Validation and calibration of DEXA body composition in mice

Robert Brommage

Department of Endocrinology, Lexicon Genetics, The Woodlands, Texas 77381

Submitted 30 October 2002 ; accepted in final form 14 May 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Validated methods of determining murine body composition are required for studies of obesity in mice. Dual-energy X-ray absorptiometry (DEXA) provides a noninvasive approach to assess body fat and lean tissue contents. Similar to DEXA analyses in other species, body fat measurements in mice show acceptable precision but suffer from poor accuracy. Because fat and lean tissues each contain various components, these inaccuracies likely result from selection of inappropriate calibration standards. Analysis of solvents showed that the PIXImus2 DEXA gave results consistent with theoretical calculations. Male mice weighing 26-60 g and having body fat percentages ranging from 3 to 49% were analyzed by both PIXImus2 DEXA and chemical carcass analysis. DEXA overestimated mouse fat content by an average of 3.3 g, and algorithms were generated to calculate body fat from both measured body fat values and the measured ratio of high- to low-energy X-ray attenuations. With calibration to mouse body fat content measured by carcass analysis, the PIXImus2 DEXA gives accurate body composition values in mice.

PIXImus; carcass analysis; obesity; dual-energy X-ray absorptiometry


WITH ADVANCES IN TECHNOLOGIES producing mice with either overexpression (transgenic) or lack of expression (knockout) of specific genes, this species has been increasingly employed to examine physiological and pathological processes (1, 23). Obesity is of particular interest, because numerous metabolic pathways are involved in body weight regulation, and obesity is a growing clinical problem in developed countries. Genetically engineered mice have provided and will continue to provide important insights into the etiology and potential treatment of human obesity (4, 13, 24, 28, 30).

Several techniques are available for in vivo measurements of body composition, and Ellis (10) has reviewed procedures used in human studies. Noninvasive techniques employed to determine body composition in mice include electrical conductivity (2, 17), magnetic resonance spectroscopy (3, 16), and dual-energy X-ray absorptiometry (DEXA) (5, 12, 15, 19, 22, 26). Although the DEXA technique is highly reproducible, validation studies in various species, including mice (19), have questioned its accuracy. There are several manufacturers of DEXA instruments, and each system employs a slightly different approach to determine body composition. Among other differences, various calibration standards and procedures are used. Because the use of tissue samples is often impractical, known masses of plastic and aluminum are usually employed as calibration standards.

Chemical carcass analysis remains the "gold standard" for determining body composition. Complete carcass analysis entails measuring tissue water content by drying, fat by extraction with organic solvents, protein and carbohydrate by chemical analyses, and bone mineral content by ashing. Because of their small size, carcass analysis in mice is relatively simple, and several groups have used this approach to measure body fat in mice following necropsy (7, 9, 20).

The goal of the present work was to employ carcass analysis to determine the accuracy of the PIXImus2 DEXA in measuring body fat in mice. Because the data agreed with previous work by Nagy and Clair (19) that the PIXImus overestimates mouse body fat content, algorithms were developed to directly calibrate the PIXImus2 by using the results of carcass analysis, and the theoretical principles underlying this calibration are described. In addition, the constant proportion of body water to lean body mass has been confirmed, and potential uses of this relationship to simplify mouse body composition studies are briefly discussed.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Two groups of male mice were examined. The first group (n = 19) consisted of C57BL/6J mice between 12 and 21 wk of age fed a high-fat diet from weaning to increase body fat content. The second group (n = 6) consisted of hybrid mice generated from an F2 cross of C57BL/6J and 129 SvEv mice that were fed a standard rodent diet from weaning. These mice ranged from 35 to 83 wk of age and were selected from a larger cohort of mice to have either low or high body fat contents. Two lean mice from this second group were fasted for 2 days to obtain very low values of body fat. No other mice were fasted, as examining fed mice is the standard procedure at Lexicon Genetics. Consequently, body composition values include the contents of the small and large intestines. Body weights for the entire population ranged from 26 to 60 g, with corresponding decapitated carcass weights of 23 to 55 g. No experimental procedures were performed on live mice.

Mice were killed by carbon dioxide asphyxiation and frozen at -20°C in airtight plastic freezer bags to prevent carcass dehydration. On the day of analysis, mouse carcasses were thawed at room temperature, decapitated, and weighed, and body composition was determined with a PIXImus2 Mouse Densitometer (GE Medical Systems, Madison, WI) using software versions 1.46 and 2.10. Results were essentially identical with these two software versions, as a linear regression analysis of body fat percentage gave a slope of 0.9996, an intercept of minus 0.02%, and an r2 value of 0.9997. All data presented are from software version 2.10. Mouse carcasses were decapitated because standard procedure during PIXI-mus analysis is to eliminate the head in body composition determinations. Decapitated carcasses were placed into double-thickness 33 x 80-mm cellulose extraction thimbles and dehydrated at 75°C in an oven until constant carcass weight was obtained, typically requiring 7 days. Lipids were extracted from dried carcasses over 20-24 h with distilled acetone in a Soxhlet apparatus and reweighed after evaporation of residual acetone. The entire decapitated carcass was analyzed without grinding, mixing, and separation into aliquots.

Body fat content is defined as the difference between dehydrated and extracted carcass weights. Lean body mass (LBM) is calculated by subtracting fat content and bone mineral content (BMC; determined by DEXA) from initial carcass weight. Body fat percentage was separately calculated, with the assumption that LBM contains 74.6% water, as follows:

To evaluate the reproducibility of the DEXA technique, one mouse was scanned ten times with repositioning. A series of solvents (paraffin oil, octanol, butanol, butyl acetate, propylene glycol, glycerol, nitromethane, and water) and canola oil were examined to evaluate the ability of the PIXI-mus2 DEXA to distinguish materials of various compositions. A square plastic container with its top and bottom removed was glued onto the PIXImus sample holder, and the liquids were added to a height of 1.4 cm. The container area (7.24 cm2) and solvent densities were used to determine the weight of solvent required to achieve a height of 1.4 cm.

The theoretical ratios of high- to low-energy X-ray mass attenuation coefficients at 40 and 80 keV for the solvent series were calculated according to the formula and elemental mass attenuation coefficients presented by Testolin et al. (27).


    RESULTS
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 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
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From theoretical considerations, the measured ratio of X-ray attenuation coefficients (DEXA ratio) is a direct function of average atomic number. As shown in Fig. 1, although the measured DEXA ratios of the solvents examined were lower than theoretical ratios, there was an excellent correlation (r2 = 0.994) between these two ratios.



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Fig. 1. Relationship of measured and theoretical dual-energy X-ray absorptiometry (DEXA) ratios (defined as the ratio of high- to low-energy X-ray attenuations) for various solvents. Theoretical DEXA ratios were calculated from the mass attenuation coefficients provided by Testolin et al. (27). Linear regression analysis gave a slope of 0.56. Dotted line represents values obtained if measured and theoretical DEXA ratios gave identical results.

 

As part of an approach to validate the carcass analysis procedure, body fat percentage was calculated on the assumption that 74.6% of LBM is water. As shown in Fig. 2, the results obtained using this assumption were virtually identical to body fat percentage determined from complete carcass analysis (with fat extraction). The results of such calculations were similar when the hydrated percentage of LBM was varied from 72 to 76%. However, a value of 74.6% gave the best fit, as indicated by the closer agreement between the two calculations.



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Fig. 2. Body fat percentage determined by carcass analysis and from assuming that 74.6% of lean body mass (LBM) is composed of water. Dotted line represents values obtained if carcass analysis and carcass dehydration measurements gave identical results.

 

Figure 3 presents the relationships between DEXA ratio and body fat percentage, both calculated by DEXA and determined by carcass analysis. The DEXA technique overestimated body fat percentage throughout the range of body fat examined. Nonetheless, there was a consistent linear relationship between DEXA ratio and body fat determined by carcass analysis (r2 = 0.94). The DEXA ratio varied between 1.25 and 1.30 for the range of body fat percentages between 3 and 49%. From the measured DEXA ratio, carcass fat percentage can be calculated directly as



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Fig. 3. Body fat percentage measured by DEXA and determined by carcass analysis as a function of DEXA ratio. For the DEXA measurements ({bullet}), body fat (%) = 1179 - 899 · DEXA ratio (r2 = 1.000). For carcass analysis ({circ}), body fat (%) = 1289-990 · DEXA ratio (r2 = 0.94).

 

For a body fat percentage of 100%, essentially identical DEXA ratios are obtained for DEXA (1.20134) and carcass (1.20136) analyses. These findings indicate that the PIXImus2 DEXA has been accurately calibrated for pure body fat. In contrast, the PIXImus2 DEXA appears to have been miscalibrated for LBM. Extrapolation of the data presented in Fig. 3 to 100% LBM (0% body fat) indicates that the DEXA ratio of 1.30242 determined by carcass analysis is lower than the DEXA ratio of 1.31264 employed by the PIXImus2 DEXA. The difference in DEXA ratios is 762-fold greater for LBM than for body fat.

The measured DEXA ratios for the solvents shown in Fig. 1 were employed to calculate theoretical body fat percentages using the equations presented in Fig. 3. The results, presented in Table 1, confirm that the PIXImus DEXA generates higher fat percentages than carcass analysis. Consistent with a miscalibration of LBM and not body fat, similar fat percentages were calculated for solvents with an atomic composition close to that of fat. As the solvents more closely mimicked LBM, calculated fat percentages diverged between estimates generated by DEXA and carcass analysis. The PIXImus2 DEXA provided a value of 9.3% body fat for water, whereas the calibration equation derived from carcass analysis gave a body fat percentage of 0.1% for water. As a further validation, canola oil analyzed in the identical fashion to the solvents was calculated to be 100.2% body fat by both approaches.


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Table 1. Body fat percentages calculated for various solvents

 

Figure 4 presents direct comparisons of body fat determined by DEXA and carcass analysis. As indicated from the data presented in Fig. 3, the DEXA technique gave higher values for body fat than carcass analysis. This overestimation of body fat averaged 3.3 ± 0.2 g (means ± SE). Because higher levels of body fat content measured by DEXA were relatively constant for various body fat contents, the overestimation of body fat percentage was greater at low body fat percentages than at higher values. From the measured DEXA body fat percentage, carcass fat percentage can be calculated directly as



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Fig. 4. Comparisons of body fat measurements by DEXA and carcass analysis. Dotted lines represent values obtained if DEXA and carcass analysis measurements gave identical results.

 

Similarly, carcass fat content can be calculated from the measured DEXA body fat content as

For a hypothetical mouse having zero body fat, DEXA measurements generate an incorrect body fat percentage of ~10%.

Because the DEXA technique overestimates body fat content, body total tissue mass and/or lean body mass (LBM) must also be incorrect. The data in Fig. 5 demonstrate that the PIXImus2 overestimates both total tissue mass and LBM. The overestimation of total tissue mass (3.3 ± 0.2 g) was identical to the overestimation of body fat content. Correct values for total tissue mass can be calculated from the DEXA data as



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Fig. 5. Comparisons of measurements by DEXA and carcass analysis, with all values expressed in g. Total tissue mass and carcass weight should be identical, because decapitated carcasses were analyzed. Dotted lines represent values obtained if DEXA and carcass analysis measurements gave identical results.

 

LBM measured by DEXA was higher by 0.5 ± 0.2 g compared with carcass analysis values, and correct values can be calculated as

The ability of the derived equations to predict body fat accurately was evaluated by calculating the differences between measured carcass fat and the difference predicted by these equations. For all 25 mice analyzed, the average errors were 1.7% for body fat percentage and 0.65 g for body fat content. Reproducibility of the DEXA measurements was determined by scanning one mouse (body fat = 15.6% by DEXA) 10 times with repositioning. Coefficients of variation were 0.016% for DEXA ratio, 0.4% for both LBM and total tissue mass, 1.2% for body fat percentage, and 1.4% for body fat content.


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
DEXA is a noninvasive technique that permits longitudinal measurements of body composition. Nonskeletal tissue is assigned to fat and lean compartments, and the differential attenuation of low- and high-energy X-rays is employed to calculate the relative proportions of fat and lean tissue. The physics of this process is well understood, as X-ray attenuation increases with increasing atomic number (21). Relative to lean tissue (water, protein, and carbohydrate), fat contains a higher proportion of carbon (atomic no. = 6) to oxygen (atomic no. = 8) and thus has lower average atomic number. Therefore, lean tissue has a higher X-ray attenuation than fat, and this differential attenuation is greater for high- than for low-energy X-rays. The ratio of high- to low-energy X-ray attenuation coefficients (DEXA ratio) is the fundamental DEXA measurement employed to calculate body composition.

GE Medical Systems recently introduced the PIXI-mus DEXA for measuring bone and body composition in mice. This absorptiometer employs a cone beam X-ray source generating energies of 35 and 80 keV and a flat 100 x 80-mm detector having individual pixel dimensions of 0.18 x 0.18 mm. Although reproducibility is acceptable, as shown previously (12, 19, 22) and confirmed in this study, the PIXImus overestimates body fat content in mice (19). This inaccuracy in body composition data prompted further validation studies and exploration of the relationship between measured DEXA ratio and chemical composition, including mouse carcasses having a wide range of fat contents.

A complication of the PIXImus cone beam DEXA not present in fan and pencil beam densitometers involves the existence of an object plane located 7 mm above the detector surface. Tissue composition is most accurately measured at this object plane. Although measured percent fat values are location invariant, measured fat, lean, and tissue masses depend on their location relative to the object plane (PIXImus manual). With these considerations, the most accurate results are presumably obtained in mice that are 1.4 cm thick. Although this value is a reasonable average body thickness, clearly neither all mice nor all parts of a single mouse are exactly 1.4 cm thick. These variations in mouse body thickness undoubtedly contribute in an incompletely characterized degree to inaccuracy and imprecision.

The data in this report demonstrate that the PIXI-mus2 DEXA reliably detects differences in the average atomic number of various solvents and that there is a linear relationship between measured DEXA ratio and mouse body fat percentage. However, although the value for pure fat is accurate, the DEXA ratio for LBM appears to have been miscalibrated. Consequently, the overestimation of body fat content by the PIXImus2 DEXA (averaging 3.3 g in this study) can be attributed to imperfect calibration rather than to any inherent fault in the instrument or technique. In principle, the body fat percentages calculated for the various solvents in Table 1 can easily be employed to check the calibrations of DEXA instruments of all manufacturers.

The slope of the relationship between measured and theoretical DEXA ratios for the various solvents, 0.56, is considerably less than unity. This discrepancy between measured and theoretical values cannot result from using mass attenuation coefficients for 40 rather than 35 keV energy X-rays to calculate theoretical DEXA ratios, as this substitution should lead to a slope greater than unity. A more important influence on measured DEXA ratios is likely to involve dispersion of the X-ray energies. Neither low nor high X-ray energies are monochromatic, and measured DEXA ratios reflect the integrated effects of a range of low and high X-ray energies.

For the practical issue of correcting mouse body fat percentages measured by the PIXImus to values obtained by carcass analysis, two equivalent approaches can be taken. By use of the algorithms derived in this study, mouse body fat percentages can be calculated directly from measured DEXA ratios (Fig. 3), or measured fat percentages can be corrected using the results of Fig. 4. Although DEXA LBM values were close to those obtained by carcass analysis, DEXA values of total tissue mass overestimated body weight, and attention must also be given to this error.

Although carcass analysis is considered the gold standard to validate DEXA body composition measurements, various laboratories employ slightly different methodologies for carcass analysis. Diethyl ether, the solvent often used for fat extraction, extracts triglycerides but not polar lipids from tissue. Triglycerides contribute 83% of total carcass lipid in control rats but 67 to 70% in rats undergoing caloric restriction with and without exercise, respectively (6). Acetone, employed to extract fat in this study, is more polar than diethyl ether and therefore presumably extracts more lipids. Nonetheless, our finding that the PIXImus2 overestimates mouse body fat by ~3.3 g is similar to findings by Nagy and Clair (19), in which diethyl ether was employed for fat extraction.

Confirmation that ~75% of the fat-free mouse carcass is composed of water (29) indicates that simple measurements of body water can be used to determine mouse body composition. As described previously (8, 25), carcass dehydration without fat extraction provides reliable body fat values. If a DEXA is unavailable, noninvasive and longitudinal body fat values can be obtained using deuterated or tritiated water "space" determinations (11, 14, 18).

In summary, the PIXImus2 DEXA provides a reproducible, noninvasive estimate of mouse body composition. Although measured values overestimate body fat and total tissue mass, these parameters can be corrected to accurate carcass analysis values by simple algorithms.


    DISCLOSURES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
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Zheng-Zheng Shi, Timothy Wilkins, Luis Freay, Laurie Minze, Juan Delmas-Mata, and Kate Combs have analyzed more than 16,600 mice by PIXImus2 DEXA as part of the high-throughput gene knockout screening process at Lexicon Genetics and shared their accumulated knowledge of this procedure.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. Brommage, Dept. of Endocrinology, Lexicon Genetics, 8800 Technology Forest Pl., The Woodlands, TX 77381 (E-mail: rbrommage{at}lexgen.com).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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