Anthropometric measurements

All participants had anthropometric assessments. Experienced researchers measured height and weight using standardized measuring methods [19]. A digital scale (graduation 100g) was used for weight measurement, and a gauge (graduation 1mm) (Seca 704, Germany) for height measurement. BMI was calculated by dividing body weight (in kilograms) by the square of height (in meters). BMI standard deviation score (BMI-SDS) was calculated according to WHO reference values. At the end of the expiratory period, the waist circumference was measured at the navel with a non-retractable soft ruler to the nearest 0.1cm [20]. Waist circumference (in centimeters) was divided by height (in centimeters) to calculate the waist to height ratio (WHtR). Body fat and skeletal muscle were measured by InBody J20 (Biospace, Korea). Percentage of body fat and percentage of skeletal muscle were calculated. After a quiet rest for 10min, blood pressure was measured three times by an electronic sphygmomanometer (Omron HBP-1300) and the mean value was taken.

Blood biochemical examination

All subjects were told to fast overnight for 12h before phlebotomy the next morning. Fasting laboratory assays included glucose, insulin, total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, alanine aminotransferase (ALT), and aspartate aminotransferase (AST).

Abdominal ultrasound examination

All subjects were examined after 12h of fasting overnight. Hepatic ultrasonography was performed by ultrasound technician to establish a diagnosis according to at least two of the following criteria: higher echogenicity higher in the liver than in the spleen or kidney; blurred hepatic vasculature; high attenuation of signals [21].

Definitions for metabolic syndrome and cardiovascular risk factors

The diagnosis of metabolic syndrome was defined as having at least three criteria with the following cut points [22]: abdominal obesity (waist circumference≥90th percentile of waist circumference of children of the same age and gender [23], elevated triglycerides (≥1.47mmol/L [130mg/dL]) [24], low HDL cholesterol (<1.03mmol/L [40mg/dL]), elevated blood pressure [systolic blood pressure (SBP) ≥90th percentile of SBP or diastolic blood pressure (DBP) ≥90th percentile of DBP of children of the same age and height and gender], and impaired fasting glucose (≥5.6mmol/L [100mg/dL]). In addition to the factors of metabolic syndrome, insulin resistance was defined according to homeostasis model assessment of insulin resistance (HOMA-IR) which was calculated as fasting insulin (mU/mL)×[fasting glucose (mmol/L)/22.5] [25]. The insulin resistance threshold was defined as ≥3.16, as described in the literature [26]. Hyperuricemia was defined as uric acid value ≥357 umol /L [27].

Comparison of biochemical parameters in children with obesity with or without NAFLD

The biochemical parameters are shown in Table 2. Children in the NASH group had higher hepatic enzymes including ALT and AST, indicating liver injury. The triglyceride levels were significantly higher in the NASH and SS groups compared with the SOB group. The uric acid concentration and HOMA-IR levels involved in the pathogenesis of NAFLD were ranked as follows: SOB < SS<NASH.

Table 2

Comparison of biochemical parameters between SOB and NAFLD

Groups	SOB	NAFLD		F/Z	p-value
		SS	NASH
ALT (U/L)	17 (9)ab	26 (21)a	106 (41)	161.92	0.001
AST (U/L)	22 (8)ab	26 (14)a	62 (26)	112.41	0.001
Uric acid (μmol/L)	352 (96)ab	406 (124)a	455 (141)	49.96	0.001
Triglycerides (mmol/L)	1.05 (0.62)ab	1.37 (0.91)	1.31 (0.90)	16.83	0.001
Cholesterol (mmol/L)	4.25±0.99a	4.33±0.89	4.59±0.88	2.29	0.103
HDL (mmol/L)	1.27 (0.37)b	1.17 (0.36)	1.18 (0.30)	18.23	0.001
Fasting glucose (mmol/L)	4.73±0.44b	4.61±0.48	4.61±0.59	3.08	0.047
Fasting insulin (mU/L)	12.71 (12.08)a	15.01 (14.39)a	21.30 (7.60)	12.39	0.002
HOMA-IR	2.77 (2.53)a	3.06 (3.00)a	4.2 (4.61)	10.39	0.006

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vs. NASH group, ^a p<0.001; vs. SS group, ^b p<0.001. Normal distribution data are expressed as the mean±SD. Non-normal distribution data are presented as median (interquartile)

SOB Simple Obese, NAFLD Nonalcoholic Fatty Liver Disease, SS Simple Steatosis, NASH Nonalcoholic Fatty Hepatitis, ALT Alanine aminotransferase, AST Aspartate aminotransferase, HDL High-density lipoprotein, HOMA-IR Homeostasis model assessment of insulin resistance

Prevalence of metabolic syndrome and cardiovascular risk factors in NAFLD compared with children without NAFLD

The occurrence rates of metabolic syndrome in the NASH and SS groups were significantly higher than in the SOB group (53.3, 49.8% vs 19.6%, p<0.001). Children with NASH had significantly elevated blood pressure and triglycerides compared with children in the SOB group, but there was no difference in the frequency of abdominal obesity, low HDL and impaired fasting glucose when dichotomous cut points were used among groups (Table 3).

Table 3

Comparisons of metabolic risk factors between obese children with NAFLD and those without liver disorder

Factors	SOB (N=148)	NAFLD		χ2	p-value
		SS (N=235)	NASH (N=45)
Abdominal obesity
Waist circumference (cm)≥p90, %	144 (97.3%)	234 (99.6%)	45 (100.0%)	4.67	0.097
Elevated blood pressure
SBP or DBP≥p90, %	62 (41.9%)a	146 (62.1%)	30 (66.7%)	17.56	0.001
Dyslipidemia
Triglycerides (mmol/L)≥1.47mmol/L, %	57 (38.5%)ab	133 (56.6%)	27 (60.%)	13.44	0.001
HDL (mmol/L)<1.03mmol/L, %	15 (10.1%)b	65 (27.7%)	8 (17.8%)	17.63	0.001
Impaired fasting glucose
Fasting Glucose ≥5.6mmol/L, %	2 (1.4%)	6 (2.6%)	2 (4.4%)	1.55	0.460
Insulin resistance
HOMA-IR (≥3.16), %	57 (38.5%)ab	110 (46.8%)a	31 (68.9%)	12.87	0.002
Hyperuricemia
Uric Acid (umol/L)≥357umol/L, %	68 (45.9%)ab	161 (68.5%)a	39 (86.7%)	32.63	0.001
Metabolic syndrome, %	29 (19.6%)ab	117 (49.8%)	24 (53.3%)	38.47	0.001

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vs. NASH group, ^a p<0.001; vs. SS group, ^b p<0.001

SOB Simple Obese, NAFLD Nonalcoholic Fatty Liver Disease, SS Simple Steatosis, NASH Nonalcoholic Fatty Hepatitis, SBP Systolic blood pressure, DBP Diastolic blood pressure, HDL High-density lipoprotein, HOMA-IR Homeostasis model assessment of insulin resistance

Further investigation indicated that the prevalence of insulin resistance (38.5% / 46.8% / 68.9%) and hyperuricemia (45.9% / 68.5% /86.7%) were steadily higher from the SOB group to the SS group to the NASH group, respectively (p<0.001). The progression of NAFLD was positively associated with cardiovascular risk factors and metabolic syndrome in these children with obesity. As shown in Fig. 1, all children in the SS and NASH groups had at least one metabolic syndrome factor. Moreover, the distribution of metabolic syndrome features in children in the NASH and SS groups was significantly (p<0.001) shifted to the right, with more features present than in children with obesity in the SOB group.

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Fig. 1

Features of metabolic syndrome in the three groups. Obese children with NASH or SS have significantly more features of metabolic syndrome compared to SOB (p<0.001)

NAFLD, cardiovascular risk and metabolic syndrome

In the present study, the NAFLD children showed higher cardiovascular risk and more metabolic syndromes than non-NAFLD children with obesity. This result is consistent to that reported in a previous case-control analysis [11]. The NAFLD children were more likely to develop abnormal glucose, insulin, triglycerides and HDL cholesterol than non-NAFLD children with matched age, sex and BMI [11]. A study covering 400 obese children also revealed that abnormal hepatic echo was associated with systolic and diastolic dysfunction [29]. Metabolic syndrome is a collection of cardiovascular risk factors that can predict diabetes and cardiovascular disease better than any single component. More and more evidence has shown that a large proportion of children with NAFLD meet the diagnostic standard of metabolic syndrome, suggesting that these children will develop diabetes and cardiovascular diseases in the future. This belief was supported by a 14-year-long Swedish cohort study of NAFLD patients diagnosed by biopsies. There was a 9% prevalence of diabetes at baseline, and most patients (78%) had developed impaired glucose tolerance or diabetes by the end of the 14-year period [18]. In the same study, the survival of NAFLD adults diagnosed by biopsy was lower than that of a matched control group, largely due to higher cardiovascular mortality [18]. Therefore, health care workers should pay more attention to the early identification of cardiovascular risk factors in the NAFLD population, which is helpful to facilitate targeted prevention and treatment strategies.

NAFLD and insulin resistance

We found that the risk of insulin resistance in the NASH group was significantly higher than that in the SS and SOB groups, and insulin resistance was a risk factor for NASH after adjusting for confounding factors. Many clinical studies suggest that insulin resistance is always associated with NAFLD in adults and children [30–33]. A biopsy-proven NAFLD study showed that 95% of children had insulin resistance [34]. In an experimental study, Bugianesi et al. found that adipose tissue is an important site for the development of insulin resistance, which can contribute to NAFLD [35]. However, the association between insulin resistance and NASH remains unclear. The pathogenesis of NAFLD is hepatocyte steatosis and hepatocyte injury, in which insulin resistance plays an important role in the development of NAFLD. As peripheral insulin resistance arouses in NAFLD patients, serum-free fatty acids, synthesized through the lipolysis of visceral fatty tissue and dietary fat, serve as the origin of hepatic triglycerides. Insulin resistance acts in metabolic disorders induced by free fatty acids and mitochondria. Lipid overload can also repress oxidative performances. Meanwhile, with the production of reactive oxygen species, cytokines are induced, inflammatory cells chemoattracted, and hepatic stellate cells activated, all leading to damage to hepatocytes [36]. Therefore, insulin resistance is involved in the occurrence and development of NAFLD and NASH. On account of the central role of insulin resistance in glucose metabolism, youth diagnosed with fatty liver need further evaluation to exclude diabetes or impaired glucose tolerance due to insulin resistance.

NAFLD and uric acid

A growing number of clinical studies have shown that hyperuricemia is related to NAFLD in both adults and children [37–39]. In a recent study of adolescents, hyperuricemia independently predicted [OR 2.5, 95% CI (1.87–2.83)] the presence of NASH after adjustment for potential confounders [40]. A biopsy-proven NAFLD study also found that the higher the serum uric acid, the greater the steatosis grade and lobular inflammation [41]. Similar to previous studies, we confirmed that obese patients with hyperuricemia had a higher risk of NASH occurrence [OR 2.98, 95% CI (1.14–7.76)]. Experimental studies observed that incubation of HepG2 cells with uric acid can increase intracellular triglycerides. The potential mechanisms may be that uric acid activates mitochondrial stress, subsequently activates endoplasmic reticulum stress and activates sterol regulatory element-binding protein 1C, while uric acid can stimulate the NOD-like receptor family pyrin domain containing 3 inflammasomes to regulate hepatic steatosis and inflammatory response in NAFLD patients [42, 43].

We are very grateful for the support from technicians in the clinical laboratory and ultrasound department of the Children’s Hospital of Nanjing Medical University. We also thank the children and parents for their participation.