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The Journal of Nutrition Vol. 128 No. 2 February 1998,
pp. 246-250
University of Georgia, Department of Foods and Nutrition, Athens, GA 30602
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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The use of dual energy X-ray absorptiometry (DXA) is increasing in animal research, but data comparing whole body composition by DXA and chemical analysis (CHEM) in rats are limited. Lean and obese Zucker female rats were used to compare DXA (QDR1000W, Hologic, Waltham, MA) values with CHEM data for percent fat (%FATDXA, %FATCHEM), lean body mass (%PROTDXA, %PROTCHEM) and bone mineral content (%BMCDXA, %ASHCHEM). Four groups of rats (n = 9) were tested for differences in body composition due to consumption of a 100 g guar gum supplement/kg to see if DXA was as sensitive as CHEM in detecting body composition differences induced by diet. The study was analyzed using a split-plot ANOVA where the main plot was a 2 × 2 factorial with phenotype (obese or lean) and treatment (guar gum or control) as the effects, and the subplot was method of detecting body composition (DXA and CHEM), which was treated as a repeated measure. Absolute values for percent fat differed significantly (P < 0.0001) between the two methods as %FATDXA was consistently higher than %FATCHEM. There was not a statistically significant difference due to method for %PROT (P = 0.13). Values for %BMCDXA were significantly (P < 0.0049) lower than %ASHCHEM values. The differences in body composition due to diet treatment were detected similarly by DXA and CHEM. Significant correlations were found between the methods (P < 0.0001) for %FAT (r = 0.99), %PROT (r = 0.96) and %BMC or ASH (r = 0.81). Bland-Altman plots showed good agreement between methods, and regression equations were developed to estimate CHEM values from DXA readings. DXA may provide an alternative method for assessing changes in whole body composition.
KEY WORDS: dual energy X-ray absorptiometry · chemical extraction · body composition · rats
Dual energy X-ray absorptiometry (DXA)3 is a method that is being used increasingly for assessment of body composition in humans and animals. In rat models, validation of DXA has been determined by comparing DXA values to chemical analysis (CHEM), with the primary aim of many of the studies being to assess bone mineral content (BMC; Jebb et al. 1996 DXA's ability to differentiate fat and fat-free soft tissue (lean body mass) and accurately estimate these components based on chemical analysis is still unclear. There is a paucity of data (Jebb et al. 1996 To date there is only one published report comparing fat mass and fat-free soft tissue mass using both DXA and CHEM (Jebb et al. 1996 The purpose of this study was to compare DXA and CHEM for body fat, lean body mass and BMC and to develop regression equations relating the two methodologies. Although absolute values may be different, high correlations between the two methods would make it possible to estimate whole body composition using DXA and provide the basis for prospective studies of body composition. A secondary purpose of this study is to assess DXA's sensitivity compared with CHEM in detecting differences in body composition due to dietary treatment.
Thirty-six female Zucker rats (18 lean and 18 obese) obtained from The University of Georgia breeding colony rat facility were used in the study. All experimental procedures using rats were approved by the University of Georgia Institutional Animal Care and Use Committee (IACUC No. A950107). Rats were housed in the animal isolation facility at 22°C in individual hanging wire-bottom cages (24.13 cm long × 20.32 cm wide × 17.78 cm high) except when rats were removed to be scanned via the DXA system. An 8-wk diet study was designed to assess the impact of a 100 g guar gum diet supplement/kg diet on changes in body composition. Rats were divided into four groups (n = 9 per group) Body composition: dual energy X-ray absorptiometry.
Rats were transported via a transport rack for the hanging cages from their usual housing location to the DXA lab. All rats were anesthetized by intraperitoneal injection of a combination of 0.01 g xylazine/L (Rompun; Mobay, Shawnee, KS) and 0.95 g ketamine hydrochloride/L (Ketaset; Aveco, Fort Dodge, IA) at a dosage of 0.1 mL/100 g of live body weight for lean rats and at 0.12-0.15 mL/100 g of live body weight rate for obese rats. Calibration of the DXA (QDR 1000W, Hologic, Waltham, MA) included a step bar with acrylic plastic (Plexiglas) and aluminum sections simulating soft tissue and bone, respectively, and the rat platform used in all scans. The rat platform is the same height as the lowest step of the calibration bar, putting the rat body in the range of the calibration of the machine. The recommended body weights for rats to be scanned range from 200 to 750 g (Hologic QDR 1000W Rat Whole Body V5.71P Software Manual). Rats then were placed in a prone position on the platform and scanned with DXA using Hologic ultra high resolution rat whole body composition software (3.28-cm-diam columinator, 186 line average/scan, 7.874 cm line spacing, 0.02996 point resolution). Each scan took 13-15 min, allowing rats' vital signs to be monitored by sight until they awoke. Scans were analyzed with ultra high resolution analysis software, and values for percent fat DXA (%FATDXA), percent lean body mass DXA (%LBMDXA) and percent BMC DXA (%BMCDXA) and body weight (BW) were recorded into a spreadsheet. After DXA measurements, all rats were killed by CO2 overdose, degutted and frozen at
Chemical analysis.
Chemical body composition analysis was performed on frozen, degutted carcasses. The method used was a modified Hartzook-Hershberger method (Hartzook and Hershberger 1963) developed by Harris et al. (1986) Statistical analysis.
For purposes of statistical analysis, %FATDXA was compared directly with %FATCHEM analysis; however, grams of lean body mass (LBM, the reading for fat-free soft tissue given by DXA) were converted to %LBM by dividing the LBM by total body weight (determined by DXA), and then, because lean tissue is ~70% water (Pond et al. 1995 Mean DXA and CHEM values and standard deviations for each treatment group for %FAT, %PROT and %BMC or ASH are summarized in Table 3. DXA and CHEM detected similarly the higher level of %FAT and lower %PROT in obese rats compared with lean rats (P < 0.0001). Absolute values for %FAT differed significantly (P < 0.0001) between the two methods, as %FATDXA was consistently higher, by ~30% than %FATCHEM. There was not a statistically significant difference due to method for %PROT (P = 0.13). Values for %BMCDXA were significantly (P <0.0049) lower than %ASHCHEM values.
The main goal of this study was to determine if DXA can be used to assess body composition in rats. This study confirmed DXA's overestimation of fat and thus questions the validity of DXA to estimate absolute values for body composition in rats. However, DXA was similar to CHEM in detecting gross differences in fat and protein between lean and obese rats. Also the high correlations (r = 0.99, 0.96 and 0.81, respectively, for %FAT, %PROT and %BMC) found between DXA and CHEM for fat, lean body mass and bone indicate that body composition can be estimated from DXA in rats weighing 200-600 g. A second goal of this study was to assess differences in body composition by DXA and CHEM as the result of guar gum diet treatment. DXA and CHEM detected differences in body composition similarly, supporting the use of DXA to estimate differences due to diet interventions.
The authors thank Andra Nelson and Ben Mullinix for advice and assistance with statistical analyses and Ronalynn Faircloth, Thomas Bass and Tonya Dalton for assistance with data summarization, manuscript review and preparation for publication.
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
). Significant correlations have been demonstrated in rats between percent ash from chemical analysis (%ASHCHEM) and percent BMC (%BMCDXA) by DXA (Casez et al. 1994, Lu et al. 1994).
) for comparing the estimates of fat and lean tissue in rats using DXA to chemical extraction values. Traditionally, extra rats have been added to a study to gain a baseline estimate of body composition by CHEM. The disadvantage is that an assumption is made that the rats killed for CHEM at the onset are representative of the intervention group used in the study. Because DXA does not require killing the rat, longitudinal assessment of body composition is possible.
). The major findings of that study were that DXA overestimated fat mass by 33% and that ASH readings by CHEM were not significantly different from BMCDXA readings. The authors concluded that the software employed did not accurately measure gross body composition compared with the "gold standard" of CHEM techniques.
METHODS AND MATERIALS
Abstract
Introduction
Methods
Results
Discussion
References
obese guar gum (OG), obese control (OC), lean guar gum (LG), and lean control (LC)and were fed for 8 wk. The study compared supplementation of 100 g/kg guar gum to a modified AIN93 purified diet (OG, LG) (Grossman et al. 1994) and AIN93 control diet (OC, LC). Diet components for the AIN93 diet (Reeves et al. 1993) were purchased from USB Biologics (Amersham), Cleveland, OH, except for vitamin and mineral mixes, which were purchased from ICN Biomedicals (Aurora, OH), and guar gum, which was purchased from Dyets (Bethlehem, PA). Food intake and body weight were monitored daily. Macroscopic visual inspection of 5-wk-old rats was used initially to determine lean and obese pairs, and baseline readings of DXA confirmed the lean/obese designation. Rats used in the study were analyzed via DXA and chemical analysis at the end of the 8-wk feeding trial.
20°C.
) for assessing body composition in rats. Four repeated measures were conducted on one rat in 1 d, and the rat was repositioned after each scan (Table 1). Coefficients of variation (CV) for body weight and %FATDXA were 0.04 and 11, respectively. The second reliability test was conducted on five different rats (4 leans and 1 obese), each scanned on three consecutive days (Table 2). The mean CV for repeated measures were 5.5-18.2, 0.9-6.9 and 1.2-11.5, respectively, for %FATDXA, %PROTDXA and %BMCDXA. These tests provided data needed to determine the numbers of observations needed to assess the reliability of DXA in measuring body composition of rats.
View this table:
Table 1.
Repeated whole body scans on one lean Zucker rat using dual energy x-ray absorptiometry (DXA)1,2
View this table:
Table 2.
Whole body scans repeated over 3 days on Zucker rats using dual energy x-ray absorptiometry (DXA)1,2
. In this method, autoclaved carcasses are blended individually, and samples of 6 mL of the rat homogenate are collected (in triplicate) for percent dry matter and percent ash determinations and 7-mL samples are collected (in triplicate) for chloroform-methanol extraction of fat. Values for percent fat CHEM (%FATCHEM), percent protein CHEM (%PROTCHEM) and percent ash CHEM (%ASHCHEM) were calculated by sample weight difference from triplicate samples using SAS (Statistical Analysis System, Cary, NC).
), %LBM was converted to an approximate %PROTDXA by multiplying %LBM values by 0.3. BMC was converted to %BMCDXA by dividing grams of BMC by carcass weight as determined by DXA.
) were used to assess relationships between the two methods and to define equations for conversion from DXA to CHEM using GB stat 5.0.6 software (Silver Spring, MD) designed for Macintosh computers. Graphs using the Bland-Altman method (Bland and Altman 1986) of evaluating degree of sameness were generated using Microsoft Excel 5.0 software (Microsoft, Redmond, WA) for Macintosh.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
View this table:
Table 3.
Body composition of female lean (L) and obese (O) Zucker rats after consumption of 100 g/kg supplemented guar gum diet (G) or control (C) diet for 8 wk as determined by dual x-ray absorptiometry (DXA) or chemical analysis (CHEM)1,2,3
View this table:
9.07 ± 1.2% (P < 0.0001) and for CHEM was 9.04 ± 0.9% (P < 0.0001). The differences in %FAT in OG and OC were 1.33 ± 1.8% for DXA and 0.69 ± 0.6% for CHEM with neither method showing a significant difference attributable to guar gum consumption.
0.44 ± 0.9% for DXA and 0.02 ± 0.7% for CHEM, and neither method detected a significant difference due to diet. In the lean rats, DXA showed a significant difference due to diet with a difference of 2.68 ± 0.6% (P < 0.0001). However, the 1.24 ± 0.9% difference detected by CHEM was not significant (P = 0.07).
0.02 ± 1.8% for CHEM (P = 0.97). The difference between LG and LC was 0.19 ± 0.1% when assessed by DXA (P < 0.004) and 0.85 ± 0.8% when assessed by CHEM (P < 0.05).
View this table:
Table 4.
Regression equations and correlation values for %FAT, %PROT and %BMC or ASH measured by chemical extraction (CHEM) and dual energy X-ray absorptiometry (DXA) for lean and obese Zucker rats using Hologic's QDR 1000 W1
are illustrated in Figure 1. These Bland-Altman plots show good agreement between DXA and CHEM, and correlation coefficients confirm this finding.
View larger version (20K):
[in a new window]
Fig 1.
The difference between dual energy X-ray absorptiometry (DXA) and chemical analysis (CHEM) plotted against the mean for %FAT, %PROTEIN and %BMC or ASH by the method of Bland and Altman (1986) . Hatched lines represent the mean ± 2 SD. %FAT (A): y = 2.29 + 0.20x, r 2 = 0.78, P < 0.05; %PROTEIN (B): y = 6.65 + 0.36x, r 2 = 0.62, P < 0.05; and %BMC or ASH (C): y = 3.03 1/3x, r 2 = 0.93, P < 0.05.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
). The overestimation of %FAT by DXA observed in the current study is consistent with that report. Jebb et al. (1996) suggested that tissue calibration of DXA with stearic acid and water is "an inappropriate substitute" for attenuation of soft tissue in vivo and may be responsible for variability in fat mass. The theory of beam hardening (i.e., lower energy photons are preferentially removed from a radiation beam compared with higher energy photons, leading to a progressive shift in spectral distribution to higher effective energies with increasing body thickness) was given for varying, nonlinear bone mineral densities (BMD) in measurements of the spine, hip and forearm of humans (Blake et al. 1992). However, this theory was based on the much greater thickness differences found in humans. Differences of rat body thickness <50 mm do not appear to contribute to the fat overestimates (Jebb et al. 1996). In the present study, rats were in the same weight range as used by Jebb et al. (1996) and differences in body thickness were all <50 mm. Therefore, body thickness was probably not a major factor contributing to overestimation by DXA.
). They found that DXA overestimated fat mass by 35.6% in larger pigs and by 234% in smaller pigs. DXA underestimated lean mass by 1.3% in larger pigs and 5.9% in smaller pigs. They found significant correlations between DXA and CHEM for %FAT (r = 0.83, P < 0.01) and %LBM (r = 0.96, P < 0.01). In another study with pigs, DXA and CHEM analysis were compared to determine the reliability for estimation of infant body composition (Picuad et al. 1996). They found that DXA overestimated fat mass (though they did not give specific ranges) with strong correlations (r = 0.97, P-value not given) to CHEM. Values for %PROT or %LBM were not reported. Svendsen et al. (1993) found no significant differences between DXA and CHEM for fat mass in seven pigs (35-95 kg). This study differs from the first two discussed because it used a Lunar DPX total body scanner with adult whole body software.
) using the Hologic QDR1000W showed high correlations between total bone mineral content using DXA vs. CHEM (r = 0.99, P < 0.0001). The differences in absolute values decreased with increasing body weight. Previous studies (Casez et al. 1994, Lu et al. 1994) used isolated bone cleared of all soft tissue and analyzed chemically, whereas the present study estimated bone composition from whole carcass analysis. The lower correlations may be due, in part, to the use of the whole carcass instead of isolated bone for chemical analysis.
and was completed in conjunction with the current study. Feeding of guar gum significantly lowered body fat in lean rats as determined by CHEM. DXA detected these changes in body fat comparably with CHEM. The LG rats gained less body fat than the LC rats, and both DXA and CHEM detected this difference (percent difference between treatments 9.04 by CHEM and 9.07 by DXA). Dietary treatment with guar gum did not result in differences in %FAT in obese rats, and this lack of difference was detected similarly by CHEM and DXA (percent difference between treatment of 0.69 by CHEM and 1.33 by DXA). The practical use of DXA to the researcher may be more for assessing changes due to intervention than for estimating absolute values; however, prospective studies are needed.
ACKNOWLEDGMENTS
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FOOTNOTES |
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Manuscript received 3 February 1997. Initial reviews completed 3 April 1997. Revision accepted 16 October 1997.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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