Department of Endocrinology, Lexicon Genetics, The Woodlands, Texas 77381
Submitted 30 October 2002 ; accepted in final form 14 May 2003
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ABSTRACT |
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PIXImus; carcass analysis; obesity; dual-energy X-ray absorptiometry
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.
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METHODS |
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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:
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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).
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RESULTS |
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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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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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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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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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Similarly, carcass fat content can be calculated from the measured DEXA
body fat content as
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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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LBM measured by DEXA was higher by 0.5 ± 0.2 g compared with carcass
analysis values, and correct values can be calculated as
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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.
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DISCUSSION |
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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.
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DISCLOSURES |
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FOOTNOTES |
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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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REFERENCES |
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