We request permission to apply for a research grant to examine cord blood samples in the ALSPAC offspring. This proposal will determine whether offspring metabolic, vascular and inflammatory phenotypes are predetermined by the time of birth. Specifically we wish to measure IGF-I, leptin, insulin, cholesterol, triglyceride, HDL-C, CRP and liver function tests in cord blood. We are aware that some analysis of cord blood has already been performed in small subsets, specifically leptin in 197 infants1, and IGF-II, soluble IGF2R, insulin, IGF-I, IGF-binding protein-1 (IGFBP-1), and IGFBP-3 in 199 infants2. Our intention is to extend the measurement of leptin, IGF-I and insulin to the remainder of the ALSPAC cohort and examine additional new analytes.

This work builds on several current studies undertaken by the applicants. First is the analysis of lipids, inflammatory markers, insulin and liver function at age 9, 15 and 17 years. To date we have measured leptin, adiponectin, insulin, lipids, CRP, liver function tests, and IL-6 in non-fasting plasma samples taken at age 9 years old. Analysis of fasting lipids, glucose, insulin, CRP, liver function tests and IL-6 at age 15 years has also recently been completed. Additionally we are currently funded to analyse fasting glucose, insulin, lipids & liver function tests in the cohort at age 17 (Lawlor PI). Secondly, the proposed study will also build on the inspection and abstraction of obstetric data from the ALSPAC cohort and the follow-up studies of the mothers, with funds available and analyses currently being completed on ALSPAC mother's fasting blood for glucose, insulin, pro-insulin, lipids and inflammatory markers now 17 years after the index pregnancy (Davey Smith and Lawlor PIs). Thirdly. the proposed study will also utilise the recently completed genome-wide association study (GWAS) of the offspring (Davey Smith PI) and the recently funded GWAS of the ALSPAC mothers (Lawlor PI), to examine the independent contributions of each genotype and their interaction in the determination of cord analytes. This combination of data, together with the additional data we plan to collect in this proposal, will allow clarification of the role of maternal lifestyle and pregnancy outcome on the offspring metabolic, vascular and inflammatory phenotypes, and whether variations in these phenotypes at birth track into later childhood and early adolescence. Additionally if we can show significant correlations between cord parameters and the same measures in later life, this then provides some rationale for future birth cohort studies to have cord measures as legitimate surrogates.

We would like permission to address the following objectives in relation to cord blood analytes:

a. Describe the distribution of IGF-I, leptin, insulin, cholesterol, triglyceride, HDL-C, CRP and liver function tests in cord blood.

b. Determine the associations of maternal weight gain in pregnancy, blood pressure change in pregnancy, maternal smoking, alcohol and dietary behaviour in pregnancy, onset of gestational diabetes/glycosuria, mode of delivery, parity and preterm birth on cord IGF-I, leptin, insulin, cholesterol, triglyceride, HDL-C, CRP and liver function tests.

c. Determine whether variation in maternal vitamin D, parathyroid hormone and calcium levels in pregnancy are associated with cord IGF-I, leptin, insulin, cholesterol, triglyceride, HDL-C, CRP and liver function tests.

d. Determine the associations of cord IGF-I, leptin, insulin, cholesterol, triglyceride, HDL-C, CRP and liver function tests with offspring birthweight, placental weight and feto-placental index.

e. Determine the prospective associations of cord IGF-I, leptin, insulin, cholesterol, triglyceride, HDL-C, CRP and liver function tests with offspring fat mass and change in fat mass and growth trajectories from birth to age 17.

f. Determine the prospective associations of cord IGF-I, leptin, insulin, cholesterol, triglyceride, HDL-C, CRP and liver function tests with the previously determined repeat measures of these analytes at age 9, 15 and 17 years.

g. Determine the prospective associations of cord IGF-I, leptin, insulin, cholesterol, triglyceride, HDL-C, CRP and liver function tests with ultrasound assessment of liver fat deposition, liver function tests, lipids, insulin and glucose at age 17.

h. Determine the prospective associations of cord IGF-I, leptin, insulin, cholesterol, triglyceride, HDL-C, CRP and liver function tests with offspring educational attainment.

i Determine the association of cord IGF-I, leptin, insulin, cholesterol, triglyceride, HDL-C, CRP and liver function tests with maternal cardiovascular risk profile in later life (lipids, carotid intimal-medial thickness, blood pressure, height, weight, BMI, fat and lean mass, insulin and glucose).

j. With available data (10,000 mothers and 3,000 offspring or larger if additional grants funded) complete a genome wide-association study of cord IGF-I, leptin, insulin, cholesterol, triglyceride, HDL-C, CRP and liver function tests

For objectives g, h, i and j this will be performed on completion of the 17 year clinic, the BHF funded follow up study of the mothers and the GWAS of the mothers (Lawlor PI for mothers follow-up clinic and for maternal GWAS and for grant that funds taking of fasting blood samples and completion of fasting glucose, insulin, lipids, liver function and inflammatory marker tests).

In order to determine the extent to which any associations of maternal characteristics with offspring cord blood levels reflect genetic, intrauterine or background socioeconomic mechanisms we will employ and compare results from three methodological approaches - multivariable regression models; within parental comparisons and Mendelian randomization studies, using maternal genotype adjusted for offspring genotype as an instrumental variable for the causal effect of an intrauterine exposure.

Request for data access to allow generation of pilot data

To facilitate the provision of pilot data for a grant submission, we request permission to examine the previously reported cord analytes of insulin, leptin and IGF-I from the In Focus group1, 2, with subsequent anthropometric and DXA measures at age 9 and 15, and repeat measures of leptin and insulin at age 9, and insulin at age 15 if available.

Background and justification of analytes

Birthweight and fetal insulin

The epidemiologic observations that smaller size or relative thinness at birth and during infancy is associated with increased rates of coronary heart disease, stroke and type 2 diabetes mellitus, in adult life have been extensively replicated3, 4. Although epidemiology studies have largely examined the association of birthweight with adult disease outcomes, because of its ready availability in many datasets, it is increasingly recognised that birth weight is most likely acting as a proxy for other exposures, including maternal diet, smoking, alcohol and other intrauterine exposures and genetic variants.

Birthweight is determined, in part, by fetal metabolic and hormonal responses to intrauterine influences. Pedersen was the first to suggest that maternal hyperglycaemia would result in excessive transfer of glucose to the fetus and the compensatory fetal hyperinsulinaemia would drive fetal growth and deposition of fetal adipose tissue5. We and others have confirmed that in maternal type 1 diabetes maternal hyperglycaemia is associated with fetal hyperinsulinaemia and increased birthweight6. We have also identified that cord insulin is the principal positive associate of the observed increases in both birthweight and neonatal adiposity in offspring of mothers with type 1 diabetes7. More recently, these positive associations have been extended to the euglycaemic range of glucose during pregnancy, with the HAPO study clearly demonstrating that there is positive linear relationship between maternal glucose and birthweight and neonatal adiposity, and that this relationship is mediated by fetal insulin production8, 9. Furthermore, there is some evidence (most convincing from populations, such as the Pima Indians, with very high prevalence of obesity and type 2 diabetes) that higher levels of maternal glucose in pregnancy are associated with greater adiposity and abnormal glucose tolerance in offspring in later life10-12 and can influence the penetrance of genetic syndromes of diabetes13. Consistent with an insulin mediated effect, fetal hyperinsulaemia has been associated with excess adiposity and elevated plasma glucose and insulin during childhood in a Caucasian population14. Given the HAPO results, it is reasonable to wonder the extent to which these associations in later life are present across the whole range of fetal insulinaemia15. Assessment of cord insulin in the ALSPAC cohort will allow us to examine the association of fetal insulin with offspring adiposity and glucose tolerance throughout childhood and adolescence.

IGF-1

Cord IGF-I is positively associated with birthweight and placental weight2, 7 and IGF-I deletion or reduced receptor expression in humans are both associated with a reduction in birthweight and placental weight16, 17. Conversely, in animal models prolonged administration of exogenous IGF-I to growth restricted fetuses substantially increases body and placental weight18. In offspring of mothers with type 1 diabetes, we have demonstrated a relationship of IGF-I and birth weight independent of insulin7, in keeping with previous observations from the ALSPAC cohort2. Furthermore, we have recently demonstrated that cord IGF-I is the principal correlate of placental sub-structure (data submitted), and that placental and fetal growth closely correlate, even if there is fetal hyperinsulinaemia19. IGF-I is also an associate of feto-placental index in offspring of mothers with type 1 diabetes19, with experimental and epidemiological studies suggesting that mismatch of placental and fetal growth are associated with postnatal abnormalities in cardiovascular, metabolic and endocrine functions 20, 21. Therefore, although IGF-I at birth does not track during childhood22, there is significant potential for an association with later development, through its associations with placental development and matching of fetal and placental growth.

Adiposity and Leptin

Cord leptin strongly correlates with maternal and neonatal adiposity7, 23-27, and also neonatal whole body bone mineral contents and estimated volumetric bone density28. Although neonatal adiposity is largely determined by maternal glycaemia9, maternal smoking has also been associated with a reduction in cord leptin independent of influences on birthweight29. Cord leptin may therefore be influenced by maternal lifestyle behaviour - potentially through an impact on both fetal and placental leptin production. Lower cord blood leptin levels, but not variation in adiponectin levels, have recently been shown to be associated with more pronounced weight gain in the first 6 months of life and higher BMI at 3 years of age30. This is consistent with studies demonstrating that early upward crossing of growth centiles is associated with an increased risk of adiposity in later life31. However, these initial results require replication and currently the relationship between cord leptin and outcomes, including adiposity and skeletal development, in later life remain to be determined.

Inflammatory Markers

CRP and ICAM-1 are increased in adults with obesity32-36 and type 2 diabetes37, 38 and similar relationships also appear to be present in childhood39. We have previously demonstrated that offspring of mothers with type 1 diabetes (OT1DM), have increased fat mass and an associated increased circulating leptin concentration40, 41. Furthermore, we have shown that CRP and ICAM-1 are not only increased in OT1DM but are associated with cord leptin and skin fold thickness42. Notably leptin was also associated with IL-6, a major stimulus for CRP production, raising the possibility that fetal adipose tissue is not only responsible for endothelial activation but induction of a pro-inflammatory phenotype which is already evident by the time of birth. Maternal HbA1c was also associated with CRP supporting a role of maternal glycaemic control in the inflammatory phenotype at birth. This finding of an elevated CRP in OT1DM and relation with maternal glycaemia has now also been replicated43, and the possibility that this extends to lower glucose values given the continuous relationship of glucose to offspring birthweight and adiposity9 requires further investigation. Inflammatory markers are also known to track in childhood44 and to be associated with later metabolic37, 38 and vascular disease45. A single study has found raised inflammatory markers in OT1DM in childhood46. We and others have demonstrated that this inflammatory phenotype is present at birth in OT1DM and that it is particularly related to fetal leptin42, 43. Collectively this raises the possibility that there is potential in-utero effects on sub-clinical inflammatory phenotypes, which directly relate to maternal glycaemia and fetal hyperinsulinaemia and adiposity.

Lipids

With respect to lipids we have also previously determined that maternal diabetes is associated with lower fetal lipids in male offspring, in particular HDL-C (thus significantly higher cholesterol to HDL-C ratio)47. Furthermore, we identified that perturbances in IGF-1 and leptin, rather than insulin - may be the major determinants of HDL-C in-utero47. Lipid metabolism and inflammation are linked via hepatic lipid metabolism with elevations of plasma triglycerides occurring during acute adult inflammatory responses48, consequently, CRP and triglyceride are positively related in children33 and adults49. We have also demonstrated that this relationship is present at birth in OT1DM, with markers; ICAM-1, CRP and IL-6 potentially all acting as independent determinants of triglyceride. The possibility that dyslipidaemia and subclinical inflammation - major determinants of adult disease may coexist at birth, requires further study, particularly if we can identify maternal antecedents which may be modified in the future.

Liver Function Tests

Non alcoholic fatty liver disease (NAFLD) has been described in children and adolescents, with estimates of the prevalence of NAFLD based on unexplained elevated levels of alanine aminotransferase (ALT) or ultrasound range from 2-3% in general paediatric populations50, 51 to between 6-30% in obese children or adolescents52-54. Prevalence estimates in general paediatric populations may underestimate the prevalence in adolescents. A recent post-mortem study provides the most robust evidence of the potential importance of NAFLD in adolescents55. In that study, of 742 US individuals (aged 2-19 years) who had died from external causes (mostly road traffic accidents) the age, gender and ethnicity standardised prevalence of NAFLD (defined as greater than 5% steatosis on histology of the post-mortem liver) was 9.6%, with 3% of the population having non alcoholic steatohepatitis. The prevalence increased with increasing age from 0.7% in those ages 2-4 to 17.3% in those aged 15-1955. A non-human primate model has recently demonstrated that a high fat maternal diet can influence offspring liver function with an increase in fetal liver fat deposition and hepatic oxidative stress - with persistence of this phenotype at 180 days postnatal56. Furthermore, it was suggested that this related to fetal lipid rather than glucose loads. Notably cord leptin and triglyceride are associated in male OT1DM47, raising the possibility that fetal liver fat deposition may occur in conjunction with fetal adiposity. Given the potential long-term importance of NAFLD, identification of whether maternal obesity is associated with derangement of LFTs at birth and if this persists through childhood is important.

Maternal cardiovascular disease

Lastly, offspring birthweight has also been inversely associated with parental mortality, with a stronger relationship seen in mothers and a dominance of cardiovascular disease57. Given the importance of fetal metabolic and hormonal responses in determining birthweight, we hypothesise that these cord analytes will be associated with maternal cardiovascular disease risk profile in later life independent of birthweight.

Methods

All biochemical analyses will be performed at Glasgow Royal Infirmary, which adheres to UK external quality control for all parameters and is Clinical Pathology Accreditation (CPA) accredited. Plasma total cholesterol, triglyceride and HDL-C will be performed by modification of the standard Lipid Research Clinics Protocol unsing enzymatic reagents for lipid determinations. LDL-C will be calculated from total cholesterol and triglyceride using the Friedwald equation. A high degree of correlation between Friedwald calculated LDL-C and directly measured LDL-C has previously been described for cord blood58. Leptin will be measured by a highly sensitive in-house ELISA with better sensitivity at lower levels than commercial assays59. Insulin will be measured by an ELISA (Mercodia), which does not cross-react with proinsulin and has a lower sensitivity of 6pmol/l, well below the (median 22.4pmol/l, IQR15.0 -38.0) we noted in control offspring in a recent study6. CRP will be measured using a high-sensitivity, 2-site enzyme-linked immunoassay42, 60. IGF-1 will be assayed using a chemiluminescence immunoassay (Nichols Institute Diagnostics, San Juan Capistrano, CA 92675, USA) using standards referenced to WHO 1st International Reference Reagent 1988 (Insulin-Like Growth Factor-1 87/518). The limit of detection is 1*0 nmol/l. Intra- and interassay coefficients of variation (CVs) were 5*5-6.8% and 5*4-7*0%. Liver enzymes will be measured by automated analyser with enzymatic methods (all CVs less than 3%). A total of 500micro-l will be required to be shipped to Glasgow for the assays due to dead space, however it is likely that only ~300micro-l will be used.

We will request in the research grant the cost of a post-doctoral statistician to be employed at the University of Bristol under the supervision of Professors Lawlor and Davey Smith for data management and completion of the statistical analysis.

References

1 Ong, K. K. L., Ahmed, M. L., Sherriff, A., Woods, K. A., Watts, A., Golding, J. and Dunger, D. B., Cord Blood Leptin Is Associated with Size at Birth and Predicts Infancy Weight Gain in Humans, J Clin Endocrinol Metab, 1999, 84: 1145-1148.

2 Ong, K., Kratzsch, J., Kiess, W., Costello, M., Scott, C. and Dunger, D., Size at Birth and Cord Blood Levels of Insulin, Insulin-Like Growth Factor I (IGF-I), IGF-II, IGF-Binding Protein-1 (IGFBP-1), IGFBP-3, and the Soluble IGF-II/Mannose-6-Phosphate Receptor in Term Human Infants, J Clin Endocrinol Metab, 2000, 85: 4266-4269.

3 Whincup, P. H., Kaye, S. J., Owen, C. G., Huxley, R., Cook, D. G., Anazawa, S., Barrett-Connor, E., Bhargava, S. K., Birgisdottir, B. E., Carlsson, S., de Rooij, S. R., Dyck, R. F., Eriksson, J. G., Falkner, B., Fall, C., Forsen, T., Grill, V., Gudnason, V., Hulman, S., Hypponen, E., Jeffreys, M., Lawlor, D. A., Leon, D. A., Minami, J., Mishra, G., Osmond, C., Power, C., Rich-Edwards, J. W., Roseboom, T. J., Sachdev, H. S., Syddall, H., Thorsdottir, I., Vanhala, M., Wadsworth, M. and Yarbrough, D. E., Birth weight and risk of type 2 diabetes: a systematic review, Jama, 2008, 300: 2886-2897.

4 Huxley, R., Owen, C. G., Whincup, P. H., Cook, D. G., Rich-Edwards, J., Smith, G. D. and Collins, R., Is birth weight a risk factor for ischemic heart disease in later life?, Am J Clin Nutr, 2007, 85: 1244-1250.

5 Pedersen, J., Diabetes and Pregnancy - Blood Sugar of Newborn Infants, In, Copenhagan, Danish Science Press Ltd, 1952.

6 Lindsay, R. S., Walker, J. D., Halsall, I., Hales, C. N., Calder, A. A., Hamilton, B. A. and Johnstone, F. D., Insulin and insulin propeptides at birth in offspring of diabetic mothers, J Clin Endocrinol Metab, 2003, 88: 1664-1671.

7 Lindsay, R. S., Hamilton, B. A., Calder, A. A., Johnstone, F. D. and Walker, J. D., The relation of insulin, leptin and IGF-1 to birthweight in offspring of women with type 1 diabetes, Clin Endocrinol (Oxf), 2004, 61: 353-359.

8 Metzger, B. E., Lowe, L. P., Dyer, A. R., Trimble, E. R., Chaovarindr, U., Coustan, D. R., Hadden, D. R., McCance, D. R., Hod, M., McIntyre, H. D., Oats, J. J., Persson, B., Rogers, M. S. and Sacks, D. A., Hyperglycemia and adverse pregnancy outcomes, N Engl J Med, 2008, 358: 1991-2002.

9 Metzger, B. E., Lowe, L. P., Dyer, A. R., Trimble, E. R., Sheridan, B., Hod, M., Chen, R., Yogev, Y., Coustan, D. R., Catalano, P. M., Giles, W., Lowe, J., Hadden, D. R., Persson, B. and Oats, J. J., Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study: Associations with Neonatal Anthropometrics, Diabetes, 2008.

10 Clausen, T. D., Mathiesen, E. R., Hansen, T., Pedersen, O., Jensen, D. M., Lauenborg, J. and Damm, P., High prevalence of type 2 diabetes and pre-diabetes in adult offspring of women with gestational diabetes mellitus or type 1 diabetes: the role of intrauterine hyperglycemia, Diabetes Care, 2008, 31: 340-346.

11 Pettitt, D. J., Aleck, K. A., Baird, H. R., Carraher, M. J., Bennett, P. H. and Knowler, W. C., Congenital susceptibility to NIDDM. Role of intrauterine environment, Diabetes, 1988, 37: 622-628.

12 Pettitt, D. J., Baird, H. R., Aleck, K. A., Bennett, P. H. and Knowler, W. C., Excessive obesity in offspring of Pima Indian women with diabetes during pregnancy, N Engl J Med, 1983, 308: 242-245.

13 Stride, A., Shepherd, M., Frayling, T. M., Bulman, M. P., Ellard, S. and Hattersley, A. T., Intrauterine hyperglycemia is associated with an earlier diagnosis of diabetes in HNF-1alpha gene mutation carriers, Diabetes Care, 2002, 25: 2287-2291.

14 Weiss, P. A., Scholz, H. S., Haas, J., Tamussino, K. F., Seissler, J. and Borkenstein, M. H., Long-term follow-up of infants of mothers with type 1 diabetes: evidence for hereditary and nonhereditary transmission of diabetes and precursors, Diabetes Care, 2000, 23: 905-911.

15 Lindsay, R. S., Many HAPO returns: maternal glycemia and neonatal adiposity: new insights from the Hyperglycemia and Adverse Pregnancy Outcomes (HAPO) study, Diabetes, 2009, 58: 302-303.

16 Woods, K. A., Camacho-Hubner, C., Savage, M. O. and Clark, A. J. L., Intrauterine Growth Retardation and Postnatal Growth Failure Associated with Deletion of the Insulin-Like Growth Factor I Gene, N Engl J Med, 1996, 335: 1363-1367.

17 Laviola, L., Perrini, S., Belsanti, G., Natalicchio, A., Montrone, C., Leonardini, A., Vimercati, A., Scioscia, M., Selvaggi, L., Giorgino, R., Greco, P. and Giorgino, F., Intrauterine Growth Restriction in Humans Is Associated with Abnormalities in Placental Insulin-Like Growth Factor Signaling, Endocrinology, 2005, 146: 1498-1505.

18 Kimble, R. M., Breier, B. H., Gluckman, P. D. and Harding, J. E., Enteral IGF-I enhances fetal growth and gastrointestinal development in oesophageal ligated fetal sheep, J Endocrinol, 1999, 162: 227-235.

19 Nelson, S. M., Freeman, D. J., Sattar, N. and Lindsay, R. S., Role of adiponectin in matching of fetal and placental weight in mothers with type 1 diabetes, Diabetes Care, 2008, 31: 1123-1125.

20 McMillen, I. C. and Robinson, J. S., Developmental Origins of the Metabolic Syndrome: Prediction, Plasticity, and Programming, Physiol. Rev., 2005, 85: 571-633.

21 Godfrey, K. M., The role of the placenta in fetal programming-a review, Placenta, 2002, 23 Suppl A: S20-27.

22 Ong, K., Kratzsch, J., Kiess, W. and Dunger, D., Circulating IGF-I Levels in Childhood Are Related to Both Current Body Composition and Early Postnatal Growth Rate, J Clin Endocrinol Metab, 2002, 87: 1041-1044.

23 Schubring, C., Kiess, W., Englaro, P., Rascher, W., Dotsch, J., Hanitsch, S., Attanasio, A. and Blum, W. F., Levels of Leptin in Maternal Serum, Amniotic Fluid, and Arterial and Venous Cord Blood: Relation to Neonatal and Placental Weight, J Clin Endocrinol Metab, 1997, 82: 1480-1483.

24 Tsai, P.-J., Yu, C.-H., Hsu, S.-P., Lee, Y.-H., Chiou, C.-H., Hsu, Y.-W., Ho, S.-C. and Chu, C.-H., Cord plasma concentrations of adiponectin and leptin in healthy term neonates: positive correlation with birthweight and neonatal adiposity, Clinical Endocrinology, 2004, 61: 88-93.

25 Schubring, C., Englaro, P., Siebler, T., Blum, W. F., Demirakca, T., Kratzsch, J. and Kiess, W., Longitudinal analysis of maternal serum leptin levels during pregnancy, at birth and up to six weeks after birth: relation to body mass index, skinfolds, sex steroids and umbilical cord blood leptin levels, Horm Res, 1998, 50: 276-283.

26 Schubring, C., Siebler, T., Kratzsch, J., Englaro, P., Blum, W. F., Triep, K. and Kiess, W., Leptin serum concentrations in healthy neonates within the first week of life: relation to insulin and growth hormone levels, skinfold thickness, body mass index and weight, Clin Endocrinol (Oxf), 1999, 51: 199-204.

27 Yajnik, C. S., Lubree, H. G., Rege, S. S., Naik, S. S., Deshpande, J. A., Deshpande, S. S., Joglekar, C. V. and Yudkin, J. S., Adiposity and Hyperinsulinemia in Indians Are Present at Birth, J Clin Endocrinol Metab, 2002, 87: 5575-5580.

28 Javaid, M. K., Godfrey, K. M., Taylor, P., Robinson, S. M., Crozier, S. R., Dennison, E. M., Robinson, J. S., Breier, B. R., Arden, N. K. and Cooper, C., Umbilical cord leptin predicts neonatal bone mass, Calcif Tissue Int, 2005, 76: 341-347.

29 Kayemba-Kay's, S., Geary, M. P., Pringle, J., Rodeck, C. H., Kingdom, J. C. and Hindmarsh, P. C., Gender, smoking during pregnancy and gestational age influence cord leptin concentrations in newborn infants, Eur J Endocrinol, 2008, 159: 217-224.

30 Mantzoros, C. S., Rifas-Shiman, S. L., Williams, C. J., Fargnoli, J. L., Kelesidis, T. and Gillman, M. W., Cord blood leptin and adiponectin as predictors of adiposity in children at 3 years of age: a prospective cohort study, Pediatrics, 2009, 123: 682-689.

31 Ekelund, U., Ong, K., Linne, Y., Neovius, M., Brage, S., Dunger, D. B., Wareham, N. J. and Rossner, S., Upward weight percentile crossing in infancy and early childhood independently predicts fat mass in young adults: the Stockholm Weight Development Study (SWEDES), Am J Clin Nutr, 2006, 83: 324-330.

32 Retnakaran, R., Hanley, A. J., Connelly, P. W., Harris, S. B. and Zinman, B., Elevated C-reactive protein in Native Canadian children: an ominous early complication of childhood obesity, Diabetes Obes Metab, 2006, 8: 483-491.

33 Wu, D.-M., Chu, N.-F., Shen, M.-H. and Wang, S.-C., Obesity, plasma high sensitivity c-reactive protein levels and insulin resistance status among school children in Taiwan, Clinical Biochemistry, 2006, 39: 810-815.

34 Ford, E. S., Ajani, U. A. and Mokdad, A. H., The Metabolic Syndrome and Concentrations of C-Reactive Protein Among U.S. Youth, Diabetes Care, 2005, 28: 878-881.

35 Weyer, C., Yudkin, J. S., Stehouwer, C. D., Schalkwijk, C. G., Pratley, R. E. and Tataranni, P. A., Humoral markers of inflammation and endothelial dysfunction in relation to adiposity and in vivo insulin action in Pima Indians, Atherosclerosis, 2002, 161: 233-242.

36 Couillard, C., Ruel, G., Archer, W. R., Pomerleau, S., Bergeron, J., Couture, P., Lamarche, B. and Bergeron, N., Circulating Levels of Oxidative Stress Markers and Endothelial Adhesion Molecules in Men with Abdominal Obesity, J Clin Endocrinol Metab, 2005, 90: 6454-6459.

37 Albertini, J. P., Valensi, P., Lormeau, B., Aurousseau, M. H., Ferriere, F., Attali, J. R. and Gattegno, L., Elevated concentrations of soluble E-selectin and vascular cell adhesion molecule-1 in NIDDM. Effect of intensive insulin treatment, Diabetes Care, 1998, 21: 1008-1013.

38 Leinonen, E., Hurt-Camejo, E., Wiklund, O., Hulten, L. M., Hiukka, A. and Taskinen, M. R., Insulin resistance and adiposity correlate with acute-phase reaction and soluble cell adhesion molecules in type 2 diabetes, Atherosclerosis, 2003, 166: 387-394.

39 Valle, M., Martos, R., Gascon, F., Canete, R., Zafra, M. A. and Morales, R., Low-grade systemic inflammation, hypoadiponectinemia and a high concentration of leptin are present in very young obese children, and correlate with metabolic syndrome, Diabetes Metab, 2005, 31: 55-62.

40 Cetin, I., Morpurgo, P. S., Radaelli, T., Taricco, E., Cortelazzi, D., Bellotti, M., Pardi, G. and Beck-Peccoz, P., Fetal Plasma Leptin Concentrations: Relationship with Different Intrauterine Growth Patterns from 19 Weeks to Term, Pediatr Res, 2000, 48: 646-651.

41 Radaelli, T., Uvena-Celebrezze, J., Minium, J., Huston-Presley, L., Catalano, P. and Hauguel-de Mouzon, S., Maternal Interleukin-6: Marker of Fetal Growth and Adiposity, Journal of the Society for Gynecologic Investigation, 2006, 13: 53-57.

42 Nelson, S. M., Sattar, N., Freeman, D. J., Walker, J. D. and Lindsay, R. S., Inflammation and Endothelial Activation Is Evident at Birth in Offspring of Mothers With Type 1 Diabetes, Diabetes, 2007, 56: 2697-2704.

43 Lindegaard, M. L., Svarrer, E. M., Damm, P., Mathiesen, E. R. and Nielsen, L. B., Increased LDL cholesterol and CRP in infants of mothers with type 1 diabetes, Diabetes Metab Res Rev, 2008, 24: 465-471.

44 Juonala, M., Viikari, J. S. A., Ronnemaa, T., Taittonen, L., Marniemi, J. and Raitakari, O. T., Childhood C-Reactive Protein in Predicting CRP and Carotid Intima-Media Thickness in Adulthood: The Cardiovascular Risk in Young Finns Study, Arterioscler Thromb Vasc Biol, 2006, 26: 1883-1888.

45 Wojakowski, W. and Gminski, J., Soluble ICAM-1, VCAM-1 and E-selectin in children from families with high risk of atherosclerosis, Int J Mol Med, 2001, 7: 181-185.

46 Manderson, J. G., Mullan, B., Patterson, C. C., Hadden, D. R., Traub, A. I. and McCance, D. R., Cardiovascular and metabolic abnormalities in the offspring of diabetic pregnancy, Diabetologia, 2002, 45: 991-996.

47 Nelson, S. M., Freeman, D. J., Sattar, N., Johnstone, F. D. and Lindsay, R. S., IGF-1 and leptin associate with fetal HDL-cholesterol at birth: examination in offspring of mothers with type 1 diabetes, Diabetes, 2007: db07-0585.

48 Gallin, J. I., Kaye, D. and O'Leary, W. M., Serum lipids in infection, N Engl J Med, 1969, 281: 1081-1086.

49 Freeman, D. J., Norrie, J., Caslake, M. J., Gaw, A., Ford, I., Lowe, G. D. O., O'Reilly, D. S. J., Packard, C. J. and Sattar, N., C-Reactive Protein Is an Independent Predictor of Risk for the Development of Diabetes in the West of Scotland Coronary Prevention Study, Diabetes, 2002, 51: 1596-1600.

50 Sathya, P., Martin, S. and Alvarez, F., Nonalcoholic fatty liver disease (NAFLD) in children, Curr Opin Pediatr, 2002, 14: 593-600.

51 Park, H. S., Han, J. H., Choi, K. M. and Kim, S. M., Relation between elevated serum alanine aminotransferase and metabolic syndrome in Korean adolescents, Am J Clin Nutr, 2005, 82: 1046-1051.

52 Strauss, R. S., Barlow, S. E. and Dietz, W. H., Prevalence of abnormal serum aminotransferase values in overweight and obese adolescents, J Pediatr, 2000, 136: 727-733.

53 Schwimmer, J. B., McGreal, N., Deutsch, R., Finegold, M. J. and Lavine, J. E., Influence of gender, race, and ethnicity on suspected fatty liver in obese adolescents, Pediatrics, 2005, 115: e561-565.

54 Fraser, A., Longnecker, M. P. and Lawlor, D. A., Prevalence of elevated alanine aminotransferase among US adolescents and associated factors: NHANES 1999-2004, Gastroenterology, 2007, 133: 1814-1820.

55 Schwimmer, J. B., Deutsch, R., Kahen, T., Lavine, J. E., Stanley, C. and Behling, C., Prevalence of Fatty Liver in Children and Adolescents, Pediatrics, 2006, 118: 1388-1393.

56 McCurdy, C. E., Bishop, J. M., Williams, S. M., Grayson, B. E., Smith, M. S., Friedman, J. E. and Grove, K. L., Maternal high-fat diet triggers lipotoxicity in the fetal livers of nonhuman primates, J Clin Invest, 2009, 119: 323-335.

57 Davey Smith, G., Hypponen, E., Power, C. and Lawlor, D. A., Offspring Birth Weight and Parental Mortality: Prospective Observational Study and Meta-Analysis, Am. J. Epidemiol., 2007, 166: 160-169.

58 Loughrey, C. M., Rimm, E., Heiss, G. and Rifai, N., Race and gender differences in cord blood lipoproteins, Atherosclerosis, 2000, 148: 57-65.

59 Wallace, A. M., McMahon, A. D., Packard, C. J., Kelly, A., Shepherd, J., Gaw, A. and Sattar, N., Plasma Leptin and the Risk of Cardiovascular Disease in the West of Scotland Coronary Prevention Study (WOSCOPS), Circulation, 2001, 104: 3052-3056.

60 Packard, C. J., O'Reilly, D. S. J., Caslake, M. J., McMahon, A. D., Ford, I., Cooney, J., Macphee, C. H., Suckling, K. E., Krishna, M., Wilkinson, F. E., Rumley, A., Lowe, G. D. O., Docherty, G., Burczak, J. D. and The West of Scotland Coronary Prevention Study, G., Lipoprotein-Associated Phospholipase A2 as an Independent Predictor of Coronary Heart Disease, N Engl J Med, 2000, 343: 1148-1155.