Diurnal variations in serum neopterin levels are associated with the pineal hormone melatonin circadian rhythm in healthy human subjects

Neopterin (2-amino-4-oxo-6[d-eritro-1,2,3,trihidroxipropil]-pteridin), is a metabolite derived from guanosine triphosphate biopterin pathway [1]. Activation of human monocytes/macrophages, on stimulation mainly with interferon-gamma secreted by stimulated T helper type 1 lymphocytes, enhances neopterin synthesis and release [2]. It has been previously reported that both immune cell number and functions may vary in the 24-hr circadian period [3, 4] as well as cytokine secretion and activity are under central neuroendocrine control [5]. The pineal gland, particularly, plays an important role in the neuroendocrine regulation of immunity through the light/dark secretion of its main hormone melatonin (N-acetyl-5-methoxytryptamine) whose highest levels occurred shortly after midnight [6]. Data about diurnal variations of neopterin serum levels are scarce. Since pineal activity plays an essential integrator role in the chronomodulation of the biological system, it might be possible that circadian production of melatonin influences the neopterin serum levels. There are no reports concerning the possible relevance of melatonin in relation to the diurnal variations of serum neopterin on healthy human subjects. Seventy subjects (37 men and 33 women), 57–73 yr of age, were selected from individuals without cardiovascular and cancer disease (by careful history, physical examination and ECG) who visited our outpatient clinic for a routine clinical and laboratory investigation. The investigation conformed with the principles outlined in the Declaration of Helsinki, and it has been approved by the ethics committee of the Hospital Universitario de Canarias (Canary Islands, Spain). Subjects had normal kidney and liver functions and were free of any sleep disorders as determined by history, actigraphy, and baseline polysomnography. All participants were screened for history depression, mania, psychosis, epilepsy, and thyroid disorders; they had no evidence of drug use by urinary toxicology screens; and they had no indication of infection or disease by blood chemistry tests. Medical history and examinations revealed no chronic or debilitating medical conditions. Subjects were studied in the coronary care unit under strictly controlled light/dark conditions. The light/dark period in the coronary unit was 14 hr (1745 ± 33 lx):10 hr (1.33 ± 0.3 lx); lights on at 07:00 hr. Venous blood samples were obtained through a catheter with a heparin lock implanted in the antecubital vein. Blood samples for neopterin and melatonin were collected at 09:00 hr (light period) and 02:00 hr (dark period). Nocturnal blood sampling was carried out by a trained nurse, in the dark when the subjects were sleeping, with the help of a minute torch with a dim red light (intensity <30 lx) avoiding any light directed to the patient’s face. Serum samples were aliquoted into several tubes and stored at )80 C until analysis. Melatonin levels in serum samples were measured using an enzyme-immunoassay kit after the samples had been extracted with chloroform (DRG Instruments GmbH, Marburg, Germany). Neopterin levels in serum samples were measured using an enzyme-immunoassay kit (DRG Instruments GmbH, Marburg, Germany). SPSS for Windows was used to perform statistical analyses. Light/dark changes in melatonin and neopterin were analyzed by a general ANOVA (SPSS Inc., Chicago, IL, USA). Mean serum light/dark levels of neopterin in relation to those of melatonin are illustrated in Fig. 1. As we expected, mean nocturnal concentrations of melatonin were