Life Sciences, Vol . 17, pp . 1211-1218 Printed in U.S .A .
Pergamon Press
THE EFFECT OF MAGNESIUM DEFICIENCY ON SERUM ALDOSTERONE IN RATS FED TWO LEVELS OF SODIUM Bernadette Merlino Soloanise and Ruth Schwanz Division of Nutritional Sciences Cornell University Ithaca, New York
14853
(Received in final forut September 8,
1975)
Summar~ Rata were fed 47 (deficient) and 606 ply (adequate) magnesium Serum corticosterone and with either 2,100 or 14,000 pPm sodium . aldosterone levels were determined by radioimmunoassay in six rats from each treatment group killed on days 7, 14, and 28 of conSerum corticosterone levels sumption of the experimental diets . were moderately, but not significantly, decreased in magnesium deficient animals . Serum eldosterone levels increased over time in the rata fed the lower sodium diet with adequate magnesium and In sodium were flu~ther elevated in magnesium deficient animals . loaded rats the increase in aldosterone levels in magnesium deficiency was leas and occurred later . Retention and urinary excretion oP sodium and potassium did not appear to be affected by magnesium status or the serum concentration of aldosterone . Possible mechanisms underlying the changes in aldosterone levels of magnesium depleted animals are discussed with reference to the known effects of magnesium deficiency on physiological functions . Several investigators have reported alterations in plasma levels or secretion rate of adrenocortical hormones in magnesium deficient animals (1-4) . In general, corticosterone secretion end plasma levels were found somewhat below control values (1-3) although a transient elevation was observed in one investigation in rats after 20 days of magnesium depletion (3) . Aldosterone secretion was reported elevated in rats on day 55 of magnesium depletion by Ginn et al (1) . Halber et al (4) also observed an increase in adrenal secretion of aldosterone in dogs magnesium depleted for 35 days, but found no deviation from normal by day 50 of deficiency . Circulating levels of aldoaterone have not been measured in magnesium deficient animals to date . That aldosterone output may be chronically elevated in magnesium deficient rats was suggested by Canon (5) who observed an increase in width of the zone glomerulosa. Concomitant hyperplasia and
1The data presented here are taken from a thesis submitted in partial fulfillment of the requirements for the Masters degree at Cornell University . 1211
1212
Serum Aldoaterone in Magnesium Deficient Rata
Vol. 17, No . 8
hypergranulation of the juxteglomerular apparatus led him to postulate that the primary stimulus to excess aldoaterone output in magnesium deficiency was reduction in colloid osmotic pressure due to reduced plasma protein levels and consequent stimulation of the renin-angiotensin system . Ia the present investigation plasma eldosterone levels were measured by competitive protein binding assay in rats at intervals through four weeks of magnesium depletion. Since, under normal circumstances, a major factor in fluencing eldosterone secretion is sodium intake end balance, magnesium deficiency was induced at two levels of dietary sodium : a relatively law adequate level sad under conditions of sodium loading. Materials and Methode Seventy-two male Sprague-Dawley rats, initially weighing about 1~+0 g were divided into four groups, and each group of eighteen animals was assigned to one of the four dietary regimens . Within sash of the dietary treatment groups, the animals were divided into three groups, each assigned to one of the three different experimental time periods, 7, 14, or 28 days, giving e total of twelve treatment combination groups, with six animals per group. Groups were arranged to contain four animals, one from each of the four diet groups at one time period. Within each group the rat with the lowest spontaneous food consumption established the amount of Pood allowed for each of the remaining three rats . The basal diet contained 15~ casein and was adequate in all nutrients (6) other than magnesium and sodium which were subsequently added to the basal diet after analysis . The experimental diets contained low or adequate (control) magnesium (47 and 606 ppm by analysis) with either 2,100 or 1+,000 p~ sodium . The animals were housed in stainless steel cages and allowed deionized water ad libitum. At the end of the experimental periods the rata were decapitated. Blood was collected from the neck, allowed to clot and centrifLiged for separation of serum. Serum samples were frozen immediately and stored at -20oC . Adrenal glands were removed, weighed and sectioned for histological examination and stained with hematoxylin and eosin . Four days prior to killing, two eaimala from each group were placed in metabolic cages for collection of feces and urine. Serum aldosterone was measured by a microprocedure based on the radioimmunoasagy developed by Ito (7) . Serum corticosterone was assayed by competitive binding to transcortin (8). Serum, urinary and fecal electro]ytes were determined by atomic absorption spectrophotametry and serum proteins by a modification of the Lowry procedure (9). The data were analyzed by two-way analysis of variance followed by Tukey's Omega (hsd) test (38) for significance of differences between means . Results Clinical signs of magnesium deficiency, initie.l],y apparent as erythema and hyperemia, were seen between days 6 and 16 of dietary treatment. After 1~F dgys of deficiency the animals developed hyperirritability, dull, thinning fur, and dermatitis around the eyes . By day 28, body weights were significantly lower in magnesium deficient animals than in pair-fed controls (Table 1) . 7~e dietary sodium level did not appear to influence the appearance or severity of deficiency signs . However, sodium loading consistently depressed weight gains of both magnesium deficient and adequately fed rats . Serum magnesium levels were significantly depressed by day 7 of magnesium depletion regardless of the dietary sodium level (Table 1) . Serum proteins increased over time in the animals fed adequate magnesium but not in magnesium deficient rats . $Y day 28 the mean difference in serum protein level between magnesium deficient and control Mg fed rats was 0.8 - 1 g~100 m1, the difference
Serum Aldosterone in Magnesium Deficient Rata
Vol. 17, No . 8
1213
being statistically significant only in the animals fed the high sodium diets (Table 1) . TABLE 1 Wei
ta
Serum
esium end Proteins in Relation to M an d Sodium Intake Meaa + sem)
esium De letion
Dietar~r treatment Days on diet
Control Mg Control Na
Low Mg Control Na
Control Mg High Na
Low Mg High Na
Weight, g.
7
1631?.6
16512 .3
164±1 .8
161±2.2
14
19813 .5
19114 .1
196±3 .3
18414.5
28
26813.8 a
242t4 .6b
263±5 .4g
23115 .4b
Serum M~
.9b
19 .9±1 .o g
9 .81.1b
7.3~.3b
20 .51o .5 a
7 .3~.5b
8.41o .6b
21 .1±1 .o g
7.51o .5b
7
21 .3to .6 a
9.o±o
l4
u .11o .6 e
28
20 .31o .6 a
Serum proteins
7
5.7
.1
(ugh
(g/100
ml)
5 .4fo .1
5.8fo .3
5.9±0 .4
14
6.41o .1 ab
6.110 .2a
6.8±o .2b
6.3fo .3~
28
7 .11o .5 a
6.3to .4 ab
7.1±o .3 a
6 .o±o .2 b
~cMeans without a common letter in their superscript are significantly different (p < 0.05) . n
= 6
with the exception of "*" where n =
5.
Adrenal weights and serum aldosterone levels are s»~pT ized in Table 2 . There were no significant differences in adrenal weights . At both levels of dietary magnesium animals fed the control sodium diet showed a progressive increase in mean serum aldosterone levels from days 7-28 . Evidently the control sodium level, although adequate according to official recommendations (10) was low enough to elicit an increased aldosterone response in growing rats . Mean serum aldosterone levels were consistently higher in magnesium deficient rats fed the control sodium diet ca~mpared to the rats fed adequate magnesium. The difference was statistically significant (P< 0.05) by day 28 of dietary treatment . A smaller, not statistically significant difference in aldosterone levels of magnesium depleted and control rats was seen only by day 28 in the animals fed the high sodium diet . Serum corticosterone levels, measured at different treatment times, were combined (Table 2) since available serum samples were limited. Levels were moderately but not significantly reduced in the magnesium deficient groups .
1214
Swum Aldoatarone in Magnesium Deficient Rata
Vol. 17, No . 8
TABLE 2 Adrenal Wei is Serum Aldosterone and Serum Corticosterone in Relation to Magnesium De letion and Sodium Intake Mean 1 aem Dietary treatment Days on diet
Control Mg Con trol Na
Low Mg Control Na
Control Mg High Na
Low Mg High Na
Adrenal weight (mg) 7
28 .710 .8
29 .8_1 .5
28 .9+2 .1
32 .712 .5
14
32 .310 .8
28 .2±1 .4
30 .5 3.0
34 .714 .7
28
37 .3*~ .7
40 .411 .6
36 .211. .6
37 .01-0.6
Serum aldoeterone (pg/ml) 59 " 7134 .6
179 .81102 .3
85 .3133 .6
101.0141 .0
14
175 " 7154 .2
249 .5166 .1
1021 11+9 .5
95 .4147.9
28
266.0~+9 .4 ab
530 .0118 .5 °
128 .0136 .8a
215.215o .9 ab
7
Serum corticoaterone 7-28 n*
(ng/ml)
279.5144.5
209 .7134 .0
318 .7151 .E
167.5134 .3
13
12
1
12
~chleana without a common letter in their superscript are significantly different (p < 0 .05) . *applies to serum corticoaterone only . each mean value .
For all other measurements n = 6 for
Neither sodium nor potassium retention, nor urinary excretion showed significant variations attributable to the level of dietary magnesium (Table 3) . Mean potassium retention appeared increased in magnesium deficient rata . However, large ataadard deviations and the observed difficulties in accurate collection of excreta indicate caution in the interpretation of this finding. Preliminary histological examination of the adrenal cortex suggested signs of increased activity in the zona glamerulosa of magnesium deficient rats fed the lower level of dietary sodium . In these sections the cells contained large, somewhat hypochromatic nuclei . The cytoplasm appeared enlarged and showed ea increase in negatively stained areas indicating the prior presence of lipid which had been removed by alcohol treatment of the sections . No differences between magnesium deficient and control groups were seen in adrenal sections taken from sodium loaded rats .
Vol . 17, No . 8
Serum Aldosterone in Magnesium Deficient Rats
1215
TART F 3
Uria bccretion and Retention of Sodium and Potassium in Relation to M esium Depletion and Sodium Intake Mean t eem. Values Obtained for Two Rats at 7, 1 , and 28 Days of Treatment were Pooled Dietary treatment Control Mg Control Na
Low Mg Control Na
Control Mg High Na
Urinary Na
18 .511 .3
17 .4 1.4
20917.5
166114 .4
Na retention ~/3 a$ys
46 .214 .0
52 " 913.9
52 .4115 .3
145 .3163.0
Urinary K mg/aey
55 .814 .4
46 .2±4 .7
57 .515 .5
45 .814 .3
K retention mg/3 days
70 .8113 .8
109 .7±17.1
63 .6116 .0
88 .4±23 .3
Parameter
~/~
Low Mg High Na
n=6 Discussion The re.dioimmnoassay for aldosterane used in this investigation involves two steps prior to protein binding : extraction into dichloromethane and subsequent separation of aldosterone from other steroids by the use of Sephadex LH-20. The original procedure required 600 a 10 mm Sephadex columns and 1-2 ml of human plasma, containing 26-737 pg aldosterone/ml (7) . The use of microcolumns used is the present study (4 x 65 mm) permitted estimation of aldosterone in 0.5 ml samples of rat serum with specificity end precision comparable to that reported previously using large columns . In our hands recovery of aldosterone after extraction sad Sephedex flltratio~n was 57 .9,112 .6, compared to 56 .5_14 and 44 .7,$17 .1 reported by Ito et e1 (7) and Waldhausl et e1 (11) respectively . Undoubtedly, the high standard deviation inherent in the assa ry contributed eubstaatislly to the large standard error of group averages . However, the expected inverse relationship between blood aldosterone and dietary sodium levels which showed clearly in these data (Table 2) suggest that the additional elevations in serum eldosterone seen in low sodiummagnesium depleted rats are a valid finding. The mechanisms by which magnesium depletion elicits excessive aldosterone output are not known. Possible mechanisms are' abnormal stimmilation of the zone glomerulosa, alterations is sensitivity of the adrenal cortex to sldosterane inducing stimuli, increased potassium balance and endorgan insensitivity to aldoeterone . The last two of these alternatives seem the least likely . While above normal sodium end water retention are consistent findings is magnesium deficient eaimAla (3,5,12-14), no observations of reduced rates of sodium reabsorption have yet been reported . Potassium balances have not previously been reported in magnesium deficient animals . Urinary excretion has been reported unchanged (15), slightly decreased (16) end significantly increased (5) . The apparent increase in potassium balance found is the present study is obscured by considerable variability, probably due to difficulties encountered in the quantitative estimation of intake and excretion . Potassium levels is skeletal muscle, heart, kidney and liver have been con-
1216
Serum Aldoaterone in Magnesium Deficient Rata
Vol . 17, No . 8
sistently found reduced in magnesium deficient animals (12-14,16,17) . This finding would argue against an increase in potassium balance . The possibility exists, however, that increased potassium balance may occur transiently in magnesium deficiency, and may be a periodic factor in the stimulation of aldosterone output . Two consistently reported consequences of magnesium depletion in rats are massive release of histamine (6,18-20) and nervous hyperreactivity (21-25) . Histamine can influence aldosterone secretion by lowering blood pressure (26,27) However, under experimental conor by stimulating release of ACTH (28,29) . ditions identical to those used in the present study (6), blood histamine levels reached a peek by day 15 of magnesium depletion. By day 30, blood histamine levels approached pre-treatment levels, whereas in the present study serum aldosterone levels on day 28 were more than twice those found an day 14 . Furthermore, increased ACTH stimulation normally causes elevations in corticosterone output . The reverse, a slight decrease in serum corticosterone levels, was seen in the present investigation . Suppression of the magnesium deficiency-induced elevation in serum aldosterone by sodium loe.ding suggests that primary alterations in magnesium deficiency may involve sites specifically concerned with osmoregulation . Cantin and Huet (30) observed extensive hyperplasia and hypertrophy of epithelial juxtaglomerular cells, without concomitant changes in the macula densa. Canon had suggested previously (5) that the major stimulus to juxtaglomerular hyperactivity in magnesium deficient rata was lowered blood and colloid osmotic pressure due to reduction in plasma proteins . An alternative suggestion is excessive neural atimu_lation of the juxtaglomerular apparatus by way of the ß-adrenergic fibers that are thought to be involved in the regulation of renin secretion (31,32) . The exact character of the hyperactivity and neuromuscular hyperexcitability in magnesium deficiency has not been systematically studied. Studies concerned with the influence of divalent canons on neural fluiction and transmission of nerve impulses have shown magnesium to inhibit release of acetylcholine (33-35) " A similar effect is suggested for rate of release of other neurotransmittor substances . The observation of Brecht et a1 (36) showing inhibition of the spontaneous release of epinephrine and norepinephrine Pram the adrenal medulla by increasing magnesium concentration is of interest in this context . If one of the generalized effects of magnesium deficiency is enhanced release of neurotransmittors at synapses and neuromuscular junctions, increased output of aldosterone may be mediated by neural hyperactivity either via the renin aldosterone system or, possibly, via neurosecretory mechanisms for stimulation of aldosterone secretion (37) . ACKNOWLEDGMINTS The authors are most grateful to Dr . William Hansel for his interest and suggestions during the couxse of this work . Thanks sre also due to Miss Mari Jensen for technical help . This work was supported (in part) by Hatch funds through the Agricultural Experiment Station, Cornea University . RRFRRF.'N
1. 2. 3.
H. E. Ginas, R. Code, T . M. McCollum, sad M . FY~egley, Endocr . 80 969-971 (1967) " C. L. Richer, R. Veineux, and P. Bois, Endocr . _82 954-958 (1968) . R. J. Elfin, W. D. Armstrong, and L . Singer, Proc . Soc. ~. Biol . Med. 542-547 (1970 "
Vol. 17, No . 8 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16, 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 . 27 . 28 . 29 . 30 . 31 . 32 . 33 . 34 . 35 . 36 . 37 . 38 .
Serum Aldosterone in Magnesium Deficient Rats
1217
A. Helber, K. Heyduk, W. Nowaczynski, H. M. Brecht, 0 . Kuchel, and J. Genest, Rea . ~. Med. ~ 336-346 (1972) . M. Canon, Lab . Invest . 22 558-568 (1970) . J. L. Greger and R. Schwartz, J. Nutr . 104 1610-1617 (1974) . T. Ito, J. Woo, R. Herring, and R . Horton,J. _ Clin . Eadocr . 34 106-112 (1972) . B . E. P. Murphy, J. Clin . Eadocr . 27 973-990 (1967) . 0 . H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randan, _ J. Biol . _Chem. ~ 265-275 (1951) . Nutrient Requirements of Laboratory Animals, 2nd revised ed . N.A .S . Washington, D. C . (1972) . W. Waldhausl, H. Haydl, and H. Friachauf, Steroids 20 727-736 (1972) . I . Maclntyre and D . Davidson, Biochem. _J . 70 ~ - 2 (1958) . L. Martindale and F. W . Heaton, Biochem. J. ~2 119-126 (1964) . F . Gunther, Ztschr . Klin . Chem . _Klirr . Biothem. _8 65-68 (1970) . W. 0 . Smith, D . J. Baxter, A. Lindner, and H. E Ginn, J. Lab . Clin . Med. 211-218 (1962) . _ A. Manitius and F. H. Epetein, J. Clin . Invest . 42 208-215 (1963) " R. Whang and L . G. Welt, _J . Clin. Inveat~2305-313 (1963) . L. F. Belanger, G. A. Van Erkel, and A. JakeroW, Science 126 29-30 (1957) . P. Bois, Br . _J . ~. Path . 44 151-155 (1963) . aiol . _44 373-377 (1966) " P. Bois and A. Beaulnes, Can. _J. E. V. Tufts and E . M. Greenberg, _J . Biol . Chew . _127 693-714 (1938) . . _J . 31 1379 -1390 (1937) " E. Watthorn and R . A. McCance, Biochem B. S . W. Smith and A. C. Field, Br . _J . Nutr.17 591-600 (1963) . J. G. Chutktnr and S. Meyers, Neurology _1963-967 (1968) . siol . 223 1407-1414 (1972) . J. G. Chutkaw and J. D. Graber, Am . J. 8 A. J. Tobia, M. D . Adams, T. S . Maya, and W. F. Bousquet, Life - Sci -. _ part 1 745-750 (1969) " A. J. Tobia, M. D . Adams, T. S . Mirya, and W. F . Bousquet, _J . Pharmac . Eux . Then . 175 619-626 (1970) . M. L. Gordon, Eadocr . ~ 13-18 (1950) . W. D. Gray and P. L. Munaon, Eadocr . 48 471-481 (1951) . M . Canon and M. Huet, Can. J. siol. Pharmacol. 51 835-844 (1973) . W. F. Ganang, Control ôf Renin Secretion , p. 17 Plenum Press, New YorkLondon (1972) . W. F, Ganong, Life Sci. ~ 1401-1414 (1974) . D . H. Jenkinson,J.- sio1 . ~ 434-444 (1957) . J. T. Hubband, S. M. Jones, and E. M. Landau, J. siol . 1~6 75-86 (1968) . R. Y. Mullen and A. Finkelstein, Ptroc . Nat . Aced . Sc . 71 923-926 (1974) . H. M. teecht, A. Helber, D. Ganten, K. Hayduk, W. Nowaczynski, and J. Genest, Proc . _Sot . ~. Biol . Med. 1~ 39-43 (1973) " A. N. Taylor and G. L. Farren, Eadocr . 70 556-566 (1962) . G. W . Snedecor and W . G. Cochran, Statistical Methods , Io~e: State Univ . Press, Ames, IoKa (1967) .
Pergamon Press
THE EFFECT OF MAGNESIUM DEFICIENCY ON SERUM ALDOSTERONE IN RATS FED TWO LEVELS OF SODIUM Bernadette Merlino Soloanise and Ruth Schwanz Division of Nutritional Sciences Cornell University Ithaca, New York
14853
(Received in final forut September 8,
1975)
Summar~ Rata were fed 47 (deficient) and 606 ply (adequate) magnesium Serum corticosterone and with either 2,100 or 14,000 pPm sodium . aldosterone levels were determined by radioimmunoassay in six rats from each treatment group killed on days 7, 14, and 28 of conSerum corticosterone levels sumption of the experimental diets . were moderately, but not significantly, decreased in magnesium deficient animals . Serum eldosterone levels increased over time in the rata fed the lower sodium diet with adequate magnesium and In sodium were flu~ther elevated in magnesium deficient animals . loaded rats the increase in aldosterone levels in magnesium deficiency was leas and occurred later . Retention and urinary excretion oP sodium and potassium did not appear to be affected by magnesium status or the serum concentration of aldosterone . Possible mechanisms underlying the changes in aldosterone levels of magnesium depleted animals are discussed with reference to the known effects of magnesium deficiency on physiological functions . Several investigators have reported alterations in plasma levels or secretion rate of adrenocortical hormones in magnesium deficient animals (1-4) . In general, corticosterone secretion end plasma levels were found somewhat below control values (1-3) although a transient elevation was observed in one investigation in rats after 20 days of magnesium depletion (3) . Aldosterone secretion was reported elevated in rats on day 55 of magnesium depletion by Ginn et al (1) . Halber et al (4) also observed an increase in adrenal secretion of aldosterone in dogs magnesium depleted for 35 days, but found no deviation from normal by day 50 of deficiency . Circulating levels of aldoaterone have not been measured in magnesium deficient animals to date . That aldosterone output may be chronically elevated in magnesium deficient rats was suggested by Canon (5) who observed an increase in width of the zone glomerulosa. Concomitant hyperplasia and
1The data presented here are taken from a thesis submitted in partial fulfillment of the requirements for the Masters degree at Cornell University . 1211
1212
Serum Aldoaterone in Magnesium Deficient Rata
Vol. 17, No . 8
hypergranulation of the juxteglomerular apparatus led him to postulate that the primary stimulus to excess aldoaterone output in magnesium deficiency was reduction in colloid osmotic pressure due to reduced plasma protein levels and consequent stimulation of the renin-angiotensin system . Ia the present investigation plasma eldosterone levels were measured by competitive protein binding assay in rats at intervals through four weeks of magnesium depletion. Since, under normal circumstances, a major factor in fluencing eldosterone secretion is sodium intake end balance, magnesium deficiency was induced at two levels of dietary sodium : a relatively law adequate level sad under conditions of sodium loading. Materials and Methode Seventy-two male Sprague-Dawley rats, initially weighing about 1~+0 g were divided into four groups, and each group of eighteen animals was assigned to one of the four dietary regimens . Within sash of the dietary treatment groups, the animals were divided into three groups, each assigned to one of the three different experimental time periods, 7, 14, or 28 days, giving e total of twelve treatment combination groups, with six animals per group. Groups were arranged to contain four animals, one from each of the four diet groups at one time period. Within each group the rat with the lowest spontaneous food consumption established the amount of Pood allowed for each of the remaining three rats . The basal diet contained 15~ casein and was adequate in all nutrients (6) other than magnesium and sodium which were subsequently added to the basal diet after analysis . The experimental diets contained low or adequate (control) magnesium (47 and 606 ppm by analysis) with either 2,100 or 1+,000 p~ sodium . The animals were housed in stainless steel cages and allowed deionized water ad libitum. At the end of the experimental periods the rata were decapitated. Blood was collected from the neck, allowed to clot and centrifLiged for separation of serum. Serum samples were frozen immediately and stored at -20oC . Adrenal glands were removed, weighed and sectioned for histological examination and stained with hematoxylin and eosin . Four days prior to killing, two eaimala from each group were placed in metabolic cages for collection of feces and urine. Serum aldosterone was measured by a microprocedure based on the radioimmunoasagy developed by Ito (7) . Serum corticosterone was assayed by competitive binding to transcortin (8). Serum, urinary and fecal electro]ytes were determined by atomic absorption spectrophotametry and serum proteins by a modification of the Lowry procedure (9). The data were analyzed by two-way analysis of variance followed by Tukey's Omega (hsd) test (38) for significance of differences between means . Results Clinical signs of magnesium deficiency, initie.l],y apparent as erythema and hyperemia, were seen between days 6 and 16 of dietary treatment. After 1~F dgys of deficiency the animals developed hyperirritability, dull, thinning fur, and dermatitis around the eyes . By day 28, body weights were significantly lower in magnesium deficient animals than in pair-fed controls (Table 1) . 7~e dietary sodium level did not appear to influence the appearance or severity of deficiency signs . However, sodium loading consistently depressed weight gains of both magnesium deficient and adequately fed rats . Serum magnesium levels were significantly depressed by day 7 of magnesium depletion regardless of the dietary sodium level (Table 1) . Serum proteins increased over time in the animals fed adequate magnesium but not in magnesium deficient rats . $Y day 28 the mean difference in serum protein level between magnesium deficient and control Mg fed rats was 0.8 - 1 g~100 m1, the difference
Serum Aldosterone in Magnesium Deficient Rata
Vol. 17, No . 8
1213
being statistically significant only in the animals fed the high sodium diets (Table 1) . TABLE 1 Wei
ta
Serum
esium end Proteins in Relation to M an d Sodium Intake Meaa + sem)
esium De letion
Dietar~r treatment Days on diet
Control Mg Control Na
Low Mg Control Na
Control Mg High Na
Low Mg High Na
Weight, g.
7
1631?.6
16512 .3
164±1 .8
161±2.2
14
19813 .5
19114 .1
196±3 .3
18414.5
28
26813.8 a
242t4 .6b
263±5 .4g
23115 .4b
Serum M~
.9b
19 .9±1 .o g
9 .81.1b
7.3~.3b
20 .51o .5 a
7 .3~.5b
8.41o .6b
21 .1±1 .o g
7.51o .5b
7
21 .3to .6 a
9.o±o
l4
u .11o .6 e
28
20 .31o .6 a
Serum proteins
7
5.7
.1
(ugh
(g/100
ml)
5 .4fo .1
5.8fo .3
5.9±0 .4
14
6.41o .1 ab
6.110 .2a
6.8±o .2b
6.3fo .3~
28
7 .11o .5 a
6.3to .4 ab
7.1±o .3 a
6 .o±o .2 b
~cMeans without a common letter in their superscript are significantly different (p < 0.05) . n
= 6
with the exception of "*" where n =
5.
Adrenal weights and serum aldosterone levels are s»~pT ized in Table 2 . There were no significant differences in adrenal weights . At both levels of dietary magnesium animals fed the control sodium diet showed a progressive increase in mean serum aldosterone levels from days 7-28 . Evidently the control sodium level, although adequate according to official recommendations (10) was low enough to elicit an increased aldosterone response in growing rats . Mean serum aldosterone levels were consistently higher in magnesium deficient rats fed the control sodium diet ca~mpared to the rats fed adequate magnesium. The difference was statistically significant (P< 0.05) by day 28 of dietary treatment . A smaller, not statistically significant difference in aldosterone levels of magnesium depleted and control rats was seen only by day 28 in the animals fed the high sodium diet . Serum corticosterone levels, measured at different treatment times, were combined (Table 2) since available serum samples were limited. Levels were moderately but not significantly reduced in the magnesium deficient groups .
1214
Swum Aldoatarone in Magnesium Deficient Rata
Vol. 17, No . 8
TABLE 2 Adrenal Wei is Serum Aldosterone and Serum Corticosterone in Relation to Magnesium De letion and Sodium Intake Mean 1 aem Dietary treatment Days on diet
Control Mg Con trol Na
Low Mg Control Na
Control Mg High Na
Low Mg High Na
Adrenal weight (mg) 7
28 .710 .8
29 .8_1 .5
28 .9+2 .1
32 .712 .5
14
32 .310 .8
28 .2±1 .4
30 .5 3.0
34 .714 .7
28
37 .3*~ .7
40 .411 .6
36 .211. .6
37 .01-0.6
Serum aldoeterone (pg/ml) 59 " 7134 .6
179 .81102 .3
85 .3133 .6
101.0141 .0
14
175 " 7154 .2
249 .5166 .1
1021 11+9 .5
95 .4147.9
28
266.0~+9 .4 ab
530 .0118 .5 °
128 .0136 .8a
215.215o .9 ab
7
Serum corticoaterone 7-28 n*
(ng/ml)
279.5144.5
209 .7134 .0
318 .7151 .E
167.5134 .3
13
12
1
12
~chleana without a common letter in their superscript are significantly different (p < 0 .05) . *applies to serum corticoaterone only . each mean value .
For all other measurements n = 6 for
Neither sodium nor potassium retention, nor urinary excretion showed significant variations attributable to the level of dietary magnesium (Table 3) . Mean potassium retention appeared increased in magnesium deficient rata . However, large ataadard deviations and the observed difficulties in accurate collection of excreta indicate caution in the interpretation of this finding. Preliminary histological examination of the adrenal cortex suggested signs of increased activity in the zona glamerulosa of magnesium deficient rats fed the lower level of dietary sodium . In these sections the cells contained large, somewhat hypochromatic nuclei . The cytoplasm appeared enlarged and showed ea increase in negatively stained areas indicating the prior presence of lipid which had been removed by alcohol treatment of the sections . No differences between magnesium deficient and control groups were seen in adrenal sections taken from sodium loaded rats .
Vol . 17, No . 8
Serum Aldosterone in Magnesium Deficient Rats
1215
TART F 3
Uria bccretion and Retention of Sodium and Potassium in Relation to M esium Depletion and Sodium Intake Mean t eem. Values Obtained for Two Rats at 7, 1 , and 28 Days of Treatment were Pooled Dietary treatment Control Mg Control Na
Low Mg Control Na
Control Mg High Na
Urinary Na
18 .511 .3
17 .4 1.4
20917.5
166114 .4
Na retention ~/3 a$ys
46 .214 .0
52 " 913.9
52 .4115 .3
145 .3163.0
Urinary K mg/aey
55 .814 .4
46 .2±4 .7
57 .515 .5
45 .814 .3
K retention mg/3 days
70 .8113 .8
109 .7±17.1
63 .6116 .0
88 .4±23 .3
Parameter
~/~
Low Mg High Na
n=6 Discussion The re.dioimmnoassay for aldosterane used in this investigation involves two steps prior to protein binding : extraction into dichloromethane and subsequent separation of aldosterone from other steroids by the use of Sephadex LH-20. The original procedure required 600 a 10 mm Sephadex columns and 1-2 ml of human plasma, containing 26-737 pg aldosterone/ml (7) . The use of microcolumns used is the present study (4 x 65 mm) permitted estimation of aldosterone in 0.5 ml samples of rat serum with specificity end precision comparable to that reported previously using large columns . In our hands recovery of aldosterone after extraction sad Sephedex flltratio~n was 57 .9,112 .6, compared to 56 .5_14 and 44 .7,$17 .1 reported by Ito et e1 (7) and Waldhausl et e1 (11) respectively . Undoubtedly, the high standard deviation inherent in the assa ry contributed eubstaatislly to the large standard error of group averages . However, the expected inverse relationship between blood aldosterone and dietary sodium levels which showed clearly in these data (Table 2) suggest that the additional elevations in serum eldosterone seen in low sodiummagnesium depleted rats are a valid finding. The mechanisms by which magnesium depletion elicits excessive aldosterone output are not known. Possible mechanisms are' abnormal stimmilation of the zone glomerulosa, alterations is sensitivity of the adrenal cortex to sldosterane inducing stimuli, increased potassium balance and endorgan insensitivity to aldoeterone . The last two of these alternatives seem the least likely . While above normal sodium end water retention are consistent findings is magnesium deficient eaimAla (3,5,12-14), no observations of reduced rates of sodium reabsorption have yet been reported . Potassium balances have not previously been reported in magnesium deficient animals . Urinary excretion has been reported unchanged (15), slightly decreased (16) end significantly increased (5) . The apparent increase in potassium balance found is the present study is obscured by considerable variability, probably due to difficulties encountered in the quantitative estimation of intake and excretion . Potassium levels is skeletal muscle, heart, kidney and liver have been con-
1216
Serum Aldoaterone in Magnesium Deficient Rata
Vol . 17, No . 8
sistently found reduced in magnesium deficient animals (12-14,16,17) . This finding would argue against an increase in potassium balance . The possibility exists, however, that increased potassium balance may occur transiently in magnesium deficiency, and may be a periodic factor in the stimulation of aldosterone output . Two consistently reported consequences of magnesium depletion in rats are massive release of histamine (6,18-20) and nervous hyperreactivity (21-25) . Histamine can influence aldosterone secretion by lowering blood pressure (26,27) However, under experimental conor by stimulating release of ACTH (28,29) . ditions identical to those used in the present study (6), blood histamine levels reached a peek by day 15 of magnesium depletion. By day 30, blood histamine levels approached pre-treatment levels, whereas in the present study serum aldosterone levels on day 28 were more than twice those found an day 14 . Furthermore, increased ACTH stimulation normally causes elevations in corticosterone output . The reverse, a slight decrease in serum corticosterone levels, was seen in the present investigation . Suppression of the magnesium deficiency-induced elevation in serum aldosterone by sodium loe.ding suggests that primary alterations in magnesium deficiency may involve sites specifically concerned with osmoregulation . Cantin and Huet (30) observed extensive hyperplasia and hypertrophy of epithelial juxtaglomerular cells, without concomitant changes in the macula densa. Canon had suggested previously (5) that the major stimulus to juxtaglomerular hyperactivity in magnesium deficient rata was lowered blood and colloid osmotic pressure due to reduction in plasma proteins . An alternative suggestion is excessive neural atimu_lation of the juxtaglomerular apparatus by way of the ß-adrenergic fibers that are thought to be involved in the regulation of renin secretion (31,32) . The exact character of the hyperactivity and neuromuscular hyperexcitability in magnesium deficiency has not been systematically studied. Studies concerned with the influence of divalent canons on neural fluiction and transmission of nerve impulses have shown magnesium to inhibit release of acetylcholine (33-35) " A similar effect is suggested for rate of release of other neurotransmittor substances . The observation of Brecht et a1 (36) showing inhibition of the spontaneous release of epinephrine and norepinephrine Pram the adrenal medulla by increasing magnesium concentration is of interest in this context . If one of the generalized effects of magnesium deficiency is enhanced release of neurotransmittors at synapses and neuromuscular junctions, increased output of aldosterone may be mediated by neural hyperactivity either via the renin aldosterone system or, possibly, via neurosecretory mechanisms for stimulation of aldosterone secretion (37) . ACKNOWLEDGMINTS The authors are most grateful to Dr . William Hansel for his interest and suggestions during the couxse of this work . Thanks sre also due to Miss Mari Jensen for technical help . This work was supported (in part) by Hatch funds through the Agricultural Experiment Station, Cornea University . RRFRRF.'N
1. 2. 3.
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Serum Aldosterone in Magnesium Deficient Rats
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