Tumor Necrosis Factor-Mediated Hypoalbuminemia in

Rabbits1
|
BERNHARD HENNIG,*f RON HONCHELf

SIMEON E. GOLDBLOMf

AND CRAIG J. McCLAIWf

"Department of nutrition and Food Science, tGraduate Center for Toxicology and Department of
Medicine, university of Kentucky and Lexington Veterans Administration Medical Center, Lexington, KY
40536

MATERIALS AND METHODS

INDEXING KEY WORDS:

albumin

Rabbit studies. New Zealand White rabbits (Clerco

Research Farms, Cincinnati, OH) having an average
weight of 3.4 kg (range: 2.6-4.2 kg) were housed in
dividually in an environmentally controlled room (tem
perature: 24 1C)with a 12-h light cycle beginning
at 0600. Rabbits were allowed food (Ralston-Purina

tumor necrosis factor

In hospitalized patients, the serum albumin concen

tration frequently is used as an indicator of protein
energy malnutrition, as an indicator of the adequacy of
nutritional support and as a predictor of morbidity and
mortality (1-10). The utility of the serum albumin value
as a prognostic indicator may be unrelated to the actual
nutritional status of the patient, but instead may be
more closely correlated with the severity of the injury
(11-14). Indeed, patients with severe trauma, such as
thermal injury or severe head injury, frequently have
0022-3166/88 $3.00 1988 American Institute of Nutrition.

'Supported in part by the Veterans Administration, National In

stitutes of Health Grant Nos. NS22712 and HL34423, a grant from
the American Heart Association, Kentucky Affiliate and the Ken
tucky Agricultural Experiment Station.
2To whom correspondence and reprint requests should be ad
dressed, at Department of Medicine, Division of Digestive Diseases
and Nutrition, University of Kentucky, Lexington, KY 40536-0084.

Received 5 April 1988. Accepted 30 August 1988.

1586

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hypoalbuminemia
at the time of hospitalization
and
continue to have low serum albumin levels in spite of
aggressive nutritional support throughout the hospital
stay (15, 16). Thus, whenever there is evidence of the
acute phase response, a fall in plasma albumin may not
be diagnostic of inadequate nutritional support (17).
One possible explanation for the initial hypoalbu
minemia observed in septic or trauma patients is that
there may be an increased leakage of albumin from the
intravascular to the extravascular space (12-14). Cy
tokines, such as interleukin-1 (IL-1) and tumor necrosis
factor (TNF), mediate many aspects of the acute phase
response observed in septic or trauma patients (18-23).
The purposes of this study were to evaluate whether
or not administration
of TNF would cause hypoalbu
minemia in healthy well-nourished animals, and to de
termine if TNF-mediated hypoalbuminemia
could be
due to increased leakage of albumin across the vascular
endothelium.
The second hypothesis was tested by
measuring the effect of TNF on transendothelial move
ment of albumin in an in vitro endothelial cell system.

ABSTRACT
The serum albumin concentration is used
clinically as an indicator of nutritional status and as a prog
nostic indicator. Critically ill patients, who display many
aspects of the acute phase response, frequently have low
serum albumin levels upon hospitalizaron. Cytokines, such
as tumor necrosis factor (TNF), mediate many aspects of
the acute phase response. One purpose of this study was
to determine if TNF administration to healthy well-nour
ished rabbits could produce hypoalbuminemia. After intra
venous administration of saline or TNF, the TNF-treated
rabbits experienced significant hypoalbuminemia which was
most prominent at 24 h and was partially corrected by 48
h. A second purpose was to evaluate the effects of TNF
treatment on transendothelial movement of albumin using
an in vitro porcine pulmonary arteryendothelial cell sys
tem. Exposure to TNF for 24 h resulted in a dose de
pendent increase in transendothelial passage of albumin.
These data suggest that the mechanisms of hypoalbu
minemia frequently observed in critically ill patients can be
explained in part by cytokine (TNF)-induced endothelial cell
injury, which results in enhanced endothelial permeability
to albumin. The hypoalbuminemia observed in many crit
ically ill patients thus may be unrelated to nutritional status,
but rather may be related to the patient's underlying disease
state. J. liutr. 119: 1586-1590,
1988.

1587

CYTOKINES AND HYPOALBUMINEMIA

pregnated polycarbonate filters (Nucleopore Corpora

tion, Pleasanton, CA) having 0.8 n-m pores and which
were glued to polystyrene
chemotactic
chambers
(ADAPS Inc., Dedham, MA). After confluent endo
thelial monolayers were exposed for 24 h to the TNFenriched media, albumin transfer across the endothelium was measured according to a previously published
procedure (24). Briefly, chemotactic chambers with at

tached monolayers were placed into 24-well culture

plates thus creating two separate compartments. Each
lower compartment contained 1.5 ml of serum-free M
199, and 0.5 ml of M 199 with 200 (xm of albumin was
added to each upper compartment. After a 1-h incu
bation period, media within each compartment were
sampled. The respective albumin concentrations were
determined by measuring the change in absorbance at
630 nm after addition of a bromcresol green reagent
solution (Sigma Chemical Company, St. Louis, MO) to
an aliquot of the sample.
Statistica! analysis. An analysis of variance table for
a repeated measures procedure was conducted for Fig
ures 1 and 2. In the presence of a significant treatment
by time interaction in Figure 1, the mean responses
between hours for each treatment were compared using
Duncan's multiple-range test procedure. Statistical sig
nificance was determined at the 0.05 level. For Figure
3, a one-way analysis of variance was conducted to
compare the mean response between doses, and then a
contrast to detect a linear trend in dose response was
constructed. Statistical significance was determined at
the 0.05 level.
RESULTS
Animal studies. Figure 1 illustrates the effect of length
of exposure to saline or to TNF on serum albumin con
centration as compared to controls. Hypoalbuminemia

O)

4-

3-

15

25

35

45

Hours after Injection

FIGURE 1 Effect of length of exposure to TNF on serum
albumin concentration. Rabbits were administered intrave
nously either saline or one of 2 doses of TNF (low dose 250,000
or high dose 1,000,000 units). Blood was collected at 0, 8, 24
and 48 h after injection. Values are mean SE,n = 4. -13, Control; -*-,
low dose; --, high dose. Injection of the
vehicle (control) caused no significant changes in serum albumin
concentration. Injection of low-dose TNF caused a significant
reduction in albumin at 24 h compared to all other time periods
(P < 0.05). Injection of high-dose TNF caused significant
(P < 0.05) hypoalbuminemia at 8, 24 and 48 h compared to
baseline. Furthermore, at 24 h, high dose TNF administration
caused a significant reduction in albumin compared to low dose
TNF administration.

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Rabbit Chow, Ralston Purina Co., St. Louis, MO) and

tap water ad libitum. At the start of the study, blood
samples were collected by inserting a 21-gauge butter
fly needle into the central artery of the ear. Using a 25gauge needle, rabbits were then given a bolus treatment
injection into the marginal vein of the ear. Treatments
were as follows: controls (n = 4) were injected with 1
ml of sterile, nonpyrogenic normal saline (Travenol
Laboratories, Deerfield, IL); the low dose group (n = 4)
was injected with 250,000 units TNF (donated by
Genentech, Inc., South San Francisco, CA) in 1 ml of
saline; and the high dose group (n = 4) was injected
with 1,000,000 units TNF in 1 ml of saline. The rTNFa
preparation had 0.50 mg TNF/ml at 5.02 x IO7 u/mg
and 200 pg/ml by Limulus Amoebocyte Lysate Assay
(Whittaker, M. A., Bioproducts, Walkersville, MD). Food
intake was measured daily throughout the study. At 8
and 24 h after injection, blood samples were collected
from the central artery of the ear. At 48 h rabbits were
injected intravenously with 3 ml of Nembutal (Abbott
Pharmaceuticals,
Inc., North Chicago, IL) and blood
samples were collected via cardiac puncture. For each
serum specimen, albumin, total protein and liver en
zymes [serum glutamate pyruvate transaminase (SGPT);
serum glutamate oxaloacetate transaminase (SGOT);
alkaline phosphatase (AP) were measured, using a Beckman Astra-8 photometric assay (Beckman Inc., Fullerton, CA). Liver enzymes were unaffected by TNF
administration.
Endothelial cell culture and materials. Endothelial
cells were isolated from porcine pulmonary arteries and
cultured as previously described (24). The basic culture
medium consisted of M 199 (GffiCO Laboratories, Grant
Island, NY) containing 10% fetal bovine serum (HyClone Laboratories, Inc., Logan, UT). The experimental
culture medium consisted of M 199 enriched with vi
tamins, amino acids (GIBCO Laboratories), 5% fetal
bovine serum and varying concentrations of TNF (0 to
1000 units TNF/ml culture medium). Cell cultures were
verified as endothelial cells by uniform morphology and
by quantitative determination of angiotensin-converting enzyme activity. Cell viability was determined by
lactate dehydrogenase (LDH) release, trypan blue ex
clusion, and morphological assessment using phasecontrast microscopy. LDH activity was assayed in the
culture medium and the cell lysates using the method
of Bergmeyer and Garvehn (25).
For the in vitro albumin transfer experiments, cells
from passages 5 through 12 were plated at a density of
3 x 10s cells/filter on 13-mm-diameter
gelatin-im

1588

HENNIG ET AL.

24

48

Hours after Injection

FIGURE 2 Effect of exposure to TNF on food intake. Food
intake was measured daily after injection of either saline or one
of 2 doses of TNF. Values are mean SE, = 4. Food intake
did not vary significantly from control values in either treatment
group.

12
O
C,

S(O

10
8

C
CO

4-

i3

2-

250

500

1000

TNF (units/ml)
FIGURE 3 Effect of various doses of TNF on subsequent transfer
of albumin across cultured endothelial cell monolayers. Cells
were washed with M 199 and then incubated for 24 hours in M
199, enriched with 5% fetal bovine serum and various doses of
TNF. After washing cells again in M 199, transendothelial move
ment of albumin was measured over a 1-h period. The concen
tration of albumin added to the upper chamber during the sub
sequent 1-h incubation was 200 JIM. Values are mean SE,
n = 6. Each dose of TNF was significantly different from each
other (P < 0.05) and there was a highly significant linear trend
in the dose response (P < 0.0001).

DISCUSSION
The serum albumin concentration frequently is used
as a biochemical marker of nutritional status for hos
pitalized patients (1-10). Many hospitalized patients
who are receiving specialized nutritional support are
being cared for in intensive care units and are among
the most critically ill. If serum albumin values are to
be used as indicators of a need for specialized nutri
tional support, then it is important to understand the
relative effects that nutritional status and the disease
state have on serum albumin levels. Several investi
gators have suggested that the initial hypoalbuminemia
observed in critically ill patients may not be due to
malnutrition but instead is related to fluid shifts and
increases in vascular permeability (14, 26, 27). Recent
studies have shown that minor elective surgery in wellnourished patients caused decreased serum albumin
levels even though adequate oral nutritional intake was
maintained (28). Fleck and co-workers demonstrated a
transcapillary leak of radioiodinated albumin in pa
tients undergoing cardiac surgery and patients in septic
shock (27). Not only can there be an initial decrease in
the serum albumin level, but also certain other proteins
such as prealbumin and transferrin may decrease after
trauma or sepsis (14). Fleck (17) and Fleck, Colley and
Myers (14) suggested that stimulation of the synthesis
of acute phase reactants such as C-reactive protein (CRP)
and the decrease in other proteins such as albumin,
may be initiated through a common mediator such as
the cytokine interleukin-1 (IL-1). Critically ill patients,
for example those with thermal injury, head injury,
sepsis, multiple trauma etc., demonstrate many aspects
of the acute phase response which is thought to be
mediated largely by the cytokines IL-1 and TNF (16,
18-22, 29). It is well documented that both IL-1 and
TNF stimulate synthesis of acute phase reactants such
as CRP, and cause a decrease in the mRNA for albumin
(30-33). However, the initial rapid decrease in serum
albumin observed in severely ill patients cannot be ex
plained by decreased albumin production alone, be-

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in the treatment groups was most prominent at 24 h

after injection,and
the albumin concentration
ap
proached that of the control group at 48 h. In addition
to the time-dependent effect of TNF on serum albumin
levels, the hypoalbuminemia
also was dependent on
the TNF dose to which the rabbits were exposed. A
maximum reduction in serum albumin concentration
was observed in the high dose group 24 h after the
intravenqus injection of 1,000,000 units of TNF.
Food intake was measured at all treatment times. As

shown in Figure 2, intravenous administration of TNF

at 2 doses of 250,000 or 1,000,000 units/ml had no
significant effect on food intake.
Cell studies. The effect of a 24-h incubation in media
enriched with various concentrations
of TNF on al
bumin transfer across cultured endothelial cell mon
olayers is shown in Figure 3. The reduced ability of the
endothelium to act as a selectively permeable barrier
to macromolecules, as measured by the increased trans
fer of albumin across cultured endothelial monolayers,
was dependent on the TNF concentrations
to which
the cultures were exposed. For example, exposure of
endothelial cells to 1000 units of TNF resulted in a
fourfold increase of albumin transfer as compared with
control cultures.

1589

CYTOKINES AND HYPOALBUMINEMIA

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ACKNOWLEDGMENT

21.

We thank Mary A. Stuart for reviewing

script.

the manu

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C. A. & YOUNG,

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cause the half-life of albumin is 18 d. Data from the

present study support the hypothesis that the cytokine
TNF, when given intravenously to otherwise healthy
rabbits, produced transient hypoalburninernia. The TNF
administration had no effect on food intake and did not
cause metabolic abnormalities such as changes in liver
enzyme activities. This is because the high dose of TNF
was quite modest and considerably less than that re
quired to produce hypotension or severe metabolic de
rangements (23, 34). Because the change in albumin
levels possibly was related to alterations in vascular
permeability, the effects of TNF on transendothelial
passage of albumin in an in vitro cell system were eval
uated next. The design of this in vitro system is so well
defined that the effects of individual substances on endothelial cell metabolism can be measured with a high
degree of accuracy. It was demonstrated that in this
system, TNF caused a dose-dependent increase in trans
endothelial movement of albumin.
We conclude that the cytokine TNF can cause tran
sient hypoalbuminemia
in well-nourished animals, and
we provide in vitro data to support the hypothesis that
this hypoalbuminemia
is caused by a leakage of albu
min from the intravascular to the extravascular space.
Thus, it is important that clinicians exercise caution
when using the serum albumin values as an indicator
of inadequacy of nutritional support in critically ill pa
tients. Clearly, in many instances the serum albumin
level of critically ill patients will reflect the underlying
disease state but will not relate necessarily to the pa
tients' nutritional state.

1590

HENNIG ET AL.

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