Determination of cyclooxygenase products and prostaglandin metabolites using high-pressure liquid chromatography and radioimmunoassay.

A method is described for measurement of the cyclooxygenase products, thromboxane,prostacyclin, and prostaglandins (PG), and several prostaglandin metabolites. The procedure involves separation of the compounds by high-pressure liquid chromatography combined with identification and estimation by serologic analysis. These combined procedures have been used to identify and estimate five such products, PGE₂, PGE₁ PGF2α, PGF_1α, and 6-keto-PGF_1α, in the culture fluids of dog kidney cells stimulated by a tumor-promoting phorbol diester. The prostaglandin metabolites, 13,14-dihydro-15-keto-PGE₂, 13,14-dihydro-15-keto-PF2_2α, 13,14-dihydro-PGE₂, and 13,14-dihydro-PGF_2α, were not found in these culture fluids.     

Purification and characterization of the gag gene products of avian-type C retroviruses by high-pressure liquid chromatography

We have developed an inexpensive procedure for bacteriophage purification suitable for small- and medium-scale preparations (up to one liter of lysate). The method consists of precipitation with polyethylene glycol 6000 and gel chromatography on a Bio-Gel A-5m column. The purity of the phage preparation is comparable to that obtained by CsCl step gradient ultracentrifugation.     

Analysis of leukotrienes by high-pressure liquid chromatography

A method is described for the partial synthesis of saturated mixed-chain phosphatidylcholines of a high degree (typically 99 mol%) of purity. This procedure has been designed to eliminate the contamination of the mixed-chain product by symmetric chain phosphatidylcholine and the mixed-chain isomer of the desired product, the two principal impurities introduced by previous techniques. This high degree of purity is obtained by employing a method designed for the complete enzymatic hydrolysis of the C-2 fatty acyl moiety in saturated symmetric phosphatidylcholines and a new technique for the acylation of lysophosphatidylcholines employing the catalyst 4-pyrrolidinopyridine. The versatility of this new procedure is illustrated with the synthesis of several saturated mixed-chain phosphatidylcholines.     

Separation of the natural retinoids by high-pressure liquid chromatography

A reverse phase high-pressure liquid chromatography system for rapid separation of various retinoids (vitamin A and its analogs) with little or no degradation is described. This method permits detection of as little as 22 pmol of retinoic acid. The procedure has been applied to the study of retinoic acid metabolism in vitamin A-deficient hamsters.     

Simultaneous analysis of substrates,products, and inhibitors of xanthine oxidase by high-pressure liquid chromatography and gas chromatography

Procedures are presented for the simultaneous analysis of hypoxanthine, xanthine, allopurinol, oxipurinol, and uric acid in standard mixtures and physiological fluids using gas chromatography (gc) or high-pressure liquid chromatography (hplc). Excellent correlation was obtained between the two methods for hypoxanthine, xanthine, oxipurinol, and uric acid. There are advantages and disadvantages to both methods. hplc requires no prior derivatization, uses isocratic elution with a buffer containing no organic solvent, and has 50- to 100-fold greater sensitivity than gc. Simpler methods of prepurification, readily adapted to clinical laboratories, can be used for hplc analysis. Although substances that are found in some urine samples from cancer patients interfere with hplc, separations by gc are not affected by these substances.     

Analysis of sugars in glycoproteins by high-pressure liquid chromatography

A method for analyzing the carbohydrate composition of glycoproteins and similar glycoconjugates by methanolysis followed by reverse-phase high-pressure liquid chromatography of the perbenzoylated methyl glycosides has been developed. As described, the method is capable of quantifying sugars in the 1- to 10-nmol range while further optimization of procedures may increase the usable sensitivity by a factor of 10 or greater. Improved yields of the sugar derivatives have been achieved by incorporating several modifications of the original methanolysis procedure. This, together with the use of high-pressure liquid chromatography rather than gas chromatography for separating the sugar derivatives, eliminates the need for empirically determined molar response ratios.     

Rapid separation of acetylated oligosaccharides by reverse-phase high-pressure liquid chromatography.

Peracetylated saccharides were separated by chromatography on a reverse-phase support, eluting with mixtures of acetonitrile-water. Gradient elution for 2.5 h gave significant separations of all linear glucose oligomers containing up to 35 sugar residues. With isocratic elution retention was exponentially related to molecular mass and only slightly affected by linkage or anomeric configuration. The presence of glucosamine in various saccharides markedly reduced their retention.     

Analysis of metallonucleoside hydrolysis by high-pressure liquid chromatography

A number of ammine complexes of transition metals in the platinum group exhibit antitumor and mutagenic activities, which probably result from in vivo metal ion coordination to nucleic acids. Coordination at N-7 purine sites on nucleic acids may induce depurination through a general acid-catalyzed cleavage of the sugar-purine bond. This possibility was tested by observing the hydrolysis of [dGuo)(NH₃)₅Ru]³⁺ under physiological conditions. Since multiple products result from this reaction, a high-pressure liquid chromatography technique was developed for separating various ammineruthenium(III) complexes with purine, pyrimidine, and nucleoside ligands. Using this technique it was possible to identify and follow the relative concentrations of several of the hydrolysis products. These data were used to develop a preliminary reaction scheme for the decomposition of (dGuo)(NH₃)₅Ru(III). The net rate constant (1.8 × 10^?6 s^?1) for the disappearance of this complex yields an upper limit for the rate of metal-induced sugar hydrolysis. While the half-life ($\text{5}\text{days}\text{< t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{< 28}\text{days}$) for the sugar hydrolysis reaction is substantially shorter than that for the free nucleoside under the same conditions, it cannot be concluded that this represents a major contribution to the mutagenicity of Ru(III) complexes.     

Purification of tyrosine hydroxylase by high-pressure liquid chromatography

Our study of tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) from rabbit adrenals has identified two major requirements which are likely to be of general application for the optimal purification and recovery of enzyme activity consequent to high-pressure liquid chromatography: (i) recovery of activity is maximized by pretreatment of the high-pressure liquid chromatography column before each use with protein to saturate high affinity, nonspecific sites exposed by the methanol used for washing, and storage of the column. (ii) Both purification and recovery are critically dependent upon the molarity of the mobile phase buffer. Examination of high-pressure liquid chromatography purified rabbit adrenal tyrosine hydroxylase by nondenaturing gel electrophoresis indicated that tyrosine hydroxylase activity was associated with one of the two protein bands in the gel. Thus, the convenient purification procedure described in this report leads to preparative amounts of tyrosine hydroxylase which is approximately 50% homogeneous.     

Racemization studies in peptide synthesis using high-pressure liquid chromatography

We have synthesized the dipeptides benzoyl-l-phenylalanyl-l-alanyl benzyl ester and benzoyl-l-phenylalanyl-l-alanyl methyl ester by both the solid-phase and solution coupling methods. A variety of coupling reagents and solvents was employed. Each coupling reaction was analyzed by high-pressure liquid chromatography for extent of racemization. Baseline separations were achieved which allowed the direct, rapid, and reproducible determination of dipeptide diastereomers. Our successes in the separation of dipeptide diastereomers indicate the future value of applying high-pressure liquid chromatography to the separation of larger peptide diastereomers.     

Determination of E. gracilis mRNA base composition using high-pressure liquid chromatography.

The purine and pyrimidine bases of E. gracilis have been separated using a high-pressure liquid chromatograph (HPLC). Each base was unambiguously identified by its characteristic elution profile and UV absorption spectrum. This method allows for the study of the base composition of mRNAs altered by pathological processes, exemplified here by the analysis and comparison of mRNAs from zinc sufficient and deficient organisms.     

Analysis of lectin properties with membrane ultrafiltration and high-pressure liquid chromatography.

A convenient method for the analysis of the binding properties of lectin with fluorogenic sugar chains is described. A lectin (concanavalin A or Datura stramonium agglutinin) was mixed with pyridylaminated sugar chains in buffer and the free chains obtained were isolated by membrane ultrafiltration. The amount of free sugar chains in the filtrate was measured by high-pressure liquid chromatography. The binding constants with the sugar chains, reaction kinetics, and other properties of these lectins were easily investigated. The method is simple and could be used to study the characteristics of any lectin in native form.     

Determination of malonaldehyde in biological materials by high-pressure liquid chromatography

A new one-dimensional agarose gel electrophoresis method for the quantitation of glycosaminoglycans in biological samples has been described. In this procedure, concanavalin A, suspended in agarose gel, interacts with glycosaminoglycans such that rocket-like precipitin lines are formed. The area of the rocket is directly proportional to the glycosaminoglycan content of the sample. This procedure permits measurement of glycosaminoglycans in amounts as low as 0.5 nmol uronic acid equivalents with a coefficient of variation of only 8%. The described method has been applied to the determination of free heparan sulfate in plasma. This method can also be used to measure all high-charge glycosaminoglycans of biological interest.     

The behaviour of peptides on reverse-phase supports during high-pressure liquid chromatography.

High-pressure ('performance') liquid chromatography has been used to investigate the reverse-phase chromatographic behaviour of peptides, ranging in length from 2 to 65 amino acid residues, which have originated from primary-sequence determinations or solution/solid-phase syntheses. By using a pyridine/formate-pyridine/acetate/propan-1-ol buffer system, as previously described [Hughes, Winterhalter & Wilson (1979) FEBS Lett. 108, 81-86], the influence of various experimental parameters were examined. (a) Peptide retention was observed to be temperature-independent between 25 and 55 degrees C. (b) The dependence of chromatographic retention on pH decreases with increasing peptide hydrophobicity. (c) Chromatographic results from C8- and C18-chain-length, as well as from 5 micrometers- and 10 micrometers-particle-size, supports were comparable. (d) The hydrophobic strength of the organic solvent in the mobile phase was observed to decrease: propan-1-ol approximately equal to propan-2-ol greater than acetonitrile much greater than methanol. (e) When gradient rates (% of buffer B/unit time) were systematically decreased, peptide retention decreased in a hyperbolic manner. Comparisons of the peptides chromatographed with respect to their measured retention properties and calculated hydrophobicities were performed by computer analysis. Deviation of peptide chromatographic behaviour was observed to be essentially independent of hydrophobicity, chain length and charge. On the basis of the measured retention properties of the chromatographed peptides, hydrophobic constants for the various amino acid side chains were determined and compared with similar constants available from the literature.     

Application of high-pressure liquid chromatography to studies of collagen production by isolated cells in culture

Techniques for assessing collagen production by cells in culture are usually based on evaluation of uptake of radiolabeled proline into collagen. Although simple in theory, this approach is often flawed because of uncertainties concerning the specific activity of labeled proline in the precursor pool for collagen synthesis. An alternative approach is to assess collagen production directly by measuring hydroxyproline in proteins secreted by cultured cells, although this has been difficult, due to the insensitivity of the methods available. Here we apply high-pressure liquid chromatography using reverse-phase elution of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole derivatives of hydroxyproline to measure collagen production by fibroblasts. The method is easy to perform and allows quantitation of hydroxyproline down to 5 pmol, making it applicable to fibroblasts in 12-well culture plates. Collagen production was shown to be constant over a period of 24 h, with a mean rate of 391 +/- 18 (SE n = 14) ng collagen/10(6) cells/h. Similar values were obtained using thin-layer chromatography and an enzyme-linked immunosorbent assay for type I collagen, but these techniques were judged to be less convenient and required additional assumptions compared with the technique described here in full.     

Analysis of derivatives of carbohydrates by high-pressure liquid chromatography

The behaviour of benzoylated derivatives of alditols, monosaccharides, disaccharides, amino sugars, and methyl glycosides in high-pressure liquid chromatography (h.p.l.c.) has been investigated. A system was devised, using the most basic equipment of a single pump and fixed-wavelength u.v. detector, which gave good separations of the components of mixtures of derivatised methyl glycosides. Fractionation of complex mixtures of many of the other benzoylated carbohydrates was achieved in less than 30 min. The 4-nitrobenzoates were less useful for routine analyses.