Dynamic Contrast-enhanced Magnetic Resonance Imaging for Patients with Breast Cancer Receiving First-line Bevacizumab and Paclitaxel.

BACKGROUND: Bevacizumab plus paclitaxel shows promise for metastatic disease; however, there is no predictive biomarker. Dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) is widely used to evaluate the efficacy of anti-angiogenic therapies. PATIENTS AND METHODS: Patients with locally advanced/metastatic breast cancer who had not received any prior chemotherapy or hormone therapy were included. DCE-MRI was conducted at baseline and after one and two cycles of bevacizumab and paclitaxel. The percentage change in the volume transfer constant (ΔKtrans) and the correlation of ΔKtrans with tumour regression and time to progression (TTP) were evaluated. RESULTS: The mean ΔKtrans from baseline after one and two cycles was -51.4% and -55.1%, respectively. Patients with ΔKtrans ≥50% displayed more tumour regression than those with ΔKtrans <50%; TTP was not significantly different. CONCLUSION: We demonstrate a decrease in blood permeability following bevacizumab and paclitaxel using DCE-MRI and a correlation between ΔKtrans and tumour regression.

Letter to the Editor: Corneoscleral Melt 50 Years after Excision of Pterygium.

PURPOSE: To report a case of corneoscleral melt that occurred 50 years after resection of pterygium with postoperative administration of mitomycin C (MMC). RESULTS: A 93-year-old woman developed acute corneal perforation and scleral melt in her left eye at 50 years after pterygium surgery with postoperative topical MMC. She underwent limbal transplantation. The anterior chamber reformed postoperatively and her intraocular pressure was normal. At 12 months after transplantation, best-corrected visual acuity was 20/500 and the graft-host junction was well apposed. CONCLUSION: This case shows that corneoscleral melt can occur even 50 years after resection of pterygium combined with postoperative topical MMC.

Changes of conjunctivochalasis after cataract surgery via a superior transconjunctival sclerocorneal incision.

The purpose of this study was to evaluate the changes in the severity of conjunctivochalasis after cataract surgery performed via a superior conjunctival sclerocorneal incision. Prospective investigation was performed in a consecutive series of 36 eyes of 36 patients aged 62-85 years who underwent phacoemulsification and implantation of a 6.0-mm foldable intraocular lens through a 2.8-mm superior sclerocorneal incision. The age, gender, medical history, ocular history, grade, refraction, and axial length were determined in all subjects. The conjunctivochalasis score (0-3) and other parameters were determined at three sites (nasal, middle, and temporal) according to the system for grading conjunctivochalasis proposed by Meller and Tseng (at baseline and at 1, 4, and 12 weeks postoperatively). The total conjunctivochalasis score (sum of the scores for the temporal, middle, and nasal regions: 0-9) increased significantly from 4.0 ± 1.9 at baseline to 4.8 ± 2.1 at 1 week postoperatively (p = 0.0048), and subsequently decreased again at 4 weeks (4.3 ± 2.0) and 12 weeks (4.0 ± 1.9). Multivariate logistic regression analysis showed that progression of conjunctivochalasis at 12 weeks was significantly associated with the axial length [odds ratio (OR) = 1.21, p = 0.0118] and with conjunctival suture placement (OR = 1.34, p = 0.0493). When cataract surgery was performed via a superior sclerocorneal incision, the severity of conjunctivochalasis at 12 weeks postoperatively was similar to that at baseline. Our findings suggest that a superior sclerocorneal incision has no influence on the progression of conjunctivochalasis after cataract surgery.

Silent information regulator T1 in aqueous humor of patients with cataract.

PURPOSE: Silent information regulator T1 (SIRT1), a member of the sirtuin family, has a preventive role in various ocular diseases. We evaluated the relations between the aqueous humor level of SIRT1 and age, sex, systemic diseases, the severity of lens opacity, and other factors. SETTING: This study was conducted at a university teaching hospital in Tokyo, Japan. DESIGN: This study was designed based on the consecutive case series. METHODS: Aqueous humor samples were obtained from 29 eyes of the 21 consecutive patients undergoing surgery for age-related cataract (ARC). SIRT1 levels were determined by enzyme-linked immunosorbent assay. RESULTS: Aqueous humor levels of SIRT1 showed a positive correlation with visual acuity (logarithm of the minimum angle of resolution) and with the severity of nuclear cataract (r=0.32 and 0.30, respectively, P<0.05). However, only visual acuity was correlated with SIRT1 according to the stepwise multiple regression analysis (P<0.05). CONCLUSION: These findings suggest that SIRT1 may have an effect on the formation of ARC, acting as a defensive factor against ARC.

Effect of aflibercept in patients with age-related macular degeneration.

The purpose of this study was to evaluate the efficacy of standard induction therapy with intravitreal aflibercept (IVA) in patients with exudative age-related macular degeneration (AMD) at 6 months after completion of induction therapy. Eleven eyes with typical AMD (tAMD) and 13 eyes with polypoidal choroidal vasculopathy (PCV) received three monthly doses of IVA (2 mg/0.05 ml in weeks 0, 4, and 8) for treatment of exudative AMD. Best-corrected visual acuity (BCVA) was measured, and optical coherence tomography was performed at baseline and at each monthly visit until 6 months after IVA. Treatment failure was defined as persistent or recurrent AMD that presented with cystoid macular edema, serous retinal detachment, and pigment epithelium detachment. Mean logMAR BCVA was improved from 0.62 ± 0.46 at baseline to 0.54 ± 0.43 at 6 months after IVA (p < 0.05). The success rate was 95.8 % at 3 months and 75.0 % at 6 months after IVA. Failure of IVA was positively associated with the absence of PVD before treatment (r = 0.35) and with the AMD type (tAMD, r = 0.43) by univariate analysis. Cox proportional hazards analysis demonstrated that the absence of PVD before treatment was associated with an increased risk of failure of IVA (OR = 33.17, p = 0.0219). Three months of induction IVA achieved a high success rate in patients with AMD monitored for up to 6 months. Factors associated with failure of IVA were the absence of PVD and the presence of tAMD. Accordingly, continuation of IVA following induction therapy may be beneficial to manage AMD in patients with tAMD or those without PVD.

Development of High-purity Certified Reference Materials for 17 Proteinogenic Amino Acids by Traceable Titration Methods.

To ensure the reliability of amino acid analyses, the National Metrology Institute of Japan of the National Institute of Advanced Industrial Science and Technology (NMIJ/AIST) has developed high-purity certified reference materials (CRMs) for 17 proteinogenic amino acids. These CRMs are intended for use as primary reference materials to enable the traceable quantification of amino acids. The purity of the present CRMs was determined based on two traceable methods: nonaqueous acidimetric titration and nitrogen determination by the Kjeldahl method. Since neither method could distinguish compounds with similar structures, such as amino acid-related impurities, impurities were thoroughly quantified by combining several HPLC methods, and subtracted from the obtained purity of each method. The property value of each amino acid was calculated as a weighted mean of the corrected purities by the two methods. The uncertainty of the property value was obtained by combining measurement uncertainties of the two methods, a difference between the two methods, the uncertainty from the contribution of impurities, and the uncertainty derived from inhomogeneity. The uncertainty derived from instability was considered to be negligible based on stability monitoring of some CRMs. The certified value of each amino acid, property value with uncertainty, was given for both with or without enantiomeric separation.

LETTER TO THE EDITOR Excision of Congenital Bilateral Persistent Pupillary Membrane in a Child with Exotropia.

PURPOSE: To report the clinical and histopathological findings of a patient who had bilateral persistent pupillary membrane with exotropia and high hyperopia. CASE REPORT: A 7-year-old boy presented with a persistent pupillary membrane in both eyes. His best corrected visual acuity (BCVA) was 20/20 in the right eye and 20/32 in the left eye with exotropia of 18 prism diopters. He underwent surgical resection of both membranes. At 5 months postoperatively, BCVA was 20/20 with final bilateral refraction of +6.5 D in both eyes. Exotropia and photophobia showed improvement immediately after surgery. Histopathological examination revealed typical features of normal iris tissue in the excised membranes. CONCLUSION: Bilateral persistent pupillary membranes were excised successfully without injury to other ocular tissues, including the crystalline lens. Surgical treatment may be required for the management of persistent pupillary membrane associated with visual impairment such as exotropia or photophobia.

A highly sensitive and accurate gene expression analysis by sequencing ("bead-seq") for a single cell.

Analyses of gene expressions in single cells are important for understanding detailed biological phenomena. Here, a highly sensitive and accurate method by sequencing (called "bead-seq") to obtain a whole gene expression profile for a single cell is proposed. A key feature of the method is to use a complementary DNA (cDNA) library on magnetic beads, which enables adding washing steps to remove residual reagents in a sample preparation process. By adding the washing steps, the next steps can be carried out under the optimal conditions without losing cDNAs. Error sources were carefully evaluated to conclude that the first several steps were the key steps. It is demonstrated that bead-seq is superior to the conventional methods for single-cell gene expression analyses in terms of reproducibility, quantitative accuracy, and biases caused during sample preparation and sequencing processes.

Specific IgG for cat allergens in patients with allergic conjunctivitis.

Immunoglobulin G (IgG) antibodies are involved in type II and type III hypersensitivity. We evaluated the relation between perennial allergic conjunctivitis and serum levels of specific IgG for cat allergens. A prospective study was conducted in patients with seasonal allergic conjunctivitis (seasonal group, n = 10), patients with perennial allergic conjunctivitis (perennial group, n = 10), and healthy control subjects (control group, n = 10). Serum levels of specific IgE and IgG for cat allergens and total tear IgE were measured, and a skin prick test was also performed. In addition, a severity score associated with allergic conjunctivitis was calculated (0-30). The positive rates and scores of for total tear IgE, serum cat-specific IgE, and serum cat-specific IgG were all higher in the seasonal and perennial groups than in the control group (all p < 0.05). Serum cat-specific IgG levels were higher in the perennial group than in the seasonal group (p = 0.0156), but there was no significant difference in the grade of cat-specific IgE between the two groups (p = 0.3008). On multivariate analysis, the mean wheal diameter for cat allergen was associated with the serum level of cat-specific IgG (not IgE) in all patients [odds ratio (OR) = 31.979, p < 0.0001]. Multivariate analysis revealed that the total objective score was strongly associated with serum cat-specific IgG (OR = 23.015, p < 0.0001). These findings suggest that specific IgG antibodies may be involved in perennial allergic symptoms caused by indoor allergens such as cat allergens.

Development of C-reactive protein certified reference material NMIJ CRM 6201-b: optimization of a hydrolysis process to improve the accuracy of amino acid analysis.

To standardize C-reactive protein (CRP) assays, the National Metrology Institute of Japan (NMIJ) has developed a C-reactive protein solution certified reference material, CRM 6201-b, which is intended for use as a primary reference material to enable the SI-traceable measurement of CRP. This study describes the development process of CRM 6201-b. As a candidate material of the CRM, recombinant human CRP solution was selected because of its higher purity and homogeneity than the purified material from human serum. Gel filtration chromatography was used to examine the homogeneity and stability of the present CRM. The total protein concentration of CRP in the present CRM was determined by amino acid analysis coupled to isotope-dilution mass spectrometry (IDMS-AAA). To improve the accuracy of IDMS-AAA, we optimized the hydrolysis process by examining the effect of parameters such as the volume of protein samples taken for hydrolysis, the procedure of sample preparation prior to the hydrolysis, hydrolysis temperature, and hydrolysis time. Under optimized conditions, we conducted two independent approaches in which the following independent hydrolysis and liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) were combined: one was vapor-phase acid hydrolysis (130 °C, 24 h) and hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) method, and the other was microwave-assisted liquid-phase acid hydrolysis (150 °C, 3 h) and pre-column derivatization liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The quantitative values of the two different amino acid analyses were in agreement within their uncertainties. The certified value was the weighted mean of the results of the two methods. Uncertainties from the value-assignment method, between-method variance, homogeneity, long-term stability, and short-term stability were taken into account in evaluating the uncertainty for a certified value. The certified value and the expanded uncertainty (k = 2) of CRM 6201-b are (40.0 ± 1.6) µmol kg(-1).

Specific IgE for wheat in tear fluid of patients with allergic conjunctivitis.

CONTEXT: Allergy to hydrolyzed wheat protein in facial soap has become a major social issue in Japan. It has been reported that the most frequent early symptoms of allergy to hydrolyzed wheat protein in soap are allergic conjunctivitis and rhinitis, while wheat-dependent exercise-induced anaphylaxis can be induced by long-term use. OBJECTIVE: We evaluated the relation between tear fluid levels of specific IgE for wheat and the features of allergic conjunctivitis. METHODS: A prospective, non-randomized, cross-sectional study was conducted in 103 patients with moderate to severe allergic conjunctivitis (allergic group) and 20 age- and sex-matched healthy control subjects (control group). Specific IgE for wheat was measured in tear fluid with an immunochromatography assay, and a skin prick test (SPT) was also performed. Symptoms (sneezing, rhinorrhea, nasal obstruction, ocular itching, and lacrimation) were assessed in each subject along with the activities of daily living (ADL) score and the total ocular symptom score for allergic conjunctivitis. A severity score (0, 1, 2, or 3) was assigned for various changes of the palpebral and bulbar conjunctiva, as well as for limbal and corneal lesions associated with allergic conjunctivitis. RESULTS: The IgE positive rate and specific IgE score were both higher in the allergic group than in the control group (71.8% versus 40.0% and 1.9 ± 0.7 versus 1.4 ± 0.5). A positive SPT for wheat was also more frequent in the allergic group than in the control group (6.8% versus 0.0%). Within the allergic group, patients with a positive SPT had higher specific IgE scores than patients with a negative SPT (3.3 ± 0.5 versus 1.8 ± 0.6, p < 0.001). In the allergic group, the wheat IgE level in tear fluid was correlated with the severity of allergic conjunctivitis symptoms, including ocular itching (r = 0.665), tearing (r = 0.672), and the total ocular symptom score (r = 0.204). Wheat IgE in tear fluid was also correlated with the severity of rhinitis symptoms, including sneezing (r = 0.610), nose blowing (r = 0.640), and nasal obstruction (r = 0.677). Furthermore, the tear fluid wheat IgE score was correlated with five objective features of allergic conjunctivitis (p < 0.05). CONCLUSIONS: These results suggest that wheat allergy may be involved in the development of allergic conjunctivitis.

Glycolysis in patients with age-related macular degeneration.

PURPOSE: Retinal adenosine triphosphate is mainly produced via glycolysis, so inhibition of glycolysis may promote the onset and progression of age-related macular degeneration (AMD). When glycolysis is inhibited, pyruvate is metabolized by lactic acid fermentation instead of entering the mitochondrial tricarboxylic acid (TCA) cycle. We measured urinary pyruvate and lactate levels in patients with AMD. METHODS: Eight patients with typical AMD (tAMD group) and 9 patients with polypoidal choroidal vasculopathy (PCV group) were enrolled. Urinary levels of pyruvate, lactate, α-hydroxybutyrate, and ß-hydroxybutyrate were measured in all patients. RESULTS: The mean urinary levels of pyruvate and lactate were 8.0 ± 2.8 and 7.5 ± 8.3 µg/mg creatinine (reference values: 0.5-6.6 and 0.0-1.6), respectively, with the mean increase over the reference value being 83.6 ± 51.1% and 426.5 ± 527.8%, respectively. In 12 patients (70.6%), the lactate/pyruvate ratio was above the reference range. Urinary levels of α-hydroxybutyrate and ß-hydroxybutyrate were decreased by -31.9 ± 15.2% and -33.1 ± 17.5% compared with the mean reference values. There were no significant differences of any of these glycolysis metabolites between the tAMD and PCV groups. Multivariate analysis revealed that none of the variables tested, including patient background factors (age, hypertension, diabetes, hyperlipidemia, cerebrovascular disease, alcohol, smoking, visual acuity, and AMD phenotype), were significantly associated with the lactate/pyruvate ratio. CONCLUSION: A high lactate/pyruvate ratio is a well-known marker of mitochondrial impairment, and it indicates poor oxidative function in AMD. Our results suggest that increased lactate levels may be implicated in the pathogenesis of AMD.

Airborne particulate matter (PM2.5) and the prevalence of allergic conjunctivitis in Japan.

OBJECTIVE: Exposure to particulate matter less than 2.5 µm in diameter (PM2.5) is associated with asthma and respiratory symptoms, but little is known about the influence of PM2.5 on allergic conjunctivitis. The purpose of this study was to examine the association of PM2.5 with outpatient attendance for allergic conjunctivitis. METHODS: We conducted a time-series analysis of the association between outpatient attendance for allergic conjunctivitis and PM2.5 levels from May to July (non-pollen season) and from August to October (the autumnal pollen season) in 2012. Air pollution data (including the levels of PM2.5, oxidants, nitric oxide, nitrogen dioxide, nitrogen oxide, carbon monoxide, methane, non-methane hydrocarbons, and total hydrocarbons) and data on the daily weather conditions (temperature, wind speed, and humidity) were collected at a centrally located monitoring station in Tokyo. We calculated weekly average values for the daily number of outpatient visits, as well as the air pollution and weather data, and used the weekly average values for analysis. RESULTS: There was a significant association between the number of outpatient visits for allergic conjunctivitis and the PM2.5 level (r=0.62, p=0.0177) from May to July, while no correlation was found between the number of outpatient visits and any variable assessed from August to November. Multivariate analysis also showed that PM2.5 was a significant predictor of the number of outpatient visits from May to July (odds ratio=9.05, p=0.0463), while there were no significant predictors of the number of outpatient visits from August to October. From May to July, PM2.5 showed a negative correlation with humidity (r=-0.53, p=0.0499). CONCLUSIONS: These findings suggest a possible role of PM2.5 in the development of allergic conjunctivitis during the non-pollen season. This association between PM2.5 and allergic conjunctivitis may have broad public health implications in relation to allergic diseases.

Sensitization to Asian dust and allergic rhinoconjunctivitis.

OBJECTIVE: Asian dust storms frequently occur in northeast Asia and the dust occasionally even spreads as far as North America during spring. Asian dust can be harmful to human health and the environment, and thus has become one of the most serious problems for Asian countries. In the present study, we evaluated sensitization to Asian dust in Japanese patients with rhinoconjunctivitis. METHODS: In March 2011, a prospective, non-randomized, cross-sectional study was conducted in 10 patients with allergic rhinoconjunctivitis (allergic group), 3 patients with atopic keratoconjunctivitis (atopic group), and 10 age- and sex-matched healthy control subjects (control group). Skin prick tests (SPT) were performed with untreated Asian dust, Asian dust extract, heat-sterilized Asian dust, silicon dioxide (SiO2), and phosphate-buffered saline (PBS). A panel of 14 allergen extracts was also tested, comprising extracts of pollens (cedar, orchard grass, ragweed, and mugwort), house dust (house dust mixture and Dermatophagoides pteronyssinus), animal dander (cat and dog), fungi (Alternaria tenuis, Candida, and Aspergillus), and foods (milk, egg, and wheat). Then the SPT-positive rate and the mean wheal diameter for each allergen were compared among the three groups. RESULTS: The SPT-positive rates for untreated Asian dust, Asian dust extract, and sterilized Asian dust were significantly higher in the allergic and atopic groups than in the control group (all p<0.05). In the allergic group, there were a significant differences of the SPT-positive rates for untreated Asian dust (70%), Asian dust extract (50%), sterilized Asian dust (20%), SiO2 (20%), and PBS (0%) (p=0.0068). The SPT response to untreated Asian dust was correlated with the mean wheal diameters for four plant pollens (r=0.71, p=0.0104) and for three fungi (r=0.57, p=0.0426). Multivariate logistic regression analysis showed that significant predictors of the SPT reaction to untreated Asian dust were the mean wheal diameter for the four plant pollen (odds ratio=2.54, p=0.0138) and that for the three fungi (odds ratio=1.84, p=0.0273). CONCLUSIONS: Asian dust may act as an adjuvant to promote allergic disease induced by inhaled allergens such as pollen and fungi.

Non-biased and efficient global amplification of a single-cell cDNA library.

Analysis of single-cell gene expression promises a more precise understanding of molecular mechanisms of a living system. Most techniques only allow studies of the expressions for limited numbers of gene species. When amplification of cDNA was carried out for analysing more genes, amplification biases were frequently reported. A non-biased and efficient global-amplification method, which uses a single-cell cDNA library immobilized on beads, was developed for analysing entire gene expressions for single cells. Every step in this analysis from reverse transcription to cDNA amplification was optimized. By removing degrading excess primers, the bias due to the digestion of cDNA was prevented. Since the residual reagents, which affect the efficiency of each subsequent reaction, could be removed by washing beads, the conditions for uniform and maximized amplification of cDNAs were achieved. The differences in the amplification rates for randomly selected eight genes were within 1.5-folds, which could be negligible for most of the applications of single-cell analysis. The global amplification gives a large amount of amplified cDNA (>100 µg) from a single cell (2-pg mRNA), and that amount is enough for downstream analysis. The proposed global-amplification method was used to analyse transcript ratios of multiple cDNA targets (from several copies to several thousand copies) quantitatively.

Influence of axial length on conjunctivochalasis.

PURPOSE: To investigate the relation between the severity of conjunctivochalasis and the axial length (AL) of the eyeball. METHODS: A prospective nonrandomized study was performed on 165 patients aged 51 to 88 years. The age, gender, medical history, ocular history, grade and other parameters of inferior conjunctivochalasis (classified as nasal, middle, and temporal), and AL were determined in all patients. Patients were divided into a short AL group (≤ 22.0 mm), a medium AL group (>22.0 to ≤ 24.0 mm), and a long AL group (>24.0 mm). RESULTS: The short AL group tended to have higher grades of temporal and nasal conjunctivochalasis compared with the medium and long AL groups. Multivariate logistic regression analysis showed that the total conjunctivochalasis score (the sum of the scores for the temporal, middle, and nasal regions) was significantly associated with the age [odds ratio (OR) = 1.32, P = 0.001] and the AL (OR = 0.81, P = 0.029). In addition, the AL was independently associated with the grade of conjunctivochalasis after adjustment for age (OR = 0.80, P = 0.020). CONCLUSIONS: This was the first assessment of the relationship between the AL and the grade of conjunctivochalasis. Our findings suggest that the severity of conjunctivochalasis is dependent on the AL, with a short AL contributing to the pathogenesis of conjunctivochalasis.

Specific IgE in tear fluid and features of allergic conjunctivitis.

PURPOSE: The level of specific class E immunoglobulins (IgE) in tear fluid is a useful diagnostic indicator for allergic conjunctivitis, but it is still unclear whether the measurement of tear fluid IgE is helpful for assessing the severity of allergic conjunctivitis. In this study, we evaluated the relation between tear fluid levels of specific IgE and features of allergic conjunctivitis. METHODS: A prospective, nonrandomized, cross-sectional study was conducted in patients with allergic conjunctivitis (n = 55, allergic group) and age- and sex-matched healthy control subjects (n = 50, control group). Levels of specific IgE for cedar pollen, cat epithelium/dander and Dermatophagoides pteronyssinus were measured in tear fluid with the Immfast Check J1®. A severity score (0, 1, 2 or 3) was assigned for various changes of the palpebral and bulbar conjunctiva, as well as for limbal and corneal lesions. RESULTS: The levels of specific IgE for both cedar pollen, and D. pteronyssinus were significantly higher in the allergic group compared with the control group (p < 0.0001), while the level of specific IgE for cat epithelium/dander showed no significant difference between the two groups (p = 0.0777). When IgE levels were divided into four classes, the classes for both D. Pteronyssinus and cat epithelium/dander IgE were correlated with four features of allergic conjunctivitis. On the other hand, no correlation was found between the class of cedar pollen IgE and any of the features of allergic conjunctivitis. CONCLUSIONS: This study demonstrated that measurement of specific IgE in tear fluid may be useful for determining the severity of allergic conjunctivitis induced by indoor allergens. Although measurement of IgE in tear fluid is only a supplemental tool for evaluating the clinical activity of allergic conjunctivitis, the test can be useful for detecting specific IgE antibodies responsible for this condition.

Intraindividual and interindividual variabilities in endogenous cortisol 6ß-hydroxylation clearance as an index for in vivo CYP3A phenotyping in humans.

This study was designed to evaluate the in vivo activity of cytochrome P450 3A (CYP3A) in 49 healthy Japanese subjects aged 22-58 years using endogenous cortisol 6ß-hydroxylation clearance [CLm(6ß)], a novel biomarker for CYP3A phenotyping. CLm(6ß) in the 49 healthy subjects was 2.40 ± 0.79 ml/min with an approximately 4-fold interindividual variability of CYP3A activity. The mean clearance in the 24 women was 2.50 ± 0.89 ml/min; the value in the women was higher than in the 25 men (2.30 ± 0.69 ml/min) by approximately 9%. We also measured the change of CLm(6ß) in 14 healthy subjects in the morning at 10:00-12:00 every 2 or 3 days over a period of 36-53 days and observed a 1.5-fold to 3.4-fold day-to-day intraindividual variability in the CYP3A activity. The mean value for CLm(6ß) in each subject for 36-53 days was 2.54 ± 0.76 ml/min (n = 14). We also evaluated the CLm(6ß) every 2 hours from 8:00-20:00 in 26 healthy subjects. The within-day intraindividual clearance variability was 1.1-fold to 2.5-fold (2.45 ± 0.91 ml/min, n = 26). No characteristic diurnal rhythms were observed in the in vivo activity of CYP3A.

Development of SI-traceable C-peptide certified reference material NMIJ CRM 6901-a using isotope-dilution mass spectrometry-based amino acid analyses.

A certified reference material (CRM) is a higher-order calibration material used to enable a traceable analysis. This paper describes the development of a C-peptide CRM (NMIJ CRM 6901-a) by the National Metrology Institute of Japan using two independent methods for amino acid analysis based on isotope-dilution mass spectrometry. C-peptide is a 31-mer peptide that is utilized for the evaluation of ß-cell function in the pancreas in clinical testing. This CRM is a lyophilized synthetic peptide having the human C-peptide sequence, and contains deamidated and pyroglutamylated forms of C-peptide. By adding water (1.00 ± 0.01) g into the vial containing the CRM, the C-peptide solution in 10 mM phosphate buffer saline (pH 6.6) is reconstituted. We assigned two certified values that represent the concentrations of total C-peptide (mixture of C-peptide, deamidated C-peptide, and pyroglutamylated C-peptide) and C-peptide. The certified concentration of total C-peptide was determined by two amino acid analyses using pre-column derivatization liquid chromatography-mass spectrometry and hydrophilic chromatography-mass spectrometry following acid hydrolysis. The certified concentration of C-peptide was determined by multiplying the concentration of total C-peptide by the ratio of the relative area of C-peptide to that of the total C-peptide measured by liquid chromatography. The certified value of C-peptide (80.7 ± 5.0) mg/L represents the concentration of the specific entity of C-peptide; on the other hand, the certified value of total C-peptide, (81.7 ± 5.1) mg/L can be used for analyses that does not differentiate deamidated and pyroglutamylated C-peptide from C-peptide itself, such as amino acid analyses and immunochemical assays.

Highly sensitive pyrosequencing system with polymer-supported enzymes for high-throughput DNA analysis.

A highly sensitive massively parallel pyrosequencing system employing a gel matrix to immobilize enzymes at high density in microreaction chambers is demonstrated. Reducing the size of microreaction chambers in a DNA analyzer is important to achieve a high throughput utilizing a commercially available detection device or camera. A high-performance system can be attained by detecting signals from one reaction chamber with one photopixel of around several micrometers by utilizing a 1:1 image magnification. However, the use of small beads immobilizing DNA has a disadvantage in detecting luminescence because only small amounts of DNA can be immobilized on the bead surfaces for sequencing. As luminescence intensity could be enhanced by increasing the luciferase density in the chambers, we overcame this difficulty by using a gel matrix to immobilize luciferase at a high concentration in the microreaction chambers. Luminescence 1 order of magnitude higher could be observed with the new method compared to the conventional method. Consequently, the chamber size and bead size immobilizing DNA could be reduced to as small as 6.5 and 4 µm, respectively. This can be successfully applied to achieving small, inexpensive, pyrosequencing systems with high throughput.