| Summary: | Hyperpolarized <sup>13</sup>C magnetic resonance imaging often uses spin-echo-based pulse sequences that are sensitive to the transverse relaxation time <i>T</i><sub>2</sub>. In this context, local <i>T</i><sub>2</sub>-changes might introduce a quantification bias to imaging biomarkers. Here, we investigated the pH dependence of the apparent transverse relaxation time constant (denoted here as <i>T</i><sub>2</sub>) of six <sup>13</sup>C-labelled molecules. We obtained minimum and maximum <i>T</i><sub>2</sub> values within pH 1–13 at 14.1 T: [1-<sup>13</sup>C]acetate (<i>T</i><sub>2,min</sub> = 2.1 s; <i>T</i><sub>2,max</sub> = 27.7 s), [1-<sup>13</sup>C]alanine (<i>T</i><sub>2,min</sub> = 0.6 s; <i>T</i><sub>2,max</sub> = 10.6 s), [1,4-<sup>13</sup>C<sub>2</sub>]fumarate (<i>T</i><sub>2,min</sub> = 3.0 s; <i>T</i><sub>2,max</sub> = 18.9 s), [1-<sup>13</sup>C]lactate (<i>T</i><sub>2,min</sub> = 0.7 s; <i>T</i><sub>2,max</sub> = 12.6 s), [1-<sup>13</sup>C]pyruvate (<i>T</i><sub>2,min</sub> = 0.1 s; <i>T</i><sub>2,max</sub> = 18.7 s) and <sup>13</sup>C-urea (<i>T</i><sub>2,min</sub> = 0.1 s; <i>T</i><sub>2,max</sub> = 0.1 s). At 7 T, <i>T</i><sub>2</sub>-variation in the physiological pH range (pH 6.8–7.8) was highest for [1-<sup>13</sup>C]pyruvate (Δ<i>T</i><sub>2</sub> = 0.95 s/0.1pH) and [1-<sup>13</sup>C]acetate (Δ<i>T</i><sub>2</sub> = 0.44 s/0.1pH). Concentration, salt concentration, and temperature alterations caused <i>T</i><sub>2</sub> variations of up to 45.4% for [1-<sup>13</sup>C]acetate and 23.6% for [1-<sup>13</sup>C]pyruvate. For [1-<sup>13</sup>C]acetate, spatially resolved pH measurements using <i>T</i><sub>2</sub>-mapping were demonstrated with 1.6 pH units accuracy in vitro. A strong proton exchange-based pH dependence of <i>T</i><sub>2</sub> suggests that pH alterations potentially influence signal strength for hyperpolarized <sup>13</sup>C-acquisitions.
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