A Human Intervention Trial Investigating the Conversion of Encapsulated Glucoraphanin to Isothiocyanates; and the Potential Link Between the Extent of the Conversion to an Individual's Gut Microbiota Phylogeny and Genotype.

Glucosinolates (GSLs) are non-volatile sulphur-containing phytochemicals (secondary metabolites) found in plant tissues of cruciferous crops, such as broccoli, cabbage and watercress. Glucosinolates accumulate in these foods with glucoraphanin being the predominant glucosinolate found in broccoli. When tissue is disrupted the glucosinolates are converted to isothiocyanates (ITCs) by the enzyme myrosinase. Sulforaphane is the predominant ITC obtained by enzymatic hydrolysis from glucoraphanin. When vegetables are cooked very thoroughly the myrosinase enzyme is denatured which results in GSLs passing through the gastrointestinal (GI) tract into the colon.

The ITCs have been shown in animal studies to exert diverse biological effects, such as anti-oxidant effects; anti-inflammatory properties; inhibition of platelet aggregation; reduction of systolic blood pressure and the reduction of cholesterol levels; all of which reduce the risk developing cardiovascular diseases. Sulforaphane has been linked to health benefits in humans, such as cancer chemoprevention and helping to maintain a healthy heart.

When cooked cruciferous vegetables, in which the myrosinase enzyme has been inactivated, are consumed, some of the microflora in the colon produce a myrosinase-like enzyme that can also convert glucosinolates to ITCs. This process of gut microbiota conversion of glucoraphanin to ITC is abolished by enteric antibiotics and bowel cleansing. There have been several in vitro studies performed with both pure and mixed cultures of bacteria confirming the ability of bacterial strains found within the human gut having the ability to metabolize GSLs in culture. It has been shown that the extent of conversion of GSLs to ITCs by human gut microbiota varies greatly between individuals but the conversion rate from repeated determinations within individuals are much more consistent. A recent study attempted to correlate the conversion of glucosinolate ex vivo by human gut bacteria to in vivo data of glucosinolate metabolism from a small number of individuals, however no specific link to individual bacterial specials was established.

Our study is a human dietary intervention in which participants will consume one capsule containing 100mg purified glucoraphanin from broccoli. The levels of glucoraphanin delivered by the capsule are similar to one to two portions of broccoli. As this is purified glucoraphanin there is no myrosinase enzyme present. All conversion of the glucoraphanin, contained within the capsule, to ITC will therefore occur by enzymes found in the gut microbiota.

The ability of the glucoraphanin in the capsule to be metabolised to ITCs by the gut microflora is unknown and will be assessed by measuring ITCs excreted in the urine. In brief each participant on the study will be requested to abstain from any glucosinolate and ITC containing foods for three days prior to dietary intervention, and for a further 24 hours during the intervention. They will be requested to fast overnight prior to the study day intervention and on the morning after the fast collect a urine sample. This will effectively constitute a negative control sample and be used as a measure of compliance with the dietary restriction. The participant will then be given the glucoraphanin capsule along with their breakfast. The dietary restriction will continue for a further 24hours, during which time the participant will collect all the urine they produce. The ITCs will be quantified in urine by HP-LC and LC-MS using validated analytical methods. Once the 24 hour urine collection is complete the dietary restriction will cease.