Magnesium deficiency in the Japanese quail

Camp. Biochem. Physiol. Vol. BA, No. 2, pp. 223-227, 1984 Printed in Great Britain

MAGNESIUM

03&S9629/84 $3.00 + 0.00 Q 1984 Pergamon Press Ltd

DEFICIENCY

IN THE JAPANESE

QUAIL

ROBERTDEDIER*, ELYETT GUEUX~ and YVES RAYSSMUIER$ *Labomtoire de Biologic Animale, ERA. 408, Universit6 de Clermont IL BP 45, 63170 Aubi&e, France flaboratoire de Pharmacofogie MLdicale et Hydrologic, U-195 Tnserm, Fact&t? de M&de&e, 6_BO1 Cteimont-f-d, France $Laboratorie des Sfaladies Metabohques, fNRA Theix 63I IQ Beaumont, France (Receit;ed I7 Junuary 1984)

Abstract-l. Morphological effects of magnesium deficiency on liver cells and general aspects of its influence on the metabolism were investigated in young quails, 2. Magnesium deficiency was characterized by a depressed growth, a high mortality rate, a decrease in hematocrit and magnesium and calcium plasma concentrations. 3. Magnesium deficiency reduced the magnesium concentration in heart by 440/,, but did not affect the concentration in liver. 4. Ultrastructural aspect of liver parenchymal ceils revealed that the number of mitochrondria per cell section was decreased and the average area of a m~tochondrion was greater in deficient quails than in control animals. The significance ofthese morpholo~~al changes was discussed in relation to disturbances in energy mctabofism of these organeiies. 5. From these results, japanese quail appeared as an interesting experimental model for studies on metabolic disturbances in magnesium deficiency.

Table

Magnesium is one of the most abundant intracellular metals which activates many enzymes in addition to being required for other cellular processes (Heaton, 1973). The effect of magnesium deficiency has been extensively studied in mammals where it produces ~trastructurai changes in tissues hke liver, heart, kidney or skeletal muscle (George and Weaton, 19X), but much less is known of its effect in birds. Young quails are very sensitive to magnesium deficiency (Tao ef al., 1983) and the present investigation was undertaken to study the detailed morphological effect of the deficiency on liver cells in addition to more general aspects of its influence on the growth and metabolism of young birds.

Ingredients* ~ ._.._-_ Casein

% 30

Starch Corn oil Glycine D-L Met&nine Mineral mixt Vitamin mix1

ss 5 0.2 0.5 5.5

the

0.5

glycine, methionine and miner~1sfront Pralabo, Paris; vitamin mix from INRA, Nouzilly, FratXe. ‘tPer kilogram mixture: CaHPO,, 893 &; NaCI, 66 g; WI, 35.5 g; MnSO,, 5.9g: ZnSO,, 3.5g; Ferric citrate, 2 g; CuCO,, 0.066 g; IKO,, 0.043 g. #Per kilogram mixture: Thiamin, 0.8 g; riboflavin, I A g; nicorinic acid, IOg; pantkenic acid, 4g; pyridoxine, 1.4g; cyanoc*balamin, Qg: folic acid, 0.4 g; biotin, 0.04 g; choline, 400 g; vitamin A, 4.8 g; vitamin D,, 3 8; vitamin E, 24 g, vitamin K, 4g; starch to 1kg.

Animals and dieis Japanese quails ~Co~~~~x cu~urnix japondca, Gerardmer strain) of both sexes, I3 days old and weighing about 25 gt were caged under controlled conditions (Ridier et al., 1983). They were randornty divided into magnesia-descent (54 animals) and control groups (30 animaIs), and fed the appropriate diets for 9 days. The basic composition of the magnesium deficient diet is given in Table 1.The magnesium content determined by anaIysis was 0.03 g/kg for the deficient group and adjusted to O.sOg/kg for the control group by the addition of magnesium sulfate. Diet and distilled water were available ad libitum. All quails were killed by decapitation, and blood was collected in tubes with heparin as anticoagulant. A sample of blood was taken for hematocrit determination and the remainder was centrifuged to separate the plasma which was used for mineral analysis. The Ever and the heart were removed and kept frazen until analysed. Fragments of the central lobe of the liver were cut for electron microscopy.

of

‘Dietary constituents were obtained from the indicated sources: casein from Louis Franqois, Paris;

MATERIAL AND METHODS

Tissue sampling

1. Composition purified diet

Analytical procedures

Magnesium and calcium were estimated in blood plasma and tissues by atomic absorption spectrophotametry (Perkin-Elmer, model 400). The plasma samples were diluted I:50 with a lanthanum chloride solution (0.1% La). Livers and hearts were weighed, then dried at 105°C for 24 hr and ashed at 550°C for 24 hr in nickel crucibles. The ashed residue was dissolved in dilme HCI before d&&on with the tanthanum solution. 223

274

Results are given as the mean k SEM and expressed as mg/g dry matter. The number of quails used is shown in parentheses. *P < 0.01,

Electron microscopy

microscopy. fragments of central lobe of iiver, l-2mmJ, were fixed in 2’1; glutaraidehyde in phosphate buffer (0.1 M, pH 7.3). The samples were rinsed in phosphate buffer and post-fixed in 24” phosphate buffered osmium t&oxide for 1hr a&4’C. After dehydration. they were embedded in Epon 8 12 (Luft, 1961). Since the hepatocyte ultrastructure varies according to their position in the hepatic lobe (Loud, 1968). semi-thin sections of each specimen were cut, stained with toiuidine blue and used for locating mid-lobular hepatocytes. Ultra-thin sections of this region of the lobe were cut with a glass knife. They were placed on electrolytic grids and stained with many1 acetate (Watson, 1958) and lead citrate (Reynolds, 1963). The grids were then covered with a thin film of carbon and examined in a Siemens IA electron microscope. For

electron

Morphometric

analwis

Twenty mid-lobular hepatocytes from four quails on each diet were analysed at a magni~~tion of x 6000. Only hepatocytes with clearly defined boundaries and those with a visible nucleus were selected. This morphometric analysis was carried out by planimetry with the help of a picture analyser which permitted estimation of different parameters such as the flat area of the cells, the nucleus and that of both single and total mitochondria in the section. The apparatus consisted of an electronic digitizer (Houston Instrument, Texas, USA) in which areas were delimited by displacement of a cursor. Calculution

Results are shown as mean f SEM. Statistical significance between mean values was estimated by the Student r-test. RESULTS EJJects ~~magnes~um deficiency on uarious parameters The parameters of magnesium deficiency are summarized in Table 2. The mean body weight of the quails fed on the deficient diet was significantly lower than that of the control group. Disturbance in moving and balance affected these birds which showed a tottering walk and high mortality rate (43%) during the experimental period. Plasma magnesium and calcium were significantly lowered in the magnesiumdeficient group and this was accompanied by a marked decrease in hematocrit.

Effect qf mugrwsium d&iency concentrations

on tissue electrolyte

The concentrations of metals in tissues from magnesium deficient and control quails are indicated in Table 3. The magnesium and calcium concentrations in the liver were not significantly modified by the magnesium content of the diet. On the other hand, the magnesium content of the heart of deficient birds was only 44% of that in controls and the calcium content tended to increase although this was not statistically significant. Effects of magnesium dt$ciency on fine .structure q/ hepatic cells Electron micrographs of liver parenchymal cells from quails fed the control diet showed the cytological characteristics previously reported by other investigators (Fig. I). The average size of the hepatic cells are not modified by magnesium deficiency. Glycogen stores, the number and the size of lipids droplets as the ultrastructural aspect of endoplasmic reticulum and the golgi complex appeared unchanged by magnesium deficiency. However, changes in the morphology of the hepato-cellular mitochondria were observed in the magnesium-deficient group (Fig. 2). Many mitochondria showed a decrease in the electron density of their matrices. Furthermore, a reduction in the number of mitochondrial profiles and an enlargement of their area were observed. These observations have been confirmed by morphometric analysis (Table 4). In the magnesiumdeficient group the mean area of individual mitochondrial sections was 1.17 prnL, which represented an increase of 307; over the control group (0.90 pm’). In the control quails, mitochondria occupied nearly 23”/, of the cytoplasm area but this percentage was significantly lowered at 19.3”,, in the magnesium deficient birds. The number of mitochondria per cell section was also decreased in the deficient animals. A quantitative measure of mitochondrial dimensions has been devised on the assumption that their general shape is that of right circular cylinders (Loud et al., 1965). In these conditions, it could seem that magnesium deficiency increased both the average

Mg deficiency

Fig. 1. Electron

micrograph

of hepatocytes

in C. coturnix japonica

from control

quails.

Glycogen

225

particles

(GI) are deposited

in

dense masses throughout the cytoplasm. Mitochondria (M) are abundant and are associated with cisternae of rough endoplasmic reticulum (RER). Nucleus (N) and lipid droplet (LD) are shown. x 6120. diameter and length of such cylinders. From the cytological observations, changes in size of mitochondria would be the result of an increase of mitochondrial matrix rather than an increase of intermembrane space. DISCUSSION

In studies on that the severity components, the privation period

magnesium deficiency, it is evident of the deficiency depends on two age of the animals during the deand the magnesium level of the diet.

Newly hatched quails, compared to 2-week-old birds, were found to be very sensitive to dietary deprivation of magnesium (Tao et al., 1983). In the present experiment when quails 13 days old were fed a diet containing 30ppm Mg, they developed rapidly the symptoms of an acute deficiency. A diet less deficient in magnesium (125 ppm) produces in quails of same age only discrete signs of magnesium deficiency (Tao et al., 1983). In our experimental conditions, magnesium deficiency induces both a large decrease in plasma

Fig. 2. Electron micrograph of hepatocytes from magnesium-deficient quails. Mitochondria (M) are enlarged and less abundant than in the cytoplasm of control quails. Some mitochondria show a decrease in the electron density of their matrices. Glycogen stores (GI) are unchanged by magnesium deficiency. Nucleus (N), lipid droplet (LD) and rough endoplasmic reticulum (RER) are identified. x 6120.

Results

are means + SEM.

,,I each goup

*P

Measurements

< 0.05.

tP

are made on 20 hepatocytes

from

four

indwdual

quails

< 0 01.

nla~nesium concentratjon and an elevated mortality. Magnesium deficiency produces also a marked decrease of hematocrit. This consequence of magnesium deficiency has been described in several animal species (Elin, 1980), but in quails it is clearly more marked and develops more rapidly. This anemia noted in magnesium deficiency could be the result of an increased rate of erythrocyte hemolysis (Tao et al., 1983). The decrease of calcium in plasma is also observed among different species fed on magnesium deficient diet. This hypocalcemia has been generally related to an decrease of responsiveness of bone to hormones stimulating Ca resorption (Rayssiguier et ul., 1982). No significant loss of liver magnesium was observed in the present experiment despite the severe hypomagnesemia. This result is in agreement with previous observations showing that the maintenance of an adequate magnesium concentration in the liver may be a priority during deficiency in this element (Martindale and Heaton, 1964). However, although the magnesium level of the liver was similar to that of the control group. cytological analysis indicated that morphological changes are present in hepatocytes of magnesium-deficient quails. Contrary to previous observations in rats (George and Heaton, 1975), the average size of hepatic cells was not modified by magnesium deficiency. Likewise, no evident changes in morphological aspects of the endoplasmic reticulum, golgi complex, content of glycogen or number of lysosomes and autophagosomes was detected in electron micrographs, although such changes have been found in rat liver in long-term magnesium deficiency (Lewicki et uf., 1979). In fact. after 9 days of deficiency the morphological disturbance affected only mitochondria and were characterized by a decrease of electron density in their matrices, an increase in their average area, a reduction in the number of mitochondrial profiles and in the cytoplasmic area occupied by these organelles. These changes in number and area of mitochondrial profiles are similar to the results of George and Heaton (1975) in rat liver after 20 days of deficiency. However, the intensity of these effects is different between the two species. In quails, the decrease in the number of mitochondrial profiles is less pronounced but the increase in average area of mitochondrial sections is greater than in rats. The calculation of average mitochondrial dimensions, devised on the assumption that their general

shape is that of right circular cylinders, indicates that mitochondrial enlargement results both in an increase of their diameter and their length. With the presence of irregularly shaped mitochondria in the liver, this assumption may lead to an underestimation of the average mitochondrial dimensions. However. as irregular shapes constituted only a low percentage of mitochondrial profiles, which did not differ between the two groups, it seems unlikely that their presence will introduce serious errors into the calculation of these mitochondrial characteristics. The increase of mitochondrial area indicates a swelling of these organelles. In different studies, it has been reported that swelling of mitochondria is often associated with an uncoupling of oxidative phosphorylation (Weinbach et ul.. 1967) and it has been demonstrated that magnesium deficiency significantly lowers the P:O ratio of liver mitochondria indicating a partial uncoupling of oxidative phosphorylation (George and Heaton, 1978). Such an impaired functioning of these organelles might contribute to the morphological change. In another connection, it is well known that the action of various uncoupling agents coincides with a loss of mitochondrial magnesium (Lee et ul., 1970). Although the liver magnesium concentration in magnesium-deficient quails was similar to that of the control group, a disturbance in the intraceIlular distribution of this cation between the different cellular fractions might occur even if the total intracellular level is not modified. Assuming that the inner membrane of mitochondria would be particularly affected by magnesium deficiency (Heaton and George, 1979; Heaton and Elie, 1983). it is possible that mitochondrial permeability has been altered. With a change in the permeability of the membrane. an increased uptake of water would reduce the electron density of the mitochondrial matrix. Hepatic ceils of magnesium deficient quails are also characterized by a decrease in number of mitochondrial profiles. From cytological examination, it does not seem that this decrease is the result of an enhanced rate of mitochondrial degradation, because electron micrographs revealed no evidence of increased lysosomal or phagocytic activity. On the other hand, the disturbance in energy metabolism affecting protein biosynthesis (George and Heaton, 1978) could explain the decrease in mitochondrial profiIes during magnesium deficiency. Mineral analysis of the liver did not show an

Mg deficiency in C. coturnix japonica increase of calcium concentration in magnesiumdeficient quails and no abnormal deposit of calcium was detectable on ultrastructural examination of hepatic cells. Magnesium deficiency in rats, however, leads to an early increase of calcium in the liver and studies on the subcellular distribution of calcium indicate a preferential deposition of calcium in the mitochondrial fraction (George and Heaton, 1975). The large decrease in magnesium concentration within the heart of magnesium-deficient quails was more severe than in other animal species fed a magnesium-deficient diet (Ebel and Gunther, 1983), and it was accompanied by an increase in heart calcium. The effect of alterations in magnesium and calcium levels on the excitability of smooth muscles is well known, in particular on cardio-vascular tissues (Altura and Altura, 1981) and the magnesium deficiency syndrome could be accompanied by sudden death (Turlapaty and Altura, 1980). Owing to the intensity of this loss in myocardial magnesium, it may be asked whether there could be a possible relationship between this depletion and the high mortality rate noted in magnesium-deficient quails. This investigation indicates the speed and severity of magnesium deficiency in the quail. Consequently, this species constitutes an interesting experimental model to study the pathology associated with magnesium deficiency. In other animal species, it has been shown that magnesium deficiency is accompanied by disturbances in lipid metabolism (Rayssiguier et al., 1981; Rayssiguier, 1984) and might thus promote atherogenesis. Owing to the short period of the deficiency, no research on the possibility of atherosclerosis has been carried out in magnesium-deficient quails. Assuming that the quail is particularly sensitive to this pathology (Radcliffe et al., 1982; Shih, 1983) this point would deserve further study. As magnesium is required for the morphological and functional integrity of varied cellular organelles, the important cellular alterations observed in our experiment would indicate severe metabolic disturbances. Acknowledgements-The authors wish to thank Dr. F. Heaton for his most helpful comments and Mrs N. Benay and Mrs S. Guerin for their expert technical assistance.

REFERENCES

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