Synthesis of Deuterium-Labeled Vitamin D Metabolites as Internal Standards for LC-MS Analysis.

Blood levels of the vitamin D3 (D3) metabolites 25-hydroxyvitamin D3 (25(OH)D3), 24R,25-dihydroxyvitamin D3, and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) are recognized indicators for the diagnosis of bone metabolism-related diseases, D3 deficiency-related diseases, and hypercalcemia, and are generally measured by liquid-chromatography tandem mass spectrometry (LC-MS/MS) using an isotope dilution method. However, other D3 metabolites, such as 20-hydroxyvitamin D3 and lactone D3, also show interesting biological activities and stable isotope-labeled derivatives are required for LC-MS/MS analysis of their concentrations in serum. Here, we describe a versatile synthesis of deuterium-labeled D3 metabolites using A-ring synthons containing three deuterium atoms. Deuterium-labeled 25(OH)D3 (2), 25(OH)D3-23,26-lactone (6), and 1,25(OH)2D3-23,26-lactone (7) were synthesized, and successfully applied as internal standards for the measurement of these compounds in pooled human serum. This is the first quantification of 1,25(OH)2D3-23,26-lactone (7) in human serum.

A novel caged Cookson-type reagent toward a practical vitamin D derivatization method for mass spectrometric analyses.

RATIONALE: 25-Hydroxylated vitamin D is the best marker for vitamin D (VD). Due to its low ionization efficiency, a Cookson-type reagent, 1,2,4-triazoline-3,5-dione (TAD), is used to improve the detection/quantification of VD metabolites by liquid chromatography/tandem mass spectrometry (LC/MS/MS). However, the high reactivity of TAD makes its solution stability low and inconvenient for practical use. We here describe the development of a novel caged Cookson-type reagent, and we assess its performances in the quantitative and differential detection of four VD metabolites in serum using LC/MS/MS. METHODS: Caged 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) analogues were prepared from 4-(4'-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione. Their stability and reactivity were examined. The optimized caged DAPTAD (14-(4-(dimethylamino)phenyl)-9-phenyl-9,10-dihydro-9,10-[1,2]epitriazoloanthracene-13,15-dione, DAP-PA) was used for LC/MS/MS analyses of VD metabolites. RESULTS: The solution stability of DAP-PA in ethyl acetate dramatically improved compared with that of the non-caged one. We measured the thermal retro-Diels-Alder reaction enabling the release of DAPTAD and found that the derivatization reaction was temperature-dependent. We also determined the detection limit and the lower limit of quantifications for four VD metabolites with DAPTAD derivatization. CONCLUSIONS: DAP-PA was stable enough for mid- to long-term storage in solution. This advantage shall contribute to the detection and quantification of VD in clinical laboratories, and as such to the broader use of clinical mass spectrometry.

An in-house centrifugation and membrane filtration technique for identifying microorganisms from positive blood culture bottles with high identification rates using matrix-assisted laser desorption ionization-Time-of-flight mass spectrometry: A preliminary report.

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is one of the most promising technologies for the identification of microbial pathogens directly from positive blood culture bottles. As blood culture bottle medium contains various nonbacterial proteins, including those derived from blood cells, pretreatment to effectively remove host cells is key for successful proteome-based identification of microorganisms. Although the Sepsityper® kit is the most widely used pretreatment protocol, its performance is not satisfactory, particularly for gram-positive isolates. We developed a new in-house protocol, the centrifugation and membrane filtration technique (CMFT), in which vacuum-filtration is coupled with differential centrifugation. We prospectively evaluated the performance of this novel method compared with that of the Sepsityper®. For gram-negative bacterial isolates, the species-level identification rates obtained with the CMFT and the Sepsityper® were comparable (98.8% vs 92.9%). By contrast, for gram-positive isolates, the performance of the CMFT was significantly better than that of the Sepsityper® (P < 0.05). Using our new protocol, 81 (95.3%) isolates were identified with a score >2.0, and 85 (100%) isolates were identified with a score >1.7, versus 46 (54.1%) and 69 (81.2%), respectively, for the Sepsityper®. These results are preliminary, but considering that this novel protocol provides notably high species-level identification rates for gram-positive isolates, it deserves assessment in a larger-scale study with a variety of platforms for MS-based identification of microorganisms.

Current Status of Proteomic Technologies for Discovering and Identifying Gingival Crevicular Fluid Biomarkers for Periodontal Disease.

Periodontal disease is caused by bacteria in dental biofilms. To eliminate the bacteria, immune system cells release substances that inflame and damage the gums, periodontal ligament, or alveolar bone, leading to swollen bleeding gums, which is a sign of gingivitis. Damage from periodontal disease can cause teeth to loosen also. Studies have demonstrated the proteomic approach to be a promising tool for the discovery and identification of biochemical markers of periodontal diseases. Recently, many studies have applied expression proteomics to identify proteins whose expression levels are altered by disease. As a fluid lying in close proximity to the periodontal tissue, the gingival crevicular fluid (GCF) is the principal target in the search for periodontal disease biomarkers because its protein composition may reflect the disease pathophysiology. Biochemical marker analysis of GCF is effective for objective diagnosis in the early and advanced stages of periodontal disease. Periodontal diseases are also promising targets for proteomics, and several groups, including ours, have applied proteomics in the search for GCF biomarkers of periodontal diseases. This search is of continuing interest in the field of experimental and clinical periodontal disease research. In this article, we summarize the current situation of proteomic technologies to discover and identify GCF biomarkers for periodontal diseases.

Ubiquitination in Periodontal Disease: A Review.

Periodontal disease (periodontitis) is a chronic inflammatory condition initiated by microbial infection that leads to gingival tissue destruction and alveolar bone resorption. The periodontal tissue's response to dental plaque is characterized by the accumulation of polymorphonuclear leukocytes, macrophages, and lymphocytes, all of which release inflammatory mediators and cytokines to orchestrate the immunopathogenesis of periodontal disease. Ubiquitination is achieved by a mechanism that involves a number of factors, including an ubiquitin-activating enzyme, ubiquitin-conjugating enzyme, and ubiquitin-protein ligase. Ubiquitination is a post-translational modification restricted to eukaryotes that are involved in essential host processes. The ubiquitin system has been implicated in the immune response, development, and programmed cell death. Increasing numbers of recent reports have provided evidence that many approaches are delivering promising reports for discovering the relationship between ubiquitination and periodontal disease. The scope of this review was to investigate recent progress in the discovery of ubiquitinated protein in diseased periodontium and to discuss the ubiquitination process in periodontal diseases.

A method for measuring serum levels of melanin-associated indole metabolites using LC-MS/MS and its application to malignant melanoma.

BACKGROUND AND AIMS: With the development of novel therapies for advanced malignant melanoma (MM), biomarkers that can accurately reflect the progression of MM are needed. Serum levels of melanin-related indole metabolites such as 5-hydroxy-6-methoxyindole-2-carboxylic acid (5H6MI2C) and 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MI2C) are potential biomarkers for MM. Here, we describe the development of a mass spectrometry (MS)-based assay to determine serum levels of 5H6MI2C and 6H5MI2C. MATERIALS AND METHODS: We developed a stable isotope dilution-selective reaction monitoring-MS protocol using liquid chromatography tandem mass spectrometry (LC-MS/MS) to measure human serum 5H6MI2C and 6H5MI2C levels. Analytical evaluations of the method were performed and the method was applied to serum samples from MM patients (n = 81). RESULTS: The method established in this study showed high reproducibility and linearity. This novel method also found that serum 6H5MI2C levels were significantly elevated in patients with metastatic MM compared to those with non-metastatic MM. Unfortunately, 5H6MI2C did not show a comparable significant difference. CONCLUSION: We successfully established measurement methods for serum 5H6MI2C and 6H5MI2C levels using LC-MS/MS. Serum 6H5MI2C levels offer a potential marker for MM.

Consumption of nonfat milk results in a less atherogenic lipoprotein profile: a pilot study.

BACKGROUND: An increase in plasma low-density lipoprotein (LDL) is a well-known risk factor in the development of atherosclerosis. Dairy consumption may lower the risk of atherosclerosis; however, studies on the effects of milk on cardiovascular risk factors are still scarce. We were interested in investigating whether the intake of milk improves the atherogenic lipoprotein profile. AIMS: We investigated the effects of consuming whole or nonfat milk on plasma lipoprotein composition in healthy Japanese subjects as a pilot study. METHODS: Normolipidemic subjects consumed 500 ml of whole milk (whole milk group; n=7) or nonfat milk (nonfat milk group; n=7) every day for 2 weeks. RESULTS: The consumption of nonfat milk resulted in a lowering of plasma triglyceride (TG) and phospholipid levels and TG level in high-density lipoprotein (HDL) and increased the plasma apolipoprotein (apo) C-III level. In addition, the TG/cholesterol ratios in HDL and LDL were significantly decreased, and LDL particles became larger. In contrast, the only changes observed following whole milk consumption were increases in the plasma levels of apoC-III and apoE. CONCLUSIONS: These findings suggest that consumption of nonfat milk, but not whole milk, may result in a less atherogenic lipoprotein profile, and that the constituents of nonfat milk may improve lipid metabolism.

Development of free 25-hydroxyvitamin D3 assay method using liquid chromatography-tandem mass spectrometry.

The free hormone hypothesis has triggered controversies regarding the measurement of free vitamin D metabolites, such as free 25-hydroxyvitamin D (25(OH)D), as a suitable indicator for total vitamin D for clinical use. This issue can be addressed by developing a precise and accurate method for free 25(OH)D measurement. In the present study, a novel assay method for free 25(OH)D3 based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. Sample preparation first involved ultrafiltration to remove vitamin D-binding protein-bound and albumin-bound 25(OH)D, followed by extraction with a column, derivatization, evaporation, dissolution, and injection into the LC-MS/MS system. The coefficient of variation of repeatability and reproducibility obtained were 3.8-4.5% and 4.8-5.9%, respectively. Satisfactory linearity (r=0.999) was obtained up to 80 pg/ml. The lower quantification limit was 0.97 pg/ml and the S/N ratio on the peak of 1.0 pg/ml sample was 24.8 (which is more than the acceptable value of 10). The recovery rate was between 84.5 and 92.4% with a negligible matrix effect (94.5-104.9%). Levels of free 25(OH)D3, but not total 25(OH)D3, in the serum of the patients with chronic kidney disease (CKD) and hepatic cirrhosis (HC) were substantially lower than those in healthy subjects. The correlation coefficient between total and free 25(OH)D3 was 0.738 in all samples, while the linear regression equations were different between the patients with CKD and HC. In conclusion, LC-MS/MS assay for free 25(OH)D3 might be useful to evaluate high-throughput methods, including ELISA.

Development of a sensitive liquid chromatography-tandem mass spectrometry method for quantification of human plasma arginine vasopressin.

BACKGROUND AND AIMS: Direct measurement of arginine vasopressin (AVP) via immunoassays is not widely conducted, mainly because of technical constraints. Liquid chromatography-tandem mass spectrometry (LC/MS/MS) has been widely used as the gold standard in clinical chemistry. Here, we aimed to develop an MS-based assay to determine human plasma AVP and compare the results with those obtained using a conventional immunoassay. MATERIALS AND METHODS: We developed a protocol using triple quadrupole MS coupled with LC for the measurement of human plasma AVP. Analytical evaluations of the method were performed, and the results obtained using LC/MS/MS and radioimmunoassay (RIA) were compared. RESULTS: The lower limit of quantification (LLOQ) for plasma AVP obtained using LC/MS/MS and RIA were 0.2 and 0.4 pg/mL, respectively. Although there was a weak overall correlation between the results obtained using the two different methods, the RIA results did not agree with the LC/MS/MS results, particularly at low concentrations. CONCLUSIONS: AVP detection through RIA is not satisfactory compared with that using LC/MS/MS. Diagnostic values of direct AVP measurements must be evaluated based on the results obtained via sensitive and accurate MS-based methods rather than those obtained through RIA.

An improved in-house lysis-filtration protocol for bacterial identification from positive blood culture bottles with high identification rates by MALDI-TOF MS.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is now a well-established method for identification of microorganisms from positive blood cultures. Pretreatments to effectively remove non-bacterial proteins are a prerequisite for successful identification, and a variety of protocols have been reported. Although commercially available kits, mainly the Sepsityper Kit, are increasingly used, the identification rates reported often are not satisfactory, particularly for Gram-positive isolates. We developed a new, in-house lysis-filtration protocol and prospectively evaluated its performance compared to the Sepsityper kit. The in-house protocol consists of three simple steps: lysis by ammonium chloride, aspiration with a syringe fitted with a 0.45-µm membrane, and centrifugation to collect microbes. The novel protocol requires only 20 min. Performance of the in-house protocol was evaluated using a total of 117 monomicrobial cases of positive blood culture. Medium from blood culture bottles was pretreated by the in-house protocol or the commercial kit, and isolated cells were subjected to direct identification by mass spectrometry fingerprinting in parallel with conventional subculturing for reference identification. The overall MALDI-TOF MS-based identification rates with score > 1.7 and > 2.0 obtained using the in-house protocol were 99.2% and 85.5%, respectively, whereas those obtained using the Sepsityper Kit were 85.4% and 61.5%, respectively. For Gram-positive cases, the in-house protocol yielded scores >1.7 and > 2.0 at 98.5% and 76.1%, respectively, whereas the commercial kit yielded these scores at 76.1% and 43.3%, respectively. Although these are preliminary results, these values suggest that this easy lysis-filtration protocol deserves assessment in a larger-scale test.

Application of the biocopolymer preparation system, rapid BACpro® II kit, for mass-spectrometry-based bacterial identification from positive blood culture bottles by the MALDI Biotyper system.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used for identification of microorganisms from positive blood cultures. Pretreatments to effectively remove non-bacterial components and selectively collect microorganisms are a prerequisite for successful identification, and a variety of home-brew and commercial protocols have been reported. Although commercially available kits, mainly the Sepsityper Kit, are increasingly used, the identification rates reported often are not satisfactory, particularly for Gram-positive isolates. We recently developed a method to collect bacteria from positive blood culture bottles using a polyallylamine-polystyrene copolymer that has been used in wastewater processing. This pretreatment protocol is now commercially available as the rapid BACpro® II kit (Nittobo Medical Co., Tokyo, Japan). The operation time required for processing using this novel kit is approximately 10 min, and the entire procedure can be completed within a biosafety cabinet. Since the performance of the rapid BACpro® II kit has not been tested using the MALDI Biotyper system, we prospectively evaluated the performance of the rapid BACpro® II kit as compared with the Sepsityper® kit. Performance of the rapid BACpro® II kit was evaluated using a total of 193 monomicrobial cases of positive blood culture. Medium from blood culture bottles was pretreated by the rapid BACpro® II kit or the Sepsityper® Kit, and isolated cells were subjected to direct identification by MS fingerprinting in parallel with conventional subculturing for reference identification. The overall MALDI-TOF MS-based identification rates with >1.7 score and >2.0 score obtained using the rapid BACpro® II kit were 99.5% and 80.8%, respectively, whereas those obtained using the Sepsityper® Kit were 89.1% and 68.4%, respectively (P < 0.05 for >1.7 and P < 0.05 for >2.0 by Pearsons's chi-square). In Gram-positive cases, the rapid BACpro® II kit gave identification rate of 100% with >1.7 score and 69.4% with >2.0 score, whereas there were 84.7% and 56.8%, respectively by the Sepsityper® Kit (P < 0.05 for >1.7). These results are preliminary, but considering that this new kit is easy to perform and the identification rates are promising, the rapid BACpro® II kit deserves assessment in a larger-scale study with a variety of platforms for MS-based bacterial identification.

Assessment by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry of the Effects of Preanalytical Variables on Serum Peptidome Profiles Following Long-Term Sample Storage.

PURPOSE: Human serum and plasma are often used as clinical specimens in proteomics analyses, and peptidome profiling of human serum is a promising tool for identifying novel disease-associated biomarkers. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is widely used for peptidomic biomarker discovery. Careful sample collection and handling are required as either can have a profound impact on serum peptidome patterns, yet the effects of preanalytical variables on serum peptidome profiles have not been completely elucidated. The present study investigated the effects of preanalytical variables, including storage temperature, duration (up to 12 months), and thawing methods, on MALDI-TOF MS-based serum peptidome patterns. EXPERIMENTAL DESIGN: Aliquots of serum samples were pretreated with weak cation exchanger magnetic beads using an automated ClinProtRobot system and then analyzed by MALDI-TOF MS. RESULTS: A number of significant differences in peak intensities were observed depending on sample processing variables. CONCLUSIONS AND CLINICAL RELEVANCE: These peaks can be used as sample quality markers to assess the effects of long-term storage on serum peptidome profiles using MALDI-TOF MS.

Increased levels of small dense low-density lipoprotein cholesterol associated with hemorheological abnormalities in untreated, early-stage essential hypertensives.

Among subfractions of low-density lipoprotein cholesterol (LDL-C), small dense LDL-C (SdLDL-C) has been highlighted as the most atherogenic lipoprotein cholesterol. The present study aimed to compare the relationship of SdLDL-C with blood viscosity, a surrogate marker for cardiovascular disease, with that of other lipid fractions with blood viscosity in essential hypertensives (EHTs). In 128 untreated, early-stage EHTs, blood viscosity was measured with a falling-ball microviscometer, and serum levels of lipid fractions were determined. Blood and plasma viscosity was significantly higher in 49 patients with dyslipidemia (fasting serum level of LDL-C > 140 mg dl(-1), triglyceride > 150 mg dl(-1) or high-density lipoprotein cholesterol (HDL-C) < 40 mg dl(-1)) compared with 79 patients without dyslipidemia, although hematocrit and RBC rigidity index 'k' did not differ between the two groups. Together, SdLDL-C, LDL-C, triglyceride and large LDL-C were positively correlated with blood viscosity, but for HDL-C, the correlation was negative. After adjusting for non-lipid variables that correlated with blood viscosity (that is, the age, body mass index, resting diastolic blood pressure, sex, hematocrit, plasma viscosity and homeostasis model of assessment of insulin resistance), SdLDL-C was most strongly associated with blood viscosity among the lipid fractions. These data suggest that SdLDL-C could strongly increase blood viscosity in EHTs.