Comparison of the quantification of caffeine in human plasma by gas chromatography and ELISA

In the present study we evaluated the precision of the ELISA method to quantify caffeine in human plasma and compared the results with those obtained by gas chromatography. A total of 58 samples were analyzed by gas chromatography using a nitrogen-phosphorus detector and routine techniques. For the ELISA test, the samples were diluted to obtain a concentration corresponding to 50% of the absorbance of the standard curve. To determine whether the proximity between the I50 of the standard curve and that of the sample would bring about a more precise result, the samples were divided into three blocks according to the criterion of difference, in modulus, of the I50 of the standard curve and of the I50 of the sample. The samples were classified into three groups. The first was composed of 20 samples with I50 up to 1.5 ng/ml, the second consisted of 21 samples with I50 ranging from 1.51 to 3 ng/ml, and the third of 17 samples with I50 ranging from 3.01 to 13 ng/ml. The determination coefficient (R² = 0.999) showed that the data obtained by gas chromatography represented a reliable basis. The results obtained by ELISA were also reliable, with an estimated Pearson correlation coefficient of 0.82 between the two methods. This coefficient for the different groups (0.88, 0.79 and 0.49 for groups 1, 2 and 3, respectively) showed greater reliability for the test with dilutions closer to I50.

Effect of perimuscular injection of Bothrops jararacussu venom on plasma creatine kinase levels in mice: influence of dose and volume

The effect of dose and volume of a perimuscular injection of Bothrops jararacussu venom on myonecrosis of skeletal muscle was studied in mice. An increase of the venom dose (0.25 to 2.0 µg/g) at a given volume (50 µl) resulted in an increase in plasma creatine kinase (CK) levels 2 h after injection. Plasma CK activity increased from the basal level of 129.27 ± 11.83 (N = 20) to 2392.80 ± 709.43 IU/l (N = 4) for the 1.0 µg/g dose. Histological analysis of extensor digitorum longus muscle 4 h after injection showed lesion of peripheral muscle fibers, disorganization of the bundles or the complete degeneration of muscle fibers. These lesions were more extensive when higher doses were injected. Furthermore, an increase in volume (12.5 to 100 µl) by dilution of a given dose (0.5 µg/g) also increased plasma CK levels from 482.31 ± 122.79 to 919.07 ± 133.33 IU/l (N = 4), respectively. These results indicate that care should be taken to standardize volumes and sites of venom injections.

Role of nitric oxide of the median preoptic nucleus (MnPO) in the alterations of salivary flow, arterial pressure and heart rate induced by injection of pilocarpine into the MnPO and intraperitoneally

We investigated the effect of L-NAME, a nitric oxide (NO) inhibitor and sodium nitroprusside (SNP), an NO-donating agent, on pilocarpine-induced alterations in salivary flow, mean arterial blood pressure (MAP) and heart rate (HR) in rats. Male Holtzman rats (250-300 g) were implanted with a stainless steel cannula directly into the median preoptic nucleus (MnPO). Pilocarpine (10, 20, 40, 80, 160 µg) injected into the MnPO induced an increase in salivary secretion (P<0.01). Pilocarpine (1, 2, 4, 8, 16 mg/kg) ip also increased salivary secretion (P<0.01). Injection of L-NAME (40 µg) into the MnPO prior to pilocarpine (10, 20, 40, 80, 160 µg) injected into the MnPO or ip (1, 2, 4, 8, 16 mg/kg) increased salivary secretion (P<0.01). SNP (30 µg) injected into the MnPO or ip prior to pilocarpine attenuated salivary secretion (P<0.01). Pilocarpine (40 µg) injection into the MnPO increased MAP and decreased HR (P<0.01). Pilocarpine (4 mg/kg body weight) ip produced a decrease in MAP and an increase in HR (P<0.01). Injection of L-NAME (40 µg) into the MnPO prior to pilocarpine potentiated the increase in MAP and reduced HR (P<0.01). SNP (30 µg) injected into the MnPO prior to pilocarpine attenuated (100%) the effect of pilocarpine on MAP, with no effect on HR. Administration of L-NAME (40 µg) into the MnPO potentiated the effect of pilocarpine injected ip. SNP (30 µg) injected into the MnPO attenuated the effect of ip pilocarpine on MAP and HR. The present study suggests that in the rat MnPO 1) NO is important for the effects of pilocarpine on salivary flow, and 2) pilocarpine interferes with blood pressure and HR (side effects of pilocarpine), that is attenuated by NO.

The biological effects of high-pressure gas on the yeast transcriptome

The aim of the present study was to examine the feasibility of DNA microarray technology in an attempt to construct an evaluation system for determining gas toxicity using high-pressure conditions, as it is well known that pressure increases the concentration of a gas. As a first step, we used yeast (Saccharomyces cerevisiae) as the indicator organism and analyzed the mRNA expression profiles after exposure of yeast cells to nitrogen gas. Nitrogen gas was selected as a negative control since this gas has low toxicity. Yeast DNA microarray analysis revealed induction of genes whose products were localized to the membranes, and of genes that are involved in or contribute to energy production. Furthermore, we found that nitrogen gas significantly affected the transport system in the cells. Interestingly, nitrogen gas also resulted in induction of cold-shock responsive genes. These results suggest the possibility of applying yeast DNA microarray to gas bioassays up to 40 MPa. We therefore think that "bioassays" are ideal for use in environmental control and protection studies.

Delayed Schwann cell and oligodendrocyte remyelination after ethidium bromide injection in the brainstem of Wistar rats submitted to streptozotocin diabetogenic treatment

Schwann cell disturbance followed by segmental demyelination in the peripheral nervous system occurs in diabetic patients. Since Schwann cell and oligodendrocyte remyelination in the central nervous system is a well-known event in the ethidium bromide (EB) demyelinating model, the aim of this investigation was to determine the behavior of both cell types after local EB injection into the brainstem of streptozotocin diabetic rats. Adult male Wistar rats received a single intravenous injection of streptozotocin (50 mg/kg) and were submitted 10 days later to a single injection of 10 µL 0.1% (w/v) EB or 0.9% saline solution into the cisterna pontis. Ten microliters of 0.1% EB was also injected into non-diabetic rats. The animals were anesthetized and perfused through the heart 7 to 31 days after EB or saline injection and brainstem sections were collected and processed for light and transmission electron microscopy. The final balance of myelin repair in diabetic and non-diabetic rats at 31 days was compared using a semi-quantitative method. Diabetic rats presented delayed macrophage activity and lesser remyelination compared to non-diabetic rats. Although oligodendrocytes were the major remyelinating cells in the brainstem, Schwann cells invaded EB-induced lesions, first appearing at 11 days in non-diabetic rats and by 15 days in diabetic rats. Results indicate that short-term streptozotocin-induced diabetes hindered both oligodendrocyte and Schwann cell remyelination (mean remyelination scores of 2.57 ± 0.77 for oligodendrocytes and 0.67 ± 0.5 for Schwann cells) compared to non-diabetic rats (3.27 ± 0.85 and 1.38 ± 0.81, respectively).

Mechanisms underlying gas exchange alterations in an experimental model of pulmonary embolism

The aim of the present study was to determine the ventilation/perfusion ratio that contributes to hypoxemia in pulmonary embolism by analyzing blood gases and volumetric capnography in a model of experimental acute pulmonary embolism. Pulmonary embolization with autologous blood clots was induced in seven pigs weighing 24.00 ± 0.6 kg, anesthetized and mechanically ventilated. Significant changes occurred from baseline to 20 min after embolization, such as reduction in oxygen partial pressures in arterial blood (from 87.71 ± 8.64 to 39.14 ± 6.77 mmHg) and alveolar air (from 92.97 ± 2.14 to 63.91 ± 8.27 mmHg). The effective alveolar ventilation exhibited a significant reduction (from 199.62 ± 42.01 to 84.34 ± 44.13) consistent with the fall in alveolar gas volume that effectively participated in gas exchange. The relation between the alveolar ventilation that effectively participated in gas exchange and cardiac output (V Aeff/Q ratio) also presented a significant reduction after embolization (from 0.96 ± 0.34 to 0.33 ± 0.17 fraction). The carbon dioxide partial pressure increased significantly in arterial blood (from 37.51 ± 1.71 to 60.76 ± 6.62 mmHg), but decreased significantly in exhaled air at the end of the respiratory cycle (from 35.57 ± 1.22 to 23.15 ± 8.24 mmHg). Exhaled air at the end of the respiratory cycle returned to baseline values 40 min after embolism. The arterial to alveolar carbon dioxide gradient increased significantly (from 1.94 ± 1.36 to 37.61 ± 12.79 mmHg), as also did the calculated alveolar (from 56.38 ± 22.47 to 178.09 ± 37.46 mL) and physiological (from 0.37 ± 0.05 to 0.75 ± 0.10 fraction) dead spaces. Based on our data, we conclude that the severe arterial hypoxemia observed in this experimental model may be attributed to the reduction of the V Aeff/Q ratio. We were also able to demonstrate that V Aeff/Q progressively improves after embolization, a fact attributed to the alveolar ventilation redistribution induced by hypocapnic bronchoconstriction.

Effect of saline infusion for the maintenance of blood volume on pulmonary gas exchange during temporary abdominal aortic occlusion

We analyzed the effects of saline infusion for the maintenance of blood volume on pulmonary gas exchange in ischemia-reperfusion syndrome during temporary abdominal aortic occlusion in dogs. We studied 20 adult mongrel dogs weighing 12 to 23 kg divided into two groups: ischemia-reperfusion group (IRG, N = 10) and IRG submitted to saline infusion for the maintenance of mean pulmonary arterial wedge pressure between 10 and 20 mmHg (IRG-SS, N = 10). All animals were anesthetized and maintained on spontaneous ventilation. After obtaining baseline measurements, occlusion of the supraceliac aorta was performed by the inflation of a Fogarty catheter. After 60 min of ischemia, the balloon was deflated and the animals were observed for another 60 min of reperfusion. The measurements were made at 10 and 45 min of ischemia, and 5, 30, and 60 min of reperfusion. Pulmonary gas exchange was impaired in the IRG-SS group as demonstrated by the increase of the alveolar-arterial oxygen difference (21 ± 14 in IRG-SS vs 11 ± 8 in IRG after 60 min of reperfusion, P = 0.004 in IRG-SS in relation to baseline values) and the decrease of oxygen partial pressure in arterial blood (58 ± 15 in IRG-SS vs 76 ± 15 in IRG after 60 min of reperfusion, P = 0.001 in IRG-SS in relation to baseline values), which was correlated with the highest degree of pulmonary edema in morphometric analysis (0.16 ± 0.06 in IRG-SS vs 0.09 ± 0.04 in IRG, P = 0.03 between groups). There was also a smaller ventilatory compensation of metabolic acidosis after the reperfusion. We conclude that infusion of normal saline worsened the gas exchange induced by pulmonary reperfusion injury in this experimental model.

Consequences of cerebroventricular insulin injection on renal sodium handling in rats: effect of inhibition of central nitric oxide synthase

In the present study, we investigated the effects of acute intracerebroventricular (icv) insulin administration on central mechanisms regulating urinary sodium excretion in simultaneously centrally NG-nitro-L-arginine methylester (L-NAME)-injected unanesthetized rats. Male Wistar-Hannover rats were randomly assigned to one of five groups: a) icv 0.15 M NaCl-injected rats (control, N = 10), b) icv dose-response (1.26, 12.6 and 126 ng/3 µL) insulin-injected rats (N = 10), c) rats icv injected with 60 µg L-NAME in combination with NaCl (N = 10) or d) with insulin (N = 10), and e) subcutaneously insulin-injected rats (N = 5). Centrally administered insulin produced an increase in urinary output of sodium (NaCl: 855.6 ± 85.1 Δ%/min; 126 ng insulin: 2055 ± 310.6 Δ%/min; P = 0.005) and potassium (NaCl: 460.4 ± 100 Δ%/min; 126 ng insulin: 669.2 ± 60.8 Δ%/min; P = 0.025). The urinary sodium excretion response to icv 126 ng insulin microinjection was significantly attenuated by combined administration of L-NAME (126 ng insulin: 1935 ± 258.3 Δ%/min; L-NAME + 126 ng insulin: 582.3 ± 69.6 Δ%/min; P = 0.01). Insulin-induced natriuresis occurred by increasing post-proximal sodium excretion, despite an unchanged glomerular filtration rate. Although the rationale for decreased urinary sodium excretion induced by combined icv L-NAME and insulin administration is unknown, it is tempting to suggest that perhaps one of the efferent signals triggered by insulin in the CNS may be nitrergic in nature.

Serum metabolic profiling of human gastric cancer based on gas chromatography/mass spectrometry

Research on molecular mechanisms of carcinogenesis plays an important role in diagnosing and treating gastric cancer. Metabolic profiling may offer the opportunity to understand the molecular mechanism of carcinogenesis and help to non-invasively identify the potential biomarkers for the early diagnosis of human gastric cancer. The aims of this study were to explore the underlying metabolic mechanisms of gastric cancer and to identify biomarkers associated with morbidity. Gas chromatography/mass spectrometry (GC/MS) was used to analyze the serum metabolites of 30 Chinese gastric cancer patients and 30 healthy controls. Diagnostic models for gastric cancer were constructed using orthogonal partial least squares discriminant analysis (OPLS-DA). Acquired metabolomic data were analyzed by the nonparametric Wilcoxon test to find serum metabolic biomarkers for gastric cancer. The OPLS-DA model showed adequate discrimination between cancer and non-cancer cohorts while the model failed to discriminate different pathological stages (I-IV) of gastric cancer patients. A total of 44 endogenous metabolites such as amino acids, organic acids, carbohydrates, fatty acids, and steroids were detected, of which 18 differential metabolites were identified with significant differences. A total of 13 variables were obtained for their greatest contribution in the discriminating OPLS-DA model [variable importance in the projection (VIP) value >1.0], among which 11 metabolites were identified using both VIP values (VIP >1) and the Wilcoxon test. These metabolites potentially revealed perturbations of glycolysis and of amino acid, fatty acid, cholesterol, and nucleotide metabolism of gastric cancer patients. These results suggest that gastric cancer serum metabolic profiling has great potential in detecting this disease and helping to understand its metabolic mechanisms.

Cardiovascular effects of the intracerebroventricular injection of adrenomedullin: roles of the peripheral vasopressin and central cholinergic systems

Our objective was to investigate in conscious Sprague-Dawley (6-8 weeks, 250-300 g) female rats (N = 7 in each group) the effects of intracerebroventricularly (icv) injected adrenomedullin (ADM) on blood pressure and heart rate (HR), and to determine if ADM and calcitonin gene-related peptide (CGRP) receptors, peripheral V1 receptors or the central cholinergic system play roles in these cardiovascular effects. Blood pressure and HR were observed before and for 30 min following drug injections. The following results were obtained: 1) icv ADM (750 ng/10 µL) caused an increase in both blood pressure and HR (DMAP = 11.8 ± 2.3 mmHg and ΔHR = 39.7 ± 4.8 bpm). 2) Pretreatment with a CGRP receptor antagonist (CGRP8-37) and ADM receptor antagonist (ADM22-52) blocked the effect of central ADM on blood pressure and HR. 3) The nicotinic receptor antagonist mecamylamine (25 µg/10 µL, icv) and the muscarinic receptor antagonist atropine (5 µg/10 µL, icv) prevented the stimulating effect of ADM on blood pressure. The effect of ADM on HR was blocked only by atropine (5 µg/10 µL, icv). 4) The V1 receptor antagonist [β-mercapto-β-β-cyclopentamethylenepropionyl¹, O-me-Tyr²,Arg8]-vasopressin (V2255; 10 µg/kg), that was applied intravenously, prevented the effect of ADM on blood pressure and HR. This is the first study reporting the role of specific ADM and CGRP receptors, especially the role of nicotinic and muscarinic central cholinergic receptors and the role of peripheral V1 receptors in the increasing effects of icv ADM on blood pressure and HR.

Influence of exercise modality on agreement between gas exchange and heart rate variability thresholds

The main purpose of this study was to investigate the level of agreement between the gas exchange threshold (GET) and heart rate variability threshold (HRVT) during maximal cardiopulmonary exercise testing (CPET) using three different exercise modalities. A further aim was to establish whether there was a 1:1 relationship between the percentage heart rate reserve (%HRR) and percentage oxygen uptake reserve (%V˙O2R) at intensities corresponding to GET and HRVT. Sixteen apparently healthy men 17 to 28 years of age performed three maximal CPETs (cycling, walking, and running). Mean heart rate and V˙O2 at GET and HRVT were 16 bpm (P<0.001) and 5.2 mL·kg-1·min-1 (P=0.001) higher in running than cycling, but no significant differences were observed between running and walking, or cycling and walking (P>0.05). There was a strong relationship between GET and HRVT, with R2 ranging from 0.69 to 0.90. A 1:1 relationship between %HRR and %V˙O2R was not observed at GET and HRVT. The %HRR was higher during cycling (GET mean difference=7%; HRVT mean difference=11%; both P<0.001), walking (GET mean difference=13%; HRVT mean difference=13%; both P<0.001), or running (GET mean difference=11%; HRVT mean difference=10%; both P<0.001). Therefore, using HRVT to prescribe aerobic exercise intensity appears to be valid. However, to assume a 1:1 relationship between %HRR and %V˙O2R at HRVT would probably result in overestimation of the energy expenditure during the bout of exercise.

Evaluation of noni (Morinda citrifolia) volatile profile by dynamic headspace and gas chromatography-mass spectrometry

Noni is a fruit that has interested the scientific community due to its medicinal and functional activities. Different products that contain noni are already in the market, but their consumption could be impaired by their distinctive unpleasant aroma and flavor. The aim of this work was to evaluate the noni pulp volatile profile by dynamic headspace and gas chromatography-mass spectrometry. Thirty seven volatile compounds were detected, mainly alcohols (63.3%), esters (26.9%), cetones (7.4%), and acids (1.2%).

(E)-2-Nonenal determination in brazilian beers using headspace solid-phase microextraction and gas chromatographic coupled mass spectrometry (HS-SPME-GC-MS)

(E)-2-nonenal is considered an important off-flavor of beer, related to the flavor of beer staling. In this study, a new method for determination of (E)-2-nonenal in beer using headspace solid-phase microextraction and gas chromatographic coupled mass spectrometry (HS-SPME-GC-MS) was developed and applied in Brazilian beer samples. The extractions were carried out in CAR-PDMS (carboxen-polydimethylsiloxane) fiber and the best results were found with 15 minutes of equilibrium and 90 minutes of extraction at 50 °C. The method was linear in the range from 0.02 to 4.0 μg.L-1 with correlation coefficient of 0.9994. The limits of detection and quantification were 0.01 and 0.02 μg.L-1, respectively. 96.5% of recovery and 4% precision (RSD) were obtained in the fortification of beer samples with 2.0 μg.L-1 of (E)-2-nonenal. The developed method proved to be simple, efficient and highly sensitive to the determination of this analyte being easily applied in the quality control of the brewery. (E)-2-nonenal was found in all beer samples analyzed with levels between 0.17 and 0.42 μg.L-1.

Characterization and classification of pequi trees (Caryocar brasiliense Camb.) based on the profile of volatile constituents using headspace solid-phase microextraction - gas chromatography - mass spectrometry and multivariate analysis

In order to determine the variability of pequi tree (Caryocar brasiliense Camb.) populations, volatile compounds from fruits of eighteen trees representing five populations were extracted by headspace solid-phase microextraction and analyzed by gas chromatography-mass spectrometry. Seventy-seven compounds were identified, including esters, hydrocarbons, terpenoids, ketones, lactones, and alcohols. Several compounds had not been previously reported in the pequi fruit. The amount of total volatile compounds and the individual compound contents varied between plants. The volatile profile enabled the differentiation of all of the eighteen plants, indicating that there is a characteristic profile in terms of their origin. The use of Principal Component Analysis and Cluster Analysis enabled the establishment of markers (dendrolasin, ethyl octanoate, ethyl 2-octenoate and β-cis-ocimene) that discriminated among the pequi trees. According to the Cluster Analysis, the plants were classified into three main clusters, and four other plants showed a tendency to isolation. The results from multivariate analysis did not always group plants from the same population together, indicating that there is greater variability within the populations than between pequi tree populations.