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Keywords:

  • Antioxidant capacity;
  • Glutathione;
  • Oxidative burst;
  • Superoxide dismutase

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

Background: Oxidative stress is an important component in the progression of chronic renal failure (CRF) and neutrophil function may be impaired by oxidative stress.

Hypothesis: Cats with CRF have increased oxidative stress and decreased neutrophil function compared with control cats.

Animals: Twenty cats with previously diagnosed renal failure were compared with 10 age-matched control cats.

Methods: A biochemical profile, CBC, urinalysis, antioxidant capacity, superoxide dismutase (SOD) enzyme activity, reduced to oxidized glutathione ratio (GSH : GSSG), and neutrophil phagocytosis and oxidative burst were measured. Statistical comparisons (2-tailed t-test) were reported as mean ± standard deviation.

Results: The CRF cats had significantly higher serum blood urea nitrogen, creatinine, and phosphorus concentrations than control cats, and significantly lower PCV and urine specific gravity than control cats. The GSH : GSSG ratio was significantly higher in the CRF group (177.6 ± 197, 61.7 ± 33; P < .02) whereas the antioxidant capacity was significantly less in the CRF group (0.56 ± 0.21, 0.81 ± 0.13 Trolox units; P < .005). SOD activity was the same in control and CRF cats. Neutrophil oxidative burst after Escherichia coli phagocytosis, measured as an increase in mean fluorescence intensity, was significantly higher in CRF cats than controls (732 ± 253, 524 ± 54; P < .05).

Conclusions: The higher GSH : GSSG ratio and lower antioxidant capacity in CRF cats is consistent with activation of antioxidant defense mechanisms. It remains to be determined if supplementation with antioxidants such as SOD beyond the level of control cats would be of benefit in cats with CRF.

Abbreviations:
ABTS

2,2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid

ACK

aluminum-chloride-potassium

CRF

chronic renal failure

DHR-1,2,3

dihydrorhodamine 1,2,3

EDTA

ethylenediaminetetraacetic acid

ESRD

end-stage renal disease

FACS

fluorescence activated cell sorting

GSH : GSSG

reduced to oxidized glutathione ratio

HBSS

Hanks balanced salt solution

M2VP

1-methyl-2-vinyl-pyridium trifluoromethane sulfonate

MFI

mean fluorescence intensity

NADPH

nicotinamide adenine dinucleotide phosphate

PBS

phosphate-buffered saline

RBC

red blood cell

SOD

superoxide dismutase

Oxidative stress is an important component in the progression of chronic renal failure (CRF) in humans. Reactive oxygen species increase vascular resistance and promote hypertension, alter NF-κB expression, and both perpetuate as well as mediate many of the deleterious affects of chronic renal inflammation in human patients.1–3 Oxidative stress increases oxidative products in the plasma and tissue of uremic patients, increases apoptosis and fibrosis within the kidney, and contributes to a reduction in erythrocyte lifespan in people with CRF.4 There is growing evidence that oxidative stress may be implicated in the pathogenesis of atherosclerosis, dialysis-related amyloidosis, malnutrition, and anemia in humans with end-stage renal disease (ESRD).5–7

The superoxide anion is a short-lived but critical free radical in the progression of renal failure. The increased concentration of the superoxide anion triggers many of the aforementioned deleterious effects of oxidative stress. Increased superoxide anion in human patients with renal failure reduces the effects of the vasodilator nitric oxide, decreases medullary blood flow and sodium excretion, and alters endothelial, podocyte, and mesangial cell function.3,8,9 Concentrations of the enzymatic antioxidant superoxide dismutase (SOD) are reported as increased, unchanged, or decreased in humans with CRF.10–12 This may result from differences in the stage or duration of disease, the severity of clinical signs, or differences in treatments and concurrent disease status.

Polymorphonuclear cell viability and function are adversely affected by oxidative stress and uremic toxins, increasing susceptibility to infection in human patients with CRF.13,14 Neutrophils also contribute to the chronic inflammation associated with renal failure progression by the production and release of myeloperoxidase and free radicals such as superoxide and hydrogen peroxide.15,16 The link between inflammation and oxidative stress in humans with CRF is well established and results in cardiovascular complications, but to date, no studies of neutrophil function in cats with CRF have been reported.17

It is hypothesized that oxidative stress in cats with CRF is significantly greater than in healthy cats, and identification of those parameters of oxidative stress that are most increased will help direct future efforts at appropriate antioxidant supplementation in this common clinical condition of cats. To date, the authors are aware of only a single study looking at the effects of antioxidant supplementation (a combination of vitamins) in cats with CRF, and the results suggested that the treatment was beneficial.18 The objective of this study was to test the hypothesis that cats with CRF have increased oxidative stress and decreased neutrophil function when compared with control cats.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

Selection of Cases

Twenty adult cats with previously diagnosed CRF were recruited from the client-owned hospital population for this study. Inclusion criteria for CRF cats included adult cats with a diagnosis of CRF from a repeatable (at least 2 separate time points) increase in serum creatinine concentration and inappropriately low urine specific gravity made >1 month before study entry, no current report of vomiting or diarrhea, and no change in diet or use of antioxidants during the month preceding entry into the study. None of the cats had received a blood transfusion before study entry. The 10 age-matched control cats were recruited from the Colorado State University veterinary student population. The inclusion criteria for the control cats included age (adult, centered around the mean of the CRF cats), no history of chronic or systemic disease or use of medication, and no current evidence of a disease on physical examination, CBC, or biochemical profile. A signed consent form was obtained and all sample acquisition and handling were in accordance with Colorado State University Animal Care and Use Committee guidelines. The cats were otherwise managed completely by the attending clinician.

A biochemical profile, CBC, urinalysis, plasma antioxidant capacity, erythrocyte lysate SOD enzyme activity, whole blood reduced to oxidized glutathione ratio (GSH : GSSG), and neutrophil phagocytosis and oxidative burst were measured.

Sample Preparation

All samples for oxidative stress assays were from heparin-preserved whole blood, processed immediately after acquisition, and stored at −80°C until analysis. Ten microliters of 1-methyl-2-vinyl-pyridium trifluoromethane sulfonate (M2VP) scavengera was added to 100 μL of whole blood in a microcentrifuge tube, vortexed, and stored for later analysis of oxidized disulfide glutathione (GSSG). Fifty microliters of whole blood was transferred to a 2nd microcentrifuge tube and stored for later analysis of GSH. Approximately 3 mL of whole blood was centrifuged at 2,500 ×g for 5 minutes at 4°C, and the plasma stored at −80°C for later analysis of antioxidant capacity. The remaining erythrocytes were washed twice in 1 × phosphate-buffered saline (PBS)b after which 10 mL of deionized water was added to a 1.5 mL volume of erythrocytes and gently agitated for 10 minutes at 4°C to lyse the red blood cells (RBC). The lysate was centrifuged at 10,000 ×g for 10 minutes and the supernatant pipetted off and stored in microcentrifuge tubes at −80°C for later analysis of RBC lysate protein content and SOD units.

All samples for neutrophil function were from heparin-preserved whole blood stored on ice and processed and analyzed within 4 hours of acquisition.

Neutrophil Oxidative Burst and Phagocytosis Assay

Lyophilized Escherichia coli (strain K-12) conjugated to Texas Redc was reconstituted with 500 μL of 2 mM sodium azide/1 × PBS, producing a working concentration of 6 × 106E. coli per microliter, and stored at −20°C. A 5 mM stock solution of dihydrorhodamine-1,2,3 (DHR-1,2,3)d was diluted to a 0.05 mM with sterile Hanks balanced salt solution (HBSS)e and stored at −20°C.

The mature neutrophil count in each sample was taken from each cat's CBC, drawn at the same time as sample acquisition for the study. A calculated volume of the E. coli-Texas Red conjugate was added to 100 μL of whole blood such that the E. coli-to-neutrophil ratio was 30 : 1, and incubated with gentle agitation in a 37°C water bath for 30 minutes. All processing was carried out in the dark after addition of the fluorescent E. coli to the mixture. For the final 15 minutes of incubation, 10 μL of 0.05 mM DHR-1,2,3 was added to the sample, resulting in a final DHR-1,2,3 concentration of 5 μM. After the complete 30-minute incubation, 4 mL of ammonium-chloride-potassium (ACK)f lysis solution was added to the sample and incubated for 5 minutes at room temperature. The mixture was centrifuged, the supernatant discarded, and the pellet washed in 1 mL HBSS. After a 2nd centrifugation, the cell pellet was resuspended in 300 μL of fluorescence-activated cell sorter (FACS)g buffer and 10 μL of trypan blue added to the cell suspension to quench any extracellular fluorescence (ie, E. coli adhered to the cell surface but not having undergone phagocytosis). The cell suspension was placed on ice and analyzed immediately with flow cytometry.19–22

Flow cytometry can simultaneously measure the fluorescence generated by neutrophil phagocytosis of E. coli (K-12 strain) conjugated to Texas Red and DHR-1,2,3 reacting with reactive oxygen species forming the fluorescent molecule rhodamine 123. This combination of markers, therefore, allows the measurement of E. coli phagocytosis and the subsequent oxidative burst by flow cytometry. Trypan blue is added to the mixture before cytometric analysis to quench any extracellular signal (E. coli adhered to the cell surface). Two distinct populations of neutrophils could be discerned after phagocytosis based on their intracellular E. coli fluorescence and oxidative burst activity; an optimal and suboptimal group.

Flow Cytometry

All samples were analyzed with a 3-laser flow cytometerh capable of rapid sample acquisition and equipped with an argon 488 nm laser. The distinct forward-by-side scatter pattern and appropriate gating paradigm allowed for the separate analysis of neutrophils.19 Unstained leukocytes were run before each assay to confirm that the autofluorescence of these cells had a mean fluorescence intensity (MFI, a unitless measure) < 101.

Measures of Oxidative Stress, Antioxidant Capacity, and SOD Enzyme Activity

The ratio of GSH : GSSG has been used previously as a measure of oxidative stress in cats.23,24 Glutathione was quantified from ethylenediaminetetraacetic acid (EDTA)-preserved blood using an enzymatic assay as described previously.25,i Briefly, the assay is run twice, once with and once without a GSH scavenging reagent, so that both GSSG and total glutathione (GSH + GSSG) can be quantified. For GSSG, a thiol-scavenging reagent, M2VP, was added to rapidly scavenge GSH and eliminate continued oxidation of GSH to GSSG after sample acquisition. Glutathione reductase, nicotinamide adenine dinucleotide phosphate (NADPH), and the chromagen 5,5-dithiobis-(2-nitrobenzoic acid), were added sequentially to the sample. Glutathione reductase converts the GSSG to GSH, which reacts with the chromagen to form a product which absorbs visible light (412 nm). The change in absorbance was measured spectrophotometrically. The rate of this reaction is proportional to the resultant GSH concentration. Total glutathione, GSH + GSSG, was quantified in EDTA preserved blood without the addition of M2VP. The concentration of GSH was determined by the difference between the total glutathione (GSH + GSSG) concentration and the GSSG concentration. The GSH : GSSG may then be calculated.

Antioxidant capacity was measured in 10 μL of plasma with a commercially available spectrophotometric assay as described previously in dogs.26,j Briefly, a ferryl myoglobin radical is formed from metmyoglobin and hydrogen peroxide. This radical oxidizes 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) to form a radical cation, ABTS+. Antioxidants suppress the production of the radical cation in a concentration dependent manner. Trolox (water-soluble vitamin E analog) serves as the control antioxidant, and the standard curve determines the millimolar concentration of Trolox units corresponding to a particular absorbance. Samples were processed in duplicate according to the manufacturer instructions and absorbance determined at a 405 nm excitation wavelength. The calibration curve was determined by averaging triplicate samples of incremental increases in Trolox concentration in buffer.

The antioxidant SOD enzyme was measured in erythrocyte lysate with a commercially available spectrophotometric assay.27,k The assay is based on the reduction of the dye WST-1 by superoxide anion to a colored WST-1 formazan product that absorbs light at 450 nm. SOD scavenges superoxide anion and thereby reduces the rate of formation of the WST-1 formazan product. The addition of xanthine to the mixture containing xanthine oxidase is used to initiate the reaction and the plate is analyzed starting immediately and at 1-minute intervals thereafter for 10 minutes. The change in absorbance was measured spectrophotometrically. The rate of this reaction is proportional to the resultant SOD activity. Samples used to determine the standard curve were run in triplicate while the CRF and control samples were an average of duplicate wells. The SOD activity was normalized to micrograms protein content of the lysate.

Statistical Analysis

A computer software programl was used for statistical analysis. Gaussian distribution was confirmed with the Kolmogorov and Smirnov test, and the difference in the means between groups was tested by an unpaired t-test assuming unequal variance with the Welsh correction. Statistical significance was set as P < .05. All data were expressed as the mean ± the standard deviation.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

Cats

The characteristics of each group are summarized in Table 1. Twenty cats with previously diagnosed CRF and 10 healthy control cats were included in the analysis. The CRF group comprised 8 spayed female and 12 neutered male cats. The control group comprised 6 spayed female and 4 neutered male cats. None of the cats in either group were purebred. The mean age for the CRF and control groups were, respectively, 14.2 ± 4.9 years (range, 1.0–23 years) compared with 13.1 ± 2.1 years (range, 10–16 years). All CRF cats had been diagnosed with their disease a minimum of 6 months before entry into the study. The majority of these cats were consuming a renal diet whereas smaller numbers of cats were receiving various other treatments including subcutaneous fluids, potassium supplementation, phosphate binders, H2-blockers, antihypertensive medication, appetite stimulants, or some combination of these. None of the CRF cats were anorectic, vomiting, or being treated with an antiemetic at the time of the study, and none of the CRF cats had received either a blood transfusion or erythropoietin before entry into the study. None of the CRF cats were known to have any other clinically relevant disease, and the thyroid hormone status of the study cats had either been determined to be normal within the previous year or was confirmed as such at the time of entry into the study. Physical examination did not identify any additional problems in the CRF group, and although subjectively assessed, the body condition score was not substantially different between the CRF and control groups, 4.4 and 5.1 of 9, respectively. The CRF cats had significantly higher blood urea nitrogen, creatinine, and phosphorus concentrations and significantly lower packed cell volume and urine specific gravity than the control cats (Table 1).

Table 1.   Summary of group characteristics for control cats and cats with renal failure.
Group SummaryAge (yrs)Body Weight (kg)BUN (mg/dL)Cr (mg/dL)Phos (mg/dL)PCV (%)U Sp Grav (unitless)
  1. There was no difference in age or body weight between control and CRF cats. The CRF cats had significantly greater serum BUN, creatinine, and phosphorus concentrations than control cats, and significantly lower PCV and urine specific gravity than control cats.

  2. yrs, years; P-value, unpaired t-test assuming unequal variance; NS, not significant; BUN, blood urea nitrogen; U Sp Grav, urine specific gravity; Phos, phosphorus; CRF, chronic renal failure.

Control (n = 10)13.14.421.11.24.136.51.056
CRF (n = 20)14.24.381.16.76.331.91.015
P-valueNSNS< .001< .03< .05< .05< .001

Oxidative Stress, Antioxidant Capacity, and Antioxidant Enzyme Activities

The GSH : GSSG of whole blood was significantly greater in cats with CRF compared with control cats. The plasma antioxidant capacity was significantly decreased in CRF compared with control cats. There was no significant difference in RBC lysate SOD enzyme activity between groups (Table 2).

Table 2.   GSH : GSSG ratio, antioxidant capacity, and SOD enzyme levels in control cats and cats with renal failure.
Parameters of Oxidative StressGSH : GSSG (unitless)Antioxidant Capacity (Trolox units/mL)SOD (U/μg)
  1. The reduced-to-oxidized glutathione ratio (GSH : GSSG) was significantly greater in cats with CRF compared to control cats, while the plasma antioxidant capacity was significantly reduced in CRF cats. There was no significant difference in superoxide dismutase enzyme levels between groups.

  2. Trolox, vit E; U/μg, SOD units per microgram lysate protein; NS, not significant; CRF, chronic renal failure.

Control (n = 10) ( ± SD)61.7 ± 3381 ± 130.088
CRF (n = 20)177.6 ± 19756 ± 210.083
P-value< .02< .005NS

Neutrophil Function by Flow Cytometry

There was no significant difference in the percent distribution of neutrophils (optimal versus suboptimal) between control cats and cats with CRF. The oxidative burst produced by the optimal neutrophils from cats with CRF was significantly greater than the burst produced by optimal neutrophils from control cats: MFI, 732 ± 253 and 524 ± 163, respectively (P < .05).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

The ratio of reduced (GSH) to oxidized (GSSG) glutathione in whole blood was significantly greater in cats with renal failure compared with age-matched healthy control cats. This GSH : GSSG ratio in CRF cats is consistent with activation of antioxidant defense mechanisms in this disease state. The GSH : GSSG ratio is significantly increased in cats given acetaminophen, and decreased in cats with liver disease.25,28 Whether the ratio increases or decreases in a particular disease may depend on the duration and severity of the oxidative stress, the primary organ or tissue involved and its role in oxidant-antioxidant equilibrium (eg, liver compared with kidney), or ongoing dietary or therapeutic treatments. The increase in the GSH : GSSG ratio seen in cats with renal failure suggests that 1 response to the oxidative stress of that condition is an upregulation of GSH-producing pathways. Whether this is a sufficient response or still represents a relative deficiency of reduced glutathione remains to be determined. Viviano et al29 found that clinically ill dogs had significantly reduced activity of GSH as measured by HPLC, and that this glutathione depletion correlated with the severity of the illness, whereas GSH concentrations between ill and healthy cats were not significantly different. The Viviano et al29 study population included only 2 cats with CRF, precluding any analysis of changes in that specific disease category. All of the cats in the current study appeared to have stable disease with only mild clinical signs, and it is possible that both GSH activity and the GSH : GSSG ratio would decrease as the severity of the renal failure increased in these cats. This question could be addressed with a longitudinal study of changes in oxidative stress parameters in cats with CRF.

The significantly decreased antioxidant capacity seen with CRF suggests a disequilibrium between oxidant processes and antioxidant defenses in cats with this disease. It may be clinically relevant that this assay measured oxidative stress in units of vitamin E (tocopherol). Hinchcliff and colleagues found significantly decreased levels of α-tocopherol in sled dogs after exercise, and Baskin and colleagues confirmed that dietary supplementation with α-tocopherol could significantly increase the plasma concentration of this antioxidant in dogs.26,30 This illustrates a relative deficiency of a specific endogenous antioxidant in a healthy dog challenged by the stress of exercise. Kittens fed a diet consisting exclusively of cooked sardines developed steatitis, and the severity of the disease appeared to correspond to significantly decreased concentrations of vitamin E.31 In the current study, cats facing the stress of CRF also appear to be deficient in vitamin E, and would potentially benefit from supplementation. Yu and Paetau-Robinson18 showed that dietary supplementation of a combination of vitamins including vitamin E to cats with CRF decreased several markers of oxidative stress.

SOD activity appeared to be the same in control and CRF cats. The superoxide anion is the reactive oxygen species most frequently responsible for initiating oxidant processes, serving as the substrate for production of additional reactive species such as hydrogen peroxide or more potent free radicals such as the hydroxyl anion, and impacting the activity of the nitrogenous free radical nitric oxide. SOD is the enzymatic antioxidant that catalyzes the dismutation of the superoxide anion to hydrogen peroxide, which can then be further reduced to water by catalase, another antioxidant enzyme.32 Although there are reports of either no change or decreased activity of SOD in renal disease, the majority of studies have found that SOD is significantly increased in humans with CRF.4 There may be species differences in oxidative metabolism that account for the disparity between cats and humans, and many of the studies in humans involve patients on dialysis, a process that may generate some degree of oxidative stress. Cats with diabetes mellitus were found to have decreased SOD activity, and normal SOD activity in cats with CRF may represent a relative deficiency or SOD activity maybe decreased in CRF cats with more advanced disease.27 Recent confirmation of the bioavailability of oral SOD supplementation in cats suggests that this remains a potential therapeutic intervention for this species and further investigation is warranted.33

Neutrophils from cats with CRF generated a significantly greater respiratory burst in response to E. coli phagocytosis than was seen in neutrophils from healthy control cats. Chronic inflammation contributes to the progression and consequences of renal disease and neutrophils are a potential source of pro-oxidants in this condition. The NADPH oxidase enzyme system produces superoxide anion as part of the respiratory burst utilized by neutrophils to kill phagocytized bacteria. Chronic inflammation could lead to an overproduction of the superoxide radical by activated neutrophils, triggering pro-oxidant processes mediated by this reactive oxygen species. Neutrophils of uremic patients show an abnormal production of ROS in response to activating stimuli.34 The increase in the magnitude of neutrophil respiratory burst in CRF cats maybe a manifestation of the oxidant-antioxidant disequilibrium within this cell type.

The consequences, progression, and complications associated with renal disease in humans are multifactorial processes involving, among other things, a state of chronic inflammation and oxidative stress. Cardiovascular disease is the major cause of death in patients with ESRD because, in part, of an increase in oxidative stress in these individuals, and an increase in pro-oxidants is an important complication of hemodialysis treatment. A variety of antioxidant therapies are being investigated for use in ESRD dialysis patients as a therapeutic strategy that maybe particularly important for dealing with hypertension or concurrent diabetes mellitus.35,36 Renal failure is a common disease of aging cats, and although cardiovascular complications of ESRD are uncommon and hemodialysis is rarely utilized in this species, it is likely that oxidative stress is an important contributing factor in the progression of this condition in cats.37 The purpose of this study was to determine whether or not oxidative stress is a component of CRF in cats, and if so, identify parameters of oxidative stress that might be amenable to therapeutic intervention.

Limitations of this study include sample size and breadth. It would be ideal to have sufficient numbers across the International Renal Interest Society stages of disease such that conclusions could be drawn regarding the most appropriate time to place antioxidant therapy into the treatment regimen for CRF cats. The study also simplifies the complexity of oxidative stress, which is the sum total of a large variety of components. Changes in the concentration of a free radical may result from changes in either or both the production and sequestration of reactive oxygen species, the interaction between antioxidants such as vitamin E and vitamin C, or the relative activity of multiple enzymes such as SOD and catalase.

The recent availability of veterinary products specifically designed to increase levels of SOD, silybinin, and vitamin E in dogs, and a preliminary study showing the effectiveness of one of these supplement in cats, now makes it possible to target a specific antioxidant deficiency in a common disease of cats with well-tolerated oral supplementation.38 It remains to be determined if supplementation with vitamin E or SOD beyond the level of control cats would be of benefit in cats with CRF.

Footnotes

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

aBioxytech GSH/GSSG-412, OXIS International Inc, Foster City, CA

bPBS, Sigma-Aldrich, St Louis, MO

cEscherichia coli (K-12 strain) BioParticles, Alexa Fluor 488 conjugate, Invitrogen, Chicago, IL

dDHR 1,2,3, Invitrogen (Molecular Probes)

eHBSS, Sigma-Aldrich

fACK, Invitrogen

gFACS, Sigma-Aldrich

hCYAN flow cytometer, DakoCytomation Summit software, Fort Collins, CO

iGSH/GSSG-412, OXIS Research, Portland, OR

jAntioxidant Assay Kit, Sigma-Aldrich

kSOD Assay Kit, Assay Designs Inc, Ann Arbor, MI

lGraphPad Prism software, San Diego, CA

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

This work was funded in part by the Winn Feline Foundation (George Sydney and Phyllis Redmond Miller Trust Grant) and the Morris Animal Foundation Student Scholar Program.

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  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References
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