9 1990 by the Humana Press, Inc. All rights of any nature, whatsoever, reserved. 0163-4984/90/2401-0049 $02.40
Effects of Zinc Deficiency on the Fatty Acid Composition and Metabolism in Rats Fed a Fat-Free Diet NAOMI KUDO,* YASUHITO NAKAGAWA, AND KEIZO WAKU
Faculty of Pharmaceutical Sciences, Teiig,o University, Sagamiko, Kanagawa 199-01, Japan ABSTRACT The effects of dietary zinc deficiency (ZD) on the composition and metabolism of the fatty acyt chains of phospholipids in rat liver were investigated with a fat-free diet. The levels of (n - 9) fatty acids such as 18:1 and 20: 3(n - 9) in liver phospholipids (PL) were significantly lower in ZD-rats (19.4% and 5.4%, respectively) than in PF-rats (25.2 and 8.3%). On the other hand, the levels of (n - 6) acids such as 18:2 and 20:4 were higher in ZD-rats (3.3 and 19.1%, respectively) than in PF-rats (2.1 and 14.9%). In order to study the metabolism of fatty acids in vivo, 14C-18:0 or 14C-18:2 was intravenously injected, and then the conversion to the respective metabolite was examined. After the injection of 14C-18:0, the radioactivity was found in 18:0 (49.3% of the total), 18:1 (33.2%), and 20:3 (n - 9) (9.1%) in liver PL in PF-rats at 24 h. In ZDrats, the radioactivity was dramatically lower in 18:1 (23.5%) and 20:3 (n - 9) (3.6%), suggesting that the conversion of 18:0 to 18:1 and 20:3 (n - 9) was strongly inhibited in ZD-rats. When 14C-18:2 was injected, the radioactivity was mainly found in 18:2, 20:3 (n 6), and 20:4. The radioactivity in 20:4 in ZD-rats was slightly higher than that in control rats. These results indicate that zinc deficiency affects the fatty acid metabolism in liver, in particular, it causes a reduction in A9 desaturase activity, when rats are fed a fat-free diet. Index Entries" Zinc deficiency; fatty acid composition; desaturasion; essential fatty acid deficiency; liver. *Author to whom all correspondence and reprint requests should be addressed.
Biological Trace Element Research
49
rot 24, 1990
50
Kudo, Nakagawa, and Waku
INTRODUCTION Several investigations have suggested a relationship between dietary zinc and essential fatty acid (EFA) metabolism (1-8). Many of the features of zinc and EFA deficiency are similar, such as skin lesions and reproductive disorders (1). Acrodermatitis enteropathica, which is regarded as a genetic zinc disorder, involves abnormal fatty acid metabolism, and the infusion of cottonseed oil partially improves it (9). In addition, treatment with EFAs attenuates the symptoms caused by zinc deficiency in rats (2,3). These results indicate that zinc may play an important role in fatty acid metabolism in mammalian tissues. Changes in the fatty acid composition have been found in the testis, mammary tissue, intestinal mucosa, plasma, skin, and liver of ZD-rats (1,2,4--6). Cunnane et al. demonstrated that the level of 18:2" was increased and that of 20:4 decreased in total phospholipids in liver and plasma of ZD-rats (6). Huang et al. showed that 18:2 accumulated in tissue phospholipids in ZD-rats, but not in control rats, when 18:2 was administered (2). These results suggest that the activity of A6 desaturase, which metabolize 18: 2 (n - 6) to 18: 3 (n - 6), was decreased in ZD-rats. Because of the relationship between the zinc status and EFA metabolism, it would be interesting to know how zinc affects fatty acid metabolism when an animal is EFA deficient. In the present study, we examined the changes in the fatty acid composition in the liver of rats fed a fat-free diet owing to zinc deficiency. In addition, the metabolism of 14C-18:0 and 14C-18:2 was studied in vivo. We also compared the effect of reduced food intake on the fatty acid composition with that of zinc deficiency, since Kramer et al. concluded that the changes in fatty acid metabolism caused by zinc deficiency were probably a result of food restriction (10).
MATERIALS AND METHODS Materials All chemicals and solvents were of analytical grade. [1-14C] Stearic acid (60 mci/mmol) and [1-14C] linoleic acid (58 mci/mmol) were purchased from Amersham International (Amersham, UK). Precoated thin layer chromatography (TLC) plates (silicagel 60, type 5721) were purchased from Merck (Darmstadt). Animals and Diets Three-wk-old male Wistar rats weighing 50-60g (Sankyo Labo Service, Co, Ltd., Tokyo, Japan) were placed in stainless-steel cages in a *The fatty acids are designated by the number of carbon atoms and double bonds. Thus, 20:4 represents eicosatetraenoic acid (arachidonic acid), which contains 20 carbons and four double bonds. Biological Trace Element Research
Vol. 24, 1990
Zinc Deficiency and/~etabolism
51
temperature-, humidity-, and light-controlled room with a 12 h light-dark cycle. The zinc deficient and fat-free diet (Clea Japan, Inc.) contained 20% egg albumin, 71.8% sucrose, 0.2% DL-methionine, 1% vitamin mix (providing, in mg/kg diet: vitamin A acetate, 24 (12000 IU); cholecarciferol, 16 (2400 IU); thiamine HNO3, 15; riboflavin, 15; pyridoxine HCI, 10; cyanocobalamine, 0.05; D-o~-tocopherol, 100: menadione (vitamin K), 2.0; o~-biotin, 0.1; DL-calcium pantothenate, 20; p-aminobenzoic acid, 100; niacin, 100; inositol, 150; folic acid, 2.0; and choline chloride, 3000) and 7% mineral mix without Zn (supplying the following concentrations (g/ kg diet): CaCO3, 13.55; CuSO4 9 5H20, 0.013; CaHPO4 ~ 2H20, 15.0; MgSO 4 ~ 7H20, 8.0; NaC1, 6.0; KH2PO 4, 17.3; Fe(C6H507) ~ 5H20, 1.9; CoCl 2 ~ 6 H20, 0.004; Ca(lOB)2, 0.015; and MnSO 4 ~4H20, 0.154). A zincadequate diet was prepared by adding zinc as Zn(CH3COO)2 ~ 2H20 to the basal diet to 50 ppm Zn. Rats were divided into three groups, with 10-12 rats in each group. The ad libitum-fed rats were allowed free access to a zinc-adequate (AL) or zinc-deficient (ZD) diet. The food restricted pair-fed (PF) rats were given the zinc-adequate diet in an amount equal to that consumed on the previous day by the ZD-rats to eliminate the effects of calorie and protein restriction. After 2 wk, all rats were killed by decapitation and their livers were immediately removed.
Zinc Determination One gram of each test diet was digested with 8 mE of 65% nitric acid for 8 h at 130~ After the samples had been diluted with double-distilled water, their zinc contents were determined by flame atomic absorption spectrophotometry with an atomic absorption spectrophotometer (Perkin-Elmer, model PE-703). The zinc content of liver was also determined as described for diet analysis.
l_J'pid Analyses Total lipids were extracted from 2 g of liver as described by Bligh and Dyer (11) and then dissolved in 2 mL of chloroform. Total phospholipids (PL), triglycerides (TG), free fatty acids, and diglycerides were separated by TLC, with development with petroleum ether:diethyl ether:acetic acid (70:30:1). Total PL and TG were scraped off from the TLC plates and mixed with 1 mL of sodium methoxide. After stirring at room temperature for 40 min, the samples were neutralized with hydrochloric acid. Fatty acid methyl esters were extracted with 2 mL of hexane 2 times and then analyzed by gas-liquid chromatography as described by Onuma et al. (12). The quantities of fatty esters in TG and PL were determined with methyl pentadecanoate as the internal standard.
In Vivo Conversion of 14C-Labeled Fatty Acids Two groups (PF and ZD) of rats were maintained for 2 wk as described above. Each rat was intravenously injected with [1J4C] stearic Biological TraceElement Research
Vol. 24, 1990
.52
Kudo, Hakagawa, and Waku
acid or [1-14C] linoleic acid (2 ~mol of fatty acid (10-14 ~ci)/100 g body weight) suspended to 0.2% of bovine serum albumin via the tail vein under light ether anesthesia. Four or 24 h after injection, the rats were killed and the total lipids in their livers were extracted. After preparation of fatty methyl esters from total PL, as described above, the samples were dissolved in chloroform:methanol (1:2) and then fractionated by high performance-liquid chromatography (HPLC) according to the modified method of Aveldano et al. (13). Each fatty acid methyl ester was separated on a reverse-phase column (Lichrosorb RP-8; Merck, Darmstadt), with elution with acetonitrile or acetonitrile:water (9:1). Each fatty methyl ester peak was detected on the basis of the absorption at 205 or 220 nm. The radioactivity of individual peaks was estimated with a liquid scintillation counter (Packard 3320).
Statistics The results are expressed as means + SD and the statistical significance of the differences between two groups was examined by means of Student's t-test.
RESULTS The effects of zinc deficiency and food restriction on the body weight gain of rats fed a fat-free diet are shown in Fig. 1. Growth was significantly retarded in the ZD- and PF-rats during the 2-wk period. The average body weights of ZD- and PF-rats were approximately 50% of that of AL-rats at the end of the experiment. The difference in body weight between PF- and ZD-rats was not significant. The zinc content in the liver of ZD-rats was reduced by 30% compared to PF-rat liver, which contained 28.98 ~g Zn/g, expressed at the concentration per gram of wet tissue (Table 1). AL-rat liver contained 21.4 ~g Zn/g, which was also less than that in PF-rats. Most fatty acids were acylated to phospholipids, which comprised about 20 times more fatty acids than TG in rat liver (Table 2). The total amounts of fatty acids acylated to phospholipids and triglycerides per gram of liver were not significantly different among AL-, PF-, and ZDrats. The fatty acid composition of liver PL is shown in Table 3(a). The decreases in the levels of 16:1, 18:0, 18:2, and 20:3 (n - 9), and the increases in the levels of 18:1 and 22:6 (n - 3) were a result of reduced food intake (AL vs PF). The proportions of 16:0 and 20:4 (n - 6) in ALrats were almost the same as those in PF-rats. When the fatty acid composition in ZD-rats were compared with that in PF-rats, the proportions of 18:0, 18:2, 20:4 (n - 6), and 22:6 were found to be high, whereas the levels of 16:1, 18:1, and 20:3 (n - 9) were decreased in ZDrats. High levels of 18: 0, 18: 2, and 20: 4, and low levels of 18:1 and 20: 3 Biological Trace Element Research
Vol. 24, 1990
Zinc Deficiency a n d Metabolism
53
120
Zn+(AL)
A
e.~J
80 O
Zn-(Z0) Zn+(PF)
T
I
I
I
4
8
12
day
Fig. 1. Effects of dietary zinc and food restriction o n the growth of rats fed a fat-free diet. G r o w t h of the rats in both the PF-(o) and ZD-(o) groups was less than that of AL-rats (4) after 3 d (P < 0.001). The b o d y weights of PF- and ZD-rats were not significantly different. The values are m e a n s _+ SD for 10 (AL and ZD) and 12 (PF) rats per group. Table 1 Zinc Concentrations in Zinc-Adequate and Zinc-Deficient Rat Liver Zinc ~g/g wet tissue Zn-adequate" (PF) Zn-deficient b (ZD) Zn-adequate c (AL)
28.98 + 3.00 (n = 12) 21.81 _+ 1.74 (n = 10)* 21.38 ___ 1.83 (n = 10)*
"PF-rats received the zinc-adequate diet (50 ppm), but limited in amount to that consumed by ZD-rats. bZD-rats received the zinc-deficient diet (
Effects of Zinc Deficiency on the Fatty Acid Composition and Metabolism in Rats Fed a Fat-Free Diet NAOMI KUDO,* YASUHITO NAKAGAWA, AND KEIZO WAKU
Faculty of Pharmaceutical Sciences, Teiig,o University, Sagamiko, Kanagawa 199-01, Japan ABSTRACT The effects of dietary zinc deficiency (ZD) on the composition and metabolism of the fatty acyt chains of phospholipids in rat liver were investigated with a fat-free diet. The levels of (n - 9) fatty acids such as 18:1 and 20: 3(n - 9) in liver phospholipids (PL) were significantly lower in ZD-rats (19.4% and 5.4%, respectively) than in PF-rats (25.2 and 8.3%). On the other hand, the levels of (n - 6) acids such as 18:2 and 20:4 were higher in ZD-rats (3.3 and 19.1%, respectively) than in PF-rats (2.1 and 14.9%). In order to study the metabolism of fatty acids in vivo, 14C-18:0 or 14C-18:2 was intravenously injected, and then the conversion to the respective metabolite was examined. After the injection of 14C-18:0, the radioactivity was found in 18:0 (49.3% of the total), 18:1 (33.2%), and 20:3 (n - 9) (9.1%) in liver PL in PF-rats at 24 h. In ZDrats, the radioactivity was dramatically lower in 18:1 (23.5%) and 20:3 (n - 9) (3.6%), suggesting that the conversion of 18:0 to 18:1 and 20:3 (n - 9) was strongly inhibited in ZD-rats. When 14C-18:2 was injected, the radioactivity was mainly found in 18:2, 20:3 (n 6), and 20:4. The radioactivity in 20:4 in ZD-rats was slightly higher than that in control rats. These results indicate that zinc deficiency affects the fatty acid metabolism in liver, in particular, it causes a reduction in A9 desaturase activity, when rats are fed a fat-free diet. Index Entries" Zinc deficiency; fatty acid composition; desaturasion; essential fatty acid deficiency; liver. *Author to whom all correspondence and reprint requests should be addressed.
Biological Trace Element Research
49
rot 24, 1990
50
Kudo, Nakagawa, and Waku
INTRODUCTION Several investigations have suggested a relationship between dietary zinc and essential fatty acid (EFA) metabolism (1-8). Many of the features of zinc and EFA deficiency are similar, such as skin lesions and reproductive disorders (1). Acrodermatitis enteropathica, which is regarded as a genetic zinc disorder, involves abnormal fatty acid metabolism, and the infusion of cottonseed oil partially improves it (9). In addition, treatment with EFAs attenuates the symptoms caused by zinc deficiency in rats (2,3). These results indicate that zinc may play an important role in fatty acid metabolism in mammalian tissues. Changes in the fatty acid composition have been found in the testis, mammary tissue, intestinal mucosa, plasma, skin, and liver of ZD-rats (1,2,4--6). Cunnane et al. demonstrated that the level of 18:2" was increased and that of 20:4 decreased in total phospholipids in liver and plasma of ZD-rats (6). Huang et al. showed that 18:2 accumulated in tissue phospholipids in ZD-rats, but not in control rats, when 18:2 was administered (2). These results suggest that the activity of A6 desaturase, which metabolize 18: 2 (n - 6) to 18: 3 (n - 6), was decreased in ZD-rats. Because of the relationship between the zinc status and EFA metabolism, it would be interesting to know how zinc affects fatty acid metabolism when an animal is EFA deficient. In the present study, we examined the changes in the fatty acid composition in the liver of rats fed a fat-free diet owing to zinc deficiency. In addition, the metabolism of 14C-18:0 and 14C-18:2 was studied in vivo. We also compared the effect of reduced food intake on the fatty acid composition with that of zinc deficiency, since Kramer et al. concluded that the changes in fatty acid metabolism caused by zinc deficiency were probably a result of food restriction (10).
MATERIALS AND METHODS Materials All chemicals and solvents were of analytical grade. [1-14C] Stearic acid (60 mci/mmol) and [1-14C] linoleic acid (58 mci/mmol) were purchased from Amersham International (Amersham, UK). Precoated thin layer chromatography (TLC) plates (silicagel 60, type 5721) were purchased from Merck (Darmstadt). Animals and Diets Three-wk-old male Wistar rats weighing 50-60g (Sankyo Labo Service, Co, Ltd., Tokyo, Japan) were placed in stainless-steel cages in a *The fatty acids are designated by the number of carbon atoms and double bonds. Thus, 20:4 represents eicosatetraenoic acid (arachidonic acid), which contains 20 carbons and four double bonds. Biological Trace Element Research
Vol. 24, 1990
Zinc Deficiency and/~etabolism
51
temperature-, humidity-, and light-controlled room with a 12 h light-dark cycle. The zinc deficient and fat-free diet (Clea Japan, Inc.) contained 20% egg albumin, 71.8% sucrose, 0.2% DL-methionine, 1% vitamin mix (providing, in mg/kg diet: vitamin A acetate, 24 (12000 IU); cholecarciferol, 16 (2400 IU); thiamine HNO3, 15; riboflavin, 15; pyridoxine HCI, 10; cyanocobalamine, 0.05; D-o~-tocopherol, 100: menadione (vitamin K), 2.0; o~-biotin, 0.1; DL-calcium pantothenate, 20; p-aminobenzoic acid, 100; niacin, 100; inositol, 150; folic acid, 2.0; and choline chloride, 3000) and 7% mineral mix without Zn (supplying the following concentrations (g/ kg diet): CaCO3, 13.55; CuSO4 9 5H20, 0.013; CaHPO4 ~ 2H20, 15.0; MgSO 4 ~ 7H20, 8.0; NaC1, 6.0; KH2PO 4, 17.3; Fe(C6H507) ~ 5H20, 1.9; CoCl 2 ~ 6 H20, 0.004; Ca(lOB)2, 0.015; and MnSO 4 ~4H20, 0.154). A zincadequate diet was prepared by adding zinc as Zn(CH3COO)2 ~ 2H20 to the basal diet to 50 ppm Zn. Rats were divided into three groups, with 10-12 rats in each group. The ad libitum-fed rats were allowed free access to a zinc-adequate (AL) or zinc-deficient (ZD) diet. The food restricted pair-fed (PF) rats were given the zinc-adequate diet in an amount equal to that consumed on the previous day by the ZD-rats to eliminate the effects of calorie and protein restriction. After 2 wk, all rats were killed by decapitation and their livers were immediately removed.
Zinc Determination One gram of each test diet was digested with 8 mE of 65% nitric acid for 8 h at 130~ After the samples had been diluted with double-distilled water, their zinc contents were determined by flame atomic absorption spectrophotometry with an atomic absorption spectrophotometer (Perkin-Elmer, model PE-703). The zinc content of liver was also determined as described for diet analysis.
l_J'pid Analyses Total lipids were extracted from 2 g of liver as described by Bligh and Dyer (11) and then dissolved in 2 mL of chloroform. Total phospholipids (PL), triglycerides (TG), free fatty acids, and diglycerides were separated by TLC, with development with petroleum ether:diethyl ether:acetic acid (70:30:1). Total PL and TG were scraped off from the TLC plates and mixed with 1 mL of sodium methoxide. After stirring at room temperature for 40 min, the samples were neutralized with hydrochloric acid. Fatty acid methyl esters were extracted with 2 mL of hexane 2 times and then analyzed by gas-liquid chromatography as described by Onuma et al. (12). The quantities of fatty esters in TG and PL were determined with methyl pentadecanoate as the internal standard.
In Vivo Conversion of 14C-Labeled Fatty Acids Two groups (PF and ZD) of rats were maintained for 2 wk as described above. Each rat was intravenously injected with [1J4C] stearic Biological TraceElement Research
Vol. 24, 1990
.52
Kudo, Hakagawa, and Waku
acid or [1-14C] linoleic acid (2 ~mol of fatty acid (10-14 ~ci)/100 g body weight) suspended to 0.2% of bovine serum albumin via the tail vein under light ether anesthesia. Four or 24 h after injection, the rats were killed and the total lipids in their livers were extracted. After preparation of fatty methyl esters from total PL, as described above, the samples were dissolved in chloroform:methanol (1:2) and then fractionated by high performance-liquid chromatography (HPLC) according to the modified method of Aveldano et al. (13). Each fatty acid methyl ester was separated on a reverse-phase column (Lichrosorb RP-8; Merck, Darmstadt), with elution with acetonitrile or acetonitrile:water (9:1). Each fatty methyl ester peak was detected on the basis of the absorption at 205 or 220 nm. The radioactivity of individual peaks was estimated with a liquid scintillation counter (Packard 3320).
Statistics The results are expressed as means + SD and the statistical significance of the differences between two groups was examined by means of Student's t-test.
RESULTS The effects of zinc deficiency and food restriction on the body weight gain of rats fed a fat-free diet are shown in Fig. 1. Growth was significantly retarded in the ZD- and PF-rats during the 2-wk period. The average body weights of ZD- and PF-rats were approximately 50% of that of AL-rats at the end of the experiment. The difference in body weight between PF- and ZD-rats was not significant. The zinc content in the liver of ZD-rats was reduced by 30% compared to PF-rat liver, which contained 28.98 ~g Zn/g, expressed at the concentration per gram of wet tissue (Table 1). AL-rat liver contained 21.4 ~g Zn/g, which was also less than that in PF-rats. Most fatty acids were acylated to phospholipids, which comprised about 20 times more fatty acids than TG in rat liver (Table 2). The total amounts of fatty acids acylated to phospholipids and triglycerides per gram of liver were not significantly different among AL-, PF-, and ZDrats. The fatty acid composition of liver PL is shown in Table 3(a). The decreases in the levels of 16:1, 18:0, 18:2, and 20:3 (n - 9), and the increases in the levels of 18:1 and 22:6 (n - 3) were a result of reduced food intake (AL vs PF). The proportions of 16:0 and 20:4 (n - 6) in ALrats were almost the same as those in PF-rats. When the fatty acid composition in ZD-rats were compared with that in PF-rats, the proportions of 18:0, 18:2, 20:4 (n - 6), and 22:6 were found to be high, whereas the levels of 16:1, 18:1, and 20:3 (n - 9) were decreased in ZDrats. High levels of 18: 0, 18: 2, and 20: 4, and low levels of 18:1 and 20: 3 Biological Trace Element Research
Vol. 24, 1990
Zinc Deficiency a n d Metabolism
53
120
Zn+(AL)
A
e.~J
80 O
Zn-(Z0) Zn+(PF)
T
I
I
I
4
8
12
day
Fig. 1. Effects of dietary zinc and food restriction o n the growth of rats fed a fat-free diet. G r o w t h of the rats in both the PF-(o) and ZD-(o) groups was less than that of AL-rats (4) after 3 d (P < 0.001). The b o d y weights of PF- and ZD-rats were not significantly different. The values are m e a n s _+ SD for 10 (AL and ZD) and 12 (PF) rats per group. Table 1 Zinc Concentrations in Zinc-Adequate and Zinc-Deficient Rat Liver Zinc ~g/g wet tissue Zn-adequate" (PF) Zn-deficient b (ZD) Zn-adequate c (AL)
28.98 + 3.00 (n = 12) 21.81 _+ 1.74 (n = 10)* 21.38 ___ 1.83 (n = 10)*
"PF-rats received the zinc-adequate diet (50 ppm), but limited in amount to that consumed by ZD-rats. bZD-rats received the zinc-deficient diet (
