Investigating the effects of an oral fructose challenge on hepatic ATP reserves in healthy volunteers: a 31P MRS study.

68 Background: Impaired homeostasis of hepatic ATP has been associated with NAFLD. An 69 intravenous fructose infusion has been shown to be an effective challenge to monitor the 70 depletion and subsequent recovery of hepatic ATP reserves using 31 P MRS. 71 Aims: The purpose of this study was to evaluate the effects of an oral rather than intravenous 72 fructose challenge on hepatic ATP reserves in healthy subjects. 73 Methods: Self-reported healthy males were recruited. Following an overnight fast, baseline 74 liver glycogen and lipid levels were measured using Magnetic Resonance Spectroscopy 75 (MRS). Immediately after consuming a 500ml 75g fructose drink (1275 kJ) subjects were 76 scanned continuously for 90 minutes to acquire dynamic 31 P MRS measurements of liver 77 ATP reserves. 78 Results: A significant effect on ATP reserves was observed across the time course (P < 79 0.05). Mean ATP levels reached a minimum at 50 minutes which was markedly lower than 80 baseline (80 ± 17% baseline, P < 0.05). Subsequently, mean values tended to rise but did not 81 reach statistical significance above minimum. The time to minimum ATP levels across 82 subjects was negatively correlated with BMI (R 2 =0.74, P < 0.005). Rates of ATP recovery 83 were not significantly correlated with BMI or liver fat levels, but were negatively correlated 84 with baseline glycogen levels (R 2 =0.7, P<0.05). 85 Conclusions: Depletion of ATP reserves can be measured non-invasively following an oral fructose challenge using 31 P MRS. BMI is the best predictor of postprandial ATP homeostasis 87 following fructose consumption.


ARTICLE ELECTRONIC WORD COUNT
is an important part of liver lipid accumulation [4,5] and it has also been suggested that an 96 inability to maintain ATP levels may prime hepatocytes to become vulnerable to injury by 97 reactive oxygen species. 98 Hepatic ATP reserves can be monitored noninvasively using 31 P magnetic resonance 99 spectroscopy (MRS) [6]. Early animal studies used this method to monitor ATP following 100 fructose injections and suggested its potential use as a diagnostic method for studying liver 101 disease [7]. A number of more recent studies have used these techniques to measure ATP 102 homeostasis following an intravenous (IV) fructose load [2,8,9]   Prior to study days subjects were asked to refrain from alcohol for 24hr. On the morning of 129 the study subjects arrived at the test centre between 7:30am and 8:00am having fasted 130 overnight.

131
On arrival, natural abundance 13 C MR spectra were acquired from the liver to determine 132 baseline hepatic glycogen levels, and localized 1 H MR spectra were acquired to determine 133 baseline hepatic lipid levels. Subjects were then asked to consume a 500ml drink of 75g 134 fructose solution (1275 kJ) within 5 minutes. Immediately following consumption, subjects were placed in the scanner and 31 P MR spectra were acquired continuously for 90 minutes to 136 assess dynamic changes in ATP and related phosphate metabolites. During the 90 minutes of 137 scanning, subjects were asked to breathe regularly and remain as still as possible and were 138 allowed to listen to the radio or music.     and were significantly below the first two points at t = 30 minutes (86 ± 14%, P < 0.05), t = 215 40 minutes (85 ± 16 %. P < 0.05) and t = 45 minutes (84 ± 14%, P < 0.005) until reaching 216 minimum at = 50 minutes (80 ± 17%, P < 0.05). There was a trend for values to recover after 217 50 minutes, but the increase was not statistical significance compared to nadir and levels 218 remained lower than baseline at the end of the study.

219
No subject showed any recovery of ATP levels during the first 6 time points (until t = 40 220 mins). The mean AUC across this period (t=0 to t=40 mins) was 232 ± 19 % h and showed a 221 strong negative correlation with BMI (R 2 = 0.65, P < 0.01).  Figure 4 shows the relationship between rate of recovery and baseline glycogen reserves, 231 which had a strong negative correlation that was statistically significant (R 2 = 0.71, P < 0.05).

232
This correlation was not observed with BMI, liver fat, or any other baseline measures. The underlying physiological hypothesis of this study is that ATP homeostasis, which 236 provides a measure of AMPK activity, acts as a biomarker for NAFLD and NASH. Rather 237 than fructose infusion, this study explored using 31 P MRS following an oral fructose 238 challenge, which is more physiological, more patient-acceptable and much simpler to  In the present study ATP levels took longer to recover compared to previous infusion studies. 256 This is probably due to the extra stages necessary to transfer fructose to the hepatic tissue,  This study showed a negative correlation between BMI and time to minimum ATP levels.

268
Given that the hepatic ATP response is a combination of depletion and recovery and fructose 269 is known to deplete ATP reserves, these findings suggest that individuals with lower BMI   In summary, this study has shown that depletion in hepatic ATP reserves following an oral 328 fructose challenge is observable using 31 P MRS in healthy subjects, allowing for a completely 329 non-invasive assessment of ATP synthesis. BMI was negatively correlated with the time to