Sialoglycan recognition is a common connection linking acidosis, zinc, and HMGB1 in sepsis.

Blood pH is tightly maintained between 7.35 and 7.45, and acidosis (pH <7.3) indicates poor prognosis in sepsis, wherein lactic acid from anoxic tissues overwhelms the buffering capacity of blood. Poor sepsis prognosis is also associated with low zinc levels and the release of High mobility group box 1 (HMGB1) from activated and/or necrotic cells. HMGB1 added to whole blood at physiological pH did not bind leukocyte receptors, but lowering pH with lactic acid to mimic sepsis conditions allowed binding, implying the presence of natural inhibitor(s) preventing binding at normal pH. Testing micromolar concentrations of divalent cations showed that zinc supported the robust binding of sialylated glycoproteins with HMGB1. Further characterizing HMGB1 as a sialic acid-binding lectin, we found that optimal binding takes place at normal blood pH and is markedly reduced when pH is adjusted with lactic acid to levels found in sepsis. Glycan array studies confirmed the binding of HMGB1 to sialylated glycan sequences typically found on plasma glycoproteins, with binding again being dependent on zinc and normal blood pH. Thus, HMGB1-mediated hyperactivation of innate immunity in sepsis requires acidosis, and micromolar zinc concentrations are protective. We suggest that the potent inflammatory effects of HMGB1 are kept in check via sequestration by plasma sialoglycoproteins at physiological pH and triggered when pH and zinc levels fall in late stages of sepsis. Current clinical trials independently studying zinc supplementation, HMGB1 inhibition, or pH normalization may be more successful if these approaches are combined and perhaps supplemented by infusions of heavily sialylated molecules.

Podocalyxin-like protein promotes gastric cancer progression through interacting with RUN and FYVE domain containing 1 protein.

Podocalyxin-like protein (PODXL), a transmembrane glycoprotein with anti-adhesive properties, is associated with an aggressive tumor phenotype and poor prognosis of several cancers. To elucidate the biological significance of PODXL and its molecular mechanism in gastric cancer (GC), we investigated the expression of PODXL in GC samples and assessed its effects on biological behaviors and the related signaling pathways in vitro and in vivo. Moreover, the possible and closely interacted partners of PODXL were identified. Our data showed that the protein or mRNA level of PODXL was significantly upregulated in tissues or serum of GC patients compared with normal-appearing tissues (NAT) or those of healthy volunteers. Overall survival (OS) curves showed that patients with high PODXL levels in tissues or serum had a worse 5-year OS. In vitro, restoring PODXL expression promoted tumor progression by increasing cell proliferation, colony formation, wound healing, migration and invasion, as well as suppressing the apoptosis. Furthermore, the PI3K/AKT, NF-κB and MAPK/ERK signaling pathways were activated. There was a significant positive correlation between PODXL and RUN and FYVE domain containing 1 (RUFY1) expression in tissues or serum. Subsequent mass spectrometry analysis, co-immunoprecipitation assays and western blot analysis identified PODXL/RUFY1 complexes in GC cells, and silencing RUFY1 expression in GC cells significantly attenuated PODXL-induced phenotypes and their underlying signaling pathways. Our results suggested that PODXL promoted GC progression via a RUFY1-dependent signaling mechanism. New GC therapeutic opportunities through PODXL and targeting the PODXL/RUFY1 complex might improve cancer therapy.

Identification of Multisialylated LacdiNAc Structures as Highly Prostate Cancer Specific Glycan Signatures on PSA.

Serum prostate-specific antigen (PSA) test is the current gold standard for screening and diagnosis of prostate cancer (PCa), while overdiagnosis and overtreatment are social problems. In order to improve the specificity and exclude a false positive diagnosis in PSA test, PCa-specific glycosylation subtypes of PSA were explored using in-depth quantitative profiling of PSA glycoforms based on mass spectrometric oxonium ion monitoring technology. As a result of analysis using sera from 15 PCa or 15 benign prostate hyperplasia (BPH) patients whose PSA levels were in the "gray zone" (4.0-10.0 ng/mL), 52 glycan structures on PSA were quantitatively observed. We found that abundance of multisialylated LacdiNAc (GalNAcß1-4GlcNAc) structures were significantly upregulated in the PCa group compared to the BPH group. A couple of those glycoforms were then extracted and subjected to establish a novel PCa-specific diagnosis model (PSA G-index). When the diagnostic power was assessed using an independent validation sample set (15 PCa and 15 BPH patients in the PSA gray zone), an AUC of PSA G-index was 1.00, while that of total PSA or PSA f/T ratio was 0.50 or 0.60, respectively. Moreover, both PSA glycoforms showed significant correlation with Gleason scores. Lectin histochemical staining analysis also showed that PCa cells overexpressed glycoproteins containing LacdiNAc and sialic acids moieties. Thus, PSA G-index could serve as not only an effective secondary screening method to exclude false positive diagnosis in PSA screening, but also a potential grading biomarker for PCa.

Detection of cerebrospinal fluid leakage by specific measurement of transferrin glycoforms.

A simple and rapid detection of cerebrospinal fluid (CSF) leakage would benefit spine surgeons making critical postoperative decisions on patient care. We have assessed novel approaches to selectively determine CSF ß2-transferrin (ß2TF), an asialo-transferrin (aTF) biomarker, without interference from serum sialo-transferrin (sTF) in test samples. First, we performed mild periodate oxidation to selectively generate aldehyde groups in sTF for capture with magnetic hydrazide microparticles, and selective removal with a magnetic separator. Using this protocol sTF was selectively removed from mixtures of CSF and serum containing CSF aTF (ß2TF) and serum sTF, respectively. Second, a two-step enzymatic method was developed with neuraminidase and galactose oxidase for generating aldehyde groups in sTF present in CSF and serum mixtures for magnetic hydrazide microparticle capture. After selectively removing sTF from mixtures of CSF and serum, ELISA could detect significant TF signal only in CSF, while the TF signal in serum was negligible. The new approach for selective removal of only sTF in test samples will be promising for the required intervention by a spine surgeon.

How accurate are blood (or breath) tests for identifying self-reported heavy drinking among people with alcohol dependence?

AIMS: Managing patients with alcohol dependence includes assessment for heavy drinking, typically by asking patients. Some recommend biomarkers to detect heavy drinking but evidence of accuracy is limited. METHODS: Among people with dependence, we assessed the performance of disialo-carbohydrate-deficient transferrin (%dCDT, ≥1.7%), gamma-glutamyltransferase (GGT, ≥66 U/l), either %dCDT or GGT positive, and breath alcohol (> 0) for identifying 3 self-reported heavy drinking levels: any heavy drinking (≥4 drinks/day or >7 drinks/week for women, ≥5 drinks/day or >14 drinks/week for men), recurrent (≥5 drinks/day on ≥5 days) and persistent heavy drinking (≥5 drinks/day on ≥7 consecutive days). Subjects (n = 402) with dependence and current heavy drinking were referred to primary care and assessed 6 months later with biomarkers and validated self-reported calendar method assessment of past 30-day alcohol use. RESULTS: The self-reported prevalence of any, recurrent and persistent heavy drinking was 54, 34 and 17%. Sensitivity of %dCDT for detecting any, recurrent and persistent self-reported heavy drinking was 41, 53 and 66%. Specificity was 96, 90 and 84%, respectively. %dCDT had higher sensitivity than GGT and breath test for each alcohol use level but was not adequately sensitive to detect heavy drinking (missing 34-59% of the cases). Either %dCDT or GGT positive improved sensitivity but not to satisfactory levels, and specificity decreased. Neither a breath test nor GGT was sufficiently sensitive (both tests missed 70-80% of cases). CONCLUSIONS: Although biomarkers may provide some useful information, their sensitivity is low the incremental value over self-report in clinical settings is questionable.

Toward standardization of carbohydrate-deficient transferrin (CDT) measurements: III. Performance of native serum and serum spiked with disialotransferrin proves that harmonization of CDT assays is possible.

Carbohydrate-deficient transferrin (CDT) is a generic term that refers to the transferrin glycoforms whose concentration in blood is temporarily increased by sustained alcohol consumption. Due to high clinical specificity, CDT was proposed as a biomarker of heavy alcohol use and has been available for about 20 years. A number of methods have been developed for CDT measurement based on different analytical techniques and principles and without any harmonization or calibration to a reference method. As a consequence, neither the reference limits nor the cut-off values have been similar across assays, hampering understanding of the diagnostic value of CDT and its routine use. This prompted the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) to initiate a Working Group on Standardization of CDT (WG-CDT). This third publication of the WG-CDT is devoted to testing the commutability of native and disialotransferrin-spiked serum panels as candidate secondary reference materials, in order to prove the harmonization potential of commercial CDT methods. The results showed that assay harmonization reduced the inter-laboratory imprecision in a network of reference laboratories running the HPLC candidate reference method. In the seven commercial methods evaluated in this study, the use of multi-level secondary calibrators of human serum origin significantly reduced the between-method imprecision. Thus, harmonization of CDT measurements by different methods can be achieved using this calibration system, opening the way for a full standardization of commercial methods against a reference method by use of certified reference materials.

Effect of transferrin glycation on the use of carbohydrate-deficient transferrin as an alcohol biomarker.

AIMS: Measurement of an alcohol-induced shift in the serum transferrin glycosylation pattern, termed carbohydrate-deficient transferrin (CDT), is used as a biomarker for sustained high alcohol consumption. The present work examined whether non-enzymatic reaction of transferrin with glucose (glycation) might interfere with the use of CDT as an alcohol biomarker. METHODS: The blood specimens were leftover volumes from the routine sample pool. Plasma and serum were collected among samples submitted for hemoglobin A1c (HbA1c) and CDT testing. Quantification of individual transferrin glycoforms in percentage of total transferrin was performed by an HPLC candidate CDT reference method. RESULTS: Incubating serum spiked with 20 or 200 mmol/l glucose caused time- and dose-dependent changes in the chromatographic profile of transferrin glycoforms, resulting in gradually wider peaks and reduced relative amounts of disialo- and trisialotransferrin. No similar chromatographic effects were seen in samples collected from diabetic patients with elevated HbA1c (>68 mmol/mol) values. These samples instead showed slightly higher mean %disialotransferrin levels (1.21%) compared with low HbA1c (<44 mmol/mol) samples (mean 1.06%; P = 0.023), pointing at a higher alcohol consumption level in the former group. Altogether ∼5% of the CDT values exceeded the cutoff. There was no significant difference in phosphatidylethanol (PEth) levels between the high and low HbA1c samples, but several (∼14%) showed elevated PEth concentrations. CONCLUSION: Glycation of serum transferrin in vivo was indicated to differ from that in vitro, and suggested not to interfere with %CDT testing by the HPLC method. The results indicated that CDT and PEth are useful as objective, complementary alcohol biomarkers to identify risky drinking also in diabetic subjects.

Relationship between advanced oxidation protein products, advanced glycation end products, and S-nitrosylated proteins with biological risk and MDR-1 polymorphisms in patients affected by B-chronic lymphocytic leukemia.

The aim of our study was to analyze the serum levels of advanced oxidation protein products (AOPPs) and advanced glycation end products (AGEs), and protein nitrosylation in patients with B-chronic lymphocytic leukemia (B-CLL). AOPPs, AGEs, and S-nitrosylated were increased in B-CLL patients. The mutation of IgVH gene, CD 38, and Zap 70 expression were not associated with increased oxidative stress. The mutant 2677GT genotype was found to be associated with higher AGEs levels with respect to wild-type genotype, while as far the C3435T MDR1 polymorphism is concerned, subjects presenting wild-type genotype showed higher values of AOPPs with respect to heterozygous genotype. Our results suggest that B-CLL is associated with oxidative stress.

High performance liquid chromatography evaluation of serum carbohydrate-deficient transferrin and more sialylated transferrin glycoforms in children.

OBJECTIVES: The evaluation of the age-specific distribution of transferrin glycoforms in paediatric patients may help in defining reference intervals which are critical for an improved and earlier diagnosis. DESIGN AND METHODS: Serum samples from 224 children (age: 2 months-14 years) were analyzed by HPLC (Bio-Rad CDT/HPLC kit) and glycoforms expressed as percentage of the total area of transferrin (Tf). RESULTS: Asialo- and Monosialo-Tf were not detectable in any patient. Medians (IQR) were respectively 0.92% (0.80-1.04%) for Disialo-Tf; 3.47% (2.69-4.18%) for Trisialo-Tf; 82.54% (81.32-83.53%) for Tetrasialo-Tf; 12.73% (11.91-14.09%) for Pentasialo-Tf. Statistically significant differences in Trisialo-Tf (p < 0.0005), Tetrasialo-Tf (p = 0.001), Pentasialo-Tf (p < 0.0005), but not in Disialo-Tf, were observed between the age groups. CONCLUSIONS: Age-specific Disialo-Tf cut-offs are not necessary. In children 1.3% and 6.4% may be suggested as upper limits of normal range to detect increases of Disialo- and Trisialo-Tf. The presence of Asialo- and Monosialo-Tf should be considered an abnormal finding and prompt further investigations.

Pregnancy and variations of carbohydrate-deficient transferrin levels measured by the candidate reference HPLC method.

AIMS: Contrasting data are available on the diagnostic accuracy of carbohydrate-deficient transferrin (CDT) during pregnancy. These differences may depend in part on how CDT was evaluated and expressed. Here, we report on variations of CDT levels in pregnant women using the high performance liquid chromatography (HPLC) candidate reference method. METHODS: Alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, mean corpuscular volume, serum transferrin, urine and serum ethyl glucuronide and CDT were measured in 64 women, self-reporting as non-alcohol abusers (age: median 34, IQR: 28-38), at different stages of normal pregnancy (gestational weeks: median 28, IQR: 8-33). CDT was expressed as percentage of disialotransferrin to total transferrin (%CDT). RESULTS: Transferrin was associated with both %CDT (r = 0.66; P < 0.001) and gestational week (r = 0.68; P < 0.001). Interestingly, %CDT was highly correlated with gestational week (r = 0.77; P < 0.001), even after controlling for the effect of transferrin. Moreover, statistically significant differences in %CDT were also evident between women grouped for pregnancy trimester (first trimester: mean 1.01% (SD 0.19); second trimester: 1.30% (SD 0.14); third trimester: 1.53% (SD 0.22); ANOVA P < 0.001). Trend analysis confirmed a proportional increase of %CDT along with pregnancy trimesters (P < 0.001). CONCLUSIONS: %CDT, measured with the HPLC candidate reference method, is independently associated with gestational week. Differently from what has been previously reported or expected, the relationship between pregnancy and CDT could be more complex. The diagnostic accuracy of CDT for detecting alcohol abuse in a legal context may be limited in pregnant women and the effect of gestational age should be considered.

Characterization of a human lymphocyte surface sialoglycoprotein that is defective in Wiskott-Aldrich syndrome.

gpL115 is a lymphocyte surface component that is deficient in patients with the X-chromosome-linked immune deficiency Wiskott-Aldrich syndrome (6). The glycoprotein nature of gpL115 is demonstrated through labeling in carbohydrate moieties by [3H]NaBH4 and its synthesis by lymphocytes through labeling with [35S]methionine. Native gpL115 adheres to wheat germ lectin-Sepharose and sialidase-treated gpL115 does not adhere, indicating that native gpL115 adheres via clusters of sialic acid residues. When tested on peanut lectin, which shows specificity for the disaccharide Gal beta 1-3GalNAc, gpL115 is nonadherent and sialidase-treated gpL115 is adherent, indicating the presence of the sequence sialic acid-Gal beta 1-3GalNAc, which is characteristic for O-linked (mucin-type, acidic-type) carbohydrates. A surface glycoprotein with all the above characteristics was found on the lymphoblastoid cell line CEM. CEM cells were used as immunogen to generate the monoclonal antibody L10, an IgG1, which binds native and sialidase-treated gpL115 . Sialidase-treatment of gpL115 significantly alters its physical properties, reducing its electrophoretic mobility and changing its behavior on isoelectrofocusing. Cumulatively, these findings indicate that gpL115 , like glycophorin of erythrocytes and GPIb of platelets, is a sialoglyco protein with significant quantities of O-linked carbohydrate. On treatment with limiting sialidase concentrations, gpL115 of normal lymphocytes is transformed into a series of partially desialylated species of decreasing electrophoretic mobility. This finding resembles the situation with lymphocytes of some Wiskott-Aldrich syndrome patients. Lymphocytes of eight Wiskott-Aldrich syndrome patients were found to be deficient in 125I-labeled gpL115 . Lymphocytes from three of these patients displayed an abnormal 125I-component of apparent mol wt 135,000.

Magnetic separation of human podocalyxin-like protein 1 (hPCLP1)-positive cells from peripheral blood and umbilical cord blood using anti-hPCLP1 monoclonal antibody and protein A expressed on bacterial magnetic particles.

Hemangioblasts are common progenitors of hematopoietic and angiogenic cells, which have been demonstrated in the mouse to possess a unique cell surface marker, podocalyxin-like protein 1 (PCLP1) (Hara, T. et al., Immunity, 11: 567-578. 1999). In this study, we prepared a novel monoclonal antibody against human PCLP1 (hPCLP1) and attempted to isolate human hematopoietic progenitor cells from umbilical cord blood and peripheral blood using nano-sized bacterial magnetic particles (BacMPs) coupled with the anti-hPCLP1 antibody. Flow cytometric analysis demonstrated that the purity of separated hPCLP1-positive cells from peripheral blood was approximately 95% whereas peripheral blood mononuclear cells contained only 0.1% PCLP1+ cells. Umbilical cord blood was demonstrated to be a better source for PCLP1+ cells than peripheral blood. These results suggest that the separation of human PCLP1+ cells using BacMPs with anti-hPCLP1 were extremely effective and may be useful as a means to prepare human hematopoietic progenitor cells.

Serum podocalyxin at 11-13 weeks of gestation in the prediction of small for gestational age neonates.

OBJECTIVE: To investigate a potential new marker for the prediction of small for gestational age (SGA) infants. STUDY DESIGN: Nested case-control study involving 280 uncomplicated pregnancies and 70 cases of SGA without pre-eclampsia. Serum podocalyxin was measured at 11-13 weeks of gestation and results were expressed in multiples of the median (MoM). The performance of screening by a combination of maternal history and podocalyxin levels was assessed with ROC curves. RESULTS: SGA was predicted by maternal age, height, South Asian ethnicity, and previous delivery without pre-eclampsia. Median podocalyxin levels were higher in affected than uncomplicated pregnancies (1.303 versus 0.994 MoM, p < 0.001). At a 10% false-positive rate, maternal history identified 40.0% of the cases (AUC = 0.74, 95%CI 0.671-0.809). The addition of podocalyxin increased the detection to 54.3% (AUC = 0.78, 95%CI 0.771-0.842, p = 0.027 for the difference in ROC curves). CONCLUSION: First-trimester podocalyxin may be useful in screening for SGA infants.

Identifying the molecular and cellular signature of cardiac dilation following myocardial infarction.

Establishing molecular and cellular indicators that reflect the extent of dilation of the left ventricle (LV) after myocardial infarction (MI) may improve diagnostic and prognostic capabilities. We queried the Mouse Heart Attack Research Tool (mHART) 1.0 for day 7 post-MI mice (age 3-9 months, untreated males and females) with serial echocardiographic data at days 0, 1, and 7 (n = 51). Mice were classified into two subgroups determined by a median fold change of 1.6 in end-diastolic dimensions (EDD) normalized to pre-MI values; n = 26 fell below (moderate; mean of 1.42 ±â€¯0.01) and n = 25 fell above this cut-off (extreme; mean of 1.79 ±â€¯0.01; p < 0.001 vs. moderate). Plasma proteomic profiling of 34 analytes measured at day 7 post-MI from male mice (n = 12 moderate and 12 extreme) were evaluated as the test dataset, and receiver operating curve (ROC) analysis was used to assess strength of biomarkers. Females (n = 6 moderate and 9 extreme) were used as the validation dataset. Both by t-test and characteristic (ROC) curve analysis, lower macrophage inflammatory protein-1 gamma (MIP-1γ), lymphotactin, and granulocyte chemotactic protein-2 (GCP-2) were identified as plasma indicators for dilation status (p < 0.05 for all). Macrophage numbers were decreased and complement C5, laminin 1, and Ccr8 gene levels were significantly higher in the LV infarcts of the extreme dilation group (p < 0.05 for all). A composite panel including plasma MIP-1γ, lymphotactin, and GCP-2, and LV infarct Ccr8 and macrophage numbers strongly mirrored LV dilation status (AUC = 0.92; p < 0.0001). Using the mHART 1.0 database, we determined that a failure to mount sufficient macrophage-mediated inflammation was indicative of exacerbated LV dilation.

Daily Fructose Traces Intake and Liver Injury in Children with Hereditary Fructose Intolerance.

BACKGROUND: Hereditary fructose intolerance (HFI) is a rare genetic disorder of fructose metabolism due to aldolase B enzyme deficiency. Treatment consists of fructose, sorbitol, and sucrose (FSS)-free diet. We explore possible correlations between daily fructose traces intake and liver injury biomarkers on a long-term period, in a cohort of young patients affected by HFI. METHODS: Patients' clinical data and fructose daily intake were retrospectively collected. Correlations among fructose intake, serum alanine aminotransferase (ALT) level, carbohydrate-deficient transferrin (CDT) percentage, liver ultrasonography, genotype were analyzed. RESULTS: We included 48 patients whose mean follow-up was 10.3 ± 5.6 years and fructose intake 169 ± 145.4 mg/day. Eighteen patients had persistently high ALT level, nine had abnormal CDT profile, 45 had signs of liver steatosis. Fructose intake did not correlate with ALT level nor with steatosis severity, whereas it correlated with disialotransferrin percentage (R2 0.7, p < 0.0001) and tetrasialotransferrin/disialotransferrin ratio (R2 0.5, p = 0.0001). p.A150P homozygous patients had lower ALT values at diagnosis than p.A175D variant homozygotes cases (58 ± 55 IU/L vs. 143 ± 90 IU/L, p = 0.01). CONCLUSION: A group of HFI patients on FSS-free diet presented persistent mild hypertransaminasemia which did not correlate with fructose intake. Genotypes may influence serum liver enzyme levels. CDT profile represents a good marker to assess FSS intake.

Association of serum podocalyxin levels with peripheral arterial disease in patients with type 2 diabetes.

OBJECTIVE: Renal podocalyxin is a marker for kidney diseases. Previous studies have shown the expression of serum podocalyxin (s-Podxl) in the endothelial cells of blood vessels. We aimed to investigate the association between s-podxl levels and peripheral arterial disease (PAD) in subjects with type 2 diabetes (T2DM). SUBJECTS AND METHODS: Serum Podxl levels were analyzed in 69 subjects with normal glucose tolerance and PAD (NGT-PAD), 120 subjects with T2DM and PAD (D-PAD) and 36 subjects with T2DM without PAD (D-NPAD). RESULTS: In D-PAD Patients, s-Podxl was significantly higher (17.67 ±â€¯20.7 ng/mL) than in D-NPAD subjects (9.97 ±â€¯5.34 ng/mL; P < 0.001). Subjects with NGT-PAD had significantly higher s-Podxl levels (15.34 ±â€¯18.21 ng/mL), than D-NPAD patients (P < 0.001). Subjects with D-PAD and medial calcific sclerosis (MCS) had significantly higher s-Podxl levels compared to the same group but without MCS (P < 0.02). In D-PAD patients, MCS (P = 0.003) and glycosylated hemoglobin (P < 0.001) were the two variables that had the strongest prediction for s-Podxl as revealed by regression analysis. Multivariate regression showed that an increase of one standard deviation in s-Podxl was associated with an odds ratio of 3.4 (95% confidence interval = 2.2-4.6, P < 0.001) for the prevalence of PAD. CONCLUSIONS: This is the first study showing an association between s-Podxl and PAD in patients with T2DM. S-Podxl was higher in D-PAD patients than in D-NPAD subjects. In NGT-PAD patients, s-Podxl was also higher than in D-NPAD patients. In patients with D-PAD, s-Podxl was positively associated with MCS.

Asbestos exposure, pleural mesothelioma, and serum osteopontin levels.

BACKGROUND: We investigated the presence of osteopontin in pleural mesothelioma and determined serum osteopontin levels in three populations: subjects without cancer who were exposed to asbestos, subjects without cancer who were not exposed to asbestos, and patients with pleural mesothelioma who were exposed to asbestos. METHODS: A group of 69 subjects with asbestos-related nonmalignant pulmonary disease were compared with 45 subjects without exposure to asbestos and 76 patients with surgically staged pleural mesothelioma. Tumor tissue was examined for osteopontin by immunohistochemical analysis, and serum osteopontin levels were measured by an enzyme-linked immunosorbent assay. RESULTS: There were no significant differences in mean (+/-SE) serum osteopontin levels between age-matched subjects with exposure to asbestos and subjects without exposure to asbestos (30+/-3 ng per milliliter and 20+/-4 ng per milliliter, respectively; P=0.06). In the group with exposure to asbestos, elevated serum osteopontin levels were associated with pulmonary plaques and fibrosis (56+/-13 ng per milliliter) but not with normal radiographic findings (21+/-5 ng per milliliter), plaques alone (23+/-3 ng per milliliter), or fibrosis alone (32+/-7 ng per milliliter) (P=0.004). Serum osteopontin levels were significantly higher in the group with pleural mesothelioma than in the group with exposure to asbestos (133+/-10 ng per milliliter vs. 30+/-3 ng per milliliter, P<0.001). Immunohistochemical analysis revealed osteopontin staining of the tumor cells in 36 of 38 samples of pleural mesothelioma. An analysis of serum osteopontin levels comparing the receiver-operating-characteristic curve in the group exposed to asbestos with that of the group with mesothelioma had a sensitivity of 77.6 percent and a specificity of 85.5 percent at a cutoff value of 48.3 ng of osteopontin per milliliter. Subgroup analysis comparing patients with stage I mesothelioma with subjects with exposure to asbestos revealed a sensitivity of 84.6 percent and a specificity of 88.4 percent at a cutoff value of 62.4 ng of osteopontin per milliliter. CONCLUSIONS: Serum osteopontin levels can be used to distinguish persons with exposure to asbestos who do not have cancer from those with exposure to asbestos who have pleural mesothelioma.

Mixed-mode chromatography for fractionation of peptides, phosphopeptides, and sialylated glycopeptides.

A mixed-mode chromatographic (MMC) sorbent was prepared by functionalizing the silica sorbent with a pentafluorophenyl (PFP) ligand. The resulting stationary phase provided a reversed-phase (RP) retention mode along with a relatively mild strong cation-exchange (SCX) retention interaction. While the mechanism of interaction is not entirely clear, it is believed that the silanols in the vicinity of the perfluorinated ligand act as strongly acidic sites. The 2.1 mm x 150 mm column packed with such sorbent was applied to the separation of peptides. Linear RP gradients in combination with salt steps were used for pseudo two-dimensional (2D) separation and fractionation of tryptic peptides. An alternative approach of using linear cation-exchange gradients combined with RP step gradients was also investigated. Besides the attractive forces, the ionic repulsion contributed to the retention mechanism. The analytes with strong negatively charged sites (phosphorylated peptides, sialylated glycopeptides) eluted in significantly different patterns than generic tryptic peptides. This retention mechanism was employed for the isolation of phosphopeptides or sialylated glycopeptides from non-functionalized peptide mixtures. The mixed-mode column was utilized in conjunction with a phosphopeptide enrichment solid phase extraction (SPE) device packed with metal oxide affinity chromatography (MOAC) sorbent. The combination of MOAC and mixed-mode chromatography (MMC) provided for an enhanced extraction selectivity of phosphopeptides and sialylated glycopeptides peptides from complex samples, such as yeast and human serum tryptic digests.

Serum podocalyxin for early detection of preeclampsia at 11-13 weeks of gestation.

Podocalyxin is expressed on endothelial surface throughout the body and is likely released into the circulation during pregnancy in association with vessel remodeling. We have recently reported that serum podocalyxin is significantly increased in preeclampsia at disease presentation. In this study we investigated whether serum podocalyxin is altered prior to clinical presentation of preeclampsia. At 11-13 weeks of gestation, serum podocalyxin was significantly elevated in women who subsequently developed preeclampsia. Multi-factorial analysis suggests that combination of serum podocalyxin with the long isoform of HtrA3 and mean arterial pressure, may provide effective detection of late-onset preeclampsia at 11-13 weeks of pregnancy.

Serum podocalyxin levels correlate with carotid intima media thickness, implicating its role as a novel biomarker for atherosclerosis.

Podocalyxin is a cell surface sialomucin, which is expressed in not only glomerular podocytes but also vascular endothelial cells. Urinary podocalyxin is used as a marker for glomerular disease. However, there are no reports describing serum podocalyxin (s-Podxl) levels. Therefore, the association between s-Podxl levels and clinical parameters were examined with 52 patients. s-Podxl level was evaluated using enzyme-linked immunosorbent assay. The median s-Podxl level was 14.2 ng/dL (interquartile range: 10.8-22.2 ng/dL). There were significant correlations (correlation coefficient: r > 0.2) of s-Podxl levels with carotid intima media thickness (IMT) (r = 0.30, p = 0.0307). Multiple logistic regression analysis showed that s-Podxl levels remained significantly associated with carotid IMT > 1 mm (OR: 1.15; 95% CI 1.02-1.31, p = 0.026) after adjustments for traditional cardiovascular risk factors such as age, sex, current smoking status, hypertension, dyslipidemias, and diabetes. In conclusion, s-Podxl is independently associated with carotid IMT and might be used as a novel biomarker for cardiovascular disease.