Clinical significance of serum hepcidin levels on early infectious complications in human leptospirosis

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1 Journal of Pharmaceutical and Biological Sciences ISSN: ; CODEN: JPBSEV Published by Atom and Cell Publishers All Rights Reserved Available online at: Original Article Clinical significance of serum hepcidin levels on early infectious complications in human leptospirosis Prabhusaran N 1*, Jeyaseelan TS 2, Sundhararajan A 3, Natarajaseenivasan K 4, Joseph PID 5 1 Postgraduate and Research Department of Microbiology, Chennai Medical College Hospital and Research Centre (SRM Group), Tiruchirapalli, India 2 Department of Microbiology, Ponniah Ramajayam Institute of Medical Sciences, Kancheepuram, India 3 Department of Biochemistry, Chennai Medical College Hospital and Research Centre (SRM Group), Tiruchirapalli, India 4 Department of Microbiology, School of Life Sciences, Bharathidasan University, Tiruchirapalli, India 5 Department of Microbiology, Karpaga Vinayaga Institute of Medical Sciences, Kancheepuram, India ABSTRACT Received: / Revised Accepted: / Published: Hepcidin is a recently identified peptide produced by hepatocytes and macrophages in response to inflammatory stimuli. This peptide plays an important role in iron metabolism and mediator of anaemia of inflammation has been hampered by the lack serum validated assays. The main aim of this study was to assess the serum concentration of hepcidin in serologically confirmed leptospirosis cases. Total of 45 individuals who are confirmed leptospirosis were included in this study. Blood was drawn for the estimation of hepcidin level and 20 healthy individuals were included as control population. Hepcidin was measured using ELISA method. A significant increase in serum hepcidin was observed in most of the cases. The drastic increase was observed in the mono leptospiral Grippotyphosa and Icterohaemorrhagiae MAT positive cases followed by Canicola, Javanica and Australis. The poly leptospiral serovar MAT positive cases showed maximum compared to control groups. The concentrations of hepcidin correlated positively with serum ferritin levels. Hepcidin is a new peptide that increases during inflammation in liver. No previous study reported on the determination of hepcidin levels in leptospirosis. The correlation of hepcidin with feritin could propose a new role in the investigation to determine the acute phase reactant protein. Hepcidin induction is part of the pathogenically important systemic inflammatory cascade triggered during leptospiral infection and may contribute to the establishment and maintenance of bacterial set point which is the strong predictor of the progression of leptospiral infection and its severity. Triggering the liver to produce hepcidin is pathogen specific, non-universal that have particular tissue tropism and elicit different systemic inflammatory responses. Further study required whether the role of hepcidin in infectious state determination, immunopathology and analyze as a biomarker for earliest diagnosis. Keywords: Hepcidin, leptospirosis, MAT, Clinical significance INTRODUCTION Leptospirosis is a contemporary zoonotic disease, where humans accidentally get the infections by exposing to infected materials and contaminated environment. 1 Rodents are the major reservoir that transmits the infection to other animals and humans. 2 It is a major occupational disease and more cases are noted. 3 Diagnosis by genus specific ELISA and serovar specific MAT are routine and culture is rare due to non-availability to perform cross adsorption agglutination test (CAAT). 4 The determination of hormonal and molecular biomarkers is very rare and need for earliest diagnosis and to avoid the organ specific inflammations. Hepcidin, a 25-amino acid peptide liver hormone, plays a crucial regulatory role in iron metabolism that also identified as an antimicrobial peptide. Elevated hepcidin has been observed in response to *Corresponding Author Address: Dr. Prabhusaran N, Postgraduate and Research Department of Microbiology, Chennai Medical College Hospital and Research Centre (SRM Group), Tiruchirapalli, India; leptoprabhu@gmail.com

2 inflammation and is speculated to be a causative factor in inflammatory anaemia due to induction of functional iron deficiency. Hepcidin has been suggested as a biomarker of anaemia of inflammation. Hepatic expression of hepcidin can be upregulated by iron lading as well as by inflammatory stimuli such as interleukin-6. 5,6,7,8 An accurate assessment of human serum hepcidin is critical to understand its role in anemia. 9,10 To date, the quantification of serum hepcidin levels by conventional immunological detection methods has proven problematic due to challenges in obtaining high quality antibodies which demonstrate good reproducibility. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) has been employed recently for more sensitive quantification of hepcidin; however, this method has high background levels and therefore less than optimal specificity. 11 In some studies, it was determined that the increased urinary hepcidin levels were detected in patients with chronic infections and severe inflammatory diseases. 12 Conversely, hepcidin deficiency has been associated with severe iron overload, further the hepcidin expression is suppressed by hypoxia and anaemia and induced by iron stores and inflammation. 13 The rapidity of the hepcidin response could be related to its proposed role as an inducer of hypferremia that would restrict the flow of essential iron to infecting microbes and slow their multiplication in tissues. 12 Some studies highlighted the analysis of classic iron markers associated to the storage process in hamsters experimentally infected by Leptospira interrogans. 14 There has been a great interest in developing a reliable and widely applicable diagnostic assay of the hormone in biological fluids even in infectious diseases. Being optimal for low molecular weight biomarkers, Mass spectrometry, ELISA are the classical and effective methods. The Prabhu et al., J Pharm Biol Sci 2016; 4(3): main objective of this study was to determine the quantitative measurements of bioactive serum hepcidin in serologically confirmed leptospirosis cases by ELISA method. MATERIALS AND METHODS Patient Criteria: A battery of 49 laboratory confirmed leptospirosis serum samples were impregnated for the determination of hepcidin levels. The samples included in this study have two stages in succession. 1. Screening to determine the responsible serogroups 2. The quantitative microscopic agglutination test (MAT) to determine the serum titre for every test antigen Among the sera, 4 serum samples are excluded have milky deposit (fat droplets), As per the guidelines, milky sera containing fat droplets should not be used. 15 Recommended serovars included: Initially all the leptospirosis cases were serologically confirmed by microscopic agglutination test (MAT) by using the battery of recommended leptospiral serovars 15,16,17 (Table 1). Locally isolated strains which often increase the sensitivity of the test compared with reference strains, can also be included in the battery of antigens. However the range of serovars should be limited to local strains in case the infection is due to a rare serovar or perhaps to a strain that is currently unknown in the region concerned. For this reason too, a saprophytic strain is included which cross reacts with human antibodies generated by a number of pathogenic serovars. It may also be necessary to add other serovars representing serogroups not included in the battery. 15,16,17 In this study, MAT was performed thereby mono and poly leptospiral serovar MAT positive cases were determined. Table 1: Serovars included for serology as per the recommendations Serogroup Serovar Strain Australis australis Ballico Autumnalis autumnalis Akiyami A Canicola canicola Hond Utrecht IV Grippotyphosa grippotyphosa Moskva V Hebdomadis hebdomadis Hebdomadis Icterohaemorrhagiae icterohaemorrhagiae RGA Javanica javanica Vedrat Batavia 46 Pomona pomona Pomona Pyrogenes pyrogenes Salinem Sejroe hardjo Hardjoprajitno Semaranga patoc Patoc 1 105

3 Inclusion and Exclusion status of hepcidin determination: All MAT confirmed leptospirosis serum samples were included in the screening of hepcidin levels. For determining the hepcidin levels in the serum samples, the following exclusion was done 1. Previously diagnosed nonhepatic cause of anemia other than iron deficiency 2. Evidence of active or occult bleeding 3. Blood transfusion with the past 4 months 4. History of malignancy, end stage of liver diseases or chronic hypoxia 5. Recent hospitalization of infection other than leptospirosis requiring antibiotics within the past 4 weeks. The control groups consisted of 20 adults (10 males and 10 females) aged 28.4 ± 6.6 years who are not having any diseases or liver disorders. Also ten liver disorder patients serum samples including hepatitis, liver cirrhosis and hepatic malignancy were also included to analyze the hepcidin levels for the comparativeness to leptospirosis positive cases. Serum hepcidin measurement: Quantitative measurement of bioactive hepcidin in serum was carried out using a sensitive competitive ELISA. 13,18 Briefly, all the serum samples were diluted appropriately, mixed with a biotinylated hepcidin analog (tracer) and added to 96 well microtitre plates coated with rabbit anti-hepcidin antibodies for 2 hours for binding and kept in dark condition. After 2 hours, wells were washed three times with 0.05% tween 20 in tris buffered saline whose ph is 8. The secondary reagent horse radish peroxidise avidin (1ng/ ml) was added and allowed this mixture to bind and blend for one hour. Then the wells were washed thrice with 0.05% tween 20 in tris buffered saline. Tetramethylbenzadine substrate was added and the reactions were stopped with 1 N H 2So 4 solution. The optical density of the reactions was determined at 450nm using Beckman Coulter DTX 880 Prabhu et al., J Pharm Biol Sci 2016; 4(3): detector. Further the sample concentrations were determined using Prism curve fitting software by comparison to a standard curve generated using synthetic hepcidin by preparing various concentrations. 13 In future all these were validated using HPLC, peptide sequencing, mass spectroscopy and bioactivity studies using HEK293 cells overexpressing ferroprotein green fluorescent protein. 19 In this study, we concentrated only on hepcidin measurements in serum other serum parameters were non measured. Ethics, Consent and Statistics: Approval for this study was obtained from the Medical ethics committee and written informed consent was also obtained from all patients. Endpoints included cumulative incidences of documented leptospiral infection and MAT positivity related to serovar analysis. Cox proportional hazard model was applied for assessing factors that potentially affect the study endpoints. 5 Leptospiral cases, control groups and other liver disorder subjects were compared. To eliminate the effect of competing risk, the cumulative incidences were assessed using standard methods described elsewhere. 5,20 The cutoff points for MAT serology and hepcidin level were chosen such that we could make optimal use of the information with a provision of subjects included. P values of < 0.05 were considered statistically significant. RESULTS Age and sex wise distribution: The demographic data (age and sex) for the 49 MAT positive leptospiral cases, 10 hepatic cases other than leptospirosis and 20 healthy control groups were analyzed. The age group of and 41 to 50 are having high numbers among leptospirosis positive cases confirmed by MAT (Table 2). This is mainly due to the occupational exposure to the contaminated environment in more time and also due to the repeated exposure. Table 2: Patients age group in different categories Age group Number of cases in different age groups vs sex among (in years) categories Leptospirosis cases (n=49) Liver disorder cases other than leptospirosis (n=10) Healthy control (n=20) M (n=26) F (n=23) M (n=7) F (n=3) M (n=10) F (n=10) (3.8) (10) 1 (10) (15.4) 3 (13) - 1 (33.3) 2 (20) 3 (30) (11.5) 3 (13) 2 (28.6) - 3 (30) 3 (30) (30.8) 6 (26.1) 3 (42.8) 1 (33.3) 2 (20) 2 (20) (26.9) 9 (39.2) 1 (14.3) 1 (33.3) 2 (20) 1 (10) (11.6) 2 (8.7) 1 (14.3) [Figure in parenthesis denoted percentages]

4 Prabhu et al., J Pharm Biol Sci 2016; 4(3): Leptospiral MAT positive results: Among the 49 clinically and genus specific ELISA confirmed leptospirosis samples included in this study, all samples showed positive to microscopic agglutination test. The common serovars against which the antibodies were present were Grippotyphosa, Icterohaemorrhagiae, Australis, Autumnalis, Canicola, Pomona and Javanica,. Other serovars including Sejroe, Pyrogens, Hebdomadis, Semaranga showed less antibody titre (1:80). Among them Grippotyphosa (85.7%) was predominant followed by Icterohaemorrhagiae (63.3%) and Australis (57.1%). The highest antibody titer was obtained in Grippotyphosa (1:5120) followed by Austumnalis (1:2560). Thus the prevalent serogroup observed among the rodents included in this study was Grippotyphosa. Others showed comparatively lower titre including Icterohaemorrhagiae (1:640), Australis (1:640), Canicola (1:320), Javanica (1:320). The microscopic agglutination test results of the leptosprial antibodies among subjects were included in tables 3. Table 3: Mono leptospiral serovars MAT positive cases Serovars Number of positive Highest titre values samples (n=49) Grippotyphosa 42 (85.7) 1:5120 Icterohaemorrhagiae 31 (63.3) 1:640 Australis 28 (57.1) 1:640 Autumnalis 14 (28.6) 1:2560 Canicola 14 (28.6) 1:320 Pomona 12 (24.5) 1:320 Javanica 12 (24.5) 1:320 Sejroe 6 (12.2) 1:80 Pyrogenes 6 (12.2) 1:80 Hebdomadis 5 (10.2) 1:80 Semaranga 5 (10.2) 1:80 [Figure in parenthesis denoted percentages] In some samples, the mixed infection was also found and the detailed description of poly leptospiral infections and its descriptive data were well analyzed. In this analysis, the highest serovar combinations are Grippotyphosa- Icterohaemorrhagiae among 28 samples, Grippotyphosa-Australis-Icterohaemorrhagiae among 14 samples and Grippotyphosa-Australis- Pomona-Canicola among 7 samples. This may be due to the exposure in various environmental conditions and animal contacts as combination risk factors. Among the 49 sera included in this study, 4 serum samples were excluded due to the presence of milky deposit (fat droplets), According to the guidelines, the milky sera containing fat droplets should not be used for the determination of hepcidin. 15 The laboratory reported that serum sample was milky white indicating the possibility of high triglycerides; 21 this may disturb the objective of the present investigation, thus we exclude these samples. Hepcidin is up-regulated during various phases of leptospirosis: The earliest events following leptospiral infection have a significant influence on subsequent disease pathogenesis. To investigate changes in hepcidin during this period, we measured hepcidin among serologically (MAT) positive leptospiral cases. On comparing with 107 mono leptospiral samples, poly leptospiral samples showed maximum elevation of the hepcidin levels. The hepcidin levels in the samples were classified into two major groups. 1. Low hepcidin groups - < 50 ng/ml 2. High hepcidin groups - 50 ng/ml 3. Among the samples, all the leptospirosis confirmed samples showed high hepcidin with maximum elevation of the hepatic peptide. A trial was done by including the milky serum samples for hepcidin determination leads to decrease in concentration. Thus it was proved that the milky serum is not subjected for determining acute phase protein. The assessment of the significance of observed levels of hepcidin was elevated and the variations were found based on the titre value of the hepcidin (Figure 1). The hepcidin elevation also varies according to the serovars of the leptospires. Variations in the acute phase protein hepcidin levels were found and were impregnated in table 4. It is clearly mentioned that according to increase in titre value the hepcidin value also increased. Also the serovar Grippotyphosa elevated maximum followed by Icterohaemorrhagiae, Australis, Autumnalis etc (Table 4).

5 Prabhu et al., J Pharm Biol Sci 2016; 4(3): Figure 1: Assessment of hepcidin levels in various leptospiral titres Table 4: Hepcidin levels versus leptospiral serovars Serovars Range of hepcidin Highest titre values Grippotyphosa ng/ml 1:5120 Icterohaemorrhagiae ng/ml 1:640 Australis ng/ml 1:640 Autumnalis ng/ml 1:2560 Canicola ng/ml 1:320 Pomona ng/ml 1:320 Javanica ng/ml 1:320 Sejroe ng/ml 1:80 Pyrogenes ng/ml 1:80 Hebdomadis ng/ml 1:80 Semaranga ng/ml 1:80 Analysis of hepcidin among liver disorder subjects: This group of subjects are affected with liver disorders. In this study, we included the patients who are having the complications of Hepatitis B infection, liver cirrhosis and hepatoma. The upregulated of hepcidin was not found elevated compared to leptospirosis samples. No such elevations were determined. However, there was a small but significant increase in hepcidin among hepatitis B infectious samples. We detected no significant perturbations in liver cirrhosis and cancer cases. Thus, leptospirosis samples elevated hepcidin and other inflammatory responses than other disorders included. The table 5 highlighted the determination of hepcidin levels in liver disorders. Table 5: Association of Hepcidin levels among liver disorders Liver disorders Range of No. of samples hepcidin supported for low hepcidin levels Hepatitis B infection (n=4) ng/ml 1 (25) 3 (75) Liver cirrhosis (n=3) ng/ml 3 (100) - Liver cancer (n=3) ng/ml 3 (100) - [Figure in parenthesis denoted percentages] 108 No. of samples supported for high hepcidin levels

6 Among the control groups all the samples showed the hepcidin range from 8 to 22 ng/ml. Some of the subjects showed less than 8 ng/ml. Thus compared to control and liver disorders, samples of serologically confirmed leptospirosis showed significant elevation of hepcidin and may be a better biomarker to understand the state of leptospirosis and its serovar predominance. DISCUSSION Using the novel validated principle, we reported the first quantitative analysis of bioactive serum hepcidin in serologically confirmed leptospiral cases. No previous report found in these combinations. The authors have the main interest of comparing the hepcidin levels in leptospirosis cases, other liver disorder cases and healthy control groups. It was observed that the leptospirosis is multi organ involvement in its pathophysiology where liver and renal systems are predominantly affected. 22,23 Hepcidin is also known as a type II acute phase protein and in chronic inflammatory conditions, increased hepcidin levels correlate with increased ferritin levels. 12 The rapidity of the hepcidin response could be related to its proposed role as an inducer of hypoferremia that would restrict the flow of essential iron to infecting microbes and slow their multiplication in tissues. 24 This host response could be particularly valuable during the earliest phases of infection, before other components of the innate and adaptive immunity are fully mobilized. In this study, we have demonstrated the serum hepcidin is progressively elevated based on the leptospiral titre values and constraint to specific serovars too. As expected from previous studies, the elevation of hepcidin REFERENCES Prabhu et al., J Pharm Biol Sci 2016; 4(3): appears multifactoial, particularly given its regulation by iron stores and other organ specific inflammations. 13,25,26 The detection of hepcidin is developed initially by quantification of hepcidin peptides by mass spectrometry and then developed by determining anti-hepcidin antibody in a competitive binding assay. 27 Some studies showed positive and significant correlation was observed between the values of serum hepcidin and other acute phase proteins including C-reactive proteins 28 but this comparison is not included in this study. When liver get affected there is an observation on the onset of anemic profile, predominantly haemolytic and regenerative, where ferric tissue sequestration tended to become chronic leptospiral infections. 14 CONCLUSION However, serum hepcidin 25 amino acid isoform (hepcidin 25) levels which are directly responsible for the biological effect, have not been documented and factors that contribute to hepcidin regulation is this disease have not been assessed. 29 Thus this investigation may be the first report in determining the hepcidin as liver biomarker in leptospirosis cases. The insights gained in this study could be clinically useful in the diagnosis and treatment of patients most at risk of iron mediated tissue damage. The hypoferremia of bacterial infections also occurred thus elevation of hepcidin determined leads to pathogen specific, not universal and rare to determine. Earliest diagnosis, appropriate antibiotic therapy to control the bacterial infections and can be considered in the patients with high hepcidin levels. Further, larger prospective studies needed to confirm our findings. 1. Ashby DR, Gale DP, Busbridge M, Murphy KG, Duncan ND, Cairns TD, Taube DH, Bloom SRm Ram FW, Chapman RS, Maxwell PH, Choi P. Kidney Int 2009; 75: Ashrafian H. Infect Immunol 2003; 71: Castagna A, Campostrini N, Zaninotto F, Girelli D. J Proteomics 2010; 73: Kemna E, Pickkers P, Nemeth E, Vad der Hoeven H, Swinkels D. Blood 2005; 106: Ganz T. J Am Soc Nephrol 2007; 18: Ganz T, Olbina G, Girelli D, Nemeth E, Westerman M. Blood 2008; 112: Gooley TA, Leisenring W, Crowley J, Storer BE. Stat Med 1999; 18: Hwang SI, Lee YY, Park JO, Norton HJ, Clemens E, Schrum LW, Bonkovsky HL. Clin Chim Acta 2011; 412: Joyce JCK, Coby MML, Erwin HJMK, Bart JB, Dorine WS. Haematologica 2009; 94: Kanda J, Mizumoto C, Kawabata H, Ichinohe T, tsuchida H, Tomosugi N, matsuo K, Yamashita K, Kondo T, Ishikawa T, Uchiyama T. Biol Blood Marrow Transplant 2009; 15: Kulaksiz H, Theilig F, Bachmann S, Gehrke SG, Rost D, Janetzko A, Cetin Y, Stremmel W. J Endocrinol 2005; 184: Li H, Rose MJ, Tran L, Zhang J, Miranda LP, James CA, Sasu BJ. J Phamacol Toxicol Methods 2009; 59: Natarajaseenivasan K, Prabhu N, Selvanayaki K, Raja SS, Ratnam S. Jpn J Infect Dis 2004; 57: Nemeth E, Valore EV, Territo M, Schiller G, Lichtenstein A, Ganz T. Blood 2003; 101:

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