Bordoni V, Bolgan I, Brendolan A, Crepaldi C, Gastaldon F, D'intini V, Pilotto L, Inguaggiato P, Bonello M, Galloni E, Everard P, Bellomo R, Ronco C
Department of Nephrology, San Bortolo Hospital, Vicenza, Italy.
Int J Artif Organs. 2003 Oct;26(10):897-905. doi: 10.1177/039139880302601005.
Pro-apoptotic molecules are generated during sepsis which may be responsible for alteration of organ function in sepsis. Removal of systemic apoptotic activity may affect recovery from sepsis. Current high flux membranes might not be sufficiently permeable to eliminate pro-apoptotic factors. We evaluated the elimination of pro-apoptotic factors induced by LPS in human whole blood by a super-permeable cellulose triacetate membrane (SUREFLUX FH 150, Nipro, Osaka, Japan) in comparison to a standard high flux cellulose triacetate membrane (UT 700, Nipro, Osaka, Japan) and a polyethersulfone plasmafilter (Bellco, Mirandola Italy) in an in vitro blood circulation. We spiked human whole blood with lipopolysaccharide from Escherichia coli (Serotype 026-86, 10 mg/ml), incubated it for 3 hours to allow cytokine generation and recirculated it at 300 ml/min for 3 hours. The UF line was first returned to the blood module at 10 min. After this, the UF was drained from 10 to 60 min at a rate of 1000 ml/h. Zero balance was obtained by re-infusion of bicarbonate buffered hemofiltration fluid. Apoptosis was assessed on U937 monocytes (incubated with plasma or ultrafiltrate) by fluorescence microscopy dyes (Hoechst 33342, propidium iodide) and annexin V flow cytometry. Caspase-3 and Caspase-8 activity was assessed on the recirculated blood monocytes by spectrophotometric methods. IL-2, IL-10 and TNFalpha were determined by commercially available ELISAs. Sieving coefficients and clearances were determined for the different cytokines. Caspase-3 and Caspase-8 were activated by LPS and remained either stable or increased during in vitro circulation. Apoptosis activity of U937 cells, when incubated with the ultrafiltrate, increased in parallel with arterial plasma values (for Uf: UT700 = 23.1%; Sureflux FH150 = 42.5%). However, by 60 min the apoptotic activity recorded with the ultrafiltrate was reduced to the levels of arterial plasma (for Uf: UT700 = 19.8%; Sureflux FH150 = 11.2%). Sieving coefficients in the super-permeable membrane were significantly higher for all measured cytokines in comparison to the standard high flux membrane (e.g. TNFalpha 0.72 vs 0.03 p < 0.001) and close to the values observed for the plasmafiltration membrane. Nevertheless protein losses measured by albumin leakage were much lower with the Sureflux filter in comparison to the plasmafilter. In conclusion, pro-apoptotic factors can be eliminated by dialytic membranes with the removal rate maximized by using super high flux dialysers which may represent a compromise between hemofiltration and plasmafiltration membranes.
促凋亡分子在脓毒症期间产生，这可能是脓毒症器官功能改变的原因。消除全身凋亡活性可能会影响脓毒症的恢复。目前的高通量膜可能没有足够的通透性来清除促凋亡因子。我们评估了一种超渗透三醋酸纤维素膜（SUREFLUX FH 150，日本大阪日机装公司）与标准高通量三醋酸纤维素膜（UT 700，日本大阪日机装公司）和聚醚砜血浆滤器（意大利米兰多拉贝尔科公司）相比，在体外血液循环中对人全血中脂多糖诱导的促凋亡因子的清除情况。我们用人全血加入大肠杆菌脂多糖（血清型026 - 86，10 mg/ml），孵育3小时以产生细胞因子，然后以300 ml/min的速度再循环3小时。超滤管路在10分钟时首先回到血液模块。在此之后，在10至60分钟以1000 ml/h的速度排出超滤液。通过回输碳酸氢盐缓冲的血液滤过液实现零平衡。通过荧光显微镜染料（Hoechst 33342、碘化丙啶）和膜联蛋白V流式细胞术对U937单核细胞（与血浆或超滤液一起孵育）进行凋亡评估。通过分光光度法对再循环血液单核细胞中的半胱天冬酶 - 3和半胱天冬酶 - 8活性进行评估。白细胞介素 - 2、白细胞介素 - 10和肿瘤坏死因子α通过市售酶联免疫吸附测定法测定。测定了不同细胞因子的筛系数和清除率。半胱天冬酶 - 3和半胱天冬酶 - 8被脂多糖激活，并且在体外循环期间保持稳定或增加。当与超滤液一起孵育时，U937细胞的凋亡活性与动脉血浆值平行增加（对于超滤液：UT700 = 23.1%；Sureflux FH150 = 42.5%）。然而，到60分钟时，超滤液记录的凋亡活性降低到动脉血浆水平（对于超滤液：UT700 = 19.8%；Sureflux FH150 = 11.2%）。与标准高通量膜相比，超渗透膜中所有测量细胞因子的筛系数显著更高（例如肿瘤坏死因子α 0.72对0.03，p < 0.001），并且接近血浆滤过膜观察到的值。然而，与血浆滤器相比，Sureflux滤器通过白蛋白泄漏测量的蛋白质损失要低得多。总之，促凋亡因子可以通过透析膜清除，使用超高通量透析器可使清除率最大化，这可能代表血液滤过膜和血浆滤过膜之间的一种折衷。
