WE in ve s tigated th e in vitro
IntroductionHypox ia and re perfusion c ause compleme nt activation in animal ex perime nts and in clinical studie s. [1][2][3][4] It c ould be show n, that after c ytological damage, contac t w ith cellular compone nts such as mitochondria, or ex c ess of hydrox yl radicals, w as re spons ible for the activation of the compleme nt syste m. 5 ,6 On the othe r hand, in vitro studies show ed a compleme nt ac tivation by ac idosis or hypox ia only. [7][8][9] In a pre vious study, w e found complement ac tivatio n induc ed by lactic acid. 10 The aim of the pre sent in v itro study w as to inve stigate: (1) w he the r lac tate itself or metabolic acidosis is re sponsible for activation, and (2) w hethe r re spiratory acidosis is also able to ac tivate the complement syste m.
Material and MethodsBlood sample s (10 ml) from 12 healthy volunte ers (6 male and 6 female, age : 28 -40 years) w e re c ollec te d and 50 IE Heparin w as adde d. Each sample w as divide d into e ight portions and plac ed in polypropylen tube s.To inve stigate the influe nce of re sp iratory as w ell as me tabolic changes of pH on anaphylatox in formation, the p ortions w e re e quilibrated w ith differe nt gas mix tures and supplemented w ith acids re sulting in marked changes in pH, pCO 2 and base de ficit ( 11 A 6 ml polyprop ylen tube (6 ml) w as filled 1.5 to 2.0 ml blood and the end of a polyprop yle n tube w ith an inne r diame te r of 3 mm w as place d into the blood a few millimetre s above the bottom of the tube . Gas mix tures (O 2 , N 2 , CO 2 ) c omposed from me dic al gas es by f low me te rs w e re put through the tube into the blood sample. Gas flow w as adjuste d to one bubble pe r second for 20 min. Foam caused by bubbling w as c ontinuou sly re move d by a suction cathete r place d above the blood level. After e quilibration, the blood samples w e re transferred into different tubes for further proce ssing.For metabolic ac idosis 10 m mol hydrochloric ac id (No. 4; Sigma-Aldrich, De isenhofe n, Ge rmany) or 5.5 m mol lactate (No: 5; Sigma-Aldrich, Deise nhofen, Ge rmany) w ere adde d per ml of blood. To achie ve compe nsated me tabolic ac idosis , the sample w as equilibrate d w ith a hypoc apnic gas mix ture for 20 min and the n 5.5 m mol lac tate per ml of blood w as added (No. 8).Blood gas analysis, potassium and lactate c onc entrations w ere me as ure d by a Radiome te r Copenhagen ABL 505 (Willich, Ge rmany) using he parinise d syringes after all samples w ere incubated at 37°C for 1 h. To stop c omplement activation after inc ubation, w e added 1 mg EDTA dissolve d in purified w ater to each sample. Plasma w as se parated by ce ntrifugation at 0962-9351/98/060417-04 $9.00 © 1998 Carfax Publishing Ltd 417
Short CommunicationMediators of Inflammation, 7, 417-420 (1998) 4°C at 3000 g for 10 min and froze n immediate ly at -80°C. Anaphylatox ins C3a and C5a w ere dete rmined as pre viously desc ribed.1 2 C3a enzyme immunoassay (EIA, Fa. Progen Biote chnik GmbH, Heide lberg, Ge rmany) sel...