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Effect of Zinc supplementation on ethanol-mediated bone alterations
Food and Chemical Toxicology 43 (2005) 1497–1505 www.elsevier.com/locate/foodchemtox
Effect of Zinc supplementation on ethanol-mediated bone alterations E. Gonza´lez-Reimers a,*, M.C. Dura´n-Castello´n a, R. Martı´n-Olivera a, A. Lo´pez-Lirola a, F. Santolaria-Ferna´ndez a, M.J. De La Vega-Prieto b, A. Pe´rez-Ramı´rez a, E. Garcı´a-Valdecasas Campelo a a
Departamento de Medicina Interna, Hospital Universitario, 38320 Tenerife, Canary Islands, Spain b Servicio de Laboratorio, Hospital Universitario, 38320 Tenerife, Canary Islands, Spain
Abstract Ethanol consumption leads to bone alterations, mainly osteoporosis. Ethanol itself may directly alter bone synthesis, but other factors, such as accompanying protein malnutrition—frequently observed in alcoholics-, chronic alcoholic myopathy with muscle atrophy, alcohol induced hypogonadism or hypercortisolism, or liver damage, may all contribute to altered bone metabolism. Some data suggest that zinc may exert beneficial effects on bone growth. Based on these facts, we analyzed the relative and combined effects of ethanol, protein malnutrition and treatment with zinc, 227 mg/l in the form of zinc sulphate, on bone histology, biochemical markers of bone formation (osteocalcin) and resorption (urinary hydroxyproline excretion), and hormones involved in bone homeostasis (insulin growth factor 1 (IGF-1), vitamin D, parathormone (PTH), free testosterone and corticosterone), as well as the association between these parameters and muscle fiber area and liver fibrosis, in eight groups of adult Sprague Dawley rats fed following the Lieber de Carli model during 5 weeks. Ethanol showed an independent effect on TBV (F = 14.5, p < 0.001), causing it to decrease, whereas a low protein diet caused a reduction in osteoid area (F = 8.9, p < 0.001). Treatment with zinc increased osteoid area (F = 11.2, p < 0.001) and serum vitamin D levels (F = 3.74, p = 0.057). Both ethanol (F = 45, p < 0.001) and low protein diet (F = 46.8, p < 0.01) decreased serum osteocalcin levels. Ethanol was the only factor independently related with serum IGF-1 (F = 130.24, p < 0.001), and also showed a synergistic interaction with protein deficiency (p = 0.027). In contrast, no change was observed in hydroxyproline excretion and serum PTH levels. No correlation was found between TBM and muscle atrophy, liver fibrosis, corticosterone, or free testosterone levels, but a significant relationship was observed between type II-b muscle fiber area and osteoid area (q = 0.34, p < 0.01). Osteoporosis is, therefore, present in alcohol treated rats. Both alcohol and protein deficiency lead to reduced bone formation. Muscle atrophy is related to osteoid area, suggesting a role for chronic alcoholic myopathy in decreased bone mass. Treatment with zinc increases osteoid area, but has no effect on TBV. 2005 Elsevier Ltd. All rights reserved. Keywords: Alcohol; Osteopenia-Muscle atrophy; Osteoporosis; Protein deficiency; Malnutrition; Zinc treatment
1. Introduction
Abbreviations: BMD, bone mineral density; BMI, body mass index; DNA, Desoxyribonucleic acid; IGF-1, insulin-like growth factor 1; IL1, interleukin 1; IL-6, interleukin 6; NS, non significant; PTH, parathormone; IA, radioimmunoanalysis; RNA, ribonucleic acid; SNK, StudentÕs-Newmann–Keuls; TBV, trabecular bone volume; Vs, versus; WHO, World Health Organization. * Corresponding author. 0278-6915/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2005.04.003
Alcoholism is a risk factor for osteoporosis. Two major reasons may explain this occurrence: the peculiar lifestyle of most alcoholics, which makes them prone to falls and traumatisms, and the association between chronic ethanol consumption and reduced bone mass. The pathogenesis of osteopenia in alcoholic subjects is not fully understood. Ethanol seems to exert a direct toxic effect on osteoblast function (Giuliani et al.,
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1999), although it also alters, directly and indirectly, bone mineral metabolism, including PTH and vitamin D homeostasis, testosterone, IGF-1, and cortisol levels. Ethanol appears to reduce bone formation and leads to decreased osteoid synthesis and decreased mineralization rate (Diamond et al., 1989) in a dose-dependent fashion (Turner, 2000). Ethanol may also increase bone resorption. Recent studies performed by Dai et al. (2000) show that ethanol induces bone loss through IL-6, which in turn activates osteoclastogenesis. Nutritional status may also be important, as well as decreased muscle mass and reduced muscle strength and physical activity. These observations are supported by several experimental and clinical studies, which have shown a relationship between a low body mass index and osteopenia and the importance of protein calorie malnutrition in the pathogenesis of the metabolic bone disease suffered by the alcoholic patient (Bendavid et al., 1996; Schurch et al., 1998; Molina-Pe´rez et al., 2000; Santolaria et al., 2000). Indeed, bone formation is impaired in protein-calorie malnutrition (Bourrin et al., 2000a), and bone resorption is also decreased, leading to a slow turn-over osteopenia (Bourrin et al., 2000b). Huuskonen et al. (2000) conclude that low dietary calcium intake, weak muscle strength and low body weight, but not alcohol consumption, are risk factors for low BMD in men. Alcoholic myopathy with muscle atrophy (Preedy and Peters, 1990; UrbanoMa´rquez et al., 1989; Romero et al., 1994; Peters et al., 1985), on which both ethanol and protein deficiency may act synergistically (Conde-Martel et al., 1992), may also exert a detrimental effect on bone mass. Alcoholics frequently show zinc deficiency (Prasad, 1991). Zinc is required for the production of many enzymes involved in protein synthesis, nucleic acid metabolism and immune function, and also plays an important role in gene transcription (Hambidge, 2000). Indeed, several transcription factors contain the so-called zinc finger domains which are important in protein-DNA or protein-RNA interactions (Dreosti, 2001). In some recent studies it has been shown that zinc supplements added to the diet may exert a positive influence on human bone linear growth (Dı´az-Go´mez et al., 2003; Merialdi et al., 2004). These results agree with former observations performed in rats or monkeys, in which zinc deficiency was associated with stunted growth and defective endochondral ossification. (Leek et al., 1984; Da Cunha Ferreira et al., 1991). Experimental data suggest that zinc stimulates bone protein synthesis and bone formation by increasing the activity of alkaline phosphatase and other key enzymes (Yamaguchi and Yamaguchi, 1986), and also reduces osteoclast activity (Kishi and Yamaguchi, 1994). Zinc may, therefore, exert an anabolic effect on bone, in some ways opposite to that exerted by ethanol. Based on these facts we investigate here the effects of zinc supplementation on bone changes induced by ethanol and protein defi-
ciency in male Wistar rats fed following the Lieber de Carli model, taking into account the possible influence of co-existing protein deficiency, myopathy, and liver fibrosis.
2. Material and methods 2.1. Animals and treatments Eighty male Sprague–Dawley rats were divided into 4 groups of 10 animals each, and treated following the Lieber de Carli model with a control diet, a 2% proteincontaining diet, a 36%-ethanol containing diet, and a 2% protein 36% ethanol containing diet (Lieber et al., 1989) with daily addition of zinc sulphate (227 mg/l zinc). Four further groups were fed identical diets and served as controls. Another group of five animals was allowed to consume the control diet ad libitum. Details about diet composition and dietary consumption are reported elsewhere (Dura´n-Castello´n et al., in press). At the end of the trial, blood was obtained by direct cardiac puncture, and centrifuged. We removed the first two lumbar vertebrae, for histomorphometric analysis, as well as the right gastroecnemius muscle for ATP-ase staining and further measurement of Type I, Type II-a and Type II-b muscle fiber areas, and the liver, for histomorphometrical assessment of the amount of liver fibrosis. 2.2. Bone histomorphometry Lumbar vertebrae were processed for undecalcified bone sample analysis. Briefly, samples were embedded in methylmetacrylate (Sigma Chemicals, St Louis, Missouri, USA), stored for 24 h at 4 C and later polymerised at 32–34 C for 3–4 days. Embedded samples were then cut in 9–12 lm thick slices with a Reichert-Jung microtome and stained with Masson-Goldner. Trabecular mineralized bone volume (TBV) and osteoid area were determined using an image analyser equipped with the program ‘‘Image Measure 4.4a’’ (Microscience Inc.), at 40· and 200·, respectively. Results are presented as percentages of the total area. 2.2.1. Liver histological analysis Liver sample slices stained with haematoxylin-eosin and Van Giesson were used for histomorphometrical analysis, which was performed with the aid of an image analyzer equipped with the program Image Measure 4.4a (Microscience Inc), calculating the total amount of fibrous tissue (at 20·). 2.2.2. Serum hormones and albumin levels Serum parathormone (PTH) was measured by radioimmunoanalysis (RIA), using an antibody directed
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towards intact rat PTH (Nichols, San Juan de Capistrano, California, USA), with a sensitivity of 1 pg/ml. Serum osteocalcin was measured by RIA, using an antibody specific for rat (Biomedical Technologies, St Stoughton, Maryland, USA; specificity for rat osteocalcin >10,000 fold bovine, monkey, human, and chicken osteocalcins; sensitivity 0.025–1.5 ng per tube). We also measured urinary creatinine and hydroxyproline, by liquid cromatography (Dawson et al., 1988). Other biochemical parameters which were used in this study include serum albumin, serum and urinary calcium and magnesium, measured by routine analytical methods, and serum insulin-like growth factor type 1 (IGF-1), free testosterone and corticosterone, measured by RIA. Details about measurement techniques are already reported (Dura´n-Castello´n et al., in press). 2.3. Statistics The Kolmogorov–Smirnov test was performed to test normality. A non-normal distribution, with marked intergroup variation, was observed for some variables, such as osteoid area, and urinary hydroxyproline, calcium, magnesium and phosphorus excretion, although these parameters showed a normal distribution within each group of animals. Therefore, SpearmanÕs rank test was used to analyse the relationships between these variables and other quantitative parameters. Differences between the experimental groups were analysed using variance analysis with further StudentÕs-Newman–Keuls (SNK) test. Independent effects of ethanol, zinc, and protein deficiency on the alterations observed, and interactions between these parameters, were analysed by twoway variance analysis. Paired t-test analysis was used to compare initial and final weight in the eight groups of rats. Differences between the analysed parameters in controls and ad libitum fed animals were analysed using the StudentÕs t test. Also, single correlation analysis
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(PearsonÕs ‘‘r’’) was performed between quantitative variables.
3. Results The results are shown in Tables 1–4. As reported elsewhere (Dura´n-Castello´n et al., in press), all the rats weighed the same at the beginning of the study and all of them, except for those in the control groups (both without and with zinc supplement), showed a significant weight loss during the experimental period. As shown in Table 1 and in Figs. 1 and 2, we found reduced TBV in rats treated with ethanol, either with or without protein restriction, and either treated or not with zinc. Two-way variance analysis revealed that ethanol (F = 14.59, p < 0.001) was the sole factor associated with decreased TBV, although a direct correlation was observed between TBV and weight change over the study period (r = 0.29, p = 0.017). The addition of zinc exerted no significant effect, although there was a trend to higher TBV values in the zinc-treated animals. A direct correlation was observed between TBV and osteocalcin (r = 0.35, p < 0.001, Fig. 3). No relationship was observed between TBV and muscle fiber area, or with liver fibrosis. Osteoid area was also lower among rats treated with ethanol, especially in those which also received a low protein diet. Indeed, a significant effect of low protein diet (F = 8.88, p = 0.004), which was additive to that of ethanol (interaction between both factors, F = 4.47, p = 0.039) was observed, as well as a direct relationship between osteoid area and weight change during the study period (rho = 0.43, p < 0.001, Fig. 4). However, treatment with zinc significantly reversed the decrease in osteoid area (F = 11.22, p = 0.001). Osteoid area showed a direct correlation with serum osteocalcin (rho = 0.41, p < 0.001, Fig. 5), IGF-1 (rho =
Table 1 Trabecular bone volume (TBV) and osteoid area (mean ± standard deviation)
Control (1) Low protein (2) Control alcoholic (3) Low protein alcoholic (4) Control zinc (5) Low protein zinc (6) Alcohol + zinc (7) Alcohol + low protein + zinc (8) F; p Differences among groups (SNK test) Main effects Interactions Ad libitum (9) Differences 1–9
TBV (%)
Osteoid (%)
22.9 ± 4.9 21.2 ± 2.1 16.0 ± 3.9 17.6 ± 4.9 22.3 ± 4.0 19.9 ± 2.2 19.3 ± 5.0 17.7 ± 4.6 F = 2.72, p = 0.016 1,5 vs 2,3,4,6,7,8 Ethanol F = 14.5, p < 0.001 – 19.6 ± 2.0 NS
1.14 ± 0.35 1.13 ± 0.41 0.90 ± 0.65 0.30 ± 0.40 1.81 ± 0.77 1.41 ± 0.44 2.60 ± 1.98 0.80 ± 0.97 F = 4.87, p = 0.001 5,7 vs 1,2,3,4,6,8 4 vs 1,2,3,5,6,7,8 Low protein, F = 8.9, p < 0.001; zinc F = 11.2, p < 0.001 Ethanol-Low protein p = 0.039 2.20 ± 0.94 NS
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Table 2 Serum corticosterone and testosterone, and urinary hydroxyproline excretion (mean ± standard deviation)
Control (1) Low protein (2) Control alcoholic (3) Low protein alcoholic (4) Control zinc (5) Low protein + zinc (6) Ethanol + zinc (7) Ethanol + zinc + low protein (8) F; p Differences among groups (SNK test) Main effects Interactions Ad libitum (9) Differences 1–9
Hydroxyproline excretion (mg/dl)
Corticosterone (ng/ml)
Free testosterone (pg/ml)
41.0 ± 26.8 38.6 ± 60.2 32.1 ± 6.9 52.7 ± 47.2 38.4 ± 31.3 49.8 ± 36.3 92.6 ± 22.9 33.5 ± 12.6 F = 2.94, p = 0.01 7 vs all the others – Ethanol/protein deficiency/zinc p = 0.007 52.9 ± 25.3 NS
413 ± 329 499 ± 162 684 ± 223 553 ± 240 450 ± 275 486 ± 229 446 ± 333 567 ± 270 F = 0.81, NS – – – 533 ± 161 NS
0.59 ± 1.23 1.51 ± 2.48 4.70 ± 9.09 0.37 ± 0.43 0.79 ± 1.63 3.69 ± 7.44 1.71 ± 2.97 1.25 ± 3.450 F = 1, NS – – – 12.32 ± 12.01 NS
Table 3 Serum IGF-1, vitamin D, osteocalcin, and parathyroid hormone (PTH) levels (mean ± standard deviation) IGF-1 (ng/ml)*
Vitamin D (ng/ml)
Osteocalcin (ng/ml)
PTH (pg/ml)
Control (1) Low protein (2) Control alcoholic (3) Low protein alcoholic (4) Control zinc (5) Low protein + zinc (6) Ethanol + zinc (7) Ethanol + zinc + low protein (8) F; p Differences among groups (SNK test) Main effects
14.93 ± 2.29 18.26 ± 2.92 6.27 ± 5.16 2.59 ± 1.88 16.78 ± 3.37 16.95 ± 4.02 4.40 ± 5.36 4.37 ± 4.30 F = 29.4, p < 0.001 1,2,5,6 vs 3,4,7,8
Ethanol/protein deficiency p = 0.04 21.22 ± 6.05 NS
49.5 ± 9.4 43.0 ± 8.2 43.2 ± 10.6 22.3 ± 9.3 58.3 ± 11.7 41.9 ± 9.2 41.0 ± 13.3 21.0 ± 8.0 F = 14.72, p < 0.001 1,5 vs 2,3,6,7,4,8 4,8 vs 1,2,3,5,6,7 Low protein F = 45 p < 0.001; ethanol F = 46.8, p < 0.001 Ethanol-Low protein p = 0.06
110.0 ± 148.1 75.4 ± 45.8 75.9 ± 30.0 99.7 ± 70.1 61.1 ± 54.9 78.6 ± 62.9 91.4 ± 47.1 105.3 ± 36.0 F = 0.59, NS –
Interactions
67.7 ± 33.1 37.8 ± 28.0 72.5 ± 13.0 17.8 ± 9.0 66.1 ± 26.4 41.3 ± 19.5 99.0 ± 36.8 38.7 ± 28.3 F = 8.80, p < 0.001 1,3,5,7 vs 2,4,6,8; 4 vs all the others Low protein F = 44 p < 0.001; zinc F = 3.74, p = 0.057 Ethanol-Low protein p = 0.02 38.7 ± 6.1 T = 2.55, p = 0.03
76.8 ± 8.8 T = 5.4, p < 0.001
80.4 ± 61.6 NS
Ad libitum (9) Differences 1–9
Ethanol F = 189 p < 0.001
–
–
NS = non significant; vs = versus; SNK = StudentÕs-Newmann–Keuls. * Excluding one outlier (42.71 ng/ml), belonging to group 6.
0.38, p = 0.001) and serum vitamin D (rho = 0.27, p = 0.02). Also, a direct relationship was observed between osteoid area and Type IIa (rho = 0.29, p = 0.03) and Type IIb (rho = 0.34, p = 0.01, Fig. 6) muscle fiber diameter, but not with the amount of liver fibrosis. In contrast with serum PTH, which had similar values in the different groups, serum osteocalcin, IGF-1 and vitamin D showed marked differences (Table 3). A low protein diet caused a decrease in serum vitamin D levels, especially when added to ethanol. On the contrary, zinc treatment tended to lessen the low-protein diet-induced reduction in serum vitamin D. In accordance with these results, serum vitamin D was directly related with weight at the end of the study (r = 0.59, p < 0.001) and with serum albumin (r = 0.36, p = 0.003) and serum osteocalcin (r = 0.40, p = 0.001).
Rats treated with a low protein diet and ethanol showed the lowest serum osteocalcin values. A synergistic interaction was detected between both factors, whereas zinc did not play any role. Highly significant relationships were observed between serum osteocalcin and weight change during the study period (r = 0.73, p < 0.001, Fig. 7) and serum osteocalcin and serum IGF-1 (r = 0.45, p < 0.001). Serum IGF-1 was lowest among rats treated with ethanol and a low protein diet; as already reported (Dura´n-Castello´n et al., in press), both factors showed an additive effect, which was not modified by treatment with zinc. Serum IGF-1 showed a significant relationship with weight at the end of the study (r = 0.23, p = 0.049), and an inverse relationship with urinary calcium excretion (rho = 0.27, p = 0.019) and serum magnesium (r = 0.30, p = 0.009).
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Table 4 Serum calcium and magnesium (mg/dl) and urinary magnesium (mg/l) and calcium excretion (mg/dl) (mean ± standard deviation) Serum calcium
Serum magnesium
Urinary magnesium
Urinary calcium
Main effects
11.4 ± 0.9 11.3 ± 0.4 11.1 ± 0.6 11.0 ± 0.8 11.4 ± 1.1 10.9 ± 0.9 11.3 ± 0.9 11.1 ± 0.6 F = 0.65, NS – –
28.2 ± 34.3 12.1 ± 19.7 12.4 ± 17.9 30.4 ± 27.0 35.8 ± 33.6 26.8 ± 42.9 22.2 ± 25.7 14.6 ± 19.5 F = 0.84, NS – –
2.08 ± 1.22 1.94 ± 1.35 2.30 ± 1.06 5.92 ± 4.52 2.26 ± 1.20 2.08 ± 1.51 3.04 ± 3.10 2.88 ± 2.11 F = 2.94, p = 0.01 4 vs all the others Ethanol F = 6.99, p < 0.01
Interactions Ad libitum (9) Differences 1–9
– 11.2 ± 0.4 NS
3.21 ± 0.58 2.60 ± 0.39 3.08 ± 0.57 2.86 ± 0.56 2.97 ± 0.53 2.31 ± 0.57 3.37 ± 0.65 3.02 ± 0.43 F = 3.69, p = 0.02 1,3,7 vs 6 Ethanol F = 5.85, p = 0.018; low protein F = 12.94, p = 0.001 – 2.76 ± 0.34 NS
– 90.0 ± 91.7 NS
– 2.88 ± 1.55 NS
Control (1) Low protein (2) Control alcoholic (3) Low protein alcoholic (4) Control zinc (5) Low protein + zinc (6) Alcohol + zinc (7) Alcohol + low protein + zinc (8) F; p
NS = non-significant.
Fig. 1. Section of the first lumbar vertebra of a control rat, stained with Masson–Goldner (20·).
We failed to find differences among the different groups with respect to corticosterone and testosterone. No significant relationships were observed between these hormones and any of the parameters analysed. In Table 4 we show the results for serum and urinary calcium and magnesium. Serum magnesium levels varied among the different groups. Low protein-fed animals, either treated with or without zinc, showed the lowest serum magnesium values. Ethanol significantly increased urinary calcium excretion. 4. Discussion This study was carried out in order to test the effect of zinc on alcohol-induced bone alterations, taking into account that protein deficiency may also play a concomitant role in its pathogenesis.
Fig. 2. Section of the first lumbar vertebra of a rat treated with ethanol and a protein-deficient diet, stained with Masson–Goldner (20·).
In recent years there have been several reports supporting the view that moderate ethanol consumption is not harmful, but even protects bone (Baron et al., 2001). The detrimental effect of ethanol would be observed in men, in contrast with a neutral or slightly beneficial effect in older women. Despite these observations, classic studies performed by Diamond et al. (1989), among others, have shown that chronic alcoholism causes osteoporosis. In this study we have shown that rats treated with ethanol following the Lieber de Carli model do develop osteopenia. We found that ethanol is solely responsible for decreased TBV, in contrast with another study performed some years ago, in which protein deficiency exerted the most important effect (Molina-Pe´rez et al., 2000). However, in this study, a relationship exists between bone mass and weight loss, also suggesting an effect of malnutrition on decreased
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osteoid (%)
TBV (%)
4.00 25.00
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2.00 15.00
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Osteoid (%)
osteoid (%)
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0.00 -100.00
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Fig. 5. Relationship between osteoid area and osteocalcin.
Fig. 3. Relationship between trabecular bone mass (TBV) and serum osteocalcin.
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Osteocalcin (ng/ml)
Osteocalcin (ng/ml)
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Weight change (g) Fig. 4. Relationship between osteoid area and weight change during the experiment.
TBV. Lalor et al. (1986) found that alcoholics affected by severe osteoporosis were heavier drinkers than those without osteoporosis and also consumed less proteins and presented lower albumin levels. Bone mass was dependent on serum albumin—which, in turn, was dependent on dietary protein ingestion—, body mass index, and dietary calcium intake. On the contrary, Diamond et al. (1989) found that ethanol consumption was the main factor associated with decreased bone for-
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Type II-b fiber area (u2) Fig. 6. Relationship between osteoid area and muscle fiber area.
mation, but the BMI of the alcoholics was in the normal range, similar to that of abstainers and of control individuals. In a study performed by our group on 181 alcoholics and 43 controls, alcoholics showed decreased osteocalcin, PTH, 25 hydroxy vitamin D and bone mass assessed by double energy X-ray absorptiometry, and a decreased BMI, lean and fat mass. The loss of bone mass was not related to the severity of alcoholism or to liver cirrhosis, but to malnutrition. However, for a similar BMI, bone loss was more severe in alcoholics
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Osteocalcin (ng/ml)
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Weight change (g) Fig. 7. Relationship between osteocalcin and weight change during the experiment.
than in controls. Bone loss was especially marked in alcoholics with irregular feeding habits (Santolaria et al., 2000). It, therefore, seems that, notwithstanding the direct and indirect effects of ethanol, protein deficiency plays a key role in the development of osteopenia in alcoholics. A low protein intake is associated with increased bone loss (Hannan et al., 2000). Other clinical studies also stress the importance of adequate nutrition and nutritional status on the maintenance of a normal bone mass (Schurch et al., 1998). Thus, in accordance with other authors (Diamond et al., 1989; Friday and Howard, 1991; Rico et al., 1987), we found in ethanol-treated rats, a decreased bone mass, related to decreased bone formation (decreased serum osteocalcin and IGF-1 levels), whereas bone breakdown was not affected (in accordance with classic observations: Diamond et al., 1989; Bikle et al., 1985; Lindholm et al., 1991). Protein deficiency exerts an independent, highly significant effect on the decrease in serum osteocalcin and IGF-1, thus supporting its importance in the impairment of bone formation. In contrast, no effect was exerted by protein deficiency on serum corticosterone or PTH, in accordance with the lack of effect on bone resorption. Despite its anabolic effect on bone, no significant effect was exerted by protein deficiency, ethanol or zinc on serum testosterone, nor was this hormone related with changes in TBV or osteoid. Although zinc did not exert any effect on TBV, it increases the osteoid area, a result in accordance with the well described anabolic effect of zinc on bone. Zinc has been shown to augment the anabolic effect of IGF-1 on osteoblasts, thus promoting formation of the extracellular matrix (Matsui and Yamaguchi, 1995). As com-
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mented previously, some experimental and clinical studies have shown that treatment with zinc promotes fetal bone growth (Merialdi et al., 2004; Dı´az-Go´mez et al., 2003), and there is also evidence from animal studies which suggest that zinc deficiency negatively affects skeletal growth (Golub et al., 1984; Da Cunha Ferreira et al., 1991). Zinc deficiency has been described in alcoholics (Prasad, 1991), a feature which may be aggravated by co-existing protein deficiency (Conde-Martel et al., 1992). It has been hypothesized that zinc deficiency might contribute to osteoporosis in alcoholics (Rico, 1990). In the present study, zinc opposed the effect of both ethanol and protein deficiency, which led to reduced osteoid proportion, suggesting a potential beneficial effect of zinc supplementation in alcoholicsÕ bone disease. Interestingly, a correlation was observed between muscle mass and osteoid area, suggesting that alcoholic myopathy plays a role in decreased bone formation. Type II muscle fiber atrophy is a well recognized feature of chronic alcoholism (Preedy and Peters, 1990; Martin and Peters, 1985; Fernandez-Sola et al., 1995; Romero et al., 1994) and, on the other hand, it is well known that muscle activity and muscle mass are main determinants of bone mass. Therefore, the association between bone formation—as measured by osteoid area—and muscle mass is expected to occur. In any case, it should be kept in mind that a significant correlation does not imply a causal relationship between the two parameters. However, in contrast with muscle alterations, liver fibrosis was not related to bone mass (although liver fibrosis is quite small using the Lieber de Carli model, even in the 2% protein-fed rats treated with ethanol (Gonza´lezReimers et al., 1996). The decrease in serum osteocalcin, IGF-1 and serum vitamin D all reflect a decreased bone formation. Interestingly, zinc treatment, although lacking an effect on IGF-1 and osteocalcin, does tend to lessen the decrease in serum vitamin D induced by ethanol and/or a low protein diet, a result consistent with the positive effect of zinc on bone formation. In contrast with the decreased levels of biochemical markers of bone formation, a decrease which depends on the additive interaction between protein deficiency and/or ethanol, no changes were observed in serum PTH, and corticosterone, whereas only those rats treated with ethanol and zinc showed an increase in hydroxyproline excretion. In contrast with the widely accepted statement that ethanol inhibits bone formation, results relating to the effect of ethanol and/or protein deficiency on bone resorption are less consistent (Molina-Pe´rez et al., 2000; Bourrin et al., 2000a,b). Thus, the results reported are in accordance with the apparent lack of effect of ethanol and protein deficiency on bone resorption. Although some reports show an inhibitory effect of zinc supplementation on bone resorption (Moonga and
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Dempster, 1995), in this study zinc had no effect on biochemical markers of bone breakdown. Several conclusions can be drawn from this study. Ethanol leads to a decreased bone mass, and this effect depends more heavily on decreased bone formation, with protein deficiency playing a secondary, but synergistic role. Bone resorption is not altered by ethanol and or protein deficiency. In accordance with its anabolic effect on bone, treatment with zinc reduces the decrease in bone formation mediated by ethanol and/or protein deficiency. This effect, however, is relatively weak, with a statistical significance for osteoid area and a trend for serum vitamin D, but not for TBV. Ethanol-mediated muscle fiber atrophy keeps a relation with osteoid area, suggesting that chronic alcoholic myopathy may be involved in decreased bone formation.
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Effect of Zinc supplementation on ethanol-mediated bone alterations E. Gonza´lez-Reimers a,*, M.C. Dura´n-Castello´n a, R. Martı´n-Olivera a, A. Lo´pez-Lirola a, F. Santolaria-Ferna´ndez a, M.J. De La Vega-Prieto b, A. Pe´rez-Ramı´rez a, E. Garcı´a-Valdecasas Campelo a a
Departamento de Medicina Interna, Hospital Universitario, 38320 Tenerife, Canary Islands, Spain b Servicio de Laboratorio, Hospital Universitario, 38320 Tenerife, Canary Islands, Spain
Abstract Ethanol consumption leads to bone alterations, mainly osteoporosis. Ethanol itself may directly alter bone synthesis, but other factors, such as accompanying protein malnutrition—frequently observed in alcoholics-, chronic alcoholic myopathy with muscle atrophy, alcohol induced hypogonadism or hypercortisolism, or liver damage, may all contribute to altered bone metabolism. Some data suggest that zinc may exert beneficial effects on bone growth. Based on these facts, we analyzed the relative and combined effects of ethanol, protein malnutrition and treatment with zinc, 227 mg/l in the form of zinc sulphate, on bone histology, biochemical markers of bone formation (osteocalcin) and resorption (urinary hydroxyproline excretion), and hormones involved in bone homeostasis (insulin growth factor 1 (IGF-1), vitamin D, parathormone (PTH), free testosterone and corticosterone), as well as the association between these parameters and muscle fiber area and liver fibrosis, in eight groups of adult Sprague Dawley rats fed following the Lieber de Carli model during 5 weeks. Ethanol showed an independent effect on TBV (F = 14.5, p < 0.001), causing it to decrease, whereas a low protein diet caused a reduction in osteoid area (F = 8.9, p < 0.001). Treatment with zinc increased osteoid area (F = 11.2, p < 0.001) and serum vitamin D levels (F = 3.74, p = 0.057). Both ethanol (F = 45, p < 0.001) and low protein diet (F = 46.8, p < 0.01) decreased serum osteocalcin levels. Ethanol was the only factor independently related with serum IGF-1 (F = 130.24, p < 0.001), and also showed a synergistic interaction with protein deficiency (p = 0.027). In contrast, no change was observed in hydroxyproline excretion and serum PTH levels. No correlation was found between TBM and muscle atrophy, liver fibrosis, corticosterone, or free testosterone levels, but a significant relationship was observed between type II-b muscle fiber area and osteoid area (q = 0.34, p < 0.01). Osteoporosis is, therefore, present in alcohol treated rats. Both alcohol and protein deficiency lead to reduced bone formation. Muscle atrophy is related to osteoid area, suggesting a role for chronic alcoholic myopathy in decreased bone mass. Treatment with zinc increases osteoid area, but has no effect on TBV. 2005 Elsevier Ltd. All rights reserved. Keywords: Alcohol; Osteopenia-Muscle atrophy; Osteoporosis; Protein deficiency; Malnutrition; Zinc treatment
1. Introduction
Abbreviations: BMD, bone mineral density; BMI, body mass index; DNA, Desoxyribonucleic acid; IGF-1, insulin-like growth factor 1; IL1, interleukin 1; IL-6, interleukin 6; NS, non significant; PTH, parathormone; IA, radioimmunoanalysis; RNA, ribonucleic acid; SNK, StudentÕs-Newmann–Keuls; TBV, trabecular bone volume; Vs, versus; WHO, World Health Organization. * Corresponding author. 0278-6915/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2005.04.003
Alcoholism is a risk factor for osteoporosis. Two major reasons may explain this occurrence: the peculiar lifestyle of most alcoholics, which makes them prone to falls and traumatisms, and the association between chronic ethanol consumption and reduced bone mass. The pathogenesis of osteopenia in alcoholic subjects is not fully understood. Ethanol seems to exert a direct toxic effect on osteoblast function (Giuliani et al.,
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1999), although it also alters, directly and indirectly, bone mineral metabolism, including PTH and vitamin D homeostasis, testosterone, IGF-1, and cortisol levels. Ethanol appears to reduce bone formation and leads to decreased osteoid synthesis and decreased mineralization rate (Diamond et al., 1989) in a dose-dependent fashion (Turner, 2000). Ethanol may also increase bone resorption. Recent studies performed by Dai et al. (2000) show that ethanol induces bone loss through IL-6, which in turn activates osteoclastogenesis. Nutritional status may also be important, as well as decreased muscle mass and reduced muscle strength and physical activity. These observations are supported by several experimental and clinical studies, which have shown a relationship between a low body mass index and osteopenia and the importance of protein calorie malnutrition in the pathogenesis of the metabolic bone disease suffered by the alcoholic patient (Bendavid et al., 1996; Schurch et al., 1998; Molina-Pe´rez et al., 2000; Santolaria et al., 2000). Indeed, bone formation is impaired in protein-calorie malnutrition (Bourrin et al., 2000a), and bone resorption is also decreased, leading to a slow turn-over osteopenia (Bourrin et al., 2000b). Huuskonen et al. (2000) conclude that low dietary calcium intake, weak muscle strength and low body weight, but not alcohol consumption, are risk factors for low BMD in men. Alcoholic myopathy with muscle atrophy (Preedy and Peters, 1990; UrbanoMa´rquez et al., 1989; Romero et al., 1994; Peters et al., 1985), on which both ethanol and protein deficiency may act synergistically (Conde-Martel et al., 1992), may also exert a detrimental effect on bone mass. Alcoholics frequently show zinc deficiency (Prasad, 1991). Zinc is required for the production of many enzymes involved in protein synthesis, nucleic acid metabolism and immune function, and also plays an important role in gene transcription (Hambidge, 2000). Indeed, several transcription factors contain the so-called zinc finger domains which are important in protein-DNA or protein-RNA interactions (Dreosti, 2001). In some recent studies it has been shown that zinc supplements added to the diet may exert a positive influence on human bone linear growth (Dı´az-Go´mez et al., 2003; Merialdi et al., 2004). These results agree with former observations performed in rats or monkeys, in which zinc deficiency was associated with stunted growth and defective endochondral ossification. (Leek et al., 1984; Da Cunha Ferreira et al., 1991). Experimental data suggest that zinc stimulates bone protein synthesis and bone formation by increasing the activity of alkaline phosphatase and other key enzymes (Yamaguchi and Yamaguchi, 1986), and also reduces osteoclast activity (Kishi and Yamaguchi, 1994). Zinc may, therefore, exert an anabolic effect on bone, in some ways opposite to that exerted by ethanol. Based on these facts we investigate here the effects of zinc supplementation on bone changes induced by ethanol and protein defi-
ciency in male Wistar rats fed following the Lieber de Carli model, taking into account the possible influence of co-existing protein deficiency, myopathy, and liver fibrosis.
2. Material and methods 2.1. Animals and treatments Eighty male Sprague–Dawley rats were divided into 4 groups of 10 animals each, and treated following the Lieber de Carli model with a control diet, a 2% proteincontaining diet, a 36%-ethanol containing diet, and a 2% protein 36% ethanol containing diet (Lieber et al., 1989) with daily addition of zinc sulphate (227 mg/l zinc). Four further groups were fed identical diets and served as controls. Another group of five animals was allowed to consume the control diet ad libitum. Details about diet composition and dietary consumption are reported elsewhere (Dura´n-Castello´n et al., in press). At the end of the trial, blood was obtained by direct cardiac puncture, and centrifuged. We removed the first two lumbar vertebrae, for histomorphometric analysis, as well as the right gastroecnemius muscle for ATP-ase staining and further measurement of Type I, Type II-a and Type II-b muscle fiber areas, and the liver, for histomorphometrical assessment of the amount of liver fibrosis. 2.2. Bone histomorphometry Lumbar vertebrae were processed for undecalcified bone sample analysis. Briefly, samples were embedded in methylmetacrylate (Sigma Chemicals, St Louis, Missouri, USA), stored for 24 h at 4 C and later polymerised at 32–34 C for 3–4 days. Embedded samples were then cut in 9–12 lm thick slices with a Reichert-Jung microtome and stained with Masson-Goldner. Trabecular mineralized bone volume (TBV) and osteoid area were determined using an image analyser equipped with the program ‘‘Image Measure 4.4a’’ (Microscience Inc.), at 40· and 200·, respectively. Results are presented as percentages of the total area. 2.2.1. Liver histological analysis Liver sample slices stained with haematoxylin-eosin and Van Giesson were used for histomorphometrical analysis, which was performed with the aid of an image analyzer equipped with the program Image Measure 4.4a (Microscience Inc), calculating the total amount of fibrous tissue (at 20·). 2.2.2. Serum hormones and albumin levels Serum parathormone (PTH) was measured by radioimmunoanalysis (RIA), using an antibody directed
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towards intact rat PTH (Nichols, San Juan de Capistrano, California, USA), with a sensitivity of 1 pg/ml. Serum osteocalcin was measured by RIA, using an antibody specific for rat (Biomedical Technologies, St Stoughton, Maryland, USA; specificity for rat osteocalcin >10,000 fold bovine, monkey, human, and chicken osteocalcins; sensitivity 0.025–1.5 ng per tube). We also measured urinary creatinine and hydroxyproline, by liquid cromatography (Dawson et al., 1988). Other biochemical parameters which were used in this study include serum albumin, serum and urinary calcium and magnesium, measured by routine analytical methods, and serum insulin-like growth factor type 1 (IGF-1), free testosterone and corticosterone, measured by RIA. Details about measurement techniques are already reported (Dura´n-Castello´n et al., in press). 2.3. Statistics The Kolmogorov–Smirnov test was performed to test normality. A non-normal distribution, with marked intergroup variation, was observed for some variables, such as osteoid area, and urinary hydroxyproline, calcium, magnesium and phosphorus excretion, although these parameters showed a normal distribution within each group of animals. Therefore, SpearmanÕs rank test was used to analyse the relationships between these variables and other quantitative parameters. Differences between the experimental groups were analysed using variance analysis with further StudentÕs-Newman–Keuls (SNK) test. Independent effects of ethanol, zinc, and protein deficiency on the alterations observed, and interactions between these parameters, were analysed by twoway variance analysis. Paired t-test analysis was used to compare initial and final weight in the eight groups of rats. Differences between the analysed parameters in controls and ad libitum fed animals were analysed using the StudentÕs t test. Also, single correlation analysis
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(PearsonÕs ‘‘r’’) was performed between quantitative variables.
3. Results The results are shown in Tables 1–4. As reported elsewhere (Dura´n-Castello´n et al., in press), all the rats weighed the same at the beginning of the study and all of them, except for those in the control groups (both without and with zinc supplement), showed a significant weight loss during the experimental period. As shown in Table 1 and in Figs. 1 and 2, we found reduced TBV in rats treated with ethanol, either with or without protein restriction, and either treated or not with zinc. Two-way variance analysis revealed that ethanol (F = 14.59, p < 0.001) was the sole factor associated with decreased TBV, although a direct correlation was observed between TBV and weight change over the study period (r = 0.29, p = 0.017). The addition of zinc exerted no significant effect, although there was a trend to higher TBV values in the zinc-treated animals. A direct correlation was observed between TBV and osteocalcin (r = 0.35, p < 0.001, Fig. 3). No relationship was observed between TBV and muscle fiber area, or with liver fibrosis. Osteoid area was also lower among rats treated with ethanol, especially in those which also received a low protein diet. Indeed, a significant effect of low protein diet (F = 8.88, p = 0.004), which was additive to that of ethanol (interaction between both factors, F = 4.47, p = 0.039) was observed, as well as a direct relationship between osteoid area and weight change during the study period (rho = 0.43, p < 0.001, Fig. 4). However, treatment with zinc significantly reversed the decrease in osteoid area (F = 11.22, p = 0.001). Osteoid area showed a direct correlation with serum osteocalcin (rho = 0.41, p < 0.001, Fig. 5), IGF-1 (rho =
Table 1 Trabecular bone volume (TBV) and osteoid area (mean ± standard deviation)
Control (1) Low protein (2) Control alcoholic (3) Low protein alcoholic (4) Control zinc (5) Low protein zinc (6) Alcohol + zinc (7) Alcohol + low protein + zinc (8) F; p Differences among groups (SNK test) Main effects Interactions Ad libitum (9) Differences 1–9
TBV (%)
Osteoid (%)
22.9 ± 4.9 21.2 ± 2.1 16.0 ± 3.9 17.6 ± 4.9 22.3 ± 4.0 19.9 ± 2.2 19.3 ± 5.0 17.7 ± 4.6 F = 2.72, p = 0.016 1,5 vs 2,3,4,6,7,8 Ethanol F = 14.5, p < 0.001 – 19.6 ± 2.0 NS
1.14 ± 0.35 1.13 ± 0.41 0.90 ± 0.65 0.30 ± 0.40 1.81 ± 0.77 1.41 ± 0.44 2.60 ± 1.98 0.80 ± 0.97 F = 4.87, p = 0.001 5,7 vs 1,2,3,4,6,8 4 vs 1,2,3,5,6,7,8 Low protein, F = 8.9, p < 0.001; zinc F = 11.2, p < 0.001 Ethanol-Low protein p = 0.039 2.20 ± 0.94 NS
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Table 2 Serum corticosterone and testosterone, and urinary hydroxyproline excretion (mean ± standard deviation)
Control (1) Low protein (2) Control alcoholic (3) Low protein alcoholic (4) Control zinc (5) Low protein + zinc (6) Ethanol + zinc (7) Ethanol + zinc + low protein (8) F; p Differences among groups (SNK test) Main effects Interactions Ad libitum (9) Differences 1–9
Hydroxyproline excretion (mg/dl)
Corticosterone (ng/ml)
Free testosterone (pg/ml)
41.0 ± 26.8 38.6 ± 60.2 32.1 ± 6.9 52.7 ± 47.2 38.4 ± 31.3 49.8 ± 36.3 92.6 ± 22.9 33.5 ± 12.6 F = 2.94, p = 0.01 7 vs all the others – Ethanol/protein deficiency/zinc p = 0.007 52.9 ± 25.3 NS
413 ± 329 499 ± 162 684 ± 223 553 ± 240 450 ± 275 486 ± 229 446 ± 333 567 ± 270 F = 0.81, NS – – – 533 ± 161 NS
0.59 ± 1.23 1.51 ± 2.48 4.70 ± 9.09 0.37 ± 0.43 0.79 ± 1.63 3.69 ± 7.44 1.71 ± 2.97 1.25 ± 3.450 F = 1, NS – – – 12.32 ± 12.01 NS
Table 3 Serum IGF-1, vitamin D, osteocalcin, and parathyroid hormone (PTH) levels (mean ± standard deviation) IGF-1 (ng/ml)*
Vitamin D (ng/ml)
Osteocalcin (ng/ml)
PTH (pg/ml)
Control (1) Low protein (2) Control alcoholic (3) Low protein alcoholic (4) Control zinc (5) Low protein + zinc (6) Ethanol + zinc (7) Ethanol + zinc + low protein (8) F; p Differences among groups (SNK test) Main effects
14.93 ± 2.29 18.26 ± 2.92 6.27 ± 5.16 2.59 ± 1.88 16.78 ± 3.37 16.95 ± 4.02 4.40 ± 5.36 4.37 ± 4.30 F = 29.4, p < 0.001 1,2,5,6 vs 3,4,7,8
Ethanol/protein deficiency p = 0.04 21.22 ± 6.05 NS
49.5 ± 9.4 43.0 ± 8.2 43.2 ± 10.6 22.3 ± 9.3 58.3 ± 11.7 41.9 ± 9.2 41.0 ± 13.3 21.0 ± 8.0 F = 14.72, p < 0.001 1,5 vs 2,3,6,7,4,8 4,8 vs 1,2,3,5,6,7 Low protein F = 45 p < 0.001; ethanol F = 46.8, p < 0.001 Ethanol-Low protein p = 0.06
110.0 ± 148.1 75.4 ± 45.8 75.9 ± 30.0 99.7 ± 70.1 61.1 ± 54.9 78.6 ± 62.9 91.4 ± 47.1 105.3 ± 36.0 F = 0.59, NS –
Interactions
67.7 ± 33.1 37.8 ± 28.0 72.5 ± 13.0 17.8 ± 9.0 66.1 ± 26.4 41.3 ± 19.5 99.0 ± 36.8 38.7 ± 28.3 F = 8.80, p < 0.001 1,3,5,7 vs 2,4,6,8; 4 vs all the others Low protein F = 44 p < 0.001; zinc F = 3.74, p = 0.057 Ethanol-Low protein p = 0.02 38.7 ± 6.1 T = 2.55, p = 0.03
76.8 ± 8.8 T = 5.4, p < 0.001
80.4 ± 61.6 NS
Ad libitum (9) Differences 1–9
Ethanol F = 189 p < 0.001
–
–
NS = non significant; vs = versus; SNK = StudentÕs-Newmann–Keuls. * Excluding one outlier (42.71 ng/ml), belonging to group 6.
0.38, p = 0.001) and serum vitamin D (rho = 0.27, p = 0.02). Also, a direct relationship was observed between osteoid area and Type IIa (rho = 0.29, p = 0.03) and Type IIb (rho = 0.34, p = 0.01, Fig. 6) muscle fiber diameter, but not with the amount of liver fibrosis. In contrast with serum PTH, which had similar values in the different groups, serum osteocalcin, IGF-1 and vitamin D showed marked differences (Table 3). A low protein diet caused a decrease in serum vitamin D levels, especially when added to ethanol. On the contrary, zinc treatment tended to lessen the low-protein diet-induced reduction in serum vitamin D. In accordance with these results, serum vitamin D was directly related with weight at the end of the study (r = 0.59, p < 0.001) and with serum albumin (r = 0.36, p = 0.003) and serum osteocalcin (r = 0.40, p = 0.001).
Rats treated with a low protein diet and ethanol showed the lowest serum osteocalcin values. A synergistic interaction was detected between both factors, whereas zinc did not play any role. Highly significant relationships were observed between serum osteocalcin and weight change during the study period (r = 0.73, p < 0.001, Fig. 7) and serum osteocalcin and serum IGF-1 (r = 0.45, p < 0.001). Serum IGF-1 was lowest among rats treated with ethanol and a low protein diet; as already reported (Dura´n-Castello´n et al., in press), both factors showed an additive effect, which was not modified by treatment with zinc. Serum IGF-1 showed a significant relationship with weight at the end of the study (r = 0.23, p = 0.049), and an inverse relationship with urinary calcium excretion (rho = 0.27, p = 0.019) and serum magnesium (r = 0.30, p = 0.009).
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Table 4 Serum calcium and magnesium (mg/dl) and urinary magnesium (mg/l) and calcium excretion (mg/dl) (mean ± standard deviation) Serum calcium
Serum magnesium
Urinary magnesium
Urinary calcium
Main effects
11.4 ± 0.9 11.3 ± 0.4 11.1 ± 0.6 11.0 ± 0.8 11.4 ± 1.1 10.9 ± 0.9 11.3 ± 0.9 11.1 ± 0.6 F = 0.65, NS – –
28.2 ± 34.3 12.1 ± 19.7 12.4 ± 17.9 30.4 ± 27.0 35.8 ± 33.6 26.8 ± 42.9 22.2 ± 25.7 14.6 ± 19.5 F = 0.84, NS – –
2.08 ± 1.22 1.94 ± 1.35 2.30 ± 1.06 5.92 ± 4.52 2.26 ± 1.20 2.08 ± 1.51 3.04 ± 3.10 2.88 ± 2.11 F = 2.94, p = 0.01 4 vs all the others Ethanol F = 6.99, p < 0.01
Interactions Ad libitum (9) Differences 1–9
– 11.2 ± 0.4 NS
3.21 ± 0.58 2.60 ± 0.39 3.08 ± 0.57 2.86 ± 0.56 2.97 ± 0.53 2.31 ± 0.57 3.37 ± 0.65 3.02 ± 0.43 F = 3.69, p = 0.02 1,3,7 vs 6 Ethanol F = 5.85, p = 0.018; low protein F = 12.94, p = 0.001 – 2.76 ± 0.34 NS
– 90.0 ± 91.7 NS
– 2.88 ± 1.55 NS
Control (1) Low protein (2) Control alcoholic (3) Low protein alcoholic (4) Control zinc (5) Low protein + zinc (6) Alcohol + zinc (7) Alcohol + low protein + zinc (8) F; p
NS = non-significant.
Fig. 1. Section of the first lumbar vertebra of a control rat, stained with Masson–Goldner (20·).
We failed to find differences among the different groups with respect to corticosterone and testosterone. No significant relationships were observed between these hormones and any of the parameters analysed. In Table 4 we show the results for serum and urinary calcium and magnesium. Serum magnesium levels varied among the different groups. Low protein-fed animals, either treated with or without zinc, showed the lowest serum magnesium values. Ethanol significantly increased urinary calcium excretion. 4. Discussion This study was carried out in order to test the effect of zinc on alcohol-induced bone alterations, taking into account that protein deficiency may also play a concomitant role in its pathogenesis.
Fig. 2. Section of the first lumbar vertebra of a rat treated with ethanol and a protein-deficient diet, stained with Masson–Goldner (20·).
In recent years there have been several reports supporting the view that moderate ethanol consumption is not harmful, but even protects bone (Baron et al., 2001). The detrimental effect of ethanol would be observed in men, in contrast with a neutral or slightly beneficial effect in older women. Despite these observations, classic studies performed by Diamond et al. (1989), among others, have shown that chronic alcoholism causes osteoporosis. In this study we have shown that rats treated with ethanol following the Lieber de Carli model do develop osteopenia. We found that ethanol is solely responsible for decreased TBV, in contrast with another study performed some years ago, in which protein deficiency exerted the most important effect (Molina-Pe´rez et al., 2000). However, in this study, a relationship exists between bone mass and weight loss, also suggesting an effect of malnutrition on decreased
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6.00
35.00
5.00
30.00
osteoid (%)
TBV (%)
4.00 25.00
20.00
3.00
2.00 15.00
1.00
10.00
0.00 20.00
40.00
60.00
80.00
20.00
40.00
6.00
5.00
5.00
4.00
4.00
Osteoid (%)
osteoid (%)
6.00
3.00
3.00
2.00
2.00
1.00
1.00
0.00
0.00 -100.00
-50.00
0.00
80.00
Fig. 5. Relationship between osteoid area and osteocalcin.
Fig. 3. Relationship between trabecular bone mass (TBV) and serum osteocalcin.
-150.00
60.00
Osteocalcin (ng/ml)
Osteocalcin (ng/ml)
50.00
Weight change (g) Fig. 4. Relationship between osteoid area and weight change during the experiment.
TBV. Lalor et al. (1986) found that alcoholics affected by severe osteoporosis were heavier drinkers than those without osteoporosis and also consumed less proteins and presented lower albumin levels. Bone mass was dependent on serum albumin—which, in turn, was dependent on dietary protein ingestion—, body mass index, and dietary calcium intake. On the contrary, Diamond et al. (1989) found that ethanol consumption was the main factor associated with decreased bone for-
500.00
1000.00
1500.00
2000.00
2500.00
Type II-b fiber area (u2) Fig. 6. Relationship between osteoid area and muscle fiber area.
mation, but the BMI of the alcoholics was in the normal range, similar to that of abstainers and of control individuals. In a study performed by our group on 181 alcoholics and 43 controls, alcoholics showed decreased osteocalcin, PTH, 25 hydroxy vitamin D and bone mass assessed by double energy X-ray absorptiometry, and a decreased BMI, lean and fat mass. The loss of bone mass was not related to the severity of alcoholism or to liver cirrhosis, but to malnutrition. However, for a similar BMI, bone loss was more severe in alcoholics
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Osteocalcin (ng/ml)
80.00
60.00
40.00
20.00
-100.00
-50.00
0.00
Weight change (g) Fig. 7. Relationship between osteocalcin and weight change during the experiment.
than in controls. Bone loss was especially marked in alcoholics with irregular feeding habits (Santolaria et al., 2000). It, therefore, seems that, notwithstanding the direct and indirect effects of ethanol, protein deficiency plays a key role in the development of osteopenia in alcoholics. A low protein intake is associated with increased bone loss (Hannan et al., 2000). Other clinical studies also stress the importance of adequate nutrition and nutritional status on the maintenance of a normal bone mass (Schurch et al., 1998). Thus, in accordance with other authors (Diamond et al., 1989; Friday and Howard, 1991; Rico et al., 1987), we found in ethanol-treated rats, a decreased bone mass, related to decreased bone formation (decreased serum osteocalcin and IGF-1 levels), whereas bone breakdown was not affected (in accordance with classic observations: Diamond et al., 1989; Bikle et al., 1985; Lindholm et al., 1991). Protein deficiency exerts an independent, highly significant effect on the decrease in serum osteocalcin and IGF-1, thus supporting its importance in the impairment of bone formation. In contrast, no effect was exerted by protein deficiency on serum corticosterone or PTH, in accordance with the lack of effect on bone resorption. Despite its anabolic effect on bone, no significant effect was exerted by protein deficiency, ethanol or zinc on serum testosterone, nor was this hormone related with changes in TBV or osteoid. Although zinc did not exert any effect on TBV, it increases the osteoid area, a result in accordance with the well described anabolic effect of zinc on bone. Zinc has been shown to augment the anabolic effect of IGF-1 on osteoblasts, thus promoting formation of the extracellular matrix (Matsui and Yamaguchi, 1995). As com-
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mented previously, some experimental and clinical studies have shown that treatment with zinc promotes fetal bone growth (Merialdi et al., 2004; Dı´az-Go´mez et al., 2003), and there is also evidence from animal studies which suggest that zinc deficiency negatively affects skeletal growth (Golub et al., 1984; Da Cunha Ferreira et al., 1991). Zinc deficiency has been described in alcoholics (Prasad, 1991), a feature which may be aggravated by co-existing protein deficiency (Conde-Martel et al., 1992). It has been hypothesized that zinc deficiency might contribute to osteoporosis in alcoholics (Rico, 1990). In the present study, zinc opposed the effect of both ethanol and protein deficiency, which led to reduced osteoid proportion, suggesting a potential beneficial effect of zinc supplementation in alcoholicsÕ bone disease. Interestingly, a correlation was observed between muscle mass and osteoid area, suggesting that alcoholic myopathy plays a role in decreased bone formation. Type II muscle fiber atrophy is a well recognized feature of chronic alcoholism (Preedy and Peters, 1990; Martin and Peters, 1985; Fernandez-Sola et al., 1995; Romero et al., 1994) and, on the other hand, it is well known that muscle activity and muscle mass are main determinants of bone mass. Therefore, the association between bone formation—as measured by osteoid area—and muscle mass is expected to occur. In any case, it should be kept in mind that a significant correlation does not imply a causal relationship between the two parameters. However, in contrast with muscle alterations, liver fibrosis was not related to bone mass (although liver fibrosis is quite small using the Lieber de Carli model, even in the 2% protein-fed rats treated with ethanol (Gonza´lezReimers et al., 1996). The decrease in serum osteocalcin, IGF-1 and serum vitamin D all reflect a decreased bone formation. Interestingly, zinc treatment, although lacking an effect on IGF-1 and osteocalcin, does tend to lessen the decrease in serum vitamin D induced by ethanol and/or a low protein diet, a result consistent with the positive effect of zinc on bone formation. In contrast with the decreased levels of biochemical markers of bone formation, a decrease which depends on the additive interaction between protein deficiency and/or ethanol, no changes were observed in serum PTH, and corticosterone, whereas only those rats treated with ethanol and zinc showed an increase in hydroxyproline excretion. In contrast with the widely accepted statement that ethanol inhibits bone formation, results relating to the effect of ethanol and/or protein deficiency on bone resorption are less consistent (Molina-Pe´rez et al., 2000; Bourrin et al., 2000a,b). Thus, the results reported are in accordance with the apparent lack of effect of ethanol and protein deficiency on bone resorption. Although some reports show an inhibitory effect of zinc supplementation on bone resorption (Moonga and
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Dempster, 1995), in this study zinc had no effect on biochemical markers of bone breakdown. Several conclusions can be drawn from this study. Ethanol leads to a decreased bone mass, and this effect depends more heavily on decreased bone formation, with protein deficiency playing a secondary, but synergistic role. Bone resorption is not altered by ethanol and or protein deficiency. In accordance with its anabolic effect on bone, treatment with zinc reduces the decrease in bone formation mediated by ethanol and/or protein deficiency. This effect, however, is relatively weak, with a statistical significance for osteoid area and a trend for serum vitamin D, but not for TBV. Ethanol-mediated muscle fiber atrophy keeps a relation with osteoid area, suggesting that chronic alcoholic myopathy may be involved in decreased bone formation.
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