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ORIGINAL RESEARCH COMMUNICATION |
1 From the Obesity Research Center, St Luke's-Roosevelt Hospital, Columbia University College of Physicians and Surgeons, New York (ZMW, WS, DPK, SH, RNP, and SBH); the Department of Applied Science, Brookhaven National Laboratory, Upton, NY (LW); the Department of Academic Affairs, Winthrop University Hospital, Mineola, NY (JFA); the Jean Mayer US Department of Agriculture, Human Nutrition Research Center on Aging at Tufts University, Boston (MEN)
2 Supported by Knoll WRISC award Prop-797-03 (MER-1296-89) and National Institutes of Health grant NIDDK 42618. 3 Reprints not available. Address correspondence to ZM Wang, Weight Control Unit, 1090 Amsterdam Avenue, 14th Floor, New York, NY 10025. E-mail: zw28{at}columbia.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Objective: The objective was to derive a theoretical cellular level TBPro mass and distribution model formulated on measured total body potassium, total body water, and bone mineral and to evaluate the new model with the IVNA method as the criterion.
Design: The new model was developed on the basis of a combination of theoretical equations and empirically derived coefficients. TBPro mass estimates with the new model were evaluated in healthy women (n = 183) and men (n = 24) and in men with AIDS (n = 84). Total body nitrogen was measured by IVNA, total body potassium by whole-body 40K counting, total body water by tritium dilution, and bone mineral by dual-energy X-ray absorptiometry.
Results: The group mean (± SD) TBPro mass estimates in healthy
women and men and men with AIDS (8.2 ± 0.9, 11.0 ± 1.8, and
10.5 ± 1.1 kg, respectively) with the new model were similar to
IVNA criterion estimates (8.9 ± 0.9, 11.1 ± 1.6, and 10.9 ± 1.2
kg, respectively). TBPro mass estimates with the new model
correlated highly with the IVNA estimates in all subjects combined (r = 0.92, P < 0.001). The new model suggests that the
composite TBPro mass within each group consists mainly of
cellular protein (75-79%) and, to a lesser extent, protein in extracellular solids (19-23%) and extracellular fluid (
2%).
Conclusion: The new model provides a non-IVNA approach for estimating protein mass and distribution in vivo.
Key Words: Body composition • nutritional assessment • total body nitrogen • total body potassium • total body water • whole-body counting
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The chemical formula for protein is assumed to be
C100H159N26O32S0.7 with a ratio of nitrogen to protein of 0.16
(4). Assuming that all body nitrogen is incorporated into protein, a total body protein (TBPro) model that can be measured
by IVNA was derived from total body nitrogen (TBN) (5, 6):
 | (1) |
0.26 mSv). Accordingly, the number of evaluated healthy subjects is relatively
small (500). The importance of protein in nutritional and physiologic research has led to alternative non-IVNA measurement methods, including model and empirical approaches. The model approach, based on measurements of TBN and total body potassium (TBK), provides estimates of TBPro and protein distribution (8, 9). Although an innovative advance at the time, the models reported by Burkinshaw et al (8) and Cohn et al (9) were later shown to have many theoretical limitations and, in some cases, were inaccurate (10, 11). James et al (12) subsequently reported another TBN-TBK model for estimating cellular and collagen proteins. Fuller et al (13) recently suggested TBPro models based on a 4-compartment approach or dual-energy X-ray absorptiometry (DXA) estimations. However, none of these earlier models were evaluated with IVNA as the criterion.
Empirical TBPro prediction formulas were developed by
using anthropometry or total body water (TBW) as the main
predictors (2, 14). Ellis et al (2) reported a high correlation
between TBPro and TBK. Using IVNA to measure TBN as the
criterion, these authors derived empirical TBPro prediction
equations based on TBK measured with the whole-body 40K
counting method. We modified the form and units (TBPro in
kg and TBK in mmol) of the equations to be consistent with
those in the present study.
 | (2) |
 | (3) |
Equations 2 and 3 show that TBK is a good predictor of TBPro, although there are no available theoretical models that provide a basis for this empirical association.
In the present study we derived a theoretical model linking protein mass and distribution with the cellular body-composition level. Available human data are then used to fit the model with empirical coefficients that require 3 estimates: TBK, TBW, and bone mineral. A simplified model with similar accuracy was also derived on the basis of measured TBK and bone mineral. We then compared TBPro estimates derived by the new model with corresponding TBPro by IVNA as the criterion.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 | (4) |
Accordingly, a 3-compartment TBPro model is derived on
the cellular level:
 | (5) |
Protein in body cell mass
BCM has been defined as a "component of body composition containing the oxygen-exchanging, potassium-rich, glucose-oxidizing, work-performing tissue" (16). BCM consists of
4 chemical components: protein, intracellular water (ICW),
intracellular fluid (ICF) minerals, and polysaccharides. BCM
protein can thus be expressed as
 | (6) |
As reported previously, BCM can be calculated as ICW/0.70, where 0.70 is the mean hydration of BCM (17). Our
recent study showed that ICF minerals are a function of ICW
and ICF minerals = 0.01617 x ICW, where 0.01617 is ICF
mineral concentration (in kg/kg H2O) (18). In addition, polysaccharides are 2% of BCM (19). Equation 6 can thus be
converted to
 | (7) |
Almost all body potassium exists in ICF and ECF and the
intracellular and extracellular potassium concentrations are relatively constant at 152 and 4 mmol/kg H2O, respectively (20).
The ICW can thus be calculated from TBK and TBW (17, 21)
via the following simultaneous equations:
 | (8) |
 | (9) |
where TBK and TBW are in mmol and kg, respectively.
Resolving these simultaneous equations, ICW and ECW can be
calculated if TBK and TBW are known:
 | (10) |
 | (11) |
By combining Equations 7 and 10, BCM protein can be expressed as a function of TBK and TBW:
 | (12) |
Protein in extracellular fluid
The ECF is a nonmetabolizing fluid that surrounds cells and
provides a medium for gas exchange, transfer of nutrients, and
excretion of metabolic end products. ECF consists of ECW, a
small amount of protein, and ECF minerals. ECF protein can be
calculated as follows:
 | (13) |
As previously reported, ECF can be expressed as ECW/0.98,
where 0.98 is the mean hydration of ECF (17). Our recent study
indicated that ECF minerals are a function of ECW and ECF
minerals = 0.009543 x ECW, where 0.009543 is ECF mineral
concentration (in kg/kg H2O) (18). Equation 13 can thus be
converted to
 | (14) |
By combining Equations 11 and 14, ECF protein can be
calculated as follows:
 | (15) |
Protein in extracellular solids
The ECS compartment consists of 2 parts: organic and
inorganic. Organic ECS include 3 types of protein (collagen,
reticular, and elastic), whereas the inorganic ECS of bone
mineral includes calcium hydroxyapatite as the major constituent. ECS are distributed in several tissues and organs, including cortical and trabecular bone, cartilage, periarticular tissue,
tendons, and fascia. In the reference man, the ECS protein is
2.08 kg (ie, 1.0 kg in cortical bone, 0.24 kg in trabecular bone,
0.18 kg in cartilage, 0.14 kg in periarticular tissue, and 0.52 kg
in tendons and fascia), and the ECS bone mineral content is
2.84 kg (ie, 2.2 kg in cortical bone, 0.50 kg in trabecular bone,
0.045 kg in cartilage, 0.037 kg in periarticular tissue, and 0.057
kg in tendons and fascia) (1). Assuming that the ratio of ECS
protein to bone mineral (ie, 2.08/2.84 = 0.732) is relatively
stable across subjects, the ECS protein compartment can be
predicted as follows:
 | (16) |
Total body protein mass
By inserting Equations 12, 15, and 16 into Equation 5,
TBPro mass can be calculated as follows:
 | (17) |
 | (18) |
Experimental approach
The subjects completed prompt-
IVNA, whole-body 40K
counting, tritium-labeled water dilution, and DXA studies. The
completed evaluations were then used to estimate TBPro mass
and distribution according to the IVNA model and new models.
Subjects
The subject pool consisted of healthy adults and men with
AIDS. The rationale for inclusion of patients with AIDS and
body weight loss was 2-fold: it provided the opportunity to
examine the new TBPro model in clinical patients, and body
weight loss might show limitations of the new TBPro model
that are not evident in healthy subjects with a normal body
weight.
The ethnically mixed healthy subjects were recruited in the New York (St Luke's-Roosevelt Hospital and Winthrop University Hospital) and Boston (Jean Mayer US Department of Agriculture Human Nutrition Research Center on Aging) areas. Each subject in the healthy group completed a medical history, a physical examination, and routine screening blood studies to exclude the presence of underlying diseases. The men with AIDS were recruited from among patients with nutritional disorders and weight loss cared for by physicians at the St Luke's-Roosevelt Hospital in New York City. The patients were ambulatory and afebrile and met the Centers for Disease Control criteria for AIDS. The study participants signed an informed consent form that was approved by the Institutional Review Boards of St Luke's-Roosevelt Hospital, Winthrop University Hospital, and the Jean Mayer US Department of Agriculture Human Nutrition Research Center on Aging. Some subject data came from our earlier unrelated body-composition studies (22, 23).
Body-composition measurements
Consenting subjects were studied after an overnight fast.
Body weight was measured to the nearest 0.1 kg (Weight
Tronix, New York) and height to the nearest 0.5 cm with the
use of a stadiometer (Holtain, Crosswell, United Kingdom).
Total body nitrogen was determined by prompt- IVNA at
Brookhaven National Laboratory with a precision (CV) of
2.7% (24). TBK was estimated at Brookhaven National Laboratory with the use of a whole-body 40K counter with a technical error of 2.4% (25). TBK was calculated as 40K/0.000118.
The tritium-labeled water dilution volume, in liters, was measured at Brookhaven National Laboratory with a precision of 1.5%. The tritium dilution volume was then converted into TBW mass by correcting nonaqueous hydrogen exchange and water density at 36 °C (TBW = 3H2O dilution volume x 0.96 x 0.994) (26). Total-body bone mineral was quantified at the 3 evaluation centers by whole-body DXA (Lunar DPX, Madison, WI). The estimated precision is 1.28% for bone mineral (27).
Statistical methods
Results are expressed as group means ± SDs unless otherwise noted. Simple linear regression analysis was applied to
describe the relation between TBPro by IVNA and TBPro
predicted by the new model. Mean differences between TBPro
by IVNA and TBPro predicted by the new model among the 3
subject groups were evaluated for statistical significance by
analysis of variance. P 
0.05 was considered statistically
significant. In Tables 1
and 3, a Bonferroni adjustment to the
significance level was made. The differences in TBPro between IVNA and the new model were related to the mean of the
2 methods as described by Bland and Altman (28) to evaluate
bias. Statistical calculations were carried out by using Microsoft EXCEL (Redmond, WA) and SPSS version 10 for
WINDOWS (SPSS Inc, Chicago).
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The healthy women ranged in age from 23 to 81 y,
in body mass from 48.0 to 82.0 kg, and in body mass index (in
kg/m2) from 18.4 to 29.8. The healthy men ranged in age from
25 to 78 y, body mass from 57.6 to 89.1 kg, and body mass
index from 20.2 to 28.4. The men with AIDS ranged in age
from 22 to 62 y, in body mass from 46.6 to 77.2 kg, and in body
mass index from 16.7 to 25.9.
There were significant differences between healthy women and
healthy men in all measures (P < 0.01-0.001), except age and
body mass index (Table 1). The men with AIDS weighed on
average 5.8 ± 7.4 kg (P = 0.009) less than the healthy men. There
were no significant differences between the 2 groups of men in
TBN, TBK, TBW, or bone mineral (Table 1; all P > 0.05).
Correlations between TBPro and TBK
There were good correlations between TBPro (kg) by IVNA
and TBK (mmol) in all 3 groups.
 | (19) |
 | (20) |
 | (21) |
The slopes and intercepts of these equations did not differ
significantly by analysis of variance, and a single prediction
equation was calculated for all of the subjects (n = 291).
 | (22) |
).
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).
New model
There were moderate-to-high correlations between TBPro by
IVNA and the new model in all 3 groups (r = 0.80 for healthy
women, 0.90 for healthy men, and 0.85 for men with AIDS; all
P < 0.001). TBPro by IVNA was also highly correlated with
estimates of TBPro by the new model when all of the subjects
were combined in one group (r = 0.92) (Table 2). Compared with
IVNA, the new model on average underestimated TBPro by 7.6%
for the healthy women, 1.0% for the healthy men, and 3.5% for
the men with AIDS (all P < 0.001). Bland-Altman analysis
indicated that the differences between TBPro by IVNA and the
new model were not significantly correlated with the mean TBPro
estimates by the 2 models for the healthy women (r = -0.06),
healthy men (r = -0.30), and men with AIDS (r = 0.15, all P >
0.05). However, a small significant bias between the methods was
observed for all subjects pooled (Table 2).
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 | (23) |
).
The use of this regression equation significantly
reduced the SEE to 0.59 kg from 0.86 kg when values directly
from the new model are used. Nevertheless, this regression
equation is empirically derived without a theoretical foundation
and should not be confused with Equation 18.
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2%, and 19-23% of TBPro(new model) in the
3 groups, respectively (Table 3).
Compared with the healthy
women, the healthy men had a higher fraction of TBPro as cellular
protein and a lower fraction as ECS protein (both P < 0.001).
Compared with the men with AIDS, the healthy men also had a
higher fraction of TBPro as cellular protein (P < 0.001) and a
lower fraction as ECS protein (P < 0.003). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mean measurement error associated with the IVNA
model can be estimated for the healthy subjects by assuming an
average TBN of the healthy men and women as shown in Table
1 and measurement precision as stated in Subjects and Methods. Accordingly,
 | (24) |
The propagated TBPro measurement error for the healthy subjects was 0.27 kg. Because IVNA is not practical for longitudinal studies and for use in children and young women, the IVNA model can be used as the reference in the present study to evaluate the new model for predicting TBPro.
TBK-TBPro association
Both potassium and protein are mainly distributed within the
intracellular compartment, and TBK was thus applied by Ellis et
al (2) as a predictor of TBPro. Our experimental results confirmed
that there were good correlations between TBPro and TBK for all
3 groups. The slopes and intercepts of the derived empirical
equations for TBPro compared with TBK for healthy women
(0.00250 and 2.79), healthy men (0.00240 and 2.63), and men
with AIDS (0.00250 and 2.56) were similar to the slope and
intercept of Ellis's equation for healthy men (0.00248 and 2.54).
Moreover, assuming that the average bone mineral value for
healthy women and men is 2.74 kg (Table 1), our derived model
(ie, Equation 18) takes the form TBPro = 0.00252 x TBK +
2.00. The slope and intercept of this formula calculated from the
new model are also similar to the results of Ellis et al in men
(0.00248 and 2.54). These observations thus cross-validate Ellis et
al's empirical prediction formula for men (Equation 2). However,
Ellis et al's prediction equation for TBPro from TBK in women
has a larger slope (0.00317) and a smaller intercept (0.95) than
those observed in the current study. We have no explanation for
this discrepancy.
New TBPro model
Because there is a lack of a strong theoretical basis for the
empirical TBK prediction formulas, we focused in the present
study on the development of a new TBPro model derived as the
combination of 3 separate protein compartments. Our results
support the new TBPro model's validity relative to the criterion
IVNA model in both the healthy adults and the men with
AIDS, although, overall, the model provided slightly lower
TBPro estimates.
The new model has 2 sources of measurement error, one
from estimates of TBK and the other from bone mineral. The
total measurement error can be evaluated in our healthy subjects by assuming an average body composition of men and
women, as shown in Table 1, and by assuming measurement
precisions as described in Methods. The total measurement
error is equal to the combination of error in TBK and bone
mineral measurement:
 | (25) |
The propagated measurement error of the new model is 0.20 kg, which is smaller than that of the IVNA-criterion model (0.27 kg). This calculation also shows that the measurement of TBK is the major source of measurement error.
The new TBPro model has a firm physiologic basis, and our results indicate that the new model can also be applied in men with AIDS. However, it is questionable whether the new model would be accurate when applied in some patient populations with significant intracellular and extracellular electrolyte disturbances (ie, in patients without constant potassium concentrations of 152 mmol/kg H2O in ICF and of 4 mmol/kg H2O in ECF). Further studies are thus needed to validate the new model in patients with various acute and chronic disease states. In addition, the reason for the systematic underestimate of mean TBPro in comparison with IVNA requires further investigation.
An important characteristic of the new model is that it provides estimates of protein distribution among BCM, ECF, and ECS compartments. On the cellular level, both BCM and ECF contain potassium and protein. Potassium can thus be used as a predictor of BCM and ECF proteins. The new model also considers the third protein compartment, ECS protein. Both bone mineral and protein are the major constituents of ECS; thus, bone mineral measured by DXA can be used as a predictor of ECS protein. At present, however, the accuracy of the protein distribution estimates cannot be evaluated, and there are no published independent estimates of protein in cellular, ECF, and ECS components. Further studies are thus needed to validate our estimates of protein distribution.
Conclusion
In the present study we developed and then validated a new
approach for estimating TBPro mass in vivo. The model,
formulated on a series of theoretical equations combined with
physiologically and empirically based coefficients, provides
TBPro estimates similar to those of the criterion IVNA method.
Our new model, which also gives an estimate of protein distribution, may also be applicable in patients with AIDS. Further
validation studies are needed in longitudinally monitored populations and in patients with various acute and chronic diseases.
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ACKNOWLEDGMENTS |
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ZMW and SBH designed the study; ZMW and SH analyzed the data; ZMW, SH, WS, and SBH wrote the manuscript; and DPK, LW, JFA, MEN, SBH, and RNP collected the data. None of the authors had any financial or personal interest in any company or organization sponsoring the research, including advisory board affiliations.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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