SERUM POSITIVITY OF ANA AND ASMA AMONG KHAT AND NONKHAT CHEWERS AS MARKERS FOR AUTOIMMUNE HEPATITIS TYPE 1

Objectives: Autoimmune hepatitis (AIH) is a necroinflammatory liver disease of unknown etiology. It has been proposed that certain herbs such as black cohosh and dai-saiko might trigger AIH. Khat is an ever green tree whose leaves have been chewed by people in Yemen where AIH is common. Aim of present study was to measure antinuclear antibodies (ANA) and anti-smooth muscle antibodies (ASMA) as serum markers for AIH type1 in healthy people who chew and do not chew khat. It also aimed to determine some other risk factors for ANA and ASMA positivity. Methods: A total of 100 healthy individuals were enrolled in this study. They were divided into: Daily khat chewers and non-khat chewers. Three ml peripheral blood was withdrawn from each participant. Blood samples were tested for ANA and ASMA using indirect immunofluorescence assay. Results: The age of khat chewers ranged from 30-60 years with mean age 40.4±7.6 years. Non-khat chewer’s age ranged from 3057 years with mean age 39.9±6.2 years. The majority of khat chewers and non-chewers were in age groups 30-39 and 40-49 years old. There is no association between khat chewing and ANA or ASMA serum positivity (χ=0.33, P=0.39 and χ=1.5, P=0.16; respectively). Khat chewing has no association with ANA and ASMA positivity. Nevertheless, ANA and ASMA positivity has an association with aging. Conclusion: Study concludes that Khat chewing has no association with ANA and ASMA positivity. Nevertheless, ANA and ASMA positivity has an association with aging.


INTRODUCTION
Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease in which the immune system attacks liver cells 1 . AIH is a relatively uncommon disease. It can occur any age but it is most common in young females. It is characterized by elevated levels of serum transaminases, seropositivity for auto antibodies, a histological picture of interface hepatitis and wellresponse to immunosuppression therapy [2][3][4][5] . According to the pattern of autoantibodies in patient's serum, AIH is subdivided into two types. Type 1 AIH is the most common form of AIH, (80% of AIH cases). It is commonly termed as a classic AIH. Type 1 AIH is mainly characterized by circulating antinuclear antibodies (ANA), anti-smooth muscle antibodies (SMA), atypical perinuclear antineutrophilic cytoplasmic antibodies (pANCA), and soluble liver antigen/liver pancreas (SLA/LP). [6][7][8][9] Type 2 AIH is a rare disorder and is mainly pediatric; average age is around 10 years. AIH type 2 is characterized by presence of liver/kidney microsomal antibodies type 1, 2 or 3 (LKM-1, 2 and 3) and liver cytosol type 1 (anti-LC-1) 6,9,10 .
The etiology of AIH remains unknown but is postulated to be related to an autoimmune process triggered by either genetic predisposition or environmental agents. AIH is strongly associated with human leukocyte antigens (HLA-DR3 and HLA-DR4, HLA-DR6) [11][12][13][14] . Environmental factors are thought to be the triggering factors for the development of AIH in genetically susceptible individuals. These environmental factors could be viruses, drugs, chemicals or certain herbs [15][16][17] . Khat (Catha edulis) is an herb its leaves have been chewed for centuries by people who live in the Eastern part of Africa and Yemen. The khat leaves chewed daily by a high proportion of the adult population in Yemen for the pleasant mild stimulant effect. 18 Khat has recently been recognized as a triggering agent for a severe form of AIH in young males in Yemen and Somalia [19][20][21][22][23] . Our study aimed to measure serum ANA and ASMA as early markers for AIH type1 in Yemeni healthy people who chew khat.

SUBJECTS AND METHODS
Our study was a cross-sectional study which was conducted for four months from March to June, 2014. It was carried out in Sana'a city, the capital of Yemen. One hundred individuals were enrolled in the study divided into two groups. First group involved 50 individuals who daily chew khat for at least five years. Second group include 50 persons who never chewed khat. According to gender, the two previous groups were subdivided into 25 males and 25 females. Participants were selected randomly from khat or social sessions. Study was approved by Faculty of Medicine and Health Sciences, University of Science and Technology. Written consent was obtained from each participant.

Inclusion criteria
Any apparently healthy individual who was equal or over 30 years did not complain any signs and symptoms and did not take any medications.

Exclusion criteria
Carriers of hepatitis viruses, pregnant women or females who ingested oral contraceptive pills were excluded from the two study groups. Data from each individual were collected using predesigned questionnaire. Three ml of venous blood was withdrawn from each person into a plain tube. The sample was allowed to clot at room temperature and was centrifuged at 3500 rpm for five minutes, and then serum was separated from each sample into Eppendorf tubes and stored at -20°C till tested. Serum ANA and ASMA were measured using indirect immunofluorescence assay on HEp2 cells according to manufacturer instructions (Innova, San Diego, USA). The nuclear staining patterns of ANA test were detected on liver and gastric cells. Nuclear staining patterns observed on theses tissues include: homogenous, centromere, speckled, peripheral and nucleolar. Sera were considered as positive for ANA when titers were >1/80 and positive for ASMA when titers were >1/40.

Data analysis
Statistical data analysis was done using SPSS (version 15). Data were presented as numbers and percentages. Significant association was measured using χ 2 and P value tests. P-value less than 0.05 was considered statistical significant.

DISCUSSION
AIH type 1 occurs primarily in adults with a female predominance. Pathogenesis of AIH type 1 is unknown, though both genetic and environmental factors are likely to predispose to the disease. Circulating ANA and ASMA are keys to the diagnosis of AIH type 1 24 . Khat chewing is proposed to cause AIH 25 . Hence our study aimed to measure serum ANA and ASMA among khat chewers and non-khat chewers as early markers for liver damage by khat chewing. This study revealed that chewing khat had no association with increased serum level of ANA or ASMA. Although Riyaz et al., 25 reported khat to be a possible cause of AIH, our findings suggest that khat chewing may have no direct role in the induction of ANA and ASMA production and imply Khat interaction with other factors such as genetic susceptibility and khat abuse in the pathogenesis of AIH. Another explanation is that Khat risks on hepatocytes may be due to other mechanisms such as hepatotoxicity or increase the rate of hepatocytes apoptosis rather than autoantibody production [26][27][28][29] . Our study showed that the positivity of ANA and ASMA tests were increasing by increase age. This result is supported by studies which reported serum ANA prevalence in the general population to be highest in elders [30][31][32] . Nevertheless, other studies found no association between serum ANA with age [33][34] . Equal males and females were enrolled in this study. No statistical differences were found between the ANA ISSN: 2456- 8058 22 CODEN (USA): UJPRA3 and ASMA results in both men and women. This finding is in agreement with other study which reported no association between ANAs and gender 31,33,35,36 . In contrast, other studies reported presence of ANA and/or ASMA to be more predominant in females than in males 30,34,35,37 . Current study demonstrates that smoking had no association with ANA and ASMA positivity. Similar results are reported by other studies.T 30,33,36,38,39 . On the other hand, this finding is in disagreement with Karabulut et al, who reported association between ANAs positivity and smoking 40 . Many khat farmers use pesticides for better khat cultivation which may have harmful effects on liver cells. Serum positivity of ANA was more frequent among participants who did not wash khat; however, the difference was not statistical significant which may be attributed to small number of the studied sample.

CONCLUSION
Khat chewing has no association with ANA and ASMA positivity. Nevertheless, ANA and ASMA positivity has an association with aging. Therefore, adults over 30 years old should be screened for ANA and ASMA positivity because such individuals are at risk for development of autoimmune diseases.

Limitations of the study
Obstacles which we encountered during the study it was difficult to determine type and amount of khat as well as time for how long chewing.

ACKNOWLEDGMENTS
This work has been supported by grants from Almughales Organization.

CONFLICTS OF INTEREST
The authors declared no conflicts of interest.