Reviews and critiques published study reporting "that tobacco glycoprotein (TGP) can be isolated from smoke condensate (CSC) or from cured Virginia bright tobacco leaves", which has "major importance in the pathogenesis of cardiovascular and pulmonary disease presumably associated with cigarette smoking". Includes headings entitled: "Introduction; II. Materials and methods; III. Activation of fibronolysis; IV. Evaluation of the complement system; V. Kinin generation; VI. Evaluation of coagulation inhibitors; Results (A. Generation of activated clotting factors by 'CON'; B. The effect of 'CON' on contact activation coagulation factors; C. The effect of 'CON' on the fibrinolytic system; D. The effect of 'CON' on antithrombin III; E. The effect of 'CON' on the complement system; [and] F. The effect of 'CON' on generation of kinins); VII. Conclusions; VIII. Summary; IX. Recommendations; [and] References". Includes five tables of study results using 'CON' per various assays. Makes recommendations regarding industry follow-up studies.
- Philip Morris Cos., Inc.
- Bick, R.L. MD
- Author (Organization)
- San Joaquin Hematology Oncology Medical Group
- Named Person
- Hillman, Dr.
- Kronick, Dr.
- Stedman, Dr.
- Named Organization
- Bureau of Biologics
- College of American Pathologists
- Council for Tobacco Research
- Cutter Diagnostics
- Dade Coagulation Diagnostics
- Dade Instruments
- Fischer Scientific Company
- Franklin Institute
- Hyland Laboratories
- Parke-Davis and Company
- Sigma Chemical Company
- World Health Organization
- Thesaurus Term
- Cardiovascular disease
- Government agency
- Health effects
- Industry sponsored research
- Research activity
- Statistical method
- Tobacco leaf constituent
- Virginia tobacco
Page 1: 1000136071
SAN JOA(~UIN HI~IATOLOGY ONCOLOGY MEDICAL GROUP
2727 Eye Street
Bakersfield, California 93301
Page 2: 1000136072
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" " o,f fibrinolysis. This system was chosen in spite of the fact that an early
.... proponents of this system have now published articles showing that the assay
.~ .... system itself is of no clinical meaning(4,5) In addition, the authors used
bovine thrombin obtained from Parke-Davis and Company (topical thrombin) in
.- phosphate buffered saline to. form their clots for the EGLT. They did not
• purify the thrombin. It is well known that Parke-Davis topical thrombin
• ': No precautions were taken to remove any of these i.e. any one of which could
obviously alter results of the EGLT. In addition, it is. well kno~ that an
• activated partial thromboplastin time or a partial thromboplastin time of ::
greater than 100 seconds has little, if any, meaning and is totally indepen- ~ ....
" " " " " " ' " ' ~ ' : ~! ~
~ i..~ ':.~"•'
: dant of any c~agulation factors or' coagulation i~hibitors in a
patient's ' '
• . ~. ,
.... ' ~ . ,,.
plasma but is, ~n the range of greater than 100 seconds, dependant
upon variables in reagents(8,9). In addition, to study kinin
. o~u-u~w~ey rat mn nodal esterase; ~
. - ....
. . . . . . ., ,, :
...... - ~
far more sophistocated s~st~ms for measuring kini:n generation
ar~avai~ab~~ '~.~ ~?~:~:.
Several other serious questions regarding this wor~ have recently
..,,• . . . . . .... ..~. • , .~ ~; ~ .~:
by Dr.'s Stedman, Kronick, ~d Hillman from the Franklin
Institute. ~ese -~:~.?.
authors have recently attempted to duplicate the work of B.eck~r~
et ai and
also found a~ditional fla~s in their work(10) Primarily, these latte~ authors ~
have found that tobacco glycoprotein could, in fact, be purified 'from pure
Virginia tobacco leaf using a polyacryl~ide gel electrophoresis system. However,
these authors are unable to purify. TC~ from cigerette smoke condensate (CSC) with-
out significant modifications in the procedure. In addition, these
"". . , ... : .~ ,, ...... .:. ~
Page 5: 1000136075
authors found that the major part of TGP-CSC was a polyacrylate contaminant
and not, in fact, pure TGP. In addition, it is known that polyacrylate can
account for all of the findings which Becket ascribes to "TGP". Specifically,
polyacrylate is known to be associat6d with thrombosis and delayed hyper-
sensitivity in mammalian, including human, tissues (11,12,15,14,15) In
evaluating the results of the work ofBecket and Dubin other serious flaws are
noted. Specifically, when reviewing Table 3 entitled The Effect ofDifferent
Concentrations of TGP-L and TGP-CSC on PTT of Normal Plasma the Table has
little, if any, meaning and is quite confusing. Firstly, most of the so-called
activated PTT's are greater than 100 seconds and therefore without sig:
meaning. In addition, it is noted that there is no correlation or linearity
between increasing concentration of TGP-L or TGP-CSC with the PTT.
fact, activation of Factor XII (or any Of the early contact activation phase
Factors) were occurring, one would see progressive linearity between concen-
tration of TGP-L/TGP-CSC and the activated PTT, i.e. with higher concentratiofi~
:Z~,- >; j, '" :-~t~:a' :~ ; -'~:5~ ~
" :. , of either TGP reagent one would see progressive shortening of the activated
:~,~i!!~",-i,ii-~ PTT (or non-activated PTT), this is a well described phenomenon
< known as the "Kingdon Assay" which is used to monitor fo~ activated clotting
. .. - '. - .- , :. • .... "'~:,::'.-,,...'~ .... ,
-:' - .'-'- ;~ :'.:O :~-"~
' factors in commerciai coagulation factor concentrates(16) The ~esults bf the
. .,-" "",U"
Becket experiments can, however, be explained by simple eomplexing of TGP-L
and TGP-ESC with Factor XII or one of the early contact activation phas~
Factors. This also is a well known phenomenon in Coagulationand will account
for highly variable and "non-linearity times" with a material which is
"compiexing with" one of the contact activation phase FactOrs; this is most
likely the case in this particular experiment~ In addition, the highly variable
Page 6: 1000136076
acid, TGP-L and TGP-CSC on PTT of normal plasma and Factor XII deficient "
plasma. None of the reagents including e11agic acid, TGP-L or TGP-CSC shorten
the clotting time of Factor XII deficient plasma~thus presumably elimi~
the possibility that any of these reagents are activating any clotting factor "
including Factor XII. Factor XII deficient plasma available from George King '
times reported by these authors with increasing concentrations of TGP-L or
,..:~t~, .? • t~-
T~P-CSC ~ake absolutely ~o sense whatsoever in light of current k~o~ledge of
the biochemistry of blood coagulation and in most instances no statistic~l
significance is obtained. Table 4 al~o raises serious quest£ons as to the
~eaning ~f t~e assag ~gste~s. ~is Table relates to the "effect
Biologicals is known to contain 1-2% of Factor XII; activation of only
1/lO00th of this would lead to shortening of a PTT performed on Factor XII
deficient plasma. In addition, when these same reagents are added to "normal
plasma" all of the times are greater than 100 seconds except when celite is
added, thus rendering the results meaningless. In addition, Table S labeled
"The Effect of Different Concentrations of Rutin-BSA on the PTT of Normal -.
• ~ .. Plasma" also raised serious questions regarding the authors ability to perform
....~ coagulation techniques. It is noted that the time changes with respect to
.... -Rutin-BSA in varying concentrations are not-statistically significant. ~n "
addition, it is noted that PBS gives a shorter time than the lowest concentration
and highest concentration BSA which makes absolutely no sense. Table 6 is
labeled "The ~ffect of TGP-L and TGP-CSC on the Thrombin Time ~f "Normal "
Plasma"; at high concentrations there is absolutely no affect. In summary, ..
the original work by Becker and Dubin raise serious questions because of using
non-recognized and non-standardized reagents which give '"normal times':' which
.. . .::: , ~.~
Page 8: 1000136078
~ by Cutter Diagnostics; this material is verified by The College of American
Pathologists to represent World Health Organization standards and totally normal
"human" plasma with respect to coagulation factor levels and levels of coaguo
lation factor inhibitors. Non-activated PTT's were utilized because of their
:~ high sensitivity to activated clotting factors; this isthe method used by
Bureau o~ Biologics (l?)to detect the presence of any ~ctivated clotting
factors in commercial therapeutic plasma fractions and is known as the ,Kingdon
Assay"(!.8). Reagents used were obtained ~rom Hyland Laboratories, a recognized
~ ' manufacturer of high quality PTT reagents~ Non-activated PTT's were per~orme~
. on Universal Coagulation. Reference Plasma which were reconstituted as follows:
Three preparations were tested: :
A. 1 ml o~ UCRP plus 0.1 ml PBS
B. 1 ml of UCRP plus 0.1 ml 4.5 mg ~ "CON"
• .~:i....~ C. 1 ml. of UCRP plus 0.1 ml 9 mg ~ "CON" "
Non-activated PTT's were performed in duplicate on all_three preparations. ..:
~" " III. ACTIVATION OF FIBRINOLYSIS
• ~•~ii Activation of fibrino.lysis was studied using fluorometric substrates by
• :-..:," ~-, the method of u e ~ and con i U ings
thetic fl etric
.,. .. substrates in: a "protopath" fluorometer supplied by Dade Instruments and Dade
.. : ..
• , . . . . • . . . ....
.. Coagulatmon DIa~nostmcs. _ . Plasmmno. gen and plasm~n assays were
perfo~ed on .~.~,..T"~.~- ~:~.~-~c~?:;:
~ "' -solution A which represents I ml of UCRP plus 0. I ml PBS, solution B: I ml
of UCRP plus 0.I ml 4.5 mg % "CON", and solution C: I ml of UCRP plus
9 mg ~ "CON". Ifactivatio~ of fibrinol~sis occurred one would see c~rculat~ng
plasmin and concomitant depletion of plasminogen. .
. " /~~~'~-
XII ASSAY W~ DONE BY METHOD OF ~TNOFF
Facto~ XII assays were perfo~ed on solutions A = 1 ml of UCRP plus 0 1 ml
Page 10: 1000136080
genic substrate(SO)on four solutions:
Solution A consisted of 1 ml of UCRP plus 0. I ml PBS.
Solution B represents 1 ml UCRP plus 0.I ml 4.5 mg % "CON".
Solution C represents 1 ml UCR~ plus 0.i ml 9 mg % "CON".
Solution D equals 1 ml of pure 9% "CON".
VI. EVALUATION OF COAGULATION INHIBITORS
Antithrombin-III assays for detection of activated clotting factors were
.... - "
performed on ~olutions A, B, C and UCRP alone (in duplicate)
" ~ " ..... (32)
and synthetic substrate methods.
A. 6eneration of Activated Clotting Factors by "CON":
~qhen "CON" was added to a Kingdon non-thrombogenic assay systeB '
(non-activated PPT), (an assay system which has been propagated by the Bureau
of Biologics as being exquisitely sensitive for detecting activated clotting
faCtors), no statistically significant changes in clotting ties 6f a Kingdon .~?-~...~
,..;-,. : thrombogenic assay performed on solution A, solution B, or so, lutio~ C was noted..
..... ~us,. it can b~ concluded that there is ~o "activation" of ~ny of the
~e E~ect o~ "CON" ~ Contact Activation Coagul~tio~ Factors:
~e~ specific ~ss~s ~ere performed on solutions A, ~, a~d C as ~e~l
_ as UCRP alone the ~ollo~ing ~es~1ts ~e~e noted:
in Factor XI, Factor X or Factor IX when specific duplicate determination
coagulation factor assays were performed on solutions A, B, C and UCRP alone.
However, there were significant differences in Factor XII assays between
..solutions A.B,C and UCRP. It was deteannined that 9 mg/% "CON"' caused an