**3. Deuterated water as a tracer of endogenous glucose and glycogen synthesis**

#### **3.1. Overview**

ditions – a critical requirement for investigating precursor-product relationships of biosyn‐ thetic pathways. This is particularly problematic for quantitative studies of gluconeogenesis based on the delivery of a labeled precursor substrate such as 14C-lactate or alanine, since both the tracee precursor and glucose product pools must be at isotopic equilibrium during

There are two approaches for quantifying plasma glucose Ra by isotope dilution. The first involves an intravenous injection of a tracer bolus and monitoring the decrease in specific activity or enrichment of plasma glucose as the tracer is being diluted by unla‐ beled glucose [75]. The dilution kinetics are best represented by more than one exponen‐ tial decay function indicating the presence of separate pools of glucose in the body with different clearance characteristics. Because of the relative simplicity of a single injection tracer delivery, this method has been applied in many fish species including kelpbass *Paralabrax clathratus* [73], seabass *D. labrax* [74], *Hoplias malabaricus* [76], common carp *C. carpio* [23]. However, this approach requires frequent blood sampling over a sustained period to adequately describe the complex tracer clearance kinetics. Limited by the num‐ ber of blood samples that could be drawn from the fish, the study described in [73] ex‐ trapolated the clearance kinetics from a smaller set of initial measurements, but the uncertainties of this approach were acknowledged. The alternative primed-infusion meth‐ od establishes a constant level of tracer in the bloodstream following an appropriate pri‐ ming dose. Under these conditions, the dilution of the tracer, measured from the ratio of specific activity or enrichment of infused label to that of blood glucose, is equal to the ratio of endogenous glucose appearance and tracer infusion rates. If glucose Ra is varia‐ ble (for example during meal ingestion) then the rate of infusion needs to be adjusted accordingly in order to maintain a constant ratio of infused glucose to blood glucose specific activity or enrichment. Compared to the bolus injection method, primed infu‐ sion measurements require fewer blood samplings; in fact the sole rationale for multiple blood samplings is to verify a constant ratio of infused to blood glucose specific activity or enrichment. Calculation of glucose Ra from steady-state isotope data is more robust compared to single injection since it is independent of the complex and often poorly de‐ fined clearance kinetics. However, a primed infusion requires catheterization of a vein to deliver the tracer over an extended period and is technically more difficult than a single bolus injection. While these procedures now allowed glucose Ra to be well determined, and by combination with dietary tracers can determine the contributions of absorbed and endogenous glucose production to glucose Ra, they do not inform the sources of en‐ dogenous glucose production. Novel methodologies of resolving the sources of glucose

H2O) have been developed and can be integrated with

abolic studies since it can be incorporated into aquarium water for an indefinite period

sult of enzyme-catalyzed exchange reactions between bulk water and metabolite hydro‐

H2O is ideally suited for fish met‐

H-enriched tank water. As a re‐

H-enrichment level of

**2.4. Measurement of glucose Ra by bolus injection and by primed infusion**

the sampling period.

256 New Advances and Contributions to Fish Biology

Ra using deuterated water (2

primed-infusion glucose Ra measurements [80, 81, 82]. 2

the fish tissue water is also fixed for the duration in the 2

and is rapidly incorporated into the fish body water such that the 2

Fish and mammals share common pathways for glucose production and consumption [86] hence the underlying principles of plasma glucose 2 H-enrichment from 2 H2O that are well described and validated for mammals [80, 87, 88] can be applied to fish. Deuterated water ( 2 H2O) is a relatively inexpensive non-radioactive tracer that can be incorporated in drinking water, or in the case of fish studies, in the tank water. It has been successfully used in hu‐ mans and other mammals for the study of hepatic intermediary metabolism in both normal and pathological conditions. It rapidly equilibrates with total body water and is distributed evenly into all tissues. It is a practical tracer for both short and long-term metabolic studies. 2 H2O is ideally suited for studying fish metabolism since it can be added to the tank water for an indefinite period, during which time it is incorporated into hepatic metabolites such as glycogen and glucose by specific enzymatic reactions in their biosynthetic pathways, as previously described for mammals. Applying these principles to free-swimming fish pro‐ vides an authentic metabolic profile that is unadulterated by anesthesia or infusion proce‐ dures that characterize the administration of classical carbon tracers.

#### **3.2. Basic principles**

Deuterium (2 H) is a stable isotope of hydrogen with a nucleus containing one proton and one neutron (the 1 H nucleus contains no neutron). In the NMR experiment, 2 H resonates at a different frequency compared to its 1 H counterpart, allowing tracer levels of 2 H to be ob‐ served in the presence of the tracee 1 H. The inherent sensitivity of 2 H (at constant field and with an equivalent number of nuclei) is about 0.9% that of 1 H. Metabolism of 2 H is not exact‐ ly equivalent to that of 1 H because of kinetic isotope effects. The strength of a chemical bond between two atoms is dependent in part on their relative masses, hence a C-2 H bond is stronger than a C-1 H for any compound. Since metabolite transformation is governed in part by breaking and formation of C-H bonds, the presence of 2 H makes the bonds harder to break thereby potentially slowing the rate of C-2 H *vs.* C-1 H transformation. This can discrim‐ inate the transformation of 2 H-enriched metabolites compared to their tracees resulting in apparently slower rates of transformation. Moreover, with bulk levels of 2 H tracers, notably 2 H2O, the aggregate isotope effects are toxic and indeed lethal to most living organisms. With tracer studies that utilize 2 H2O, toxicity from isotope effects is minimized by substitut‐ ing a relatively low proportion of 1 H by 2 H (<10%). Furthermore, discrimination against 2 H incorporation into metabolites via enzymatic reactions is minimized when the reaction that transfers 2 H from water to the metabolite hydrogen is reversible. Most of the enzymatic steps of intermediary metabolism are reversible with extensive exchange of precursor and product, and under these conditions, discrimination of 2 H incorporation via kinetic isotope effects is not significant. Indeed, when 2 H-discrimination is observed, it informs the unidir‐ ectionality of a particular enzymatic step.

These caveats notwithstanding, 2 H2O is an inexpensive tracer that is easily delivered in‐ to body water by immersion of fish in 2 H-enriched water. It has been successfully used in humans [89, 90, 91] and other mammals [82, 92, 93], for study of hepatic carbohy‐ drate metabolism in physiological and pathophysiological conditions. The methodology was pioneered in humans [80, 87, 94] and was rapidly adopted by others in tissues [81, 88, 95]. As previously discussed, G6P is a common precursor to both glycogen and glu‐ cose and in the presence of 2 H2O, G6P is labeled with 2 H in several positions due to the incorporation of that isotope via exchange with body water. It rapidly equilibrates with total body water and distributes homogeneously within tissues and the body wa‐ ter 2 H-enrichment level can be maintained indefinitely [96]. In fish, incorporation of 2 H from a 5%-enriched saltwater tank into plasma water is rapid, reaching more than 1% in 15 minutes, half of the enrichment of the tank water within 1 hour and approaching that of the tank water after 6h [85].

For studies of carbohydrate metabolism, the 2 H-enrichment distribution of plasma glucose from 2 H2O is established according to origin of the G6P precursor (Figure 2.).

> **Figure 2.** Metabolic model representing gluconeogenesis in the liver with special detail to the labeling with deuteri‐ um in position 2 (2H2) and 5 (2H5) of the glucose molecule. Some metabolic intermediates were omitted for clarity. Abbreviations are as follows: G6P - glucose 6-phosphate; F6P - fructose 6-phosphate; F16P2 - fructose 1,6-bisphos‐ phate; G3P - glyceraldehyde 3-phosphate; DHAP - dihydroxyacetone phosphate; PEP - phosphoenolpyruvate; OA - ox‐

> shown in Figure 2. Therefore, enrichment of plasma glucose or hepatic glycogen in po‐ sition 5 reflects the contribution of gluconeogenic fluxes to endogenous glucose produc‐

> > **H enrichment analysis**

Many improvements on the measurement of stable isotope tracer enrichment and analy‐ sis of positional labeling information have been largely driven by the development of nu‐ clear magnetic resonance (NMR) and mass spectrometry (MS) technologies. Choosing between these methods depends on the kind of enrichment information that is required from the experiment, the available sample size and access to instrumentation. MS techni‐

tion or indirect pathway contributions to hepatic glycogen synthesis.

H-Enrichment in position 5 of G6P occurs at the level of triose phosphates as

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259

aloacetate; 2H2O - deuterated water.

**3.3. NMR and MS methods for 2**

Besides, 2

If produced from gluconeogenic substrates (gluconeogenic amino acids, pyruvate or glycerol) enrichment in position 2 (H2) is obligatory since conversion of F6P to G6P (fa‐ cilitated by G6P-isomerase) is part of the gluconeogenic pathway. In mammals, G6P-F6P exchange is extensive and essentially complete hence the hepatic G6P pool is quantitatively enriched in H2 regardless of its origin. This means that newly-synthe‐ sized glycogen from G6P is also enriched in this position, regardless of whether the G6P was derived via the direct or indirect pathway [93, 97]. There is some evidence that in fish, hepatic G6P-isomerase activity is sensitive to the nutritional state [43]. In common carp *C. carpio* hepatic G6P-isomerase activity decreased in direct relation to feeding rates [98, 99] but there is no information on how G6P-isomerase activity is modified during the fasting to feeding transition. One possible explanation is that in‐ duction of G6P-isomerase activity by feeding is slow compared to activation of glyco‐ gen synthesis fluxes. Under these conditions, G6P-isomerase activity could be a rateliming step for indirect pathway synthesis of glycogen, at least in the initial stages of refeeding. In principle, sub-maximal G6P-isomerase activity could limit the glycolytic metabolism of G6P derived from glucose and favor its conversion to glycogen via the direct pathway or its utilization by the pentose phosphate pathway.

**Figure 2.** Metabolic model representing gluconeogenesis in the liver with special detail to the labeling with deuteri‐ um in position 2 (2H2) and 5 (2H5) of the glucose molecule. Some metabolic intermediates were omitted for clarity. Abbreviations are as follows: G6P - glucose 6-phosphate; F6P - fructose 6-phosphate; F16P2 - fructose 1,6-bisphos‐ phate; G3P - glyceraldehyde 3-phosphate; DHAP - dihydroxyacetone phosphate; PEP - phosphoenolpyruvate; OA - ox‐ aloacetate; 2H2O - deuterated water.

Besides, 2 H-Enrichment in position 5 of G6P occurs at the level of triose phosphates as shown in Figure 2. Therefore, enrichment of plasma glucose or hepatic glycogen in po‐ sition 5 reflects the contribution of gluconeogenic fluxes to endogenous glucose produc‐ tion or indirect pathway contributions to hepatic glycogen synthesis.

#### **3.3. NMR and MS methods for 2 H enrichment analysis**

2

transfers 2

ter 2

from 2

With tracer studies that utilize 2

258 New Advances and Contributions to Fish Biology

ing a relatively low proportion of 1

effects is not significant. Indeed, when 2

ectionality of a particular enzymatic step.

to body water by immersion of fish in 2

These caveats notwithstanding, 2

cose and in the presence of 2

that of the tank water after 6h [85].

For studies of carbohydrate metabolism, the 2

H2O, the aggregate isotope effects are toxic and indeed lethal to most living organisms.

incorporation into metabolites via enzymatic reactions is minimized when the reaction that

steps of intermediary metabolism are reversible with extensive exchange of precursor and

in humans [89, 90, 91] and other mammals [82, 92, 93], for study of hepatic carbohy‐ drate metabolism in physiological and pathophysiological conditions. The methodology was pioneered in humans [80, 87, 94] and was rapidly adopted by others in tissues [81, 88, 95]. As previously discussed, G6P is a common precursor to both glycogen and glu‐

H2O, G6P is labeled with 2

the incorporation of that isotope via exchange with body water. It rapidly equilibrates with total body water and distributes homogeneously within tissues and the body wa‐

from a 5%-enriched saltwater tank into plasma water is rapid, reaching more than 1% in 15 minutes, half of the enrichment of the tank water within 1 hour and approaching

If produced from gluconeogenic substrates (gluconeogenic amino acids, pyruvate or glycerol) enrichment in position 2 (H2) is obligatory since conversion of F6P to G6P (fa‐ cilitated by G6P-isomerase) is part of the gluconeogenic pathway. In mammals, G6P-F6P exchange is extensive and essentially complete hence the hepatic G6P pool is quantitatively enriched in H2 regardless of its origin. This means that newly-synthe‐ sized glycogen from G6P is also enriched in this position, regardless of whether the G6P was derived via the direct or indirect pathway [93, 97]. There is some evidence that in fish, hepatic G6P-isomerase activity is sensitive to the nutritional state [43]. In common carp *C. carpio* hepatic G6P-isomerase activity decreased in direct relation to feeding rates [98, 99] but there is no information on how G6P-isomerase activity is modified during the fasting to feeding transition. One possible explanation is that in‐ duction of G6P-isomerase activity by feeding is slow compared to activation of glyco‐ gen synthesis fluxes. Under these conditions, G6P-isomerase activity could be a rateliming step for indirect pathway synthesis of glycogen, at least in the initial stages of refeeding. In principle, sub-maximal G6P-isomerase activity could limit the glycolytic metabolism of G6P derived from glucose and favor its conversion to glycogen via the

H2O is established according to origin of the G6P precursor (Figure 2.).

direct pathway or its utilization by the pentose phosphate pathway.

H-enrichment level can be maintained indefinitely [96]. In fish, incorporation of 2

H from water to the metabolite hydrogen is reversible. Most of the enzymatic

H by 2

product, and under these conditions, discrimination of 2

H2O, toxicity from isotope effects is minimized by substitut‐

H (<10%). Furthermore, discrimination against 2

H-discrimination is observed, it informs the unidir‐

H-enriched water. It has been successfully used

H-enrichment distribution of plasma glucose

H2O is an inexpensive tracer that is easily delivered in‐

H incorporation via kinetic isotope

H in several positions due to

H

H

Many improvements on the measurement of stable isotope tracer enrichment and analy‐ sis of positional labeling information have been largely driven by the development of nu‐ clear magnetic resonance (NMR) and mass spectrometry (MS) technologies. Choosing between these methods depends on the kind of enrichment information that is required from the experiment, the available sample size and access to instrumentation. MS techni‐ 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‐ crease in the molecular mass, (i.e. 2 H and 13C) cannot be resolved, placing limitations on 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 throughput measurements.

**4. Conclusions**

standing of fish nutritional physiology.

\*Address all correspondence to: iviegas@ci.uc.pt

**Acknowledgements**

tiveness Programme.

**Author details**

John Griffith Jones2

Portugal

**References**

1103.

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‐

Advances and Applications of Tracer Measurements of Carbohydrate Metabolism in Fish

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261

We would like to thank Fundação para a Ciência e Tecnologia (FCT) for funding the project "Optimizing carbohydrate utilization in farmed sea-bass through metabolic profiling" (PTDC/EBB-BIO/098111/2008) through the programme COMPETE - Operational Competi‐

1 Department of Life Sciences, Faculty of Sciences and Technology, University of Coimbra,

[1] Roush W. Zebrafish Embryology Builds Better Model Vertebrate. Science 1996;272:

[2] Lieschke GJ, Currie PD. Animal models of human disease: zebrafish swim into view.

[3] Berman JN, Kanki JP, Look AT. Zebrafish as a model for myelopoiesis during em‐

2 CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Portugal

3 CFE - Centre for Functional Ecology, University of Coimbra, Portugal

bryogenesis. Experimental Hematology 2005;33: 997-1006.

Nature Reviews Genetics 2007;8: 353-367.

Ivan Viegas1,2,3, Rui de Albuquerque Carvalho1,2, Miguel Ângelo Pardal1,3 and

Following a simple method based on a LC-MS/MS procedure for quantifying plasma [6,6-2 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 gluconeogenic contribution include the incomplete incorporation of 2 H into sites other than position 5, as seen by the tendency for lower enrichments in positions 1, 3, 4, 6*R* and 6*S* com‐ pared to position 5 by 2 H-NMR analysis [85].

Analysis of 2 H enrichment by 2 H NMR spectroscopy is a method with much lower sensitivi‐ ty compared to MS, requiring 5-50 μmol of analyte in the typical experimental setting for 2 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 are present at ~100% natural abundance and are observed by NMR include 1 H and 31P and 23Na. Isotopes that are more rare in nature such as 2 H (0.015% of hydrogen) and 13C (1.11% of carbon) can also be observed at natural abundance levels, but molecules that are enriched to higher levels from 2 H- or 13C-enriched precursors can be measured against this background. 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 [102, 103, 104]. This is particularly important for analysis of carbohydrate 2 H enrichment, which feature highly crowded hydrogen signals that are poorly resolved by the inherently small dispersion of 2 H signals (~15% of 1 H signals).
