EFFECTS OF ALCOHOL ON BONE MINERAL AND MECHANICAL PROPERTIES OF BONE IN MALE RATS

Fredrik Nyquist1,*, Henrik Düppe1, Karl J. Obrant1, Lennart Bondeson2 and Lars Nordsletten3

1 Departments of Orthopaedics and
2 Pathology, Malmö University Hospital, SE-205 02 Malmö, Sweden and
3 Surgical Research, Rikshospitalet, The National Hospital, University of Oslo, Norway

Received 16 February 2001; in revised form 20 June 2001; accepted 30 July 2001


    ABSTRACT
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
— The effect of ethanol on bone mineral is poorly understood. In this study we have investigated whether ethanol affects bone mineral content (BMC), bone mineral density (BMD) measured by dual energy X-ray absorptiometry, and the mechanical properties of the tibia and femora in male Sprague–Dawley rats without histopathological signs of liver disease or nutritional deficit. Thirty-five male rats were fed a liquid diet containing 15% ethanol and glucose. An equivalent iso-volumetric amount of glucose-containing liquid was fed to the controls (n = 35). After an initial difference in weight, we found no difference in weight gain from week 1 to week 6. All animals were killed at 6 weeks. We found no evidence of ethanol-induced liver disease in a histopathological evaluation. The BMD and BMC were found to be lower in the ethanol group. No differences between the groups were found in the mechanical properties or in the length and size of the femora. We suggest that alcohol may have a toxic effect on bone in male rats known not to suffer from any histopathological hepatic lesions.


    INTRODUCTION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The cause of osteoporosis is a multifactorial entity in which alcohol consumption is known to play a part (Rico, 1990Go; Moniz, 1994Go). The concept of how alcohol affects bone metabolism is still not fully elucidated. Earlier studies have shown that, in animals, a chronic alcohol intake can cause defects in mineralization (Saville, 1965Go; Turner et al., 1987Go, 1988Go). In rats treated with alcohol, Peng et al. (1982, 1988, 1991) found a decreased bone strength. Kusy et al. (1989) also found that the femora of rats fed an alcohol liquid for 4 weeks not only was weaker, but also could absorb less energy. In the above-mentioned studies, and in other published rat model studies, the histopathological absence of liver disease has not fully been evaluated, and thus the direct effect of alcohol on bone mineral and bone strength could have been overestimated. The purpose of the present study was therefore to assess, in an in vivo model, whether ethanol has any short-term effects on bone mineral, measured by dual energy X-ray absorptiometry (DEXA), or the mechanical properties of bone in male rats.


    MATERIALS AND METHODS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Animals
Seventy, 70-day-old, male Sprague–Dawley rats (Møllegard, Copenhagen, Denmark) with a mean weight of 389 g (range 355–425) were randomly divided into two groups. One group (n = 35) were fed ad libitum a liquid containing ethanol and glucose and the other group (n = 35) were fed a liquid containing only glucose. Both groups had free access to laboratory pellets (R3; Lactamin, Vadstena, Sweden) (1.1% calcium, 0.8% phosphorus; 315 kcal/100 g, each pellet 3 g). The amount of consumed pellets was determined and recorded each day.

The weight of each animal was recorded at the start, after 8 days and at the end of the experiment. The animals were housed alone in wire-topped metal cages (420 x 260 mm wide, 150 mm high) in a room with a 12-h light/12-h dark cycle, with a relative humidity of 50–60%. The total study time was 6 weeks. The animals' nocturnal activity was observed and recorded by the laboratory technicians. The daily amount of liquid consumed by each animal was recorded. The experiments conformed to the Swedish Council of Animal Research code for the care and use of animals for experimental purposes.

Ethanol administration
Initially the group of animals being given ethanol was fed an ethanol–glucose liquid of 5% (v/v) ethanol and 260 g glucose in 890 ml water. The amount of ethanol was successively increased to 10% and finally to 15% on day 8. The same amount of alcohol (%) has previously been used in several studies (Jänicke-Lorenz and Lorenz, 1984Go; Pierce and Perry, 1991Go). The serum ethanol level was measured once a week throughout the study period. A liquid containing an equivalent iso-volumetric amount of glucose was fed to the controls. The volume of the liquid as well as the amount of ethanol (g) consumed by each animal each day were recorded.

Bone measurements
At our bone metabolic research laboratory the total bone mineral area density (BMD), expressed in g/cm2, total bone content (BMC) expressed as gram (g) and the total bone calcium (g) of each animal were measured by the dual-energy X-ray absorptiometry technique after the animals were killed, using the Lunar® DPX small animal software, version 1.0 D. The total body content was measured. The precision was calculated to <2%. The measurements were also corrected for weight differences that existed between the groups.

Liver histopathology
The animals were killed after 6 weeks and livers from all animals were then carefully excised, after the DEXA measurement, and their weights recorded. The liver was frozen, sectioned and stained with Oil Red. A histopathological examination was then performed at the Department of Pathology, Malmö University Hospital, Malmö, Sweden. The investigator looked for an increase in the amount of fat, increased inflammatory activity and fibrosis. This was a ‘blind’ study, as the investigator did not know which group of animals the liver had been taken from.

Mechanical testing
After the DEXA measurement, the right femora and tibia were excised and the size and length of the femora were measured, from the trochanteric region to the lateral femoral condyle, with a micrometer. This was done to detect any differences in bone growth during the study period. The tibia and femora were then stored at –20°C until mechanical testing. Before the mechanical testing, the bones were thawed and cleared of soft tissues.

The femoral shaft and the neck were fractured in a hydraulic testing device using a loading rate of 0.095 radians/s. First, the shaft was fractured 19 mm above the knee joint in a three-point ventral bending test, the fulcrum being the centre of rotation in the test system. Thereafter the necks were fractured in a combined bending and compression test. The tibia was then tested in three-point ventral bending (Nordsletten and Ekeland, 1993Go; Nordsletten et al., 1994Go).

Load–deflection curves were recorded on line in Work Bench Mac Software (Strawberry Tree Incorporated, Sunnyvale, CA, USA). Ultimate bending moment, energy absorption, stiffness and deflection at fracture were read out directly or calculated from the computer readings. The ultimate bending moment was taken as the product of the ultimate load and the moment-arm. Energy absorption was the area under the load– deflection curve. Bending stiffness was defined as the slope of the linear elastic part of the curve, and was read directly from the computer. Deflection was the distance on the x-axis from the point of intersection of the linear portion of the load–deflection curve to the point of failure. The term strength in relation to the results was defined according to Burstein et al. (1971). The coefficient of variation (CV) of the apparatus, for testing steel rods to 45° deflection, is ~1%. The precision is therefore high but the CV in the present study (resected rat bones) was estimated to be 15%. This is mainly due to the biological variation in the bone (Nordsletten and Ekeland, 1993Go).

Statistics
All data are presented as means ± SD. Statistical analyses were done using the Macintosh Statistica software. The data followed a normal distribution and the unpaired Student's t-test and multiple analysis of covariance (MANCOVA) were used for detecting between-group differences. A significance level of P < 0.05 was adopted.


    RESULTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The results in Table 1Go show that, in spite of the randomization into two groups, there was a difference in initial weight between the groups. After an initial difference in weight gain over the first week, there was no significant difference in weight gain after the 6 week experimental period. No significant differences in the amount of pellets consumed were noted between the groups (average daily consumption of 20 g pellets/day in each group).


View this table:
[in this window]
[in a new window]
 
Table 1. Body weight gain, liquid consumption, liver weight and the length of the femora in male rats fed ethanol and their controls during a 6-week study
 
In the ethanol group, the mean total consumption of ethanol was 137 g (124–148), corresponding to 3.4 ± 0.2 g/day (3.1–3.7). The serum alcohol level was in all animals above 0.1% (100 mg/dl) during the whole experiment.

The nocturnal activity of the animals was observed and recorded; no evident differences were found.

The total liquid consumption is also presented in Table 1Go. The total and daily consumption of liquid was significantly less in the group on the ethanol diet (P < 0.001). Although there were no differences in the average daily energy consumption between the two groups (ethanol-fed rats 51 kcal/day: 23 kcal/day from ethanol and 28 kcal/day from glucose-water, i.e. versus glucose-fed rats 46 kcal/day).

The liver weights are presented in Table 1Go; no differences were found between the groups. We were unable to detect any signs of ethanol-induced liver disease in the histopathological evaluation. There were also no differences in the length of the femora between the two groups.

The BMD, BMC and total calcium content are presented in Table 2Go. We found a significantly lower BMD, and BMC and calcium content among the rats fed the ethanol diet (P < 0.001) both before and after correction for weight.


View this table:
[in this window]
[in a new window]
 
Table 2. Total body bone mineral density (BMD), total body mineral content (BMC) and total body calcium in male rats fed an ethanol-containing diet for 6 weeks and their controls
 
The mechanical properties are presented in Table 3Go. No differences were found between the two groups when bending moment, energy absorption, stiffness and deflection were calculated.


View this table:
[in this window]
[in a new window]
 
Table 3. Mechanical results at failure of the right tibia, right femoral shaft and neck in male rats fed an ethanol-containing diet for 6 weeks and their controls
 

    DISCUSSION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The dose-dependency of alcohol-induced derangements of bone and mineral metabolism is unknown. In male chronic alcoholics, a daily consumption of ethanol exceeding 120 g/day (corresponding to 1.7 g/kg/day) has been reported to have a negative impact on bone mineral density and biochemical markers of bone metabolism (Saville, 1965Go; Laitinen and Välimäki, 1991Go; Nyquist et al., 1996Go). It has also been shown by Laitinen et al. (1991) that prolonged moderate ethanol intake of 60 g/day impairs osteoblastic function, leading to lowered serum levels of osteocalcin. It is not always possible to extrapolate results from human studies to animals: in this study among male rats, the daily consumption of ethanol was 3.4 ± 0.2 g/day (corresponding to 7.9 g/kg/day). This amount of ethanol exceeds by far the ingested amount of ethanol consumed by the alcoholics in the studies mentioned above and is at the same level as previous animal studies (Jänicke-Lorenz and Lorenz, 1984Go; Pierce and Perry, 1991Go). The aim of the present study was to investigate if ethanol would have any effects on BMD and bone strength. We were interested in a possible direct effect on bone tissue of high intake of ethanol for a short period of time, which is why we chose a 15% ethanol concentration in a liquid diet.

Additionally, confounding variables, such as dietary deficiencies and liver damage, are known to interfere with bone metabolism. Thus, in previous studies (Lalor et al., 1986Go; Diamond et al., 1989Go; Sampson et al., 1996Go; Hogan et al., 1997Go) the impact of alcohol itself may have been overestimated. In this study, we looked at the short-term effects of high ethanol doses over a time span which is sub-optimal for liver disease development. To detect any liver derangements, a histopathological examination was performed. This is the first time such an investigation has been done in rat models studying the adverse effects of ethanol on bone. The dietary differences are of major importance and in this study we found no significant differences in the amount of pellets consumed. The similar weight increase (weeks 1–6) and the similar bone growth (femoral length) indicate no signs of malnutrition. Thus, we believe that even malnutrition and caloric deprivation, as confounding effects on bone mass, can be ruled out.

The animals fed a liquid containing ethanol/glucose had a significantly lower day-to-day consumption of liquid. The reason for this change in behaviour could be multifactorial — the increase in caloric intake among animals receiving ethanol/ glucose, the central nervous system effects of ethanol and a certain dislike of the taste of ethanol among animals could be part of such an explanation. However, these differences in liquid consumption did not have any effects on weight increase or the length of the femora.

Earlier studies have shown that, in animals, chronic ethanol intake can cause defects in mineralization (Saville and Lieber, 1965Go; Turner et al., 1987Go, 1988Go), and other studies have indicated that these defects might be related to load-bearing and/or activity (Preedy et al., 1991Go). Since in the present study with the given amount of ethanol there was no evidence of difference in the nocturnal activity of the animals, we also believe that such a confounder can be ruled out, and that ethanol thus has an effect on the BMD and BMC.

In the present study, we found no significant difference in the results of the biomechanical tests. In vivo, it seems that energy absorption capacity would be the most relevant measure for resistance to fracture in the dynamic activities of everyday life (Nordsletten and Ekeland, 1993Go). In dissected bones, bending moment is considered the most appropriate measure of bone strength (Hayes, 1991Go). Neither of these entities showed any alteration in this study. There was a slight, but not significant, decrease in bending stiffness among rats fed an ethanol liquid. Our findings in this study are consistent with the consensus in the literature on ethanol-induced osteopenia (Bikle et al., 1985Go; Klein, 1997Go; Nyquist et al., 1999Go), but it appears that inhibition of new bone formation during bone remodelling was less affected than the inhibition that occurs during bone healing (Nyquist et al., 1999Go), and therefore the mechanical properties of bone were accordingly less affected. Another explanation could be that bone metabolism in Sprague– Dawley male rats is more resistant to ethanol than earlier suggested by Hogan et al. (1997) and Sampson et al. (1997) in studies performed on female Sprague–Dawley rats. To elucidate this, further studies are needed.

The ideal situation would have been to have a pretreatment bone mass value; this would have even further strengthened our suggestion that ethanol has a toxic effect on bone. However, in this study, we were unable to measure the pretreatment bone mass value, since DEXA measurements, using the Lunar® DPX small animal software, is a time-consuming procedure (>60 min) and therefore the animals have to be anaesthetized during the whole measurement. To anaesthetize the animals for such a long period of time would certainly have caused some death in both groups and there would also have been a potential risk for hepatic lesions.

The influence of ethanol on bone was, presently, studied with DEXA measurement and biomechanical testing. In the DEXA measurements, we found a decrease in BMD and BMC, but were unable to detect any negative impact of ethanol on the biomechanical characteristics of male rat femora and tibiae. The DEXA technique is a more sensitive measure, with a lower degree of variation, so that small changes in bone quality are easier to detect with such a technique. Perhaps the differences in the mechanical properties of bone would have been more evident in a study performed over a longer period of time.

In the present study, we found ethanol to have a negative impact on bone mineral content, but the mechanical properties were not influenced. Although it is not always possible to extrapolate data from animal studies, our data could possibly suggest that the direct toxic effect of ethanol accounts to some extent for the well-known increased risk of fractures in alcoholics. Other side-effects of ethanol, such as repetitive trauma associated with drunkenness, malnutrition and liver disease may be the dominant cause for the high fracture incidence among abusers.


    ACKNOWLEDGEMENTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Financial support was obtained from the Swedish Medical Research Council, the Lund University Research Funds, the Swedish Council for Planning and Coordination of Research, and the Greta and Johan Kock Foundation.


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


    REFERENCES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Bikle, D. D., Genant, H. K., Cann, C., Recker, R. R., Halloran, B. P. and Strewler, G. J. (1985) Bone disease in alcohol abuse. Annals of Internal Medicine 103, 42–48.[ISI][Medline]

Burstein, A. H., Currey, J. D., Frankel, V. H. and Reilly, D. T. (1971) The ultimate properties of bone tissue: the effects of yielding. Journal of Biomechanics 4, 155–158.[ISI][Medline]

Diamond, T., Stiel, D., Lunzer, M., Wilkinson, M. and Posen, S. (1989) Ethanol reduces bone formation and may cause osteoporosis. American Journal of Medicine 86, 282–288.[ISI][Medline]

Hayes, W. C. (1991) Biomechanics of cortical and trabecular bone: implications for assessment of fracture risk. In Basic Orthopaedic Biomechanics, Mow, M. V. and Hayes, W. C. eds, pp. 102–110. Raven Press, New York.

Hogan, H. A., Sampson, H. W., Cashier, E. and Ledoux, N. (1997) Alcohol consumption by young actively growing rats: a study of cortical bone histomorphometry and mechanical properties. Alcoholism: Clinical and Experimental Research 21, 809–816.[ISI][Medline]

Jänicke-Lorenz, J. and Lorenz, R. (1984) Alcoholism and fracture healing — a radiological study in rat. Archives of Orthopaedic and Trauma Surgery 103, 286–289.[ISI][Medline]

Klein, R. F. (1997) Alcohol-induced bone disease: impact of ethanol on osteoblast proliferation. Alcoholism: Clinical and Experimental Research 21, 392–399.[ISI][Medline]

Kusy, R., Hirsch, P. F. and Peng, T. C. (1989) Influence of ethanol on stiffness, toughness and ductility of femurs of rats. Alcoholism: Clinical and Experimental Research 13, 185–189.[ISI][Medline]

daddyLaitinen, K. and Välimäki, M. (1991) Alcohol and bone. Calcified Tissue International 49, (Suppl.), S70–S73.

Laitinen, K., Lamberg-Allardt, C., Tunninen, R., Karonen, S. L., Ylikahri, R. and Välimäki, M. (1991) Effects of 3 weeks' moderate alcohol intake on bone and mineral metabolism in normal men. Bone and Mineral 13, 139–151.[ISI][Medline]

Lalor, B. C., France, M. W., Powell, D., Adams, P. H. and Counihan, T. B. (1986) Bone and mineral metabolism in chronic alcohol abuse. Quarterly Journal of Medicine, New Series 59, 497–511.

Moniz, C. (1994) Alcohol and bone. British Medical Journal 50, 67–75.

Nordsletten, L. and Ekeland, A. (1993) Muscle contraction increases the structural capacity of the lower leg: an in vivo study in the rat. Journal of Orthopaedic Research 11, 299–304.[ISI][Medline]

Nordsletten, L., Madsen, J. E., Almaas, R., Rootwelt, T., Halse, J., Konttinen, Y. T., Hukkanen, M. and Santavirta, S. (1994) The neuronal regulation of fracture healing — effects of sciatic nerve resection in rat tibia. Acta Orthopaedica Scandinavica 65, 299–304.[ISI][Medline]

Nyquist, F., Ljunghall, S., Berglund, M. and Obrant, K. J. (1996) Biochemical markers of bone metabolism after short and long term ethanol withdrawal in alcoholics. Bone 19, 51–54.[ISI][Medline]

Nyquist, F., Halvorsen, V., Madsen, J. E., Nordsletten, L. and Obrant, K. J. (1999) Ethanol and its effects on fracture healing and bone mass in male rats. Acta Orthopaedica Scandinavica 70, 212–216.[ISI][Medline]

Peng, T. C., Barner, S. C., Frye, G. D. and Crenshaw, M. A. (1982) Evidence of a toxic effect of ethanol on bone in rats. Alcoholism: Clinical and Experimental Research 6, 96–99.[ISI][Medline]

Peng, T. C., Kusy, R., Hirsch, P. F. and Hagaman, J. R. (1988) Ethanol-induced changes in morphology and strength of femurs of rats. Alcoholism: Clinical and Experimental Research 12, 655–659.[ISI][Medline]

Peng, T. C., Lian, J. B., Hirsch, P. F. and Kusy, R. P. (1991) Lower serum osteocalcin in ethanol-fed rats. Journal of Bone and Mineral Research 6, 107–115.[ISI][Medline]

Pierce, R. O. and Perry, A. (1991) The effect of ethanol on bone mineral. Journal of the National Medical Association 83, 505–508.[ISI][Medline]

Preedy, V. R., Baldwin, D. R., Keating, J. W. and Salisbury, J. R. (1991) Bone collagen, mineral and trace element composition, histomorphometry and urinary hydroxyproline excretion in chronically-treated alcohol-fed animals. Alcohol and Alcoholism 26, 39–46.[ISI][Medline]

Rico, H. (1990) Alcohol and bone disease. Alcohol and Alcoholism 25, 345–352.[ISI][Medline]

Sampson, H. W., Perks, N., Champney, T. H. and DeFee, B. (1996) Alcohol consumption inhibits bone growth and development in young actively growing rats. Alcoholism: Clinical and Experimental Research 20, 1375–1384.[ISI][Medline]

Sampson, H. W., Chaffin, C., Lange, J. and DeFee, II B. (1997) Alcohol consumption by young actively growing rats: a histomorphometric study of cancellous bone. Alcoholism: Clinical and Experimental Research 21, 352–359.[ISI][Medline]

Saville, P. D. (1965) Changes in bone mass with age and alcoholism. Journal of Bone and Joint Surgery 47A, 492–499.

Saville, P. D. and Lieber, C. S. (1965) Effect of alcohol on growth, bone density and muscle magnesium in the rat. Journal of Nutrition 87, 477–484.[ISI][Medline]

Turner, R. T., Greene, V. S. and Bell, N. H. (1987) Demonstration that ethanol inhibits bone matrix synthesis and mineralisation in the rat. Journal of Bone and Mineral Research 2, 61–66.[ISI][Medline]

Turner, R. T., Aloia, R. C., Segal, L. D., Hannon, K. S. and Bell, N. H. (1988) Chronic alcohol treatment results in disturbed vitamin D metabolism and skeletal abnormalities in animals. Alcoholism: Clinical and Experimental Research 12, 159–162.[ISI][Medline]





This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Request Permissions
Google Scholar
Articles by Nyquist, F.
Articles by Nordsletten, L.
PubMed
PubMed Citation
Articles by Nyquist, F.
Articles by Nordsletten, L.