Effect of dietary supplementation with conjugated linoleic acid on ...

European Journal of Clinical Nutrition (2005) 59, 432–440

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ORIGINAL COMMUNICATION Effect of dietary supplementation with conjugated linoleic acid on markers of calcium and bone metabolism in healthy adult men L Doyle1, C Jewell1, A Mullen3, AP Nugent3, HM Roche3 and KD Cashman1,2* 1 Department of Food and Nutritional Sciences, University College, Cork, Ireland; 2Department of Medicine, University College, Cork, Ireland; and 3Unit of Nutrition, Department of Clinical Medicine, Trinity Centre for Health Sciences, St. James’ Hospital, Dublin, Ireland

Introduction: Conjugated linoleic acid (CLA) has been shown to positively influence calcium and bone metabolism in experimental animals and cells in culture, but there are limited human data available. Objective: To investigate the effect of CLA supplementation on biomarkers of calcium and bone metabolism in healthy adult males. Design: The study consisted of a double-blind, placebo-controlled trial in which 60 healthy adult males (aged 39–64 y) were randomly assigned to receive daily either 3.0 g CLA isomer blend (50:50% cis-9,trans-11:trans-10,cis-12 isomers) or a palm/bean oil blend (placebo) for 8 weeks. Urine and blood samples were collected at weeks 0 and 8 and were analysed for biomarkers of calcium and bone metabolism. Results: Supplementation with CLA or placebo for 8 weeks had no significant effects on markers of bone formation (serum osteocalcin and bone-specific alkaline phosphatase) or bone resorption (serum C-telopeptide-related fraction of type 1 collagen degradation products, urinary N-telopeptide-related fraction of type 1 collagen degradation products, urinary pyridinoline and deoxypyridinoline), or on serum or urinary calcium levels. Baseline levels of these biochemical parameters were similar in both groups of subjects. While the placebo had no effect, CLA supplementation resulted in a three-fold increase (Po0.00001) in cis9,trans-11 CLA isomer in total plasma lipids. Conclusion: Under the conditions tested in this double-blind, placebo-controlled trial in adult men, a CLA supplement of mixed isomers did not affect markers of calcium or bone metabolism. Further investigation of the effects of CLA on calcium and bone metabolism in other gender- and age-groups is warranted. Sponsorship: Irish Government.

European Journal of Clinical Nutrition (2005) 59, 432–440. doi:10.1038/sj.ejcn.1602093 Published online 26 January 2005 Keywords: conjugated linoleic acid; biomarkers of bone metabolism; human subjects

Introduction Conjugated linoleic acid (CLA) is a collective term describing a mixture of positional and geometric isomers of linoleic

*Correspondence: KD Cashman, Department of Food and Nutritional Sciences, University College, Cork, Ireland. E-mail: [email protected] Guarantor: KD Cashman. Contributors: LD contributed to design, execution, analysis and writing of the study. CJ, AM, and APN contributed to the execution and analysis of the study. KDC and HMR contributed to design, analysis and writing of the study. Received 11 February 2004; revised 19 July 2004; accepted 14 October 2004; published online 26 January 2005

acid, with conjugated double bonds, which may be of cis or trans configuration at positions 9 and 11 or 10 and 12 (Lawson et al, 2001; Roche et al, 2001). While much of the attention over the last 5 y has focused on the possible beneficial effects of CLA on body composition, lipoprotein metabolism, inflammation, and carcinogenesis (see reviews by Kritchevsky, 2000; Roche et al, 2001; Belury, 2002; Albers et al, 2003), recently attention has focused on a possible beneficial effect on calcium (Ca) absorption and bone health. Park et al (1997) have reported that dietary supplementation with CLA in experimental mice led to a reduction in whole-body fat and an increase in body protein, water, and

Conjugated linoleic acid and bone metabolism L Doyle et al

433 ash; the increase in whole-body ash content suggests that CLA may enhance bone mineralization and protect against bone loss. This contention was supported by the findings that bone ash was higher in the tibia of CLA-fed chicks compared with control animals (Cook et al, 1997). ThielCooper et al (2001) recently found that pigs whose diets were supplemented with 0.5 or 1.0% CLA had increased bone weight compared to control unsupplemented animals and those who received 0.12 or 0.25% CLA. CLA has been shown to have a direct effect on the metabolic activity of bone cells. Watkins et al (1997) found that a dietary source of anhydrous butterfat (a rich natural source of CLA) stimulated the rate of bone formation in young growing chicks by modulating prostaglandin (PG) E2 production in bone. PGE2 plays an important role in the local regulation of bone formation and bone resorption (Marks & Miller, 1993). In experimental rats, dietary supplementation with CLA lowered ex vivo PGE2 production in bone organ culture from ovariectomized rats as well as reducing the levels of urinary pyridinium crosslinks (markers of bone resorption) (Kelly & Cashman, 2004). In young rats, on the other hand, CLA supplementation, while lowering ex vivo PGE2 production in bone organ culture, either reduced (Li et al, 1999) or had no effect on markers of bone formation (Kelly et al, 2003), and had no effect on markers of bone resorption (Kelly et al, 2003). CLA supplementation did, however, significantly increase the efficiency of intestinal Ca absorption in the young rats fed a n-3 polyunsaturated fatty acids (PUFA)-rich diet (Kelly et al, 2003). This effect on Ca absorption in experimental animals has been confirmed in studies using the human intestinallike Caco-2 cell model (which has been shown to be a useful in vitro model for predicting Ca absorption in human subjects; Fleet & Wood, 1999). For example, chronic exposure (2–3 weeks) of these cells to specific isomers of CLA can stimulate transepithelial Ca transport, predominantly by modulation of the paracellular route of absorption (Jewell & Cashman, 2003a, b). In this way, therefore, CLA may indirectly influence bone mass by making more Ca available for calcification. There have been only two studies which investigated the effect of CLA supplementation on bone mass in humans. Kreider et al (2002) investigated the effect of CLA supplementation (with 6.2 g CLA/day as mixed isomers) for 28 days on body composition and markers of catabolism and immunity in human male athletes undertaking resistance training. Despite suggestive trends for an increase in bone mass in CLA treated athletes, CLA supplementation did not promote statistically significant changes in bone mass or other aspects of body composition, strength or in markers of catabolism and immunity. However, this study had very small subject numbers (n ¼ 11–12 per group) and was of short duration (28 days) for investigation of effects on bone mass. More recently, Gaullier et al (2004) reported their findings of a 1 y CLA intervention study in healthy overweight adults (n ¼ 59–61 per group). While the effect on body fat mass was the primary outcome, they also reported effects on bone

mineral mass as a secondary outcome. Supplementation with CLA isomers (50% cis-9,trans-11 and 50% trans-10,cis12; 3.4–3.6 g/day), as triacylglycerol-bound CLA, had no effect on bone mineral mass over 12 months, whereas CLA isomers, as free fatty acids, reduced bone mineral mass slightly but significantly (P ¼ 0.01) from month 0 to 12. Despite the fact that the rate of bone turnover is a major determinant of bone mass, and animal studies have shown effects of CLA on bone metabolism, there has been no study on the effects CLA on Ca and bone metabolism in human subjects. Therefore, the aim of the present study was to investigate the effect of CLA supplementation on several biochemical markers of Ca and bone metabolism in healthy adult males, using a randomized, double-blind, placebocontrolled study design.

Materials Subjects A total of 60 healthy adult males (mean age 49.1 (range 39– 64 y) were recruited from among personnel of University College, Cork (n ¼ 30) and Trinity College and St James’s Hospital, Dublin (n ¼ 30), Ireland. The mean weight, height and body mass index (BMI) are provided in Table 1. All subjects adhered to the following inclusion criteria: BMI o33 kg/m2, fasting haemoglobin 4120 g/l, fasting plasma glucose o6.0 mmol/l, fasting plasma cholesterol o6.8 mmol/l, fasting plasma triglycerides (TAG) o2.3 mmol/l, fasting g-glutamyl transferase o60 IU. Subjects were without any history of bone or articular disease, and with no intake of medicine that could affect bone or cartilage metabolism. Additional exclusion criteria included chronic illness or taking fatty acid nutritional supplements. Subjects were requested to avoid excessive alcohol intake for the duration of the study and to avoid vigorous exercise the day preceeding blood and urine sampling. Otherwise, subjects were free to maintain their usual physical activity levels and practises.

Ethical considerations Before participation in this study, all subjects signed an informed consent document approved by the Clinical Research Ethics Committee of the Cork Teaching Hospitals (Cork subjects) or the Ethics Committee of the Federated Dublin Voluntary Hospitals (Dublin subjects). Table 1 Characteristics of the group of apparently healthy adult male volunteers (n ¼ 60) selected for the CLA supplementation trial (mean values with their standard deviations, and range)

Age (y) Height (m) Weight (kg) BMI (kg/m2)

Mean

s.d.

Range

49.1 1.79 85.3 26.6

6.2 0.08 11.1 2.8

39–64 1.60–2.00 65–110 20.3–32.9

European Journal of Clinical Nutrition


434 Design The study consisted of a double-blind, placebo-controlled, randomized trial of the effect of CLA supplementation of the usual diet (‘CLA-supplemented diet’) vs ‘un-supplemented diet (placebo)’ for 56 days on biochemical markers of Ca and bone metabolism in healthy adult males. Subjects were randomly assigned to the unsupplemented (placebo) or CLA-supplemented groups. During the 56-day experimental period, each subject received, in addition to their usual diet, either 3.0 g (six 0.625 g capsules) of CLA isomer blend (50% cis-9,trans-11 isomer and 50% trans10,cis-12 isomer (50:50 CLA), as triacylglycerol-bound)/day (CLA-supplemented) or palm and bean oil blend/day (placebo). The dosage was chosen on the basis that levels between B2 and 4 g CLA/day have been used in several human studies of CLA and body composition, plasma lipids, and glucose, insulin and/or leptin levels (see review by Terpstra, 2004). The CLA supplement (Clarinolt) and placebo capsules were kindly supplied by Loders Croklaan, B.V., Wormerveer, The Netherlands. The fatty acid composition of the CLA and placebo capsules are shown in Table 2. The CLA supplements contained a high level of linoleate; however, we chose not to use a placebo high in linoleate because van Dokkum et al (1983) showed that increasing the linoleate level in the diet of young men participating in a mineral balance study significantly reduced faecal Ca, suggesting a stimulation of Ca absorption. Therefore, the oils mixture used for the placebo was chosen because it was not particularly rich in linoleate and provided a range of fatty acids reflective of those consumed in the Irish diet. Study compliance was 98% using a pill count and was also measured by the incorporation of the cis-9,trans-11 isomer of CLA into total plasma lipids. The duration of the intervention (8 weeks) was chosen on the basis that most of the studies that have investigated the effect of CLA on body composition, plasma lipids, and glucose, insulin and/or

Table 2 Capsule composition of the 50:50 cis-9,trans-11-trans-10,cis12 isomer blend and the control treatment, blend of palm and bean oila Ingredient

50:50 CLA isomer blend

Control (blend of palm and bean oil)

Fatty acid composition (g/100 g fatty acids) 16:0 4.2 18:0 2.2 18:1 10.4 18:2 1.5 20:1 3.0 SFA 1.2

35.5 4.4 34.1 17.9 0.1 2.3

CLA isomers cis-9,trans-11 CLA trans-10,cis-12 CLA

0.2 ND

37.4 35.1

CLA, conjugated linoleic acid; SFA, saturated fatty acid; ND, not detected. a Materials kindly supplied by Loders Croklaan, B.V., Wormerveer, The Netherlands, and independently analysed by Consult-Us Laboratories Ltd, Glanmire, Co. Cork, Ireland.


leptin levels (see review by Terpstra, 2004) were of 4–12 weeks in duration, together with the fact that we and others have shown that levels of biochemical markers of bone turnover can be altered by dietary intervention/supplementation in as little as 2–4 weeks (Ginty et al, 1998a, b; Lin et al, 2003; Harrington et al, 2004). Subjects were instructed to collect fasting first void morning urine samples between 07 00 and 09 00 hours for 2 days immediately preceding the supplementation period (baseline) and for the last 2 days of the supplementation period. In addition, after an overnight fast, a blood sample (20 ml) was taken at 09 00 hours on the day immediately prior to starting the supplementation period and on the final (56th) day of supplementation period. Dietary analysis Nutrient intakes were estimated using a semiquantitative food-frequency questionnaire (Rimm et al, 1992). Nutrient intakes were calculated using data from McCance and Widdowson’s Composition of Foods, 5th Edition food nutrient data base (Holland et al, 1995).

Collection and preparation of samples Subjects were supplied with suitable collection containers for urine samples and asked to collect fasting first void morning urine samples as outlined above. Portions of urine were stored at 201C from the morning of collection until required for analysis. Blood was collected by venepuncture into vacutainer tubes containing either no additive or citrate and processed to serum and plasma, respectively, which were immediately stored at 801C until required.

Experimental techniques Urinary pyridinoline and deoxypyridinoline. Samples were analysed in duplicate using an automated analysis system (Gilson ASPEC (Auotmated Sample Preparation with Extraction Columns); Gilson S.A., Villiers-le-Bel, France). Extracted samples were linked to a gradient HPLC system comprising a Gilson 321 pump and a Shimadzu RF-10AXL fluorescence detector (Shimadzu Scientific Instruments Inc., Columbia, MD, USA). In brief, portions of pooled urine (500 ml) were first hydrolysed with an equal volume of 12 M HCl at 1071C for 18 hours. The crosslinks from urine hydrolysates were then extracted with cellulose partition chromatography, with the use of an internal standard (acetylated pyridinoline (PYD); MetraBiosystems Ltd, Wheatley, Oxon., UK) (Pratt et al, 1992). The acetylated PYD was used in accordance with the method described by Calabresi et al (1994) and Robins et al (1994). The crosslink contents of urine samples were quantified by external standardization using a commercially available PYD-deoxypyridinoline (DPD) HPLC calibrator (MetraBiosystems Ltd). The intra-assay CV for PYD and DPD measured as the variation between 10 chromatograms obtained between column regenerations as


435 described by Colwell et al (1993) were 5 and 3% respectively. The interassay CV for PYD and DPD were 9 and 11% respectively. Urinary type I collagen cross-linked N-telopeptides. Type I collagen cross-linked N-telopeptides (NT ) was measured in the urine samples by an ELISA (Osteomarks, Ostex International, Inc., WA, USA). The intraassay CV was 5%. Interassay variation was avoided by analysing all samples from an individual in the same run. Urinary creatinine. Creatinine was determined in urine samples using a diagnostic kit (Metra Creatinine Assay Kit, Catalogue No. 8009, Quidel Corporation, CA, USA). The intraassay CV was 1.6%. Interassay variation was avoided by analysing all samples from an individual in the same run. Serum type-I collagen degradation product levels. Levels of degradation products of C-terminal telopeptides of type-I collagen (CTX-1) were measured in serum samples using a recently developed enzyme-linked immunosorbent assay (Serum CrossLapst One Step ELISA; Osteometer Biotech A/S, Herlev, Denmark). The intraassay CV was 5.2%. Interassay variation was avoided by analysing all samples from an individual in the same run. Urinary and serum calcium levels. Ca was analysed in duplicate in urine and serum samples by atomic absorption spectrophotometry (Spectr AA-600, Varian Australia Ltd, Victoria, Australia) after appropriate dilution with LaCl3 solution (5 g/l, BDH Ltd, Poole, Dorset, UK). A range of Ca standards was used to obtain a Ca calibration curve. The intra- and interassay CV for Ca was 2.8 and 7.8%, respectively. The accuracy of mineral analysis was assured in each analytical run by appropriate recovery of mineral in samples of a reference serum (Seronorm Trace Elementst: Serum, Nycomed, Olso, Norway) and a reference urine (Seronorm Trace Elementst: Urine, Nycomed). Serum osteocalcin and bone-specific alkaline phosphatase. Bone-specific alkaline phosphatase (EC 3.1.3.1) levels were measured in serum samples using a recently developed ELISA (Alkphase-Bt, MetraBiosystems Inc., CA, USA). The intra-assay CV was 4.5%. Osteocalcin levels were measured in serum samples using an ELISA (N-MIDt, Osteometer Biotech, Osteopark, Denmark). The intra-assay CV was 11%. Interassay variation for both serum osteocalcin and bonespecific alkaline phosphatase was avoided by analysing all samples from an individual in the same run. Serum 25-hydroxyvitamin D (25(OH)D). 25(OH)D levels were measured in serum samples using a recently developed ELISA (OCTEIAs 25-Hydroxy Vitamin D, Immuno Diagnostic Systems, Ltd, Boldon, UK). The intra- and interassay CV were 5.9 and 6.6%, respectively. The quality and accuracy of

the serum 25(OH)D analysis in this laboratory was assured on an ongoing basis by participation in the Vitamin D External Quality Assessment Scheme (DEQAS, Charing Cross Hospital, London, UK). Serum 25(OH)D cutoff values for defining vitamin D status as adequate, marginally deficient or severely deficient were: 440, 25–40 and o25 nmol/l, respectively (Vieth, 1999). GC analysis of total plasma lipid fatty acid composition. Total plasma lipid were isolated using the method derived by Folch et al (1957). Lipid present in the organic phase was dried using a vortex evaporator (AGB Scientific, Dublin, Ireland). When samples were dried, they were flushed with N2, sealed to prevent lipid oxidation and stored at 201C. Methyl esters of total plasma lipids were prepared by adding 0.5 ml 0.01 M NaOH in dry methanol. Samples were vortexed and flushed with N2 and then placed in a heating block at 601C for 15 min. Boron trifluoride (0.75 ml) was then added to mixture, samples were vortexed and incubated at 601C for 15 min. Lipids were extracted three times using 0.5 ml hexane. Samples were dried in a vortex evaporator and stored under N2 at 201C until analysis. The fatty acid methyl ester composition of total plasma lipids for CLA analysis was analysed using a Shimadzu GC14A GLC (Mason Technologies, Dublin, Ireland) that was fitted with a Shimadzu C-16A integrator. A CP Sil 88 fused silica column (50 m 0.22 mm, 0.2 file thickness; Chrompack Ltd, Middelburg, The Netherlands) was fitted. N2 was used as a carrier gas. Conditions for the GC analysis of plasma lipids were as follows. An initial column temperature of 1201C increased at 81C/min to 1801C. Column temperature was held at 1801C for 40 min. Column temperature was increased at 41C/min to 2201C and was held at 2201C for 15 min. Peaks were identified using a fatty acid methyl ester standard spiked with known concentrations of the cis9,trans-11 and trans-10,cis-12 isomers of CLA. Fatty acids were identified by retention times compared with standard and fatty acid compositions and were calculated as % total fatty acids.

Statistical methods Prior to the start of the experiment, the required sample size at a ¼ 0.05 and b ¼ 0.80 was calculated (Dallal, 1990) using the variability around the mean serum CTX-1 levels in healthy adults and a selected minimum detectable percent difference (ie, delta) in this bone biomarker between groups of 16%. A value of 16% was chosen as a meaningful difference in the absence of reported data on the magnitude of the reduction in serum CTX-1 levels following CLA supplementation in humans. For example, Lin et al (2003) recently reported reductions of 16–18% in plasma CTX-1 levels in adults adhering to a dietary intervention (the Dietary Approached to Stop Hypertension (DASH) diet) for 30 days. Data are presented as means with their standard deviations (s.d.). Data for all variables were normally European Journal of Clinical Nutrition


436 distributed and allowed for parametric tests of significance. Repeated-measured ANOVA examining the treatment time interaction was used to investigate changes in biochemical markers of Ca metabolism and bone turnover following CLA intervention. ‘Centre’ was also included as a variable in the model, but there were no significant (P40.05) centre effects. Post hoc statistical analysis (Student’s t-tests) was used to ascertain which data points were significantly different when repeated-measures ANOVA showed a significant treatment time interaction. Student’s t-tests were also used to test for statistical differences in baseline biochemical variables between groups as well as differences in changes in these variables over the 8 weeks of supplementation between groups.

throughout the present study. Similarly, levels of Ca and Cr, as well as NTx/Cr, PYD/Cr and DPD/Cr in urine remained unchanged in both supplementation groups throughout the present study. Baseline serum 25(OH)D levels showed that none of the men had severe vitamin D deficiency (ie, o25 nmol 25(OH)D/l), but 20% of subjects had marginal vitamin D deficiency (ie, 25–40 nmol 25(OH)D/l). The number of men who were marginally vitamin D-deficient in the CLA-supplemented group was twice that in the placebo group. Therefore, the statistical analysis was performed again after omitting the 12 men with marginal vitamin D deficiency from the data sets, but the findings were unaltered (data not shown). Fatty acid composition of total plasma lipids is presented in Table 4. There was a three-fold increase (Po0.00001) in cis-9,trans-11 isomer of CLA present in total plasma lipids in the CLA group after 8 weeks of supplementation. The trans10,cis-12 isomer was undetectable in most samples. There was no significant increase in cis-9,trans-11 isomer of CLA in the placebo group. There was a significant (Po0.05) increase in C18:3 n-3 in the placebo group over 8 weeks of treatment. There was a significant (Po0.05) increase in C20:5 n-3 in CLA group over 8 weeks of treatment.

Results There were no significant differences in age, weight, height or BMI between subjects from the two study centres. In addition, baseline biochemical variables were similar in both groups of subjects (data not shown). All 60 subjects completed the dietary intervention. The effect of fatty acid supplementation for 8 weeks on biochemical markers of Ca metabolism and bone turnover is shown in Table 3. Unpaired t-tests showed no significant differences in serum Ca, 25(OH)D, CTX-1 (a marker of bone resorption), osteocalcin and bone-specific alkaline phosphatase levels (markers of bone formation), or urinary Ca, creatinine (Cr), NTx/Cr, PYD/Cr and DPD/Cr (markers of bone resorption) levels between both study groups (ie, CLA and placebo) at week 0. Serum CTX-1, Ca, 25(OH)D, osteocalcin and bone-specific alkaline phosphatase levels remained unchanged in both supplementation groups

Discussion In the present study, the levels of four different markers of bone resorption (three urinary-based and one serum-based (CTX-1)) and two markers of bone formation in healthy adults were unaffected by supplementation with CLA (50:50) for 8 weeks. There is no other published data on the effect of CLA on Ca and/or bone metabolism in human subjects with which to compare our findings.

Table 3 Effects of dietary supplementation using an isomeric blend of conjugated linoleic acid (CLA) and a palm and bean oil mixture (3 g/day) for 8 weeks on biochemical markers of calcium and bone metabolism (mean values with their standard deviations) Control (palm and bean oil) (n ¼ 29) Week 0

Week 8

50:50 CLA group (n ¼ 31) Week 0

Mean

s.d.

Mean

s.d.

Serum Ca (mmol/l) 25 (OH) D (mmol/l) Osteocalcin (mg/l) Alkphase-B (U/l) CTX-1 (mg/l)

2.26 59.1 13.5 23.5 0.48

0.09 22.3 3.1 5.0 0.27

2.24 55.2 12.1 23.1 0.46

0.09 17.1 3.0 5.4 0.24

Urine Creatinine (Cr) (mmol//) Ca (mmol/mmol Cr) NTx (nM BCE/mmol Cr) PYD (nmol/mmol Cr) DPD (nmol/mmol Cr)

11.9 0.27 39.4 16.2 9.5

5.3 0.19 19.1 4.7 2.9

12.4 0.25 36.1 16.0 8.9

4.7 0.11 10.3 4.6 2.7

Week 8

Statistical significance of variance ratio ( P), effects of: Trt

Time

Trt Time

2.23 0.10 2.23 0.09 64.6 20.8 60.7 20.0 13.0 3.3 12.2 3.4 21.7 5.6 22.0 6.2 0.37 0.14 0.37 0.14

0.519 0.265 0.815 0.318 0.144

0.317 0.093 0.105 0.962 0.395

0.391 0.968 0.388 0.222 0.546

13.2 5.1 12.1 4.1 0.21 0.08 0.21 0.09 34.6 17.7 34.0 18.0 15.0 4.2 15.2 4.3 8.8 2.7 8.7 2.7

0.664 0.189 0.390 0.359 0.529

0.501 0.479 0.241 0.990 0.233

0.102 0.424 0.399 0.574 0.439

Mean

s.d.

Mean

s.d.

Trt, treatment; Trt Time, treatment by time interaction; Alkphase-B, bone-specific alkaline phosphatase; 25(OH)D, 25-hydroxyvitamin D; CTX-1, C-terminal telopeptides of type-I collagen; Cr, creatinine; NTx, Type I collagen cross-linked N-telopeptides; BCE, Bone collagen equivalents; PYD, pyridinoline; DPD, deoxypyridinoline.



437 Table 4 Fatty acid composition of total plasma lipids (g/100 g total plasma lipids) as a result of dietary supplementation using conjugated linoleic acid (CLA) and a palm and bean oil mixture (3 g/day) for 8 weeksa (mean values with their standard deviations) Control (palm and bean oil) Week 0 Fatty acid

b

16:0 16:1 18:0 18:1 18:2 n-6 18:3 n-6 18:3 n-3 cis-9, trans-11 CLA 20:0 20:1 20:2 20:3 n-6 20:4 n-6 20:5 n-3 22:6 n-6

50:50 CLA group Week 8

Week 0

Week 8

Mean

s.d.

Mean

s.d.

Mean

s.d.

Mean

s.d.

25.86 3.39 7.82 23.11 27.75 0.39 0.86 0.17 0.07 2.18 0.17 1.29 5.46 0.46 0.81

0.88 0.30 0.26 1.32 1.81 0.09 0.08 0.05 0.03 1.81 0.05 0.16 0.35 0.12 0.16

25.96 3.08 8.29 23.59 28.62 0.40 1.02w 0.24 0.08 0.50 0.10 1.22 5.72 0.49 0.68

0.58 0.22 0.27 0.65 0.99 0.08 0.08 0.05 0.04 0.08 0.05 0.05 0.35 0.13 0.22

25.21 3.14 8.10 21.67 28.98 0.46 0.94 0.23 0.08 1.22 0.09 1.25 5.59 0.36 0.68

0.39 0.21 0.56 1.20 1.03 0.10 0.06 0.06 0.05 0.51 0.04 0.06 0.26 0.14 0.21

26.23 3.20 8.66 22.32 28.38 0.28 1.42 0.71z 0.18 0.39 0.11 1.19 5.19 0.70w 1.01

0.46 0.21 0.21 0.88 1.08 0.07 0.49 0.06 0.06 0.07 0.04 0.07 0.20 0.24 0.26

Repeated measures ANOVA shows significant treatment time interaction: wPo0.05; zPo0.00001. a For details of supplements, subjects and procedures, see Tables 1 and 2 and the ‘Methods’ section. b trans-10, cis-12 CLA isomer undetectable in most samples, results not presented.

Various biochemical markers are available for the assessment of bone formation and bone resorption. Urinary excretion of the collagen telopeptide crosslinking molecules, such as PYD and DPD have been used as resorption markers for over a decade (Robins et al, 1996). Urinary levels of Ntelopeptide-related fraction of type 1 collagen degradation products (NTx) and C-telopeptide-related fraction of type 1 collagen degradation products (CTX-1) have also been used in more recent years (Gertz et al, 1994; Rosenquist et al, 1998). The present study had sufficient power to detect a minimum of 20, 22 and 26% reduction in urinary DPD, PYD and NTx, respectively. Measurement of circulating CTX-1 levels, which have also been shown to reflect the rate of bone resorption (Christgau et al, 1998; Rosenquist et al, 1998; Peichl et al, 2001), have been reported to be have greater precision and lower variability than urinary-based markers (Rosen et al, 2000). The present study had sufficient power to detect a minimum of 14% reduction in serum CTX-1. This magnitude of response of serum CTX-1 to dietary intervention was recently reported in subjects placed on the DASH (Dietary Approaches to Stop Hypertension) diet for 30 days (16–18% reductions; Po0.0001) compared to that in subjects on the control diet (Lin et al, 2003). As mentioned already, there have been only two studies which investigated the effect of CLA supplementation on bone mass in humans, neither selecting it as a primary outcome measure. Kreider et al (2002) investigated the effect of CLA supplementation (with 6.2 g CLA/day as mixed isomers) for 28 days on bone mass together with other aspects of body composition and markers of catabolism and immunity in human male athletes undertaking resistance

training. Despite suggestive trends for an increase in bone mass ( þ 1.1%; P ¼ 0.08) in CLA-treated athletes relative to athletes taking placebo (olive oil), CLA supplementation did not promote statistically significant changes in bone mass or other aspects of body composition, strength or in markers of catabolism and immunity. However, this study had very small subject numbers (n ¼ 11–12 per group) and was of short duration (28 days) for investigation of effects on bone mass. More recently, Gaullier et al (2004) reported their findings of a 1 y CLA intervention study in healthy overweight adults (n ¼ 59–61 per group). While the effect on body fat mass was the primary outcome, they also reported effects on bone mineral mass as a secondary outcome. Supplementation with CLA isomers (50% cis-9,trans-11 and 50% trans-10,cis12; 3.4–3.6 g/day), as triacylglycerol-bound CLA, had no effect on bone mineral mass over 12 months, whereas CLA isomers, as free fatty acids, reduced bone mineral mass slightly but significantly (by 1.4%; P ¼ 0.01) from month 0 to 12. The apparent lack of effect of CLA, especially triacylglycerol-bound CLA, on bone mass could be explained by a lack of effect on Ca metabolism and bone turnover, as evidenced in the present study. Evidence for an effect of CLA on bone from studies with experimental animals is mixed. For example, Li et al (1999) showed that CLA supplementation (10 g/kg diet) of young growing male rats reduced bone mineralization, possibly by reducing the biosynthesis of PGE2 by bone, while Kelly et al (2003) found no effect of CLA supplementation, at the same dietary level, on markers of bone formation or bone resorption in young growing male rats, despite a reduction in biosynthesis of PGE2. Kelly & Cashman (2004) did, European Journal of Clinical Nutrition


438 however, report that CLA reduced markers of bone resorption (urinary pyridinium crosslinks) in ovariectomized rats. Watkins et al (1997) found that a dietary source of anhydrous butterfat (a rich natural source of CLA) stimulated the rate of bone formation in young growing chicks. Similarly, the effect of CLA on bone mass, ash or mineral content in studies with experimental animals is also conflicting, with some studies showing an increase with CLA (Cook et al, 1997 (chicks); Park et al, 1997 (mice); Thiel-Cooper et al, 2001 (pigs)), while others found no effect (Li et al, 1999; Kelly et al, 2003; Kelly & Cashman, 2004 (rats) and Demaree et al, 2002; Ostrowska et al, 2003 (pigs)). As already mentioned, CLA is a collective term describing a mixture of positional and geometric isomers of linoleic acid, with conjugated double bonds, which may be of cis or trans configuration at positions 9 and 11 or 10 and 12 (Lawson et al, 2001). Isomer-specific effects of CLA have been reported for some biological variables. For example, Noone et al, (2002) reported that the 50:50 CLA supplements reduced plasma TAG concentrations, while another isomer blend of CLA (80% cis-9,trans-11 isomer and 20% trans-10,cis-12 isomer (80:20 CLA)) had no effect, and 80:20 CLA reduced VLDL-cholesterol, while 50:50 CLA had no effect. Therefore, it is tempting to suggest that the lack of effect of CLA on bone may be related, at least in part, to the isomer composition of the CLA supplement used (50:50 vs 80:20 CLA). However, Park et al (1999) demonstrated that enhanced whole-body ash in experimental mice was associated with feeding trans-10,cis-12 CLA isomer rather than the cis-9,trans-11 or trans-9,trans-11 CLA isomers. Cusack et al (2003) in a preliminary report of studies of osteoblast-like SaOS2 cells in culture, showed that PGE2 biosynthesis was significantly reduced by the trans-10,cis-12 CLA isomer, but not by the cis-9,trans-11 isomer. Isomer-specific effects have also been reported for intestinal Ca absorption. For example, Jewell and Cashman (2003a) reported a stimulatory effect of the trans-10,cis-12 isomer of CLA on paracellular Ca absorption in Caco-2 cells in culture, but not with the cis-9,trans-11 isomer. Therefore, the trans-10,cis-12 isomer of CLA would appear to be more bioactive for bone metabolism and intestinal Ca absorption, even though it is the less prevalent naturally occurring isomer in foods (Lawson et al, 2001). The content of the trans-10,cis-12 isomer of CLA in the 50:50 isomer blend of CLA used in the present study was over double that found in the 80:20 CLA isomer blend used in some studies mentioned above. While half of the CLA in the supplement used in the present study was provided as the trans-10,cis-12 isomer of CLA, this isomer was not incorporated efficiently into plasma lipids, in agreement with the findings of Noone et al (2002). This may relate to the isomer being metabolized, via desaturation and elongation pathways (Sebedio et al, 1997), or being more easily oxidized due to its structure (Martin et al, 2000). However, irrespective of the reasons for the lack of incorporation into plasma lipids, a low exposure European Journal of Clinical Nutrition

of bone cells to circulating trans-10,cis-12 isomer of CLA (potentially the more bone-active isomer of CLA) might be one explanation of the lack of effect of CLA supplementation on bone metabolism in the present study. Intestinal cells, on the other hand, would be exposed to high luminal concentrations of trans-10,cis-12 isomer of CLA. However, CLA had no effect on indices of Ca metabolism in healthy adult males in the present study. There has been no other report of the effect of CLA on Ca metabolism in humans. Kelly et al (2003) recently showed that CLA supplementation (containing cis-9,trans-11 and trans-10,cis-12 CLA isomers in a 50:50 ratio) significantly increased the efficiency of intestinal Ca absorption in young growing rats fed a n-3 PUFA-rich diet, but not in rats fed a n-6 PUFA-rich diet. A preliminary investigation of the dietary ratio of n-3 to n-6 fatty acids in a subset of subjects (n ¼ 30) participating in the present study showed that the diet was rich in n-6 fatty acids (B8.6:1 n-6:n-3), which may have limited any potential stimulatory effect of CLA on intestinal Ca absorption. The apparent lack of agreement of the effect of CLA on bone between these human studies and some animal trials, may relate to the dosages used. Pariza (2004) has suggested that levels used in animal studies (typically around 1.5% CLA) is a level B30 times greater than humans would ingest at 3 g/day. Therefore, it is possible that dosages much greater than that used in the present study and that of Gaullier et al (2004) (ie, 3–3.6 g/day of active isomers) may positively influence bone, but this would need to be researched. Kelly and Cashman (2004) recently showed that while supplementation of the diet with 0.5% and 1.0% CLA reduced markers of bone resorption in ovariectomized rats, a dietary level of 0.25% (still five-times greater than humans would ingest at 3 g CLA/day) had no effect. In conclusion, the findings of the present study, which was a comprehensive investigation of the effect of supplementation with a blend of CLA isomers on several markers of bone turnover in human subjects, suggest that, under the conditions tested, supplementation with mixed isomers of CLA for 8 weeks did not influence bone or Ca metabolism in healthy adult males. However, the dose-related effect of CLA (alone and in combination with a diet high in n-3 fatty acids) on bone turnover as well as on intestinal Ca absorption per se (preferably using stable isotope technology) would need to be investigated. Furthermore, given recent evidence of an antiresorptive effect of CLA in bone of ovariectomised rats (Kelly & Cashman, 2004) further investigation of the effect of CLA on bone metabolism in human subjects is warranted, and especially in postmenopausal women.

Acknowledgements This work was supported by funding made available by the Irish Government under the National Development Plan 2000–2006.


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