Liquid chromatography method for the analysis of adenosine compounds

A newly available chromatography column packing material that employs hybrid particle technology was used to improve the analysis of adenosine compounds. Using a TBAS buffer/acetonitrile gradient this material permits separation of etheno-adenosine compounds in less than 4 min with excellent resolution and sensitivity (50 fmol). Variability of compound quantification is small (coefficients of variation 0.23+/-0.14% for 50 pmol and 1.70+/-0.53% for 0.5 pmol). The new method is well suited for the analysis of adenosine compounds in small biological samples and permits a high sample throughput in autosampler setups with high precision and reproducibility.     

Plasma melatonin concentration in neonatal northern elephant seals,Mirounga angustirostris

The development of pineal function in northern elephant seals was examined in an attempt to understand the physiological basis for previously observed high daytime levels of melatonin in neonatal southern elephant seals. Pineal glands from four northern elephant seal pups, estimated age less than 1 week, weighed 3.0 ± 0.80 g, which was significantly less than that previously found in southern elephant seals (4.6 ± 0.35 g). Midday concentrations of plasma melatonin in pups averaged more than 3000 pmol/l in the first 5 days post-partum, but declined rapidly to less than 400pmol/l after day 9. Daytime melatonin levels in northern elephant seals tended to be lower than in southern elephant seals, although they were very high compared with other species. A circadian cycle of plasma melatonin concentration was observed in newborn northern elephant seals, with levels of 3000–5000 pmol/1 during the day, rising to more than 10,000 pmol/1 late in the dark phase. Soon after weaning at 4 weeks of age, daytime and night-time levels were in the range 60–100 pmol/1 and 100–400 pmol/1, respectively. When approximately 10 weeks old, most samples were in the range 100–400 pmol/1 with no discernible difference between day and night levels. The results do not support the hypothesis that the pineal gland is involved in thermogenesis in new-born southern elephant seals. Instead, the very active pineal gland may contribute to energy conservation, by lowering body temperature, particularly at night. As physical insulation is acquired by the deposition of blubber, the mechanism is not required and melatonin falls to adult levels.     

A simple spectrophotometric method for measurement of nematode populations.

A convenient automated method for measuring inorganic phosphate based on the malachite green reaction with a phosphomolybdate complex has been developed. Less than 100 pmol of inorganic phosphate can be readily quantitated by the method which utilizes standard AutoAnalyzer equipment. Inorganic phosphate is measured in sample volumes of less than 0.1 ml and without interference by a number of phosphorylated metabolic intermediates or nucleotides. This methodology is especially useful in the analysis of hydrolytic processes involving phosphorylated substrates.     

Changes in adrenal and testicular activity monitored by salivary sampling in males throughout marathon runs

Measurement of cortisol and testosterone in saliva samples provided by marathon runners at 6.4 km (4-mile) intervals has been used for monitoring acute changes in adrenal and testicular activity, and the changes compared with mean values in timed samples on five rest days. The collection of mixed whole saliva was well accepted; the missed sample rate in the 8 runners in the Cardiff marathon was less than 10%. On rest days, salivary cortisol and testosterone were within the normal male range and showed a circadian rhythm; mean values at 08.00 h (23.5 nmol L-1; 258 pmol L-1, p less than 0.001, p less than 0.001 respectively) were higher than at 22.00 h (2.8 nmol L-1; 130 pmol L-1). In samples collected at 09.00 h, immediately prior to the Cardiff marathon, cortisol (25.1 nmol L-1) and testosterone (304 pmol L-1) were higher than the mean values (14.9 nmol L-1; 209 pmol L-1) on non-run days. Concentrations of both steroids increased during the marathon; testosterone peaked (442 pmol L-1) at 21 miles, whereas cortisol continued to increase, being maximal (87.9 nmol L-1) at 30 min after completion of the run. Four of the runners in the Cardiff marathon also participated in the Bristol marathon and the changing patterns in salivary hormones were strictly comparable. Salivary sampling would appear to be of value in monitoring acute and rhythmic changes in endocrine function in marathon runners. The temporal relationship between changes in salivary cortisol and testosterone are consistent with direct inhibition of testicular secretion by high cortisol concentrations.     

Potent vasoactive properties of endothelin 1 in human skin.

The effect of the endothelial cell-derived peptide endothelin 1 was investigated in human skin. Intradermal injection of endothelin 1 (1-100 pmol) caused a dose-dependent area of pallor that was associated with a significant reduction in basal skin blood flow, measured by laser-Doppler blood flowmeter (with 1 pmol endothelin, P = 0.012, analysis of variance). The coadministration of endothelin 1 (1-100 pmol) with the neuropeptide vasodilator calcitonin gene-related peptide (CGRP) inhibited the vasodilator response to CGRP (10 pmol) by up to 82.7 +/- 9.2% (with 100 pmol endothelin, P less than 0.001). The response of the prostanoid vasodilator prostaglandin E2 (10 pmol) was inhibited by endothelin in a similar manner. In addition to the vasoconstrictor effects, endothelin 1 produced a dose-dependent flare that surrounded the area of pallor, and this was associated with a significant increase in blood flow (P less than 0.05) within the flare area. The H1 antagonist terfenadine (120 mg po) significantly reduced the flare area associated with endothelin 1: flare 5 min after intradermal endothelin (10 pmol, placebo treated), 668 +/- 405 mm2; terfenadine treated, 201 +/- 257 mm2 (P less than 0.05). The flare was also significantly attenuated when endothelin (10 pmol) was injected into local anesthetic-treated skin. Thus intradermal injection of endothelin in humans causes long-lasting vasoconstriction at the site of injection and a surrounding flare. Results suggest that the flare component is partially histamine dependent and the result of an axon reflex. This study demonstrates the potent activity of endothelin in human skin. It is possible that endothelin could be relevant to the local response of skin to injury.     

Determination of anti-HIV drug concentration in human plasma by MALDI-TOF/TOF

The antiretroviral therapeutic drug monitoring is becoming increased in clinical care to determine the best dosage regimen adapted to each patient. Here, the determination of the anti-HIV drugs lamivudine, lopinavir, and ritonavir concentration in the plasma of HIV-infected patients by MALDI-TOF/TOF is reported. The volume of the plasma sample was 600 microL. Plasma samples were extracted by solid-phase (divinylbenzene and N-vinylpyrrolidone) and evaporated in a water bath under a nitrogen stream. The extracted samples were reconstituted with methanol (100 microL), mixed (1:1) with a saturated matrix solution (4-hydroxybenzoic acid in 50% acetonitrile-0.1% trifluoracetic acid), and spotted onto the MALDI-TOF/TOF sample target plate. The lamivudine, lopinavir and ritonavir concentration was determined by standard additions analysis. Regression of standard additions was linear over the anti-HIV drug concentration ranges explored (lamivudine, 0.010-1.0 pmol/microL; lopinavir and ritonavir, 0.0025-0.50 pmol/microL). Moreover, emtricitabine (i.e., the fluorinated analog of lamivudine) was used as the internal standard to determine the lamivudine concentration. The calibration curve was linear on the emtricitabine concentration ranging between 0.050 and 5.0 pmol/microL. The absolute recovery ranged between 80 and 110%. Values of the lamivudine, lopinavir and ritonavir concentration determined by MALDI-TOF/TOF are in excellent agreement with those obtained by HPLC-UV and HPLC-MS/MS. MALDI-TOF/TOF experiments allowed also the detection of the ritonavir metabolite R5. Zidovudine was undetectable by MALDI-TOF/TOF analysis because also the minimal laser intensity may induce the anti-HIV drug photolysis. The MALDI-TOF/TOF technique is useful to determine very low concentrations of anti-HIV drugs (0.0025-0.010 pmol/microL).     

Rapid and sensitive determination of protein-nitrotyrosine by ELISA: Application to human plasma

3-Nitrotyrosine (3NT) is known as an important indicator of nitrosative stress and has been linked to various diseases. Our aim was to develop an indirect ELISA (enzyme-linked immunosorbent assay) method suitable for the detection of protein-bound 3NT in clinical plasma and serum samples. Nitrated protein standards and reduced protein standards were prepared. Limit of detection was determined for standards; recovery and reproducibility were determined for human plasma samples. The limit of detection for this method is 1.82±0.56 pmol/mg protein. Mean recovery of standards was 95%. 3NT concentration in plasma samples of obese and normal weight subjects was determined to be between 2 pmol/mg and 19 pmol/mg. No time-consuming sample preparation or expensive laboratory equipment is required, and applied antibodies are commercially available. Sensitivity, rapid analysis time, possibilities of high throughput applications and small sample volumes make this ELISA attractive for use in clinical laboratories.     

Automated determination of orotic acid, uracil and pseudouridine in urine by high-performance liquid chromatography with column switching

A column-switching high-performance liquid chromatographic method, requiring no sample preparation apart from filtration, is described for quantification of urinary orotic acid, uracil and pseudouridine. The analyses were carried out using a reversed-phase octadecylsilane-bonded column for sample clean-up and a cation-exchange column for separation; 5–20 ]sml samples of urine were directly analysed, and more than 100 samples could be analysed consecutively. Each sample required only 30 min. Detection limits of these compounds were 5 pmol. Creatinine-related urinary uracil excretion was lowest in the newborn period (17.3 ± 14.4 μmol/g of creatinine). A patient with partial ornithine transcarbamylase deficiency and his mother usually excreted a high level of uracil during the period of normal orotic acid excretion and normal serum ammonia level.     

Ascorbic acid analysis using high-performance liquid chromatography with coulometric electrochemical detection

A method for the detection of ascorbic acid using high-performance liquid chromatography with coulometric electrochemical detection and a technique for stabilization of the vitamin are described. Since less than 1 pmol of ascorbic acid can be detected, this assay provides significantly greater sensitivity than nearly all of the currently available procedures. Stabilization of 10 pmol or less of ascorbic acid at room temperature for up to 4 h and for several weeks at -70 degrees C facilitates storage of a large number of samples and measurement of ascorbic acid using an automated sampling device. This method was used to quantitate the amounts of ascorbic acid in human polymorphonuclear leukocytes and bovine adrenomedullary chromaffin granules. The calculated concentrations found for human neutrophils (1.35 mM) and bovine chromaffin granules (10.0 mM) are in agreement with previously published data. The assay is suitable for the determination of ascorbic acid in biological samples where only a small amount of tissue is available or very low amounts of ascorbic acid are found. This method is the first application of coulometric electrochemical detection to ascorbic acid HPLC analysis.     

Microsequence analysis of peptides and proteins. VI. A continuous flow reactor for sample concentration and sequence analysis

We have designed and tested a continuous flow reactor (CFR) for microsequence analysis of peptides and proteins. The CFR forms the site for immobilization of the peptide or protein substrate and automated Edman chemistry. The CFR was constructed from 0.125-in.-o.d., 0.0625-in.-i.d. Teflon tubing (length 2-3 cm) containing 5-10 mg of Polybrene-coated, spherical, porous silica (100-200-micron particle size). The silica is retained in the CFR with porous Teflon filters (Zitex) at the bed bottom and optionally at the bed top. The i.d. of the CFR was selected for a tight press fit when 0.0625-in.-o.d. Teflon lines are inserted at the top and bottom of the CFR. This design allows the replacement of the existing cartridge/glass fiber disk found in conventional microsequencers with a CFR with a minimal amount of changes. The advantages of the CFR over the previous design include a lower background or noise level and no need to precycle Polybrene before sample application, and the entire unit is inexpensive and therefore disposable. We believe that the decrease in noise, especially the decrease in the commonly observed diphenylthiourea peak, is due to the more direct flow path and relative absence of unswept area in the CFR. Several standard peptides and proteins were sequenced in the CFR to demonstrate the improved results. A direct comparison to the cartridge/glass fiber disk design demonstrated less background and higher initial and repetitive yields for the CFR. An additional advantage is the ability to directly concentrate samples on CFRs containing reverse-phase packing. We have successfully concentrated 1.0-ml samples (200 pmol) onto 5 mg of octyldecylsilyl-derivatized silica in yields of 95-100%. The resulting samples were microsequenced after addition of Polybrene-coated silica to the CFR with high initial and repetitive yields. This methodology promises to improve sample handling and microsequence analysis of low picomole amounts of peptides and proteins.     

Simultaneous determination of six adenyl purines in human plasma by high-performance liquid chromatography with fluorescence derivatization

A sensitive method was developed for the simultaneous determination of six adenyl purines in human plasma by high-performance liquid chromatography. The adenyl purines (adenine, adenosine, AMP, ADP, ATP and cyclic AMP) were derivatized using 2-chloroacetaldehyde for fluorescence detection, and the reaction and separation conditions were reinvestigated to improve sensitivity for small volume sample analysis. Each derivatized purine was separated on a Capcell Pack SG120A™ column with mobile phase consisting of 0.05 M citric acid–0.1 M dipotassium hydrogen phosphate (pH 4.0)–methanol (97+3). The detection limits were 100–1000 fmol/ml by fluorescence detection, some 500 times better than previous reports. The proposed method was applied to determine adenyl purines in human plasma. The purine levels were as follows: ATP (9.2–22.2 pmol/ml), ADP (5.5–22.2 pmol/ml), AMP (0.8–3.2 pmol/ml). Other purines, adenine, adenosine, cAMP were lower than 0.1 pmol/ml.     

A one-vial method for routine extraction and quantification of free fatty acids in blood and tissue by HPLC

A one-vial procedure has been developed to quantify free fatty acids in human blood serum and rat heart tissue. To allow routine analysis the system has been constructed to allow simultaneous processing of nine samples (Praechromat). The free fatty acids are extracted with Freon 11 and then derivatized to coumarin esters prior to HPLC. The Freon 11 extraction of the free fatty acids is rapid and complete. Neither hydrolytic degradation of natural fatty acid esters nor oxidative damage of unsaturated fatty acids was observed. Fifteen free fatty acids (FFA) were routinely quantified by isocratic elution with high reproducibility (SD less than 4%) and good recovery (0.1 mM FFA: 98-100%, 0.02 mM: 91-105%). The free fatty acids could be determined in the range from 20 pmol to 20 nmol.     

Effects of high-intensity cycle exercise on sympathoadrenal-medullary response patterns

Plasma proenkephalin peptide F immunoreactivity and catecholamines were examined on separate days in nine healthy males before and after maximal exercise to exhaustion at four intensities [36, 55, 73, and 100% of maximal leg power (MLP)] by use of a computerized cycle ergometer. The mean duration of 36, 55, 73, and 100% MLP was 3.31, 0.781, 0.270, and 0.1 min, respectively. All intensities were greater than those eliciting peak O2 uptake for the individual subjects. Blood samples were obtained before, immediately after exercise, and 5 and 15 min after exercise. Significant (P less than 0.05) increases in plasma peptide F immunoreactivity (i.e., from mean resting value of 0.18 to 0.43 pmol/ml) were observed immediately after exercise at 36% MLP. Significant increases in plasma epinephrine were observed immediately after exercise at 36% MLP (i.e., from mean resting value of 2.22 to 3.11 pmol/ml) and 55% MLP (i.e., from mean resting value of 1.67 to 2.98 pmol/ml) and 15 min after exercise at 100% MLP (i.e., from mean resting value of 1.92 to 3.88 pmol/ml). Significant increases for plasma norepinephrine were observed immediately after exercise (36, 55, 73, and 100% MLP), 5 min after exercise (36, 55, and 73% MLP), and 15 min after exercise (36% MLP). Increases in whole blood lactate were observed at all points after exercise for 36, 55, and 73% MLP and 5 min after exercise for 100% MLP. These data show that brief high-intensity exercise results in differential response patterns of catecholamines and proenkephalin peptide F immunoreactivity.     

Amniotic fluid testosterone and testosterone glucuronide levels in the determination of foetal sex

Unconjugated testosterone levels were assayed in 351 amniotic fluid samples obtained at 15-19 weeks gestation. The median values for unconjugated testosterone in the 166 female foetuses and 185 male foetuses were 137 and 712 pmol/l respectively. Sixteen amniotic fluid samples from male foetuses had unconjugated testosterone levels lower than the highest female unconjugated testosterone value (361 pmol/l). Testosterone glucuronide was measured in amniotic fluid from 48 female and 55 male foetuses. There was a significant sex difference in the median values of testosterone glucuronide between female (median 160 pmol/l, range 64-465 pmol/l) and male (median 817 pmol/l, range 68-3707 pmol/l) amniotic fluid specimens (P less than 0.001). Of the sixteen male foetuses with amniotic fluid unconjugated testosterone levels in the female range, 12 had amniotic fluid testosterone glucuronide levels within the male testosterone glucuronide range of values. Hence used in conjunction with unconjugated testosterone, testosterone glucuronide increased the predictive accuracy of foetal sexing from 95.4 to 98.9%. Testosterone sulphate was measured in 24 female and 25 male amniotic fluid samples. There was no Testosterone sulphate was measured in 24 female and 25 male amniotic fluid samples. There was no significant difference between female (median 2591 pmol/l) and male (median 2964 pmol/l) testosterone sulphate levels.     

Monoclonal antibodies toward a stable cyclic AMP-C8 antigen.

A cyclic AMP antigen was prepared utilizing a stable thioether linkage from C8 of the purine moiety. Monoclonal antibodies were raised toward the cyclic AMP-C8-diphtheria toxoid antigen and found to be of high specificity, with only very low cross-reactivity against related compounds, less than 0.03% against cGMP being the most significant cross-reaction. The stability of the thioether linkage enabled the preparation of an enzyme tracer, which was used in an ELISA. The assay had an effective working range from 0.1 to 100 pmol cAMP. Due to the structure of the antigen, the need for sample derivatization was eliminated.     

Determination of imipramine and seven of its metabolites in human liver microsomes by a high-performance liquid chromatographic method

The metabolism of the widely used antidepressant drug imipramine is subject to marked interindividual variation. A sensitive and specific reversed-phase high-performance liquid chromatography method for the simultaneous determination of imipramine and seven of its metabolites in human liver microsomal preparations was developed. These metabolites include 10-hydroxy-desipramine, 10-hydroxyimipramine, 2-hydroxydesipramine, 2-hydroxyimipramine, desipramine, didesmethylimipramine, and imipramine N-oxide. The detection limit for imipramine and the metabolites was approximately 20 pmol. At concentrations of 100 and 500 pmol per tube, the reproducibility showed a coefficient of variation less than 10%, except for the 2-hydroxy-desipramine (16%), 2-hydroxyimipramine (15%), and imipramine N-oxide (17%), all three at 100 pmol per tube. Linear standard curves were obtained for all the compounds within a concentration range of 50 to 1000 pmol per tube. This assay will provide a tool to assess the contribution of different enzymes to the formation of imipramine metabolites.     

A fluorometric method for measuring ethoxycoumarin O-deethylase activity by reversed-phase high performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method that uses an on-line change in the protonation state of the nonfluorescent product to yield a fluorescent derivative that is detected by fluorometry was developed for the determination of 7-ethoxycoumarin O-deethylase activity. Tissue samples (1-20 micrograms protein) were incubated with 7-ethoxycoumarin, and 7-hydroxycoumarin metabolite was extracted in chloroform. Following drying under nitrogen, the extract was resuspended in methanol (10-100 microliters) and an aliquot of 5-20 microliters was directly injected into a C8 Nova-Pak column. Isocratic separation of hydroxycoumarin was achieved using a mobile phase consisting of methanol:1% acetic acid, 35:65, v/v, pH 3.5, at a flow rate of 1 ml/min. Following chromatographic separation, samples were derivatized with 1.0 N NaOH prior to fluorescent measurements. The detection limit for 7-hydroxycoumarin was less than 1 pmol, with a mean recovery from the incubates of 96.4 +/- 2.3%. This HPLC-fluorometric method was linear up to at least 400 pmol of 7-hydroxycoumarin and could accurately detect metabolite formation in incubates containing control liver microsomes with less than 0.05 microgram total protein. The method also allowed determinations of cytochrome P450-dependent function in extrahepatic tissues of rats, including individual segments of gastrointestinal epithelium and brain, as well as in cultured cells, such as HepG2 cells, in which microsomal protein yield is very small. The wide range of linearity afforded by this method allows a reliable estimation of cytochrome P450-dependent function in samples containing varying concentrations of protein.     

A direct and sensitive determination of histamine in acid-deproteinized biological samples by high-performance liquid chromatography

A convenient method for the routine measurement of histamine (HA) in biological samples was developed. This method does not require any preliminary purification or concentration of HA, and features high sensitivity, specificity, and reliability. The method consists of the direct application of the acid-deproteinized sample to high-performance liquid chromatography on a sulfonated polystyrene column with detection by means of a postcolumn fluorogenic reaction with o-phthaladehyde. The detection limit was found to be 0.1 pmol (signal-to-noise ratio = 3). The coefficient of variation for measurements of 10 pmol of standard histamine was 1.1%. Each chromatography takes only 10 min and therefore more than 50 samples can be measured in a day. The high sensitivity of the method allows it to be applied even to samples of very low HA concentration such as human plasma without any procedure for concentration of the sample, and further, only 0.1 ml of the sample is necessary for determination. The method was applied to compare the HA levels of the whole blood and plasma of man and various animals. Applications of the method to the supernatant of rat peritoneal mast cell incubates and to extracts of mouse brain and stomach are also described.     

Purification of proctolin-binding proteins from the foregut of the insect Blaberus craniifer.

A membrane protein that specifically binds the insect neuropeptide proctolin was purified using standard chromatography from cockroach foregut membranes. Proctolin-binding sites were efficiently solubilized with either the nonionic detergent digitonin or the zwitterionic detergent Chaps, as indicated by the specific binding of 3H-proctolin to solubilized samples. A solubilized sample obtained from 1600 foregut membranes was subjected to a five-step chromatographic purification including chromatofocusing, anion-exchange and size-exclusion chromatographies. The final size-exclusion separation resulted in the isolation of approximately 100 pmol of purified proctolin-binding proteins, eluting as a single peak at approximately 74 kDa. Analysis of the purified sample using SDS/PAGE and silver staining showed two bands at 80 kDa and 76 kDa. Densitometric analysis of the gel indicated that each band contained approximately 7-8 microg of protein, suggesting that one band corresponds to the proctolin-binding activity. Proctolin-binding proteins were thus purified 1800-fold using standard chromatography.     

A sensitive capillary GC assay for the determination of sparteine oxidation products in microsomal fractions of human liver

A sensitive method for the assay of sparteine oxidase activity in vitro by microsomal fractions of human liver is described. The activity of sparteine oxidase was assessed by the formation of 2- and 5-dehydrosparteines, which were estimated by capillary gas chromatography with N2-FID detection. The limit of detection of the two metabolites, 2- and 5-dehydrosparteine, was 10 pmol (2.3 ng) per sample. Sparteine oxidase activity was linear with microsomal protein concentration ranging from 25 to 200 ug and with incubation times between 5 and 60 minutes. Omission of NADPH on incubation under an atmosphere of carbon monoxide inhibited formation of both metabolites, thus indicating that aforementioned metabolites arise in reaction catalyzed by cytochrome P-450. In three liver samples from humans classified as extensive (EM) metabolizers the formation of 2- and 5-dehydrosparteines was observed, 2-dehydrosparteine being the major metabolite. In these samples sparteine oxidase activity was characterised by Vmax = 136 +/- 53 pmol/min/mg and Km = 44 +/- 12 microM for 2-dehydrosparteine formation. For 5-dehydrosparteine formation the following values were obtained: Vmax = 57 +/- 18 pmol/min/mg and Km = 42 +/- 26 microM. In a liver sample from a poor metabolizer (PM) only the formation of 2-dehydrosparteine was detected with the method of analysis used. In this sample a great increase in Km (Km PM = 3033 microM) was noted, while Vmax was very similar to those obtained for 2-dehydrosparteine formation in EM subjects (Vmax PM = 147 pmol min/mg).