Synthesis of hepatic secretory proteins in normal adults
consuming a diet marginally adequate in protein
Alan A.
Jackson1,
Gary
Phillips1,
Irene
McClelland1, and
Farook
Jahoor2
1 Institute of Human Nutrition, University of
Southampton, Southampton S016 6YD, United Kingdom; and 2 United
States Department of Agriculture/Agricultural Research Service,
Children's Nutrition Research Center, Department of Pediatrics, Baylor
College of Medicine, Houston, Texas 77030-2600
 |
ABSTRACT |
The plasma concentration
and hepatic synthesis rates of albumin, transthyretin, very low-density
lipoprotein apolipoprotein B-100 (VLDL-apoB-100), high-density
lipoprotein apolipoprotein A-1, fibrinogen,
1-antitrypsin, and
haptoglobin were measured in six normal adults before and after
consuming a protein intake of 0.6 g · kg body
wt
1 · day
1 for 7 days. The
synthesis of hepatic proteins was measured from the incorporation of
[2H5]- phenylalanine, following
prime/continuous infusion, using plasma VLDL-apoB-100 isotopic
enrichment to represent the precursor pool. Synthesis of albumin
declined by 50% (P < 0.001) following the
lower-protein diet, VLDL-apoB-100 declined by 20% (P < 0.001), and apoA-1 declined by 16% (P < 0.05). By
contrast, synthesis increased for fibrinogen (50%, P < 0.05) and haptoglobin (90%, P < 0.001). This
pattern of change, with decreased synthesis of nutrient transport
proteins and increased formation of acute-phase proteins, suggestive of
a low-grade inflammatory response, was accompanied by increased plasma
concentration of the inflammatory cytokine interleukin 6 (30%,
P < 0.05). The pattern of change in the synthesis of
hepatic secretory proteins following 7 days on the low-protein diet may
be of functional relevance for lipid transport and the capacity to cope
with stress.
protein synthesis; phenylalanine; albumin; transthyretin; very
low-density lipoprotein apolipoprotein B-100; high-density lipoprotein
apolipoprotein A-1; fibrinogen;
1-antitrypsin; haptoglobin
 |
INTRODUCTION |
THE DIETARY REQUIREMENT
FOR protein in adults has been based on the level of consumption
that enables body weight and nitrogen balance to be maintained
(10). Thus the average requirement for protein is 0.6 g · kg
1 · day
1, and when
allowance is made for the variation among individuals, the dietary
recommendation has been set at 0.75 g · kg
1 · day
1. The
habitual protein intake is usually much greater than this, and hence
adaptive processes are brought into play if nitrogen balance is to be
achieved when normal adults consume protein at the recommended level.
Whereas it is necessary to achieve nitrogen balance, it is important
that adequate function is also maintained. It is possible that to make
the metabolic adaptations that are required for the achievement of
nitrogen balance, other important metabolic functions cannot be
sustained at an optimal level (17, 36). Indeed, given that
all adaptations carry a cost, it has been suggested that to achieve
nitrogen balance on marginal intakes of protein, there must be some
sacrifice of metabolic function (39). This limitation of
metabolic function may not be evident for the maintenance of normal
homeostasis but would become more obvious when the metabolic capacity
of an individual is challenged, thereby reducing the ability to cope
with stress.
In individuals who are chronically adapted to low dietary
intakes, the metabolic adaptations consist of a combination of changes (5, 39). For example, a reduction in metabolic activity
through a decrease in muscle mass, which is probably combined with a
fall in the intensity of metabolic activity in the liver (37,
38). It is clear that during the response to stress, there are
substantial changes in hepatic function with major modifications in the
proteins that are synthesised and secreted (24). Whereas,
under normal circumstances, the liver is active in the synthesis and
secretion of proteins that play an important role in the transport of
nutrients around the body, this is altered in the face of significant
stress. There is a reduction in the synthesis and secretion of
nutrient-transport proteins, with a shift to increased synthesis and
secretion of the positive acute-phase proteins, with the liver being
able to exert differential control over the synthesis and secretion of individual proteins (21, 24). We hypothesised that when
faced with a reduced intake of protein from the diet, a part of the adaptive metabolic response would be a decrease in the synthesis and
secretion of hepatic secretory proteins. We were interested to know
whether any change in synthesis was shared equally among different
proteins. In the present study, we have measured the rates of synthesis
of several hepatic secretory proteins, especially those involved in
lipid transport and in the acute-phase response to stress, in normal
adults while they were consuming their habitual diet and following a
period when they had consumed a diet that provided a low but adequate
intake of protein.
 |
METHODS |
The study protocol was approved by the joint ethical committees
of Southampton University Hospital NHS Trust and South West Hampshire
Health Authority. Signed informed consent was obtained from each
subject after the nature of the study had been explained. The studies
were carried out in the metabolic ward of the Institute of Human
Nutrition at Southampton General Hospital.
Subjects.
Six normal healthy subjects (3 males, 3 females) were recruited from
the staff of the Clinical Nutrition and Metabolism Unit, University of
Southampton. They were in good health based on a complete medical
history and physical examination. The physical characteristics of all
subjects are shown in Table 1. They were within the normal range of ideal body weight. Before the first study,
each subject completed a 3-day weighed record of all food consumed to
determine their habitual patterns of consumption of energy and protein.
After an overnight fast, the subjects presented to the Metabolic Unit
at 07:30 AM, and resting energy expenditure was measured by indirect
calorimetry (Delatatrac, Datex Instrumentarium, Helsinki, Finland). The
study protocol lasted for 6 h. After completion of the first
study, each subject was provided with meals to consume at home over the
next 7 days. The diets were structured to provide the habitual level of
energy consumption and a protein intake of ~40 g/day, that is ~0.61
g · kg
1 · day
1. On the 8th
day, the subjects presented to the Metabolic Unit at 07:30 AM and the
study protocol was repeated. The female subjects were studied during
the preluteal phase (days 10-12) of their menstrual
cycle (18).
Isotope infusion protocol.
The rates of synthesis of the hepatic secretory proteins were measured
from the rates of incorporation of
[2H5]phenylalanine into the proteins, using
plasma very low density lipoprotein apolipoprotein B-100
(VLDL-apoB-100) isotopic enrichment at plateau to represent the
isotopic enrichment of the phenylalanine precursor pool from which the
liver synthesized the other plasma proteins (21). A
sterile solution of [2H5]phenylalanine
(Cambridge Isotope Laboratories, Woburn, MA) prepared in 4.5 g/l NaCl
was infused to measure the rates of synthesis of the four nutrient
transport proteins albumin, transthyretin, VLDL-apoB-100, and
high-density lipoprotein apolipoprotein A-1 (HDL-apoA-1) and three
positive acute-phase proteins fibrinogen, haptoglobin, and
1-antitrypsin.
After a 10-h overnight fast, the weight and height of each subject were
measured and venous catheters were inserted under local anaesthesia
into each arm. One catheter was used for infusion of isotope and the
other for blood sampling. The hand and forearm with the sampling
catheter were wrapped in a heating pad to arterialize venous blood. A
sterile solution of [2H5]phenylalanine was
infused continuously for 6 h at 4 µmol · kg
1 · h
1 through the catheter in one
forearm after a priming dose of 4 µmol/kg was injected. A sample of
blood (6 ml) was drawn before the start of the infusion and at hourly
intervals throughout the infusion.
To estimate plasma volume, each subject was administered a dose
of 5 mg/kg of Evans blue dye by intravenous injection, and a blood
sample was withdrawn before and 10 min later (13).
Sample analyses.
Blood was drawn in prechilled tubes (containing
Na2EDTA and a cocktail of sodium azide,
merthiolate, and soybean trypsin inhibitor) and immediately centrifuged
at 1,000 g for 15 min at 4°C. The plasma was removed and
stored at
70°C for later analysis.
Plasma amino acid concentrations were determined using reverse-phase
high-performance liquid chromatography (Pico-Tag, Waters, Millipore,
Milford, MA). Plasma interleukin 6 (IL-6) concentrations were measured
by standard enzyme-linked immunosorbent assay using ELISA kits
(Quantikine, R&D Systems, Minneapolis, MN).
Plasma concentrations of six proteins (albumin, HDL-apoA-1,
transthyretin,
1-antitrypsin, haptoglobin, and fibrinogen) were measured by radial immunodiffusion using NL RID kits (The Binding Site,
San Diego, CA). The concentration of VLDL-apoB-100 was measured as the
apoB-100 concentration in the VLDL supernatant by radial immunodiffusion, as previously described by Egusa et al.
(8). VLDL was removed from 1 ml of plasma by
ultracentrifugation at 30,000 g for 2 h at a density of
10.6 g/ml, and apoB-100 was extracted with isopropanol. EDTA-NaBr
buffer (1 ml, 0.1012 molal, density = 1.0063 g/ml) was
overlayed on to 1 ml of plasma in a 13 × 51-mm polycarbonate
centrifuge tube (Beckman Instruments, Palo Alto, CA) and spun at
100,000 rpm in a Beckman TL-100 ultracentrifuge for 4 h at 22°C.
The tubes were carefully placed on a rack under a light against a black
background, and the grayish upper layer (3-4 mm) was carefully
removed with a micropipette and transferred to a clean tube. The
supernatant was concentrated by transferring into centricon 30 microconcentrators, 2 ml of the buffer were added, and it was spun at
5,900 rpm for 15 min at 25°C. The concentrated sample was brought to
a volume of 0.5 ml with buffer, and an equal volume of 2-propanol was
added; the mixture was vortexed and allowed to stand overnight at room
temperature. The solution was centrifuged, and the protein precipitate
was washed twice with 2-propanol. The VLDL-apoB-100 precipitates were
dried and hydrolyzed in 1 ml of 6 mol/l HCl for 24 h at 110°C,
and the amino acids were purified by cation-exchange chromatography
(Dowex 50 H+ form). To determine whether the apoB-100 was
contaminated by chylomicron-associated apoB-48, a sample was divided
into two aliquots. For one aliquot, the apoB-100 was isolated by
isopropanol precipitation as described by Egusa et al.
(8). For the other aliquot, the supernatant was reacted
with human apoB-100 antibody overnight, the complex was precipitated
and washed, and the protein was separated from the antibody on a 5%
SDS-PAGE gel using pure apoB-100 as the standard. The gel band
corresponding to the protein was cut, washed, dried, and hydrolyzed,
and the amino acid was released, purified, and derivatized. The isotope
enrichment of the phenylalanine was determined. It was 3.67 mol%
excess in the antibody extracted specimen and 4.0 mol% excess in the
specimen extracted with isopropanol.
Albumin was extracted from plasma with acidified ethanol,
fibrinogen was extracted as fibrin by thrombin precipitation, and VLDL-apoB-100 was separated by ultracentrifugation and isopropanol precipitation. The high-density lipoprotein (HDL) fraction was separated on a 1.21 g/ml NaBr-EDTA gradient by ultracentrifugation at
450,000 g and 22°C for 16 h (21).
Transthyretin, haptoglobin, and
1-antitrypsin were isolated from
plasma by sequential immunoprecipitation with anti-human transthyretin,
haptoglobin, and
1-antitrypsin (Behring, Somerville, NJ) as
previously described (23). The immunoprecipitates and
protein precipitates were subjected to SDS-gel electrophoresis to
separate the particular protein from its specific antibody and to
separate apolipoprotein A-1 from HDL. A pure standard of the protein
(Sigma, St. Louis, MO) and low molecular weight standards (Bio-Rad
Laboratories, Richmond, CA) were also included in the gel
(23). After the bands corresponding to the protein
standard were stained with Coomassie brilliant blue dye, they were cut
out and washed several times. The dried protein precipitates and gel
bands were hydrolyzed in 6 mol/l HCl at 110°C for 12 h. Amino
acids released from hydrolysis of the proteins, plasma amino acids were
extracted by cation-exchange chromatography, and the tracer-to-tracee
ratio of the phenylalanine was determined by negative chemical
ionization gas chromatography-mass spectrometry on a Hewlett-Packard
5988A GC/MS (Palo Alto, CA). The amino acid was converted to the
n-propyl ester, heptafluorobutyramide derivative, and
phenylalanine isotope ratio was determined by monitoring ions at m/z
383 to 388.
Calculations and statistics.
The fractional synthesis rates (FSR) of all proteins were calculated
with the precursor-product equation
where IRt2
IRt1 is the increase
in isotope ratio of albumin (or transthyretin, HDL-apoA-1, fibrinogen,
haptoglobin,
1-antitrypsin)-bound phenylalanine over the period
t6-t4 h of the
infusion, and IRpl is the plateau isotope ratio of
VLDL-apoB-100-bound phenylalanine. In this calculation, the plateau
tracer/tracee ratio of VLDL-apoB-100-bound phenylalanine in plasma is
assumed to represent the tracer/tracee ratio of the intrahepatic
phenylalanine pool from which albumin and the other secretory proteins
are synthesized (21). Steady-state tracer/tracee ratio was
obtained by finding the average of the individual tracer/tracee ratio
values after the tracer/tracee ratio-time curve reached a plateau (Fig.
1). Plateau was defined as follows: the
tracer/tracee ratio at each time point was normalized to the last value
obtained at the end of the 6-h infusion. These values were then
analyzed by linear regression against time of infusion as the
independent variable. Plateau was verified when the slope of the
normalized tracer/tracee ratio/time line was not significantly
different from zero. The criterion was made stringent by setting the
level of significance at P < 0.25. In the case of
VLDL-apoB-100, the FSR was calculated, as described by Lichtenstein et
al. (26), from the rate of incorporation of
[2H5]phenylalanine into the protein during
the rise to a plateau and the isotopic enrichment of the protein at
plateau.

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Fig. 1.
Six normal adults received a prime and continuous
intravenous infusion of [2H5]phenylalanine
for 6 h, and samples of blood were taken at hourly intervals. The
enrichment of phenylalanine was measured in plasma free phenylalanine
( ) and in phenylalanine isolated from circulating very
low-density lipoprotein apolipoprotein B-100 (VLDL-apoB-100;
). The values are means ± SE, and the enrichment
in the 2 pools was significantly different at each time point.
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The absolute intravascular synthesis rate (ivASR) of albumin (or
transthyretin, HDL-apoA-1, fibrinogen, haptoglobin,
1-antitrypsin) was estimated as the product of FSR and the intravascular mass of the
protein: ivASR
(mg · kg
1 · day
1) = intravascular protein mass × FSR; where the intravascular mass of
a protein is the product of the plasma volume and the plasma
concentration of the particular protein.
The plasma volume of each subject was calculated by the
dye-dilution technique as described by Gibson and Evans
(13).
The standard steady-state equation was used to calculate the flux of
phenylalanine in the circulation: flux = (IRInf
1)/IRplat × D; where IRInf and
IRplat are the isotope ratios of the tracer amino acid in
the infusate and in plasma at isotopic steady state and D is the rate
of infusion of the tracer (in µmol · kg body wt
1 · h
1). The units of flux are
micromoles per kilogram per hour.
Data are expressed as means ± SE for each group. Differences
between the first and second study period were assessed by paired t-test. A probability of 5% (P < 0.05) was
taken to represent statistical significance.
 |
RESULTS |
All subjects were able to keep to the reduced protein diet
without problems (as shown in Table 1) for the 7 days before
study 2, and energy consumption was maintained at ~10.5
MJ/day with protein consumption reduced from 92 to 40 g/day.
Therefore, protein consumption was decreased from 1.4 g · kg
1 · day
1, or 15% of
dietary energy, to 0.6 g · kg
1 · day
1, or 6.5% of
dietary energy. On the marginal protein diet, carbohydrate provided
50% of energy and lipid provided 44% of energy.
As shown in Table 2, the plasma
concentration of three amino acids increased significantly from
study 1 to study 2. These were alanine (36%),
glycine (26%), and serine (17%). The plasma flux of phenylalanine
decreased significantly from 48 to 39 µmol · kg
1 · h
1 between
studies 1 and 2. The plateau level of enrichment
in plasma phenylalanine during the two studies is shown in Fig.
2, and a comparison is drawn with the
plateau level of enrichment in phenylalanine in the VLDL-apoB-100
isolated from plasma. For both studies 1 and 2,
enrichment in plasma was significantly higher than in VLDL-apoB-100, by
~50%, with the ratio of tracer/tracee in VLDL-apoB-100 to plasma phenylalanine being 0.6 in both studies.

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Fig. 2.
Six normal adults received a prime and continuous intravenous
infusion of [2H5]phenylalanine for 6 h
before (study 1) and after (study 2) consuming a
diet that provided a marginal level of protein for 7 days. The plateau
enrichment of [2H5]phenylalanine in both the
free plasma pool and bound as VLDL-apoB-100 was determined toward the
end of the infusion period.
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Table 3 shows the plasma
concentration, the FSR, and the absolute synthesis rate (ASR) of
albumin in the two studies. The plasma concentration was not different
between the two studies, but after 7 days on the low-protein diet, both
FSR and ASR were significantly reduced by ~40%. The table also shows
that the absolute values for FSR and ASR are different depending on
whether the plateau enrichment of free phenylalanine in plasma or the
phenylalanine that is bound in VLDL-apoB-100 is used in the calculation
to represent the enrichment in the precursor pool from which amino
acids are drawn for the synthesis of albumin. The values are 60%
greater when the phenylalanine enrichment in VLDL-apoB-100 is used to represent the precursor pool. We have assumed that the enrichment in
VLDL-apoB-100 better represents the enrichment within hepatocytes, and,
therefore, this has been used for all subsequent calculations (6, 21). Clearly, this will make an important difference to the ASRs, which are derived for individual proteins. Importantly, it
will not affect the relative change between two states, as, for
example, shown for albumin between studies 1 and
2, Table 3.
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Table 3.
The concentration, FSR, and ASR of plasma albumin in 6 normal adults
before and following consuming a diet that provided a marginal level of
protein for 7 days
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Figure 3 shows the plasma concentrations,
FSRs, and ASRs for three nutrient-transport proteins, transthyretin,
and VLDL-apoB-100 and HDL-apoA-1 before and after 7 days consuming the
reduced protein diet. For transthyretin, plasma concentration, FSR, and
ASR are not different before and after the controlled dietary
intervention. For VLDL-apoB-100, there was a significant decrease in
plasma concentration of 20% after the lower protein diet, associated with no change in FSR, but a similar significant reduction in ASR of
20%. In contrast, for HDL-apoA-1, there were significant reductions in
plasma concentration (6%), FSR (13%), and ASR (16%).

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Fig. 3.
Six normal adults received a prime and continuous
intravenous infusion of [2H5]phenylalanine
for 6 h before (study 1, open bars) and following
(study 2, hatched bars) consuming a diet that provided a
marginal level of protein for 7 days to determine the fractional (FSR)
and absolute synthesis rates (ASR) and the concentration of plasma
transthyretin, VLDL-apoB-100, and high-density lipoprotein
apolipoprotein A-1 (HDL-apoA-1). Values are means ± SE, with
significant differences determined by paired t-test;
*P < 0.05; **P < 0.001.
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The response in the hepatic secretory proteins that have been
characterized as positive acute-phase proteins is shown in Fig. 4. There were no changes in plasma
concentration, FSR, or ASR for
1-antitrypsin. For fibrinogen and
haptoglobin, although there were no changes in plasma concentration,
there were significant differences in synthesis rates. On the reduced
protein diet, the FSR for fibrinogen was significantly increased by
38%, and the ASR was increased by 50%. For haptoglobin, the
changes in synthesis in response to the reduced protein diet were even
more marked, with highly significant increases in FSR (78%) and ASR
(90%).

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Fig. 4.
Six normal adults received a prime and continuous
intravenous infusion of [2H5]phenylalanine
for 6 h before (study 1, open bars) and following
(study 2, hatched bars) consuming a diet that provided a
marginal level of protein for 7 days to determine the FSR and ASR and
the concentration of plasma fibrinogen, haptoglobin, and
1-antitrypsin. Values are means ± SE, with significant
differences determined by paired t-test; *P < 0.05; **P < 0.001.
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Figure 5 shows the concentration of
interleukin (IL)-6 in plasma before and after the consumption of the
low protein diet for 7 days, at which time the plasma concentration had
increased by ~30%.

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Fig. 5.
Plasma interleukin 6 (IL-6) in 6 normal adults measured
before (open bar) and following (hatched bar) consuming a diet that
provided a marginal level of protein for 7 days. Values are means ± SE, with significant differences determined by paired
t-test; *P < 0.05.
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 |
DISCUSSION |
The objective of this study was to determine the extent to which
there were identifiable changes in the pattern and rates of synthesis
of hepatic secretory proteins when normal adults consumed a diet
containing a marginal but adequate level of protein. The data show that
although normal adults appear to maintain good health in the short term
when the protein consumed is reduced to the requirement level, there
were significant changes in phenylalanine flux and the synthesis
rates of hepatic secretory proteins. These changes were similar for men
and women. For most of the proteins studied, albumin, transthyretin,
fibrinogen, haptoglobin, and
1-antitrypsin, there was no change in
plasma concentration. There were, however, modest but significant falls
in the plasma concentration of VLDL-apoB-100 and HDL-apoA-1. The
picture was very different when the rates of synthesis for the
different proteins were considered. The rates at which albumin,
VLDL-apoB-100, and HDL-apoA-1 were being synthesized all decreased
substantially (by 13-40%). By contrast, there was a marked
increase in the rate of synthesis of fibrinogen and haptoglobin, with
no change for
1-antitrypsin. This pattern of change is similar to
that usually identified with a stress or the acute-phase response: that
is a reduction in the formation of nutrient transport proteins and
enhanced synthesis of positive acute-phase proteins. Although there was
no overt evidence to suggest a change in the health or wellbeing of the subjects between the two study periods, the plasma concentration of the
inflammatory cytokine IL-6 increased significantly, by 30%, between
studies 1 and 2.
Remarkably, along with a reduction in the synthesis of albumin, there
was a significant reduction in both the concentration and synthesis
rates for VLDL-apoB-100 and HDL-apoA-1 on the lower protein diet. So
far as we are aware, this is the first report of a significant effect
of dietary protein on either the concentration or rates of synthesis of
VLDL-apoB-100 and HDL-apoA-1, which play a central role in the movement
of lipid around the body. Thus the present data raise the possibility
that the movement of lipid around the body, either as triacylglycerides
or bound to albumin as nonesterified fatty acids, might be modulated by
the level of protein consumed. Studies in vitro show an inverse
relationship between the concentration of amino acids in the medium and
the rate at which HepG2 cells secrete apoB-100, with specific amino acids having a particularly marked effect (7, 40). It is almost invariable that reducing the dietary protein from 90 to 40 g/day
means a substantial reduction in the consumption of meat protein
(20), and this will also mean a change in other aspects of
the macronutrient composition of the diet, especially the pattern of
lipids consumed. After 4 wk on a diet enriched with fish oil, there was
a decrease in the pool size of VLDL-apoB-100, no change in FSR, but a
30% decrease in the ASR (4). Therefore, although it is
not possible to relate the change in the synthesis of these two
proteins directly to the reduced dietary protein, it is important that
their synthesis was modified by a pattern of dietary change that is
widely seen across habitual intakes (20).
It was surprising to find that for two of the proteins, fibrinogen and
haptoglobin, there was a highly significant increase in both the FSR
and ASR. The plasma concentration of fibrinogen in the young subjects
reported here was very similar to that reported previously, and when
allowance is made for the method of derivation (see above), the FSR is
similar for the two studies (12). We do not know of any
other reports for the rate of haptoglobin or
1-antitrypsin synthesis
in normal adults. It is not clear why there should have been an
increase in the synthesis rate for fibrinogen and haptoglobin but not
1-antitrypsin, although the general pattern of overall change is
similar to that usually associated with an inflammatory stimulus.
It has been suggested that the pattern of proteins synthesized by the
liver is determined by the availability of amino acids relative to the
pattern of amino acid composition of individual proteins. Thus, for
example, if in vitro hepatocytes are to maintain the rate of albumin
secretion, they have to be provided with an unusual mixture of amino
acids, especially rich in the nonessential amino acids glycine,
glutamine, and arginine (16). Similarly, it has been
suggested that different patterns of synthesis require different mixes
of amino acids (19) and that the formation of acute-phase
proteins in particular requires relatively large amounts of the
aromatic amino acids (35). In the present study, there was
no change in the plasma concentration of most amino acids between the
two study periods, but there was a significant increase in three amino
acids: alanine, glycine, and serine (Table 2). This pattern of change
in the amino acid profile with a relative increase in the nonessential
amino acids compared with the essential amino acids is similar to that
reported in rat studies when the energy intake is maintained but the
protein intake is reduced (27). As the protein in the diet
is progressively reduced, the concentration of nonessential amino acids
in the circulation increases, especially alanine, glycine, and serine,
and at the same time, there is a progressive fall in albumin
concentration (27, 28). In the present study, there was no
change in the plasma concentration of phenylalanine, but there was a
significant reduction in the plasma flux of the amino acid by ~20%
on the marginal protein intake. The flux of this essential amino acid
was not maintained as well as that of nonessential amino acids
(14). Thus the concentration of individual amino acids in
plasma cannot be taken to reflect changes in flux. It is likely that on
the marginal level of protein consumption, the flux of amino acids
better reflects the availability of specific amino acids to maintain
adequate rates of synthesis for hepatic secretory proteins. However, on
the basis of the pattern of change in plasma amino acid concentration
and the reduced flux of phenylalanine seen in the present study, this
mechanism seems insufficient to explain the changes observed.
An alternative explanation for the observed changes in hepatic protein
synthesis would be that the lower protein intake is associated with a
low-grade inflammatory response leading to a reduction in the synthesis
of albumin, VLDL-apoB-100, and HDL-apoA-1 and an increase in the
synthesis of fibrinogen and haptoglobin. The synthesis and secretion of
apolipoprotein B is inhibited in vitro by IL-6 (31). The
finding of a significant increase in the plasma concentration of IL-6
after 1 wk of the lower protein diet would give support to the proposal
of an ongoing low-grade inflammatory process. There is a significant
increase in mRNA for IL-6 in the small intestine and peripheral blood
mononuclear cells of rats placed on a zero-protein diet for 14 days
(29). Purified human apolipoprotein A-1 inhibits the
endotoxin-stimulated release of cytokines, and, therefore, a lower
level of production of HDL-apoA-1 would likely contribute to enhance a
low-grade inflammatory state (11). These possibilities
require further investigation but together raise the possibility that
adaptation to a lower protein diet predisposes to a low-grade
inflammatory state and hence a potentially atherogenic metabolic set.
In the "Nurses' Health Study," there was a significant
relationship between protein consumption and the risk of ischaemic
heart disease, with a reduction in risk of ~25% for women in the
highest fifth of protein consumption (15). This
relationship remained when allowance had been made for other possible
confounding variables such as smoking, the pattern and amount of
different dietary fatty acids, and other lifestyle characteristics. The
relationship was present for both animal and vegetable proteins.
However, the relationship was demonstrated over a range of protein
consumption far higher than that in the present study: from 15% of
total energy in the lowest fifth to 24% of total energy in the highest
fifth. Nevertheless, in a meta-analysis of prospective studies that
related inflammatory factors with coronary heart disease, there was an
80% increase in risk for the top compared with the lowest third of
fibrinogen concentration and a 50% increased risk for a 4-g/l
reduction in plasma albumin concentration (8). Therefore,
the pattern of change in fibrinogen and albumin synthesis identified in
this study on the lower protein diet would, if reflected in changes in
concentration, fit a profile associated with increased risk of
ischemic heart disease.
There is a considerable literature on the factors that determine
the plasma concentration, synthesis, and distribution of albumin from
experimental work in animals and humans (33). Much of the
work on albumin kinetics explores the effect of extreme dietary
manipulations or the response to different pathological states but very
little on changes within the normal range of protein intake. There is
downregulation of the albumin transcription gene in the rat during
starvation or while consuming a very low-protein diet (25, 30,
34). In adult humans, the FSR for albumin varies from 5 to 11%
per day, depending on dietary history and clinical state
(1-3). The synthesis rate is within a similar range
during pregnancy and in older people (12, 32). When plasma-free phenylalanine was used to estimate the enrichment in
the precursor pool in the present work, FSR was 8.2% per day on the
habitual protein intake and 5.1% per day on the marginal protein
intake, similar to other reports. However, when the enrichment in bound
phenylalanine in VLDL-apoB-100 was used to represent enrichment in the
precursor pool (6, 21, 26), albumin FSR decreased from
13.7% per day on the habitual diet to 8.1% per day on the marginal
protein diet.
The present work has shown that in normal adults, the rate of
albumin synthesis is probably 50% greater than estimated in most
published reports. After 7 days consuming a diet that is marginally
adequate in protein, normal adults appear to maintain good health.
However, adaptation to the diet is associated with reduced protein
turnover and changes in the pattern of hepatic secretory proteins that
are probably of functional importance and carry a significant metabolic
cost. There was a significant reduction in the synthesis of
albumin, VLDL-apoB-100, and HDL-apoA-1 with a significant increase in
the synthesis of fibrinogen and haptoglobin. This pattern of change has
similarities to that expected during a low-grade inflammatory response,
a suggestion that is supported by the finding of a significant increase
in the circulating level of IL-6. There is the need to determine in
greater detail the extent of the functional changes of this level of
protein consumption, the basis of the change in response, and the level of consumption at which such potentially damaging effects are not observed.
 |
ACKNOWLEDGEMENTS |
We thank our subjects for agreeing to participate in this study,
and we are grateful to M. Frazer and M. del Rosario for skilled technical assistance.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: A. A. Jackson, Institute of Human Nutrition, Univ. of Southampton, Level C
(113) West Wing, Southampton General Hospital, Tremona Rd,
Southampton, SO16 6YD, UK.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12 December 2000; accepted in final form 8 August 2001.
 |
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