ブックタイトル教育医学 J.Educ.Health Sci.　第63巻　第3号　通巻 第289号

概要

教育医学 J.Educ.Health Sci.　第63巻　第3号　通巻 第289号

Change in Oral Antimicrobial Peptides and Neuroendocrine Response by Intense Exerciseby extraction of a saliva sample from the cotton.The whole saliva volume (ml) was estimated byweighing to the nearest milligram and the salivadensity was assumed to be 1.0 g ml -1 . The salivaflow rate (mlmin -1 ) was represented as the wholesaliva volume. The saliva supernatant was storedat -80℃for subsequent assays.4. Saliva analysisAn enzyme-linked immunosorbent assay (ELISA)was used for measurement of the saliva concentrationsof HBD-2 (Humanβ-Defensin 2 ELISAKit, Phoenix Pharmaceuticals Inc., Burlingame,CA), LL-37 (Human LL-37 ELISA Test Kit, Hycultbiotechnology, Uden, The Netherlands), IgA(EIA-sIgA Test, Medical and Biological LaboratoriesCo., Ltd., Nagoya, Japan), and cortisol (ParameterCortisol Assay, R&D Systems, Minneapolis,MN), respectively. The present cortisol assaymeasured both the free and corticosteroid-bindingglobulin-bound forms of cortisol 23) .The quantification of HBD-2 and LL-37 insaliva was carried out using an enzyme-linkedimmunosorbent assay ? ELISA according tothe manufacturer?s instructions. Briefly, 100μl(0.25μg/ml) of specific antibody (anti-HBD-2or anti-LL-37) was added to the 96-well polystyreneELISA plates and incubated overnight (4℃).After washing four times with PBST (PBS with0.05% Tween 20), 300μl of a blocking solution(1% BSA in PBST) was added to the wellsand incubated for 1 h at room temperature. Theplates were washed and 100μl of the samplesor standards were added into the respective wellsin duplicate and the plates were incubated for 2h. After washing, 100μl of detection antibody(0.5μg/ml) was applied to the wells and the plateswere incubated for 2 h. After this period, the plateswere washed and 100μl of streptavidin-conjugatedhorseradish peroxidase (1:2000 in PBST)was added to the respective wells and incubatedfor 30 minutes. Colorimetric reactions were developedusing o-phenylenediamine in the presenceof 0.02% H2O2. Color development was stoppedusing H2SO4 (2N) and measured by an ELISAreader (OD 490 nm).The interassay coefficients of variation wereless than 15, 10, 7.4, and 21.1% for the HBD-2,LL-37, IgA, and cortisol assays, respectively. Theminimum detections limit for the HBD-2, LL-37, IgA, and cortisol assays were 7.815 pg ml-1,0.14 ng ml-1, 0.06μgml-1, and 0.030 ng ml-1,respectively. All the samples were assayed induplicate and an average of the absorbance valueswas used as the representative value.Secretion rate of each parameter in saliva wascalculated by multiplying the saliva flow rate bythe saliva concentration of each component. Thesaliva total protein concentration was analyzedusing the Lowry assay (24) (bicinchoninic acid(BCA) assay; Pierce, Rockford, IL) standardizedto bovine serum albumin (BSA), according to themanufacturer's protocol. The saliva protein secretionrate was calculated by multiplying the salivaflow rate by the saliva total protein concentration.Saliva osmolality was determined by freezingpoint depression using a cryoscopic osmometer(Osmomat 030, Gonotec, GbBH, Berlin, Germany)calibrated with a 300 mOsmol kg -1 NaClsolution. The interassay coefficients of variationfor the osmolality assay were less than 1%. Theamount of each parameter-to-protein ratio andthat-to-osmolality ratio was also obtained by dividingthe absolute concentration of each componentin saliva by the saliva protein concentrationsor by saliva osmolality.5. Blood analysisSupernatant of blood samples were immediatelytransferred into disodium EDTA-treated tubes formeasurement of the plasma norepinephrine concentration.The test tubes were then centrifugedat 3,000 rpm for 15 min at 4℃immediately aftercollection, and the blood samples were stored at- 80℃until assay. The norepinephrine level wasdetermined by a high-pressure liquid chromatog-? 230 ?