The primary purpose of the present review was to determine if the level of scientific evidence available for potential human health benefits of conjugated linoleic acid (CLA) is sufficient to support health claims on foods based on naturally CLA-enriched milk. Health claims on foods in Europe must now be selected from community lists of approved claims or be the subject of a scientific dossier to gain approval(1). In order to gain approval, the scientific evidence must be based on human studies, with human intervention studies accorded a higher weighting(1). Cows' milk contains predominantly the cis-9, trans-11 isomer of CLA (c9, t11-CLA). Naturally CLA-enriched milk is defined for the purpose of the present review as milk obtained from grass-feeding regimens that have proven to result in higher levels of c9, t11-CLA than do conventional feeding regimens (see below).
‘Conjugated linoleic acid‘ is a term used to describe a mixture of positional and geometric isomers of linoleic acid containing conjugated double bonds. It is a group of naturally occurring fatty acids synthesised as intermediates in the biohydrogenation of linoleic and linolenic acid in the rumen of animals, and thus is predominantly found in dairy products and ruminant meat(Reference Chin, Liu and Storkson2). It can also be synthesised by industrial partial hydrogenation or alkali-isomerisation of linoleic acid(Reference Banni3). CLA includes twenty-eight possible isomers, with two of these – cis-9, trans-11-CLA (c9, t11-CLA) and trans-10, cis-12-CLA (t10, c12-CLA) – being known to possess biological activity(Reference Pariza, Park and Cook4). Commercially available CLA supplements usually contain c9, t11-CLA and t10, c12-CLA at a ratio of approximately 1:1. The majority of CLA in the human diet occurs as c9, t11-CLA, with this isomer accounting for 85–90% of the total CLA content in dairy products(Reference Lock and Bauman5).
CLA was first discovered in 1932, by scientists at the University of Reading (UK) who were investigating seasonal variation in the vitamin content of milk(Reference Christie, Sebedio, Christie and Adlof6). Interest in the potential health benefits of CLA was later sparked by the identification of CLA's anti-carcinogenic activity in vitro, in extracts from fried ground beef(Reference Ha, Grimm and Pariza7). Since then, numerous studies and reviews have investigated the potential health benefits of CLA, with purported health benefits including anti-cancer, anti-atherogenic, anti-adipogenic, anti-diabetogenic, anti-inflammatory and effects on bone health, at least in vitro.
CLA is present in relatively low quantities (mg) in meat and dairy products(Reference Chin, Liu and Storkson2). Estimated dietary intakes from 3 d diet records in the USA are 176 mg total CLA per d for men, with slightly lower intakes for women (104 mg/d). However, in the UK, intake of c9, t11-CLA was estimated to be 97.5 mg/d(Reference Ritzenthaler, McGuire and Falen8). Furthermore, this may vary depending on the method used to assess dietary intake(Reference Mushtaq, Heather and Hunter9). In recent times, there has been a surge of interest in increasing the concentration of CLA in food in order to increase dietary CLA intake. Cows' milk fat is the richest natural source of CLA(Reference Parodi, Sebedio, Christie and Adlof10); therefore, interest has focused on the potential for naturally increasing the c9, t11-CLA content of milk and dairy products. Levels of CLA in milk fat ranging from 2 to 37 mg/g fat have been recorded and are due to numerous factors(Reference Parodi, Yurawecz, Mossoba, Kramer, Pariza and Nelson11). The composition of the animals' diet is a major factor, with cows that graze on fresh pasture having higher concentrations of CLA in their milk fat than those grazing on hay or concentrates(Reference Dhiman, Anand and Satter12). However, cows that are fed the same diet can demonstrate large intra-individual variation in CLA levels, which may be due to differences in metabolism and the rumen microflora responsible for biohydrogenation(Reference Parodi, Sebedio, Christie and Adlof10). Altitude, breed and lactation age can also influence CLA levels(Reference Parodi, Sebedio, Christie and Adlof10). Research in the UK has shown that there is no difference between the content of CLA in milk from organic and conventional farms(Reference Ellis, Innocent and Grove-White13). Furthermore, it appears that processing of dairy products causes insignificant changes in CLA levels, particularly compared with the large variations in CLA levels due to diet and intra-individual variation(Reference Parodi, Sebedio, Christie and Adlof10).
Much research has been carried out on strategies to manipulate the diets of cows to produce CLA-rich milk, which can then be used to make CLA-rich dairy products. Supplementing the diets of cows with plant oils rich in linoleic or linolenic acid (such as sunflower-seed, soyabean or linseed oil) is known to cause an increase in the concentration of c9, t11-CLA in milk fat(Reference Stanton, Murphy, McGrath, Sebedio, Christie and Adlof14). A study which evaluated the characteristics of naturally CLA-enriched ultra-high temperature (UHT) milk, butter and cheese reported that although the sensory profiles of the CLA-enriched products were different from those of control products, subjects did not rate the overall impression and flavour as being different(Reference Jones, Shingfield and Kohen15). It has also been shown that consumption of naturally CLA-enriched dairy products for 6 weeks, at similar levels to which conventional dairy products are habitually consumed in the UK, increases c9, t11-CLA concentration in plasma lipids(Reference Burdge, Tricon and Morgan16). Together these data show that it is feasible and acceptable to increase c9, t11-CLA intake in the human diet by producing naturally CLA-enriched dairy products for consumption.
The overall purpose of the present review was to examine the current literature in relation to c9, t11-CLA and human health benefits with the focus on, in particular, milk and dairy products where CLA content has been enhanced by natural feeding regimens. As there are relatively few studies on enhanced dairy products and in order to identify potential opportunities for future research on c9, t11-CLA, studies on synthetic CLA isomers were also considered but not subject to exhaustive review. Much of the interest in CLA has been provoked by promising results from animal and in vitro studies and in order to put this in context, an overview of these studies is provided although this does not represent a complete picture of the large body of literature.
Initially, reviews were identified from PubMed and used to provide an overview of the key areas of interest. Subsequently, Medline, Embase and evidence-based medicine (EBM) reviews (including Cochrane) were searched via OvidSP (Wolters Kluwer, Alphen aan den Rijn, The Netherlands) using the terms ‘CLA’, ‘conjugated linoleic acid’ and ‘dairy’, both separately and together. Thus, for all databases, this yielded:
(a) 535 for ‘CLA’ (subheading: ‘conjugated linoleic acid’);
(b) 41 760 for ‘dairy’ (subheadings: ‘dairy products’ and ‘milk’);
(c) Combining both searches above yielded fifty-six papers;
(d) Separate searches with the above databases for ‘cis-9, trans-11’ yielded 13 525 papers;
(e) Medline was searched for ‘cis-9, trans-11’; only 4476 papers were found and the introduction of ‘human’ reduced the number of papers to 1378. Further specification to linoleic acids, conjugated yielded 348;
(f) Embase (n 9002) – narrowed to ‘humans’ and ‘CLA’ (n 120);
(g) EBM reviews (n 37) – narrowed to ‘humans’ and ‘CLA’ (n 35).
The searches were merged using a reference manager programme and duplicates removed, with a total of 538, the abstracts were then examined to determine whether the studies were relevant to the present review. A total of sixty-six human studies utilising observational, randomised control trials and crossover designs, published up to July 2011, were included in the present review. References within studies were also checked for completeness. Reviews on animal studies were identified to provide an overview and then key references followed up individually.
Conjugated linoleic acid and cancer
Since the initial identification of CLA from grilled minced beef and its anticarcinogenic effects on skin cancer tumours in mice(Reference Ha, Grimm and Pariza7), the intervening years have provided a cascade of studies and reviews examining the anticarcinogenic properties of CLA. The mechanisms relating to anticarcinogenic properties of CLA are largely unresolved; CLA may act by antioxidant mechanisms, pro-oxidant cytotoxicity, inhibition of nucleotide and protein synthesis, reduction of cell proliferative activity and inhibition of both DNA–adduct formation and carcinogen activation(Reference Parodi17–Reference Kelley, Hubbard and Erickson19). The studies examined in these reviews have identified potential beneficial effects of CLA on colorectal, breast and prostate cancer, with the majority of evidence from animal and in vitro studies.
CLA has shown consistent anticarcinogenic effects against several types of experimental cancer(Reference Belury20) including breast cancer(Reference Ip, Chin and Scimeca21, Reference Ip, Singh and Thompson22). A review by Kelley et al. (Reference Kelley, Hubbard and Erickson19) examined the literature in terms of the effects of studies where purified isomers of CLA were administered. Results from in vitro studies suggest that the effects of the two isomers of CLA vary according to the cancer model examined. In the majority of studies, c9, t11-CLA did not inhibit tumour growth, whereas t10, c12-CLA demonstrated inhibitory effects in studies using mouse and human mammary tumour cell lines. The t10, c12-CLA isomer also inhibited cell growth in colon and gastric cancer cell lines. However, c9, t11-CLA was more potent than t10, c12-CLA in colon cell lines where both isomers were examined, though the optimal concentration level varied between studies (50 μmol/l and 200 μmol/l)(Reference Palombo, Ganguly and Bistrian23, Reference Beppu, Hosokawa and Tanaka24). Subsequent work by Yasui et al. (Reference Yasui, Suzuki and Kohno25) also confirmed the chemopreventive effect of c9, t11-CLA against pre-initiation (dose-dependent) as well as post-initiation stages of colorectal carcinogenesis (doses ≤ 1% of diet).
Overall, in studies using animal models of cancer, the purified c9, t11-CLA isomer reduced tumorigenesis in six studies and showed no effect in two others(Reference Kelley, Hubbard and Erickson19). Similarly, the t10, c12-CLA isomer decreased tumorigenesis in six studies, but in contrast increased tumorigenesis in two studies. Interestingly, three studies included in the present review found similar effects on the reduction of mammary tumours when a naturally enriched butter(Reference Ip, Banni and Angioni26) and synthetic isomers of c9, t11-CLA were fed to rats(Reference Lavillonniere, Chajes and Martin27) and mice(Reference Hubbard, Lim and Erickson28). Though more recent work suggests that t10, c12-CLA stimulates mammary tumours in a mouse model, where the gene erbB2/her2 is over-expressed, application of c9, t11-CLA showed no apparent effects(Reference Ip, McGee and Masso-Welch29). The same paper also demonstrated that the reduction in tumours was in the same order of magnitude irrespective of whether the CLA source was natural or synthetic. The authors of this paper suggest that it would be prudent to avoid supplements containing t10, c12-CLA in those at risk of developing breast cancer in which the erbB2/her2 gene is over-expressed (observed in 20–30% of human breast cancers), whereas supplements containing c9, t11-CLA may be safe and efficacious in breast cancer prevention(Reference Ip, McGee and Masso-Welch29). However, due to the differences in proliferation of tumours by the site of cancer, combining results may not elicit the true effects of CLA as an anti-carcinogenic agent, though in vitro and animal studies do demonstrate potential benefits.
The manifestation of cancer is not a practical end-point in human studies, combined with the numerous genetic and environmental risk factors for different cancers. Consequently, the majority of studies relating to CLA and cancer in humans are observational studies, particularly on breast cancer (Table 1). Dietary and serum CLA was shown to be significantly lower in postmenopausal cases of breast cancer compared with controls, thus suggesting a protective effect of CLA(Reference Aro, Mannisto and Salminen30). In a continuation of this study, breast adipose concentrations of CLA were not significantly different between cases and controls(Reference Chajes, Lavillonniere and Ferrari31). Furthermore, there was no association between breast adipose tissue CLA concentration and prognostic factors of breast cancer or occurrence of metastases during a 7.5-year follow-up period(Reference Chajes, Lavillonniere and Maillard32). In the Netherlands Cohort Study on Diet and Cancer, intake of milk and milk products and meat products, as major sources of dietary CLA, showed no relationship with breast cancer incidence in postmenopausal patients(Reference Voorrips, Brants and Kardinaal33). This could be attributed to the fact that there were no significant differences in total CLA intake between cancer cases and controls(Reference Voorrips, Brants and Kardinaal33). The null association between breast cancer risk and intake of CLA was also demonstrated in a large epidemiological study in Sweden(Reference Larsson, Bergkvist and Wolk34). In contrast, in the same cohort, women who consumed four or more servings of high-fat dairy foods per d (including whole milk, full-fat cultured milk, cheese, cream, soured cream and butter) had a lower risk of developing colorectal cancer(Reference Larsson, Bergkvist and Wolk35). It has been suggested that a higher intake of c9, t11-CLA confers a reduced risk of a specific type of breast cancer tumour in premenopausal women. However, further investigation is warranted, as the sample size was small(Reference McCann, Ip and Ip36).
F, female; c9, t11, cis-9, trans-11; ER, oestrogen receptor; M, male; RCT, randomised controlled trial; t10, c12, trans-10, cis-12.
Recently, one small cross-over study examined colon cancer markers after subjects (n 15) consumed milk naturally enriched with c9, t11-CLA or synthetically enriched with t10, c12-CLA or normal milk as a control(Reference Farnworth, Chouinard and Jacques37). There were large variations in the responses to supplementation across all three groups (NS), therefore all data were combined and a significant decrease in enzyme activity β-glucosidase, nitroreductase and urease; P < 0·01 between day 0 and day 56 was observed. The authors stated that this was important due to links between enzymic activity and the production of carcinogens. However, it is important to note that the main aim of the study was to examine the effects of CLA-enriched milk on lipid metabolism and body composition(Reference Venkatramanan, Chouinard and Jacques38).
Currently the evidence for the anti-carcinogenic properties of CLA in human subjects is limited to observational studies, with broader epidemiological evidence not specifically focusing on CLA but rather on milk and dairy products. The World Cancer Research Fund & Association for International Cancer Research report reviewed the available evidence on the consumption of milk and links with cancer(39). The report concluded that milk probably protects against colorectal cancer, whereas there is limited evidence suggesting that cheese is a cause of colorectal cancer. There is also limited evidence suggesting that consumption of milk conveys a protective effect against bladder cancer. In contrast, diets high in Ca are a probable cause of prostate cancer, but there is limited evidence suggesting that high consumption of milk and dairy products is a cause of prostate cancer(39). Currently the evidence available is confusing, with suggestions that the effects are dependent on the site of the cancer due to the complex nature of diet, environment and nutrient interactions. However, a substantial amount of further work is required to fully elucidate the potential anti-carcinogenic properties of CLA in humans.
Conjugated linoleic acid and body composition
The overwhelming increases in the proportion of overweight and obesity in the world have been the focus of much debate and research. Currently two-thirds of the UK adult population are classified as overweight or obese (BMI > 25 kg/m2)(Reference Craig and Mindell40). Obesity is a multifaceted disorder, largely driven by its co-morbidities including type 2 diabetes, insulin resistance and CVD. To date feasible and sustainable approaches to prevent further increases in overweight and obesity, let alone attenuate it, have remained largely elusive. More recently, obesity has been recognised as a state of chronic or low-grade systemic inflammation, due to the abnormal circulating levels of inflammatory molecules, including TNFα, leptin and IL-6, which are secreted by adipose tissue(Reference Forsythe, Wallace and Livingstone41).
Studies in animals have shown that feeding CLA at levels of 0.5–1% of the diet reduces body fat in mice, chickens, hamsters, rats and pigs(Reference Wahle, Heys and Rotondo42). The most substantial decreases in body fat have been observed in mice, where CLA at levels of 0.5% of the diet has been shown to lower body fat by 40 to 80%(Reference Wahle, Heys and Rotondo42). This effect is thought to be attributable to the t10, c12-CLA isomer, as the greatest body fat reductions in mice were observed with a CLA mix with a higher proportion of t10, c12-CLA than c9, t11-CLA(Reference Brown and McIntosh43). Also, in vitro, t10, c12-CLA prohibits TAG accumulation in cultures of differentiating human preadipocytes, whereas c9, t11-CLA increases TAG content(Reference Brown and McIntosh43). Evidence suggests that this effect may be due in part to a reduction in lipid uptake by adipocytes due to effects of CLA on stearoyl-CoA desaturase and lipoprotein lipase activity(Reference Pariza, Park and Cook4).
Promising evidence from animal studies led to an array of human intervention studies being carried out investigating the effect of CLA on body composition in normal weight, overweight and obese subjects. The majority of these studies used a 50:50 (c9, t11-CLA and t10, c12-CLA) CLA mix, and results have been inconsistent. Almost all of these studies have shown no effect on body weight; however, some have reported reduced body fat mass (BFM) following supplementation with CLA, as discussed in detail below.
The body composition studies conducted in normal-weight adults (Table 2) have supplemented with 0.7–5.5 g 50:50 CLA/d, for 4–16 weeks, and of those which measured BFM, some have reported non-significant changes(Reference Petridou, Mougios and Sagredos44, Reference Lambert, Goedecke and Bluett46, Reference Nazare, de la Perriere and Bonnet47, Reference Brown, Trenkle and Beitz50, Reference Tricon, Burdge and Kew79, Reference Kreider, Ferreira and Greenwood175), and others have reported BFM reductions of 4% up to 20%(Reference Mougios, Matsakas and Petridou51–Reference Raff, Tholstrup and Toubro56). However, it is important to note that in some of the studies that have reported significant BFM reductions in normal-weight adults, subjects were involved in physical training throughout the supplementation periods, which may potentially be a confounder(Reference Thom, Wadstein and Gudmundsen53–Reference Pinkoski, Chilibeck and Candow55).
CLA mixture, 50:50 cis-9, trans-11- and trans-10, cis-12-CLA; M, male; F, female; RCT, randomised controlled trial; BFM, body fat mass; c9, t11, cis-9, trans-11; t10, c12, trans-10, cis-12; LBM, lean body mass.
* Subjects exercising.
In overweight and obese human subjects (Table 3), 50:50 CLA given at doses of 1.7–6.8 g/d, over periods of 4 to 104 weeks, has resulted in non-significant BFM changes in some instances(Reference Steck, Chalecki and Miller57–Reference Joseph, Jacques and Plourde63), and reductions of 3–15% in other studies(Reference Brown, Trenkle and Beitz50, Reference Steck, Chalecki and Miller57–Reference Joseph, Jacques and Plourde63, Reference Malpuech-Brugère, Verboeket-van de Venne and Mensink77, Reference Risérus, Vessby and Arnlöv78). The greatest reduction in BFM (14.8%) was observed in a study of patients on blood pressure-lowering medication, who were supplemented with 4.5 g 50:50 CLA/d for 8 weeks(Reference Zhao, Zhai and Wang71). In apparently healthy adults, the greatest reduction in BFM (6%) was observed in the study by Gaullier et al. (Reference Gaullier, Halse and Hoye66) which was of 104 weeks' duration, and supplemented with 3.4 g 50:50 CLA/d. One study in children found that body fat gain was attenuated during prepubertal growth in 6–10-year-olds supplemented with 3.0 g 50:50 CLA/d(Reference Racine, Watras and Carrel73). However, in a few cases it has been noted that the largest reduction in BFM occurs in the lower body (for example, legs)(Reference Raff, Tholstrup and Toubro56, Reference Gaullier, Halse and Hoivik67). Furthermore, some studies have reported increases in lean body mass (LBM) with CLA supplementation(Reference Steck, Chalecki and Miller57, Reference Blankson, Stakkestad and Fagertun64, Reference Gaullier, Halse and Hoivik67). In the study by Blankson et al. (Reference Blankson, Stakkestad and Fagertun64) increased LBM was only observed in the group which significantly increased their level of intensive physical training during the intervention, hence it is possible that the observed effects were, at least partially, due to increased physical activity and not CLA supplementation.
CLA mixture, 50:50 cis-9, trans-11- and trans-10, cis-12-CLA; F, female; M, male; RCT, randomised controlled trial; BFM, body fat mass; SAD, sagittal abdominal diameter; t10, c12, trans-10, cis-12; c9, t11, cis-9, trans-11; LBM, lean body mass.
Interestingly, in another study, overweight subjects receiving 3.2 g of 50:50 CLA per d over a 6-month period, including the Christmas period, demonstrated a lower rate of weight gain and a 4% reduction in BFM compared with control(Reference Watras, Buchholz and Close69). A study of subjects with type 2 diabetes supplemented with 6 g of 50:50 CLA per d for 8 weeks found that plasma concentration of t10, c12-CLA, but not c9, t11-CLA, was inversely associated with body weight, suggesting that t10, c12-CLA is the active CLA isomer in relation to weight change(Reference Belury, Mahon and Banni74). This is in agreement with evidence from animal studies which also points to the t10, c12-CLA isomer as being the CLA isomer which elicits BFM reductions. A meta-analysis concluded that CLA, at a dose of 3.2 g/d, produces a modest body fat loss in humans of about 0.09 kg/week, with the relationship being linear up to 6 months(Reference Whigham, Watras and Schoeller75). This may be partly explained by the isomer- and tissue-specific effects of CLA, whereby c9, t11-CLA was found to be increased in skeletal muscle and t10, c12-CLA was incorporated into adipose tissue TAG in a subset of healthy non-obese participants(Reference Goedecke, Rae and Smuts76).
In addition to studies examining effects of CLA mixes, a number of studies have investigated the effects of individual CLA isomers on body composition. Findings from these studies show that consumption of 0.59–3 g c9, t11-CLA per d or 0.6–3.4 g t10, c12-CLA per d does not change body composition(Reference Risérus, Arner and Brismar60, Reference Malpuech-Brugère, Verboeket-van de Venne and Mensink77–Reference Tricon, Burdge and Kew79).
Currently, only three studies have been carried out which have fed subjects naturally CLA-enriched dairy products and investigated the effects on body composition(Reference Tricon, Burdge and Jones45, Reference Brown, Trenkle and Beitz50, Reference Desroches, Chouinard and Galibois80). In the study by Desroches et al. (Reference Desroches, Chouinard and Galibois80), sixteen normolipidaemic overweight and obese men consumed butter naturally enriched with CLA (c9, t11-CLA; 2.59 g/d), or non-enriched control butter (0.24 g/d), for 4 weeks each in a cross-over design, and results showed no changes in body composition. Tricon et al. (Reference Tricon, Burdge and Jones45) fed thirty-two healthy normolipidaemic men either naturally CLA-enriched or control dairy products (UHT full-fat milk, butter and cheese (1.42 v. 0.15 g c9, t11-CLA/d) in a 6-week cross-over study. Similarly, no changes in body weight were observed; however, body composition was not the primary outcome of this study, but rather blood lipid profile. No changes in body composition were observed when subjects consumed beef and dairy products naturally enriched with 1.17 g CLA/d for 56 d(Reference Brown, Trenkle and Beitz50). Also, with all of these studies it is important to note that the durations (4–8 weeks) were relatively short for investigating effects on body composition.
There are many possible explanations for the lack of reproducibility in studies of CLA's effect on body composition between animals and humans. These include age, sex, genetic predisposition to fat accumulation and differences in experimental design(Reference Plourde, Jew and Cunnane81). It is interesting to note that although animal studies have evaluated the effects of CLA on weight gain over time in growing animals, the majority of human studies tend to investigate whether CLA affects weight or fat loss only in adults.
Conjugated linoleic acid, lipid metabolism and atherosclerosis
CVD are the leading cause of mortality globally(82) and so modification of key risk factors such as LDL-cholesterol or blood TAG are key targets (for example, in the UK(83)). The impact of dietary fat and specific fatty acids on blood lipids has been a focus of research at least since Keys et al.'s early epidemiological work(Reference Keys, Aravanis and Blackburn84), so it is not surprising that the effect of CLA on blood lipids has been investigated.
Evidence from animal studies in rabbits, hamsters and mice has suggested that CLA has the potential to modulate plasma lipid metabolism and make an impact on the development and regression of cholesterol-induced atherosclerotic plaques(Reference Mitchell and McLeod85).
In rabbits, mixed-isomer CLA, fed at levels of 0.1–1% of diet over periods of 13 to 22 weeks, has been shown to reduce cholesterol deposition in the aorta(Reference Lee, Kritchevsky and Pariza86) and result in significant regression of established atherosclerotic lesions(Reference Kritchevsky, Tepper and Wright87). Furthermore, mixed-isomer CLA at a lower dose (0.05%) has been shown to be sufficient to decrease lesion development in rabbits(Reference Kritchevsky, Tepper and Wright88). Supplementation with either c9, t11-CLA or t10, c12-CLA results in similar reductions in lesion development to that seen with mixed-CLA isomer supplementation(Reference Kritchevsky, Tepper and Wright89).
Studies in hamsters that have supplemented with CLA over periods of 6–12 weeks, using different CLA isomers and doses, have shown mixed results, but there is evidence of improvements in lipid profile(Reference Nicolosi, Rogers and Kritchevsky90, Reference Gavino, Gavino and Leblanc91). In addition, there is some indication that CLA in conjunction with a lower-fat diet may reduce atherosclerotic lesions in the hamster(Reference Mitchell and McLeod85). It has been suggested that t10, c12-CLA may be the protective isomer in relation to lipid profile, as in the study by Gavino et al. (Reference Gavino, Gavino and Leblanc91), a CLA mix, but not the c9, t11-CLA isomer, improved the lipid profile of hamsters.
In mice, studies with supplemental CLA carried out over periods of 4–20 weeks, using different CLA isomers and doses, have also shown mixed results(Reference Mitchell and McLeod85). There has been one promising report of CLA (80:20 blend of c9, t11-CLA and t10, c12-CLA) resulting in marked regression of atherosclerotic lesions in apoE mice(Reference Toomey, Harhen and Roche92). In addition, there is some evidence of opposing effects of CLA isomers, with one study in mice showing c9, t11-CLA decreasing and t10, c12-CLA increasing atherosclerotic lesion area(Reference Arbones-Mainar, Navarro and Guzman93).
Further to the above studies which have supplemented animals' diets with commercial CLA preparations, studies have been carried out to investigate the anti-atherogenic effects of inclusion of dairy foods, and other foods such as eggs, naturally enriched with CLA, into the diets of animals(Reference Lock, Horne and Bauman94–Reference Franczyk-Żarów, Kostogrys and Szymczyk98). The results of these studies have shown that CLA can improve plasma lipid profile and decrease atherosclerosis-related biomarkers. Overall, at present there is no general consensus as to the effect of CLA supplementation on lipids or atherosclerosis in animals. Furthermore, most animal studies that have suggested protective anti-atherogenic effects have generally provided CLA doses greater than those achievable in the human diet.
Despite much investigation, the precise mechanisms by which CLA affects lipid metabolism and adipose tissue are not fully elucidated. However, it is thought that CLA modulates energy expenditure, apoptosis, fatty acid oxidation, lipolysis and lipogenesis(Reference House, Cassady and Eisen99). As discussed in the previous section, the t10, c12-CLA isomer is thought to exert effects on body composition, partly due to a reduction in lipid uptake by adipocytes due to effects of CLA on stearoyl-CoA desaturase and lipoprotein lipase activity(Reference Pariza, Park and Cook4).
In humans epidemiological studies on dietary CLA intakes and prevalence of atherosclerosis have not been carried out to date. However, over the past decade, numerous human intervention studies have investigated the effect of CLA on lipids and other markers of atherosclerotic risk (Table 4), the results of which have been highly inconsistent, possibly due to the use of different isomers and varying doses. The majority of these studies have used commercial mixed- or pure-isomer CLA preparations, at levels of 1.7 to 6.8 g/d, over periods of 4 to 13 weeks, and have not shown any overall effect on plasma lipid or lipoprotein concentrations, compared with placebo, in normal-weight and overweight subjects(Reference Petridou, Mougios and Sagredos44, Reference Lambert, Goedecke and Bluett46, Reference Brown, Trenkle and Beitz50, Reference Smedman and Vessby52, Reference Herrmann, Rubin and Häsler61–Reference Blankson, Stakkestad and Fagertun64, Reference Racine, Watras and Carrel73, Reference Risérus, Vessby and Arnlöv78, Reference Benito, Nelson and Kelley100–Reference Engberink, Geleijnse and Wanders105). However, one study did report significant within-group reductions in total cholesterol and LDL-cholesterol with doses of 1.7 and 3.4 g CLA/d(Reference Blankson, Stakkestad and Fagertun64), but it was stated that the reductions were not clinically important.
CLA mixture, 50:50 cis-9, trans-11- and trans-10, cis-12-CLA; M, male; F, female; RCT, randomised controlled trial; tChol, total cholesterol; c9, t11, cis-9, trans-11; t10, c12, trans-10, cis-12.
* Same study with results reported over two papers.
Some studies have reported that supplementation with commercial CLA preparations can have a negative effect on the lipid profile. For example, a significant decrease in HDL-cholesterol was observed on supplementing with 3.4 g t10, c12-CLA per d in obese men with the metabolic syndrome(Reference Risérus, Arner and Brismar60), and in healthy subjects who were supplementing their diets with 0.7–1.4 g CLA mix per d(Reference Mougios, Matsakas and Petridou51). There is some evidence to suggest that CLA (mixtures and individual isomers) can induce lipid peroxidation(Reference Risérus, Vessby and Arnlöv78, Reference Raff, Tholstrup and Basu103); however, it is not known whether this effect of CLA could be pro-atherogenic in humans.
In contrast, other studies have shown a positive effect of CLA, with 3 g 50:50 CLA mix per d lowering fasting TAG, and 3 g 80:20 CLA mix per d decreasing VLDL, in healthy subjects(Reference Noone, Roche and Nugent106). Furthermore, 3 g 50:50 CLA per d was shown to significantly increase HDL-cholesterol and significantly decrease LDL:HDL-cholesterol in patients with type 2 diabetes(Reference Moloney, Yeow and Mullen107). Consumption of foods enriched with 26.8 g CLA/d led to a significant positive effect on HDL concentration and a significant lowering of LDL-cholesterol(Reference Wanders, Brouwer and Siebelink49). Interestingly, Tricon et al. (Reference Tricon, Burdge and Kew79) observed divergent responses in plasma lipids with CLA supplementation, with t10, c12-CLA (0.6–2.5 g/d) increasing LDL:HDL-cholesterol and total:HDL-cholesterol and c9, t11-CLA (0.59–2.38 g/d) decreasing these ratios, with no dose-dependent effect observed. Elevated cholesterol ratios of LDL:HDL and total:HDL-cholesterol are independent risk factors for CHD(Reference Ridker, Stampfer and Rifai108, Reference Lewington, Whitlock and Clarke109).
Recently, the effects of consuming dairy products, naturally rich in CLA or naturally enriched with CLA, on lipids in human subjects have been examined in four studies(Reference Tricon, Burdge and Jones45, Reference Brown, Trenkle and Beitz50, Reference Desroches, Chouinard and Galibois80, Reference Sofi, Buccioni and Cesari110). Three of these studies manipulated cows' diets to produce dairy products naturally enriched with CLA(Reference Petridou, Mougios and Sagredos44, Reference Brown, Trenkle and Beitz50, Reference Desroches, Chouinard and Galibois80). In the study by Desroches et al. (Reference Desroches, Chouinard and Galibois80), normolipidaemic overweight and obese men consumed butter naturally enriched with CLA (c9, t11-CLA; 2.59 g/d), or non-enriched control butter (0.24 g/d), for 4 weeks. Results showed plasma lipid subfraction levels (VLDL, LDL and HDL) were not significantly different between the two treatments; however, consumption of the non-enriched butter resulted in a significantly greater reduction of total cholesterol, total:HDL-cholesterol and LDL:HDL-cholesterol compared with the CLA-enriched butter, a result which was contradictory to the hypothesis. Tricon et al. (Reference Tricon, Burdge and Jones45) fed healthy normolipidaemic men either naturally CLA-enriched or control dairy products (UHT full-fat milk, butter and cheese (1.42 v. 0.15 g c9, t11-CLA per d)) in a 6-week cross-over study. Overall, lipid subfractions were not affected; however, a small but significant increase in LDL:HDL-cholesterol was observed. These results were similar to findings by Brown et al. (Reference Brown, Trenkle and Beitz50) where consumption of beef and dairy products rich in CLA (1.17 g CLA/d) for 56 d did not alter blood lipid profile.
A small, cross-over study in ten healthy subjects found that consumption of cheese made from naturally CLA-rich sheep's milk (0.25 g c9, t11-CLA per d) for 10 weeks had no effect on plasma lipids, as compared with consumption of a regular cows' cheese(Reference Sofi, Buccioni and Cesari110). The daily intake of CLA was confirmed as being 0.25 g c9, t11-CLA in correspondence with the author. It is important to note that using cows' milk cheese as a control was not ideal, due to the fact that it has a very different fatty acid profile compared with sheep's cheese. Overall these studies have shown no significant effect of treatment with dairy products naturally rich in CLA or naturally enriched with CLA on plasma lipids.
Dairy products which are naturally enriched in CLA are also higher in trans-vaccenic acid (trans-18 : 1), lower in SFA content, and slightly higher in n-3 PUFA content than conventional dairy products, due to the feeding strategies employed for enrichment(Reference Jones, Shingfield and Kohen15). It has been suggested that consuming trans-fatty acids impairs the lipid profile by lowering HDL-cholesterol and raising LDL-cholesterol levels(Reference Lichtenstein, Ausman and Jalbert111). Whether the content of trans-vaccenic acid in naturally CLA-enriched dairy products could counteract the potential benefit of CLA on the lipid profile unclear. The current evidence examining the intake of trans-fatty acids from animal sources and associations with CHD presents a confusing picture, particularly given the higher than typically consumed levels of trans-fatty acids used within studies(Reference Willett, Stampfer and Manson112–Reference Mensink116). However, it is unclear whether the partial conversion of trans-vaccenic to c9, t11-CLA in human intestines, liver and adipose tissue promotes adverse or beneficial effects on lipid profile(Reference Mozaffarian, Katan and Ascherio113, Reference Turpeinen, Mutanen and Aro117).
The reason for the inconsistent and mostly neutral results in relation to the effects of CLA on lipids in human studies compared with animal studies is unclear. However, it is important to note that while animal studies examined the effect of using CLA to supplement hyperlipidaemic animals that were eating atherogenic diets, human studies examined the effect of supplementing diets of normolipidaemic subjects with CLA. Furthermore, it is conceivable that the anti-atherosclerotic effects of CLA observed in animal studies may be due to mechanisms other than effects on lipids, for instance anti-inflammatory effects, as atherosclerosis is an inflammatory disease.
Conjugated linoleic acid, inflammation and immune effects
Inflammation underlies a wide range of conditions. For example, as noted above, obesity is now recognised as a state of chronic or low-grade systemic inflammation, due to the abnormal circulating levels of inflammatory molecules, including TNFα, leptin and IL-6, which are secreted by adipose tissue(Reference Forsythe, Wallace and Livingstone41). In addition, inflammation is central to atherosclerosis(Reference Libby, Ridker and Hansson118) and the metabolic syndrome(Reference Gade, Schmit and Collins119).
In vitro studies have shown that CLA has anti-inflammatory effects. CLA (CLA mix, or c9, t11-CLA or t10, c12-CLA) is associated with a lower mRNA expression of the inflammatory mediators cyclo-oxygenase-2, TNFα, and inducible NO synthase, and decreases production of induced PGE2, NO, IL-6 and IL-1β in mouse macrophage cells(Reference Yu, Correll and Vanden Heuvel120). The c9, t11-CLA isomer inhibits induced eosinophil activation, decreases transcription of TNFα, IL-6 and IL-12 in Caco-2 cells and enhances IL-10 production in murine dendritic cells(Reference Jaudszus, Foerster and Kroegel121–Reference Reynolds, Loscher and Moloney123). Furthermore, both c9, t11-CLA and t10, c12-CLA reduce PGE2 and thromboxane B2 concentrations in human macrophages(Reference Stachowska, Dolegowska and Dziedziejko124).
Animal studies have been carried out to determine if CLA exerts anti-inflammatory effects in vivo; however, results to date have been inconsistent. Three animal studies have found a CLA mix to be anti-inflammatory(Reference Changhua, Jindong and Defa125–Reference Noto, Zahradka and Ryz127). Obese rats fed 1.5% CLA mix for 8 weeks were found to have less adipose TNFα mRNA expression; however, other markers of inflammation did not change(Reference Noto, Zahradka and Ryz127). Butz et al. (Reference Butz, Li and Huebner126) reported that mice fed 0.5% CLA mix for 3 weeks had less plasma TNFα compared with mice on a control diet. In pigs fed 2% CLA mix for 14 d, a decrease in induced elevation and mRNA expression of pro-inflammatory cytokines (IL-6 and TNF-α), and an increase in an anti-inflammatory cytokine (IL-10) were observed. Furthermore, a molecular aspect of the same study determined t10, c12-CLA to be the main isomer to which the anti-inflammatory effect can be attributed(Reference Changhua, Jindong and Defa125). However, in contrast to these findings, two studies have established t10, c12-CLA to have pro-inflammatory effects, where mice fed 0.5% t10, c12-CLA for 14 d showed induced pro-inflammatory cytokine transcripts in white adipose tissue(Reference LaRosa, Miner and Xia128), and short-term supplementation with t10, c12-CLA in mice also increased pro-inflammatory cytokine gene expression in a study(Reference Poirier, Shapiro and Kim129).
Human intervention studies have investigated the effect of CLA (both commercial preparations and naturally CLA-enriched dairy products) on various biomarkers of inflammation (Table 5). Results to date have been mixed, with most studies either showing an increase in inflammatory markers, or no change. Three studies that have supplemented subjects with a CLA mixture at doses of 4.2 to 6.4 g/d, over periods of 12 to 16 weeks, have found increases in plasma levels of C-reactive protein (CRP)(Reference Steck, Chalecki and Miller57, Reference Smedman, Basu and Jovinge130, Reference Tholstrup, Raff and Straarup131). There were no significant effects on inflammatory markers including CRP and a range of interleukins when subjects were supplemented with 4 to 4.5 g CLA mixture/d(Reference MacRedmond, Singhera and Attridge132, Reference Stickford, Mickleborough and Fly133). Two studies with CLA added to foods showed no effect on plasma CRP levels; however, the duration of these trials was relatively short (5 and 8 weeks)(Reference Joseph, Jacques and Plourde63, Reference Raff, Tholstrup and Basu103). Furthermore, two crossover studies that provided c9, t11-CLA at doses of 4 g/d(Reference Sluijs, Plantinga and de Roos62) or 0.6–2.4 g/d and 0.6–2.5 g/d t10, c12-CLA(Reference Tricon, Burdge and Kew134), for 6 months and 8 weeks respectively, observed no change in plasma CRP concentrations.
CLA mixture, 50:50 cis-9, trans-11- and trans-10, cis-12-CLA; F, female; DTH, delayed-type hypersensitivity; t10, c12, trans-10, cis-12; M, male; RCT, randomised controlled trial; c9, t11, cis-9, trans-11; CRP, C-reactive protein; VCAM, circulating vascular adhesion molecule; PBMC, peripheral blood mononuclear cell; ICAM, intercellular adhesion molecule; LT, leucotriene; E-selectin, endothelial leucocyte adhesion molecule; PAI, plasminogen activator inhibitor; MCP, monocyte chemoattractant protein; FVII-C, factor VII coagulant; EDN, eosinophil-derived neurotoxin; GM-CSF, granulocyte macrophage colony-stimulating factor; IFN, interferon; ACE, angiotensin-converting enzyme; ECP, eosinophil cationic protein; LTC4-E4, cysteinyl 4-series leukotrienes.
Supplementation with t10, c12-CLA at doses of 3–3.4 g/d for 12–13 weeks has produced inconsistent results. A study in obese men with the metabolic syndrome found increased plasma CRP levels; on the other hand, a study in overweight men and women demonstrated no effect on plasma CRP, or on other markers of inflammation(Reference Ramakers, Plat and Sebedio135, Reference Risérus, Basu and Jovinge136). In the case of c9, t11-CLA, supplementation with similar doses (3 g) for similar durations (12–13 weeks) has also resulted in contrasting results, with one study reporting increased excretion of a pro-inflammatory marker (15-keto-dihydro-PGF2α) in obese subjects(Reference Risérus, Vessby and Arnlöv78), and another study reporting no effect on a range of pro-inflammatory markers in overweight subjects(Reference Ramakers, Plat and Sebedio135).
As described in the previous section, the effect of feeding subjects dairy products which are naturally enriched in c9, t11-CLA (due to the manipulation of diets of cows) has been investigated in two studies to date(Reference Tricon, Burdge and Jones45, Reference Desroches, Chouinard and Galibois80). In these studies, daily doses of 1.4–2.6 g c9, t11-CLA were fed for durations of 4–6 weeks, and no changes in plasma CRP concentrations and other inflammatory markers were observed. In contrast, a study by Sofi et al. (Reference Sofi, Buccioni and Cesari110) found that consumption of sheep cheese, naturally rich in CLA (0.25 g c9, t11-CLA per d), for 10 weeks decreased circulating levels of the pro-inflammatory cytokines IL-6, IL-8 and TNFα, compared with consumption of a control cows' cheese. However, as noted above, this study was small, poorly controlled and may not have been adequately powered for the multiple variables measured.
Some studies have investigated other immune effects in addition to inflammation. A study where the diets of young women were supplemented with a CLA mixture at 3.9 g/d for 9 weeks found that no indices of immune status were affected (such as the number of circulating leucocytes; granulocytes; monocytes; lymphocytes and their subsets; lymphocytes proliferation in response to phytohaemagglutinin and influenza vaccine; and serum influenza antibody titres)(Reference Kelley, Taylor and Rudolph137). However, the sample size was small, at seventeen. In a larger study, with fifty-five subjects, Nugent et al. (Reference Nugent, Roche and Noone138) found that either a 50:50 CLA mixture or an 80:20 CLA mixture at about 2 g/d had minimal effects on lymphocytes and cytokines, and had no additional benefit on immune function compared with linoleic acid. CLA supplementation has also been linked to reduced symptoms of birch pollen allergy(Reference Turpeinen, Ylönen and von Willebrand104) and improved airway hyper-responsiveness in asthmatics(Reference MacRedmond, Singhera and Attridge132). However, a second study in asthmatics found no attenuation of airway inflammation or bronchoconstrictive response(Reference Stickford, Mickleborough and Fly133).
However, Song et al. (Reference Song, Grant and Rotondo139) found that supplementing twenty-eight males and females with 3 g CLA 50:50 for 12 weeks had beneficial effects on immune function as it decreased pro-inflammatory cytokines (TNFα and IL-1β) and increased an anti-inflammatory cytokine (IL-10). Furthermore, Tricon et al. (Reference Tricon, Burdge and Kew134) found that supplementing men with 0.6 to about 2.5 g of either c9, t11-CLA or t10, c12-CLA per d decreased mitogen-induced T lymphocyte activation dose-dependently (however, lymphocytes and cytokines were unaffected). Mullen et al. (Reference Mullen, Moloney and Nugent140) showed that 2.2 g CLA 50:50 per d for 8 weeks decreased stimulated peripheral blood mononuclear cell IL-2 secretion, but did not affect other markers including plasma levels of IL-6, CRP, fibrinogen or TNFα, in thirty men.
Overall, studies investigating the effect of CLA (both supplements and naturally CLA-enriched products) on immune indices and inflammation provide inconsistent results.
Conjugated linoleic acid, insulin resistance and diabetes
In addition to the potential anti-atherogenic, anti-obesity and anti-inflammatory properties of CLA, the effects on diabetes have also been examined. As previously stated, increases in overweight and obesity have been concurrent with increases in type 2 diabetes, which is characterised by insulin resistance and occurs as a result of excess adipose tissue. A 5% reduction in body weight has been shown to decrease insulin resistance in overweight and obese subjects(Reference Brown and McIntosh43, Reference Taylor and Zahradka141, Reference Belury142). Therefore the observed modest reductions in body weight with CLA mixtures at 3 g/d may also improve insulin resistance.
Overall, the results from both animal and in vitro work are conflicting, with the effects of CLA on insulin resistance examined in addition to other outcomes (atherogenic and obesogenic properties). The vast majority of studies have examined the effects of CLA isomer mixtures, though some results do suggest isomeric differences(Reference Roche, Noone and Sewter143). In a mouse model, feeding a diet rich in t10, c12-CLA induced insulin resistance whereas c9, t11-CLA improved lipid metabolism without impairing insulin action(Reference Moloney, Toomey and Noone144) by possible mediation of the pro-inflammatory state(Reference Houseknecht, Vanden Heuvel and Moya-Camarena145). Similarly, studies with male Zucker diabetic fatty (ZDF) rats feeding a 50:50 blend of CLA reduced glucose and insulin concentrations(Reference Ryder, Portocarrero and Song146), although the diet with 91% c9, t11-CLA showed no effect(Reference Tsuboyama-Kasaoka, Takahashi and Tanemura147). In contrast, in another mouse model of diabetes, a blend of CLA isomers induced marked lipodystrophic insulin resistance and glucose tolerance(Reference Halade, Rahman and Fernandes148, Reference Halade, Rahman and Fernandes149). In the same strain of young and ageing mice, supplementation with the individual isomers or a CLA mix demonstrated divergent responses(Reference Halade, Rahman and Fernandes148, Reference Halade, Rahman and Fernandes149). Supplementation, with c9, t11-CLA elicited no effects on indices of insulin resistance, plasma insulin and glucose, whereas supplementation with t10, c12-CLA or a CLA mix increased plasma glucose, insulin and homeostasis model assessment of insulin resistance (HOMA-IR). However, during an intravenous glucose tolerance test, mice supplemented with c9, t11-CLA eliminated glucose faster than the control, t10, c12-CLA- or CLA mix-fed mice(Reference de Roos, Rucklidge and Reid150). These data highlight the importance of not just measuring plasma glucose and insulin, as true effects may only be apparent when more robust measures of insulin resistance are used.
One group has used a proteomics approach for eliciting the interactions between CLA isomers and diseases in an animal model(Reference de Roos and McArdle151). Proteomic techniques measure changes in the protein complement of a biological system and enable modelling of biological processes in response to dietary interventions(Reference de Roos, Rucklidge and Reid150). In a study with apoE mice consuming 7% c9, t11-CLA or t10, c12-CLA or control (linoleic acid), results suggested that c9, t11-CLA exerted anti-diabetic effects due to altered expression of markers, whereas t10, c12-CLA asserted pro-diabetic effects(Reference Risérus, Arner and Brismar60, Reference Risérus, Vessby and Arnlöv78, Reference Risérus, Smedman and Basu152). Overall, this study suggests that c9, t11-CLA potentially contributes to a less severe inflammatory response or protection against the development of atherosclerosis. However, conducting a trial in human subjects would be prohibitively expensive and require a rigorously controlled protocol in order to examine the effects of CLA supplementation on protein structure and function.
Currently the anti-diabetic properties of CLA in human subjects (Table 6) cannot be fully determined, as few studies are undertaken using rigorous measures of insulin resistance such as the hyperinsulinaemic–euglycaemic clamp(Reference Moloney, Yeow and Mullen107, Reference Ahren, Mari and Fyfe153) or the oral glucose tolerance test(Reference Brown, Trenkle and Beitz50, Reference Raff, Tholstrup and Toubro56, Reference Syvertsen, Halse and Hoivik58, Reference Gaullier, Halse and Hoye65–Reference Gaullier, Halse and Hoivik67, Reference Noone, Roche and Nugent106, Reference Tarnopolsky, Zimmer and Paikin154, Reference Eyjolfson, Spriet and Dyck155). Indeed the majority of results on the anti-diabetic properties of CLA relate to studies where only fasting plasma or serum glucose or insulin have been measured, are not the main focus of the study and typically have small sample sizes. Given these limitations, it is perhaps not surprising that the overall results show no effects of CLA supplementation(Reference Raff, Tholstrup and Toubro56, Reference Herrmann, Rubin and Häsler61, Reference Sluijs, Plantinga and de Roos62, Reference Gaullier, Halse and Hoye65–Reference Gaullier, Halse and Hoivik67, Reference Norris, Collene and Asp70, Reference Zhao, Zhai and Wang71, Reference Turpeinen, Ylönen and von Willebrand104, Reference Noone, Roche and Nugent106, Reference MacRedmond, Singhera and Attridge132, Reference Tarnopolsky, Zimmer and Paikin154) or consumption of CLA-enriched products(Reference Tricon, Burdge and Jones45, Reference Joseph, Jacques and Plourde63, Reference Racine, Watras and Carrel73, Reference Naumann, Carpentier and Saebo102, Reference Raff, Tholstrup and Basu103) on glucose and insulin. However, supplementing with a CLA mixture has shown beneficial effects on insulin resistance in healthy male subjects(Reference Eyjolfson, Spriet and Dyck155) and type 2 diabetic subjects(Reference Belury, Mahon and Banni74). In contrast, a negative effect on insulin resistance was reported in type 2 diabetic patients; however, this may have been due to the bias in the glucose tolerance between the supplementation and placebo groups and may not have been due to CLA supplementation(Reference Moloney, Toomey and Noone144).
c9, t11, cis-9, trans-11; t10, c12, trans-10, cis-12; M, male; F, female; RCT, randomised controlled trial; OGTT, oral glucose tolerance test; CLA mixture, 50:50 cis-9, trans-11- and trans-10, cis-12-CLA; EGC, hyperinsulinaemic-euglycaemic clamp; HOMA-IR, homeostasis model assessment of insulin resistance; QUICKI, quantitative insulin sensitivity check index.
A recent study also found increased insulin resistance in older obese subjects, but no effects of combined CLA–n-3 supplementation in lean or obese younger subjects or older lean subjects(Reference Ahren, Mari and Fyfe153). Supplementation with the individual isomers, c9, t11-CLA or t10, c12-CLA increased insulin resistance (+15%) in obese men with the metabolic syndrome(Reference Risérus, Arner and Brismar60, Reference Risérus, Vessby and Arnlöv78), whereas a CLA isomer mixture did not affect insulin resistance(Reference Risérus, Arner and Brismar60). Furthermore, lipid peroxidation increased relative to placebo when the individual isomers were administered, but the differences did not remain significant when adjusted for changes in lipid peroxidation(Reference Risérus, Arner and Brismar60, Reference Risérus, Vessby and Arnlöv78). The authors of these papers suggest that irrespective of the CLA isomer, CLA-induced lipid peroxidation may mediate insulin resistance. However, further work is required, particularly studies where the hyperinsulinaemic–euglycaemic clamp is utilised(Reference Risérus, Smedman and Basu152, Reference Risérus156). The conflicting responses to increased CLA intake in both human and animal studies do not currently imply compelling anti-diabetic properties of CLA. Thus, studies should be designed that provide rigorous measures of insulin resistance in subjects of varying age groups and weight status(Reference Alberti, Eckel and Grundy157).
Conjugated linoleic acid and bone health
Bone is a complex tissue system whereby the skeleton is continually renewed through the resorption (breakdown) of existing bone and the formation of new bone (remodelling). Peak bone mass in humans usually occurs late in the second or early in the third decade of life with a progressive decline in bone mineral density starting in the fourth decade of life for both men and women(Reference Prentice, Schoenmakers and Laskey158). Bone modelling (children and young adults) or remodelling (adults) is influenced by many factors including nutritional status, hormones and mechanical loading. One of the consequences of low bone turnover or remodelling is the development of osteoporosis, particularly in white, postmenopausal women. In the UK, the costs of osteoporosis to the National Health Service are estimated at £2.3 billion per year or £6 million per d, with almost 3 million individuals diagnosed with osteoporosis(159). Thus, strategies that attenuate decreases in bone mass are of great importance, with much of research focused on Ca, vitamin D, protein and vitamin K intakes(Reference Lanham-New160). However, other nutrients, including CLA, have been the focus of research due to influences on bone mass and metabolism(Reference Bhattacharya, Banu and Rahman18, Reference Roy and Antolic161–Reference Watkins and Seifert163).
The majority of work on CLA and bone metabolism has been conducted using human cells and animal models, particularly those reflecting postmenopausal women. Supplementation studies have demonstrated decreased PGE2 production in rats, but results were dependent on the CLA concentration levels(Reference Kelly and Cashman164–Reference Watkins, Shen and McMurtry168). PGE2 is an important factor in the regulation of bone metabolism, including bone formation as well as bone resorption(Reference Prentice, Schoenmakers and Laskey158). PGE2 production increases in postmenopausal bone loss due to oestrogen deficiency(Reference Prentice, Schoenmakers and Laskey158). CLA may also stimulate Ca absorption, thus making more Ca available for bone formation(Reference Kelly and Cashman164, Reference Jewell and Cashman169). Recently, Park et al. (Reference Park, Pariza and Park170) reanalysed previous studies in mice and showed that extra Ca (0.66%) in the diet improved CLA effects on bone mass in male, but not female mice. A recent review concluded that based on the current evidence from in vitro and animal studies the addition of CLA, overall, improves bone strength and density(Reference Roy and Antolic161). However, the majority of studies currently published were conducted using CLA isomer mixtures. Only two studies have examined the differences between the c9, t11 and t10, c12 isomers and found no direct effects on bone, but rather attenuation of parathyroid hormone concentration(Reference Weiler, Austin and Fitzpatrick-Wong171, Reference Weiler, Fitzpatrick and Fitzpatrick-Wong172).
Whilst there are numerous publications examining the effects of CLA and bone formation in cell and animal models, studies in human subjects are lacking (Table 7). Data from an observational study showed that in postmenopausal women dietary intake of CLA was a weak but significant predictor of Ward's triangle bone mineral density(Reference Brownbill, Petrosian and Ilich173). The same study also found that subjects with above median intake of CLA had higher bone mineral density of the forearm. In contrast, supplementation with a CLA mix (3.0–3.4 g/d) did not affect bone formation or resorption in healthy lean, overweight, obese men and women(Reference Gaullier, Halse and Hoye65–Reference Gaullier, Halse and Hoivik67, Reference Doyle, Jewell and Mullen174). A further two studies in young and elderly subjects who completed resistance training in addition to CLA supplementation (6 g/d) also demonstrated no change in bone mineral density and bone mass(Reference Tarnopolsky, Zimmer and Paikin154, Reference Kreider, Ferreira and Greenwood175). Brown et al. (Reference Brown, Trenkle and Beitz50) reported no change in bone mineral content when subjects consumed a CLA-enriched diet, although the study duration was only 56 d, an insufficient length of time for observing changes in bone mineral content. In children, significantly less bone mineral content accretion occurred in the CLA supplemented after 7 months(Reference Racine, Watras and Carrel73); however, the reasons are not fully elucidated. Currently, the only human study to demonstrate a positive association between CLA supplementation(5 g/d) and bone found a decrease in bone resorption markers and increase in LBM(Reference Pinkoski, Chilibeck and Candow55). However, this study did not identify whether the increases in LBM were due to increased muscle or bone mass and whether it was an artifact of the 7-week resistance training programme. Since there are relatively few human studies (four out of seven studies where bone was not the primary outcome examined), the lack of consistency in protocols, measurement of bone metabolites and small sample sizes hinder a clear conclusion between the effects of CLA and bone.
CLA mixture, 50:50 cis-9, trans-11- and trans-10, cis-12-CLA; M, male; RCT, randomised controlled trial; BMD, bone mineral density; F, female; BMC, bone mineral content; OGTT, oral glucose tolerance test.
The overall evidence from the studies examined here demonstrates a lack of definitive and reproducible results, particularly in relation to the consumption of naturally enriched CLA products, as the number of published studies is low relative to the number on synthetic supplements. The majority of randomised controlled trials are conducted with CLA supplements, with varying mixtures of isomers and dosage levels. However, the evidence from animal studies is promising, but extrapolation from animal to human studies is difficult due to the differences in the amount of CLA used. For example, in animal studies the observed benefits of CLA on bone are between 0.1–1% CLA of total weight of diet(Reference Brownbill, Petrosian and Ilich173). For men consuming on average 3.0 kg food and beverages per d, this is equivalent to 3–30 g CLA/d; for women consuming about 2.2 kg food and beverages per d, this equates to 2.2–22 g CLA/d(Reference Kant and Graubard176). In addition, given the differences in study protocols, relatively small sample sizes and other methodological issues (including measurement of dietary CLA intakes(Reference Ritzenthaler, McGuire and Falen8) and accurate measurement of body composition), it is not surprising that there is a lack of consensus on what health claims could be applicable to CLA, either natural or synthetic products. Current submissions on CLA health claims to the European Food Safety Authority (EFSA) include seven for body weight/LBM, two on immune function, two on antioxidant properties and one relating to insulin. The present review suggests that the only possible candidate would be in relation to the synthetic t10, c12-CLA isomer and reductions in body fat.
The present review was prepared as part of a project funded by Scottish Enterprise (Glasgow, UK). Nino Binns Consulting provides consultancy in nutrition and food regulation to a variety of commercial clients.
T. A. M. and E. M. K. drafted the review. J. M. W. W., N. B. and M. B. E. L. commented on the review.
There are no conflicts of interest.