Origina6a

Role of Lipid Peroxidation and Vitamin E in Magnesium Deficiency

1 1 1 2 2 \ T. Giinther ), J. Vonnann ), V. Hollriegl ), G. Disch ), H.-G. Classen )

Zusammenfassung Summary Resume Es wurde untersucht, ob Lipidperoxidation an The aim of the study was to ascertain whether Le but de la presente etude etait de verifier den Erscheinungen des Mg-Mangels beteiligt lipid peroxidation is involved in the effects of l'eventuelle implication de la peroxydation ist. Dazu wurde 115 g schweren mannlichen Mg deficiency. Male Wistar rats, weighing 115 des lipides dans les effets des deficits en ma­ Wistarratten eine Kontroll-, eine Mg-arme, g, were fed a control, an Mg-deficient, an Mg­ gnesium. Des rats Wistar males pesant 115 g eine Mg-arme plus Vitamin E-arme und eine deficient plus vitamin E-reduced and an Mg­ ont re<;u pendant 11 semaines les differents Mg-arme Vitamin E-reiche Diat 11 Wochen deficient-vitamin E-supplemented diet for 11 regimes alimentaires suivants: regime temoin, lang verftittert. Anschlief:lend wurden Vita­ weeks. At the end of the experiment, vitamin regime carence en Mg, regime carence en Mg min E, Malondialdehyd und der Mineralge­ E. malondialdehyde and mineral content were et pauvre en vitamine E et, enfin, regime halt in Serum, Leber, Herz, Nieren, Aorta, measured in serum, liver, heart, kidney, aorta, carence en Mg et supplemente en vitamine E. Hoden und Skelettmuskel gemessen. testis and skeletal muscle. Mg deficiency re­ A la fin de !'experimentation, les auteurs ont Mg Mange[ verminderte den Vitamin E-Ge­ duced vitamin E in serum and all examined mesure les taux seriques, hepatiques, cardia­ halt im Serum und alien untersuchten Gewe­ tissues. Vitamin E supplementation of Mg­ ques, renaux, aortiq ues, testiculaires et muscu­ ben. Vitamin E-Supplementation erhi:ihte den deficient rats produced only a slight increase losquelettiques de vitamine E, d'aldehyde a-Tocopherolgehalt besonders in der Leber. in a-tocopherol levels, except for the liver, malonique et de substances minerales. Mg-Mangel erh6hte den Malondialdehyd­ which stored a considerable amount. Le deficit magnesique a reduit les concentra­ Gehalt, besonders bei Vitamin E-reduzierter Mg deficiency increased malondialdehyde, tions de vitamine E dans le serum et dans to us Diat. Die durch Mg-Mangel hervorgerufenen particularly in rats also fed the low vitamin E les tissus examines. Une supplementation en Veranderungen im Mineralgehalt der Gewe­ diet. Mgdeficiency-induced alterations in min­ vitamine E chez les animaux carences en Mg a be, die Erytheme, die malign en T-Zellympho­ eral content of the tissues, erythema, develop­ entralne une augmentation des taux d'a-toco­ me, Leukamien und Tumore wurden durch ment of malignant T cell lymphoma, leukemia pherol, en particulier dans le foie. Les altera­ Vitamin E-S upp lementation nicht verhindert. and development of tumors were not pre­ tions de la teneur tissulaire en substances Nurdie durch Mg-Mangel entstandenen Hau­ vented by vitamin E supplementation. Only minerales, les erythemes, les lymphomes des tulzerationen wurden durch Vitamin E signifi­ skin ulcerations in Mg deficien~)' were signifi­ cellules T, les leucemies et les effets tumorige­ kant vermindert. cantly reduced by vitamin E. nes induits par la carence magnesique n'ont pas ete minimises par la supplementation en vitamine E, n'a reduit significativement

Introduction content, cardiac necroses and in­ lead to the increased ea-dependent creased collagen content in heart and release of some hormones, such as Significant effects of experimental neuromuscular hyperexcitability [1, 2, catecholamines, prostaglandins and Mg deficiency in rats are transient 3, 4, 5]. related substances [6]. erythema and edema, skin lesions, The basic pathobiochemical mechan­ Moreover,inMg deficiency, increased reduced growth, development of ma­ ism ofMg deficiency may be an altera­ lipid peroxidation (LPO) was found lignant T-celllymphoma and leuke­ tion of the Mg-Ca-antagonism in the in isolated liver mitochondria [7] and mia, development of intestinal tumors, extracellular fluid and at membranes in liver in vivo [8]. Mg deficiency­ calcification ofkidneys and large blood 2 due to reduced extracellular Mg ' con­ induced cardiac necrosis was effec­ vessels, changed intracellular mineral centration [6]. This effect is an explan­ tively reduced in a dose-dependent ation for the Mg deficiency-increased manner by simultaneous adminis­ cell membrane permeability and ion tration of vitamin E to Mg deficient 1 ) Institute of Molecular Biology and Bio­ turnover and the increased effect of Syrian hamsters [9 ]. Since vitamin E is chemistry, Free University of Berlin, FRG 2 hormones on smooth muscle cells act­ ) Department of Pharmacology and Toxi­ one of the most effective scavengers 2 2 cology of Nutrition, ing via Ca + influx and rise in [Ca +]i. of free radicals [10], these results sug­ University of Hohenheim, Stuttgart, FRG The altered Mg-Ca ratios may also gest that free radical mechanisms are

Magnesium-Bulletin 14, 2 (1992) 57 Lipid Peroxidation in Magnesium Deficiency

involved in Mg deficiency-induced per cage Type Ill) were kept at a VitaminE was determined by its fluor­ myocardial injury. light-dark cycle from 8 a.m. to 8 p.m. escence in hexane extracts according The same relationship may hold for at a temperature of 21 ± 1 oc and a to Taylor et al [20]. human ischemic heart disease (IHD). relative humidity of 50--60 % for 11 Malondialdehyde (MDA) was deter­ Epidemiological studies revealed a weeks. Body weight was measured mined by a variation of the thiobar­ strong inverse correlation between weekly. bituric acid (TBA) method [21, 22]. A IHD mortality and the serum level of At the end of the experiment the rats 20% homogenate in 150 mmol/1 KCl vitamin E, whereas the classic risk were anesthesized with nembutal (or serum) was diluted 1:1 (v/v) with factors cholesterol and diastolic blood (50 mglkg i.p.) and blood, liver, kid­ 5 % trichloroacetic acid (TCA) and pressure had only a moderate asso­ neys, heart, testes and aorta were centrifuged for 5 min at 13 000 x g. ciation with IHD [11]. Alteration of taken, frozen in liquid nitrogen and 500 j..tl TBA (1%, pH7) was added to ion permeability and LPO, adversely stored at 20 oc. 500 j..tl supernatant and heated at 95 oc affected by Mg deficiency, may act Intestines were inspected for intes­ for 15 min. After cooling, the probes in accord. An increase in intra­ tinal tumors [17]. Thymus was in­ were extracted with 3 ml1-butanol by cellular Ca2~ can enhance LPO [12]. spected formalignent T celllymphoma vortexingfor 30 sec and centrifugation Mg deficiency-increased release of [18], removed and weighed. Leuco­ at 2100 g for 15 min. MDA in the catecholaminesmayincrease LPO [13] cyte content was countedinlymphoma butanol phase was measured fluor­ and Mg deficiency-induced alteration bearing rats by means of a Neubauer ometrically (Perkin Elmer LS 50, ex­ of fatty acids in phospholipids may chamber. citation: 532 nm, emission: 553 nm, slit increase LPO. Increased LPO may Blood was centrifuged at 1000 g for width: 5 nm). lead to an increase in membrane per­ 5 min. The concentrations of Mg, Ca, The calibration curve was prepared meability [14] and may enhance the and Fe in serum were measured by with malonaldehyde tetraethylacetal effects of Mg deficiency. Since LPO atomic absorption spectrophotometry (Sigrna ), which was treated in the same may be a general pathological mechan­ (AAS). way. Statistical analysis was performed ism [14], LPO may play a significant Portions of the livers and hearts were as indicated in the tables. role in the pathology ofMg deficiency. freeze-dried. Dried liver was powd­ To clarify the realtionship between ered in a plastic mortar, freeze-dried Mg deficiency and LPO, we fed a low hearts were powdered by means of a Results and Discussion vitamin E diet, and a vitamin E sup­ vibrating steel ball (Mikro-Dismem­ plemented diet to rats on a Mg de­ brator, B. Braun, Melsungen, FRG). ficient diet. For determination of Na, K, Mg, Ca, Vitamin E content (tab. 2) and Fe in livers and hearts, freeze­ In serum and all investigated tissues, dried, powdered tissue was ashed in vitamin E content was reduced by MateriaJs and Methods the Plasma Processor 200-E (Tech­ Mg deficiency. This reduction was es­ nics, Miinchen, FRG). The ash was pecially pronounced in serum and After obtaining approval of local dissolved in 0.1 N HCl. N a and K liver. When Mg deficiency was com­ authorities and the Animal Protection were measured by flame photometry bined with vitamin E reduction, the Committee, the experiment with 175 (Klina, Beckman), Mg, Ca and Fe vitamin E content was drastically re­ male Wistarrats, weighing115 g(Inter­ were measured by AAS (Philips, duced, particularly in liver. After feed­ fauna, Tuttlingen, FRG) was under­ SP9). ing the vitamin E-supplemented diet taken. Males were used, because of An aliquot of freeze-dried, powdered to Mg-deficient rats, vitamin E con­ their greater susceptibility to free-radi­ hearts was taken for determination of tent in serum and tissues was some­ cal-induced hepatotoxicity [15]. A collagen content by means ofmeasure­ what higher than in controls. How­ control diet (described elsewhere [16]) ment of hydroxyproline according to ever, in liver, vitamin E was markedly was fed to 25 rats (group A); Mg­ Stegemann [19]. increased. deficient diet was fed to 150 rats, de­ These results indicate that liver cells vided into 3 groups: group B (Mg store vitamin E, and this store can deficiency alone), group C (also vita­ Tab.l: ContentofMg. Ca,FeandvitaminEin be more depleted in vitamin E de­ control (A), Mg-deficient (B), Mg-deficient min E reduced), and group D (fed plus vit E-reduced (C) and Mg-deficient-vit E­ ficiency as compared with other excess vitamin E supplements). Omis­ supplemented (D) diets. tissues. sion of Mg and vitamin E from the Dietary Zn deficiency also reduces control diet produced the respective the vitamin Econtentofratserum, the deficient diets (provided in pellets by Mg (mmol/kg) 26.5 :1.3 .:1.7 3.1 only source studied [23]. The authors Ssniff (Soest, FRG). Tab. 1 provides Ca (mmol/kg) 272 249 249 247 suggested that absorption and trans­ the Mg, Ca, Fe and vitamin E contents Fe (mmol/kg) 3.24 3.29 2.97 3.10 port of tocopherol by the intestinal of the diets. Food and deionized water mucosa and the blood transport sys­ 325 325 92 6965 were provided ad libitum. The rats (5 tem may be affected [23]. However,

58 Magnesium-Bulletin 14,2 (1992) Lipid Peroxidation in Magnesium Deficiency

Tab. 2: Vitamin E content of serum (in llmol/1 a-tocopherol) and various tissues (in llffiOI a­ our results on MDA (tab. 3), showing tocopherol/kg wet weight) of control (A),Mg-deficient (B), Mg-deficient plus vitE-reduced (C) the reciprocal behavior of vitamin E and Mg-deficient-vitE-supplemented (D) rats. and MDA (fig.l ), suggests rather that reduction of vitamin E is caused by its A B c D destruction due to free radicals. Since lipid peroxidation due to oxygen free Serum 42.6 + 3.7 (10) 26.0 + 2.4 (10)b 5.92 + 0.67 (10)c 50.8 .!:. 3.7 (10) radicals is also increased by Zn de­ Liver 111.7 + 7.2 (6) 63.0 + 1.8 (6)c 4.30 1.30 (6)c 465.5 + 53.4 (6)c ficiency [24, 25), the reduction of Heart 108.5 + 6.5 (6) 83.2 + 3.6 (6)b 19.92 +. 2.30 (6) c 135.0 .:::. 7.3 (6)a plasma vitamin E by Zn deficiency may also be caused by its destruction Kidney 57.0 + 2.9 (6) 46.0 1.6 (6)b 10.74 + 1.76 75.7 .:::. 4.9 due to free radicals. Supporting this Testis 77.1 + 4.8 (8) 58.0 + 3.8 (10)b 16.69.:::. 1.97 (6) c 82.2 6.2 (6) .:': concept is the increased formation of Skeletal . 43 1 + 2.2 (10) 32.1 10.24 + 1.28 (6)c 65.5 4.7 (6)b muscle .:::. 1.8 .:. free radicals by Fe-loading, and the increased cardiac and hepatic Fe con­ tent in Mg deficiency (see tab. 8, 12). Mean + SEM. Number cf rats in brackets. Significant d1fferences to control rats by unpaired Student's t-test. a, p < 0.05; b, p < 0.01; c, p < 0.001. Malondialdehyde content (tab. 3) 3.0 MDA content, which was much higher in kidney than in other tissues, was increased by Mg deficiency (group ...... 2.5 \ B), particularly when also low in vita­ s:: . \ "'. \ .....Ch .,.. min E (group C). After vitamin E QJ .t( •• supplementation ofMg-deficient rats 31: \ ·. 2.0 \ \ (groupD),MDAcontentwasreduced ...... \ QJ \ to control values. Thus, MDA is reci­ 31: \ \ \ procally correlated to the free radical Ch 1.5 \ ~ \ \ scavenger vitamin E, indicating in­ ...... \ \ A 0 \ creasedformation of oxygen free radi­ e a', 0 - 1.0 \ cals and increased LPO in Mg defi­ ..:! \ \ ciency, which can be normalized by \ \ excess vitamin E. Alternatively, the < ~ 0 0.5 \ ::::E \ formation of oxygen free radicals may A be unchanged but scavenging of oxy­ 0.0 gen free radicals may be reduced in 0 50 100 150 Mg deficiency. So far, there is no in­ dication for this possibility. In any vitamin E [umol a-tocopherol/kg wet weight] case, there is an increased action of Fig. 1: Negative correlation between MDA (LPO) and vitamin E content of heart (0. r = oxygen free radicals in Mg deficiency. -0.9765), testis (il, r =-0.9789) and skeletal muscle (D, r = -0.9926). Data represent means of A more detailed analysis of the MDA group A, B and C from Table 2 and 3. and vitamin E contents of the tissues Tab. 3: Malondialdehyde content of serum (in llffiOI/1) and various tissues (in llmol/kg weight) from group A, B and C revealed an of control (A), Mg-deficient (B), Mg-deficient plus vitE-reduced (C) and Mg-deficient-vitE­ excellent negative correlation between supplemented (D) rats. MDA and vitamin E for vitamin E contents up to 50-110 f..lll101Jkg wet A B c D weight,dependingonthetissue(fig. 1). The negative correlation was much Serum 0.071 + 0.012 {8) 0.127 + 0.008 (10)b 0.108;+: 0.006 (lO)a 0.088 ;+: 0.005 (10) less profound when the values of vita­ Liver 0. 70 + 0.05 (6) 2.03 + 0.42 (6) a 15.34 + 2.63 (6)c 0.80 + 0.08 (6) min E-supplemented rats were in­

Heart 1. 06 + 0.03 (6) 1. 82 0.22 (9)b 2.65 + 0.35 (10)c 1.35 + 0.14 (8) cluded (data not shown). It appears, thus, that with high tissue vitamin E Kidney 8.25 + 0.82 (10) 23.16 .!:. 2.50 (10)c 34.68 ;+: 3.58 (10)c 10.99 + 1.69 (10) levels (exceeding a threshold), there Testis 0.32 + 0.02 (8) 1.21 + 0.20 (8)c 2.11 + 0.31 (6) c 0.31 + 0.06 (6) is not a further effect on MDA pro­ Skeletal . 0 59 + 0.04 (9) 1.03 + 0.17 (10)b 2.57 + 0.60 (6)b 0.99 + 0.12 (8)b duction. Increased oxygen radical for­ muscle mation and LPO in Mg deficiency may be mediated by increased intra­ Mean + SEM. Number of rats in brackets. Significant differences to control rats by unpaired Student's t-test. cellular contents of Fe and Ca, in­ a, p < 0.05; b, p < 0.01; c, p < 0.001. creased release of catecholamines and

Magnesium-Bulletin 14, 2 (1992) 59 Lipid Peroxidation in Magnesium Deficiency

2 changed contents of polyunsaturated and sequestration of Ca + in the intra­ Thus, Mg deficiency may increase the fatty acids of membrane phospho­ cellular compartments [36]. This ef­ action of oxygen free radicals and LPO lipids. Moreover, in Mg deficiency the fect may be caused either by altered by various mechanisms leading to in­ synthesis of prostaglandins, parti­ phospholipids ( e.g.lysophosphatides) creased [Ca2+]i [14], and on the other cularly of thromboxane Az, in associ­ within the cell membrane or by the hand, increased [Ca2+]i can increase ation with increased formation of oxy­ products of LPO. LPO [12]. gen radicals, was increased. For de­ Increased generation of oxygen radi­ The effects of LPO with respect to 2 tailed literature and discussion ofthese cals decreased the Ca content of car­ increased Ca + influx are similar to the pathobiochemical mechanisms see diac sarcoplasmic reticulum by en­ effects of Mg deficiency. Ref. [6, 7, 8]. hancing passive Ca permeability of Thus, some of the effects of Mg de­ The MD A levels in serum and tissues this organelle [26, 37]. In heart muscle ficiency may be caused by increased represent steady state concentrations cells, oxygen free radicals increased LPO. When this mechanism plays a 2 2 and not formation of MDA, because [Ca + ]i via a rise in N a+/Ca ' exchange role in Mg deficiency, it can be ex­ MDA is rapidly metabolized by mito­ and altered the electrical function of pectedthattheeffectsofMgdeficiency chondrial aldehyde dehydrogenase myocardial cells [38]. On the other are augmented by additional vitamin and is excreted by the kidneys [27,28]. hand, an increase in Na+/Ca2+can en­ E reduction and may be reduced by Moreover, the formation of free radi­ hance the generation of oxygen free additional vitamin E supplementation. cals in Mg deficiency may be higher radicals [39]. At reduced extracellular To test the premise that free radicals than indicated by LPO and MDA Mg concentration as in Mg deficiency and LPO influence Mg-deficiency­ formation, because a part of free radi­ this increase in [Ca 2+]i can occur via an induced changes, the effect of vitamin cals was scavenged by vitamin E, lead­ increase in [Na+]i and/or by altered E deficiency and excess, in the 2 2 ing to destruction of vitamin E in Mg extracellular Mg +/Ca + interaction [40, presence of Mg deficiency was deter­ deficiency (tab. 2 see above). Oxygen 41,42]. mined. free radicals or lipid peroxyl radicals g transform vitamin E to its phenoxy 600 radical [29], followed by resonance stabilization [30] or secondary re­ actions of the vitamin E radical. Prob­ ably, renal excretion of MDA and 500 enrichment of MDA (potentially by binding to protein) may be respon­ sible for the high level of MDA in kidneys. Comparison of our MDA values with 400 those of other investigators [31, 32, 33] showed, that in serum they were lower by the factor 80 [31] or 50 [32, 33] and in liver by the factor 5 [33]. In an extensive discussion on MDA deter­ 300 mination it was stated that MDA con­ centration in plasma is less than 0.1 !lJUol/1 [34] as found by our method. Of particular importance is the ques­ 200 tion whether LPO or some of its pro­ ducts may contribute to the patho­ logical effects of Mg deficiency. As shown with cultured fibroblasts, the products of LPO such as linoleic 100~ acid peroxide, MDA and 4-hydroxy­ nonenal are cytotoxic at concen­ trations above 10' 7 molll and vitamin E can protect against peroxidation OL___ L_ __ L_ __ L_ __ L_ __ ~--~------~------~--' damages [35]. In hepatocytes, LPO can change the 0 1 2 3 4 5 6 7 8 9 10 11 functions ofverapamil-and nifedipine­ weeks 2 sensitive Ca + channels, resulting in a Fig. 2: Growth of rats fed control ( 0 ), Mg-deficient Mg-deficient plus vitamin E-reduced net influx of Ca 2+, an increase in [Ca2+], (*)and Mg-deficient-vitamin E-supplemented (8) diet.

60 Magnesium-Bulletin 14,2 (1992) Lipid Peroxidation in Magnesium Deficiency

Erythema Tab. 4: Body weight, weight of thymus and left testis of control (A), Mg-deficient (B), Mg­ deficient plus vitE-reduced (C) and Mg-defieient-vitE-supplemented (D) rats at the end of the When serum Mg concentration was experiment. drastically reduced after feeding an Mg-deficient diet to rats, the animals A B c 0 developed edema due to liberation of c histamine from mast cells [43]. Body weight 520 + 2 306 + 2 316 + 2 c 281 + 2 c In the present experiments all rats developed erythema (seen particularly Thymus 0.85 + 0.02 0.46 + 0.01 c 0.47 + 0.01 c 0.40 + 0.01 c from red ears) with a peak at day 7 Testis 1.73 + 0.01 1.95 + 0.02 c 2.01 + 0.01 c 2.12 + 0. 02 c after starting with the Mg-deficient diet, and at day 12 the erythema had disappeared in all groups (data not Values in g. Mean + SEM of 20 rats in each grou::J. Significant differences to control rats by unpa1red Student's t-test. · shown). c, p < 0.001. There was no significant difference in erythema development among the pair-fed groups were omitted. Weight of the vitamin E-supplemented Mg­ experimental groups, indicating that gain was severely reduced in Mg de­ deficient diet was somewhat lower erythema may be caused by altered ficiency. Additional reduction of vita­ than in the other diets (tab. 1). extracellular Mg-Ca antagonism and min E did not significantly change ea-dependent histamine release, in­ body weight, indicating that the Mg Weight of thymus and testis (tab. 4) dependent of oxygen free radicals. content of the diets was the growth­ During Mg deficiency the weight of limiting factor. the thymus was reduced, but the body Body weight (fig. 2) In agreement with this conclusion, the weight/thymus weight remained con­ Since the study explored onlywhether growth-rate of the vitamin E-supple­ stant. the effects of Mg deficiency can be mented Mg-deficient group was some­ The weight of the testes of Mg-de­ influenced by vitamin additional what lower, because the Mg content ficient and Mg-deficient plus vitamin

Tab. 5: Skin ulcerations of control (A), Mg-deficient (B), Mg-deficient plus vitE-reduced (C) and Mg-deficient-vitE-supplemented (D) rats.

A B c D

day 0 1 2 0 1 2 0 1 2 0 1 2

0 0/25 0/50 0/50 0/50

14 24/24 0/24 0/24 41/49 5/49 3/49b 42/49 4/49 3/49c 43/46 3/46 0/46

24 24/24 0/24 0/24 42/49 4/48 3/49a 35/47 9/47 3/47 8 44/46 1/46 1/46g

31 24/24 0/24 0/24 43/49 3/49 3/49° 37/47 6/47 4/47d 44/46 1/46 1/46f

36 24/24 0/24 0/24 42/49 6/49 1/498 39/47 4/47 4/47d 43/46 2/46 1/46f

42 24/24 0/24 0/24 43/49 4/49 2/498 38/47 6/47 3/47d 42/44 1/44 1/44f

0, no ulcerations; 1, light ulcerations; 2, severe ulcerations. Volues represent number of rats I total number of rats. Differences to a total of 25 or 50 reflects number of dead rats per group.

For statistical analysis light and severe ulcerations were taken together. Statistical analysis was performed according to Chi 2 -test. Significance between group A and B: a, p < 0.10 b, p < 0.05 Significance between group A and C: c, p < 0.10 d, p < 0.05 e, p < 0.01 Significance between group C and D: f, p < 0.05 g, p < 0.01

Magnesium-Bulletin 14, 2 (1992) 61 Lipid Peroxidation in Magnesium Deficiency

Tab. 6: Development of thymus lymphoma and abdominal tumors. Development oftumors (tab. 6) A, Control. B. Mg-deficient rats. C, Mg-deficient plus vitE-reduced rats. D, Mg-deficient-vitE­ supplemented rats. In preceding experiments [17] a tumor­ likeconnective tissue proliferation was Weight of Weight of Number of Diameter of abdom­ Group lymphoma (g) spleen (g) leucocytes (~l-1) inal tumor (cm) observed in the intestine of Mg-de­ ficient rats. In the present experiment A with less severe Mg deficiency, we did not find this alteration. B However, we observed 3 tumors 0 10.3 3.1 5.800 originating from the muscle layer of 10.0 4.7 14.000 the abdominal wall. There was also 3.500 1.5 3.200 1.0 one tumor in Mg-deficient vitamin E­ supplemented rats. Therefore, it re­ D 4.7 0.8 4.200 9.5 4.4 80.000 mains open whether the development 8.3 3.5 21.600 of these tumors is dependent on free 3.300 1.5 radicals and MDA, which have been implicated in tumor development I'l group A and 8, there were no tumors. The values in the same line belong to [27, 46]. The mechanism of tumor de­ the same rat. (2 rats with lymphoma and 2 rats with abdominal tumors in group 0, 3 ::cats with lymphoma and 1 rat with abdominal tumor in group 0). velopment in Mg deficiency is un­ known. E-reduced rats was not reduced but malignant T cell lymphoma of the slightly increased. Since reduction of thymus (18). In the present experi­ testis weight is characteristic of vita­ ment the rate of lymphoma was lower Electrolyte and mineral content of min E deficiency [44], this result shows (3.5 % ), the reasons probably being serum and tissues that the vitamin E content of the vita­ the following: min E-reduced diet was still sufficient In our preceding experiments we used Serum (tab. 7) to prevent classic vitamin E deficiency female rats, whose body weight at symptoms, although the reduced con­ start of the experiment was lower (70- Mg concentration of all groups, fed tents of vitamin E in serum and tissues 80 g) and a more severe Mg deficiency the Mg-deficient diet was markedly of group C led to a further increase in had been induced. reduced, indicating severe Mg de­ MDA compared to group B. In parallel to the development of ficiency. Serum Ca concentration was thymus lymphoma, the spleen was not significantly changed, but serum Skin lesions (tab. 5) enlarged and the number of leuco­ Fe was somewhat reduced (tab. 7). During Mg deficiency, some of the cytes in blood was increased. Since Since the Fe content of some tissues Mg-deficientrats (group B) developed the vitamin E-supplemented Mg-de­ was increased (tab. 8, 9, 12) and Fe ulcerations of the skin, the pathologic ficient rats (group D) had even more excretion is minimal, the reduction of mechanism of which is unknown. In thymus lymphomata than did the Mg­ serum Fe by Mg deficiency may be the Mg-deficient plus vitamin E-re­ deficient vitamin E low rats (group caused by intracellular Fe uptake. duced group (C) the number of ul­ C), it appears that free radicals do not Possibly, cellular Fe uptake which is cerations was higher than in group B play a role in the development of Mg performed by internalization of the and in the Mg-deficient vitamin E­ deficiency-induced thymus lymph­ Fe transferrin complex [47] is increased supplemented group (D), the number oma. in Mg deficiency. of ulcerations was lower than in group Band C. It appears that free radicals are in­ Tab. 7: Mg, Caand Fe concentration in serum of control (A), Mg-deficient (B), Mg-deficient plus volved in the development of skin vitE-reduced (C) and Mg·deficient-vitE-supplemented (D) rats. ulcerations. Possibly, increased num­ bers ofleucocytes, which produce free A 8 c 0 radicals [45], increase the need for vitamin E, which scavenge free radi­ Mg (mmo1/1) 0.75 .:':. O.C2 (10) 0.21 .:':. 0.01 (29)c 0.18 .:':. 0.01 (25)c 0.17 + 0.01 (23) c cals. ea (mmol/1) 2.47 + 0.05 (10) 2.53 .:':. 0.02 (10) 2.47 0.01 (10) 2.46 + 0.08 (10)

8 Development of thymus lymphoma Fe (~mol/1) 39.9 + 1. 4 (23) 33.3 + 2. 3 (11) ~ 31.0 + 3.0 (12)" 32.9 3.0 (12) (tab. 6)

Mean + SEM. Nurr.ber cf rats in brackets. SignificanL differences to control rats by In chronic severe experimental Mg unpaired Student's -::-test. deficiency, 13% of the rats developed a, p < 0.05; c, p < 0.001.

62 Magnesium-Bulletin 14, 2 (1992) Lipid Peroxidation in Magnesium Deficiency

Tab. 8.: K, Mg, Ca, Fe and hydroxyproline content in heart of control (A),Mg-deficient (B), Mg­ Kidney (tab. 10) deficient plus vitE-reduced (C) and Mg-deficient-vitE-supplernented (D) rats. Renal calcification, a typical con­ A 8 c D sequence of Mg deficiency in rats, was confirmed. Neither lowering dietary K 195 + 3 (6) 17C + 3 (10)c 182 + 3 (9)b 175 + 4 vitamin E nor providing an excess had a significant effect on kidney Ca con­ Mg 36.91 0.35 (8) 34.42 + 0.39 34.31+ 0.37 (9)c 33.69 + 0.37 (1G)c tent, indications that renal calcification Fe 6.12 + 0.15 (8) 6.84 0.09 (10)c 6.94 ~ 0.10 (9)c 7.C9 0.16 (10)c is an effect of lowered Mg/Ca ratio, 8 Ca 1.93 + 0.07 (8) 2.07 + 0.04 (10) 2.11 ~ 0.06 (9) 2.:.:~ ~ 0.03 (10) and is not caused by free radicals. Hydroxy- + 0.11 (8) 1.49 + 0.15 (8) + ore line 1.50 1.40 0.13 0.17 (9) Skeletal muscle (tab. 11) Values in mcnol/kg dry Number of rats in brackets. In Mg-deficient rats the skeletal Student; 1 s t-test. a, p < 0.05; b, p < 0.01; c, p < 0.001. muscle contents of Na and Ca were increased and the contents of K and Heart (tab. 8) tent among the experimental groups Mg were decreased. These findings, (tab. 8). Preceding experiments only which confirmed earlier studies (re­ During Mg deficiency, the contents of showed a strong increase in the myo­ view: 48), were not influenced by vita­ K and Mgwere decreased by approxi­ cardial hydroxyproline content, when min E reduction or supplementation. mately 10 % and the contents of Fe Mg-deficient rats were additionally Therefore, the alterations in mineral and Ca were increased by approxi­ chronically stressed, thus increasing content which are caused by in­ mately 10 %, confirming findings in catecholamine release (49, 50, 51], creased membrane permeability are other studies [48]. not caused by free radicals. Neither low nor high dietary vitamin Aorta (tab. 9) E influenced Mg-deficiency-induced alterations in mineral content. There­ Cardiac injury may also result from Liver (tab. 12) fore, under the conditions of the ischemia resulting from vascular calci­ Mg deficiency-induced alterations of present experiment the increased level fication and atherosclerosis such as Na, K, Ca and Mg content in liver of oxygen free radicals did not lead to has been induced by chronic Mg defi­ were less than in other tissues [8, 48] or cardiac electrolyte alterations. A ciency [52]. In addition, also LPO may did not occur at all [48]. However, the different result was found with Mg­ play a role in atherosclerosis [10, 53]. Mg deficiency-induced increase of Fe deficientSyrianhamstersin which vita­ As shown in tab. 9, Mg deficiency was greater in liver than in other min E supplementation could reduce caused a 10% decrease in cellular Mg tissues [8, 54]. the extent of cardiac lesions (9]. Prob­ content, a 20% increase in Ca and a 10 In the present Mg deficiency ex­ ably, the Syrian hamster is more sen­ to 30 % increase in Fe content. periment there were no significant sitive to Mg deficiency and LPO and/ However, vitamin E reduction and alterations in Na, K, Mg and Ca con­ or may release more catecholamines vitamin E supplementation had no tent. However, the Fe content in liver than the rat strain used in the present significant influence on the Mg de­ was much higher than in preceding experiment. ficiency-induced alterations. From this experiments, probably because of the In agreement with a lack of severe result it can be concluded that under longerexperimentalperiod(ll weeks cardiac injury in the present experi­ the conditions of the present experi­ compared to 2 weeks [54] or 4 weeks ment, there was no alteration in car­ ment, LPO had no effect on the alter­ [8]). diac hydroxyproline (collagen) con- ations of aortic mineral content. Additional vitamin E reduction and vitamin E supplementation had no significant effect on Fe content. Thus, Tab. 9: Mg, Ca and Fe content of aorta from control (A), Mg-deficient (B), Mg-deficient plus it can be concluded that it was the Mg vitE-reduced (C) and Mg-deficient-vitE-supplernented (D) rats. deficiency, not the free radicals, that induced the increase in liver Fe. Pos­ A 8 c 0 sible mechanisms may be: 1. Increased instability and de­ Mg 7.00 + 0.13 (24) 6.36 + 0.14 (21)b 6.34 ~ 0.17 (20)b (20) b 6.44 ~ 0.12 struction of erythrocytes [55], the Cs 8.86 .::. 0.18 (24) 10.48 + 0.31 (21) c 10.77 0.25 (21)c 10.63 :':. 0.27 (21)c released Fe being stored in the 8 Fe 2.40 + 0.14 (22) 2.68 + 0_23 (18} 3.10 + 0.16 (19) 2. 77 0.22 (10) liver. 2. Increased intestinal Fe absorption

because in experiments with mouse 1 Values in rnmoc/kg dry SEI-1. Number cf rats in brac~<:ets. Significant: duodenal fragments, addition of · differences ~o con~rol by Student's r.-test. a, p < 0. 05; b, p < 0. 01; c, p ( 0.001. Mg inhibited 59Fe uptake [56].

Magnesium-Bulletin 14, 2 (1992) 63 Lipid Peroxidation in Magnesium Deficiency

Tab.lO: Mg and Ca content of kidney from control (A), Mg-deficient (B), Mg-deficient plus vitE­ Fe is essential for oxygen radical for­ reduced (C) and Mg-deficient-vitE-supplemented (D) rats. mation) and the availability of pro­ tective substances (e.g. vitamin E) de­ A 8 c termine the level of oxygen free radi­ cals, LPO and MD A. The major part Mg 31.7 + 0.7 (B) 30.3 + 0.4 (10) 30.5 + 0.5 (10) 30.6 + 0.6 (10) of Fe stored in the liver of Mg de­ Ca 6.7 + 0.4 (10) 33.6 + 11.5 (6)a 56.0 + 18.7 (9)a 118.7 + 45.6 (7)a ficient rats may be bound as ferritin and hemosiderin which is less active in radical formation [58]. Values in mmal/kg dry weight. Mean + SE1'~. Number of rats in brackets. Significan~ differences to control ra-cs by unpaired Student 1 s t-test. An additional finding was the differ­ a, p < 0. 05. ent hepatic mineral content from Mg­ deficient rats which had developed a thymus lymphoma (group E). In these Tab. 11: Na, K, Mg and Ca content of skeletal muscle (M. gastrocnemius) from control (A), Mg­ livers, K, Mg and Fe content were deficient (B), Mg-deficient plus vitE-reduced (C) and Mg-deficient-vitE-supplemented (D) rats. increased and Ca content was reduced compared to Mg-deficient rats with­ A 8 c 0 out thymus lymphoma. Further ex­ periments are needed to explain this (8) b (8)a (9)c Na 90.8 + 3.0 (9) 108.8 + 4. 9 118.9 + 7.5 120.8 + 3.3 effect. K 304.9 + 9.9 (9) 268.4 + 8.3 (9) a 257.1 + 13.5 (9)a 266.3 + 7.5 (7) b

Mg 40.7 + 0.6 (10) 36.5 + 1. 4 (10)a 36.9 + 0.9 (10.) b 26 1 + 1. 0 (~)b Conclusion Ca 5.83 + 0.57 (10) 6.76 + 0.60 (10) 7.49 + 0.38 (10)a 7.39 + 0.38 (9)a In Mg deficiency, the basic rate of MDA produced by LPO via oxygen free radicals was increased, depend­ Values in mmol/kg dry weight. Mean~ SEM. Numbe~ of rats in brackets. Significant differences to con~rol rats by unpaired Student 1 s t-test. ing on vitamin E nutrition. Mg de­ a, p < 0. 05; b, p < 0. 01; c, p < 0. 001. ficiency reduced the content of vita­ 3. Increased cellular non-transfer­ 4. Increased uptake of Fe-transfer- min E in serum and tissues, probably rin-Fe (NT-Fe) uptake. Although rin by liver. by an increased formation of free radi­ NT-Fe in serum is less than 1% of The slightly reduced serum Fe con­ cals. However, only in Mg deficiency­ total Fe, it is more rapidly taken up centration in groups B, C, D is com­ induced skin ulcerations, vitamin E by hepatocytes than transferrin­ patible with the last possibilities. and free radicals may be involved. bound Fe [57]. NT-Fe uptake was There was no correlation between There was a negative correlation be­ effectively inhibited by Zn2+ and MDA (tab. 3) and Fe in liver and heart tween MD A, which is formed by oxy­ 2 Mn + [57]. In analogy, at reduced (tab. 8, 12). Therefore, MDA content gen free radicals, and vitamin E con­ extracellular Mg2+, as in Mg de­ is not a simple function of Fe content tent in the tissues. Thus, in Mg de­ ficiency,moreNT-Femaybetaken and oxygen free radicals. However, ficiency and particularly in Mg de­ up. the type of Fe binding (weakly bound ficiencyplus vitaminE reduction, there

Tab. 12: Na, K, Mg, Ca and Fe content of liver from control (A), Mg-deficient (B), Mg-deficient plus vitE-reduced (C) and Mg-deficient-vitE­ supplemented (D) rats. Group E contains livers from rats which had developed thymus lymphoma.

A B c D E

Na 102.8 + 8.1 (10) 112.4 + 5.3 (10) 114.2 + 8.0 (10) 116.8 + 5 (10)

K 333.6 + 3.9 (16) 343.7 + 3.9 (12) 339.9 + 4.7 (12) 342 + 4.2 (11) 444.3 + 3.6 (4) c

11g 29.10 + 0.25 (16) 28.81 + 0.49 (12) 27.64 + 0.47 (12)b 28.26 + 0.31 (11)a 33.33 + 1.56 (4) c

Ca 1.95 + 0.09 (16) 1.88 + 0.08 (12) 1. 82 + 0.06 (12) 1. 85 + 0. 04 (11) 1.34 + 0.06 (4)b

Fe 7.24 + 0.39 (16) 21.13 + 2.85 (12)c 16.37 + 1.115 (12)c 19.26 I J. 37 (1l)c 34.89 + 4.12 (4)c

Values in mmol/kg dry weight. Mean + SEM. Number of rats in brackets. Significant differences to control rats by unpal'red Student's t-test. a, p < 0.05; b, p < 0.01; c, p < 0.001.

64 Magnesium-Bulletin 14, 2 (1992) Lipid Peroxidation in Magnesium Deficiency

was an increased formation of MDA from Mg-deficient rats. J. Trace E!em. reactivity in human plasma and urine. due to an increased action of oxygen Electrol. Hlth. Dis. 3 (1989) 213-216. Analyt. Biochem. 91 (1978) 250-257. free radicals. However, most effects [8] Giinther, T.; Vormann, 1.; Hollriegl, V.; [23] Bunk, M. 1.; Dnistrian, A. M.; Schwartz, Gossrau, R.: Effect of Mg deficiency and ofMg deficiency were independent of M. K.; Rivlin, R. S.: Dietaryzincdefiency salicylate on lipid peroxidation in vivo. decreases plasma concentrations of vita­ vitamin although the level of vita­ Magn.-Bull. 13 (1991) 26-29. min E. Proc. Soc. Exp. Bioi. Med. 190 min E was in the range of correlation [9] Freedman, A. M.; Atrakchi, A. H.; Cas­ (1989) 379-384. to MDA. This means, that there was sidy, M. M.; Weglicki, W. 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Magnesiwn-Bulletin 14, 2 (1992) 65 Lipid Peroxidation in Magnesium Deficiency

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66 Magnesium-Bulletin 14,2 (1992)