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For the analysis of the spectra of complex biofluids, preprocessing methods play a crucial role in rendering the subsequent data analyses more robust and accurate. Normalization is a preprocessing method, which accounts for different dilutions of samples by scaling the spectra to the same virtual overall concentration. In the field of [sup.1]H NMR metabonomics integral normalization, which scales spectra to the same total integral, is the de facto standard. In this work, it is shown that integral normalization is a suboptimal method for normalizing spectra from metabonomic studies. Especially strong metabonomic changes, evident as massive amounts of single metabolites in samples, significantly hamper the integral normalization resulting in incorrectly scaled spectra. The probabilistic quotient normalization is introduced in this work. This method is based on the calculation of a most probable dilution factor by looking at the distribution of the quotients of the amplitudes of a test spectrum by those of a reference spectrum. Simulated spectra, spectra of urine samples from a metabonomic study with cyclosporin-A as the active compound, and spectra of more than 4000 samples of control animals demonstrate that the probabilistic quotient normalization is by far more robust and more accurate than the widespread integral normalization and vector length normalization.

The assignment of significantly changed NMR signals, which were identified with the help of multivariate models, to individual metabolites in biofluids is a manual and tedious task requiring knowledge in chemometrics and NMR spectroscopy. Metabolite projection analysis, introduced in this work, allows automatic linking of multivariate models with metabolites by skipping the level of manual NMR signal identification. The method depends on the projection of sets of metabolite NMR spectra from a database into PCA or PLS models of NMR spectra of biofluid samples. Metabolites that are significantly changed can be identified graphically in metabolite projection plots or numerically as projected virtual concentration. The method is demonstrated together with a newly introduced algorithm for refined nonequidistant binning using a metabonomics study with amiodarone as administered drug. Amiodarone can induce phospholipidosis in the lung and liver, which is accompanied by associated organ toxicity in these organs. It is shown how metabolite projection analysis allows easy and fast tentative assignment of all structures of metabolites whose concentrations in the urine samples significantly changed upon dosage. These metabolites had also been identified previously by manually interpreting the multivariate models and spectra. Among these metabolites, phenylacetylglycine was also identified as being significantly increased. This metabolite has recently been proposed as urinary biomarker for phospholipidosis.

A new triple-resonance (TXI) ([sup.1]H, [sup.13]C, [sup.15]N) high-resolution nuclear magnetic resonance (NMR) capillary probe with 2.5-[micro]L NMR-active sample volume ([V.sub.obs]) was built and tested for applications with mass- and volume-limited samples and for coupling of microbore liquid chromatography to NMR. This is the first microliter probe with optimized coil geometry for use with individual capillary tubes with an outer diameter of 1 mm. The 90[degrees] pulse lengths of the 1-mm microliter probe were below 2 [micros]s for proton, below 8 [micro]s for carbon, and below 20 [micros]s for nitrogen, and a spectral line width at signal haft-height below 1 Hz was obtained. Compared to a conventional 5-mm probe, the new 600-MHz 1-mm TXI microliter probe with z-gradient shows an increase in mass sensitivity by a factor of 5, corresponding to a 25-fold reduction in measuring time. The consumption of costly deuterated solvent is reduced by at least 2 orders of magnitude. The 1-mm TXI microliter probe with z-gradient allows the measurement of one-dimensional [sup.1]H NMR and two-dimensional heteronuclear NMR spectra with a few nanomoles (micrograms) of compound with high sensitivity, speed, and quality. This is a breakthrough for discrete sample NMR spectroscopy with paramount importance for structure elucidation in natural compound chemistry and metabolic research. It offers also advantages for linking chromatographic methods to NMR in an industrial environment. Capillary tube NMR may find new applications in areas where high sample throughput is essential, e.g., in the quality control of large sample arrays from parallel chemistry, screening, and compound depositories. It has the potential to increase the sample throughput by 1 order of magnitude or more if new hardware for fast sample handling and exchange becomes available.

A study of the relaxational behavior of proteins using Lipuri and Szabo's model-free approach reveals that the granulocyte colony stimulating factor backbone has a discrete structure of limited conformational flexibilities in its four helices. The long loop connecting helices C and D displays substantial fluctuations in comparison with the overall rotational correlation time of the entire molecule.

Single-chain Fv fragment (ScFv) backbone revealed a structure that was well-defined while the linker of the ScFv fragment demonstrated rapid internal motion, relative to the rotational correlation time of the entire molecule. Fv fragments, the smallest functional antibody units that have an approximate molecular mass of 26 kDa, are heterodimers of the variable heavy chain and variable light domains.