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Milk and blood ghrelin level in diabetics
Nutrition 23 (2007) 807– 811 www.elsevier.com/locate/nut
Applied nutritional investigation
Milk and blood ghrelin level in diabetics Suleyman Aydin, Ph.D.a,*, Hikmet Geckil, Ph.D.b, Fikret Karatas, Ph.D.c, Emir Donder, M.D.d, Selahattin Kumru, M.D.e, Ebru Celik Kavak, M.D.e, Ramis Colak, M.D.d, Yusuf Ozkan, M.D.d, and Ibrahim Sahin, M.S.f a
Department of Biochemistry and Clinical Biochemistry, School of Medicine, Firat University, Firat Medical Center, Elazig, Turkey b Molecular Biology Division, Department of Biology, Inonu Univeristy, Malatya, Turkey c Department of Chemistry, Firat University, Elazig, Turkey d Department of Endocrinology–Internal Medicine, Firat University, Medical School (Firat Medical Center), Elazig, Turkey e Department of Obstetrics and Gynecology, Firat University, Medical School, Elazig, Turkey f Department of Biology, Faculty of Arts and Sciences, Firat University, Elazig, Turkey Manuscript received May 11, 2007; accepted August 21, 2007.
Abstract
Objective: Besides its presence in various tissues, ghrelin has recently been shown to be present in blood and breast milk. No previous studies, however, have evaluated the level of this hormone under the condition of pregestational and gestational diabetes mellitus (P-GDM and GDM, respectively). This study was undertaken to show whether a relation exists between serum and milk ghrelin levels in lactating mothers with and without diabetes. Methods: Venous blood was obtained from four groups of women (age range 22–37 y): GDM lactating (n ⫽ 12), P-GM lactating (n ⫽ 3), healthy non-diabetic lactating (n ⫽ 14), and healthy non-lactating (n ⫽ 14). Colostrum and mature milk samples were collected just before suckling. The ghrelin level was determined by radioimmunoassay and high-performance liquid chromatography. Results: Radioimmunoassay results showed that women with GDM and P-GDM had greater than two-fold lower colostrum and serum levels of ghrelin than did lactating women with no GDM at 2 d after parturition. The GDM and non-diabetic groups at 15 d after delivery, however, showed similar levels of ghrelin in mature milk and serum. High-performance liquid chromatographic results indicated that in serum the deacylated form of ghrelin was 18-fold higher than the acylated form. Furthermore, in milk the acylated form of ghrelin was 24-fold that of the active form. Conclusion: These results indicate that mothers with GDM have a substantial (greater than two-fold) decrease in their serum and colostral ghrelin levels. This is, however, a temporary effect lasting only up to early postparturition (2 d after delivery). This peptide hormone restores to completely normal levels at day 15 of parturition, but not P-GDM. The significance of these results in terms of the health of the mother and her newborn, however, has yet to be determined. © 2007 Elsevier Inc. All rights reserved.
Keywords:
Ghrelin; Lipopeptide; Breast milk; Colostrum; Gestational diabetes mellitus
Introduction Human breast milk contains a number of bioactive substances with high nourishment and healthy benefit for the newborn. Especially growth- and development-related effects last into adulthood [1]. Among several peptides with * Corresponding author. Tel.: ⫹90-533-493-4643; fax: ⫹90-424-2379138. E-mail address: [email protected] (S. Aydin). 0899-9007/07/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2007.08.015
this kind of function, ghrelin is relatively newly discovered one. Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, was originally discovered in extracts of rat and human stomach, where it is localized in the endocrine X/A-like (ghrelin cells) cells of the gastric mucosa [2], and is known to act as a signal for food intake [3–5]. This peptide hormone is composed of 28 amino acid residues, bearing a serine (rarely threonine) residue on the third position, where a modification (acylation) is essential for its function. The acylated (octanoylated) form is the
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S. Aydin et al. / Nutrition 23 (2007) 807– 811
active form, whereas the deacylated form is known as the inactive form of ghrelin. Both forms are present in various tissues and blood, where they have important physiologic roles [3,5]. One of the main differences between these two forms is that only the deacylated form can pass the bloodbrain barrier [6]. In terms of milk ghrelin, however, the “active” form is physiologically more important for infant development [4,5]. Breast milk contains a variety of biologically active nutrients of immediate benefit to the newborn infant [1]. Milk hormones function to transiently regulate the activity of a variety of tissues including endocrine organs until the neonate’s own endocrine systems begin to function [7]. In the case of ghrelin, this hormone might function in the growth and development of the neonate in addition to providing nourishment because it directly regulates bone formation [8]. Gestational diabetes mellitus (GDM), a condition for carbohydrate intolerance during pregnancy, is a widespread medical complication of pregnancy. Women with a history of GDM are more likely to develop type 2 diabetes compared with their counterparts who remain normoglycemic during pregnancy [9]. Indeed, among 15 diabetic women in this study, 3 had type 2, or pregestational, diabetes (P-GDM) that needed no insulin treatment. Although low ghrelin is associated with type 2 diabetes mellitus [10], no previous studies have evaluated the level of this hormone in breast milk in individuals with GDM and P-GDM. In a related study [11], however, fasting serum ghrelin levels in women with GDM was measured and compared with those in normal pregnant and non-pregnant women. Another report measured plasma ghrelin in infants with insulin-dependent diabetic mothers [12]. Recent studies from this laboratory [13] and others [14] have shown that ghrelin is produced and secreted by human breast tissue. There have been contradictory reports [13,14] regarding plasma and breast milk levels of ghrelin, an issue that needs to be clarified. The purpose of this study was two-fold: 1) determine the acylated (active) form of ghrelin by radioimmunoassay (RIA) in serum and milk from women with GDM; and 2) evaluate the high-performance liquid chromatographic (HPLC) results of milk and serum ghrelin levels to clarify the controversial results reported for this hormone.
venous blood glucose measurement 1 h later. Women with blood glucose levels higher than 140 mg/dL were considered to have a positive result. Women with a positive test result were asked to return for a 3-h, diagnostic, 100-g oral glucose tolerance test within the next week. The tolerance test was performed in the morning after a 12-h overnight fast and 3 d of a 150- to 200-g (minimum) carbohydrate diet. GDM was diagnosed if two or more values of the tolerance test equalled or exceeded the thresholds proposed by the American Diabetes Association (ADA) [15]. All participants were healthy pregnant women and had no pregnancy complications (i.e., pre-eclampsia, hypertension, infection, or metabolic or surgical complications except for GDM). In our study population, there were no high-risk pregnancies for GDM (according to ADA criteria). All lactating women had full-term natural deliveries without any health problems. All took no medications 10 d before and during the sampling. The mean age of these two groups (lactating) was 27 y (range 24 –37 y). The control group (non-lactating) women were all healthy and exclusion criteria for the controls were as follows: pregnancy, use of any drugs, alcohol consumption of more than one drink per day, diabetes (even family history), use of tobacco products (past and present), regular intense exercise (⬎15 min of aerobics three times per week), chronic medical illness, history of abdominal surgery, history of gastrointestinal diseases, and family history of obesity. The mean age of this group was 26 y (range 19 –34 y). All subjects (lactating and control women) were advised not to eat, smoke, or drink (except water) overnight before collection of colostral milk, mature milk, and blood samples. Thus, they came to the hospital after an overnight fast and the blood specimens were drawn before breakfast. Approximately 2 mL of breast milk and 5 mL of blood were taken from the lactating women on day 2 (colostral milk) and 15 (mature milk) after delivery. The same amount of blood was also drawn from the non-lactating controls during the follicular phase of menses. The blood was centrifuged at 4000 rpm for 5 min and the serum was stored at ⫺70°C. Colostrum and mature milk were separated by centrifugation of samples twice at 4000 rpm for 10 min. In the first cycle of the centrifugation of milk, a thick fat layer on the top of the tube was removed with a sterile tooth pick, and each supernatant was divided into three aliquots and stored at ⫺70°C until the assay was performed.
Materials and methods Hormone assay The study was carried out in volunteers whose written consent was taken before the study together with the institutional ethical committee approval of the study protocol (dated June 29, 2005, issue no. 153). Sampling was from 12 lactating GDM, 3 lactating P-GDM, 14 lactating healthy control, and 14 non-lactating women. In this study, a twostep screening approach for GDM was used. In this method, pregnant women were given a standard, 1-h, 50-g glucose challenge test between gestational weeks 24 and 28, with a
Serum, colostrum, and mature milk ghrelin levels were measured using a commercially available RIA kit (Linco Research, St. Charles, MO, USA). This RIA uses an antibody that recognizes active epitopes of ghrelin. The colostrum and mature milk active ghrelin measurements were validated as previously described [16]. All samples were read with a gamma counter (MultiGamma 1261, LKBWallac, Turku, Finland). Ghrelin concentrations were cal-
S. Aydin et al. / Nutrition 23 (2007) 807– 811
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Fig. 1. High-performance liquid chromatographic analysis of the deacylated (white arrows) and acylated (black arrows) forms of ghrelin: (a) pure ghrelin standard, (b) serum, and (c) milk.
culated from standard curves generated in the same way as with ghrelin. The ghrelin levels were also determined using an HPLC system. HPLC was performed at room temperature on a Cecil liquid chromatographic system (Series 1100, Cecil, Cambridge, United Kingdom) consisting of a sample injection valve (Cotati 7125) with a 20-L sample loop, an ultraviolet spectrophotometric detector (Cecil 68174), an integrator (HP 3395), and an SGE Walkosil 11 5Cl8 RS column (SGE Deutschland GmbH, Griesheim, Germany), 5-m particle size, 12.0-nm pore size (15.0 ⫻ 4.6-mm inner diameter) with a mobile (1 mL/min) phase consisting of a 50-mM NaClO4 solution at room temperature. The HPLC injected ghrelin mixture contained 26.7 fmol/mL (90 pg/ mL) of acylated (active) ghrelin and 110 fmol/mL (370 pg/mL) of deacylated ghrelin. A three-fold serum physiologic dilution of samples (milk and serum) was injected as with the standard ghrelin solution. To protect ghrelin from proteolytic action (if any), 20 L (200 kallikrein inactivator units) of aprotinin was added per milliliter of sample. The chromatographic graphs of serum and milk are shown in Figures 1b and 1c, respectively. Statistical analysis Statistical analysis was done using SPSS 12 (SPSS Inc., Chicago, IL, USA). The comparison between groups was determined by the Mann-Whitney U test. The same group parameters were determined by Wilcoxon’s test correlation analysis method. The data are expressed as arithmetic means ⫾ standard deviation (SD). P ⬍ 0.05 was considered significant.
Ghrelin was determined in colostral milk (milk taken at 2 d after delivery), mature milk (day 15 of parturition), and blood serum taken at these respective times. Results showed that women with GDM had greater than two-fold lower colostrum and serum levels of ghrelin than did lactating women without GDM at 2 d after parturition. Both groups at 15 d after delivery, however, showed similar levels of ghrelin in mature milk and serum. The serum ghrelin level of the control group (non-lactating women) was similar to that of lactating women. The active ghrelin concentrations in colostrums from women with and without GDM were 7.75 fmol/mL (26.1 pg/mL, from a molecular mass of 33 371 for ghrelin ) and 19.0 fmol/mL (64.1 pg/mL), respectively. These figures were 16.1 fmol/mL (54.1 pg/mL) and 16.47 fmol/mL (55.5 pg/mL), respectively in mature milk. Serum levels of ghrelin also showed a similar trend in both groups (Table 2). Women with GDM had a ghrelin level of 8.81 fmol/mL (29.7 pg/mL) 2 d after parturition, whereas women with no GDM had a level of 18.8 fmol/mL (63.3 pg/mL). The serum ghrelin levels of both groups at 15 d after delivery, however, were similar (17.7 and 16.7 fmol/mL, respectively). The birth weights of neonates from the control, GDM, and P-GDM mothers were 3.12 ⫾ 0.33, 65 ⫾ 0.37, and 3.40 ⫾ 0.40 kg, respectively.
Table 1 Demographic features of subjects Parity*
Gestational age (wk)†
BMI (kg/m2)
Age (y)
2 3 2 2
37.5 ⫾ 1.3 37.3 ⫾ 1.4 38.2 ⫾ 1.5
29.32 ⫾ 2.0 31.2 ⫾ 2.2 29.6 ⫾ 1.8 22.6 ⫾ 2.4
27 ⫾ 3.6 32 ⫾ 3.0 27 ⫾ 3.6 26 ⫾ 4.9
Results
Lactating women GDM P-GDM Non-diabetic Non-lactating women (control)
The demographic characteristics of the study groups are presented in Table 1. The active ghrelin levels of the three study groups (lactating women with GDM, normal lactating, and non-lactating women) are summarized in Table 2.
BMI, body mass index; GDM, gestational diabetes mellitus; P-GDM, pregestational diabetes mellitus * Total number of births given. † Gestational weeks of groups of women at time of delivery were not significantly different.
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Table 2 Active ghrelin level in serum, colostrum, and mature milk from the study groups Ghrelin (fmol/mL)
Lactating women GDM P-GDM Non-diabetic Non-lactating women (control)
Colostrum
Mature milk
Serum-1
Serum-2
7.75 ⫾ 2.2 8.48 ⫾ 1.8‡ 18.99 ⫾ 2.7§ —
16.06 ⫾ 3.2* 9.54 ⫾ 3.2‡ 16.47 ⫾ 3.3§ —
8.81 ⫾ 2.2 9.22 ⫾ 1.9‡ 18.78 ⫾ 2.9§ 17.80 ⫾ 4.9储
17.50 ⫾ 2.39† 7.88 ⫾ 1.80‡ 16.72 ⫾ 2.86§ —
GDM, gestational diabetes mellitus; P-GDM, pregestational diabetes mellitus; Serum-1, blood serum from lactating women at 2 d after parturition and from controls; Serum-2, blood serum from lactating women at 15 d after parturition * P ⬍ 0.05, colostrum versus serum in lactating GDM women. † P ⬍ 0.05, serum-1 and colostrum versus serum-2 in lactating GDM women. ‡ P ⬍ 0.05, colostrum and serum-1 versus mature milk and serum-2 in lactating P-GDM women. § P ⬍ 0.05, colostrum and serum-1 versus mature milk and serum-2 in lactating GDM women. 储 P ⬍ 0.05, control serum versus colostrum and serum-1 in lactating GDM women.
An HPLC analysis was also carried out to determine serum and milk ghrelin levels (Table 3). Serum and milk (mature) ghrelin levels were detected in women after delivery (15 d after parturition). The retention times were 5.896 and 15.906 min for active and deacylated ghrelin forms, respectively (Fig. 1). The lowest detectable level of active ghrelin with this method was determined to be 11 ⫾ 2 pg/mL, and the lowest detectable level of deacylated ghrelin was 14 ⫾ 3 pg/mL. In serum, the deacylated form of ghrelin (range 286 – 406 fmol/mL, which corresponds to 965–1368 pg/mL) was 18-fold higher than the acylated (active) form (range 35–98 pg/mL). In milk the inactive deacylated form (range 122–234 fmol/mL or 412–788 pg/mL) was 24-fold that of the active (acylated) form (range 4.45– 8.90 fmol/mL, corresponding to 15–30 pg/mL; Table 3). In RIA analysis the lowest detectable level for active ghrelin in milk was 1.92 fmol/mL (6.5 ⫾ 2 pg/mL). The intra- and interassay percentage coefficients of variation for milk active ghrelin were 9.8 and 13.4, respectively, with a mean recovery of 92%. No significant correlations were observed between ghrelin levels and maternal body mass index (calculated as kilograms divided by square meters), which was measured and calculated after delivery.
control group of normal individuals. This abnormally low level of ghrelin in mothers with GDM returns to normal after 15 d of parturition, when they acquire the normoglycemic condition. Our study population consisted of mothers with GDM (n ⫽ 12) and P-GDM (type 2, n ⫽ 3). However, all diabetic women’s blood glucose levels were controlled through a diet regimen and none used insulin. When postterm (15 d after giving birth) blood glucose levels of these three women were monitored, serum glucose levels were not normal (119 mg/dL for the first part of the experiment and 128 mg/dL for the second part). In other words, these diabetic mothers continued to have abnormally low levels of ghrelin (in their serum and milk) even 15 d after delivery. These results show that the diabetic condition is one of the main causes of decreased ghrelin levels. These findings are also in accordance with reported ghrelin levels in saliva of diabetic subjects [16]. However, there is no similar work in the literature reporting active ghrelin levels in the milk and serum of individuals with GDM. This experiment carried out with RIA analysis was also carried out with HPLC analysis. This type of analysis was, however, carried out on lactating mothers without GDM after delivery (15 d after giving birth). With this method, the level of acylated (active) ghrelin detected in serum was 19 fmol/mL (64 pg/mL) compared with 4.96 fmol/mL (16.7
Discussion To the best of our knowledge this is the first study to report colostrum, mature milk, and serum active ghrelin levels in the lactating women with GDM and P-GDM, lactating women without GDM, and non-lactating women (control). Ghrelin levels in colostrum and serum from women with GDM were similar after day 2 of parturition. At this period the serum and colostrum levels of ghrelin were similar in the lactating non-GDM group. The level of ghrelin in this group, however, was two-fold higher than in the lactating GDM group. These results are in good agreement with those of Katsuki et al. [10] who found that serum active ghrelin in diabetic subjects was lower than that in a
Table 3 High-performance liquid chromatographic analysis of two forms of ghrelin from serum and milk of women after parturition (15 d after birth) Ghrelin (pg/mL)
Serum Milk
Acylated
Deacylated
Total
64 ⫾ 22* (19 fmol/mL) 23 ⫾ 6† (6.8 fmol/mL)
1161 ⫾ 150* (344 fmol/mL) 548 ⫾ 123† (163 fmol/mL)
1225 ⫾ 177* (363 fmol/mL) 571 ⫾ 128† (169 fmol/mL)
* P ⬍ 0.05, serum versus acylated, deacylated, and total. P ⬍ 0.05, milk versus acylated, deacylated, and total.
†
S. Aydin et al. / Nutrition 23 (2007) 807– 811
pg/mL) detected with RIA. These figures were 6.82 fmol/mL (23 pg/mL) and 4.89 fmol/mL (16.47 pg/mL) in milk, respectively. Also, when compared with our previous reports [13], the HPLC result of this study confirmed that the total ghrelin level in blood was greater than two-fold higher than that in the milk (Table 3). These results confirm our previous findings and are somehow contradictory to that of Kierson et al. [14] who measured ghrelin using an RIA method that measured all epitopes of ghrelin and the levels in breast milk were still higher than previously published data on total plasma ghrelin levels. The discrepancy between those studies and the present study seems to be related to differences in methods of measurement. In the present HPLC analysis, total ghrelin levels in milk were more consistent with those of Kierson et al. [14]. However, the serum levels were much higher than previously published reports of “normal plasma ghrelin levels” [17]. Perhaps this has to do with lactation and/or differences between serum and plasma. This is a discrepancy that warrants further investigation.
Conclusion The results from this study show that lactating mothers with GDM have two-fold lower serum and colostral ghrelin levels than do lactating mothers without GDM. However, this lasts up to day 2 of parturition and the level of this hormone resumes normal levels about 2 wk after parturition. It is assumed that the physiologic impact of ghrelin in blood may directly affect the appetite of the mother, and in milk it may be involved in the healthy development of the newborn. The significance of these results in terms of the health of the mother and the newborn has yet to be clarified.
References [1] Hamosh M. Bioactive factors in human milk. Pediatr Clin North Am 2001;48:69 – 86.
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[2] Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999;402:656 –9. [3] Kojima M, Kangawa K. Ghrelin: structure and function. Physiol Rev 2005;85:495–522. [4] Aydin S, Ozkan Y, Caylak E, Aydin S. Ghrelin and its biochemical functions. J Med Sci 2006;26:272– 83. [5] Aydin S. Discovery of ghrelin hormone: research and clinical applications. Turk J Biochem 2007;32:76 – 89. [6] Banks WA, Tschop M, Robinson SM, Heiman ML. Extent and direction of ghrelin transport across the blood-brain barrier is determined by its unique primary structure. J Pharmacol Exp Ther 2002; 302:822–7. [7] McGill HC Jr, Mott GE, Lewis DS, McMahan CA, Jackson EM. Early determinants of adult metabolic regulation: effects of infant nutrition on adult lipid and lipoprotein metabolism. Nutr Rev 1996; 54:S31– 40. [8] Fukushima N, Hanada R, Teranishi H, Fukue Y, Tachibana T, Ishikawa H, et al. Ghrelin directly regulates bone formation. J Bone Miner Res 2005;20:790 – 8. [9] Lee AJ, Hiscock RJ, Wein P, Walker SP, Permezel M. Gestational diabetes mellitus: clinical predictors and long-term risk of developing type 2 diabetes: a retrospective cohort study using survival analysis. Diabetes Care 2007;30:878 – 83. [10] Katsuki A, Urakawa H, Gabazza EC, Murashima S, Nakatani K, Togashi K, et al. Circulating levels of active ghrelin is associated with abdominal adiposity, hyperinsulinemia and insulin resistance in patients with type 2 diabetes mellitus. Eur J Endocrinol 2004;151: 573–7. [11] Palik E, Baranyi E, Melczer Z, Audikovszky M, Szocs A, Winkler G, Cseh K. Elevated serum acylated (biologically active) ghrelin and resistin levels associate with pregnancy-induced weight gain and insulin resistance. Diabetes Res Clin Pract 2007;76:351–7. [12] Ng PC, Lee CH, Lam CW, Wong E, Chan IH, Fok TF. Plasma ghrelin and resistin concentrations are suppressed in infants of insulin-dependent diabetic mothers. J Clin Endocrinol Metab 2004;89:5563– 8. [13] Aydin S, Aydin S, Ozkan Y, Kumru S. Ghrelin is present in human colostrum, transitional and mature milk. Peptides 2006;27:878 – 82. [14] Kierson JA, Dimatteo DM, Locke RG, Mackley AB, Spear ML. Ghrelin and cholecystokinin in term and preterm human breast milk. Acta Paediatr 2006;95:991–5. [15] American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care 2006;29(suppl 1)1. [16] Aydin S. A comparison of ghrelin, glucose, alpha-amylase and protein levels in saliva from diabetics. J Biochem Mol Biol 2007;40:29 –35. [17] Soriano-Guillén L, Barrios V, Chowen JA, Sánchez I, Vila S, Quero J, Argente J. Ghrelin levels from fetal life through early adulthood: relationship with endocrine and metabolic and anthropometric measures. J Pediatr 2004;144:30 –5.
Applied nutritional investigation
Milk and blood ghrelin level in diabetics Suleyman Aydin, Ph.D.a,*, Hikmet Geckil, Ph.D.b, Fikret Karatas, Ph.D.c, Emir Donder, M.D.d, Selahattin Kumru, M.D.e, Ebru Celik Kavak, M.D.e, Ramis Colak, M.D.d, Yusuf Ozkan, M.D.d, and Ibrahim Sahin, M.S.f a
Department of Biochemistry and Clinical Biochemistry, School of Medicine, Firat University, Firat Medical Center, Elazig, Turkey b Molecular Biology Division, Department of Biology, Inonu Univeristy, Malatya, Turkey c Department of Chemistry, Firat University, Elazig, Turkey d Department of Endocrinology–Internal Medicine, Firat University, Medical School (Firat Medical Center), Elazig, Turkey e Department of Obstetrics and Gynecology, Firat University, Medical School, Elazig, Turkey f Department of Biology, Faculty of Arts and Sciences, Firat University, Elazig, Turkey Manuscript received May 11, 2007; accepted August 21, 2007.
Abstract
Objective: Besides its presence in various tissues, ghrelin has recently been shown to be present in blood and breast milk. No previous studies, however, have evaluated the level of this hormone under the condition of pregestational and gestational diabetes mellitus (P-GDM and GDM, respectively). This study was undertaken to show whether a relation exists between serum and milk ghrelin levels in lactating mothers with and without diabetes. Methods: Venous blood was obtained from four groups of women (age range 22–37 y): GDM lactating (n ⫽ 12), P-GM lactating (n ⫽ 3), healthy non-diabetic lactating (n ⫽ 14), and healthy non-lactating (n ⫽ 14). Colostrum and mature milk samples were collected just before suckling. The ghrelin level was determined by radioimmunoassay and high-performance liquid chromatography. Results: Radioimmunoassay results showed that women with GDM and P-GDM had greater than two-fold lower colostrum and serum levels of ghrelin than did lactating women with no GDM at 2 d after parturition. The GDM and non-diabetic groups at 15 d after delivery, however, showed similar levels of ghrelin in mature milk and serum. High-performance liquid chromatographic results indicated that in serum the deacylated form of ghrelin was 18-fold higher than the acylated form. Furthermore, in milk the acylated form of ghrelin was 24-fold that of the active form. Conclusion: These results indicate that mothers with GDM have a substantial (greater than two-fold) decrease in their serum and colostral ghrelin levels. This is, however, a temporary effect lasting only up to early postparturition (2 d after delivery). This peptide hormone restores to completely normal levels at day 15 of parturition, but not P-GDM. The significance of these results in terms of the health of the mother and her newborn, however, has yet to be determined. © 2007 Elsevier Inc. All rights reserved.
Keywords:
Ghrelin; Lipopeptide; Breast milk; Colostrum; Gestational diabetes mellitus
Introduction Human breast milk contains a number of bioactive substances with high nourishment and healthy benefit for the newborn. Especially growth- and development-related effects last into adulthood [1]. Among several peptides with * Corresponding author. Tel.: ⫹90-533-493-4643; fax: ⫹90-424-2379138. E-mail address: [email protected] (S. Aydin). 0899-9007/07/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2007.08.015
this kind of function, ghrelin is relatively newly discovered one. Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, was originally discovered in extracts of rat and human stomach, where it is localized in the endocrine X/A-like (ghrelin cells) cells of the gastric mucosa [2], and is known to act as a signal for food intake [3–5]. This peptide hormone is composed of 28 amino acid residues, bearing a serine (rarely threonine) residue on the third position, where a modification (acylation) is essential for its function. The acylated (octanoylated) form is the
808
S. Aydin et al. / Nutrition 23 (2007) 807– 811
active form, whereas the deacylated form is known as the inactive form of ghrelin. Both forms are present in various tissues and blood, where they have important physiologic roles [3,5]. One of the main differences between these two forms is that only the deacylated form can pass the bloodbrain barrier [6]. In terms of milk ghrelin, however, the “active” form is physiologically more important for infant development [4,5]. Breast milk contains a variety of biologically active nutrients of immediate benefit to the newborn infant [1]. Milk hormones function to transiently regulate the activity of a variety of tissues including endocrine organs until the neonate’s own endocrine systems begin to function [7]. In the case of ghrelin, this hormone might function in the growth and development of the neonate in addition to providing nourishment because it directly regulates bone formation [8]. Gestational diabetes mellitus (GDM), a condition for carbohydrate intolerance during pregnancy, is a widespread medical complication of pregnancy. Women with a history of GDM are more likely to develop type 2 diabetes compared with their counterparts who remain normoglycemic during pregnancy [9]. Indeed, among 15 diabetic women in this study, 3 had type 2, or pregestational, diabetes (P-GDM) that needed no insulin treatment. Although low ghrelin is associated with type 2 diabetes mellitus [10], no previous studies have evaluated the level of this hormone in breast milk in individuals with GDM and P-GDM. In a related study [11], however, fasting serum ghrelin levels in women with GDM was measured and compared with those in normal pregnant and non-pregnant women. Another report measured plasma ghrelin in infants with insulin-dependent diabetic mothers [12]. Recent studies from this laboratory [13] and others [14] have shown that ghrelin is produced and secreted by human breast tissue. There have been contradictory reports [13,14] regarding plasma and breast milk levels of ghrelin, an issue that needs to be clarified. The purpose of this study was two-fold: 1) determine the acylated (active) form of ghrelin by radioimmunoassay (RIA) in serum and milk from women with GDM; and 2) evaluate the high-performance liquid chromatographic (HPLC) results of milk and serum ghrelin levels to clarify the controversial results reported for this hormone.
venous blood glucose measurement 1 h later. Women with blood glucose levels higher than 140 mg/dL were considered to have a positive result. Women with a positive test result were asked to return for a 3-h, diagnostic, 100-g oral glucose tolerance test within the next week. The tolerance test was performed in the morning after a 12-h overnight fast and 3 d of a 150- to 200-g (minimum) carbohydrate diet. GDM was diagnosed if two or more values of the tolerance test equalled or exceeded the thresholds proposed by the American Diabetes Association (ADA) [15]. All participants were healthy pregnant women and had no pregnancy complications (i.e., pre-eclampsia, hypertension, infection, or metabolic or surgical complications except for GDM). In our study population, there were no high-risk pregnancies for GDM (according to ADA criteria). All lactating women had full-term natural deliveries without any health problems. All took no medications 10 d before and during the sampling. The mean age of these two groups (lactating) was 27 y (range 24 –37 y). The control group (non-lactating) women were all healthy and exclusion criteria for the controls were as follows: pregnancy, use of any drugs, alcohol consumption of more than one drink per day, diabetes (even family history), use of tobacco products (past and present), regular intense exercise (⬎15 min of aerobics three times per week), chronic medical illness, history of abdominal surgery, history of gastrointestinal diseases, and family history of obesity. The mean age of this group was 26 y (range 19 –34 y). All subjects (lactating and control women) were advised not to eat, smoke, or drink (except water) overnight before collection of colostral milk, mature milk, and blood samples. Thus, they came to the hospital after an overnight fast and the blood specimens were drawn before breakfast. Approximately 2 mL of breast milk and 5 mL of blood were taken from the lactating women on day 2 (colostral milk) and 15 (mature milk) after delivery. The same amount of blood was also drawn from the non-lactating controls during the follicular phase of menses. The blood was centrifuged at 4000 rpm for 5 min and the serum was stored at ⫺70°C. Colostrum and mature milk were separated by centrifugation of samples twice at 4000 rpm for 10 min. In the first cycle of the centrifugation of milk, a thick fat layer on the top of the tube was removed with a sterile tooth pick, and each supernatant was divided into three aliquots and stored at ⫺70°C until the assay was performed.
Materials and methods Hormone assay The study was carried out in volunteers whose written consent was taken before the study together with the institutional ethical committee approval of the study protocol (dated June 29, 2005, issue no. 153). Sampling was from 12 lactating GDM, 3 lactating P-GDM, 14 lactating healthy control, and 14 non-lactating women. In this study, a twostep screening approach for GDM was used. In this method, pregnant women were given a standard, 1-h, 50-g glucose challenge test between gestational weeks 24 and 28, with a
Serum, colostrum, and mature milk ghrelin levels were measured using a commercially available RIA kit (Linco Research, St. Charles, MO, USA). This RIA uses an antibody that recognizes active epitopes of ghrelin. The colostrum and mature milk active ghrelin measurements were validated as previously described [16]. All samples were read with a gamma counter (MultiGamma 1261, LKBWallac, Turku, Finland). Ghrelin concentrations were cal-
S. Aydin et al. / Nutrition 23 (2007) 807– 811
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Fig. 1. High-performance liquid chromatographic analysis of the deacylated (white arrows) and acylated (black arrows) forms of ghrelin: (a) pure ghrelin standard, (b) serum, and (c) milk.
culated from standard curves generated in the same way as with ghrelin. The ghrelin levels were also determined using an HPLC system. HPLC was performed at room temperature on a Cecil liquid chromatographic system (Series 1100, Cecil, Cambridge, United Kingdom) consisting of a sample injection valve (Cotati 7125) with a 20-L sample loop, an ultraviolet spectrophotometric detector (Cecil 68174), an integrator (HP 3395), and an SGE Walkosil 11 5Cl8 RS column (SGE Deutschland GmbH, Griesheim, Germany), 5-m particle size, 12.0-nm pore size (15.0 ⫻ 4.6-mm inner diameter) with a mobile (1 mL/min) phase consisting of a 50-mM NaClO4 solution at room temperature. The HPLC injected ghrelin mixture contained 26.7 fmol/mL (90 pg/ mL) of acylated (active) ghrelin and 110 fmol/mL (370 pg/mL) of deacylated ghrelin. A three-fold serum physiologic dilution of samples (milk and serum) was injected as with the standard ghrelin solution. To protect ghrelin from proteolytic action (if any), 20 L (200 kallikrein inactivator units) of aprotinin was added per milliliter of sample. The chromatographic graphs of serum and milk are shown in Figures 1b and 1c, respectively. Statistical analysis Statistical analysis was done using SPSS 12 (SPSS Inc., Chicago, IL, USA). The comparison between groups was determined by the Mann-Whitney U test. The same group parameters were determined by Wilcoxon’s test correlation analysis method. The data are expressed as arithmetic means ⫾ standard deviation (SD). P ⬍ 0.05 was considered significant.
Ghrelin was determined in colostral milk (milk taken at 2 d after delivery), mature milk (day 15 of parturition), and blood serum taken at these respective times. Results showed that women with GDM had greater than two-fold lower colostrum and serum levels of ghrelin than did lactating women without GDM at 2 d after parturition. Both groups at 15 d after delivery, however, showed similar levels of ghrelin in mature milk and serum. The serum ghrelin level of the control group (non-lactating women) was similar to that of lactating women. The active ghrelin concentrations in colostrums from women with and without GDM were 7.75 fmol/mL (26.1 pg/mL, from a molecular mass of 33 371 for ghrelin ) and 19.0 fmol/mL (64.1 pg/mL), respectively. These figures were 16.1 fmol/mL (54.1 pg/mL) and 16.47 fmol/mL (55.5 pg/mL), respectively in mature milk. Serum levels of ghrelin also showed a similar trend in both groups (Table 2). Women with GDM had a ghrelin level of 8.81 fmol/mL (29.7 pg/mL) 2 d after parturition, whereas women with no GDM had a level of 18.8 fmol/mL (63.3 pg/mL). The serum ghrelin levels of both groups at 15 d after delivery, however, were similar (17.7 and 16.7 fmol/mL, respectively). The birth weights of neonates from the control, GDM, and P-GDM mothers were 3.12 ⫾ 0.33, 65 ⫾ 0.37, and 3.40 ⫾ 0.40 kg, respectively.
Table 1 Demographic features of subjects Parity*
Gestational age (wk)†
BMI (kg/m2)
Age (y)
2 3 2 2
37.5 ⫾ 1.3 37.3 ⫾ 1.4 38.2 ⫾ 1.5
29.32 ⫾ 2.0 31.2 ⫾ 2.2 29.6 ⫾ 1.8 22.6 ⫾ 2.4
27 ⫾ 3.6 32 ⫾ 3.0 27 ⫾ 3.6 26 ⫾ 4.9
Results
Lactating women GDM P-GDM Non-diabetic Non-lactating women (control)
The demographic characteristics of the study groups are presented in Table 1. The active ghrelin levels of the three study groups (lactating women with GDM, normal lactating, and non-lactating women) are summarized in Table 2.
BMI, body mass index; GDM, gestational diabetes mellitus; P-GDM, pregestational diabetes mellitus * Total number of births given. † Gestational weeks of groups of women at time of delivery were not significantly different.
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Table 2 Active ghrelin level in serum, colostrum, and mature milk from the study groups Ghrelin (fmol/mL)
Lactating women GDM P-GDM Non-diabetic Non-lactating women (control)
Colostrum
Mature milk
Serum-1
Serum-2
7.75 ⫾ 2.2 8.48 ⫾ 1.8‡ 18.99 ⫾ 2.7§ —
16.06 ⫾ 3.2* 9.54 ⫾ 3.2‡ 16.47 ⫾ 3.3§ —
8.81 ⫾ 2.2 9.22 ⫾ 1.9‡ 18.78 ⫾ 2.9§ 17.80 ⫾ 4.9储
17.50 ⫾ 2.39† 7.88 ⫾ 1.80‡ 16.72 ⫾ 2.86§ —
GDM, gestational diabetes mellitus; P-GDM, pregestational diabetes mellitus; Serum-1, blood serum from lactating women at 2 d after parturition and from controls; Serum-2, blood serum from lactating women at 15 d after parturition * P ⬍ 0.05, colostrum versus serum in lactating GDM women. † P ⬍ 0.05, serum-1 and colostrum versus serum-2 in lactating GDM women. ‡ P ⬍ 0.05, colostrum and serum-1 versus mature milk and serum-2 in lactating P-GDM women. § P ⬍ 0.05, colostrum and serum-1 versus mature milk and serum-2 in lactating GDM women. 储 P ⬍ 0.05, control serum versus colostrum and serum-1 in lactating GDM women.
An HPLC analysis was also carried out to determine serum and milk ghrelin levels (Table 3). Serum and milk (mature) ghrelin levels were detected in women after delivery (15 d after parturition). The retention times were 5.896 and 15.906 min for active and deacylated ghrelin forms, respectively (Fig. 1). The lowest detectable level of active ghrelin with this method was determined to be 11 ⫾ 2 pg/mL, and the lowest detectable level of deacylated ghrelin was 14 ⫾ 3 pg/mL. In serum, the deacylated form of ghrelin (range 286 – 406 fmol/mL, which corresponds to 965–1368 pg/mL) was 18-fold higher than the acylated (active) form (range 35–98 pg/mL). In milk the inactive deacylated form (range 122–234 fmol/mL or 412–788 pg/mL) was 24-fold that of the active (acylated) form (range 4.45– 8.90 fmol/mL, corresponding to 15–30 pg/mL; Table 3). In RIA analysis the lowest detectable level for active ghrelin in milk was 1.92 fmol/mL (6.5 ⫾ 2 pg/mL). The intra- and interassay percentage coefficients of variation for milk active ghrelin were 9.8 and 13.4, respectively, with a mean recovery of 92%. No significant correlations were observed between ghrelin levels and maternal body mass index (calculated as kilograms divided by square meters), which was measured and calculated after delivery.
control group of normal individuals. This abnormally low level of ghrelin in mothers with GDM returns to normal after 15 d of parturition, when they acquire the normoglycemic condition. Our study population consisted of mothers with GDM (n ⫽ 12) and P-GDM (type 2, n ⫽ 3). However, all diabetic women’s blood glucose levels were controlled through a diet regimen and none used insulin. When postterm (15 d after giving birth) blood glucose levels of these three women were monitored, serum glucose levels were not normal (119 mg/dL for the first part of the experiment and 128 mg/dL for the second part). In other words, these diabetic mothers continued to have abnormally low levels of ghrelin (in their serum and milk) even 15 d after delivery. These results show that the diabetic condition is one of the main causes of decreased ghrelin levels. These findings are also in accordance with reported ghrelin levels in saliva of diabetic subjects [16]. However, there is no similar work in the literature reporting active ghrelin levels in the milk and serum of individuals with GDM. This experiment carried out with RIA analysis was also carried out with HPLC analysis. This type of analysis was, however, carried out on lactating mothers without GDM after delivery (15 d after giving birth). With this method, the level of acylated (active) ghrelin detected in serum was 19 fmol/mL (64 pg/mL) compared with 4.96 fmol/mL (16.7
Discussion To the best of our knowledge this is the first study to report colostrum, mature milk, and serum active ghrelin levels in the lactating women with GDM and P-GDM, lactating women without GDM, and non-lactating women (control). Ghrelin levels in colostrum and serum from women with GDM were similar after day 2 of parturition. At this period the serum and colostrum levels of ghrelin were similar in the lactating non-GDM group. The level of ghrelin in this group, however, was two-fold higher than in the lactating GDM group. These results are in good agreement with those of Katsuki et al. [10] who found that serum active ghrelin in diabetic subjects was lower than that in a
Table 3 High-performance liquid chromatographic analysis of two forms of ghrelin from serum and milk of women after parturition (15 d after birth) Ghrelin (pg/mL)
Serum Milk
Acylated
Deacylated
Total
64 ⫾ 22* (19 fmol/mL) 23 ⫾ 6† (6.8 fmol/mL)
1161 ⫾ 150* (344 fmol/mL) 548 ⫾ 123† (163 fmol/mL)
1225 ⫾ 177* (363 fmol/mL) 571 ⫾ 128† (169 fmol/mL)
* P ⬍ 0.05, serum versus acylated, deacylated, and total. P ⬍ 0.05, milk versus acylated, deacylated, and total.
†
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pg/mL) detected with RIA. These figures were 6.82 fmol/mL (23 pg/mL) and 4.89 fmol/mL (16.47 pg/mL) in milk, respectively. Also, when compared with our previous reports [13], the HPLC result of this study confirmed that the total ghrelin level in blood was greater than two-fold higher than that in the milk (Table 3). These results confirm our previous findings and are somehow contradictory to that of Kierson et al. [14] who measured ghrelin using an RIA method that measured all epitopes of ghrelin and the levels in breast milk were still higher than previously published data on total plasma ghrelin levels. The discrepancy between those studies and the present study seems to be related to differences in methods of measurement. In the present HPLC analysis, total ghrelin levels in milk were more consistent with those of Kierson et al. [14]. However, the serum levels were much higher than previously published reports of “normal plasma ghrelin levels” [17]. Perhaps this has to do with lactation and/or differences between serum and plasma. This is a discrepancy that warrants further investigation.
Conclusion The results from this study show that lactating mothers with GDM have two-fold lower serum and colostral ghrelin levels than do lactating mothers without GDM. However, this lasts up to day 2 of parturition and the level of this hormone resumes normal levels about 2 wk after parturition. It is assumed that the physiologic impact of ghrelin in blood may directly affect the appetite of the mother, and in milk it may be involved in the healthy development of the newborn. The significance of these results in terms of the health of the mother and the newborn has yet to be clarified.
References [1] Hamosh M. Bioactive factors in human milk. Pediatr Clin North Am 2001;48:69 – 86.
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[2] Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999;402:656 –9. [3] Kojima M, Kangawa K. Ghrelin: structure and function. Physiol Rev 2005;85:495–522. [4] Aydin S, Ozkan Y, Caylak E, Aydin S. Ghrelin and its biochemical functions. J Med Sci 2006;26:272– 83. [5] Aydin S. Discovery of ghrelin hormone: research and clinical applications. Turk J Biochem 2007;32:76 – 89. [6] Banks WA, Tschop M, Robinson SM, Heiman ML. Extent and direction of ghrelin transport across the blood-brain barrier is determined by its unique primary structure. J Pharmacol Exp Ther 2002; 302:822–7. [7] McGill HC Jr, Mott GE, Lewis DS, McMahan CA, Jackson EM. Early determinants of adult metabolic regulation: effects of infant nutrition on adult lipid and lipoprotein metabolism. Nutr Rev 1996; 54:S31– 40. [8] Fukushima N, Hanada R, Teranishi H, Fukue Y, Tachibana T, Ishikawa H, et al. Ghrelin directly regulates bone formation. J Bone Miner Res 2005;20:790 – 8. [9] Lee AJ, Hiscock RJ, Wein P, Walker SP, Permezel M. Gestational diabetes mellitus: clinical predictors and long-term risk of developing type 2 diabetes: a retrospective cohort study using survival analysis. Diabetes Care 2007;30:878 – 83. [10] Katsuki A, Urakawa H, Gabazza EC, Murashima S, Nakatani K, Togashi K, et al. Circulating levels of active ghrelin is associated with abdominal adiposity, hyperinsulinemia and insulin resistance in patients with type 2 diabetes mellitus. Eur J Endocrinol 2004;151: 573–7. [11] Palik E, Baranyi E, Melczer Z, Audikovszky M, Szocs A, Winkler G, Cseh K. Elevated serum acylated (biologically active) ghrelin and resistin levels associate with pregnancy-induced weight gain and insulin resistance. Diabetes Res Clin Pract 2007;76:351–7. [12] Ng PC, Lee CH, Lam CW, Wong E, Chan IH, Fok TF. Plasma ghrelin and resistin concentrations are suppressed in infants of insulin-dependent diabetic mothers. J Clin Endocrinol Metab 2004;89:5563– 8. [13] Aydin S, Aydin S, Ozkan Y, Kumru S. Ghrelin is present in human colostrum, transitional and mature milk. Peptides 2006;27:878 – 82. [14] Kierson JA, Dimatteo DM, Locke RG, Mackley AB, Spear ML. Ghrelin and cholecystokinin in term and preterm human breast milk. Acta Paediatr 2006;95:991–5. [15] American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care 2006;29(suppl 1)1. [16] Aydin S. A comparison of ghrelin, glucose, alpha-amylase and protein levels in saliva from diabetics. J Biochem Mol Biol 2007;40:29 –35. [17] Soriano-Guillén L, Barrios V, Chowen JA, Sánchez I, Vila S, Quero J, Argente J. Ghrelin levels from fetal life through early adulthood: relationship with endocrine and metabolic and anthropometric measures. J Pediatr 2004;144:30 –5.