Di-(2-ethylhexyl) phthalate suppresses IL-12p40 production by GM-CSF-dependent macrophages via the PPARα/TNFAIP3/TRAF6 axis after lipopolysaccharide stimulation.

Activation of peroxisome proliferator-activated receptor α (PPARα) by di-(2-ethylhexyl) phthalate (DEHP) has an anti-inflammatory effect. This study investigated the potential combined influence of PPARα, tumor necrosis factor α-induced protein 3 (TNFAIP3/A20), and tumor necrosis factor receptor-associated factor 6 (TRAF6) on interleukin (IL)-12p40 production by macrophages exposed to DEHP and stimulated with lipopolysaccharide (LPS). LPS upregulated IL-12p40 expression by granulocyte-macrophage colony-stimulating factor-dependent macrophages (on day 9 of culture), whereas adding DEHP to cultures significantly attenuated the response of IL-12p40 to LPS stimulation. PPARα protein was also reduced by DEHP. Interestingly, transfection of macrophages with small interfering RNA (siRNA) duplexes for PPARα, TNFAIP3/A20, or dual oxidase 2 restored the response of IL-12p40 protein to LPS stimulation in the presence of DEHP. siRNAs for various protein kinase Cs (PKCs) (α, ß, γ, or δ) also restored IL-12p40 production by macrophages exposed to LPS and DEHP. While LPS upregulated both IL-12p40 and TNFAIP3/A20 production, adding DEHP to cultures dramatically reduced IL-12p40 and TNFAIP3/A20 levels. Silencing of PKCα reduced TNFAIP3/A20 production, whereas PKCγ siRNA (but not PKCß or δ siRNA) significantly increased TNFAIP3/A20. TRAF6 was also attenuated by macrophages with DEHP. The PPARα/TNFAIP3/TRAF6 axis may have an important role in the mechanism through which DEHP reduces IL-12p40 production by LPS-stimulated macrophages.

Midkine as a prognostic biomarker in oral squamous cell carcinoma.

The aim of this study was to evaluate serum midkine (S-MK) concentrations as a prognostic tumour marker in oral squamous cell carcinoma (OSCC). We measured S-MK concentrations in patients with OSCC and healthy volunteers. In addition, we performed real-time quantitative reverse transcription-PCR analysis and immunohistochemistry with fresh tumour samples. To determine whether S-MK concentrations have prognostic value, we performed survival analyses with clinical information by using the log-rank test. Serum midkine concentrations were significantly higher in patients with OSCC than in healthy controls (P<0.001). Serum midkine concentrations were also significantly increased in early-stage OSCC compared with those of healthy individuals (P<0.001). In addition, immunohistochemistry allowed identification of overexpressed MK protein in OSCC tissues. MK mRNA showed higher expression in OSCC samples compared with normal mucosal samples. Patients in high S-MK groups showed a significantly lower 5-year survival rate compared with patients in low S-MK groups (P<0.05). The increased S-MK concentrations in early-stage OSCC were strongly associated with poor survival. Serum midkine concentrations may thus be a useful marker not only for cancer screening but also for predicting prognosis of OSCC patients.

Homogeneous assay for measuring low-density lipoprotein cholesterol in serum with triblock copolymer and alpha-cyclodextrin sulfate.

We have developed a fully automated method for measuring LDL-cholesterol (LDL-C) in human serum without the need for prior separation, using a nonionic surfactant, polyoxyethylene-polyoxypropylene block copolyether (POE-POP), and a sodium salt of sulfated cyclic maltohexaose, alpha-cyclodextrin sulfate. Of the surfactants tested, POE-POP with a higher molecular mass of the POP block and a greater hydrophobicity reduced the reactivity of cholesterol in lipoprotein fractions; the reactivity in descending order was LDL >> VLDL > chylomicron approximately HDL. Gel filtration chromatographic studies revealed that POE-POP removed lipids selectively from the LDL fraction and allowed them to participate in the cholesterol esterase-cholesterol oxidase coupling reaction system. By contrast, alpha-cyclodextrin sulfate reduced the reactivity of cholesterol, especially in chylomicrons and VLDL. A combination of POE-POP with alpha-cyclodextrin sulfate provided the required selectivity for the determination of LDL-C in serum in the presence of magnesium ions and a small amount of dextran sulfate without precipitating lipoprotein aggregates. There was a good correlation between the results of LDL-C assayed by the proposed method and the beta-quantification reference method involving 161 sera with triglyceride concentrations ranging from 0.3 to 22.6 mmol/L.

A novel automated assay for malate dehydrogenase isoenzymes.

We have developed a new automated method for the determination of malate dehydrogenase (MDH) isoenzymes in serum employing guanidine hydrochloride. Our proposed method showed good reproducibility; within-run precision coefficient of variations (CVs) were less than 2.5 (mean 13.6-42.9 U/L) for total MDH (T-MDH) and less than 6.7% (mean 6.3-23.5 U/L) for mitochondrial MDH (m-MDH) (n = 10). The upper detection limit of the proposed method exhibited good linearity up to 1,000 U/L for both T-MDH and m-MDH. In the proposed m-MDH reagent, the presence of up to 2,000 U/L of cytosolic MDH(c-MDH) activity had no effect on the outcome of m-MDH assay. Results of our proposed method (y) correlated well with those of the electrophoretic method (x) giving the regression equation: y = 1.46 x + 6.87 (N = 30); r = 0.99. Normal concentrations of various anticoagulants and bilirubin did not affect the assay results. Both ascorbic acid and glucose exhibited a slight positive interference with the proposed assay. Clinically, we found that m-MDH activity in serum had greater diagnostic predictive value than T-MDH activity for judging successful outcome of reperfusion therapy; the prognosis was poor when the m-MDH/T-MDH ratio was greater than 20%.

Measurement of Lp(a) with a two-step monoclonal competitive sandwich ELISA method.

OBJECTIVE: To evaluate the results of Lipoprotein (a)[Lp(a)] measurements by a competitive two-step monoclonal enzyme-linked immuno sorbent assay method comparing them with those by a conventional ELISA. METHODS: Serum having various isoforms of Lp(a) and purified Lp(a) were assayed using the method described here and commercially available kits. The reference range was determined with the use of 324 normal subjects by means of calculation from Lp(a) results of logarithmic transformation. RESULTS: Our method takes advantage of a competitive reaction between fixed antibody and free antibody to Lp(a), having the detection range up to 1000 mg/L with the lowest detection limit of 2 mg/L. The anti-Lp(a) monoclonal antibody employed in the assay system reacts uniformly with all phenotypes of Lp(a) but showing very low cross-reactivity for plasminogen and LDL. Within-run and between-run precisions were excellent, giving CVs of 2.9 and 4.0% with mean values of 145 and 635 mg/L, respectively. In comparison of the results by our method with those by a polyclonal method (Biopool) or a monoclonal antibody method (Terumo), they correlated well; Y (our method) = 0.99 x (polyclonal method, Biopool) - 1.9, r = 0.994 (n = 60), and Y = 0.94 X(monoclonal method, Terumo) -9.8, r = 0.97 (n = 60), respectively. The reference range was 105.9 +/- 25.4 mg/L, the difference between the sexes was not significant. CONCLUSION: Our method has proven highly accurate and specific. It is applicable with auto analyzer because it does not require such a pre-dilution step as is necessary for Lp(a) determination by conventional ELISA assay. Accordingly, we can conclude that our test method is workable for both clinical laboratories and mass screening.

Direct measurement of high-density lipoprotein cholesterol in serum with polyethylene glycol-modified enzymes and sulfated alpha-cyclodextrin.

We have developed an automated method for measuring high-density lipoprotein (HDL)-cholesterol in serum without prior separation, using polyethylene glycol (PEG)-modified enzymes and sulfated alpha-cyclodextrin. When cholesterol esterase and cholesterol oxidase enzymes were modified with PEG, they showed selective catalytic activities towards lipoprotein fractions, with the reactivity increasing in the order: low-density lipoprotein < very-low-density lipoprotein approximately chylomicron < HDL. In the presence of magnesium ions, alpha-cyclodextrin sulfate reduced the reactivity of cholesterol, especially in chylomicrons and very-low-density lipoprotein, without the need for precipitation of those lipoprotein fractions. The combination of PEG-modified enzymes with alpha-cyclodextrin sulfate provided selectivity for the determination of HDL-cholesterol in serum in the presence of a small amount of dextran sulfate without the need for precipitation of lipoprotein aggregates. The results of the HDL-cholesterol assayed in serum by this direct method correlated well with those obtained by precipitation-based methods and also that by an ultracentrifugation method.

Comparison of monoclonal and polyclonal enzyme-linked immunoabsorbent (ELISA) assays for serum Lp(a) and differences in reactivities to Lp(a) phenotypes.

We have prepared a monoclonal antibody to Lipoprotein(a)[Lp(a)] and have used it to develop an ELISA test for assaying Lp(a) in serum. The monoclonal antibody employed in the assay system reacts uniformly with S1, S2, S3 and B phenotypes of isoforms, and no cross-reaction with plasminogen at a concentration of 100 mg/dL was observed. Results of the monoclonal ELISA assay were similar to those obtained with a polyclonal antibody ELISA method and demonstrated a correlation coefficient, r = 0.99 with the equation for the regression line: Y(proposed) = 1.06 x (polyclonal antibody reference ELISA test) = 0.36 (N = 51). Inter- and intra-assay precision(CVs) of the monoclonal ELISA assay were between 2.2-3.6% at a mean Lp(a) concentration range of 19.1-68.2 mg/dL,(N = 12). Assay results of various standards were compared by both monoclonal and polyclonal antibody ELISA tests. We observed some discrepancies between expected concentrations and the polyclonal antibody ELISA assay results, which is thought to be more uniformly reactive to the various Lp(a) phenotypes. The monoclonal antibody employed in our proposed method reacts uniformly with Lp(a) phenotypes, and the assay exhibits excellent sensitivity, specificity, and accuracy and is well suited for clinical use.

Simultaneous automated measurement of serum total CK and CK-MM isoform ratio in serum.

We automated a two-step kinetic procedure for determining serum CK-MM isoform ratio using an immunoinhibition method. By measuring the total CK activity and the residual CK activity (serum CK-MM isoform) remaining after the inhibition by tissue CK-MM isoform specific monoclonal antibody reagent (CK-M01) the CKMM isoform ratio is calculated using the difference between total CK and residual CK activities divided by the residual CK activity. Linearities of total CK and residual CK assays were < or = 7750 U/L and 2,500 U/L, respectively; within-run CVs of isoform ratio (N = 10) were 2.8 and 7.0% (mean 0.14 and 0.60), respectively. The MM3/MM1 isoform ratio obtained with the proposed method (X) correlated well with the results of electrophoretic method (Y) according to the equation: Y = 0.98X-0.3, r = 0.988. The normal reference range of isoform ratios obtained by assaying 1,222 serum samples from healthy subjects was 0.09-0.75. The isoform ratio increased after onset of chest pain, peaking at 2-6 hr thereafter. A mean isoform ratio of 1.86 was obtained with serum sample from 86 patients diagnosed as having an acute myocardial infarction (AMI). This method is accurate and highly sensitive, as the detection and early diagnosis of AMI can be completed in 10 min.

Preliminary findings on the variation of serum apolipoprotein levels in neural degenerative disorders.

We measured six apolipoproteins (AI, AII, B, CII, CIII, and E) in the serum of patients with several kinds of neural diseases [diabetic neuropathy and neural degenerative disorders (motor neuron degenerative disorders, spinocerebellar degeneration, Parkinson's disease)], comparing them to the age-matched healthy controls using the immunoturbidimetric method. Statistically significant decreases of serum apo-AI, apo-A-II and increases of apo-CIII, apo-E were observed in neural degenerative diseases; and, particularly, higher apo-B and apo-CII concentrations were observed in diabetic neuropathy. Most neural degenerative disease showed lower apo-AII. However, in motor neuron degenerative disorders, higher apo C-II and apo-E were seen. Lower apo-AI was seen in spinocerebellar degeneration, and lower apo-AII was seen in Parkinson's disease. Higher apo-B, CII, and E levels were observed in females with spinocerebellar degeneration and Parkinson's disease, and lower apo-AII was seen in males with spinocerebellar disease.

[Evaluation of serum troponin T measurement in acute myocardial infarction].

A monoclonal solid phase enzyme immunoassay has been developed for the detection of human troponin T. The serum troponin T levels in healthy subjects gave 0.05 +/- 0.06 ng/ml in total (n = 176), 0.06 +/- 0.07 ng/ml in males (n = 79) and 0.03 +/- 0.05 ng/ml in females. Within-run and between-run precision (CVs) of the assay were less than 5%. Various common interferents tested did not affect on the assay, but higher titer of rheumatoid factor, and anti-coagulants such as EDTA, heparin oxalate and citrate affected the assay. In all patients with defined acute myocardial infarction, serum troponin T levels increased 7 to 10 folds the upper reference range within 6 hours after the onset of chest pains and maximum elevation of serum troponin T level was at around 20 hours and its levels remained elevated for 7 to 20 days. Specificity and sensitivity for acute myocardial infarction was 92.4% and 100%, respectively. The results indicated that troponin T measurement improved the diagnostic efficiency for the detection of myocardial necrosis as compared with conventionally used cardiac enzymes and was an effective tool for the confirmation of the reperfusion by PTCA and PTCR.

Measurement of serum myoglobin by a turbidimetric latex agglutination method.

We evaluated an immunoturbidimetric quantitation for serum myoglobin by the latex agglutination method using an automated biochemical analyzer. This method is rapid, specific, accurate, precise, and has wide dynamic range. The total assay time is 10 min and is performed at 37 degrees C with continuous monitoring at 570 nm. The assay results were compared with radioisotopic immunoassay results and showed a good correlation coefficient, r = 0.99; Y = 0.98 x + 9.3; N = 79. Sera from healthy adults has a myoglobin concentration in the range of 15-80 ng/ml(N = 362). Sex- and age-related differences were observed. The serum myoglobin levels in males and elderly people showed higher concentration than in females and younger people. The peak elevation of serum myoglobin compared with other cardiac markers was observed within 6 hours after onset of chest pain as well as the CK-isoform ratio (MM3/MM1). All of the serum from 21 patients with definite acute myocardial infarction showed increased serum myoglobin levels (100-1200 ng/ml) upon admission and within 6 hours. The results suggest that assays for serum myoglobin levels are helpful in the early diagnosis of acute myocardial necrosis.

[Immunoinhibitory determination of CK-MM subbands by monoclonal antibody].

The measurement of CK-MM isoform by monoclonal antibody and its clinical application were summarized. The tissue type of CK-MM3 isoform was completely inhibited, MM2 isoform was about 57% and the serum type of CK-MM1 isoform was not inhibited by use of monoclonal antibody. This method enabled selective immunoinhibitory measurement of tissue type of CK-MM isoform. The heat lability and inhibition by EDTA suggested that it is a metal-depending enzyme. The analysis of MM isoform offers a promising alternate, non-invasive method to detect and follow up AMI and successful reperfusion. The MM3/MM1 ratio has been found to peak at about 2 to 6 hours, making it a quicker responding parameter to AMI than MM3 alone and significantly more responsive than the total CK or CK-MB. An MM3/MM1 ratio greater than 1.0 appears to be a practical cutoff point for detection of the pathological release of MM3 from tissue. Reperfusion therapy should be instituted within about 2 to 4 hours of AMI onset.

[Clinical significance of creatine kinase isoform assay by a new reagent based on immunoinhibition method].

Clinical significance of creatine kinase (CK) isoform assay by a new diagnostic reagent based on immunoinhibition method was evaluated. In the method, a monoclonal antibody (CKM-G 01), which inhibits MM3 but not MM1 isoform, was used. Serial serum samples from 143 acute myocardial infarction (AMI) patients were examined in hospitals. Isoform ratio (I.R.: defined as follows; inhibited CK activity/uninhibited CK activity) increased most rapidly (whose peak appeared 8.5 h after onset of AMI), compared with total CK (18.3 h) and CK-MB (16.1 h). In 61 patient samples collected within 4 h from onset, I.R. in 25 samples (41%) exceeded reference interval, whereas total CK in only 17 samples (28%) did it. Reference interval of the I.R. was 0.42 +/- 0.33 (mean +/- SD) in 1,246 normal subjects. These results show that the assay of CK isoforms by the reagents based on immunoinhibition method is useful for earlier detection of AMI.

Measurement of human ventricular myosin light chain-1 by monoclonal solid-phase enzyme immunoassay in patients with acute myocardial infarction.

A monoclonal solid-phase enzyme immunoassay has been developed for the detection of human serum ventricular myosin light chain-1. Cross-reactivity of this with human skeletal muscle myosin was observed, but the enzyme immunoassay with the sera of patients with acute myocardial infarction gave similar results with radioimmunoassay. The human ventricular myosin light chain-1 levels in the healthy subjects were 0.2-6.6 ng/ml in males and 0.2-4.1 ng/ml in females. Within-run and between-run precision (CVs) of the assays was on the order of 2.3-4.7% and 4.3-8.7, respectively. Sensitivity of the assay was 1.0 ng/ml, and working range was 5-100 ng/ml. In all patients with define acute myocardial infarction, serum ventricular myosin light chain-1 levels increased three- to ten-fold the upper reference range within 6 hr after the onset of chest pains. Two types of subtrend were discovered: its levels remain elevated for 3-4 days and its levels increased and then decreased 1-2 days after the initial rise but became elevated again for the next 4-7 days after the onset of chest pain, which is in contrast to the case with all conventionally used biochemical cardiac markers.

Automated immunoturbidimetric analysis of six plasma apolipoproteins: correlation with radial immunodiffusion assays.

We measured six apolipoproteins (apo AI, AII, B, CII, CIII, and E) by turbidimetric method using an automatic discrete biochemical analyzer and commercially available antisera. The turbidimetric method was compared with the single radial immunodiffusion method. Linearity for serum apolipoprotein assay by the automated turbidimetric method was better than by the single immuno-diffusion method. The linearity by the turbidimetric method was 2.5 G/L for AI, 1.0 G/L for AII, 4.5 G/L for B, 0.12 G/L for CII, 0.3 G/L for CIII, and 0.12 G/L for E. The presence of high concentrations of bilirubin (up to 0.15 G/L) and hemoglobin (up to 50 G/L) interfered with apolipoprotein measurement. Comparison of the immunoturbidimetric and the single radial immunodiffusion (SRID) methods showed excellent coefficients of correlation, r = 0.963, 0.896, 0.846, 0.936, 0.972, and 0.937 for apo AI, AII, B, CII, CIII, and E, respectively. Reference ranges for the six apolipoproteins were determined by using sera from 450 healthy subjects and were 1.4 +/- 0.3 G/L for AI and 0.3 +/- 0.01 for E. The observed levels of AII (P less than 0.001), B (P less than 0.01), and CIII (P less than 0.01) were significantly higher in males. The serum levels of apo B, CII, and E showed a gradual increase with age which was more prominent in females than in males. The levels of apo AI, AII decreased significantly over an 11 day period in 22 patients with myocardial infarction.

Proteolytic measurement of mitochondrial aspartate aminotransferase in human serum.

A new proteolytic measurement of serum mitochondrial aspartate aminotransferase was evaluated using cytosolic aspartate aminotransferase inactivating protease. Some of the proteases, such as, alpha-chymotrypsin, subtilisin and cytosolic aspartate aminotransferase inactivating protease 401 from Streptomyces species, also specifically inactivated cytosolic aspartate aminotransferase, but not mitochondrial, aspartate aminotransferase. The protease 401 was the most heat stable for storage and showed a higher inactivation rate for cytosolic aspartate aminotransferase--up to 7000 IU/L--more than 200-fold the upper limit. The coefficient of variation of the proteolytic method was less than 10%. Results by the present method correlated with those by the immunochemical method (r = 0.970) and the regression curve was Y = 0.95X + 1.60 (Y: immunochemical method; X: proteolytic method). In the present assay system, reference values for mitochondrial aspartate aminotransferase activity in 500 healthy people ranged from 2.0-7.2 U/L (mean 3.8 U/L).

New automated measurement of mitochondrial aspartate aminotransferase with use of protease 401.

Total mitochondrial aspartate aminotransferase (EC 2.6.1.1), the sum of apo- and holo-mitochondrial aspartate aminotransferase activity in human serum, was measured by using a proteolytic method: inactivation of cytosolic aspartate aminotransferase with cytosolic aspartate aminotransferase-inactivating protease 401 from Streptomyces violaceochromogenes. Cytosolic aspartate aminotransferase is completely inactivated, and apo-mitochondrial aspartate aminotransferase is completely activated by pyridoxal 5'-phosphate within 5 min. Results by the proposed method correlated well with those by an immunochemical method (r = 0.994, n = 145) and showed excellent inhibitory activity of the protease for holo- and apo-cytosolic aspartate aminotransferase up to 5000 U/L and activation of mitochondrial apo-aspartate aminotransferase up to 2000 U/L in the presence of 100 mumol of pyridoxal 5'-phosphate per liter. Within-run Cvs were good (1.13-7.49%). Mean values for total mitochondrial aspartate aminotransferase and apo-mitochondrial aspartate aminotransferase activities in serum of the healthy subjects were 4.8 (SD 0.9) and 1.8 (SD 0.8) U/L, respectively (n = 154). Various common interferents tested did not affect this assay.

Optimal conditions for protease use in the assay of serum mitochondrial aspartate aminotransferase.

The optimal conditions for selective proteolytic inactivation of cytosolic aspartate aminotransferase (c-AST) to determine mitochondrial aspartate aminotransferase (m-AST) in serum were studied. Protease 401 was found to be effective over a pH range of 6.0-10.0. A pH of 9.5 with 0.5% albumin in the reagent mixture was determined to be optimal for inactivation of c-AST and preservation of m-AST, lactic dehydrogenase (LDH), and malic dehydrogenase (MDH) in the assay procedure. The presence of serum endogenous protein inhibitors such as alpha 1-antitrypsin and alpha 2-macroglobin did not inhibit protease 401.

Elevated serum levels of neuron specific enolase in lymphoid leukaemia.

Using an enzyme-linked immunosorbent assay (ELISA), we measured the levels of neuron-specific enolase (NSE) in the sera of 100 normal controls, 31 patients with adult T cell leukaemia (ATL), 13 patients with acute lymphoblastic leukaemia (ALL), 19 patients with acute non-lymphoblastic leukaemia (ANLL) and 17 patients with human T lymphotropic type I (HTLV-I) associated myelopathy (HAM). Levels higher than 5 ng/ml were considered as positive for NSE, since this value was almost 2 standard deviations (S.D) above the mean of normal controls. The rates of positive NSE levels were 55 percent in patients with ATL, 54 percent in patients with ALL, 11 percent in patients with ANLL and 0 percent in patients with HAM. High NSE levels fell to near normal levels in patients with ATL and ALL responding to cytotoxic therapy. It is concluded from these results that NSE may be a useful tumour marker for lymphoid malignancies.