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Metabolomic investigations of American oysters using H-NMR spectroscopy.

Tikunov AP, Johnson CB, Lee H, Stoskopf MK, Macdonald JM - Mar Drugs (2010)

Bottom Line: Mitochondrial metabolism appeared most pronounced in the adductor muscle with elevated levels of carnitine facilitating ß-oxidation, and ATP, and phosphoarginine synthesis, while glycogen was elevated in the mantle/gills and stomach/digestive gland.A biochemical schematic is presented that relates metabolites to biochemical pathways correlated with physiological organ functions.This study identifies metabolites and corresponding (1)H NMR peak assignments for future NMR-based metabolomic studies in oysters.

View Article: PubMed Central - PubMed

Affiliation: Joint Department of Biomedical Engineering, NC State University and UNC Chapel Hill, Chapel Hill, NC 27599, USA. tikunov@email.unc.edu

ABSTRACT
The Eastern oyster (Crassostrea virginica) is a useful, robust model marine organism for tissue metabolism studies. Its relatively few organs are easily delineated and there is sufficient understanding of their functions based on classical assays to support interpretation of advanced spectroscopic approaches. Here we apply high-resolution proton nuclear magnetic resonance ((1)H NMR)-based metabolomic analysis to C. virginica to investigate the differences in the metabolic profile of different organ groups, and magnetic resonance imaging (MRI) to non-invasively identify the well separated organs. Metabolites were identified in perchloric acid extracts of three portions of the oyster containing: (1) adductor muscle, (2) stomach and digestive gland, and (3) mantle and gills. Osmolytes dominated the metabolome in all three organ blocks with decreasing concentration as follows: betaine > taurine > proline > glycine > ß-alanine > hypotaurine. Mitochondrial metabolism appeared most pronounced in the adductor muscle with elevated levels of carnitine facilitating ß-oxidation, and ATP, and phosphoarginine synthesis, while glycogen was elevated in the mantle/gills and stomach/digestive gland. A biochemical schematic is presented that relates metabolites to biochemical pathways correlated with physiological organ functions. This study identifies metabolites and corresponding (1)H NMR peak assignments for future NMR-based metabolomic studies in oysters.

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The entire 1H NMR spectrum (bottom center) of the adductor muscle block of the oyster. The various portions of the spectrum are displayed (a–g), each scaled to the largest peak in that portion. The peak at 0 ppm is the external standard TSP (trimethylsilyl propionate) and pD = 7.0 (pH = 7.04) (see Experimental Section). *—13C labeled acetate (f) was added as a reference for the 13C spectroscopy (not shown).
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f2-marinedrugs-08-02578: The entire 1H NMR spectrum (bottom center) of the adductor muscle block of the oyster. The various portions of the spectrum are displayed (a–g), each scaled to the largest peak in that portion. The peak at 0 ppm is the external standard TSP (trimethylsilyl propionate) and pD = 7.0 (pH = 7.04) (see Experimental Section). *—13C labeled acetate (f) was added as a reference for the 13C spectroscopy (not shown).

Mentions: Figure 2 shows several expanded regions of the 1H spectrum of the oyster muscle block and vertical scales of each spectral region were increased for better definition of smaller peaks. 1H NMR spectra of the organ block extracts were comprised of over a hundred peaks (Figure 2), corresponding to low molecular weight acid-soluble endogenous metabolites. Thirty-seven of the most abundant are listed in Table 1. Although several metabolite classes were observed, the spectra were dominated by the amino acids and its organic acid derivatives, all of which are known to serve as osmolytes [10,11,20,21], including: betaine [3.25 (s) and 3.89 (s) ppm], taurine [3.25 (s) and 3.41 (t) ppm] and glycine [3.54 (s) ppm] (s: singlet, t: triplet, Figure 2). Two other osmolytes of lower peak areas and presumably lower concentrations are homarine [4.35 (s), 7.95 (dd), 8.02 (d), 8.53 (dd), 8.71 (d) ppm, d: doublet, dd: double doublet, m: multiplet] and proline [1.99 (m), 2.06 (m), 2.34 (m), 3.33 (dt), 3.41 (dt), and 4.12 (dd) ppm, dt: double triplet]. The relative abundance of the various osmolytes relative to all other small molecules is rapidly approximated by comparison of peak heights in the 1H NMR spectrum (Figure 2), using the innate quantitative nature of NMR spectroscopy. Clearly, a major portion of the Eastern oyster’s energy is devoted to dealing with its saline environment, and maintaining normal intracellular and blood volumes using molecules that can double as carbon sources for intermediary metabolism, and/or are involved in multiple biochemical functions. For example, in addition to glycine and proline, all amino acids act as osmolytes [9–11,20,22–24].


Metabolomic investigations of American oysters using H-NMR spectroscopy.

Tikunov AP, Johnson CB, Lee H, Stoskopf MK, Macdonald JM - Mar Drugs (2010)

The entire 1H NMR spectrum (bottom center) of the adductor muscle block of the oyster. The various portions of the spectrum are displayed (a–g), each scaled to the largest peak in that portion. The peak at 0 ppm is the external standard TSP (trimethylsilyl propionate) and pD = 7.0 (pH = 7.04) (see Experimental Section). *—13C labeled acetate (f) was added as a reference for the 13C spectroscopy (not shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2992993&req=5

f2-marinedrugs-08-02578: The entire 1H NMR spectrum (bottom center) of the adductor muscle block of the oyster. The various portions of the spectrum are displayed (a–g), each scaled to the largest peak in that portion. The peak at 0 ppm is the external standard TSP (trimethylsilyl propionate) and pD = 7.0 (pH = 7.04) (see Experimental Section). *—13C labeled acetate (f) was added as a reference for the 13C spectroscopy (not shown).
Mentions: Figure 2 shows several expanded regions of the 1H spectrum of the oyster muscle block and vertical scales of each spectral region were increased for better definition of smaller peaks. 1H NMR spectra of the organ block extracts were comprised of over a hundred peaks (Figure 2), corresponding to low molecular weight acid-soluble endogenous metabolites. Thirty-seven of the most abundant are listed in Table 1. Although several metabolite classes were observed, the spectra were dominated by the amino acids and its organic acid derivatives, all of which are known to serve as osmolytes [10,11,20,21], including: betaine [3.25 (s) and 3.89 (s) ppm], taurine [3.25 (s) and 3.41 (t) ppm] and glycine [3.54 (s) ppm] (s: singlet, t: triplet, Figure 2). Two other osmolytes of lower peak areas and presumably lower concentrations are homarine [4.35 (s), 7.95 (dd), 8.02 (d), 8.53 (dd), 8.71 (d) ppm, d: doublet, dd: double doublet, m: multiplet] and proline [1.99 (m), 2.06 (m), 2.34 (m), 3.33 (dt), 3.41 (dt), and 4.12 (dd) ppm, dt: double triplet]. The relative abundance of the various osmolytes relative to all other small molecules is rapidly approximated by comparison of peak heights in the 1H NMR spectrum (Figure 2), using the innate quantitative nature of NMR spectroscopy. Clearly, a major portion of the Eastern oyster’s energy is devoted to dealing with its saline environment, and maintaining normal intracellular and blood volumes using molecules that can double as carbon sources for intermediary metabolism, and/or are involved in multiple biochemical functions. For example, in addition to glycine and proline, all amino acids act as osmolytes [9–11,20,22–24].

Bottom Line: Mitochondrial metabolism appeared most pronounced in the adductor muscle with elevated levels of carnitine facilitating ß-oxidation, and ATP, and phosphoarginine synthesis, while glycogen was elevated in the mantle/gills and stomach/digestive gland.A biochemical schematic is presented that relates metabolites to biochemical pathways correlated with physiological organ functions.This study identifies metabolites and corresponding (1)H NMR peak assignments for future NMR-based metabolomic studies in oysters.

View Article: PubMed Central - PubMed

Affiliation: Joint Department of Biomedical Engineering, NC State University and UNC Chapel Hill, Chapel Hill, NC 27599, USA. tikunov@email.unc.edu

ABSTRACT
The Eastern oyster (Crassostrea virginica) is a useful, robust model marine organism for tissue metabolism studies. Its relatively few organs are easily delineated and there is sufficient understanding of their functions based on classical assays to support interpretation of advanced spectroscopic approaches. Here we apply high-resolution proton nuclear magnetic resonance ((1)H NMR)-based metabolomic analysis to C. virginica to investigate the differences in the metabolic profile of different organ groups, and magnetic resonance imaging (MRI) to non-invasively identify the well separated organs. Metabolites were identified in perchloric acid extracts of three portions of the oyster containing: (1) adductor muscle, (2) stomach and digestive gland, and (3) mantle and gills. Osmolytes dominated the metabolome in all three organ blocks with decreasing concentration as follows: betaine > taurine > proline > glycine > ß-alanine > hypotaurine. Mitochondrial metabolism appeared most pronounced in the adductor muscle with elevated levels of carnitine facilitating ß-oxidation, and ATP, and phosphoarginine synthesis, while glycogen was elevated in the mantle/gills and stomach/digestive gland. A biochemical schematic is presented that relates metabolites to biochemical pathways correlated with physiological organ functions. This study identifies metabolites and corresponding (1)H NMR peak assignments for future NMR-based metabolomic studies in oysters.

Show MeSH