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Background The measurement of hemoglobin A1c (HbA1c) is important for diagnosing diabetes mellitus as well as assessing glycemic control in diabetic patients. Commutable whole blood certified reference materials (CRMs) are needed in the measurement of HbA1c for method validation and/or as quality controls. Methods We developed three levels of hemolyzed whole blood CRMs for HbA1c. The certified values were determined using liquid chromatography-isotope dilution tandem mass spectrometry method (LC-IDMS/MS) where two “signature” hexapeptides of HbA1c and hemoglobin A0 (HbA0) were used as the calibration standards. The concentrations of the hexapeptide solutions were determined by amino acid analysis by the LC-IDMS/MS method using amino acid CRMs as the calibration standards. The commutability study was conducted by measuring 25 patient specimens and the whole blood CRMs by both LC-IDMS/MS method and various routine methods using six different clinical analyzers. Results The certified values were determined to be 35.1±2.0, 50.3±1.9 and 65.8±2.6 mmol/mol, respectively. These CRMs showed good commutability on five of the six clinical analyzers but showed poor commutability on one of the clinical analyzers that used similar method as two other analyzers where good commutability was observed. Conclusions With certified target values based on metrological traceability and good commutability on most of the clinical analyzers, the developed whole blood CRMs can be used for method validation or as quality control materials in the measurement of HbA1c. The commutability study results also underscored the need of commutability testing of clinical CRMs using various clinical analyzers.

Reference measurement procedures are essential for the accurate measurement of urea in human serum. The widely used gas chromatography-isotope dilution mass spectrometry (GC–IDMS) involves tedious derivatization processes. We developed and validated a simple liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS) method for the measurement of urea in human serum. In this method, 13C,15N2-urea was spiked into serum samples as the internal standard, and pure urea certified reference material (CRM) was used as the calibration standard to ensure the traceability of the measurements to the International System of Units (SI). The sample cleanup procedure involved a simple protein precipitation step, followed by centrifugation and filtration. The developed LC-IDMS/MS method was found to be accurate and highly reproducible. The coefficient of variation (CV) was generally below 1%. Good accuracy was demonstrated by measuring serum CRMs from two National Metrology Institutes (NMIs) with relative deviations from the certified values being close to or below 1%, and measurement uncertainties of 2.0%–2.8%. The method was used to assign the reference values of serum urea in ten cycles of an external quality assessment (EQA) program for clinical laboratories over a period of four years. The robust mean of the reported results from clinical laboratories using automated analyzers were close to the assigned values from LC-IDMS/MS method. With good accuracy and precision, as well as simple sample cleanup procedure, the developed LC-IDMS/MS method can be used as a reference measurement procedure for the measurement of urea in human serum.

The development of reference measurement methods for hemoglobin A1c (HbA1c) is important for quality assurance in diabetes management. The IFCC reference method using purified proteins as calibration standards is the recommended accuracy-based reference method for the standardization of HbA1c measurement. We developed a highly precise and accurate liquid chromatography-isotope-dilution tandem mass spectrometry (LC-IDMS/MS) procedure, which can serve as an alternative accuracy-based method for HbA1c measurement. In this method, enzymatic proteolysis was applied to sample preparation, followed by LC-IDMS/MS measurement of hemoglobin A0 (HbA0) and HbA1c, using two "signature" hexapeptides for calibration. The concentrations of the signature hexapeptide calibration solutions were, in turn, determined using a hydrolysis method with HCl, followed by LC-IDMS/MS measurement using amino acid solutions as calibration standards. These solutions were gravimetrically prepared from pure amino acid certified reference materials (CRMs). The developed LC-IDMS/MS method was used in participation in an IFCC ring trial for reference laboratories (RELA 2013 and 2014) for HbA1c, where our results were compared with those using the IFCC reference method. The deviations were found to be 0.4-1.7 mmol mol(-1) [or 0.04-0.16% in National Glygohemoglobin Standardization Program (NGSP) units], revealing good comparability with the IFCC reference method. The relative expanded uncertainty of the LC-IDMS/MS was in the range of 2.6% to 2.8% (1.6% to 2.2% after converting to NGSP units). With excellent method precision, good comparability with the IFCC reference method, and a small measurement uncertainty, the developed LC-IDMS/MS method may be used as an alternative accuracy-based reference method for HbA1c measurement.

Required metrological tools, such as higher order reference measurements procedures, pure substance and matrix certified reference materials, are established for small well defined molecules. Difficulties still remain in the provision of SI traceable standards in the area of larger biomolecules such as peptides/proteins. The provision of Primary Calibration Reference Services has been identified as a core technical competency for National Measurement Institutes (NMIs). A concept has been elaborated by the Focus Group I on peptide/protein purity for the strategic planning of ongoing Key Comparisons of the Protein Analysis Working Group (PAWG) within the Comité Consultatif pour la Quantité de Matière (CCQM). The assignment of the mass fraction content of high purity peptide/protein materials will be the subject of the CCQM-K115/P55.2 comparison series to directly support NMI services and certified reference materials currently being provided by NMIs.

Triple-negative breast cancer (TNBC) is characterized by unique aggressive behavior and lack of targeted therapies. Among the various molecular subtypes of breast cancer, it was observed that TNBCs express elevated levels of sphingosine kinase 1 (SPHK1) compared to other breast tumor subtypes. High levels of SPHK1 gene expression correlated with poor overall and progression- free survival, as well as poor response to Doxorubicin-based treatment. Inhibition of SPHK1 was found to attenuate ERK1/2 and AKT signaling and reduce growth of TNBC cells in vitro and in a xenograft SCID mouse model. Moreover, SPHK1 inhibition by siRNA knockdown or treatment with SKI-5C sensitizes TNBCs to chemotherapeutic drugs. Our findings suggest that SPHK1 inhibition, which effectively counteracts oncogenic signaling through ERK1/2 and AKT pathways, is a potentially important anti-tumor strategy in TNBC. A combination of SPHK1 inhibitors with chemotherapeutic agents may be effective against this aggressive subtype of breast cancer.

The CCQM Organic Analysis Working Group (OAWG) agreed on a Track A comparison for the measurement of polar organic in biological matrix as part of its 10-year strategic plan. As a model for this comparison, two polar clinical biomarkers: urea and uric acid, in human serum were chosen. This comparison was designed to enable participating National Metrology Institutes (NMIs) or Designated Institutes (DIs) to demonstrate their measurement capabilities in the determination of analytes with molecular mass of 50 to 500 g/mol, having the polarity pKOW > 2 in the range of 10 to 2,000 mg/kg in a biological matrix such as human serum, blood and urine. Two pools of human serum materials with different concentration levels of urea and uric acid were used as the comparison materials. Fifteen NMIs/DIs participated in this Track A comparison. All of them submitted the results for urea and 14 NMIs/DIs submitted the results for uric acid. With the exception of one NMI, all participating institutes employed isotope dilution mass spectrometry (IDMS) for the measurement of both urea and uric acid. Protein precipitation, liquid-liquid extraction and/or clean-up were applied, followed by instrumental analyses using GC-HRMS, GC-MS/MS, GC-MS, LC-HRMS, LC-MS/MS, LC-MS or HPLC-DAD. The Laplacian weighted medians were used as the Key Comparison Reference Values (KCRVs). The assigned KCRVs were the weighted medians of 13 results for urea (both serum pools), 10 results for uric acid (Serum I) and 11 results for uric acid (Serum II). Urea (Serum I) was assigned a KCRV of 1,486.0 mg/kg with a standard uncertainty of 9.0 mg/kg, urea (Serum II) was assigned a KCRV of 334.7 mg/kg with a standard uncertainty of 1.8 mg/kg, uric acid (Serum I) was assigned a KCRV of 136.50 mg/kg with a standard uncertainty of 0.98 mg/kg, and uric acid (Serum II) was assigned a KCRV of 39.39 mg/kg with a standard uncertainty of 0.11 mg/kg. The degree of equivalence (with the KCRV) and its uncertainty were calculated for each result. The majority of the participating institutes in CCQM-K109 demonstrated their capabilities in the measurement of the clinical markers in the biological matrix using IDMS. KEY WORDS FOR SEARCH Clinical biomarkers; urea; uric acid; human serum, IDMS Main text To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

The CCQM OAWG 2017 CCQM-K142 "Comparison of CRMs and Value-Assigned Quality Controls: Urea and Uric Acid in Human Serum or Plasma" is the third Model 2 Key Comparison coordinated by the OAWG directly testing the chemical measurement services provided to customers by National Metrology Institutes (NMIs) and Designated Institutes (DIs) through certified reference materials (CRMs). CRMs certified for urea and/or uric acid content in human serum or plasma were compared using measurements made on these materials under repeatability conditions. Four NMIs/DIs submitted 10 CRMs certified for urea; five NMIs/DIs submitted 12 CRMs certified for uric acid. These materials represent most of the higher-order reference materials available then for these clinically important measurands. Uncertainty-weighted generalised distance regression was used to establish the Key Comparison Reference Function (KCRF) relating the CRM certified values to the repeatability measurements. The urea results for all 10 CRMs were considered to be equivalent at the 95 % level of confidence and were used to define the KCRF for urea. The uric acid result for one of the 12 CRMs was found to be non-equivalent: the submitting NMI re-evaluated the result and withdrew the material from use in defining the KCRF for uric acid. The remaining 11 CRMs were used to define the KCRF for uric acid. Monte Carlo methods were used to estimate 95 % level-of-confidence coverage intervals for the relative degrees of equivalence of materials, %d ± U95 (%d), and of the participating NMIs/DIs, %D ± U95 (%D). For the urea materials, the %D ± U95(%D) intervals were within (-3 to 5) % of the consensus results. For the uric acid materials from four of the five NMIs/DIs, the %D ± U95(%D) intervals were within (-4 to 5) % of the consensus results. These results demonstrated that with the exception of one material, the participating institutions could value-assign CRMs for urea and/or uric acid in human serum and plasma. KEY WORDS FOR SEARCH Certified reference materials; urea; uric acid; human serum; key comparison reference function Main text To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

To achieve fast and accurate analysis of carbamazepine in surface water, we developed a novel porous membrane-protected micro-solid-phase extraction (μ-SPE) method, followed by liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS) analysis. The μ-SPE device (∼0.8 × 1 cm) was fabricated by heat-sealing edges of a polypropylene membrane sheet to devise a bag enclosing the sorbent. The analytes (both carbamazepine and isotope-labelled carbamazepine) were first extracted by μ-SPE device in the sample (10 mL) via agitation, then desorbed in an organic solvent (1 mL) via ultrasonication. Several parameters such as organic solvent for pre-conditioning of μ-SPE device, amount of sorbent, adsorption time, and desorption solvent and time were investigated to optimize the μ-SPE efficiency. The optimized method has limits of detection and quantitation estimated to be 0.5 ng L −1 and 1.6 ng L −1 , respectively. Surface water samples spiked with different amounts of carbamazepine (close to 20, 500, and 1600 ng L −1 , respectively) were analysed for the validation of method precision and accuracy. Good precision was obtained as demonstrated by relative standard deviations of 0.7% for the samples with concentrations of 500 and 1600 ng kg −1 , and 5.8% for the sample with concentration of 20 ng kg −1 . Good accuracy was also demonstrated by the relative recoveries in the range of 96.7%–103.5% for all samples with uncertainties of 1.1%–5.4%. Owing to the same chemical properties of carbamazepine and isotope-labelled carbamazepine, the isotope ratio in the μ-SPE procedure was accurately controlled. The use of μ-SPE coupled with IDMS analysis significantly facilitated the fast and accurate measurement of carbamazepine in surface water.

A transition metal carbonyl species, Cr(CO)(4)dpp, has been successfully attached to bare silver nanoparticles prepared by laser ablation of a metal foil in ethanol. Transmission electron microscopy (TEM) images have shown that at least a portion of the silver nanoparticles have been capped by the chromium species, and ligand shells corresponding to Cr(CO)(4)dpp multilayer adsorption onto the silver nanoparticles of 30-50 nm diameter have been observed. The detection of the strongest Raman-active nu(CO) band of Cr(CO)(4)dpp at 2004 cm(-1) revealed that the species has been adsorbed without decomposition. The time-of-flight secondary ion mass spectrometry (TOF-SIMS) signals recorded of the chromium-capped silver nanoparticles were also consistent with the nondecomposition adsorption process. Density functional calculations have been used to reproduce the Raman spectrum using Ag(7)(+) as a model surface. A large binding energy of about 122 kJ/mol has also been computed between silver and nitrogen atoms thus lending support to Cr(CO)(4)dpp being chemisorbed onto the silver surface.

Under the auspices of the Protein Analysis Working Group (PAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) a key comparison, CCQM-K115, was coordinated by the Bureau International des Poids et Mesures (BIPM) and the Chinese National Institute of Metrology (NIM). Eight Metrology Institutes or Designated Institutes and the BIPM participated. Participants were required to assign the mass fraction of human C-peptide (hCP) present as the main component in the comparison sample for CCQM-K115. The comparison samples were prepared from synthetic human hCP purchased from a commercial supplier and used as provided without further treatment or purification. hCP was selected to be representative of the performance of a laboratory's measurement capability for the purity assignment of short (up to 5 kDa), non-cross-linked synthetic peptides/proteins. It was anticipated to provide an analytical measurement challenge representative for the value-assignment of compounds of broadly similar structural characteristics. The majority of participants used a peptide impurity corrected amino acid analysis (PICAA) approach as the amount of material that has been provided to each participant (25 mg) is insufficient to perform a full mass balance based characterization of the material by a participating laboratory. The coordinators, both the BIPM and the NIM, were the laboratories to use the mass balance approach as they had more material available. It was decided to propose KCRVs for both the hCP mass fraction and the mass fraction of the peptide related impurities as indispensable contributor regardless of the use of PICAA, mass balance or any other approach to determine the hCP purity. This allowed participants to demonstrate the efficacy of their implementation of the approaches used to determine the hCP mass fraction. In particular it allows participants to demonstrate the efficacy of their implementation of peptide related impurity identification and quantification. More detailed studies on the identification/quantification of peptide related impurities and the hydrolysis efficiency revealed that the integrity of the impurity profile of the related peptide impurities obtained by the participant is crucial for the impact on accuracy of the hCP mass fraction assignment. The assessment of the mass fraction of peptide impurities is based on the assumption that only the most exhaustive and elaborate set of results is taken for the calculation of the KCRVPepImp. The KCRVPepImp for the peptide related impurity mass fractions of the material was 83.3 mg/g with a combined standard uncertainty of 1.5 mg/g. Inspection of the degree of equivalence plots for the mass fraction of peptide impurities and additional information obtained from the peptide related impurity profile indicates that in many cases only a very small number of impurities have been identified and quantified resulting in an underestimation of the peptide related impurity mass fractions. The approach to obtain a KCRVhCP for the mass fraction of hCP is based on a mass balance calculation that takes into account the most exhaustive and elaborate set of results for the peptide related impurities KCRVPepImp, the TFA mass fraction value, water and other minor counter ions obtained by the coordinating laboratories. Differences in the quality of the results obtained for both peptides related impurity mass fractions and hCP mass fractions are better weighted and reflected in smaller uncertainties. The KCRVhCP for CCQM-K115 is 801.8 mg/g with a corresponding combined standard uncertainty of 3.1 mg/g. In general, mass balance approaches show smaller uncertainties than PICAA approaches and the majority of results obtained by the PICAA approach are in agreement because of larger corresponding uncertainties. Main text To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

To the Editor: We have developed an improved reference measurement method for hemoglobin A1c (Hb A1c)1 based on liquid chromatography–isotope dilution–tandem mass spectrometry (LC-ID-MS/MS) with traceability to the SI unit. The method has a small measurement uncertainty (MU) and gives results in good agreement with the accuracy-based IFCC reference method. Kaiser et al. (1) demonstrated the possibility of using ID-MS for Hb A1c measurement, which involved the proteolysis of hemoglobins using endoproteinase Glu-C and hydrolysis of hexapeptide calibration standards using HCl. However, a large MU and significant difference in results compared with the IFCC reference method were observed. Using the same approach, we developed an improved procedure based on LC-ID-MS/MS and used the method for participation in an IFCC ring trial for reference laboratories (RELA 2013) for Hb A1c, in which our results for 2 lyophilized samples were compared against those from the IFCC reference method, including 6 approved IFCC network laboratories for Hb A1c. Key steps in our ID-MS procedure to preserve the unbroken traceability to …

Sepsis ProtectionSepsis is a serious medical condition characterized by an uncontrolled inflammatory response to infection. Sepsis often results in organ failure and/or death, and current treatments are not very effective.Puneetet al.(p.1290) now show that the enzyme sphingosine kinase 1 (SphK1) may represent an important therapeutic target for the treatment of sepsis. SphK1 expression increased on human phagocytes in response to bacterial products and was also highly expressed on phagocytes from septic patients. Inhibition of SphK1 reduced the production of inflammatory mediators in vitro by human phagocytes stimulated with bacterial products. In vivo, pretreatment with small interfering RNA against SphK1 or a specific SphK1 inhibitor protected mice from death in two lethal models of sepsis. Protection was also seen when mice were treated with the SphK1 inhibitor up to 8 hours after sepsis induction, and this protection was enhanced if mice were given a broad-spectrum antibiotic.

Sphingolipid-metabolizing enzymes control the critical balance of the cellular levels of sphingolipids, including the apoptotic inducing ceramide (Cer) and the proliferative inducing sphingosine 1-phosphate (S1P). The production of S1P, catalyzed by the action of sphingosine kinases (SPHKs), is known to be critical for many cellular processes. However, it is suggested that SPHK, and/or its catalytic product S1P, plays critical roles in various diseases including autoimmune diseases, cancer, and allergies. However, there is a great limitation of specific pharmacological inhibitors for SPHKs. In this paper, we describe a novel and stereoselective method of synthesizing SPHKs inhibitors. We generated a number of novel compounds and identified a number of specific inhibitors of human SPHKs. These compounds demonstrated inhibition of SPHKs at micromolar concentrations, making them more potent than dimethylsphingosine (DMS), a well-known inhibitor of SPHKs. In particular, one of the inhibitors was found to be selective toward a particular isoform of SPHK.

Objectives: Sphingosine kinase 1 (SphK1) phosphorylates the membrane sphingolipid, sphingosine, to sphingosine-1-phosphate (S1P), an oncogenic mediator, which drives tumor cell growth and survival. Although SphK1 has gained increasing prominence as an oncogenic determinant in several cancers, its potential as a therapeutic target in colon cancer remains uncertain. We investigated the clinical relevance of SphK1 expression in colon cancer as well as its inhibitory effects in vitro. Methods: SphK1 expression in human colon tumor tissues was determined by immunohistochemistry and its clinicopathological significance was ascertained in 303 colon cancer cases. The effects of SphK1 inhibition on colon cancer cell viability and the phosphoinositide 3-kinase (PI3K)/Akt cell survival pathway were investigated using a SphK1-selective inhibitor—compound 5c (5c). The cytotoxicity of a novel combination using SphK1 inhibition with the chemotherapeutic drug, 5-fluorouracil (5-FU), was also determined. Results: High SphK1 expression correlated with advanced tumor stages (AJCC classification). Using a competing risk analysis model to take into account disease recurrence, we found that SphK1 is a significant independent predictor for mortality in colon cancer patients. In vitro, the inhibition of SphK1 induced cell death in colon cancer cell lines and attenuated the serum-dependent PI3K/Akt signaling. Inhibition of SphK1 also enhanced the sensitivity of colon cancer cells to 5-FU. Conclusion: Our findings highlight the impact of SphK1 in colon cancer progression and patient survival, and provide evidence supportive of further development in combination strategies that incorporate SphK1 inhibition with current chemotherapeutic agents to improve colon cancer outcomes.