COMBINED EFFECTS OF STEROIDS, ETHANOL AND PROTEIN DEFICIENCY ON TISSUE CONTENT AND URINARY AND FAECAL EXCRETION OF ZINC, COPPER AND IRON

E. González-Reimers,*, F. Santolaria-Fernández, P. Garrido-Benedicto, M. C. Durán-Castellón, L. Galindo-Martín1, A. Martínez-Riera, J. Viña-Rodríguez and M. J. de La Vega-Prieto2

Servicio de Medicina Interna, Hospital Universitario de Canarias, Tenerife,
1 Dpto. de Química Analítica, Universidad de La Laguna, Tenerife and
2 Servicio de Laboratorio, Hospital Universitario de Canarias, Tenerife, Canary Islands (Spain)

Received 30 May 2001; in revised form 31 July 2001; accepted 25 August 2001


    ABSTRACT
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study was performed in order to determine the relative and combined effects of ethanol, a low protein diet and steroid treatment on bone, muscle, liver, and urinary and faecal excretion of zinc, copper and iron in 64 rats divided into eight groups treated following the Lieber–DeCarli liquid diet technique, with and without dexamethasone, 1 mg/l. Steroids showed a lack of effect on liver zinc, but enhanced ethanol- and low protein-mediated liver iron overload when both factors were combined. Steroids also increased muscle copper, iron and zinc, and bone copper, especially in the low protein, ethanol-fed rats.


    INTRODUCTION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Zinc is an essential element involved in protein synthesis (it is a cofactor of several DNA and RNA polymerases; Hsu, 1980Go). Low liver zinc levels are associated with enhanced prolyl hydroxylase activity (Rojkind and Dunn, 1979Go), a situation which favours liver collagen deposition (Anttinen et al., 1982Go); collagen breakdown is also inhibited by low liver zinc levels. In addition, zinc shows antioxidant properties (Powell, 2000Go). Therefore, zinc supplementation has been considered an efficient therapy for liver cirrhosis (Anttinen et al., 1982Go) and other disorders related to heavy alcohol consumption (Menzano and Carlen, 1994Go). Also, zinc supplementation may inhibit bone resorption (Moonga and Dempster, 1995Go), an eventually important effect in chronic alcoholics, who show variable degrees of osteoporosis (Diamond et al., 1989Go).

Other elements, such as copper and iron, may also be involved in several biochemical abnormalities observed in alcoholic liver injury. Iron overload is observed in alcoholics, especially when protein malnutrition coexists (Chapman et al., 1982Go; Lesage et al., 1983Go), and may lead to lipid peroxidation, a key step in the development of alcoholic liver disease (Lieber, 1980Go; Poli, 2000Go). Although some authors, such as our group (González-Reimers et al., 1998Go), have failed to observe ethanol-induced alterations of copper metabolism, copper accumulation may lead to liver injury (Sternlieb, 1980Go), whereas copper deficiency may affect scavenging of free radicals (Ziedenberg-Cherr et al., 1990Go) and thus promote oxidative injury (Saari, 2000Go).

Steroids have been employed to treat alcoholic hepatitis (Carithiers et al., 1989Go), especially when liver failure is severe (Maddrey et al., 1978Go) in non-bleeding patients (Imperiale and McCullough, 1990Go). Although not widely studied, some reports point to an increase of liver zinc (Fontaine et al., 1989Go) or testicular zinc (Nair et al., 1998Go) after treatment with steroids, and other observations reported increased serum iron levels after treatment with steroids (Smith et al., 1986Go). Some of these effects are opposite to those caused by ethanol consumption. It was therefore considered important to analyse in the present work the relative and combined effects of ethanol and steroids on trace element metabolism, also taking into account the effect of protein malnutrition, which is frequently associated with ethanol consumption (Mendenhall et al., 1984Go).


    MATERIALS AND METHODS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and treatments
Sixty-four male Sprague–Dawley rats were divided into eight groups of eight animals each. The control rats (group 1) received the Lieber–DeCarli (1989) control diet (Dyets Inc., Bethlehem, PA, USA), containing 18% protein and 1 kcal/ml; a second group was fed an isocaloric, 36% ethanol-containing diet (group 2); the third group was fed an isocaloric, 2% protein-containing diet (group 3); and the fourth group was fed an isocaloric diet containing 2% protein and 36% ethanol (group 4); groups 5–8 received identical treatment to groups 1–4 but additionally dexamethasone, which was dissolved in the diet at a dosage of 1 mg/l. As liquid mean diet consumption was ~55 ml/day, the daily dexamethasone dosage was ~55 µg/day (~0.18 mg/kg/day), which is roughly equivalent to a dose of 1 mg/kg/day prednisone. Due to the loss of weight experienced by the ethanol and/or low protein-fed animals with steroids, dosage was reduced to 75% at the 3rd week and to 50% at the 6th week, and also to 75% at the 6th week to the control animals + steroids, so that the amount of steroids/kg remained approximately the same during the whole study in the four groups treated with steroids. This treatment was necessary because, previously, most rats treated with either the ethanol and/or low protein diet plus steroids without dosage reduction died at about the 5th week of the experiment; necropsy studies revealed multiple abscesses in lung, liver, and pleura; cultures of the abscess content were uniformly positive for Corynebacterium kutscheri, a well-known pathogen of laboratory animals.

Those rats receiving the ethanol-containing protein-deficient diet + steroids consumed the diet ad libitum, and the same amount consumed by these animals was then given to the other groups. This pair-feeding 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, steroid-treated, ethanol-containing diet. The mean daily amount of diet consumed is shown in Table 1Go. There were slight differences between the amount of diet consumed by the different groups, which became statistically significant when the control and the low protein-fed groups were compared with the group consuming the protein-deficient, steroid-treated, ethanol-containing diet. Another group of five animals was allowed to consume the control diet ad libitum. The amount of diet consumed by these animals was 79.85 ± 2.35 kcal/day, significantly more than the amount consumed by the study groups.


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Table 1. Serum albumin, weight change, and daily diet consumption
 
All four diets contained the same amount of iron (8.8 mg/l), copper (1.5 mg/l) and zinc (7.5 mg/l), so the experimental animals consumed approximately 400 µg zinc, 80 µg copper and 500 µg iron daily, amounts which are adequate for normal development (Underwood, 1977Go).

All but two animals were alive at the end of the experiment, 8 weeks later. One day before killing (with pentobarbital), the rats were placed in metabolic cages, and 24-h urine and faeces were collected. Blood was obtained by direct cardiac puncture, and centrifuged. The right femur was also removed.

Trace element determinations
Bone, liver, muscle and faecal samples destined for trace element analysis were dehydrated in a furnace at 100°C for 4–7 days, weighed, and then dissolved in 65% HNO3 (Merck p.a.) and 10% H2O2, in order to digest organic material. The digestion solutions were quantitatively transferred to volumetric flasks and diluted to 10 ml with ultrapure water (Milli-Q OM-140 deionization system). Urine samples were diluted 1:1 with ultrapure water (Milli-Q OM-140 deionization system). The weight of the dehydrated liver samples ranged from 170 to 1310 mg, that of the muscles from 86 to 860 mg, that of the bones from 111 to 540 mg, and that of faeces from 150 to 3550 mg.

All the measurements were performed with the aid of a Varian Spectra AA spectrophotometer (Victoria, Australia) by flame atomic absorption spectrophotometry. Detection limits for these elements were 0.009 for Zn, 0.026 for Cu and 0.039 p.p.m. for Fe.

Other determinations
We also measured serum albumin and serum and urinary creatinine by routine analytical methods.

Statistics
Differences between the experimental groups were analysed using analysis of variance (ANOVA) with further Student– Newman–Keuls test. Independent effects of ethanol, steroids and protein deficiency and interactions between these parameters (two by two and the three altogether) were analysed by means of two-way ANOVA. Also, single correlation analyses (Pearson's r) were performed between quantitative variables.


    RESULTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Body weight and serum albumin concentration
These are shown in Table 1Go. The only rats which gained weight during the experiment were the control ones, whereas weight loss was observed in the other groups (Table 1Go). The weight loss was especially marked in the low protein-fed animals with steroids. Indeed, both the three factors (steroids, alcohol and a low protein diet) exerted significant, independent effects on final weight, with significant interactions existing between steroids and the other two factors, especially between steroids and the low protein diet.

Serum albumin (Table 1Go) was lower in the low protein-fed animals, especially without steroids. Rats treated with steroids showed a trend to higher serum albumin values, a low protein diet exerting a significant negative effect (F = 22.8), whereas steroids exerted a positive one (F = 6.7).

Zinc
Steroids led to an accumulation of zinc in muscle (Table 2Go), counteracting the lowering effect of both ethanol and a low protein diet. A similar result was observed in the liver (Table 3Go): the ethanol, 2% protein-fed, steroid-treated rats showed the highest liver zinc values. Similar changes were observed in bone (Table 2Go), low protein-fed rats both with and without ethanol treated with steroids showing the highest bone zinc values, whereas treatment with ethanol and a low protein diet or ethanol and steroids led to the lowest bone zinc values.


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Table 2. Muscle and bone iron, copper and zinc contents
 

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Table 3. Liver content and faecal excretion of copper, zinc and iron
 
As the low protein-fed animals treated with steroids and ethanol lost most weight during the study (Table 1Go), it is not surprising that a negative correlation was observed between muscle zinc and weight difference (r = -0.41, P < 0.001). No differences were observed regarding both urinary (Table 4Go) and faecal (Table 3Go) excretion of zinc.


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Table 4. Urinary excretion of copper, zinc and iron
 
Iron
Low protein-fed animals, either with or without concomitant ethanol administration, and especially with steroids, showed the highest liver iron values (Table 3Go); with both ethanol (F = 4.2), but especially low protein (F = 13) exerting independent effects. Similar results were observed in muscle (Table 2Go), the highest values being observed in the ethanol, 2% protein, steroid-treated group, although steroids, but not alcohol, exerted independent effects on muscle iron, together with a low protein diet.

Significant negative correlations were observed between muscle iron and weight difference (r = -0.49, P < 0.001) and muscle iron and serum albumin (r = -0.28, P = 0.03), and also between weight difference and liver iron (r = -0.32, P = 0.005).

No differences were observed in faecal (Table 3Go) and urinary (Table 4Go) iron excretion between the eight groups, nor regarding bone iron (Table 2Go). A significant correlation was observed between liver iron and muscle iron (r = 0.26, P < 0.04).

Copper
Steroids tended to lower liver copper, with control steroid rats showing the lowest copper values (Table 3Go). However, this effect seemed to be counteracted by both a low protein diet (F = 10.4) and ethanol (F = 10). However, unusually high liver copper values were observed in control animals, even significantly higher than those of the animals fed ad libitum. Steroid- and ethanol-treated, 2% protein-fed animals showed the highest bone and muscle copper values (Table 2Go). In both cases, steroids exerted significant independent effects, together with a low protein diet in the case of bone, and together with a low protein diet and ethanol in the case of muscle copper.

Muscle copper (r = -0.44) and bone copper (r = -0.37, P < 0.01 in both cases) were inversely related to weight loss; a negative correlation was also observed between bone copper and urinary copper excretion (r = -0.36, P = 0.01)

Correlations between the tissue contents of iron, copper and zinc are shown in Table 5Go. Strong correlations were observed between muscle zinc and muscle iron and copper, and between muscle copper and muscle iron. Urinary zinc excretion correlated with urinary iron (r = 0.30, P < 0.05) and copper excretion (r = 0.33, P < 0.01). Also, a significant correlation was observed between urinary copper and urinary iron (r = 0.35, P < 0.01). Total iron excretion was significantly higher in those groups treated with ethanol, whereas a trend to lower values was observed in the groups treated with steroids. Both factors (steroids and ethanol) exerted independent, opposite effects.


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Table 5. Correlations between the tissue contents of copper, zinc and iron
 

    DISCUSSION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The results obtained in the non-steroid-treated groups in the present study are similar to those previously reported by us (González Reimers et al., 1998Go). To our knowledge, no other study has dealt with the combined effects of steroids and ethanol on trace element concentrations in diverse organs, and only a few have analysed the combined effects of ethanol and protein deficiency on muscle (González-Reimers et al., 1993Go) and bone trace elements, higher bone Zn values having been reported in ethanol-fed rats (Preedy et al., 1991Go).

Some authors have observed that steroids may lead to liver zinc accumulation (Fontaine et al., 1989Go), whereas others have observed an accumulation of zinc in testes after steroid treatment (Nair et al., 1998Go). The accumulation of zinc in the liver would be beneficial for the alcoholic, taking into account the aforementioned effects of zinc on collagen synthesis and breakdown. In our study, however, we failed to find a beneficial effect of steroids on liver zinc, except perhaps in the rats treated with steroids and ethanol and fed a low protein diet, in which liver zinc was slightly increased. Thus, steroids do not seem to modify significantly ethanol- and/or low protein-induced alterations in liver zinc content.

Despite a slightly lower diet consumption, we also observed an increase of zinc in bone and muscle of rats treated with steroids, especially in the ethanol and low protein diet (in the case of muscle and bone), and also in the low protein diet without ethanol (in bone). The relationship between increased zinc and the expected changes in bone and muscle is unclear: steroids increase muscle and bone protein breakdown, and thus favour protein wastage (Afifi and Bergman, 1969Go), a clinical situation classically related to zinc depletion via increased urinary excretion (Fell et al., 1973Go). Indeed, in another study performed by our group using the Lieber–DeCarli model, muscle zinc was associated to type II fibre diameter with serum albumin (González-Reimers et al., 1993Go). Urinary zinc excretion corrected by creatinine showed a non-significant trend to higher values in those groups which showed the highest bone and muscle zinc contents, although absolute zinc excretion showed a trend to lower values in these groups. It is also important to keep in mind that, since the pair-feeding method was carried out every 2 days, in the last weeks of the experiment the rats which lost more weight received more zinc, copper and iron (and ethanol)/body weight than those rats which did not lose weight. This could explain, at least in part, the increased levels of the elements analysed in the low protein-fed animals with steroids. This is a factor which is impossible to control using the Lieber–DeCarli experimental method, but in any case, the dosage of zinc (and other elements) contained in the Lieber–DeCarli diets is appropiate for the ideal weight, so it would be unwise to underfeed the rats in certain elements only because they are truly undernourished. However, two-way ANOVA show that steroids exert independent, significant effects on muscle copper, iron and zinc, as well as on bone copper, increasing tissue concentrations of all the three elements, although the mechanisms by which steroids could cause these effects are unknown.

In confirmation of several earlier reports, we have found significantly higher liver and muscle iron in the low protein-fed animals. The liver iron content was indeed higher in the ethanol, 2% protein-fed animals than in the controls (a result in accordance with observations performed in children with Kwashiorkor; Ashour et al., 1999Go), but even higher when steroids were added to the low protein-fed, ethanol-treated animals. Accumulation of iron in the liver exerts deleterious effects in the alcoholic, since it promotes lipid peroxidation (Bacon et al., 1983Go; Shaw et al., 1988Go) and enhances prolyl hydroxylase activity (Brissot et al., 1987Go). However, the effect of steroids on the other groups was the opposite, and so two-way ANOVA failed to show any effect of steroids on liver iron. In any case, liver alterations are more marked when ethanol and protein deficiency coexist (Conde-Martel et al., 1992Go), so steroids may even increase liver damage in this group.

In the present study, we have observed unusually high liver copper values in the control population, a result which distorts the relative uniformity of the values observed in the other groups. Indeed, although ANOVA yielded statistically significant differences between the mean copper values of all the groups taken together, post hoc analysis revealed that the differences only exist between the control group and all the others. There is no satisfactory explanation for this result, especially considering the differences which also exist between the ad libitum-fed group and the control group. Overall, there was a non-significant trend to higher copper values, both in liver and in muscle, in those animals treated with steroids. The absence of significant increases in liver copper after steroid administration has been also described in rats (Fontaine et al., 1992Go). Moreover, there are disparate results regarding the effect of ethanol and/or protein deficiency on liver copper: most authors have failed to find any effect (Zarski et al., 1985Go; González Reimers et al., 1998Go), although increased liver copper has been also described (Sternlieb, 1980Go), and low serum copper has been observed in marasmic children (Ashour et al., 1999Go; Tatli et al., 2000Go).

In summary, steroids show a lack of effect on liver zinc, and seem to enhance ethanol and low protein-mediated liver iron overload when both factors are combined. Steroids also increase muscle copper, iron and zinc, and bone copper, especially in low protein, ethanol-fed rats.


    FOOTNOTES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
* Author to whom correspondence should be addressed. Back


    REFERENCES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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