Introduction
Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder affecting about 10–15% of people in the developed world and 1 in 26 people worldwide. IBS is characterised by a group of symptoms that commonly include abdominal pain, bloating and changes in the consistency of bowel movements. These symptoms are known to persist over a long period of time, sometimes for years. Symptoms can be debilitating and can negatively affect a persons quality of life. IBS, as well as other disorders, such as Crohn’s disease, congenital diarrhoea, idiopathic secretory diarrhoea, abdominal radiation induced diarrhoea, post gastrectomy or post cholecystectomy diarrhoea, post vagotomy diarrhoea, microscopic colitis, short bowel syndrome and bacterial infection are all associated with large shifts in bile acid profiles. In instances of pathogenic bacterial infection, Clostridioides difficile infection
(CDI), is particularly prone to bile acid profile shifts.
Bile acids are amphipathic molecules with a steroid backbone that are actively secreted by the liver into bile. Upon ingestion of a food source, these molecules are released into the upper portion of the small intestine where they facilitate the emulsifying process and form micelles; thereby aiding host lipases and other fat-degrading enzymes for digestion and assimilation of dietary fats. Bile acids are then reabsorbed in the lower portion of the small intestine and enter the enterohepatic circulation to be returned to the liver.
This cycle of secretion and absorption results in the accumulation of bile acids within the body, referred to as the bile acid pool. Approximately 5% of the bile acid pool escapes reabsorption per cycle and is lost into the large intestine and finally into the faeces. It is fluctuations in the levels of these excreted bile acids that often indicate underlying disorders, such as IBS, Crohn’s disease etc.
Methods to analyse samples of this type have consequently been costly and inefficient. Engevik et al have investigated alternate pathways to monitor and test for these fluctuations that could have a considerable effect on future diagnostics.
Repurposing Dried Blood Spot (DBS) Devices
In the haematology field, clinicians routinely use dried blood spot (DBS) analysis to assess blood-related compounds as a means to screen infants for metabolism-based disorders. This process involves a finger or heel prick to produce a blood sample, which is subsequently spotted onto a filter paper. The filter paper is then dried at ambient temperature. Once dried, DBS filters can be stored and shipped to laboratories for testing at ambient temperature without the need for costly packaging or temperature controlled transport. The DBS system has existed since the 1960’s and is considered an easy, convenient and cost-efficient way to collect, store and ship samples.
The ease and practicalities of Dried Blood Spot analysis led the researchers to a feasibility study of using a similar process for dried faecal spot (DFS) based analysis of faecal bile acids, with an overall aim to improve the diagnostic process for patients and practitioners.
DFS samples were generated from the faeces of the following cohorts: healthy individuals (control), individuals with diarrhoea and patients with CDI. All samples were weighed and prepped into 2-mL fast prep tubes with 1mL of ice-cold methanol and water at a ratio of 1:1. Once processed, the clarified faecal extract supernatant was applied to the two sample ports integrated into the Capitainer B qDBS device, sealed and allowed to dry overnight. These samples were then shipped at ambient temperature to Baylor College of Medicine/Texas Children’s Hospital, Houston, Texas and stored for 4 months at ambient temperature.
Comparative samples were also produced simultaneously. Equally prepared samples of the clarified faecal extract supernatant were frozen and shipped on dry ice then immediately stored at -80°C for 4 months.
Both DFS and frozen extracts were examined by Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) using a targeted bile analysis featuring unlabelled bile acid standard mixes and deuterated bile acid internal standard (IS) mixes. Mass spectrometry is considered the most accurate method for the measurement of faecal bile acids; the results of which were astounding. Investigators measured a variety of primary and secondary bile acids from both sampling methods, resulting in a strong positive correlation between the two, and amongst all three cohort types.
Discussion
The data produced by Engevik et al strongly suggests that DFS successfully retains bile acids from stool samples at ambient temperature allowing for LC-MS/MS-based downstream bioanalytical applications. These findings have brought about a number of promising advantages. The costs for both storage and shipping have reduced dramatically due to the ambient temperature and stability of the samples. Patient benefits have also greatly improved. Individuals could potentially collect stool samples in the privacy of their own homes and ship the qDBS device to a bioanalytic laboratory for analysis without the need to attend a clinic. This would be particularly beneficial to rural communities or developing countries. In theory, DFS devices could be shipped from anywhere in the world. A further postulation by researchers is the remote sampling possibilities of individuals exposed to the most extreme environments, such as the Antarctic or even outer space. This technique could also be used to examine bile acid profiles of non-human media. For instance livestock studies, where facilities are located a considerable distance from the sampling source.
The work carried out has also produced some unexpected findings. Investigators discovered β-muricholic acid (β-MCA) in four samples. MCAs are typically scarce in humans, but not entirely absent. They tend to be present in infant faeces, with concentrations dissipating into adulthood. MCA is known to be an inhibitor for C. difficile spore germination. It is unclear what role MCA plays in human gut bacteria interactions and consequently the relationship with C. difficile infections. It is certain that the data provided by this investigation warrants further studies.
This study focused solely on bile acids, but there are a wide range of host and microbial-derived compounds present in the human gut. DFS technology could potentially be used to examine any of these compounds that are non-volatile and are stable in the dried state. The mirroring of DFS with original bile acid profiles attained via frozen extracts highlights the utility of DFS as a legitimate methodology. Continuing studies will become more prevalent and discerning as the library of standards and mixes from facilities such as Cambridge Isotope Laboratories Inc. becomes more expansive.