*2.3. Laboratory Determinations*

− Blood samples were drawn from the patients and controls enrolled in the study, under basal conditions using a holder-vacutainer system. The blood samples were centrifuged at 3000× *g* for ten minutes and the supernatant was frozen at −80 ◦C. The hemolyzed or lactescent samples were rejected.

The serum levels of sialic acid were determined using resorcinol-chlorohydric acid. Blue chromophore was extracted with n-butyl/n-butanol acetate and the optical density was measured at 580 nm with the Sigma reactive (SIALICQ Kit) and the BS3000 analyzer (SINNOWA Medical Science and Technology, Nanjing, China). The results were expressed as mg/dL.

The determination of lipid-bound sialic acid (LSA) levels was performed as follows: 50 µL serum was diluted with 150 µL of cold distilled water. There was added 3 mL of chloroform:methanol (*v*/*v*) 2:1, at 4 ◦C. The extraction and partition were made after adding 0.5 mL of cold distilled water. After separating the phases by centrifugation, sialic acid was titred with resorcinol-chlorhidric acid.

The serum levels of beta-galactoside 2,6-sialyltransferase I (ST6GalI) (E.C.2.4.99.1) were assessed by ELISA method-sandwich variant (IBL. Co., Ltd., 27762 kit, Okayama, Japan) using a Tecan analyzer (Männedorf, Switzerland). The method is sensible (0.20 ng/mL), reproducible (95–97%), both intra-assay coefficient of variation (CV) and inter-assay CV are less than 15%. It has large limits of detection (1.09–70 ng/mL). The technique uses two kinds of highly specific antibodies. The colorimetric evaluation of the final product was made at a wavelength of 450 nm. The concentration of ST6GalI in the samples was determined by comparing the optical density of the samples to the standard curve. The results were expressed as ng/mL serum.

The serum levels of human sialidase-3 (NEU3) (E.C.3.2.1.18) were assessed by ELISA method-sandwich variant (Mybiosource. Cat. No:MBS 9368355) using a Tecan analyzer (Männedorf, Switzerland). The method is sensible (0.1ng/mL), reproducible (95–97%), both intra-assay CV and inter-assay CV are less than 15%. It has large limits of detection (0.25 ng/mL–8 ng/mL). The colorimetric evaluation of the final product was made at a

wavelength of 450 nm. The concentration of NEU3 in the samples was determined by comparing the optical density of the samples to the standard curve. The results were expressed as ng/mL serum.
