Formerly named Non-alcoholic fatty liver disease (NAFLD) is now known as Metabolic Associated Steatotic Liver Disease (MASLD), wich  falls into a spectrum of liver diseases characterized mainly by macrovesicular fatty degeneration that occurs in the absence of significant alcohol consumption ( 20-30 g pure alcohol per day or under 200g pure alcohol / week) or other liver abnormalities(Ramesh, 2005; Adams, 2005; Balistreri, 2006; Radu, 2008). 

 The spectrum of disease is composed of three clinical-pathological entities that are in fact evolutionary stages of the disease: 

1. Fatty liver disease: is characterized by predominant presence in hepatocytes of  fatty acids and triglycerides macrovezicule

2. Steatohepatitis: fatty liver disease associated with a necro-inflammatory process, Mallory bodies  and incipient fibrosis

3. Cirrhosis: characterized by architectural changes in liver fibrosis and inflammatory infiltration associated steatosis (Ziamanesh, 2023; Leuscher, 2006).

Non-alcoholic fatty liver disease is caused by multiple factors and variations, of which most view  in practice are: 

-Nutritional causes 

-Drugs

-Metabolic or genetic diseases

-Syndromes characterized by insulin resistance

-Exposure to toxins 

Both liver steatosis  and steatohepatitis are associated with insulin resistance. It was postulated by many authors that the development towards steatohepatitis requires additional pathophysiological abnormalities, thus reaching the so-called hypothesis of double strokes (the "two-hits" hypothesis). We are currently considering that the final road to development of  steatohepatitis is  oxidative stress in hepatocytes. Oxidative status of a cell is determined by the balance between pro-and anti-oxidant processes. Thus, the oxidative stress may occur when reactive oxygen species (ROS) are generated in hepatocytes but also when there is a breakdown of antioxidants. This singularity of steatosis is also associated with increased levels of 3-nitrotyrozin, a product of lipid peroxidation. This indicates the presence of oxidative stress in non-alcoholic fatty liver hepatocytes.  The first "hit" appears to be the storage of fat in hepatocytes, which is  the substrate of  liver fat degeneration or hepatic steatosis. This will further increase the sensitivity of the body towards  the second "hit". Fatty liver disease is  keeping up the appearance until  the second "hits" which consists in appearance of one risk factors like  surgery on the intestine, drugs, development of type 2 diabetes. An important role in this scenario appears to be owned by the sudden release of free fatty acids in circulation. So non-alcoholic steatohepatitis occurs in patients already suffering from diabetes, central obesity, weight loss, unexpected factors which mobilizes free fatty acids and precipitate their storage in the liver (Leuscher, 2006).

Motivation of the Study

Both in U.S. and Western Europe non-alcoholic fatty liver steatotic disease is a topic increasingly more discussed and studied. In our country for now there is not any  large study to evaluate the incidence of disease among the general population. Increasing prevalence of metabolic syndrome and type 2 diabetes among patients in conjunction with the close links between these diseases and non-alcoholic fatty liver may presume the existence of a fairly large number of undiagnosed patients. More detailed study of the epidemiology and etiopathogenesis of  this disease in our geographical area can only be helpful in understanding and implicitly reducing morbidity and mortality factors of the Romanian  population. Because of the rising incidence in the general population, finding more cases of evolution from non-alcoholic fatty liver to cirrhosis and liver cancer  is increasing. Therefore I think that this work can provide important data on the characteristics of disease in our geographical area and may be a prerequisite to the holding of larger studies in order to correct diagnosis and initiating treatment.

Aim of the Study

In the context of major importance as a public health problem, especially in the light of comorbidities and complications that involve non-alcoholic fatty liver disease, the aim of this study is to highlight correlations between  clinical and biological features and the particularities of the disease among patients in our geographical area. 

We conducted a prospective observational investigation on patients with ultrasound appearance of hepatic steatosis without significant alcohol consumption (< 200gr/week) and without  chronic hepatitis B or C infection. I watched  the degree of steatosis, the associated diseases and  family  history, assessing the degree of liver fibrosis by non-invasive methods, the study of cytokines, the presence of insulin resistance, diabetes mellitus, metabolic syndrome or  abdominal obesity, trying to point out correlations between clinical aspects and laboratory examinations (imaging, biochemical). We also tried to identify any features of this non-alcoholic fatty liver patients in our  geographical area (frequency and type of risk factors, clinical aspects and forms, associated diseases, biochemical characteristics). The data were analyzed versus a control group. The studied group  was selected from consecutive patients admitted in Medical Clinics I and II, Gastroenterology, Endocrinology, Diabetes and Metabolic Diseases,  Cardiology of Clinical County Hospital Sibiu and Medical Department - CF Gen. Hospital  Sibiu.

Study inclusion criteria

Group A: 125 patients with ultrasound appearance of fatty liver  without serological markers of HVB or HVC viral infection, without significant alcohol consumption ( less than 200 g pure alcohol/week), without clinical or biological aspect of hemochromatosis or other liver pathology. 

Control group

34 individuals with normal ultrasound appearance of liver without serological markers of infection with hepatitis B or C, without significant alcohol consumption (less than 200 g pure alcohol/week).

Exclusion criteria 

Patients with ultrasound appearance of fatty liver with  alcohol use over 200g/week  or known with serological markers of infection with HVB/HVC, with the clinical/biological aspect of hemochromatosis, auto-immune hepatitis or other liver pathology. We have made these next several  examinations:

 -Abdominal ultrasound : liver appearance confirming steatosis, portal vein size, presence or absence of biliary vesicular lithiasis or ascites, spleen size, ultrasound appearance of pancreas.

- waist / hip ratio

-body mass index calculation (BMI)

-biochemical examinations - blood count, blood glucose, lipid profile: total cholesterol, HDL-cholesterol, triglycerides, tests for detection of inflammation (ESR, fibrinogen, CRP) ,  global test of liver function (aspartate aminotransferase - AST, alanine aminotransferase-ALT, LDH , alkaline phosphatase, total and direct bilirubin, serum protein electrophoresis, Time Quick); sideremia + /- ferritin serum, cytokines: interleukin 6 (IL6), IL8, C reactive protein  (CRP), TNF-alpha, Erythropoietin (EPO)

- virological tests in order to confirm absence of infection with hepatitis B and C. Patients were asked to complete an questionnaire regarding:

-lifestyle, age, gender, environment of origin (Urban / Rural)

-food behavior 

-degree of physical activity daily

-ethanol consumption, smoking

-collateral and personal history (diabetes, obesity, ischemic cardiovascular disease, hypertension, dyslipi-daemia, atherosclerosis with different localization, hypothyroidism),

-current symptoms (dyspeptic digestive events, asthenia, fatigue, neurological manifestations of hepatic encephalopathy)

-medication until the study.

We performed in all group A  patients calculation of prediction for existence of liver fibrosis using non-invasive markers currently recognized (APRI score, Forns score, AP Index, AST / ALT ratio, Fib 4, BARD Score), correlations between blood glucose levels, BMI, platelets, age and degree of fibrosis.The data were  processed and analyzed using statistical analysis programs available.

Analyzing patients depending on the environment of origin indicates that most of them come from urban areas (69%) and women are represented in a percentage of  70.4 (n = 88).  In the total hospitalization patients  were predominantly of urban origin. Predominance of  female gender in the group of patients studied is explained by the high prevalence of women in the total number of patients hospitalized during the selection of cases period. In terms of distribution according to age, age group best represented was  between 50-59 years, following groups 40-49 and 70-79 years, the youngest patient was  26 years while the oldest was 81years old. The results show that 70.4% (n = 88) of patients were middle-aged women (40-59 years) and only 29.6% (n = 37) were men, the most common age group being between 50-59 years. We also represented the most common symptoms, which are dominated by asthenia, fatigue and flatulence. Over 50% of patients said they felt in the last years some  intermittent  small  pain in righ  hypochondrium. The most common diseases from heredocolaterale history were cardiovascular  illness and diabetes mellitus. Distribution according to lifestyle reveal a significant percentage of patients that recognize a sedentary lifestyle (79.2%), low or medium fruit and vegetables intake , nutrition with high carbohydrates / high lipids load.

Hypertension, Diabetes Mellitus and Metabolic Syndrome

We determined blood pressure, fasting glucose level and we have examined patients in group A according to the criteria for inclusion in the current definitions of metabolic syndrome in order to assess the prevalence of metabolic syndrome in the group of patients with non-alcoholic fatty liver. We found a  significant proportion of patients with at least one component of metabolic syndrome: dyslipidaemia-72%, obesity-66% and  hypertension-60%.  Also, over one quarter of patients present chronic kidney disease ( 26.4% have  glomerular filtration rate < 60ml/min) or chronic ischemic heart disease (51%). Of the total of  76 patients with fatty liver disease and hypertension, most lie within the age groups 50-59 years (38.15%) and 70-79 years (23.68%), the majority being women (n=53). We  also found a large number of patients comprising at least 2 of the components of metabolic syndrome, most cases female, also  because  they  prevailed in the selection of the plot. Regarding the patients with diabetes mellitus , the most represented age groups were 50-59 years (n = 23) and 70-79 years (n = 12), 34 patients (27.2%) with the combination of already known comorbidities : type 2 diabetes + dyslipidemia. The metabolic syndrome is not only a risk factor for the occurrence of non-alcoholic fatty liver disease but is also an essential element in the evolution of the disease towards aggressive forms, as steatohepatitis.  Considering the two accepted definitions of metabolic syndrome,  we obtained slightly different results in terms of number of patients who fit the criteria for the definition of the syndrome:

- WHO definition – we found  23 patients with type 2 diabetes, triglycerides> 150 and BP ≥ 140/90mmHg, and 25 patients with type 2 diabetes, BMI> 30 and BP ≥ 140/90mmHg, 21 patients with type 2 diabetes, BMI> 30 and triglycerides> 150 mg%.

-  ATP III  definition - 34 patients with triglycerides> 150, blood pressure and waist high above the normal range, 32 patients (20 women) with fasting blood glucose> 100, high blood pressure and triglycerides, 43 patients (29 women) with fasting blood sugar > 100, triglycerides> 150 mg% and waist circumference higher the normal  values (88cm in women and 102 cm in men). Analysis of the results obtained allowed significant correlations between blood glucose, age, triglycerides, BMI, waist-hip ratio  and  ALT in patients with metabolic liver steatosis. Waist/hip ratio, Body mass index (BMI)  and Obesity 

In assessing patients with diabetes, hypertension, obesity or metabolic syndrome, waist – hip ratio  and body mass index are crucial. One explanation of metabolic syndrome, and maybe NAFLD is the  regional distribution of fat. Thus, it was proved that predominantly central (or abdominal)  obesity is a better sensitive marker for insulin resistance  than total body fat.   (Radu, 2008) (Balistreri, 2006) (Watanabe , 2015). I found in the group of patients of fatty liver   a high prevalence of  elevated waist / hip ratio  above the normal range (78 women with values > 0.85 and 33 men with waist / hip > 0.9). Most patients with hypertension or diabetes mellitus in group A presents values of  waist-hip higher than the normal range, regardless of age or sex. A total of 82 patients (65.6%) in group A meet the definition of obesity, presenting values of  BMI ≥ 30kg/m2. Linking transaminase (ALT) with other laboratory items we  observed a close positive correlation with GGT (r = 0.33) which signifies the synergic linear action of liver enzymes and cholestasis within the disorder. Meanwhile, the correlation between age and waist / hip ratio (r = 0.22) draws attention to the increased risk of abdominal obesity with increasing age, this type of obesity, as we know, being an important and independent marker of cardiovascular risk. Also  it  is not to be neglected the  combination between blood sugar and age (r = 0.25), as a  marker of the evolution of patients towards an  alterated  carbohydrate metabolism, insulin  resistance or type 2 diabetes with increasing age, all of them being  risk factors for developing non-alcoholic fatty  liver. 

Assesment of  Renal Function

There are studies on diabetic patients that  have shown that the risk of chronic kidney disease is 69% higher in patients with type 2 diabetes and steatohepatitis compared with diabetic patients without liver damage. (Targher, 2008). We proposed the evaluation of renal function in patients with non-alcoholic fatty liver. In the 125 patients of group A we used for calculating the renal filtration ratio,  the MDRD formula (Modifi-cation of Diet in Renal Disease Study equation) currently applied in  Nefrology for determining the degree of glomerular filtration. 

GFR = 186.3 x serum creatinine-1.154*age-0.203* 0.742 (♀) ml/min/1.73 m2 (Levey, 1999) (Radu, C. G., 2008)

Evaluation of renal function using MDRD formula  demonstrate the existence of a significant percentage of people who show impaired glomerular filtration, 49 patients (39.2%) with GFR between 60-80 ml / min/1.73m2, while 33 patients (26.4%) presented an important degree of  kidney failure with GFR <60 ml/min/1.73m2. Biological mechanisms by which the  fatty liver  may increase risk of chronic kidney disease in type 2 diabetes are not fully understood, but the most obvious explanation is that the findings simply reflect the coexistence of already known risk factors for chronic kidney disease. In addition, liver disease may worsen insulin resistance and hyperglycemia, which in turn contributes to the progression of renal disease. 

Non-invasive Liver Fibrosis Assesment

Since it is known that patients with fatty liver and advanced fibrosis are prone to evolution to the final stages of the disease, namely to cirrhosis,  and liver biopsy is invasive, expensive and marked by multiple complications, there is now an increasingly higher concern about  finding reliable methods of non-invasive diagnosis of the degree of inflammation and liver fibrosis.  Considering the quite low  predictivity of ALT, GGT and ultrasound as well as risk and variability of liver biopsy results, non-invasive assessment of fibrosis using batteries of biochemical tests seems to be one of good  solutions for correct  future assesment of non-alcoholic   steatohepatitis. In this study it was not possible an histological assessment of liver fibrosis by liver biopsy puncture because  the patients refused the intervention. Therefore we decided to assess the degree of liver fibrosis by non-invasive methods, using clinical data and serum markers. We used  8 non-invasive methods of calculating the degree of liver fibrosis that are  currently available, in order to assess all the 125 patients in group A and 34 subjects in the control group, namely: AST / ALT ratio, Forns index, FIB 4, API, ASPRI, APRI  and Fatty Liver Index, BARD score. In patients of group A we calculate the FIB-4 and found that 124 cases (99.2%) had values <1.4, which suggests absence of severe fibrosis (average of 0.482), and only one case presenting an  index of  1.8 (not fitting into evaluation). The control group obtained a mean Fib-4 of 0.312 and  no case with score > 1.45. In steatosis patients  the calculated Forns index  obtained an average of 3.78097 (± 1.573306). A total of 78 patients (62.4%) had a Forns score <4.2, 44 patients (35.2%) had a score between 4.2 and 6.9, while only 3 patients obtained a score of over 6.9 which suggests significant liver fibrosis. For the  control group we obtained a mean  Forns index of 2.15, only 2 people with scores between 4.2 and 6.9, and no case of score > 6.9.  In patients with fatty liver disease was found an average ratio of AST: ALT of 1.087252742. Specifically, 69 patients had a score <1 (55.2%), 50 patients had a score between 1-2 (40%) and only 6 patients a score above 2. In group A  all subjects had APRI values <1.4, with an average of 0.341, suggesting the absence of liver fibrosis F> 2 in this group of patients. Also, in all cases the control group we obtained a score Apri <1.  Prevalence of API score> 1.5 in group A was 119 patients (95.2%) with predominent values between 4 and 8, which shows the presence of a certain degree of liver fibrosis, while in  the control group only 11 (32%) subjects had an API score> 1.5, with values predominent 2 and 3. Of patients in group A, 102 have achieved a score ASPRI<5, which excludes the presence of liver cirrhosis and 23 patients had values between 5-12, not fitting into evaluation. Fatty Liver Index (FLI) or calculation of  steatosis  prediction index is an Italian researchers simple unpatented formula, including triglyceride levels (mg / dl), BMI (kg/m2), GGT (U / L) and waist circumference (cm) resulting in a numerical value.  One result of FLI >  60 signify a possibility of over 85% for  having fatty liver disease while a  FLI value below 30 means  more than 86% probability of not having fatty liver. Calculation of FLI in patients from group A showed that only 13 subjects had values of FLI score below 30, but in these patients ultrasound showed that  fatty liver was mild with minimal posterior attenuation, most patients =  77.6% (n = 97) having FLI values > 60, confirming also by  this method the presesnce of liver steatosis. The FLI evaluation  in the control group resulted in  only 2 cases of FLI > 60 of the 34 subjects examined, 79.4% with values of FLI <30, confirming the absence of steatosis. BARD Score aims to identify patients with non-alcoholic fatty liver without significant liver fibrosis. BARD score can vary between 0 and 4 points. A score of 2-4 points was associated with degree of fibrosis F3 - F4, respectively with significant fibrosis. BARD score calculation results in patients with fatty liver in group A confirm the presence of a significant degree of hepatic fibrosis in 97 patients, ie 77.6% of the 125 subjects selected. We also found significant negative correlations between platelet count and liver fibrosis as presented in Table 8. Interleukins, Inflammation markers, Endogenous Erythropoietin 

We aimed to study if there was any correlation between IL6, TNF, CRP and hepatic biochemical tests. Also, we planned to study whether EPO level correlates with other markers of inflammation or liver biochemical tests in patients with non-alcoholic fatty liver disease. Determination of IL6, IL8, TNFα, EPO and PCR was performed in 43 patients, respectively 43.4% in group A and in all patients in the control group. 

The article complies with the ethical rules of research in the hospitals were the patients were recruited.  

This  prospective observational investigation studied 125 Romanian patients with  metabolic associated fatty liver disease-MASLD. The results pointed out correlations between clinical aspects and laboratory examinations.  There are original contributions to the study of this disease by analysis of cytokines involved in disease maintenance (EPO, CRP, IL 6, TNF, but also by assessing the cardiovascular risk of patients with metabolic fatty liver steatosis using two methods widely recognized (Framingham and SCORE risk). The results of this study confirm data from literature according to which MASLD is a disease more common than originally thought and is accompanied by multiple comorbidities, metabolic syndrome being the  most important constellation of disorders present in these cases. The results justify continuous actions of prevention, correction and treatment of obesity and associated factors addressed towards all sections of the population by promoting physical exercise (the cheapest and effective treatment for steatotic liver disease and metabolic syndrome) and habits of healthy eating (increased consumption of fiber, fruits and vegetables) in order to prevent cardiometabolic morbidities. We should not forget that metabolic associated steatosis proved to be cause of liver cryptogenic cirrhosis and many fatty liver patients presented some degree of liver fibrosis. A significant proportion of these illnesses could be avoided by adopting a healthier lifestyle. Cardiovascular risk of subjects with MASLD is extremely high and often neglected, doctors being concerned about the digestive tract   pathology. Information and involvement of health professionals at all levels is yet not sustained nor sufficient and sadly enough, the addressability, adherence and compliance of patients to change their lifestyle is far from an acceptable threshold. Many patients with cardiovascular disease are neglected in terms of existence for associated pathologies, so the study reiterates the assertion that Metabolic Associated Liver Steatosis can be considered the hepatic component of Metabolic Syndrome.

Sincere acknowledgments for my former internal medicine professor, regretted doctor Mircea Deac, MD (RIP) and for the staff of Internal Medicine ward from Clinical County Hospital of Sibiu that made possible this research. 

The author declare not to have any conflict of interest.