Effects of Copper Status on Neutrophil Function, Superoxide Dismutase, and Copper Distribution in Steers 1
z.
XIN, D. F. WATERMAN, R. W. HEMKEN, and R. J. HAR~ON Department of Animal ScIence University of Kentucky Lexington 40546
Cu status affected its distribution in the tissues and related enzyme activities as well as bactericidal function of neutrophils. (Key words: copper, superoxide dismutase, neutrophil, bovine)
ABSTRACT
Twelve Holstein steers in a completely randomized block design ~ere fed either a basal diet (concentrate:siIage or hay at a DM ratio of 35:65) plus Cu sulfate at 20 ppm of Cu (Cu-supplemented diet) or a basal diet plus ammonium molybdate to obtain 10 ppm of Mo (Cu-depleting diet) on a DM basis in the whole diet for 8 mo. Supplemental Mo was utilized in the Cu-depleting diet to develop a Cu-deficient group. Molybdenum slowly accumulated in the liver in the group fed the Cu-depleting diet. Copper concentrations in the liver and polymorphonuclear neutrophiIs decreased.in the Cu-deficient group compared WIth the Cu-sufficient group. Plasma Cu concentration did not change during the trial for the Cu-sufficient group. In the Cudeficient group, plasma Cu concentrations increased during the first 3 mo of the trial, then declined, and remained unchanged for the last 5 mo. Superoxide dismutase activities in red blood cells, polymorphonuclear neutrophiIs, and whole blood decreased in the Cu-deficient group. Phagocytic capacity was not affected by Cu status, but killing capacity was decreased by low Cu status in the Cu-deficient group by the end of the trial. Glutathione peroxidase activity was unaffected by Cu status. Clinical symptoms of Cu-deficiency were not observed in this trial; there was no evidence of blood hemoglobin or BW gain difference between the two groups. In this study,
Abbreviation key: +Cu = copper-sufficient treatment group, -Cu = copper-deficient treatment group, HBSS = Hanks balanced salt s?lution, PMN = polymorphonuclear neutroph?, RBC = erythrocytes, SOD = superoxide dismutase. INTRODUCTION
Received January 30, 1991. Accepted April 1, 1991. IThis manuscript (91-5-13) is published with the approval of the director of the Kentucky Agricultural Experiment Station. 1991 J Dairy Sci 74:3078-3085
Copper plays a variety of important biological roles in animals through several Cu-dependent enzymes. Relationships between dietary Cu level, related enzyme activities, and cellular and immune functions recently have attracted considerable interest among scientists. Superoxide dismutase (SOD), one of the most important Cu-dependent enzymes, i~ associated functionally with Cu in different tissues (15). The relationship between the activity of SOD and the concentration of Cu has been observed in erythrocytes (RBC) of beef calves and peripheral blood leucocytes of ewes (10, 14). Recently, researchers have observed a decreased leucocyte Cu concentration in human patients with healing problems compared with those of healthy individuals (20). The reduction in leucocyte-killing capacity on Candida albicans also was reported in Cu-deficient ewes (10) and steers (2). However, effects of Cu status on neutrophil bactericidal action on pathogens causing diseases in ruminants have not been studied. In studying Cu deficiency, one of the difficulties researchers often encountered was selection of an indicator that could be used to evaluate the true Cu status of subjects. Liver Cu level commonly has been considered to be
3078
3079
COPPER STATUS AND NEUlROPHILS
a good indicator of systemic Cu status, but it has certain limitations for general use because of the inconvenient sampling and sample preparation procedures involved. Plasma Cu concentration commonly has been used in Cu studies. However, plasma Cu concentration can be misleading under some circumstances (6). The determination of SOD in RBC was more reliable than the determination of total Cu in the blood (14). However, comparison of sensitivity of different indicators to Cu status and interrelationship between intracellular Cu level, SOD activities, and cell functions are not well known. The objectives of this trial were to compare the sensitivity of different parameters to Cu status and to evaluate the influence of Cu status on SOD activities in different tissues and neutrophil function in dairy steers. MATERIALS AND METHODS Animals and Experimental Design
dextrose solution as anticoagulant were centrifuged at 1000 x g for 15 min. The plasma, buffy coat, and top third of the RBC pellet were aspirated and discarded. The remaining cell pellet was resuspended in Hanks balanced salt solution (HBSS) and transferred to siliconized centrifuge tubes. Red blood cells then were lysed by adding a hypotonic salt solution (.03 M NaQ). The tubes were centrifuged (500 x g, 2.5 min) after isotonicity was restored by adding a hypertonic salt solution (.63 M NaCl). The RBC lysate was harvested into polystyrene tubes and then frozen (-15°C) for subsequent SOD assay. The cell pellet was washed with and resuspended in HBSS. The differential cell count was performed from a Wright's stained cytospin smear (Cytospin 2, Shandon Southern Instruments, Inc., Sewickley, PA). Cell concentration was determined with a hemocytometer, and cell viability was determined by trypan blue exclusion. The final PMN concentration was adjusted to 5 x 1()6 cells/ml with HBSS. The procedure for preparing Staphylococcus aureus was a modification of the method described by Shuster (19). Briefly, culture of S. aureus 305 (ATCC 29740) was added to brainheart infusion broth medium and incubated in a shaking water bath at 37"C for 4 h to achieve log-phase growth. The S. aureus culture then was centrifuged (500 x g, 15 min), washed twice with diluting buffer (.24 roM KH2P04, .12 roM Na2C03, .94 roM MgCl2, pH 7.2), then resuspended in HBSS. Concentration of S. aureus was determined using a Petroff-Hausser
Twelve Holstein steers averaging 211 kg initial BW were assigned to one of two treatments in a completely randomized block design for an 8-mo trial. Treatments were 1) basal diet (Table 1) containing alfalfa haylage and concentrate (in the first 5 mo) or fescue hay and concentrate (in the last 3 mo) in an approximate 65:35 DM ratio plus 20 ppm of Cu supplementation (OM basis) as Cu sulfate (Cu-supplemented diet, +Cu treatment) and 2) basal diet plus 10 ppm of Mo supplementation as ammonium molybdate (Cu-depleting diet, -Cu treatment). The addition of Mo in the -Cu group was designed to develop a low Cu status according to the observation of Phillippo et al. (16). All steers individually were fed silage or hay twice daily at intake just below ad libitum level and concentrate once a day.
Item
mix
Alfalfa haylage
Sampling Proceclures and Analytical Methods
CP, % ADF, % NDF, %
11.7 2.7
18.7 38.9
Blood samples were collected via jugular vein once prior to treatment and monthly after the beginning of treatment. The procedure described by Carlson and Kaneko (4) for isolating polymmphonuclear neutrophils (PMN) was used with some modifications. Briefly, blood samples with 10% acid-eitrate-
TABLE 1. Nutrient composition of concentrate mix, silage, and haylage (DM basis).l
Coucentrate
11.0
48.1 1.34 .40.06
1.32
Ca, %
Na, % K, % S, % Cu, ppm
Mo, ppm ppm
ZD,
.61 2.08 .28.27 6.0 1.1 49.0
12.5 NA2 40.5
Fescue hay
14.9 32.1 67.5 .99
.32 2.29 .30 24.0
1.9 130.0
IMeasured in the commercial laboratory. 2Yalue not available.
Ioumal of Dairy Science Yol.
74, No.9, 1991
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XIN ET AL.
Bacteria Counter (1/400 nun2 , 1/50 nun deep, C. A. Hausser & Son, Philadelphia, PA) and adjusted to 4 x 107 cells/ml. Polymorphonuclear neutrophils were incubated with viable log-phase S. aureus at an 8:1 bacteria:PMN ratio for the determination of bactericidal capacity. Suspensions containing .1 ml of serum collected from healthy calves, .5 ml of HBSS, and .2 ml of PMN (5 x 1()6 cells/ml) in treatment tubes (polypropylene, 17 x 100 nun, Becton Dickinson & Co., Rutherford, NJ) and .1 ml of serum and .7 ml of HBSS in the control tubes were preincubated at 37·C in the shaking water bath (10 oscillations/min) for 30 min. Following preincubation, .2 ml of S. aureus (4 X 107/ml) were added to each tube, and incubation was continued for another 65 min. Following incubation, .1 ml of incubation mixture was taken and added to a tube containing 2 drops of HBSS and 1 drop of serum. A .1-ml sample taken from each tube was placed in the cytospin. Slides were stained (Diff-Quik, American Scientific Products, McGaw Park, ll..) and obselVed microscopically for the determination of percentage of PMN phagocytosing and average number of S. aureus per phagocytosing PMN. After sampling for phagocytosis activity from each tube, the incubation mixture was disrupted with a microultrasonic cell disrupter (K.ontes, Scientific Glassware/Instmments, Vineland, NJ). A .1-ml aliquot of cell-disrupted incubation mixture was diluted with buffer and plated on trypticase soy agar by spreading .1-ml on each of three plates. Following incubation at 37·C for approximately 48 h, the average colony-forming units per milliliter was determined on both control and sample plates for calculating percentage killing of S. aureus by PMN. Superoxide dismutase activity was measured using a modification of the procedure described by Jones and Suttle (10) and McCord and Fridovich (15). Briefly, assays were carried out in 3 ml of final reaction solution containing .375 ml of standard (b0vine kidney SOD, Sigma, St. Louis, MO) or sample (Wldiluted leucocyte lysate after three free~thaw cycles, RBC lysate with dilution of 1:10 sample:lO mM phosphate buffer, pH 7), and 2.55 ml of 10 mM carbonate buffer (pH 10.2, containing 347 ml of .1 M sodium carbonate, 152 ml of .1 M sodium bicarbonate, Ioumal of Dairy Science Vol. 74, No.9, 1991
5 ml of 40 roM xanthine in .IN sodium hydroxide, and 20 ml of 10 roM iodonitrotetrazolium violet in 11 % ethanoVL). Just before mixing, .075 ml of xanthine oxidase (.3 unitsl ml) was added. Absorbance was read continuously for 5 min on a spectrophotometer (Response, UV-VIS Spectrophotometer, Gilford, Oberlin, OR) at a wavelength of 500 run at room temperature. The changing rate of absorbance in the linear portion of the curve was used to determine SOD activity. Concentrations of SOD in samples were determined by comparison with the calibration CUlVe from standards containing .5 to 2 units/.375 ml of phosphate buffer. Glutathione peroxidase activity in whole blood was determined spectrophotometrically by modified procedures (11, 18) on the same spectrophotometer as used for SOD assay. One unit of enzyme activity represents the oxidation of 1 mol of NADPH/ min at 25"C. Hemoglobin was assayed by the hemoglobin-cyanide method (procedure number 525, Sigma Diagnostics, St. Louis, MO). Copper in samples of plasma and liver, which were prepared with a modified wet-ashing procedure using two cycles of nitric acid and one cycle of hydrogen peroxide (30%) digestion, was analyzed by electrothermal atomization atomic absorption spectrophotometry (RCG-21DO on Perkin-Elmer, Model 560, Norwalk, Cf). Liver Mo was analyzed by using a catalytic assay on an autoanalyzer according to the procedure described by Eivazi et al. (7). Concentrate samples were taken at the feed mill after being mixed. Forage samples were taken every month and composited before shipping for laboratory analysis. Nutrient composition of diets (Table 1) was determined by commerciallabo-
mary. AlI data were analyzed statistically using the general linear models procedure of SAS (17). The least significant difference procedure was performed to compare treannent means at each sampling time. Significance was declared at P < .01 unless otherwise noted. RESULTS AND DISCUSSION
Copper status and distribution are illustrated in Table 2. Liver Co concentration increased with Co supplementation in the +Co treatment group and decreased with Mo addition in the
3081
COPPER STATUS AND NEUI'ROPffiLS
TABLE 2. Effects of Cu status on Cu distribution in the tissues, liver Mo, hemoglobin (Hb), and BW gain (BWG) after 8 mo. Parameters
Treatment1 +Cu
-Cu
PMN Cu (pg!107)
Liver Cu
Plasma Cu
Liver Mo
Hb
ijl.g!g)
ij1g/ml)
(ppm)
(g/dl)
13.0" 5.1b
305.8a 18.1b
.81 a .78a
3.61 ab 15.98a
9.77a 9.88 a
BWG
a,~eans with different superscripts in the same column differ (P < .01).
l+cu
=Cu-sufficient group fed Cu-supplcmentcd diet; -eu =Cu-deficient group fed Cu-dcpleting diet by using Mo.
treatment. Liver Cu concentration declined sharply during the first 5 mo and slowly over the last 3 mo of the trial in the -Cu treatment (Figure 1). The rate of change in liver Cu may reflect the change of forage sources, in which alfalfa haylage fed in the first 5 mo contained less Cu and fescue hay fed in the last 3 mo contained more Cu. Liver Cu concentration in the -Cu treatment by the end of the trial reached a level that was considered to be approximately borderline between deficiency and sufficiency (8). Because liver Cu concentration can reflect Cu absorption and status of the animal according to studies (3, 6, 23), it is concluded that a Cu-adequate or inadequate status was established in the two treatment groups by mo 8 in this trial. Several studies (1, 13) suggested that the mechanism by which Mo depletes Cu is that Mo forms a complex with S and protein. This Mo-S-protein complex decreases Cu absorption by Cuchelating action in the rumen, changes Cu -Cu
360 E
280
:Ii
240
c
.5 :s
()
:;
>
::::;
1.60 _
+Cu
~
-Cu
1.40
320
a. a.
distribution in the liver, or increases excretion of Cu in the feces. Plasma Cu concentrations were not affected by treatment in the last period of the trial but were elevated in the first 3 mo (P < .05) in the -Cu group (Figure 2). This agrees with observations reported in sheep and guinea pigs (20). The increase in plasma Cu may be due to formation of Cu-Mo-ceruloplasmin complex in the blood, which is a measurable but biologically unavailable form of Cu. This is one of the processes that may result in liver Cu depletion by high dietary Mo (12). Plasma Cu concentration commonly has been used to examine Cu deficiency in animals. However, plasma Cu concentration can be misleading under at least three circumstances: 1) when total Cu concentration in plasma is affected by other minerals such as Mo, as shown in this study; 2) when ceruloplasmin, which binds 90% of the total plasma Cu, is elevated significantly during inflammatory stress; and 3) when diseases that affect the
...
1.20
:s
1.00
E
....
+Cu
.......
-Cu
4
5
6
7
.....
'
"
,j
u
200
•E •• is:
160 120
.80
.60 .40
80
.20
40
.0
0
..
D
.. ' ...•.
-0-
o
0 125
2
3
8
8
Month Of Treatment
Figure 1. Mean concentration of liver Cu in steers fed Cu-supplemented (+Cu) and Cu-depleting (-eu) diets during 8 mo of supplementation of Cu or Mo.
Month Of Treatment
Figure 2. Mean concentration of plasma Cu in steers fed Cu-supplemented (+Cu) and Cu-depleting (-eu) diets during 8 mo of supplementation of Cu or Mo. Journal of Dairy Scieoc.e Vol. 74, No.9, 1991
3082
XIN ET AL.
TABLE 3. R.espooscs in neutrophil functions and saperoxide dismutase (SOD) activities after 8 mo. Treatment
SA:p1
+Cu --eu
4.7 a
5.oa
a,"Means
pp2
PK3
SODRBc4
SODWBcS
SODWB 6
('l&)
('l&)
(U/mg Hb)
(U)
(U/mI)
57.8a
27.7a
.wab
.65a
20.4a 15.4b
58.Sa
17.3b
.31 b
.36
with different supersaipts in the same column differ (P < .01).
JNumber of Staphylococcus
QJlTe1IS
eoguIfed per phaBocytosiDg polymorpbonuc1ear IIe11trophil (PMN).
2Percentage of PMN phagocytosing. 3Percentage of killing of S. aureus by PMN.
4-rhe SOD activities in red blood cell lysate as units (U) per milligram of hemoglobin (lIb). SThe SOD activities in neutrophil as unilS/IO,OOO,OOO cells. 6o:rhc
SOD activities in whole blood lysate as units of activity per milliliter.
synthesis of hepatic export proteins, such as albumin or transferrin, influence ceruloplasmin level independently of Co status. The Co concentration in neutrophils was lower in -eo group compared with the +Co group by the last sampling period. This change may be due to decrease in the labile Co pool, which is complexed with amino acids or is in the finnly bound fonn of SOD as observed in RBe (6). When decrease in granulocyte Co concentration occurs, cell functions may be impaired. Researchers observed that human patients with decreased healing times tended to have lower leucocyte Co concentrations (21). In this trial, Co concentration in the neutrophils was lowered before Co status reached borderline or deficiency levels. Liver Mo concentration was increased by Mo supplementation in the -Co group compared with the +Co group, which agrees with earlier observations (5). Molybdenum accumulated in the liver was probably in the fonn of thiomolybdate, a complex consisting of Mo, S, and albumin (9). It is postulated that Mo deposited in the liver may decrease Co level in the liver by at least two mechanisms. Molybdenum may decrease Co absorption from the gastrointestinal tract via a chelating mechanism (1), or it may alter the distribution of Co in the tissues and fecal excretion (13). Olange of liver Mo concentration with time is' presented in Figure 3. Liver Mo level in -eo group increased rapidly in the 1st roo and slowly in the remaining time of the experiment. Liver Mo level in the +Co group consistently was low and tended to decline with Journal of Dairy Science Vol 74. No.9. 1991
time, indicating a possible antagonistic reaction between Co and Mo in the liver during Co supplementation. Blood total hemoglobin and BW gain were not different between treatments (fable 2), indicating that clinical Co deficiency was not reached in this trial. During clinical Co deficiency, hemoglobin synthesis and BW gain nonnally are depressed (22). Effects on PMN functions and SOD glutathione peroxidase activities are presented in Table 3. Neither the number of S. aureus in each engulfing PMN nor the percentage of total PMN that phagocytosed S. aureus was affected by Co status. However, the bactericidal capacity of PMN expressed as percentage of S. aureus killed by PMN in this experiment
20 18 E
Do Do
S
14
12
.5
10 8
0
.,•.
:J
.eu £ZZl -eu
16
a 2
_
6
" 2
0
o
1 2 Month Of Treetment
5
Figure 3. Mean concentration of liver Mo in steers fed Co-supplemented (+Cu) and Cu-depleting (--eu) diets during the 8 mo of supplementation of Co or Mo.
3083
COPPER STATIJS AND NEUI'ROPHILS 70
-0-
+Cu ....... -Cu
1.00
50
ii'
40
l/I.
30
-0-
.90 .80
60
~ ....
20
......
j.~--e ..................... ......•........•
10
-Cu
.
.70 0
+Cu
/°"0
~
.60
.50 .40 . 30 .20 .10
. ..........•........•
.0
0 2
0
3
4
5
6
7
o
8
2
345
6
7
8
Month Of Treatment
Month Of Treatment
fed diets supplemented with Cu (iCu) or Mo (-CD) during 8 mo.
Figure 6. Mean superoxide dismutase activities in neutrophils in steers fed Cu-supplemented (+Cu) and Cudepleting (-CD) diets during 8 mo of supplementation of Cu or Mo.
was decreased in the --Cu group. This agreed with observations in ewes (10). The changes of bactericidal capacity of PMN with time (Figure 4) showed a significant difference between treatments only in the last month of the experiment. We suggest that fluctuation was caused mainly by variation in the ratio of S. aureus to PMN in the incubation mixture from assay to assay. However, this conclusion should not be affected because a relative rather than absolute difference between treatment groups was examined. Polymorphonuclear neutrophils are one of the most important phagocytes in defending the body against the
invasion of pathogenic microorganisms. Once the functions of PMN are impaired, the animals may be more susceptible to various infections. Hence, Co deficiency may lead to increased occurrence of certain diseases in animals. Superoxide dismutase activities in RBC lysate, PMN, and whole blood were decreased by low Co status in the -en group compared with the +Co group, which agrees with data of Jones and Suttle (10). The decrease in SOD activity of PMN may be responsible for the reduction in bactericidal capacity of PMN. This impaired bactericidal function may be caused by a decline in protec-
Figure 4. Mean percentage killing of Staphylococcus
aureus by polymorphonuclear neotrophils in vitro in steers
1.00
:S 0
.90 .80
E
.70
a0
•
21
.60 .50 .40
...
12 9 6 3
a
E
C
.30
c:i
.20
~
0
III
30 27 24 •
+Cu •.••. -Cu
~"--o............... -........,-~
:z:
....:l
-0-
.." ....
. °.... ................ .
.10
~:
-0-
+Cu
~o-o~
'.- _. -.. --.... --...
oL-----L---'---""--'--.......-
.0
o
2 3 4 5 Month Of Treatment
7
8
_.•-. -Cu
o
2
3
4
-""
.......-
5
6
0_0.
-..----
........--7 8
Month Of Treatment
Figure 5. Mean superoxide dismutase (SOD) activities in red blood cell lysate in steers fed Cu-supplemented (iCu) and Cu-depleting (-CD) diets during 8 mo of supplementation of Cu or Mo.
Figure 7. Mean superoxide dismutase (SOD) activities in whole blood lysate in steers fed Cu-supplemented (iCu) and Cu-depleting (-CD) diets during 8 mo of supplementation of Cu or Mo. Journal of Dairy Science Vol. 74, No.9, 1991
3084
XIN ET AL.
tion of phagocytic cells from oxidation mediated by oxygen-free radicals during the bactericidal process (10) or a decrease in supply of H2~ to myeloperoxidase-halide system to produce microbial killing agents such as hypochlorite ion and hydroxyl radicals (to). The pattern of changes in SOD activities in RBC, PMN, and whole blood (Figure 5, 6, 7) corresponds to the decline in liver Cu concentrations in the --Cu group (Figure 1) over the last period and the decline in percentage S. aureus killed during the last sampling time of the trial (Table 3). Changes in SOD activity of whole blood were less dramatic and had a more consistent pattern in comparison with the other parameters. This may be due to less variation in sample preparation. Furthermore, SOD activities in whole blood were affected at the 2nd mo of treatment, which was earlier than any other parameter except for liver Cu concentration. Therefore, SOD activities may be pragmatically important for surveying Cu status in the field as well as serving as an indicator of Cu deficiency. More research on the relationship between Cu status and standard SOD quantity or other equivalent values must be conducted before the practice can be accepted. One possible approach is to make a pooled control sample that is from Cu-sufficient animals and to compare its SOD result with that in the questionable sample in each assay. Thus, variations from assay to assay or from time to time will not affect the relative comparison between Cu-sufficient control sample and questionable samples from the field to decide whether or not the animal is Cu-deficient. Glutathione peroxidase activity was not affected by Cu status or changes in SOD activities in this experiment. CONCLUSIONS
Results from this study have shown that SOD activities in blood parameters and intracellular Cu concentration in P:MN will decrease when Cu status is low or deficient based on liver Cu concentration. Therefore, definition of functional Cu deficiency .may need to be specified further under different physiological or pathological conditions. Based on the observations in this study, it is of potential importance to use SOD as a screening criterion for diagnosing Cu deficiency by 10urnal of Dairy Science Vol. 74, No.9, 1991
using a Cu-sufficient control sample in each assay. Results from this study also have shown that low Cu status can decrease intracellular SOD activity and neutrophil bactericidal capability in dairy steers. The level of ceruloplasmin, a Cu-containing C12-globulin possessing potent antioxidant activity and modulation of tissue injury associated with the production of oxygen metabolites by phagocytes, also can be reduced by low Cu status from other studies. Hence, low Cu status in animals may contribute partially to an increase in susceptibility to infections and consequent tissue injuries as in the case of mastitis in dairy cows. Therefore, we suggest that the relationship between Cu status under certain physiological conditions and disease susceptibility or tissue injury needs to be investigated further. REFERENCES 1 Allen, 1. D., and 1. M Gawthorne. 1987. Involvement of the solid phase of rumen digesta in the interaction between copper, molybdenum and sulphur in sheep. Br. 1. Nutr. 58:265. 2 Boyne, R., and 1. R. Arthur. 1981. Effects of selenium and copper deficiency on neutrophil function in cattle. 1. Comp. Pathol. 91:271. 3 Bremner, I. 1980. Page 23 in Biological roles of copper. Elsevic- Appl. Sci. Publ., New York, NY. 4 Carlson, G. P., and 1. 1. Kaneko. 1973. Isolation of leucocytes from bovine peripheral blood. Proc. Soc. Exp. BioI. Med. 142:853. 5 Cook, G. A., A. L. Lesperance, V. R. Bobman, and E. H. 1ensen. 1966. Inten'elationsbip of molybdenum and certain factors to the development of the molybdenum toxicity syndrome. 1. Anim. Sci. 25:96. 6 Davis, G. K., and W. Mertz. 1987. Copper. Trace elements in human and animal nutrition. 5th ed. Academic Press, Inc., San Diego, CA. 7 Eivazi, P., 1. L. Sims, and 1. Crutchfield. 1982. Determination of molybdenum in plant materials using a rapid, automated method. Commun. Soil Sci. Plant Anal. 13(2):135. 8 Gooneralne, R, and D. Christensin. 1985. Gestation age and maternal-fetal liver copper levels in the b0vine. Page 334 in Trace elements in man and animals-lEMA 5. Commonw. Agric. Bur., FIlIIIham Royal, Engl. 9 Hynes, M, M. Lamand, G. Montel. and 1. Mason. 1984. Some studies on the metabolism and the effects on Me- and S-labelled thiomolybdates after intravenous infusion in sheep. Br. 1. Nutr. 52:149. 10 Jones, D. G., and N. P. Suttle. 1981. Some effects of copper deficiency on leucocyte function in sheep and cattle. Res. Vet. Sci. 31:151. 11 Lawrence, R. A., and R. P. Bark. 1976. Glutathione peroxidase activity in selenium deficient rat liver. Biochem. Biophys. Res. Commun. 71(2):952.
COPPER STATUS AND NEUTROPmLS 12 Mason, J. 1978. The relationship between copper, molybdenum and sulphur in ruminant and n0nruminant animals: a review. Vet. Sci. Commun. 2:85. 13 Mason, J., M. Lamand, 1. C. Tressel, and G. Mulryan. 1988. Studies of the changes in systemic copper metabolism and excretion produced by the intravenous administration of trithiomolybdate in sheep. Br. J. Nutr. 59:289. 14 Masters, H. G., G. M. Smith, and R H. Casey. 1985. Page 575 in Trace elements in man and aniInal&TBMA 5. Commonw. Agric. Bur., Farnham Royal, Engl. 15 McCord, J. M., and I. Fridovich. 1969. Superoxide dismutase, an enzymic function for erythrocuperin. 1. BioI. Cbem. 244:6049. 16 Phillippo, M., W. R Humphries, and T. Atkinson. 1987. The effect of dietary molybdenum and iron on copper status, puberty, fertility and oestrous cycles in cattle. J. Agric. Sci. (Carob.) 109:321. 17 SAS$ User's Guide: Basics. 1985. SAS IIlst, Inc., Cary, NC. 18 Scholz, R. W., and L. J. Hutehinsin. 1979. Distribu-
3085
tion of glutathione peroxidase activity and selenium in the blood of dairy cows. Am. J. Vet. Res. 40:245. 19 Shuster, D. E. 1985ln vitro effects of anti-inflammatory agents on bovine neutrophils stimulated with opsonized Staphylococcus aureus. M. S. Thesis, Univ. Kentucky, Lexington. 20 Smith, B.S.W., and H. Wright 1975. Effect of dietary Mo and Cu metabolism, evidence for the involvement of Mo in abnormal binding of Cu to plasma proteins. Clin. Chim. Acta 62:55. 21 Thomas, A. J., V. W. Bunker, L. J. Rinks, N. Sodha, M. A. Mullee, and B. E. Clayton. 1988. Energy, protein, zinc and copper status of twenty-one elderly inpatients: analyzed dietary intake and biochemical indices. Br. J. Nutr. 59:181. 22 Ward, G. M. 1978. Molybdenum toxicity and hypocuprosis in ruminants: a review. J. Anim. Sci. 46: 1078. 23 Xin, Z., W. 1. Silvia, R. W. Hemken, W. B. Tucker, and J. A. Boling. 1989. Effects of copper status on LH secretion in dairy steers. J. Anim. Sci. 67(Suppl. 1): 320.(Abstr.)
Journal of Dairy Science Vol. 74, No.9, 1991
z.
XIN, D. F. WATERMAN, R. W. HEMKEN, and R. J. HAR~ON Department of Animal ScIence University of Kentucky Lexington 40546
Cu status affected its distribution in the tissues and related enzyme activities as well as bactericidal function of neutrophils. (Key words: copper, superoxide dismutase, neutrophil, bovine)
ABSTRACT
Twelve Holstein steers in a completely randomized block design ~ere fed either a basal diet (concentrate:siIage or hay at a DM ratio of 35:65) plus Cu sulfate at 20 ppm of Cu (Cu-supplemented diet) or a basal diet plus ammonium molybdate to obtain 10 ppm of Mo (Cu-depleting diet) on a DM basis in the whole diet for 8 mo. Supplemental Mo was utilized in the Cu-depleting diet to develop a Cu-deficient group. Molybdenum slowly accumulated in the liver in the group fed the Cu-depleting diet. Copper concentrations in the liver and polymorphonuclear neutrophiIs decreased.in the Cu-deficient group compared WIth the Cu-sufficient group. Plasma Cu concentration did not change during the trial for the Cu-sufficient group. In the Cudeficient group, plasma Cu concentrations increased during the first 3 mo of the trial, then declined, and remained unchanged for the last 5 mo. Superoxide dismutase activities in red blood cells, polymorphonuclear neutrophiIs, and whole blood decreased in the Cu-deficient group. Phagocytic capacity was not affected by Cu status, but killing capacity was decreased by low Cu status in the Cu-deficient group by the end of the trial. Glutathione peroxidase activity was unaffected by Cu status. Clinical symptoms of Cu-deficiency were not observed in this trial; there was no evidence of blood hemoglobin or BW gain difference between the two groups. In this study,
Abbreviation key: +Cu = copper-sufficient treatment group, -Cu = copper-deficient treatment group, HBSS = Hanks balanced salt s?lution, PMN = polymorphonuclear neutroph?, RBC = erythrocytes, SOD = superoxide dismutase. INTRODUCTION
Received January 30, 1991. Accepted April 1, 1991. IThis manuscript (91-5-13) is published with the approval of the director of the Kentucky Agricultural Experiment Station. 1991 J Dairy Sci 74:3078-3085
Copper plays a variety of important biological roles in animals through several Cu-dependent enzymes. Relationships between dietary Cu level, related enzyme activities, and cellular and immune functions recently have attracted considerable interest among scientists. Superoxide dismutase (SOD), one of the most important Cu-dependent enzymes, i~ associated functionally with Cu in different tissues (15). The relationship between the activity of SOD and the concentration of Cu has been observed in erythrocytes (RBC) of beef calves and peripheral blood leucocytes of ewes (10, 14). Recently, researchers have observed a decreased leucocyte Cu concentration in human patients with healing problems compared with those of healthy individuals (20). The reduction in leucocyte-killing capacity on Candida albicans also was reported in Cu-deficient ewes (10) and steers (2). However, effects of Cu status on neutrophil bactericidal action on pathogens causing diseases in ruminants have not been studied. In studying Cu deficiency, one of the difficulties researchers often encountered was selection of an indicator that could be used to evaluate the true Cu status of subjects. Liver Cu level commonly has been considered to be
3078
3079
COPPER STATUS AND NEUlROPHILS
a good indicator of systemic Cu status, but it has certain limitations for general use because of the inconvenient sampling and sample preparation procedures involved. Plasma Cu concentration commonly has been used in Cu studies. However, plasma Cu concentration can be misleading under some circumstances (6). The determination of SOD in RBC was more reliable than the determination of total Cu in the blood (14). However, comparison of sensitivity of different indicators to Cu status and interrelationship between intracellular Cu level, SOD activities, and cell functions are not well known. The objectives of this trial were to compare the sensitivity of different parameters to Cu status and to evaluate the influence of Cu status on SOD activities in different tissues and neutrophil function in dairy steers. MATERIALS AND METHODS Animals and Experimental Design
dextrose solution as anticoagulant were centrifuged at 1000 x g for 15 min. The plasma, buffy coat, and top third of the RBC pellet were aspirated and discarded. The remaining cell pellet was resuspended in Hanks balanced salt solution (HBSS) and transferred to siliconized centrifuge tubes. Red blood cells then were lysed by adding a hypotonic salt solution (.03 M NaQ). The tubes were centrifuged (500 x g, 2.5 min) after isotonicity was restored by adding a hypertonic salt solution (.63 M NaCl). The RBC lysate was harvested into polystyrene tubes and then frozen (-15°C) for subsequent SOD assay. The cell pellet was washed with and resuspended in HBSS. The differential cell count was performed from a Wright's stained cytospin smear (Cytospin 2, Shandon Southern Instruments, Inc., Sewickley, PA). Cell concentration was determined with a hemocytometer, and cell viability was determined by trypan blue exclusion. The final PMN concentration was adjusted to 5 x 1()6 cells/ml with HBSS. The procedure for preparing Staphylococcus aureus was a modification of the method described by Shuster (19). Briefly, culture of S. aureus 305 (ATCC 29740) was added to brainheart infusion broth medium and incubated in a shaking water bath at 37"C for 4 h to achieve log-phase growth. The S. aureus culture then was centrifuged (500 x g, 15 min), washed twice with diluting buffer (.24 roM KH2P04, .12 roM Na2C03, .94 roM MgCl2, pH 7.2), then resuspended in HBSS. Concentration of S. aureus was determined using a Petroff-Hausser
Twelve Holstein steers averaging 211 kg initial BW were assigned to one of two treatments in a completely randomized block design for an 8-mo trial. Treatments were 1) basal diet (Table 1) containing alfalfa haylage and concentrate (in the first 5 mo) or fescue hay and concentrate (in the last 3 mo) in an approximate 65:35 DM ratio plus 20 ppm of Cu supplementation (OM basis) as Cu sulfate (Cu-supplemented diet, +Cu treatment) and 2) basal diet plus 10 ppm of Mo supplementation as ammonium molybdate (Cu-depleting diet, -Cu treatment). The addition of Mo in the -Cu group was designed to develop a low Cu status according to the observation of Phillippo et al. (16). All steers individually were fed silage or hay twice daily at intake just below ad libitum level and concentrate once a day.
Item
mix
Alfalfa haylage
Sampling Proceclures and Analytical Methods
CP, % ADF, % NDF, %
11.7 2.7
18.7 38.9
Blood samples were collected via jugular vein once prior to treatment and monthly after the beginning of treatment. The procedure described by Carlson and Kaneko (4) for isolating polymmphonuclear neutrophils (PMN) was used with some modifications. Briefly, blood samples with 10% acid-eitrate-
TABLE 1. Nutrient composition of concentrate mix, silage, and haylage (DM basis).l
Coucentrate
11.0
48.1 1.34 .40.06
1.32
Ca, %
Na, % K, % S, % Cu, ppm
Mo, ppm ppm
ZD,
.61 2.08 .28.27 6.0 1.1 49.0
12.5 NA2 40.5
Fescue hay
14.9 32.1 67.5 .99
.32 2.29 .30 24.0
1.9 130.0
IMeasured in the commercial laboratory. 2Yalue not available.
Ioumal of Dairy Science Yol.
74, No.9, 1991
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XIN ET AL.
Bacteria Counter (1/400 nun2 , 1/50 nun deep, C. A. Hausser & Son, Philadelphia, PA) and adjusted to 4 x 107 cells/ml. Polymorphonuclear neutrophils were incubated with viable log-phase S. aureus at an 8:1 bacteria:PMN ratio for the determination of bactericidal capacity. Suspensions containing .1 ml of serum collected from healthy calves, .5 ml of HBSS, and .2 ml of PMN (5 x 1()6 cells/ml) in treatment tubes (polypropylene, 17 x 100 nun, Becton Dickinson & Co., Rutherford, NJ) and .1 ml of serum and .7 ml of HBSS in the control tubes were preincubated at 37·C in the shaking water bath (10 oscillations/min) for 30 min. Following preincubation, .2 ml of S. aureus (4 X 107/ml) were added to each tube, and incubation was continued for another 65 min. Following incubation, .1 ml of incubation mixture was taken and added to a tube containing 2 drops of HBSS and 1 drop of serum. A .1-ml sample taken from each tube was placed in the cytospin. Slides were stained (Diff-Quik, American Scientific Products, McGaw Park, ll..) and obselVed microscopically for the determination of percentage of PMN phagocytosing and average number of S. aureus per phagocytosing PMN. After sampling for phagocytosis activity from each tube, the incubation mixture was disrupted with a microultrasonic cell disrupter (K.ontes, Scientific Glassware/Instmments, Vineland, NJ). A .1-ml aliquot of cell-disrupted incubation mixture was diluted with buffer and plated on trypticase soy agar by spreading .1-ml on each of three plates. Following incubation at 37·C for approximately 48 h, the average colony-forming units per milliliter was determined on both control and sample plates for calculating percentage killing of S. aureus by PMN. Superoxide dismutase activity was measured using a modification of the procedure described by Jones and Suttle (10) and McCord and Fridovich (15). Briefly, assays were carried out in 3 ml of final reaction solution containing .375 ml of standard (b0vine kidney SOD, Sigma, St. Louis, MO) or sample (Wldiluted leucocyte lysate after three free~thaw cycles, RBC lysate with dilution of 1:10 sample:lO mM phosphate buffer, pH 7), and 2.55 ml of 10 mM carbonate buffer (pH 10.2, containing 347 ml of .1 M sodium carbonate, 152 ml of .1 M sodium bicarbonate, Ioumal of Dairy Science Vol. 74, No.9, 1991
5 ml of 40 roM xanthine in .IN sodium hydroxide, and 20 ml of 10 roM iodonitrotetrazolium violet in 11 % ethanoVL). Just before mixing, .075 ml of xanthine oxidase (.3 unitsl ml) was added. Absorbance was read continuously for 5 min on a spectrophotometer (Response, UV-VIS Spectrophotometer, Gilford, Oberlin, OR) at a wavelength of 500 run at room temperature. The changing rate of absorbance in the linear portion of the curve was used to determine SOD activity. Concentrations of SOD in samples were determined by comparison with the calibration CUlVe from standards containing .5 to 2 units/.375 ml of phosphate buffer. Glutathione peroxidase activity in whole blood was determined spectrophotometrically by modified procedures (11, 18) on the same spectrophotometer as used for SOD assay. One unit of enzyme activity represents the oxidation of 1 mol of NADPH/ min at 25"C. Hemoglobin was assayed by the hemoglobin-cyanide method (procedure number 525, Sigma Diagnostics, St. Louis, MO). Copper in samples of plasma and liver, which were prepared with a modified wet-ashing procedure using two cycles of nitric acid and one cycle of hydrogen peroxide (30%) digestion, was analyzed by electrothermal atomization atomic absorption spectrophotometry (RCG-21DO on Perkin-Elmer, Model 560, Norwalk, Cf). Liver Mo was analyzed by using a catalytic assay on an autoanalyzer according to the procedure described by Eivazi et al. (7). Concentrate samples were taken at the feed mill after being mixed. Forage samples were taken every month and composited before shipping for laboratory analysis. Nutrient composition of diets (Table 1) was determined by commerciallabo-
mary. AlI data were analyzed statistically using the general linear models procedure of SAS (17). The least significant difference procedure was performed to compare treannent means at each sampling time. Significance was declared at P < .01 unless otherwise noted. RESULTS AND DISCUSSION
Copper status and distribution are illustrated in Table 2. Liver Co concentration increased with Co supplementation in the +Co treatment group and decreased with Mo addition in the
3081
COPPER STATUS AND NEUI'ROPffiLS
TABLE 2. Effects of Cu status on Cu distribution in the tissues, liver Mo, hemoglobin (Hb), and BW gain (BWG) after 8 mo. Parameters
Treatment1 +Cu
-Cu
PMN Cu (pg!107)
Liver Cu
Plasma Cu
Liver Mo
Hb
ijl.g!g)
ij1g/ml)
(ppm)
(g/dl)
13.0" 5.1b
305.8a 18.1b
.81 a .78a
3.61 ab 15.98a
9.77a 9.88 a
BWG
a,~eans with different superscripts in the same column differ (P < .01).
l+cu
=Cu-sufficient group fed Cu-supplcmentcd diet; -eu =Cu-deficient group fed Cu-dcpleting diet by using Mo.
treatment. Liver Cu concentration declined sharply during the first 5 mo and slowly over the last 3 mo of the trial in the -Cu treatment (Figure 1). The rate of change in liver Cu may reflect the change of forage sources, in which alfalfa haylage fed in the first 5 mo contained less Cu and fescue hay fed in the last 3 mo contained more Cu. Liver Cu concentration in the -Cu treatment by the end of the trial reached a level that was considered to be approximately borderline between deficiency and sufficiency (8). Because liver Cu concentration can reflect Cu absorption and status of the animal according to studies (3, 6, 23), it is concluded that a Cu-adequate or inadequate status was established in the two treatment groups by mo 8 in this trial. Several studies (1, 13) suggested that the mechanism by which Mo depletes Cu is that Mo forms a complex with S and protein. This Mo-S-protein complex decreases Cu absorption by Cuchelating action in the rumen, changes Cu -Cu
360 E
280
:Ii
240
c
.5 :s
()
:;
>
::::;
1.60 _
+Cu
~
-Cu
1.40
320
a. a.
distribution in the liver, or increases excretion of Cu in the feces. Plasma Cu concentrations were not affected by treatment in the last period of the trial but were elevated in the first 3 mo (P < .05) in the -Cu group (Figure 2). This agrees with observations reported in sheep and guinea pigs (20). The increase in plasma Cu may be due to formation of Cu-Mo-ceruloplasmin complex in the blood, which is a measurable but biologically unavailable form of Cu. This is one of the processes that may result in liver Cu depletion by high dietary Mo (12). Plasma Cu concentration commonly has been used to examine Cu deficiency in animals. However, plasma Cu concentration can be misleading under at least three circumstances: 1) when total Cu concentration in plasma is affected by other minerals such as Mo, as shown in this study; 2) when ceruloplasmin, which binds 90% of the total plasma Cu, is elevated significantly during inflammatory stress; and 3) when diseases that affect the
...
1.20
:s
1.00
E
....
+Cu
.......
-Cu
4
5
6
7
.....
'
"
,j
u
200
•E •• is:
160 120
.80
.60 .40
80
.20
40
.0
0
..
D
.. ' ...•.
-0-
o
0 125
2
3
8
8
Month Of Treatment
Figure 1. Mean concentration of liver Cu in steers fed Cu-supplemented (+Cu) and Cu-depleting (-eu) diets during 8 mo of supplementation of Cu or Mo.
Month Of Treatment
Figure 2. Mean concentration of plasma Cu in steers fed Cu-supplemented (+Cu) and Cu-depleting (-eu) diets during 8 mo of supplementation of Cu or Mo. Journal of Dairy Scieoc.e Vol. 74, No.9, 1991
3082
XIN ET AL.
TABLE 3. R.espooscs in neutrophil functions and saperoxide dismutase (SOD) activities after 8 mo. Treatment
SA:p1
+Cu --eu
4.7 a
5.oa
a,"Means
pp2
PK3
SODRBc4
SODWBcS
SODWB 6
('l&)
('l&)
(U/mg Hb)
(U)
(U/mI)
57.8a
27.7a
.wab
.65a
20.4a 15.4b
58.Sa
17.3b
.31 b
.36
with different supersaipts in the same column differ (P < .01).
JNumber of Staphylococcus
QJlTe1IS
eoguIfed per phaBocytosiDg polymorpbonuc1ear IIe11trophil (PMN).
2Percentage of PMN phagocytosing. 3Percentage of killing of S. aureus by PMN.
4-rhe SOD activities in red blood cell lysate as units (U) per milligram of hemoglobin (lIb). SThe SOD activities in neutrophil as unilS/IO,OOO,OOO cells. 6o:rhc
SOD activities in whole blood lysate as units of activity per milliliter.
synthesis of hepatic export proteins, such as albumin or transferrin, influence ceruloplasmin level independently of Co status. The Co concentration in neutrophils was lower in -eo group compared with the +Co group by the last sampling period. This change may be due to decrease in the labile Co pool, which is complexed with amino acids or is in the finnly bound fonn of SOD as observed in RBe (6). When decrease in granulocyte Co concentration occurs, cell functions may be impaired. Researchers observed that human patients with decreased healing times tended to have lower leucocyte Co concentrations (21). In this trial, Co concentration in the neutrophils was lowered before Co status reached borderline or deficiency levels. Liver Mo concentration was increased by Mo supplementation in the -Co group compared with the +Co group, which agrees with earlier observations (5). Molybdenum accumulated in the liver was probably in the fonn of thiomolybdate, a complex consisting of Mo, S, and albumin (9). It is postulated that Mo deposited in the liver may decrease Co level in the liver by at least two mechanisms. Molybdenum may decrease Co absorption from the gastrointestinal tract via a chelating mechanism (1), or it may alter the distribution of Co in the tissues and fecal excretion (13). Olange of liver Mo concentration with time is' presented in Figure 3. Liver Mo level in -eo group increased rapidly in the 1st roo and slowly in the remaining time of the experiment. Liver Mo level in the +Co group consistently was low and tended to decline with Journal of Dairy Science Vol 74. No.9. 1991
time, indicating a possible antagonistic reaction between Co and Mo in the liver during Co supplementation. Blood total hemoglobin and BW gain were not different between treatments (fable 2), indicating that clinical Co deficiency was not reached in this trial. During clinical Co deficiency, hemoglobin synthesis and BW gain nonnally are depressed (22). Effects on PMN functions and SOD glutathione peroxidase activities are presented in Table 3. Neither the number of S. aureus in each engulfing PMN nor the percentage of total PMN that phagocytosed S. aureus was affected by Co status. However, the bactericidal capacity of PMN expressed as percentage of S. aureus killed by PMN in this experiment
20 18 E
Do Do
S
14
12
.5
10 8
0
.,•.
:J
.eu £ZZl -eu
16
a 2
_
6
" 2
0
o
1 2 Month Of Treetment
5
Figure 3. Mean concentration of liver Mo in steers fed Co-supplemented (+Cu) and Cu-depleting (--eu) diets during the 8 mo of supplementation of Co or Mo.
3083
COPPER STATIJS AND NEUI'ROPHILS 70
-0-
+Cu ....... -Cu
1.00
50
ii'
40
l/I.
30
-0-
.90 .80
60
~ ....
20
......
j.~--e ..................... ......•........•
10
-Cu
.
.70 0
+Cu
/°"0
~
.60
.50 .40 . 30 .20 .10
. ..........•........•
.0
0 2
0
3
4
5
6
7
o
8
2
345
6
7
8
Month Of Treatment
Month Of Treatment
fed diets supplemented with Cu (iCu) or Mo (-CD) during 8 mo.
Figure 6. Mean superoxide dismutase activities in neutrophils in steers fed Cu-supplemented (+Cu) and Cudepleting (-CD) diets during 8 mo of supplementation of Cu or Mo.
was decreased in the --Cu group. This agreed with observations in ewes (10). The changes of bactericidal capacity of PMN with time (Figure 4) showed a significant difference between treatments only in the last month of the experiment. We suggest that fluctuation was caused mainly by variation in the ratio of S. aureus to PMN in the incubation mixture from assay to assay. However, this conclusion should not be affected because a relative rather than absolute difference between treatment groups was examined. Polymorphonuclear neutrophils are one of the most important phagocytes in defending the body against the
invasion of pathogenic microorganisms. Once the functions of PMN are impaired, the animals may be more susceptible to various infections. Hence, Co deficiency may lead to increased occurrence of certain diseases in animals. Superoxide dismutase activities in RBC lysate, PMN, and whole blood were decreased by low Co status in the -en group compared with the +Co group, which agrees with data of Jones and Suttle (10). The decrease in SOD activity of PMN may be responsible for the reduction in bactericidal capacity of PMN. This impaired bactericidal function may be caused by a decline in protec-
Figure 4. Mean percentage killing of Staphylococcus
aureus by polymorphonuclear neotrophils in vitro in steers
1.00
:S 0
.90 .80
E
.70
a0
•
21
.60 .50 .40
...
12 9 6 3
a
E
C
.30
c:i
.20
~
0
III
30 27 24 •
+Cu •.••. -Cu
~"--o............... -........,-~
:z:
....:l
-0-
.." ....
. °.... ................ .
.10
~:
-0-
+Cu
~o-o~
'.- _. -.. --.... --...
oL-----L---'---""--'--.......-
.0
o
2 3 4 5 Month Of Treatment
7
8
_.•-. -Cu
o
2
3
4
-""
.......-
5
6
0_0.
-..----
........--7 8
Month Of Treatment
Figure 5. Mean superoxide dismutase (SOD) activities in red blood cell lysate in steers fed Cu-supplemented (iCu) and Cu-depleting (-CD) diets during 8 mo of supplementation of Cu or Mo.
Figure 7. Mean superoxide dismutase (SOD) activities in whole blood lysate in steers fed Cu-supplemented (iCu) and Cu-depleting (-CD) diets during 8 mo of supplementation of Cu or Mo. Journal of Dairy Science Vol. 74, No.9, 1991
3084
XIN ET AL.
tion of phagocytic cells from oxidation mediated by oxygen-free radicals during the bactericidal process (10) or a decrease in supply of H2~ to myeloperoxidase-halide system to produce microbial killing agents such as hypochlorite ion and hydroxyl radicals (to). The pattern of changes in SOD activities in RBC, PMN, and whole blood (Figure 5, 6, 7) corresponds to the decline in liver Cu concentrations in the --Cu group (Figure 1) over the last period and the decline in percentage S. aureus killed during the last sampling time of the trial (Table 3). Changes in SOD activity of whole blood were less dramatic and had a more consistent pattern in comparison with the other parameters. This may be due to less variation in sample preparation. Furthermore, SOD activities in whole blood were affected at the 2nd mo of treatment, which was earlier than any other parameter except for liver Cu concentration. Therefore, SOD activities may be pragmatically important for surveying Cu status in the field as well as serving as an indicator of Cu deficiency. More research on the relationship between Cu status and standard SOD quantity or other equivalent values must be conducted before the practice can be accepted. One possible approach is to make a pooled control sample that is from Cu-sufficient animals and to compare its SOD result with that in the questionable sample in each assay. Thus, variations from assay to assay or from time to time will not affect the relative comparison between Cu-sufficient control sample and questionable samples from the field to decide whether or not the animal is Cu-deficient. Glutathione peroxidase activity was not affected by Cu status or changes in SOD activities in this experiment. CONCLUSIONS
Results from this study have shown that SOD activities in blood parameters and intracellular Cu concentration in P:MN will decrease when Cu status is low or deficient based on liver Cu concentration. Therefore, definition of functional Cu deficiency .may need to be specified further under different physiological or pathological conditions. Based on the observations in this study, it is of potential importance to use SOD as a screening criterion for diagnosing Cu deficiency by 10urnal of Dairy Science Vol. 74, No.9, 1991
using a Cu-sufficient control sample in each assay. Results from this study also have shown that low Cu status can decrease intracellular SOD activity and neutrophil bactericidal capability in dairy steers. The level of ceruloplasmin, a Cu-containing C12-globulin possessing potent antioxidant activity and modulation of tissue injury associated with the production of oxygen metabolites by phagocytes, also can be reduced by low Cu status from other studies. Hence, low Cu status in animals may contribute partially to an increase in susceptibility to infections and consequent tissue injuries as in the case of mastitis in dairy cows. Therefore, we suggest that the relationship between Cu status under certain physiological conditions and disease susceptibility or tissue injury needs to be investigated further. REFERENCES 1 Allen, 1. D., and 1. M Gawthorne. 1987. Involvement of the solid phase of rumen digesta in the interaction between copper, molybdenum and sulphur in sheep. Br. 1. Nutr. 58:265. 2 Boyne, R., and 1. R. Arthur. 1981. Effects of selenium and copper deficiency on neutrophil function in cattle. 1. Comp. Pathol. 91:271. 3 Bremner, I. 1980. Page 23 in Biological roles of copper. Elsevic- Appl. Sci. Publ., New York, NY. 4 Carlson, G. P., and 1. 1. Kaneko. 1973. Isolation of leucocytes from bovine peripheral blood. Proc. Soc. Exp. BioI. Med. 142:853. 5 Cook, G. A., A. L. Lesperance, V. R. Bobman, and E. H. 1ensen. 1966. Inten'elationsbip of molybdenum and certain factors to the development of the molybdenum toxicity syndrome. 1. Anim. Sci. 25:96. 6 Davis, G. K., and W. Mertz. 1987. Copper. Trace elements in human and animal nutrition. 5th ed. Academic Press, Inc., San Diego, CA. 7 Eivazi, P., 1. L. Sims, and 1. Crutchfield. 1982. Determination of molybdenum in plant materials using a rapid, automated method. Commun. Soil Sci. Plant Anal. 13(2):135. 8 Gooneralne, R, and D. Christensin. 1985. Gestation age and maternal-fetal liver copper levels in the b0vine. Page 334 in Trace elements in man and animals-lEMA 5. Commonw. Agric. Bur., FIlIIIham Royal, Engl. 9 Hynes, M, M. Lamand, G. Montel. and 1. Mason. 1984. Some studies on the metabolism and the effects on Me- and S-labelled thiomolybdates after intravenous infusion in sheep. Br. 1. Nutr. 52:149. 10 Jones, D. G., and N. P. Suttle. 1981. Some effects of copper deficiency on leucocyte function in sheep and cattle. Res. Vet. Sci. 31:151. 11 Lawrence, R. A., and R. P. Bark. 1976. Glutathione peroxidase activity in selenium deficient rat liver. Biochem. Biophys. Res. Commun. 71(2):952.
COPPER STATUS AND NEUTROPmLS 12 Mason, J. 1978. The relationship between copper, molybdenum and sulphur in ruminant and n0nruminant animals: a review. Vet. Sci. Commun. 2:85. 13 Mason, J., M. Lamand, 1. C. Tressel, and G. Mulryan. 1988. Studies of the changes in systemic copper metabolism and excretion produced by the intravenous administration of trithiomolybdate in sheep. Br. J. Nutr. 59:289. 14 Masters, H. G., G. M. Smith, and R H. Casey. 1985. Page 575 in Trace elements in man and aniInal&TBMA 5. Commonw. Agric. Bur., Farnham Royal, Engl. 15 McCord, J. M., and I. Fridovich. 1969. Superoxide dismutase, an enzymic function for erythrocuperin. 1. BioI. Cbem. 244:6049. 16 Phillippo, M., W. R Humphries, and T. Atkinson. 1987. The effect of dietary molybdenum and iron on copper status, puberty, fertility and oestrous cycles in cattle. J. Agric. Sci. (Carob.) 109:321. 17 SAS$ User's Guide: Basics. 1985. SAS IIlst, Inc., Cary, NC. 18 Scholz, R. W., and L. J. Hutehinsin. 1979. Distribu-
3085
tion of glutathione peroxidase activity and selenium in the blood of dairy cows. Am. J. Vet. Res. 40:245. 19 Shuster, D. E. 1985ln vitro effects of anti-inflammatory agents on bovine neutrophils stimulated with opsonized Staphylococcus aureus. M. S. Thesis, Univ. Kentucky, Lexington. 20 Smith, B.S.W., and H. Wright 1975. Effect of dietary Mo and Cu metabolism, evidence for the involvement of Mo in abnormal binding of Cu to plasma proteins. Clin. Chim. Acta 62:55. 21 Thomas, A. J., V. W. Bunker, L. J. Rinks, N. Sodha, M. A. Mullee, and B. E. Clayton. 1988. Energy, protein, zinc and copper status of twenty-one elderly inpatients: analyzed dietary intake and biochemical indices. Br. J. Nutr. 59:181. 22 Ward, G. M. 1978. Molybdenum toxicity and hypocuprosis in ruminants: a review. J. Anim. Sci. 46: 1078. 23 Xin, Z., W. 1. Silvia, R. W. Hemken, W. B. Tucker, and J. A. Boling. 1989. Effects of copper status on LH secretion in dairy steers. J. Anim. Sci. 67(Suppl. 1): 320.(Abstr.)
Journal of Dairy Science Vol. 74, No.9, 1991
