LOW PROTEIN DIET AUGMENTS HYPERPROTEINEMIA
PRODUCED BY REPEATED INJECTIONS OF
HOMOLOGOUS PLASMA
EVIDENCE FOR A DYNAMIC EQUILIBRIUM BETWEEN FOOD, PLASMA,
AND TISSUE PROTEINS*
Bx RUSSELL L. HOLMAN, M.D.
(From the Department of Pathology, University of North Carolina, Chapel Hill, and the
Department o]Laboratories, Watts Hospital, Durham, North Carolina)
(Received for publication, August 3, 1942)
During recent years the use of plasma transfusions has become widespread
and increasingly so of late as a result of war conditions. More and more
the need for really large amounts of plasma in treating certain types of cases
is being appreciated. The data presented in this paper should be of interest
to those engaged in studying the quantitative aspects of such therapy. Fur-
ther these data have certain theoretical impfications that bear upon the more
general problems of plasma protein formation and protein metabolism.
Hyperproteinemia can regularly be produced in dogs, and presumably in
other species of animals, by repeated injections of plasma obtained from
homologous donors. Almost none of the injected protein escapes in the urine
as protein, nor is it eliminated quantitatively as increased urinary or fecal N2.
While no direct measurements have been made in dogs, Addis' results following
intraperitoneal injections of serum in rats (1) indicate that the protein content
of all of the viscera and tissues is increased. The greatest increment is in the
serum, next in the liver, but all organs and tissues show a definite increase in
protein content.
Practically all of the experiments to date (2-6) have been carried out with
the recipient of the plasma or serum injections maintained in a fasting state or
receiving only sugar, or sugar and fat, by mouth. A priori one would expect
that plasma injections in an animal maintained on a full diet or a high protein
diet would yield summation effects and result in a more marked hyperpro-
teinemia. In the experiments reported below this does not happen; in fact, a
significantly higher hyperproteinemia is produced when the animals are main-
tained on a low protein diet than when a high protein diet is given. This
seeming paradox is open to a number of interpretations but all of them seem
to imply an equilibrium between food, plasma, and tissue proteins.
* This work has been aided by a grant from The John and Mary R. Marlde Foun-
dation.
519
520 DIET AND I~3~:'EI~I-M-~.NTALH'YPERPROTEINEMIIA
Methods
All of the dogs were healthy adult mongrels. Small dogs (about 5.0 kilos) were
chosen so that increase of the plasma protein level could be accomplished without
using too great quantities of blood. They were kept in individual cages and had
free access to water at all times. One group was fed a high protein diet, the other
group a low protein diet.
The high protein diet consisted of lean beef--25 gin. per kg. per day--to which 1
gin. of the salt mixture (7) was added.
The low protein diet consisted of: calves' liver (raw wet weight) 32 parts, cane
sugar 25 parts, corn starch 25 parts, butter 12 parts, and cod liver oil 6 parts. 1 gin.
of salt mixture (7) and 5 gin. of kaolin were thoroughly mixed with each day's diet.
Enough tomato juice was added to make a pasty mixture of which each gram con-
tained 3 calories. The diet was fed in amounts to furnish 75 calories per kg. per day.
Essentially this diet is a low protein diet with 7 per cent of its caloric value derived
from protein, 50 per cent from carbohydrate, and 43 per cent from fat.
All of the dogs in both groups consumed 100per cent of the diet each day.
The methods used in making the dogs hyperproteinemic have been published in
detail (2, 8). Briefly these consisted of bleeding a donor--a sufficient number of
large dogs (15 to 25 kg.) were used so that anemia did not develop--about 200 cc.
into a flask containing 2.5 cc. of a saturated solution of trisodium citrate, centrifuging
the citrated blood in 100 cc. centrifuge tubes at 3000 R.P.M. for 30 minutes, with-
drawing the plasma (usually 100 to 110 cc.) with suction, warming it to 40C., and
injecting it into one of the external jugular veins (about 10 minutes being required
for each injection). This procedure was repeated daily, 6 days per week, for 3 to
4 weeks.
Duplicate micro Kjeldahl analyses of total N, N.P.N. (the filtrate from 10 per cent
trichloroacetic acid precipitation) and albumin plus N.P.N. (the filtrate from 22 per
cent sodium sulfate precipitation by Howe's method) served as the basis for calculat-
ing the blood level studies. All of the recorded studies were made on hematocrit
samples (using 2.0 cc. of 1.4 per cent sodium oxalate and 10 to 13 cc. of blood), and
the "final samples" were taken at least 18 hours after the last injection of donor's
plasma.
EXPERIMENTAL OBSERVATIONS
The experimental data are summarized in Tables I and II.
In dogs consuming a high protein diet (Table I) the intravenous injection
of plasma obtained from healthy donor dogs, amounting in all to five or six
times the quantity of plasma protein in circulation at the start, resulted in
only a moderate increase in the concentration of the blood plasma proteins.
The average increase was 20 per cent. As would be expected, there was a fall
in the hematocrit reading but this was not great--ranging from about 5
per cent in dog 35-693 to about 28 per cent in dog 40-60 and averaging 18
per cent. "Final" blood and plasma volume studies made in isolated cases
in this series agree with previous observations (2, 3) that these are increased
RUSSELL L. HOLMAN 521
only about 15 per cent; and this increase--which is almost entirely in the plasma
volume---accounts in part at least for this fall in hematocrit value. There were
no significant changes in the albumin:globulin ratio, plasma N.P.N., or in
body weight.
TABLE I
Plasma Injections in Dogs on High Protein Diet
Dog No.
35-693
40-59
40-60
40-67
Body
weight
l--
~g. [ kg.
2.7 12.7
6.3] S.8
6.5 6.3
4.81 4.3
No. of injections
19 in 24 days
18 " 21 "
24 " 28 "
20 " 24 "
~-o Plasma Albumin/ N P N
protein Hematocrit
.~. concen- globulin ' ' " reading
,~ tration
co. gm./lO0 co. mg./lO0 co. per cent
4355 6.6 8.2 27 27 41 39
15951 7.5 8.9 1.2 ] 1.3 36 36 58 44
2665[ 7.2 8.7 1.7 [ 1.4 30 30 57 41
2080h72 8.310.910.8 / 40 ]32 s3 39
* Initial value--before first plasma injection.
Finalvalue--18to 24hours after last plasma injection.
TABLE II
Plasma Injections in Dogs on Low Protein Diet
Body
Dog No. weight
I.* F.:~
kg. kg.
39-28 6.7 5.1
39-34 4.5 4.4
39-40 6.3 6.5
39-45 7.1 7.3
40-50 4.7 4.8
40-63 8.1 9.2
40-80 5.7 5.8
No. of injections
24 in 28 days
22 " 28 "
17 " 21 "
22 " 26 "
; 24 " 28 "
24 " 28 "
18 " 21 "
CC.
2225
262~
2010
2210
188(
2960
1840
Plasma
protein
concen-
tration
L [ F.
gm./lO0 co.
7.2 9.9
6.5 9.4
7.0 8.8
7.1 9.2
6.6 10.0
6.0 8.0
6.7 9.4
Albumin/
globulin
I. F.
1.3 0.7
1.3 1.0
1.2 0.9
1.9 0.9
1.2 1.1
1.3 1.0
N.P.N. Hema~crit
rea~ng
.IF. LIF.
mg.~O0 cc. per cent
41 23 36 32
30 41 43 40
33 32 51 42
36 31 54 43
41 25 55 49
28 28 48 36
31 24 48 38
* Initial value--before first plasma injection.
Final value--18 to 24 hours after last plasma injection.
In dogs consuming a low protein diet (Table II) the intravenous injectio~
of comparable amounts of plasma---obtained in most instances from the same
donor dogs--resulted in a more marked increase in the concentration of the
plasma proteins. The average increase was 40 per cent, or twice as great as in
the group of dogs maintained on high protein diet. Changes in the hematocrit
readings were in the same direction and of the same order of magnitude in
522 DIET AND EXPERIMENTAL HYPERPROTEINEMIA
both groups. In the group on the low protein diet (Table II) the albumin:
globulin ratio fell in every instance and in some of the dogs (e.g. 40-50) this
change was marked. The average reduction in this ratio in the six dogs
on which it was determined was about 30 per cent. There were no significant
changes in plasma N.P.N. or in body weight.
Following cessation of the daily injections, the plasma protein level returned
to approximately normal in about 2 weeks regardless of diet.
DISCUSSION
A dynamic equilibrium between food, plasma, and tissue proteins was postu-
lated by Holman, Mahoney, and Whipple in 1934 (2). The evidence which
has accumulated since that time has lent support to this view. The subject
has recently been reviewed by Madden and Whipple (9) and by Whipple (10).
Briefly stated this concept implies that food protein, absorbed from the
gastrointestinal tract as amino acids, can be fabricated into units (or aggre-
gates) in one portion of the body for utilization in another part of the body.
During transport these units (or aggregates) constitute part of the plasma
proteins. It is probable that a large portion of this synthesis takes place
in the liver, but other tissues are undoubtedly involved, e.g.antibody (globulin)
formation by lymph nodes and insulin production by the islets of Langerhans,
and it is not improbable that much of this equilibrium is maintained in the
blood which as an organ is about four times the size of the liver. This concept
also implies that neither the capillary endothelium nor the cell membrane is
impermeable to these units or aggregateswhich by the usually employed meth-
ods are classed as proteins. Direct measurement of lymph protein in all parts
of the body and the rapid rate of restoration of plasma protein following acute
severe hemorrhage or plasmapheresis lend support to both of these assumptions.
This view in no way invalidates the Starling hypothesis, but merely adds an
adaptive mechanism that makes it more dynamic.
At first glance it might seem that the data presented in this paper do not
support the hypothesis of an equilibrium. It might be contended that the
more protein going into the body by whatever route, the greater should be the
concentration in the plasma and in the tissues. Within narrow limits this is
true, for by high or low protein feeding the concentration of plasma protein
can be raised or lowered by 5 to 10 per cent of the normal. It must be remem-
bered, however, that the nitrogen balance can be established with widely
Varying intakes, and that under these wide variations in the intake the body
holds tenaciously to a rather limited zone of concentration of plasma protein
(5.5 per cent to 7.0 per cent). Marked reductions in diet or great physical
removals (plasmaphereses) must be carried out in order to lower significantly
the plasma protein concentration. The same is true of hyperproteinemla--
relatively large quantities of donor's plasma totalling several times the amount
RTJSSELL L. HOLM_AN 523
actually in circulation at the start of the experiment must be injected before
significant elevations in the plasma protein concentration are effected. During
the first week of such injections the increment in the concentration of the
plasma protein is slight, and it is only during the later weeks that significant
amounts of the injected protein pile up in the circulation. These amounts are
greater with low protein feeding, presumably because less of the necessary
"chemicals" to maintain the normal equilibrium is supplied. High protein
feeding on the other hand maintains the normal equilibrium better by supplying
more of these necessary "chemicals."
Qualitative differences in the utilization of the injected protein under
varying conditions of dietary protein are indicated by the fact that in the
animals maintained on a low protein diet the albumin:globulin ratio is de-
creased; whereas there is no significant change in this ratio in the animals
maintained on a high protein diet. Most of the difference in the extent of the
hyperproteinemia in the two groups can be accounted for by the greater
increase in globulin in the group maintained on a low protein diet. It could
be argued that the more marked hyperproteinemla in this group is necessary
to maintain the same osmotic pressure relationships. This is a teleological
argument and is not supported by available data, for albumin is not decreased
and there is no need for a further increment in globulin. Rather the data
point to an upset in the normal equilibrium with the accumulation of an excess
of globulin. Whatever the true explanation happens to be, it seems definite
from the experiments here reported that the hyperproteinemia following
repeated plasma injections is greater in animals maintained on a low protein
diet than in animals maintained on a high protein diet.
The promptness with which the plasma proteins return to normal level after
cessation of injections or withdrawals despite high or low protein feeding serves
to emphasize the abnormality that must be present in certain cases of Bright's
disease in which "optimum" feeding fails to influence materially the hypopro-
teinemia, and in some cases of multiple myeloma in which extreme hyperglobu-
linemia persists even during inanition and fasting. The comparative rareness
of these abnormalities serves in turn to emphasize the stability of the normal
mechanism which must be of the nature of an equilibrium between food,
plasma, and tissue proteins.
The data in this paper do not embrace the tissue proteins except indirectly
in the figures on body weight. In previous publications (2-4, 8, 9) the part
played by the tissue proteins in this equilibrium has been discussed.
The experimental findings in this paper and the general thesis of an equilib-
rium have received partial confirmation by other workers using the opposite
approach to the problem, namely treatment of hypoproteinemia occurring in
human cases of "nephrosis" or induced in experimental animals by plas-
mapheresis or extremely low protein feeding. Liu and Chu (11), Keutmann
524 DIET AND EXPERIMENTAL HYPERPROTEINEMIA
and Bassett (12), and Farr (13) have all found an "optimum" intake of about
3.0 gin. of protein per kg. per day. Greater intake resulted in less retention.
Whipple and his coworkers (10) found, "In general, food proteins are better
used when given alone and in moderate amounts. Larger protein intake
yields a lower per cent return of plasma protein."
The "ideal" protein to combat hypoproteinemia has not yet been defined,
but it is reasonable to predict on the basis of the findings reported in this paper
that when it is defined quantitative as well as qualitative factors will play an
important part in the utilization of that protein. It is possible that this "ideal"
protein may prove to be a more or less specific substance that controls the
normal equilibrium between food, plasma, and tissue proteins. Possibly the
repeatedly confirmed observation (9, 14, 15) that serum protein is the most
potent of all the proteins that have been tested thus far for combating hypo-
proteinemia means that more of this hypothetical substance is present in
serum.
SUMMARY
1. In 4 dogs maintained on a high protein diet (lean meat) repeated in-
travenous injections of plasma obtained from healthy donor dogs (18 to 24
injections during the course of 3 to 4 weeks, totalling 1595 to 4355 cc.--averag-
ing 1800 cc. when figured on the basis of a 5 kg. dog) resulted in a mean increase
in the plasma protein concentration of 20 per cent (from 7.1 per cent to 8.5
per cent).
2. In 7 dogs maintained on a low protein diet (only 7 per cent of total caloric
value derived from protein) almost identical injections of donor's plasma
caused an average increase in the plasma protein concentration of 40 per cent
(from 6.7 per cent to 9.4 per cent).
3. The albumin: globulin ratio in the group on the low protein diet showed
an average fall of 30 per cent (from 1.4 to 0.9) while in the group on the high
protein diet the change in this ratio was insignificant (from 1.3 to 1.2).
4. In all dogs in both groups there was a consistent fall in the hematocrit
value of about 15 to 20 per cent (from 49 to 40, or 18 per cent) which can be
explained in part at least by the increase in plasma volume of about 15 per cent.
5. There were no significant changes in body weight or in plasma N.P.N.
CONCLUSIONS
1. Hyperproteinemia produced by repeated daily injections of homologous
plasma is more marked in dogs maintained on a low protein diet than it is
when comparable amounts of plasma are injected into dogs maintained on a
high protein diet.
2. This seeming paradox is interpreted as additional evidence for a dynamic
equilibrium between food, plasma, and tissue proteins.
RUSSELL L. IIOLMAN ~25
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