Peculiarities of biochemical blood parameters and cytokine profile in patients with non-alcoholic fatty liver disease depending on the form of hypothyroidism

Background. The purpose of the study was to investigate the biochemical blood peculiarities and cytokine profile in non-alcoholic fatty liver disease (NAFLD) patients depending on the form of hypothyroidism. Material and methods. The study involved 188 NAFLD patients (average age 53.60 ± 12.34 years). Among the examined individuals 44 of them had diagnosed hypothyroidism in addition to NAFLD (20 patients had subclinical form and 24 patients had manifest hypothyroidism). A comparison group consisted of 144 NAFLD patients with thyroid normal functional activity. The control group consisted of 45 healthy individuals represented by their age and gender similar to the patients of the studied groups. Biochemical blood parameters, tumor necrosis factor α, interleukin-10, leptin, adiponectin blood levels were investigated in the observed patients and healthy individuals. Results. Total lactate dehydrogenase blood activity in NAFLD patients with subclinical and manifest hypothyroidism was found to be 13.5 % increased compared to the NAFLD patients with normal functional activity of the thyroid gland (p = 0.02 and p = 0.01, respectively). Higher alkaline phosphatase blood activity by 12.0 % (p = 0.03) was recorded in NAFLD and manifest hypothyroidism patients as compared to the patients with intact thyroid gland. Leptin blood concentration in NAFLD patients with subclinical as well as manifest hypothyroidism was 35.7 % and 72.1 % increased compared to NAFLD patients with normal thyroid functional activity (p = 0.04 and p = 0.009, respectively). Adiponectin blood level in NAFLD patients with manifest hypothyroidism was 2.1 lower (p = 0.004) in comparison with NAFLD patients with thyroid normal functional activity and 50.0 % lower (p = 0.009) as compared to the NAFLD patients with subclinical hypothyroidism. NAFLD and manifest hypothyroidism patients showed greater vertical size of the liver measured by midclavicular line on average by 9.1 mm (p = 0.004) as compared to NAFLD patients with unchanged thyroid gland functional activity and by 8.6 mm (p = 0.04) in comparison with the NAFLD patients with subclinical hypothyroidism. Conclusions. There were found higher total lactate dehydrogenase activity and leptin blood level in NAFLD patients with subclinical and manifest hypothyroidism and higher alkaline phosphatase activity and lower adiponectin blood level in NAFLD patients with manifested hypothyroidism as compared to NAFLD patients with normal functional activity of the thyroid gland. A significant increase in liver vertical size measured by midclavicular line was observed in NAFLD patients with manifest hypothyroidism as compared to the patients with normal thyroid function or subclinical hypothyroidism.


Introduction
In recent decades, a significant increase in the prevalence of nonalcoholic fatty liver disease (NAFLD) is observed, which occurs in about one third of adults in Western Europe and North America and in 15 % of Asian population [3].The abovementioned is associated with a growing number of individuals with obesity, dyslipidemia, type 2 diabetes and metabolic syndrome.In addition to the foregoing factors the conditions with occurring NAFLD association are now actively discussed.Among them hypothyroidism plays an important role, which even in subclinical form is associated with an increased risk of NAFLD development [2,5].Some scientists indicate the level-dependent relationship between the blood concentration of thyroid stimulating hormone (TSH) and NAFLD progression [9].At the same time, free thyroxine (T 4 ) blood content inversely correlates with the degree of hepatic steatosis [10].These Том 13, № 3, 2017 Міжнародний ендокринологічний журнал, p-ISSN 2224-0721, e-ISSN 2307-1427 Клінічна тиреоїдологія /Clinical Thyroidology/ associations are possible due to active influence of thyroid hormones on metabolism of lipids, carbohydrates, proteins and energy exchange [16].
A. Perra et al. studies demonstrates that triiodothyronine (T 3 ) produces a strong inhibitory effect on fatty liver development and promotes regression of already formed steatosis [17].T 3 also increases the expression of several genes involved in the processes of lipogenesis in the liver: acyl-CoA synthetase 5 gene, fatty acids transport protein gene, glucose-6-phosphate dehydrogenase gene [4,6].The positive effect of T 3 on hepatic steatosis is also realized through NADP-dependent sirtuin deacylase 1, which stimulates the oxidation of fatty acids in the liver [19].The abovementioned indicates that in addition to oxidation stimulation, thyroid hormones inhibit the pathways contributing to the accumulation of lipids in the liver and stimulate the expenditure of lipids from their depot [8].In case of their in sufficiency the conditions promoting accumulation of lipidsin the liver appear with subsequent addition of inflammatory processes in it.
The purpose of the study was to investigate the biochemical blood peculiarities and cytokine profile in NAFLD patients depending on the form of hypothyroidism.

Material and methods
The study involved 188 NAFLD patients (average age 53.6 ± 12.34 years).Among observed patients 102 (54.3 %) were males, 86 (45,7 %) -females.Among the examined individuals 44 of them in addition to NAFLD were diagnosed with hypothyroidism inclu ding 20 patients with subclinical form and 24 patients with manifest hypothyroidism.144 NAFLD patients with normal functional activity of the thyroid gland served as a comparison group.The control group consisted of 45 healthy individuals represented by their age and gender similar to the patientsof the studied groups.
Diagnosis of NAFLD was verified according to the Order of the Ministry of Public Health of Ukraine N 826 from 11.06.2014,adaptive clinical guidelines based on evidence "Nonalcoholic fatty liver disease" (2014) [1] and EASL-EASD-EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease (2016) [5].Diagnosis of hypothyroidism was verified according to clinical recommendations for diagnosis of hypothyroidism of the European Thyroid Association (2013) [15], the American Association of Clinical Endocrinologists and the American Thyroid Association (2012) [7].Compensation of hypothyroidism was determined on the basis of clinical symptoms and indicators of thyroid profile: TSH and free T 4 , T 3 blood concentrations.
In order to exclude viral etiology ofliver disease all of the patients were tested on possible hepatitis B and C infections with the help of polymerase chain reaction method.In all of the examined patients potentially dangerous in take of alcohol (consumption more than 30 g of ethanol per day for males, and more than 20 g of ethanol daily for females) and prolonged administration of hepatotoxic drugs were excluded [1,5].It should be noted, that detailed analysis of anamnestic data of the observed patients indicated, that the consumption of alcohol drinks in lower than the above mentioned amounts, happened less than once a week, which eliminates the influence of ethanol as a possible etiological factor of liver damage.
All of the patients and healthy individuals underwent general comprehensive clinical, laboratory and instrumental diagnostic investigations.An informed consent was obtained from all the participants.Blood samples were obtained in the morning before taking meal from the antecubital vein.5 % solution of disodium salt of ethylene diamine tetraacetate was used as an anticoagulant.Biochemical studies were performed on the blood biochemical analyzer "Accent-200" ("Cormay SA", Poland).The range of indicators of biochemical blood analysis included: total bilirubin and its fractions, uric acid, total protein and albumin, urea, creatinine, plasma enzyme activity (aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), alkaline phosphatase (AP), gamma-glutamyl transferase (GGT)).
Type of data distribution was determined by comparing the arithmetic mean, median and mode, and using Shapiro-Wilkie test.To determine the statistical differences between two independent groups Mann-Whitney test was applied.P-values < 0.05 (p < 0.05) were considered statistically significant.

Results
Patients with combined liver and thyroid pathologies admitted more pronounced complaints on pain and feeling of heaviness in the right subcostal area, frequent nausea, decreased overall health and greater general weakness, worse clinical course of the disease as compared to NAFLD patients with normal functional activity of the thyroid gland.An objective examination of these patients revealed frequent moderate pain and feeling of heaviness in the right subcostal area, hepatomegaly, presence of xanthomas, xanthelasmas and telangiectasis.
Investigations of biochemical blood tests showed significantly greater glucose, uric acid, urea, higher AST, ALT, GGT activities in the blood of NAFLD patients with normal functional activity of the thyroid gland as well as in NAFLD patients with thyroid hypofunction (table 1).Total LDG plasma activity in NAFLD patients with subclinical and manifest hypothyroidism prevailed proper indicator in NAFLD patients with normal functional activity of the thyroid gland by 13.5 % (p = 0.02 and p = 0.01 respectively).Significantly higher AP plasma activity by 12.0 % (p = 0.03) was observed in NAFLD patients and manifest hypothyroidism as compared to

Notes: p 1 -significance of differences as compared to the indicators in the group of healthy people; p 2significance of differences as compared to the indicators in NAFLD patients with normal functional activity of the thyroid gland.
NAFLD patients and normal functional activity of the thyroid gland, indicating an increased severity of cholestasis [14].The above-mentioned is probably associated with a decreased T 4 blood concentration in NAFLD patients with manifest hypothyroidism, since it is known that thyroxin promotes relaxation of Oddi sphincter, however its decreased plasma concentration is accompanied with the sphincter hypertonicity causing the development of cholestasis [11].Clinically these changes of the enzymic activity were reflected in frequent complaints of NAFLD patients with subclinical and especially manifest hypothyroidism on periodic bitter taste in the mouth, nausea, worse overall health, general weakness, headache, torpid course of the disease.
The TNF-α plasma content was more than two times higher in the examined patients of all groups as compared to healthy individuals indicating increase in the activity of inflammation processes [13].However, the analysis of IL-10 plasma levels in the observed patients and healthy individuals did not reveal any statistically significant differences (table 2).
Leptin plasma concentration in NAFLD patients with subclinical as well as manifest hypothyroi dism dominated over the proper parameter in NAFLD patients with normal thyroid functional activity by 35.7 % (p = 0.04) and 72.1 % (p = 0.009), respectively (table 2).The adiponectin blood level was significantly 2.1 times (p = 0.004) lower in NAFLD patients with manifest hypothyroidism as compared to patients of the comparison group.Moreover, in NAFLD patients with manifest forms of hypothyroidism adiponectin blood content was lower by 50.0 % (p = 0.009) in comparison with the proper indicator in NAFLD patients with subclinical hypothyroidism.

Discussion
Similar changes in adiponectin blood concentration in patients with various forms of NAFLD as compared to the healthy people were found by Z.M. Younossi et al. [20].G. Li et al. demonstrated that low adiponectin blood content was associated with the progression of steatohepatitis [12], indicating an active observation and possible prophylaxis acquired in order to prevent NAFLD progression in patients with hypothyroidism.Our findings are indicative of the formation of adipokine imbalance in the observed patients, characterized by an increased leptin plasma concentration against the ground of a decreased adiponectin blood level [18].Клінічна тиреоїдологія /Clinical Thyroidology/ All observed NAFLD patients both with normal functional activity of the thyroid gland and concomitant subclinical or manifest hypothyroidism determined increased right and left liver lobes sizes, which is typical for NAFLD [14].In particular, in NAFLD patients with manifest hypothyroidism a significantly greater vertical size of the liver measured by the midclavicular line on an average 9.1 mm (p = 0.004) was observed as compared to NAFLD patients with unchanged functional activity of the thyroid gland and 8.6 mm (p = 0.04) in comparison with NAFLD patients with subclinical hypothyroidism (table 3).The mentioned enlargement of the right liver lobe was accompanied bymore pronounced sensation of heaviness in the right subcostal area, pain during liver palpation.No significant changes in the sizes of the liver left lobe in NAFLD patients of different studied groups were found.

Conclusions
1. Higher total lactate dehydrogenase plasma activity was investigated in NAFLD patients with subclinical and manifest hypothyroidism, and greater alkaline phosphatase activity in the blood -in those with manifest hypothyroidism as compared to proper indicators in NAFLD patients with normal functional activity of the thyroid gland.
2. Elevated leptin plasma level was observed in NAFLD patients with both subclinical and manifest forms of hypothyroidism, and lower adiponectin blood level -in NAFLD patients with manifest hypothyroidism in comparison with NAFLD patients with intact thyroid gland.
3. An increased vertical size of the liver right lobe was observed in NAFLD patients with manifest hypothyroidism as compared to proper parameter in NAFLD patients with normal thyroid function or subclinical hypothyroidism.

Conflicts of interests.
Authors declare the absence of any conflicts of interests that might be construed to influence the results or interpretation of their manuscript.

Plasma level
Healthy individuals, n = 20