Next Article in Journal
Effect of Marine Polyunsaturated Fatty Acids on Biofilm Formation of Candida albicans and Candida dubliniensis
Next Article in Special Issue
NMR-Based Metabolomic Investigations on the Differential Responses in Adductor Muscles from Two Pedigrees of Manila Clam Ruditapes philippinarum to Cadmium and Zinc
Previous Article in Journal
Demospongic Acids Revisited
Previous Article in Special Issue
Symbiodinium—Invertebrate Symbioses and the Role of Metabolomics
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Marine Drugs
Volume 8
Issue 10
10.3390/md8102578
marinedrugs-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Metabolomic Investigations of American Oysters Using 1H-NMR Spectroscopy

by
Andrey P. Tikunov
1,2,*,
Christopher B. Johnson
3,
Haakil Lee
1,2,
Michael K. Stoskopf
1,2,4 and
Jeffrey M. Macdonald
1,2
1
Joint Department of Biomedical Engineering, NC State University and UNC Chapel Hill, Chapel Hill, NC 27599, USA
2
Environmental Medicine Consortium, NC State University, 4700 Hillsborough St., Raleigh, NC 27606, USA
3
Department of Surgery, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425, USA
4
Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough St., Raleigh, NC 27606, USA
*
Author to whom correspondence should be addressed.
Mar. Drugs 2010, 8(10), 2578-2596; https://doi.org/10.3390/md8102578
Submission received: 27 August 2010 / Revised: 22 September 2010 / Accepted: 30 September 2010 / Published: 8 October 2010
(This article belongs to the Special Issue Metabolomic Approaches to Marine Organisms)
Browse Figures
Versions Notes

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 (1H 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 1H NMR peak assignments for future NMR-based metabolomic studies in oysters.

1. Introduction

Metabolites, as the end products of metabolism, represent the functional responses of a cell. Their characterization can provide insight into the underlying mechanisms of genomic or environmental actions on metabolism. The marine environment is varied and dynamic, providing a vast diversity of physical and chemical challenges to metabolism, making the study of the metabolites of marine organisms particularly fruitful for scientists interested in comparative physiology, pharmacology and toxicology. Organ specific metabolic fingerprints can establish time dependent assessments for interpreting functional adaptations to environmental and nutritional challenges using either invasive tissue extraction from multiple individuals or non-invasive longitudinal observation of the same individual. NMR spectroscopy and magnetic resonance imaging (MRI) permit non-invasive monitoring of the metabolome.
Metabolomics is an emerging discipline that is based on the measurement and study of all low molecular weight endogenous metabolites to establish an overview of the metabolic status of a biological system. High-resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy-based metabolomics measures the metabolic profiles of cells, tissues, biofluids, and even whole organisms, employing computer-assisted pattern recognition techniques to identify metabolic differences in response to experimentally induced perturbations [1–4]. This technique has several important advantages for studies of marine organisms including the ability to provide unbiased, rapid, and cost-effective analytical data on the concentrations of a wide range of small-molecule metabolites simultaneously. The spectral peaks are quantitative representations of the number of protons in the respective molecule. The analytical method is unbiased because all proton-containing molecules in the tissue extract are equally represented in the 1H NMR spectrum regardless of polarity, extinction coefficients, or ionization properties commonly biasing alternative analytical methods [5]. Therefore, unlike other separation methods (i.e., chromatography, electrophoresis), which are dependent on detection methods (i.e., UV-Vis, electrochemical, mass spectrometry) requiring previous knowledge of analytes and the use of concentration calibrants for each compound studied, 1H NMR spectroscopy detects unknown environmental contaminants and their biological effects on the metabolome as a quantitative change in the NMR spectral pattern of a tissue or organ extract.
“The resolution in 1-D NMR is often inadequate for unambiguous structure identification and isotope quantification in crude cell or tissue extracts. Spreading NMR signals over a plane results in an enormous increase in resolution, with the added advantage that molecular fragments can be visualized according to the type of experiment performed.” (From Fan and Lane 2008) [6,7]. However, 2-D NMR measurements require significantly more time to acquire than 1-D and although 2-D increases resolution of co-resonating peaks, the fine detail required for determining coupling constants is less accurate in 2-D NMR. The intrinsic sensitivity of NMR spectroscopy depends on the observed nucleus, metabolite concentrations, experimental conditions and the resolution desired. The standard 2D NMR spectroscopic protocol for small molecule structure elucidation, is typically obtaining a 1H-1H TOCSY followed by a 1H-13C HSQC [6,7]. The TOCSY determines the chemical backbone of a molecule by correlating peaks in the 1D 1H NMR spectrum across the second dimension. The 1H-13C HSQC correlates the resonances in the 1D 1H NMR spectrum to those in the 1D 13C NMR spectrum.
The Eastern oyster (Crassostrea virginica) is the basis of the United States oyster industry along the Atlantic and Gulf of Mexico coast [8]. It has been the subject of many studies examining biomass, industrial productivity and biochemical composition [9–15]. Oysters have several readily separated organs with defined functions, making it a good model for whole organism metabolism using non-invasive 1H magnetic resonance spectroscopy (MRS) and MRI. For example, there is a single, relatively large muscle that can be easily isolated from the rest of the oyster tissue using surface coil localized MRS [16,17] or chemical shift imaging (CSI) of the whole oyster permitting the determination of the metabolic contribution of entire organ systems (see Lee et al., this special edition of Marine Drugs). Therefore, longitudinal MRS or CSI studies of chemical dynamics, target organ disease or intoxication can be more easily interpreted [1–4]. However, a complete metabolic baseline of each organ or organ group is essential to evaluate the effects of treatments in future studies. In this study we identified major compounds for the functional organ blocks of the Eastern oyster by dividing the body into three functional parts: (i) muscle (movement), (ii) GI tract with digestive gland (digestion), and (iii) gills with mantle (respiration and electrolyte balance). Muscle, with its biological role of motor function, metabolically converts stored chemical energy to movement. The GI tract in conjunction with the digestive gland performs digestion and energy assimilation. It is also the site of the most active bacterial metabolism in the healthy oyster [18,19]. The mantle and gills perform the physiological functions of oxygen transfer, osmotic regulation, and shell creation.
In the present study, we applied 1H-NMR-based metabolomic analysis to the Eastern oyster. Differences in the metabolic profile of three dissected organ blocks were compared: (1) muscle, (2) mantle including gills, and (3) the gastrointestinal (GI) tract including the stomach and digestive gland. The goals of the study were to: (i) use NMR to metabolically profile the Eastern oyster (C. virginica) for future studies, (ii) ascertain how the metabolic profiles differ among the various organs (muscle, mantle including gills, and GI tract including digestive gland) and (iii) assess the potential of NMR-based metabolomics as a rapid and informative screening tool for monitoring changes in tissue metabolism.

2. Results and Discussion

2.1. Anatomical definition of oyster body parts

To evaluate organ block metabolic baselines, after one day of acclimation in a closed system of filtered natural seawater, oysters were dissected into the three organ blocks, and metabolites from those organ blocks were extracted with perchloric acid. 1H NMR spectra of each organ block extract were obtained (see Experimental Section). Figure 1 displays a 1H MRI image of the oyster body illustrating the anatomical landmarks delineating the three dissected organ blocks and their corresponding 1H spectra. The spectra from all three organ blocks appeared similar, with three major peaks representing two osmolytes, betaine (at 3.25 and 3.89 ppm) and taurine (3.25 and 3.41 ppm). On closer examination of the smaller peaks, the GI/digestive gland and mantle/gills spectra are quite similar, but the muscle block spectrum has additional peaks, for example the amino acid glycine (3.54 ppm).

2.2. Identification of the metabolites on 1D 1H spectrum

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].
The most abundant amino acids in the oyster muscle block spectrum are the: (1) neutral zwitterions such as proline showing multiplets centered at 1.99 ppm and 2.06 ppm, alanine with a doublet at 1.46 ppm (C-3) and quartet at 3.76 ppm (C-2), and glycine with a singlet at 3.75 ppm; (2) the acidic amino acids such as glutamate and aspartate; (3) the basic amino acid arginine [1.68 (m), 1.90 (m), 3.23 (t), 3.74 (t) ppm]; (4) the branched chain amino acids including isoleucine, leucine, and valine; and (5) the aromatic amino acids tyrosine and phenylalanine. Table 1 lists the additional chemical shifts.
In the oyster, the amino acid arginine has a biochemical role similar to that of creatine in mammals, as an energy store of high energy phosphagen that can be transferred to ADP to form ATP. The nucleotides ATP and ADP are also identified (Figure 2). Arginine clearly would play a role in muscle with regard to energy metabolism, but in the GI tract with digestive gland block, arginine is also the final intermediate in the urea cycle before formation of urea via arginase. The Eastern oyster releases 0.32–0.86 μmoles/g tissue/day of urea [14].

2.3. 2D 1H NMR spectra of oyster muscle

Although the 1H-13C HSQC spectrum is not presented in this study, it was performed to confirm the identity of the peaks in the 1D 1H NMR spectrum and was correlated to the natural abundance 13C (see Experimental Section).
To identify metabolites in the 1D 1H NMR spectra, a 2D 1H-1H TOCSY spectrum of the adductor muscle block extract was obtained and is shown in Figure 3. Proline is one of the main osmolytes in Eastern oyster, but unlike betaine, glycine, or homarine, it is not uniquely identified in the 1D 1H spectra. However, the 2D spectrum clearly shows that the scalar coupled multiplets at 2.00, 2.07, 2.34, and 4.12 ppm are correlated to one another and match those resonances of proline (Figure 3). The TOCSY also identified two other osmolytes, hypotaurine [2.63 (t), 3.36 (t)], or 2-aminosulfinic acid, and ß-alanine [2.55 (t), 3.18 (t)], or 2-aminocarboxylic acid. These are organic acids with very similar structure creating similar 1H NMR spectral patterns in the TOCSY with two correlated triplets (Figure 3). Hypotaurine is an intermediate in the catabolism of cysteine to taurine (2-aminosulfonic acid). The osmolyte, ß-alanine, is a beta amino acid, wherein there is no alpha carbon, rather the carboxylic acid moiety is bound to the beta carbon. The identities of three other important amino acids, threonine, arginine, and glutamate were confirmed.
The 2D TOCSY spectrum of oyster muscle also identified carnitine. Carnitine is an essential factor in fatty acid metabolism functioning to transport fat into the mitochondria of muscle cells [29]. l-acetylcarnitine, an acetyl ester of carnitine, facilitates movement of acetyl CoA into the matrices of mammalian mitochondria during the oxidation of fatty acids [30,31]. There are peaks at 2.43, 3.21, 3.42 and 4.56 ppm corresponding to carnitine.
As mentioned above, arginine is used as phosphoarginine in muscle as a pool of a high energy phosphagen and its C-3 (1.90 ppm) and C-4 (1.68 ppm) peaks are distinguished from the peaks from C-3 methylene and methyne groups from the branched chain amino acids, leucine [1.66 ppm (m)], and isoleucine [1.96 ppm (m)], respectively. Although these resonances are dominated by arginine, there is some contribution from leucine and isoleucine as resolved in the 2D TOCSY spectrum.
Finally, the large multiplet near the proline resonances around 2.13 ppm could be due to glutamine, glutamate, or the glutamyl moiety of glutathione. The concentration of glutathione in oyster has been reported to be 0.8–1 mmol/g wet weight [15,32]. Glutathione is an antioxidant and tripeptide (gamma-glutamylcysteinylglycine) [33], while glutamate is an anaplerotic amino acid derived from the Krebs cycle. Although glutathione synthesis could be up-regulated during times of stress, we do not detect such high concentrations of glutathione in the 1D 1H muscle spectrum (Figure 2) or other organ blocks, rather an order-of-magnitude higher concentration of glutamate. During times of high ammonia accumulation, glutamate will form glutamine via glutamine synthetase, or xenobiotic stress, it will form glutathione. Therefore, for future toxicological or metabolomic studies, it will be important to identify this relatively large multiplet at 2.13 ppm. The TOCSY confirms that the resonance near 2.13 ppm is from the C-3 of glutamate and is correlated to its C-4 at 2.34 ppm, which co-resonates with C-4 of proline’s in the 1D spectrum (Figure 2), and is correlated to the alpha carbon C-2 of glutamate at 3.75 ppm (Figure 3).
In the aromatic region, the TOCSY helps confirm homarine and ATP/ADP resonance, but also that of trigonelline, or methylnicotinate. Trigonelline is an alkaloid found in plants, such as coffee, and animals, first being identified in anemones over half a century ago [34].

2.4. Differences in metabolites from the three oyster organ blocks

1D 1H NMR spectra from the three dissected organ blocks of the oyster are compared in Figure 4. Three regions of the spectra (−0.5 to 3.2 ppm; 3.2 to 4.3 ppm; and 4.3 to 9.2 ppm) ranging from the aromatic (9.2 ppm) to the aliphatic (0.5 ppm) are scaled to the largest peak in that region to visualize the smaller peaks. Osmolytes have a constant ratio across the organ blocks. Therefore, these peaks (betaine, taurine, homarine, marked with asterisk (*)) were used as an internal reference in Figure 4, to calibrate the Y-scale across organ blocks (Figure 4). Figure 5 is a graph of the molar ratio (Experimental Section for calculation) of chosen metabolites in the three different extracted organ blocks. Marine mollusks, similar to other marine invertebrates, use large amounts of nitrogenous solutes, such as free amino acids and their catabolites, as the major intracellular osmolytes to protect against the high and fluctuating extracellular osmolarity of their environment [35]. Betaine and taurine, which are glycine and methionine/cysteine catabolites, respectively [9–11,20,22–24], were an order of magnitude more abundant than any other metabolite (Figure 5).
Although taurine (2-aminosulfonic acid) is often referred to as an amino acid [9,10,13,14] even in recent literature [36–38], it does not contain a zwitterion containing an amine and carboxylic acid that sandwich an alpha carbon, and thus cannot make peptides (amide linkages) to be incorporated into protein. Taurine, is also known as 2-aminoethanesulphonic acid and classified as a ß-amino acid, according to Mendel and Bradley [39]. Taurine was first reported in mollusks in 1845 by Karsten [40]. First thought to be non-essential in mammals, it is now known to be essential during development in the cat [41], and involved in a variety of biological processes such as bile salt formation, osmoregulation, oxidative stress inhibition, immunomodulation, diabetes and atherosclerosis. It is one of the most abundant organic acids in animal tissues, but it is not found in plants with the exception of some algae [42]. In C. gigas it has been shown to increase with salinity, clearly serving an osmoregulatory role [37], but its anti-oxidant, and storage role for sulfur amino acids is still unclear.
The free amino acids make up large fractions of the metabolome of marine invertebrates [23,24,43]. The total concentration of amino acids in muscle homogenate of C. virginica is reported to be 0.035 M to 0.164 M, depending on salinity [25]. Free amino acids predominantly contribute to the intracellular pool of osmolytes in all the molluscan species investigated, and these are typically proline, glycine, glutamate, and alpha and ß-alanine [44]. We found these five amino acids to be the most abundant in all tissues (Figure 5). In C. gigas, free amino acids as well as the beta-amino acids, β-alanine and taurine, increased in some cases 10-fold with increasing environmental salinity [37]. One of these amino acids discovered to increase with salinity is threonine, and this was the only amino acid found in higher concentrations in the gills in our study as compared to the muscle (Figure 5).
Homarine, another osmolyte of high concentration [34,45–47] first extracted from lobster by Hoppe-Seyer in 1933, is slightly lower in the oyster muscle block than in other tissues (Figure 5—note log scale of graph). The concentrations of these compounds in Figure 5 are consistent between the different organ blocks (muscle, mantle with gills and GI tract with digestive gland). Monitoring of osmolyte distribution and kinetics may be useful for the study of mechanisms of adaptation to tidal and seasonal salinity changes.
Depending on season, glycogen comprises 20–40% of dry flesh weight of the pacific oyster [48], and similar percentages of the C. virginica [26]. It has been shown that major glycogen storage tissues in mollusks are the mantle and digestive gland, and there is a much lower concentration of glycogen in muscle [49,50]. Our work supports these reported data, showing that the glycogen peak (at 5.42 ppm) is significantly smaller in the muscle block than in the other two organ blocks (Figure 4). The concentration of glycogen in the muscle block is approximately 5–7 times lower than that found in the GI with digestive gland and the mantle with gills organ blocks (see Figure 5).
Presented data is a snapshot of oyster metabolites based on samples collected from Taylor creek, Beaufort NC, in spring 2009. Changes of glycogen concentration and distribution across tissues depend on season for the Eastern oyster C. virginica. The biochemical cycle in bivalves shows glycogen storage activity during favorable trophic conditions, followed by mobilization and conversion of these reserves during the maturation period [51–53]. This cycle was confirmed in the Pacific oyster C. gigas [54–56], and the blue mussel M. edulis [57]. Turnover of stored glycogen is correlated with the annual reproductive cycle and food availability [51,58,59]. Glycogen metabolism pathways are controlled by glycogen synthetase, hemolymph glucose concentration, and feeding conditions [60]. Gabbott (1975) suggested that vitellogenesis takes place at the expense of stored glycogen reserves in the blue mussel, M. edulis [51] and this was later demonstrated in C. gigas [61]. Glucose incorporation into glycogen was first studied in the flat oyster Ostrea edulis by Fando et al. [62], who reported that gill or mantle tissues incorporated significantly more glucose than muscle tissue by a factor of 5. This generally agrees with our data.
Other metabolites showing significant differences across organ blocks in our study were ATP. Adenine, guanine, and uracil nucleotides have been measured in extracts of C. gigas tissue [63]. As is found in most forms of animal and microbial life, the oyster contained adenosine-5′ phosphates (AMP, ADP, and ATP) in greatest abundance, with concentrations of 32.0, 15.2, and 2.4 micromoles/100 g, respectively [13]. Other investigators have reported the concentration of ATP in muscle tissue to be 3–4-fold higher than in mantle tissue [64,65]. In our studies, the resolved ATP peaks are located at 6.15 ppm and 8.3 ppm respectively, and are clearly evident in the muscle organ block 1H NMR spectrum. They are evanescent on the mantle/gill and GI/digestive gland spectra (Figure 4). ATP concentrations in the mantle/gill organ block and GI/digestive gland organ block were at least 100-fold lower than those in the muscle organ block (Figure 5, ATP). This is logical because muscle would be expected to be the main ATP consumer and by extension producer in the oyster body per unit tissue weight. The muscle performs mechanical movement using chemical energy stored in the phosphoanhydride bond of ATP. It is interesting that arginine does not correlate with ATP in its distribution across the organ blocks in our study, being relatively constant throughout all three blocks. Arginine is one of the more abundant free amino acids of oyster (Figure 5). Phosphoarginine replaces phosphocreatine in mollusks as the high energy phosphagen used for ATP regeneration [16,17]. However, it also plays a role as a pool for nitrogen and can detoxify ammonia in a manner similar to creatine in mammals [66]. Perhaps the ubiquitous distribution of arginine in the oyster is related to biochemical and physiological roles other than energetics.
Glycine can enter the Krebs cycle through the glyoxylate pathway and also participate in glycolysis through the formation of serine [67]. Glycine can serve as a 2 carbon energy source in both pathways, ultimately generating ATP. Glycine also acts as an osmolyte and is a precursor of betaine, but it may be its role in energetics that is be responsible for its elevated concentration in muscle (Figure 5) [68,69].
We believe our study is the first report of carnitine in oysters. Our finding is confirmed by the 2D TOCSY NMR spectra peaks at 3.2, 4.56 ppm (carnitine). These explicit peaks are easily seen in the muscle block spectrum and negligible in the mantle/gill and GI/digestive gland organ block spectra (Figure 4). The carnitine concentration in the muscle organ block, where we would expect more mitochondria per unit of tissue, is approximately 3–5 times higher than in the other two organ block extracts. Based on this, we propose that carnitine, known to be important in the metabolism of fatty acids [30,31] may be a useful marker of mitochondrial activity.
This study forms the foundation for future metabolic studies of oysters and identifies key metabolically important compounds in each of three organ blocks. Figure 6 is a biochemical schematic representation of the compounds we identified and their metabolic relationships displayed overlaying an MRI image of the Eastern oyster representing the relationships between anatomy, physiological function and the relative abundance of metabolites. Biochemically the distribution of the metabolites we identified corresponds well with expected organ block functions. For example, mitochondrial metabolism would be expected to be elevated in muscle, and if this is the case, the concentrations of carnitine, a fatty acid carrier for ß-oxidation, and ATP, the end-product of oxidative phosphorylation and glycolysis should also be elevated. It is logical to find glycogen stored in the gill/mantle and GI/digestive gland organ blocks, and utilized extensively in the muscle organ block. The metabolite separation found in our studies suggests the use of MRI with spectroscopic imaging could be used effectively to investigate the real-time dynamics of metabolic changes in response to environmental changes and exposure to toxins.

3. Experimental

Unless otherwise stated, all chemicals used in this study were obtained from Sigma (St. Louis, MO, USA). Eastern Oysters (Crassostrea virginica) (50–70 g) were collected from Taylor Creek near Beaufort, NC, and fasted for one day in filtered natural seawater prior to the experiments. A single oyster is presented throughout this manuscript as a means of comparison, although a total of five oysters were analyzed by NMR spectroscopy and MRI. Measurements were prepared in April–May 2009. Seawater was obtained from Bogue Sound at the Center for Marine Sciences and Technology (CMAST), and filtered through a 0.2 μm filter then used immediately in the experiments. After fasting the oyster was quickly opened, and three organ blocks were dissected (muscle; GI with digestive gland; and mantle with gills) rapidly and immediately frozen in liquid nitrogen. Each organ block was pulverized individually using a stainless steel mortar and pestle with constant addition of liquid nitrogen. The crushed organ block was then transferred to a 50 cc polyethylene disposable centrifuge tube and weighed. Perchloric acid was added 2-to-1 v:w (volume:weight) and the mixture vortexed for 1 min, before being incubated overnight in a refrigerator (4 °C). The mixture was then centrifuged at 4500 g for 5 min, and the supernatant collected. The pH of the supernatant was adjusted with potassium hydroxide to alkaline pH between pH 7–7.4. The supernatant was then centrifuged again at 4500 g for 5 min to remove any precipitate. The supernatant was then lyophilized in 50 cc polyethylene disposable tube and stored at −80 °C in a plastic cryovial until spectroscopy was performed. For NMR spectroscopy the lyophilized powder from the perchloric acid extraction for each of the three organ blocks was dissolved in 0.7 mL deuterium oxide with 0.2% TSP and transferred to a 5 mm NMR tube. 1H spectra were acquired at 25 °C. 1H spectra were acquired on a 16.4 T Varian INOVA spectrometer (700 MHz 1H frequency) equipped with a 5 mm indirect cold probe. 1H spectra of extracts were acquired using a total repetition time (TR) of 12.65 s, and a 90° flip angle. Data were analyzed using an ACD Labs 9.0 1D NMR Processor (ACD Labs). 1H spectra were zero-filled to 32,000 points, and line broadened using a 0.5 Hz exponential Gaussian function. Chemical shifts presented in Table 1 were obtained from the Human Metabolome Database ( http://www.hmdb.ca). Concentrations were calculated from the 1H spectrum by comparing peak areas to TSP peaks as previously described [70]. Molar ratio was defined as peak area divided by the sum of all peak areas in the spectrum, excluding the water peak area. NMR determination of Molar ratios is a conventional and validated method for metabolic profiling [71,72].
The 2D NMR spectra were acquired on a 16.4 T Varian INOVA spectrometer (700 MHz 1H frequency) equipped with a 5 mm indirect HCN probe. The z-filtered 1H-1H TOCSY (Total Correlation Spectroscopy) data were acquired with TR = 2.48 s, nt (number of transients) = 16, and the number of indirect dimension increments (ni) was 64. Data was linearly predicted in 3 × N in the indirect dimension and zero-filled to 2,000 points. Data was processed with SpinWorks 3.1.7, Copyright © 2010 Kirk Marat University of Manitoba. The 1H-1H heteronuclear single quantum coherence (HSQC) data was acquired using a Heteronuclear Overbodenhausen experiment using REVINEPT with spectral conditions described previously [73].
MR imaging and spectroscopy were performed using a Varian 4.7T INOVA MRI system (Varian, Inc., Palo Alto, CA, USA) with a 33 cm horizontal bore magnet equipped with a 20 cm inside diameter gradient coil insert with integrated shims (Resonance Research Inc, Billerica, MA, Model BFG-300/200). The maximum gradient strength was 300 mT/m. At this field strength, the resonant frequencies of proton and carbon were 200 and 50 MHz, respectively. The images were collected using a multi-slice spin echo sequence with TR/TE (time of repetition/time of echo) settings of 2,000 ms/8 ms (proton-density), 300 ms/8 ms (T1-weighted), and 2,000 ms/30 ms (T2-weighted), respectively. The number of averages in each case was two. Therefore the image acquisition times were 512 (proton-density), 77 (T1) and 512 (T2) seconds. The field of view was 8 cm × 4 cm with 256 and 128 pixels acquired, producing 0.31 × 0.31 mm pixel resolution for each image. Experimental conditions are described in a recently published paper [74].

4. Conclusions

Metabolomic NMR analysis of three organ blocks of the Eastern oyster identified over 32 major compounds and provides a basis for future metabolomic studies of the oyster. The compounds with the highest concentrations most likely serve roles in osmolarity regulation in addition to any other roles. The oyster adductor muscle has elevated concentrations of metabolites involved in mitochondrial energy metabolism such as carnitine, ATP, glycine, and alanine. Carnitine was identified for the first time in oysters. The relatively equal concentrations of arginine across the three organ blocks suggest a role as a high energy phosphagen storage molecule in the form of phosphoarginine and perhaps importance in the urea cycle in the stomach and digestive gland. High concentrations of glycogen in the stomach and digestive gland block most likely reflect its use as a glucose storage mechanism.
In conclusion, this is the first comprehensive NMR analysis of the Eastern oyster. Now that all major peaks in the 1H NMR spectrum of organ blocks of the Eastern oyster have been identified, the effects of the environment, age, genetics, and disease on the metabolome can be studied.

Acknowledgements

This work was supported in part by an instrumentation grant (2006-IDG-1012) from the North Carolina Biotechnology Center. These studies were conducted at the Marine Magnetic Resonance Imaging and Spectroscopy Facility (MMRISF) at the Center for Marine Sciences and Technology (CMAST) in Morehead City, NC, USA.
  • Samples Availability: Available from the authors.

References

  1. Hines, A; Oladiran, GS; Bignell, JP; Stentiford, GD; Viant, MR. Direct sampling of organisms from the field and knowledge of their phenotype: Key recommendations for environmental metabolomics. Environ. Sci. Technol 2007, 41, 3375–3381. [Google Scholar]
  2. Jones, OAH; Spurgeon, DJ; Svendsen, C; Griffin, JL. A metabolomics based approach to assessing the toxicity of the polyaromatic hydrocarbon pyrene to the earthworm Lumbricus rubellus. Chemosphere 2008, 71, 601–609. [Google Scholar]
  3. Tuffnail, W; Mills, GA; Cary, P; Greenwood, R. An environmental 1H NMR metabolomic study of the exposure of the marine mussel Mytilus edulis to atrazine, lindane, hypoxia and starvation. Metabolomics 2009, 5, 33–43. [Google Scholar]
  4. Viant, MR; Rosenblum, ES; Tjeerdema, RS. NMR-based metabolomics: a powerful approach for characterizing the effects of environmental stressors on organism health. Environ. Sci. Technol 2003, 37, 4982–4989. [Google Scholar]
  5. Willard, HH; Merritt, LL, Jr; Dean, JA; Settle, FA, Jr. Belmont, CA, Ed.; Instrumental Methods of Analysis, 7th ed; Wadsworth: Belmont, CA, USA, 1988. [Google Scholar]
  6. Fan, TWM. Metabolite profiling by one- and two-dimensional NMR analysis of complex mixtures. Prog. Nucl. Magn. Reson. Spectrosc 1996, 28, 161–219. [Google Scholar]
  7. Fan, TWM; Lane, AN. Structure-based profiling of metabolites and isotopomers by NMR. Prog. Nucl. Magn. Reson. Spectrosc 2008, 52, 69–117. [Google Scholar]
  8. Sabatini, P; Josupeit, H. Bivalves: Commodity Update; FAO GLOBEFISH: Rome, Italy, 2007. [Google Scholar]
  9. Allen, K. Amino acids in the Mollusca. Integr. Comp. Biol 1961, 1, 253–261. [Google Scholar]
  10. Allen, K. The effect of salinity on the amino acid concentration in Rangia cuneata (Pelecypoda). Biol. Bull 1961, 121, 419–424. [Google Scholar]
  11. Burg, MB; Ferraris, JD. Intracellular organic osmolytes: function and regulation. J. Biol. Chem 2008, 283, 7309–7313. [Google Scholar]
  12. Collicutt, JM; Hochachka, PW. The anaerobic oyster heart: coupling of glucose and aspartate fermentation. J. Comp. Physiol. B Biochem. Syst. Environ. Physiol 1977, 115, 147–157. [Google Scholar]
  13. Hammen, CS. Metabolism of the oyster, Crassostrea virginica. Integr. Comp. Biol 1969, 9, 309–318. [Google Scholar]
  14. Hammen, CS; Miller, HF. Nitrogen excretion of Crassostrea virginica. Comp. Biochem. Physiol 1966, 17, 1199–1200. [Google Scholar]
  15. Ringwood, AH; Conners, DE; DiNovo, A. The effects of copper exposures on cellular responses in oysters. Mar. Environ. Res 1998, 46, 591–595. [Google Scholar]
  16. Fan, TWM; Higashi, RM; Macdonald, JM. Emergence and recovery response of phosphate metabolites and intracellular pH in intact Mytilus edulis as examined in situ by in vivo 31P-NMR. Biochim. Biophys. Acta 1991, 1092, 39–47. [Google Scholar]
  17. Higashi, RM; Fan, TWM; Macdonald, JM. Monitoring of metabolic responses of intact Haliotis (abalones) under salinity stress by 31P surface probe localized NMR. J. Exp. Zool 1989, 249, 350–356. [Google Scholar]
  18. Harris, JM. The presence, nature, and role of gut microflora in aquatic invertebrates: a synthesis. Microb. Ecol 1993, 25, 195–231. [Google Scholar]
  19. Kueh, CSW; Chan, K-y. Bacteria in bivalve shellfish with special reference to the oyster. J. Appl. Microbiol 1985, 59, 41–47. [Google Scholar]
  20. Ballantyne, JS; Moyes, CD. The effects of salinity acclimation on the osmotic properties of mitochondria from the gill of Crassostrea virginica. J. Exp. Biol 1987, 133, 449. [Google Scholar]
  21. Powell, EN; Kasschau, M; Chen, E; Koenig, M; Pecon, J. Changes in the free amino acid pool during environmental stress in the gill tissue of the oyster, Crassostrea virginica. Comp. Biochem. Physiol. A Physiol 1982, 71, 591–598. [Google Scholar]
  22. Carr, WES; Netherton, JC, III; Gleeson, RA; Derby, CD. Stimulants of feeding behavior in fish: analyses of tissues of diverse marine organisms. Biol. Bull 1996, 190, 149–160. [Google Scholar]
  23. Henry, RP; Mangum, CP. Salt and water balance in the oligohaline clam, Rangia cuneata III. Reduction of the free amino acid pool during low salinity adaptation. J. Exp. Zool 1980, 211, 25–32. [Google Scholar]
  24. Henry, RP; Mangum, CP; Webb, KL. Salt and water balance in the oligohaline clam, Rangia cuneata II. Accumulation of intracellular free amino acids during high salinity adaptation. J. Exp. Zool 1980, 211, 11–24. [Google Scholar]
  25. Lynch, MP; Wood, L. Effects of environmental salinity of free amino acids of Crassostrea virginica gmelin. Comp. Biochem. Physiol 1966, 19, 783–790. [Google Scholar]
  26. Sidwell, VD; Loomis, AL; Grodner, RM. Geographic and monthly variation in composition of oysters. Mar. Fish Rev 1979, 41, 13–17. [Google Scholar]
  27. Pierce, SK; Rowland-Faux, LM; O’Brien, SM. Different salinity tolerance mechanisms in Atlantic and Chesapeake Bay conspecific oyster: glycine betaine and amino acid pool variations. Mar. Biol 1992, 113, 107–115. [Google Scholar]
  28. Foreman, RA, III; Ellington, WR. Effects of inhibitors and substrate supplementation on anaerobic energy metabolism in the ventricle of the oyster, Crassostrea virginica. Comp. Biochem. Physiol. B 1983, 74, 543–547. [Google Scholar]
  29. Bremer, J. Carnitine-metabolism and functions. Physiol. Rev 1983, 63, 1420–1480. [Google Scholar]
  30. Bremer, J. Carnitine in intermediary metabolism. J. Biol. Chem 1963, 238, 2774–2779. [Google Scholar]
  31. Fritz, IB. Carnitine and its role in fatty acid metabolism. Adv. Lipid. Res 1963, 1, 285–334. [Google Scholar]
  32. Conners, DE; Ringwood, AH. Effects of glutathione depletion on copper cytotoxicity in oysters (Crassostrea virginica). Aquat. Toxicol 2000, 50, 341–349. [Google Scholar]
  33. Lamoureux, GL; Rusness, DG; Dolphin, D; Poulson, R; Avramovic, O. Dolphin, D, Poulson, R, Avramovic, O, Eds.; The role of glutathione and glutathione-S-transferases in pesticide metabolism, selectivity, and mode of action in plants and insects. In Glutathione: Chemical, Biochemical and Medical Aspects; John Wiley & Sons: New York, NY, USA, 1989; pp. 153–196. [Google Scholar]
  34. Ackermann, D. Uber das vorkommen von homarin, trigonellin und einer neuen base anemonin in der anthozoe anemonia-sulcata. Hoppe-Seyler’s Z. Physiol. Chem 1953, 295, 1–9. [Google Scholar]
  35. Yancey, PH; Clark, ME; Hand, SC; Bowlus, RD; Somero, GN. Living with water stress: evolution of osmolyte systems. Science 1982, 217, 1214–1222. [Google Scholar]
  36. Hu, YH; Lin, CL; Huang, YW; Liu, PE; Hwang, DF. Dietary amino acid taurine ameliorates liver injury in chronic hepatitis patients. Amino Acids 2008, 35, 469–473. [Google Scholar]
  37. Lee, N-H; Han, K-N; Choi, K-S. Effects of salinity and turbidity on the free amino acid composition in gill tissue of the Pacific oyster Crassostrea gigas. J. Shellfish Res 2004, 23, 129–133. [Google Scholar]
  38. Schuller-Levis, GB; Park, E. Taurine: new implications for an old amino acid. FEMS Microbiol. Lett 2003, 226, 195–202. [Google Scholar]
  39. Mendel, LB; Bradley, HC. Experimental studies on the physiology of the mollusks-third paper. Am. J. Physiol 1906, 17, 167–176. [Google Scholar]
  40. Karsten, H. Disquisitio microscopia et chemica hepatis et bilis crustaceorum et molluscorum. Nova acta Academiae Caesareae Leopoldino-Carolinae Germanicae Naturae Curiosorum 1845, 21, 295–326. [Google Scholar]
  41. Markwell, PJ; Earle, KE. Taurine: an essential nutrient for the cat. A brief review of the biochemistry of its requirement and the clinical consequences of deficiency. Nutr. Res 1995, 15, 53–58. [Google Scholar]
  42. Bouckenooghe, T; Remacle, C; Reusens, B. Is taurine a functional nutrient? Curr. Opin. Clin. Nutr. Metab. Care 2006, 9, 728–733. [Google Scholar]
  43. Toyohara, H; Ikeda, M; Goto, C; Sawada, H; Hosoi, M; Takeuchi, K; Hayashi, I; Imamura, S; Yamashita, M. Osmo-responsive expression of oyster amino acid transporter gene and analysis of the regulatory region involved. Fish. Sci 2005, 71, 465–470. [Google Scholar]
  44. Somero, GN; Bowlus, RD. Hochachka, PW, Ed.; Osmolytes and metabolic end products of molluscs: the design of compatible solute systems. In The Mollusca. Environmental Biochemistry and Physiology; Academic Press: New York; NY, USA, 1988; pp. 77–100. [Google Scholar]
  45. Affeld, S; Wagele, H; Avila, C; Kehraus, S; Konig, GM. Distribution of homarine in some Opisthobranchia (Gastropoda: Mollusca). Bonner Zoologische Beitrage 2006, 55, 181–190. [Google Scholar]
  46. Gasteiger, EL; Gergen, JA; Haake, PC. A study of the distribution of homarine (N-methyl picolinic acid). Biol. Bull 1955, 109, 345–346. [Google Scholar]
  47. Polychronopoulos, P; Magiatis, P; Skaltsounis, AL; Tillequin, F; Vardala-Theodorou, E; Tsarbopoulos, A. Homarine, a common metabolite in edible Mediterranean molluscs: Occurrence, spectral data and revision of a related structure. Nat. Prod. Res 2001, 15, 411–418. [Google Scholar]
  48. Linehan, LG; O’Connor, TP; Burnell, G. Seasonal variation in the chemical composition and fatty acid profile of Pacific oysters (Crassostrea gigas). Food Chem 1999, 64, 211–214. [Google Scholar]
  49. Berthelin, C; Kellner, K; Mathieu, M. Storage metabolism in the Pacific oyster (Crassostrea gigas) in relation to summer mortalities and reproductive cycle (West Coast of France). Comp. Biochem. Physiol. B, Biochem. Mol. Biol 2000, 125, 359–369. [Google Scholar]
  50. Lenoir, F; Robbins, I; Mathieu, M; Lubet, P; Gabbott, PA. Isolation, characterization and glucose metabolism of glycogen cells (=vesicular connective-tissue cells) from the labial palps of the marine mussel Mytilus edulis. Mar. Biol 1989, 101, 495–501. [Google Scholar]
  51. Gabbott, PA. Barnes, HB, Ed.; Storage cycles in marine bivalve molluscs: a hypothesis concerning the relationship between glycogen metabolism and gametogenesis. In Ninth European Marine Biology Symposium; Aberdeen University Press: Aberdeen, UK, 1975; pp. 191–211. [Google Scholar]
  52. Goddard, CK; Martin, AW. Wilbur, KM, Yonge, CM, Eds.; Carbohydrate metabolism. In Physiology of Mollusca; Academic Press: New York, NY, USA, 1966; Volume 2, pp. 275–308. [Google Scholar]
  53. Walne, PR. The seasonal variation of meat and glycogen content of seven populations of Oysters Ostrea edulis L. and a review of the literature. Fish. Invest. Lond. Ser. II 1970, 26, 1–35. [Google Scholar]
  54. Deslous-Paoli, JM; Heral, M. Biochemical composition and energy value of Crassostrea gigas (Thunberg) cultured in the bay of Marennes-Oleron. Aquat. Living Resour 1988, 1, 239–249. [Google Scholar]
  55. Patrick, S; Faury, N; Goulletquer, P. Seasonal changes in carbohydrate metabolism and its relationship with summer mortality of Pacific oyster Crassostrea gigas (Thunberg) in Marennes-Oleron bay (France). Aquaculture 2006, 252, 328–338. [Google Scholar]
  56. Perdue, JA; Beattie, JH; Chew, KK. Some relationships between gametogenic cycle and summer mortality phenomenon in the Pacific oyster (Crassostrea gigas) in Washington State. J. Shellfish Res 1981, 1, 9–16. [Google Scholar]
  57. Maurer, D; Borel, M. Croissance, engraissement et cycle sexuel de Crassostrea gigas dans le Bassin d’Arachon: comparaison des huitres agees de 1 et 2 ans. Haliotis 1985, 15, 125–134. [Google Scholar]
  58. Mathieu, M; Lubet, P. Storage tissue metabolism and reproduction in marine bivalves: a brief review. Invertebr. Reprod. Dev 1993, 23, 123–129. [Google Scholar]
  59. Ruiz, C; Abad, M; Sedano, F; Garcia-Martin, LO; Sanchez Lopez, JL. Influence of seasonal environmental changes on the gamete production and biochemical composition of Crassostrea gigas (Thunberg) in suspended culture in El Grove, Galicia, Spain. J. Exp. Mar. Biol. Ecol 1992, 155, 249–262. [Google Scholar]
  60. Gabbott, PA; Whittle, MA. Glycogen synthetase in the sea mussel Mytilus edulis L.—II. Seasonal changes in glycogen content and glycogen synthetase activity in the mantle tissue. Comp. Biochem. Physiol. B, Biochem. Mol. Biol 1986, 83, 197–207. [Google Scholar]
  61. Berthelin, CH; Fievet, B; Leclerc, G; Germain, P; Kellner, K; Mathieu, M. In vivo and in vitro approaches to the analysis of glycogen metabolism in the Pacific oyster, Crassostrea gigas. J. Shellfish Res 2003, 22, 715–720. [Google Scholar]
  62. Fando, JJ; Garcia-Fernandez, MC; Candela, JL. Glycogen metabolism in Ostrea edulis (L.)— factors affecting glycogen synthesis. Comp. Biochem. Physiol. B 1972, 43, 807–814. [Google Scholar]
  63. Wylie, V; Smith, M. Nucleotides of the Pacific oyster Crassostrea gigas (Thunberg). Biochem. Cell Biol 1964, 42, 1347–1351. [Google Scholar]
  64. Michaelidis, B; Haas, D; Grieshaber, MK. Extracellular and intracellular acid base status with regard to the energy metabolism in the oyster Crassostrea gigas during exposure to air. Physiol. Biochem. Zool 2005, 78, 373–383. [Google Scholar]
  65. Yokoyama, Y; Sakaguchi, M; Kawai, F; Kanamori, M. Changes in concentration of ATP-related compounds in various tissues of oyster during ice storage. Nippon Suisan Gakk 1992, 58, 2125–2136. [Google Scholar]
  66. Van Pilsum, JF; Stephens, GC; Taylor, D. Distribution of creatine, guanidinoacetate and the enzymes for their biosynthesis in the animal kingdom. Biochem. J 1972, 126, 325–345. [Google Scholar]
  67. Nakada, HI; Friedmann, B; Weinhouse, S. Pathways of glycine catabolism in rat liver. J. Biol. Chem 1955, 216, 583–592. [Google Scholar]
  68. Lehninger, AL; Nelson, DL; Cox, MM. Lehninger Principles of Biochemistry; W.H. Freeman: New York, NY, USA, 2005. [Google Scholar]
  69. Stryer, L. Biochemistry; W.H. Freeman: New York, NY, USA, 1995; pp. 86–88. [Google Scholar]
  70. Dewar, BJ; Keshari, K; Jeffries, R; Dzeja, P; Graves, LM; Macdonald, JM. Metabolic assessment of a novel chronic myelogenous leukemic cell line and an imatinib resistant subline by 1H NMR spectroscopy. Metabolomics 2010, 6, 439–450. [Google Scholar]
  71. Fan, TWM; Colmer, TD; Lane, AN; Higashi, RM. Determination of metabolites by 1H NMR and GC: analysis for organic osmolytes in crude tissue extracts. Anal. Biochem 1993, 214, 260–271. [Google Scholar]
  72. Fan, TWM; Higashi, RM; Lane, AN; Jardetzky, O. Combined use of 1H-NMR and GC-MS for metabolite monitoring and in vivo 1H-NMR assignments. Biochim. Biophys. Acta 1986, 882, 154–167. [Google Scholar]
  73. Keshari, KR; Kurhanewicz, J; Jeffries, RE; Wilson, DM; Dewar, BJ; Van Criekinge, M; Zierhut, M; Vigneron, DB; Macdonald, JM. Hyperpolarized 13C spectroscopy and an NMR-compatible bioreactor system for the investigation of real-time cellular metabolism. Magn. Reson. Med 2010, 63, 322–329. [Google Scholar]
  74. Lee, H; Tikunov, A; Stoskopf, MK; Macdonald, JM. Application of chemical shift imaging to marine sciences. Mar. Drugs 2010, 8, 2369–2383. [Google Scholar]
Marinedrugs 08 02578f1 1024
Figure 1. A sagittal 1H MRI image displaying the anatomical identification of the dissected oyster organ blocks outlined with dashed margins: (1) muscle, (2) GI tract (with digestive gland) and (3) mantle (with gills). The 1H NMR spectra of the perchloric acid extracts from the three dissected organ blocks border the MRI image.
Figure 1. A sagittal 1H MRI image displaying the anatomical identification of the dissected oyster organ blocks outlined with dashed margins: (1) muscle, (2) GI tract (with digestive gland) and (3) mantle (with gills). The 1H NMR spectra of the perchloric acid extracts from the three dissected organ blocks border the MRI image.
Marinedrugs 08 02578f1
Marinedrugs 08 02578f2 1024
Figure 2. 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).
Figure 2. 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).
Marinedrugs 08 02578f2
Marinedrugs 08 02578f3 1024
Figure 3. The 2D 1H-TOCSY of the adductor muscle block of an oyster. Boxed regions correlate the various resonances of alanine, arginine, aspartate, carnitine, glutamate, homarine, hypotaurine, proline, threonine, and tyrosine. C-1 resonances of glucose and glycogen are also labeled. The correlated resonances and coupling constants of the unknown is shown in Table 1.
Figure 3. The 2D 1H-TOCSY of the adductor muscle block of an oyster. Boxed regions correlate the various resonances of alanine, arginine, aspartate, carnitine, glutamate, homarine, hypotaurine, proline, threonine, and tyrosine. C-1 resonances of glucose and glycogen are also labeled. The correlated resonances and coupling constants of the unknown is shown in Table 1.
Marinedrugs 08 02578f3
Marinedrugs 08 02578f4 1024
Figure 4. 1H NMR spectra of the three different oyster organ blocks, scaled to the betaine peak at 3.89 ppm.
Figure 4. 1H NMR spectra of the three different oyster organ blocks, scaled to the betaine peak at 3.89 ppm.
Marinedrugs 08 02578f4
Marinedrugs 08 02578f5 1024
Figure 5. The molar ratios of selected biochemicals among the three organ blocks.
Figure 5. The molar ratios of selected biochemicals among the three organ blocks.
Marinedrugs 08 02578f5
Marinedrugs 08 02578f6 1024
Figure 6. Anatomic and metabolic relationships of biochemicals identified from the 1H NMR spectra as they relate to the physiology of the Eastern oyster.
Figure 6. Anatomic and metabolic relationships of biochemicals identified from the 1H NMR spectra as they relate to the physiology of the Eastern oyster.
Marinedrugs 08 02578f6
Table
Table 1. List of compounds identified in the 1H NMR spectrum of the oyster muscle block, and their respective chemical shifts. (s: singlet, d: doublet, t: triplet, dd: doublet of doublets, m: multiplet, br.s.: broad singlet).
Table 1. List of compounds identified in the 1H NMR spectrum of the oyster muscle block, and their respective chemical shifts. (s: singlet, d: doublet, t: triplet, dd: doublet of doublets, m: multiplet, br.s.: broad singlet).
MetabolitesChemical shift and peak shape, ppmConcentrationReference
Amino acids
Alanine1.46 (d), 3.76 (m)1.30 ± 0.27 *[21]
Arginine1.68 (m), 1.90 (m), 3.23 (t), 3.74 (t)0.16–0.24 †[25]
Aspartate2.66 (dd), 2.79(dd), 3.87 (dd)2.59 ± 0.72 *[21]
Glutamate2.08 (m), 2.34 (m), 3.74 (t)1.30 ± 0.19 *[21]
Glutamine2.12 (m), 2.44 (m), 3.75 (t)0.53 ± 0.13 *[21]
Glycine3.54 (s)1.29 ± 0.29 *[21]
Histidine7.81 (s) 7.04 (s), 3.99 (dd), 3.24/3.15 (dd)0.089–0.255 †[25]
Isoleucine0.92 (t), 1.00 (d), 1.26(m), 1.44 (m), 1.96 (m), 3.66 (d)0.001–0.008 †[25]
Leucine0.94 (d), 0.96 (d), 1.66 (m), 3.71 (t)0.005–0.014 †[25]
Phenylalanine3.98 (m), 7.31 (d), 7.36 (t), 7.41 (m)0.006–0.033 †[25]
Proline1.99 (m), 2.06 (m), 2.34 (m), 3.33 (dt), 3.41 (dt), 4.12 (dd)0.020–0.582 †[25]
Serine3.84 (m), 3.96 (m)0.023–0.057 †[25]
Threonine1.33 (d), 3.578 (d), 4.25 (m)0.009–0.036 †[25]
Tyrosine6.89 (d), 7.19 (d)0.005–0.046 †[25]
Valine0.98 (d), 1.03 (d), 2.25 (m), 3.59 (d)0.004–0.018 †[25]
Energy related
α-Glucose3.23 (dd), 3.40 (m), 3.46 (m), 3.52 (dd), 3.73 (m), 3.82 (m), 3.88 (dd), 4.63 (d), 5.22 (d)n/a-
ß-Glucose4.64 (d)n/a-
Glycogen3.40 (m), 3.60 (m), 3.80 (m), 3.96 (br. s.), 5.40 (br. s.)0.467–6.920 ‡[26]
ADP4.15 (m), 4.16 (m), 4.57 (m), 5.94 (m), 8.29 (s), 8.54 (s)n/a-
ATP4.21 (m), 4.28 (m), 4.39 (m), 4.51 (m), 4.62 (t), 6.13 (d), 8.24 (s), 8.53(s)n/a-
Osmolytes/Organic acids
Betaine3.25 (s), 3.89 (s)230 ± 30 □[27]
ß-alanine2.55 (t), 3.18 (t)0.25 ± 0.13 *[21]
Homarine4.35 (s), 7.95 (dd), 8.02 (d), 8.53 (dd), 8.71 (d)n/a-
Hypotaurine2.64 (t), 3.36 (t)n/a-
Taurine3.25 (s), 3.41 (t)16.37 ± 3.39 *[21]
Krebs cycle intermediates
Succinate2.41 (s)0.733 ± 0.288 □[28]
Fatty Acid Metabolism
Acetoacetate2.22 (s), 3.41 (m)n/a-
Carnitine2.43 (dd), 3.21 (s), 3.42 (m), 4.56 (s)n/a-
Alkaloids
Trigonelline4.43 ppm (s), 8.08 ppm (t), 8.84 ppm (t), 9.12 ppm (s)n/a-
Unknown Resonances
Unknown #11.1 ppm (s)
Unknown #21.25 ppm (s)
Unknown #32.88 (m)(Jab = 7.67 Hz, Jbc = 7.75), 3.44 (m), 3.62 (m)(Jab = 7.63 Hz, Jbc = 11.12), 3.82 (m)
Unknown #42.93 (d) or two (s)
Unknown #53.15 ppm (s)
Unknown #63.23 ppm (s)
Unknown #74.51 (t) (Jab = 4.41 Hz)
Unknown #87.73 ppm (s)
‡:glycogen values range of oysters, Crassostrea virginica, harvested monthly for 1 year from three areas (Alabama, Louisiana and Maryland), expressed in mg of glycogen per 100 g of oyster tissue;
*:the concentration of amino acids in the free amino acid pool in Eastern Oyster, the concentration means are in μmoles amino acid per mg protein;
†:the range of free amino acid concentrations in the adductor muscle of C. virginica from various salinities (3.4–26.7‰), expressed as μmoles amino acid/mg Kjeldahl nitrogen;
□:the mean of betaine concentration in the gills of Atlantic oyster, expressed as μmole/g dry wt;
□:the level of succinate in the ventricles of C. virginica under aerobic conditions (μmole/g wet wt).

Share and Cite

MDPI and ACS Style

Tikunov, A.P.; Johnson, C.B.; Lee, H.; Stoskopf, M.K.; Macdonald, J.M. Metabolomic Investigations of American Oysters Using 1H-NMR Spectroscopy. Mar. Drugs 2010, 8, 2578-2596. https://doi.org/10.3390/md8102578

AMA Style

Tikunov AP, Johnson CB, Lee H, Stoskopf MK, Macdonald JM. Metabolomic Investigations of American Oysters Using 1H-NMR Spectroscopy. Marine Drugs. 2010; 8(10):2578-2596. https://doi.org/10.3390/md8102578

Chicago/Turabian Style

Tikunov, Andrey P., Christopher B. Johnson, Haakil Lee, Michael K. Stoskopf, and Jeffrey M. Macdonald. 2010. "Metabolomic Investigations of American Oysters Using 1H-NMR Spectroscopy" Marine Drugs 8, no. 10: 2578-2596. https://doi.org/10.3390/md8102578

Article Metrics

Back to TopTop