**Jennifer Gjerde \*, Marian Kjellevold, Lisbeth Dahl, Torill Berg, Annbjørg Bøkevoll and Maria Wik Markhus**

Institute of Marine Research, P.O box 1870 Nordnes, 5817 Bergen, Norway; Marian.Kjellevold@hi.no (M.K.); Lisbeth.Dahl@hi.no (L.D.); Torill.Berg@hi.no (T.B.); Annbjorg.Bokevoll@hi.no (A.B.); Maria.Wik.Markhus@hi.no (M.W.M.)

**\*** Correspondence: Jennifer.Gjerde@hi.no; Tel.: +4793652137

Received: 12 July 2020; Accepted: 27 July 2020; Published: 29 July 2020

**Abstract:** Vitamin D deficiency in pregnant women and their offspring may result in unfavorable health outcomes for both mother and infant. A 25hydroxyvitamin D (25(OH)D) level of at least 75 nmol/L is recommended by the Endocrine Society. Validated, automated sample preparation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were used to determine the vitamin D metabolites status in mother-infant pairs. Detection of 3-Epi25(OH)D3 prevented overestimation of 25(OH)D3 and misclassification of vitamin D status. Sixty-three percent of maternal 25(OH)D plasma levels were less than the recommended level of 25(OH)D at 3 months. Additionally, breastmilk levels of 25(OH)D decreased from 60.1 nmol/L to 50.0 nmol/L between six weeks and three months (*p* < 0.01). Furthermore, there was a positive correlation between mother and infant plasma levels (*p* < 0.01, r = 0.56) at 3 months. Accordingly, 31% of the infants were categorized as vitamin D deficient (25(OH)D < 50 nmol/L) compared to 25% if 3-Epi25(OH)D3 was not distinguished from 25(OH)D3. This study highlights the importance of accurate quantification of 25(OH)D. Monitoring vitamin D metabolites in infant, maternal plasma, and breastmilk may be needed to ensure adequate levels in both mother and infant in the first 6 months of infant life.

**Keywords:** breastmilk; infant; mother; plasma; vitamin D metabolites; 3-Epi25(OH)D3; 25-hvdroxyvitamin

### **1. Introduction**

Vitamin D plays an important role in bone metabolism. It regulates the calcium and phosphate in the body, making it important for muscle, tooth, and growth development [1]. It may also play an important role in immune system regulation [2,3]. The source of vitamin D in Norwegian diets is in the form of vitamin D2 (ergocalciferol) from plants and vitamin D3 (cholecalciferol) from fish, butter, and eggs [4]. Vitamin D2 and D3 bind to the Vitamin D carrier protein (DBP) before transported to the liver for hydroxylation, producing 25-hydroxyvitamin (25(OH)) D3 and 25(OH)D2. The 25(OH)D2 is derived solely from the diet/supplements and 25(OH)D3 is either derived from the diet/supplements or synthesized in the skin. The C3 epimer forms of vitamin D3 have less affinity toward vitamin D protein and even lower affinity for vitamin D receptors compared to 25(OH)D3. The 3-Epi25(OH)D3 binds to vitamin D receptor (VDR) at 2–3% the affinity of 25(OH)D3 [5]. Studies have also shown reduced ability in inducing calcium transport and reduced gene expression in the human colonic carcinoma cell line, Caco-2 [6–8]. Determining the concentration of 25(OH)D3 and 3-Epi25(OH)D3 separately is therefore important due to possible difference of effectiveness. However, further studies on the function and source of 3-Epi25(OH)D3 in humans are warranted.

Vitamin D3 has a plasma half-life of 4 to 12 h [9,10] and a circulating half-life of 12 to 24 h [11]. On the other hand, 25(OH)D2 and 25(OH)D3 have longer half-lives, 13 and 15 to 25 days, respectively [12–15]. The 25(OH)D is further hydroxylated to 1,25-dihydroxyvitamin D (1,25(OH)2D), the most potent physiologically active metabolite with a relatively short half-life of 4 to 6 h [16]. This suggests that measurements of 25(OH)D, 25(OH)D2, and 25(OH)D3 are better indicators of vitamin D status in the blood. Holick et al. have published a set of guidelines for the evaluation of Vitamin D deficiencies [17]. Blood 25(OH)D levels < 50 nmol/L are considered 25(OH)D-deficient, levels between 50 and 75 nmol/L are considered 25(OH)D-insufficient, and >75 nmol/L are considered 25(OH)D-sufficient [17–20].

Methods which only measure 25(OH)D3 and not 3-Epi25(OH)D3 may cause overestimation of 25(OH)D3 because both analytes would be determined as 25(OH)D3. To obtain accurate measurements of vitamin D levels in mothers and infants, a highly selective and sensitive quantification method for measuring vitamin D metabolites in blood and breastmilk is needed [21]. Thus, in this study, simple, sensitive, and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) methods were used for determination of 25(OH)D2, 25(OH)D3, and 3-epi-25-hydroxyvitamin D3 (3-Epi25(OH)D3) in plasma, breastmilk, and infant formula. An automated sample preparation involving protein-crash and solid-phase extraction techniques was applied to ensure simple sample treatment, and reduced time and labor.

During pregnancy, the main source of vitamin D for the fetus is the mother, through the umbilical cord. Studies have reported correlation between maternal and infant cord blood 25(OH)D concentrations [22–25]. Thus, mothers with sufficient 25(OH)D during pregnancy can provide sufficient cord blood concentrations of 25(OH)D crossing the placenta [26,27]. However, vitamin D metabolites levels < 50 nmol/L have been observed in pregnant women and their offspring [28–30]. Pregnant women and infants are highly susceptible to vitamin D deficiency which has been associated with adverse health outcomes, such as pre-eclampsia, perinatal complications, postpartum depression, spontaneous abortion, emergency cesarean section delivery, oligohydramnios, polyhydramnios, and gestational diabetes [31,32]. As for the infants, vitamin D deficiency may cause a small-for-gestational age condition, preterm birth, low birth weight, stunting, impaired fetal bone formation, and rickets [31–35]. Accordingly, Norwegian maternal vitamin D < 30 nmol/L has been associated with lower offspring peak bone mass [36]. Personalized vitamin D supplementation during pregnancy and lactation has been suggested [37]. Monitoring vitamin D deficiency in Norwegian mother-infant pairs before and after childbirth is therefore of importance.

The aim of this study was to validate selective and sensitive LC-MS/MS methods for the analysis of vitamin D metabolites with automated sample preparation. These methods were applied to determine the concentration levels of Vitamin D metabolites in mother-infant pairs during the first six months of breastfeeding. Detection and quantification of 3-Epi25(OH)D3 allows evaluation of the impact of 3-Epi25(OH)D3 when assessing vitamin D metabolites status in plasma and breastmilk. The relationships between plasma vitamin D metabolites levels in mother-infant pairs and breastmilk levels were also examined.

#### **2. Materials and Methods**

#### *2.1. Study Population and Design*

From January 2016 until February 2017, pregnant women were recruited to participate in a two-armed randomized controlled intervention trial involving cod intake in pregnancy [38]. The study was registered on ClinicalTrials.gov (NCT02610959) in 17 November 2015. All participants were recruited through the womens' clinic at Haukeland University Hospital in Norway. A total of 137 pregnant women were included in this secondary analysis. Details regarding the main study have been described elsewhere [38].

#### *2.2. Biological Samples and Laboratory Analysis*

Blood samples were obtained from mother-infant pairs. The mothers were requested to provide a sample of breastmilk at six weeks and three months postpartum. Breastmilk was collected at the beginning, middle, and end of a chosen feed. The three samples were then stored in a freezer until pick-up by study investigators or submission during the third-month follow-up visit. Samples were placed in freezer packs during transport. Upon arrival in the laboratory, samples were stored at –80 ◦C until analyzed as a pooled sample. Meanwhile, blood sampling was conducted in mothers and infants at three and six months postpartum. Plasma samples from the participants were obtained by collecting blood into BD Vacutainer ® K2E 5.4 mg vials (Franklin Lakes, NJ, USA), centrifuged (1000–1300<sup>×</sup> *<sup>g</sup>*, 20 ◦C, 10 min) within 30 min, and the supernatant was stored at –80 ◦C until analyzed.

#### *2.3. Laboratory Analysis*

#### 2.3.1. Chemical and Reagents

The standards 25(OH)D3, 25(OH)D2, 3-Epi25(OH)D3 and internal standard D6-25(OH)D3-(26,26,26,27,27,27-D6) were obtained from Cerilliant (Round Rock, TX, USA). The internal standards 25(OH)D2-(6,19,19-d3) and 3-Epi25(OH)D3-(6,19,19-d3), zink sulfate monohydrate, formic acid (analytical grade) and ammonium acetate were purchased from Sigma-Aldrich (St Louis, MO, USA).

#### 2.3.2. Sample Preparation

All sample preparation and extraction process were performed using robotic a Dual Head MultiPurpose Sampler (MPS XL) equipped with an Anatune CF-100 Centrifuge Option, MicroLiter ITSP Option and Active WashStation [39,40] Using an automated system, samples were prepared by adding 80 μL of internal standards to aliquots of 200-μL samples. Protein-crash, centrifugation, and solid-phase extraction techniques were automated using MPS XL (Anatune, Cambridge, UK). The samples were precipitated with 200 μL zinc sulfate and 500 μL methanol. Samples were then vortexed and centrifuged for 5 min. An aliquot of 500 μL of supernatant was loaded for solid phase extraction and eluted with 40 μL methanol. High purity water (18.2 million ohms, MΩ x cm) was added to the eluted sample prior to LC-MS/MS injection.

#### 2.3.3. LC-MS/MS Procedure
