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Copper Deficiency-lnduced Changes in the Fatty Acid Composition of Mitochondrial and Microsomal Membranes of Rat Liver P. S. BALEVSKA, E. M. RUSSANOV,* and P. I. STANCHEV Institute of Physiology, Bulgarian Academy of Sciences, bL 23, Acad. G. Bonchev St., I 113 Sofia, Bulgaria Received August 20, 1984; Accepted April t, 1985

ABSTRACT The effects of copper deficiency on the fatty acid composition of mitochondrial and microsomal phospholipids in rat liver were studied. Copper deficiency was induced by a milk powder diet. To evaluate the effect of the milk diet on the fatty acid pattern of mitochondrial and microsomal phospholipids, one group of rats was fed Cusupplemented powdered milk. A decrease in the relative proportion of linoleic acid and an increase in the level of oleic and docosahexaenoic acids in membrane phospholipids were found in this group. However, no changes in the fatty acid pattern characteristic of essential fatty acid deficiency were observed. Dietary copper deficiency produced a significant decrease in the relative amounts of linoleic and arachidonic acids, as well as an increase in the docosahexaenoic acid content in both mitochondrial and microsomal membranes compared to the nondeficient controls. The disproportionate quantities of polyunsaturated fatty acids are discussed with a view to the disturbances of membrane function in copper deficiency. Index Entries: Copper deficiency and fatty acid membrane composition; milk diet, and Cu-adequate diet; effect of Cu-supplemented milk diet on fatty acid composition of liver mitochondrial and microsomal membranes; mitochondrial membranes, Cu-deficiencyinduced changes in fatty acid composition of; microsomal membranes, Cu-deficiency-induced changes in fatty acid composition of.

*Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research

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Balevska, Russanov, and Stanchev

INTRODUCTION Copper deficiency in the liver cell results in a decrease in the electron transport in mitochondria, disturbances of oxidative phosphorylation, and inhibition of the activity of many enzymes of the respiratory chain (1,2). There is also evidence for a decrease in the levels of cytochromes, SH-groups (3), phospholipids (4) and particularly of cardiolipin in copper-deficient mitochondria (5), which could partly explain the changes in the basic mitochondrial functions. We have also observed that dietary copper deficiency in rats intensifies lipid peroxidation processes in liver mitochondria and microsomes (6). Copper deficiency has been reported to decrease the activity of some microsomal enzymes in the liver (7). All these changes provoked by copper deficiency have focused attention on the fatty acid composition of phospholipids because of their importance to the properties and functions of biomembranes. There are data that the degree of unsaturation of fatty acids in phospholipids influences the permeability properties of m e m b r a n e s (8-10), the activity of membranebound enzymes, and the electron transport system components (11,12). Little attention was devoted to the effect of copper deficiency on the fatty acid composition of subcellular membranes. Gallagher and Reeve (4) have found an increase in the levels of linoleic, arachidonic, and docosahexaenoic acids and a decrease in the amount of oleic acid in liver mitochondria from copper-deficient rats. Other authors have reported a decrease of the 9-desaturase activity in liver microsomes as well as a decrease of the mono-unsaturated:saturated ratios for C16 and C18 fatty acids from subcutaneous adipose tissue of rats maintained on a semisynthetic copper-deficient diet (13). The present investigation was undertaken to study the changes in the fatty acid composition of phospholipids in the most important liver subcellular organelles, the mitochondria and the endoplasmic reticulum, induced in rats by a copper-deficient milk diet.

MATERIALS AND METHODS Animals and Diets Adult male Wistar rats, weighing approximately 150 g, were used. The animals were divided into three groups (five rats per group). The first group consisted of rats fed a pelleted natural ingredient diet (standard lab chow) commonly used to maintain rat colonies. The second group of rats was fed Cu-supplemented powder milk, containing 15 mg/kg Cu 2§ in the form of CuSO4"SH20. The third group, Cu-deficient rats, received powdered milk (0.6 mg/kg copper, by analysis) and glass redistilled water. Copper content was the only difference between the milk diets. Food allowed to the animals of Group I was limited to ensure equicaloric consumption of food for the three groups of rats. All animals were maintained on the diets for 4 wk. Biological Trace Element Research

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Isolation of Liver I~Iitochondria and lVlicrosomes The rats w e r e sacrificed by decapitation. The livers were immediately excised and washed in a chilled 0.25M sucrose solution that was previously passed through a Dowex 50W resin column to reduce traces of copper and adjusted to p H 7.4 with HC1. Mitochondria and microsomes were prepared by differential centrifugation at 4~ (14). Protein content was estimated by the m e t h o d of Lowry et al. (15). Cytochrome c oxidase was assayed according to Sottocasa et al. (16).

Lipid Extraction and Analysis The total lipids of subcellular organelles were extracted with chloroform-methanol (1:2, v/v) (17) to which butylated hydroxytoluene (0.005%, w/v) had been a d d e d to prevent peroxidative damage to the unsaturated lipids (18). The phospholipids were purified by column chromatography on silicic acid (70-230 mesh, Merck, Darmstadt, Germany), d u r i n g t h e m with methanol. The fatty acid moieties of phospholipids were transmethylated with methanol at 65~ for 1 h using chlorotrimethylsilane (Merck, Darmstadt, Germany) as a catalyst. The resulting methyl esters were extracted with hexane, evaporated to dryness, redissolved in a suitable volume, and injected into gas chromatograph. A Perkin-Elmer Model 3920B gas chromatograph (Perkin-Elmer Corporation, Norwalk, CT) equipped with dual-column and dual-flame ionization detectors was used. Analyses were carried out on glass columns (184 x 0.2 cm) packed with 10% diethylene glycol succinate on Celite, 80-100 mesh. The oven temperature was p r o g r a m m e d as follows: at 150~ for 30 min and then from 150 to 170~ at a rate of 2~ Carrier-gas flow rate (N2) w a s 40 mL/min. Fatty acid methyl esters were identified by their relative retention times compared with k n o w n standards. Quantitative analysis was m a d e on the basis of the peak areas. The data were assessed for statistical significance using Student's t-test.

RESULTS Milk is considered to be a well-balanced food with respect to proteins, carbohydrates, and fats, containing sufficient amounts of vitamins and mineral salts. It is, however, low in copper and that is w h y milk diets are frequently used to induce copper deficiency in rats (4,19). Rats fed a p o w d e r e d milk diet for 4 w k do not develop anemia. Copper deficiency is manifested by a considerable decrease in the liver copper content (by 55-60%), in an inhibition of the cuproenzyme activities, and in some changes in liver functions (1,2,6,19). In the present experiments, cytochrome c oxidase activity was considered to be an index of copper status and a 56% decrease in this activity was detectable in experimental rats in comparison with that of controls (594 + 40 ng-atoms O2/min/mg Biological Trace Element Research

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TABLE 1 Fatty Acid Composition of the Diets" Fatty acid 14:0 b 16:0 16:1 18:0 18:1 18:2

Standard lab chow

Powdered milk diet

1.0 19.2 1.8 5.2 26.2 46.6

14.3 36.0 4.9 11.4 30.6 2.4

"Fatty acid composition was determined by gas chromatography as described in "Materials and Methods." Values are % composition by weight and are 9 the average of three determinations. bNumber of C a t o m s : n u m b e r of double bonds.

in the Cu-deficient group and 1379 _+ 59 and 1310 + 65 rig-atoms O2/min/mg in the control groups fed a standard lab chow and Cus u p p l e m e n t e d milk, respectively). From Table 1 it is seen that the milk diet differed in the fatty acid composition from the standard lab chow. The main difference consisted in the significantly higher relative percentage of saturated fatty acids and in the lower percentage of linoleic acid. Similar fatty acid patterns of lipids isolated from cow milk have been demonstrated by other authors (20). For the daily consumption of milk powder (about 20 g/rat), the milk lipid content (4% by analysis) and the relative percentage of the linoleic acid in lipids (2.4%), it becomes evident that each rat receives nearly 19 mg linoleic acid daily. According to Mohrhauer and Holman (21), this amount is lower than the adequate dose necessary for satisfying the requirements of essential fatty acids in rats. To evaluate the effect of the milk diet on the fatty acid composition of mitochondrial and microsomal phospholipids one group of rats was maintained on a C u - s u p p l e m e n t e d milk diet and the results were c o m p a r e d with those obtained with animals fed a standard lab chow (Tables 2 and 3). The changes in the percentage of n-6 series of fatty acids w e r e not as great as expected considering the dietary lipid composition. The proportions of linoleic acid in mitochondria and microsomes accounted for 75 and 65%, of those of the controls and the relative arachidonate content was not changed. The levels of oleic and docosahexaenoic acids in both subcellular structures were elevated. Some decrease in the palmitoleic acid content in mitochondria was observed. These findings are consistent with the data s h o w i n g that diets rich in saturated fatty acids (22) as well as diets poor in essential fatty acids (21) considerably increase the proportions of oleic acid in liver lipids. However, the other biochemical parameters generally associated with an essential fatty acid deficiency, namely an increase in the levels of Biological Trace Element Research

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TABLE 2 Fatty Acid C o m p o s i t i o n (mot%) of P h o s p h o l i p i d s Isolated from Liver Mitochondria of Rats Fed C o p p e r - A d e q u a t e and Copper-Deficient Diets C o p p e r - a d e q u a t e diets Fatty acid 16:0 ~ 16:1 16:2 18:0 18:1 18:2 18:3 20:3 20:4 20:5 22:4 22:5 22:6 18 : 2/20: 4 Double-bond index c DBI/S r a t i d

Standard

Cu-supplemented

lab c h o w

milk

19.6 -+ 1.0 b 1.7 -+ 0.1 1.0 + 0.2 23.0 _ 1.0 6.8 -+ 0.3 20.1 + 1.1 Trace to 0.2 0.7 • 0.1 19.7 • 0.6 1.1 • 0.3 Trace to 0.4 0.7 ~ 0.2 4.2 • 0.3

19.1 1.2 1.3 22.6 11.8 15.2 Trace 0.8 18.0 1.3 Trace 0.8 6.9

- 1.0 -+- 0.1 + 0.1 • 0.9 ___ 0.5 • 0.8 to 0.3 + 0.1 • 0.5 • 0.2 to 0.6 • 0.3 + 0.5

Copper-deficient diet P*

P o w d e r e d milk

P**

-

Copper deficiency-induced changes in the fatty acid composition of mitochondrial and microsomal membranes of rat liver.

The effects of copper deficiency on the fatty acid composition of mitochondrial and microsomal phospholipids in rat liver were studied. Copper deficie...
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