Lymphedema and vitamins’
The
American Journal of Clinical Nutrition 26: FEBRUARY 1973, pp. 185-190. Printed
in U.S.A. 185
Ethel F#{246}ldi-8#{246}rcs#{a2n4d6}k M. F#{246}ldi
In 1 97
1, the unexpected fact has been
described (1) that surgically induced
acute
experimental lymphedema in the rat flourished
with the usual
laboratory diet rich in
vitamins, can be treated successfully by
the
administration of various vitamins, e.g., pyndoxine,
pantothenic
acid, and particularly, a
highly active representative of the vitamin
P
family,2 coumarin (5 , 6-benzo-a-pyron).
These data, obtained by means of
plethysmographic
assessment of the volume of
lymphedema, were confirmed
by histology
(2, 3).
Based on these results, the question has
to be
raised whether an inadequate supplying
of the organism with vitamins would
aggravate
lymphedema. After having obtained an
affirmative answer to this
question, therapeutic
trials were performed in
avitaminotic
lymphedematous animals.
Material and methods
Experiments
were performed in 150 male Wistar
rats (body weights given in Fig. 1). The
animals
were divided into three groups.
Group 1
Group 1 comprised rats
fed ad libitum an artificial
diet rich in vitamins (Table 1). On the
56th
day, these animals were divided into five subgroups.
Subgroup 1.1 (n
= 10). From the 56th to the
63rd day, the rats were given daily ip
injections of
saline, 10 mI/kg body wt. On the 60th day, the
rats were
anesthetized with nembutal (50 mg/kg
ip). Preoperative volume of the neck
and head was
measured by electronic plethysmography by means
of the
apparatus constructed by Bundschuh (Griesheim,
W. Germany). This was
followed by a
radical cervical lymphatic blockage. From a
midline
incision reaching from the mandibula to the
sternum, lymph nodes
were carefully prepared and
ligated. Plethysmographic measurements were
repeated
on the 63rd day.
Subgroup 1.2 (n = 10). In this subgroup,
the
procedure was the same as in subgroup 1.1, with
just one difference.
Instead of saline, the rats were
treated with the following vitamins in
milligrams
per kilogram body weight: vitamin B1, 40; lactoflavin,
23;
niamid, 160; pyridoxine, 16; pantothenic
acid, 240; biotin, 2 and
cyanocobalamin, 80 pg/kg
body weight.
Subgroup 1.3 (n 10). Procedure was
the same
as in subgroups 1.1 and 1.2 with one difference; i.e.,
the
animals were treated with 25 mg/kg coumarin
(5 ,
6-benzo-alpha-pyron).
Subgroup 1.4 (,z 10). Procedure differed from
that
used in subgroups 1.1, 1.2, and 1.3 in one
respect; the rats were treated
with 500 mg/kg tnhydroxy-
ethyl-rutin.
Subgroup 1.5 (n 10). In this
subgroup, the
same procedure was followed as with subgroup 1.1
except
that instead of a cervical lymphatic blockage,
only a sham operation was
performed.
Group 2
Rats were fed a diet (ad libitum) that was poor
in
B-complex vitamins (Table 1). On the 35th day,
the rats were divided into
five subgroups. (This day
was chosen instead of the 56th because
vitamin
B deficiency was already marked and the state of
the rats
deteriorated rapidly.)
Subgroup 2.1 (n 10). From the 35th to the
42nd
day, the rats were given daily ip injections of
saline. On the 39th day,
they were treated as those
in subgroup 1 . 1 . Plethysmographic
measurements
were repeated on the 42nd day.
Subgroup 2.2 (n 10). In this
subgroup, procedure
was the same as in subgroup 2.1, with one
exception.
Instead of saline, the rats were treated
with the same vitamins as those in
subgroup 1.2.
Subgroup 2.3 (n 10). The rats in this group
were treated
with coumarin; otherwise the procedure
was the same as in subgroup 2.1 and
2.2.
Subgroup 2.4 (n = 10). Our procedure differed
from that used in
subgroups 2.1, 2.2, and 2.3 in
one respect, these rats were treated with
trihydroxyethyl-
rutin.
Subgroup 2.5 (n = 10). In this subgroup,
the
procedure was the same as in subgroup 2.1 except
that instead of a
lymphatic blockage, only a sham
operation was performed.
Group 3
Rats
in this group were fed the Sherman-LaMer-
Campbell diet (4), free of vitamin
P but supplemented
with ascorbic acid (Table 1). This group
1 From the
Lymphological Research Laboratory,
Schaper and Br#{252}mmer,
Salzgitter-Ringelheim, West
Germany.
2 The controversial views concerning
vitamin P
are handled in the Discussion.
Downloaded from www.ajcn.org by
on May 7, 2009
300. -
200.
100.
0 ‘ı 2 3 14 5 6 7 8
FIG. 1.
Changes of body weight in the three groups.
186
F#{212}LDI-B#{246}RCS#{246A}KND FOLDI
E_Eı
ı ı
I weeks ı
also was
fed ad libitum. On the 56th day, the rats
were divided into five
subgroups.
Subgroup 3.1 (n = 10). From the 56th to the
63rd day, the rats
were given saline. On the 60th
day, they were handled as those belonging to
subgroup
I . 1 . Plethysmographic measurements were
repeated on the 63rd
day.
Subgroup 3.2 (,i = 10). In this subgroup, procedure
was the same as
in subgroup 3.1 but instead of
saline, the rats were treated with the same
vitamins
as those in subgroup 1.2.
Subgroup 3.3 (n 10). These rats were
given
coumanin in addition to the treatment received by
subgroups 3.1 and
3.2.
Subgroup 3.4 (,z 10). The procedure for these
rats differed from
that used in subgroups 3.1, 3.2,
and 3.3 in one respect: they were treated
with tnhydroxy-
ethyl-rutin.
Subgroup 3.5 (ii = 10). With this subgroup
also,
a sham operation was performed instead of
lymphatic blockage.
Otherwise, the procedure was
the same as with subgroup 3.1.
Within the
three groups, volumes of lymphedemas
were expressed as the difference
between
the postoperative (V2) and the preoperative (V1)
TABLE
1
volumes. In order to compare data of the three
groups with each other,
it was necessary to eliminate
the differences existing in the V1 values.
Therefore,
volumes of lymphedemas (L) were expressed
with the
formula:
\.ı - \rl
L:=100ı
Statistical analysis was performed by
appropriate
forms of Student’s t test.
Results
The extent of
lymphedema was significantly
higher in both avitaminotic,
salinetreated
subgroups (subgroup 2. 1 and 3.1)
than in subgroup 1.1
(normal diet) (Figs.
2, 3). In group 2, hair loss, rhagades, and
a
markedly diminished growth rate could be
observed (Fig. 1). There were
no such signs
in group 3.
A statistically significant protective
effect
Composition of diets used in group I to LII
Group I (Altromin ı
1,000) Group II (Altromin C 1,000 without Group III
(ShermanıLaMerıCampbelI)C
B vitamins)
Casein
Rice starch-flour
Soja
oil
Cellulose
powder
Minerals
Vitamins#{176}
Percent
22
64
2
4
6
2
Casein
Rice
starch-flour
Soja oil
Cellulose
powder
Minerals
Vitaminsb
I
Percent
22 Butter
64 Milk
powder
2 Yeast powder
4 I
NaCI
6
2
Percent
10
30
59
1
a Vitamins: DL-methionine; 1,200 mg; vitamin A, 15,000 lU; vitamin Dı, 500 IU; vitamin E, 150 mg;
vitamin K,, 10 mg; vitamin B1 . HCI, 20 mg; vitamin B2, 20 mg; vitamin
B3. HCI, 15 mg; vitamin B12, 30 ı.
tg;
pantothenic acid, 50 mg;
nicotinic acid, 50 mg; cholinchlorid, I ,000 mg; folic acid, 10 mg; biotin,
200,ug;
inositol, 100 mg; p-aminobenzoic acid, 100 mg; and ascorbic acid, 20
mg.
b Vitamins: vitamin A, 15,000 LU; vitamin Dı, 500 IU; vitamin E, 150 IU;
vitamin K3, 10 mg; and
ascorbic acid, 20 mg.
The mixture was heated for 1
hr at 100 C. After cooling down, 100 mg ascorbic acid was added to
each 100
g of the dry food. Daily consumption of food/rat 30 g. Daily consumption of
vitamin C/rat
- 25 mg.
Downloaded from www.ajcn.org by on May 7,
2009
Prot.ct#{233}Cı,f ,f.ct
.5
LYMPHEDEMA AND VITAMINS 187
FIG. 4.
Protective effects of various treatments.
BVK = vitamin B complex.
21i
terized by a generalized increase in blood
capillary permeability. In
vitamin B6 deficiency,
brought about by the administration
of
desoxypyridoxine, even the permeability
20 of the blood-brain barrier is
increased (6). In
group 2 of these series, a B-complex avitaminosis
has
been induced; its effect on the
blood capillaries can certainly be
expected
15. to be even more pronounced. With plasma
proteins leaking out
of the blood capillaries,
lymphatic blockage will, of course, lead to
a
more extensive lymphedema than in normal
ıo. animals with a
physiological degree of protein
permeability.
On the other hand,
permeability of the
Fio. 2. Extent of lymphedema (percent volume
increase
after lymphatic blockage); 1) in rats fed a
normal diet (subgroup 1.1); 2)
in rats fed the Sherman-
LaMer-Campbell diet (subgroup 2.1); 3) in
rats
with a vitamin B complex deficiency (subgroup
3.1).
could be achieved
against lymphedema in
rats fed the normal diet by coumarin and by
vitamin
B complex treatment; against lymphedema
aggravated by B-avitaminosis, by
FIG. 3. Lymphedema in a rat fed a normal diet
coumarin treatment, and in
P-avitaminosis (subgroup 1.1; left) and in a rat in a vitamin Pby
coumarin
treatment (Fig. 4). deficiency state (subgroup 3.1;
right).
Discussion
After lymphatic blockage, lymph will
accumulate in
the occluded lymphatics. Stagnating
lymph, however, will dilate them.
The
high intralymphatic pressure will induce
“mural insufficiency” (5),
i.e., induce the
diffusion of lymph through the wall of lymph
vessels,
and will force open interendothelial
junctions in initial lymphatics; here
too,
lymph will escape back into the tissues. At
the same time, plasma
protein molecules,
steadily escaping from the blood capillaries,
will
accumulate in the interstice. They are,
normally, removed by lymphatics.
Lymphedema,
finally, will be the result of raised
oncotic pressure in the
interstitial fluid.
It is well known that beriberi is
characDownloaded
from www.ajcn.org by on May 7, 2009
188 FOLDI-BORCSOK
AND FıLDI
FIG. 5. Vitamin P family.
lymph vessels may eventually be
increased
by B-complex avitaminosis leading to a mural
insufficiency of
lymph drainage, a hypothesis
awaiting elucidation.
Normal connective
tissue cells incorporate
and digest plasma proteins; in this way,
they
become colloidosmotically inactive (7, 8).
In B-complex avitaminosis
quite a number
of the most important mitochondrial
enzyme systems are
severely damaged. In
lymphedema of normally fed animals, the
extent of
lymphedema may be limited to an
appreciable extent through this
mechanism.
The possibility that plasma protein catabolism
in these
connective tissue cells
could be subnormal or absent may well be
a
further factor meriting additional studies.
The matter of vitamin P has
been heavily
debated since 1936, when Szent-Gy#{246}rgyi (9)
described
the fact that experimental scurvy
is caused not only by a deficiency in
ascorbic
acid (discovered by the same author) but
also by a concomitant
deficiency in vitamin
P. Based on the original studies of
Szent-
Gy#{246}rgyi and on those of Javillier and Lavollay
(10), we are
now aware of the fact that
vitamin P is not just one chemical
compound,
as e.g., vitamin B1. It is rather similar to
vitamin K, which
is not a single molecule
either. Just as various molecules
possess
vitamin K activity, other chemically related
compounds possess
qualitatively, but not
quantitatively, the same action on
blood
capillaries; if they are entirely absent from
the food, blood
capillary permeability and
fragility will increase (Fig. 5). The
administration
of the diet described by Sherman,
LaMer and Campbell (4),
which is free of
vitamin P and vitamin C, produces an increase
in blood
capillary fragility and permeability
not only in the guinea pig, but
in
the rat, which is also able to synthesize
ascorbic acid. It has been
shown by Benk#{246}
et al. (11) that in rats fed this diet,
cerebral
edema will appear. V#{225}rkonyi and co-workers
(12) have shown
that cerebral edema is
caused by an increased permeability of
the
blood-brain barrier, as indicated by the
Evans-blue fluorescence
method.
In sharp contrast to Clark and MacKay
(13) who consider “many of
the physiologic
and pharmacologic nonspecific effects which
they (i.e.,
members of the vitamin P family)
are said to exert” as an “alarm
reaction,”
we share the arguments of Javillier and
Lavolley (10): “Any
organic substance,
which is contained in natural food and is
responsible
for the maintenance of a physiologic
equilibrium is a vitamin. The
question
to be answered is, whether there exist, in
the
OH
ı__%
Ct$.O.P,yd?OmyCffVfeıf#{232}C octd
(CoumOrH’ıc
eOd)
FLAVONE
FLAVANONE
COrO
ANTHOCYANtDIN
(f$S.yIsuıı’
soft
ıOO
1ı ı COUMARIN w
toctons of Do coumorWuc aced
or U binzo-
-pyrofı
HO ı...OH ı ı OH
H
PHLORETIN
0
(Oı
0
FLAVONOL
0
FLAVANONOL
FL AVAN
0
0
ı‘#{176}iııi(ır
HO -Jıı
,O-CH2CH2 -OH
OH-H2C.H2C- O ı
o.cH2cH2-OM
I O-C12H50
OH 0 TROXERUTIN
CHALCONE
Downloaded from
www.ajcn.org by on May 7, 2009
LYMPHEDEMA AND VITAMINS 189
normal animal
and human food, substances,
small amounts of which are maintaining
blood
capillary fragility and permeability
at normal levels. If such is the
case-and
such actually it is-these substances are
vitamin P substances .
. . .“
In the present series, the extent of lymphedema
was significantly
increased by feeding
the Sherman et al. diet, supplemented (in
spite of
the rat’s ability to synthesize this
vitamin) with ascorbic acid. This fact
can
readily be explained, as in the case of Bcomplex
avitaminosis, by an
increased protein
permeability of the blood capillaries.
Again, there
arises the possibility that permeability
of lymph vessels may be
increased
too.
The therapeutic effect of B-complex vitamins
and of
vitamin P (coumarin) treatment
in animals fed the usual laboratory
diet
(group 1) enriched in various vitamins may
have the following
explanation:
The treatment may 1) prevent the normal
leakage of plasma
protein molecules through
blood capillaries; 2) it may cause
collateral
lymphatics to open more rapidly; and 3)
it may increase the
catabolism of plasma proteins
by connective tissue cells. From what
is
known of the passage of protein molecules
through continuous,
nonfenestrated blood
capillaries, it would seem that the first
possibility
is highly unlikely. The second possibility
is unlikely too.
We feel the last theory
to be the correct answer.
Two questions arise in
connection with
the therapeutic trials performed in rats suffering
from
B-complex avitaminosis. a) The
ineffectiveness of the vitamin B
complex
therapy is a rather unexpected finding. As a
rule, the
consequences of any avitaminosis
are swept away by the administration of
the
missing vitamin. But, as bone destruction
brought about by mechanical
strain in vitamin
D avitaminosis cannot be cured by the
administration of
vitamin D, so we assume
that vascular and/or cellular lesions
brought
about by B avitaminosis of long duration are,
in combination with
lymphatic blockage, too
serious as to be influenced by the
vitamin
therapy in the manner and the dosage employed
in our experiments.
b) The effectiveness
of vitamin P (only the protective effect
of coumarin
was biologically and statistically
significant) therapy in lymphedema of
rats
suffering of B-complex avitaminosis is a surprising
finding, but an
analogous result has
already been described by F#{246}ldiand
Zolt#{225}n
(14). The authors induced a combined pyridoxine-
pantothenic
acid deficiency in the rat
by the administration of the two
specific
antivitamins, deoxypyndoxine and
omegamethylpantothenate.
Specific deterioriation
of the function of the
central nervous system
could be effectively prevented by coumarin.
To
quote another well-known fact, consequences
of A-avitaminosis can be
treated
effectively by the administration of vitamin
C. The most
effective therapeutic agent
against lymphedema aggravated by the
administration
of the Sherman-LaMer-Campbell
diet was coumarin.
We
are, of course, aware of the fact that
these results can be interpreted by
the
assumption of some unidentified pharmacological
effect of the
vitamins used in our
experiments. Dosage-response curves,
and
autoradiographic histology could bring the
definite answer to this
question. The fact that
various vitamins exert a therapeutic effect
in
lymphedema and that a deficiency in various
vitamins aggravate it
cannot be regarded as
an argument in favor of a specific
interrelationship
between vitamins and lymphedema.
Axeirod ( 1 5) has
described quite similar
relationships between vitamins and
immunologic
responses. Coumarin has been found
to be effective in the
treatment of some types
of lymphedema in the human being too (J.
S.
Calnan and J. J. Pflug, personal cornmunication).
Based on these studies, we
propose the
hypothesis that in the etiopathornechanism
of some of those
gigantic lymphedemas leading
to massive destruction in tropical
countries,
dietetic factors may play an aggravating
role but, we are, of
course, aware of the
fact that the present work was perfornied in
the rat
and, at the present state of our
knowledge, there is no evidence that
the
described mechanism exists in man.
Summary
Both B-complex
avitaminosis and P avitaminosis
aggravate lymphedema in the rat.
A member
of the vitamin P family, coumarin
Downloaded from www.ajcn.org by on May 7,
2009
190 F#{212}LDI-BORCSOK AND FOLD!
(devoid of any antithrombotic
activity)
proved to be a highly active therapeutic agent
against both
forms of avitaminosis-aggravated
lymphedemas. ı
References
1.
B#{246}RCS#{246E}K.,, K. F#{246}LDI, 0. WITTLINGER AND
M. FoLD!. Zur
therapeutischen Beeinflussung
des akuten experimentellen
lymphostatischen
Odems mit Vitaminen, vitaminartigen Naturstoffen
sowie
mittels Massage. Angiologica 8:
31, 1971.
2. CASLEY-SMITH, J. R., M.
FOLD! AND O. T.
ZOLTAN. The treatment of acute lymphedema
with
pantothenic acid and pynidoxine: an electron
microscopical investigation.
Lympliology
2: 63, 1969.
3. FOLDI-BORCSOK, E., J. R. CASLEY-SMITH
AND
M. FOLD!. The treatment of experimental
lymphedema. Angiologica 9:
92, 1972.
4. SHERMAN, H. C., V. K. LAMER AND H. L.
CAMPBELL. Quantitative
determination of the
antiscorbutic vitamin (vitamin C). I. Am.
Chem. Soc.
44: 165, 1922.
5. FOLD!, M. Diseases of Lymphatics and Lymph
Circulation.
Springfield, Illinois: Thomas, 1969.
6. QUADBECK, G., H. R. LANDMANN, W.
SACHSSEE
AND I. SCHMIDT. Der Einfluss von Pyrithioxin
auf die
Blut-Hirnschranke. Med. Exptl. 7: 144,
1962.
7. JANCSO, M. Speicherung.
Budapest: Akademieverlag,
1955.
8. MANC!NI, R. E. Connective tissue and
serum
proteins. Intern. Rev. Cytol. 14: 193, 1963.
9. BENTSATH, A., ST.
RUSZNYA.K AND A. SZENTGYORGYI.
Vitamin nature of flavonoids. Nature
138:
798, 1936.
10. JAVILLIER, M., AND J. LAVOLLAY. Les substances
agissant
sun las resistance et la permeabilit#{233}
capillaires et la notion de
vitamin P. Helv.
Chim. Ada 29: 1283, 1946.
11. BENKO, S., M. GABOR, T.
VARKONY!, A. ANTAL
AND M. FOLD!. Brain edema and subpleural
hemorrhage in
experimental P-avitaminosis.
Physiol. Chem. Phys. 2: 110, 1970.
12.
VARKONYI, T., A. ANTAL, M. GABOR AND S.
BENKO. Investigation of the
blood-brain barrier
permeability in experimental P-avitaminosis.
13.
CLARK, W. G., AND E. M. MACKAY. The
absorption and excretion of rutin and
related
flavonoid substances. J. Am. Med. Assoc. 143:
1411, 1950.
14.
FOLD!, M., AND 0. T. ZOLTAN. Die Wirkung
eines Mangels an
Pantothens#{228}ure und Pyridoxin
auf die Funktion des
Zentralnervensystems
und dessen Beeinflussung durch Cumanin.
Drug Res.
20: 1618, 1970.
15. AXELROD, A. E. Immune processes in vitamin
deficiency
states. Am. J. Clin. Nutr. 24: 265,
1971.
Downloaded from www.ajcn.org
by
on May 7, 2009
The
American Journal of Clinical Nutrition 26: FEBRUARY 1973, pp. 185-190. Printed
in U.S.A. 185
Ethel F#{246}ldi-8#{246}rcs#{a2n4d6}k M. F#{246}ldi
In 1 97
1, the unexpected fact has been
described (1) that surgically induced
acute
experimental lymphedema in the rat flourished
with the usual
laboratory diet rich in
vitamins, can be treated successfully by
the
administration of various vitamins, e.g., pyndoxine,
pantothenic
acid, and particularly, a
highly active representative of the vitamin
P
family,2 coumarin (5 , 6-benzo-a-pyron).
These data, obtained by means of
plethysmographic
assessment of the volume of
lymphedema, were confirmed
by histology
(2, 3).
Based on these results, the question has
to be
raised whether an inadequate supplying
of the organism with vitamins would
aggravate
lymphedema. After having obtained an
affirmative answer to this
question, therapeutic
trials were performed in
avitaminotic
lymphedematous animals.
Material and methods
Experiments
were performed in 150 male Wistar
rats (body weights given in Fig. 1). The
animals
were divided into three groups.
Group 1
Group 1 comprised rats
fed ad libitum an artificial
diet rich in vitamins (Table 1). On the
56th
day, these animals were divided into five subgroups.
Subgroup 1.1 (n
= 10). From the 56th to the
63rd day, the rats were given daily ip
injections of
saline, 10 mI/kg body wt. On the 60th day, the
rats were
anesthetized with nembutal (50 mg/kg
ip). Preoperative volume of the neck
and head was
measured by electronic plethysmography by means
of the
apparatus constructed by Bundschuh (Griesheim,
W. Germany). This was
followed by a
radical cervical lymphatic blockage. From a
midline
incision reaching from the mandibula to the
sternum, lymph nodes
were carefully prepared and
ligated. Plethysmographic measurements were
repeated
on the 63rd day.
Subgroup 1.2 (n = 10). In this subgroup,
the
procedure was the same as in subgroup 1.1, with
just one difference.
Instead of saline, the rats were
treated with the following vitamins in
milligrams
per kilogram body weight: vitamin B1, 40; lactoflavin,
23;
niamid, 160; pyridoxine, 16; pantothenic
acid, 240; biotin, 2 and
cyanocobalamin, 80 pg/kg
body weight.
Subgroup 1.3 (n 10). Procedure was
the same
as in subgroups 1.1 and 1.2 with one difference; i.e.,
the
animals were treated with 25 mg/kg coumarin
(5 ,
6-benzo-alpha-pyron).
Subgroup 1.4 (,z 10). Procedure differed from
that
used in subgroups 1.1, 1.2, and 1.3 in one
respect; the rats were treated
with 500 mg/kg tnhydroxy-
ethyl-rutin.
Subgroup 1.5 (n 10). In this
subgroup, the
same procedure was followed as with subgroup 1.1
except
that instead of a cervical lymphatic blockage,
only a sham operation was
performed.
Group 2
Rats were fed a diet (ad libitum) that was poor
in
B-complex vitamins (Table 1). On the 35th day,
the rats were divided into
five subgroups. (This day
was chosen instead of the 56th because
vitamin
B deficiency was already marked and the state of
the rats
deteriorated rapidly.)
Subgroup 2.1 (n 10). From the 35th to the
42nd
day, the rats were given daily ip injections of
saline. On the 39th day,
they were treated as those
in subgroup 1 . 1 . Plethysmographic
measurements
were repeated on the 42nd day.
Subgroup 2.2 (n 10). In this
subgroup, procedure
was the same as in subgroup 2.1, with one
exception.
Instead of saline, the rats were treated
with the same vitamins as those in
subgroup 1.2.
Subgroup 2.3 (n 10). The rats in this group
were treated
with coumarin; otherwise the procedure
was the same as in subgroup 2.1 and
2.2.
Subgroup 2.4 (n = 10). Our procedure differed
from that used in
subgroups 2.1, 2.2, and 2.3 in
one respect, these rats were treated with
trihydroxyethyl-
rutin.
Subgroup 2.5 (n = 10). In this subgroup,
the
procedure was the same as in subgroup 2.1 except
that instead of a
lymphatic blockage, only a sham
operation was performed.
Group 3
Rats
in this group were fed the Sherman-LaMer-
Campbell diet (4), free of vitamin
P but supplemented
with ascorbic acid (Table 1). This group
1 From the
Lymphological Research Laboratory,
Schaper and Br#{252}mmer,
Salzgitter-Ringelheim, West
Germany.
2 The controversial views concerning
vitamin P
are handled in the Discussion.
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on May 7, 2009
300. -
200.
100.
0 ‘ı 2 3 14 5 6 7 8
FIG. 1.
Changes of body weight in the three groups.
186
F#{212}LDI-B#{246}RCS#{246A}KND FOLDI
E_Eı
ı ı
I weeks ı
also was
fed ad libitum. On the 56th day, the rats
were divided into five
subgroups.
Subgroup 3.1 (n = 10). From the 56th to the
63rd day, the rats
were given saline. On the 60th
day, they were handled as those belonging to
subgroup
I . 1 . Plethysmographic measurements were
repeated on the 63rd
day.
Subgroup 3.2 (,i = 10). In this subgroup, procedure
was the same as
in subgroup 3.1 but instead of
saline, the rats were treated with the same
vitamins
as those in subgroup 1.2.
Subgroup 3.3 (n 10). These rats were
given
coumanin in addition to the treatment received by
subgroups 3.1 and
3.2.
Subgroup 3.4 (,z 10). The procedure for these
rats differed from
that used in subgroups 3.1, 3.2,
and 3.3 in one respect: they were treated
with tnhydroxy-
ethyl-rutin.
Subgroup 3.5 (ii = 10). With this subgroup
also,
a sham operation was performed instead of
lymphatic blockage.
Otherwise, the procedure was
the same as with subgroup 3.1.
Within the
three groups, volumes of lymphedemas
were expressed as the difference
between
the postoperative (V2) and the preoperative (V1)
TABLE
1
volumes. In order to compare data of the three
groups with each other,
it was necessary to eliminate
the differences existing in the V1 values.
Therefore,
volumes of lymphedemas (L) were expressed
with the
formula:
\.ı - \rl
L:=100ı
Statistical analysis was performed by
appropriate
forms of Student’s t test.
Results
The extent of
lymphedema was significantly
higher in both avitaminotic,
salinetreated
subgroups (subgroup 2. 1 and 3.1)
than in subgroup 1.1
(normal diet) (Figs.
2, 3). In group 2, hair loss, rhagades, and
a
markedly diminished growth rate could be
observed (Fig. 1). There were
no such signs
in group 3.
A statistically significant protective
effect
Composition of diets used in group I to LII
Group I (Altromin ı
1,000) Group II (Altromin C 1,000 without Group III
(ShermanıLaMerıCampbelI)C
B vitamins)
Casein
Rice starch-flour
Soja
oil
Cellulose
powder
Minerals
Vitamins#{176}
Percent
22
64
2
4
6
2
Casein
Rice
starch-flour
Soja oil
Cellulose
powder
Minerals
Vitaminsb
I
Percent
22 Butter
64 Milk
powder
2 Yeast powder
4 I
NaCI
6
2
Percent
10
30
59
1
a Vitamins: DL-methionine; 1,200 mg; vitamin A, 15,000 lU; vitamin Dı, 500 IU; vitamin E, 150 mg;
vitamin K,, 10 mg; vitamin B1 . HCI, 20 mg; vitamin B2, 20 mg; vitamin
B3. HCI, 15 mg; vitamin B12, 30 ı.
tg;
pantothenic acid, 50 mg;
nicotinic acid, 50 mg; cholinchlorid, I ,000 mg; folic acid, 10 mg; biotin,
200,ug;
inositol, 100 mg; p-aminobenzoic acid, 100 mg; and ascorbic acid, 20
mg.
b Vitamins: vitamin A, 15,000 LU; vitamin Dı, 500 IU; vitamin E, 150 IU;
vitamin K3, 10 mg; and
ascorbic acid, 20 mg.
The mixture was heated for 1
hr at 100 C. After cooling down, 100 mg ascorbic acid was added to
each 100
g of the dry food. Daily consumption of food/rat 30 g. Daily consumption of
vitamin C/rat
- 25 mg.
Downloaded from www.ajcn.org by on May 7,
2009
Prot.ct#{233}Cı,f ,f.ct
.5
LYMPHEDEMA AND VITAMINS 187
FIG. 4.
Protective effects of various treatments.
BVK = vitamin B complex.
21i
terized by a generalized increase in blood
capillary permeability. In
vitamin B6 deficiency,
brought about by the administration
of
desoxypyridoxine, even the permeability
20 of the blood-brain barrier is
increased (6). In
group 2 of these series, a B-complex avitaminosis
has
been induced; its effect on the
blood capillaries can certainly be
expected
15. to be even more pronounced. With plasma
proteins leaking out
of the blood capillaries,
lymphatic blockage will, of course, lead to
a
more extensive lymphedema than in normal
ıo. animals with a
physiological degree of protein
permeability.
On the other hand,
permeability of the
Fio. 2. Extent of lymphedema (percent volume
increase
after lymphatic blockage); 1) in rats fed a
normal diet (subgroup 1.1); 2)
in rats fed the Sherman-
LaMer-Campbell diet (subgroup 2.1); 3) in
rats
with a vitamin B complex deficiency (subgroup
3.1).
could be achieved
against lymphedema in
rats fed the normal diet by coumarin and by
vitamin
B complex treatment; against lymphedema
aggravated by B-avitaminosis, by
FIG. 3. Lymphedema in a rat fed a normal diet
coumarin treatment, and in
P-avitaminosis (subgroup 1.1; left) and in a rat in a vitamin Pby
coumarin
treatment (Fig. 4). deficiency state (subgroup 3.1;
right).
Discussion
After lymphatic blockage, lymph will
accumulate in
the occluded lymphatics. Stagnating
lymph, however, will dilate them.
The
high intralymphatic pressure will induce
“mural insufficiency” (5),
i.e., induce the
diffusion of lymph through the wall of lymph
vessels,
and will force open interendothelial
junctions in initial lymphatics; here
too,
lymph will escape back into the tissues. At
the same time, plasma
protein molecules,
steadily escaping from the blood capillaries,
will
accumulate in the interstice. They are,
normally, removed by lymphatics.
Lymphedema,
finally, will be the result of raised
oncotic pressure in the
interstitial fluid.
It is well known that beriberi is
characDownloaded
from www.ajcn.org by on May 7, 2009
188 FOLDI-BORCSOK
AND FıLDI
FIG. 5. Vitamin P family.
lymph vessels may eventually be
increased
by B-complex avitaminosis leading to a mural
insufficiency of
lymph drainage, a hypothesis
awaiting elucidation.
Normal connective
tissue cells incorporate
and digest plasma proteins; in this way,
they
become colloidosmotically inactive (7, 8).
In B-complex avitaminosis
quite a number
of the most important mitochondrial
enzyme systems are
severely damaged. In
lymphedema of normally fed animals, the
extent of
lymphedema may be limited to an
appreciable extent through this
mechanism.
The possibility that plasma protein catabolism
in these
connective tissue cells
could be subnormal or absent may well be
a
further factor meriting additional studies.
The matter of vitamin P has
been heavily
debated since 1936, when Szent-Gy#{246}rgyi (9)
described
the fact that experimental scurvy
is caused not only by a deficiency in
ascorbic
acid (discovered by the same author) but
also by a concomitant
deficiency in vitamin
P. Based on the original studies of
Szent-
Gy#{246}rgyi and on those of Javillier and Lavollay
(10), we are
now aware of the fact that
vitamin P is not just one chemical
compound,
as e.g., vitamin B1. It is rather similar to
vitamin K, which
is not a single molecule
either. Just as various molecules
possess
vitamin K activity, other chemically related
compounds possess
qualitatively, but not
quantitatively, the same action on
blood
capillaries; if they are entirely absent from
the food, blood
capillary permeability and
fragility will increase (Fig. 5). The
administration
of the diet described by Sherman,
LaMer and Campbell (4),
which is free of
vitamin P and vitamin C, produces an increase
in blood
capillary fragility and permeability
not only in the guinea pig, but
in
the rat, which is also able to synthesize
ascorbic acid. It has been
shown by Benk#{246}
et al. (11) that in rats fed this diet,
cerebral
edema will appear. V#{225}rkonyi and co-workers
(12) have shown
that cerebral edema is
caused by an increased permeability of
the
blood-brain barrier, as indicated by the
Evans-blue fluorescence
method.
In sharp contrast to Clark and MacKay
(13) who consider “many of
the physiologic
and pharmacologic nonspecific effects which
they (i.e.,
members of the vitamin P family)
are said to exert” as an “alarm
reaction,”
we share the arguments of Javillier and
Lavolley (10): “Any
organic substance,
which is contained in natural food and is
responsible
for the maintenance of a physiologic
equilibrium is a vitamin. The
question
to be answered is, whether there exist, in
the
OH
ı__%
Ct$.O.P,yd?OmyCffVfeıf#{232}C octd
(CoumOrH’ıc
eOd)
FLAVONE
FLAVANONE
COrO
ANTHOCYANtDIN
(f$S.yIsuıı’
soft
ıOO
1ı ı COUMARIN w
toctons of Do coumorWuc aced
or U binzo-
-pyrofı
HO ı...OH ı ı OH
H
PHLORETIN
0
(Oı
0
FLAVONOL
0
FLAVANONOL
FL AVAN
0
0
ı‘#{176}iııi(ır
HO -Jıı
,O-CH2CH2 -OH
OH-H2C.H2C- O ı
o.cH2cH2-OM
I O-C12H50
OH 0 TROXERUTIN
CHALCONE
Downloaded from
www.ajcn.org by on May 7, 2009
LYMPHEDEMA AND VITAMINS 189
normal animal
and human food, substances,
small amounts of which are maintaining
blood
capillary fragility and permeability
at normal levels. If such is the
case-and
such actually it is-these substances are
vitamin P substances .
. . .“
In the present series, the extent of lymphedema
was significantly
increased by feeding
the Sherman et al. diet, supplemented (in
spite of
the rat’s ability to synthesize this
vitamin) with ascorbic acid. This fact
can
readily be explained, as in the case of Bcomplex
avitaminosis, by an
increased protein
permeability of the blood capillaries.
Again, there
arises the possibility that permeability
of lymph vessels may be
increased
too.
The therapeutic effect of B-complex vitamins
and of
vitamin P (coumarin) treatment
in animals fed the usual laboratory
diet
(group 1) enriched in various vitamins may
have the following
explanation:
The treatment may 1) prevent the normal
leakage of plasma
protein molecules through
blood capillaries; 2) it may cause
collateral
lymphatics to open more rapidly; and 3)
it may increase the
catabolism of plasma proteins
by connective tissue cells. From what
is
known of the passage of protein molecules
through continuous,
nonfenestrated blood
capillaries, it would seem that the first
possibility
is highly unlikely. The second possibility
is unlikely too.
We feel the last theory
to be the correct answer.
Two questions arise in
connection with
the therapeutic trials performed in rats suffering
from
B-complex avitaminosis. a) The
ineffectiveness of the vitamin B
complex
therapy is a rather unexpected finding. As a
rule, the
consequences of any avitaminosis
are swept away by the administration of
the
missing vitamin. But, as bone destruction
brought about by mechanical
strain in vitamin
D avitaminosis cannot be cured by the
administration of
vitamin D, so we assume
that vascular and/or cellular lesions
brought
about by B avitaminosis of long duration are,
in combination with
lymphatic blockage, too
serious as to be influenced by the
vitamin
therapy in the manner and the dosage employed
in our experiments.
b) The effectiveness
of vitamin P (only the protective effect
of coumarin
was biologically and statistically
significant) therapy in lymphedema of
rats
suffering of B-complex avitaminosis is a surprising
finding, but an
analogous result has
already been described by F#{246}ldiand
Zolt#{225}n
(14). The authors induced a combined pyridoxine-
pantothenic
acid deficiency in the rat
by the administration of the two
specific
antivitamins, deoxypyndoxine and
omegamethylpantothenate.
Specific deterioriation
of the function of the
central nervous system
could be effectively prevented by coumarin.
To
quote another well-known fact, consequences
of A-avitaminosis can be
treated
effectively by the administration of vitamin
C. The most
effective therapeutic agent
against lymphedema aggravated by the
administration
of the Sherman-LaMer-Campbell
diet was coumarin.
We
are, of course, aware of the fact that
these results can be interpreted by
the
assumption of some unidentified pharmacological
effect of the
vitamins used in our
experiments. Dosage-response curves,
and
autoradiographic histology could bring the
definite answer to this
question. The fact that
various vitamins exert a therapeutic effect
in
lymphedema and that a deficiency in various
vitamins aggravate it
cannot be regarded as
an argument in favor of a specific
interrelationship
between vitamins and lymphedema.
Axeirod ( 1 5) has
described quite similar
relationships between vitamins and
immunologic
responses. Coumarin has been found
to be effective in the
treatment of some types
of lymphedema in the human being too (J.
S.
Calnan and J. J. Pflug, personal cornmunication).
Based on these studies, we
propose the
hypothesis that in the etiopathornechanism
of some of those
gigantic lymphedemas leading
to massive destruction in tropical
countries,
dietetic factors may play an aggravating
role but, we are, of
course, aware of the
fact that the present work was perfornied in
the rat
and, at the present state of our
knowledge, there is no evidence that
the
described mechanism exists in man.
Summary
Both B-complex
avitaminosis and P avitaminosis
aggravate lymphedema in the rat.
A member
of the vitamin P family, coumarin
Downloaded from www.ajcn.org by on May 7,
2009
190 F#{212}LDI-BORCSOK AND FOLD!
(devoid of any antithrombotic
activity)
proved to be a highly active therapeutic agent
against both
forms of avitaminosis-aggravated
lymphedemas. ı
References
1.
B#{246}RCS#{246E}K.,, K. F#{246}LDI, 0. WITTLINGER AND
M. FoLD!. Zur
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lymphostatischen
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31, 1971.
2. CASLEY-SMITH, J. R., M.
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VARKONYI, T., A. ANTAL, M. GABOR AND S.
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