Assay of human fibrinopeptides by high-performance liquid chromatography

Fibrinopeptides A, AP, and B, desarginine fibrinopeptide B, and a previously unknown peptide corresponding to B beta 3-14 were resolved within 10 min by an HPLC technique using an isocratic solvent system (22% acetonitrile in 0.1% trifluoroacetic acid) and a 0.46 X 10-cm Spherisorb ODS-2 (3-micron) octadecylsilane column. Fibrinopeptides A and AY eluted in the same peptide peak. The method was used to evaluate a carboxypeptidase which converts fibrinopeptide B into its desarginine form. Fifty percent inhibition of this activity occurred at 1.7 mM epsilon-aminocaproic acid (EACA). At saturating substrate concentrations the rates of total fibrinopeptides A and B release were unaffected by 125 mM EACA, a concentration at which the carboxypeptidase activity is completely inhibited.     

Assay of 2-naphthol in human urine by high-performance liquid chromatography

This paper describes a novel liquid chromatographic method for the quantitation of 2-naphthol in human urine. Urine samples were extracted after enzymatic hydrolysis of glucuronides and sulfates; 2-naphthol was then separated using reversed-phase high-performance liquid chromatography. The corresponding detection limits were 0.04 ng/ml for the standard sample in acetonitrile and 0.13 ng/ml for urine samples. The level of urinary 2-naphthol in 100 Korean shipyard workers was analyzed using this new method. The level ranged from 0.21 ng/ml (0.26 μmol/mol creatinine) to 34.19 ng/ml (59.11 μmol/mol creatinine), and the mean±standard deviation was 5.08 ng/ml (6.60 μmol/mol creatinine)±5.75 ng/ml (9.22 μmol/mol creatinine). The mean±standard deviation of urinary 2-naphthol level of smokers, 7.03 ng/ml (8.49 μmol/mol creatinine)±6.16 ng/ml (10.23 μmol/mol creatinine), was significantly higher than that of non-smokers, 2.49 ng/ml (4.10 μmol/mol creatinine)±3.92 ng/ml (7.03 μmol/mol creatinine).     

Assay of mouse liver uroporphyrinogen decarboxylase by reverse-phase high-performance liquid chromatography

A method for the estimation of hepatic uroporphyrinogen decarboxylase activity employing reverse-phase HPLC is described. Mouse liver homogenate in 0.25 M sucrose was pretreated with a suspension of cellulose phosphate and then centrifuged to remove hemoglobin and debris. The supernatant was used as the enzyme source. Incubations were acidified, oxidized, and centrifuged only before analysis of the porphyrins formed, using a Spherisorb ODS column and a gradient solvent system constructed from methanol/lithium citrate mixtures. Coproporphyrinogen formation by BALB/c mouse liver supernatant was estimated as about 5.0 and 9.1 pmol/min/mg protein from uroporphyrinogens I and III, respectively, at 10 microM substrate concentration and pH 6.8. Decarboxylation of pentacarboxyporphyrinogens (the last step in coproporphyrinogen formation) proved to be easily measured. Coproporphyrinogen formation from pentacarboxyporphyrinogen III abd (20 microM) at pH 6.8 was about 109 pmol/min/mg protein. Pentacarboxyporphyrinogen I was not as good a substrate as III abd but was decarboxylated faster at pH 5.4 than at 6.8, and at the lower pH and at 10 microM concentration of substrate 42 pmol of coproporphyrinogen was formed/min/mg protein. These results compared favorably with those obtained by previously published procedures involving time-consuming extraction and esterification steps.     

Assay of galactosyltransferase by high-performance liquid chromatography

Galactosyltransferase catalyzes transfer of galactose from UDP-galactose to glucose or N-acetylglucosamine with resultant formation of galactosides and UDP. In this new assay galactosyltransferase activity is measured by determining UDP by isocratic high-performance liquid chromatography on an amino-bonded column monitored spectrophotometrically. Concurrently, unreacted UDP-galactose and breakdown products arising from UDP-galactose (UMP and uridine) are also determined. The new technique does not require radioactive substrates, permits usage of saturating concentrations of UDP-galactose, and provides monitoring of side reactions.     

Assay of aspartylglycosylaminase by high-performance liquid chromatography

An aspartylglycosylaminase assay based on high-performance liquid chromatographic analysis of the substrate aspartylglucosamine and product aspartate is described. Aspartylglucosamine and aspartate are derivatized with phenylisothiocyanate and resolved by reverse-phase chromatography. The detection limit for the compounds is 2 pmol. The method can be used for analysis of aspartylglycosylaminase activity in crude cell extracts and tissue samples.     

Assay for beta-ureidopropionase by high-performance liquid chromatography

A sensitive assay for beta-ureidopropionase based on derivatization of the reaction product beta-alanine with phenylisothiocyanate has been developed. Purification of the resulting phenylthiocarbamoyl-beta-alanine is achieved on a LiChrospher 100 C18 reversed-phase high-performance liquid chromatography column using an isocratic elution system. Phenylthiocarbamoyl-beta-alanine is detected by its absorbance at 245 nm and quantitated by automatic peak integration referring to a calibration curve. This technique offers a high degree of sensitivity as beta-alanine quantities in the picomole range can be identified. N-Carbamoyl-beta-alanine, the natural substrate of beta-ureidopropionase, does not interfere with the described assay system. The enzymatic reaction is linear for an incubation time of 45 min with enzyme concentrations of 3.2 micrograms/ml.     

Assay of ornithine aminotransferase by high-performance liquid chromatography

Ornithine aminotransferase has been measured previously with a spectrophotometric assay and with a radioactive assay. We report here an isocratic reverse phase high-performance liquid chromatography assay which measures Δ¹-pyrroline-5-car?ylic acid, the reaction product. This assay offers the advantages of sensitivity and convenience.     

Assay of cardiolipin peroxidation by high-performance liquid chromatography

Commercial preparations of bovine cardiolipin (diphosphatidylglycerol) in chloroform solution contain substantial amounts of oxidation products. These oxidized derivatives, characterized by the presence of varying amounts of hydroperoxides and conjugated dienes, can be separated from unoxidized cardiolipin by normal phase high-performance liquid chromatography (HPLC) using UV detection. When purified cardiolipin is subjected to autoxidation in aqueous media, oxidation products of similar HPLC properties are produced. Storage of cardiolipin in chloroform induces both autoxidation and hydrolysis whereas storage in ethanol and other solvents does not. It is recommended not to use chloroform for the long-term storage of cardiolipin.     

Assay of the glutathione-synthesizing enzymes by high-performance liquid chromatography

We report a new convenient assay of the activity of gamma-glutamylcysteine synthetase (EC 6.3.2.2) and glutathione synthetase (EC 6.3.2.3) in crude microbial extracts as well as in purified enzyme preparations. The assay is based on the quantitative analysis of the reaction products by high-performance liquid chromatography after derivatization of the thiol group with 5,5'-dithiobis-(2-nitrobenzoic acid) as described by J. Reeve, J. Kuhlenkamp, and N. Kaplowitz [(1980) J. Chromatogr. 194, 424-428]. In addition, the procedure yields information on basal levels of gamma-glutamylcysteine and glutathione in crude microbial extracts. The two enzymes responsible for glutathione biosynthesis can be determined in parallel under the same chromatographic conditions. No prior separation from substrates and by-products is necessary. Product formation is linear with time for at least 30 min between 0.03 and 12 mU for both enzymes. Even in crude extracts 0.2-0.5 nmol of products formed can be detected with certainty. The method was found to be sensitive and highly reproducible.     

Assay of human transthyretin-bound holo-retinol-binding protein with reversed-phase high-performance liquid chromatography

We describe a reversed-phase high-performance liquid chromatographic method for the determination of vitamin A-transporting (holo) transthyretin-bound (TTR) retinol-binding protein (RBP) concentrations in serum or plasma. Holo-TTR—RBP and free retinol derived primarily from free RBP are consistently observed with this chromatographic method. Holo-TTR—RBP concentrations determined by this method are highly correlated to holo-TTR—RBP concentrations measured by chromatography. This method has the advantage of using less expensive columns and having peak areas which are more proportional to their true concentrations in plasma, as determined by comparison to purified protein spectrophotometry and radial immunodiffusion. The percentage of RBP circulating as holo-TTR—RBP decreased significantly as the total concentration of RBP or retinol increased. Because purified holo-TTR—RBP did not dissociate under these chromatographic conditions, this suggests that more vitamin A circulates as holo-free RBP or free retinol in the blood of people with high serum RBP.     

Simultaneous determination of testosterone metabolites in liver microsomes using column-switching semi-microcolumn high-performance liquid chromatography

A sensitive and selective column-switching semi-microcolumn high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of testosterone and eight of its metabolites (6alpha-, 6beta-, 16alpha-, 16beta-, 7alpha-, 2alpha-, and 2beta-hydroxytestosterone, and androstenedione) in liver microsomes. After incubation for 10 min, testosterone and its metabolites were extracted from the microsomes with ethyl acetate, and the extract was evaporated to dryness. The residue was dissolved in the mobile phase and loaded onto the HPLC system. The analytes were first concentrated in a precolumn and subsequently transferred to the analytical column, where they were separated using linear gradient elution. A UV detector set at 254 nm was used to detect the analytes. This newly developed method clearly separated TES and the metabolites with high resolution and was found to be reproducible with intra- and interday variability of <10.7%. This method has been subsequently used to determine the testosterone hydroxylation activities catalyzed by 15 different recombinant CYP isozymes. The results confirmed the formation of stereoselectively hydroxylated metabolites by each CYP isozyme.     

Assay of human platelet guanylate cyclase activity by high-performance liquid chromatography with fluorescence derivatization

A selective and sensitive high-performance liquid chromatography (HPLC) method with fluorescence derivatization for the assay of guanylate cyclase (GC) activity is described. GTP and cGMP, which are the substrate and the product of GC, respectively, and other guanine-containing compounds are selectively converted by the reaction with (3,4-dimethoxyphenyl)glyoxal to the fluorescent derivatives. The derivatives were separated by reversed-phase HPLC. The limit of detection at a signal-to-noise ratio of 3 for cGMP was 10 fmol on the column. The sensitivity of this method was less than that of the conventional radioisotopic method, but this method is simple and convenient. Human platelet GC activity was measured, and the effects of some compounds were investigated.     

Assay for aliphatic amino acid decarboxylases by high-performance liquid chromatography

A sensitive and rapid assay for aliphatic amino acid decarboxylases based on separation of the product from the substrate by ion-pairing reversed-phase high-performance liquid chromatography and subsequent fluorometric detection has been developed. The resolution of substrates and products of seven amino acid decarboxylases, namely, arginine, aspartate, 2,6-diaminopimelate, histidine, glutamate, lysine, and ornithine decarboxylase, is complete within 15 to 35 min of isocratic elution. The limit of detection for the product is 40 pmol. The applicability of the procedure was assessed with glutamate decarboxylase. The formation of the product 4-aminobutyrate proved to be linear with time and protein concentration. The method allows the time course of the reaction to be followed in a single assay and works well with crude extracts of bacteria or tissues.     

Assay for ADP-ribosyl cyclase by reverse-phase high-performance liquid chromatography.

Cyclic ADP-ribose (cADPR), a natural metabolite of beta-NAD(+), is a second messenger for Ca(2+) signaling in T cells. As a tool for purification and identification of ADP-ribosyl cyclase(s) in T cells, a sensitive and specific enzymatic assay using 1,N(6)-etheno-NAD(+) as substrate was developed. A major problem-the sensitivity of 1,N(6)-etheno-cADPR toward the extraction medium perchloric acid-was solved by replacing the perchloric acid extraction procedure of nucleotides by a filtration step. Standard compounds for the HPLC analysis of ADP-ribosyl cyclases and NAD(+)-glycohydrolases, e.g., 1,N(6)-etheno-cADPR, 1,N(6)-etheno-ADPR, and 1,N(6)-etheno-AMP, were produced by ADP-ribosyl cyclase from Aplysia californica and dinucleotide pyrophosphatase. The assay was applied to subcellular fractions prepared from human Jurkat T cells. As a result ADP-ribosyl cyclase and NAD(+)-glycohydrolase activity could be detected and precisely quantified in different subcellular fractions indicating the presence of different isoenzymes in T cells.     

Assay of soluble guanylate cyclase activity by isocratic high-performance liquid chromatography

A HPLC method alternative to labelled or unlabelled procedures was developed for the assay of guanylate cyclase (GC) activity. The substrate (GTP) and the product (cGMP) of the enzymatic reaction were separated in the isocratic mode on a μBondapak C₁₈ column. The activity of GC was linearly dependent on the amount of cGMP produced in the presence of sodium nitroprusside. This approach was applied to follow the purification of GC from bovine lung and to evaluate its stability in different storage conditions.     

Quantitation of free sphingosine in liver by high-performance liquid chromatography

Conditions were established for the extraction of free sphingosine from liver and the separation and quantitation of this and other long-chain (sphingoid) bases (e.g., sphingosine, sphinganine, phytosphingosine, and homologs) by reverse-phase high-performance liquid chromatography (HPLC). The long-chain bases were extracted with chloroform and methanol and then treated with base to remove interfering lipids. After preparation of the o-phthalaldehyde derivatives, the long-chain bases could be separated using C18 columns eluted isocratically with methanol:5 mM potassium phosphate, pH 7.0 (90:10). The HPLC analyses took 15 to 20 min per sample and had lower limits of detection in the picomole range. Quantitation was facilitated by using a 20-carbon long-chain base homolog as an internal standard. The utility of the method was demonstrated with rat liver, providing the first quantitation of free sphingosine in this tissue of approximately 7 nmol/g wet wt.     

Radiochemical high-performance liquid chromatography detection of arginine metabolism in human endothelial cells

Arginine is a semi-essential amino acid that plays an important role in the regulation of metabolic processes associated with several pathological/physiological conditions. In the vasculature, it mainly exerts its biological functions as a substrate of two alternative pathways: the conversion to nitric oxide (NO) by nitric oxide synthase (NOS) and the breakdown to urea and ornithine by arginase. To determine arginine metabolism, in the current study we propose an original radiochemical technique that allows the simultaneous monitoring of NOS and arginase activation within intact cells. Taking advantage of this method, we show here the consequences of different experimental conditions known to modulate endothelial homeostasis on arginine metabolism.     

Assay of nicotinamide deamidase activity using high-performance liquid chromatography

A rapid, simple and reproducible method has been developed for the determination of nicotinamide deamidase activity using high-performance liquid chromatography (HPLC). Nicotinic acid (NA) liberated from nicotinamide (NAA) after a 15-min enzyme reaction was determined directly by HPLC without further separation steps. Both NA, the product, and NAA, the substrate were separated by reversed-phase ion-pair isocratic chromatography and detected at 261 nm. The present method could be applied to the measurement of deamidase activity in crude cell extracts prepared from several bacterial strains. The Michaelis constant of nicotinamide deamidase in Enterobacter agglomerans was 36 μM for NAA. This method is useful for the measurement of nicotinamide deamidase from various sources.