Profile
International Journal of Pediatrics & Neonatal Care Volume 1 (2015), Article ID 1:IJPNC-110, 4 pages
http://dx.doi.org/10.15344/2455-2364/2015/110
Research Article
Type 4 Retinol Binding Protein as a Marker of Hepatic Steatosis in Children and Adolescents with Type 2 Diabetes

Ana Carolina Hill de Tito1, Hanna Marrodán García1, Miguel Klünder Klünder2, María Teresa Valadez Reyes3, Fengyang Huang4, Mario Molina Díaz1, Lubia Velázquez López5 and Patricia Medina Bravo1*

1Department of Endocrinology, Hospital Infantil de México Federico Gómez, Mexico
2Department of Community Health Research, Hospital Infantil de México Federico Gómez, Mexico
3Department of Radiology, Hospital Infantil de México Federico Gómez, Mexico
4Department of Pharmacology, Hospital Infantil de México Federico Gómez, Mexico
5Clinical Epidemiology Research Unit, Hospital Regional General No. 1 Carlos McGregor Sánchez-Navarro, Instituto Mexicano del Seguro Social, Mexico
Dr. Patricia Medina Bravo, Endocrinology Department, Hospital Infantil de México Federico Gómez, Dr. Márquez No. 162, Colonia Doctores. Delegación Cuauhtémoc, C. P. 06720, Mexico, Tel: +5252289917; E-mail: drapatty_75@hotmail.com
24 March 2015; 20 November 2015; 24 November 2015
de Tito ACH, García HM, Klünder MK, Reyes MTV, Huang F, et al. (2015) Type 4 Retinol Binding Protein as a Marker of Hepatic Steatosis in Children and Adolescents with Type 2 Diabetes. Int J Pediatr Neonat Care 1: 110. doi: http://dx.doi.org/10.15344/2455-2364/2015/110

Abstract

Background: Type 4 retinol binding protein (RBP4) is a novel adipocytokine that has been related with insulin resistance (IR). In pediatric population, RBP4 levels are higher in obese subjects compared with lean subjects. In adults with type 2 diabetes (T2D) a positive correlation has been found between RBP4 levels and hepatic steatosis (HS). There is scarce information on RBP4 levels in children and adolescents with T2D. The aim of our study was to evaluate RBP4 levels in T2D adolescents and its association to HS.
Methods: 34 T2D adolescents were included. Fasting glucose, glicated hemoglobin (HBA1c), lipid profile, and RBP4 levels were determined. The degree of HS was evaluated by hepatic Doppler-ultrasound. Results: Of the 34 patients, 64.7% were female. Mean age was 14.37±2.27 years and HbA1c was 9.27± 3.27%. 33.3% did not have steatosis (NHS), 41.7% had mild HS (MHS) and 25% had severe HS (SHS). No differences were found between the 3 groups in age, weight, BMI, waist, years of DM2, and Tanner stage. ALT, AST and RBP4 concentrations were higher in patients with SHS compared with those with MHS and NHS (p=0.04). There was a positive correlation between HS and RBP4 levels (r=0.487, p=0.043).
Conclusion: In children and adolescents with T2D RBP4 levels are associated with the degree of HS. This adipocytokine could be used as an HS serum marker.


1. Introduction

Type 2 diabetes (T2D) in children and adolescents is emerging as a health problem worldwide due to the global epidemic of obesity, accounting for as many as 46% of new cases in Latin populations [1]. In the majority of these cases, chronic complications can be detected since its diagnosis, such as hypertension, nephropathy, neuropathy or dyslipidemia [2]. Hepatic steatosis (HS) and non-alcoholic steatohepatitis (NASH), is seldom searched for in these patients, although it has been reported in up to 78% of adults with T2D [3] and it is considered the most common liver pathology in adults and children [4].

The gold-standard for the diagnosis of HS is liver biopsy, but this is a costly procedure that is not exempt of possible severe complications. This is why, in the last decade many other non-invasive diagnostic methods have been used instead. Some imaging modalities include ultrasound, computed tomography or magnetic resonance imaging, have been used with excellent results [5]. The hepatic ultrasound has reported a 100% sensitivity in detecting more than 33% liver fat and a positive predictive value of 62%. Other authors have used different serum markers and biochemical scales to diagnosis HS, such as the SteatoTest and fatty liver index [6].

RBP4 is a member of the lipocalin family of proteins with 201 amino acids. It is mainly expressed in liver and adipose tissue, and it transports retinol and binds to transthyretin, which prevents its renal excretion [7]. Studies in humans have linked this molecule with obesity and insulin resistant (IR) states, such as the metabolic syndrome (MS) or T2D [8]. Its also been associated with increased levels of visceral fat [9]. In pediatric population, RBP4 is elevated in obese patients, and it showed a positive correlation with body mass index (BMI), higher visceral fat, different components of the MS and some serum markers of systemic inflammation [10]. In recent years, the relationship between this molecule and HS has been evaluated, finding positive correlations with increased liver enzymes in several studies [11]. RBP4 levels are also increased in, obese and non obese T2D patients, compared with non-diabetic weight matched controls [12,13].

There is scarce information with regard to the presence of HS in T2D in pediatric population, and to the expression of this adipcytokine in these patients. The aim of this study was to identify the frequency of HS in T2D children and adolescents and evaluate the RBP4 serum levels, and its relationship to HS.

2. Materials & Method

A cross sectional study was conducted in 34 T2D children and adolescents aged 8 –18 years attending the outpatient diabetic clinic at the Hospital Infantil de Mexico Federico Gomez (Mexico City, Mexico). All patients were diagnosed according to the American Diabetes Association criteria [14,15], and were on multiple dose dietary treatment according to age, BMI and Tanner stage and either metformin (41%) or insulin (24%), or a combination of both (35%). Exclusion criteria were evidence of thyroid dysfunction, hepatic, renal or liver disease, chronic or acute infections, micro or macrovascular complications. The patients were divided into 3 groups according to the degree of HS on ultrasound for their analysis: No HS (NHS) (n=13), mild HS (MHS) (n=15) and severe HS (SHS) (n=6) patients. Parental informed written consent and child assent were obtained from all participants. The protocol was approved by the Institutional Review Board of the Hospital Infantil de Mexico Federico Gomez.

3. Measurements

All patients with help from their parents completed a brief questionnaire regarding age, sex, age at diagnosis, pharmacologic treatment and presence of complications or other diseases. Weight, height, body mass index (BMI), waist circumference, blood pressure and Tanner stage was evaluated by a trained pediatric endocrinologist. A blood sample was taken, after an overnight fast, to determine glucose, glycosilated hemoglobin A1c (HbA1c), total cholesterol, high density lipoprotein cholesterol (HDL-c), low density lipoprotein cholesterol (LDL-c), triglycerides, hepatic enzymes (ALT, AST and GGT) and RBP4 levels.

Serum glucose, ALT, AST and GGT concentrations as well as HbA1c, total cholesterol, HDL-c and triglycerides were measured with the Siemens Dimension RXLMAX System (Siemens Healthcare Diagnostics). The LDL-c levels were calculated using the Friedewald formula modified by De Long. RBP4 levels were measured by ELISA (Millipore Linco-Research kit). Coefficient of variation for this determination was 7.7%.

To evaluate the presence of HS, an hepatic Doppler ultrasound (Siemens, 5.5Hz) was performed to all patients by a trained pediatric radiologist, taking into consideration 3 aspects to determine the degree of hepatic steatosis: 1) echo levels of liver and kidney parenchyma, 2) echo penetration into liver (diaphragm visibility) and 3) clarity of liver blood vessel structures (veins) [modified form Tominaga 1995] [ 16]. Each of these was given a value from 0 to 3, zero being of normal characteristics, and 3 being completely abnormal. The 3 values were added and the degree of HS was determined by the total score in this manner: 0: NHS, 1-3:MHS and >4:SHS, and thus patients categorized into the 3 different groups.

4. Data Analyses

Data are expressed as mean ± SD or median (maximum and minimum value) if the data were skewed distributed. Statistical analyses were performed using analysis of variance or Kruskall Wallis test for comparison. Pearson correlation test was used to examine relationships between variables. All analyses were conducted using the Stata SE V. 11. All p-values≤0.05 were considered statistically significant.

5. Results and Discussion

Clinical and antrophometric features of the 3 groups are depicted in Table 1. No differences were found between the 3 groups in age, age at diagnosis, weight, height, BMI waist circumference (WC), blood pressure and Tanner stage.

table 1
Table 1: Clinical and antrophometric features in patients with T2D.

With regards to the level of disease control, the HbA1c levels and lipid profile were similar between the 3 groups (p>0.05). There were a significant increase in the levels of ALT and AST in the SHS (p=0.002 and p=0.001 respectively). The levels of GGT did not have a statistical difference, but the level in patients with SHS was twice as high as the other 2 groups (Table 2). RBP4 levels were also higher in the SHS group (p=0.04). RBP4 showed a positive correlation with HS (r=0.487, p=0.043) and ALT levels (r=0.367, p=0.035). Table 3.

table 2
Table 2: Biochemical characteristics in patients with T2D.
table 3
Table 3: Correlation analyses between RBP4 and biochemical variables.

The prevalence of HS in T2D adult patients varies widely, ranging from 21 to 78%, and the presence of diabetes is a strong independent predictor of progression to fibrosis [17]. There are very few reports concerning to the presence of this disease in children and adolescents with T2D. Dean and Sellers [18] studied 49 patients, from 8 to 18 years of age, and reported that 44% had elevation in ALT, AST or both. Nadeau, et al [19] reported elevation in ALT in 48% of 115 adolescent patients with T2D. In our study the frequency of HS was 61%, with 17% showing signs of SHS in the ultrasound. Only 20% had high levels of ALT, and non were higher than twice the normal value. This data shows the presence of HS in many patients with normal levels of liver enzymes, and could indicate the need for an earlier detection, either by ultrasound or with some other serum marker, to avoid the progression to steatohepatitis.

In our study we found that RBP4 correlates with the degree of HS in children and adolescents with T2D. These results are in agreement to those published by Romanowska, et al [13], who found elevation of this adipocytokine in obese pediatric patients with NASH compared with normal weight controls, and who also found a positive correlation between RBP4 levels and degree of HS by ultrasound (r=0.45;P=0.05). Shaker O et al [12] published data on RBP4 in T2D adult patients, and also found a positive correlation between fatty liver and RBP4 (r=0.7, P=0.001). We also found a positive correlation between ALT and RBP4. Conroy et al [10], also found this relationship in pediatric nondiabetic population. They suggest that RBP4 could be used as an early serum marker of HS, as do we. Our study has some potential limitations. First, its cross-sectional nature does not allow us to establish a causal relationship between RBP4 levels and HS. Second, our small sample size. Nevertheless, to our knowledge, no previous information exists regarding the behavior of in adolescents with T2D and its association to HS.

6. Conclusion

In conclusion, we think that this adipocytokine could be a useful marker of HS not only in obese pediatric patients, but also in pediatric patients with T2D.

Competing Interests

The authors declare that they have no competing interests.

Author Contributions

PM, ACHT, LV conceived of the study, participated in its design and coordination. PM, MTV, MK, HM, MM, FH performed the statistical analysis and revised the manuscript critically. All authors were involved in drafting the manuscript. All of them read and approved the final version of the manuscript.


References

  1. Writing Group for the SEARCH for Diabetes in Youth Study Group1, Dabelea D, Bell RA, D'Agostino RB Jr, Imperatore G, et al. (2007) Incidence of diabetes in youth in the United States. JAMA 297: 2716-2724 [CrossRef] [Google Scholar] [PubMed]
  2. Pinhas-Hamiel O, Zeitler P (2007) Acute and chronic complications of type 2 diabetes mellitus in children and adolescents. Lancet 369: 1823-1831 [CrossRef] [Google Scholar] [PubMed]
  3. Clark JM, Diehl AM (2002) Hepatic steatosis and type 2 diabetes mellitus. Curr Diab Rep 2: 210-215 [Google Scholar] [PubMed]
  4. Loomba R, Sirlin CB, Schwimmer JB, Lavine JE (2009) Advances in pediatric nonalcoholic fatty liver disease. Hepatology 50: 1282-1293 [CrossRef] [Google Scholar] [PubMed]
  5. Saadeh S, Younossi ZM, Remer EM, Gramlich T, Ong JP, et al. (2002) The utility of radiological imaging in nonalcoholic fatty liver disease. Gastroenterology 123: 745-750 [CrossRef] [Google Scholar] [PubMed]
  6. Guiu B, Crevisy-Girod E, Binquet C, Duvillard L, Masson D, et al. (2012) Prediction for steatosis in type-2 diabetes: clinico-biological markers versus 1H-MR spectroscopy. Eur Radiol 22: 855-863 [CrossRef] [Google Scholar] [PubMed]
  7. Kotnik P, Fischer-Posovszky P, Wabitsch M (2011) RBP4: a controversial adipokine. Eur J Endocrinol 165: 703-711 [CrossRef] [Google Scholar] [PubMed]
  8. Esteve E, Ricart W, Fernández-Real JM (2009) Adipocytokines and insulin resistance: the possible role of lipocalin-2, retinol binding protein-4, and adiponectin. Diabetes Care 32 Suppl 2: S362-367 [CrossRef] [Google Scholar] [PubMed]
  9. Kelly KR, Kashyap SR, O'Leary VB, Major J, Schauer PR, (2010) Retinolbinding protein 4 (RBP4) protein expression is increased in omental adipose tissue of severely obese patients. Obesity 18: 663-666 [CrossRef] [Google Scholar] [PubMed]
  10. Conroy R, Espinal Y, Fennoy I, Accacha S, Boucher-Berry C, et al. (2011) Retinol binding protein 4 is associated with adiposity-related co-morbidity risk factors in children. J Pediatr Endocrinol Metab 24: 913-919 [CrossRef] [Google Scholar] [PubMed]
  11. Huang SC, Yang YJ (2013) Serum retinol-binding protein 4 is independently associated with pediatric NAFLD and fasting triglyceride level. J Pediatr Gastroenterol Nutr 56: 145-150 [CrossRef] [Google Scholar] [PubMed]
  12. Shaker O, El-Shehaby A, Zakaria A, Mostafa N, Talaat S, et al. (2011) Plasma visfatin and retinol binding protein-4 levels in patients with type 2 diabetes mellitus and their relationship to adiposity and fatty liver. Clin Biochem 44: 1457-1463 [CrossRef] [Google Scholar] [PubMed]
  13. Romanowska A, Lebensztejn DM, Skiba E, Tarasów E, Kaczmarski M (2011) Retinol binding protein-4 as a serum biomarker of intrahepatic lipid content in obese children--preliminary report. Acta Biochim Pol 58: 35-38 [CrossRef] [Google Scholar] [PubMed]
  14. Angulo P, Keach JC, Batts KP, Lindor KD (1999) Independent predictors of liver fibrosis in patients with nonalcoholic steatohepatitis. Hepatology 30: 1356-1362 [CrossRef] [Google Scholar] [PubMed]
  15. American Diabetes Association (2012) Standards of medical care in diabetes--2012. Diabetes Care 35 Suppl 1: S11-63 [CrossRef] [Google Scholar] [PubMed]
  16. MacAllister RJ, Parry H, Kimoto M, Ogawa T, Russell RJ, et al. (1996) Regulation of nitric oxide synthesis by dimethylarginine dimethylaminohydrolase. Br J Pharmacol 119: 1533-1540 [CrossRef] [Google Scholar] [PubMed]
  17. Tominaga K, Kurata JH, Chen YK, Fujimoto E, Miyagawa S, et al. (1995) Prevalence of fatty liver in Japanese children and relationship to obesity. An epidemiological ultrasonographic survey. Dig Dis Sci 40: 2002-2009 [CrossRef] [Google Scholar] [PubMed]
  18. Dean H, Sellers E (2001) Steatohepatitis in children with type 2 diabetes mellitus. Diabetes 2001 50: A378 [Google Scholar]
  19. Nadeau KJ, Klingensmith G, Zeitler P (2005) Type 2 diabetes in children is frequently associated with elevated alanine aminotransferase. J Pediatr Gastroenterol Nutr 41: 94-98 [CrossRef] [Google Scholar] [PubMed]