Association between Breast Milk Mineral Content and Maternal Adherence to Healthy Dietary Patterns in Spain: A Transversal Study

foods Article Association between Breast Milk Mineral Content and Maternal Adherence to Healthy Dietary Patterns in Spain: A Transversal Study Cristina Sánchez 1, Cristina Fente 2,* , Rocío Barreiro 2, Olga López-Racamonde 2,3, Alberto Cepeda 2and Patricia Regal 2 1Pharmacy Faculty, Campus Monteprincipe, San Pablo-CEU University, 28668 Madrid, Spain; [email protected] 2Department of Analytical Chemistry, Nutrition and Bromatology, Santiago de Compostela University, 27002 Lugo, Spain; rocio.barreir[email protected] (R.B.); [email protected] (O.L.-R.); [email protected] (A.C.); [email protected] (P.R.) 3Midwifery Service at San Roque Health Center, SERGAS (Servizo Galego de Saúde), 27002 Lugo, Spain *Correspondence: [email protected]; Tel.: +34-600-942349 Received: 18 April 2020; Accepted: 17 May 2020; Published: 20 May 2020   Abstract: The composition of breast milk is influenced by many factors, some of which dependent on the mother and others on the child. Changes in lactation and other factors depending on the mother’s physiology and anthropometric characteristics, as well as her nutritional status and diet, are of key importance. Breast milk minerals have been extensively studied with highly uneven results. In this work, a comparison will made with data across the world. To understand the factors that might explain the disparity, several minerals (Na, K, Ca, P, Mg, Fe, Se and I) have been analyzed using ICP-MS in a set of human milk samples (n=75). The samples had an identical geographical origin (Galicia, in northwestern Spain) but different lactation circumstances, including maternal anthropometric data, lactating time, newborn sex and maternal adherence to healthy dietary patterns (Mediterranean Diet, MD, or Atlantic Diet, AD). The required concentrations of essential elements reported in the literature are similar to those found in these Spanish women. A univariate approach revealed that factors such as lactating time, body mass index (BMI) and newborn sex have a significant influence in breastmilk mineral content. According to multivariate linear regression analysis, minerals in milk are particularly associated with lactating time, but also with newborn sex, maternal BMI, age and diet pattern in some cases. More precisely, these results suggest that the iron and selenium concentrations in the milk of Galician donors may be positively influenced by maternal adherence to AD and MD, respectively. Keywords: breast milk; ICP-MS; minerals; lactating time; Mediterranean Diet; Atlantic Diet 1. Introduction Maternal eating habits are important during lactation. In this period, the child needs energy and fundamental nutrients, not only to maintain life, but also to achieve rapid growth and the development of his tissues, organs and functions [1]. The Lancet series on Breastfeeding [ 2 ] and the publication Nurturing the Health and Wealth of Nations: The Investment Case for Breastfeeding [ 3 ] present some of the latest efforts to disseminate the almost indisputable scientific evidence supporting the benefits of breastfeeding for infants, for the mother, and even to lay the foundations for healthier and more prosperous societies. During breastfeeding, maternal diet determines not only the mother’s nutritional status and, therefore, her health, but also that of the child. One of the latest systematic reviews [ 4 ] includes a Foods 2020,9, 659; doi:10.3390/foods9050659 www.mdpi.com/journal/foods Foods 2020,9, 659 2 of 15 wide range of studies on a broad spectrum of foods which can vary the nutritional characteristics of maternal milk. The main conclusion of this work is that the information available on this subject is scarce and diversified. This means that most of the evidence currently used in clinical practice to make recommendations is limited to studies that report only indirect associations. Therefore, further studies on the role of maternal diet in milk composition are needed. The Mediterranean Diet (MD) is a dietary pattern that includes a high intake of olive oil, fruit, nuts, vegetables, legumes, moderate fish intake, poultry, red meat, wine and dairy products. It also features low consumption of processed meats and refined sugars. MD is intimately linked to the Mediterranean lifestyle, including Spain [ 5 ]. Many studies have been published about the health benefits of adherence to the MD pattern [ 6 – 8 ]. This style of diet could have several advantages during lactation, related to heart-healthy fatty acids and iron profile, along with dietary antioxidant factors and the low oxidative load of the foods consumed [9]. The dietary pattern of Northern Portugal and the Galicia region (northwest Spain) shares common characteristics with the Mediterranean Diet, but certain particularities suggest a typical Atlantic pattern that has been called the Southern European Atlantic Diet (SEAD) or Atlantic Diet (AD) [ 10 ]. The consumption of fish three/four times a week, fruits and vegetables, milk and dairy products, the moderate consumption of meat and the simplicity in the preparation of food, to maintain the quality of the raw materials and, therefore, the nutritional value, are characteristics of this type of diet. This style of eating could have many health advantages related to its supply of omega-3 fatty acids, vitamin D and other vitamins, Ca, Fe and other minerals [11–13]. In this work, the mineral content (Na, K, Ca, P, Mg, Fe, Se and I) was determined in a set of human milk samples donated by Spanish mothers living in the northwest of Spain. Data on maternal anthropometric characteristics, lactating time, adherence to healthy dietary patterns, i.e., MD or AD, and newborn sex were also collected and analyzed in order to determine the influence of these factors on the mineral content of breast milk from an identical geographical origin. 2. Materials and Methods 2.1. Samples and Data Collection This work belongs to an observational cross-sectional study designed to investigate the breast milk composition of lactating mothers living in the north-west of Spain (Galicia). This region was chosen for the characterization of breast milk mineral composition on the basis of its proximity to the research group and also the coexistence of two healthy dietary patterns in a relatively small area. Recruitment, milk sampling and data collection were done by applying convenience sampling in collaboration with midwifery service and a local breastfeeding association. The study was registered in ClinicalTrials.gov with the identification number NCT03245697. This study was approved by the Galician Clinical Research Ethics Committee (Approval code 2016/280) and adhered to the principles of the Helsinki Declaration of 1975, as revised in 1983. Written informed consent was obtained from all participants. Milksampleswere donated by 85lactatingwomen. Themotherswere interviewed and prescreened for the following exclusion criteria: mothers with acute or chronic diseases, suffering from any metabolic disorders, gestation of less than 36 weeks, abusers of drugs or alcohol. All babies were healthy and growing well. For the study, 75 of those mothers were selected. These nursing women were surveyed about personal data: age, medicines, tobacco use, anthropometric data (body weight, height and BMI), childbirth (date of delivery, natural or C-section and sex) and lactation (lactating time as months after delivery). Additionally, mothers were asked about their dietary habits to determine the degree of adherence to the MD or AD. A short questionnaire of 14 items (Mediterranean Diet Adherence Screener, MEDAS), validated for the Spanish population by the Mediterranean Diet Prevention group (PREDIMED), was used [ 14 , 15 ]. To get the score, each positive response relative to the MD was assigned a value of +1, or 0 when the question received a negative response. The sum of the values Foods 2020,9, 659 3 of 15 obtained for the 14-item questionnaire was used to appraise the MD adherence of participants [ 15 ]. Also, a short, 9-item questionnaire (SEAD) was used to determine the level of adherence to an Atlantic diet [ 12 , 16 ]. Human milk samples were collected at home by the mothers. Approximately 25 mL milk was extracted via manual expression or breast pump, before breastfeeding the child, and collected into a sterile plastic tube. The samples were stored at 3 to 5 ◦ C and delivered to the laboratory within 12 h. All samples were collected before the first morning breastfeeding or two hours after the last session, when the child breastfeeds at night. 2.2. ICP-MS Method Analytical-gradereagents and chemicals wereusedinthepreparationofallsolutions. Milksamples (5 mL) were digested with 8 mL HNO 3 69% (Hiperpur, Panreac) and 2 mL 33% H 2 O 2 (Panreac) using the microwave digestion method (Milestone, Ethos1 Plus) (190 ◦ C for 15 min at 1000 W). Once digested, the resulting solutions were cooled and diluted to a final volume of 50 mL with H 2 O (Milli-Q). Determination of mineral contents (Na, K, Ca, P, Mg, Fe and Se) was carried out by inductively coupled plasma-mass spectrometry (ICP-MS) (Agilent 7700 × , with a sample introduction system formed by a Micromist glass low-flow nebulizer, a double-pass glass spray chamber with Peltier system (2aC) and quartz torch. The operating parameters are given in Table 1. Table 1. ICP-MS parameters setting. Plasma Parameters Ion Lenses (V) Octopole Parameters (V) RF Power (W) 1550 Extract 1 0 OctP RF 180 Sample Depth 8 Extract 2 −175 OctP Bias −18 Carrier Gas (L/min) 1.1 Omega Bias −100 Reaction Cell (mL/min) Nebulizer Pump (rps) 0.1 Omega Lens 12.6 He Gas 3.6 S/C Temp (◦C) 2 Cell Entrance −40 Detector Parameters Cell Exit −60 Discriminator (mV) 4.5 Deflect 0.4 Analog HV (V) 1730 Plate Bias −60 Pulse HV (V) 954 QP Bias −15 Digestion and determination of iodine in the milk samples was also done by ICP-MS following the protocol European Standard EN 15111 [ 17 ]. In both methods, two blanks and a certified reference material (Reference material 1549 Not-Fat milk power, NIST) were included in each digestion batch. Working standard solutions were prepared by dilution of stock standard solutions (Panreac) to the desired concentration in NO 3 H-H 2 O in the same proportion as the samples. Calibration curves ranges (5 points) were selected to match the expected concentrations for all elements in milk samples. The correlation coefficients for each element were equal to or greater than 0.9999. 2.3. Statistics A data analysis was performed using SPSS version 24 statistical computer software (SPSS, Chicago, IL, USA). All data were assessed for normality using a Kolmogorov-Smirnov test, and for equality of variances using the Levene’s test. Accordingly, a Student’s ttest or Mann-Whitney U test was used to detect differences between the two independent groups of samples constructed on the basis of cut-off values for lactating time, BMI and newborn sex. For all tests, p<0.05 was considered significant. Further, toassessassociationsbetweenmineralsandmaternal factors, multivariatelinear regression analysis was used (stepwise method), where mineral levels were the dependent variables, and the dietary scores (MD or AD) and maternal characteristics (lactating time, BMI, maternal age, and newborn sex) were considered as independent variables. Variables that did not show a normal distribution were log-transformed (natural logarithms). The final models were further evaluated with the aid of their respective normal probability plots of residuals. Two-sided pvalues <0.05 were considered statistically significant. Foods 2020,9, 659 4 of 15 3. Results and Discussion 3.1. Maternal Characteristics Among the 85 initial donors, 75 were selected for this study, all of whom were healthy, nursing mothers from the northwest of Spain. Three donors said they were smokers at the time of sampling. Only 9 women (12%) underwent a C-section delivery and 60% were primiparous. Significant data on gestation and on maternal and newborn characteristics are provided in Table 2. Continuous data are expressed as mean and standard deviation (SD), and categorical variables are expressed as percentages. Table 2. Pregnancy and maternal characteristics, continuous and categorical variables (n=75). Maternal Data Mean Median SD Min Max Pregnancy Time (weeks) 39.91 40.00 1.29 36.00 42.29 Maternal Age (years) 35.50 35.00 3.99 27.00 46.00 Maternal Height (m) 1.65 1.65 0.05 1.53 1.77 Maternal BMI (kg/m2)24.11 23.34 3.88 17.99 35.03 Lactating time (months) 8.02 3.72 10.43 0.77 58.97 MD adherence (score) 9.00 8.00 2.26 2.00 12.00 AD adherence (score) 3.87 4.00 1.52 0.00 7.00 Infant gender: n♂(%)/n♀(%) 33 (44.00)/42 (56.00) C-section delivery (%) 12.00 Time lactation <6 months (%) 64.00 Parity number 1st child (%) 60.00 3.2. Galician Human Milk Composition Compared with Worldwide In recent years, the number of publications on the mineral composition of breast milk has increased. Many of these studies have been conducted in different populations with very different lifestyles and diets. It is possible that these variables influence the disparate results found in the literature. The next three tables present the mineral concentrations in mature human milk collected in different countries, according to the scientific literature, with each table containing representative minerals from three categories [ 18 ]: (1) minerals such as calcium, phosphorus and magnesium, that are regulated by homeostatic mechanisms in maternal serum (Table 3); (2) electrolytes, including sodium and potassium, which are determined by a gradient of electrical potential in the secreting cell (Table 4); and (3) trace elements, such as iron, selenium or iodine, which exist in very low concentrations in human milk (Table 5). Data since 1980 have been included, and works have been selected from populations in different parts of the world, including Spain. The table also reflects the concentrations obtained for these minerals in the breast milk of Galician women analyzed in this study. Table 3. Mineral regulated by homeostatic mechanisms [ 18 ] reported in mature human milk in different populations. Mineral Authors, Year, Reference, Country Mean SD Range Units Calcium Gross et al., 1980, [19], USA 249.8 mg/L Fransson & Lonnerdal, 1983, [20], USA 279 28.6 mg/L Dewey & Lonnerdal, 1983, [21], USA 253.0 mg/L Garza et al., 1983, [22], USA 213.4 mg/L Feeley et al., 1983, [23], USA 262.0 mg/L Dewey et al., 1984, [24], USA 229.4 40.1 mg/L Butte et al., 1984, [25], USA 154.9 mg/L WHO & IAEA, 1989, [26] 220.0–300.0 mg/L Allen et al., 1991, [27], USA 280.7 20.1 mg/L Dorea et al., 1999, [28], Gambia 208.93 24 mg/L Friel et al., 1999, [29], USA 279.6 mg/L Foods 2020,9, 659 5 of 15 Table 3. Cont. Mineral Authors, Year, Reference, Country Mean SD Range Units Yamawaki et al., 2005, [30], Japan 249.0 16.52 mg/L Mastroeni et al., 2006, [31], Brazil 250 31.0 mg/L Thacher et al., 2006, [32], Nigeria 186 41 mg/L Shi et al., 2011, [33], China 334.00 70.00 mg/L Björklund et al., 2012, [34], Sweden 305.00 45.00 mg/L Andrade et al., 2104, [35] Brazil 142.30 21.60 mg/L Klein et al., 2017, [36], USA 268.7 59.34 mg/L Klein et al., 2017, [36], Namibia 143.83 64.67 mg/L Klein et al., 2017, [36], Poland 227.06 36.72 mg/L Klein et al., 2017, [36], Argentina 231.79 37.47 mg/L Aumeistere et al., 2017, [37], Latvia 227.52–398.34 mg/L Perrin et al., 2017, [38], USA 194.0 mg/L Butts et al., 2018, [39], New Zealand 275–309 mg/L Sabatier et al., 2019, [40], Switzerland 286 47 mg/L Daniels et al., 2019, [41], Indonesia 247–300 mg/L Present study, 2019, NW Spain 279.99 136.44–463.26 mg/L Phosphorus Gross et al., 1980, [19], USA 150.8 mg/L Feeley et al., 1983, [23], USA 133.0 mg/L Butte et al., 1984, [25], USA 144.4 mg/L Mastroeni et al., 2006, [31], Brazil 137.0 20.0 mg/L Thacher et al., 2006, [32], Nigeria 126.81 32.99 mg/L Sabatier et al., 2019, [40], Switzerland 148 30 mg/L Daniels et al., 2019, [41], Indonesia 119–145 mg/L Present study, 2019, NW Spain 126.46 73.00–219.57 mg/L Magnesium Gross et al., 1980, [19], USA 26.2 mg/L Fransson & Lonnerdal, 1983, [20], USA 35.04 2.46 mg/L Dewey & Lonnerdal, 1983, [21] USA 31.7 mg/L Feeley et al., 1983, [23], USA 50 mg/L Dewey et al., 1984, [24], USA 31.2 5.6 mg/L Butte et al., 1984, [25] USA 34.6 mg/L WHO & IAEA, 1989, [26] 29.00–38.00 mg/L Allen et al., 1991, [27], USA 38.7 2.4 mg/L Friel et al., 1999, [29], USA 29.1 mg/L Yamawaki et al., 2005, [30], Japan 28.33 4.16 mg/L Mastroeni et al., 2006, [31], Brazil 29.9 5.00 mg/L Shi et al., 2011, [33], China 37.00 10.00 mg/L Björklund et al., 2012, [34], Sweden 28.00 4.80 mg/L Andrade et al., 2104, [35], Brazil 39.80 4.20 mg/L Aumeistere et al., 2017, [37], Latvia 25.73–49.52 mg/L Butts et al., 2018, [39], New Zealand 10.2–30.8 mg/L Sabatier et al., 2019, [40], Switzerland 32 7 mg/L Daniels et al., 2019, [41], Indonesia 26.3–35.9 mg/L Present study, 2019, NW Spain 33.22 19.86–50.83 mg/L Calcium is the second most abundant mineral in breast milk [ 18 ]. EFSA expert opinion [ 42 ] considers that the average calcium intake suitable for most babies in the first half year of life is 200 mg per day. This could be achieved with an intake of 800 mL of milk per day, if that milk has a calcium concentration of 250 mg/L. In 2017, a comparison was published in PLoS ONE [ 36 ] between countries with varying degrees of development (Argentina, Namibia, Poland, and the United States). The results, in terms of calcium concentration, showed a high degree of variability among the different populations. Calcium concentrations were significantly lower in the Namibian population than elsewhere, i.e., while the mean calcium concentration in U.S. breast milk was 278.62 mg/L, in Namibia, it was 143.83 [ 36 , 39 ]. Foods 2020,9, 659 6 of 15 These results are consistent with previous data, in which calcium was found to be one of most variable essential elements among different populations (Nigeria 186 [ 32 ]; Gambia 208.93 [ 28 ]; Brazil 250 [ 31 ]; Japan, 250 [ 30 ]; Sweden 305 mg/L [ 34 ]) [ 39 ]. The authors of reported similar calcium concentrations in New Zealand, depending on ethnicity, i.e., ranging from 275 to 309 mg/L. In the present study, the average calcium concentration was 279.99 mg/L, very close to the countries with economies similar to that of Spain, and higher than the levels reported for countries with lower levels of development. Low phosphorus breast milk concentrations have been associated with improved calcium absorption and a number of other benefits for the newborn [ 43 ]. Data in different areas of the world show less disparity than those obtainedfor calcium, which is in line with the observations of Björklund et al. [34] , who referred to a fine regulation of the P content in breast milk. Only Sabatier et al. [40] found values in some samples below 100 and above 200 mg/L, although the mean was 148 mg/L. The same observation was made in this work, i.e., the phosphorus concentration ranged from 73.00 to 219.57, with a mean of 126.46 mg/L. The average Ca/P ratio was 2.29. Table 4. Electrolyte concentration [18] reported in mature human milk in different populations. Electrolyte Authors, Year, Reference, Country Mean SD Range Units Sodium Gross et al., 1980, [19], USA 215.3 mg/L Dewey & Lonnerdal, 1983, [21], USA 185.9 mg/L Garza et al., 1983, [22], USA 131.9 mg/L Dewey et al., 1984, [24], USA 94.6 51.4 mg/L Butte et al., 1984, [25], USA 154.9 mg/L WHO & IAEA, 1989, [26] 90.0–130.0 mg/L Allen et al., 1991, [27], USA 154.8 28.50 mg/L Wack et al., 1997, [44], USA 144.5–124.5 mg/L Yamawaki et al., 2005, [30], Japan 120.67 16.50 mg/L Mastroeni et al., 2006, [31], Brazil 205.0 156.00 mg/L Björklund et al., 2012, [34], Sweden 217.00 77.00 mg/L Roy et al., 2014, [45], India 527.40 199.00 mg/L Aumeistere et al., 2017, [37], Latvia 58.56–256.38 mg/L Perrin et al., 2017, [38], USA 67.9 mg/L Sabatier et al., 2019, [40], Switzerland 235.0 237 mg/L Daniels et al., 2019, [40], Indonesia 101–193 mg/L Present study, 2019, NW Spain 176.94 44.75–942.67 mg/L Potasium Gross et al., 1980, [19], USA 582.5 mg/L Dewey & Lonnerdal, 1983, [21], USA 457.2 mg/L Dewey et al., 1984, [24], USA 397.7 71.0 mg/L WHO & IAEA, 1989 [26] 410.0–550.0 mg/L Allen et al., 1991 [27], USA 542.9 48.2 mg/L Wack et al., 1997, [44], USA 504.4–448.8 mg/L Yamawaki et al., 2005, [30], Japan 437.33 7.57 mg/L Mastroeni et al., 2006, [31], Brazil 462 84 mg/L Shi et al., 2011, [33], China 540.00 146 mg/L Björklund et al., 2012, [34], Sweden 633.00 40 mg/L Aumeistere et al., 2017, [37], Latvia 445.33–736.71 mg/L Perrin et al., 2017, [38], USA 363.7 mg/L Sabatier et al., 2019, [40], Switzerland 575 92 mg/L Daniels et al., 2019, [41], Indonesia 402–499 mg/L Present study, 2019, NW Spain 456.62 342.98–622.25 mg/L Magnesium is the second most abundant intracellular cation after sodium. Its concentrations in breast milk are regulated by homeostatic mechanisms which ensure magnesium stability, regardless of constitutional, metabolic or dietary variables. The concentrations of this essential element reported in the literature (see Table 2) are very similar among all populations. The values ranged in all cases between 10 and 50 mg/L, although in most studies, the concentrations were close to 30. In this study, the average concentration among Galician mothers was 33.22. In an EFSA report [ 42 ], the mean was 31 mg/L. The panel considers that, assuming an intake of 800 mL of milk per day, an average Foods 2020,9, 659 7 of 15 magnesium concentration of 31 mg/L would provide 25 mg of that mineral per day, which is suitable for most babies during the first half year of life. Electrolyte concentrations (sodium, potassium and chlorine) in human milk are determined by the gradient of electrical potential in the secreting cell rather than by maternal nutritional status, being lower in mature milk than in calostrum [ 46 ]. Some researchers have reported increases in sodium levels, and occasionally chloride, of 5 to 40 times in human milk associated with emotional stress, mastitis and decreased milk production [ 47 ]. In a WHO/IAEA report [ 26 ], sodium values were between 60 and 170 mg/L, while for EFSA [ 42 ], values ranged from 140 to 160 mg/L. In the literature, values were higher than 200 mg/L [ 19 , 31 , 34 , 37 , 40 ]. The authors observed an average of 176.94, albeit with wide variability. The daily intake of 150 mg (achieved for example with a daily intake of 800 mL of 120 mg/L sodium concentration milk) is appropriate for most babies in the first year of life [42]. Table 5. Trace element concentration [18] reported in mature human milk in different populations. Mineral Authors, Year, Reference, Country Mean SD Range Units Iron Fransson & Lonnerdal, 1983, [20], USA 0.36 0.19 mg/L Dewey & Lonnerdal, 1983, [21], USA 0.2 mg/L Garza et al., 1983, [22], USA 0.3 mg/L Feeley et al., 1983, [48], USA 0.76 mg/L Dewey et al., 1984, [24], USA 0.2 0.1 mg/L Gunshin et al., 1985, [49], Japan 0.32 0.16 mg/L WHO & IAEA, 1989 [26] 0.07–0.35 mg/L Domellöf et al., 2004, [50], Honduras 0.29 0.21 mg/L Yamawaki et al., 2005, [30], Japan 1.19 2.51 mg/L Mastroeni et al., 2006, [31], Brazil 0.90 0.5 mg/L Hannan et al., 2009, [51], USA 0.5 1.0 mg/L Shi et al., 2011, [33], China 0.50 0.2 mg/L Mello-Neto et al., 2012, [52], Brazil 0.3 0.2 mg/L Björklund et al., 2012, [34], Sweden 0.33 0.19 mg/L Roy et al., 2014, [45], India 0.162 0.06 mg/L Andrade et al., 2104, [35], Brazil 0.27 0.4 mg/L Klein et al., 2017, [36], USA 1.27 0.26 mg/L Klein et al., 2017, [36], Namibia 1.53 0.86 mg/L Klein et al., 2017, [36] Poland 1 0.15 mg/L Klein et al., 2017, [36], Argentina 0.99 0.21 mg/L Aumeistere et al., 2017, [37], Latvia 0.31 mg/L Perrin et al., 2017, [38], USA 0.20 mg/L Sabatier et al., 2019, [40], Switzerland 0.44 0.26 mg/L Daniels et al., 2019, [41], Indonesia 0.33 mg/L Peixoto et al., 2019, [53] Brazil 0.01–0.52 mg/L Present study, 2019, NW Spain 0.23 0.06–0.75 mg/L Selenium WHO & IAEA, 1989 [26] 13.0–33.0 µg/L Casey et al., 1989, [54], USA 7–20.0 µg/L Krachler et al., 1998, [55], Austria 17 µg/L Zachara & Pilecki, 2000, [56], Poland 8.81–11.58 µg/L Navarro-Blasco & Alvarez-Galindo, 2004, [ 57 ], Spain 16.3 4.7 µg/L Yamawaki et al., 2005, [30], Japan 15.3 2.5 µg/L Özdemir et al., 2008, [58], Turkey 68.63 7.78 µg/L Hannan et al., 2009, [51], USA 15.9 4.1 µg/L Shi et al., 2011, [33], China 15.0 6.0 µg/L Björklund et al., 2012, [34], Sweden 13.00 2.6 µg/L Butts et al., 2018, [39], New Zealand 13–16 µg/L Sabatier et al., 2019, [40], Switzerland 15.0 4.2 µg/L Daniels et al., 2019, [41], Indonesia 8.7–12.9 µg/L Peixoto et al., 2019, [53] Brazil 2.5–70.6 µg/L Present study, 2019, NW Spain 15.13 4.37–148.97 µg/L Iodine Fernández Sánchez et al., 2007, [59], Spain 144 93.2 µg/L Dasgupta et al., 2008, [60], USA 110 µg/L Leung et al., 2009, [61], USA 51.4 µg/L Hannan et al., 2009, [51], USA 47.8 17.1 µg/L Andersson et al., 2010, [62], Switzerland 49 µg/L Andersen et al., 2014, [63], Denmark 83 µg/L Mekrungcharas et al., 2014, [64], Thailand 129.7 µg/L Sabatier et al., 2019, [40] Switzerland 87 41 µg/L Present study, 2019, NW Spain 95.44 11.25–247.63 µg/L Foods 2020,9, 659 8 of 15 Regarding potassium, the WHO/IAEA [ 26 ] showed differences among mothers and among countries. Samples from Hungary and Sweden had very high values, while samples from Nigeria showed quite low levels. In this review (see Table 2), only samples described by Dewey et al. [ 24 ] and Perrin et al. [38] had averages below 400 mg/L. EFSA [42] considers that the appropriate intake for most children up to 6 months of age is 400 mg per day, i.e., with a daily consumption of 800 mL of breast milk, a concentration of 500 mg/L would be required. The average for Galician mothers was 456.62 mg/L. EFSA [ 42 ] considers that the mean iron intake from breast milk (0.3 mg per day for a concentration of 0.35 mg/L and an intake of 800 mL/day) is generally sufficient to ensure that the iron status in the first half year of life remains within a healthy range [ 65 ]. However, it should be noted that for formula-fed infants and some infants after 4 to 6 months of age, an intake equivalent to this value is not sufficient to keep the iron status within the normal range. The iron concentrations observed in many studies (including the present one, 0.23 mg/L) were below 0.35 mg/L. Nevertheless, the absorption of iron from breast milk is somehow greater than its absorption from infant formula or complementary foods fortified with iron [ 66 ]. In addition, babies have been shown to have the ability to regulate iron absorption when iron reserves decrease [ 50 , 67 ] this ability makes babies more resistant to iron deficiency than a simple factorial approach would predict [42]. Breastfed infants rely on breast milk iodine to meet their physiological needs, which, in turn, depends on the mother’s iodine status, as well as on other factors [ 68 ]. According to the EFSA [ 42 ], the concentration of iodine in the mature breast milk of European women is in the range of 50 to 100 µ g/L. Assuming a milk intake of 0.8 L per day, between 40 and 80 µ g of iodine per day would be consumed. The panel considers that an iodine intake of 90 µ g per day is suitable for most children in the first half year of life. In our review, the levels of iodine in most populations were not taken into account, with results only being reported from countries with high standards of living and other developing countries. The concentrations of iodine ranged from 49 µ g/L in Swiss women [ 63 ] to 144 µ g/L in Spanish women, as sampled by Fernández Sánchez [59]. In the present study, the mean value was 95.44 µg/L. Globally, there are large differences in soil selenium content, and therefore, in food availability. A wide range of selenium concentrations has been observed in human milk, depending on the amount of selenium consumed by the mother from natural foods. The concentrations obtained differ from one country to another [ 69 ]. The WHO/IAEA [ 26 ] reported average values ranging from 13 to 33 µ g/L. Samples from the Philippines had very high values, while in Sweden, values were relatively low. According to EFSA [ 42 ], selenium concentrations in the breast milk of European women ranged from 3 to 84 µ g/L, with an average value of 16.3 µ g/L. The panel considers that 12.5 µ g average intake per day of Selenium in breast milk (i.e., 16 µ g/L × 0.8 L) is suitable for most infants in the first half year of life. In this review, the highest concentrations of selenium were found in samples from Turkey [ 58 ]. In Galician women, an average concentration of 15.13 µg/L was observed with wide variability, i.e., a little lower than what the EFSA [43] considers optimum. 3.3. Influential Factors on Breast Milk Mineral Content Milk production is a complex process conditioned by maternal and child factors. The mineral content is quite variable, and the study of influencing factors is very important for lactation management and to provide adequate dietary advice. In order to establish the possible relationship of categorical factors with the mineral content of breast milk, univariate statistical analyses were performed. Samples were divided into two groups for each category: less and more than 6 months of continued breastfeeding; BMI greater than or equal to 25 kg/m 2 ; and infant gender. The sodium, potassium, calcium, magnesium, phosphorus, iron, selenium and iodine contents across factor categories and significant differences are presented in Table 6. Samples were divided into two groups according to lactation time (LT) because, at 6 months, babies normally start complementary feeding. Only the Mg and K content in both groups of mothers, according to LT, followed a normal distribution. There aren’t significant differences when lactation is prolonged more than 6 months. The results obtained for Mg in the sampled women are homogeneous, Foods 2020,9, 659 9 of 15 in line with D ó rea [ 70 ], who refers to the homeostatic regulation of Mg to explain the limited variability in the concentration of magnesium in breast milk. With regard to potassium, although different values have been found in different parts of the world, among Galician women, there was no difference during lactation, whether it is more or less prolonged. A nonparametric Mann-Whitney U test was used for the other minerals. Using this statistical approach, significant differences between early and late lactation ( ≥ 6 months) were found for Ca, Ca/P ratio and I content. Calcium and phosphorus are major minerals that are tightly regulated in maternal serum. In milk, calcium is linked to casein and citrate, or occurs as ionic calcium; the concentration of the latter is more stable, which suggests a homeostasis similar to that of calcemia [ 71 ]. The findings in this work were consistent with those of other studies that observed a gradual decline of calcium in maternal milk with the advance of maternal lactation [ 38 , 72 , 73 ]. No significant differences in phosphorus content were found throughout lactation. This could be explained by the fine regulation of P content in breast milk [ 34 ]. Therefore, the differences found in the Ca/P ratio (i.e., declining in milk after 6 months of lactation) are due to the decrease of calcium. Feeley et al. [ 56 ] observed significant differences in Fe content with lactating time. As in this work, other authors such as Arnaud et al. [ 74 ] or Emmett and Rogers [ 75 ] reported, however, a constant level of iron throughout lactation. The iodine content in breast milk showed significant differences between the samples from less than six months and those with longer lactation times. Dror and Allen [ 76 ] also saw a gradual decline in iron levels in mature milk across longitudinal studies. Table 6. Sodium, potassium, calcium, magnesium, phosphorus, iron, selenium and iodine content in breast milk, as well Ca/P ratio, presented as mean, standard deviation and range across different groups of samples (lactating time or LT, BMI, newborn sex). Mineral Statistic LT <6 Months (n=48) LT ≥6 Months (n=27) BMI <25 kg/m2 (n=41) BMI ≥25 kg/m2 (n=34) Infant Gender ♂ (n=33) Infant Gender ♀ (n=42) Na (mg/L) Mean 133.53 256.37 132.37 * 205 * 212.39 * 131.30 * SD 55.16 242.31 66.72 156.08 200.60 30 Range 44.75–303.88 46.81–942.67 44.75–371.21 69.71–942.67 44.75–389.21 68.04–942.67 K (mg/L) Mean 465.37 438.03 449.00 476.86 450.42 463.60 SD 64.57 60.29 58.49 77.01 65.54 282.96 Range 345.57–622.25 342.98–550.27 345.57–579.69 342.98–622.25 353.34–622.25 342.98–599.71 Ca (mg/L) Mean 295.15 * 251.94 * 281.93 275.09 282.96 278.69 SD 55.18 58.24 59.72 56.90 63.94 55.28 Range 196.1–463.26 136.44–383.5 192.60–463.26 136.44–382.50 197.81–381.38 136.44–463.26 P (mg/L) Mean 126.79 127.02 133.38 120 122.38 * 132.66 * SD 26.56 34.10 29.47 30.07 32.35 23.44 Range 79.82–177.95 73–219.57 83.93–210.57 73.00–177.95 82.50–177.95 73–219.57 Ca/P (ratio) Mean 2.39 * 2.07 * 2.17 2.39 2.41 * 2.13 * SD 0.55 0.54 0.55 0.60 0.65 0.42 Range 1.34–4.44 1.16–3.36 1.29–4.44 1.16–3.44 1.29–3.14 1.16–4.44 Mg (mg/L) Mean 32.96 33.63 33.38 33.20 34.56 31.78 SD 6.82 5.75 7.08 4.85 6.43 6.28 Range 19.86–50.83 20.77–44.78 19.86–50.83 20.77–44.78 19.86–44.78 20.77–50.83 Fe (mg/L) Mean 0.22 0.24 0.22 0.24 0.21 0.24 SD 0.10 0.17 0.09 0.15 0.10 0.14 Range 0.10–0.45 0.06–0.75 0.06–0.42 0.06–0.75 0.06–0.45 0.06–0.74 Se (µg/L) Mean 11.41 21.99 11.60 12.07 18.95 10.66 SD 5.00 33.55 5.65 4.85 27.44 4.21 Range 4.37–25.85 6.18–148.97 4.37–28.13 5.30–148.97 4.37–21.14 6.18–148.97 I (µg/L) Mean 110.92 * 75.27 * 104.83 97 96.03 99.16 SD 53.61 61.61 62.09 53.36 68.01 52.72 Range 26.71–233.19 11.25–247.63 11.25–247.63 26.71–233.19 26,71–247.63 11.25–233.19 Significant differences between groups (*). Concerning maternal anthropometric characteristics (BMI), there are significant differences in Na levels in breast milk, with higher concentrations in women with BMIs greater than or equal to 25. This fact can potentially be explained by taking into account a possible failure in prolonged lactation (more than 12 months) in 7 women with BMIs greater than 25. Sodium content increases have been