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Immunology & Immunological Disorders Poster Session






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Cytotoxicity of EDTA Used in Biological Samples: Effect on Some Breast-milk Studies


Contact Person: Michael O. Ogundele (mogundel@yahoo.com)


Results

EFFECT OF EDTA ON THE HUMAN BREASTMILK pH
EDTA caused a significant fall in the pH of all breast-milk samples (Table 1). This was partly reversed by de-fattening the milk samples through high-speed centrifugation.

Table 1: Effect of EDTA on pH changes in human breast-milk. Results expressed as average SD.
Whole-milk alone Whole-milk + PBS After centrifugation Whole-milk + edta After centrifugation
Colostrum 7.60 0.40 7.63 0.34 7.70 0.65 6.80 0.35 7.00 0.74
Transitional milk 7.44 0.50 7.46 0.55 7.66 0.45 6.93 0.23 7.12 0.61
Mature milk 7.29 0.43 7.20 0.47 7.40 0.49 6.97 0.36 7.05 0.42

Table 2. Effects of EDTA on human breast-milk cell count. Results expressedas cells X 104/ml (Average SD).
Milk sample Fresh Whole-milk + PBS After 12 hours on ice Fresh Whole-milk + edta
Colostrum 62 8.5 22 6.0 40 5.5
Transitional milk 7.5 2.0 2.5 1.25 4.0 2.5
Mature milk 4.0 2.5 1.5 1.0 2.25 1.5

TOXIC EFFECTS OF EDTA ON BREASTMILK CELLS
EDTA caused a significant loss of cells in fresh milk samples, compared to parallel samples containing an equivalent volume of PBS (Table 2). A similar progressive loss of milk cells was observed during in-vitro storage of milk samples on ice for 12 hours or longer.

EFFECT OF EDTA ON THE MEASURABLE PROTEIN CONTENT OF HUMAN BREASTMILK
The total protein content of HBM was increased in the EDTA-containing samples compared to those with equivalent volumes of PBS (Table 3). This increased level of protein was reversed or diminished by de-fattening the breast-milk samples, suggesting that the source of the excess measurable protein was the milk lipid fraction. All de-fatted milk samples contained less measurable total protein in comparison to whole-milk samples.

Table 3. Effects of EDTA on the total protein concentration of different human breast-milk samples. Results expressed as mg/ml (Average SD)
Milk sample Whole-milk + EDTA Whole-milk + PBS Centrifuged EDTA sample
(20,000g)
Colostrum 16.82 2.5 14.30 2.8 12.66 3.0
Transitional milk 12.56 1.3 10.09 1.5 8.94 1.7
Mature milk 13.70 2.2 9.51 0.5 9.06 1.5

Table 4. Comparative cytotoxicity of EDTA on sensitized sheep and unsensitized rabbit erythrocytes in the presence of human breast-milk and different buffer compositions. Results expressed as % haemolysis (Average SD).
Unsensitized rabbit RBC Sensitized sheep RBC
Volume of De-fatted Milk 0 10L 50L 0 10L 50L
Mg 10mM, Ca 0.015mM 0 0.9 0.14 9 2.3 0 0.8 0.3 6.5 1.4
Mg 10mM, Ca 0.015mM + EDTA 22.4 5.4 79 15 98 10 47.5 6.8 223 17 282 12.5
Mg 25mM, Ca 0.015mM 0 0.9 0.2 8 3.1 0 0.8 0.3 0.8 0.2
Mg 25mM, Ca 0.015mM + EDTA 115 21 ++++++ +++++ 135 20 ++++++ +++++
EDTA at a final concentration of 10mM in GVB++ buffer (Ca 0.015mM and Mg 1mM) with 40l of RBC at 5x108 cells/ml in a total reaction volume of 375 l.
+++++ Excessive haemolysis spectrophotometrically not measurable.

EFFECTS OF EDTA ON COMPLEMENT-INDUCED HAEMOLYSIS OF HUMAN BREASTMILK
When employed in CH50 haemolytic assays, different buffer compositions containing 10mM EDTA consistently showed an enhanced haemolysis ofthe sensitized SRBC, compared to the those containing no EDTA, irrespective of the original level of actual complement activity (Table 4). This concentration of EDTA used in the buffers compares with 20mM used as Complement inhibitor agent used in collected fresh milk samples. When GVB2- containing 0.01M EDTA was used as the buffer for stopping the complement activity reaction, the level of haemolysis was also increased in comparison to EDTA-depleted GVB2- buffer (Table 5).

Table 5 Effect of EDTA in the stopping solution on the degree of complement-induced and -independent haemolysis of sensitized SRBC by human breast-milk samples
20 of Whole-milk 40l of Whole-milk
Stopped with GVB-EDTA Stopped with GVB2- Stopped with GVB-EDTA Stopped with GVB2-
Colostrum 11.2 1.3 0.7 0.62 30.6 26.7 1.8 0.93
Transitional milk 36.9 22.8 0.87 0.61 57.5 37.5 1.0 0.9
Mature milk 21.3 11.7 1.7 2.13 43.3 28.9 1.47 2.5
Results expressed as % haemolysis (Average SD) SRBC = sheep red blood cells

COMPARATIVE CYTOTOXIC EFFECT OF EDTA ON RABBIT AND SHEEP ERYTHROCYTES
The unsensitized rabbit red blood cells were more sensitive to the toxic effects of EDTA in the presence of breast-milk samples, when compared to sensitized sheep erythrocytes (Table 4). In the absence of EDTA, the enhanced susceptibility of sensitized sheep erythrocytes could be attributed to the activation of complement system components of HBM.

In the presence of 0.01M EDTA, a brownish discolouration of the "ghost" red blood cells as well as the reaction mixture supernatant occurred in both cases, after an incubation period of 1 hr at 37C. This was probably a result of oxidation of haemoglobin to methaemoglobin.

EFFECTS OF CALCIUM AND MAGNESIUM ON THE TOXIC ACTIVITY OF EDTA
EDTA failed to cause any degree of haemolysis of rabbit RBC in excess of that in the buffer. Ca2+ caused a dose dependent increase in EDTA-induced haemolysis up to a maximum of 5mM. Above this concentration, it caused a progressive inhibition of the EDTA-induced haemolysis in a dose-dependent manner, approaching the baseline buffer level .

Figure 1. Effect of calcium and magnesium cations on the cytotoxicity of EDTA in GVB2+ buffer a b .

Mg2+ ions on the other hand, consistently caused a dose-dependent enhancement of EDTA cytotoxicity.

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Ogundele, M.O.; (1998). Cytotoxicity of EDTA Used in Biological Samples: Effect on Some Breast-milk Studies. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Available at URL http://www.mcmaster.ca/inabis98/immunology/ogundele0175/index.html
© 1998 Author(s) Hold Copyright