IJPBS JOURNAL VOLUME 4 ISSUE 2 2014 by jaypalreddy g

STUDY OF PROSTATIC LESION FOR A PERIOD OF FIVE YEARS B. Rajashekar Reddy1, Rameswarapu Suman Babu1 and Sujatha.P*2 *1

Associate professor, Associate professor, Assistant professor Department of Pathology, Department of Anatomy, Mediciti Institute of Medical Sciences, Ghanpur, Ranga Reddy District-501401. Telangana, India. *2 Department of Biochemistry, Katuri Medical college, Guntur. A.P

*Corresponding Author Email: drsujathapasula@gmail.com

ABSTRACT Early detection and management of prostate cancer is an important public health problem in all industrialized countries, where the relative rate of the elderly population is rapidly increasing. Aims of the study is to identify the incidence of the premalignant and malignant conditions and Study of high grade prostatic intraepithelial neoplasia and its association with Prostatic hyperplasia and carcinoma.The present study was undertaken in the Upgraded department of pathology, King George Hospital, Andhra medical college for a period of five years from January 2002 to December 2006. A total of 340 cases evaluated, 277(81.47%) were benign, 11(3.23%) were premalignant and 52 (15.29%) malignant lesions. The ratio of benign to malignant lesions were 5.5: 1 ; Benign prostatic hyperplasia and prostrate carcinoma were the two principal conditions account for majority of all prostrate diseases. Among premalignant lesions only high grade prostatic intraepithelial lesion is seen in association with prostatic carcinoma (40%).

KEY WORDS Prostate cancer (PC), High grade prostatic intraepithelial neoplasia(HGPIN), Benign prostatic hyperplasia(BPH)

INTRODUCTION The term “prostate” was originally derived from the Greek word “prohistani”, meaning “to stand in front of”, and has been attributed to Herophilus of 1 Alexandria who used the term in 335 B.C . Early detection and management of prostate cancer (PC) is an important public health problem in all industrialized countries, where the relative rate of the elderly population is rapidly increasing. Diseases of the prostate are common causes of morbidity in adult males and show wide geographical and ethnic variations in incidence and mortality worldwide. There are several benign proliferations and normal histoanatomical structures of prostate which mimic malignancy and their awareness is essential to avoid diagnostic pitfalls. Currently, two premalignant lesions have been recognized: prostatic intraepithelial neoplasia (PIN) and atypical adenomatous hyperplasia (AAH), there is a need to determine their significance as there are many important unanswered questions. International Journal of Pharmacy and Biological Sciences

The present study aims at highlightening on all these issues. Aims of study are lesions of prostate during a five year period to identify the incidence of the premalignant and malignant conditions. Study of high grade prostatic intraepithelial neoplasia and its association with Prostatic hyperplasia and Prostatic carcinoma.

MATERIAL AND METHODS The present study was undertaken in the Upgraded department of pathology, King George Hospital, Andhra medical college for a period of five years from January 2002 to December 2006. Specimens were obtained from the patients with prostatic lesions who underwent transurethral resection of prostate (TURP) or radical prostatectomy attending to the department of surgery and urology. The study comprises a retrospective analysis of 187 cases received from January 2002 to May 2004 and prospective analysis of

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. 153 cases received from June 2004 to December 2006. The material obtained is embedded as per the guidelines suggested in Ackerman’s surgical pathology (9th edition, 2004). All the prostatic specimens fixed in 10% formalin were received. They were weighed

and subjected to a careful detailed gross examination, then fixed in 10% buffered formalin for 24 hours, after fixation bits were given from represent areas and processed routinely. Sections were prepared after routine processing and embedding, and then stained with Haematoxylin and Eosin.

RESULTS Of the 340 cases, 277 cases had BPH, 8 cases had BPH with HGPIN, 3 cases had BPH with AAH, while 50 cases had prostate cancer, and 2 cases had secondary carcinomas in prostate extending from bladder.

AGE

BPH

31-40 41-50 51-60 61-70 71-80 81-90 TOTAL

3 31 109 98 35 1 277

TABLE-I: Age Wise Distribution of Lesions PREMALIGNANT MALIGNANT BPHBPH– MALIGANT MALIGNANT HGPIN AAH** WITH HGPIN 0 0 0 0 2 0 0 0 4 3 2* 12 1 0 19 6 1 0 10* 2 0 0 1 0 8 3 32 20

TOTAL

3(0.9%) 33(9.7%) 130(38.2%) 124(36.4%) 48(14.1%) 2(0.58%) 340(100%)

* Secondary carcinomas-prostate; one case in each category; n=2 **Atypical adenomatous hyperolasia (AAH) The maximum incidence of prostatic lesions was in the sixth and seventh decades. Maximum incidence of benign and premalignant lesions was sixth decade and malignant lesions were in the seventh decade. TABLE-II: Hgpin Association with Benign Vs Malignant and Nature Of Specimen LESION TURP PROSTATECTOMY TOTAL BENIGN WITH HGPIN 4 4 8 MALIGNANT WITH HGPIN 6 14 20 TOTAL 10 18 28 Changes of HGPIN were observed most commonly in prostatectomy specimens when compare to TURP chips TABLE-III: INCIDENCE OF VARIOUS MALIGNANT LESIONS TYPE OF MALIGNANCY PRIMARY ADENOCARCINOMA VARIANTS OF PROSTATIC ADENOCARCINOMA SECONDARY CARCINOMAS Transitional cell carcinoma -1 Signet ring carcinoma-1 TOTAL

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NO. OF CASES 50 (96.15%) 0 2 (3.84%)

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. TABLE-IV: Distribution of Gleason Grade GLEASON GRADE 1 2 3 4 5 TOTAL

TURP 0 4 8 2 0 14(28%)

PROSTATECTOMY 0 4 15 15 2 36(72%)

TOTAL 0 8(16%) 23(46%) 17(34%) 2(4%) 50(100%)

Figure-1: TURP specimen displaying grey white bits of prostatic tissue

Figure-2: Prostatic cancer Gleasons pattern

Gleasons pattern-3 varying size and shape of the gland

DISCUSSION Benign prostatic hyperplasia and prostrate carcinoma were the two principal conditions that involve the prostrate and account for majority of all prostrate diseases. In the similar study done by Xess A et. al2, Indira Gandhi Institute of Medical Sciences, Sheikhpura, Patna, out of 98 cases, 52% were adenocarcinomas and 42.8% cases were benign prostatic hyperplasia. A study done by Tay KP et. al3 in Singapore found that the prevalence of BPH was higher, similar to the finding of the present study. In the present study, patients aged from 30-90 years were observed with majority of BPH occurring in the age group of 5070years, with a mean age of 58 years.

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Gleasons pattern-3C cribriform glands In the study conducted by Ro J Y et al., the age range was 60-80 years with a mean age of 64 years4. Where as in the study conducted by Shapiro et al., mean age was 73 years. In the study conducted by J.T. Animet al., the age range was 33-98 years with a mean age of 63 years5. All these studies indicate that BPH has significantly associated with age and the present study correlated well with the above studies. In the present study comprising mainly TURP chips 3(1%) cases of AAH was reported. Study by Qian J, Bostwick DG et al., in totally embedded radical prostatectomies with prostate cancer , AAH was identified in 23% of the cases, and was more frequent in transitional zone than in the non transitional zone. AAH showed a weak but significant association.

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SL.NO 1. 2. 3. 4.

TABLE- V: Comparision of Various Studies Studies Prostates With Prostates Carcinoma Carcinoma Mc Neal and Bostwick 33% 4% Troncoso et al 72.1% 17.9% Desai et al 85.24% 0% Present study 40% 2.77%

All the studies indicate that HGPIN was the most commonly observed premalignant lesion, thereby suggesting it to be likely precursor of prostatic carcinoma. The incidence of HGPIN in transurethral resection of prostate is relatively uncommon with the studies reporting a rate of 2.3% and 2.8% respectively. Study by Pacelli A, Bostwick DG., reported 4.2% of HGPIN in TUPR chips. Including 2.8% of those with BPH and 10.2% of those with cancer and BPH. In the present study, the incidence of HGPIN in transurethral resection of prostate was 4.29% coinciding with the above studies. In the present study, the most common incidence of benign and pre-malignant lesions was in the 6th decade (40.3% & 61.2% respectively), with a mean age of 58 years. Prostatic carcinoma was in 7th decade (50%), with a mean age of 66years. Premalignant lesions were preceded by a decade as compare to malignant lesions, with a mean age of 8 years difference. Kovi et al., demonstrated that the prevalence of PIN in malignant glands increased with age, and that these lesions appeared to predate the onset of carcinoma by more than 5 years6. Lee et al studied ultrasound guided biopsies of hypo echoic lesions and observed, the mean age of those with PIN (65years) was significantly lower than the age of men with cancer (70years) 7, findings consistent with these studies. Gleason grading and scoring criteria, most common grade was Grade-3 (46%) closely followed by Grade4. Most common Gleason score was 7 (28%) closely followed by 6 (22%). According to WHO classification of tumours. Pathology & Genetics (2002), Gleason pattern 3 is the most common pattern and Gleason scores 6 and 7 are the most common scores and include the majority of the tumours in most studies.

Without

CONCLUSION In a total of 15,800 surgical biopsies, 340 were prostatic biopsies giving an incidence of 2.15%. The ratio of benign to malignant lesions were 5.5: 1. Premalignant lesions were preceded by a decade (mean-58y) as compare to malignant lesions (mean66y), with a mean age of 8 years difference. Among premalignant lesions only high grade prostatic intraepithelial lesion is seen in association with prostatic carcinoma (40%). In prostatic adenocarcinoma Gleason grade-3, Gleason score -7 were most commonly encountered. Direct spread from bladder carcinoma is the most common type of secondary’s in prostate.

REFERENCES 1.

6. 7.

Roger S Kirby, Timothy J Christmas & Brawer, M.K. prostate cancer: second edition, Mosby International Ltd 2001. Xess, Singh M, Raghwendra KH, sharma HP, Sahi SKProstate specific antigen as tumour marker; relationship with histologic grading. Indian J pathol Microbiol 2001 July; 44 (3): 261-4 Tay KP, Chin CM, Lim PH; Prostate Screening- The Singapore experience, Int. J. Urol. 1996 Mar; 3(2):1027 (ISSN: 0919-8172) Ro JY, Sachin AA, Ayala AG. ”Tumours and tumorous conditions of the male genital tract” In: Fletcher CDM. Diagnostic histopathology of tumours. Edinburgh, st Churchill Livingstone, 1 Ed, 1995, 521-563. J.T. Anim, benign disorders of the prostate: a histopathological study Ann Saudi Med 1998; 18(1):2227. Kovi J et al., Large acinar atypical hyperplasia and carcinoma of the prostate, Cancer; 1998; 61; 555. Lee F, Torp Pederson ST et al., Use of transrectal ultrasound and prostate specific antigen in the diagnosis of prostatic intraepithelial neoplasia, Urology; 1989; 24;4-8.

*Corresponding Author: Sujatha.P* Email: drsujathapasula@gmail.com

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MEASUREMENT OF CREATINE KINASE LEVEL IN SUDANESE CHILDREN WITH CEREBRAL MALARIA, CENTRAL REGION, SUDAN Shamseldin M. Ahmed, Tarig Guma2, Ahmed Kamal3, Gad Allah Modawe4* 1

Department of Clinical Chemistry, Faculty of Medical Laboratory Sciences, Gezira University, Wad Medani, Sudan. 2 Department of Physiology, Faculty of Medicine, Omdurman Islamic University, Omdurman, Sudan 3 Department of Biochemistry, Faculty of Dental Medicine, Gezira University, Wad Medani, Sudan. 4 Department of Biochemistry, Faculty of Medicine, Omdurman Islamic University, Omdurman, Sudan.

*Corresponding Author Email: gadobio77@hotmail.com

ABSTRACT Background: Malaria is a global disease, and it is a major health problem in Sudan, associated with many complications that increase morbidity and mortality. Objective: This study was aimed to measure the creatine kinase (CK) level in Sudanese children with cerebral malaria. Materials and Methods: This is prospective crosssectional hospital based study, was done in sixty three children with cerebral malaria (CM) from Kosti, Rabak, Sinnar, Singa, Rufaa and Medani Teaching hospitals, central region Sudan, during May-October, 2012. Fifty two with cerebral malaria and eleven children affected with uncomplicated complicated. The age of children was ranged between (2-12 years). Data was collected through, clinical evaluation form and designed questionnaire of pediatricians and cerebral malaria was confirmed microscopically, serum concentration of creatine kinase, was measured by spectrophotometer. Results: The mean levels of CK is increased in children with cerebral malaria ten hours after admission (488.99 u/L) but decreases 24-48 hours after admission(227 Âľ/L), and also decreases in those with uncomplicated malaria (p= 0.001). Conclusion: Children with elevated urea levels on admission or those who experienced mean low level of creatine kinase subsequently were more likely to die.

KEY WORDS Creatine Kinase, Cerebral malaria, Central Sudan.

INTRODUCTION Malaria continues to be a major health problem in Sub-Saharan Africa. In general, the disease is endemic in more than 90 countries and is responsible for about 500 million cases and more than 1 million deaths each year (1). In Sudan, malaria was determined as one of the most devastating problem that led to loss of 2,877,000 DALYs in the year 2002 and was identified as a common cause of fever (2, 3). Cerebral malaria (CM) is estimated to affect more than 785000 children who are younger than 9 years in sub-Saharan Africa every year, the case fatality rate even with optimal therapy, is 15-30% (4). Children who survive CM may be left with permanent neurological sequence, including seizures, acquired language disorders, motor deficits and problems with International Journal of Pharmacy and Biological Sciences

memory and attention (5). Skeletal muscle damage is common in malaria. In order to investigate the relationship between serum creatine kinase (CK) and myoglobin levels, muscle histology, and renal function of plasmodium falciparum malaria, serum creatine kinase levels were estimated in patients with uncomplicated malaria. Muscle appears to be an important site for P-falciparum sequestration, which could contribute to metabolic and renal complication (6). Cerebral malaria (CM) is a serious complication of plasmodium falaciparum infection that contributes significantly to morbidity and mortality worldwide. Primarily young children develop a diffuse potentially rapidly invisible encephalopathy associated with loss of consciousness, seizures and few localizing neurological sign; other complication as severe anemia or respiratory distress increase the tragic Gad Allah Modawe* et al

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outcome (7). Pathophysiology of malaria infection needs two phases to complete the disease picture: one that involves the liver (exoerythrocytic phase), and one that involves red blood cells or erythrocytes (erythrocytic phase). When an infected mosquito pierces a person's skin to take a blood meal, sporozoites in the mosquito's saliva enter the bloodstream and migrate to the liver where they infect hepatocytes, multiplying asexually and asymptomatically for a period of 8–30 days (8). In humans, malaria is caused by P. falciparum, P. malariae, P. ovale, P. vivax and P. knowlesi. (9,10). Among those infected, P. falciparum is the most common species identified (~75%) followed by P. vivax (~20%)(11) Although P. falciparum traditionally accounts for the majority of deaths,(12) recent evidence suggests that P. vivax malaria is associated with potentially life-threatening conditions about as often as with a diagnosis of P. falciparum infection (13). The histopathological hallmark of cerebral malaria is engorgement of cerebral capillaries and venules with parasitised red blood cells (PRBCs) and non-paratised RBCs (NPRBCs)(14).Some authors think that cerebral malaria has features of a diffuse encephalomyelitis (15). There are several serious complications of malaria. Infection with P. falciparum may result in cerebral malaria, a form of severe malaria that involves encephalopathy. It is associated with retinal whitening, which may be a useful clinical sign in distinguishing malaria from other causes of fever (16) Splenomegaly, severe headache, hepatomegaly (enlarged liver), hypoglycemia, and hemoglobinuria with renal failure may occur (17). No documented data was found to estimate the ceratine kinase level among Sudanese children infected with cerebral malaria. So, this study aimed to measure the creatine kinase (CK) level.

MATERIALS AND METHODS This is cross-sectional study was carried out in central Sudan Gezira irrigation scheme and cities nearby where malaria is endemic. The study population was children diagnosed as case of malaria who are admitted to teaching hospitals of major cities in this region, selected randomly that include: Kosti, Rabak, Sinnar, Singa, Rufaa and Wad Medani teaching

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Int J Pharm Biol Sci. hospitals. The study was conducted during the period between May – October 2012. The sample of this was taken by simple random sample from the pediatrics wards of the selected hospitals. Sixty three child were selected , then the divided into two strata, fifty two of them are eligible to WHO criteria of cerebral malaria, and eleven (11) child affected with un complicated malaria, their age was ranged between (2- 12 years). Cerebral malaria was diagnosed clinically by pediatrician and confirmed microscopically by blood film for malaria using Giemsa stain. First 2ml of venous blood sample were taking from all patients; after 4-10 hours of the onset of admission, and second blood sample was taken after 24 hours of the onset of the admission. The first blood sample was used to measure hemoglobin (Hb), and to estimate total white blood cell count (TWBC), blood glucose, serum urea and creatine kinase (CK) levels. The second venous blood sample was used to analyze creatine kinase (CK) level. The personal data, history of presenting complain and family history was taken by designed questionnaire. All biochemical measurements were analyzed by photometric method using spectrophotometer device. Clinical examination and findings were achieved by pedestrians in the ward. Children with CM were enrolled if they were admitted to these hospitals and met the World Health Organization criteria for cerebral malaria (CM) which include; coma (Blantyre coma scale 2 or Glasgow coma scale 8), P. falciparum on blood smear, and no other cause for coma. Children were considered to have uncomplicated malaria (UM) if they had signs and symptoms of malaria (fever, chills, vomiting, and headache), P. falciparum infection on blood smear, and no evidence of malaria complications (e.g., seizures, respiratory distress, severe anemia, or coma) or other acute illness. All steps of this study were implemented after taking the informed consent by the child’s parent and pediatricians. Statistical analysis was considered as frequency and means for all parameters and outcomes using the SPSS Software program for widows, version11 to find the mean difference between two groups at p-value ≥ 0.05 for confidence interval (C.I) = 95%.

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RESULTS Table (1): Mean of Serum Urea, Blood glucose and Creatine Kinase (CK) levels in Sudanese children with cerebral malaria, Central Sudan Parameter Mean Reference values Serum urea 35.8 mg/dL 15-45 mg/dL Random blood glucose level 98.8 mg/dL 60-110 mg/dL CK(4-10)hrs after admission 488.99 µ/L † CK(24-48)hrs after admission 227 µ/L † † Reference values have not been established for patients that are less than 6 years of age

Table (2): Mean of hemoglobin (Hb) and white blood cell count (WBCC) in Sudanese children with cerebral malaria, Central region, Sudan: Parameter Mean Reference values Hemoglobin(Hb) 8.23 mg/dL 12-16 mg/dL WBCs Count 4700/ mm3 >1100/ mm3

Table (3): The outcome of Sudanese children with Cerebral Malaria (CM) by means of some hematological and biochemical tests, central region, Sudan. Outcome of CM No. Mean Mean Mean Mean Mean Hb Level TWBC blood Blood C.K Level glucose urea 3 Recovered 43 8.85 mg/dL 3.6 mm 98.6 35.5 472 µ/L mg/dL mg/dL Died 9 18.1 mg/dL 3.16 mm3 96.2 61.2 1679 µ/L mg/dL mg/dL Total 52

Table (4): Mean ± Std of Creatine Kinase (CK), Hemoglobin (Hb) and Blood urea Levels among children with cerebral malaria (CM) and children with un complicated malaria (UCM) of Sudanese children, Central region, Sudan. CM UCM Parameter (n=43) (n= 9) P-value mean ± Std mean ± Std CK ( u/L) 740 ± 488.99 192 ± 198.55 0.001‫٭‬ Hb (mg/dL) 8.23 ±1.46 11.11 ± 1.08 0.000‫٭‬ Blood urea( mg/dL) 35.83 ±13.67 26.18 ± 4.94 0.025‫٭‬

‫ ٭‬significant mean difference

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Figure (1): Shows the Frequency (%) of family history of epilepsy and convulsion of Sudanese children suffered malaria and admitted to pediatrics ward, central region, Sudan.

Frequency (%) 100 80 60 40 20 0 Family history of febrile convulsion

No family history of febrile convulsion

Family history No family of epilepsy history epilepsy

Figure (2): Shows the outcome of Sudanese children suffered malaria and admitted to pediatrics wards, central region, Sudan

Recovered children Dead children

DISCUSSION Cerebral malaria is notable disease among children in central Sudan, where malaria is endemic resulting in raising the prevalence of mortality and morbidity (Figure 2). Because CM is severe illness; it characterized by wide range of metabolic disorders elicits spectrum changes in the blood biochemistry among study children (Table 3). In general, the function of enzymes is to accelerate biological reactions involved in all chemical transformation reaction in the body (18, 19). Creatine kinase is one of enzymes indeed.

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Regarding the effect of cerebral malaria on the activity of creatine kinase enzyme, scientific background confirmed that there is no clear role of creatine kinase in malaria infection, but it may be due to the decrease in the ATP levels that due to loss of heat energy in malarial fever. In the present study the mean levels of CK increased in children with Cerebral Malaria whereas it decreases in those with uncomplicated malaria (p= 0.001). The mean level of CK is increased in children with Cerebral Malaria ten hours after admission (488.99 Âľ/L) but decreases 2448 hours after admission (227 Âľ/L).

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Regarding the manifestations of cerebral malaria as convulsion, blood glucose level, hemoglobin levels (Table 4) of this study are similar to the results of studies in African children with cerebral malaria (20) Sudanese children like African children growing up in malaria’s endemic areas, concludes that severe falciparum malaria usually manifests as seizures, impaired consciousness, or metabolic acidosis presenting as respiratory distress or severe anaemia (20). But, African children rarely develop renal failure or pulmonary edema. A combination of clinical and laboratory abnormalities particularly low hemoglobin level can identify a group of children with cerebral malaria who are most at risk of dying and require intensive care and also they are candidates for especial forms of therapy. Both groups in the study showed normal values for serum urea, blood glucose level. Children with elevated urea levels on admission or those who experienced mean low level of creatine kinase subsequently were more likely to die. Most of children in the study (Figure 2) haven’t history of febrile or epileptic convulsions.

10.

11.

12.

13.

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4. 5.

Ranque, S., Poudiougou, B., Traore, A., Keita, M., Oumar, A.A, Sapeukui, and Marquest, S. (2008). Lifethreatening malaria in Africa children: a prospective study in a messoendemic urban setting. Pediatr Infect. Dis. J., 27 (2): 130-135. Abdalla SI, Malik EM, Ali KM: The burden of malaria in Sudan: incidence, mortality and disability – adjusted life – years. Malar J 2007, 6:97. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text Malik EM, Saeed OK: Malaria in Sudan: past, present and the future. Gazera Journal of Health Sciences 2004, 1:47-53. Publisher Full Text Return to text. Turner, G. (1997). Cerebral malaria Brain Pathol., 7 (1): 569-582. Murphy, S.C. and Breman J.G. (2001). Gaps in the children malaria burden in Africa: Cerebral malaria, neurological sequence, anaemia, respiratory distress, hypoglycemia and complication of pregnancy. An J. Trop. Med. Hyg., 64: (1-2 suppl): 57-67. Carter, J.A., Lees, J.A., Gona, J.K., Muria, G. and Rimba, K. (2006). Severe falciparum malaria and acquired childhood language disorder. Dev. Med. Child Neurol., 48(1): 51-57.

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Idro, R., Carter, J.P., Fegan, G. and Nevill, B.G. (2006). Risk factor for persisting neurological and cognitive impairments following cerebral malaria. Arch Dischild, 91(2): 142-148. Bledsoe GH (2005). "Malaria primer for clinicians in the United States". Southern Medical Journal 98 (12): 1197–204; quiz 1205, 1230. doi:10.1097/01.smj.0000189904.50838.eb. PMID 16440920. Mueller I, Zimmerman PA, Reeder JC (2007). "Plasmodium malariae and Plasmodium ovale—the "bashful" malaria parasites". Trends in Parasitology 23 (6): 278–83. doi:10.1016/j.pt.2007.04.009. PMC 3728836. PMID 17459775. Collins WE (2012). "Plasmodium knowlesi: A malaria parasite of monkeys and humans". Annual Review of Entomology 57: 107–21. doi:10.1146/annurev-ento121510-133540. PMID 22149265. Nadjm B, Behrens RH (2012). "Malaria: An update for physicians". Infectious Disease Clinics of North America 26 (2): 243–59. doi:10.1016/j.idc.2012.03.010. PMID 22632637. Sarkar PK, Ahluwalia G, Vijayan VK, Talwar A (2009). "Critical care aspects of malaria". Journal of Intensive Care Medicine 25 (2): 93–103. doi:10.1177/0885066609356052. PMID 20018606. Baird JK (2013). "Evidence and implications of mortality associated with acute Plasmodium vivax malaria". Clinical Microbiology Reviews 26 (1): 36–57. doi:10.1128/CMR.00074-12. PMC 3553673. PMID 23297258. MacPherson GG, Warrell MJ, White NJ, et al. (1985) Human cerebral malaria. A quantitative ultrastructural analysis of parasitized erythrocyte sequestration. Am J Pathol 119:385–401. Toro G, Roman G (1978) Cerebral malaria. A disseminated vasculomyelinopathy. Arch Neurol 35:271–275. Beare NA, Lewallen S, Taylor TE, Molyneux ME (2011). "Redefining cerebral malaria by including malaria retinopathy". Future Microbiology 6 (3): 349–55. doi:10.2217/fmb.11.3. PMC 3139111. PMID 21449844. Bartoloni A, Zammarchi L (2012). "Clinical aspects of uncomplicated and severe malaria". Mediterranean Journal of Hematology and Infectious Diseases 4 (1): e2012026. doi:10.4084/MJHID.2012.026. PMC 3375727. PMID 22708041. Bairoch, A. (2000). The enzyme database in 2000. Nucleic acid Res., 28(1): pp. 304-315. Smith, A.L (1997). Oxford dictionary of biochemistry and molecular biology. Oxford University Press.

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. 20. Marsh K, Forster D, Waruiru C, et al. (1995) Indicators of life-threatening malaria in African children. N Engl J Med 332:1399â&#x20AC;&#x201C;1404.

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PARADOXICAL INCREASE IN CYSTATIN-C LEVELS IN CONVENTIONAL HEMODIALYSIS Uma Maheshwari.K*, Santhi Silambanan1 & Jothi Malar.R2 1

*Assistant professor, Department of Biochemistry, Chettinad hospital & research institute, Professor& HOD, Department of Biochemistry, Sri Ramachandra medical college & research institute. 2 Professor, Department of Biochemistry, Sri Ramachandra medical college & research institute.

*Corresponding Author Email: druma.neha@gmail.com

ABSTRACT The conventional, low flux (LF) dialyser allows the removal of small molecular solutes like urea and creatinine. The removal of middle molecules (molecular weight between 500D and 60,000 D, which is nearly the size of albumin) is relatively low. Cystatin C has attractive characteristics as a representative middle molecule. Objective: To determine per dialysis Cys C reduction ratio (RR) in low flux group and to compare it with urea, marker of dialysis adequacy. Methods: Thirty seven patients were subjected to conventional, low flux dialysis. Serum urea and Cystatin C were measured pre and post dialysis. Cystatin C was measured by latex enhanced Immuno turbidimetry. Results: The URR is72.273±14.686% in low flux group & the Cys CRR is -9.7 ± 6.7 %. Discussion: The paradoxical increase in Cystatin C in the low flux group shows the ineffective clearance of middle molecules by low flux dialysers which is associated with dialysis related morbidity & mortality. Hence, Cys CRR could be applied as a surrogate marker for the inadequacy of dialysis.

KEY WORDS Cystatin C, Middle molecules, low flux dialysis.

INTRODUCTION The uremic syndrome is attributable to the progressive retention of a large number of compounds, which are called uremic retention solutes or uremic toxins. They interfere negatively with physiologic function. They include not only small plasma solutes, but also protein bound solutes and middle molecules [1] (molecular weight between 500D and 60,000D, which is nearly the size of albumin). Ineffective clearance of middle molecules results in dialysis related amyloidosis in long term dialysis patients. There is an increased incidence of carpal tunnel syndrome and osteoarticular lesions. Development of erythropoietin resistance, dyslipidemia, especially decreased HDL cholesterol, increased triglyceride levels, and accumulation of advanced glycosylation end-products which have been implicated in the pathogenesis of atherosclerosis are the other morbidities associated with long term treatment with conventional dialysers [2].

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Serum creatinine and urea are small molecules that are commonly measured to monitor renal function in patients with chronic kidney disease. The use of serum urea is recommended by the Kidney Disease Outcomes Quality Improvement (KDOQI) clinical practice guideline to assess dialysis clearance [3]. It has a molecular weight of 60 D [4]. The urea reduction ratio that is commonly used show only the removal of small solutes by conventional hemodialysis, the removal of middle molecules is not known. Cystatin C is a single non-glycosylated polypeptide chain consisting of 120 amino acid residues with a molecular mass of 13kDa, which is in the middle molecular range [5]. It is produced by all nucleated cells [6], freely filtered at the glomerulus and virtually all is reabsorbed and metabolised by proximal tubular cells [7-10]. Several studies have suggested that CyC is useful as a marker of hemodialysis toxin removal, since it has the attractive features as a representative middle molecule [11, 12].

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This study was conducted to assess the efficacy of Cystatin C in assessing the middle molecular clearance by conventional hemodialysis.

MATERIALS AND METHODS The study was approved by the human ethics committee of Sri Ramachandra medical college & research institute, Chennai, India and written consent was obtained from all the participants. A total set of 37 patients of both sexes were selected. All the patients were subjected to conventional, low flux hemodialysis. The dialyzers used were F6HPS for Low Flux group (Fresenius medical care). All patients undergoing maintenance hemodialysis at SRMC, three times per week, with age group above 18years were included. Patients with thyroid dysfunction, malignancy, steroid therapy, HIV infection and Pregnancy were excluded from the study. All the blood samples were collected before and after the second HD session of the week, according to the guidelines for HD adequacy [13]. Blood samples were collected in tubes without additional anticoagulant and allowed to stand at room temperature for 30 minutes. Then, the samples were centrifuged to collect serum, which were stored at -70⁰C until assayed. Urea nitrogen was measured using Urease–GLDH method on the Biolis premium

Int J Pharm Biol Sci. 24i (Tokyo Boeki Medical System) analyzer according to the manufacturer’s procedure. Serum Cystatin C was measured by latex enhanced Immuno turbidimetry on the same analyzer according to the manufacturer’s procedure (14). The efficacy of dialysis was then assessed by calculating the reduction ratio for serum creatinine as shown below: Urea reduction ratio (URR) = 100* (1-Ui / Uo) where Ui & Uo represent post dialysis and pre-dialysis serum urea levels. The same formula is used for the calculation of urea and Cystatin C reduction ratios. SPSS 10 statistical software was used for the analysis of the results. Student’s T test was used for the analysis of the pre and post dialysis samples of urea and Cystatin C.

RESULTS There is a statistically significant decrease in urea levels from the pre dialysis value (97.865±42.508 mg/dl) to the post dialysis value (26.756±17.742 mg/dl) with P value of 0.000 (Table.1, Fig.1). There is a statistically significant increase in Cystatin-C levels from pre dialysis value (5.242±0.604 mg/L) to the post dialysis value (5.709±0.606 mg/L) with P value of 0.000(Table.1, Fig.2).

Table 1: Represents the urea and Cystatin C levels in patients undergoing low flux hemodialysis Pre dialysis Post dialysis % No. of patients 37 37 Urea (mg/dl) 97.865±42.508 26.756±17.742 Cystatin C (mg/l) 5.242±0.604 5.709±0.606 URR 72.273±14.686 CysCRR -9.78±6.705

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Uma Maheshwari.K * et al

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. Fig 1: Shows the bar diagram of pre dialysis and post dialysis values of urea in low flux hemodialysis

UREA 97.865

PREDIALYSIS

100

POST DIALYSIS mg/dl

80 60

26.756

40 20 0 PREDIALYSIS

POST DIALYSIS

Fig 2: Shows the pre dialysis and post dialysis values of cystatin C in low flux hemodialysis

CYSTATIN C 5.709

5.8

PRE DIALYSIS

POST DIALYSIS

mg/L

5.6 5.4

5.242

5.2 5 PRE DIALYSIS

POST DIALYSIS

DISCUSSION Krishnamurthy N et al., has shown a high statistically significant increase in the mean values of cystatin C with a CysCRR of -38% in the low flux group. Thysell et al., also showed a paradoxical increase in the Cys C level after dialysis (26.8Âą 14.4%) in low efficiency hemodialysis [12]. Montini et al., have found in their study, an increase in serum cystatin C in anuric patients during peritoneal dialysis. The pore size of the low flux membrane is 1.5nm which does not allow the removal of middle molecules like cystatin C. The electrostatic interaction International Journal of Pharmacy and Biological Sciences

between the microproteins and other plasma proteins adsorbed onto the dialyser membrane hinders the filtration of these molecules. Cystatin C is strongly cationic and the charged nature of the molecule hinders its filtration. [15]. Small amounts of serum Cystatin C are adsorbed or sieved through the cuprophane membranes, rendering their kinetics still more complex [12]. The rise in cystatin C is attributable to the nature of the dialysing membrane and the composition of the dialysate. The rise in Cystatin C could be attributed to the effect of hemo concentration which occurs during

Uma Maheshwari.K * et al

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci.

dialysis. Hence, Cystatin C could be used as a surrogate marker for the inadequacy of conventional LF dialysis. Further large scale studies are required for assessing the middle molecular clearance by high flux dialysers to reduce dialysis related morbidity & mortality.

ACKOWLEDGEMENTS: NIL

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Vanholder R, De smet R, Glorieux G, et al. Review on uremic toxins: classification, concentration, and interindividual variability. Kidney Int.2003; 63:19341943. Edith M. Simmons, B. Blake Weathersby, Stephen D. Clyne et al., High- Flux and High-Efficiency Procedures; Principles and Practice of Dialysis: 5th edition National kidney foundation: 2006 updates clinical practice guidelines and recommendations. Hemodialysis adequacy, peritoneal dialysis adequacy and vascular access. Am j kidney Dis 48[suppl1]: S1S322, 2006. Cheung AK: Hemodialysis and hemodiafiltration. ln: primer on kidney diseases, 4th edition, edited by Greenberg A, Philadelphia, Elsevier Saunders, 2005, pp 464-476. Newman DJ, Thakkar H, Edwards RG, Wilkie M, White T, Grubb AO, et al. Serum cystatin C measured by automated immunoassay: A more sensitive marker of changes in GFR than serum creatinine. Kidney Int 1995; 47: 312-8. Prigent A: Monitoring renal function and limitations of renal function tests. Semin Nucl Med 38:32-46, 2008.

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Mussap M, Plebani M. Biochemistry and clinical role of human cystatin C. Crit Rev Clin Lab Sci.2004; 41: 467550. Rule AD, Berstralh EJ, Slezak JM, Bergert J, Larson TS. Glomerular filtration rate estimated by cystatin C among different clinical presentations. Kidney Int2006; 69: 399-405. Shilpak MG, Praught ML, Sarnak MJ. Update on cystatin C: new insights into the importance of mild kidney dysfunction. Curr Opin Nephrol Hypertens.2006; 15: 270-275. Knight EL, Verhave JC, Spiegelman D, et al. Factors influencing serum cystatin C levels other than renal function and the impact on renal function measurement. Kidney Int.2004; 65: 1416-1421. Campo A, Lanfranco G, Gramaglia L, Goia F, Cottino R, Giusto V.could plasma cystatin C be useful as a marker of hemodialysis low molecular weight protein removal? Nephron Clin Pract.2004; 98: c79-c82. Thysell H, Grubb A, Lindholm T, Ljunggren L, Martensson L. Cystatin C: a new marker of biocompatibility or a good marker for the redistribution of LMW proteins during hemodialysis? ASAIO Trans.1988; 34: 202-204. Hemodialysis adequacy 2006 work group. Clinical practice guidelines for hemodialysis adequacy, update 2006. Am J Kidney Dis.2006; 48[suppl 1]: S2-S90. Filler G, Bokenkam A, Hofmann W et al.,Cystatin C assay Krishnamurthy N, Arumugasamy K, Anand U et al., Effect of Hemodialysis on Circulating Cystatin C levels in Patients with End Stage Renal Disease. Indian Journal of Clinical Biochemistry, 2010/25: 43-46.

*Corresponding Author: Uma Maheshwari.K Email: druma.neha@gmail.com

International Journal of Pharmacy and Biological Sciences

Uma Maheshwari.K * et al