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
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ABSTRACT |
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INTRODUCTION |
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MATERIALS AND METHODS |
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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 5060%. 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 ethanolglucose 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, 1984; Pierce and Perry, 1991
). 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, 1993; Nordsletten et al., 1994
).
Loaddeflection 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 loaddeflection 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, 1993).
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.
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RESULTS |
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The nocturnal activity of the animals was observed and recorded; no evident differences were found.
The total liquid consumption is also presented in Table 1. 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 1; 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 2. 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.
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DISCUSSION |
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Additionally, confounding variables, such as dietary deficiencies and liver damage, are known to interfere with bone metabolism. Thus, in previous studies (Lalor et al., 1986; Diamond et al., 1989
; Sampson et al., 1996
; Hogan et al., 1997
) 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 16) 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, 1965; Turner et al., 1987
, 1988
), and other studies have indicated that these defects might be related to load-bearing and/or activity (Preedy et al., 1991
). 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, 1993). In dissected bones, bending moment is considered the most appropriate measure of bone strength (Hayes, 1991
). 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., 1985
; Klein, 1997
; Nyquist et al., 1999
), 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., 1999
), 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 SpragueDawley 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.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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REFERENCES |
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