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Supplement: Waltham International Symposium

Evaluation of Body Composition in Dogs by Isotopic Dilution Using a Low-Cost Technique, Fourier-Transform Infrared Spectroscopy

Unité de nutrition et endocrinologie, Ecole nationale vétérinaire, Nantes, France and ^* Unité de physicochimie des macromolécules, Institut national de la recherche agronomique, Nantes, France

²To whom correspondence should be addressed. E-mail: pnguyen{at}vet-nantes.fr.

KEY WORDS: • body composition • isotopic dilution • deuterium • infrared spectroscopy

EXPANDED ABSTRACT

Evaluation of body composition is indicative of the nutritional status of animals and therefore of crucial interest for the clinician. Among available techniques, assessment of total body water (TBW) (3) by isotopic dilution has been used for a long time for indirect evaluation of body composition and energy expenditure in live animals (1). The isotopic enrichment can be assayed in plasma water after injection of a known dose of labeled water. TBW measurement then allows the calculation of fat-free mass (FFM), on the assumption that FFM hydration is constant at around 73.2% (2,3).

Three isotopes are commonly used as water labels: deuterium (D), tritium and 18-oxygen (¹⁸O). Because of proton exchange with organic compounds in the body, ¹⁸O is known to provide the best evaluation of TBW (4), although at a prohibitive cost. Moreover, isotopes are classically assayed by isotopic ratio mass spectrometry (IRMS), an expensive and time-consuming technique requiring a highly skilled staff. Fourier-transform infrared spectroscopy (FTIR) allows quicker assessment of deuterium concentration and requires less-expensive analytical material. This technique makes it possible to quantify O—D bonds in the mid-infrared spectrum by measuring absorbance, not only at 2500 cm^-1 but throughout the absorption spectrum (2650–2400 cm^-1). Moreover, the use of principal-component analysis (PCA) saves time by allowing the assay to be performed on crude plasma after brief sample preparation.

The FTIR technique described here is inexpensive and easy to run, allowing rapid and reliable determination of body composition by a deuterium assay in dog plasma. The first part of the study attempted to calibrate IR data by IRMS measurement of deuterium concentration. FFM values obtained for the dilution spaces of deuterium, as assessed by FTIR (FFM-Nd) and ¹⁸O by IRMS (FFM-No), were compared and correlated to establish an equation for correcting the overestimation of FFM-Nd ascribed to proton exchange. The second part of the study validated the FTIR technique by comparing FFM-No and FFM-Nd, as corrected by the equation, in another panel of dogs.

	MATERIALS AND METHODS

TOP MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Animals

Twenty-three dogs of different breeds (Miniature Poodle, Beagle, medium and giant Schnauzer, Labrador Retriever, Weimar Pointer and Great Dane) and weights (4 to 52 kg) were used in this study. The animals were housed in accordance with the regulations for animal welfare of the French Ministry of Agriculture and Fisheries. The experimental protocols adhered to European Union guidelines and were approved by the Animal Use and Care Advisory Committee of the University of Nantes. Animals were deprived of food and water for 12 h before experiments.

Experimental procedures

    First part of the study. The protocol was designed to establish an equation correlating FFM deduced from the dilution space of ¹⁸O and deuterium. Fifteen dogs were injected subcutaneously with 150 mg/kg D₂O (99.9% D/H; Euriso-top, Gif-sur-Yvette, France) and 400 mg/kg H₂¹⁸O (10.5% ¹⁸O/O; Rotem Industries, Israel). Blood samples were collected before and 5 h after injection. Another injection of 500 mg/kg D₂O was then performed, and a blood sample was taken after another 5-h period. Plasma deuterium was assayed by FTIR on a Vector 22-type spectroscope (Brüker SA, Wissembourg, France) and on IRMS as previously described (5). Plasma ¹⁸O was assessed by IRMS (5).

    Second part of the study. To validate the equation obtained in the first part of the study, eight dogs were injected with 500 mg/kg D₂O (99.9%) and 400 mg/kg H₂¹⁸O (10.5%). Blood was collected before and 5 h after injection. Deuterium and ¹⁸O were assayed by FTIR and IRMS, respectively.

Plasma samples were frozen at -20°C in sealed vials until analysis. Before deuterium assessment by FTIR, samples were centrifuged for 5 min at 2500 x g.

FTIR analysis

Plasma deuterium was analyzed using a liquid sampling cell system (Perkin–Elmer, Courtaboeuf, France) equipped with matched CaF₂ windows with 0.1-mm pathlength. The cell was overfilled with crude plasma and placed in the spectroscope. Mid-infrared spectra (4000–400 cm^-1) were taken under the following conditions: 4 cm^-1 resolution, 200 scans. Spectra were then processed (smoothing and normalization) and restricted to the region of interest (2650–2400 cm^-1) using OPUS software (Brücker). PCA was applied to IR data to extract the deuterium concentration-dependent component. Samples of deuterium concentration obtained from IRMS were attributed to their respective IR data.

Fat-free mass calculation

TBW was deduced from the dilution space of the isotopes. FFM was calculated as TBW/0.744, that is, the FFM hydration rate originally described for dogs (6).

Statistical analysis

PCA, a multivariate statistical technique, was applied to IR data (8) to assess several variables that are in fact linear combinations of original variables. Briefly, these synthetic variables ("principal components"), which are not correlated, highlight the main variations observed in the data. Our analysis also indicated eigenvectors (coefficients of linear combinations) and eigenvalues (percentages of total variance described by the components, which are subsequently ranked according to this percentage). Thus, the first principal component reveals the main information in the data (e.g., deuterium concentration), whereas the second contains only noise. Similarity maps (graphical representations plotted from the scores of two principal components) allow samples to be compared by taking into account the main information extracted from the original data.

Deuterium assays by FTIR and IRMS were compared using parametric (Pearson) correlation. The nonparametric Wilcoxon’s signed rank test was used to compare FFM-No and FFM-Nd calculated before and after correction by the equation.

	RESULTS

TOP MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Mathematical treatment of plasma IR spectra

Raw IR spectra from dog plasma enriched with various concentrations of D₂O were smoothed, cut in the region of interest (2650–2400 cm^-1) and normalized (Fig. 1). The deuterium concentration measured by IRMS was attributed to each spectrum. The PCA similarity map (Fig. 2) showed that principal component 1 depended on deuterium concentration, whereas principal component 2 related to another source of spectrum variation. The deduced eigenvector for the first principal component indicated that maximum absorbance in the 2650–2400 cm^-1 region was reached near 2500 cm^-1, which corresponded to the stretching vibration of the O—D bond and represented 98% of total inertia.

View larger version (9K): [in this window] [in a new window]   FIGURE 1 Infrared spectra from dog plasma with natural D2O abundance (standard line) or 500 mg/kg enrichment (bold line). Spectra were smoothed, restricted to the region of interest and normalized.

View larger version (15K): [in this window] [in a new window]   FIGURE 2 Principal-component analysis similarity map of infrared spectral data from dog plasma enriched with 0 (a), 150 (b) and 500 mg/kg (c) deuterium oxide. Principal component 1 (x-axis) is closely related to deuterium oxide enrichment and was therefore the only component used to calculate deuterium concentration.

After PCA, a regression curve was calculated only in terms of the first component (Fig. 3). FFM-Nd was then deduced and correlated with FFM-No. The equation obtained [FFM-No = 0.9866(FFM-Nd) - 0.1622, r² = 0.997] was used in the next step of the study.

View larger version (13K): [in this window] [in a new window]   FIGURE 3 Prediction equation for FTIR deuterium assay: D2O (FTIR) = 0.9907 D2O (IRMS) + 5.86 (r2 = 0.991, P < 0.001, = 46.1 ppm, n = 29).

FFM-Nd corrected by the equation and FFM-No (obtained from eight dogs) were compared (Table 1). No statistical differences were observed between the values (Wilcoxon’s signed rank test).

	DISCUSSION

TOP MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The results of Burkholder and Thatcher (8) with IR spectroscopy were poorly correlated with both IRMS and direct body weight measurement, probably because measurement was limited to a single absorbance frequency. In another study, algorithmic processing of spectra showed a very high correlation between FTIR and IRMS for the deuterium assay in human biological fluids (saliva, urine and plasma) (9). Plasma, the most convenient fluid for our study in dogs, needed only simple preparation (centrifugation) compared to the water extraction and purification steps required with other techniques such as IRMS, NMR and gas-phase FTIR (10).

In our study, deuterium was assayed by FTIR to determine TBW and FFM, and the results were compared with those obtained with an ¹⁸O assay by IRMS. The hydration rate used to calculate FFM is usually 73.2%. However, FFM hydration depends on many factors such as species, age and the method used [see Wang et al. (3) for a review], which led us to adopt the hydration rate of 74.4% described by Harrison et al. (6). FFM-Nd and FFM-No were strongly correlated (r² = 0.997) and showed good relative agreement (slope = 0.9866) and a low y-intercept value. Son et al. (11), who compared body fat in dogs as calculated from dual-energy X-ray absorptiometry (DXA) and D₂O dilution and as assayed by NMR, also observed good relative agreement between the methods (regression slope = 1.04), but found considerable differences in absolute values. Our study compared FFM as deduced from the dilution rates of deuterium and ¹⁸O, a method not dependent on proton exchange and therefore likely to be more accurate (4). Indeed, the Nd/No ratio has been calculated as 1.034 in humans (12), which leads to an overestimation of TBW with the deuterium dilution rate. In dogs, deuterium space overestimates TBW by 21.8% (8). Our method appears to rectify FFM-Nd values, and no significant differences were noted between corrected FFM-Nd and FFM-No.

The mean overall relative difference between corrected FFM-Nd and FFM-No was 3.91 ± 0.93%. This can be considered acceptable unless in some cases it could be as high as 8.6%. Assessment of FFM and body fat content is notably interesting for the follow-up of variation in body composition of obese subjects, in which relative differences would be lower. As the group size was limited, factors eventually involved like the degree of adiposity could not be taken into account. Independently of the accuracy of the correction, the value we measured using FTIR deuterium determination was closer to the reference value (FFM-No) than values reported by others using RMN (11) or IRMS (8).

It may be concluded that FTIR, compared to IRMS, is a rapid, easy-to-run, low-cost method for estimating body composition. The simple experimental procedure described here (one injection, two blood samples) allows routine analysis as well as field trials (which are not possible with other techniques such as DXA).

	ACKNOWLEDGMENTS

 
The authors are grateful to Samuel Ninet, Philippe Bleis and Alexandre Vincent for technical assistance.

	FOOTNOTES

 
¹ Presented as part of the Waltham International Symposium: Pet Nutrition Coming of Age held inVancouver, Canada, August 6–7, 2001. This symposium and the publication of symposium proceedings were sponsored by the Waltham Centre for Pet Nutrition. Guest editors for this supplement were James G. Morris, University of California, Davis, Ivan H. Burger, consultant to Mars UK Limited, Carl L. Keen, University of California, Davis, and D’Ann Finley, University of California, Davis.

³ Abbreviations used: DXA, dual-energy X-ray absorptiometry; FFM, fat-free mass; FFM-Nd, fat-free mass deduced from the dilution rate of deuterium-labeled water; FFM-No, fat-free mass deduced from the dilution rate of ¹⁸O-labeled water; FTIR, Fourier-transform infrared spectroscopy; IRMS, isotopic ratio mass spectrophotometry; NMR, nuclear magnetic resonance; PCA, principal-component analysis; ppm, part per million (µg/g); TBW, total body water.

	LITERATURE CITED

TOP MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

1. Lifson, N., Gordon, G. B. & McClintock, R. (1955) Measurement of total dioxide production by means of D₂¹⁸O. J. Appl. Physiol. 7:704-710.[Free Full Text]

2. Pace, N. & Rathburn, E. N. (1945) Studies on body composition. III. The body water and chemically combined nitrogen content in relation to fat content.. J. Biol. Chem. 158:685-691.[Free Full Text]

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4. Schoeller, D. A., Santen, E. V., Peterson, D. W., Dietz, W., Jaspan, J. & Klein, P. D. (1980) Total body water measurement in humans with ¹⁸O- and ²H-labelled water. Am. J. Clin. Nutr. 33:2286-2293.

5. Pouteau, E. B., Mariot, S. M., Martin, L. J., Dumon, H. J., Mabon, F. J., Krempf, M. A., Robins, R. J., Darmaun, D. H., Naulet, N. A. & Nguyen, P. G. (2001) Rate of carbon dioxide production and energy expenditure in fed and food-deprived adult dogs determined by indirect calorimetry and isotopic methods. Am. J. Vet. Res. (in press).

6. Harrison, H. E., Darrow, D. C. & Yannet, H. (1936) The total electrolyte content of animals and its probable relation to the distribution of body water. J. Biol. Chem. 113:515-529.[Free Full Text]

7. Robert, P., Devaux, M.-F. & Bertrand, D. (1996) Beyond prediction: extracting relevant information from near infrared spectra. J. Near Infrared Spectrosc. 4:75-84.

8. Burkholder, W. J. & Thatcher, C. D. (1998) Validation of predictive equations for use of deuterium oxide dilution to determine body composition of dogs. Am. J. Vet. Res. 59:927-937.[Medline]

9. Jennings, G., Bluck, L., Wright, A. & Elia, M. (1999) The use of infrared spectrophotometry for measuring body water spaces. Clin. Chem. 45:1077-1081.[Abstract/Free Full Text]

10. Backus, R. C., Havel, P. J., Gingerich, R. L. & Rogers, Q. R. (2000) Relationship between serum leptin immunoreactivity and body fat mass as estimated by a novel gas-phase Fourier transform infrared spectroscopy infrared deuterium dilution method in cats. Am. J. Vet. Res. 61:796-801.[Medline]

11. Son, H. R., d’Avignon, A. & Laflamme, D. P. (1998) Comparison of dual energy X-ray absorptiometry and measurement of total body water content by deuterium oxide dilution for estimating body composition in dogs. Am. J. Vet. Res. 59:529-532.[Medline]

12. Coward, W. A., Ritz, P. & Cole, T. J. (1994) Revision of calculations in the doubly labeled water method for measurement of energy expenditure in humans. Am. J. Physiol. Endocrinol. Metab. 267:E805-E807.[Abstract/Free Full Text]

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