Does Alcohol Affect Vitamin D
Alcoholic Myopathy: Vitamin D Deficiency is Related to Muscle Fibre Atrophy in a Murine Model
E. González-Reimers, 1 Departamento de Medicina Interna , Hospital Universitario, Ofra s/n 38320 La Laguna , Tenerife, Canary Islands , Spain *Corresponding author: Tel: +-922-678600; Fax: +-922-319279; E-mail: egonrey@ull.es Search for other works by this author on: 1 Departamento de Medicina Interna , Hospital Universitario, Ofra s/n 38320 La Laguna , Tenerife, Canary Islands , Spain Search for other works by this author on: 1 Departamento de Medicina Interna , Hospital Universitario, Ofra s/n 38320 La Laguna , Tenerife, Canary Islands , Spain Search for other works by this author on: 1 Departamento de Medicina Interna , Hospital Universitario, Ofra s/n 38320 La Laguna , Tenerife, Canary Islands , Spain Search for other works by this author on: 2 Departamento de Fisiología , Universidad deLa Laguna , Tenerife, Canary Islands , Spain Search for other works by this author on: 1 Departamento de Medicina Interna , Hospital Universitario, Ofra s/n 38320 La Laguna , Tenerife, Canary Islands , Spain Search for other works by this author on: 1 Departamento de Medicina Interna , Hospital Universitario, Ofra s/n 38320 La Laguna , Tenerife, Canary Islands , Spain Search for other works by this author on:
Received:
03 November 2009
Revision received:
02 February 2010
Accepted:
05 February 2010
Published:
26 February 2010
Abstract
Aims: Chronic myopathy has been described in alcoholics, characterized by atrophy of type II fibres, and vitamin D deficiency. Low serum vitamin D levels are frequent in alcoholics. The possibility exists that serum vitamin D levels are related to muscle changes in a murine experimental model. Methods: Histological analysis of the right gastrocnemius muscle was performed in four groups of adult Sprague-Dawley rats, sacrificed after 5 weeks of treatment following the Lieber–DeCarli model. We studied the association between muscle histological changes and the activity of glutathione peroxidase (GPX), superoxide dismutase (SOD) and lipid peroxidation products (malondialdehyde); parathyroid hormone (PTH), insulin-like growth factor 1 (IGF-1), free testosterone, 1,25 dihydroxyvitamin D3 (vitamin D) and corticosterone; and serum calcium and magnesium. Results: Alcoholic animals showed type IIa and IIb fibre atrophy, especially the low-protein-fed ones, an effect dependent on protein deficiency. A significant relationship was observed between serum vitamin D levels and IIa fibre area (ρ = 0.56, P = 0.002), and also, as a trend, between vitamin D and type IIb fibre area (ρ = 0.39, p = 0.053); between vitamin D and muscle GPX (ρ = 0.40, P = 0.025) and SOD activities (ρ = 0.43, P = 0.012). Muscle GPX activity was significantly related with type I fibre area (ρ = 0.49, P = 0.01) and muscle SOD, with type IIa fibre area (ρ = 0.38, P = 0.045). Serum testosterone was also related with type IIa fibre area (ρ = 0.61, P < 0.001). No relation was observed between serum PTH, corticosterone, or IGF-1 and fibre area PTH and antioxidant systems. Multiple regression analysis disclosed that the only parameter independently related with type IIa fibre area was serum vitamin D. Conclusion: Low vitamin D levels are related to muscle fibre atrophy, and altered levels of muscle antioxidant enzymes could play a role in alcoholic myopathy.
Introduction
Alcoholic myopathy affects 50–60% of alcoholics ( Peters et al., 1985; Urbano-Márquez et al., 1989; Preedy and Peters, 1990; Preedy et al., 1994; Romero et al., 1994). Usual manifestations include chronic muscle wasting — some patients may lose up to 30% of their muscle mass — and reduced muscle strength. Type II fibre atrophy, especially type IIx fibre atrophy, is a practically universal finding, whereas type I fibres are usually not affected. This is accompanied by a reduction in RNA content in skeletal muscle, a feature already observed after 1 week of treatment in experimental models, at least in young rats ( Preedy et al., 1990). Clinical and experimental data support the concept that malnutrition probably contributes to chronic alcoholic myopathy ( Conde et al., 1992; Romero et al., 1994; Nicolas et al., 2003; Durán Castellón et al., 2005).
Several mechanisms may be involved in the pathogenesis of alcoholic myopathy. Ethanol is a potent inhibitor of muscle protein synthesis ( Preedy et al., 2001), something which also happens in situations of protein malnutrition ( Svanberg et al., 2000). Protein synthesis is more intensely decreased in type II fibres than in type I fibres. In contrast, the effect of ethanol on protein breakdown is less well known. However, it has been shown that ethanol reduces protein catabolism ( Koll et al., 2002), an effect also observed in chronic starvation and protein malnutrition (Mitch and Goldberg, 1996). Moreover, increased muscle fibre apoptosis has been shown in alcoholics with myopathy ( Fernández-Sola et al., 2003). In addition to the direct effects of ethanol, ethanol and/or protein deficiency-mediated alterations of some hormones, such as insulin-like growth factor 1 (IGF-1), testosterone, or corticosterone, may also exert deleterious effects on muscle.
Ethanol exerts profound effects on calcium homeostasis and vitamin D metabolism. Many researchers ( Turner et al., 1988; Diamond et al., 1989; Laitinen and Välimäki, 1991; Lindholm et al., 1991; Peris et al., 1994; Santori et al., 2008, among many others), in studies designed to analyse ethanol-related bone alterations, have reported low 25 hydroxyvitamin D3 (25OH vitamin D) and/or low 1,25 dihydroxyvitamin D3 (1,25 (OH)2 vitamin D) levels in alcoholics or ethanol-treated animals. Besides possible effects on impaired vitamin D intake ( Manari et al., 2003), absorption or synthesis, related to both direct and indirect effects of ethanol and/or to the peculiar style of life of the alcoholic patient (Pitts and Van Thiel, 1986), it has been recently shown that ethanol impairs renal production of 1,25 (OH)2 vitamin D, affecting both synthesis and metabolic inactivation of 1,25 (OH)2 D3 ( Shankar et al., 2008). This effect is related to ethanol-induced increased oxidative damage and leads to reduced plasma 1,25 (OH)2 vitamin D levels.
In the last decade, it was shown that a vitamin D receptor (VDR) is present in several tissues (Demay, 2003), including human skeletal muscle ( Bischoff et al., 2001). Binding of 1,25 dihydroxyvitamin D to its receptor activates a genomic pathway which ultimately leads to muscle cell proliferation and differentiation (Ceglia, 2008), to an increase in the synthesis of calmodulin ( Drittanti et al., 1990) and to changes in intracellular calcium content, which facilitate muscle function. Calmodulin is highly sensitive to oxidative stress (Sharp and Tomer, 2007), so functional alterations of this protein occur in relation with oxidative damage, leading to altered intracellular calcium homeostasis and muscle contractility. Thus, both vitamin D deficiency and increased peroxidation may act synergistically impairing calmodulin function and muscle structure and contractility. Indeed, a link between low serum 1,25 (OH)2 vitamin D deficiency and muscle weakness and atrophy has been reported in different conditions (Russell, 1994; Ahmed et al., 2009; Albany and Servetnyk, 2009; Al-Said et al., 2009). Thus, since it has been shown that ethanol consumption leads to a decrease in vitamin D levels and vitamin D deficiency exerts a deleterious effect on muscle, it is possible that vitamin D deficiency is related to alcoholic myopathy. Based on these facts, in the present study we analyse the relation of 1,25 (OH)2 vitamin D and muscle fibre area in rats fed ethanol and a protein deficient diet following the Lieber–DeCarli model ( Lieber et al., 1989). Since oxidative damage may also play a role in alcoholic myopathy ( Preedy et al., 2002), and vitamin D may exert antioxidant effects (Chatterjee, 2001), we also analyse the relations between vitamin D levels, muscle malondialdehyde (MDA) content and muscle antioxidant enzyme activity.
Materials And Methods
Animals and treatments
Forty adult (∼60 days old) male Sprague-Dawley rats were divided into four groups of 10 animals each. The control rats (group 1) received the Lieber–DeCarli ( Lieber et al., 1989) control diet (Dyets Inc., Bethlehem, Pennsylvania, USA) containing 18% protein and 1 kcal/ml; a second group was fed an isocaloric, 2% protein-containing diet. The third group was fed with 36% ethanol- and 18% protein-containing diet, and the fourth one, 2% protein- and 36% ethanol-containing diet. All the diets contain 400 IU vitamin D/1000 kcal. A pair-feeding procedure was followed, trying to adjust the amount given to the different rats to that consumed by the rats which ate the least. However, this was only partially fulfilled, since total mean consumption, at the end of the study, 5 weeks later, was less in the rats fed ethanol low-protein diet (Table 1). Another group of five animals was allowed to consume the control diet ad libitum. The amount of diet consumed by these animals was 83.99 ± 1.27 kcal/day, significantly (P < 0.001) more than the amount consumed by the study groups. All the four diets contain the same amount of calories (1 kcal/ml).
Table 1
Initial and final weight, serum albumin and diet consumption by the groups of experimental animals (mean ± standard deviation; groups 1–4, in brackets)
| Weight at beginning (g) | Final weight (g) | Mean daily consumption (kcal) | Serum albumin (g/dl) | |
|---|---|---|---|---|
| Control (1) | 299 ± 10 | 288 ± 11 | 54.1 ± 3.5 | 3.88 ± 0.32 |
| Low protein (2) | 303 ± 13 | 227 ± 15 | 51.9 ± 5.2 | 3.11 ± 0.20 |
| Control alcoholic (3) | 298 ± 4 | 261 ± 15 | 48.7 ± 2.2 | 3.73 ± 0.27 |
| Low-protein alcoholic (4) | 299 ± 5 | 191 ± 13 | 42.3 ± 3.3 | 3.18 ± 0.30 |
| F, P | F = 0.7, NS | F = 82.7, P < 0.001 | F = 15.58, P < 0.001 | F = 18.11, P < 0.001 |
| Differences among groups (SNK test) | 1 vs 2, 3, 4; 2 vs 3, 4; 3 vs 4 | 1 vs 3, 4; 4 vs 1, 2, 3 | 1, 3 vs 2, 4 | |
| Main effects | Ethanol F = 44, P < 0.001; protein deficiency F = 193, P < 0.001 | Ethanol F = 29.56, P < 0.001; protein deficiency F = 8.97, P = 0.005 | Protein deficiency F = 48.04, P < 0.001 | |
| Interactions | NS | NS | NS | NS |
| Ad libitum (5) | 302 ± 11 | 341 ± 21 | 84 ± 1.5 | 3.80 ± 0.14 |
| Differences 1–5 | NS | t = 6.3, P < 0.001 | t = 17.87, P < 0.001 | NS |
| Weight at beginning (g) | Final weight (g) | Mean daily consumption (kcal) | Serum albumin (g/dl) | |
|---|---|---|---|---|
| Control (1) | 299 ± 10 | 288 ± 11 | 54.1 ± 3.5 | 3.88 ± 0.32 |
| Low protein (2) | 303 ± 13 | 227 ± 15 | 51.9 ± 5.2 | 3.11 ± 0.20 |
| Control alcoholic (3) | 298 ± 4 | 261 ± 15 | 48.7 ± 2.2 | 3.73 ± 0.27 |
| Low-protein alcoholic (4) | 299 ± 5 | 191 ± 13 | 42.3 ± 3.3 | 3.18 ± 0.30 |
| F, P | F = 0.7, NS | F = 82.7, P < 0.001 | F = 15.58, P < 0.001 | F = 18.11, P < 0.001 |
| Differences among groups (SNK test) | 1 vs 2, 3, 4; 2 vs 3, 4; 3 vs 4 | 1 vs 3, 4; 4 vs 1, 2, 3 | 1, 3 vs 2, 4 | |
| Main effects | Ethanol F = 44, P < 0.001; protein deficiency F = 193, P < 0.001 | Ethanol F = 29.56, P < 0.001; protein deficiency F = 8.97, P = 0.005 | Protein deficiency F = 48.04, P < 0.001 | |
| Interactions | NS | NS | NS | NS |
| Ad libitum (5) | 302 ± 11 | 341 ± 21 | 84 ± 1.5 | 3.80 ± 0.14 |
| Differences 1–5 | NS | t = 6.3, P < 0.001 | t = 17.87, P < 0.001 | NS |
ANOVA analysis was performed first (F, P file), and if differences were statistically significant, an SNK test was performed to assess differences among groups. In the case of significant differences, main effects (of ethanol and/or protein deficiency) and interactions between both parameters were also disclosed by means of a two-way ANOVA. A comparison between the parameters of the control group (1) and the ad libitum-fed ones (group 5; Student's t-test) was also performed. NS, non-significant; vs, versus.
Table 1
Initial and final weight, serum albumin and diet consumption by the groups of experimental animals (mean ± standard deviation; groups 1–4, in brackets)
| Weight at beginning (g) | Final weight (g) | Mean daily consumption (kcal) | Serum albumin (g/dl) | |
|---|---|---|---|---|
| Control (1) | 299 ± 10 | 288 ± 11 | 54.1 ± 3.5 | 3.88 ± 0.32 |
| Low protein (2) | 303 ± 13 | 227 ± 15 | 51.9 ± 5.2 | 3.11 ± 0.20 |
| Control alcoholic (3) | 298 ± 4 | 261 ± 15 | 48.7 ± 2.2 | 3.73 ± 0.27 |
| Low-protein alcoholic (4) | 299 ± 5 | 191 ± 13 | 42.3 ± 3.3 | 3.18 ± 0.30 |
| F, P | F = 0.7, NS | F = 82.7, P < 0.001 | F = 15.58, P < 0.001 | F = 18.11, P < 0.001 |
| Differences among groups (SNK test) | 1 vs 2, 3, 4; 2 vs 3, 4; 3 vs 4 | 1 vs 3, 4; 4 vs 1, 2, 3 | 1, 3 vs 2, 4 | |
| Main effects | Ethanol F = 44, P < 0.001; protein deficiency F = 193, P < 0.001 | Ethanol F = 29.56, P < 0.001; protein deficiency F = 8.97, P = 0.005 | Protein deficiency F = 48.04, P < 0.001 | |
| Interactions | NS | NS | NS | NS |
| Ad libitum (5) | 302 ± 11 | 341 ± 21 | 84 ± 1.5 | 3.80 ± 0.14 |
| Differences 1–5 | NS | t = 6.3, P < 0.001 | t = 17.87, P < 0.001 | NS |
| Weight at beginning (g) | Final weight (g) | Mean daily consumption (kcal) | Serum albumin (g/dl) | |
|---|---|---|---|---|
| Control (1) | 299 ± 10 | 288 ± 11 | 54.1 ± 3.5 | 3.88 ± 0.32 |
| Low protein (2) | 303 ± 13 | 227 ± 15 | 51.9 ± 5.2 | 3.11 ± 0.20 |
| Control alcoholic (3) | 298 ± 4 | 261 ± 15 | 48.7 ± 2.2 | 3.73 ± 0.27 |
| Low-protein alcoholic (4) | 299 ± 5 | 191 ± 13 | 42.3 ± 3.3 | 3.18 ± 0.30 |
| F, P | F = 0.7, NS | F = 82.7, P < 0.001 | F = 15.58, P < 0.001 | F = 18.11, P < 0.001 |
| Differences among groups (SNK test) | 1 vs 2, 3, 4; 2 vs 3, 4; 3 vs 4 | 1 vs 3, 4; 4 vs 1, 2, 3 | 1, 3 vs 2, 4 | |
| Main effects | Ethanol F = 44, P < 0.001; protein deficiency F = 193, P < 0.001 | Ethanol F = 29.56, P < 0.001; protein deficiency F = 8.97, P = 0.005 | Protein deficiency F = 48.04, P < 0.001 | |
| Interactions | NS | NS | NS | NS |
| Ad libitum (5) | 302 ± 11 | 341 ± 21 | 84 ± 1.5 | 3.80 ± 0.14 |
| Differences 1–5 | NS | t = 6.3, P < 0.001 | t = 17.87, P < 0.001 | NS |
ANOVA analysis was performed first (F, P file), and if differences were statistically significant, an SNK test was performed to assess differences among groups. In the case of significant differences, main effects (of ethanol and/or protein deficiency) and interactions between both parameters were also disclosed by means of a two-way ANOVA. A comparison between the parameters of the control group (1) and the ad libitum-fed ones (group 5; Student's t-test) was also performed. NS, non-significant; vs, versus.
Five animals died during the experiment, four alcohol-treated animals and one fed a low protein, ethanol-containing diet. At the end of the experiment, blood was obtained by direct cardiac puncture, and centrifuged. The right gastrocnemius muscle was removed. Part of this was used for the histological and histochemical analyses. In the remaining portion, glutathione peroxidase (GPX) and superoxide dismutase (SOD) enzyme activities were determined. Also, MDA and hydroxyalkenals were measured, as a global estimation of lipid peroxidation (LPX).
These groups of rats form part of a more ample experiment designed with the aim to analyse the effects of zinc supplementation on ethanol and/or protein deficiency-mediated bone and muscle alterations, which results were already published ( Durán Castellón et al., 2005; González Reimers et al., 2005).
This study was approved by the ethical committee of our hospital (Hospital Universitario de Canarias).
Enzyme activities
Cu/Zn SOD activity
Muscle samples were washed with NaCl (0.9%) containing 0.16 mg/ml of heparin to remove red cells, dried on absorbent paper and weighed, followed by homogenization in cold 0.9% NaCl. The homogenate was centrifuged at 13,000 g for 15 min at 4°C. The assay was performed on the supernatant. The Cu/Zn SOD assay kit uses autooxidation of 5, 6, 6, 11 tetrahydro 3, 9, 19 trihydrobenzofluorene, a chromophere which absorbs maximally at 525 nm, provided by Calbiochem (San Diego, California, USA). The activity was calculated directly from the rate of sample versus the average of blank control, using the ratio table provided in the Calbiochem kit.
Se dependent GPX activity
The GPX assay measures activity indirectly, using the oxidation of NADPH to NADP recording the absorbance at 340 nm (A340) (Calbiochem, San Diego, California, USA). The reaction is initiated by the addition of tertbutyl hydroperoxide to a solution containing reduced glutathione, glutathione reductase and NADPH and the sample (containing GPX). The oxidation of NADPH to NADP is accompanied by a decrease in absorbance at 340 nm. The rate of the decrease in absorbance is directly proportional to GPX activity in the sample. Muscle samples were washed with NaCl (0.9%), following the protocol previously described (for SOD activity). The homogenate was centrifuged at 8000 g for 15 min at 4°C. The assay was performed on the supernatant. The activity was calculated using the sample data and calculation sheet provided by the Calbiochem kit (1 mU/ml = 1 nmol NADPH/min/ml = A340/min/0.0062).
LPX products
The Calbiochem LPX assay is based on the formation of a chromogen with MDA and 4-hydroxyalkenals (4-HNE) at 45°C. The stable chromogen presents a maximal absorbance (assessed by spectrophotometry) at 586 nm. Muscle samples were washed with NaCl (0.9%) following the protocol previously described. The homogenate was centrifuged at 3000 g for 10 min at 4°C. The assay was performed in the supernatant. The standard curves for MDA 4-HNE were prepared using the solutions purchased from Calbiochem (San Diego, California, USA), which contain 10 mM 4-hydroxynonenal as the diethylacetal, in acetonitrile, and 10 mM MDA as a bis (dimethyl acetal) in 20 mM Tris–HCl buffer, ph 7.4.
The precision of SOD (intra-assay = 7.60%; inter-assay = 10.20%) and GPX (intra-assay = 5.36%; inter-assay = 8.76%) enzyme activities and MDA concentration (intra-assay = 1.67%; inter-assay = 2.82%) were calculated using the variation coefficient (in percent) of repeated measurements of control samples.
Histological analysis
Samples of the right gastrocnemius muscle were obtained for histochemical and histomorphometrical analysis. A 3–5-mm-thick cross-section was cut from the middle portion (belly) of the muscle, rapidly frozen in liquid nitrogen and stored at−80°. Sections were cut at 8 μm using a cryostat at−40°. Fibres were classified according to the acid–alkali sensitivity (pH 4.3, 4.6, 9.6) of the ATPases in the myosin isoenzymes: type I (slow oxidative), type IIa (fast oxidative glycolytic) and type IIb (fast glycolytic) fibres. A morphometric study was performed at a magnification of ×200 to study the area of the fibres. The form factor (sphericity) was also estimated. Only fibres with a form factor >0.9 were measured, with the aid of a VIDS-II image analyser. In some cases, freezing artefacts or excessive obliquity of the muscle sections precluded accurate estimation of the area of the muscle fibres. Thus, valid muscle histomorphometrical analysis was carried out in seven controls, eight low-protein-fed animals, six ethanol-fed animals and eight ethanol and low-protein-fed animals, as well as in the five ad libitum-fed animals.
Serum hormones, calcium, magnesium and albumin levels
We measured:
-
Serum IGF-1, with a sensitivity of 0.06 ng/ml (Nichols, San Juan de Capistrano, CA). The standard curve for IGF-1 determination was generated using DNA-recombinant IGF-1 standard. This standard is calibrated against WHO 1st International Reference Reagent 1988. Recovery for IGF-1 ranged from 92.9 to 106.8%.
-
Serum free testosterone (with a sensitivity of 0.15 pg/ml) by radioimmunoanalysis (RIA). Calibrators for testosterone, containing 0, 0.55, 2.5, 9, 25 and 50 pg/ml, were directly purchased from Diagnostic Products Corporation (DPC), Los Angeles, CA. Recovery ranged from 95 to 105%.
-
Serum corticosterone, highly specific for rat corticosterone, with a sensitivity of 5.7 ng/ml, by RIA (DPC). Recovery for corticosterone ranged from 90 to 103%. Calibrators for corticosterone, containing 0, 20, 50, 100, 200, 500, 1000 and 2000 ng/ml, were also purchased directly from DPC.
We also measured serum 1,25 dihydroxyvitamin D3 by high performance liquid chromatography, and serum parathyroid hormone (PTH) by RIA, using an antibody directed towards intact rat PTH (Nichols, San Juan de Capistrano, California, USA), with a sensitivity of 1 pg/ml.
Serum albumin, calcium and magnesium were determined by routine analytical methods.
Statistics
Differences between the experimental groups were analysed using analysis of variance (ANOVA) with further Student–Newman–Keuls (SNK) test. Independent effects of ethanol and protein deficiency on the alterations observed, and interactions between these parameters, were analysed by two-way ANOVA. Paired t-test analysis was used to compare initial and final weight in the four groups of rats. Differences of the analysed parameters between controls and ad libitum-fed animals were analysed using Student's t-test. Also, single correlation analysis (Pearson's r) was performed between quantitative variables. If variables were not normally distributed, non-parametric tests, such as Mann–Whitney's U-test, Kruskall–Wallis and Spearman's correlation analysis, were used.
In order to identify which parameters were independently related with muscle fibre atrophy, we performed stepwise multiple regression analysis between type IIa and type IIb fibre area and albumin, vitamin D and other hormones, weight at the end of the study, dietary consumption and consumption of ethanol or a low-protein diet.
Results
Results are shown in Tables 1–3.
Table 2
Muscle fibre area (in micro square) and muscle GPX, SOD and MDA (mean ± standard deviation, and if non-parametric distribution, median and interquartile range are also in brackets)
| Type I fibre | Type IIa fibre | Type IIb fibre | |
|---|---|---|---|
| Control (1) | 1867 ± 207 | 1876 ± 314 | 1569 ± 419 |
| Low protein (2) | 1669 ± 186 | 1382 ± 424 | 1555 ± 536 |
| Control alcoholic (3) | 1773 ± 428 | 1896 ± 482 | 1526 ± 121 |
| Low-protein alcoholic (4) | 1617 ± 343 | 1550 ± 372 | 1131 ± 322 |
| F, P | F = 0.69, NS | F = 3.35, P = 0.035 | F = 2.83, P = 0.06 (NS) |
| Differences among groups (SNK test) | |||
| Main effects | Low protein F = 7.93, P = 0.009 | ||
| Interactions | NS | NS | NS |
| Ad libitum (5) | 2238 ± 254 | 1661 ± 352 | 1973 ± 238 |
| Differences 1–5 | t = 2.78, P = 0.02 | NS | NS |
| Muscle GPX (mU/mg) | Muscle SOD (IU/mg) | Muscle MDA (nmol/g tissue) | |
| Control (1) | 5.12 ± 3.96 3.91 (1.55–9.03) | 10.50 ± 10.09 | 28.4 ± 30.3 |
| Low protein (2) | 4.44 ± 6.35 2.08 (1.60–3.85) | 7.20 ± 8.01 | 17.7 ± 5.8 |
| Control alcoholic (3) | 7.48 ± 4.42 6.92 (3.42–11.82) | 22.31 ± 13.35 | 59.8 ± 53.8 |
| Low-protein alcoholic (4) | 1.93 ± 1.33 1.41 (1.13–2.38) | 11.32 ± 6.91 | 77.7 ± 37.8 |
| F, P | KW = 7.75, P = 0.05 | F = 3.34, P = 0.02 | F = 5.82, P = 0.003 |
| Differences among groups (SNK test) | 3 vs 1, 2, 4 | 1 vs 4; 2 vs 3, 4 | |
| Main effects | Ethanol F = 5.13, P = 0.03; low protein F = 4.06, P = 0.053 | Ethanol F = 15.45, P < 0.001 | |
| Interactions | NS | NS | NS |
| Ad libitum (5) | 1.06 ± 0.22 1.10 (0.84–1.25) | 1.68 ± 0.46 | 5.62 ± 0.94 |
| Differences 1–5 | Z = 2.20, P = 0.019 | t = 2.61, P = 0.031 | NS |
| Type I fibre | Type IIa fibre | Type IIb fibre | |
|---|---|---|---|
| Control (1) | 1867 ± 207 | 1876 ± 314 | 1569 ± 419 |
| Low protein (2) | 1669 ± 186 | 1382 ± 424 | 1555 ± 536 |
| Control alcoholic (3) | 1773 ± 428 | 1896 ± 482 | 1526 ± 121 |
| Low-protein alcoholic (4) | 1617 ± 343 | 1550 ± 372 | 1131 ± 322 |
| F, P | F = 0.69, NS | F = 3.35, P = 0.035 | F = 2.83, P = 0.06 (NS) |
| Differences among groups (SNK test) | |||
| Main effects | Low protein F = 7.93, P = 0.009 | ||
| Interactions | NS | NS | NS |
| Ad libitum (5) | 2238 ± 254 | 1661 ± 352 | 1973 ± 238 |
| Differences 1–5 | t = 2.78, P = 0.02 | NS | NS |
| Muscle GPX (mU/mg) | Muscle SOD (IU/mg) | Muscle MDA (nmol/g tissue) | |
| Control (1) | 5.12 ± 3.96 3.91 (1.55–9.03) | 10.50 ± 10.09 | 28.4 ± 30.3 |
| Low protein (2) | 4.44 ± 6.35 2.08 (1.60–3.85) | 7.20 ± 8.01 | 17.7 ± 5.8 |
| Control alcoholic (3) | 7.48 ± 4.42 6.92 (3.42–11.82) | 22.31 ± 13.35 | 59.8 ± 53.8 |
| Low-protein alcoholic (4) | 1.93 ± 1.33 1.41 (1.13–2.38) | 11.32 ± 6.91 | 77.7 ± 37.8 |
| F, P | KW = 7.75, P = 0.05 | F = 3.34, P = 0.02 | F = 5.82, P = 0.003 |
| Differences among groups (SNK test) | 3 vs 1, 2, 4 | 1 vs 4; 2 vs 3, 4 | |
| Main effects | Ethanol F = 5.13, P = 0.03; low protein F = 4.06, P = 0.053 | Ethanol F = 15.45, P < 0.001 | |
| Interactions | NS | NS | NS |
| Ad libitum (5) | 1.06 ± 0.22 1.10 (0.84–1.25) | 1.68 ± 0.46 | 5.62 ± 0.94 |
| Differences 1–5 | Z = 2.20, P = 0.019 | t = 2.61, P = 0.031 | NS |
ANOVA analysis was performed first (F, P file), and if differences were statistically significant, an SNK test was performed to assess differences among groups. In the case of significant differences, main effects (of ethanol and/or protein deficiency) and interactions between both parameters were also disclosed by means of a two-way ANOVA. A comparison between the parameters of the control group (1) and the ad libitum-fed ones (group 5; Student's t-test) was also performed. Given the non-parametric distribution of muscle GPX, Kruskall–Wallis (KW) and Mann–Whitney's U-test were used in this case. NS, non-significant.
Table 2
Muscle fibre area (in micro square) and muscle GPX, SOD and MDA (mean ± standard deviation, and if non-parametric distribution, median and interquartile range are also in brackets)
| Type I fibre | Type IIa fibre | Type IIb fibre | |
|---|---|---|---|
| Control (1) | 1867 ± 207 | 1876 ± 314 | 1569 ± 419 |
| Low protein (2) | 1669 ± 186 | 1382 ± 424 | 1555 ± 536 |
| Control alcoholic (3) | 1773 ± 428 | 1896 ± 482 | 1526 ± 121 |
| Low-protein alcoholic (4) | 1617 ± 343 | 1550 ± 372 | 1131 ± 322 |
| F, P | F = 0.69, NS | F = 3.35, P = 0.035 | F = 2.83, P = 0.06 (NS) |
| Differences among groups (SNK test) | |||
| Main effects | Low protein F = 7.93, P = 0.009 | ||
| Interactions | NS | NS | NS |
| Ad libitum (5) | 2238 ± 254 | 1661 ± 352 | 1973 ± 238 |
| Differences 1–5 | t = 2.78, P = 0.02 | NS | NS |
| Muscle GPX (mU/mg) | Muscle SOD (IU/mg) | Muscle MDA (nmol/g tissue) | |
| Control (1) | 5.12 ± 3.96 3.91 (1.55–9.03) | 10.50 ± 10.09 | 28.4 ± 30.3 |
| Low protein (2) | 4.44 ± 6.35 2.08 (1.60–3.85) | 7.20 ± 8.01 | 17.7 ± 5.8 |
| Control alcoholic (3) | 7.48 ± 4.42 6.92 (3.42–11.82) | 22.31 ± 13.35 | 59.8 ± 53.8 |
| Low-protein alcoholic (4) | 1.93 ± 1.33 1.41 (1.13–2.38) | 11.32 ± 6.91 | 77.7 ± 37.8 |
| F, P | KW = 7.75, P = 0.05 | F = 3.34, P = 0.02 | F = 5.82, P = 0.003 |
| Differences among groups (SNK test) | 3 vs 1, 2, 4 | 1 vs 4; 2 vs 3, 4 | |
| Main effects | Ethanol F = 5.13, P = 0.03; low protein F = 4.06, P = 0.053 | Ethanol F = 15.45, P < 0.001 | |
| Interactions | NS | NS | NS |
| Ad libitum (5) | 1.06 ± 0.22 1.10 (0.84–1.25) | 1.68 ± 0.46 | 5.62 ± 0.94 |
| Differences 1–5 | Z = 2.20, P = 0.019 | t = 2.61, P = 0.031 | NS |
| Type I fibre | Type IIa fibre | Type IIb fibre | |
|---|---|---|---|
| Control (1) | 1867 ± 207 | 1876 ± 314 | 1569 ± 419 |
| Low protein (2) | 1669 ± 186 | 1382 ± 424 | 1555 ± 536 |
| Control alcoholic (3) | 1773 ± 428 | 1896 ± 482 | 1526 ± 121 |
| Low-protein alcoholic (4) | 1617 ± 343 | 1550 ± 372 | 1131 ± 322 |
| F, P | F = 0.69, NS | F = 3.35, P = 0.035 | F = 2.83, P = 0.06 (NS) |
| Differences among groups (SNK test) | |||
| Main effects | Low protein F = 7.93, P = 0.009 | ||
| Interactions | NS | NS | NS |
| Ad libitum (5) | 2238 ± 254 | 1661 ± 352 | 1973 ± 238 |
| Differences 1–5 | t = 2.78, P = 0.02 | NS | NS |
| Muscle GPX (mU/mg) | Muscle SOD (IU/mg) | Muscle MDA (nmol/g tissue) | |
| Control (1) | 5.12 ± 3.96 3.91 (1.55–9.03) | 10.50 ± 10.09 | 28.4 ± 30.3 |
| Low protein (2) | 4.44 ± 6.35 2.08 (1.60–3.85) | 7.20 ± 8.01 | 17.7 ± 5.8 |
| Control alcoholic (3) | 7.48 ± 4.42 6.92 (3.42–11.82) | 22.31 ± 13.35 | 59.8 ± 53.8 |
| Low-protein alcoholic (4) | 1.93 ± 1.33 1.41 (1.13–2.38) | 11.32 ± 6.91 | 77.7 ± 37.8 |
| F, P | KW = 7.75, P = 0.05 | F = 3.34, P = 0.02 | F = 5.82, P = 0.003 |
| Differences among groups (SNK test) | 3 vs 1, 2, 4 | 1 vs 4; 2 vs 3, 4 | |
| Main effects | Ethanol F = 5.13, P = 0.03; low protein F = 4.06, P = 0.053 | Ethanol F = 15.45, P < 0.001 | |
| Interactions | NS | NS | NS |
| Ad libitum (5) | 1.06 ± 0.22 1.10 (0.84–1.25) | 1.68 ± 0.46 | 5.62 ± 0.94 |
| Differences 1–5 | Z = 2.20, P = 0.019 | t = 2.61, P = 0.031 | NS |
ANOVA analysis was performed first (F, P file), and if differences were statistically significant, an SNK test was performed to assess differences among groups. In the case of significant differences, main effects (of ethanol and/or protein deficiency) and interactions between both parameters were also disclosed by means of a two-way ANOVA. A comparison between the parameters of the control group (1) and the ad libitum-fed ones (group 5; Student's t-test) was also performed. Given the non-parametric distribution of muscle GPX, Kruskall–Wallis (KW) and Mann–Whitney's U-test were used in this case. NS, non-significant.
Table 3
Serum vitamin D and PTH, magnesium and calcium, IGF-1, testosterone and corticosterone levels (mean ± standard deviation; given the non-parametric distribution of testosterone, median and interquartile range are also given for this parameter)
| Vitamin D (ng/ml) | PTH (pg/ml) | Magnesium (mg/dl) | Calcium (mg/dl) | |
|---|---|---|---|---|
| Control (1) | 67.7 ± 33.1 | 110.0 ± 148.1 | 3.21 ± 0.58 | 11.44 ± 0.91 |
| Low protein (2) | 37.8 ± 28.0 | 75.4 ± 45.8 | 2.60 ± 0.39 | 11.26 ± 0.39 |
| Control alcoholic (3) | 72.5 ± 13.0 | 75.9 ± 30.0 | 3.08 ± 0.57 | 11.13 ± 0.60 |
| Low-protein alcoholic (4) | 17.8 ± 9.0 | 99.7 ± 70.1 | 2.86 ± 0.56 | 10.97 ± 0.77 |
| F, P | F = 9.40, P < 0.001 | F = 0.30, NS | F = 2.50, NS | F = 0.75, NS |
| Differences among groups (SNK test) | 1, 3 vs 2, 4 | |||
| Main effects | Low protein F = 25.3, P < 0.001 | |||
| Interactions | NS | NS | NS | NS |
| Ad libitum (5) | 38.7 ± 6.1 | 80.4 ± 61.6 | 2.76 ± 0.34 | 11.24 ± 0.44 |
| Differences 1–5 | t = 2.55, P = 0.03 | NS | NS | NS |
| Serum IGF-1 (ng/ml) | Serum free testosterone (pg/ml) | Serum corticosterone (ng/ml) | ||
| Control (1) | 14.93 ± 2.29 | 0.59 ± 1.23 0.15 (0.10–0.35) | 413 ± 329 | |
| Low protein (2) | 18.26 ± 2.92 | 1.51 ± 2.48 0.26 (0.10–2.16) | 499 ± 162 | |
| Control alcoholic (3) | 6.76 ± 5.48 | 4.70 ± 9.09 1.10 (0.51–7.18) | 684 ± 223 | |
| Low-protein alcoholic (4) | 2.59 ± 1.88 | 0.37 ± 0.43 0.16 (0.10–0.67) | 553 ± 240 | |
| F, P | F = 48.6, P < 0.001 | KW = 7.5, NS | F = 1.55, NS | |
| Differences among groups (SNK test) | 1 vs 2, 3, 4; 2 vs 3, 4; 3 vs 4 | |||
| Main effects | Ethanol F = 122, P < 0.0001 | |||
| Interactions | Ethanol–low protein F = 12, P = 0.002 | NS | NS | |
| Ad libitum (5) | 21.22 ± 6.05 | 12.32 ± 12.01 5.39 (2.73–25.39) | 533 ± 161 | |
| Differences 1–5 | t = 2.98, P = 0.011 | Z = 2.84, P = 0.003 | t = 0.77, NS | |
| Vitamin D (ng/ml) | PTH (pg/ml) | Magnesium (mg/dl) | Calcium (mg/dl) | |
|---|---|---|---|---|
| Control (1) | 67.7 ± 33.1 | 110.0 ± 148.1 | 3.21 ± 0.58 | 11.44 ± 0.91 |
| Low protein (2) | 37.8 ± 28.0 | 75.4 ± 45.8 | 2.60 ± 0.39 | 11.26 ± 0.39 |
| Control alcoholic (3) | 72.5 ± 13.0 | 75.9 ± 30.0 | 3.08 ± 0.57 | 11.13 ± 0.60 |
| Low-protein alcoholic (4) | 17.8 ± 9.0 | 99.7 ± 70.1 | 2.86 ± 0.56 | 10.97 ± 0.77 |
| F, P | F = 9.40, P < 0.001 | F = 0.30, NS | F = 2.50, NS | F = 0.75, NS |
| Differences among groups (SNK test) | 1, 3 vs 2, 4 | |||
| Main effects | Low protein F = 25.3, P < 0.001 | |||
| Interactions | NS | NS | NS | NS |
| Ad libitum (5) | 38.7 ± 6.1 | 80.4 ± 61.6 | 2.76 ± 0.34 | 11.24 ± 0.44 |
| Differences 1–5 | t = 2.55, P = 0.03 | NS | NS | NS |
| Serum IGF-1 (ng/ml) | Serum free testosterone (pg/ml) | Serum corticosterone (ng/ml) | ||
| Control (1) | 14.93 ± 2.29 | 0.59 ± 1.23 0.15 (0.10–0.35) | 413 ± 329 | |
| Low protein (2) | 18.26 ± 2.92 | 1.51 ± 2.48 0.26 (0.10–2.16) | 499 ± 162 | |
| Control alcoholic (3) | 6.76 ± 5.48 | 4.70 ± 9.09 1.10 (0.51–7.18) | 684 ± 223 | |
| Low-protein alcoholic (4) | 2.59 ± 1.88 | 0.37 ± 0.43 0.16 (0.10–0.67) | 553 ± 240 | |
| F, P | F = 48.6, P < 0.001 | KW = 7.5, NS | F = 1.55, NS | |
| Differences among groups (SNK test) | 1 vs 2, 3, 4; 2 vs 3, 4; 3 vs 4 | |||
| Main effects | Ethanol F = 122, P < 0.0001 | |||
| Interactions | Ethanol–low protein F = 12, P = 0.002 | NS | NS | |
| Ad libitum (5) | 21.22 ± 6.05 | 12.32 ± 12.01 5.39 (2.73–25.39) | 533 ± 161 | |
| Differences 1–5 | t = 2.98, P = 0.011 | Z = 2.84, P = 0.003 | t = 0.77, NS | |
ANOVA analysis (or in the case of testosterone, Kruskall–Wallis test) was performed first (F, P file), and if differences were statistically significant, an SNK test was carried out to assess differences among groups. In the case of significant differences, main effects (of ethanol and/or protein deficiency) and interactions between both parameters were also disclosed by means of a two-way ANOVA. A comparison between the parameters of the control group (1) and the ad libitum-fed rats (group 5; Student's t-test or Mann–Whitney's U-test in the case of testosterone) was also performed.
Table 3
Serum vitamin D and PTH, magnesium and calcium, IGF-1, testosterone and corticosterone levels (mean ± standard deviation; given the non-parametric distribution of testosterone, median and interquartile range are also given for this parameter)
| Vitamin D (ng/ml) | PTH (pg/ml) | Magnesium (mg/dl) | Calcium (mg/dl) | |
|---|---|---|---|---|
| Control (1) | 67.7 ± 33.1 | 110.0 ± 148.1 | 3.21 ± 0.58 | 11.44 ± 0.91 |
| Low protein (2) | 37.8 ± 28.0 | 75.4 ± 45.8 | 2.60 ± 0.39 | 11.26 ± 0.39 |
| Control alcoholic (3) | 72.5 ± 13.0 | 75.9 ± 30.0 | 3.08 ± 0.57 | 11.13 ± 0.60 |
| Low-protein alcoholic (4) | 17.8 ± 9.0 | 99.7 ± 70.1 | 2.86 ± 0.56 | 10.97 ± 0.77 |
| F, P | F = 9.40, P < 0.001 | F = 0.30, NS | F = 2.50, NS | F = 0.75, NS |
| Differences among groups (SNK test) | 1, 3 vs 2, 4 | |||
| Main effects | Low protein F = 25.3, P < 0.001 | |||
| Interactions | NS | NS | NS | NS |
| Ad libitum (5) | 38.7 ± 6.1 | 80.4 ± 61.6 | 2.76 ± 0.34 | 11.24 ± 0.44 |
| Differences 1–5 | t = 2.55, P = 0.03 | NS | NS | NS |
| Serum IGF-1 (ng/ml) | Serum free testosterone (pg/ml) | Serum corticosterone (ng/ml) | ||
| Control (1) | 14.93 ± 2.29 | 0.59 ± 1.23 0.15 (0.10–0.35) | 413 ± 329 | |
| Low protein (2) | 18.26 ± 2.92 | 1.51 ± 2.48 0.26 (0.10–2.16) | 499 ± 162 | |
| Control alcoholic (3) | 6.76 ± 5.48 | 4.70 ± 9.09 1.10 (0.51–7.18) | 684 ± 223 | |
| Low-protein alcoholic (4) | 2.59 ± 1.88 | 0.37 ± 0.43 0.16 (0.10–0.67) | 553 ± 240 | |
| F, P | F = 48.6, P < 0.001 | KW = 7.5, NS | F = 1.55, NS | |
| Differences among groups (SNK test) | 1 vs 2, 3, 4; 2 vs 3, 4; 3 vs 4 | |||
| Main effects | Ethanol F = 122, P < 0.0001 | |||
| Interactions | Ethanol–low protein F = 12, P = 0.002 | NS | NS | |
| Ad libitum (5) | 21.22 ± 6.05 | 12.32 ± 12.01 5.39 (2.73–25.39) | 533 ± 161 | |
| Differences 1–5 | t = 2.98, P = 0.011 | Z = 2.84, P = 0.003 | t = 0.77, NS | |
| Vitamin D (ng/ml) | PTH (pg/ml) | Magnesium (mg/dl) | Calcium (mg/dl) | |
|---|---|---|---|---|
| Control (1) | 67.7 ± 33.1 | 110.0 ± 148.1 | 3.21 ± 0.58 | 11.44 ± 0.91 |
| Low protein (2) | 37.8 ± 28.0 | 75.4 ± 45.8 | 2.60 ± 0.39 | 11.26 ± 0.39 |
| Control alcoholic (3) | 72.5 ± 13.0 | 75.9 ± 30.0 | 3.08 ± 0.57 | 11.13 ± 0.60 |
| Low-protein alcoholic (4) | 17.8 ± 9.0 | 99.7 ± 70.1 | 2.86 ± 0.56 | 10.97 ± 0.77 |
| F, P | F = 9.40, P < 0.001 | F = 0.30, NS | F = 2.50, NS | F = 0.75, NS |
| Differences among groups (SNK test) | 1, 3 vs 2, 4 | |||
| Main effects | Low protein F = 25.3, P < 0.001 | |||
| Interactions | NS | NS | NS | NS |
| Ad libitum (5) | 38.7 ± 6.1 | 80.4 ± 61.6 | 2.76 ± 0.34 | 11.24 ± 0.44 |
| Differences 1–5 | t = 2.55, P = 0.03 | NS | NS | NS |
| Serum IGF-1 (ng/ml) | Serum free testosterone (pg/ml) | Serum corticosterone (ng/ml) | ||
| Control (1) | 14.93 ± 2.29 | 0.59 ± 1.23 0.15 (0.10–0.35) | 413 ± 329 | |
| Low protein (2) | 18.26 ± 2.92 | 1.51 ± 2.48 0.26 (0.10–2.16) | 499 ± 162 | |
| Control alcoholic (3) | 6.76 ± 5.48 | 4.70 ± 9.09 1.10 (0.51–7.18) | 684 ± 223 | |
| Low-protein alcoholic (4) | 2.59 ± 1.88 | 0.37 ± 0.43 0.16 (0.10–0.67) | 553 ± 240 | |
| F, P | F = 48.6, P < 0.001 | KW = 7.5, NS | F = 1.55, NS | |
| Differences among groups (SNK test) | 1 vs 2, 3, 4; 2 vs 3, 4; 3 vs 4 | |||
| Main effects | Ethanol F = 122, P < 0.0001 | |||
| Interactions | Ethanol–low protein F = 12, P = 0.002 | NS | NS | |
| Ad libitum (5) | 21.22 ± 6.05 | 12.32 ± 12.01 5.39 (2.73–25.39) | 533 ± 161 | |
| Differences 1–5 | t = 2.98, P = 0.011 | Z = 2.84, P = 0.003 | t = 0.77, NS | |
ANOVA analysis (or in the case of testosterone, Kruskall–Wallis test) was performed first (F, P file), and if differences were statistically significant, an SNK test was carried out to assess differences among groups. In the case of significant differences, main effects (of ethanol and/or protein deficiency) and interactions between both parameters were also disclosed by means of a two-way ANOVA. A comparison between the parameters of the control group (1) and the ad libitum-fed rats (group 5; Student's t-test or Mann–Whitney's U-test in the case of testosterone) was also performed.
All the rats weighed the same (∼300 g) at the beginning of the study (Table 1). All the groups except for the control group showed a significant weight loss over the experimental period. Both low-protein diet and ethanol exerted independent, significant effects on weight loss, although no additive effect between the two factors was found. Serum albumin was lower in the low-protein-fed groups than in the others; only a low-protein diet (F = 48, P < 0.001) exerted independent effects on serum albumin. As shown in Table 2, we found muscle fibre atrophy, especially type IIa fibre atrophy, in the low-protein-fed animals, an effect only dependent on a low-protein diet (F = 7.93, P = 0.009). Type IIb fibre atrophy was also observed, but only in the ethanol-treated, low-protein-fed animals, which showed a lower fibre area than the other groups, although differences were not statistically significant. No change was observed in type I fibres. Muscle MDA was significantly raised in ethanol and ethanol 2% protein-fed animals, whereas a decrease in GPX and SOD was also observed in these animals (Table 2).
In Table 3, we show serum levels of hormones, calcium and magnesium. Whereas no differences were observed regarding PTH, calcium and magnesium, serum vitamin D levels were significantly lower among the animals fed a low-protein diet, especially in the ethanol-treated, low-protein-fed animals. Only a low-protein diet exerted a significant effect on serum vitamin D levels (Table 3). Serum IGF-1 levels were also significantly lower among in the ethanol and ethanol 2% protein-fed animals.
Significant relationships were observed between serum vitamin D levels and type IIa muscle fibre area (ρ = 0.56, P = 0.002; Fig. 1), and also, as a trend, between vitamin D and type IIb muscle fibre area (ρ = 0.39, P = 0.053). Indeed, both type IIa (Z = 2.45, P = 0.013) and type IIb (Z = 2.32, P = 0.02) fibre areas were significantly smaller in animals with the lowest vitamin D quartile compared with those with the highest quartile. A significant relationship was also observed between testosterone and type IIa muscle fibre area (ρ = 0.61, P < 0.001).
Fig. 1
This figure shows the relationships between serum vitamin D levels and muscle fibre area (type IIa and IIb) in the experimental groups of animals (the ad libitum-fed animals are excluded).
Fig. 1
This figure shows the relationships between serum vitamin D levels and muscle fibre area (type IIa and IIb) in the experimental groups of animals (the ad libitum-fed animals are excluded).
When we performed a stepwise multiple regression analysis comparing type IIa muscle fibre as dependent variable with other variables analysed in the study, such as 1,25 dihydroxyvitamin D, IGF-1, testosterone, corticosterone, dietary consumption, weight difference, serum albumin, PTH, MDA, SOD, GPX, ethanol treatment and feeding a 2% protein-containing diet, we observed that the only parameter which was independently related with type IIa fibre area was 1,25 (OH)2 vitamin D (beta = 0.602, t = 3.54, P = 0.002). On the other hand, including the same variables in a multiple regression analysis with type IIb fibre types, the daily amount of diet consumed was the only parameter related to type IIb fibre area (beta = 0.452, t = 2.21, P = 0.04).
Serum 1,25 (OH)2 vitamin D levels were also significantly related with muscle GPX activity (ρ = 0.40, P = 0.025) and SOD activity (ρ = 0.43, P = 0.012), with dietary consumption (ρ = 0.49, P = 0.004), weight loss during the experiment (ρ = 0.74, P < 0.001) and serum albumin (ρ = 0.50, P = 0.003). However, by stepwise multiple regression analysis, introducing the variables weight change during the experiment, serum albumin, dietary consumption, ethanol treatment and feeding a 2% protein-containing diet, only weight change during the experiment was independently related with 1,25 (OH)2 vitamin D levels (beta = 0.73, t = 6.01, P < 0.001).
Muscle GPX activity was significantly related with type I fibre area (ρ = 0.49, P = 0.01) and muscle SOD, with type IIa fibre area (ρ = 0.38, P = 0.045).
On the contrary, no relation was observed between serum PTH and fibre area or PTH and antioxidant systems, but a significant relationship was observed between PTH and serum magnesium (r = 0.37, P = 0.031). Also, serum magnesium was related to type IIa muscle fibre area (r = 0.39, P = 0.038), and serum calcium to type IIb fibre area (r = 0.41, P = 0.038). Corticosterone and testosterone did not show any relations, except for a negative one between testosterone and MDA (ρ = −0.58, P < 0.001).
Discussion
The main objective of this study was to assess if there is a relationship between fibre muscle area and serum 1,25 (OH)2 vitamin D levels in a murine model of alcoholic myopathy. We have followed the Lieber–DeCarli model, since it is important to control for protein deficiency, a frequently observed condition in the alcoholic patient. We have prolonged our experiment for 5 weeks, and in this period we have observed a substantial atrophy of type II fibres; other researchers have maintained rats under treatment with alcohol during 6 weeks ( Salisbury et al., 1992) or more ( Conde et al., 1992), but it has been shown that decreased muscle protein synthesis and RNA content — features of alcoholic myopathy ( Reilly et al., 2000) — ensue already in the first week of treatment ( Preedy et al., 1990). The convenience to adjust the amount consumed by all the groups of animals to that consumed by those which ate the least necessary implies that the control group is not a true control, but a calorie-restricted one. Several hormones, such as IGF-1, testosterone and vitamin D, muscle GPX and SOD and type I fibre diameter — which was not significantly affected by ethanol or protein deficiency — showed differences between the control groups and the ad libitum-fed one. However, the pair-feeding process may be difficult to carry out when several groups are fed simultaneously. Indeed, in our model, rats fed the low protein ethanol-containing diet consumed significantly less than the other groups, so an effect of caloric deficiency on the results obtained cannot be excluded. However, given the considerable loss of weight suffered by these rats, the amount of ethanol in relation to body weight consumed by the four groups fed the ethanol-containing diets was similar.
We found atrophy of type IIa and also type IIb fibres. Other subtle alterations are described in alcoholic myopathy ( Salisbury et al., 1992; Romero et al., 1994), and atrophy is considered as a non-specific feature of alcoholic myopathy ( Fernández-Sola et al., 1995), since it may also appear in other situations such as malnutrition, corticosteroid excess or peripheral neuropathy. However, all these three features may be seen in alcoholics, so type II fibre atrophy is a usual finding in these patients.
In this study, we found a direct relationship between type IIa fibre area and 1,25 (OH)2 vitamin D levels. As commented before, the effect of vitamin D on muscle is an out-of-discussion, well-known effect, a proximal myopathy having been described in patients affected by osteomalacia (Boland, 1986). Adult individuals with vitamin D deficiency usually show predominantly type II muscle fibre atrophy (Ceglia, 2008). In one study, treatment with vitamin D and calcium supplements led to an increase in type IIa fibre area in aged patients ( Sorensen et al., 1979), a result confirmed in a more recent study by Sato et al. (2005) who also found a significant relationship between vitamin D levels and type II fibre area in women affected by stroke. On the other hand, experiments with VDR null mice have shown that muscle fibre diameters of mice are smaller than the wild type, affecting all the three types of muscle fibres ( Endo et al., 2003). All these effects may be due in part to the well-described actions of vitamin D on muscle calcium uptake, increasing it; phosphate transport across the cell membrane, increasing it and promoting muscle cell proliferation and differentiation (Boland, 1986; Walters et al., 1987). Muscle intracellular calcium levels regulate muscle contraction. The effect of vitamin D on muscle calcium is in part mediated by calmodulin ( Drittanti et al., 1990), a molecule highly sensitive to oxidative stress, on which oxidative damage causes conformational changes and altered function (Sharp and Tomer, 2007).
In 1989, Hickish et al. failed to find any relation between muscle strength in alcoholics and serum vitamin D levels ( Hickish et al., 1989). In our study, it is clear that a relation exists between vitamin D and muscle fibre area, involving type II fibres, especially, in accordance with Sorensen et al. (1979), with type IIa fibres. The low number of animals in the experimental groups in which both muscle histology and serum data (1,25 (OH)2 vitamin D and other hormones) are available constitutes a shortage of this study, and perhaps some results are subjected to a type II error, but relationships between muscle fibre area and 1,25 (OH)2 vitamin D levels are highly significant, and moreover, the stepwise multiple regression analysis identifies vitamin D as the main factor related to type IIa fibre atrophy. It is important to keep in mind that this is a statistical result, and perhaps, other factors (not assessed in this study), and not a direct effect of vitamin D, may explain the relation between vitamin D and fibre area. Another shortage of our study is the fact that the pair-feeding process was not successfully fulfilled. Indeed, vitamin D was related with the amount consumed, but multivariate analysis showed that only weight loss was independently related with vitamin D status. However, this data raises the possibility that malnutrition plays a main role in vitamin D deficiency and, probably, in muscle fibre atrophy. But, as commented, by multivariate analysis, the only parameter which was related to muscle fibre atrophy was vitamin D, not weight loss during the experiment. The recent observation of a direct effect of ethanol on 1,25 (OH)2 synthesis ( Shankar et al., 2008) also points that there is a real, direct effect of ethanol on vitamin D levels, independent of nutritional status. In any case, the aim of this study was not to discern the mechanisms underlying vitamin D deficiency in this experimental model, but showing that a relationship exists between vitamin D levels and type II muscle fibre area.
Interestingly, no relation was found between PTH and muscle area, a result in accordance with a study on post-menarcheal adolescent girls, in whom vitamin D was related with muscle power and force ( Ward et al., 2009), in contrast with the negative effect of PTH upon jump velocity. Our results are also according with previous, unpublished observations on more than 100 alcoholic patients in whom a significant relationship was observed between vitamin D levels and lean mass and handgrip strength, suggesting a role of vitamin D in alcoholic myopathy, in a similar way as in other situations of vitamin D deficiency (Russell, 1994; Ahmed et al., 2009; Albany and Servetnyk, 2009; Al-Said et al., 2009). In addition, vitamin D may yield antioxidant properties (Chatterjee, 2001). Interestingly, in our study there was a significant relationship between serum vitamin D levels and muscle GPX and SOD activities, well-known systems involved in the antioxidant defence. Also, a significant relationship was observed between antioxidant activity and muscle fibre area. All these data suggest that oxidative damage may be involved in alcoholic myopathy, in accordance with previous results, reported by other authors ( Fernández-Sola et al., 2002; Fujita et al., 2002) and by ourselves ( Durán Castellón et al., 2005).
Patients with PTH excess may also show muscle weakness and fatigue, and also atrophy of type II muscle fibres ( Patten et al., 1974). However, in our study, no relation was observed. Moreover, several researchers have obtained disparate results regarding PTH in alcoholics — low, normal or increased (Laitinen and Välimäki, 1991; Bikle et al., 1993; García-Valdecasas-Campelo et al., 2006; Santori et al., 2008) — so it does not seem that serum PTH may play a role on muscle myopathy, at least in our study. However, interestingly, serum magnesium, an element involved in muscle function, was related with type II muscle fibre area. It is important to consider that muscle magnesium is related to muscle strength ( Aagaard et al., 2002) and it is decreased in alcoholics ( Abbott et al., 1994; Aagaard et al., 2003), so our results are in a certain way, in accordance with these data.
Therefore, we conclude that there is a relation between low vitamin D levels and muscle fibre atrophy in a murine model of alcoholic myopathy, raising the possibility that low vitamin D may play a role in alcoholic myopathy. Further studies are needed to reject or confirm this hypothesis.
Conflict of interest statement. None declared.
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