Masayuki Totani

A useful ELISA system for human liver-type arginase, and its utility in diagnosis of liver diseases.

OBJECTIVES To develop a new ELISA system for liver-type arginase using monoclonal antibodies against the enzyme, and to verify the utility of the arginase in diagnosis of hepatic disorders. DESIGN AND METHODS We have developed an enzyme-linked immunosorbent assay (ELISA), using two kinds of monoclonal antibodies (Mo6G3 and Mo9C5) for human liver-type arginase as the first and second antibodies respectively. We have also developed a new method to eliminate the influence of erythrocyte-derived arginase contamination in hemolytic samples. This ELISA was applied to specimens received from patients with acute and chronic hepatic disease and also patients who had undergone partial hepatectomy. RESULTS This assay is sensitive and reproducible for the measurement of liver-type arginase in the sera of patients with liver dysfunction, and enabled us to detect enzyme concentrations as low as 27 pmol/L without any processing of the samples. The assay showed within-run coefficients of variation (CV) ranging from 1.9 to 4.1% and between-day CV from 3.6 to 5.1% for arginase concentrations varying from 57.1 to 1200 pmol/L. The recovery was 113% (mean) with a range of 96 to 129%. These antibodies reacted strongly with both recombinant and native liver-type arginases, while, to some extent, with erythrocyte-derived arginase. Correction for erythrocyte-derived arginase contamination in hemolytic samples was, however, easily made by assaying peroxidase-like activity of hemoglobin. From the view of a limited localization of arginase in the liver, the marked increase of the enzyme in serum reflects initiation of liver injury, while the rapid decrease reflects termination of the damage. Such quick normalization in circulating liver-type arginase indicated another merit of the enzyme in diagnosis of liver diseases. CONCLUSIONS The changes in circulating liver-type arginase level could be helpful not only in the diagnosis of liver diseases but also subsequent treatment of the patients with liver damage.

Liver-type arginase in serum during and after liver transplantation: a novel index in monitoring conditions of the liver graft and its clinical significance.

We quantified liver-type arginase in sera of 47 patients undergoing partial liver transplantation with use of an ELISA method. The level of liver-type arginase fluctuated slightly beyond the normal range in successful liver recipients, while it changed more drastically or precipitously in unsuccessful ones, accompanying or unaccompanying elevation of AST and ALT levels. A higher elevation pattern of the arginase level (above 100 ng ml-1) was observed in each of the unsuccessful recipients with critical condition, except for one patient. Other hepatic markers (LDH, ALP, and T-BIL) remained relatively unchanged until the terminal stage of deceasing patients. The finding that the liver-type arginase emerged in large quantity in the blood stream immediately after reperfusion of the liver graft indicates that the enzyme leaks out of hepatocytes damaged, presumably, by storage in the absence of circulation. A half-life of the liver-type arginase in the human blood was estimated to be 1 h, that is clearly shorter than that of AST. The short half-life of the arginase appears to be ascribable, at least partly, to formation of an immune complex with circulating autoantibody which appears in many liver recipients. These results suggest that liver-type arginase behaves uniquely in the serum among many hepatic enzymes, and could serve as a distinct marker of hepatic lesions, particularly during and after liver transplantation.

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.

Salivary indicators of protein nutritional status in the elderly

Abstract Protein-energy malnutrition (PEM) is a serious nutritional problem in hospitalized elderly patients. Serum albumin, transthyretin (prealbumin), and total protein have been used as biochemical indicators of protein nutritional status, but taking blood from the elderly is difficult and invasive. We therefore assessed the possibility of using saliva or urine as noninvasive materials to estimate serum concentrations of albumin, transthyretin and total protein. Blood, saliva and urine were collected from 32 hospitalized elderly (aged 68–97 y) and the correlation between the concentrations of albumin, transthyretin and total protein in serum and those in saliva or urine were evaluated. There was no correlation among them, but the concentrations of albumin and transthyretin adjusted for the concentration of IgG in saliva were significantly correlated with the concentrations of albumin and transthyretin in serum (albumin: R 2 = 0.308, p=0.0010, transthyretin: R 2 = 0.433, p

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.

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.

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.

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-β-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 εN-2,4-dinitrophenyl-L-lysine and transferred to polystyrene balls coated with anti-human IgA α-chain. Bound β-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.

Effects of Fish or Beef Ingestion in Common Japanese Diet with Low Dietary Linoleic Acid on Plasma and Erythrocyte Membrane Fatty Acid Composition in Young Adult Females.

脂肪酸組成の異なる油脂を含む食事の摂取が血漿等の脂肪酸組成に及ぼす影響を検討するため, 女子学生12名を6名ずつのA, B2群に分け, クロスオーバー試験(A群: 魚肉-牛肉, B群: 牛肉-魚肉) により魚肉または牛肉を主菜とする日常的食材を用いた実験食を各5日間摂取させた。実験食摂取による血漿および赤血球膜中脂肪酸組成の変動は以下のとおりである。1) 血漿中脂肪酸組成は, 魚肉食によりA, B両群ともEPA, DHAが増加し, リノール酸, オレイン酸が減少した。牛肉食では, A, B両群ともオレイン酸が増加し, EPA, DHAが減少した。これらの変化は実験食切り替え後短期間で起こるため, A群10日目の変動はB群5日目の変動と, B群10日目の変動はA群5日目の変動と類似のパターンを示した。2) 赤血球膜中では, A, B両群とも最初の5日間でオレイン酸が増加し, リノール酸, パルミチン酸, DHAが減少した。実験食の切り替えによるクロスオーバーの影響は少なかった。その結果, 10日目の変動はA群, B群とも類似パターンとなった。3) 以上のことから, 血漿中脂肪酸組成変動に対する carry-over effect は大きくなく, 摂取脂質によって短期間に絶えず影響を受ける傾向があるのに対して, 赤血球膜中脂肪酸組成は, 短期間では摂取脂質よりもLAの低値によって影響される傾向のあることが明らかになった。

Sensitive Enzyme Immunoassay for Anti-β-Lactoglobulin IgG in Serum

A sensitive enzyme immunoassay for anti-β-lactoglobulin immunoglobulin G (IgG) in serum is described. Serum containing anti-β-lactoglobulin IgG was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-β-lactoglobulin conjugate and β-lactoglobulin-peroxidase conjugate. The complex formed from the three components was trapped onto polystyrene balls coated with anti-2,4-dinitrophenyl group IgG, eluted with ε-2,4-dinitrophenyl-L-lysine and transferred to polystyrene balls coated with anti-human IgG-γ-chain IgG. Bound peroxidase activity was determined by fluorometry. This enzyme immunoassay was 100- to 1000-fold more sensitive and more reliable than the enzyme-linked immunosorbent assay (ELISA). Anti-β-lactoglobulin IgG was detected in 91% of healthy subjects using this method.