Exclusive breast feeding is the best choice for full term infant nutrition. The role of fat and fatty acids (FA) in human breast milk as main source of energy and essential lipid soluble vitamins and in optimum infant development was shown in many studies. ¹,²,³ Poly unsaturated fatty acids (PUFA) such as Linoleic acid (LA) (18: 2n-6) and α-Linolenic acid (ALA) (18:3n-3) are precursors of long chain poly unsaturated fatty acids (LCPUFA) such as arachidonic acid (AA; 20:4n- 6) and Docosohexaanoic acid (DHA; 22:6n-3). They are necessary for growing brain and the retina during perinatal development. ²,⁴

It is well established that current and long-term mother′s diet, individual and diurnal variations in fatty acid synthesis, genetic factors, stage of lactation influences on fatty acid composition of breast milk. ¹,²,⁴ And due to various dietary habits and life styles worldwide, fatty acid composition of breast milk is different not only within countries but also within regions of a country., ¹,⁵,⁶,⁷,⁸,⁹ In a study among German women, the percentage of AA and DHA was 0.7% and 0.46% of total fatty acid in mature breast milk, respectively. ¹⁰ In another study, DHA increased with consumption fatty sea fish or by supplementation with fish oil.

In Iran, little is known of the FA composition in the milk of Iranian women and its relations to their diet. A study in the west of Iran showed the fat content and the total PUFA in milk samples of 52 women were 3.8 ± 1.5% and 16.5%, respectively. ¹¹

The aim of our study was to determine the content of fat and AA and DHA of breast milk as two major LC-PUFA connected with favorable outcomes in human infants and to associate these results to the mother′s diet such as carbohydrate, protein, calorie intake and lipid composition of colostrum as first food of newborns.

This cross-sectional study was done among pregnant mothers who came to health care centers for last prenatal care in Isfahan, Iran. They were selected by convenient sampling. Pregnant women with gestational age between 37 to 42 weeks, without chronic disease and drug use (only routine supplements use of iron and folic acid) included the study. Neonates with congenital anomaly, intrauterine growth retard and small for gestational age were excluded from the study.

The Research and Ethics Committee of Faculty of Medicine, Isfahan University of Medical Sciences approved the study. Written consent was obtained from all mothers. They completed one-day food record questionnaire to determine their current food intake and energy, protein, carbohydrates, fat and fatty acids using nutrition software.

After giving a clean plastic disposable storage box, the researcher explained mothers to bring 20cc of their breast milk in it to neonatal screening clinic after delivery. In Iran all neonates was screened for congenital hypothyroidism in 1-5 days after delivery. Then the samples were delivered to Pharmacy School of Isfahan University of Medical Science.

Breast milk sampling and fatty acid analysis

When the mothers were in the post-partum ward, 8 to 72 hours after delivery, 2 to 5 ml of colostrum was collected by hand into tubes. They were kept in an ice box and sent within half an hour to the collaborating health centre for freezing at -20°C until analysis, which was performed at the laboratory of NNFTRI in Tehran. The milk samples were homogenized by Vortex (Heidolph Vortex Shaker REAX 1. 220 V. 30 W Germany) at 2400 rpm for 30 sec.

Direct FAME synthesis

About 1 ml of milk were placed into a 16 × 125 mm screw-cap Pyrex culture tube to which 1.0 mL of the C19:0 internal standard (1 mg of C19:0/mL of MeOH), 0.7 mL of 10 N KOH in water, and 5.3 mL of MeOH were added. The tube was incubated in a 55°C water bath for 1.5 h with vigorous hand shaking for 5 s every 20 min to properly permeate, dissolve, and hydrolyze the sample. After cooling below room temperature in a cold tap water bath, 0.58 mL of 24 N H ₂ SO ₄ in water was added. The tube was mixed by inversion and with precipitated K ₂ SO ₄ present was incubated again in a 55°C water bath for 1.5 h with hand-shaking for 5 s every 20 min. After FAME synthesis, the tube was cooled in a cold tap water bath, 2 milliliters of hexane was added, and the tube was vortex-mixed for 2 min on the vortex. The tube was centrifuged (Rotina 35 R, Hettich, Tuttingen, Germany) for 2 min at 5000 rpm at 4°C and Na ₂ SO ₄ (1 g) was added to the upper phase contained fatty acid methyl esters (FAME) and it was removed to clean test tubes.

Gas chromatography (GC)

The vial was capped and placed at −20°C until GC analysis; 1 μl of the esterified sample was injected into the gas chromatography (GC-CP3800, Varian, USA) equipped with a flame ionization detector and a fused silica capillary column 50 m × 0.25 mm (I.D. CP-SIL 88 for FAME) supplied with 0.2 μm stationary phase (Varian Chrompack, USA-Netherlands). Oven temperature was maintained at 100°C for 2 min, then increased to 10°C/min to 175°C and held there for 15 minutes followed increase at 5°C/min to 205°C and followed increase 3°C/min to 220°C and kept there for 24.5 min (total time: 60 min). The injector and the detector temperatures were set at 260°C and 270°C, respectively. Helium was the carrier gas with a split ratio of 30: 1 and a column flow of 0.5 ml/min.

Fatty acids were identified by comparing the retention times of FAME with a standard FAME mixture (SUPELCO 37 Comp. FAME mix, (595, North Harrison Road, Bellefonte, Pennsylvania (PA) 16823-0048, USA). All samples were analysed in duplicate. The results were expressed as weight/weight area percent. The values were given as means (SD).

Statistical analysis

Results were evaluated with SPSS software version 17.0. Descriptive analysis was done to determine the frequency, the mean ± SD of some variables. Pearson correlation test was used to test the correlations between total fatty acids and mothers diet composition.

The data of 86 out of 91 samples were complete. The mean of age and gestational age was 28.37 ± 5.55 years old and 38.7 ± 1.2 weeks, respectively. Mother′s diet composition is presented in Table 1.{Table 1}

The content of fat was 2.17 ± 1.22 g/100 ml breast milk. AA (20:4n-6) and DHA (22:6n-3) made 0.8 ± 0.4% and 0.3 ± 0.2% of total fatty acids.

The correlations between fat content of breast milk and some components of mother′s diet were presented in Table 2.{Table 2}

Our results suggested that the content of fat in 100 cc of breast milk is lower than many studies which reported its average between 3.8-3.9 g/100 ml. ¹² We did not find any study in Iran that evaluated the fat content of breast milk in first 5 days of lactation. In a study in the west of Iran, the fat content in the milk of 52 healthy women was reported 3.8 ± 1.5% in the 45 ^th to 155 ^th day of their lactation. ¹¹ Another study in the north west of Iran in Tabriz among mothers with exclusively breastfed infants aged 90-120 days showed the mean fat content of breast milk samples was 3.52 ± 1.41 g/ dl). It was well established that total fat content of breast milk increased with the duration of lactation. ²,⁴,¹³

In a study in Germany, the content of 4n-6 and 6n-3 in 38 term mothers was reported 0.72% and 0.45% of total fatty acids in 5 days of their lactation. ¹⁰

In our study, the milk AA content was lower in comparison with Western part of Iran. In other studies it was shown that the percentage of LC-PUFA of both n-6 and n-3 derivatives decrease during the first month after child birth. ²,⁴ In addition, we found association between AA content of breast milk and mother′s diet composition. Many studies showed that the milk AA content may be less sensitive to maternal dietary intakes and it is synthesized from essential fatty acids in mammary glands. ¹,²,⁴

The content of DHA in our study is low in comparison to other studies. It was reported to be low as 0.2 wt.% in Italy to as high as 1.1 wt.% in ST.Luica in other investigations. ⁸,⁹ No correlation was found between breast milk DHA and DHA of mother′s diet did not confirm the influences of the diet of lactating women. Many studies showed that consumption of fatty sea fish or supplementation with fish oil increased the DHA content of breast milk. ²,⁴,⁹,¹⁴

These differences might be due to using different methods in extraction of lipids and fatty acids of breast milk and different programs for mother′s diet analysis. In addition, genetic and geographical differences, which documented as important factors on fat content of breast milk, should be considered. ²,⁴

WHO recommended AA/DHA ratio should be 2:1 in colostrums. In well developed country, this ratio varies from 5:1 to 15:1. ⁴ It was 2.6:1 in our study higher than WHO recommendation.

Although the AA/DHA ratio in our study is suitable, but the content of DHA is nearly low. Because of its important role in infant brain development interventions to improve dietary habits in before and during pregnancy is needed. It was shown that in regions where fish consumption is high, the DHA content of breast milk is higher. ⁹,¹⁵

In this study, we found no association between measured breast milk fat content and daily energy intake, carbohydrate, protein, linoleic acid, linolenic acid, saturated fatty acid, PUFA, monounsaturated fatty acid, and cholesterol of mother′s diet. For mature milk, the correlation of carbohydrate of mother′s diet and fat content was shown in Tabriz. ¹⁶ But there are many researches which showed no correlation between breast fat content and energy intake, carbohydrate, protein of mothers′ diet in mature milk and colostrums. ¹⁷,¹⁸,¹⁹ Breast feeding should be promoted for prevention of chronic diseases, ²⁰ and consumption of foods rich in fatty acids should be encouraged for women in reproductive age group.

Using the one-day questionnaire is one of the limitations of the study. This questionnaire determined the current dietary of pregnant mothers. It was well established that not only current diet, but also long term dietary habit influences the human breast milk fatty acid composition especially DHA.

Dietary habits of women in reproductive age group should be improved, with special emphasis on the fatty acid content of breast milk. Consumption of oily fish, nuts, seeds, whole grains, egg, dairy, and dark green, leafy vegetables may be helpful in this regard. This may have long-term impact on health promotion and disease prevention.