A survey of the reactivity of in vitro diagnostic bilirubin reagents developed in Japan using artificially prepared bilirubin materials: A comparison of synthetic delta, unconjugated, and taurine-conjugated bilirubin.

BACKGROUND: In vitro diagnostic bilirubin reagents based on oxidation with bilirubin oxidase or vanadic acid for total and direct-reacting bilirubin are widely used in Japan; however, their reactivity to unconjugated and conjugated bilirubin and delta bilirubin has not been completely disclosed by manufacturers. We used artificially prepared bilirubin materials to investigate the reactivity with four in vitro diagnostic bilirubin reagents. METHODS: Porcine unconjugated bilirubin solution, chemically synthesized ditaurobilirubin solution, and chemically synthesized delta bilirubin solution were used as surrogates of naturally occurring unconjugated bilirubin, conjugated bilirubin, and delta bilirubin, respectively. The total bilirubin and direct-reacting bilirubin concentrations were measured by three bilirubin oxidase methods and one vanadic acid method, and the observed concentrations were compared with those obtained by the diazo-based reference measurement procedure. RESULTS: The unconjugated bilirubin and delta bilirubin concentrations were similar when any of the four in vitro diagnostic bilirubin reagents were used during total bilirubin measurement. This was consistent with reference measurement procedure and exhibited a converged inter-method variation. Compared with reference measurement procedure, significantly low ditaurobilirubin concentrations were observed by the in vitro diagnostic bilirubin reagents despite the converged inter-method variation. In delta bilirubin measurement, some reagents reacted doubtfully with unconjugated bilirubin, while showed lower ditaurobilirubin concentrations than its corresponding total bilirubin concentration. Reactivity with delta bilirubin was different for each method including reference measurement procedure. Some reagents were developed to react less with delta bilirubin and others to strongly react with delta bilirubin. CONCLUSIONS: We revealed the reactivity of IVD-TB and IVD-DB reagents to artificially prepared bilirubin materials, and their consistency with reference measurement procedure. The delta bilirubin data results vary depending on the reagents used.

Surveillance evaluation of the standardization of assay values for serum total 25-hydroxyvitamin D concentration in Japan.

Background To assess the vitamin D nutritional status, serum total 25-hydroxyvitamin D (25(OH)D) concentration is measured. We used six automated 25(OH)D immunoassays (AIAs) available in Japan and certified by the Vitamin D Standardization Program (VDSP) at the U.S. Center for Disease Control and Prevention to assess the concordance of the assay results. Methods Serum total 25(OH)D concentrations in SRM 972a and 20 serum samples from patients were determined using three liquid chromatography-tandem mass spectrometry (LC-MS/MS) and six AIAs (pilot study), and an additional 110 serum samples were assessed by the six AIAs (surveillance study). The assay bias from the results of LC-MS/MS by Chiba University or consensus values (i.e. average of six AIAs) was estimated using the procedure described in CLSI document EP09-A3. Results LC-MS/MS at Chiba University could completely separate 25(OH)D2, 25(OH)D3 and 3-epi-25(OH)D3, and the observed values including total 25(OH)D in SRM 972a were all within ±1·SD of the assigned values. All AIAs produced results greater than ±3·SD. In the pilot study, four of the six AIAs had an average percentage bias, as estimated by confidence interval (CI), larger than ±5% (acceptance criterion in CLSI); the bias converged from -6.5% to 3.2% after adjustment by LC-MS/MS. In the surveillance study, 25(OH)D concentrations in AIAs all adjusted to LC-MS/MS converged within ±5% from consensus values. However, some AIAs showed negative or positive bias from the consensus values. Conclusions Current AIAs in Japan continue to lack standardization. Manufacturers should implement quality assurance strategies so that their values more closely align to those of standard reference material 972a.

Dynamic mobility of immunological cells expressing S100A8 and S100A9 in vivo: a variety of functional roles of the two proteins as regulators in acute inflammatory reaction.

The immunological properties of rat S100A8 (r-S100A8) and S100A9 (r-S100A9) in immune cells are poorly understood. Enzyme-linked immunosorbent assay (ELISA) for r-S100A9 enabled us to discuss the differential functional roles of the two proteins, and their localization in the cells was observed microscopically. Recombinant human S100A8 (rh-S100A8) or S100A9 (rh-S100A9) were intravenously administrated into rats with LPS-induced liver damage. ELISA was used to measure the serum concentration of S100A9 in the rats. Western blotting and a preparative ELISA were used to prove specificity and avidity of monoclonal antibodies for r-S100A8 and r-S100A9. Immunohistochemical staining was carried out to visualize intracellular localization of the two proteins in the immune cells using the antibodies. When rh-S100A8 was intravenously injected in the rats (B group), the serum concentration of r-S100A9 apparently decreased as compared with that of the positive control rats (A group). The activities of AST, ALT, and LD in the rat sera (B group) also significantly went down in comparison with those of the rats (A group). Although both the S100A8 and S100A9 were abundantly expressed in activated immune cells, quite difference of not only their intracellular localization but also distribution of the cells expressing the two proteins was microscopically observed. In the rats (B group), less number of the immune cells or less amount of r-S100A8 and r-S100A9 in the cells than those of the rats (A group) was also seen. The r-S100A8 could serve as a regulator of acute inflammatory reaction in the rats with LPS-induced damage.

Commutability of National Institute of Standards and Technology standard reference material 1955 homocysteine and folate in frozen human serum for total folate with automated assays.

BACKGROUND: The aim of the present study was to evaluate standard reference material (SRM) 1955 commutability as a reference material for serum folate using automated methods. We also designed so as to reduce the intermethod variability present in different automated methods. METHODS: Using a microbiological assay related to the 'information value' of SRM 1955 as a comparison method, we investigated the possibility of standardization for the assay values of serum folate as measured by the automated methods (Access, Centaur and Elecsys). In the assay of 50 patient sera by these automated methods, we corrected observed values by the SRM 1955 and compared with comparison values. RESULTS: The observed values of SRM 1955 Levels I, II and III were within or outside (but near) a 95% prediction interval obtained from patient sera by the automated methods. The normalized residuals obtained from SRM 1955 were within ±3.0 (in SD units), which enabled us to conclude that the SRM 1955 had a physicochemical characterization similar to native serum. Twelve patients were assessed as hypofolataemia (<6.0 ng/mL) and 38 patients as normal (≥6.0 ng/mL). Before correction, folate levels in six of 12 patients were lower than 6.0 ng/mL, and those in seven of 38 patients were higher than 6.0 ng/mL with the automated methods. After correction, low levels were found in four of 12 patients, and normal levels were found in 33 of 38 patients. CONCLUSIONS: The use of SRM 1955 would help to reduce the intermethod variability present in different automated methods for serum folate measurement.

Identification of serum proteins that bind with S100A8, S100A9 and S100A8/A9: clinical significance of using proteins for monitoring the postoperative condition of liver recipients.

BACKGROUND: Serum proteins that non-specifically bind with human S100A8/A9 (h-S100A8/A9) have been proposed. Our aim was to isolate and identify these proteins, and verify their clinical significance for monitoring the postoperative condition of liver recipients, and further to discuss the transportation of human fibronectin (h-FN) with h-S100A8/A9 and its functional role in vivo. METHODS: To isolate the serum proteins, recombinant human S100A8, S100A9 and S100A8/A9 affinity columns were used. Proteins were identified by mass spectrometry. Two enzyme-linked immunosorbent assays (ELISA) were used to measure h-S100A8/A9 and h-FN in the sera of liver recipients. Flow cytometry was employed to detect h-S100A8/A9 and h-FN on immunological cells. Western blotting was used to confirm serum constituents using antibodies specific to each constituent. RESULTS: One of the proteins was identified with h-FN, and its fluctuation pattern in the serum of the recipient was in contrast to that of CRP. Flow cytometry showed a positive reaction for h-S100A8/A9 and h-FN on neutrophils and monocytes, indicating that both proteins exist on these cells. CONCLUSIONS: The h-FN could be transported with S100A8/A9 in blood and/or on immunological cells, and effectively prevent further attack by various internal oxidants or repair damaged liver tissue in vivo.

[Possible mechanism for regulation of inflammatory responses with the S100A8/A9 protein].

We have described a possible mechanism for the regulation of excessive inflammatory responses with S100A8/A9 protein in damaged rat livers. Recombinant human S100A8(r-S100A8) and S100A9 (r-S100A9) were expressed in E. coli cells, and their heterodimer (r-S100A8/A9) with 90% approximate purity was also prepared successfully. The effect of the r-S100A8/A9 on suppression of acute inflammatory changes in rat livers with LPS-induced damage was microscopically observed. Indeed, the liver damage diminished as the dose of the r-S100A8/A9 increased, and the minimum requirement of the protein was estimated to be 1,000 microg/rat in this study. Observation of superoxide anions was positively observed in control rats treated with LPS alone, but almost not in the livers of rats treated with the r-S 100A8/A9 1h after injection of LPS. This fact strongly suggests that the r-S100A8/A9 could indirectly suppress production of such internal oxidants according to unknown pathway (s) in acute inflammation. Expression of mRNAs of several kinds of inflammatory cytokines, such as TNF-alpha, IL-6 and IL-1beta, was also significantly suppressed, which was of much note. Therefore, the possibility that the r-S100A8/A9 partly inhibits the process of signal transduction of inflammatory responses in the immunological cells leading to down regulation of inflammatory changes in vivo was suggested in this study. Conclusively, S100A8/A9 is not necessarily an inflammatory-induced factor, and preferably effective on suppression of excessive inflammatory reaction in vivo dose-dependently, although the mechanism is still unclear.

[Possibility of formation of the S100A8/A9-proinflammatory cytokine complexes in vivo in acute inflammation and their functional roles].

We previously hypothesized that S100A8/A9 binds with several kinds of proinflammatory cytokines, such as TNF-alpha, IL-6 and IL-1beta, to form the S100A8/A9-proinflammatory cytokine complexes in vivo in acute inflammation, leading to subsidence of inflammatory responses. Our goal was to verify the presence of these complexes in liver tissues of rats with lipopolysaccharide (LPS) induced damage. We firstly prepared two kinds of the full-length cDNA encoding amino acid sequences of human S100A8 and S100A9 proteins, and constructed their pCold-I expression vectors. The recombinant S100A8 and S100A9 were successfully expressed in E. coli, and then purified by Ni-agarose columns, respectively. The S100A8/A9 was noncovalently synthesized in 2.0 mol/1 Tris-NaOH solution (pH 12) using the purified S100A8 and S100A9. After purification, this heterodimer (1 mg) was intraperitoneally injected into a rat 1h after injection of LPS. Two kinds of ELISA systems were used to detect the S100A8/A9-inflammatory cytokine complexes in the rat liver tissue. As determined by the ELISA-A and B, the reaction was apparently positive and quantitative. Immunohistochemistry provided such complexes-positive cells in the liver with damage. The S100A8/A9-positive cells almost corresponded to the cytokines-positive ones morphologically, strongly suggested the presence of the S100A8/A9-proinflammatory cytokine complexes. In conclusion, the possibility that these complexes were formed in vivo and accumulated to the immunological cells, such as macrophages and/or activated neutrophils, was indicated. Our effort is currently addressed to isolate the S100A8/A9-proinflammatory cytokine complexes using biochemical techniques, and to comprehensively resolute their clinical significance in the differential diagnosis of inflammatory diseases.

Intrinsic function of S100A8/A9 complex as an anti-inflammatory protein in liver injury induced by lipopolysaccharide in rats.

BACKGROUND: We hypothesized that the S100A8/A9 complex is effective in the suppression of acute inflammatory changes. METHODS: To clarify such a functional role of the S100A8/A9 complex in acute inflammatory disorder, the complex purified from human leukocytes (approx. 1 mg) was intraperitoneally injected into rats 1.0 or 3.5 h after an injection of lipopolysaccharide (LPS). RESULTS: The serum concentrations of interleukin-6 (IL-6) and nitric oxide (NOx) were significantly decreased in the treated rats. Conversely, when anti-S100A8/A9 complex IgG was injected into the tail blood vessel of a rat 1.0 h after the injection of LPS, the serum concentration of IL-6 increased slightly, indicating that the antibody immunoregulatorily blocked the activity of the complex as an anti-inflammatory protein in vivo. In addition, the S100A8/A9 complex bound non-specifically with interleukin-1beta (IL-1beta), IL-6 and TNF-alpha in vitro, suggesting that the complex could bind with these cytokines in vivo. A large number of endogenous S100A8/A9 complex-positive cells that accumulated in the inflamed region in the liver 6 h after the injection of LPS were microscopically observed, while apparent inflammatory changes were not found microscopically in other organs, such as the kidney, lung and spleen. In rats treated with the S100A8/A9 complex, neither acute inflammatory changes nor S100A8/A9 complex-positive cells were also observed microscopically in the liver tissue. CONCLUSIONS: These findings suggest that the S100A8/A9 complex indirectly suppresses the overproduction of NOx from activated neutrophils and/or macrophages by neutralizing the activity of pro-inflammatory cytokines. Thus, the S100A8/A9 complex may play an important role in the suppression of acute inflammation by modulating the vital activity of pro-inflammatory cytokines in vivo.

Values of water-soluble vitamins in blood and urine of Japanese young men and women consuming a semi-purified diet based on the Japanese Dietary Reference Intakes.

We investigated the levels of water-soluble vitamins except for vitamin B6 in the blood and urine of Japanese college male (n = 10) and female (n = 10) students. They consumed for 7 d a semi-purified diet based on Japanese Dietary Reference Intakes to assess the Recommended Dietary Allowances (RDA) for water-soluble vitamins and to present some new normal values for blood and urine levels of water-soluble vitamins in Japanese. The blood and the 24-h urine samples were collected on the last day of the experiment and measured. The values of total vitamin B1 in whole blood, total vitamin B2 in whole blood, total cyanocobalamin in serum, total nicotinamide in whole blood, total pantothenic acid in whole blood, total folates in serum, total biotin in serum, and ascorbic acid in plasma were 104+/-17 pmol/mL (mean+/-SD), 216+/-25 pmol/mL, 0.34+/-0.05 pmol/mL, 59.1+/- 5.0 nmol/ mL, 2.45+/-0.37 nmol/mL, 15.6+/-4.6 pmol/mL, 8.3+/-0.5 pmol/mL, and 62+/-10 nmol/mL, respectively, in males, and 90+/-23, 234+/-18, 0.67+/-0.20, 61.9+/-6.0, 2.48+/-0.30, 30.2+/-8.6, 8.4+/-0.3, and 67+/-14, respectively, in females. There was a significant difference in the values of cyanocobalamin and total folates between men and women. The urinary excretion of vitamin B1, vitamin B2, cyanocobalamin, sum of the catabolic metabolites of nicotinamide, pantothenic acid, folates, biotin, and ascorbic acid were 665+/-114 nmol/d, 562+/-325 nmol/d, 93+/-31 pmol/d, 84+/-26 micromol/d, 9.3+/-2.3 micromol/d, 19.4+/-2.8 nmol/d, 83+/-18 pmol/d, and 148+/-51 micromol/d, respectively, in males, and 495+/-212, 580+/-146, 145+/-49, 83+/-19, 16.9+/-1.3, 22.7+/-2.7, 83+/-23, and 140+/-51, respectively, in females. There was a significant difference in the urinary excretion of cyanocobalamin, pantothenic acid and total folates between men and women. These values will be useful for the nutritional assessment of water-soluble vitamins for Japanese, although the examination period was short. In future, an experiment with various age groups and re-evaluation by repeated experiments will provide more accurate values.

Immune complex transfer enzyme immunoassay for anti-ovalbumin IgA in serum.

BACKGROUND: An immune complex transfer enzyme immunoassay for antiovalbumin immunoglobulin A (IgA) was developed. METHODS: Serum-specific antibody was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-ovalbumin conjugate and ovalbumin-beta-D-galactosidase conjugate. The complex formed from the three components was trapped onto polystyrene balls coated with anti-2,4-dinitrophenyl group immunoglobulin G, eluted with epsilonN-2,4-dinitrophenyl-L-lysine and transferred to polystyrene balls coated with anti-human IgA alpha-chain. Bound beta-D-galactosidase activity was determined by fluorometry. RESULTS & CONCLUSIONS: The detection limit of this method for the measurement of specific anti-ovalbumin IgA was 9 fmol/tube, which was 20-fold lower than that of the enzyme-linked immunosorbent assay (ELISA). Because serum interference with this method was lower than that with the ELISA, the detection limit of this method was 300-fold lower than that by the ELISA. Anti-ovalbumin IgA was detected in 100% of healthy subjects, which was confirmed by pre-incubation with an excess amount of ovalbumin.