Effectiveness of mid-infrared spectroscopy for the prediction of cow milk metabolites.

Proton nuclear magnetic resonance (1H NMR) spectroscopy is acknowledged as one of the most powerful analytical methods with cross-cutting applications in dairy foods. To date, the use of 1H NMR spectroscopy for the collection of milk metabolic profile is hindered by costly and time-consuming sample preparation and analysis. The present study aimed at evaluating the accuracy of mid-infrared spectroscopy (MIRS) as a rapid method for the prediction of cow milk metabolites determined through 1H NMR spectroscopy. Bulk milk (n = 72) and individual milk samples (n = 482) were analyzed through one-dimensional 1H NMR spectroscopy and MIRS. Nuclear magnetic resonance spectroscopy identified 35 milk metabolites, which were quantified in terms of relative abundance, and MIRS prediction models were developed on the same 35 milk metabolites, using partial least squares regression analysis. The best MIRS prediction models were developed for galactose-1-phosphate, glycerophosphocholine, orotate, choline, galactose, lecithin, glutamate, and lactose, with coefficient of determination in external validation from 0.58 to 0.85, and ratio of performance to deviation in external validation from 1.50 to 2.64. The remaining 27 metabolites were poorly predicted. This study represents a first attempt to predict milk metabolome. Further research is needed to specifically address whether developed prediction models may find practical application in the dairy sector, with particular regard to the screening of dairy cows' metabolic status, the quality control of dairy foods, and the identification of processed milk or incorrectly stored milk.

Nuclear magnetic resonance spectroscopy to investigate the association between milk metabolites and udder quarter health status in dairy cows.

Nuclear magnetic resonance spectroscopy was applied to investigate the association between milk metabolome and udder quarter health status in dairy cows. Mammary gland health status was defined by combining information provided by traditional somatic cell count (SCC) and differential SCC (DSCC), which expresses the percentage of neutrophils and lymphocytes over total SCC. Quarter milk samples were collected in triplicate (d 1 to 3) from 10 Simmental cows, 5 defined as cases and 5 defined as controls according to SCC levels at d 0. A total of 120 samples were collected and analyzed for bacteriology, milk composition, SCC, DSCC, and milk metabolome. Bacteriological analysis revealed the presence of mostly coagulase-negative staphylococci in quarter milk samples of cows defined as cases. Nuclear magnetic resonance spectra of all quarter samples were first analyzed using the unsupervised multivariate approach principal component analysis, which revealed a specific metabolomic fingerprint of each cow. Then, the supervised cross-validated orthogonal projections to latent structures discriminant analysis unquestionably showed that each cow could be very well identified according to its milk metabolomic fingerprint (accuracy = 95.8%). The comparison of 12 different models, built on bucketed 1-dimensional NOESY spectra (noesygppr1d, Bruker BioSpin) using different SCC and DSCC thresholds, corroborated the assumption of improved udder health status classification ability by joining information provided by both SCC and DSCC. Univariate analysis performed on the 34 quantitated metabolites revealed lower levels of riboflavin, galactose, galactose-1-phosphate, dimethylsulfone, carnitine, hippurate, orotate, lecithin, succinate, glucose, and lactose, and greater levels of lactate, phenylalanine, choline, acetate, O-acetylcarnitine, 2-oxoglutarate, and valine, in milk samples with high somatic cells. In the 5 cases, results of the udder quarter with the highest SCC compared with its symmetrical relative were in line with quarter-level findings. Our study suggests that increased SCC is associated with changes in milk metabolite fingerprint and highlights the potential use of different metabolites as novel indicators of udder health status and milk quality.

Grazing affects metabolic pattern of individual cow milk.

Effective traceability tools able to characterize milk from pasture are important to safeguard low-input farming systems, niche dairy products, and local traditions. The aims of the present study were to investigate the ability of proton nuclear magnetic resonance (1H NMR) spectroscopy to discriminate between milk produced from cows before and after the beginning of the grazing season, and to assess the effects of grazing on milk metabolites. The research trial involved a single alpine holding with 72 lactating cows. Individual milks were repeatedly sampled from the same animals before (i.e., d -3 and -1) and after (i.e., d 2, 3, 7, 10, and 14) the onset of the grazing period. One-dimensional 1H NMR spectra of milk extracts were collected through a Bruker spectrometer. Random forest discriminant analysis was applied to 1H NMR spectra to predict the period of collection for each sample. Data concerning the relative abundance of milk metabolites were analyzed through a linear mixed model, which included the fixed effects of period of sampling, cow breed, stage of lactation, and parity, and the random effect of cow nested within breed. The random forest model exhibited great accuracy (93.1%) in discriminating between samples collected on d -3, -1, 2, and 3 and those collected on d 7, 10, and 14. Univariate analysis performed on the 40 detected metabolites highlighted that milk samples from pasture had lower levels of 14 compounds (with fumarate being the most depressed metabolite) and greater levels of 15 compounds (with methanol and hippurate being the most elevated metabolites). Results indicate that milk 1H NMR spectra are promising to identify milk produced in different conditions. Also, our study highlights that grazing is associated with significant changes of milk metabolic profile, suggesting the potential use of several metabolites as indicators of farm management.

Solving the crystal structure of human calcium-free S100Z: the siege and conquer of one of the last S100 family strongholds.

The X-ray structure of human apo-S100Z has been solved and compared with that of the zebrafish calcium-bound S100Z, which is the closest in sequence. Human apo-S100A12, which shows only 43% sequence identity to human S100Z, has been used as template model to solve the crystallographic phase problem. Although a significant buried surface area between the two physiological dimers is present in the asymmetric unit of human apo-S100Z, the protein does not form the superhelical arrangement in the crystal as observed for the zebrafish calcium-bound S100Z and human calcium-bound S100A4. These findings further demonstrate that calcium plays a fundamental role in triggering quaternary structure formation in several S100s. Solving the X-ray structure of human apo-S100Z by standard molecular replacement procedures turned out to be a challenge and required trying different models and different software tools among which only one was successful. The model that allowed structure solution was that with one of the lowest sequence identity with the target protein among the S100 family in the apo state. Based on the previously solved zebrafish holo-S100Z, a putative human holo-S100Z structure has been then calculated through homology modeling; the differences between the experimental human apo and calculated holo structure have been compared to those existing for other members of the family.

Identification of a serum-detectable metabolomic fingerprint potentially correlated with the presence of micrometastatic disease in early breast cancer patients at varying risks of disease relapse by traditional prognostic methods.

BACKGROUND: Prognostic tools in early breast cancer are inadequate. The evolving field of metabolomics may allow more accurate identification of patients with residual micrometastases. PATIENTS AND METHODS: Forty-four early breast cancer patients with pre- and postoperative serum samples had metabolomic assessment by nuclear magnetic resonance. Fifty-one metastatic patients served as control. Differential clustering was identified and used to calculate individual early patient 'metabolomic risk', calculated as inverse distance of each early patient from the metastatic cluster barycenter. Metabolomic risk was compared with Adjuvantionline 10-year mortality assessment. RESULTS: Innate serum metabolomic differences exist between early and metastatic patients. Preoperative patients were identified with 75% sensitivity, 69% specificity and 72% predictive accuracy. Comparison with Adjuvantionline revealed discordance. Of 21 patients assessed as high risk by Adjuvantionline, 10 (48%) and 6 (29%) were at high risk by metabolomics in pre- and postoperative settings, respectively. Of 23 low-risk patients by Adjuvantionline, 11 (48%) preoperative and 20 (87%) postoperative patients were at low risk by metabolomics. CONCLUSIONS: This study identifies metabolomic discrimination between early and metastatic breast cancer. Micrometastatic disease may account for metabolomic misclassification of some early patients as metastatic. Metabolomics identifies more patients as low relapse risk compared with Adjuvantionline. Further exploration of this metabolomic fingerprint is warranted.

Interaction of cobalt-bovine carbonic anhydrase with the acetate ion.

The visible spectra of solutions of cobalt(II) bovine carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) with increasing amounts of acetate have allowed the determination of the apparent inhibition constants and of the extrapolated limit spectra of the completely bound enzyme. The electronic spectra have been interpreted on the basis of the presence of five coordinate adduct species. 13C and 1H NMR spectra have also been recorded and discussed on the basis of the metal enzyme-acetate type of interactions. Titrations by means of NMR spectroscopy of the acetate-cobalt(II) bovine carbonic anhydrase with p-toluenesulfonamide and azide indicate the existence of two binding sites for the acetate group.

New applications of paramagnetic NMR in chemical biology.

The methodological accessibility to solution structure and dynamic investigation of paramagnetic metallobiomolecules has afforded the ability to tackle the redox pairs of electron transfer proteins of which at least one is paramagnetic, to study the orientation effects of high magnetic fields on paramagnetic biomolecules, and finally to study the role of metal-based cofactors in protein folding and stability.

Crystal structure of the catalytic domain of human matrix metalloproteinase 10.

The catalytic domain of matrix metalloproteinase-10 (MMP-10) has been expressed in Escherichia coli and its crystal structure solved at 2.1 A resolution. The availability of this structure allowed us to critically examine the small differences existing between the catalytic domains of MMP-3 and MMP-10, which show the highest sequence identity among all MMPs. Furthermore, the binding mode of N-isobutyl-N-[4-methoxyphenylsulfonyl]glycyl hydroxamic acid (NNGH), which is one of the most known commercial inhibitors of MMPs, is described for the first time.

Transient versus steady state NOE in paramagnetic molecules Cu2Co2SOD as an example.

Truncated, steady state and transient NOE experiments have been performed on bovine Cu2Co2 superoxide dismutase. The effectiveness of the different NOE experiments in the general case of paramagnetic macromolecules is discussed. It is concluded that steady state NOEs give superior results. The validity of the two spins approximation is discussed, and NOE values for a fully coupled set of nuclei have been calculated. Transient NOE experiments, when properly performed, confirm the previous assignment of the hyperfine shifted signals in Cu2Co2SOD based on steady state NOE measurements [(1989) Inorg. Chem. 28, 4650] and eliminate any further reason for controversy on an important issue as the assignment of the 1H NMR signals of protons of metal-coordinated imidazoles.

ePHOGSY experiments on a paramagnetic protein: location of the catalytic water molecule in the heme crevice of the oxidized form of horse heart cytochrome c.

The hydration properties of the oxidized form of horse heart cytochrome c have been studied by 1H NMR spectroscopy. Application of ePHOGSY (enhanced protein hydration observed through gradient spectroscopy) experiments over a paramagnetic molecule provided firm spectroscopic evidence of the presence of a water molecule in the heme crevice. A few intermolecular NOEs have been used to locate the water molecule at about 0.65 nm away from the iron atom and to compare the position observed in solution with that observed in the crystal structure and in solution for the reduced state. The resulting picture is that there is a detectable movement of the water molecule upon oxidation.

The D13C variant of Bacillus schlegelii 7Fe ferredoxin is an 8Fe ferredoxin as revealed by 1H-NMR spectroscopy.

The N-terminal cluster binding motif Cys8XXXXXXXCys16....Cys49 of Bacillus schlegelii 7Fe ferredoxin, which provides the ligands to the [Fe3S4]+ cluster, was modified by the mutation Asp13 --> Cys. The mutant D13C is expressed in Escherichia coli as an 8Fe ferredoxin, with NMR properties similar to those of clostridial-type ferredoxins. The full assignment of the hyperfine shifted resonances indicates that Cys13 serves as ligand to the new fourth iron atom in the N-terminal cluster despite the atypical binding sequence CysXXXXCysXXCys....Cys. The C alpha-C beta-S-Fe dihedral angles of all cysteine ligands to the two [Fe4S4]2+ clusters of the D13C variant are similar to those observed in other 8Fe and 4Fe ferredoxins.

2D 1H NMR studies of oxidized 2(Fe4S4) ferredoxin from Clostridium pasteurianum.

Oxidized ferredoxin from Clostridium pasteurianum, containing two Fe4S4 clusters, has been investigated using 2D 1H NMR spectroscopy at 600 MHz. 2D NMR experiments allowed complete assignment of the sixteen isotropically shifted signals corresponding to the beta-CH2 protons of the eight metal coordinated cysteines. Geminal connectivities of Cys beta-CH2 protons were identified through magnitude COSY experiments and confirmed through 2D NOESY experiments. A few additional signals could be assigned to the corresponding alpha-CH protons. The importance of 2D experiments to achieve firm assignments of isotropically shifted signals in paramagnetic metalloproteins is stressed.

Determination of the [Fe4S4]Cys4 cluster geometry of Desulfovibrio africanus ferredoxin I by 1H NMR spectroscopy.

1D and 2D 1H NMR studies of the Fe4S4 cluster containing ferredoxin I from Desulfovibrio africanus have been carried out with the aim of determining the geometry of the cluster linkages with the 4 Cys side chains that bind the cluster. This required the Cys beta CH resonances of the oxidised protein to be sequence-specifically and stereo-specifically assigned, and this was accomplished by a combination of TOCSY and NOE measurements, allied to model building based on X-ray structures of related ferredoxins. An analysis of the estimated hyperfine shifts of the Cys beta CH resonances with a Karplus-type equation relating the shifts to iron-sulfur-beta carbon-beta proton dihedral angles, taken together with the relative relaxation rates of the two beta CH2 resonances, estimated from their linewidths, then allowed the iron-sulfur-beta-carbon-alpha-carbon dihedral angles to be determined. A novel representation of the NMR data is presented which shows that the cluster dihedral angles are uniquely determined by the NMR data. The analysis reveals that the dihedral angles for D. africanus ferredoxin I are similar to the corresponding angles of other ferredoxins even though there are differences in their 1H NMR spectra. The sequence-specific and stereospecific assignments have been extended by analogy to the related Fe4S4-containing D. gigas ferredoxin I, and the stereospecific assignments to the Fe4S4-containing Thermococcus litoralis ferredoxin.

Cross correlation between the dipole-dipole interaction and the Curie spin relaxation: the effect of anisotropic magnetic susceptibility.

Cross-correlated relaxation caused by the interference of nuclear dipole-dipole interaction and the Curie spin relaxation (DD-CSR cross relaxation) is generalized to treat the case of anisotropic magnetic susceptibility, including the important case where the latter originates from zero-field splitting. It is shown that the phenomenon of DD-CSR cross relaxation is absolutely general and to be expected under any electronic configuration. The results of the generalization are presented for a model system, and the consequences for paramagnetic metalloproteins are illustrated with an example of cerium(III)-substituted calbindin. The effects of the magnetic anisotropy are found to be substantial.

Solution structure calculations through self-orientation in a magnetic field of a cerium(III) substituted calcium-binding protein.

Within the frame of a research aimed at characterizing paramagnetic metal ions capable of inducing self-orientation of metalloproteins in solution, we have studied the complex of the 75-amino-acid calcium-binding protein calbindin D(9k) with one Ce(III) ion (CaCeCb). Backbone (15)N-(1)H (1)J values have been determined for CaCeCb at two different magnetic fields. The above values showed a distinct dependence on the magnetic field, which is caused by the partial orientation of the molecule in solution. The difference in the values at the two magnetic fields provides structural constraints, which have been used to refine the structure of CaCeCb. The refined structure showed an improvement in terms of the number of residues falling in favored regions of the Ramachandran plot. The comparison of the molecular magnetic susceptibility tensor, obtained from the (15)N-(1)H (1)J values, with the magnetic susceptibility tensor of the metal, obtained from pseudocontact shifts, showed that the orientation of the molecule in solution is mainly determined by the Ce(III) ion. This paper shows that Ce(III), like low-spin Fe(III) in hemoproteins, is sufficiently magnetically anisotropic to induce self-orientation to an extent which can be exploited for solution structure determination.

A low frequency 1H-NMR external unit for the analysis of large foodstuff samples.

An inexpensive external unit that allows the use of a commercial high-resolution NMR spectrometer as a very low frequency instrument is described. The external unit is phase coherent, the pulse timing being given by the parent spectrometer. With the exception of the probe, the external unit does not contain any tuned elements. This permits easy change of frequency in the range 100 kHz-1 MHz. The external unit may be appropriately employed in food science where, in several cases, low frequency is desirable. An application to hen shell eggs at the frequency of 700 kHz is described.

Protein hydration and location of water molecules in oxidized horse heart cytochrome c by (1)H NMR.

The hydration properties of the oxidized form of horse heart cytochrome c have been studied by (1)H NMR spectroscopy. Two-dimensional, homonuclear ePHOGSY-NOESY experiments are used to map water-protein interactions. The detected NOEs reveal interactions between nonexchangeable protein protons and both water protons and labile protein protons which exchange with water protons. Among the many water molecules apparent in the X-ray structure, three have been identified with a residence time longer than 300 ps. One of them is located inside the distal heme cavity, in the deepest part of a hydration pathway extending toward the surface. The identification of hydrophilic regions and detection of three long-lived water molecules settles some ambiguities and provides a better representation of the water-protein interactions in oxidized cytochrome c.

Development of NMR instrumentation to achieve excitation of large bandwidths in high-resolution spectra at high field.

A prototype 2.5-mm (1)H high-resolution probe for an 18.8-T (800 MHz) nuclear magnetic resonance spectrometer has been designed, together with a dedicated amplifier capable of delivering up to 1 kW of power. This probe permits a 90 degrees pulse length of 2 mus to be achieved at 300 W, corresponding to an excitation bandwidth of +/-125 kHz. Probe performances were tested on samples commonly used for this purpose as well as on protein and paramagnetic model compound samples. It is shown that this probe is useful for a wide range of applications at high magnetic field, especially in the study of systems characterized by very broad and far-shifted resonances and in experiments that require high-power radiofrequency irradiation.

Spectral characterization of vanadium-transferrin systems.

The preparation procedure of vanadium(III) transferrin and its stability are confirmed to be as previously reported. The electronic spectra of vanadium(III), oxovanadium(IV), and vanadium(V) transferrin derivatives are comparatively discussed. A band in the near infrared of the oxovanadium(IV) derivative is observed for the first time.

Investigation of the system cobalt(II) bovine carbonic anhydrase B-trichloroacetaldehyde.

The interactions between hydrated trichloroacetaldehyde and cobalt(II)bovine carbonic anhydrase B have been investigated as a function of pH by means of electronic spectroscopy of FT nmr spectroscopy. The hydrated aldehyde is bound to the metal ion and its apparent affinity constant is pH dependent with a bell-shaped profile. The kinetic parameters of the dissociation process have also been determined.