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Relative and Combined Effects of Ethanol and Protein Deficiency on Zinc, Iron, Copper, and Manganese Contents in Different Organs and Urinary and Fecal Excretion
Alcohol, Vol. 16, No. 1, pp. 7–12, 1998 © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0741-8329/98 $19.00 1 .00
PII S0741-8329(97)00156-0
Relative and Combined Effects of Ethanol and Protein Deficiency on Zinc, Iron, Copper, and Manganese Contents in Different Organs and Urinary and Fecal Excretion E. GONZALEZ-REIMERS,* A. MARTINEZ-RIERA,* F. SANTOLARIA-FERNANDEZ,* A. MAS-PASCUAL,* F. RODRIGUEZ-MORENO,* L. GALINDO-MARTIN,† M. MOLINA-PEREZ* AND N. BARROS-LOPEZ* * Dpto. de Medicina Interna, Hospital Universitario de Canarias, La Laguna, Tenerife, Canary Islands, Spain † Dpto. de Química Analítica, Universidad de La Laguna, Tenerife, Canary Islands, Spain Received 1 July 1997; Accepted 13 October 1997 GONZALEZ-REIMERS, E., A. MARTINEZ-RIERA, F. SANTOLARIA-FERNANDEZ, A. MAS-PASCUAL, F. RODRIGUEZ-MORENO, L. GALINDO-MARTIN, M. MOLINA-PEREZ AND N. BARROS-LOPEZ. Relative and combined effects of ethanol and protein deficiency on zinc, iron, copper, and manganese contents in different organs and urinary and fecal excretion. ALCOHOL 16(1) 7–12, 1998.—The relative contribution of protein deficiency to the altered metabolism of certain trace elements in chronic alcoholics is not well defined, so this study was performed to analyse the relative and combined effects of ethanol and protein deficiency on liver, bone, muscle, and blood cell content of copper, zinc, iron, and manganese, and also on serum levels and urinary and fecal excretion of these elements in four groups of eight animals each that were pair-fed during 8 weeks with a nutritionally adequate diet, a 36% (as energy) ethanol-containing isocaloric diet, a 2% protein isocaloric diet, and a 36% ethanol 2% protein isocaloric diet, respectively, following the Lieber–DeCarli model. Five additional rats were fed ad lib the control diet. Protein malnutrition, but not ethanol, leads to liver zinc depletion. Both ethanol and protein malnutrition cause muscle zinc depletion and increase urinary zinc and manganese excretion, whereas ethanol also increases urinary iron excretion and liver manganese content. No differences were observed regarding copper metabolism. © 1998 Elsevier Science Inc. Ethanol Protein malnutrition Trace element excretion
Manganese
Iron
Zinc
Copper
Liver
Muscle
Bone
coholic animals, low zinc levels enhance prolylhydroxylase activity (1,39) and inhibit collagenase, thus favouring collagen deposition. In addition, low hepatic zinc would impair Cu/Zn superoxide-dismutase function, thus allowing free radicals to damage hepatocyte function and structure, leading to hepatocyte necrosis and fibrosis. Indeed, in miniature pigs treated with alcohol, the activity of Cu/Zn superoxide-dismutase is reduced (51), and it has been also shown that zinc supplementation was associated with increased concentration of metallothionein and a decrease in lipid peroxidation, and also with decreased activity of prolylhydroxilase in alcoholic rats (7).
SOME elements, such as iron, copper, zinc, and manganese, among others, may play key roles in certain alterations observed in alcoholic patients. There is controversy regarding the relative and combined effects of ethanol, malnutrition, and associated liver cirrhosis on the metabolism of some of these elements. This is the case of zinc (20,26), an element whose depletion is important in the pathogenesis of liver injury, although it is unclear whether zinc supplementation has a place in the treatment of alcoholic liver disease (1,23). Zinc depletion may exert deleterious effects on the enzymatic pathways involved in collagen synthesis and breakdown: in al-
Requests for reprints should be addressed to Dr. González-Reimers, Dpto. de Medicina Interna, Hospital Universitario de Canarias, La Laguna, Tenerife, Canary Islands, Spain.
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GONZALEZ-REIMERS ET AL.
A greater body of knowledge exists regarding iron metabolism: iron accumulates in the liver in chronic alcoholics, especially in those with superimposed protein–calorie malnutrition (10,25,42,49). Copper excess may be responsible for lipid peroxidation (44) and hepatocyte damage, whereas copper depletion, which may be due to ethanol consumption (16), may affect scavening of free radicals (52). Manganese may become affected in chronic alcoholism and/or in the frequently accompanying protein–calorie malnutrition (16,29), and is also involved in the enzymatic pathways of collagen synthesis and in superoxide dismutase activity (39,52). The effects of ethanol and malnutrition on the distribution of these elements in organs other than the liver are largely unknown, but may also yield clinical importance (47). Therefore, we have performed the present study to analyse the relative and combined effects of ethanol and protein deficiency on liver, bone, muscle, and blood cells content of copper, zinc, iron, and manganese, and also on serum levels and urinary and fecal excretion of these elements. METHOD
Thirty-two male Wistar rats were divided into four groups of eight animals each. The control rats received the Lieber– DeCarli control diet (27,28) (Dyets Inc., Bethlehem, PA) containing 18% protein and 1 kcal/ml; a second group consisted of another eight animals fed an isocaloric, 36% ethanolcontaining diet; the third group received an isocaloric, 2% protein-containing diet; and the fourth group received an isocaloric, 2% protein- and 36% ethanol-containing diet. The amount of iron (8.8 mg/l), manganese (13.5 mg/l), copper (1.5 mg/l), and zinc (7.5 mg/l) was the same in the four diets. Those rats receiving the alcohol, protein-deficient diet consumed the diet ad lib, and the same amount consumed by these animals was then given to the other groups. This pairfeeding process was repeated every 2 days, always adjusting the amount of liquid diet received by the other groups to that consumed by the animals fed the protein-deficient, ethanolcontaining diet. The mean daily amount of diet consumed was (in ml or kcal, mean 6 SD): 54.71 6 1.6 ml for the controls, 54.75 6 2.2 ml for the alcoholic animals, 53.65 6 1.7 ml for the low-protein, ethanol-fed animals, and 52.80 6 1.3 ml for the low-protein-fed animals. Thus, our animals consumed approximately 400 mg zinc, 700 mg manganese, 80 mg copper, and 500 mg iron daily, amounts that are adequate (27,45) for normal development. Another group of five animals each was allowed to consume the control diet ad lib. The amount of diet consumed by
these animals was 78.65 6 2.7 kcal/day, significantly more than the amount consumed by the study groups. All the animals were alive at the end of the experiment, 8 weeks later. At this time, animals were anaesthetized by pentobarbital and sacrificed. One day before sacrifice, the rats were placed in metabolic cages, and 24-h urine and feces were collected. Blood was obtained by direct cardiac puncture and centrifuged. We measured serum albumin and creatinine by routinary analytical methods, and the aforementioned trace elements in serum and blood cells as described below. We also removed liver and muscle (right gastroecnemius) samples, and the right femur. Trace Element Determination Samples of liver, muscle, bone, packed blood cells, and feces were dehydrated in a furnace at 100°C for 4–7 days. Dry weight after this procedure ranged from 116 to 737 mg for liver samples, 69 to 317 mg for bones, 275 to 1574 mg for muscles, 64 to 1211 mg for packed blood cells, and 536 to 1200 mg for feces. These samples were then dissolved in 65% nitric acid (Merck p.a.) and 10% hydrogen peroxide (Merck p.a.), to digest organic material. The digestion solutions were quantitatively transferred to volumetric flasks, and diluted to 10 ml with ultrapure water (prepared using a Milli-Q OM-140 deionisation system). Serum samples were deproteinized with 10% trichloroacetic acid and centrifuged. Trace elements were determined in the supernatant. Five milliliters of each of the urine samples was diluted to 10 ml with ultrapure water. Flame atomic absorption spectrophotometry was used to determine liver, serum, and urine trace element concentrations. These analyses were performed with the aid of a Varian Spectra spectrophotometer (Victoria, Australia); detection limits for these elements are 0.021 ppm (mg/ml) for Mn, 0.009 ppm for Zn, 0.026 ppm for copper, and 0.039 ppm for iron. Statistics Differences between the experimental groups were analysed using analysis of variance (ANOVA) with further SNK test. Independent effects and interactions were analysed by means of two-way ANOVA. Also, single correlation analyses (Pearson’s r) were performed between quantitative variables. Data of the ad lib group were compared with those of the control group using Student’s t-test.
TABLE 1 SERUM ALBUMIN AND WEIGHT DIFFERENCE IN THE ANIMALS STUDIED
Group 1 (control) Group 2 (ethanol) Group 3 (low prot.) Group 4 (ethanol & low prot.) Variance analysis SNK test Ad lib group
Serum albumin (g/dl)
Initial Weight (g)
Final Weight (g)
2.88 6 0.25 2.61 6 0.44 2.13 6 0.35 2.35 6 0.20 F 5 7.78 p , 0.001 1, 2 vs. 3; 1 vs. 4 2.82 6 0.25
309 6 12 310 6 16 311 6 15 310 6 18 NS
335 6 32 323 6 16 219 6 18 193 6 14 F 5 80 p , 0.001 1 vs. 3, 4; 2 vs. 3, 4 415 6 8
Values are mean 6 SD. SNK, Student–Newman–Keuls.
304 6 27
ETHANOL AND PROTEIN DEFICIENCY AND TRACE ELEMENTS
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TABLE 2 ZINC DISTRIBUTION IN THE DIFFERENT GROUPS
Liver Muscle Bone R. cell Plasma (mg/dl) Urine (Zn/creat.)( mg 3 10/mg) Urine (total exc.) (mg/day) Fecal 200 (mg/day)
Control
Ethanol
2% Protein
2% Protein 1 Ethanol
86.6 6 4.9 73.4 6 6.5 191.8 6 12.7 24 6 3.8 100 6 12.3 74 6 1.7 5.9 6 1.4 200 6 26.6
97.7 6 5.7 60.8 6 3.1 98.7 6 43.6 25.5 6 4.2 96 6 6.4 43 6 0.6 3.2 6 0.4 206.8 6 51.8
68.8 6 6.8 76.4 6 5.6 157.8 6 21.7 43.9 6 13.9 101.7 6 6.4 1.8 6 0.6 1.3 6 0.4 92.8 6 20.2
73.8 6 7.8* 8.5 6 1.3† 119.3 6 30.5 30.8 6 3.6 75.6 6 0.3 147 6 3† 3.1 6 0.8* 88.6 6 30.3*
Values are mean 6 SE (mg/g dry tissue). R. cell, packed red blood cells; creat., creatinine; exc., excretion. *p , 0.05. †p , 0.001.
RESULTS
Results regarding serum albumin and weight difference are shown in Table 1. As expected, both group of rats fed the low-protein and the ethanol 1 low-protein diets lost weight, results that are in accordance with those of other studies (18). Results regarding trace elements are shown in Tables 2–5. Both the protein-deficient and the protein-deficient, ethanol-fed animals showed significantly lower liver zinc concentrations, whereas protein deficiency decreased liver manganese content, an effect that was counteracted by ethanol. Liver zinc content was related to weight difference (r 5 0.51, p 5 0.003) and serum albumin (r 5 0.35, p , 0.05); liver manganese also correlated with this last parameter (r 5 0.36, p , 0.05) and with liver zinc (r 5 0.36, p , 0.05). Protein deficiency exerted an independent, significant effect on liver zinc content (p 5 0.008), whereas ethanol exerted an independent, significant effect on liver manganese (p 5 0.004). Low muscle zinc values were observed in the ethanoltreated animals and, especially in the ethanol 2% protein-fed animals, both ethanol and protein deficiency exerted independent, significant effects. Muscle content of the other trace elements analysed showed no differences between the four groups of animals. We failed to find differences between the four groups of animals regarding bone and blood cell zinc, copper, manganese, and iron contents. Also, although there was a trend to lower serum zinc levels in the ethanol 2% protein-fed animals, no significant differences were observed regarding serum levels of any of the elements analysed.
Urinary zinc excretion (as Zn/creatinine index) was significantly higher in the ethanol 2% protein-fed animals, with both ethanol and protein deficiency exerting independent, significant effects. Total zinc excretion [zinc content (ppm) 3 diuresis] was significantly lower in all the experimental groups, but especially in the low-protein-fed animals, when compared with the controls (p 5 0.007), differences still remaining statistically significant when this parameter is corrected by body weight (p 5 0.04). Zinc excretion (as Zn/ creatinine) inversely correlated with muscle zinc (r 5 20.44, p , 0.01). Although urinary copper/creatinine index was slightly higher in the ethanol 2% protein-fed animals, these differences did not exist when total copper excretion or total copper excretion/body weight were considered. Urinary manganese excretion (as urinary manganese/creatinine) was significantly higher in both the 2% protein-fed and the ethanol 2% protein-fed animals; also, total urinary manganese output/body weight was significantly higher in the two groups mentioned before (p 5 0.002). Inverse correlations were observed between manganese excretion (as Mn/creatinine) with final weight (r 5 20.53, p 5 0.002), weight difference (r 5 20.54, p 5 0.002), and serum albumin (r 5 20.39, p 5 0.034). Urinary iron excretion (as urinary Fe/creatinine) was much higher in the ethanol-fed animals, and also total urinary iron excretion (p , 0.001) and total urinary iron excretion/body weight (p , 0.01). Fecal zinc concentration was similar in the four groups of animals, but 24-h zinc excretion was significantly lower in
TABLE 3 COPPER DISTRIBUTION IN THE DIFFERENT GROUPS
Liver Muscle Bone R. cell Plasma (mg/dl) Urine (Cu/creat.) (mg 3 10/mg) Urine (total exc.) (mg/day) Fecal (mg/day)
Control
Ethanol
2% Protein
2% Protein 1 Ethanol
10.4 6 1.5 4.2 6 0.7 5.8 6 0.8 2.5 6 0.4 179.3 6 13.2 23 6 0.6 2 6 0.5 28.9 6 3.8
9.0 6 1.7 3.3 6 0.6 4.0 6 0.8 4.7 6 1.9 146.5 6 9.3 4.3 6 1.2 2.9 6 0.3 30.9 6 8.8
7.0 6 0.6 4.6 6 0.4 6.2 6 0.5 5.4 6 2.3 164.5 6 13.4 23 6 0.6 1.5 6 0.3 12.3 6 2.9
8.1 6 0.9 5.1 6 1.0 6.9 6 1.1 4.1 6 0.7 192.0 6 56.3 71 6 2.1* 1.7 6 0.5 12.7 6 3.1*
Values are mean 6 SE (mg/g dry tissue). R. cell, packed red blood cells; creat., creatinine; exc., excretion. *p , 0.05.
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GONZALEZ-REIMERS ET AL. TABLE 4 MANGANESE DISTRIBUTION IN THE DIFFERENT GROUPS
Liver Muscle Bone R. cell Urine (Mn/creat.) (mg 3 10/mg) Urine (total exc.) (mg/day) Fecal (mg/day)
Control
Ethanol
2% Protein
2% Protein 1 Ethanol
14.3 6 1.6 2.3 6 1.1 14.9 6 2.0 4.1 6 1.8 3.1 6 0.5 2.6 6 0.4 246.3 6 47.3
18.3 6 1.3 0.7 6 0.1 12.2 6 1.9 4.9 6 1.9 1.8 6 1.0 1.6 6 0.9 227.7 6 41.2
11.8 6 1.7 1.2 6 0.1 13.8 6 1.1 64.7 6 40.1 4.1 6 0.5 3.2 6 0.3 132.6 6 26.7
17.2 6 1.2† 1.7 6 0.4 16.5 6 3.1 60.3 6 21.4‡ 14.8 6 3.5‡ 3.1 6 0.3 120.9 6 24.2*
Values are mean 6 SE (mg/g tissue). R. cell, packed red blood cells; creat., creatine; exc., excretion. *p , 0.05. †p , 0.01. ‡p , 0.001.
both the 2% protein-fed and in the ethanol 2% protein-fed animals, although not when 24-h excretion was corrected by weight. Exactly the same happened with fecal copper, manganese, and iron excretion. No differences were observed between the ad lib group and the control group regarding any of the parameters analysed. DISCUSSION
In this study we have analysed the relative and combined effects of ethanol and protein deficiency on the distribution of manganese, zinc, copper, and iron in some organs that become importantly affected by ethanol consumption, such as liver, muscle, or bone. We have also analysed the effects of these factors on fecal and urinary excretion of these elements, keeping in mind that fecal excretion includes not only the nonabsorbed fraction of the amount ingested but also biliary excretion (especially in the case of copper) and gastrointestinal secretion (especially in the case of manganese), and that dermal loss in the sweat, hair, and nails also contributes to iron and zinc excretion (45). Data derived from zinc analysis show that ethanol consumption with an adequate control diet does not significantly alter zinc distribution, leading only to a slight, nonsignificant decrease in bone and muscle zinc and to reduced urinary zinc excretion. However, significant changes are indeed observed when ethanol and protein deficiency are combined, leading to very low liver and muscle concentrations, to high urinary ex-
cretion, and to low fecal excretion. Although low serum and liver zinc levels and increased urinary zinc wastage are wellknown features in alcoholic cirrhosis (30,31,33,37,38), together with reduced zinc absorption (46), only a few works, to our knowledge, deal with alcohol-mediated zinc content alterations in other organs, higher bone zinc in ethanol-treated rats (35), and normal erythrocyte zinc in alcoholic cirrhosis (37). On the other hand, in a previous study we have reported that the ethanol 2% protein-fed animals showed the lowest muscle zinc content, which was 12.82 6 9.28 mg/g dry tissue (18) (i.e., similar to that obtained in the present study). These animals also showed the highest values of urinary zinc excretion in relation to creatinin excretion. Urinary zinc may reflect protein catabolism (15), a fact that probably accounts for the high zinc excretion observed in these rats. High liver manganese content has been described in alcoholics (30,48), probably because of impaired biliary excretion. However, liver damage is not intense in rats following the Lieber–de Carli model during 8 weeks (12), so the slight, nonsignificantly increased liver manganese content observed both in the ethanol-fed [in accordance with other studies performed in mice (19)] and in the ethanol 2% protein-fed animals is probably independent of altered biliary excretion. On the other hand, manganese depletion is commonly observed in malnutrition (17). In our animals, indeed, urinary manganese excretion was inversely related to body weight and serum albumin, and was significantly higher in the 2% protein ethanolfed animals; moreover, liver manganese reached the lowest
TABLE 5 IRON DISTRIBUTION IN THE DIFFERENT GROUPS
Liver Muscle Bone R. cell Plasma (mg/dl) Urine (Fe/creat.) (mg 3 10/mg) Urine (total exc.) (mg/day) Fecal (mg/day)
Control
Ethanol
2% Protein
2% Protein 1 Ethanol
461.6 6 58.1 64.4 6 5.6 93.1 6 30.4 1204.8 6 318.9 45.7 6 12.1 3.1 6 0.8 2.4 6 0.5 284.2 6 54.8
1019.2 6 592.6 71.9 6 10.7 33.3 6 10.4 1486.9 6 310.6 47.6 6 13.0 20 6 4.0 15.4 6 3.3 212.9 6 55.6
480.9 6 58.9 77.6 6 4.8 82.6 6 14.4 1231.5 6 263.1 44.2 6 13.1 5.0 6 1.0 3.2 6 1.0 75.8 6 26.4
1425.0 6 532.4 83 6 16 120.7 6 39.0 1163 6 320.0 25.3 6 9.4 8.6 6 2.0† 1.8 6 0.3† 93.8 6 23.4*
Values are mean 6 SE (mg/g dry tissue). R. cell, packed red blood cells; creat., creatinine; exc., excretion. *p , 0.01. †p , 0.001.
ETHANOL AND PROTEIN DEFICIENCY AND TRACE ELEMENTS value in the low-protein-fed animals, a result in accordance with results obtained in rats fed a 6% protein-containing diet (11). The increased urinary loss of manganese was compensated by the decreased fecal loss that was present in these last animals. Ethanol enhances manganese absorption, although other cations, as iron, whose absorption increases in alcoholics, compete with manganese absorption (24). In our study, although fecal manganese excretion is slightly reduced in alcoholics, this reduction is significant both in the 2% protein-fed and in the ethanol 2% protein-fed animals, pointing to an enhanced intestinal absorption. Although there is general agreement in the observation of raised liver copper levels in cholestatic syndromes (44) and in alcoholic cirrhosis (43), controversy exists about the effect of ethanol on liver copper changes (16,21), as well as regarding serum copper levels in alcoholic patients with or without liver damage (20,36). Zidenberg-Cherr et al. (51) have found decreased zinc/copper superoxide-dismutase levels in alcoholtreated guinea pigs. In previous studies we have found that serum copper is raised in noncirrhotic alcoholics (38), and that copper increases in the liver of growing alcoholized mice (19), and in rats fed a protein-deficient diet (11), but we and other authors have failed to find alterations in copper in rat liver (21), muscle (18) or bone (35), and in blood (11) or urine (38) of nonalcoholic cirrhotics. In the present study we failed to find any difference in copper tissue distribution between the four groups analysed, except for a slight increase in the urinary copper/creatinin index in the 2% protein ethanol-fed animals, differences that were not present if total copper excretion was considered; fecal excretion was also lower in the low-protein groups. We have found that ethanol increases urinary iron loss, a fact that seems to be independent of protein deficiency. There
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is a trend to lower bone iron content in alcoholic rats—a result in disagreement with that reported by others (35)—and a trend to higher liver iron content both in the ethanol and ethanol 2% protein-fed animals. Liver iron overload has been observed in 30% of alcoholics with chronic liver disease (10), and may be involved in the progression of liver injury (2,3,5,6). Pathogenesis of hepatic iron overload remains unclear; proposed mechanisms include increased absorption (14) [although this is uncertain (9)], ingestion of excessive iron in alcoholic beverages, excessive hepatic uptake of transferrinbound iron (8) and/or ferritin (48) [although controversy also exists regarding these aspects (34,50)], and iron mobilization from ferritin due to the effect of superoxide radicals (41). Ethanol promotes liver and cerebellar uptake of iron from plasma (40) and leads to an increase in liver iron in mice (19); in our study, only the 2% protein-fed, ethanol-treated animals, with high liver iron content, showed a trend to lower plasma iron levels. However, in other works, ethanol does not influence iron overload and liver fibrosis provoked by experimental iron overload (32), and other authors have failed to show any effect of ethanol on intestinal iron absorption and liver iron uptake (4). In our study we have not found that protein deprivation per se is responsible for liver iron overload, in contrast with the results of another study in which iron overload ensued after feeding rats with a 6% protein-containing diet (11). Liver siderosis has been described in kwashiorkor (22), perhaps due to impaired synthesis of transport proteins (13). Thus, we conclude that protein malnutrition, but not ethanol, leads to liver zinc depletion. Both ethanol and protein malnutrition cause muscle zinc depletion and increase urinary zinc and manganese excretion. Ethanol also increases urinary iron excretion and liver manganese content.
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12 18. González-Reimers, E.; Conde-Martel, A.; Santolaria-Fernández, F.; Martínez-Riera, A.; Rodríguez-Moreno, F.; González-Hernández, T.; Castro Alemán, V. V.: Relative and combined roles of ethanol and protein malnutrition on muscle zinc, copper, iron and magnesium. Alcohol Alcohol. 28:311–318; 1993. 19. González-Reimers, E.; Rodríguez-Moreno, F.; Castro-Alemán, V.; Santolaria-Fernández, F.; Galindo-Martín, L.; Ferres-Torres, R.; Conde-Martel, A.: Effect of ethanol on hepatic iron, copper, zinc, and manganese contents in the male albino mouse. Drug Alcohol Depend. 26:195–198; 1990. 20. Hartoma, T. R.; Sotaniemi, E. A.; Pelkonen, O.; Ahlqvist, J.: Serum zinc and copper and indices of drug metabolism in alcoholics. Eur. J. Clin. Pharmacol. 12:147–151; 1977. 21. Hopf, G.; Bocker, R.; Biscoff, J.; Werner, M. G.; Estler, C. J.: Investigation into the combined effects of ethanol and cadmium on rat liver and kidneys. Arch. Toxicol. 64:470–473; 1990. 22. Jackson, A. A.: Severe undernutrition in Jamaica. Acta Paediatr. Scand. 323:43–51; 1986. 23. Labadie, M.; Arnaud, J.; Enkaoua, W.; Zarski, J. P.; Tricot, F.; Vaysee, J.; Beaugrand, M.; Favier, A.: Effect of zinc supplementation in alcoholic cirrhosis. A double-blind randomized study. Trace Elem. Electrolytes 11:23–28; 1994. 24. Leach, R. M.: Metabolism and function of manganese. In: Prasad, A. S.; Oberleas, D., eds. Trace elements in human health and disease. New York: Academic Press; 1976:235–247. 25. Lesage, G. D.; Baldus, W. P.; Fairbanks, V. F.; Baggenstoss, A. H.; McCall, J. T.; Moore, S. B.; Taswell, H. F.; Gordon, H.: Hemochromatosis: Genetic or alcohol-induced? Gastroenterology 84: 1471–1477; 1983. 26. Lieber, C. S.: The influence of ethanol on nutritional status. Nutr. Rev. 46:241–254; 1988. 27. Lieber, C. S.; DeCarli, L. M.; Sorrell, M.: Experimental methods of ethanol administration. Hepatology 10:501–510; 1989. 28. Lieber, C. S.; DeCarli, L. M.: Liquid diet technique of ethanol administration: 1989 update. Alcohol Alcohol. 24:197–211; 1989. 29. Mendenhall, C. L.; Anderson, S.; Weesner, R. E.; Goldberg, S. J.; Crolic, K. A.: Protein-calorie malnutrition associated with alcoholic hepatitis. Am. J. Med. 76:211–222; 1984. 30. Milman, N.; Laursen, J.; Podenphant, J.; Asnaes, S.: Trace elements in normal and cirrhotic human liver tissue I. Iron, copper, zinc, selenium, manganese, titanium and lead measured by X-ray fluorescence spectrometry. Liver 6:111–117; 1986. 31. Navarro-Rodríguez, M. C.; Sosa-Henríquez, M.; Font de MoraTurón, A.; Novoa, F. J.; Gómez-Díaz, J.; Betancor-León, P.: Trace elements in chronic alcoholism. Trace Elem. 3:164–167; 1986. 32. Olynyk, J.; Hall, P.; Reed, W.; Williams, P.; Kerr, R.; Mackinnon, M.: A long-term study of the interaction between iron and alcohol in an animal model of iron overload. J. Hepatol. 22:671–676; 1995. 33. Poo, J. L.; Rosas-Romero, R.; Rodríguez, F.; Silencio, J. L.; Muñoz, R.; Bourges, H.; Uribe, M.: Serum zinc concentrations in two cohorts of 153 healthy subjects and 100 cirrhotic patients from Mexico City. Dig. Dis. 13:136–142; 1995. 34. Potter, B. J.; McHugh, T. A.; Beloqui, O.: Iron uptake from transferrin and asialotransferrin by hepatocytes from chronically alcohol-fed rats. Alcohol. Clin. Exp. Res. 16:810–815; 1992. 35. Preedy, V. R.; Baldwin, D. R.; Keating, J. W.; Salisbury, J. R.:
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PII S0741-8329(97)00156-0
Relative and Combined Effects of Ethanol and Protein Deficiency on Zinc, Iron, Copper, and Manganese Contents in Different Organs and Urinary and Fecal Excretion E. GONZALEZ-REIMERS,* A. MARTINEZ-RIERA,* F. SANTOLARIA-FERNANDEZ,* A. MAS-PASCUAL,* F. RODRIGUEZ-MORENO,* L. GALINDO-MARTIN,† M. MOLINA-PEREZ* AND N. BARROS-LOPEZ* * Dpto. de Medicina Interna, Hospital Universitario de Canarias, La Laguna, Tenerife, Canary Islands, Spain † Dpto. de Química Analítica, Universidad de La Laguna, Tenerife, Canary Islands, Spain Received 1 July 1997; Accepted 13 October 1997 GONZALEZ-REIMERS, E., A. MARTINEZ-RIERA, F. SANTOLARIA-FERNANDEZ, A. MAS-PASCUAL, F. RODRIGUEZ-MORENO, L. GALINDO-MARTIN, M. MOLINA-PEREZ AND N. BARROS-LOPEZ. Relative and combined effects of ethanol and protein deficiency on zinc, iron, copper, and manganese contents in different organs and urinary and fecal excretion. ALCOHOL 16(1) 7–12, 1998.—The relative contribution of protein deficiency to the altered metabolism of certain trace elements in chronic alcoholics is not well defined, so this study was performed to analyse the relative and combined effects of ethanol and protein deficiency on liver, bone, muscle, and blood cell content of copper, zinc, iron, and manganese, and also on serum levels and urinary and fecal excretion of these elements in four groups of eight animals each that were pair-fed during 8 weeks with a nutritionally adequate diet, a 36% (as energy) ethanol-containing isocaloric diet, a 2% protein isocaloric diet, and a 36% ethanol 2% protein isocaloric diet, respectively, following the Lieber–DeCarli model. Five additional rats were fed ad lib the control diet. Protein malnutrition, but not ethanol, leads to liver zinc depletion. Both ethanol and protein malnutrition cause muscle zinc depletion and increase urinary zinc and manganese excretion, whereas ethanol also increases urinary iron excretion and liver manganese content. No differences were observed regarding copper metabolism. © 1998 Elsevier Science Inc. Ethanol Protein malnutrition Trace element excretion
Manganese
Iron
Zinc
Copper
Liver
Muscle
Bone
coholic animals, low zinc levels enhance prolylhydroxylase activity (1,39) and inhibit collagenase, thus favouring collagen deposition. In addition, low hepatic zinc would impair Cu/Zn superoxide-dismutase function, thus allowing free radicals to damage hepatocyte function and structure, leading to hepatocyte necrosis and fibrosis. Indeed, in miniature pigs treated with alcohol, the activity of Cu/Zn superoxide-dismutase is reduced (51), and it has been also shown that zinc supplementation was associated with increased concentration of metallothionein and a decrease in lipid peroxidation, and also with decreased activity of prolylhydroxilase in alcoholic rats (7).
SOME elements, such as iron, copper, zinc, and manganese, among others, may play key roles in certain alterations observed in alcoholic patients. There is controversy regarding the relative and combined effects of ethanol, malnutrition, and associated liver cirrhosis on the metabolism of some of these elements. This is the case of zinc (20,26), an element whose depletion is important in the pathogenesis of liver injury, although it is unclear whether zinc supplementation has a place in the treatment of alcoholic liver disease (1,23). Zinc depletion may exert deleterious effects on the enzymatic pathways involved in collagen synthesis and breakdown: in al-
Requests for reprints should be addressed to Dr. González-Reimers, Dpto. de Medicina Interna, Hospital Universitario de Canarias, La Laguna, Tenerife, Canary Islands, Spain.
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8
GONZALEZ-REIMERS ET AL.
A greater body of knowledge exists regarding iron metabolism: iron accumulates in the liver in chronic alcoholics, especially in those with superimposed protein–calorie malnutrition (10,25,42,49). Copper excess may be responsible for lipid peroxidation (44) and hepatocyte damage, whereas copper depletion, which may be due to ethanol consumption (16), may affect scavening of free radicals (52). Manganese may become affected in chronic alcoholism and/or in the frequently accompanying protein–calorie malnutrition (16,29), and is also involved in the enzymatic pathways of collagen synthesis and in superoxide dismutase activity (39,52). The effects of ethanol and malnutrition on the distribution of these elements in organs other than the liver are largely unknown, but may also yield clinical importance (47). Therefore, we have performed the present study to analyse the relative and combined effects of ethanol and protein deficiency on liver, bone, muscle, and blood cells content of copper, zinc, iron, and manganese, and also on serum levels and urinary and fecal excretion of these elements. METHOD
Thirty-two male Wistar rats were divided into four groups of eight animals each. The control rats received the Lieber– DeCarli control diet (27,28) (Dyets Inc., Bethlehem, PA) containing 18% protein and 1 kcal/ml; a second group consisted of another eight animals fed an isocaloric, 36% ethanolcontaining diet; the third group received an isocaloric, 2% protein-containing diet; and the fourth group received an isocaloric, 2% protein- and 36% ethanol-containing diet. The amount of iron (8.8 mg/l), manganese (13.5 mg/l), copper (1.5 mg/l), and zinc (7.5 mg/l) was the same in the four diets. Those rats receiving the alcohol, protein-deficient diet consumed the diet ad lib, and the same amount consumed by these animals was then given to the other groups. This pairfeeding process was repeated every 2 days, always adjusting the amount of liquid diet received by the other groups to that consumed by the animals fed the protein-deficient, ethanolcontaining diet. The mean daily amount of diet consumed was (in ml or kcal, mean 6 SD): 54.71 6 1.6 ml for the controls, 54.75 6 2.2 ml for the alcoholic animals, 53.65 6 1.7 ml for the low-protein, ethanol-fed animals, and 52.80 6 1.3 ml for the low-protein-fed animals. Thus, our animals consumed approximately 400 mg zinc, 700 mg manganese, 80 mg copper, and 500 mg iron daily, amounts that are adequate (27,45) for normal development. Another group of five animals each was allowed to consume the control diet ad lib. The amount of diet consumed by
these animals was 78.65 6 2.7 kcal/day, significantly more than the amount consumed by the study groups. All the animals were alive at the end of the experiment, 8 weeks later. At this time, animals were anaesthetized by pentobarbital and sacrificed. One day before sacrifice, the rats were placed in metabolic cages, and 24-h urine and feces were collected. Blood was obtained by direct cardiac puncture and centrifuged. We measured serum albumin and creatinine by routinary analytical methods, and the aforementioned trace elements in serum and blood cells as described below. We also removed liver and muscle (right gastroecnemius) samples, and the right femur. Trace Element Determination Samples of liver, muscle, bone, packed blood cells, and feces were dehydrated in a furnace at 100°C for 4–7 days. Dry weight after this procedure ranged from 116 to 737 mg for liver samples, 69 to 317 mg for bones, 275 to 1574 mg for muscles, 64 to 1211 mg for packed blood cells, and 536 to 1200 mg for feces. These samples were then dissolved in 65% nitric acid (Merck p.a.) and 10% hydrogen peroxide (Merck p.a.), to digest organic material. The digestion solutions were quantitatively transferred to volumetric flasks, and diluted to 10 ml with ultrapure water (prepared using a Milli-Q OM-140 deionisation system). Serum samples were deproteinized with 10% trichloroacetic acid and centrifuged. Trace elements were determined in the supernatant. Five milliliters of each of the urine samples was diluted to 10 ml with ultrapure water. Flame atomic absorption spectrophotometry was used to determine liver, serum, and urine trace element concentrations. These analyses were performed with the aid of a Varian Spectra spectrophotometer (Victoria, Australia); detection limits for these elements are 0.021 ppm (mg/ml) for Mn, 0.009 ppm for Zn, 0.026 ppm for copper, and 0.039 ppm for iron. Statistics Differences between the experimental groups were analysed using analysis of variance (ANOVA) with further SNK test. Independent effects and interactions were analysed by means of two-way ANOVA. Also, single correlation analyses (Pearson’s r) were performed between quantitative variables. Data of the ad lib group were compared with those of the control group using Student’s t-test.
TABLE 1 SERUM ALBUMIN AND WEIGHT DIFFERENCE IN THE ANIMALS STUDIED
Group 1 (control) Group 2 (ethanol) Group 3 (low prot.) Group 4 (ethanol & low prot.) Variance analysis SNK test Ad lib group
Serum albumin (g/dl)
Initial Weight (g)
Final Weight (g)
2.88 6 0.25 2.61 6 0.44 2.13 6 0.35 2.35 6 0.20 F 5 7.78 p , 0.001 1, 2 vs. 3; 1 vs. 4 2.82 6 0.25
309 6 12 310 6 16 311 6 15 310 6 18 NS
335 6 32 323 6 16 219 6 18 193 6 14 F 5 80 p , 0.001 1 vs. 3, 4; 2 vs. 3, 4 415 6 8
Values are mean 6 SD. SNK, Student–Newman–Keuls.
304 6 27
ETHANOL AND PROTEIN DEFICIENCY AND TRACE ELEMENTS
9
TABLE 2 ZINC DISTRIBUTION IN THE DIFFERENT GROUPS
Liver Muscle Bone R. cell Plasma (mg/dl) Urine (Zn/creat.)( mg 3 10/mg) Urine (total exc.) (mg/day) Fecal 200 (mg/day)
Control
Ethanol
2% Protein
2% Protein 1 Ethanol
86.6 6 4.9 73.4 6 6.5 191.8 6 12.7 24 6 3.8 100 6 12.3 74 6 1.7 5.9 6 1.4 200 6 26.6
97.7 6 5.7 60.8 6 3.1 98.7 6 43.6 25.5 6 4.2 96 6 6.4 43 6 0.6 3.2 6 0.4 206.8 6 51.8
68.8 6 6.8 76.4 6 5.6 157.8 6 21.7 43.9 6 13.9 101.7 6 6.4 1.8 6 0.6 1.3 6 0.4 92.8 6 20.2
73.8 6 7.8* 8.5 6 1.3† 119.3 6 30.5 30.8 6 3.6 75.6 6 0.3 147 6 3† 3.1 6 0.8* 88.6 6 30.3*
Values are mean 6 SE (mg/g dry tissue). R. cell, packed red blood cells; creat., creatinine; exc., excretion. *p , 0.05. †p , 0.001.
RESULTS
Results regarding serum albumin and weight difference are shown in Table 1. As expected, both group of rats fed the low-protein and the ethanol 1 low-protein diets lost weight, results that are in accordance with those of other studies (18). Results regarding trace elements are shown in Tables 2–5. Both the protein-deficient and the protein-deficient, ethanol-fed animals showed significantly lower liver zinc concentrations, whereas protein deficiency decreased liver manganese content, an effect that was counteracted by ethanol. Liver zinc content was related to weight difference (r 5 0.51, p 5 0.003) and serum albumin (r 5 0.35, p , 0.05); liver manganese also correlated with this last parameter (r 5 0.36, p , 0.05) and with liver zinc (r 5 0.36, p , 0.05). Protein deficiency exerted an independent, significant effect on liver zinc content (p 5 0.008), whereas ethanol exerted an independent, significant effect on liver manganese (p 5 0.004). Low muscle zinc values were observed in the ethanoltreated animals and, especially in the ethanol 2% protein-fed animals, both ethanol and protein deficiency exerted independent, significant effects. Muscle content of the other trace elements analysed showed no differences between the four groups of animals. We failed to find differences between the four groups of animals regarding bone and blood cell zinc, copper, manganese, and iron contents. Also, although there was a trend to lower serum zinc levels in the ethanol 2% protein-fed animals, no significant differences were observed regarding serum levels of any of the elements analysed.
Urinary zinc excretion (as Zn/creatinine index) was significantly higher in the ethanol 2% protein-fed animals, with both ethanol and protein deficiency exerting independent, significant effects. Total zinc excretion [zinc content (ppm) 3 diuresis] was significantly lower in all the experimental groups, but especially in the low-protein-fed animals, when compared with the controls (p 5 0.007), differences still remaining statistically significant when this parameter is corrected by body weight (p 5 0.04). Zinc excretion (as Zn/ creatinine) inversely correlated with muscle zinc (r 5 20.44, p , 0.01). Although urinary copper/creatinine index was slightly higher in the ethanol 2% protein-fed animals, these differences did not exist when total copper excretion or total copper excretion/body weight were considered. Urinary manganese excretion (as urinary manganese/creatinine) was significantly higher in both the 2% protein-fed and the ethanol 2% protein-fed animals; also, total urinary manganese output/body weight was significantly higher in the two groups mentioned before (p 5 0.002). Inverse correlations were observed between manganese excretion (as Mn/creatinine) with final weight (r 5 20.53, p 5 0.002), weight difference (r 5 20.54, p 5 0.002), and serum albumin (r 5 20.39, p 5 0.034). Urinary iron excretion (as urinary Fe/creatinine) was much higher in the ethanol-fed animals, and also total urinary iron excretion (p , 0.001) and total urinary iron excretion/body weight (p , 0.01). Fecal zinc concentration was similar in the four groups of animals, but 24-h zinc excretion was significantly lower in
TABLE 3 COPPER DISTRIBUTION IN THE DIFFERENT GROUPS
Liver Muscle Bone R. cell Plasma (mg/dl) Urine (Cu/creat.) (mg 3 10/mg) Urine (total exc.) (mg/day) Fecal (mg/day)
Control
Ethanol
2% Protein
2% Protein 1 Ethanol
10.4 6 1.5 4.2 6 0.7 5.8 6 0.8 2.5 6 0.4 179.3 6 13.2 23 6 0.6 2 6 0.5 28.9 6 3.8
9.0 6 1.7 3.3 6 0.6 4.0 6 0.8 4.7 6 1.9 146.5 6 9.3 4.3 6 1.2 2.9 6 0.3 30.9 6 8.8
7.0 6 0.6 4.6 6 0.4 6.2 6 0.5 5.4 6 2.3 164.5 6 13.4 23 6 0.6 1.5 6 0.3 12.3 6 2.9
8.1 6 0.9 5.1 6 1.0 6.9 6 1.1 4.1 6 0.7 192.0 6 56.3 71 6 2.1* 1.7 6 0.5 12.7 6 3.1*
Values are mean 6 SE (mg/g dry tissue). R. cell, packed red blood cells; creat., creatinine; exc., excretion. *p , 0.05.
10
GONZALEZ-REIMERS ET AL. TABLE 4 MANGANESE DISTRIBUTION IN THE DIFFERENT GROUPS
Liver Muscle Bone R. cell Urine (Mn/creat.) (mg 3 10/mg) Urine (total exc.) (mg/day) Fecal (mg/day)
Control
Ethanol
2% Protein
2% Protein 1 Ethanol
14.3 6 1.6 2.3 6 1.1 14.9 6 2.0 4.1 6 1.8 3.1 6 0.5 2.6 6 0.4 246.3 6 47.3
18.3 6 1.3 0.7 6 0.1 12.2 6 1.9 4.9 6 1.9 1.8 6 1.0 1.6 6 0.9 227.7 6 41.2
11.8 6 1.7 1.2 6 0.1 13.8 6 1.1 64.7 6 40.1 4.1 6 0.5 3.2 6 0.3 132.6 6 26.7
17.2 6 1.2† 1.7 6 0.4 16.5 6 3.1 60.3 6 21.4‡ 14.8 6 3.5‡ 3.1 6 0.3 120.9 6 24.2*
Values are mean 6 SE (mg/g tissue). R. cell, packed red blood cells; creat., creatine; exc., excretion. *p , 0.05. †p , 0.01. ‡p , 0.001.
both the 2% protein-fed and in the ethanol 2% protein-fed animals, although not when 24-h excretion was corrected by weight. Exactly the same happened with fecal copper, manganese, and iron excretion. No differences were observed between the ad lib group and the control group regarding any of the parameters analysed. DISCUSSION
In this study we have analysed the relative and combined effects of ethanol and protein deficiency on the distribution of manganese, zinc, copper, and iron in some organs that become importantly affected by ethanol consumption, such as liver, muscle, or bone. We have also analysed the effects of these factors on fecal and urinary excretion of these elements, keeping in mind that fecal excretion includes not only the nonabsorbed fraction of the amount ingested but also biliary excretion (especially in the case of copper) and gastrointestinal secretion (especially in the case of manganese), and that dermal loss in the sweat, hair, and nails also contributes to iron and zinc excretion (45). Data derived from zinc analysis show that ethanol consumption with an adequate control diet does not significantly alter zinc distribution, leading only to a slight, nonsignificant decrease in bone and muscle zinc and to reduced urinary zinc excretion. However, significant changes are indeed observed when ethanol and protein deficiency are combined, leading to very low liver and muscle concentrations, to high urinary ex-
cretion, and to low fecal excretion. Although low serum and liver zinc levels and increased urinary zinc wastage are wellknown features in alcoholic cirrhosis (30,31,33,37,38), together with reduced zinc absorption (46), only a few works, to our knowledge, deal with alcohol-mediated zinc content alterations in other organs, higher bone zinc in ethanol-treated rats (35), and normal erythrocyte zinc in alcoholic cirrhosis (37). On the other hand, in a previous study we have reported that the ethanol 2% protein-fed animals showed the lowest muscle zinc content, which was 12.82 6 9.28 mg/g dry tissue (18) (i.e., similar to that obtained in the present study). These animals also showed the highest values of urinary zinc excretion in relation to creatinin excretion. Urinary zinc may reflect protein catabolism (15), a fact that probably accounts for the high zinc excretion observed in these rats. High liver manganese content has been described in alcoholics (30,48), probably because of impaired biliary excretion. However, liver damage is not intense in rats following the Lieber–de Carli model during 8 weeks (12), so the slight, nonsignificantly increased liver manganese content observed both in the ethanol-fed [in accordance with other studies performed in mice (19)] and in the ethanol 2% protein-fed animals is probably independent of altered biliary excretion. On the other hand, manganese depletion is commonly observed in malnutrition (17). In our animals, indeed, urinary manganese excretion was inversely related to body weight and serum albumin, and was significantly higher in the 2% protein ethanolfed animals; moreover, liver manganese reached the lowest
TABLE 5 IRON DISTRIBUTION IN THE DIFFERENT GROUPS
Liver Muscle Bone R. cell Plasma (mg/dl) Urine (Fe/creat.) (mg 3 10/mg) Urine (total exc.) (mg/day) Fecal (mg/day)
Control
Ethanol
2% Protein
2% Protein 1 Ethanol
461.6 6 58.1 64.4 6 5.6 93.1 6 30.4 1204.8 6 318.9 45.7 6 12.1 3.1 6 0.8 2.4 6 0.5 284.2 6 54.8
1019.2 6 592.6 71.9 6 10.7 33.3 6 10.4 1486.9 6 310.6 47.6 6 13.0 20 6 4.0 15.4 6 3.3 212.9 6 55.6
480.9 6 58.9 77.6 6 4.8 82.6 6 14.4 1231.5 6 263.1 44.2 6 13.1 5.0 6 1.0 3.2 6 1.0 75.8 6 26.4
1425.0 6 532.4 83 6 16 120.7 6 39.0 1163 6 320.0 25.3 6 9.4 8.6 6 2.0† 1.8 6 0.3† 93.8 6 23.4*
Values are mean 6 SE (mg/g dry tissue). R. cell, packed red blood cells; creat., creatinine; exc., excretion. *p , 0.01. †p , 0.001.
ETHANOL AND PROTEIN DEFICIENCY AND TRACE ELEMENTS value in the low-protein-fed animals, a result in accordance with results obtained in rats fed a 6% protein-containing diet (11). The increased urinary loss of manganese was compensated by the decreased fecal loss that was present in these last animals. Ethanol enhances manganese absorption, although other cations, as iron, whose absorption increases in alcoholics, compete with manganese absorption (24). In our study, although fecal manganese excretion is slightly reduced in alcoholics, this reduction is significant both in the 2% protein-fed and in the ethanol 2% protein-fed animals, pointing to an enhanced intestinal absorption. Although there is general agreement in the observation of raised liver copper levels in cholestatic syndromes (44) and in alcoholic cirrhosis (43), controversy exists about the effect of ethanol on liver copper changes (16,21), as well as regarding serum copper levels in alcoholic patients with or without liver damage (20,36). Zidenberg-Cherr et al. (51) have found decreased zinc/copper superoxide-dismutase levels in alcoholtreated guinea pigs. In previous studies we have found that serum copper is raised in noncirrhotic alcoholics (38), and that copper increases in the liver of growing alcoholized mice (19), and in rats fed a protein-deficient diet (11), but we and other authors have failed to find alterations in copper in rat liver (21), muscle (18) or bone (35), and in blood (11) or urine (38) of nonalcoholic cirrhotics. In the present study we failed to find any difference in copper tissue distribution between the four groups analysed, except for a slight increase in the urinary copper/creatinin index in the 2% protein ethanol-fed animals, differences that were not present if total copper excretion was considered; fecal excretion was also lower in the low-protein groups. We have found that ethanol increases urinary iron loss, a fact that seems to be independent of protein deficiency. There
11
is a trend to lower bone iron content in alcoholic rats—a result in disagreement with that reported by others (35)—and a trend to higher liver iron content both in the ethanol and ethanol 2% protein-fed animals. Liver iron overload has been observed in 30% of alcoholics with chronic liver disease (10), and may be involved in the progression of liver injury (2,3,5,6). Pathogenesis of hepatic iron overload remains unclear; proposed mechanisms include increased absorption (14) [although this is uncertain (9)], ingestion of excessive iron in alcoholic beverages, excessive hepatic uptake of transferrinbound iron (8) and/or ferritin (48) [although controversy also exists regarding these aspects (34,50)], and iron mobilization from ferritin due to the effect of superoxide radicals (41). Ethanol promotes liver and cerebellar uptake of iron from plasma (40) and leads to an increase in liver iron in mice (19); in our study, only the 2% protein-fed, ethanol-treated animals, with high liver iron content, showed a trend to lower plasma iron levels. However, in other works, ethanol does not influence iron overload and liver fibrosis provoked by experimental iron overload (32), and other authors have failed to show any effect of ethanol on intestinal iron absorption and liver iron uptake (4). In our study we have not found that protein deprivation per se is responsible for liver iron overload, in contrast with the results of another study in which iron overload ensued after feeding rats with a 6% protein-containing diet (11). Liver siderosis has been described in kwashiorkor (22), perhaps due to impaired synthesis of transport proteins (13). Thus, we conclude that protein malnutrition, but not ethanol, leads to liver zinc depletion. Both ethanol and protein malnutrition cause muscle zinc depletion and increase urinary zinc and manganese excretion. Ethanol also increases urinary iron excretion and liver manganese content.
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