Camp. ~iochem. Pbysioi. Vol. 8?A, No. 3, pp. 561-564, 1987 Printed in Great Britain
03~9629/87 $3.00+ 0.00 0 1987Pergamon Journals Ltd
THE POSTNATAL DEVELOPMENT COPPER AND CERULOPLASMIN Department
OF SERUM ZINC, IN THE HORSE
J. 7-J. BELL, J. M. LOPEZ and K. D. BARTON of Physiological Sciences, University of Florida, Gainesville, Florida 32610-0137, USA. Telephone: (904) 392-l 841 (Received 9 September 1986)
Abstract-l. Serum samples were collected from ten foals at predete~n~ times during the first 12 months following birth and zinc and copper concentrations and ceruloplasmin activity were evaluated. 2. Serum zinc concentrations were found to be quite variable with respect to age (range = 67-95 &dl). 3. Serum copper concentrations increased in a linear fashion from day 0 to day 28 before levelling off at 190-247 pg/dl. 4. Ceruloplasmin activity was found to correlate with the concentration of serum copper (r = 0.92) and reached a plateau at an activity of 30-38 IU by day 28.
activity was expressed in international units (IU) as defined by Rice (1962). To evaluate age-related changes in copper, zinc and ceruloplasmin, all data, expressed as the mean + SE, were subjected to analysis using Duncan’s multiple range test. The 0.05 level of probability was used as the criterion of significance.
INTRODUffION Although copper and zinc are essential trace elements in mammals, there appear to he special requirements associated with pregnancy. Even relatively short periods of zinc deficiency during pregnancy have been associated with central nervous system malformations in the developing rat (Warkany and Petering, 1972) and depressed immune function in the immature mouse (Beach et al., 1982). In many species, it has been found that both the concentration of serum copper and ceruloplasmin activity are low in the neonate and increase to adult levels over variable time periods (Keen et al., 1981; Amer et al., 1973; Gomez-Garcia and Matrone, 1967; Schenker et al., 1972; Terao and Owen, 1977). The current study was undertaken to follow the changes in serum concentrations of zinc and copper as well as the activity of ceruloplasmin during the first year of life in the foal, an animal which is apparently very susceptible to both excesses and deficiencies of both zinc and copper.
RESULTS The effect of postnatal development on the concentration of serum zinc is shown in Fig. 1. Although
MATERIALS AND METHODS Ten newborn foals from the University of Florida Horse Research Unit were used in this study. Blood samples were collected from the animals on the day of birth (day 0) and at 1, 3, 5, 7, 14 and 28 days and 2, 4, 6, 9 and 12 months after birth. Following centrifugation of the blood, serum was removed and stored at -20°C in polypropylene tubes pending analysis. For the measurement of copper and zinc, serum was deproteinized with 5% trichloroaoztic acid followed by ~nt~fugation. The deproteinized samples were then analyzed using an atomic absorption spectrophotometer (Perkin-Elmer model 2380). Zinc was determined using the flame mode, whereas copper was determined using an HGA-400 electrothermal graphite furnace with deuterium arc background correction, Commercially available standard metal solutions were used. Ce~oplasmin activity was measured by the method of Houchin (1958) using pphenylenediamine dihydrochloride as the substrate. The
there was a tendency for zinc concentrations to fall during the first 24 hr after birth, a significant although transient increase was seen at days 3 and 5. From day 7 to month 12, serum zinc concentrations remained relatively constant at approximately 75 ,ug/dl. The developmental pattern for serum copper concentrations was quite different (Fig. 2). Although the copper concentration was only 36 pgg/dl at day 0, it increased steadily during the first month of extrauterine life, reaching a value of 233 pg/dl by day 28. Although some fluctuations were seen during the next 11 months, concentrations remained between 190 and 247 /.ig/d]. The developmental pattern observed for serum ceruloplasmin activity (Fig. 3) paralleled that of serum copper. Activity increased IO-fold during the first 28 days following birth, from a low value of 3.3 IU at day 0 to 34.1 IU at day 28. As was the case with the serum copper concentration, ceruloplasmin activity remained relatively constant for the next 11 months, ranging from 29.7 to 37.5 IU. When the apparent relationship between the concentration of serum copper and ceruloplasmin activity was examined by linear regression (Fig. 4), a strong correlation was found to exist. When all the data points were included, a correlation coefficient (r) of 0.92 was found. When the data were divided into two subgroups by age, days O-28 and months 2-12, correlation coefficients (r) of 0.93 and 0.65, respectively, were generated.
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Fig. 4. Serum copper concentration and ceruloplasmin activity in 10 foals at 12 time periods (days 0, 1, 3, 5, 7, 14, 28, months 2, 4, 9, 12) during the first year of extrauterine life. Solid circles represent animals aged between 0 and 28 days, whereas the asterisks represent animals aged between 2 and 12 months. The line was fitted to all the points with a correlation coefficient (r) of 0.92. DISCUSSION
The zinc concentrations measured in these foals compared closely with those determined in species such as ferrets (Straube et al., 1980), dogs (Keen et al., 1981; Fisher, 1977) and man (Henkin et al., 1971; Shaw, 1979). However, it has been found in man and dogs that serum zinc concentrations increase with age to a certain maximum and then decrease before stabilizing (Keen et al., 1981; Shaw, 1979). In the present study, it was not really surprising that we failed to observe a similar trend, because the study was only conducted over the first 12 months of extrauterine life, while in the dog, the maximum zinc concentration was not reached until 7.5 years (Keen et al., 1981). In humans, the maximum concentration was not reached until between 7 and 20 years of age (Shaw, 1979). In addition, the increase in zinc concentration observed with aging was gradual and not of very large magnitude in either species. The copper concentrations measured in these foals were higher than those measured previously in horses (Gunson et al., 1982) rats (Terao and Owen, 1977), humans (Henkin et al., 1971) and dogs (Keen et al., 1981; Fisher, 1977) lower than those measured in calves (Amer et al., 1973) and comparable to those measured in swine (Gomez-Garcia and Matrone, 1967). Copper and ceruloplasmin levels have been shown to increase with age in a variety of species. Copper concentrations reach adult levels at about 1 month of age in the rat (Terao and Owen, 1977) swine and cattle (Amer et al., 1973; Gomez-Garcia and Matrone, 1967) human infants (Hillman, 1981) and as shown in the present study, in the foal. In all of these studies, ceruloplasmin activity increased in parallel with the concentration of copper. Neither parameter increased in neonatal pigs fed copperdeficient diets (Gomez-Garcia and Matrone, 1967) or in premature humans with an increased copper intake (Hillman et al., 1981) indicating that ceruloplasmin
synthesis in the young animal is a maturationdependent process which may be relatively insensitive to dietary influences. Many factors have been documented to affect copper and zinc concentrations. Zinc concentrations fall in humans and dogs in a number of disease states
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(Rosner and Gorflen, 1968; Fisher, 1977) and increase with increasing ambient temperature in the dog (Keen et al., 1981). Copper concentrations rise in some disease states in the dog (Fisher, 1977), and fall with zinc toxicosis in rats (L’Abbe and Fischer, 1984) and horses (Gunson et al., 1982). Recent evidence suggests there may be a link between low serum copper (and possibly high serum zinc) and osteochondrosis in suckling foals (Bridges et al., 1984). A complete study of all the factors affecting the concentrations of these two essential elements has not been undertaken in the horse or any other mammalian species. In conclusion, the results of the present study indicate that during the first year of extrauterine life, concentrations of zinc in horse serum are variable and apparently unrelated to the age of the animal; whereas, serum copper and its major transport protein, ceruloplasmin, increase in parallel from birth through day 28. The factors responsible for regulation of copper and ceruloplasmin in the neonatal horse remain to be resolved. Acknowledgements-The authors would like to acknowledge the assistance of Drs John Harvey, Richard Asquith and the University of Florida Horse Research Unit for making serum samples available for this study. REFERENCES
Amer M. A., St. Laurent G. T. and Brisson G. J. (1973) Supplemental copper and selenium for calves: effects upon ceruloplasmin activity and liver copper concentration. Can. J. Physiol. Pharmac. 51, 649-653. Beach R. S., Gershwin M. E. and Hurley L. S. (1982) Gestational zinc deprivation in mice: persistence of immunodeficiency for three generations. Science 218, 469-47 1. Bridges C. H., Womack J. E., Harris E. D. and Scrutchheld W. L. (1984) Considerations of copper metabolism in osteochondrosis of suckling foals. J. Am. Vet. Med. Ass. 185, 173-178. Fisher G. L. (1977) Effects of disease on copper and zinc values in the beagle Am. J. Vet. Res. 38, 935-940. Gomez-Garcia C. G. and Matrone G. (1967) Copper metabolism in the early postnatal period of the piglet. J. Nutr. 92, 237-244. Gunson D. E., Kowalczyk D. F., Shoop C. R. and Ramberg C. F. (1982) Environmental zinc and cadmium pollution associated with generalized osteochondrosis, osteoporosis and nephrocalcinosis in horses. J. Am. Vet. Med. Ass. 150, 295-299. Henkin R. I., Marshall J. R. and Meret S. (1971) Maternal-fetal metabolism of copper and zinc at term. Am. J. Obstet. Gynecol. 110, 131-134. Hillman L. S. (1981) Serial serum copper concentration in premature and SGA infants during the first three months of life. J. Pediat. 98, 305-308. Hillman L. S., Martin L. and Fiori B. (1981) Effect of oral copper supplement on serum copper and ceruloplasmin concentration in premature infants. J. Pediat. 98, 311-313. Houchin 0. B. (1958) A rapid calorimetric method for the quantitative determination of copper oxidase activity (ceruloplasmin). Clin. Chem. 4, 519-523. Keen C. L., Lonnerdal B. and Fisher G. L. (1981) Seasonal variations and the effects of age on serum copper and zinc values in the dog. Am. J. Vet. Res. 42, 347-350. L’Abbe M. R. and Fischer P. W. F. (1984) The effects of dietary zinc on the activity of copper-requiring metalloenzymes in the rat. J. Nutr. 114, 823-828.
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Shaw J. C. L. (1979) Trace. elements in the fetus and young infant. J. Dis. Child. 133, 126&1268. Straube E. F., Schuster N. H. and Sinclair A. J. (1980) Zinc toxicity in the ferret. J. Come. Path. 90. 355-361. Terao T: and Owen C. A. (1677) Copper metabolism in pregnant and postpartum rat and pups. Am. J. Physiol. 232, E172-E179. Warkany J. and Petering H. G. (1972) Congenital malformations of the central nervous system in rats produced by maternal zinc deficiency. Teratology 5, 319-334.