Polycythaemia in Infants of Diabetic Mothers: β-hydroxybutyrate Stimulates Erythropoietic Activity

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1 The Journal of International Medical Research 2011; 39: Polycythaemia in Infants of Diabetic Mothers: β-hydroxybutyrate Stimulates Erythropoietic Activity H CETIN, M YALAZ, M AKISU AND N KULTURSAY Department of Paediatrics, Ege University Medical School, Izmir, Turkey This study tested whether elevated maternal b-hydroxybutyrate (b-ohb) levels contribute to polycythaemia in infants of diabetic mothers. Pregnant diabetic women (n = 27) and non-diabetic controls (n = 20) and their singleton infants were included. Maternal glycosylated haemoglobin and b-ohb levels were studied at weeks gestation; levels were significantly higher in mothers with diabetes than in controls. Birth weights and cord blood levels of insulin and fetal haemoglobin were significantly higher in infants from diabetic mothers compared with control infants, as were haematocrit levels in venous blood samples taken from each infant at 4 h following delivery. Cord blood erythropoietin levels were similar in both groups. There was a positive strong correlation between maternal b-ohb levels and polycythaemia in newborn infants, indicating that b-ohb could activate erythropoiesis independently from intrauterine hyperinsulinaemia and/or erythropoietin levels, and may be important in the pathogenesis of polycythaemia in infants born to diabetic mothers. KEY WORDS: POLYCYTHAEMIA; b-hydroxybutyrate; INFANTS OF DIABETIC MOTHERS; FETAL HAEMOGLOBIN; ERYTHROPOIESIS Introduction Diabetes mellitus (DM) is an important metabolic disturbance influencing carbohydrate, lipid and protein metabolism, the effects of which can be profound in pregnancy. 1,2 Polycythaemia occurs frequently at birth in infants of diabetic mothers, 3 5 presenting an increased risk for hyperviscosity, renal vein thrombosis, cardiac failure and necrotizing enterocolitis in the infant. The condition may also be associated with hyperbilirubinaemia, resistant hypoglycaemia, hypocalcaemia and hypomagnesaemia in these infants. 5 The exact mechanism of how polycythaemia develops in neonates remains to be identified. Several studies have shown that increased erythropoiesis in infants born to diabetic mothers might be related to intrauterine hypoxia, due to hyperinsulinism and hyperglycaemia. 6 8 Elevated cord plasma erythropoietin (Epo) levels have been reported in sheep and in human studies, 9 11 although Epo levels in cord blood are generally normal in infants of diabetic mothers whose glycaemic control is well maintained throughout pregnancy. 12 Earlier work has shown that DM in 815

2 pregnancy alters serum lipid and lipoprotein composition in infants at birth. 13 Ketone body metabolism is important in diabetic pregnancy both for the mother and fetus, with β-hydroxybutyrate (β-ohb), an important ketone body, freely transported to the fetus. 14,15 In sheep, maternal infusion of β-ohb significantly decreased fetal partial pressure of arterial oxygen and increased fetal lactate concentrations and heart rate. 9 In humans, studies have demonstrated that serum levels of β-ohb were significantly raised in diabetic pregnant women, compared with levels in non-diabetic pregnant controls. 16,17 Bard and Prosmanne 18 suggested that infants born to diabetic mothers synthesized significantly higher levels of fetal haemoglobin (HbF), compared with infants born to non-diabetic controls. In the normal fetus, a switch from production of HbF to adult haemoglobin occurs between 28 and 34 weeks gestation. 19 Butyric acid and butyrate derivates act specifically on the promoter of the δ-globin chain gene to stimulate fetal haemoglobin production. 20,21 Perrine et al. 22 found a significant delay in the switch from δ-globin to β-globin in full-term infants born to diabetic mothers, compared with the infants of non-diabetic mothers. 22 In light of these findings the present study was designed to investigate the effects of maternal levels of β-ohb, glycosylated haemoglobin (HbA 1c ) and fetal concentrations of insulin and Epo on fetal erythropoiesis and HbF synthesis in infants born to diabetic mothers. Patients and methods PATIENTS Pregnant women with or without diabetes and their singleton infants were studied at the Department of Obstetrics and Gynaecology, Ege University Medical School, Izmir, Turkey, between March 2002 and August Women with known DM that predated the pregnancy (pregestational DM) and those with gestational DM at weeks gestation, confirmed according to oral glucose tolerance test results and World Health Organization (WHO) criteria, 1 were included. The control group comprised agematched non-diabetic pregnant women, selected at random from the same institute. The exclusion criteria for mothers and infants in this study were: pre-eclampsia; birth weight lower than the 10th percentile for gestational age; 5-min Apgar score < 6; cord blood or arterial blood ph < 7.1 taken shortly after delivery; encephalopathy consisting of stupor, hypotonia, abnormal neonatal reflexes; presence of haemolytic disease, major congenital abnormalities; and cyanotic heart disease. The study was approved by the Ethical Committee of the Medical Faculty of Ege University. Verbal informed consent was obtained from all participants. ULTRASOUND SCANS AND BLOOD TESTS From 24 weeks gestation, all mothers had serial monthly ultrasound examinations, using standard hospital equipment, to monitor fetal growth. At the end of the 34th and 36th weeks of gestation (mean ± SE 34.9 ± 0.5 weeks), maternal blood was obtained by venipuncture in order to examine overnight fasting blood glucose, plasma β-ohb and serum HbA 1c concentrations. The mean values for plasma β-ohb levels measured at 34 and 36 weeks gestation (mean ± SE 34.9 ± 0.5 weeks) were also calculated. All babies were delivered between 37 and 39 weeks gestation at the Department of Obstetrics and Gynaecology, Ege University Medical School. Immediately after delivery, umbilical venous blood (cord blood) was collected and plasma glucose, plasma insulin, plasma 816

3 Epo, neonatal haemoglobin concentrations and HbF levels were measured. Venous blood samples, drawn into heparinized capillary tubes, were obtained from the infants 4 h after delivery. These samples were centrifuged for 5 min and neonatal haematocrit was measured: levels > 65% were considered to show polycythaemia. Maternal and neonatal plasma glucose concentrations were measured with the Roche Hitachi 902 Auto-analyser (Roche Diagnostics, Indianapolis, IN, USA). Serum HbA 1c levels were measured using the thiobarbituric acid calorimetric assay (Nycocard Reader II; Axis-Shield AS, Oslo, Norway). Plasma samples for β-ohb analysis were tested in duplicate using a direct electrode assay (Abbott Laboratories MediSense Products, Bedford, MA, USA), test range 0 6 mm/ml, sensitivity 0.1 mm/ml. Plasma insulin in newborn infants was measured by radioimmunoassay using a standard kit (Linco Research, St Charles, MO, USA). Plasma Epo levels were measured by enzyme-linked immunosorbent assay (ELISA Epo kit; Biomerica, Irvine, CA, USA). HbF values were determined by using highperformance liquid chromatography (Bio- Rad Variant Haemoglobin Analyser; Bio-Rad, Tokyo, Japan). STATISTICAL ANALYSES All values are presented as mean ± SD. Statistical analyses were carried out using the SPSS statistical package, version 13.0 (SPSS Inc., Chicago, IL, USA) for Windows. All data from both groups were compared using an unpaired Student s t-test. The association between studied parameters was compared using Pearson s correlation coefficient. A P-value < 0.05 was considered to be statistically significant. Results In total, the study included 47 pregnant women (27 diabetic, 20 non-diabetic controls) and their singleton infants (Table 1). Eight of the women were insulindependent and had known pregestational DM; 19 had gestational DM. The 20 controls had normal glucose metabolism, according to their oral glucose tolerance test results and WHO criteria, at weeks gestation. Maternal fasting plasma glucose levels, serum HbA 1c and plasma β-ohb concentrations, cord blood haemoglobin, TABLE 1: Demographic characteristics of pregnant women with diabetes (gestational or preexisting diabetes) and pregnant controls, and their singleton infants Mothers with diabetes Controls Characteristic (n = 27) (n = 20) Women with pre-existing diabetes 8 Women with gestational diabetes 19 Maternal age (years) 32.2 ± ± 2.5 Infants gestational age at birth (weeks) 39.1 ± ± 0.7 Infants birth weight (g) 3936 ± 681 a 3356 ± 174 Infants gender (female/male) 15/12 10/10 Type of delivery Caesarean section Normal spontaneous 11 8 Data presented as mean ± SD or number of subjects. a P < 0.01 versus control; Student s t-test. 817

4 HbF, cord plasma Epo and insulin concentrations, and neonatal venous haematocrit levels measured during the study period are presented in Table 2. Maternal fasting blood glucose levels, HbA 1c and β-ohb concentrations were significantly higher in women with diabetes than in nondiabetics at the end of weeks gestation (P < 0.05, P < 0.05 and P < 0.001, respectively; Table 2). Elevated Epo levels were found in two cords from infants of mothers with diabetes. Twelve women with diabetes were hyperinsulinaemic; however, elevated Epo levels were observed in only one hyperinsulinaemic infant. Polycythaemia was observed in six (22%) infants of diabetic mothers; three had hyperinsulinaemia and only one had an elevated Epo level. No polycythaemic infants were born to nondiabetic mothers. Cord blood haemoglobin, HbF levels and venous neonatal haematocrit values were significantly higher in infants born to diabetic mothers compared with control infants (P < 0.01 all comparisons). Cord plasma insulin levels were significantly higher in samples from infants born to diabetic mothers compared with control infants (P < 0.05), while no significant between-group difference in cord plasma levels of Epo was observed. In mothers with diabetes, a good correlation was found between maternal β- OHB concentrations and neonatal haemoglobin, venous neonatal haematocrit and HbF values (r = 0.489, r = and r = 0.575, respectively; Table 3). No correlation was observed between cord plasma insulin levels and neonatal haemoglobin, venous TABLE 2: Blood parameter results from pregnant women with diabetes (gestational or pre-existing diabetes) and pregnant controls, and their singleton infants Mothers with diabetes Controls Statistical Blood parameter (n = 27) (n = 20) significance a Maternal FBG (mg/dl) ± ± 2.93 P < 0.05 (72 118) (68 102) Maternal HbA 1c (%) 6.14 ± ± 0.19 P < 0.05 ( ) ( ) Maternal β-ohb (mm/ml) 0.48 ± ± 0.06 P < ( ) (0 0.2) Cord Hb (g/dl) ± ± 0.7 P < 0.01 ( ) ( ) Neonatal venous Hct (%) ± ± 1.95 P < 0.01 (49 68) (44 53) Cord HbF (%) ± ± 2.18 P < 0.01 (67 96) (64 83) Cord plasma Epo (miu/ml) 12.7 ± ± 1.93 NS (4 20.5) (9 15.6) Cord plasma insulin (miu/ml) ± ± 0.97 P < 0.05 (7 87) (6 9) Maternal samples taken at weeks gestation. Cord samples taken at delivery; neonatal venous Hct taken 4 h postdelivery. Data are presented as mean ± SD (range). a Student s t-test, FBG, fasting blood glucose; HbA 1c, glycosylated haemoglobin; β-ohb, β-hydroxybutyrate; Hb, haemoglobin; Hct, haematocrit; HbF, fetal haemoglobin; Epo, erythropoietin; NS, not statistically significant (P > 0.05). 818

5 neonatal haematocrit and HbF values in infants from mothers with diabetes (r = 0.045, r = and r = 0.211, respectively). There was no correlation between maternal β-ohb levels and HbA 1c and cord plasma insulin levels in infants of diabetic mothers (r = and r = 0.019, respectively). Discussion Polycythaemia is an important problem that is observed in some infants born to diabetic mothers: fetal hyperinsulinaemia and elevated Epo levels, due to intrauterine chronic hypoxia, may cause polycythaemia in these infants. 10,11 No consistent correlation between plasma Epo levels and poly cythaemia has been reported in human 6 8 or animal studies Some polycythaemic infants have been reported as having normal Epo levels, whereas others with elevated Epo levels were not considered to be polycythaemic. 6 8 Similarly, of the six polycythaemic infants reported in the present study, only one had an elevated Epo level and three had hyperinsulinaemia. Thus, the aetiology of polycythaemia is not simply accounted for by hyperinsulinaemia and elevations in Epo. The strong correlation between maternal β-ohb concentration and neonatal venous haematocrit level observed in the present study suggests that elevated ketone bodies are implicated in polycythaemia occurring in infants born to diabetic mothers. Ketone levels increase in diabetic women, especially during the third trimester of pregnancy, and cross the placenta to the fetus seen as a strong correlation between maternal and fetal β-ohb levels Fetal ketone bodies were not determined in the present study due to the riskiness of the sampling procedure. Fasting blood glucose, plasma β-ohb and serum HbA 1C levels were all significantly higher in diabetic pregnant women, compared with non-diabetic pregnant controls, in the present study. There were no correlations between neonatal venous haematocrit levels and cord plasma insulin and Epo levels, whereas a positive correlation was seen between neonatal venous haematocrit levels and maternal β-ohb levels. In infants of diabetic mothers, butyrate can lead to intrauterine chronic hypoxia, thereby causing elevated Epo levels. 9 Experimental studies have also indicated that butyrate can directly increase erythropoiesis without affecting Epo levels. 23,24 Thus, butyrate and its derivates could cause an increase in erythropoietic activity that is unrelated to Epo. 18,20 22,24 These findings appear to support the present study, in that polycythaemia can occur without a concomitant elevation in Epo levels. In vitro studies have shown that TABLE 3: Pearson s correlation coefficients (r) between maternal b-ohb levels in pregnant women with diabetes (gestational or pre-existing diabetes) and pregnant controls (n = 47), and markers of fetal haematopoiesis in their singleton infants Maternal b-ohb Fetal haematopoiesis marker r Statistical significance a Cord Hb concentration P = Venous Hct P = Cord HbF P = β-ohb, β-hydroxybutyrate; Hb, haemoglobin; Hct, haematocrit; HbF, fetal haemoglobin. a Pearson s correlation coefficient. 819

6 proliferation and maturation of erythroid progenitors are both increased by butyrate and its derivates Cappellini et al. 24 and Reinhardt et al. 26 have shown that soluble transferrin receptors, as a parameter of erythropoietic activity, are increased by experimental application of butyrate. Pace et al. 27 detected significant elevations in reticulocyte count and haematocrit levels following short-chain fatty acid infusion. As shown above, maternal plasma β-ohb could play an important role in the pathogenesis of polycythaemia in infants born to diabetic mothers. Similar to data reported in the literature, 28,29 in the present study, elevated HbF levels were observed in the infants of diabetic mothers. Perrine et al. 22 showed that there was a delay to the globin gene switch and decreased β-globin levels in infants from diabetic mothers. In vitro studies have also shown that the β-globin switch was inhibited by butyrates, 30,31 and that butyrate and its derivates activate proliferation of early erythroid progenitors, which are HbF synthesizing cells. 20,32 35 A positive correlation between elevated β-ohb and HbF levels in infants born to mothers with diabetes was observed in the present study. In fetal development, the presence of butyrate acts as a transcriptional regulator of the γ-globin chain gene, therefore γ-globin synthesis persists. There were some limitations to the present study. The number of subjects included was small and the blood analysis of pregnant women was only performed at weeks gestation. Larger studies are needed to verify the results of the present study. In conclusion, the present study indicates that elevated maternal plasma ketone levels, as a result of insufficient control of diabetes in pregnancy, affects erythropoiesis during fetal development. Butyrates may increase the proliferation of erythroid progenitors thereby causing polycythaemia and elevated HbF levels in infants born to diabetic mothers. Thus, monitoring maternal plasma butyrate levels, together with fasting blood glucose and HbA 1c testing in diabetic women during pregnancy are recommended for the prevention of polycythaemia in their infants. Conflicts of interest The authors had no conflicts of interest to declare in relation to this article. Received for publication 16 October 2010 Accepted subject to revision 10 December 2010 Revised accepted 28 March 2011 Copyright 2011 Field House Publishing LLP References 1 Cunningham FG, Gant NF, Leveno KJ, et al (eds): Diabetes. In: Williams Obstetrics, 21st edn. New York: McGraw-Hill, 2001; pp Di Cianni G, Miccoli R, Volpe L, et al: Intermediate metabolism in normal pregnancy and in gestational diabetes. Diabetes Metab Res Rev 2003; 19: Tyrala EE: The infant of the diabetic mother. Obstet Gynecol Clin North Am 1996; 23: Schwartz R, Teramo KA: Effects of diabetic pregnancy on the fetus and newborn. Semin Perinatol 2000; 24: Mimouni F, Tsang RC, Hertzberg VS, et al: Polycythemia, hypomagnesemia, and hypocalcemia in infants of diabetic mothers. Am J Dis Child 1986; 140: Weintrob N, Karp M, Hod M: Short- and longrange complications in offspring of diabetic mothers. J Diabetes Complications 1996; 10: Perrine SP, Greene MF, Lee PD, et al: Insulin stimulates cord blood erythroid progenitor growth: evidence for an aetiological role in neonatal polycythaemia. Br J Haematol 1986; 64: Shannon K, Davis JC, Kitzmiller JL, et al: Erythropoiesis in infants of diabetic mothers. Pediatr Res 1986; 20: Miodovnik M, Skillman CA, Hertzberg V, et al: Effect of maternal hyperketonemia in hyperglycemic pregnant ewes and their fetuses. Am J Obstet Gynecol 1986; 154: Philips AF, Dubin JW, Matty PJ, et al: Arterial 820

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