Abstract
Purpose A relationship between liver diseases and serum vitamin B12 levels was observed in previous reports. The purpose of this study was to determine if a similar relationship existed between vitamin B12 and nonalcoholic fatty liver disease (NAFLD), a common chronic liver disorder.
Materials and Methods A total of 45 consecutive patients with NAFLD formed the NAFLD group, whereas 30 healthy controls (HC) formed the HC group. The subjects in all of the groups were of similar age and body mass index (BMI). A fatty liver is described in 3 ultrasonographic grades. Fasting blood samples were obtained, and serum vitamin B12 levels were measured. In addition, liver enzymes including aspartate aminotransferase, alanine aminotransferase (ALT), and alkaline phosphatase, and folic acid and other serum parameters were evaluated. The Mann-Whitney U test, χ2 test, and Spearman correlation analysis were used to compare the vitamin B12 levels and other serum parameters in both groups.
Results The mean ± SD age and BMI of the NAFLD were 47.2 ± 11.2 and 28.8 ± 3.5. The mean ± SD age and BMI of the HC were 47.1 ± 8.8 and 27.7 ± 2.9, respectively. The serum aspartate aminotransferase and ALT levels of the patients with NAFLD were statistically higher compared with those of the controls (P = 0.001). The levels of vitamin B12 and folate were statistically lower in the NAFLD patients compared with those of the controls (P < 0.05). We found that there was a reduction of vitamin B12 levels, especially in grade 2 to grade 3 hepatosteatosis. In addition, in the Spearman correlation analysis between the vitamin B12 levels and ALT, the grade of fatty liver and the liver dimension were found to have an important negative correlation.
Conclusion The serum vitamin B12 levels were significantly lower in the patients with NAFLD than in those of the control group; however, these still remain in the reference range. Consequently, low vitamin B12 levels may be associated with NAFLD especially in grade 2 to grade 3 hepatosteatosis.
Nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum of liver disease from bland steatosis to steatosis with inflammation and liver cell death (steatohepatitis). Pathogenesis in NAFLD is characterized by fat deposition, inflammation, and fibrosis of the liver. Steatohepatitis occurs owing to multiple factors-chiefly, insulin resistance leading to accumulation of free fatty acids-and other mechanisms such as oxidative stress, lipid peroxidation, endotoxins, iron overload, and so on. Various cytokines such as tumor necrosis factor and interleukins (IL-6 and IL-8) are responsible for inflammation. Tumor necrosis factor mainly affects the triglyceride synthesis in the liver.1
Vitamin B12 (cobalamin) is a water-soluble cobalt-containing vitamin with an important role in biochemical processes referred to as single carbon transfers. Vitamin B12 is a cofactor for at least 3 enzymes that carry out these types of reactions, acting as a transitional carrier of the single carbon group. It is reported that several liver diseases such as acute hepatitis, cirrhosis, hepatocellular carcinoma, and metastatic liver disease are associated with major changes in plasma vitamin B12 concentrations.2Although these pathologies can lead to change plasma levels vitamin B12 by different pathways, no clear effect of NAFLD has been established. Several studies reported that there might be a relationship between insulin resistance of both levels of vitamin B12 and NAFLD.3,4For this reason, we hypothesized that there may be a relationship between vitamin B12 levels and NAFLD.
The criterion standard for establishing diagnosis and severity of NAFLD is liver biopsy, but ıt is invasive and expensive. A diagnosis of NAFLD is commonly made using the ultrasonography (US) imaging technique because of its lower cost and lack of known risks.5Hamaguchi et al.6reported that US has a high sensitivity (91.7%) and specificity (100%) of fatty liver detection. We investigated the vitamin B12 levels in patients with NAFLD. The aim of this study was to investigate a possible relationship between the levels of vitamin B12 and NAFLD defined ultrasonographically.
MATERIALS AND METHODS
A total of 45 consecutive patients with NAFLD formed the NAFLD group, ranging in age from 31 to 66 years (mean ± SD, 47.2 ± 11.2; 21 women and 24 men), whereas 30 healthy controls (HCs) formed the HC group, ranging in age from 32 to 61 years (mean ± SD, 47.1 ± 8.8; 19 women, 11 men). The study was made between April 2009 and September 2009 in the Medical Faculty of Dumlupinar University. The subjects in all of the groups were of similar age and body mass index. For the diagnoses of NAFLD, and to rule out other possible liver diseases, both the NAFLD group and the HC group were evaluated by abdominal ultrasonography (US), clinical and laboratory findings. The time interval between US and the laboratory study was less than 1 week. Apart from vitamin B12, the liver enzymes including aspartate aminotransferase, alanine aminotransferase (ALT), and alkaline phosphatase, as well as the total cholesterol, triglycerides, serum lipoproteins, glucose, folic acid and hepatitis markers, were evaluated. In addition, body mass index (calculated as weight in kilograms divided by the square of the height in meters) and the waist circumference (measured with a tape measure just above the umbilicus) were calculated.
The study was approved by the Medical Ethics Committee of the Medical School, Dumlupinar University, and informed consent was obtained from all of the patients within the study groups. We took a detailed history of all the patients and we included only the patients who never took alcohol. Members of the HC group did not have any known disease or symptoms. We excluded patients with diabetes mellitus and those who had a reported history of known liver disease, malignant disease, active infections, or a fatty liver that was drug induced, for instance, by steroids. In addition, patients who had a positive hepatitis B and C tests (viral hepatitis) and biliary disease were excluded from the study.
A radiologist performed abdominal sonographic examinations using convex-type real-time electronic scanners (3- to 5-MHz convex-type transducer, Logiq 5-Expert, General Electric (GE) Healthcare). The severity of steatosis was graded as mild, moderate, or severe using the criteria described previously.7,8Right kidney echogenicity was used for the determination of liver parenchyma echogenicity. With the same kidney cortex and liver parenchyma echogenicity, it is evaluated as normal, no fatty liver (grade 0; Fig. 1). Mild (grade 1): minimal diffuse increase in hepatic echogenicity and normal visualization of the diaphragm and intrahepatic vessel borders. Moderate (grade 2): moderate grade diffuse increase in hepatic echogenicity and slightly impaired visualization of intrahepatic vessels and the diaphragm. Severe (grade 3): apparent increase in echogenicity and poor or nonvisualization of the hepatic vessels and diaphragm. The posterior segment of the right hepatic lobe is difficult to display.9
Statistical Analysis
The Mann-Whitney U test and the χ2 test were used to compare the vitamin B12 levels and other serum parameters in both groups. All of the results are expressed as mean ± SD. Whereas cases with the fatty liver were accepted as 1, 2, and 3 according to the grade, control groups were accepted as grade 0, and they were compared by one-way analysis of variance methods. In addition, to determine the relationship between the vitamin B12 levels, the liver function tests, and the ultrasonographic liver findings (grade of fatty liver and liver dimension), a Spearman correlation analysis was done. Data were analyzed using SPSS for Windows 11.0 software. P < 0.05 was considered to be statistically significant.
RESULTS
The results of the main parameters that were studied in both of the groups are presented in Table 1. The serum aspartate aminotransferase and ALT levels of the patients with NAFLD were statistically higher compared with those of the controls (P = 0.001) but the levels of glucose, alkaline phosphatase, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglycerides did not differ in both groups. However, the levels of vitamin B12 (reference range, 191-663 pg/mL) and folate (reference range, 4.6-18.7 ng/mL) were statistically lower in the patients with NAFLD compared with those of the controls (P < 0.05).
According to the US results of the liver, grade 1 hepatosteatosis (fatty liver) was detected in 33.3% of the 45 patients (n = 15: 9 men and 6 women), and grade 2 and grade 3 hepatosteatosis were detected in 28.9% (n = 13: 7 men and 6 women) and 37.8% (n = 17; 8 men and 9 women) of the patients, respectively. A larger liver dimension was detected in the patients with NAFLD when compared to the controls (P = 0.001). In our study, we found that there was a reduction of vitamin B12 levels, especially in grade 2 to grade 3 hepatosteatosis. The vitamin B12 levels according to the grade (degree) of fatty liver are presented in Table 2. In addition, the association between the grade of ultrasonographic fatty liver and the vitamin B12 levels are presented in Table 3. Here, statistically significant difference was present between grade 0 and grades 1, 2, and 3 hepatosteatosis (P < 0.05). In the Spearman correlation analysis between the vitamin B12 levels and ALT, the grade of fatty liver and liver dimension were found to have an important negative correlation, respectively ([r = −289, P = 0.016], [r = −399, P = 0.001], [r = −430, P = 0.001]).
DISCUSSION
Our study shows that there was a reduction of vitamin B12 levels, especially in grade 2 to grade 3 hepatosteatosis, despite normal serum vitamin B12 levels. Hepatosteatosis is a condition characterized by triglyceride accumulation within the cytoplasm of hepatocytes. Nonalcoholic fatty liver disease involves a spectrum of diseases ranging from steatosis to nonalcoholic steatohepatitis, which in its most severe form can lead to cirrhosis and liver failure. The pathophysiology of NAFLD still has not been completely elucidated.10Nevertheless, the "two-hit hypothesis" is the most widely accepted model to explain the development of NAFLD and the progression from simple steatosis to nonalcoholic steatohepatitis. The "first hit" is the accumulation of lipids in the hepatocytes, and insulin resistance is the key pathogenic factor for the development of hepatic steatosis. The "second hit" leads to hepatocyte injury, inflammation, and fibrosis. Oxidative stress and subsequent lipid peroxidation, proinflammatory cytokines, adipokines, and mitochondrial dysfunction are factors that initiate the second hit.10,11
Ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI) can be used to detect fatty livers. Ultrasound has been used most often in epidemiologic studies owing to its advantages. Computed tomography and MRI can accurately detect and quantify the amount of steatosis in patients with a higher sensitivity and specificity rates. However, these methods have limitations because they use radiation (for CT), are expensive, and cannot be used in patients with implantable devices or claustrophobia (for MRI).12We used the US imaging technique because of its lower cost and lack of known risks and the highly sensitive/specific in detecting moderate and severe fatty liver.
In the human body, vitamin B12 exists in 2 essential coenzyme forms, methylcobalamin and 5'-deoxyadenosylcobalamin.13,14Upon entering the stomach, vitamin B12 is bound to haptocorrin and transferred to the duodenum where intrinsic factor (IF), arriving from the stomach, binds vitamin B12. The IF-vitamin B12 complex is absorbed via the IF-B12 receptor, and vitamin B12 is subsequently bound to transcobalamin II (TC II) and released into the circulation. Transcobalamin II facilitates the transport of vitamin B12 in blood to various tissues. Holo-TC II (TC II saturated with vitamin B12) transports the vitamin B12 to the cell via specific transcobalamin receptors at the cell surface.2
Because the liver plays an essential role in the storage of vitamin B12, it is not amazing that liver diseases are associated with major changes in serum vitamin B12 levels. Although several studies have been reported that liver pathologies, including acute hepatitis and metastatic liver, disease elevate the level of plasma vitamin B12, the effect of chronic liver disease on it is controversial. Cylwik et al.15reported that the vitamin B12 levels in patients of alcohol abuse are significantly higher than those of healthy subjects, despite normal serum vitamin B12 levels. Fragasso et al.16reported that the serum vitamin B12 levels may decrease in some alcohol-dependent patients with megaloblastic anemia, although these still remain in the reference range. Ermens et al.2reported that in liver cirrhosis, plasma vitamin B12 levels can reach high values depending on the severity of the disease. However, Holdsworth et al.17reported that a significant number of cirrhotic patients have lower serum vitamin B12 level in their study. They advocate that rapid cellular necrosis releases less of the vitamin into the blood owing to severe tissue depletion. Our results with the patients, especially in grade 2 to grade 3 hepatosteatosis, can be supported with their explanation. Our alternative explanation is that insulin resistance may be associated with low-level serum vitamin B12 in patients with NAFLD. Moreover, a current study reported that there may be an association between insulin resistance and low vitamin B12 level.3In addition, it was shown that there had been a relationship between insulin resistance and NAFLD.4On the other hand, cellular, physiological, clinical, and epidemiological studies strongly support a reciprocal relationship between endothelial dysfunction and insulin resistance.18Furthermore, several studies have shown that hepatocytes did not carry TC II receptors and that uptake of vitamin B12 by the liver is mediated by endothelial cells.2,19Therefore, endothelial dysfunction can contribute to low vitamin B12 levels in patients with NAFLD via unknown mechanisms. As a result, all these mechanisms could be explained to a relationship between low vitamin B12 levels and NAFLD.
This study was limited by the lack of a histological confirmation of the diagnosis of fatty liver. Abdominal US is highly sensitive/specific in detecting moderate and severe fatty liver, but its sensitivity is reduced when hepatic fat infiltration on the biopsy is less than 33%.20In addition, its sensitivity is reduced in the morbidly obese and also in those with small amounts of fatty infiltration and operator dependent.21,22We could not evaluate Holo-TC II insulin levels owing to technical insufficiency.
In conclusion, the data suggest that serum vitamin B12 levels may decrease in patients with NAFLD. The decrease in vitamin B12 levels may be associated with insulin resistance and endothelial dysfunction. Setola et al.20reported that vitamin B12 treatment improved insulin resistance and endothelial dysfunction, along with decreasing homocysteine levels. Therefore, vitamin B12 treatment in hepatosteatosis should also be reassessed by an initial screening of patients for NAFLD. However, further studies are needed to determine the role that the serum vitamin B12 level has on hepatosteatosis and steatohepatitis more clearly.