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. Author manuscript; available in PMC: 2009 Feb 1.
Published in final edited form as: Environ Res. 2007 Nov 9;106(2):219–225. doi: 10.1016/j.envres.2007.09.008

No effect of cigarette smoking dose on oxidized plasma proteins

Chih-Ching Yeh a,b, R Graham Barr c,d, Charles A Powell c, Sonia Mesia-Vela c, Yuanjia Wang e, Nada K Hamade b, John HM Austin f, Regina M Santella b,*
PMCID: PMC2268894  NIHMSID: NIHMS40706  PMID: 17996865

Abstract

Cigarette smoking is a major source of oxidative stress. Protein carbonyls have been used as a biomarker of oxidative stress because of the relative stability of carbonylated proteins and the high protein concentration in blood. Increased levels of carbonyl groups have been found in serum proteins of smokers compared to nonsmokers. However, neither the dose effect of current cigarette smoke nor other predictors of oxidative stress have been studied. Hence, we used an ELISA (Enzyme-Linked Immunosorbent Assay) to evaluate plasma protein carbonyls in smokers recruited in the Early Lung Cancer Action Project (ELCAP) program. The lung cancer screening program enrolled current and former smokers age 60 years and over without a prior cancer diagnosis. A total of 542 participants (282 men and 260 women) completed a baseline questionnaire and provided blood samples for the biomarker study. Protein oxidation was measured by derivatization of the carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH) and ELISA quantitation of the DNPH group. Current smoking status was confirmed with urinary cotinine. The mean (± SD) protein carbonyl level was 17.9 ± 2.9 nmol carbonyls/ml plasma. Protein carbonyls did not differ significantly by gender. Carbonyl levels were higher among current than former smokers, but these differences did not attain statistical significance, nor did differences by urine cotinine levels, pack-years, pack/day among current smokers, and smoking duration. In a multiple regression analysis, higher protein carbonyl levels were independently associated with increasing age (0.59 nmol/ml increase per 10 years, 95% CI 0.14, 1.05, p = 0.01), African-American vs. white race/ethnicity, (1.30 nmol/ml, 95% CI 0.4, 2.19, p =0.008), and lower educational attainment (0.75 nmol/ml, 95% CI 0.12, 1.38, p = 0.02). Although we found no significant difference between current versus past cigarette smoking and protein carbonyls in this older group of smokers, associations were found for age, ethnicity and educational attainment. Our results indicate that the measurement of plasma carbonyls by this ELISA technique is still an easy and suitable method for studies of diseases related to oxidative stress.

Keywords: Oxidative stress, Cigarette smoking, Protein carbonyls, Biomarker, ELCAP

1. Introduction

Cigarette smoke contains over 4000 different chemicals, 400 of which are proven carcinogens. Cigarette smoke also contains various oxidants such as oxygen free radicals (Pryor et al., 1983) and volatile aldehydes (O’Neill et al., 1994), which are probably the major causes of damage to biomolecules. Epidemiological studies have identified and randomized clinical trials have confirmed that cigarette smoking is an important risk factor for cardiovascular disease, chronic obstructive pulmonary disease, as well as for lung cancer and other malignancies (Shah & Helfant, 1988; Frank, 1993; Bartecchi et al., 1994). Oxidative stress is prominent among the hazardous effects of cigarette smoke and entails lipid peroxidation (Frei et al., 1991), protein oxidation (Reznick et al., 1992), and DNA damage (Kiyosawa et al., 1990). Increased level of 8-hydroxyguanine (8-OH-dG), a biomarker of oxidative DNA damage, in human leukocytes, lung tissues, and urine, have been observed in smokers compared to nonsmokers (Kiyosawa et al., 1990; Loft et al., 1992; Asami et al., 1996; Asami et al., 1997).

Proteins are major targets for reactive oxidants in cells and oxidized proteins that accumulate during aging, oxidative stress and in some pathological conditions (Berlett & Stadtman, 1997; Stadtman & Berlett, 1997). Exposure of proteins to reactive oxygen species results in modification of amino acid residues, which alters protein structure and function (Berlett & Stadtman, 1997). Previous studies have demonstrated that in vitro exposure of plasma to gas-phase cigarette smoke leads to the rapid accumulation of plasma protein carbonyls (Reznick et al., 1992). Increased levels of protein carbonyls have also been found in globin and serum proteins of smokers compared to nonsmokers (Lee et al., 1998; Marangon et al., 1999; Pignatelli et al., 2001).

Carbonyl groups in proteins, determined as DNPH (2,4-dinitrophenylhydrazine) derivatives, have been analyzed as a biomarker of oxidative damage of proteins (Berlett & Stadtman, 1997). In addition, a sensitive ELISA (Enzyme-Linked Immunosorbent Assay) method has been developed for measurement of protein carbonyls (Buss et al., 1997). This biomarker may be more useful than others for larger studies since proteins are present in higher concentrations in blood than is DNA, and only small amounts of blood (<50 μl) are required for analysis.

Although increased levels of protein carbonyls are found among smokers compared to nonsmokers, the dose effect of cigarette smoke on oxidative stress has not been studied. We hypothesized that smokers who currently use more cigarettes would have greater oxidative stress and have higher levels of plasma protein carbonyls, as a biomarker for oxidative stress. Hence, we used the ELISA method to evaluate plasma protein carbonyls in subjects who were recruited into a lung cancer screening cohort.

2. Materials and methods

2.1. Study population

Participants were recruits at the Columbia University site of the New York Early Lung Cancer Action Project (ELCAP)(Henschke et al., 2006). This lung cancer screening program enrolled current and former smokers with 10 or more pack-years, age 60 and over, without a prior cancer diagnosis, who were willing to undergo screening for lung cancer with low-dose chest computed tomography (CT). The protocol specified baseline and one-year follow-up examinations, which each comprised of a low-dose CT, an interviewer-administered questionnaire, and a smoking intervention for current smokers. The Columbia University site recruited ELCAP participants into a study on biomarkers and emphysema, the EMphysema and Cancer Action Project (EMCAP). These participants completed additional questionnaires on smoking history and spirometry. Blood and urine samples were also collected in 2001-2 and stored at -80°C until analysis. Participants at the Columbia site were specifically consented for biomarker and genetic analyses, and the Columbia University Institutional Review Board approved all study activities.

Overall, 557 current and former smokers were enrolled at Columbia University. Of these, 557 (100%) completed the baseline questionnaire and CT scan, and 542 (97%) provided biological specimens.

2.2. Protein carbonyl measurement

The levels of plasma protein carbonyl groups were assessed using a noncompetitive ELISA (Buss et al., 1997), with minor modifications (Marangon et al., 1999). After determination of protein concentration (BCA-1 Protein Assay Kit, Sigma, St. Louis, MO), plasma samples were adjusted to a protein concentration of 4 mg/ml. Samples were derivatized with 2,4-dinitrophenylhydrazine (Sigma) and adsorbed to Maxisorb 96 well plates (Nunc, Life Technologies, Eggenstein, Germany). After blocking with 0.1% Tween 20, protein carbonyls were detected using a polyclonal rabbit anti-dinitrophenyl antibody (Molecular Probes Inc., 1:1500 for 1 h at 37°C) and horseradish peroxidase-conjugated secondary antibody (Amersham International, 1:4000 for 1 h at 37°C). Immunoreactivity was determined by measuring the conversion of 3,3′,5,5′-tetramethylbenzidine (TMB)(Sigma) at 450 nm after termination of the reaction with sulfuric acid. Hypochlorous acid-oxidized bovine serum albumin was used as external standard (Buss et al., 1997). Results were expressed as nmol carbonyl/ml plasma. The assay for the total carbonyl content had an interbatch coefficient of variation of 6.8% (n=16).

Cotinine levels were measured with kits from Orasure Technologies, Inc, Bethlehem, PA, as directed by the manufacturer. Smoking status was defined as current if the urinary cotinine level was ≥500 ng/mL or if the participant self-reported current smoking. Smoking status was defined as past if the participant denied current smoking and the urinary cotinine level was < 500 ng/mL. The urinary creatinine was assayed using a Creatinine kit from Sigma (St. Louis, MO; Cat. No. 555-A).

2.3. Statistical Analysis

The data on general characteristics or protein carbonyl levels were expressed as mean ± SD. The differences between strata for the general characteristics were examined by Student t-test and one-way analysis of variance (ANOVA) for two or more than two strata, respectively. We conducted trend tests to evaluate the dose effect of these characteristics on the level of protein carbonyls. Significant variables identified from the univariate analyses were included as covariates in multiple regression models. All analyses were performed using the SAS statistical package (version 8.2 for windows; SAS Institute, Inc., Cary, NC) and p<0.05 was used as the criterion for statistical significance.

3. Results

3.1. General characteristics of participants in the EMCAP Study

The general characteristics of the participants in the EMCAP study are summarized in table 1. Subjects were nearly equally divided by gender (mean age, 67 years). Among the participants, approximately 74% were Caucasian and half attained college or advanced degrees. The mean cumulative cigarette smoke exposure was 49 pack-years, a mean years smoked of 39 years and a mean daily cigarette use of 1.1 packs among current smokers. The median urinary cotinine was much higher in current smokers than in former smokers (1772 vs. 23 ng/mL). Participants had a mean body mass index of 27.

Table 1.

General characteristics of participants in the EM CAP Study at baseline

Characteristic N=541 % Mean(SD)
Sex
Male 281 52
    Female 260 48
Mean age 541 67 (5)
Ethnicity
    White 398 74
    Hispanic 49 9
    Black 42 8
    Asian/Pacific 52 10
Educational Attainment
    High school degree 250 46
    College degree 148 27
    Graduate degree 143 26
Smoking History
    Former 317 59
    Current 224 41
Mean pack-years 541 49 (26)
Mean years smoked 541 39 (12)
Mean packs/day among current smokers 541 1.1 (0.6)
Median urine cotinine (ng/ml)(IQR)
    Former smokers 317 23 (45)
    Current smokers 224 1772(1048)
Mean BMI 541 27 (5)
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3.2. Level of oxidized plasma proteins stratified by general characteristics

Among all participants, the mean (± SD) protein carbonyl level was 17.9 ± 2.9 nmol carbonyl/ml plasma. The levels of protein carbonyl were significant higher among participants who were older, black, and obese and significantly lower among those with advanced degrees (Table 2). We also found that females had marginally higher carbonyl content in their plasma than males (p=0.09). Although carbonyl levels were higher among current than former smokers, these differences did not attain statistical significance, nor did observed differences by pack-years, packs per day among current smokers, and smoking duration. Smoking status measured by cotinine levels did not alter the results. The middle quintile of cotinine/creatinine ratio was significantly elevated, although no linear trend across quintiles of cotinine/creatinine ratio was observed. A U-shaped relationship with BMI was observed, and obese subjects had significantly higher levels of protein carbonyl compared to normal-weight subjects.

Table 2.

Level of oxidized plasma proteins stratified by general characteristics

Characteristic n nmol carbonyIs/ml plasma (mean ± SD) Age-adjusted beta (95%CI) p-value
Sex
   Male 281 17.7 ± 2.8 Ref
   Female 260 18.1 ± 3.0 0.417 (-0.071, 0.905) 0.094
Age range (years)
   59, <65 245 17.7 ± 2.9 Ref
   65, <70 147 17.8 ± 3.1 0.065 (-0.529, 0.658) 0.83
   >70 149 18.3 ± 2.7 0.623 (0.032, 1.215) 0.039
   p for trend 0.012
Ethnicity
   White 398 17.8 ± 2.9 Ref
   Hispanic 49 17.3 ± 2.9 -0.428 (-1.279, 0.423) 0.32
   Black 42 19.5 ± 2.8 1.645 (0.731,2.558) 0.0004
   Asian 52 17.9 ± 2.8 0.061 (-0.770, 0.891) 0.89
Educational Attain
   High School 250 18.1 ± 2.7 Ref
   College Degree 148 18.0 ± 3.1 -0.167 (-0.753, 0.419) 0.58
   Graduate Degree 143 17.4 ± 3.0 -0.759 (-1.352, -0.166) 0.012
   p for trend 0.076
Smoking Status
   Former 317 17.9 ± 2.9 Ref
   Current 224 17.9± 2.9 0.155 (-0.345, 0.655) 0.54
Pack-years
   <30 132 18.0 ± 2.9 Ref
   >30 <50 185 17.7 ± 2.9 -0.209 (-0.858, 0.441) 0.53
   >50 224 18.0 ± 2.9 0.014 (-0.610, 0.638) 0.97
   p for trend 0.99
Years smoked (years)
   <30 113 17.7 ± 2.7 Ref
   30, <45 221 17.9 ± 2.8 0.374 (-0.288, 1.037) 0.27
   >45 207 17.9 ± 3.1 0.181 (-0.486, 0.849) 0.59
   p for trend 0.39
Current packs per day
   0 317 17.8 ± 2.9 Ref
   <1 59 18.3 ± 2.6 0.492 (-0.313, 1.30) 0.23
   1, <1.5 94 18.1 ± 3.1 0.313 (-0.356, 0.982) 0.36
   >1.5 62 17.6 ± 2.8 -0.148 (-0.943, 0.647) 0.72
   p for trend 0.88
Urine cotinine level (ng/mL)
   <100 289 17.8 ± 2.9 Ref
   100, <500 41 18.6 ± 3.6 0.762 (-0.188, 1.711) 0.116
   ≥500 201 17.9± 2.8 0.142 (-0.384, 0.667) 0.597
   p for trend 0.82
Cotinine/creatinine ratio
   <14 107 17.4 ± 2.8 Ref
   >14, <40 101 17.7 ± 2.8 0.257 (-0.527, 1.041) 0.520
   >40, <300 108 18.6± 3.0 1.170 (0.400, 1.938) 0.003
   >300, <1500 103 18.2± 3.3 0.817 (0.038, 1.596) 0.040
   >1500 111 17.5± 2.6 0.170 (-0.595, 0.934) 0.663
   p for trend 0.56
BMI (kg/m2)
   <20 32 18.3 ± 3.7 0.678 (-0.405, 1.761) 0.22
   20, <25 192 17.6 ± 2.8 Ref
   25-30 196 17.9 ± 2.9 0.338 (-0.239, 0.916) 0.25
   ≥30 121 18.2 ± 2.8 0.717 (0.053, 1.381) 0.034
   p for trend 0.087
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3.3. Multivariate regression coefficients and their standard errors for oxidized protein

Multiple regression analysis showed no association between current and former cigarette smoking and protein carbonyl levels was found after adjustment for covariates (Table 3). However, higher protein carbonyl levels were independently associated with increasing age (0.59 nmol/ml increase per 10 years, 95% CI 0.14, 1.05, p=0.01), African-American vs. white race/ethnicity, (1.30 nmol/ml, 95% CI 0.4, 2.19, p=0.008), lower educational attainment (0.75 nmol/ml, 95% CI 0.12, 1.38, p=0.02) and middle quintile of cotinine/creatinine ratio (1.17 nmol/ml, 95% CI 0.40, 1.95, p=0.003). Differences in protein carbonyl levels by BMI were not statistically significant after adjustment for covariates.

Table 3.

Multivariate regression coefficients (in nmole carbonyls/ml plasma) and their standard errors (S.E.) for oxidized protein*

Characteristic β** S.E. p-value
Intercept 13.290 1.635 <0.0001
Age (per 10-year increase) 0.59 0.23 0.010
Sex
  Female vs. Male 0.260 0.266 0.328
Ethnicity
  Hispanic vs. White -0.583 0.456 0.217
  Black vs. White 1.299 0.491 0.008
  Asian vs. White 0.241 0.460 0.600
Educational Attainment
  College vs High School Degree -0.028 0.312 0.929
  Graduate vs High School Degree -0.751 0.321 0.020
  p-trend for education 0.089
Smoking status (current vs former) 0.730 0.578 0.207
Years smoked (per 10 years) 0.052 0.11 0.637
Self-reported packs per day -0.0971 0.196 0.621
Urine cotinine level -0.00008 0.0001 0.488
Packyears -0.001 0.005 0.767
Cotinine Creatinine Ratio (CCR)
  14<CCR<40 vs 0<CCR<14 0.241 0.399 0.546
  40<CCR<300 vs 0<CCR<14 1.174 0.395 0.003
  300<CCR<1500 vs 0<CCR<14 -0.005 0.631 0.994
  1500<CCR vs O<CCR<14 -0.581 0.672 0.388
BMI (kg/m2)
  0<BMI<20 vs 20≤BMI<25 0.312 0.561 0.578
  25≤BMI<30 vs 20≤BMI<25 0.257 0.297 0.388
  30≤BMI vs 20≤BMI<25 0.557 0.3456 0.108
  p-trend for BMI 0.155
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*

Adjusted for variables in the table.

**

β is change in plasma carbonyl (nmole/ml plasma) per unit change in characteristic.

4. Discussion

Although we found no association between cotinine-confirmed current and former cigarette smoking status and protein carbonyls in this older group of long-term smokers and exsmokers, associations were found for age, ethnicity and educational attainment. The results obtained indicate that the measurement of plasma carbonyls by this ELISA technique is still relatively easy and suitable for large studies of oxidative stress-related disease.

Previous studies have found that smokers have higher contents of protein carbonyls in globin and serum proteins than nonsmokers (Lee et al., 1998; Marangon et al., 1999; Pignatelli et al., 2001). Using the colorimetric assay, Lee et al. examined the chemopreventive effect of antioxidants on cigarette smoke-induced oxidative stress among 15 smokers and 5 nonsmokers (Lee et al., 1998). They found a significantly elevated carbonyl level in globin from smokers compared to nonsmokers (2.56 vs. 1.59 nmol/mg protein, p<0.01). A study using the ELISA method showed that smokers have higher plasma protein carbonyls than individually matched nonsmokers (N=22) (Marangon et al., 1999). The levels of oxidized serum proteins measured by western-blot assay were also significantly associated with smoking status, whereas no difference was seen between heavy and light smokers (Pignatelli et al., 2001). The protein carbonyl level among the older and heavier smokers in the present study (17.9 nmol carbonyl/ml plasma) was comparable to our breast cancer study (28.3% controls were age > 65 years) using the same ELISA method (under revision). Although no significant association was seen between protein carbonyl levels and cigarette smoking status, we did find smokers have higher carbonyl levels than nonsmokers (protein carbonyls among health controls were 16.6, 16.2 and 15.7 nmol carbonyl/ml plasma for current, former and never smokers, respectively). Because the participants enrolled in our study were current and former smokers, we could not explore the difference between smokers and nonsmokers. Nevertheless, our findings are in agreement with the latest report and our breast cancer study that found no significant dose effect of smoking on protein carbonyls (Pignatelli et al., 2001).

We found the protein carbonyl levels were significantly higher among those who were elderly, African-American, less well educated and obese. Oxidative modifications of intracellular proteins that accrue during aging have been suggested to play a key role in the causation of senescence-associated losses in physiological functions (Stadtman, 1992; Berlett & Stadtman, 1997; Stadtman & Berlett, 1997; Stadtman, 2001). Addition of carbonyl-containing adducts to the side chains of amino acid residues is the most well characterized, age-associated, post-translational structural alteration in proteins (Stadtman, 1992; Berlett & Stadtman, 1997; Stadtman & Berlett, 1997; Stadtman, 2001). The results in the present study of a positive association between plasma protein carbonyl level and increasing age supports the results cited in these review articles.

Oxidative stress is different among ethnic groups. Numerous studies have reported that biomarkers for oxidative stress, including plasma F2-isoprostanes (Lopes et al., 2003), serum C-reactive protein (Lee & Jacobs, 2005) and coenzyme Q10 (Miles et al., 2003) were greater in African Americans than in whites. In addition, the prevalence of diseases related to oxidative stress-related endothelial dysfunction, such as hypertension and diabetes mellitus, is considerably greater in blacks than whites (Kalinowski et al., 2004). Whether these differences are due to social or biological factor is not clear. We also found that educational attainment, as an inexact measure of socioeconomic status (SES), was significantly related to plasma protein carbonyl level. Our results suggest that SES may be an explanation for the observed differences by race/ethnicity and educational level.

Protein carbonyl level was significantly elevated in subjects in the middle quintile of the cotinine/creatinine ratio, although no dose response trend across quintiles was observed. Without a plausible biologic mechanism, this result may be due to chance. We also observed that plasma protein carbonyl concentrations were elevated among obese subjects, although the differences were not statistically significant in multivariate analysis. Oxidative stress is considered to be one of the main causes of molecular damage to cellular and tissue structures and is known to be increased in patients with diabetes mellitus (Baynes, 1991; Giugliano et al., 1996). Studies have also provided evidence that obesity may be associated with defective antioxidant status and enhanced lipid peroxidation (Davi et al., 2002; Ozata et al., 2002). Because carbonyl content is a good biomarker for oxidative stress, our finding is reasonable. A recent study has reported an increase in reactive oxygen species-induced damage in lipids, proteins, and amino acids in the obese compared with normal subjects (Dandona et al., 2001). They also found that plasma protein carbonyl levels were reduced after 4 weeks of caloric restriction.

Increased oxidative stress in smokers has been shown by measuring various biomarkers, including lipid peroxidation products in plasma (Reilly et al., 1996; Miller et al., 1997), oxidized DNA bases in leukocyte DNA and urine (Kiyosawa et al., 1990; Asami et al., 1996; van Zeeland et al., 1999) and F2-isoprostanes in plasma and urine (Morrow et al., 1995; Reilly et al., 1996). It would be worthwhile to further study the correlation between these biomarkers and levels of oxidized proteins in relation to smoking habits.

This study has a number of strengths, including relatively large size, precise measurement of protein carbonyls, and confirmation of current smoking status by urinary cotinine. Limitations included lack of a nonsmoking control group. We therefore could not determine if protein carbonyls were higher among ever smokers than never smokers, although the mean values in both current and former smokers suggest this to be the case compared to our breast cancer study and previously published studies of never smokers (Pignatelli et al., 2001). The cross-sectional design meant that we could not rule out reverse causality, and it is possible that smokers who were particularly susceptible to smoking and had elevated levels of protein carbonyls quit smoking before enrollment in the study. The health status and nutritional information of the participants were not assessed, which are likely to bias our results. Elevated protein oxidation have been associated with neurological and inflammatory disease, and some antioxidants may decrease oxidative stress (Mayne, 2003). However, we found no association of protein carbonyl with chronic obstructive pulmonary disease (COPD) and the adjustment for information on antioxidant supplementation use for a subgroup (n=134) of our participants also did not alter results (data not show). Finally, protein carbonyls were measured on specimens stored for several years at -80°C; it is not certain whether protein oxidized during storage. However, one study measuring protein carbonyl in saliva showed that neither fresh or frozen conditions altered the carbonyl values (Nagler et al., 2000).

Although proteins are major targets for oxidative damage in vivo (Berlett & Stadtman, 1997; Davies et al., 1999), the modified proteins in human plasma have not been extensively measured as possible biomarkers of oxidative stress in relation to human nutrition, disease status, and life-style. The ELISA method described in this study is sensitive and specific for oxidized proteins (Dalle-Donne et al., 2003). In addition, this marker may be more useful than others for larger studies since proteins are present in higher concentrations in blood than is DNA, small amounts of plasma (<20 μl) are required for analysis, and oxidized proteins are relatively stable, allowing for its more sensitive detection. Studies to investigate the effects of antioxidants and interaction between cigarette smoking and protein carbonyls on disease risk are warranted.

Acknowledgments

This study was supported by grants ES09089, CA013696 and HL075476 from the National Institutes of Health.

Footnotes

Participants at the Columbia site were consented for biomarker and genetic analyses, and the Columbia University Institutional Review Board approved all study activities.

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