EFFECT OF NICKEL DEFICIENCY ON FATTY ACID COMPOSITION OF TOTAL LIPIDS AND INDIVIDUAL PHOSPHOLIPIDS IN BRAIN AND ERYTHROCYTES OF RATS

NutritionResearch,Vol. 17, No. 1, pp. 137–147, 1997 Copyright01996 Elsevier Science Inc. Printedin the USA. All rightsreserved 0271-5317/97 $17.00 + al

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ELSEVIER

PIIS0271-5317(%)00240-0

EFFECT OF NICKEL DEFICIENCY ON FATTY ACID COMPOSITIONOF TOTAL LIPIDS AND INDIVIDUALPHOSPHOLIPIDSIN BRAIN AND ERYTHROCYTESOF RATS G.I. Stangl (Dr.) and M. Kirchgessner(Prof.Dr.Dr.h.c.mult.)* Institut fuer Ernaehrungsphysiologie,Technische Universitaet MuenchenWeihenstephan, 85350 Freising, Germany

ABSTRACT The present investigation was designed to examine the effect of nickel deficiency on fatty acid composition of total lipids and individual phospholipids in brain and erythrocyte membranes of rats. Therefore, a study over two generations was conducted feeding a nickeldeficient diet containing 13 ,ugikg nickel or a nickeladequate control diet supplemented with 1 mgkg nickel. Male 7 week old rat pups from the second offkpring were studied. The concentrations of individual brain phospholipids did not differ between the nickel-deficient and the nickel-adequate group, whereas in erythrocyte membranes the concentration of phosphatidylethanolamine (PE) diacyl was reduced in consequence of nickel depletion. Moreover, nickel deficiency slightly altered fatty acid composition of total lipids and individual phospholipids both in brain and erythrocytes. The changes in fatty acids were more marked in erythrocytes than in brain, and mainly concerned phosphatidylcholine (PC). The fatty acid composition in total lipids of brain was hardly affected by nickel. In brain individual phospholipidsnickel deficiency slightly, but significantly elevated the proportions of polyunsaturated fatty acids (PUFA), and in the case of PC additionally increased the levels of monounsaturatedfatty acids (MUFA) at the extent of saturated fatty acids (SFA). Total lipids of erythrocytes from nickeldeprived rats had a significantly higher proportion of total MUFA than nickel-adequate rats, and a somewhat lower proportion of total (n-6) PUFA. Individual phospholipids in erythrocytes from nickel-deficient pups tended to have lower PUFA levels than nickel-adequate rats, and had additionally inconsistently altered levels of SFA and MUFA in PC and PE plasmalogen, respectively. Moreover, individual erythrocyte phospholipids from nickel-deficient rats tended to have lower 20:4/18:2 ratios than nickel-adequate rats. Inc. @@@t@19%llscviersCkme KEY WORDS: Nickel Deficiency, Brain, Erythrocyte, Phospholipids, Fatty Acids, Rat

*Reprint requests to: Prof. Dr. Dr. h.c. mult. M. Kirchgeasner, Institut fuer Emaehrungsphysiologie, Technische Universitaet Muenchen-Weihenstephan,85350 Freising, Germany

137

138

G.1.STANGLand M. KIRCHGESSNER

INTRODUCTION Nickel deficiency has been shown to cause general disturbances in lipid metabolism of animals [1,2,4,4,5,6,7]. Resulting from these experiments lipid level in serum and liver was altered due to nickel deficiency. Additionally, nickel-deprived rats had a decreased activity of glucose 6 phosphate dehydrogenase, a key enzyme in lipogene+s [8]. Moreover, Nielsen and coworkers [1] observed lower levels of the lipid-boun$$hosphoms lnnlck!l-defic~ent~h~cks and Pu~~arkateta~o [9] presented evidence that ATP and N1 are part of a reaction that converts phosphatidic acid to pyrophosphatidic acid, which is a precursor of phosphatidylserine biosynthesis by rat brain microsomes. Those results obtained from several experiments elucidate that nickel plays an important role in lipid metabolism, and particularly in regulation of lipid concentrations in tissues and synthesis of phospholipids. The ultrastructural changes noted during nickel deficiency suggests that nickel may play also a role in the structure and function of membranes [1,2], not at least, because nickel binds more strongly to membranes than calcium [10]. Due to those findings the present study was conducted in order to find out whether nickel is involved in synthesis of phospholipids, a structural component of all membranes, or may influence the fatty acid composition of the most important individual phospholipids. The target tissuea for these measurements were brain and erythrocyte membranes. In order to intensify nickel deficiency in the second generation, rat dams from the first generation were mated tsvo times in order to become successively pregnant, which might depress body nickel. All measurements were taken from the pups of the second offkpring, raised for a total of 7 weeks with the nickeldeficient or nickel-adequate diet. MATERIALSAND METHODS Animals and Diets In this experiment weaned female SPF Sprague-Dawley rats (WIGA GmbH, Sulzfeld, Germany) with an average body weight of 36 ? 0.6 g were fed two diets differing in nickel concentration. For that purpose the rats were divided into 2 groups of 12 rats each. The animals were fed a nickel-deficient diet containing 13 @kg or a nickel-adequatecontrol diet supplemented with 1 mg/kg nickel as NiSO “ 6H20 (analyzed nickel content 967 ,ug/kg). All the other components of the diet remaine#constant. All diets were fortified with recommended amounts of vitamins and minerals according to AIN diet [11]. The composition of the basal diet is given in Table 1. After 7 weeks rats were mated to produce the first offspring. Two weeks subsequentto the lactation period the dams were paired again to produce the second offspring. In order to intensify signs of nickel deprivation male rats of the second successive generation (24 animals per group) were raised for a total of 7 weeks with the nickeldeficient or nickel-adequate control diet. All measurements described in the present study were taken from the 7 weeks old pups from second offspring. The diet was prepared from natural feedstuff low in nickel. Ingredients with high nickel content were treated by the following procedures. Nickel in casein was removed with EDTA using a modified method of Davis et al. [12]. Nutritive fiber (cellulose) was acid washed by a method of Sunderman et al. [13]. All the other dietary ingredients were obtained in the purest known form in respect to nickel contamination and newly obtained ingredients were monitored for nickel concentration. The rats were individually housed in a controlled environment, in Macrolon cages in a room maintained at 24°C with a humidity of 60%. All animals were kept under conditions of controlled lighting with a daily 12 h-light/dark cycle, and had flee access to food and drinking water. At the end of the experimental period of 7 weeks pups from the second offspring were food deprived for 12 hours and killed by decapitation after a light anesthesia with diethyl ether. Blood for obtaining erythrocytes was collected into heparinized tubes, and brain was carefully excised.

NICKELDEFICIENCY AND PHOSPHOLIPIDS

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Lipid Extraction and Analysis Determination of the amounts of major phospholipidclasses was carried out as described by Eder et al. [14]. Erythrocytes were washed three times with physiological saline by centrifugation and resuspendation. Then erythrocytes were hemolyzed by adding distilled water and freezing. Erythrocyte ghosts were washed three times with tris buffer (11 mmol/L) and were then extracted with a hexane/isopropanolmixture (3:2, v/v, containing BHT as antioxidant).Also lipids from total brain were extracted with hexane/isopropanolmixture (3:2, v/v, containing BHT as antioxidant) as described by Hara and Radin [15]. The phospholipidclasses of the extractes were separated by high performance liquid chromatography,collected with a fraction collector (model 201, Gilson, Villiersle-Bel, France) and then methylated with boron fluoride/methanolreagent. Fatty acid methyl esters were separated using a capillary column gas chromatographicsystem (SICHROMAT 2-8, Siemens, Karlsruhe, Germany) fitted with a programmed temperature vaporizer (PTV), a CP-Sil 88 WCOT fused silica column (50 m “ 0.25 mm I.D., film thickness 0.2 ,um, Chrompack, Middleburg, The Netherlands), a flame ionization detector (FID) and an integrator (D-2500, Merck-Hitachi, Darrnstadt, Germany). Conditions of chromatographic separation were as described by Eder et al. [16]. Fatty acid methyl esters were identified using heptadecanoic acid methyl ester as internal standard. The amounts of individual phospholipid classes were calculated by the amount of ita bound fatty acids. Statistical Analysis The effect of nickel concentration in the diet was tested for statistical significance (pcO.05) by the Student’sttest. All data in the present text are expressed as means * standard error of means (SEM). TABLE 1 Compositionof the Basal Diet*

Ingredient Casein, fat fke# Corn starch Sucrose Fiber (cellulose)$ Soybean oil Coconut oil Vitamin mixture** Mineral mixture~ DL-methionine

glkg diet

200 328 300 30 50 30 20 40 2

*Diet analyz%dhad approximately 13pg nickel/kg. #Casein Wmremov~ of nickel by EDTA accordingto the method of Davis et al. [12]. ~~utritive fiber (cellulose) was acid washed according to the method of Sundermanet al. [13]. Vitamins per kg diet: 4000 IU all-trans retinol; 1000 IU cholecalciferol; 150 mg all-rac-cw tocopherol; 1.47 mg menadione sodium bisulfite; 5 mg thiamin ”HCL; 7.20 mg riboflavin; 6.00 mg pyridoxine “HCL; 15 mg Ca pantothenate; 30.0 mg nicotinic acid; 1.38 g choline chloride; 0.2 mg $#icacid;0.2mgD-biotin; 25mgcyanocobalamin; sucrose to20g. Minerals per kg diet: Na HP04“ 2 H20 3.15 g; KH2P04 3.82 g; KC14.77 g; MgC12“6 H20 4.24 g; CaC03 12.49 g; Fe3 04 “ 7 H O 174.22 mg; ZnSO -7 H O 131.9 mg; CUSO “7 H O 4&mg” o~3 Na2M00 mg ~a2~;~%12~$~ 23.6 mg; Mn~04 “5 H20 30.8 mg; K?0.26mg; NiS04” ; ~~ O ~. ‘g; 37.76Na2*013” mg; H3 032. 5H?i00”50mg; 6 mg; NaF 2.21 cK13”6w0’”61mg mg; (C 3COO)2Pb .3 H$O O.1 3 mg; “ su&ose to“4d“g.

140

G.1.STANGLand M. KIRCHGESSNER

RESULTS At the end of the experimental period after a total of 7 weeks nickeldeprived animals from the second offspring tended to have a lower body weight gain than nickel-adequatecontrol animals (184 t 19 vs. 197 f 32 g, p
Ni-deficient

Ni-adequate

Brain Phosphatidylserine Phosphatidylethanolamine(diacyl) Phosphatidylcholine

mg/g tissue 3.19 k 0.18 2.98 ? 0.13 4.49 ? 0.29 4.38 ? 0.31 7.70 k 0.42 7.16 * 0.35

Erythrocyte Phosphatidylethanolamine(diacyl) Phosphatidylethanolamine(plasmalogen) Phosphatidylcholine

mg/g membranes 0.17 t 0.00 0.19 * 0.00+ 0.20 t 0.01 0.21 t 0.02 1.33 t 0.05 1.45 t 0.05

*Data are means t SEM, n=24 for Ni-depletion group and n=23 for Ni-adequate group.xmeans were compared by Student’st test; ‘significantly different means (pcO.05). Brain was remarkable for high levels of polyunsaturatedfatty acids (PUFA), particularly (n3) PUFA in phosphatidylserine (PS) and PE. Fatty acid composition of total brain lipids and PC is shown in Table 3. Nickel deficiency had only slight effects on the proportions of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and PUFA of total lipids in brain. Rata fed a nickeldeficient diet tended to have higher total SFA proportions in brain than nickel-adequaterats, whereas total MUFA level did not differ between the groups. The proportionof PUFA in total lipids of brain was somewhat lowered in nickel-deprivedrats, and caused by a reduction of 22:4 (n-6) and 22:6 (n-3). Alterations in fatty acid composition of individual phospholipidswere strongest in brain PC. In brain PC, nickel-deficient rats had significantly lower total SFA levels and higher total MUFA proportions than nickel-adequate rats. This effect was mainly due an increase in 18:1 at the extent of 16:0. Nickel deficiency caused an elevation of (n-6) and (n-3) PUPA proportions,which was particularly due to a rise in 18:2 (n-6), 20:4 (n-6) and 22:6 (n-3). Fatty acid composition of brain PS and PE was demonstrated in Table 4. Total SFA and MUFA level in brain PS remained unchanged due to dietary nickel supply, whereas the proportionof total (n-6) PUFA in PS and PE of nickel-deprived rats tended to be higher than that of nickel-adequaterats. Table 5 shows the fatty acid composition of total lipids and PC in erythrocyte membranes. Erythrocyte lipids from nickel-deprived rats had a significantly higher MUFA level than erythrocyte

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141

lipids from nickel-adequate rats. This was mainly due to a rise in 18:1. The most striking elevations were found in individual PUPA levels. Total lipids in erythrocytes from nickeldeprived rats tended to have a lower proportion of PUFA than those from nickel-adequate rats, which was due to a TABLE 3 Proportionsof Saturated, Monounsaturatedand PolyunsaturatedFatty Acids in Total Lipids and phosphatidylcholine of Brain from 7 Week C)~X#s Fed a Nickeldeficient or Nickel-adequate

Fatty acid

Total lipids Ni-deficient Ui-adequate mol/loo

Phosphatidylcholine Ni-deficient Ni-adequate mol fatty acids

SFA 14:o 16Z O 18:O 20:o 22:o 24:O Total

0.31 * 20.28 * 19.20 * 0.56 * 0.53 * 0.73 * 41.61 *

0.03 0.11 0.08 0.01 0.01 0.02 0.15

0.35 * 20.02 * 19.19 * 0.54 * 0.51 * 0.68 * 41.30 *

0.02 0.07s 0.04 0.01 0.01+ 0.02s 0.10s

43.08 * 14.04 i 0.32 * 0.30 * 0.27 * 57.69 t

0.56 0.11 0.01 0.01 0.01 0.51

44.94 * 14.14 * 0.29 + 0.29 * 0.27 * 59.65 i

0.51+ 0.16 0.01S 0.02 0.02 0.46+

MUFA 16:1 18:1 20:1 22:1 24:1 Total

0.54 t 19.67 i 1.51 * 0.22 * 1.00 * 22.94 *

0.01 0.14 0.02 0.00 0.02 0.16

0.56 * 19.56 * 1.44 * 0.22 * 0.97 * 22.76 i

0.01 0.11 0.02+ 0.00 0.02 0.15

0.75 i 26.44 * 1.10 * 0.13 * 0.11 * 28.52 k

0.01 0.26 0.01 0.01 0.00 0.26

0.80 * 25.67 * 0.99 * 0.15 i 0.12 * 27.73 t

0.02+ 0.27+ 0.03+ 0.01 0.01 0.27+

1.17 * 0.56 i 0.60 i 8.87 i 5.74 i 0.65 * 17.38 f

0.02 0.01 0.01 0.06 0.08 0.02 0.08

1.14 * 0.54 * 0.60 * 8.67 * 6.15 * 0.56 & 17.45 *

0.02 0.01 0.01 0.03+ 0.05+ 0.02+ 0.06

1.40 * 0.55 * 0.32 * 5.36 * 0.69 * 0.21 * 8.53 *

0.05 0.03 0.01 0.13 0.02 0.01 0.18

1.27 * 0.53 i 0.30 * 4.90 * 0.62 * 0.19 * 7.81 *

0.03+ 0.03 0.01 0.12+ 0.02+ 0.01 0.16+

0.73 * 0.13 * 0.22 * 16.82 * 18.07 i 35.45 *

0.01 0.01 0.01 0.16 0.16 0.18

0.72 t 0.15 * 0.21 * 17.26 * 18.49 * 35.94 *

0.01 0.01 0.01s 0.20S 0.19S 0.19s

0.72 * 0.06 * 0.12 * 4.05 * 4.94 * 13.47 *

0.02 0.00 0.01 0.11 0.12 0.29

0.67 i 0.06 i 0.10 i 3.70 i 4.52 i 12.33 i

0.03 0.00 0.01 0.11+ 0.12+ 0.24+

1.82 * 7.61 + 23.12 * 13.61 + 23.61 *

0.02 0.13 0.49 0.24 0.50

1.82 * 7.66 * 24.18 * 14.12 i 24.68 f

0.01 0.14 0.55 0.26 0.56

2.03 i 3.89 t 5.72 * 4.76 * 5.97 *

0.03 0.11 0.18 0.12 0.18

2.16 * 3.91 * 5.75 * 4.80 * 5.99 *

0.04+ 0.11 0.27 0.13 0.28

PUFA (n-6) PUFA 18:2 20:2 20:3 20:4 22:4 22:5 Total (n-3) PUFA 18:3 20:5 22:5 22:6 Total Total PUFA SFA/MUFA 20:4/18:2 22:6/18:3 n-6D/18:2 n-3D/18:3

*Data are means ? SEM, n=24for Ni-depletion group and n=23 for Ni-adequate group.Fmeans were compared by Student’s ttest; ‘significantly different means (p
G.1.STANGLand M. KIRCHGESSNER

142

significant reduction in 20:4 (n-6) and 22:5 (n-3) level. The most marked alterations in fatty acids occurred in PC. Total SFA level in erythrocyte PC of nickeldeprived rats was somewhat higher than that of nickel-adequate rats, and caused by a significant rise of 14:0, 16:0 and 18:0. MUFA levels in PC remained unchanged by dietary nickel supply. The most marked variations also occurred within the PUFA group. Both the proportions of (n-6) and (n-3) PUFA were somewhat TABLE 4 Proportions of Saturated, Monounsaturatedand PolyunsaturatedFatty Acids in Phosphatidylethanolamineand Phosphatidylserineof Brain fro~ Nicke’deficient or Nickel-adequate Diet J~weekO1dRasFeda Fatty acid

Phosphatidylethanolamine Ni-deficient Ni-adequate

Phosphatidylserine Ni-deficient Ni-adequate

mol/100 mol fatty acids SFA 16:O 18:O 20:o 22:o 24:O Total MUFA 16:1 18:1 20:1 24:1 Total

9.74 i 32.45 * 0.37 + 0.26 +

0.14 0.25 0.01 0.01

9.94 * 32.56 * 0.36 + 0.26 *

0.20 0.41 0.02 0.02

42.82 + 0.31

43.12 f 0.50

0.46 * 0.02 14.94 * 0.14 1.42 i O.02

0.49 i 0.03 15.14 * 0.09 1.32 t 0.03+

16.81 .tO.16

16.95 + 0.11

0.72 i 0.02 0.48 i 0.05

0.75 * 0.03 0.43 + 0.03

3.33 * 39.61 + 0.84 i 1.17 * 1.66 * 46.61 *

0.19 0.42 0.06 0.09 0.07 0.50

3.27 + 39.83 i 0.77 * 1.04 + 1.63 t 46.54 i

0.15 0.51 0.04 0.05 0.12 0.55

0.63 i 17.93 * 1.24 * 0.56 * 20.36 +

0.06 0.30 0.04 0.08 0.34

0.75 i 18.45 i 1.26 i 0.51 i 20.96 +

0.07 0.21 0.05+ 0.06 0.27

0.49 * 0.48 f 0.88 * 4.48 * 2.98 + 1.41 i 10.71 *

0.03 0.05 0.04 0.15 0.05 0.06 0.14

0.42 * 0.40 i 0.90 * 4.46 f 2.72 i 1.29 + 10.19 *

0.02+ 0.03 0.07 0.11 0.05+ 0.05 0.13+

0.03 0.02 0.04 0.35 0.36 0.46

0.34 * 0.50 * 0.27 * 21.20 * 22.31 i 32.50 *

0.02+ 0.04 0.02~ 0.42 0.42 0.50

0.05 0.69 2.78 1.22 2.88

2.24 * 11.10 * 67.38 i 23.37 f 69.82 *

0.05 0.53 4.31+ 1.08 4.39+

PUFA n-6 18:2 n-6 20:2 n-6 20:3 n-6 20:4 n-6 22:4 n-6 22:5 n-6 Total n-6 n-3 18:3 n-3 20:5 n-3 22:5 n-3 22:6 n-3 Total n-3 Total

0.26 20.21 20.95 40.36

i * * *

0.06 0.25 0.26 0.32

0.26 * 20.24 * 20.96 f 39.93 *

0.07 0.32 0.32 0.53

0.42 * 0.51 t 0.34 * 21.05 * 22.32 * 33.03 *

SFA/MUFA 20:4/18:2 22:6/18:3 n-6D/18:2 n-3D/18:3

2.55 i 18.35 * 43.25 t 25.75 i 43.81 t

0.03 0.52 0.96 0.69 0.97

2.54 * 17.48 * 45.51 * 24.31 + 46.08 *

0.03 0.69 1.97 0.98 2.00

2.31 i 9.95 * 53.84 * 21.44 + 56.02 *

12.99 * 3.63 i 1.05 * 19.42 +

0.20 0.05 0.03 0.19

0.47 * 0.01

12.80 + 3.47 * 0.95 * 18.97 *

0.25 0.06+ 0.03+ 0.31

0.46 * 0.02

*Data are means f SEM, n=24 for Ni-depletion group and n=23 for Ni-adequate group.#means were compared by Student’s ttest; ‘significantly different means (p
NICKELDEFICIENCY AND PHOSPHOLIPIDS

143

reduced in erythrocytes of rats fed a nickel-deficient diet relative to rats with an adequate nickel supply. This reduction mainly concerned 20:4 (n-6), 22:5 (n-3) and 22:6 (n-3). The fatty acid composition of PE diacyl in the erythrocyte membrane was hardly influenced by dietary nickel (Table 6). PE plasmalogen which was remarkable for the high 20:4 (n-6) level had a significantly increased proportion of 18:1 and reduced levels of individual long-chainPUPA in consequenceof a nickel depletion. Moreover, erythrocytes born nickel-deficient rats had slightly reduced 20:4/18:2 ratios compared to erythrocytes from nickel-adequate rats. TABLE 5 Proportionsof Saturated, Monounsaturatedand PolyunsaturatedFatty Acids in Total Lipids and Phosphatidylcholineof Erythrocytes from 7 Week 01 Rats Fed a Nickel-deficientor Nickeladequate Diet*$ > Fatty acid

Total Lipids Ni-deficient Ni-adequate

Phosphatidylcholine Ni-deficient Ni-adequate

mol/100 mol fatty acids SFA 14:o 16:O 18:O 24:O Total

2.14 * 42.97 i 10.75 i 1.57 * 57.43 *

0.13 0.62 0.18 0.11 0.68

2.20 * 41.51 i 11.23 i 1.32 * 56.26 *

0.12 0.95 0.24 0.07S 0.99

MUFA 14:1 16:1 18:1 24:1 Total

1.43 i 14.36 t 1.52 + 17.32 i

0.06 0.27 0.09 0.30

1.39 i 13.58 * 1.35 * 16.32 i

0.07 0.30S 0.08 0.37+

8.58 * 0.48 + 0.50 * 8.29 f 3.13 t 1.10 * 22.08 +

0.33 0.02 0.04 0.55 0.24 0.05 0.71

8.72 * 0.50 * 0.55 * 10.65 * 2.82 * 1.17 i 24.40 *

0.73 * 2.44 * 3.17 * 25.25 t

0.05 0.21 0.20 0.74

0.94 * 2.07 * 3.02 t 27.42 k

PUFA n-6 18:2 n-6 20:2 n-6 20:3 n-6 20:4 n-6 22:4 n-6 22:5 n-6 Total n-6 n-3 22:5 n-3 22:6 n-3 Total n-3 Total SFA/MUFA 20:4/1s:2 n-6D/18:2

3.34 * 0.07 0.98 * 0.07 1.56 i 0.08

1.72 i 0.06 57.14 * 0.55 11.13 i 0.16

1.40 * 0.05+ 53.42 + 1.08+ 12.20 i 0.27+

69.99 + 0.55

67.01 + 0.98S

0.75 * 0.03 1.10 i 0.03 11.22 + 0.26

0.75 * 0.03 0.98 * 0.04S 11.30 * 0.21

13.07 i 0.27

13.02 f 0.24

0.25 0.02 0.05 1.05+ 0.25 0.06 1.16S

10.74 * 0.40 + 0.43 * 4.54 * 0.19 * 0.12 * 16.41 t

0.36 0.02 0.03 0.34 0.01 0.01 0.62

11.20 * 0.48 + 0.41 * 6.63 + 0.21 * 0.16 * 19.09 +

0.25 O.O1+ 0.03 0.65+ 0.01 0.02 0.87S

0.08+ 0.15 0.17 1.22

0.11 * 0.43 * 0.54 i 16.94 f

0.01 0.04 0.05 0.66

0.17 * 0.71 * 0.88 * 19.97 *

0.02+ 0.11s 0.12s 0.98S

3.47 * 0.07 1.21 i 0.11s 1.75 * 0.12

5.40 * 0.11 0.42 t 0.03 0.49 * 0.03

5.19 * 0.14 0.58 * 0.05S 0.65 + 0.05S

*Data are means * SEM, n=24 for Ni-depletion group and n=23 for Ni-adequate group.#means were compared by Student’s t test; ‘significantly different means (pcO.05); %omewhat different means (p
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144

DISCUSSION Thisstudy has shown that nickel deficiency influences fatty acid compositionof total lipids and individual phospholipids both in brain and erythrocytes. Moreover, the present results revealed that brain lipids were more resistant to dietary nickel supply than erythrocyte lipids, regarding the fatty acid composition. This could be also found in several other studies with rats [18,19,20] and piglets [21] showing that dietary modifications can alter fatty acid pattern of several tissues, but not of brain. It appears that brain endeavors to minimize fatty acid changes. Therefore, brain may be capable of buffering fatty acid changes by minimizing or promoting the transport of fatty acids across the blood-brain barrier [22]. This apparent regulation of fatty acids in the rat brain may potentially be of important functional significance for enzyme activities, cellular response, ion transport and membrane electrophysiology.

The present findings demonstrate that both in brain and erythrocytes the most alterations in fatty acid composition occurred in PC, which correspond with results from other studies showing TABLE 6 Proportions of Saturated, Monounsaturatedand PolyunsaturatedFatty Acids in Phosphatidylethanolamine(Diacyl) and Phosphatidylethanolamine(Plasmalogen)~f~rythroeytes from 7 Week Old Rats Fed a Nickeldeficient or Nickel-adequateDiet ~ Fatty acid

Phosphatidylethanolsmine diacyl Ni-deficient Ni-adequate

Phosphatidylethanolamine plasmalogen Ni-deficient Ni-adequate

mol/100 mol fatty acids SFA 16:O 18:o Total

26.79 i 0.80 14.83 * 0.48 41.62 + 1.22

24.81 * 1.16 14.12 t 0.47 38.93 * 1.52

23.26 t 1.33 13.41 * 0.99 36.68 i 2.21

MUFA 18:1 Total

20.99 * 0.36 20.99 + 0.36

21.50 * 0.28 21.50 * 0.28

8.32 * 0.38 8.32 * 0.38

11.12 i 19.85 * 2.17 i 1.20 i 34.35 *

0.32 0.91 0.10 0.09 1.10

10.32 * 22.50 f 2.26 * 1.04 * 36.12 *

0.19 1.32 0.19 0.11 1.36

2.41 36.08 7.08

1.28 * 1.76 i 3.04 * 37.39 i

0.11 0.14 0.21 1.18

1.37 * 2.08 * 3.45 * 39.57 *

0.11 0.23 0.30 1.60

PUFA n-6 18:2 n-6 20:4 n-6 22:4 n-6 22:5 n-6 Total n-6 n-3 22:5 n-3 22:6 n-3 Total n-3 Total SFA/MUFA 20:4/18:2 n-6D/18:2

2.00 * 0.08 1.80 * 0.08 2.11 * 0.09

1.81 * 0.07 2.19 * 0.135 2.51 * 0.145

18.84 i 1.49 11.22 + 0.98 30.05 i 2.42 6.19 i 0.40+ 6.19 i 0.40+

1.47 i 0.11 47.04 * 2.07

2.25 i 40.61 i 8.41 i 2.19 + 53.46 i

0.20 1.93 0.36S 0.15+ 2.09

4.79 t 3.17 * 7.97 * 55.00 *

5.92 * 4.38 k 10.30 i 63.76 i

0.40 0.30+ 0.64S 2.63

* 0.20 * 1.76 k 0.31

0.29 0.20 0.46 2.47

4.45 * 0.22 17.12 * 1.35 21.21 * 1.67

4.93 -i0.41 21.24 * 2.21 26.88 k 2.74

*Data are means t SEM, n=24 for Ni-depletion group and n=23 for Ni-ade uategroup.smeans # different were compared by Student’s ttest; ‘significantly different means @
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that PC reacts stronger to various dietary influence than other individual phospholipids [23,24,25]. Several fatty acid alterations occurred within the group of PUPA. Nickel deficiency particularly reduced long-chain PUPA levels and the 20:4/18:2 ratios in erythrocytes,which were indicativeof a depressed desaturation of essential fatty acids. The same alterations could be observed in the individual hepatic phospholipids of nickel-deficient rats [26]. Long-chain PUPA are synthesized from essential fatty acids by the action of A-6 desaturase, an iron-containingenzyme [27]. Earlier studica with rats clearly demonstrated that nickel deficiency affects iron status by impairing iron absorption from the intestine [1,28]. In the present study, nickel-deficient rats also had a lower iron status than nickel-adequate control rats, as indicated by a somewhat reduced concentrationof iron in serum and significantly reduced hematological variables, including erythrocyte count, hemoglobin concentration and hematocrit [17]. The same variables also have been shown to be affected by iron deficiency [29]. Since iron-deficiency also has been shown to cause a reduction in desaturation products [30,31,32,33], it is therefore possible that some of the observed alterations in fatty acid composition in nickeldeficient rats are due to the reduced iron status in those rats. However, it is uncertain, whether these slight changes in fatty acid composition influence regulation of cell function, by altering physiochemical characteristic like microviscosity or fluidity of the membrane lipid matrix which in turn can influence the conformation,mobility and function of a wide variety of intrinsic and extrinsic membrane-bound proteins [34,35]. However, nickel deficiency aIso led to some changes in fatty acid profile e.g. an elevation of MUFA proportions, which were not indicative of a reduced fatty acid desaturation. Therefore, it was assumed that nickel itself has any specific function in metabolism of fatty acids. Since nickel is involved in structure and function of membranes [1,2] it is also possible that membranes compensate a lack of nickel by altering the fatty acid composition. Although, nickel has been shown to be involved in the in vitro synthesis of brain PS [9], nickel deficiency did not have any influence on the concentrations of individual phospholipids including PS, PC and PE in brain. Therefore, it was assumed that either reduced nickel concentrations in body of nickel-deficient rats are still sufficient to synthesize PS, or Ni could be in vivo replaced by other elements, in this respect. In contrast, the concentration of PE diacyl in erythrocytes from nickel-deficient rats was reduced by 11% compared with the control level, which supports the finding of Nielsen [36], showing that phospholipids, an integral part of membranes, were depressed by nickel deficiency. REFERENCES 1.

Nielsen FH, Myron DR, Givand SH, Ollerich DA. Nickel deficiency and nickel-rhodium interactions in chicks. J NutT1975a; 105:1607-19.

2. Nielsen FH, Myron DR, Givand SH, Zimmerman TJ, Ollerich DA. Nickel deficiency in rats. J Nutr 1975b; 105:1620-30.

. 3.

Kirchgessner M, Schnegg A 1981Biochemical and physiologicaleffects of Nideficiency. In: Nriagu JO Nickel in the environment, New York: John Wiley, pp 635-52.

4. Spears JW, Hatfield EE. 1980Role of nickel in the ruminant nutrition. In: Anke M, Schneider HJ, Briickner C 3rd Spurenelement-Symposium, Nickel, Vol. 3, Friedrich Schiller Univ, Jena, pp 47-53.

5. Spears JW, Hatfield EE, Fahey GC Jr. Nickel depletion in the growing ovine. Nutr Rep Int 1978; 18:621-9.

6. Anke M, Griin M, Kronemann H 1980aThe capacity of different organs to indicate the nickel level. In: Anke M, Schneider HJ, Bruckner C %d Spurenelement-Symposium,Nickel. Vol. 3, Friedrich Schiller Univ., Jena, pp 237-44.

146

7.

G.L STANGLand M. KIRCHGESSNER Anke M, Kronemann H Groppel B, Hennig ~ Meissner D, Schneider HJ 1980bThe influence of nickeldeficiency on growth, reproduction,longevity and different biological parameters of goats. In: Anke ~ Schneider HJ, Briickner C 3rd Spurenelement-Symposium, Nickel. Vol. 3, Friedrich Schiller Univ., Jena, pp 3-10.

8. Kirchgessner M, Schnegg A. Malate dehydrogenaseand gIucose-6-phosphatedehydrogenase activity in livers of Ni-deficient rats. Bioinorg Chem 1976;6:155-61.

9. Pullarkat R& Sbaschnig-AglerM, Reha H. Biosynthesis of phosphatidylserinein rat brain microsomes. Biochim Biophys Acta 1981;663:117-23. 10. Hagiwara S, Takashashie K. Surface density of calcium ions and calcium spikes in the barnacle muscle fiber membrane. J Gen Physiol 1%7; 50:583-601. 11.

Reeves PG, Nielsen FH, Fahey GC Jr. AIN-93 purified diets for laboratoryrodents: final report of the American Institute of Nutrition ad hoc writing Committee on the reformulation of the AIN-76A rodent diet. J Nutr 1993; 123:1939-51.

12.

Davis PN, Norris LC, Kratzer FH. Iron deficiency studies in chick using treated isolated soybean protein diets. J Nutr 1%2; 78:445-53.

13.

Sunderman FW Jr, Nomoto S, Morang R, Nechay MW, Burke CN, Nielsen SW. Nickel deprivation in chicks. J Nutr 1972; 102:259-68.

14.

Elder~ Reichlmayr-Lais AM, KirchgeasnerM. Simultaneousdeterminationof amounts of major phospholipid classes and their fatty acid composition using high-performanceliquid chromatographyand gas chromatography.J Chromatogr 1992; 598:33-42.

15.

Hara ~ Radin SN. Lipid extraction of tissues with a low-toxicitysolvent. Anal Biochem 1978;90:420-6.

16. Eder ~ Reichlmayr-Lais AM, Kirchgessner M. Gas chromatographicanalysis of fatty acid methyl esters: avoiding discrimination by programmedtemperaturevaporizing injection.J Chromatogr 1991; 588:265-72. 17.

Stangl GI, Kirchgessner M. Effect of nickel deficiency on various metabolic parametemof rats. J Anim Physiol a Anim Nutr 1996 (in press).

18.

Witting LA Harvey CC, Century B, Horwitt MK. Dietary alterations of fatty acids of erythrocytes and mitochondria of brain and liver. J Lipid Res 1961;2:412-8.

19. Gibson RAj McMurchie IN, Charnock JS, Kneebone GM. Homeostaticcxmtrolof membrane fatty acid composition in the rat after dietary lipid treatment. Lipids 1984; 19:942-51. 20.

Reichlmayr-Lais AM, Stangl GI, KirchgessnerM, Eder K. Fatty acid compositionof brain and heart of rats fed various dietary oils. Nutr Res 1994; 16:829-40.

21.

Hrboticky N, Mackinnon MJ, Innis SM. Effect of a vegetable oil formula rich in Iinoleicacid on tissue fatty acid accretion in the brain, liver, plasma, and erythrocytesof infant piglets. Am J Clin Nutr 1990; 51:173-82.

22.

Chaudiere J, C16mentM, Driss F, BoumeJM. Unaltered brain membran= after prolonged intake of highly oxidizable long-chain fatty acids of the (n-3) series. Neurosci Lett 1987; 82:233-9.

23< Renooij W, Van Golde LMG,Zwaal RF~ Van Deenen LLM. Topological asymmetryof phospholipidmetabolism in rat erythrocyte membranes. Evidence for flip-flop of lecithin. Eur J Biochem 1976; 61:53-8.

NICKELDEFICIENCY AND PHOSPHOLIPIDS

147

24.

Eder ~ Kirchgessner M. Levels of polyunsaturatedfatty acids in tissues from zincdeficient rats fed a linseed oil diet. Lipids 1994; 29:839-44.

25.

Eder ~ Kirchgessner M. Activities of liver microsomal fatty acid desaturasea in zincdeficient rats force-fed diets with a coconut oil/safflower oil mixture or linseed oil. Biol Trace Elem Res 1995; 48:215-29.

26.

Stangl GI, Kirchgessner M. Nickel deficiency alters liver lipid metabolism in rats. J Nutr 1996 (in press).

27.

Okayasu T, Nagao M, Ishibashi T, Irnai Y. Purification and partial characterizationof linoleoyl COAdesaturase from rat liver microsomes.Arch Biochem Biophys 1981;206:21-42.

28.

Schnegg ~ Kirchgessner M. Zur Absorption und Verfiigbarkeitvon Eisen bei NickelMangel. Int Z Vit Emahrungsforsch 1976;46:96-9.

29.

Lee YH, Layman D& Bell RR. Glutathione peroxidase activity in iron-deficientrats. J Nutr 1981; 111:194-200.

30.

Cunnane SC, McAdoo KR. Iron intake influences essential fatty acid and lipid compositionof rat plasma and erythrocytes. J Nutr 1978; 117:1514-9.

31.

Eder ~ Kirchgessner M, Kralik A. The effect of trace element deficiency (iron, copper, zinc, manganese and selenium) on hepatic fatty acid composition in the rat. Trace Elem a Electrol 1996; 13:1-7.

32.

Johnson SB, Kramer TR, Briske-AndersonM, Holman RT. Fatty acid pattern of tissue phospholipids in copper and iron deficiencies. Lipids 1989; 24:141-5.

33.

Sherman AR, Bartholmey SJ, Perkins EG. Fatty acid patterns in iron-deficientmaternal and neonatal rats. Lipids 1982; 17:639-43.

34.

Stubbs CD, Smith AD. The modification of mammalian membrane polyunsaturatedfatty acid composition in relation to membrane fluidity and function. Biochim Biophys Acta 1984; 779:89-137.

35.

Brenner RR. Effect of unsaturated acids on membrane structure and enzyme kinetics. Prog Lipid Res 1984; 23:69-96.

36.

Nielsen FH 1974 E.ssentiallvand function of nickel. In: Hoekstra WG. Suttie JW. Ganther HE Mertz W Trace elemen~metabolism in animals -2, Baltimore: University Park Press, pp 381-95.

Accepted for publicationAugust 5, 1996.