Reversed-phase High-performance Liquid Chromatography Analysis Research Articles

Ontogeny of diazepam-binding inhibitor-related peptides (endozepines) in the rat brain

Benzodiazepine receptors are expressed very early in the brain during embryonic life, suggesting that endogenous ligands for these receptors may play an important role during ontogenesis in the central nervous system. In the present study, the distribution and characterization of diazepam-binding inhibitor-related peptides (endozepines) in the rat brain was investigated during embryonic and postnatal development using an antibody raised against the biologically active region of the precursor molecule. Immunohistochemical labelling showed that, in newborn rats, endozepine-like immunoreactivity was present in ependymal cells of the hypothalamus. Although the number of positive cells increased by day 5, the intensity of the immunoreaction in each cell diminished. In 15-day-old rats, both the number of endozepine positive cells and the intensity of the immunoreaction increased in the ependymal layer. At day 40, a dense accumulation of immunoreactive tanycytes and glial cells was observed in the median eminence and the arcuate nucleus. Endozepines were detected by radioimmunoassay in all regions of the brain as early as embryonic day 18. The concentration of endozepine-related peptides increased in the hypothalamus and olfactory bulb during late gestation. Between birth and postnatal day 5, the levels of endozepines decreased two- to four-fold in all brain regions studied. Thereafter, endozepine concentration increased gradually until day 25. Reversed-phase high-performance liquid chromatography analysis of tissue extracts revealed that the olfactory bulb, pituitary, hypothalamus and cerebellum contained only one immunoreactive peak eluting at 39 min (peak C). In the telencephalon two peaks were observed: peak C and a second one eluting at 34 min (peak B). Peak B was present as early as embryonic day 20 and the ratio peak B/peak C gradually increased until day 25. At day 25 peak B was also detected in hippocampus, medulla oblongata, cortex and striatum extracts. In any brain region, no immunoreactivity co-eluting with the octadecaneuropeptide was observed. Sephadex G-50 gel filtration of hypothalamus extracts of 25-day-old animals, confirmed the existence of only one immunoreactive compound with an apparent molecular weight of 10,000. In the telencephalon two major species were resolved, with apparent molecular weights of 10,000 and 8800, and a minor one of 6500 mol. wt. In conclusion, the present study shows that endozepines are expressed in the rat brain as early as embryonic day 18 and the amount of endozepine-like material increases rapidly during the two days preceding birth. The results also indicate that diazepam-binding inhibitor is processed to different molecular forms depending on the brain region. Taken together these data support the concept that endozepines may play important functions during brain development.

Reversed-phase high-performance liquid chromatographic analysis of hydroxyproline and proline from collagen by derivatization with dabsyl chloride

A high-performance liquid chromatographic method for the analysis of hydroxyproline and proline has been developed. The method is based on the derivatization of the secondary amino group with dabsyl-chloride after blocking of the primary amino group with o-phthalaldehyde. Dabsyl-hydroxyproline and dabsyl-proline were separated from other amino acids by high-performance liquid chromatography in the gradient elution mode, and eluted at 10.27 and 16.02 min, respectively. The correlations between the peak areas of dabsyl-hydroxyproline and dabsyl-proline were linear in the range from 20–200 pmol, with equations y = 1.10 x − 0.80 ( r = 0.999) and y = 1.12 x − 0.52 ( r = 0.999), respectively. The method was applied to the analysis of rat tail collagen, and the contents of hydroxyproline and proline were 1.55 ± 0.04 and 2.03 ± 0.04 nmol/μg, respectively.

New ultra-micro high-performance liquid chromatographic method for determining the γ chain composition of hemoglobin F in normal adults

The difficulty in isolating the minute quantity of Hb F (<1%) present in the red blood cells of normal adults greatly complicates the determination of its γ chain composition. We have developed a rapid ultra-micro high-performance liquid chromatographic (HPLC) method and used it to analyze the γ chain composition of Hb F in 47 adults with Hb F levels between 0.1–3.4%. The method involves the isolation of Hb F from as little as 50 μl of whole blood on an analytical size cation-exchange HPLC column, followed by concentration in a Centricon micro concentrator unit and by reversed-phase HPLC analysis. The entire procedure can be completed in one day and 3–4 analyses can be made simultaneously. We reanalyzed the blood samples from 22 subjects with known β-globin gene cluster haplotypes, and confirmed the association of high, low, and very low Gγ levels with haplotypes A, B, and C, respectively. Also included are the results of DNA sequence analyses of the Gγ and β promoters, and of the locus-control-region hypersensitive site-2 (LCR-HS-2) of the β-globin gene cluster in five subjects homozygous for haplotypes A, B or C; the data obtained failed to provide a satisfactory explanation for all the variations in the Gγ levels that have been observed.

Role of eicosanoids in vasopressin-induced calcium mobilization in A7r5 vascular smooth muscle cells.

The role of arachidonic acid (AA) and its metabolites in vasopressin (AVP)-induced calcium mobilization in A7r5 aortic smooth muscle cells was explored by intracellular calcium monitoring, [14C]AA labeling, and high-performance liquid chromatography (HPLC) techniques. In fura 2-loaded A7r5 cells, AA potentiated AVP-stimulated increase in intracellular free Ca2+ ([Ca2+]i). The cyclooxygenase inhibitor indomethacin reduced both the AA- and AVP-induced influx of extracellular Ca2+. AVP-induced [Ca2+]i transients were not altered by lipoxygenase inhibitors but were reduced in a dose-dependent fashion by ketoconazole, an inhibitor of cytochrome P-450 monooxygenases. Among several epoxygenase metabolites of AA tested, 5,6-epoxyeicosatrienoic acid potentiated AVP-induced [Ca2+]i transients. Reverse-phase HPLC analysis of lipid extracts from A7r5 cells prelabeled with [14C]AA isolated a radioactive peak that did not coelute with established products of cyclooxygenase-, lipoxygenase-, or cytochrome P-450-catalyzed oxidations of AA. This peak was significantly increased after AVP stimulation and was completely blocked by preincubation with ketoconazole. Thus the stimulation of V1-vascular AVP receptors of A7r5 cells triggers several cytoplasmic signaling pathways involving AA metabolite formation through the cyclooxygenase and epoxygenase pathways.

A Simple and Quick Solid Phase Extraction and Reversed Phase HPLC Analysis of Some Tropane Alkaloids in Feedstuffs and Biological Samples

Abstract A simple and rapid reversed - phase high performance liquid chromatographic (HPLC) method using an ultraviolet detector for the analysis of tropane alkaloids in feedstuffs and biological samples is presented. Hyoscyamine, scopolamine and the internal standard bamifylline are adsorbed on a C18 cartridge employing the solid - phase liquid extraction technique for sample clean up and subsequent separation of those compounds and internal standard from endogenous interfering compounds and preconcentration. Chromatographic analysis is achieved on a Lichrosorb RP-18 10μm reversed phase column and the mobile phase is an isocratic mixture of acetonitrile, methanol and 0.05 M ammonium acetate (20.9 : 27.9 : 51.2) at a flow rate of 1.30 ml/min. The edited alkaloids are detected at 210 nm. The retention time is 3.990 min for scopolamine, 6.934 min for hyoscyamine and 8.750 min for the internal standard bamifylline. The correlation of the integrated peak area ratios of scopolamine and hyoscyamine to internal ...

Staphylococcus aureus toxic shock syndrome toxin 1 and Streptococcus pyogenes erythrogenic toxin A modulate inflammatory mediator release from human neutrophils

We studied the influence of staphylococcal toxic shock syndrome toxin 1 and streptococcal erythrogenic (pyrogenic) toxin A (ETA) on intact and digitonin-permeabilized human polymorphonuclear granulocytes (PMNs). As was shown by reversed-phase high-performance liquid chromatography analysis, toxic shock syndrome toxin 1 or ETA alone, in the absence of any additional stimulus, did not induce the generation of the chemoattractant leukotriene B4 (LTB4) from PMNs in a wide range of concentrations. In addition, pretreatment of intact PMNs with either toxin potentiated formyl-methionyl-leucyl-phenylalanine (fMLP)- and washed Staphylococcus aureus cell-induced generation of LTB4 in a time- and dose-dependent manner. This increase included LTB4 as well as its inactive omega-oxidated compounds. Further studies revealed evidence that toxin exposure was accompanied by enhanced cellular receptor expression for fMLP as well as for LTB4. The intrinsic GTPase activity of membrane fractions was modulated by both toxins. Short-term incubation with ETA increased the GTPase activity of PMNs up to 141%. Inhibitory effects were obtained when GTP-binding protein functions were stimulated with sodium fluoride (NaF). In addition, specific binding of Gpp(NH)p to GTP-binding protein was inhibited by both toxins during the first 10 min of incubation and was restored at later times of incubation. Our data therefore suggest that both toxins significantly affect the signal transduction pathways of human PMNs, which results in immunomodulatory functions.

Dual microcolumn immunoaffinity liquid chromatography: an analytical application to human plasma proteins.

Dual-column microcolumn immunoaffinity chromatography was accomplished by coupling immunoaffinity (IAC) and reversed-phase high-performance liquid chromatography (RP-HPLC) analysis columns. The IAC support was prepared by hydrophobically adsorbing the appropriate antibody onto an octyl silica-based stationary phase contained in a 250-microns-l.d. fused-silica microcolumn. Once the antigen was captured, the antigen-subtracted human plasma could be sent to the RP-HPLC microcolumn for plasma profiling. The antigen was quantitated by desorbing it from the IAC column and transferring it to the RP-HPLC system. Human plasma profiles were generated after removal of albumin, and transferrin and alpha 1-antitrypsin were quantitated with nanogram sensitivity. All analyses were accomplished with less than 1 microL of human plasma.

Total solid-phase synthesis and prolactin-inhibiting activity of the gonadotropin-releasing hormone precursor protein and the gonadotropin-releasing hormone associated peptide.

The human gonadotropin-releasing hormone precursor protein, pHGnRH (Met-23-Ile69) (preproGnRH), and three of its fragment peptides, pHGnRH (Asp14-Ile69) (gonadotropin-releasing hormone associated peptide--GAP), pHGnRH (Phe38-Ile69), and pHGnRH (Ser47-Ile69), were assembled in a stepwise solid-phase cosynthesis employing Boc/Bzl tactics and an optimized acylation schedule which included recoupling steps with hexafluoro-2-propanol to help overcome the aggregation of the pendant peptide chains of the peptidoresin during difficult couplings. Reversed-phase high-performance liquid chromatography (HPLC) purification yielded products which were characterized by analytical reversed-phase HPLC, ion-exchange chromatography, capillary zone electrophoresis, SDS-polyacrylamide gel electrophoresis, and ion-spray mass spectrometry to reveal a high degree of homogeneity. Biological characterization demonstrated that only GAP stimulated luteinizing hormone and follicle-stimulating hormone release from primary cultures of rat anterior pituitary cells, while GAP, pHGnRH (Phe38-Ile69), and preproGnRH all inhibited prolactin release, with the latter being the most potent at concentrations comparable to bromocryptine. However, only GAP and pHGnRH (Phe38-Ile69) were able to displace a labeled gonadotropin-releasing hormone agonist from binding to rat pituitary membrane preparations. This first demonstration of significant biological activity with a precursor protein also suggests that the gonadotropin-releasing and prolactin release-inhibiting functions of GAP are not mediated through the same pituitary receptors.

COOH-terminal methylation of lamin B and inhibition of methylation by farnesylated peptides corresponding to lamin B and other CAAX motif proteins.

Previous reports from this laboratory have demonstrated that lamin B is reversibly methylesterified in a cell cycle-dependent manner. The site of this methylation, however, was not identified. In this report, we describe a single major methylated product obtained following reversed-phase high-performance liquid chromatographic analysis of peptides generated by proteolytic digestion of lamin B from rat liver nuclear envelopes. This peptide was retained on a lamin B COOH-terminal-specific antibody-affinity column, and COOH-terminal localization was confirmed by amino acid sequencing. Two other COOH-terminal peptides were found but were not methylated and differed in sequence by at least a single residue from the methylated peptide, indicating the existence of two lamin B gene products. Tetrapeptides, representing the putative mature COOH termini of lamin B, K-ras-2A, and unprocessed lamin A, were synthesized with or without farnesyl modification of the COOH-terminal cysteines. All three farnesylated peptides served as substrates for the partially purified lamin B methyltransferase with apparent Km values of 4.5, 0.69, and 21 microM, respectively. Nonfarnesylated peptides were not substrates for the enzyme. The three farnesylated peptides were also effective to varying degrees at inhibiting the methylation of lamin B and other cellular proteins in cell lysates.

High-performance liquid chromatography of corynomycolic acids as a tool in identification of Corynebacterium species and related organisms.

A high-performance liquid chromatography (HPLC) study of 307 strains of Corynebacterium species and related taxa revealed that strains classified as "Corynebacterium aquaticum"; "Corynebacterium asperum"; and Centers for Disease Control (CDC) groups 1, 2, A-3, A-4, A-5, B-1, B-3, E, F-2, and I-2 as well as some unidentified coryneforms do not contain any corynomycolic acids; therefore, they should not be included in the genus Corynebacterium. Such an HPLC method of identification permitted the correct assignment to the genus Rhodococcus of two unpigmented strains of coryneform bacteria whose mycolic acid profiles were comparable to those of Rhodococcus equi. Bacteria belonging to CDC groups ANF-1, ANF-3, F-1, G-1, G-2, and I-1, as well as some other Corynebacterium sp. strains, yielded corynomycolic acid HPLC patterns related to those of Corynebacterium species. Either similarities or differences were observed in the corynomycolic acid profiles of Corynebacterium species tested after culture on sheep blood agar and/or sheep blood agar supplemented with Tween 80, which demonstrated that identification at the species or group level is possible. However, Corynebacterium striatum and CDC group I-1 bacteria as well as CDC group G-1 and group G-2 bacteria had indistinguishable HPLC patterns. Conversely, some variations were observed within some species as Corynebacterium xerosis, C. striatum, and Corynebacterium minutissimum. The evaluation procedure of this HPLC method by mass spectrometry analysis of isolated eluted peaks revealed that analytical reverse-phase HPLC alone does not provide any structural information, since isomers with identical polarities coeluted as a single peak. Nevertheless, HPLC is a rapid and reliable method for identification of corynomycolic acid-containing bacteria in the clinical microbiological laboratory.

Peptidoglycan composition in heterogeneous Tn551 mutants of a methicillin-resistant Staphylococcus aureus strain.

It was shown that Tn551 inactivation of two chromosomal (so-called auxiliary) loci other than the mec gene result in a dramatic reduction of methicillin resistance and decreased cell wall turnover and autolytic capacity in a methicillin-resistant Staphylococcus aureus strain (de Jonge, B. L. M., de Lencastre, H., and Tomasz, A. (1990) J. Bacteriol. 173, 1105-1110). To understand the mechanistic basis of these phenomena we have examined the status of the autolytic enzymes and the muropeptide composition of peptidoglycan using reversed-phase high-performance liquid chromatography and mass spectral analyses. While no differences could be detected in the number of autolytic hydrolases, the mutants showed major changes in peptidoglycan composition. Nine prominent muropeptides of the parental strain each carrying a pentaglycyl substituent were missing from the cell wall of one group of mutants. The second mutant lacked four parental muropeptides which were composed of the unsubstituted disaccharide pentapeptide and its alanyl-tetraglycine derivative. The auxiliary genes are genetic determinants involved with the biosynthesis of peptidoglycan precursors, the presence of which in the cell wall may be needed for optimal cell wall turnover.

Studies on stress metabolites: XIV. High-performance liquid chromatographic analysis of cruciferous phytoalexins using complex ternary mobile phase gradients

A reversed-phase high-performance liquid chromatographic analysis is cruciferous phytoalexins, antimicrobial compounds synthesized de novo by plants after exposure to microorganisms, was performed using an acetonitrile-methanol-water complex solvent gradient system. A well resolved chromatogram of thirteen phytoalexins and three related indole metabolites isolated from cruciferous plants was obtained by this method. Accumulation of phytoalexins in Pseudomonas cichorii-inoculated turnip tissue was followed by this high-performance liquid chromatographic analysis.

Chromatographic analysis of low-molecular-mass copper-binding ligands from the crab species Syclla serrata and Portunis pelagicus

Two copper-binding proteins and a zinc-binding ligand were isolated from the hepatopancreas of the crab Portunus pelagicus. The copper-binding proteins behave similarly to those from the crabs Carcinus maenas and Scylla serrata, and were shown to be metallothioneins. Reversed-phase high-performance liquid chromatographic (HPLC) analysis confirmed the relative purity of both proteins with only cross-contamination between the two different forms of metallothioneins, and offers a good method to separate the two forms of metallothioneins. The vast difference in the retention times (and hence the hydrophobicity) in reversed-phase HPLC indicates that the two proteins could be conformationally very different.

Characterization of serine protease-derived metabolites of big endothelin in the cytosolic fraction from human polymorphonuclear leukocytes.

We have characterized by bioassay and reversed-phase high-performance liquid chromatography analysis (rp-HPLC) the conversion of human big endothelin-1 (bET-1) to endothelin-1 (ET-1) by the cytosolic fraction from human polymorphonuclear leukocytes (PMNs). Either the general serine protease inhibitor 3,4-dichloroisocoumarin (DCI; 50 microM) or the selective elastase inhibitor ONO-5046 (100 microM) blocked the formation of ET-1 from bET-1. Interestingly, human leukocyte elastase formed some of the same products from bET-1 as the PMN cytosol, but generated negligible amounts of ET-1. However, coincubation of the elastase-derived fragments of bET-1 with the PMN cytosol in the presence of ONO-5046 resulted in a 17-fold increase in the formation of ET-1, indicating that an elastase-derived intermediate of bET-1 was subsequently cleaved by a soluble protease(s) to form mature ET-1. We have identified by electrospray-mass spectrometry (ESMS) analysis this intermediate as bET-1(1-22). Analysis of bET-1 digestion by human leukocyte cathepsin G revealed the formation of a biologically active metabolite chromatographically distinct from ET-1, identified as bET-1(1-31) by ESMS. These findings indicate the presence of complex enzyme systems in human PMNs capable of activating bET-1.

Analysis of major tomato xylem organic acids and PITC-derivatives of amino acids by RP-HPLC and UV detection

Major amino acids and organic acids in xylem exudates of tomato plants were separated by reversed phase high performance liquid chromatography (RP-HPLC) and quantified by UV detection. Before separation, amino acids were converted into their phenylisothiocyanate (PITC) derivatives. In a single run, Asp, Glu, Ser, Gln, His, Thr, Ala, Tyr, Val, Met, Cys, Ile, Leu, Phe, and Lys could be separated and detected down to the pmol level. Unresolved peaks were obtained for Asn and Gly and for Arg and Pro. For organic acid analysis, exudates were pre-treated by perfusion over a prepacked Adsorbex SCX cation exchange column, to eliminate exudate amino acids. Elution recoveries for organic acids were close to 100%. The exudate organic acids were separated by ion suppression RP-HPLC chromatography, and peaks could be resolved for L-malic acid, malonic acid, maleic acid, citric acid and fumaric acid, down to the pmol level. UV signals for exudate ascorbic acid, and succinic acid were below the limits of detection. Determination of oxalic acid and tartaric acid was impossible, due to the presence of the exudate salt peak in the chromatogram. The results indicate the potential of the methods applied, and show the applicability of RP-HPLC analysis for the determination of both amino acids and organic acids in xylem exudates.

Quantitative variation in high molecular weight glutenin subunit 7 in some Canadian wheats

Gradient SDS-PAGE analysis of some Canadian wheat cultivars and breeders' lines revealed that there was variation in the mobility of the high molecular weight (HMW) glutenin subunit 7. One group of cultivars/lines had a subunit 7 band that was comparable to that found in the cultivar Chinese Spring, whereas another group had a slightly more mobile band comparable with the subunit 7 band in Bezostaya. This latter band was designated as HMW glutenin subunit 7* in order to differentiate it from the subunit 7 component present in cultivars such as Chinese Spring. Reversed-phase high-performance liquid chromatographic (RP-HPLC) analysis showed that subunits 7 and 7* had comparable elution times. Quantitatively, the proportion of subunit 7 relative to the total amount of HMW glutenin subunits was significantly higher (41·2± 1·5 %) than subunit 7* (27·1 ±0·9 %). The relative proportions were consistent within each group of cultivars as indicated by low coefficients of variation, 3·6 and 3·4 %, respectively. Similarly, the absolute peak area, on a per cent protein basis, was significantly higher for subunit 7 than for subunit 7*. Subunit 7* was found in combination with subunit 9 or subunit 8 or a variant of subunit 8 (as determined by RP-HPLC) designated subunit 8*. This indicated that there are at least three alleles controlling this subunit. Subunit 7 was present in combination with subunits 8 or 8*, and thus there are at least two alleles controlling this subunit. Subunit 7 may be associated with greater dough strength and decreased extractability of gluten proteins. The identification, therefore, of these subunits could be useful for selection purposes in a wheat breeding program.

Deamidation at asparagine-88 in recombinant human interleukin 2.

Reversed-phase high-performance liquid chromatography (RP-HPLC) and protein-chemical analysis revealed that only Asn88 in recombinant human interleukin 2 (rIL-2) is liable to be deamidated during a long period of storage in aqueous solutions (pH 5.0) at 25 degrees C, even though there are eight asparagine and six glutamine residues. The deamidation occurred more easily at 40 degrees C than at 25 degrees C but did not occur at all at temperatures below 5 degrees C. The biological activity of Asn88-deamidated rIL-2 was found to be almost the same as that of intact rIL-2, whereas its isoelectric point (pI 7.6) is different from that of intact rIL-2 (pI 7.9).

Reversed-phase high-performance liquid chromatographic analysis of branched-chain keto acid hydrazone derivatives: optimization of techniques and application to branched-chain keto acid balance studies across the forearm

A sensitive method of quantifying branched-chain keto acids in plasma and whole blood samples is described. It is based on the separation by ion-pair reversed-phase liquid chromatography of 2,4-dinitrophenylhydrazine derivatives with ultraviolet detection. The sample clean-up steps that are usually required for reversed-phase high-performance liquid chromatography are eliminated. A reduction in ketoisocaproate isomer formation is obtained by incubation of derivatives in ice. The method is reproducible (coefficient of variation 2%, n = 5, at the 200-pmol level) and the ultraviolet response is linearly related to branched-chain keto acid concentration. Recoveries are high (>95%). Other keto acids do not co-elute with branched-chain keto acids. Because of its sensitivity and precision, this method can be proposed for whole blood branched-chain keto acid balance studies across organs.

Avidin protein-conjugated column for direct injection analysis of drug enantiomers in plasma by high-performance liquid chromatography

A new concept in high-performance liquid chromatography supports is proposed for the direct injection analysis of drug enantiomers in plasma. The new supports are designed with disuccinimidyl suberate as a hydrophobic internal region, and avidin protein as a hydrophilic and bulky surface region. Plasma proteins are excluded by the avidin phase and are eluted immediately from the column, whereas low-molecular-mass analytes can penetrate the surface region and interact with disuccinimidyl suberate. Enantiomers interact differentially with avidin, and are thereby separated. This column was used in reversed-phase high-performance liquid chromatographic analysis to determine ketoprofen enantiomers in plasma by direct injection. The recovery of racemic drug from plasma was almost 100%.

Improved high-performance liquid chromatographic resolution of the geometric isomers of 6-hydroxy-4-(1-hydroxy-1-methylethyl)-1-cyclohexene-1-ethanol and by-products with β-cyclodextrin

The aim of this study was to optimize the high-performance liquid chromatographic separation of (6 S,4 R)-(−)-6-hydroxy-4-(1-hydroxy-1-methylethyl)-1-cyclohexene-1-ethanol and its potential impurities to determine them in the active ingredient and in pharmaceutical formulations for purity and stability analysis. A comparison of conventional normal- and reversed-phase high-performance liquid chromatographic analysis and a method employing β-eyclodextrin was made. The reversed-phase analysis without β-cyclodextrin was undoubtedly unsuitable for an acceptable high-performance liquid chromatographic separation. On the other hand the other two methods were more selective and showed good precision, accuracy and linearity in the range investigated. However the use of β-cyclodextrin as eluent modifier with a reversed-phase was preferable for its improved selectivity and also for allowing the use of a non-toxic and less expensive eluent. Also, the limits of detection and quantitation obtained for the impurities made the β-cyclodextrin method very suitable for purity and stability analysis.