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Alpha tocopherol quinone (αTQ): A potent inhibitor of platelet function
ALPHA TOCOPHEROL QUINONE (~TQ): A POTENT INHIBITOR OF PLATELET FUNCTION GUNDU H. R. RAO, CHADWICK A. Cox, JONATHAN M. GERRARD
and JAMESG. WHITE Health Sciences Center, University of Minnesota, Minneapolis, Minnesota 55455, U.S.A.
INTRODUCTION
Vitamin E (~t-tocopherol) is a required nutrient first identified in wheat germ 50 years ago and studied intensively since that time because of its potent influence on oxidationreduction reactions. Results of clinical trials have suggested that vitamin E, which has been shown to inhibit platelet aggregation,~ 0,~2,~4,~5,20.27 may reduce the incidence of thrombotic disorders, intermittent claudication, a'7'9' ~l. ~a, 1a cerebral arteriosclerosis, 22.23 and possibly, angina pectoris and coronary artery disease. 1'~9'2~ As a result, it is extremely important to identify the specific mechanisms through which vitamin E protects the vascular system and blood cells from damage or activation. During the course of our studies of vitamin E and platelets, it was discovered that the oxidation product of vitamin E, vitamin E quinone (VEQ), was a more potent inhibitor of platelet function than the parent compound. However, our initial studies were limited by the complex procedures ordinarily required to prepare vitamin E quinones. In this report, we describe a simple procedure for preparing and characterizing the purity of ~-tocopherol quinones and summarize the influence of these compounds, compared to vitamin E, on platelet function. MATERIALS AND METHODS
Arachidonic acid as sodium salt was obtained from NuChek Prep, Elysian MN. ~-Tocopherol and quinone derivatives were a gift from Dr. Takhita Oki, of Elasi Co., Tokyo, Japan. Injectable adrenalin and topical thrombin were from Parke Davis Co., Detroit MI. Acid soluble collagen was secured from Worthington Biochem, Corp. Freehold, N J, and calcium ionophore, A23187 was obtained from Calbiochem-Behring Corp., Lajolla, CA. Radiochemicals were obtained from the following companies: [1-14C] arachidonic acid (t4C-AA) New England Nuclear, Boston, MA, [2-t4C "] 5-hydroxy tryptamine from Amersham Corp., Arlington Heights, IL. A Waters Associates model 204 high pressure liquid chromatograph (HPLC) was used for the separation of products. A 30 crn stainless steel column, packed with C18 #Bondapak TM,was used for the analytical work and the solvent used for eluting compounds was pure acetonitrile. A Hewlett Packard 3385 A automation system was used to obtain electronic integration of the peaks. Oxidized products of vitamin E, the parent compound and authentic standards, were subjected to electron impact mass spectrometry by the direct inlet technique using a LKB 9000 mass spectrometer equipped with a PDP-Se data processor.
Preparation of Vitamin E Quinone VEQ was prepared by oxidation of 100 mg of ~-tocopherol with 5-10 drops of concentrated nitric acid at room temperature for 2-3 min. The oxidized material was thoroughly rinsed several times with distilled water until the pH of the rinse water was greater than 5. The residue was then dissolved in absolute ethanol. 549
550
G . H . R . Rao et al.
Studies on Platelet Function Blood was obtained from healthy adult volunteers and processed by the standard methods established in this laboratory to obtain platelet-rich-plasma (PRP). '~'2'* Aggregation of PRP was monitored by a dual-channel Payton aggregometer. Secretion of I'~C-5HT was measured by a modification ~6 of the method of Jerushalmy and Zucker. 8 Platelet ultrastructure and biochemical studies were done by the methods described previously from this laboratory. 16't7'25 Platelet phospholipase A2 activity was determined by measuring the amount of ~4C-AA released from the membranes stimulated by calcium ionophore. 2 Cyclooxygenase activity was assayed by following the conversion of ~'C-AA. 6 RESULTS AND DISCUSSION
Characterization of Vitamin E Quinone The oxidation product of vitamin E prepared by the method described in this paper, on analysis by HPLC and UV spectrometry, revealed that the major product was a quinone closely resembling ~t-tocopherol quinone. Mass spectral analysis of the compound showed major ion peaks at 446, 221 and 178, and matched exactly the fragmentation pattern of authentic ~t-tocopherol quinone (Fig. l).
Biolo~lical Activity of Vitamin E Quinone Vitamin E (VE), VE acetate, VE nicotinate, VEQ and other quinone derivatives of VE were tested for their inhibitory activity against platelet aggregation. Though all of these compounds showed some inhibition of platelet function, VEQ was the most effective blocker and more potent than the parent compound. Therefore, detailed studies on the mechanism of inhibition of platelet function were carried out with VEQ. At a concentration of 2 mM, VEQ inhibited platelet aggregation as well as release of I'*C-SHT
M A 8 8 SPECTRA OF AUTHENTIC
a - T O C O P H E R O L QUINONE
(UPPER) AND VITAMIN E QUINONE 180LATED FROM THE LIQUID CHROMATOGRAPH (LOWER) d-TQ (a- Tocol)herolquinone) I~
~
CH3
CH3
] ~
o
C~t~
1446 721
~a
>~
......[.~.~
i~ g I~
.
~.*.~.~
~
~.~. . ,~. . . ~ ............
,.
~
~
~
,,,~.;
~.
~
~o
~oo
.....
..
~
~ IIIII .~ ~ ~ ~
~ N
. ... ~. . .. .~. .,.~. . i. ., . . . . . . . . . . . . . . . . . . . . . . . . . . .~t~ ....
~o
~oo
~o
~o
J~ L J _ ~o
~oo
~_~
~1.1, . .
4~o
~
Melee/Charge Ratio FIG. 1. Mass spectra of vitamin E Quinone and authentic :t-tocopherol quinone, Fragmentation pa.tterns of the major ion peaks of vitamin E quinone matched those of :t-tocopherol quinone.
Alpha tocopherol quinone [~TQ}
551
TABLE I. Effect of VEQ on Platelet Aggregation and the Release of I~C-Serotonin Per cent release" Without b With ~ VEQ VEQ Control P R P P R P + Collagen (30/~g/ml) PRP + Epinephrine
5 _+ 0.2 50 _ 1.0
-6 _+ 1.0
32 _ 2.0
7 _ 0.8
49 _+ 2.0
8 _+ 0.8
55 _4- 1.0
I1 _+ 1.0
49 _ 1.0
5 _+ 0.6
65 +_ 2.0
12 _ 0.4
(5 u~) PRP + ADP
(5 um PRP + Arachidonate (0.45 raM) P R P + Thrombin (0.2/~/ml) P R P + A23187
(1/~) ~Mean and the standard error (n = 3). bPRP samples incubated with VEQ, upon stimulation with aggregating agents did not show any aggregation response, whereas, the samples without VEQ responded with irreversible aggregation. Vitamin E Quinone at 2ram concentration effectively blocked the release of l~C-serotonin from the platelets in response to all the aggregating agents tested.
(Table l) induced by aggregating agents such as collagen (30/zg ml), epinephrine (5/ZM), adenosine diphosphate (3/~M), thrombin (0.2/~/ml), arachidonate (0.45 mM) and calcium ionophore A23187 (1/zM). Effect of VEQ on Platelet Morpholooy and Biochemistry Platelets exposed to 2 mM concentration of VEQ had normal levels of serotonin and adenine nucleotides. Upon washing and resuspension, the treated platelets aggregated in a normal fashion in response to several aggregating agents. Examination of VEQ treated platelets by electron microscopy revealed no apparent effect of the agent on discoid shape or fine structure. Studies on Arachidonic Acid Release and Transformation VEQ significantly blocked the release of ~4C-AA from platelet membranes when labeled plate!ets were stirred with calcium ionophore, A23187. However, it had no significant effect on the transformation of 14C-AA by the cyclooxygenase enzyme to thromboxane (Table 2). TABLE 2. Influence of VEQ on Platelet Phospholipase A 2 and Cyclo-Oxygenase Enzymes
Washed platelets Washed platelets + 14C-AA Washed platelets Washed platelets
+ 14C-AA + VEQ (2 mM) + A~3187 (10pM) + VEQ + A23187
Per cent of AA converted to thromboxane B2
Per cent release of AA from membranes ~
25 _ 6.0 20 _+ 5.0
---
---
16.6 _+ 0.2 1.0
_
0.4
"Mean and the standard error (n -- 3). Vitamin E Quinone significantly blocked the release of 14C-AA from platelet membranes in response to stimulation by calcium ionophore A23187. However, it had no significant effect on the transformation of arachidonic acid to thromboxane A2.
552
G . H . R . Rag et HI. SUMMARY
Vitamin E Quinone prepared by the oxidation of VE with concentrated nitric acid yielded a single major product. Analysis of this product showed that it was g-tocopherol quinone. Studies on its biological activity confirmed our earlier observations, that it is a potent inhibitor of platelet function. The mechanism of inhibition of aggregation and release reaction seems to be through the inhibition of phospholipase activity. Inhibition was achieved without any deleterious effect on the morphological or biochemical characteristics of platelets.
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 1I. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23~ 24. 25. 26. 27.
ANDERSON,T. W. Can. Med. Assoc. J. Ii0, 401-406 (1974). BILLS,T. K., SMITH,J. B. and SILVER,M. J. Biochim. Biophys. Acta 424, 303-314 (1976). BOYD,A. M. and MARK, J. Angiolooy 14, 198-207 (1963). CLAWSON,C. C. and WHITE,J. G. Am. d. Pathol. 65, 367-380 (1971). Cox, A. C., RAO, G. H. R., GERRARD,J. M. and WroTE J. G. Blood (In press). HAMBERG,M. and SAMUELSSON,B. Proc. Natl. Acad. Sci. 71, 3400-3404 (1974). IIYO, K., ABE, K., KARIYA,S., KIMURA,H., KUSABA,T., KUSUNOKI,R., SAKU,J., SOE./IMA,K., NAKAKURA,S., NAKAMURA,K., NAKAYAMA,Y., NOTOMI,M., HANADA,T., MIYAZAKI,T. and FURUKAWA,I. Jpn. Heart .I. 18, 277-286 (1977). JERUSHALMY,Z. and ZUCKER,M. B. Thromb. Diath. Haemorrh. 15, 413-421 (1966). KRAUSE,D. In Tocophero~ Oxygen and Biomembranes, p. 333, Elsevier/North-Holland Biomedical Press Amsterdam, 1978. LAKE,A. M., STUART,M. J. and OSKI, F. A. 2. Pediatr. 90, 722-725 (1977). LIVINGSTONE,P. D. and JONES,C. Lancet 2, 602-604 (1958). MACHLIN,L. J., FILIPSKI, R., WILLIS, A. L., KUHN, D. C. and BRIN, M. Proc. Soc. Exp. Biol. Med. 149, 275-277 (1975). NAITO,C., OHTA, A., OHMURE,R., OKANIWA,H., OGAWA,K., GOTO, K., KOMWA,H. and NAKADA,F. lguku No Ayumi 91,628-636 (1974). NOROOV,A.. and STROM, E. J. Lipid Res. 16, 386-391 (1975). RAO, G. H. R., GERRARD,J. M., EATON,J. W. and WHITE, J. G. Photochem. Photobiol. 28, 845-850 (1978). RAO, G. H. R., WHITE, J. G., JACH1MOWICZ,A. A. and WITKOP, C. J., JR..J. Lab. Clin. Med. 84, 839-850 (1974). RAO, G. H. R., WHITE, J. G., JACHIMOWICZ,A. A. and WITKOP, C. J., JR. J. Lab. Clin. Med. 87, 129-137 (1976). RATCLIFFE,A. M. Lancet 2, 1128-1130 (1949). RINZLER,S. H., BAKST,H. BENIAMIN,Z. H., BOBB,A. L. and TR~VELL,J. Circulation 1,288-293 (1950). STEINER,M. and ANASTASl,J. J. Clin. Invest. 57, 732-737 (1976). TOONE,W. M. N. Eng. J. Med. 289, 979-980 (1973). VOGELSANG,A. and SHUTE, E. V. Nature 157, 772-773 (1946). VOGELSANG,A., SHUTE, E. V. and SHUTE,W. E. Med. Rec. 160, 91-96 (1947). WHITE, J. G. Blood 31,604-622 (1968). WHITE, J. G. Am. J. Pathol. 69, 439-458 (1972). WI~IITEJ. G., RAO, G. H. R. and ESTENSENR. D. Am. J. Pathol. 75, 301-312 (1974). WHITE,J. G., RAO, G. H. R. and GERRARD,J. M. Am. J. Path. 88, 387-402 (1977).
and JAMESG. WHITE Health Sciences Center, University of Minnesota, Minneapolis, Minnesota 55455, U.S.A.
INTRODUCTION
Vitamin E (~t-tocopherol) is a required nutrient first identified in wheat germ 50 years ago and studied intensively since that time because of its potent influence on oxidationreduction reactions. Results of clinical trials have suggested that vitamin E, which has been shown to inhibit platelet aggregation,~ 0,~2,~4,~5,20.27 may reduce the incidence of thrombotic disorders, intermittent claudication, a'7'9' ~l. ~a, 1a cerebral arteriosclerosis, 22.23 and possibly, angina pectoris and coronary artery disease. 1'~9'2~ As a result, it is extremely important to identify the specific mechanisms through which vitamin E protects the vascular system and blood cells from damage or activation. During the course of our studies of vitamin E and platelets, it was discovered that the oxidation product of vitamin E, vitamin E quinone (VEQ), was a more potent inhibitor of platelet function than the parent compound. However, our initial studies were limited by the complex procedures ordinarily required to prepare vitamin E quinones. In this report, we describe a simple procedure for preparing and characterizing the purity of ~-tocopherol quinones and summarize the influence of these compounds, compared to vitamin E, on platelet function. MATERIALS AND METHODS
Arachidonic acid as sodium salt was obtained from NuChek Prep, Elysian MN. ~-Tocopherol and quinone derivatives were a gift from Dr. Takhita Oki, of Elasi Co., Tokyo, Japan. Injectable adrenalin and topical thrombin were from Parke Davis Co., Detroit MI. Acid soluble collagen was secured from Worthington Biochem, Corp. Freehold, N J, and calcium ionophore, A23187 was obtained from Calbiochem-Behring Corp., Lajolla, CA. Radiochemicals were obtained from the following companies: [1-14C] arachidonic acid (t4C-AA) New England Nuclear, Boston, MA, [2-t4C "] 5-hydroxy tryptamine from Amersham Corp., Arlington Heights, IL. A Waters Associates model 204 high pressure liquid chromatograph (HPLC) was used for the separation of products. A 30 crn stainless steel column, packed with C18 #Bondapak TM,was used for the analytical work and the solvent used for eluting compounds was pure acetonitrile. A Hewlett Packard 3385 A automation system was used to obtain electronic integration of the peaks. Oxidized products of vitamin E, the parent compound and authentic standards, were subjected to electron impact mass spectrometry by the direct inlet technique using a LKB 9000 mass spectrometer equipped with a PDP-Se data processor.
Preparation of Vitamin E Quinone VEQ was prepared by oxidation of 100 mg of ~-tocopherol with 5-10 drops of concentrated nitric acid at room temperature for 2-3 min. The oxidized material was thoroughly rinsed several times with distilled water until the pH of the rinse water was greater than 5. The residue was then dissolved in absolute ethanol. 549
550
G . H . R . Rao et al.
Studies on Platelet Function Blood was obtained from healthy adult volunteers and processed by the standard methods established in this laboratory to obtain platelet-rich-plasma (PRP). '~'2'* Aggregation of PRP was monitored by a dual-channel Payton aggregometer. Secretion of I'~C-5HT was measured by a modification ~6 of the method of Jerushalmy and Zucker. 8 Platelet ultrastructure and biochemical studies were done by the methods described previously from this laboratory. 16't7'25 Platelet phospholipase A2 activity was determined by measuring the amount of ~4C-AA released from the membranes stimulated by calcium ionophore. 2 Cyclooxygenase activity was assayed by following the conversion of ~'C-AA. 6 RESULTS AND DISCUSSION
Characterization of Vitamin E Quinone The oxidation product of vitamin E prepared by the method described in this paper, on analysis by HPLC and UV spectrometry, revealed that the major product was a quinone closely resembling ~t-tocopherol quinone. Mass spectral analysis of the compound showed major ion peaks at 446, 221 and 178, and matched exactly the fragmentation pattern of authentic ~t-tocopherol quinone (Fig. l).
Biolo~lical Activity of Vitamin E Quinone Vitamin E (VE), VE acetate, VE nicotinate, VEQ and other quinone derivatives of VE were tested for their inhibitory activity against platelet aggregation. Though all of these compounds showed some inhibition of platelet function, VEQ was the most effective blocker and more potent than the parent compound. Therefore, detailed studies on the mechanism of inhibition of platelet function were carried out with VEQ. At a concentration of 2 mM, VEQ inhibited platelet aggregation as well as release of I'*C-SHT
M A 8 8 SPECTRA OF AUTHENTIC
a - T O C O P H E R O L QUINONE
(UPPER) AND VITAMIN E QUINONE 180LATED FROM THE LIQUID CHROMATOGRAPH (LOWER) d-TQ (a- Tocol)herolquinone) I~
~
CH3
CH3
] ~
o
C~t~
1446 721
~a
>~
......[.~.~
i~ g I~
.
~.*.~.~
~
~.~. . ,~. . . ~ ............
,.
~
~
~
,,,~.;
~.
~
~o
~oo
.....
..
~
~ IIIII .~ ~ ~ ~
~ N
. ... ~. . .. .~. .,.~. . i. ., . . . . . . . . . . . . . . . . . . . . . . . . . . .~t~ ....
~o
~oo
~o
~o
J~ L J _ ~o
~oo
~_~
~1.1, . .
4~o
~
Melee/Charge Ratio FIG. 1. Mass spectra of vitamin E Quinone and authentic :t-tocopherol quinone, Fragmentation pa.tterns of the major ion peaks of vitamin E quinone matched those of :t-tocopherol quinone.
Alpha tocopherol quinone [~TQ}
551
TABLE I. Effect of VEQ on Platelet Aggregation and the Release of I~C-Serotonin Per cent release" Without b With ~ VEQ VEQ Control P R P P R P + Collagen (30/~g/ml) PRP + Epinephrine
5 _+ 0.2 50 _ 1.0
-6 _+ 1.0
32 _ 2.0
7 _ 0.8
49 _+ 2.0
8 _+ 0.8
55 _4- 1.0
I1 _+ 1.0
49 _ 1.0
5 _+ 0.6
65 +_ 2.0
12 _ 0.4
(5 u~) PRP + ADP
(5 um PRP + Arachidonate (0.45 raM) P R P + Thrombin (0.2/~/ml) P R P + A23187
(1/~) ~Mean and the standard error (n = 3). bPRP samples incubated with VEQ, upon stimulation with aggregating agents did not show any aggregation response, whereas, the samples without VEQ responded with irreversible aggregation. Vitamin E Quinone at 2ram concentration effectively blocked the release of l~C-serotonin from the platelets in response to all the aggregating agents tested.
(Table l) induced by aggregating agents such as collagen (30/zg ml), epinephrine (5/ZM), adenosine diphosphate (3/~M), thrombin (0.2/~/ml), arachidonate (0.45 mM) and calcium ionophore A23187 (1/zM). Effect of VEQ on Platelet Morpholooy and Biochemistry Platelets exposed to 2 mM concentration of VEQ had normal levels of serotonin and adenine nucleotides. Upon washing and resuspension, the treated platelets aggregated in a normal fashion in response to several aggregating agents. Examination of VEQ treated platelets by electron microscopy revealed no apparent effect of the agent on discoid shape or fine structure. Studies on Arachidonic Acid Release and Transformation VEQ significantly blocked the release of ~4C-AA from platelet membranes when labeled plate!ets were stirred with calcium ionophore, A23187. However, it had no significant effect on the transformation of 14C-AA by the cyclooxygenase enzyme to thromboxane (Table 2). TABLE 2. Influence of VEQ on Platelet Phospholipase A 2 and Cyclo-Oxygenase Enzymes
Washed platelets Washed platelets + 14C-AA Washed platelets Washed platelets
+ 14C-AA + VEQ (2 mM) + A~3187 (10pM) + VEQ + A23187
Per cent of AA converted to thromboxane B2
Per cent release of AA from membranes ~
25 _ 6.0 20 _+ 5.0
---
---
16.6 _+ 0.2 1.0
_
0.4
"Mean and the standard error (n -- 3). Vitamin E Quinone significantly blocked the release of 14C-AA from platelet membranes in response to stimulation by calcium ionophore A23187. However, it had no significant effect on the transformation of arachidonic acid to thromboxane A2.
552
G . H . R . Rag et HI. SUMMARY
Vitamin E Quinone prepared by the oxidation of VE with concentrated nitric acid yielded a single major product. Analysis of this product showed that it was g-tocopherol quinone. Studies on its biological activity confirmed our earlier observations, that it is a potent inhibitor of platelet function. The mechanism of inhibition of aggregation and release reaction seems to be through the inhibition of phospholipase activity. Inhibition was achieved without any deleterious effect on the morphological or biochemical characteristics of platelets.
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 1I. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23~ 24. 25. 26. 27.
ANDERSON,T. W. Can. Med. Assoc. J. Ii0, 401-406 (1974). BILLS,T. K., SMITH,J. B. and SILVER,M. J. Biochim. Biophys. Acta 424, 303-314 (1976). BOYD,A. M. and MARK, J. Angiolooy 14, 198-207 (1963). CLAWSON,C. C. and WHITE,J. G. Am. d. Pathol. 65, 367-380 (1971). Cox, A. C., RAO, G. H. R., GERRARD,J. M. and WroTE J. G. Blood (In press). HAMBERG,M. and SAMUELSSON,B. Proc. Natl. Acad. Sci. 71, 3400-3404 (1974). IIYO, K., ABE, K., KARIYA,S., KIMURA,H., KUSABA,T., KUSUNOKI,R., SAKU,J., SOE./IMA,K., NAKAKURA,S., NAKAMURA,K., NAKAYAMA,Y., NOTOMI,M., HANADA,T., MIYAZAKI,T. and FURUKAWA,I. Jpn. Heart .I. 18, 277-286 (1977). JERUSHALMY,Z. and ZUCKER,M. B. Thromb. Diath. Haemorrh. 15, 413-421 (1966). KRAUSE,D. In Tocophero~ Oxygen and Biomembranes, p. 333, Elsevier/North-Holland Biomedical Press Amsterdam, 1978. LAKE,A. M., STUART,M. J. and OSKI, F. A. 2. Pediatr. 90, 722-725 (1977). LIVINGSTONE,P. D. and JONES,C. Lancet 2, 602-604 (1958). MACHLIN,L. J., FILIPSKI, R., WILLIS, A. L., KUHN, D. C. and BRIN, M. Proc. Soc. Exp. Biol. Med. 149, 275-277 (1975). NAITO,C., OHTA, A., OHMURE,R., OKANIWA,H., OGAWA,K., GOTO, K., KOMWA,H. and NAKADA,F. lguku No Ayumi 91,628-636 (1974). NOROOV,A.. and STROM, E. J. Lipid Res. 16, 386-391 (1975). RAO, G. H. R., GERRARD,J. M., EATON,J. W. and WHITE, J. G. Photochem. Photobiol. 28, 845-850 (1978). RAO, G. H. R., WHITE, J. G., JACH1MOWICZ,A. A. and WITKOP, C. J., JR..J. Lab. Clin. Med. 84, 839-850 (1974). RAO, G. H. R., WHITE, J. G., JACHIMOWICZ,A. A. and WITKOP, C. J., JR. J. Lab. Clin. Med. 87, 129-137 (1976). RATCLIFFE,A. M. Lancet 2, 1128-1130 (1949). RINZLER,S. H., BAKST,H. BENIAMIN,Z. H., BOBB,A. L. and TR~VELL,J. Circulation 1,288-293 (1950). STEINER,M. and ANASTASl,J. J. Clin. Invest. 57, 732-737 (1976). TOONE,W. M. N. Eng. J. Med. 289, 979-980 (1973). VOGELSANG,A. and SHUTE, E. V. Nature 157, 772-773 (1946). VOGELSANG,A., SHUTE, E. V. and SHUTE,W. E. Med. Rec. 160, 91-96 (1947). WHITE, J. G. Blood 31,604-622 (1968). WHITE, J. G. Am. J. Pathol. 69, 439-458 (1972). WI~IITEJ. G., RAO, G. H. R. and ESTENSENR. D. Am. J. Pathol. 75, 301-312 (1974). WHITE,J. G., RAO, G. H. R. and GERRARD,J. M. Am. J. Path. 88, 387-402 (1977).