Also, inside the RV there were placed a few (5–7) 2 mm diameter glass beads that helped to damp liquid motion when pyruvic acid first entered. The pH of the injected substrate was automatically measured during the dissolution procedure using a pH electrode (ASP200-2-1M-BNC, Active robots Ltd., Radstock, UK) placed in the RV. The pH electrode was connected to a custom built amplifier that had a variable output voltage in response 3-Methyladenine mw to changes in pH, see Supplementary

information. The amplifier was connected to an analogue to digital converter input on the microcontroller. By titrating 45 mg pyruvic acid against 2.0 M sodium hydroxide over a pH range of 1.8-–13.0, voltage versus pH was plotted and used to generate a linear calibration equation. The pH electrode could also be calibrated from within the Arduino software by measuring the electrode voltage in 3 different buffer solutions (pH 4, pH 7, pH 10) and calculating a linear

equation for pH versus voltage. Also connected into the RV was a 6 mm O.D. pipe connected to a vacuum pump (GAST GF3, Gast Manufacturing Inc., MI) to reduce back pressure during transfer of hyperpolarized solution. The vacuum pump was gated on/off by the HyperSense DNP polarizer. Injection volumes for each species are limited to ensure that the circulation Sirolimus in vivo of the animals is not overloaded. The physical constraints of an MRI scanner require a long length of cannula line for i.v. injections, resulting in a significant dead volume that contributes

to the injection volume. This is problematic where hyperpolarized signal is limited. If, for example, saline occupies the dead volume of the cannula then, during injection, its volume must be considered part of the dose and yet it does Neratinib cell line not contribute to the measured hyperpolarized signal. To increase the percentage the hyperpolarized compound contributes to the injected volume, the dead volume was reduced by splitting the cannula into two pathways without introducing additional dead volume; one pathway was then used as a waste stream for clearing the dead space volume whilst the other was used for drug administration into the animal. Flow direction was computer-controlled by valves. A fluid diverter cannula was constructed using two types of tubing: 0.96 mm O.D. polyethylene tube (Portex, Smiths Medical, St. Paul, MN), hereby referred to as ‘small tube’ and 1.0 mm I.D. Tygon tube (Cole-Parmer, London, UK), hereby referred to as ‘large tube’. Tygon tubing was used as its mechanical properties permit multiple compressions without permanent damage. A 19 gauge Luer hub was drilled to enlarge its inner diameter to 2 mm, see Fig. 3, into which the ends of three 30 mm lengths of small tubing were inserted to ensure the hole was almost completely occupied; one was used for the waste pathway, one for the animal pathway which was inserted into a rat vein, whilst the third one was unused and blanked off. The tubes were then sealed to the Luer hub with glue.