Era, Seiichi

Hayashi, Tomoya

Murakami, Masataka

Tokushima University Institutional Repository

INTRODUCTIONOne of structural characteristics in human serumalbumin is a single reactive free sulfhydryl group inposition 34. In extracellular fluid such as serum andinterstitial fluid, albumin is a mixture of oxidizedforms and reduced form having free sulfhydryl group(1). The oxidized form of albumin is induced by oxi-dization of free sulfhydryl group due to several oxi-dants such as cystine, oxidized glutathione and hy-drogen peroxide. It has been reported that the sulf-hydryl group in albumin is highly oxidized in aging,exercise and renal dysfunctions, having attractedclinical application as an extracellular marker of oxi-dative stress (2-4). However, the oxidization mecha-nism including the site within organ still remainsto be explained. The isolated rat salivary gland (5)allows us to sample venous effluent from the organ,interstitial fluid and saliva, thus useful for under-standing the site of oxidization through capillary,across vascular wall or through paracellular route.In this study, to clarify the oxidization mechanismof albumin in the living system, we isolated a ratsubmandibular gland and perfused it vascularly withthe perfusate containing commercial human albu-min. Then, the collected samples were analyzed bya specially designed system of high-performanceliquid chromatography (HPLC) which could sepa-rate albumin into reduced and oxidized forms (2, 3).MATERIALS AND METHODSThe isolation and perfusion of rat submandi-bular gland was followed with the procedure ofMurakami, et al. (5). The isolated gland was per-fused with HEPES buffered solution containing 2%commercial human albumin and a secretory stimu-lant (carbachol) for secretion of saliva. After onsetof secretory stimulation, venous effluent, intersti-tial fluid and saliva were collected to microcen-trifuge tubes. Each sample was immediately storedat -80until HPLC measurements. HPLC meas-urements were carried out by solvent gradient elu-tion with increasing ethanol concentration from 0to 10% in 0.05 M sodium acetate - 0.40 M sodiumsulfate (pH 4.85) by using of an anion - exchangeHPLC column. Samples were injected to the HPLCsystem without chemical modification for detec-tion of reduced and oxidized forms of albumin.RESULTS AND DISCUSSIONHuman albumins were able to detect in venouseffluent, interstitial fluids and saliva from isolatedrat submandibular gland. Under 1.0 μM carbacholstimulation during 60 min, the concentrations oftotal albumin in venous effluent, interstitial fluids,and saliva were 315.52.5, 195.112.7 and 1.0EXPANDED ABSTRACTPreliminary study of modification of serum albumin throughparacellular pathway of rat submandibular glandTomoya Hayashi1, Masataka Murakami2, and Seiichi Era31Department of Physiology, Meiji University of Integrative Medicine, Kyoto, Japan ; 2Nano-structurePhysiology, National Institute of Physiological Science, Okazaki, Japan ; and 3Department of Physi-ology and Biophysics, Gifu University Graduate School of Medicine, Gifu, JapanKeywords : salivary gland, albumin, paracellular pathwayJ. Med. Invest. 56 Suppl. : 393-394, December, 2009Received for publication October 17, 2009 ; accepted October24, 2009.Address correspondence and reprint requests to TomoyaHayashi, PhD, Department of Physiology, Meiji University of Inte-grative Medicine, Hiyoshi -cho, Nantan-shi, Kyoto 629-0392,Japan and Fax : +81-771-72-0326.The Journal of Medical Investigation Vol. 56 Supplement 20093930.3 μM, respectively (n=5). The concentration insaliva was approximately 0.05-0.7% of albumin inthe perfusate. Despite the evidence that the rat sali-vary gland has no intrinsic secretion system of hu-man albumin, small amount of the albumin in salivawas detected. Additionally, variation in the concen-trations was large, which indicated that albumin con-centration in saliva might depend on individual dif-ferences in rat. These facts suggested that the hu-man albumin in saliva might be passed through theparacellular pathway such as tight junction in ratsubmandibular gland. In this study, albumin couldbe separated to three fractions of reduced form, re-versible oxidized form and irreversible oxidizedform. In time course in saliva secretion by 20 min,concentrations of total albumin in saliva were 0.70.2, 0.50.1 and 0.70.2 μM during 0-20 min, 20-40 min and 40-60 min from onset of carbacholstimulation, respectively (n=10). Redox states in al-bumin in the same time course were measured. Afraction of irreversible oxidized form which wasmixed with sulfenic acid, sulfinic acid and sulfonicacid, was increased as compared with same form ofalbumin in the perfusate. The irreversible oxidizedforms were 9.52.1, 15.64.1 and 13.53.6% of to-tal albumin in saliva during 0-20, 20-40 and 40-60min from onset of 1.0 μM carbachol stimulation,respectively (n=10). The well known pathway forsulfenic acid formation is the reaction of a thiol withoxidants such as hydrogen peroxide and peroxyni-trite (6). In this study, increase of the oxidized formwhich was mixed with sulfenic acid, sulfinic acid andsulfonic acid suggested that reactive oxygen speciessuch as hydrogen peroxide oxidized directly freesulfhydryl group of albumin in intercellular spacesin salivary gland. In conclusion, several evidencesstrongly suggested that the human albumin in sa-liva might be passed through tight junction in therat submandibular gland and oxidative modificationof the albumin might be occurred through theparacellular pathway.REFERENCES1. Sogami M, Nagaoka S, Era S, Honda M,Noguchi K : Resolution of human mercapt- andnonmercaptalbumin by high-performance liq-uid chromatography. Int J Pept Protein Res 24 :96-103, 19842. Hayashi T, Era S, Kawai K, Imai H, NakamuraK, Onda E, Yoh M : Observation for redox stateof human serum and aqueous humor albuminfrom patients with senile cataract. Pathophysi-ology 6 : 237-243, 20003. Soejima A, Matsuzawa N, Hayashi T, KimuraR, Ootuska T, Fukuoka K, Yamada A,Nagasawa T, Era S : Alteration of redox state ofhuman serum albumin before and after hemo-dialysis. Blood Purif 22 : 525-529, 20044. Musante L, Bruschi M, Candiano G, PetrettoA, Dimasi N, Boccio PD, Urbani A, Rialdi G,Ghiggeri GM : Characterization of oxidationend product of plasma albumin ‘in vivo’. Bio-chem Biophys Res Commun 349 : 668-673,20065. Murakami M, Shachar-Hill B, Steward MC,Hill AE : The paracellular component of waterflow in the rat submandibular salivary gland. JPhysiol 537 : 899-906, 20016. Carballal S, Radi R, Kirk MC, Barnes S,Freeman BA, Alvarez B : Sulfenic acid forma-tion in human serum albumin by hydrogenperoxide and peroxynitrite. Biochemistry 42 :9906-9914, 2003T. Hayashi, et al. Modification of albumin in rat saliva394

Preliminary study of modification of serum albumin through paracellular pathway of rat submandibular gland

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Preliminary study of modification of serum albumin through paracellular pathway of rat submandibular gland

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