Analytical techniques used for multivariate analysis of endogenous metabolites in biological systems (e.g., metabolomics, metabonomics) must be capable of accurately and selectively monitoring many known and unknown molecules that span a diverse chemical spectrum and over extremely large dynamic concentration ranges. Mass spectrometric (MS) and electrochemical array (EC-Array) detection have been widely used for multi-component analysis with applicability to low-level (fmol) metabolites. Described here are practical considerations and results obtained with the combined use of EC-Array and MS for HPLC-based multivariate metabolomic analysis. Data presented include the study of changes in rat urinary metabolite profiles associated with xenobiotic toxin exposure analyzed by HPLC using water:acetonitrile binary gradient conditions and post-column flow splitting between EC-Array and MS detectors. Results show complementary quantitative and qualitative analysis and the ability to differentiate sample groups consistent with xenobiotic-induced histopathological changes. The potential applicability of this hyphenated technique for biomarker elucidation through measurement of redox active compounds that are commonly associated with disease pathology and xenobiotic toxicity is discussed. The use of EC reactor cells in series with MS is also presented as a means of producing likely metabolites to facilitate structural elucidation and confirmation. (J Am Soc Mass Spectrom 2004, 15, 1717-1726

Recent studies have shown that substitution of Ala for one or more Phe residues in calmodulin (CaM) imparts a temperature-sensitive phenotype to yeast (Ohya, Y., and Botstein, D. (1994) Science 263, 963-966). The Phe residue immediately preceding the first Ca(2+) ligand in site III of CaM (Phe-92) was found to be of particular importance because the mutation at this position alone was sufficient to induce this phenotype. In the present work we have studied the functional and structural consequences of the Phe-92 --> Ala mutation in human liver calmodulin. We found that the mutant (CaMF92A) is incapable of activating phosphodiesterase, and the maximal activation of calcineurin is reduced by 40% as compared with the wild type CaM. Impaired regulatory properties of CaMF92A are accompanied by an increase in affinity for Ca(2+) at the C-terminal domain. To investigate the structural consequences of the F92A mutation, we constructed four recombinant C-terminal domain fragments (C-CaM) of calmodulin (residues 78-148): 1) wild type (C-CaMW); 2) Ala substituted for Phe-92 (C-CaMF92A); 3) cysteine residues introduced at position 85 and 112 to lock the domain with a disulfide bond in the Ca(2+)-free (closed) conformation (C-CaM85/112); and 4) mutations 2 and 3 combined (C-CaM85/112F92A). The Cys-containing mutants readily form intramolecular disulfide bonds regardless whether Phe or Ala is present at position 92. The F92A mutation causes a decrease in stability of the domain in the absence of Ca(2+) as indicated by an 11.8 degree C shift in the far UV circular dichroism thermal unfolding curve. This effect is reversed by the disulfide bond in the C-CaM85/112F92A mutant. The C-CaMW peptide shows a characteristic Ca(2+)-dependent increase in solvent-exposed hydrophobic surface which was monitored by an increase in the fluorescence of the hydrophobic probe 1,1'-bis(4-anilino)-naphthalene-5,5'-disulfonic acid. The fluorescence increase induced by C-CaMF92A is approximately 45% lower than that induced by C-CaMW suggesting that the F92A mutation causes a decrease in the accessibility of several hydrophobic side chains in the C-terminal domain of CaM in the presence of Ca(2+). The Cys-85-Cys-112 disulfide bond causes a 10- or 5.9-fold decrease in Ca(2+) affinity depending on whether Phe or Ala is present at position 92, respectively, suggesting that coupling between Ca(2+) binding and the conformational transition is weaker in the absence of the phenyl ring at position 92. Our results indicate that Phe-92 makes an important contribution to the Ca(2+)-induced transition in the C-terminal domain of CaM. This is most likely the reason for the severely impaired regulatory properties of the CaM mutants having Ala substituted for Phe-92.

Low-density lipoproteins (LDLs) in plasma are constructed from a single molecule of apolipoprotein B-100 (apoB) (M(r) 512,000) in association with lipid [approximate M(r) (2-3) x 10(6)]. LDL oxidation is an important process in the development of atherosclerosis, and can be imitated by the addition of Cu2+ ions. Synchrotron X-ray scattering of LDL yields curves without radiation damage effects at concentrations close to physiological. The radius of gyration RG for preparations of LDL from different donors ranged between 12.1 and 16.0 nm, with a mean of 13.9 nm. At 4 degrees C, the distance distribution curve P(r) indicated a maximum dimension of 25-27 nm for LDL, a peak at 19.5 nm which corresponds to a surface shell of protein and phospholipid head groups in LDL, and submaxima between 1.7 and 13.5 nm, which correspond to an ordered lipid core in LDL. LDL from different donors exhibited distinct P(r) curves. For oxidation studies of LDL by X-rays, data are best obtained at 4 degrees C at a concentration of > or = 2 mg of LDL protein/ml together with controls based on non-oxidized LDL. LDL oxidation (2 mg of apoB/ml) was studied at 37 degrees C in the presence of 6.4, 25.6 and 51.2 mu of Cu2+/g of apoB. Large changes in P(r) were reproducibly observed in the inter-particle distance range between 13 and 16 nm shortly after initiation of oxidation. This corresponds to the phospholipid hydrocarbon in LDL, which has either increased in electron density during oxidation or become increasingly disordered. After 25 h, the structural changes subsequently spread to regions of the P(r) curves assigned to surface apoB and the central core of cholesteryl esters and triacyl-glycerols. Lipid analyses were carried out under the same solution conditions. The alpha-tocopherol and beta-carotene antioxidant contents of LDL were consumed within 1-2 h. Analyses of the formation of thiobarbituric acid-reactive substances and lipid hydroperoxides indicated that arachidonic acid was preferentially oxidized before the maximal formation of lipid hydroperoxides at 8-12 h after initiation of oxidation. High-performance TLC showed that phosphatidylcholine was continuously converted into lysophosphatidylcholine during oxidation, which is consistent with the early changes in the X-ray P(r) curves. The neutral core lipids became modified only after 12-15 h of oxidation. The combination of X-ray scattering structural analyses with biochemical analyses shows that the oxidation of LDL first affects the outer shell of surface phospholipid, then it spreads towards damage of apoB and the internal neutral lipid core of LDL.

Low-density lipoproteins (LDL) in plasma are constructed from a single molecule of apolipoprotein B-100 (M(r) 512000) in association with lipid (approximate M(r) 2-3 x 10(6)). The gross structure was studied using an updated pulsed-neutron camera LOQ with an area detector to establish the basis for the interpretation of structural changes seen during dynamic studies of LDL oxidation. Neutron-scattering data for LDL in 100% 2H2O buffers emphasize their external appearance. Guinier analysis on a continuous-flux neutron camera D17 revealed pronounced concentration-dependences in the radius of gyration, RG, and the intensity of forward scattering, I(0) (equivalent to the M(r) of LDL) between 0.5 and 11 mg of LDL protein/ml. LDL preparations from different donors gave different RG values. When extrapolated to zero concentration, RG values ranged between 8.3 and 10.6 nm and were linearly correlated with M(r), which is consistent with a spherical structure. The distance-distribution function P(r) in real space showed a single maximum at 9.1-10.9 nm, which is just under half the observed maximum dimension of 23.1 +/- 1.2 nm expected for a spherical structure. The neutron radial-density function p(r) exhibited a plateau of high and featureless density at the centre of LDL. LDL can be modelled by a polydisperse assembly of spheres with two internal densities and a mean radius close to 10.0 nm in a normal distribution of radii with a standard deviation of 2.0 nm. The data are consistent with recent electron-microscopy and ultracentrifugation data.(ABSTRACT TRUNCATED AT 250 WORDS)

The oxidative modification of low-density lipoproteins (LDL) is recognized to be a key event in the development of atherosclerotic plaques on artery walls. The characteristics of LDL oxidized by cells of the artery wall can be imitated by the addition of Cu2+ ions to initiate lipid peroxidation in LDL. Neutron scattering of LDL in 2H2O buffers enables the time course of changes in the gross structure of LDL during oxidation to be continuously monitored under conditions close to physiological. Oxidation of LDL [2 mg of apolipoprotein B (apoB) protein/ml] was studied in the presence of 6.4, 25.6 and 51.2 mumol of Cu2+/g of apoB by incubation at 37 degrees C for up to 70 h. Neutron Guinier analyses showed that the radius of gyration RG (indicative of size) and the forward-scattered intensity at zero angle I(0) (indicative of M(r)) continuously increased during oxidation, indicating that LDL had aggregated. Both the rate of aggregation and the change in RG and I(0) values after 10 and 50 h increased with Cu2+ concentration. Distance-distribution functions P(r) showed that, within 4 h, the maximum dimension of LDL increased from 23 to 55 nm. The P(r) curves of oxidatively modified LDL exhibited two peaks at 10-12 nm and 26 nm. The 10-12 nm peak corresponds to native LDL, and the 26 nm peak is assigned to the initial formation of LDL dimers and trimers and their progression to form higher oligomers. The growth of the 26 nm peak depended on Cu2+ concentration. Particle-size-distribution functions Dv(r) suggested that the polydisperse spherical structure of LDL ceased to exist after 30 h, at which point the LDL samples underwent a phase separation. Related, but not identical, changes in the I(Q) and P(r) curves were observed when native LDL was self-aggregated by brief vortexing. Parallel assessment of LDL protein modification by SDS/PAGE showed increased aggregation and degradation of apoB with increased Cu2+ concentrations, and that the main apoB protein band had diminished after 2-8 h, depending on the amount of Cu2+ added. The uptake and degradation of oxidized 125I-labelled LDL by mouse peritoneal macrophages occurred maximally within the first 10 h, and increased in proportion to the Cu2+ concentration. ApoB protein broke down within the first 10 h of oxidation, and this is the period when scavenger receptors on macrophages can recognize and internalize oxidized LDL. Within 10 h, the protein-lipid interactions responsible for the spherical LDL structure became destabilized by protein fragmentation.(ABSTRACT TRUNCATED AT 400 WORDS)