Bengtsson B G, Kiessling K H, Smith-Kielland A, Mørland J
Eur J Biochem. 1981 Sep 1;118(3):591-7. doi: 10.1111/j.1432-1033.1981.tb05560.x.
Suspensions of enzymatically prepared hepatocytes from starved rats were separated according to their buoyant density at 12 degrees C in linear, isosmotic gradients of metrizamide, centrofuged at low speed for a relatively short time. The recovery of cell protein was 86%. Hepatocytes of high viability formed a single band around 1.10 g/cm3 and were recovered as four density populations (P1-P4) form low to high density, respectively. The content of protein was significantly lower in population P1, while the content of neutral fat or the averaged cell size was similar in the various populations. The specific activity of alanine aminotransferase increased in the order P1-P4. The distribution of this enzyme within the intact liver acinus obtained by others indicate that a partial separation of periportal and perivenous hepatocytes had occurred. The activity patterns of lactate dehydrogenase, glutamate dehydrogenase, isocitrate dehydrogenase (NADP+) and pyruvate kinase, also with known intra acinar distributions, supported this conclusion. The hepatocytes showed signs of shrinkage after separation, but since they retained a normal ultrastructure, most enzyme activities and viability, the present technique was regarded superior to previous procedures of hepatocyte separation by density. The degree of separation was calculated from an equation (see Appendix), and the periportal/perivenous ratio for parameters measured in density populations can be obtained. The specific activity of phosphofructokinase, alcohol dehydrogenase and aldehyde dehydrogenase showed no differences between populations. However, the ratio high-Km/low-Km aldehyde dehydrogenase increased in the order P4-P1.
将饥饿大鼠经酶法制备的肝细胞悬液，于12℃在甲泛葡胺的线性等渗梯度中，根据其浮力密度进行分离，以相对较低的速度离心较短时间。细胞蛋白回收率为86%。高活力的肝细胞在1.10 g/cm³左右形成一条带，并分别以四个密度群体（P1 - P4）从低密度到高密度回收。群体P1中的蛋白含量显著较低，而中性脂肪含量或平均细胞大小在不同群体中相似。丙氨酸转氨酶的比活性按P1 - P4的顺序增加。其他人获得的完整肝腺泡内该酶的分布表明，门周和中央静脉周围肝细胞已部分分离。乳酸脱氢酶、谷氨酸脱氢酶、异柠檬酸脱氢酶（NADP⁺）和丙酮酸激酶的活性模式，同样具有已知的腺泡内分布，支持了这一结论。分离后肝细胞出现收缩迹象，但由于它们保留了正常的超微结构、大多数酶活性和活力，因此本技术被认为优于以往的密度法分离肝细胞的方法。分离程度由一个方程式计算得出（见附录），并且可以得到密度群体中所测参数的门周/中央静脉周围比率。磷酸果糖激酶、乙醇脱氢酶和醛脱氢酶的比活性在各群体之间没有差异。然而，高Km/低Km醛脱氢酶的比率按P4 - P1的顺序增加。
