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Most biological effects of zinc deficiency corrected by γ-linolenic acid (18: 3ω6) but not by linoleic acid (18: 2ω6)
193
Atherosclerosis, 41 (1982) 193-207 0 Elsevier/North-Holland Scientific
Publishers,
Ltd.
MOST BIOLOGICAL EFFECTS OF ZINC DEFICIENCY CORRECTED BY y-LINOLENIC ACID (18 : 3~6) BUT NOT BY LINOLEIC ACID (18 : 2~6) Y.S.
HUANG,
Clinical Quebec (Received (Revised, (Accepted
S.C. CUNNANE
*, D.F. HORROBIN
Research Institute of Montreal, HZW lR7 (Canada)
and J. DAVIGNON
110 Pine Avenue,
West, Montreal,
25 March, 1981) received 8 June, 1981) 9 June, 1981)
--.
---.
-
Summary Zinc deficiency mimics many of the clinical features of essential fatty acid (EFA) deficiency in rats. Since zinc appears to be needed for the A-6-desaturase step in EFA metabolism, experiments were conducted to determine whether bypassing this step with y-linolenic acid (18 : 3~6) would alleviate the biological effects induced by a zinc-deficient diet. In pair-fed rats over a period of 5 weeks the deficient diet impaired growth and changed the relative weights of internal organs. It also induced hypolipidemia but had little effect on the fatty acid composition of tissue lipids. Daily subcutaneous injection of primrose oil containing 10% 18 : 306 reversed most of the effects of zinc deficiency on tissue weights, growth and plasma lipids. In contrast, injection of safflower oil, which has a similar content of linoleic acid (18 : 2~6) but is devoid of 18 : 306, had only a partial effect on some tissue weight changes. Neither oil affected the plasma fatty acid pattern, but both of them increased liver triglyceride concentrations. They also elevated the proportion of 18 : 2~6 in liver, skin and epididymal fat. The latter effects were not observed in the absence of zinc deficiency. Supplementing the diet of EFA-deficient animals with an excess of zinc in their drinking water did not affect the typical tissue fatty-acid pattern of EFA deficiency. It is suggested that several of the manifestations of zinc deficiency are mediated by a relative state of EFA deficiency attributable, at least in part, to a reduced conversion of 18 : 2~6 to 18 : 3~6 resulting in an accumulation of 18 : 206 in tissues. These findings are consistent with a role of zinc as a cofactor in the ASdesaturase enzyme reaction. The suggested role of zinc in essential fatty This manuscript was taken in part from a dissertation submitted to the Graduate School of McGill University by Stephen Cunnane in partial fulfillment of the requirements for the degree of Doctor of Philosophy, and was supported by grants from the Medical Research Council of Canada and the Quebec Heart Foundation. * Research fellow of Le Cons&l de la Recherche en SantC du QuBbec.
OOZl-9150/82/0000-0000/$02.75
@ 1982
Elsevier/North-Holland
Scientific
Publishers,
Ltd.
194
acid metabolism may be of significance in understanding acids protect against cardiovascular disease in general. Key words:
Essential ciency
fatty
acid deficiency
-
Linoleic -
acid -
how essential
y-Linolenic
acid -Zinc
fatty
defi-
_--__
Introduction The evidence for an interaction between zinc and essential fatty acid (EFA) metabolism has been recently reviewed [l]. The main points are: (1) many of the features of zinc and EFA deficiency are similar, (2) the features of either zinc or EFA deficiency are exaggerated when the diet is also deficient in the other nutrient, (3) treatment of pregnant rats with drugs which block EFA conversion to prostaglandins (PCs) produces changes that are remarkably similar to those brought about by zinc deficiency in pregnancy [2], (4) treatment of zincdeficient rats [3] and humans [4] with vegetable oils rich in EFAs attenuates the effects of zinc deficiency on growth and other parameters. In previous studies [3,5,6] we have shown that primrose oil is able to prevent the development of some features of zinc deficiency. When primrose oil and safflower oil were compared in male rats, the former was considerably more effective [3,5]. The total EFA content of these two oils is almost identical (Table l), but whereas the safflower oil contains 83% linoleic acid (18 : 2w6), the primrose oil contains 74% 18 : 206 and 10% y-linolenic acid (18 : 306). The latter is formed from 18 : 206 by the A-6-desaturase enzyme system [ 7,8]. Studies in cats, which lack this desaturase, have demonstrated that 18 : 2~6 is largely inert as an EFA when this transformation cannot take place [9,10], and therefore cats must rely on a dietary source of 18 : 3~6 for prostaglandin synthesis. Since there is evidence that zinc could be needed as a cofactor in the A8desaturase enzyme activity [ll], experiments were conducted to determine whether bypassing this step in EFA metabolism would alleviate the biological effects induced by a zinc-deficient diet. We took advantage of the difference in 18 : 3~6 content between the two oils with similar 18 : 206 content, namely primrose and safflower, and report here their effects in pair-fed zincdeficient rats on growth, organ weight, liver and plasma lipid concentrations as well as on the total fatty acid pattern of plasma, liver and other organs. These studies were considered to be of importance in view of a possible involvement of zinc deficiency in the pathogenesis of atherosclerosis [ 12-151. Zinc therapy has been used to treat some of the symptoms of obliterative vascular disease and cardiac insufficiency [12,16]. An increase in the ratio of zinc to copper in the diet has been suggested to be related to hypercholesterolemia in humans [17]. Increased essential fatty acid intake in humans significantly lowers plasma low and very low density lipoproteins, decreases the thrombogenie properties of blood platelets as well as the susceptibility to sodiuminduced hypertension ,[ 181. The intake of 18 : 206 has also been shown in epidemiological studies to be negatively correlated with blood pressure and serum sodium concentrations in man [19]. Thus, a study of the possible effects of
195
zinc deficiency on lipid metabolism in general and essential fatty acid metabolism in particular is relevant to furthering our understanding of the pathogenesis of atherosclerosis and heart disease in general. Material and Methods Zinc deficiency
experiment
The purpose of this experiment was to compare the effect of linoleic acid in the presence (primrose oil) and absence (safflower oil) of y-linolenic acid on the biological effects of zinc deficiency. Weanling male Wistar rats weighing about 50 g were separated into 4 groups of 7 rats and maintained on a zinc-deficient diet (containing less than 3 ppm zinc, other constituents of diet are detailed in Table 1) obtained from Ziegler Bros., Gardners, PA, over a period of 5 weeks and deionized water ad libitum. The first group (ZD) received this diet alone. For the second group (Z) the deionized water was supplemented with zinc (10 pg/ml of zinc as ZnS04 * 7 HzO, Baker). The rats of the third group (ZDP) received 250 pi/day of primrose oil (Agricultural Holdings, London, England) by subcutaneous injection. The last group (ZDS) was given the same amount of commercial safflower oil by the same route. Both oils contained less than 2 ppm-zinc, so that the oilsupplemented rats received less than 20 pg-zinc from oils for the duration of the experiment. The pair-feeding was done on all treated rats by giving an amount of food equal to the average daily intake of the untreated zinc-deficient rats (ZD). The rats were killed by decapitation after 5 weeks and blood was collected from the neck vessels. Heart, lungs, thymus, liver, spleen, left kidney, adrenal, testis and epididymal fat pad were excised, rinsed with cold saline, blotted and weighed. Liver, epididymal fat pad and a piece of skin removed from the top of the hind paw were frozen on dry ice and stored for subsequent lipid analysis. The fatty acid composition of the diet and oils is given in Table 1. TABLE
1
PERCENTAGE
FATTY
ACID
COMPOSITION
OF
ZINC-DEFICIENT
DIET
AND
SUPPLEMENTED
OILS
Fatty
acid
Zinc-deficient
diet
a
Oils Primrose
14
:O
16
:O
16
:l
18
10
18
:l
18
: 2~3 : 3w3 : 3~6
60.9
18 18
a Zinc corn
0.2
deficient
and Tomarelli)
Safflower
0.1
12.6
starch,
oil
0.2
5.7
5.0
0.1
-
0.1
1.5
0.9
1.3
23.3
8.8
9.3
74.2
82.7
10.3
-
1.2
1.4
-
diet 10%
(Ziegler
corn
and
0.3%
oil
oil.
Bros., 3%
choline
Gardner,
cellulose chloride.
PA)
powder,
contained 0.5%
vitamin
20% mix
egg
white
(Murphy).
solids. 4%
31% mineral
sucrose. mix
31.2%
(Bernhart
196
Primrose and safflower
oil supplementation
in normal rats
This study was carried out after the completion of the above experiment to determine whether the accumulation of linoleic acid following primrose oil and safflower oil injections noted for the zinc-deficient rats could also be produced in normal rats. Three groups of six 50-g weanling male Wistar rats were fed Purina laboratory rat chow plus tap water ad libitum for 4 weeks. The first group (C) served as control, the second group (CP) received 250 /.d/day of primrose oil in a single subcutaneous injection and the third group (CS) received the same daily amount of safflower oil. The rats were killed and blood, liver and epididymal fat pad were obtained, as in the previous experiment, for lipid studies. Zinc supplementation
in essential fatty acid deficiency
The purpose of this experiment was to determine whether an excess of dietary zinc could modify the abnormal fatty acid pattern induced by EFA deficiency. Weanling male Wistar rats were divided into 3 groups of 6 animals and fed for 10 weeks, either a Purina rat chow plus tap water ad libitum (C), or an EFA-deficient diet (Teklad test diets, Madison, WI). The latter were fed either with deionized drinking water given ad libitum (group ED) or with deionized water containing a supplement of zinc (10 pg/ml of zinc as ZnS04 * 7 HzO) (group EDZ). The rats were killed and blood, liver, skin and epididymal fat pad were removed for lipid analyses as in the first experiment. The EFA-deficient diet consisted of 20% casein, 68.5% sucrose, 5% medium chain triglycerides (66% of fatty acids were capric and 33% lauric acids), 2% fiber, 3.5% minerals, and 1% vitamin mix. Both control and EFA-deficient diets contained an adequate amount of zinc (~40 ppm, as determined by our analysis). For all 3 experiments the rats were housed in pairs in stainless steel-topped plastic cages to avoid contamination from the zinc that is present in galvanized steel cages. A wire rack was placed at the bottom of the cages over and above woodchips used to absorb moisture so that coprophagy did not occur. Lipid analyses Fatty acid analyses were applied to samples of plasma, liver, epididymal fat and skin. Trunk blood was collected into test tubes containing 1 mg EDTA per ml blood and centrifuged at 4°C to separate the plasma. Aliquots of plasma and samples of liver and epididymal fat were homogenized and extracted with 20 volumes of chloroform--methanol (2 : 1, v/v) by the method of Folch et al. [ 201. The concentration of cholesterol [21] and triglycerides [22] in liver was assayed with a Technicon Autoanalyzer. Plasma cholesterol and triglycerides were measured enzymatically with an Abbott Bichromatic Analyzer (ABA100). Methyl esters of fatty acids (FAME) were prepared directly from the total lipid extract by BF,-methanolysis [23]. Samples of skin were cut into small pieces and saponified with alkaline methanol. After acidification, fatty acids were extracted into hexane and methylated with BF,-methanol.
197
FAME were analyzed by gas-liquid chromatography on an F&M model 402 apparatus at 185°C using a glass column (6 ft X 4 mm ID) packed with 10% silar-lOC on loo-120 mesh Gas-Chrom Q (Applied Science Laboratories, State College, PA). The percentage of each fatty acid was assessed with a HewlettPackard automatic integrator (Model 3373B). Zinc analyses
Analysis of the zinc content of plasma, adrenal tissue and liver tissue samples and of various diets and oil supplementations was performed by atomic absorption spectrophotometry, using a Perkin-Elmer 403 [ 51. The dried and weighed tissue samples were wet digested in 1 ml concentrated HN03 for 48 h. An aliquot of the tissue-acid solution was then diluted 1 : 10 with deionized water and analyzed. Plasma zinc was determined directly following acidification and a 1 : 10 dilution with deionized water. The oils were analyzed for zinc by dissolving them in iso-amylacetate-methanol (1 : 1) and measuring them directly with appropriate reference standards and recovery assessment. Using the methods described above, recoveries of known amounts of zinc added to solutions of digested liver or adrenal tissue samples varied from 94 to 102% and those of plasma ranged between 98 and 102%. Statistics
The differences between all possible pairs of means of groups were compared with the Student-Newman-Keuls multiple range test [ 241. Results Zinc deficiency
study
At the end of the experiment the zinc-deficient (ZD) animals exhibited retarded growth, hunched posture, keratosis of the paws, the tail, and the nasal, genital and eye regions, alopecia, and bleeding from the paws and nose. Animals treated with safflower oil (ZDS) were almost identical clinically except for a minor improvement of the skin. Animals treated with primrose oil (ZDP) appeared considerably healthier and showed only partial growth retardation. The zinc-supplemented rats (Z) appeared normal in all respects and served as the control group. Plasma zinc levels (Table 2) were significantly depressed in all ZD groups as compared to group Z (P < 0.05). However, neither adrenal, nor liver zinc content were significantly altered by the ZD conditions nor by the oil treatment. The body weight changes are shown in Fig. 1. The relative weights of the various organs, expressed per 100 g body weight, are given in Table 2. When expressed in this way, the heart, lung, kidney, and testis of the ZD animals all showed a moderate increase in weight although in absolute terms these organs were smaller because of the overall size of the animals. The relative hypertrophy of the adrenal was more striking (2.4 times the control weight). In contrast there was a marked atrophy of the thymus (a 75% reduction in weight) and of the epididymal fat pad (a 55% reduction). Liver weight was unaffected.
198 TABLE
2
EFFECT
OF
WEIGHTS TION
ZINC
(Z).
(g/lOOg
OF
PRIMROSE
body
ZINC-DEFICIENT
weight
OF
(ZD)
Tissue Body
OIL
weights)
(g)
(P)
OR
SAFFLOWER
VARIOUS
OIL
TISSUES
AND
(S) SUPPLEMENTATION ON
PLASMA
ZINC
ON
RATS
ZD
Z
59
163
ZDS
ZDP 104
a
Liver
3.10
3.43
Heart
0.44
0.36
Lung
0.81
0.54
a.b
71 b.c
4.29
a.b
3.02
a
0.37
a
0.38
c a
a
0.63
a
0.77
b a.b
0.60
0.41
a
0.44
a
0.51
0.83
0.65
a
0.61
a
0.76
Spleen
0.22
0.26
Thymus
0.09
0.30
a
0.24
a
0.12
b.c
0.18
0.46
a
0.34
a,b
0.16
b.c
17.8
b.=
Kidney
e
Testis
e
Epididymis Adrenal Plasma
e
e (mg/lOO zinc
g BW)
(pug/100
8.4
19.8 32
ml)
f 5
153 at P < 0.05
a Significantly
different
from
group
ZD
b Significantly
different
from
group
Z at P < 0 05
c Significantly
different
from
group
ZDP
d Significantly
different
from
group
ZD
e Only
left-side
organs
were
THE
CONCENTRA-
at P <
f
0.28
a
12.4
lad
45
(ZD
YS Z,
(Z vs ZDP.
0.05
(ZDP
ZDP,
0.24
a.b ? 6
35
f 5
ZDS).
ZDS).
vs ZDS).
at P < 0.01.
weighed.
Safflower oil treatment had little effect on the organ weight differences caused by zinc deficiency. Only the heart weight was normalized, whereas the kidney weight was partly reduced towards normal. In contrast, primrose oil treatment substantially shifted almost all organ weights towards normal (Table 2). Heart, lung, kidney, testis, spleen and epididymal fat pad relative
180
160
I-
g
120
*
ZOP
& 0 m
100
80 20s 60
20
40
1 I
2
4
3
5
WEEKS Fig.
1.
cient mented of
Effects
(ZD)
ZDP
of
rats.
(Z) group
rats
supplementation
Growth after
after
of
the
3 weeks
4 weeks
with ZDP, (P <
primrose
ZDS 0.01).
(P < 0.05).
The
and
oil ZD
Growth mean
(P)
groups of the weights
or safflower was ZD
less
and
i SD
oil than
ZDS
(S)
on
the
growth
that
of
the
pair-fed
rats was significantly
(n = 7) are given.
of
zinc-defi-
zinc-suppleless than
that
199
weights were normal in the ZDP group. Adrenal hypertrophy and thymus atrophy were also partly corrected. In addition, the relative liver weight was significantly increased by primrose oil treatment and approached normal values in absolute terms (5.59 g in Z, 4.47 g in ZDP and 1.84 g in ZD) and the growth rate of the animals was significantly higher in this ZDP group than in the ZD animals, both at the 4th and the 5th week (Fig. 1). The effects of the various treatments on plasma and liver lipids are given in Table 3. Both plasma cholesterol and triglyceride concentrations were significantly lowered by zinc deficiency. Primrose oil but not safflower oil treatment could reverse this effect. Liver cholesterol was practically unaffected in any of the experimental conditions but the liver triglyceride content was reduced in the ZD animals, and markedly increased by both primrose oil and safflower oil administration. The fatty acid profile of a total lipid extract of various tissues is shown in Table 4. All zinc-deficient animals (ZD, ZDP, ZDS groups) had a lower proportion of arachidonic acid (20 : 4~6) in plasma lipids than the zinc-supplemented group (Z). In contrast, the proportion of 20 : 406 in liver lipids was higher in the ZD group than in any other group; primrose oil or safflower oil supplement reversed this effect. There was a small but significant increase in the 18 : 206 content of plasma and liver lipids in the ZD group as compared to the pair-fed Z group. This was not observed in adipose tissue and skin (Table 4). On the other hand there was a striking increase in the liver 18 : 206 content induced by primrose oil and safflower oil administration. A similar accumulation of linoleic acid was noted in lipids of both the epididymal fat and the skin (Table 4). These were accompanied by reciprocal changes in other fatty acid such as stearic acid (18 : 0) and 20 : 406, which varied with the tissue examined. A selective increase was also noted in 18 : 0 and 20 : 4~6 proportions of adipose tissue lipids. Because of the marked atrophy of this tissue in the ZD group this could be an artefact related to the relatively high content of connective tissue. In none of the fatty acid profiles studied was there an abnormally high level of the triene/tetraene ratio, so that there was no indication of a frank EFA deficiency state.
TABLE
3
EFFECT
OF
PRIMROSE
CONCENTRATIONS RATS
(MEAN
OR
SAFFLOWER
CHOLESTEROL
OIL
(CH)
ZD
Z
ON
TRIGLYCERIDES
(TG)
ZDP
PLASMA IN
AND
LIVER
ZINC-DEFICIENT
ZDS
(mg/dl)
CH
66
f 14
102
+13a
TG
51
? 10
83
f12a
Liver
SUPPLEMENTATION
AND
+ SD)
Lipid Plasma
OIL OF
75 104
+11
66
+lSb
k3ga
56
+ 20 b-c
(mg/g)
CH
4.6
f
0.2
4.2
+
0.5
TG
1.2
+
0.1
2.0
f
0.6
a Significantly
different
from
group
ZD
5 Significantly
different
from
group
Z at P < 0.05.
at P <
c Significantly
different
from
group
ZDP
at P <
0.05. 0.05.
4.1f a
7.3
0.5a t
2.8
a.b
4.7
*
1.0
8.5
f
3.0
a.b
FATTY
29.2
different
26.6
20.5 0.2
C Significantly
: 4
20
23.9 1 .o
14.9
different
: 2 : 3
18 20
12.4
10.0
22.5
different
: 1
18
8.9
20.9
Z
b Significantly
: 0
18
ZD
Plasma
composition.
IN
from
from
b
group
group
group
24.0
22.0 0.8
17.0
9.3
24.6
LIVER,
ZDP
0.05.
24.8
20.5 0.2
at P <
9.9
21.8
20.2
ZD
Liver
0.05.
16.7
15.9 0.2
11.2
25.9
21.8
Z
(ZDS)
EPIDIDYMAL
OIL
0.05.
at P <
b
Z at P <
ZD
23.9
23.0 1.1
15.9
10.4
23.2
ZDS
SAFFLOWER
PLASMA,
OR
ZDP
from
(ZDP)
ACIDS
OIL
a Significantly
: 0
16
acid
Fatty
Percentage
PRIMROSE
MAJOR
(Z),
4
TABLE
a
a
21.5
17.8
30.6 0.6
13.4
15.6 a.b a
=.b
b
AND
ZDP
FAT
12.8
37.5 0.2
12.4
17.2
17.1
a
a.b c
b
b
TOTAL
ZDS
SKIN
31.8
30.5
8.4
18.8
ZD
33.9
32.5
1.7
=
a
fat
-
45.7 -
25.1
2.2
19.6
ZDP
a.b
a.b
a
a
ZINC-DEFICIENT
29.0
Z
IN
Epididymal
LIPIDS
a.b
53.0 -
-
a.b
25.1
a
12.2
0.8
19.1
23.2
9.3
19.6 3.6
15.1
Skin
11.9
0.4
21.4
23.5
7.0
22.4
Z
SUPPLEMENTED
ZD a7b.c
(ZD)
ZDS
RATS
3.9
0.3
51.4
19.1
3.8
13.7
ZDP
a.b
a.b
b
a.b
a.b
WITH
c
a.b
6.9
0.6
44.1
a.b
0
lg.0 a.b
1.0
14.0
ZDS
ZINC
201
Primrose
oil and safflower
oil supplemenation
in normal rats
This experiment showed no increase in the relative proportion of 18 : 206 in plasma, liver or adipose tissue lipids (Table 5) of normal animals injected subcutaneously with either primrose or safflower oil. The only difference in fatty acid composition was observed in the epididymal fat. CS rats had a smaller proportion of 18 : 0 whereas CP rats had a smaller proportion of palmitic acid (16 : 0). Growth rate and organ weights were not different among the 3 groups in this experiment. Zinc supplementation
in essential fatty acid deficiency
Feeding an EFA-deficient diet resulted in a growth curve significantly lower from control by the 2nd week, with a plateau after the 5th week. A diffuse, orange-brown pigmentation of the fur developed within 3 weeks and alopecia became apparent after the 5th week. The growth rate of zinc-supplemented EFAdeficient animals (EDZ) was identical to that of the EFAdeficient group (ED) until the 7th week. After that, the growth tended to be more stunted in the ED group although the difference did not reach statistical significance even at the 10th week. The discoloration of the fur was more apparent in the ED group at 10 weeks and the alopecia more severe. On the other hand the fatty acid composition of plasma, liver, skin and epididymal fat lipids did not differ between the ED and the EDZ groups (Table 6). As expected, oleic acid (18 : 109) was markedly raised and linoleic acid (18 : 2~6) markedly reduced in the EFA-deficient animals. The rise in eicosatrienoic acid (20 : 3w9) and the reduction in arachidonic acid (20 : 4~6) in plasma, liver and skin lipids resulted in the expected increase in the triene/tetraene ratio, the conventional hallmark of established EFA deficiency. Not only did zinc supplementation fail to improve the fatty acid pattern of EFA deficiency, but the triene/tetraene ratio actually tended to be higher in the zinc-supplemented animals.
TABLE
5
MAJOR TROL
FATTY RATS
Percentage Fatty
16 18 18 18 20 20
ACIDS
(C),
IN
PLASMA,
SUPPLEMENTED
LIVER WITH
AND
PRIMROSE
EPIDIDYMAL OIL
(CP)
FAT OR
TOTAL
SAFFLOWER
LIPIDS OIL
FROM
CON-
(CS)
composition.
acid
:0 :0 :1 :2 :3 :4
a Significantly
PI%llla
Liver
C
CP
cs
20.1
21.1
20.9
17.1
14.5
13.9
14.4
22.8
C
Epididymal CP
fat
CS
C
CP
17.3
17.2
18.7
13.5
22.0
21.9
3.9
4.4
cs a
23.2
a
2.1
a
12.2
12.3
13.4
10.4
9.5
9.2
29.5
29.8
29.2
19.8
18.8
18.7
14.6
13.6
13.8
33.2
34.2
31.6
0.9 23.8 different
1.1 24.7 from
group
0.6
0.5
0.4
0.4
22.8
27.1
27.0
26.8
C at P <
0.05.
from
0.6
2o:a”
b 20
: 3~6
different
0.04
for control
:4 : 3120 : 4
a Significantly
20
20.2
5.2
: 2
20
30.2
12.2
25.9
:
18
18
and 20
a
a
a
a
: 3w9
for
the group
0.70
10.7
6.6
12.9
10.7
1
27.9
21.8
ED
C
Plasma
SUPPLEMENTATION
: 0
ZINC
: 0
OF
18
acid
6
16
Fatty
EFFECT
TABLE
a
a
a
a
ED
and
C at P <
0.74
10.9
1.4
5.2
30.2
11.1
26.5
EDZ
(EDZ)
7.4
EDZ
a
0.55
17.8
9.1
5.0
19.4
was made.
is notsignificantly
0.44
21.5
7.9
5.0
17.3
no comparison
but
0.04
20.9
0.9
17.5
18.2
22.3
a
a
ED.
5.4
different
50.8
29.3 32.9
from
7.1
a
25.4
2.1
20.1
19.4
EDZ 21.8
19.7
19.3
ED
fat
COMPOSITION
ED
ACID
C
FATTY
C
PERCENTAGE
Epididymal
THE
Liver
EDZ.
0.05,
ON
a -
5.0
51.5
3.7
24.7
a
a
TISSUE
EDZ
OF
0.03
14.2
0.28
6.8
1.4
4.7 0.4
36.5
22.0
13.5
26.0
ED
a
a
a
a
0.32
5.3
1.7
5.1
42.7
8.2
25.3
EDZ
IN EFA-DEFICIENT
16.4
9.9
22.1
C
Skill
LIPIDS
a
a
a
a
(ED)
RATS
N 0 p3
203
Discussion Our study reproduced the well known biological effects of zinc deficiency including dermal changes [ 1,2], marked atrophy of thymus [ 251, and hypocholesterolemia [26,27]. Reversal of several of these effects by primrose oil but not by safflower oil supplementation lends substantial support to the idea that zinc profoundly affects the metabolism of essential fatty acids. Previous research on zinc and essential fatty acid interactions had not identified a specific biochemical effect of zinc deficiency on fatty acid metabolism. The studies [1,3,5] suggesting an effect of EFA supplementation on zinc deficiency receive support from the present results, which indicate for the first time a possible mechanism for the effect of zinc on the metabolism of essential fatty acids. Changes in total fatty acid patterns in tissues must be interpreted with caution, especially with regard to the acids present in small amounts. The fatty acids are found free or esterified with glycerides, cholesterol or various phospholipid fractions, and the levels of even two such closely related acids as 20 : 3~6 and 20 : 4~6 may fluctuate quite independently in these various fractions [ 281. Much work needs to be done on fatty acid transfers among the fractions and an absence of change in overall levels may mask substantial movement among the individual fractions. Similarly, results obtained at one point in time cannot give definitive informatioh as to whether, say, a higher proportion of a particular acid in a fraction is due to increased entry into or reduced mobilization from that fraction. In this study, the Z group (receiving the zinc-deficient diet but supplemented with zinc in the drinking water) served as a control group among the ZD animals. Thus the lower 20 : 406 in the ZD, ZDP and ZDS than in the Z group probably reflected an effect of the zinc deficiency per se. Changes in 20 : 406 and 20 : 3~6, the precursors of the prostaglandins, cannot be interpreted in relation to their effects on PG synthesis without knowing which specific esters are the usual prostaglandin precursors in a particular tissue. There is some evidence that the 20 : 4~6 and 20 : 3~6 used for prostaglandin synthesis are derived from minor and as yet unidentified lipid fractions [ 29,301 since large changes in prostaglandin production may occur without detectable changes in the major 20 : 3~6 and 20 : 4~6 pools. The present results therefore cannot be used either to support or refute the ideas that zinc is necessary either for 20 : 406 [l] or for 20 : 3~6 [31] mobilization. With these reservations in mind, a number of conclusions may be drawn from the present study. Two lines of evidence point to a deficiency of A-6-desaturase activity in the ZD animals and lend support to the concept that zinc ions might be an essential cofactor for this enzyme (see Fig. 2). (1) The most striking fatty acid change was the substantial elevation of 18 : 2~6 in liver, epididymal fat and skin in the ZD animals given primrose or safflower oil. These changes did not occur in the normal animals given primrose or safflower oil. This argues for the idea that ZD rats, unlike normal animals, cannot metabolize the large amounts of 18 : 2~6 in the primrose or safflower oil. This defect was not as striking when the Z and ZD groups, which did not
204 P-LINOLEOYL -GLYCEROL Zn----a
ABSORPTION t
LINOLEATE
I - LINOLENATE
(l&l:3 OeJ
ARACHDONATE (204
<
DIHOMO -)’ - LINOLENATE
OSJ
(20:3
PROSTAGLANDINS ( 1 SERIES)
PROSTAGLANOINS ( 2 SERIES J Fig. 2. Outline of the pathway of EFA action of zinc on the A-Gdesaturase.
06 J
metabolism
to prostaglandins.
indicating
the proposed
sites of
receive extra 18 : 2~6, are compared. Nevertheless, the proportion of 18 : 206 in liver lipid (Table 4) was significantly higher in the ZD than in the Z group. This also suggested that relatively limited stores of 18 : 2~6 in the pair-fed zinc-supplemented rats may have been more rapidly metabolized. Since 18 : 2~6 is normally metabolized to 18 : 3~6 by A-gdesaturase, a partial block at this level in the absence of dietary zinc and in conditions of 18 : 206 overload should lead to a tissue accumulation of 18 : 206, as’was observed. (2) Dietary zinc deficiency was found to induce profound changes in body growth, organ weights and lipid concentrations of both plasma and liver. Many of these changes were either improved or completely reversed by primrose oil administration but not by safflower oil. Apart from the y-linoleic acid (18 : 3~6) in the primrose oil the fatty acid composition of the two oils is virtually identical (Table 1). This suggests that 18 : 3~6, which by-passes the need for A-6desaturase, is the key factor. Thus many of the biological effects of zinc deficiency could be attributed to a reduced transformation of the essential fatty acid 18 : 206 to 18 : 3~6 secondary to a deficit at the A-6-desaturase step. This is in accordance with the findings that primrose oil is much more effective than safflower oil in alleviating the signs of EFA deficiency in the cat, which lacks A-6-desaturase entirely [ 9,101. The precise mode of interaction of zinc with A-6desaturase remains a matter of conjecture. It may be connected with the NADP/NADPH cycle which affects the activity of A8desaturase since zinc has been shown to have a regulatory function on this cycle [ 111. A second possible site of action at which zinc deficiency could affect EFA metabolism is the intestinal wall. Absorption of triglycerides has been shown to be significantly decreased in zinc deficiency [32]. 18 : 2~6 absorption in the small intestine has also been reported to depend on alkaline phosphatase, a
205
zinc-dependent enzyme whose function may be decreased in zinc deficiency [33]. This point is also supported by the fact that the ZD diet contained 10 g of corn oil (45% 18 : 206)/100 g of diet, an amount normally sufficient to provide adequate EFA. Therefore, aside from a tissue-dependent effect of zinc deficiency related to A-6desaturase, the exogenous fatty acids in the primrose and safflower oils may have exerted their beneficial effect because they were injected subcutaneously and therefore by-passed the intestinal absorption process. Our findings also raise questions about the definition of EFA deficiency. The biological effects of zinc deficiency which are reversed by the administration of 18 : 3w6-rich primrose oil could be operatively ascribed to a “relative” state of EFA deficiency in the absence of any change in the triene/tetraene ratio. Indeed, in none of the ZD groups was there any substantial elevation of the triene/tetraene ratio, the accepted biochemical indicator of EFA deficiency. Two possibilities may be entertained: subtle changes in the formation of 18 : 3~6 are sufficient to induce specific biological effects before any alteration in the triene/tetraene ratio is detected; or the ratio may be unaffected in zinc deficiency because other desaturases are also affected (notably the A-6-desaturase) thus preventing the formation of 20 : 309 and the rise in the triene/tetraene ratio. In either case these concepts could prove of the utmost importance in the analysis and understanding of EFA deficiency states. If what matters in EFA deficiency is lack of the longer-chain acids and in particular of their prostaglandin metabolites, then the triene/tetraene ratio alone becomes inadequate as a measure of such deficiency. A rise in this ratio simply indicates a specific lack of EFAs of the 18 : 2~6 series. Defects in the desaturation and elongation stages which also affected the formation of 20 : 3w9 could thus be associated with severe functional EFA deficiency without any detectable change in the triene/tetraene ratio. Other biochemical measures of EFA deficiency based on measurements of such acids as 20 : 3~6 and 20 : 4~6 in the key lipid fractions are therefore urgently needed. The changes in thymus and adrenal weight are of particular interest. Thymus atrophy was profound in the ZD group, as is to be expected from the key role of zinc in the thymus and in the immune system [34]. Primrose oil, but not safflower oil, restored adrenal and thymus weight to nearly normal values (there was no significant difference for these weights between group Z and ZDP). This suggests that modulation of EFA metabolism may be the most important aspect of the effect of zinc on the immune system, possibly because it will lead to inadequate formation of prostaglandin El, a substance which seems to be particularly important in T lymphocytes [ 341. The rise in adrenal weight might be related to failure of an adrenal secretion and a consequent compensatory hypertrophy. The possibility that the rise in adrenal corticosterone output which can occur in zinc deficiency could account for the thymic atrophy has previously been investigated [35]. The conclusion was that the greater part of the thymus atrophy was not secondary to increased adrenal activity. The lack of effect of zinc on the growth and fatty acid profiles of EFA-deficient rats strongly suggests that zinc has no effect on body growth and maintenance of the dermis unless EFA are present in the tissues. Furthermore, it
206
confirms that the similarity of zinc and EFA deficiencies is not coincidental but that the two have a significant interaction. As noted earlier, the relationship between zinc and 18 : 2~6 metabolism is of potential importance to the understanding of dietary influences on atherosclerosis. In order for 18 : 206 to be effective as an antithrombotic, antihypertensive agent [18,19] its metabolism to 18 : 306 must be assured. Our data would suggest that zinc is necessary for this conversion to proceed normally. In addition to its apparent role in 18 : 206 metabolism, zinc is thought to increase the synthesis of prostaglandin El from 20 : 3~6 [31]. Essential fatty acid supplementation favors an increase in tissue 20 : 3~6 over 20 : 4~6 [ 361, with a consequent increase in prostaglandin E, synthesis, particularly in platelets [37]. Prostaglandin El is both an anti-aggregatory and also a vasodilatory prostaglandin [38]. Hence, the beneficial effect of essential fatty acids in the prevention or reduction of hypertension, atherosclerosis, and other forms of heart disease [18,19] may depend to a large extent on an adequate availability of zinc. Acknowledgements The skilful technical assistance of M. Paquette, L. Boulet and M. Tremblay is gratefully acknowledged. The advice of Dr. S. Lussier-Cacan on the nutritional aspect of this study is deeply appreciated. References 1 Bettger. W.J., Reeves, P.G., Moscatelli, E.A. and O’Dell. B.L., Interaction of zinc and essential fatty acids in the rat, J. Nutr., 109 (1979) 480. 2 O’Dell, B.L., Reynolds. G. and Reeves, P.G., Analogous effects of.zinc deficiency and aspirin toxicity in the pregnant rat, J. Nutr., 107 (1977) 1222. 3 Cunnane. S.C., Honobin, D.F., Ruf. K.B. and Sella, G.E.. Prevention of dietary effects of zinc deficiency by administration of essential fatty acids, J. Physiol. (Land.). 296 (1979) 113P. 4 Cash, R. and Berger, C.L.. Acrodermatitis enteropathica - Defective metabolism of essential fatty acids. J. Pediat., 74 (1969) 717. 5 Cunnane, S.C. and Horrobin, D.F.. Parental linoleic and gamma-linolenic acids ameliorate the gross effects of zinc deficiency, Proc. Sot. Exp. Biol. Med., 164 (1980) 583. 6 Curmane, S.C., Horrobin, D.F., Sella. G.E. and Ruf, K.B., Essential fatty acid supplementation inhibits the effect of dietary zinc deficiency. Adv. Prostag. Thromboxane Res., 8 (1980) 1797. 7 Brenner, R.R., The oxidative desaturation of unsaturated fatty acids in animals. Mol. Cell. Biochem.. 3 (1974) 42. 8 Brenner, R.R.. Metabolism of endogenous substrates by microsomes, Drug Metab. Rev., 6 (1977) 155. 9 Rivers, J.P.W., Sinclair. A.J. and Crawford, M.A., Inability of the cat to desaturate essential fatty acids, Nature (Land.), 258 (1975) 171. 10 Frank& T.L. and Rivers, J.P.W.. The nutritional and metabolic impact of gamma-linolenic acid on cats deprived of animal lipid, Brit. J. Nutr.. 39 (1978) 227. 11 Chvapil, M., Ludwig, J.C., Sipes, I.G. and Misiorowski, R.L., Inhibition of NADPH oxidation and related drug oxidation in liver microsomes by zinc. Biochem. Phannacol.. 25 (1976) 1787. 12 Henzel, J.H.. Holtmann, B.. Keitzer, F.W., De Weese. M.S. and Lichti, E., Trace elements in atherosclerosis. efficacy of zinc medication as a therapeutic modality. In: D.D. Hemphill (Ed.), Trace Substances in Environmental Health. Vol. 2, University of Missouri. Columbia, MO. 1969, p. 83. 13 Pories. W.J. and Strain, W.H., Zinc sulphate therapy in surgical patients. In: W.I. PO&S. W.H. Strain, J.M. Hsu and R.L. Woolsey (Eds.), Clinical Applications of Zinc Metabolism, C.C. Thomas, Springfield, IL, 1974, p.139. 14 Volkov, N.F., Cobalt, manganese and zinc content of blood and internal organs of patients with atherosclerosis, Jer. Arkh., 34 (1962) 52. 15 Netsky, M.G., Harrison, W.W.. Brown. M. and Benson, C.. Tissue zinc and human disease -Relation
207 of zinc content 16 17
18
19
20 21 22 23 24 25 26 27
28 29 30 31
32 33
34
35 36 37 38
of kidney
liver and lung to atherosclerosis
and hypertension,
Amer. J. Clin. Path.,
51
(1969) 358. Kaindl, F., Kuhn, P.. Holzhey, P. and Niederberger, M., Herztherapie mit Zink-Protamin-Glucagon, Verh. Dtsch. Gee.. Inn. Med., 78 (1972) 1099. Klevay, L.M., Interactions among dietary copper, zinc and the metabolism of cholesterol and phospholipids. In: H.G. Hockstra, J.W. Suttie, H.E. Ganther and W. Mertz (Eds.), Trace Element Metabolism in Animals, Vol. 2. University Park Press, Baltimore, MD. 1974, p. 553. Vergroesen, A.J., de Deckere, E.A.M., Ten Hoor, F. and Hornstra. G., Cardiovascular effects of linoleic acid. In: C. Galli and P. Avogaro (Eds.). Progress in Food and Nutrition Science, Vol. 4, Pergaman, New York. NY, 1980. p. 13. Oster. P., Arab, L.. Schellenberg, B., Kohlmeier, M. and Schlierf, G.. Linoleic acid and blood pressure. In: C. Galli and P. Avogaro (Eds.), Progress in Food and Nutrition Science, Vol. 4, Pergamon. New York, NY, 1980, p. 39. Folch, J., Lees, M. and Sloane-Stanley, H., A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem.. 226 (1957) 497. Block. W.D.. Jarrett, K.J. and Levine, L.B.. An improved automated determination of serum total cholesterol with a single color reagent, Clin. Chem.. 12 (1966) 681. Kraml. M. and Cosyns, L., A semi-automated determination of serum triglycerides, Clin. Biochem., 2 (1969) 373. Morrison, W.R. and Smith, L.M.. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride--methanol. J. Lipid Res., 5 (1964) 600. Zar, J.H.. Multiple comparisons. In: Biostatistical Analysis. Prentice-Hall Inc., Englewood Cliffs, NJ, 1974, p. 151. Fraker, P.J., Haas. S.M. and Luecke. R.W.. Effect of zinc deficiency on the immune response of the young adult A/J mouse, J. Nutr., 107 (1977) 1889. Patel, P.B.. Chung, R.A. and Lu. J.Y., Effect of zinc deficiency on serum and liver cholesterol in female rats, Nutr. Rep. Internat., 12 (1975) 205. Burch, R.E., Williams, R.V.. Hahn, H.K.L. Jottor, M.M. and Sullivan, J.F., Serum and tissue enzyme activity and trace-clement content in response to zinc deficiency in the pig, Clin. Chem., 21 (1975) 568. Marcus, A.J., Ullman. H.L. and Safier. L.B., Lipid composition of subcellular particles of human blood platelets, J. Lipid Res.. 10 (1969) 108. Crawford, M.A.. Denton, J.P.. Hassam, A.G., Lynn, J., Marples. P., Stevens, P. and Willis, A.L.. Levels of prostaglandins and their precursors in EFA deficiency, Brit. J. Pharmacol.. 63 (1978) 363P. Legarde, M.. Dechavanne, M., Rigaud. M. and Durand, J., Bass’ level of human platelet prostaglandins - PGEl is more elevated than PGE2, Prostaglandins, 17 (1979) 685. Manku, M.S.. Horrobin. D.F., Karmazyn, M. and Cunnane. S.C., Prolactin and zinc effects on rat vascular reactivity - Possible relationship to dihomogammahnolenic acid and to prostaglandin synthesis, Endocrinology. 104 (1979) 774. Koo, S.I. and Turk. D.E., Effect of zinc deficiency on intestinal transport of triglycerides in the rat, J. Nutr.. 107 (1977) 909. Muira. S., Study on the fat absorption and transportation into intestinal lymph of rats -Differences in the absorption of saturated and unsaturated long chain fatty acids and the role of intestinal alkaline phosphatase. Nippon Shokakibyo Gukkai Sasshi, 76 (1979) 871. Horrobin, D.F., Manku. M.S., Oka, M., Morgan, R.O., Cunnane, S.C., Ally. A.L., Ghayur. T., Schweitzer. M. and Karmali, R.A., The nutritional regulation of T lymphocyte function, Med. Hypotheses, 5 (1979) 969. De Pasquale-Jardieu. P. and Fraker, P.J.. The role of corticosterone in the loss in immune function in the zinc deficient A/J mouse, J. Nutr., 109 (1979) 1847. Holman, R.T., Polyunsaturated fatty acid profiles in human disease, Progr. Clin. Biol. Res., (1981) In press. Stone, K.J., Willis, A.L., Hart, M., Kirtland. S.J.. Kernoff, P.B.A. and McNichol, G.P., The metabolism of dihomo-gamma-linolenic acid in man, Lipids, 14 (1979) 174. Mitchell, J.R.A., Prostaglandins in vascular disease - A seminal approach. Brit. Med. J.. 282 (1981) 590.
Atherosclerosis, 41 (1982) 193-207 0 Elsevier/North-Holland Scientific
Publishers,
Ltd.
MOST BIOLOGICAL EFFECTS OF ZINC DEFICIENCY CORRECTED BY y-LINOLENIC ACID (18 : 3~6) BUT NOT BY LINOLEIC ACID (18 : 2~6) Y.S.
HUANG,
Clinical Quebec (Received (Revised, (Accepted
S.C. CUNNANE
*, D.F. HORROBIN
Research Institute of Montreal, HZW lR7 (Canada)
and J. DAVIGNON
110 Pine Avenue,
West, Montreal,
25 March, 1981) received 8 June, 1981) 9 June, 1981)
--.
---.
-
Summary Zinc deficiency mimics many of the clinical features of essential fatty acid (EFA) deficiency in rats. Since zinc appears to be needed for the A-6-desaturase step in EFA metabolism, experiments were conducted to determine whether bypassing this step with y-linolenic acid (18 : 3~6) would alleviate the biological effects induced by a zinc-deficient diet. In pair-fed rats over a period of 5 weeks the deficient diet impaired growth and changed the relative weights of internal organs. It also induced hypolipidemia but had little effect on the fatty acid composition of tissue lipids. Daily subcutaneous injection of primrose oil containing 10% 18 : 306 reversed most of the effects of zinc deficiency on tissue weights, growth and plasma lipids. In contrast, injection of safflower oil, which has a similar content of linoleic acid (18 : 2~6) but is devoid of 18 : 306, had only a partial effect on some tissue weight changes. Neither oil affected the plasma fatty acid pattern, but both of them increased liver triglyceride concentrations. They also elevated the proportion of 18 : 2~6 in liver, skin and epididymal fat. The latter effects were not observed in the absence of zinc deficiency. Supplementing the diet of EFA-deficient animals with an excess of zinc in their drinking water did not affect the typical tissue fatty-acid pattern of EFA deficiency. It is suggested that several of the manifestations of zinc deficiency are mediated by a relative state of EFA deficiency attributable, at least in part, to a reduced conversion of 18 : 2~6 to 18 : 3~6 resulting in an accumulation of 18 : 206 in tissues. These findings are consistent with a role of zinc as a cofactor in the ASdesaturase enzyme reaction. The suggested role of zinc in essential fatty This manuscript was taken in part from a dissertation submitted to the Graduate School of McGill University by Stephen Cunnane in partial fulfillment of the requirements for the degree of Doctor of Philosophy, and was supported by grants from the Medical Research Council of Canada and the Quebec Heart Foundation. * Research fellow of Le Cons&l de la Recherche en SantC du QuBbec.
OOZl-9150/82/0000-0000/$02.75
@ 1982
Elsevier/North-Holland
Scientific
Publishers,
Ltd.
194
acid metabolism may be of significance in understanding acids protect against cardiovascular disease in general. Key words:
Essential ciency
fatty
acid deficiency
-
Linoleic -
acid -
how essential
y-Linolenic
acid -Zinc
fatty
defi-
_--__
Introduction The evidence for an interaction between zinc and essential fatty acid (EFA) metabolism has been recently reviewed [l]. The main points are: (1) many of the features of zinc and EFA deficiency are similar, (2) the features of either zinc or EFA deficiency are exaggerated when the diet is also deficient in the other nutrient, (3) treatment of pregnant rats with drugs which block EFA conversion to prostaglandins (PCs) produces changes that are remarkably similar to those brought about by zinc deficiency in pregnancy [2], (4) treatment of zincdeficient rats [3] and humans [4] with vegetable oils rich in EFAs attenuates the effects of zinc deficiency on growth and other parameters. In previous studies [3,5,6] we have shown that primrose oil is able to prevent the development of some features of zinc deficiency. When primrose oil and safflower oil were compared in male rats, the former was considerably more effective [3,5]. The total EFA content of these two oils is almost identical (Table l), but whereas the safflower oil contains 83% linoleic acid (18 : 2w6), the primrose oil contains 74% 18 : 206 and 10% y-linolenic acid (18 : 306). The latter is formed from 18 : 206 by the A-6-desaturase enzyme system [ 7,8]. Studies in cats, which lack this desaturase, have demonstrated that 18 : 2~6 is largely inert as an EFA when this transformation cannot take place [9,10], and therefore cats must rely on a dietary source of 18 : 3~6 for prostaglandin synthesis. Since there is evidence that zinc could be needed as a cofactor in the A8desaturase enzyme activity [ll], experiments were conducted to determine whether bypassing this step in EFA metabolism would alleviate the biological effects induced by a zinc-deficient diet. We took advantage of the difference in 18 : 3~6 content between the two oils with similar 18 : 206 content, namely primrose and safflower, and report here their effects in pair-fed zincdeficient rats on growth, organ weight, liver and plasma lipid concentrations as well as on the total fatty acid pattern of plasma, liver and other organs. These studies were considered to be of importance in view of a possible involvement of zinc deficiency in the pathogenesis of atherosclerosis [ 12-151. Zinc therapy has been used to treat some of the symptoms of obliterative vascular disease and cardiac insufficiency [12,16]. An increase in the ratio of zinc to copper in the diet has been suggested to be related to hypercholesterolemia in humans [17]. Increased essential fatty acid intake in humans significantly lowers plasma low and very low density lipoproteins, decreases the thrombogenie properties of blood platelets as well as the susceptibility to sodiuminduced hypertension ,[ 181. The intake of 18 : 206 has also been shown in epidemiological studies to be negatively correlated with blood pressure and serum sodium concentrations in man [19]. Thus, a study of the possible effects of
195
zinc deficiency on lipid metabolism in general and essential fatty acid metabolism in particular is relevant to furthering our understanding of the pathogenesis of atherosclerosis and heart disease in general. Material and Methods Zinc deficiency
experiment
The purpose of this experiment was to compare the effect of linoleic acid in the presence (primrose oil) and absence (safflower oil) of y-linolenic acid on the biological effects of zinc deficiency. Weanling male Wistar rats weighing about 50 g were separated into 4 groups of 7 rats and maintained on a zinc-deficient diet (containing less than 3 ppm zinc, other constituents of diet are detailed in Table 1) obtained from Ziegler Bros., Gardners, PA, over a period of 5 weeks and deionized water ad libitum. The first group (ZD) received this diet alone. For the second group (Z) the deionized water was supplemented with zinc (10 pg/ml of zinc as ZnS04 * 7 HzO, Baker). The rats of the third group (ZDP) received 250 pi/day of primrose oil (Agricultural Holdings, London, England) by subcutaneous injection. The last group (ZDS) was given the same amount of commercial safflower oil by the same route. Both oils contained less than 2 ppm-zinc, so that the oilsupplemented rats received less than 20 pg-zinc from oils for the duration of the experiment. The pair-feeding was done on all treated rats by giving an amount of food equal to the average daily intake of the untreated zinc-deficient rats (ZD). The rats were killed by decapitation after 5 weeks and blood was collected from the neck vessels. Heart, lungs, thymus, liver, spleen, left kidney, adrenal, testis and epididymal fat pad were excised, rinsed with cold saline, blotted and weighed. Liver, epididymal fat pad and a piece of skin removed from the top of the hind paw were frozen on dry ice and stored for subsequent lipid analysis. The fatty acid composition of the diet and oils is given in Table 1. TABLE
1
PERCENTAGE
FATTY
ACID
COMPOSITION
OF
ZINC-DEFICIENT
DIET
AND
SUPPLEMENTED
OILS
Fatty
acid
Zinc-deficient
diet
a
Oils Primrose
14
:O
16
:O
16
:l
18
10
18
:l
18
: 2~3 : 3w3 : 3~6
60.9
18 18
a Zinc corn
0.2
deficient
and Tomarelli)
Safflower
0.1
12.6
starch,
oil
0.2
5.7
5.0
0.1
-
0.1
1.5
0.9
1.3
23.3
8.8
9.3
74.2
82.7
10.3
-
1.2
1.4
-
diet 10%
(Ziegler
corn
and
0.3%
oil
oil.
Bros., 3%
choline
Gardner,
cellulose chloride.
PA)
powder,
contained 0.5%
vitamin
20% mix
egg
white
(Murphy).
solids. 4%
31% mineral
sucrose. mix
31.2%
(Bernhart
196
Primrose and safflower
oil supplementation
in normal rats
This study was carried out after the completion of the above experiment to determine whether the accumulation of linoleic acid following primrose oil and safflower oil injections noted for the zinc-deficient rats could also be produced in normal rats. Three groups of six 50-g weanling male Wistar rats were fed Purina laboratory rat chow plus tap water ad libitum for 4 weeks. The first group (C) served as control, the second group (CP) received 250 /.d/day of primrose oil in a single subcutaneous injection and the third group (CS) received the same daily amount of safflower oil. The rats were killed and blood, liver and epididymal fat pad were obtained, as in the previous experiment, for lipid studies. Zinc supplementation
in essential fatty acid deficiency
The purpose of this experiment was to determine whether an excess of dietary zinc could modify the abnormal fatty acid pattern induced by EFA deficiency. Weanling male Wistar rats were divided into 3 groups of 6 animals and fed for 10 weeks, either a Purina rat chow plus tap water ad libitum (C), or an EFA-deficient diet (Teklad test diets, Madison, WI). The latter were fed either with deionized drinking water given ad libitum (group ED) or with deionized water containing a supplement of zinc (10 pg/ml of zinc as ZnS04 * 7 HzO) (group EDZ). The rats were killed and blood, liver, skin and epididymal fat pad were removed for lipid analyses as in the first experiment. The EFA-deficient diet consisted of 20% casein, 68.5% sucrose, 5% medium chain triglycerides (66% of fatty acids were capric and 33% lauric acids), 2% fiber, 3.5% minerals, and 1% vitamin mix. Both control and EFA-deficient diets contained an adequate amount of zinc (~40 ppm, as determined by our analysis). For all 3 experiments the rats were housed in pairs in stainless steel-topped plastic cages to avoid contamination from the zinc that is present in galvanized steel cages. A wire rack was placed at the bottom of the cages over and above woodchips used to absorb moisture so that coprophagy did not occur. Lipid analyses Fatty acid analyses were applied to samples of plasma, liver, epididymal fat and skin. Trunk blood was collected into test tubes containing 1 mg EDTA per ml blood and centrifuged at 4°C to separate the plasma. Aliquots of plasma and samples of liver and epididymal fat were homogenized and extracted with 20 volumes of chloroform--methanol (2 : 1, v/v) by the method of Folch et al. [ 201. The concentration of cholesterol [21] and triglycerides [22] in liver was assayed with a Technicon Autoanalyzer. Plasma cholesterol and triglycerides were measured enzymatically with an Abbott Bichromatic Analyzer (ABA100). Methyl esters of fatty acids (FAME) were prepared directly from the total lipid extract by BF,-methanolysis [23]. Samples of skin were cut into small pieces and saponified with alkaline methanol. After acidification, fatty acids were extracted into hexane and methylated with BF,-methanol.
197
FAME were analyzed by gas-liquid chromatography on an F&M model 402 apparatus at 185°C using a glass column (6 ft X 4 mm ID) packed with 10% silar-lOC on loo-120 mesh Gas-Chrom Q (Applied Science Laboratories, State College, PA). The percentage of each fatty acid was assessed with a HewlettPackard automatic integrator (Model 3373B). Zinc analyses
Analysis of the zinc content of plasma, adrenal tissue and liver tissue samples and of various diets and oil supplementations was performed by atomic absorption spectrophotometry, using a Perkin-Elmer 403 [ 51. The dried and weighed tissue samples were wet digested in 1 ml concentrated HN03 for 48 h. An aliquot of the tissue-acid solution was then diluted 1 : 10 with deionized water and analyzed. Plasma zinc was determined directly following acidification and a 1 : 10 dilution with deionized water. The oils were analyzed for zinc by dissolving them in iso-amylacetate-methanol (1 : 1) and measuring them directly with appropriate reference standards and recovery assessment. Using the methods described above, recoveries of known amounts of zinc added to solutions of digested liver or adrenal tissue samples varied from 94 to 102% and those of plasma ranged between 98 and 102%. Statistics
The differences between all possible pairs of means of groups were compared with the Student-Newman-Keuls multiple range test [ 241. Results Zinc deficiency
study
At the end of the experiment the zinc-deficient (ZD) animals exhibited retarded growth, hunched posture, keratosis of the paws, the tail, and the nasal, genital and eye regions, alopecia, and bleeding from the paws and nose. Animals treated with safflower oil (ZDS) were almost identical clinically except for a minor improvement of the skin. Animals treated with primrose oil (ZDP) appeared considerably healthier and showed only partial growth retardation. The zinc-supplemented rats (Z) appeared normal in all respects and served as the control group. Plasma zinc levels (Table 2) were significantly depressed in all ZD groups as compared to group Z (P < 0.05). However, neither adrenal, nor liver zinc content were significantly altered by the ZD conditions nor by the oil treatment. The body weight changes are shown in Fig. 1. The relative weights of the various organs, expressed per 100 g body weight, are given in Table 2. When expressed in this way, the heart, lung, kidney, and testis of the ZD animals all showed a moderate increase in weight although in absolute terms these organs were smaller because of the overall size of the animals. The relative hypertrophy of the adrenal was more striking (2.4 times the control weight). In contrast there was a marked atrophy of the thymus (a 75% reduction in weight) and of the epididymal fat pad (a 55% reduction). Liver weight was unaffected.
198 TABLE
2
EFFECT
OF
WEIGHTS TION
ZINC
(Z).
(g/lOOg
OF
PRIMROSE
body
ZINC-DEFICIENT
weight
OF
(ZD)
Tissue Body
OIL
weights)
(g)
(P)
OR
SAFFLOWER
VARIOUS
OIL
TISSUES
AND
(S) SUPPLEMENTATION ON
PLASMA
ZINC
ON
RATS
ZD
Z
59
163
ZDS
ZDP 104
a
Liver
3.10
3.43
Heart
0.44
0.36
Lung
0.81
0.54
a.b
71 b.c
4.29
a.b
3.02
a
0.37
a
0.38
c a
a
0.63
a
0.77
b a.b
0.60
0.41
a
0.44
a
0.51
0.83
0.65
a
0.61
a
0.76
Spleen
0.22
0.26
Thymus
0.09
0.30
a
0.24
a
0.12
b.c
0.18
0.46
a
0.34
a,b
0.16
b.c
17.8
b.=
Kidney
e
Testis
e
Epididymis Adrenal Plasma
e
e (mg/lOO zinc
g BW)
(pug/100
8.4
19.8 32
ml)
f 5
153 at P < 0.05
a Significantly
different
from
group
ZD
b Significantly
different
from
group
Z at P < 0 05
c Significantly
different
from
group
ZDP
d Significantly
different
from
group
ZD
e Only
left-side
organs
were
THE
CONCENTRA-
at P <
f
0.28
a
12.4
lad
45
(ZD
YS Z,
(Z vs ZDP.
0.05
(ZDP
ZDP,
0.24
a.b ? 6
35
f 5
ZDS).
ZDS).
vs ZDS).
at P < 0.01.
weighed.
Safflower oil treatment had little effect on the organ weight differences caused by zinc deficiency. Only the heart weight was normalized, whereas the kidney weight was partly reduced towards normal. In contrast, primrose oil treatment substantially shifted almost all organ weights towards normal (Table 2). Heart, lung, kidney, testis, spleen and epididymal fat pad relative
180
160
I-
g
120
*
ZOP
& 0 m
100
80 20s 60
20
40
1 I
2
4
3
5
WEEKS Fig.
1.
cient mented of
Effects
(ZD)
ZDP
of
rats.
(Z) group
rats
supplementation
Growth after
after
of
the
3 weeks
4 weeks
with ZDP, (P <
primrose
ZDS 0.01).
(P < 0.05).
The
and
oil ZD
Growth mean
(P)
groups of the weights
or safflower was ZD
less
and
i SD
oil than
ZDS
(S)
on
the
growth
that
of
the
pair-fed
rats was significantly
(n = 7) are given.
of
zinc-defi-
zinc-suppleless than
that
199
weights were normal in the ZDP group. Adrenal hypertrophy and thymus atrophy were also partly corrected. In addition, the relative liver weight was significantly increased by primrose oil treatment and approached normal values in absolute terms (5.59 g in Z, 4.47 g in ZDP and 1.84 g in ZD) and the growth rate of the animals was significantly higher in this ZDP group than in the ZD animals, both at the 4th and the 5th week (Fig. 1). The effects of the various treatments on plasma and liver lipids are given in Table 3. Both plasma cholesterol and triglyceride concentrations were significantly lowered by zinc deficiency. Primrose oil but not safflower oil treatment could reverse this effect. Liver cholesterol was practically unaffected in any of the experimental conditions but the liver triglyceride content was reduced in the ZD animals, and markedly increased by both primrose oil and safflower oil administration. The fatty acid profile of a total lipid extract of various tissues is shown in Table 4. All zinc-deficient animals (ZD, ZDP, ZDS groups) had a lower proportion of arachidonic acid (20 : 4~6) in plasma lipids than the zinc-supplemented group (Z). In contrast, the proportion of 20 : 406 in liver lipids was higher in the ZD group than in any other group; primrose oil or safflower oil supplement reversed this effect. There was a small but significant increase in the 18 : 206 content of plasma and liver lipids in the ZD group as compared to the pair-fed Z group. This was not observed in adipose tissue and skin (Table 4). On the other hand there was a striking increase in the liver 18 : 206 content induced by primrose oil and safflower oil administration. A similar accumulation of linoleic acid was noted in lipids of both the epididymal fat and the skin (Table 4). These were accompanied by reciprocal changes in other fatty acid such as stearic acid (18 : 0) and 20 : 406, which varied with the tissue examined. A selective increase was also noted in 18 : 0 and 20 : 4~6 proportions of adipose tissue lipids. Because of the marked atrophy of this tissue in the ZD group this could be an artefact related to the relatively high content of connective tissue. In none of the fatty acid profiles studied was there an abnormally high level of the triene/tetraene ratio, so that there was no indication of a frank EFA deficiency state.
TABLE
3
EFFECT
OF
PRIMROSE
CONCENTRATIONS RATS
(MEAN
OR
SAFFLOWER
CHOLESTEROL
OIL
(CH)
ZD
Z
ON
TRIGLYCERIDES
(TG)
ZDP
PLASMA IN
AND
LIVER
ZINC-DEFICIENT
ZDS
(mg/dl)
CH
66
f 14
102
+13a
TG
51
? 10
83
f12a
Liver
SUPPLEMENTATION
AND
+ SD)
Lipid Plasma
OIL OF
75 104
+11
66
+lSb
k3ga
56
+ 20 b-c
(mg/g)
CH
4.6
f
0.2
4.2
+
0.5
TG
1.2
+
0.1
2.0
f
0.6
a Significantly
different
from
group
ZD
5 Significantly
different
from
group
Z at P < 0.05.
at P <
c Significantly
different
from
group
ZDP
at P <
0.05. 0.05.
4.1f a
7.3
0.5a t
2.8
a.b
4.7
*
1.0
8.5
f
3.0
a.b
FATTY
29.2
different
26.6
20.5 0.2
C Significantly
: 4
20
23.9 1 .o
14.9
different
: 2 : 3
18 20
12.4
10.0
22.5
different
: 1
18
8.9
20.9
Z
b Significantly
: 0
18
ZD
Plasma
composition.
IN
from
from
b
group
group
group
24.0
22.0 0.8
17.0
9.3
24.6
LIVER,
ZDP
0.05.
24.8
20.5 0.2
at P <
9.9
21.8
20.2
ZD
Liver
0.05.
16.7
15.9 0.2
11.2
25.9
21.8
Z
(ZDS)
EPIDIDYMAL
OIL
0.05.
at P <
b
Z at P <
ZD
23.9
23.0 1.1
15.9
10.4
23.2
ZDS
SAFFLOWER
PLASMA,
OR
ZDP
from
(ZDP)
ACIDS
OIL
a Significantly
: 0
16
acid
Fatty
Percentage
PRIMROSE
MAJOR
(Z),
4
TABLE
a
a
21.5
17.8
30.6 0.6
13.4
15.6 a.b a
=.b
b
AND
ZDP
FAT
12.8
37.5 0.2
12.4
17.2
17.1
a
a.b c
b
b
TOTAL
ZDS
SKIN
31.8
30.5
8.4
18.8
ZD
33.9
32.5
1.7
=
a
fat
-
45.7 -
25.1
2.2
19.6
ZDP
a.b
a.b
a
a
ZINC-DEFICIENT
29.0
Z
IN
Epididymal
LIPIDS
a.b
53.0 -
-
a.b
25.1
a
12.2
0.8
19.1
23.2
9.3
19.6 3.6
15.1
Skin
11.9
0.4
21.4
23.5
7.0
22.4
Z
SUPPLEMENTED
ZD a7b.c
(ZD)
ZDS
RATS
3.9
0.3
51.4
19.1
3.8
13.7
ZDP
a.b
a.b
b
a.b
a.b
WITH
c
a.b
6.9
0.6
44.1
a.b
0
lg.0 a.b
1.0
14.0
ZDS
ZINC
201
Primrose
oil and safflower
oil supplemenation
in normal rats
This experiment showed no increase in the relative proportion of 18 : 206 in plasma, liver or adipose tissue lipids (Table 5) of normal animals injected subcutaneously with either primrose or safflower oil. The only difference in fatty acid composition was observed in the epididymal fat. CS rats had a smaller proportion of 18 : 0 whereas CP rats had a smaller proportion of palmitic acid (16 : 0). Growth rate and organ weights were not different among the 3 groups in this experiment. Zinc supplementation
in essential fatty acid deficiency
Feeding an EFA-deficient diet resulted in a growth curve significantly lower from control by the 2nd week, with a plateau after the 5th week. A diffuse, orange-brown pigmentation of the fur developed within 3 weeks and alopecia became apparent after the 5th week. The growth rate of zinc-supplemented EFAdeficient animals (EDZ) was identical to that of the EFAdeficient group (ED) until the 7th week. After that, the growth tended to be more stunted in the ED group although the difference did not reach statistical significance even at the 10th week. The discoloration of the fur was more apparent in the ED group at 10 weeks and the alopecia more severe. On the other hand the fatty acid composition of plasma, liver, skin and epididymal fat lipids did not differ between the ED and the EDZ groups (Table 6). As expected, oleic acid (18 : 109) was markedly raised and linoleic acid (18 : 2~6) markedly reduced in the EFA-deficient animals. The rise in eicosatrienoic acid (20 : 3w9) and the reduction in arachidonic acid (20 : 4~6) in plasma, liver and skin lipids resulted in the expected increase in the triene/tetraene ratio, the conventional hallmark of established EFA deficiency. Not only did zinc supplementation fail to improve the fatty acid pattern of EFA deficiency, but the triene/tetraene ratio actually tended to be higher in the zinc-supplemented animals.
TABLE
5
MAJOR TROL
FATTY RATS
Percentage Fatty
16 18 18 18 20 20
ACIDS
(C),
IN
PLASMA,
SUPPLEMENTED
LIVER WITH
AND
PRIMROSE
EPIDIDYMAL OIL
(CP)
FAT OR
TOTAL
SAFFLOWER
LIPIDS OIL
FROM
CON-
(CS)
composition.
acid
:0 :0 :1 :2 :3 :4
a Significantly
PI%llla
Liver
C
CP
cs
20.1
21.1
20.9
17.1
14.5
13.9
14.4
22.8
C
Epididymal CP
fat
CS
C
CP
17.3
17.2
18.7
13.5
22.0
21.9
3.9
4.4
cs a
23.2
a
2.1
a
12.2
12.3
13.4
10.4
9.5
9.2
29.5
29.8
29.2
19.8
18.8
18.7
14.6
13.6
13.8
33.2
34.2
31.6
0.9 23.8 different
1.1 24.7 from
group
0.6
0.5
0.4
0.4
22.8
27.1
27.0
26.8
C at P <
0.05.
from
0.6
2o:a”
b 20
: 3~6
different
0.04
for control
:4 : 3120 : 4
a Significantly
20
20.2
5.2
: 2
20
30.2
12.2
25.9
:
18
18
and 20
a
a
a
a
: 3w9
for
the group
0.70
10.7
6.6
12.9
10.7
1
27.9
21.8
ED
C
Plasma
SUPPLEMENTATION
: 0
ZINC
: 0
OF
18
acid
6
16
Fatty
EFFECT
TABLE
a
a
a
a
ED
and
C at P <
0.74
10.9
1.4
5.2
30.2
11.1
26.5
EDZ
(EDZ)
7.4
EDZ
a
0.55
17.8
9.1
5.0
19.4
was made.
is notsignificantly
0.44
21.5
7.9
5.0
17.3
no comparison
but
0.04
20.9
0.9
17.5
18.2
22.3
a
a
ED.
5.4
different
50.8
29.3 32.9
from
7.1
a
25.4
2.1
20.1
19.4
EDZ 21.8
19.7
19.3
ED
fat
COMPOSITION
ED
ACID
C
FATTY
C
PERCENTAGE
Epididymal
THE
Liver
EDZ.
0.05,
ON
a -
5.0
51.5
3.7
24.7
a
a
TISSUE
EDZ
OF
0.03
14.2
0.28
6.8
1.4
4.7 0.4
36.5
22.0
13.5
26.0
ED
a
a
a
a
0.32
5.3
1.7
5.1
42.7
8.2
25.3
EDZ
IN EFA-DEFICIENT
16.4
9.9
22.1
C
Skill
LIPIDS
a
a
a
a
(ED)
RATS
N 0 p3
203
Discussion Our study reproduced the well known biological effects of zinc deficiency including dermal changes [ 1,2], marked atrophy of thymus [ 251, and hypocholesterolemia [26,27]. Reversal of several of these effects by primrose oil but not by safflower oil supplementation lends substantial support to the idea that zinc profoundly affects the metabolism of essential fatty acids. Previous research on zinc and essential fatty acid interactions had not identified a specific biochemical effect of zinc deficiency on fatty acid metabolism. The studies [1,3,5] suggesting an effect of EFA supplementation on zinc deficiency receive support from the present results, which indicate for the first time a possible mechanism for the effect of zinc on the metabolism of essential fatty acids. Changes in total fatty acid patterns in tissues must be interpreted with caution, especially with regard to the acids present in small amounts. The fatty acids are found free or esterified with glycerides, cholesterol or various phospholipid fractions, and the levels of even two such closely related acids as 20 : 3~6 and 20 : 4~6 may fluctuate quite independently in these various fractions [ 281. Much work needs to be done on fatty acid transfers among the fractions and an absence of change in overall levels may mask substantial movement among the individual fractions. Similarly, results obtained at one point in time cannot give definitive informatioh as to whether, say, a higher proportion of a particular acid in a fraction is due to increased entry into or reduced mobilization from that fraction. In this study, the Z group (receiving the zinc-deficient diet but supplemented with zinc in the drinking water) served as a control group among the ZD animals. Thus the lower 20 : 406 in the ZD, ZDP and ZDS than in the Z group probably reflected an effect of the zinc deficiency per se. Changes in 20 : 406 and 20 : 3~6, the precursors of the prostaglandins, cannot be interpreted in relation to their effects on PG synthesis without knowing which specific esters are the usual prostaglandin precursors in a particular tissue. There is some evidence that the 20 : 4~6 and 20 : 3~6 used for prostaglandin synthesis are derived from minor and as yet unidentified lipid fractions [ 29,301 since large changes in prostaglandin production may occur without detectable changes in the major 20 : 3~6 and 20 : 4~6 pools. The present results therefore cannot be used either to support or refute the ideas that zinc is necessary either for 20 : 406 [l] or for 20 : 3~6 [31] mobilization. With these reservations in mind, a number of conclusions may be drawn from the present study. Two lines of evidence point to a deficiency of A-6-desaturase activity in the ZD animals and lend support to the concept that zinc ions might be an essential cofactor for this enzyme (see Fig. 2). (1) The most striking fatty acid change was the substantial elevation of 18 : 2~6 in liver, epididymal fat and skin in the ZD animals given primrose or safflower oil. These changes did not occur in the normal animals given primrose or safflower oil. This argues for the idea that ZD rats, unlike normal animals, cannot metabolize the large amounts of 18 : 2~6 in the primrose or safflower oil. This defect was not as striking when the Z and ZD groups, which did not
204 P-LINOLEOYL -GLYCEROL Zn----a
ABSORPTION t
LINOLEATE
I - LINOLENATE
(l&l:3 OeJ
ARACHDONATE (204
<
DIHOMO -)’ - LINOLENATE
OSJ
(20:3
PROSTAGLANDINS ( 1 SERIES)
PROSTAGLANOINS ( 2 SERIES J Fig. 2. Outline of the pathway of EFA action of zinc on the A-Gdesaturase.
06 J
metabolism
to prostaglandins.
indicating
the proposed
sites of
receive extra 18 : 2~6, are compared. Nevertheless, the proportion of 18 : 206 in liver lipid (Table 4) was significantly higher in the ZD than in the Z group. This also suggested that relatively limited stores of 18 : 2~6 in the pair-fed zinc-supplemented rats may have been more rapidly metabolized. Since 18 : 2~6 is normally metabolized to 18 : 3~6 by A-gdesaturase, a partial block at this level in the absence of dietary zinc and in conditions of 18 : 206 overload should lead to a tissue accumulation of 18 : 206, as’was observed. (2) Dietary zinc deficiency was found to induce profound changes in body growth, organ weights and lipid concentrations of both plasma and liver. Many of these changes were either improved or completely reversed by primrose oil administration but not by safflower oil. Apart from the y-linoleic acid (18 : 3~6) in the primrose oil the fatty acid composition of the two oils is virtually identical (Table 1). This suggests that 18 : 3~6, which by-passes the need for A-6desaturase, is the key factor. Thus many of the biological effects of zinc deficiency could be attributed to a reduced transformation of the essential fatty acid 18 : 206 to 18 : 3~6 secondary to a deficit at the A-6-desaturase step. This is in accordance with the findings that primrose oil is much more effective than safflower oil in alleviating the signs of EFA deficiency in the cat, which lacks A-6-desaturase entirely [ 9,101. The precise mode of interaction of zinc with A-6desaturase remains a matter of conjecture. It may be connected with the NADP/NADPH cycle which affects the activity of A8desaturase since zinc has been shown to have a regulatory function on this cycle [ 111. A second possible site of action at which zinc deficiency could affect EFA metabolism is the intestinal wall. Absorption of triglycerides has been shown to be significantly decreased in zinc deficiency [32]. 18 : 2~6 absorption in the small intestine has also been reported to depend on alkaline phosphatase, a
205
zinc-dependent enzyme whose function may be decreased in zinc deficiency [33]. This point is also supported by the fact that the ZD diet contained 10 g of corn oil (45% 18 : 206)/100 g of diet, an amount normally sufficient to provide adequate EFA. Therefore, aside from a tissue-dependent effect of zinc deficiency related to A-6desaturase, the exogenous fatty acids in the primrose and safflower oils may have exerted their beneficial effect because they were injected subcutaneously and therefore by-passed the intestinal absorption process. Our findings also raise questions about the definition of EFA deficiency. The biological effects of zinc deficiency which are reversed by the administration of 18 : 3w6-rich primrose oil could be operatively ascribed to a “relative” state of EFA deficiency in the absence of any change in the triene/tetraene ratio. Indeed, in none of the ZD groups was there any substantial elevation of the triene/tetraene ratio, the accepted biochemical indicator of EFA deficiency. Two possibilities may be entertained: subtle changes in the formation of 18 : 3~6 are sufficient to induce specific biological effects before any alteration in the triene/tetraene ratio is detected; or the ratio may be unaffected in zinc deficiency because other desaturases are also affected (notably the A-6-desaturase) thus preventing the formation of 20 : 309 and the rise in the triene/tetraene ratio. In either case these concepts could prove of the utmost importance in the analysis and understanding of EFA deficiency states. If what matters in EFA deficiency is lack of the longer-chain acids and in particular of their prostaglandin metabolites, then the triene/tetraene ratio alone becomes inadequate as a measure of such deficiency. A rise in this ratio simply indicates a specific lack of EFAs of the 18 : 2~6 series. Defects in the desaturation and elongation stages which also affected the formation of 20 : 3w9 could thus be associated with severe functional EFA deficiency without any detectable change in the triene/tetraene ratio. Other biochemical measures of EFA deficiency based on measurements of such acids as 20 : 3~6 and 20 : 4~6 in the key lipid fractions are therefore urgently needed. The changes in thymus and adrenal weight are of particular interest. Thymus atrophy was profound in the ZD group, as is to be expected from the key role of zinc in the thymus and in the immune system [34]. Primrose oil, but not safflower oil, restored adrenal and thymus weight to nearly normal values (there was no significant difference for these weights between group Z and ZDP). This suggests that modulation of EFA metabolism may be the most important aspect of the effect of zinc on the immune system, possibly because it will lead to inadequate formation of prostaglandin El, a substance which seems to be particularly important in T lymphocytes [ 341. The rise in adrenal weight might be related to failure of an adrenal secretion and a consequent compensatory hypertrophy. The possibility that the rise in adrenal corticosterone output which can occur in zinc deficiency could account for the thymic atrophy has previously been investigated [35]. The conclusion was that the greater part of the thymus atrophy was not secondary to increased adrenal activity. The lack of effect of zinc on the growth and fatty acid profiles of EFA-deficient rats strongly suggests that zinc has no effect on body growth and maintenance of the dermis unless EFA are present in the tissues. Furthermore, it
206
confirms that the similarity of zinc and EFA deficiencies is not coincidental but that the two have a significant interaction. As noted earlier, the relationship between zinc and 18 : 2~6 metabolism is of potential importance to the understanding of dietary influences on atherosclerosis. In order for 18 : 206 to be effective as an antithrombotic, antihypertensive agent [18,19] its metabolism to 18 : 306 must be assured. Our data would suggest that zinc is necessary for this conversion to proceed normally. In addition to its apparent role in 18 : 206 metabolism, zinc is thought to increase the synthesis of prostaglandin El from 20 : 3~6 [31]. Essential fatty acid supplementation favors an increase in tissue 20 : 3~6 over 20 : 4~6 [ 361, with a consequent increase in prostaglandin E, synthesis, particularly in platelets [37]. Prostaglandin El is both an anti-aggregatory and also a vasodilatory prostaglandin [38]. Hence, the beneficial effect of essential fatty acids in the prevention or reduction of hypertension, atherosclerosis, and other forms of heart disease [18,19] may depend to a large extent on an adequate availability of zinc. Acknowledgements The skilful technical assistance of M. Paquette, L. Boulet and M. Tremblay is gratefully acknowledged. The advice of Dr. S. Lussier-Cacan on the nutritional aspect of this study is deeply appreciated. References 1 Bettger. W.J., Reeves, P.G., Moscatelli, E.A. and O’Dell. B.L., Interaction of zinc and essential fatty acids in the rat, J. Nutr., 109 (1979) 480. 2 O’Dell, B.L., Reynolds. G. and Reeves, P.G., Analogous effects of.zinc deficiency and aspirin toxicity in the pregnant rat, J. Nutr., 107 (1977) 1222. 3 Cunnane. S.C., Honobin, D.F., Ruf. K.B. and Sella, G.E.. Prevention of dietary effects of zinc deficiency by administration of essential fatty acids, J. Physiol. (Land.). 296 (1979) 113P. 4 Cash, R. and Berger, C.L.. Acrodermatitis enteropathica - Defective metabolism of essential fatty acids. J. Pediat., 74 (1969) 717. 5 Cunnane, S.C. and Horrobin, D.F.. Parental linoleic and gamma-linolenic acids ameliorate the gross effects of zinc deficiency, Proc. Sot. Exp. Biol. Med., 164 (1980) 583. 6 Curmane, S.C., Horrobin, D.F., Sella. G.E. and Ruf, K.B., Essential fatty acid supplementation inhibits the effect of dietary zinc deficiency. Adv. Prostag. Thromboxane Res., 8 (1980) 1797. 7 Brenner, R.R., The oxidative desaturation of unsaturated fatty acids in animals. Mol. Cell. Biochem.. 3 (1974) 42. 8 Brenner, R.R.. Metabolism of endogenous substrates by microsomes, Drug Metab. Rev., 6 (1977) 155. 9 Rivers, J.P.W., Sinclair. A.J. and Crawford, M.A., Inability of the cat to desaturate essential fatty acids, Nature (Land.), 258 (1975) 171. 10 Frank& T.L. and Rivers, J.P.W.. The nutritional and metabolic impact of gamma-linolenic acid on cats deprived of animal lipid, Brit. J. Nutr.. 39 (1978) 227. 11 Chvapil, M., Ludwig, J.C., Sipes, I.G. and Misiorowski, R.L., Inhibition of NADPH oxidation and related drug oxidation in liver microsomes by zinc. Biochem. Phannacol.. 25 (1976) 1787. 12 Henzel, J.H.. Holtmann, B.. Keitzer, F.W., De Weese. M.S. and Lichti, E., Trace elements in atherosclerosis. efficacy of zinc medication as a therapeutic modality. In: D.D. Hemphill (Ed.), Trace Substances in Environmental Health. Vol. 2, University of Missouri. Columbia, MO. 1969, p. 83. 13 Pories. W.J. and Strain, W.H., Zinc sulphate therapy in surgical patients. In: W.I. PO&S. W.H. Strain, J.M. Hsu and R.L. Woolsey (Eds.), Clinical Applications of Zinc Metabolism, C.C. Thomas, Springfield, IL, 1974, p.139. 14 Volkov, N.F., Cobalt, manganese and zinc content of blood and internal organs of patients with atherosclerosis, Jer. Arkh., 34 (1962) 52. 15 Netsky, M.G., Harrison, W.W.. Brown. M. and Benson, C.. Tissue zinc and human disease -Relation
207 of zinc content 16 17
18
19
20 21 22 23 24 25 26 27
28 29 30 31
32 33
34
35 36 37 38
of kidney
liver and lung to atherosclerosis
and hypertension,
Amer. J. Clin. Path.,
51
(1969) 358. Kaindl, F., Kuhn, P.. Holzhey, P. and Niederberger, M., Herztherapie mit Zink-Protamin-Glucagon, Verh. Dtsch. Gee.. Inn. Med., 78 (1972) 1099. Klevay, L.M., Interactions among dietary copper, zinc and the metabolism of cholesterol and phospholipids. In: H.G. Hockstra, J.W. Suttie, H.E. Ganther and W. Mertz (Eds.), Trace Element Metabolism in Animals, Vol. 2. University Park Press, Baltimore, MD. 1974, p. 553. Vergroesen, A.J., de Deckere, E.A.M., Ten Hoor, F. and Hornstra. G., Cardiovascular effects of linoleic acid. In: C. Galli and P. Avogaro (Eds.). Progress in Food and Nutrition Science, Vol. 4, Pergaman, New York. NY, 1980. p. 13. Oster. P., Arab, L.. Schellenberg, B., Kohlmeier, M. and Schlierf, G.. Linoleic acid and blood pressure. In: C. Galli and P. Avogaro (Eds.), Progress in Food and Nutrition Science, Vol. 4, Pergamon. New York, NY, 1980, p. 39. Folch, J., Lees, M. and Sloane-Stanley, H., A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem.. 226 (1957) 497. Block. W.D.. Jarrett, K.J. and Levine, L.B.. An improved automated determination of serum total cholesterol with a single color reagent, Clin. Chem.. 12 (1966) 681. Kraml. M. and Cosyns, L., A semi-automated determination of serum triglycerides, Clin. Biochem., 2 (1969) 373. Morrison, W.R. and Smith, L.M.. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride--methanol. J. Lipid Res., 5 (1964) 600. Zar, J.H.. Multiple comparisons. In: Biostatistical Analysis. Prentice-Hall Inc., Englewood Cliffs, NJ, 1974, p. 151. Fraker, P.J., Haas. S.M. and Luecke. R.W.. Effect of zinc deficiency on the immune response of the young adult A/J mouse, J. Nutr., 107 (1977) 1889. Patel, P.B.. Chung, R.A. and Lu. J.Y., Effect of zinc deficiency on serum and liver cholesterol in female rats, Nutr. Rep. Internat., 12 (1975) 205. Burch, R.E., Williams, R.V.. Hahn, H.K.L. Jottor, M.M. and Sullivan, J.F., Serum and tissue enzyme activity and trace-clement content in response to zinc deficiency in the pig, Clin. Chem., 21 (1975) 568. Marcus, A.J., Ullman. H.L. and Safier. L.B., Lipid composition of subcellular particles of human blood platelets, J. Lipid Res.. 10 (1969) 108. Crawford, M.A.. Denton, J.P.. Hassam, A.G., Lynn, J., Marples. P., Stevens, P. and Willis, A.L.. Levels of prostaglandins and their precursors in EFA deficiency, Brit. J. Pharmacol.. 63 (1978) 363P. Legarde, M.. Dechavanne, M., Rigaud. M. and Durand, J., Bass’ level of human platelet prostaglandins - PGEl is more elevated than PGE2, Prostaglandins, 17 (1979) 685. Manku, M.S.. Horrobin. D.F., Karmazyn, M. and Cunnane. S.C., Prolactin and zinc effects on rat vascular reactivity - Possible relationship to dihomogammahnolenic acid and to prostaglandin synthesis, Endocrinology. 104 (1979) 774. Koo, S.I. and Turk. D.E., Effect of zinc deficiency on intestinal transport of triglycerides in the rat, J. Nutr.. 107 (1977) 909. Muira. S., Study on the fat absorption and transportation into intestinal lymph of rats -Differences in the absorption of saturated and unsaturated long chain fatty acids and the role of intestinal alkaline phosphatase. Nippon Shokakibyo Gukkai Sasshi, 76 (1979) 871. Horrobin, D.F., Manku. M.S., Oka, M., Morgan, R.O., Cunnane, S.C., Ally. A.L., Ghayur. T., Schweitzer. M. and Karmali, R.A., The nutritional regulation of T lymphocyte function, Med. Hypotheses, 5 (1979) 969. De Pasquale-Jardieu. P. and Fraker, P.J.. The role of corticosterone in the loss in immune function in the zinc deficient A/J mouse, J. Nutr., 109 (1979) 1847. Holman, R.T., Polyunsaturated fatty acid profiles in human disease, Progr. Clin. Biol. Res., (1981) In press. Stone, K.J., Willis, A.L., Hart, M., Kirtland. S.J.. Kernoff, P.B.A. and McNichol, G.P., The metabolism of dihomo-gamma-linolenic acid in man, Lipids, 14 (1979) 174. Mitchell, J.R.A., Prostaglandins in vascular disease - A seminal approach. Brit. Med. J.. 282 (1981) 590.