Whole blood glutathione peroxidase activity in Standardbred broodmares supplemented with vitamin E and selenium

Abstract

The objective of this study was to determine the effect of either oral supplementation of sodium selenite and alpha-tocopherol or intramuscular administration of a commercial preparation of selenium (Se) and vitamin E to Standardbred broodmares with low (21.0 ± 18.1 IU/g Hb) whole blood glutathione peroxidase (GPx) activity. Ten mares received 3 weekly intramuscular (IM) doses, whereas 9 mares were orally supplemented daily for 10 wk. Blood samples were collected via jugular venipuncture and the whole blood GPx activity was measured at each sampling time for the 32-week period. Both oral and intramuscular administration resulted in a marked increase in whole blood GPx activity in both groups of mares.

Introduction

Selenium (Se) is an essential micronutrient and co-factor of antioxidant enzymes that prevents oxidative damage to cells. Selenium is an essential component of gluthatione peroxidase (GPx), a cytosolic Se-containing enzyme that catalyzes the reduction of hydrogen peroxide (H₂O₂) or organic hydroperoxides to water or the corresponding alcohols using reduced glutathione (1).

Similarly, vitamin E (Vit E), particularly alpha-tocopherol, is a fat-soluble antioxidant that scavenges lipid peroxyl radicals interfering with lipid oxidation chain reactions in cell membranes thereby protecting lipid peroxidation (2). In combination, Se and Vit E work synergistically to prevent or neutralize cellular oxidative stress by detoxifying and inhibiting the formation of lipid hydroperoxides. Deficiencies of either of these antioxidants can therefore lead to an increased requirement for the other, as the body compensates for reduced antioxidant capacity.

Diseases associated with selenium deficiency occur in animals grazing on farms with Se-deficient soils, with resulting Se-deficient grains and forages (3–5). Selenium-deficient soils are found in many parts of the world, including the United States, Canada, Australia, New Zealand, and Europe (6). A survey of the Se content of feedstuffs grown in 52 counties throughout Ontario showed that 79% of grains (n = 450) and 86% of forages (n = 415) contained less then 0.10 ppm Se and some samples contained 0.03 ppm or less (7).

It has been determined that serum Se levels in Ontario horses depend on the origin of the fodder and location of the horse’s farm (8,9). Selenium deficiencies are therefore most often seen on farms using solely locally grown feeds (8,10). As clinical signs arising from deficiencies of Vit E and Se can be severe, prevention of these deficiencies is critical in equine nutritional management (6).

The Se status of mammals has been assessed by measuring an Se-containing enzyme, GPx (EC 1.11.1.9) (11,12). Glutathione peroxidase plays a major and important role in the defensive function against oxidative stress and many of the nutritional effects of Se can be explained by its role in GPx (11).

In horses, the activity of GPx in blood has been shown to be related to blood Se concentration and dietary Se intake. Glutathione peroxidase activity in erythrocytes is considered a suitable indicator of Se status in horses (4,10,12–14). In pasture-fed horses, the activity of GPx appears to depend on the locality where the horses are grazed (12).

It has been shown that Se supplementation increases serum Se and whole blood GPx activity (15,16). Injectable and oral supplementation of both Se and Vit E, at the recommended doses, results in adequate circulating levels; however, regular assessment of the serum or plasma concentration is recommended. Dietary supplementation of both is also generally considered safer, although Se toxicity can occur that can result in hoof and haircoat abnormalities (6).

Nutritional muscular dystrophy (NMD) in foals, also known as white muscle disease or dystrophic myodegeneration, is a noninflammatory degenerative disease that has been associated primarily with a deficiency of Se and to a lesser extent of Vit E (5,8,12,17,18). Most cases occur in foals during the first 2 wk of life, affecting the skeletal and cardiac muscles (4,5,17,19).

Selenium deficiency is accompanied by low activity of Se-containing enzymes such as GPx. A linear relationship between whole blood GPx activity and whole blood Se concentrations has been reported in horses (10,12,14). In the present study, the whole blood GPx activity was positively associated with the serum Se concentration in Standardbred horses in southern Ontario (r = 0.83). The whole blood GPx activities ranged from 25 to 180 IU/g hemoglobin (Hgb) and serum Se concentrations ranged from 0.04 to 0.25 μg/g, which is consistent with similar correlation coefficient reported in horses (12–14,20). It was hypothesized that supplementation with Se and Vit E would increase the whole blood GPx activity in mares.

Therefore, the objective of this study was to determine the effect of either oral supplementation of sodium selenite and alpha-tocopherol or intramuscular administration of a commercial preparation of Se and Vit E to Standardbred broodmares with low GPx values.

Materials and methods

Farm

In a previously published study, blood samples were obtained from 193 Standardbred brood mares located on 8 farms in southern Ontario (21,22). The mares were categorized based on whether they were residents or non-residents of the farm. The resident mares were those mares that remained on that farm for the full year, whereas non-resident mares were only on the farm for approximately 2 to 3 mo for breeding purposes.

On 1 of the 8 farms (farm 7), the resident mares had significantly lower whole blood GPx activities than the non-resident mares. The resident mares on this farm were fed hay and grain grown on the farm and did not receive any mineral supplementation other than access to salt blocks in the paddocks.

Animals

A total of 19 adult Standardbred broodmares located on farm 7 were included in the study. All 19 mares grazed together in the same paddocks during the study period. Prior to supplementation, the mean whole blood GPx activity of the group of mares (N = 19) was 21.0 ± 18.1 IU/g Hb, which was markedly lower than the mean GPx values (70.5 ± 34.9 IU/g Hb) determined in a previous study in 193 Standardbred broodmares in southern Ontario (21,22).

Experimental procedures were approved by the Animal Care Committee of the University of Guelph and conformed to the standards of the Canadian Council on Animal Care.

Oral supplementation

Ten of the 19 mares were supplemented with a commercial preparation of Vit E and Se (Alphasel Powder; Armitage Carroll, London, Ontario). This supplement was added to the basal diet and provided an additional 2 mg of Se and 1000 units of Vit E per day. The daily oral supplementation was discontinued after 10 wk and whole blood GPx activity was monitored for the following 32 wk.

Intramuscular administration

The remaining 9 mares on farm 7 that did not receive oral Vit E and Se supplementation were administered a commercial preparation of Vit E and Se (E-SE Injectable; Merck Animal Health, Rahway, New Jersey, USA) by deep intramuscular (IM) injection in the cervical muscles using an 18G × 2-inch needle every 7 d for 3 consecutive weeks. Each injection provided 25 mg of Se and 680 units of Vit E. Whole blood GPx activity was then monitored for 32 wk following the first intramuscular injection of Vit E and Se.

Blood sampling

Blood samples were collected via jugular venipuncture prior to IM administration bi-weekly for 10 wk. The mares were then injected at weekly intervals for a total of 3 injections. After the first injection, they were sampled weekly for 8 wk, then at 20, 26, 28, 31, 37, 39, and 42 wk. The oral-supplemented group was sampled bi-weekly until supplementation was discontinued. Blood was collected weekly for the following 8 wk and then at 20, 26, 28, 30, 37, 39, and 42 wk. Samples were collected using Vacutainer EDTA-containing tubes and serum tubes, transported in ice, and processed within 3 h after collection. The whole blood GPx activity was measured at each sampling time for the 30-week period post-supplementation.

Whole blood glutathione peroxidase activities

A phosphate buffer solution was prepared and the pH adjusted to 7.0. Reduced glutathione (110 g) was added to 62.5 mL of the phosphate buffer solution. After mixing, 2.5 mL of the mixture was discarded. To the remaining 60 mL, 100 μg of glutathione reductase and 400 μL of water, and 12.5 mg of nicotinamide adenine dinucleotide phosphate (NADPH) and 500 mL of water were added. This reaction mixture was gently mixed and 1 mL of the solution was transferred into each cuvette along with 50 μL of blood lysate. Prior to reading the samples, 50 μL of tert-butyl hydroxyperoxidase (t-BHP) solution (8 mM) was added to each cuvette.

The samples, standards, and blanks were all read on an LKB 8600 Reaction Rate Analyzer (LKB-Produkter AB, S-16/25 Bromo, Sweden). The spectrometer was set at 340 nm, using a blue filter, normal aperture, and cuvette compartment and cuvettes at 25°C. The samples were read 1/min and the results reported as microliters (μL) of glutathione peroxidase activity. The glutathione peroxidase activity measured was used to calculate the activity per grams of hemoglobin (μ/g Hb) per sample. The repeatability of the technique for measuring GPx activity was investigated by testing 1 blood sample 60 times in a single automated run. A comparison between runs was conducted during the routine analysis by using duplicate samples and a standard bovine blood sample with each run.

Results

Oral supplementation

The mean ±SD whole blood GPx value in the 10 mares supplemented daily for 10 wk with oral sodium selenite and Vit E increased above the previously reported means (70.5 ± 34.9 IU/g Hb) for broodmares in Southern Ontario (Figure 1). The mean ±SD whole blood GPx activity reached a mean of 80.0 ± 12.4 IU/g Hb at 3 wk following the end of oral supplementation and remained elevated for 8 wk. The whole blood GPx activity began to gradually decrease at approximately 20 wk after supplementation was discontinued.

Figure 1.

Mean whole blood glutathione peroxidase (GPx) activity (IU/g Hb) in 10 Standardbred mares supplemented with oral vitamin E and sodium selenite daily for 10 wk (−10 to 0 weeks). Following the cessation of the supplementation, mean GPx activity was determined for the next 32 wk. Each circle represents the mean ± SD whole blood GPx activity.

Intramuscular administration

The mean whole blood GPx increased from 27.9 ± 18.1 IU/g Hb to 72.1 ± 8.3 IU/g Hb at 16 wk following the first IM dose. The activity of GPx in blood samples collected from the mares receiving IM supplementation is shown in Figure 2.

Figure 2.

Mean whole blood glutathione peroxidase (GPx) activity (IU/g Hb) in 9 Standardbred broodmares injected with 25 mg selenium and 680 units vitamin E by intramuscular injection at weekly intervals for 3 wk (arrows). Mean GPx activity was determined for the following 32 wk. Each circle represents the mean ± SD whole blood GPx activity.

Discussion

A marked increase in whole blood GPx activity occurred in both groups of mares, those that received dietary sodium selenite and Vit E for 10 wk and those that received 3 weekly injections of Vit E and Se (Figures 1, 2). The orally supplemented group had higher whole blood GPx values than the mares that received 3 weekly injections of Vit E and Se.

The dietary supplementation of selenium recommended by the National Research Council (NRC) (1 mg/d 500 kg horse) or greater (2.5 to 3 mg/d 500 kg horse) increases Se status and GPx activity significantly (23,24). Direct feeding of the daily requirements is preferred since voluntary Se intake, i.e., salt blocks, can be unreliable. Furthermore, the consumption of certain elements such as sulfur and copper can interfere with the gastrointestinal absorption of Se or physiologic stress conditions such as pregnancy, lactation, heavy exercise, or growth periods can increase the need for antioxidant capacity and therefore require higher Se levels (1).

It is also suspected that mature horses can sustain suboptimal concentrations of blood and liver selenium for months before showing clinical signs associated with this deficiency. Some horses can be clinically asymptomatic despite low blood selenium concentrations or can experience periods of subclinical disease. These horses potentially have suboptimal immune responses and suboptimal reproductive function, and pregnant mares may be at risk of dystocia and abortion or produce foals with Se-associated deficiency (6).

The mares that received 3 intramuscular injections at weekly intervals reached a higher whole blood GPx activity of 72.0 ± 8.3 IU/g Hb at week 16 after the first intramuscular injection. Previous studies have shown that parenteral Se administration results in a rapid increase in Se levels and GPx activity in mares (25). Parenteral Vit E and Se is the preferred route of administration for treating clinically deficient animals.

As affected animals may require prolonged treatment that sometimes extends over multiple weeks, repeated doses are administered to achieve clinical improvement and resolution of clinical signs. Complete recovery can take months, and permanent fibrosis and atrophy of damaged musculature can occur. Repeated IM injections can be painful however and injection site reactions have been reported (6). Dietary supplementation is safe and effective at maintaining adequate levels and preventing the development of clinical conditions associated with antioxidant deficiencies.

Previous studies in horses have also shown that the GPx activity increases after 28 d of Se supplementation, although this increased GPx activity only became statistically significant after 56 d of supplementation (23). It is notable that some studies have not observed an increase in plasma GPx activity with Se supplementation (16). This lack of response to Se supplementation has been attributed to an adequate amount of Se in the environment of the diet of the horses and that horses in these studies had already reached a plateau (16,23,26).

Previous studies have reported that whole blood GPx activity in horses required at least 3 wk to respond to Se supplementation (27) and approximately 5 to 6 wk to increase significantly (10,15,27). This delayed response has also been noted in previous Se supplemented trials (10,12,15,23,27). It has been proposed that this delayed response is a result of Se being incorporated into erythrocyte GPx only during erythropoiesis and attributed to the time necessary for newly formed red blood cells (RBC) to become a substantial proportion of the mature RBC population (10,15).

The amount of erythrocyte GPx is dependent on the amount of Se that is available for synthesis. Maylin et al (10) also reported that dietary Se had a marked effect on blood GPx activity, which increased from 15.2 to 29.3 units after 11 wk of 1 mg Se supplementation in their feed. Similarly, in this study, although the increases in whole blood GPx activity occurred following Se supplementation, they did not occur immediately, as observed in another study (15).

In a study to determine the lifespan of red blood cells in horses, Carter et al (28) administered 0.2 mg (1 mCi) of ⁷⁵selenomethionine intravenously to 3 Standardbred mares and recorded the lifespan of labelled red blood cells for 250 d after the radio-selenium injection. Their study reported a red cell lifespan of 155 ± 10 days of 75 Se-selenomethionine radiolabelled equine erythrocytes of Standardbred mares (28). In this study, the whole blood GPx activity observed in the broodmares following IM administration of Se and alpha-tocopherol closely parallels that previously reported (28).

The present study also demonstrated that continuous Se supplementation is required to maintain adequate GPx enzymatic activity as an indicator of Se status (Figure 1). Important limitations of this study include the lack of a comprehensive assessment of the antioxidative status of the supplemented mares. Measurement of other factors, such as serum Se and Vit E levels, oxidative stress markers, immune function indicators, and vitamin E-specific parameters would have enhanced the evaluation of the effects of Vit E and Se supplementation.

A comparative statistical analysis could not be carried out between the supplemented groups, as many variables, such as length of supplementation, doses, intervals, and sampling schemes, differed between groups. However, the study was not designed to test the effect of dietary versus parenteral supplementation of Vit E and Se in this group of broodmares.

In addition, only a small group of Standardbred broodmares from a single farm were enrolled in this study. The results from parenteral and oral administration of Vit E and Se to a selected group of mares at a single location may not reflect those of a larger, heterogeneous, and geographically diverse population. Furthermore, mares may have higher antioxidant demands according to their physiological status, which may affect the observations of this study.

Selenium toxicity is rare but can potentially result in hoof and haircoat abnormalities. A maximum daily dose of selenium is therefore recommended, and serum plasma concentration should be assessed regularly.

In summary, oral and intramuscular injection of Se resulted in a significant increase in GPx activity in both groups of mares in this study. As observed in previous studies, however, there is a delay in the increase in whole blood GPx activity due to the incorporation of Se during erythropoiesis.