Distribution of calcium, magnesium and inorganic phosphate in ...

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Jul 20, 1984 - Dryer RL, Tammes AR, Routh JI (1957) The determination of phosphorus and phosphatase withN-phenyl-p-phenylenediamine. J Biol ChemĀ ...
Journal of Comparative S y,ooicBi~ and Environ-

J Comp Physiol B (1985) 155:347-352

Physiology B ~Physiology '' 9 Springer-Verlag 1985

Distribution of calcium, magnesium and inorganic phosphate in plasma of estradiol-17fl treated rainbow trout Bj6rn T. Bj6rnsson and Carl Haux Department of Zoophysiology, University of G6teborg, Box 250 59, S-400 31 G6teborg, Sweden Accepted July 20, 1984

Summary. Freshwater rainbow trout, Salmo gairdneri, were injected with different doses of estradiol17fi in order to induce the synthesis of a protein, regarded as identical to vitellogenin. The plasma levels of free and protein-bound calcium, magnesium and inorganic phosphate were studied in control and estradiol-17fl treated fish, using an ultrafiltration method. Estradiol-17fl caused a dosedependent increase in plasma vitellogenin levels, which strongly correlated to protein-bound levels of calcium and magnesium in plasma. Calcium and magnesium were bound to vitellogenin in a ratio of 9:1, which was considerably higher than the protein-binding ratio of these ions in normal plasma (5.2:1). The dose-dependent increase in total plasma levels of calcium, magnesium and inorganic phosphate during estradiol-17fl treatment was solely due to an increase in the protein-bound fraction of these ions. It is concluded that the physiologically important plasma levels of free calcium, magnesium and inorganic phosphate are effectively regulated at normal levels during vitellogenin synthesis.

Introduction Calcium, magnesium and inorganic phosphate are present in blood plasma in three forms: free ions; undissociated complexes with bicarbonate, citrate, CaHPO 4 and MgHPO~; and protein-bound forms (Walser 1961; Ebel and Gfinther 1980). Since the pioneering work by McLean and Hastings (1935), the ionic form of plasma calcium has been considered to be the physiologically active component (Dacke 1979). A similar fundamental role has been assumed for the ionic form of magnesium, although the distribution of magnesium in plasma

has received little attention (Walser 1961; Ebel and Gfinther 1980; Speich et al. 1981). As the complexed form normally constitutes a small fraction, the measurement of the ionic and complexed forms collectively, as the ultrafiltrable or free fraction, can often be used when the distribution between the physiologically active and the protein-bound forms are studied. In teleosts, the control of the total plasma calcium has attracted extensive interest, but information on the distribution of calcium fractions is scarce (Pang 1973; Dacke 1979). The plasma levels of free and protein-bound calcium in freshwater teleosts are similar to those in mammals (Bailey 1957; Walser 1961 ; Chan and Chester Jones 1968; Fenwick 1974; Yaron et al. 1980; Copp and Ma 1981; Wendelaar Bonga 1981; Bj6rnsson and Hansson 1983). The importance of measuring the free fraction of plasma calcium, when studying calcium regulation in teleosts, is becoming increasingly evident, especially since changes in the free calcium fraction are not necessarily reflected in the level of total plasma calcium (Wendelaar Bonga 1981; Bj6rnsson and Hansson 1983). The distribution of inorganic phosphate in teleost plasma has been measured by Bailey (1957) and Urist (t962), but the distribution of plasma magnesium has not been reported for freshwater teleosts. Administration of estradiol-17fl to teleosts increases the total plasma level of calcium by inducing the synthesis in the liver of a female-specific lipophosphoprotein, vitellogenin, which binds calcium in considerable amounts when present in the blood (De Vlaming et al. 1980). Although total plasma calcium can increase greatly during vitellogenesis, it appears that the free plasma calcium levels remain essentially unchanged (Bailey 1957; Urist and Schjeide 1961 ; C h a n and Chester Jones 1968; Yaron et al. 1980). In addition, the plasma

348

B.T. Bj6rnsson and C. Haux : Effects of estradiol on ion distribution in rainbow trout

distribution of inorganic phosphate can be affected during vitellogenesis (Bailey 1957). The main objective of this study was to establish a comprehensive picture of the distribution of calcium, magnesium and inorganic phosphate in the plasma of normal and estradiol-a7fi treated rainbow trout. A further aim was to clarify the relationship between these ion species and protein in normal and vitellogenin-rich plasma.

Table 1. Data compilation of plasma calcium distribution in

freshwater teleosts and humans Species

Carassius auratus a Anguilla anguilla,

n

Ca T mM

Ca v mM

Ca~ mM

Ca c mM

> 17

2.52

1.60 (63)

9

2.46

1.73 (70) 1.38 (56) 0.35 (14)

5

2.61

1.84 (71) 1.38 (53) 0.45 (18)

10 9

2.48 3.76

1.61 (64) 1.36 (55) 0.25 (9) 1.23 (37)

10

2.82

1.52 (54)

24

2.80

1.45 (52)

30 15

2.53 2.23

1.23 (55)

10

2.48

1.36 (55) 1.18 (48) 0.16 (7)

yellow b Anguilla anguilla,

silver b

Materials and methods Fish. Juvenile rainbow trout, Salmo gairdneri, less than a year

old and with a body weight of 62 +__2 g, were obtained from a hatchery (Antens Laxodling AB) close to G6teborg. The fish were acclimated for one week in basins with filtered, aerated and recirculating freshwater at a temperature of 8-10 ~ The fish were not fed before or during the experiments, which took place in November-December. The photoperiod was 12 h light/ 12 h dark. Experimental design. Estradiol (estradiol-17fl; 1,3,5(10)-estratriene-3,17fl-diol; Sigma Chemical Co.) was finely dispersed in peanut oil (30, 300, and 3,000 gg-ml-1), using an ultrasonic bath. The experimental fish were injected intraperitoneally with 0.2 ml of the appropriate estradiol concentration, while controls received peanut oil only. Three experiments were performed, and at the start of each the fish were divided into four groups with four to six animals in each, which were injected with vehicle, 0.1, 1 and 10 mg estradiol-kg-1, respectively. The first experiment (I) was terminated after five days. In the second experiment (II), which was terminated after 10 days, one additional injection was made after five days to yield a total dose of 0, 0.2, 2 and 20 mg estradiol.kg-1, respectively. In the third experiment (III), sampling was performed after 15 days, and two additional injections were made after five and 10 days to yield a total of 0, 0.3, 3 and 30 mg estradiol.kg-1, respectively. Immediately after the first injection, each group of fish was transferred to a separate glass aquarium containing 50 1 of noncirculating, aerated freshwater at a temperature of 8-10 ~ The water was renewed every third day. Sampling and analytical methods. At the termination of each

experiment, the fish were stunned by a blow on the head, weighed, and about 1 ml of blood withdrawn from the caudal vessels using a heparinized syringe. Plasma was obtained by centrifugation, and 0.3 ml of each plasma sample was immediately ultrafiltered, employing the Amicon Micropartition System (MPS-I). This ultrafiltration method has been evaluated in detail for calcium and magnesium, and found to have good precision and accuracy (D'Costa and Cheng 1983). Sampling and centrifugation were performed at 15 ~ in all experiments, and plasma and ultrafiltrate samples were stored at - 2 0 ~ until analyzed. The liver of each fish was weighed and the liver somatic index (LSI) calculated as a percentage of the corresponding body weight (liver wt-body wt- t. 100). The concentrations of calcium and magnesium in plasma and ultrafiltrates were determined by atomic absorption spectroscopy (Perkin-Elmer 303). The concentrations of total protein and inorganic phosphate in plasma and ultrafiltrates were analyzed according to the methods described by Henry et al. (1957) and Dryer et al. (1957), respectively.

Anguilla rostrata ~ Mirogrex terraesanctae d Oncorhynchus nerca e Gasterosteus aculeatus f Salmogairdneri g Salmo gairdneri h Homo sapiens i

1.29 (5J)

The number of animals is denoted by n. Total, free, ionic and complexed plasma levels are denoted by T, F, I and C, respectively. Concentrations are expressed as means, with the percentage of the total plasma concentration given in parenthesis " b c a e f

Bailey (1957) Chan and Chester Jones (1968) Fenwick (1974) Yaron et al. (1980) Copp and Ma (1981) Wendelaar Bonga (1981) BjSrnsson and Hansson (1983) h All control fish in the present study i Walser (1961)

The protein-bound fraction (B) of calcium and magnesium in plasma was calculated as the difference between the total plasma concentration (T) and the concentration (F) found in the corresponding ultrafiltrate sample. Statistical and mathematical analysis. The dose-dependency of

the parameters studied was evaluated by testing equality against ordered alternatives (Lehman 1975). The testing was two-tailed, and the level of significance was set at P