# Nano-HPLC-HRMS Analysis to Evaluate Leptin Level in Milk Samples: A Pilot Study
## 1 Introduction  
Leptin is a lipophilic protein of 167 amino acids with a molecular weight of about 16 kDa secreted by adipocytes. It is a protein hormone discovered in 1994 during a study related to obesity in mice [1]. The gene encoding for the protein is expressed on the surface of adipocytes cell membrane; leptin serum concentration levels are related to adipose tissue and food intake. The protein is involved in different organism activities; the most significant is to inform the brain with a negative feedback that regulates food intake. Moreover, the hormone modulates angiogenesis, fertility, and bone formation [2]. Recently, leptin was also related to human breast feeding and obesity since infants that are breastfed showed lower probability to develop pediatric obesity compared with newborn nots fed with human breast milk [3; 4; 5; 6; 7; 8; 9]. In light of this consideration, leptin could be considered to be a biomarker of health. Human milk is characterized by a complex composition which includes nutrients, hormones, and several bioactive components that could influence infant growth, development, and well-being later in life [10]. Leptin is one of the proteins present in mammalian breast milk. These play a fundamental role in infant growing and development and their intake through breast feeding isimportant. In particular, human milk contains many other elements, such as lipids, carbohydrates, vitamins, minerals, growing factors, and immunity/stem cells. All of these compounds make human milk a unique vital resource for the newborn. Moreover, human milk changes its composition during lactation: colostrum, produced from the first to the sixth day after birth, has a high concentration of antibodies and nutrients (proteins); transition milk, formed from about six days until 10 to 14 d after birth, shows elevated amount of fat and lactose (concentration of proteins is reduced); and finally the mature milk, composed by high amount of fat and lactose too and low proteins content [11]. A recent systematic review of literature tried to provide evidence on the effect of leptin, ghrelin, Insulin Growth Factor 1, adiponectin and insulin on infants' and children's growth and body composition. The available data reveals conflicting findings concerning the potential role of all these hormones on modeling growth and fat mass apposition and health outcomes later in life [12]. However, in animal models, leptin, when ingested as a component of breast milk, is increasingly recognized as playing a role in the postnatal programming of a healthy phenotype in adulthood age [13; 14]. We recently reported that breast milk leptin and maternal BMI could influence infant serum leptin values, and this could be of interest to explain the beneficial effect of breast feeding on preventing obesity in children [15]. Leptin concentration in human whole milk shows different concentration among lactation period: from 0.63 ng\(\cdot\)mL\({}^{-1}\) to 32.7 ng\(\cdot\)mL\({}^{-1}\), 2-42 and 60-120 d after birth respectively [16; 17]. It was reported that breastfed infants have a higher leptin values that formula fed ones [18; 19]. However, since breast feeding is not always possible or practical, in this case the newborn is fed with infant formulas. These are defined by Commission Directive 2006/141/EC of 22 December 2006 as \"the only processed foodstuff which wholly satisfies the nutritional requirements of infants during the first months of life until the introduction of appropriate complementary feeding [...]. The essential composition of infant formulas must satisfy the nutritional requirements of infants in good health as established by generally accepted scientific data\" [20]. The European Commission Directive 2006/141/EC lists minimum and maximum concentrations of the compounds contained in infant formulas, such as proteins, lipids, vitamins, mineral salts, and so on. Most of the infant formula products are produced with dairy products; the most important is certainly bovine milk, which contains bovine leptin, appropriately processed.