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Journal of the American College of Nutrition, Vol. 24, No. 3, 177-181 (2005)
Published by the American College of Nutrition

Association between Dietary Conjugated Linoleic Acid and Bone Mineral Density in Postmenopausal Women

Rhonda A. Brownbill, PhD, RD, Mary Petrosian, BS and Jasminka Z. Ilich, PhD, RD, FACN

University of Connecticut School of Allied Health, Storrs, Connecticut

Address reprint requests to: Jasminka Z. Ilich, Ph.D., University of Connecticut, Division of Health and Human Development, School of Allied Health, 358 Mansfield Rd., U-101, Storrs, CT 06269. E-mail: Jasminka.Ilich{at}uconn.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 CONCLUSION
 ACKNOWLEDGMENTS
 REFERENCES
 
Objective: To determine if dietary conjugated linoleic acid (CLA) is associated with bone mineral density (BMD) of different skeletal sites in postmenopausal women.

Methods: A cross-sectional analysis in 136 Caucasian, healthy, postmenopausal women, mean age 68.6 years. BMD and soft tissue were assessed by dual energy x-ray abosorptiometry (DXA). Energy, calcium, protein, fat, CLA and other relevant nutrients were estimated using 3 day dietary records. Supplement use was recorded as well. Current and past physical activity were determined using the Allied Dunbar National Fitness Survey for older adults.

Results: CLA (63.1 ± 46.8 mg, mean ± SD) was a significant predictor of Ward’s triangle BMD (p = 0.040) in a multiple regression model containing years since menopause (18.5 ± 8.4 y), lean tissue, energy intake (1691 ± 382 kcal/day) dietary calcium (873 ± 365 mg), protein (70.6 ± 18.6 g), fat (57.9 ± 23.9 g), zinc (19.2 ± 13.6 mg), and current and past physical activity, with R2adj = 0.286. Subjects were also divided into groups below (Group 1) and above (Group 2) the median intake for CLA. Group 2 had higher BMD in the forearm, p = 0.042, and higher BMD in the hip, lumbar spine and whole body, however statistical significance was not reached.

Conclusion: These findings indicate dietary CLA may positively benefit BMD in postmenopausal women. More studies are warranted examining the relationship between dietary CLA and BMD.

Key words: conjugated linoleic acid, hip bone mineral density, lumbar spine bone mineral density, postmenopausal women


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 CONCLUSION
 ACKNOWLEDGMENTS
 REFERENCES
 
Conjugated linoleic acid (CLA) refers to a group of positional and geometric isomers of linoleic acid. The most abundant geometric isomer in food is the c9t11-CLA isomer, referred to as rumenic acid [1,2]. CLA occurs naturally in many foods, though the primary sources are foods from ruminant animals such as beef, lamb and dairy products [2]. CLA in food can vary depending on the animal’s diet, the breed and maturity of the animal, but generally the levels range from 3–7 mg/g fat [2]. In the past two decades CLA has been attributed to have many potential health benefits including acting as an antiadipogenic, antidiabetogenic, anticarcinogenic and an antiatherosclerotic agent [1,3,4]. Recently some other characteristics were attributed to CLA such as enhancing immunity and bone formation [1]. The majority of studies showing these effects were conducted in animals using synthetic forms of CLA which are not identical to isomers found naturally in foods. Generally the amounts given to observe all the above benefits were between 0.1%–1% of the total weight of the diet [1,3]. An average woman is estimated to eat about 2 1/2 lbs of food a day [5], therefore 0.1–1% of her diet would amount to about 1.14–11.4 g of CLA a day. Given that dietary CLA intakes in men and women are reported to not exceed 500 mg a day [6], theoretically the health benefits attributed to CLA would not likely be observed by diet alone, but would require supplementation.

Among all potential health benefits of CLA, its effect on bone is probably the least studied. The few studies that have investigated the effects of CLA on bone metabolism were conducted in animals and/or were of short duration. Current evidence in animals suggests CLA may help decrease bone loss through its ability to lower levels of prostaglandins (PG) in bone tissue [7,8] or by enhancing calcium absorption [9]. Prostaglandin E2 (PGE2), the primary PG affecting bone metabolism, is synthesized from arachidonic acid (an n-6 fatty acid) through the action of the enzyme, cyclooxygenase. PGE2 can stimulate and inhibit bone formation and resorption depending on its concentration in bone, as well as facilitate bone formation through its action on insulin like growth factor (IGF) [10]. Excessive production of PGE2 can lead to decreased bone formation, and therefore, decreased bone mass [8]. It is suggested CLA can inhibit PGE2 production by moderating cyclooxygenase activity or its expression [8].

It is not known whether CLA from diet and/or supplements can actually reduce bone loss since there are no longitudinal studies assessing its effects on bone mass or fracture risk. Evidence about the relationship between dietary or supplemental CLA and BMD is murky, in both humans and animals. To date, researchers have only attempted to define the mechanism of how CLA may affect calcium and bone metabolism using animal models, but the mechanism is still not clear. To the best of our knowledge, there are no studies evaluating the relationship between dietary CLA and bone mass in humans. Therefore, we investigated whether dietary CLA is related to BMD in various skeletal sites in postmenopausal women.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 CONCLUSION
 ACKNOWLEDGMENTS
 REFERENCES
 
Subjects
This cross-sectional study consisted of 136 community dwelling, non-smoking, Caucasian postmenopausal women, age (57.4–88.6 y) free of chronic diseases (including severe osteoporosis defined as a t-score below –3.5 for the hip and spine) and medications (including hormone replacement therapy) known to affect bone. Data were collected during one visit. The recruitment and detailed description of subjects were reported previously [11]. The Institutional Human Subjects Review Board approved study protocol and subjects signed informed consent.

Anthropometry and Bone Densitometry
Weight and height were measured by standard procedures in indoor clothes without shoes, and were used to calculate body mass index (BMI kg/m2). BMD (g/cm2) was measured by dual X-ray absorptiometry with a Lunar DPX-MD instrument (GE Medical Systems, Madison, WI, USA) using specialized software for whole body, lumbar spine, femur (neck, trochanter, Ward’s triangle, shaft, and total) and forearm (including radius and ulna), as described previously [12]. The whole body scan was used to determine total lean tissue. Quality assurance was performed daily and coefficients of variation using our densitometer were reported previously [12].

Dietary Assessment
Subjects were instructed by a registered dietitian to record 3 days of dietary intake, including 2 weekdays and one weekend day, as reported previously [11]. Subjects were interviewed to clarify portion sizes, food brands and preparation. Mineral and vitamin supplements were also recorded. Dietary records were analyzed by Food Processor, version 7.4 (ESHA Research, Salem, Oregon, USA) for all nutrients including vitamins, minerals, though only energy, calcium, protein, fat and zinc intake were used for this analysis. Average daily CLA content was calculated from an estimate based on mg of CLA per gram of fat in all meat, seafood and dairy foods consumed by our population [2]. Our subjects consumed a total of 25 different types of meat, seafood and dairy products containing CLA. Vegetable oils, which contain small amount of CLA (<1 mg/g fat), and foods containing vegetable oils and dairy products were not used in calculating CLA intake because the amount of CLA in these foods could not be determined. Table 1 lists a sampling of foods rich in CLA.


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  		Table 1. Conjugated Linoleic Acid Content in Various Foods* Used for Calculation

 
Physical Activity
Physical activity (PA) was assessed using the Allied Dunbar National Fitness Survey for older adults [13] and was used as a confounder in statistical analysis. Average number of hours spent per day in recreational and/or sport activities were used as a measure of current physical activity. Past activity was defined as percent of adult life since age 18 engaged in recreational and sport activities for at least three times a week for at least three months of the year. The detailed description of PA assessment in this population was reported earlier [14].

Data Analysis
Data analysis was conducted with SPSS statistical software for Windows (version 12.0) and all variables were checked for normality. Pearson’s correlation coefficients (r) were calculated as preliminary evaluation to determine the degree of association between BMD and CLA. Multiple regression models controlled for years since menopause, lean tissue, energy, dietary calcium, protein, fat, zinc, current and past physical activity; variables known to affect bone in general and in this population [11], were used to investigate the relationship between CLA and BMD of various skeletal sites. Foods containing CLA such as red meat, also contain protein and zinc which are both related to bone mass [11], and were therefore included as confounders in analysis. Subjects were also divided into groups below (Group 1) and above (Group 2) the median for CLA intake. Analysis of covariance (ANCOVA) using the above confounders was used to determine significant group differences between CLA and BMD. The accepted level of statistical significance was p ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
CONCLUSION
 ACKNOWLEDGMENTS
 REFERENCES
 
Tables 2 and 3 list subjects’ descriptive characteristics. The average BMI (26.0 ± 3.8 kg/m2) indicated subjects were borderline overweight. The range of dietary CLA was comparable to the intake in other populations [6] and the average energy, total calcium, protein, fat and zinc intake were considered adequate for this age group. Subjects spent almost half of their adult life engaged in regular physical activity expressed as past activity, and were currently engaged in about 1 hour a week in recreational and/or sport activities. All variables were normally distributed and without big outliers except for CLA which was negatively skewed and therefore log transformed for all analyses.


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  		Table 2. Descriptive Characteristics of Subjects

 

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  		Table 3. Dietary Intake and Physical Activity of Subjects

 
All sites of hip BMD were positively correlated with CLA, Pearson’s r ranged from 0.179–0.217 (p ranged from 0.014–0.045). In a multiple regression model (containing years since menopause, energy, dietary calcium, fat, zinc and current physical activity) lean tissue, protein, past physical activity and CLA were significant predictors of Ward’s triangle BMD (Table 4). The model accounted for 28.6% of the total variance in BMD. CLA alone accounted for 4.0% of the total variance in Ward’s Triangle BMD based on the partial coefficient of determination (p < 0.05). When subjects were divided into below (group 1) and above (group 2) the median intake of CLA, those in group 2 had significantly higher BMD in the total forearm (ulna and radius), p = 0.042. Group 2 also had higher BMD in the Ward’s triangle, lumbar spine and whole body, based on means adjusted for years in menopause, lean tissue, energy, dietary calcium, protein, fat, zinc, current and past physical activity, though the difference did not reach statistical significance (Fig. 1).


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  		Table 4. Multiple Regression Model with Ward’s Triangle BMD (g/cm2) as Dependent Variable*

 


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  		Fig. 1. Adjusted* mean BMD of various skeletal sites for below (Group 1) and above (Group 2) the median dietary CLA intake**.

*Means adjusted for years since menopause, lean tissue, energy, dietary calcium, protein, fat, zinc and current and physical activity.

**Results for the hip, spine and whole body did not reach significance, p ranged from 0.065–0.326.

 

   DISCUSSION
 
This is the first study to show that dietary CLA is positively associated with BMD in postmenopausal women. Our results suggest an average intake of only 63 mg of dietary CLA a day may benefit hip and forearm BMD. In multiple regression models controlled for several confounders known to influence bone density including calcium, CLA accounted for 4% of the total variance in Ward’s triangle BMD. Vitamin D, though known to influence bone mass was not included as a confounder since it did not show a relationship with BMD of any bone site in this population [11].

Though we found subjects with higher dietary intakes of CLA to have higher BMD in several skeletal sites (suggesting a possible decrease in PGE2 production), statistical significance was only reached in the forearm using ANCOVA. This could be due to several factors. First, CLA was calculated from an estimate based on amounts in meat, seafood and dairy products. CLA in food can vary widely depending on the breed of the animal and the season. Other foods which contain very small amounts of CLA such as vegetable oils were not included which could have led to a small underestimation of dietary CLA, though it is unlikely this would have changed the significance of our results. Second, our sample size was limited to 136 women. With a larger amount of subjects, significance in ANCOVA may have been reached. We used 3 days of dietary records to calculate an average daily CLA intake; it is possible longer dietary records may have resulted in a more accurate representation of CLA intake.

The exact mechanism of how PGE2 stimulates bone formation and resorption is not fully understood. It is hypothesized that PGE2 at low levels increases the production of IGF and regulates IGF binding protein expression, which stimulates bone formation [15]. At high levels, PGs can inhibit collagen synthesis and reduce mRNA levels in bone, which inhibits the function of osteoblasts [10]. In rats, it appears the effects of CLA on bone are dependent on the levels of polyunsaturated fatty acids (PUFA), omega 3 and omega 6 (n-3 and n-6) fatty acids, in the diet [16]. Both n-3 and n-6 fatty acids influence PG and cytokine formation, and therefore, bone metabolism [17]. Watkins et al. [7] found chicks fed butter fat (a rich source of CLA) had a higher rate of bone formation compared to chicks given an n-6 rich diet. The increased rate of bone formation was attributed to a reduced level of arachidonic acid and PGE2 production as well as higher levels of IGF-1 in bone [8]. Watkins hypothesized that the reduced rate of PGE2 production from CLA might be due to a competitive inhibition of n-6 PUFA elongation that results in a lower amount of available substrate for cyclooxygenase, the enzyme necessary for PGE2 production [7]. CLA may also directly or indirectly alter cyclooxygenase-2 (the inducible form of cyclooxygenase) activity or expression, and therefore affect PGE2 production [7].

A few studies focusing on the effects of CLA on lean and fat tissue found small improvements in bone mass. Thiel-Copper et al. [18] found 26 kg pigs fed CLA until their weight reached 114 kg (slaughtering weight), had a linear increase in leg bone mass (p < 0.05) as the concentration of CLA increased in the diet from 0–1%. Pigs fed 0.5% and 1% CLA diets had more leg bone mass than did controls or pigs fed 0.12 and 0.25% CLA diets. A study in men conducting resistance training found supplementing the diet with 6 g/day of CLA for 28 days increased total body bone mineral content from 2,492 g to 2,516 g [19]. It appears, based on the above evidence that CLA may help to decrease the production of PGE2 to a more optimal level favoring the production of IGF to stimulate bone formation.


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 CONCLUSION
ACKNOWLEDGMENTS
 REFERENCES
 
Our results show dietary CLA is positively associated with Ward’s triangle and total forearm BMD and possibly in other skeletal sites in postmenopausal women. Additional cross-sectional and longitudinal studies are needed using different populations and more precise estimates of dietary CLA to assess its effects on both BMD and fracture risk. It is possible diets with higher levels of CLA may have a greater benefit on BMD.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 CONCLUSION
 ACKNOWLEDGMENTS
REFERENCES
 
This work was funded in part by the NRI/USDA 2001-00836, Donaghue Medical Research Foundation DF98-056, University of Connecticut Office for Sponsored Programs and Mission Pharmacal®, San Antonio, TX, USA.

Received April 27, 2004. Accepted November 30, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 CONCLUSION
 ACKNOWLEDGMENTS
 REFERENCES
 

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  16. Li Y, Seifert MF, Ney DM, Grahn M, Grant AL, Allen GD, Watkins BA: Dietary conjugated linoleic acids alter serum IGF-1 and IGF binding protein concentrations and reduce bone formation in rats fed (n-6) or (n-3) fatty acids.J Bone Miner Res14 :1153 –1162,1999 .[Medline]
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