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- PMC7307891
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Braz J Med Biol Res. 2020; 53(8): e9268.
Published online 2020 Jun 19. doi:10.1590/1414-431X20209268
PMCID: PMC7307891
PMID: 32578717
J.A.X. Silva,1,2 A.V.P. Albertini,1,2 C.S.M. Fonseca,3 D.C.N. Silva,4 V.C.O. Carvalho,3 V.L.M. Lima,3 A. Fontes,5 E.V.L. Costa,1,2 and R.A. Nogueira1,2
Author information Article notes Copyright and License information PMC Disclaimer
Abstract
Leptospirosis is a zoonotic disease caused by bacteria of the genusLeptospira, which can cause lipid changes in theerythrocyte membrane. Optical tweezers were used to characterize rheologicalchanges in erythrocytes from patients with leptospirosis in the late stage.Biochemical methods were also used for quantification of plasma lipid,erythrocyte membrane lipid, and evaluation of liver function. Our data showedthat the mean elastic constant of erythrocytes from patients with leptospirosiswas around 67% higher than the control (healthy individuals), indicating thatpatient’s erythrocytes were less elastic. In individuals with leptospirosis,several alterations in relation to control were observed in the plasma lipids,however, in the erythrocyte membrane, only phosphatidylcholine showed asignificant difference compared to control, increasing around 41%. With respectto the evaluation of liver function of individuals with leptospirosis, there wasa significant increase in levels of alanine transaminase (154%) and aspartatetransaminase (150%), whereas albumin was 43.8% lower than control (P<0.01).The lecithin-cholesterol acyltransferase fractional activity was 3.6 times lowerin individuals with leptospirosis than in the healthy individuals (P<0.01).The decrease of the erythrocyte elasticity may be related to the changes oferythrocyte membrane phospholipids composition caused by disturbances that occurduring human leptospirosis, with phosphatidylcholine being a strong candidate inthe erythrocyte rheological changes.
Keywords: Leptospirosis, Lipid composition of membrane, Erythrocyte elasticity, Optical tweezers, Erythrocyte membrane
Introduction
Leptospirosis is a bacterial disease caused by Leptospira speciesthat are transmitted to human beings and animals through contact with soils andwater contaminated with the urine of infected animals, predominantly rodents (1). This zoonosis occurs worldwide, but theincidence is highest in the tropical regions, being a disease with a great impact onthe public health of the tropics (1–4). Leptospirosis is an infection with a broadgeographical distribution due to the large spectrum of mammalian hosts that houseand excrete the leptospires from their renal tubules (5,6). Outbreaks intropical regions arise after heavy rainfall and flooding (1).
Leptospires are spirochetes that include saprophytic species (6 species with 60serovars) and pathogenic species (13 species with more than 260 serovars) (7). Rodents are the main leptospirosiscarriers, however, other wild or domestic animals can also be carriers, such assmall marsupials, cattle, pigs, and dogs (3,7). The causative microorganismis capable of infecting the mammalian hosts through abraded skin and mucousmembranes, and disseminating through the bloodstream to several organs (2).
Human leptospirosis presents clinical manifestations divided into two stages: thefirst stage or acute phase (septicemic phase) and the second stage or immune phase(4). The septicemic phase ischaracterized by fever, chills, headache, myalgia, skin rash, cough, anorexia,nausea, and vomiting (1,4,6). The immune orsevere phase is known as Weil's disease and is characterized by jaundice, pulmonaryhemorrhage, meningitis, liver, and acute kidney injury (1,3,6,7).
Deformability is defined as the matter capacity to change its shape in response to anapplied force, which is a particularity of soft matter including red blood cells.Deformability can involve a change in cell curvature, a uniaxial deformation, or anarea expansion (8). Thus, red blood cells areable to pass through capillaries with a smaller diameter than their size and tocarry out their role as gas carriers between blood and tissues (8,9).Alterations in erythrocyte deformability have been associated with various diseasessuch as malaria, sickle cell anemia, diabetes, and hereditary disorders (9). Studies have shown that erythrocytedeformability is reduced in some diseases (10,11) and altered in patientswith liver disease (12).
Cell deformability can be studied using optical tweezers (11,13,14). Optical tweezers are able to provideinformation on electrical and mechanical properties of red blood cells (13). Some studies have reported the use ofoptical tweezers to evaluate the erythrocyte elasticity as a way to investigate thedeformability (11,13,14).
In this work, the optical tweezers were used to capture human erythrocytes andmeasure their elastic constant in patients in late stage leptospirosis. Biochemicaltechniques of identification and dosage (lipid composition of plasma anderythrocytes, some liver enzymes, and albumin) were used to establish possiblerelationships between lipid profiles of blood plasma and of the erythrocyte membranewith its elasticity.
Material and Methods
Collection and processing of blood samples
This study was approved by the Ethics Committee of the Hospital UniversitárioOswaldo Cruz (HUOC), Recife, Brazil (CAAE-11524813.3.0000.5207). Blood sampleswere obtained from patients fasted for 12 h at HUOC. In a period of six months,only three patients presented the late stage of the disease (characterized byjaundice, respiratory failure, and kidney failure), for this reason just threeblood samples were collected. Despite this, a great number of cells wereanalyzed. For control, 10 blood samples of healthy individuals were used.Peripheral blood was collected in vacuum tubes (Vacutainer, USA) containinganticoagulant EDTA-K3+ (1.8 mg/mL) to perform the biochemical assays.Additionally, blood was also collected in tubes without anticoagulant to obtainserum for the biomechanical experiment (assay with optical tweezers). The bloodsamples were processed over a period of up to 4 h and centrifuged at 2,500g for 15 min at 4°C (Sorvall RC6, Thermo Fisher Scientific,USA). Then, a portion of blood was separated to evaluate erythrocyte elasticity.The remaining erythrocytes were washed 4 times with 4.5 mL of saline and againcentrifuged at 2,500 g for 15 min at 4°C. The erythrocytepellets were then used for lipid extraction.
Measuring erythrocyte elasticity
The measurement of erythrocyte elasticity was carried out according to Moura etal. (13). The optical tweezers systemconsisted of a laser beam (λ=1064 nm, IPG Photonics, USA) focused on themicroscope (Axio Lab, Carl Zeiss Microscopy GmbH, Germany) using a 100×objective lens, NA=1.25. The microscope was equipped with a motorized stage(Prior Scientific Instruments Ltd., UK) and an image capture system controlledby a computer. Cells from patients with leptospirosis (n=73 cells) and controlindividuals (n=222 cells) were captured by optical tweezers and dragged againstblood serum at six different velocities (140 to 315 µm/s). The images of theerythrocytes deformation in the serum, according to the drag velocities, allowedus to measure their elastic constants. To determine the erythrocyte elasticity,the following equation was used (13):
where ΔL = L - L0 is the cell length deformation(L0 is the length of the cell before dragging), η is serumviscosity that is measured by an Ostwald viscometer, μ is the elastic constant(also called apparent elasticity), and ν is the velocity. Zeq is aparameter for the relative position of the erythrocyte to the Neubauer chamber,defined as follows: 1/Zeq = 1/Z1 + 1/Z2.Z1 and Z2 are, respectively, the distance from theerythrocyte to the bottom and the top of a Neubauer chamber, in our caseZeq = 25 μm. The value μ is calculated from a plot of cell lengthL vs the drag velocity ν, since the viscosity of the serum (η),the initial length (L0), and Zeq are known. The celllength L was measured from the video images of the cells using Image-Pro Plussoftware (Media Cybernetics, USA).
Lipid composition of plasma and erythrocyte membrane
Lipids were extracted from blood plasma and from erythrocyte membrane withchloroform:methanol (2:1, v/v), as described by Folch et al. (15) and Lima et al. (16). Aliquots of plasma and erythrocyte extracts wereconcentrated under a stream of N2. The isolation of the plasmaphospholipids was performed by one-dimensional thin-layer chromatography with asolvent mixture containing chloroform:acetone:methanol:acetic acid:water(50:20:10:10:5, v/v). The isolation of the erythrocytemembrane phospholipids was performed by two-dimensional thin-layerchromatography on silica H containing 2.5% (w/w) of magnesium acetate. Themobile phase consisted, at the first dimension, of a solvent mixture containingchloroform:methanol:ammonia (65:35:5, v/v), and the second dimension wasperformed in a solvent system containing chloroform:acetone:methanol:aceticacid:water (50:20:10:10:5, v/v). Phosphatidylcholine (PC), sphingomyelin (SpM),phosphatidylethanolamine (PE), and lysophosphatidylcholine (LPC) were visualizedafter exposure to iodine vapor, labeled according to the relative mobility ofthe standards, and scraped from the silica tubes. These individual phospholipidsamples were digested with 0.3 mL of 99.9% sulfuric acid by heating at 180°C for2 h. The tubes were allowed to cool and after drops of 30%H2O2 were added, the tubes were again heated for 2 h(16,17). The inorganic phosphorus present in the phospholipids wasquantified by the method of Bartlett (18). The total phospholipids were quantified from extracts of plasma anderythrocytes. Absorbance was measured spectrophotometrically at 735 nm.
The determination of triglyceride (TG), total cholesterol (TC), and high-densitylipoprotein cholesterol (HDL-C) levels was performed by chemical and enzymaticmethods using Labtest assay kit (Labtest Diagnostics Ltd., Brazil).
For the determination of triglycerides, Labtest kit used a lipoprotein lipase,glycerol kinase, glycerolphosphate oxidase, and peroxidase that act on thetriglycerides of the sample, thus producing a quinoneimine (maximum absorbanceat 505 nm). The concentration of triglycerides was determined from theabsorbance ratio of the sample and standard solution.
Cholesterol was measured by the presence of antipyrilquinonimine. This product isobtained from the sample cholesterol by the action of cholesterol esterase,cholesterol oxidase, and peroxidase. After the formation of antipyrilquinonimine(maximum absorbance at 500 nm), it was quantified on the spectrophotometer.
HDL cholesterol was quantified in the supernatant after sample centrifugation.The colorimetric test, containing phosphotungstic acid and magnesium chloride,allowed the measurement of the endpoint reaction at 500 nm.
The levels of low-density lipoprotein cholesterol (LDL-C) and very-low-densitylipoprotein cholesterol (VLDL-C) were determined by the Friedewald equation(19).
Liver function was evaluated measuring the alanine transaminase (ALT), aspartatetransaminase (AST), albumin, and lecithin-cholesterol acyltransferase (LCAT)activity. AST, ALT, and albumin were measured with the Labtest assay kit(Labtest Diagnostics Ltd.).
The test for ALT is based on the catalytic action of ALT that transfers thealanine amino group to ketoglutarate, with the formation of glutamate andpyruvate. Pyruvate was reduced to lactate by lactate dehydrogenase, whilecoenzyme NADH was oxidized to NAD. As NADH coenzyme oxidation, monitoredphotometrically (reduction in absorbance at 340 nm), is directly proportional toALT activity in the sample, it was possible to measure its concentration.
The test to measure AST is based on the catalytic action of AST, which transfersthe amino group from aspartic acid to ketoglutarate, with the formation ofglutamate and oxalacetate. Oxalacetate was reduced to malate by malatedehydrogenase, while coenzyme NADH was oxidized to NAD. The reduction inabsorbance at 340 nm due to NADH coenzyme oxidation was monitoredphotometrically and is directly proportional to AST activity in the sample.
The measurement of albumin was done by binding albumin to bromocresol green dye.The color formed in the reaction was measured colorimetrically between 600 and640 nm and it is proportional to the amount of albumin in the sample.
The LCAT activity was determined according to the method of Stokke and Norum(20), which uses a radioactivesubstrate. The substrate was prepared by adding 20 µL of(14C)-cholesterol (2 µCi) to a 5% (w/v) solution of human serumalbumin in 1.0 mL of phosphate buffer saline (0.2 M), pH 7.4. The(14C)-free cholesterol and (14C)-esterified cholesterolwere separated by thin-layer chromatography on silica G plates and removed fromthe chromatography plate to scintillation tubes. After the addition ofscintillation fluid, the radioactivity of the samples was counted in the liquidscintillation counter Packard - Tri-Carb 2100TR (PerkinElmer Life Sciences,USA). The unit (U) of enzyme activity is reported as the percentage of freecholesterol (FC) converted to cholesterol ester (esterified cholesterol - EC)per hour.
Statistical analysis
The biochemical data are reported as the mean±SE (standard error). Theerythrocyte elasticity data are reported as the mean±SD (standard deviation). Toanalyze the biochemical data, Student's t-test was used and forerythrocyte elasticity data, a non-parametric Mann-Whitney test was used with asignificance level of 5% (P=0.05).
Results
Erythrocyte elasticity
The images were taken from a pool of cells from controls (n=222) and patientswith leptospirosis (n=73). Figure 1A(erythrocytes from the control group individuals) and Figure 1B (erythrocytes from patients with leptospirosis)show typical erythrocyte elongations when dragged by the optical tweezer withvelocities of 140, 245, and 315 µm/s. The different velocities are from the samesample. This figure shows that erythrocytes of the control individuals are morestretched out than the patients.
Representative erythrocyte elongations from controls (A)and patients with leptospirosis (B) obtained in experimentswith optical tweezers. The dragging velocities used were 140, 245, and315 µm/s. Scale bar: 2 µm.
Figure 2 shows the boxplot of elasticitiesfor patient and control erythrocytes. The optical tweezers experiments showedthat the average values of erythrocyte elastic constant obtained from patientsand healthy individuals were (6.5±2.5)×10-4 dyn/cm and(3.9±1.9)×10-4 dyn/cm, respectively. Erythrocyte elasticconstants were statistically different (P<0.05) between patients withleptospirosis and healthy individuals. The value of the erythrocyte elasticconstant is inversely related to its stretching length. Under the analysis ofoptical tweezers, when the cell stretching length is longer, the value of theelastic constant is smaller.
Box plot of erythrocyte elasticity for controls and patients withleptospirosis. The black lines represent the median of the elasticconstant of erythrocyte for control and patients with leptospirosis.*P<0.05 (Mann-Whitney test).
The results showed that the red blood cells of leptospirosis patients had higherelastic constant values and, thus, a shorter stretch length. In other words,elasticity was reduced in diseased red blood cells.
Lipid content of plasma and erythrocyte membrane
Table 1 shows the lipid composition ofblood plasma and erythrocyte membrane of healthy individuals and patients withleptospirosis. Patients with leptospirosis exhibited a reduction of 40.1% forTC, 58.0% for HDL-C, and 69.4% for LDL-C compared to the control group. TG andVLDL-C were higher in patients with leptospirosis, with an increase of 88.4 and130%, respectively, in relation to the control. Moreover, the ratio of TC/HDL-Cshowed an increase of 42.8% in patients with leptospirosis.
Table 1
Lipid composition of plasma and erythrocyte membrane andbiochemical analysis of liver function of control subjects andpatients with leptospirosis.
| Controls (n=10) | Patients (n=3) | P | |
|---|---|---|---|
| Plasma | |||
| TC (mg/dL) | 172.40±27.96 | 103.33±9.26 | 0.0018 |
| HDL-C (mg/dL) | 41.63±7.83 | 17.47±3.89 | 0.0004 |
| LDL-C (mg/dL) | 101.69±19.90 | 31.13±14.35 | 0.0002 |
| VLDL-C (mg/dL) | 23.77±10.15 | 54.80±7.03 | 0.0009 |
| TG (mg/dL) | 145.40±40.94 | 274.00±35.17 | 0.0002 |
| PC (%) | 64.62±2.05 | 70.55±0.67 | 0.0001 |
| PE (%) | 7.60±0.58 | 15.46±0.39 | 0.0001 |
| SpM (%) | 19.34±1.05 | 9.37±0.09 | 0.0001 |
| LPC (%) | 8.45±0.01 | 6.63±0.05 | NS |
| FC (%)# | 20.90±2.93 | 38.03±4.40 | 0.005 |
| EC (%)# | 79.10±2.95 | 61.97±4.45 | 0.0051 |
| Erythrocyte membranes | |||
| PC (%) | 33.43±7.25 | 47.06±4.74 | 0.0117 |
| PE (%) | 31.31±11.02 | 27.40±1.75 | NS |
| SpM (%) | 28.48±6.21 | 20.66±2.86 | NS |
| LPC (%) | 6.77±2.36 | 4.88±1.25 | NS |
| Liver-specific analysis | |||
| Albumin (g/dL) | 4.50±0.29 | 2.53±0.30 | 0.0001 |
| ALT (U/L) | 25.20±10.28 | 64.00±17.05 | 0.0004 |
| AST (U/L) | 23.90±4.07 | 59.67±15.53 | 0.0001 |
Data are reported as mean±SD. Student's t-testwas used. TC: total cholesterol; HDL-C: high density lipoproteincholesterol; LDL-C: low density lipoprotein cholesterol; VLDL-C:very low density lipoprotein cholesterol, TG: triglycerides: PC:phosphatidylcholine; PE: phosphatidylethanolamine, SpM:sphingomyelin; LPC: lysophosphatidylcholine; FC: freecholesterol; EC: esterified cholesterol; ALT: alaninetransaminase; AST: aspartate transaminase; NS: not significant.#n=3 for controls and patients for FC and EC.
Patients with leptospirosis presented a rise compared to the control group of9.2% for plasma PC, 40.8% for erythrocyte membrane PC, and 103.4% for plasma PE.On the other hand, the plasma SpM of patients was 51.6% lower than the control.The plasma and erythrocyte membrane LPC values did not show a significantdifference between groups, as well as the PE and SpM of the erythrocytemembrane.
Biochemical data of liver function
In individuals with leptospirosis, a significant increase in levels of ALT (154%)and AST (150%) were found compared to the control group. The increased level ofthese two liver enzymes is related to liver dysfunction.
The albumin level of patients was 43.8% lower compared to the control group(P=0.0001), which can indicate an abnormality in liver function.
The fractional LCAT activity was 3.6 times lower in individuals withleptospirosis than in healthy individuals (control) (P<0.0001). Inindividuals with leptospirosis, an activity of 0.11±0.07 U was obtained,compared to 0.40±0.03 U in the control group. FC was 20.9% in the control groupand 38.03% in the patients, whereas EC was 79.1% and 61.97%, respectively. Thevalues mentioned above indicated that a decrease of the LCAT activity promotedan increase in free cholesterol in patients with leptospirosis.
Discussion
The deformability of erythrocytes plays an essential role in the transport of gasesvia blood vessels (8,9,11,13,14).It is a rheological property related to viscoelasticity of both the cell membraneand cytosol that can be altered by certain diseases (10). Garnier et al. (10) used theHanss hemorheometer to measure erythrocyte deformability and observed a higher indexof rigidity in diabetic patient’s samples, whilst Agrawal et al. (11) observed a reduction of deformability indiabetic patient’s erythrocytes using optical tweezers.
Optical tweezers can be used for investigating red blood cell rheology. Our studyshowed that the erythrocyte elastic constant of patients with leptospirosis washigher compared to the control indicating that their erythrocytes were more rigidthan the cells of healthy individuals. Santoro et al. (21) observed a lower erythrocyte osmotic fragility in dogs withLeptospira interrogans, which suggests higher erythrocyterigidity, or lesser deformability, than the control (21). A decrease in the erythrocyte deformability causes a significantincrease in microvascular flow resistance and blood viscosity (8).
The TG level was increased for patients with leptospirosis, and this result wascompatible with data reported by Peces (22),Liberopoulos et al. (23), and Gazi et al.(24). The levels of LDL-C and TC weredecreased, confirming the results described by Liberopoulos et al. (23) and Gazi et al. (24). These authors have shown a correlation, direct or inverse,between lipids and lipoproteins and cytokine concentrations. Liberopoulos et al.(23) described an increase ofinterleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in severe leptospirosis.IL-6 is capable of stimulating the expression of genes responsible for LDLreceptors, decreasing LDL-C, while TNF-α stimulates hepatic TG synthesis. Accordingto Liberopoulos et al. (23), IL-6 isresponsible for the reduction of TC and this reduction was also observed in ourstudy. Various inflammatory cytokines (IL-1β, IL-6, IL-12, TNF-α) are increased inleptospirosis as an immune response to the infection, however, this increase may behigher in severe stages of the disease (25).
Vanaja et al. (26) observed an elevation ofTG, VLDL, and phospholipids, and a reduction of HDL in young guinea pigs infected byLeptospira interrogans serovars australis,canicola, and icterohaemorrhagiae. These authors alsonoted an increasing trend of cholesterol and LDL for pigs with serovaricterohaemorrhagiae. Gazi et al. (24) and Cisternas and Milstein-Kuschnaroff (27) have also reported that the HDL-C level isreduced, whereas the VLDL-C level is increased in patients with leptospirosis, as wehave observed. Cisternas and Milstein-Kuschnaroff (27) have reported the LDL-C level to be decreased.
Changes in liver function promoted by leptospirosis are normally found in the severephase (1,3,6,7). Liver injury caused by leptospirosis implicates inmodifications of biochemistry parameters as ALT and AST; normally both enzymes areincreased in the course of the disease (29)and our results were in agreement.
Albumin is the most abundant protein in plasma and predominantly synthesized in theliver (29,30). Its reduced values, as observed in this study, mean alterations inliver function (29). In the advanced stage ofleptospirosis, as the liver becomes the target of this disease, changes in thesynthesis of liver enzymes and proteins are expected. Gancheva (30) reported hypoalbuminemia in leptospirosis,in addition to hypoproteinemia, and elevated ALT and AST values.
In liver disease, there is an alteration in the erythrocyte membrane lipidcomposition that is associated with abnormalities in the composition of plasmalipoproteins. Such changes can lead to modifications in the erythrocyte shape anddeformability (12). We observed that plasmalevels of PC and PE were increased whilst SpM was decreased in patients withleptospirosis. Meanwhile, we did not observe a significant difference in the valuesof PE and SpM in erythrocyte membrane. Owen et al. (12) reported an increase in the PC fraction in erythrocyte membrane ofpatients with liver disease, while the proportion of PE and SpM were reduced inthese patients compared to normal subjects. Furthermore, we did not identifydifferences in the LPC levels in plasma and erythrocyte membrane of patients withleptospirosis.
Shiraishi et al. (31) reported a decrease inerythrocyte deformability and erythrocyte membrane fluidity, abnormal lipidcompositions, and increased PC:SpM ratio in patients with alcoholic liver disease.The lipid domain fluidity of the erythrocyte membrane is determined by the molarratio of cholesterol to phospholipid, degree of unsaturation of phospholipid acylchains, and the PC/SpM ratio (32). Ourresults showed that the phospholipid constitution of the erythrocyte membrane ofpatients with leptospirosis was not significantly altered, except for PC, whose meanvalue was 40.8% higher in patients than controls. Thus, the increase of PC seemed tocontribute to the decrease of erythrocyte deformability, while there was nosignificant alteration for other phospholipids. However, according to Borochov etal. (33), PC forms highly fluid lipidregions, while sphingomyelin induces rigidity. Kuypers et al. (32) showed that changes in the molecular species composition ofPC alter the morphology and erythrocyte deformability. The replacement of native PCwith 1-palmitoyl, 2-arachidonoyl PC resulted in lower osmotic fragility, whereasreplacement with 1,2-dipalmitoyl PC led to higher osmotic fragility.
LCAT is an enzyme synthesized by the liver that plays a part in the cholesteroltransport process by converting FC to EC to form mature HDL (34). EC generated by LCAT is more hydrophobic than FC and,thus, migrates into the hydrophobic core of the lipoprotein, resulting in theconversion of pre-β-HDL into α-HDL (35). Weobserved a fractional LCAT activity reduction in patients with leptospirosis.Cisternas and Milstein-Kuschnaroff (27) alsoreported a reduction in fractional LCAT rate in patients with leptospirosis inrelation to the control. LCAT activity, as well as the concentration of thismolecule, is directly proportional to the HDL-C levels (35). Therefore, the decrease of enzyme activity is associatedwith the HDL-C reduction, since LCAT is present in the HDL (34,35). This process ofcholesterol esterification in plasma by LCAT is essential for cholesterol uptakefrom the liver. A large amount of EC formed by LCAT is exchanged with triglyceridesthrough the mediated process by cholesteryl ester transfer protein intoapolipoprotein B containing lipoproteins that are finally catabolized by the liver.Alternatively, HDL-cholesteryl esters are taken-up by the liver through scavengerreceptor class B member 1 (35,36). This is the clearance process of FC fromplasma lipoproteins. Our study showed a reduction of LCAT activity that reflected ina decrease of EC levels in patients with leptospirosis, resulting in an increase inFC.
Summarizing our results, the optical tweezers showed that erythrocyte elasticity wasmodified by leptospirosis. Also, leptospirosis promoted lipid concentration changesin plasma. However, in the erythrocyte membrane, only PC was increased, whichprobably had a contribution in the decrease of erythrocyte elasticity.
Acknowledgments
This work was supported by the Centro de Apoio a Pesquisa (CENAPESQ)/UFRPE,Laboratório de Imunopatologia Keizo Asami (LIKA), Departamento de Bioquímica andDepartamento de Biofísica e Radiobiologia da UFPE, Hospital Universitário OswaldoCruz (HUOC)/Universidade de Pernambuco (UPE), Coordenação de Aperfeiçoamento dePessoal de Nível Superior (CAPES), Fundação de Amparo a Ciência e Tecnologia dePernambuco (FACEPE), and Conselho Nacional de Desenvolvimento Científico eTecnológico (CNPq). A special thanks to Dr. José Anchieta de Brito for helping toelaborate the project. We also thank Iracema Correia Silva of the Arquivos Médicosdo HUOC and Núcleo de Epidemiologia do HUOC.
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