Serum Carnitine Level and Its Associated Factors in Patients With Chronic Viral Hepatitis

Azin Nassiri; Simin Dashti-Khavidaki; Hossein Khalili*; Mohsen Nassiri-Toosi; Alireza Abdollahi

Disclosures

Future Virology. 2014;9(4):373-383. 

In This Article

Methods

Participants

This cross-sectional case–control study was performed in patients with chronic viral hepatitis (HBV and/or HCV) who were referred to the hepatitis outpatient clinic of Imam-Khomeini Hospital Complex affiliated to Tehran University of Medical Sciences, Iran, during a 12-month recruitment period. Patients with confirmed chronic viral hepatitis based on the serological markers and liver biopsy were included. Patients with a recent history of taking supplements containing carnitine during the past 3 months or receiving medications that alter carnitine metabolism (e.g., valproate, phenobarbital, carbamazepine and zidovudine) were excluded from the study.

Age- and sex-matched healthy volunteers from the individuals' family with similar dietary regimens were selected as the control group. In total, 86 healthy volunteers and 86 patients (41 patients with hepatitis B, 42 patients with hepatitis C and three patients with hepatitis B and C coinfection) completed the study.

Questionnaire

The questionnaire of the study consisted of three sections; demographic data (age, sex, weight and height for measuring BMI), data regarding hepatic disease (type of viral hepatitis, route of transmission and if receiving, treatment regimen and duration of therapy) and the third part consisted of questions regarding the nutritional status of the patients. BMI was calculated for each participant (both patients and healthy controls) by means of height and weight. Participants were classified into six nutritional groups based on their BMI:[50] underweight (BMI <18.5 kg/m2); normal weight (18.5 ≤ BMI ≤ 24.9 kg/m2); overweight (25 ≤ BMI ≤ 29.9 kg/m2); obesity class I (30 ≤ BMI ≤ 34.9 kg/m2); obesity class II (35 ≤ BMI ≤ 39.9 kg/m2); and morbid obese (BMI ≥ 40 kg/m2).

For evaluation of carnitine dietary content in the participants' daily meals, a list of carnitine-rich containing foods such as beef steak, ground beef, milk, chicken breast, ice cream and whole wheat bread was prepared. Participants were asked to determine how often and how much in a week, they consume each of the foods. Then the approximate daily carnitine intake was calculated for each participant individually based on the carnitine food content.[51]

Sampling

Blood samples of all participants were collected after an overnight fasting. The venous blood samples were centrifuged and the separated serum fractions were stored at -70°C until measurement of carnitine levels.

A double antibody sandwich ELISA kit was used to measure total serum carnitine level. For assay procedures, standards obtained through serial dilution and samples were added to the wells that were coated with human carnitine antibody. Biotinylated carnitine antibody and streptavidin horseradish peroxide were added to each well and incubated for 60 min at 37°C. After Chromogen A & B solution were added to each well, an apparent color change occurred and the stop solution terminated the ongoing reaction. For the final measurement, the blank well (which contained nothing but Chromogen A & B) was used to calibrate the spectrophotometer under 450 nm wavelength. According to standard concentrations and the corresponding optical density values, the standard curve linear regression equation was calculated. Carnitine concentrations of the samples were calculated by applying the optical density values of the samples on the regression equation. Serum carnitine level less than 40 μmol/l was considered as carnitine deficiency.[52]

Other paraclinical information of the patients such as serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), bilirubin, albumin, creatinine, total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL) and triglyceride (TG), blood urea nitrogen (BUN), and complete blood count were collected from the patients' medical records.

The study protocol was approved by the local ethics committee of Tehran University of Medical Sciences. All participants signed written informed consent forms.

Statistical Analysis

All statistical analyses were performed using SPSS 18 software (IBM, NY, USA). Means ± standard deviations were calculated for continuous variables and percentage for categorical variables. All data were examined for normality by the Kolmogorov–Smirnov test. Independent t-test was used to compare means of variables with normal distribution while variables with non-normal distribution were compared by the Mann–Whitney test. χ2 or Fisher exact test were used to analyze probable associations between categorical variables. Uni- and multi-variate ana-lysis were used for determining serum carnitine level associated factors. P-values less than 0.05 were considered to be significant.

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