Preparation of permeability solution and TiO2 capsules
To assess that the volunteers had normal intestinal permeability, an iso-osmolar test sugar solution (300 mmol/kg) containing 0.5 g D-xylose, 1 g L-rhamnose, 0.2 g 3-O-methyl-D-glucose and 5 g lactulose was prepared and 100 ml administered orally per subject. Gelatine capsules, containing 50 mg pharmaceutical/food grade anatase TiO2 (Kronos® 1171; reported manufacturer’s d50 of 260 nm, Fagron UK,) were manufactured by St Thomas’ Hospital Pharmacy (London).
Conduct of the study
Following ethical approval (EC01/037) and informed consent, eight healthy (self-reported) volunteers were recruited; seven completed the study as blood could not be withdrawn from the cannula of one subject. First, subjects were provided with the permeability solution, 1 l of deionised water and a 2 l urine bottle containing the preservative thiomerasol. Subjects were asked not to consume any dairy products from lunchtime the day before the test. At 7.00 am following an overnight fast, subjects emptied their bladders and provided a baseline urine sample in 50 ml universal tubes. They then ingested the 100 ml test sugar solution and during the following two hours they were only allowed to drink deionised water. Urine was collected thereafter for 5 h in the 2 l urine bottles (i.e. until 12.00 pm).
At 9.00 am, a peripheral venous cannula was inserted and a 5 ml baseline blood sample taken. Immediately following this, subjects ingested two capsules each containing 50 mg of pharmaceutical/food grade TiO2 (total 100 mg) with 250 ml water - one capsule was taken immediately following the other. Blood samples were then taken at baseline (0), at 30 min, and at 1, 1.5, 2, 3, 6, 8 and 10 h post TiO2 ingestion. Blood samples were heparinised and analysed as described below.
Normal drinking water was provided throughout the study and meals, free from added TiO2, were provided at 10.00 am (Breakfast: wholemeal toast with salted butter and strawberry jam), 1.00 pm (Lunch: chicken and green pepper stir fry with boiled rice, fresh fruit and a small packet of plain crisps) and 4.00 pm (Mid-afternoon snack: chocolate biscuit and/or fresh fruit). Tea and coffee with milk and/or sugar were allowed as desired from 11 am onwards.
Identification of TiO2 in blood by dark field microscopy
One drop of blood (~45 μl) from the syringe following sampling (i.e. non-heparinised) was placed on a clean, freshly opened glass slide. Superfrost plus slides were used to allow cell adherence. The blood was spread thinly with a clean slide to aid monolayer formation and a cover slip was then sealed in place with nail vanish to prevent sample drying. The slide was examined by light microscopy first at x 100 magnification and then x 400. Random areas were visualised at x 400 using a dark-field condenser to allow the titanium dioxide particles to be easily observed as bright white discrete particles in contrast, for example, to cell debris which was much less bright and less punctate, and generally translucent. The estimation of particles within each field was based on four reflectance grades; namely 0, 1, 2 and 3, and the operator was blinded to sample codes. A reflectance grade of 0 indicated 5 or less particles/field, a grade of 1 showed 5 to 10 particles/field, a grade of 2 showed 10 to 20 particles/field, and a grade of 3 indicated > 20 particles/field. Reflectance grades, rather than absolute numbers, were used for rapid data acquisition and because of the semi-quantitative nature of detection. Visualisation of TiO2 particles by dark field microscopy could only be carried out in five out of seven subjects due to clotting of blood in two subjects.
Measurement of Ti by Inductively Coupled Plasma- Mass Spectrometry (ICP-MS)
Aliquots of collected heparinised blood samples were acid digested using sub-boiling nitric acid diluted with Milli-Q water and high-purity hydrogen peroxide . Total Ti concentrations were determined by High-Resolution ICP-MS at the Department of Physical and Analytical Chemistry, University of Oviedo, Spain, using an Element 2 (Thermo Fisher Scientific, Waltham, MA, USA) working at medium resolution (RS = 4000) to analyse the isotope of 47Ti [15, 16]. For quantification, the method of isotope dilution was applied . Measurement of Ti was carried for time points 0–10 h except in two subjects where samples at 8 h in both, and 10 h in one, could not be collected.
The estimation of particle numbers present in blood films and the levels of Ti following ingestion of TiO2 were assessed using a two-tailed paired t–test. The relationship between reflectance grades (i.e. TiO2 particles within blood) and the levels of Ti measured by ICP-MS was determined using a Spearman correlation test. For all tests, significance was taken at p < 0.05.