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

我们进一步试图确定存在于一名体温过低患者血液中的未知冷源(S)。患者的血清被分成两个组分，高疏水组分(HA)和低疏水组分(LA)，使用商用疏水亲和层析色谱柱。LA组分中的白蛋白含量是HA组分的1/5。当将每一组分注射到大鼠体内时，每3只大鼠中就有1只产生明显的低温。凝胶电泳法显示，健康对照组血清中未见LA组分的条带。然后提取谱带，直接进行质谱分析。根据对人类基因组数据库的搜索，在该条带中鉴定出两种类型的蛋白质片段。用胰酶处理A和B两种类型的蛋白质，并将样品注射到大鼠体内。5只大鼠中有2只经胰酶处理的蛋白A经腹腔注射后出现体温过低。凝胶电泳法显示，体温过低的患者血清中有一条特殊的条带，尽管银染仅能轻微识别这些条带。用不同的方法用胰酶处理蛋白A，其中一种方法明显增强了凝胶电泳法的特异性条带。然而，这些样品几乎没有引起大鼠体温过低。在不同的系列研究中，我们调查了患者血清中的蛋白酶活性，并观察到其活性水平似乎比年龄匹配的健康人高2倍。因此推测患者血清中较高的蛋白水解酶活性提高了蛋白A分解代谢的水平，这可能具有低温作用。然而，需要进一步的深入研究来确定体温过低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.