Purification of [125I]-vasoactive intestinal peptide by reverse-phase HPLC

VIP was labeled with sodium ¹²⁵iodide, and ¹²⁵I-VIP was purified by reverse-phase high performance liquid chromatography. Optimal separations of ¹²⁵I-VIP and unlabeled VIP were obtained using two C₁₈-Novapak columns in series and a gradient of acetonitrile in triethylamine phosphate for elution. The specific activity of the ¹²⁵I-VIP was 1.99±0.21 Ci/μmole, approaching the maximum specific activity of monoiodinated VIP (2.26 Ci/μmole). Radioimmunoassay and radioreceptorassay for VIP were more sensitive (2.6-fold, and 2.5-fold, respectively) using ¹²⁵I-VIP purified by HPLC compared to ¹²⁵I-VIP obtained from an open-end cellulose column. These results demonstrate the advantage of preparing purified ¹²⁵I-VIP by HPLC for the accurate assay of VIP and VIP-receptors in tissues and biological fluids.     

Trp7,Leu8]LH-RH in reptilian brain

Luteinizing hormone-releasing hormone (LH-RH) immunoreactive peptides in acetic acid extracts of lizard (Cordylis nigra) brain were studied by high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera. Four different LH-RH immunoreactive peptides were detected. The major form co-eluted with salmon brain LH-RH, [Trp7,Leu8]LH-RH, in a cation exchange and three reverse phase HPLC systems which were specifically designed to separate a range of LH-RH analogues. The interaction of this major LH-RH immunoreactive peptide with a number of antisera directed against different regions of mammalian, chicken and salmon LH-RH was similar to the relative interaction of [Trp7,Leu8]LH-RH with these antisera. These data strongly indicate that the major form of lizard brain LH-RH is identical to salmon brain LH-RH [( Trp7,Leu8]LH-RH). The three additional molecular forms of immunoreactive LH-RH in lizard brain appear to differ from mammalian LH-RH in the middle to C-terminal region of the molecule.     

Quantitative distribution of calcitonin gene-related peptide in the rat central nervous system

Using an antiserum directed against human calcitonin gene-related peptide (hCGRP), which fully cross reacts with rat CGRP, a sensitive radioimmunoassay was developed. The antiserum was characterized by displacement curve characteristics and high performance liquid chromatography. The assay was applied to rat brain tissue and the concentration of CGRP for 48 microdissected brain areas is presented. Highest levels (1000–4500 fmol/mg protein) were found in the central amygdaloid, caudate putamen, and spinal trigeminal nerve nucleus and tract, substantia gelatinosa, and the dorsal horn of the spinal cord. Moderate levels (200–600 fmol/mg protein) were found in the bed nucleus of the stria terminalis, the subfornical organ, the paraventricular, arcuate, dorsomedial, dorsal parabrachial, ambiguus and tractus solitarii nuclei and in the median eminence. These results coincide with those previously obtained by immunohistochemistry. The widespread distribution in the brain suggests involvement of CGRP in a variety of behavioral functions.     

Purification of monoiodinated vasointestinal peptide (M125I-VIP) by high pressure liquid chromatography (HPLC)

In our developed reverse phase high performance liquid chromatography, four forms of M¹²⁵I-VIP have been isolated free from unlabled VIP and other iodinated components. The quicker eluting M¹²⁵I-VIP forms (oxidised and reduced) have a consistently and significantly low non-specific binding with specific target cells of VIP (HT-29) as compared to the late eluting forms of VIP. The retention time is considerably increased when the molecule of VIP is fully iodinated.     

猕猴桃籽有机成分的初步研究

对猕猴桃籽的水溶粗提物进行了清除H2O2的实验,结果表明粗提物对H2O2有清除能力,提示猕猴桃籽具有抗过氧化、延缓衰老作用的能力。目前大量废弃的猕猴桃籽有待合理开发利用,但其有机成分尚未见报道。本实验进行了猕猴桃籽的成分预分析,结果表明,猕猴桃籽含有生物碱、氨基酸、蛋白质、还原糖、油脂等,可能含有酚类、皂苷、三萜和甾醇等,不含有鞣质。利用高效液相色谱技术对其可能的有效成份进行了分离和纯化,得到3种纯的待鉴定的化合物。     

高效液相色谱法测定红景天根中红景天甙的含量

建立了一种高效液相色谱法测定红景天根中红景天甙的含量。色谱柱为Hypersil—C18（5μm,150mm×4．6mm）,甲醇-水为流动相,梯度洗脱,柱温25℃,在250nm波长处检测。研究结果表明：红景天甙的检测限为50μg/L,线性范围为4—40mg／L,回归方程为Y=5．6348X-4．0931,r=0．9996,加样回收率为98．05％。方法操作简便、快速、准确。     

Using HPLC pigment analysis to investigate phytoplankton taxonomy: the importance of knowing your species

Phytoplankton microscopic enumerations and HPLC analyses of their pigments were performed weekly for a complete year at a coastal station in the English Channel. The taxonomic composition of the phytoplankton community was assessed using the HPLC results combined with the mathematical tool CHEMTAX in two different ways. Firstly, without using the species level taxonomic information obtained at the microscopic level (blind analyses), and secondly by including the information from the microscopic taxonomic analysis (directed analyses). The results indicate that, due to the particular pigment composition of some species (for example, the dinoflagellate, Karenia mikimotoi and the haptophyte, Phaeocystis pouchetii), a blind analysis would result in very significant errors in the taxonomic determination of the bloom events at this station. Major blooms of Karenia mikimotoi and P. pouchetii were mistaken for blooms of diatoms on the basis of a blind HPLC-CHEMTAX analysis. Only with the information from the microscopic observations was it possible to obtain an accurate representation of the phytoplankton community.Communicated by H.-D. Franke     

真核细胞基因工程产物人生长激素的纯化研究

应用反相高效液相色谱法（ＲＰ－ＨＰＬＣ）对真核细胞基因表达产物人生长激素进行了纯化研究,发现在７１．６７％的乙晴浓度下人生长激素可有效地与其它杂蛋白分离,乙晴与人生长激素的短时间接触不影响其放射免疫活性．     

胶原蛋白降解物高效液相色谱/质谱联用分析

利用高效液相色谱／质谱联用技术分析了胶原蛋白Ⅱ和Ⅰ经变性和酶解处理得到的多肽混合物,比较研究了两种类型胶原蛋白降解物之间的区别,利用液相色谱质谱连用技术识别出了不同类型胶原蛋白中的特征肽段。胶原蛋白在50℃处理3h可溶解于盐溶液,凝胶过滤分析结果表明,热变性的胶原蛋白分子量在10万以上,因此在热变性过程中胶原蛋白的三螺旋结构被破坏但没有明显的随机降解。利用胰蛋白酶对胶原蛋白进行了酶切处理,在酶用量为2％,pH 8.1~8.2,温度37℃条件下,处理20h后胶原蛋白降解较完全,多肽混合物的平均分子量在1万以下。利用液质联用技术研究Ⅱ型和Ⅰ型胶原蛋白降解物中的多肽,通过多级质谱分析了胶原蛋白降解物多肽的序列。结果表明,不同类型的胶原蛋白酶解后的多肽混合物中存在特定序列的特征肽段,利用特征肽段进行胶原蛋白类型识别是可行的。     

Determination of choline and ethanolamine plasmalogens in human plasma by HPLC using radioactive triiodide (1-) ion (125I3-)

For the purpose of developing highly sensitive and convenient determination of plasmalogens, the high-performance liquid chromatography (HPLC) method using radioactive iodine ((125)I) was investigated. Radioactive triiodide (1-) ion ((125)I(3)(-)), which is an actual iodine form capable of reacting with vinyl ether bond ([bond]CH(2)[bond]O[bond]CH[double bond]CH[bond]) of plasmalogens, could be safely and efficiently produced by oxidizing a commercial radioactive sodium iodine (Na(125)I) with hydrogen peroxide (H(2)O(2)) under acid condition (pH 5.5-6.0), which is called iodine-125 reagent. I(3)(-) specifically reacted with plasmalogens at the molar ratio of 1:1 in methanol, and 1 or 2 mol of plasmalogens was involved in the binding with iodine per iodine atom, resulting in the formation of stable iodine-binding phospholipids. The HPLC system with Diol column and acetonitrile/water as a mobile phase was available for separating iodine-binding phospholipids from nonbinding free iodine and for separately eluting iodine-binding phospholipids derived from choline and ethanolamine plasmalogens. Using iodine-125 reagent (1.85 MBq/ml), plasmalogens were detectable at high sensitivity of 10,000-15,000 cpm/nmol, which is more than 1000-fold higher sensitivity than the classical determination with nonradioactive iodine. Plasmalogen concentrations in human plasma were measured with the HPLC system and determined as, on average, 129.1+/-31.3 microM (n=8) in a 1.2 content ratio of choline to ethanolamine plasmalogens, a concentration that nearly agrees with the value reported previously.     

Synthesis of [I]iodoDPA-713: A new probe for imaging inflammation

[¹²⁵I]IodoDPA-713 [¹²⁵I]1, which targets the translocator protein (TSPO, 18 kDa), was synthesized in seven steps from methyl-4-methoxybenzoate as a tool for quantification of inflammation in preclinical models. Preliminary in vitro autoradiography and in vivo small animal imaging were performed using [¹²⁵I]1 in a neurotoxicant-treated rat and in a murine model of lung inflammation, respectively. The radiochemical yield of [¹²⁵I]1 was 44 ± 6% with a specific radioactivity of 51.8 GBq/μmol (1400 mCi/μmol) and >99% radiochemical purity. Preliminary studies showed that [¹²⁵I]1 demonstrated increased specific binding to TSPO in a neurotoxicant-treated rat and increased radiopharmaceutical uptake in the lungs of an experimental inflammation model of lung inflammation. Compound [¹²⁵I]1 is a new, convenient probe for preclinical studies of TSPO activity.     

Effects of Valeriana Officinalis Extracts on [3H]Flunitrazepam Binding,Synaptosomal [3H]GABA Uptake,and Hippocampal [3H]GABA Release

Extracts of Valeriana officinalis have been used in folkloric medicine for its sedative, hypnotic, tranquilizer and anticonvulsant effects, and may interact with -aminobutyric acid (GABA) and/or benzodiazepine sites. At low concentrations, valerian extracts enhance [³H]flunitrazepam binding (EC₅₀ 4.13 × 10^–10 mg/ml). However, this increased [³H]flunitrazepam binding is replaced by an inhibition at higher concentrations (IC₅₀ of 4.82 × 10^–1 mg/ml). These results are consistent with the presence of at least two different biological activities interacting with [³H]flunitrazepam binding sites. Valerian extracts also potentiate K⁺ or veratridine-stimulated release of radioactivity from hippocampal slices preloaded with [³H]GABA. Finally, inhibition of synaptosomal [³H]GABA uptake by valerian extracts also displays a biphasic interaction with guvacine. The results confirm that valerian extracts have effects on GABA_A receptors, but can also interact at other presynaptic components of GABAergic neurons.     

Photooxidation products of 7,12-dimethylbenz[a]anthracene.

The principal products of the photooxidation of 7,12-dimethylbenz[a]-anthracene (DMBA) in aqueous solutions by photooxidation induced by laboratory lighting have been characterized by high performance liquid chromatograms (HPLC), ultraviolet and mass spectrograms and by comparisons with authentic samples. The products identified were the 7,12-epidioxy-7,12-dihydro-7-12-dimethyl-, 7,12-dione, 7-hydroxymethyl-12-methyl-, 12-hydroxymethyl-7-methyl-, 7-formyl-12-methyl-, 12-formyl-7-methyl-, and 12-hydroxy-12-methyl-7-one derivatives of benz[a]-anthracene. The HPLC profile of products is similar to that obtained from oxidation of DMBA by 'one-electron' reagents, singlet oxygen, or liver microsomal metabolism. The first points of attack are the 7- and 12- positions. The mechanism of photooxidation appears to be generation of singlet oxygen by photodynamic effect of DMBA. None of the products is photosensitizing, however.     

Measurement of choline acetyltransferase with [14C]acetate by a cycling procedure

A multiple enzyme and multisubstrate cycling system is described for the radiometric determination of cholineacetyltransferase (ChAT) activity in crude tissue homogenates. The methods employs [¹⁴C]acetate coupled with the enzymes acetate kinase (AK) and phosphotransacetylase (PTA) for the generation of [¹⁴C]acetyl CoA. By recycling it was possible to avoid product inhibition of ChAT by CoA, ATP was maintained constant by rephosphorylation of ADP. Kinetics of the individual enzyme reactions were studied and the parameters obtained were used to select appropriate conditions to maintain linearity of varying amounts ChAT activity over a sixty minute time course. The sensitivity of the method is limited only by the specific activity of commercially available isotope labeled acetate.Special issue dedicated to Dr. O. H. Lowry.     

Purification of endogenous inhibitors of [3H]flunitrazepam binding from bovine brain

Endogenous substances which inhibited the binding of [³H]flunitrazepam ([³H]FNZ) to bovine synaptosomal membranes have been purified from the hot acetic acid extracts of the bovine brain. Three peaks of inhibitory activity were obtained by Sephadex G-10 gel chromatography. Two of the peaks (Peak 2, and Peak 3) which had lower molecular weights that that of peak 1 were identified as inosine and hypoxanthine by TLC methods. Another peak (Peak 1) was further purified to homogeneity using both cation and anion ion-exchange chromatography and the following two-step reversed-phase HPLC. The purified substance inhibited the [³H]FNZ binding dose-dependently and competitively but did not have an effect on the binding of the peripheral-type BZ ligand [³H]Ro 5-4864. It was also shown that the substance was heat-stable and resistant to proteolytic degradation (trypsin, -chymotrypsin, pronase). However, a significant loss of inhibitory activity to [³H]FNZ binding was observed after acid hydrolysis. Molecular weight estimates based on gel filtration methods were less than 500 dalton, and the maximal ultraviolet absorption peak was at 314 nm. These results suggest that this substance is a new endogenous ligand for the central BZ receptor and may play an important role in regulating the GABAergic tone in the central nervous system.     

湖北小连翘中化学成分的高效液相色谱与电喷雾离子化质谱联用分析

湖北小连翘(Hypericum hubeiense L.H.Wu et D.P.Yang)是2004年在我国发现的一个金丝桃属(Hypericum)植物(藤黄科)新种.利用高效液相色谱与质谱联用技术对该植物中主要化学成分进行了分离分析,初步鉴定出金丝桃素类、黄酮类等12种化学成分.同时,以金丝桃素为外标,采用HPLC法在590 nm波长下测定了金丝桃素类成分的含量.     

Synthesis of a novel N[bond]O-interglycosidic disaccharide

The carbohydrate subunits carrying an N-O-interglycosidic bond play a very important role in the biological activity of the enediyne antibiotics. Condensation of O-(alpha- and beta-D-glucopyranosyl)hydroxylamine (5a and 5b) with the hex-3-ulopyranoside (6) furnished methyl 4,6-O-benzylidene-2,3-dideoxy-3-(2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyloxy)imino-alpha- and beta-D-erythro-hexopyranoside (7a and 7b). Stereoselective reduction of the Cz.dbnd6;N bond of 7a and 7b with sodium cyanoborohydride resulted in the formation of the required protected N-O-interglycosidic disaccharides (8a and 8b). Finally, catalytic hydrogenation of 8a afforded methyl 2,3-dideoxy-3-(alpha-D-glucopyranosyloxy)amino-alpha-D-ribo-hexopyranoside (9a). Under similar conditions the beta anomer 8b underwent decomposition.     

用HPLC法测定发酵液中的生物素含量

采用高效液相色谱法测定发酵液中生物素的含量。色谱柱为Symmetry C18,流动相为V(甲醇):V(pH 2.5磷酸缓冲溶液)=14:86,检测波长为210 nm,柱温为35℃。结果表明,样品进样量在5-150μg/mL范围内线性关系良好,加样回收率在95%以上,相对标准偏差RSD为6.6%,最低检测限为0.01μg/mL。该方法简便、快捷、准确、重现性好,可用于发酵液中生物素的含量测定。     

Measurement of muramic acid in marine sediments by high performance liquid chromatography

Bacterial biomass in marine sediments may be estimated from the amount of muramic acid present. A method for determining muramic acid by high performance liquid chromatography is described, which is simpler and faster than other methods. Muramic acid is released from sediment by acid hydrolysis, and assayed as an o-phthaldialdehyde derivative.