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An 18-month follow-up study on the influence of smoking on blood antioxidant status of teenage girls in comparison with adult male smokers in Korea
APPLIED NUTRITIONAL INVESTIGATION
An 18-Month Follow-up Study on the Influence of Smoking on Blood Antioxidant Status of Teenage Girls in Comparison With Adult Male Smokers in Korea Sun H. Kim, PhD, Jodi L. Ensunsa, MS, Qin Yan Zhu, PhD, Jung S. Kim, PhD, Ho S. Shin, PhD, and Carl L. Keen, PhD From the Departments of Food Service Management and Nutrition, Physical Education, and Environmental Education, Kongju National University, Kongju, Korea; and the Departments of Nutrition and Nutrition and Internal Medicine, University of California at Davis, Davis, California, USA OBJECTIVES: The influence of cigarette smoking on blood antioxidant status in teenage girls with a history of short-term smoking was followed over 18 mo. METHODS: Data obtained from female senior high school students (ages 14 to 18 y) in Korea were compared with data obtained from adult male smokers (ages 36 to 51 y) with a long history of smoking and living in the same geographic areas as the teenage subjects. A smoker was a person who had smoked at least three cigarettes a day for at least 1 y for teenagers (n ⫽ 35) or at least 10 cigarettes a day for at least 13 y for adults (n ⫽ 20). Serum, urine, and anthropometric data were obtained from teenagers every 6 mo over an 18-mo period. Samples were collected once from adults. Data were analyzed by Student’s t test and Fisher’s protected least significant difference test for comparing smokers and non-smokers and for analyzing period effects in each group. RESULTS: Serum nicotine and cotinine concentrations were higher in smokers than in non-smokers. Blood pressures were higher in teenage (at 0 and 12 mo) and adult smokers than in non-smokers. Extracellular superoxide dismutase activities and concentrations of serum vitamin C and folate were lower in smokers in the teenage (at 0, 12, or 18 mo) and adult groups. Serum ceruloplasmin activities and thiobarbituric acid-reactive substance production were not influenced by smoking. In adults, serum copper concentrations were higher in smokers than in non-smokers. This parameter for teenagers did not change consistently throughout the study. CONCLUSIONS: Similar to adults, cigarette smoking by teenagers has a negative effect on oxidant defense systems. Nutrition 2004;20:437– 444. ©Elsevier Inc. 2004 KEY WORDS: smoking, extracellular superoxide dismutase, vitamin C, folate, mineral, teenage short-term smokers
INTRODUCTION Tobacco smoke contains numerous compounds emitted in gases and condensed tar particles, many of which are oxidants and pro-oxidants capable of producing reactive oxygen species.1 The increased production of reactive oxygen species by smoke can produce a condition of oxidative stress that can result in the oxidation of lipids, induction of DNA single-strand breakage, inactivation of certain proteins, and the disruption of biological membranes.2,3 Increased oxidative stress has been suggested to play a major role in the pathogenesis of several smoking-related
This work was supported by a 2001 grant from Kongju National University in Korea and grant DK-35747 from the National Institutes of Health in the United States. Correspondence to: Sun Hyo Kim, PhD, Department of Food Service Management and Nutrition, Kongju National University, Shinkwan-dong, Kongju, Chungnam-do, 314-701, Republic of Korea. E-mail: shkim@ kongju.ac.kr Nutrition 20:437– 444, 2004 ©Elsevier Inc., 2004. Printed in the United States. All rights reserved.
diseases such as cancer, cardiovascular diseases, and oral diseases.4 – 6 The damage that can be caused by free radicals is normally minimized by biological antioxidant systems including enzymatic and non-enzymatic systems. Important antioxidant enzymes include copper (Cu) and zinc (Zn) superoxide dismutase (SOD), manganese SOD, ceruloplasmin (Cp), selenium glutathione peroxidase, glutathione reductase, and catalase. Non-enzymatic antioxidants include vitamin C, ␣-tocopherol, provitamin A carotenoids, and urate.1,7,8 In adult smokers, several arms of the oxidant defense system have been reported to be impaired relative to oxidant defense systems in non-smokers. It has been reported that adult smokers can have significantly lower erythrocyte CuZnSOD activities than non-smokers.9 Similarly, concentrations of serum vitamin C and vitamin E have been reported to be lower in adult smokers than in non-smokers.10 Lower serum vitamin C concentrations in smokers are thought to be a consequence of greater vitamin C turnover in response to a sustained oxidant load and lower vitamin C intakes by heavy smokers.1 Similar to vitamin C, serum folate concentrations are often lower in adult smokers than in non-smokers because 0899-9007/04/$30.00 doi:10.1016/j.nut.2004.01.008
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folate catabolism is increased under conditions of oxidative stress.11 Collectively, these findings support the association of long-term smoking with increased oxidative stress that can affect an individual’s oxidant defense system and overall nutrition status. With respect to serum mineral concentrations, several investigators have reported that Zn concentrations are often reduced in adult smokers,12 whereas Cu concentrations typically are increased.13,14 Changes in Zn and Cu homeostases in smokers have been postulated to contribute to some of the long-term negative effects associated with smoking and hypertension. It is not known whether these changes in Zn and Cu are acute effects that occur shortly after the initiation of smoking or are secondary to the development of chronic diseases related to smoking such as hypertension. Hypozincemia and hypercupremia can be characteristic of hypertension independent of smoking.12 Although smoking is recognized to be a significant health problem in adult subjects,15 the health consequences of smoking in teenagers who have a short-term smoking history are less well understood. The frequency of tobacco use by teenagers is increasing annually, especially in developing countries in Asia, such as Korea.16 It could be argued that cigarette smoking by teenage girls poses particular health problems because this population group is often undernourished due to dieting. Suboptimal nutritional status during periods of rapid growth and development can have consequences that persist into adulthood.17 In light of these findings, we performed the current study to observe the longitudinal effects of smoking on antioxidant status in female teenagers with a short-term smoking history. Female teenage smokers were sampled at 6-mo intervals over an 18-mo period. Results from these subjects were compared with data from nonsmoking teenage girls and from men with a long-term smoking history.
MATERIALS AND METHODS Subjects Teenage subjects were selected from a population of senior high school girls (ages 14 to 18 y) in Korean urban and rural areas located in the Chungnam province. The subjects attended one of six senior high schools selected in these two areas. All subjects were healthy and reported no use of illegal drugs during the study period. They were interviewed for their tobacco use and that of their families. They were questioned with respect to the average number of cigarettes smoked per day and when they started, or stopped, smoking. Serum nicotine and cotinine concentrations were determined to confirm their answers regarding smoking status. If the subject’s responses regarding her smoking habits and her serum nicotine and cotinine concentrations coincided, the girl was included in the study. Fifty smokers who had smoked continually for at least 1 y and who had no experience of stopping smoking were identified at the beginning of study. Fifty non-smokers were identified who reported neither previous smoking experience nor exposure to passive smoking at home at the beginning of the study. The subjects were selected by quota structure and followed over an 18-mo study period. They were interviewed for smoking habits, including average number of cigarettes smoked per day at 6-mo intervals (period A, 0 mo; period B, 6 mo; period C, 12 mo; and period D, 18 mo). They were not treated with any other intervention during the study period. Some subjects moved during the study; refused to give blood and urine samples; or stopped smoking during the study periods. Thirty-five subjects in the smoking and non-smoking groups completed the 18-mo study. At the end of the study, smokers had a mean age of 16.5 ⫾ 0.1 y (range, 14 –18 y), with a period of 2.5 ⫾ 0.2 y (1– 4.5 y) of smoking, and smoked 7.6 ⫾ 0.4 (3–12) cigarettes a day. Non-smokers had a mean age of 15.6 ⫾ 0.1 y (15–17 y). The percentage of subjects who finished the study completely during the 18-mo period relative to the total
Nutrition Volume 20, Number 5, 2004 number of selected subjects at the beginning of study was 70. The girls in the smoking group indicated that they had some concern that they would be stigmatized by being recognized as smokers. To reduce their anxiety, all records were coded and kept confidential. Adult male subjects, as a positive control group, were selected from a male population living in the same Korean urban and rural areas as the teenage subjects of this study. At the beginning of the current study, we attempted to select an adult female smoking group, but these women expressed considerable anxiety that they would be stigmatized by being recognized as smokers and thus refused to participate. As a consequence, we selected men as a positive control group. All subjects were healthy and reported no use of illegal drugs during the study period. They were interviewed for their tobacco use; average number of cigarettes smoked per day; initial age when they started smoking; and whether they have smoked continually. Analysis of serum nicotine and cotinine concentrations combined with results of the interview were done to determine the eligibility of subjects. Twenty adult male smokers (44.7 ⫾ 0.9 y; range, 36 –51 y) who had smoked at least 10 cigarettes a day (18.8 ⫾ 1.4, 10 –30) for at least 13 y (23.5 ⫾ 1.1 y, 13–30 y) were identified. Twenty adult male non-smokers (46.5 ⫾ 1.2 y, 38 –56 y) who reported no previous smoking experience were identified; adult subjects in both groups were selected by quota structure. Adult male subjects were studied once during the study period because they declined to continue a longitudinal study. The adults were not treated with any other intervention.
Analytical Methods Fasting blood samples were collected from teenage subjects at 6-mo intervals during the 18-mo study period. Blood samples were collected once from all adult subjects. Subjects were asked not to smoke for 12 h before sampling to exclude the effects of acute smoking on the blood parameters that were studied. An aliquot of blood was used for measuring the hematocrit. Remaining blood samples were centrifuged at 1700g for 20 min at 4°C. The serum was used for the assessment of nicotine, cotinine, vitamin C, folate, thiobarbituric acid-reactive substances (TBARS), Zn and Cu concentrations; and extracellular (EC) SOD and Cp activities. Serum samples were stored at ⫺80°C until analyzed. Urine samples were collected from teenage subjects at 6-mo intervals; and samples were collected once from adult subjects. Urine samples were used for measuring Zn, Cu, and creatinine concentrations. Samples were stored at ⫺80°C until analyzed. For the analysis of nicotine and cotinine, serum was mixed with an internal standard (di-isopropyl-amine dodecane; DIPA 12, Sigma, St. Louis, MO, USA) in 0.1 M KOH solution and ether. The mixture was saturated with NaCl and the ether phase was injected into a Hewlett-Packard 6890 gas chromatograph equipped with a 30-m ⫻ 0.32-mm HP-5 MS capillary column. The injector temperature was set at 260°C, and the detector temperature was set at 300°C. Nicotine and cotinine concentrations were determined by comparison with a commercial standard.18 EC SOD (EC 1.15.1.1) activity was analyzed as described by Paik et al.19 Serum Cp (EC 1.16.3.1) activity was measured as described by Schosinsky et al.20 Serum TBARS production was measured according to a fluorometric method;21 values are reported as nanomoles of malondialdehyde per milliliter of serum. Serum vitamin C analysis was performed as follows: Serum was mixed with 2% metaphosphoric acid (1:2) and centrifuged at 13 000g for 10 min at 4°C; the supernatant was removed and mixed with dithiothreitol and allowed to incubate at room temperature for 60 min. Samples were then mixed with a mobile phase (0.1 M acetate buffer, pH 5.0), and the solution was filtered and injected into a Hewlett-Packard 1090 II/M high-performance liquid chromatograph with an inner diameter of 100 ⫻ 3.2 mm and a column size of 3 m.22 Serum folate concentrations were measured with a radioassay procedure using a solid-phase no-boil
Nutrition Volume 20, Number 5, 2004 folate kit (Diagnostic Product Corporation, Los Angeles, CA, USA). Serum and urine Zn and Cu concentrations were analyzed by inductively coupled argon plasma-atomic emission spectrophotometry (Trace Scan ICP, Thermal-Jarrell Ash Corp., Franklin, MA, USA) after dilution with 1 N HNO3. For the analysis of serum mineral concentrations, the procedure of protein denaturation followed by discarding the denatured pellet was used before reading samples on the inductively coupled argon plasma-atomic emission spectrophotometer. However, for the analysis of urine mineral concentrations, samples were read directly without the denaturation and discarding of protein. Urine creatinine concentrations were measured by colorimetric determination at 500 nm using a diagnostic creatinine reagent kit (Sigma). Urine mineral concentrations are expressed as micromoles per millimole of creatinine. Anthropometric Measurement Teenage subjects’ heights were measured annually by public health teachers at the subjects’ schools. Body weights and blood pressures of teenagers were measured at 6-mo intervals during the 18-mo period by the nursing staff in a medical center. Adult subjects’ heights, body weights, and blood pressures were measured once by a nursing staff in a medical center. Heights and body weights of teenage and adult subjects were measured on an electrical scale that could measure heights and body weights concurrently while subjects were shoeless and wearing light clothing. Blood pressures were determined with a sphygmomanometer and expressed as the mean value of two measurements taken 3 to 5 min apart. Dietary Survey At the beginning of the study, a subset of the teenage subjects (14 to 18 y) in each group (n ⫽ 35) was randomly selected from the larger population and asked to complete a dietary study. They were asked to record 2 d during the week of all food intakes by using the 24-h recall method for cross checking. All subjects in each group selected at the beginning of the dietary survey completed the dietary records. A computer-aided nutritional program developed by the Korean Nutrition Society was used for the nutrition interview by researchers and for the analysis of dietary nutrient intakes. Nutrient intakes were compared with the Korean recommended dietary allowance (RDA)23 for girls ages 14 to 18 y. Statistical Analysis Data are presented as mean ⫾ standard error of the mean. Data were analyzed with unpaired Student’s t test for comparing serum antioxidant status, urine mineral concentrations, anthropometric measurements, and dietary nutrient intakes between smokers and non-smokers for teenage and adult subjects, respectively, at each sampling period. The influence of the sampling period on these parameters in each teenage group was analyzed by the F test; if there was a significant influence of sampling period, Fisher’s protected least significant difference test was applied again. Differences were considered to be statically significant at P ⬍ 0.05. All statistical analyses were conducted with StatView 4.1 (Abacus Concepts, Berkeley, CA, USA).
RESULTS The mean age for teenage subjects was significantly higher in smokers than in non-smokers (P ⬍ 0.001), but the ages in these two groups were developmentally similar (Table I). The mean age of adult subjects also was similar between smokers (44.7 ⫾ 0.9 y)
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and non-smokers (46.5 ⫾ 1.2 y). Mean heights for teenage subjects were higher in smokers than in non-smokers at 12 mo (P ⬍ 0.05), but these values did not change significantly over 12 mo in the smoking and non-smoking groups. Mean body weights of the teenage subjects were similar between the two groups, and these values did not change significantly over 18 mo in the smoking and non-smoking groups. Mean heights and body weights of adult subjects were similar in the two groups. Blood pressure values for teenage and adult subjects were within the normal range for their age groups; diastolic and systolic blood pressures were significantly higher in teenage smokers than in teenage non-smokers at periods A (P ⬍ 0.001) and C (P ⬍ 0.05). Blood pressure values of the teenage subjects changed inconsistently throughout the 18-mo period. For adult subjects, diastolic and systolic blood pressures were higher in smokers than in non-smokers; there was a significant difference for systolic blood pressure (P ⬍ 0.01). Hematocrit values of teenage and adult subjects during the entire study period were within the normal range; however, values for the smokers in teenage subjects (at periods B and C) and for the adult subjects were significantly higher than those for their non-smoking counterparts (P ⬍ 0.05). The average smoking period of teenage smokers was 2.5 y and that of adult subjects was 23.5 y (Table II). The average number of cigarettes smoked daily by teenage smokers changed from 8 (twofifths of a pack/d) to10 (half a pack/d) cigarettes a day over the 18-mo period. Adult smokers had an average of 18.8 cigarettes a day (nine-tenths to one pack/d). Serum nicotine and cotinine concentrations in teenage (P ⬍ 0.01 to 0.001) and in adult subjects (P ⬍ 0.001) were higher in smokers than in non-smokers throughout the study period (Table III). Serum nicotine and cotinine concentrations in the teenage smoking group showed inconsistent changes over the study period. Reported nutrient intakes for teenage subjects are shown in Table IV. Daily energy intakes were significantly lower in smokers than in non-smokers (P ⬍ 0.01); ratios of mean energy intake to the Korean RDA for the same age (2100 kcal/d)23 were within 75% of the RDA for smokers and close to the RDA for nonsmokers. Reported intakes of vitamins A and C (P ⬍ 0.001) were lower in smokers than in non-smokers. In particular, vitamin C intakes by smokers were less than 65% of the Korean RDA for the same age (70 mg/d),23 whereas vitamin C intakes in non-smokers were higher than the RDA. Vitamin C intakes on an energy basis were 0.03 and 0.04 mg/kcal for smokers and non-smokers, respectively. Niacin intakes were significantly lower in smokers than in non-smokers (P ⬍ 0.001); vitamin B1 and B2 intakes were similar in the two groups. Calcium and iron intakes in both groups were less than 65% of the Korean RDA23 for the same age. EC SOD activities of teenage smokers were lower (⬇50%) in smokers than in non-smokers at period D (P ⬍ 0.001); similarly, the values for adult subjects were lower (46%) in smokers than in non-smokers (P ⬍ 0.01; Table V). Serum Cp activities were similar in the two groups for teenage and adult subjects. Serum vitamin C concentrations in teenage subjects were significantly lower in smokers than in non-smokers at periods A (31%) and C (30%; P ⬍ 0.001). Adult smokers also had lower serum vitamin C concentrations than did non-smokers (36%; P ⬍ 0.001). Serum folate concentrations were lower in teenage smokers than in teenage non-smokers (55%; P ⬍ 0.001) at period D. Adult smokers had lower serum folate concentrations than did non-smokers (38%; P ⬍ 0.05). Serum TBARS production was similar in smokers and non-smokers for teenage and adult groups at all time points. In teenagers, serum Zn concentrations were similar in smokers and non-smokers at periods A and C but significantly higher in smokers than in non-smokers at periods B (16%) and D (21%; P ⬍ 0.001; Table VI). For adult subjects, serum Zn concentrations were similar in the smoking and non-smoking groups. Serum Cu concentrations were similar in the two groups for teenage subjects, but these values for adult subjects were significantly higher (14%) in smokers than in non-smokers (P ⬍ 0.01).
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Nutrition Volume 20, Number 5, 2004 TABLE I. GENERAL CHARACTERISTICS OF SUBJECTS* Teenagers
Variables Age (y) Height (cm) Period A Period C Body weight (kg) Period A Period B Period C Period D Blood pressure (mmHg) Diastolic Period A Period B Period C Period D Systolic Period A Period B Period C Period D Hematocrit (%) Period B Period C Period D
Adults
Smokers (n ⫽ 35)
Non-smokers (n ⫽ 35)
t Test between groups
Smokers (n ⫽ 20)
Non-smokers (n ⫽ 20)
16.5 ⫾ 0.1
15.6 ⫾ 0.1
P ⬍ 0.001
44.7 ⫾ 0.9
46.5 ⫾ 1.2
160.5 ⫾ 1.0 162.2 ⫾ 1.0
158.7 ⫾ 1.0 159.2 ⫾ 1.0
P ⬍ 0.05
169.6 ⫾ 1.0
169.3 ⫾ 1.6
52.4 ⫾ 1.4 52.3 ⫾ 1.3 52.6 ⫾ 1.4 51.8 ⫾ 1.2
51.2 ⫾ 1.4 51.2 ⫾ 0.8 52.0 ⫾ 0.9 50.8 ⫾ 0.8
67.9 ⫾ 1.7
68.3 ⫾ 1.9
70.2 ⫾ 1.7a 63.8 ⫾ 1.7b 62.4 ⫾ 1.5b 73.9 ⫾ 1.7a
62.6 ⫾ 1.4a 63.8 ⫾ 1.0a 58.0 ⫾ 1.1b 72.3 ⫾ 1.2c
P ⬍ 0.001
82.3 ⫾ 2.4
77.7 ⫾ 2.2
109.3 ⫾ 2.1 104.4 ⫾ 2.3 103.5 ⫾ 1.6 108.9 ⫾ 2.1
98.3 ⫾ 1.9ab 102.4 ⫾ 1.7a 98.0 ⫾ 1.1b 109.6 ⫾ 1.2c
P ⬍ 0.001
124.1 ⫾ 2.6
113.1 ⫾ 2.0
38.8 ⫾ 0.4 38.7 ⫾ 0.5 39.3 ⫾ 0.6
37.4 ⫾ 0.5a 37.3 ⫾ 0.4a 39.2 ⫾ 0.4b
P ⬍ 0.05 P ⬍ 0.05
44.5 ⫾ 0.7
42.8 ⫾ 0.5
t Test between groups
P ⬍ 0.05 P ⬍ 0.01
P ⬍ 0.01
* Mean ⫾ standard error of the mean. Means with different superscript letters in a column differ significantly by Fisher’s protected least significant difference test at P ⬍ 0.05. Period A, first sampling of teenagers at 0 mo; Period B, second sampling of teenagers at 6 mo; Period C, third sampling of teenagers at 12 mo; Period D, fourth sampling of teenagers at 18 mo.
Urine mineral concentrations adjusted by urine creatinine concentrations are shown in Table VII. Urine Zn concentrations were similar in smokers and non-smokers for teenage subjects at period A and for adult subjects but were significantly higher (47%) in smokers than in non-smokers at period D for teenage subjects (P ⬍ 0.001). Urine Cu concentrations were similar in smokers and non-smokers for teenage subjects throughout the study period and for adult subjects.
TABLE II. SMOKING PERIOD AND AMOUNT AND THEIR RANGES IN TEENAGE AND ADULT SMOKERS Teenage smokers (n ⫽ 35) Variables
DISCUSSION In the current study, the smoking status of teenage and adult subjects was markedly different: 8.8 (range, 3–20) cigarettes a day for 2.5 y (1– 4.5 y) for teenage smokers versus 18.8 (10 –30) cigarettes a day for 23.5 y (13–30 y) for adult smokers; thus, the teenage smokers can be regarded as milder shorter-term smokers than adult smokers. As could be predicted, serum nicotine (144%) and cotinine (49%) concentrations were higher in adult smokers (nicotine, 92.3 ⫾ 12.6 ng/mL; cotinine, 281.9 ⫾ 20.0 ng/mL) than in teenage smokers (nicotine, 37.8 ⫾ 2.0 ng/mL; cotinine, 188.8 ⫾ 16.0 ng/mL; P ⬍ 0.001). However, mean serum nicotine and cotinine concentrations of teenage subjects were highly variable and not positively related to the reported number of cigarettes smoked per day over the 18-mo period. Mean heights of teenage
Smoking period (y) Number of cigarettes smoked per day Period A Period B Period C Period D
Adult smokers (n ⫽ 20)
Mean ⫾ SEM
Range
Mean ⫾ SEM
Range
2.5 ⫾ 0.2
1–4.5
23.5 ⫾ 1.1
13–30
7.6 ⫾ 0.4a* 8.4 ⫾ 0.5a 8.7 ⫾ 0.6ab 10.3 ⫾ 0.9b
3–12 3–13 3–15 3–20
18.8 ⫾ 1.4
10–30
* Means with different superscript letters in a column differ significantly by Fisher’s protected least significant difference test at P ⬍ 0.05. Period A, first sampling of teenagers at 0 mo; Period B, second sampling of teenagers at 6 mo; Period C, third sampling of teenagers at 12 mo; Period D, fourth sampling of teenagers at 18 mo; SEM, standard error of the mean.
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TABLE III. SERUM NICOTINE AND COTININE CONCENTRATIONS* Teenagers
Variables Nicotine (ng/mL) Period A Period B Period C Period D Cotinine (ng/mL) Period A Period B Period C Period D
Adults
Smokers (n ⫽ 35)
Non-smokers (n ⫽ 35)
t Test between groups
28.4 ⫾ 4.8a 43.1 ⫾ 4.3b 39.4 ⫾ 3.4b 35.4 ⫾ 1.6ab
2.6 ⫾ 1.1a 28.1 ⫾ 2.3b 27.4 ⫾ 1.2b 23.3 ⫾ 0.5c
P P P P
⬍ ⬍ ⬍ ⬍
137.0 ⫾ 24.7a 206.0 ⫾ 23.7bc 163.2 ⫾ 13.4ac 248.6 ⫾ 21.4b
0.0 ⫾ 0.0a 114.9 ⫾ 15.0b 29.4 ⫾ 2.5c 45.1 ⫾ 2.8c
P P P P
⬍ ⬍ ⬍ ⬍
Smokers (n ⫽ 20)
Non-smokers (n ⫽ 20)
t Test between groups
0.001 0.01 0.001 0.001
92.3 ⫾ 12.6
8.1 ⫾ 1.6
P ⬍ 0.001
0.001 0.01 0.001 0.001
281.9 ⫾ 20.0
79.6 ⫾ 7.9
P ⬍ 0.001
* Mean ⫾ standard error of the mean. Means with different superscript letters in a column differ significantly by Fisher’s protected least significant difference test at P ⬍ 0.05. Period A, first sampling of teenagers at 0 mo; Period B, second sampling of teenagers at 6 mo; Period C, third sampling of teenagers at 12 mo; Period D, fourth sampling of teenagers at 18 mo.
subjects were higher in smokers than in non-smokers, whereas mean body weights were similar between the two groups. These results suggested that, for teenage subjects, smokers tend to be slimmer than non-smokers. In general, teenage smokers, especially girls, express more concerns regarding their body weight, so they make a great attempt to reduce dietary nutrient intakes, including energy.24,25 In the current study, for teenage subjects, dietary intakes of energy by smokers were lower (83%) than those by non-smokers or the Korean RDA.23 This parallels our previous findings.26 The difference in energy intakes of teenage subjects in the current study was greater than the ratio of weight for height
between smokers and non-smokers. This suggests the possibility of under-reporting of dietary intakes by smoking girls. Although we attempted to obtain accurate diet records from all the teenage subjects, it is likely that individuals who were in the smoking group reported markedly lower energy intakes than did those in the non-smoking group because of a heightened concern in these teenage smokers over body weight. It is worth noting that, on an energy basis, micronutrient intakes of teenage girls in the two groups of the current study were similar, so we do not think the low serum concentrations of vitamin C observed in the teenage smoking group can be attributed to diet alone. In addition, given similar hematocrits noted in the teenage smoking group, the over-
TABLE IV. DIETARY NUTRIENT INTAKES OF TEENAGE SUBJECTS*
Daily nutrient intakes
Percentage of daily nutrient intakes to the Korean RDA
Nutrient
Korean RDA†
Smokers (n ⫽ 35)
Non-smokers (n ⫽ 35)
Smokers (n ⫽ 35)
Non-smokers (n ⫽ 35)
Energy (kcal) Carbohydrate (g) Fat (g) Protein (g) Vitamin A (g RE) Vitamin B1 (mg) Vitamin B2 (mg) Niacin (mg NE) Vitamin C (mg) Calcium (mg) Iron (mg)
2100 ND ND 60–65 700 1.1 1.3 14 70 800 16
1578.6 ⫾ 96.9§ 239.6 ⫾ 14.1§ 46.2 ⫾ 3.0 54.4 ⫾ 4.7‡ 484.0 ⫾ 46.8 1.1 ⫾ 0.1 0.8 ⫾ 0.1 0.4 ⫾ 0.9㛳 45.0 ⫾ 4.3㛳 337.0 ⫾ 40.2 9.1 ⫾ 0.8
1896.0 ⫾ 64.9 290.6 ⫾ 10.1 51.1 ⫾ 2.6 67.8 ⫾ 2.6 557.6 ⫾ 45.2 1.2 ⫾ 0.1 1.0 ⫾ 0.1 15.6 ⫾ 0.7 72.7 ⫾ 5.9 423.2 ⫾ 23.5 9.9 ⫾ 0.4
75.2 ⫾ 4.6§ — — 90.5 ⫾ 7.8‡ 69.1 ⫾ 6.7 95.8 ⫾ 6.3 64.5 ⫾ 4.5 74.2 ⫾ 6.6㛳 64.4 ⫾ 6.1㛳 42.1 ⫾ 5.0 56.8 ⫾ 5.1
90.3 ⫾ 3.1 — — 112.9 ⫾ 4.4 79.7 ⫾ 6.5 106.7 ⫾ 5.0 75.7 ⫾ 3.8 111.3 ⫾ 5.2 103.8 ⫾ 8.5 52.9 ⫾ 3.0 61.9 ⫾ 2.5
* Mean ⫾ standard error of the mean. † The RDA used in this table were obtained from the Korean RDA23 for girls ages 14 to 18 y. ‡ P ⬍ 0.05. § P ⬍ 0.01. 㛳 P ⬍ 0.001. ND, not determined; NE, niacin equivalent; RDA, recommended daily allowance; RE, retinol equivalent.
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Nutrition Volume 20, Number 5, 2004 TABLE V.
ACTIVITIES OF EC SOD AND SERUM CERULOPLASMIN AND CONCENTRATIONS OF SERUM VITAMIN C, FOLATE, AND TBARS* Teenagers
Variables EC SOD (U/mL)† Ceruloplasmin (U/L) Period A Period B Period C Period D Vitamin C (mg/dL) Period A Period B Period C Period D Folate (ng/mL)† TBARS (MDA nM/mL) Period A Period B Period C Period D
Adults
Smokers (n ⫽ 35)
Non-smokers (n ⫽ 35)
t Test between groups
Smokers (n ⫽ 20)
Non-smokers (n ⫽ 20)
t Test between groups
8.54 ⫾ 1.16
17.56 ⫾ 2.04
P ⬍ 0.001
8.27 ⫾ 1.33
15.25 ⫾ 1.28
P ⬍ 0.01
91.71 ⫾ 4.74a 110.34 ⫾ 3.97b 106.85 ⫾ 4.29bc 97.96 ⫾ 4.25ac
90.28 ⫾ 3.48a 107.39 ⫾ 3.88b 106.15 ⫾ 2.94b 93.02 ⫾ 3.19a
76.84 ⫾ 3.35
72.60 ⫾ 6.63
0.88 ⫾ 0.05a 1.29 ⫾ 0.06b 1.03 ⫾ 0.06ac 1.11 ⫾ 0.07c 7.06 ⫾ 0.36
1.27 ⫾ 0.07a 1.15 ⫾ 0.07a 1.46 ⫾ 0.05b 0.96 ⫾ 0.05c 15.69 ⫾ 0.70
P ⬍ 0.001
0.57 ⫾ 0.05
0.89 ⫾ 0.07
P ⬍ 0.001
P ⬍ 0.001
8.82 ⫾ 0.78
14.29 ⫾ 1.75
P ⬍ 0.05
2.90 ⫾ 0.15a 3.09 ⫾ 0.23a 1.07 ⫾ 0.05b 1.08 ⫾ 0.04b
3.62 ⫾ 0.16a 2.79 ⫾ 0.13b 1.07 ⫾ 0.07c 1.02 ⫾ 0.03c
P ⬍ 0.01
2.11 ⫾ 0.09
2.16 ⫾ 0.09
P ⬍ 0.001
* Mean ⫾ standard error of the mean. Means with different superscript letters in a column differ significantly by Fisher’s protected least significant difference test at P ⬍ 0.05. † Values for period D. ‡ P ⬍ 0.05. § P ⬍ 0.01. 㛳 P ⬍ 0.001. EC SOD, extracellular superoxide dismutase; Period A, first sampling of teenagers at 0 mo; Period B, second sampling of teenagers at 6 mo; Period C, third sampling of teenagers at 12 mo; Period D, fourth sampling of teenagers at 18 mo; TBARS, thiobarbituric acid-reactive substances
all nutritional status of the smoking group (with the obvious exception of vitamin C and folate) may have been similar to that of the non-smoking group; this tendency could be seen in the adult subjects of the current study. Mean blood pressures in the teenage and adult subjects were in the normal range for their age groups; however, smokers had higher diastolic and systolic blood pressures at 0 and 12 mo for teenage subjects, and adult smokers also had higher systolic blood pressures than did non-smokers. These observations suggest that the seeds of hypertension may be sown even with short-term smoking; thus, smoking may be initiating vascular damage, even in teenagers.5 Although mechanisms of smoking-induced hypertension are poorly understood, hypertension alters trace element metabolism in heavy long-term smokers. For example, hypozincemia and hypercupremia are often observed in adult smokers secondary to the acute-phase response that is triggered by tissue damage.12 For our population of adult male smokers, hypercupremia was evident, but hypozincemia was not. Interestingly, in our population of female teenagers, serum Zn concentrations and urinary excretions of Zn/creatinine were higher in smokers than in non-smokers. Based on this finding, for teenage smokers in the current study, the lack of changes in mineral metabolism may have been due to the process of adaptation that may take place in young smokers, in whom the antioxidant systems may be able to counteract oxidant factors. Differences in smoking status, sex, and nutritional status of Zn and Cu also may explain the difference in mineral metabolism altered by smoking between the two age groups of the current study. However, the nutrition status of Zn and Cu for teenage and adult subjects cannot be fully discussed at
this point because data on the Zn and Cu contents of conventional Korean foods are not available. EC SOD activities were lower in smokers than in non-smokers. That these reductions in EC SOD activities were functionally important is suggested by our findings of lower serum vitamin C concentrations in smokers versus non-smokers in the teenage (at 0 and 12 mo) and adult subjects. These results suggest that shortterm smoking can induce oxidative stress similarly to long-term smoking. Some previous findings also show that erythrocyte CuZnSOD activities are lower in smokers than in non-smokers for adults9 and teenagers.26 However, serum Cp activities and serum lipid peroxidation, as measured by the production of TBARS, were not increased by smoking in teenage and adult subjects. These results support the concept that the overall health of the subjects in the current study may have been better than in previous reported studies.2 Clinical studies on smokers have indicated adverse effects of smoking on vitamin C metabolism, and several investigators have reported that serum vitamin C concentrations are lower in cigarette smokers than in non-smokers for subjects ages 17 to 50 y.27 Lower serum vitamin C concentrations in smokers may be due to several factors including decreased vitamin C consumption, impaired vitamin C absorption, or an increased turnover of vitamin C.1 Similar to our previous findings,26 intakes of vitamin C in the teenage group were lower in smokers than in non-smokers in the current study. This altered vitamin C metabolism also was found in adult smokers of the current study, although their diet was not surveyed. Similar to the findings for vitamin C, serum folate concentrations were lower in smokers than in non-smokers for teenage and adult
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TABLE VI. CONCENTRATIONS OF SERUM ZINC AND COPPER Teenagers
Variables Zinc (M/L) Period A Period B Period C Period D Copper (M/L) Period A Period B Period C Period D
Adults
Smokers (n ⫽ 35)
Non-smokers (n ⫽ 35)
14.7 ⫾ 0.3a 11.9 ⫾ 0.2b 12.8 ⫾ 0.4b 12.8 ⫾ 0.2b
14.0 ⫾ 0.3a 10.3 ⫾ 0.3b 12.5 ⫾ 0.3c 10.6 ⫾ 0.2b
13.1 ⫾ 0.3 12.6 ⫾ 0.4 13.2 ⫾ 0.4 13.5 ⫾ 0.4
12.8 ⫾ 0.4a 11.7 ⫾ 0.4b 13.1 ⫾ 0.3a 12.6 ⫾ 0.3ab
t Test between groups
P ⬍ 0.001
Smokers (n ⫽ 20)
Non-smokers (n ⫽ 20)
13.9 ⫾ 0.7
14.1 ⫾ 0.7
14.1 ⫾ 0.4
12.4 ⫾ 0.3
t Test between groups
P ⬍ 0.001 P ⬍ 0.01
* Mean ⫾ standard error of the mean. Means with different superscript letters in a column differ significantly by Fisher’s protected least significant difference test at P ⬍ 0.05. † P ⬍ 0.01. ‡ P ⬍ 0.001. Period A, first sampling of teenagers at 0 mo; Period B, second sampling of teenagers at 6 mo; Period C, third sampling of teenagers at 12 mo; Period D, fourth sampling of teenagers at 18 mo
subjects of the current study. Previous studies also have shown that smokers have lower circulating folate concentrations than nonsmokers.11 The lower serum folate concentrations observed in teenage and adult smokers may be related to a lower intake of fruits and vegetables by the smokers and to an increased turnover of folate secondary to oxidative stress.11,28 In the current study, we could not assess the folate intake of subjects because the folate contents of conventional Korean foods have not been reported. Inadequate folate nutriture among women of childbearing age has been recognized as a risk factor for the birth defects known as neutral tube defects, which can occur at the beginning of the pregnancy.29 Overall parameters on physical growth, smoking habits, and blood antioxidant status of teenage subjects did not change consistently through the study; these results may be related to seasonal effect, change of dietary nutrient intakes, and/or alterations in smoking habits over the study period.
Collectively, these findings suggest that cigarette smoking has a negative effect on numerous arms of the oxidant defense system for teenage and adult subjects based on higher blood pressures, lower EC SOD activities, and lower serum vitamin C and folate concentrations in smokers than in non-smokers, although alteration of mineral metabolism by smoking was not observed, especially in teenage subjects. Lower antioxidant status and inadequate folate status observed in the current teenage smokers parallel our previous findings for teenage smokers smoking more heavily and longer than the current smokers (16.0 ⫾ 1.1 cigarettes a day, 10 –25 cigarettes a day, for 2.9 ⫾ 0.2 y, 1.5– 4.1 y).26 The increased oxidative stress that can be associated with direct effects of oxidants in cigarette smoke and consequences of lower antioxidant status associated with smoking may represent risk factors for the development of chronic diseases in teenage and adult smokers. The low concentrations of serum folate in female teenage smokers
TABLE VII. CONCENTRATIONS OF URINE ZINC AND COPPER* Teenagers
Variables Zinc (M/mM creatinine) Period A Period D Copper (M/mM creatinine) Period A Period D
Smokers (n ⫽ 35)
Adults
Non-smokers (n ⫽ 35)
t Test between groups
1.112 ⫾ 0.219 0.789 ⫾ 0.053
0.797 ⫾ 0.148‡ 0.537 ⫾ 0.049
P ⬍ 0.001
0.010 ⫾ 0.006†‡ 0.021 ⫾ 0.001
0.007 ⫾ 0.003 0.018 ⫾ 0.001
* Mean ⫾ standard error of the mean. † Difference between two periods in each group. ‡ P ⬍ 0.05. Period A, first sampling of teenagers at 0 mo; Period D, fourth sampling of teenagers at 18 mo.
Smokers (n ⫽ 20)
Non-smokers (n ⫽ 20)
0.613 ⫾ 0.101
0.517 ⫾ 0.066
0.009 ⫾ 0.001
0.011 ⫾ 0.001
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suggest that these girls may be at increased risk for several potential health complications. In conclusion, the current results emphasize that oxidative stress can be induced in short-term smokers, similarly to long-term adult smokers and teenagers who smoke heavily.26
ACKNOWLEDGMENTS The authors thank Joel Commisso and Greenie Hu at the University of California at Davis in the United States for excellent technical assistance. They appreciate the skillful assistance of Young Sang Park and In Sook Jeong at Kongju Medical Center in Korea for collecting blood and urine samples from the subjects, and they thank Principal Moon Ha Lee and the many teachers, including Min Hyung Lee, Tak Yeon Won, Jong Suk Jeong, Jun Ho Kim, Chul Ryu, and One Joo Lee, at Korean senior high schools that teenage subjects attended for recruiting and managing the subjects of the current study throughout the 18-mo period. They also appreciate the patient and responsible help of the many teenage and adult subjects.
REFERENCES 1. Alberg AJ. The influence of cigarette smoking on circulating concentrations of antioxidant micronutrients. Toxicology 2002;180:121 2. Durak I, Elgun S, Kemal BN, et al. Effects of cigarette smoking with different tar content on erythrocyte oxidant/antioxidant status. Addict Biol 2002;7:255 3. Liu X, Lu J, Liu S. Synergistic induction of hydroxyl radical-induced DNA single-strand breaks by chromium(VI) compound and cigarette smoke solution. Mutat Res 1999;440:109 4. Therriault MJ, Proulx LI, Castonguay A, Bissonnette EY. Immunomodulatory effects of the tobacco-specific carcinogen, NNK, on alveolar macrophages. Clin Exp Immunol 2003;132:232 5. Michael PR. Cigarette smoking, endothelial injury and cardiovascular disease. Int J Exp Pathol 2000;81:219 6. Reibel J. Tobacco and oral diseases. Update on the evidence, with recommendations. Med Princ Pract 2003;12(suppl):22 7. Blokhina O, Virolainen E, Fagerstedt KV. Antioxidant, oxidative damage and oxygen deprivation stress: a review. Ann Bot 2003;91:179 8. Kelly GS. The interaction of cigarette smoking and antioxidants. Part 2: alphatocopherol. Altern Med Rev 2002;7:500 9. Yildiz L, Kayaoglu N, Aksoy H. The changes of superoxide dismutase, catalase and glutathione peroxidase activities in erythrocytes of active and passive smokers. Clin Chem Lab Med 2002;40:612 10. Abou-Seif MAM. Blood antioxidant status and urine sulfate and thiocyanate levels in smokers. J Biochem Toxicol 1996;11:133
Nutrition Volume 20, Number 5, 2004 11. Walmsley CM, Bates CJ, Prentice A, Cole TJ. Relationship between cigarette smoking and nutrient intakes and blood status indices of older people living in the UK: further analysis of data from the National Diet and Nutrition Survey of people aged 65 years and over, 1994/95. Public Health Nutr 1999;2:199 12. Keen CL, Clegg MS, Ferrell F, Hunter GC, Dubick MA. Hypertension induced alterations in copper and zinc metabolism: a link to vascular disease?. In: Sorenson JRJ, ed. Biology of copper complexes. Clifton, NJ: Humana Press, 1987:141 13. Dubick MA, Keen CL. Influence of nicotine on tissue trace element concentrations and tissue antioxidant defense. Biol Trace Elem Res 1991;31:97 14. Kocyigit A, Erel O, Gur S. Effects of tobacco smoking on plasma selenium, zinc, copper and iron concentrations and related antioxidative enzyme activities. Clin Biochem 2001;34:629 15. Dietrich M, Block G, Norkus EP, et al. Smoking and exposure to environmental tobacco smoke decrease some plasma antioxidants and increase ␥-tocopherol in vivo after adjustment for dietary antioxidant intakes. Am J Clin Nutr 2003;77:160 16. Suh I, Jee SH, Kim SY, et al. The changing pattern of cigarette smoking of students in junior and senior high schools in Korea: 1988 –1997. Korean J Epidemiol 1998;20:257 17. Gibson RS, Heath AL, Ferguson EL. Risk of suboptimal iron and zinc nutriture among adolescent girls in Australia and New Zealand: causes, consequences, and solutions. Asia Pac J Clin Nutr 2002;11:S543 18. Voncken P, Schepers G, Schafer KH. Capillary gas chromatographic determination of trans-3⬘-hydroxy-cotinine simultaneously with nicotine and cotinine in urine and blood samples. J Chromatogr 1989;479:410 19. Paik HY, Joung HY, Lee JY, et al. Serum extracellular superoxide dismutase activity as an indicator of zinc status in humans. Biol Trace Elem Res 1999;69:45 20. Schosinsky KH, Lehmann HP, Beeler MF. Measurement of ceruloplasmin from its oxidase activity in serum by use of -dianisidine dihydrochloride. Clin Chem 1974;20:1556 21. Yagi K. Assay for blood plasma or serum. Methods Enzymol 1984;105:328 22. Kenneth KP, Trevithick JR. High performance liquid chromatographyelectrochemical detection of antioxidants in vertebrate lens: glutathione, tocopherol and ascorbate. Methods Enzymol 1994;233:523 23. Recommended dietary allowances for Koreans, 7th ed. Seoul: Korean Nutrition Society, 2000 24. Dowdell EB. Urban seventh graders and smoking: a health risk behavior assessment. Issues Compr Pediatr Nurs 2002;25:217 25. Lim WK, Kim SH. Cigarette smoking habits among teenage girls living in a rural community in Korea. Korean J Nutr 2000;33:755 26. Kim SH, Kim JS, Shin HS, Keen CL. Influence of smoking on markers of oxidative stress and serum mineral concentrations in teenage girls in Korea. Nutrition 2003;19:240 27. Wei W, Kim Y, Boudreau N. Association of smoking with serum and dietary levels of antioxidants in adults: NHANES III, 1988 –1994. Am J Public Health 2001;91:258 28. Mansoor MA, Kristensen O, Hervig T, et al. Low concentrations of folate in serum and erythrocytes of smokers: methionine loading decreases folate concentrations in serum of smokers and nonsmokers. Clin Chem 1997;43:2192 29. Refsum H. Folate, vitamin B12, and homocysteine in relation to birth defects and pregnancy outcome. Br J Nutr 2001;85:S109
An 18-Month Follow-up Study on the Influence of Smoking on Blood Antioxidant Status of Teenage Girls in Comparison With Adult Male Smokers in Korea Sun H. Kim, PhD, Jodi L. Ensunsa, MS, Qin Yan Zhu, PhD, Jung S. Kim, PhD, Ho S. Shin, PhD, and Carl L. Keen, PhD From the Departments of Food Service Management and Nutrition, Physical Education, and Environmental Education, Kongju National University, Kongju, Korea; and the Departments of Nutrition and Nutrition and Internal Medicine, University of California at Davis, Davis, California, USA OBJECTIVES: The influence of cigarette smoking on blood antioxidant status in teenage girls with a history of short-term smoking was followed over 18 mo. METHODS: Data obtained from female senior high school students (ages 14 to 18 y) in Korea were compared with data obtained from adult male smokers (ages 36 to 51 y) with a long history of smoking and living in the same geographic areas as the teenage subjects. A smoker was a person who had smoked at least three cigarettes a day for at least 1 y for teenagers (n ⫽ 35) or at least 10 cigarettes a day for at least 13 y for adults (n ⫽ 20). Serum, urine, and anthropometric data were obtained from teenagers every 6 mo over an 18-mo period. Samples were collected once from adults. Data were analyzed by Student’s t test and Fisher’s protected least significant difference test for comparing smokers and non-smokers and for analyzing period effects in each group. RESULTS: Serum nicotine and cotinine concentrations were higher in smokers than in non-smokers. Blood pressures were higher in teenage (at 0 and 12 mo) and adult smokers than in non-smokers. Extracellular superoxide dismutase activities and concentrations of serum vitamin C and folate were lower in smokers in the teenage (at 0, 12, or 18 mo) and adult groups. Serum ceruloplasmin activities and thiobarbituric acid-reactive substance production were not influenced by smoking. In adults, serum copper concentrations were higher in smokers than in non-smokers. This parameter for teenagers did not change consistently throughout the study. CONCLUSIONS: Similar to adults, cigarette smoking by teenagers has a negative effect on oxidant defense systems. Nutrition 2004;20:437– 444. ©Elsevier Inc. 2004 KEY WORDS: smoking, extracellular superoxide dismutase, vitamin C, folate, mineral, teenage short-term smokers
INTRODUCTION Tobacco smoke contains numerous compounds emitted in gases and condensed tar particles, many of which are oxidants and pro-oxidants capable of producing reactive oxygen species.1 The increased production of reactive oxygen species by smoke can produce a condition of oxidative stress that can result in the oxidation of lipids, induction of DNA single-strand breakage, inactivation of certain proteins, and the disruption of biological membranes.2,3 Increased oxidative stress has been suggested to play a major role in the pathogenesis of several smoking-related
This work was supported by a 2001 grant from Kongju National University in Korea and grant DK-35747 from the National Institutes of Health in the United States. Correspondence to: Sun Hyo Kim, PhD, Department of Food Service Management and Nutrition, Kongju National University, Shinkwan-dong, Kongju, Chungnam-do, 314-701, Republic of Korea. E-mail: shkim@ kongju.ac.kr Nutrition 20:437– 444, 2004 ©Elsevier Inc., 2004. Printed in the United States. All rights reserved.
diseases such as cancer, cardiovascular diseases, and oral diseases.4 – 6 The damage that can be caused by free radicals is normally minimized by biological antioxidant systems including enzymatic and non-enzymatic systems. Important antioxidant enzymes include copper (Cu) and zinc (Zn) superoxide dismutase (SOD), manganese SOD, ceruloplasmin (Cp), selenium glutathione peroxidase, glutathione reductase, and catalase. Non-enzymatic antioxidants include vitamin C, ␣-tocopherol, provitamin A carotenoids, and urate.1,7,8 In adult smokers, several arms of the oxidant defense system have been reported to be impaired relative to oxidant defense systems in non-smokers. It has been reported that adult smokers can have significantly lower erythrocyte CuZnSOD activities than non-smokers.9 Similarly, concentrations of serum vitamin C and vitamin E have been reported to be lower in adult smokers than in non-smokers.10 Lower serum vitamin C concentrations in smokers are thought to be a consequence of greater vitamin C turnover in response to a sustained oxidant load and lower vitamin C intakes by heavy smokers.1 Similar to vitamin C, serum folate concentrations are often lower in adult smokers than in non-smokers because 0899-9007/04/$30.00 doi:10.1016/j.nut.2004.01.008
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folate catabolism is increased under conditions of oxidative stress.11 Collectively, these findings support the association of long-term smoking with increased oxidative stress that can affect an individual’s oxidant defense system and overall nutrition status. With respect to serum mineral concentrations, several investigators have reported that Zn concentrations are often reduced in adult smokers,12 whereas Cu concentrations typically are increased.13,14 Changes in Zn and Cu homeostases in smokers have been postulated to contribute to some of the long-term negative effects associated with smoking and hypertension. It is not known whether these changes in Zn and Cu are acute effects that occur shortly after the initiation of smoking or are secondary to the development of chronic diseases related to smoking such as hypertension. Hypozincemia and hypercupremia can be characteristic of hypertension independent of smoking.12 Although smoking is recognized to be a significant health problem in adult subjects,15 the health consequences of smoking in teenagers who have a short-term smoking history are less well understood. The frequency of tobacco use by teenagers is increasing annually, especially in developing countries in Asia, such as Korea.16 It could be argued that cigarette smoking by teenage girls poses particular health problems because this population group is often undernourished due to dieting. Suboptimal nutritional status during periods of rapid growth and development can have consequences that persist into adulthood.17 In light of these findings, we performed the current study to observe the longitudinal effects of smoking on antioxidant status in female teenagers with a short-term smoking history. Female teenage smokers were sampled at 6-mo intervals over an 18-mo period. Results from these subjects were compared with data from nonsmoking teenage girls and from men with a long-term smoking history.
MATERIALS AND METHODS Subjects Teenage subjects were selected from a population of senior high school girls (ages 14 to 18 y) in Korean urban and rural areas located in the Chungnam province. The subjects attended one of six senior high schools selected in these two areas. All subjects were healthy and reported no use of illegal drugs during the study period. They were interviewed for their tobacco use and that of their families. They were questioned with respect to the average number of cigarettes smoked per day and when they started, or stopped, smoking. Serum nicotine and cotinine concentrations were determined to confirm their answers regarding smoking status. If the subject’s responses regarding her smoking habits and her serum nicotine and cotinine concentrations coincided, the girl was included in the study. Fifty smokers who had smoked continually for at least 1 y and who had no experience of stopping smoking were identified at the beginning of study. Fifty non-smokers were identified who reported neither previous smoking experience nor exposure to passive smoking at home at the beginning of the study. The subjects were selected by quota structure and followed over an 18-mo study period. They were interviewed for smoking habits, including average number of cigarettes smoked per day at 6-mo intervals (period A, 0 mo; period B, 6 mo; period C, 12 mo; and period D, 18 mo). They were not treated with any other intervention during the study period. Some subjects moved during the study; refused to give blood and urine samples; or stopped smoking during the study periods. Thirty-five subjects in the smoking and non-smoking groups completed the 18-mo study. At the end of the study, smokers had a mean age of 16.5 ⫾ 0.1 y (range, 14 –18 y), with a period of 2.5 ⫾ 0.2 y (1– 4.5 y) of smoking, and smoked 7.6 ⫾ 0.4 (3–12) cigarettes a day. Non-smokers had a mean age of 15.6 ⫾ 0.1 y (15–17 y). The percentage of subjects who finished the study completely during the 18-mo period relative to the total
Nutrition Volume 20, Number 5, 2004 number of selected subjects at the beginning of study was 70. The girls in the smoking group indicated that they had some concern that they would be stigmatized by being recognized as smokers. To reduce their anxiety, all records were coded and kept confidential. Adult male subjects, as a positive control group, were selected from a male population living in the same Korean urban and rural areas as the teenage subjects of this study. At the beginning of the current study, we attempted to select an adult female smoking group, but these women expressed considerable anxiety that they would be stigmatized by being recognized as smokers and thus refused to participate. As a consequence, we selected men as a positive control group. All subjects were healthy and reported no use of illegal drugs during the study period. They were interviewed for their tobacco use; average number of cigarettes smoked per day; initial age when they started smoking; and whether they have smoked continually. Analysis of serum nicotine and cotinine concentrations combined with results of the interview were done to determine the eligibility of subjects. Twenty adult male smokers (44.7 ⫾ 0.9 y; range, 36 –51 y) who had smoked at least 10 cigarettes a day (18.8 ⫾ 1.4, 10 –30) for at least 13 y (23.5 ⫾ 1.1 y, 13–30 y) were identified. Twenty adult male non-smokers (46.5 ⫾ 1.2 y, 38 –56 y) who reported no previous smoking experience were identified; adult subjects in both groups were selected by quota structure. Adult male subjects were studied once during the study period because they declined to continue a longitudinal study. The adults were not treated with any other intervention.
Analytical Methods Fasting blood samples were collected from teenage subjects at 6-mo intervals during the 18-mo study period. Blood samples were collected once from all adult subjects. Subjects were asked not to smoke for 12 h before sampling to exclude the effects of acute smoking on the blood parameters that were studied. An aliquot of blood was used for measuring the hematocrit. Remaining blood samples were centrifuged at 1700g for 20 min at 4°C. The serum was used for the assessment of nicotine, cotinine, vitamin C, folate, thiobarbituric acid-reactive substances (TBARS), Zn and Cu concentrations; and extracellular (EC) SOD and Cp activities. Serum samples were stored at ⫺80°C until analyzed. Urine samples were collected from teenage subjects at 6-mo intervals; and samples were collected once from adult subjects. Urine samples were used for measuring Zn, Cu, and creatinine concentrations. Samples were stored at ⫺80°C until analyzed. For the analysis of nicotine and cotinine, serum was mixed with an internal standard (di-isopropyl-amine dodecane; DIPA 12, Sigma, St. Louis, MO, USA) in 0.1 M KOH solution and ether. The mixture was saturated with NaCl and the ether phase was injected into a Hewlett-Packard 6890 gas chromatograph equipped with a 30-m ⫻ 0.32-mm HP-5 MS capillary column. The injector temperature was set at 260°C, and the detector temperature was set at 300°C. Nicotine and cotinine concentrations were determined by comparison with a commercial standard.18 EC SOD (EC 1.15.1.1) activity was analyzed as described by Paik et al.19 Serum Cp (EC 1.16.3.1) activity was measured as described by Schosinsky et al.20 Serum TBARS production was measured according to a fluorometric method;21 values are reported as nanomoles of malondialdehyde per milliliter of serum. Serum vitamin C analysis was performed as follows: Serum was mixed with 2% metaphosphoric acid (1:2) and centrifuged at 13 000g for 10 min at 4°C; the supernatant was removed and mixed with dithiothreitol and allowed to incubate at room temperature for 60 min. Samples were then mixed with a mobile phase (0.1 M acetate buffer, pH 5.0), and the solution was filtered and injected into a Hewlett-Packard 1090 II/M high-performance liquid chromatograph with an inner diameter of 100 ⫻ 3.2 mm and a column size of 3 m.22 Serum folate concentrations were measured with a radioassay procedure using a solid-phase no-boil
Nutrition Volume 20, Number 5, 2004 folate kit (Diagnostic Product Corporation, Los Angeles, CA, USA). Serum and urine Zn and Cu concentrations were analyzed by inductively coupled argon plasma-atomic emission spectrophotometry (Trace Scan ICP, Thermal-Jarrell Ash Corp., Franklin, MA, USA) after dilution with 1 N HNO3. For the analysis of serum mineral concentrations, the procedure of protein denaturation followed by discarding the denatured pellet was used before reading samples on the inductively coupled argon plasma-atomic emission spectrophotometer. However, for the analysis of urine mineral concentrations, samples were read directly without the denaturation and discarding of protein. Urine creatinine concentrations were measured by colorimetric determination at 500 nm using a diagnostic creatinine reagent kit (Sigma). Urine mineral concentrations are expressed as micromoles per millimole of creatinine. Anthropometric Measurement Teenage subjects’ heights were measured annually by public health teachers at the subjects’ schools. Body weights and blood pressures of teenagers were measured at 6-mo intervals during the 18-mo period by the nursing staff in a medical center. Adult subjects’ heights, body weights, and blood pressures were measured once by a nursing staff in a medical center. Heights and body weights of teenage and adult subjects were measured on an electrical scale that could measure heights and body weights concurrently while subjects were shoeless and wearing light clothing. Blood pressures were determined with a sphygmomanometer and expressed as the mean value of two measurements taken 3 to 5 min apart. Dietary Survey At the beginning of the study, a subset of the teenage subjects (14 to 18 y) in each group (n ⫽ 35) was randomly selected from the larger population and asked to complete a dietary study. They were asked to record 2 d during the week of all food intakes by using the 24-h recall method for cross checking. All subjects in each group selected at the beginning of the dietary survey completed the dietary records. A computer-aided nutritional program developed by the Korean Nutrition Society was used for the nutrition interview by researchers and for the analysis of dietary nutrient intakes. Nutrient intakes were compared with the Korean recommended dietary allowance (RDA)23 for girls ages 14 to 18 y. Statistical Analysis Data are presented as mean ⫾ standard error of the mean. Data were analyzed with unpaired Student’s t test for comparing serum antioxidant status, urine mineral concentrations, anthropometric measurements, and dietary nutrient intakes between smokers and non-smokers for teenage and adult subjects, respectively, at each sampling period. The influence of the sampling period on these parameters in each teenage group was analyzed by the F test; if there was a significant influence of sampling period, Fisher’s protected least significant difference test was applied again. Differences were considered to be statically significant at P ⬍ 0.05. All statistical analyses were conducted with StatView 4.1 (Abacus Concepts, Berkeley, CA, USA).
RESULTS The mean age for teenage subjects was significantly higher in smokers than in non-smokers (P ⬍ 0.001), but the ages in these two groups were developmentally similar (Table I). The mean age of adult subjects also was similar between smokers (44.7 ⫾ 0.9 y)
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and non-smokers (46.5 ⫾ 1.2 y). Mean heights for teenage subjects were higher in smokers than in non-smokers at 12 mo (P ⬍ 0.05), but these values did not change significantly over 12 mo in the smoking and non-smoking groups. Mean body weights of the teenage subjects were similar between the two groups, and these values did not change significantly over 18 mo in the smoking and non-smoking groups. Mean heights and body weights of adult subjects were similar in the two groups. Blood pressure values for teenage and adult subjects were within the normal range for their age groups; diastolic and systolic blood pressures were significantly higher in teenage smokers than in teenage non-smokers at periods A (P ⬍ 0.001) and C (P ⬍ 0.05). Blood pressure values of the teenage subjects changed inconsistently throughout the 18-mo period. For adult subjects, diastolic and systolic blood pressures were higher in smokers than in non-smokers; there was a significant difference for systolic blood pressure (P ⬍ 0.01). Hematocrit values of teenage and adult subjects during the entire study period were within the normal range; however, values for the smokers in teenage subjects (at periods B and C) and for the adult subjects were significantly higher than those for their non-smoking counterparts (P ⬍ 0.05). The average smoking period of teenage smokers was 2.5 y and that of adult subjects was 23.5 y (Table II). The average number of cigarettes smoked daily by teenage smokers changed from 8 (twofifths of a pack/d) to10 (half a pack/d) cigarettes a day over the 18-mo period. Adult smokers had an average of 18.8 cigarettes a day (nine-tenths to one pack/d). Serum nicotine and cotinine concentrations in teenage (P ⬍ 0.01 to 0.001) and in adult subjects (P ⬍ 0.001) were higher in smokers than in non-smokers throughout the study period (Table III). Serum nicotine and cotinine concentrations in the teenage smoking group showed inconsistent changes over the study period. Reported nutrient intakes for teenage subjects are shown in Table IV. Daily energy intakes were significantly lower in smokers than in non-smokers (P ⬍ 0.01); ratios of mean energy intake to the Korean RDA for the same age (2100 kcal/d)23 were within 75% of the RDA for smokers and close to the RDA for nonsmokers. Reported intakes of vitamins A and C (P ⬍ 0.001) were lower in smokers than in non-smokers. In particular, vitamin C intakes by smokers were less than 65% of the Korean RDA for the same age (70 mg/d),23 whereas vitamin C intakes in non-smokers were higher than the RDA. Vitamin C intakes on an energy basis were 0.03 and 0.04 mg/kcal for smokers and non-smokers, respectively. Niacin intakes were significantly lower in smokers than in non-smokers (P ⬍ 0.001); vitamin B1 and B2 intakes were similar in the two groups. Calcium and iron intakes in both groups were less than 65% of the Korean RDA23 for the same age. EC SOD activities of teenage smokers were lower (⬇50%) in smokers than in non-smokers at period D (P ⬍ 0.001); similarly, the values for adult subjects were lower (46%) in smokers than in non-smokers (P ⬍ 0.01; Table V). Serum Cp activities were similar in the two groups for teenage and adult subjects. Serum vitamin C concentrations in teenage subjects were significantly lower in smokers than in non-smokers at periods A (31%) and C (30%; P ⬍ 0.001). Adult smokers also had lower serum vitamin C concentrations than did non-smokers (36%; P ⬍ 0.001). Serum folate concentrations were lower in teenage smokers than in teenage non-smokers (55%; P ⬍ 0.001) at period D. Adult smokers had lower serum folate concentrations than did non-smokers (38%; P ⬍ 0.05). Serum TBARS production was similar in smokers and non-smokers for teenage and adult groups at all time points. In teenagers, serum Zn concentrations were similar in smokers and non-smokers at periods A and C but significantly higher in smokers than in non-smokers at periods B (16%) and D (21%; P ⬍ 0.001; Table VI). For adult subjects, serum Zn concentrations were similar in the smoking and non-smoking groups. Serum Cu concentrations were similar in the two groups for teenage subjects, but these values for adult subjects were significantly higher (14%) in smokers than in non-smokers (P ⬍ 0.01).
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Nutrition Volume 20, Number 5, 2004 TABLE I. GENERAL CHARACTERISTICS OF SUBJECTS* Teenagers
Variables Age (y) Height (cm) Period A Period C Body weight (kg) Period A Period B Period C Period D Blood pressure (mmHg) Diastolic Period A Period B Period C Period D Systolic Period A Period B Period C Period D Hematocrit (%) Period B Period C Period D
Adults
Smokers (n ⫽ 35)
Non-smokers (n ⫽ 35)
t Test between groups
Smokers (n ⫽ 20)
Non-smokers (n ⫽ 20)
16.5 ⫾ 0.1
15.6 ⫾ 0.1
P ⬍ 0.001
44.7 ⫾ 0.9
46.5 ⫾ 1.2
160.5 ⫾ 1.0 162.2 ⫾ 1.0
158.7 ⫾ 1.0 159.2 ⫾ 1.0
P ⬍ 0.05
169.6 ⫾ 1.0
169.3 ⫾ 1.6
52.4 ⫾ 1.4 52.3 ⫾ 1.3 52.6 ⫾ 1.4 51.8 ⫾ 1.2
51.2 ⫾ 1.4 51.2 ⫾ 0.8 52.0 ⫾ 0.9 50.8 ⫾ 0.8
67.9 ⫾ 1.7
68.3 ⫾ 1.9
70.2 ⫾ 1.7a 63.8 ⫾ 1.7b 62.4 ⫾ 1.5b 73.9 ⫾ 1.7a
62.6 ⫾ 1.4a 63.8 ⫾ 1.0a 58.0 ⫾ 1.1b 72.3 ⫾ 1.2c
P ⬍ 0.001
82.3 ⫾ 2.4
77.7 ⫾ 2.2
109.3 ⫾ 2.1 104.4 ⫾ 2.3 103.5 ⫾ 1.6 108.9 ⫾ 2.1
98.3 ⫾ 1.9ab 102.4 ⫾ 1.7a 98.0 ⫾ 1.1b 109.6 ⫾ 1.2c
P ⬍ 0.001
124.1 ⫾ 2.6
113.1 ⫾ 2.0
38.8 ⫾ 0.4 38.7 ⫾ 0.5 39.3 ⫾ 0.6
37.4 ⫾ 0.5a 37.3 ⫾ 0.4a 39.2 ⫾ 0.4b
P ⬍ 0.05 P ⬍ 0.05
44.5 ⫾ 0.7
42.8 ⫾ 0.5
t Test between groups
P ⬍ 0.05 P ⬍ 0.01
P ⬍ 0.01
* Mean ⫾ standard error of the mean. Means with different superscript letters in a column differ significantly by Fisher’s protected least significant difference test at P ⬍ 0.05. Period A, first sampling of teenagers at 0 mo; Period B, second sampling of teenagers at 6 mo; Period C, third sampling of teenagers at 12 mo; Period D, fourth sampling of teenagers at 18 mo.
Urine mineral concentrations adjusted by urine creatinine concentrations are shown in Table VII. Urine Zn concentrations were similar in smokers and non-smokers for teenage subjects at period A and for adult subjects but were significantly higher (47%) in smokers than in non-smokers at period D for teenage subjects (P ⬍ 0.001). Urine Cu concentrations were similar in smokers and non-smokers for teenage subjects throughout the study period and for adult subjects.
TABLE II. SMOKING PERIOD AND AMOUNT AND THEIR RANGES IN TEENAGE AND ADULT SMOKERS Teenage smokers (n ⫽ 35) Variables
DISCUSSION In the current study, the smoking status of teenage and adult subjects was markedly different: 8.8 (range, 3–20) cigarettes a day for 2.5 y (1– 4.5 y) for teenage smokers versus 18.8 (10 –30) cigarettes a day for 23.5 y (13–30 y) for adult smokers; thus, the teenage smokers can be regarded as milder shorter-term smokers than adult smokers. As could be predicted, serum nicotine (144%) and cotinine (49%) concentrations were higher in adult smokers (nicotine, 92.3 ⫾ 12.6 ng/mL; cotinine, 281.9 ⫾ 20.0 ng/mL) than in teenage smokers (nicotine, 37.8 ⫾ 2.0 ng/mL; cotinine, 188.8 ⫾ 16.0 ng/mL; P ⬍ 0.001). However, mean serum nicotine and cotinine concentrations of teenage subjects were highly variable and not positively related to the reported number of cigarettes smoked per day over the 18-mo period. Mean heights of teenage
Smoking period (y) Number of cigarettes smoked per day Period A Period B Period C Period D
Adult smokers (n ⫽ 20)
Mean ⫾ SEM
Range
Mean ⫾ SEM
Range
2.5 ⫾ 0.2
1–4.5
23.5 ⫾ 1.1
13–30
7.6 ⫾ 0.4a* 8.4 ⫾ 0.5a 8.7 ⫾ 0.6ab 10.3 ⫾ 0.9b
3–12 3–13 3–15 3–20
18.8 ⫾ 1.4
10–30
* Means with different superscript letters in a column differ significantly by Fisher’s protected least significant difference test at P ⬍ 0.05. Period A, first sampling of teenagers at 0 mo; Period B, second sampling of teenagers at 6 mo; Period C, third sampling of teenagers at 12 mo; Period D, fourth sampling of teenagers at 18 mo; SEM, standard error of the mean.
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TABLE III. SERUM NICOTINE AND COTININE CONCENTRATIONS* Teenagers
Variables Nicotine (ng/mL) Period A Period B Period C Period D Cotinine (ng/mL) Period A Period B Period C Period D
Adults
Smokers (n ⫽ 35)
Non-smokers (n ⫽ 35)
t Test between groups
28.4 ⫾ 4.8a 43.1 ⫾ 4.3b 39.4 ⫾ 3.4b 35.4 ⫾ 1.6ab
2.6 ⫾ 1.1a 28.1 ⫾ 2.3b 27.4 ⫾ 1.2b 23.3 ⫾ 0.5c
P P P P
⬍ ⬍ ⬍ ⬍
137.0 ⫾ 24.7a 206.0 ⫾ 23.7bc 163.2 ⫾ 13.4ac 248.6 ⫾ 21.4b
0.0 ⫾ 0.0a 114.9 ⫾ 15.0b 29.4 ⫾ 2.5c 45.1 ⫾ 2.8c
P P P P
⬍ ⬍ ⬍ ⬍
Smokers (n ⫽ 20)
Non-smokers (n ⫽ 20)
t Test between groups
0.001 0.01 0.001 0.001
92.3 ⫾ 12.6
8.1 ⫾ 1.6
P ⬍ 0.001
0.001 0.01 0.001 0.001
281.9 ⫾ 20.0
79.6 ⫾ 7.9
P ⬍ 0.001
* Mean ⫾ standard error of the mean. Means with different superscript letters in a column differ significantly by Fisher’s protected least significant difference test at P ⬍ 0.05. Period A, first sampling of teenagers at 0 mo; Period B, second sampling of teenagers at 6 mo; Period C, third sampling of teenagers at 12 mo; Period D, fourth sampling of teenagers at 18 mo.
subjects were higher in smokers than in non-smokers, whereas mean body weights were similar between the two groups. These results suggested that, for teenage subjects, smokers tend to be slimmer than non-smokers. In general, teenage smokers, especially girls, express more concerns regarding their body weight, so they make a great attempt to reduce dietary nutrient intakes, including energy.24,25 In the current study, for teenage subjects, dietary intakes of energy by smokers were lower (83%) than those by non-smokers or the Korean RDA.23 This parallels our previous findings.26 The difference in energy intakes of teenage subjects in the current study was greater than the ratio of weight for height
between smokers and non-smokers. This suggests the possibility of under-reporting of dietary intakes by smoking girls. Although we attempted to obtain accurate diet records from all the teenage subjects, it is likely that individuals who were in the smoking group reported markedly lower energy intakes than did those in the non-smoking group because of a heightened concern in these teenage smokers over body weight. It is worth noting that, on an energy basis, micronutrient intakes of teenage girls in the two groups of the current study were similar, so we do not think the low serum concentrations of vitamin C observed in the teenage smoking group can be attributed to diet alone. In addition, given similar hematocrits noted in the teenage smoking group, the over-
TABLE IV. DIETARY NUTRIENT INTAKES OF TEENAGE SUBJECTS*
Daily nutrient intakes
Percentage of daily nutrient intakes to the Korean RDA
Nutrient
Korean RDA†
Smokers (n ⫽ 35)
Non-smokers (n ⫽ 35)
Smokers (n ⫽ 35)
Non-smokers (n ⫽ 35)
Energy (kcal) Carbohydrate (g) Fat (g) Protein (g) Vitamin A (g RE) Vitamin B1 (mg) Vitamin B2 (mg) Niacin (mg NE) Vitamin C (mg) Calcium (mg) Iron (mg)
2100 ND ND 60–65 700 1.1 1.3 14 70 800 16
1578.6 ⫾ 96.9§ 239.6 ⫾ 14.1§ 46.2 ⫾ 3.0 54.4 ⫾ 4.7‡ 484.0 ⫾ 46.8 1.1 ⫾ 0.1 0.8 ⫾ 0.1 0.4 ⫾ 0.9㛳 45.0 ⫾ 4.3㛳 337.0 ⫾ 40.2 9.1 ⫾ 0.8
1896.0 ⫾ 64.9 290.6 ⫾ 10.1 51.1 ⫾ 2.6 67.8 ⫾ 2.6 557.6 ⫾ 45.2 1.2 ⫾ 0.1 1.0 ⫾ 0.1 15.6 ⫾ 0.7 72.7 ⫾ 5.9 423.2 ⫾ 23.5 9.9 ⫾ 0.4
75.2 ⫾ 4.6§ — — 90.5 ⫾ 7.8‡ 69.1 ⫾ 6.7 95.8 ⫾ 6.3 64.5 ⫾ 4.5 74.2 ⫾ 6.6㛳 64.4 ⫾ 6.1㛳 42.1 ⫾ 5.0 56.8 ⫾ 5.1
90.3 ⫾ 3.1 — — 112.9 ⫾ 4.4 79.7 ⫾ 6.5 106.7 ⫾ 5.0 75.7 ⫾ 3.8 111.3 ⫾ 5.2 103.8 ⫾ 8.5 52.9 ⫾ 3.0 61.9 ⫾ 2.5
* Mean ⫾ standard error of the mean. † The RDA used in this table were obtained from the Korean RDA23 for girls ages 14 to 18 y. ‡ P ⬍ 0.05. § P ⬍ 0.01. 㛳 P ⬍ 0.001. ND, not determined; NE, niacin equivalent; RDA, recommended daily allowance; RE, retinol equivalent.
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Nutrition Volume 20, Number 5, 2004 TABLE V.
ACTIVITIES OF EC SOD AND SERUM CERULOPLASMIN AND CONCENTRATIONS OF SERUM VITAMIN C, FOLATE, AND TBARS* Teenagers
Variables EC SOD (U/mL)† Ceruloplasmin (U/L) Period A Period B Period C Period D Vitamin C (mg/dL) Period A Period B Period C Period D Folate (ng/mL)† TBARS (MDA nM/mL) Period A Period B Period C Period D
Adults
Smokers (n ⫽ 35)
Non-smokers (n ⫽ 35)
t Test between groups
Smokers (n ⫽ 20)
Non-smokers (n ⫽ 20)
t Test between groups
8.54 ⫾ 1.16
17.56 ⫾ 2.04
P ⬍ 0.001
8.27 ⫾ 1.33
15.25 ⫾ 1.28
P ⬍ 0.01
91.71 ⫾ 4.74a 110.34 ⫾ 3.97b 106.85 ⫾ 4.29bc 97.96 ⫾ 4.25ac
90.28 ⫾ 3.48a 107.39 ⫾ 3.88b 106.15 ⫾ 2.94b 93.02 ⫾ 3.19a
76.84 ⫾ 3.35
72.60 ⫾ 6.63
0.88 ⫾ 0.05a 1.29 ⫾ 0.06b 1.03 ⫾ 0.06ac 1.11 ⫾ 0.07c 7.06 ⫾ 0.36
1.27 ⫾ 0.07a 1.15 ⫾ 0.07a 1.46 ⫾ 0.05b 0.96 ⫾ 0.05c 15.69 ⫾ 0.70
P ⬍ 0.001
0.57 ⫾ 0.05
0.89 ⫾ 0.07
P ⬍ 0.001
P ⬍ 0.001
8.82 ⫾ 0.78
14.29 ⫾ 1.75
P ⬍ 0.05
2.90 ⫾ 0.15a 3.09 ⫾ 0.23a 1.07 ⫾ 0.05b 1.08 ⫾ 0.04b
3.62 ⫾ 0.16a 2.79 ⫾ 0.13b 1.07 ⫾ 0.07c 1.02 ⫾ 0.03c
P ⬍ 0.01
2.11 ⫾ 0.09
2.16 ⫾ 0.09
P ⬍ 0.001
* Mean ⫾ standard error of the mean. Means with different superscript letters in a column differ significantly by Fisher’s protected least significant difference test at P ⬍ 0.05. † Values for period D. ‡ P ⬍ 0.05. § P ⬍ 0.01. 㛳 P ⬍ 0.001. EC SOD, extracellular superoxide dismutase; Period A, first sampling of teenagers at 0 mo; Period B, second sampling of teenagers at 6 mo; Period C, third sampling of teenagers at 12 mo; Period D, fourth sampling of teenagers at 18 mo; TBARS, thiobarbituric acid-reactive substances
all nutritional status of the smoking group (with the obvious exception of vitamin C and folate) may have been similar to that of the non-smoking group; this tendency could be seen in the adult subjects of the current study. Mean blood pressures in the teenage and adult subjects were in the normal range for their age groups; however, smokers had higher diastolic and systolic blood pressures at 0 and 12 mo for teenage subjects, and adult smokers also had higher systolic blood pressures than did non-smokers. These observations suggest that the seeds of hypertension may be sown even with short-term smoking; thus, smoking may be initiating vascular damage, even in teenagers.5 Although mechanisms of smoking-induced hypertension are poorly understood, hypertension alters trace element metabolism in heavy long-term smokers. For example, hypozincemia and hypercupremia are often observed in adult smokers secondary to the acute-phase response that is triggered by tissue damage.12 For our population of adult male smokers, hypercupremia was evident, but hypozincemia was not. Interestingly, in our population of female teenagers, serum Zn concentrations and urinary excretions of Zn/creatinine were higher in smokers than in non-smokers. Based on this finding, for teenage smokers in the current study, the lack of changes in mineral metabolism may have been due to the process of adaptation that may take place in young smokers, in whom the antioxidant systems may be able to counteract oxidant factors. Differences in smoking status, sex, and nutritional status of Zn and Cu also may explain the difference in mineral metabolism altered by smoking between the two age groups of the current study. However, the nutrition status of Zn and Cu for teenage and adult subjects cannot be fully discussed at
this point because data on the Zn and Cu contents of conventional Korean foods are not available. EC SOD activities were lower in smokers than in non-smokers. That these reductions in EC SOD activities were functionally important is suggested by our findings of lower serum vitamin C concentrations in smokers versus non-smokers in the teenage (at 0 and 12 mo) and adult subjects. These results suggest that shortterm smoking can induce oxidative stress similarly to long-term smoking. Some previous findings also show that erythrocyte CuZnSOD activities are lower in smokers than in non-smokers for adults9 and teenagers.26 However, serum Cp activities and serum lipid peroxidation, as measured by the production of TBARS, were not increased by smoking in teenage and adult subjects. These results support the concept that the overall health of the subjects in the current study may have been better than in previous reported studies.2 Clinical studies on smokers have indicated adverse effects of smoking on vitamin C metabolism, and several investigators have reported that serum vitamin C concentrations are lower in cigarette smokers than in non-smokers for subjects ages 17 to 50 y.27 Lower serum vitamin C concentrations in smokers may be due to several factors including decreased vitamin C consumption, impaired vitamin C absorption, or an increased turnover of vitamin C.1 Similar to our previous findings,26 intakes of vitamin C in the teenage group were lower in smokers than in non-smokers in the current study. This altered vitamin C metabolism also was found in adult smokers of the current study, although their diet was not surveyed. Similar to the findings for vitamin C, serum folate concentrations were lower in smokers than in non-smokers for teenage and adult
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TABLE VI. CONCENTRATIONS OF SERUM ZINC AND COPPER Teenagers
Variables Zinc (M/L) Period A Period B Period C Period D Copper (M/L) Period A Period B Period C Period D
Adults
Smokers (n ⫽ 35)
Non-smokers (n ⫽ 35)
14.7 ⫾ 0.3a 11.9 ⫾ 0.2b 12.8 ⫾ 0.4b 12.8 ⫾ 0.2b
14.0 ⫾ 0.3a 10.3 ⫾ 0.3b 12.5 ⫾ 0.3c 10.6 ⫾ 0.2b
13.1 ⫾ 0.3 12.6 ⫾ 0.4 13.2 ⫾ 0.4 13.5 ⫾ 0.4
12.8 ⫾ 0.4a 11.7 ⫾ 0.4b 13.1 ⫾ 0.3a 12.6 ⫾ 0.3ab
t Test between groups
P ⬍ 0.001
Smokers (n ⫽ 20)
Non-smokers (n ⫽ 20)
13.9 ⫾ 0.7
14.1 ⫾ 0.7
14.1 ⫾ 0.4
12.4 ⫾ 0.3
t Test between groups
P ⬍ 0.001 P ⬍ 0.01
* Mean ⫾ standard error of the mean. Means with different superscript letters in a column differ significantly by Fisher’s protected least significant difference test at P ⬍ 0.05. † P ⬍ 0.01. ‡ P ⬍ 0.001. Period A, first sampling of teenagers at 0 mo; Period B, second sampling of teenagers at 6 mo; Period C, third sampling of teenagers at 12 mo; Period D, fourth sampling of teenagers at 18 mo
subjects of the current study. Previous studies also have shown that smokers have lower circulating folate concentrations than nonsmokers.11 The lower serum folate concentrations observed in teenage and adult smokers may be related to a lower intake of fruits and vegetables by the smokers and to an increased turnover of folate secondary to oxidative stress.11,28 In the current study, we could not assess the folate intake of subjects because the folate contents of conventional Korean foods have not been reported. Inadequate folate nutriture among women of childbearing age has been recognized as a risk factor for the birth defects known as neutral tube defects, which can occur at the beginning of the pregnancy.29 Overall parameters on physical growth, smoking habits, and blood antioxidant status of teenage subjects did not change consistently through the study; these results may be related to seasonal effect, change of dietary nutrient intakes, and/or alterations in smoking habits over the study period.
Collectively, these findings suggest that cigarette smoking has a negative effect on numerous arms of the oxidant defense system for teenage and adult subjects based on higher blood pressures, lower EC SOD activities, and lower serum vitamin C and folate concentrations in smokers than in non-smokers, although alteration of mineral metabolism by smoking was not observed, especially in teenage subjects. Lower antioxidant status and inadequate folate status observed in the current teenage smokers parallel our previous findings for teenage smokers smoking more heavily and longer than the current smokers (16.0 ⫾ 1.1 cigarettes a day, 10 –25 cigarettes a day, for 2.9 ⫾ 0.2 y, 1.5– 4.1 y).26 The increased oxidative stress that can be associated with direct effects of oxidants in cigarette smoke and consequences of lower antioxidant status associated with smoking may represent risk factors for the development of chronic diseases in teenage and adult smokers. The low concentrations of serum folate in female teenage smokers
TABLE VII. CONCENTRATIONS OF URINE ZINC AND COPPER* Teenagers
Variables Zinc (M/mM creatinine) Period A Period D Copper (M/mM creatinine) Period A Period D
Smokers (n ⫽ 35)
Adults
Non-smokers (n ⫽ 35)
t Test between groups
1.112 ⫾ 0.219 0.789 ⫾ 0.053
0.797 ⫾ 0.148‡ 0.537 ⫾ 0.049
P ⬍ 0.001
0.010 ⫾ 0.006†‡ 0.021 ⫾ 0.001
0.007 ⫾ 0.003 0.018 ⫾ 0.001
* Mean ⫾ standard error of the mean. † Difference between two periods in each group. ‡ P ⬍ 0.05. Period A, first sampling of teenagers at 0 mo; Period D, fourth sampling of teenagers at 18 mo.
Smokers (n ⫽ 20)
Non-smokers (n ⫽ 20)
0.613 ⫾ 0.101
0.517 ⫾ 0.066
0.009 ⫾ 0.001
0.011 ⫾ 0.001
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Kim et al.
suggest that these girls may be at increased risk for several potential health complications. In conclusion, the current results emphasize that oxidative stress can be induced in short-term smokers, similarly to long-term adult smokers and teenagers who smoke heavily.26
ACKNOWLEDGMENTS The authors thank Joel Commisso and Greenie Hu at the University of California at Davis in the United States for excellent technical assistance. They appreciate the skillful assistance of Young Sang Park and In Sook Jeong at Kongju Medical Center in Korea for collecting blood and urine samples from the subjects, and they thank Principal Moon Ha Lee and the many teachers, including Min Hyung Lee, Tak Yeon Won, Jong Suk Jeong, Jun Ho Kim, Chul Ryu, and One Joo Lee, at Korean senior high schools that teenage subjects attended for recruiting and managing the subjects of the current study throughout the 18-mo period. They also appreciate the patient and responsible help of the many teenage and adult subjects.
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Nutrition Volume 20, Number 5, 2004 11. Walmsley CM, Bates CJ, Prentice A, Cole TJ. Relationship between cigarette smoking and nutrient intakes and blood status indices of older people living in the UK: further analysis of data from the National Diet and Nutrition Survey of people aged 65 years and over, 1994/95. Public Health Nutr 1999;2:199 12. Keen CL, Clegg MS, Ferrell F, Hunter GC, Dubick MA. Hypertension induced alterations in copper and zinc metabolism: a link to vascular disease?. In: Sorenson JRJ, ed. Biology of copper complexes. Clifton, NJ: Humana Press, 1987:141 13. Dubick MA, Keen CL. Influence of nicotine on tissue trace element concentrations and tissue antioxidant defense. Biol Trace Elem Res 1991;31:97 14. Kocyigit A, Erel O, Gur S. Effects of tobacco smoking on plasma selenium, zinc, copper and iron concentrations and related antioxidative enzyme activities. Clin Biochem 2001;34:629 15. Dietrich M, Block G, Norkus EP, et al. Smoking and exposure to environmental tobacco smoke decrease some plasma antioxidants and increase ␥-tocopherol in vivo after adjustment for dietary antioxidant intakes. Am J Clin Nutr 2003;77:160 16. Suh I, Jee SH, Kim SY, et al. The changing pattern of cigarette smoking of students in junior and senior high schools in Korea: 1988 –1997. Korean J Epidemiol 1998;20:257 17. Gibson RS, Heath AL, Ferguson EL. Risk of suboptimal iron and zinc nutriture among adolescent girls in Australia and New Zealand: causes, consequences, and solutions. Asia Pac J Clin Nutr 2002;11:S543 18. Voncken P, Schepers G, Schafer KH. Capillary gas chromatographic determination of trans-3⬘-hydroxy-cotinine simultaneously with nicotine and cotinine in urine and blood samples. J Chromatogr 1989;479:410 19. Paik HY, Joung HY, Lee JY, et al. Serum extracellular superoxide dismutase activity as an indicator of zinc status in humans. Biol Trace Elem Res 1999;69:45 20. Schosinsky KH, Lehmann HP, Beeler MF. Measurement of ceruloplasmin from its oxidase activity in serum by use of -dianisidine dihydrochloride. Clin Chem 1974;20:1556 21. Yagi K. Assay for blood plasma or serum. Methods Enzymol 1984;105:328 22. Kenneth KP, Trevithick JR. High performance liquid chromatographyelectrochemical detection of antioxidants in vertebrate lens: glutathione, tocopherol and ascorbate. Methods Enzymol 1994;233:523 23. Recommended dietary allowances for Koreans, 7th ed. Seoul: Korean Nutrition Society, 2000 24. Dowdell EB. Urban seventh graders and smoking: a health risk behavior assessment. Issues Compr Pediatr Nurs 2002;25:217 25. Lim WK, Kim SH. Cigarette smoking habits among teenage girls living in a rural community in Korea. Korean J Nutr 2000;33:755 26. Kim SH, Kim JS, Shin HS, Keen CL. Influence of smoking on markers of oxidative stress and serum mineral concentrations in teenage girls in Korea. Nutrition 2003;19:240 27. Wei W, Kim Y, Boudreau N. Association of smoking with serum and dietary levels of antioxidants in adults: NHANES III, 1988 –1994. Am J Public Health 2001;91:258 28. Mansoor MA, Kristensen O, Hervig T, et al. Low concentrations of folate in serum and erythrocytes of smokers: methionine loading decreases folate concentrations in serum of smokers and nonsmokers. Clin Chem 1997;43:2192 29. Refsum H. Folate, vitamin B12, and homocysteine in relation to birth defects and pregnancy outcome. Br J Nutr 2001;85:S109