Florian Wulfert

Proposed minimum reporting standards for data analysis in metabolomics

The goal of this group is to define the reporting requirements associated with the statistical analysis (including univariate, multivariate, informatics, machine learning etc.) of metabolite data with respect to other measured/collected experimental data (often called meta-data). These definitions will embrace as many aspects of a complete metabolomics study as possible at this time. In chronological order this will include: Experimental Design, both in terms of sample collection/matching, and data acquisition scheduling of samples through whichever spectroscopic technology used; Deconvolution (if required); Pre-processing, for example, data cleaning, outlier detection, row/column scaling, or other transformations; Definition and parameterization of subsequent visualizations and Statistical/Machine learning Methods applied to the dataset; If required, a clear definition of the Model Validation Scheme used (including how data are split into training/validation/test sets); Formal indication on whether the data analysis has been Independently Tested (either by experimental reproduction, or blind hold out test set). Finally, data interpretation and the visual representations and hypotheses obtained from the data analyses.

Direct detection of peptides and small proteins in fingermarks and determination of sex by MALDI mass spectrometry profiling

Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI MS) can detect and image a variety of endogenous and exogenous compounds from latent fingermarks. This opportunity potentially provides investigators with both an image for suspect identification and chemical information to be used as additional intelligence. The latter becomes particularly important when the fingermark is distorted or smudged or when the suspect is not a previously convicted offender and therefore their fingerprints are not present in the National Fingerprint Database. One of the desirable pieces of intelligence would be the sex of the suspect from the chemical composition of a fingermark. In this study we show that the direct detection of peptides and proteins from fingermarks by MALDI MS Profiling (MALDI MSP), along with the multivariate modeling of the spectra, enables the determination of sex with 85% accuracy. The chemical analysis of the fingermark composition is expected to additionally provide information on traits such as nutritional habits, drug use or hormonal status.

Oral processing assessed by M-mode ultrasound imaging varies with food attribute.

Ultrasonic imaging was used to quantify oral movements made during the oral processing of foods while subjects assessed the intensity of the sensory attributes, thick, creamy, sweet and bitter. A series of four stimuli were prepared with high and low viscosities and high and low sweetness. Over five sessions, subjects (N=8) were asked to consume 5 ml spoonfuls of each of the stimuli while holding an ultrasound probe beneath their chin so as to produce a mid-line sagital image of the floor of the mouth and tongue. In the first session, subjects were asked to swallow the sample. In subsequent sessions, subjects were asked to rate one of the attributes, thickness, sweetness, creaminess or bitterness, in random order. The resulting video recordings were subjected to an image-processing algorithm to quantify the amount of intra-oral manipulation performed. The results demonstrated that oral movements varied with attribute, especially in the period during which the bulk of the food is typically processed and the following swallowing phase. The foods sweetness affected oral movements especially during the bulk phase, whereas the foods viscosity primarily affected movements during the following swallowing and clearance phases.

Multiple protein extract microarray for profiling human food-specific immunoglobulins A, M, G and E

Existing food immunoglobulin (Ig) tests require large volumes of serum, are limited to one immunoglobulin class, are not amenable to high throughput analysis and only give a limited picture of the immunological response to food antigens. Conversely a new generation of Component Resolved Diagnostic systems using pure proteins is highly specific and totally dependent on the availability of the protein in its recombinant or natural origin form. Here we demonstrate a proof-of-concept of a microarray test based on protein extracts of food components. Our approach relies on innovations on three different fronts: the novelty of using arrayed food samples sequentially extracted with detergent and chaotropic agents, the ability to measure four different Ig classes simultaneously and the ability to analyse the generated data via a suitable bioinformatics/statistical analysis interface. This approach combines high numerical power of microarrays with automation, high throughput analysis and enables detailed investigation of the Ig profiles to food antigens. The prototype shown contains extracts of approximately 350 food ingredients that cover most of the food products found in the UK. Here we showed that the use of a sequential extraction technique to solubilise and then denature food samples has its benefits in the assessment of variations in antigenicity when tested with human sera. A patient dependent degree of class specificity was observed with human sera (IgG specificity correlates well with IgA>IgM>>>>>IgE). Besides generating a simultaneous profile for IgA, IgM, IgG and IgE the array system has shown good discrimination between challenge responders in atopic and non-atopic individuals. Poly- and mono-specific IgE responders were easily identified. The mathematical modelling of specific IgE content showed good correlations when compared with established IgE antibody testing assay (UniCAP). Although in its proof-of-principle stages, the immune profiling technique described here has the potential to provide unique insights into exposure/sensitization and establish relationships between specific immunoglobulin classes and subclasses against food protein antigens. In further developments, the immune profiling technique could also be extended to other related areas such as parasite and bacterial gut infection. Full analyses of large longitudinal and retrospective clinical trials are on going to determine the positive and negative predictive values of the technique.

Predicting in vivo starch digestibility coefficients in newly weaned piglets from in vitro assessment of diets using multivariate analysis

The study was based on correlating a dataset of in vivo mean starch digestibility coefficients obtained in the immediate post-weaning phase of piglets with a range of dietary in vitro variables. The paper presents a model that predicts (R2 0.71) in vivo average starch digestibility coefficients in the 0.5 small-intestinal region of newly weaned piglets fed cereal-based diets using seven in vitro variables describing starch properties that are fundamentally associated with the quality of feed materials, i.e. hydration, structure and amylolytic digestion. The variables were: Rapid Visco Analyser (RVA; measures the viscosity of materials when sheared under defined hydration and temperature regimens); RVA end viscosity; RVA (gelatinisation) peak viscosity; DeltaH (gelatinisation enthalpy that provides an estimate of helical order or degree of crystallinity in starch); water solubility index (WSI; that denotes the amount of soluble polysaccharides released from starch granules to the aqueous phase); grain endogenous amylase (concentration of endogenous alpha-amylase in cereals, assessed by pasting cereal flours in 25 g of AgNO3, an amylase inhibitor v. water using RVA).

Measurement of ischaemia–reperfusion in patients with intermittent claudication using NMR-based metabonomics

Intermittent claudication has proved to be a good in vivo model for ischaemia–reperfusion. For assessment of ischaemia–reperfusion damage, the known biochemical markers all have disadvantages with respect to sensitivity and interference with other physiological events. In this work, we studied the metabolic effects of ischaemia–reperfusion in patients with intermittent claudication, and the effects of vitamin C and E intervention, using both traditional biochemical measurements and 1H‐NMR‐based metabonomics on urine and plasma. The 1H‐NMR spectra were subjected to multivariate modelling using principal components discriminant analysis, and the observed clusters were validated using joint deployment of univariate analysis of variance and Tukey–Kramer honestly significant difference (HSD) testing. The study involved 14 patients with intermittent claudication and three healthy volunteers, who were monitored during a walking test, before and after a vitamin C/E intervention, and after a washout period. The effect of exercise was only observable for a limited number of biochemical markers, whereas 1H NMR revealed an effect in line with anaerobic ATP production via glycolysis in exercising (ischaemic) muscle of the claudicants. Thus, the beneficial effect of vitamins C and E in claudicants was more pronounced when observed by metabonomics than by traditional biochemical markers. The main effect was more rapid recovery from exercise to resting state metabolism. Furthermore, after intervention, claudicants tended to have lower concentrations of lactate and glucose and several other citric acid cycle metabolites, whereas acetoacetate was increased. The observed metabolic changes in the plasma suggest that intake of vitamin C/E leads to increased muscle oxidative metabolism. Copyright

Data handling for interactive metabolomics: tools for studying the dynamics of metabolome-macromolecule interactions

All published metabolomics studies investigate changes in either absolute or relative quantities of metabolites. However, blood plasma, one of the most commonly studied biofluids for metabolomics applications, is a complex, heterogeneous mixture of lipoproteins, proteins, small organic molecules and ions which together undergo a variety of possible molecular interactions including metal complexation, chemical exchange processes, micellular compartmentation of metabolites, enzyme-mediated biotransformations and small-molecule-macromolecule binding. In particular, many low molecular weight (MW) compounds (including drugs) can exist both ‘free’ in solution and bound to proteins or within organised aggregates of macromolecules. To study the effects of e.g. disease on these interactions we suggest that new approaches are needed. We have developed a technique termed ‘interactive metabolomics’ or i-metabolomics. i-metabolomics can be defined as: “The study of interactions between low MW biochemicals and macromolecules in heterogeneous biosamples such as blood plasma, without pre-selection of the components of interest”. Standard 1D NMR experiments commonly used in metabolomics allow metabolite concentration differences between samples to be investigated because the intensity of each peak depends on the concentration of the compound in question. On the other hand, the instrument can be set-up to measure molecular interactions by monitoring the diffusion coefficients of molecules. According to the Stokes–Einstein equation, the diffusion coefficient of a molecule is inversely proportional to its effective size, as represented by the hydrodynamic radius. Therefore, when low MW compounds are non-covalently bound to proteins, the observed diffusion coefficient for the compound will be intermediate between those of its free and bound forms. By measuring diffusion by NMR, the degree of protein binding can be estimated for either low MW endogenous biochemicals or xenobiotics. This type of experiment is referred to as either Diffusion-Ordered Spectroscopy (DOSY) or Diffusion-Edited Spectroscopy, depending on the type of post-acquisition data processing applied to the spectra. Results presented in this paper demonstrate approaches for the non-selective modelling of metabolite-macromolecule interactions (i-metabolomics), whilst additionally highlighting some of the all too frequently ignored issues associated with interpretation of data derived from profiling of blood plasma.

Prediction of tolerance in children with IgE mediated cow's milk allergy by microarray profiling and chemometric approach

The sera of a retrospective cohort (n=41) composed of children with well characterized cows milk allergy collected from multiple visits were analyzed using a protein microarray system measuring four classes of immunoglobulins. The frequency of the visits, age and gender distribution reflected real situation faced by the clinicians at a pediatric reference center for food allergy in São Paulo, Brazil. The profiling array results have shown that total IgG and IgA share similar specificity whilst IgM and in particular IgE are distantly related. The correlation of specificity of IgE and IgA is variable amongst the patients and this relationship cannot be used to predict atopy or the onset of tolerance to milk. The array profiling technique has corroborated the clinical selection criteria for this cohort albeit it clearly suggested that 4 out of the 41 patients might have allergies other than milk origin. There was also a good correlation between the array data and ImmunoCAP results, casein in particular. By using qualitative and quantitative multivariate analysis routines it was possible to produce validated statistical models to predict with reasonable accuracy the onset of tolerance to milk proteins. If expanded to larger study groups, the array profiling in combination with the multivariate techniques show potential to improve the prognostic of milk allergic patients.

Nutrimetabolomics: development of a bio-identification toolbox to determine the bioactive compounds in grape juice.

BACKGROUND Grape juice and related products have previously been associated with many health benefits, such as protection against cardiovascular disease. Current consensus is that the polyphenols are the likely bioactive species in these products. RESULTS Extracts of commercially available grape juices exhibited biological antioxidant activities ranging from 19.30 to 3099.51 µM trolox equivalents, as determined by cell-based assay in which J774 macrophages were stimulated with lipopolysaccaride at a concentration of 100 µg/ml for 1 h. Partial least-squares regression was then used to determine covariance between the antioxidant activity and 400 MHz (1)H NMR spectral profiles using models with R(2)X and R(2)Y values of 0.64 and 0.95, respectively, using three latent variables: the Q(2)(cum) was 0.63. Hydroxycinnamic acids and their derivatives were identified as being the most positively correlated with the antioxidant activity. CONCLUSION The work presented here describes a strategy for the bioinformatic linkage of plant metabolomic data with in vitro biological activity as an initial step towards determining structure-activity relationships.

Identification and monitoring of intermediates and products in the acrylamide pathway using online analysis.

Acrylamide formation under controlled processing conditions was studied in a starch matrix by analyzing volatile compounds in the gas phase using online mass spectrometry. Compounds were identified using mass spectral analysis, authentic standards, and the labeling patterns from isotopically labeled asparagine and sugars. Acrylamide, 3-aminopropanamide, methylpyrazine, 3-oxopropanamide, and aminopropan-2-one were assigned to the ions at m/ z 72, 89, 95, 88, and 74, respectively. Ion m/ z 60 was proposed as the transamination product of glyoxal, but labeling experiments did not support this assignment. Temporal formation of acrylamide and related compounds was studied in 51 samples containing asparagine and selected sugars or carbonyls. Data from the experiments were analyzed to investigate correlations between the amounts of acrylamide, intermediates, and pyrazines formed. A strong correlation between 3-aminopropanamide and acrylamide was found in all samples, whereas other correlations were reactant specific. Preliminary multiway analysis of the data identified temporal similarities in the ion profiles and showed that dynamic monitoring can follow the production and utilization of intermediates leading to acrylamide.