Rooyakkers D R, van Eijk H M, Deutz N E
Department of Surgery, University of Limburg, Maastricht, The Netherlands.
J Chromatogr A. 1996 Apr 12;730(1-2):99-105. doi: 10.1016/0021-9673(95)01113-7.
Enteral intake of a mixture of inert, non-metabolic monosaccharide and disaccharide probes, followed by measurement of their urinary probe ratio, is a well known method to investigate gut permeability. However, most applications lack sensitivity, thus a large amount of especially the disaccharide lactulose has to be ingested. This may cause diarrhoea, which influences the outcome of the test. Recently, a new fluorescent label 9-fluorenylethyl chloroformate hydrazine (FMOC-hydrazine) was introduced, which reacts with reducing sugars to form stable and highly fluorescent single peak derivatives in organic medium. We applied this reagent to develop a sensitive measurement of reducing sugar probes in aqueous samples (e.g., urine). The presented method has a linear response for each sugar derivative between 1 and 1250 pmol with an R2 ranging from 0.9997 for lactulose to 0.9999 for rhamnose. The limit of detection, calculated as a signal-to-noise ratio of three, was 0.05 pmol for lactulose and 0.01 pmol for rhamnose, xylose and 3-O-methyl-D-glucose, corresponding to urine concentrations of 0.11 micromol/l for lactulose and 0.02 micromol/l for rhamnose, xylose and 3-O-methyl-D-glucose. Compared to other tests, the limit of detection is very low. This enabled a reduction in the enteral intake of the disaccharide lactulose from 6-10 g to 1.5 g, thereby minimizing the chance of introducing diarrhoea. The coefficient of variation was below 3% both in standards and urine samples. After spiking the urine with the saccharides a recovery of 102% for lactulose, 101% for rhamnose, xylose and 3-O-methyl-D-glucose was found. In order to evaluate the presented method we compared the lactulose rhamnose ratio measured in urine of healthy human volunteers and kept the ingested dose in agreement with literature values. Furthermore, the ratio was measured after 3, 6 and 9 h to establish the minimal response time required to measure correct ratios. We found that even after 3 h the ratio was stable at a value of 0.0133 which is comparable to literature values (0.008-0.052).
口服惰性、非代谢性单糖和二糖探针混合物，随后测量其尿中探针比率，是一种研究肠道通透性的知名方法。然而，大多数应用缺乏敏感性，因此必须摄入大量的特别是二糖乳果糖。这可能会导致腹泻，从而影响测试结果。最近，一种新的荧光标记物9-芴基乙基氯甲酸酯肼（FMOC-肼）被引入，它与还原糖反应，在有机介质中形成稳定且具有高荧光的单峰衍生物。我们应用这种试剂开发了一种在水性样品（如尿液）中灵敏测量还原糖探针的方法。所提出的方法对每种糖衍生物在1至1250 pmol之间具有线性响应，R2范围从乳果糖的0.9997到鼠李糖的0.9999。以信噪比为3计算的检测限，乳果糖为0.05 pmol，鼠李糖、木糖和3-O-甲基-D-葡萄糖为0.01 pmol，分别对应于乳果糖尿浓度为0.11 μmol/l，鼠李糖、木糖和3-O-甲基-D-葡萄糖尿浓度为0.02 μmol/l。与其他测试相比，检测限非常低。这使得二糖乳果糖的口服摄入量从6 - 10克减少到1.5克，从而将引入腹泻的可能性降至最低。标准品和尿液样品中的变异系数均低于3%。在尿液中加入糖类后，发现乳果糖的回收率为102%，鼠李糖、木糖和3-O-甲基-D-葡萄糖的回收率为101%。为了评估所提出的方法，我们比较了健康人类志愿者尿液中测量的乳果糖与鼠李糖比率，并使摄入剂量与文献值一致。此外，在3、6和9小时后测量该比率，以确定测量正确比率所需的最短响应时间。我们发现，即使在3小时后，该比率也稳定在0.0133，与文献值（0.008 - 0.052）相当。
