CN111103423B - 一种检测肠道菌群代谢气体的呼气试验方法 - Google Patents
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Abstract
本发明提供一种检测肠道菌群代谢气体的呼气试验方法,受试者空腹12小时后首先测试空腹的呼气H2、CH4与H2S的浓度,然后服用可通过菌群代谢产生H2、CH4与H2S的底物,在服用底物1‑3小时内每20‑30分钟测试一次呼气的H2、CH4与H2S浓度,根据服用底物前后的呼气浓度对肠道菌群代谢相关的疾病进行诊断。所述底物含葡萄糖或乳果糖、乳酸盐或苹果酸盐与硫酸氨基葡萄糖或硫酸锌,三种成分的重量比范围为10:1:2~10:1:0.5。本发明方法采用的检测装置为一种可同时检测呼气H2、CH4、H2S与CO2的装置,H2与CH4的检测限与精度为1‑2ppm,H2S的检测限与精度为1‑2ppb,呼气时动态监测鼻呼气CO2并在CO2达到平台期后采样分析鼻呼气的H2、CH4、H2S。
Description
技术领域
本发明涉及肠道菌群代谢气体的呼气检测领域。
背景技术
H2、CH4与H2S是肠菌群代谢产生的重要气体。约70%的肠道细菌可酵解糖类底物生成乙酸与H2,并作为前体或供体被产甲烷菌(Methane producing bacteria,下文简称MPB)与硫酸盐还原菌(Sulfate reducing bacteria,下文简称SRB)用于进一步生成CH4与H2S。在肠道菌群平衡时的正常生理状态,呼气中H2与CH4的浓度量级是ppm, H2S仅为ppb。对肠道菌群失衡的病理状态,例如对小肠细菌过度生长(下文简称SIBO)相关的胃肠道疾病,在服用葡萄糖或乳果糖底物后3小时内,呼气H2与CH4会升高超过10ppm或几倍,而呼气H2S浓度的升高则不到10%或几个ppb (Hydrogen sulphide in exhaled breath: a potentialbiomarker for small intestinal bacterial overgrowth in IBS,J. Breath Res. 10(2016) 026010)。
呼气H2与CH4本底浓度高且对SIBO升高显著,临床容易检测,已广泛用于SIBO及相关疾病临床常检,而呼气H2S本底浓度低且对SIBO升高不明显,临床不易检测,目前尚无临床实用的技术与专利等文献报道。因此,2017年的《胃肠道疾病氢与甲烷呼气检测的北美共识》(Hydrogen & Methane-based Breath testing in gastrointestinal disorders-The north American consensus),在规范胃肠道疾病H2与CH4呼气检测技术的基础上,要求开发推广肠道气H2S、包括H2、CH4与H2S同时呼气检测的新技术。
本发明的目的是开发一种对肠道菌群代谢气体,包括H2、CH4与H2S的呼气检测的临床实用新技术。
发明内容
胃肠道疾病的呼气H2与CH4试验需要服用葡萄糖或乳果糖的底物。这些富含C与H元素的底物被过度生长的菌群(SIBO)酵解产生临床容易检测的呼气H2与CH4,但由于缺乏S元素而不能产生临床容易检测的H2S。因此,需要设计或选择一种新的底物,通过肠道菌群代谢,既能产生H2,作为产H2S与CH4的供体或前体,又能产生临床上可以检测具有诊断意义的H2S。
可以考虑两种底物,一种包括葡萄糖或乳果糖与无机硫酸盐或有机硫化物,另一种是含有H、C与S等元素的有机化合物。排除人体不能服用或对人体有副作用的这两类底物后,我们采用尚沃医疗H2、CH4、H2S、CO2与O2呼气测定的纳库仑呼气分析仪,来评估筛选合适的呼气试验底物。该仪器的H2与CH4呼气测定的检测限与精度为1ppm,H2S呼气测定的检测限与精度接近1ppb。按2017北美共识推荐的标准进行测试,测试时先憋气10-15秒,然后呼气到呼气末测定,并同时测定呼气末的CO2值,对呼气H2、CH4与H2S的测定值按呼气末CO2的测定值校准。用来评估筛选底物时,先测试空腹呼气浓度,服用底物后每20或30分钟测试一次底物呼气浓度,·根据底物与空腹呼气浓度差值的最大值超过SIBO诊断阈值的标准选择合适的底物:对腹泻或便秘为主的功能性胃肠病或肠易激综合征患者,参照呼气H2或CH4浓度差值必须超过10ppm诊断标准,要求H2S浓度差值的最大值大于10ppb。
通过呼气实验首先发现,通常市场上可服用的含硫药物或补充剂,如B族维生素、硫酸锌或硫酸镁、硫酸氨基葡萄糖等,在医嘱或正常的剂量范围内服用1-3小时内,对腹泻为主的肠应激综合征志愿者,在排除了肠息肉与结肠癌患者后,呼气H2S的本底或空腹浓度不到10ppb,服用无乳果糖或甚至乳果糖加上含硫物质的的底物后的升高不超过5ppb,不具临床意义或诊断价值。这些试验说明,用于H2、CH4与H2S呼气试验的底物不是一个对乳果糖或葡萄糖与含硫化合物简单的加和关系,也不是采用现有含C、H与S元素化合物的直接应用。
进一步的试验研究发现,SRB代谢产生H2S是一个相比菌群产生H2以及MPB代谢产生CH4更为复杂的过程,需要研究很多影响及限制因素或者需要模拟肠道菌群代谢产生H2S的微生态环境,包括(1)激活、促进或维持SRB活性的碳源、氮源与pH等因素,(2)提供前体或供体用于SRB产生H2S的适量的糖或碳氢化合物,(3)平衡SRB生成H2S与MPB生成CH4对同一H2源竞争的糖、碳源与硫化物的组成。
实验发现,葡萄糖、乳果糖或同时含有C、H与S元素的化合物,如硫酸氨基葡萄糖虽然可以提供SRB代谢所必须的碳源,但作用十分有限,在原来的底物中加入可提供有效碳源的乳酸盐或苹果酸盐后,可产生更多的H2S;使呼气H2S的浓度进一步升高。而且,乳酸盐可配合硫酸盐氨基葡萄糖或硫酸铵使用,其中的硫酸盐可作为SRB生成H2S的底物,而氨基葡萄糖或铵还可提供所需的氮源,虽然硫酸氨基葡萄糖的葡萄糖也可用来产生H2并作为维持SRB活性的碳源,但其作用十分有限,除非非常大的剂量,否则仍需要葡萄糖或乳果糖用来提供生成H2S的H2源,用乳酸盐提供SRB活性的碳源,用含硫化合物提供生成H2S的供体。试验优选的含硫化合物为硫酸锌与硫酸氨基葡萄糖。
呼气H2与CH4的本底浓度统计上低于10ppm, 为获得对SIBO相关疾病有诊断价值的呼气检测结果,北美共识推荐用75g葡萄糖或10g乳果糖产生超过10ppm升高的呼气H2或CH4。呼气H2S的本底浓度统计上低于10ppb,为获得超过10ppb H2S浓度升高的检测结果,试验优选的乳果糖、硫酸氨基葡萄糖或硫酸锌与乳酸盐或苹果酸盐的比例范围是10:0.5~2:1。在这个比例条件下,呼气H2S在服用底物后的升高统计上可以超过10 ppb。
此外,试验还发现鼻呼气末测定要优于目前普遍采用的口呼气末测定。在服用底物后,我们发现口腔H2S可能由于口腔硫酸盐还原菌对残留在口腔的底物或其它物质的作用有10ppb的浓度变化,会影响到口呼气H2S测定的重复性与可靠性。采用鼻呼气测定,并同时保证在鼻呼气CO2达到平台后采样分析,而不是采用到达平台后的CO2进行校正,可以消除口腔H2S的影响,而且口与鼻呼气H2或CH4并无显著差异。
综合本发明进行的大量实验研究,我们提出一种检测肠道菌群代谢气体的呼气试验方法:空腹12小时后首先测试空腹的呼气H2、CH4与H2S的浓度,然后服用可通过菌群代谢产生H2、CH4与H2S的底物,在服用底物后1-3小时内每20-30分钟测试一次呼气的H2、CH4与H2S浓度,根据服用底物前后的呼气的H2、CH4与H2S浓度对肠道菌群代谢相关的疾病进行诊断。
本发明提出的方法服用的底物,是一种可通过菌群代谢产生H2、CH4与H2S的底物,该底物含葡萄糖或乳果糖、乳酸盐或苹果酸盐与硫酸氨基葡萄糖或硫酸锌,三种成分的重量比范围为10 : 1 : 2 ~ 10 : 1 : 0.5。有两个优选方案:乳果糖、乳酸盐与硫酸锌,重量比为10 : 1 : 0.6;乳果糖、苹果酸盐与硫酸氨基葡萄糖,重量比为10 : 1 : 1.5。
本发明方法采用的检测装置为一种可同时检测呼气H2、CH4、H2S与CO2的装置,H2与CH4的检测限与精度为1-2ppm, H2S的检测限与精度为1-2ppb,该装置除口呼气外还具有鼻呼气的采样功能,动态监测鼻呼气CO2, 在鼻呼气CO2达到平台期后采样分析鼻呼气的H2、CH4、H2S。
本发明的技术方案的应用,克服了现有技术存在的呼气H2S本底浓度低且对SIBO升高不明显、临床不易检测等问题,通过对服用底物的创新性设计,使得通过肠道菌群代谢,既能产生H2,作为产H2S与CH4的供体或前体,又能产生临床上可以检测具有诊断意义的H2S,从而使呼气H2、CH4、H2S检测技术应用于肠道代谢相关疾病的临床常规检测得以实现。
具体实施方式
为了使本发明的目的,技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明,应当理解,此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明。
我们按照以下要求进行肠道菌群代谢气体的呼气试验:
1)呼气分析仪器:尚沃医疗H2、CH4、H2S、CO2与O2呼气测定的纳库仑呼气分析仪。该仪器的H2与CH4呼气测定的检测限与精度为1ppm,H2S呼气测定的检测限与精度接近1ppb。按2017北美共识推荐的标准进行测试,测试时先憋气10-15秒,然后呼气到呼气末测定,并同时测定呼气末的CO2值,对呼气H2、CH4与H2S的测定值按呼气末CO2的测定值校准。
2)测试人群:长期腹泻为主与无任何胃肠道症状的志愿者。
3)测试前准备:禁食12小时后。测试期间禁止剧烈运动、吸烟及其他饮食。
4)测试过程及数据记录:受试者使用呼气分析仪器进行憋气15秒后呼气,测试时间点记录为0min,并记录对应的空腹H2、H2S及CH4测试值;空腹呼气结束后,口服不同底物(具体实施例1-4中底物),然后每20分钟进行憋气15秒后呼气,测试时间点分别记录为20min、40min、60min、80min、120min,并记录每个时间点对应的H2、H2S及CH4测试值。
5)测试结果分析:测试期间所检测的结果,其中任一口服底物后时间点的H2S测试值减去空腹基线H2S测试值,其差值记录为∆H2S。
6)预期要求:口服底物后,经测试人呼出气体中H2S含量升高,较空腹时呼出气体H2S升高大于10ppb。即∆H2S≧10ppb。
具体实施例1:
服用底物:10g乳果糖
测试结果记录表:
测试结果分析:产生的的硫化氢升高小于10ppb。
具体实施例2:
服用底物:1.5g硫酸氨基葡萄糖
测试结果记录表:
测试结果分析:产生的的硫化氢升高小于10ppb。
具体实施例3:
服用底物: 10g乳果糖+1g乳酸盐+0.6g硫酸锌
测试结果记录表:
测试结果分析:产生的的硫化氢升高大于10ppb。
具体实施例4:
服用底物: 10g乳果糖+1g苹果酸盐+1.5g硫酸氨基葡萄糖
测试结果记录表:
测试结果分析:产生的的硫化氢升高大于10ppb。
本发明不限于显示和描述的实施例,但是任何变化和改进都在所附权利要求书的保护范围内。
Claims (6)
1.一种可通过菌群代谢产生H2、CH4与H2S的底物在制备用于检测肠道菌群代谢气体的试剂盒中的用途,其特征在于,所述可通过菌群代谢产生H2、CH4与H2S的底物含葡萄糖或乳果糖、乳酸盐或苹果酸盐与硫酸氨基葡萄糖或硫酸锌,三种成分的重量比范围为10 : 1 :2 ~ 10 : 1 : 0.5;受试者空腹12小时后首先测试空腹的呼气的H2、CH4与H2S浓度,然后服用可通过菌群代谢产生H2、CH4与H2S的底物,在服用底物1-3小时内每20-30分钟测试一次呼气的H2、CH4与H2S浓度。
2.如权利要求1所述一种可通过菌群代谢产生H2、CH4与H2S的底物在制备用于检测肠道菌群代谢气体的试剂盒中的用途,其特征在于,所述可通过菌群代谢产生H2、CH4与H2S的底物为乳果糖、乳酸盐与硫酸锌,三者的重量比为10 : 1 : 0.6。
3.如权利要求1所述一种可通过菌群代谢产生H2、CH4与H2S的底物在制备用于检测肠道菌群代谢气体的试剂盒中的用途,其特征在于,所述可通过菌群代谢产生H2、CH4与H2S的底物为乳果糖、苹果酸盐与硫酸氨基葡萄糖,三者的重量比为10 : 1 : 1.5。
4.如权利要求1所述一种可通过菌群代谢产生H2、CH4与H2S的底物在制备用于检测肠道菌群代谢气体的试剂盒中的用途,其特征在于,所述方法采用的检测装置为一种可同时检测呼气H2、CH4、H2S与CO2的装置。
5.如权利要求4所述一种可通过菌群代谢产生H2、CH4与H2S的底物在制备用于检测肠道菌群代谢气体的试剂盒中的用途,其特征在于,所述检测装置的H2与CH4的检测限与精度为1-2ppm, H2S的检测限与精度为1-2ppb。
6.如权利要求4所述一种可通过菌群代谢产生H2、CH4与H2S的底物在制备用于检测肠道菌群代谢气体的试剂盒中的用途,其特征在于,所述检测装置在呼气时动态监测鼻呼气CO2并在CO2达到平台期后采样分析鼻呼气的H2、CH4、H2S。
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