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Long-chain n–6 polyunsaturated fatty acids in breast milk decrease the risk of HIV transmission through breastfeeding ^1,2,3

¹ From the Departments of Nutrition (EV, JF, HC, and WWF), Immunology and Infectious Diseases (INK), and Epidemiology (EV and WWF), Harvard School of Public Health, Boston, MA; the Department of Community Health, Brown University, Providence, RI (AB); and the Departments of Microbiology and Immunology (SA) and Pediatrics and Child Health (KM), Muhimbili University College of Health Sciences, Dar es Salaam, Tanzania

² Supported by the National Institute of Child Health and Human Development (R01 HD045134).

³ Reprints not available. Address correspondence to E Villamor, Department of Nutrition, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115. E-mail: evillamo{at}hsph.harvard.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Background: Breastfeeding accounts for a sizable proportion of infant HIV infections. Some fatty acids (FAs) are potent immunomodulators with virucidal activity, and their primary source in breastfed children is breast milk.

Objectives: The aims of the study were to examine whether the percentage weight concentration of FAs in breast milk was associated with the risk of mother-to-child transmission (MTCT) of HIV by breastfeeding and with shedding of cell-free virus (CFV) or cell-associated virus (CAV) in breast milk.

Design: We conducted a case-control study nested within a cohort of HIV-infected Tanzanian women and children. We matched 59 incident breastfeeding MTCT cases to 59 nontransmitting controls based on the child's age at sample collection. We quantified FAs, CFV, and CAV in a breast milk sample collected before the infant's first positive HIV test.

Results: After adjustment for indicators of maternal HIV disease stage, the risk of MTCT was inversely related to 11c,14c-eicosadienoic acid [odds ratio (OR) for quartile 4 compared with quartile 1: 0.21; P for trend = 0.04], arachidonic acid (OR: 0.21; P for trend = 0.03), and dihomo--linolenic acid (OR: 0.24; P for trend = 0.03); the latter 2 were also linearly, inversely related to virus shedding in breast milk. Lauric acid and pentadecanoic acid were associated with increased MTCT, whereas trans FAs were related to higher CAV and CFV.

Conclusion: Increasing concentrations of long-chain n–6 polyunsaturated FAs in breast milk might reduce the risk of MTCT.

Key Words: Fatty acids • breast milk • HIV • mother-to-child transmission • arachidonic acid • breastfeeding • viral load

	INTRODUCTION

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By the end of 2006 there were 2.3 million HIV-infected children in the world, at least 2 million of whom were living in sub-Saharan Africa (1). In the absence of interventions, up to 40% of all infant infections in this region is due to mother-to-child transmission (MTCT) through breastfeeding. Although complete avoidance of breastfeeding would theoretically prevent MTCT by breast milk, this option is usually not acceptable, feasible, affordable, sustainable, or safe in the countries most heavily hit by the HIV epidemic. In such settings, exclusive breastfeeding appears to carry a lower risk of postnatal HIV transmission than does early mixed feeding (2). Thus, the World Health Organization (WHO) has recently recommended that HIV-infected women exclusively breastfeed for up to 6 mo or until the conditions for optimal replacement feeding are met (WHO, HIV and Infant Feeding Technical Consultation, Geneva, Switzerland, October 25–27, 2006). In the context of this recommendation, it is critical to identify potentially modifiable factors that contribute to the risk of HIV transmission through breast milk.

Fatty acids (FAs) are milk constituents with strong immunomodulating potential (3). In breastfed children, breast milk is the main source of essential polyunsaturated FAs (PUFAs; -linolenic acid and linoleic acid) and their n–3 and n–6 long-chain derivatives (LCPUFAs). Studies indicate that LCPUFAs, including docosahexaenoic acid and arachidonic acid, are essential for the development of T cell function in infants (4). In addition, several experiments have suggested that arachidonic acid and other free FAs exhibit virucidal activity against encapsulated viruses in vitro (5, 6). Given that HIV shedding in breast milk is a strong predictor of transmission (7-10) and considering the noted immunomodulatory and virucidal potential of FAs, we hypothesized that the percentage weight concentration of FAs in breast milk could be associated with the risk of MTCT, possibly through modulating the concentration of cell-free or cell-associated HIV in milk. We examined this hypothesis by conducting a nested case-control study among HIV-infected women and their children in Tanzania.

	SUBJECTS AND METHODS

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Study population
We conducted a case-control study nested within the cohort of women and children who participated in the Tanzania Trial of Vitamins. In that randomized, double-blind, placebo-controlled trial, 1078 HIV-infected women were recruited during pregnancy to investigate the effects of daily vitamin A, multivitamin (B, C, and E), or both supplements on MTCT and HIV disease progression. The study design and methods were previously described in detail (10, 11). At recruitment, we obtained information on the women's sociodemographic characteristics, clinical and immunologic HIV disease stage, including a CD4 cell count, and potential risk factors for transmission of HIV. During follow-up, we collected data on the outcome of pregnancy, clinical progression of HIV disease, morbidity, and mortality of both the mothers and their children.

Information on vertical transmission of HIV was obtained by testing blood samples collected from the child at birth, 6 wk after birth, and every 3 mo thereafter. In children who were <18 mo of age, HIV status was tested by polymerase chain reaction with the use of the Amplicor HIV detection kit (Roche Diagnosis Systems, Branchburg, NJ). When children became 18 mo of age, we determined HIV status with the use of enzyme-linked immunosorbent assay and confirmed positive results by Western blot. Children who tested negative by 6 wk and positive thereafter were presumed to have been infected by breastfeeding. At delivery and at 3 monthly intervals thereafter, we collected 10 mL breast milk from either breast by manual expression. It was not possible to standardize the time from collection since the last nursing; however, the FA composition of breast milk does not change during feeding, between breasts, or by time from nursing, and a single sample taken at any time is deemed to be adequate for FA composition analyses (12, 13).

The study was approved by the Human Subjects Committee of the Harvard School of Public Health and the Research and Publications Committee of Muhimbili University College of Health Sciences. All participants provided informed consent before enrollment.

Study design
We defined cases to be women who transmitted HIV-1 to their infants through breast milk. Sixty-one such incident cases were identified. We selected one breast milk sample per case to be tested for FA concentrations and for cell-free and cell-associated HIV-1. We aimed to select samples that had been collected before the child's first HIV-positive test. Such samples were not available in 2 cases; therefore, the final number of cases considered was 59. In 38 cases, a sample was available between 4 and 24 wk before the first positive test; in 11 cases, the only breast milk sample available was at the same time as the child's first positive test (n = 8) or within the prior 4 wk (n = 3) and in another 10 cases, the sample was collected >24 wk before the first positive test.

We selected one control per case from the pool of mothers whose child's final HIV status was negative after the end of breastfeeding. Controls were individually matched to cases on the time since delivery at which the breast milk sample was collected ± 1 wk. Typically, 2–3 potential control samples per case were identified, from which one was randomly selected.

Laboratory methods
Breast milk samples were immediately separated into two 5-mL aliquots on collection. One aliquot was centrifuged at 1500 x g for 12 min at 4 °C to obtain a cell-free aqueous milk fraction and a milk-cell pellet, and the other was kept as whole milk. All samples were stored in Dar es Salaam at –70 °C and later transported in hermetic containers filled with liquid nitrogen to the Harvard School of Public Health in Boston.

FAs were quantified in the whole-milk aliquots. The samples were thawed at room temperature for 30 min and shaken to ensure homogeneity. FAs were extracted from a hexane and isopropanol (3:2 by vol) and sodium sulfate (6%) mixture containing the sample and were esterified with methanol and acetyl chloride as described by Lillington et al (14). After esterification, the methanol and acetyl chloride were evaporated, and the FA methyl esters were redissolved in isooctane. The methyl esters were measured by gas-liquid chromatography under the following conditions: fused silica, capillary cis or trans column (100 m x 250 mm internal diameter, with a 0.20-µm film; SP2560; Supelco, Bellefonte, PA); splitless injection port at 240 °C; hydrogen carrier gas at a constant flow of 1.3 mL/min; Hewlett-Packard (now Agilent) Model GC 6890 FID gas chromatograph with 7673 Autosampler injector (Palo Alto, CA); 1 µL injected sample; temperature program of 90–170 °C at 10 °C/min, 170 °C for 5 min, 170–175 °C at 5 °C/min, 175–185 °C at 2 °C/min, 185–190 °C at 1 °C/min, 190–210 °C at 5 °C/min, 210 °C for 5 min, 210–250 °C at 5 °C/min, and 250 °C for 10 min. Peak retention times and area percentages of total FAs were identified by injecting known standards (NuCheck Prep, Elysium, MN) and were analyzed with CHEMSTATION A.08.03 software (Agilent Technologies, Santa Clara, CA). Fifty-one known FAs were identified.

Cell-associated HIV load in milk was quantified from the milk-cell pellet. We washed the cell pellets with phosphate-buffered saline solution and extracted genomic DNA with the use of the QIAamp Blood Kit (Qiagen, Valencia, CA). Real-time polymerase chain reaction that used the FastStart DNA Master SYBR Green I mix and the LightCycler instrument (Roche Diagnostics) was performed to quantify cell-associated HIV, according to a previously described protocol (7). Proviral load was expressed as the proportion of HIV-infected cells per 10 000 cells. Cell-free viral RNA was isolated from the aqueous milk fraction with the use of the high Pure Viral RNA Kit (Roche Diagnostics, Indianapolis, IN). We implemented an ultrasensitive protocol of the Amplicor HIV Monitor test, version 1.5 (Roche Diagnostics) to quantify cell-free HIV, as detailed previously (7).

Statistical analyses
We compared the baseline characteristics of cases and controls with the signed-rank test for continuous variables and the McNemar test for categorical variables. The associations between breast milk FAs and breastfeeding transmission of HIV were examined by fitting conditional logistic regression models with case-control status as the outcome. Predictors included ordinal categories representing quartiles for each FA, expressed as the percentage of total fat. Adjusted odds ratios and 95% CIs were estimated from models that included CD4 cell counts and clinical stage of HIV disease at baseline according to the WHO staging system (15). Given that vitamin A supplementation increased the risk of breastfeeding transmission in this population (11), assignment to the vitamin A arm of the trial was also introduced as an adjustment variable. To test for dose-response associations between the concentration of FAs in breast milk and transmission, we introduced an ordinal variable representing the distribution quartiles as continuous into the regression models and tested its statistical significance by use of the Wald test.

To examine the associations between FA percentage weight concentrations and HIV shedding in milk, we defined high cell-free or high cell-associated HIV concentrations dichotomously, with the use of the median among the cases as the cutoff (7). The proportions of women with high cell-free virus (CFV) or cell-associated virus (CAV) were compared across FA quartiles, irrespective of case-control status, with the use of the Cochran-Armitage test for trend. Adjusted tests for trend were derived from multivariate binomial regression models that included an ordinal predictor representing FA quartiles and indicator variables for baseline CD4 cell counts, clinical stage of HIV disease, and assignment to vitamin A supplements. SAS software (version 9.1; SAS Institute, Cary, NC) was used for all analyses.

	RESULTS

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Compared with controls, cases had significantly lower CD4 cell counts at baseline and shed greater amounts of both CVF and CAV in the index breast milk sample before transmission (Table 1). The mean percentage weight concentration of pentadecanoic acid (15:0) was significantly higher in cases than in controls, whereas concentrations of several n–6 LCPUFAs, including 11c,14c-eicosadienoic acid (20:2n–6), dihomo--linolenic acid (20:3n–6), and arachidonic acid (20:4n–6), were significantly higher in controls (Table 2).

Table 3

Table 4

	DISCUSSION

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Breast milk FAs are known to have strong immunomodulating properties, some of which could be relevant to explain the apparent protective effect of gondoic acid and n–6 LCPUFAs on HIV transmission that we currently report. Individual breast milk FAs, released from triacylglycerols by the infant's gastric and lingual lipases, may exhibit direct antimicrobial activity in the intestinal tract (16). Some in vitro and ex vivo studies indicate that LCPUFAs, including arachidonic acid, could effectively inactivate enveloped viruses possibly by dissolving the virions' fatty envelopes (5, 6, 17). In addition, n–6 LCPUFAs inhibited HIV-1 reverse transcriptase in one experiment (18). The microbicidal potential of breast milk lipids is not limited to viruses but also includes bacteria and other microorganisms (19). In addition to a potential direct anti-HIV effect in the infant's gastrointestinal tract, FAs might decrease the risk of MTCT through protection against other pathogens that could disrupt mucosal barriers and facilitate HIV infection (20). This mechanism would be particularly relevant in the context of mixed feeding, which is associated with the highest risk of MTCT (21).

If the protective effect of FAs on MTCT is likely to occur in the infant's gastrointestinal tract, the associations between breast milk FA percentage weight concentrations and transmission may not necessarily be accompanied by decreases in breast milk HIV shedding. However, we did observe significant inverse associations between CFV and the percentage weight concentrations of dihomo--linolenic acid and arachidonic acid. These FAs, released from milk triacylglycerols by milk lipoprotein lipase, could have exerted some virucidal activity in the milk samples after collection; however, a negative association between lauric acid and viral load was not observed, in contrast to previous in vitro experiments (22). It is unclear whether a virucidal effect of FAs in milk is in the causal pathway of the observed associations with MTCT, because viral load and FAs were measured in the same sample, opening a possibility for reverse causality. In addition, not all the associations between FAs and viral load in milk were consistent with those previously reported in vitro. Alternatively, an LCPUFA-mediated decrease in viral load could represent beneficial effects of these FAs on the immunologic status of the mother. Although no evidence from intervention trials is found on the effect of LCPUFAs on HIV disease progression, one observational study suggested that advanced HIV disease stage was associated with significant decreases in the content of linoleic acid and arachidonic acid in cell membranes of CD4 lymphocytes (23).

We observed an increase in the risk of HIV transmission in relation to higher breast milk percentage weight concentrations of 2 saturated FAs, lauric acid and pentadecanoic acid. The nature of these associations is uncertain, particularly because lauric acid (22) and its monoglyceride form, monolaurin (24), were reported to exhibit virucidal activity against encapsulated viruses in vitro. Breast milk lauric acid and other 10–14-carbon FAs are thought to be primarily synthesized de novo in the mammary gland (25), and their production appears to be stimulated by low-fat and high-carbohydrate diets (26). However, odd-carbon saturated fats in milk, including pentadecanoic acid, could originate from maternal diet and indicate higher consumption of dairy products (27). We cannot rule out that the associations observed between these 2 FAs and MTCT be confounded by dietary factors on which we do not have information.

Whether the protective associations between n–6 LCPUFAs and MTCT could also be confounded by unmeasured dietary factors is less likely, given that the amounts of LCPUFAs in milk are metabolically regulated, as suggested by studies showing similar or greater breast milk concentrations of n–6 LCPUFAs in women with low dietary intake of preformed n–6 LCPUFA sources compared with those with higher consumption of such foods (28). Thus, compared with other FAs, secretion of n–6 LCPUFAs in milk seems to depend less on maternal dietary intake and more on mobilization from adipose stores and endogenous synthesis.

The adverse effects of trans FAs on blood lipids, inflammatory markers, and cardiovascular health are well documented (29). We found that the trans FA percentage weight content of breast milk was directly related to increased CAV shedding, and we also observed a marginally significant positive association with CFV. Although trans FAs were not linearly related to the risk of MTCT, the point estimate odd ratios for the third and fourth quartiles of trans FAs in breast milk seemed to be in the direction of increased risk compared with the lower categories. trans FAs in breast milk are considered to be a direct reflection of maternal dietary intake (30), and their concentrations vary across populations according to the amount of partially hydrogenated fats consumed. In the Tanzanian women, total trans FAs accounted for 1.4% of milk FAs, which is less than the 4.4% and 5.3% averages reported from Germany (31) and Canada (30), respectively. It is possible that an increase in MTCT because of trans fats in breast milk could be detectable at higher concentrations of trans fats than those found in our study population.

Evidence suggests that trans fats could interfere with the synthesis of arachidonic acid and other LCPUFAs by inhibiting 6- and possibly 5-desaturases (32). It is tempting to speculate that the greater risk of MTCT observed in women with lower LCPUFAs could represent an adverse effect of trans FAs; however, negative relations between trans FAs and LCPUFAs in milk have not been shown (33). Whether trans FAs could directly impair cellular immunity or accelerate HIV disease progression through their proinflammatory activity is yet to be determined.

In summary, we found that MTCT was associated with the percentage weight concentration of several FAs in breast milk. Of note, 3 n–6 LCPUFAs, including dihomo--linolenic acid and arachidonic acid, were strongly related to lower risk. The extent to which these associations have a causal interpretation is uncertain, given the observational nature of our study. One question raised by the findings is whether the FA profile in breast milk could have been affected by maternal HIV disease progression, and whether the observed relations with MTCT could be partly confounded by advanced stage of HIV, a strong predictor of transmission. Previous studies in adults showed that HIV infection (34) or advanced AIDS (35) were related to lower concentrations of linoleic acid (34), dihomo--linolenic acid, and arachidonic acid (34, 35) and higher concentrations of saturated FAs in plasma (34). It is therefore conceivable that HIV-related changes in maternal diet or body composition could alter the FA profile in breast milk. Although we cannot completely rule out residual confounding, our analyses were controlled for maternal CD4 cell counts and clinical stage of disease in an effort to decrease variability in the FA profile of breast milk that was potentially due to advanced maternal disease stage.

If these associations are found to be causal, the public health implications could be substantial. Despite the existence of regulatory mechanisms to keep the secretion of n–6 LCPUFAs in breast milk relatively constant, one intervention study suggests that supplementation of lactating women with combined arachidonic acid, EPA, and docosahexaenoic acid increases the concentration of arachidonic acid in milk (36). Among HIV-infected women, the WHO currently recommends exclusive breastfeeding for up to 6 mo when replacement feeding is not acceptable, feasible, affordable, sustainable, or safe (WHO, HIV and Infant Feeding Technical Consultation, Geneva, Switzerland, October 25–27, 2006). Therefore, interventions aimed at increasing n–6 PUFAs in milk during the first 6 mo of lactation might contribute to further decrease the burden of MTCT among HIV-infected women who choose to exclusively breastfeed. The efficacy and safety of such interventions need to be tested in randomized clinical trials.

Identifying sources of variability in the breast milk concentration of n–6 LCPUFAs among HIV-infected women will be a relevant future research question. Although some of those sources of variability may be environmental, genetic differences in the metabolic efficiency of the enzymes that intervene in the synthesis of LCPUFAs also need consideration (37). Additional studies are also warranted to elucidate the potential role of other FAs on HIV transmission. We noted protective trends against transmission and CFV shedding for EPA, but our statistical power may have been limited to detect statistically significant associations. It is also relevant to study whether the FA status of the children is related to their risk of becoming infected with HIV. At the time of our study, the majority of infants were likely to be receiving mixed feeding; therefore, the concentration of breast milk FAs may not completely reflect their total dietary intake. Additional research efforts are needed to examine the impact of FAs on HIV disease progression and immunity.

	ACKNOWLEDGMENTS

 
The author's responsibilities were as follows—EV and INK: designed the case-control study; EV (principal investigator of the Vitamins, Breast milk HIV shedding, and Child Health study): carried out the data analyses, interpreted the results, and wrote the initial draft of the manuscript; INK: quantified viral and proviral loads in breast milk; SA, KM, WWF (principal investigator of the Tanzania Trial of Vitamins study), and EV: participated in the study implementation in the field; JF, AB, and HC: carried out the analyses of fatty acids in breast milk and contributed to data interpretation. All authors participated in writing the final draft of the manuscript. None of the authors had any conflicts of interest.

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