¹H and ³¹P magnetic resonance spectroscopy in a rat model of chronic hepatic encephalopathy: in vivo longitudinal measurements of brain energy metabolism

Abstract

Chronic liver disease (CLD) leads to a spectrum of neuropsychiatric disorders named hepatic encephalopathy (HE). Even though brain energy metabolism is believed to be altered in chronic HE, few studies have explored energy metabolism in CLD-induced HE, and their findings were inconsistent. The aim of this study was to characterize for the first time in vivo and longitudinally brain metabolic changes in a rat model of CLD-induced HE with a focus on energy metabolism, using the methodological advantages of high field proton and phosphorus Magnetic Resonance Spectroscopy (¹H- and ³¹P-MRS). Wistar rats were bile duct ligated (BDL) and studied before BDL and at post-operative weeks 4 and 8. Glutamine increased linearly over time (+146 %) together with plasma ammonium (+159 %). As a compensatory effect, other brain osmolytes decreased: myo-inositol (-36 %), followed by total choline and creatine. A decrease in the neurotransmitters glutamate (-17 %) and aspartate (-28 %) was measured only at week 8, while no significant changes were observed for lactate and phosphocreatine. Among the other energy metabolites measured by ³¹P-MRS, we observed a non-significant decrease in ATP together with a significant decrease in ADP (-28 %), but only at week 8 after ligation. Finally, brain glutamine showed the strongest correlations with changes in other brain metabolites, indicating its importance in type C HE. In conclusion, mild alterations in some metabolites involved in energy metabolism were observed but only at the end stage of the disease when edema and neurological changes are already present. Therefore, our data indicate that impaired energy metabolism is not one of the major causes of early HE symptoms in the established model of type C HE.

Appendix

Calculations of energy metabolism parameters

Intracellular pH was calculated based on the difference in resonance frequencies between Pi and PCr signals (δ_Pi) in ppm (parts per million) using the Henderson-Hasselbalch equation

$$ \mathrm{p}\mathrm{H} = 6.75 + \log \left[\left({\updelta}_{\mathrm{Pi}} - 3.29\right)/\left(5.68 - {\updelta}_{\mathrm{Pi}}\right)\right] $$

(1)

(Petroff and Prichard 1985).

Similarly, [Mg²⁺] was assessed from the difference in resonance frequencies between β-ATP and PCr signals (δ_β-ATP) in ppm using equation

$$ {\mathrm{pMg}}^{2+}=4.24 - \log \left[{\left({\updelta}_{\upbeta -\mathrm{A}\mathrm{T}\mathrm{P}} + 18.58\right)}^{0.42}/{\left(-15.74 - {\updelta}_{\upbeta -\mathrm{A}\mathrm{T}\mathrm{P}\ }\right)}^{0.84}\right] $$

(2)

(Iotti et al. 1996).

ADP concentration was calculated as

$$ \left[\mathrm{A}\mathrm{D}\mathrm{P}\right] = \left(\left[\mathrm{A}\mathrm{T}\mathrm{P}\right]\times \left[\mathrm{C}\mathrm{r}\right]\right)/\left(\left[\mathrm{P}\mathrm{C}\mathrm{r}\right]\times {\mathrm{K}}_{\mathrm{CK}}\right) $$

(3)

with an apparent equilibrium constant of biochemical creatine kinase reaction (K_CK) (where the reactant concentrations are the sum of all the ionic and metal complex species at specified pH and [Mg²⁺]) adapted for each particular pH and [Mg²⁺] of each individual rat as described previously (Golding et al. 1995). [ATP], [Cr] and [PCr] were measured by ¹H- and ³¹P-MRS, pH and [Mg²⁺] from Eqs. (1) and (2) respectively.

The phosphorylation potential (PP) representing the immediately available high energy phosphate pool was calculated as

$$ \mathrm{P}\mathrm{P} = \left[\mathrm{A}\mathrm{T}\mathrm{P}\right]/\left(\left[\mathrm{A}\mathrm{D}\mathrm{P}\right]\times \left[\mathrm{Pi}\right]\right) $$

(4)

(Veech et al. 1979).

The percentage of the maximal rate of ATP biosynthesis (v/V_max-ATP) was calculated according to

$$ \mathrm{v}/{\mathrm{V}}_{\max -\mathrm{A}\mathrm{T}\mathrm{P}}=1/\left(1 + 0.2/\left[\mathrm{A}\mathrm{D}\mathrm{P}\right]\kern0.5em +0.13/\left[\mathrm{Pi}\right]\right) $$

(5)

(Veech et al. 1979; Nioka et al. 1987).

Finally the relative rate of CK reaction (v/V_max-CK) was determined from

$$ \mathrm{v}/{\mathrm{V}}_{\max -\mathrm{C}\mathrm{K}} = \left(\left[\mathrm{A}\mathrm{D}\mathrm{P}\right]/\left(\left[\mathrm{A}\mathrm{D}\mathrm{P}\right]+{\mathrm{K}}_{\mathrm{m}-\mathrm{A}\mathrm{D}\mathrm{P}}\right)\right)\times \left(\left[\mathrm{P}\mathrm{C}\mathrm{r}\right]/\left(\left[\mathrm{P}\mathrm{C}\mathrm{r}\right]+{\mathrm{K}}_{\mathrm{m}-\mathrm{P}\mathrm{C}\mathrm{r}}\right)\right) $$

(6)

(Veech et al. 1979), with K_{m- ADP} = 0.8 mmol/l and K_m-PCr = 5.0 mmol/l (Kuby et al. 1954).