Chemerin and Vaspin as Noninvasive Biomarkers in the Pathogenesis and Diagnosis of Non- Alcoholic Fatty Liver Disease

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1 Ameican Jounal of Medicine and Medical Sciences 2019, 9(1): 7-13 DOI: /j.ajmms Chemein and Vaspin as Noninvasive Biomakes in the athogenesis and Diagnosis of Non- Alcoholic Fatty Live Disease Saha Mohamed Ismail 1, Naglaa Atef Elgendy 2, Zeinab H. El Sayed 1,*, Aida Ahmed Abd El Hameed 3, Inass Hassan Ahmad 4, Azza Ali Althoqapy 5 1 Depatment of Intenal Medicine, Faculty of Medicine Gils, Al-Azha Univesity, Caio, Egypt 2 Depatment of Topical Medicine, Faculty of Medicine Gils, Al- Azha Univesity, Caio, Egypt 3 Depatment of Clinical athology, Faculty of Medicine Gils, Al Azha Univesity, Caio, Egypt 4 Depatment of Endocinology, Faculty of Medicine Gils, Al- Azha Univesity, Caio, Egypt 5 Depatment of Micobiology and Immunology, Faculty of Medicine Gils, Al Azha Univesity, Caio, Egypt Abstact Backgound: Many challenges facing the accuate diagnosis of pogession of Nonalcoholic fatty live disease (NAFLD). Live biopsy is the only accuate test. Chemein and vaspin ae adipokines that may play a ole in the pathogenesis of simple steatosis and Nonalcoholic steatohepatitis (NASH). We aimed to evaluate the ole of chemein and vaspin in pathogenesis and diagnosis of simple steatosis and NASH as non-invasive biomakes in obese patients. Methods: Thity obese NAFLD patients included in ou study wee divided into two goups: simple steatosis (n=fifteen patients), and NASH (n=fifteen patients). Anothe fifteen nonobese subjects who wee appaently healthy seved as a contol goup (CG). We detemined seum of chemein, vaspin, insulin, alanine tansaminase (ALT), aspatate aminotansfease (AST), fasting blood glucose and HOMA-IR. Results: Chemein was significantly elevated in the simple steatosis and NASH goups compaed to CG, but thee wee no significant diffeences between simple steatosis and NASH. ositive associations with chemein wee obseved fo BMI, fasting insulin levels, tiglyceides, total cholesteol, HOMA-IR, ALT, and AST. Vaspin was significantly deceased in simple steatosis and NASH goups, compaed to the CG, while thee wee no significant diffeences between simple steatosis and NASH. Thee was no coelation of vaspin with all the paametes in the simple steatosis and NASH goups. Conclusion: We confimed that the chemein, and not the vespin levels, is stongly associated with obese NAFLD. Chemein and vespin may play a ole in the pathogenesis of NAFLD, but they do not play a ole in the pogession of simple steatosis. Keywods Biomakes, Chemein, Non-alcoholic fatty live disease, athogenesis, Vaspin 1. Intoduction Nonalcoholic fatty live disease (NAFLD) is the most common chonic live disease all ove the wold (1) and is commonly associated with obesity, dyslipidemia and insulin esistance (2). It usually has no symptoms, in most cases, and it diagnosed accidentally when investigations wee pefomed fo othe easons [3, 4]. On the bases of disease seveity, NAFLD is divided into simple steatosis and a moe sevee fom called nonalcoholic steatohepatitis (NASH), which pogesses to live fibosis and cihosis * Coesponding autho: zeinabhelmy77@yahoo.com (Zeinab H. El Sayed) ublished online at Copyight 2019 The Autho(s). ublished by Scientific & Academic ublishing This wok is licensed unde the Ceative Commons Attibution Intenational License (CC BY). faste than simple steatosis [5]. The patients with NASH ae at a high isk of developing cadiovascula disease, chonic kidney disease, and type 2 diabetes mellitus, besides live complications [6]. Although many studies ecognize that NASH is an impotant cause of decompensated live disease and hepatocellula cacinoma, the diagnosis by noninvasive techniques gives a suboptimal pefomance. The live biopsy is still the only accuate way fo the diagnosis and diffeentiation between simple steatosis and NASH, but it is not possible to outinely cay out biopsies, and biopsies ae associated with isks [7]. Theefoe, we need new, accuate, and noninvasive biomakes to help in the ealy diagnosis of NAFLD and NASH. Chemein and vaspin ae adipokines poduced by the live cells and adipose tissue [8]. They have a stong elation with obesity, insulin esistance, and atheoscleosis. Recently, thee is gowing evidence that chemein and

2 8 Saha Mohamed Ismail et al.: Chemein and Vaspin as Noninvasive Biomakes in the athogenesis and Diagnosis of Non- Alcoholic Fatty Live Disease vaspin may play a ole in the pogession fom simple steatosis to inflammation and fibosis [9]. Howeve, an accuate assessment of the ole of chemein and vaspin in the diagnosis is still lacking. Theefoe, the aim of ou study is to evaluate the ole of chemein and vaspin in the pathogenesis and diagnosis of simple steatosis and NASH as non-invasive makes in obese patients. 2. atients and Methods Ou study included thity obese NAFLD patients who visited the topical and intenal medicine outpatient s clinics between Novembe 2017 and Mach The patients wee diagnosed and divided into two goups; a simple steatosis goup and a NASH goup, based on the evidence of fatty changes on abdominal ultasonogaphy [10], and on the HAIR scoe [11] (hypetension, elevation of alanine tansaminase (ALT), insulin esistance), to the exclusion of live diseases of othe etiologies. Thee wee anothe fifteen appaently healthy non-obese paticipants seving as a contol goup. Infomed witten consent was obtained fom all paticipants and was conducted accoding to the declaation of Helsinki. Ou study was appoved by the local Ethics Committee at Al-Azha Univesity. Contol goup: thei age anged fom 35 to 70 yeas with a mean ± SD (50.20±9.10) yeas. BMI anged between 20<25 kg/m 2. All wee females. Simple steatosis goup: this includes fifteen obese (BMI>30 kg/m 2 ) patients who had nomal seum AST and ALT levels. Thei age anged between 40 to 74 yeas with a mean ±SD (47.93±4.31) yeas. All of them wee females. NASH goup: this included fifteen obese (BMI 30 kg/m 2 ) NASH patients who had a pesistent elevation of seum ALT and AST. Thei age anged fom 35 to 70 yeas with a mean ±SD (50.20±9.10). Ten of them wee females. Exclusion citeia: We exclude all patients who had acute o chonic hepatitis, chonic enal disease, patients who had eceived metfomin, had a malignancy, active infection o consumed alcohol. Methods: A full compehensive histoy and a full clinical examination wee taken fo all paticipants. Anthopometic measuements: Individual weight was measued/kg and height was measued/cm. Body mass index (BMI) was calculated by Body weight(kg) (Height)2(m) Laboatoy tests: A complete blood count, alanine tansaminase (ALT) Iu/l, aspatate aminotansfease (AST)Iu/l, gamma-glutamyl tansfease, seum albumin g/dl, anti-hepatitis C vius antibodies, and hepatitis B suface antigen. Fasting blood suga mg/dl, seum tiglyceides, total cholesteol, blood uea, and seum ceatinine wee done at Al Zahaa hospital. Seum vaspin ng/ml, seum chemein ng/ml, and fasting seum insulin level uiu/ml wee pefomed at Al-Azha Univesity Cente fo Vius Reseach and Studies. Insulin esistance, it was estimated by the homeostasis model assessment fo insulin esistance (HOMA-IR) Fasting insulin level (IAU/ML) fasting glucose level (mg) 450 Abdominal ultasound and its evaluation wee pefomed by an expeienced adiologist using a hilips- Affinti 70 G. Sample collection Fom each subject and contol, fasting peipheal venous blood samples wee collected unde complete aseptic conditions. Seven ml of venous blood wee withdawn and divided as follows: A- 2ml anticoagulated with EDTA was used immediately fo a complete blood count. This was pefomed by the fully automated cell counte Sysmex KX-2IN. B- 5ml was put in a plan tube, left to clot then centifuged at 1000xg fo 5 minutes and seum was sepaated. at of the sea wee used immediately fo outine laboatoy tests; anti-hepatitis C vius antibodies HCVAb, hepatitis B suface antigen HBSAg and HIV antibody, cholesteol, tiglyceide, kidney function test, live enzymes (ALT, AST) fasting blood suga, total cholesteol (TC), tiglyceide (TG), kidney function test (all chemisty investigation wee done on Cobase c311 auto analyze using (Roche-eagent kits.hitachi, Japan)). The emaining seum sample was emoved in aliquots and stoed at -70 C until assays of adepokines (chemein & vaspin) and seum insulin stoed at -20 C. Adipokines and Insulin measuement: Seum levels of adipokines (chemein, andvaspin) and seum insulin wee measued by enzyme-linked immune-sobent assay (ELISA) uses Sandwich ELISA as the method using commecial kits: Human visceal adipose-specific seine potease inhibito (vaspin) ELISA kit (Cat No. In- Hu3180: China Bioneovan Co., Ltd.), and Human chemein ELISA kit (Cat No. In- Hu1852: China Bioneovan Co., Ltd.) Human insulin ELISA kit (Cat No.10801: USA efect Ease Biotech (Beijing) Co., Ltd.) (16. Biefly, standads, samples and contols wee pipetted into wells, pecoated with specific Antibody, and any vaspin o chemein and insulin pesent was bound to the immobilized antibody. Afte emoving any unbound substances. Afte washing, Hoseadish eoxidase (HR) conjugated specific antibody fo vaspin o chemein o insulin was added to the wells. Following wash to emove any unbound conjugate-enzyme eagent, a substate solution (TMB) was added to the wells and colo developed in popotion to the amount of vaspin o chemein o insulin

3 Ameican Jounal of Medicine and Medical Sciences 2019, 9(1): bound in the initial step. The colo development was stopped and the intensity of the colo was measued (Read at 450nm wave length using Stat-Fax 2100, AWARENESS TECHNOLOGY INC.ELISA eade). A standad cuve was pepaed elating colo intensity to the concentation. The levels wee calculated fom the standad cuve coesponding to the measued optical density. The esults wee expessed as ng/ml fo vaspin, ng/ml, pg/ml fo chemein, µiu/ml fo insulin. The inta- and inte-assay coefficient of vaiation (CV) of vaspin and chemein was anged fom < 10% to < 12%, espectively. The assay ange fo vaspin is ( ng/ml). The assay ange fo chemein is (20pg/mL-1200 pg /ml). The sensitivity of insulin was 2 µiu/ml. Expected fo insulin: adult (nomal) ( µiu/ml). Diabetic (type2) ( µiu/ml) Statistical Methods Collected and ecoded data wee analyzed using the statistical package fo social science (IBM SSS), vesion 23. Quantitative data wee expessed as a mean± standad deviation. A test to compae quantitative data of moe than two goups was pefomed by the analysis of vaiance test. 3. Results Table 1 shows the compaative study between all goups by using ANOA test accoding the clinical and anthopometic data. The simple steatosis goup (n=15 patients), 12 of them had T2DM, 9 had hypetiglyceidemia, and 10 had hypecholesteolemia. The NASH goup (n=15 patients), 10 had T2DM, 11 had hypetiglyceidemia, and 10 had hypecholesteolemia. Thee wee significant diffeences in age, weight, height, and BMI between the goups. In simple steatosis goup BMI, Tiglyceides, total cholesteol, FBG, insulin levels, and HOMA-IR, (34.59 ±3.75, ±43.58, ±55.43, ±108.68, 13.43±8.37, 12.07±21.13, 4.27±1.86, and 0.71±0.47, espectively), wee significant elevated when compaed with the contol goup (23.86±2.19, 81.13±6.98, ±11.03, ±10.26, 3.11±2.00, 0.68±0.42, 6.87±6.19, and 0.25±0.19, espectively), (p= 0.001; p= 0.012, p= 0.003, p= 0.001, p= 0.001, p= 0.046, espectively). But thee was no significant diffeence accoding to ALT and AST levels (p1= 0.103, p1= 0.429, espectively), as shown in table 2. Table 1. Clinical and anthopometic data of the thee goups by ANOVA test. Data ae expessed as means ± SD aametes Sex: Male (N/ %) Female (N/%) Contol (n-15) 2(13.3%) 13(86.7%) Simple steatosis (n=15) 0(0.0%) 15(100.0%) NASH (n-15) 5(33.3%) 10(66.7%) Value Significant 0.040* S Age/yea ± ± ± NS Weight/kg ± ± ± * S Height/cm ± ± ± * S BMI (Kg/M 2 ) 20< 25 (Kg/ M 2 ) ± (Kg/ M 2 ) ± (Kg/ M 2 ) ± * S T2DM Hype-tiglycedemia Hype-cholestolmia BMI: body mass index; T2DM: Type 2 diabetes mellitus; *: significant. Table 2. The compaative study between contol and simple steatosis goups aametes Contol (n-15) Simple steatosis (n=15) BMI (Kg/M 2 ) ± ± * Tiglyceides mg/dl ± ± * Total cholesteol mg/dl ± ± * ALT Iu/l ± ± AST Iu/l ± ± FBG mg/dl ± ± * Insulin uiu/ml 3.11 ± ± * HOMA-IR 0.68 ± ± * Vaspin ng/ml 6.87 ± ± * Chemein ng/ml 0.25 ± ± * BMI: body mass index; ALT: alanine tansaminase; AST: aspatate aminotansfease; FBG: fasting blood glucose; HOMA-IR: homeostasis model of assessment-insulin esistance; *: significant.

4 10 Saha Mohamed Ismail et al.: Chemein and Vaspin as Noninvasive Biomakes in the athogenesis and Diagnosis of Non- Alcoholic Fatty Live Disease Table 3. The compaative study between simple steatosis and NASH goups aametes Simple steatosis (n=15) NASH (n-15) 2 BMI (Kg/M 2 ) 30 (Kg/ M 2 ) ± (Kg/ M 2 ) 34.33± Tiglyceides mg/dl ± ± * Total cholesteol mg/dl ± ± ALT Iu/l 22.53± ± * AST Iu/l 24.27± ± * FBG mg/dl ± ± Insulin uiu/ml 13.43± ± * HOMA-IR 12.07± ± Vaspin ng/ml 4.27± ± Chemein ng/ml 0.71± ± BMI: body mass index; ALT: alanine tansaminase; AST: aspatate aminotansfease; FBG: fasting blood glucose; HOMA-IR: homeostasis model of assessment-insulin esistance; *: significant. Table 4. The coelation study of Vaspin and Chemein with othe paametes in the simple steatosis goup and NASH goup aametes Simple steatosis goup Chemein NASH goup Simple steatosis goup Vaspin NASH goup Age/ yea Weight/ kg Height/ mete BMI * * Tiglyceides mg/dl * * Total cholesteol mg/dl * * FBG mg/dl ALT Iu/l * * AST Iu/l * * Insulin level uiu/ml * * HOMA-IR * * BMI: body mass index; ALT: alanine tansaminase; AST: aspatate aminotansfease; FBG: fasting blood glucose; HOMA-IR: homeostasis model of assessment-insulin esistance; *: significant. Figue 1. A. Vaspin statuses in goup s studies. B. Chemein statuses in goup s studies

5 Ameican Jounal of Medicine and Medical Sciences 2019, 9(1): Table 2 also shows that the vaspin level was significantly deceased in the simple steatosis goup (4.27± 1.86) compaed to the healthy contol goup (6.87±6.19) (p=0.035). While thee was no significant diffeence when compaing the simple steatosis goup (4.27± 1.86) with the NASH goup (3.37±3.61) (p=0.398) (table 3, Fig, 1A). Regading chemein, thee was a significant incease of chemein in the simple steatosis goup (0.71± 0.47) compaed to the healthy contol goup (0.25±0.19) (p=0.002) (table 2, Fig1B). But thee was a non-significant diffeence when compaing between the NASH goup (0.78±0.48) and the simple steatosis (0.71±0.47) goup (p=0.578) (Table 3, Fig. 1B). Table 4 shows that the simple steatosis and NASH-elated paametes, BMI, fasting insulin levels, tiglyceides, total cholesteol, and HOMA-IR, wee positively coelated with the seum chemein in the simple steatosis goup and the NASH goup. Also, thee was a positive association of chemein with ALT and AST, wheeas no association was detected with fasting plasma glucose (Table 4). The coelation study of vaspin with BMI, fasting insulin levels, tiglyceides, total cholesteol, ALT, AST and HOMA-IR evealed no association in the simple steatosis goup o the NASH goup (Table 4). 4. Discussion The data egading the ole of chemein and vaspin in NAFLD is limited, paticulaly in obese patients. The adipokines ae in balance when the adipose tissue is in the nomal ange, but the balance tends to be disupted when the adipose tissues expand. Ou study aimed to evaluate the ole of chemein and vaspin in the pathogenesis and diagnosis of simple steatosis and NASH as non-invasive makes in obese patients. Ou esults demonstated that the ciculating chemein was significantly elevated and vaspin was significantly educed in the obese simple steatosis and NASH goups compaed to non-obese healthy contol subjects. Howeve, both chemein and vaspin fo simple steatosis and NASH patients wee not significantly diffeent. In addition, chemein was positively coelated with body mass index, tiglyceide, total cholesteol, ALT, AST, insulin level, and HOMA-IR in the obese simple steatosis and NASH goups. Howeve, we did not find any coelation of vaspin with body mass index, tiglyceide, total cholesteol, ALT, AST, insulin level, and HOMA-IR in the obese simple steatosis and NASH goups. These findings indicate that obese patients with elevated chemein and educed vaspin ae at an inceased isk of developing NAFLD. Theefoe, the chemein and vaspin play impotant oles in the pathogenesis of NAFLD. This esult is in accodance with othe studies that found that NAFLD was associated with high ciculating chemein and low ciculating vaspin levels [12-14]. Futhemoe, a ecent study by Kukla et al. [15] showed an incease in the chemein level in mobidly obese NAFLD women, and the chemein level in hepatic vein samples was highe than that in systemic ciculation in cihotic patients, which indicated that the chemein was poduced by the live as by the adipose tissue. Anothe study epoted that chemein was appositively coelated with total cholesteol, tiglyceides, fasting insulin and the high sensitive C-eactive potein [16, 17]. Theefoe, chemein appeas to be elated to some featues of the metabolic syndome, and plays a ole in the pathogenesis of NAFLD [18]. This could be explained by obesity being a state of low-gade subclinical systemic inflammation, with the pesence high ciculating pion, inflammatoy cytokines; this inflamed adipose tissue might play a ole in inceased chemein secetion by adipocytes [19]. Moeove, chemein induces the initiation of the innate immune esponse and inflammatoy pocesses by attacting macophages. Theefoe, the po-inflammatoy and immune-modulating popeties of chemein may be closely associated with simple steatosis pathogenesis [18]. The elevation of chemein with NAFLD may be explained by the adipocytes being esponsible fo stoing excess caloies. Theefoe, any enegy imbalance leads to adipocyte hypeplasia, which leads to inceased ciculatoy pion-inflammatoy cytokines [19]. Futhemoe, elevated seum chemein may incease the insulin esistance though the educing effect of chemein on the insulin ecepto signaling cascades, which leads to insulin esistance o aggavates it; this was confimed by the association between chemein and HOMA-IR [20]. On the othe hand, few authos epoted no significant diffeence in chemein levels between NAFLD and the contol goups [21]. Howeve, Buechle et al [22] epoted that the chemein was educed in hepatic cihosis. Moeove, anothe study found no significant coelations between seum chemein levels with fasting blood glucose and HOMA-IR [23]. Ou study indicated that chemein plays a ole in the pogession of simple steatosis to NASH and anothe study suggested that the chemein plays a ole in hepatocyte ballooning degeneation in NAFLD patients, which indicates a possible connection between live chemein and the development of NASH [24]. Howeve, in ou study, we did not find that chemein plays any ole in the pogession of simple steatosis to NASH. In the case of vaspin, a study by olygons et al [25] found low s of ciculating vaspin in NAFLD patients compaed to the contol, but the amount of ciculating vaspin was simila in simple steatosis and NASH patients, which was in line with ou esult. While Kukla M. et al [26] found the amount of vaspin was less in simple steatosis patients compaed to the contol, but thee was a significant incease in vaspin in obese (BMI 40) NASH and in advanced live fibosis patients compaed to simple steatosis. Also, they [26] did not find any association between vaspin and lipid paametes in NAFLD. Unfotunately, ou study did not include live fibosis o cihosis. Howeve, Genc et al. [27] showed thee was no significant diffeence in the seum concentation of vaspin between patients with NAFLD and

6 12 Saha Mohamed Ismail et al.: Chemein and Vaspin as Noninvasive Biomakes in the athogenesis and Diagnosis of Non- Alcoholic Fatty Live Disease healthy contols. On the othe hand, some authos found highe vaspin levels in NAFLD patients compaed to healthy contols [28]. Theefoe, ou esult with egad to vaspin evealed that a low level of vaspin was a isk facto fo NAFLD development, which could be explained by the anti-inflammatoy and anti-oxidant effect of vaspin [29]. The limitation of ou study: 1. The numbe of patients in the study sample was small. 2. A limitation aose fom the NAFLD patients who disageed to pefom a live biopsy. 5. Conclusions Ou study confimed that the chemein level, and not the vespin level, is stongly associated with obese NAFLD patients and metabolic syndome elements. Both vespin and chemein may play a ole in the pathogenesis of NAFLD, but they did not play a ole in the pogession fom simple steatosis to NASH. Theefoe, we cannot depend on eithe of the pevious biomakes to diffeentiated NASH fom simple steatosis, and a live biopsy emains the moe accuate method fo diagnosis of NASH. Futhe studies will be equied: (I) to undestand why some simple steatosis patients pogess to NASH, and anothe to lean why some simple steatosis patients can pogess. (II) Additional study is needed with a lage numbe of patients which also contain lean non-diabetic NAFLD patients. ACKNOWLEDGEMENTS The authos would like to thank the staff of the adiological depatment, who was helpful in pefoming and intepeting the abdominal ultasound. REFERENCES [1] Eickson SK (2009). Nonalcoholic fatty live disease. J Lipid Res. 50: [2] Chalasani N, Younossi Z, Lavine JE, Chalton M, Cusi K, Rinella M. et al. (2012). The diagnosis and management of nonalcoholic fatty live disease: actice guidance fom the Ameican Association fo the Study of live disease. Gastoenteology. 142 (7): [3] EASL-EASD-EASO (2016). 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