SDS-PAGE-Based Quantitative Assay of Hemolymph Proteins in Honeybees: Progress and Prospects for Field Application.

In human and veterinary medicine, serum proteins are considered to be useful biomarkers for assessing the health and nutritional status of the organism. Honeybee hemolymph has a unique proteome that could represent a source of valuable biomarkers. Therefore, the aims of this study were to separate and identify the most abundant proteins in the hemolymph of worker honeybees to suggest a panel of these proteins that could represent useful biomarkers for assessing the nutritional and health status of the colonies and, finally, to analyze them in different periods of the year. Four apiaries were selected in the province of Bologna, and the bees were analyzed in April, May, July, and November. Thirty specimens from three hives of each apiary were sampled and their hemolymph was collected. The most represented bands obtained after 1D sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were cut from the gel, and the proteins were identified using an LC-ESI-Q-MS/MS System. A total of twelve proteins were unmistakably identified; the two most abundant proteins were apolipophorin and vitellogenin, which are known biomarkers of bee trophic and health status. The two other proteins identified were transferrin and hexamerin 70a, the first being involved in iron homeostasis and the second being a storage protein. Most of these proteins showed an increase from April to November, mirroring the physiological changes of honeybees during the productive season. The current study suggests a panel of biomarkers from honeybee hemolymph worth testing under different physiological and pathological field conditions.

Iron Speciation and Iron Binding Proteins in Arthrospira platensis Grown in Media Containing Different Iron Concentrations

Cyanobacteria are characterized by high iron content. This study investigated the effects of varying iron concentrations (1, 5, and 10 mg L-1) in the culture media on the biochemical composition and the iron bioaccumulation and speciation in Arthrospira platensis F&M-C256. Iron content measured in biomasses varied from 0.35 to 2.34 mg g-1 dry weight depending on the iron concentration in the culture media. These biomasses can be considered of interest for the production of spirulina-based supplements with low and high iron content. Iron speciation was studied using size exclusion chromatography followed by atomic absorption spectrometry and proteomic analysis. The role of C-phycocyanin as an iron binding protein was also investigated. Overall, the present results provide a better understanding of iron metabolism in cyanobacteria and a foundation for further studies.

Urinary proteomic profiles of prostate cancer with different risk of progression and correlation with histopathological features.

Prostate cancer (PCa) is the most common tumor in men with extremely variable outcome, varying from latent or indolent form to very aggressive behavior. High grade tumors, expansions exceeding the prostatic capsule into the surrounding soft tissues and spreading through lymph vascular channels, represent the most consistent unfavorable prognostic factors. However, accuracy in the prediction of the disease progression is sometimes difficult. Along with new molecular diagnostic techniques and more accurate histopathological approaches, proteomic studies challenge to identify potential biomarkers predictive of PCa progression. In our study we analyzed the urinary proteomes of 42 patients affected by PCa through two-dimensional electrophoresis associated with mass spectrometry. Proteomic profiles were correlated to histopathological features including pTNM stage and tumor differentiation in order to provide new promising markers able to define more accurately the PCa aggressiveness and driving new therapeutic approaches.

Comparison of pregnenolone sulfate, pregnanolone and estradiol levels between patients with menstrually-related migraine and controls: an exploratory study.

BACKGROUND: Neurosteroids affect the balance between neuroexcitation and neuroinhibition but have been little studied in migraine. We compared the serum levels of pregnenolone sulfate, pregnanolone and estradiol in women with menstrually-related migraine and controls and analysed if a correlation existed between the levels of the three hormones and history of migraine and age. METHODS: Thirty women (mean age ± SD: 33.5 ± 7.1) with menstrually-related migraine (MM group) and 30 aged- matched controls (mean age ± SD: 30.9 ± 7.9) participated in the exploratory study. Pregnenolone sulfate and pregnanolone serum levels were analysed by liquid chromatography-tandem mass spectrometry, while estradiol levels by enzyme-linked immunosorbent assay. RESULTS: Serum levels of pregnenolone sulfate and pregnanolone were significantly lower in the MM group than in controls (pregnenolone sulfate: P = 0.0328; pregnanolone: P = 0.0271, Student's t-test), while estradiol levels were similar. In MM group, pregnenolone sulfate serum levels were negatively correlated with history of migraine (R2 = 0.1369; P = 0.0482) and age (R2 = 0.2826, P = 0.0025) while pregnenolone sulfate levels were not age-related in the control group (R2 = 0.04436, P = 0.4337, linear regression analysis). CONCLUSION: Low levels of both pregnanolone, a positive allosteric modulator of the GABAA receptor, and pregnenolone sulfate, a positive allosteric modulator of the NMDA receptor, involved in memory and learning, could contribute either to headache pain or the cognitive dysfunctions reported in migraine patients. Overall, our results agree with the hypothesis that migraine is a disorder associated with a loss of neurohormonal integrity, thus supporting the therapeutic potential of restoring low neurosteroid levels in migraine treatment.

Identification of candidate proteomic markers in the serum of medication overuse headache patients: An exploratory study.

PURPOSE OF THE STUDY: The pathophysiological mechanism of medication overuse headache is uncertain; no distinctive markers have been described right now. The aim of this study was to conduct proteomic analyses on serum samples from patients with medication overuse headache and healthy individuals. Specifically, mono- (SDS-PAGE) and two-dimensional gel electrophoresis (2-DE) followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) were used to evaluate changes in serum proteins. MAIN FINDINGS: By SDS-PAGE, four over-expressed bands were revealed in patients, compared to controls. 2-DE combined with LC-MS/MS analysis allowed confirmation of some proteins preliminarily detected by SDS-PAGE: Hemopexin, alpha-1-acid glycoprotein 1, apolipoprotein A4 and haptoglobin. Moreover, other differential proteins were isolated, mostly increased in MOH patients: Alpha-1-antitrypsin, immunoglobulin heavy constant alpha 1, retinol binding protein and transthyretin. Only one protein, immunoglobulin kappa constant, was decreased in the patients' group. CONCLUSIONS: The investigation of the serum proteome can offer a better understanding about biological mechanisms underlying medication overuse headache. Specifically, medication overuse headache shares some serum biochemical markers with chronic pain conditions. Further studies might uncover the relevance of these proteins in medication overuse headache.

Serum levels of allopregnanolone, progesterone and testosterone in menstrually-related and postmenopausal migraine: A cross-sectional study.

BACKGROUND: Reduced blood or cerebrospinal fluid levels of allopregnanolone are involved in menstrual cycle-linked CNS disorders, such as catamenial epilepsy. This condition, like menstrually-related migraine, is characterized by severe, treatment-resistant attacks. We explored whether there were differences in allopregnanolone, progesterone and testosterone serum levels between women with menstrually-related migraine (MM, n = 30) or postmenopausal migraine without aura who had suffered from menstrually-related migraine during their fertile age (PM, n = 30) and non-headache control women in fertile age (FAC, n = 30) or post-menopause (PC, n = 30). METHODS: Participants were women with migraine afferent to a headache centre; controls were female patients' acquaintances. Serum samples obtained were analyzed by HPLC-ESI-MS/MS. RESULTS: In menstrually-related migraine and postmenopausal migraine groups, allopregnanolone levels were lower than in the respective control groups (fertile age and post-menopause) (p < 0.001, one-way analysis of variance followed by Tukey-Kramer post-hoc comparison test) while progesterone and testosterone levels were similar. By grouping together patients with migraine, allopregnanolone levels were inversely correlated with the number of years and days of migraine/3 months (p ≤ 0.005, linear regression analysis). CONCLUSION: Decreased GABAergic inhibition, due to low allopregnanolone serum levels, could contribute to menstrually-related migraine and persistence of migraine after menopause. For the management of these disorders, a rise in the GABAergic transmission by increasing inhibitory neurosteroids might represent a novel strategy.

Heparin-induced lipoprotein precipitation apheresis in dyslipidemic patients: A multiparametric assessment.

Low-density lipoprotein (LDL) apheresis (LA) selectively eliminates lipoproteins containing apolipoprotein B 100 (ApoB100) on patients affected by severe dyslipidemia. In addition to lowering lipids, LA is thought to exert pleiotropic effects altering a number of other compounds associated with atherosclerosis, such as pro- and anti-inflammatory cytokines or pro-thrombotic factors. More knowledge needs to be gathered on the effects of LA, and particularly on its ability to modify blood components other than lipids. We performed a multiparametric assessment of the inflammatory, metabolic and proteomic profile changes after Heparin-induced lipoprotein precipitation (H.E.L.P.) apheresis on serum samples from nine dyslipidemic patients evaluating cholesterol and lipoproteins, plasma viscosity and density, metabolites, cytokines, PCSK9 levels and other proteins selectively removed after the treatment. Our results show that H.E.L.P. apheresis is effective in lowering lipoprotein and PCSK9 levels. Although not significantly, complement and inflammation-related proteins are also affected, indicating a possible transient epiphenomenon induced by the extracorporeal procedure.

Essential (Mg, Fe, Zn and Cu) and Non-Essential (Cd and Pb) Elements in Predatory Insects (Vespa crabro and Vespa velutina): A Molecular Perspective.

The recent introduction of the Asian yellow-legged hornet, Vespa velutina, into Europe has raised concern regarding the threat to honeybees and the competition with the European hornet, Vespa crabro. The aim of this study was to investigated essential (Mg, Fe, Zn, Cu) and non-essential (Cd and Pb) elements in these two species. Element concentrations were determined in the whole body and separately in the head, thorax and abdomen using atomic absorption spectrometry (AAS). The changes in essential element concentration and speciation during metamorphosis were also studied using size exclusion chromatography followed by AAS and proteomic analysis. In both species, the essential elements were more concentrated in the abdomen due to the presence of fat bodies. Magnesium, Fe and Zn concentrations were significantly higher in V. crabro than in V. velutina and could have been related to the higher aerobic energy demand of the former species required to sustain foraging flight. Low concentrations of Cd and Pb were indicative of low environmental exposure. The concentration and speciation of essential elements, particularly Fe, varied among the developmental stages, indicating a modification of ligand preferences during metamorphosis. Overall, the results in the present study provide a better understanding of the hornet metal metabolism and a foundation for additional studies.

Evaluation of potential cardiovascular risk protein biomarkers in high severity restless legs syndrome.

Restless legs syndrome (RLS) is a common sensorimotor disorder that, in case of severe symptoms, can be very distressing and negatively interfere with quality of life. Moreover, increasing evidences associate RLS with higher risk of cerebrovascular and cardiovascular disease (CVD). The purpose of this study was to quantify two proteins, previously identified by proteomics and potentially linked with CVD risk, namely kininogen-1 (KNG1) and alpha-1-antitrypsin (A1AT), in primary RLS patients at high severity grade (HS-RLS) in comparison to healthy control subjects. Proteins were quantified through enzyme-linked immunosorbent assay (ELISA) in plasma samples from 14 HS-RLS patients and 15 control individuals. The two groups were closely matched for age and gender. The expression level of KNG1 resulted significantly higher (p < 0.001), while A1AT was significantly decreased (p < 0.05) in HS-RLS patients compared to controls, confirming the relationship between these proteins and the disease severity. Furthermore, in patients group the association between the protein concentrations and the following parameters was further evaluated: age, disease onset and diagnosis, scores obtained from the RLS rating scales (Epworth Sleepiness Scale, Pittsburgh Sleep Quality Index, Beck Depression Inventory) and smoking habit. All the considered variables resulted independent of protein levels, so the disease can be reasonably considered the main cause of protein changes. As emerged from the literature, high levels of KNG1 and low amounts of A1AT seem to be related with a highest probability to develop CVD. Consequently, these proteins may be reliable candidate biomarkers of CVD risk in patients with RLS at high severity grade.

Exploration of candidate serum biomarkers potentially related to the chronic pain condition in Medication-overuse headache.

BACKGROUND: Medication Overuse Headache (MOH) is a prevalent and disabling disorder resulting from the overuse of analgesic drugs, triptans or other acute headache medications. In previous proteomic studies, several proteins have been found at high concentrations in the urine of MOH patients and in the serum of rats with neuropathic pain. The aim of this study was to compare the serum levels of lipocalin-type Prostaglandin D2 synthase (L-PGDS), Vitamin D-binding protein (VDBP), apolipoprotein E (APOE) and apolipoprotein A1 (APOA1) in MOH patients and healthy individuals, further exploring their relationship with cutaneous pain thresholds (CPTs) in the territories innervated by the trigeminal nerve. METHODS: Sixty-nine MOH patients and 42 age- and sex-matched healthy volunteers were enrolled in the study. Von Frey-like filaments were applied to the skin territories innervated by the trigeminal nerve, to determine the CPTs. L-PGDS, VDBP, APOE and APOA1 were quantified in the serum by Enzyme-linked Immunosorbent Assay (ELISA). Clinical and laboratory data were collected. Comparisons between MOH patients and healthy individuals were performed using independent t test or χ2 test. To correlate serum proteins with CPTs, Pearson correlation coefficient or Spearman's rank correlation coefficient were used. RESULTS: CPTs were lower among MOH patients. L-PGDS, VDBP and APOE had significantly different serum concentrations between groups (p < 0.01), but no correlation was found with CPTs. APOA1 serum concentrations did not differ between patients and healthy individuals. CONCLUSIONS: L-PGDS, VDBP and APOE had abnormal serum levels in MOH patients, confirming their alteration in some conditions of chronic headache and neuropathic pain. However, they had no relationship with CPTs. The in-depth study of serum proteins represents a promising approach for a better understanding of MOH, as well as the detection of candidate biomarkers for chronic headache or the risks associated with overuse medications.

Analysis of protein expression in periodontal pocket tissue: a preliminary study.

BACKGROUND: The periodontal disease is caused by a set of inflammatory disorders characterized by periodontal pocket formation that lead to tooth loss if untreated. The proteomic profile and related molecular conditions of pocket tissue in periodontally-affected patients are not reported in literature. To characterize the proteomic profile of periodontally-affected patients, their interproximal periodontal pocket tissue was compared with that of periodontally-healthy patients. Pocket-associated and healthy tissue samples, harvested during surgical therapy, were treated to extract the protein content. Tissues were always collected at sites where no periodontal-pathogenic bacteria were detectable. Proteins were separated using two-dimensional gel electrophoresis and identified by liquid chromatography/mass spectrometry. After identification, four proteins were selected for subsequent Western Blot quantitation both in pathological and healty tissues. RESULTS: A significant unbalance in protein expression between healthy and pathological sites was recorded. Thirty-two protein spots were overall identified, and four proteins (S100A9, HSPB1, LEG7 and 14-3-3) were selected for Western blot analysis of both periodontally-affected and healthy patients. The four selected proteins resulted over-expressed in periodontal pocket tissue when compared with the corresponding tissue of periodontally-healthy patients. The results of Western blot analysis are congruent with the defensive and the regenerative reaction of injured periodontal tissues. CONCLUSIONS: The proteomic analysis was performed for the first time directly on periodontal pocket tissue. The proteomic network highlighted in this study enhances the understanding of periodontal disease pathogenesis necessary for specific therapeutic strategies setting.

Validation of potential candidate biomarkers of drug-induced nephrotoxicity and allodynia in medication-overuse headache.

BACKGROUND: Medication-overuse headache (MOH) is a chronic disorder that results from the overuse of analgesics drugs, triptans or other acute headache compounds. Although the exact mechanisms underlying MOH remain still unknown, several studies suggest that it may be associated with development of "central sensitization", which may cause cutaneous allodynia (CA). Furthermore, the epidemiology of drug-induced disorders suggests that medication overuse could lead to nephrotoxicity. The aim of this work was to confirm and validate the results obtained from previous proteomics studies, in which we analyzed the urinary proteome of MOH patients in comparison with healthy non-abusers individuals. METHODS: MOH patients were divided into groups on the basis of the drug abused: triptans, non-steroidal anti-inflammatory drugs (NSAIDs) and mixtures, (mainly containing indomethacin, paracetamol and, in some cases, caffeine). Healthy subjects, with a history of normal renal function, were used as controls. In this study, four proteins that were found differentially expressed in urine, and, on the basis of the literature review, resulted related to kidney diseases, were verified by Western Blot and Enzyme-linked Immunosorbent Assay (ELISA); Prostaglandin-H2 D-synthase (PTGDS), uromodulin (UROM), alpha-1-microglobulin (AMBP) and cystatin-C (CYSC). RESULTS: Western blot analysis allowed to validate our previous proteomics data, confirming that all MOH patients groups show a significant over-excretion of urinary PTGDS, UROM, AMBP and CYSC (excluding triptans group for this latter), in comparison with controls. Moreover, the expression of PTGDS was further evaluated by ELISA. Also by this assay, a significant increase of PTGDS was observed in all MOH abusers, according to 2-DE and Western blot results. CONCLUSIONS: In this study, we confirmed previous findings concerning urinary proteins alterations in MOH patients, identified and demonstrated the over-expression of PTGDS, UROM, AMBP, and CYSC, particularly in NSAIDs and mixtures abusers. Over-expression of these proteins have been related to renal dysfunction and probably, PTGDS, to the development of CA. The detection and confirmation of this proteins pattern represent a promising tool for a better understanding of potential nephrotoxicity induced by drugs overuse and may enhance awareness related to the MOH-associated risks, even in absence of clinical symptoms.

Inflammation: an important parameter in the search of prostate cancer biomarkers.

BACKGROUND: A more specific and early diagnostics for prostate cancer (PCa) is highly desirable. In this study, being inflammation the focus of our effort, serum protein profiles were analyzed in order to investigate if this parameter could interfere with the search of discriminating proteins between PCa and benign prostatic hyperplasia (BPH). METHODS: Patients with clinical suspect of PCa and candidates for trans-rectal ultrasound guided prostate biopsy (TRUS) were enrolled. Histological specimens were examined in order to grade and classify the tumor, identify BPH and detect inflammation. Surface Enhanced Laser Desorption/Ionization-Time of Flight-Mass Spectrometry (SELDI-ToF-MS) and two-dimensional gel electrophoresis (2-DE) coupled with Liquid Chromatography-MS/MS (LC-MS/MS) were used to analyze immuno-depleted serum samples from patients with PCa and BPH. RESULTS: The comparison between PCa (with and without inflammation) and BPH (with and without inflammation) serum samples by SELDI-ToF-MS analysis did not show differences in protein expression, while changes were only observed when the concomitant presence of inflammation was taken into consideration. In fact, when samples with histological sign of inflammation were excluded, 20 significantly different protein peaks were detected. Subsequent comparisons (PCa with inflammation vs PCa without inflammation, and BPH with inflammation vs BPH without inflammation) showed that 16 proteins appeared to be modified in the presence of inflammation, while 4 protein peaks were not modified. With 2-DE analysis, comparing PCa without inflammation vs PCa with inflammation, and BPH without inflammation vs the same condition in the presence of inflammation, were identified 29 and 25 differentially expressed protein spots, respectively. Excluding samples with inflammation the comparison between PCa vs BPH showed 9 unique PCa proteins, 4 of which overlapped with those previously identified in the presence of inflammation, while other 2 were new proteins, not identified in our previous comparisons. CONCLUSIONS: The present study indicates that inflammation might be a confounding parameter during the proteomic research of candidate biomarkers of PCa. These results indicate that some possible biomarker-candidate proteins are strongly influenced by the presence of inflammation, hence only a well-selected protein pattern should be considered for potential marker of PCa.

Proteomic analisys of protein extraction during hemofiltration with on-line endogenous reinfusion (HFR) using different polysulphone membranes.

In end-stage renal disease patients, extracorporeal dialytic therapy is not able to prevent the accumulation of toxins related to the uremic syndrome, a severe complication that increases morbidity and mortality rate. In this paper, hemoFiltration with on-line Reinfusion (HFR) architecture is used to evaluate the effect of a more permeable membrane on the extraction of medium-high molecular weight molecules. The aim of this study was to compare two polysulphone membranes for convective chamber: polyphenylene High Flux (pHF) and polyphenylene Super High-Flux (pSHF). Fourteen patients were subjected to HFR with pHF and pSHF membranes and ultra filtrate (UF) samples were collected to evaluate molecular weight cut-off (MWCO) and to identify extracted proteins. Furthermore, image analysis software was used in order to evaluate change in protein extraction during the dialysis. The quantification of four proteins by immunoassay demonstrates a higher permeability of pSHF membrane. Two-dimensional electrophoresis (2-DE) gels showed, for both membranes, the greater number of protein spots at 235 min. Some of the identified proteins, involved in nephropathic disease complications, were compared to assess differences in extraction during dialytic treatment by PDQuest analysis. UF proteomic analysis demonstrated a different behavior for the two membranes; pHF membrane was more permeable at the beginning of HFR treatment (15 min), while pSHF membrane at the end of treatment (235 min). Proteomic analysis is a suitable approach to investigate the behavior of different membranes during dialysis. Results indicated that pSHF membrane offers the higher permeability, and showed higher efficiency in removal of middle molecules related to uremic syndrome.

Non-bacterial protein expression in periodontal pockets by proteome analysis.

OBJECTIVES: To compare the proteomic profile of inter-proximal pocket tissues with inter-proximal healthy tissues in the same subject to reveal proteins associated with periodontal disease in sites where periodontopathogenic bacteria were not detectable. METHODS: Twenty-five healthy patients, with moderate-to-advanced chronic periodontitis and presenting with at least one intra-bony defect next to a healthy inter-proximal site were enrolled. The periodontal defects were treated with osseous resective surgery, and the flap design included both the periodontal pockets and the neighbouring inter-proximal healthy sites. Pocket-associated and healthy tissues were harvested for proteomic analyses. RESULTS: Fifteen proteins were differently expressed between pathological and healthy tissues. In particular, annexin A2, actin cytoplasmic 1, carbonic anhydrase 1 & 2; Ig kappa chain C region (two spots) and flavinreductase were overexpressed, whereas 14-3-3 protein sigma and zeta/delta, heat-shock protein beta -1 (two spots), triosephosphateisomerase, peroxiredoxin-1, fatty acid-binding protein-epidermal, and galectin-7 were underexpressed in pathological tissue. CONCLUSIONS: The unbalanced functional network of proteins involved could hinder adequate tissue response to pathogenic noxa. The study of periodontal pocket tissue proteomic profile would be crucial to better understand the pathogenesis of and the therapeutic strategies for periodontitis.

Discovery by a proteomic approach of possible early biomarkers of drug-induced nephrotoxicity in medication-overuse headache.

BACKGROUND: Medication-overuse headache (MOH) is a chronic headache condition that results from the overuse of analgesics drugs, triptans, or other antimigraine compounds. The epidemiology of drug-induced disorders suggests that medication overuse could lead to nephrotoxicity, particularly in chronic patients. The aim of this work was to confirm and extend the results obtained from a previous study, in which we analyzed the urinary proteome of 3 MOH patients groups: non-steroidal anti-inflammatory drugs (NSAIDs), triptans and mixtures abusers, in comparison with non-abusers individuals (controls). METHODS: In the present work we employed specialized proteomic techniques, namely two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry (MS), and the innovative Surface-Enhanced Laser Desorption/Ionization Time-of-Flight mass spectrometry (SELDI-TOF-MS), to discover characteristic proteomic profiles associated with MOH condition. RESULTS: By 2-DE and MS analysis we identified 21 over-excreted proteins in MOH patients, particularly in NSAIDs abusers, and the majority of these proteins were involved in a variety of renal impairments, as resulted from a literature search. Urine protein profiles generated by SELDI-TOF-MS analysis showed different spectra among groups. Moreover, significantly higher number of total protein spots and protein peaks were detected in NSAIDs and mixtures abusers. CONCLUSIONS: These findings confirm the presence of alterations in proteins excretion in MOH patients. Analysis of urinary proteins by powerful proteomic technologies could lead to the discovery of early candidate biomarkers, that might allow to identify MOH patients prone to develop potential drug overuse-induced nephrotoxicity.

The omics in migraine.

The term omics consist of three main areas of molecular biology, such as genomics, proteomics and metabolomics. The omics synergism recognise migraine as an ideal study model, due to its multifactorial nature. In this review, the plainly research data featuring in this complex network are reported and analyzed, as single or multiple factor in pathophysiology of migraine. The future of migraine biomolecular research shall be focused on networking among these different and hierarchical disciplines. We have to look for its Ariadne's tread, in order to see the whole painting of migraine molecular biology.

Research of Prostate Cancer Urinary Diagnostic Biomarkers by Proteomics: The Noteworthy Influence of Inflammation.

Nowadays, in the case of suspected prostate cancer (PCa), tissue needle biopsy remains the benchmark for diagnosis despite its invasiveness and poor tolerability, as serum prostate-specific antigen (PSA) is limited by low specificity. The aim of this proteomic study was to identify new diagnostic biomarkers in urine, an easily and non-invasively available sample, able to selectively discriminate cancer from benign prostatic hyperplasia (BPH), evaluating whether the presence of inflammation may be a confounding parameter. The analysis was performed by two-dimensional gel electrophoresis (2-DE), mass spectrometry (LC-MS/MS) and Enzyme-Linked Immunosorbent Assay (ELISA) on urine samples from PCa and BPH patients, divided into subgroups based on the presence or absence of inflammation. Significant quantitative and qualitative differences were found in the urinary proteomic profile of PCa and BPH groups. Of the nine differentially expressed proteins, only five can properly be considered potential biomarkers of PCa able to discriminate the two diseases, as they were not affected by the inflammatory process. Therefore, the proteomic research of novel and reliable urinary biomarkers of PCa should be conducted considering the presence of inflammation as a realistic interfering element, as it could hinder the detection of important protein targets.

Quantification of p-cresol sulphate in human plasma by selected reaction monitoring.

Chronic renal failure patients accumulate in the blood molecules that are normally excreted into the urine. p-Cresol Sulphate (pCS), the most representative retained toxin, shows a high level of toxicity. Therefore, its quantification could represent a prediction factor to determine the risk of endothelial dysfunction and cardiovascular complication and response to the haemodialysis treatment. The aim of this study was to evaluate the suitability of the multiple reaction monitoring (MRM) technique in order to improve the sensibility, the selectivity and the timing of pCS detection in a small amount of plasma. Deproteinized plasma of uremic patients was concentrated and dissolved in liquid chromatography (LC) mobile phase solution. pCS was quantified by LC coupled to tandem mass spectrometry (LC-MS/MS) on a triple-quadrupole mass spectrometer. Selective and sensitive detection of pCS was achieved by selecting the specific parent ion and monitoring two specific fragment ions. The MRM assay was carried out using the following transitions: m/z 187 → 80.00 and m/z 187 → 107.00. A good linearity was observed for each calibration curve. The intra-day and inter-day results showed a good precision and repeatability. The percentage recoveries indicate an optimal selectivity of the analytical method. The MRM assay to quantify pCS in a small amount of human plasma is rapid, highly sensitive, selective and with a good repeatability.

Proteomic analysis of urine in medication-overuse headache patients: possible relation with renal damages.

Medication-overuse headache (MOH) is a chronic disorder associated with overuse of analgesic drugs, triptans, non-steroidal anti-inflammatory drugs (NSAIDs) or other acute headache compounds. Various epidemiologic investigations proved that different drug types could cause nephrotoxicity, particularly in chronic patients. The aim of the present work was to analyze, by a proteomic approach, the urinary protein profiles of MOH patients focusing on daily use of NSAIDs, mixtures and triptans that could reasonably be related to potential renal damage. We selected 43 MOH patients overusing triptans (n = 18), NSAIDs (n = 11), and mixtures (n = 14), for 2-30 years with a mean daily analgesic intake of 1.5 ± 0.9 doses, and a control group composed of 16 healthy volunteers. Urine proteins were analyzed by mono-dimensional gel electrophoresis and identified by mass spectrometry analysis. Comparing the proteomic profiles of patients and controls, we found a significantly different protein expression, especially in the NSAIDs group, in which seven proteins resulted over-secreted from kidney (OR = 49, 95% CI 2.53-948.67 vs. controls; OR = 11.6, 95% CI 0.92-147.57 vs. triptans and mixtures groups). Six of these proteins (uromodulin, α-1-microglobulin, zinc-α-2-glycoprotein, cystatin C, Ig-kappa-chain, and inter-α-trypsin heavy chain H4) were strongly correlated with various forms of kidney disorders. Otherwise, in mixtures and in triptans abusers, only three proteins were potentially associated to pathological conditions (OR = 4.2, 95% CI 0.33-53.12, vs. controls). In conclusion, this preliminary proteomic study allowed us to define the urinary protein pattern of MOH patients that is related to the abused drug. According with the obtained results, we believe that the risk of nephrotoxicity should be considered particularly in MOH patients who abuse of NSAIDs.