Abstract: In this research, we evaluated the effect of Slow Release Theophylline (SRT) therapy on lipoprotein a, cholesterol and triglycerides in children with bronchial asthma to asses their risk for atherosclerotic coronary heart disease. The study included 38 asthmatic children (26 males and 12 females) aged 6-13 years (mean±SD was 8.37±2.17) receiving SRT, mean duration of therapy was 10±2.5 mo. (Group I). Another 30 asthmatic children of the same age and sex but not receiving SRT (Group II) were included. Twenty healthy children of the same age and sex, not asthmatics, were recruited in the study as controls (Group III). All children were subjected to history taking, medical examination and assessment of serum level of lipoprotein a (Lp a), total cholesterol (Tc), HDL-c, LDL-c, VLDL-c and triglycerides. Theophylline level was assessed in the Group I. No statistically significant difference was found between asthmatics receiving SRT and controls as regards Lp (a), Tc, HDL-c, LDL-c, HDL-c/LDL-c, VLDL-c or triglycerides. HDL-c and HDL-c/LDL-c was significantly higher in asthmatics receiving SRT than asthmatics that did not (p = 0.002 and 0.009, respectively). No correlation was detected between serum theophyllin level and different lipid parameters apart from triglycerides and VLDL-c levels (was significantly positively correlated (r = 0.57, p = 0.04)). So, we can conclude that slow release theophylline therapy did not appear to alter lipid profile of asthmatic children receiving it and those children are not at increased risk for atherosclerotic coronary heart disease.
INTRODUCTION
Atherosclerosis in childhood has a slowly progressive course and its clinical features usually become prominent in middle ages. Hypercholesterolemia is one of the major risk factors for the development of atherosclerosis. A clear correlation exists between hypercholesterolemia in childhood and atherosclerotic lesions extending into adulthood (Uzuner et al., 2002).
Risk factors that have been identified for arteriosclerosis of the coronary arteries are hyperlipidemia and a decrease in high density lipoprotein cholesterol (HDL-c) level (Onaka, 1993) and increased low-density lipoprotein cholesterol (LDL-c). Lipoprotein a (Lp a) is a unique lipoprotein which is considered as an additional risk factor (independent of LDL-c) for Coronary Heart Disease (CHD) (Natoobhai and Sailesh, 1997). Some considered it a coronary risk factor only in the presence of traditional risk factors (Barghash et al., 2004). Its clinical importance is derived from its role in atherogenesis (Bahar et al., 2003). Lp (a) contributes to atherothrombotic risk by multiple mechanisms that include impaired fibronolysis, increased cholesterol deposition in the arterial wall and enhanced oxidation of low density lipoprotein cholesterol (Stein and Rosenson, 1997).
The previous data has raised the interest to screen children for hypercholesterolemia (Yagupsky et al., 1992). The American Heart Association (AHA) recommended in July 2002 that children should have checked their blood cholesterol by the age of five (Marshall et al., 2002).
Coffee drinking has been associated with increased serum cholesterol level in some, but not all studies (Sun et al., 2001). Theophylline, structurally an almost identical methylxanthine, is a bronchodilator utilized for many decades as bronchial asthma therapy, may induce similar changes. Theophylline remained the most widely prescribed anti- asthma drug worldwide in spite of other anti asthma medication (Barnes and Pauwels, 1994). The prevalence of asthma among Egyptian children aged 3-15 years was estimated to be 8.2% (Kheders, 1998; EL-Hefny et al., 1991).
In this study we tried to evaluate the effect of Slow Release Theophylline (SRT) therapy on different lipid parameters (lipoprotein a, cholesterol and triglycerides) in children with bronchial asthma to asses their risk for atherosclerotic coronary heart disease.
MATERIALS AND METHODS
The study included 38 asthmatic children (26 males and 12 females) aged 6-13 years (mean±SD was 8.37±2.17) receiving SRT (Group I) for at least 4 months before the study. Another 30 asthmatic children of the same age and sex but not receiving SRT (Group II) for at least 4 months before the study were also included. All asthmatic cases were recruited from allergy clinic, pediatric hospital-Cairo University, Cairo-Egypt. Asthma duration was between 3-12.5 years. All patients were receiving other asthma therapy (B2 agonist's preparation by inhalation or orally for the control of acute symptoms and inhaled corticosteroid therapy). Another 20 healthy children of the same age and sex were included in the study as a control group (Group III). All controls were recruited from pediatric clinic, National Research Centre. They have no symptoms or signs of bronchial asthma or family history of allergic diseases. The study done in the period, from February 2005 till December 2005.
All children were subjected to complete medical history (children with acute illnesses or chronic disease, or a history of cardiovascular, renal, liver disease or known history of hyperlipidemia or diabetes mellitus were excluded. Children with positive family history of premature CHD were also excluded). All patients and control were subjected to physical examination, including anthropometric measures (height, weight and calculation of body mass index (those with BMI >95th centile for age and sex were excluded), general examination (those with hypertension were excluded). System examinations with special emphasis on the respiratory system were performed. Measurement of Peak Expiratory Flow Rate (PEFR) using Mini-Right peak flow meter. The best reading from three forced expirations was recorded.
Skin prick tests were performed using a battery of common environmental allergens (Dome Hollisteir extracts) for asthmatic children. Chest X-rays were done to exclude any other chest condition.
Laboratory investigations: (Done at Clinical pathology laboratory, National Research Centre, Cairo, Egypt). After overnight fasting, blood samples were obtained from all children. Complete blood picture (for eosinophilic count) was assessed. Serum was separated by centrifugation and stored at -20°C. Total serum IgE was assessed for all children using IMx Microparticle Enzyme Immunoassay (MEIA).
Lipid parameters including total serum cholesterol (Tc), HDL-c, LDL-c, triglycerides and Lipoprotein (a) (Lp a ) were assessed for all children. Total serum cholesterol and Lipoprotein cholesterol contents were determined by the enzymatic-calorimetric method using cholesterol esterase, cholesterol oxidase, peroxidase and a chromagen (Allain et al., 1974). For measuring HDL-c, the major lipoproteins were precipitated using heparin-Mn (II) Leaving only HDL-c in solution. The precipitated lipoproteins were sedimented by centrifugation and the clear HDL-c containing supernatant recovered for cholesterol analysis. LDL-c was calculated {Total cholesterol TC- (HDL-c + triglycerides/5)}. HDL-c/LDL-c was calculated. There is no simple direct way to measure VLDL-c so, it is usually calculated as percentage of triglycerides level (VLDL-c = triglycerides/5) (Mayo, clinic, 2006).
Serum triglycerides levels were determined by the enzymatic-calorimetric method using lipoprotein lipase-glycerokinase, glycerophosphate oxidase and a chromogen, as described (Fossati and Prencipe, 1982).
Lipoprotein a was measured using INNO test Lp (a), an enzyme immunoassay (ELISA) from IMMUNOGENETICS with a monoclonal anti Lp (a) as the solid phase antibody and a sheep anti- apoB polyclonal antibody labelled with horse radish peroxidase.
For those receiving SRT therapy (Group I), blood sample was obtained at the time of the expected peak serum theophylline concentration (12 h after evening dose or 9 h after a morning dose at a steady state). Theophylline level was assessed in this group to check drug level and to ensure compliance among those children receiving it. Serum theophylline level was measured by a competitive fluorescence polarization immunoassay (TDX; Abott Laboratories Diagnostic Division, North Chicago, III).
Statistical analysis: SPSS for windows, version 7.0 computer program was used for statistical analysis. A p-value of less than 0.05 was considered statistically significant. One-way analysis of variance followed by post-hoc comparisons procedures were used to compare between 3 or more independent means. The t-test was used to compare between 2 independent means. Non parametric tests: Kruskal-Wallis Test and Mann-Whitney U-test were used when parametric tests couldn’t be used. Pearson Correlation Coefficient r was used to measure the linear relationship between two quantitative, normally distributed variables, while Spearman’s rho correlation coefficient was used when data were not normally distributed or have ordered categories.
RESULTS
The clinical and biochemical characteristics of all studied cases presented as ranges (Min and Max) and percentage were summarized in Table 1. The mean age (years) for cases receiving SRT was 8.37±2.17, for cases not receiving SRT was 9.1±1.97 and for controls was 8.78±2.3 (Not presented in the Table). The mean duration for asthma (years) was 6.71±2.57 in group receiving SRT and 6.33±2.92 in group not receiving it (Not presented in the Table).
| Table 1: | Clinical and biochemical characteristics of all studied cases |
| SRT Slow Release Theophylline, HDL-c high density lipoprotein cholesterol, LDL-c low density lipoprotein cholesterol, VLDL-c very low density lipoprotein cholesterol | |
| Table 2: | Skin tests for common inhalant and food allergens among all asthmatic children |
| SRT: Slow Release Theophylline | |
| Table 3: | Correlations between lipid parameters and different variables among asthmatic children |
| *p<0.05: Significant, Tc: Total cholesterol, HDL-c: High Density Lipoprotein cholesterol, LDL-c: Low Density Lipoprotein cholesterol, VLDL-c: Very Low Density Lipoprotein cholesterol, GP: Group | |
The mean duration for use of theophylline therapy (SRT) was 10±2.5 months and its mean serum level was 13.5±3.25 mcg mL-1 (Not presented in the Table).
Skin tests details for common inhalant and food allergens for asthmatic children were summarized in Table 2.
Details of different lipid parameters among all studied groups were shown in Fig. 1. No statistically significant difference was detected between asthmatics receiving SRT and controls as regards all lipid parameters lp (a), Tc, HDL-c, LDL-c, HDL-c/LDL-c, VLDL-c or triglycerides. Asthmatics without SRT had significant lower Tc level than controls (p = 0.02) and also less than asthmatics with SRT but not statistically significant. Both asthmatics with SRT and controls had significant higher levels of HDL-c and HDL-c /LDL-c ratio than asthmatics not receiving SRT (p = 0.002 and 0.009, respectively).
Triglycerides and VLDL-c were significantly positively correlated with age in group receiving SRT (p = 0.03). Total cholesterol was significantly positively correlated with age in group not receiving SRT (p = 0.049) (Table 3).
Table 4 showed the correlation between SRT and different lipid parameters in group receiving SRT. Theophylline level was only significantly positively correlated with triglycerides and VLDL-c (r = 0.57, p = 0.04).
| Fig. 1: | Lipid parameters among studied groups. HDL-c = High Density Lipoprotein cholesterol, LDL-c = Low Density Lipoprotein cholesterol, VLDL-c= Very Low Density Lipoprotein cholesterol. X-axis: Represent studied groups and Y-axis: Represent lipid parameters |
| Table 4: | Correlation between Theophylline level and lipid parameters |
| *p<0.05: significant | |
Low density lipoprotein cholesterol (LDL-c) was statistically significantly lower in male asthmatics (110.40±15.89) than female asthmatics (133±27.92) (p = 0.02). High density lipoprotein cholesterol (HDL-c) were higher in male asthmatics (33.66±11.99) than female asthmatics (24.29±8.02) but not statistically significant (p = 0.07) (Not presented in Table).
DISCUSSION
Determination of metabolic effects of theophylline on lipid parameters may contribute to the under standing of the effects of other methyl-xanthines, including caffeine on lipid metabolism. Several studies had demonstrated a clear association between heavy drinking of coffee or other caffeine consumption and increased risk of coronary artery disease (Grobbee et al., 1990). A dose- response relation between coffee consumption and both total cholesterol and LDL-c was identified (Sun et al., 2001).
Cholesterol level in our study showed no statistical difference between asthmatics receiving SRT and controls but it was significantly lower in asthmatics not receiving SRT than controls (p = 0.02) and less than asthmatics receiving SRT but didn’t reach statistical significance (Fig. 1). This agreed with (Shenoi et al., 1992). Yagupsky et al. (1992) reported higher levels of Tc in asthmatics receiving theophylline therapy. Uzuner et al. (2002) found increased total cholesterol in asthmatics receiving SRT than controls (contrary to our result) and than asthmatics with no SRT (agreed with our result). In our study cases and controls were not homogenous as regards socioeconomic status and geographical area and hence general diet may be the cause of lowering of Tc in asthmatics not receiving SRT.
Low density lipoprotein cholesterol is the major cholesterol carrier. It acts as a transporter for cholesterol and triglycerides to various cells and tissues throughout the body (Alexander et al., 2006). Too much LDL-c in blood can slowly build up in the wall of arteries feeding heart and brain, together with other substances; it can form hard deposits (AHA, 2006). LDL-c showed no statistical significant difference between asthmatics receiving SRT and controls or those not receiving SRT in our work (Fig. 1). This agreed with Schanen et al. (2005) in adults and Bahar et al. (2003) in cases with Obstructive Lung Disease (OLD) as they found no difference between cases and controls. On the contrary Uzuner et al. (2002) found increased mean LDL-c in patients receiving SRT and concluded that long term SRT may increase the risk for atherosclerotic CHD.
High density lipoprotein cholesterol binds and removes cholesterol from the cell membranes and its level relates negatively to the risk of atherosclerotic coronary artery disease in adults (Hamsten, 1988). No statistically significant difference was found between asthmatics receiving SRT and controls as regards HDL-c and HDL-c/LDL-c (Fig. 1). HDL-c and HDL-c/LDL-c were significantly lower in Group II (not receiving SRT) than Group I (receiving theophylline) and than controls (p = 0.02 and 0.009, respectively) (Fig. 1). On the contrary (Shenoi et al., 1992) found higher HDL-c in asthmatics than controls. Bahar et al. (2003) found no difference between cases with obstructive lung disease and controls. This controversy could be explained by different dietary habits or geographical distribution. Another explanation is that, in our study, HDL-c in male asthmatics were higher (33.66±11.99) than female asthmatics (24.29±8.02) but not statistically significant (p = 0.07) and most of Group I and III were males which may explain higher HDL-c among them than Group II. Also, increase percentage of males in Group I and controls than Group II (Table 1) with significant lower LDL-c in males (110.40±15.89) than females (133±27.92) (p = 0.02) could explain higher HDL-c/LDL-c in Group I and controls. Sammal et al. (1988) had shown that acute bacterial and viral infections in adults cause decrease in HDL-c in acute phase. HDL-c level is decreased in all species during acute-phase response (Hardardottir et al., 1995). Also stress-like acute myocardial infarction and burns had been reported to cause decrease in HDL-c and Tc levels (Clpin and Price, 1988). The previous data (viral infections and stress) could explain reduction of HDL-c in Group II than controls but did not explain the difference between Group I and II. The rise in HDL-c and HDL-c/LDL-c in Group I may reflect effect of SRT. It was reported that theophylline at serum concentration 10-20 μg mL-1 range modestly increase total cholesterol from 140-160 mg dL-1, HDL-c from mean 36-50 mg dL-1 and HDL-c/LDL-c from mean 0.5-0.7 (Purdue Pharmaceutical Product, 2004). Yagupsky et al. (1992). found significant higher HDL-c in asthmatics receiving SRT, with conclusion that SRT don’t increase atherosclerotic risk. Group II had very low HDL-c (Table 1 and Fig. 1) by the American standards, in age Group 1-14 the normal level is 35-84 mg dL-1 (John and Michael, 1992) so, SRT may protect asthmatics against atherosclerosis.
No statistical significant difference was found between asthmatics (receiving SRT or not) and controls as regards triglycerides (Fig. 1). This agreed with Bahar et al. (2003) among cases with obstructive lung disease.
Lipoprotein (a) excess has been identified as a powerful predictor of premature atherosclerotic vascular disease in several studies (Mohler and Rader, 2000) and elevated plasma lipoprotein (a) and cardiac events showed a modest but significant association in various clinical studies (Buechler et al., 2001). In our work Lp (a) showed no statistical significant difference between asthmatics receiving SRT and controls or asthmatics not receiving theophylline (Fig. 1). This disagreed with Yagupsky et al. (1992) as they found higher Lp (a) with SRT therapy and Mutius et al. (1998) who reported that serum lipoprotein (a) was positively related to asthma. Plasma levels of Lp (a) are genetically determined and increase slightly with age and vary by race (Joseph and Frolkis, 1999) which may explain variability in results.
Only age was positively correlated with triglycerides level in Group I (p = 0.03) and cholesterol level in Group II (0.049) (Table 3) as eating more fat increase by ageing.
In conclusion, slow release theophylline therapy did not appear to alter lipid profile of asthmatics receiving it and those children are not at increased risk for hypercholesterolemia or premature atherosclerotic coronary heart disease than general population.