The current study attempts to separate Glu from Gln, quantify several cerebral metabolites using Profit algorithm and correlate the metabolite changes with NP tests in patients with MHE. Our results demonstrate that Profit fitting of L-COSY data, from both GE and Siemens 1.5T scanner, was able to quantify Glu and Gln reliably. There are no reports of separating these metabolites while post-processing the 1.5T and 3T MRS data. Hence, this finding is first of its kind. The mI ratio was found to be the most significant variable among other MRS ratios and NP domains. We found significant impairment in cognitive speed, motor function, executive function and global domain scores in the patient group. The strong correlations between the significantly altered MRS ratios and NP domains imply that the metabolite ratio changes not only are clinically relevant but also may be responsible for the neuropsychological manifestations.
Following is a brief discussion of each metabolite demonstrating significant difference in MHE patients via Profit processing of L-COSY, its putative role in the brain, and possible implication in hepatic encephalopathy. We discuss each metabolite, if and how, multidimensional MRS data- sets acquired on the GE and Siemens 1.5T MR scanners, add to the basis of our understanding of the pathogenesis of MHE.
Neuronal activity triggers various responses that act together to adapt and deliver the energy substrates for local neuronal needs. The neuronal activity is determined primarily by electric (rate of action potential firing) and synaptic activity. Glu released from glutamatergic synapses is taken up by the astrocytes in cotransport with sodium, thereby increasing intracellular sodium and stimulating the sodium-potassium pump (Pellerin and Magistretti 1997
), which increases the astrocytic adenosine-triphosphate (ATP) consumption, and hence links the astrocytic glucose metabolism to neuronal glutamate release (Magistretti et al. 1999
). Since the blood-brain barrier is quite impermeable to Glu, almost all brain Glu is synthesized from Glc, Gln, or from the degradation of proteins (Gruetter et al. 1994
It is generally understood that in MHE, the increased brain concentration of ammonia is correlated with increased permeability of the blood-brain barrier (BBB) to ammonia (Lockwood et al. 1991a
) and the increase of Gln in astrocytes since ammonia is metabolized via glutamine synthetase (GS) mediated amidation of Glu into Gln (Berl et al. 1962
). This increase of Gln could contribute to the decrease of total brain concentration of Glu in hyperammonemia long known in rats with acute hyperammonemia of acute liver failure (ALF) induced by thioacetamide (Bosman et al. 1990
). Decreased Glu has also been seen in patients with cirrhosis who died in hepatic coma (Lavoie et al. 1987
However, in 1D proton MRS, it is very difficult to separate the resonances of Glu from Gln and quite often the two are reported together as Glx. Thus, the increase of Gln is quite often foreshadowed by the decrease in Glu and the total peak of Glx is reported as an increase. With the ProFit algorithm used in 2D proton MRS, we clearly see a significant decrease in Glu and an increase in Gln as shown in and in keeping with the ammonia detoxification hypothesis. Reduction in Glu could also be partly due to reduced synthesis from Glc and the influence of ammonia on Glu pools other than the astrocytes (Zwingmann et al. 2003
Several lines of evidence have also shown that increased ammonia exposure can lead to decreased uptake of Glu by the astrocytes (Vaquero and Butterworth 2006
). This could lead to reduced local Glc oxidation. In addition, astrocyte swelling (discussed later) is an important feature of both acute and chronic liver failure and this can potentially lead to a change in transport processes.
Cho is important for normal membrane function, lipid transport, and methyl metabolism. It is a precursor of betaine, used by the kidney to maintain water balance and by the liver as a source of methyl groups for methionine formation (Koc et al. 2002
). In the central nervous system, it is an important precursor of choline-containing membrane phospholipids such as phosphatidylcholine (PtdCho) and sphingomyelin in neurons and glial cells and of acetylcholine (ACho) in cholinergic neurons.
The pool of total choline in the rat brain is large; however, 90% of the total choline is bound in the phospholipids of the cell membranes such as PtdCho and a further 9% in hydrophilic metabolites such as PCh and GPC. Similarly in the rat blood plasma, 99% of total choline is present in choline-containing phospholipids, and only 1% is present as free and the hydrophilic choline metabolites (Klein et al. 1993
). Free choline, in contrast to other choline-containing metabolites, is the only well-defined pathway for choline supply from the blood into the brain. There is no data indicating major transport of choline-containing phospholipids into and out of the brain under physiological conditions (Pardridge et al. 1979
In an in vivo
proton MRS correlation study of the Cho peak with in vitro
chemical measures of choline-containing compounds, the proton MRS Cho peak strongly correlated with free Cho and hydrophilic metabolites such as PCh (membrane synthesis) and to some extent with GPC (membrane degradation) but not with membrane-bound, PtdCho (Miller et al. 1996
Unfortunately, 1D proton MRS in vivo
techniques cannot differentiate the trimethyl protons of choline from those of PCh and GPC as their signals are separated by less than 0.03 ppm (Loening et al. 2005
). The 31
P resonances of PCh and GPC, on the other hand, differ from one another by ~ 3.5 ppm and can be used for better quantification (Bhujwalla et al. 1999
). However, the improved sensitivity of 1
H nuclei for detection, makes a strong case for using proton MRS. 2D proton MRS with ProFit algorithm enables separation for GPC and the total choline peaks.
In human cerebral neoplasms, it has been shown both in vivo
and in vitro
P MRS that the phospholipid composition of cell membranes is changed (Kaibara et al. 1998
). This change in phospholipid metabolism is seen in two resonances: the phosphomonoester (PME) and the phosphodiester (PDE) (Griffiths et al. 1983
). The aqueous extracts of a tissue, when analyzed in vitro
by high resolution 31
P MRS, can show the two phospholipid precursors, PCh and PE in the PME signal while the PDE signal includes the degradation products GPC and glycerophosphoethanolamine (GPE) (Evanochko et al. 1984
). In tumors, the PME resonance has been found to be increased, a change explained by an accumulation of PCh and PE (Solivera et al. 2009
In HE, the typical 31
P MR spectrum contains resonances assigned to PME, inorganic phosphate (Pi), PDE, phosphocreatine, γATP, αATP, and βATP, all providing information on energy metabolism. However, no single consensus has emerged from these studies although Taylor-Robinson group has consistently demonstrated reduction in PME and PDE to βATP ratio (Taylor-Robinson et al. 1994
; Taylor-Robinson et al. 1999
; Patel et al. 2000
Our 2D proton MRS results show a pattern similar to that observed by the Taylor-Robinson group, i.e., a reduction in GPC (although not reaching significance perhaps, due to high standard deviation) as well as a significant decline in total choline including free choline, PCh and GPC in patients when compared to healthy controls (see ). In addition, a significant decline is also observed in PE, clearly pointing to a decrease in the phospholipid metabolism in MHE. This is a new insight not decipherable solely from the data in 1D proton MRS and points to the need for techniques such as 2D proton MRS to tease out these inferences.
mI is an organic osmolyte presumed to serve as a compensatory tool of astrocytes to buffer ammonia-induced increase in glutamine within the astrocytes, which can lead to swelling. The swelling can have implications on transporters such as Glu. However, effective compensation shifts takes time for astrocyte volume homeostasis. ALF as opposed to chronic liver failure happens too rapidly to allow this compensatory shift and hence, mI/Cr ratio is not significantly different in patients with ALF compared to the healthy controls. Since the application of MRS in MHE, it has been shown in 1D MRS that mI concentration is significantly reduced in patients to maintain the astrocytic volume. With the application of 2D MRS, we were able to verify this finding as well as significant reduction of a similar osmolyte, sI belonging to the inositol group in these patients.
GSH is a primary antioxidant and plays an important role in oxidative stress protection in living cells. GSH is a tripeptide, consisting of glycine, cysteine, and glutamate. GSH is quite difficult to observe via 1D proton MRS even though its concentration is not very low (~1–3 mM) because of severe overlap with much more intense signals of creatine, glutamate, and aspartate. 2D proton MRS can clearly delineate GSH and with the ProFit algorithm, we have been able to quantify its concentration. Studies of GSH levels in blood and urine, have shown that subjects with risk factors of stroke have relatively low levels of GSH and patients with acute ischemic stroke develop elevated blood levels of GSH during the first hours to days post ictus (Zimmerman et al. 2004
In our study of MHE, we found a general trend towards increase of GSH in patients compared to controls. However, the changes were found to be non-significant possibly due to technical difficulties at 3T. In liver dysfunction, like in stroke, time may be of significance in how the cells cope with stress and what the resultant systemic consequence may be in acute and chronic cases. Hence, it would be quite useful to employ 2D proton MRS in cases of ALF to see the levels of GSH and compare it with our findings.
Tau is another metabolite not observable via 1D proton MRS. It is one of the most abundant free amino acids in the brain and is localized in both neuronal and glial compartments. Autopsied brain tissue from patients who died of hepatic coma has shown significantly decreased Tau in the prefrontal cortex (Butterworth 1996
). Opposite to this, our results show an increasing trend in Tau in patients with MHE compared to healthy controls using both GE and Siemens 1.5T MRS data. The biochemical significance of this finding is unclear. It is unknown if the subjects considered in the autopsied results were in the early stages (MHE) or late stages of HE. At the same time, in our L-COSY study, the 2D cross peak of Tau occurs very close to the diagonal and as a result could be effected by line-width changes from a patient to another. To understand all these more detailed study is required.
Glc is the major energy substrate for the brain that is degraded by glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation (Chih and Roberts 2003
). Neuronal activity and local Glc use and consumption are strongly correlated with the local blood flow (Clarke and Sokoloff 1999
). Brain Glc transport involves the breaching of two barriers: the BBB formed by the capillary endothelial cells, which are effectively connected together by tight junctions, and the barrier formed by the plasma membranes of neurons and astrocytes to which Glc must be delivered. The transport parameters of BBB Glc transport as well as the passage of many other substances including ions and water can be modulated by various extracellular and intracellular messengers, and also by pathologic stimuli involving metabolic, osmotic, or oxidative stress (Leybaert 2005
In MHE, Lockwood et al have reported via positron emission tomography (PET) that for whole-slice cerebral blood flow (CBF) and cerebral metabolic rate of glucose (CMRglc
), the values were not different in this group of patients versus healthy controls. However, for both CBF and CMRglc
, there was a highly significant difference in the pattern of flow and metabolism. Higher values for both flow and metabolism were observed in the cerebellum, thalamus, and caudate in patients and lower values in the cortex (Lockwood et al. 1991b
). Weissenborn et al. (2007)
have also reported via PET both CMRglc
in patients with MHE. They found similar alteration in cerebral glucose utilization as reported by Lockwood et al. (Lockwood et al. 1991b
) except for a decrease in glucose metabolism in the motor cortex and stable or increased glucose utilization rate within the frontolateral cortex. When it comes to MRS, again 1D proton MRS is unable to differentiate the resonances of Glc. In our 2D MRS study, in the frontal cortex, we did not observe any significant differences in Glc concentration in the MHE patients compared to the healthy controls.