effects of dietary molybdenum on the metabolism of copper and molybdenum in young cattle

BIOINORGANIC CHEMISTRY 6,1 l-28 (1976) 11

Effects of Dietary Molybdenum on the Metabolism of Copper and Molybdenum in Young CattIe *t

GERALD L. FISHER,* CHARLES A. HJERPE,§ and CHARLES W. QUALLSS Radiobiology Laboratory, University of California, Davis, California 95616

ABSTRACT

On separate occasions young cattle were injected intrarumenally with 9 9 MO or 6eCu or intravenously with 64Cu. The metabolism of the isotopes were coqpared to evaluate metabolic changes associated with molybdenum-induced copper deficiency_ Molybdenum-99 metabolism was the same in both controls and experimental subjects. Marked differences in plasma kinetics were observed following intrarumenal 64Cu injection, with experimental animals displaying earlier plasma appearance and maintaining higher plasma IeveIs than did controls. Similarly, higher plasma levels, more rapid plasma reappearan= and greater fecal excretion were observed following intravenous injection of ‘*Cu in the experimental animals than in controls_ A mechanism involving abnormal ceruloplasmin synthesis associated with the molybdenum-copper interaction is proposed_

Copper deficiency in ruminants associated with high dietary molybdenum (molybdenosis) has been reported throughout the world. The interaction of molybdenum with copper absorption and/or utilization is further complicated by dietary sulfur levels which may either antagonize or ameliorate the interaction_ In his review of the literature, Underwood [l] indicated that pastures containing 4-6 ppm copper and with low levels of molybdenum and sulfur can meet the dietary requirements of cattle and cross-bred sheep. Pope [Z] and Todd [ 31 have indicated that MO IeveIs as Iow as 2 ppm may affect copper metabolism, leading to increased dietary copper requirements.

The Cu-Mo-S04 metabolic interactions are complicated, and the biologic effects depend on the initial copper status of the animal. Huisingh et al. [4] have proposed a mechanism to account for the Cu-MO-SO4 interactions in ruminants.

*Jointly supported by funds from General Research Support +5SOlRR05457-13 and Livestock Disease Research Laboratory, School of Veterinary Medicine, University of California, Davis, California, and U.S. E_R_D.A_

Wresented in part at the Symposium on Molybdenum in the Environment, Denver, Cal., 16-19 June, 1975.

fRadiobiclogJ% Laboratory, University of California, Davis, California. pSchoo1 of Veterinary Medicine, University of California, Davis, California.

0 American Elsevier Publishing Company, Inc., 1976

12 G. L. FISHER, C. A. HJERPE AND C. W. QUALLS

According to their theory, molybdenum interacts with copper in the rumen or in tissues to form a Cu-MoOa complex in which the copper is not biologically available. This complex is thought to be excreted in the urine and possibly in the feces_ Sulfate is thought to interact with copper absorption through microbial reduction of sulfate to sulfide and subsequent formation of insoluble copper sulfide. Molybdate may limit sulfate reduction by competition with sulfate membrane transport or by direct inhibition of the sulfate-reducing process in rumen microorganisms. Sulfate may decrease molybdate absorption by com- peting for common protein carriers and increase its excretion by inhibition of molybdate absorption in the kidney distal tubtrIes_ Suttle IS] and Underwood 161 recently have proposed that formation of the highly insoluble cupric thiomolybdate (CuMo&) in the rumen may be the mechanism associated with this disorder.

The clinical signs of molybdenum-induced copper deficiency are variable and appear to depend on species, age, duration of dietary exposure, and initial copper status [ 7 J _ More commonly reported clinical signs in cattle are diarrhea, poor growth, achromotrichia, and alopecia, as well as improper bone mineraliza- tion, leading to lameness and spontaneous fractures [ I]_ Anemia has also been reported to be-associated with molybdenosis. However, this condition is not commonly encountered in field cases but rather in laboratory experiments with very high levels of dietary molybdenum.

We have observed young cattle with lameness associated with enlargement and swelling of the distal metacarpal and metatarsal physeal regions [7, 81. Histologic and radiographic evaluation indicated that these lesions were similar to those of rickets, and were probably not the same bone disorder reported in primary copper deficiency in dogs, pigs, and sheep [8] _ Recently, knee joint deformities similar to those in cattle have been observed in young men in India [9) _ This human disorder is thought to be associated with a molybdenum- induced copper deficiency_

This paper describes 64Cu and “MO metabolism in young cattle with molybdenum-induced copper deficiency and discusses an alternative theory to those cited for the mechanism of molybdenum-induced copper deficiency.

MATERiALS AND METHODS

Bxperimental animals were obtained from a ranch located in the northern portion of California’s San Joaquin Valley. The pastures were predominantly DalIis grass (Pizspalum di.tzfatum) and bermuda grass (Cynadon dactylon),

together with lesser quantities of Iadino ciover (Ttifolium repens) and narrowleaf birdsfoot trefoil (Lotus tenuti). The ranch was stocked with 50 cross-bred beef cows and their calves, 50 yearling cattle, and 2 mature bulls. The cattle were supplemented only with plain salt in block form and, during the winter, with hay cut from the pastures during the summer.

Signs of copper deficiency appeared in April of 1974 as pathologic bone fractures, primarily in the scapula or humerus of nursing calves. During the next

64CU AND “MO METABOLISM IN YOUNG CATTLE 13

7 months, bone fractures occurred in a 2-year-old bull, in 2 yearlings, and in 20 of the 50 nursing calves. -Most affected calves were 6- to IO-no-old, although some were as young as 2 months.

Three calves affected with fractures of the scapula and/or humerus were examined on September 19, 1974. Hair coarseness was the only clinical evidence of copper deficiency observed in these 3 calves and was not present in the rest of the herd. The cattle were generally in good condition, with normal hair coat texture and color and feces of normal consistency. Enlarged metacarpal and metatarsal epiphyses were not observed. Anemia was not present in calves affected with bone fractures, as evidenced by normal blood hematocrits and hemoglobin concentrations, although the erythrocytes were microcytic and were in the low-normal range with respect to hemoglobin concentration and content. The diagnosis of molybdenum-induced copper deficiency was based on the presence of extremely IOW liver copper concentrations of less than 10 ppm (normat = 200 ppm D-M_) in 2 of the 3 calves affected with pathologic fractures, on subnormal serum copper levels (average from 15 calves of 0.45 ppm vs. normal of 0.87 ppm) in clinically unaffected calves, and on the presence of abnormal copperrmolybdenum relationships and normal calcium:phosphorus relationships in the pasture and hay. Pasture analysis indicated 5.3 ppm Cu and 3.6 ppm MO.

Two calves were obtained from this ranch for subsequent “MO and “4Cu metabolic studies. Throughout the course of the studies the animals were maintained in indoor metabolic stanchions. The experimental subjects were approximately 6 months of age and the controk were 8-mo-old at the start of the study. Body weights were determined throughout the course of the study_ The two copper-deficient calves were fed rations of hay cut the previous year from the described pastures, while two control calves were fed-alfalfa hay during the study period- All animals were provided water from one source. Feed and water were provided ad libitum. Hay and serum samples were analyzed for Cu by atomic absorption spectroscopy, for MO by farmation of the thiocyanate complex and subsequent calorimetric analysis, and for SO4 by th& Nishita- Johnson cotorimetric technique IlO] _ The hay analyses for both the experimeu- tal and control diets are summarized in Table 1_

Since the hay fed during the experiment was different from the initial pasture diet, interpretation of the results must consider the fact that the experimental subjects were initially on a pasture diet containing 3.6-ppm MO and 5.3-ppm Cu while these metabolism studies were conducted after the animals had been maintained for 2 months on hay cut from the same pasture one year prior and containing 1.8-ppm MO and 4.7-ppm Cu.

Copper-64 ( 12.75 hr half-life) as the nitrate (purity > 99%) and molybdenum- 99 (66.6 hr half-life) in the form of (?!&)+MoO~ (pWty > 99%) were obtained from International Chemical and Nuclear Corp. (Waltham, Mass_)_ In separate studies, 4 mCi of 64 Cu was administered intravenously (i-v.) and 8 mCi of 64 Cu intrarumenaily (i.r.1 to each animal. In a third study, 2 mCi molybdenum-99 Was injected into the rumen of each animal. The ” MO was administered 25 days prior to the 64Cu intrarumenal injection and 32 days prior to the 64Cu


TABLE 1

Copper, Molybedum, and Sulfur (as Sulfate) Concentrations (dry matter basis) in Treatment and Control Diets

cu MO

(ppm) (ppm) so4-S

(ppm)

Treatment diet Control diet Water

4.7 1.8 1520 8.4 1150 NA= K 52

“Not analyzed.

intravenous injection. The isotopes were diluted in physiologic saline solution and administered by catheters which were flushed with saline solutions after delivery of the isotopes. The specific activities of the 64Cu and “Mu10 were 4.6 mCi 64Cu/mg Cu and 0.12 mCi “Mo/mg MO. Thus the stable copper administered to each animal were 0.87-mg Cu and 1.74mg Cu for tbe intravenous and intrarumenal injections, respectively. The stable molybdenum administered to each animal was 16.8 mg.

Blood for plasma samples was obtained from the jugular vein at 5 mm, 1,2,4, 8, 16, 24, 48, 72, 96, and 120 hr after administration of each isotopes; an additional sample was taken at 30-min after the i-v. administration. Feces and urine were collected continuously and weighed at 12-hr intervals for up to six days; aliquots were taken for counting

Five-milliliter plasma samples were analyzed in a Nuclear-Chicago Automatic Gamma Counting system with a well-type scintillation counter and a NaI(T1) crystal of nearly 4 7~ geometry and a thickness of 7-6 cm. The activities of the fecal and urine samples were determined in a laboratory-fabricated counting system, utilizing two large NaI(T1) detectors (IO-cm diameter X lO-cm thick) of 1.3 n geometry and a multichannel analyzer_

Data were reported only for samples with net count rates greater than 10 cpm. In the calculation of percent of dose in total pIasma, the plasma volume was obtained by using hematocrit data and assuming the total blood volume of the animal to be 0.077 liters per kg of body weight I1 13. Body weights used in calculating total plasma volumes are reported in Table 2.

RESULTS

IntrarumenaJ ’ ?Mo Exposure

The “MO plasma kinetics for the four calves (Fig. 1) showed little difference between the experimental subjects and control animals. The isotopes first appeared in the p&ma of 3 of the 4 animaIs 4 hr after dosing, and peak


TABLE 2

Body Weights (kg) of Experimental Subjects and Controls Used in the Calculation of Total Plasma Volumes

Experimental Treatment

“MO 64cu

(i.r.F (i.r., i.v.)b

Subject 1 83 94 Subject 2 153 168 Control 1 172 225

Control 2 189 218

Oi_r. = intrarumenally. Xv. = intravenously_

0.400

0.300

9 0.200

z Q

2 0.100 a 0.080

g 0.060

s 0.040

5 0.030 0

I& 0.020

, *

0.010 0.006 %o-IR PLASMA KINETICS 0.006

0.004

0.003 0 8 16 24 36 46 60 72 64 96 108 120

TIME c HOURS) POST INTRARUUENAL INJECTION

FIG. 1. Plasma levels of “MO in experimental subjects and controls as a function of time after intrarumenal injection.

16 G. L_ FISHER, C. A_ HJERPE AND C. W. QUALLS

concentrations ranging from approximately 0.2% to 0.7% of the dose occved at 24 hr. Although one control animal (control 2) had substantially lower peak “MO concentrations, the other control had a peak concentration similar to those of the two experimental subjects. After 24 hr ail animals showed similar plasma “MO levek and disappearance kinetics_ Molybdenum-99 excretion was observed in the fast I2-hr fecal samples in 3 of the 4 anhnals (Fig. 2). Excretion of the isotope peaked with approximately 15% of the dose in the feces 24 to 48 hr after dosing. Urinary excretion of “MO was frost detectable in the 24&r sample (Fig_ 3), with control 2 showing lower levels than the other three animals. In general, maximal urinary excretion of “MO, approximately 2% of the dose, occurred at 2448 hr. The apparent diurnal rhythm of urinary excretion (Fig. 3) for “MO is probably attributable to the pattern of urination;

the fecal excretion data show no suggestion of such a pattern. The average cumulative urinary excretion to 144 hr of “MO administered intrarumenally was 15% + 4.6% (k S-D.) of the fecal excretion_

Although control 2 appeared to metabolize “MO differently than the other animaIs early in the experiment, the cumulative fecal and urinary exptions (Table 3 and 4) were similar for all animals. Cumulative fecal excretion at 144 hr

60.0 . s . . I a l a 1 a 1

40.0 - ?do-IR FECAL EXCRETION 30.0r 7

20.0 -

10.0 1 8.0-

z? 6.0 -

g 4.0 -

= 3_0-

z 2-G

& g I.01

0.8 -

0.6 -

Subject i&

co.1 + ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ 0 I2 24 36 48 60 72 84 96 108 I20 132 144

TIME (HOURS1 POST INTRARUYENAL INJECTION

FIG. 2. Fecal excretion of **MO in experimental subjects and controls as a function of time after intrarumenal injection

64CU AND ggMO METABOLISM IN YOUNG CATTLE 17

after dosing was 65% to 68% of the dose, and cumulative urinary excretion at 144 hours was 7.4 to 13.9% of the dose with no difference attributable to either

group.

10.0

6.0 Uo- IR URINARY EXCRETION

6.0

4.0

3.0

g 2.0

z 5 2 0.8 I .o

- % 0.6

c 0.4

0.3

co.1 0 12 24 36 46 60 72 64 96 106 120 132 144

TIME (HOURS) POST INTRARUYENM INJECTION

FIG. 3. Urinary excretion of 99hio in experimental subjects and controls as a function time after in&arume& injection_

TABLE 3

Levels of 9g MO in 12-IIr Fecal Collections after Intrarumenal Injection

of

Time Subject 1 Subject 2 Control 1 Control 2 after dosing % of cum. % % of cum. lo % of cum_ % % of cum. 70

(hr) dose of dose dose of dose dose of dose dose of dose

12 I.33 I.33 0.47 o-47 0.66 0.66 0.00 0.00 24 13.79 15.12 21-13 21.60 13.96 14.62 6.84 6.84 36 12.89 28.01 15.45 37.05 17.95 32.57 1339 20.23 48 17.10 45.11 15.16 52.2 1 14.15 46-72 13.65 33.88 60 7.15 52.26 4.9 1 57.12 7.17 53.89 14.45 48.33 72 5.09 57.35 3.04 60.16 6.13 60.02 6.43 54.76 84 4.38 61.73 2.11 62.27 3.31 63.33 4.89 59.65 96 2.57 64.30 1.04 63.3 I 1.09 64.42 2.36 62.01

108 1.53 65.83 0.61 63.92 0.77 65.19 1.44 63.45 120 1.05 66.88 0.52 64.44 0.52 65.71 0.90 64.35 132 0.68 67.56 0.17 64.61 0.30 66.01 0.45 64.80 144 0.67 68.23 0.17 64.78 0.27 66.28 0.34 65.14


TABLE 4

Levels of “MO in 12-Hr Urine Collections after Intrarumenal Injection

Time Subject 1 Subject 2 Control 1 Control 2 after dosing % of cum- % % of cum_ % % of cum. % % of cum. %

(W dose of dose dose of dose dose of dose dose of dose

12 o-00 o-00 0.0’7 0.07 0.00 0.00 0.03 0.03 24 1.84 1.84 2.47 2.54 1.91 1.91 0.34 O-37 36 1.43 3.27 2.85 5.39 4.26 6.17 1.75 2.12 48 -a S-226 2.36 7-75 2-19 8.36 1.30 3-42 60 0.55 5.77 0.84 8.59 1.37 9.73 0.69 4.11 72 0.6 I 6-38 O-46 9.05 1.67 11.40 1.02 5.13 84 0.38 6.76 0.57 9.62 0.62 12.02 0.75 5.88 96 0.40 7.16 0.3 1 9.93 OS2 12.54 0.34 6.22

108 0.28 7.44 0.26 10.19 0.26 12.80 0.24 6.46 120 O-17 7.6 1 o-22 10.41 0.63 13.43 0.35 6.81 132 0.19 7.80 0.12 10.53 0.15 13.58 0.19 7.00 144 0.18 7.98 0.18 10.71 0.32 13.90 0.37 7.37

USample lost. &stimated value (195% of dose) by averaging 48-hr samples from other animals due to

accidenta disposal_

Intrammena! 6 4Cu Exposure

The plasma kinetics (Fig. 4) for intrarumenaIly injected 64 Cu indicate striking differences between controls and experimental subjects_ As early as 4 hr after dosing, the experimental subjects show higher plasma levels of 64Cu than do controls_ These differences are maintained in later plasma measurements, with 48-hr values of 0.6% and 0.8% of the dose for the experimental subjects vs approximately 0.2% for the controls_ These data indicate that the experimental subjects absorbed 64 Cu more rapidly and maintained higher levels in plasma than did the controls.

The overall fecal excretion pattern of 64Cu (Fig. 5) is similar for the two groups aIthough the 12&r sampIe does indicate higher Ievek for the experimental subjects (Table 5). Maximal fecal excretion occurred 2448 hr postinjection, sin&r to the “MO results. Cumulative fecal excretion to 144 hr postinjection was simiIar for all a5mals and ranged from 75% to 83% of the administered dose. Urinary excretion of the isotope (Fig. 6) showed a similar pattern for all animals and was relatively constant from 24 to 96 hr postinjection The cumulative urinary excretion of 6 4 Cu by the experimental subjects was generally


O-006 .. PLASMA KINETICS : 0.004 0.003 -

0.002”” ’ ’ ’ ’ ’ ’ * 0 12 24 36 48 60 72 84 96

TIME (HOURS) POST INTRARUYENAL INJECTION

FIG. 4. Plasma levels of 64Cu in experimental subjects and controls as a function of time after intrarumenal injection_

lower than that of the controls (Table 6) with cumulative urinary excretions of 0.18% and 0.27% of the dose for the experimental group vs. 0.43% and 0.32% for the controls. The average cumulative urinary excretion to 96 hr of 64Cu administered intrarumenally was 0.38% 2 0.15% (2 S. D.) of the fecal excretion. Thus, partitioning between urine and feces of intrarumenally administered 64C~ was very different than that for “MO.

Intravenous 64Cu Exposure

Intravenous injection of 64 Cu resulted in marked differences in plasma kinetics (Fig. 7) between the two groups. The experimental subjects and controls both demonstrate rapid plasma clearance of the isotope_ However, minimum


FIG. 5_ Fecal excretion of 6 * Cu in experimental subjects and controls as after intrarumenal injection.

0 I2 24 36 48 60 72 84 96 108 120

TIME ( HOURS1 POST INTRARUYENAL INJECTION

a function

TABLE 5

Levels of 6 4 Cu in 12-Hr Fecal Collections after intrarumenal Injection

of time

Time Subject 1 Subject 2 Control I Control 2

after dosing % of cum_ % % of cum. % % of cum. % % of cum. %

GM dose of dose dose of dose dose of dose dose of dose

12 0.98 0.98 2.22 2.22 0.20 0.20 0.25 O-25

24 18.36 19.34 24.70 26.92 14.16 24-36 25.68 25.93 36 IS.00 34.34 19.07 45.99 23-32 37.68 17.09 43.02 48 22.53 56.87 20.04 66.03 18.25 55.93 17.11 60.13

60 7-46 64-33 8.42 74.45 7.30 63.23 7.47 67.61 72 7-78 72.10 4.16 78.6 I 5.11 68.34 7-26 74.87 84 4.43 76.54 2.12 80-73 3.04 71.38 2.41 77.28

96 2.52 79.05 1.40 82.13 1.96 73.34 2.11 79.39 108 1.31 80.36 0.56 82.69 0.96 74.30 0.96 80.35

.120 0.70 81.06 0.44 83.13 0.69 74.99 0.70 81.05

64CU AND ?vIO METABOLISM IN YOUNG CATTLE 21

0.200

t

‘%u- IR URINARY EXCRETION

0.100

o.oao - 0.060 -

$I 0.040 -

5 0.030 -

s 0.020 -

: 0 u. 0.010 - ; 0.008 -

0.006 r

0.004

0.002 i

0.003

: 0.001 L ’ ’ ’ ’ ’ ’ ’ ’

0 12 24 36 40 60 72 04 96 106 120

TIME (HOURS) POST INTRARUYENAL INJECTION

FIG. 6. Urinary excretion of 64Cu in experimental subjects and controls as a function of time after intrarumenal injection.

plasma levels are higher in experimentals (7% and 9%) than in controls (2.2% and 2.5%) and appear to occur earlier than in the controls (2.v.s. 8 hr postinjection). Furthermore, although the pattern of plasma reappearance is similar for both

groups, the experimental subjects display a more rapid reappearance and maintain higher levels (12% to 15% of the administered dose) than-do controls (2.5% to 3.2%). These resuhs agree with and accentuate the differences observed for the 64Cu intrarumena1 plasma kinetics.

The feca1 excretion (Fig. 8) of intravneously injected 64Cu is greater for the experimental subjects than for the controls (Table 7), with 120-hr cumulative fecal excretions of 9.2% and 6_6% of the dose for the experimental subjects and 3.9% and 3.3% for the controls. These differences appear to be consistent throughout the study with the widest differences occurring between 24 and 48 br after dosing. Urinary excretion (Fig. 9) for the experimental subjects (0.36% and 0.32%) was initially less than controls (0.95% and 1-l 1%). However, this difference was not maintained throughout the study. The differences in the 72-hr cumulative urinary excretion (Table 8) of 64Cu between the experimental animals (0.61% and 0.63% of the dose) and the controls (1.25% and 1.35%) are attributable solely to the greater urinary excretion of 64Cu by controls in the fit 12 hr.

22 G. L. FISHER, C. A. HJFXPE AND C. W. QUALLS

TABLE 6

Levels of 64Cu in 12-Hr Urine Collections after Intrarumenal Injection

Time Subject 1 Subject 2 Control 1 Control 2 after dosing % of cum % % of cum_ % % of cum_ % % of cum. % (w dose of dose dose of dose dose of dose dose of dose

12 0.002 0.002 0.001 0.001 0.002 0.002 0.002 24 0.02 1 O-023 0.019 0.020 0.046 0.048 0.057 36 0.016 0.038 0.09 1 0.111 0.107 0.155 O-05 1 48 0.032 0.070 0.075 0.186 0.085 0.240 0.040 60 0.04 1 0.111 0.023 0.210 0.041 0.281 0.030 72 0.034 0.145 0.023 0.232 0.058 0.339 0.073 84 0.010 0.155 0.008 0.24 1 0.016 0.355 0.018 96 0.021 0.177 0.029 0.270 0.072 0.427 0.052

60

2

I 0 8 16 24 36 48 60 72 84 96

TIME (HOURS1 POST INTRAVENOUS INJECTION

0.002 0.059 0.110 0.149 0.180 0.253 0.27 1 0.323

FIG_ 7. Phsma levels of 64Cu in experimental subjects and controls as a function of time after intravenous injection_


TABLE 7

Levels of 64CU in 12-Hr Fecal Collections after Intravenous Injection

Time Subject 1. Subject 2 Control I Control 2 after dosing % of cum. % F/o of cum. % % of cum. % % of cum. %

(hr) dose of dose dose of dose dose of dose dose of dose

12 0.36 0.36 0.50 0.50 0.56 0.56 0.34 0.34 24 0.98 1.34 0.90 1.40 0.56 1.12 0.52 0.86 36 0.89 2-24 0.59 1.98 0.28 1.40 0.19 1.04 48 0.98 3.22 0.54 2.52 0.25 1.65 0.23 1.27 60 0.84 4.06 0.58 3.10 0.24 1.89 0.18 1.45 72 1.20 5.26 0.65 3.75 0.28 2.16 0.26 1.72 84 0.70 5.96 0.58 4.33 0.39 2.55 0.25 1.97 96 1.08 7-04 0.84 s-17 0.39 2.93 0.45 2.42

108 1.11 8.15 0.60 5.77 0.54 3.48 0.42 2.84 120 1.10 9.24 0.82 6.58 0.44 3.92 0.49 3.34

2.0. , 1 , , , ( , L

‘%u -IV FECAL EXCRETION

0.11 ’ * . ’ ’ I 1 1 I

0 12 24 36 48 60 72 84 96 108 120

TIME ( HOURS) POST INTRAVENOUS INJECTION

FIG. 8. Fecal excretion of 6 4Cu in experimental subjects and controls as a function of time after intravenous injection_

SERUM COPPER LEVELS

Stable serum copper levels (SCL) were determined at various times throughout the course of the tracer studies (Fig. IO). Throughout the studies, subject 1 had a SCL similar to that of controls and greater than that of subject 2_ Despite these

24 G. I.. FISHER, C_ A_ HJERPE AND C. W_ QUAILS

differences in SCL’s, both experimental animals showed similar copper metabolism in both tracer studies. No apparent explanation can account for the decrease in SCL’s observed on February 11 and 16_

DISCUSSION

This work indicates that levels of dietary molybdenum as low as 1.8 ppm may affect copper metabolism in young cattle, since the level of Cu in the hay should supply sufficient Cu to meet the dietary requirements [ 11. These findings are similar to those of Pope [2] and Todd [31 who indicate that 2 ppm of MO may affect copper metabolism in ruminants_ It is interesting to note that at the time of i-v. 64Cu injection one of the experimental subjects had higher serum copper

2.00 L i I I L I I

“%u - IV URINARY EXCRETION

0.40 -

0.30 -

Y 5 0.20 -

r

z x

k 0.10 -

ap 0.08 -

0.06 -

0.04 -

0.03 -

0.02 -

0.01 1 I I

0 12 24 36 48 60 72 84

TIME ( HOURS1 POST INTRAVENOUS INJECTION

FIG_ 9. Urinary excretion of 64Cu in experimental subjects and controls as a function of time after intravenous injection_

64CU AND 99M0 METABOLISM IN YOUNG CATTLE 25

TABLE 8

Levels of 6 4CU in 12-Hr Urine CoIIections after Intravenous Injection

Time Subject 1 Subject 2 Control 1 Control 2 after dosing % of cum. % % of cum_ % % of cum. % % of cum. % (br) dose of dose dose of dose dose of dose dose of dose

12 0.355 0.355 0.326 0.326 0.946

24 0.086 0.440 0.146 0.472 0.117 36 0.042 0.483 0.05 1 0.523 0.064 48 0.043 0.525 0.036 0.559 0.045 60 0.038 OS64 O-029 0.589 0.038 72 0.047 0.611 0.041 0.630 0.038 84 0.03 1 0.642 0.035 0.665 -n 96 -Lx 0.040 0.705 _a

0.946 1.105 1.105 1.063 0.095 1.200 1.126 0.054 1.254 1.171 0.039 l-293 1.209 0.023 1.316 1.247 O-035 1.351

-cl -a

aCount rate < IO cpm.

SERUM COPPER LEVEL (ppfn) 1.10 1 I

. 0.60 -

I 8 - I

9 14 31L 3 7, II I$

January February

FIG. 10. Serum copper levels in experimental subjects and controls as a function of time throughout the study.


levels than controls. Thus, although marked differences were observed in the metabolism of 64Cu, the SCL did not reflect the copper status of this animal.

The metabolism of g9Mo after intrarumenal injection was similar for experimental subjects and controls. The IeveIs of Cu and Mo in the two diets did not alter MO metabolism- These resuhs agree with those of Miller et al. [ 111 who admirnistered rumen doses in gelatin capsuIes of 9gM~ to yearling cattle. They reported pIasma concentrations and kinetics similar to ours, with peak concentrations occurring 2448 hr after dosing. They also reported average 72-hr cumuIative urinary and fecal excretion values of 1 O-O% and 61.6% of dose, which are only s@htIy higher than our 72-hr values (Tables 3 and 4) of 8_0% and 58.1%. The differences observed between these resuhs and those of Miher et al. may have been associated with the specific activities of the 99Mo used. In our work the intrarumenaI injection of 2 mCi “MO contained 16.8-mg stable molybdenum, whereas Miller’s 99Mo had a higher specific activity, such that 2 mCi 9 9 MO contained only 1.2-mg stabIe molybdenum

Chapman and BeII [ 121 have studied the plasma kinetics and fecal and urinary excretion of 64Cu in various chemical forms administered oraliy and intravenously to normal Hereford steers. Oral dosing with 61C~(NO~)a resulted in average 96-hr cumuIative fecal excretions of 85.5% of the dose compared to our findings for ir. exposure of 78.5% average cumulative fecal excretion_ ALSO, they report 96-hr average cumulative urinary excretions of 0.098% compared to our find&s of 0.22% for experimental calves and 0.38% for controls. They aIso report 48-hr plasma levels (% of dose/liter of plasma) of 0.039% compared to our fmdings of 0.02% for both control animals and 0.07% and 0.17% for the experimental subjects. Our findings of higher relative absorption of 64Cu compared to their work was probably reIated to the route of administration (oral vs. intrarumenaI injection) and their administration of a greater amount of stable copper (80 mg vs. 1.7 mg). For intravenous injection of 64C~(N03)a Chapman and Bell report 96hr cumuIative 64Cu excretion of 2.5% of dose in feces and 0.62% in urine compared to our 96hr findings of 2.7% and 6.1% in feces and 72-hr resuits of 1.30% and 0.62% in urine from controls and experimental subjects, respectiveIy.

This study also provides useful Information about the relative efficiency of intrarumenaI vs_ intravenous copper supplementation in cattle. The 120-hr cumulative fecal excretion ranged from 75% to 83% of the intrarumenal dose compared to 3.3-g-2% of the intravenous dose- These results indicate that rumenaIIy-administered copper compared to intravenously administered copper, is poorly absorbed and rapidly excreted.

The 64Cu metabolism data may provide insight into the mechanism of the Mo-Cu interaction_ in both the ir_ and i-v_ studies, plasma 64Cu levels were higher and cumulative urinary excretion Iower in the experimental animals than in the controls_ The secondary reappearance curve of 64Cu in the plasma of the iv_ Injected anImaIs is presumabIy associated with ceruIoplasmin (Cp) synthesis, as other investigators have reported similar curves due to Cp synthesis in both man and the rat [ 13 1. if a Cu-MO complex was formed in the rumen or tissues of these animals as Huisingh et aL’s 141 theory would suggest, it apparently was not


excreted in the urine. Furthermore, the higher plasma levels of 64Cu from the ir. exposure and the reappearance of 64Cu observed in the iv. exposure suggest that the levels of MO in the experimental diet did not decrease copper absorption of utilization for Cp synthesis. Also the higher levels of fecal 64Cu in the experimental calves indicate increased catabolism of organically bound 64 Cu and subsequent biliary excretion_

Marcilese et al. [ 141, in their studies of 64Cu metabolism in sheep as a function of MO + SO4 levels in the diet, reported slower clearance of i-v. injected 64Cu in sheep fed a basal diet (12-14 ppm CU and low sulfate) supplemented with SO-ppm MO and 0_4% SO4 compared with that of control animals fed the basal diet alone or supplemented with 0.4% SO4 _ On the basis of the amount of 64Cu in the liver, they reported a threefold greater 64Cu fecal excretion in their treated animals vs. controls_ They showed no 64 Cu reappearance curve for either controls or treated animals and suggested a reduced use of 64Cu for ceruloplasmin synthesis. These authors suggest in agreement with the previously summarized theories of Huisingh et al. that a CU-MO complex may be formed which either inhibits copper transport to liver cells or interacts through metabolic antagonism to prevent synthesis of Cp and copper storage molecules. However, our results indicate that the 64Cu is available for ceruloplasmin synthesis_ These differences may be associated with the diets and perhaps species used in these studies. Our cattle at the time of i-v. 64Cu injection were on hay of low-normal copper status (4.7 ppm) and relatively low MO (1.8 ppm), while the sheep were on normal copper diets (12-14 ppm) supplemented with very high molybdenum (50 ppm). The major point of difference between our results and those of Marcilese et al. is the question of extent of Cp synthesis. Marcilese et al. determined 64Cu-labeled Cp levels indirectly by measuring the rate of catabolism of labeled serum fractions. The 64Cu-labeled plasma from the high-M0 sheep was cleared much more rapidly than the 64Cu-labeled plasma from the controls.

-4 hypothesis that would explain observations of Marcilese et al_ as well as those in this cattle study is that MO does not interfere with Cu transport to liver, but interferes with the synthesis of Cp leading to a molecule that is more rapidly catabolized than normal. One possibility is the resultant synthesis of apocerulo plasmin-like substances, which are catabolized more rapidly than normal holoceruloplasmin. Such abnormal compounds have been reported in the plasma of copper-deficient rats by Holtzman and Gaumnitz [ 15, 163 _ They showed that the apoceruloplasmin in copper-deficient rat plasma contained less than 10% of the copper of holoprotein. This apoprotein is catabolized more rapidly than Cp and is not stabilized in plasma by copper although it is stabilized by copper at the time of liver synthesis_ Similarly, it has been shown that asialo-Cp is rapidly removed by liver [ 17, 18]_ The cleavage of two of the twelve terminal asialic acid chains of Cp by neuraminidase has resulted in rapid liver removal of asialo-Cp in rabbits, even though the asialo-Cp had the same properties as Cp with regard to optical density, copper content, hexose content, and enzyme activity as measured by paraphenylenediamine oxidase. Thus it is possible that the higher Ievels of 64Cu in both the plasma and feces of the sheep and cattle


results from a greater percentage of 64Cu being utilized in the synthesis of altered Cp molecules, such as apo- or asial*Cp, which are more rapidly catabolized and subsequently excreted via bile to feces. Although this work does not support the formation of an insoluable copper compound in the animals studied, such a compound may be formed in the rumen at higher dietary sulfur and molybdenum Ievels. Both the synthesis of rapidIy catabolized forms of ceruloplasmin as well as rumenal formation of insoIubIe copper compounds, such as copper thiomolybdate, could be contributory mechanisms leading to copper loss in molybdenosis. Further work is necessary to evaluate this hypothesis. If correct, then fecal loss of absorbed copper is the major route of copper loss in molybdenum-induced copper deficiencies of cattle on marginal copper diets.

The authors gratefuily acknowledge the technical assistance of B. A. Prentice and S. R. Paape, the editorial assistance of .i_ Azevedo, and the clerka assistance of C. C. DiBartola. One of us (C. W. A.) was supported by a U. S. Public Health Service grant (TOI-GMOO.537-IS).

1.

2. _ 3.

4. 5 6.

87:

9. 10. ll- i2. 13. 14.

15. 16. 17.

REFERENCES

E, 3. Underwood, Trace Elements in Human and Animal Nutrition, Academic Press, New York (1975), Chapters 3 and 4. A_ L_ P0pe.J. Anim Sci 33,1332 (1971). J. R Todd, in Proceedings of the Symposium on Molybdenum in the Environment,

Denver (1975), in press. T. Huisingh, G. G. Gomez, and G. hiatrone, Fed. Proc. 3fl921 (1973). N_ F_ SuttIe,&oc_ Nun. Sot. 33,299 (Iq74)). E. J. Underwood, in Proceedings of the Symposium on Molybdenum in the

Environment, Denver (1975), in press. B. P. Smith, G. L. Fisher, P. W. Poulos, and hi. R Irwin,JA. Y.MA. 166,682 (1975). M. R. irwin, P. W. PouIos, B. P. Smith, and G. L. Fisher, J. Camp. Pathol. 84, 611

(1974). k K- Agarwal,fVew Scientist 30,260 (1975). H. Nishita and C. M. Jobnson,Anal. Chem 24,736 (1952). J_ K- Miller, B_ R Mos, M_ C. Be& and W. W. Sneed,J. Anim Sci 34,846 (1972). EL L. Chapman, Jr. and M. C. Be&J. Anim Sci 22,82 (1973). G. W. Evans, PhysioL Rev. 53,535 (1973)_ N. A. Marcilese, C. B. Ammerman. R hi. VaIeseccbI, B_ G. Danavant, and G. K. Davis, L Nutr. 99,177 (1969). N. A. Iioltzman and B. M_ Gaumnitz, J. BioL Chem 245,235O (1970). N- A. Holtzman and B. hi_ Gaumnitz, J. BioL Chem 245,2354 (1970). C. J. k Van den Hamer, A. G. MoreII, I. H. Scheinberg, J. Hickman, and G. Ashwell.J. -_ _ - SroL C?zem. 245,4397 (1970).

18. -A_ G- MoreIl, G. Gregoriadis, I. H. Scheinberg, J. Hickman, and G. AshweII, J. Biol. Chem. 246,146l (i971).

Received 27August 1975

effects of dietary molybdenum on the metabolism of copper and molybdenum in young cattle

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