Association of serum omentin levels with cardiac autonomic neuropathy in patients with type 2 diabetes mellitus: a hospital-based study

Patients

We recruited 146 patients with diabetes who were evaluated for microvascular complications
at the diabetes clinic of Soonchunhyang University Bucheon Hospital from April 2012
to February 2014. Among them, those with type 1 diabetes, and those with severe illness,
such as heart failure, liver cirrhosis with ascites, severe infection, malignancy,
uncontrolled hyperthyroidism or a history of arrhythmia, and were not available fasting
serum samples or clinical data from medical records were excluded. Finally, this study
included 97 patients with T2DM (men 56, women 41, mean age: 57.6 years). We reviewed
detailed demographic data, biochemical data, and clinical and treatment histories
from patient medical records. The smoking status of the subjects was classified as
being a non-smokers or smokers (former or current). All patients were informed of
the purpose of the study and written consent was obtained. The study was approved
by the Institutional Review Board of Soonchunhyang University School of Medicine,
Bucheon Hospital.

Measurement of serum omentin

Blood samples were taken after overnight fasting. Serum was separated and stored at
?80 °C until analyzed. Serum omentin levels were measured using a commercially available
enzyme-linked immunosorbent assay (ELISA; RD Systems, Minneapolis, MN, USA). Serum
insulin and C-peptide levels were measured by radioimmunoassay (Immunotech, Prague,
Czech Republic).

Cardiac autonomic neuropathy and microvascular complications

CAN was assessed by an autonomic function test (AFT). CAN was assessed by five standard
cardiovascular reflex tests according to Ewing’s protocol 18]. The patients were asked to fast for 12 h before the test, and to avoid taking antihypertensive
agent such as beta-blocker, caffeine, nicotine, or antihistamines. Three of these
measurements mainly assess parasympathetic function : heart rate responses to deep
breathing (beat-to-beat variation), to standing (30:15 ratio), and to the Valsalva
maneuver. The other two tests mainly assess sympathetic function: blood pressure responses
to standing and to a sustained handgrip. The heart rate response to deep breathing,
standing, and the Valsalva maneuver were assessed automatically from ECG recordings
using the DICAN evaluation system (Medicore Co. Ltd, Korea). The results of each of
the above five tests for the detection of CAN were classified into one of three categories
based on the severity of abnormality detected, and each of them was given a definite
point value as described by Bellavere et al. 19]. The severity of CAN was quantified from the summation of the points obtained from
each of the five tests, where each test was given a point value of 0, 0.5, or 1 if
it yielded normal, borderline, or abnormal values, respectively. Consequently, the
minimum and maximum numbers of autonomic neuropathy points were 0 and 5, respectively.
CAN was defined as the presence of at least two abnormal tests or an autonomic neuropathy
points total ?2.

Diabetic nephropathy (DN) was defined using albuminuria, which was measured by radioimmunoassay
(Immunotech). An albumin excretion rate (AER) 20 ?g/min or urine albumin 30 mg/g
creatinine were the criteria for normoalbuminuria; an AER in the range of 20–200 ?g/min
or urine albumin 30–300 mg/g creatinine, microalbuminuria; and an AER ?200 ?g/min
or urine albumin ?300 mg/g creatinine as overt proteinuria. Patients were considered
to have nephropathy if they had microalbuminuria or overt proteinuria.

Diabetic retinopathy (DR) was evaluated by experienced ophthalmologists while the
patients’ pupils were dilated. If needed, fluorescein angiography was performed. Patients
were classified as normal, or as having non-proliferative or proliferative retinopathy;
patients were considered to have retinopathy if they were in the non-proliferative
or proliferative stage.

Diabetic peripheral neuropathy (DPN) was diagnosed in subjects displaying two or more
of the following features: typical subjective neuropathic symptoms determined using
the Michigan Neuropathy Screening Instrument (MNSI), insensitivity to a 10-g monofilament,
abnormal pin-prick sensation, and abnormal current perception threshold (CPT), or
a nerve conduction study. CPT was performed with a Neurometer CPT/C (Neurotron, Inc.,
Baltimore, MD, USA).

An automated device (VP-1000; Colin, Japan) was used to measure arterial PWV and the
ABI. Insulin resistance status was evaluated by the homeostasis model assessment-insulin
resistance (HOMA-IR) index. The HOMA-IR was calculated using the formulae: [fasting
insulin (uIU/mL) × fasting blood glucose (mmol/L)]/22.5. The HOMA-IR score was available
in only 89 patients not receiving exogenous insulin.

Statistical analysis

Statistical analysis was performed using SPSS 14.0 (SPSS Inc, Chicago, IL, USA). Data
are reported as mean ± standard deviation (SD) for normally distributed variables,
as median (interquartile range) for non-normally distributed variables or as number
of participants (percentages). Statistical differences in demographic and clinical
characteristics between groups by gender were evaluated by means of Chi square test
or Fisher’s exact test for categorical variables and Student’s t test or Wilcoxon’s ranksum test for continuous variables. Before the t test, Shapiro–Wilk
test for normality and Levene’s homogeneity of variance test was conducted. Non-normally
distributed variables, that is, omentin, hsCRP, triglyceride and HOMA-IR, were natural-logarithm-transformed
before analysis. The significance of the mean differences in parameters among the
tertiles of omentin levels was evaluated with one-way ANOVA. Post-hoc comparison for
tertiles of omentin groups was performed with the adjusted p-value using Tukey’s HSD
method.

The correlation of serum omentin levels and other clinical variables was assessed
by Pearson’s correlation coefficient or Spearman’s rank correlation as appropriate.
In addition, partial correlations were computed after adjustment for age, mean PWV,
and CAN points. To determine the particular aspects of anthropometric, biochemical,
and metabolic parameters that are related to CAN point, simple linear regression was
conducted for each explanatory variable. On the result of simple linear regression,
multiple linear regression analyses were performed to check the original relationship
of each variable and CAN point when the other variables were adjusted. The significance
of the relationship between CAN point and each explanatory variable was evaluated
with a t-test for each regression coefficient. An F test was used to test the significance
of the proportion of variance in CAN point (R
²
) that was explained in the model, the regression model which included the subset
of explanatory variables that were at least marginally significant (p  0.15).