James R. Larkin

Recent advances in SALDI-MS techniques and their chemical and bioanalytical applications

Although laser desorption mass spectrometry was introduced in the 1960s, the potential of laser mass spectrometry was not realised until the introduction of matrix-assisted laser desorption/ionisation (MALDI) in the 1980s. The technique relies on light-absorbing compounds called matrices that are co-crystallised with the analyte to achieve high ionisation and desorption efficiencies. MALDI offers a lot of advantages and is an indispensable tool in macromolecule analysis. However, the presence of the matrix also produces a high chemical background in the region below m/z 700 in the mass spectrum. Surface-assisted laser desorption/ionisation (SALDI) substitutes the chemical matrix of MALDI for an active surface, which means that matrix interference can be eliminated. SALDI mass spectrometry has evolved in recent years into a technique with great potential to provide insight into many of the challenges faced in modern research, including the growing interest in “omics” and the demands of pharmaceutical science. A great variety of materials have been reported to work in SALDI. Examples include a number of nanomaterials and surfaces. The unique properties of nanomaterials greatly facilitate analyte desorption and ionisation. This article reviews recent advances made in relation to carbon- and semiconductor-based SALDI strategies. Examples of their environmental, chemical and biomedical applications are discussed with the aim of highlighting progression in the field and the robustness of the technique, as well as to evaluate the strengths and weaknesses of individual approaches. In addition, this article describes the physical and chemical processes involved in SALDI and explains how the unique physical and electronic properties of nanostructured surfaces allow them to substitute for the matrix in energy transfer processes.

A type 2 biomarker separates relapsing-remitting from secondary progressive multiple sclerosis

Objective: We tested whether it is possible to differentiate relapsing-remitting (RR) from secondary progressive (SP) disease stages in patients with multiple sclerosis (MS) using a combination of nuclear magnetic resonance (NMR) metabolomics and partial least squares discriminant analysis (PLS-DA) of biofluids, which makes no assumptions on the underlying mechanisms of disease. Methods: Serum samples were obtained from patients with primary progressive MS (PPMS), SPMS, and RRMS; patients with other neurodegenerative conditions; and age-matched controls. Samples were analyzed by NMR and PLS-DA models were derived to separate disease groups. Results: The PLS-DA models for serum samples from patients with MS enabled reliable differentiation between RRMS and SPMS. This approach also identified significant differences between the metabolite profiles of each of the MS groups (PP, SP, and RR) and the healthy controls, as well as predicting disease group membership with high specificity and sensitivity. Conclusions: NMR metabolomics analysis of serum is a sensitive and robust method for differentiating between different stages of MS, yielding diagnostic markers without a priori knowledge of disease pathogenesis. Critically, this study identified and validated a type II biomarker for the RR to SP transition in patients with MS. This approach may be of considerable benefit in categorizing patients for treatment and as an outcome measure in future clinical trials. Classification of evidence: This study provides Class II evidence that serum metabolite profiles accurately distinguish patients with different subtypes and stages of MS.

Glucose-Induced Down Regulation of Thiamine Transporters in the Kidney Proximal Tubular Epithelium Produces Thiamine Insufficiency in Diabetes

Increased renal clearance of thiamine (vitamin B1) occurs in experimental and clinical diabetes producing thiamine insufficiency mediated by impaired tubular re-uptake and linked to the development of diabetic nephropathy. We studied the mechanism of impaired renal re-uptake of thiamine in diabetes. Expression of thiamine transporter proteins THTR-1 and THTR-2 in normal human kidney sections examined by immunohistochemistry showed intense polarised staining of the apical, luminal membranes in proximal tubules for THTR-1 and THTR-2 of the cortex and uniform, diffuse staining throughout cells of the collecting duct for THTR-1 and THTR-2 of the medulla. Human primary proximal tubule epithelial cells were incubated with low and high glucose concentration, 5 and 26 mmol/l, respectively. In high glucose concentration there was decreased expression of THTR-1 and THTR-2 (transporter mRNA: −76% and −53% respectively, p<0.001; transporter protein −77% and −83% respectively, p<0.05), concomitant with decreased expression of transcription factor specificity protein-1. High glucose concentration also produced a 37% decrease in apical to basolateral transport of thiamine transport across cell monolayers. Intensification of glycemic control corrected increased fractional excretion of thiamine in experimental diabetes. We conclude that glucose-induced decreased expression of thiamine transporters in the tubular epithelium may mediate renal mishandling of thiamine in diabetes. This is a novel mechanism of thiamine insufficiency linked to diabetic nephropathy.

The longitudinal cerebrospinal fluid metabolomic profile of amyotrophic lateral sclerosis.

Neurochemical biomarkers are urgently sought in ALS. Metabolomic analysis of cerebrospinal fluid (CSF) using proton nuclear magnetic resonance (1H-NMR) spectroscopy is a highly sensitive method capable of revealing nervous system cellular pathology. The 1H-NMR CSF metabolomic signature of ALS was sought in a longitudinal cohort. Six-monthly serial collection was performed in ALS patients across a range of clinical sub-types (n = 41) for up to two years, and in healthy controls at a single time-point (n = 14). A multivariate statistical approach, partial least squares discriminant analysis, was used to determine differences between the NMR spectra from patients and controls. Significantly predictive models were found using those patients with at least one years interval between recruitment and the second sample. Glucose, lactate, citric acid and, unexpectedly, ethanol were the discriminating metabolites elevated in ALS. It is concluded that 1H-NMR captured the CSF metabolomic signature associated with derangements in cellular energy utilization connected with ALS, and was most prominent in comparisons using patients with longer disease duration. The specific metabolites identified support the concept of a hypercatabolic state, possibly involving mitochondrial dysfunction specifically. Endogenous ethanol in the CSF may be an unrecognized novel marker of neuronal tissue injury in ALS.

NMR-Based Metabolomics Separates the Distinct Stages of Disease in a Chronic Relapsing Model of Multiple Sclerosis

Relapsing experimental allergic encephalomyelitis (Cr-EAE) is commonly used to explore the pathogenesis and efficacy of new therapies for MS, but it is unclear whether the metabolome of Cr-EAE is comparable to human multiple sclerosis (MS). For MS, the diagnosis and staging can be achieved by metabolomics on blood using a combination of magnetic resonance spectroscopy and partial least squares discriminant analysis (PLS-DA). Here, we sought to discover whether this approach could be used to differentiate between sequential disease states in Cr-EAE and whether the same metabolites would be discriminatory. Urine and plasma samples were obtained at different time-points from a clinically relevant model of MS. Using PLS-DA modelling for the urine samples furnished some predictive models, but could not discriminate between all disease states. However, PLS-DA modelling of the plasma samples was able to distinguish between animals with clinically silent disease (day 10, 28) and animals with active disease (day 14, 38). We were also able to distinguish Cr-EAE mice from naive mice at all-time points and control mice, treated with complete Freund’s adjuvant alone, at day 14 and 38. Key metabolites that underpin these models included fatty acids, glucose and taurine. Two of these metabolites, fatty acids and glucose, were also key metabolites in separating relapsing-remitting MS from secondary-progressive MS in the human study. These results demonstrate the sensitivity of this metabolomics approach for distinguishing between different disease states. Furthermore, some, but not all, of the changes in metabolites were conserved in humans and the mouse model, which could be useful for future drug development.

Thiamine in diabetic nephropathy: a novel treatment modality? Reply to Alkhalaf A, Kleefstra N, Groenier KH et al. [letter]

To the Editor: We thank Alkhalaf and co-authors for their comment on our recent paper in Diabetologia [1]. We are grateful for their interest in our work and the recognition of its potential importance in stimulating further research, which may lead to a significant improvement in the treatment of diabetic nephropathy. They have made several comments on issues that could not be dealt with in our short communication [2]. We now address these comments. The urinary albumin excretion (UAE) data in Table 1 of our paper are the median (minimum–maximum) values of the 24 h UAE values for the treatment groups at the study times indicated. The data illustrated in Fig. 1a are the median values of data distributions of 24 h UAE change from baseline deduced for each patient. These data sets are not the same: one is absolute UAE values (distributions summarised in Table 1) and the other is of changes in UAE from baseline for each patient (medians of changes shown in Fig. 1a). This, and the non-parametric distribution of the data sets, accounts for the different values for the difference between median UAE after 3 months of treatment and at baseline (−13.6 mg/24 h) that can be deduced from Table 1 and the median of 24 h UAE changes from baseline (−17.7 mg/24 h) shown in Fig. 1a for the thiamine treatment group. Computing a data distribution of analyte change from baseline is a strategy often employed where there is large dispersion of baseline data, and the response to therapy (rather than change in absolute value) is of greatest interest. Change in UAE was the primary endpoint of our study. There was a trend for UAE data during the posttreatment washout period to show a continuing decrease beyond that achieved in the treatment period. We stated that the decrease in UAE in the post-treatment period was ‘maintained’, as the trend for further decrease was not statistically significant. This is not a concern, but rather it might be expected that patients have continued benefit during the washout period from the thiamine supplementaDiabetologia (2009) 52:1214–1216 DOI 10.1007/s00125-009-1334-7

Anti-inflammatory Microglia/Macrophages As a Potential Therapeutic Target in Brain Metastasis

Brain metastasis is a common complication of cancer patients and is associated with poor survival. Histological data from patients with brain metastases suggest that microglia are the major immune population activated around the metastatic foci. Microglia and macrophages have the ability to polarize to different phenotypes and to exert both tumorigenic and cytotoxic effects. However, the role of microglia/macrophages during the early stages of metastatic growth in the brain has not yet been determined. The aim of this study was to profile microglial/macrophage activation in a mouse model of breast cancer brain metastasis during the early stages of tumor growth, and to assess the role of the anti-inflammatory microglial/macrophage population, specifically, during this phase. Following intracerebral injection of 5 × 103 4T1-GFP mammary carcinoma cells into female BALB/c mice, robust microglial/macrophage activation around the 4T1 metastatic foci was evident throughout the time-course studied (28 days) and correlated positively with tumor volume (R2 = 0.67). Populations of classically (proinflammatory) and alternatively (anti-inflammatory) activated microglia/macrophages were identified immunohistochemically by expression of either induced nitric oxide synthase/cyclooxygenase 2 or mannose receptor 1/arginase 1, respectively. Temporally, levels of both pro- and anti-inflammatory cells were broadly stable across the time-course. Subsequently, selective depletion of the anti-inflammatory microglia/macrophage population by intracerebral injection of mannosylated clodronate liposomes significantly reduced metastatic tumor burden (p < 0.01). Moreover, increased levels of apoptosis were associated with tumors in clodronate liposome treated animals compared to controls (p < 0.05). These findings suggest that microglia/macrophages are important effectors of the inflammatory response in the early stages of brain metastasis, and that targeting the anti-inflammatory microglial/macrophage population may offer an effective new therapeutic avenue for patients with brain metastases.

Are brain and heart tissue prone to the development of thiamine deficiency

Thiamine deficiency is a continuing problem leading to beriberi and Wernickes encephalopathy. The symptoms of thiamine deficiency develop in the heart, brain and neuronal tissue. Yet, it is unclear how rapid thiamine deficiency develops and which organs are prone to development of thiamine deficiency. We investigated these issues in a thiamine deficient animal model. Twenty-four male Lewis rats were fed a thiamine deficient diet, which contained 0.04% of normal thiamine intake. Six control rats were fed 200 μg of thiamine per day. Every week a group of six rats on the thiamine-deficient diet was sacrificed and blood, urine and tissue were stored. Blood and tissue transketolase activity, thiamine and thiamine metabolites were measured and PCR of thiamine transporter-1 (ThTr-1) was performed. Transketolase activity was significantly reduced in red blood cells, liver, lung, kidney and spleen tissue after two weeks of thiamine deficient diet. In brain tissue, transketolase activity was not reduced after up to four weeks of thiamine deficient diet. The amount of thiamine pyrophosphate was also significantly conserved in brain and heart tissue (decrease of 31% and 28% respectively), compared to other tissues (decrease of ~70%) after four weeks of thiamine deficient diet. There was no difference between tissues in ThTr-1 expression after four weeks of thiamine deficient diet. Despite the fact that the heart and the brain are predilection sites for complications from thiamine deficiency, these tissues are protected against thiamine deficiency. Other organs could be suffering from thiamine deficiency without resulting in clinical signs of classic thiamine deficiency in beriberi and Wernickes encephalopathy.

Covalent assembly of nanoparticles as a peptidase-degradable platform for molecular MRI.

Ligand-conjugated microparticles of iron oxide (MPIO) have the potential to provide high sensitivity contrast for molecular magnetic resonance imaging (MRI). However, the accumulation and persistence of non-biodegradable micron-sized particles in liver and spleen precludes their clinical use and limits the translational potential of MPIO-based contrast agents. Here we show that ligand-targeted MPIO derived from multiple iron oxide nanoparticles may be coupled covalently through peptide linkers that are designed to be cleaved by intracellular macrophage proteases. The synthesized particles possess potential characteristics for targeted MRI contrast agents, including high relaxivity, unappreciable sedimentation, clearance from circulation and no overt toxicity. Importantly, we demonstrate that these particles are rapidly degraded both in vitro and in vivo, and that the targeted probes can be used for detection of inflammation in vivo using MRI. This approach provides a platform for molecular MRI contrast agents that is potentially more suitable for translation to humans.

Severe thiamine deficiency complicated by weight loss protects against renal ischaemia-reperfusion injury in rats

Injury due to reperfusion after prior ischaemia (IRI) is an important cause of delayed graft function after renal transplantation [1]. Studies in dogs and rats suggest that thiamine is protective against IRI in heart and brain [2–4]. It has been argued that many organs, including kidneys are deficient in thiamine at the moment of transplantation [4]. We aimed to investigate the effect of severe tissue thiamine deficiency on ischaemia-reperfusion injury in rat kidneys.