Perspective of the thesis

The aim of the research detailed in this thesis was to establish a dynamic method for measuring gut health based on the ‘classical’ dual sugar absorption test for intestinal permeability22_{, with the view that it could eventually be used to test the effects of food}

ingredients or neutraceutical agents on gut health.

The procedure was to firstly choose/identify an appropriate method, to suitably refine the method, and to check that the refined method was able to accurately and reproducibly quantify the permeability probes in urine. A review of the literature indicated that a number of methodologies have been used to quantify urinary excretion of the sugar probes which included enzymatic assays23, 24_{, calorimetric assays}25 _{and chromatography}26_.

Following careful consideration of the limitations of each method, high performance liquid chromatography (HPLC) was chosen as the preferred technique27_{. A range of columns, i.e.}

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monosaccharide H+ _{- columns, cation exchange columns and anion exchange columns, and}

detectors such as absorbance detectors (ultraviolet, photodiode array), refractive index, florescence and mass spectroscopic detectors for HPLC have been previously used. A simple, reliable, reproducible, cost effective method that did not involve derivitization of analytes was required. Accordingly, the methodology was standardized and calibrated by using an appropriate column and detector system that optimally detected urinary saccharidic probe sugars.

Moreover, a test procedure was further developed that standardized fluid intake and optimized urine sampling time to refine the permeability test. A literature search showed that differing volumes of fluid have been administered during the course of the test28_{; in some studies food was allowed two hrs after the test solution was administered}29-31_,

while other study protocols used different urine collection periods that ranged between 2 - 24 hrs32, 33 _{and probes with similar molecular weights which were used interchangeably}34

at different doses22, 35_{. Such discrepancies in the study protocol precluded meaningful}

comparisons between studies. This is due to the fact that the volume of fluid36_{, its nutrient}

content37, 38 _{and its consumption along with solids}39, 40 _{influence GI residence time, the}

absorption and consequently the quantities of the sugar probes recovered in urine. Hence a classical/static test protocol was designed to restrict fluid intake to 700 ml over a six hr urine collection period. This was done in order to limit the reported effect of diuresis on the urinary recovery of the sugar probes. No food was allowed during the study period. Urine samples were collected every half-hour over a six hr period so as to allow the dynamics of absorption of each sugar to be tracked and the recovery period used in clinical tests to be optimized.

It is noteworthy that in reports multiple sugar probes having similar molecular weights were used and were at times administered simultaneously34_{. Given that the}

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permeability depends on the diffusion of molecules through simple pores, it is likely that the probes will compete for pore space. Thus the quantities of the various probes that are recovered will depend on the number and relative proportion of pores available for

absorption. Ideally the sugar probes are water soluble, hydrophilic, lipophobic, non-metabolized and readily quantified in urine22_{. However some probes had been shown}

to be absorbed by active transport41, 42_{, available through dietary sources}43_{, have brush}

border activity41 _{and need to be radio labelled}22_{. Hence after reviewing the potential}

limitations of the commonly used paracellular and transcellular marker probes, lactulose and mannitol were chosen. As these sugars are degraded in the colon by enteral microflora44_{, they can be used to optimally assess small intestinal permeability. The}

dynamics of absorption of lactulose and mannitol were determined and compared to two other probes that are often used interchangeably with these sugars45_.

The sensitivity of the lactulose mannitol test was further explored using a standardized noxious stimulus that has been reported to increase intestinal permeability to larger probe sugars. Various exogenous agents that are reported to increase tight junction permeability were reviewed. The findings indicated that the effects of ethanol/alcohol consumption and cigarette smoking on permeability are not limited to the small intestine, with contradictory reports to the effect of smoking on tight junction permeability46, 47_.

Therefore a non-steroidal anti-inflammatory drug seemed like the practical choice given that the drug could be chosen based on its safety profile, general use and the effect on the small intestine. Previous studies have used drug such as indomethacin, ibuprofen, naproxen, sulindac; however, aspirin was selected as it met the criteria and is well researched.

A half-hourly urine sample collection regime was instituted, both with and without the aspirin challenge, to enable the dynamics of absorption of each sugar to be determined.

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Previous studies assessed permeability based on the ratio of the two sugars derived from cumulative samples. The half-hourly regime allowed the identification of the timing at which the peak in urinary excretion occurred. Moreover it allowed the rate of excretion of the sugars over the six hr period to be traced. Following this the suitability of a single dose of aspirin as an appropriate stimulus to augment the test was investigated. It was conducted on the premise that were aspirin to alter GI residence time or the physicochemical properties of the sugars, it could alter patterns of excretion of the sugars.

In a similar manner the administration of a nutrient during the test could also affect GI motility patterns. Therefore nutrients could decrease the proportion of sugar probes that are absorbed in the small bowel and increase the proportion in the large bowel48_{. They}

could also reduce transit of a substance present in high concentration from the stomach to the large bowel to enhance absorption by increasing residence time in the small intestine. Alternatively, nutrients could increase residence time of gut contents in the lower bowel to engender extensive fermentative digestion.

The dynamic aspirin augmented test was therefore standardized based on the timing at which the sugars resided in the small intestine. Based on the temporal patterns of excretion of each sugar, three phases were identified, which likely corresponded to the emptying of the column of digesta from the stomach; to the small intestine; and its subsequent movement into the proximal colon over the six hour collection period. These time period’s over which the sugars resided in the stomach, small intestine and colon were confirmed using a wireless motility capsule, SmartPill®. The non-invasive technique was chosen over several other expensive and protracted methods49, 50 _{such as the paracetamol}

absorption test51, 52 _{(gastric transit),} 13_CO

2 acetate breath test53, 54 (gastric and small bowel transit), scintigraphy49, 55 _{(whole gut transit) and radio opaque markers}56 _{(whole gut and}

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ambient pH, temperature and pressure recordings from the intestinal lumen49 _obtained

from the SmartPill.

Finally, the ability of the dynamic standardized lactulose mannitol test to determine the effect of food ingredients and neutraceuticals on gut health was assessed. This was important if the effects of substances that promote gut health by decreasing permeability to noxious materials using the test are to be meaningfully compared.