- Email: [email protected]
Effects of Environmental Tobacco Smoke on Serum Levels of Acute Phase Proteins in Schoolchildren
PREVENTIVE MEDICINE ARTICLE NO.
25, 617–624 (1996)
0097
Effects of Environmental Tobacco Smoke on Serum Levels of Acute Phase Proteins in Schoolchildren MASAYUKI SHIMA, M.D.,
AND
MOTOAKI ADACHI, M.D.
Department of Public Health, Chiba University School of Medicine, Chiba 260, Japan
Background. Few biological changes due to longterm exposure to environmental tobacco smoke (ETS) have been identified. This study examined the relationship between exposure to ETS and serum levels of acute phase proteins among children. Methods. The concentrations of the third component of complement (C3c), haptoglobin (Hp), a1 acid glycoprotein (a1AG), and ceruloplasmin (Cp) were determined in serum samples obtained from 480 schoolchildren. Exposure to ETS was assessed from responses to questionnaires. Results. Serum levels of C3c, Hp, and a1AG were significantly higher among boys living in households in which there were smokers other than their parents than among boys with no smokers in their family or those in households where only the father smoked. Levels of C3c, Hp, and a1AG in relation to the level of ETS exposure were significantly increased among boys exposed to ETS of 11 or more cigarettes per day. Among girls, increased a1AG levels were associated with exposure to high levels of ETS, but no differences were found in any of the other proteins. Cp levels were not affected by exposure to ETS among either boys or girls. Conclusions. These findings suggest that the serum levels of C3c, Hp, and a1AG among schoolchildren may reflect different levels of exposure to ETS. Boys seemed to be more susceptible to ETS exposure than girls. q 1996 Academic Press, Inc. Key Words: environmental tobacco smoke; acute phase proteins; the third component of complement; haptoglobin; a1 acid glycoprotein; ceruloplasmin.
nary function,3 and bronchial hyperresponsiveness4 have been reported. Urinary cotinine levels have often been used as an indicator of exposure to ETS.5,6 Recent findings have shown a relationship between emergency room admissions for asthma and urinary levels of cotinine.7 In ETS, nicotine is present in the gaseous phase and, on inhalation, is immediately metabolized to cotinine.5 Therefore, urinary cotinine only serves as an index of short-term brief exposure to ETS, and does not appear to reflect constant states of exposure.6 At present, no biological marker has yet been identified which could be used to determine the effect of long-term exposure to ETS on human health. Acute phase proteins (APP) are a group of serum protein components that increase in response to the presence of foreign substances invading the body.8 APP such as haptoglobin (Hp) and ceruloplasmin (Cp) are reported to be increased among smokers,9,10 but the effects of exposure to ETS on serum protein components were not considered in these studies. To evaluate the effects of various environmental factors on respiratory health, we have been conducting a series of epidemiologic surveys among schoolchildren from various areas of Japan.11 In the present study, we measured concentrations of serum APP, the third component of complement (C3c), Hp, a1 acid glycoprotein (a1AG), and Cp, among schoolchildren living in an area of relatively low air pollution, to examine the relationship between serum protein levels and exposure to ETS in the households. METHODS
INTRODUCTION
The effects of exposure to environmental tobacco smoke (ETS) on the respiratory system in children have been extensively investigated.1 Increased respiratory symptoms of bronchial asthma,2 lower levels of pulmoAddress reprint requests to Masayuki Shima, Department of Public Health, Chiba University School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260, Japan. Fax: /81 43-226-2070; E-mail: [email protected].
Subjects The study subjects were 654 first- to third-grade children attending two elementary schools (ages 6–9 years) in Chiba Prefecture, Japan. The two schools were located on the coast of Tokyo Bay, in adjacent school districts. The annual average concentrations of sulfur dioxide, nitrogen dioxide, and suspended particulate matter in 1992, measured at a monitoring station close to these schools, were 6 ppb, 18 ppb, and 37 mg/
617
/ a203$$2163
08-21-96 19:30:51
pma
0091-7435/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
AP: PM
618
SHIMA AND ADACHI
m3, respectively, below Japanese ambient air quality standards. In November 1992, a standard respiratory symptom questionnaire, a modified Japanese version of ATSDLD-78-C,12 was sent to all subjects. The questionnaire was filled out by either parent or guardians. Incomplete questionnaires were returned with a request to complete them.13 Altogether, 651 (99.5%) completed questionnaires were returned. The questionnaire consisted of questions about the respiratory symptoms and medical history of the child, the smoking habits of the household members, and certain characteristics of the home, such as the type of heating. If any members of the household were smokers, the respondent was asked to note the number of cigarettes that each smoked per day in the presence of the child. According to the responses to the questionnaire, children who had experienced two or more episodes of wheezing accompanied by dyspnea, or who had been under medical treatment for bronchial asthma during the past 2 years, were considered to have asthma or wheezing symptoms. Those who had been diagnosed as having eczema, atopy, allergic rhinitis, or pollinosis by a physician, or who had received hyposensitization therapy, were considered to have a history of allergic diseases. Blood Collection and Laboratory Measurements In January 1993, venous blood samples were collected from 483 children (73.9%) whose parents or guardians had given their written consent. Children with acute infections or symptoms, such as cold or fever, the day before and the day of examination were excluded (n Å 35). Consent for venipuncture was not given by the remaining parents (n Å 136). All venipuncture was performed between 9 AM and 2 PM, and the blood specimens were centrifuged on the same day. All serum samples were kept frozen at 0807C until analysis. C3c, Hp, a1AG, and Cp were determined by a nephelometric method14 using a Behring nephelometer analyzer (Behringwerke AG, Marburg, Germany). Data Analysis In 1 of the 483 children, complete data on exposure to ETS were not available. Also, the volume of blood collected from two subjects was insufficient for measurement of all the proteins. Thus, the final sample for analysis included 480 children (school A, 161 children; school B, 319). The serum levels of each protein were first compared in relation to gender, school grade, familial smoking habits, asthma or wheezing symptoms, allergic diseases, feeding method in infancy, and heating type in the home. Then, the mean values adjusted for all variables were calculated separately by sex, using the gen-
/ a203$$2163
08-21-96 19:30:51
pma
eral linear models. Significance was evaluated by the analysis of variance, followed by Scheffe´’s method. As for the level of exposure to ETS, the subjects were categorized into three groups: children in households in which there were no smokers; those in which there were smokers, but the total number of cigarettes smoked in the presence of the child was 10 or less a day; and those in which the total number of cigarettes smoked in the presence of the child was 11 or more a day. The mean values of serum proteins in these groups, adjusted for the above variables, except familial smoking habits, were compared by Scheffe´’s method, and the trends in their alterations in relation to the level of exposure to ETS were evaluated. Statistical analyses were conducted using SYSTAT package programs.15 RESULTS
The initial characteristics of the study subjects are shown in Table 1. The percentage of girls was slightly lower among children from whom blood samples were obtained than among those whose samples were unavailable, although the difference was not significant. There were no differences between responders and nonresponders in the smoking habits of families, the prevalence of asthma or wheezing, their histories of allergic diseases, and the other characteristics. Comparison according to school showed that the percentage of homes with vented-type heating in winter was higher in school A than in school B, but their familial smoking habits and the other demographic characteristics were similar. There was no significant difference between the children of the two schools in the prevalence of asthma, wheezing, or allergic diseases. Moreover, since the concentrations of all the measured serum proteins also showed a similar distribution between the school groups, data were pooled and analyzed together. The mean concentrations of each serum protein in relation to various factors are shown in Table 2. Cp levels were significantly higher among boys than among girls, but there were no significant differences in C3c, a1AG, and Hp levels between boys and girls. All the serum protein levels were highest in the first graders. Hp levels decreased with higher grades, but C3c, a1AG, and Cp levels were lower among second graders than among third graders. Serum a1AG levels were higher among children in which both father and mother were smokers than among those in households where only the father smoked. In addition, a1AG levels were higher among children living in households with smokers other than their parents (including households in which one or both of the parents also smoked), than among children living with nonsmokers and those in households where only the father smoked, respectively. However, concen-
AP: PM
619
ENVIRONMENTAL TOBACCO SMOKE AND SERUM PROTEINS
TABLE 1 Initial Characteristics (%) of the Study Subjectsa Children whose blood samples were available
Characteristics Gender Boy Girl Grade 1st 2nd 3rd Familial smoking habits None smoke Father only smokes Mother only smokes Both smoke Others smoke Asthma or wheezing Yes No Allergic diseases Yes No Feeding method in infancy Breast Bottle Mixed Heating type in the home Vented Unvented
Remainder of children (n Å 171)
School A (n Å 161)
School B (n Å 319)
54.7 45.3
50.2 49.8
0.404
43.9 56.1
0.096
30.4 33.5 36.0
35.1 30.7 34.2
0.585
34.5 34.5 31.0
0.642
32.9 41.6 1.9 11.2 12.4
24.5 48.3 1.6 9.1 16.6
0.233
32.2 48.0 1.8 7.6 10.5
0.436
12.4 87.6
14.4 85.6
0.646
14.0 86.0
1.000
59.6 40.4
55.5 44.5
0.443
50.3 49.7
0.163
32.3 21.1 46.6
34.5 27.6 37.9
0.144
30.4 24.0 45.6
0.544
32.9 67.1
22.9 77.1
0.024
28.7 71.3
0.611
P valueb
P valuec
a
Complete data were not available for 3 children. School A versus school B, by x2 test. c All responders (schools A and B) versus nonresponders, by x2 test. b
trations of the other serum proteins showed no significant differences in relation to familial smoking habits. All the serum protein levels were slightly higher among children with asthma or wheezing symptoms than among those without these symptoms, although the differences were not significant. Hp levels, however, were significantly lower among children with a history of allergic diseases than among those without. The levels of C3c, a1AG, and Cp showed no differences with a history of allergic diseases. But, with regard to the feeding method in infancy and the heating type in the home, there were no differences in all the serum protein levels. The multivariate adjusted mean levels of the serum proteins by sex are shown in Table 3. After adjustment for multiple variables using the general linear models, the levels of all the proteins showed no difference in relation to grades of boys and girls. The levels of C3c, Hp, and a1AG were significantly higher among boys living with smokers other than their parents than among boys living with nonsmokers and those in households where only the father smoked. There were no
/ a203$$2163
08-21-96 19:30:51
pma
significant differences in Cp levels in relation to familial smoking habits. The levels of Hp and a1AG were higher among boys with no history of allergic diseases than among those with such a history. None of the proteins showed any differences in relation to asthma or wheezing. Among girls, however, there were no significant effects of these factors on any of the serum proteins. The adjusted mean levels of the serum proteins in relation to the level of exposure to ETS are shown in Fig. 1. Boys exposed to ETS from 11 cigarettes or more a day showed the highest levels of all serum proteins. Among these boys, both C3c and Hp levels were significantly higher than levels among boys living with nonsmokers, and a1AG levels were also significantly higher than those among boys with nonsmokers in the households and boys exposed to ETS from 10 cigarettes or less a day. There were no significant differences in concentrations of any of the proteins between boys exposed to ETS from 10 cigarettes or less a day and those living with nonsmokers. Testing for trends showed that the levels of C3c, Hp, and a1AG among boys increased
AP: PM
620
SHIMA AND ADACHI
TABLE 2 Serum Levels of Complement C3c, Haptoglobin, a1 Acid Glycoprotein, and Ceruloplasmin among Children in Relation to Various Factorsa
Gender Boy Girl Grade 1st 2nd 3rd Familial smoking habits None smoke Father only smokes Mother only smokes Both smoke Others smoke Asthma or wheezing Yes No Allergic diseases Yes No Feeding method in infancy Breast Bottle Mixed Heating type in the home Vented Unvented
n
C3c (mg/dl)
248 232
80.7 (0.8) 79.0 (0.9)
99.5 (4.5) 107.4 (4.7)
77.0 (1.6) 72.6 (1.6)
33.7 (0.4)b 32.3 (0.4)
161 152 167
80.9 (1.0) 78.4 (1.1) 80.2 (1.0)
116.9 (5.6)c 100.1 (5.7) 93.1 (5.5)
79.0 (2.0) 72.4 (2.0) 73.1 (1.9)
34.0 (0.4)d 31.7 (0.5) 33.3 (0.4)e
131 221 8 47 73
80.1 78.7 84.2 79.1 83.1
94.3 102.1 102.0 116.9 114.7
73.3 72.4 85.1 81.0 80.4
33.2 32.8 35.3 33.3 33.2
66 414
80.9 (1.6) 79.7 (0.7)
107.1 (8.8) 102.7 (3.5)
77.7 (3.1) 74.4 (1.2)
34.3 (0.7) 32.9 (0.3)
273 207
80.7 (0.8) 78.7 (0.9)
96.0 (4.3) 113.0 (4.9)h
73.3 (1.5) 77.0 (1.7)
33.2 (0.3) 32.8 (0.4)
162 122 196
81.1 (1.0) 78.7 (1.2) 79.6 (0.9)
107.9 (5.6) 101.0 (6.5) 101.0 (5.1)
75.7 (2.0) 73.9 (2.3) 74.8 (1.8)
33.6 (0.4) 32.5 (0.5) 33.0 (0.4)
126 354
81.1 (1.2) 79.4 (0.7)
101.3 (6.4) 104.0 (3.8)
74.0 (2.2) 75.2 (1.3)
33.3 (0.5) 33.0 (0.3)
(1.2) (0.9) (4.7) (1.9) (1.5)
Haptoglobin (mg/dl)
(6.2) (4.8) (25.2) (10.4) (8.3)
a1 AG (mg/dl)
(2.2) (1.7) (8.7) (3.6) f (2.9) f,g
Ceruloplasmin (mg/dl)
(0.5) (0.4) (2.0) (0.8) (0.7)
a
Values are means, with SEM shown in parentheses. P õ 0.01 compared with girls. c P õ 0.05 compared with third graders. d P õ 0.01 compared with second graders. e P õ 0.05 compared with second graders. f P õ 0.05 compared with children in households where only the father smoked. g P õ 0.05 compared with children living with nonsmokers. h P õ 0.05 compared with children with a history of allergic diseases. b
significantly with the intensity of exposure to ETS. No differences were found in Cp levels. Similarly among girls, a1AG levels also were highest in those exposed to ETS from 11 cigarettes or more a day, significantly higher than among girls exposed to ETS from 10 cigarettes or less a day. Serum Hp levels were also highest among girls exposed to ETS from 11 cigarettes or more a day, although the difference was not significant. We found no differences in C3c and Cp levels in relation to the level of exposure to ETS. Testing for trends indicated that exposure to ETS was not related to levels of any of the serum proteins among girls. DISCUSSION
Increases in peripheral leukocyte counts in smokers have been recognized for many years, suggesting that
/ a203$$2163
08-21-96 19:30:51
pma
cigarette smoking triggers an inflammatory response in the body.16,17 APP are a group of serum proteins that increase in response to inflammatory stimuli or tissue injury, and raised levels of serum APP, such as Hp and Cp, have been found among smokers.9,10 However, cigarette smoking has not shown a consistent association with APP in serum; it has been reported that the levels of all APP examined, except for C-reactive protein, were unaffected by smoking.18 Furthermore, the changes in serum APP levels in response to ETS exposure have not been reported. In the present study, we measured the levels of C3c, Hp, a1AG, and Cp in the serum of schoolchildren and examined the relationship between APP levels and exposure to ETS in their households. Serum C3c is involved in a variety of biological activities by virtue of its nonspecific binding to antigen–antibody complex, reflecting the immune response in the
AP: PM
621
ENVIRONMENTAL TOBACCO SMOKE AND SERUM PROTEINS
TABLE 3 Multivariate Adjusted Mean Levels of Serum Complement C3c, Haptoglobin, a1 Acid Glycoprotein, and Ceruloplasmin among Children in Relation to Various Factorsa n
C3c (mg/dl)
Haptoglobin (mg/dl)
a1 AG (mg/dl)
Ceruloplasmin (mg/dl)
Boys Grade 1st 2nd 3rd Familial smoking habits None smoke Father only smokes Mother only smokes Both smoke Others smoke Asthma or wheezing Yes No Allergic diseases Yes No
88 71 89
86.3 (2.2) 82.1 (2.2) 87.0 (2.2)
128.2 (11.6) 108.8 (11.6) 111.9 (11.5)
88.4 (4.3) 79.4 (4.3) 84.0 (4.2)
36.5 (0.9) 33.7 (0.9) 36.2 (0.9)
68 110 4 30 36
78.2 78.3 97.2 82.3 89.8
88.8 94.2 122.6 129.7 146.3
71.8 72.5 93.7 88.5 93.2
33.6 33.7 39.0 34.9 36.2
39 209
85.7 (2.6) 84.6 (1.7)
125.7 (13.8) 107.0 (8.8)
86.6 (5.0) 81.3 (3.2)
36.3 (1.1) 34.7 (0.7)
144 104
85.3 (2.0) 85.1 (2.2)
100.7 (10.2) 131.9 (11.2)c
79.8 (3.7) 88.1 (4.1)d
35.1 (0.8) 35.8 (0.9)
78.2 (2.2) 76.9 (2.1) 77.3 (2.1)
115.1 (12.3) 96.3 (11.7) 89.8 (12.0)
77.0 (4.0) 73.0 (3.8) 72.1 (3.9)
33.5 (0.9) 31.7 (0.9) 32.8 (0.9)
63 111 4 17 37
81.7 80.0 72.1 75.3 78.3
105.3 116.0 81.6 104.6 94.5
76.3 74.3 77.4 70.9 71.3
33.1 33.0 33.1 32.4 31.8
27 205
77.8 (2.7) 77.2 (1.5)
99.2 (15.3) 101.6 (8.7)
75.1 (5.0) 73.0 (2.8)
33.2 (1.2) 32.1 (0.7)
129 103
78.9 (1.8) 76.0 (2.0)
95.6 (10.5) 105.2 (11.7)
73.1 (3.4) 75.0 (3.8)
33.2 (0.8) 32.2 (0.9)
(1.9) (1.7) (6.9) (2.7) (2.5)b
(10.0) (8.8) (35.7) (13.8) (12.9)b
(3.7) (3.2) (13.1) (5.1) (4.7)b
(0.8) (0.7) (2.9) (1.1) (1.0)
Girls Grade 1st 2nd 3rd Familial smoking habits None smoke Father only smokes Mother only smokes Both smoke Others smoke Asthma or wheezing Yes No Allergic diseases Yes No
73 81 78
(1.9) (1.6) (6.1) (3.3) (2.2)
(11.0) (9.3) (34.8) (18.7) (12.7)
(3.6) (3.0) (11.4) (6.1) (4.1)
(0.8) (0.7) (2.7) (1.4) (1.0)
a Values are means adjusted for all variables listed in Table 2, calculated using the general linear model, with SEM shown in parentheses. There were no differences in all values in relation to the feeding method in infancy and the heating type in the home. b P õ 0.01 compared with children living with nonsmokers and those in households where only the father smoked, respectively. c P õ 0.01 compared with children with a history of allergic diseases. d P õ 0.05 compared with children with a history of allergic diseases.
body, and is recognized as an APP.8 Stiller-Winkler et al.19 reported that serum C3c levels tended to increase among residents of areas with high air pollution levels. They found that the effect of smoking on C3c levels as a confounding factor was small among adults; however, the effect of exposure to ETS among children was not considered. In the present study, we observed a significantly increased level of serum C3c among boys living in households in which family members other than their parents smoked. C3c levels were also higher among boys exposed to ETS from 11 cigarettes or more a day, while no association was found between exposure to ETS and C3c levels among girls.
/ a203$$2163
08-21-96 19:30:51
pma
Hp, an APP binding hemoglobin, is associated with respiratory diseases such as bronchial asthma and chronic obstructive lung disease.20 Decreased Hp levels have been reported in the serum of those with allergic diseases.21 In the present study, we also found that serum Hp levels were lower among children with a history of any allergic disease than among those without, and this difference was significant for boys. There was no difference in Hp levels with the presence of asthma or wheezing. However, the number of subjects reporting these symptoms may have been too small to detect such differences. Kauffmann et al.9 reported that serum Hp levels were associated with smoking, and
AP: PM
622
SHIMA AND ADACHI
FIG. 1. Serum levels of (A) complement C3c, (B) haptoglobin, (C) a1 acid glycoprotein, and (D) ceruloplasmin among schoolchildren in relation to the level of exposure to ETS. All values are means { SEM adjusted for school grade, asthma or wheezing, allergic diseases, feeding method in infancy, and heating type, calculated using the general linear models.
that these levels were particularly high among heavy smokers (those who smoked 20 cigarettes or more a day). The serum Hp levels among children in our study were higher among both boys and girls exposed to ETS from 11 cigarettes or more a day, and ETS exposure was significantly associated with Hp levels among boys. a1AG is another APP which serves as a sensitive marker of inflammatory disease, since it participates in the modulation of immune reaction in the body. Kauffmann et al.9 reported that serum a1AG levels were high among smokers. On the contrary, Das18 found no change in a1AG levels in relation to smoking, although the number of subjects was small. In our study, serum a1AG levels among boys increased significantly with the level of exposure to ETS. Among girls, high levels of a1AG were also related to exposure to ETS from 11 cigarettes or more a day, while the
/ a203$$2163
08-21-96 19:30:51
pma
levels among girls exposed to 10 cigarettes or less a day and those among girls living with nonsmokers were similar. Cp is an APP that exhibits anti-complement activity. Galdston et al.10 and Bridges et al.17 reported that serum Cp levels were higher among smokers than among nonsmokers. However, we detected no relationship of serum Cp levels to either familial smoking habits or to the intensity of exposure to ETS among either boys or girls. Duthie et al.23 suggested that Cp levels were correlated with plasma copper concentration, and that the elevated levels reflected increased copper intake from cigarette smoke. This effect may not occur with ETS exposure. It has been established that APP elevation is an important index of the existence of an inflammatory process.8,22 Our study subjects were general schoolchildren, and those with infections or acute symptoms
AP: PM
ENVIRONMENTAL TOBACCO SMOKE AND SERUM PROTEINS
were excluded to determine the relationship between serum APP levels and ETS exposure. However, there were no differences for household and health characteristics between children from whom blood samples were obtained and the remainder. The physiological mechanism of the actual APP alteration remains unclear, but APP has been reported to be involved in a protective role and in an antioxidant activity in the body.10,22 Therefore, an APP elevation in response to ETS exposure may indicate the state of host defense against tobacco smoke. The effect of exposure to tobacco smoke in utero should be also considered, but we have no information of smoking mothers during pregnancy. Matthews and Soothill24 and Chandra25 reported that the complement was activated after bottle feeding in infants, suggesting an effect of cow’s milk on serum protein concentrations. We found no differences in any of the serum protein components in relation to the method of feeding during infancy. Since our subjects were schoolchildren, the feeding method in infancy may no longer have had an effect on serum protein levels. The effects of ETS exposure on health have been investigated in many epidemiologic studies.2 – 4,26,27 Most of these studies have obtained information about exposure levels to ETS via questionnaire, and have used the presence or absence of smokers in the household and the number of cigarettes smoked in assessing exposure to ETS. Our comparisons of serum protein levels with familial smoking habits showed that the levels were significantly higher among boys living with smokers other than their parents. If a smoker other than the parents lived in the household, it was usually the grandfather. The intensity of the child’s exposure to ETS from the grandfather was assumed to be considerable, since grandfathers spend most of their time at home. Many reports have described the health effects of maternal smoking on children,2 – 4,26 whereas the effects of paternal smoking have not been widely recognized, since fathers often smoke at their workplace or outside the house, smoking less in the presence of their child. Friedman et al.27 showed that a considerable number of nonsmoking spouses of smokers were not exposed to ETS at all. Thus, it is necessary to determine both familial smoking habits and the amount of smoking in the presence of the subject. In the present study, if there were any smokers in the household, we asked the respondent to note the number of cigarettes that each smoked per day in the presence of the child. In addition, whenever a questionnaire was not complete, we requested the parents or guardians to complete them. Thus, we took measures to ensure that the reported level of exposure to ETS was fairly reliable.13 The use of biological markers, such as cotinine in urine or serum, must also be considered as an objec-
/ a203$$2163
08-21-96 19:30:51
pma
623
tive means of assessing the level of exposure to ETS.28,29 However, to date, since no markers are known to reflect long-term exposure to ETS, we opted for a detailed questionnaire to gain the pertinent epidemiologic information.5,27 Further study is needed to establish such a method for accurately determining exposure levels to ETS. In conclusion, the present study showed that serum levels of C3c, Hp, and a1AG were significantly higher among boys exposed to high levels of ETS than among those exposed to low levels, suggesting that changes in the levels of these APP may reflect differences in exposure levels to ETS. Among girls, however, we found no significant changes in APP, except in a1AG. In previous studies, girls seemed to be more susceptible to indoor air pollution.11,30 Our results, on the contrary, showed that the effects of exposure to ETS on serum APP levels appeared to be greater among boys. This finding may, however, have been reflected by the small number of girls exposed to high levels of ETS. It has been shown that the pattern of the APP response is often sex dependent.8 The changes in serum APP in response to inflammation have been reported to be greater among males than among females.31 Therefore, to determine the significance of changes in these serum APP, the involvement of various factors, including sexrelated differences, should be further evaluated. REFERENCES 1. Holt PG, Turner KJ. Respiratory symptoms in the children of smokers: an overview. Eur J Respir Dis 1984;Suppl 133:109– 20. 2. Murray AB, Morrison BJ. The effect of cigarette smoke from the mother on bronchial responsiveness and severity of symptoms in children with asthma. J Allergy Clin Immunol 1986;77:575– 81. 3. Wang X, Wypij D, Gold DR, Speizer FE, Ware JH, Ferris BG Jr, et al. A longitudinal study of the effects of parental smoking on pulmonary function in children 6–18 years. Am J Respir Crit Care Med 1994;149:1420–5. 4. Forastiere F, Agabiti N, Corbo GM, Pistelli R, Dell’Orco V, Ciappi G, et al. Passive smoking as a determinant of bronchial responsiveness in children. Am J Respir Crit Care Med 1994;149:365– 70. 5. Jarvis M, Tunstall-Pedoe H, Feyerabend C, Vesey C, Salloojee Y. Biochemical markers of smoke absorption and self reported exposure to passive smoking. J Epidemiol Community Health 1984;38:335–9. 6. Henderson FW, Reid HF, Morris R, Wang OL, Hu PC, Helms RW, et al. Home air nicotine levels and urinary cotinine excretion in preschool children. Am Rev Respir Dis 1989;140:197–201. 7. Ehrlich R, Kattan M, Godbold J, Saltzberg DS, Grimm KT, Landrigan PJ, et al. Childhood asthma and passive smoking: urinary cotinine as a biomarker of exposure. Am Rev Respir Dis 1992;145:594–9. 8. Kushner I, Mackiewicz A. Acute phase proteins as disease markers. Dis Markers 1987;5:1–11. 9. Kauffmann F, Frette C, Annesi I, Oryszczyn MP, Dore MF, Neukirch F. Relationships of haptoglobin level to FEV1, wheez-
AP: PM
624
SHIMA AND ADACHI
ing, bronchial hyper-responsiveness and allergy. Clin Exp Allergy 1991;21:669–74. 10. Galdston M, Levytska V, Schwartz MS, Magnusson B. Ceruloplasmin: increased serum concentrations and impaired antioxidant activity in cigarette smokers, and ability to prevent suppression of elastase inhibitory capacity of alpha1-proteinase inhibitor. Am Rev Respir Dis 1984;120:258–63. 11. Shima M, Nitta Y, Adachi M. Association of outdoor and indoor nitrogen dioxide with pulmonary function in schoolchildren. J Epidemiol 1994;4:137–46. 12. Ferris BG. Epidemiology standardization project. II. Recommended respiratory disease questionnaires for use with adults and children in epidemiological research. Am Rev Respir Dis 1978;118 Suppl 6:7–53. 13. Adachi M, Kobayashi M, Iwasaki A, Nitta Y, Yoshida R. A new children’s respiratory disease questionnaire. 2. Study on noncompletion in self-completion method [in Japanese]. Jpn J Public Health 1983;30:581–8. 14. Bienvenu J, Sann L, Bienvenu F, Lahet C, Divry P, Cotte J, et al. Laser nephelometry of orosomucoid in serum of newborns: reference intervals and relation to bacterial infections. Clin Chem 1981;27:721–6. 15. Wilkinson L. SYSTAT: Statistics, version 5.2 edition. Evanston (IL): SYSTAT, Inc., 1992. 16. Petitti DB, Kipp H. The leukocyte count: associations with intensity of smoking and persistence of effect after quitting. Am J Epidemiol 1986;123:89–95. 17. Bridges RB, Wyatt RJ, Rehm SR. Effects of smoking on inflammatory mediators and their relationship to pulmonary dysfunction. Eur J Respir Dis 1986;69 Suppl 146:145–52. 18. Das I. Raised C-reactive protein levels in serum from smokers. Clin Chim Acta 1985;153:9–13. 19. Stiller-Winkler R, Kra¨mer U, Fiedler E, Ewers U, Dolgner R. C3c concentrations in sera of persons living in areas with different levels of air pollution in Northrhine–Westphalia (Federal Republic of Germany). Environ Res 1989;49:7–19.
/ a203$$2163
08-21-96 19:30:51
pma
20. Kauffmann F, Kleisbauer JP, Cambon-de-Mouzon A, Mercier P, Constans J, Blanc M, et al. Genetic markers in chronic air-flow limitation: a genetic epidemiologic study. Am Rev Respir Dis 1983;127:263–9. 21. Piessens MF, Marien G, Stevens E. Decreased haptoglobin levels in respiratory allergy. Clin Allergy 1984;14:287–93. 22. Lubega J, Davies TJ. A comparison of serum mucoprotein with serum a1 acid glycoprotein, haptoglobin, and a1 antitrypsin assays in monitoring inflammatory bowel disease. Clin Chim Acta 1990;188:59–70. 23. Duthie GG, Arthur JR, James WPT. Effects of smoking and vitamin E on blood antioxidant status. Am J Clin Nutr 1991; 53:1061S–3S. 24. Matthews TS, Soothill JF. Complement activation after milk feeding in children with cow’s milk allergy. Lancet 1970; ii:893 – 5. 25. Chandra RK. Prospective studies of the effect of breast feeding on incidence of infection and allergy. Acta Paediatr Scand 1979;68:691–4. 26. Vedal S, Schenker MB, Samet JM, Speizer FE. Risk factors for childhood respiratory disease: analysis of pulmonary function. Am Rev Respir Dis 1984;130:187–92. 27. Friedman GD, Petitti DB, Bawol RD. Prevalence and correlates of passive smoking. Am J Public Health 1983;73:401–5. 28. Coultas DB, Samet JM, McCarthy JF, Spengler JD. Variability of measures of exposure to environmental tobacco smoke in the home. Am Rev Respir Dis 1990;142:602–6. 29. Pe´rez-Stable E, Benowitz NL, MarıB n G. Is serum cotinine a better measure of cigarette smoking than self-report? Prev Med 1995;24:171–9. 30. Hasselblad V, Humble CG, Graham MG, Anderson HS. Indoor environmental determinants of lung function in children. Am Rev Respir Dis 1981;123:479–85. 31. Bosanquet AG, Chandler AM, Gordon AH. Effects of injury on the concentration of a1-macroglobulin and a2-macroglobulin in the plasmas of male and female rats. Experientia 1976; 32: 1348 – 9.
AP: PM
25, 617–624 (1996)
0097
Effects of Environmental Tobacco Smoke on Serum Levels of Acute Phase Proteins in Schoolchildren MASAYUKI SHIMA, M.D.,
AND
MOTOAKI ADACHI, M.D.
Department of Public Health, Chiba University School of Medicine, Chiba 260, Japan
Background. Few biological changes due to longterm exposure to environmental tobacco smoke (ETS) have been identified. This study examined the relationship between exposure to ETS and serum levels of acute phase proteins among children. Methods. The concentrations of the third component of complement (C3c), haptoglobin (Hp), a1 acid glycoprotein (a1AG), and ceruloplasmin (Cp) were determined in serum samples obtained from 480 schoolchildren. Exposure to ETS was assessed from responses to questionnaires. Results. Serum levels of C3c, Hp, and a1AG were significantly higher among boys living in households in which there were smokers other than their parents than among boys with no smokers in their family or those in households where only the father smoked. Levels of C3c, Hp, and a1AG in relation to the level of ETS exposure were significantly increased among boys exposed to ETS of 11 or more cigarettes per day. Among girls, increased a1AG levels were associated with exposure to high levels of ETS, but no differences were found in any of the other proteins. Cp levels were not affected by exposure to ETS among either boys or girls. Conclusions. These findings suggest that the serum levels of C3c, Hp, and a1AG among schoolchildren may reflect different levels of exposure to ETS. Boys seemed to be more susceptible to ETS exposure than girls. q 1996 Academic Press, Inc. Key Words: environmental tobacco smoke; acute phase proteins; the third component of complement; haptoglobin; a1 acid glycoprotein; ceruloplasmin.
nary function,3 and bronchial hyperresponsiveness4 have been reported. Urinary cotinine levels have often been used as an indicator of exposure to ETS.5,6 Recent findings have shown a relationship between emergency room admissions for asthma and urinary levels of cotinine.7 In ETS, nicotine is present in the gaseous phase and, on inhalation, is immediately metabolized to cotinine.5 Therefore, urinary cotinine only serves as an index of short-term brief exposure to ETS, and does not appear to reflect constant states of exposure.6 At present, no biological marker has yet been identified which could be used to determine the effect of long-term exposure to ETS on human health. Acute phase proteins (APP) are a group of serum protein components that increase in response to the presence of foreign substances invading the body.8 APP such as haptoglobin (Hp) and ceruloplasmin (Cp) are reported to be increased among smokers,9,10 but the effects of exposure to ETS on serum protein components were not considered in these studies. To evaluate the effects of various environmental factors on respiratory health, we have been conducting a series of epidemiologic surveys among schoolchildren from various areas of Japan.11 In the present study, we measured concentrations of serum APP, the third component of complement (C3c), Hp, a1 acid glycoprotein (a1AG), and Cp, among schoolchildren living in an area of relatively low air pollution, to examine the relationship between serum protein levels and exposure to ETS in the households. METHODS
INTRODUCTION
The effects of exposure to environmental tobacco smoke (ETS) on the respiratory system in children have been extensively investigated.1 Increased respiratory symptoms of bronchial asthma,2 lower levels of pulmoAddress reprint requests to Masayuki Shima, Department of Public Health, Chiba University School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260, Japan. Fax: /81 43-226-2070; E-mail: [email protected].
Subjects The study subjects were 654 first- to third-grade children attending two elementary schools (ages 6–9 years) in Chiba Prefecture, Japan. The two schools were located on the coast of Tokyo Bay, in adjacent school districts. The annual average concentrations of sulfur dioxide, nitrogen dioxide, and suspended particulate matter in 1992, measured at a monitoring station close to these schools, were 6 ppb, 18 ppb, and 37 mg/
617
/ a203$$2163
08-21-96 19:30:51
pma
0091-7435/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
AP: PM
618
SHIMA AND ADACHI
m3, respectively, below Japanese ambient air quality standards. In November 1992, a standard respiratory symptom questionnaire, a modified Japanese version of ATSDLD-78-C,12 was sent to all subjects. The questionnaire was filled out by either parent or guardians. Incomplete questionnaires were returned with a request to complete them.13 Altogether, 651 (99.5%) completed questionnaires were returned. The questionnaire consisted of questions about the respiratory symptoms and medical history of the child, the smoking habits of the household members, and certain characteristics of the home, such as the type of heating. If any members of the household were smokers, the respondent was asked to note the number of cigarettes that each smoked per day in the presence of the child. According to the responses to the questionnaire, children who had experienced two or more episodes of wheezing accompanied by dyspnea, or who had been under medical treatment for bronchial asthma during the past 2 years, were considered to have asthma or wheezing symptoms. Those who had been diagnosed as having eczema, atopy, allergic rhinitis, or pollinosis by a physician, or who had received hyposensitization therapy, were considered to have a history of allergic diseases. Blood Collection and Laboratory Measurements In January 1993, venous blood samples were collected from 483 children (73.9%) whose parents or guardians had given their written consent. Children with acute infections or symptoms, such as cold or fever, the day before and the day of examination were excluded (n Å 35). Consent for venipuncture was not given by the remaining parents (n Å 136). All venipuncture was performed between 9 AM and 2 PM, and the blood specimens were centrifuged on the same day. All serum samples were kept frozen at 0807C until analysis. C3c, Hp, a1AG, and Cp were determined by a nephelometric method14 using a Behring nephelometer analyzer (Behringwerke AG, Marburg, Germany). Data Analysis In 1 of the 483 children, complete data on exposure to ETS were not available. Also, the volume of blood collected from two subjects was insufficient for measurement of all the proteins. Thus, the final sample for analysis included 480 children (school A, 161 children; school B, 319). The serum levels of each protein were first compared in relation to gender, school grade, familial smoking habits, asthma or wheezing symptoms, allergic diseases, feeding method in infancy, and heating type in the home. Then, the mean values adjusted for all variables were calculated separately by sex, using the gen-
/ a203$$2163
08-21-96 19:30:51
pma
eral linear models. Significance was evaluated by the analysis of variance, followed by Scheffe´’s method. As for the level of exposure to ETS, the subjects were categorized into three groups: children in households in which there were no smokers; those in which there were smokers, but the total number of cigarettes smoked in the presence of the child was 10 or less a day; and those in which the total number of cigarettes smoked in the presence of the child was 11 or more a day. The mean values of serum proteins in these groups, adjusted for the above variables, except familial smoking habits, were compared by Scheffe´’s method, and the trends in their alterations in relation to the level of exposure to ETS were evaluated. Statistical analyses were conducted using SYSTAT package programs.15 RESULTS
The initial characteristics of the study subjects are shown in Table 1. The percentage of girls was slightly lower among children from whom blood samples were obtained than among those whose samples were unavailable, although the difference was not significant. There were no differences between responders and nonresponders in the smoking habits of families, the prevalence of asthma or wheezing, their histories of allergic diseases, and the other characteristics. Comparison according to school showed that the percentage of homes with vented-type heating in winter was higher in school A than in school B, but their familial smoking habits and the other demographic characteristics were similar. There was no significant difference between the children of the two schools in the prevalence of asthma, wheezing, or allergic diseases. Moreover, since the concentrations of all the measured serum proteins also showed a similar distribution between the school groups, data were pooled and analyzed together. The mean concentrations of each serum protein in relation to various factors are shown in Table 2. Cp levels were significantly higher among boys than among girls, but there were no significant differences in C3c, a1AG, and Hp levels between boys and girls. All the serum protein levels were highest in the first graders. Hp levels decreased with higher grades, but C3c, a1AG, and Cp levels were lower among second graders than among third graders. Serum a1AG levels were higher among children in which both father and mother were smokers than among those in households where only the father smoked. In addition, a1AG levels were higher among children living in households with smokers other than their parents (including households in which one or both of the parents also smoked), than among children living with nonsmokers and those in households where only the father smoked, respectively. However, concen-
AP: PM
619
ENVIRONMENTAL TOBACCO SMOKE AND SERUM PROTEINS
TABLE 1 Initial Characteristics (%) of the Study Subjectsa Children whose blood samples were available
Characteristics Gender Boy Girl Grade 1st 2nd 3rd Familial smoking habits None smoke Father only smokes Mother only smokes Both smoke Others smoke Asthma or wheezing Yes No Allergic diseases Yes No Feeding method in infancy Breast Bottle Mixed Heating type in the home Vented Unvented
Remainder of children (n Å 171)
School A (n Å 161)
School B (n Å 319)
54.7 45.3
50.2 49.8
0.404
43.9 56.1
0.096
30.4 33.5 36.0
35.1 30.7 34.2
0.585
34.5 34.5 31.0
0.642
32.9 41.6 1.9 11.2 12.4
24.5 48.3 1.6 9.1 16.6
0.233
32.2 48.0 1.8 7.6 10.5
0.436
12.4 87.6
14.4 85.6
0.646
14.0 86.0
1.000
59.6 40.4
55.5 44.5
0.443
50.3 49.7
0.163
32.3 21.1 46.6
34.5 27.6 37.9
0.144
30.4 24.0 45.6
0.544
32.9 67.1
22.9 77.1
0.024
28.7 71.3
0.611
P valueb
P valuec
a
Complete data were not available for 3 children. School A versus school B, by x2 test. c All responders (schools A and B) versus nonresponders, by x2 test. b
trations of the other serum proteins showed no significant differences in relation to familial smoking habits. All the serum protein levels were slightly higher among children with asthma or wheezing symptoms than among those without these symptoms, although the differences were not significant. Hp levels, however, were significantly lower among children with a history of allergic diseases than among those without. The levels of C3c, a1AG, and Cp showed no differences with a history of allergic diseases. But, with regard to the feeding method in infancy and the heating type in the home, there were no differences in all the serum protein levels. The multivariate adjusted mean levels of the serum proteins by sex are shown in Table 3. After adjustment for multiple variables using the general linear models, the levels of all the proteins showed no difference in relation to grades of boys and girls. The levels of C3c, Hp, and a1AG were significantly higher among boys living with smokers other than their parents than among boys living with nonsmokers and those in households where only the father smoked. There were no
/ a203$$2163
08-21-96 19:30:51
pma
significant differences in Cp levels in relation to familial smoking habits. The levels of Hp and a1AG were higher among boys with no history of allergic diseases than among those with such a history. None of the proteins showed any differences in relation to asthma or wheezing. Among girls, however, there were no significant effects of these factors on any of the serum proteins. The adjusted mean levels of the serum proteins in relation to the level of exposure to ETS are shown in Fig. 1. Boys exposed to ETS from 11 cigarettes or more a day showed the highest levels of all serum proteins. Among these boys, both C3c and Hp levels were significantly higher than levels among boys living with nonsmokers, and a1AG levels were also significantly higher than those among boys with nonsmokers in the households and boys exposed to ETS from 10 cigarettes or less a day. There were no significant differences in concentrations of any of the proteins between boys exposed to ETS from 10 cigarettes or less a day and those living with nonsmokers. Testing for trends showed that the levels of C3c, Hp, and a1AG among boys increased
AP: PM
620
SHIMA AND ADACHI
TABLE 2 Serum Levels of Complement C3c, Haptoglobin, a1 Acid Glycoprotein, and Ceruloplasmin among Children in Relation to Various Factorsa
Gender Boy Girl Grade 1st 2nd 3rd Familial smoking habits None smoke Father only smokes Mother only smokes Both smoke Others smoke Asthma or wheezing Yes No Allergic diseases Yes No Feeding method in infancy Breast Bottle Mixed Heating type in the home Vented Unvented
n
C3c (mg/dl)
248 232
80.7 (0.8) 79.0 (0.9)
99.5 (4.5) 107.4 (4.7)
77.0 (1.6) 72.6 (1.6)
33.7 (0.4)b 32.3 (0.4)
161 152 167
80.9 (1.0) 78.4 (1.1) 80.2 (1.0)
116.9 (5.6)c 100.1 (5.7) 93.1 (5.5)
79.0 (2.0) 72.4 (2.0) 73.1 (1.9)
34.0 (0.4)d 31.7 (0.5) 33.3 (0.4)e
131 221 8 47 73
80.1 78.7 84.2 79.1 83.1
94.3 102.1 102.0 116.9 114.7
73.3 72.4 85.1 81.0 80.4
33.2 32.8 35.3 33.3 33.2
66 414
80.9 (1.6) 79.7 (0.7)
107.1 (8.8) 102.7 (3.5)
77.7 (3.1) 74.4 (1.2)
34.3 (0.7) 32.9 (0.3)
273 207
80.7 (0.8) 78.7 (0.9)
96.0 (4.3) 113.0 (4.9)h
73.3 (1.5) 77.0 (1.7)
33.2 (0.3) 32.8 (0.4)
162 122 196
81.1 (1.0) 78.7 (1.2) 79.6 (0.9)
107.9 (5.6) 101.0 (6.5) 101.0 (5.1)
75.7 (2.0) 73.9 (2.3) 74.8 (1.8)
33.6 (0.4) 32.5 (0.5) 33.0 (0.4)
126 354
81.1 (1.2) 79.4 (0.7)
101.3 (6.4) 104.0 (3.8)
74.0 (2.2) 75.2 (1.3)
33.3 (0.5) 33.0 (0.3)
(1.2) (0.9) (4.7) (1.9) (1.5)
Haptoglobin (mg/dl)
(6.2) (4.8) (25.2) (10.4) (8.3)
a1 AG (mg/dl)
(2.2) (1.7) (8.7) (3.6) f (2.9) f,g
Ceruloplasmin (mg/dl)
(0.5) (0.4) (2.0) (0.8) (0.7)
a
Values are means, with SEM shown in parentheses. P õ 0.01 compared with girls. c P õ 0.05 compared with third graders. d P õ 0.01 compared with second graders. e P õ 0.05 compared with second graders. f P õ 0.05 compared with children in households where only the father smoked. g P õ 0.05 compared with children living with nonsmokers. h P õ 0.05 compared with children with a history of allergic diseases. b
significantly with the intensity of exposure to ETS. No differences were found in Cp levels. Similarly among girls, a1AG levels also were highest in those exposed to ETS from 11 cigarettes or more a day, significantly higher than among girls exposed to ETS from 10 cigarettes or less a day. Serum Hp levels were also highest among girls exposed to ETS from 11 cigarettes or more a day, although the difference was not significant. We found no differences in C3c and Cp levels in relation to the level of exposure to ETS. Testing for trends indicated that exposure to ETS was not related to levels of any of the serum proteins among girls. DISCUSSION
Increases in peripheral leukocyte counts in smokers have been recognized for many years, suggesting that
/ a203$$2163
08-21-96 19:30:51
pma
cigarette smoking triggers an inflammatory response in the body.16,17 APP are a group of serum proteins that increase in response to inflammatory stimuli or tissue injury, and raised levels of serum APP, such as Hp and Cp, have been found among smokers.9,10 However, cigarette smoking has not shown a consistent association with APP in serum; it has been reported that the levels of all APP examined, except for C-reactive protein, were unaffected by smoking.18 Furthermore, the changes in serum APP levels in response to ETS exposure have not been reported. In the present study, we measured the levels of C3c, Hp, a1AG, and Cp in the serum of schoolchildren and examined the relationship between APP levels and exposure to ETS in their households. Serum C3c is involved in a variety of biological activities by virtue of its nonspecific binding to antigen–antibody complex, reflecting the immune response in the
AP: PM
621
ENVIRONMENTAL TOBACCO SMOKE AND SERUM PROTEINS
TABLE 3 Multivariate Adjusted Mean Levels of Serum Complement C3c, Haptoglobin, a1 Acid Glycoprotein, and Ceruloplasmin among Children in Relation to Various Factorsa n
C3c (mg/dl)
Haptoglobin (mg/dl)
a1 AG (mg/dl)
Ceruloplasmin (mg/dl)
Boys Grade 1st 2nd 3rd Familial smoking habits None smoke Father only smokes Mother only smokes Both smoke Others smoke Asthma or wheezing Yes No Allergic diseases Yes No
88 71 89
86.3 (2.2) 82.1 (2.2) 87.0 (2.2)
128.2 (11.6) 108.8 (11.6) 111.9 (11.5)
88.4 (4.3) 79.4 (4.3) 84.0 (4.2)
36.5 (0.9) 33.7 (0.9) 36.2 (0.9)
68 110 4 30 36
78.2 78.3 97.2 82.3 89.8
88.8 94.2 122.6 129.7 146.3
71.8 72.5 93.7 88.5 93.2
33.6 33.7 39.0 34.9 36.2
39 209
85.7 (2.6) 84.6 (1.7)
125.7 (13.8) 107.0 (8.8)
86.6 (5.0) 81.3 (3.2)
36.3 (1.1) 34.7 (0.7)
144 104
85.3 (2.0) 85.1 (2.2)
100.7 (10.2) 131.9 (11.2)c
79.8 (3.7) 88.1 (4.1)d
35.1 (0.8) 35.8 (0.9)
78.2 (2.2) 76.9 (2.1) 77.3 (2.1)
115.1 (12.3) 96.3 (11.7) 89.8 (12.0)
77.0 (4.0) 73.0 (3.8) 72.1 (3.9)
33.5 (0.9) 31.7 (0.9) 32.8 (0.9)
63 111 4 17 37
81.7 80.0 72.1 75.3 78.3
105.3 116.0 81.6 104.6 94.5
76.3 74.3 77.4 70.9 71.3
33.1 33.0 33.1 32.4 31.8
27 205
77.8 (2.7) 77.2 (1.5)
99.2 (15.3) 101.6 (8.7)
75.1 (5.0) 73.0 (2.8)
33.2 (1.2) 32.1 (0.7)
129 103
78.9 (1.8) 76.0 (2.0)
95.6 (10.5) 105.2 (11.7)
73.1 (3.4) 75.0 (3.8)
33.2 (0.8) 32.2 (0.9)
(1.9) (1.7) (6.9) (2.7) (2.5)b
(10.0) (8.8) (35.7) (13.8) (12.9)b
(3.7) (3.2) (13.1) (5.1) (4.7)b
(0.8) (0.7) (2.9) (1.1) (1.0)
Girls Grade 1st 2nd 3rd Familial smoking habits None smoke Father only smokes Mother only smokes Both smoke Others smoke Asthma or wheezing Yes No Allergic diseases Yes No
73 81 78
(1.9) (1.6) (6.1) (3.3) (2.2)
(11.0) (9.3) (34.8) (18.7) (12.7)
(3.6) (3.0) (11.4) (6.1) (4.1)
(0.8) (0.7) (2.7) (1.4) (1.0)
a Values are means adjusted for all variables listed in Table 2, calculated using the general linear model, with SEM shown in parentheses. There were no differences in all values in relation to the feeding method in infancy and the heating type in the home. b P õ 0.01 compared with children living with nonsmokers and those in households where only the father smoked, respectively. c P õ 0.01 compared with children with a history of allergic diseases. d P õ 0.05 compared with children with a history of allergic diseases.
body, and is recognized as an APP.8 Stiller-Winkler et al.19 reported that serum C3c levels tended to increase among residents of areas with high air pollution levels. They found that the effect of smoking on C3c levels as a confounding factor was small among adults; however, the effect of exposure to ETS among children was not considered. In the present study, we observed a significantly increased level of serum C3c among boys living in households in which family members other than their parents smoked. C3c levels were also higher among boys exposed to ETS from 11 cigarettes or more a day, while no association was found between exposure to ETS and C3c levels among girls.
/ a203$$2163
08-21-96 19:30:51
pma
Hp, an APP binding hemoglobin, is associated with respiratory diseases such as bronchial asthma and chronic obstructive lung disease.20 Decreased Hp levels have been reported in the serum of those with allergic diseases.21 In the present study, we also found that serum Hp levels were lower among children with a history of any allergic disease than among those without, and this difference was significant for boys. There was no difference in Hp levels with the presence of asthma or wheezing. However, the number of subjects reporting these symptoms may have been too small to detect such differences. Kauffmann et al.9 reported that serum Hp levels were associated with smoking, and
AP: PM
622
SHIMA AND ADACHI
FIG. 1. Serum levels of (A) complement C3c, (B) haptoglobin, (C) a1 acid glycoprotein, and (D) ceruloplasmin among schoolchildren in relation to the level of exposure to ETS. All values are means { SEM adjusted for school grade, asthma or wheezing, allergic diseases, feeding method in infancy, and heating type, calculated using the general linear models.
that these levels were particularly high among heavy smokers (those who smoked 20 cigarettes or more a day). The serum Hp levels among children in our study were higher among both boys and girls exposed to ETS from 11 cigarettes or more a day, and ETS exposure was significantly associated with Hp levels among boys. a1AG is another APP which serves as a sensitive marker of inflammatory disease, since it participates in the modulation of immune reaction in the body. Kauffmann et al.9 reported that serum a1AG levels were high among smokers. On the contrary, Das18 found no change in a1AG levels in relation to smoking, although the number of subjects was small. In our study, serum a1AG levels among boys increased significantly with the level of exposure to ETS. Among girls, high levels of a1AG were also related to exposure to ETS from 11 cigarettes or more a day, while the
/ a203$$2163
08-21-96 19:30:51
pma
levels among girls exposed to 10 cigarettes or less a day and those among girls living with nonsmokers were similar. Cp is an APP that exhibits anti-complement activity. Galdston et al.10 and Bridges et al.17 reported that serum Cp levels were higher among smokers than among nonsmokers. However, we detected no relationship of serum Cp levels to either familial smoking habits or to the intensity of exposure to ETS among either boys or girls. Duthie et al.23 suggested that Cp levels were correlated with plasma copper concentration, and that the elevated levels reflected increased copper intake from cigarette smoke. This effect may not occur with ETS exposure. It has been established that APP elevation is an important index of the existence of an inflammatory process.8,22 Our study subjects were general schoolchildren, and those with infections or acute symptoms
AP: PM
ENVIRONMENTAL TOBACCO SMOKE AND SERUM PROTEINS
were excluded to determine the relationship between serum APP levels and ETS exposure. However, there were no differences for household and health characteristics between children from whom blood samples were obtained and the remainder. The physiological mechanism of the actual APP alteration remains unclear, but APP has been reported to be involved in a protective role and in an antioxidant activity in the body.10,22 Therefore, an APP elevation in response to ETS exposure may indicate the state of host defense against tobacco smoke. The effect of exposure to tobacco smoke in utero should be also considered, but we have no information of smoking mothers during pregnancy. Matthews and Soothill24 and Chandra25 reported that the complement was activated after bottle feeding in infants, suggesting an effect of cow’s milk on serum protein concentrations. We found no differences in any of the serum protein components in relation to the method of feeding during infancy. Since our subjects were schoolchildren, the feeding method in infancy may no longer have had an effect on serum protein levels. The effects of ETS exposure on health have been investigated in many epidemiologic studies.2 – 4,26,27 Most of these studies have obtained information about exposure levels to ETS via questionnaire, and have used the presence or absence of smokers in the household and the number of cigarettes smoked in assessing exposure to ETS. Our comparisons of serum protein levels with familial smoking habits showed that the levels were significantly higher among boys living with smokers other than their parents. If a smoker other than the parents lived in the household, it was usually the grandfather. The intensity of the child’s exposure to ETS from the grandfather was assumed to be considerable, since grandfathers spend most of their time at home. Many reports have described the health effects of maternal smoking on children,2 – 4,26 whereas the effects of paternal smoking have not been widely recognized, since fathers often smoke at their workplace or outside the house, smoking less in the presence of their child. Friedman et al.27 showed that a considerable number of nonsmoking spouses of smokers were not exposed to ETS at all. Thus, it is necessary to determine both familial smoking habits and the amount of smoking in the presence of the subject. In the present study, if there were any smokers in the household, we asked the respondent to note the number of cigarettes that each smoked per day in the presence of the child. In addition, whenever a questionnaire was not complete, we requested the parents or guardians to complete them. Thus, we took measures to ensure that the reported level of exposure to ETS was fairly reliable.13 The use of biological markers, such as cotinine in urine or serum, must also be considered as an objec-
/ a203$$2163
08-21-96 19:30:51
pma
623
tive means of assessing the level of exposure to ETS.28,29 However, to date, since no markers are known to reflect long-term exposure to ETS, we opted for a detailed questionnaire to gain the pertinent epidemiologic information.5,27 Further study is needed to establish such a method for accurately determining exposure levels to ETS. In conclusion, the present study showed that serum levels of C3c, Hp, and a1AG were significantly higher among boys exposed to high levels of ETS than among those exposed to low levels, suggesting that changes in the levels of these APP may reflect differences in exposure levels to ETS. Among girls, however, we found no significant changes in APP, except in a1AG. In previous studies, girls seemed to be more susceptible to indoor air pollution.11,30 Our results, on the contrary, showed that the effects of exposure to ETS on serum APP levels appeared to be greater among boys. This finding may, however, have been reflected by the small number of girls exposed to high levels of ETS. It has been shown that the pattern of the APP response is often sex dependent.8 The changes in serum APP in response to inflammation have been reported to be greater among males than among females.31 Therefore, to determine the significance of changes in these serum APP, the involvement of various factors, including sexrelated differences, should be further evaluated. REFERENCES 1. Holt PG, Turner KJ. Respiratory symptoms in the children of smokers: an overview. Eur J Respir Dis 1984;Suppl 133:109– 20. 2. Murray AB, Morrison BJ. The effect of cigarette smoke from the mother on bronchial responsiveness and severity of symptoms in children with asthma. J Allergy Clin Immunol 1986;77:575– 81. 3. Wang X, Wypij D, Gold DR, Speizer FE, Ware JH, Ferris BG Jr, et al. A longitudinal study of the effects of parental smoking on pulmonary function in children 6–18 years. Am J Respir Crit Care Med 1994;149:1420–5. 4. Forastiere F, Agabiti N, Corbo GM, Pistelli R, Dell’Orco V, Ciappi G, et al. Passive smoking as a determinant of bronchial responsiveness in children. Am J Respir Crit Care Med 1994;149:365– 70. 5. Jarvis M, Tunstall-Pedoe H, Feyerabend C, Vesey C, Salloojee Y. Biochemical markers of smoke absorption and self reported exposure to passive smoking. J Epidemiol Community Health 1984;38:335–9. 6. Henderson FW, Reid HF, Morris R, Wang OL, Hu PC, Helms RW, et al. Home air nicotine levels and urinary cotinine excretion in preschool children. Am Rev Respir Dis 1989;140:197–201. 7. Ehrlich R, Kattan M, Godbold J, Saltzberg DS, Grimm KT, Landrigan PJ, et al. Childhood asthma and passive smoking: urinary cotinine as a biomarker of exposure. Am Rev Respir Dis 1992;145:594–9. 8. Kushner I, Mackiewicz A. Acute phase proteins as disease markers. Dis Markers 1987;5:1–11. 9. Kauffmann F, Frette C, Annesi I, Oryszczyn MP, Dore MF, Neukirch F. Relationships of haptoglobin level to FEV1, wheez-
AP: PM
624
SHIMA AND ADACHI
ing, bronchial hyper-responsiveness and allergy. Clin Exp Allergy 1991;21:669–74. 10. Galdston M, Levytska V, Schwartz MS, Magnusson B. Ceruloplasmin: increased serum concentrations and impaired antioxidant activity in cigarette smokers, and ability to prevent suppression of elastase inhibitory capacity of alpha1-proteinase inhibitor. Am Rev Respir Dis 1984;120:258–63. 11. Shima M, Nitta Y, Adachi M. Association of outdoor and indoor nitrogen dioxide with pulmonary function in schoolchildren. J Epidemiol 1994;4:137–46. 12. Ferris BG. Epidemiology standardization project. II. Recommended respiratory disease questionnaires for use with adults and children in epidemiological research. Am Rev Respir Dis 1978;118 Suppl 6:7–53. 13. Adachi M, Kobayashi M, Iwasaki A, Nitta Y, Yoshida R. A new children’s respiratory disease questionnaire. 2. Study on noncompletion in self-completion method [in Japanese]. Jpn J Public Health 1983;30:581–8. 14. Bienvenu J, Sann L, Bienvenu F, Lahet C, Divry P, Cotte J, et al. Laser nephelometry of orosomucoid in serum of newborns: reference intervals and relation to bacterial infections. Clin Chem 1981;27:721–6. 15. Wilkinson L. SYSTAT: Statistics, version 5.2 edition. Evanston (IL): SYSTAT, Inc., 1992. 16. Petitti DB, Kipp H. The leukocyte count: associations with intensity of smoking and persistence of effect after quitting. Am J Epidemiol 1986;123:89–95. 17. Bridges RB, Wyatt RJ, Rehm SR. Effects of smoking on inflammatory mediators and their relationship to pulmonary dysfunction. Eur J Respir Dis 1986;69 Suppl 146:145–52. 18. Das I. Raised C-reactive protein levels in serum from smokers. Clin Chim Acta 1985;153:9–13. 19. Stiller-Winkler R, Kra¨mer U, Fiedler E, Ewers U, Dolgner R. C3c concentrations in sera of persons living in areas with different levels of air pollution in Northrhine–Westphalia (Federal Republic of Germany). Environ Res 1989;49:7–19.
/ a203$$2163
08-21-96 19:30:51
pma
20. Kauffmann F, Kleisbauer JP, Cambon-de-Mouzon A, Mercier P, Constans J, Blanc M, et al. Genetic markers in chronic air-flow limitation: a genetic epidemiologic study. Am Rev Respir Dis 1983;127:263–9. 21. Piessens MF, Marien G, Stevens E. Decreased haptoglobin levels in respiratory allergy. Clin Allergy 1984;14:287–93. 22. Lubega J, Davies TJ. A comparison of serum mucoprotein with serum a1 acid glycoprotein, haptoglobin, and a1 antitrypsin assays in monitoring inflammatory bowel disease. Clin Chim Acta 1990;188:59–70. 23. Duthie GG, Arthur JR, James WPT. Effects of smoking and vitamin E on blood antioxidant status. Am J Clin Nutr 1991; 53:1061S–3S. 24. Matthews TS, Soothill JF. Complement activation after milk feeding in children with cow’s milk allergy. Lancet 1970; ii:893 – 5. 25. Chandra RK. Prospective studies of the effect of breast feeding on incidence of infection and allergy. Acta Paediatr Scand 1979;68:691–4. 26. Vedal S, Schenker MB, Samet JM, Speizer FE. Risk factors for childhood respiratory disease: analysis of pulmonary function. Am Rev Respir Dis 1984;130:187–92. 27. Friedman GD, Petitti DB, Bawol RD. Prevalence and correlates of passive smoking. Am J Public Health 1983;73:401–5. 28. Coultas DB, Samet JM, McCarthy JF, Spengler JD. Variability of measures of exposure to environmental tobacco smoke in the home. Am Rev Respir Dis 1990;142:602–6. 29. Pe´rez-Stable E, Benowitz NL, MarıB n G. Is serum cotinine a better measure of cigarette smoking than self-report? Prev Med 1995;24:171–9. 30. Hasselblad V, Humble CG, Graham MG, Anderson HS. Indoor environmental determinants of lung function in children. Am Rev Respir Dis 1981;123:479–85. 31. Bosanquet AG, Chandler AM, Gordon AH. Effects of injury on the concentration of a1-macroglobulin and a2-macroglobulin in the plasmas of male and female rats. Experientia 1976; 32: 1348 – 9.
AP: PM