New improved radiometabolite analysis method for [18F]FTHA from human plasma: a test-retest study with postprandial and fasting state

Abstract

Background: Fatty acid uptake can be measured using PET and 14-(R,S)-[¹⁸F]fluoro-6-thia-heptadecanoic acid ([¹⁸F]FTHA). However, the relatively rapid rate of [¹⁸F]FTHA metabolism significantly affects kinetic modeling of tissue uptake. Thus, there is a need for accurate chromatographic methods to analyze the unmetabolized [¹⁸F]FTHA (parent fraction). Here we present a new radiometabolite analysis (RMA) method, with comparison to a previous method for parent fraction analysis, and its use in a test-retest clinical study under fasting and postprandial conditions. We developed a new thin-layer chromatography (TLC) RMA method for analysis of [¹⁸F]FTHA parent fraction and its radiometabolites from plasma, by testing stationary phases and eluent combinations. Next, we analyzed [¹⁸F]FTHA, its radiometabolites, and plasma radioactivity from subjects participating in a clinical study. A total of 17 obese or overweight participants were dosed with [¹⁸F]FTHA twice under fasting, and twice under postprandial conditions and plasma samples were obtained between 14 min (mean of first sample) and 72 min (mean of last sample) post-injection. Aliquots of 70 plasma samples were analyzed using both methods, enabling head-to-head comparisons. We performed test-retest and group comparisons of the parent fraction and plasma radioactivity.

Results: The new TLC method separated seven [¹⁸F]FTHA radiometabolite peaks, while the previous method separated three. The new method revealed at least one radiometabolite that was not previously separable from [¹⁸F]FTHA. From the plasma samples, the mean parent fraction value was on average 7.2 percentage points lower with the new method, compared to the previous method. Repeated [¹⁸F]FTHA investigations on the same subject revealed reproducible plasma SUV and parent fractions, with different kinetics between the fasted and postprandial conditions.

Conclusions: The newly developed improved radio-TLC method for [¹⁸F]FTHA RMA enables accurate parent fraction correction, which is required to obtain quantitative data for modelling [¹⁸F]FTHA PET data. Our test-retest study of fasted and postprandial conditions showed robust reproducibility, and revealed clear differences in the [¹⁸F]FTHA metabolic rate under different study settings.

Trial registration: EudraCT No: 2020-005211-48, 04Feb2021; and Clinical Trials registry NCT05132335, 29Oct2021, URL: https://classic.

Clinicaltrials: gov/ct2/show/NCT05132335 .

Keywords: Metabolic imaging; Parent fraction; Radio-TLC; Radiometabolite analysis; [18F]FTHA.

Fig. 1

Representative radiochromatograms of the 14 min plasma supernatant (continuous line) and [¹⁸F]FTHA standard sample (dashed line) using the previous method (A) and new method (B). The intensities of the standard and plasma samples were normalized

Fig. 2

Percentages of unchanged [¹⁸F]FTHA out of all radioactivity in the sample. (A, B) Parent fraction (mean and SD) analyzed using the previous method (blue) and new method (red), during fasted visits (n = 25 fasting visits and previous method; n = 13 fasting visits and new method) (A) and postprandial visits (n = 26 postprandial visits and previous method; and n = 11 postprandial visits and new method) (B). The representative parent fraction curves were generated using the new method and from multiple visits of a single subject; twice during fasting, indicated by dashed blue and red lines, and twice during postprandial, represented by black and green lines (C)

Fig. 3

Representative percentage of radioactivity over time of [¹⁸F]FTHA and its radiometabolites (M1–M7) in plasma, from plasma samples obtained during postprandial imaging, using the previous method (A) and the new method (B)

Fig. 4

Correlation of parent fraction results (%) between the two tested methods. Parent fraction values are plotted from all the subjects analyzed with both the previous and new methods (n = 5 for fasted visits; n = 5 for postprandial visits, data collected from four different subjects)

Fig. 5

(A) Plasma time-activity curves (TACs) from collected blood samples during fasted visits (continuous line) and postprandial visits (dashed line). (B) Parent fraction-corrected TACs using the new radiometabolite analysis (RMA) method. Data presented are mean and SD. n = 34 for fasted, and n = 34 for postprandial visits