Nonalcoholic steatohepatitis is associated with an atherogenic lipoprotein subfraction profile

Cardiovascular disease (CVD) is prevalent in individuals with nonalcoholic fatty liver disease (NAFLD). Three community-based studies from the United States and Japan have demonstrated an increased CVD risk in individuals with radiographic evidence of NAFLD when compared to those without NAFLD [1–3]. Individuals with histologically defined nonalcoholic steatohepatitis (NASH) have also been shown to have an increased CVD mortality when compared to the general population [4, 5]. Thus, individuals with both radiographic NAFLD and histologically defined NASH are at increased risk for CVD and CVD-related mortality.

Multiple factors likely contribute to the increased CVD risk seen in those with NASH including hyperinsulinemia, impaired thrombolysis and alterations in lipid and lipoprotein metabolism. To date studies of lipids in NASH have been largely limited to standard lipid profiles which include total cholesterol, triglycerides, high density lipoproteins (HDL) cholesterol and a calculated, rather than direct, measurement of low density lipoprotein (LDL) cholesterol. While these parameters are accepted markers of CVD risk, it has long been recognized that these measures provide only a crude index of disease pathophysiology and recent evidence shows that additional information can be gained from measurements of lipoprotein particles and their subfractions [6–8].

Ion Mobility (IM) is an innovative method that uses gas-phase differential electrophoretic macromolecular mobility to distinguish lipoprotein particles as a function of their size and to directly and rapidly quantify their plasma levels [9, 10]. We hypothesized that IM would reveal important differences in the lipoprotein subfraction profiles of individuals with obesity with and without NASH that may contribute to the increased CVD seen in NASH.

This cohort study included consecutively enrolled patients who underwent weight loss surgery (WLS) at the Bon Secours Health System between 2010 and 2012. Subjects were assessed by a treating clinician for weight, height, BMI and co-morbidities. Diabetes mellitus was defined by a fasting glucose ≥ 126 mg/dL, HbA1C >6.5% or known diagnosis of diabetes. Hypertension was defined by a blood pressure ≥135/85 or undergoing treatment for hypertension. Individuals on lipid lowering therapy were excluded. No individual experienced significant weight loss prior to surgery.

All subjects undergoing WLS had a standard of care liver biopsy at the time of surgery. Liver biopsies were reviewed by single blinded hepatopathologist (JM) and assigned a score for grade of steatosis (grade 0 = <5% steatosis; 1 = 5-33%; 2 = 33-66%; 3 = >66%), hepatocyte ballooning (0 = no ballooning; 1 = few; 2 = many), and lobular inflammation per 200 × (0 = no foci; 1 = <2 foci; 2 = 2–4 foci; 3 = >4 foci), as described by Kleiner et al. [11] NAFLD activity score (NAS) is a sum of the scores for steatosis grade, lobular inflammation and hepatocyte ballooning and ranges from 0–8. Fibrosis stage was assigned according to the modified Brunt stage (stage 0, 1a, 1b, 1c, 2, 3, or 4) [11]. Non-NASH was defined by the presence of grade 0 or greater not meeting criteria for NASH. NASH was defined as lobular inflammation > =1, hepatocyte ballooning > =1 and steatosis grade > =1 [12].

The present study reveals several important novel differences in the lipid profiles on patients with and without NASH. Patients with NASH were found to have a significantly smaller LDL peak diameter than those without NASH. In addition, we found that LDL-IVb was significantly higher in those with NASH. LDL-IVb is the smallest subclass of LDL particles with a diameter of 22.0-23.3 nm. LDL-IVb represents a relatively minor subfraction of LDL, accounting for approximately 10% of LDL particles. However, a study by Williams et al. found that LDL-IVb was the single best predictor among lipoproteins of increased coronary artery stenosis [13]. Stenosis change was six times more rapid among individuals with LDL-IVb levels in the highest quartile than those with the lowest LDL-IVb levels. This study suggested that a considerable portion of CVD risk attributed to small dense LDL may be driven by very small LDL particles. Thus, our finding of increased LDL-IVb levels in NASH patients may contribute to the increased CVD experienced by this group.

HDL is composed of 5 subclasses including the small in size HDL3a, HDL3b, HDL3c, as well as the large HDL2a and HDL2b [13]. Decreased HDL2b content relative to HDL3b and HDL3c is associated with an increased CVD risk [14, 15]. We found that, in individuals with NASH, HDL2b was significantly lower in those without NASH and it is possible that this is related to the increased CVD seen in NASH.

Several important limitations in the present study must be acknowledged. Our group included all individuals undergoing weight loss surgery and thus the findings may not be generalizable to the full spectrum of NAFLD and NASH patients. Finally, our study is limited by small size and predominance of white women. Future studies of increased size and diversity as well as evaluation of lipoprotein particle differences in patients with normal liver histology compared to those with steatosis and NASH are needed.

In conclusion, the present study suggests that individuals with NASH may have a more atherogenic lipoprotein subfraction profile than those without fatty liver disease.