2.1. Study Design and Population

We conducted a prospective, case series, single-center study of nursing mothers and a cross-sectional study of IF in University Clinical Hospital of Santiago de Compostela (Spain), shown in Figure 1. The inclusion criteria of nursing mothers were maternal age > 18 years old, without chronic disease and without taking nutrient supplements. All potential participants were introduced to this research and invited to join the study during prenatal and postnatal care at our institution. After receiving prior written informed consent, HM samples (5–10 mL) were obtained in 3 different periods of lactation during the first 6 months after birth: colostrum during the first 3–4 days of lactation, intermediate milk up to 7–10 days, and later mature milk, both in mothers of full-term; and later mature in mothers of premature newborns. Samples of full-term colostrum (n = 70), intermediate HM (n = 70), mature HM (n = 70) and preterm mature HM (n = 100) were collected between 1 January 2018 and 30 June 2019. In addition, we made a comparison group with 30 IF samples, selecting the brands used in our institution for children under 1 year of age, that account for 30% of the total brands sold in Spain, and classified into 4 groups: starter formulas (n = 13), continuation formulas (n = 10) (both milk protein-based formulas), hydrolyzed formulas (n = 5), and formulas for preterm infants (n = 2).

Study design.

Concentrations of the elements in milk were analyzed at the Laboratory of Analytical Chemistry, Nutrition and Bromatology of the University of Santiago de Compostela. Elements were classified into 3 groups: minerals (n = 5), including calcium (Ca), potassium (K), magnesium (Mg), sodium (Na), phosphorus (P); essential trace elements (n = 9), including cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), iodine (I), manganese (Mn), molybdenum (Mo), selenium (Se), zinc (Zn); and toxic trace elements (n = 21), including silver (Ag), aluminum (Al), arsenic (As), barium (Ba), beryllium (Be), cadmium (Cd), cesium (Cs), gallium (Ga), mercury (Hg), lithium (Li), nickel (Ni), lead (Pb), platinum (Pt), rubidium (Rb), antimony (Sb), tin (Sn), strontium (Sr), titanium (Ti), thallium (Tl), uranium (U), and vanadium (V).

Data were collected on medical, social, environmental, and demographic factors that may influence the composition of HM. For each participating mother, age, weight gain during pregnancy (excessive weight gain ≥16 kg) [21], residency, and smoking and drinking status were evaluated at the end of pregnancy. Gestational age and birth weight were recorded for all newborns.

Study approval was obtained from the Research Ethics Committees of Galicia (2017/082) and all the samples collected were analyzed exclusively for the purpose of the present study.