Biochemical Analysis of Mineral Metabolism and Central Bone Mineral Density in 157 Adult Women

EDWIN SEVER BECHIR1, MARA CARSOTE2*, MIHAELA JANA TUCULINA3, MARILENA BATAIOSU3, IONELA TEODORA DASCALU3, MIHAELA RAESCU4, RADU RICA3, CONSTANTIN DAGUCI3, LUMINITA DAGUCI3, ANCA PREDESCU3, OANA CELLA ANDREI5, RAZVAN MERCUT6, CRISTIAN NIKY CUMPATA4 1University of Medicine and Pharmacy, Dental Medicine Faculty,38 Gheorghe Marinescu Str., 540139, Targu Mures, Romania 2Csrol Davila University of Medicine and Pharamacy, Department of Endocrinology, C.I.Parhon National Institute of Endocrinology, 34-38 Aviatorilor Ave, 37 Dionisie Lupu Str., 020021, Bucharest, Romania 3University of Medicine and Pharmacy , Faculty of Dental Medicine, 2-4 Petru Rares Str., 200349, Craiova, Romania 4Titu Maiorescu University, Dental Medicine Faculty, 67A Gheorghe Petrascu Str., 031593, Bucharest, Romania 5 Carol Davila University of Medicine and Pharmacy, Faculty of Dental Medicine, 37 Dionisie Lupu Str., 020021, Bucharest, Romania 6University of Medicine and Pharmacy Craiova, Faculty of Medicine, 2-4 Petru Rares Str., 200349, Craiova, Romania

This is a cross-sectional retrospective study of observational type. 157 menopausal subjects were included. A number of N1=89 were younger of 60 years old (also included) and a number of N2=68 were older than 60 years old. Median of age was of 55 years, respective 66 years.The biochemical parameters like total and ionic serum calcium, serum magnesium, and phosphorus between the two groups N1-N2 were similar (p>0.05).The median values of mentioned chemical elements were within normal limits.The bone turnover markers were not statistically significant different between N1 and N2. 25OHD was found deficient in both populations, irrespective of age. DXA-BMD and T-score N1-N2 difference was statistical significant for all the four central sites. Biochemical mineral parameters seem not to be influenced by the cut off of 60 years in menopausal women aged between 40 and 80 years. Yet, a large prevalence of hypovitaminosis D is identified regardless the age without secondary PTH raise. The statistical significant results are for BMD and T-score for all the four central sites.

Experimental part Method and subjects Study design
This is a cross-sectionalretrospective study of observational type. The study was conductedbetween 2016 and 2017. The patients were evaluated for different medical conditions but the population was not pre-selected for calcium anomalies presentation, neither for skeletal anomalies (apparently healthy regarding potential dysfunctions of mineral metabolism).

Material (patients)
The clinical evaluation of the subjects included the medical background in order to evaluate the inclusion and exclusion criteria, fasting morning blood assays and 24hours urinary calcium assessment. Also, each patient had a central DXA (Dual-Energy X-Ray Absortiometr y) performed at the following levels: lumbar spine (from first to fourth vertebra), left hip (for total hip and femoral neck areas), and third distal radius level at non-dominant arm. DXA analysis provided BMD (Bone Mineral Density) at the four central sites: lumbar, total hip, femoral neck, distal third radius and derived T-scores and Z-score (which are directly provided by the DXA machine, a GE Lunar Prodigy device) according to WHO criteria [8].
Inclusion criteria were: adult female, menopausal status for at least one year (without current or prior estrogens replacement therapy), age between 40 and 80 years, informed written consent.
Exclusion criteria are: confirmed cancers of any pattern, including primary or secondary bone neoplasia; lack of complete panel of investigations including central DXA at the four mentioned sites, specific medication for fracture risk reduction (previous supplements with vitamin D and calcium are not quantified and thus allowed for this study).

The blood biochemical assays
Calcium (an alkaline earth metal having the atomic number 20, situated at fourth period) is tested in daily human practice at blood and urinary level. [10] There are two types of blood-derived values for every day practice: total serum calcium and ionic serum calcium. Total calcium (CaT) is based on a correction (mg/dL) according to the formula: CaT = measured serum CaT (mg/dL) + 0.8[4-measured serum albumin (g/dL)]. [10] Serum ionic calcium (CaI) levels are calculated based on CaT and circulating total proteins (TP) based on formula: CaI = [6xCaT (mg/dL) -TP(g/dL)/3]: [TP (g/dL) + 6]. [10] The normal serum values and the method of detection are displayed in table 1. 24-h urinary calcium (24-h Ca) is measured on a urinary sample covering an entire day. (Table  1) Also, the mineral metabolism includes the assessment of serum phosphorus (P) represent the chemical element associating the 15 atomic number (third period) and clinically tested as introduced in table 1 [11]. Moreover, magnesium (Mg) is a chemical element (alkaline earth metal) having the atomic number 12 (third period) (table 1) [12].
The activity of bone cells is reflected by bone turnover markers: of formation -osteocalcin (also named G1 protein of the bone), P1NP (aminoterminalpropeptide of type I collagen), and alkaline phosphatase(AP; this is ahomodimeric protein enzyme serving as basic phosphatase which requires alkaline pH for optimal function. [13]The bone resorption marker is serum CrossLaps which is C-terminal telopeptide (a named derived from carboxy-terminal collagen crosslinks) [14] (table 1).
The endocrine control of mineral metabolism is reflected by calcifediol (25-hydroxycholecalciferol) or 25OHD and parathormone (PTH) assays [15,16]. 25OHD offers the best reflection of vitamin D status which is regulated based on a negative feedback with PTH (table 1) [15,16].

Statistical tests
Statistical analysis introduced features as mean, standard deviation (SD), and median. The statistical significant results are considered at p<0.05 (for functions as ttest).

Results and discussions
157 menopausal subjects were included in the study. A number of N1=89 were younger of 60 years old (also included) and a number of N2=68 were older than 60 years old ( fig. 1)    This is a study of chemical assays involving the mineral metabolism and central DXA in menopausal women (N=157) of above (N1) and over 60 years old (N2). The cut-off of 60 years is important in skeletal evaluation as well as others cardio-metabolic features [17].
Limits of the study are worth to be mentioned: lack of correlation data with calcium and vitamin D supplements; also the menopausal status might influence the bone profile as DXA and bone turnover markers, an effect that has not been quantified in the study.

Conclusions
Biochemical mineral parameters seem not to be influenced by the cutoff of 60 years in menopausal women aged between 40 and 80 years. Yet, a large prevalence of hypovitaminosis D is identified regardless the age without secondary PTH raise. The statistical significant results are for BMD and T-score for all the four central sites analysed at central DXA. Abbreviations AP = Alkaline Phosphatase BMD = Bone Mineral Density BMD = Bone Mineral Density CaT = serum total calcium CaI = serum ionic calcium DXA = Dual-Energy X-Ray Absortiometry P = phosphorus PTH = parathormone TP = total proteins SD = standard deviation 25OHD = 25-hydroxyvitamin D 24-h Ca = 24-hours urinary calcium .