**4. Conclusions**

ques quantify metabolite enrichment by resolving heavier labeled molecules from lighter unlabeled ones. For most MS instruments, the presence of two isotopes with similar in‐

multiple isotope studies. Positional enrichment can be inferred from fragmentation and analysis of the mass of the daughter fragments (MS-MS). Nevertheless, as fragmentation is dependent on the molecule's chemical structure, the label of interest may or may not be isolated. Chemical derivatization of metabolites is often used to facilitate fragmenta‐ tion and positional enrichment analysis [87]. MS is highly sensitive and can quantify en‐ richments from submicromole to picomole amounts of analyte and with appropriate signal calibration and sample purification safeguards, it can be configured for high

Following a simple method based on a LC-MS/MS procedure for quantifying plasma

position 5, as seen by the tendency for lower enrichments in positions 1, 3, 4, 6*R* and 6*S* com‐

ty compared to MS, requiring 5-50 μmol of analyte in the typical experimental setting for

H2O studies (0.5-5.0 % body water enrichment). However, in addition to being nondestruc‐ tive to the sample, NMR provides a much higher level of positional enrichment information, allows enrichment from multiple stable isotope tracers to be selectively observed, and can provide a global analysis of metabolite enrichments from a complex mixture of metabolites, such as cellular extracts, biological fluids, and intact tissues [101]. This technique relies on the ability of atomic nuclei with odd mass and/or atomic number to align if subjected to an external magnetic field. When irradiated with a certain frequency signal the nuclei in a mol‐ ecule can change their alignment and the energy frequency at which this occurs can be measured and displayed as an NMR spectrum. Common biologically relevant nuclei that

carbon) can also be observed at natural abundance levels, but molecules that are enriched to

Since isotopes resonate at a specific frequency, its signals can be uniquely isolated from any other isotope that may be present. Derivatization of the target molecule can be used to pro‐ vide a more heterogeneous chemical environment therefore improving signal dispersion

which feature highly crowded hydrogen signals that are poorly resolved by the inherently

H signals).

H- or 13C-enriched precursors can be measured against this background.

gluconeogenic contribution include the incomplete incorporation of 2

H-NMR analysis [85].

are present at ~100% natural abundance and are observed by NMR include 1

[102, 103, 104]. This is particularly important for analysis of carbohydrate 2

H2]glucose enrichment [100] which does not require glucose derivatization, analysis of glucose can be performed on a few microliters of blood, either whole or as a dried spot on filter paper. This means it can be applied to any size fish and can also be used for repeated sampling of the same fish [85]. This LC-MS/MS measurement provides the mole percent en‐ richment (MPE) of the glucose molecule, equivalent to the sum of all seven positional en‐ richments. The principal uncertainties of utilizing plasma MPE levels as a marker of

H and 13C) cannot be resolved, placing limitations on

H NMR spectroscopy is a method with much lower sensitivi‐

H into sites other than

H and 31P and

H enrichment,

H (0.015% of hydrogen) and 13C (1.11% of

crease in the molecular mass, (i.e. 2

260 New Advances and Contributions to Fish Biology

throughput measurements.

pared to position 5 by 2

higher levels from 2

small dispersion of 2

H enrichment by 2

23Na. Isotopes that are more rare in nature such as 2

H signals (~15% of 1

Analysis of 2

2

[6,6-2

There is a compelling need to better understand the metabolism of carbohydrates by fish in general and aquaculture species in particular. Stable-isotope tracer methodologies have evolved such that safe, inexpensive and practical measurements of carbohydrate metabo‐ lism may be directly performed on naturally feeding fish in the aquaculture setting. These studies have great potential for informing the efficacy of novel dietary supplements in spar‐ ing the conversion of feed protein to carbohydrate as well as improving our general under‐ standing of fish nutritional physiology.
