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Effect of fat on the vitamin K requirement of the chick. Davies, Ronald Edgar 1956

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THE EFFECT OF FAT ON THE VITAMIN K REQUIREMENT OF THE CHICK by RONALD EDGAR DAVIES A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN AGRICULTURE in the Department of Poultry Science le accept this thesis as conforming to the standard required from candidates for the degree of MASTER OF SCIENCE IN AGRICULTURE. Members of the Department of Poultry Science. THE UNIVERSITY OF BRITtSH COLUMBIA April, 1956. ACKNOWLEDGEMENT The author wishes to thank Professor Jacob Biely, Head of the Department of Poultry Science, for providing the facilities used in this study, and for his encouragement and interest throughout the course of the work. He would also like to express his appreciation to Mrs. B. E. March, of the Department of Poultry Science, who supervised the planning and execution of the experiments. Thanks are also due to the following members of the Department of Poultry Science: Mrs. f. E. Marsden and Mr. S. P. Touchburn, who assisted in carrying out the ,!prothrombin time" determinations} and Mr. R. E. Salmon, who prepared the photographs of the figures. The generosity of Gordon Young (B. C.) Ltd., who donated the beef tallovf used in these experiments, is grate-fully acknowledged. 1. ABSTRACT A study has bean made of the relationship of dietary fat to the vitamin K requirement of the chick. In these experiments, the vitamin K status of chicks was measured by determining the blood "prothromMn time" by the method of Quick (1936) as modified by Almquist (1941). I t was possible to produce a "prothrombin deficiency" i n chicks by feeding diets which consisted of unextracted natural ingredients. The extent of this deficiency was affected by the maternal supply of vitamin K. In some cases the coagulation defect was sufficiently severe to cause f a i r l y heavy mortality from haemorrhaging. Certain fats, when added to the diet, protected the chick from a vitamin K deficiency. Beef tallow, hydrogenated cottonseed o i l , and to a lesser extent cottonseed o i l and hydrogenated animal fat a l l shortened blood "prothrombin time" of chicks, when added to a vitamin K deficient diet. These fats prevented the haemorrhaging which sometimes resulted from vitamin K depletion. Herring o i l generally did not shorten "prothrombin time" unless the vitamin K deficient diet was also deficient i n f o l i c acid. The effect of fats on "prothrombin time" was apparently not due to the vitamin K content of the fat, nor to an increased absorption of vitamin K resulting from increased bile secretion. Oral administration of o i l s shortly before drawing blood did not affect "prothrombin time" of blood from vitamin K deficient chicks; this indicates that lipaemia per se did not cause the shortening i n "prothrombin time" observed after feeding a high-fat diet. Free fatty acids added to a vitamin K deficient diet did not affect "prothrombin time" to a significant degree. Addition of choline to a vitamin K deficient diet did not affect "prothrombin time". This observation agrees with those of fi e l d and Dam (194-5) but disagrees with those of Honorato and Moline (1942) . the addition of methionine also failed to affect "prothrombin time". Herring o i l also has a shortening effect when added to such a diet, but not when added to a diet containing adequate folic acid. Cholic acid added to a vitamin K deficient diet caused a lengthened "prothrombin time." Desoxycholic acid had a similar effect in one test, but had no effect in another case. Injection of chicks with the surface active agent Triton WR-1339 likewise lengthened "prothrombin time." Triton had no effect, however, when added to blood jjj vitro. Breast muscle and brain tissue differed in the thromboplastic properties of their extracts. The breast muscle preparation required the addition of phospholipid for maximum thromboplastic activity. Acetone partly extracted and partly destroyed the thromboplastic agent of breast muscle, but did not affect that of brain tissue. Although bile salts may affect blood phospholipid levels, their effect on blood "prothrombin time" was not due to the sensitivity of the breast muscle thromboplastic agent to phospholipid. The *'prothrombin time" of blood, as determined with breast muscle thromboplastic agent, is not necessarily a measure of the vitamin K supplied to the chick. TABLE OF CONTENTS Introduction I Review of Literature II Experimental 1 Experiment I 2 Results 2 Experiment II Test 1 2(a) Results 3 Test 2 3 Results 4 Experiment III 4 Experiment IV Test 1 5 Results 6 Test 2 6 Results 7 Discussion of Results 7 Experiment V 10 Test 1 11 Results 11 Test 2 12 Results 12 Experiment VI 12 Results 13 Experiment VII 13 Results 14 Discussion of Results 14 Experiment VIII 17 Results 13 Discussion of Results 18 Experiment IX 21 Results 21 "^1 — <. — Discussion of Results 22 Experiment X 25 Experimental 33 Test 1 33 Results 33 Test 2 34 Results • 35 Test 3 35 (a) 36 Results 36 (b) 36 Results 36 Test 4 37 Results 37 Test 5 38 Results 39 Test 6 39 Results 40 Test 7 40 Results 42 Discussion of Results *< 42 Experiment XI 46 Results .. • 46 Discussion of Results .. 47 Conclusions 48 Appendix Tables (43) Figures (4) Bibliography. I. THE EFFECT OF FAT ON THE VITAMIN K REQUIREMENT OF THE CHICK Introduction Vitamin K, the anti-haemorrhagic vitamin, is widely distributed in vegetable feedstuffs. Moreover, i t is the only vitamin of the fat-soluble group which is synthesized by intestinal bacteria. Thus the appearance within recent years of a specific deficiency of this vitamin is of considerable interest. One clue to the appearance of such a deficiency disease at this time is the fact that i t parallels the increasing use of high-energy feeds. In rations based on the Connecticut formula, fibre content is reduced to a minimum. This has resulted in a marked decrease in the use of green feed, the richest natural source of vitamin K. Concurrently, the screw-press method for extracting o i l from soybeans has been replaced by solvent extraction. The o i l content of soybean meal, a staple ingredient of poultry feeds, has been reduced from 5% to less than 1$, with a corresponding reduction of fat-soluble vitamins. The increasing use of antibiotics and sulpha drugs in poultry feeds has also been implicated. Many of the sulpha drugs and broad-spectrum antibiotics have been shown to suppress the coliform organisms of the intestine. Escherichia coli and related species are the principal bacterial producers of vitamin K. The use of high-energy, low-fibfce feeds containing anti-infective sulpha drugs and antibiotics has greatly increased the growth rate of chicks. At the same time the amount of feed consumed per pound of gain has decreased. II. It appears, therefore, that while the metabolic requirements for growth have been increasing, the supply of vitamin K from the diet and from intestinal synthesis has been decreasing. At the present time, fats are being used to increase the energy content of poultry feeds. Addition of fat generally decreases the amount of feed consumed per pound of gain, and might therefore be expected to decrease the intake of dietary vitamin K per pound of gain. It is also possible that the dietary requirement of the chick for Vitamin K may be affected by the level of fat which is fed. It has been shown that the requirement for some of the B-complex vitamins may be influenced by dietary fat. The fact that vitamin K is fat soluble suggests that, like vitamins A and D, absorption of vitamin K from the intestine may be affected by ingested fat. In consideration of the above observations, a study has been made of the relationship of dietary fats to the vitamin K requirement of chicks. REVIEW OF LITERATURE Vitamin K (Koagulations vitamine) is the name given by Dam (1935) to a dietary essential which prevented a haemorrhagic condition of chicks. The active principle was isolated from alfalfa by Dam et aj, (1939). Almost simultaneously McKee jet al (1939) reported isolation of two different active compounds, one from alfalfa and one from fish meal. They showed that the basic structure of the two vitamers involved a naphthoquinone nucleus, with a methyl substituent at the 2- position and a hydrocarbon side-chain at the III. 3- position. In vitamin K-^, isolated from alfalfa, the side-chain is a phytyl group. Vitamin K.,, isolated from fish meal, contains the larger difarnesyl group. Vitamin is not a constituent of the fish meal itself, but is a bacterial product which accumulates during putrefaction (Almquist and Stokstad, 1935). The synthetic vitamin (menadione) contains only the 2-methyl-l, A-naphthoquinone nucleus, with no substituent at the 3 position. It appears to have the same effectiveness, on a molar basis, as the two naturally occurring vitamers, although this belief is questioned by Quick and Collentine (:195l). Phthiocol (2-methyl-3-hydroxy-l, A-naphthoquinone) was early shown to have vitamin K activity (Almquist and Klose, 1939). Thus i t appeared that the 2-methyl naphthoquinone nucleus was necessary for vitamin K activity. Vitamin (2-methyl-l, A-naphthalenediol diacetate) was believed to function by con-version to the quinoid structure ±Q vivo. More recently, however, several vitamin K active compounds have been discovered vshich do not appear to be readily convertible to the naphthoquinone structure. Notable among these are A-amino-2-methyl-l-naphthol hydrochloride, 2-methyl-l, 5-naphthalenediamine-2 hydrochloride, and particularly 2, 6-dimathylbenzoquinone (Hunter and Shepherd, 1955). Quick (1935, 1936, 1937, 1938) developed a method of measuring blood prothrombin, by means of which he demonstrated that prothrombin is reduced in vitamin K deficiency. A similar reduction was demonstrated in cases of biliary dysfunction, icterus and certain types of digestive dis-turbances. These conditions were shown to involve impaired absorption of vitamin K. "Sweet clover poisoning", a haemorrhagic disease of cattle which consume spoiled sweet clover, was also shown to involve reduced blood IV. prothrombin. The active agent in the clover is dicoumarol (3,3'-methylene-bis (4-hydroxycoumarin)) (Stahman, Reubner and Link, (1941) which bears a limited structural analogy to two naphthoquinone nuclei joined through a methyl group. For this reason i t has been considered to act as a specific antimetabolite interfering with the utilization of vitamin K. Dicoumarol, and its derivative Tromexan (ethyl acetate substituted for the linking methyl) have been used therapeutically to lessen the danger of thrombosis following surgery. The mode of action of these and related drugs has been reviewed recently by Douglas (1955). Certain observations related to the use of these anticoagulant drugs have provided indirect evidence that prothrombin is not the only coagulation factor affected by a vitamin K deficiency. Quick, Hussey and Collentine (1952) showed that the "hypoprothrombinaemia" resulting from a vitamin K deficiency does not differ from that caused by dicoumarol therapy. De Nicola (1953) stated that the principal deficiency produced in dicoumarol therapy involves the newly identified Factor VII, with a relatively unimpor-tant change in the prothrombin level. Thus i t may be a defect in the production of Factor VII, rather than of prothrombin, which produces the haemorrhagic state in vitamin K deficiency. It is well established that the site of vitamin K action is in the liver (Bririkhouse and Warner, 1940; Carter, Chambers and Warner, 1949). Recently Witte and Dirnberger (1953) showed that several coagulation factors, including prothrombin and probably including Factor VII, are synthesized in the liver. Thus the site of vitamin K action provides no direct evidence as to which of these factors i s actually dependent on the vitamin. V. The chemical estimation of vitamin K activity i s relatively difficult. The chemical procedures are based on the reactions of the quinone or naphthoquinone group. Thus, in the method of Scudi and Buhs (1941), the quinone is reduced by catalytic hydrogenation to the hydroquinone, which in turn is reacted with 2,6-dichlorophenol indophenolj the extent of the reaction is measured photometrically. Kofler (1945) developed a fluorometric procedure involving condensation of the naphthoquinone with o-phenylenediamine. These procedures are not sufficiently specific for assay of heterogeneous materials. As yet no microhiological method has been evolved. The procedure most commonly used for vitamin K measurement is the biological assay developed by Almquist (1941)• Chicks are depleted of prothrombin by feeding a vitamin K free diet (polished rice, ether extracted fish meal and brewer* s yeast, vitamins and minerals). The test substance is administered orally in an o i l solution for four days. Within 24 hours of the last dose blood is drawn by decapitation of the chick, and prothrombin level i s measured by a modification of the method of Quick (1936). The values so obtained are compared with those of chicks receiving graded levels of mena-dione. Almquist (1952) has developed a formula relating vitamin K intake to prothrombin level over the range of biological activity. In the experiments here reported this procedure was modified somewhat. The rations used consisted of unextracted natural ingredients, and the test substances were in most cases incorporated directly into the feed. The objective of the experiments was not to assess "fee vitamin K content of the fat, but rather to determine what effect, i f any, dietary fat would have on the vitamin K requirement of the chick. VI. An increased riboflavin requirement in the presence of high levels of dietary fat has been reported in rats (Mannering, Orsine and Elvehjem, 1944) and dogs (Potter, Axelrod and Elvehjem, 1942). Mannering, Eipton and Elvehjem (1941) suggested that this effect might be due to a reduction in synthesis of riboflavin by the intestinal bacteria. On the other hand, Couch .§£ ai (1949) reported that high-fat diets stimulated intestinal synthesis of biotin in the fowl. Vitamin K, like riboflavin and biotin, is synthesized in the intestinal tract of birds. In this respect i t is unique among fat-soluble vitamins. The difficulties encountered in attempts to produce a vitamin K deficiency in rats (Black jgi al, 1942; Kornberg, Daft and Sebrell, 1944) indicate the importance of the intestinal source of this vitamin. The common coliform organises, particularly Escherichia c o l i f are important vitamin K. synthesizers (Orla-Jensen et al, 1942). Any effect which fat might have on intestinal microflora, therefore, might be reflected in the vitamin K status of the chick. The report by Couch et (1949) that fat stimulates biotin synthesis suggests a possible secon-dary effect. Welch and Iright (1943) have reported that biotin shortened the clotting time of blood from vitamin K deficient rats. This effect is presumably due to increased synthesis of vitamin K in the intestine. Some reported effects of fat, on the other hand, are not due to an effect on the microflora. Several workers have reported a shortening in clotting time of blood of various species following administration of fats (Fullerton, Davie and Anastopoulos, 1953; Duncan and Waldron, 1949)• Waldron, Beidelman and Duncan (1951) have reviewed effects of fat on blood coagulation; both a local haemostatic and a systemic effect are believed to be involved. The effect of fat on blood coagulation is characterized by a shortening of VII. coagulation time for a period beginning shortly after consumption of fat, and lasting for 4-6 hours. In most cases there is l i t t l e apparent change in "prothrombin time". It is not likely therefore that vitamin K is affected. The elevated blood l i p i d level following fat ingestion may be related to the shortened clotting time, although Boulanger (1952) did not find any change in coagulability of the blood of laying turkey hens despite the appearance of lipaemia. Pellegrino and Solinas (1952) found no change in coagulability of blood after injecting synthetic oestrogens into rabbits. In the following experiments the whole blood clotting time of the birds was not determined. The effects of fat on coagulation time reported above may not therefore be applicable to these tests, since another part of the clotting mechanism may be involved. 1. EXPERIMENTAL The general experimental procedure followed in a l l chick tests was as follows: Day-old New Hampshire chicks were individually wing-banded and treated with Lederle 1s ocular live-virus Newcastle Disease vaccine, ^he chicks were distributed at random into groups in Jamesway electrically heated battery brooders. Room t e m P e r a t u r e was thermostatically controlled at 70° F. Water and experimental rations were provided ad l i b . Water troughs were cleaned daily to prevent bacterial synthesis of vitamin K. After a period of from eighteen to thirty-three days chicks from each lot"were bled by venipuncture into tubes calibrated at 1.0 ml. and containing 0.1 ml. M/lO sodium oxalate. "Prothrombin time" was deter-mined by the Quick (1936) method as modified by Almquist (1941).. The "prothrombin time" of blood from a bird receiving a commercial ration was recorded for comparison of different preparations of the thrombo-plastic agent. The procedure for the determination of "prothrombin time" was as follows* Thromboplastic agent* A normal bird was killed by bleeding. Ten grams of breast muscle was freed from a l l visible blood vessels, minced, and macerated in a mortar with Ottawa sand and lOnml. of 0.85$ saline. The mixture was centrifuged and filtered through coarse paper (Whatman #4)• The volume was made up to 200 ml. with 0.85$ saline, and an equal volume of M/40 CaCl Q was added. A fresh solution was prepared for each series 2 of determinations. 0.2 ml. of this preparation was added to 0.1 ml. of oxalated whole blood in a flat-bottomed vial 15 x 45 mm. A stopwatch was started as the agent was added. The tube was repeatedly tilted in a water bath (38.5-39.0° C.) until a gelatinous film appeared on the bottom. The" time required for this film to form was recorded as the "prothrombin time". EXPERIMENT I. This experiment was designed to indicate whether or not light pressed herring o i l would affect "prothrombin time" in chicks receiving a basal vitamin K deficient diet, with and without supplements of dried cereal grass and synthetic vitamin K (menadione). Both male and female chicks from two sources were used. The dams of one group were receiving a diet containing no green feed, ushile the dams of the other group were receiving a diet supplemented with 5% dehydrated cereal grass. The rations used in this experiment are shown in table 1:1. The control ration (ration 1) was essentially a commercial type chick starter diet but contained no green feed. In rations 3 and 6, contain-ing 5% dried cereal grass, protein level was adjusted by reduction of wheat and soybean meal levels. In rations 4, 5 and 6, 8% of commercial light pressed herring o i l replaced the 8$ cerelose contained in the control ration. "Prothrombin time" of five male and five female chicks from each group was measured at 26 and 27 days respectively. Re suit si The mean "prothrombin time" of five birds from each ration, sex and maternal source is shown in table 1:2. 4 statistical analysis of the results is shown in table 1:3. Supplements of cereal grass and of vitamin K were equally effective in shortening "prothrombin time". ^heir effect was not modified by addition of herring o i l to the ration or by the source of chicks used. On the other hand, both source of chicks and addition of herring o i l affected the response to rations which did not include a supplementary source of vitamin K. Chicks whose dams did not receive cereal grass had significantly longer blood "prothrombin time" (p<.05) when fed the urisupplemented ration than did chicks from dams receiving 5$ cereal grass. When herring o i l was added to the unsupplemented diet, "prothkombifntime" of chicks was prolonged. This effect was significant (p «s.05) with chicks whose dams received cereal grass, and highly significant (p<.Ol) with chicks whose dams did not receive cereal grass. The response of males and females was similar in a l l cases, although the males tended to be more variable. EXPERIMENT II - Test 1. . Earlier work at this laboratory (Burdett, 1955) has demon-strated that herring o i l will aggravate a folic acid deficiency in corn-fish meal rations. & second experiment was set up to determine whether or not the effect of herring o i l on blood "prothrombin time" was modified by the level of folic acid in the diet. simplified diet consisting mainly of wheat and herring meal was used 3. (table .2:1). Weights of individual chicks were recorded at 28 days. "Prothrombin time" of nine or ten birds per lot was measured at 29 days. The results of Experiment I indicated that i t was easier to produce a vitamin K deficiency in chicks when the dams were, fed diets containing no green feed. For this reason, chicks used in this experiment were from dams receiving no green feed. The same source of chicks was also used in a l l subsequent experiments, except where otherwise stated. Results* The growth response to folic acid (table 2:2) indicated that the basal ration was deficient in this vitamin. The effect of folic acid supplementation on "prothrombin time" i s less clear (table 2:3). Statistical analysis of this data (table 2:A) indicated that while neither folic acid nor herring o i l , singly, had a signifi-cant influence on "prothrombin time", a combination of the two produced a statistically significant shortening effect. Supplementation with vitamin K shortened "prothrombin time". This effect was not modified by the addition of folic acid or of herring o i l . Test 3, The previous test was repeated. In the second case "prothrombin time" was measured at 33 days. Individual weights of chicks were 4. recorded at 28 days. Results* Growth response to folic acid supplementation was similar to that in the previous test (table 2:2). Both folic acid and herring o i l significantly shortened "prothrombin time" (tables 2:3, 2*4). A combination of folic acid and herring o i l produced a highly significant shortening effect. Vitamin K shortened "prothrombin time" in a l l cases; the effect was not modified by addition of folic acid or herring o i l . EXPERIMENT III. The experiment reported here was part of another study being carried on at this laboratory. It was designed to investigate the effect of various fats on the folic acid requirement of chicks. During the course of the experiment, a severe haemorrhagic condition was observed in some of the chicks. Experimental rations are shown in table 3:1. They were similar to those used in Experiment II except tha,t corn replaced wheat. Fats used in this test were cottonseed o i l , hydrogenated cottonseed o i l ^ and hydrogenated animal fat^ (m.p. 55° C ) . The hydrogenated cottonseed o i l was melted for addition to the feed. The hydrogenated animal fat, a dry granular product, was incorporated directly, without melting. These fats were added at the 8$ level, with"and without folic acid. J,Broth-rombin time" of ten birds from groups receiving each experimental ration was determined at 28 days. Results: Mortality from haemorrhaging was high in groups receiving the basal ration, and the basal ration supplemented with folic acid. There 1 - Primex: a product of Procter and Gamble Ltd. 2 - Rydropoid: courtesy of Bowman Feed Products, Inc., Holland, Michigan. 5 was no haemorrhaging observed in any of the chicks receiving fat. "Prothrombin time" measurements gave values which were in accord with the above observations (table 3s2). Each of the fats produced a short-ening in "prothrombin time" which was statistically highly significant table (3 i 3 )• Hydrogenated cottonseed o i l was more effective than the other fats in this respect. The 28-day weights (table 3i4) indicated that the basal ration was deficient in folic acid. Nevertheless, folic acid alone had no effect on "prothrombin time". Folic acid plus hydrogenated animal fat, however, produced a significantly greater effect than did the hydrogenated animal fat alone. EXPERIMENT IV - Test 1. The results of the previous experiments indicated that the effect of fat on "prothrombin time" depended on the fat used and on the composi-tion of the ration. Lockhart, Sherman and Harris (1942) produced hypo-prothrombinaemia in rats by feeding fats containing high levels of dihydrosxystearic acid. Mineral o i l produced a similar effect (Elliot, Isaacs and Ivy, 1940). On the other hand, Duncan and Waldron (1949) reported a shortening in whole blood clotting time of dogs after administration of corn o i l , olive o i l or cow's cream. They considered that the activation of prothrombin was accelerated. Honorato and Moline (1942) found a relationship between vitamin K and methyl donors. They reported that choline shortened "prothrombin time" in chicks receiving a diet deficient in both vitamin K and choline. In the present experiment the effect on "prothrombin time" of two different fats added to an all-vegetable diet was observed. Fats tested were herring o i l and stablized beef tallow^-. The effect of supplementing the basal ration with folic acid and choline chloride was also observed. The rations used in this experiment are shown in table 4*1* "Prothrombin time" of 8-10 chicks per lot was determined at 23 days. Results: Folic acid, choline chloride and herring o i l , either alone or in any combination of the three, failed to influence "prothrombin time" of blood (table 4*2). On the other hand beef.tallow, either alone or in combination with the two vitamins, decreased "prothrombin time" to a statistically significant degree (table 4*3). Neither choline nor folic acid influenced this shortening effect, which was less than that obtained by vitamin K supplementation. Microbiological assay of the feed revealed that the folic acid level of the basal diet exceeded the requirement of the chick for this vitamin (N.R.C., 1955). Test 2. Procedure: This test was similar to test 1, except that supplementary vitamin B and meat scrap were incorporated into the ration (table 4:1). The effect of supplementary choline and methionine in the presence and absence of beef tallow was observed by determination of average weight of a l l birds, and "prothrombin time" of ten birds per group, at 23 days. 1 - Sta-fat: courtesy Gordon Young, Ltd., Vancouver, B . C . 7. Results: The group receiving supplementary vitamin K had unexpectedly long "prothrombin times" (table 4*2). Re-examination of the ration formulae revealed that vitamin K had been inadvertently added at only one-tenth the required level (N.R.C., 1955). Although the addition of methionine stimulated growth (table 4« 4), neither methionine nor choline affected "prothrombin time.'" Beef tallow, either alone or in combination with choline or methionine, produced a shortening effect which was statistically highly significant (table 4*3), and which far exceeded the effect of 0.02 mg. vitamin K per pound of feed. There appeared to be slight differences between mean "prothrombin time" of birds receiving beef tallow with and without added choline, methionine and vitamin K. These groups continued to receive their experimental diets for an additional week, to observe whether or not the differences would increase. The mean "prothrombin time" of 11-12 birds per group at 30 days was identical in a l l cases (table 4> 2). DISCUSSION OF RESULTS It appears from the results of the foregoing experiments that the effect of dietary fat on "prothrombin time11 in the chick is extremely variable, depending on the fat used and on the composition of the test diet. When the amount of vitamin K supplied to the chick is marginal, herring o i l appears to act as a stress agent. This was demonstrated in Experiment I, where the carryover of the vitamin was apparently a critical factor even after the chicks reached four weeks of age. Under these circumstances herring o i l had an unfavorable effect, lengthening 8. "prothrombin time" significantly. The effect was more severe in the case of chicks whose dams were depleted of vitamin K. On the other hand, when the diet fed was actually deficient in vitamin K the effect of herring o i l on "prothrombin time" varied. When the vitamin K deficient diet was also low in folic acid, herring o i l shortened ^ prothrombin time" (Experiment II tests 1 and 2). SShen the folic acid level was adequate, on the other hand, herring o i l appeared to have no effect (Experiment IV test 1). Supplementation of the vitamin K deficient diet with folic acid alone did not generally affect "prothrombin time", although i t did produce a significant shortening effect in Experiment II test 2. Other fats produced a dramatic shortening in '"prothrombin time*1 and adequately protected the birds from the haemorrhagic tendency sometimes caused by the depletion diet. Cottonseed o i l , hydrogenated cottonseed o i l , hydrogenated animal fat and a stabilized animal fat a l l produced this effect. The hydrogenated cottonseed o i l produced a greater shortening of "prothrombin time" than did the cottonseed o i l . In the case of hydrogenated animal fat, the addition of folic acid produced a further significant shortening, although folic acid alone was without effect. Honorato and Moline (1942) reported that when chicks fed diets deficient in vitamin K and choline received supplementary choline, the "prothrombin time" was reduced, field and Dam (1945) were unable to duplicate this result. In the present experiments neither choline nor methionine supplementation affected "prothrombin time" (Experiment IV). 9. The diets used were not seriously deficient in choline, judging by the growth rates. However, methionine supplementation did produce a growth response. 10. EXPERIMENT V. The general design of the f i r s t four experiments approximated that of the biological assay for vitamin K suggested by Almquist (1941). Groups of chicks were fed a basal diet deficient in vitamin K, and the basal diet was supplemented with the test substances. "Prothrombin time" of blood from these chicks was measured after 3-4 weeks. Since "prothrom-bin time" , under the conditions specified by Quick (1936), was considered to be a measure of the level of blood prothrombin, and since prothrombin was considered to be the coagulation factor affected by vitamin K, the test was thought to reflect the amount of vitamin K contained in the test substance. With the fats used in the above experiments, three effects were therefore possible. The fat might contain appreciable quantities of the vitamin. In the case of herring o i l and beef tallow this would be surprising, since animal products generally are considered to be very poor sources of the vitamin. Alternatively, the fats might affect the intestinal absorption of the fat-soluble vitamin. A third possibi-l i t y is an effect of dietary fat on microbiological synthesis of vitamin K in the digestive tract. To test the possibility that the fats contained appreciable quantities of vitamin K a series of experiments were carried out to observe the effect of treatment of the fats with heat and alkali. Vitamin K is labile under these conditions, and therefore should be destroyed by this treatment. 11. Test 1. Procedure: Herring o i l was heated to 75° C. and % of 20% aqueous KOH was added slowly with stirring. The mixture was allowed to cool, and the o i l phase removed by decanting through cheesecloth. This process removed most of the soaps formed, and left the greater part of the KOH in the aqueous phase. The "alkali-treated" o i l was incorporated into a corn-soybean meal diet (table 5:1) at 0>, and 3% levels. An untreated sample of the same o i l was incorporated at the same levels. A sample of herring o i l which had been stored at room temperature for a year was also included at the 8$ level, to test the effect of oxidation. These rations and a basal control diet were fed to eight groups of chicks as in previous tests. Individual weights were recorded at 23 days. "'Prothrombin time" of five birds from each group was measured at eighteen days, and of an additional ten birds from each group at 25 days. Results: The treated and untreated oils had similar effects on growth rate (table 5 :2) , producing a slight and identical depression at the 6% and 8$ levels. The stored o i l produced a more marked depression ingrowth. No significant differences in. " prothrombin time" were observed at 18 days (tables 5*3, 5 :4) . After 25 days, however, "prothrombin time" was significantly shorter in a l l groups receiving untreated o i l than in the comparable groups receiving treated o i l , although four of these six groups did not differ significantly from the control group. The stored herring o i l did not affect "prothrombin time". 12. Test 2. Procedure: Three fats were treated with alkali as described in the previous test. The fats used in this case were herring o i l , beef tallow and cottonseed o i l . The treated fats and untreated samples of each were incorporated into the basal ration at 2% and b% levels. The basal ration had the same composition as that used in the previous test (table 5:1). •"Prothrombin time" of ten birds from each group was measured at 19 days. Results: The degree of depletion of these chicks, as indicated by the "prothrombin time" (table 5*3), was less than that in previous experi-ments. This was presumably due to the fact that the depletion period (19 days) was shorter than usual. Few differences between groups were observed. Statistical analysis of the results (table 5*4) revealed the only significant effects to be the lengthening effect of 2% treated tallow and 6% untreated herring o i l , and the shortening effect of 6% treated tallow. 2% treated tallow produced significantly longer times than 2% untreated tallow, -while the b% level of treated tallow or herring o i l produced significantly shorter times than did the untreated samples. EXPERIMENT VI. Procedure: Cottonseed o i l had previously produced a decreased "proth-rombin time" in a corn-fish meal diet (Experiment III), but failed to do so in a corn-soya diet (Experiment V - Test 2). This experiment 13. was designed to observe whether or not alkali treatment would affect the response to cottonseed o i l in a corn-fish meal diet, and whether or not folic acid would affect such a response. The basal ration (table 6:1) was of similar composition to that used in Experiment III. There was some loss from haemorrhaging in groups 1 and 2 (basal and &®sal +• folic acid, respectively). "Prothrombin time" of a l l surviving chicks (ll-20pper lot) was determined at 23 days. Results: Cottonseed o i l , treated and untreated, produced a statisti-cally significant reduction in'"prothrombin time" (tables 6:2, 6:3). Although folic acid produced a slight reduction in a l l cases, these differences were not statistically significant. No explanation can be offered for the prolonged "prothrombin time" of chicks receiving vitamin K, since this vitamin was included in the diet at the usual level of 0.2 mg./lb. EXPERIMENT VII. The previous experiments indicated that, with the possible exception of herring o i l , the fats used did not shorten "prothrombin time" because of their vitamin K, content. This experiment was designed to observe whether a pure fatty acid or a mixture of fatty acids would produce a shortening effect. Oleic acid and mixed fish liver fatty acids were incorporated into a corn-fish meal diet (see table 3:1) at the level. In addition, to observe whether an o i l fed just prior to drawing blood would produce a reduction in "prothrombin time", u . fifteen chicks which had received the deficient basal diet were treated with 1.0 ml. of cottonseed o i l by oral pipetting on the 17th day. After one hour blood was drawn from a l l chicks for "prothrombin time" deter-mination. Results: Mean "prothrombin time" values are shown in table 7:2. The mixed fatty acids and oleic acid produced a shortening effect which was not, however, statistically significant (table 7:2). Orally administered cottonseed o i l produced a lengthening effect which was statistically significant. This was surprising, since other oils administered to dogs in this way had been reported to accelerate thrombin formation (Duncan and Waldron, 1949). Two other oils were tested at different times for their effect on "prothrombin time" when administered orally. Corn o i l , which had been reported by Duncan and Waldron (1949) to shorten the whole 'blood clotting time of dogs, was tested on two occasions by administrations to chicks which had received corn-fish meal depletion diets. Herring o i l was tested on chicks receiving a wheat-fish meal diet. Blood was drawn for "prothrombin time" determination 1-2 hours after administration of the o i l . The results are summarized in table 7:4* Neither o i l appeared to influence "prothrombin time". • DISCUSSION OF RESULTS It is unlikely that vitamin K would survive the treatment used in these experiments. It is unstable in the presence of heat, light or alkali (Greaves and Schmidt, 1937: McKee ej al, 1939). Nevertheless, 15. only herring o i l showed a consistent difference between treated and untreated samples (Experiment V test 1). Since a considerable quantity of soap was formed during the treatment of herring o i l , the effect of removal of free fatty acids may have been an important factor in pro-ducing this difference. The fats used in Experiment V, test 2, failed to produce the marked shortening effect noted in some previous experiments. This was also true, to a lesser extent, in experiment IV, test 1. In both of these cases the ration was a corn-soybean meal diet. In experiment IV, test 2, where meatmeal replaced part of the soybean meal, and in Experiment VI where fish meal replaced a l l the soybean meal, the effect of fat on "prothrombin time" was much more marked. Oleic acid and mixed fish liver fatty acids appeared to produce some slight shortening of "prothrombin time", but this effect was not statistically significant. This might suggest that the effect of the fats was in fact due to vitamin K content, and that the treatment with alkali failed to destroy i t . However, an examination of the results in Experiment IV, test 2, shows that 8$ beef tallow had an effect far greater than that of 0.02 mg. menadione per pound of feed. To account for its activity in terms of vitamin or K 2 content the fat would have to contain more than 150 micrograms 1^ or 185 micrograms K 2 per 100 gm. fat. In other words, i t would have to have a vitamin K content approaching that of alfalfa, the most concentrated natural source known (Wooster, 1954). 16. This observation implies that the effect of dietary fat on "'prothrombin time" is not related to its vitamin K content. A direct effect on intestinal absorption of the vitamin is possible. However, such an effect does not appear to be consistent with the fact that higher melting fats were generally more effective in shortening "prothrombin time." ^he digestibility of fats generally decreases with increasing melting point (Deuel, 1955)• Alternatively, dietary fat might improve absorption of the vitamin by increasing the production of bile. Another possible explanation could be that the fat content of the diet affects microbiological synthesis of the vitamin. None of these explanations can account for the unexpected . lengthening of "prothrombin time" when cottonseed o i l was administered by pipette. This effect is not likely to be mediated by vitamin K, since even i f synthesis of prothrombin was completely blocked no significant drop would be expected in one hour. (Quick and Collentine, 1951, found that prothrombin is about 80% catabolized in ZU hours). Neither corn o i l nor herring o i l produced an effect comparable to that of cottonseed o i l when administered by pipette. 17. EXPERIMENT VIII. Greaves and Schmidt (1937) and Greaves (1939) showed that the haemorrhagic tendency which follows' bile duct ligation in rats can be prevented by feeding bile. Bile itself has been shown to contain l i t t l e vitamin K activity (Greaves, 1939). These workers therefore concluded that bile stimulated the absorption of vitamin K. The ingestion of fats tends to stimulate the secretion of bile (Hawk, Oser and Summerson, 1947). One effect of dietary fats, there-fore, might be to improve vitamin K absorption through increased bile sectetion. To investigate this possibility, bile salts were fed to chicks receiving diets deficient i n vitamin K. Bile salts which have been shown to be present in the chicken include cholic acid, chenodesoxy-cholic acid, an as yet unidentified C H 0, acid, and the conjuga-27 40 o tion products of these acids with taurine (Deuel, 1955)• In the present series of tests, cholic acid was the f i r s t bile salt studied. In test 1, a wheat-fish meal diet (table 8-1) with and without the addition of 0.5$ of cholic acid was fed to chicks for 21 days. At the end of this period blood "prothrombin time" was measured. In the second test the effect of cholic acid and of beef tallow, alone and in combination, when added to a corn-fish meal diet (table 8:1) was studied. "Prothrombin time" was measured at 21 days. Schmidt (1938) inferred from studies of other fat-soluble vitamins that desoxycholic acid was the bile salt responsible 'T. 18. for vitamin K absorption. In the third test the effects of cholic acid, desoxycholic acid, and two levels of vitamin K (menadione) incorporated into a corn-fish meal diet (table 8:1) were compared. "Prothrombin time" was determined at 24 days. Results: The mean "prothrombin time" values for these three tests are summarized in table .8:2. Statistical analyses of the results are shown in table 8:3. Cholic acid produced an unexpected lengthening in "prothrombin time" in a l l three cases. In the f i r s t test this effect was statistically highly significant. In the second and third tests, with fewer birds used, the effect was not significant. Beef tallow produced a marked reduction in "prothrombin time" which was not modified in any way "b^r the addition of cholic acid. Desoxycholic acid produced . a slight shortening in prothrombin time" -which was not, however, statistically significant. DISCUSSION OF RESULTS. Desoxycholic acid has been shown to be the bile component necessary for the absorption of other fat soluble vitamins (Greaves and Schmidt, 1935) • The cholic acid of vitamin was absorbed in choledo-chocolonostomized rats (Kohrn and Schmidt, 1939), although i t had been demonstrated that bile was necessary for the absorption of this vitamin (Greaves, 1939). Fantl, Nelson and Lincoln (1951) showed that water-insoluble diacetyl 2-methylnaphthoquinone was absorbed in the absence of bile, but explained this on the basis of the relatively small size of the molecule. 19. If the effect of fat had been to increase the production of bile and thus to facilitate absorption of vitamin K, a similar effect might be expected of bile acids added to the ration. Even i f the specific bile acids studied in this experiment were not directly involved in vitamin K absorption, the administration of these acids would increase the secretion of other bile acids. "Bile salts absorbed from the intestine have a marked power to stimulate bile formation " (Hawk, Oser and Summerson, 1947). Since the addition of bile acids to the diet failed to shorten "prothrombin time", i t does not appear that increased bile secretion can explain the effect of fats on "prothrombin time*1. The effect of cholic acid in lengthening "prothrombin time" might be due to supression of bacteria synthesizing vitamin K. However, Best and Taylor (1945) state that "bile will serve as a suitable culture medium for microorganisms.11 Cholic acid and desoxycholic acid have been shown to differ in their effects on blood l i p i d level. In a series of studies, Byers and Friedman showed that cholic acid is the bile salt responsible for the hypercholesteraemia and hyperlipaemia which follows bile- duct ligation, and that other bile salts do not produce this effect (Byers, Friedman and Michaelis, 1950} Friedman, Byers and Michaelis, 1951} Friedman and Byers, 1951, 1952} Byers and Friedman, 1952). Swell e$ al (1953) showed that dietary cholic or taurocholic acids produced hypercholes-teraemia in rats receiving high-fat, high-cholesterol diets, while desoxycholic acid had considerably less effect. T Q determine whether 20. a direct effect on blood fat level could be responsible for the effect of cholic acid on "prothrombin time", a surface active agent known to induce hyperlipaemia in chicks was studied. 21. EXPERIMENT IX. The surface active agent Triton WR-1339^  has been shown to produce lipaemia in chicks (March and Biely, 1955)• Three tests were conducted to determine whether this effect is reflected in the "prothrombin time". In the f i r s t test, five chicks which had been fed a depletion diet for 24 days received 1.0 ml. of the surface active agent by sub-cutaneous injection. After 31 hours blood was drawn from these chicks and from five untreated chicks for "prothrombin time" determination. Similarly, in the second test "prothrombin time" of five birds injected with 1.0 ml. Triton and of four control birds was determined AO hours after injection of Triton. The birds were treated at 22 days. In the-'third test, 0.5 ml. Triton was injected into each of five birds at 21 days. "Prothrombin time" of these and of five untreated birds was determined after 42 hours. A wheat-fish meal ration-was used to deplete the birds of vitamin K in the f i r s t test. In the second and third tests a corn-fish meal diet was used. Results: "Prothrombin time" was prolonged in the chicks which had received Triton injections (table 9*1). In the fi r s t test this effect was statistically highly significant (table 9:2). In the second and third tests, however, due to the small numbers of birds used and to the extreme variability within groups, the differences were not statistically significant. 1 - A polymeric p-isooctyl polyoxyethylene. Courtesy of Rohm and Haas.Company, Philadelphia. 22. Test 4. To observe whether or not i t s effect on "prothrombin time" was a direct result of its presence in the blood, Triton was added to blood and to the thromboplastic agent in vitro. -A solution of 2.5$ Triton in 0.85$ saline ("Triton-saline") was added to blood or to the clotting agent at graded levels previous to the determination of "prothrombin time". Comparable dilutions were made with saline. 0.1 ml., 0.2 ml., 0.4 ml., and 1.0 ml. of saline or of Triton-saline were added to 1.0 ml. samples of normal blood. Similar additions of Triton-saline were made to 1.0 ml. samples of the clotting agent; these diluted agents were tested with undiluted blood. Results: The final concentration of the added saline or Triton-saline in the combined solution of blood+clotting agent, together with the "prothrombin time" of each solution, is shown in table 9:3. There is no apparent difference between the effect of Triton added to the blood or to the clotting agent, and that of saline alone. DISCUSSION OF RESULTS The effect of Triton WR-1339 in raising blood li p i d level has been demonstrated with ruminants (Chung and Shaw, 1951), mice (Cornforth e& a l . 1951)» rats (Friedman and Byers, 1953) rabbits (Kellner, Correll and Ladd, 1951) and chicks (March and Biely, 1955)• Several of these workers demonstrated an elevated blood cholesterol level; Friedman and Byers stated that the effect of Triton was similar to, but greater than that of cholic acid. The effect on "prothrombin time" observed in the present experiments was also similar to that of cholic acid. 23. Thus cholic acid and T riton, both of vrhich have been shown to cause elevation of blood lipid level, caused a lengthening of "prothrom-bin time". Oral administration of cottonseed o i l , which would be expected to produce alimentary lipaemia, likewise lengthened "prothrombin time". On the other hand, oral administration of corn o i l or herring o i l had no effect. Oral administration of corn o i l , olive o i l or cream accelerated prothrombin activation i n dogs (Duncan and Waldron, 1949). The administration of oestrogens, which would also be expected to elevate blood lipid, did not affect blood coagulation time of rabbits (Pellegrino and Solinas, 1952). Boulanger (1952) found that the lipaemia of turkey hens during the laying season did not influence blood coagulation. The elevation of blood l i p i d level per se cannot adequately explain the effects of Triton or of cholic acid in the present experiments. From the results of the chick tests i t is apparent that dietary fat has a complex effect on the one-stage "prothrombin time" of chicks, fed vitamin & deficient diets. The effect is dependent on the fat, and on the composition of the diet in which i t is used. It does not appear that any of the fats, with the possible exception of herring o i l , con-tained appreciable quantities of vitamin K. The possibility of increased microbiological synthesis of the vitamin cannot be dismissed, ssince herring o i l and corn o i l failed to affect "prothrombin time" when administered shortly before blood was drawn. On the other hand, oral administration of cottonseed o i l and injection of a surface active agent both caused a lengthening of Jlprothrombin time". These latter effects, at least, cannot be accounted for by an -effect on the microflora. 24. Moreover, i f the fat produced conditions which were favorable for active growth of bacteria, i t i s difficult to understand why the addition of 0.5$ cholic acid failed to influence in any way the effect of 8$ beef tallow. Cholic acid, with its strong surface action, could reasonably be expected to modify conditions in the intestine. An effect on intestinal absorption of the vitamin through increased bile secretion appears unlikely, since a high level of desoxycholic acid produced l i t t l e i f any effect on "prothrombin time." 25. EXPERIMENT X. The variations in "prothrombin time" observed in the previous experiments were not a reflection of the vitamin K content of the diet. The shortened "prothrombin time" which resulted when fat was included in the diet was not caused by increased bile secretion with resultant improved vitamin K absorption. The effect of Triton on "prothrombin time" probably could not have resulted from decreased intestinal synthesis of vitamin K. In the case of Triton, at least, i t appeared that some in vivo change was involved. The "prothrombin time" method used in these experiments was studied with a view to determining whether variables other than vitamin K could be expected to affect "prothrombin time". The recent litera-ture pertaining to blood coagulation suggested that several variables might be involved. Since much of this work deals with the coagulation of human blood, the possibility of species differences must be recognized. The Quick one-stage prothrombin test i s based on the following theory of coagulation: Thromboplastin i-Ca Prothrombin > Thrombin Thrombin Fibrinogen > Fibrin It is assumed that i f "thromboplastin" (tissue extract from brain, lung or thymus) and fibrinogen are present in excess, and Ca^ *" in optimum concentration (0.008 M), the clotting time is directly proportional to 26. the level of prothrombin present (Quick 1935, 1936, 1937). This concept of blood coagulation has received much criticism for more than a decade. Certain unexplained characteristics of the coagulation mechanism in this test led to the postulation of another component of the reaction. • This component has been variously named Thrombogen (Nolf, 1945), prothrombin accelerator (Fantl and Nance, 1946) , accelerator globulin (or Ac globulin) (Ware, Guest and Seegers, 1947) ,. prothrombokinase (Milstone, 1948), plasma prothrombin conversion factor (PPCF) (Stefanini, 1951), labile factor (Quick, 1943), plasmatic co-factor of thromboplastin (PCT) (Honorato, 1947), pro-accelerin and accelerin (Owren, 1951), and factors V and VI (Owren, 1947). Though there is considerable confusion in the use of nomenclature by different authors, the above-named compounds have certain characteristics in common. A l l are relatively labile, especially in the presence of oxalate. They are adsorbed bp aluminum hydroxide, are present in plasma and absent from serum. They are therefore generally classified as one compound (Biggs, 1951} De Nicola, 1953). In this report the nomenclature adopted by Ssoth Biggs and De Nicola, who refer to this compound or group of compounds as Factor V, will be followed. Another factor or group of factors involved in the Quick "prothrombin time" test includes co-thromboplastin (Mann, 1949), proconvertin-convertin (Owren, 1952), prothrombin conversion factor (PCF) (Jacox, 1949), prothrombinogen (Quick, 1949), serum prothrombin conversion accelerator (SPCA) (Alexander .§& al, 1949), and factor VII (Koller, Loeliger and DUckert, 1951). Once again following the 27. pattern of De Nicola (1953) and Macfarlane and Biggs (1955) this group will be referred to as Factor VII in this report. Factor Vii is relatively stable in fresh and stored blood, is not adsorbed by aluminum hydroxide, and is present in both plasma and serum. It is again emphasized that the factors referred to above are directly involved in the Quick "prothrombin time" test. Other newly-identified factors such as anti-hemophillic globulin (AHG), Christmas factor, platelet factor-(serotonin), and the postulated plasma thromboplastin antecedent (PTA) will be referred to in connection with the newer theories of blood coagulation, but are not directly involved in the one-stage prothrombin assay method under study. The classical theory of blood coagulation (Schmidt, 1895} Horowitz, 1905) has already been mentioned. The steps involved are s t i l l accepted as being a part of the true coagulation mechanism. However, the single term "thromboplastin" in the earlier theory has since been shown to include a number of important steps not previously recognized and not yet fully understood. If blood is drawn from a vein with precautions to avoid contamination from damaged tissue, coagulation will occur normally in from five to ten minutes. Further precautions (silicone-treated or collodion-lined glassware) to avoid the presence of a "wettable" surface will delay this coagulation indefinitely. Normal coagula-tion i s believed to be due to agglutination and rupture of the blood platelets on a wettable surface, with the release of a thromboplastic substance (Ware, Fahey and Seegers, 1943), or a substance involved 28. in thromboplastin formation (Biggs and Macfarlane, 1953). The addition of extracts of brain tissue, lung, thymus (Quick, 1936), or muscle (Almquist, 1941) will accelerate the reaction, presumably due to their thromboplastic action. Other compounds having such an effect include the venoms of certain snakes, particularly Russel1 s viper (Lamb, 1903). Seegers (1949) reported some activation of purified prothrom-bin by 25$ sodium citrate, and an almost quantitative conversion by the use of certain substituted diphenyl sulfones. This report is particularly interesting since i t shows the possibility of activation of prothrombin to thrombin in the absence of ionic calcium. The removal of calcium from blood by precipitation as the citrate or oxalate i s a common method of preventing jln. vitro coagulation of blood. In the absence of calcium, thrombin formation is blocked. It has been shown that snake venom requires the presence of li p i d to act as a thromboplastic agent (Macfarlane, 1938). Quick (1936) found that extraction with organic solvents reduced the activity of brain thromboplastin, and suggested the rupture of a lipoprotein. However Lein and Lein (1948) reported two thromboplastic agents in tissue, one protein and one l i p i d in nature. Moreover the brain thromboplastic agent used by Biggs (1951) is extracted repeatedly with acetone: the action of this agent i s not dependent on the amount of li p i d present. The steps preceding the activation of prothrombin are not yet understood. It is clear that the reactions in whole blood 29. coagulation .in vitro are not identical with those of the Quick "prothrombin time" test, since "'Thromboplastin' formation occupies most of the 'clotting time'" (Macfarlane, 1955). Macfarlane and Biggs (1955) postulate the following series of reactions for coagula-tion of mammalian blood: on contact with a foreign surface an interaction i s i n i t i a t e d which involves the platelets, antihaemophilic globulin (AHG) and the Christinas factor. Schematically, Christmas factor AHG 4 platelets ^ Intermediate product Ca 4 4" Factor VII Intermediate product + Factor V Thromboplastin Ca*+ Thromboplastin, Ca44> Prothrombin Thrombin Thrombin Fibrinogen )> Fibrin (Composite scheme from Macfarlane and Biggs (1955), Biggs (1955), and Macfarlane (1955)). Marbet and l i n t e r s t e i n (1954) suggest that clotting involves five steps or phases: Prephase - agglutination and vasoconstriction by serotonin from platelets. F i r s t principal phase - thromboplastin formation. Second principal phase - thrombin formation. Third principal phase - f i b r i n formation. Postphase - retraction and fi b r i n o l y s i s . I t i s important to note that both these groups use the term "thromboplastin" i n a new sense. I t i s now considered to be a 30. "potent but transitory" agent produced by a series of reactions, and not a constituent of tissues or of platelets, as earlier believed. "Tissue extract" is believed to be similar to the hypothetical "intermediate product", in its thromboplastic action (Biggs, Douglas and Macfarlane, 1953). Andreenko and Kudryashov (1955) have recently reported that the thromboplastic activity of the blood is increased when vitamin B^ Is injected into white rats. Graham £L (195A) have suggested that platelets and antihaemophilic globulin are also involved in the "prothrombin time" test. If the above scheme is accepted as a working hypothesis for the coagulation of avian blood i t is readily apparent that the Quick "prothrombin" assay includes steps two to four, with the substitution of tissue extract for the "intermediate product". Thus the test will measure variations in any or a l l of Factor V, -Factor VII, prothrombin, Ca'' or fibrinogen. Deficiencies of Factor V or -Factor VII may be hereditary or acquired. They may also be affected by development of inhibitors of thromboplastin formation (Macfarlane and Biggs, 1955)• The measurement of prothrombin is further complicated by the indica-tion thatprothrombin deficiency may not cause much lengthening of the one-stage method" (Biggs and Douglas, 1953). De Nicola (1953) states that variations in prothrombin time are correlated with variations in Factor VII. Several other assay methods for prothrombin have been devel-oped in an attempt to overcome these difficulties. Most are classified as"two-stage" methods as distinct from Quick*s "one-stage" assay. Of 31. this type are the procedures of Warner, Brinkhous and Smith (1936), Herbert (1940), Ware and Seegers (1949), Sternberger (1947), and Biggs (1951). In these methods diluted plasma is incubated with tissue extract and samples are removed periodically for addition to a fibrinogen solution. When the clotting time of the fibrinogen reaches a minimum the conversion of prothrombin to thrombin i s considered to be complete. These methods separate steps two and three from step four in the postulated scheme. They have the advantage of measuring the rate of prothrombin activation and of assaying maximum thrombin level, neither of which i s accomplished by the one-stage technique. Unfortunately the maximum thrombin level is not an accurate assay of prothrombin concentration, particularly in cases of delayed activation, due to the presence of an antithrombic agent in plasma (Quick, 1944). Thus the thrombin present at any given time is a measure of thrombin produced minus that which has been inactivated by anthrombin (Biggs, 1951) • %en a deficiency of either •factor V or Factor VII exists, the rate of activation will be reduced. Anti-thrombin will then have a greater opportunity to inactivate the thrombin produced, resulting in a lower final concentration than i f the activation rate were normal. This delay can be eliminated by adding concentrates of the deficient factors, and at the same time a distinction may be made between the two. Factor V is present in aluminum hydroxide-treated normal plasma, while prothrombin and Factor VII are adsorbed and 32. removed, factor VII, which i s not consumed i n coagulation, i s present i n normal serum, while Factor V and prothrombin are not. Thus the addition of either or both, serum or Al(OH) adsorbed plasma w i l l 3 provide the missing activators (Biggs and Macfarlane, 1953). From the foregoing i t i s apparent that a quantitative measure of prothrombin l e v e l cannot be made by the one-stage test. An assess-ment of the level of this constituent requires that a two-stage test be performed i n the presence of an excess of Factors V and VII. I f thrombin level (asmmeasured by the time required to clot fibrinogen) i s plotted against time, the area under the curve gives a reasonable estimate of the prothrombin originally present (Biggs and Macfarlane, 1953). However, i n most cases i t i s the presence or absence of a coagulation defect which i s to be determined. For this purpose the simple one-stage test i s of great value provided i t i s regarded as an empirical measure of several factors, rather than as a quantitative measure of prothrombin. A possible explanation for the results obtained i n the present series of experiments i s suggested by Macfarlane's (1938) observation that a thromboplastic agent prepared from the venom of Russel* s viper i s influenced by blood l i p i d l e v e l . This effect, which can be overcome by addition of l e c i t h i n to the agent, i s not evident i n the thromboplastic preparation from brain tissue. A series of tests was performed to determine whether or not the breast muscle preparation used i n the previous tests differed significantly from brain extract i n thromboplastic properties. 33. EXPERIMENTAL "Prothrombin-deficient" blood was obtained from chicks 20-35 days of age which had been depleted of vitamin K by the method previously described. A corn-fish meal diet was used in a l l cases (see table 3:1). Normal blood was obtained from chicks which had been receiving a commercial diet containing green feed. Test 1. The breast muscle clotting agent described previously was diluted with 0.85$ saline and with a 1$ emulsion of soybean lecithin (mixed soybean phospholipids) in 0.85$ saline. 10$, 20$, 30$, 40$, 50$ and 67$ of lecithin-saline and of saline was added to the normal preparation. An equal volume of M/40 C a C l 2 w a s 8 - 1 8 0 a d d e d t o provide the same calcium ion concentration in each solution. "Prothrombin time" of blood from a normal and a vitamin K deficient bird was measured with the normal agent and with each dilution. Results: The composition of each clotting agent, together with observed "prothrombin time", is shown in table 10:2. The results are illustrated graphically in figure 1. Addition of saline alone pro-duced progressive increases in "prothrombin, time", with only minor irregularities. Addition of lecithin-saline, on the other hand, caused a decreased ^prothrombin time", at the lower concentrations, in both normal and "prothrombin-deficient1 blood. As the amount of added lecithin was increased, the "prothrombin time" also increased, but at 34. no time was i t as long as that of the comparable saline dilution. In the case of the blood from the vitamin K deficient chick, the clotting agent diluted with twice its volume of lecithin-saline (ignoring the M/AO CaCl^ content) produced a "prothrombin time" shorter than that pro-duced by the undiluted agent. Test 2. Procedure: Brain tissue thromboplastic agent prepared by the method of Herbert (1940) is not sensitive to blood li p i d level, according to Biggs (1951)'. A preparation of this type was subjected to saline and to lechithin-saline dilution. The procedure followed for preparation of the agent was as follows5 •Fresh chicken brain was macerated in four times its volume of acetone in a mortar. The acetone was filtered off and discarded. This procedure was repeated four times. The final granular powder was allowed to dry. For use, 0.5 gm. of the powder was suspended in 5.0 ml. of 0.85% saline and incubated, with occasional shaking, for 15 minutes at 38° c . The mixture was centri-fuged briefly, and the supernatant diluted to five times its volume with 0.85% saline. The determination of "prothrombin time" as described by Biggs and Macfarlane (1953) differed from the method used with breast muscle thromboplastic agent in that the agent was added to the blood sample separately rather than with M/AO CaCl^. That is, 0.1 ml. of clotting agent was added to 0.1 ml. of blood in a vial. The mixture was warmed 35. briefly in a water bath at 38,5-39.0°C. 0.1 ml. M/40 CaCl 2 was then added and a stopwatch was started at the same time. As in the previously described method, the time required for formation of a definite fibrin film was recorded as the "prothrombin time". The clotting agent described above was diluted by the addition of saline or of lecithin-saline to make up 10$, 20$, 40$, 50$ and 6^ of the final solutions. These dilutions were then used to measure "prothrombin time" of blood from a normal and a vitamin K deficient chick. Results* "Prothrombin times" are shown in table 10:3, and are illustrated graphically in figure 2. The results differ considerably from those obtained with breast muscle thromboplastic agent (test 2). In the case of the brain tissue preparation, the addition of increasing quantities of lecithin-saline produced a small but steady increase in "prothrombin time" in blood from both the normal and the vitamin K deficient chick. On the other hand, saline dilution up to one-third the original concentration failed to affect "prothrombin time". Test 3. In tests 1 and 2 the methods of "prothrombin time" determina-tion differed. In the fi r s t test, Ca^ ** d thromboplastic agent we're added to the blood as one solution. In the second test the Ca hh was added to a mixture of blood and thromboplastic agent. In test 3 11. ' the effect of the method of adding CafT on "prothrombin time*" was studied. 36. (a) A solution of breast muscle thromboplastic agent, prepared as described previously, was diluted with the same quantities of saline and of lecithin-saline as in the previous two tests. To an aliquot from each dilution was added an equal volume of M/AO CaCl "Prothrombin time" was determined by (i) adding 0.2 ml. of agent + CaCl 2 solution to 0.1 ml. of blood, and (ii) adding 0.1 ml. of M/AO CaCl 2 to 0.1 ml. of blood 4 °*1 m1' o f a S e n t ' Results: "Prothrombin time" values are shown in tabid 10:3, and are illustrated in figure 3. The results are similar to those in test 2, except that addition of CaT^ to the thromboplastic agent appeared to increase the effect of lecithin in lengthening "prothrombin time". (b) Breast muscle thromboplastic agent was prepared in the manner previously described except that M/AO CaCl 2 was not added. Saline and lecithin-saline dilutions with and without added C a C l 2 were prepared and "prothrombin time" determinations carried out as described in test 3 (a). "Calcium time" of blood samples was also measured, by adding 0.1 ml. saline or lecithin-saline and 0.1 ml. M/AO CaCl^ to 0.1 ml. blood. Results: "Prothrombin time" values are shown in table 10:3, and are illustrated in figure 4« The results were somewhat more variable than in previous tests, but the pattern was similar to that of test &. Addition 37. of CaCl^ to the clotting agent rather than to the blood appeared to increase the effect of dilution i n a l l casesj under these conditions l e c i t h i n had much less effect i n shortening "prothrombin time". In the determination of "calcium time", when no thromboplastic agent was added, blood samples containing lecithin-saline had shorter clotting times than those containing saline only. Test 4. In order to obtain a high concentration of l e c i t h i n i n an undiluted thromboplastic agent, the 1$ lecithin-saline emulsion was used i n place of saline for the extraction of tissue thromboplastic agents. These extracts were compared to saline extracts from the same tissue. The thromboplastic agents used i n this test were breast muscle extract and dried brain tissue extract, prepared as described previously, and the same agents prepared by using lecithin-saline i n place of saline throughout. The same weight of tissue was used for preparations with and without l e c i t h i n . These extracts were used to measure "prothrombin time" of blood samples from five normal and five vitamin K deficient chicks. CaCl was added separately to the blood -f clotting agent. 2 . Results. The individual and mean "prothrombin time" values, as determined using each agent, are shown i n table 10:4« I t i s apparent that l e c i t h i n affected the response to the breast muscle preparations, particularly i n testing the blood of vitamin K deficient chicks. On the other hand, l i t t l e i f any effect of l e c i t h i n on the brain tissue preparation was observed. The "sensitivity" of the breast muscle preparation appeared to be reduced by lecithin. That is, mean "prothrombin time" of the normal birds was 21 seconds when measured with saline extract of breast muscle; for the vitamin K deficient birds the mean value was 61 seconds. When lecithin-saline was used as solvent, the value for normal birds was 17 seconds, and for vitamin K deficient birds, only 36 seconds. The brain powder extract was much less affected. Mean "prothrombin time" figures with saline and lecithin-saline prepara-tions were 14 and 16 seconds, respectively, for normal blood, and 53 and 50 seconds respectively for "prothrombin-deficient" blood. It is possible, of course, that the use of a phospholipid solution for extraction of breast muscle produced a more powerful thromboplastic solution than did the normal saline extraction. Test 5. It was desired to determine whether or not the methods of preparation of tissue thromboplastic agents affected the response of the agents to lecithin, fresh breast muscle was.therefore dried with acetone according to the procedure described previously for brain tissue. The powder thus obtained was prepared for use as follows; 0.5 gm. powder was extracted with 5 ml. or 0.85% saline, as for dried brain tissue. 4s a preliminary test to determine the optimum concentration of the agent, the resultant solution, and dilutions of the extract at 50%, 20%, 10% and 5% of i t s original concentration 3 9 . (saline used as diluent) were used to determine the "prothrombin time" of normal blood. The "calcium time" (0.1 ml. saline 4- 0.1 ml. M/AO CaCl^ added to 0.1 ml. blood) was also determined. The following thromboplastic agents were used to determine "prothrombin time" of blood samples from five normal birds: saline extract of fresh breast musclej saline extract of fresh brain tissue;n saline extract of dried breast muscle powder: saline extract of dried brain tissue powder; and the corresponding lecithin-saline extracts in each case. The extracts of dried breast muscle powder were diluted 1:5 as in the case of extracts of dried brain tissue. 0.1 ml. M/40 CaCl^ was added separately in making the determinations. Results: The preliminary assay of the dried breast muscle powder indicated no thromboplastic activity (table 10:5)• Individual and mean "prothrombin time" values of blood from five normal birds confirmed this observation (table 10:6). The decrease in mean "prothrombin time" when breast muscle powder in lecithin-saline was used may be a function of lecithin alone or i t may be an effect of the solvent action of lecithin. Lecithin again shortened the "prothrom-bin time" values obtained with the fresh breast muscle preparation. A slight prolongation of "prothrombin time" was noted on addition of lecithin to either brain preparation. Test 6. Procedure: The thromboplastic solutions prepared from fresh and from dried tissue represented different tissue content on a dry-weight basis. 40. In other words, the extract from 10 gm. fresh breast muscle, which contained approximately 3. gm. dry matter, was made up to 200 cc. of solution, representing l-»5 gm. dry inatter/lOO ml. In the case of dried brain tissue the extract from 0.5 gm. was made up to 25 ml., represen-ting 2.0 gm. dry matter /100 ml. To determine whether or not the difference i n the amount of dry matter extracted would account for the difference i n unit thromboplastic ac t i v i t y of the extract, extracts of breast muscle were prepared at 4/3 the usual concentration. Saline and lecithin-saline extracts of breast muscle and of dried brain powder were diluted 1:10, 1:100, 1:1000 and 1:10000 with the appropriate diluent. "Brothrombin time" of blood from a normal and from a vitamin K deficient chick was measured using each dilution of each preparation. '"Calcium time" was also measured. Results 1 The more concentrated breast muscle extract f a i l e d to show thromboplastic a c t i v i t y equal to that of dried brain tissue extract (table 1 0 : 8 ) . Serial dilution of the clotting agents showed that brain extract retained some ac t i v i t y when diluted 1:1000, while breast muscle extract did not have any effect at that dilution. Addition of l e c i t h i n reduced the effectiveness of flooth agents. Lecithin-saline extract of breast muscle had no effect at dilutions greater than 1:10, while the lecithin-saline extract of dried brain powder appeared to retain slight a c t i v i t y at the 1:100 dilution. Test 7» Quick (1936) reported that tissue thromboplastic agents were destroyed by extraction with organic solvents. In the present experiments breast muscle failed to show thromboplastic activity after extraction with acetone. The activity of brain tissue, on the other hand, was not affected by this treatment. In order to determine whether the thromboplastic component of breast muscle was destroyed or was extracted by acetone, the activity of the acetone extract was tested, five grams of fresh breast muscle was dried with five 20 ml. portions of acetone. The dried extracted powder weighed 1.5 gm. The acetone washings were evaporated under reduced pressure at room temperature until the odour of acetone could no longer be detected. The volume of the acetone extract was 3.3 ml. In the method of Biggs and Macfarlane (1953) the thrombo-plastic extract is prepared from 0.5 gm. of dried tissue. Thus one-third of the powder prepared from breast muscle in the present test was sufficient to prepare a thromboplastic solution by that method. If a l l activity had been extracted by the acetone, therefore, one-third of the concentrated acetone extract should have contained sufficient activity to prepare.a thromboplastic solution. Following this reasoning, two thromboplastic solutions were prepared, '^or the f i r s t of these, 0.5 gm. of dried breast muscle was extracted with 5 ml. saline, as described previously. The second solution was pre-pared by diluting one-third of the concentrated acetone extract ( l . l ml.) to 5 ml. with saline. The thromboplastic activity of these two solutions was studied by using each of them to measure "prothrom-bin, time" of a sample of normal blood. The "calcium time" of the 42. blood sample was measured for comparison. Results* 4 S in the previous test, the extract of acetone-dried breast muscle had no thromboplastic activity. "Prothrombin time" using this solution was 76 seconds, compared to a "calcium time" of 75 seconds (averages of duplicate determinations). The agent prepared from the acetone extract, on the other hand, showed some activity, producing a "prothrombin time" measurement of 43 seconds. third thromboplastic agent was prepared by combining equal volumes of each of the previous two agents. "Prothrombin time" using this solution was 60 seconds. Since "prothrombin time" increases as the concentration of the thromboplastic agent exceeds the optimum level (Biggs and Macfarlane, 1953), the solution prepared from the acetone extract was diluted 1:5 with saline. "Prothrombin time" using the diluted agent was 48 seconds. DISCUSSION OF RESULTS The breast muscle thromboplastic agent differed from the agent in brain tissue in several respects. The brain tissue preparation was not generally affected by dilution to one-third i t s normal concentra-tion. This dilution gave an extract equivalent to 1 gm. dry matter 'extracted by 150 ml. saline. Breast muscle thromboplastic agent, on the other hand, was appreciably weakened by the addition of as l i t t l e as 20% saline, equivalent to 1 gm. dry matter extracted by 100 ml. Thus the concentration of thromboplastic activity per unit dry matter was apparently greater in brain tissue than in breast muscle. 43. A second difference between the two agents was evident in their reaction to the addition of lecithin. An extract of acetone-dried brain tissue suffered a gradual loss of activity as increasing concentrations of lecithin were added. That this was not a direct effect of dilution is shown by the fact that equal additions of saline produced no effect. On the other hand, the activity of the extract from fresh breast muscle was increased by lecithin. This latter effect was particularly noticeable in blood with a prolonged "prothrombin time" from vitamin K deficient chicks. Macfarlane (1938) showed that the thromboplastic activity of the venom of Russel1s viper was dependent upon blood l i p i d level unless lecithin was added. Biggs, Douglas and Macfarlane (1953) showed that lipoid level i s a factor in the thromboplastic activity of trypsin. The difference in the response of brain tissue extract and of breast muscle extract to lecithin is probably due to the fact that brain contains considerably more phospholipid than does skeletal muscle. In the rat phospholipid makes up 21-27$ of the dry matter of brain, and only 6-9$ of the dry matter of skeletal muscle (Deuel, 1955). Since phospholipids are insoluble in acetone they would not be removed by the drying process in the present tests. Presumably there is an optimum phospholipid level for thromboplastic activity, since the addition of lecithin solution to the brain extract reduced its activity. •Quick (1936) reported that the effectiveness of tissue thrombo-plastic agents was reduced by extraction with organic solvents, possibly due to the rupture of a lipoprotein. In the present series of tests i t was found that extraction of the tissue with acetone extracted and partially destroyed the agent present in breast muscle, but did not affect that present in brain tissue. On a dry-weight basis the unit activity of acetone-dried brain tissue appeared to be at least equal to that of fresh brain tissue. This i s surprising, since%ick found the agent in brain tissue to be more labile in the presence of organic solvents than was the agent in other tissues. The difference may be due to the fact that Quick used solvents which would extract phospholipids. The possibility of differences between mammalian and avian tissues must also be considered. The addition of lecithin appeared to reduce the sensitivity of breast muscle thromboplastic agent in the test for vitamin K deficiency. The addition of lecithin shortened average "prothrombin time" by 15 seconds (25$) with breast muscle thromboplastic agent, and by only 3 seconds (6$) with brain tissue thromboplastic agent. It was not possible to judge which method of assessment gave a more accurate indication of a haemorr-hagic tendency. The addition of Ca*"*" to the thromboplastic agent rather than to a mixture of blood and thromboplastic agent generally resulted in prolonged "prothrombin time" measurements. This treatment reduced the ability of leciuthin to shorten "prothrombin time" measurements with the breast muscle agent. Dilution of the two agents indicated that the brain preparation had about ten times the thromboplastic activity of the breast muscle 45. preparation. In the presence of lecithin, the minimum concentration of either agent which showed thromboplastic activity was about 10 times as great as when lecithin was not added. It appeared that some of the thromboplastic activity of breast muscle was extracted by acetone. The greater part, however, was destroyed during the acetone treatment. The thromboplastic solution prepared from the acetone extract of breast muscle represented a concentration of 10 gm. dry matter extracted per 100 ml. agent, compared to the usual saline extract of fresh muscle which represents a concentration of 1.5 gm. dry matter per 100 ml. Nevertheless, this highly concentrated acetone extract showed less activity than the saline extract. That this low potency was not the result of too high a concentration of thromboplastic agent i s shown by the fact that dilution of the solution decreased i t s activity. "Reconstituting" the breast muscle agent, by adding the saline extract to the acetone extract, failed to restore normal activity. The mixed solution, in fact, had less activity than did the acetone extract alone. This loss of activity was not the result of the 1:2 dilution of the acetone extract, since 1:5 dilution of this extract with saline gave a smaller loss of activity. No explanation is offered for the decreased activity when the two extracts were combined. EXPERIMENTAL There appeared to be a possibility that some of the effects on "prothrombin time" observed in the present series of chick tests were the result of sensitivity of the thromboplastic agent to the blood l i p i d level. An experiment was designed to examine this possibility. 46. EXPERIMENT XI. The effect on "prothrombin time" of two bile salts added to a vitamin K deficient ration was measured using two different tissue thromboplastic agents. Cholic acid and desoxycholic acid were added to a corn-fish meal ration (table 11*1)' and fed to chicks as in previous tests. Blood for "prothrombin time" determinations was drawn at 24 days. The two thromboplastic agents used were the fresh breast muscle extract used in the previous series of chick tests, and the extract of dried brain tissue described above. Results: "Prothrombin time" of 7-9 chicks from each group, measured with the two thromboplastic agents, is shown in table 11:2. It is not known why the experimental diet failed to produce a satisfactory depletion of vitamin K in this test? As far as i s known, conditions were the same as those prevailing i n previous tests, in which a marked depletion was generally reached within 3 weeks. The brain tissue thromboplastic preparation appeared to have lost some of its activity. The dry powder had been prepared two months previously, and had not showed any decrease in activity during the intervening period. Biggs and Macfarlane (1953) state that the activity of the powder is not lost in three months at room temperature. Both cholic and desoxycholic acids produced lengthening effects as measured by both agents. The effect of cholic acid was statistically significant with brain tissue, but not significant with breast muscle: that of desoxycholic acid was significant in each case (table l i s 3 ) . 47. DISCUSSION OF RESULTS The effect of the bile acids on "prothrombin time" was not dependent on the thromboplastic agent used. I t appears unlikely, therefore, that their effect can be ascribed to a change i n blood l i p i d level to which the breast muscle was sensitive. The effect of bile salts could be due to some adverse effect on microbiological synthesis of vitamin K, although i t i s not apparent from this explanation why cholic acid should have fai l e d to modify the effect of beef tallow on "prothrombin time" (table 8*2). CONCLUSIONS A series of experiments was conducted to observe the effect of dietary fat on the vitamin K requirement of the chick. The criterion of vitamin K status used in these experiments was the "prothrombin time" of the blood. It was possible to produce a '"prothrombin deficiency" in chicks by feeding diets which consisted of unextracted natural ingredients. The extent of this deficiency was affected by the maternal supply of vitamin K. In some.cases the coagulation defect was sufficiently severe to cause fairly heavy mortality from haemorrhaging. Certain fats, when added to the diet, protected the chick from a vitamin K deficiency. Beef tallow, hydrogenated cottonseed o i l , and. to a lesser extent cottonseed o i l and hydrogenated animal fat a l l shortened blood "prothrombin time" of chicks, when added to a vitamin K deficient diet. These fats prevented the haemorrhaging which sometimes resulted from vitamin K depletion. Herring o i l generally did not shorten "prothrombin time" unless the vitamin K deficient diet was also deficient in folic acid. The effect of fats on "prothrombin time" was apparently not due to the vitamin K content of the fat, nor to an increased absorption of vitamin K resulting from increased bile secretion. Oral administration of oils shortly before drawing blood did not affect "prothrombin time" of blood from vitamin K deficient chicks: this indicates that lipaemia per se did not cause the shortening in 49. "prothrombin time" observed after feeding a high-fat diet. Free fatty-acids added to a vitamin K deficient diet did not affect "prothrombin time" to a significant degree. Addition of choline to a vitamin K deficient diet did not affect "prothrombin time". This observation agrees with those of Field and Dam (1945) hut disagrees with those of Honorato and Moline (1942). The addition of methionine also failed to>affect "prothrombin time". Addition of folic acid to a diet deficient in both vitamin K and folic acid sometimes shortened "prothrombin time". Herring o i l also had a shortening effect when added to such a diet, but not when added to a diet containing adequate folic acid. Cholic acid added to a vitamin K deficient diet caused a lengthened "prothrombin time". Desoxycholic acid had a similar effect in one test, but had no effect in another case. Injection of chicks with the surface active agent Triton WR-1339 likewise lengthened "prothrombin time". Triton had no effect, however, when added to blood j j i vitro. Breast muscle and brain tissue differed in the thromboplastic properties of their extracts. The breast muscle preparation required the addition of phospholipid for maximum thromboplastic activity. Acetone partly extracted and partly destroyed the thromboplastic agent of breast muscle, but did not affect that of brain tissue. Although bile salts may affect blood phospholipid levels, their effect on blood "prothrombin time" was not due to the sensitivity of the breast muscle extract to phospholipid. The "prothrombin time" of blood, as determined with breast muscle thromboplastic agent, is not necessarily a measure of the vitamin K supplied to the chick. A P P E N D I X TABLE 1*1. Experimental Diets - Experiment I. Ingredients common to a l l Rations Ingredient % Ground Wheat 42.25 Ground Yellow Corn 5.0 Pulverized Oats 5.0 Soybean Oil Meal (44$) 22.0 Herring Meal 5*0 Meat Meal 5.0 Bone Meal 1.0 Limestone 1.0 Salt (Iodized) 0.5 Feeding Oil (2200A, 300 D j 0.25 4 0.0125 Riboflavin 0.2 gm. Niacin 1.0 gm. Calcium Pantothenate 0.5 gm. Folic Acid 0.035 gm. 87.0 Composition of Individual Nations. Ingredient Pounds per Ration Premix (as above) 1 2 3 4 5 6 52.2 52.2 52.2 52.2 52.2 52.2 Cerelose 4.8 4.8 4.8 Soybean Oil Meal 0.9 0.9 — — 0.9 0.9 Ground Wheat 2.1 2.1 2.1 2.1 Menadione 0.05gm. 0.05gm. Dehydrated Cereal Grass 3.0 3.0 Herring Oil — - 4.8 4.3 4.3 60.0 60.0 60.0 60.0 60.0 60.0 TABLE -1:2. Mean "Prothrombin Time" - Experiment 1 (•Figures in parentheses represent number of birds tested) Group 1. Males Depleted Stock Undepleted Stock Females Depleted Stock Undepleted Stock 2. Males Depleted Stock Undepleted Stock Females Depleted Stock Undepleted Stock 3. Males Depleted Stock Undepleted Stock Females Depleted Stock Undepleted Stock A* Males Depleted Stock Undepleted Stock Females Depleted Stock Undepleted Stock 5. Males Depleted Stock Undepleted Stock Females Depleted Stock Undepleted Stock 6. Males Depleted Stock Undepleted Stock Females Depleted Stock Undepleted Stock Control Bird Supplement to Basal Ration None Menadione Dehydrated Cereal Grass Herring Oil Herring Oil Menadione Herring Oil Dehydrated Cereal Grass. Mean "Prothrombin Time" in Seconds.  39 31 35 30 28 31 31 29 30 27 28 30 A9 37 AO 35 28 30 .31 29 30 28 29 29 25 5) 5) 5) 5) 5) 5) 5) 5) 5) 5) 5) 5) 5) 5) 5) 5) 5) 5) 5) 5) 5) 5) 5) 5) TABLE 1:3 Analysis of Variance - Experiment I Males. Source of Variation SS df Variance l\£tt Total 3424 59 Rations 1897 5 379 22.9 Jte Source of Chicks 216 1 216 12.5 i t Rations X Source _ 4 i 5 - 5 93 5.4 Error 346 48 17.6 Least Difference required for Significance = / / 2 X Error Variance X "t" for group d.f, / tr Individuals / Group MJ * L.S.D. rations 4 6 L.S.D. sources 2 3 L.S.D. rations X sources 7 12 Females. Source of Variation SS df Variance iifii Total 934 59 Rations 547 5 109 20.8 M Source of Chicks 52 1 52 9.9 A* Rations X Source 82 _5 16 3.0 * Error 253 49 5.25 J * L.S.D. Rations 2 3 L.S.D. sources 1 2 L.S.D. rations X sources 4 7 TABLE 2:1. Experimental Diets - Experiment II Ingredients common to a l l rations: Ingredient % Ground % e a t 70.75 Herring Meal 19.0 Salt (Iodized) 0.5 Limestone 1.25 Choline Chloride (25%) 0.25 Feeding Oil (2250 A, 300 D) 0.25 10.0 gm. 0.16 gm. 0.50 gm. 0.80 gm. Composition of Individual Rations: Ration # Amount per Ration Premix (lbs) (as above) Cerelose (lbs) Herring Oil (lbs) Folic Acid (gms.) Menadione (gms.) 1. 46.0 4.0 2. 46.0 4.0 0.018 3. 46.0 4.0 0.045 4. 46.O 4.0 0.018 0.045 5. 46.0 4.0 6. 46.O 4.0 0.018 7. 46.O 4.0 3T— 0.045 8. 46.0 — — 4.0 0.018 0.045 MnS(D Riboflavin Calcium Pantothenate Niacin 92.0 TABLE 2:2 Mean ^ eights of Chicks - Experiment.II. (Figures in parentheses indicate number of birds per group) Supplement to Weight in grams Group # Basal Ration Test 1 Test 2 (23 days) (2$ days) 1 None 262 (15) 207 (18) 2 Folic acid 306 (14) 257 (18) 3 Menadione 217 ( 9) 238 (17) A Folic acid + Menadione 281 (15) 257 (18) 5 Herring o i l 215 (11) 214 (16) 6 Herring o i l * Folic acid 273 (15) 242 (17) 7 Herring o i l •*• Menadione . 233 (15) 206 (14) 8 Herring o i l •»• Folic acid * Menadione 323 (16) 257 (17) TABLE 2:3 Mean "Prothrombin Time" - Experiment II (Figures in parentheses indicate number of birds per group) Supplement to "Prothrombin Time" Group # Basal Ration in seconds. Test 2 (33 days) 1 None 62 (10) 92 (10) 2 Folic Acid 63 (10) 72 (10) 3 Menadione 38 (9) 38 ( 6) 4 Folic Acid t-Menadione 34 (10) 44 ( 9) 5 Herring o i l 52 (10) 72 (10) 6 Herring o i l +• Folic acid 44 (10) 64 ( 9) 7 Herring o i l r Menadione 36 (10) 40 (10) 8 Herring o i l r Folic acid + Menadione 34 (10) . 36 ( 9) Control Bird 30 30 1 TABLE 2:A Analysis of "Prothrombin Time" Variance - Experiment II Source of Variation Total Treatment Error L.S.D. treatments Test 1. SS 25242 14997 df 78 _2 71 Variance 1463 211 15 21 6.93 AA Source of Variation Total Treatment Error L.S.D. treatments Test 2 . SS 41625 28055 13570 df 72 65 variance 4007 208 16 23 il £11 19.2 Aft TABLE 2:5 Effect of Herring O i l on "Prothrombin Time" - Experiment II (Figures i n Parentheses indicate number of birds treated) "Prothrombin Time" i n Seconds Basal Diet r 8% Basal Diet Herring on 1 45 (14) 44 (15) 2 36 (10) 40 (10) TABLE 3:1 Experimental Diets - Experiment III. Ingredients common to a l l rations: Ingredient % Ground Yellow Corn 71.5 Herring Meal 18.75 Iodized Salt 0.5 Limestone 1.25 Choline Chloride (25%) 0.25 Feeding Oil (2250 A, 30b D) 0.25 MnSO^  10.0 gm. Riboflavin 0.16 gm. Calcium Pantothenate 0.50 gm. Niacin 0.80 gm. 92.5 Composition of Individual Rations in Pounds* Hydrogenated Hydrogenated Premix Cottonseed Cottonseed Animal fat Folic ierelose Oil Oil Acid (gms, Ration (as above) 1. 55.5 2. 55.5 3. 55.5 4. 55.5 5. 55.5 6. 55.5 7. 55.5 8. 55.5 4.5 4.5 0.021 4.5 0.021 4.5 0.021 4.5 0.021 TABLE 3:2. Mean "Prothrombin Time" - Experiment III. (Figures in parentheses indicate number of birds tested) Group Supplement to "Prothrombin Time" Basal Diet Seconds. 1. None 50 (10) 2. Folic Acid 50 (10) 3. Cottonseed Oil 34 (10) 4 . Cottonseed Oil T F o l i c A c i d 2 9 ( 1 0) 5. Hydrogenated Cottonseed Oil 23 (10) 6 . Hydrogenated Cottonseed Oil + Folic Acid 21 (10) 7. Hydrogenated Animal Fat 31 (10) 8. Hydrogenated Animal Fat 4. Folic Acid 24 (10) Control Bird 24 TABLE 3:3 Analysis df "Prothrombin Time" Variance - Experiment III. Source of Variation SS df Variance .ft. Total Treatment Error L.S.D. Treatments 12085 9215 2870 79 _2 72 1316 39 33.7 3k 6 TABLE 3:4 Mean 28-day Weights of Chicks - Experiment III (Figures in Parentheses indicate number of birds per lot) Supplement to Mean Body Group Basal Diet Weightc-O grams) 1 None 239 IA 240 240 2 Folic Acid 324 2A J12 318 y. Cottonseed Oil 256 3A 258 257 4 Cottonseed Oil + Folic Acid 330 44 ML 327 5 Hydrogenated Cottonseed Oil 241 54 222 259 6 Hydrogenated Cottonseed Oil *• 335 6A Folic Acid .3J1 333 7 Hydrogenated Animal Fat 233 7A 244 238 8 Hydrogenated Animal Fat * 3A Folic Acid 310 .318 314 14) 15) 18) 16) 19) 21) 19) 21) 20) 22) 22) 21) 20) 19) 21) 22) TABLE 4:1 Experimental Diets - Experiment IV. Ingredients Common to a l l Rations: Ingredient Ground Yellow Corn Soybean O i l Meal (44%) Meat Meal Limestone Bone Meal Salt (Iodized) Feeding O i l (2250 A, 300 D) MnSO Riboflavin Calcium Pantothenate Niacin Folic acid B 12 Test 1 % 51.5 37.0 1.0 .2.0 0.5 10.0 gm. 0.16 gm. 0.50 gm. 0.80 gm. Vitamin A (10,000 IU/gm.) Vitamin D (1650 ICU/gm.) 92.0 20.0 81.3 gm. gm. Ration Test 1 1 2 3 4 5 6 7 8 9 10 11 12 13 Composition of Individual Diets i n Pounds: Choline Folic Premix Beef Herring Chloride Acid (see abov^Cerelose Tallow O i l (25% gms) (gms) 36.3 36.3 36.3 36.3 36.8 36.8 36.3 36.8 36.8 36.8 36.3 36.3 36.8 3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.2 72.64 72.64 72.64 72.64 72.64 72.64 0.014 0.014 0.014 0.014 0.014 0.014 Test 2 & 46.25 30.0 10.0 1.0 0.5 0.25 6 . 0 gm. 0.16 gm. 0.50 gm. 1.00 gm. 0.035 gm. 0.0004 gm. 88.0 Methio- Mena-nine dione (gmg) IsmL 0.036 1 35.2 4.3 2 35.2 4.8 • • • - T ~ T T 3 35.2 4.3 4 35.2 4.8 5 35.2 4.3 6 35.2 4.8 7 35.2 4.3 3 35.2 4.8 24.00 24.0 4 5 . 4 4 5 . 4 0.0014 0.0014 TABLE 4:2 Mean ^ Prothrombin Time" Values - Experiment IV (Figures in Parentheses indicates; number of birds treated) Supplement to Mean "Prothrombin Time" in Seconds Basal Diet.  Test 1 Test 2. days 23 days 30 dars None 67 (10) 134 (10) Herring Oil 78 ( 9) ; Beef Tallow 45 ( 9) 33 (10) 24 (12) Choline Chloride 64 ( 9) 141 (10) Herring Oil •- Choline 62 (10) Beef Tallow t- Choline 47 ( 9) 30 (10) 24 (12) Folic Acid 73 ( 9) Herring Oil r Folic 80 (10) Beef Tallow r Folic 43 (10) Choline r Folic Acid ' 68 ( 9) — - — -Choline r Folic Herring Oil 76 ( 9) — Choline * Folic *• Beef Tallow 45 ( 8) i Menadione 30 ( 8) 103 (10) Menadione r Beef Tallow — — 27 (10) 23 (12) Methionine — 135 (10) Methionine * Beef T allow 28 (10) 23 (11) Control Bird 31 28 24 TABLE 4:3 Analysis of "Prothrombin Time" Variance Experiment IV Test 1. Source of Variation Total Treatment Error SS 51304 27270 24034 df 118 8.15 109.85 Variance 3346 219 15.28 kk L.S.D. Treatments 16 23 Test 2. Source of Variation Total Treatment Error L.S.D. Treatments SS 250439 203925 &65H df 79 -2 72 Variance 29132 646 _5i 26 1& 37 45.1 kk TABLE 4:4 Mean Weight at 23 davs - Experiment IV Test 2. Group 1 2 3 4 5 6 7 8 Supplement to Basal Diet None Menadione Choline chloride Methionine Beef Tallow Tallow r Menadione Tallow t- Choline Tallow + Methionine Mean Body Weight in Grams. 220 (21) 205 (22) 221 (19) 243 (20) 214 (22) 205 (22) 222 (22) 244 (21) TABLE 5:1 - Experimental Diets - Experiment V. Ingredients Common to A l l Rations: Ingredient Ground Yellow Corn Soybean Oil Meal Salt (Iodized) Limestone Bone Meal 51.5 37.0 0.5 1.0 2.0 MnSO Riboflavin Calcium Pantothenate Niacin Vitamin A (10,000 IU/gm) Vitamin D (1650 ICU/gm) Choline Chloride (25$) folic Acid Composition of Individual rations in Pounds: gm gm 10.0 0.16 0.50 gm 0.80 gm 20.0 81.8 181.6 gm gm gm 0.635 gm. Ration #. Test 1, Premix (as above) Cerelose Herring Oil alkali not Cottonseed Oil Beef Tallow alkali not alkali not treated traa,t. oldtreated treatedtreat' d. treat 1 d. 1 27.6 ^4 2 27.6 1.2 1.2 3 27.6 0.6 1.8 4 27.6 2.4 5 27.6 1.2 1.2 6 27.6 0.6 1.8 7 27.6 2.4 8 27.6 2.4 Test 2. 1 2 3 4 5 6 7 8 9 10 11 12 13 36.3 36.3 36.3 36.8 36.8 36.8 36.3 36.8 36.8 36.8 36.8 36.8 36.8 3.2 2.4 0.8 2.4 0.8 2.4 0.3 2.4 0.8 2.4 0.3 2.4 0.8 0.8 2.4 0.3 2.4 — 0.3 - 2.4 0.3 2.4 0.3 2.4 0.3 2.4 14 36.8 3.2 plus 0.036 gm. menadione. TABLE 5:2 . ... . .. Mean Body Weight &t 23 days - Experiment V Test 1. (Figures in Parentheses indicate number of birds per group) Supplement to Group Basal Ration Mean Body Weight.(Gms.) 1 None 210 (19) 2 1$ Alkali-treated Herring Oil 214 (17) 3 6% Alkali-treated Herring Oil 194 (18) 4 8% Alkali-treated Herring Oil 195 (18) 5 4% Untreated Herring Oil 217 (18) 6 6% Untreated Herring Oil 199 (20) 7 8% Untreated Herring Oil 199 (19) 8 8% Stored Herring Oil 182 (17) TABLE 5*3 Mean "Prothrombin Time" Values - Experiment V (Figures in Parentheses indicate number of birds tested) Supplement to. Basal Ration Mean "Prothrombin Time11 in Seconds. None 2% Treated Herring Oil 4% Treated Herring Oil 6% Treated Herring Oil 8% Treated Herring Oil 2% Untreated Herring Oil 4$ Untreated Herring Oil b% Untreated Herring Oil 8% Untreated Herring Oil 8% Stored Herring Oil 2% Treated Beef Tallow Al ( 9) 6% Treated Beef Tallow 26 ( 8) 2% Untreated Beef Tallow 30 (10) 6% Untreated Beef Tallow 36 (10) 2% Treated Cottonseed Oil 35 (10) 6% Treated Cottonseed Oil 30 (10) 2% Untreated Cottonseed Oil 38 (10) 6% Untreated Cottonseed Oil 36 ( 9) Menadione 21 ( 7) Test 1 Test 2 18 days 2? day? 19 davs 65 (5) 77 (10) 38 ( 9) _ — — — — 50 (9) 71 (5) 87 (10) 67 (5) 97 (10) 39 ( 9) 71 (5) 80 (10) 47 (10) 71 ~(5) 65 (10) 76 (5) 66 (10) 49 (10) 56 (5) 60 (10) 60 (5) 72 (10) Control Bird 30 36 23 TABLE 5:4 Analysis of "Prothrombin Time" Variance - Experiment V  Test 1-18 days. Source of Variation Total Treatment Error SS 9010 1Z62 7548 df 39 _Z 32 Variance 209 236 n-pu 0.88 Test 1 - 25 days. Source of Variation Total Treatment Error L.S.D. treatments SS 26622 1Q843 15779 df 79 JL. 72 Variance 1549 219 15 l£ 22 7.07 kk Test 2 - 19 days. Source of Variation Total Rations Error L.S.D. treatments SS 17570 8032 9538 df 129 13 116 Variance 618 83 10 14 u f II 7.44 A* TABLE 6:1 Experimental Diets - Experiment VI Ingredients Common to A l l Rations* Ingredient Ground Yellow Corn Herring Meal Salt (Iodized) Limestone Choline Chloride (25%) Feeding O i l (2250 A, 300 D) MnSO/ Riboflavin Calcium Pantothenate Niacin Composition of Individual Rations: 71.5 18.75 0.5 1.25 0.25 0.25 92.5 10.0 gm. 0.16 gm. 0.50 gm. 0.80 gm. Premix - lbs. Cerelose Ration S (ag above) . lbs. Cottonseed Oil-treated lbs. „ Cottonseed Oil-untreated lbs. F o l i c Acid (gms) 1. 37.0 3.0 2. 37.0 3.0 3. 37.0 A. 37.0 5. 37.0 6. 37.0 7. 37.0 3.0 3.0 3.0 3.0 3.0 — 0.008 gm. Menadione 0.01A 0.01 A 0.014 TABLE 6:2 Mean "Prothrombin Time" Values - E x p f t r l m a T i t VI t(Figures in parentheses indicate number of birds tested) Supplement to Mean "Prothrombin Group Basal Ration Time" in Seconds. 1 None 131 (11) 2 Folic Acid 114 (14) 3 Cottonseed Oil 80 (19) 4 Cottonseed Oil 4- Folic Acid 65 (18) 5 Alkali Treated Cottonseed O i l 71 ; (19) 6 Alkali Treated CSO «• Folic A cid 64 (18) 7 Menadione 58 (20) Control Bird 22 TABLE 6:3 Analysis of '"Prothrombin Time" Variance - Experiment VI tSource of Variation SS df Variance " f n Total 182995 137 Treatment 76728 .. • 7 10961 13.4 kk Error 106267 130 817 L.S.D. treatments _5i 37 56 TABLE 7*1 Experimental Diets - Experiment VII Ingredients Common to a l l Rations* Ingredient Jk Ground Yellow Corn Herring Meal Salt (Iodized). Limestone Choline Chloride {25%) Feeding Oil (2250 A, 300 D) MnSO, Riboflavin Calcium Pantothenate Niacin 71.50 18.75 0.5 1.25 0.25 0.25 92.5 10.0 gms. 0.16 gm. 0.50 gm. 0.80 gm. Composition of Individual Rations in Pounds* Ration # 1. 2. 3. Premix (as above) 27.75 18.5 18.5 Cerelose 2.25 Oleic Acid 1.50 Mixed Fish Liver Fatty Acids  1.50 TABLE 7*2 Mean "Prothrombin Time" Values - Experiment VII (Figures in parentheses indicate number of birds tested) Supplement to Mean "Prothrombin Group Basal Ration Tjme" in Seconds 1. None 80 (15) 2. 0le£cx Acid 72 (15) 3. Mixed Fatty Acids 63 (15) A. Cottonseed Oil administered 1 hour before Blood was Drawn 99 (15) Control Bird 24 TABLE 7:3 Analysis of '"Prothrombin Time" Variation - Experiment VII Source of Variation Total Treatment Error. L.S.D. treatments SS 39078 10981 28097 24 Df 59 56 Variance 3660 502 n m 18 7.29 kk TABLE 7:4 Effect of Orallig Administered @il on "Prothrombin Time" (figures in parentheses indicate number of chicks tested) Basal Test Ration 1 Wheat-Fishmeal 2 Corn-Fishmeal 3 Corn-F ishmeal Treatment Herring Oil Corn Oil Corn Oil "Prothrombin Time" (Seconds) Untreated Treated Control Chicks Chicks 48 (4) 399 ( 4) 104 (5) 44 (4) 383 (5) 94 (5) Bird 26 31 22 TABLE 8:1 Experimental Diets - Experiment VIII Ingredients Common to A l l Rations Test 1 As in Table 2:1, 0.35 mg./lb. folic acid added. Test 2 As in Table 3:1, 0.5% ground corn subtracted. Test 3.— As in Table 3:1, 7.5% ground corn added. Composition of Individual Rations -Beef Cholic Desoxy-Premix Cerelose Tallow Acid cholic Acid Menadione Ration (as above) (pounds) (pounds) (grams) (grams) (grams) (pounds) Test 1 1 18.4 1.6 2 18.4 1.6 45.4 — -Test 2 1 18.4 1.6 :  2 18.4 1.6 45.4 3 18.4 1.6 4 18.4 1.6 45.4 Test 3 1 20.0 2 20.0 45.4 • 3 20.0 45.4 i 20.0 0.00036 5 20.0 0.0072 TABLE 8:2 Mean "Prothrombin Time" Values - Experiment VIII (Figures in parentheses indicate number of birds tested) Supplement to Basal Ration None Cholic Acid Desoxycholic Acid Beef Tallow Beef Tallow 4- Cholic Acid Menadione (l/lO requirement) Menadione (2 X requirement) Control Bird Mean "Prothrombin Time" in Seconds Test 3 Testll A5 ( U ) 67 (15) Test 2 127 (8) 166 (3) 35 (10) 35 (10) 26 26 78 (10) 109 (10) 58 (10) A3 (10) 25 ( 9) 28 TABLE 8:3 Analysis of "Prothrombin Time" Variance - Experiment VIII Test 1. Source of Variation SS Total 9659 Treatment 3366 Error 6293 L.S.D. treatments df 27 _1 26 Variance 3366 2A2 JSk 13 18 13.9 AA Test :2. Source of Variation SS Total . 39937 Treatment 6202 Error . 33785 df 15 _1 14 Variance 6202 2A13 2.6 Test 3. Source of Variation SS Total 93802 Treatment A0591 Error 53211 L.S.D. treatments df 48 _4 44 Variance 10148 1209 36 51 8.4 4 4 TABLE 9:1 Mean "Prothrombin Time" Values - Experiment IX (Figures in parentheses indicate number of birds tested) Eation Fed Test 1 Wheat-fishmeal Test 2 Corn-fishmeal Test 3 Corn-fishmeal Mean "Prothrombin Time - Seconds Triton-treated Untreated Control Bir4t 50 (5) 1182 (5) 211 (5) 37 (5) 399 (4) 104 (5) 24 31 22 TABLE 9:2 Analysis of "Prothrombin Time" Variance - Experiment IX Test 1 Source of Variation SS df Variance H I f" Total Treatment Error 589 166 9 1 8 423 21 20.1 kk L.S.D. treatment 2k 8 2& 13 Test 2 Source of Variation SS df Variance U £11 Total Treatment Error 3536201 1361550 2174651 3 1 7 1361550 310644 4.4 Test 3 Source of Variation SS df Variance II £ M Total Treatment Error 116800 23515 88285 9 1 8 28515 11036 2.58 TABLE 9:3 Effect of Triton Wfi-1339 in vitro on Blood "Prothrombin Time1 0.85% Saline or 2.5% Triton in 0.85% Saline Added to Blood or Clotting Agent. Concentration of Added Saline or "Prothrombin Time" Triton-saline as % of Final Snlytfon in Seconds  Triton-saline Added to Blood 3.0% 37 5/6% 38 9.5% 36 16.7% A5 Saline Added to Blood 3.0% AO 5.6% 36 9.5% 38 16.7% AA Triton-saline Added to Clotting Agent 6.1% 38 11.1% AO 19.1% A3 33.3% 46 Nothing Added 32 TABLE 10:1 Effect of Lecithin on Breast Muscle- Thromboplastic Agent 0.85$ Saline or 1% Lec&ininu in 0.85$ Saline plus an Equal Volume of M/40 Ga01o Added to Tissue Extract. '• '•• $ of Saline or of Lecithin-saline added Ignoring CaCl^ solution Saline Added to Make 20$ . 30$ 40$ 50$ 67$ Lecithin-saline Added to Make 10$ 20$ 30$ 40$ 50$ 67$ Undiluted Agent "Prothrombin Time" in Seconds Normal Vitamin K Deficient 31 32 35 36 38 23 22 22 24 30 33 24 82 87 100 94 118 49 43 44 48 51 56 62 TABLE 10:2 Effect of Lecithin on Dried Brain Thromboplastic Agent 0.85$ Saline or 1$ Lecithin in 0.85$ Saline added to Tissue Extract. $ of Saline or of Lecithin-saline added Saline Added to Make 10$ 20$ 30$ 40$ 50$ 67$ Lecithin-saline Added to Make 10$ 20$ 30$ 40$ 50> 67£ Undiluted Tissue Extract. "Prothrombin Time" in Seconds. Normal Vitamin K Deficient 16 16 15 16 16 16 16 17 18 18 20 21 16 38 38 38 38 38 39 42 42 46 47 51 57 38 TABLE 10s3 Effect of Preincubation with CaClg o n Response of Tissue Extracts to Lecithin "Prothrombin Time" in Seconds Breast Muscle Extract D r i e d B r a l » Extract GaCi2 Added to Blood CaCl 2 Added to Agent C a C l 2 A d d e d t o B l o o d C a C 1 2 t o A 3 e n t Normal Vitamin K Normal Vitamin K Normal Vitamin K Normal Vitamin K Blood Deficient Blood Deficient Blood Deficient Blood Deficient Blood Blood Blood Blood Saline Added to Make 10% 34 50 32 68 16 40 19 40 20% 27 54 34 78 16 42 19 42 30% 29 56 32 86 16 40 19 38 40% 34 64 39 105 18 41 19 38 50% 31 63 34 89 17 41 20 39 67% 32 72 39 122 18 43 21. 42 Lecithin-saline Added to Make 10% ' 19 34 25 44 17 43 19 42 20% 19 35 27 49" 16 44 20 44 30% 20 38 27 58 19 46 21 50 40% 20 39 30 61 22 48 24 57 50% 22 48 30 67 23 50 28 63 67% 27 65 34 82 23 54 27 75 Undiluted Agent 26 50 26 54 17 42 18 38 "Calcium Time" with Saline 96 10' 105 10' Not Tested "Calcium Time" with Lecithin-Saline 93 9,13" 88 8'46" TABLE 10:4 Comparison of Activity of Four Thromboplastic Agents Blood Sample # 1 2 3 4 5 6 7 8 9 10 Breast Muscle Thromboplastic Agent Prepared with Saline Normal Vitamin K Blood Deficient Blood 20 22 21 22 22 57 47 77 60 64 Prepared with Lecithin-Saline Normal Vitamin K Blood Deficient Blood, 18 16 16 18 17 33 30 47 35 36 Dried Brain Tissue Thromboplastic A gent Prepared with Saline Normal Blood 14 14 13 15 14 Vitamin K Deficient Blood 50 38 76 46 54 Prepared with Leeifchin-Saline Normal Vitamin K Blood Deficient Bjpod  15 17 16 16 15 47 33 73 45 51 Mean Value 21 61 17 36 14 53 16 50 TABLE 10:5 Thromboplastic Activity of Acetone-dried B r e a s t Muscle Clotting Agent Diluted in Saline Tested with Oxalated Normal Blood Concentration of Original "Prothrombin Ime" .Extract - % Seconds 100 145 50 167 20 . 138 10 217 5 138 "Calcium Time" 130 TABLE 10:6 Comparison of Thromboplastic Properties of Extracts of Fresh and of Acetone Dried Breast Muscle Tissue and Brain Tissue Tested with Normal Blood Fresh Breast Muscle Fresh Brain Tissue Dried Breast Muscle Dried Brain T issue "Calcium1" Saline Lecithin- Saline Lecithin- Saline Lecithin- Saline Lecithin-* Bird # Time Extract Saline Extract Saline Extract Saline Extract Saline Extract Extract Extract Extract 1 122 25 18 20 24 126 80 13 18 2 145 26 23 23 26 139 70 13 18 3 146 30 23 20 27 194 193 14 16 4 200 35 27 23 29 173 136 15 21 5 im 2$ 2k 21 166 16 18 Mean 151 29 23 23 27 155 129 14 18 TABLE 10*7 Relative Thromboplastic Activity of Fresh Breast Muscle and Dried Brain Tissue on a Dry-weight Basis  "Prothrombin Time11 in Seconds Dried Brain Thromboplastic Agent Final Concentration of Agent Agent Diluted with Saline 100% 10% 1% 0.1% 0.01% "Calcium Time" with Saline Normal Blood 17 21 AO 7A 86 87 Breast Muscle Thromboplastic Agent (1 1/3 X Normal Strength) Vitakin K Deficient Blood Normal Blood Vitakin K Deficient BlQQd 32 48 177 328 6» 6« .27 40 62 87 91 92 66 123 246 6« 6' 6' Agent Diluted with Lecithin-Saline 100% 22 10% 51 1% * 90 0.1% 98 0.01%. 104 "Calcium Time" with Lecithin-Saline 103 81 37 85 202 81 240 326 108 318 345 112 328 350 112 338 340 108 324 TABLE 11:1 Experimental Diets - Experiment XI Ingredients Common to A l l Rations As in Table 3«1, 0.5% ground corn subtracted. Composition of Individual Rations in Pounds: Ration Premix (as above) Supplement 1 23 2# Cerelose 2 23 2# Cerelose 57 gm. Cholic Acid. 3 23 2# Cerelose *• 57 gm. Desoxycholic Acid. TABLE 11:2 Mean "Prothrombin Time" - Experiment XI (Figures in parentheses indicate number of birds tested) Supplement to "Prothrombin Time" (Seconds Group Basal Ration Breast Muscle Brain Tissue Agent Agent 1 None 24 (8) 32 (8) 2 . Cholic Acid 29 (9) 42 (9) 3 Desoxycholic Acid 32 (8) 45 (8) Control Bird 20 24 TABLE 11;3 Analysis of "Prothrombin Time" Variance - Experiment XI Brain Tissue Thromboplastic Agent Source of Variation SS df Total Treatments Error L.S.D. Treatments 2413 1604 24 _£ 22 10 Variance 15 404 73 •tt £« 5.53 * Breast Muscle Thromboplastic Agent Source of Variation Total Treatments Error L.S.D. Treatments SS 225 567 6 df 24 22 Variance 138 26 9 U£ir 5.31 k i o 2 0 3 0 ho 5 0 6 o % of Phospholipid Solution or of Saline Added to Thromboplaatle Agent FIGURE I. E f f e c t of phospholipid on breast muscle thromboplastic agent. "Prothrombin time" of blood from normal b i r d (lower curve) and vitamin K d e f i c i e n t b i r d (upper curve). X-0 --x S a l i n e Added -o 1% P h o s p h o l i p i d i n S a l i n e Added x x x X o io 20 30 ko 50 6o % of P h o s p h o l i p i d S o l u t i o n or of S a l i n e Added to Thromboplastic Agent FIGURE 2. E f f e c t of p h o s p h o l i p i d on b r a i n t i s s u e thromboplastic agent. "Prothrombin time" of blood from normal b i r d (lower curve)' and Vitamin K d e f i c i e n t b i r d (upper curve)\ go -X Saline Added (a) 6 0 •a c o o <D CO a> •H ««> o- -o 1% Phospholipid i n Saline Added (a) x — x Saline Added (b) o o 1% Phospholipid i n Saline Added (b) B o u X! O u 20 -o— . X---X— -o X 10 20 30 U0 50 S of Phospholipid Solution or of Saline Added to Thromboplastic Agent FIGURE 3. E f f e c t of method of calcium a d d i t i o n on response of b r a i n t i s s u e thromboplastic agent to phospholipid. "Prothrombin time" of blood from normal b i r d (lower curves) and vitamin K d e f i c i e n t b i r d (upper curves). (a) Calcium added to blood. (b) Calcium added to thromboplastic agent 6 0 120 100 ?S0 o o o CD "60 -x Saline Added (a) -o 1% Phospholipid i n Saline Added (a) x x Saline Added (b) ,x. o o \% Phospholipid i n Saline Added (b)' ,_x' io 20 30 U-o 50 6o $ of Phospholipid Solution or of Saline Added to Thromboplastic Agent FIGURE k. E f f e c t of method of calcium a d d i t i o n on response of breast muscle thromboplastic agent to phospholipid. "Prothrombin time" of blood from normal b i r d (lower curves)' and vitamin K d e f i c i e n t b i r d (upper curves) 1. (a) Calcium added to blood. (b) Calcium added to thromboplastic agent. BIBLIOGRAPHY Alexander, B., A. Devries, R. Goldstein and G. Landwehr, 1949. 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