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Obesity Research Center, St. Luke's-Roosevelt Hospital, Columbia University College of Physicians and Surgeons, New York, NY
2 To whom correspondence should be addressed. Email: ZW28{at}Columbia.edu.
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ABSTRACT |
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 TOP ABSTRACT SUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • nutritional assessment • body composition • bone mineral • whole-body counting
Protein is a body component affected by many physiological and pathological processes, including growth, development, nutritional status, physical activity, and diseases. Alterations in protein can, in turn, serve as a biomarker of relevant physiological and pathological conditions.
Although total body nitrogen (TBN)3 measured by in vivo neutron activation (IVNA) analysis can be applied as the criterion for total body protein (TBPro) estimation, application is limited, especially for measurements in children and adolescents, due to radiation exposure (1). Therefore, the estimation of TBPro in healthy children and adolescents remains impractical or difficult, and there are only a few reports in the literature regarding protein mass in pediatrics (2–4).
We recently reported a model for predicting TBPro (in kg) from total body potassium (TBK, in mmol) and bone mineral (Mo, in kg): TBPro = 0.00252 x TBK + 0.732 x Mo (5). This model is based on observations that both potassium and protein distribute mainly within the intracellular compartment and that intracellular potassium and protein concentrations are relatively stable. In addition to this physiological model, we developed an empirical TBPro prediction method from fat-free mass (FFM, in kg) in healthy adults, TBPro = 0.199 x FFM – 0.45 (6).
Although the TBK-Mo TBPro model was validated in healthy adults and patients with several chronic diseases (5,7), it is not known whether the model is applicable in pediatric subjects. The aims of the present study were to first develop a TBK-independent TBPro model as a practical reference for estimating TBPro mass, and then to evaluate the applicability of the TBK-Mo models in children and adolescents. In addition, we evaluated the applicability of the adult FFM TBPro method in children and adolescents.
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SUBJECTS AND METHODS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Total body nitrogen measured by IVNA is currently the criterion for TBPro estimation (1). However, IVNA is inappropriate for use in children and adolescents because of the associated radiation exposure (0.26 mSv). A 6-component (6-C) approach was thus developed and applied as a practical criterion in the present study. Body weight (BW) can be expressed as the sum of fat, total body water (TBW), TBPro, Mo, soft-tissue mineral (Ms), and glycogen (Gly) (8). Accordingly,
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A 6-C TBPro model was derived from Eq. 1,
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All of the major components in Eq. 2 are now measurable or calculable. Fat mass can be measured by a multicompartment model: fat = 2.748 x BV – 0.699 x TBW + 1.129 x Mo – 2.051 x BW, where BV is body volume (in L) by air displacement plethysmography; TBW (in kg) is measured by 2H2O dilution; Mo (in kg) is measured by dual-energy X-ray absorptiometry (DXA); and BW is in kg (9). In addition, Ms (in kg) can be calculated from total body water (in kg) as Ms = 0.0129 x TBW (9); and Gly (in kg) can be calculated from total body protein (in kg) as Gly = 0.044 x TBPro (10). Equation 2 can thus be converted as,
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or
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A 6-C TBPro model can thus be derived and applied as the criterion in the present study,
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FFM TBPro model
Our previous study indicated that FFM is the strongest TBPro predictor, explaining 
79% of the observed between-individual variation in subjects (6). A simple prediction equation was derived for healthy adults, TBPro (kg) = 0.199 x FFM (kg) – 0.45. This empirical equation was used in the present study to predict TBPro for healthy children and adolescents.
Subjects. Adolescents and children >5 y old completed a medical evaluation including a physical examination and standard blood studies to exclude any medical conditions except overweight. Only subjects recruited for our current grant (NIDDK PO1-42618) were included in the study. Subjects participated in recreational physical activities and were not actively engaged in any sports training programs. All study subjects or their parents signed an informed consent. This investigation was approved by St. Luke's-Roosevelt Hospital's Institutional Review Board.
Body composition measurements. Subjects reported in the morning after an overnight fast and wore a hospital gown with no shoes. Body weight was measured to the nearest 0.1 kg with a Weight Tronix digital scale, and height was measured to the nearest 0.5 cm with a Holtain wall-mounted stadiometer. After completion of the baseline anthropometric measurements, subjects were evaluated with whole-body 40K counting for TBK, 2H2O dilution for TBW, air displacement plethysmography for body volume, and DXA for Mo.
Total body potassium.
There are 3 isotopes of potassium in nature, as well as in the human body, with isotopic abundances of 0.0118% 40K, 93.1% 39K, and 6.9% 41K. A whole-body counter was used to detect the 1.46 MeV 
-ray of the naturally radioactive 40K.
The raw counts in a subject were corrected for counting efficiency using a 40K phantom with a weight equivalent to the subject's weight (11). The corrected 40K activity was converted to TBK. The St. Luke's 4
instrument was installed in 1967, and updated and recalibrated in the 1990s. The reproducibility was improved from ± 4% to ± 2.3% for subjects with body weights as low as 20 kg (12).
Total body water. The deuterium dilution volume was measured in liters after an oral D2O dose of 0.1 gm of D2O/kg body weight. A saliva sample was collected 3 h later, and the D2O concentration was measured on an infrared analyzer (13). The measurement precision of the method is 2.1% (14). The deuterium dilution volume was then converted into TBW mass (in kg) by correcting for water density at 36°C (i.e., TBW = deuterium dilution volume x 0.9937). Total body water measured by deuterium oxide is not different from the nonaquatic phase corrected TBW by tritium (3H2O) dilution in our laboratory (15).
Body volume. Body volume was measured by air displacement plethysmography using the BOD POD system (Life Measurements Instruments) (16,17). Use of the BOD POD in children was described previously (18,19). Subjects did not consume food and water and did not exercise for at least 2 h before testing. Minimal clothing was worn into the chamber, either underwear or a tight-fitting bathing suit, and a swim cap. Subjects were asked to remove their jewelry. Two measurements were taken for each subject and an average was applied in the study. The subject's thoracic gas volume was estimated during normal tidal breathing using a tube connected to the BOD POD breathing circuit system. Thoracic gas volume was determined as the subject puffed gently into the tube while the tube was mechanically obstructed. A detailed description of the procedure was given in an earlier report (17). The BOD POD body density measurement has a CV of 0.8% in our laboratory.
Bone mineral. Bone mineral content (BMC) was estimated with a GE Lunar DXA system using software versions 6.6 and 6.7. BMC measured by DXA represents ashed bone. Each gram of bone mineral (Mo) produces 0.9582 g of ashed bone due to loss of labile components including bound water and CO2 with combustion. BMC was thus converted to Mo as Mo = BMC/0.9582. The CV of Mo as measured by DXA is 1.3% (20,21).
Fat-free mass.
Fat-free mass was calculated as the difference between body weight and fat mass measured by a multicompartment model,
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Statistical analysis
Results are expressed as the group mean and standard deviation (mean ± SD). Student's t tests were used to compare the physical characteristics of boys and girls and to compare TBPro values calculated by the different methods. Linear regression analysis was applied to describe the relations between TBK and TBPro, and between TBPro estimates by different models. The differences in TBPro estimates between models were examined using Bland-Altman analysis (22). Statistical calculations were conducted using Microsoft Excel for Windows and SPSS v12. Differences with P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Correlations between TBPro and TBK.
Within each gender group, there were correlations between TBPro (kg) by the 6-C model and TBK (mmol), TBPro = 0.0030 x TBK + 0.71; r = 0.96, P < 0.001 for the boys; and TBPro = 0.0030 x TBK + 0.25; r = 0.92, P < 0.001 for the girls. The intercepts and slopes of a linear regression model did not differ between boys and girls (gender, P = 0.75; and gender by TBK interaction, P = 0.58). A single prediction equation was then calculated for all subjects pooled:
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with an r = 0.96, P < 0.001, SEE = 0.85 kg; n = 62 (Fig. 1A). In addition, a multiple regression analysis in which TBPro was the dependent variable and TBK was the major independent variable showed that age was not associated with TBPro.
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TBK-Mo model. Total body protein mass calculated by the 6-C model was 7.76 ± 2.94 kg in boys, 5.86 ± 1.47 kg in girls, and 7.02 ± 2.98 kg in the combined sample subjects. There were significant correlations observed for TBPro estimated by the 6-C model and the TBK-Mo model for each gender separately and for all subjects (boys, r = 0.97; girls, r = 0.93; and all subjects pooled, r = 0.94; all P < 0.001; Table 2, Fig. 1B). TBPro estimates did not differ between the 2 models (7.02 ± 2.98 kg by the 6-C model vs. 6.82 ± 2.95 kg by the TBK-Mo model, with mean difference 0.20 ± 0.86 kg; P = 0.074).
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DISCUSSION |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Six-component TBPro Model. Because of its unique role as an essential element of protein, the assessment of total body nitrogen by IVNA has been used classically as the criterion for measurement of total body protein mass. However, IVNA is not advised for healthy children and adolescents, and is prohibited for these subjects in the United States due to radiation exposure.
In recent years, multicomponent models were developed for measuring TBPro mass. Siconolfi and colleagues (24) first estimated TBPro based on a 3-component (3-C) model in which TBPro could be assessed by using estimates of TBW, body volume, and body weight. However, these authors did not provide a complete equation for deriving TBPro and we were thus unable to evaluate their 3-C model in the current study.
The 3-C model concept advanced by Siconolfi et al. (24) was extended in later studies. Fuller et al. (25) developed a 4-component (4-C) TBPro model: TBPro = 3.05 x BW – 0.290 x TBW – 2.734 x BMC – 2.74 x BV. In the present study a 6-C model for estimating TBPro (i.e., Eq. 3) was developed.
The multicomponent model approach provides an alterative method for making estimates of TBPro for body composition laboratories in which IVNA or whole-body 40K counters are unavailable. Unfortunately, we were unable to evaluate all of the existing multicomponent models, including 3-C, 4-C, and 6-C models against the IVNA-TBN method in children and adolescents.
TBK-Mo TBPro model. Both potassium and protein are distributed primarily within the intracellular compartment. The validated TBK-Mo model developed from healthy adults is based on relatively stable intracellular potassium and protein concentrations (5,26). If the intracellular potassium and protein concentrations in children and adolescents are the same as those observed in adults, the TBK-Mo model should also be applicable for use in pediatrics.
Ellis et al. (4) used a set of assumptions to predict TBPro from TBK: 150 mmol/L for the intracellular potassium concentration, 4 mmol/L for the extracellular potassium concentration, 0.16 kg N/kg protein, and 2.17 mmol K/g N for lean tissue. Based on these assumptions, a TBPro prediction equation was derived by Ellis et al. (4) from TBK: TBPro (kg) = {[(1/0.16) x (1/2.17)] x 10–3} x TBK = 0.00288 x TBK.
Our results confirm that TBPro and TBK are correlated in both healthy boys and girls. The slopes and intercepts of the empirical derived equations for TBPro vs. TBK were 0.0030 and 0.71 for boys, and 0.0030 and 0.25 for girls. These observations are similar to Ellis's empirical prediction formula for adult women with a slope of 0.00317 and an intercept of 0.95 (27). This observation supports the applicability of the TBK-Mo model in children and adolescents.
As a theoretically derived model, the TBK-Mo model should work well at different body composition laboratories, and in both adults and children. We recently evaluated the TBK-Mo model in adult subjects studied in New York and Melbourne. The TBPro estimates did not differ in the New York and Melbourne studies between the IVNA criterion and the TBK-Mo model in healthy adults and patients with AIDS, liver disease, chronic renal failure, and growth hormone deficiency (5,7). The present study shows the applicability of the adult TBK-Mo model in healthy children and adolescents. However, we do not yet know whether the TBK-Mo model can be applied reliably in children with acute and chronic diseases. In general, the TBK-Mo model could be applied in patients with disease if the intracellular ratio of potassium to protein remained stable and unchanged from that in healthy subjects. However, this deduction warrants evaluation in future studies.
FFM TBPro model. DXA can measure body fat and fat-free mass, and protein is one of the major components of FFM. Clinical studies designed to measure TBPro will benefit if TBPro can be measured using DXA because DXA are now widely available and relatively less expensive to purchase. Because the fraction of FFM as protein is often assumed to be stable, we derived an empirical equation for predicting TBPro from FFM with DXA measurements (6). Although the mean fractions of FFM as protein do not differ between healthy groups, there was a moderate variation in the range of TBPro/FFM within each group. For example, the ratios of TBPro to FFM were 0.194 ± 0.017 with a CV of 8.8% for healthy men and 0.186 ± 0.025 with a CV of 13.4% for healthy women (7). In the children and adolescents in the present study, the ratio of TBPro to FFM was 0.197 ± 0.024 with a CV of 12.0%. Therefore, FFM alone may not be a good predictor of TBPro mass in adults and children.
Study limitations and future directions.
Although the mean difference in TBPro for the population of boys and girls tested was 0, there was a relatively large discrepancy in estimates by the TBK-Mo and 6-C models for a few individuals (Fig. 2A). However, we did not find any connection between the discrepancy and biological factors such as age, gender, body weight, and adiposity. Therefore, these discrepancies are likely caused by measurement errors.
With the TBK-Mo prediction model, whole-body 40K counting provides a noninvasive approach for estimating TBPro that is applicable in a wide range of subjects including healthy children and adults, and patients with selected chronic diseases. However, the prohibitive construction costs of whole-body counters limit their widespread application, so that there are now only 30 whole-body counters throughout the world.
It is not known whether our TBK-Mo model gives accurate estimates in newborns. Further studies of newborns and children <5 y old are warranted to explore the associations between TBK and TBPro. This future research has the potential of providing a noninvasive TBPro prediction method for newborns. Further studies are also required to evaluate the TBK-Mo model in pediatric patients with chronic diseases.
In conclusion, we previously developed a TBPro prediction model from TBK and Mo in healthy adults (TBPro = 0.00252 x TBK + 0.732 x Mo). The present study provides evidence that the same model can be used to estimate TBPro in healthy pediatric subjects 
5 y old.
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FOOTNOTES |
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3 Abbreviations used: BMC, bone mineral content; BV, bone volume; BW, body weight; 6-C model, 6-component total body protein model; DXA, dual X-ray absorptiometry; FFM, fat-free mass; FFM model, total body protein prediction model by fat-free mass; Gly, glycogen; IVNA, in vivo neutron activation; Mo, bone mineral; Ms, soft tissue mineral; TBK, total body potassium; TBK-Mo model, total body protein model by total body potassium and bone mineral; TBN, total body nitrogen; TBPro, total body protein; TBW, total body water.
Manuscript received 29 July 2005. Initial review completed 29 August 2005. Revision accepted 28 December 2005.
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LITERATURE CITED |
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