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New analytical methods for the determination of free asparagine （Asn）, which is a precursor of acrylamide, in grains were developed using LC-MS and LC-MS/MS. Asn was extracted from a sample with 5％ （w/v） aqueous trichloroacetic acid solution, appropriately diluted with 0.1％ （v/v） formic acid solution, and then analyzed by LC-MS or LC-MS/MS. HPLC separation was performed by isocratic elution on a Penta Fluoro Phenyl （PFP） column using 0.1％ （v/v） formic acid and acetonitrile mixture as the mobile phase. The calibration curve was linear in the range of 0.005-0.1 μg/mL. The mean recoveries from potato starch, non-glutinous rice flour and whole wheat flour ranged from 95.4 to 100.9％, repeatability （RSD） ranged from 0.9 to 6.0％, and within-laboratory reproducibility （RSDwr） ranged from 2.8 to 7.1％. Limits of quantitation （LOQs） were 7 mg/kg for potato starch, and 5 mg/kg for non-glutinous rice flour. In addition, an inter-laboratory study was performed in 10 laboratories using 5 kinds of grains （non-glutinous brown rice flour, corn flour, strong flour, whole wheat flour, and whole rye flour）, which naturally contained free asparagine. The HORRAT R values ranged from 0.4 to 1.0. These results are within the range of the procedural manual of the Codex Alimentarius Commission, confirming the effectiveness of the developed procedures.

In order to validate an HPLC-UV method for the determination of free asparagine, which is a precursor of acrylamide, in grains using dansyl derivatization, an inter-laboratory study was performed in 9 laboratories using 5 kinds of grains (non-glutinous brown rice flour, corn flour, strong flour, whole wheat flour, and whole rye flour), which naturally contain free asparagine. The relative standard deviations for repeatability (RSDr) and reproducibility (RSDr) were in the ranges of 0.5-2.2 and 2.3-5.9%, respectively. The HorRat values ranged from 0.4 to 0.6. These results were within the range of the procedural manual of the Codex Alimentarius Commission, and therefore the method is effective.

A novel analytical method was developed for the determination of free asparagine (Asn), which is a precursor of acrylamide, in grains. Asn was extracted from a sample with 5% (w/v) aqueous trichloroacetic acid solution, and the crude extract was cleaned up using a reversed-phase solid-phase cartridge. The cleaned extract was derivatized with dansyl chloride and analyzed by HPLC-UV. HPLC separation was performed by gradient elution on a ODS column using 0.01 mol/L ammonium acetate and acetonitrile mixture as the mobile phase. The calibration curve was linear in the range of 0.5-100 μg/mL. The mean recoveries from potato starch, non-glutinous rice flour and whole wheat flour ranged from 97.7 to 102.6%, the repeatability (RSDs) ranged from 0.8 to 2.0%, and the within-laboratory reproducibility (RSDwr) ranged from 1.4 to 6.2%. Limits of quantitation (LOQs) were 13 mg/kg for potato starch and 4 mg/kg for non-glutinous rice flour. The quantitative values obtained for about 15 kinds of grains using this developed method were approximately equal to those obtained with an automatic amino acid analyzer.

Site-specific drug delivery to bone is considered achievable using acidic amino acid (L-Asp or L-Glu) homopeptides known as acidic oligopeptides. We found that fluorescence-labeled acidic oligopeptides containing six or more residues bound strongly to hydroxyapatite, which is a major component of bone, and were selectively delivered to and retained in bone after systemic administration. We explored the applicability of this result for drug delivery by conjugation of estradiol and levofloxacin with an L-Asp hexapeptide. We also similarly tagged enzymes (tissue-nonspecific alkaline phosphatase, β-glucuronidase, and N-acetylgalactosamine-6-sulfate sulfatase) and decoy receptors (endogenous secretory receptor for advanced glycation end products and etanercept) to assess whether these would improve therapeutic efficacy. The L-Asp hexapeptide-tagged drugs, including enzymes and decoy receptors, were efficiently delivered to bone in comparison with the untagged drugs. An in vivo experiment confirmed the efficacy of L-Asp hexapeptide-tagged drugs on bone and joint disorders, although there was some loss of bioactivity of estradiol and levofloxacin in vitro, suggesting that the acidic hexapeptide was partly removed by hydrolysis in the body after delivery to bone. It was expected that the ester linkage to the hexapeptide would be susceptible to hydrolysis in situ, releasing the drug from the acidic oligopeptide. These results support the usefulness of acidic oligopeptides as bone-targeting carriers for therapeutic agents. We present some pharmacokinetic and pharmacological properties of the L-Asp hexapeptide-tagged drugs.

For understanding the three-dimensional mechanisms that control potency and selectivity of the ligand binding at the atomic level, we have analysed opioid receptor-ligand interaction based on the receptor's 3D model. The analyses of the interactions of some opioid ligands with the predicted ligand binding sites are consistent with the results of the affinity labeling experiments. On the basis of the three-dimensional structures of the putative binding sites in the opioid receptors, a potent kappa agonist KT-95 was designed in our laboratory.

Two specific and sensitive assays have been developed for the measurement of as-hCG in this study. These assay systems are immunoradiometric assays which utilize peanut lectin, Arachis hypogaea (PNA) or caster bean lectin, Ricin communis agglutinin (RCA-I), to selectively extract as-hCG and then directly quantify with purified and radiolabeled antiserum against hCG beta COOH terminal peptide (beta-CTP). PNA is highly specific for the terminal carbohydrate linkage Gal beta-(1----3)-GalNAc which is only exposed in hCG when the O-serine linked oligosaccharide of its unique beta-CTP region are desialylated. RCA-I is also specific for the terminal carbohydrate beta-D-Galp., especially the linkage of Gal beta-(1----4)-GlcNAc, which is exposed in hCG when the N-asparagine linked oligosaccharide of its alpha and beta subunits are desialylated. The assays (PNA-R525 and RCA-I-R525) are capable of reliably and accurately measuring as little as 0.01 and 0.002 pmoles as-hCG/ml without interference from hCG, respectively (crossreactivity: 0.1% and 0.03%). The urine concentrates of a patient with choriocarcinoma and a normal pregnant woman were fractionated by Sephadex G-100 gel filtration and total hCG and as-hCG levels were measured. When the fractions were analysed by beta-CTP RIA, there were two peaks of immunoreactive hCG in the urine of a patient with choriocarcinoma. The bigger molecular fractions were hCG of native size, and 16% of which was desialylated. The smaller molecular fraction was beta-CTP, and practically all of that was desialylated. Whereas, beta-CTP could not be observed, and only 1.5% of hCG of native size was desialylated in the urine of a normal pregnant woman. Although the absolute levels of as-hCG in the urine specimens increased as total hCG increased, the proportion of as-hCG decreased as total hCG concentration increased. The proportion of as-hCG in urine specimens from choriocarcinoma patients was higher than specimens from patients with hydatidiform mole (including invasive mole) in both these lectin immunoradiometric assay (LIRMA) systems. Also, the proportion of as-hCG in urine specimens from molar patients was higher than those from normal pregnant women using PNA as extracting reagent. Whereas, when RCA-I was used, there was no significant difference between specimens from molar patients and those from normal pregnant women.(ABSTRACT TRUNCATED AT 400 WORDS)