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Biomedical subjects

K Inagaki

Publications and source records attributed to K Inagaki.

At least 19 recordsLinked to original sources
PubMed2005

Novel approach on the risk assessment of oxidized fats and oils for perspectives of food safety and quality. I. Oxidized fats and oils induces neurotoxicity relating pica behavior and hypoactivity.

Food poisoning caused by deteriorated fat and oil in instant noodles was first reported in Japan approximately 40 years ago. In these cases, many people developed neurotoxic symptoms such as emesis and discomfort. The degree of oxidation of the fat and oil in the instant noodles that induced food poisoning was at least 100 meq/kg in peroxide value (PV). No general toxicity studies with animals, however, have examined the toxicity of fat and oil oxidized to that extent. In this study, pica behavior, a behavior characterized by eating a nonfood material such as kaolin and that relates to the degree of discomfort in animals, and alterations of locomotor activity of rats eating deteriorated fat and oil were measured. The groups fed fat and oil with at least 138.5 meq/kg PV consumed significantly more kaolin compared to the control group. Furthermore, rats that ate deteriorated fat and oil with at least 107.2 meq/kg PV had significantly decreased locomotor activity compared to control rats. These phenomena suggest that oxidized fat and oil with at least 100 meq/kg PV induce neurotoxicity. The toxicity of oxidized fat and oil has only been addressed using general toxicity tests, but the present results reveal the importance of evaluating toxicity by using other measures.

Animals↗
PubMed2005

Efficacy of periodontal disease and tooth loss to screen for low bone mineral density in Japanese women.

The relationship between oral indicators and bone mineral density (BMD) has been studied by many investigators, with mixed and complex results. The purpose of the present cross-sectional study was to evaluate the associations of periodontal conditions and tooth loss with metacarpal BMD (m-BMD) in a community-based cohort and the usefulness of tooth count as a potential screening tool to detect low BMD. Subjects were 356 Japanese women (171 premenopausal, mean age 37.9+/-8.0 years; 185 postmenopausal, mean age 63.3+/-7.7 years). Periodontal status was evaluated by the Community Periodontal Index of Treatment Needs (CPITN). m-BMD was measured by computerized X-ray densitometry. The proportion of subjects with periodontitis (CPITN 3 or 4) increased as m-BMD decreased. The odds ratio (OR) of osteopenia or osteoporosis in relation to periodontitis was 3.2 (95% confidence interval [CI], 2.0--5.3). After adjustment for age and menopausal status, the OR was 2.0 (95% CI, 1.1--3.7). Among postmenopausal women, those having fewer than 20 teeth were 1.6 times more likely to have low m-BMD than those having more than 20 teeth (chi-square for trend in postmenopausal group, 4.27; P<0.05). Receiver-operating curve (ROC) analysis indicated that number of teeth remaining or CPITN score had a greater than 50/50 chance to correctly identify women with osteoporosis or osteopenia, but the areas under the curve (0.72 and 0.67, respectively) are considered less than highly accurate screening tools. These results indicate that periodontitis and tooth loss after menopause may be useful indicators of m-BMD loss in Japanese women.

Absorptiometry, Photon↗
PubMed2005

DeltaPKC-mediated activation of epsilonPKC in ethanol-induced cardiac protection from ischemia.

Previous studies have demonstrated that acute ethanol exposure induces activation of delta protein kinase C (deltaPKC) and epsilonPKC, and mimics ischemic preconditioning via epsilonPKC activation. However, the role of deltaPKC isozyme in ischemia and reperfusion is still controversial. Here, we investigated the role of deltaPKC in ethanol-induced cardioprotection using a selective deltaPKC activator (psideltaRACK), or inhibitor (deltaV1-1), and a selective epsilonPKC inhibitor (epsilonV1-2) in isolated mouse hearts. Mice were injected intraperitoneally or by gavage with ethanol, regulators of delta and epsilonPKC or an adenosine A1 receptor blocker (DPCPX). Isolated perfused mouse hearts were subjected to a 30-min global ischemia and a 120-min reperfusion, ex vivo. Injection of 0.5 g/kg ethanol 1 h, but not 10 min, before ischemia reduced infarct size and CPK release. Pretreatment with epsilonV1-2 abolished this ethanol-induced cardioprotection. Pretreatment with deltaV1-1 induced cardioprotection when injected with ethanol (0.5 g/kg) 10 min before ischemia, but deltaV1-1 partly inhibited ethanol-induced cardioprotection when injected with ethanol 1-h before the onset of ischemia. psideltaRACK injection 1 h, but not 10 min, before ischemia induced cardioprotection and translocation of epsilonPKC from the cytosol to the particulate fraction. Pretreatment with DPCPX or epsilonV1-2 inhibited psideltaRACK-induced cardioprotection and translocation of epsilonPKC. Therefore, activation of deltaPKC-induced by ethanol or by the deltaPKC activator is cardioprotective, provided that sufficient time passes to allow deltaPKC-induced activation of epsilonPKC, an A1 adenosine receptor-dependent process.

Animals↗
PubMed2004

Effect of cabergoline treatment on Cushing's disease caused by aberrant adrenocorticotropin-secreting macroadenoma.

The present case involves a 47-yr-old woman with Cushing's disease due to pituitary macroadenoma. The patient had suffered from hypertension and obesity for two yr. Her serum cortisol levels were moderately elevated throughout the observation period, and dexamethasone failed to suppress the cortisol secretion. Plasma ACTH levels were markedly high (>100 pg/ml) and did not respond to CRH provocation. Gel filtration analysis of the patient's plasma detected the existence of big ACTH molecules, which eluted with a peak of authentic 1-39 ACTH. Cranial magnetic resonance imaging (MRI) revealed a 3 cm pituitary tumor occupying the sellar region and right cavernous sinus with diffuse enhancement by gadolinium. The pituitary mass was removed by transsphenoidal surgery, and was pathologically identified as compatible to ACTH-producing pituitary adenoma by immunohistochemistry. RT-PCR analysis of total cellular RNA extracted from the resected adenoma revealed a relatively high expression level of dopamine D2 receptor (D2R) mRNA. Therefore, a long-acting D2R agonist, cabergoline (0.25 to 0.5 mg/week), was administered for the remnant adenoma, which gradually reduced ACTH levels in 90 days. In addition, cranial MRI exhibited shrinkage of the remnant pituitary mass after a 6-month treatment with cabergoline. This case demonstrates the efficacy of cabergoline to treat Cushing's disease caused by pituitary macroadenoma secreting aberrant ACTH molecules.

Adenoma↗
PubMed2003

Determination of tributyltin in marine sediment: Comité Consultatif pour la Quantité de Matière (CCQM) pilot study P-18 international intercomparison.

The capabilities of National Metrology Institutes (NMIs-those which are members of the Comité Consultatif pour la Quantité de Matière (CCQM)of the CIPM) and selected outside "expert" laboratories to quantitate (C(4)H(9))(3)Sn(+) (TBT) in a prepared marine sediment were assessed. This exercise was sanctioned by the 7th CCQM meeting, April 4-6, 2001, as an activity of the Inorganic Analysis Working Group and was jointly piloted by the Institute for National Measurement Standards of the National Research Council of Canada (NRC) and the Laboratory of the Government Chemist (LGC), UK. A total of 11 laboratories submitted results (7 NMIs, and 4 external labs). Two external laboratories utilized a standard calibration approach based on a natural abundance TBT standard, whereas all NMIs relied upon isotope dilution mass spectrometry for quantitation. For this purpose, a species specific (117)Sn-enriched TBT standard was supplied by the LGC. No sample preparation methodology was prescribed by the piloting laboratories and, by consequence, a variety of approaches was adopted by the participants, including mechanical shaking, sonication, accelerated solvent extraction, microwave assisted extraction and heating in combination with Grignard derivatization, ethylation and direct sampling. Detection techniques included ICP-MS (with GC and HPLC sample introduction), GC-MS, GC-AED and GC-FPD. Recovery of TBT from a control standard (NRCC CRM PACS-2 marine sediment) averaged 93.5+/-2.4% ( n=14). Results for the pilot material averaged 0.680+/-0.015 micro mol kg(-1) ( n=14; 80.7+/-1.8 micro g kg(-1)) with a median value of 0.676 micro mol kg(-1). Overall, performance was substantially better than state-of-the-art expectations and the satisfactory agreement amongst participants permitted scheduling of a follow-up Key comparison for TBT (K-28), a Pilot intercomparison for DBT (P-43), and certification of the test sediment for TBT content and its release as a new Certified Reference Material (HIPA-1) with a TBT content of 0.679+/-0.089 micro mol kg(-1) (expanded uncertainty, k=2, as Sn) (80.5+/-10.6 micro g kg(-1)).

Journal Article↗
PubMed2001

Calcium handling and sarcoplasmic-reticular protein functions during heart-failure transition in ventricular myocardium from rats with hypertension.

The objective of this study was to determine the primary event that occurs in Ca2+-regulatory sarcoplasmic-reticular (SR) proteins during subacute transition from concentric/mechanically-compensated left ventricular (LV) hypertrophy to eccentric/decompensated hypertrophy. Using Dahl salt-sensitive rats with hypertension, changes of myocardial contraction, intracellular Ca2+ transients, SR Ca2+ uptake, protein levels of SR Ca2+ ATPase (SERCA2), phospholamban, and calsequestrin (CSQ), and mRNA levels of SERCA2 and CSQ were serially determined and compared between the established stage of LV hypertrophy (LVH) and the subsequent stage of overt LV dysfunction (CHF). In LVH, isolated LV papillary muscle preparations showed an equal peak-tension level and a mild prolongation of the isometric tension decay compared to those of age-matched controls. The Ca2+ transients as measured by aequorin were unchanged. The Ca2+ uptake of isolated SR vesicles and the protein/mRNA levels of SR proteins were also equivalent to those of the controls. In contrast, in CHF, the failing myocardium showed a further prolongation of the contraction time course and a 39% reduction of the peak-tension development. The Ca2+ transients showed changes consisting of a decrease in the peak level and a prolongation of the time course. In addition, the SR Ca2+ uptake was decreased by 41%. Despite these functional changes, the protein and mRNA levels of the SR components remained equivalent to those of the age-matched controls. Thus, in this hypertensive animal, 1) at the LVH stage, myocardial contractility and intracellular capability to regulate Ca2+ remained normal; 2) at the CHF stage, impaired SR Ca2+ handling and the subsequent reduction of myocardial contraction were in progress; and 3) impairments of SR function occurred at the post-translational protein level rather than at the transcriptional/translational levels. Our findings support the role of SR proteins as the primary determinant of the contractile dysfunction that occurs during the heart-failure transition; however, post-translational modulators of these SR elements may also be critical.

Aequorin↗
PubMed2001

Differential effects of angiotensin II versus endothelin-1 inhibitions in hypertrophic left ventricular myocardium during transition to heart failure.

BACKGROUND: In view of their mutual crosstalk, the roles of angiotensin II (Ang II) and endothelin-1 (ET-1) in the myocardium are assumed to be synergistic and supplemental. METHODS AND RESULTS: In the phase of compensated left ventricular (LV) hypertrophy of Dahl salt-sensitive rats, Ang II peptide and the ACE mRNA in the LV were increased by 1.6- and 3.8-fold, respectively. In contrast, ET-1 peptide and the preproET-1 mRNA remained unchanged. In subsequent congestive heart failure (CHF), Ang II and ACE mRNA did not show further increases. But ET-1 and the mRNA were increased de novo by 5.3- and 4.1-fold, respectively. In ascending aorta-banded rats, the local activations of Ang II and ET-1 also showed a differential time course between LV hypertrophy and CHF. Long-term treatments of Dahl salt-sensitive rats with temocapril (an ACE inhibitor) and with bosentan (a mixed ET receptor blocker) equally improved long-term survival. Temocapril reduced the LV/body weight ratio and ameliorated LV fractional shortening. Conversely, although bosentan equally improved fractional shortening, it did not reduce the increase in LV mass. Combined treatment with these 2 drugs further ameliorated the animal's survival without additional decreases in systolic pressure. CONCLUSIONS: The pathophysiological roles in the myocardium during the transition to CHF differ qualitatively between Ang II and ET-1. Thus, long-term therapy with a combination of ACE inhibition and ET antagonism may provide a new approach for heart failure in humans.

Angiotensin II↗
PubMed2001

Endothelin-1 has a unique oxygen-saving effect by increasing contractile efficiency in the isolated rat heart.

BACKGROUND: The effect of endothelin (ET)-1 on cardiac energetics is not fully understood. METHODS AND RESULTS: In isolated, coronary-perfused rat hearts, we measured left ventricular contractility index (E(max)), pressure-volume area (PVA), and myocardial oxygen consumption (MVO(2)) before and after administration of ET-1 (1x10(-)(9) mol/L). ET-1 increased E(max) by 48+/-16% (P<0.01) and the total MVO(2) by 24+/-11% (P<0.01). The MVO(2)-PVA relations were linear both before and after ET-1 (r>0.99). ET-1 shifted MVO(2)-PVA upward, increasing the MVO(2) intercept by 24+/-13%. At the same time, ET-1 decreased the slope (S), with 1/S (contractile efficiency) being 46+/-5% before and 56+/-5% after ET-1 (P<0.01). ET-1-induced increases in E(max) and in contractile efficiency were abolished by an ET(A) receptor blocker (S-0139) but not by an ET(B) blocker (BQ-788). Although high [Ca(2+)] perfusion increased E(max) and the intercept to the same extent as ET-1, it did not change S. N(G)-Nitro-L-arginine (an inhibitor of nitric oxide synthase) increased the coronary perfusion pressure as much as ET-1, but S again remained unchanged. Dimethylamyloride (Na(+)/H(+) exchanger inhibitor) partially blocked the positive inotropic effect of ET-1 but not the ET-1-induced increase in the contractile efficiency. CONCLUSIONS: Agonistic effects of ET-1 on the ET(A) receptor economized the chemomechanical conversion efficiency of the left ventricular unit myocardium by a mechanism independent of the Na(+)/H(+) exchanger. This unique oxygen-saving effect of ET-1 may play an adaptive role in the failing myocardium, in which local accumulation of ET-1 is present.

Amiloride↗
PubMed2001

Inhibition of cell growth and spreading by stomach cancer-associated protein-tyrosine phosphatase-1 (SAP-1) through dephosphorylation of p130cas.

SAP-1 (stomach cancer-associated protein-tyrosine phosphatase-1) is a transmembrane-type protein-tyrosine phosphatase that is abundant in the brain and certain cancer cell lines. With the use of a "substrate-trapping" approach, p130(cas), a major focal adhesion-associated phosphotyrosyl protein, has now been identified as a likely physiological substrate of SAP-1. Expression of recombinant SAP-1 induced the dephosphorylation of p130(cas) as well as that of two other components of the integrin-signaling pathway (focal adhesion kinase and p62(dok)) in intact cells. In contrast, expression of a substrate-trapping mutant of SAP-1 induced the hyperphosphorylation of these proteins, indicating a dominant negative effect of this mutant. Overexpression of SAP-1 induced disruption of the actin-based cytoskeleton as well as inhibited various cellular responses promoted by integrin-mediated cell adhesion, including cell spreading on fibronectin, growth factor-induced activation of extracellular signal-regulated kinase 2, and colony formation. Finally, the enzymatic activity of SAP-1, measured with an immunocomplex phosphatase assay, was substantially increased by cell-cell adhesion. These results suggest that SAP-1, by mediating the dephosphorylation of focal adhesion-associated substrates, negatively regulates integrin-promoted signaling processes and, thus, may contribute to contact inhibition of cell growth and motility.

Animals↗
PubMed2001

Stage-specific differential activation of mitogen-activated protein kinases in hypertrophied and failing rat hearts.

Mitogen-activated protein kinases (MAPKs) are involved in the early development of cardiac hypertrophy, but their roles in chronic left ventricular hypertrophy (LVH) are unclear. We studied the angiotensin (Ang) II-induced cardiac MAPK activation of the hypertensive Dahl salt-sensitive (DS) rats in the subacute developing LVH stage, the chronic compensated LVH stage, and the congestive heart failure (CHF) stage. In the isolated, coronary-perfused heart preparation, Ang II infusion (1x10(-6)mol/l) activated extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38-MAPK in the LV myocardium. No substantial differences were observed in the Ang II-induced ERK activation between the normotensive control DS rats and the hypertensive DS rats in either stage. In contrast, the Ang II-induced activation of JNK and p38-MAPK was augmented in the subacute LVH stage of the hypertensive DS rats, but then progressively attenuated in the chronic LVH and CHF stages. Chronic treatment with an angiotensin converting enzyme inhibitor, temocapril (20 mg/kg/day), ameliorated the responsiveness of the JNK/p38-MAPK activation, suggesting that the decreased JNK/p38-MAPK activation is a consequence of negative feedback regulation for the activated cardiac renin-angiotensin system in chronic LVH and CHF. Thus, the Ang II-induced activation of multiple cardiac MAPK pathways are differentially regulated, depending on the stages of chronic hypertrophic process. The JNK and p38-MAPK activation may be involved in the early development of adaptive LVH. However, the responsiveness of the cardiac JNK/p38-MAPK pathways progressively decreased in chronic LVH and CHF under the chronic activation of tissue renin-angiotensin system.

Angiotensin II↗
PubMed2001

Low metacarpal bone density, tooth loss, and periodontal disease in Japanese women.

The relationship between periodontitis and systemic bone mineral density in Japanese women is undetermined. We tested the hypothesis that periodontitis was more frequent in women with low metacarpal bone mineral density (m-BMD). Subjects were 190 Japanese women (89 premenopausal, 101 post-menopausal). Periodontal status was evaluated according to the Community Periodontal Index of Treatment Need (CPITN). M-BMD was measured by computed x-ray densitometry. The proportion of subjects with periodontitis (CPITN > or = 3) increased as m-BMD decreased in pre-menopausal (18.2%, 36.9%, and 66.6% in the normal, borderline, and very low m-BMD groups, p < 0.02) and post-menopausal women (41.5%, 54.8%, 60%, and 68.4% in the normal, borderline, low, and very low m-BMD groups, p < 0.05). Among post-menopausal women, those with very low m-BMD had fewer teeth present than women with normal m-BMD (19.9+/-7.2 vs. 25.1+/-4.1, p < 0.01). These results indicate that m-BMD loss is associated with periodontitis in Japanese women, and with tooth loss after menopause.

Adult↗
PubMed2001

Purification and characterization of an acid trehalase from Acidobacterium capsulatum.

We purified an acid trehalase (EC 3.2.1.28, alpha,alpha'-trehalose glucohydrolase) from an acidophilic bacterium, Acidobacterium capsulatum. The enzyme was homogeneous based on polyacrylamide gel electrophoresis, and was composed of a single polypeptide chain with a molecular mass of 57 kDa. Maximum trehalase activity was observed at pH 2.5. The acid trehalase exhibited an apparent K(m) of 1.0 mM for trehalose at 30 degrees C and pH 3.0. The trehalase was located in the periplasmic space. The activity of the enzyme was activated by 1.0 mM MnCl2 or CoCl2, and inhibited by 1.0 mM PbCl2, HgCl2, NiCl2, p-chloromercuribenzoate, N-ethylmaleimide, monoiodoacetate, or EDTA. The enzyme showed high specificity for trehalose. It was found that an equimolar mixture of alpha-D-glucose and beta-D-glucose was formed on hydrolysis of trehalose by the trehalase.

Journal Article↗
PubMed2001

Effect of repeated local minocycline administration on periodontal healing following guided tissue regeneration.

BACKGROUND: Infection after a periodontal surgical site has been prepared for guided tissue regeneration (GTR) is one of the common complications that can compromise healing. The purpose of this study was to assess the effect of repeated local antimicrobial therapy following GTR for improving clinical attachment gains, and to histologically evaluate the various cell populations and bacterial contamination of the retrieved expanded polytetrafluoroethylene membrane (ePTFE). METHODS: Forty periodontal intrabony defects in 40 patients were treated by a flap procedure that included the use of ePTFE membranes to allow GTR. Patients were randomly assigned to 2 treatment groups: 20 patients were treated with the ePTFE alone (control group), and the other 20 were treated with the ePTFE combined with the administration of a weekly repeated local application of minocycline ointment for 8 weeks after membrane placement (test group). The membranes were retrieved 6 weeks after the initial surgery and sectioned serially in a coronal-apical plane. The sections were then divided into 9 fields and examined by light microscopy for the presence of inflammatory cells and oral bacteria. Clinical measurements were taken at the time of baseline examination and at a 6-month follow-up examination after removal of the ePTFE. RESULTS: At the 6-month follow-up examination, control and test groups showed significant improvement; i.e., reduction in the probing depth and increased clinical attachment gain compared with the values at the baseline examination. However, the mean clinical attachment gain of the test group (3.0+/-0.3 mm) was significantly (P = 0.03) greater than that of the control group (2.0+/-0.5 mm). Histologically, the total number of the cells of both groups was similar. In both groups, mononuclear cells were dominant and fibroblasts, neutrophils, and plasma cells were rarely encountered. There was a tendency for the number of macrophages to be somewhat higher in the control group. The total number of bacteria in the test group was significantly less than that in the control group. The number of bacteria in both control and test groups decreased toward the apical portion. CONCLUSIONS: In the present study, clinical attachment gain of intrabony defects following GTR was favorable with repeated local administration of minocycline ointment. However, a complete microbial eradication was not achieved.

Administration, Topical↗
PubMed2000

SHPS-1 regulates integrin-mediated cytoskeletal reorganization and cell motility.

The transmembrane glycoprotein SHPS-1 binds the protein tyrosine phosphatase SHP-2 and serves as its substrate. Although SHPS-1 has been implicated in growth factor- and cell adhesion-induced signaling, its biological role has remained unknown. Fibroblasts homozygous for expression of an SHPS-1 mutant lacking most of the cytoplasmic region of this protein exhibited increased formation of actin stress fibers and focal adhesions. They spread more quickly on fibronectin than did wild-type cells, but they were defective in subsequent polarized extension and migration. The extent of adhesion-induced activation of Rho, but not that of Rac, was also markedly reduced in the mutant cells. Activation of the Ras-extracellular signal-regulated kinase signaling pathway and of c-Jun N-terminal kinases by growth factors was either unaffected or enhanced in the mutant fibroblasts. These results demonstrate that SHPS-1 plays crucial roles in integrin-mediated cytoskeletal reorganization, cell motility and the regulation of Rho, and that it also negatively modulates growth factor-induced activation of mitogen-activated protein kinases.

Animals↗
PubMed2000

Wound healing acceleration of a novel transforming growth factor-beta inducer, SEK-1005.

The studies were carried out to elucidate the effect of a novel cyclic peptide, SEK-1005 (C(45)H(70)N(8)O(13)), on wound healing. SEK-1005 (4-10 microg/wound) applied topically significantly accelerated the healing of a full-thickness wound on the dorsal skin of a rat. In a healing-impaired mouse, the peptide (2-10 microg/wound) had more potent activity, exerting an effect comparable to that of basic fibroblast growth factor (FGF). However, SEK-1005 (0.1-100 ng/ml) scarcely promoted the proliferation of cultured fibroblasts (NIH3T3 cells) while basic FGF (0.2-5 ng/ml) showed marked mitogenic activity. SEK-1005 (2-10 microg/wound) significantly increased the topical production of transforming growth factor (TGF)-beta1, a cytokine that is known to accelerate wound healing. This activity was closely correlated with the wound-repairing effect. From the above, SEK-1005 can be considered as a new type of wound healing agent with potent TGF-beta1-inducing activity.

3T3 Cells↗
PubMed2000

Noncompetitive, reversible inhibition of aminoacylase-1 by a series of L-alpha-hydroxyl and L-alpha-fluoro fatty acids: ligand specificity of aspergillus oryzae and porcine kidney enzymes.

L-lactate and L-beta-phenyllactate have been identified in the culture broth of Streptomyces sp. KY-11 as reversible noncompetitive inhibitors of Aspergillus oryzae aminoacylase-1 and porcine kidney aminoacylase I. A series of alpha-hydroxyl acids (DL-R-CH(OH)-COOH, R = Et, n-pro, n-butyl, n-pentyl, n-hexyl) also inhibited the two enzymes in reversible noncompetitive kinetics, and the inhibition potency (-log K(i)) increased with the increased hydrophobicity of the R group. The two eukaryotic enzymes showed distinct preferences to the ligand alpha-alkyl group, and the fungus enzyme was inhibited by L-beta-phenyllactate (R = benzyl) 10(3)-fold more potently than the mammalian enzyme. L-alpha-Fluoro-beta-phenyl-propionate and its D-isomer were used to show that the L-configuration of the alpha-substituent was important for potent inhibition of both the enzymes. The fungus aminoacylase-1 steeply decreased the affinity to alpha-fluoro- and alpha-hydroxy-n-caproate as pH was raised from 7 to 11, whereas the mammalian enzyme retained the affinity to these ligands under alkaline conditions. These results suggest that A. oryzae aminoacylase-1 has an acidic residue that interacts with -OH or -F, while the mammalian enzyme would have a basic residue that recognizes the alpha-substituents.

Alkylation↗
PubMed2000

Simultaneous analysis of anthranilic acid derivatives in pharmaceuticals and human urine by high-performance liquid chromatography with isocratic elution.

A high-performance liquid chromatographic (HPLC) method for simultaneous determination of mefenamic acid (MFA), flufenamic acid (FFA) and tolfenamic acid (TFA) is presented for application to pharmaceuticals and human urine. Isocratic reversed-phase HPLC was employed for quantitative analysis using tetra-pentylammonium bromide (TPAB) as an ion-pair reagent. Urine samples were purified by solid-phase extraction using a silica-based strong anion-exchanger, Bond-Elut SAX cartridge. The HPLC assay was carried out using a Wakosil ODS 5C18 column (5 microm, 150x4.6 mm I.D.). The mobile phase consisted of 1.9 g of TPAB dissolved in 1:1 of a mixture of acetic acid-sodium acetate buffer solution, pH 5.0, and acetonitrile (11:9, v/v). The calibration curves of MFA, FFA and TFA showed good linearity in the concentration range of 33-167 microg/ml with a wavelength of 280 nm for pharmaceuticals, and in the low concentration range (1.7-30.1 microg/ml) with a wavelength of 230 nm for biological fluids. The correlation coefficients were better than 0.9999 in all cases. The lower limits of detection (defined as a signal-to-noise ratio of about 3) were approximately 2 ng for MFA, 3.5 ng for FFA and 2.5 ng for TFA. The procedure described here is rapid, simple, selective and is suitable for routine analysis of pharmaceuticals and pharmacokinetic studies in human urine samples.

Calibration↗