Analytical chemical studies on high-performance recognition and detection of bio-molecules in life

In order to understand the mechanism for maintaining life of animals based on the search of dynamics of biomolecules, I have developed several sensitive and selective methods for their quantification. Using the methods of derivatization with the developed benzofurazan fluorogenic reagents (4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), ammonium 7-fluoro-2,1,3-benzoxadiazole 4-sulfonate (SBD-F) and etc.) followed by high-performance liquid chromatography (HPLC)--fluorescence detection, a certain kind of biological and clinical importance was demonstrated of chiral bio-molecules (D-amino acids, D-lactic acid and so on), peptides and proteins. The proposed method (derivatization with SBD-F, isolation of the fluorescent proteins by two-dimensional HPLC, enzymatic digestion and identification of the altered proteins by HPLC-mass spectrometry (MS)/MS with database-searching algorithm) for proteomics studies revealed the changed proteins in the islets of Langerhans of the dexamethazone-induced diabetic rats. An importance of catecholamine metabolism on the blood pressure regulation was also suggested by the method of HPLC-chemiluminescence detection of catecholamines and their 3-O-methylmetabolites. A new field of Analytical Chemistry, i.e., Bio-Analytical Chemistry, was also proposed.     

Selective production of fungal beauveriolide I or III by fermentation in amino acid-supplemented media

Beauveriolides I and III, cyclic depsipeptides composed of L-Phe, L-Ala, D-Leu and (3S,4S)-3-hydroxy-4-methyloctanoic acid, and L-Phe, L-Ala, L-allo-Ile and (3S,4S)-3-hydroxy-4-methyloctanoic acid, respectively, were previously isolated from the culture broth of fungal Beauveria sp. FO-6979 as inhibitors of macrophage foam cell formation. To improve the production of these compounds by fermentation, the culture conditions were studied. The production of both beauveriolides was increased five to ten folds by fermentation in the culture media containing tryptone. Further study revealed that addition of L-Leu/L-Ile, but not D-Leu/D-allo-Ile, to the culture medium yielded a high and selective production of beauveriolide I or III. As a result, regardless of their separation difficulty due to the similar physico-chemical properties, a large amount of beauveriolide I or III was prepared from the culture broth obtained from L-Leu- or L-Ile-supplemented fermentation, respectively, by one step purification using silica gel column chromatography.     

Selective haptenation of cellular or extracellular protein by chemical allergens: association with cytokine polarization

Sensitizing chemicals can cause different forms of allergy, allergic contact dermatitis, or sensitization of the respiratory tract. These discrete types of chemicals induce in mice qualitatively divergent immune responses; contact allergens provoke preferential type 1 responses, whereas respiratory allergens stimulate selective type 2 responses. We have questioned whether the ability of chemicals to initiate polarized immune responses is in part a function of the nature of their association with protein. Cytokine secretion profiles provoked following topical exposure of BALB/c mice to dinitrochlorobenzene (DNCB), dinitrofluorobenzene (DNFB), fluorescein isothiocyanate (FITC), trimellitic anhydride (TMA), and dinitrobenzenesulfonyl chloride (DNBSCl) were compared with the distribution of covalent binding to U937 cells and/or to serum proteins in vitro. DNCB and DNFB each provoked a type 1 cytokine secretion profile, with high levels of IFN-gamma, but relatively low levels of type 2 cytokines IL-4, -5, and -10. The converse selective type 2 phenotype was seen following equivalent exposure to TMA, FITC, or DNBSCl. Each chemical bound covalently to U937 cells and to serum proteins, when incubated with cells or serum alone. When incubated with cells and serum together, DNCB and DNFB bound selectively to cellular protein, whereas TMA, FITC, and DNBSCl bound selectively to serum. These investigations show that the distribution of antigen formation of chemical allergens in an in vitro model system segregates with the type of cytokines secreted from activated lymph node cells in an in vivo mouse model. Chemical allergens that stimulate type 1 cytokine secretion profiles bind selectively to cellular proteins, whereas others that provoke type 2 cytokine profiles bind preferentially to serum proteins.     

Flow cytometric detection and analysis of tailless sperm caused by sonication or a chemical agent

Flow cytometric analysis has been developed to detect tailless sperm with heads detached from the tails at the neck position. When isolated tailless sperm suspension was subjected to flow cytometry, a second sperm population appeared alongside the normal sperm population on light scatter-histogram. The percentage of this second sperm population (85.2%) was in good agreement with that for the tailless sperm (88.7%) determined microscopically, indicating that the second sperm population would correspond to tailless sperm population in the light scatter-histogram. Rates for tailless sperm determined by flow cytometry significantly correlated with those estimated microscopically following exposure of sperm to either sonication (r = 0.94, P < 0.01), or nitrobenzene (r = 0.80, P < 0.01). The results indicated the utility of the light scatter-histogram in flow cytometry as a simple and convenient procedure for the detection of tailless sperm induced by chemical compounds.     

Peptide hormones and sport: misuse and detection.

In 1989 the Medical Commission of the International Olympic Committee (IOC) introduced the new doping class of 'peptide hormones and analogues,' which include human chorionic gonadotrophin (hCG) and related compounds, adrenocorticotrophic hormone (ACTH), human growth hormone (hGH), all the releasing factors of these listed hormones, and erythropoietin (Epo). Currently there are no IOC approved definitive tests for these hormones but highly specific immunoassays combined with suitable purification techniques may be sufficient to warrant IOC approval. The importance of measuring hCG and luteinizing hormone (LH) in the control of testosterone misuse is discussed and strategies for the detection of hGH, ACTH and Epo administration are suggested.     

Nature and reactivity of platinum-pyrimidine blues with biomacromolecules

The reaction of platinum-pyrimidine complexes with a range of biomacromolecules was studied using circular dichroic (CD) and viscometric techniques. The reaction of platinum-uracil blue with DNA is characterized by a small increase in the positive CD transition at 275 nm and a large decrease in the amplitude of the negative band at 245 nm; these data are consitent with a change to a conformational state characteristic of the premelt stage as well as of partial denaturation respectively. Concomitant with denaturation, the viscosity of DNA changes from 74 to 45 dl/g. Part of this interaction with DNA may involve intercalation of the platinum-uracil complex since it will competitively displace ethidium bromide from the DNA. Alternatively, the displacement of ethidium bromide may be the result of denaturing the nucleic acid. The platinum complex also reacts instantaneously with single and double stranded synthetic nucleic acids, RNA, whole ribosomes and proteins. Platinum-uracil causes nearly complete destruction of the secondary structure of bovine serum albumin which was used as a model for protein interaction. Using computer analysis of the CD data, the helical content of serum albumin decreased from 70 to 10 per cent. Carbon-13 nuclear magnetic resonance (¹³C-n.m.r.) spectra of the platinum-thymine blue complex revealed that this complex consists of a multiplicity of isomers. Because of this and the complex nature of its reactions with selected biomacromolecules, it is doubtful that a precise mechanism of action for antitumor activity can be established.     

四消丸HPLC指纹图谱及化学模式识别研究

目的:建立四消丸HPLC指纹图谱方法,为评价其质量提供参考。方法:采用Waters Symmetry Shield TM RP₁₈色谱柱;乙腈(A)-0.1%磷酸溶液(B)为流动相,梯度洗脱;流速1.0mL·min^-1;柱温30℃;检测波长254nm;进样量10μL。通过相似度评价、聚类分析、主成分分析和偏最小二乘判别分析对6家生产企业不同批次的四消丸样品进行质量评价和分析。结果:6家生产企业17批样品共标定24个共有峰,相似度为0.747~0.972,并对24个色谱峰进行了药味归属,同时指认了12个色谱峰;通过聚类分析及主成分分析将所有样品分为2类;偏最小二乘判别分析筛选出包括大黄素甲醚、大黄素葡萄糖苷、大黄素等在内的8种质量差异标志物。结论:该方法操作简便、准确,可用于四消丸的质量控制和评价研究。     

龙生蛭胶囊的HPLC指纹图谱及其化学模式识别研究

目的 建立龙生蛭胶囊的HPLC指纹图谱,结合化学模式识别方法进行质量评价。方法 采用HPLC法建立龙生蛭胶囊的指纹图谱,进行相似度评价,确定共有峰;对测定结果进行层次聚类分析和主成分分析,并结合正交偏最小二乘–判别分析对样品进行模式识别,以VIP值大于1为标准筛选影响龙生蛭胶囊质量的差异性成分。结果 19批龙生蛭胶囊样品的HPLC指纹图谱共标定了20个共有峰,相似度均大于0.95,指认了9个共有峰,分别为槲皮素、没食子酸、原儿茶酸、紫丁香苷、绿原酸、芍药苷、刺五加苷E、异嗪皮啶、毛蕊异黄酮葡萄糖苷。19批样品可分为2类;前4个主成分的累积方差贡献率为85.504%;以VIP值大于1为标准,筛选出8个主要峰,13、8、2（没食子酸）、3（原儿茶酸）、1（槲皮素）、14（芍药苷）、10、11号峰。结论 建立了龙生蛭胶囊更全面、系统的质量评价和分析方法,为龙生蛭胶囊的质量标准提高提供理论依据。     

高效液相色谱法测定脑活素注射液中的氨基酸含量及肽图分析

本文采用高效液相色谱法分析研究了进口药品脑活素注射液中的氨基酸含量及其低分子肽的肽图。试验结果反映了该药品的内在质量,同时提出控制质量的可靠方法.     

使用手性冠醚的高效液相色谱法研究伯胺类药物的手性分离

使用手性柱Crownpak CR( )的高效液相色谱(HPLC)法对11种伯胺类药物进行拆分，结果有10种药物被手性分离。实验结果表明：柱温下降，洗脱时间增大，分离度Rs值明显提高，而甲醇浓度增大，分离度有所减小。     

Fast and Selective Assay of l-Ascorbic Acid in Rose Hips by RP-HPLC Coupled with Electrochemical and/or Spectrophotometric Detection

A simple, fast, and selective method for the assay of L-ascorbic acid in rose hips was developed using RP-HPLC. Ascorbic acid was extracted with 2% metaphosphoric acid and sample clean-up was optimized with C18 disposal extraction cartridges. Use of 0.5% metaphosphoric acid as the mobile phase completely suppressed the dissociation of ascorbic acid. Sufficient retention was achieved so that the addition of ion-pairing agents was not necessary. The column effluent was monitored by spectrophotometric (242 nm) and electrochemical detectors (550 mV vs Ag/AgCl); the electrochemical detector was found to be both more selective and more sensitive. By coup-ling the two detectors in series, a true two channel detection with on-line validation was achieved. Reproducible results (r.s.d. 1-4%) were obtained from assays of several commercially available products, as well as from fresh rose hips, and revealed considerable differences in ascorbic acid content, ranging from 0.03 to 1.3%.     

苯乙酮酸的高特异性生物转化合成

从20株酵母菌中筛得一株解脂假丝酵母（Candida lipolytica）SIPI0201，可高特异性转化苯甘氨酸为苯乙酮酸。经优化转化条件后表明当温度30℃、pH9．0、Ca^2＋浓度0．75mmol／L，并于转化培养初始阶段加入底物，转化率可达47．9％，较优化前提高91％。该菌株具有较好的底物特异性和选择性。     

金莲花药材UPLC指纹图谱及模式识别研究

目的采用超高效液相色谱法(UPLC)建立金莲花药材指纹图谱的质量评价方法。方法以3种不同基源的金莲花药材为研究对象,采用UPLC法,以Acquity UPLC BEH C18(100 mm×2.1 mm,1.7μm)为色谱柱,乙腈-体积分数0.1%磷酸溶液为流动相进行梯度洗脱,流速为0.3 m L·min-1,检测波长为340 nm,温度为45℃。结果对38批样品进行测定,建立了金莲花药材的UPLC标准指纹图谱。采用对照品指认了荭草素-2″-O-β-L-半乳糖苷、荭草苷及牡荆苷3个色谱峰。采用聚类分析和主成分分析法对指纹图谱数据进行了模式识别研究,可很好地区分金莲花、短瓣金莲花和阿尔泰金莲花。结论所采用UPLC法建立金莲花药材的指纹图谱专属性强、方法快速、简便、可靠,可用于金莲花药材的质量控制及综合评价。     

Selective impairment in the recognition of anger induced by diazepam

Rationale: Facial expressions appear to be processed by at least partially separable neuro-cognitive systems. Given this functional specialisation of expression processing, it is plausible that these neurocognitive systems may also be dissociable pharmacologically. Objective: The present study therefore compared the effects of diazepam (15 mg) with placebo upon the ability to recognise emotional expressions. Methods: A double blind, independent group design was used to compare the effects of diazepam and matched placebo in32 healthy volunteers. Participants were presented morphed facial expression stimuli following a paradigm developed for use with patients with brain damage and asked to name one of the six basic emotions (sadness, happiness, anger, disgust, fear and surprise). Results: Diazepam selectively impaired subjects’ ability to recognise angry expressions but did not affect recognition of any other emotional expression. Conclusions: The findings are interpreted as providing further support for the suggestion that there are dissociable systems responsible for processing emotional expressions. It is suggested that these findings may have implications for understanding paradoxical aggression sometimes elicited by benzodiazepines. Received: 27 May 1999 / Accepted: 7 July 1999     

Reactivity of chemical respiratory allergens in the Peroxidase Peptide Reactivity Assay

Sensitizing chemicals are commonly associated primarily with either skin or respiratory sensitization. In the Direct Peptide Reactivity Assay (DPRA), when compared with skin sensitizers, respiratory allergens have been demonstrated to selectively react with lysine rather than cysteine. The Peroxidase Peptide Reactivity Assay (PPRA) has been developed as a refinement to the DPRA. The PPRA incorporates dose–response analyses, mass spectroscopy for peptide detection and a horseradish peroxidase–hydrogen peroxide enzymatic system, increasing the potential to identify pro-haptens. In the investigations reported here, the PPRA was evaluated to determine whether it provides advantages for the identification of respiratory allergens. Twenty respiratory sensitizers, including five predicted to be pre-/pro-haptens were evaluated. The PPRA performed similarly to the DPRA with respect to identifying inherently reactive respiratory sensitizers. However, three respiratory sensitizers predicted to be pre-/pro-haptens (chlorhexidine, ethylenediamine and piperazine) were non-reactive and the general selectivity of the respiratory allergens for lysine was lost in the PPRA. Overall, the data indicate that the PPRA does not provide an advantage over the DPRA for discriminating allergens as either contact or respiratory sensitizers. Nevertheless, the PPRA provides a number of refinements to the DPRA that allow for an enhanced characterization of reactivity for both classes of chemical allergens.     

The direct Peptide reactivity assay: selectivity of chemical respiratory allergens

It is well known that some chemicals are capable of causing allergic diseases of the skin and respiratory tract. Commonly, though not exclusively, chemical allergens are associated with the selective development of skin or respiratory sensitization. The reason for this divergence is unclear, although it is hypothesized that the nature of interactions between the chemical hapten and proteins is influential. The direct peptide reactivity assay (DPRA) has been developed as a screen for the identification of skin-sensitizing chemicals, and here we describe the use of this method to explore whether differences exist between skin and respiratory allergens with respect to their peptide-binding properties. Known skin and respiratory sensitizers were reacted with synthetic peptides containing either lysine (Lys) or cysteine (Cys) for 24h. The samples were analyzed by HPLC/UV, and the loss of peptide from the reaction mixture was expressed as the percent depletion compared with the control. The potential for preferential reactivity was evaluated by comparing the ratio of Lys to Cys depletion (Lys:Cys ratio). The results demonstrate that the majority of respiratory allergens are reactive in the DPRA, and that in contrast to most skin-sensitizing chemicals, preferentially react with the Lys peptide. These data suggest that skin and respiratory chemical allergens can result in different protein conjugates, which may in turn influence the quality of induced immune responses. Overall, these investigations reveal that the DPRA has considerable potential to be incorporated into tiered testing approaches for the identification and characterization of chemical respiratory allergens.     

Chiral recognition in gas chromatographic resolution of amino acid enantiomers

Studies of selectivity of hydrogen bond formation in chiral solute-solvent systems have been performed by¹H and¹³C nuclear magnetic resonance techniques. These data are correlated with the results of gas chromatographic investigations of the same systems. Interactions between the optically active solvent(N-(N-benzoyl-L-amino acid)-anilide) and optically active solute(N-trifluoroacetyl-L-alanyl isopropyl ester) were examined. NMR evidence indicated that hydrogen bonding interaction occurred between two N-H portion and on peptidyl carbonyl portion in stationary phase and solute molecule on three points. The association constants of solvent-solute interaction were calculated and the structure of the diastereomeric association complex between N-(N-benzoyl-L-valyl)-anilide and N-TFA-L-alanyl isopropyl ester was proposed.     

西洋参药材皂苷类成分HPLC-UV-ELSD特征图谱及模式识别研究

采用HPLC-UV-ELSD串联技术,建立西洋参药材皂苷类成分HPLC-UV-ELSD特征图谱的质量控制方法。色谱柱为Agilent Extend-C18(250 mm×4.6 mm,5μm),乙腈-水二元梯度洗脱模式,流速为1.0 mL·min-1,检测波长为203 nm,漂移管温度为106.5℃,空气流速为2.9 L·min-1,对20批西洋参药材进行HPLC-UV-ELSD测定,采用聚类分析和主成分分析对特征图谱数据进行了模式识别研究,并指认了人参皂苷Rg1、Re、Rb1、Rc、Rb2、Rb3、Rd及拟人参皂苷F11等8个色谱峰。所建立的西洋参皂苷类成分特征图谱特征性和专属性强,方法快速、简便、可靠,可用于西洋参质量控制及综合评价。