Reappraisal of serum retinol-binding protein as a surrogate marker for retinol and discovery of a novel retinol estimation formula.

BACKGROUND & AIMS: Serum retinol (ROH) is commonly used for population level assessment of vitamin A status. High-performance liquid chromatography (HPLC) is considered most accurate method for measuring ROH. However, with the technical difficulty of using HPLC for routine assays, serum retinol-binding protein (RBP) measured by immunological assays is expected to be a surrogate marker for ROH, with reports of a close correlation between serum RBP and ROH. Nevertheless, RBP is not commonly tested to assess vitamin A status with concerns over RBP alterations under various physiopathological conditions. Thus, we reappraised the extent to which RBP could be used as a surrogate marker in representative disorders that alter serum RBP levels. As a related marker, diagnostic utility of transthyretin (TTR) was also evaluated. METHODS: To evaluate the reliability of ROH and RBP assays, specimen stability was assessed in terms of (1) storage at 25, 4, -20, and -80 °C for 1-28 days, (2) five-cycle freeze-thawing, and (3) fluorescent light exposure for 1-14 days. Sources of variation (sex, age, body mass index [BMI], and drinking habits) and reference intervals for ROH, RBP, and TTR were determined in 617 well-defined healthy individuals. To investigate the influence of disorders that affect serum RBP, patients with five diagnostic groups were enrolled: 26 with chronic kidney disease (CKD); 13 with various malignancies in advanced stages (AdM), 12 with acute bacterial infections (ABI), 6 with liver cirrhosis (LC), and 26 with simple obesity (BMI ≥ 27 kg/m2). RESULTS: The stability of RBP and ROH in serum was confirmed under all conditions. In healthy individuals, serum ROH, RBP, and TTR were appreciably high in males with a slight increase in proportion to age and BMI. The major-axis regression line between RBP (x) and ROH (y) in healthy individuals was y = x, with a correlation coefficient of 0.986. In the LC, AdM, and ABI groups, similar strong correlations were observed; however, the regression lines were shifted slightly rightward from the healthy group line, indicating a positive bias in estimating ROH. Interestingly, the same analyses between TTR and ROH revealed similar strong linear relationships in all groups; however, the regression line of each group showed a leftward (opposite) shift from the healthy group line. Based on these observations, we developed a novel regression model composed of RBP and TTR, which gave much improved accuracy in estimating ROH, even under these pathological conditions. CONCLUSIONS: The perfect RBP-ROH correlation in healthy individuals indicates the utility of RPB as a surrogate marker for ROH. Nevertheless, under RBP-altered conditions, a slight overestimation of ROH is inevitable. However, when the TTR was tested together, the bias can be corrected almost perfectly using the novel ROH estimation formula comprising RBP and TTR.

The complexity of kidney disease and diagnosing it - cystatin C, selective glomerular hypofiltration syndromes and proteome regulation.

Estimation of kidney function is often part of daily clinical practice, mostly done by using the endogenous glomerular filtration rate (GFR)-markers creatinine or cystatin C. A recommendation to use both markers in parallel in 2010 has resulted in new knowledge concerning the pathophysiology of kidney disorders by the identification of a new set of kidney disorders, selective glomerular hypofiltration syndromes. These syndromes, connected to strong increases in mortality and morbidity, are characterized by a selective reduction in the glomerular filtration of 5-30 kDa molecules, such as cystatin C, compared to the filtration of small molecules <1 kDa dominating the glomerular filtrate, for example water, urea and creatinine. At least two types of such disorders, shrunken or elongated pore syndrome, are possible according to the pore model for glomerular filtration. Selective glomerular hypofiltration syndromes are prevalent in investigated populations, and patients with these syndromes often display normal measured GFR or creatinine-based GFR-estimates. The syndromes are characterized by proteomic changes promoting the development of atherosclerosis, indicating antibodies and specific receptor-blocking substances as possible new treatment modalities. Presently, the KDIGO guidelines for diagnosing kidney disorders do not recommend cystatin C as a general marker of kidney function and will therefore not allow the identification of a considerable number of patients with selective glomerular hypofiltration syndromes. Furthermore, as cystatin C is uninfluenced by muscle mass, diet or variations in tubular secretion and cystatin C-based GFR-estimation equations do not require controversial race or sex terms, it is obvious that cystatin C should be a part of future KDIGO guidelines.

Different interaction of onset age and duration of type 1 diabetes on the dynamics of autoantibodies to insulinoma-associated antigen-2 and zinc transporter 8.

AIMS/INTRODUCTION: This study aimed to investigate the dynamics associated with autoantibodies to insulinoma-associated antigen-2 (IA-2A) and zinc transporter 8 (ZnT8A) relating to the onset age and disease duration in patients with type 1 diabetes. METHODS: Using bridging-type enzyme-linked immunosorbent assay, IA-2A, ZnT8A and glutamic acid decarboxylase autoantibodies were evaluated in 269 patients with type 1 diabetes (median onset age 18.2 years, range 0.8-86 years; median diabetes duration 7 years, range 0-58 years). We then compared the prevalence of these autoantibodies among the different age groups, along with the duration of diabetes using the Cochran-Armitage trend test and multivariate logistic regression analysis. RESULTS: The prevalence of IA-2A, ZnT8A and glutamic acid decarboxylase autoantibodies in patients with duration of ≤3 years was 41.1, 36.7 and 72.2%, respectively, with 80.0% expressing one or more of these autoantibodies. This prevalence declined according to the disease duration (P < 0.005). Both IA-2A and ZnT8A were more frequently observed in younger patients, whereas glutamic acid decarboxylase autoantibodies was more common in older patients. Multivariate logistic regression analysis showed that there was a significant interaction between the onset age and duration of diabetes in patients diagnosed when aged ≤10 years regarding all anti-islet autoantibodies (P < 0.05). However, for patients diagnosed in the middle tertile (aged 11-30 years), the interaction was significant only for ZnT8A, and for those with late-onset diabetes (aged ≥31 years) only for IA-2A. CONCLUSIONS: The current study showed that the rate of disappearance of anti-islet autoantibodies is faster in patients aged ≤10 years, and that even though both proteins are localized in the insulin granule membrane, humoral autoimmunity to IA-2 and ZnT8 differs according to the age of onset.

Clinical significance of serum complement factor 3 in patients with type 2 diabetes mellitus.

AIMS: Although serum complement factor 3 (C3) is an acute phase reactant mainly synthesized in the liver, several recent studies have shown high C3 gene expression in adipose tissue (AT). However, the relationship between C3 and AT levels has not been fully clarified in type 2 diabetes mellitus (T2DM) patients. METHODS: A total of 164 T2DM patients (109men and 55 women) participated in this cross-sectional study. A computed tomography scan was performed to measure visceral, subcutaneous, and total AT. The correlation between these factors and C3 levels was examined using Pearson's correlation analysis. A multivariate regression model was used to assess an independent determinant associated with C3 levels after adjusting the explanatory variables (i.e., all ATs [visceral, subcutaneous, and total], and clinical features [sex, age, body mass index, waist circumference, glycated hemoglobin, duration of diabetes, systolic blood pressure, diastolic blood pressure, aspartate aminotransferase levels, alanine aminotransferase levels, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, log(triglyceride levels), estimated glomerular filtration rate, and log(high-sensitivity C-reactive protein levels)]). RESULTS: Serum C3 levels were correlated with visceral, subcutaneous, and total AT among both men (r=0.505, p<0.001; r=0.545, p<0.001; r=0.617, p<0.001, respectively) and women (r=0.396, p=0.003; r=0.517, p<0.001; r=0.548, p<0.001, respectively). In the multivariate regression model, the association between total AT and C3 levels remained significantly positive (ß=0.490, p<0.001). CONCLUSIONS: Serum C3 levels are associated with visceral, subcutaneous, and total AT in T2DM patients. Furthermore, C3 levels seem to be a marker for overall adiposity rather than regional adiposity.

Relationship Between Serum High-Sensitivity C-Reactive Protein and Myocardial Infarction in a General Japanese Population.

BACKGROUND: Due to ethic differences in its serum levels, clinical applicability of high-sensitivity C-reactive protein (hsCRP) to the primary prevention of atherosclerotic events has not completely been established in Japanese people whose hsCRP levels are lower than in Western people. This study investigated the relationship between hsCRP and myocardial infarction (MI) in general Japanese people. METHODS: In relation to hsCRP, the incidence of MI was determined in a multiregional population-based prospective cohort study (n = 6,637; mean age 54.9 years; 2,513 men/4,124 women). RESULTS: Fifty-six cases of MI were confirmed during a follow-up period of 10.7 years. The cut-off levels of hsCRP between the highest quartile (fourth quartile) and the other quartiles combined were 0.368 mg/l in men and 0.279 mg/l in women. The hazard ratio (HR) of the highest quartile for MI was significantly greater than that of the other quartiles combined (multivariate-adjusted HR: 2.07, 95% confidence interval: 1.03-4.15) in men, but not in women (1.03, 0.35-2.21). CONCLUSIONS: In this population, serum hsCRP measurement predicted MI in men, but not in women. Under the low hsCRP level, a method of applicability of hsCRP to a risk assessment for preventing MI among Japanese people should be further explored.

Collaborative derivation of reference intervals for major clinical laboratory tests in Japan.

OBJECTIVES: Three multicentre studies of reference intervals were conducted recently in Japan. The Committee on Common Reference Intervals of the Japan Society of Clinical Chemistry sought to establish common reference intervals for 40 laboratory tests which were measured in common in the three studies and regarded as well harmonized in Japan. METHODS: The study protocols were comparable with recruitment mostly from hospital workers with body mass index ≤28 and no medications. Age and sex distributions were made equal to obtain a final data size of 6345 individuals. Between-subgroup differences were expressed as the SD ratio (between-subgroup SD divided by SD representing the reference interval). Between-study differences were all within acceptable levels, and thus the three datasets were merged. RESULTS: By adopting SD ratio ≥0.50 as a guide, sex-specific reference intervals were necessary for 12 assays. Age-specific reference intervals for females partitioned at age 45 were required for five analytes. The reference intervals derived by the parametric method resulted in appreciable narrowing of the ranges by applying the latent abnormal values exclusion method in 10 items which were closely associated with prevalent disorders among healthy individuals. Sex- and age-related profiles of reference values, derived from individuals with no abnormal results in major tests, showed peculiar patterns specific to each analyte. CONCLUSION: Common reference intervals for nationwide use were developed for 40 major tests, based on three multicentre studies by advanced statistical methods. Sex- and age-related profiles of reference values are of great relevance not only for interpreting test results, but for applying clinical decision limits specified in various clinical guidelines.

Call for the use of a common equation for glomerular filtration rate estimation in East and South-East Asia.

BACKGROUND: Estimated glomerular filtration rate (eGFR) is currently calculated using various equations and serum creatinine (Scr) value measured by different assays. Differences among these eGFRs deserve further study. METHODS: Volunteers from eight Asian regions (n=3283; age 20-65 years, 1454 men, 1829 women) were recruited. The Chronic Kidney Disease Epidemiology Collaboration equation (EPI), Modification of Diet in Renal Disease Study equation (MDRD) for Japanese (MDRDJap) and MDRD for Chinese (MDRDChi) were selected. Jaffe and enzymatic assays were used to measure Scr. Six eGFRs were obtained for each volunteer: EPI equation using Scr value of enzymatic assay (EPI/E) and Jaffe assay (EPI/J); MDRDJap equation using Scr value of the two assays (MDRDJap/E, MDRDJap/J); and MDRDChi equation using Scr value of the two assays (MDRDChi/E, MDRDChi/J). RESULTS: Neither Scr nor eGFR showed significant regional difference. We compared eGFR calculated using the same equation but with different assays. The medians (2.5%, 97.5%) of eGFR difference were 2.0 (-7, 14) mL/min/1.73 m(2) for EPI, 3.0 (-12.0, 18.0) mL/min/1.73 m(2) for MDRDJap, and 5.0 (-18, 30) mL/min/1.73 m(2) for MDRDChi. We also compared eGFR calculated using different equations but with the same assay. The medians (2.5%, 97.5%) of eGFR difference were 11 (-6, 56) mL/min/1.73 m(2) between MDRDChi/E and EPI/E; 26 (9, 35) mL/min/1.73 m(2) between EPI/E and MDRDJap/E; and 39 (22, 65) mL/min/1.73 m(2) between MDRDChi/E and MDRDJap/E, respectively. CONCLUSIONS: eGFR difference caused by using different equations is much larger than that caused by using different Scr assays. A common equation for GFR estimation is encouraged for use in Asians.

A reference system for urinary albumin: current status.

BACKGROUND: Increased urinary excretion of albumin reflects kidney damage and is a recognized risk factor for progression of renal and cardiovascular disease. Considerable inter-method differences have been reported for both albumin and creatinine measurement results, and therefore the albumin-to-creatinine ratio. Measurement accuracy is unknown and there are no independent reference measurement procedures for albumin and no reference materials for either measurand in urine. METHODS: The National Kidney Disease Education Program (NKDEP) Laboratory Working Group and the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) have initiated joint projects to facilitate standardization of urinary albumin and creatinine measurement. RESULTS: A candidate LC-MS/MS reference measurement procedure for urinary albumin and candidate reference materials for urinary albumin and creatinine has been developed. The status of validations of these reference system components is reported. CONCLUSIONS: The development of certified reference materials and reference measurement procedures for urinary albumin will enable standardization of this important measurand.

[Fundamental and clinical aspects of cystatin C].

Recent progress of fundamental and clinical studies on cystatin C was reviewed. Most of key studies are indebted to prof. Grubb A and his groups. International contributions from Japanese research work are included here. The protein is a basic low molecular weight protein of 13,300 with 120 amino acid residues and pI 9.3, functioning as a cysteine protease inhibitor. With an introduction of ERM-DA471, international reference material for serum cystatin C, global standardization for immunoassay systems has been much facilitated. No serious problems are present in the pre-analytical stage. Serum reference intervals are properly set in all Asian populations including Japanese with age and gender-related differences. The protein is a powerful serum intrinsic marker for glomerular filtration rate. Estimated glomerular filtration rate (eGFRcysC) in coupled with eGFRCr will definitely be a clinical routine for early detection and prevention of altered kidney function and cardiovascular events in general population. Genetic tests clinically indicated include hereditary cystatin C amyloid angiopathy (L68Q) and adult macular degeneration (A25T) although their frequency is extremely low.

Preparation of highly purified monomeric human serum albumin as secondary reference material for standardization of urinary albumin immunoassays.

BACKGROUND: International external quality assessments have shown variation in results of urinary albumin among various immunoassays. A well-defined candidate reference material for urine albumin (cRM-UA) was prepared to improve standardization. METHODS: cRM-UA was prepared from a commercially available preparation of human serum albumin by using gel-filtration HPLC. The value was assigned by transfer from ERM-DA470 using immunoassay systems qualified based on the linearity and variability observed in dilution tests of pooled urine and the calibrators. Effectiveness of recalibration using the cRM-UA was evaluated by measuring 129 urine specimens. RESULTS: The cRM-UA had a monomeric albumin peak which accounted for 98.9% of the total area by gel filtration HPLC. The lyophilized preparation of the cRM-UA had suitable homogeneity, and short- and long-term stability. Nine of 14 immunoassays met the criteria were used for value assignment. The assigned concentration was 225.1±9.11 mg/l [mean±U: expanded uncertainty with k=2] when reconstituted with 3.00 ml of purified water on weight basis. Recalibration of 7 qualified immunoassays using the cRM-UA resulted in between-method CV of 6.6%. CONCLUSIONS: The cRM-UA was successful in achieving standardization of urine albumin results among 7 immunoassays which possess performance attributes representing uniform reactivity to both cRM-UA and clinical urine samples.

Questões atuais relativas à dosagem e à descrição da excreção urinária de albumina / Current issues in measurement and report of urinary albumin excretion

ANTECEDENTES: A excreção urinária de albumina indica lesão nos rins e é reconhecida como fator de risco para a progressão das doenças renal e cardiovascular. A dosagem da albumina urinária chama a atenção sobre a necessidade clínica de relatos de resultados precisos e claramente descritos. O National Kidney Disease Education Program e a Federação Internacional de Química Clínica e Medicina Laboratorial (IFCC) reuniram-se para avaliar o estado atual das questões pré-analíticas, analíticas e pós-analíticas que afetam as dosagens da albumina na urina e para identificar as áreas que necessitam de melhorias. CONTEÚDO: A química da albumina na urina não é completamente compreendida. Diretrizes atuais recomendam a utilização da relação albumina/creatinina (RAC) como substituta para a coleta de amostras cronometradas de urina, frequentemente inadequadas. Os resultados da RAC são afetados pela preparação do paciente, pela hora do dia da coleta das amostras e não é padronizada. Foram relatadas consideráveis diferenças intermétodos para a dosagem tanto de albumina quanto de creatinina, mas a verdade é desconhecida, porque não existem procedimentos de referência para a dosagem de albumina e não há materiais de referência para qualquer um desses analitos na urina. Os intervalos de referência recomendados para a RAC não consideram as grandes diferenças intergrupos na excreção da creatinina (por exemplo, relacionadas com diferenças em idade, sexo e etnia), nem o aumento contínuo no risco relacionado com a excreção de albumina. DISCUSSÃO: Necessidades clínicas foram identificadas para a padronização de (a) métodos de coleta da urina, (b) dosagens de albumina e de creatinina na urina com base em um sistema de referência completo, (c) relatórios dos resultados dos testes e (d) intervalos de referência para a RAC.

BACKGROUND: Urinary excretion of albumin indicates kidney damage and is recognized as a risk factor for progression of kidney disease and cardiovascular disease. The role of urinary albumin measurements has focused attention on the clinical need for accurate and clearly reported results. The National Kidney Disease Education Program and the IFCC convened a conference to assess the current state of preanalytical, analytical, and postanalytical issues affecting urine albumin measurements and to identify areas needing improvement. CONTENT: The chemistry of albumin in urine is incompletely understood. Current guidelines recommend the use of the albumin/creatinine ratio (ACR) as a surrogate for the error-prone collection of timed urine samples. Although ACR results are affected by patient preparation and time of day of sample collection, neither is standardized. Considerable intermethod differences have been reported for both albumin and creatinine measurement, but trueness is unknown because there are no reference measurement procedures for albumin and no reference materials for either analyte in urine. The recommended reference intervals for the ACR do not take into account the large intergroup differences in creatinine excretion (e.g., related to differences in age, sex, and ethnicity) nor the continuous increase in risk related to albumin excretion. DISCUSSION: Clinical needs have been identified for standardization of (a) urine collection methods, (b) urine albumin and creatinine measurements based on a complete reference system, (c) reporting of test results, and (d) reference intervals for the ACR.

[Antimicrobial susceptibility of clinical isolates of aerobic Gram-positive cocci and anaerobic bacteria in 2006].

The activity of antibacterial agents against aerobic Gram-positive cocci (26 species, 1022 strains) and anaerobic bacteria (23 species, 184 strains) isolated from clinical specimens in 2006 at 16 clinical facilities in Japan were studied using either broth microdilution or agar dilution method. The ratio of methicillin-resistant strains among Staphylococcus aureus and Staphylococcus epidermidis was 53.0% and 65.8%, suggesting that resistant strains were isolated at high frequency. Vancomycin (VCM) and quinupristin/dalfopristin (QPR/DPR) had good antibacterial activity against methicillin-resistant S. aureus and methicillin-resistant S. epidermidis, with MIC90s of < or = 2 micrcog/mL. The ratio of penicillin (PC) intermediate and resistant strains classified by mutations of PC-binding proteins among Streptococcus pneumoniae was 87.6%. Ceftriaxone, cefpirome, cefepime, carbapenem antibiotics, VCM, teicoplanin, linezolid(LZD) and QPR/DPR had MIC90s of < or = 1 microg/mL against PC-intermediate and resistant S. pneumoniae strains. Against all strains of Enterococcus faecalis and Enterococcus faecium, the MICs of VCM and TEIC were under 2 microg/mL, and no resistant strain was detected, suggesting that these agents had excellent activities against these species. 10.9% of E. faecalis strains or 3.5% of E. faecium strains showed intermediate or resistant to LZD. 24.4% of E. faecium strains showed intermediate or resistant to QPR/DPR. Against all strains of Clostridium difficile, the MIC of VCM were under 1 microg/mL, suggesting that VCM had excellent activity against C. difficile. Carbapenems showed good activity against Peptococcaceae, Bacteroides spp., and Prevotella spp. However since several strains of Bacteroides fragilis showed resistant to carbapenems and the susceptibility of this species should be well-focused in the future.

[Antimicrobial susceptibility of clinical isolates of aerobic Gram-negative bacteria in 2006].

We determined MICs of antibacterial agents against 1280 clinical strains of aerobic Gram-negative bacteria (19 genus or species) isolated at 16 Japanese facilities in 2006. MICs were determined using mostly broth microdilution method and antibacterial activity was assessed. Strains producing extended-spectrum beta-lactamases (ESBL) accounted for 3.7% of Escherichia coli, 2.7% of Klebsiella spp., and 11.4% of Proteus spp. Notably, 18.8% of Proteus mirabilis was found to produce ESBL higher than 16.7% in 2004. This result was higher extremely than other species. Among Haemophilus influenzae, only 1.2% produced beta-lactamase and 62.8% that increased compared with 57.7% in 2004, were beta-lactamase-negative ampicillin-resistant strains when classified by penicillin-binding protein 3 mutation. Although few antibacterial agents against Pseudomonas aeruginosa have potent activity, only three agents--doripenem, ciprofloxacin, and tobramycin-showed an MIC90 of 4 microg/mL. Of all P aeruginosa strains, 5.7% were resistant to six or more agents of nine antipseudomonal agents, a decrease compared to 8.7% in 2004. Against other glucose-non-fermentative Gram-negative bacteria, the activity of most antibacterial agents was similar to that in 2004.

Current issues in measurement and reporting of urinary albumin excretion.

BACKGROUND: Urinary excretion of albumin indicates kidney damage and is recognized as a risk factor for progression of kidney disease and cardiovascular disease. The role of urinary albumin measurements has focused attention on the clinical need for accurate and clearly reported results. The National Kidney Disease Education Program and the IFCC convened a conference to assess the current state of preanalytical, analytical, and postanalytical issues affecting urine albumin measurements and to identify areas needing improvement. CONTENT: The chemistry of albumin in urine is incompletely understood. Current guidelines recommend the use of the albumin/creatinine ratio (ACR) as a surrogate for the error-prone collection of timed urine samples. Although ACR results are affected by patient preparation and time of day of sample collection, neither is standardized. Considerable intermethod differences have been reported for both albumin and creatinine measurement, but trueness is unknown because there are no reference measurement procedures for albumin and no reference materials for either analyte in urine. The recommended reference intervals for the ACR do not take into account the large intergroup differences in creatinine excretion (e.g., related to differences in age, sex, and ethnicity) nor the continuous increase in risk related to albumin excretion. DISCUSSION: Clinical needs have been identified for standardization of (a) urine collection methods, (b) urine albumin and creatinine measurements based on a complete reference system, (c) reporting of test results, and (d) reference intervals for the ACR.

Recovery of susceptibility to penicillin G in clinical isolates of Streptococcus pneumoniae despite increased accumulation of pbp gene alterations.

Two hundred consecutive clinical isolates of Streptococcus pneumoniae isolated in 2005 and 2006 were analysed for susceptibility to various antimicrobials, pbp gene alterations and macrolide resistance gene expression (2007 analysis) and the results were compared with previous data (2003 analysis). The average minimum inhibitory concentration (MIC) of penicillin G in isolates with 1a(m)/2x(m)/2b(m) decreased from 1.135+/-0.503 mg/L in the 2003 analysis to 0.872+/-0.540 mg/L in the 2007 analysis (P=0.0046). The prevalence of isolates with 1a(m)/2x(m)/2b(m) increased from 30.5% to 32.3%, but the difference was not statistically significant (P=0.6979). The prevalence of isolates with a clarithromycin MIC > or = 1.0mg/L increased from 65.9% to 80.0% (P=0.0005). Isolates expressing ermB increased from 46.6% to 62.6% (P=0.0004). We conclude that the decrease in penicillin resistance of S. pneumoniae does not correlate with a decrease in pbp mutations; on the contrary, the prevalence of isolates with pbp mutations increased. A decrease in penicillin resistance in S. pneumoniae with pbp mutations appears to explain the present results regarding the recovery of penicillin susceptibility. Our results suggest that the spread of mutated pbp genes among S. pneumoniae itself is not responsible for acquisition of the penicillin-resistant phenotype. Use of beta-lactams, especially oral cephalosporins, appears to be responsible for the acquisition of penicillin resistance.

Evaluation of adsorption of urine cystatin C to the polymer materials on the microplate by an antigen capture enzyme-linked immunosorbent assay.

BACKGROUND: Cystatin C is a low molecular weight protein of 13 kDa with an isoelectric point of 9.3. Its adsorption on the urine sampling containers may cause the underestimation of cystatin C levels. We newly developed an antigen capture enzyme-linked immunosorbent assay (ELISA) of sandwich method for measurement of adsorbed level. METHODS: We used a polystyrene microplates with 3 different polymers. These include high hydrophobic, low hydrophobic, and hydrophilic materials. Using the same microplate, the absorbed protein was measured by an antigen Capture ELISA, and calibration was conducted by an ordinary ELISA. RESULTS: In normal urine the concentrations of absorbed cystatin C levels to the 3 materials at day 1 were 0.50, 0.32-0.84 microg/l (median, interquartile range), 0.28, 0.21-0.37 microg/l, and <0.08, <0.08-0.09 microg/l in high hydrophobic, low hydrophobic, and high hydrophilic material, respectively. The absorption rate was 6%, 3%, and 1%, respectively. The adsorption is dependent on urine pH. It changes reciprocally with urine protein concentration. In pathologic urine, the absolute absorption level was <0.08 microg/l on the median, and the adsorption ratio (absorption level/urine level) was much less than 0.5% of that in normal urine. CONCLUSION: In the clinical setting, the absorption of cystatin C to sample containers is negligible since the rate of adsorption is low both in normal and pathologic urine. The material with high hydrophilic surface processing may be used for other proteins when interaction of the proteins with surface material affects the value to clinical decision.

Efficacy of titanium dioxide photocatalyst for inhibition of bacterial colonization on percutaneous implants.

The purpose of this study was to evaluate the efficacy of titanium dioxide photocatalyst in inhibition of bacterial colonization on percutaneous implants. Titanium dioxide photocatalyst was prepared by direct oxidization of pure titanium substrate, and a comparative study with pure titanium was performed. The bactericidal ability of the photocatalyst was examined using methicillin-resistant Staphylococcus aureus (MRSA) suspensions in a colony-forming assay according to the Japanese Industrial Standards committee standard. After exposing the MRSA suspension on sample plates to ultraviolet A (UVA) light, the number of surviving bacteria was estimated. Next, an animal model for inhibition of colonization was examined in vivo. Pins were inserted into the femurs of rabbits, were infected with 10(8) colony-forming units of MRSA suspension, and were illuminated with UVA light for 60 min daily; the number of colonizing bacteria was estimated after 7 days. The bactericidal ability of the photocatalyst was apparent after 60 min, when the bacteria had almost disappeared. The number of colonizing bacteria on photocatalytic pins was decreased significantly in vivo. The photocatalyst was effective even against resistant bacterial colonization. Clinically, the incidence of percutaneous implant infection such as pin tract infection in external fixation could be reduced using the titanium photocatalyst.

Sources of variation of commonly measured serum analytes in 6 Asian cities and consideration of common reference intervals.

BACKGROUND: In a previous study to determine the feasibility of common reference intervals in Asia, we found significant differences among populations from 6 cities. In this study, we attempted to define the sources of these differences. METHODS: We enrolled 580 healthy volunteers (279 men, 301 women, 20-62 years old), after a selection process that was based on the Clinical and Laboratory Standards Institute guidelines, and used a lifestyle questionnaire. All sera were obtained at a basal state and frozen at -80 degrees C until the collective assay was done. We measured 21 basic chemical analytes and 10 serum proteins. RESULTS: We used 3-level nested ANOVA to separate the variation (SD) into between-city (SD-city), between-sex (SD-sex), between-age (SD-age), and between-individual (SD-indiv) components. SD-indiv corresponds to one-quarter of the "pure" reference interval obtained after removing variations due to city, sex, and age. The SD-sex to SD-indiv ratio was >0.8 for creatinine, urate, retinol-binding protein, and transthyretin. We observed high SD-city to SD-indiv ratios, ranging from 0.4 to 0.7, for 11 analytes including lactate dehydrogenase (LDH), electrolytes, IgG, and complement components and SD-age to SD-indiv ratios >0.4 for LDH, alkaline phosphatase, and total cholesterol. Multiple regression analysis demonstrated several other relevant sources of variation, including body mass index, alcohol consumption, and cigarette smoking, although their contributions were generally smaller than those for sex, region, or age. CONCLUSION: We observed unacceptably large regional differences in measured values of some analytes even after adjustment for age, sex, and lifestyle variables. Genetic and environmental factors may account for the residual differences.

Urinary protein 1/Clara cell 16 concentrations and lung functions in male subjects with pneumoconiosis.

BACKGROUND: Protein 1 (P1)/Clara cell 16 kDa protein (CC16, previously named CC10), a potentially immunosuppressive protein secreted by non-ciliated cells of the tracheobronchial epithelium, has been found to be a new useful lung-specific biomarker in several pathological lung conditions. Particularly, urinary P1 (uP1) may reflect the altered lung functions in pneumoconiosis. METHODS: We investigated the relationship between uP1 values and lung functions in 31 non-smoking pneumoconiotic males (mean age 73 years) with a history of dust exposure work in shipbuilding. The protein was measured using an originally prepared enzyme-linked immunosorbent assay system. The forced expiratory volume in 1 s % (FEV(1.0)%) and % vital capacity (%VC) were tested with a spirometer. RESULTS: The mean values of uP1 were 4.62 +/- 4.82 (mean +/- standard deviation) ng/mol creatinine. A univariable correlation test showed a significant positive correlation between uP1 and %VC (r = 0.356, P = 0.049). Also, a multiple regression analysis, when adjusted for age, disease duration, FEV(1.0)% and %VC, showed a significant correlation of uP1 with %VC (beta = 0.467, P = 0.030). CONCLUSION: The results suggest that a decreased uP1, corroborated by a decreased %VC, may be the result of damage to secretory cells. Measurement of uP1 may become a possible index of fibrotic changes in pneumoconiosis.