Damipipecolin and damituricin, novel bioactive bromopyrrole alkaloids from the Mediterranean sponge Axinella damicornis

Two new bromopyrrole alkaloids, damipipecolin (1) and damituricin (2), have been isolated from the Mediterranean sponge Axinella damicornis, and their structures established through spectroscopic methods. Compounds 1 and 2 extend the structural variety of the so far known pyrrole alkaloids; in these compounds, the 4-bromopyrrole 2-carboxylic acid is directly condensed with a non-protein cyclic alpha-amino acid, the (2R, 4R)-trans-4-hydroxypipecolic acid and (2R, 4R)-cis-N,N'-dimethyl-4-hydroxyproline (D-turicine) in 1 and 2, respectively. Compounds 1 and 2 were found to display a modulating effect of the serotonin receptor activity in vitro.     

Phospholipase A2 activities with a plasmalogen substrate in brain and in neural tumor cells: a sensitive and specific assay using pyrenesulfonyl-labeled plasmenylethanolamine

We have developed a new assay method for phospholipase A2 (EC 3.1.1.4.), towards ethanolamine plasmalogen using pyrenesulfonyl-labeled plasmenylethanolamine as the substrate. This procedure is sensitive to about 3 pmol/ml per min and is absolutely specific for plasmalogen. In this method, the product of phospholipase A2, pyrenesulfonyl-labeled lysoplasmalogen, is hydrolyzed to aldehyde and labeled glycerophosphoethanolamine with hydrochloric acid exposure, and after TLC separation, the pyrenesulfonyl-glycerophosphoethanolamine is quantitated spectrofluorometrically. The excitation and emission wave lengths were 340 and 376 nm, respectively. The activity of bovine brain homogenate was 44.1 +/- 6.47 pmol/min per mg protein (n = 3). Among bovine brain subcellular fractions, the distribution and specific activity of the enzymes were highest in cytosol (38.7 +/- 1.58% and 102.6 +/- 16.2 pmol/min per mg protein, n = 3). The activities of neural tumor cells, PC12 pheochromocytoma, Neuro2A and SKNSH neuroblastoma and U1242MG glioblastoma, were 34.4 +/- 6.83 (n = 5), 7.05 +/- 0.97 (n = 4), 5.25 +/- 1.69 (n = 5), and 9.68 +/- 1.35 (n = 4), pmol/min per mg protein (M +/- S.E.M.), respectively.     

PAF-induced synthesis of tetraenoic and pentaenoic leukotrienes in the isolated rabbit lung

In an isolated rabbit lung model, we tested the hypothesis that platelet-activating factor (PAF)-induced leukotriene (LT) synthesis is critically dependent on the free precursor fatty acid supply and the possible substitution of arachidonic acid (AA) by eicosapentaenoic acid (EPA). To augment the intravascular polymorphonuclear neutrophils (PMNs) in the isolated lung, human PMNs were infused into the pulmonary artery. LTs and hydroxyeicosatetra(penta)enoic acids were quantified with HPLC techniques. Application of PAF (5 microM) or AA (10 microM) provoked the generation of limited quantities of 4-series LTs and 5-hydroxyeicosatetraenoic acid (total sum of 5-lipoxygenase products approximately 7 and approximately 27 pmol/ml in lungs both with and without infused PMNs, respectively). Combined administration amplified 5-lipoxygenase product formation, with a predominance of cysteinyl-LT synthesis in lungs both without (total sum approximately 67 pmol/ml) and, much more strikingly, with (total sum approximately 308 pmol/ml) an infusion of neutrophils. EPA (10 microM) elicited exclusive generation of 5-series LTs and 5-hydroxyeicosapentaenoic acid (total sum approximately 82 pmol/ml). Dual stimulation with PAF and EPA provoked amplification of EPA-derived 5-lipoxygenase product formation, again with predominance of cysteinyl-LTs in lungs without (total sum approximately 224 pmol/ml) and, in particular, with (total sum approximately 545 pmol/ml) preceding microvascular PMN entrapment. Combined application of PAF, AA, and EPA resulted in the synthesis of LTs derived from both fatty acids, with a predominance of 5-series products. We conclude that the PAF-evoked 5-lipoxygenase product formation in the neutrophil-harboring lung capillary bed is critically dependent on intravascular precursor fatty acid supply, with EPA representing the preferred substrate compared with AA. PMN-related transcellular eicosanoid synthesis is suggested to underlie the predominant generation of cysteinyl-LTs. The supply of n-3 versus n-6 precursor fatty acid may thus have a major impact on inflammatory mediator generation.     

Determination of d-fenfluramine,d-norfenfluramine and fluoxetine in plasma,brain tissue and brain microdialysate using high-performance liquid chromatography after precolumn derivatization with dansyl chloride

A HPLC method is described for the simultaneous determination of d-fenfluramine (FEN), d-norfenfluramine (NF) and fluoxetine (FLX) using fluorometric detection after precolumn derivatization with dansyl-chloride. The method has limits of quantitation of 200 fmol for FEN and NF, 500 fmol for FLX in brain microdialysate, and 1 pmol for NF and FEN, and 2 pmol for FLX in plasma. Brain tissue standards were linear between 5 and 200 pmol/mg for all three compounds. The inter-assay variability (relative standard deviation) was 6.6%, 6.9% and 9.3% for FEN, 4.6%, 3.7% and 7.9% for NF and 10.4%, 4.9% and 12.2% for FLX, for brain microdialysate (2 pmol/μl), plasma (2 pmol/ μl) and brain tissue (50 pmol/mg), respectively. Intra-assay variability was always lower, typically several times lower than inter-assay variability. Extraction recovery was 108% and 48% for FEN, 105% and 78% for NF and 94% and 45% for FLX, in plasma (2 pmol/μl) and brain tissue (5 pmol/mg), respectively. Due to the stability of the dansyl-chloride derivatives this method is well suited for an autoinjector after manual derivatization with dansyl chloride at room temperature for 4 h.     

Presence and Measurement of Imidazoleacetic Acid, a γ-Aminobutyric Acid Agonist, in Rat Brain and Human Cerebrospinal Fluid

Imidazoleacetic acid (IAA) was unequivocally demonstrated in rat brain, human CSF, and human plasma by a gas chromatographic-mass spectrometric method that can reliably quantify as little as 8 pmol, i.e., 1 ng. Owing to tautomerism of the imidazole ring, IAA and [15N, 15N]IAA, the internal standard, each formed two chromatographically distinct isomers after derivatization of the ring nitrogens with either ethyl chloroformate or methyl chloroformate. The isomers of n-butyl(N-ethoxycarbonyl)imidazole acetate and n-butyl(N-methoxycarbonyl)imidazole acetate were identified by analysis with methane chemical ionization and electron impact ionization of molecular and fragment ions. The levels (mean +/- SEM) of free IAA were 140 +/- 14 pmol/g and 2.7 +/- 0.2 pmol/ml in brains of untreated rats and human lumbar CSF, respectively. Mean levels of IAA in brains of anesthetized rats, perfused free of blood, did not differ significantly from mean levels of anesthetized, nonperfused controls or from untreated rats. The source or sources of IAA in brain and CSF are unknown. Because IAA is a potent agonist at gamma-aminobutyrate receptors, it merits examination as a regulator in brain.     

Determination of d-fenfluramine, d-norfenfluramine and fluoxetine in plasma, brain tissue and brain microdialysate using high-performance liquid chromatography after precolumn derivatization with dansyl chloride

A HPLC method is described for the simultaneous determination of d-fenfluramine (FEN), d-norfenfluramine (NF) and fluoxetine (FLX) using fluorometric detection after precolumn derivatization with dansyl-chloride. The method has limits of quantitation of 200 fmol for FEN and NF, 500 fmol for FLX in brain microdialysate, and 1 pmol for NF and FEN, and 2 pmol for FLX in plasma. Brain tissue standards were linear between 5 and 200 pmol/mg for all three compounds. The inter-assay variability (relative standard deviation) was 6.6%, 6.9% and 9.3% for FEN, 4.6%, 3.7% and 7.9% for NF and 10.4%, 4.9% and 12.2% for FLX, for brain microdialysate (2 pmol/μl), plasma (2 pmol/ μl) and brain tissue (50 pmol/mg), respectively. Intra-assay variability was always lower, typically several times lower than inter-assay variability. Extraction recovery was 108% and 48% for FEN, 105% and 78% for NF and 94% and 45% for FLX, in plasma (2 pmol/μl) and brain tissue (5 pmol/mg), respectively. Due to the stability of the dansyl-chloride derivatives this method is well suited for an autoinjector after manual derivatization with dansyl chloride at room temperature for 4 h.     

Quantification of the alpha- and gamma-tocopherol metabolites 2,5,7, 8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman and 2,7, 8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman in human serum.

alpha- and gamma-tocopherol are the major vitamin E compounds found in human blood and tissues. The metabolites are 2,5,7, 8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC) and 2,7,8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman (gamma-CEHC, LLU-alpha), respectively. alpha-CEHC is excreted mainly as glucuronide or sulfate conjugates in the urine. Here we describe a sensitive and reliable method to analyze alpha- and gamma-CEHC in human serum. The concentration of alpha-CEHC in human serum is in the range of 5-10 pmol/ml but increases significantly up to 200 pmol/ml upon supplementation with RRR-alpha-tocopherol. About one-third of the alpha-CEHC circulating in the blood is present as a glucuronide conjugate. Baseline levels of gamma-CEHC are about 50 to 85 pmol/ml.     

Determination of mevalonate in blood plasma in man and rat. Mevalonate "tolerance" tests in man.

A method is described for the determination of mevalonate in ultrafiltrates of blood plasma. The method depends on the phosphorylation of mevalonate with [gamma-32P]ATP and mevalonate kinase to 5-[32P]phosphomevalonate, and the subsequent isolation of the 5-[32P]phosphomevalonate together with known amounts of added 5-phospho[14C]mevalonate by ion-exchange chromatography. The 32P/14C ratio in the isolated 5-phosphomevalonate is a linear function of the mevalonate content of the samples. The smallest amount that can be determined is 1--2 pmol. The fasting level in human plasma varied between 20 and 75 pmol/ml. Human red blood cells absorb mevalonate from plasma relatively slowly; their maximum storage capacity is about 1.3 pmol/10(6) red cells. An oral and intravenous "mevalonate tolerance test" in man is described that can be carried out with 200 and 30 mumol. respectively, of the unlabeled (RS)-mevalonate in a 70-kg man. Beer and wine contain mevalonate at a concentration of 3--8 microns, too low to provide a significant amount of mevalonate even for heavy drinkers. The mevalonate content of the plasma from the blood of the vena cava inferior of male rats varied between 81 and 502 pmol/ml and is positively related to the levels of liver 3-hydroxy-3-methylgultaryl-CoA reductase, suggesting that the liver is probably the main source of mevalonate circulating in blood. The plasma of renal venous blood contained only 33--85% as much mevalonate as the arterial plasma.     

Chemiluminescent simultaneous determination of phosphatidylcholine hydroperoxide and phosphatidylethanolamine hydroperoxide in the liver and brain of the rat.

The quantification of phospholipid hydroperoxides in biological tissues is important in order to know the degree of peroxidative damage of membrane lipids. For this purpose, optimal conditions for the chemiluminescent simultaneous assay of phosphatidylcholine hydroperoxide (PCOOH) and phosphatidylethanolamine hydroperoxide (PEOOH) in rat liver and brain were determined. A chemiluminescence detection-high performance liquid chromatography (CL-HPLC) method that incorporates cytochrome c and luminol as a post-column hydroperoxide-specific luminescent reagent was used (Miyazawa et al. 1987. Anal. Lett. 20: 915-925; Miyazawa. 1989. Free Radical Biol. Med. 7: 209-217). An n-propylamine-bound silica column with hexane-2-propanol-methanol-water 5:7:2:1 (v/v/v/v) (flow rate 1.0 ml/min) as eluant was used to determine both PCOOH and PEOOH, which were separated from each other and from other lipids and lipid-soluble antioxidants. High reproducibility and sensitivity as low as 10 pmol hydroperoxide-O2 were observed with a mixture of 10 micrograms/ml cytochrome c and 2 micrograms/ml luminol in 50 mM borate buffer (pH 10.0, flow rate 1.1 ml/min) as luminescent reagent and a post-column mixing joint temperature of 40 degrees C. Using the established analytical conditions, it was confirmed that both PCOOH (1324 +/- 122 pmol/g liver, 114 +/- 18 pmol/g brain, mean +/- SD) and PEOOH (728 +/- 89 pmol/g liver, 349 +/- 60 pmol/g brain, mean +/- SD) are present in the liver and brain of Sprague-Dawley rats bred on a slightly modified AIN-76A semisynthetic diet for 3 months. The phospholipid hydroperoxide content in the rat liver was shown to be affected by dietary oils, but not significantly affected in the brain.     

Simultaneous measurement of dolasetron and its major metabolite, MDL 74,156, in human plasma and urine

A selective and sensitive analytical method for the simultaneous measurement of dolasetron (I) and its major metabolite, MDL 74,156 (II), in human plasma and urine samples has been developed using a structural analogue, MDL 101,858, as internal standard (I.S.). The compounds were extracted from plasma and urine using solvent extraction after the addition of the I.S. Chromatographic separation was carried out on a reversed-phase HPLC column and detection and quantification was by fluorescence with excitation and emission wavelengths of 285 and 345 nm, respectively. Linear responses were obtained over concentration ranges of 5 to 1000 pmol/ml for plasma samples and 20 to 1000 pmol/ml for urine samples with correlation coefficients for the calibration curves exceeding 0.999 in all cases. Intra-day and inter-day reproducibility yielded limits of quantification of 10 pmol/ml for I and 5 pmol/ml for II in plasma and 50 pmol/ml for I and II in urine. The method has been applied to the simultaneous analysis of both compounds in plasma and urine samples coming from clinical pharmacokinetic studies.     

Quantitative determination of dehydroepiandrosterone fatty acyl esters in human female adipose tissue and serum using mass spectrometric methods

Dehydroepiandrosterone-fatty acyl esters (DHEA-FAE) are naturally occurring water-insoluble metabolites of DHEA, which are transported in plasma exclusively by lipoproteins. To find out whether DHEA, like estradiol, might be stored in adipose tissue in FAE form, we set up a mass spectrometric method to quantify DHEA-FAE and free DHEA in human adipose tissue and serum. The method consists of chromatographic purification steps and final determination of hydrolyzed DHEA-FAE and free DHEA, which was carried out by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our results showed that no detectable amounts of DHEA-FAE could be found in adipose tissue although 32-178 pmol/g of free DHEA were determined by GC-MS and LC-MS/MS. The DHEA-FAE concentrations in serum quantified by GC-MS were 1.4±0.7 pmol/ml in premenopausal women (n=7), and 0.9±0.4 pmol/ml in postmenopausal women (n=5). Correspondingly, the free DHEA concentrations were 15.2±6.3 pmol/ml and 6.8±3.0 pmol/ml. In addition, the mean proportions of DHEA-FAE of total DHEA (DHEA-FAE+free DHEA) in serum were 8.6% and 11.2% in pre- and postmenopausal women, respectively. Serum DHEA-FAE concentration was below quantification limit for LC-MS/MS (signal-to-noise ratio, S/N=10), while free DHEA concentrations varied between 5.8 and 23.2 pmol/ml. In conclusion, the proportion of DHEA-FAE of total DHEA in serum was approximately 9%. However, in contrast to our previous findings for estradiol fatty acid esters in adipose tissue which constituted about 80% of total estradiol (esterified+free), the proportion of DHEA-FAE of total DHEA was below 5%. Four to ten times higher concentrations of free DHEA were quantified in adipose tissue compared to those in serum.     

Intracoronary and systemic release of the atrial natriuretic factor and cyclic-guanosine monophosphate during coronary angioplasty

Summary This study was undertaken to investigate intracoronary production and systemic release of the atrial natriuretic factor (ANF) and cyclic-guanosine monophosphate (c-GMP) during coronary angioplasty (PTCA). three coronary blood samples were collected, through a balloon catheter, from the area distal to the lesion: before balloon inflation, at maximum inflation and 5 min later. Four additional venous samples were collected: before PTCA, and 5 min, 2 h and 24 h after the procedure. Local intracoronary c-GMP production increased from the baseline level of 7.5±0.9 pmol/ml to 11.1±1.3 pmol/ml at maximum balloon inflation (p<0.01) and decreased 5 min later to 9.5 ±1.0 pmol/ml (p=NS). In contrast, intracoronary ANF production failed to show any significant change at any time during the procedure. Peripheral venous ANF levels increased from 79.1±11.1 pmol/ml to 99.9±16.6 pmol/ml 5 min after balloon inflation (p<0.05) and gradually decreased 2 h (91.9±13.6 pmol/ml) and 24 h (85.6±10.4 pmol/ml) after the procedure. Similarly, peripheral venous c-GMP levels increased from 11.3±1.7 pmol/ml before PTCA to 14.9±1.9 pmol/ml 5 min after balloon inflation (p<0.05), and then gradually decreased 2 h (10.8±1.4 pmol/ml) and 24 h (8.2±1.4 pmol/ml) after the procedure (p<0.01 and <0.0001 compared to the peak value, respectively). In conclusion, acute vessel occlusion and distension during balloon inflation stimulates intracoronary c-GMP production without affecting ANF release.     

Metabolic conversion of intra-amniotically-injected deuterium-labeled essential fatty acids by fetal rats following maternal n-3 fatty acid deficiency

Accumulation of polyunsaturated fatty acids (PUFA) in the fetal brain is accomplished predominantly via a highly selective flow of docosahexaenoic acid (22:6n-3, DHA) and arachidonic acid (20:4n-6, AA) through the placenta. Little is known regarding the endogenous capability of the fetus to generate its own DHA and AA from lower homologues such as linolenic (18:3n-3, ALA) and linoleic (18:2n-6, LA) acids, respectively. Deuterium-labeled d5-ALA and d5-LA at millimolar concentrations were injected directly into the amniotic fluid in order to investigate maternal-independent metabolic conversion of the stable isotopes in brain and liver of the fetus near delivery. After 48 h under adequate maternal diet, the levels of d5-ALA metabolites in the fetal brain and fetal liver were 45 ± 2.2 pmol/mg and 86 ± 4 pmol/mg of which 79% and 63.6% were comprised of d5-DHA. At this time point, incorporation of d5-LA metabolites was 103 ± 5 pmol/mg and 772 ± 46 pmol/mg for brain and liver, of which 50% and 30% were comprised of d5-AA. Following sustained maternal dietary ALA deficiency, the levels of total d5-ALA derived metabolites in the fetal brain and fetal liver were increased to 231 pmol/mg and 696 pmol/mg of which 71% and 26% were comprised of d5-DHA. From the time course and relative rates of d5-ALA precursor displacement by d5-DHA in cellular phosphoglycerides, it is concluded that the fetal rat brain can generate its own DHA from its d5-ALA precursors particularly under dietary stress.     

Quantification of bioactive acylethanolamides in rat plasma by electrospray mass spectrometry

We developed a high-performance liquid chromatography/mass spectrometry (HPLC/MS) method for the identification and quantification of anandamide, an endogenous cannabinoid substance, and other fatty acid ethanolamides (AEs) in biological samples. Using a mobile-phase system of methanol/water and gradient elution, we achieved satisfactory resolution of all major AEs, including anandamide, palmitylethanolamide (PEA), and oleylethanolamide (OEA). Electrospray-generated quasi-molecular species were used as diagnostic ions and detected by selected ion monitoring (SIM). Synthetic deuterium-labeled AEs were used as internal standards, and quantification was carried out by isotope dilution. A linear correlation (r2 = 0.99) was observed in the calibration curves for standard AEs over the range 0-0.5 nmol. Detection limits between 0.1 and 0.3 pmol per sample and quantification limits between 0.5 and 1.2 pmol per sample were obtained. The method was applied to the quantification of anandamide, PEA, and OEA in plasma prepared from rat blood collected either by cardiac puncture or by decapitation. After cardiac puncture, AE levels were in the low-nanomolar range: anandamide, 3.1 +/- 0.6 pmol/ml; PEA, 9.4 +/- 1.6 pmol/ml; OEA, 9.2 +/- 1.8 pmol/ml (mean +/- SE, n = 9). By contrast, after decapitation AEs were dramatically elevated (anandamide, 144 +/- 13 pmol/ml; PEA, 255 +/- 55 pmol/ml; OEA, 175 +/- 48 pmol/ml). Thus, disruptive procedures of blood collection may result in gross overestimates in the concentrations of circulating AEs.     

Concentration Gradients of Free and Total γ-Aminobutyric Acid and Homocarnosine in Human CSF: Comparison of Suboccipital and Lumbar Sampling

Abstract: Concentrations of free and total γ-aminobutyric acid (GABA) and homocarnosine were determined in sequential aliquots of the first 30 ml of CSF obtained by lumbar puncture in five patients. Rostrocaudal gradients were calculated and compared to gradients estimated by determining concentrations of these substances in CSF obtained by simultaneous suboccipital and lumbar punctures in four more patients. In the lumbar fractions study, rostrocaudal mean gradients of 0.36, 36, and 21 pmol/ml for free GABA, total GABA, and homocarnosine, respectively, were calculated. In the suboccipital/lumbar study, gradients of 0.33, 30, and 24 pmol/ml for free GABA, total GABA, and homocarnosine, respectively, were estimated. These results indicate that valid comparison of CSF concentrations of these substances is restricted to similar fractions and suggest that in CSF the substances originate largely from brain rather than from peripheral sources.     

Determination of carbon monoxide (CO) in rodent tissue: effect of heme administration and environmental CO exposure

Carbon monoxide (CO), produced endogenously during heme degradation, is considered a messenger molecule in vascular and neurologic tissues. To study this role, it is important to determine CO concentration in target tissues pre- and post-perturbations. Here, we describe a sensitive and reproducible method, which is linear and accurate, and provide some examples of its application for quantitation of CO concentrations in tissues pre- and post-perturbations. Tissues from adult rats and mice were sonicated (20% w/w), and volumes representing 0.04-8 mg fresh weight (FW) were incubated at 0 degrees C for 30 min with sulfosalicylic acid. CO liberated into the headspace was quantitated by gas chromatography. Tissue CO concentrations (mean+/-SD, pmol CO/mg FW) were as follows: blood (47+/-10, 45+/-5), muscle (4+/-4, 10+/-1), kidney (5+/-2, 7+/-2), heart (6+/-3, 6+/-1), spleen (11+/-3, 6+/-1), liver (4+/-1, 5+/-1), intestine (2+/-1, 4+/-2), lung (2+/-1, 3+/-1), testes (1+/-1, 2+/-1), and brain (2+/-1, 2+/-0) in untreated rat (n=3) and mouse (n=5), respectively. Between the rat and the mouse, only CO concentrations in the muscle and spleen were significantly different (p0.05). Endogenous CO generation, after administration of heme arginate to mice (n=3), increased CO concentrations by 0-43 pmol/mg FW. Exposure of mice (n=3) to 500 ppm CO for 30 min yielded significantly elevated CO concentrations by 4-2603 pmol/mg FW in all tissues over the native state. While blood had the highest CO concentration for all conditions, muscle, kidney, heart, spleen, and liver, all rich in hemoglobin and/or other CO-binding hemoproteins, also contained substantial CO concentrations. Intestine, lung, testes, and brain contained the lowest CO concentrations.     

Assay of leucine aminopeptidase activity in vitro using large-pore reversed-phase chromatography with fluorescence detection

A chromatographic method for determination of leucine aminopeptidase (LAP) activity in complex matrices is described. L-Leucine-beta-naphthylamide was used as the substrate and its hydrolytic product, beta-naphthylamine, was monitored by fluorescence at 280 nm excitation and 400 nm emission wavelengths. Under optimized conditions, the components in the incubation mixture were baseline separated and eluted out of a large-pore (300 angstroms) reversed-phase C4 column (RPC4) within 15 min with a non-linear gradient elution of methanol (0.05% (v/v) trifluoroacetic acid additive). The detection limit of the hydrolytic product reached 0.35 pmol at three time signal-to-noise (S/N) ratio with 5 microl sample injection. The method showed a wide dynamic range for quantitation of both the hydrolytic product (10 ng/ml to 80 microg/ml) and LAP (0.1-46.0 microg/ml) with correlation coefficient larger than 0.998 and reproducibility <3 and 10% R.S.D. (n=3), respectively. A fairly broad range of incubation time could be selected within 1 h. The LAP activities and concentrations in rabbit serum, tears, and mouse lens homogenates were determined to be 41.8 (0.3 mg/ml), 2.8 (40.0 microg/ml), and 1.6 pmol/(microl min) (17.5 microg/ml), respectively, with reproducibility of 2-9% R.S.D. (n=3) and intra- and inter-day variation for the retention time of the hydrolytic product being <1% R.S.D. (n=3). The results indicate that the present method is rapid and sensitive as compared to the conventional one.     

Correlation between pentosidine and endothelin-1 in subjects undergoing chronic hemodialysis.

Advanced glycation end-products (AGEs), which accumulate in the blood and tissues of patients with chronic renal failure (CRF) undergoing chronic hemodialysis, play an important role in the pathogenesis of uremic complications. Endothelin 1 (ET1), a 21-amino acid peptide with vasoconstricting and mitogenic properties, is an important factor in the endothelial dysfunction occurring in uremia. The circulating levels of both AGEs and ET1 have been reported to be increased in chronic renal failure. In the present study we evaluated the possible relationship between pentosidine and ET1 plasma levels in CRF patients undergoing chronic hemodialysis treatment. The plasma concentrations of "free" and bound pentosidine (HPLC methods) and endothelin-1 (RIA method) were measured before the hemodialysis session in 40 nondiabetic CRF patients (22 males and 18 females; 54+/-3 years) on chronic hemodialysis for at least 1 year. Forty age- and sex-matched normal subjects served as a control group. In hemodialyzed patients, the overall pentosidine residues and pentosidine-free adduct plus pentosidine-free adduct bound reversibly to protein levels (24.9+/-2.04 pmol/mg protein and 110.5+/-5.9 pmol/ml, respectively) were significantly higher than those recorded in normal subjects (2.0+/-0.2 pmol/mg protein and 0.7+/-0.2 pmol/ml, respectively ). Endothelin-1 was also significantly (p<0.01) increased in CRF patients (10.6+/-0.4 pmol/ml in CRF patients and 2.7+/-0.3 pmol/ml in normal subjects). A significant positive correlation (p<0.01) was seen between "total" pentosidine (pentosidine residues and pentosidine-free adduct plus pentosidine-free adduct bound reversibly to protein) levels and endothelin-1 plasma values. The correlation between pentosidine and endothelin-1 provides further evidence that some AGEs exert a detrimental effect on the vascular endothelium, thereby contributing to the hypertension and other cardiovascular damage seen in CRF patients.     

An automatic method of 4-hydroxyproline determination convenient for large series

A continuous automatic method involving alkaline hydrolysis, neutralization with sulfuric acid, and specific detection of 4-hydroxyproline is described. The main parameters of the method are studied. This technique is capable of detecting 10 μmol/liter of 4-hydroxyproline linked in peptidic sequence with acceptable repeatability (SD/mean = 0.05). The automatic alkaline hydrolysis yield is 67% for soluble type I collagen. The method enables the analysis of 500 samples in 24 h.     

Effect of a test meal,duodenal acidification,and tetragastrin on the plasma concentration of β-endorphin-like immunoreactivity in man

The effects of various test materials on plasma β-endorphin-like immunoreactivity (β-EpLI) were investigated in man using a specific radioimmunoassay developed by the authors. Plasma β-EpLI was determined after extraction by the acid/acetone method (recovery 73±5%). The intraassay and interassay coefficients of variation were 5.0% and 7.6%, respectively. The plasma concentrations of human β-EpLI in normal subjects were 11.6±4.0 pmol/l for men (n=23) and 10.7±4.8 pmol/l for women (n=27). Ingestion of a test meal (150 g of Campbell's condensed meat soup) resulted in a biphasic rise in plasma β-EpLI from the basal level of 4.4±1.0 pmol/l to 29.2±1.9 pmol/l after 5 min and 24.8±6.7 pmol/l after 90 min. Intraduodenal infusion of 115 ml of 0.1 M HCl over 10 min increased the plasma β-EpLI level from 8.7±0.5 pmol/l to 15.5±0.4 pmol/l at 10 min after the start of infusion, but the level rapidly returned to the initial value after the end of the infusion. Intramuscular injection of 4 μg/kg body weight of tetragastrin markedly stimulated gastric acid output and β-EpLI release, but pretreatment with 10 mg of histamine H₂ receptor antagonist inhibited the gastric acid output and plasma β-EpLI release induced by tetragastrin.These results indicate that β-EpLI release is stimulated by ingestion of meat soup, duodenal acidification and tetragastrin administration. It is suggested that gastric acid participates, at least in part, in postprandial release of β-EpLI, probably from the gastrointestinal tract.