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© 2005 The American Society for Nutritional Sciences J. Nutr. 135:422-430, March 2005

Human Nutrition and Metabolism

Consumption of Conjugated Linoleic Acid (CLA) from CLA-Enriched Cheese Does Not Alter Milk Fat or Immunity in Lactating Women1,2

Kristin L. Ritzenthaler, Michelle K. McGuire3, Mark A. McGuire*, Terry D. Shultz, Alfred E. Koepp, Lloyd O. Luedecke, Travis W. Hanson*, Nairanjana Dasgupta{dagger} and Boon P. Chew

Department of Food Science and Human Nutrition, Washington State University, Pullman, WA 99164-6376; * Department of Animal and Veterinary Sciences, University of Idaho, Moscow, ID 83844-2330; and {dagger} Program in Statistics, Washington State University, Pullman, WA 99164-3144

3To whom correspondence should be addressed. E-mail: smcguire{at}wsu.edu.


   ABSTRACT
TOP
 ABSTRACT
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Isomers of conjugated linoleic acid (CLA) decreased milk fat, altered immunity, and reduced the risk for cardiovascular disease (CVD) in some animals. The major form of CLA in the human diet is c9,t11–18:2 (rumenic acid; RA). We studied the effects of high RA consumption on plasma and milk RA concentration, milk composition, immunity, and CVD risk factors in lactating women (n = 36) assigned to 1 of 3 treatments: control, low CLA cheese (LCLA; 160 mg RA/d), or high CLA cheese (HCLA; 346 mg RA/d). The increase in plasma RA concentration between baseline and 8 wk in women consuming HCLA cheese was significantly greater than that of controls. At study completion (8 wk), milk RA concentration among women consuming HCLA cheese was greater (P < 0.05) than that of controls (0.37 vs. 0.26% of fatty acids). Treatment did not affect milk fat, protein, or lactose concentrations, immune indices (e.g., plasma T-helper cells and interleukin-2), or measured risk factors for CVD (e.g., plasma triacylglyceride and cholesterol). In summary, consumption of a RA-enriched cheese modestly increased plasma and milk RA concentrations without affecting total milk fat, plasma and milk indices of immunity, or selected risk factors for CVD.

KEY WORDS: • conjugated linoleic acid • milk fat • immunity • human milk • lactation

Conjugated linoleic acids (CLA)4 are geometric and positional isomers of linoleic acid (c9,c12–18:2) that occur naturally in dairy products and ruminant meats as a result of rumen biohydrogenation and endogenous conversion from trans-vaccenic acid [t11–18:1; (1,2)]. To date, little is known about the physiologic role of CLA in humans. However, numerous animal studies indicate that CLA may influence diverse physiologic functions and promote health with regard to cancer, heart disease, diabetes, bone formation, growth modulation, and immunity (3–9).

The 2 major CLA isomers with known physiologic activities are c9,t11–18:2 (rumenic acid, RA), found in dairy and beef products, and t10,c12–18:2, a predominant isomer in CLA supplements. Animal studies suggest that RA is the primary isomer responsible for CLA’s anticarcinogenic properties (10,11), whereas t10,c12–18:2 is the isomer linked to alterations in lipid metabolism (12), including milk fat depression (MFD) in ruminants (13).

Combined results from 2 human studies (14,15) indirectly suggest that t10,c12–18:2, but not RA, can decrease milk lipid concentrations in lactating women. Park and colleagues (14) investigated the effects of high vs. low dairy fat intake in a randomized, crossover study in lactating women; treatments were 7 d long. Consumption of a high dairy fat diet resulted in a greater milk lipid concentration compared with a low dairy diet [4.6 ± 0.5 vs. 3.8 ± 0.2% milk lipid, respectively (14)]. Although consumption of dairy products increased milk RA concentrations, RA intake was not correlated with milk lipid content, suggesting that alterations in milk lipid concentrations were not a result of enhanced dietary RA intake. Instead, the authors hypothesized that lower milk fat during the low dairy period was due to the substitution of trans fatty acid–containing partially hydrogenated vegetable oils (e.g., margarine) for dairy fat (e.g., butter). In addition, results from another study (15) showed that consumption of a commercial CLA mixture containing similar amounts of RA and t10,c12–18:2 reduced milk lipid in humans. Such results are supported by animal studies and suggest that MFD is linked to the intake of t10,c12–18:2 and not RA. These findings are of public health relevance because decreased milk lipid might decrease energy intake by breast-fed infants. Thus, it is important to confirm that increased dietary intake of RA (found naturally in dairy and beef products) does not decrease milk lipid concentration in humans.

An effect of CLA on various functions of the immune system was also demonstrated in a number of animal species. For example, supplementation with CLA was associated with increased lymphocyte proliferation in mice, rats, and pigs (3,16–18). Studies in pigs indicated that CLA supplementation increased peripheral CD8 lymphocyte subsets (18). Other studies showed that CLA supplementation influenced immune system components in rats and mice (19–21). Conversely, Kelley and colleagues (22) examined the effect of CLA supplementation on the immune status of healthy young women and found no effect. The discrepancy between animal and human data, as well as the dearth of human intervention studies in this area, highlight the importance of further investigation.

A protective effect of dietary CLA intake on risk of heart disease was also reported in a number of animal studies (5,7,23,24). However, data from human intervention trials suggested no beneficial effects (25–27). Once more, the lack of agreement between animal and human data suggests that further investigation is warranted.

The study described here was designed primarily to investigate the effect of dietary RA consumption on human milk lipid content. Secondarily, we were interested in studying the effect of RA intake on immune system variables and cardiovascular disease (CVD) risk factors in lactating women. We utilized a natural food (i.e., cheese) instead of commercial CLA supplements, because pure isomers of CLA were not available in quantities required for human intervention trials. We tested the hypotheses that consumption of RA-enriched cheese would have the following effects: 1) no effect on milk lipid, protein or lactose, but increase RA concentration of the milk, 2) enhancement of the immune response, and 3) no effect risk factors for CVD.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Subjects and study design. Healthy breast-feeding women (n = 44) between 1 and 10 mo postpartum were recruited from southeastern Washington and northwestern Idaho. All procedures were approved by the Washington State University and the University of Idaho Institutional Review Boards. Potential subjects completed a health history questionnaire, and subjects were screened for the absence of selected metabolic abnormalities using a fasting blood chemistry profile, which tested for liver function, plasma blood urea nitrogen, total protein, albumin, and iron concentrations. Potential subjects were excluded if they smoked, had allergies, were lactose intolerant, or had unusual dietary habits, eating disorders, or a recent history of dieting. Women taking vitamin-mineral supplements were asked to discontinue supplementation for 1 mo before the study and during the 8-wk intervention period.

The study was conducted between March and December 1999, and each subject participated for an 8-wk period. Upon enrollment, women were blocked by sets of 3 and assigned randomly to 1 of 3 groups: control, low CLA cheese (LCLA), or high CLA cheese (HCLA). The control group consumed no additional cheese during the study. Milk was obtained from the Washington State University herd and used for producing the LCLA cheese. Milk used for production of the HCLA cheese was produced by cows fed diets containing greater levels of forage (50% forage, 50% grain) and linoleic acid [soybean oil 9.5% of dry matter (28)]. This feeding regimen resulted in a milk containing a naturally enhanced concentration of RA (28). "Low CLA" and "high CLA" milks were made into experimental cheddar cheeses using standard procedures (Table 1). The overall fatty acid composition of the 2 experimental cheeses was quite different. However, the high CLA cheese contained twice as much RA as the low CLA cheese. Also, the term "CLA" in the remainder of this paper reflects the sum of CLA isomers identified (c9,t11–18:2, t10,c12–18:2, t9,t11–18:2 and t10,t12–18:2), despite the fact that dietary intake data include only the c9,t11–18:2 and t10,c12–18:2 isomers. The term 18:2 will be used in the remainder of this manuscript to refer to linoleic acid (c9,c12–18:2), as opposed to other 18:2 isomers (e.g., RA).


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  		TABLE 1 Fatty acid composition of LCLA and HCLA cheeses1

 
Subjects selected for cheese consumption (i.e., LCLA and HCLA groups) consumed 113 g of experimental cheese daily for 8 wk; the cheese was weighed by study personnel and delivered to subjects weekly. Subjects and investigators were unaware of the specific group to which women were assigned if they were to consume the experimental cheeses; however, both subjects and investigators were aware of which women were assigned to the control group. The LCLA and HCLA cheese contained 5.8 and 10.9 mg CLA/g lipid, respectively. Thus, the LCLA group received an additional 218 and 160 mg/d of CLA and RA, respectively, from the LCLA cheese. The HCLA group consumed an additional 407 and 346 mg/d of CLA and RA, respectively, from the HCLA cheese.

    Sample collection. Blood samples were collected from fasting subjects between 0645 and 1000 h on the last day of baseline (BL) and wk 2, 4, and 8. Milk was collected by complete breast expression using an electric breast pump on the same mornings before or immediately after blood collection. Subjects were asked to pump milk from the same breast throughout the study; subjects thoroughly emptied the breast at least 2 h before sample collection. Milk samples were collected and stored in polypropylene containers (29). Immediately after sample collection, milk collected for secretory IgA (sIgA) analysis was refrigerated, and milk collected for macronutrient analysis was frozen. Blood and milk samples were transported within 3 h of collection to the laboratory and either utilized immediately (immune analyses) or stored at –80°C. For sIgA analysis, fresh milk samples were centrifuged (1000 x g, 30 min, 10°C) to remove lipid and cellular components, and skim milk was stored at –20°C.

    Estimation of infant milk consumption. Infant milk consumption was estimated by weighing the infant before and after each feeding session at BL and at study completion using an electronic infant scale. The initial weight was subtracted from the final weight at each feeding to determine milk intake during the respective nursing session. Collectively, values from all feeding sessions within 24 h were used to estimate total daily milk intake by the infant.

    Anthropometric assessment. Maternal and infant weights (± 0.1 kg) were measured using electronic scales. Maternal height and infant length (±0.1 cm) were assessed using a wooden height board. All measurements were made during BL and the 8-wk time point. BMI was calculated as weight (kg) divided by height squared (m2).

    Dietary assessment. Subjects recorded 3-d weighed dietary records using portable electronic scales before blood collection during BL, 2, 4, and 8 wk for estimation of current dietary intake. At BL and 8 wk, chronic CLA intake was also estimated using a modified version of a previously utilized semiquantitative FFQ (30). Data collected by dietary records and FFQ were evaluated using a computerized nutrient database (Food Processor®, Version 7.02; ESHA Research) modified by us to contain quantities of total CLA and RA [mg/g fat, (30)] as well as the fatty acid composition of the LCLA and HCLA experimental cheeses (Table 1).

    Milk lipid. Milk lipid was extracted using a modified procedure of Ingalls and colleagues (31). For quantitative analysis of total milk lipid, the extraction procedure was repeated and completed with a final column rinse of 200 µL chloroform:methanol. For total milk lipid quantification, samples were completely dried under nitrogen and kept in a desiccator for 48 h before the final weight was determined. A 2nd set of milk samples was extracted for further fatty acid analyses.

    Plasma and milk fatty acids. Plasma and milk lipids were extracted and methylated using standard procedures (32,33). Samples were analyzed on a gas chromatograph (Hewlett Packard 6890, Agilent Technologies) equipped with a capillary column (Resteck #10641; 60 m, 0.25 mm i.d. with 0.5 µm film thickness; Stabelwax®; Restek). Helium was used as carrier gas at a constant flow mode with linear velocity set at 20 cm/s. The flame ionization detector was heated to 260°C, and detector gas flows were set at 40, 450, and 49 mL/min for hydrogen, compressed air, and nitrogen, respectively. Samples (1 µL) were injected in the splitless mode (injection temperature 260°C), with an initial oven temperature of 50°C and 4 min hold time increased by 10°C/min to 200°C and at 2°C/min to the final temperature of 240°C. The identities of fatty acid peaks were established by comparing retention times to a 14-component C8-C24 fatty acid methyl ester standard mixture (Sigma-Aldrich Chemical), C6 and C19 (Nu-Chek-Prep), a CLA mixture (Matreya), pure t10,c12–18:2 (Matreya), and an anhydrous milk fat reference standard (CRM 164; European Community Bureau of Reference). A pooled plasma sample set was run with each assay, and all samples were assayed in duplicate. Interassay CV for 18:2 and RA were 5.1 and 14.9% in plasma, and 4.0 and 21.4% in milk, respectively.

    Milk protein and lactose. Milk protein was analyzed spectrophotometrically using a procedure for human milk described by Polberger and Lönnerdal (34). Bovine serum albumin was used as a standard (Protein Assay Kit II, Bio-RadTM). Milk lactose was analyzed spectrophotometrically as described by Polberger and Lönnerdal (34). A pooled sample was analyzed with each assay, and all samples were analyzed in duplicate. Interassay CVs for protein and lactose were 2.1 and 3.8%, respectively.

    Blood lymphocyte proliferation. Peripheral blood lymphocytes collected at BL, wk 4, and 8 were isolated by ficol gradient centrifugation, washed, and resuspended to 2 x 106 cells/mL in RPMI 1640 supplemented with 10% fetal bovine serum, 100 kU/L penicillin and 100 mg/L streptomycin (Sigma-Aldrich Chemical). The assay was performed in triplicate in the presence of pokeweed mitogen (5 mg/L final concentration), concanavalin A (10 mg/L) or phytohemagglutinin (30 mg/L) (Sigma-Adrich Chemical) and cell proliferation determined by [3H]-thymidine (1 µCi/well; ICN-Pharmaceuticals) uptake (35). Data are expressed as adjusted dpm (dpm of mitogen-stimulated cultures minus dpm of unstimulated cultures).

    Plasma interleukin 2. Interleukin-2 production by concanavalin A–stimulated peripheral blood mononuclear cells was analyzed by ELISA (Cytokine DirectTM Human interleukin 2 ELISA, Intergen®).

    Immunophenotyping. Populations of blood T-helper (CD3+, CD4+), T-cytotoxic (CD3+, CD8+), B (CD3–, CD19+) and natural killer (NK) (CD3–, CD16+, CD56+) cells were analyzed (Caltag) by flow cytometry (FACSCaliber, Becton Dickinson) as previously described (36). Lymphocyte populations were gated using CD45-fluorescein thiocyanate/CD14-phycoerythrin markers. Data analyses were performed using matching isotypic Ig controls.

    Milk sIgA. sIgA was measured by single radial immunodiffusion using goat antiserum against human sIgA (ICN-Pharmaceuticals) as previously described (37).

    Plasma triacylglycerides (TAG) and cholesterol (C). Total cholesterol (TC), HDL-C and TAG concentrations were quantified using an enzymatic, end-point determination method utilizing an automated Roche Hitachi 917 analyzer (Roche Diagnostics). Concentrations of LDL-C and VLDL-C were determined by calculation from TC and HDL-C (38).

    Statistical analyses. Descriptive statistics and ANOVA procedures were performed using MinitabTM Statistical Software (Release 13.0). To include unstructured and autoregressive tests in addition to compound symmetry for repeated-measures analyses, data were also analyzed using the General Linear Models procedure of the Statistical Analysis System (SAS, Release 8.01). To test for any seasonal variations that could potentially affect the immune system, subjects were blocked in groups of 3 at the time of enrollment. However, no effect of block for any measurement was found; thus, the following variables were included in the final ANOVA model: treatment (control, LCLA, HCLA), time (BL and 2, 4, 8 wk), subject (treatment), and time x treatment.

Women were also divided into 2 groups depending on whether they gained or lost weight during the study. The "weight loss" group (n = 20) lost 1.5 ± 0.3 kg (range: 0.1–3.8 kg), and the "weight gain" group (n = 16) gained 1.4 ± 0.4 kg (range: 0.2–5.6 kg). Mean BMI did not differ significantly between these groups at BL (24.4 ± 0.6 vs. 27.6 ± 1.7 kg/m2 for weight loss and gain groups, respectively). Women were also divided into low and high BMI groups (21.7 ± 0.6 vs. 29.9 ± 0.9 kg/m2, respectively), but this variable did not relate to any outcome that we measured. All group mean comparisons were made within and between control and treatment groups utilizing Tukey’s multiple comparison test. To meet assumptions of normality, data were log transformed when necessary.

Multiple regression analyses were performed to determine whether treatment and/or time postpartum were related to infant milk RA intake. In addition, the independent and interactive effects of maternal RA intake and plasma RA concentrations on changes ({Delta}) in plasma RA, milk RA, and milk lipid during the study were investigated. The analyses were performed in 2 ways with the following independent variables included in each model: 1) BL dietary or BL plasma RA, BL BMI, and treatment, and 2) changes ({Delta}) in dietary RA, plasma RA, body weight, and BL BMI. Changes ({Delta}) represent differences in specified variables between 8 wk and BL. Last, to investigate the relations among diet, plasma, and milk RA as well as milk lipid at individual time periods, maternal BMI and RA intake or plasma RA were included in the model.

For all analyses, main effects were considered significant at P ≤ 0.05. Interaction terms were considered significant at P ≤ 0.1. Data presented in the text represent means ± SEM.


   RESULTS
TOP
 ABSTRACT
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    Description of subjects. A total of 36 women and their infants completed the study. Of the 8 subjects who did not complete the study, 3 discontinued due to perceived constipation of the nursing baby, 1 disliked the amount and monotony of cheese consumption, 1 withdrew for personal reasons, 2 were not compliant, and 1 subject moved during the study. The control and intervention groups each contained equal numbers of subjects (n = 12) participating in the study, although data of 1 subject (in the LCLA group) were collected only through wk 4 and were included in the analyses when possible. At baseline, there were no differences among treatment groups for any demographic or anthropometric variable (Table 2). Furthermore, treatment had no effect on maternal weight or BMI at the conclusion of the study, nor did it influence change in infant weight or length (data not shown).


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  		TABLE 2 Demographic and anthropometric variables of women and infants in control, LCLA, and HCLA groups at baseline1

 
    Maternal macronutrient and fatty acid intake. At BL, dietary intake variables did not differ by treatment group (Table 3). However, there was a significant treatment x time interaction for the percentage of energy (%en) derived from carbohydrates and fat as well as saturated fat and cholesterol. The %en derived from carbohydrates decreased (P < 0.05) in both cheese treatment groups during the first 2 wk with no further changes thereafter, although this variable was not different between control and treatment groups at any time period. The %en derived from fat increased (P < 0.05) in the LCLA group by 2 wk and in the HCLA group by 8 wk with no differences observed between control and intervention groups at any time period. During the initial 2 wk, consumption of experimental cheeses increased (P < 0.05) the intake of saturated fat by 53 and 49% in the LCLA and HCLA groups, respectively. At study completion, intake of saturated fat was higher (P = 0.05) in the HCLA group compared with the control group, with no other differences observed among groups. Compared with BL, the intake of cholesterol was higher (P ≤ 0.05) at study completion in the HCLA group. There was no effect of treatment on intakes of monounsaturated and polyunsaturated fats. There was an effect of time on the %en derived from protein (P < 0.05). Carbohydrate intake (g/d) decreased over time, regardless of treatment, resulting in overall lower carbohydrate intake at 8 wk compared with BL (257 ± 16 vs. 319 ± 17 g/d, P < 0.05). Interestingly, in spite of the inclusion of an energy-dense food (i.e., cheese), total energy intake did not change throughout the study in either intervention group.


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  		TABLE 3 Maternal daily dietary intakes of macronutrients, fatty acid classes, and fatty acids of lactating women in control, LCLA, and HCLA groups1

 
There was a significant treatment x time interaction for intakes of 12:0, 14:0, and 16:0, as well as independent treatment and time effects for 18:0. Because intakes of 12:0, 14:0, 16:0, and 18:0 remained unchanged in the control group but increased significantly in the intervention groups, increased intakes of these fatty acids probably reflected intake of the experimental cheeses as opposed to other dietary changes of subjects.

As expected, there was a treatment x time interaction for CLA and RA intakes (Table 3). At 2 wk, intakes of CLA and RA had increased by 102 and 95%, respectively, in the LCLA group and 191 and 210%, respectively, in the HCLA group. Dietary CLA and RA intakes were higher (P < 0.005) in the HCLA compared with the LCLA and control groups at 2, 4, and 8 wk. The LCLA group also consumed more (P ≤ 0.05) CLA and RA than did the control group at 4 and 8 wk.

Chronic CLA and RA intakes were also assessed by FFQ at BL and 8 wk. At BL, chronic CLA and RA intakes did not differ among treatment groups and were estimated to be 263 ± 28 and 204 ± 22 mg/d, respectively. Overall estimates of chronic intakes were higher (P < 0.05) than estimates of current intakes (3-d dietary records) at BL (204 ± 22 and 145 ± 13 mg/d, respectively). Treatment was effective in raising estimated chronic CLA and RA intakes (FFQ) at 8 wk. At study completion, chronic dietary CLA and RA intakes were estimated to be 464 ± 71 and 344 ± 56 mg/d, respectively, for the LCLA group and 566 ± 39 and 470 ± 36 mg/d, respectively for the HCLA group. At study completion, estimates of chronic intake (FFQ) did not differ from estimates of current intake (dietary records).

    Immunity-related micronutrient intake. There were no treatment x time or independent treatment effects on selected nutrients reported to influence immunity (e.g., vitamins A, B-6, C, and E, carotenoids, zinc, and selenium; data not shown). However, there was a significant effect of time on zinc intake. Zinc intake of all groups increased from 12.3 ± 0.8 to 14.8 ± 0.9 mg/d between BL and 2 wk (P < 0.05) and remained unchanged thereafter. For the majority of the selected nutrients, intakes were greater than or equal to current Dietary Reference Intake recommendations (39–41). Vitamin E intake (8.3 ± 0.4 {alpha}-tocopherol equivalents, mg/d) was the exception with intakes below the current Recommended Dietary Allowance (41).

    Plasma TAG, cholesterol, and fatty acid concentrations. There were no interactive or independent effects of treatment and time on plasma TAG, TC, HDL-C, LDL-C, or VLDL-C concentrations (data not shown). A significant treatment by time interaction was observed for plasma concentrations of 15:0 and 18:0 (Table 4). However, there were no pairwise differences among treatment groups or time periods for these fatty acids. There was an effect of time on plasma concentrations of 14:0, 16:0, 17:0, and 18:2 (c9,c12). Plasma concentrations of 14:0 and 16:0 exhibited no consistent patterns within or among groups. Plasma concentration of 17:0 increased (P < 0.05) over the course of the study in all 3 groups. The plasma concentration of 18:2 (c9,c12) tended (P = 0.06) to decrease between BL and 4 wk.


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  		TABLE 4 Plasma fatty acids in lactating women in control, LCLA, and HCLA groups1

 
There were no treatment or time effects on plasma concentrations of any identified CLA isomer. Although adjusting for BL concentrations resulted in a significant treatment x time interaction for RA, none of the pairwise comparisons between adjusted values were significant. The addition of the "weight group" variable to the statistical model revealed a significant effect of this variable (P < 0.05) on plasma RA, showing that women who gained weight had higher (P < 0.006) plasma RA concentrations at 8 wk than women who lost weight during the study. Further analysis using multiple regression indicated that HCLA treatment influenced changes in plasma RA; the change in plasma RA concentration between BL and 8 wk in women consuming HCLA cheese was greater (P < 0.05) than that of controls. This relation remained after controlling for RA intake (current and chronic) and maternal BMI at BL.

    Milk composition. There were no significant interactive or independent effects of treatment or time on milk lipid, protein and lactose concentrations (data not shown). Overall, milk lipid was 3.3 ± 0.4 g/100 g, and protein and lactose concentrations were 7.3 ± 0.1 and 70.4 ± 0.8 g/L, respectively. Adjustment for maternal BMI, plasma RA concentration, and energy intake did not influence these results.

There was a treatment x time interaction on milk concentrations of 18:0 and RA (Table 5). Milk 18:0 increased (P ≤ 0.05) in the LCLA group from BL to 4 wk; a similar trend (P = 0.06) was observed for the HCLA group during the first 2 wk. Milk RA concentration of the HCLA group tended (P = 0.06) to increase between BL and 2 wk and was higher (P < 0.05) than in the LCLA group at 2 and 4 wk. After adjusting for BL data, the treatment x time interaction remained significant; results of pairwise comparisons suggested an increase (P < 0.05) only within the HCLA group between BL and 2 wk. Further analysis using multiple regression indicated that after controlling for BL maternal BMI and RA intake (current and chronic) or plasma RA, the effect of treatment remained significant. After adjusting for these variables, women in both treatment groups had higher milk RA concentrations throughout the study compared with controls.


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  		TABLE 5 Milk fatty acids in lactating women in control, LCLA, and HCLA groups1

 
There were independent effects of treatment and time on milk 14:0, 15:0, and 18:2 concentration. Milk 14:0 and 15:0 increased by 4 and 2 wk, respectively, in the LCLA group. Overall, milk 18:2 (c9,c12) was higher (P < 0.01) in the control compared with the LCLA group, and its concentration decreased (P < 0.005) between BL and 4 wk. A treatment effect was observed for 18:3; women in the control group had higher (P < 0.05) concentrations than LCLA women. There was an effect of time on 12:0, 16:0, 17:0, and t9,t11/t10,t12–18:2; 12:0 was lower (P < 0.05) at BL compared with 8 wk, and 17:0 was lower (P < 0.05) at BL compared with 4 wk. Milk concentration of 16:0 increased among LCLA women between BL and 4 wk. Milk t9,t11/t10,t12–18:2 was higher (P < 0.05) at 2 and 4 wk compared with BL.

    Infant CLA and RA intakes from breast milk. There were no interactive or independent effects of treatment or time on infant milk intake (data not shown). Overall, infant milk intake was 769.1 ± 28.7 g/d. The interaction between dietary group and time was not significant for either infant intake of t10,c12–18:2 or total CLA. However, an independent effect of time existed for intake of t10,c12–18:2 and total CLA from breast milk, which decreased from 8.7 ± 1.0 to 6.5 ± 0.8 mg/d (P < 0.05) between BL and 8 wk and from 113.0 ± 5.9 to 86.9 ± 5.6 mg/d (P < 0.01) for t10,c12–18:2 and total CLA, respectively. Interestingly, although total CLA intake from milk decreased slightly in all groups, only in the control group was this significant; infant CLA intake in controls decreased from 114.5 ± 15.5 mg/d at BL to 68.0 ± 9.3 mg/d at 8 wk (P < 0.05).

Furthermore, no interactive or independent effects of treatment or time were observed for infant intakes of RA and t9,t11/t10,t12–18:2 from breast milk, but a similar decrease (P < 0.05) in infant RA intake among infants nursed by control women was observed. Daily intakes of RA and t9,t11/t10,t12–18:2 from breast milk were estimated to be 74.8 ± 6.7 and 17.1 ± 1.1 mg/d, respectively. Note that the calculation used to estimate daily milk CLA intake is based on milk lipid concentration from a single milk sample taken in the morning and should be interpreted cautiously because diurnal fluctuation of milk lipid is not accounted for by this calculation.

    Relations among body composition, RA intake, plasma and milk RA concentrations, and milk lipid. Changes in weight and RA intake over the course of the study as well as BL maternal BMI were evaluated as possible predictors of change in plasma and change in milk RA concentrations. Changes in RA intake positively predicted (P < 0.05) change in plasma RA concentrations; the relation was not affected by maternal BMI or change in weight. Furthermore, the interaction between change in weight and change in RA intake negatively predicted change in milk RA concentration. A separate analysis indicated that among women who had increased their RA intake throughout the study (n = 31), those women who lost weight, had the largest increase of RA intake, or experienced a combination of both weight loss and increase in RA intake, had the largest increase in milk RA during the study. In addition, change in plasma RA positively predicted change in milk RA. Again, the relation was not affected by maternal BMI or change in weight. No significant predictors of change in milk lipid were found using this model.

To further investigate the relation among diet, plasma and milk RA, as well as milk lipid, exploratory regression analyses were conducted within each time period. Rumenic acid intake positively predicted plasma and milk RA (P < 0.005 and 0.05, respectively) at 2 wk and milk RA (P < 0.05) at 8 wk. Although controlling for maternal BMI did change the relation between RA intake and plasma RA at 2 wk, a positive (P < 0.05) correlation between BMI and plasma RA existed at 8 wk. Furthermore, plasma RA correlated positively with milk RA at 2 and 4 wk; controlling for maternal BMI did not modify this relation. No relations among maternal BMI, RA intake, and plasma RA were apparent in explaining the variation in total milk lipid at any time. The adjusted R2 of all discussed multiple regression models ranged from 0.22 to 0.26.

Finally, we investigated whether maternal weight gain/loss was related to milk fatty acid concentration at 8 wk. Analysis showed that there were no marked differences in any milk fatty acid (including CLA) between the "weight gain" and "weight loss" groups and no difference between these groups in regard to dietary fatty acid intake, including CLA.

    Immune indices. A significant treatment x time interaction was observed for T-cytotoxic cells; cell percentages in the control group decreased (P < 0.05) between BL and 4 wk and again at 8 wk (Table 6). An effect of time was observed for NK cells; overall, NK cell percentages decreased (P < 0.05) between BL and 4 wk. There was neither a treatment x time interaction nor independent effects of treatment or time for any of the remaining immune indices.


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  		TABLE 6 Effect of dietary intervention (control, LCLA and HCLA) on selected immune system components in lactating women1

 

   DISCUSSION
TOP
 ABSTRACT
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Consumption of HCLA cheese resulted in a greater increase in plasma RA concentration compared with the control and LCLA groups. Consumption of RA-enriched cheese increased milk RA concentration only after controlling for RA intake at BL or plasma RA concentration and maternal BMI at BL. Treatment did not affect milk lipid, lactose, or protein concentrations. Commonly assessed CVD risk factors, such as plasma TC, TAG and lipoprotein cholesterol concentrations also were unaffected by treatment. Taken together, these findings somewhat support our initial research hypotheses that 1) consumption of RA-enriched cheese would increase milk RA concentration without affecting milk lipid content and 2) increased RA intake would not be associated with an increase in CVD risk factors. However, contrary to our initial hypothesis, selected immune indices were not enhanced by consumption of RA-enriched cheese.

Rumenic acid intake at BL and during the study was similar to data reported previously in human CLA intervention studies (14,15,22,27). Furthermore, BL plasma RA and milk RA concentrations are in agreement with BL values reported in U.S. women (14,27), but milk RA was slightly lower than reports of German and Canadian women (42,43), most likely reflecting contrasting chronic dietary habits among countries. Similarities in RA intakes at BL, plasma and milk RA concentrations, and RA intake during cheese intervention suggest that data presented here are comparable to those of previous human CLA studies.

Although consumption of RA-enriched cheese increased RA intake, enhancement of plasma RA as a result of cheese intervention was observed only when considering changes in plasma RA concentrations over the course of the study (BL to 8 wk), rather than at individual time points (i.e., 2, 4, 8 wk). Because the HCLA group consumed an additional 346 mg RA/d, these findings were somewhat surprising. In contrast, plasma RA concentration of lacto-ovovegetarian males (n = 9) increased significantly after a 4-wk period of cheese supplementation, providing 178 mg RA/d (44), an amount that is very similar to that consumed by women in the LCLA cheese group (160 mg/d). On the other hand, data show that regardless of treatment, the largest increase in RA intake was related to the greatest plasma RA response, explaining why consumption of only the HCLA cheese successfully increased plasma RA concentrations. Interestingly, nursing mothers who gained weight throughout the study had higher plasma RA concentrations at study completion, a relation that was not influenced by differences in RA intake. Subjects gaining weight also had higher (P < 0.05) concentrations of plasma lipids (i.e., TC, TAG and VLDL-C). Because CLA is present in various plasma lipid fractions (i.e., TAG, cholesteryl ester, and phospholipase), higher plasma RA concentrations in women gaining weight may simply be a reflection of higher plasma lipid concentrations (45,46). Future studies are warranted to elucidate the relations among energy balance, RA metabolism, and nutrient partitioning.

Consumption of experimental cheeses also modestly increased milk RA concentrations. Changes in milk RA concentrations were also related to corresponding changes in plasma RA. Moreover, regardless of treatment, women who lost weight and increased RA intake experienced the largest increase in milk RA concentrations. These results provide evidence that RA consumption and weight loss, most likely due to adipose tissue fatty acid mobilization, contribute to increased milk RA concentration. In contrast, BMI was not a predictor of milk RA concentrations. However, considering that BMI represents only a weak indicator of body composition, future studies should utilize accurate measurements of the percentage of body fat to gain insight into the role of body composition in regulating plasma and milk RA concentrations. Note that we did not quantify concentrations of trans-vaccenic acid, a major precursor of RA in ruminant milk lipid (2) and human plasma (47). Thus, a more complete interpretation of the relation between RA intake and plasma/milk RA concentrations requires the consideration of both lipid mobilization as an important source of plasma and/or milk RA and endogenous RA production.

Although the response of milk RA to dietary intervention was less than what was originally expected, comparisons with published data support our results. For example, in a previous study of 20 lactating women, a significant correlation between maternal fatty acid intake and milk fatty acid concentrations was found only for the PUFA 18:2 (48). Furthermore, Scopesi and colleagues (49) reported that a significant correlation between maternal intake and milk fatty acids in mature milk existed only for PUFA. Taken together with recent findings that RA shows metabolic characteristics of trans- and cis-18:1 positional isomers rather than 18:2 (50), the expected response of milk RA to maternal RA intake is not similar to 18:2 (c9,c12).

Using the assumption that 10% of dietary fatty acids are transferred to human milk (51), we estimated the approximate contribution of maternal RA intake to milk RA concentration. These calculations suggested that at BL, ~17 and 18% of daily infant RA intake in the LCLA and HCLA groups, respectively, originated from current maternal RA intake. In contrast, at study completion, the contribution of current dietary RA to total milk RA intake by the infants had risen to 40 and 57%, for the LCLA and HCLA groups, respectively. These results either do not support the widely held believe that 10% of dietary fatty acids are transferred into the milk, or they indicate a shift in dietary RA partitioning between mammary and adipose tissues. To fully understand the mechanisms regulating RA partitioning, further research should strive to utilize stable isotope methodologies.

Our data support the hypothesis that increased consumption of RA, the major CLA isomer in milk lipid, does not influence milk fat. These conclusions remain robust even after controlling for BMI, RA intake, and plasma RA. Our findings support the hypothesis of Park and colleagues (14) that the decreased milk lipid content present during low dairy consumption did not result from lower RA intake during this period. Furthermore, because milk fat decreased in lactating women after supplementation with a commercially available CLA supplement, which contained equal amounts of the RA and t10,c12–18:2 isomers (547 and 560 mg/d, respectively), our data indirectly support the proposition that t10,c12–18:2 is likely the isomer responsible for MFD in humans (15). Consequently, our results strongly agree with animal data that RA is not the CLA isomer linked to MFD in lactating animals. Furthermore, other milk macronutrients such as protein and lactose were also unchanged by treatment. These findings are of physiologic importance, not only because milk fat is the major source of energy and the only source of essential fatty acids to exclusively breast-fed infants, but also provide evidence that consumption of CLA in the form of a dairy product during lactation poses no risk to nursing infants in regard to energy intake.

Immune indices including lymphocyte proliferation, selective lymphocyte subsets, interleukin 2, and sIgA were not altered, thus supporting the findings of Kelley and colleagues (22,52) who investigated the effect of CLA supplementation in young healthy women (n = 17) participating in a more controlled metabolic study. Moreover, no changes in lymphocyte proliferation, NK cell activity, and cytokine production were reported by Albers and colleagues (53) who administered CLA supplements (1.7 g/d, 50% RA and t10,c12–18:2; 80% RA and 20% t10,c12–18:2) to healthy men (n = 71, 30–70 y) over a 12-wk period. Consequently, our results are in agreement with human studies that consumption of RA at this level has no measurable effect on the immune system.

Although the daily addition of 113 g of cheddar cheese to diets of lactating women resulted in a significant increase in the %en from fat, the increase was compensated for in part by a decrease in energy from carbohydrates. Thus, cheese consumption led to a redistribution of energy derived from these macronutrient classes. Furthermore, the intervention also increased saturated fat and cholesterol intakes. Despite the potential risk of CVD associated with increased intake of saturated fatty acids (54), TAG, TC, and LDL- and HDL-C concentrations remained unaffected by cheese treatment and were within the optimal/desirable range reported by the NIH (55). Thus, increased cheese consumption, regardless of cheese type, was not in any way detrimental as reflected by CVD risk factors assessed in these lactating subjects.

In summary, consumption of RA-enriched cheese over a period of 8 wk did not affect either human milk macronutrient content or CVD risk factors. Furthermore, immune indices remained unaffected by treatment. Maternal consumption of RA-enriched cheese inhibited the significant decline in infant RA intake from human milk that occurred in control subjects. The lack of agreement between human and animal studies in the area of immunity and CVD warrants further investigation. Potential long-term benefits to lactating women and breast-fed infants are not known at this time, although it is possible that exposure to CLA during these periods of growth and development might influence long-term risk of a variety of chronic diseases including mammary cancer and obesity.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Teri Wong and Philip Mixter for their expertise with immune analyses and Julie Woods, Nicole Anderson, and Emily Wiens for technical assistance and data entry.


   FOOTNOTES
 
1 Presented at Experimental Biology 2002 in New Orleans, LA and published in part as [Ritzenthaler, K. L., McGuire, M. K., Shultz, T. D., Luedecke, L. O., Koepp, A., Chew, B., Dasgupta, N., Hanson, T. W. & McGuire M. A. (2002) Consumption of conjugated linoleic acid (CLA)-enriched cheese increases milk and plasma CLA but does not alter total milk lipids, immunity or risk factors for heart disease during lactation. FASEB J. 16: A662 (abs.)]. Back

2 Supported in part by grants from Dairy Management, Incorporated and the Washington State Dairy Products Commission. Back

4 Abbreviations used: BL, baseline; C, cholesterol; CLA, conjugated linoleic acid; CVD, cardiovascular disease; %en, percentage of energy; HCLA, high CLA cheese; LCLA, low CLA cheese; MFD, milk fat depression; NK, natural killer; RA, rumenic acid; sIgA, secretory IgA; TAG, triacylglyceride; TC, total cholesterol. Back

Manuscript received 19 December 2003. Initial review completed 24 February 2004. Revision accepted 10 December 2004.


   LITERATURE CITED
TOP
 ABSTRACT
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
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