Acta Pzdiatr Scand 64: 709-717, 1975

QUANTITATIVE DETERMINATION O F IMMUNOGLOBULINS, LYSOZYME, AND CERTAIN ELECTROLYTES IN BREAST MILK DURING T H E ENTIRE PERIOD O F LACTATION, DURING A 24-HOUR PERIOD, AND IN MILK FROM T H E INDIVIDUAL MAMMARY GLAND BIRGIT PEITERSEN, LEIF BOHN and HENNING ANDERSEN From the Children’s Hospital, Fuglehakken and the Department of Clinical Chemistry, Bispebjerg Hospital, Copenhagen, Denmark

ABSTRACT. Peitersen, B., Bohn, L., and Andersen, H. (The Children’s Hospital, Fuglebakken and the Department of Clinical Chemistry, Bispebjerg Hospital, Copenhagen, Denmark). Quantitative determination of immunoglobulins, lysozyme, and certain electrolytes in breast milk during the entire period of lactation, for a 24-hour period, and in milk from the individual mammary gland. Acta Paediatr Scand, 64: 709, 1975.-During a period commencing at birth and lasting for up to 27 months 193 milk samples have been collected from 29 mothers. The IgA globulin content was high immediately after birth, averaging 2.7 arb.U, decreasing to 0.3 arb.U within the Fist 2 to 3 weeks after birth, then remaining almost constant for the rest of the lactational period. In the case of IgG globulin, similar results were obtained, but the quantity was much smaller. IgM globulin was demonstrated in small quantities during the fiist 3 weeks of lactation. The lysozyme content varied considerably during the whole lactafional period. Individual variations were found for all the immunoglobulins, while the concentration in the individual woman varied only slightly from day to day following in other respects the pattern described above. In 19 mothers IgA, IgG, IgM, lysozyme and electrolyte content were determined in serum and in milk from the right and the left breast on the same day. No difference in content was found between milk from the left and the right mammary gland. A positive correlation was found between the concentrations of IgA and sodium chloride in milk, between those of IgG in milk and serum, and between those of lysozyme in milk and serum. No variations were registered during the individual breast feeding, nor for the 24-hour period as a whole.

KEY WORDS: Immunoglohulins, lysozyme, electrolytes, human milk, daily variations, mammary gland

In recent years the proteins of breast milk, especially immunoglobulins, have been reviewed with increasing interest. Only a few publications on quantitative determinations have appeared, while the number of investigations concerned with qualitative determinations is much larger ( 3 , 7, 8, 9, 10, 1 1 , 22). During the period 1959-61 Hanson and coworkers found 14 immunologically different precipitates in colostrum, i.e. the milk col46-752875 Aclu Pzdiurr Scund 64

lected during the first 96 hours after birth, 13 of these precipitates being present also in milk collected later in the lactational period. Other investigations (18, 23, 31) have corroborated this, and at present at least 30 different antigens have been demonstrated in colostrum, 15 of which were also found in milk collected at later stages in the lactational period. 18 out of the 30 antigens were similar to those found in serum (12, 14, 2 2 ) , whereas Acta Pediatr Scand &I

710

B . Peitersen et al.

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tion. In all quantitative determinations IgA is the predominant immunoglobulin in breast milk constituting 90 % of all immunoglobulins in early colostrum and in milk from the first month of lactation. IgG was found in considerably smaller amounts, and IgM was found in determinable quantities only during the first week of lactation (2, 25, 27). Finally, Underwood et al. 1970 (31) examined the protein content in breast milk from Pakistani women, the milk being collected within a period from the 6th week to 24 months after birth. A distinct' fall in protein content from the 6th week to the 6th month after birth was registered. Following this the protein concentration remained stable for the following 12 months, increasing slightly during the period 18 to 24 months after birth. This late rise was not statistically significant. Several investigations concerning the content of electrolytes, proteins, amino acids and minerals in breast milk have been published (4, 5 , 6 , 19,21), but only Chodirker et al. (4) have carried out a simultaneous investigation of the serum content of these substances in the women in question. IgA, IgG, and IgM as well as lysozyme and electrolytes have been determined in milk collected simultaneously

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at least 12 were specific for milk (13, 14, 22). Furthermore, pre-colostrum, i.e. milk secreted 3 to 4 weeks prior to death, contained fewer antigen substances, but in late precolostrum and in colostrum the antigen content was almost identical, both containing IgM in determinable quantities, while only traces were found in milk collected after the third week of lactation. On the other hand, IgG and, especially, IgA are found in colostrum as well as in milk from the later stages of lacta-

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tation, which has been divided into groups. The number of samples within each group is indicated by N . The concentration of IgA is given in arbitrary Units based on the mass concentration values (gil) given in the instructions from Behring Werke.

Immunoglobulins, lysozyme and electrolytes in breast milk

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Table 1. Total secretions of IgA, IgG, I g M and lysozyme per 24 hours in 9 different women IgA is given in arbitrary units based on mass concentration values &/I) given in the instructions from Behring Werke

Lysozyme (mg/day) Patient I days 4-16 280 9.6 326 10.6 I99 13.2 152 9.2 286 14.0 226 11.4 2 78 9.4 404 11.0 Patient 2 , days 2-10 228 9.8 145 14.4 159 19.9 252 28.3 187 21.3 Patient 3 , days 2-24 1315 7.1 333 5.9 268 4.4 238 3.8 195 2.1 211 2.7 I65 2.2 210 2.6 Patient 4 , days 3-18 550 13.6 318 10. I 416 14.0 I72 6.9 258 11.7 306 15.0 262 15.2 242 L5.8 154 9.3 85 4.4 Patient 5 , days 4-14 657 19.2 426 16.5 263 16.4 223 15.1 23 1 18.9 120 18.0 ~

17.7 20.3 11.6 8.4 19.6 15.6 12.4 18.4

20.4 23.1 21.6 14.2 26.8 21.6 18.8 33 .O

10.2 12.3 11.2 21.3 14.1

21.0 44.7 65.1 72.8 89.4

21.5 7.2 5.2 2.8 1.2 I .4 2. I 1.5

31.1 39.2 50.2 51.0 36.8 44.1 31.8 41.6

14.8 9.9 12.8 5.4 7.9 8.8 6.4 traces traces traces

21.8 20.4 31.8 15.3 25.2 32.4 35.2 38.4 19.3 8.1

52.8 33.9 15.6 14.7 16.2 19.4

12.0 8.4 10.4 10.2 11.2 13.5

from the left and the right mammary gland, respectively, and in serum. Until recently little was known of the 24hour variations in the secretion of immunoglobulins in breast milk in the individual woman, and the present study has, therefore, been extended in a n attempt to elucidate this question. MATERIAL AND METHOD Lysozyme was determined by electrophoresis in antibody-containing agar (17). and two-dimensional im-

Patient 6 , days 3-20 1 626 28.8 680 15.2 338 8.0 507 traces 600 12.8 410 9.7 432 9.7 358 7.7 285 7.3 Patient 7, days 4-22 818 6.3 543 6.3 405 7.8 125 3.0 258 6.4 268 6.0 374 5.8 229 4.2 189 2.8 Patient 8 , days 24-60 3 I4 90.0 282 114.0 3 10 86.0 282 84.0 270 81.9 222 81.4 475 90.2 250 83.6 255 88.0 Patient 9 , days 26-60 404 51.6 306 57.6 327 48.0 344 32.4 345 28.6 445 43.5 513 45.0 639 40.5 584 44.8 412 51.2

24.6 12.0 traces traces traces traces traces traces traces

18.0 10.0 5.8 13.2 24.0 23.4 18.9 16.0 14.4

22.6 13.2 16.1 2.8 5.4 5.2 6.2 3.9 3.0

6.0 9.9 11.4 3.6 9.8 11.0 15.2 12.0 9.5

traces traces traces traces traces traces traces traces traces

38.0 34.0 42.0 26.0 16.8 33.0 79.2 46.2 39.6

traces traces traces traces traces traces traces traces traces traces

9.6 10.8 traces 40.8 14.3 16.5 25.5 25.5 65.6 38.4

munodiffusion in antibody-containing agar was used to determine the content of IgA, IgG, and IgM (16, 20). Sodium ion and potassium ion were determined by flame photometry, chloride ion titremetrically. The accuracy for IgA, IgG, and IgM was established by use of standard human serum from Behring Werke AG. Marburg-Lahn. This standard was also used for I g A . although milk IgA contains a secretory piece lacking in serum IgA. Hence, the results are given in arbitrary units based on mass concentration values (g/l) given i n the instruction from Behring Werke. For lysozyme a freeze-dried, purified human lysozyme from a patient with monocytic leucemia was used. The amino acid content and part of the lysozyme used is given in ref. 26. The precision was from 2.7 to 6.1 % for the imAcra Pediatr Scand 64

B . Peitersen et al.

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ing the procedure morning, noon, and night on the same day, and repeating it again on 5 different days within the first and second month of lactation. Finally, samples of milk were collected from each breast separately in 19 women. Serum samples as well as the milk samples from the individual patient were collected at the same time on the same day, while the time in relation to the period of lactation varied from woman to woman. Determinations of sodium ion, potassium ion, chloride ion, IgA, IgG, IgM, and lysozyme were carried out for all samples.

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munoglobulins and lysozyme. For the electrolytes the precision, expressed as coefficient of variation, was 1 % . Commencing with the first 4 days after birth, i.e. colostrum, and continuing up to 27 months after birth, 193 milk samples representing the entire period have been collected from 29 mothers. Each mother supplied a varying number of milk samples. viz. from I to 16, collected at different times during the entire period of lactation. However, none of the mothers supplied milk for periods longer than 3 months in all. as seen in Fig. I . The mothers were specially selected. some because the child was hospitalized immediately after birth, the remainder being mothers supplying milk to the Centre for Breast Milk at the Children’s Hospital, Fuglebakken. In the latter group, lactation was maintained longer than usual. The majority of the samples were collected early in the lactational period, while 4 mothers were able to supply milk for periods exceeding one year after birth. One of the women collected milk samples in the beginning, half-way. and at the end of a breast-feeding, repeatActu P z d i u t r Scund

64

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Fig. 4 . IgA concentration in milk from 9 different mothers.

Samples are all taken from first 2 months of lactational period. IgA concentration is given in arbitrary Units (see test). C H = m o t h e r no. I . A-A=no. 2. A-A=no. 3 . 0-O=no. 4. 0-Uno. 5. -.=no. 6. x-=no. 7. X-=no. 8. %.=no. 9.

Immunoglobulins, lysozyme and electrolytes in breast milk

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reaching an almost constant value of 0.3 arb. Ull after 2 to 3 weeks. Towards the end of the lactational period the content increases slightly. These findings hold true for the individual mother as well as for the material as a whole. It is remarkable that the individual variations in IgA concentration are so great, while the concentration in the individual case varies very little within the above-mentioned pattern. The changes in the IgA concentration in

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Fig. 6 . IgM content during period of lactation, which has been divided into groups. The number of samples within each group is indicated by N .

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breast milk simply during the lactational period are not merely a question of dilution, as is evident from Table 1 and Figs. 3 and 4. Here the total 24-hour secretion of IgA has been calculated in 10 mothers, and the total secretion is seen to be high in colostrum while a decrease soon sets in, i.e. in the course of 1-2 weeks. Following this the content remains more o r less constant for the remainder of the lactational period. It should be noted that the

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714 B . Peitersen et al.

strated, which condition remains unchanged for the remajnder of the lactational period. In 3 out of the 4 mothers supplying milk for more than one year after birth, determinable quan-

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Fig. 7. Lysozyme content during period of lactation. The material has been divided into groups and the number of samples within each group indicated by N . In the period 15 to 27 months of lactation, lysozyme was found in only 3 samples, the values varying considerably.

increase in concentration found during the last phase of lactation is due, mainly, to the decreasing amount of milk, as no corresponding increase in the total secretion is registered. IgG is also found in breast milk during the entire lactational period. Fig. 5 shows the concentration to be high in colostrum but considerably lower than the IgA concentration, the mean value being 0.08 g/l, ranging from 0.03 g/l to 0.25 g/l. Following this the content decreases rapidly, and 2 to 3 weeks after birth the secretion is stabilized on the 0.03 g/1 level, increasing slightly towards the last weeks of the lactational period. Also in the case of IgG the individual differences are considerable, while the concentration in the individual case follows the pattern outlined above. However, the total 24-hour secretion of IgG in the individual mother is far more constant right from the start of the lactation, as Table 1 shows. IgM is found in relatively small amounts in breast milk and in determinable quantities only during the first 3 weeks of the lactational Acra Pediatr Scond 64

as in the material as a whole (Fig. 7). No definite correlation was found regarding intervals within the lactational period, or regarding the amount of milk secreted, as shown in Table 1 . The results of the determination of IgA, IgG, IgM, lysozyme, and electrolytes in milk from the right and the left breast, and in serum, appear in Table 2. It should be noted that all serum concentrations were within the normal range for adults for the analytical method in question. Analysing all data by the Spearman Ranking Test, the following conclusions may be drawn. A significant, positive correlation was found between the following Concentrations: ( I ) The concentrations of sodium and chloride ion in milk @