Imaging Mass Spectrometry for Visualization of Drug and Endogenous Metabolite Distribution: Toward In Situ Pharmacometabolomes
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- Sugiura, Y. & Setou, M. J Neuroimmune Pharmacol (2010) 5: 31. doi:10.1007/s11481-009-9162-6
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It is important to determine how a candidate drug is distributed and metabolized within the body in early phase of drug discovery. Recently, matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS; also referred to as mass spectrometry imaging) has attracted great interest for monitoring drug delivery and metabolism. Since this emerging technique enables simultaneous imaging of many types of metabolite molecules, MALDI-IMS can visualize and distinguish the parent drug and its metabolites. As another important advantage, changes in endogenous metabolites in response to drug administration can be mapped and evaluated in tissue sections. In this review, we discuss the capabilities of current IMS techniques for imaging metabolite molecules and summarize representative studies on imaging of both endogenous and exogenous metabolites. In addition, current limitations and problems with the technique are discussed, and reports of progress toward solving these problems are summarized. With this new tool, the pharmacological research community can begin to map the in situ pharmacometabolome.
Keywordsimaging mass spectrometryMALDIpharmacometabolome
Workflow of imaging mass spectrometry
Current IMS for small organic compounds
- 1.Mapping the metabolism of an administered drug and the distribution of its metabolites by IMS is attracting much attention because of its advantages over conventional imaging techniques, as will be discussed below. In this area, a number of studies on antipsychotics, cancer drugs, antianxiety drugs, and hypnotics have been reported (summarized in Table 2).Table 2
Imaging/detection of small metabolite molecules in tissues by MALDI and related ionization techniques
Cardiolipins (Wang et al. 2007)
Imatinib (cancer drug; Cornett et al. 2008)
Vinblastine (cancer drug; Trim et al. 2008)
Banoxantrone (cancer drug; Atkinson et al. 2007)
Triacylglycerols (Astigarraga et al. 2008)
Diacylglycerols (Astigarraga et al. 2008)
Fatty acids (Zhang et al. 2007)
Amino acids (Li et al. 2008)
Among the endogenous metabolites, lipids have been intensively investigated. Studies describing detection and imaging of complex lipids (e.g., glycerophospholipids and glycosphingolipids) and simple lipids (e.g., cholesterol, acylglycerides, and fatty acids) have been reported. In addition, other metabolites with superior ionization efficiency such as heme, which is a prosthetic group consisting of an iron atom contained in the center of a porphyrin ring, have also been investigated, even in an African ritual art object (Mazel et al. 2007; summarized in Table 2).
We will describe a representative research application in imaging both endogenous and exogenous metabolites.
IMS for exogenous drugs
IMS for endogenous metabolites: phospholipids
Establishment of IMS application for endogenous small metabolites also benefits the fields of toxicology and pharmacology because it will provide an insight into metabolic changes linked to drug administration that are unwanted and deleterious side effects or toxicity. This research field is expanding at an increasing rate, and IMS methodologies for various types of metabolites are developing. Below, we summarize IMS applications for endogenous metabolites.
MALDI-IMS for profiling (Jackson et al. 2005b, 2007a; Jones et al. 2006; Rujoi et al. 2004) and visualizing distribution (Garrett et al. 2006; Hayasaka et al. 2008b; McLean et al. 2007) of endogenous lipids such as phospholipids is the best established application; these lipids can also be imaged with secondary ion mass spectrometry (Colliver et al. 1997; Monroe et al. 2005; Ostrowski et al. 2004; Touboul et al. 2005). This is because phospholipids are ionized efficiently for the following reasons: first, a large fraction—for example, more than 60% by dry weight—of brain tissue consists of lipids. Second, these compounds have an easily ionizable structure; phospholipids, particularly phosphatidylcholines (PCs), contain a phosphate group and trimethylamine that are easily charged (Pulfer and Murphy 2003).
IMS for endogenous metabolites: gangliosides
Previous biochemical studies have revealed that the LCB of the brain ganglioside species has either 18 or 20 carbon atoms (i.e., C18- or C20-sphingosine), and C20-sphingosine (C20-LCB species) is present in significant amounts only in the central nervous system (Jungalwala et al. 1979; Sambasivarao and McCluer 1964; Schwarz et al. 1967; Sonnino and Chigorno 2000). Its content increases significantly in rodents and humans throughout life (Mansson et al. 1978; Palestini et al. 1990, 1991). The C20-LCB gangliosides are of great interest because of their characteristic brain specificity and their dramatic increase during the organism’s life span. However, lack of a visualization technology for specific detection and visualization of C18 and C20 gangliosides has left us incapable of determining their precise tissue distribution. Antibodies to some oligosaccharide moieties are available for visualizing the molecular species with different constituent oligosaccharides (Kotani et al. 1993), but such immunological methods cannot detect the differences in the ceramide structure hidden in the lipid bilayer.
Detection of gangliosides in mouse hippocampus
[M + Na-2H]−
[M + K-2H]−
[M + 2Na-3H]−
[M + Na + K-3H]−
[M + 2 K-3H]−
Current limitations and perspective
Current limitation and perspective of IMS for small molecules
Sample preparation (ionization)
High ionization efficiency of the targeted molecule is required
Optimization of sample preparation procedure (Prideaux et al. 2007)
MS measurement (ion separation)
Multiple compounds often share the same nominal mass in low-m/z region
Imaging with FTICR instrument (Cornett et al. 2008)
Development of organic matrix-free ionization method (such as use of nanoparticles (Jackson et al. 2007a; Taira et al. 2008), ME-SALDI (Liu et al. 2008), DIOS (Liu et al. 2007), and NIMS (Northen et al. 2007))
The sample preparation procedure must be optimized to maximize detection efficiency of the targeted molecule.
It is necessary to optimize several parameters of the sample preparation procedure including matrix selection and solvents used to extract analytes from tissues, according to the physical and chemical properties of the targeted molecule. In fact, previous studies have shown that, due to the nature of MALDI, successful detection of different molecules of interest can depend on the sample preparation procedure for the following reason. Since tissues and cells are the subjects of MALDI-IMS, the sample cleanup procedure is limited, whereas, in traditional MS, analyte molecules are generally extracted and separated from crude samples by gas chromatography or high-performance liquid chromatography. When such a crude sample is subjected to MS, numerous molecular species compete for ionization, and molecules that are easily ionized reach the detector preferentially while suppressing the ionization of other molecules, causing severe ion suppression effects (Annesley 2003; Gharahdaghi et al. 1996; Krause et al. 1999). In fact, using a mouse whole-body section coated with analyte drugs, Stoeckli and colleagues have found small regions (although less than 5% of the total region) in which the drugs could not be detected, presumably because of ion suppression effects (Stoeckli et al. 2006). Thus, optimizing the sample condition so that the analyte molecule present in the crude mixture can be efficiently ionized is an important issue. For this purpose, sample preparation has a critical role. In particular, it is helpful to perform a preliminary experiment using a reference drug because the choice of a suitable matrix compound, as well as optimizing the composition of the matrix solution, can improve the ionization efficiency of the molecules of interest.
Advanced MS analysis methods should be applied to separate multiple compounds with the same nominal mass.
We also note that elimination of matrix-derived ions is also effective for reducing the overlap of mass peaks from multiple compounds, thereby obtaining specific information in a targeted analysis. To achieve this, several important studies have been published in which organic matrix-free ionization was developed, such as the use of nanoparticle-based ionization (Jackson et al. 2007a; Taira et al. 2008), matrix-enhanced surface-assisted desorption/ionization (Liu et al. 2008), desorption/ionization on silicon (Liu et al. 2007), and nanostructure-initiator mass spectrometry (Northen et al. 2007).
IMS on small molecules has opened a new frontier in pharmacology and toxicology. As discussed above, MALDI-IMS provides an attractive alternative for monitoring drug distribution within animal models, with much faster results and lower cost than the traditional WBA method. Furthermore, its unique capability to simultaneously image many types of molecules enables distinct visualization of a parent drug and its metabolites. This advantage is important for pharmacological and toxicological study because we can learn whether the intact compound reaches desirable or undesirable organs.
As another application, we can also localize numerous endogenous metabolites such as lipids; this will benefit not only biological research but also pharmacological research since metabolite reactions in response to drug administration may be assessed.
Herein, we have discussed the promising capabilities of IMS, as well as the importance of the experimental protocol, particularly the sample preparation step. As introduced here, with attention paid to some technical points, MALDI-IMS provides valuable information for exploring the in situ pharmacometabolome that could not be obtained by any other existing technique: high selectivity, rapid acquisition, and parallel acquisition of multiple analytes. We expect that continued improvement in the experimental protocol, as well as in the MS instrumentation, will further expand the capability of this emerging technique. In conclusion, we hope this review will help readers to explore IMS as a new tool in their research fields.