The aim of this study was to investigate the difference in accumulation of AGEs (advanced glycation end-products) in the tissues of individuals who smoke in comparison with individuals who do not as well as of individuals who consume caffeine versus those who do not, and finally to examine the probable differences concerning participants' haemodynamic parameters.

Materials and methods Study population The study population consisted of students of Health Care Professions who accepted to participate in the study and gave their informed consent for their participation. The population study included healthy volunteers without cardiovascular problems, other chronic or systemic diseases. Participants were divided into 3 intervention groups: the first group are non-smokers who consume caffeine on daily basis, the second group are smokers who do not consume caffeine on daily basis, and the third group are smokers who consume caffeine on daily basis. Additionally, there was a control group consisting of non smoking students and no caffeine users on daily basis.

Methodology A complete medical history with information about the health status of the subjects, drug use and the main risk factors for cardiovascular conditions was recorded. Details concerning smoking and drinking habits, caffeine intake and other medical conditions and conditions from the individual and family history were retrieved, by filling-in self-reporting questionnaires. Information about dietary habits and physical activity of the subjects was also recorded. Body weight and height of all participants were measured. Also, participants were informed that on the day of the scheduled measurements, they should not have breakfast nor smoke for 2 hours (for smokers) prior to measurements and finally abstain from any physical activity. Inclusion criteria in the group of smokers were cigarette smoking of more than 10 cigarettes/day for at least 2 years.

Skin- autofluorescence measurement Tissue AGEs accumulation was assessed using a validated skin-autofluorescence (skin-AF) device, namely AGE Reader. In short, the AGE reader illuminates a skin surface of approximately 4 cm2, guarded against surrounding light, with an excitation light source between 300 and 420 nm (peak excitation ~370 nm). Skin-AF was measured at the volar side of the lower arm at approximately 10-15 cm below the elbow fold. Care had been taken to perform the measurement at normal skin site (without visible vessels, scars, lichenification, or other skin abnormalities). The investigators performed three measurements with a 5min interval between them and the investigators calculated the mean value of three measurements. For the individuals who smoked, these measurements were repeated 2 hours after the smoking of one cigarette and for individuals who consumed caffeine, measurements were repeated after the intake of 100 mg caffeine (delivered as a beverage). This was done to check whether there was a change in accumulation of AGEs following cigarette smoking and caffeine intake.

Haemodynamic parameters' measurement The haemodynamic parameters' measurement included measurement of cardiac output, stroke volume, systolic and diastolic blood pressure, mean pressure, pulse rate, interbeat intervals, arterial compliance and systemic vascular resistance. These measurements were performed with the Finometer device (FMS, Finapress Medical System, BV, Netherlands), while the data were recorded by using the Beatscope, a specially designed software, to transfer data to the PC and to process pressure waveform files. The measurements were performed by using a non-invasive method via a finger cuff, which was wrapped around the finger of the individual, who was in a lying down position for 10 min before the measurements in a room of a sound-attenuated, temperature-controlled environment, in order to ensure that all measurements will be performed under constant and stable conditions. Subsequently, the haemodynamic parameters' measurement was performed for 15 min in a sitting position. Additionally, for smokers the measurements were repeated after 15-30 min since smoking one cigarette and after 45 min for individuals who consumed caffeine.

Inclusion Criteria:

• Healthy individuals who smoke ≥ 10 cigarettes per day
• Healthy non smoking individuals consuming caffeine on daily basis
• Healthy individuals, smoking and consuming caffeine on daily basis

Exclusion Criteria:

• Individuals suffering from any chronic disease (diabetes mellitus, cardiovascular diseases, etc)