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Journal of the American College of Nutrition, Vol. 23, No. 1, 79-84 (2004)
Published by the American College of Nutrition

Original Research

Validation of Bone Mass and Body Composition Measurements in Small Subjects with Pencil Beam Dual Energy X-Ray Absorptiometry

Winston W. K. Koo, MBBS, FRACP, FACN, Mouhanad Hammami, MD and Elaine M. Hockman, PhD

Departments of Pediatrics, Obstetrics and Gynecology (W. W. K. K., M. H.)
Computing and Information Technology (E. M. H.), Wayne State University, Detroit, Michigan

Address correspondence to: Dr. Winston Koo, Hutzel Hospital, Department of Pediatrics, 4707 St. Antoine Blvd, Detroit MI 48201. e-mail: wkoo{at}wayne.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS AND SUBJECTS
 RESULTS
 CONCLUSION
 REFERENCES
 
Objective: To validate the most widely reported dual energy X-ray absorptiometry (DXA) technique for the measurement of bone mass and body composition in human infants with a piglet model.

Methods: Duplicate scans were obtained in 13 piglets (1950g to 21100g) using a whole body densitometer (Hologic QDR 2000 plus, Hologic Inc., Waltham, MA) operated in the pencil-beam mode on a two platform (aluminum platform overlying a foam table pad) system. DXA measurements that included total weight, bone mineral content, fat and lean mass were compared with carcass weight and chemical analysis for ash and calcium content, fat and lean mass.

Results: Measurements from duplicate DXA scans were nearly perfectly correlated (r = 0.98 to 1.00). DXA measurements were strongly predictive of scale weight and chemical composition for all piglets (adjusted r2 = 0.93 to 1.00, intraclass reliability coefficients = 0.943 to 0.999, p < 0.001 for all comparisons) although DXA bone mineral content consistently underestimated carcass ash and calcium content. Measured values from heavier piglets were not significantly different from values predicted from the lighter piglets’ data. Slopes from regression based on lighter versus heavier piglets were not significantly different except for the bone mineral content with carcass ash or calcium content.

Conclusion: Our study validated the use of pencil beam DXA and its ability to determine relative changes in bone mass and body composition measurements over a much greater range of body weight than previous reports although its use as a direct indicator of nutrient requirement may be limited.

Key words: pig, ash, calcium, fat, lean tissue, bone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS AND SUBJECTS
 RESULTS
 CONCLUSION
 REFERENCES
 
Dual energy X-ray absorptiometry (DXA) technique for the measurement of bone mass and body composition with piglets have been validated independently by multiple investigators [1–3]. DXA is now generally accepted as the standard for measuring bone mass and the preferred means to measure soft tissue composition in small subjects [4] with an extensive body of literature on DXA measurements in human infants during the first 2 years [4–17]. However, it is well known that differences in hardware (X ray voltage, generation of higher and lower energy spectra using switching kilovolt or K-edge filters, different detectors) and in software (algorithm for edge detection, assumptions regarding distribution of soft tissue above bone), techniques for scan acquisition and scan analysis, and imaging geometry (pencil beam DXA using a pinhole collimator coupled to a single detector versus fan beam DXA using a slit collimator coupled to a multidetector array) can result in deviations in DXA measurements. In addition, small human and animals have body composition significantly different from adults and require modifications in scan acquisition technique such as the use of an aluminum platform to improve system linearity, and in scan analysis with specific algorithm [1–4,18–20]. Thus validation studies for DXA measurement must be specific for DXA hardware, software and techniques of scan acquisition and analysis.

The most widely used DXA technique for the studies during infancy is based on the pencil beam instrument that switches voltage to generate high and low energy spectra [1–19]. Validation studies for this pencil beam technique were based on software algorithm generated from small piglets (<8 kg body weight) using the two platform system (an aluminum platform overlying a foam table pad) with an external calibration standard then followed by carcass chemical analysis [1–3]. Cross validation of the software with magnetic resonance imaging and three-dimensional chemical shift imaging [3] were also performed. However, there are no data to validate its use in subjects with greater body weights. This study aims to repeat the validation studies using a piglet model over a greater range of body weights using the currently recommended techniques for scan acquisition and analysis. In addition, we aim to determine whether heavier piglets (>8 kg) require a separate analytic algorithm for DXA measurement compared to lighter piglets studied in the original reports [1–3].


   METHODS AND SUBJECTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND SUBJECTS
RESULTS
 CONCLUSION
 REFERENCES
 
DXA scans
All measurements were performed using a whole body densitometer (Hologic QDR 2000 plus, Hologic Inc., Bedford, MA) operated in the pencil beam mode. A two platform system (aluminum platform overlying a foam table pad) and a manufacturer supplied external calibration standard (placed beside the piglet parallel with the long axis of the animal at a level below the head of the animal) were used for all scan acquisition. Each piglet was placed on a cotton blanket in the prone position with front and hind limbs extended. The long axis of the animal was positioned at the midline of the platform with the snout approximately 5 cm from the cranial end of the platform. Each piglet was covered with a cotton blanket and a disposable diaper was used in larger piglets to prevent soiling. The piglets were sedated with sodium pentobarbital and sodium thiopental for the scan acquisition. Duplicate scans were obtained with commercial software (v5.71p) validated with previous studies [1–3] and each scan was reviewed by the same operator (MH) to assure that no movement artifact was evident [18] prior to scan analysis.

Animal Study
Thirteen domestic swine piglets (1950g to 21100g) commercially obtained (J&M Farms, Lansing, MI) were studied. There were 8 piglets with weights <8 kg (4508 +/- 1625g, mean +/- SD) and 5 piglets with weights >8 kg (14564 +/- 4211g). Each animal was weighed using an electronic scale (Seca, Toledo scale company, Toledo OH) immediately prior to DXA scanning and euthanized immediately upon completion of the in vivo study procedures. The study protocol was approved by the Animal Investigation Committee at Wayne State University.

Ashing
Immediately after euthanasia, piglets were stored frozen at -20°C until processing for tissue analysis. Details of animal processing including drying and homogenization of whole carcass have been described elsewhere [1,21]. The dried homogenate from each piglet was placed in individual polypropylene bag, vigorously shaken and then stored at -70°C. To obtain the ash weight, three aliquots were taken from separate depths of each bag containing the homogenate and ashed in a computer-controlled temperature regulated muffle furnace (Isotemp muffle furnace, 650 series, Fisher Scientific, Pittsburgh, PA) at 500°C. The average coefficient of variation (CV) of triplicate samples from each piglet for ash measurement was 2.7%.

Chemical Analysis
All chemical analyses were performed on three aliquots of dried homogenate from each piglet obtained as described above. The drying procedure also was repeated prior to chemical analysis as reported elsewhere [1,21].

Nitrogen was measured by the micro-Kjeldahl method [22]. In our laboratory, the recovery of nitrogen using the National Institute of Standards and Technology (NIST, US Department of Commerce, Gaithersburg, MD) certified standard reference material was 101.7 +/- 2.06 %. The average CV of nitrogen measured from triplicate samples from each piglet was 2.4%. Total body content of crude protein was determined as total carcass nitrogen x 6.25, and the lean mass was calculated as the crude protein content plus total body water. The latter was taken as the difference between wet and dry weight.

Fat was measured as total lipid extracted with various proportions of chloroform, methanol and water [23]. In our laboratory, the recovery of lipid was 98.5 +/- 1.46% from commercial vegetable oil. The average CV of total lipid measured from triplicate samples for each piglet was 2.1%.

Each aliquot of ash was dissolved in nitric acid prior to the measurement of calcium by atomic absorption spectrophotometry [24]. In our laboratory, the CV for calcium measurements was <1%. The measured calcium content of the NIST certified bone ash was 38.98 +/- 0.71% by weight compared to the expected values of 38.18 +/- 0.13%. The average CV of calcium measured from triplicate samples for each piglet was 1.1%.

Statistical Analyses
Equivalence of the duplicate DXA measurements was determined by correlation and paired t test. The applicability of the current DXA algorithm for the measurement of bone mineral content, fat mass, lean mass and total weight of all piglets studied was determined using regression analysis. The average values of these measurements from duplicate DXA scans served as the independent variables whilst the carcass ash and calcium content, fat mass, lean tissue mass, and scale weights of the piglets with and without covering served as the dependent variables. The level of agreement between specific DXA measurements and the corresponding validation measurements was determined by intraclass reliability. The precision error was determined by the method of Gluer et al [25].

To test the applicability of the lighter piglet equation, the measured values from heavier piglets were compared to the values predicted from the lighter piglet data, i.e., the regression equations derived from only the lighter (<8 kg) piglets were applied to the measurements from the heavier (>8 kg) piglets. These comparisons were computed for carcass ash and calcium content, fat and lean mass, and scale weight of the piglet.

Whether a separate equation for the DXA measurement of heavier piglets might result in a better prediction for direct carcass measurements was determined by comparing the slopes of the regression equation for DXA prediction of carcass analysis in lighter piglets versus those obtained from heavier piglets, using the F ratio [26].

Unless otherwise indicated, all values were expressed as mean +/- SD. All statistical tests were performed using SPSS 11.5 for Windows (SPSS Inc, Chicago, IL), and a p value of <0.05 was used to judge significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND SUBJECTS
 RESULTS
CONCLUSION
 REFERENCES
 
Measurements from duplicate DXA scans were nearly perfectly correlated and not significantly different with respect to means (Table 1). DXA measurements were highly predictive of scale weight with and without covering and chemical composition for all piglets studied (Table 2). Intraclass reliability coefficient between DXA measured total weight with scale weight was 0.999 regardless whether the weight of the covering was taken into account, between DXA measured BMC with its surrogate from chemical analysis (carcass ash and calcium) were 0.9432 and 0.9482 respectively, and between DXA lean and fat mass with carcass derived lean and fat mass were 0.9996 and 0.9973 respectively. The relationship of bone mineral density with carcass ash showed adjusted r2 of 0.903 and 0.920 respectively. The precision error for duplicate measurements of piglets with average weight of 8459g (range 640–21100g) were 0.2, 2.0, 1.2, 1.2, 9.1 and 0.8% for total weight, bone mineral content, bone area, bone mineral density, fat and lean mass respectively.


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  		Table 1. Duplicate Pencil Beam Dual Energy X-Ray Absorptiometry Measurements in 13 Piglets

 

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  		Table 2. Dual Energy X-Ray Absorptiometry (DXA) Prediction for Scale Weight and Carcass Chemical Analysis Based on All Piglets

 
The DXA equation generated from lighter piglets can be used to predict the bone mass and body composition of heavier piglets (Table 3). DXA bone mineral density predicted carcass ash or calcium content were 300 +/-68g and 139 +/- 33g respectively, and were significantly lower (p = 0.002 and 0.01 respectively) than the corresponding measured values of 459 +/- 100g and 196 +/- 56g respectively. The slopes of the regression equations generated from the lighter versus those from heavier piglets were not significantly different except for the relationship between bone mineral content and carcass ash or calcium content (Table 4).


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  		Table 3. Application of Regression Equation from Lighter (<8 kg) to the Heavier (>8 kg) Piglets

 

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  		Table 4. Comparison of the Slope of Regression Equations between Lighter (<8 kg) and Heavier (>8 kg) Piglets

 

   DISCUSSION
 
Pencil beam DXA is the most widely used DXA technique in the measurement of bone mass and body composition in infants. It allows the non-invasive assessment of both bone and soft tissue components and the potential for assessing the nutrient requirement of the developing human as well as the interaction among nutritional, physiological, and biochemical functions in health and disease states. However, multiple technical issues are shown to affect DXA measurement in small subjects. For example, scan acquisition without the use of aluminum platform [27], scan acquisition and analysis with prototype software algorithm prior to v5.64p [5,11,15,19,27], or the presence of movement artifacts [18] can result in spurious DXA measurements in small subjects. Inconsistencies in the techniques used in clinical studies have contributed to the discrepancies in the literature and confounded the ability to interpret the validity of clinical or research studies. For example, the use of prototype versions of the DXA software [5,11,15], scan acquisition without the aluminum platform [5,11], no documentation whether the aluminum platform was used [13,14] or quality of the scan [13,14], in particular the presence of movement artifact, and the use of a combination of prototype and validated DXA software within and among different subjects [16,17].

To date, almost all DXA studies in human infants are based on the use of pencil beam DXA instruments that switched voltage to provide alternate high and low energy spectra and the software validated and cross validated with small piglets <8 kg in weight [1–3]. It is not known whether the same scan acquisition technique and the current software is applicable for the study of larger subjects although many investigators have extended the DXA studies in human infants to include older infants with much greater body mass in cross sectional [7,8] or longitudinal [14–17] design. In order to avoid another potential pitfall in generating spurious results, it is imperative to perform validation studies in subjects with a much greater range of body weights, bone mass and body composition measurements.

Our data show excellent predictive value of pencil beam DXA measurements for carcass chemical analysis and we have documented the feasibility of using the current software and scanning technique for small subjects over a much greater range of body weight, bone mass and body composition than previous reports [1–3]. Similar to earlier reports [1–3], our data also showed pencil beam DXA measurements underestimated the carcass ash and calcium content. It is not surprising that bone mineral density has poorer predictive value for carcass ash and calcium since the current and previous [1–3] validation studies are based on absolute quantities from carcass analysis rather than density values. In any case, our findings provide assurance that appropriately collected DXA measurements in clinical studies using the same type of instrument and software with standardized scan acquisition and analysis techniques, can be used for comparative nutritional, biochemical and physiologic studies on bone mass and body composition throughout infancy.

Optimal measurement of bone mass ideally would require the use of separate equations for lighter and heavier infants since there were differences in the slopes of DXA bone mineral content prediction for carcass ash and calcium content. However, further improvement in DXA measurements with the use of different equation for heavier subjects is likely to be minimal in view of the persistent underestimation of carcass ash and calcium content regardless of the piglet weight. There are other practical difficulties in the development of separate equations for the study of larger infants, for example, the determination of the exact age or weight of the infant for transition from one DXA equation to another, and that the piglets’ growth in bone mass and DXA measured bone mineral content and bone mineral density are not uniformly proportional across different regions of the carcass [28,29].

The recent validation and cross validation of fan beam DXA technique for the measurement of bone mass and body composition in small subjects [21,29] offer the potential for more accurate and faster DXA studies compared to pencil beam DXA. However, the use of fan beam DXA is associated with significantly higher cost and could necessitate the continued use of pencil beam DXA in some centers at least for the foreseeable future. Furthermore, the need to maintain the same DXA technique [30] whether it is pencil- or fan- beam DXA for longitudinal studies ensures the data would be useful to the understanding of current and future publications. Additional cross validation studies utilizing a specific format [30] would be necessary during transition from pencil- to fan-beam DXA technique.


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND SUBJECTS
 RESULTS
 CONCLUSION
REFERENCES
 
The current pencil beam DXA technique can be used for subjects with a large range of body weight, bone mass and body composition with excellent reproducibility when the data are generated according to specific techniques described in this report. Our data support the validity of appropriately generated clinical DXA data throughout infancy including those from older and heavier infants and ongoing longitudinal studies. However, the role of pencil beam DXA appears primarily to be the determination of relative changes in bone mass and body composition measurements during infancy. The persistent underestimation of bone mass and the possibility of improved DXA measurement with the use of different equations for heavier subjects would limit its ability to determine nutrient requirements in infants.

Received June 20, 2003. Accepted October 15, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND SUBJECTS
 RESULTS
 CONCLUSION
 REFERENCES
 

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