Aspects of transition cow metabolomics-Part I: Effects of a metaphylactic butaphosphan and cyanocobalamin treatment on the metabolome in liver, blood, and urine in cows with different liver metabotypes.

Dairy cows in modern production systems are at risk to develop metabolic disorders during the transition period. Reasons for individual differences in susceptibility, as well as the underlying pathomechanisms, are still only partially understood. The development of metaphylactic treatment protocols is needed. In this context, an on-farm prospective 3-fold blinded randomized study involving 80 German Holstein cows was performed throughout 1 yr. The trial involved a thorough recording of the production and clinical traits, clinical chemistry, and liver biopsies and blood and urine sampling at d 14 (mean: 12 d, range: 1-26 d) antepartum (AP), and d 7 (7, 4-13) and 28 (28, 23-34) postpartum (PP) for metabolomics analyses. Two groups received a treatment with butaphosphan and cyanocobalamin (BCC) at either the dosage recommended by the manufacturer or the double dosage (5 or 10 mL/100 kg of body weight 10% butaphosphan and 0.005% cyanocobalamin (Catosal, Bayer Animal Health), n = 20 in each group, parity: 4.2 ± 2.0 and 3.4 ± 1.3, respectively (mean ± SD)] and one group a placebo treatment (NaCl 0.9%, n = 40, parity: 4.0 ± 1.9). The animals were treated at 6 time points (7, 6, and 5 d AP, and 1, 2, and 3 d PP) via intravenous injection. Mass spectroscopy-based targeted metabolomics analysis of blood plasma and liver samples were performed using the AbsoluteIDQ p180 kit (Biocrates Life Sciences), whereas the urine samples were analyzed by nuclear magnetic resonance spectroscopy. Statistical analysis was performed using multivariate [partial least squares discriminant analysis (PLS-DA)] and univariate methods (linear mixed model). Multivariate data analysis (PLS-DA plots) of the liver metabolome revealed 3 different metabotypes (A = medium, B = minor, C = large alterations in liver metabolome profile between AP and PP status). Metabotype B animals were characterized by higher PP lipomobilization (stronger PP body condition decrease and higher blood bilirubin, fatty acids, gamma-glutamyltransferase, and triglyceride levels) and a higher occurrence of transition cow diseases, compared with the animals in metabotype C. Analysis of the feeding data showed that the period of metabotype B animals (calving in a distinct time frame) was characterized by a decreased grass silage quality. The PP liver metabolome of the metabotype C animals was characterized by higher concentrations of AA, acylcarnitines, lysoPC and sphingomyelins compared with metabotype B. For the metaphylactic treatment with BCC a dose-dependent effect was confirmed, differing between the metabotypes. In all matrices and metabotypes at various time points significant treatment effects were observed, with different profiles in clinical chemistry and as well in metabolomics data. The most clear-cut treatment effect was observed in metabotype B in the liver at 7 d PP, characterized by an increase in several acylcarnitines and phosphatidylcholines, indicating a more efficient influx and oxidation of fatty acids in mitochondria and thereby an increase in energy supply and more efficient triglyceride export in the liver. The results from the liver metabolomics analysis support the application of an indication-based metaphylactic treatment with BCC.

Aspects of transition cow metabolomics-Part III: Alterations in the metabolome of liver and blood throughout the transition period in cows with different liver metabotypes.

The liver plays a central role in the postpartum (PP) energy metabolism of the transition dairy cow; however, studies describing the liver metabolome during this period were lacking. The aim of the presented study was therefore to compare the alterations in the liver and blood metabolome of transition dairy cows. For this purpose, an on-farm trial with 80 German Holstein cows (mean lactation number: 3.9; range: 2-9) was performed, with thorough documentation of clinical traits and clinical chemistry, as well as production data. Liver biopsies and blood samples were collected at d 14 (mean: 12 d, range: 1-26 d) antepartum (AP), d 7 (7, 4-13) and 28 (28, 23-34; mean, earliest-latest) PP for targeted mass spectroscopy-based metabolomics analysis using the AbsoluteIDQ p180 kit (Biocrates Life Sciences). Statistical analysis was performed using multivariate (partial least squares discriminant analysis) as well as univariate methods (linear mixed model). Multivariate data analysis of the liver metabolome revealed 3 different metabotypes (A = medium, B = minor, C = large alterations in the liver metabolome profile between AP and PP). In metabotype C, an increase of almost all acylcarnitines, lysophosphatidylcholines (lysoPC), sphingomyelins, and some phosphatidylcholines (PC, mainly at 7 d PP) was observed after calving. In contrast to metabotype C, the clinical data of the metabotype B animals indicated a higher PP lipomobilization and occurrence of transition cow diseases. The liver metabolome profile of these animals most likely mirrors a failure of adaptation to the PP state. This strong occurrence of metabotypes was much less pronounced in the blood metabolome. Additionally, differences in metabolic patterns were observed across the transition period when comparing liver and blood matrices (e.g., in different biogenic amines, acylcarnitines and sphingolipids). In summary, the blood samples at 7 d PP showed lower acylcarnitines and PC, with minor alterations and a heterogeneous pattern in AA, biogenic amines, and sphingomyelins compared with 14 d AP. In contrast to 7 d PP, the blood samples at 28 PP revealed an increase in several AA, lysoPC, PC, and sphingomyelins in comparison to the AP state, irrespective of the metabotype. In the liver biopsies metabotype B differed from metabotype C animals ante partum by following metabolites: higher α aminoadipic acid, lower AA, serotonin, taurine, and symmetric dimethylarginine levels, lower or higher concentrations of certain acylcarnitines (higher: C2, C3, C5, C4:1; lower: C12:1, C14:1-OH, C16:2), and lower lysoPC (a C16:0, C18:0, C20:3, C20:4) and hexose levels. In blood samples, fewer differences were observed, with lower serotonin, acylcarnitine C16:2, lysoPC (a C16:0, C17:0, C18:0 and C18:1), PC aa C38:0, and PC ae C42:2. The results show that the use of only the blood metabolome to assess liver metabolism may be hampered by the fact that blood profiles are influenced by the metabolism of many organs, and metabolomics analysis from liver biopsies is a more suitable method to identify distinct metabotypes. Future studies should investigate the stability and reproducibility of the metabotype and phenotypes observed, and the possible predictive value of the metabolites already differing AP between metabotype B and C.

The involvement of pro-inflammatory cytokines in nephrogenic systemic fibrosis - a mechanistic hypothesis based on preclinical results from a rat model treated with gadodiamide.

The evidence for the potential involvement of gadolinium-based contrast agents (GBCAs) in the pathomechanism of nephrogenic systemic fibrosis (NSF), a rare but serious disease occurring in patients with severe or end-stage renal failure, has grown due to recent epidemiological and preclinical research. Nevertheless there is still uncertainty with regard to the prevailing patho-physiological processes that may lead to NSF. To examine the potential mechanism of the fibrotic skin changes we applied a recently published rat model of NSF for investigations into serum markers for inflammation. For this purpose male Wistar rats were treated either once, three, or eight times with a daily intravenous injection of 2.5 mmol/kg gadodiamide, the drug substance of the magnetic resonance imaging (MRI) agent Omniscan. Clinical observations, hematology, clinical pathology, histopathology including electron microscopy and gadolinium (Gd) determination in serum, skin, femur and liver tissue, and a multiplexed analysis of 70 protein serum markers were performed. Gd was detectable in the skin, femur, and liver of the gadodiamide-treated rats 6h after the first administration. Macroscopic skin changes, appearing as reddening and early scab formation, were observed in one animal after the third daily administration and affected all animals after 8 daily administrations. Microscopy revealed dermal infiltrations after three administrations, progressing towards inflammatory lesions, ulcerations and crusts. Among the investigated serum marker panel 13 cytokines were significantly (p<0.01) elevated 6 h after the first injection, and eight stayed elevated over all time points: the monocyte chemotactic proteins MCP-1 and MCP-3, the macrophage inflammatory proteins MIP-1beta and MIP-2, the tumor necrosis factor TNF-alpha, the extracellular matrix regulator tissue inhibitor of metalloproteinase type 1 (TIMP-1), the vascular epithelial growth factor (VEGF) and osteopontin. The latter cytokine is of particular interest, since this matrix cellular glycoprotein is involved in the regulation of dystrophic calcification but also plays a role as a chemoattractant for dendritic cells, macrophages and T-lymphocytes, which in turn activate inflammatory pathways. Reflecting the physiological role of osteopontin, we hypothesize that Gd release from the GBCA-complex leads to the formation of insoluble Gd-deposits subsequently eliciting a physiological response similar to that seen during dystrophic calcification, i.e. an up-regulation of osteopontin and chemoattractant cytokines. Concomitant increase in vascular permeability caused by MIP-1, TNF-alpha and VEGF may lead to extravasation of chelated Gd or Gd-deposits. The inherent persistence of the Gd-deposits may subsequently result in an overactivation of pro-inflammatory pathways progressing towards overt skin effects.

Contrast media: future aspects.

In spite of the dramatic development in CT, there was no major breakthrough in the iodinated contrast media development. New agents based on hybrid between MRI and CT compounds may be a new innovative alternative. This new approach may also open new indications such as radiotherapy.

Hydrophilic cyanine dyes as contrast agents for near-infrared tumor imaging: synthesis, photophysical properties and spectroscopic in vivo characterization.

We have synthesized a group of glucamine and gluosamine-substituted cyanine dyes structurally related to indocyanine green (ICG) and have characterized these compounds with regard to their potential as contrast agents for biomedical optical imaging. The compounds reported herein exhibit increased hydrophilicity and less plasma protein binding (< 50%), and are thus expected to have different pharmacokinetic properties compared with ICG. Furthermore, we measured enhanced fluorescence quantum yields (7-15%) in a physiological environment with respect to ICG. For the derivative with the highest hydrophilicity (5a) the efflux from tumor and normal tissue was monitored by intensity-modulated diffuse optical spectroscopy after intravenous injection into tumor-bearing rats. In comparison with ICG, 5a exhibited a considerably enhanced tissue-efflux half-life (73 min versus less than 10 min for ICG in tumor tissue), a two-fold higher initial tissue absorption coefficient compared to ICG, and finally, it generated an elevated tumor-to-tissue concentration gradient up to 1 h after injection. In conclusion, compounds such as 5a are promising contrast agents for optical imaging, and could facilitate highly sensitive and specific detection of breast cancer or other malignancies by utilizing mechanisms similar to contrast-enhanced magnetic resonance imaging or computerized tomography.

Macromolecular contrast agents for optical imaging of tumors: comparison of indotricarbocyanine-labeled human serum albumin and transferrin.

Macromolecules accumulate in solid tumors and can thus be used as carriers for the delivery of attached contrast agents to tumors. We report the synthesis and use of serum protein-dye conjugates consisting of transferrin (Tf) or human serum albumin (HSA) and an indotricarbocyanine (ITCC) derivative as contrast agents for the optical imaging of tumors. The compounds were characterized with respect to their photophysical properties and tested in vitro for their ability to bind to tumor cells and in vivo for their potential to delineate experimental tumors. In contrast to HAS-ITTC, Tf-ITCC showed receptor-mediated uptake by HT29 human colon cancer cells in vitro. After intravenous injection into HT29 tumor-bearing nude mice both compounds induced increased fluorescence contrast of tumors in vivo. After 24 h the contrast between tumor and normal tissue was significantly higher for Tf-ITCC than for HAS-ITCC. Dye-induced fluorescence was found to be predominantly located in perinecrotic areas of the tumor. Furthermore, Tf-ITCC produced fluorescence of viable tumor cells, whereas HAS-ITCC fluorescence was recorded along connective tissue. We conclude that ITCC-labeled Tf and HSA can serve as macromolecular contrast agents for the optical imaging of tumors, with Tf-ITCC showing higher efficiency.

[Contrast media for optical mammography]. / Kontrastmittel für die optische Mammographie.

Near infrared imaging is a non-invasive imaging modality which uses light of a spectral range between 650 and 1000 nm for tissue transillumination. Near infrared photons can penetrate by diffusion into tissue up to several centimeters due to low absorption by intrinsic chromophors (e.g. hemoglobine). During the last years optical mammography has become field of growing interest. Several prototypes of near infrared scanners have been developed for clinical studies in order to obtain transillumination images of the breast. The results indicated, that discrimination of tumors located up to several centimeters below the tissue surface (e.g. breast tumors) from surrounding healthy tissue did not succeed with sufficient specificity. The diagnostic potential of near infrared imaging can be enhanced using dyes as contrast agents. Specially designed cyanine dye derivatives are potential contrast agents because of their suitable optical properties and pharmacokinetic behaviour leading to an increased tumor fluorescence in animals after intravenous injection.