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1 Sokoto Journal of Medical Laboratory Science 2017; 2(1): Original Research SJMLS-2(1) Pancreatic enzymes activities in Human Immunodeficiency virus-1 infected subjects in Benin City, Nigeria. Emokpae, M.A. * 1, Nwokedi D.O. 1 Department of Medical Laboratory Science, School of Basic Medical Sciences, College of Medical Sciences, University of Benin, Benin City, Nigeria 1. Author for correspondence*: mathias.emokpae@uniben.edu/ Abstract Pancreatitis is a known complication of human immunodeficiency virus-1 (HIV-1) infection even in this era of highly active anti-retroviral therapy (HAART). There is however conflicting report regarding the activity of serum amylase, lipase and urine amylase in HIV-1 positive subjects on HAART. Despite the fact that pancreatitis has been reported in HIV-1 infection, serum levels of these enzymes are rarely routinely evaluated as part of the clinical assessment of these patients. The aim of this study was to evaluate the activities of serum amylase, lipase and urinary amylase in HIV-1 infected subjects. A total of 150 participants comprising of 100 HIV-1 positive subjects (40 males and 60 females) and 50 HIV-1 negative controls (20 males and 30 females) were enrolled in the study. Serum amylase, lipase and urine amylase activities were assayed by spectrophotometric methods using commercially available reagent kits. Cluster of differentiation 4 (CD4 + ) cell counts were determined using BD FACSCount system (Becton and Dickinson s Company, California, USA). Statistically significant higher levels (p<0.001) of serum amylase, lipase and urine amylase (p<0.05) were observed in HIV-1 positive subject on HAART and HAART naïve compared with HIV-1 negative controls. Serum amylase correlated positively with CD4 + cell count in HIV-1 positive subjects on HAART. HIV-1 positive subjects evaluated in the study had higher activity levels of pancreatic enzymes. Pancreatitis should always be considered in HIV-1 positive subjects especially in those with abdominal pain. Therefore, the pancreatic enzymes activities of the HIV-1 positive subjects should be routinely evaluated in this group of subjects. Keywords: Human Immunodeficiency virus-1 infection, serum amylase, lipase and urine amylase. Introduction Human immunodeficiency virus-1 (HIV-1) infection is one of the most prevalent chronic infectious diseases, causing significant morbidity and mortality worldwide. According to the World Health Organization estimation, 3.2 million persons are infected with HIV/AIDS in Nigeria, giving a prevalence of 3.2% (UNAIDS, 2014). Studies have shown that apart from symptomatic increase in pancreatic enzymes, unexplained elevation of pancreatic enzymes without clinical evidence of pancreatitis have been reported (Argiris et al., 1999; Bonacini, 1991; Dowell et al., 1990; Schwartz and Brandt, 1989). Whereas some authors attributed the elevation of the pancreatic enzymes to pancreatic pathology as a result of opportunistic infections (Argiris et al., 1999) and HIV virus (Moore and Schneider, 2013). Others however, attributed such elevation to the use of antiretroviral (monotherapy) drugs (Maxson et al., 1992). The introduction of highly active antiretroviral therapy (HAART) in the treatment of the disease has reduced mortality and morbidity of the infected subjects. It is a combination of drugs with different actions. They are divided into 5 classes which includes Nucleoside/Nucleotide reverse transcriptase Page 58 SJMLS Volume 2, Number 1 March, 2017

2 ISSN: SJMLS inhibitors (NRTIs/NtRTIs), Non-nucleotide reverse transcriptase inhibitors (NNRTIs), Fusion inhibitors (FI), Integrase inhibitors (II) and Protease inhibitors (PI) (Teixeira et al., 2011). They help to reduce morbidity and to prolong the lives of persons living with HIV, enhance immunity by increasing CD4 count, achieve rapid and sustained suppression of viral load and also reduce the risk of transmission from mother to child (Anderson et al., 2008; Reisler et al., 2005). Despite the fact that HAART has been effective in the management of HIV, HIV-infected patients continue to have multiple potential risk factors for subclinical or clinical pancreatic involvement (Reisler et al., 2005; Moore et al., 2001). In some studies, it was reported that HAART has a wide range of adverse effects, which also includes drug-induced pancreatitis (Teixeira et al., 2011). Elevated amylase and lipase as a result of long- term antiretroviral therapy can lead to acute pancreatitis which is potentially a life-threatening condition that is characterized clinically by abdominal pain, nausea and vomiting (Sah et al., 2012). The effects of HAART on the levels of pancreatic enzymes activities in HIV-1 infected subjects are not completely known. Earlier studies have reported elevated levels of pancreatic enzymes in HIV-1 positive subjects (Argiris et al., 1999; Cappell, 1994; Bonacini, 1991; Dowell et al., 1990 and Schwartz and Brandt, 1989). Recently, a contradicting observation was reported by Szoke and Colleagues (2016). They observed that the frequency of pancreatic amylase increase did not significantly differ between HIV-1 positive subjects on HAART and HIV-1 positive HAART naïve. Also, it was observed that in some HIV-1 positive subjects with elevated serum amylase, the lipase activity was normal. Since the earlier publications that reported elevated levels of pancreatic enzymes were conducted either before the introduction of antiretroviral drugs or in those that were on antiretroviral monotherapy, this present study seeks to investigate the activities of pancreatic amylase and lipase in HIV-1 positive subjects on HAART and HAART naïve. Materials and Methods Selection of study participants: All study participants who met the inclusion criteria were randomly selected from the anti-retroviral therapy clinic of Central Hospital, Benin City from May to August The participants comprised of 50 confirmed HIV-1 positive individuals receiving HAART (20 males and 30 females with age range of 20-55years), 50 newly diagnosed HIV-1 positive HAART naïve (20 males and 30 females with age range of years) and 50 HIV-1 negative apparently healthy individuals (20 males and 30 females with age range of years). Ethical consideration: The protocol of the study was reviewed and approved by the Edo State Ministry of Health (ethical code HM.1208/112 dated 12 th May 2016). Inclusion and exclusion criteria Confirmed HIV-1 positive subjects who were on routine visit to the anti-retroviral clinic of the Central Hospital and who gave verbal informed consent were included in the study. HIV negative subjects who had gastrointestinal illness or infection, those who did not give consent or self-reported HIV-1 positive individuals as well as those with AIDS were excluded. Sample Collection Clinical data with other relevant demographic information were obtained. Under strict aseptic precautions, 5ml of venous blood was obtained from each participant and 2mL was transferred into EDTA container which was used for CD4 cell count using FASCount auto-analyzer (Becton Dickinson, USA) while the remaining 3mL was emptied into a plain tube. The sample collected in the plain tube was allowed to clot at room temperature and was centrifuged at 1000g for 5 minutes. The serum was separated into another plain tube and kept frozen at - 20 o C until analyzed. The amylase and lipase activities were assayed using commercially available reagent kits (Agape Diagnostics, Switzerland). Random urine samples were also collected in universal containers and were used for the assay of urine amylase activity. The reference ranges of serum amylase were 30-86U/L, lipase 20-80U/L and urine amylase was U/L. SJMLS Volume 2, Number 1 March, 2017 Page 59

3 Quality Control: In order to ensure accurate and precise results, quality control sera were included in the assays. The control sera used were: ACUSERA Premium plus (RANDOX, UK) (Cat No: AY1580): For amylase with a measuring range of U/L. ACUSERA Premium 3 (Cat No: LI3837), for lipase with measuring range of U/L and BD FACSCount control beads for CD4 + cell counts (0 beads/ μl; 250 beads/ μl and 1000 beads/ μl). Statistical analysis Statistical analysis was done using the statistical package for social sciences (SPSS) version All values were expressed as Mean ± Standard error of the mean. The Students -test and analysis of variance (ANOVA) were used to compare the mean values of the observed measured parameters between the groups. Pearson correlation coefficient was used to test the association between CD4 + cell counts with enzymes. A p-value of 0.05 was considered to be statistically significant. Results The results are presented in tables 1, 2 and 3. Table 1 shows that the serum amylase and lipase activities were significantly higher (p<0.001) in HIV-1 positive on HAART than HAART naïve and controls. Also, serum amylase and lipase activities were significantly higher (p<0.001) in HIV-1 positive HAART naïve compared with controls. Urine amylase activity was significantly higher (p<0.05) in HIV-1 positive subjects on HAART compared with HIV-1 positive HAART naïve and control subjects. There was no significant difference in the measured enzymes activities between male and female HIV-1 positive subjects (table 2). Table 3 shows that the CD4 + cell count correlated positively with serum amylase while serum lipase and urine amylase activities showed no significant correlation with CD4 + count. Table 1: Comparison of measured enzyme activities in HIV-1 positive individuals on HAART, HAARTnaïve and controls (mean±sem). Measured enzyme HIV-1 positive HIV-1 positive HIV-1 negative p-value activities subjects on subjects control subjects HAART (n=50) HAART naïve (n=50) (n=50) Serum amylase (U/L) 128±5.0 a 84±4 a 63± Serum lipase (U/L) 83±4 a 69±3 a 55± Urine amylase (U/L) 178±14 b 128±8 c 136± CD4 + count(cells/µl) 488.1±12.7 ab 298.8±9.9 a 789.9± a=p<0.001; b=p<0.05; c=p>0.05 Table 2: Comparison of measured enzyme activities between male and female HIV-1 positive subjects (mean±sem). Measured enzyme activities HIV-1positive HIV-1 positive p-value male subjects female subjects (n=40) (n=60) Serum amylase (U/L) 96±6 98± Serum lipase (U/L) 69±3 74± Urine amylase (U/L) 160±12 142± CD4 + count(cells/µl) 248±10 344± Page 60 SJMLS Volume 2, Number 1 March, 2017

4 ISSN: SJMLS Table 3: Correlation of CD4 + cell count with measured enzyme activities Measured enzyme activities R-values p-value Serum amylase Serum lipase Urine amylase Discussion Amylase and lipase are enzymes that are essential for health and wellbeing of human beings as they are involved in the digestion of carbohydrates and lipids respectively. Pancreatitis is a common complication among HIV- 1 infected individuals on HAART and a common cause of morbidity and occasional mortality (Dassopoulos and Ehrenpreis, 1999). The conflicting reports in literature regarding the activity levels of pancreatic enzymes in HIV-1 positive individuals were attributed to inconsistencies in the collection and analysis of clinical and laboratory data as well as lack of long- term follow-up (Raza et al., 2013). It was therefore suggested that pancreatitis among HIV-1 infected subjects on HAART require frequent revisits (Cappell and Hassan, 1993). In this study, serum amylase and lipase activities were significantly higher in HIV-1 positive subjects than controls. Also, the activities of serum amylase and lipase were significantly higher (p<0.001) in HIV-1 positive subjects on HAART than HIV-1 positive HAART naïve subjects. The observed higher enzyme activities (serum amylase and lipase) in HIV-1 positive subjects observed in this study is in agreement with previous studies (Manfrendi et al., 2004; Moore et al., 2001; Argiris et al., 1999; Cappell, 1994 and Maxson et al., 1992) which indicated a significant increase in serum amylase and serum lipase in HIV-positive subjects and attributed the cause of the increase to HIV infection and medications used in the treatment of opportunistic infections. The reasons for the significantly higher activities levels of serum amylase and serum lipase in HIV-1 positive naïve subjects are not completely clear. It was reported that HIV-1 infection may cause elevation of pancreatic enzymes even without HAART treatment. Elevation of pancreatic enzymes activities was reported in subjects suffering from an acute HIV-1 infection with HIV negative serology but very high viral load (Moore and Schneider, 2013). This was an indication that the observed elevation of pancreatic enzymes may be due to HIV infection (Moore and Schneider, 2013). On the other hand, our observation is at variance with a previous report (Szoke et al., 2016). These authors observed that the frequency of pancreatic amylase increase was not significantly different between HIV-1 positive and the general population (HIV-1 negative individuals). In the same vein, they reported that there was no significant difference between HIV-1 positive subjects on HAART and HIV-1 positive HAART naïve. They further observed that in about 48% of HIV-1 positive subjects with high serum amylase activity, lipase was normal suggesting that the increased amylase was not of pancreatic origin (Szoke et al., 2016). Our observation may suggest the presence of silent pancreatic involvement which was indicated by higher serum amylase and lipase activities than controls. In a study of biochemical assessment of pancreatic disease in HIV infected men, it was observed that total amylase is a poor indicator of pancreatic disease. Pancreatic amylase was therefore suggested as a better marker than traditional total amylase especially when lipase activity is added. The addition of lipase assay could improve the biochemical identification of HIV-1 subjects with possible pancreatic disease (Hancock et al., 1997). The significantly higher levels of serum amylase, lipase and urine amylase in HIV-1 positive subjects on HAART compared to HIV-positive HAART naïve subjects may be due to the effects of HAART drugs. The participants were on combination therapy, which included Lamivudine, Nevirapine and Zidovudine, as well as Efavirenz, Lamivudine and Tenofovir. Previous studies have associated pancreatitis with Nucleosidase Reverse Transcriptase inhibitors (NRTIs) especially Didanosine and Stavudine (Dassopoulos and SJMLS Volume 2, Number 1 March, 2017 Page 61

5 Ehrenpreis, 1999 and Cappell and Hassan, 1993). Other authors have questioned the relationship between pancreatitis and use of combination HAART therapy. Raza and Colleagues (2013) reported that only one third of their study participants with pancreatitis were on HAART and only a handful of the subjects were on pneumocystis carinii pneumonia (PCP) prophylaxis. This may suggest other risk factors other than HAART use that are contributing to the development of acute pancreatitis in HIV-1 infection (Oliveira et al., 2014). Our observation of higher pancreatic enzymes levels in HIV-1 infected subjects on HAART compared to HAART naïve is consistent with previous studies (Marques et al., 2015; Chehter, 2014 and Chehter et al., 2000). These authors reported in autopsy studies that morphological changes in endocrine pancreas of patients who were taking HAART did take place. The observed changes in pancreas particularly in the number and volume of pancreatic islets in individuals who were on HAART compared to HAART naïve (Chehter, 2014 and Chehter et al., 2000). Reduction in zymogen granules in more than 50% of the arcinar cells, parenchymal atrophy, steatosis and nuclear abnormalities were also reported in HAART naïve HIV-1 patients (Chehter et al., 2000 and Chehter, 1997). Previous studies observed that patients who used Zidovudine in combination regimen had more than two-fold pancreatic enzymes elevation (Agiris et al., 1999). Also, other studies attributed the elevation of serum amylase and lipase activities in HIV-1 positive subjects on HAART to antiretroviral treatment (drugs- induced pancreatitis or HAART toxicity) (Manfrendi et al., 2004; Moore et al., 2001). There was no gender dimorphism in the activities of the measured enzymes since no significant difference in the measured variables between males and female (p>0.05) was observed. This is in agreement with a previous study (Smith et al., 2008), the authors observed no gender dimorphism in pancreatic enzymes activities. This is in contrast with another study (Onochie et al., 2007), where the authors observed a significantly higher levels of serum amylase activity among the male HIV-1 positive subjects on HAART than their female counterparts. Conversely, a significantly higher levels of pancreatic enzymes activities was reported among females than males (p<0.05) (Riedel et al., 2008). A positive correlation was observed between serum amylase (r = 0.386, p = 0.001) and CD4 + count. This indicated that serum amylase increased with increasing levels of CD4 + count. The positive correlation is in agreement with a study that was carried out elsewhere in Nigeria (Onochie et al., 2007). It was reported that HAART succeeded in improving the immune status of the patients by increasing their CD4 + count at the expense of the pancreas which was affected negatively by the drug use. In this study, it was observed that while antiretroviral treatment was efficacious in improving the immune system of the infected subjects, it could be harmful, due to its toxic effects and including HAART drug-induced pancreatitis. This can be seen in the significant elevation of pancreatic enzymes activities (even though the levels of the measured enzymes were within reference range) in HIVpositive subjects on HAART. HIV-1 positive HAART naïve subjects are also vulnerable to pancreatitis since enzymes activities were also elevated in this group of participants. Conclusion Based on the results of this study, it could be concluded that HIV-1 positive subjects had higher levels of pancreatic enzymes activities, which may be due to HAART use and HIV infection. The pancreatic enzymes activities of the HIV-1 positive patients should be routinely monitored, especially in patients on HAART and those showing symptoms of pancreatitis. References Anderson, F., Thomson, S.R., Clarke, D.L. and Loots, E. (2008). Acute pancreatitis: demographics, aetiological factors and outcomes in a regional hospital in South Africa. South African Journal of Surgery; 46(3): Argiris, A., Mathur-Wagh, U., Wilet, I. and Mildvan, D. (1999). Abnormalities of serum Page 62 SJMLS Volume 2, Number 1 March, 2017

6 ISSN: SJMLS amylase and lipase in HIV-positive patients. American Journal of Enterology; 94(5): Bonacini, M. (1991). Pancreatic involvement in human immunodeficiency virus infection. Journal of Clinical Gastroenterology; 13: Cappell, M.S. and Hassan, T. (1993). Pancreatic disease in AIDS: a review. Journal of Clinical Gastroenterology; 17(3): Cappell, M.S. (1994). The pancreas in AIDS. In: Broder, S., Merigan, T.C and Bolognesi, D. eds. Textbook of AIDS Medicine. Williams and Wilkins, Baltimore: Chehter, E.Z., Longo, M.A., Laudanna, A.A., Duarte, M.I. (2000). Involvement of the pancreas in AIDS: a prospective study of 109 post-mortems. AIDS; 14: Chehter, E.Z. (1997). Acquired Immunodeficiency Syndrome (AIDS) and pancreas: a prospective study of clinical and pathological features [PhD Thesis]: Faculty of Medicine, USP. São Paulo. Chehter, E.Z (2014). AIDS and Pancreas in the HAART era: a cross sectional study. International Archive of Medicine; 6:28. Dassopoulos, T. and Ehrenpreis, E.D. (1999). Acute pancreatitis in human immunodeficiency virus infected patients: a review. American Journal of Medicine; 107: Dowell, S.F., Holt, E.A. and Murphy, F.K. (1996). Pancreatitis associated with human immunodeficiency virus infection: A matched case- control study. Journal of Infectious Diseases; 92: Hancock, M.R., Smith, N.A., Hawkins, D.A., Gazzard, B. and Ball, S.G. (1997). Biochemical Assessment of pancreatic disease in Human Immunodeficiency Virus infected men. Journal of Clinical Pathology; 50: Manfredi, R. and Calza, L. (2008). HIV infection and the pancrease : risk factors and potential management guidelines. International Journal of STD and AIDS; 19(2): Manfredi, R., Calza, L. and Chiodo, F. (2004). A case-control study of HIV-associated pancreatic abnormalities during HAART era. Focus on emerging risk factors and specific management. European Journal of Medical Research; 9(12): Marques, L.M., Hurtado, R.M.Y. and Chehter, E.Z. (2015). HIV and pancreas in the HAART Era: Endocrinological patterns. Journal of Pancreas; 16(16): Maxson, C.J., Greenfield, S.M. and Turner, J.L. (1992). Acute pancreatitis as a common complication of 29, 3 9-dideoxyinosine therapy in the acquired immunodeficiency syndrome. The American Journal of Gastroenterology. 87: Moore, J.R. and Schneider, S.M. (2013). Acute Human Immunodeficiency Virus (HIV) infection presenting with fever, elevated amylase/lipase and haematologic abnormalities. Journal of Emergency Medicine; 44(5): e341- e344. Moore, R.D., Keruly, J.C. and Chaisson, R.E. (2001). Incidence of pancreatitis in HIVinfected patients receiving nucleoside reverse transcriptase inhibitor drugs. AIDS; 15: Oliveira, N.M., Ferreira, F.A.Y., Yonamine, R.Y. and Chehter, E.Z. (2014). Antiretroviral drugs and acute pancreatitis in HIV/AIDS patients: Is there any association? A literature review. Einstein; 12(1): Onochie, A.U., Okaka, N.C., Onyenekwe, C. C., Meludu, S.C., Ukibe, N., Igwegbe, A.O. and Ilika, A. (2007). Pattern of serum amylase activity in HIV-positive seropositive subjects on antiretroviral therapy. Journal of Biomedical Investigation. 5(1): Raza, S., Chaudhry, N.A., Brown, J.D., Aghaiem S., Rezai, D., Khan, A., Tan, P.D.L. and Berger, B.J. (2013). To study the clinical, biochemical and radiological feactures of acute pancreatitis in HIV and AIDS. Journal of Clinical Medicine Research; 5(1): Reisler, R.B., Murphy, R.L., Redfield, R.R. and Parker, R.A. (2005). Incidence of pancreatitis in HIV-1-infected individuals enrolled in 20 adult AIDS clinical trials group studies: lessons learned. Journal of Acquired Immune Deficiency Syndrome; 39(2): SJMLS Volume 2, Number 1 March, 2017 Page 63

7 Riedel, D.J., Gebo, K.A., Moore, R.D., Lucas, G.M. (2008). A ten-year analysis of the incidence and risk factors for acute pancreatitis requiring hospitalization in an urban HIV clinical cohort. AIDS Patient Care STDS. 22(2): Riedel, D.J., Gebo, K.A., Moore, R.D. and Lucas, G.M. (2008). A ten-year analysis of the incidence and risk factors for acute pancreatitis requiring hospitalization in an urban HIV clinical cohort. AIDS; 22 (2): Sah, R. P., Garg, P. and Saluja, A.K. (2012). Pathogenic mechanisms of acute pancreatitis. Current Opinion in Gastroenterology; 28: Schwartz, M.S. and Brandt, L.J. (1989). The spectrum of pancreatic disorders in patients with the acquired immune deficiency syndrome. American Journal of Gastroenterology; 84: Smith, C.J., Olsen, C.H., Mocroft, A., Viard, J.P., Staszewski, S. and Panos, G. (2008). The role of antiretroviral therapy in the incidence of pancreatitis in HIV- positive individuals in the EuroSIDA study. AIDS; 22: Szoke, D., Ridolfo, A., Valente, C., Galli, M. and Panteghini, M. (2016). Frequency of Pancreatic hyperamylasaemia in Human immunodeficiency virus-positive patients in the Highly Active Antiretroviral Therapy Era. American Journal of Clinical Pathology; 145(1): Teixeira, C., Gomes, J. R., Gomes, P., Maurel, F. and Barbault, F. (2011). Viral surface glycoproteins, gp120 and gp 41, as potential drug targets against HIV-1, brief overview one quarter of a century past the approval of Zidovudine. European Journal of Medicinal Chemistry; 46: UNAIDS (2013). AIDS by the numbers. Available from Page 64 SJMLS Volume 2, Number 1 March, 2017