Goro Kominami

Serum Soluble Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 Levels Are Elevated in Acute Coronary Syndrome A Novel Marker for Early Diagnosis

Background—Markers of cardiac injury, including troponin-T (TnT), are used to diagnose acute coronary syndrome (ACS); however, markers for plaque instability may be more useful for diagnosing ACS at the earliest stage. Lectin-like oxidized LDL receptor-1 (LOX-1) appears to play crucial roles in the pathogenesis of atherosclerotic plaque rupture and ACS onset. LOX-1 is released in part as soluble LOX-1 (sLOX-1) by proteolytic cleavage. Methods and Results—We examined serum sLOX-1 levels in 521 patients, consisting of 427 consecutive patients undergoing coronary angiography, including 80 ACS patients, 173 symptomatic coronary heart disease patients, 122 patients with significant coronary stenosis without ischemia, and 52 patients without apparent coronary atherosclerosis plus 34 patients with noncardiac acute illness and 60 patients with noncardiac chronic illness. Time-dependent changes in sLOX-1 and TnT levels were analyzed in an additional 40 ACS patients. Serum sLOX-1 levels were significantly higher in ACS than the other groups and were associated with ACS as shown by multivariable logistic regression analyses. Given a cutoff value of 1.0 ng/mL, sLOX-1 can discriminate ACS from other groups with 81% and 75% of sensitivity and specificity, respectively. sLOX-1 can also discriminate ACS without ST elevation or abnormal Q waves and ACS without TnT elevation from non-ACS with 91% and 83% of sensitivity, respectively. Peak values of sLOX-1 in ACS were observed earlier than those of TnT. Conclusions—sLOX-1 appears to be a useful marker for early diagnosis of ACS.

Mechanism of the drug interaction between valproic acid and carbapenem antibiotics in monkeys and rats.

The Ministry of Health and Welfare, Japan banned coadministration of carbapenems, such as panipenem/betamipron (PAPM), meropenem (MEPM), and valproic acid (VPA) because clinical reports have indicated that the coadministration caused seizures in epileptic patients due to lowered plasma levels of VPA. In this study, we have clarified the mechanism of the drug-drug interaction using PAPM, MEPM, and doripenem [S-4661; (+)-(4R,5S,6S)-6-[(1R)-1-hydroxyethyl]-4-methyl-7-oxo-3-[[(3S,5S)-5-[(sulfamoylamino)methyl]-3-pyrrolidinyl]thio]-1-azabicyclo[3.2.0]hept-2-ene-2-caboxylic acid monohydrate], a newly synthesized carbapenem. In vitro experiments using monkey liver slices suggested that the apparent synthetic rate of VPA glucuronide (VPA-G) increased in the presence of carbapenems. However, no such increase was observed in the experiment using monkey liver microsomes. Although no increase of uridine 5′-diphosphate d-glucuronic acid was found in monkey liver slices in the presence of carbapenems, potent inhibitory activity of carbapenems for the hydrolysis of VPA-G was found in monkey and rat liver homogenate. In vivo hydrolysis of VPA-G was clearly shown by the existence of VPA in plasma after dosing of VPA-G to rats, and its inhibition by carbapenems was also clearly shown by the negligible levels of VPA in rat plasma after coadministration of carbapenems and VPA-G. These results clearly indicate one of the important causes of drug interaction as follows: carbapenems would inhibit the hydrolytic enzyme, which is involved in the hydrolysis of VPA-G to VPA, resulting in a decrease of plasma concentration of VPA.

Soluble lectin-like oxidized LDL receptor-1 (sLOX-1) as a sensitive and specific biomarker for acute coronary syndrome—Comparison with other biomarkers

BACKGROUND Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) appears to be involved in atherosclerotic plaque vulnerability and rupture. Circulating soluble LOX-1 (sLOX-1) levels are dramatically elevated in patients with acute coronary syndrome (ACS), and its diagnostic sensitivity and specificity is superior to high-sensitivity C-reactive protein (hs-CRP). In this study, we have compared the diagnostic value of sLOX-1 for ACS with those of troponin T (TnT) and heart-type fatty acid binding protein (H-FABP). METHODS One hundred and seven patients who underwent coronary angiography (CAG), including 18 ACS and 89 non-ACS patients were enrolled. Peripheral blood samples were obtained during the emergent or elective CAG. The non-ACS group consisted of 30 patients with normal CAG, 30 stable angina pectoris patients controlled by medical treatment, and 29 patients with stable angina who required elective coronary revascularization (percutaneous coronary intervention or coronary artery bypass graft). RESULTS Age, gender, lipid profiles, or prevalence of diabetes, smoking, or hypertension were not significantly different between ACS and non-ACS. These factors did not significantly affect blood sLOX-1 levels. Circulating sLOX-1, TnT, and H-FABP levels were significantly higher in ACS than non-ACS. Area under the curve (AUC) values of the receiver-operating characteristic curves were 0.948, 0.704, and 0.691 for sLOX-1, TnT, and H-FABP, respectively. In a TnT-negative (<0.03 ng/mL) subgroup, the AUC values for sLOX-1 and H-FABP were 0.848 and 0.476, respectively. CONCLUSION Circulating sLOX-1 is a more sensitive and specific biomarker for ACS than TnT and H-FABP, and provides additional diagnostic values when measured in combination with TnT.

High-throughput miniaturized immunoassay for human interleukin-13 secreted from NK3.3 cells using homogenous time-resolved fluorescence

A miniaturized immunoassay for human interleukin-13 (IL-13) using homogeneous time-resolved fluorescence (HTRF) has been developed. In this assay, IL-13 which was secreted from NK3.3 cells stimulated with interleukin-2 (IL-2) was detected by measuring the time-resolved fluorescence after adding a mixture of three reagents, biotinylated anti-IL-13 monoclonal antibody, europium cryptate (fluorescence donor)-labeled different anti-IL-13 monoclonal antibody and crosslinked allophycocyanin (fluorescence acceptor)-conjugated with streptavidin in a 384-well assay plate. The detection limit of IL-13 using this immunoassay was estimated to be less than 600 pg/ml and IL-13 levels measured by this method were very close to those measured by enzyme linked immunosorbent assay (ELISA; the correlation coefficient was 0.9535). The proposed assay requires only a fourth of the quantities of all reagents compared with the assay using a conventional 96-well microtiter plate. Furthermore, there is no need to transfer the culture supernatant to another assay plate and wash the plate. Therefore, this miniaturized immunoassay is economical and efficient and is particularly suitable for high-throughput drug screening.

Generation of monoclonal antibodies against a soluble form of lectin-like oxidized low-density lipoprotein receptor-1 and development of a sensitive chemiluminescent enzyme immunoassay

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), expressed prominently in atherosclerotic lesions, is cleaved and released as a soluble LOX-1 (sLOX-1), which is a specific biomarker to diagnose acute coronary syndrome (ACS) at an early stage. Although sLOX-1 levels in patients blood were successfully measured with our previously established enzyme-linked immunosorbent assay (ELISA), the assay was not sensitive enough to detect normal serum levels of sLOX-1 in healthy human subjects. We therefore developed sensitive and specific monoclonal antibodies (mAbs) against sLOX-1 in order to establish a more sensitive immunoassay. Mice were immunized with recombinant human LOX-1 extracellular domain. mAbs were subsequently generated by standard myeloma cell fusion techniques with a novel screening method using time-resolved fluorescence immunoassay. Using two anti-human sLOX-1 mAbs and alkaline phosphatase as a label, a sandwich chemiluminescent enzyme immunoassay (CLEIA) was developed. In total, nine mAbs were obtained. The dissociation constant (K(d)) values of these mAbs for sLOX-1 were 0.12-1.32 nM. Characteristics of these mAbs were estimated and the best combination for CLEIA was selected. The newly established CLEIA could determine sLOX-1 levels as low as 8 pg/mL, and thus, was sensitive enough to measure serum sLOX-1 levels in normal human subjects and to evaluate subtle differences. Values for sLOX-1 measured by monoclonal CLEIA and polyclonal ELISA were highly correlated (r(2)=0.7594, p<0.0001). Area under the curve values of the receiver-operating characteristic curves in detecting ACS were 0.948 and 0.978 for monoclonal CLEIA and polyclonal ELISA, respectively. Thus, a more sensitive sLOX-1 CLEIA was established using newly developed mAbs against sLOX-1. In addition to its advantage in early diagnosis of ACS, this assay may also be useful in predicting cardiovascular disease risk in disease-free subjects.

On-line immunoaffinity extraction followed by high-performance liquid chromatography and radioimmunoassay for a novel retinobenzoic acid, AM-80, in human plasma.

On-line coupled immunoaffinity chromatography and reversed-phase high performance liquid chromatography (on-line IAC-HPLC) with detection by radioimmunoassay (RIA) was developed for 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-carbamoyl] benzoic acid (Am-80) in human plasma. A 0.05-mL sample was directly loaded onto an immunoaffinity pre-column packed with immobilized polyclonal antibodies against Am-80. The immunoaffinity extract was then automatically introduced to reversed-phase HPLC column for separation by column switching. The immunoreactivity in the HPLC-eluted fraction was measured by competitive RIA. The within-assay precision and accuracy of the method were 4.0–10.6% and −6.0–+8.3%, respectively, and between-assay precision and accuracy were 6.8–11.9% and −16.0–+9.0%, respectively. The limit of quantitation was 0.5 ng/mL in human plasma. The proposed method yields highly purified analyte in complex matrices, detects it with high sensitivity, not only by radioimmunoassay, but also by other methods, such as mass-spectrometry, and can be used for routine assay of many samples without manual sample extraction and purification.

Radioimmunoassay for a novel lignan-related hypocholesterolemic agent, S-8921, in human plasma after high-performance liquid chromatography purification and in human urine after immunoaffinity extraction

The novel compound methyl-1-(3,4-dimethoxyphenyl)-3-(3-ethylvaleryl)-4-hydroxy-6,7,8- trimethoxy-2-naphthoate (S-8921) has hypocholesterolemic activity in animals and is expected to exhibit a similar activity in human. Reversed-phase high-performance liquid chromatography (HPLC) separation followed by radioimmunoassay (RIA) for human plasma samples (HPLC-RIA) and immunoaffinity extraction (IAE) followed by RIA for human urine samples (IAE-RIA) were developed for investigation of S-8921 behavior in clinical studies. For the RIA, antisera from rabbit and a radioiodine-labelled S-8921 were prepared by immunizing a conjugate of S-8921 with bovine serum albumin and by the Bolton and Hunter method, respectively. HPLC-RIA using a semi-micro column was very sensitive, that is a 0.05 ng/ml limit of quantitation in human plasma, and specific for unchanged form of S-8921. IAE-RIA using a centrifugal filtration tube completely eliminated the matrix effect of human urine, and was very feasible. The limit of quantitation was 0.10 ng/ml. RIA detection following HPLC or IAE proved to be very useful for the pharmaceutical analysis of extremely low drug concentrations in body fluids.

Radioimmunoassay for a novel benzodiazepine inverse agonist, S-8510, in human plasma and urine.

A radioimmunoassay (RIA) was developed for a new benzodiazepine inverse agonist, 2-(isoxazol-3-yl)-3,6,7,9-tetrahydroimidazo [4,5-d] pyrano [4,3-b] pyridine monophosphate monohydrate (S-8510), in human plasma and urine. For competitive RIA, three amino derivatives of S-8510 were labelled by the Bolton and Hunter method and rabbit antisera were prepared using three immunogens, conjugates of three carboxyl derivatives of S-8510 with bovine serum albumin. All combinations of the labelled antigens and antisera were examined and homologous combinations were selected for the competitive RIA. One of the three homologous combinations had the best selectivity after investigations of cross-reactivity using 12 related compounds and was very sensitive for S-8510. Next, a pretreatment for biological samples was developed using mixed mode solid-phase extraction (SPE) column followed by the RIA (SPE/RIA). The assay recoveries for human plasma and urine were both excellent and the limits of quantitation were extremely low, 80 and 200 pg ml(-1), respectively. Human plasma samples and urine samples after administration of this drug were successfully measured by the SPE/RIA. No cross-reactive metabolites were detected in any fractions after RP-HPLC separation of the plasma samples. The RIA using carefully selected antiserum and labelled antigen was highly specific for unchanged S-8510. To simplify the RIA procedure, a scintillation proximity assay (SPA) using the same labelled antigen and antiserum was developed for analyzing S-8510 in human plasma and found to be very promising.

Immunoenzymometric assay for recombinant methioninase in biological fluids.

Immunoassay for recombinant methioninase (rMETase), an anti-cancer agent, in biological sample was developed. Antisera were produced by immunizing rabbits with rMETase. The antisera were evaluated using radioiodine-labeled rMETase and good antisera for sensitive immunoassay were obtained. Horseradish peroxidase (HRP) was coupled to reduced IgG of K232 antiserum through bridging agent, N-(epsilon -maleimidocaproyloxy) sulfosuccinimide ester (sulfo-EMCS), to prepare enzyme-labeled antibody. IgG fraction of K231 antiserum was immobilized on microplate well. Two-site sandwich immunoenzymometric assay (IEMA) was developed using these antibodies and had good standard curve between 0.4 and 12 ng per well. For determination of rMETase in mouse plasma, sample was diluted 100-fold with dilution buffer containing protease inhibitors, because about 10% of rMETase immunoreactivity was lost for 2 h at room temperature. rMETase in mouse plasma could be determined by the proposed method in the range of 0.5-8 microg/ml and the method was validated. This novel IEMA, in substitution for the measurement of its enzyme activity, should be very useful not only for preclinical studies of rMETase but also for the clinical studies.

Radioimmunoassay and gas chromatography/mass spectrometry for a novel antiglaucoma medication of a prostaglandin derivative, S-1033, in plasma

A radioimmunoassay (RIA) and a gas chromatographic/mass spectrometric (GC/MS) method for a new antiglaucoma medicament, the prostaglandin derivative sodium (5Z, 9 alpha, 11 alpha, 13E)-9,11-dihydroxyprosta-5, 13-dienoate (S-1033), in human and rabbit plasma were investigated. For a competitive RIA, antisera from rabbit and radioiodine-labeled S-1033 were prepared by immunizing a conjugate of S-1033 with bovine serum albumin and by the Bolton and Hunter method, respectively. Pretreatment by C18 solid-phase extraction (SPE) for rabbit plasma sample and further purification by high-performance liquid chromatography (HPLC) for human plasma samples followed by the RIA (SPE/RIA and HPLC/RIA, respectively) were developed. The assay recoveries of SPE/RIA and HPLC/RIA were both excellent and the limits of quantitation were 320 and 10 pg ml-1, respectively. GC/MS for plasma samples after solid-phase extraction and thin-layer chromatographic purification was also developed using deuterium-labeled S-1033 as internal standard. The limit of quantitation was 100 pg ml-1 in human or rabbit plasma. Rabbit plasma samples after administration of this drug were measured by SPE/RIA and GC/MS and the assay results from both methods agreed well. The SPE/RIA, HPLC/RIA and GC/MS assay methods were suitable for measuring samples from preclinical studies, clinical studies and cross-validation, respectively.