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

我们进一步尝试识别存在于低体温患者血液中的未知冷原。将患者血清分为高疏水（HA）和低疏水（LA）两部分，用商用柱进行疏水亲和层析。LA部分的白蛋白含量是HA部分的1/5。当将每种成分注射到大鼠体内时，每3只大鼠中就有1只出现明显的低体温。凝胶电泳显示，LA部分存在健康对照血清中未见的条带。然后，用质谱法直接对谱带进行分析。根据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。将两种蛋白（分别命名为A和B）用胰蛋白酶处理后，将样品注射到大鼠体内。5只大鼠中有2只腹腔注射胰蛋白酶处理过的蛋白A。样品的凝胶电泳显示了在低温患者血清中特有的条带的存在，尽管这些条带只有通过银染色才能略微识别出来。用胰蛋白酶处理蛋白A的方法多种多样，其中一种方法明显增强了凝胶电泳的特异带。然而，这些样品几乎没有引起大鼠体温过低。在不同系列的研究中，我们调查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。由此推测，患者血清蛋白酶活性高，蛋白A分解代谢物水平升高，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低温物质。

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.