Early proton MRS studies generally reported only the three principal metabolites. Studies performed at shorter TE provide the opportunity to measure additional metabolites relevant to the investigation of brain metabolism. Glutamate and glutamine (resolved at 1.5 Tesla as the combined signal GLX) and myo-inositol (Ins) may be measured using short TE sequences whilst GABA can be measured with advanced pulse sequences that make use of J-modulation effects. As magnet B0 field strengths and scanner hardware generally improve these metabolites will be measured with increasing reliability and further compounds will also become resolvable.
1.7.6.2.1 GABA, glutamate and glutamine
The metabolism of GABA, glutamate and glutamine has been discussed in a previous section (Section 1.2). All are visible to 1H MRS performed at short TE, although visualisation of the spectral peaks is made more complicated by overlap with other more dominant peaks and with each other and most researchers have been
obliged to report the combined signal of glutamate plus glutamine (GLX) or to employ more sophisticated methodologies such as spectral editing or J-resolved experiments to measure them directly. There are so far very few reports of the separate measurement of glutamate and glutamine in humans although Petroff has reported separate measurement in the occipital lobes of human volunteers using spectral editing(Petroff et al., 2000) and Garcia has reported separate values in human patients with epilepsy taking valproate examined on a 3T system(Garcia et al., 2009). Several groups have reported on measurement of GABA. Petroff, Rothman and co-workers have published extensively using a spectral editing sequence. The findings of this group are considered in Section 1.8.
1.7.6.2.2 Myo-inositol
Myo-Inositol (Ins) [(cis-1,2,3,5-trans-4,6-cyclohexanehexol], is a carbocyclic polyol that is the core component of the increasingly important phosphoinositide family of phospholipids. Phosphoinositides act as substrates for the generation of potent intracellular second messengers and as activators of other signalling proteins (for review see (Hammond and Schiavo, 2007). The central nervous system possesses relatively high concentrations of myo-inositol as well as the means to synthesize it from glucose 6 phosphate. Myo-Inositol serves not only as a precursor molecule for inositol lipid synthesis, but also as a physiologically important osmolyte.
Figure 1.7.61H MRS spectrum for myo-Inositol [from (Govindaraju et al., 2000)]
In the first stage of phosphoinositide synthesis Ins is combined with diacylglycerol to form phosphotidyl inositol (PI) with later phosphorylation at the 3, 4 or 5 position
of the inositol ring. The major phosphoinositide in many of the above activities is phospatidylinositol 4,5-bisphosphate (PI(4,5)P2). PI(4,5)P2 is hydrolysed by
PI(4,5)P2 specific phospholipase C (PLC) to yield Ins(1,4,5)P3 (IP3) and diacylglycerol (DAG). IP3 acts as a second messenger to mobilize Ca2+ from intracellular Ca2+ stores, whereas DAG activates C1-domain-containing proteins, such as protein kinase C (PKC) and MUNC-13 which plays a pivotal role in controlling synaptic vesicle formation(Brose and Rosenmund, 2002;Irvine, 2003). Furthermore, PI(4,5)P2 is also phosphorylated at position 3 to yield PI(3,4,5)P3, another important second messenger(Vanhaesebroeck et al., 2001).
Over the past decade a large number of membrane transport proteins have been shown to be sensitive to the action of phosphoinositides in the plasma membrane. These proteins include voltage-gated potassium and calcium channels, ion channels that mediate sensory and nociceptive responses, epithelial transport proteins and ionic exchangers. Each of the regulatory lipids is also under multifaceted regulatory control(Hammond and Schiavo, 2007). Phosphoinositide modulation of membrane proteins in neurons often has a dramatic effect on neuronal excitability and synaptic transmitter release. In astrocytes phosphoinositides have been implicated in the generation of intracellular [Ca2+] oscillations and inter cellular signalling(Nedergaard et al., 2003;Nahorski et al., 2003).
The PI system has been the subject of much speculation in parallel with the development of a new understanding of the role of astrocytes in nervous system tissue(Nedergaard et al., 2003;Oberheim et al., 2006). Glial cells have been shown to form non-overlapping domains bordered by small blood vessels. The glial foot processes overlap these blood vessels and also the synaptic terminals of neurons included within the functional domain. This anatomy places the astrocyte at the centre of the interaction between glia, neurons and the blood brain barrier. In this position astrocytes have been shown to modulate synaptic activity, take up and recycle neurotransmitter compounds released at the synaptic terminal, and to regulate the flow of molecules such as water and perhaps glucose through their close association with the blood brain barrier. Current understanding of this functional unit has encouraged speculation that astrocyte function (and dysfunction) may explain a wide range of neurological conditions(De Keyser J. et al., 2008). In particular the phenomenon of cortical spreading depression may be explainable in
terms of astrocytic Ca2+ activation with associated synaptic terminal inhibition and this observation together with the release of cyclo-oxygenase by activated astrocytes across the blood brain barrier causing a reactive hyperaemia may neatly explain many of the manifestations of migraine. Epilepsy associated with K+ channel disorders would also be a potential consequence of primarily astrocytic dysfunction.
Myo-inositol is visible to proton MRS at magnetic field strengths of 1.5T as a single peak at ~3.5 ppm. Early experiments have indicated that this signal is predominantly derived from Ins within the glial pool(Brand et al., 1993) although inositol concentrations in some neuronal populations may equal or exceed those observed for glia.
Interest in the measurement of Ins in humans developed in the field of psychiatry in the late 1980s with the observation that lithium administration is associated with reduction in measurable Ins levels leading to the proposal of an “inositol depletion” theory as a suggested mechanism for the therapeutic effect of lithium(Berridge et al., 1982;Berridge and Irvine, 1989;Berridge et al., 1989). Lithium reduces Ins levels by acting as an uncompetitive inhibitor of inositol monophosphatase (IMPase) resulting in an accumulation of inositol monophosphates and a reduction in Ins concentrations. Subsequent MRS experiments in vivo revealed that Ins decreases following lithium treatment(Moore et al., 1999) and that bipolar patients may have elevated pre-treatment levels of Ins(Davanzo et al., 2001). Later work by O’Donnell(O'Donnell et al., 2003) in rats and and in vitro work by Williams(Williams et al., 2002) showed that both lithium and sodium valproate decrease the concentration of Ins, despite the fact that valproate is not known to have any effect on the IMPase enzyme. Ding and Greenberg later reported a study of the effects of valproate and lithium in saccharoyces cerevisiae yeast. They found that the addition of either of these agents brought about a decrease in PI synthesis and to a lesser but still significant degree, the steady state relative PI composition(Ding and Greenberg, 2003).
Other work has focussed on the role of Ins as an osmolyte in glial cells. Ins is taken up against a steep concentration gradient(Biden and Wollheim, 1986) via the high affinity sodium myoinositol co-transporter (SMIT) which appears to be highly expressed in astrocytes(Kwon et al., 1992). Chronic exposure to various cell types to
hypertonicity leads to up regulation of SMIT(Ibsen and Strange, 1996). This effect protects the cell since it increases the intracellular Ins concentrations and thus osmolarity without disrupting cellular functioning. Both the activity of SMIT and the expression of its mRNA in astrocytes are down regulated in astrocytes with chronic ingestion of lithium over a period of 8 days (which is similar to the time required for efficacy of the drug).
1.7.6.2.3 Lactate
Lactate metabolism has also been discussed in Section 1.2. Lactate signal is visible to 1H MRS. The spin-spin coupling of lactate means that the lactate doublet is in phase at TEs of 270ms and mostly in phase at TE less than about 60ms but inverted at TE = 135ms. This characteristic pattern has been utilised in a number of studies that have reported the presence of visible elevation of lactate in ischemia, status epilepticus and mitochondrial encephalopathy(Fazekas et al., 1995;Saunders, 2000;McKnight, 2004;Bianchi et al., 2007). Elevation in lactate has also been reported in healthy volunteers following functional activation(Sappey-Marinier et al., 1992). As noted above for glutamate, glutamine and GABA, two dimensional acquisition techniques are sometimes used to better visualise the lactate peak.