Experimental Physiology (1996), 81, 1007-1020
Printed in Great Britain


       BLOOD AND MILK PROLACTIN AND THE RATE
    ...
1008                     D. B. COX, R. A. OWENS AND P. E. HARTMANN

Hartmann, 1981; De Carvalho, Robertson, Friedman & Kla...
PROLACTIN AND MILK SYNTHESIS IN WOMEN                                            1009

    Table 1. Details of the subject...
1010                      D. B. COX, R. A. OWENS AND P. E. HARTMANN

Milk production
   The 24 h milk production was measu...
PROLACTIN AND MILK SYNTHESIS IN WOMEN                                        1011

Statistical analysis
   Mixed model reg...
1012                         D. B.   COX. R. A. OWENS                     AND P. E. HARTMANN


                       400 ...
PROLACTIN AND MILK SYNTHESIS IN WOMEN                                                             1013

      40 -Mother A...
1014                        D. B. COX, R. A. OWENS AND P. E. HARTMANN


                           30       (20)          ...
PROLACTIN AND MILK SYNTHESIS IN WOMEN                                                 1015


 Table 2. Linear regression a...
1016                   D. B. COX, R. A. OWENS AND P. E. HARTMANN

infants breastfed on demand from 1 to 6 months of lactat...
PROLACTIN AND MILK SYNTHESIS IN WOMEN                                   1017
possible that the effect of prolactin was del...
1018                     D. B. COX, R. A. OWENS AND P. E. HARTMANN

Autocrine control of milk synthesis
   Recent research...
PROLACTIN AND MILK SYNTHESIS IN WOMEN                                     1019
                                           ...
1 020                    D. B. COX, R. A. OWENS AND P. E. HARTMANN

LAWRENCE, R. A. (1989). Breastfeeding; A Guide for the...
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Blood And Milk Prolactin And The Rate Of Milk Synthesis In Women

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Blood And Milk Prolactin And The Rate Of Milk Synthesis In Women

  1. 1. Experimental Physiology (1996), 81, 1007-1020 Printed in Great Britain BLOOD AND MILK PROLACTIN AND THE RATE OF MILK SYNTHESIS IN WOMEN DAVID B. COX, ROBYN A. OWENS* AND PETER E. HARTMANN* Departments of Biochemistry and * Computer Science, The University of Western Australia, Nedlands, WA 6907, Australia (MANUSCRIPT RECEIVED 18 APRIL 1996, ACCEPTED 16 JULY 1996) SUMMARY In women, the concentration of prolactin in the plasma increases in response to nipple stimulation. This response has led to the assumption that prolactin influences the rate of milk synthesis. To investigate this hypothesis we have measured 24 h milk production, the short-term (between breastfeeds) rates of milk synthesis and the concentration of prolactin in the blood and breastmilk, from 1 to 6 months of lactation in eleven women. Over the long term, the 24 h milk production remained constant (means + S.E.M.): 708 + 54-7 g/24 h (n = 11) and 742 + 79-4 g/24 h (n = 9) at 1 and 6 months, respectively. The average short-term rate of milk synthesis (calculated from the increase in breast volume between breastfeeds; means + S.E.M.) did not change: 23 + 3 5 ml/h (n = 23) and 23 + 3 4 ml/h (n = 21) at 1 and 6 months, respectively. However, significant variation in the short-term rate of milk synthesis (from <5-8 to 90 ml/h) was found both between breasts, measured concurrently (coefficient of variation, c.v. = 72 %), and within the same breast, measured over consecutive breastfeeds (c.v. = 85 %). The basal and suckling-stimulated concentrations of prolactin in the plasma (means + S.E.M.) declined from 1 to 6 months (basal, from 119 + 93 to 59 + 29 ,ug/l; peak, from 286 + 109 to 91 + 44 ug/1). In contrast, the concentration of prolactin in milk was much lower than in plasma, and decreased only slightly from 1 to 6 months of lactation (fore-milk, from 26.4 + 10 to 23 3 + 98 8ug/l; hind-milk, from 18-9 + 5.1 to 13 2 + 6-3 jtg/l). The concentration of prolactin in the milk was related to the degree of fullness of the breast, such that the concentration was highest when the breast was full. We found no relationship between the concentration of prolactin in the plasma and the rate of milk synthesis in either the short or long term. However, the relationship between the concentration of prolactin in milk and the degree of fullness of the breast suggests that the internalization of prolactin, after binding to its receptor, may be restricted when the alveolus is distended with milk. INTRODUCTION There is strong evidence for the involvement of the endocrine system, and prolactin in particular, in milk synthesis in women and other mammals (Cowie, Forsyth & Hart, 1980). In women, suppression of prolactin secretion with bromocriptine immediately after the delivery of the infant inhibited lactogenesis II (the onset of copious milk secretion) (Kulski, Hartmann, Martin & Smith, 1978), whereas augmentation of the concentration of prolactin in the plasma with sulpiride on day 2 postpartum increased total milk production (suckling plus additional expressed milk) over the subsequent 3 days of lactation (Aono, Shioji, Aki, Hirota, Nomura & Kurachi, 1979). Prolactin is secreted into the blood in response to the suckling stimulus (Noel, Suh & Frantz, 1974; Tyson, Khojandi, Huth & Andreassen, 1975) and increased suckling frequency during galactopoiesis (established lactation) results in an elevation of the basal concentration of prolactin in the plasma (Delvoye, Demaegd, Delogne-Desnoeck & Robyn, 1977; Gross & Eastman, 1983). In addition, increased suckling frequency is also associated with increased milk production (Hennart, Delogne-Desnoeck, Vis & Robyn, 1981; Rattigan, Ghisalberti & Downloaded from Exp Physiol (ep.physoc.org) by guest on August 22, 2009
  2. 2. 1008 D. B. COX, R. A. OWENS AND P. E. HARTMANN Hartmann, 1981; De Carvalho, Robertson, Friedman & Klaus, 1983) and is recommended to women as a practical means of increasing an inadequate supply of milk (Phillips, 1991). In this connection, drugs which increase prolactin secretion also have been recommended for women with infants who are failing to thrive (Lawrence, 1989). Clinically, it has been assumed from this circumstantial evidence that the suckling-evoked release of prolactin provides the stimulus to the breast to make more milk (Renfrew, Fisher & Arms, 1990). Studies in Western countries have shown that the magnitude of the suckling-evoked increase in the concentration of prolactin in the plasma declines over the first 6 months of lactation (Noel et al. 1974; Tyson et al. 1975). However, this has not been related to concomitant measurements of milk production. In selected Chinese mothers with 'adequate' milk production at 40 and 60 days of lactation, Huang, Zheng & Qian (1987) reported no significant difference in milk production, although there was a significant decrease in the prolactin response to suckling from 40 to 60 days. Prolactin also has been measured in milk, and while Healy, Rattigan, Hartmann, Herington & Burger (1980) found significant relationships between the concentration of prolactin in the milk and the concentrations of lactose, a-lactalbumin and total protein in milk, there are no reports on either the relationship between the concentration of prolactin in the milk and milk production, or the prolactin response to suckling and the short-term rates of milk synthesis in women. In order to determine the importance of prolactin in the control of milk synthesis, we have investigated the relationships between the concentrations of prolactin in the blood or milk and milk synthesis, in women who were breastfeeding on demand at 1, 2, 4 and 6 months of lactation. METHODS Subjects Mothers (n= 11) of healthy infants breastfed on demand (Table 1) provided informed consent to participate in the study, which was approved by The University of Western Australia, Committee for Human Rights. The mothers were recruited through either the Nursing Mothers' Association of Australia, or private health care centres. The mothers were familiarized with the study and the operation of the equipment before any measurements were made. Measurements of the short-term rates of milk synthesis and the collection of blood began between 09.00 and 10.00 h, and continued for two to three breastfeeds, depending on the frequency of breastfeeding and the mother's time constraints. The measurements were all performed within 1 week of 1, 2, 4 and 6 months postpartum, except for mother H, who was studied at 3 months instead of 4 months. In addition, measurements were not made on mothers D and I at 6 months, since their infants were completely weaned by this time. Blood samples were collected, and the short-term rates of milk synthesis were measured, at the Department of Biochemistry, while the 24 h milk production was measured in the volunteers' homes. The volunteers maintained their normal pattern of breastfeeding at all times. Plasma prolactin Soft TouchTm Lancets (Boehringer Mannheim Australia, Castle Hill, NSW, Australia) were used to collect finger prick blood samples into heparinized haematocrit tubes (Chase Instruments Corp., Glens Falls, NY, USA) before, and 45 min after (to give an approximation of the maximum concentration of prolactin in the plasma following nursing; Noel et al. 1974), the start of a breastfeed. The tubes were centrifuged to separate the plasma, which was collected and frozen at -20 °C until analysis. The prolactin concentration was determined using the RIABEAD II kit (Abbott Australasia, Diagnostics Division, North Ryde, NSW, Australia). Recovery of a known amount of prolactin added to plasma samples was 1024 + 058 % (mean + S.E.M.; n = 10). Downloaded from Exp Physiol (ep.physoc.org) by guest on August 22, 2009
  3. 3. PROLACTIN AND MILK SYNTHESIS IN WOMEN 1009 Table 1. Details of the subjects and patterns of infant milk intake 24 h individual breast milk production Time after Fully or partly Feeding Subject Parity birth breastfeeding frequency Left breast Right breast (months) (breasts/24 h) (g/24 h) (g/24 h) A 1 1 Fully 7 330 603 2 Fully 6 335 320 4 Fully 9 366 587 6 Partly 8 487 363 B 1 1 Fully 13 400 527 2 Fully 16 498 558 4 Fully 13 357 569 6 Fully 13 346 558 C 3 1 Fully 10 206 470 2 Fully 9 191 263 4 Fully 12 331 549 6 Fully 14 462 697 D 1 1 Fully 11 332 539 2 Fully 8 357 395 4 Partly 12 219 516 E 3 1 Fully 7 329 517 2 Fully 8 321 400 4 Fully 7 382 446 6 Fully 7 551 485 F 2 1 Fully 8 439 430 2 Fully 8 492 398 4 Fully 8 417 310 6 Partly 7 551 627 G 2 1 Fully 12 452 602 2 Fully 11 418 532 4 Fully 11 325 530 6 Partly 10 255 335 H 1 1 Fully 9 425 508 2 Fully 10 344 653 3 Fully 7 483 546 6 Partly 10 545 450 I 2 1 Fully 7 211 190 2 Fully 6 312 708 4 Partly 8 289 328 J 2 1 Fully 11 224 380 2 Fully 11 248 218 4 Fully 9 329 298 6 Fully 11 237 307 K 1 1 Fully 11 591 372 2 Fully 9 373 582 4 Partly 9 258 469 6 Partly 9 136 265 Downloaded from Exp Physiol (ep.physoc.org) by guest on August 22, 2009
  4. 4. 1010 D. B. COX, R. A. OWENS AND P. E. HARTMANN Milk production The 24 h milk production was measured by maternal test weighing (Arthur, Hartmann & Smith, 1987). Due to the difficulty in defining a breastfeed for demand-fed infants, we have expressed the frequency of breastfeeding in terms of the number of times the infant suckled on a breast that resulted in milk removal (breasts/24 h). Mothers usually fed their infants from one breast during a breastfeeding episode (over 80 %); however, if the baby had been fed from both breasts during a breastfeeding episode, we classified this as two breasts, for the calculation of feeding frequency. Short-term rates of milk synthesis The Computerized Breast Measurement (CBM) system (Daly, Kent, Huynh, Owens, Alexander, Ng & Hartmann, 1992; Daly, Owens & Hartmann, 1993 b) was used to determine changes in breast volume, allowing calculation of the short-term rates of milk synthesis. In addition to the apparatus described by Daly et al. (1992), a JVC-1200 Colour Special Effects Generator (Television Communications, Malaga, WA, Australia) was introduced to assist the mothers to resume the same position for each measurement, by allowing a stored and a live video image, each at half-density, to be superimposed. At the beginning of each session, an image of the breast was captured and stored. This image was then retrieved to a monitor with live video input. The mothers then moved their bodies until the painted lines encircling the breast in the live image were exactly superimposed on the same lines in the stored image. The breast volume of mothers A-G was determined before and after each breastfeed, and, when time permitted, during the interval between breastfeeds. Short-term rates of milk synthesis were calculated from the changes in breast volume over time (Daly et al. 1992, 1993 b). The accuracy of these measurements was determined by comparing the changes in breast volume over a breastfeed with the changes in maternal weight over the same breastfeed, corrected for evaporative water loss (Arthur et al. 1987). The regression coefficients ranged from 0 83 to 0 96 for individual mothers with the pooled regression (0.92) being similar to that described previously (Daly et al. 1993 b). Short-term rates of milk synthesis were not calculated for mothers H-K because the volume of their breasts could not be measured reproducibly by the CBM system, due to large occlusion zones (Daly et al. 1992). Milk prolactin Mothers A-G expressed milk samples from each breast, before (fore-milk) and after (hind-milk) feeding their infants, both during the measurements of the short-term rates of milk synthesis and during the 24 h period over which milk production was measured. These samples were stored at -20 °C until analysis. Milk samples were defatted by centrifugation at 10000g for 10 min. Milk prolactin was measured in the defatted milk samples, using the RIABEAD II kit, by directly substituting defatted milk for plasma in the company-supplied protocol. Recovery of a known amount of prolactin added to milk samples was 102 7 + 1.6 % (mean + S.E.M.; n = 7). Milk fat content Since 98 % of milk fat is present as triacylglycerols (Jensen, Bitman, Carlson, Couch, Hamosh & Newburg, 1995), we determined the fat content of breast milk as the esterified fatty acid content (Stern & Shapiro, 1953; Atwood & Hartmann, 1992; Daly, Di Rosso, Owens & Hartmann, 1993a). Samples of breast milk were thawed, warmed to 37 °C and mixed thoroughly. Aliquots (2-5 ,ul) were taken from each sample, placed into 600 ,ul of redistilled ethanol and mixed vigorously. This mixture and standards (2.5 ,ul triolein (Sigma T-7140) in 600#,1 of redistilled ethanol) were assayed in duplicate. The interassay coefficient of variation was 7-26 % (n = 20) and the detection limit was 3.26 g/l. Degree offullness of the breast Fat content was determined in fore- and hind-milk collected at each breastfeed over a 24 h period. The relationship between the milk fat content and the degree of emptying of the breast (Daly et al. 1993a) was used to calculate the degree of fullness of the breast. Milk samples containing the maximum and minimum milk fat concentration were designated as having degrees of emptying one and zero, respectively (see Daly et al. 1993 a). By fitting the curve described by Daly et al. (1993 a), the fat content of a milk sample could be used to determine the degree of emptying of the breast at the time the sample was collected. This was converted to the degree of fullness of the breast by subtracting the degree of emptying from one. Downloaded from Exp Physiol (ep.physoc.org) by guest on August 22, 2009
  5. 5. PROLACTIN AND MILK SYNTHESIS IN WOMEN 1011 Statistical analysis Mixed model regression analysis was performed using SAS release 6.07 (SAS Institute Inc., Cary, NC, USA, 1992). Student's paired t test and linear regression analysis were performed using the Abacus Concepts, StatView SE+GraphicsTM software package (Abacus Concepts, Inc., Berkeley, CA, USA, 1987). Coefficient of variation (c.v.) is the standard deviation expressed as a percentage of the mean. All values are expressed as means + S.E.M. unless otherwise stated. RESULTS Subjects The eleven women recruited for this study all fully breastfed their infants to 2 months of age. By 6 months only four of the mothers were fully breastfeeding their infants (Table 1). A long- term medical condition may have influenced the feeding behaviour of infant I through lactation, while a routine immunization made infant A unsettled at 2 months and may have influenced the measurement of milk production. Plasma prolactin concentration After statistical analysis to control for individual variation, there was a significant decline in the basal concentration of prolactin in the plasma (prolactin concentration at least 90 min after a breastfeed) from 119 + 19-1 jug/l (n = 24) and 121 + 18 3 jug/l (n = 32) at I and 2 months, respectively, to 71 + 81 ,ug/l (n = 31) at 4 months and 59 + 5.7 jug/l (n = 26) at 6 months (mixed model analysis; P = 0.0001; Fig. 1). At 1 month postpartum the mean concentration of prolactin in the plasma 45 min after the commencement of feeding (suckling-stimulated concentration) was 286 + 22 6 jtg/l (n = 23) and by 2, 4 and 6 months, this had decreased significantly, to 218 + 24.1 jug/l (n = 31), 139 + 16 4 jug/l (n = 30) and 91 + 8-7 jug/l (n = 26), respectively, after statistical analysis to control for individual variation (mixed model analysis; P = 0.0001; Fig. 1). Similarly, following statistical analysis to control for individual variation, the magnitude of the increase in the suckling-stimulated concentration of prolactin in the plasma declined significantly, from 172 + 29-0 j/tg/l (n = 24) at 1 month to 98 + 21-5 jug/l (n = 32), 70 + 131 ,ug/l (n = 29) and 31 + 5.0 jtg/l (n = 25) at 2, 4 and 6 months, respectively (mixed model analysis; P = 0-000 1; Fig. 1). Milk production The frequency of breastfeeding did not change, being 9 6 + 0-65 breasts/24 h (n = 11) at 1 month, 9 3 + 0-84 breasts/24 h (n = 11) at 2 months, 9 6 + 0 64 breasts/24 h (n = 11) at 4 months and 9 9 + 0-82 breasts/24 h (n = 9) at 6 months of lactation. The mean duration of the breastfeeds at 1 month was not significantly different to the mean duration of the breastfeeds at 6 months: 30 3 + 1 6 min (n = 113) and 25-0 + 4 1 min (n = 99), respectively. The mean milk production at 1, 2, 4 and 6 months was 708 + 54-7 g/24 h (n = 11), 732 + 53.2 g/24 h (n = 11), 735 + 34.7 g/24 h (n = 11) and 742 + 79.4 g/24 h (n = 9), respectively. Controlling for variation due to individual mothers, no significant difference was found in milk production between different stages of lactation (mixed model analysis; P = 0 77; Fig. 1). A positive relationship was found between total milk production and feeding frequency (linear regression analysis; correlation coefficient, r2 = 0.40, P = 0 008, n = 42). Downloaded from Exp Physiol (ep.physoc.org) by guest on August 22, 2009
  6. 6. 1012 D. B. COX. R. A. OWENS AND P. E. HARTMANN 400 800 300 22) ( T600£ I _ ~~~~~~~~(31) E 200 400 (24) (32) Q~~~~~~~~~~~(0 (30) E 0 (26) - 100 (2)200 2 0 LJ0 1 2 4 6 Time postpartum (months) Fig. 1. Immunoreactive prolactin determined in plasma samples collected from the eleven mothers (at 1, 2 and 4 months), and from nine mothers (at 6 months), immediately before suckling (U) and 45 min after the commencement of suckling (LO). Number of observations is shown in parentheses. Twenty-four hour milk production (ml/24 h) of the same mothers determined by test weighing (e). Results are mean values + S.E.M. Short-term rates of milk synthesis The rate of milk synthesis in individual breasts ranged from a minimum detectable rate of 5 8 ml/h to 90 ml/h. At 1, 2, 4 and 6 months postpartum, the mean rate of milk synthesis was 23 + 3.5 mI/h (n = 23), 20 + 3.0 ml/h (n = 26), 20 + 3.8 ml/h (n = 24) and 23 + 3 4 ml/h (n = 21), respectively. Controlling for variation due to the individual breast and the mother, there was no significant change in the average rate of milk synthesis over the duration of the study (mixed model analysis; P = 0.95). The rates of milk synthesis within a breast could be grouped into two categories: first, those where the rates of milk synthesis remained the same following consecutive breastfeeds, and second, those where the rates of milk synthesis changed following consecutive feeds. Out of the thirty-three pairs of consecutive measurements of the rates of milk synthesis within the same breast (unilateral), eleven pairs exhibited significant changes over time (95 % confidence intervals for the rates of milk synthesis did not overlap). Similarly, out of twenty-four pairs of rates of milk synthesis determined simultaneously for both breasts (bilateral), eleven exhibited significant differences between breasts (see examples in Fig. 2). The c.v. for the absolute differences between the unilateral rates of milk synthesis was 85 %, and between bilateral rates of milk synthesis c.v. was 72 %. Milk prolactin concentration The concentration of prolactin in milk was measured in seven mothers on samples collected in conjunction with the measurements of the short-term rates of milk synthesis. The concentration of prolactin in fore-milk was 26 4 + 2 29 jug/l (n = 19) at 1 month, 26 4 + 3.43 ,ug/l (n = 22) at 2 months, 18 7 + 1 38 ,tg/l (n = 24) at 4 months and 23-3 + 2-25 ,ug/l Downloaded from Exp Physiol (ep.physoc.org) by guest on August 22, 2009
  7. 7. PROLACTIN AND MILK SYNTHESIS IN WOMEN 1013 40 -Mother A 30- 20- 4 30 - Mo)ther B 1n 20 v1- 10E :1o .4 50 . ,T~~ID v: *-5 40 -n 30 - 30 20 10 [ 10- . 0. 10.00 12.00 14.00 16.00 10.00 12.00 14.00 16.00 Time of day (h) Time of day (h) 30 Mother D _ 20 - .w -z 10 Lili 1I 30 ar- 30~ 20 -4 20 4 a 10 10I - - 10. l.00 12.00 14.00 16.00 10.00 12.00 14.00 16.00 Time of day (h) Time of day (h) Fig. 2. Examples of' the range of within-day variation in the rate of milk synthesis for the left (C1) and right (a) breast measured at 4 months of lactation. The height of the histogram bars represents the average rate of milk synthesis between measurements of breast volume (width of histogram bars). The bars with arrows indicate the start, duration and finish of' breastfeeding episodes within a breast. In mothers A (right breast), B (right breast) and D (left breast), breast volume also was measured in the interval between breastfeeds, and the rate of milk synthesis was calculated for each interval. No measurement was made for mother C on the left breast between 10.30 and 14.00 h, and mother D's infant required breastfeeding before the initial breast volume could be determined for the left breast. (n = 19) at 6 months (Fig. 3). The concentration of prolactin in hind-milk was 18 9 + 1.23 jug/l (n = 17) at 1 month, 147 + 198,ug/l (n = 20) at 2 months, 12.9 + 071 ug/l (n = 19) at 4 months and 13 2 + 1 45 lug/l (n = 19) at 6 months (Fig. 3). The concentration of prolactin in fore-milk was significantly higher than that in hind-milk (Student's paired t test; P = 0 0001), and when the variation due to the mothers was controlled for, the concentration of prolactin in the milk declined from 1 to 6 months of lactation (mixed model analysis; fore- milk, P = 0 049; hind-milk, P = 0.012). Comparative analysis No significant relationship was found between the volume of milk removed during a feed and the rate of milk synthesis after that feed (linear regression analysis; r2 = 0 042, P = 0.75). No relationship was found between the basal or the suckling-stimulated concentration of Downloaded from Exp Physiol (ep.physoc.org) by guest on August 22, 2009
  8. 8. 1014 D. B. COX, R. A. OWENS AND P. E. HARTMANN 30 (20) (23) ~~~~ i ~~~~~~~~(20) i (18) (27) 120 2 (21) (22) ~(21) I-5 10 0 2 ~~~46 Time postpartum (months) Fig. 3. Immunoreactive prolactin determined in milk saimples collected from seven mothers (at 1, 2 and 4 months; mothers A-G), and six mothers (at 6 months; mothers A-C and E-G). immediately before (U) and immediately after suckling (O) on the same day as the plasma samples depicted in Fig. 1. Number of observations in parentheses. Results are mean values + S.E.M. prolactin in plasma and the short-term rates of milk synthesis, in either breast, immediately after that breastfeed (mixed model analysis; basal, P = 0-55; suckling-stimulated, P = 0-93) or after the next breastfeed (mixed model analysis; basal, P = 0-23; suckling-stimulated, P = 0.48). No significant relationships were found between the concentration of prolactin in the milk and the short-term rate of milk synthesis before or after a breastfeed. In addition, no significant relationship was found between the concentration of prolactin in plasma and the concentration of prolactin in milk, controlled for variation due to the individual breast, the mother and the stage of lactation (mixed model analysis; fore-milk vs. basal concentration of prolactin in the plasma, P = 0-45; hind-milk vs. basal concentration of prolactin in the plasma, P = 0.65). The concentration of prolactin in milk samples collected simultaneously with the measurement of milk production was negatively related to the fat content and positively related to the degree of fullness of the breast, when both were included in the same statistical model, which controlled for variation due to the individual breast and the stage of lactation (mixed model analysis; fat content, P = 0.0001; degree of fullness, P = 0.0001). Although the degree of fullness was calculated from the fat content of the sample and the maximum and minimum fat content over the 24 h period, using the relationship described by Daly et al. (1993 a), it is important to note that the statistical model demonstrated a relationship between the degree of fullness of the breast and the concentration of prolactin in the milk, independent of the milk fat content. We have analysed mothers and individual breasts separately by linear regression analysis (Table 2). For six of the mothers, the degree of fullness of the right breast explained 37-67 % of the variation in the concentration of prolactin in milk (Table 2), while for the left breasts of five of the mothers, 21-56 % of the variation in the concentration of prolactin in milk was explained. Of the seven women, only mother D consistently showed no relationship Downloaded from Exp Physiol (ep.physoc.org) by guest on August 22, 2009
  9. 9. PROLACTIN AND MILK SYNTHESIS IN WOMEN 1015 Table 2. Linear regression analyses of the relationships between milk prolactin and the degree of breast fullness, within each breast and within each mother Right breast Left breast Both breasts Mother Probability Correlation Probability Correlation Probability Correlation A 0.0005 0-62 0.95 0-0028 0.12 0.073 B 0 0009 0.65 0 0059 0.37 0.0001 0 44 C 0.0032 0.45 00004 0.56 00001 049 D 0.65 0013 050 0.036 0.012 0.54 E 0.0006 0-67 0 032 0 21 0 0001 0 37 F 0.0059 0.37 00049 0-38 0.0002 0.33 G 00039 0.46 0011 0.36 0.0001 040 C* 0.0033 0.25 0.010 0.39 0.0007 0.22 Asterisk indicates analysis of samples from mother C obtained from a previous study (Daly et al. 1993 b), in which the degree of breast emptying was determined using the CBM system. For this analysis, degree of breast emptying was converted into degree of breast fullness, by subtraction of the degree of emptying from one. between milk prolactin and degree of fullness (Table 2). Subject C also participated in a previous study (Daly et al. 1993 b) when she was feeding a 5-month-old infant. We have measured the prolactin concentrations in milk samples collected during that study and compared them with the degree of fullness of the breasts determined directly using the CBM system. In this case, the degree of fullness explained 25 and 39 % of the variation in the concentration of prolactin in milk, on the right and left sides, respectively (Table 2). Furthermore, the decrease in the concentration of prolactin from the fore- to hind-milk was significantly related to the volume of milk removed from the breast during a feed (Student's paired t test; P = 0.014). DISCUSSION Plasma prolactin and milk synthesis We found a significant, progressive decrease, from 1 to 6 months of lactation, in the concentration of prolactin in blood plasma, both before (basal) and 45 min after (suckling- stimulated) the commencement of breastfeeding (Fig. 1). Despite this decline, basal prolactin at 6 months postpartum was still higher than the concentration reported for non-lactating women at 6 months postpartum (Gross & Eastman, 1983). Similar decreases in basal prolactin during lactation have been found for women in the USA during the first month of lactation and from 1 to 7 months of lactation (Noel et al. 1974; Tyson et al. 1975; Battin, Marrs, Fleiss & Mishell, 1985), in Zaire from approximately I to 30 months of lactation (Hennart et al. 1981) and in the Philippines from 1 to 20 months of lactation (Gross, Haynes, Eastman, Balderrama-Guzman & del Castillo, 1980). Basal prolactin, at 1 month of lactation, ranged from as low as 15 + 1.4 ug/l (n = 3) (Noel et al. 1974) to 119 + 19.1 ,ug/l (n = 24) measured in the present study (Fig. 1). This variation may be explained by differences in the frequency of breastfeeding, which ranged from four to six times per 24 h up to 2 months of lactation and from two to four times per 24 h up to 7 months of lactation for American mothers feeding to a strict routine (Noel et al. 1974), to 9 6 + 0-65 feeds per 24 h (n = 11) for Downloaded from Exp Physiol (ep.physoc.org) by guest on August 22, 2009
  10. 10. 1016 D. B. COX, R. A. OWENS AND P. E. HARTMANN infants breastfed on demand from 1 to 6 months of lactation in the present study (Table 1) and nine to ten feeds per 24 h for at least the first 12 months of lactation in Zairean women (Hennart et al. 1981). Furthermore, Gross et al. (1980) found a significant correlation between the frequency of breastfeeding and the basal concentration of prolactin in both amenorrhoeic and menstruating lactating Filipina women. Since the clearance of prolactin from the plasma can take up to 180 min (Noel et al. 1974), more than eight feeds per 24 h may not allow the concentration of prolactin to fully decline before the next breastfeed and this could explain the elevated basal concentration of prolactin associated with more frequent breastfeeding. We found no change in milk production from 1 month (708 + 54.7 ml/24 h (n = 11)) to 6 months (742 + 79.4 ml/24 h (n = 9)) of lactation (Fig. 1). Similar milk intakes have been reported for longitudinal studies in the USA by Neville, Keller, Seacat, Lutes, Neifert, Casey, Allen & Archer (1988) (range, 739 + 47-3 ml/24 h (n = 12) to 787 + 24 4 ml/24 h (n = 13)) and Dewey & Lonnerdal (1983) (range, 673 + 48.0 ml/24 h (n = 16) to 896 + 36.8 ml/24 h (n = 1)). However, concurrent prolactin measurements were not made in the earlier studies. In the present study, while milk production remained relatively constant until 6 months of lactation, the concentration of prolactin in plasma declined (Fig. 1). This is consistent with Huang et al. (1987), who reported that between 40 and 60 days postpartum there was no change in milk production (1160 + 102 and 1125 + 108 g/24 h, respectively), while there was a decline in the concentration of prolactin in the plasma (from 3975 + 533 mU/I in 13 women to 2695 + 334 mU/l in 15 women; P < 0 05) in mothers selected from groups of women (n = 24 and 26, respectively) on the basis of 'adequate' milk volume. Therefore, 24 h milk production was not controlled by either the basal or suckling-stimulated concentration of prolactin in the blood. Practical experience suggests that, within women, there is considerable variation in milk production between breasts (Mobbs, 1990; Phillips, 1991). We have found that milk production from right breasts was significantly higher than that from left breasts (mixed model analysis; P= 0021; Table 1), which is consistent with Daly et al. (1993 b), who reported asymmetric milk production. Whereas almost all of the literature presents only the combined milk production for both breasts, the above findings stress the importance of assessing each breast independently for investigation of the mechanisms controlling milk production in women. We have investigated the possibility of a relationship between the concentration of prolactin in the plasma and the short-term rate of milk synthesis (i.e. the rate of milk synthesis between breastfeeds). We found no significant change in the mean short-term rate of milk synthesis from 1 to 6 months of lactation (mixed model analysis; P = 0.95) and there was no relationship between the mean short-term rate of milk synthesis and either the basal or suckling-stimulated concentration of prolactin in the plasma. In agreement with Daly et al. (1993b), there was considerable variation in the short-term rate of milk synthesis both between breasts (c.v. = 72 %) and within the same breast (c.v. = 85 %0) (see examples in Fig. 2). Nevertheless, these variations in the short-term r.ate of milk synthesis immediately following a breastfeed were not related to variations in either the basal or suckling-stimulated concentration of prolactin in the plasma, controlled for variation due to the individual breasts, the mothers and the stage of lactation (mixed model analysis; basal concentration, P = 0.55; suckling-stimulated concentration, P = 0 93). Since prolactin reaches a peak concentration in blood approximately 45 min after the beginning of a breastfeed (Noel et al. 1974), it was Downloaded from Exp Physiol (ep.physoc.org) by guest on August 22, 2009
  11. 11. PROLACTIN AND MILK SYNTHESIS IN WOMEN 1017 possible that the effect of prolactin was delayed until after the subsequent breastfeed. However, there were no relationships between the short-term rate of milk synthesis following the subsequent breastfeed and either the basal or suckling-stimulated prolactin from the previous breastfeed, controlled for variation due to the individual breasts and the stage of lactation (mixed model analysis; basal concentration, P = 0.23; suckling-stimulated concentration, P = 0.48). The bilateral variation in the short-term rates of milk synthesis following successive breastfeeds (Fig. 2) indicates that the rate of milk synthesis within one breast was independent of the rate of milk synthesis in the other breast. For example, whereas the rate of milk synthesis in one breast was higher than in the other breast after one breastfeed, the reverse was often observed after the next breastfeed (see Fig. 2, mother B). Overall, we found that ten out of thirteen sets of bilaterally paired rates of milk synthesis exhibited significant reversals in the rate of milk synthesis occurring following consecutive breastfeeds. Similarly, Daly et al. (1993 b) found significant variations in the short-term rate of milk synthesis between breasts within women. These findings clearly demonstrate that milk synthesis is controlled independently in each breast. Since each breast would be expected to be exposed to the same concentration of blood prolactin, asymmetry in milk production and bilateral variations in the rate of milk synthesis are inconsistent with changes in the concentration of prolactin in the blood directly regulating either the amount of milk produced or the short-term rate of milk synthesis. Nevertheless, threshold concentrations of prolactin in blood are essential for both the initiation (Kulski et al. 1978) and maintenance of lactation (Cowie et al. 1980). Thus, for blood prolactin to have an effect on the regulation of milk synthesis, it would have to act through a locally selective mechanism within individual breasts. Milk prolactin and milk synthesis The concentration of prolactin in milk (Fig. 3) was significantly lower (P = 0.049) than its concentration in blood plasma (Fig. 1), in agreement with previous reports for milk prolactin at 2 weeks (Healy et al. 1980) and 4 weeks (Yuen, 1988) of lactation. The concentration of prolactin in the defatted milk varied during suckling, with fore-milk having a significantly higher concentration of prolactin than the hind-milk (Fig. 3). Yuen (1988) compared twenty- one paired samples of fore- and hind-milk, collected from women between 7 and 88 days of lactation, and found a similar difference between fore- and hind-milk. Noilin (1979) reported that there was cyclic movement of prolactin into the lactocytes (mammary secretory epithelial cells) of the rats, with the highest rate of prolactin entry into the lactocyte occurring when the alveolus was empty, and decreasing as the alveolus filled with newly secreted milk. This lead Yuen (1988) to speculate that the variation in the concentration of prolactin between fore- and hind-milk of women may be a result of the cyclic entry of prolactin into the lactocyte in response to alveolar distension and contraction. This hypothesis is consistent with the relationship between the concentration of prolactin in the milk and the degree of fullness of the breast, providing that there is limited mixing of milk in the ducts, permitting the formation of a concentration gradient for milk prolactin from the alveoli to the nipple. Since prolactin mRNA has been detected in rat lactocytes (Steinmetz, Grant & Malven, 1993), the possibility that de novo synthesis of prolactin contributed to the differences in the concentration of prolactin in fore- and hind-milk cannot be dismissed. Downloaded from Exp Physiol (ep.physoc.org) by guest on August 22, 2009
  12. 12. 1018 D. B. COX, R. A. OWENS AND P. E. HARTMANN Autocrine control of milk synthesis Recent research suggests that autocrine mechanisms appear to control the rate of milk synthesis in rats (Heesom, Souza, Ilic & Williamson, 1992), goats (Peaker & Wilde, 1987; Wilde, Calvert, Daly & Peaker, 1987), marsupials (Sharman, 1970) and women (Prentice, Addey & Wilde, 1989; Daly et al. 1993 b). A 7-6 kDa component of the whey fraction of goats' milk (feedback inhibitor of lactation, FIL) has been shown to inhibit the synthesis and secretion of milk by stopping membrane trafficking between the endoplasmic reticulum and the Golgi body (Wilde, Addey, Boddy-Finch & Peaker, 1995). Proteins similar to FIL have been identified in the milk of other species, including cows (Wilde et al. 1995), tammar wallabies (Nicholas, Wilde, Bird, Hendry, Tregenza & Warner, 1995) and women (Prentice et al. 1989). Bennett, Knight & Wilde (1990) found that, when applied to mouse lactocytes, a 10-30 kDa whey fraction from goats' milk, containing the autocrine inhibitor (2 times concentrated relative to milk), reduced the number of receptors for prolactin on the basal membrane of the lactocyte, thereby reducing prolactin binding. A reduction in the number of prolactin receptors is also consistent with the cyclic movement of prolactin into the lactocytes reported by Noilin (1979) and the higher concentration of prolactin in fore-milk (providing fore-milk represents milk secreted immediately following a breastfeed). Daly et al. (1993 b) found that the short-term rate of milk synthesis was higher when most of the available milk had been removed from the breast. While we did not find a relationship between the short-term rate of milk synthesis and the concentration of prolactin in the milk, it is of interest that we did find a relationship between the degree of fullness of the breast and the concentration of prolactin in milk. Indeed, the only mother (mother D) who consistently showed no relationship between milk prolactin and the degree of fullness experienced problems with an over supply of milk early in lactation, suggesting a lack of response to local feedback inhibition of milk synthesis. Consequently, as milk accumulates within the mammary gland, the binding of prolactin to its membrane receptors, signal transduction and subsequent movement of prolactin into the lactocyte during receptor-ligand uncoupling are likely to be reduced by the action of FIL. Such a mechanism would permit asynchronous milk synthesis between breasts both in the short and long term. In summary, our investigations show that changes in the concentration of prolactin in the blood could not directly regulate milk synthesis in women. They do not, however, preclude prolactin from having a permissive role in milk synthesis. The variations in the rate of milk synthesis, both unilaterally and bilaterally, are indicative of autocrine control mechanisms regulating milk synthesis. Furthermore, the gradient for prolactin in the milk and the known requirements of prolactin for the synthesis of milk (Cowie et al. 1980) suggest that the control of milk synthesis may be by an interplay of autocrine inhibitors and endocrine effectors within individual breasts. Since infants consume irregular quantities of milk at irregular times during the day (Hartmann, Sherriff & Kent, 1995), this interplay would allow the breast to regulate milk synthesis such that the variable demands of the infant can be met, with minimal wastage of maternal resources. We offer special thanks to the volunteer subjects, their families and the Nursing Mothers' Association of Australia. We thank Drs R. Parsons and H. Vu of The University of Western Australia, Department of Public Health, Biostatistics Consulting Unit, for their valuable statistical assistance; Ms Y. Ge for modifications to the Shape(© Measurement System and Ms B. Lovelock and Ms J. C. Kent for their technical assistance. The project was supported by the National Health and Medical Research Council. Downloaded from Exp Physiol (ep.physoc.org) by guest on August 22, 2009
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