Comparison between two methods of ionized calcium measurement in newborns

First submission on 05/01/13; last submission on 05/01/13; accepted for publication on 30/05/13; published on 20/10/13 1. Biomedical scientist; coordinator of the medical laboratory of Hospital Estadual Mário Covas. 2. Doctorate in Biochemistry from Instituto de Química da Universidade de São Paulo (IQ-USP); associate professor at Universidade Federal de São Paulo (UNIFESP), campus Diadema. 3. Doctorate in Clinical Analysis from Faculdade de Ciências Farmacêuticas da USP (FCF-USP); associate professor at UNIFESP, campus Diadema. 4. Professor of Biochemistry at IQ-USP; full professor at UNIFESP, campus Diadema. 5. Master’s degree in Pathophysiology at Faculdade de Medicina da USP (FMUSP); professor at Universidade de Santo Amaro (UNISA). 6. Master’s degree in Public Health from Hospital Emílio Ribas; assistant professor at Instituto de Pesquisa e Educação em Saúde de São Paulo (IPESSP). 7. Doctorate in Internal Medicine from UNIFESP; full professor at Faculdade de Medicina do ABC (FMABC). 8. Post doctoral research in Clinical Biochemistry from Roche Center for Medical Genomics-Basel (RCMG); coordinator of the medical laboratory of FMABC; associate professor at UNIFESP, campus Diadema. ABStrACt


introDuCtion
Calcium is an essential mineral in the human body, involved in several important metabolic processes, such as blood clotting, muscle excitability and contraction, transmission of nerve impulses, enzyme activation, second messenger formation and hormone secretion, with the main feature of mineralization of bone and teeth (9) .Calcium is present in the plasma in three forms: the free ionized calcium (iCa) constitutes about 50% of the total; 35% to 40% are bound to proteins, mainly to albumin; and 10% to 15% are complexed with low-molecular-weight ligands such as bicarbonate, phosphate, lactate, citrate, and others (8) .
Though poorly understood, the regulation of calcium homeostasis in the newborn has been of considerable interest.At birth, the iCa level in cord blood exceeds that in maternal blood.Recent findings show that parathyroid hormone-related peptide plays an important role in transferring calcium across the placenta into the fetal circulation.During the early days of life, the iCa level progressively decreases in normal infants, so that by the second or third day of life, the level is lower than that found in older infants and children.In most normal full-term infants the iCa level returns to normal by 10 days of life.In some infants the plasma iCa falls to pathologically low levels, and tetany or convulsions may occur (6) .
In almost all Brazilian laboratories, only total calcium is routinely measured, and iCa concentration is indirectly calculated based on total calcium, total protein and albumin concentrations.Errors associated with the measurement of those and other variables, such as pH, contribute to the difficulty of producing useful formulae to calculate iCa indirectly.Direct measurement of iCa by potentiometry is the method of choice for iCa determination, but it is an expensive analysis (8) .Therefore, the aim of this study was to evaluate whether iCa measured by potentiometry (direct measurement) correlates with iCa calculated by formulae (indirect measurement) in newborns.

Subjects
After parents' consent, 50 newborns underwent the protocol previously approved by the Ethics Committee.The criteria included newborns, but excluded those showing marked malnutrition, and/or premature babies.The group comprised male (n = 26) and female (n = 24) 9-day-old (mean) newborns, from NeoMater Hospital, in São Bernardo do Campo, São Paulo, Brazil.

Determination of iCa
After at least a four-hour fast, an anaerobic venous blood sample was drawn between 7 and 9 a.m.The samples were analyzed in duplicate at the hospital laboratory.The samples for albumin, total protein and total calcium were collected using gel heparin tubes.They were measured colorimetrically using Selectra II analyzer.A serum sample was taken for the direct measurement of iCa, which was carried out by potentiometry using Omni C apparatus.
All instruments and methods were found to be in good working order based on the manufacturer's descriptions and accumulated quality-control data.Among numerous formulae proposed to obtain serum iCa, the following formula was chosen because it is the most frequently used in Brazil

Statistical analysis
The statistical differences between mean values were assessed by Student's t test.The Pearson's correlation coefficient and its level of significance were calculated by the bivariate correlation procedure.

Patients' characteristics
The Table lists the mean values for age, both directly and indirectly measured iCa, total calcium, albumin and total protein of the newborns.All newborns were in good health.

Association between directly obtained serum iCa and calculated iCa
The

DiSCuSSion
This study is important because profound changes in iCa concentration seem to occur in newborns when the calcium supply from the mother is removed at birth, and a related hypocalcemia is produced.Thus, there is a demand for a reliable assessment of Comparison between two methods of ionized calcium measurement in newborns iCa measurement in newborns.Moreover, it is necessary to verify if the iCa measurement obtained by potentiometry can be correlated with the iCa calculated by formulae (indirect measurement) in newborns.To our knowledge, few studies pursue those aims (6) .
Once iCa is the most important physiologic fraction of calcium, it is necessary to measure it directly or to estimate it from measurements of total calcium, total protein, and albumin.Both methods, however, have limitations that must be understood for the appropriate interpretation of iCa levels in the clinical setting (5) .
Precision in iCa measurement was revolutionized after the introduction of ion-selective electrodes; however, the clinical application of this technique was initially limited and delayed by its cost, susceptibility to errors, need prevent CO 2 losses from the sample, and control of pH (7) .Advances in technology for direct measurement of iCa have decreased the cost and improved its availability in the clinical setting since the 1980s (1) .A number of limitations remained, however, particularly in the outpatient setting, including the technical challenge of equipment maintenance, frequent electrode replacement with associated downtime, and redundancy of instrumentation and personnel, all leading to increased costs (2,4) .
We think that iCa measurement can be systematically performed, directly or indirectly, in newborns because it was possible to verify a significant correlation between both methods.Also, our mean levels for iCa are consistent with previously published values (4) .Perhaps, in sicker newborns with malnutrition and low albumin, the calculated iCa cannot be used.
It is possible to conclude that there is a significant correlation between the iCa values obtained using Omni C apparatus and the calculated ones.We believe that both methods can be used in newborns.

Figure
shows that iCa measured by potentiometry (direct measurement) correlates with iCa calculated by formulae (indirect measurement) in newborns (r = 0.609, p < 0.001).
figurE -Pearson's correlation between ionized calcium obtained by potentiotiometry (Omni C) and obtained by formulae (calculated)