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.

A standard to report biological variation data studies - based on an expert opinion.

There is a need for standards for generation and reporting of Biological Variation (BV) reference data. The absence of standards affects the quality and transportability of BV data, compromising important clinical applications. To address this issue, international expert groups under the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) have developed an online resource (https://tinyurl.com/bvmindmap) in the form of an interactive mind map that serves as a guideline for researchers planning, performing and reporting BV studies. The mind map addresses study design, data analysis, and reporting criteria, providing embedded links to relevant references and resources. It also incorporates a checklist approach, identifying a Minimum Data Set (MDS) to enable the transportability of BV data and incorporates the Biological Variation Data Critical Appraisal Checklist (BIVAC) to assess study quality. The mind map is open to access and is disseminated through the EFLM BV Database website, promoting accessibility and compliance to a reporting standard, thereby providing a tool to be used to ensure data quality, consistency, and comparability of BV data. Thus, comparable to the STARD initiative for diagnostic accuracy studies, the mind map introduces a Standard for Reporting Biological Variation Data Studies (STARBIV), which can enhance the reporting quality of BV studies, foster user confidence, provide better decision support, and be used as a tool for critical appraisal. Ongoing refinement is expected to adapt to emerging methodologies, ensuring a positive trajectory toward improving the validity and applicability of BV data in clinical practice.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Mitochondrial coenzyme Q10 determination by isotope-dilution liquid chromatography-tandem mass spectrometry.

BACKGROUND: Coenzyme Q10 (CoQ10) is an essential part of the mitochondrial respiratory chain. Unlike most other respiratory chain disorders, CoQ10 deficiency is potentially treatable. We aimed to develop and validate an accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of mitochondrial CoQ10 in clinical samples. METHODS: We used mitochondria isolated from muscle biopsies of patients (n = 166) suspected to have oxidative phosphorylation deficiency. We also used fibroblast mitochondria from 1 patient with CoQ10 deficiency and 3 healthy individuals. Samples were spiked with nonphysiologic CoQ10-[(2)H6] internal standard, extracted with 1-propanol and with ethanol and hexane (2 mL/5 mL), and CoQ10 quantified by LC-MS/MS. The method and sample stability were validated. A reference interval was established from the patient data. RESULTS: The method had a limit of quantification of 0.5 nmol/L. The assay range was 0.5-1000 nmol/L and the CVs were 7.5%-8.2%. CoQ10 was stable in concentrated mitochondrial suspensions. In isolated mitochondria, the mean ratio of CoQ10 to citrate synthase (CS) activity (CoQ10/CS) was 1.7 nmol/U (95% CI, 1.6-1.7 nmol/U). We suggest a CoQ10/CS reference interval of 1.1-2.8 nmol/U for both sexes and all ages. The CoQ10/CS ratio was 5-fold decreased in fibroblast mitochondria from a patient with known CoQ10 deficiency due to recessive prenyl (decaprenyl) diphosphate synthase, subunit 2 (PDSS2) mutations. CONCLUSIONS: Normalization of mitochondrial CoQ10 concentration against citrate synthase activity is likely to reflect most accurately the CoQ10 content available for the respiratory chain. Our assay and the established reference range should facilitate the diagnosis of respiratory chain disorders and treatment of patients with CoQ10 deficiency.

Collagen degradation by tumor-associated trypsins.

In normal soft tissues, collagen is degraded primarily by collagenases from the matrix metalloproteinase family. Yet, collagenase-like activity of tumor-associated isoforms of other enzymes might be involved in cancer invasion as well. In the present study, we systematically examined collagen degradation by non-sulfated isoforms of trypsins, which were proposed to possess such an activity. We found that non-sulfated trypsin-1, -2, and -3 were able to cleave non-helical and unfolded regions of collagen chains but not the intact triple helix, similar to sulfated trypsins produced by the pancreas. Trypsin-2 sulfation did not affect the cleavage rate either. An apparent triple helix cleavage by tumor-associated trypsin-2 reported earlier likely occurred after triple helix unfolding during sample denaturation for gel electrophoresis. Nevertheless, tumor-associated trypsins might be important for releasing collagen from fibers through telopeptide cleavage as well as for degrading unfolded collagen chains, e.g. after initial cleavage and destabilization of triple helices by collagenases.

Trypsin-2 enhances carcinoma invasion by processing tight junctions and activating ProMT1-MMP.

Enhanced proteolysis and altered tight junction (TJ) proteins associate with carcinoma invasion. We hypothesized that trypsin-2, a tumor-associated serine proteinase, induces tongue carcinoma invasion by activating pro-membrane type-1 matrix metalloproteinase (MT1-MMP) and disturbing the TJs. The effects of invasion were analyzed using trypsin-2 over-expressing human tongue squamous cell carcinoma cells (Try2-HSC-3) in vitro and in vivo. The invasion of Try2-HSC-3 cells was increased in mouse xenografts and human organotypic model. Trypsin-2 activated proMT1-MMP, as well as altered the expression of TJ protein claudin-7. In conclusion, trypsin-2 over-expression enhanced tongue carcinoma cell invasion by various genetic and proteolytic mechanisms.

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.

Specific immunoassay reveals increased serum trypsinogen 3 in acute pancreatitis.

BACKGROUND: Trypsinogen 3 is a minor trypsinogen isoform in the pancreas. In contrast with trypsin 1 and 2, trypsin 3 degrades pancreatic secretory trypsin inhibitor, which may lead to an excess of active trypsin and acute pancreatitis (AP). We developed an immunoassay for trypsinogen 3 and studied whether an assay of serum trypsinogen 3 is of clinical utility in the diagnosis of AP. METHODS: Monoclonal antibodies were generated using recombinant human trypsinogen 3 as the antigen and used to establish a sandwich-type immunoassay. We analyzed serum trypsinogen 3 concentrations in 82 patients with AP and 63 patients with upper abdominal pain (controls). The reference interval was determined using serum samples from 172 apparently healthy individuals. RESULTS: The measuring range of the trypsinogen 3 assay was 1.0-250 µg/L. Intra- and interassay CVs were <11%, and cross-reactivity with other trypsinogen isoenzymes was <0.1%. The median trypsinogen 3 concentration in serum from healthy individuals was <1.0 µg/L, and the upper reference limit was 4.4 µg/L. We observed increased trypsinogen 3 concentrations in patients with mild (median 9.5 µg/L) and severe (15.0 µg/L) AP; in both groups, the concentrations were significantly higher than in controls (median <1.0 µg/L) (P < 0.0001). In ROC analysis, the area under the curve of trypsinogen 3 for separation between AP and controls was 0.90 (P < 0.0001). CONCLUSIONS: We established for the first time a specific immunoassay for trypsinogen 3 using monoclonal antibodies. Patients with AP were found to have increased serum concentrations of trypsinogen 3. The availability of this assay will be useful for studies of the clinical utility of trypsinogen 3.

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).

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.

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.

Human trypsinogens in the pancreas and in cancer.

This study led to the development of monoclonal antibodies and time-resolved immunofluorometric methods recognizing human trypsinogen-1 and -2, respectively. Using these methods in normal sera the concentration of trypsinogen-1 was found to be higher than that of trypsinogen-2. However, in acute pancreatitis the concentration of serum trypsinogen-2 was 50-fold higher than in controls, whereas the difference in trypsinogen-1 concentration was only 15-fold. Serum samples from patients who had undergone pancreatoduodenectomy contained trypsinogen-2, while trypsinogen-1 was detected in only one of nine samples. Furthermore, in human ovarian cyst fluids tumor-associated trypsinogen-2 (TAT-2) is the predominant isoenzyme and in mucinous cyst fluids the levels of TAT-2 were associated with malignancy. These results suggest that (i) trypsinogen-2 could be used as a diagnostic marker for acute pancreatitis, (ii) its expression is not restricted to the pancreas, and (iii) TAT could be involved in ovarian tumor dissemination and breakage of tissue barriers. In ion exchange chromatography, isoelectric variants of the trypsinogen isoenzymes were seen. Mass spectrometric analysis of these revealed that pancreatic trypsinogens are sulfated at tyrosine 154 (Tyr154), whereas TAT-2 from a colon carcinoma cell line is not. Tyr154 is located within the primary substrate binding pocket of trypsin. Thus, Tyr154 sulfation is likely to influence substrate binding. The previously known differences in charge and substrate binding between pancreatic and tumor-associated trypsinogens are suggested to be caused by sulfation of Tyr154 in pancreatic trypsinogens.