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Thromboplastic Activity of Acetone-Dehydrated Chicken Brain Powder Extracts After Repeated Extraction and Dilution1
Thromboplastic Activity of Acetone-Dehydrated Chicken Brain Powder Extracts After Repeated Extraction and Dilution 1 YIN-SHAN SHUM AND PAUL GRIMINGER Department of Nutrition, Rutgers University, New Brunswick, New Jersey 08903 (Received for publication July 2, 1971)
POULTRY SCIENCE 51: 402-407,
A
CETONE-EXTRACTED brain powder from vitamin K-deficient chickens, when used as a source of thromboplastic activity in the one-stage prothrombin time determination, will promote slower prothrombin times with a given plasma than identically prepared brain powder from vitamin K-sufficient chicks (Griminger, 1963). This activity differential is due, at least in part, to the presence or absence of vitamin K-dependent clotting factors in the residual blood of the chicken brains used as a source of thromboplastin (Griminger et al., 1970). In the course of experimental work leading to this conclusion it was noted that a second saline suspension of certain acetone-extracted chicken brain powders had more thromboplastic activity than the first, suggesting the presence of inhibitors, and also that a certain amount of thromboplastic activity was present even after repeated extractions 1
Paper of the Journal Series, New Jersey Agricultural Experiment Station, New Brunswick. This project was supported, in part, by U.S.P.H.S., N.I.H. Grant HE-10997. A preliminary report was presented at the 1971 F.A.S.E.B, meetings (Fed. Proc. 30: 479).
1972
with saline. The existence of inhibitors in thromboplastic preparations had been suggested by Deutsch et al. (1964). These authors discarded the first saline extract of their brain powder thromboplastin preparation, claiming it to contain undesirable proteins which were apparently removed during the first suspension in saline. Hecht et al. (1957) have also commented on the existence of an inhibitor, sphingosine, which is a constituent of brain cerebrosides and sphingomyelin. This report illustrates the presence of the inhibitor (s) and shows the results of a study of the effect of various factors such as repeated extraction, dilution, and addition of plasma on the thromboplastic activity of chicken brain powder. MATERIALS AND METHODS
Vitamin K-deficient and normal chicks were obtained by feeding a vitamin K-free basal diet, or the same diet supplemented with 10 mg. vitamin Kj. (phylloquinone, as 1% beadlet)/kg. feed to female crossbred chicks (N. H. c? X Columbian $ ) to three weeks of age. The composition of the basal diet has been given previously (Griminger
402
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ABSTRACT The second saline extract (E2) of acetone-dehydrated chicken brain powder contained less than one fifth of protein and phosphorus, compared with the first extract (Ei). Thromboplastic activity (TPA) of E 2 from vitamin K-deficient donors was significantly higher than of Ei. Response with vitamin K-sufficient donors varied. Ei and Ei from normal chicks contained significantly more protein than those from vitamin K-deprived ones. P content differed significantly in Ei only. Differences in TPA could not be quantitatively attributed to differences in protein and P content. TPA of brain powder, repeatedly extrasted with saline, declined, as did protein and P content. The addition of normal chick plasma returned TPA of repeatedly extracted brain powders to level found in fresh samples. Extracting samples of equal quantity of brain powder with increasing volumes of saline yielded higher TPA than when saline extract from a single brain powder sample was diluted with saline to produce, in effect, the same dilution ratios.
403
BRAIN THROMBOPLASTIC ACTIVITY
RESULTS AND DISCUSSION When a given sample of brain powder from vitamin K-deficient donor birds was twice extracted with saline, the second extract had a higher thromboplastic (TP) activity than the first one (Table 1). There was a slight decrease in activity with brain powders from vitamin K-normal donor birds (Table 2). However, in results not shown here, there was either no change or a slight increase in TP activity when ABPs
TABLE 1.—Prothrombin times and protein and phosphorus content of the 1st and 2nd saline
extracts of acetone-dehydrated brain powders from vitamin K-free chicks
Lot
A B C D E F G H I
J
Average 1 2
Donor bird plasma prothrombin time
Prothrombin time with normal plasma (sec.)
Total P (meg.) ^
Total protein (mg.)2
Protein/P ratio
Range (sec.)
1
21
1
2
1
2
1
2
31.4-40.8 45.0-50.0 50.4-52.7 53.4-57.1 57.6-59.5 59.5-60.9 61.1-66.1 66.2-67.5 70.1-77.4 81.5-85.8
15.6 16.7 18.8 20.9 17.4 17.6 19.3 20.6 17.1 18.0
16.3 15.6 16.5 16.2 15.5 15.8 16.6 17.4 15.4 16.2
2.72 3.14 3.18 2.89 3.14 2.58 3.08 2.80 3.25 3.17 2.99
.54 .50 .48 .71 .52 .50 .52 .43 .50 .50 .52
173 163 169 166 166 161 179 136 177 178 167
26 27 35 24 26 26 26 21 25 25 26
15.7 19.2 18.8 17.5 19.0 16.0 17.2 20.6 18.4 17.9 18.0
20.9 18.6 13.8 28.9 19.6 18.8 19.9 20.9 19.9 20.0 19.8
1—first saline extract; 2—second saline extract. Total protein or P extracted from 25 mg. samples of brain powder with 2.5 ml. saline.
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from normal donors were subjected to a second saline extraction. Regardless of the change in TP activity, there was a great reduction in the amount of both the protein and P present in the second extract, as compared with the first one. These data strongly suggest that brain powder preparations contain an inhibitor or inhibitors, those from vitamin K-deprived birds apparently having higher concentrations than those from the normal ones. The inhibitor(s) seems to have a high degree of solubility in saline and thus can be readily removed after the first extraction, resulting in a higher TP activity in the second extract. There seemed to be slightly, but consistently, more protein and P in the normal brain powder extracts (i.e., saline extracts of brain powders from vitamin K-normal donors), than in those from vitamin K-deficient chicks. The difference in P concentration between the first saline extracts of ABP from normal and vitamin K-deficient birds was significant at the 0.05 level. The difference in protein content of the first saline extracts of the two types of ABP was highly significant (P < 0.01), while that of the second extracts was significant at the
et al., 1970) except that all feeds contained 5% refined corn oil and 0.02% antioxidant. The same paper also described the preparation of the acetone-dehydrated brain powder (ABP), the extraction (activation) of thromboplastically active material from the ABP, as well as the onestage prothrombin time determination. Analyses of phosphorus (as a measure of phospholipids) and of protein were carried out in some of the experiments. In the determination of phosphorus content, the method by Bartlett (1959) as modified by Dodge and Phillips (1967) was essentially followed, while that for protein content was based on the procedure of Lowry et al. (1951), as modified by Miller (1958).
404
YIN-SHAN SHUM AND P. GRIMINGER
TABLE 2.—Prothrombin times and protein and phosphorus content of the 1st and 2nd saline
extracts of acetone-dehydrated brain powders from normal chicks
Lot
A B C D E F Average 1 2
Donor bird plasma prothrombin time
Prothrombin time with normal plasma (sec.)
Total protein (mg.)2
Protein/P ratio
Total P (meg.)2
Range (sec.)
1
21
1
2
1
2
1
2
9.6-10.2 10.4-10.5 10.8-11.0 11.1-11.3 11.4-12.2 12.2-13.1
12.6 11.9 12.2 12.3 13.8 13.0
13.5 13.8 13.7 13.2 14.7 14.6
3.36 3.19 3.71 3.73 3.61 3.19 3.46
.56 .64 .67 .64 .56 .57 .61
189 171 180 182 175 180 179
29 28 25 30 25 26 27
17.8 18.7 20.6 20.5 20.6 17.7 19.3
19.3 22.5 26.5 21.6 22.7 21.7 22.3
1—first saline extract; 2—second saline extract. Total protein or P extracted from 25 mg. samples of brain powder with 2.5 ml. saline.
was concluded to contain a thromboplastic activity-enhancing moiety. This was confirmed by the finding that the one-time addition of normal plasma (equivalent of IS microliters of plasma per brain) to brain powders after 5 extractions restored their thromboplastic activity to a level comparable to that in fresh extracts (Figure 2). It is assumed that brain powders from normal as well as from vitamin K-deficient chickens contain a phospholipid-protein complex. It is possible that this complex is not identical in the two types of brain powder. This complex seems to be exhausted very slowly, as seen from the fact that even after 10 extractions it had sufficient thromboplastic activity to clot plasma within 30 seconds. Aside from this phospholipid-protein complex, one or more prothromboplastic factor(s), possibly factors VII and X or their avian equivalents, reside in the brain powder of vitamin K-normal birds, as well as in the plasma of such chicks (Griminger et al., 1970). These pro thromboplastic factors may also be present in the ABP from vitamin K-deficient chicks; if so, however, only in very small quantities. On the other hand, an inhibitor(s), which may be a protein, a phospholipid, or a combination of the two, is present in higher concentration in brain powder from vita-
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0.02 level. The decrease in extracted P, from the first to the second saline extracts, was greater than the decrease in protein; thus, the protein/P ratio in the second extract was higher than in the first one. In spite of these observations we failed to establish any correlation between the thromboplastic activity and protein or P content, or protein/P ratio. When brain powder samples were repeatedly extracted with saline, TP activity, as well as protein and P content, gradually declined (Figure 1). If it is assumed that "thromboplastin" is a phospholipid-protein complex, it is surprising that after 8 or 9 extractions such minute amounts of protein and P as remained still elicited a prothrombin time response shorter than 30 seconds. It was also observed that the amount of P and protein extracted decreased in successive extractions at a decreasing rate. In many of these re-extraction experiments the ABPs from normal and vitamin K-free donor chicks tended to have the same thromboplastic activity by the time the fourth or fifth extractions were reached. In a previous report (Griminger et al., 1970) we had found that adding blood or plasma from a normal bird to ABP extracts from vitamin K-free birds shortened their prothrombin time, and thus normal plasma
405
BRAIN THROMBOPLASTIC ACTIVITY
r °
a.
.7 28 600
.6 24 .5 20
500
400
.4 • 16 .3
12
.2
8
.1
4
2
4 6 8 No. of Extractions
10
FIG. 1. Effect of re-extraction on prothrombin time, protein and phosphorus content of brain powder.
„„.,
-l) -si £•
tftt.
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700-
K-def.
t 6th a x t r s c c
600"
\. \
t 50C-
\
n
1
I
^. I \
\\
~"-"0
400-
• b
\Xo 2
4 6 8 No. of Extractions
10
FIG. 2. Decline of thromboplastic activity of brain powders upon repeated extraction, and recovery by the addition of normal plasma.
K-dencient donor birds after 4 or 5 extractions. Table 3 summarizes the results obtained when the same source of brain powder was subjected to three dilution experiments. When the thromboplastin extract from a single sample of brain powder was continually diluted with saline, the TP activity declined rapidly. This was to be expected, because in this instance both the prothromboplastic factor and the phospholipid component were depleted. The second type of dilution, in which the prothromboplastic factor was supplied by adding appropriate amounts of normal plasma to the extract from one sample of brain powder while it was being diluted so that it was the phospholipid component alone that was being depleted, yielded more active extracts than the first type at equal dilution ratios. However, in the third dilution experiment, when
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min K-deficient donors than in those from normal donors. The prothrombin time of the first extract represents the net result of the interaction of these three factors. Due to its high solubility, the inhibitor (s) is essentially removed after the first saline extraction, and thus, at this point, the amount of the prothromboplastic factor in conjunction with the phospholipid-protein complex are the determining factors for thromboplastic activity. In repeated extractions of the same brain powder, this prothromboplastic factor in ABP from normal donors is exhausted very rapidly to the level found in those from vitamin K-free birds. Consequently a clot is produced, using either type of brain extract, by the phospholipid-protein complex in the absence of the prothromboplastic factor, or in the presence of a very low concentration of the factor. This hypothesis may explain the essentially equal activity of thromboplastin extracts of ABP from normal and vitamin
406
YIN-SHAN SHUM AND P. GRIMINGER
TABLE 3.—Effect
of dilution of phospholipid-protein (PLP) and prothromboplastic factor (s) (PTF) in brain powders on their thromboplastic activity Prothrombin time with normal plasma (sec.)
Dilution ratio
_
1 2
_
+
14.3 1 18.6 22.1 30.0 40.7 65.9
18.9 25.1 30.4 38.3 50.4 80.0
1 2 4 8 16 32
Dilution of PLP and PTF using a fresh sample of brain powder for each dilution.
Dilution of PLP alone in extract from one sample of brain powder. (normal plasma added) 2
Dilution of PLP and PTF in extract from one sample of brain powder
_
+
+
16.3 18.7 20.9 24.3 34.5 36.0
19.2 29.1 25.4 29.7 35.0 48.4
16.0 13.8 16.2 22.4 29.8 46.1
18.0 13.5 15.1 21.9 30.6 43.9
samples of equal quantities of brain powder were extraced with multiple volumes of saline to produce, in effect, the same type of dilution as in the first one, more active extracts were obtained. It was suspected that a solubility or contact factor might have been involved in this unusual phenomenon. This prompted us to analyze for P and protein both in the first and second thromboplastin extracts in this type of dilution. The data in Table 4 show that in the first extract more protein and phosphorus were extracted when the volume of saline was doubled. The prothrombin time, however, was increased, which could be due to the lower P and protein concentration per unit volume of extract. Still larger volumes of saline did not increase the total amount of P and protein in the extract. In
the second extract, there was no significant difference in the amount of phosphorus or protein extracted with various volumes of saline. It was previously shown that the P and protein concentration of the first extract was at least 5 times as high as that of the second one. Therefore, one may speculate that with the same volume of saline used to extract a given amount of brain powder, due to limitations in solubility, only a certain quantity of P or protein is dissolved out in the first extract; thus, more phospholipid and protein could be extracted if a larger volume of saline were used. On the other hand, as mentioned previously, still larger volumes of saline did not increase further the amount of P and protein extracted. Apparently, there is no simple explanation for the solubility behav-
TABLE 4.—Prothrombin times, protein and phosphorus content of 1st and 2nd extracts of a given amount of brain powder extracted with increasing volumes of saline1 Prothrombin time (sec.)
1 4
Total P (meg.)'
1
2
1
22
21.0 28.2 27.5 29.5 91.0 91.8
16.3 16.3 16.5 18.7 21.0 27.0
16.8 19.8 24.6 20.5 43.3 42.8
11.8 13.4 15.7 18.4 20.2 22.1
1
2
1.99 .39 2.75 .41 2.32 .31 2.38 .37 2.48 .44 2.45 .26
1
Protein/P ratio
+
+
+
Dilution ratio
1 2 4 8 16 32
Total Protein (mg.)3
2
2.09 .47 2.78 .45 2.77 .42 2.78 .41 2.42 .44 2.56 .26
+
1
2
1
2
1
2
1
2
122.8 142.0 139.6 142.0 140.8 136.0
20.3 15.2 15.0 15.3 15.5 10.2
117.8 146.0 141.2 145.4 149.2 124.8
21.1 18.5 19.4 20.5 19.0 17.0
16.2 19.4 16.6 16.8 17.6 18.0
19.4 27.0 20.9 24.1 28.6 25.0
17.8 19.1 19.6 19.2 16.2 20.5
22.3 24.4 21.8 20.0 23.3 15.1
Source of brain powder: (—) vitamin K-deficient chicks; (+) normal chicks. 1 —first saline extract; 2 —second saline extract. »Total protein or P content when 20 mg. samples of brain powder were extracted with multiple volumes of saline.
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Source of brain powders: (—) vitamin K-deficient chicks; (+) normal chicks. Normal plasma was added starting with dilution ratio 2.
BRAIN THROMBOPLASTIC ACTIVITY
ior of a system as complex as the one involved here. Further experiments are planned to separate and identify the three interacting factors, namely the prothromboplastic plasma factor, the phospholipid component and the inhibitor (s), and to test their dependence on vitamin K nutriture. SUMMARY
extract from a single brain powder sample. A hypothesis was proposed to explain some of these observations. REFERENCES Bartlett, G. R., 1959. Phosphorus assay in column chromatography. J. Biol. Chem. 234: 466-468 Deutsch, E., K. Irsigler and H. Lomoschitz, 1964. Studien iiber Gewebethromboplastin. I. Reinigung, chemische Charakterisierung und Trennung in einen Eiweiss- und Lipoidanteil. Thromb. Diath. Haemorrh. 12: 12-34 Dodge, J. T., and G. B. Phillips, 1967. Composition of phospholipids and of phospholipid fatty acids and aldehydes in human red cells. J. Lipid Res. 8: 667-675 Griminger, P., 1963. The influence of previous vitamin K nutrition on thromboplastic activity of brain extract. Poultry Sci. 42: 784-786 Griminger, P., Y. S. Shum and P. Budowski, 1970. Effect of dietary vitamin K on avian brain thromboplastin activity. Poultry Sci. 49: 16811686 Hecht, E., R. H. Landaburu and W. H. Seegers, 1957. Sphingosine as inhibitor of prothrombin activation with thromboplastin or with threone. Am. J. Physiol. 189: 203-208 Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall, 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275 Miller, G. L., 1958. Protein determination for large numbers of samples. Anal. Chem. 3 1 : 964
NEWS AND NOTES (Continued from page 401) berger Hall, Kansas State University, Manhattan, Kansas 66502. Louisiana—Cooperative Extension Service, Knapp Hall, Louisiana State University, Baton Rouge, Louisiana 70803. New Mexico—Department of Agricultural Information, Drawer 3A1, New Mexico State University, Las Cruces, New Mexico 88001. Oklahoma—Audio-Visual Center, 216 Library Annex, Oklahoma State University, Stillwater, Oklahoma 74074. Texas—Department of Agricultural Information, Agricultural Extension Service, Texas A and M University, College Station, Texas 77843.
Pierre C. Boucher has been named Chief of the Poultry Marketing Programs Branch of the Consumer and Marketing Service of the U. S. Department of Agriculture. He fills the position left vacant when Richard C. Larkin retired earlier this year. Mr. Boucher's agricultural experience began on the family poultry farm in Penacok, New Hampshire. Later he served as a County Agricultural Agent, the Director of the Division of Poultry and Poultry Products for the Massachusetts Department of Agriculture, a U. S. Department of Agriculture News Reporter, and most recently Contract Officer for the Marketing Programs Branch. He majored in poultry science and agronomy at
(Continued on page 418)
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Saline extracts of ABP from vitamin Knormal chicks had slightly higher concentrations of protein and phosphorus than those from vitamin K-deficient ones. The second saline extract of ABP from vitamin K-deficient chicks had higher TP activity than the first one, though its protein and phosphorus concentration was much lower, as was also true with ABP from normal birds. Further extractions of ABP resulted in a gradual decrease in TP activity, as well as in protein and phorphorus content. TP activity could be restored to the original level by adding normal chick plasma. Extracting separate brain powder samples with increasing volumes of saline yielded more active extracts than the equivalent ones obtained by continued dilution of the
407
POULTRY SCIENCE 51: 402-407,
A
CETONE-EXTRACTED brain powder from vitamin K-deficient chickens, when used as a source of thromboplastic activity in the one-stage prothrombin time determination, will promote slower prothrombin times with a given plasma than identically prepared brain powder from vitamin K-sufficient chicks (Griminger, 1963). This activity differential is due, at least in part, to the presence or absence of vitamin K-dependent clotting factors in the residual blood of the chicken brains used as a source of thromboplastin (Griminger et al., 1970). In the course of experimental work leading to this conclusion it was noted that a second saline suspension of certain acetone-extracted chicken brain powders had more thromboplastic activity than the first, suggesting the presence of inhibitors, and also that a certain amount of thromboplastic activity was present even after repeated extractions 1
Paper of the Journal Series, New Jersey Agricultural Experiment Station, New Brunswick. This project was supported, in part, by U.S.P.H.S., N.I.H. Grant HE-10997. A preliminary report was presented at the 1971 F.A.S.E.B, meetings (Fed. Proc. 30: 479).
1972
with saline. The existence of inhibitors in thromboplastic preparations had been suggested by Deutsch et al. (1964). These authors discarded the first saline extract of their brain powder thromboplastin preparation, claiming it to contain undesirable proteins which were apparently removed during the first suspension in saline. Hecht et al. (1957) have also commented on the existence of an inhibitor, sphingosine, which is a constituent of brain cerebrosides and sphingomyelin. This report illustrates the presence of the inhibitor (s) and shows the results of a study of the effect of various factors such as repeated extraction, dilution, and addition of plasma on the thromboplastic activity of chicken brain powder. MATERIALS AND METHODS
Vitamin K-deficient and normal chicks were obtained by feeding a vitamin K-free basal diet, or the same diet supplemented with 10 mg. vitamin Kj. (phylloquinone, as 1% beadlet)/kg. feed to female crossbred chicks (N. H. c? X Columbian $ ) to three weeks of age. The composition of the basal diet has been given previously (Griminger
402
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ABSTRACT The second saline extract (E2) of acetone-dehydrated chicken brain powder contained less than one fifth of protein and phosphorus, compared with the first extract (Ei). Thromboplastic activity (TPA) of E 2 from vitamin K-deficient donors was significantly higher than of Ei. Response with vitamin K-sufficient donors varied. Ei and Ei from normal chicks contained significantly more protein than those from vitamin K-deprived ones. P content differed significantly in Ei only. Differences in TPA could not be quantitatively attributed to differences in protein and P content. TPA of brain powder, repeatedly extrasted with saline, declined, as did protein and P content. The addition of normal chick plasma returned TPA of repeatedly extracted brain powders to level found in fresh samples. Extracting samples of equal quantity of brain powder with increasing volumes of saline yielded higher TPA than when saline extract from a single brain powder sample was diluted with saline to produce, in effect, the same dilution ratios.
403
BRAIN THROMBOPLASTIC ACTIVITY
RESULTS AND DISCUSSION When a given sample of brain powder from vitamin K-deficient donor birds was twice extracted with saline, the second extract had a higher thromboplastic (TP) activity than the first one (Table 1). There was a slight decrease in activity with brain powders from vitamin K-normal donor birds (Table 2). However, in results not shown here, there was either no change or a slight increase in TP activity when ABPs
TABLE 1.—Prothrombin times and protein and phosphorus content of the 1st and 2nd saline
extracts of acetone-dehydrated brain powders from vitamin K-free chicks
Lot
A B C D E F G H I
J
Average 1 2
Donor bird plasma prothrombin time
Prothrombin time with normal plasma (sec.)
Total P (meg.) ^
Total protein (mg.)2
Protein/P ratio
Range (sec.)
1
21
1
2
1
2
1
2
31.4-40.8 45.0-50.0 50.4-52.7 53.4-57.1 57.6-59.5 59.5-60.9 61.1-66.1 66.2-67.5 70.1-77.4 81.5-85.8
15.6 16.7 18.8 20.9 17.4 17.6 19.3 20.6 17.1 18.0
16.3 15.6 16.5 16.2 15.5 15.8 16.6 17.4 15.4 16.2
2.72 3.14 3.18 2.89 3.14 2.58 3.08 2.80 3.25 3.17 2.99
.54 .50 .48 .71 .52 .50 .52 .43 .50 .50 .52
173 163 169 166 166 161 179 136 177 178 167
26 27 35 24 26 26 26 21 25 25 26
15.7 19.2 18.8 17.5 19.0 16.0 17.2 20.6 18.4 17.9 18.0
20.9 18.6 13.8 28.9 19.6 18.8 19.9 20.9 19.9 20.0 19.8
1—first saline extract; 2—second saline extract. Total protein or P extracted from 25 mg. samples of brain powder with 2.5 ml. saline.
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from normal donors were subjected to a second saline extraction. Regardless of the change in TP activity, there was a great reduction in the amount of both the protein and P present in the second extract, as compared with the first one. These data strongly suggest that brain powder preparations contain an inhibitor or inhibitors, those from vitamin K-deprived birds apparently having higher concentrations than those from the normal ones. The inhibitor(s) seems to have a high degree of solubility in saline and thus can be readily removed after the first extraction, resulting in a higher TP activity in the second extract. There seemed to be slightly, but consistently, more protein and P in the normal brain powder extracts (i.e., saline extracts of brain powders from vitamin K-normal donors), than in those from vitamin K-deficient chicks. The difference in P concentration between the first saline extracts of ABP from normal and vitamin K-deficient birds was significant at the 0.05 level. The difference in protein content of the first saline extracts of the two types of ABP was highly significant (P < 0.01), while that of the second extracts was significant at the
et al., 1970) except that all feeds contained 5% refined corn oil and 0.02% antioxidant. The same paper also described the preparation of the acetone-dehydrated brain powder (ABP), the extraction (activation) of thromboplastically active material from the ABP, as well as the onestage prothrombin time determination. Analyses of phosphorus (as a measure of phospholipids) and of protein were carried out in some of the experiments. In the determination of phosphorus content, the method by Bartlett (1959) as modified by Dodge and Phillips (1967) was essentially followed, while that for protein content was based on the procedure of Lowry et al. (1951), as modified by Miller (1958).
404
YIN-SHAN SHUM AND P. GRIMINGER
TABLE 2.—Prothrombin times and protein and phosphorus content of the 1st and 2nd saline
extracts of acetone-dehydrated brain powders from normal chicks
Lot
A B C D E F Average 1 2
Donor bird plasma prothrombin time
Prothrombin time with normal plasma (sec.)
Total protein (mg.)2
Protein/P ratio
Total P (meg.)2
Range (sec.)
1
21
1
2
1
2
1
2
9.6-10.2 10.4-10.5 10.8-11.0 11.1-11.3 11.4-12.2 12.2-13.1
12.6 11.9 12.2 12.3 13.8 13.0
13.5 13.8 13.7 13.2 14.7 14.6
3.36 3.19 3.71 3.73 3.61 3.19 3.46
.56 .64 .67 .64 .56 .57 .61
189 171 180 182 175 180 179
29 28 25 30 25 26 27
17.8 18.7 20.6 20.5 20.6 17.7 19.3
19.3 22.5 26.5 21.6 22.7 21.7 22.3
1—first saline extract; 2—second saline extract. Total protein or P extracted from 25 mg. samples of brain powder with 2.5 ml. saline.
was concluded to contain a thromboplastic activity-enhancing moiety. This was confirmed by the finding that the one-time addition of normal plasma (equivalent of IS microliters of plasma per brain) to brain powders after 5 extractions restored their thromboplastic activity to a level comparable to that in fresh extracts (Figure 2). It is assumed that brain powders from normal as well as from vitamin K-deficient chickens contain a phospholipid-protein complex. It is possible that this complex is not identical in the two types of brain powder. This complex seems to be exhausted very slowly, as seen from the fact that even after 10 extractions it had sufficient thromboplastic activity to clot plasma within 30 seconds. Aside from this phospholipid-protein complex, one or more prothromboplastic factor(s), possibly factors VII and X or their avian equivalents, reside in the brain powder of vitamin K-normal birds, as well as in the plasma of such chicks (Griminger et al., 1970). These pro thromboplastic factors may also be present in the ABP from vitamin K-deficient chicks; if so, however, only in very small quantities. On the other hand, an inhibitor(s), which may be a protein, a phospholipid, or a combination of the two, is present in higher concentration in brain powder from vita-
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0.02 level. The decrease in extracted P, from the first to the second saline extracts, was greater than the decrease in protein; thus, the protein/P ratio in the second extract was higher than in the first one. In spite of these observations we failed to establish any correlation between the thromboplastic activity and protein or P content, or protein/P ratio. When brain powder samples were repeatedly extracted with saline, TP activity, as well as protein and P content, gradually declined (Figure 1). If it is assumed that "thromboplastin" is a phospholipid-protein complex, it is surprising that after 8 or 9 extractions such minute amounts of protein and P as remained still elicited a prothrombin time response shorter than 30 seconds. It was also observed that the amount of P and protein extracted decreased in successive extractions at a decreasing rate. In many of these re-extraction experiments the ABPs from normal and vitamin K-free donor chicks tended to have the same thromboplastic activity by the time the fourth or fifth extractions were reached. In a previous report (Griminger et al., 1970) we had found that adding blood or plasma from a normal bird to ABP extracts from vitamin K-free birds shortened their prothrombin time, and thus normal plasma
405
BRAIN THROMBOPLASTIC ACTIVITY
r °
a.
.7 28 600
.6 24 .5 20
500
400
.4 • 16 .3
12
.2
8
.1
4
2
4 6 8 No. of Extractions
10
FIG. 1. Effect of re-extraction on prothrombin time, protein and phosphorus content of brain powder.
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FIG. 2. Decline of thromboplastic activity of brain powders upon repeated extraction, and recovery by the addition of normal plasma.
K-dencient donor birds after 4 or 5 extractions. Table 3 summarizes the results obtained when the same source of brain powder was subjected to three dilution experiments. When the thromboplastin extract from a single sample of brain powder was continually diluted with saline, the TP activity declined rapidly. This was to be expected, because in this instance both the prothromboplastic factor and the phospholipid component were depleted. The second type of dilution, in which the prothromboplastic factor was supplied by adding appropriate amounts of normal plasma to the extract from one sample of brain powder while it was being diluted so that it was the phospholipid component alone that was being depleted, yielded more active extracts than the first type at equal dilution ratios. However, in the third dilution experiment, when
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min K-deficient donors than in those from normal donors. The prothrombin time of the first extract represents the net result of the interaction of these three factors. Due to its high solubility, the inhibitor (s) is essentially removed after the first saline extraction, and thus, at this point, the amount of the prothromboplastic factor in conjunction with the phospholipid-protein complex are the determining factors for thromboplastic activity. In repeated extractions of the same brain powder, this prothromboplastic factor in ABP from normal donors is exhausted very rapidly to the level found in those from vitamin K-free birds. Consequently a clot is produced, using either type of brain extract, by the phospholipid-protein complex in the absence of the prothromboplastic factor, or in the presence of a very low concentration of the factor. This hypothesis may explain the essentially equal activity of thromboplastin extracts of ABP from normal and vitamin
406
YIN-SHAN SHUM AND P. GRIMINGER
TABLE 3.—Effect
of dilution of phospholipid-protein (PLP) and prothromboplastic factor (s) (PTF) in brain powders on their thromboplastic activity Prothrombin time with normal plasma (sec.)
Dilution ratio
_
1 2
_
+
14.3 1 18.6 22.1 30.0 40.7 65.9
18.9 25.1 30.4 38.3 50.4 80.0
1 2 4 8 16 32
Dilution of PLP and PTF using a fresh sample of brain powder for each dilution.
Dilution of PLP alone in extract from one sample of brain powder. (normal plasma added) 2
Dilution of PLP and PTF in extract from one sample of brain powder
_
+
+
16.3 18.7 20.9 24.3 34.5 36.0
19.2 29.1 25.4 29.7 35.0 48.4
16.0 13.8 16.2 22.4 29.8 46.1
18.0 13.5 15.1 21.9 30.6 43.9
samples of equal quantities of brain powder were extraced with multiple volumes of saline to produce, in effect, the same type of dilution as in the first one, more active extracts were obtained. It was suspected that a solubility or contact factor might have been involved in this unusual phenomenon. This prompted us to analyze for P and protein both in the first and second thromboplastin extracts in this type of dilution. The data in Table 4 show that in the first extract more protein and phosphorus were extracted when the volume of saline was doubled. The prothrombin time, however, was increased, which could be due to the lower P and protein concentration per unit volume of extract. Still larger volumes of saline did not increase the total amount of P and protein in the extract. In
the second extract, there was no significant difference in the amount of phosphorus or protein extracted with various volumes of saline. It was previously shown that the P and protein concentration of the first extract was at least 5 times as high as that of the second one. Therefore, one may speculate that with the same volume of saline used to extract a given amount of brain powder, due to limitations in solubility, only a certain quantity of P or protein is dissolved out in the first extract; thus, more phospholipid and protein could be extracted if a larger volume of saline were used. On the other hand, as mentioned previously, still larger volumes of saline did not increase further the amount of P and protein extracted. Apparently, there is no simple explanation for the solubility behav-
TABLE 4.—Prothrombin times, protein and phosphorus content of 1st and 2nd extracts of a given amount of brain powder extracted with increasing volumes of saline1 Prothrombin time (sec.)
1 4
Total P (meg.)'
1
2
1
22
21.0 28.2 27.5 29.5 91.0 91.8
16.3 16.3 16.5 18.7 21.0 27.0
16.8 19.8 24.6 20.5 43.3 42.8
11.8 13.4 15.7 18.4 20.2 22.1
1
2
1.99 .39 2.75 .41 2.32 .31 2.38 .37 2.48 .44 2.45 .26
1
Protein/P ratio
+
+
+
Dilution ratio
1 2 4 8 16 32
Total Protein (mg.)3
2
2.09 .47 2.78 .45 2.77 .42 2.78 .41 2.42 .44 2.56 .26
+
1
2
1
2
1
2
1
2
122.8 142.0 139.6 142.0 140.8 136.0
20.3 15.2 15.0 15.3 15.5 10.2
117.8 146.0 141.2 145.4 149.2 124.8
21.1 18.5 19.4 20.5 19.0 17.0
16.2 19.4 16.6 16.8 17.6 18.0
19.4 27.0 20.9 24.1 28.6 25.0
17.8 19.1 19.6 19.2 16.2 20.5
22.3 24.4 21.8 20.0 23.3 15.1
Source of brain powder: (—) vitamin K-deficient chicks; (+) normal chicks. 1 —first saline extract; 2 —second saline extract. »Total protein or P content when 20 mg. samples of brain powder were extracted with multiple volumes of saline.
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Source of brain powders: (—) vitamin K-deficient chicks; (+) normal chicks. Normal plasma was added starting with dilution ratio 2.
BRAIN THROMBOPLASTIC ACTIVITY
ior of a system as complex as the one involved here. Further experiments are planned to separate and identify the three interacting factors, namely the prothromboplastic plasma factor, the phospholipid component and the inhibitor (s), and to test their dependence on vitamin K nutriture. SUMMARY
extract from a single brain powder sample. A hypothesis was proposed to explain some of these observations. REFERENCES Bartlett, G. R., 1959. Phosphorus assay in column chromatography. J. Biol. Chem. 234: 466-468 Deutsch, E., K. Irsigler and H. Lomoschitz, 1964. Studien iiber Gewebethromboplastin. I. Reinigung, chemische Charakterisierung und Trennung in einen Eiweiss- und Lipoidanteil. Thromb. Diath. Haemorrh. 12: 12-34 Dodge, J. T., and G. B. Phillips, 1967. Composition of phospholipids and of phospholipid fatty acids and aldehydes in human red cells. J. Lipid Res. 8: 667-675 Griminger, P., 1963. The influence of previous vitamin K nutrition on thromboplastic activity of brain extract. Poultry Sci. 42: 784-786 Griminger, P., Y. S. Shum and P. Budowski, 1970. Effect of dietary vitamin K on avian brain thromboplastin activity. Poultry Sci. 49: 16811686 Hecht, E., R. H. Landaburu and W. H. Seegers, 1957. Sphingosine as inhibitor of prothrombin activation with thromboplastin or with threone. Am. J. Physiol. 189: 203-208 Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall, 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275 Miller, G. L., 1958. Protein determination for large numbers of samples. Anal. Chem. 3 1 : 964
NEWS AND NOTES (Continued from page 401) berger Hall, Kansas State University, Manhattan, Kansas 66502. Louisiana—Cooperative Extension Service, Knapp Hall, Louisiana State University, Baton Rouge, Louisiana 70803. New Mexico—Department of Agricultural Information, Drawer 3A1, New Mexico State University, Las Cruces, New Mexico 88001. Oklahoma—Audio-Visual Center, 216 Library Annex, Oklahoma State University, Stillwater, Oklahoma 74074. Texas—Department of Agricultural Information, Agricultural Extension Service, Texas A and M University, College Station, Texas 77843.
Pierre C. Boucher has been named Chief of the Poultry Marketing Programs Branch of the Consumer and Marketing Service of the U. S. Department of Agriculture. He fills the position left vacant when Richard C. Larkin retired earlier this year. Mr. Boucher's agricultural experience began on the family poultry farm in Penacok, New Hampshire. Later he served as a County Agricultural Agent, the Director of the Division of Poultry and Poultry Products for the Massachusetts Department of Agriculture, a U. S. Department of Agriculture News Reporter, and most recently Contract Officer for the Marketing Programs Branch. He majored in poultry science and agronomy at
(Continued on page 418)
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Saline extracts of ABP from vitamin Knormal chicks had slightly higher concentrations of protein and phosphorus than those from vitamin K-deficient ones. The second saline extract of ABP from vitamin K-deficient chicks had higher TP activity than the first one, though its protein and phosphorus concentration was much lower, as was also true with ABP from normal birds. Further extractions of ABP resulted in a gradual decrease in TP activity, as well as in protein and phorphorus content. TP activity could be restored to the original level by adding normal chick plasma. Extracting separate brain powder samples with increasing volumes of saline yielded more active extracts than the equivalent ones obtained by continued dilution of the
407