Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
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PROGRESSIVE PH CHANGES AND CYTOLYSIS INDUCED IN STORED BREAST-MILK
In-vitro storage of both the mature HBM and colostrum was associated with a progressive lipolysis and release of FFA, as evidenced by a steady fall in pH values and rising levels of degree of haemolyis, using rabbit erythrocytes as target cells (Table 1).
Table 1 Correlation of pH changes and hemolysis caused by refrigerated mature breast-milk samples with the duration of storage a b

No. of storage days      0            1            3            7           14             21            28
pH                  7.29 ± 0.40  7.20 ± 0.54  7.25 ± 0.35  6.95 ± 0.24  6.61 ± 0.20    6.28 ± 0.42   6.37 ± 0.32
% haemlysis          1.5 ± 0.3    7.5 ± 1.4   16.4 ± 3.2   51.2 ± 4.5    123 ± 10.6     119 ± 9.5

Figure 1 Effect of temperature and storage duration on lipolysis-induced hemolysis by mature milk in GVB2- buffer .

Similar results were obtained for colostrum samples. EFFECT OF CALCIUM AND MAGNESIUM ON LIPOLYSIS-INDUCED HEMOLYSIS IN GVB2+ BUFFER Presence of divalent calcium and magnesium cations greatly enhanced the cytolytic effects of stored HBM, when tested both at 0°C and 37°C. After 24-48 hours of refrigeration, an average haemolysis of 2.5% was caused by colostrum samples in GVB2-, in comparison to 102% in the standard GVB2+ buffer at 37°C, and 9% at 0°C (Table 2). Similarly, an average haemolysis of 1.5% was caused by mature milk samples in GVB2-, in comparison to 45% in the standard GVB2+ buffer at 37°C (Table 1 and Fig. 2). Increasing concentrations of calcium in the reaction buffer, up to 50mM, inhibited the degree of lipolysis-induced haemolysis by the stored breast-milk samples, while increasing levels of magnesium had the opposite effect .

Figure 2 Effect of combining increasing levels of Mg++ cations with high (50mM) or low (0.15mM) levels of Ca++ on lipolysis-induced hemolysis by mature milk refrigerated for 48 hours in GVB++ at 37°C

Figure 3 Effect of combining increasing levels of Mg++ cations with high (50mM) or low (0.15mM) levels of Ca++ on lipolysis-induced hemolysis by colostrum refrigerated for 48 hours in GVB++ at 37°C .

An attempt was made to determine the most favourable combination of cations which would maximally inhibit undesirable effects of lipolyis-induced cytolysis in the reaction of the CH50 whole complement assays, with its own peculiar needs of at least 1mM of magnesium. The minimum level of haemolysis was found with the combination of 1mM of magnesium and 50mM of calcium in GVB2+ buffer (Table 2). Table 2 Comparison of different Ca++/Mg++ combinations and temperatures on lipolysis-induced hemolysis in GVB++ buffer a b Concentration of Ca++/Mg++ in GVB++ Ca2+ 50mM, Mg2+ 1mM Ca2+ 50mM, Mg2+ 25mM Ca2+ 0.15mM, Mg2+ 1mM Ca2+ 0.15mM, Mg2+ 25mM 0°C 2.5 ± 0.7 3.5 ± 1.2 9 ± 2.5 50 ± 4.8 37°C 27.5 ± 3.4 50 ± 4.5 102 ± 15.3 134 ± 9.5 a Using colostrum samples after 48 hours of refrigeration b Results expressed as average ± SD

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