Dietary Vitamin C Intake and Concentrations in the

Human and Clinical Nutrition

Dietary Vitamin C Intake and Concentrations Body Fluids and Cells of Male Smokers and Nonsmokers1 MAI H. BUI, ALAIN SAUTY, * FABIENNE COLLETf

in the

AND PHILIPPE LEÃœENBERGER*

Swiss Vitamin Institute, Institute of Biochemistry, University of Lausanne, 1066 Epalinges, Switzerland; *Department of Medicine, Pulmonary Division, Faculty of Medicine, University of Lausanne, CHUV,7007 Lausanne, Switzerland; and /Dietetics Unit, University of Lausanne, CHUV,7077 Lausanne, Switzerland

INDEXING KEY WORDS:

â€¢humans â€¢tobacco smoke â€¢bronchoalveolar lavage fluid â€¢alveolar macrophage

ascorbic acid

A variety of oxidizing agents in tobacco smoke may be considered as contributing to the pathogenesis of emphysema (1, 2). Antioxidants may be important in the prevention of cigarette smoke-induced free radical injury (3). Such systems include intracellular enzymes

'Supported by research funds of Pulmonary Division (CHUV).

0022-3166/92 $3.00 Â©1992 American Institute of Nutrition. Received 19 February 1991. Accepted 10 July 1991.

312

Downloaded from jn.nutrition.org by guest on July 10, 2011

such as Superoxide dismutase, catalase and glutathione peroxidase and the free radical scavengers tocopherol, ascorbic acid, carotenes and certain trace minerals. The role of these various agents in protecting the lungs from the effects of cigarette smoke remains un clear, although there is circumstantial evidence that ascorbic acid might be important in detoxifying the oxidants inhaled in cigarette smoke. The concentration of ascorbic acid has been found to be lower in the serum of smokers compared with nonsmokers (4, 5), but it is not clear to what extent this reflects differences in dietary intake (6). According to Kallner et al. (7), smokers require a 40% higher daily intake than nonsmokers to maintain comparable serum levels. Bolton-Smith et al. (8) showed that both vitamin C dietary and serum values were lower in smokers than nonsmokers. However, serum levels may not be the best indicator of vitamin C status. Indeed, McGowan et al. (9) demonstrated that the alveolar macrophages of smokers contain greater amounts of vitamin C than those of nonsmokers, and that the rate of accumulation of ascorbate and dehydroascorbate is greater in the alve olar macrophages from smokers. From animal studies, there is also evidence that the extracellular fluid lining of the alveolus, which comes into close contact with cigarette smoke, is enriched with ascorbic acid (10,11 ). The present study deals with an analysis of the ascorbic acid content of various bio logical fluids (plasma, mononuclear leukocytes, alveo lar macrophages and bronchoalveolar lavage) and an assessment of dietary ascorbic acid intake from a rela tively homogeneous group of healthy male smokers and nonsmokers.

ABSTRACT Inhaled cigarette smoke releases a vari ety of oxidizing agents. Ascorbic acid is recognized as an important biological antioxidant. To better charac terize the antioxidant protective role of ascorbic acid, a comparison of ascorbic acid concentrations in plasma, leukocytes, bronchoalveolar lavage fluid and alveolar macrophages from a homogeneous group of healthy male smokers (n = 10) and nonsmokers (n = 14) was investigated. The resulting ascorbic acid con tents were (means Â±so) 91 Â±25 (n = 10) and 87 Â±25 (n = 14) umoI/L in plasma, 2.09 Â±0.62 (n = 7) and 2.12 Â±0.77 (n = 11) umol/109 cells in mononuclear leukocytes, 3.2 Â±2.2 (n = 10) and 1.7 Â±1.5 (n = 13) umol/L in bronchoalveolar lavage fluid and 3.4 Â±2.3 (n = 8) and 1.6Â± 1.3 (n = 6) umol/109 cells in alveolar macrophages from smokers and nonsmokers, respec tively. Mean daily dietary vitamin C intake was 116 + 68 and 107 Â±59 mg/d for smokers and nonsmokers, respectively. The ascorbic acid contents of bronchoalveolar lavage [3.9 Â±1.9 |imol/L (n = 8)] and alveolar macrophages [4.1 Â±2.1 umol/10 cells (n = 6)] of smokers consuming 15 to 20 cigarettes/d were significantly higher (P < 0.05) than those of nonsmokers. The increased content of ascorbic acid in bronchoalveolar lavage and in alveolar macrophages of smokers compared with nonsmokers may reflect a defensive mechanism against free radical species de rived from cigarette smoke. J. Nutr. 122: 312-316, 1992.

ANTIOXIDANT

ASCORBIC ACID IN SMOKERS AND NONSMOKERS

MATERIALS AND METHODS

analysis by HPLC. Preparation of peripheral mononuclear leukocytes. The mononuclear cells were isolated on a FicollHypaque gradient (Pharmacia AB, Uppsala, Sweden) and washed three times in Hank's balanced salt solution. Cells were counted in a hemocytometer and the viabil ity was assessed by trypan blue dye exclusion. The differential count was determined on a centrifuge prep aration stained by May-GrÃ¼nwald-Giemsa (Siegfried SA, Zofingen, Switzerland). Bronchoalveolar lavage. Bronchoalveolar lavage was performed according to recent recommendations (15). After premedication with 0.5 mg of atropine and

occasionally with 2.5 mg of midazolam intravenously and topical anesthesia with 10 g/L oxybuprocaine, a flexible fiber-optic bronchoscope was wedged into a subsegmentai bronchus of the right medium lobe and 50 mL of sterile 9 g/L NaCl at 37Â°Cwere injected and slowly aspirated by a syringe until a total of 200 mL was instilled. The recovered bronchoalveolar lavage fluid was then passed through a single sheet of gauze and centrifuged to separate cellular and noncellular compo nents. Alveolar macrophages. Using 10-20 mL of Hank's balanced salt solution each time, the cell pellets were washed twice by centrifugation. Viability was assessed by measuring the ability of alveolar macrophages to exclude 2 g/L trypan blue. Only samples that contained >70% of viable cells were used. The bronchoalveolar lavage supernatant were passed through a Millipore filter (0.2 urn) (Millipore, Bedford, MA) and then stored at -70Â°Cin small-volume aliquots until analysis with out any further processing. Extraction of ascorbic acid. Plasma ascorbic acid was extracted by adding 900 uL of 0.35 mol/L perchloric acid to 100 \iL of plasma, mixing and centrifuging at 1000 x g, 4Â°Cfor 5 min. A 15-uL aliquot was injected immediately for HPLC analysis. Ascorbic acid was extracted from mononuclear cells (3 x IO6 viable cells) and alveolar macrophages (2 x IO6 viable alveolar macrophages) by adding 1.0 mL of 0.35 mol/L perchloric acid to the washed cell pellets. The cells were homogenized by vortex mixing, allowed to stand for 10 min at 4Â°Cand centrifuged at 1000 x g for 15 min. The upper layer was removed and stored at -70Â°Cin small-volume aliquots until ascorbic acid anal ysis by HPLC. Ascorbic acid assay. Ascorbic acid was assayed as previously described (16) with the following modifica tion. The HPLC system was equipped with a pulsation damper setting on between the pump and the automatic injector. An electrochemical detector Coulochem Model 5 100 from ESA (Bedford, MA) was used. Flow rate was set at 1 mL/min and ascorbic acid was detected at a potential +0.4 V. Ascorbic acid stock standard (250 mg/L) was prepared by dissolving 25 mg of ascorbic acid (Merck, Darmstadt, Germany) in 100 mL of 50 mmol/L perchloric acid. Ascorbic acid standards of 200, 400, and 600 ug/L in 50 mmol/L perchloric acid were freshly prepared daily. Perchloric acid (50 mmol/L) was pre pared by dissolving 100 mg sodium EDTA in 900 mL of twice-distilled deionized water, adding 4.3 mL of ana lytical grade perchloric acid (70 g/100 g, Merck) and diluting to 1000 mL with twice-distilled deionized water. Each ascorbic acid determination was a mean of triplicate injections on HPLC; samples were kept at 4Â°C until injection. The standard deviation of replicates was Retention

times of ascorbic acid and uric acid were


Sub feet groups. The volunteers (10 male smokers age 27 Â±6 y and 14 male nonsmokers age 26 Â±4 y) were recruited from Lausanne Hospital (CHUV) employees and medical students. All were healthy according to screening by medical history and a physical examina tion, and none were currently taking vitamin supple ments or drugs. Written informed consent was obtained from all the subjects. The procedure used in this study was approved by the ethics committee of the Internal Medicine Department, Lausanne Hospital and was con ducted between December 1989 and March 1990. Assessment of diet and smoking habit. Dietary in take was assessed by a history method questionnaire on the day of lavage. This questionnaire was derived from the questionnaire originally designed by the Nutrition Unit of INSERM (12). The method was previously tested against weighted intake (13). Interviews were conducted by the same qualified dietician for the entire study. Respondents were asked to report their average fre quency per week at which foods were consumed for each meal of the day, along with the usual portion size of that food for that meal. A 2-wk period was considered. In addition, all the volunteers took one of the two main meals at Lausanne Hospital cafeteria, from which we determined portion size. Complementary questions on the annual frequency of consumption of a wide variety of fruits and vegetables were included. The food and beverages were chosen to be a reasonably complete listing of the commonly consumed food, taking account of occasional excess and seasonal habits. These records were coded in terms of 115 food items and the mean daily vitamin C intake was calculated using the food tables of Paul and Southgate (14), adapted by us for local dietary patterns. A questionnaire on smoking habits was adminis tered. It included the usual number and brand of ciga rettes currently smoked per day and smoking histories. Plasma. From each fasting subject a venous blood sample was collected in a heparinized tube on the day of the dietary record and lavage. Samples were centrifuged within 15 min and 100-uL plasma aliquots were stored at -70Â°Cin closed plastic tubes until ascorbic acid

313

314

BUI ET AL.

4.2 and 9.8 min, respectively. Quantification was based on peak heights using an external standard method. A linear response was observed from 0.2 to 2.0 mg/L. The lower detection limit was 100 ng/L. Ascorbic acid iden tification was based on retention behavior and cochromatography with the reference compound. Recoveries of ascorbic acid from plasma, leukocytes, bronchoalveolar lavage and alveolar macrophages were 97.7 Â± 3.4%. There was no interference with the ascorbic acid peak. The ascorbic acid content in biological fluids was found to be stable for >3 mo at -70'C.

TABLE 1

Characteristics of the smokers and nonsmokers and their daily total energy and dietary vitamin C intake^ (n 10)27 yTotal Age,

Â±6120-37)9960Â±4(21-35)10020

kfPercentage energy intake,

24704670-16610)15Â±2(13-17)32 Â± Â±3260(6780-13370)15Â±2

energyProtein, of %Fat,

Statistical methods. Statistical differences between the groups of smokers and nonsmokers and coefficients of correlation of polynomial regression were calculated using a commercial software package (Stat Works, Cricket Software, Philadelphia, PA). Data are presented as means Â±so. The difference between the two groups (smokers and nonsmokers) was tested using unpaired Student's Ã® test. A P value of 0.05 or less was considered

cigarettes/dVitamin

statistically

mg/dRelative C intake,

Â±5(23^2)49

%Alcohol,

Â±6(40-60)4Â±3*11-11)18Â±4(10-25)116 1*(0-3)|n

%Smoking,

13)0107 -

intake,mg-d~l-kg vitamin C

body wt'1Smokers

RESULTS Ascorbic acid levels in cells. The separation of granulocytes and monocellular cells was considered satisfac tory. On average, the mononuclear layer was composed of lymphocytes and monocytes in a proportion of 2:1. The total cell counts for alveolar macrophages were considered higher in smokers compared with nonsmok ers (38.8 Â±22.8 x IO6 and 13.3 Â±6.0 x IO6 cells, respec tively). The differentiation cell count in bronchoalveolar lavage was 84.6 Â±6.0% alveolar macro phages, 9.3 Â±5.4% lymphocytes and 1.2 Â±0.6% neutrophils for smokers, and 68.4 Â±12.7% alveolar macrophages, 24.1 Â±6.3% lymphocytes and 3.5 Â±2.2% neutrophils for nonsmokers. In the case of bronchoalveolar lavage, the recovery of instilled saline solution was similar for smokers and nonsmokers (58.6 Â±14.5% and 60.7 Â±17.6%, respec tively). Clinical results. As shown in Table 1, there were no significant differences in the ages, total energy intake or estimated mean daily dietary intake of vitamin C be tween the smokes and nonsmokers. Dietary intake of vitamin C did not differ between the groups when adjustment was made for body weight. Although total energy intake was similar for smokers and nonsmokers, the percentage of energy derived from alcohol was sig nificantly higher in the smokers than in the nonsmokers (P = 0.002). The smokers consumed an average of 18 cigarettes per day and the nonsmokers were complete abstainers. In regard to dietary intake, there was a positive but statistically nonsignificant correlation between dietary and plasma ascorbic acid. The mean ascorbic acid con-

Â±68(49-268)1.6Â±59(32-258)1.5


significant.

%Carbohydrate,

Â±0.9(0.8-^.6)Nonsmokers(rj-14)26 Â±0.7(0.4^.9)

Values are means Â± so, with ranges in parentheses. 'Significantly different between smokers and nonsmokers. Student's t test (P 0.002).

tent of plasma, mononuclear leukocytes, bronchoalveo lar lavage and alveolar macrophages for the smokers and nonsmokers are reported in Table 2. Alveolar macro phages from both smokers and nonsmokers were con taminated by lymphocytes. Ascorbic acid levels in alveolar macrophages were calculated from the differ ence between the ascorbic acid content of alveolar mac rophages and that of lymphocytes. The ascorbic acid concentrations in plasma and mononuclear leukocytes were similar for smokers and nonsmokers. The correlation coefficients between plasma and mononuclear leukocyte ascorbic acid were r = 0.797 (P < 0.05) for smokers {n = 7), r = 0.558 (nonsignificant) for nonsmokers (n = 11) and r = 0.618 (P < 0.01) for both smokers and nonsmokers (n = 18). The ascorbic acid contents in bronchoalveolar lavage and alveolar macrophages from smokers and nonsmok ers in correlation with their smoking habits are summa rized in Table 3. Within the group of smokers and nonsmokers, ascorbic acid concentrations in bronchoalveolar lavage and alveolar macrophages tended to increase as the number of cigarettes per day increased up to 15 cigarettes,- at heavier consumption levels there was an apparent trend of decreasing ascorbic acid levels in both bronchoalveolar lavage and alveolar macrophages. Using polynomial regression (order 2) analyses plotting difference between ascorbic acid bronchoalveolar lavage and alveolar macrophage con-

ANTIOXID ANT ASCORBIC ACID IN SMOKERS AND NONSMOKERS

315

TABLE 3

TABLE 2

Ascorbic acid content in human plasma, mononuclear leucocytes, bronchoalveolar lavage and alveolar macrophages1

Smokers Plasma ascorbic acid,\imol/LMononuclear

Nonsmokers Â±25(44-136)(n

Ascorbic acid in bronchoalveolar lavage and alveolar macrophages from smokers and nonsmokers as a function of the number of cigarettes consumed per day1

Cigarettes per day

Bronchoalveolar lavage

Alveolar macrophages

(\mtolll09 cell) 10)2.09 14)2.1210.77(1.06-3.37)(n-11)1.711.5(0.3-4.4)(n (]unol/L) 0.62(1.03-2.84)|n Â± 0101520251.711.5(0.3-^.4)(n -7)3.2 13)0.3(n-1)4.712.7*(2.3-7.7)In 2.2(0.3-7.6)(n Â±

leucocytesascorbic acid,\unol/109 cellsBronchoalveolar lavageascorbic acid,\unol/LAlveolar macrophageascorbic acid,\wiol/W9 cells91125(55-132)|n

10)3.4 13)1.611.3(0.1^.0)(n-6) 2.3(0.7-6.5)(n-8)87 Â±

-3)3.411.4*(1.3^.7)(n-5)0.7(a-H1.611.3(0.1^3.0)(n-6

tents in smokers with ascorbic acid bronchoalveolar lavage and alveolar macrophage mean values of nonsmokers against the number of cigarettes per day, corre lation coefficients were r = 0.694 (P < 0.01) and 0.627 (P < 0.1),respectively. The ascorbic acid content of bron choalveolar lavage [3.9Â±1.9umol/L [n = 8)]and alveolar macrophages [4.1Â±2.1umol/109 cells (n =6)]of smokers consuming 15 to 20 cigarettes were significantly higher (P < 0.05) than those of nonsmokers.

DISCUSSION This study showed no significant difference in plasma ascorbic acid concentrations between healthy male smokers and nonsmokers who have a relatively high dietary intake of vitamin C. Similar observations were reported in smoking and nonsmoking subjects with a dietary vitamin C intake >100 mg/d (17) and in those who regularly consume vitamin preparations con taining vitamin C (18).This lack of difference compared with some other studies (4, 5, 7) may be explained by a relative dietary excess at these high levels of intake resulting in saturation of plasma. Although the sample size was small, and all the methods of dietary assess ment are subject to wide errors (19), the high levels of ascorbic acid intake as calculated from the history method questionnaire seem to be in good agreement with the high levels in plasma, which are considered to reflect recent intakes. Mononuclear leukocyte ascorbic acid concentrations are believed to reflect long-term ascorbic acid status (20).The values of mononuclear leukocyte ascorbic acid reported here are in good agreement with the literature (20, 21). There was a significant correlation between plasma and leukocyte ascorbic acid contents, which was

'Values are means 1 so, with ranges and number of smokers and nonsmokers in parentheses. 'Mean differences between broncho alveolar lavage ascorbic acid and alveolar macrophage ascorbic acid in nonsmokers and smokers consuming 15 to 20 cigarettes per day were significantly different, Student's t test (P < 0.05).

especially apparent in smokers. In smokers, despite an oxidant stress, levels of ascorbic acid observed in mononuclear leukocytes were not decreased. We are not aware of any previous reports of ascorbic acid content in bronchoalveolar lavage fluid. The inter esting finding from this present study is the relatively higher (though weakly significant) level of ascorbic acid in the bronchoalveolar lavage from smokers. This could not be attributed to a differential recovery of instilled saline solution, and moreover the percent recovery re ported here was similar to that noted by Pacht et al. (22). The higher level of ascorbic acid in the alveolar fluid of smokers may result from the greater number of alveolar macrophages in smokers compared with nonsmokers as reported here and by other investigators (22-24). The alveolar macrophages may release greater amounts of ascorbic acid into the alveolar fluid. Ascorbic acid, which scavenges reactive free-radical species from to bacco smoke, changes to the ascorbate free radical, then, by the loss of an unpaired electron to dehydroascorbic acid. Dehydroascorbic acid can be reduced back to as corbate in the cell membrane. Moreover, McGowan et al. (9) demonstrated that the alveolar macrophages of human smokers accumulate higher levels of vitamin C than those of nonsmokers, but that alveolar macro phages in both smokers and nonsmokers have a similar capacity for reducing internalized dehydroascorbic acid to ascorbic acid. A greater amount of ascorbic acid in the alveolar macrophages of smokers compared with non-


'Values are means 1 SD,with ranges and number of smokers and nonsmokers in parentheses.

316

BUI ET AL.

ACKNOWLEDGMENTS The authors wish to thank Caroline Bolton-Smith and Henri Isliker for useful suggestions. LITERATURE CITED 1. fanoff, A., Carp, H., Laurent, P. & Raju, L. (1983) The role of oxidative processes in emphysema. Am. Rev. Respir. Dis. 127: 2. Heffner, I. E. & Repine, I. E. (1989) State of the an: pulmonary strategies of antioxidant defense. Am. Rev. Respir. Dis. 140: 531-554. 3. Fridovich, I. & Freeman, B. (1986) Antioxidant defenses in the lung. Annu. Rev. Physiol. 48: 693-702. 4. Pelletier, O. (1977) Vitamin C and tobacco. Int. J. Vitam. Nutr. Res. Suppl. 16: 147-149.

5. Mahalko, ]. R., Johnson, L. K., Gallagher, S. K. & Milne, D. B. (1985) Comparison of dietary histories and seven-day food records in a nutritional assessment of older adults. Am. J. Clin. Nutr. 42: 542-553. 6. Hornig, D. H. & Glatthaar, B. E. (1985) Vitamin C and smoking. Int. J. Vitam. Nutr. Res. Suppl. 27: 139-155. 7. Kallner, A B., Hartmann, D. & Hornig, D. H. (1981) On the requirements of ascorbic acid in man: steady-state turnover and body pool in smokers. Am. J. Clin. Nutr. 34: 1347-1355. 8. Bolton-Smith, C, Casey, C. E., Gey, K. F., Smith, W.C.S. A. Tunstall-Pedoe, H. (1991) Antioxidant vitamin intake assessed using a food frequency questionnaire: correlation of biochemical markers in smokers and nonsmokers. Br. J. Nutr. 65: 337-346. 9. McGowan, S. E., Parenti, C. M., Hoidal, J. R. & Niewoehner, D. E. (1984) Ascorbic acid content and accumulation by alveolar macrophages from cigarette smokers and nonsmokers. J. Lab. Clin. Med. 104: 127-134. 10. Willis, R. M. & Kratzing, C. C. (1974) Ascorbic acid in rat lung. Biochem. Biophys. Res. Commun. 59: 1250-1253. 11. Snyder, A., Skoza, L. & Kikkawa, Y. (1983) Comparative removal of ascorbic acid and other airway substances by sequential bronchoalveolar lavages. Lung 161: 111-121. 12. Cubeau, J. & Pequignot, G. (1980) The quantitative alimentary questionnaire technique used by the I.N.S.E.R.M. nutrition de partment, (author's translation) Rev. Epidemiol. SantÃ©Publique 28(3): 367^72. 13. Cubeau, J. & Pequignot, G. (1976) Feasibility survey on the reliability of questioning on past eating habits in a group of men. (author's translation) Rev. Epidemiol. SantÃ©Publique 24(1): 6167. 14. Paul, A. A. & Southgate, D.A.T. (1978) McCance and Widdowson's The Composition of Foods, 4th ed. Her Majesty's Stationary Office, London, U.K. 15. Report of the European Society of Pneumology Task Group BAL (1989) Technical recommendations and guidelines for bronchoalveolar lavage. Eur. Respir. J. 2: 561-585. 16. Bui, M. (1985) Ascorbic acid and related compounds. In: Modern Chromatographie Analyses of the Vitamins (De Leenheer, A. P., Lambert, W. E. & De Ruyter, M.G.M., eds.), vol. 30, pp. 267-301. Marcel Dekker, New York, NY and Basel, Switzerland. 17. Yeung, D. L. (1976) Relationship between cigarette smoking, oral contraceptives, and plasma vitamins A, E, C and plasma triglyc Ã©ridesand cholesterol. Am. J. Clin. Nutr. 29: 1216-1221. 18. Ritzel, G. & Brubacher, G. (1977) Vitamin C and tobacco. Int. J. Vitam. Nutr. Res. Suppl. 16: 171-183. 19. Cameron, M. E. & Van Staveren, W. A. (1988) Manual on Meth odology for Food Consumption Studies. Oxford University Press, New York, NY. 20. Evans, R. M., Currie, L. & Campbell, A. (1982) The distribution of ascorbic acid between various cellular components of blood, in normal individuals, and its relation to the plasma concentration. Br. J. Nutr. 47: 473-482. 21. Schorah, C. J. (1981) The level of vitamin C reserves required in man: towards a solution to the controversy. Proc. Nutr. Soc. 40: 147-154. 22. Pacht, E. R., Kaseki, H., Mohammed, I. R., Cornwell, D. G. & Davis, W. B. (1986) Deficiency of vitamin E in the alveolar fluid of cigarette smokers. ]. Clin. Invest. 77: 789-796. 23. Hunninghake, G. W. & Cristal, R. G. (1983) Cigarette smoking and lung destruction. Accumulation of neutrophils in the lungs of cigarette smokers. Am. Rev. Respir. Dis. 128: 833-838. 24. Hoidal, J. R., Fox, R. B., LeMarbe, P. A., Pern, R. & Repine, J. E. (1981) Altered oxidative metabolic responses in vitro of alveolar macrophages from asymptomatic cigarette smokers. Am. Rev. Resp. Dis. 123: 85-89.


smokers was found here, in agreement with McGowan et al. (9), although the observed values were lower and did not reach significance when the group was taken as a whole; they were significant, however, for the moder ately heavy smokers. Thus the number of cigarettes smoked and exposure to other environmental oxidants may have contributed to differences between the sub jects in their level of antioxidant stress in the high vitamin C-intake group. In smokers, ascorbic acid con centrations in bronchoalveolar lavage and alveolar mac rophages show variations when correlated with cigarette consumption, the significance of which re mains undetermined. When compared with nonsmokers, ascorbic acid concentrations in bronchoalveolar lavage and alveolar macrophages of smokers tended to be higher. This suggests that the alveolar fluid and alveolar macrophages are the first line of defense against the oxidants liberated by tobacco smoke, rather than the peripheral mononuclear leukocytes. The variability between subjects, which may partly reflect the differing number of cigarettes or smoking behavior and the relatively small numbers in each group, may account for the only marginal statistically significant differences between the two groups. Never theless, the observed positive relationship between the ascorbic acid levels in the bronchoalveolar lavage and alveolar macrophages of smokers and the number of cigarettes consumed (15, 20) suggests an adaptive pro cess to elevated levels of antioxidant stress. A protective effect against free radicals may decrease with large num ber of cigarettes and/or when the vitamin C intake is abnormally low, as in chronic alcoholism and malnutri tion. These hypotheses need to be further investigated on a larger sample size. The relative importance of other antioxidants such as vitamin E, carotenes and riboflavin also needs to be considered in future studies.