Materials and Methods

Horses—Horses considered for inclusion in the case group were those suspected and subsequently confirmed by a veterinarian to have MND at the Hospital for Animals at Cornell University or on the premises where they resided. Confirmation was made in accordance with the published histopathologic criteria¹² for detecting MND in the CNS of euthanized horses or by examination of biopsy specimens collected from the spinal accessory nerve or the sacrocaudalis medialis muscle of the tail. Horses considered for inclusion in the control group had no apparent clinical signs of MND and resided on the same premises from which a case originated or were selected from those admitted to the hospital at the same time as a case. Candidate controls with any neurologic signs were excluded. All histologic examinations were performed by one of the investigators (ADL) or by staff of the Cornell University Pathology Department.

Erythrocyte preparation and SOD activity determination—Blood samples were collected into evacuated tubes containing heparin and kept on ice during the procedure. The samples were mixed thoroughly and centrifuged at 2,500 × g for 10 minutes at 4°C. Plasma was removed with a pipette, transferred into another tube (for protein determination), and frozen at −70°C. Plasma volume in the original tubes was replaced with an equal volume of saline solution (30.82 mL of 1M NaCl and 169.18 mL of water). Tubes were then centrifuged as before, and the supernatant was removed and replaced with an equal volume of saline solution. Tubes were centrifuged again, and the supernatant was removed and replaced with an equal volume of distilled water. Finally, tube contents were mixed thoroughly, covered, and frozen at −70°C for a minimum of 2 hours.

Extraction of SOD1 from erythrocytes and determination of SOD1 activity were performed as described elsewhere.¹³ Briefly, hemolysates were thawed at 37°C in a water bath and mixed gently. A 2.5-mL aliquot of each sample was transferred into a glass tube. Under a chemical hood, 1 mL of Tschushihashi reagent (3:2 [vol/vol] ethanol-chloroform) was added and the contents mixed with a vortex device for 30 seconds. Two milliliters of distilled water was added, and the tube contents were again mixed with the vortex device to obtain a thick precipitate. Samples were then incubated at 37°C in a shaking water bath for 20 minutes and centrifuged at 4,800 × g (4°C) for 10 minutes to yield a precipitate. The supernatant was recovered with a transfer pipette into a 5-inch segment of dialysis tubing.^a Dialysis buffer (sodium acetate–acetic acid buffer) was added to fill, and the tubes were closed with dialysis clips. Dialysis was performed overnight on a stir plate in a cold room (approx 4°C), and the dialysate was transferred via pipette into 15-mL glass tubes the following day. Dialysates were centrifuged at 4,800 × g (4°C) for 15 minutes. The supernatant was divided into aliquots into 1.5-mL tubes and frozen at −70°C.

The procedure by which erythrocyte SOD1 activity was measured relies on the oxidation of NADPH, which is easily measured.¹³ The method consists of a chemical reaction sequence that generates superoxide from molecular oxygen in the presence of EDTA, manganese (II) chloride, and mercaptoethanol. Oxidation of NADPH depends on the availability of superoxide anions in the medium. As soon as SOD is added to the assay mixture, this enzyme brings about the inhibition of nucleotide oxidation. Therefore, at high SOD activity, the absorbance at 340 nm remains unchanged; in the control sample (no SOD), absorbance decreases at a predictable rate. Each set of assays included its own control sample, which consisted of a cuvette in which the volume of tested sample was replaced with an equal volume of the medium used for enzyme solutions.

For each SOD assay, 137 samples were thawed at room temperature (approx 22°C) and centrifuged for 15 to 20 seconds at 4,800 × g. Tubes were then placed on ice. For each tested cuvette, the following solutions were subsequently added in via pipette: 890 μL of triethanolamine-diethanolamine HCl buffer, 40 L of NADPH, 25 μL of EDTA-MnCl₂, and 10 μL of sample (or solvent for control samples). Cuvettes were incubated at room temperature for exactly 5 minutes. Then, 100 μL of mercaptoethanol was added to each cuvette and the mixture was agitated by pipette. Solution absorbance was read at 340 nm with a spectrophotometric kinetics assay,^b and the decrease in absorbance over time was monitored for 20 minutes.

Measurements of the relative rates of absorbance in the control and sample cuvettes were made on the linear portion of their respective reaction curves 5 to 10 minutes after the addition of mercaptoethanol. The percentage inhibition was determined by dividing the sample rate by the control rate and multiplying by 100.

The amount of SOD1 required to inhibit the rate of NADPH oxidation of the control sample by 50% was defined as 1 U. A calibration curve for internal control sample was produced by testing concentrations (0.5, 1.0, 1.5, 2.0, 2.5, 5.0 μg/10 μL) of purified bovine SOD.^c The percentage inhibition of samples was converted to units of SOD1 expressed in half-maximal inhibitions; the units were obtained by substituting for activity of SOD1 in the calibration curve in the sample as estimated from the following equation derived from the calibration curve of SOD1 activity (provided by the manufacturer):

View Expanded

where x is activity of SOD1 in the calibration curve and y is the percentage inhibition. Afterward, SOD1 activity was calculated by dividing x by 1.75 μg, which is the amount of pure enzyme yielding 50% inhibition (ie, 1 U).

Determination of plasma protein concentrations via the Lowry method—The harvested plasma samples were removed from the freezer, warmed slowly, and centrifuged at 2,000 × g for 20 seconds. Then, 0.1 mL of 1% copper sulfate pentahydrate, 0.1 mL of 2% tartrate, and 10 mL of 2% Na₂CO₃ in 0.1N NaOH were mixed to make enough alkaline copper solution for 10 assays. For the standard curve, standards of bovine serum albumin were prepared in concentrations of 0.5, 1.0, 1.5, and 2.0 μg/10 L of H₂O in 1.5-mL sterile tubes. Two hundred milliliters of each standard solution was transferred to 1.5-mL sterile tubes, and 1 mL of alkaline copper solution was added to each. Tube contents were mixed with a vortex device for 20 seconds and then left to stand at room temperature for 10 minutes. One hundred microliters of phenol solution (2 mL of phenol in 2 mL of water) was added to each tube, and tube contents were mixed with a vortex device for 20 seconds. Tubes were left to stand at room temperature for 30 minutes.

Absorbance of each sample was determined at 600 nm. Linear regression was performed to establish the relationship between absorbance and protein concentration and obtain a standard curve. The total protein concentration in each sample was determined via the standard curve that was extrapolated through testing of various concentrations (0.5, 1.0, 1.5, and 2.0 g/10 μL) of bovine serum albumin.^c Subsequently, the ratio of SOD activity to the total protein concentration was computed for each sample.

Determination of plasma β-carotene and α-tocopherol concentrations—Plasma was harvested from the whole blood samples, and 1-mL aliquots of plasma were transferred to sterile polypropylene screw-cap microtubes with neoprene O rings^d containing an antioxidant mixture (100 mL of an ethanol mixture of propylgallate and EDTA) and frozen at −75°C until testing. Plasma concentrations of β-carotene and α-tocopherol were measured at the Animal Health Diagnostic Laboratory at Cornell University by means of high-performance liquid-liquid partition chromatography. Analytes of interest were detected by spectrophotometry (450 nm for 1.38 minutes for β-carotene and molecular fluorescence emission at 330 nm for 7.05 minutes for α-tocopherol) with a tandem arrangement of a variable-wavelength UV detector and a spectrofluorometric detector.

Determination of plasma GSHPx activity—Plasma GSHPx activity was determined at the Animal Health Diagnostic Laboratory at Cornell University by use of a modification of the method reported elsewhere.¹⁴ Activity was measured as the production of nicotinamide adenine dinucleotide phosphate by the action of glutathione reductase on oxidized glutathione in the presence of NADPH.

Determination of plasma lipid hydroperoxide concentrations—Plasma concentrations of lipid hydroperoxides were measured with a colorimetric, quantitative assay for lipid hydroperoxides.^e The assay was performed in accordance with the manufacturer's protocol. For each sample, 2 microcentrifuge tubes were used: one labeled as test and the other as blank. Ten microliters of enzyme (catalase [3,800 U/mL in 10mM PBS solution]; pH, 7.0) was added to each tube. Ninety microliters of the sample was then added to each tube. Tubes were mixed gently at room temperature for 2 minutes. Ten microliters of reducing agent (20mM tris [2-carboxyethyl] phosphine HCl in deionized water) was added to each sample blank tube. Ten microliters of deionized water was added to each sample test tube. Tube contents were then mixed with a vortex device for 30 seconds and incubated for 30 minutes at room temperature. Nine hundred microliters of working reagent (1 volume of color developer [25mM ferrous ammonium sulfate in 2.5M H₂SO₄ under inert gas, mixed with 100 volumes of chromogen, 125μM xylenol orange, and o-cresolsulfonphthalein-3′3″-bis [methylliminodiacetic acid]] in methanol with 4mM butylated hydroxytoluene) was added to each tube. Tubes were then mixed with a vortex device for 30 seconds and incubated for 60 minutes at room temperature. Tubes were centrifuged at 12,000 × g for 10 minutes. Samples were transferred to spectrophotometric cuvettes and the contents measured at an absorbance of 560 nm.

Concentration of lipid hydroperoxides was determined by use of the following equation:

View Expanded

where LOOH is the concentration of lipid hydroperoxides in μM, net A560 is the net absorbance at 560 nm, ϵ is 0.043 lμM^−-1 cm^−-1, and δ is the dilution factor, which was equal to 11.2 (1.010 mL/0.090 mL).

Statistical analysis—Statistical software^f was used for all analyses. Summary statistics are reported as mean ± SD. Comparisons between cases and controls with respect to age, sex, and breed were made with the 2-sample t test (age) and univariate logistic regression (breed and sex). The significance of differences between groups in the mean inhibition of erythrocyte SOD1 activities; erythrocyte SOD1 activity; plasma concentrations of total proteins, vitamins E and A, and lipid hydroperoxidases; and plasma GSHPx activity was evaluated via the Student t test. The standard curve for SOD1 activity was estimated through linear regression analysis with appropriate transformation to improve prediction. The outcome (dependent) variable was optical density, and the predictor variable was the dilution of SOD1 activity. A value of P < 0.05 was considered significant for all analyses.

Results

Animals—Eighty-eight horses with MND and 49 horses without MND of various breeds, ages, and sexes were enrolled in the study. No significant differences in these intrinsic factors were evident between cases and controls (Table 1).

Erythrocyte SOD1 activity—Although it appeared that the controls had higher erythrocyte SOD1 activity than did horses with MND, no significant difference was found in the mean inhibition percentage between the groups (Table 2). Controls had more variability in the inhibition of SOD1 activity as measured by the coefficient of variation (53%) than did cases (23%). Controls appeared to have higher mean SOD1 activity than did cases; however, there was no significant difference in the mean activities between the 2 groups.

Plasma analyte concentrations—No significant difference in the mean plasma total protein concentrations was found between controls and cases (Table 2). The ratio of mean erythrocyte SOD1 activity to plasma total protein concentration in the case group was 0.624, and the ratio in the control group was 0.660; the difference between these ratios was not significant.

Horses with MND had a significantly (P < 0.001) lower plasma concentration of vitamin E than did controls (Table 1). There were no significant differences in plasma vitamin A concentration or erythrocyte GSHPx activity between groups. Horses with MND had a higher plasma concentration of lipid hydroperoxides than did controls.

Discussion

Antioxidants play a major role in mitigating the damage associated with the excessive production of free radicals.¹⁵ Superoxide dismutase 1 is expressed in virtually every cell of all organisms more complex than bacteria and is highly conserved across species. The enzyme exists in 3 forms: cytoplasmic (SOD1), mitochondrial (SOD2), and extracellular (SOD3).¹⁰ As an antioxidant, SOD1 detoxifies the free radical superoxide anion by converting it to hydrogen peroxide and hence reduces the risk of oxidative stress in humans with familial ALS.¹⁶

In the present study, SOD1 activity was lower in horses with versus without MND, but the difference was not significant. This finding is consistent with those of other studies¹¹ regarding the apparent lack of polymorphisms in the SOD1 gene in horses with MND. It is also consistent with findings in humans with sporadic ALS, who do not differ significantly from others with respect to SOD1 activity.^17,18 Because polymorphisms exist in the human SOD1 gene in familial ALS patients, SOD1 activity has been found to differ between certain patients with and without ALS. On the other hand, polymorphisms have not been detected in the equine SOD1 gene and SOD1 activity does not appear to differ between horses with and without MND.¹¹ The epidemiological features of the disease in horses have been compared with those of sporadic ALS and nonfamilial ALS, and the findings for SOD1 are similar.^4,14 Evidence is strong for the role of the mutation in the SOD1 gene in familial ALS, but the mechanism by which this mutation may contribute to the disease is not fully understood.^9,15,19

Although there was no significant difference in erythrocyte SOD1 activity between horses with MND and controls, the groups differed in plasma vitamin E concentration. Vitamin E is essential for the integrity and optimum function of several systems in the body, including nervous, immune, reproductive, muscular, and circulatory systems, and its concentration has been found experimentally to be associated with MND in horses.^5,19 The vitamin is a known antioxidant that helps in the neutralization of free radicals and hence is used to manage some of the cases ALS and prevent adverse consequences of the disease.^20–22 This antioxidant activity blocks the chain reaction of lipid peroxidation by scavenging the intermediate peroxyl radical that is produced in the reaction.²²

In the present study, plasma vitamin A concentration and GSHPx activity did not differ between horses with and without MND, which is in agreement with the findings of our experimental studies.^3,5 Studies^17,23–25 of humans with familial or sporadic ALS yielded similar results. Vitamin A was used as a marker of oxidative stress in the study horses because of its hypothesized potential as an antioxidant; however, its role in this regard is still debated. Although the roles of vitamin A in vision, development, and general health are known, the impact of this antioxidant on MND development is yet to be established.^15,17,26

Glutathione peroxidase influences cellular health by contributing to the regulation of the redox system and protecting against ROS.²⁴ The selenium-containing enzyme GSHPx breaks down the lipid peroxides produced by metabolic processes, and the reaction relies on the oxidation of the reduced glutathione to oxidized glutathione. Hence, this enzyme is important for mitigating the cell damage associated with the presence of superoxide radicals.

Various methods to assess lipid peroxidation have been described.^23,25,27,28 In the study reported here, the degree of oxidative burden was measured through assessment of the degree of lipid peroxidation, which can be inferred from plasma lipid hydroperoxide concentration.²⁷ We found significant differences in oxidative stress between horses with and without MND, suggesting that oxidative stress may play a role in the etiology of MND and may be a trait shared with its human counterpart, ALS.^9,15,25 Reported values for by-products of lipid oxidation in healthy male humans determined with a colorimetric lipid peroxide method similar to the one we used range from 4.8 to 10.4M.²⁸

Because the present study was the first in which plasma lipid hydroperoxide concentrations were determined in horses under field conditions, no reference values were available. The range of these concentrations was wider than that of humans, which might be attributed to inclusion of controls with other conditions that might affect the generation of ROS and hence lipid peroxidation. It might also have been attributable to differences in the stage of the clinical disease or the type of dietary supplementation the horses received. Our experience is that owners supplement the diet of affected horses with vitamin E at different concentrations.^6,29 The high variability in lipid hydroperoxide concentration could lend credence to the fact that MND is multifactorial, involving several intrinsic and management factors that might influence the results of peroxidation testing at the time of blood sample collection.

Oxidative stress is unlikely the main cause of MND, but it is plausible that it contributes to disease development. One of the factors associated with equine MND and its human counterpart, ALS, is increasing age. The production of ROS also increases with age, primarily because of an increase in leakage from the mitochondrial respiratory chain.¹³ These molecules may affect the permeability of the blood-brain barrier, exposing the CNS to neurotoxic substances. The study groups of horses did not differ significantly with respect to age. In another study,³⁰ we found preliminary evidence of the relationship between oxidative stress and blood-brain barrier permeability. Additional research should help to better characterize this relationship.

In a previous study,³¹ a high correlation between plasma and CNS concentrations of α-tocopherol were identified in horses with MND and controls, with control values significantly higher than case values. Given reported findings of the endothelial accumulations of lipopigment granules in the spinal cord tissues from horses with MND,⁴ it is not unreasonable to speculate that the CNS of affected horses is affected by oxidative stress. Although a lack of association between erythrocyte SOD1 activity and MND was found in the present study, evidence is mounting for the role of oxidative stress in MND development on the basis of the observed vitamin E deficiency and the high plasma peroxide concentrations of peroxides in affected horses.³² The findings contribute to the evidence for the involvement of oxidative stress in the risk of MND; however, the role of oxidative stress in the pathogenesis of MND remains to be fully elucidated.