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Bioassay of a hemostatic factor from peanuts
ARCHIVES
OF
BIOCHEMISTRY
AND
Bioassay
89, 27&280 (1960)
BIOPHYSICS
of a Hemostatic
H. B. BOUDREAUX, From the Department
Factor
R. M. BOUDREAUX of Zoology,
Louisiana
State
from
Peanuts
AND MARTHA University,
Baton
BRANDON
Rouge,
Louisiana
AND
VERNON From the Southern
L. FRAMPTON Regional
Research
Received
AND LOUISE Laboratory,1
February
New
S. LEE
Orleans,
Louisiana
8, 1960
Extreme vasoconstriction is induced on the transection of arteries of the cheek pouch of bilaterally adrenalectomized male hamsters that have ingested an absolute alcohol extract of defatted peanuts. This is compared with slight or moderate vasoconstriction observed with control bilaterally adrenalectomized male hamsters. Apparently the vasoconstrictor from peanuts is released from storage in the blood platelets during their viscous metamorphosis in the formation of the platelet plug. The active factor is also myotonic to smooth muscles. INTRODUCTION
The existence of a hemostatic factor in peanuts is indicated in the report by Boudreaux and Frampton (1) of remissions of clinical symptoms of hemophilia in several AHF hemophiliacs when they consumed peanut products during hemophilia attacks. Additional evidence for the presence of a hemostatic factor in peanuts has developed in the course of a search for a bioassay for the peanut constituent which is responsible for this apparent hemostasis. METHODS
AND
RESULTS
METHODS The preliminary studies of the effects of ingested peanut products on blood vessels indicated that use could be made of the cheek pouch of the golden hamster [Microcricetus aura& (Waterhouse)] in an investigation of hemostasis induced by peanut products. The cheek pouch can be exposed and the effects of severing blood vessels in the pouch membrane may be observed under the 1 One of the Laboratories of the Southern zation Research and Development Division, cultural Research Service, U. S. Department Agriculture.
UtiliAgriof
microscope. Male golden hamsters of the Louisiana State University strain were used. The animals were anesthetized with Nembutal for observations, and laid on a paraffined balsa wood platform, one end of which was hollowed out to form a shallow moat. A half-inch square Plexiglass window was built into the bottom of the moat [a modification of the technique of Lutz et al. (2)]. This permitted the transillumination of the exposed cheek pouch. The head of the hamster under study was laid on the edge of the depressed portion of the platform, and the exposed cheek pouch was spread out over the window and anchored to the bottom of the moat where the exposed cheek pouch could be washed with Ringer’s solution while observations were made on the blood vessels with the aid of a stereoscopic microscope. Most of the investigations reported here were carried out with an extract prepared from lightly roasted peanuts. The skin-free cotyledons and germs were flaked. The oil was extracted from the flaked material with commercial hexane. The resulting meal (moisture content of about 10%) was then exhaustively extracted with 95% ethyl alcohol at room temperature. The residue was discarded. The alcohol content was reduced by evaporation at room temperature and low-temperature vacuum distillation. The final drying of the extract was by lyophilization. This powder, which
276
HEMOSTATIC represented about 3.5% of the whole peanuts, is referred to in this paper as the primary extract. Where the primary extract was used, it was dissolved in water (100 mg./ml.) and administered orally with the aid of a blunted No. 18 hypodermic needle and syringe at the rate of 50 mg./day/lOO g. body a-eight in three doses 8 hr. apart. The hamsters were held vertically and drank from the tip of the needle. Vessel bleeding time for one pouch of each of the control and experimental animals was determined after 4-5 days on the primary extract regimen. The feeding of the groups was then reversed so that, whenever possible, each animal served as his own control, and the bleeding time of the vessels in the opposite cheek pouches was determined. Approximately ten transections were made at random in each pouch. The reduction in the bleeding time, as measured in these experiments, was too variable for usefulness in a bioassay of the hemostatic factor. There was observed, however, an enhanced vasoconstriction at the site of the transection of the blood vessels in the animals receiving the primary extract. This enhanced vasoconstriction at the site of the transection in the primary extract-fed animals may have been partly masked by the effect of 5.hydroxytryptamine, since this myotonic amine is reported to be released from platelets during the formation of the platelet plug (3) and to induce vasoconstriction of the transected vessel. Accordingly, hamsters were bilaterally adrenalectomized, since adrenalectomy markedly reduces the 5.hydroxytryptamine content of the platelets (4). One group of the adrenalectomized hamsters received regular laboratory chow, while the other received an aqueous solution of the primary extract (at a rate of 75 mg./day/lOO g. body weight) for 2 days in addition to laboratory chow. One cheek pouch from each was exposed, and the main artery was transected in each successively toward the base. Note was taken of the degree of vasoconstriction during and following the formation of the platelet plug at the end of the severed artery. The feeding of the two groups was then reversed so that, whenever possible, each animal served as his own control. RESULTS Data recorded in Table I for 130 vasoconstriction responses show that vasoconstriction was much more pronounced in the animals receiving the primary extract. The symbol (-), in this table and in t’he succeeding t,able and in the figures, indicates no
FACTOR
277
vasoconstriction response of a transection after the establishment of the platelet plug; (i-) indicates a mild vasoconstriction; (+ +) indicates a moderate vasoconstriction; (t + + ) indicates an extreme vasoconstrict,ion. Under this last classificabion, the vasoconstrict’ion spread up the artery from the site of the cut. It was so strong that a segment of the cut, vessel almost disappeared from view. The same marked enhancement of the vasoconstriction occurred whether the administrat’ion of t’he primary extract preceded or followed the control period. Completely adrenalectomized hamsters died in 4-5 days. The hamsters surviving more than 7 days lived long enough for repeat experiments. The results with these animals were always the same. Those animals that survived more than 7 days were found, on autopsy, to have retained fragments of adrenal cortex. Subsequent adrenalectomies were performed with the intent of leaving a small bit of the cort’ex and of removing all of the medullary portion. The vasoconstriction response of these animals to the primary extract was excellent, as may be seen from the data presented in histogram form in Fig. 1. These data represent 130 transections in 16 cheek pouches. It was observed that the response to the primary extract was most marked between 1 and 5 hr. after t’he last administration of the concentrate. Experiments involving intraperitoneal injection of the primary extract were carried out with four partially adrenalectomized (demedullated) hamsters. Two inject,ions were given to each animal at t’he rate of 20 mg./lOO g. body weight 3 hr. apart. One cheek pouch from each was exposed under anesthesia l-3 hr. later, and the main cheek pouch artery was transected in the manner indicated above. The opposite cheek pouch was exposed and observed for vasoconstrict’ion 2 days later. Intraperitoneal injection produced the same effects as oral administration, as may be seen from the data for 32 transections, as recorded in the histogram in Fig. 2. Actually, the animals respond to single injections of 20 mg./lOO g. body weight, as may be seen from the data recorded in Table
278
BOUDREAUX,
BOUDREAUX,
OF
ORAL
ADMINISTRATION
AND
LEE
PRIMARY
OF
CONCENTRATE ON VASOCONSTRICTION SECTED CHEEK POGCH ARTERIES ECTOMIZED HAMSTERS Number
FRAMPTON
I
TABLE EFFECT
BRANDON,
of vasoconstriction
OF
IN TRANADRENAL-
responses
on
-
Animal No.
Treatment with 25 mg./ 1(IO g. per dose, 3 times pel r day. 2 days before examination
Days since 4d-XC
83b 83 84 856 85 86 87b 87 107 109 111 123 124 126 127 128 129 130 133 134" 134 135 136 137 138 139 141 142b 143
Degree of vasoconstrictiaP
-
6 28
-
-
0 0 0
3
0 -
0
10 9 -
7
78
El
CONTROL ANIMALS
-+t
::
6
78
6
0
-
2 18 6 12 -
2 2 -
-
2
DEGREE OF VASOCONSTRICTION
1
5 8 3
0 0 0
0 1 7 7 5 0
1. Effect of oral administration of the primary extract on vasoconstriction in transected cheek pouch arteries of partially adrenalectomized (demedullated) hamsters. (-) No vasoconstriction after establishment of the platelet plug; (+) mild local constriction at the site of the cut; (++) moderate vasoconstriction at the site of the cut; (+++) extreme vasoconstriction spreading from the site of the cut up the artery, and which occurs to a degree that a segment of the vessel, as viewed in the stereoscopic microscope, almost disappears. FIG.
-
3 3 2
-
-
-
-
0 0
9
5 3 7
6 9 3 3 3 3
0 2
-
-
-
0 0
-
-
3
8 7
8 7
3 3 3 1 4 3
1
-
-
3
-
0
6
3 2
9 3 6
-
Degree of vasoconstriction’
-
-
-
-
Sum of vasoconstriction responses.
7 8 -
-
-
7 1 6 3 2 6 0 10 -
0 0
3 3 3
-
4 4 4 7 8 4 9 2
Days since Ad-X
-I-
-
-
-
3 8
0 2 0 1 0 0 4 0
3 3 -
1 2
0 0 -
5 26 2 2 8 3 3 3 -
2
:t -+t
No treatment for at least 2 days before examination
1 5 4 3 5 9
8 3 0 0 0 1
-
-
5 5 0
7 5 5
72 -
92 -
1 0 5
19
-
a The symbol (-) represents no vasoconstriction after establishment of platelet plug; (+) represents mild local constriction at site of cut; (++) represents stronger constriction near site of cut; (+++) represents extreme constriction spreading from site of cut up the artery to the
II where two partially adrenalectomized hamsters were used. These data were obtained in the manner indicated immediately above; one cheek pouch was exposed and examined for vasoconstriction in 2-3 hr., while the other pouch was exposed for observation 2-3 days later. It was observed that the intraperitoneal administration of the primary extract produced a cramplike response, in which the animal would stretch and arch its back for a few seconds. point of almost disappearance of a segment of the vessel. 6 Survived long enough to be tested a second time; adrenalectomy not complete. c Adrenalectomy.
HEMOSTATIC
IOIOIO'OioiOIOw!OIO-
DEGREE OF VASOCONSTRICTION
FIG. 2. Effects
of intraperitoneal administration of the primary extract on the vasoconstriction in transected cheek pouch arteries of partially adrenalectomized (demedullated) hamsters. (-) No vasoconstriction after establishment of the platelet plug; (+) mild local constriction at the site of the cut; (++) moderate constriction; (+++) extreme constriction. TBBLE
II
RESULTS OF INTRAPERITONEAL INJECTION OF PEANUT CONCENTRATE ON THE VASOCONSTRICTION OF CUT ARTERIES IN CHEEK POUCHES OF PARTIALLY ADRENALECTOMIZED (DEMEDULLATED) HAMSTERS
279
FACTOR
intestinal cramp produced when the active material was administered intraperitoneally, it seemedpossible that the factor in the primary extract might be similar in action to 5-hydroxytryptamine and be myotonic for smooth muscles. Excised hamster duodenum muscle suspended in 75 ml. of Tyrode’s solution at 39°C. contracted strongly when 0.4 ml. of a 10% aqueous solution of the primary extract was added to the bath. A typical kymograph tracing of the muscle responseis shown in Fig. 3(c). Included also in Fig. 3 are the kymograph tracings obtained from successive responses of a single muscle preparation to 0.4 mg. 5-hydroxytryptamine (5)(b), to 0.5 mg. of the absolute alcohol extract of the primary extract (d), and to 40 mg. of t’he residue remaining after the primary extract was extracted with absolute alcohol (e). It is apparent that the active factor is removed quantitatively from the primary extract with absolute ethyl alcohol. DISCUSSION
It was indicat’ed in the report by Boudreaux and Frampton (1) that the hemostatic effect of peanut flour and of the primary extract on hemophiliacs is not becauseof any change in the clotting mechanism, but that the hemostasis must be related to blood vessel response,following trauma. While the evidence presented in this communication
-
Number
of vasoconstriction
responses
on
T
Intraperitoneal njection, 20 mg./lOC mg. per dose. Intensity of vasoconstriction
*i%?
Two or nvxe days
following injection. Intensity of vasoconstriction
-
_---
212
224
1.5 2.0
001 006
+
-
+++++
9 4
-
136 271
+
-0
TIME
+++++
0 0
In view of the very strong vasoconstriction induced in the cheek pouch arteries of the partially adrenalectomized animals by the primary extract, as well as the apparent
IN
L
SECONDS
FIG. 3. Kymograph tracings obtained with a hamster duodenum muscle preparation: (a) resting muscle; (b) contraction induced by 0.4 mg. of 5-hydroxytryptamine; (c) contraction induced by 0.04 g. of primary extract; (d) contraction induced by 0.005 g. of the absolute alcohol extract of the primary extract; (e) contraction induced by 40 mg. of the residue from the absolute alcohol extract of the primary extract.
280
BOUDREAUX,
BOUDREAUX,
BRANDON,
does not necessarily identify the vasoconstrictor-myotonic factor in peanuts with the hemostatic agent which seems to give relief in hemophilia, it is noted that preparations which give the responses reported in this paper also give relief from hemophilia symptoms. The impression gained from the observations made in the course of this study is that the peanut factor has a physiological behavior similar to that of 5-hydroxytryptamine in being bound in the platelets and released from them, during the viscous metamorphosis of the platelets in the construction of the platelet plug, to cause the vasoconstriction.
FRAMPTON
AND
LEE
ACKNOWLEDGMENTS Support for that part of the research conducted at Louisiana State University was received from the New Orleans Chapter of the National Hemophilia Foundation and from the Graduate School of the University. The interest and helpful suggestions offered by Dr. B. Jackson are gratefully acknowledged. REFERENCES
1. BOUDREAUX, H.B.,
AND FRAMPTON, V.L.,Na186, 469 (1960). 2. LUTZ, R.B., FULTON,G. P., AND AKERS, R.P., Exptl. Med. Surg. 8, 258 (1950). 3. ZUCKER, M. B., AND BORRELLI, J., J. Appl. Physiol. 7, 432 (1955). 4. DEMAIO, D., Science 129, 1678 (1959). ture
OF
BIOCHEMISTRY
AND
Bioassay
89, 27&280 (1960)
BIOPHYSICS
of a Hemostatic
H. B. BOUDREAUX, From the Department
Factor
R. M. BOUDREAUX of Zoology,
Louisiana
State
from
Peanuts
AND MARTHA University,
Baton
BRANDON
Rouge,
Louisiana
AND
VERNON From the Southern
L. FRAMPTON Regional
Research
Received
AND LOUISE Laboratory,1
February
New
S. LEE
Orleans,
Louisiana
8, 1960
Extreme vasoconstriction is induced on the transection of arteries of the cheek pouch of bilaterally adrenalectomized male hamsters that have ingested an absolute alcohol extract of defatted peanuts. This is compared with slight or moderate vasoconstriction observed with control bilaterally adrenalectomized male hamsters. Apparently the vasoconstrictor from peanuts is released from storage in the blood platelets during their viscous metamorphosis in the formation of the platelet plug. The active factor is also myotonic to smooth muscles. INTRODUCTION
The existence of a hemostatic factor in peanuts is indicated in the report by Boudreaux and Frampton (1) of remissions of clinical symptoms of hemophilia in several AHF hemophiliacs when they consumed peanut products during hemophilia attacks. Additional evidence for the presence of a hemostatic factor in peanuts has developed in the course of a search for a bioassay for the peanut constituent which is responsible for this apparent hemostasis. METHODS
AND
RESULTS
METHODS The preliminary studies of the effects of ingested peanut products on blood vessels indicated that use could be made of the cheek pouch of the golden hamster [Microcricetus aura& (Waterhouse)] in an investigation of hemostasis induced by peanut products. The cheek pouch can be exposed and the effects of severing blood vessels in the pouch membrane may be observed under the 1 One of the Laboratories of the Southern zation Research and Development Division, cultural Research Service, U. S. Department Agriculture.
UtiliAgriof
microscope. Male golden hamsters of the Louisiana State University strain were used. The animals were anesthetized with Nembutal for observations, and laid on a paraffined balsa wood platform, one end of which was hollowed out to form a shallow moat. A half-inch square Plexiglass window was built into the bottom of the moat [a modification of the technique of Lutz et al. (2)]. This permitted the transillumination of the exposed cheek pouch. The head of the hamster under study was laid on the edge of the depressed portion of the platform, and the exposed cheek pouch was spread out over the window and anchored to the bottom of the moat where the exposed cheek pouch could be washed with Ringer’s solution while observations were made on the blood vessels with the aid of a stereoscopic microscope. Most of the investigations reported here were carried out with an extract prepared from lightly roasted peanuts. The skin-free cotyledons and germs were flaked. The oil was extracted from the flaked material with commercial hexane. The resulting meal (moisture content of about 10%) was then exhaustively extracted with 95% ethyl alcohol at room temperature. The residue was discarded. The alcohol content was reduced by evaporation at room temperature and low-temperature vacuum distillation. The final drying of the extract was by lyophilization. This powder, which
276
HEMOSTATIC represented about 3.5% of the whole peanuts, is referred to in this paper as the primary extract. Where the primary extract was used, it was dissolved in water (100 mg./ml.) and administered orally with the aid of a blunted No. 18 hypodermic needle and syringe at the rate of 50 mg./day/lOO g. body a-eight in three doses 8 hr. apart. The hamsters were held vertically and drank from the tip of the needle. Vessel bleeding time for one pouch of each of the control and experimental animals was determined after 4-5 days on the primary extract regimen. The feeding of the groups was then reversed so that, whenever possible, each animal served as his own control, and the bleeding time of the vessels in the opposite cheek pouches was determined. Approximately ten transections were made at random in each pouch. The reduction in the bleeding time, as measured in these experiments, was too variable for usefulness in a bioassay of the hemostatic factor. There was observed, however, an enhanced vasoconstriction at the site of the transection of the blood vessels in the animals receiving the primary extract. This enhanced vasoconstriction at the site of the transection in the primary extract-fed animals may have been partly masked by the effect of 5.hydroxytryptamine, since this myotonic amine is reported to be released from platelets during the formation of the platelet plug (3) and to induce vasoconstriction of the transected vessel. Accordingly, hamsters were bilaterally adrenalectomized, since adrenalectomy markedly reduces the 5.hydroxytryptamine content of the platelets (4). One group of the adrenalectomized hamsters received regular laboratory chow, while the other received an aqueous solution of the primary extract (at a rate of 75 mg./day/lOO g. body weight) for 2 days in addition to laboratory chow. One cheek pouch from each was exposed, and the main artery was transected in each successively toward the base. Note was taken of the degree of vasoconstriction during and following the formation of the platelet plug at the end of the severed artery. The feeding of the two groups was then reversed so that, whenever possible, each animal served as his own control. RESULTS Data recorded in Table I for 130 vasoconstriction responses show that vasoconstriction was much more pronounced in the animals receiving the primary extract. The symbol (-), in this table and in t’he succeeding t,able and in the figures, indicates no
FACTOR
277
vasoconstriction response of a transection after the establishment of the platelet plug; (i-) indicates a mild vasoconstriction; (+ +) indicates a moderate vasoconstriction; (t + + ) indicates an extreme vasoconstrict,ion. Under this last classificabion, the vasoconstrict’ion spread up the artery from the site of the cut. It was so strong that a segment of the cut, vessel almost disappeared from view. The same marked enhancement of the vasoconstriction occurred whether the administrat’ion of t’he primary extract preceded or followed the control period. Completely adrenalectomized hamsters died in 4-5 days. The hamsters surviving more than 7 days lived long enough for repeat experiments. The results with these animals were always the same. Those animals that survived more than 7 days were found, on autopsy, to have retained fragments of adrenal cortex. Subsequent adrenalectomies were performed with the intent of leaving a small bit of the cort’ex and of removing all of the medullary portion. The vasoconstriction response of these animals to the primary extract was excellent, as may be seen from the data presented in histogram form in Fig. 1. These data represent 130 transections in 16 cheek pouches. It was observed that the response to the primary extract was most marked between 1 and 5 hr. after t’he last administration of the concentrate. Experiments involving intraperitoneal injection of the primary extract were carried out with four partially adrenalectomized (demedullated) hamsters. Two inject,ions were given to each animal at t’he rate of 20 mg./lOO g. body weight 3 hr. apart. One cheek pouch from each was exposed under anesthesia l-3 hr. later, and the main cheek pouch artery was transected in the manner indicated above. The opposite cheek pouch was exposed and observed for vasoconstrict’ion 2 days later. Intraperitoneal injection produced the same effects as oral administration, as may be seen from the data for 32 transections, as recorded in the histogram in Fig. 2. Actually, the animals respond to single injections of 20 mg./lOO g. body weight, as may be seen from the data recorded in Table
278
BOUDREAUX,
BOUDREAUX,
OF
ORAL
ADMINISTRATION
AND
LEE
PRIMARY
OF
CONCENTRATE ON VASOCONSTRICTION SECTED CHEEK POGCH ARTERIES ECTOMIZED HAMSTERS Number
FRAMPTON
I
TABLE EFFECT
BRANDON,
of vasoconstriction
OF
IN TRANADRENAL-
responses
on
-
Animal No.
Treatment with 25 mg./ 1(IO g. per dose, 3 times pel r day. 2 days before examination
Days since 4d-XC
83b 83 84 856 85 86 87b 87 107 109 111 123 124 126 127 128 129 130 133 134" 134 135 136 137 138 139 141 142b 143
Degree of vasoconstrictiaP
-
6 28
-
-
0 0 0
3
0 -
0
10 9 -
7
78
El
CONTROL ANIMALS
-+t
::
6
78
6
0
-
2 18 6 12 -
2 2 -
-
2
DEGREE OF VASOCONSTRICTION
1
5 8 3
0 0 0
0 1 7 7 5 0
1. Effect of oral administration of the primary extract on vasoconstriction in transected cheek pouch arteries of partially adrenalectomized (demedullated) hamsters. (-) No vasoconstriction after establishment of the platelet plug; (+) mild local constriction at the site of the cut; (++) moderate vasoconstriction at the site of the cut; (+++) extreme vasoconstriction spreading from the site of the cut up the artery, and which occurs to a degree that a segment of the vessel, as viewed in the stereoscopic microscope, almost disappears. FIG.
-
3 3 2
-
-
-
-
0 0
9
5 3 7
6 9 3 3 3 3
0 2
-
-
-
0 0
-
-
3
8 7
8 7
3 3 3 1 4 3
1
-
-
3
-
0
6
3 2
9 3 6
-
Degree of vasoconstriction’
-
-
-
-
Sum of vasoconstriction responses.
7 8 -
-
-
7 1 6 3 2 6 0 10 -
0 0
3 3 3
-
4 4 4 7 8 4 9 2
Days since Ad-X
-I-
-
-
-
3 8
0 2 0 1 0 0 4 0
3 3 -
1 2
0 0 -
5 26 2 2 8 3 3 3 -
2
:t -+t
No treatment for at least 2 days before examination
1 5 4 3 5 9
8 3 0 0 0 1
-
-
5 5 0
7 5 5
72 -
92 -
1 0 5
19
-
a The symbol (-) represents no vasoconstriction after establishment of platelet plug; (+) represents mild local constriction at site of cut; (++) represents stronger constriction near site of cut; (+++) represents extreme constriction spreading from site of cut up the artery to the
II where two partially adrenalectomized hamsters were used. These data were obtained in the manner indicated immediately above; one cheek pouch was exposed and examined for vasoconstriction in 2-3 hr., while the other pouch was exposed for observation 2-3 days later. It was observed that the intraperitoneal administration of the primary extract produced a cramplike response, in which the animal would stretch and arch its back for a few seconds. point of almost disappearance of a segment of the vessel. 6 Survived long enough to be tested a second time; adrenalectomy not complete. c Adrenalectomy.
HEMOSTATIC
IOIOIO'OioiOIOw!OIO-
DEGREE OF VASOCONSTRICTION
FIG. 2. Effects
of intraperitoneal administration of the primary extract on the vasoconstriction in transected cheek pouch arteries of partially adrenalectomized (demedullated) hamsters. (-) No vasoconstriction after establishment of the platelet plug; (+) mild local constriction at the site of the cut; (++) moderate constriction; (+++) extreme constriction. TBBLE
II
RESULTS OF INTRAPERITONEAL INJECTION OF PEANUT CONCENTRATE ON THE VASOCONSTRICTION OF CUT ARTERIES IN CHEEK POUCHES OF PARTIALLY ADRENALECTOMIZED (DEMEDULLATED) HAMSTERS
279
FACTOR
intestinal cramp produced when the active material was administered intraperitoneally, it seemedpossible that the factor in the primary extract might be similar in action to 5-hydroxytryptamine and be myotonic for smooth muscles. Excised hamster duodenum muscle suspended in 75 ml. of Tyrode’s solution at 39°C. contracted strongly when 0.4 ml. of a 10% aqueous solution of the primary extract was added to the bath. A typical kymograph tracing of the muscle responseis shown in Fig. 3(c). Included also in Fig. 3 are the kymograph tracings obtained from successive responses of a single muscle preparation to 0.4 mg. 5-hydroxytryptamine (5)(b), to 0.5 mg. of the absolute alcohol extract of the primary extract (d), and to 40 mg. of t’he residue remaining after the primary extract was extracted with absolute alcohol (e). It is apparent that the active factor is removed quantitatively from the primary extract with absolute ethyl alcohol. DISCUSSION
It was indicat’ed in the report by Boudreaux and Frampton (1) that the hemostatic effect of peanut flour and of the primary extract on hemophiliacs is not becauseof any change in the clotting mechanism, but that the hemostasis must be related to blood vessel response,following trauma. While the evidence presented in this communication
-
Number
of vasoconstriction
responses
on
T
Intraperitoneal njection, 20 mg./lOC mg. per dose. Intensity of vasoconstriction
*i%?
Two or nvxe days
following injection. Intensity of vasoconstriction
-
_---
212
224
1.5 2.0
001 006
+
-
+++++
9 4
-
136 271
+
-0
TIME
+++++
0 0
In view of the very strong vasoconstriction induced in the cheek pouch arteries of the partially adrenalectomized animals by the primary extract, as well as the apparent
IN
L
SECONDS
FIG. 3. Kymograph tracings obtained with a hamster duodenum muscle preparation: (a) resting muscle; (b) contraction induced by 0.4 mg. of 5-hydroxytryptamine; (c) contraction induced by 0.04 g. of primary extract; (d) contraction induced by 0.005 g. of the absolute alcohol extract of the primary extract; (e) contraction induced by 40 mg. of the residue from the absolute alcohol extract of the primary extract.
280
BOUDREAUX,
BOUDREAUX,
BRANDON,
does not necessarily identify the vasoconstrictor-myotonic factor in peanuts with the hemostatic agent which seems to give relief in hemophilia, it is noted that preparations which give the responses reported in this paper also give relief from hemophilia symptoms. The impression gained from the observations made in the course of this study is that the peanut factor has a physiological behavior similar to that of 5-hydroxytryptamine in being bound in the platelets and released from them, during the viscous metamorphosis of the platelets in the construction of the platelet plug, to cause the vasoconstriction.
FRAMPTON
AND
LEE
ACKNOWLEDGMENTS Support for that part of the research conducted at Louisiana State University was received from the New Orleans Chapter of the National Hemophilia Foundation and from the Graduate School of the University. The interest and helpful suggestions offered by Dr. B. Jackson are gratefully acknowledged. REFERENCES
1. BOUDREAUX, H.B.,
AND FRAMPTON, V.L.,Na186, 469 (1960). 2. LUTZ, R.B., FULTON,G. P., AND AKERS, R.P., Exptl. Med. Surg. 8, 258 (1950). 3. ZUCKER, M. B., AND BORRELLI, J., J. Appl. Physiol. 7, 432 (1955). 4. DEMAIO, D., Science 129, 1678 (1959). ture