Bronshteyn V L, Steponkus P L
Department of Soil, Crop and Atmospheric Sciences, Cornell University, Ithaca, New York 14853.
Biophys J. 1993 Nov;65(5):1853-65. doi: 10.1016/S0006-3495(93)81250-5.
Differential scanning calorimetry (DSC) was used to determine the amount of water that freezes in an aqueous suspension of multilamellar dipalmitoylphosphatidylcholine (DPPC) liposomes. The studies were performed with dehydrated suspensions (12-20 wt% water) and suspensions containing an excess of water (30-70 wt% water). For suspensions that contained > or = 18 wt% water, two ice-formation events were observed during cooling. The first was attributed to heterogeneous nucleation of extraliposomal ice; the second was attributed to homogeneous nucleation of ice within the liposomes. In suspensions with an initial water concentration between 13 and 16 wt%, ice formation occurred only after homogeneous nucleation at temperatures below -40 degrees C. In suspensions containing < 13 wt% water, ice formation during cooling was undetectable by DSC, however, an endotherm resulting from ice melting during warming was observed in suspensions containing > or = 12 wt% water. In suspensions containing < 12 wt% water, an endotherm corresponding to the melting of ice was not observed during warming. The amount of ice that formed in the suspensions was determined by using an improved procedure to calculate the partial area of the endotherm resulting from the melting of ice during warming. The results show that a substantial proportion of water associated with the polar headgroup of phosphatidylcholine can be removed by freeze-induced dehydration, but the amount of ice depends on the thermal history of the samples. For example, after cooling to -100 degrees C at rates > or = 10 degrees C/min, a portion of water in the suspension remains supercooled because of a decrease in the diffusion rate of water with decreasing temperature. A portion of this supercooled water can be frozen during subsequent freeze-induced dehydration of the liposomes under isothermal conditions at subfreezing storage temperature Ts. During isothermal storage at Ts > or = -40 degrees C, the amount of unfrozen water decreased with decreasing Ts and increasing time of storage. After 30 min of storage at Ts = -40 degrees C and subsequent cooling to -100 degrees C, the amount of water associated with the polar headgroups was < 0.1 g/g of DPPC. At temperatures > -50 degrees C, the amount of unfrozen water associated with the polar headgroups of DPPC decreased with decreasing temperature in a manner predicted from the desorption isotherm of DPPC. However, at lower temperatures, the amount of unfrozen water remained constant, in large part, because the unfrozen water underwent a liquid-to-glass transformation at a temperature between -50 degrees and -140 degrees C.
差示扫描量热法（DSC）用于测定多层二棕榈酰磷脂酰胆碱（DPPC）脂质体水悬浮液中冻结的水量。研究使用了脱水悬浮液（含水量12 - 20 wt%）和含水量过量的悬浮液（含水量30 - 70 wt%）。对于含水量≥18 wt%的悬浮液，冷却过程中观察到两个结冰事件。第一个归因于脂质体外冰的异相成核；第二个归因于脂质体内冰的均相成核。在初始水浓度为13 - 16 wt%的悬浮液中，仅在温度低于 -40℃发生均相成核后才出现结冰。在含水量<13 wt%的悬浮液中，DSC检测不到冷却过程中的结冰现象，然而，在含水量≥12 wt%的悬浮液中观察到升温过程中冰融化产生的吸热峰。在含水量<12 wt%的悬浮液中，升温过程中未观察到对应冰融化的吸热峰。通过使用改进的程序计算升温过程中冰融化产生的吸热峰的部分面积来确定悬浮液中形成的冰量。结果表明，与磷脂酰胆碱极性头基团结合的大量水分可通过冷冻诱导脱水去除，但冰量取决于样品的热历史。例如，以≥10℃/min的速率冷却至 -100℃后，悬浮液中的一部分水由于温度降低导致水的扩散速率下降而保持过冷状态。在随后脂质体在低于冰点的储存温度Ts下等温冷冻诱导脱水过程中，这部分过冷水的一部分会结冰。在Ts≥ -40℃等温储存期间，未冻结水的量随Ts降低和储存时间增加而减少。在Ts = -40℃储存30分钟并随后冷却至 -100℃后，与极性头基团结合的水量<0.1 g/g DPPC。在温度>-50℃时，与DPPC极性头基团结合的未冻结水的量随温度降低而减少，其变化方式符合DPPC解吸等温线的预测。然而在较低温度下，未冻结水的量在很大程度上保持恒定，因为未冻结水在 -50℃至 -140℃之间的温度下发生了液 - 固转变。
