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19"/6, OUR EXPERIENCE WITH THE EARLY DETECTION OF SMALL RESPIRATORY DISEASES (Ni~, Yougosla~,ia) In order to establish which method o] examining ~ulmonk: b appropriate for early dia~nosls o/small r, splratory airway di~ease~, w, ~de ex~i~tio~ o~ several ~ou~ by ~ing di]jeren~ ap~r~t~¢s and di[fsr~nt I~sts. " The first group c~ted o[ 1517 young men. We exami~d them by MMFR and FEV~ and estab~d th~ MMFR 25-751FVC had d~covered double number with ~ssened ~aes then In the [oarth ~roup a hundred men. aged 50. were e~amined by plethysmo- graphy and me~uremen~ o~ closing volume and me establbhed the results o~ MEF 25 and ~ 50 ~ the besl. ~ well ~ IGV wh~e ~ and Rs did not show ~uffic~at s~ceptibility /or small respiratory ai~ay di~e~es. ~ /or e/o~a~ volu~ it showed incre~ing for all. especially In 1956, Duhois intr~!uced the direct measurement of the resistance respiratory airway by using phthysmography. For some time it was thought that the best way of early detection of respiratory disease was measurement of resistance in it. Soon it was discovered thaf a disease of respiratory airway can be very significant although the finds of splro~ merry are normal.-This statement mostly refers .to'small respiratory diseases. In the year 1950 and 1958, McLean explained pathogenesis of em- physema and attached great importance to pathological changes in ter- minal respiratory bronchiole~. Obliteration and obmration of small respi- ratory airway is a matter under great discussion. It is known that resistance in small respiratory airway is ten or more times weaker than the one in big respiratory airway and being like that, i~ is of little influence in total pulmonary resistance. Because of that fact we cannot discover ~the disease of small respiratory airway by measurement of its resistance. On the occasion of routine spirometric examination of small respiratory airway diseases, classical test of a spirometry are almost always of a normal value. During small respiratory airway, diseases~ besides changes in bron- chioles, peribronchiai changes appear as Well and lessening of pulmonary T!04231211

522 Basni~ Mil,,nka et coll. recoil comes as a mmfifestation of that. So. the measurement of com- pliance, residual volume, FRG and other speeial test were nlade in order to discover small respiratory disease as soon as possible.' The test which shows how quickly lungs are empty during" ma_ximal expiration in the unit of time with re]atiou to volume is the best one. FEVI, dynamic forced expiratory vtllume per second, is kllowB as a good one. At forced expiration (FEF) speed V max. of 100 % to 75'% of VC depends on expiratory musculature previously. V max. 75'°]~ to 25 o-~ of VC depends higher degree on resistance ill respiratory airway. The longer expiration, closer to the end of VC (25 %) the greater dependance of flow on the state of structures and patbophyslologlcal factors of small respiratory airway. FEV~ is significant for big respirator)' airways but is not enough noticeable for small one, because closing of small respiratory airway, at small volumes, appears at the end of the first second. In order to mesure small respiratory volumes and closing volumes as well a lot of more or less promising test have been used in recent years. Its value is different~ so we tried to examine them on own material and compare their values. MF-,THOD AND MATERIAL We used the best known tests for early diagnosis of small respiratory airway diseases. We compared the results of the tests for four different groups of examined persons. In the first group we compared the results of examination of the young men who live in mountainous areas with tile youth who live in the city: all of them were the same age. We compared two groups of persons, smokers and non smokers, age 30 and 50, as well. We used mostly maximal midexpiratory flow rate 25-75 % of FVC test, discovered by Fowler and Leuallen in 1955, then a curve expiring volume and its MEF 25 and 50% of F\rC. The group of workers in metal industry, aged 50, was examined plethysrno~aphieally with the use of closing volume. We used apparatuses of the type - Vitalograph, Jones Co., Pneumo- datascreen and Plethysmograph (Jaeger Co.). Closing volume was done by the helium bolus method with the apparatus of Godart Co. Spirometric finds of 1517 young men, who live in mountainous area, aged 18-19, were shown on the table I. We used Vitalograph. MMFR was counted from the curve of FVC. It is seen on the table that values of FEW were lessened under 80 % for 10% examined persons, 19 % persons have lessened values of parameter under 3,51/s. It can be sedn by comparing these two tests that MMFR was lessened with l0 % persons who have normal find of FEV~. It can be deduced by this comparison that the medium segment shows doubled number with suspicion to the beginni~ cion of slavian in tim group li datascre. Thi smokers values o persons . 50% w~ with M1- with The degree o (who sm, FEW is of values. • is closer Whe is seen was lesse. seen by of the te, . all paran, The same bral; get Co. az method. Whe~. smokers value$ are smokers al percentage non smoke The r, the same, considerab] than with ~S for Tile as well as T104231212

of corn- in order ~est one. s a good ~- of VG 25 % of ay. The ~ndance of small t enough .[rway, at volunles in recent material -espiratory • different on of the ho live in groups of We used discovered ne and its examined ., Pneumo- was done ~o. inous area, h. MMFR that values ~ons, 19 % an be seen .% persons comparison :ion to the Early detection of small respiratory diseases 523 beginning of obstructive disease. Our results (19 % young men under suspi- cion of having early stadium of disease) is on accord with other Yougo- slavian authors. ]n the second .group we examined young men of the santo age as in the first group but in co.ntrast to them, the young men of the second group live in a polluted air city. The apparatus Vitalograph and Pneumo- datascreen were used. The results of both do not diverge essentially. The table II shows that 66 young men were examined, 51 non smokers and 15 smokers with 10-90 cigarettes daily. If we compare the values of the tests it can be seen that FEW was lessened with 10 % person~, and MEF 25 % with 35~ ones. It is obviously seen that MEF 50 % was also lessened with 35 % examined persons and the same was with MEF 25 %. Tlae values with smokers were lessened for 10'% than with non smokers. "/'he third group consists of 150 clerks who live in a city a high degree of air pollution. There were 79 non smokers and 71 smokers (who smoke 20 cigarettes daily). They were about SI years old (tab. IIl), FEX,a is ahnost of the same value with both groups. From distribution of values, which is not shown here it can be seen that FEVt of smokers is closer to the lower linfit than it is for non smokers. When MEF-FE, F 25 of smokers and nora smokers are compared, it is seen that the values are smaller for 24% for smokers, and MEF 50 was lessened for 21'%. MEF was lessened for 16 % smokers. It was seen hy distribution of values under 80 % FEW and MEF that values of the tests were lessened more for smokers than for non smokers for all parameters. The 4th group consist of metal workers, age 50, 57 smokers~ 35 non smokers and 8 of them gave up smoking ten years age. All work in the same branch of industry. Examination were made by plethysanograph Jae- ger Co. and closing volume by apparatus of Godart Co. with helium bolus method. When the diseases of small respiratory airway with smokers and non smokers were compared, it was seen on the table that the significant values are just for IGV with P ~ 0.005. When other parameters between smokers and non smokers were compared it was shown that, concerning percentage, all values with smokers were in same degree smaller than with non smokers. The results of VG and F.EVx for smokers and non smokers are almost the same, but the values of parameters MEF 50 and MEF 25 were lessened considerably. MEF 25 and MEF 50 with smokers was lessened for 10 % than with non smokers and MF~F 50 with smokers was smaller for 90 %, as for ex-smokers, it was lessened minimally (tab. IV). "]'he resistance was greater with smokers for than with non smokers as well as IGV, which is greatly significant with smokers. Ti04231213

524 Bosn[d Mi[anka ct coll. Data ]rom comparLton between FEV~ and M. ~o zheor ]00 + 20 80 -- 60 6O -- 45 FEVz (tot~! = 1517) 1363 121 33 89.8 8 2.2 < z.4 2 0.15 1.7 1 29 lo MMFR (Max3mul Midexpiratory Flowrate = Fowler). O.Itz 24 30.3 ,, No smoker 51 TAnLZ lI. -- Data [rom compar,oo 15 Pred. val. Pred. val. Meas. va|. MMFR (25- <2.5 l/s % 20 1.45 46 38 14 42.5 lung [unction tests (. "VC FEVt -- . MEF.,.__~ Smoker Ill. -- Data from compt VC FEV] N. ~, 71 Pred, val. Mess. val. Pred. val. Meas. 4.74 4.80 4.a3 4.79 % SD 102 80 99 % 3.8Z ~ 4.30 113 3.9 4.06 104 lung function tezts (/ 8.87 8.71 7.40 T104231214

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526 I I I i Val regard t. the diffi of smok, show th for sma Discuss In in direcl proporti, EPI intrabrol RFe Oppo but I~ l are small moves ir~ condition, close earl to close close mot units is Closing and it In tl examinin.. seen that or they Orl an av early dia,_ wkhdraw The FEV~. O. examined the value- We it is evide in the fir, With age and • question susceptibh T1042312I 6

Early detection of small respiratory diseases 527 Values of closing volume are not statistically significant and with regard to 1~ercentage of medium values between smokers and non smokers, the difference is in the benefit of ~nokers in small degree. C11osing volume of ~mokers is 25,4-9 ~o of VC with standard deviation of 6.q4.. These results show that whole closing volume was increased which obviously speaks for small respiratory, airway disease. ]~'ISC'USSION In 1967, Mead established that MEF for any pulmonary volume is in directly proportion with elastic pulmonary pressure and in inverted proportion with the nfidbetween pressure of alveolas. EPP (Equal Pressure Point) is a place where, during expiring, positive intrabronchial pressure and intrapleural pressure equalize and their forces are opposite. During'quiet breathing EPP is in a big respiratory airways but during forced expiration it moves towards small respiratory airway. In 1969 Macklem affirms that EPP of a healthy r/~an moves if volumes are smaller than 25'% of VC. But with obstruction of RT and RSm EPP moves into Rt already with larger pulmonary volumes. These are basic conditions which increase resistance in small respiratory, airways so they close earlier and that is why we get greater IGV and GV. Airways begin to close with 46 % of total pulmonary capacity -- Sutherland asserts. They close more and more with fall of pulmonary volume. A half of pulmonar5, units is closed for RV. Early closing has been measured by closing volume. Closing volume shows greater closing during fall of pulmonic elasticity, and it can be seen in diseases of small respiratory airways. In the comment of our table, when we compared their values got by examining different groups with different apparatuses, it is obviously seen that the values of routine parameter are on the limits of normality or they were partially narrowed with smokers, 10 % of theoretical values on an average at the most. But this comparison shows that the tests for early diagnosis of small respiratory airway diseases have considerably great withdraw all from nonaaal values (predicted). The table I shows that MMFR was lessened almost doubly than FEV~. On the table II FEF 25 and FEF 50 were lessened for 35% examined persons, while FEVa was lessened just for 10 %. In this way, the values of both tests can be seen. We compare both parameters by comparing those two groups, so, it is evident that FEF 25 and FEF 50 are considerably lower than MMFR in the first group. With regard to the fact that these are the young men of thg same age and the difference is pritty considerable, the most probable is the question of susceptibility of the test. It is hard to say if MMFR is less susceptible than FEF 25 and FEF 50 or it is the matter of greater part T!04231217

.528 BosniE Milanka ct coll. of persons from city area that I~ave the heginning disease than the ones from mountainous area. If we consider the values of the tests in the table III and distrihutive values, we see that FEVt is of equal values with smokers and non smokers, hut the values with smoker.~ are somewhat smaller. The values with smokers are lower for 20 % that with non smokers. that can be seen by comparing ]:EF 25 and FEF The 4th group was the oldest of all groups, hut as they work metal industry about 30 years, it was normal to expect worse results. There is no great significance between smokers and non smokers, hut average values show that smokers have worse test values. IGV and Rt are the only ones significant statistically, while closing volume is not, but anyway, it is increased three times with all examined persons, slightly more with smokers. It is seen in thi~ group that the routine tests are closer to normal values while other tests (FEF 25,' FEF 50. IGV, Rt, Rs and CV) as more susceptible parameters for detection of changes in small respiratory airway, more susceptible and they incline to pathological values in higher degree. This examination justified, more or less, other authors assertion about possibility of early diagnosis of small respiratory airway diseases, and especially this examination told the difference hetween smokers and non smokers and all others which are open to pathologic agents. Both young or old men can be obstructive persons. The conclusion of our work is that the shown parametres confirmed justification to be used for early diagnosis of disease and that they are mostly the same. Some of them fit mass use, others are just for clinical work, Hospital [or Lung Disease: Knez Selo, 18.000 Ni~, Yougoslarla (Prim.: Bos.~d dr. A: One h. 66 carpet ~, [actory wet .411 we disease due gu?n~lTary l't to deterrah, workers ana The r, parameters Finally I NTROD UCT: All int appearing other dust: A rex" Diseases of Extrinsic I" dusts was (2~ %). In the of respirato inhalation , the same p. and the res (*) Parth T!04231218

mucosa! m[crosomes from treated rats in a dose-dependenl fashion. Maximal stimulation of benzo(a}pyrene hydroxylation activity (160% above control) was achieved with doses be- tween 250 and 375 ,ug of penlagastrin per kg body weight, which was in good agreement with the amount of pentagastrin reported to stimulate maximal DNA synthesis in the colonic mucosa (18). As shown in Table 1, the hydroxylation of benzo(a)pyrene and a variety of drugs is stimulated by pentagastrin pretreat- merit but to different degrees, The rate of hydroxylation of some drugs, especially ethylmorphine, is stimulated after only a single treatment with pentagastrin. Highest activities were observed after 3 days of treatment. The hydroxylation rates of ethylmorphlne and benzo(a)pyrene were most responsive to pentagastrin pretreatment, Colonic cytochrome P-450 content was almost doubled and cytochrome P-450 reductaae activity was increased by pentagastrin pretreatment. Having shown the dosage and time dependence of the pen- tagastrin induction of mucosal drug metabolism system activi- ties, it was important to determine whether pentagastrin induc- tion involves the synthesis of new enzyme protein as has already been shown for phenobarbital induction of the liver drug metabolism system (7) and for the emergence of drug metabolism activities in neonatal hepatocytes (4}. The inhibitor of protein synthesis, cycloheximide, was used to examine this possibility. As shown in Table 2, cycloheximide injected 45 min before pentagastrin for 3 days completely prevented the induc- tion of drug metabolism in the colon by pentagastrin as judged by the elimination of pentagastrin-dependent increases over the untreated controls in cytochrome P-450 content, cyto- chrome c reductase activity, or the rate of hydroxylation of any of a variety of substrates. These data suggest a role for protein synthesis in induction of the colonic drug metabolism system by pentagastrin. Induction of Hydroxylatlon Activities by Cholecystokinin and Secretin, The data in the experiments described above show that pentagastrin, a pentapeptide hormone containing the minimum amino acid sequence required for the physiolog- ical activities of gastrin (1 1), markedly increases drug metab- olism activities in the colon through a process involving protein synthesis. Of the other major gastrointestinal hormones, cho- lecystokinin has an almost identical COOHoterminal sequence to that of gastrin. The terminal pentapeptide is the same in the 2 hormones. Secretin, on the other hand, has more sequence Table 1 Induction of colon microsomal drug metabolism system activities by pentagastrin Rals received injections of pentagastrm, (250 Fg/kg/day) for I, 2, or 3 days and were sacrificed 16 hr after the last injection. Microsomes were 10repared from the colonic mucosa and assayed for the catalytic activity usir~g various substrates. The data are reported as I~ercenlage of increase in activity above the control treated only with 0.9% NaCI solution. Control levels are given in Table 2. % of increase over control Hydroxylation activities Day I Day 2 Day 3 eenzphetamine 0 13 17 Ethylmorphine 28 134 278 ~-Nitroanisole 6 36 65 p-Nitrophenetole 0 13 22 Benzo(a)pyrene 0 8 e3 System components Cytochrome c reductase 5 22 29 Cytochrome P-450 content 0 0 77 Benzo(a)pyrene Hydroxylation and Gastrointestinal Hormones Table 2 Effect of cycloheximlde on inducb'on of colon mtcrasomal drug metaDol~sm activities by pentagastnn Animals received either a single i.p. injechon of Denlagastrin (250 g/kg) for 3 days or 2 Lp. injections of cyclohex=mide {1 mg/kg) plus one injechon of pentagastrin for 3 days. Control rats received vehmle only. The animals were sacrificed, and the colon microsomes were assayed as described. Cyclo- hexlm- Cyclo- ~de + hex~m- Penta- penta- Aclivities Conlrol Ide gastrm gastrln Benzphetamine=' 0.48 0.47 0.56 0.27 Ethylmorpilinea 0.35 0.52 1.32 0.26 p-Nitroanisoleb 1.42 2.34 0.72 p-Nitrophenetoleb 1.04 0.0£] 1.27 0.62 Benzo(a)pyrenec 3°50 0.03 6.40 2.20 System components Cytochrome c reductase°' 26.7 t B.6 34.4 21.7 CyIochrome P-450° 13 23 6 a nmol formaldehyde liberated per mit~ per rag. b nmol p-nilrophenol liberated per rain per rag. c pmot 3-bydroxybenzo(a)pyrene produced per rain per rag. d nmoI cytochrome c reduced per rain per rag. e pmol cytochrome P.-450 per rag. homology with pancreatic glucagon than with gastrin. Thus, it was important to compare the actions of these gastrointestinal hormones with those of pentagastrin. As shown in Table 3, both secretin and cholecystokinin octapeptide have marked effects on the drug metabolism activities of the colon. Cyto- chrome P-450-specific content was increased 2.5-fold by se- cretin and 1.5-fold by cholecystokinin. With the exception of its effects on the rates of O-dealkylation, cholecystokinin af- fects the colon drug metabolism activities in the same direction as does pentagastrin. Cholecystokinin stimulates benzpheta- mine hydroxylation a little more and elhylmorphine hydroxyl- ation a little less than does pentagastrin. The main point of difference, however, between these highly similar polypeptides is the marked lowering of O-dealkylation activity after treatment with cholecystokinin, whereas pentagastrin pretreatment elicits a modest increase in O-dealkylation of p-nitroanisole and p- nitrophenetole. Secretin, which is known to inhibit the physio- logical effects of gastrin (31), stimulates the colon microsomal hydroxylation of most substrates, with the exception of benz- phetamine and ethylmorphine. Moreover, the stlmulatory effect of secretin on O-dealkylation is greater than that of pentagas- trim The effect of the gastrointestinal hormones on hepatic drug metabolism system activities is shown in Table 4. In general, some of the effects of these hormones on the liver system parallel their effects on the colon system, while others do not. O-Dealkylation activity in the liver is not significantly affected by any gastrointestinal hormone. Pentagastrin does not signif- icantly alter ethylmorphine metabolism in liver, and cholecys- tokinin does not alter benzphetamine metabolism. Quantita- tively, however, the liver responds to these hormones less well in terms of the percentage of increase than does the colon. Nonetheless, the control liver activities are higher than the induced colon activities. Effects of Tissue Substances on Colon and Liver Micro- somal Drug Metabolism System Activities. Pentagastrin, se- cretin, and cholecystokinin show some pronounced specificity in the hydroxylation activities that they preferentially induce inter se. They also seem to affect liver and colon systems APRIL 1981 1409 T!04231221

W. F. Fang and H. We Strobel differentially. In order to examine further this possible specific- ity, we tested several tissue substances for their effects on drug metabolism activity. (Tissue substances, like hormones, have a broad range of effects but are produced by several tissues.) We chose 5-hydroxytryptamine (serotonin) and its precursor 5-hydroxytryptophan, 1 6,16-dimethylprostaglandin E2 (a stable prostaglandin derivative), and reserpine (a plant alkaloid) for this study and determined the appropriate dos- ages. The effects of these tissue substances on colon drug metabolism activity are shown in Table 5, and the effects on liver drug metabolism activity are shown in Table 6. None of the tissue substances induces O-dealkylation in either the liver or colon systems. In fact, the lowering of activity in pretreated animals is statistically significant in most cases. Reserpine pretreatment significantly stimulates colonic ethylmorphine de- methylation. 16,16-Dimethylprostaglandin E2, on the other hand, markedly stimulates colonic benzphetamine hydroxyl- ation (2.5-fold) but inhibits liver benzphetamine hydroxylation. Table 3 Induction of colon drug metabolism activities by gastrointestinal hormones Animals received injections el a single dose of secretin (27 Fg/kg), cholecysloklnin octapeptide (20 ~.g/ kg), or pentagastrin (250 ~g/kg) for 3 days as described in Table 1. Microsemal preparations el the liver and colon were assayed tot various drug metabolism activities. Hydroxylation activities Control Pentagastrin Secretin Cholecystoklnln Benzphetaminea 0,48 -~- 0.02b 0.56 + 0.03 0.19 "+- 0.04¢ 0.74 + 0.04c Ethylmorphinea. 0,35 .'*- 0.03 1.32 ± 0.15c 0.32 ± 0.02 0.45 ± 0.05 p-Nitroanisole° 1.42 ± 0.10 2.34 __. 0.42 t.94 + 0.28 0.39 ± 0.03c p-Nitrophenetoled 1.04 + 0.17 1.27 --. 0.16 2.26 +_ 0.03c 0.54 ± 0.05c Benzo(a)pyrenet 3.5 ± 0.7 6.4 "*- 0.50c 6.0 ± 0.4c 7.0 ± 0.4e System components Cylochrome P-450 contentg 13 ± 2.0 23.0 ± 2c 31.0 ± 3c 21 Cytochrome c reductase/~ 26.8 _ 2.8 34.4 ± 2.2 33.8, ± 1.9 28.6 '= nmol formaldehyde liberaled per rain per mg. b Average ± S.D. of at least 3 separate determinations of duplicale assay mixture. c Significantly changed from control group by unpaired t tee1 for at least 3 separate determinations at p < 0.05. ~ nmol p-nitrcphenol liberated per rain per rag, e pmol 3-hydroxybenzo(a)pyrene produced I~er mln !Per rag. pmol/mg o! colon m~crosomal protein; nmol/mg of hver mmrosemal protein. o nmol cytochrome c reduced per rain per rag. Table 4 Induction of liver drug metabolism activities by gastrointestinal hormones Experimental details are described in Table 3. All activities are expressed in units described in Table 3 except for the cytochrome P--450 content which is reported as nmol/m9. Hydroxylation activities Control Pentagastrin Secretin Cholecystokinin Benzphetamine 5.28 ± 0,25e 6.49 ± O,4ta 5.06 ¢ 0.11~ 5.51 ± 0,42a Ethylmorphine 8.34 ± 0.56 8.61 ± 0.77 9.66 ± 0.84c 7.36 "* 0.82 p-.Nitroanisole 3.50 _ 0.66 2,05 ± 0.16 2.96 ± 0.13 1,89 ~ 0.23d p-Nitrophenetole 3.36 + 0.21 3.87 ± 0.46 3.37 ± 0.27 4.0 _ 0.82 eenzo(a)pyrene 130.0 ± 10.0 320.0 ± 40.0c 248.02 ± 21.83d 370.0 ± 30.0c System componenls Cytochrome P.-450 content 0.76 ± 0.05 0.91 ".*- 0.04 0.97 ± 0.03 0.83 ± 0.06 Cytochrome c reductase t53.4 ± 22.9 276.6 ± 24.6d 154.6 ± 5.3 236.3 __. 21 Average :1: S.D. as in Table 3. Averages of 2 delerminations of 5 animals each --. variation from the average. ~Significant at p < O.01. Signlflcanlly changed Irom control group by unpaired t test for at least 3 separate determinations at p < 0.05. Table 5 Effects o! tissue substances on colon drug metabolism system Animals were i~retreated with 16.16-dimethylprostaglandin Ez, serotonin, 5-hydroxyoL-tryptophan, or reserpine, and the colon microsomes were assayed for drug metabolism activities. Experimental details are described in Table 1. All activities are expressed in unite described for the colon system in Table 3. Dimethyl-prosta- 5-Hydroxy-L-tryp- Hydroxylation activities Control glandin E= Serotonin tophan Reserpine eenzphetamlne 0.48 +_. 0.02~= 1.18 ± 0.16b 0.39 ± 0.03 0.57 ± 0.02b 0.51 ± 0.03 Ethylmorphine 0.35 ± 0.03 0.37 ± 0.06 0.44 "+- 0.07b 0.62 ± 0.07° 0.65 ± 0.06~' />-Nitroanisole 1.42 ± 0.10 0.24:1:: 0.05c 0.27 ± 0.17c 0.286 ~. 0.02c 0.26 ± 0.03c p--Nttrophenetole 1.04 ± 0.17 0.66 ± 0.06 0.08 ± 0.01c 0.28 0.03b 0.54 .+- 0.08b Benzo(e)pyrene 3.50 :l: 0.70 5.0 ± 0.80 4.40 ± 0.80 5.0 -=- 0.40 7.3 ± 0.50b System components Cytochrome P-,450 content 13 d: 2 17" +--. 2 11 ± 1 10 ± 1 15 ± 2 Cytochrome c reductase 25.7 ± 2.8 34.8 ± 2.2 24.8 ± 1.5 31.7 ± 2.6 15,7 ± 1.1b 1410 • Average ± S.D. as in Table 3. b Significantly changed from control group by unpaired t lest for at least 3 separate determinations at p < 0.05. c Significant at ~ < 0.01. CANCER RESEARCH VOL 41 Ti04231222