Buoncristiani U, Galli F, Rovidati S, Albertini M C, Covarelli C, Carobi C, Di Paolo N, Canestrari F
Nephrology and Dialysis Unit, R. Silvestrini Hospital, Perugia, Italy.
Perit Dial Int. 1996 Sep-Oct;16(5):511-8.
Using the erythrocyte as a model for other kinds of cells not directly exposed to peritoneal dialysis (PD) solutions, we investigated the tolerance of the cell metabolism to lactate and bicarbonate buffers.
We studied, in vivo (in two groups of 5 PD patients each) and in vitro, the Embden-Meyerhof pathway (EMP) because it represents a potential target for the unphysiological effects of lactate or bicarbonate buffers. The EMP is the main glucose-utilizing route in the red blood cell (RBC), producing energy and reducing power.
The enzymatic activities of the key steps in the glycolytic pathway and the energy charge (EC), determined by the levels of phosphorylated adenine nucleotides, were investigated spectrophotometrically and by high performance liquid chromatography (HPLC) in two groups of patients undergoing lactate (L-group) and bicarbonate (B-group) PD, respectively. The in vitro effects of both bicarbonate and lactate buffers on some EMP enzyme activities and energy production were determined. Cellular pH (pHi) was also investigated.
The B-group showed an EC value near the control levels, while in the L-group a significantly lower EC value was observed (t-test: p < 0.05 vs both B-group and controls). The key enzymes in the EMP, and in particular hexokinase, were higher in the L-versus B-group (p < 0.03 for the comparison of the Hk mean values). As demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the bound form of glyceraldehyde-3-phosphate dehydrogenase (G-3-PD), an inactive form of this EMP enzyme, was significantly higher in the L-group with respect to the B-group (p < 0.004). In the in vitro experiments, high lactate concentrations acutely inhibited the key enzymatic steps of glycolysis, producing a significant decrease in glucose consumption and adenosine triphosphate production. These effects were not observed when bicarbonate was used in the incubations. Both in vivo and in vitro lactate, but not bicarbonate, induce a significant drop in pHi (p < 0.05). Decreased levels of pHi like those observed in the lactate-incubated RBC were demonstrated to be able to inhibit G-3-PD activity (25 +/- 2%) here used as an indicator of the actual decrease in pH.
This study provides evidence for a damaging action of lactate with respect to bicarbonate buffer on the RBC metabolism. This condition was demonstrated observing a cell energy depletion, which coincides in vitro with an acute EMP impairment; the lactate accumulation together with the consequent lowering of pHi seem to be responsible for this effect, which was not observed when bicarbonate was used instead of lactate.
以红细胞作为未直接接触腹膜透析（PD）溶液的其他类型细胞的模型，我们研究了细胞代谢对乳酸盐和碳酸氢盐缓冲液的耐受性。
我们在体内（两组，每组5名PD患者）和体外研究了糖酵解途径（EMP），因为它代表了乳酸盐或碳酸氢盐缓冲液非生理性作用的一个潜在靶点。EMP是红细胞（RBC）利用葡萄糖的主要途径，可产生能量和还原能力。
分别通过分光光度法和高效液相色谱法（HPLC），研究了两组接受乳酸盐（L组）和碳酸氢盐（B组）腹膜透析的患者糖酵解途径关键步骤的酶活性以及由磷酸化腺嘌呤核苷酸水平决定的能荷（EC）。测定了碳酸氢盐和乳酸盐缓冲液对一些EMP酶活性和能量产生的体外影响。还研究了细胞内pH值（pHi）。
B组的EC值接近对照水平，而L组的EC值显著更低（t检验：与B组和对照组相比，p < 0.05）。EMP中的关键酶，尤其是己糖激酶，L组高于B组（Hk平均值比较，p < 0.03）。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析表明，EMP酶甘油醛-3-磷酸脱氢酶（G-3-PD）的结合形式（该酶的一种无活性形式）在L组中相对于B组显著更高（p < 0.004）。在体外实验中，高浓度乳酸盐急性抑制糖酵解的关键酶步骤，导致葡萄糖消耗和三磷酸腺苷产生显著减少。当在孵育中使用碳酸氢盐时未观察到这些效应。体内和体外，乳酸盐而非碳酸氢盐均导致pHi显著下降（p < 0.05）。如在乳酸盐孵育的RBC中观察到的那样，降低的pHi水平被证明能够抑制G-3-PD活性（25 +/- 2%），这里将其用作实际pH下降的指标。
本研究为乳酸盐相对于碳酸氢盐缓冲液对RBC代谢的损害作用提供了证据。观察到细胞能量消耗证明了这种情况，在体外这与急性EMP损伤一致；乳酸盐积累以及随之而来的pHi降低似乎是造成这种效应的原因，当使用碳酸氢盐而非乳酸盐时未观察到这种效应。
