Data were analyzed using the NutriQuanti On-line Computerized System [13]. Dietary intakes were adjusted according to total energy intake, calculated by the residual method [14] and to intra-individual variation [15]. The recommendations proposed by the dietary reference intakes were employed in the estimation of Ca and Mg intake. The probability of inadequate Ca and Mg intake was determined

selleck compound from the ratio D/SDD, where D is the difference between the average intake by an individual and the estimated average requirement (EAR) according to age and physiological state (pregnancy), and SDD is the standard deviation of D, calculated by taking into account the SD of the intake distribution of the reference group and the SD of the data obtained from the 4-day food record [16], [17] and [18]. Blood and 24-hour urine samples were employed in the assessment of Ca and Mg status. Venus blood samples were collected Palbociclib from participants after 8 hours of fasting and transferred to demineralized tubes containing anticoagulant. Plasma and erythrocytes were separated by centrifugation, and the erythrocytes were washed 3 times in NaCl solution (0.9%, w/v) before

re-centrifugation. Participants were requested to collect a 24-hour urine sample on the day before blood collection. Urine was collected in demineralized bottles from 6 am (including morning urination) to 6 am the following day, and samples were stored at − 20°C until analysis. Bone resorption was evaluated from the amount of type I collagen C-telopeptides (CTX) in plasma as determined using Serum CrossLaps enzyme-linked immunosorbent assay kits (Nordic Bioscience Diagnostics A/S, Herlev, Denmark). The level of CTX was obtained by extrapolating the average of duplicate readings against a standard curve constructed in the concentration range 0 to 2.988 ng/mL. The normal range for plasma

CTX in women was taken to be 0.112 to 0.738 ng/mL [19]. The levels of Mg in plasma and erythrocytes, and the excretion of Ca and Mg in urine, were determined by flame atomic absorption spectroscopy (AAnalyst 100; Perkin Elmer, Norwalk, CT, USA). La2O3 was added to all standard and sample solutions prior SPTLC1 to analysis. Standard curves were constructed using CaCl2 or MgCl2 (Titrisol; Merck, Darmstadt, Germany) in the concentration range 0.05 to 5 μg/mL [20]. The certified standard Trace Element Serum L1 (Seronorm, Billingstad, Norway) was used for plasma analyses, while urine and erythrocyte pools were employed as secondary standards. All items of glassware employed in the analyses were demineralized. In the absence of specific reference data for pregnant women, normal adult values were adopted for urinary Ca excretion (3.74-7.50 mmol/L) [21], urinary Mg excretion (3.00-5.00 mmol/L) and erythrocyte Mg (1.65-2.65 mmol/L) [22]. The normal range for plasma Mg in pregnant women was taken as 0.63 to 0.91 mmol/L [23].