The European biological variation study (EuBIVAS): Biological variation data for testosterone, follicle stimulating hormone, prolactin, luteinizing hormone and dehydroepiandrosterone sulfate in men.

BACKGROUND: Knowledge of biological variation (BV) of hormones is essential for interpretation of laboratory tests and for diagnostics of endocrinological and reproductive diseases. There is a lack of robust BV data for many hormones in men. METHODS: We used serum samples collected weekly over 10 weeks from the European Biological Variation Study (EuBIVAS) to determine BV of testosterone, follicle-stimulating hormone (FSH), prolactin, luteinizing hormone (LH) and dehydroepiandrosterone sulfate (DHEA-S) in 38 men. We derived within-subject (CVI) and between-subject (CVG) BV estimates by CV-ANOVA after trend, outlier, and homogeneity analysis and calculated reference change values, index of individuality (II), and analytical performance specifications. RESULTS: The CVI estimates were 10 % for testosterone, 8 % for FSH, 13 % for prolactin, 22 % for LH, and 9 % for DHEA-S, respectively. The IIs ranged between 0.14 for FSH to 0.66 for LH, indicating high individuality. CONCLUSIONS: In this study, we have used samples from the highly powered EuBIVAS study to derive BV estimates for testosterone, FSH, prolactin, LH and DHEA-S in men. Our data confirm previously published BV estimates of testosterone, FSH and LH. For prolactin and DHEA-S BV data for men are reported for the first time.

Biological variation of inflammatory and iron metabolism markers in high-endurance recreational athletes; are these markers useful for athlete monitoring?

OBJECTIVES: To deliver biological variation (BV) data for serum hepcidin, soluble transferrin receptor (sTfR), erythropoietin (EPO) and interleukin 6 (IL-6) in a population of well-characterized high-endurance athletes, and to evaluate the potential influence of exercise and health-related factors on the BV. METHODS: Thirty triathletes (15 females) were sampled monthly (11 months). All samples were analyzed in duplicate and BV estimates were delivered by Bayesian and ANOVA methods. A linear mixed model was applied to study the effect of factors related to exercise, health, and sampling intervals on the BV estimates. RESULTS: Within-subject BV estimates (CVI) were for hepcidin 51.9 % (95 % credibility interval 46.9-58.1), sTfR 10.3 % (8.8-12) and EPO 27.3 % (24.8-30.3). The mean concentrations were significantly different between sex, but CVI estimates were similar and not influenced by exercise, health-related factors, or sampling intervals. The data were homogeneously distributed for EPO but not for hepcidin or sTfR. IL-6 results were mostly below the limit of detection. Factors related to exercise, health, and sampling intervals did not influence the BV estimates. CONCLUSIONS: This study provides, for the first time, BV data for EPO, derived from a cohort of well-characterized endurance athletes and indicates that EPO is a good candidate for athlete follow-up. The application of the Bayesian method to deliver BV data illustrates that for hepcidin and sTfR, BV data are heterogeneously distributed and using a mean BV estimate may not be appropriate when using BV data for laboratory and clinical applications.

The prevalence of hemoglobin Tacoma in Finland detected by HbA1c capillary electrophoresis.

Previous studies have identified occasional cases of heterozygous Hb Tacoma in areas that have attracted Finnish immigrants, especially in Sweden and North America, but large studies of this slightly unstable beta variant in vitro have not been carried out. Here we determined the prevalence of hemoglobin variants across Finland. A total of 5059 samples from 11 different hospital districts were analyzed using HbA1c capillary electrophoresis and reviewed for atypical profiles (HbA1c, Capillarys 3 Tera, Sebia). 38 heterozygous Hb Tacoma cases were found (0.75%). The prevalence was highest in South Ostrobothnia (2.0%), located in western Finland, and second highest in the neighboring provinces (1.0-1.4%), but only two districts were devoid of variants. Heterozygous Hb Tacoma was confirmed by genetic testing (NM_000518.5(HBB):c.93G > T (p.Arg31Ser)). In addition, five other variants were found, suggestive of HbE, Hb Helsinki (two cases) and an alpha variant, as interpreted from the electropherograms. The fifth variant, belonging to the South Ostrobothnian cohort, remained unknown at the time of the initial analyses, but was later interpreted as homozygous Hb Tacoma and confirmed by hemoglobin fraction analysis (Hemoglobin(E), Capillarys 3 Tera). In a subsequent retrospective study of the electropherograms of routine HbA1c diagnostics, altogether nine homozygous Hb Tacoma cases were identified in South Ostrobothnia. While heterozygous Hb Tacoma is usually an incidental finding, it interferes with several HbA1c assays. The present study is the first demonstration of homozygous Hb Tacoma. The clinical presentations of homozygous Hb Tacoma are not known and need to be addressed in future studies.

Changing Immunochemistry Platforms: Thyroid Function Test Comparison and Reference Intervals Based on Clinical Needs.

BACKGROUND: Diagnosis of thyroid dysfunction relies on thyroid stimulating hormone (TSH), free thyroxine (FT4), and free tri-iodothyronine (FT3) tests against valid reference intervals (RIs). We changed the immunoassay platform from Abbott Architect to Siemens Atellica and aimed to establish Atellica RIs based on laboratory information system (LIS) patient data. METHODS: Atellica thyroid hormone immunoassays were verified against those of Architect. Real-life patient results were retrieved from LIS. A single result per patient dataset was used to establish the RIs by the indirect method. RESULTS: Atellica and Architect assays correlated well but Atellica showed a positive bias between 13% and 53%, the largest for FT4. Variations of the Atellica assays were ≤4%. The 95% Atellica RIs were 0.4-3.8 mU/L for TSH, 0.9-1.6 ng/dL for FT4, and 227-416 pg/dL for FT3. Considering the accumulating clinical experience with Atellica, the RIs for clinical use were adjusted as 0.5-4.0 mU/L, 0.9-1.8 ng/dL, and 169-409 pg/dL, respectively. CONCLUSIONS: We verified thyroid hormone RIs for Atellica by the indirect method for the first time. Our model proved reliable for selecting results of presumably healthy individuals from LIS data. Critical review of the RIs with local endocrinologists is essential.

Long-term within- and between-subject biological variation of 29 routine laboratory measurands in athletes.

OBJECTIVES: Within- and between-subject biological variation (BV) estimates have many applications in laboratory medicine. However, robust high-quality BV estimates are lacking for many populations, such as athletes. This study aimed to deliver BV estimates of 29 routine laboratory measurands derived from a Biological Variation Data Critical Appraisal Checklist compliant design in a population of high-endurance athletes. METHODS: Eleven samples per subject were drawn from 30 triathletes monthly, during a whole sport season. Serum samples were measured in duplicate for proteins, liver enzymes, lipids and kidney-related measurands on an Advia2400 (Siemens Healthineers). After outlier and homogeneity analysis, within-subject (CVI) and between-subject (CVG) biological variation estimates were delivered (CV-ANOVA and log-ANOVA, respectively) and a linear mixed model was applied to analyze the effect of exercise and health related variables. RESULTS: Most CVI estimates were similar or only slightly higher in athletes compared to those reported for the general population, whereas two- to three-fold increases were observed for amylase, ALT, AST and ALP. No effect of exercise and health related variables were observed on the CVI estimates. For seven measurands, data were not homogeneously distributed and BV estimates were therefore not reported. CONCLUSIONS: The observation of higher CVI estimates in athletes than what has been reported for the general population may be related to physiological stress over time caused by the continuous practice of exercise. The BV estimates derived from this study could be applied to athlete populations from disciplines in which they exercise under similar conditions of intensity and duration.

Comparison of Serum and Urinary 5-Hydroxyindoleacetic Acid as Biomarker for Neuroendocrine Neoplasms.

CONTEXT: Patients with serotonin-secreting neuroendocrine neoplasms (NENs) have increased serum 5-hydroxyindoleacetic acid (5HIAA) concentrations. Serum 5HIAA thus serves as a biomarker in NEN. OBJECTIVE: To evaluate an improved tandem mass spectrometric serum 5HIAA assay for diagnosis and follow-up of NEN in a clinical cohort. DESIGN: A retrospective study during 2016-2018 at the Diagnostic Center and Department of Endocrinology at Helsinki University Hospital, Finland. METHODS: Detailed patient data was obtained from 116 patients. Serum 5HIAA was analyzed by 2 different liquid chromatography with tandem mass spectrometry (LC-MS/MS) assays with samples prepared either by protein precipitation or solid phase extraction. Twenty-four-hour urine 5HIAA samples (n = 33) were analyzed by amperometric LC, and the results were compared. Specificity and sensitivity were calculated by receiver operating characteristic (ROC) analysis. RESULTS: We achieved 5 to10 000 nmol/L linearity and ≤2.5% variation with our new serum 5HIAA assay. In ROC analysis, the area under curve was 85% by serum assays [upper reference limit (URL) value 123 nmol/L] and 88% by the 24-h urine 5HIAA assay (URL value of 47.1 µmol), respectively. A difference (P < 0.001) between patients with active NEN and patients in remission was found by all 5HIAA assays. CONCLUSION: Serum 5HIAA by LC-MS/MS after protein precipitation performs equally well for the diagnosis of NEN as urinary 5HIAA LC assay. The outcome and sensitivity for serum and 24-h urine assays are convergent. Due to much more reliable and convenient sampling, we recommend serum instead of 24-h urine 5HIAA for diagnosis and follow-up of NEN patients.

Mitochondrial Coenzyme Q10 Determination Via Isotope Dilution Liquid Chromatography -Tandem Mass Spectrometry.

Coenzyme Q10 (CoQ10) is an essential part of the mitochondrial respiratory chain . Here, we describe an accurate and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of mitochondrial CoQ10 in isolated mitochondria . In the assay, mitochondrial suspensions are spiked with CoQ10-[2H9] internal standard (IS), extracted with organic solvents and CoQ10 quantified by LC-MS/MS using multiple reaction monitoring (MRM).

Orexin-A measurement in narcolepsy: A stability study and a comparison of LC-MS/MS and immunoassays.

BACKGROUND: Orexin-A and -B are neuropeptides involved in sleep-wake regulation. In human narcolepsy type 1, this cycle is disrupted due to loss of orexin-producing neurons in the hypothalamus. Cerebrospinal fluid (CSF) orexin-A measurement is used in the diagnosis of narcolepsy type 1. Currently available immunoassays may lack specificity for accurate orexin quantification. We developed and validated a liquid chromatography mass spectrometry assay (LC-MS/MS) for CSF orexin-A and B. METHODS: We used CSF samples from narcolepsy type 1 (n = 22) and type 2 (n = 6) and non-narcoleptic controls (n = 44). Stable isotope-labeled orexin-A and -B internal standards were added to samples before solid-phase extraction and quantification by LC-MS/MS. The samples were also assayed by commercial radioimmunoassay (RIA, n = 42) and enzymatic immunoassay (EIA, n = 72) kits. Stability of orexins in CSF was studied for 12 months. RESULTS: Our assay has a good sensitivity (10 pmol/L = 35 pg/mL) and a wide linear range (35-3500 pg/mL). Added orexin-A and -B were stable in CSF for 12 and 3 months, respectively, when frozen. The median orexin-A concentration in CSF from narcolepsy type 1 patients was <35 pg/mL (range < 35-131 pg/mL), which was lower than that in CSF from control individuals (98 pg/mL, range < 35-424 pg/mL). Orexin-A concentrations determined using our LC-MS/MS assay were five times lower than those measured with a commercial RIA. Orexin-B concentrations were undetectable. CONCLUSIONS: Orexin-A concentrations measured by our LC-MS/MS assay were lower in narcolepsy type 1 patients as compared to controls. RIA yielded on average higher concentrations than LC-MS/MS.

Optimizing hepcidin measurement with a proficiency test framework and standardization improvement.

Objectives: Hepcidin measurement advances insights in pathophysiology, diagnosis, and treatment of iron disorders, but requires analytically sound and standardized measurement procedures (MPs). Recent development of a two-level secondary reference material (sRM) for hepcidin assays allows worldwide standardization. However, no proficiency testing (PT) schemes to ensure external quality assurance (EQA) exist and the absence of a high calibrator in the sRM set precludes optimal standardization. Methods: We developed a pilot PT together with the Dutch EQA organization Stichting Kwaliteitsbewaking Medische Laboratoriumdiagnostiek (SKML) that included 16 international hepcidin MPs. The design included 12 human serum samples that allowed us to evaluate accuracy, linearity, precision and standardization potential. We manufactured, value-assigned, and validated a high-level calibrator in a similar manner to the existing low- and middle-level sRM. Results: The pilot PT confirmed logistical feasibility of an annual scheme. Most MPs demonstrated linearity (R2>0.99) and precision (duplicate CV>12.2%), although the need for EQA was shown by large variability in accuracy. The high-level calibrator proved effective, reducing the inter-assay CV from 42.0% (unstandardized) to 14.0%, compared to 17.6% with the two-leveled set. The calibrator passed international homogeneity criteria and was assigned a value of 9.07±0.24 nmol/L. Conclusions: We established a framework for future PT to enable laboratory accreditation, which is essential to ensure quality of hepcidin measurement and its use in patient care. Additionally, we showed optimized standardization is possible by extending the current sRM with a third high calibrator, although international implementation of the sRM is a prerequisite for its success.

Comparison of LC-MS/MS and chemiluminescent immunoassays for immunosuppressive drugs reveals organ dependent variation in blood cyclosporine a concentrations.

INTRODUCTION: Life-long monitoring of immunosuppressive drugs (ISDs) in blood is essential after organ transplantation. However, the ISD concentrations vary depending on the assay employed. ISDs are strongly bound to cytoplasmic proteins in erythrocytes in circulation. Therefore, the relatively rapid sedimentation of blood cells in whole blood samples may affect the results when using liquid handling robots. METHODS: We used 1115 blood samples from outpatients and ward patients with kidney (n = 373), liver (n = 101), heart (n = 29) and bone marrow (n = 155) transplant. Whole blood samples were pretreated by protein precipitation. Alternatively, the samples were hemolyzed by freezing prior to precipitation. ISDs were analyzed by a 2-plexing liquid chromatography tandem mass spectrometry (LC-MS/MS) assay and commercial chemiluminescent microparticle immunoassays (CMIA). RESULTS: The difference between the two sample preparation practices was negligible (<2%). Overall, the measured ISD concentrations in patient samples were lower by LC-MS/MS than by CMIA. The difference was the largest (20.2%) and the smallest (9.1%) in samples from liver and from heart transplant patients, respectively. CONCLUSIONS: CMIA overestimates blood ISD concentrations as compared to LC-MS/MS. The extent of the difference was found to be organ transplant dependent. The ISDs can be quantitatedeither from intact or hemolyzed blood samples.

Plasma bradykinin concentrations during septic shock determined by a novel LC-MS/MS assay.

BACKGROUND: Bradykinin is an important mediator of inflammation and vascular permeability and could have an important role in the development of septic shock. Measurement of bradykinin by immunological methods may suffer from interference and lack of specificity. We developed and validated a liquid chromatography mass spectrometry assay (LC-MS/MS) for plasma bradykinin. METHODS: We used plasma samples from healthy volunteers (n = 19) and patients with septic shock (n = 47). Stable isotope bradykinin internal standard was added to samples before solid-phase extraction and quantification by LC-MS/MS. Stability of bradykinin was studied for 12 months. RESULTS: Our assay has good sensitivity (0.1 nmol/l) and a wide linear range (0.1-1000 nmol/l). Bradykinin added to plasma was stable for 12 months at -20 °C when a mixture of protease inhibitors was added at sampling but degraded during repeated freezing and thawing. Bradykinin concentration in plasma from septic shock patients (<0.1-0.6 nmol/l) did not change significantly during shock and recovery but differed slightly from that in healthy individuals (0.5-1.1 nmol/l). CONCLUSIONS: Our bradykinin assay was successfully used to determine bradykinin concentrations in plasma samples. Intensive care unit patients with septic shock had low concentrations of plasma bradykinin during both shock and recovery phases.

Serum Serine Peptidase Inhibitor Kazal-Type 1, Trypsinogens 1 to 3, and Complex of Trypsin 2 and α1-Antitrypsin in the Diagnosis of Severe Acute Pancreatitis.

OBJECTIVES: We explored prediction of severe acute pancreatitis (AP) and development of organ dysfunction (OD). METHODS: Serum concentrations of serine peptidase inhibitor Kazal type 1 (SPINK1), trypsinogen 1, trypsinogen 2, and trypsinogen 3, complex between trypsin 2 and α1-antitrypsin, serum C-reactive protein, creatinine, and pancreatic amylase were measured in 239 AP patients with disease onset within 72 hours. RESULTS: SPINK1 distinguished most accurately patients who later developed severe AP. The area under the receiver operating characteristic curve for SPINK1 was 0.742, followed by trypsinogen 2 (0.726), complex between trypsin 2 and α1-antitrypsin (0.657), creatinine (0.656), trypsinogen 1 (0.652), trypsinogen 3 (0.557), and C-reactive protein (0.499). With a cutoff of 166 µg/L, SPINK1 had a specificity of 93%, a sensitivity of 48%, and diagnostic odds ratio of 11.52. In multivariate logistic regression analysis, only SPINK1 was an independent predictor of severe AP among patients presenting without OD on admission (P < 0.001). CONCLUSIONS: Plasma levels of the biomarkers and creatinine correlated with the severity of AP and development of OD. In patients presenting without OD at admission, SPINK1 was an independent marker for later development of severe AP.

Provisional standardization of hepcidin assays: creating a traceability chain with a primary reference material, candidate reference method and a commutable secondary reference material.

Background Hepcidin concentrations measured by various methods differ considerably, complicating interpretation. Here, a previously identified plasma-based candidate secondary reference material (csRM) was modified into a serum-based two-leveled sRM. We validated its functionality to increase the equivalence between methods for international standardization. Methods We applied technical procedures developed by the International Consortium for Harmonization of Clinical Laboratory Results. The sRM, consisting of lyophilized serum with cryolyoprotectant, appeared commutable among nine different measurement procedures using 16 native human serum samples in a first round robin (RR1). Harmonization potential of the sRM was simulated in RR1 and evaluated in practice in RR2 among 11 measurement procedures using three native human plasma samples. Comprehensive purity analysis of a candidate primary RM (cpRM) was performed by state of the art procedures. The sRM was value assigned with an isotope dilution mass spectrometry-based candidate reference method calibrated using the certified pRM. Results The inter-assay CV without harmonization was 42.1% and 52.8% in RR1 and RR2, respectively. In RR1, simulation of harmonization with sRM resulted in an inter-assay CV of 11.0%, whereas in RR2 calibration with the material resulted in an inter-assay CV of 19.1%. Both the sRM and pRM passed international homogeneity criteria and showed long-term stability. We assigned values to the low (0.95±0.11 nmol/L) and middle concentration (3.75±0.17 nmol/L) calibrators of the sRM. Conclusions Standardization of hepcidin is possible with our sRM, which value is assigned by a pRM. We propose the implementation of this material as an international calibrator for hepcidin.

Comparison of serum serotonin and serum 5-HIAA LC-MS/MS assays in the diagnosis of serotonin producing neuroendocrine neoplasms: A pilot study.

BACKGROUND: Serotonin (5-hydroxytyramine) is a mediator of gastrointestinal smooth muscle contraction, and is secreted by neuroendocrine neoplasms (NENs). We developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for serum serotonin to be used in NEN diagnostics and follow-up. METHODS: We used serum samples from healthy volunteers (n = 31) and patients suspected or monitored for NEN (n = 98). Serotonin-D4 internal standard was added to samples before solid phase extraction (SPE) and quantification by LC-MS/MS. The effects of sample handling and preparation on serotonin stability were studied. Finally, we established a provisional reference range for serum serotonin and compared our assay with serum 5-hydroxyindoleacetic acid (5-HIAA) for detection of NENs. RESULTS: Our assay is sensitive and has a wide linear range (10-10,000 nmol/l). Serum serotonin is stable for 7 days at room temperature and for 3 months at -20 °C. Sampling temperature is not critical. Normal range for serum serotonin was 270-1490 nmol/l. We found that serum serotonin and 5-HIAA performed equally well as diagnostic tests for NENs. CONCLUSIONS: Our LC-MS/MS assay for serum serotonin is well suited for clinical research and patient diagnostics. Our results confirm that it can complement 5-HIAA in diagnosis of NENs.

An immunocapture-LC-MS-based assay for serum SPINK1 allows simultaneous quantification and detection of SPINK1 variants.

Pancreatic secretory trypsin inhibitor Kazal type 1 (SPINK1) is a 6420 Da peptide produced by the pancreas, but also by several other tissues and many tumors. Some mutations of the SPINK1 gene, like the one causing amino acid change N34S, have been shown to confer susceptibility to recurrent or chronic pancreatitis. Detection of such variants are therefore of clinical utility. So far SPINK1 variants have been determined by DNA techniques. We have developed and validated an immunocapture-liquid chromatography-mass spectrometric (IC-LC-MS) assay for the detection and quantification of serum SPINK1, N34S-SPINK1, and P55S-SPINK1. We compared this method with a time-resolved immunofluorometric assay (TR-IFMA) for serum samples and primer extension analysis of DNA samples. We used serum and DNA samples from patients with acute pancreatitis, renal cell carcinoma, or benign urological conditions. With the help of a zygosity score calculated from the respective peak areas using the formula wild-type (wt) SPINK1/(variant SPINK1 + wt SPINK1), we were able to correctly characterize the heterozygotes and homozygotes from the samples with DNA information. The score was then used to characterize the apparent zygosity of the samples with no DNA characterization. The IC-LC-MS method for SPINK1 was linear over the concentration range 0.5-1000 µg/L. The limit of quantitation (LOQ) was 0.5 µg/L. The IC-LC-MS and the TR-IFMA assays showed good correlation. The median zygosity score was 1.00 (95% CI 0.98-1.01, n = 11), 0.55 (95% CI 0.43-0.61, n = 14), and 0.05 (range 0.04-0.07, n = 3) for individuals found to be wt, heterozygous, and homozygous, respectively, for the N34S-SPINK1 variant by DNA analysis. When DNA samples are not available, this assay facilitates identification of the N34S- and P55S-SPINK1 variants also in archival serum samples.

Toward Worldwide Hepcidin Assay Harmonization: Identification of a Commutable Secondary Reference Material.

BACKGROUND: Absolute plasma hepcidin concentrations measured by various procedures differ substantially, complicating interpretation of results and rendering reference intervals method dependent. We investigated the degree of equivalence achievable by harmonization and the identification of a commutable secondary reference material to accomplish this goal. METHODS: We applied technical procedures to achieve harmonization developed by the Consortium for Harmonization of Clinical Laboratory Results. Eleven plasma hepcidin measurement procedures (5 mass spectrometry based and 6 immunochemical based) quantified native individual plasma samples (n = 32) and native plasma pools (n = 8) to assess analytical performance and current and achievable equivalence. In addition, 8 types of candidate reference materials (3 concentrations each, n = 24) were assessed for their suitability, most notably in terms of commutability, to serve as secondary reference material. RESULTS: Absolute hepcidin values and reproducibility (intrameasurement procedure CVs 2.9%-8.7%) differed substantially between measurement procedures, but all were linear and correlated well. The current equivalence (intermeasurement procedure CV 28.6%) between the methods was mainly attributable to differences in calibration and could thus be improved by harmonization with a common calibrator. Linear regression analysis and standardized residuals showed that a candidate reference material consisting of native lyophilized plasma with cryolyoprotectant was commutable for all measurement procedures. Mathematically simulated harmonization with this calibrator resulted in a maximum achievable equivalence of 7.7%. CONCLUSIONS: The secondary reference material identified in this study has the potential to substantially improve equivalence between hepcidin measurement procedures and contributes to the establishment of a traceability chain that will ultimately allow standardization of hepcidin measurement results.

Emerging Roles of SPINK1 in Cancer.

BACKGROUND: Tumor-associated trypsin inhibitor (TATI) was originally isolated from the urine of a patient with ovarian cancer. It was later shown to be produced by many other tumors and several normal tissues. It had earlier been isolated from the pancreas and was hence called pancreatic secretory trypsin inhibitor (PSTI). It belongs to a family of protease inhibitors presently called serine peptidase inhibitor Kazal type (SPINK). In the SPINK family TATI/PSTI is SPINK1, which is the name used in this review. CONTENT: In addition to being a protease inhibitor, SPINK1 also acts as an acute-phase reactant and a growth factor. Furthermore, it has been shown to modulate apoptosis. Overexpression of SPINK1 predicts an unfavorable outcome in several cancers and determination of SPINK1 in serum can be used to identify patients at increased risk of aggressive disease. Thus serum SPINK1 can be used as a prognostic tumor marker. Because SPINK1 acts as a growth factor and an inhibitor of apoptosis in some cancers, it has also been suggested that it can be a therapeutic target in cancer. However, because SPINK1 is the major physiological inhibitor of trypsin, inhibition of SPINK1 may increase the risk of pancreatitis. SUMMARY: Taking into account the many functions of SPINK1, assessing the role of SPINK1 in cancer has several potentially important clinical applications ranging from a biomarker to a potential new target for cancer therapy.

Interleukin-6 increases expression of serine protease inhibitor Kazal type 1 through STAT3 in colorectal adenocarcinoma.

Inflammation promotes colorectal cancer (CRC) tumorigenesis, but the underlying molecular mechanisms are still being uncovered. Proinflammatory cytokine interleukin-6 (IL-6) stimulates survival signaling in CRC; inflammatory signals also regulate production and activity of proteases and their inhibitors. Over-expression of serine protease inhibitor Kazal type 1 (SPINK1) predicts an unfavorable outcome in colon cancer. The SPINK1 gene contains an IL-6 responsive element, suggesting it could act as an acute phase reactant. We assessed the connection between IL-6 and SPINK1, and the function and mechanism of this signaling. Our results show that Colo205 and HT-29 cells express and secrete SPINK1, and both fibroblast-derived and recombinant IL-6 further increased the SPINK1 levels. Concurrently CRC cells augmented the IL-6 production in fibroblasts. In CRC tissues cancer cells were positive for SPINK1, whereas IL-6 was found in stromal cells. In Colo205 cells IL-6 also stimulated the secretion of trypsin-1 and -2, the key targets of SPINK1 protease inhibition, whereas in HT-29 cells trypsin-1 and -2 levels remained constantly low. Functionally, both IL-6 and SPINK1 increased the motility of the CRC cells. Mechanistically, IL-6 activated the canonical STAT3 pathway and inhibition of STAT3 phosphorylation decreased the levels of SPINK1, trypsin-1 and -2. Taken together, our results indicate a novel link between inflammatory signals originating from the tumor microenvironment and increased SPINK1 levels. This finding has potential therapeutic implications for targeted therapy, as it confirms that SPINK1 acts as an acute phase reactant and that it participates in the paracrine crosstalk with the tumor microenvironment of colon cancer. © 2015 Wiley Periodicals, Inc.

Preanalytical validation and reference values for a mass spectrometric assay of serum vanillylmandelic acid for screening of catecholamine secreting neuroendocrine tumors.

BACKGROUND: Urinary vanillylmandelic acid (VMA) is used to diagnose and monitor catecholamine secreting neuroendocrine tumors (NETs). We developed and validated a new liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for determination of serum VMA. METHODS: We used serum samples from healthy volunteers (n=314) and patients suspected for NET (n=36). Deuterated VMA as an internal standard was added to samples before solid phase extraction (SPE) and LC-MS/MS analysis. We studied the effects of sample storage, sampling device and a meal on serum VMA and metanephrine concentrations. Diurnal variation and age-dependent reference intervals were established. The diagnostic performance was compared with a urinary HPLC assay for VMA and metanephrines and a serum metanephrine LC-MS/MS assay. RESULTS: Serum VMA is stable at least for one day at +4°C, seven days at room temperature and 98 days at -20°C. Type of sampling device was not critical, but elevated serum VMA occurs after a meal (p = 0.031). Serum VMA increased with age. Therefore, we suggest clinical cut-off values of 62 nmol/L, 80 nmol/L and 108 nmol/L for age groups 18-50 yrs, 51-70 yrs and > 70 yrs, respectively. Comparison between a urinary VMA HPLC assay and serum VMA LC-MS/MS assay showed good correlation. CONCLUSIONS: Our LC-MS/MS assay is fast and sensitive and suits well for use in a clinical laboratory. Compared to 24-h urine collection our serum assay enables well controlled sampling and convenient preanalytical steps.