Iron is tightly connected to oxygen homeostasis and erythropoiesis. Our aim was to better understand how hypoxia regulates iron acquisition for erythropoiesis in humans, a topic relevant to common hypoxia-related disorders. Fortyseven healthy volunteers participated in the HIGHCARE project. Blood samples were collected at sea level and after acute and chronic exposure to high altitude (3400-5400 m above sea level). We investigated the modifications in hematocrit, serum iron indices, erythropoietin, markers of erythropoietic activity, interleukin-6, and serum hepcidin. Hepcidin decreased within 40 hours after acute hypoxia exposure (P < .05) at 3400 m, reaching the lowest level at 5400 m (80% reduction). Erythropoietin significantly increased (P < .001) within 16 hours after hypoxia exposure followed by a marked erythropoietic response supported by the increased iron supply. Growth differentiation factor-15 progressively increased during the study period. Serum ferritin showed a very rapid decrease, suggesting the existence of hypoxia-dependent mechanism(s) regulating storage iron mobilization. The strong correlation between serum ferritin and hepcidin at each point during the study indicates that iron itself or the kinetics of iron use in response to hypoxia may signal hepcidin down-regulation. The combined and significant changes in other variables probably contribute to the suppression of hepcidin in this setting. (Blood. 2011; 117(10):2953-2959) IntroductionIron is an essential element required as a cofactor for proteins managing oxygen transport, such as hemoglobin, myoglobin, and cytochromes. Hypoxia, which occurs in a broad range of pathologic or environmental conditions, is primarily sensed in vertebrates by the hypoxia inducible family of transcription factors (hypoxia inducible factors), which activate or repress genes involved in adaptations to low oxygen pressure. 1 In the erythropoietic compartment, which contains approximately 70% of the body iron, hypoxia stimulates erythropoiesis and promotes hemoglobin synthesis, which largely depends on iron availability. [2][3][4][5][6] Accordingly, both hypoxia and anemia induce the synthesis of erythropoietin (EPO) and are the 2 main signals that increase iron absorption independently of iron stores. [6][7][8][9] Thus, iron metabolism is tightly connected to oxygen homeostasis and erythropoiesis.The liver peptide hepcidin, codified by HAMP, is the key regulator of iron homeostasis. It acts by inhibiting intestinal iron absorption and release by macrophages, and its production is regulated by different stimuli, including inflammatory cytokines, 10 iron, 11 erythropoietic activity, 12-14 anemia, and hypoxia, 15-17 mainly at the transcriptional level as described in recent reviews. 18,19 Anemia could mediate hepcidin suppression through multiple mechanisms, including increased EPO or erythropoietic activity, increased iron demand, or liver hypoxia. 3 The nature of the erythropoietic regulator of hepcidin is still uncharacterized but may include one or more prote...

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Renal cell carcinoma (RCC) is one of the major causes of cancer death and is radio- and chemoresistant. Urine of 29 healthy subjects and 39 clear cell RCC patients were analyzed using the ClinProt technique to search for possible biomarkers for early RCC diagnosis. A cluster of three signals (marker A= at m/z 1827 ± 8 Da, marker B = 1914 ± 8 Da and marker C = 1968 ± 8 Da) was able to discriminate patients from controls. A receiver operating characteristic curve analysis showed values of area under the curve (AUC) higher than 0.9 for marker A and B, corresponding to a sensitivity of 85-90% and a specificity of 90%, while marker C gave a lower AUC (0.84) corresponding to sensitivity of 70% and specificity of 100%. The combination of three markers lead to an improvement in diagnostic efficacy, with specificity and sensitivity of 100% and 95%, respectively, in the training test and of 100% and of 85% in the test experiment. The efficacy of this cluster of signals to distinguish RCC patients grouped by tumor stage showed a sensibility of 100% for patients at the primary tumor 1 stage. One of the signals present in the cluster was identified as a fragment of Tamm-Horsfall protein.

MALDI imaging mass spectrometry (IMS) is a unique technology to explore the spatial distribution of biomolecules directly on tissues. It allows the in situ investigation of a large number of small proteins and peptides. Detection of high molecular weight proteins through MALDI IMS still represents an important challenge, as it would allow the direct investigation of the distribution of more proteins involved in biological processes, such as cytokines, enzymes, neuropeptide precursors and receptors. In this work we compare the traditional method performed with sinapinic acid with a comparable protocol using ferulic acid as the matrix. Data show a remarkable increase of signal acquisition in the mass range of 20k to 150k Th. Moreover, we report molecular images of biomolecules above 70k Th, demonstrating the possibility of expanding the application of this technology both in clinical investigations and basic science.

Our results confirmed the possible role of MALDI-IMS in the search for diagnostic targets of PTC and follicular lesions, which could be applied in larger trials aimed at the identification of proteins, convertible to cost-effective diagnostic tools such as immunohistochemistry. These tests could be used to analyse in vivo cytological smears, improving the preoperative diagnosis of indeterminate thyroid nodules.

Matrix-Assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) is a molecular technology that allows simultaneous investigation of the content and spatial distribution of molecules within tissue. In this work, we examine different classes of detergents, the anionic sodium dodecyl sulfate (SDS), the nonionic detergents Triton X-100, Tween 20 and Tween 80, and the zwitterionic 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) for use in MALDI IMS of analytes above m/z 4000. These detergents were found to be compatible with MALDI MS and did not cause signal suppression relative to non-detergent applications and did not produce interfering background signals. In general, these detergents enhanced signal acquisition within the mass range m/z 4–40 000. Adding detergents into the matrix was comparable with the separate application of detergent and matrix. Evaluation of spectra collected from organ-specific regions of a whole mouse pup section showed that different detergents perform optimally with different organs, indicating that detergent selection should be optimized on the specific tissue for maximum gain. These data show the utility of detergents towards enhancement of protein signals for on-tissue MALDI IMS analysis.

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