Dependence of exhaled breath composition on exogenous factors, smoking habits and exposure to air pollutants.

Non-invasive disease monitoring on the basis of volatile breath markers is a very attractive but challenging task. Several hundreds of compounds have been detected in exhaled air using modern analytical techniques (e.g. proton-transfer reaction mass spectrometry, gas chromatography-mass spectrometry) and have even been linked to various diseases. However,the biochemical background for most of compounds detected in breath samples has not been elucidated; therefore, the obtained results should be interpreted with care to avoid false correlations. The major aim of this study was to assess the effects of smoking on the composition of exhaled breath. Additionally, the potential origin of breath volatile organic compounds (VOCs) is discussed focusing on diet, environmental exposure and biological pathways based on other's studies. Profiles of VOCs detected in exhaled breath and inspired air samples of 115 subjects with addition of urine headspace derived from 50 volunteers are presented. Samples were analyzed with GC-MS after preconcentration on multibed sorption tubes in case of breath samples and solid phase micro-extraction (SPME) in the case of urine samples. Altogether 266 compounds were found in exhaled breath of at least 10% of the volunteers. From these, 162 compounds were identified by spectral library match and retention time (based on reference standards). It is shown that the composition of exhaled breath is considerably influenced by exposure to pollution and indoor-air contaminants and particularly by smoking. More than 80 organic compounds were found to be significantly related to smoking, the largest group comprising unsaturated hydrocarbons (29 dienes, 27 alkenes and 3 alkynes). On the basis of the presented results, we suggest that for the future understanding of breath data it will be necessary to carefully investigate the potential biological origin of volatiles, e.g., by means of analysis of tissues, isolated cell lines or other body fluids. In particular, VOCs linked to smoking habit or being the results of human exposure should be considered with care for clinical diagnosis since small changes in their concentration profiles(typically in the ppt(v)­ppb(v) range) revealing that the outbreak of certain disease might be hampered by already high background.

Body mass index in a large cohort of patients assigned to age decades between <20 and ≥80 years: relationship with cardiovascular morbidity and medication.

OBJECTIVE: There is an ongoing debate about the relationship between obesity and morbidity in the elderly, the clinical relevance of overweight and obesity in older patients and the need or harms of treatment. The main purpose of our study was to investigate whether a higher BMI is associated with a worse cardiovascular risk in all age groups, especially in the older ones. SUBJECTS AND DESIGN: We performed a retrospective evaluation of clinical data from 3926 patients who visited a medical outdoor center for diagnostic and/or therapeutic interventions in the period from January 1995 to July 2010. Patients were assigned to eight age groups of one decade from <20 years to ≥80 years. RESULTS: The Body Mass Index (BMI) of our patients showed a continuous increase with increasing age with peak values in the age decade 61-70 years (26.29 ±4.42 kg/m2). This was paralleled by an increase in cardiovascular events and need for continuous medication, demonstrating peak values in the age decade 61-70 years (22.3% in the female and 24.7% in the male group). In all age decades up to 80 years the BMI values were higher in patients with events compared to those without it. multivariable linear regression analysis - including confounding variables (blood pressure, fasting glucose, HDL-cholesterol, triglycerides, physical activity, smoking) - revealed for all age groups a strong positive relation of BMI and a negative relation of fat free mass (FFM) to the probability for a cardiovascular event and need for medication. CONCLUSION: In all age groups, the percentage of cardiovascular events was directly correlated with the BMI. Having in mind the transition to an aging society, therapeutic and preventive strategies should, therefore, include weight management strategies also for the elderly.

Breath acetone-aspects of normal physiology related to age and gender as determined in a PTR-MS study.

The present study was performed to determine the variations of breath acetone concentrations with age, gender and body-mass index (BMI). Previous investigations were based on a relatively small cohort of subjects (see Turner et al 2006 Physiol. Meas. 27 321-37). Since exhaled breath analysis is affected by considerable variation, larger studies are needed to get reliable information about the correlation of concentrations of volatiles in breath when compared with age, gender and BMI. Mixed expiratory exhaled breath was sampled using Tedlar bags. The concentrations of a mass-to-charge ratio (m/z) of 59, attributed to acetone, were then determined using proton transfer reaction-mass spectrometry. Our cohort, consisting of 243 adult volunteers not suffering from diabetes, was divided into two groups: one that fasted overnight prior to sampling (215 volunteers) and the other without a dietary control (28 volunteers). In addition, we considered a group of 44 healthy children (5-11 years old).The fasted subjects' concentrations of acetone ranged from 177 ppb to 2441 ppb, with an overall geometric mean (GM) of 628 ppb; in the group without a dietary control, the subjects' concentrations ranged from 281 ppb to 1246 ppb with an overall GM of 544 ppb. We found no statistically significant shift between the distributions of acetone levels in the breath of males and females in the fasted group (the Wilcoxon-Mann-Whitney test yielded p = 0.0923, the medians being 652 ppb and 587 ppb). Similarly, there did not seem to be a difference between the acetone levels of males and females in the group without a dietary control. Aging was associated with a slight increase of acetone in the fasted females; in males the increase was not statistically significant. Compared with the adults (a merged group), our group of children (5-11 years old) showed lower concentrations of acetone (p < 0.001), with a median of 263 ppb. No correlation was found between the acetone levels and BMI in adults. Our results extend those of Turner et al's (2006 Physiol. Meas. 27 321-37), who analyzed the breath of 30 volunteers (without a dietary control) by selected ion flow tube-mass spectrometry. They reported a positive correlation with age (but without statistical significance in their cohort, with p = 0.82 for males and p = 0.45 for females), and, unlike us, arrived at a p-value of 0.02 for the separation of males and females with respect to acetone concentrations. Our median acetone concentration for children (5-11 years) coincides with the median acetone concentration of young adults (17-19 years) reported by Spanel et al (2007 J. Breath Res. 1 026001).

The analysis of healthy volunteers' exhaled breath by the use of solid-phase microextraction and GC-MS.

We analysed breath and inhaled room air samples from 39 healthy volunteers (28 non-smokers, 8 smokers and 3 ex-smokers) by SPME-GC-MS. Mixed expiratory and indoor air samples were collected in freshly cleaned Tedlar bags. Eighteen millilitres of each sample were transferred into sealed, evacuated glass vials, preconcentrated by solid-phase microextraction (SPME, carboxen/polydimethylsiloxane) and investigated by gas chromatography with mass spectrometric detection (GC-MS). For the unequivocal identification of potential marker compounds, pure calibration mixtures of reference compounds (depending on commercial availability) were prepared to determine the retention time and mass spectra with respect to our analytical setting. Applying the adapted SPME-GC/MS method with limit of detection in the high ppb range (0.05-15.00 ppb), we succeeded in identifying altogether 38 compounds with concentrations in exhaled breath being at least 50% higher than concentration in inhaled air. From these 38 compounds, 31 were identified not only by the spectral library match but also by retention time of standards. A comparison of retention times and spectrum obtained for standards and determined compounds was performed. We found hydrocarbons (isoprene, 2-pentene, 2-methyl-1-pentene, benzene, toluene, p-cymene, limonene, 2,4-dimethylheptane, n-butane), ketones (acetone, hydroxypropanone, methylvinyl ketone), ethers (dimethyl ether, 1,3-dioxolane), esters (ethyl acetate), aldehydes (propanal, hexanal, heptanal, acrolein) and alcohols (ethanol, 2-metoxyethanol, isopropyl alcohol, 2,2,3,3- tetramethylcyclopropanemethanol, 3,4-dimethylcyclohexanol). Proper identification of compounds in different cohorts of patients and volunteers is the base for further investigation of origin, biochemical background and distribution of potential breath biomarkers.

The metabolic syndrome as a link between smoking and cardiovascular disease.

OBJECTIVE: Smoking is associated with a significant increase in the cardiovascular risk. The possible relationship of smoking with insulin resistance might further enhance the cardiovascular risk of the patients and is therefore of great clinical interest. DESIGN, SETTING AND SUBJECTS: We have retrospectively evaluated data of 3804 non-diabetic men attending a medical outdoor clinic. Clinical [body mass index (BMI), percentage of body fat, waist-to-hip ratio] and laboratory results were compared between smokers (n = 124) and non-smokers (n = 1915) without cardiovascular disease, as well as between smokers (n = 759) and non-smokers (n = 1006) with cardiovascular disease. RESULTS: Smokers without clinically manifest cardiovascular disease revealed significantly higher fasting glucose (5.8 +/- 0.6 mmol/l) and triglyceride levels (1.8 +/- 0.9 mmol/l) than non-smokers (fasting glucose: 5.1 +/- 0.7 mmol/l, p < 0.010; triglycerides: 1.5 +/- 0.8 mmol/l, p < 0.030). The adverse metabolic profile of smokers was even more pronounced in patients with cardiovascular disease. An age-matched analysis of smokers could demonstrate that cardiovascular patients revealed higher BMI values (27.3 +/- 2.4 kg/m2) and a higher percentage of body fat (25.5 +/- 5.5%) than those without cardiovascular disease (BMI: 25.7 +/- 2.2 kg/m2, p < 0.010; percentage of body fat: 23.0 +/- 5.5%, p < 0.030). CONCLUSION: In men with and without clinically manifest cardiovascular disease, smoking was associated with a metabolic profile indicating a higher degree of insulin resistance.

The influence of obesity on the frequency and distribution of medication.

Obesity is a serious health problem in industrialized countries and is associated with a significant increase in total health care costs. Only few data are available about the costs of drug therapies in patients with an increased body weight treated under clinical routine procedures. Such data could support efforts to intensify obesity prevention and treatment programmes in order to reduce comorbidities and costs. We have evaluated body mass index (BMI), diagnosis, and medication in 3360 outpatients (2175 women and 1185 men; mean age: 56.7 +/- 17.5 years). All patients underwent physical examinations, including BMI determination, and provided a detailed record concerning medication. In 1809 patients, the percentage of body fat content was measured with a bioimpedance method (OMRON BF 302 body fat monitor). Continuous variables were compared using the t-test or Wilcoxon U-test. Frequency distributions were compared using chi-squared tests. With respect to BMI, most of the patients (n = 1793; 53 %) were overweight or obese, 1349 (40 %) showed a normal BMI and 218 (7 %) a low BMI. The majority of cardiovascular (61 %), rheumatological (61.1 %) and metabolic (60.4 %) medication was administered to overweight and obese patients. Parallel findings could be obtained by analysing the percentage of body fat and the frequency of medication. Overall, 82.5 % of all medication was given to patients with a body fat content >20 %. Our results support the importance of weight-reduction programmes in order to prevent an overall increase in the costs of medication as a consequence of overweight and obesity.

Tumour necrosis factor-alpha plasma levels in elderly patients with Type 2 diabetes mellitus-observations over 2 years.

AIMS: The cytokine tumour necrosis factor-alpha (TNF-alpha) is involved in the development of obesity-linked insulin resistance. TNF-alpha plasma levels rise with increasing age and might thus also be related to metabolic control in Type 2 diabetes mellitus. We have studied the relationship of TNF-alpha plasma levels to glycaemic control in elderly patients with Type 2 diabetes over 2 years. METHODS: Clinical and laboratory data of 53 patients (26 women, 27 men) with Type 2 diabetes (mean age 71.6 +/- 5.6 years) were regularly evaluated over 2 years, and the relationship to anti-diabetic treatment regimens analysed. TNF-alpha plasma level was measured by a solid-phase enzyme amplified sensitivity immunoassay. RESULTS: TNF-alpha plasma levels increased significantly from 16.2 +/- 9.6 pg/ml at baseline to 28.0 +/- 13.8 pg/ml after 2 years (P = 0.028). HbA1c values also increased from 6.4 +/- 1.2% to 7.7 +/- 1.6% (P = 0.046). Mean body mass index of the patients remained almost constant, while a moderate increase in the percentage of body fat (34.5 +/- 7.0% to 35.3 +/- 6.9%; P= 0.061) and in waist-hip ratio was observed (0.86 +/- 0.04 to 0.88 +/- 0.04; P= 0.052). After adjustment for covariates multivariate analysis demonstrated that TNF-alpha plasma levels are positively related to the HbA1c values of the whole study population at the baseline control and after 2 years. TNF-alpha also revealed a positive correlation to the percentage of body fat. CONCLUSIONS: In elderly patients with Type 2 diabetes TNF-alpha plasma levels revealed a continuous increase during an observation period of 2 years. This increase in TNF-alpha plasma levels might add another aspect to the worsening of glycaemic control in the progression of Type 2 diabetes.

Tumour necrosis factor-alpha plasma level in patients with type 1 diabetes mellitus and its association with glycaemic control and cardiovascular risk factors.

OBJECTIVES: Diabetic patients reveal a significant increase in their cardiovascular risk. Beside glycaemic control and management of established risk factors, determination of cytokines, like serum levels of tumour necrosis factor-alpha (TNF-alpha), might offer a tool to determine patients at high risk. The cytokine TNF-alpha reveals a complex relationship with diabetes. It is involved in beta-cell damage leading to type 1 diabetes, causes insulin resistance associated with obesity and is of influence in the formation of atherosclerotic vascular lesions. We were interested in the possible association of this cytokine with metabolic control and cardiovascular risk factors in patients with type 1 diabetes. DESIGN AND SUBJECTS: TNF-alpha plasma levels were determined in 44 outdoor patients (15 women, 29 men) with type 1 diabetes mellitus (mean duration 11.2 +/- 8.7 years) and in 24 healthy controls by use of a solid phase enzyme amplified sensitivity immunoassay (TNF-alpha ELISA, Biosource Fleurus, Belgium). None of our study participants suffered from inflammatory or other concurrent diseases. Relationships between variables were evaluated by non-parametric Spearman correlation coefficients. RESULTS: TNF-alpha plasma levels were significantly higher in diabetic patients (19.3 +/- 7.5 pg mL-1) than in non-diabetic subjects (11.1 +/- 5.8 pg mL-1; P < 0. 023), and revealed a significant positive correlation with glycated haemoglobin (HbA1c) (r = 0.43; P < 0.004) and fructosamine (r = 0. 31; P < 0.049) values, and a negative correlation with HDL cholesterol (r = -0.36; P < 0.018) and apoAI-levels (r = -0.37; P < 0.015). These relationships could be observed in patients with a duration of diabetes for more than 5 years, as well as in patients with a shorter duration of diabetes. In the male group, TNF-alpha plasma levels revealed a significant positive correlation with plasma levels of thiobarbituric acid reacting substances (r = 0.61; P < 0.001). Plasma levels of thiobarbituric acid reacting substances showed a positive correlation with the duration of diabetes (r = 0. 58; P < 0.008), as well as with the serum levels of the vascular adhesion molecules intercellular adhesion molecule (ICAM) (r = 0.34; P < 0.051) and vascular cell adhesion molecule (VCAM) (r = 0.30; P < 0.052). CONCLUSIONS: Our data indicate that TNF-alpha plasma levels are increased in type 1 diabetes mellitus and reveal a significant association with metabolic long-term control parameters, HbA1c and fructosamine for glycaemic control, and HDL cholesterol for triglyceride metabolism, as well with lipid peroxidation.

The correlation of office blood pressure and 24-hour ambulatory measurements in hypertensive patients - comparison between non-pharmacological treatment and antihypertensive medication.

Blood pressure control in many hypertensive patients remains imperfect, also because routine office blood pressure can only give limited information about diurnal variations and nocturnal dipping. It was the aim of our evaluation to study the efficacy of antihypertensive therapy and the correlation between repeated office blood pressure values and 24-hour ambulatory measurements in hypertensive outdoor patients treated by life-style modification and antihypertensive medication. Clinical data and blood pressure values in 343 outdoor patients who were admitted to the medical centre for diagnostic and therapeutic procedures in hypertension were evaluated. Database was created from 1991 to 1998. The study population (mean age 59.5 +/- 11.6 years) comprised 153 men and 190 women, 141 (41%) were treated by life-style modification, 202 (59%) received antihypertensive medication. 57 patients showed symptoms of a metabolic syndrome, 62 suffered from manifest diabetes mellitus type 2. - Repeated office blood pressure measurements showed a significant positive correlation to the systolic and diastolic values obtained by 24-hour blood controls. While diastolic night minima revealed a positive correlation to office measurements (R = 0. 211; P <0.05), systolic night minima showed no correlation to office pressure control. In the whole study population and in subgroups (metabolic syndrome, diabetes mellitus) patients under antihypertensive medication still revealed significantly higher mean 24-hour systolic blood pressure values (140.5 +/- 16.9 mm Hg) than patients treated by life-style modification (133.0 +/- 14.4 mm Hg; P <0.001). Diastolic day- and night-time difference (dipping) was less pronounced in patients with antihypertensive medication. For appropriate antihypertensive therapy 24-hour blood pressure measurements are thus of advantage to repeated office controls especially to optimize medication for high systolic blood pressure values and adapt therapy to the nocturnal decrease of blood pressure values (dipping).

Prognostic value of antibody titre to heat-shock protein 65 on cardiovascular events.

Accumulating evidence suggests that the immune system is involved in atherogenesis, such as the correlation of the antibody titre to heat shock protein (hsp) with atherosclerotic lesions in the carotid and coronary arteries. Because the prognostic value of the hsp antibody titre for future cardiovascular events has not been evaluated until now, we performed a follow-up study on 195 subjects without a history of established cardiovascular risk factors (e.g. hypercholesterolaemia, diabetes, smoking), recruited for hsp antibody titre determination in 1995. Cardiovascular events were defined as unstable angina with the need for hospitalisation, myocardial infarction, re-vascularisation (PTCA, bypass), stroke and cardiovascular death. Among 79 men with coronary artery disease defined by coronary angiography, hsp antibody titres were significantly higher in those with future cardiovascular events (467.0 +/- 56.3) than in patients without further events (351.0 +/- 23.3; p < 0.049). Because anti-hsp-antibody titres might be of prognostic value for coronary artery disease, patients with an increased hsp antibody titre should obtain intensive management of classical risk factors.

Changes of serum antibodies to heat-shock protein 65 in coronary heart disease and acute myocardial infarction.

Accumulating evidence indicates the involvement of heat shock proteins (hsp), a family of stress-inducible proteins, in atherosclerosis. For carotid atherosclerosis an association with an increase in hsp65 antibodies has been demonstrated. To investigate whether such antibodies are also associated with coronary heart disease (CHD) and acute myocardial infarction (MI), an age- and sex-matched study with patients suffering from CHD (n = 114) and MI (n = 89) and healthy controls (n = 76) was performed. All study participants (n = 279) were consecutively recruited according to typical diagnostic criteria. Determination of antibody titres to hsp65 was performed by an enzyme-linked immunosorbent assay (ELISA). Hsp65 antibody titres in CHD showed a significant increase compared to the healthy control group (P = 0.029), however, hsp65 antibody titres were found to be significantly lower in acute MI, compared to CHD (P = 0.005). Alteration in hsp65 antibody titres showed no correlation to established cardiovascular risk factors, e.g. serum total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides, blood pressure, smoking, alcohol intake and body weight. In conclusion, serum concentrations of hsp65 antibodies were elevated independently in coronary heart diseases and declined in patients with acute myocardial infarction, indicating a possible involvement of the antibodies in the pathogenesis of this disease.

Long-term effect of intensified insulin treatment on lipid parameters in diabetes mellitus type I.

Premature atherosclerosis is often found in patients with diabetes mellitus (DM) type I, and alterations in lipid metabolism seem to play an important role in the development of this complication. Intensified insulin therapy improves glycemic control parameters significantly. To evaluate the effect of this optimized insulin treatment (OIT) not only on glycemic control, but also on plasma lipids, 24 patients with DM type I (19 men and 5 women, 18 to 61 years) were switched from a standard insulin therapy to a regimen of OIT which has been maintained for more than 3 years now. After 2 years on OIT a reduction of HbA1c values from 8.1% to 7.5% (p less than 0.01) was accompanied by an increase in HDL cholesterol from 52 to 67 mg/dl (p less than 0.05) and a decrease of triglyceride levels from 319 to 67 mg/dl (p less than 0.001). At the end of the second year on OIT some of the patients exhibited a reversal of the favorable trend in HbA1c and lipid values. Intensified instructions regarding the implementation of OIT were therefore repeated and resulted in a renewed improvement of overall HbA1c, HDL cholesterol and triglyceride levels to 6.43%, 67 mg/dl, and 78 mg/dl, respectively. Our findings underline the value of OIT not only for glycemic control, but also for the control of plasma lipids considered to be major risk factors for coronary artery disease.