Hydration of fat-free body mass:

new physiological modeling approach

ZIMIAN WANG,1 PAUL DEURENBERG,2 WEI WANG,1 ANGELO PIETROBELLI,1
RICHARD N. BAUMGARTNER,3 AND STEVEN B. HEYMSFIELD1
1Obesity Research Center, St. Luke’s-Roosevelt Hospital, Columbia University College of Physicians

and Surgeons, New York, New York 10025; 2Department of Human Nutrition and Epidemiology,
Wageningen Agricultural University, 6700 EV Wageningen, The Netherlands; and 3Clinical Nutrition
Research Center, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131

Wang, ZiMian, Paul Deurenberg, Wei Wang, Angelo
Pietrobelli, Richard N. Baumgartner, and Steven B.
Heymsfield. Hydration of fat-free body mass: new physiologi-
cal modeling approach. Am. J. Physiol. 276 (Endocrinol.
Metab. 39): E995–E1003, 1999.—Water is an essential com-
ponent of living organisms, and in adult mammals the
fraction of fat-free body mass (FFM) as water is remarkably
stable at ⬃0.73. The stability of FFM hydration is a corner-
stone of the widely used water isotope dilution method of
estimating total body fat. At present, the only suggested
means of studying FFM hydration is by experimental total
body water (TBW) and FFM measurements. Although devia-
tions from the classical hydration constant are recognized, it
is unknown if these are explainable physiological aberrations
and/or methodological errors. Moreover, many questions re-
lated to hydration stability prevail, including body mass and
age effects. These unresolved questions and the importance of
the TBW-fat estimation method led us to develop a cellular
level FFM hydration model. This physiological model reveals
that four water-related ratios combine to produce the ob-
served TBW-to-FFM ratio. The mean and range of FFM
hydration observed in adult humans can be understood with
the proposed physiological model as can variation in the
TBW-to-FFM ratio over the human life span. An extension of
the model to the tissue-organ body composition level confirms
on a theoretical basis a small but systematic decrease in
hydration observed in mammals ranging in body mass by a
factor of 105. The present study, the first to advance a
physiological hydration model, provides a conceptual frame-
work for the TBW-fat estimation method and identifies
important areas that remain to be studied.
total body water; body composition
THE SHREW AND THE WHALE, both mammals, share in
common a similar hydration of fat-free body mass
(FFM). Defined as the ratio of total body water (TBW)
to FFM (TBW/FFM) and measured by in vitro chemical
analysis, the mean ⫾ SD hydration is 0.739 ⫾ 0.015 for
nine mammals, including mouse, rat, hamster, Rhesus
monkey, baboon, goat, sheep, gray seal, and human (25,
27, 29, 31, 36). The importance of TBW/FFM is that
estimation of TBW by dilution methods allows deriva-
tion of total body fat from the following equations: body
mass ⫺ TBW/0.73 or body mass ⫺ 1.37 ⫻ TBW (30).
Studies of body composition (16), energy balance (12),
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and thermoregulation (29) in mammals often rely on
fat estimates by the TBW method. No other body
composition method applied in vivo is capable of provid-
ing fat estimates in such a wide range of mammals,
which differ in body mass by a factor of 105. Moreover,
the assumed stable TBW/FFM serves as the basis of
dual-energy X-ray absorptiometry and hydrodensitom-
etry body composition models (26) and TBW-derived
FFM prediction models used to calculate other body
composition components, such as skeletal muscle
(0.50 ⫻ FFM), body cell mass (0.57 ⫻ FFM), total body
protein (0.18 ⫻ FFM), and resting energy expenditure.
Although FFM hydration is generally assumed con-
stant in mammals, including humans, some deviations
from a TBW/FFM of ⬃0.73 are recognized. Newborn
humans and other mammals share in common a high
FFM hydration (⬃0.81) (15). Similarly, FFM hydration
may be increased in elderly humans (13, 33) and in
obese humans (34) and monkeys (20). Moreover, Pitts
and Bullard (27) reported a small but statistically
significant decline in TBW/FFM observed across mam-
mals of increasing body mass when TBW and FFM
were directly quantified in autopsied animals caught in
the wild. Animals with outer exoskeletons, such as the
armadillo, had particularly low FFM hydration levels
(0.70–0.71, Ref. 27).
The question thus arises: is FFM hydration of ⬃0.73
a biological constant in adult mammals, reflecting
underlying physiological regulation? Alternatively, is
the value of 0.739 ⫾ 0.015 observed across mammals a
coalescence of several independent factors that gener-
ally result in a TBW/FFM of ⬃0.73 with only minimal
variability? The importance of the TBW-fat estimation
method in the field of body composition research led us
to explore these questions.
At present, the only suggested means of studying
FFM hydration is by experimental TBW and FFM
measurements. Both in vitro and in vivo experimental
approaches in general have two primary limitations
(36). First, a large population sample is necessary to
explore the full range of FFM hydration for each
mammalian species. Second, even small errors in mea-
suring TBW and FFM may have a significant effect on
the magnitude of calculated TBW/FFM, which only
varies by several percent under normal physiological
conditions.
The long-term aim of our research is to establish the
molecular and physiological determinants of FFM hy-
dration magnitude and variability. A new strategy of
investigating FFM hydration, which differs from the
E995

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E996 FAT-FREE BODY MASS HYDRATION
earlier experimental approach, was applied in the
present report. Our approach was to develop a FFM
hydration model at the cellular body composition level.
The nature of living organisms first becomes manifest
at this level, and we reasoned that FFM hydration
could be effectively modeled at this level. The model
was then used to examine individually the cellular level
determinants of FFM hydration in an attempt to
establish if any regulatory processes underlie the widely
observed TBW/FFM of ⬃0.73. Organs and tissues have
cellular and molecular level components as their basis,
and a useful approach when making interspecies hydra-
tion comparisons is to analyze TBW/FFM with a tissue-
organ level model. We introduce this concept in a final
section of the paper when we explore the stability of
hydration across a wide range of mammals.
HYDRATION MODEL
The developed hydration model is based on the
five-level body composition model that indicates that
the ⬃40 major components in humans and mammals
can be organized into atomic, molecular, cellular, tissue-
organ, and whole body levels (37). At the cellular body
composition level, body mass consists of cells, extracel-
lular fluid (ECF), and extracellular solids (ECS). The
cellular component can be further divided into fat and
body cell mass (BCM), defined as a ‘‘component of body
composition containing the oxygen-exchanging, potas-
sium-rich, glucose-oxidizing, work-performing tissue’’
(22)
body mass ⫽ cells ⫹ ECF ⫹ ECS
⫽ fat ⫹ BCM ⫹ ECF ⫹ ECS
FFM can thus be expressed as the sum of three cellular
level components
FFM ⫽ BCM ⫹ ECF ⫹ ECS
Similarly, TBW can be expressed as the sum of intracel-
lular water (ICW) and extracellular water (ECW)
TBW ⫽ ICW ⫹ ECW
Based on Eqs. 2 and 3, FFM hydration can be expressed
as
ICW ⫹ ECW
TBW/FFM ⫽
BCM ⫹ ECF ⫹ ECS
This is the primary FFM hydration model on the
cellular body composition level. In the next stage of
model development, our aim was to resolve Eq. 4 into
relevant compartment ratios.
Body cell mass and extracellular fluid consist of
aqueous and solid compartments (Fig. 1), and both
components can be expressed as hydration ratios,
BCM ⫽ ICW/a and ECF ⫽ ECW/b, where a and b are
the fractions of body cell mass and extracellular fluid as
water, respectively. In addition, extracellular solids can
be expressed as a function of TBW as follows: ECS ⫽
c ⫻ TBW ⫽ c ⫻ (ICW ⫹ ECW), where c is the ratio of
ECS to TBW (ECS/TBW). Equation 4 can thus be
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Fig. 1. Cellular body composition level model of fat-free body mass
(FFM), which contains three components: body cell mass (BCM),
extracellular fluid (ECF), and extracellular solids (ECS). ICW, intra-
cellular water compartment of BCM; ECW, extracellular water
compartment of ECF.
converted into
ICW ⫹ ECW
TBW/FFM ⫽ (5)
ICW/a ⫹ ECW/b ⫹ c ⫻ (ICW ⫹ ECW)
Intracellular water and extracellular water are interre-
lated compartments of body water, and extracellular
water can be expressed as a function of intracellular
water as follows: ECW ⫽ (E/I) ⫻ ICW, where E/I is the
ratio of extracellular water to intracellular water. Equa-
tion 5 can be converted and simplified to a secondary
cellular level FFM hydration model as
ICW ⫹ ICW ⫻ (E/I)
TBW/FFM ⫽
ICW/a ⫹ ICW ⫻ (E/I)/b
(1) ⫹ c ⫻ [ICW ⫹ ICW ⫻ (E/I)]
(6)
1 ⫹ E/I
⫽
1/a ⫹ 1/b ⫻ (E/I) ⫹ c ⫹ c ⫻ (E/I)
Equation 6 reveals that FFM hydration is determined
(2) by four factors: hydration of body cell mass (a), hydra-
tion of extracellular fluid (b), ratio of extracellular
solids to TBW (c), and ratio of extracellular water to
intracellular water (E/I). Equation 6 indicates that
(3) hydration of FFM can be analyzed from aqueous and
solid compartments, and this approach allows us to
explore the four individual hydration determinants, a,
b, c, and E/I, in healthy humans.
MODEL COEFFICIENTS
(4)
Physiological aspects and the magnitude and varia-
tion range for each of the four hydration determinants
are presented in this section. Sources of body composi-
tion information based on ‘‘Reference Man’’ data (32)
are presented in Table 1.
Cellular hydration: ratio a. Prebiotic conditions in
the primordial ocean are hypothesized to have allowed
development of eobionts, protoplasmic masses with the
capacity to divide but that lacked cell walls (1). These
primordial cells purportedly had an aqueous proto-
plasm identical in electrolyte and mineral composition
to that of the early oceans (1, 21). The present opera-
tional concept is that formation of prokaryotic cells
along with appropriate membrane pumps led to stable
 FAT-FREE BODY MASS HYDRATION E997

Table 1. Reference data used to develop cellular hydration must be larger than that in red blood
model coefficients cells and smaller than that in skeletal muscle cells. The
present study thus assumed a ⫾1% variation range
Reference Calculated Reference about the assumed mean whole body cellular hydration
Data Data (Page)
of 0.70 (i.e., 0.693–0.707).
Body weight, kg 70 32 (280) Extracellular fluid hydration: ratio b. Extracellular
Total body fat, kg 13.5 32 (280) fluid is a nonmetabolizing component that surrounds
FFM, kg 56.5
Total body potassium, g 140 32 (312) cells and provides a medium for gas exchange, transfer
Body cell mass, kg 29.8 16 of nutrients, and excretion of metabolic end products.
Total body water, kg 42 32 (280) Extracellular fluid is distributed into two main compart-
Extracellular water, kg 18 32 (280) ments, with about one-sixth as plasma in the intravas-
Intracellular water, kg 24 32 (280)
E/I 0.75
cular space and the remaining five-sixths as interstitial
Plasma, kg 3.1 32 (280) fluid in the extravascular space (Table 1). Extracellular
Plasma-to-extracellular fluid ratio 0.17 fluid consists of water, protein, and minerals, with
Dry, fat-free skeleton, kg 4.4 32 (64) water accounting for ⬃94% of plasma and ⬃99% of
Ash in skeleton, kg 2.9 32 (64) interstitial fluid (14, 21). In the present investigation,
Ash-to-dry, fat-free skeleton ratio 0.659
Total body calcium, kg 1.0 32 (296) extracellular fluid hydration was assumed to be equal
Bone mineral-to-calcium ratio 4.81 2, 8 to ⬃0.98 (i.e., a proportional mix of plasma and intersti-
ECS-to-bone mineral ratio 1.18 8 tial fluid), with a range of ⬃0.97–0.99 that reflects
ECS-to-total body calcium ratio 5.68 extreme plasma and interstitial fluid proportions.
ECS, kg 5.68
ECS-to-TBW ratio 0.135
The highly hydrated extracellular fluid component,
FFM hydration 0.743 which accounts for ⬃32% of FFM (Table 1), has a major
effect on observed hydration levels. It is also evident
Fat-free body mass (FFM) ⫽ body weight ⫺ total body fat; body cell
mass (in kg) ⫽ total body potassium (in g) ⫻ (1,000/39.1) ⫻ 0.00833.
that any change in the proportional relationship be-
E/I ⫽ extracellular water/intracellular water; ECS, extracellular tween extracellular fluid with water fraction ⬃0.98 and
solids; TBW, total body water. ECS/total body calcium ⫽ (ECS/bone body cell mass with water fraction ⬃0.70 will result in
mineral) ⫻ (bone mineral/calcium); FFM hydration ⫽ TBW/FFM. changes in FFM hydration.
Ratios a and b represent body cell mass hydration
intracellular hydration, electrolyte content, and osmo- and extracellular fluid hydration, respectively. The
lality even as the surrounding ocean changed composi- water contents of body cell mass and extracellular fluid
tion with an influx of sodium leached from igneous appear to be maintained remarkably stable within
rocks and an efflux of potassium through silicate forma- individuals, between subjects, and even between mam-
tion (6, 21). Accession of land by animals required preser- mals. What are the regulatory mechanisms that control
vation of the ‘‘milieu interieur’’ with the ‘‘private ocean’’ and maintain extracellular fluid and intracellular hy-
extracellular fluid maintained by renal mechanisms. dration and thus preserve the stable milieu interieur?
Although an agreed-on physical model for cellular Most mammals maintain an extracellular fluid osmo-
water is still lacking (1, 6, 19), empirical observations lality of ⬃300 mosmol/kgH2O. The main determinant of
reveal striking similarities in the hydration and electro- extracellular fluid osmolality is sodium and associated
lyte content of prokaryotic and eukaryotic cells that anions. There are two parallel regulatory systems,
relate to their common evolutionary heritage. By weight, antidiuretic hormone and renin-angiotensin-aldoste-
cells from Escherichia coli to mammals consist of ⬃70% rone, which maintain extracellular fluid osmolality,
water even though there is a 100-fold difference in the sodium concentration, and extracellular water content
volumes of bacterial and mammalian cells. extremely stable in health (Fig. 2). An inference from
The ‘‘typical’’ mammalian cell contains 70% water,
18% protein, 5% phospholipids, 1% inorganic ions (e.g.,
K⫹, Na⫹, Mg2⫹, Cl⫺ ), 1.35% RNA and DNA, 2% polysac-
charides, and 3% miscellaneous small metabolites (1).
In the present investigation, cellular hydration was
thus assumed to be a mean of 0.70, representing a
typical mammalian cell.
Body cell mass includes water, protein, and minerals
in all cell types, and water is the largest chemical
compartment of body cell mass (22, 36). Because differ-
ent cell types have specific functions, it is not surprising
that they may differ with respect to their precise
chemical composition, including water fraction. For
example, the intracellular water fraction of red blood
cells is relatively low and varies between 0.65 and 0.68.
In contrast, skeletal muscle cells, accounting for about Fig. 2. Model depicting regulation of mammalian total body water
two-thirds of body cell mass, have water fractions of content, sodium concentration, and fluid osmolality. ADH, antidi-
0.718–0.728 in healthy dogs (10, 18). Whole body uretic hormone; ICF, intracellular fluid.

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E998 FAT-FREE BODY MASS HYDRATION
these well-established relationships is that extracellu-
lar fluid hydration [i.e., extracellular water-to-extracel-
lular fluid ratio (ECW/ECF)] is stable within individu-
als, species, and perhaps across all mammals.
The intracellular fluid compartment is separated
from extracellular fluid by a selectively permeable
membrane (Fig. 2). Sodium content of intracellular
fluid is maintained low by limiting membrane effects
and the sodium-potassium-ATPase pump (28). Water
flows freely across cell membranes, and hence intracel-
lular fluid also maintains a stable osmolality of ⬃300
mosmol/kgH2O (11). Cellular hydration and cell volume
are thus maintained stable through the same regula-
tory mechanisms as for extracellular fluid. Mainte-
nance of stable cell volume is the highest organismic
priority, and even small volume changes constitute a
potent signal for modifying cell metabolism and gene
expression (17, 19, 35).
The implication of these well-established homeo-
static regulatory mechanisms is that extracellular fluid
hydration and cellular hydration are maintained ex-
tremely stable in healthy mammals, including humans
(11, 24). Accordingly, it is likely that factors a and b
vary minimally in healthy adult subjects.
ECS/TBW: ratio c. Extracellular solids are also a
nonmetabolizing component that consists of organic
and inorganic compounds. The organic extracellular
solids include three types of fiber: collagen, reticular,
and elastic. Inorganic extracellular solids, with calcium
hydroxyapatite 5[Ca3(PO4 )2]3Ca(OH)26 as the major con-
stituent, represent ⬃66% of dry bone matrix (Table 1).
Because there is no water in the extracellular solids
component, larger proportional contributions to FFM
correspondingly lower observed levels of FFM hydra-
tion.
ECS/TBW can be estimated from previous studies.
Cohn et al. (7) measured total body calcium by in vivo
neutron activation analysis. Assuming constant bone
mineral-to-total body calcium and extracellular solids-
to-bone mineral ratios, the authors calculated extracel-
lular solids from total body calcium (8). ECS/TBW was
similar between young and old adult men (0.15 vs. 0.15)
and women (0.16 vs. 0.14; Ref. 9). ECS and TBW in
Reference Man are 5.68 and 42 kg, respectively, with
ECS/TBW ⫽ 0.135 (Table 1). Ratio c, ECS/TBW, was
assumed in the present study to range between 0.12
and 0.16 with a mean of ⬃0.14 for a healthy adult.
Although extracellular hydration and body cell mass
hydration are physiologically regulated, as noted ear-
lier, there is no direct regulatory linkage between water
and extracellular solids. Moreover, extracellular solids
and closely related bone are minimally developed in the
newborn at a time when the fraction of FFM as water is
high. This suggests that ratio c, considered over the
whole life span, may be age dependent in humans, and
this possibility needs to be explored. Some mammals,
such as the armadillo, have chitenous shells that are
included in the extracellular solids component, and
ratio c would correspondingly be larger in magnitude
relative to other mammals.
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E/I. Unlike the three other model components, many
physiological factors are known to change relative
extracellular and intracellular water distribution over
the life span, such as growth, gender, exercise, fluid
intake, and sweating (16). Children have a larger
fraction of small young cells and a larger extracellular
fluid-to-cell mass ratio than do adults. The large ratio of
extracellular fluid to cell mass in children permits
rapid movement of nutrients from extracellular fluid to
cells and of end products from cells to extracellular
fluid (16). Obesity, acquired immunodeficiency syn-
drome, chronic renal failure, edema with malnutrition,
and sepsis may also cause overhydration and an in-
crease in E/I. Conversely, diseases or conditions associ-
ated with dehydration may decrease E/I. Hence, there
exists no direct physiological regulation of relative
water distribution, and E/I varies widely in health and
disease.
There is no method for directly measuring total body
intracellular water, and it is often calculated as the
difference between TBW and extracellular water. A
number of dilution techniques (e.g., bromide, sulfate,
and inulin; Refs. 16, 30) and total body chlorine mea-
sured by in vivo neutron activation analysis are applied
for extracellular water estimation (38). However, the
available methods, based on different assumptions,
may vary in their estimates of extracellular water. The
observed E/I thus varies according to the applied
method.
We evaluated E/I in the present study with TBW and
total body potassium (see APPENDIX ) in 384 healthy
adults (220 men and 164 women). These group charac-
teristics (mean ⫾ SD) were age, 45 ⫾ 20 yr; body mass,
64.1 ⫾ 11.9 kg; and body mass index, 22.5 ⫾ 2.7 kg/m2.
TBW measured by tritium dilution was 38.1 ⫾ 9.0 kg,
total body potassium measured by whole body 40K
counting was 3,132 ⫾ 903 mmol, and the calculated E/I
was 0.97 ⫾ 0.20. Although the mean E/I is close to 1.0
for the whole group, a significantly larger E/I was
present in women (1.07 ⫾ 0.22) than in men (0.82 ⫾
0.16, P ⬍ 0.001). In the present study, the E/I was thus
assumed to range between 0.58 (mean ⫺ 1.96 ⫻ SD)
and 1.36 (mean ⫹ 1.96 ⫻ SD) with a mean of 0.97 for
healthy adults.
MODEL FEATURES
Although investigators have expressed interest in
FFM hydration for over 50 years, fundamental ques-
tions remain unanswered. Why do healthy young adult
humans demonstrate a relatively stable mean magni-
tude of FFM hydration of ⬃0.73? Why does hydration in
adult humans vary within a narrow range? Does this
observed range primarily represent biological varia-
tion? Does body size in mammals influence FFM hydra-
tion? In this section, we demonstrate how the proposed
model can be used to explore these fundamental ques-
tions.
Why is FFM hydration in adult humans relatively
stable at ⬃0.73? The proposed cellular level model
indicates that FFM hydration is a function of four
determinants, i.e., TBW/FFM ⫽ f (a, b, c, E/I), and the
 FAT-FREE BODY MASS HYDRATION
approximate mean value of each determinant is known
as described above (i.e., a ⫽ ⬃0.70, b ⫽ ⬃0.98, c ⫽
⬃0.14, and E/I ⫽ ⬃0.97). The mean TBW/FFM can
therefore be predicted for healthy young adult humans
according to Eq. 6
TBW/FFM
1 ⫹ 0.97
⫽ row range for adults (31, 36). However, it is unknown if
1/0.70 ⫹ (1/0.98) ⫻ 0.97 ⫹ 0.14 ⫹ 0.14 ⫻ 0.97
⫽ 0.73
The model-predicted mean FFM hydration is similar to
that in in vitro studies on human cadavers (0.737) and
in Reference Man as suggested by Brozek et al. (0.737)
and Snyder et al. (0.741; Refs. 4, 32). In addition, the
calculated TBW/FFM is almost identical to that in
other mammals (0.739 ⫾ 0.015, coefficient of variation ⫽
2.0%) ranging in average body mass from 0.036 kg for
mice to 214 kg for gray seals, indicating hydration
stability between species (31, 36).
Can the relative constancy of FFM hydration be
explained with the proposed cellular level model? Even
though small changes (e.g., ⫾1%; Table 2) in cellular
hydration (a) and extracellular fluid hydration (b)
would have relatively large effects on FFM hydration
(i.e., ⬃0.5%), these two determinants are maintained
stable by physiological mechanisms in humans and
other mammals. The ratio of extracellular solids to
TBW (c) is also stable in adult humans, although a
change in ratio c of ⫾1% would cause a corresponding
FFM hydration change of ⫾0.1% (Table 2).
Water distribution (i.e., E/I) is highly variable within
subjects over time and between subjects. The cellular
level model (Eq. 6) can thus be simplified, assuming
constant ratios a, b, and c, for discussion purposes to a
model that applies in young adults
1 ⫹ E/I
TBW/FFM ⫽ (7)
1.569 ⫹ 1.16 ⫻ (E/I)
Equation 7 indicates that both numerator and denomi-
nator contain E/I terms that have the same operational
symbol (⫹) and similar coefficients (1 and 1.16). This
mathematical feature indicates that relative changes
in water distribution have only a small effect on
TBW/FFM. For example, when E/I increases by 50%
(e.g., from 0.80 to 1.20), TBW/FFM, according to Eq. 7,
Table 2. The influence of 1% increase of the 4
determinants on FFM hydration
1% ⌬ ⌬(TBW/FFM)/
Determinant Mean Increase (TBW/FFM) 0.7312
a 0.70 0.7070 0.0039 0.53%
b 0.98 0.9898 0.0027 0.37%
c 0.14 0.1414 ⫺0.0007 ⫺0.10%
E/I 0.97 0.9797 0.0006 0.076%
When hydration of body cell mass (a) ⫽ 0.70, hydration of extracel-
lular fluid (b) ⫽ 0.98, ratio of ECS to TBW (c) ⫽ 0.14, and ratio of
extracellular water to intracellular water (E/I) ⫽ 0.97, the calculated
the TBW-to-FFM ratio (TBW/FFM) ⫽ 0.7312.
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E999
increases by only 3% (from 0.721 to 0.743). Hence,
although E/I is highly variable between subjects or in
the same subject over long time periods, the impact of
this variability on the observed FFM hydration is
relatively small.
Why does FFM hydration in adult humans vary
within a narrow range? Both in vitro and in vivo studies
demonstrate that FFM hydration varies within a nar-
these variations are explainable physiological devia-
tions and/or methodological errors.
As indicated above, each of the four cellular level
determinants may vary within an assumed range for
young adults: a, from 0.69 to 0.71; b, from 0.97 to 0.99;
c, from 0.12 to 0.16; and E/I from 0.58 to 1.36. Ratios a,
b, and E/I are in direct proportion, and c is in inverse
proportion, to TBW/FFM magnitude. One can thus
estimate the range of FFM hydration if the four deter-
minants take their extreme values. When a is 0.69, b is
0.97, c is 0.16, and E/I is 0.58, TBW/FFM may reach its
low value according to Eq. 6
TBW/FFM
1 ⫹ 0.58
⫽
1/0.69 ⫹ (1/0.97) ⫻ 0.58 ⫹ 0.16 ⫹ 0.16 ⫻ 0.58
⫽ 0.69
When a is 0.71, b is 0.99, c is 0.12, and E/I is 1.36,
TBW/FFM may reach its high value
TBW/FFM
1 ⫹ 1.36
⫽
1/0.72 ⫹ (1/0.99) ⫻ 1.36 ⫹ 0.12 ⫹ 0.12 ⫻ 1.36
⫽ 0.77
The predicted variation range of FFM hydration for
healthy young adult humans is thus approximately
from 0.69 to 0.77. This range is similar to the results of
in vitro human cadaver studies (0.68–0.81; Refs. 31,
36). The proposed model thus indicates that the ob-
served variation in FFM hydration can be attributed
primarily to variation in the four cellular level determi-
nants.
An interesting observation reported by Pitts and
Bullard (27) was that mammals with a hard chitenous
shell, such as the armadillo, had particularly low
hydration levels (0.70–0.71). These exoskeleton-clad
animals would be expected to have a disproportionately
large extracellular solids component and a high ratio c.
A high ratio c, according to the proposed cellular level
model, would cause a low hydration of FFM. This
prediction is supported in the Pitts and Bullard study.
Of the four cellular level model ratios, E/I is the only
factor that changes substantially in adult humans. The
influence of E/I on FFM hydration is illustrated in Fig.
3. Figure 3, middle, shows the hypothetical normal
state for water distribution and hydration: E/I and
TBW/FFM are ⬃1.0 and ⬃0.73, respectively. If E/I
increases for any physiological or pathological reason,
E1000 FAT-FREE BODY MASS HYDRATION
Fig. 3. Effect of ECW-to-ICW ratio (E/I) change
on FFM hydration [total body water-to-FFM ratio
(TBW/FFM)]. Middle, normal E/I of ⬃1.0 with
TBW/FFM of ⬃0.73. Increase in E/I (e.g., ECW
increase and ICW decrease, E/I ⬎ 1.2) may cause
a TBW/FFM of ⬎0.74 (top). In contrast, a de-
crease in E/I (e.g., ECW decrease and ICW in-
crease, E/I ⬍ 0.8) may cause a TBW/FFM of
⬍0.72 (bottom).
as shown in Fig. 3, top, this may cause a small increase
in FFM hydration (e.g., when E/I ⬎ 1.2, TBW/FFM ⬎
0.74). In contrast, if E/I decreases for physiological or
pathological reasons, as shown in Fig. 3, bottom, this
may cause a low FFM hydration (e.g., when E/I ⬍ 0.8,
TBW/FFM ⬍ 0.72).
Does growth influence FFM hydration? Previous stud-
ies indicate that FFM hydration is significantly influ-
enced by biological factors such as growth (16). Moul-
ton, in his classical investigation (23), summarized
chemical analysis results of nine mammals, including
mouse, rat, guinea pig, rabbit, cat, dog, pig, cattle, and
human. At birth, all mammals show a high FFM
hydration (⬃0.81) and low concentrations of protein
and mineral. FFM hydration then rapidly declines, and
protein and mineral content increase from early life
until chemical maturity is reached.
A reasonable question thus arises: can the proposed
cellular level model be applied in modeling the relation-
ship between FFM hydration and growth? Of the four
determinants of FFM hydration, ratios a, which equals
⬃0.70, and b, which equals ⬃0.98, can be assumed for
modeling purposes to be stable throughout life. The
cellular level hydration model (Eq. 6) can therefore be
simplified to
1 ⫹ E/I
TBW/FFM ⫽
1.429 ⫹ c ⫹ (1.020 ⫹ c) ⫻ (E/I)
Equation 8 shows that ratio c changes in inverse
proportion to, and E/I changes directly with, FFM
hydration. Based on Reference Children data (15), ratio
c is very low at birth (i.e., ⬃0.07) and then increases
rapidly to adolescence (i.e., ⬃0.14). In contrast, E/I is
maximal (i.e., ⬃1.7) at birth and then decreases rapidly
to ⬃1.0 in adults.
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We were thus able to predict the change of FFM
hydration during growth. At birth, when c is ⬃0.07 and
E/I is ⬃1.7, predicted FFM hydration, according to Eq.
8, is 0.81. The theoretical FFM hydration then de-
creases to 0.73 for adults when c is ⬃0.14 and E/I is
⬃1.0. This trend is similar to measured changes in
FFM hydration, 0.810 at birth and 0.746 for 10-yr-old
boys (15). As indicated by Eq. 8, both an increase in
ratio c and a decrease in E/I cause a rapid decline in
FFM hydration during growth.
Does body size influence FFM hydration in mam-
mals? A pervasive finding in the biological literature is
that mammals share in common a FFM hydration of
⬃0.73 (31, 36). Although this observation generally has
ample support, there are notable deviations. Pitts and
Bullard’s classic study (27) examined FFM hydration in
a wide range of mammals captured in their native
habitat. The investigators noted a small but consistent
decrease in TBW/FFM with increasing FFM from mouse
to cattle. The empirical equation derived by Pitts and
Bullard is
log(TBW/FFM)
⫽ ⫺0.1308 ⫺ 0.0076 ⫻ logFFM; r ⫽ ⫺0.764 (9)
or TBW/FFM ⫽ 0.7399 ⫻ FFM⫺0.0076
(8) where FFM is in kilograms. According to Eq. 9, FFM
hydration is higher in mouse (0.760, FFM 0.03 kg) than
in monkey (0.726, FFM 12 kg) and human (0.718, FFM
55 kg). Can the mammalian FFM hydration of ⬃0.73 in
general and specifically the small downward trend with
increasing body size be theoretically explained?
One approach to examining this question is to extend
our analysis of FFM hydration from the cellular level to
the tissue-organ level. Whole body FFM hydration can
 FAT-FREE BODY MASS HYDRATION E1001

be calculated by summing the water and FFM of Table 4. Potential influence of biological factors on
individual organs and tissues cellular level model determinants and FFM hydration
兺Wi Cellular Level Model Determinants
TBW/FFM ⫽
兺FFMi Biological Factors a b c E/I TBW/FFM
(10)
兺[(W/M)i ⫻ Mi ] Growth = = > << <<
⫽ Aging = = = > (?) > (?)
兺[(FFM/M)i ⫻ Mi ] Adiposity > = = = > > (?)
Gender (females
where Mi is individual organ-tissue mass, Wi and FFMi vs. males) = = = > >
represent water mass and fat-free mass of individual Body size > = = > = (?) <
organ-tissue, and (W/M)i and (FFM/M)i is the fraction Acute or chronic
catabolic illness < (?) = < > >
of individual organ-tissue mass as water and FFM,
respectively. Both cadaver and in vivo measurements =, No obvious influence on cellular level model determinants and
by computerized axial tomography or magnetic reso- FFM hydration; >, positive influence on cellular level determinants
and FFM hydration; <, negative influence on cellular level determi-
nance imaging show that individual organ-tissue mass nants and FFM hydration; ?, association speculated or evidence
can be expressed as a function of body mass among limited.
mammals ranging in body mass from rat to elephant (2,
5, 12). The general relationship between organ-tissue mass of Reference Man. It is assumed that the fractions
mass (M ) and body mass (BM) is of individual organ-tissue mass as water (W/M)i and as
fat-free mass (FFM/M)i are similar among adult mam-
M ⫽ k ⫻ BMm (11) mals. According to the known (W/M)i, (FFM/M)i, k, and
m for individual organs-tissues (Table 3), we derived
where k is constant and m is a scaling exponent. Most the following model for characterizing FFM hydration
organs, including liver, kidneys, brain, heart, and lung, from body mass
occupy a decreasing fraction of body mass (i.e., m ⬍ 1)
as body size increases. Skeletal muscle is almost di-
rectly proportional to body mass (i.e., m ⫽ 1): skeletal log(TBW/FFM)
muscle is 0.468 ⫻ BM0.99 (5). In contrast, bone and ⫽ ⫺0.1198 ⫺ 0.0102 ⫻ logBM; r ⫽ ⫺1.0 (13)
adipose tissue occupy increasing fractions of body mass
(i.e., m ⬎ 1) as body size increases (Table 3). The or TBW/FFM ⫽ 0.7589 ⫻ BM ⫺0.0102

tissue-organ level FFM hydration model (Eq. 10) can be

converted to where BM is in kilograms. According to Eq. 13, FFM
hydration is 0.784 for mouse (BM ⫽ 0.04 kg), 0.738 for
兺[(W/M)i ⫻ (k ⫻ BMm)i] monkey (BM ⫽ 15 kg), and 0.727 for humans (BM ⫽ 70
TBW/FFM ⫽ (12) kg). Although there is a small systematic difference in
兺[(FFM/M)i ⫻ (k ⫻ BMm)i]
FFM hydration prediction, Pitts and Bullard’s (27)
The (W/M)i and (FFM/M)i values for 14 organs and empirical formula (Eq. 9) and our prediction model (Eq.
tissues of Reference Man are shown in Table 3 (32). 13) show the same trend, with FFM hydration decreas-
These organs and tissues account for 87.3% of the body ing minimally but systematically with a remarkable
increase in animal size by a factor of 105. The tissue-
organ level FFM hydration model thus provides a basis
Table 3. Organ or tissue mass-to-body mass for the small downward trend in hydration as a func-
relationships in mammals tion of body mass observed by Pitts and Bullard in
mammals living within their natural habitats.
Organ or Tissue (W/M )i (FFM/M )i Organ or Tissue ⫽ k ⫻ BMm

Liver 0.7222 0.9333 0.0491 ⫻ BM0.70 POTENTIAL RESEARCH AREAS

Kidney 0.7742 0.9484 0.0089 ⫻ BM0.71
Brain 0.7857 0.8929 0.1025 ⫻ BM0.71 There are many important unanswered questions
Adrenal 0.5714 0.7429 0.0003 ⫻ BM0.80 related to FFM hydration. We have selected several of
Heart 0.7273 0.9000 0.0131 ⫻ BM0.88 these questions to highlight the need for more research
Thyroid 0.7500 0.9000 0.0001 ⫻ BM0.92 in this important area. Some examples of unresolved or
Lung 0.7800 0.9901 0.0092 ⫻ BM0.92
Gut 0.7917 0.9383 0.075 ⫻ BM0.94
incompletely understood aspects of FFM hydration in
Skin 0.6154 0.9000 0.106 ⫻ BM0.94 health and disease are summarized in Table 4. Table 4
Skeletal muscle 0.7857 0.9779 0.468 ⫻ BM0.99 also suggests directional changes in each of the four
Spleen 0.7778 0.9839 0.003 ⫻ BM1.02 cellular level model determinants and their collective
Blood 0.8000 0.9935 0.069 ⫻ BM1.02 impact on FFM hydration. Experiments can be de-
Bone 0.1700 0.9900 0.061 ⫻ BM1.09
Adipose tissue 0.1533 0.2000 0.075 ⫻ BM1.19 signed to test the validity of the hypotheses presented
in Table 4.
Information on relationship between organ or tissue and body mass Although our general FFM hydration models (e.g.,
is based on Refs. 5 and 12, and information on fraction of individual
organ or tissue mass as water [(W/M )i ] and as FFM [(FFM/M )i ] is
Eqs. 4, 6, 10, and 12) are mathematically valid for
based on Ref. 32 (modified). BM, body mass (kg); k, constant; m, various species, many biological assumptions regard-
scaling exponent. ing the four cellular level model determinants (i.e., a, b,

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E1002 FAT-FREE BODY MASS HYDRATION
c, and E/I) were made in the discussions that followed.
There remain opportunities for verifying these assump-
tions and expanding on the concepts presented in this
report.
How might the four cellular level model determi-
nants be compared between individuals or across
groups? Body cell mass hydration, ratio a, requires
primarily in vitro analysis. Extracellular fluid hydra-
tion, ratio b, can best be inferred from analysis of easily
obtained blood samples. The third ratio, c (⫽ ECS/
TBW), can be evaluated from bone mineral or total body
ash. Bone mineral can be measured in vivo with
dual-energy X-ray absorptiometry (26), and bone or
total body ash can be evaluated in vitro in animal or
human cadavers. TBW is easily quantified with labeled
isotopes in vivo (30) or by desiccation in vitro. Finally,
E/I can be calculated from total body potassium and
water masses as in this report (see APPENDIX ).
FFM hydration was reexamined with the proposed
cellular level model in the present study. There are also
several other classic body composition constants, such
as the ratio of total body potassium to FFM (⬃68
mmol/kg FFM) and FFM density (⬃1.10 g/cm3 ), that
are presently used in body composition research. These
assumed stable constants, as well as FFM hydration,
form the cornerstone of widely used body composition
methods, and the origin of their constancy is of funda-
mental scientific interest. Similar cellular level models
could be developed that may be useful in improving
understanding of these widely used body composition
constants.
CONCLUSION
The empirical relationship between TBW and FFM
in mammals has been recognized for over five decades.
The relative constancy of FFM hydration led to the
widely used TBW method of estimating fatness in
mammals ranging widely in body size. Deviations from
‘‘constant’’ hydration in earlier reports were often viewed
as aberrations or methodological errors. The present
study, to our knowledge, is the first effort aimed at
providing a physiological basis for FFM hydration, and,
in so doing, our developed model provides new insights
into earlier, poorly understood phenomena, such as
why hydration is high in newborns. The model and our
accompanying review identify important potential re-
search areas and present several testable hypotheses.
Moreover, our review of earlier reports identified little
research on FFM hydration outside of mammals (36).
Many opportunities still exist for advancing understand-
ing and application of the TBW-fat estimation method,
even though it is among the earliest body composition
methods.
APPENDIX
Water Distribution Measurement
The water distribution was measured based on total potas-
sium and water (15). It is known that almost all body
potassium exists in intracellular water (ICW) and extracellu-
lar water (ECW), and given m and n as the potassium
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concentrations in intracellular and extracellular fluid, respec-
tively, the following simultaneous equations may be written
TBW ⫽ ICW ⫹ ECW (A1)
TBK ⫽ m ⫻ ICW ⫹ n ⫻ ECW (A2)
Because TBK and TBW are measurable, ECW and ICW can
be solved as
ECW ⫽ (m ⫻ TBW ⫺ TBK)/(m ⫺ n) (A3)
ICW ⫽ (TBK ⫺ n ⫻ TBW)/(m ⫺ n) (A4)
The ratio of ECW to ICW can thus be calculated as
E/I ⫽ (m ⫻ TBW ⫺ TBK)/(TBK ⫺ n ⫻ TBW) (A5)
Previous studies reported similar intracellular potassium
concentrations (m) in mammals: 150–160 mmol/kgH2O (21),
150 ⫾ 7.2 (SD) mmol/l (22), 152 mmol/kgH2O (21), and 159
mmol/kgH2O (28). In the present investigation m was as-
sumed as 155 mmol/kgH2O. The potassium concentration in
extracellular fluid (n) is much lower than m and close to the 5
mmol/kgH2O reported in previous studies. Equation A5 can
thus be expressed as
E/I ⫽ (155 ⫻ TBW ⫺ TBK)/(TBK ⫺ 5 ⫻ TBW) (A6)
where TBW is in kilograms and TBK is expressed in milli-
moles. Water volume, estimated by 3H2O dilution in the
present study, was estimated to overestimate TBW by 4%
(30, 36).
This research was supported by National Center for Research
Resources Grant RR-00645 and National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-42618.
Address for reprint requests and other correspondence: ZM. Wang,
Weight Control Unit, 1090 Amsterdam Ave., 14th Floor, New York,
NY 10025 (E-mail: ZW28@Columbia.edu).
Received 14 August 1998; accepted in final form 19 February 1999.
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