Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

BREAST-MILK SAMPLES
Breast-milk samples were voluntarily donated by lactating mothers at various postpartum (PP) periods, by manual expression or a breast pump. The samples were placed in sterile plastic tubes and transported to the laboratory on ice and processed immediately or stored in small aliquots at -70° or -80°C until use. One set of milk samples was collected in EDTA solution (to a final concentration of 20mM), while the other set of samples from the same mother was collected in an equivalent volume of PBS buffer only. The samples were classified according to postpartum period of lactation as follows (Lewis-Jones et al, 1985); colostrum (1-4 days PP), transitional milk (5-30 days PP) and mature milk (>30 days PP).

STORAGE OF MILK SAMPLES
Two samples each of colostrum and mature milk were refrigerated for up to 28 days, after which they were collected and either immediately processed or stored at -70 or -80°C in small aliquots until processed.

MEASUREMENT OF PH VALUES
Readings of pH values were taken using a glass electrode, with combined sensing and reference elements, inserted into the samples, connected to a research pH meter. The Radiometer (PHM84), (Radiometer, Copenhagen, Denmark), was capable of measuring temperature-dependent pH values, to an accuracy of three-decimal places. The Radiometer was calibrated daily, before and after sample measurements, by measuring its response to buffers of known pH.

REAGENTS AND BUFFERS
Standard Gelatin-veronal-buffered saline containing 0.15mM of Ca++ and 1mM of Mg++ (GVB2+), as well as GVB2-- were prepared as described (12). In addition, GVB2- containing different concentrations of calcium and magnesium ions were prepared for the haemolytic assays.

RABBIT RED BLOOD CELLS AND THE HAEMOLYTIC ASSAY
Non-sensitized rabbit erythrocytes (ORBO 20/21) were obtained from Behringwerke, Marburg, Germany. Using a micro-modification of the standard complement haemolytic assay (Mayer, 1971), 40µl of 5 x 108 the rabbit red blood cells/ml were added to Veronal buffer containing different concentrations of calcium and magnesium ions, to obtain a total reaction volume of 400µl. The milk samples were pre-diluted 1:20 with either GVB2+ or GVB2- and 100µl included in the reaction mixture. The reaction mixture was incubated for 60 minutes at 0°C (on ice) or at 37°C (in water bath), with occasional mixing. The reaction was stopped by addition of 1ml of cold GVB--. The cells were pelleted after the reaction by centrifugation at 12000g for 5 minutes and 200µl of each supernatant was pipetted into a 96 well flat microtitre plate (Greiner, Frickenhausen, Germany) and the amount of released haemoglobin recorded by a plate spectrophotometer (MR 700 microplate reader, Dynatech Instruments, Inc. Torrance, California) at a test wavelength of 402 against a reference wavelength of 612. Average of three readings were taken for each sample.

From the values of optical densities (OD) obtained, the degree of haemolysis produced by a given volume of HBM was obtained from the equation:

Y = Test OD - spontaneous lysisOD - HBM blank OD X 100 H2O lysis OD - spontaneous lysis OD

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