改良亚甲基蓝法测定血浆硫化氢

Modified methylene blue method for measurement of hydrogen sulfide level in plasma

硫化氢(hydrogen sulfide,H2S)作为新的气体信号分子参与多系统生理及病理生理功能的调节,并受到学术界广泛关注。但迄今为止,血液中H2S浓度的检测依旧是本领域的一大难题。本文旨在建立一种简易的改良亚甲基蓝分光光度计法,提高亚甲基蓝法检测H2S浓度的敏感性。亚甲基蓝法测定血浆H2S时,大量血浆蛋白会影响测定结果。本方法先加入过量醋酸锌溶液使血浆中游离H2S、HS、S2形成ZnS沉淀,并使血浆蛋白变性析出;加入5mol/L NaOH强碱溶解变性的蛋白,而ZnS沉淀不溶于强碱;离心去除大量蛋白质;再加入0.2%N,N-二甲基对苯二胺盐酸盐溶液使ZnS完全溶解,用三氯乙酸沉淀剩余蛋白质,最后用分光光度计法测定H2S浓度。结果显示,各种离子甚至硫酸根离子以及硫代硫酸根离子不影响该方法检测的特异性。而向样本中添加4.5%白蛋白可降低检测数值,表明H2S与蛋白质结合影响本方法的检测,而且该方法不能检测全部与蛋白结合的H2S。该方法回收率为81.9%,表明该方法的精确度基本达到要求。利用该方法检测到65名健康志愿者的冻存血浆样本H2S浓度为(13.93±4.98)μmol/L,且SD大鼠新鲜和冻存后血浆H2S浓度之间没有明显差异。本方法能够较为准确地检测血浆中H2S的含量,具有稳定性好,灵敏度高,操作简单,需要样本少,可实现高通量等优点,能够用于临床及基础研究中血浆H2S的检测。

In past decade, hydrogen sulfide （H2S） as a novel gasotransmitter, covered many fields in biological and medical research. However, there is no effective, convenient and high-throughput method for determination of circulatory H2S until now. Here, we aim to develop an easy method for measurement of circulatory H2S by modified methylene blue method. In the present study, we added Zn2＋ to plasma sample to deposit H2S, HS- and S2- , as well as plasma protein, then used NaOH to re-dissolve plasma protein. ZnS deposition was re-dissolved by the addition of N, N-dimethyl-p-phenylenediamine, and the remnant protein was deposited by trichloroacetic acid. After centrifugation, ferriammonium sulfate was added to the supernatant fluid to generate methylene blue, which was analyzed by spectrophotometer at 665 nm. Using the present method, we found that most ions including sulfate and thiosulfate did not affect detection of H2S concentration, but albumin （physiological concentration） reduced the detection value, which suggested the binding of serum albumin and a certain amount of H2S. The relative recovery ratio of present method is 81.9%, which implies that the method is relative accurate for the determination of H2S concentration in plasma or serum. H2S levels of frozen plasma samples from 65 healthy volunteers detected by the present method were （13.93 ± 4.98） μmol/L. There was no obvious difference between the detection values of fresh and frozen samples from the same SD rats. These results suggest the modified methylene blue assay is stable,sensitive, convenient and high-throughput. The method can be used to analyze the circulatory H2S in clinical and basic research.