8. Sample collection
A total of 21 healthy subjects were recruited for this study, 13 male and 8 female individuals.
The mean age of the volunteers was 35± 10 years (range 28–51 years) and the body weight
between 50 and 105 kg; 7 subjects were non-smokers while the remaining were smokers.
Semen samples were collected by masturbation after 3 days of abstinence.
Unstimulated whole saliva samples were collected randomly during the day. (5–6 ml)
Urine samples were collected randomly during the day in a urine sterile box.
Venous blood was collected in sterile plastic vials.
All the collected BFs (saliva, semen, blood, and urine) were centrifuged at 13 000 rpm and 4
C for 10 min in order to separate the serum from the blood, separate the seminal fluid from
the semen, and remove large proteins, cells, and solid debris from the urine and saliva
samples.
Approximately 1 ml of the supernatant of all samples was dried using a Speed Vacuum
Concentrator (Eppendorf) and immediately stored at –80 C.
Furthermore, aliquots of venous blood were disposed in a permeable support (tissue) and were
allowed to coagulate; the spot was then eluted using 2 ml 0.9% NaCl solution overnight at 37
C and then stored at –80c
Before NMR analysis, frozen samples were reconstituted in 660 ml of D2O with 0.77 mM of TSP as
internal standard and then transferred into 5-mm NMR tube
