux from Glucose to Glutamate in the Rat Brain in vivo as Determined by ¹-Observed, ¹³C-Edited NMR Spectroscopy

Fitzpatrick

Hetherington

Behar

et al. 1990

J Cereb Blood Flow Metab

249

221

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Summary:The rate of incorporation of carbon from [1-13C]glucose into the and [3-CH21 of cerebral glutamate was measured in the rat brain in vivo by IH_ observed, 13C-edited (POCE) nuclear magnetic resonance (NMR) spectroscopy. Spectra were acquired every 98 s during a 6O-min infusion of [l-13C]glucose. Complete time courses were obtained from six animals. The measured intensity of the unresolved resonances of glu tamate and glutamine increased exponentially during the infusion and attained a steady state in -20 min with a first-order rate constant of 0. 130 ± 0.010 min -I (t1/2 = 5. 3 ± 0. 5 min). The appearance of the [3-13CH21 resonance in the POCE difference spectrum lagged behind that of the [4-13CH21 resonance and had not reached steady state at the end of the 60-min infusion (t1/2 = 26. 6 ± 4. 1 min). The increase observed in 13C-labeled glutamate represented isotopic enrichment and was not due to a change in the Cerebral glutamate functions as a neurotransmit ter, is necessary for cerebral ammonia detoxifica tion, and is directly related to cerebral energy me tabolism (see Cooper and Plum, 1987, and ref. therein). Glutamate metabolism occurs in at least two morphologically distinct cellular compartments (Hertz, 1979). A small, rapidly turning over pool of glutamate, thought to be the precursor of brain glu- 170 total glutamate concentration. The glucose infusion did not affect the levels of high-energy phosphates or intrac ellular pH as determined by 31p NMR spectroscopy. Since glucose carbon is incorporated into glutamate by rapid exchange with the tricarboxylic acid (TCA) cycle intermediate a-ketoglutarate, the rate of glutamate label ing provided an estimate of TCA cycle flux. We have determined the flux of carbon through the TCA cycle to be = 1.4 fLmol g -1 min -I. These experiments demon strate the feasibility of measuring metabolic fluxes in vivo using 13C-labeled glucose and the technique of 1 H observed, 13C-decoupled NMR spectroscopy. Key Words: Brain metabolism-[1-13C]Glucose-Glutamate Glycolysis-Nuclear magnetic resonance-Tricarboxylic acid cycle-Two-dimensional heteronuclear spectros copy.tamine, is located within the astrocytes. The pool of glutamate localized to the neurons is larger (at least 80% of total brain glutamate) and has a slower turn over rate than the astrocytic glutamate pool. Each glutamate pool is in equilibrium with a distinct tri carboxylic acid (TCA) cycle (Van den Berg et aI., 1969; Berl et aI., 1970). The flow of carbon from glucose to glutamate and the turnover time of the glutamate pool depend both on the rate of glycolysis and on the rate of entry of glucose carbon into the TCA cycle. Although turnover rates for glutamate have been estimated by radioactive tracer and sta ble isotope studies (Berl et aI., 1962(Berl et aI., , 1970 Cooper et aI., 1988) there has been no acceptable way to measure these rates in vivo.The turnover of cerebral glutamate and other me tabolites can be determined in vivo by monitoring the site-specific incorporation of l 3C using l 3C_ en...

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Section: Discussionsupporting

confidence: 88%

The Flux from Glucose to Glutamate in the Rat Brain in vivo as Determined by ¹-Observed, ¹³C-Edited NMR Spectroscopy

Fitzpatrick

Hetherington

Behar

et al. 1990

J Cereb Blood Flow Metab

249

221

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“…2a: a-aЈ, b-bЈ) from the methyl group of (3-13 C) lactate molecules. The ratio of intensities of the external doublet of doublets to the internal doublet allows the fractional 13 C enrichment in the lactate C3 carbon to be determined (19).…”

Section: Resultsmentioning

confidence: 99%

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

Rodrigues

Cerdán

2005

Magnetic Resonance in Med

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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.

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“…However, direct measurement of lactate and alanine is difficult due to the presence of overlapping lipid resonances and the intense water signal also adds a dynamic range problem. Recently, several techniques have been reported for measuring lactate by tH NMR in perfused organs and in vivo [4][5][6][7][8][9][10][11], The objective of this work is to extend these techniques to simultaneously measure t3C-enrichment in lactate and alanine in hearts supplied with [3-t3C]pyruvate by proton double quantum metabolite specific spectroscopy.…”

Section: Introductionmentioning

confidence: 99%

Direct observation of lactate and alanine by proton double quantum spectroscopy in rat hearts supplied with [3‐¹³C]pyruvate

Zhao¹,

Sherry

Malloy

1992

FEBS Letters

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The °C-fractional enrichments in the lactate and alanine methyl carbon positions were determined by tH NMR spectroscopy of e~tracts of rat hearts t'~rfused with various coneentlations of [3-L'~C]pyruvate + unlabeled glucose or acetate, In general, the ~C-fraetional enrichment of the alaaine methyl carbon pool paralleled the t~C-fractional enrichment of the aeetyl.CoA which entered the TCA cycle (as determined by ~C-isotopomer analysis) while the t~C.fractional enrichment of the lactate methyl carbon was always significantly lower, consistent with a pool of lactate which does not mix with exogeneous [3-~'C]pyruvate, This has also been examined in intact, perfuscd, KCI-arrasted rat hearts supplied with [3-t'~C]pyravatc by proton doable quantum metabolite specific spectroscopy (MSS). A comparison of MSS spectra of intact hearts with one pulse spectra of extracts of those same hearts indicates there is a sizeable non-enriched pool of lactate in the intact hearts which is not visible by NMR spectroscopy.

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1H-Observe/13C-decouple spectroscopic measurements of lactate and glutamate in the rat brain in vivo.

Cited by 219 publications

References 19 publications

The Flux from Glucose to Glutamate in the Rat Brain in vivo as Determined by ¹-Observed, ¹³C-Edited NMR Spectroscopy

The Flux from Glucose to Glutamate in the Rat Brain in vivo as Determined by ¹-Observed, ¹³C-Edited NMR Spectroscopy

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

Direct observation of lactate and alanine by proton double quantum spectroscopy in rat hearts supplied with [3‐¹³C]pyruvate

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1H-Observe/13C-decouple spectroscopic measurements of lactate and glutamate in the rat brain in vivo.

Cited by 219 publications

References 19 publications

The Flux from Glucose to Glutamate in the Rat Brain in vivo as Determined by 1-Observed, 13C-Edited NMR Spectroscopy

The Flux from Glucose to Glutamate in the Rat Brain in vivo as Determined by 1-Observed, 13C-Edited NMR Spectroscopy

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

Direct observation of lactate and alanine by proton double quantum spectroscopy in rat hearts supplied with [3&#x2010;13C]pyruvate

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The Flux from Glucose to Glutamate in the Rat Brain in vivo as Determined by ¹-Observed, ¹³C-Edited NMR Spectroscopy

The Flux from Glucose to Glutamate in the Rat Brain in vivo as Determined by ¹-Observed, ¹³C-Edited NMR Spectroscopy

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

Direct observation of lactate and alanine by proton double quantum spectroscopy in rat hearts supplied with [3‐¹³C]pyruvate