Effect of collection, transport, processing and storage of blood specimens on the activity of lysosomal enzymes in plasma and leukocytes

This study was designed to evaluate the effect of different conditions of collection, transport and storage on the quality of blood samples from normal individuals in terms of the activity of the enzymes ß-glucuronidase, total hexosaminidase, hexosaminidase A, arylsulfatase A and ß-galactosidase. The enzyme activities were not affected by the different materials used for collection (plastic syringes or vacuum glass tubes). In the evaluation of different heparin concentrations (10% heparin, 5% heparin, and heparinized syringe) in the syringes, it was observed that higher doses resulted in an increase of at least 1-fold in the activities of ß-galactosidase, total hexosaminidase and hexosaminidase A in leukocytes, and ß-glucuronidase in plasma. When the effects of time and means of transportation were studied, samples that had been kept at room temperature showed higher deterioration with time (72 and 96 h) before processing, and in this case it was impossible to isolate leukocytes from most samples. Comparison of heparin and acid citrate-dextrose (ACD) as anticoagulants revealed that ß-glucuronidase and hexosaminidase activities in plasma reached levels near the lower normal limits when ACD was used. In conclusion, we observed that heparin should be used as the preferable anticoagulant when measuring these lysosomal enzyme activities, and we recommend that, when transport time is more than 24 h, samples should be shipped by air in a styrofoam box containing wet ice.

Characterization of mannose-binding lectin plasma levels and genetic polymorphisms in HIV-1-infected individuals

INTRODUCTION: The present study investigated the association between mannose-binding lectin (MBL) gene polymorphism and serum levels with infection by HIV-1. METHODS: Blood samples (5mL) were collected from 97 HIV-1-infected individuals resident in Belém, State of Pará, Brazil, who attended the Special Outpatient Unit for Infections and Parasitic Diseases (URE-DIPE). CD4+ T-lymphocyte count and plasma viral load were quantified. A 349bp fragment of exon 1 of the MBL was amplified via PCR, using genomic DNA extracted from controls and HIV-1-infected individuals, following established protocols. MBL plasma levels of the patients were quantified using an enzyme immunoassay kit. RESULTS: Two alleles were observed: MBL*O, with a frequency of 26.3% in HIV-1-infected individuals; and the wild allele MBL*A (73.7%). Similar frequencies were observed in the control group (p > 0.05). Genotype frequencies were distributed according to the Hardy-Weinberg equilibrium in both groups. Mean MBL plasma levels varied by genotype, with statistically significant differences between the AA and AO (p < 0.0001), and AA and OO (p < 0.001) genotypes, but not AO and OO (p = 0.17). Additionally, CD4+ T-lymphocytes and plasma viral load levels did not differ significantly by genotype (p > 0.05). CONCLUSIONS: The results of this study do not support the hypothesis that MBL gene polymorphism or low plasma MBL concentrations might have a direct influence on HIV-1 infection, although a broader study involving a large number of patients is needed.

A malaria merozoite surface protein (MSP1)-structure, processing and function

Merozoite surface protein-1 (MSP-1, also referred to as P195, PMMSA or MSA 1) is one of the most studied of all malaria proteins. The proteins. The protein is found in all malaria species investigated and structural studies on the gene indicate that parts of the molecule are well-conserved. Studies on Plasmodium falciparum have shown that the protein is in a processed form on the merozoite surface, a result of proteolytic cleavage of the large percursor molecule. Recent studies have identified some of these cleavage sites. During invasion of the new red cell most of the MSP1 molecule is shed from the parasite surface except for a small C-terminal fragment which can be detected in ring stages. Analysis of the structure of this fragment suggests that it contains two growth factor-like domains that may have a functional role.

Effects of erosion and deposition on particle size distribution of deposited farmland soils on the chinese loess plateau

Particle size distribution (PSD) in the soil profile is strongly related to erosion, deposition, and physical and chemical processes. Water cycling and plant growth are also affected by PSD. Material sedimented upstream of the dam constructions formed large areas of deposited farmland (DF) soils on the Chinese Loess Plateau (CLP), which has been the site of the most severe soil erosion in the world. Two DFs without tillage on the CLP were chosen to study the combined effect of erosion and check dams on PSD. Eighty-eight layers (each 10 cm thick) of filled deposited farmland (FDF) soils and 22 layers of silting deposited farmland (SDF) soils of each studied soil profile were collected and 932 soil samples were investigated using laser granulometry. The particle sizes were stratified in both DFs based on soil properties and erosion resistance. The obtained results of clay and silt fractions showed similar horizontal distribution, indicating parallel characteristics of erosion and deposition processes. Fine sand represented the largest fraction, suggesting the preferential detachment of this fraction. The most erodible range of particle sizes was 0.25-0.5 mm, followed by 0.2-0.25 mm in the studied soil profiles. The correlation between particle size and soil water contents tended to increase with increasing water contents in FDF. Due to the abundant shallow groundwater, the relationship between particle size and soil water content in SDF was lost. Further studies on PSD in the DF area are needed to enhance the conservation management of soil and water resources in this region.

Changes in the isoflavone profile and in the chemical composition of tempeh during processing and refrigeration

The objective of this work was to analyze changes in the isoflavone profile, determined by high performance liquid chromatography, at different processing stages and after refrigeration of tempeh. For tempeh production, clean soybean grains from cultivars BR 36 (low isoflavone content) and IAS 5 (high) were dehulled, and the separated cotyledons were hydrated and then cooked in boiling water for 30 min. Spores of the fungus Rhizopus microsporus var. oligosporus were inoculated in the cooked and cooled cotyledons, and incubated at 32ºC for 6, 12, 18, and 24 hours in perforated polypropylene bags, for fermentation. The resulting tempeh was stored at 4ºC for 6, 12, 18, and 24 hours. After 24-hour fermentation, isoflavone glucosides were 50% reduced, and the aglycone forms in the tempeh from both cultivars was increased. The malonyl forms reduced 83% after cooking. Less than 24 hours of refrigeration did not affect the isoflavone profile of tempeh from either cultivar, which is a good indicator of its quality. The tempeh maintains the high and low isoflavone content of the cultivars, which indicates that cultivar differences in this trait should be considered when processing tempeh.

Maintenance requirement and deposition efficiency of lysine in pigs

The objective of this work was to determine the maintenance requirement and the deposition efficiency of lysine in growing pigs. It was used the incomplete changeover experimental design, with replicates over time. Twelve castrated pigs with average body weight (BW) of 52±2 kg were kept in metabolism crates with a controlled temperature of 22ºC. The diets were formulated to supply 30, 50, 60, and 70% of the expected requirements of standardized lysine, and provided at 2.6 times the energy requirements for maintenance. The trial lasted 24 days and was divided into two periods of 12 days: seven days for animal adaptation to the diet and five days for sample collection. The increasing content of lysine in the diet did not affect dry matter intake of the pigs. The amount of nitrogen excreted was 47% of the nitrogen intake, of which 35% was excreted through feces and 65% through urine. The estimated endogenous losses of lysine were 36.4 mg kg-1 BW0.75. The maintenance requirement of lysine for pigs weighing around 50 kg is 40.4 mg kg-1 BW0.75, and the deposition efficiency of lysine is 90%.

Calibration of angles of nozzles and deposition of an axial-flow sprayer on dwarf cashew

The study aimed to determine an optimum angle for the nozzles axial-flow sprayers a deposition for better vertical distribution focused on cashew. In laboratory tests were conducted adjusting the angle of the nozzle axial-flow sprayers. The experimental design was randomized blocks in a 2x3 factorial with four replications. The treatment for this test were two settings (with and without the adjustment of the angles of the nozzles ) and tree application volumes 273, 699 and 954 L ha-¹.The study was conducted in an orchard of dwarf cashew, with eight years of age. It was concluded that the volumetric distribution profile showed better vertical distribution uniformity when the angles of the nozzles were regulated for the canopy, the adjustment of the angles of the nozzles for the canopy provided greater deposition of droplets, the increased volume of application resulted in higher depositions in the leaves of the crop.

Clinical, hematological, total plasma protein and fibrinogen parameters of magellanic penguins ( Spheniscus magellanicus ) pre- and post-rehabilitation

Abstract: Magellanic penguins (Spheniscus magellanicus) usually arrive in poor body conditions at Brazilian beaches during the winter. Hematology provides valuable information about clinical and immunity status of the animals. The aims of this study were to determine the hematologic, total plasma protein (TPP) and fibrinogen profiles of young and adult magellanic penguins in PROAMAR and CETAS-SC, relating these results with the state of health and survival possibility of the animals. In Paraná 14 animals were evaluated in pre and eight in post-rehabilitation and 29 animals were evaluated in Santa Catarina after rehabilitation. Before rehabilitation, all animals showed weakness. In hematological exams of these animals, we found that anemia was present in 83% of the penguins that died and 50% of those which survived. The heterophils/lymphocytes (H/L) ratio was 3.87±0.57 in animals that died, significantly higher than the average of 2.20±0.30 for animals that survived. These two parameters are useful to assess the survival possibility of animals to rehabilitation. The body condition score was positively correlated with hematocrit and TPP, and negatively correlated with H/L ratio. After rehabilitation, the values were similar to other animals of the family Spheniscidae, with averages ranging from 1.64 to 1.90x106 erythrocytes/μL; 43.38 to 48.80% of hematocrit; 12.45 to 13.52g/dL of hemoglobin; 8,684 to 14,011 leukocytes/μL; 4,767 to 8,041 heterophils/μL; 3,215 to 4,951 lymphocytes/μL; 95 to 655 eosinophils/μl; 179.8 to 277.9 monocytes/μL; 141 to 184.9 basophils/μL; and 1.26 to 1.74 of H/L ratio. These parameters can therefore be used as reference values and release parameters for young and adult Magellanic penguins in captivity on the rehabilitation centers.

Plasma clearance and biodistribution of oxidatively modified 99mTc-ß-VLDL in rabbits

The biodistribution and removal from plasma (measured as fractional clearance rate, FCR, per hour) of native and oxidatively modified 99mtechnetium-labeled ß-very low density lipoprotein (99mTc-ß-VLDL) were investigated in hypercholesterolemic (HC) and control (C) three-month old New Zealand rabbits. The intracellular accumulation of ß-VLDL labeled with 99mTc was studied in vitro in THP-1 cells and monocyte-derived macrophages isolated from rabbits. After intravenous injection into C rabbits, copper-oxidized ß-VLDL (99mTc-ox-ß-VLDL) was cleared from the circulation faster (0.362 ± 0.070/h) than native ß-VLDL (99mTc-nat-ß-VLDL, 0.241 ± 0.070/h). In contrast, the FCR of 99mTc-ox-ß-VLDL in HC rabbits was lower (0.100 ± 0.048/h) than that of 99mTc-nat-ß-VLDL (0.163 ± 0.043/h). The hepatic uptake of radiolabeled lipoproteins was lower in HC rabbits (0.114 ± 0.071% injected dose/g tissue for 99mTc-nat-ß-VLDL and 0.116 ± 0.057% injected dose/g tissue for 99mTc-ox-ß-VLDL) than in C rabbits (0.301 ± 0.113% injected dose/g tissue for 99mTc-nat-ß-VLDL and 0.305 ± 0.149% injected dose/g tissue for 99mTc-ox-ß-VLDL). The uptake of 99mTc-nat-ß-VLDL and 99mTc-ox-ß-VLDL by atherosclerotic aorta lesions isolated from HC rabbits (99mTc-nat-ß-VLDL: 0.033 ± 0.012% injected dose/g tissue and 99mTc-ox-ß-VLDL: 0.039 ± 0.017% injected dose/g tissue) was higher in comparison to that of non-atherosclerotic aortas from C rabbits (99mTc-nat-ß-VLDL: 0.023 ± 0.010% injected dose/g tissue and 99mTc-ox-ß-VLDL: 0.019 ± 0.010% injected dose/g tissue). However, 99mTc-nat-ß-VLDL and 99mTc-ox-ß-VLDL were taken up by atherosclerotic lesions at similar rates. In vitro studies showed that both monocyte-derived macrophages isolated from rabbits and THP-1 macrophages significantly internalized more 99mTc-ox-ß-VLDL than 99mTc-nat-ß-VLDL. These results indicate that in cholesterol-fed rabbits 99mTc-ox-ß-VLDL is slowly cleared from plasma and accumulates in atherosclerotic lesions. However, although the extent of in vitro uptake of 99mTc-ox-ß-VLDL by macrophages was high, the in vivo accumulation of this radiolabeled lipoprotein by atherosclerotic lesions did not differ from that of 99mTc-nat-ß-VLDL.

Plasma testosterone and 11-ketotestosterone levels of male pacu Piaractus mesopotamicus (Cypriniformes, Characidae)

The levels of testosterone (T) and 11-ketotestosterone (11-KT) of the South American pacu Piaractus mesopotamicus were determined by radioimmunoassay during two stages of the reproductive cycle, i.e., resting and maturation, and the gonadosomatic index (GSI) was calculated. The highest levels of T and 11-KT were reached during the maturation stage (T = 2400 ± 56 pg/ml; 11-KT = 2300 ± 60 pg/ml) and lower levels were maintained during the resting period. The rise in androgen levels occurred with the appearance of spermatozoa in the maturation stage, when GSI was highest

Association of apolipoprotein E polymorphism with plasma lipids and Alzheimer's disease in a Southern Brazilian population

Apolipoprotein E (protein: apo E; gene: APOE) plays an important role in the multifactorial etiology of both Alzheimer's disease (AD) and lipid level concentrations. The polymerase chain reaction (PCR) was used to investigate the APOE gene polymorphism in 446 unrelated Caucasians, among them 23 AD patients, and 100 Afro-Brazilians living in Porto Alegre, Brazil. The frequencies of the APOE*2, APOE*3 and APOE*4 alleles were 0.075, 0.810 and 0.115 in Caucasians and 0.075, 0.700 and 0.225 in Afro-Brazilians, respectively (c2 = 8.72, P = 0.013). A highly significant association was observed between the APOE*4 allele and AD in this population-based sample. The APOE*4 frequency in AD patients (39%) was about four times higher than in the general Caucasian population (11.5%). The influence of each of the three common APOE alleles on lipid traits was evaluated by the use of the average excess statistic. The E*2 allele is associated with lower levels of triglycerides and of total and non-HDL cholesterol in both men and women. Conversely, the E*4 allele is associated with higher levels of these traits in women only. The effect of APOE alleles was of greater magnitude in women.

Plasma free and total carnitine measured in children by tandem mass spectrometry

Free and total carnitine quantification is important as a complementary test for the diagnosis of unusual metabolic diseases, including fatty acid degradation disorders. The present study reports a new method for the quantification of free and total carnitine in dried plasma specimens by isotope dilution electrospray tandem mass spectrometry with sample derivatization. Carnitine is determined by looking for the precursor of ions of m/z = 103 of N-butylester derivative, and the method is validated by comparison with radioenzymatic assay. We obtained an inter- and intra-day assay coefficient of variation of 4.3 and 2.3, respectively. Free and total carnitine was analyzed in 309 dried plasma spot samples from children ranging in age from newborn to 14 years using the new method, which was found to be suitable for calculating reference age-related values for free and total carnitine (less than one month: 19.3 ± 2.4 and 23.5 ± 2.9; one to twelve months: 28.8 ± 10.2 and 35.9 ± 11.4; one to seven years: 30.7 ± 10.3 and 38.1 ± 11.9; seven to 14 years: 33.7 ± 11.6, and 43.1 ± 13.8 µM, respectively). No difference was found between males and females. A significant difference was observed between neonates and the other age groups. We compare our data with reference values in the literature, most of them obtained by radioenzymatic assay. However, this method is laborious and time consuming. The electrospray tandem mass spectrometry method presented here is a reliable, rapid and automated procedure for carnitine quantitation.

Effect of an aqueous extract of Scoparia dulcis on blood glucose, plasma insulin and some polyol pathway enzymes in experimental rat diabetes

The effects of an aqueous extract of the plant Scoparia dulcis (200 mg/kg) on the polyol pathway and lipid peroxidation were examined in the liver of streptozotocin adult diabetic male albino Wistar rats. The diabetic control rats (N = 6) presented a significant increase in blood glucose, sorbitol dehydrogenase, glycosylated hemoglobin and lipid peroxidation markers such as thiobarbituric acid reactive substances (TBARS) and hydroperoxides, and a significant decrease in plasma insulin and antioxidant enzymes such as glutathione peroxidase (GPx), glutathione-S-transferase (GST) and reduced glutathione (GSH) compared to normal rats (N = 6). Scoparia dulcis plant extract (SPEt, 200 mg kg-1 day-1) and glibenclamide (600 µg kg-1 day-1), a reference drug, were administered by gavage for 6 weeks to diabetic rats (N = 6 for each group) and significantly reduced blood glucose, sorbitol dehydrogenase, glycosylated hemoglobin, TBARS, and hydroperoxides, and significantly increased plasma insulin, GPx, GST and GSH activities in liver. The effect of the SPEt was compared with that of glibenclamide. The effect of the extract may have been due to the decreased influx of glucose into the polyol pathway leading to increased activities of antioxidant enzymes and plasma insulin and decreased activity of sorbitol dehydrogenase. These results indicate that the SPEt was effective in attenuating hyperglycemia in rats and their susceptibility to oxygen free radicals.

Plasma concentrations and placental immunostaining of interleukin-10 and tumornecrosis factor-α as predictors of alterations in the embryo-fetal organism and the placental development of diabetic rats

Interleukin-10 (IL-10) appears to be the key cytokine for the maintenance of pregnancy and inhibits the secretion of inflammatory cytokines such as tumor necrosis factor-α (TNF-α). However, there are no studies evaluating the profile of these cytokines in diabetic rat models. Thus, our aim was to analyze IL-10 and TNF-α immunostaining in placental tissue and their respective concentrations in maternal plasma during pregnancy in diabetic rats in order to determine whether these cytokines can be used as predictors of alterations in the embryo-fetal organism and in placental development. These parameters were evaluated in non-diabetic (control; N = 15) and Wistar rats with streptozotocin (STZ)-induced diabetes (N = 15). At term, the dams (100 days of life) were killed under anesthesia and plasma and placental samples were collected for IL-10 and TNF-α determinations by ELISA and immunohistochemistry, respectively. The reproductive performance was analyzed. Plasma IL-10 concentrations were reduced in STZ rats compared to controls (7.6 ± 4.5 vs 20.9 ± 8.1 pg/mL). The placental scores of immunostaining intensity did not differ between groups (P > 0.05). Prevalence analysis showed that the IL-10 expression followed TNF-α expression, showing a balance between them. STZ rats also presented impaired reproductive performance and reduced plasma IL-10 levels related to damage during early embryonic development. However, the increased placental IL-10 as a compensatory mechanism for the deficit of maternal regulation permitted embryo development. Therefore, the data suggest that IL-10 can be used as a predictor of changes in the embryo-fetal organism and in placental development in pregnant diabetic rats.