Analysis of unknown cryogens existing in the blood of a hypothermic patient

我们进一步尝试识别低温患者血液中存在的未知冷冻剂。将患者血清分为高疏水性（HA）和低疏水性（LA）两个组分，用商业疏水亲和色谱柱。LA级分中的白蛋白量是HA级分中白蛋白量的1/5。当将每种组分注射到大鼠中时，其在3只大鼠中的1只中产生明显的低温。LA组分的凝胶电泳显示存在健康对照血清中未观察到的条带。然后，提取条带并通过质谱法直接分析。通过对人类基因组数据库的检索，在该条带中鉴定出两种类型的蛋白质片段。用胰蛋白酶处理两种类型的蛋白质（命名为A和B），并将样品注射到大鼠体内。腹膜内注射胰蛋白酶处理的蛋白质A在5只大鼠中的2只中产生体温过低。样品的凝胶电泳显示存在低温患者血清中特有的条带，尽管这些条带仅通过银染色略微识别。用胰蛋白酶处理蛋白A，有一种方法明显增强了凝胶电泳中的特异性条带。然而，样品几乎没有引起大鼠体温过低。在不同系列的研究中，我们研究了患者血清的蛋白酶活性，并观察到活性水平似乎比年龄匹配的健康人高2倍。因此，推测患者血清的高蛋白酶活性提高了蛋白A催化剂的水平，其可能具有低温作用。然而，需要进一步深入的研究来确定低温患者血液中存在的冷冻剂。

We further attempted to identify unknown cryogen(s) existing in the blood of a hypothermic patient. The patient's serum was divided into two fractions high-hydrophobic (HA) and low-hydrophobic fractions (LA), with a commercial column for hydrophobic affinity chromatography. The amount of albumin in the LA fraction was 1/5 of that in the HA fraction. When each fraction was injected into rats, it produced clear hypothermia in one out of 3 rats. Gel electrophoresis of the LA fraction showed that the presence of bands which were not seen in the serum of healthy controls. Then, the bands was taken and directly analyzed by mass spectrometry. According to the search of human genome database, fragments of two types of proteins were identified in the band. The twp types of proteins (named A and B) were treated with trypsin and the samples were injected into rats. Intraperitoneal injections of the trypsin-treated protein A produced hypothermia in 2 rats out of 5. Gel electrophoresis of the sample showed the presence of bands which were peculiarly seen in the hypothermic patient's serum, although the bands were slightly recognized only by silver staining. Protein A was treated with trypsin in various methods and one method overtly enhanced the specific bands in gel electrophoresis. However, the samples hardly induced hypothermia in rats. In different series of study, we investigated protease activities of the patient's serum, and observed that the activity level appeared to be greater by 2 times than that of age-matched healthy humans. It is therefore speculated that the high protease activity of the patient's serum elevates the levels of protein A catabolites, which may have a cryogenic role. However, further intensive studies are needed to identify the cryogens existing in the hypothermic patient's blood.