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Dietary selenium effects on milk eicosanoid concentration in dairy cows during coliform mastitis
PROSTAGLANDINS
DIETARY SELENIUM EFFECTS ON MILK EICOSANOID CONCENTRATION DAIRY COWS DURING COLIFORM MASTITIS
IN
J.F. Maddox, C.C. Reddy, R.J. Bberhart, and R.W. Schola The Penneylvania State University Department of Veterinary Science University Park, PA. 16802
ABSTRACT The effect of seleniumdeficiency on the productprofile of arachidonicacid oxidation by enzymaticpathwaysin Holstein cows with experimentally-induced coliform mastitis was investigated. The animals were fed dairy rations containing 0.05 mg Se/kg dry matter, with the supplemented group receiving additional Se to increase the dietary concentration to approximately 0.35 mg Se/kg dry matter. Cows were inoculated intracisternally with 30 colony-forming-units of EschericI~ia coli at 1416 weeks of lactation. Eicosanoids and bacteria numbers were recorded at various intervals of time for 60 h postinoculation. Milk from cows fed the Se-depleted diet had significantly higher (~~0.05) concentrations of TXJ3, between 24 and 48 h and 6-keto-PGF,, between 24 and 60 h postinoculation. Milk PGE, concentration was significantly higher in the Se-deficient group at 24 h, whereas LTB, was higher between 36 and 60 h postinoculation in the Se-deficient cows (~~0.05). Milk bacteria numbers were significantly higher between 16 and 24 h postinoculation in the Sedeficient group and three of the four cows in this group required euthanasia, whereas all four cows in the Se-supplemented group recovered without therapeutic intervention. These data indicate marked effects of dietary Se on milk eicosanoid The changes in eicosanoid concentrations in response to an E. coli infection. concentrations may be associated with the altered pathogenesis and outcome of mastitis in a Se-deficient state. INTRODUCTION Selenium (Se) is recognized as an essential micronutrient and an integral component of the mammalian enzyme, Se-dependent glutathione peroxidase (SeGSH-Px). Se-GSH-Px plays an important role in the modulation of hydroperoxide concentration in tissues, thus preventing peroxidative damage to cellular membranes and organelles. Dietary Se concentration has been positively correlated to tissue SeGSH-Px activity in different species (1,2). Se status has been shown to influence nonspecific, humoral and cellular immune system response and has been the subject of recent reviews (3,4). Previous research in our laboratory indicates that Se status is positively correlated with mammary resistance to infection. For example, Erskine e d (5) observed that Se-deficient (-Se) cows experimentally challenged with Eschmichia coli in the mammary gland had higher bacteria counts in milk, a slower somatic cell response and an increased duration of infection compared to the Sesupplemented (+Se) group. In addition, neutrophils isolated from the milk of -Se
OCTOBER 1991 VOL. 42 NO. 4
369
PROSTAGLANDINS cows had reduced bactericidal activity and released large amounts of hydrogen peroxide compared to neutrophils from +Se cows (6). Se-GSH-Px has been shown to influence the activity of various enzymes associated with the arachidonic acid cascade (7,8,9). Arachidonic acid metabolites have pervasive actions on immune cells, which may activate or inhibit immune function and inflammatory response. In the non-specific immune response, which is the principal system active in coliform mastitis, polymorphonuclear leukocyte (FMNL) function and activity are highly influenced by eicosanoids. LTB, in particular, is a potent PMNL modulator, inducing chemotaxis (10-12) and chemoactivation (1214), as well as degranulation (15,16). Cyclooxygenase products appear to have varying effects on PMNL activation; PGE, was shown to inhibit superoxide anion production by human PMNL (17,18). In contrast, TXA, was suggested to be a promotor of neutrophil H202 production in ischemic tissue (19). In the investigation of the role of eicosanoids in acute mastitis, several studies have demonstrated elevated eicosanoid concentrations in milk (20-23). Therefore, by modulating the activities of key enzymes involved in PG and LT biosynthesis, SeGSH-Px may influence the response to bacterial infection. The purpose of the present study was to examine the influence of dietary Se on the production of arachidonic acid metabolites during experimentally-induced acute coliform mastitis in dairy cows.
MATERIALS
AND METHODS
Animals and diets: Eight Holstein cows in their second or third lactation were used in this study. For animals fed the Se-depleted diet, 4 cows were maintained on a standard dairy ration prepared from locally grown corn, soybean meal, oats, silage, hay and a vitamin-mineral supplement, which had a Se concentration of approximately 0.05 mg/kg of dry matter for the total ration. Another group of 4 cows was fed the same diet supplemented with 6 mg Se/cow/day as sodium selenite to bring the Se content of the diet to approximately 0.35 mg/kg of dry matter. The animals had been maintained on their respective diets for at least one year. Blood samples were obtained from all animals each month to monitor blood Se concentration (24). Bacterial inoculation and sample collection: Approximately 30 colony forming units (CFU) of E. cofi (MacDonald 487) in 2 ml phosphate-buffered saline solution (PBSS) were aseptically inoculated in one rear quarter of the mammary gland of each cow at 14-16 weeks of lactation. Quarters selected for use in the trial had been free of bacterial infection during the current lactation as determined by weekly microbiological culture of milk samples. Starting at the time of bacterial inoculation, milk samples were collected every 4 h for 24 h to quantitate bacteria and somatic cells and every 12 h for eicosanoid measurement Subsequent samples were collected at 12 h intervals from 36 to 84 h following inoculation. For eicosanoid measurement, 50 ml of milk was collected into a vial containing a final concentration of 50 PM indomethacin (Sigma, St. Louis, MO) and 50 PM nordihydroguaiaretic acid (Sigma, St. Louis, MO) to inhibit further
OCTOBER 1991 VOL. 42 NO. 4
PROSTAGLANDINS metabolism of arachidohic acid. Samples were cooled to 4°C and the eicosanoids were extracted within 4 h of collection. Milk bacteriologic culture and somatic cell counts: Milk samples were thoroughly mixed and serially-diluted in 0.15M PBSS. Dilutions were plated (0.1 ml) on blood agar and incubated overnight at 37°C. Milk bacteria concentrations were calculated from CFU counts on the agar plates. Milk somatic cell counts were determined with a Coulter Counter (Coulter Electronics, Hialeah, FL) (25). Extraction and measurement of arachidonic acid metabolites in milk: A two-step solid phase extraction method was employed, which is a modification of the method in the radioimmunoassay (RIA) instruction manual provided with New England Nuclear (Boston, MA) RIA kits. The milk fat and cells were separated by centrifugation at 27,000g for 15 min and 20-25 ml aliquots of the supernatant were acidified with 0.5 ml 6N HCl to precipitate proteins and the precipitate was removed by centrifugation at 27,000g for 10 min. Two to 5 ml of this supernatant were applied to a preprimed Bakerbond 500 mg C,, cartridge (J.T. Baker, Phillipsburg, NJ) and washed with 5 ml acidified water (pH 3.5). Eicosanoids were eluted from the column with 2 ml ethyl acetate into a tube containing 2 ml hexane. The hexane:ethyl acetate mixture was applied to a preprimed Bakerbond 500 mg silica column (J.T. Baker, Phillipsburg, NJ) and subsequently washed with 1 ml of each of the following solvents: hexane:ethyl acetate (80:20), hexane:ethyl acetate (60:40), and hexane:ethyl acetate:methanol(60:40:2). Finally, eicosanoids were eluted from the cartridge with 2 ml of methanol and stored at -20°C until analysis. Radiolabelled internal standards were included for all metabolites extracted and recoveries were calculated. For RIA, methanol was evaporated under nitrogen and the sample resuspended in assay buffer. Each sample was analyzed for PGE, 6keto-PGF,,, TXB, and LTB, using commercially available R_IAkits from New England Nuclear @‘GE, and LTB,) and Advanced Magnetics (6-keto-PGF,, and TXB; Cambridge, MA). Statistical analvses: Data collected for milk bacteria numbers, somatic cell counts and eicosanoid concentrations were subjected to analyses of variance using the general linear models procedure and treatment means were compared by Duncan’s multiple-range tests (26). Means were compared between dietary Se groups only within each sampling time for PGE, TXB, LTB,, 6-keto-PGF,, and Log,, milk bacteria counts. Student t test was used for comparison of mean blood selenium values between the two dietary groups at the time of bacterial infusion. Statistical significance for all comparisons was determined at a P value <0.05. RESULTS The mean blood Se concentration of the -Se cows (0.07 + 0.01 pg/ml) was significantly lower than that of the +Se cows (0.24 + 0.01 pg/ml) at the time of bacterial challenge (P
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Systemic symptoms of coliform mastitis manifest in all cows inoculated included anorexia, neutropenia, pyrexia (data not shown) and recovery of E. cdi from the mammary gland. Mean milk somatic cell counts were less than 2 x 10’ of milk preinfusion, increased to more than 1 x 16 (~45% PMNL) at 16 h postinoculation remained at this level through 60 h postinoculation. clinical data,
numbers in the milk were the most notably different bacteria concentrations observed in the -Se cows at 16,2O and 24 h postinoculation. bacteria concentration bacteria concentration required euthanasia due to persistent
anorexia and hypocalcemic
increased dramatically
observed at 24 h postinoculation
0
E %i
21,000
-
19,000
-
15,000
-
continued up to 48 h postinoculation.
12,000 -
a s!l
9,000 -
F 6,000 -
3,000
-
0
12
24
36
46
60
Hours Postinoculation
Fig 1. Mean concentration & SEM) of TXB, in milk following intramammary inoculation. ‘P
E. coIi
Milk PGE, concentration changed very little in the +Se cows, but increased exponentially in the -Se cows between 12 and 24 h postinoculation to a peak concentration of 7000 pg/ml, which was significantly higher than in the +Se cows (fig. 2).
OCTOBER 1991 VOL. 42 NO. 4
PROSTAGLANDINS
7000
-
6000
-
+Secows
12
0
36
24
46
60
Hours Postinoculation Fig 2. Mean concentration h SEM) of PGE, in milk following intramammary inoculation. *P
E. coli
The 6-keto-PGF,, concentration in milk increased only in the -Se cows, and was significantly higher than the +Se cows between 24 and 60 h postinoculation (fig. 3).
2500
-
h 5
*
+Se COWS A
. ....
: ; 8'
:I
-Secows 2000
-
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;
g Y cb
1000
*
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: :
1500--
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.*...,
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4
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t
-
500
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0
t 0
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: *
12
a
24
36
48
60
Hours Postinoculation Fig 3. Mean concentration (+SEM) of 6-keto-PGF,, in milk following intramammary E. coli inoculation. *P
OCTOBER 1991 VOL. 42 NO. 4
The LTB, concentration was also significantly higher in the milk from the -Se cows than in the +Se group, but this occurred later (after 36 h) in the postinoculation period than the other metabolites (fig. 4).
1600
1200 h 2E
1000
4 $
600
5
600
0
t
I
0
12
24
36
46
60
Hours Postinoculation Fig 4. Mean concentration c_LSEM) of LTB, in milk following intramammary inoculation. V’cO.05 between the two dietary groups at time point indicated.
E. coli
DISCUSSION The present study was based on the premise that biosynthesis of PG’s and LT’s is altered during the Se-deficient state in dairy cows and that this might be one of the underlying mechanisms for the pathogenesis of mastitis. To elucidate this mechanism, we have systematically analyzed milk eicosanoid concentrations in -Se cows and compared them with the values obtained for +Se cows during the course of experimentally-induced coliform mastitis. Milk from the -Se cows contained significantly higher concentrations of PGE, 6-keto-PGF,,, TXB, and LTB, than milk from the +Se cows at several time points The higher eicosanoid concentrations were associated with postinoculation. significantly greater numbers of bacteria in the milk of the -Se cows. The increased milkbacteria concentration may be due to impaired killing abilities of the -Se PMNL. Previous studies have shown inhibited degranulation and microtubule function (27) and decreased killing of E. coli by neutrophils from -Se animals (6). It is conceivable that the increased number of bacteria released greater quantities of endotoxin and increased liberation of inflammatory mediators such as histamine, serotonin and arachidonic acid, which might have escalated inflammation and tissue damage. In turn, in the Se-GSH-Px depleted state, higher tissue hydroperoxide concentration may have aided the activation of cyclooxygenase and
374
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PROSTAGLANDINS lipoxygenase enzymes., It is known that these enzymes have an obligatory requirement for fatty acid hydroperoxides for maintenance of maximal activity (28 30). Thus, as a result of increased substrate availability and enzyme activity, the eicosanoid levels in the milk of the -Se cows were all significantly higher than in the +Se cows. Though other studies have shown decreased production of LTB, (31,32) and PGI, (33) in cells from -Se animals, it is likely that the quantities of arachidonic acid liberated due to tissue damage in the -Se cows obscured the more subtle effects of a Se-GSH-Px deficiency on individual metabolites of the arachidonic acid cascade. This is supported by the fact that the rate-limiting step in eicosanoid biosynthesis in viva is the release of esterified arachidonic acid from cell membranes by phospholipases (34,35). The clinical symptoms and sequelae (anorexia, hypocalcemia) from E. coli inoculation were much more severe in the -Se cows than the +Se cows, and euthanasia was necessary for three of the four -Se cows. The severity of symptoms could be at least partially accounted for by the increased eicosanoid concentrations because several studies implicate these eicosanoids in the pathogenesis of inflammatory and endotoxin-induced diseases @O-23,36,37). However, other Se effects on immune response (3,4) may also be responsible for contributing to the markedly different outcomes of the two dietary groups from E. coli inoculation of the mammary gland.
ACKNOWLEDGEMENTS This research was partially supported by a grant from the Pennsylvania Department of Agriculture and by grants from the United States Department of Agriculture (CRSR-23171) and Public Health Service (Rol 31245).
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Showell, H.J., P.H. Naccache, I’. Borgeat, S. Picard, I’. Ballerand, E.L. Becker and RI. Sha’afi. Characterization of the secretory activity of leukotriene B, toward rabbit neutrophils. J. Immunol. m:811-816, 1982.
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Szczeklik, A., R.J. Gryglewski, M. Wandzilak M. Grzywacz, J. Radwan and J. Musial. Effects of prostanoids on generation of superoxide ions by human neutrophils. In: Prostaglandins in Clinical Research. (H. Sinzinger and K. Schror, eds.) Alan R. Liss, Inc., New York, 1987, pp.215-219.
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Gryglewski, R.J., A. Szczeklik and M. Wandzilak. The effect of six prostaglandins, prostacyclin and Iloprost on generation of superoxide anions by human polymorphonuclear leukocytes stimulated by zymosan or formylmethionyl-leucyl-phenylalanine. Biochem. Pharm. &4209-4213, 1987.
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Paterson, I.S., J.M. Klausner, G. Goldman, L. Kobzik Hechtman. Thromboxane mediates the ischemia induced burst (Abst.). FASEB J. aA908, 1989.
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Anderson, K.L., H. Kindahl, A. Petroni, A.R. Smith and B.K. Gustafsson. Arachidonic acid metabolites in milk of cows during acute coliform mastitis. Am. J. Vet. Res. 46:1573-1577, 1985.
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Zia, S., S.N. Giri, J. Cullor, P. Emau, B.I. Osburn and R.B. Bushnell. Role ofeicosanoids, histamine and serotonin in the pathogenesis of Klebsiella pneumoniae induced bovine mastitis. Am. J. Vet. Res. @1617-1625,1987.
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Rose, D.M., S.N. Giri, S.J. Wood and J.S. Cullor. Role of leukotriene B, in the pathogenesis of Klebsiella pnerlmoniae-induced bovine mastitis. Am. J. Vet. Res. a915-918, 1989.
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Whetter P.A. and D.E. Ullrey. Improved fluorometric selenium. J Assoc Off Anal Chem, a927-930, 1978.
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Statistics,
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method
SAS Institute,
J., R.E. Harris, R.L. Baihner and L.A. function in glutathione peroxidase-deficient J. Reticula. Sot. 259-66, 1980.
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Aziz, E. and P.H. Klesius. Effect of selenium deficiency on caprine polymorphonuclear leukocyte production of leukotriene B4 and its neutrophil chemotactic activity. Am. J. Vet. Res. %426-428, 1986.
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Gairola, C. and H.H. Tai. Selective inhibition of leukotriene Bd biosynthesis in rat pulmonary alveolar macrophages by dietary selenium deficiency. Biochem. Biophys. Res. Comm. 132:397-403, 1985.
33.
Funk, C.D., W. Boubez and W.S. Powell. Effects of selenium-deficient diets on the production of prostaglandins and other oxygenated metabolites of arachidonic acid and linoleic acid by rat and rabbit aortas. Biochim. Biophys. Acta 921:213-220, 1987.
34.
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Vonkeman, H. and D.A. Van Dorp. The action of prostaglandin synthetase on Zarachidonyl-lecithin. Biochim. Biophys. Acta &~430-432, 1968.
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Ball, H.A., J.A. Cook, WC. Wise and P.V.’ Halushka. Role of thromboxane,prostaglandins and leukotrienes in endotoxic and septic shock. Int. Care Med. =116-126, 1986.
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Editor:
378
W.E.M.
Lands
Received:
5-17-91
acid, and superoxide
Accepted:
7-29-91
OCTOBER 1991 VOL. 42 NO. 4
DIETARY SELENIUM EFFECTS ON MILK EICOSANOID CONCENTRATION DAIRY COWS DURING COLIFORM MASTITIS
IN
J.F. Maddox, C.C. Reddy, R.J. Bberhart, and R.W. Schola The Penneylvania State University Department of Veterinary Science University Park, PA. 16802
ABSTRACT The effect of seleniumdeficiency on the productprofile of arachidonicacid oxidation by enzymaticpathwaysin Holstein cows with experimentally-induced coliform mastitis was investigated. The animals were fed dairy rations containing 0.05 mg Se/kg dry matter, with the supplemented group receiving additional Se to increase the dietary concentration to approximately 0.35 mg Se/kg dry matter. Cows were inoculated intracisternally with 30 colony-forming-units of EschericI~ia coli at 1416 weeks of lactation. Eicosanoids and bacteria numbers were recorded at various intervals of time for 60 h postinoculation. Milk from cows fed the Se-depleted diet had significantly higher (~~0.05) concentrations of TXJ3, between 24 and 48 h and 6-keto-PGF,, between 24 and 60 h postinoculation. Milk PGE, concentration was significantly higher in the Se-deficient group at 24 h, whereas LTB, was higher between 36 and 60 h postinoculation in the Se-deficient cows (~~0.05). Milk bacteria numbers were significantly higher between 16 and 24 h postinoculation in the Sedeficient group and three of the four cows in this group required euthanasia, whereas all four cows in the Se-supplemented group recovered without therapeutic intervention. These data indicate marked effects of dietary Se on milk eicosanoid The changes in eicosanoid concentrations in response to an E. coli infection. concentrations may be associated with the altered pathogenesis and outcome of mastitis in a Se-deficient state. INTRODUCTION Selenium (Se) is recognized as an essential micronutrient and an integral component of the mammalian enzyme, Se-dependent glutathione peroxidase (SeGSH-Px). Se-GSH-Px plays an important role in the modulation of hydroperoxide concentration in tissues, thus preventing peroxidative damage to cellular membranes and organelles. Dietary Se concentration has been positively correlated to tissue SeGSH-Px activity in different species (1,2). Se status has been shown to influence nonspecific, humoral and cellular immune system response and has been the subject of recent reviews (3,4). Previous research in our laboratory indicates that Se status is positively correlated with mammary resistance to infection. For example, Erskine e d (5) observed that Se-deficient (-Se) cows experimentally challenged with Eschmichia coli in the mammary gland had higher bacteria counts in milk, a slower somatic cell response and an increased duration of infection compared to the Sesupplemented (+Se) group. In addition, neutrophils isolated from the milk of -Se
OCTOBER 1991 VOL. 42 NO. 4
369
PROSTAGLANDINS cows had reduced bactericidal activity and released large amounts of hydrogen peroxide compared to neutrophils from +Se cows (6). Se-GSH-Px has been shown to influence the activity of various enzymes associated with the arachidonic acid cascade (7,8,9). Arachidonic acid metabolites have pervasive actions on immune cells, which may activate or inhibit immune function and inflammatory response. In the non-specific immune response, which is the principal system active in coliform mastitis, polymorphonuclear leukocyte (FMNL) function and activity are highly influenced by eicosanoids. LTB, in particular, is a potent PMNL modulator, inducing chemotaxis (10-12) and chemoactivation (1214), as well as degranulation (15,16). Cyclooxygenase products appear to have varying effects on PMNL activation; PGE, was shown to inhibit superoxide anion production by human PMNL (17,18). In contrast, TXA, was suggested to be a promotor of neutrophil H202 production in ischemic tissue (19). In the investigation of the role of eicosanoids in acute mastitis, several studies have demonstrated elevated eicosanoid concentrations in milk (20-23). Therefore, by modulating the activities of key enzymes involved in PG and LT biosynthesis, SeGSH-Px may influence the response to bacterial infection. The purpose of the present study was to examine the influence of dietary Se on the production of arachidonic acid metabolites during experimentally-induced acute coliform mastitis in dairy cows.
MATERIALS
AND METHODS
Animals and diets: Eight Holstein cows in their second or third lactation were used in this study. For animals fed the Se-depleted diet, 4 cows were maintained on a standard dairy ration prepared from locally grown corn, soybean meal, oats, silage, hay and a vitamin-mineral supplement, which had a Se concentration of approximately 0.05 mg/kg of dry matter for the total ration. Another group of 4 cows was fed the same diet supplemented with 6 mg Se/cow/day as sodium selenite to bring the Se content of the diet to approximately 0.35 mg/kg of dry matter. The animals had been maintained on their respective diets for at least one year. Blood samples were obtained from all animals each month to monitor blood Se concentration (24). Bacterial inoculation and sample collection: Approximately 30 colony forming units (CFU) of E. cofi (MacDonald 487) in 2 ml phosphate-buffered saline solution (PBSS) were aseptically inoculated in one rear quarter of the mammary gland of each cow at 14-16 weeks of lactation. Quarters selected for use in the trial had been free of bacterial infection during the current lactation as determined by weekly microbiological culture of milk samples. Starting at the time of bacterial inoculation, milk samples were collected every 4 h for 24 h to quantitate bacteria and somatic cells and every 12 h for eicosanoid measurement Subsequent samples were collected at 12 h intervals from 36 to 84 h following inoculation. For eicosanoid measurement, 50 ml of milk was collected into a vial containing a final concentration of 50 PM indomethacin (Sigma, St. Louis, MO) and 50 PM nordihydroguaiaretic acid (Sigma, St. Louis, MO) to inhibit further
OCTOBER 1991 VOL. 42 NO. 4
PROSTAGLANDINS metabolism of arachidohic acid. Samples were cooled to 4°C and the eicosanoids were extracted within 4 h of collection. Milk bacteriologic culture and somatic cell counts: Milk samples were thoroughly mixed and serially-diluted in 0.15M PBSS. Dilutions were plated (0.1 ml) on blood agar and incubated overnight at 37°C. Milk bacteria concentrations were calculated from CFU counts on the agar plates. Milk somatic cell counts were determined with a Coulter Counter (Coulter Electronics, Hialeah, FL) (25). Extraction and measurement of arachidonic acid metabolites in milk: A two-step solid phase extraction method was employed, which is a modification of the method in the radioimmunoassay (RIA) instruction manual provided with New England Nuclear (Boston, MA) RIA kits. The milk fat and cells were separated by centrifugation at 27,000g for 15 min and 20-25 ml aliquots of the supernatant were acidified with 0.5 ml 6N HCl to precipitate proteins and the precipitate was removed by centrifugation at 27,000g for 10 min. Two to 5 ml of this supernatant were applied to a preprimed Bakerbond 500 mg C,, cartridge (J.T. Baker, Phillipsburg, NJ) and washed with 5 ml acidified water (pH 3.5). Eicosanoids were eluted from the column with 2 ml ethyl acetate into a tube containing 2 ml hexane. The hexane:ethyl acetate mixture was applied to a preprimed Bakerbond 500 mg silica column (J.T. Baker, Phillipsburg, NJ) and subsequently washed with 1 ml of each of the following solvents: hexane:ethyl acetate (80:20), hexane:ethyl acetate (60:40), and hexane:ethyl acetate:methanol(60:40:2). Finally, eicosanoids were eluted from the cartridge with 2 ml of methanol and stored at -20°C until analysis. Radiolabelled internal standards were included for all metabolites extracted and recoveries were calculated. For RIA, methanol was evaporated under nitrogen and the sample resuspended in assay buffer. Each sample was analyzed for PGE, 6keto-PGF,,, TXB, and LTB, using commercially available R_IAkits from New England Nuclear @‘GE, and LTB,) and Advanced Magnetics (6-keto-PGF,, and TXB; Cambridge, MA). Statistical analvses: Data collected for milk bacteria numbers, somatic cell counts and eicosanoid concentrations were subjected to analyses of variance using the general linear models procedure and treatment means were compared by Duncan’s multiple-range tests (26). Means were compared between dietary Se groups only within each sampling time for PGE, TXB, LTB,, 6-keto-PGF,, and Log,, milk bacteria counts. Student t test was used for comparison of mean blood selenium values between the two dietary groups at the time of bacterial infusion. Statistical significance for all comparisons was determined at a P value <0.05. RESULTS The mean blood Se concentration of the -Se cows (0.07 + 0.01 pg/ml) was significantly lower than that of the +Se cows (0.24 + 0.01 pg/ml) at the time of bacterial challenge (P
OCTOBER 1991 VOL. 42 NO. 4
371
Systemic symptoms of coliform mastitis manifest in all cows inoculated included anorexia, neutropenia, pyrexia (data not shown) and recovery of E. cdi from the mammary gland. Mean milk somatic cell counts were less than 2 x 10’ of milk preinfusion, increased to more than 1 x 16 (~45% PMNL) at 16 h postinoculation remained at this level through 60 h postinoculation. clinical data,
numbers in the milk were the most notably different bacteria concentrations observed in the -Se cows at 16,2O and 24 h postinoculation. bacteria concentration bacteria concentration required euthanasia due to persistent
anorexia and hypocalcemic
increased dramatically
observed at 24 h postinoculation
0
E %i
21,000
-
19,000
-
15,000
-
continued up to 48 h postinoculation.
12,000 -
a s!l
9,000 -
F 6,000 -
3,000
-
0
12
24
36
46
60
Hours Postinoculation
Fig 1. Mean concentration & SEM) of TXB, in milk following intramammary inoculation. ‘P
E. coIi
Milk PGE, concentration changed very little in the +Se cows, but increased exponentially in the -Se cows between 12 and 24 h postinoculation to a peak concentration of 7000 pg/ml, which was significantly higher than in the +Se cows (fig. 2).
OCTOBER 1991 VOL. 42 NO. 4
PROSTAGLANDINS
7000
-
6000
-
+Secows
12
0
36
24
46
60
Hours Postinoculation Fig 2. Mean concentration h SEM) of PGE, in milk following intramammary inoculation. *P
E. coli
The 6-keto-PGF,, concentration in milk increased only in the -Se cows, and was significantly higher than the +Se cows between 24 and 60 h postinoculation (fig. 3).
2500
-
h 5
*
+Se COWS A
. ....
: ; 8'
:I
-Secows 2000
-
___4___
;
g Y cb
1000
*
*
*.. ______.-----_ +
: :
1500--
a a
*. ..
.*...,
:: : :
4
....
* ---___ ‘"‘..__A
t
-
500
:++
0
t 0
-..___
: *
12
a
24
36
48
60
Hours Postinoculation Fig 3. Mean concentration (+SEM) of 6-keto-PGF,, in milk following intramammary E. coli inoculation. *P
OCTOBER 1991 VOL. 42 NO. 4
The LTB, concentration was also significantly higher in the milk from the -Se cows than in the +Se group, but this occurred later (after 36 h) in the postinoculation period than the other metabolites (fig. 4).
1600
1200 h 2E
1000
4 $
600
5
600
0
t
I
0
12
24
36
46
60
Hours Postinoculation Fig 4. Mean concentration c_LSEM) of LTB, in milk following intramammary inoculation. V’cO.05 between the two dietary groups at time point indicated.
E. coli
DISCUSSION The present study was based on the premise that biosynthesis of PG’s and LT’s is altered during the Se-deficient state in dairy cows and that this might be one of the underlying mechanisms for the pathogenesis of mastitis. To elucidate this mechanism, we have systematically analyzed milk eicosanoid concentrations in -Se cows and compared them with the values obtained for +Se cows during the course of experimentally-induced coliform mastitis. Milk from the -Se cows contained significantly higher concentrations of PGE, 6-keto-PGF,,, TXB, and LTB, than milk from the +Se cows at several time points The higher eicosanoid concentrations were associated with postinoculation. significantly greater numbers of bacteria in the milk of the -Se cows. The increased milkbacteria concentration may be due to impaired killing abilities of the -Se PMNL. Previous studies have shown inhibited degranulation and microtubule function (27) and decreased killing of E. coli by neutrophils from -Se animals (6). It is conceivable that the increased number of bacteria released greater quantities of endotoxin and increased liberation of inflammatory mediators such as histamine, serotonin and arachidonic acid, which might have escalated inflammation and tissue damage. In turn, in the Se-GSH-Px depleted state, higher tissue hydroperoxide concentration may have aided the activation of cyclooxygenase and
374
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PROSTAGLANDINS lipoxygenase enzymes., It is known that these enzymes have an obligatory requirement for fatty acid hydroperoxides for maintenance of maximal activity (28 30). Thus, as a result of increased substrate availability and enzyme activity, the eicosanoid levels in the milk of the -Se cows were all significantly higher than in the +Se cows. Though other studies have shown decreased production of LTB, (31,32) and PGI, (33) in cells from -Se animals, it is likely that the quantities of arachidonic acid liberated due to tissue damage in the -Se cows obscured the more subtle effects of a Se-GSH-Px deficiency on individual metabolites of the arachidonic acid cascade. This is supported by the fact that the rate-limiting step in eicosanoid biosynthesis in viva is the release of esterified arachidonic acid from cell membranes by phospholipases (34,35). The clinical symptoms and sequelae (anorexia, hypocalcemia) from E. coli inoculation were much more severe in the -Se cows than the +Se cows, and euthanasia was necessary for three of the four -Se cows. The severity of symptoms could be at least partially accounted for by the increased eicosanoid concentrations because several studies implicate these eicosanoids in the pathogenesis of inflammatory and endotoxin-induced diseases @O-23,36,37). However, other Se effects on immune response (3,4) may also be responsible for contributing to the markedly different outcomes of the two dietary groups from E. coli inoculation of the mammary gland.
ACKNOWLEDGEMENTS This research was partially supported by a grant from the Pennsylvania Department of Agriculture and by grants from the United States Department of Agriculture (CRSR-23171) and Public Health Service (Rol 31245).
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Editor:
378
W.E.M.
Lands
Received:
5-17-91
acid, and superoxide
Accepted:
7-29-91
OCTOBER 1991 VOL. 42 NO. 4