the target of the present invention is to create and apply the methods in which biological substances are used that react specifically and scavenge the following : g ) withholding the trace elements cadmium , copper , manganese , iron etc . which are inhaled during smoking . a ) there is selection of appropriate scavengers , like hemoglobin or lysates of erythrocytes or any substance which contains stereospecifically bound iron b ) there is selection of scavengers which contain porphyrin ring with iron ( e . g . protoporphyrin ) c ) there is selection of scavengers which contain porphyrin ring that does not necessarily contain iron d ) there is selection of scavengers which contain porphyrin ring complexed with other metals , e . g . mg 2 + , cu 2 + e ) a biotechnical process will be designed for the enrichment of common conventional materials which are presently used in the production of cigarette filters which will contain the above mentioned biological substances - scavengers . the pivotal idea in this invention lies in the concept that impregnation of common conventional cigarette filters and / or filters containing activated charcoal can be enriched with the biological substances , characterized by the presence of metal ions fe 2 + , cu 2 + , mg 2 + complexed with the porphyrin ring , as well as fe 2 + bound stereospecifically to protein molecules , thus allowing the noxious compounds contained in the cigarette to be withheld before the smoker inhales the cigarette smoke . this fact is the main characteristic of the present invention and consists of an undeniable innovation with great feasible industrial applications . this invention was prepared in the following way in light of its applicability to industrial production levels : a solution of 1 mg / ml of hemoglobin and / or lysate of erythrocytes in phosphate buffered saline solution ( pbs ) with a ph of 7 . 4 was prepared and added to 100 mg of activated charcoal . they were incubated for 30 min at room temperature and filtered through a s & amp ; s carl schleicher & amp ; schuell co u . s . a . filter paper . the quantity of the non - absorbed hemoglobin was estimated in the filtrate spectrophotometricaly . the charcoal enriched with hemoglobin was left to dry at room temperature . a quantity of 200 mg of dry charcoal enriched with hemoglobin was sandwiched between two common filters so that all cigarette smoke drawn through comes into contact with the active . groups of the molecules ( fe 2 + , fe 3 + , -- sh , -- nh 2 ) ( fig2 ). these compatible materials are now ready to be used for the manufacturing of the new cigarette filters which we will refer to from now on as biological filters . alternatively hemoglobin can be replaced by biological substances characterized by the presence of metal ions fe 2 + , cu 2 + , mg 2 + complexed with the porphyrin ring , as well as fe 2 + bound stereospecifically to protein molecules , such as transferin , catalase , protoporphyrine , cytochrome c , chlorophyll . alternatively , a solution of 5 mg / ml of hemoglobin and / or lysate of erythrocytes in phosphate buffered saline solution ( pbs ) with a ph of 7 . 4 was prepared and scanned at 25 ° c . using an acta beckman recording spectrophotometer . an absorbance peak was consistently observed at 540 nm and 575 nm ( smith , r . p ., kruszyma , h . j . pharmacol . exper . ther . 191 , 557 - 563 , 1974 ). common conventional cigarette filters were impregnated with these solutions and they were air dried at 25 - 35 ° c . these compatible materials are now ready to be used for the manufacturing of the new cigarette filters which we will refer to from now on as biological filters . these new biological filters ensure that the smoke which is inhaled comes completely into contact with the active groups of the hemoglobin molecules and / or lysates of the filter without changing the physical properties or the taste of the cigarette smoke . for aesthetic reasons a small part ( 3 mm ) of a conventional filter can be adapted to the visible end of the biological filter . a solution of 5 mg / ml of protoporphyrin in buffer solution ( pbs ) ph 7 . 4 was prepared , and scanned at 25 ° c . using an acta beckman recording spectrophotometer . excitation of protoporphyrin with ultra violet light ( 498 - 408 ) produced an orange - red fluorescence between 620 - 630 nm . the conventional filters were then impregnated ( soaked ) with the above solution and dried with hot air ( 25 - 35 ° c .). alternatively a 5 mg / ml solution of transferine in pbs ph 7 . 4 is scanned using the acta beckman recording spectrophotometer . the ferric - transferine shows a characteristic spectrum of 470 nm . the above methods for impregnating the currently used conventional filters was used . alternatively a 5 mg / ml solution of catalyse in pbs ph 7 . 4 is prepared . the above method for the preparation of the biological filter is to be followed . alternatively a 5 mg / ml solution of cytochrome c in pbs ph 7 . 4 is prepared . the above method for the preparation of the biological filter is to be followed . alternatively a 5 mg / ml of chlorophyll in pbs ph 7 . 4 is prepared . the above method for the preparation of the biological filter is to be used . alternatively the above mentioned biological substances are sandwiched between two common filters in solid form so that all cigarette smoke drawn through the filter comes into contact with the active groups of the molecules ( fe 2 + , fe 3 + , -- sh , -- nh 2 ). the various biological substances used to enrich the conventional filters have been shown to retain the toxic compounds ( no , co , free radicals , h 2 o 2 , aldehydes and trace elements and nitrosocompounds ) from cigarette smoke in varying degrees as can be seen in the table below : ______________________________________ nitroso - free alde - com - trace no co radicals h . sub . 2 o . sub . 2 hydes pounds elementsscavengers % % % % % % % ______________________________________hemoglobin 90 90 90 80 90 90 95transferin 85 90 60 60 60 75 50catalase 85 90 90 90 80 80 80protoporphirin 85 90 70 80 70 75 80cytochrome c 85 80 70 80 60 60 70chlorophyll 15 10 40 15 10 10 80______________________________________ the degree of retention of the highly damaging substances of the cigarette smoke was obtained , and the smoke of the cigarette ( 20 ml ) filtered through a biological filter was compared with that filtered through a conventional filter ( 20 ml ). only 1 ml cigarette smoke drawn through the conventional filter was compared with 40 ml of cigarette smoke drawn through a biological filter . it appears that the biological filters have 40 times the capability of retaining the trace elements as compared to conventional filters . in the following detailed experimental description representative results are shown so as to better comprehend the activity of these biological substances . a ) identification of no contained in cigarette smoke using the chemiluminescence method : no was identified using the luminol enhanced chemiluminescence method as described in the experimental section . fig3 and 4 illustrate a typical experiment of no identification and estimation , as well as its scavenging after the passage of cigarette smoke through the biological filter . it appears that more than 90 % of the no is retained by the hemoglobin . the effectiveness of the biological filter is apparent in retaining and neutralizing the no which has been implicated in toxic reactions both in lung cells and in lung fluids especially when it is involved in the formation of the strong oxidant onoo -- b ) identification of free radicals contained in cigarette smoke using the chemiluminescence method : the free radicals in cigarette smoke were identified by the chemiluminescence response caused by the system lucigenine / damco after its reaction with the free radicals . fig5 shows a characteristic peak taken within 2 seconds of the chemiluninescence response which was inhibited 100 % after the passage of the cigarette smoke through a biological filter . the retention of the free radicals by the biological filters implies that there will be reduction of oxidative stress in the alveolar macrophages which is caused by conventional cigarette smoke . c ) identification of h 2 o 2 contained in cigarette smoke using the chemiluminescence method : h 2 o 2 was estimated by the chemiluminescence response produced by the system isoluminol / microperoxidase . fig6 shows the characteristic peak of cheniluminescence due to the presence of h 2 o 2 in cigarette smoke . in the presence of catalase ( 100 units / ml ) the chemiluminescence response was inhibited approximately 90 %. when the cigarette smoke passed through a biological filter an 80 % inhibition of the chemiluminescence response was observed . the isoluminol / microperoxidase system is specific for the identification of h 2 o 2 . the free radicals contained in cigarette smoke evoke a minute chemiluminescence response after their interaction with isoluminol . this minute chemiluminescence appears to be approximately 10 % of the total chemiluminescence caused by h 2 o 2 in the presence of free radicals since catalase inhibits the maximum chemiluminesent response up to 90 %. the retention of h 2 o 2 apparently reduces both the oxidative stress and the production of no by the alveolar macrophages . d ) identification of trace elements and aldeydes contained in cigarette smoke using the enzymatic system luciferine / luciferase . trace elements contained in the cigarette smoke were identified by their capacity to stimulate the luciferase activity . fig7 depicts : 1 ) the chemiluminescence response caused by the oxidation of luciferine in the presence of atp , 2 ) the enhanced chemiluminescence response in the presence of cd 2 + ions ( 0 . 5 mg ), 3 ) the enhanced chemiluminescence response in the presence of cu 2 + ions ( 0 . 5 mg ), 4 ) the enhanced chemiluminescence response caused by cigarette smoke ( 1 ml ) and 5 ) the inhibition of chemiluminescence response ( with respect to that caused by the cigerette smoke ) caused by 40 ml cigarette smoke when passed through the biological cigarette filter . it is obvious that the chemiluminescence response caused by trace elements contained in conventional cigarette smoke are more than 40 times higher than those passed through a biological filter . the withholding of trace elements by the biological filters may have both short term and long term effects . short term effects could entail the inhibition of redox reactions from taking place in the lung ( fe , mn ) and long term effects could entail inhibition of damages to the constituents and substances in the blood ( cd ). the aldehydes contained in cigarette smoke were identified and estimated using the same enzymatic system luciferine / luciferase in the absence of atp . aldeydes are capable of causing oxidation of luciferine . fig8 shows a characteristic chemiluminescence response which could last for more than an hour . this chemiluminescence response was inhibited 100 % when the cigarette smoke used had been passed through the biological filter , suggesting that the effectiveness of the biological filter to withhold the toxic aldehydes is substantial . the identification of nitrosocompounds contained in cigarette smoke was obtained by estimating the slow release of no from nitrosocompounds after their treatment with h 2 o 2 . as shown in fig9 a peak chemiluminescence response was obtained at approximately 900 seconds . passage of the cigarette smoke through a biological filter showed a 90 % inhibition in the chemiluminescence response observed and its peak was taken at approximately 1200 seconds . the slow release of no by sodium nitroprusside ( snp ) after its treatment with h 2 o 2 is also shown . fig1 shows the slow release of no from both : the nitrosocompounds diethyl nitrosamine and dimethyl nitrosamine ; and from hemoglobin enriched with nitrosocompounds from cigarette smoke treated with h 2 o 2 . it is clear that the no release by the nitrosocompounds of the cigarette smoke , which have formed adducts with hemoglobin , follow the same pattern of no release as the nitrosocompounds diethylnitrosamine and dimethyl nitrosamine . fig1 shows the release of no by the nitrosocompounds of the cigarette smoke which have formed adducts with hemoglobin after the hemoglobin - nitrosocompound adducts were irradiated with uvb ( 100 mj / cm 2 ) for one minute . the no release was estimated in the presence of h 2 o 2 and gave a chemiluminescence response at 1 second . the gradual rise observed in fig1 is due to the effect of h 2 o 2 on hemoglobin ( fenton reaction ). in vitro experiments were carried out with the help of a special chamber that was created in our lab , and which is shown in fig1 . the teflon membrane , separating the two compartments in the chamber , is permeable to gas no and impermeable to no 2 -- and onoo --. unchallenged lung macrophages isolated as described in the experimental section were suspended in hbss buffer solution ( 1 × 10 6 cells / ml ) and placed in the a compartment of the chamber . in compartment b of the chamber 2 . 5 ml griess reagent or sulfanilamide / scopoletin reagent is placed . the no , released by macrophages in compartment a , diffuses through the teflon membrane into compartment b . and binds with the griess and / or sulfamide / scopoletin reagents where it remains trapped . this indicates that lung macrophages produce gas no . the amount of no now present in compartment b was then determined spectrophotometrically or fluorophotometrically . the quantities of onoo -- and no 2 -- contained in compartment a of the chamber were also determined using the griess and / or sulfanilamide / scopoletin reagents . the above experiments were repeated after challenging the macrophages with cigarette smoke before placing them in compartment a . the results , as depicted in fig1 , show that cigarette smoke decreases the amount of no produced whilst increasing production of onoo -- in lung macrophages , indirectly indicating the tremendous production of both no and o 2 -- which interact to form onco --. repetition of the above experiments using biological filters ( i . e . in which cigarette smoke was drawn through a biological filter ) showed that the biological substances used , produce the same quantities of no 2 -- and onoo -- in compartment a and similar quantities of no in compartment b as would macrophages not challenged with cigarette smoke . in this context , the components of the griess reaction were also used to examine the kinetics of nitrosation by intermediate ( s ) generated during the no / o 2 reaction in aqueous solution at physiologigal ph . addition of cigarette smoke ( 50 ml ) to a 100 mm phosphate solution ph 7 . 4 containing 25 mm sulfanilamine and 2 . 5 mm n -( 1 - naphthyl ethylenediamine dihydrochloride ( nedd ) generated an absorption at λmax = 496 mm indicative of the characteristic azo product resulting from nitration . it is worthwhile to consider the implications of the present observations vis - a - vis the expected reactivities of no under physiological relevant conditions , where maximal concentrations of no in the cellular microenvironment are estimated to be in the range of 0 . 5 - 10 μm . the no concentrations are dramatically increased during cigarette smoking with detrimental effects on the lung cells . the results on the effects of cigarette smoke on the oxidative stress of lung macrophages are illustrated in fig1 . estimations of the oxidative stress using t - bhp , showed that cigarette smoke causes twice the oxidative stress that unchallenged macrophages do . when the cigarette smoke was passed through a biological filter the oxidative stress observed was similar to that of unchallenged lung macrophages . it is thus clearly indicated the elimination of the oxidative stress induced by cigarette smoke on macrophages . the cigarette smoke is now free of the substances that cause oxidative stress on lung macrophages . h 2 o 2 produced by macrophages challenged by cigarette smoke show more that 10 times the production rate as those macrophages not challenged . the use of a biological filter show a decrease in h 2 o 2 production by 90 % ( fig1 ) as compared to conventional filters . it is obvious that as cigarette smoke induces oxidative stress in the macrophages it increases the production of toxic h 2 o 2 by these cells . the amount of cyclic gmp produced by the no released by alveolar macrophages was determined using the chamber shown in fig1 where soluble guanylate cyclase was placed in compartment a and alveolar macrophages were placed in compartment b . the quantities of no produced by the macrophages were determined over a period of 50 minutes with and without cells challenged with cigarette smoke . marcrophages challenged by cigarette smoke ( 10 ml ) released approximately ten times less the amount of no with respect to the untreated cells thus showing 10 times less production of cyclic gmp . the above procedure was repeated using cigarette smoke passed through a biological filter . it was shown a non statistically significant difference with respect to unchallenged macrophages ( control ) ( fig1 ). the accumulation of no in compartment b was increased more than 5 times when the alveolar macrophages were treated with h 2 o 2 ( 5 mm ) fig1 . this suggests that h 2 o 2 increases the production of no by a positive feedback mechanism . the l - arginine / no pathway in macrophages is consistent with the concept that cigarette smoke causes the release of no / onoo --. co presence in cigarette smoke was determined using the biological method based on the stimulation of soluble guanylate cyclase by co . introduction of hbss saturated with cigarette smoke into compartment a of the chamber , in the presence of superoxide so as to neutrilize no , and the introduction of soluble guanylate cyclase into compartment b resulted in an increase in cyclic gmp production due to co diffusing from compartment a to compartment b . passage of cigarette smoke through a biological filter reduces the amount of cyclic gmp produced by approximately 80 % ( fig1 ). the above data indicates that the noxious substances nox and co contained in cigarette smoke are retained and neutralized by the biological filters . a ) we first confirmed the presence of no and onco -- in exhaled cigarette smoke . human volunteers smoking a cigarette bearing a conventional filter no present in the exhaled cigarette smoke was identified after the introduction of the exhaled smoke into an acid solution ( 50 ml ) ph 4 . no concentration was estimated by the lyminol enhanced chemiluminescence method described in the experimental section , using standard curves made by commercial no . no concentration was found to be 0 . 045 mm . the experiments were repeated using biological filters and the no concentration in the inhaled smoke was approximately 70 % lower compared with the conventional filter ( fig1 ). concentration of onoo -- was determined using a solution of naoh 1 . 2m which showed an increase in absorption at 303 nm ( fig1 ) ( ε 303nm = 1670m - 1 cm - 1 ). our experiments showed that during smoking the exhaled smoke contains large quantities of onoo -- ( passage of 50 ml exhaled smoke into 5 ml naoh 1 . 2m yielded a solution of 0 . 9 mm onoo --). the ratio of no / onoo -- in the exhaled smoke was determined to be 1 : 20 . therefore it appears that nox in the lung is transformed to onoo -- when it reacts with superoxide in the lung . superoxide is released from both macrophages and redox reactions occuring in the lung during smoking . cigarette smoke drawn by a pump does not contain onoo --, however a quantity of nox reacts with superoxide or oxygen to form nitrite ions ( no 2 --). onoo -- is formed only when cigarette smoke enters the lungs . the use of biological filters reduces the exhaled quanities of no and onoo -- by 70 %. b ) onoo -- reacts with bicarbonate ions of the human erythrocytes according to the reaction the bicarbonate radical oxidizes luminol as well as aromatic and heterocyclic molecules . alternatively onoo -- may peroxidize bicarbonate to peroxybicarbonate another strong oxidizing species . on the other hand superoxide dismutase ( sod ) catalyzes the nitration by onoo -- and a wide range of phenolics including tyrosine in proteins . thus there are several potential mechanisms by which bicarbonate and sod could influence the overall reactivity of onoo -- in the cells . the presence of onoo -- formed in the lungs by inhaled cigarette smoke , exhibits a dramatic increase in the oxidative stress in erythrocytes which was detected by a chemiluminescence response occuring within 5 seconds . the same experiment conducted using a biological filter resulted in an almost 100 % inhibition of oxidative stress in human erythrocytes ( fig2 ). hemoglobin or eurythrocyte lysates exposed to onoo -- ( contained in the exhaled cigarette smoke ) caused the abolition of the two peaks at 540 and 575 nm normally observed in hemoglobin . the results of a representative experiment similar to the one described above was performed in 12 volunteers and is shown in fig2 . when hemoglobin and / or lysate were exposed to a small quantity of exhaled smoke ( 10 ml ) a shift of the peaks from 540 and 575 to 525 and 555 nm was observed consistent with the formation of nitrosyl hemoglobin . the experiments were repeated using biological filters . the peaks observed maintained their characteristic wavelengths . d ) aldehydes were identified in the exhaled cigarette smoke from human volunteers by their characteristic chemiluminescence peak . the experiments were repeated using biological filters and a 90 % reduction of the chemiluminescent response , was observed as compared to a maximum chemiluminescence response observed when using a conventional filter ( fig2 ). it is obvious that the biological filters withhold and neutralize the aldehydes in cigarette smoke whilst retaining the oxidants , thus apparently inhibiting the initiation of redox reactions from taking place in the lung which would result in the production of endogenous aldehydes . e ) free radicals were identified in the exhaled cigarette smoke , from human volunteers by their characteristic chemiluminescence peak . human volunteers used cigarettes bearing conventional and biological filters . they were advised to exhaled cigarette smoke ( 50 ml ) in an acid solution ( 0 . 01n hcl ) ( 50 ml ) ph : 6 and the chemiluminescence response was taken after 5 min and 60 min . at ph : 6 the exhaled onoo -- is spontaneously decomposed . within 5 min there was a 160 % increase of the chemiluminescence response in the exhaled smoke passed through a conventional filter as compared to cigarette smoke passed through a biological filter ( fig2 ). when the saturated by the exhaled smoke acid solution was left for an hour the difference in the chemiluminescence response increased from 160 % to 250 % ( fig2 ). this is consistent with the concept that redox reactions are taking place continuously in the cigarette smoke through the quinone radicals and produce a series of activated oxygen species that can cause biological damage . our studies have shown that alveolar macrophages possess an endogenous no synthase , like other cells , and are capable of releasing no / onco -- for prolonged time periods following exposure to cigarette smoke . furthermore , once no begins to be released by these cells , the production of no becomes self supporting even after the stimulus is removed . such a reaction accounts for the ability of the cigarette smoke derived no to stimulate alveolar macrophages in releasing no and onoo -- for a period of several hours after the removal of the stimulus . such a reaction may be initiated by the production of h 2 o 2 in the lungs upon stimulation of alveolar macrophages by cigarette smoke . h 2 o 2 may stimulate no synthase activity of the lung cells to produce no and onoo -- for a time period of more than an hour after the removal of the stimuli . our experiments indeed showed that passage of cigarette smoke through a biological filter resulted in a 90 % reduction ( as compared to a conventional letter ) of the oxidative stress in the rat alveolar macrophages . an onoo -- radical formed in the lungs may possibly attack and inactivate the a1 - proteinase inhibitor ( a1pi ). inhibition of the a1pi in human lungs often causes emphysema in which lung capacity is reduced . statistical evidence indicates that smoking predisposes one to the development of emphysema ( southon , p . a ., pwis , g ., free radicals in medicine . involvement in human disease . mayo clin . proc . 63 : 390 - 408 , 1988 ). in in vivo experiments performed in 12 volunteers smokers a 90 % reduction of the exhaled no / onoo -- was shown when the inhaled cigarette smoke was passed through a biological filter . oxygen free radicals have also been implicated in the pathogenesis of iga immune complex induced alveolitis . pretreatment of animals with superoxide dismutase , catalase , the iron chelator desferioxamine , or the hydroxyl radical scavenger dmso , supresses the development of lung injury . in contrast , the lungs of untreated positive control animals are characterized by the presence of increased numbers of alveolar macrophages . interstitial edema and hemorrhage are also present . furthermore , in this model of lung injury , the l - arginine is also highly protective as demonstrated by reduced : vascular permeability ; vascular hemorrhage ; and injury to vascular endothelial and alveolar epithelial cells . these findings suggest that the macrophages are the source of the damage causing no , o2 --, h 2 o 2 and oh compounds ( mullingan , m . s ., jonhson , k . j ., ward , p . a ., in : &# 34 ; biological oxidants : generation and injurious consequences &# 34 ; ( eds . cochrane , c . g ., and gilbrone , m . a ., jr . academic press 157 - 172 , 1992 ). the retention and neutralization of the oxidants contained in the cigarette smoke by the biological filters may play a significant role in reducing the activity of the redox enzymes which are directly related to the oxidative stress in the lung cells . biological filters drastically reduce the oxidative stress caused by inhaled cigarette smoke . oxidative stress in the lung macrophages and endothelial cells of the lung vessels may be induced by no , nox oxygen radicals and / or aldehydes contained in the cigarette smoke . furthermore the retention of aldehydes and trace elements ( especially of cd ) by the biological filters may have considerable long term effects in preserving the plasma antioxidants and in inhibiting the development of artherosclerosis . hemoglobin contains several neutrophilic centers which undergo covalent reactions with electrophiles . these centers induce the n - terminal valine residues of the α - and β - chain , the n1 and n3 atoms of histidine residues and the sulphydryl group of cystein residues . the carcinogenic nitrosocompound 4 -( methylnitrosamino )- 1 -( 3 - pyridyl )- 1 - butanone ( nnk ) present in tobacco is transferred to the smoke during burning of cigarette and its levels in mainstream smoke could vary from 4 to 1700 ng per cigarette nnik can form adducts with hemoglobin ( hecht , s . s ., karan , s ., and carmella , s . g ., in : &# 34 ; human carcinogen expose &# 34 ; eds . garmer , r . c ., farmer , p . b ., steel , g . i ., and wright , a . s .) irl press pp . 267 - 274 , 1991 ). clearly the only way to avoid tobacco - related diseases is to refrain from tobacco chewing and smoking . however , the statistics on current smokers , indicate that a strong case can be made for the need to reduce exposure to tobacco carcinogens and to modify their mode of action . principal approaches toward this goal are : 1 ) modification of tobacco products , 2 ) inhibition of the metabolic activation of tobacco carcinogens and their endogenous formation by certain micro - and macro nutrients and chemopreventing agents and 3 ) retention of tobacco carcinogens using specific filters which will be adapted in the tobacco of the cigarettes . our invention using biological substances for the manufacturing of biological filters finally concerns the discovery that nitrosocompounds present in the inhaled cigarette smoke are withheld by the biological substances protecting the health not only of the smokers but of the non - smokers as well .
this invention refers to a method of withholding noxious compounds contained in cigarette smoke which were up to today insufficiently retained by conventional cigarette filters . the method described specifically refers to the enrichment of common convention filters with biological substances of the metal ions complexed with porphirin ring as well as fe 2 + ions stereospecifically bound to protein molecules , either separately or in combinations . the enrichment of these conventional filters with the abovementioned biological substances alters neither the physical properties of the cigarette smoke nor the physical properties of the filter itself .