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Decreased adrenomedullin and total nitrite levels in breast milk of preeclamptic women
Clinical Biochemistry 37 (2004) 146 – 148
Decreased adrenomedullin and total nitrite levels in breast milk of preeclamptic women Mustafa Baki Cekmen, a,* Ayse Balat, b Ozcan Balat, c Fuat Aksoy, c Muhittin Yurekli, d Ayse Binnur Erbagci, e and Saime Sahinoz f a Department of Biochemistry, Medical Faculty, Kocaeli University, I˙zmit, Turkey Department of Pediatric Nephrology, Medical Faculty, Gaziantep University, Gaziantep, Turkey c Department of Obstetrics & Gynecology, Medical Faculty, Gaziantep University, Gaziantep, Turkey d Department of Molecular Biology, Medical Faculty, I˙no¨nu¨ University, Malatya, Turkey e Department of Biochemistry, Medical Faculty, Gaziantep University, Gaziantep, Turkey f Department of Public Health, Medical Faculty, Gaziantep University, Gaziantep, Turkey b
Received 17 July 2003; received in revised form 14 October 2003; accepted 15 October 2003
Abstract Objective: To observe the adrenomedullin (AM) and total nitrite levels in the milk of preeclamptic and normal pregnant women. Design and methods: Fifteen women with preeclampsia and 15 normal pregnant women were included in the study. Total nitrite was quantitated by Griess reaction, while AM was measured by HPLC. Results: The levels of AM and total nitrite in colostrum and 30th-day breast milk were decreased in preeclamptics. Total nitrite levels (Amol/l) were 56.09 F 11.18 vs. 82.20 F 12.01, P < 0.05, in colostrum of preeclamptics and controls, respectively. The level of total nitrite was 37.75 F 12.10 vs. 53.28 F 10.25, P < 0.05, in 30th-day milk of same patients. AM levels (pg/ml) were 11.18 F 1.11 vs. 16.59 F 1.24, P < 0.0001, in colostrum of preeclamptics and controls, respectively. In 30th-day milk of same patients, AM levels were 8.41 F 1.39 vs. 12.18 F 1.48, P < 0.005, respectively. Conclusion: This report shows for the first time that human milk has decreased levels of AM and total nitrite in preeclampsia. D 2003 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Adrenomedullin; Breast milk; Nitric oxide; Preeclampsia
Introduction Nitric oxide (NO) is synthesized from endothelium and has an important role in control of vascular tonus [1,2]. It is also synthesized in a variety of species and tissues. Recently, the presence of the L-arginine/nitric oxide pathway in the endothelium and smooth muscle of the human internal mammary artery has been described [3]. Preeclampsia is a syndrome that is associated with many pathophysiological features that could lead to upregulation of nitric oxide synthase (NOS) expression, and in turn NO production in placenta of preeclamptic women. It has been shown that placental NOS activity and NO end-products (total nitrite) were significantly higher * Corresponding author. Kocaeli Universitesi Faku¨ltesi Temel Bilimler Bo¨lu¨mu¨, Biyokimya Ad, 41900 Derince-I˙zmı˙t, Turkey. Fax: +90-2623216847. E-mail address: [email protected] (S. Sahinoz).
in villous homogenate of preeclamptic placenta compared to that of normal pregnancy, and directly related to the severity of this syndrome [4], while another study [5] showed decreased nitric oxide synthase activities in placental villi of preeclamptic women. Adrenomedullin (AM) is a potent vasodilator and natriuretic peptide, originally purified from human pheochromocytoma [6]. It has been detected in the adrenal medulla, heart, lung, and kidney as well as in plasma and urine. The main source of circulating AM is considered to be the vasculature. AM and its receptor are prominently expressed by vascular endothelial cells (ECs) and smooth muscle cells (VSMCs). AM may function as a circulating hormone and an autocrine – paracrine mediator involved in the regulation of the cardiovascular system, blood pressure, immune response, and renal function [6,7]. Recent investigations have also identified AM in amniotic fluid and fetal membranes and have hypothesized that AM may play a role in the maintenance of human pregnan-
0009-9120/$ - see front matter D 2003 The Canadian Society of Clinical Chemists. All rights reserved. doi:10.1016/j.clinbiochem.2003.10.010
M.B. Cekmen et al. / Clinical Biochemistry 37 (2004) 146–148
cy [8,9]. AM levels were found to be significantly increased at term in preeclamptic women, and it has been postulated that AM modulates the physiologic response of women to preeclampsia, as it modulates human response to other conditions characterized by vasospasm [10]. Milk contains substances that have a critical role in the growth and development of the newborn. Recently, an experimental study showed that the expression of adrenomedullin (AM) in mouse mammary gland, showing AM immunoreactivity in the milk within the ducts from lactating glands [11]. After this, Pio et al. [12] demonstrated that human milk contains high levels of AM, and that AM has a direct growth effect on intestinal cells. However, to our knowledge, there is no study on the AM and NO levels in breast milk of preeclamptic women in the literature. We conducted this study to observe the AM and NO levels in the milk of preeclamptic and normal pregnant women.
147
and 2.5% phosphoric acid) (Sigma Chemical Co., St, Louis, MO) was then added to 1 ml of the specimens. Absorbance was read at 545 nm after a 30-min incubation. Standard curves were prepared with known concentrations (1– 100 Amol/l) of sodium nitrite. The coefficients of variation were 4.20 –5.62% for intra- and inter-assay precision, respectively. Measurement of adrenomedullin level After extraction and purification, samples were applied to Supelcosil C18 columns (Cecil 100HPLC). Loaded material was eluted 60% acetonitrile in 0.1% trifluoroacetic acid [14]. Rat adrenomedullin (1 – 50) (Phoenix Pharmaceuticals, Inc.) was used as the standard in the determination of AM levels. The coefficients of variation were 2.40 – 3.90% for intra- and inter-assay precision, respectively.
Statistical analysis Materials and methods Fifteen women with preeclampsia (mean age 28.9 F 5.8 years), and 15 normal pregnant women (mean age 27.0 F 2.9 years) were included in the study. Normal pregnants were free of kidney disease when included from the clinic. All controls had a normal physical examination and blood pressure and normal serum electrolytes, BUN, creatinine levels at the time of study. A total of 60 samples of breast milk were obtained from 30 (15 with preeclampsia, 15 with normal pregnancy) lactating Turkish women during postpartum days 1 – 30. The samples were all collected just before regular breast feeding by manual expression at 7– 9 am. The breast milk samples (1– 10 ml each, average 5 ml) were expressed into sterile polystyrene centrifuge tubes and were stored at 40jC until assayed. Measurement of total nitrite level NO is a labile molecule and has a short half-life that is rapidly converted to nitrite (NO2 ) and nitrate (NO3 ). For this reason, total nitrite (NO2 + NO3 ) level in biological fluids is usually suggested as an indicator of NO production [13]. We deproteinized 300 Al of human milk by adding 600 Al of 75 mmol/l ZnSO4 solution, stirring, and centrifuging at 10 000 g for at least 1 min at room temperature, after which 600 Al of 55 mmol/l NaOH was added. Again, the solution was stirred and centrifuged at 1000 g for 3 min and the supernatant was recovered. Total nitrite was quantitated by the Griess reaction after incubation of samples with Escherichia coli reductase to convert NO3 to NO2 [13]. Griess reagent (1 ml, 1% sulfanilamide, 0.1% naphthylene diamine hydrochloride,
The results are provided as means F SD. Statistical significance was determined at P < 0.05. Mann –Whitney U and Kolmogrov – Smirnov 2-sample tests are used for comparison of groups. Statistical analysis was performed with Statistical Package for the Social Sciences for Windows (SPSS, version 10.0).
Results The results of AM and NO are shown in Table 1. The levels of AM and total nitrite in colostrum and 30th-day breast milk were decreased in preeclamptics. Total nitrite levels (Amol/l) were 56.09 F 11.18 vs. 82.20 F 12.01, P < 0.05, in colostrum of preeclamptics and controls, respectively. The level of total nitrite was 37.75 F 12.10 vs. 53.28 F 10.25, P < 0.05, in 30th-day milk of same patients. AM levels (pg/ml) were 11.18 F 1.11 vs. 16.59 F 1.24, P < 0.0001, in colostrum of preeclamptics and controls, respectively. AM levels were 8.41 F 1.39 vs. 12.18 F 1.48, P < 0.005, respectively, in 30th-day milk of same patients.
Table 1 Total nitrite and adrenomedullin levels in milks of preeclamptic and controls Samples
Total nitrite levels (Amol/l)*
Adrenomedullin levels (pg/ml)**
1
56.09 37.75 82.20 53.28
11.18 F 1.11 8.41 F 1.39 16.59 F 1.24 12.18 F 1.48
Preeclampsia—colostrum Preeclampsia—30th-day milk 3 Control—colostrum 4 Control—30th-day milk 2
F F F F
11.18 12.10 12.01 10.25
* 1 – 3, P < 0.05; 2 – 4, P < 0.05; 1 – 2, P < 0.0001; 3 – 4, P < 0.0001. ** 1 – 3, P < 0.0001; 2 – 4, P < 0.005; 1 – 2, P < 0.0001; 3 – 4, P < 0.0001.
148
M.B. Cekmen et al. / Clinical Biochemistry 37 (2004) 146–148
Discussion
References
This report shows for the first time that human milk has decreased levels of AM and NO in preeclampsia compared with normal pregnants. It has been known that many of the human hormones and growth factors present in milk exceed their concentrations found in plasma [15], and the concentrations of them are usually higher in colostrum than in milk [16,17], like our findings. However, the decreased levels of both AM and NO in colostrum and 30th-day milk of preeclamptics are interesting because some of the studies in the literature [5] suggest that a possible defect in NO pathway may be responsible for the clinical findings of preeclampsia. AM levels were also reported to be lower in women with preeclampsia compared with normotensive controls [18]. In an another study, AM levels were found to be increased in women with preeclampsia, suggesting that it may be part of a compensatory response to the vasoconstriction of preeclampsia [10]. But, as the authors stressed [10], these studies were not comparable with each other because of the differences in methodology and study protocol. However, recently, it has been shown that AM mRNA level in placenta specimens of women with preeclampsia is also decreased [19]. Further research will be necessary to elucidate clearly the role of AM in preeclampsia. Although we could not measure the plasma levels of AM and NO simultaneously in plasma, it probably reflects the low plasma levels of NO and AM. Recently, Pio et al. [12] demonstrated that milk has a growth-promoting activity on human small intestinal epithelial cell line (Int-407). They suggest that since MoAbG6 partially blocks the milk-induced growth, AM may be one of the growth factors present in milk. AM has also been described as an agent with antimicrobial activity against gastrointestinal microorganisms [20,21]. This activity could be important in the protection of the neonate against gastroenteritis produced by intestinal pathogens. Since some peptides are absorbed from the neonatal gastrointestinal tract and appear intact in plasma [22], AM could also exert actions in the modulation of tissue growth as well as in the regulation of the immune system. Considering all these important effects of this peptide, it would be interesting to follow these patients’ children regarding the tendency of infection, necrotizing enterocolitis, and the rate of mortality. In conclusion, this report shows for the first time that human milk has decreased levels of AM and NO in preeclampsia compared with normal pregnants, and probably decreased AM and NO levels may tend these mothers’ children to infection. However, this hypothesis should be clarified by further studies.
[1] Kone BC. Nitric oxide in renal health and disease. Am J Kidney Dis 1997;30:11 – 333. [2] Farrell AJ, Blake DR. Nitric oxide. Ann Rheum 1996;55:7 – 20. [3] Qi X, Chen D, Nottin R, Mace L, Herve P, Weiss M. Evidence of Larginine/nitric oxide pathway in endothelium and smooth muscle of human internal mammary artery. Biochem Biophys Res Commun 1993;195:90 – 6. [4] Shaamash AH, Elsonosy ED, Zakhari MM, Radwan SH, El-Dien HM. Placental nitric oxide synthase (NOS) activity and nitric oxide (NO) production in normal pregnancy, pre-eclampsia and eclampsia. Int J Gynaecol Obstet 2001;72:127 – 33. [5] Morris NH, Sooranna SR, Learmont JG, Poston L, Ramsey B, Pearson JD, et al. Nitric oxide synthase activities in placental tissue from normotensive, pre-eclamptic and growth retarded pregnancies. Br J Obstet Gynaecol 1995;102:711 – 4. [6] Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H, et al. Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun 1993;192:553 – 60. [7] Samson WK. Adrenomedullin and the control of fluid and electrolyte homeostasis. Annu Rev Physiol 1999;61:363 – 89. [8] DiLorio R, Marinoni E, Scavo D, Letzia C, Cosmi E. Adrenomedullin in pregnancy. Lancet 1997;349:328. [9] DiLorio R, Marinoni E, Cosmi E. Adrenomedullin in pre-eclampsia. Lancet 1998;351:676. [10] Lauria MR, Standley CA, Sorokin Y, Yelian FD, Cotton DB. Adrenomedullin levels in normal and preeclamptic pregnancy at term. J Soc Gynecol Investig 1999;6:318 – 21. [11] Jahnke GD, Miller MJ, Martinez A, Montuenga L, Cuttitta F. Adrenomedullin expression in the mouse mammary gland: evidence for the mature form in milk. J Mol Endocrinol 1997;19:279 – 89. [12] Pio R, Martinez A, Elsasser TH, Cuttitta F. Presence of immunoreactive adrenomedullin in human and bovine milk. Peptides 2000;21: 1859 – 63. [13] Bories PN, Bories C. Nitrate determination in biological fluids by an enzymatic one-step assay with nitrate reductase. Clin Chem 1995;41: 904 – 7. [14] Mant CT, Hudges RS, editors. Separations, Analysis, and Conformation. High performance liquid chromatography of peptides and proteins. Boston: CRC Press; 1991. [15] Grosvenor CE, Picciano MF, Baumrucker CR. Hormones and growth factors in milk. Endocr Rev 1992;14:710 – 28. [16] Corps AN, Brown KD, Rees LH, Carr J, Prosser CG. The insulin-like growth factor I content in human milk increases between early and full lactation. J Clin Endocrinol Metab 1988;67:25 – 9. [17] Ducroc R, Rubio S, Garzon B, Brunel-Riveau B, Couraud JY. Immunoreactive substance P and calcitonin-gene-related peptide (CGRP) in rat milk and in human milk and infant formulas. Am J Clin Nutr 1995;62:554 – 8. [18] Hata T, Miyazaki K, Matsui K. Decreased circulating adrenomedullin in pre-eclampsia. Lancet 1997;350:1600. [19] Knerr I, Dachert C, Beinder E, Metzler M, Dotsch J, Repp R, et al. Adrenomedullin, calcitonin gene-related peptides and their receptors: evidence for a decreased placental mRNA content in preeclampsia and HELLP syndrome. Eur J Obstet Gynecol Reprod Biol 2002; 101:47 – 53. [20] Allaker RP, Zihni C, Kapas S. An investigation into the antimicrobial effects of adrenomedullin on members of the skin, oral, respiratory tract and gut microflora. FEMS Immunol Med Microbiol 1999;23:289 – 93. [21] Walsh T, Martinez A, Peter J, Unsworth E, Cuttitta F. Antimicrobial activity of adrenomedullin and its gene-related peptides. Clin Infect Dis 1996;23:896. [22] Thornburg W, Matrisian L, Magun B, Koldovsky O. Gastrointestinal absorption of epidermal growth factor in suckling rats. Am J Physiol 1984;246:G80 – 5.
Acknowledgements We thank all mothers who provided milk samples.
Decreased adrenomedullin and total nitrite levels in breast milk of preeclamptic women Mustafa Baki Cekmen, a,* Ayse Balat, b Ozcan Balat, c Fuat Aksoy, c Muhittin Yurekli, d Ayse Binnur Erbagci, e and Saime Sahinoz f a Department of Biochemistry, Medical Faculty, Kocaeli University, I˙zmit, Turkey Department of Pediatric Nephrology, Medical Faculty, Gaziantep University, Gaziantep, Turkey c Department of Obstetrics & Gynecology, Medical Faculty, Gaziantep University, Gaziantep, Turkey d Department of Molecular Biology, Medical Faculty, I˙no¨nu¨ University, Malatya, Turkey e Department of Biochemistry, Medical Faculty, Gaziantep University, Gaziantep, Turkey f Department of Public Health, Medical Faculty, Gaziantep University, Gaziantep, Turkey b
Received 17 July 2003; received in revised form 14 October 2003; accepted 15 October 2003
Abstract Objective: To observe the adrenomedullin (AM) and total nitrite levels in the milk of preeclamptic and normal pregnant women. Design and methods: Fifteen women with preeclampsia and 15 normal pregnant women were included in the study. Total nitrite was quantitated by Griess reaction, while AM was measured by HPLC. Results: The levels of AM and total nitrite in colostrum and 30th-day breast milk were decreased in preeclamptics. Total nitrite levels (Amol/l) were 56.09 F 11.18 vs. 82.20 F 12.01, P < 0.05, in colostrum of preeclamptics and controls, respectively. The level of total nitrite was 37.75 F 12.10 vs. 53.28 F 10.25, P < 0.05, in 30th-day milk of same patients. AM levels (pg/ml) were 11.18 F 1.11 vs. 16.59 F 1.24, P < 0.0001, in colostrum of preeclamptics and controls, respectively. In 30th-day milk of same patients, AM levels were 8.41 F 1.39 vs. 12.18 F 1.48, P < 0.005, respectively. Conclusion: This report shows for the first time that human milk has decreased levels of AM and total nitrite in preeclampsia. D 2003 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Adrenomedullin; Breast milk; Nitric oxide; Preeclampsia
Introduction Nitric oxide (NO) is synthesized from endothelium and has an important role in control of vascular tonus [1,2]. It is also synthesized in a variety of species and tissues. Recently, the presence of the L-arginine/nitric oxide pathway in the endothelium and smooth muscle of the human internal mammary artery has been described [3]. Preeclampsia is a syndrome that is associated with many pathophysiological features that could lead to upregulation of nitric oxide synthase (NOS) expression, and in turn NO production in placenta of preeclamptic women. It has been shown that placental NOS activity and NO end-products (total nitrite) were significantly higher * Corresponding author. Kocaeli Universitesi Faku¨ltesi Temel Bilimler Bo¨lu¨mu¨, Biyokimya Ad, 41900 Derince-I˙zmı˙t, Turkey. Fax: +90-2623216847. E-mail address: [email protected] (S. Sahinoz).
in villous homogenate of preeclamptic placenta compared to that of normal pregnancy, and directly related to the severity of this syndrome [4], while another study [5] showed decreased nitric oxide synthase activities in placental villi of preeclamptic women. Adrenomedullin (AM) is a potent vasodilator and natriuretic peptide, originally purified from human pheochromocytoma [6]. It has been detected in the adrenal medulla, heart, lung, and kidney as well as in plasma and urine. The main source of circulating AM is considered to be the vasculature. AM and its receptor are prominently expressed by vascular endothelial cells (ECs) and smooth muscle cells (VSMCs). AM may function as a circulating hormone and an autocrine – paracrine mediator involved in the regulation of the cardiovascular system, blood pressure, immune response, and renal function [6,7]. Recent investigations have also identified AM in amniotic fluid and fetal membranes and have hypothesized that AM may play a role in the maintenance of human pregnan-
0009-9120/$ - see front matter D 2003 The Canadian Society of Clinical Chemists. All rights reserved. doi:10.1016/j.clinbiochem.2003.10.010
M.B. Cekmen et al. / Clinical Biochemistry 37 (2004) 146–148
cy [8,9]. AM levels were found to be significantly increased at term in preeclamptic women, and it has been postulated that AM modulates the physiologic response of women to preeclampsia, as it modulates human response to other conditions characterized by vasospasm [10]. Milk contains substances that have a critical role in the growth and development of the newborn. Recently, an experimental study showed that the expression of adrenomedullin (AM) in mouse mammary gland, showing AM immunoreactivity in the milk within the ducts from lactating glands [11]. After this, Pio et al. [12] demonstrated that human milk contains high levels of AM, and that AM has a direct growth effect on intestinal cells. However, to our knowledge, there is no study on the AM and NO levels in breast milk of preeclamptic women in the literature. We conducted this study to observe the AM and NO levels in the milk of preeclamptic and normal pregnant women.
147
and 2.5% phosphoric acid) (Sigma Chemical Co., St, Louis, MO) was then added to 1 ml of the specimens. Absorbance was read at 545 nm after a 30-min incubation. Standard curves were prepared with known concentrations (1– 100 Amol/l) of sodium nitrite. The coefficients of variation were 4.20 –5.62% for intra- and inter-assay precision, respectively. Measurement of adrenomedullin level After extraction and purification, samples were applied to Supelcosil C18 columns (Cecil 100HPLC). Loaded material was eluted 60% acetonitrile in 0.1% trifluoroacetic acid [14]. Rat adrenomedullin (1 – 50) (Phoenix Pharmaceuticals, Inc.) was used as the standard in the determination of AM levels. The coefficients of variation were 2.40 – 3.90% for intra- and inter-assay precision, respectively.
Statistical analysis Materials and methods Fifteen women with preeclampsia (mean age 28.9 F 5.8 years), and 15 normal pregnant women (mean age 27.0 F 2.9 years) were included in the study. Normal pregnants were free of kidney disease when included from the clinic. All controls had a normal physical examination and blood pressure and normal serum electrolytes, BUN, creatinine levels at the time of study. A total of 60 samples of breast milk were obtained from 30 (15 with preeclampsia, 15 with normal pregnancy) lactating Turkish women during postpartum days 1 – 30. The samples were all collected just before regular breast feeding by manual expression at 7– 9 am. The breast milk samples (1– 10 ml each, average 5 ml) were expressed into sterile polystyrene centrifuge tubes and were stored at 40jC until assayed. Measurement of total nitrite level NO is a labile molecule and has a short half-life that is rapidly converted to nitrite (NO2 ) and nitrate (NO3 ). For this reason, total nitrite (NO2 + NO3 ) level in biological fluids is usually suggested as an indicator of NO production [13]. We deproteinized 300 Al of human milk by adding 600 Al of 75 mmol/l ZnSO4 solution, stirring, and centrifuging at 10 000 g for at least 1 min at room temperature, after which 600 Al of 55 mmol/l NaOH was added. Again, the solution was stirred and centrifuged at 1000 g for 3 min and the supernatant was recovered. Total nitrite was quantitated by the Griess reaction after incubation of samples with Escherichia coli reductase to convert NO3 to NO2 [13]. Griess reagent (1 ml, 1% sulfanilamide, 0.1% naphthylene diamine hydrochloride,
The results are provided as means F SD. Statistical significance was determined at P < 0.05. Mann –Whitney U and Kolmogrov – Smirnov 2-sample tests are used for comparison of groups. Statistical analysis was performed with Statistical Package for the Social Sciences for Windows (SPSS, version 10.0).
Results The results of AM and NO are shown in Table 1. The levels of AM and total nitrite in colostrum and 30th-day breast milk were decreased in preeclamptics. Total nitrite levels (Amol/l) were 56.09 F 11.18 vs. 82.20 F 12.01, P < 0.05, in colostrum of preeclamptics and controls, respectively. The level of total nitrite was 37.75 F 12.10 vs. 53.28 F 10.25, P < 0.05, in 30th-day milk of same patients. AM levels (pg/ml) were 11.18 F 1.11 vs. 16.59 F 1.24, P < 0.0001, in colostrum of preeclamptics and controls, respectively. AM levels were 8.41 F 1.39 vs. 12.18 F 1.48, P < 0.005, respectively, in 30th-day milk of same patients.
Table 1 Total nitrite and adrenomedullin levels in milks of preeclamptic and controls Samples
Total nitrite levels (Amol/l)*
Adrenomedullin levels (pg/ml)**
1
56.09 37.75 82.20 53.28
11.18 F 1.11 8.41 F 1.39 16.59 F 1.24 12.18 F 1.48
Preeclampsia—colostrum Preeclampsia—30th-day milk 3 Control—colostrum 4 Control—30th-day milk 2
F F F F
11.18 12.10 12.01 10.25
* 1 – 3, P < 0.05; 2 – 4, P < 0.05; 1 – 2, P < 0.0001; 3 – 4, P < 0.0001. ** 1 – 3, P < 0.0001; 2 – 4, P < 0.005; 1 – 2, P < 0.0001; 3 – 4, P < 0.0001.
148
M.B. Cekmen et al. / Clinical Biochemistry 37 (2004) 146–148
Discussion
References
This report shows for the first time that human milk has decreased levels of AM and NO in preeclampsia compared with normal pregnants. It has been known that many of the human hormones and growth factors present in milk exceed their concentrations found in plasma [15], and the concentrations of them are usually higher in colostrum than in milk [16,17], like our findings. However, the decreased levels of both AM and NO in colostrum and 30th-day milk of preeclamptics are interesting because some of the studies in the literature [5] suggest that a possible defect in NO pathway may be responsible for the clinical findings of preeclampsia. AM levels were also reported to be lower in women with preeclampsia compared with normotensive controls [18]. In an another study, AM levels were found to be increased in women with preeclampsia, suggesting that it may be part of a compensatory response to the vasoconstriction of preeclampsia [10]. But, as the authors stressed [10], these studies were not comparable with each other because of the differences in methodology and study protocol. However, recently, it has been shown that AM mRNA level in placenta specimens of women with preeclampsia is also decreased [19]. Further research will be necessary to elucidate clearly the role of AM in preeclampsia. Although we could not measure the plasma levels of AM and NO simultaneously in plasma, it probably reflects the low plasma levels of NO and AM. Recently, Pio et al. [12] demonstrated that milk has a growth-promoting activity on human small intestinal epithelial cell line (Int-407). They suggest that since MoAbG6 partially blocks the milk-induced growth, AM may be one of the growth factors present in milk. AM has also been described as an agent with antimicrobial activity against gastrointestinal microorganisms [20,21]. This activity could be important in the protection of the neonate against gastroenteritis produced by intestinal pathogens. Since some peptides are absorbed from the neonatal gastrointestinal tract and appear intact in plasma [22], AM could also exert actions in the modulation of tissue growth as well as in the regulation of the immune system. Considering all these important effects of this peptide, it would be interesting to follow these patients’ children regarding the tendency of infection, necrotizing enterocolitis, and the rate of mortality. In conclusion, this report shows for the first time that human milk has decreased levels of AM and NO in preeclampsia compared with normal pregnants, and probably decreased AM and NO levels may tend these mothers’ children to infection. However, this hypothesis should be clarified by further studies.
[1] Kone BC. Nitric oxide in renal health and disease. Am J Kidney Dis 1997;30:11 – 333. [2] Farrell AJ, Blake DR. Nitric oxide. Ann Rheum 1996;55:7 – 20. [3] Qi X, Chen D, Nottin R, Mace L, Herve P, Weiss M. Evidence of Larginine/nitric oxide pathway in endothelium and smooth muscle of human internal mammary artery. Biochem Biophys Res Commun 1993;195:90 – 6. [4] Shaamash AH, Elsonosy ED, Zakhari MM, Radwan SH, El-Dien HM. Placental nitric oxide synthase (NOS) activity and nitric oxide (NO) production in normal pregnancy, pre-eclampsia and eclampsia. Int J Gynaecol Obstet 2001;72:127 – 33. [5] Morris NH, Sooranna SR, Learmont JG, Poston L, Ramsey B, Pearson JD, et al. Nitric oxide synthase activities in placental tissue from normotensive, pre-eclamptic and growth retarded pregnancies. Br J Obstet Gynaecol 1995;102:711 – 4. [6] Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H, et al. Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun 1993;192:553 – 60. [7] Samson WK. Adrenomedullin and the control of fluid and electrolyte homeostasis. Annu Rev Physiol 1999;61:363 – 89. [8] DiLorio R, Marinoni E, Scavo D, Letzia C, Cosmi E. Adrenomedullin in pregnancy. Lancet 1997;349:328. [9] DiLorio R, Marinoni E, Cosmi E. Adrenomedullin in pre-eclampsia. Lancet 1998;351:676. [10] Lauria MR, Standley CA, Sorokin Y, Yelian FD, Cotton DB. Adrenomedullin levels in normal and preeclamptic pregnancy at term. J Soc Gynecol Investig 1999;6:318 – 21. [11] Jahnke GD, Miller MJ, Martinez A, Montuenga L, Cuttitta F. Adrenomedullin expression in the mouse mammary gland: evidence for the mature form in milk. J Mol Endocrinol 1997;19:279 – 89. [12] Pio R, Martinez A, Elsasser TH, Cuttitta F. Presence of immunoreactive adrenomedullin in human and bovine milk. Peptides 2000;21: 1859 – 63. [13] Bories PN, Bories C. Nitrate determination in biological fluids by an enzymatic one-step assay with nitrate reductase. Clin Chem 1995;41: 904 – 7. [14] Mant CT, Hudges RS, editors. Separations, Analysis, and Conformation. High performance liquid chromatography of peptides and proteins. Boston: CRC Press; 1991. [15] Grosvenor CE, Picciano MF, Baumrucker CR. Hormones and growth factors in milk. Endocr Rev 1992;14:710 – 28. [16] Corps AN, Brown KD, Rees LH, Carr J, Prosser CG. The insulin-like growth factor I content in human milk increases between early and full lactation. J Clin Endocrinol Metab 1988;67:25 – 9. [17] Ducroc R, Rubio S, Garzon B, Brunel-Riveau B, Couraud JY. Immunoreactive substance P and calcitonin-gene-related peptide (CGRP) in rat milk and in human milk and infant formulas. Am J Clin Nutr 1995;62:554 – 8. [18] Hata T, Miyazaki K, Matsui K. Decreased circulating adrenomedullin in pre-eclampsia. Lancet 1997;350:1600. [19] Knerr I, Dachert C, Beinder E, Metzler M, Dotsch J, Repp R, et al. Adrenomedullin, calcitonin gene-related peptides and their receptors: evidence for a decreased placental mRNA content in preeclampsia and HELLP syndrome. Eur J Obstet Gynecol Reprod Biol 2002; 101:47 – 53. [20] Allaker RP, Zihni C, Kapas S. An investigation into the antimicrobial effects of adrenomedullin on members of the skin, oral, respiratory tract and gut microflora. FEMS Immunol Med Microbiol 1999;23:289 – 93. [21] Walsh T, Martinez A, Peter J, Unsworth E, Cuttitta F. Antimicrobial activity of adrenomedullin and its gene-related peptides. Clin Infect Dis 1996;23:896. [22] Thornburg W, Matrisian L, Magun B, Koldovsky O. Gastrointestinal absorption of epidermal growth factor in suckling rats. Am J Physiol 1984;246:G80 – 5.
Acknowledgements We thank all mothers who provided milk samples.