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Journal of Biomechanics 41 (2008) 20–24 www.elsevier.com/locate/jbiomech www.JBiomech.com
Acute and chronic dose of alcohol affect the load carrying capacity of long bone in rats D.V. Raia,, Gaurav Kumara, Priyamvada Tewaria, D.C. Saxenab a
Department of Biophysics, Basic Medical Sciences Block, Panjab University, Chandigarh 160014, India Department of Food Technology, Sant Longowal Institute of Engineering and Technology, Longowal 148106, Punjab, India
b
Accepted 2 August 2007
Abstract In the present investigation, the effects of acute and chronic dose of alcohol were evaluated on mechanical properties of long bones of Sprague Dawley rats. In ‘‘acute study’’, 18 animals were divided into three groups containing six animals each, i.e. Group A: control animals, normal saline was given to them intraperitoneally for the period of 5 days; Group B: treated animals, given 20% (v/v) absolute alcohol and Group C: treated animals, given 30% (v/v) absolute alcohol, by same route and time duration. In ‘‘chronic study’’, also, 18 animals were divided into three groups containing six animals each, i.e. Group A: control animals, normal saline was given to them intraperitoneally for the period of 6 weeks; Group B: treated animals, given 20% (v/v) absolute alcohol and Group C: treated animals, given 30% (v/v) absolute alcohol by same route and time duration. A significant increase was observed in bone weight of animals taking 20% alcohol but there was decrease in the same for 30% alcohol in case of acute study. For chronic study, there was a decrease in bone weight for both treated groups. During acute study, breaking strength of bone was increased in case of 20% alcohol administration but a slight decrease was shown in the same for 30% alcohol group as compared to control animals. Breaking strength of long bone in the case of chronic study was decreased in case of both groups taking alcohol, i.e. 20% and 30%. The present document is useful in understanding the functional load carrying capacity of bone during alcoholism. r 2007 Elsevier Ltd. All rights reserved. Keywords: Alcohol; Acute and chronic doses; Bone strength
1. Introduction Bone is a living tissue that undergoes continuous changes and replacements (i.e. remodeling) even after a person has attained full stature. Bone mineralization takes place in different ways. One of the stages is the creation of environment conductive to mineralization. There are changes in extracellular calcium and phosphate concentration and activities of phosphoproteins, glycoproteins and alkaline phosphatase. Mechanical strength of bone tissue depends mainly on the nanosize apatite crystals. It is generally considered that the mineral resists compression and the collagen fibers withstand torsion and tension. The orientation of these fibers seems to be load related it is considered as the most important determinant of Corresponding author. Tel.: +91 172 2534127.
E-mail addresses:
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[email protected] (D.V. Rai). 0021-9290/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jbiomech.2007.08.002
mechanical properties of bones and is considered the determining factor of its strength which depends on its mineral content (Singh and Rai, 1979; Singh et al., 1985). There are evidences that indicate that ethanol promotes osteoporosis through alteration of both the production and resorption arms of bone remodeling. Evidences also suggest that bone mineral density (BMD) is correlated with bone strength and fracture risk (Veenland et al., 1997). Alcohol is recognized to be an important risk factor for bone disorders. Overwhelming evidence from human and laboratory animal studies shows that chronic heavy drinking has detrimental effects on the skeleton. The effect of ethanol in BMD is poorly understood. Studies have shown that alcohol consumption modulates and interferes with bone metabolism. Alcohol abuse and its dependence are associated with lower BMD. However, there are scanty of reports suggesting that moderate alcohol consumption would be beneficial to bone structure (Diez et al., 1997).
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Clinical studies also demonstrate the association of chronic alcohol consumption and increased risk of fractures. These points, forever is not well understood in the case of acute treatment. Loss of bone mineral content is thus the major cause of increased susceptibility of bone to fractures, i.e. reduced bone strength caused in case of chronic alcoholism (Addolorato et al., 1997; Turner, 2000). Alcohol abuse and its serious medical and social consequences represent a major public health problem in many areas of the world. As per reports of World Health Organization (WHO Report, 2004) there are nearly 2 billion people worldwide who consume alcoholic beverages. It has also found that 76.3 million people suffer alcoholic disorders. This fact has stimulated the study of effects of alcohol in man and in animals. It seems that ethanol may be involved in several pathways directly or indirectly, through profound derangements in metabolic, hormonal and nutritional mechanisms (Nordmann et al., 1990). It also appears evident that these consequences and adaptive changes differ between acute and chronic alcohol administrations and in many cases are dose dependent. However, there is evidence for a positive effect of moderate alcohol consumption on bone. Several studies have also shown that chronic alcohol consumption influences the bone metabolism. Abuse of alcohol is known to derange the bone metabolism and to cause osteoporosis. The rate of bone loss varies from bone to bone, also with age, sex, race and nutritional conditions (Keiver and Weinberg, 2004; Wheeler and Lewis, 1977). Long-term alcohol consumption can interfere with bone growth and remodeling, resulting in decreased bone density and increased risk of fracture. Osteoporosis is a common disease characterized by a generalized reduction in BMD, microarchitectural deterioration of bone tissue (Morii and Genant, 1997; Sobti et al., 2006) resulting in the fragility and porosity of bone and its susceptibility to fractures (Kanis et al., 2004). These effects may be exerted directly or indirectly through the many cell types, hormones, and growth factors that regulate bone metabolism. In addition to providing structural support, bone is a major storage depot for calcium and other minerals. The small intestine absorbs calcium from ingested food, and the kidneys excrete excess calcium. An adequate concentration of calcium in the bloodstream is required for the proper functioning of nerves and muscle. The body monitors calcium concentration and responds through the action of hormones, vitamins, and local growth factors to regulate the distribution of calcium between blood and bone. Alcohol may disrupt this balance by affecting the hormones that regulate calcium metabolism as well as the hormones that influence calcium metabolism indirectly (e.g. steroid reproductive hormones and growth hormone) (Sampson, 1997). It was shown by many researchers that chronic and heavy alcohol consumption is known to contribute to low bone mass, decreased bone formation, an increased incidence of fractures, and delays in fracture healing. It was also evident in some studies that acute and moderate
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alcohol consumption strengthens the bone. However, conflicting results have been reported as well. This discrepancy may be explained in part by different study designs. The mechanisms through which ethanol promotes bone loss are currently not well understood. The relationship between the alcohol and strength of the bone has remained of interest in biomechanics. Our objective is to find out the relationship between alcoholism and bone strength. In this study we have observed the effects of acute and chronic doses of alcohol in different concentrations on long bones of rats. To monitor this, we have observed the bone weight (femur and tibia) of animals after sacrificing them. We have also observed the mechanical properties of bones by observing the breaking strength of bones. 2. Materials and methods 2.1. Chemicals used Absolute alcohol was purchased in the form of Dehydrated Alcohol B.P. from Bengal Chemicals and Pharmaceuticals Ltd., Kolkatta, India.
2.2. Animal model and experimental conditions Male Sprague–Dawley rats were procured from the Central Animal House of Punjab University, Chandigarh. The animals were housed in polypropylene cages bedded with sterilized rice husk. They were given free access to clean drinking water (tap water) and standard animal pellet diet (Ashirwad Industries, Kharar, Punjab, India), throughout the experiment. The temperature of the animal room was maintained at 2171 1C, humidity 50–60% and dark and light cycle as 12:12 fv. The experimental protocols were approved by the Institutional Ethics Committee and conducted according to Indian National Science Academy Guidelines for the use and care of experimental animals. The rats were acclimatized to experimental conditions for 1 week after which various treatments were employed.
2.3. Treatment of animals 2.3.1. Acute study Eighteen rats were taken and divided into three groups containing six animals each. In this first group, Group A (control animals), normal saline was administered to them intraperitoneally for a period of 5 days. In, Group B (treated animals) and Group C (treated animals), respectively, 20% ethanol and 30% ethanol were given by same route. The study was terminated upon 6 days of treatment. 2.3.2. Chronic study Eighteen rats were taken and divided into three groups containing six animals each. In Group A (control animals) normal saline was given. In Group B (treated animal) and Group C (treated animals) 20% ethanol and 30% ethanol were given respectively, intraperitoneally for the period of 6 weeks, respectively. The study was terminated upon 6 weeks of treatment. 2.3.3. Mechanical testing At the termination of both of the above-said studies, rats from all groups were killed and their long bones, i.e. femur and tibia were removed. The relative bone weight was calculated for each bone using the formula in relative bone weight ¼ bone weight 100/body weight. Bone marrow was flushed out with normal saline after giving a cut on diaphyseal ends of the bone. The bone samples were then kept in normal saline for the period of 24 h. After that the bone samples were placed in
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autoclave for 1 h. After this, breaking strength of all the samples was measured by using Texture Analyzer (Model TA-XT2i, Stable Micro Systems, UK). In this assembly, the bone is placed horizontally on the stationary probe (consisting of two plates which are separated by just a few centimeters). Another probe which is movable (shaped like a knife) moves down at a constant velocity and breaks the bone sample into two pieces. The instrument is attached to a computer system which records the breaking force. The force required to break the bone sample is expressed as breaking strength (Gupta et al., 2005).
300 Breaking strength (N)
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**
250
*
200 150
**
Femur Tibia
100 50
3. Results 0
3.1. Acute study The data shows that there is an increase in bone weight at low doses, i.e. 20% (v/v) absolute alcohol. But at high doses, i.e. for 30% (v/v) absolute alcohol, there is a decrease in bone weight (see Table 1). Similar trend is also observed for breaking strength. It is found to be higher in the case of low alcohol dose, i.e. for 20% (v/v) absolute alcohol. However, as the amount of alcohol is increased, i.e. for 30% (v/v) absolute alcohol, the breaking strength decreased (see Fig. 1).
Gp. A
Gp. B Groups
Gp. C
Data: Mean + − Standard Deviation (n=6) Statistically significant differences are represented by: (p < 0.01) - * and (p < 0.001) - ** - for femur. (p < 0.001) - ** - for tibia But for Group B there is not a statistically significant difference with (p < 0.118), this is may be due to random sample variability
Fig. 1. Breaking strength of femur and tibia in acute alcohol treatment.
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Here, bone weight is observed to decrease in both treated groups as compared to control animals (see Table 1). Same trend is also observed in breaking strength. Here, the breaking strength decreased in 20% (v/v) absolute alcohol group and 30% (v/v) absolute alcohol group, compared to control groups. It is observed that the breaking strength decreases as amount and duration of alcohol administration gets higher as shown in Fig. 2. 3.3. Discussion In the present study evidence is provided to show that alcoholism in rats induces changes in bone mechanical properties. In our study, a significant increase in breaking strength of bones of 20% alcohol dose is shown in the case
Table 1 Relative bone weight in acute and chronic alcohol treatment Groups
Femur (gms.)
Tibia (gms.)
Acute alcohol treatment Group A: control Group B: 20% alcohol Group C: 30% alcohol
0.8870.12 1.0570.23* 1.0270.14*
0.4870.14 0.7270.08* 0.6970.05*
Chronic alcohol treatment Group A: control Group B: 20% alcohol Group C: 30% alcohol
0.6870.03 0.2370.01* 0.1970.01*
0.35770.02 0.26370.01* 0.26270.01*
Note: Data: mean7S.D. (n ¼ 6). Statistically significant differences are represented by (po0.05)—* for femur and (po0.01)—* for tibia in acute alcohol treatment. (po0.001)— * for femur and tibia in chronic alcohol treatment.
Breaking Strength (N)
3.2. Chronic study
100 80 *
60
* *
40
*
Femur Tibia
20 0
Gp. A
Gp. B Gp. C Groups Data: Mean + Standard Deviation (n=6) Statistically significant differences are represented by: (p < 0.001) - * for femur and tibia.
Fig. 2. Breaking strength of femur and tibia in chronic alcohol treatment.
of acute study. However, there was decrease in the case of 30% alcohol. Same trend is also observed for bone weight. Further, in the case of acute doses, bone weight increases, but it decreases when the amount of alcohol gets higher. In the present work it is observed that as the amount of ethanol is increased it adversely affects the breaking strength of bones (Diez et al., 1997). It suggests that alcohol increases calcitonin secretion acutely. Calcitonin is an inhibitor of bone resorption and may be the mechanism by which moderate alcohol intake protects bone structure. This study is in tune with the studies that conclude that moderate alcohol intake can be beneficial to the bone structure (e.g. Rico, 1990). Ethanol impairs mainly osteoblastic activity and that results in reduced bone formation and mineralization
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(Medras and Jankowska, 2000). The results in the case of chronic alcoholism have shown that there is a considerable decrease in breaking strength of long bones. Here, bone weight is observed to decrease in both treated groups as compared to control animals. Same trend is also be observed in breaking strength. As the amount of alcohol is increased, the breaking force decreases progressively. This decrease can be attributed to chronic consumption of ethanol shown by Nyquist et al. (1999). This leads to lowering of bone mass thereby reducing the bone weight. It was also demonstrated that chronic ethanol feeding of rats causes deficiency in bone matrix synthesis and mineralization, resulting in inferior microarchitectural and mechanical properties of the bone (Baran et al., 1980; Turner and Burr, 1993; Sampson, 1997; Sampson et al., 1998). Our experimental results also showed the lowering of bone strength with increase in alcohol dose. This is in tune with the studies which conclude that high intake of alcohol confers a substantial risk for fractures in human beings (Kanis et al., 2004). The combined effect of alcohol on osteoblasts and the calcitropic hormones plus acidosis (due to alcohol) influence bone changes and increase the incidence of bone fractures. Excessive alcohol intake is a well recognized cause of secondary osteoporosis (Kogawa and Wada, 2005; Holbrook and Barrette, 1993). High doses of alcohol can raise parathyroid hormone (PTH) levels. PTH is an important regulator of body’s calcium and phosphorus levels. Their PTH levels can remain elevated. That puts a strain on the body’s calcium reserves. Since the bones are a major calcium reserve, alcohol can cause loss of calcium from the bones. It can also interfere with liver enzymes that are necessary for converting the inactive form of vitamin D into the active form. Without sufficient active vitamin D, body cannot absorb calcium from gastrointestinal tract. Cortisol, the stress hormone, reduces the work of osteoblasts and so less bone is formed. It also increases the work of osteoclasts and so more bone is resorbed (removed). This double action of lowered bone formation and greater bone resorption will reduce over all bone density (Suter, 2005; Elmali et al., 2002). So, alcohol has effect on cortisol for lowering bone strength. High dose of alcohol also inhibits bone formation, reduces bone mass and impairs fracture healing in experimental animals and disrupts cell signaling in cultured osteoblasts. Bone is a two-component composite material in which the mineral phase (mainly hydroxyapatite) confers the strength and stiffness, and the organic matrix (mainly Type I collagen) primarily influences the toughness of‘ bone (Zioupos and Currey, 1994). While mineral and collagen contribute to the bone’s competency, as do microarchitecture (e.g. porosity and trabecular connectivity), macrostructure (e.g. curvature of diaphysis and thickness of cortical shell), and in vivo microdamage (e.g. microcracks and diffuse cracks), their interaction with water is equally important to the mechanical behavior of bone. Thus, bone
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is also a fluid-imbibed material in which the distribution of water affects the mechanical properties of bone (Rai and Singh, 2000; Rai et al., 1986). Numerous studies have shown that alcohol consumption interferes with bone metabolism (Diez et al., 1994). Abuse of alcohol is known to derange bone metabolism and cause osteoporosis. Ethanol mainly impairs osteoblastic activity that results in reduced bone formation and mineralization. Loss of mineral content is thus the major cause of increased susceptibility of bone fractures, i.e. reduced bone strength caused in the case of chronic alcoholism. Chronic ethanol feeding of rats causes deficiency in bone matrix and mineralization (Sampson et al., 1998). Mineral deficit in bone as in the case of alcoholism and pathological conditions like osteomalacia have similarity in that as both there is an increase in water content. Bone water is a significant contributor to explain mechanical properties of bone. Bone’s mechanical properties are associated with the degree of mineralization and thus with water content (Timmins and Wall, 1977). The present study is carried out to monitor the effect of acute and chronic alcohol doses on the mechanical properties of long bones. Our primary purpose is to evaluate the modulation in bone strength caused by alcohol consumption. Alcohol abuse leads to deranged bone metabolism and mineralization leading to a change in water content which affects the mechanical properties of bones. 4. Conclusion As a result of acute doses of alcohol bone strength may increase. But as amount and time duration of alcohol administration gets higher, bone strength may decrease. We hope that the present data are helpful in understanding the bone strength in relation to alcohol ingestion during alcoholism. Conflict of interest statement
As per reports of WHO there are nearly 2 billion people worldwide who consume alcoholic beverages. It has also found that 76.3 million people are under the influence of alcoholic disorders. Alcohol is recognized to be an important risk factor for bone disorders. The effect of ethanol in BMD is poorly understood. This is the primary reason which has sloped over mind to this area. Interaction mechanism of alcohol in demineralization of bone is not A well-known established pathway. The loss of mineral from the bone finally influenceS the composition are further how this relates to the mechanical strength is not well documented. In the past studies, various scientists have worked on same area but this study includes the detection of mechanical strength of bone. All authors have perfectly reviewed the manuscript.
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The work done was purely novel and the data are original.
References Addolorato, G., Capristo, E., Greco, A.V., Stefanini, G.F., Gasbarrini, G., 1997. Energy expenditure, substrate oxidation and body compositions in the subjects with chronic alcoholism: new findings from metabolic assessment. Alcoholism: Clinical and Experimental Research 21, 962–967. Baran, D.T., Teitelbaum, S.L., Bergfeld, M.A., Parker, G., Cruvant, E.M., Avioli, L.V., 1980. Effect of alcohol ingestion on bone and mineral metabolism in rats. American Journal of Physiology 238, E507–E510. Diez, A., Puig, J., Serrano, S., Marinoso, L.L., Bosch, J., Marrugat, J., Mellibovsky, L., Nogues, X., Knobel, H., Aubia, J., 1994. Alcohol induced bone disease in the absence of severe chronic liver damage. Journal of Bone and Mineral Research 9, 825–831. Diez, A., Serrano, S., Cucurull, J., Marinoso, L.L., Bosch, J., Puig, J., Nogues, X., Aubia, J., 1997. Acute effects of ethanol on mineral metabolism and trabecular bone in Sprague Dawley rats. Calcified Tissue International 61, 168–171. Elmali, N., Ertern, K., Ozen, S., Inan, M., Baysal, T., Guner, G., Bora, A., 2002. Fracture healing and bone mass in rats fed on liquid diet containing ethanol. Alcoholism: Clinical and Experimental Research 26, 509–513. Gupta, R., Koul, A., Rai, D.V., 2005. Effect of Alcohol on Mechanical Properties of Bone, Biomechanics. Anamaya Publisher, New Delhi, India, pp. 228–233. Holbrook, T.L., Barrette, C.E., 1993. A prospective study of alcohol consumption and bone mineral density. British Medical Journal 306, 1506–1509. Kanis, J.A., Johnell, O., Oden, A., De Laet, C., Mellstrom, D., 2004. Epidemology of osteoporosis and fracture in men. Calcified Tissue International 75, 90–99. Keiver, K., Weinberg, J., 2004. Effect of duration of alcohol consumption on calcium metabolism and bone in fetal rats. Alcoholism: Clinical and Experimental Research 28, 456–467. Kogawa, M., Wada, S., 2005. Osteoporosis and alcohol intake. Clinical Calcium 15 (1), 102–105. Medras, M., Jankowska, E.A., 2000. The effect of alcohol on bone mineral density in men. Prezegl Lek 57 (12), 743–746. Morii, H., Genant, H.K., 1997. Statement on the diagnosis and management of osteoporosis from the Consensus Development Conference at the Second International conference on osteoporosis, Osaka. Bone and Mineral Metabolism 16, 206–214. Nordmann, R., Riviere, C., Rouach, H., 1990. Ethanol induced lipid peroxidation and oxidative stress in extrahepatic tissue. Alcoholism: Clinical and Experimental Research 25, 163–168.
Nyquist, F., Karlsson, M.K., Obrant, K.J., Nilsson, J.A., 1999. Osteopenia in alcoholics after tibia shaft fractures. Alcohol and Alcoholism 32, 599–604. Rai, D.V., Singh, K.V., 2000. Effect of mineral loss on the elemental composition and thermostability of bone collagen. Journal of Punjab Academic Science 2 (1), 15–18. Rai, D.V., Behari, J., Saha, S., 1986. The effect of mineral deficient diet on the structural and mechanical properties of long bones. In: Saha, S. (Ed.), Biomedical Engineering V Recent Development. Pergmon Press, New York, pp. 456–460. Rico, H., 1990. Alcohol and bone disease. Alcohol and Alcoholism 25, 345–352. Sobti, R.C., Gathwan, K.H., Mittal, P.K., Sharma, V.L., Rai, D.V., 2006. Histological, ultrastructural and infrared spectroscopic studies on bone of mice intoxicated with nickel salts. Journal of Cytology and Genetics 7, 109–119. Sampson, H.W., 1997. Alcohol, osteoporosis and bone regulating hormones. Alcoholism: Clinical and Experimental Research 21 (3), 400–403. Sampson, H.W., Chaffin, C., Lange, J., Defee, B., 1998. Alcohol consumption by young actively growing rats: a histomorphometric study of cancellous bone. Alcoholism: Clinical and Experimental Research 21 (2), 352–359. Suter, P.M., 2005. Is alcohol consumption a risk factor for weight gain and obesity? Critical Review on Clinical and Laboratory Science 42 (3), 197–227. Singh, S., Rai, D.V., 1979. Physical characteristics of piezoelectric materials of Biological origin. Journal of Pure and Applied Ultrasonics 1 (4), 94–97. Singh, S., Behari, J., Rai, D.V., 1985. Elastic constants relating to two phase bone system. Journal of Pure and Applied Ultrasonics 7 (1), 6–9. Timmins, P.A., Wall, J.C., 1977. Bone water. Calcified Tissue Research 23 (1), 1–5. Turner, R.T., 2000. Skeletal response to alcohol. Alcoholism: Clinical and Experimental Research 24 (11), 1693–1701. Turner, C.H., Burr, D.B., 1993. Basic biomedical measurements of bone: a tutorial. Bone 14, 595–608. Veenland, J.F., Link, I.M., Konermenn, W., Meier, N., Grashuis, J.L., Gelseme, E.S., 1997. Unraveling the role of structure and density in determining vertebral bone strength. Calcified Tissue International 61, 474–479. WHO Global Status Report on Alcohol, 2004. World Health Organization Department of Mental Health and Substance Abuse Geneva 2004, 1–94. Wheeler, E.J., Lewis, D., 1977. An X-ray study of paracrystalline nature of bone apatite. Calcified Tissue International 24, 243–248. Zioupos, P., Currey, J.D., 1994. The extent of micro cracking and the morphology of micro cracks in damaged bone. Journal of Material Science 29 (4), 978–986.