Anales de la RANM

58 A N A L E S R A N M R E V I S T A F U N D A D A E N 1 8 7 9 SERUM MYELOPEROXIDASE AND PARKINSON´S DISEASE Fernández-Espejo E An RANM. 2022;139(01): 56 - 66 the modified Hoehn-Yahr staging, and the Interna- tional Parkinson and Movement Disorder Society- Sponsored revision of the Unified Parkinson’s Disease Rating Scale (MDS-UPDRS). Antipar- kinsonian medication was expressed as levodopa equivalent dose (LED, mg per day), according to an established formula (27). Individuals suffering from liver, renal, hemato- logical, and cardiovascular dysfunctions, as well as malabsorption, dementia, autoimmune diseases, acquired immunodeficiency syndrome, cancer, infectious conditions or taking statins or anti-inflam- matory drugs were excluded from the study (peroxi- dase markers may be altered in such conditions). Since alcohol, tobacco and caffeine can also alter oxidative biomarkers in biofluids, all participants were non-alcohol drinkers, non-smokers, and non-coffee drinkers, according to established criteria (28, 29). 2.3. Single-photon emission computed tomography study SPECT studies were performed according to a standardized protocol. All SPECT scans were performed, quantitatively analyzed, and visually assessed by expert physicians at the Service of Nuclear Medicine. The radioligand 123 I-Ioflupane was used for classifying the extent and degree of dopaminergic cell loss. 123 I-Ioflupane has a high binding affinity for presynaptic dopamine- transporter or DAT (30-34). Four hours after intravenous injection of 185 MBq of 123 I-Ioflupane, scans were acquired on a double-head gamma- camera (Symbia-T2, Siemens) using high-resolu- tion and low energy collimators, 180º, 128 projec- tions, 30 s per view, and a 128 x 128 matrix. Data were reconstructed by filtered backprojec- tion, transferred to a workstation for quantitative analysis (Syngo software). All individual patient studies were co-registered automatically to a mean template derived from studies of healthy volunteers. This template was used to define a 3-dimensional ROI map for the striatum, the caudate nucleus, the putamen, and the occipital cortex (OC) as reference region. Specific binding ratio (SBR) of the radioli- gand was calculated by substracting the mean counts per pixel in the occipital cortex (OC), from the mean counts per pixel in the basal ganglia nucleus of reference (ROI) and dividing the results by the mean counts per pixel in the OC ([ROI-OC]/OC). SBR was calculated for the 4 striatal regions (left caudate, right caudate, left putamen, right putamen), as well as the striatum (left striatum, right striatum). The relation between observed SBR versus SBR as expected for individual’s age and gender was calculated. Afterwards, this value was transformed into percentage reduction of SBR (%rSBR) as follows: %rSBR = (100-([observed SBR/expected SBR] x 100)). Expected SBR value was calculated by using the formulas proposed by the European multicenter database of healthy controls for [123I]FP-CIT SPECT (ENC-DAT study, promoted by the European Association of Nuclear Medicine) (33, 34), as follows: Females: Expected SBR in the striatum = 7.15 - (0.03 x age in years) Expected SBR in the caudate nucleus = 7.232 - (0.0273 x age) Expected SBR in the putamen = 7.116 - (0.0339 x age) Males: Expected SBR in the striatum = 6.75 - (0.03 x age) Expected SBR in the caudate nucleus = 6.8 - (0.02733 x age) Expected SBR in the putamen = 6.702 - (0.0339 x age) Theminimum putamen (calculated as the minimum SBR value from either the left or right putamen) was determined, and a subject was considered to have a normal DAT binding if minimum putamen SBR was >80% versus SBR as expected for age and gender. A subject was considered to have a deficit in DAT binding if the putamenal SBR was less than 65% as predicted for individual’s age and gender (26). Regarding subjects with percentage reduction of SBR between 65 and 80% relative to normal, visual interpretation was used to determine eligibility, following established criteria (25, 26, 32-34). 2.4. Serum and cerebrospinal fluid collection and ELISA analysis Myeloperoxidase was measured in blood serum and the cerebrospinal fluid. Blood was obtained by venipuncture. Five ml of blood were collected in gel-coated tubes to obtain serum (BD Vacuotainer, Madrid). Serum was centrifuged at 3000 rpm during 10 min, and then it was frozen at -80ºC in 0.5 ml aliquots. CSF was collected by expert physicians by using lumbar puncture. Two ml of CSF were collected, aliquoted, coded, and rapidly frozen at -80ºC. A 0.5-ml fresh collection was employed to observe the absence of traumatic puncture, and to quantify red cells (CSF with >500 red cells/microL was discarded). Serum and CSF aliquots were unfrozen and sonicated with homogenizing solution (150 mM NaCl, 50 mM HEPES, 1 mM phenyl- methylsulfonil-fluoride, 0.6µm leupeptin, 1% Triton X-100, pH 7.4). Myeloperoxidase concentration was evaluated with an Enzyme-linked Immunosorbent Assay kit (Myeloperoxidase Human Instant ELISA™ Kit, BMS2038INST, ThermoFisher Scientific, USA), following manufacturer’s instructions. The sensiti- vity of the ELISA assay is 26 pg/ml. Each sample was analyzed in duplicate (serum, 1/50 dilution; CSF, undiluted). 2.5. Statistics Two groups were compared with the Student’s t test (quantitative variables) or the χ 2 test (dichot-