Subjects and the sera collection
According to ICD–10, 3 psychiatrists identified major depression patients without psychotic symptoms (DP; 4 women and 2 men, aged from 48 to 62, average 53 years-old), manic patients without psychotic symptoms (MA; 2 women and 4 men, aged from 45 to 62, average 46 years-old) and schizophrenia patients (SZ; 3 women and 3 men, aged from 33 to 59, average 43 years-old) from the inpatients and the outpatients of Department of Neuropsychiatry, Akita University Hospital. They were medicated with antidepressants, lithium carbonate, or atypical antipsychotics for 4–12 weeks. They did not suffer from physical diseases at the identification. These patients and healthy volunteers not-suffering from the psychoses (Positive Control; 3 women and 3 men, aged from 28 to 62, average 38 years-old) agreed to participate in the present study, under the intensive informed consent with preservation of their anonymity and guarantee of the withdrawal agreement. A 2 ml of venous blood was individually collected from their arm vein by a medical doctor, under watching of the other medical doctors. The sera were pooled and restored at 4 ºC. All of these procedures were conditioned in accordance with Clinical Study Ethics Committee, Graduate School of Medicine, Akita University (the approval number: 2042).
Humoral lipid fractionation
Humoral lipid fractionation was performed as previously described [4]. Briefly, 1.25 ml of chloroform and 2.5 ml of methanol were added to each 1 ml of the pooled serum. The solution was intensively mixed for 3 min and incubated for 10 min at room temperature (RT). Then, 1.25 ml of chloroform was added to the solution, and followed by intensive mixing for 30 s. A 1 ml of water was added to the solution, and followed by intensive mixing for another 30 s. The mixture was then centrifuged at 150 gravities for 10 min at RT. The lower chloroform layer was collected, and the solvent chloroform was evaporated at RT. The extracted lipids were then suspended in 1 ml of water. The solution was applied to 0.5 ml of an ion exchanger DE–52 (Whatman Co., Maidstone, UK) column, which had been saturated with 10 mM NaHCO3, pH8.3, and washed with water. Samples were eluted with 0.5 ml consecutive washes of 50, 100, 150, 200, 250, and 300 mM NaCl. Fractions eluted with 50, 100, 150, and 250 mM NaCl were then diluted to 1 ml with water as the present samples.
Sulfate-radical elimination
Stress-coping humoral glycolipids fractionated with 100 and 250 mM NaCl are sulfated. Sulfate-radical was eliminated from the glycolipids for measuring the terminal sugar-chain reactivity as previously described [4]. Briefly, lipids were extracted again from 800 µl of the sample solutions by using methanol-chloroform method as described above. The extracted lipids were added 400 µl of the reagent containing silyl-agents of TMS-HT kit (Tokyo Chemical Industry Co. Tokyo, Japan), and then, incubated at 90 ℃ for 3 h. The solutions were added 800 µl water, and intensively mixed for 30 s.
Measurement of the glycolipid production
Bipolar glycolipids attach to plastic plates in 50 % ethanol solution. A modified Enzyme-Linked ImmunoSorbent Assay (ELISA) was performed for measuring the glycolipids production as previously described [4]. Briefly, the sample solution obtained from the fraction eluted with 50 or 150 mM NaCl, the sulfate-radical-eliminated sample solution obtained from the fraction eluted with 100 or 250 mM NaCl, and physiological saline (PS) as Negative Control, were prepared to 50 % ethanol solution. 100 µl of the solution was poured into a well of a 96-well plastic plate (Sumitomo-Bakelite Co., Tokyo, Japan). The ELISA was performed with the use of 300 µl of 5 % bovine serum albumin (Sigma-Aldrich Co., St. Louis, MO, USA) as a blocker, a biotinized-lectin of Macckia amurensis recognizing Sialalpha2–3Gal, that of Recinus communis recognizing Galbeta1–4GlcNAc, that of Dolichos biflorus recognizing GalNAcalpha1–3GalNAc or that of Aleuria aurentia recognizing Fucalpha1–2Glc, peroxidase-conjugated-avidin (Seikagaku Co., Tokyo, Japan), and the coloring kit (Sumitomo Bakelite Co.). Then, the light absorbance was measured at the dual wavelength of 450/655 nm. The ELISA procedure was individually performed on different 5 plates.
Statistical analyses
Mann-Whitney U-test was used for detecting difference from Positive Control. A p<0.05 was considered as a significant difference.