Andrew A. Vaughan

Is Serum or Plasma More Appropriate for Intersubject Comparisons in Metabolomic Studies? An Assessment in Patients with Small-Cell Lung Cancer

In clinical analyses, the most appropriate biofluid should be analyzed for optimal assay performance. For biological fluids, the most readily accessible is blood, and metabolomic analyses can be performed either on plasma or serum. To determine the optimal agent for analysis, metabolic profiles of matched human serum and plasma were assessed by gas chromatography/time-of-flight mass spectrometry and ultrahigh-performance liquid chromatography mass spectrometry (in positive and negative electrospray ionization modes). Comparison of the two metabolomes, in terms of reproducibility, discriminative ability and coverage, indicated that they offered similar analytical opportunities. An analysis of the variation between 29 small-cell lung cancer (SCLC) patients revealed that the differences between individuals are markedly similar for the two biofluids. However, significant differences between the levels of some specific metabolites were identified, as were differences in the intersubject variability of some metabolite levels. Glycerophosphocholines, erythritol, creatinine, hexadecanoic acid, and glutamine in plasma, but not in serum, were shown to correlate with life expectancy for SCLC patients, indicating the utility of metabolomic analyses in clinical prognosis and the particular utility of plasma in relation to the clinical management of SCLC.

IMMOBILISED REAGENTS FOR OPTICAL HEAVY METAL IONS SENSING

Abstract Many ligands such as 1-nitroso-2-naphthol (NN), 4-(2-pyridylazo)resorcinol (PAR), 2,4-dinitrosoresorcinol (DNR) and 1-(2-pyridylazo)-2-naphthol (PAN) have been investigated in the development of optical sensors for heavy metal ions found in aquatic environments which include Co2+, Cu2+, Ni2+, Fe3+, Cd2+, Zn2+, Pb2+ and Hg2+. The reagents were immobilised by physical adsorption on to polymeric materials such as XAD-4, XAD-7 and Dowex ion-exchange resins, and were found to exhibit chromic characteristics in the absence and presence of metal ions in solution. Reflectance measurements are used here. Parameters such as pH, measurement wavelength and reagent concentration have been optimised for a given metal ion, and the reversibility/regenerability characteristics of the sensing reagent have been studied together with the limit of detection and the concentration range producing linear response for the metal ion.

Optical fibre reflectance sensors for the detection of heavy metal ions based on immobilised Br-PADAP

Abstract Optical fibre based reflectance sensors for the detection of heavy metal ions have been developed based on the use of the immobilised reagent Br-PADAP. Two methods of immobilisation, namely physical adsorption and membrane entrapment, have been employed and their relative merits have been discussed. Br-PADAP immobilised onto XAD-4 shows a reproducible and reversible response to heavy metal ions and a good limit of detection (31 ppb for Zn(II) with an analysis time of 6 min). Br-PADAP/PVC membranes show fairly quick and reproducible responses, but are irreversible. Br-PADAP/XAD-4 is proposed as a suitable reagent phase for use in a multi-metal ion sensor.

Optical ammonia sensing films based on an immobilized metalloporphyrin

Optical sensing films for ammonia gas have been developed by immobilizing zinc 5,10,15,20-tetraphenylporphine (ZnTPP) in silicone rubber. The detection of ammonia can be achieved using both absorbance and fluorescence emission measurements. Spectral changes were observed due to the coordination of NH3 molecules to the ZnII ion in the immobilized metalloporphyrin. Effects of other immobilization matrices on sensor properties were investigated. ZnTPP-silicone films were found to show a reasonably high sensitivity to ammonia, with a linear range of 0–6 mg m–3(0–8.5 ppm). The detection limit for ammonia in nitrogen was 0.5 mg m–3(0.7 ppm). The equilibrium response time of the films was approximately 4 min and the reaction with ammonia was reversible. ZnTPP films were also found to be sensitive to triethylamine.

Liquid Chromatography–Mass Spectrometry Calibration Transfer and Metabolomics Data Fusion

Metabolic profiling is routinely performed on multiple analytical platforms to increase the coverage of detected metabolites, and it is often necessary to distribute biological and clinical samples from a study between instruments of the same type to share the workload between different laboratories. The ability to combine metabolomics data arising from different sources is therefore of great interest, particularly for large-scale or long-term studies, where samples must be analyzed in separate blocks. This is not a trivial task, however, due to differing data structures, temporal variability, and instrumental drift. In this study, we employed blood serum and plasma samples collected from 29 subjects diagnosed with small cell lung cancer and analyzed each sample on two liquid chromatography-mass spectrometry (LC-MS) platforms. We describe a method for mapping retention times and matching metabolite features between platforms and approaches for fusing data acquired from both instruments. Calibration transfer models were developed and shown to be successful at mapping the response of one LC-MS instrument to another (Procrustes dissimilarity = 0.04; Mantel correlation = 0.95), allowing us to merge the data from different samples analyzed on different instruments. Data fusion was assessed in a clinical context by comparing the correlation of each metabolite with subject survival time in both the original and fused data sets: a simple autoscaling procedure (Pearsons R = 0.99) was found to improve upon a calibration transfer method based on partial least-squares regression (R = 0.94).

A workflow for bacterial metabolic fingerprinting and lipid profiling: application to Ciprofloxacin challenged Escherichia coli

The field of lipidomics focuses upon the non-targeted analysis of lipid composition, the process of which follows similar routines to those applied in conventional metabolic profiling, however lipidomics differs with respect to the sample preparation steps and chosen analytical platform applied to the sample analysis. Conventionally, lipidomics has applied analytical techniques such as direct infusion mass spectrometry and more recently reverse phase liquid chromatography–mass spectrometry, for the detection of mono-, di-, and tri-acyl glycerols, phospholipids, and other complex lipophilic species such as sterols. The field is rapidly expanding, especially with respect to the clinical sciences where it is known that changes of lipid composition, especially phospholipids, are commonly associated with many disease processes. As a proof of principle study, a small number of Escherichia coli isolates were selected on the basis of their sensitivity to a second generation fluoroquinolone antibiotic, known as Ciprofloxacin (E. coli isolates 161 and 171, non-ST131 isolates, which are resistant and sensitive respectively: E. coli isolates 160 and 173, ST131 sequence isolates which are resistant and susceptible respectively). It has been proposed that Ciprofloxacin may be a surface active drug that interacts at the surface-water interface of the phospholipid bi-layer where the head groups reside. Further, antibiotic resistance through intracellular exclusion is known to result in remodelling of the phospholipid membrane. Therefore, to study the effects of Ciprofloxacin on both susceptible and resistant bacterial strains, lipid profiling would present an informative approach. Control and antibiotic challenged cultures for each of the isolates were compared for changes in metabolite and lipid composition as detected by FT-IR spectroscopy and RP-UHPLC–MS, and appraised with a variety of chemometric data analysis approaches. The developed bacterial lipidomics workflow was deemed to be highly reproducible (with respect to the employed technical and analytical routines) and led to the detection of a large array of lipid classes as well as highlighting a range of significant lipid alterations that differed in regulation between susceptible and resistant E. coli isolates.