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A STUDY ABOUT THE CHANGES IN LEVELS OF

PANCREATIC ENZYMES IN CASES OF CHRONIC


RENAL FAILURE

DISSERTATION

Submitted to The Tamilnadu Dr.M.G.R Medical

University in partial fulfilment of the requirements for the

Degree of MD Biochemistry

Branch XIII

2011-2014
CERTIFICATE
Certified that the dissertation entitled “A STUDY ABOUT THE CHANGES IN

LEVELS OF PANCREATIC ENZYMES IN CASES OF CHRONIC RENAL

FAILURE” is a bonafide record of the work done by Dr.V.S. Deepa under our guidance

during her post graduate study during the period of 2011-2014 under THE TAMILNADU

Dr.M.G.R. MEDICAL UNIVERSITY, CHENNAI, in partial fulfillment for the degree of

MD in BIOCHEMISTRY, BRANCH XIII. It has not been submitted (partial or full) for the

award of any other degree or diploma.

Dr. S. Jaya, (Guide) Dr. J. K. Sathya Sudha Devi, (Co-Guide)


Professor, Professor & HOD,
Department of Biochemistry, Department of Biochemistry,
Sree Mookambika Institute of Medical Science, Sree Mookambika Institute of Medical Science,
Kulasekaram. Kulasekaram.
Kulasekaram. Kulasekaram

Kulasekaram

Department Of Biochemistry
Sree Mookambika Institute Of Medical Science
Kulasekaram
AKNOWLEDGEMENT

I bow to the almighty god, for showering upon me his blessings that gave me the courage
to venture out this thesis.

I extend my profound sense of gratitude to Dr. J.K. Sathya Sudha Devi, Professor and
Head of the Department of Biochemistry for her, direction, co-operation, constant
encouragement and immense patience with me at every step of this endeavour.

I am extremely thankful to my guide Dr.S.Jaya, Professor, Department of Biochemistry


for her valuable guidance & earnest support from the very beginning of the course.

I am very grateful to Dr.Velayuthan Nair, M.B.B.S, M.S. Chairman and


Dr.Rema.V.Nair, M.B.B.S, M.D, DGO Director, Sree Mookambika Institute of Medical
Science for providing the central lab facilities to accomplish my thesis work.

I am extremely thankful to Dr.Pethuru,MD, Assistant Professor, Department of


Community Medicine, Sree Mookambika Institute of Medical Science, Kulasekaram for
providing me with the timely statistical analysis involved in the study.

I take this opportunity to thank all the faculty members of the Department of Biochemistry,
Sree Mookambika Institute of Medical Science, Kulasekaram for the valuable support and
encouragement rendered by them.

With deep sense of gratitude, I remember the love, support, and encouragement I received
from my family for being with me throughout.
CONTENTS

Sl. No. Index Page No

1 List of Abbreviations

2 List of tables

3 List of graphs

4 List of colour plates

5 List of Appendices

6 Abstract

7 Introduction

8 Aims and Objectives

9 Review of literature

10 Materials and methods

11 Results and observations

12 Discussion

13 Summary and conclusion

14 Bibliography
LIST OF ABBREVIATIONS

CRF Chronic Renal Failure

GFR Glomerular Filtration Rate

ml/mt Milli litre / minute

ml/mt/m2 Milli litre per minute per metre square

CKD Chronic Kidney Disease

AFR Acute Renal failure

ESRD End Stage Renal Disease

AER Albumin excretion

EGFR Estimated Glomerular Filtration Rate

USA United States of America

% Percentage

GN Glomerulo Nephritis

DM Diabetes Mellitus

SLE Systemic Lupus Erythematosis

RF Renal Failure

MM Middle Molecule

BUN Blood Urea Nitrogen

CO2 Carbon di Oxide

G-6-P DH Glucose-6-Phosphate Dehydrogenase

RBC Red Blood Corpuscles

FSH Follicle Stimulating Hormone

LH Luteinizing Hormone

GH Growth Hormone

ECF Extra Cellular Fluid


GIT Gastro Intestinal Tract

mmol/lit Milli mole / liter

mmol Milli mole

PTH Parathyroid Hormone

CCK Cholecystokinin

S.amylase Serum Amylase

K.Da Kilo Dalton

RES Reticulo Endothelial System

U/L Units per liter

SU Somogyi Units

Hr Hour

ml Milli litre

CU Caraway Unit

Mg/dl Milligram per deciliter

ng/day Nanogram per day

Cam Amylase clearance

Ccr Creatinine clearance

Mg% Milligram percentage

mmol/L Millimole per litre

µL Microliter

IU/L International units per litre

CNP Chloro nitro phenol

nm Nanometer

ERCP Endoscopic Retrograde Cholangio Pancreatography


LIST OF TABLES

Table No. Title

Table 1 Comparison between mild and moderate renal failure

Table 2 Comparison between moderate and severe renal failure

Table 3 Comparison between mild and severe renal failure

Table 4 Correlation of serum amylase and serum urea in various study groups

Table 5 Correlation of serum lipase and serum urea in various study groups

Table 6 Correlation of serum amylase and serum creatinine in various study groups

Table 7 Correlation of serum lipase and serum creatinine in various study groups

Correlation of amylase clearance and serum creatinine in various study


Table 8
groups

𝐶𝑎𝑚
Table 9 Correlation of ratio and serum creatinine in various study groups
𝐶𝑐𝑟

Table 10 Correlation of serum calcium and serum creatinine in various study groups

Correlation of serum phosphorous and serum creatinine in various study


Table 11
groups

Table 12 Correlation of serum phosphorous and serum calcium in various study groups
LIST OF GRAPHS

Graph No. Title

Graph 1 Comparison of serum amylase in various study groups

Graph 2 Comparison of urinary amylase in various study groups

Graph 3 Comparison of Cam in various study groups


𝐶𝑎𝑚
Graph 4 Comparison of 𝐶𝑐𝑟
in various study groups

Graph 5 Comparison of serum lipase in various study groups

Graph 6 Comparison of serum calcium in various study groups

Graph 7 Comparison of serum phosphorous in various study groups


LIST OF COLOUR PLATES

Colour Title

Plate No

CP1 Collection of venous blood sample

CP2 Various blood collection tubes

CP3 Table top centrifuge

CP4 Seperation of serum

CP5 Semi auto analyzer

CP5 Structure of alpha amylase

CP6 Action of alpha amylase

CP7 Mechanism of action of lipase


LIST OF APPENDICES

Appendix No. Title

Appendix 1 Ethical Committee Certificate

Patient Information Sheet

English
Appendix 2
Tamil

Malayalam

Patient Consent Sheet

English
Appendix 3
Tamil

Malayalam

Appendix 4 Patient proforma


ABSTRACT
ABSTRACT

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INTRODUCTION
Introduction

CHRONIC RENAL FAILURE (CRF)

CRF is characterized by a progressive and generally irreversible decline CFR. Kidney

failure and be defined as either.

1. A level of GFR < 60 ml/mt/1.73 m2 for more than three months which is accompanied

in

most cases by signs and symptoms of uraemia (or)

2. Structural or functional abnormalities of the kidney with normal or decreased GFR in

the

beginning, but progresses to decreased GFR with time1.

CRF in caused by many diseases out of which glomerulonephritis the most important

cause.

Increase in the blood levels of non-protein nitrogenous substances is referred to as

azotemia which is the hallmark of Kidney failure. When azotemia is associated with signs

and symptoms of end stage renal failure it is termed as uraemic syndrome which is the

terminal manifestation of renal failure. It is characterized by failure of renal excretory

functions as well as metabolic and endocrine abnormalities.

It is also associated with electrolyte disturbances, anaemia,

atherosclerosis and hypertension leading to cardiovascular dysfunctions, poor immunity due

to leucocyte dysfunctions altered calcium metabolism leading to renal osteodystrophy,

gastrointestinal abnormalities causing anorexia, nausea, vomiting and even gastrointestinal

bleeding, neuromuscular abnormalities, myopathy and very rarely acute pancreatitis also.

Pancreatitis occurs with a high frequency in uraemic patients. Hemodialysis patients

are more likely to suffer from triglyceridemia which is a predisposing factor for pancreatitis.
Introduction

Diagnosis of these complications of CRF is also important as we can prevent the

further worsening of the condition of the CRF patient. Most of the complications of CRF can

be diagnosed based on clinical signs and symptoms. Some symptoms of CRF like abdomen

pain and vomiting can also be due to the development of acute pancreatitis in these patients.

Therefore pancreatic enzymes like amylase and lipase should be quantitated, which will show

a marked raise in their levels, when compared to the minimal raise of these enzymes in CRF,

if acute pancreatitis had complicated the CRF.

Even though acute pancreatitis is a complication of CRF, the incidence and

aetiopathology of acute pancreatitis complicating CRF is not known in our population. So the

present study was done to study the variations in pancreatic enzymes levels in CRF patients

without already known pancreatitis and also to determine the incidence of acute pancreatitis

in CRF patients.
AIMS AND OBJECTIVES
Aims and objectives

The main aim of the study is to investigate about the changes in levels of pancreatic

enzymes in cases of chronic renal failure.

The objectives of the study include the following:

a)To detect the incidence and prevalence of acute pancreatitis in known CRF patients

with or without dialysis.

b) To find out the diagnostic value of serum amylase by measuring the rate of

amylase clearance to the rate of creatinine clearance in relation to the stages of chronic renal

failure. (mild, moderate and severe).

C) To evaluate if serum creatinine, serum amylase and serum lipase have any

correlation with the severity of CRF(mild, moderate and severe)


REVIEW OF LITERATURE
Review of literature

THE KIDNEYS

Human Kidneys converts over 1700 litres of blood to about 1-1.5 litres of

concentrated fluid called urine per day. During this process, the Kidneys also serve to

excrete the waste products of metabolism, regulate the body’s water and salt, and maintain

appropriate acid base balance of plasma and serves as an endocrine organ secreting some

hormones like erythropoietin, calcitriol, renin and prostaglandins.

CHRONIC RENAL FAILURE

Chronic Kidney disease in explained as 1

i. Kidney damage for more than 3 months. It is defined by structural or functional

abnormalities of the Kidney with or without decrease in GFR and is manifested by

 Pathological abnormalities

 Markers of Kidney damage including abnormalities in the composition

of blood/urine/abnormalities in imaging tests.

ii. GFR less than 60ml/min/1.7m2 for more than 3 months with or without Kidney

damage.

Presence of CKD should be established based on the presence of Kidney damage and

the level of Kidney function (GFR) irrespective of the diagnosis. Based on the extent of

kidney injury kidney failure can be classified as follows.

Acute renal failure: A sudden loss of kidney function caused by an illness, an injury,
(ARF) or a toxin that stresses the kidneys (Kidney function may
recover)

Chronic Kidney Disease: A long and usually slow process were the kidney loses its ability
(CKD) to function

End-stage renal disease: When the kidneys have completely and permanently shut down
(ESRD)
Review of literature

BASED ON eGFR CHRONIC KIDNEY DISEASE IS STAGED AS;

Stage eGFR Description(Renal structure and function) Predominant

(mL/min/1.73 m2) AER status

1 ≥ 90 Kidney damage with normal/high GFR Micro

2 60-89 Kidney damage with mild reduction in GFR Micro

3 30-59 Kidney damage with moderate reduction in GFR Macro

4 15-29 Kidney damage with severe reduction in GFR Macro

5 <15 Kidney failure

AER = albumin excretion; eGFR = estimated glomerular filtration rate

PREVALENCE AND INCIDENCE OF CKD

Based on the data of National Health and Nutrition Examination Survey (NHANES)

reported in 2003, in the USA the estimated number of adults with CKD, staged 1, 2 and 3

was 10-11% of the total population.

The prevalence of CRF was predicted to be about 500 to 1000 patients per million

population2.

AETIOLOGY

The Kidney may be affected by a number of progressive diseases which can destroy

the nephrons and cause signs and symptoms of CRF. CRF is a syndrome which results from
Review of literature

progressive and irreversible destruction of nephrons regardless of the cause. In many patients

the condition progresses over a number of years and sometimes it is impossible to determine

the underlying renal disease.

The aetiological spectrum of CRF differs in different parts of the world.

Primary glomerulonephritis is the commonest cause of CRF in developing countries

of the world, whereas diabetic nephropathy is emerging as the most common cause of CRF in

developed countries where life expectancy of diabetes has increased considerably as a result

of better diabetic care3.

Diabetic and hypertensive nephropathy are the leading underlying aetiologies of both

CRF and ESRD4.

IMPORTANT CAUSES OF CRF

i. Primary Glomerulo Nephritis - Proliferative GN

- Focal glomerulosclerosis

- Membranous GN

- Membranoproliferative GN

- Crescentic

ii. Secondary glomerulopathy - Systemic diseases

(DM, SLE, Amyloidosis,


Vasculitis)

iii. Interstitial Renal diseases - Chronic Interstitial nephritis

- Chronic Pyelonephritis

- Reflux nephropathy
Review of literature

iv. Hypertensive Renal disease - Nephrosclerosis

- Renal artery stenosis

v. Obstructive nephropathy - Urinary Calculus disease

vi. Heredo familial renal disease - Autosomal dominant polycystic

Kidney Disease

- Medullary cystic disease

- Alport’s syndrome

PATHOPHYSIOLOGICAL CHANGES

1) Impairment in Concentration and dilution of urine

2) Impaired excretion and conservation of sodium.

3) Decreased excretion of Potassium with hyperkalemia is often the immediate life

threatening consideration in the management of patients with RF.

4) Excretion of acid.

5) Calcium, phosphate, vitamin D or bone homeostasis.

6) Impaired erythropoietin products leading to renal anaemia.

7) Impaired excretion of many substances and metabolites that act as uraemic toxins.

PROGRESSIVE NEPHRON LOSS AND ADAPTATIONS

In CRF the number of nephrons goes on decreasing with passage of time. As the

nephrons continue to be lost, the remaining undamaged or less severely damaged nephrons

undergo certain changes. Experimental studies have shown that single nephron GFR in the

remaining intact nephrons tends to increase and they undergo compensatory hypertrophy. At
Review of literature

this stage of the disease, GFR can be maintained normal despite the reduced number of

nephrons until the limits of increase of single nephron GFR is reached.

These beneficial compensatory adaptations in residual renal function ultimately have

harmful systemic and renal effects. These have been termed as trade off hypothesis5.

There is some evidence that supports the adaptive increase in phosphate excretion by

the nephron that typically occurs in CRF. It is thought to be mediated at least in part by

parathormone and the levels of this hormone have found to rise progressively throughout the

course of CRF. During the course of CRF, the hyperparathyroidism causes hyperplasia of the

parathyroid. The consequence of this secondary hyperparathyroidism however extends far

beyond the promotion of increased fractional phosphate excretion rates.

MIDDLE MOLECULE HYPOTHESIS (MM) 6

The discrepancy between the severity of symptoms and degree of azotemia seems to

be most marked in patients who are treated with maintenance peritoneal dialysis. Even

though the BUN and serum creatinine levels are high, the symptoms of uremia are mild and

peritoneal dialysis patients may be less prone to develop peripheral neuropathy than the

hemodialysis patients. Thus it is observed that the toxicity is related to the accumulation of

higher molecular weight substances which are cleared more readily by peritoneal dialysis

than by hemodialysis.

The peritoneal membrane is more permeable to solutes of middle molecular weight

(Approximately 500-3000 Daltons) when compared to hemodialysis membranes. According

to the middle molecule hypothesis, shortening the duration of dialysis will jeopardize the

removal of these compounds despite the same clearance of small solutes.

The MM hypothesis has a number of shortcomings. First the efficiency with which

dialysis corrects the uraemic syndrome argues in favor of low molecular weight solutes
Review of literature

playing the pathophysiological pre-eminent role. Second is that most solutes are of low

molecular weight and are not of MM size.

In summary the MM hypothesis remains controversial, despite a great deal of

research.

URAEMIC TOXINS7

Most of the manifestation of uraemic syndrome is due to the accumulation of toxic

products of normal or abnormal metabolism, most of them are products of protein and amino

acid metabolism.

Urea is the

nitrogen containing metabolic product of protein catabolism. It accounts for more than 75%

of non-protein nitrogenous substances excreted. An increase in plasma urea concentration is

known as uremic state. Some of the clinical abnormalities like anorexia, vomiting, malaise

and headache are contributed by urea itself. Urea exerts toxic effects in cells either directly

or indirectly when it is converted to ammonia and CO2, principally by bacterial urease.

Creatinine is the final product of phosphocreatine catabolism.

Measurement of its excretion rates are also used as indicators of kidney function. Serum

creatinine concentration is mostly used as an index of renal function.

Additional categories of nitrogenous excretory products include,

By products of proteins and amino acid metabolism:

-Guanidino compounds

-Guanidine

-Urates and hippurates

-End products of Nucleic acid metabolism

-Polyamines
Review of literature

-Phenols

-Benzoates

-Indoles

GUANIDINO COMPOUNDS

A Guandino compound as uremic toxins is still a controversy as it is difficult to

measure its plasma and tissue concentrations.

Guandino acetic acid, guanidine propionic acid and  - guanidine butyric acid can

cause auto hemolysis of RBC and inhibit G-6-P DH invitro.

In uraemic patients, plasma levels correlate inversely with erythrocyte glutathione

concentration, suggesting loss of protective effect of glutathione against hemolysis. Other

guanidine compounds may have neurotoxic effects.

Guanidine and MM have been related to peripheral neuropathy

while  -guanidino butyric acid, tauro cyanine, homoarginine and  -Keto -  -

guanidinovaleric acid may lower the seizure threshold.

ENDOCRINE DYSFUNCTION IN CRF6

Nature of Defect Hormonal defect

1. Diminished Production of Decreased erythropoietin

renal hormones production Decreased

conversion of 25-hydroxy

vitamin D3 to 125 – dihydroxy

vitamin D3
Review of literature

2. Hormonal hyper secretion Hyperparathyroidism and

to re-establish homeostasis secretion of natriuretic hormone

3. Decreased metabolic FSH, LH, Prolactin, GH etc

clearance of hormone

4. Defective tissue conversion Thyroxin to triiodothyronine, 25-

of prohormone to hormone hydroxy vitamin D3 to 125

dihydroxy vitamin D3

5. Decreased hormone Testosterone

production

6. End organ unresponsiveness Insulin, Parathyroid hormone

FLUID, ELECTROLYTE AND ACIDBASE DISORDERS DUE TO URAEMIA7

i. Sodium and water homeostasis

In most patients with stable CRF, the total body contents of sodium and water are

increased.

The underlying etiologic disease process may itself disrupt glomerulotubular balance

and promote sodium retention and ECF volume expansion. Such ECF volume expansion

contributes to hypertension, which in turn further accelerates progression of nephron injury.

When water intake exceeds the capacity of free water clearance, it leads to

hyponatraemia. Patients with CRF also have impaired renal mechanism for conserving

sodium and water.

ii. Potassium homeostasis7

In CRF the decline in GFR is associated with a proportionate decline in urinary

potassium excretion. Potassium excretion in the GIT is increased.


Review of literature

Hyperkalemia may be present in constipation, augmented dietary intake and protein

catabolism, hemolysis, hemorrhage, transfusion of stored RBC, Metabolic acidosis, following

consumption of certain medicines that inhibit potassium entry into the cells or potassium

secretion in distal nephrons.

Conditions like diabetic nephropathy and certain renal tubular acidosis are associated

with earlier and severe disruption of potassium secretary mechanism in distal nephrons.

Hypokalemia is uncommon in CRF and rarely occurs due to reduced dietary

potassium intake or excessive diuretic therapy or gastrointestinal loses.

METABOLIC ACIDOSIS

Advancing renal failure is associated with total urinary net daily acid excretion

limited to 30-40 m. mole and an anion gap of upto 20 m.mol/litre with a reciprocal fall in

plasma bicarbonate. In most patients, the metabolic acidosis is mild.

A LIST OF URAEMIC TOXINS 7, 8

 Creatine

 Creatinine

 Urates and hippurates

 End products of aliphatic amino acid metabolism

 End products of aromatic amino acid metabolism

 Tryptophan- Tyrosine- Phenylalanine

 Advanced glycation end products

 Inhibitors of ligand protein binding

 Glucurono conjugates and aglycones

 Inhibitors of somatomedin and insulin action.


 Review of literature

DIFFERENT PHASES OF CRF;

PHASE1-DECREASED RENAL RESERVE

In this phase the kidney function is almost normal. The basal GFR may be

normal or sometimes even elevated, but a rise in response to a protein challenge

which is expected is attenuated. Patient might not be symptomatic or have any

significant biochemical variations. Only creatinine clearance may change which is

lower than normal but above 50 ml/min.

PHASE 2-MODERATE RENAL INSUFFICIENCY

The Ccr is below 50 ml/min in this stage. The patient may usually present with

nocturia, decreased appetite and mild anemia.

PHASE 3-SEVERE RENAL INSUFFICIENCY

The Ccr at this stage is approximately 10- 15 ml/min. Serum creatinine rises to

5.5 to 7 mg/dl. This stage is symptomatic stage when anemia becomes severe

accompanied by metabolic acidosis, hypocalcemia, hyponatraemia and

hypochloremia.

PHASE 4-STAGE OF UREMIA

Now the renal function is only about 5-10 % of the normal. Serum creatinine

is more than 8 mg/dl. The patient is symptomatic with symptoms referred to all

organs. Hypocalcemia becomes frequent along with anemia, bleeding and bone

disease.

PHASE 5-END STAGE RENAL DISEASE

ESRD occurs when the fall in GFR is 5 to 10% of the normal, ie ≤3

ml/min. At this stage the patient requires renal replacement therapy.


Review of literature

SERUM AND URINARY CREATININE

These values are necessary in the study of amylase as the amylase clearance values

are compared with that of creatinine clearance. Reference ranges of serum creatinine are, 9

Serum or Plasma

Men : 0.8 – 1.3 mg/100ml

Women : 0.6 – 1.0 mg/100 ml.

Normal ranges of urine Creatinine is 0.5-2.0 ng/day10

Normal creatinine clearance is 95 – 105 ml/min.

(Creatinin e) Urine x volume of urine


Ccr =
(Creatinin e) serum x Time

PANCREAS

The pancreas is a flattened, elongated and soft organ which is oblique and is about 12

to 20 cm in length and lies behind the peritoneum of posterior abdominal wall.

PANCREATITIS

Acute pancreatitis can be defined clinically by a patient who presents with, -

Symptoms like epigastric pain, vomiting etc.

-An elevated serum amylase or serum lipase more than three times the upper normal

limit.

AETIOLOGY:

1) Obstructive causes:

-Gall stones (commonest)

-Stenosis of Sphincter of Oddi


Review of literature

-Ampullary/pancreatic tumors

-Parasites, clots or foreign bodies lodged in the ampulla

2) Toxic causes:

 -Alcohol

 -Drugs

 Immunosuppressants like azathioprine,6-mercaptopurine etc

 cyclosporine

 tacrolimus

 trimethoprim-sulfamethoxasole

 IV/aerosolized pentamidine

 antiviral drugs like 2,3-dideoxy inosine

 sulfasalazine

 oral 5-aminosalicylic acid

 tetracycline

 scorpion venom

3) Metabolic causes:

 hyperlipidemia

 hypercalcemia

4) Infectious causes:

 viral infections

 mumps

 coxsackie virus
Review of literature

 hepatitis B

 cytomegalovirus

 rubella

 HIV

 bacterial infections

 salmonella

 shigella

 hemorrhagic

 Escherichia coli

 Legionella

 Leptospira

 brucella

 parasitic infections

 ascaris lumbricoides can cause biliary pancreatitis

5) Autoimmune causes:

 sclerosing pancreatitis

 inflammatory bowel disease

 systemic autoimmune diseases

6) Genetic causes:

 mutation in cystic fibrosis transmembrane regulator

 mutation in trypsinogen gene

7) Iatrogenic causes:

 ERCP
Review of literature

coronary artery bypass

8) Neoplastic:

 primary pancreatic tumors

9) Vascular causes:

 ischaemia

 cholesterol emboli

 vasculitis

10) Other causes:

 trauma

 peptic ulcer

 childhood disease like sickle cell anemia

 annular pancreas

 duodenal diverticulum

 chronic renal failure

 food allergy

 ectopic pancreatic tissue

STAGES OF PANCREATITIS

Acute pancreatitis can be divided into two stages:

The first stage is the inflammatory stage which lasts for one week. During this

inflammatory stage the extent of severity of the disease is related to extra pancreatic organ

failure, which is considered as secondary effect of inflammatory response of the patient

which is triggered by acinar cell injury. During this first stage the patient has signs and
symptoms like fever, increased pulse rate, decreased blood pressure, leukocytosis and

respiratory distress. This is called Systemic Inflammatory Response Syndrome (SIRS).The

pathology involves multiple cytokines like PAF, TNF and interleukins. During this

inflammatory stage complications are rare. 75% to 80% of the patients recover at this stage

itself and only approximately 20%of the patients will enter the second stage of the disease.

The second stage of acute pancreatitis is more prolonged which may last from weeks

to months. Necrotization may also occur when the process is called necrotizing pancreatitis.

During this second stage the morbidity of the disease may be high due to organ failure,

infected necrosis etc. Patients suffering from second stage acute pancreatitis are also prone

for complications due to surgical intervention.

Acute pancreatitis can be classified according to the severity of the disease as follows:

(a) Mild- No peripancreatic complications and no organ failure.

(b) Moderate- Sterile peripancreatic complications or transient organ failure.

(c) Severe- Infectious peripancreatic complications or persistent organ failure.

(D) Critical- Infectious peripancreatic complications and persistent organ failure.

PATHOPHYSIOLOGY OF PANCREATITIS

The pathophysiology of acute pancreatitis involves the activation and release of

pancreatic enzymes in the interstitium which can cause auto digestion of the pancreas which

could be followed by multiple organ dysfunctions.

The basic changes which occur are:

a) Proteolytic destruction of the pancreas.

b) Necrosis of blood vessels.

c) Necrosis of fat.
All the above changes are accompanied by an inflammatory reaction. Interstitial

oedema is present in early stages. Later on frank necrosis of the endocrine and exocrine tissue

occurs.

The pancreatitis inducing substances blocks the secretion of zymogen granules from

the acinar cells which causes fusion of zymogen granules with the intracellular proenzyme

trypsinogen which forms intracellular trypsin and causes cellular auto digestion. The excess

trypsin generated exhausts the pancreatic secretory trypsin inhibitors inside the acinar cell

and the alpha-2-macroglobulin (nonspecific antiprotease) in the interstitium. Free trypsin also

activates other zymogen and also a cascade system of protease like the complement system,

coagulation, fibrinolysis and Kallikereum-kinin.

Trypsin also activates the complement system C5 which has a major role in

inflammatory cells formation. As the macrophages and neutrophils are more and more

stimulated, there is local spillage of arachidonic acid metabolites, clot promoting factors,

cytokines, platelet activating factors, Proteolytic and lipolytic enzymes. When all the above

factors exceed the scavenging capacity of antioxidant systems of the body, they interact with

the microcirculation of the pancreas resulting in thrombosis and hemorrhage.

Endothelial cell damage activates the local coagulation system by trypsin resulting in

interstitial oedema and leads to the formation of fibrin-platelet thrombus within the pancreatic

microcirculation.

Thus the activation of pancreatic enzymes lead to exacerbation of inflammation,

finally resulting in widening of regional necrosis.

COMPLICATIONS OF ACUTE PANCREATITIS

1) Myocardial depression is caused as acinar cells release myocardial depressant factor.


2) Acute Respiratory Distress Syndrome due to increased permeability of alveolar capillary

membrane. Pulmonary leucostasis and Proteolytic enzymes like elastase also trigger

ARDS.

3) Pleural effusion due to transfer of peripancreatic exudates through peridiaphramatic

lymphatic plexus.

4) Oliguria and renal failure result due to hypovolemia and deposition of fibrin in the

glomerular capillaries leading to acute tubular necrosis.

5) DIC due to photolytic effects of circulating trypsin.

As mentioned earlier there are many causes for acute pancreatitis. Even though it is

rare, chronic renal failure can also precipitate an attack of acute pancreatitis 11. Acute

pancreatitis can itself precipitate an attack of acute renal failure12. Acute renal failure

resulting from renal tubular necrosis is occasionally observed particularly in fulminant

pancreatitis13 which is due to fluid imbalance14.

Pancreatic disease may occur as one of the complications in renal failure patients,

although the exact incidence and pathogenesis of acute pancreatitis is not known15.

Acute pancreatitis in renal failure may be due to excess PTH, which would stimulate

calcium uptake by acinar cells. This altered calcium within the cell could lead to release of

the enzyme trypsinogen which is activated to trypsin within the pancreas causing lysis with

functional and histologic alterations16.Thus pancreatitis is initiated.

Increased gut hormones levels are found in renal failure patients. Cholecystokinin

(CCK) is also increased CCK can induce pancreatitis by causing increased secretion of

enzymes by the pancreas. It induces caspase activation and mitochondrial alterations in the

pancreatic acinar cells17.


Malignant hypertension, which is one of the complications of renal failure is also said

to precipitate pancreatitis. The pancreas is one of the organs most commonly affected with

the necrotizing lesions of malignant hypertension18.

It is possible that anatomical abnormalities of the pancreas like pancreatic ectasia,

interstitial inflammation and fibrosis, acinar ductal metaplasia, ductal or periductal

proliferation may make the patient with the end stage renal disease more susceptible to the

development of acute pancreatic disease when exposed to a variety of physiologic and non-

physiologic influences.

Several tests are used to evaluate the pancreatic exocrine function. Of this estimation

of pancreatic enzymes in body fluids is widely used as a screening test for acute pancreatitis.

Serum amylase, the most commonly estimated enzyme is found to be increased not only in

pancreatitis, but also in other conditions as the enzyme is also found in other organs in

addition to pancreas like salivary glands, liver, kidney, fallopian tubes etc.

Lipase determination exhibits good sensitivity and excellent specificity. Other

enzymes like trypsinogen19, elastase, and phospholipase A220 also have been found to be

elevated in acute pancreatitis. Trypsin and elastase remains elevated longer than that of S.

amylase.

Symptoms
Abdominal pain,
Nausea and vomiting

Serum Amylase Pancreatitis unlikely

Normal
Consider;Macroamylas
Serum Lipase emia, renal failure,
ischemic bowel,
Determine cause of parotitis, etc.
the pancreatitis

Evaluate pancreatitis
Severity
Fig 1. Evaluation of Pancreatitis
Review of literature

AMYLASE

Amylase is an important starch splitting enzyme which is increased in pancreas

abnormalities. In 1916, stock found that amylase activity in blood and urine was a sensitive

and reliable test for various pancreatic disorders21.

HISTORY

The term amylase is coined from the Greek word “Amylon” which means starch

splitting, originally it was named diastase by Payen and Persoz when they precipitated it from

Malt22.

Diastase was derived from the word diastasis meaning separation. Megendic found

amylase in the year 1846 and Foster measured it quantitatively in animals23.

DEFINITION

Alpha amylase is an enzyme that acts on starch, glycogen and other polysaccharides24.

It acts in a random fashion splitting the alpha-1,4- glucosidic bonds of a polysaccharide.

Pancreatic and salivary amylases are alpha amylases, while beta amylases are of plant origin

(Alpha 1, 4, glucon maltohydrolase).

CHEMISTRY

Amylase has molecular weight of 55.4 K.Da and acts at an optimum pH of 7.0. It

requires Calcium and chloride for optimum enzyme activity.

AMYLASE ISOENZYMES

Amylase activity can be detected in many tissues. Therefore the presence of amylase

in a tissue does not necessarily imply that it is the source of the enzyme. Contributions from
Review of literature

several organs are responsible for the serum amylase and the diseases that involve tissues and

viscera other than pancreas can also cause S1 amylase elevations.

Berk and his coworkers discovered that discrepancies in the findings were dependent

upon the method used. On the basis of polyacrylamide gel electrophoresis, Sephadex

chromatography and Isoelectrical focusing, it is believed that serum and urinary amylases can

be fractioned into two principle isoenzymes25, 26. The first is pancreatic type (P – Type) and

the second salivary type (S-Type) which chromatographically resembles the amylase derived

from organ extracts of pancreas and salivary glands respectively. Each isoenzyme may be

composed of slightly different subunits as evidenced by chromatographic mobility27, 28.

Many evidences obtained from normal subject, pancreatectamized dogs and

individuals with acute pancreatitis suggest that p- type isoamylase in serum and urine is

derived from pancreas29.

S-type is not only from the salivary glands but also from other organs30. In normal

serum the S-type component may be from other organs like fallopian tubes, ovaries, lungs,

prostate and liver31, 32.

Another type of isoamylase described was X type which may be probably derived

from S-type isoamylase. This isoamylase was found in normal urine, in sera of cancer lung

patients and also in human milk33.

In a normal person, the total serum amylase activity results from both pancreatic and

salivary type isoamylases, the pancreatic type was nearly 50% of the total isoamylase

activity34.
Review of literature

The two major isoenzymes are similar to each other except a slight variation in aminoacid

composition35. The diversity of isoenzymes observed within the pancreatic and salivary

isoamylase are due to occurance of several genetic alleles. Multiple species with each allelic

family are then formed by post translational modification35 such as glycosylation,

deglycosylation and deamidation. Many minor isoenzyme forms found by electrophoresis

are believed to be the result of post translational deamidation. In addition, some patients

demonstrate abnormally migrating isoamylases that contain sialic acid residues which are not

normally present in human amylase isoenzymes. Three to six minor P-isoamylases (P1 – P6)

and four to five minor S-isoamylases (S1 – S5) are known. The normal pattern that is usually

seen is P2 S236.

P3 minor isoamylase is seen in acute pancreatitis and in renal failure37.It is considered

as a more specific marker of acute pancreatitis than serum amylase alone.Hyperamylasemia

is associated with an increase in P3 isoamylase only when pancreatitis is present38.

METABOLISM

From the metabolism of P and S-type isoamylases in Baboon, an animal in which the

serum level and renal clearance of amylase are similar to those of man39. Duanne et al has

found out that40:

1. Most of the amylase was cleared by an external mechanism probably by the Reticulo

Endothelial System (RES) and only around 24% of amylase is cleared by urinary

excretion.

2. As the half-lives of these enzymes are approximately 130 minutes the metabolic

clearance of this enzyme is rapid.


Review of literature

3. Renal clearance of pancreatic isoamylase is almost 80% more rapid than the clearance

of salivary isoamylase.

Renal excretion of amylase takes place by glomerular filtration and tubular

reabsorption41, 42.

Serum amylase is regarded as the single most important practical diagnostic test in

acute pancreatitis. A raise in the level of serum amylase at least three times that of upper

normal limit favors the diagnosis of acute pancreatitis.

Serum amylase rises within 12 hours after the onset of acute pancreatitis and becomes
43
normal within 3to 5 days . The rise may be due to Trans peritoneal absorption of the

enzyme44, 45
and other factors like changes in the metabolism and renal secretion. Serum

amylase levels return to normal levels within 3 to 5 days usually46. Sometimes normalization

occurs rapidly indicating early resolution of the disease or extensive pancreatic necrosis or

acute exacerbation of chronic pancreatitis46, 47.

Also when pancreatitis is associated with triglyceridemia serum amylase

determination may be spuriously normal. Persistant hyperamylasemia if present suggests

ongoing inflammation or complications such as pancreatic pseudocyst or abscess48, 49.

Now it has been found that urinary amylase estimation in acute pancreatitis is more

diagnostic of the disease than serum amylase as it rises relatively earlier and higher and

persists longer50. Contrary to a normal individual who does not show any variation in urinary

amylase output within 24 hours period51 wide fluctuation in the rate of urinary amylase

excretion is seen in acute pancreatitis in a short period of time or within two hours which did

not bear any relation to the clinical condition of the patient51. Hence when one hour urine

amylase may be raised intermittently, the 24 hours urine amylase may be normal, so a raised
Review of literature

24 hour urinary amylase is significant but is misleading if normal and to be accurate in cases

of acute pancreatitis one hour urinary amylase determination every 8 to 12 hours is preferred

which is more practical and gives fewer false negative data.

Urinary amylase excretion is more sensitive index of acute pancreatitis52.This is due

to a temporary disruption of reabsorption of amylase by the tubules. This is believed to be

produced by the toxic substances which are released from the inflamed pancreas.

When serum levels are increased marginally or where amylase values have to be

correlated with clinical findings, it will be advantageous, if amylase clearance values are

studied. In an attempt to do this, Levitt et al studied in detail the amylase clearance and other

related values53.

CLEARANCE OF AMYLASE (Cam)

After knowing the serum and urinary amylase values, the rate of amylase clearance

(Cam) can be calculated as it also helps in the diagnosis of various diseases elaborated. To

calculate the Cam, the following formula can be used.

amylase (Urine) x volume of urine


Cam =
Amylase (serum) x Time

According to Michael D. Levitt, the average normal cam is 2-9 ml/minute which is increased

in pancreatitis.

Cam / Ccr Ratio

This ratio can be obtained directly from the amylase and creatinine clearance values

but in those cases where timed urine collection is not possible the ratio can be obtained from

the following formula.


Review of literature

Cam (Amylase) Urine x (Creatinin e) Serum


% x100
Ccr (Amylase) serum x (Creatinin e) Urine

According to Levitt et al Normal Cam/Ccr ratio is 2.30.097 (Range 1-5%)

In acute pancreatitis the Kidney clears amylase at a markedly increased rate due to

which Cam/Ccr is increased54, which persists even after the serum amylase returns to normal,

because urinary amylase takes a longer time to return to normal. So this ratio is said to be a

Cam
sensitive indicator of pancreatitis55. The cause of increased ratio is due to transient
Ccr

reduction in renal tubular reabsorption of amylase56. The nature of the tubular defect is not

yet fully clarified; however renal tubular dysfunction may not be the only explanation for this

increased clearance. Renal tubular dysfunction may reflect transient inhibition by a

pancreatic enzyme or toxins or possibly interference with normal tubular reabsorption of

amylase by the presence of other low molecular weight proteins that are released in acute

pancreatitis as well as in other catabolic illness57.

Cam/Ccr ratio is also increased in diabetic Ketoacidosis54. Among the possibilities

that have been postulated are hyperglucagonemia causing renal excretion of amylase, an

increased amount of low molecular weight proteins competing with amylase for renal tubular

absorption increases the S- type rather than P–type isoamylase and interference by Ketone

bodies with the measurement of serum creatinine in diabetic ketoacidosis.

NORMAL VALUES AND THEIR MODE OF EXPRESSION

P iso-enzyme in new born is around only 3% of adult range. The serum activity
Review of literature

increases with age and reaches the adult range at around 8 months of age58. The S isoamylase

in newborn is around 32% of adult range. It begins to increase by 3 – 4 months of age and

reaches adult levels (about 80 U/L) at around 5 Years of age59.

The normal values of serum and urinary amylase according to various methods of

estimation are as follows:

SOMOGYI METHOD

A Somogyi Unit is defined as a unit that is a measure of the hydrolyzing action of

serum amylase on starch and that is equivalent to the amount of enzyme in 100 milliliters of

blood serum required to produce 1 milligram of glucose when acting on a standard starch

solution under defined conditions.

The values of serum and urinary amylase by this method are expressed in

Somogyi Units (SU) per 100 ml. The normal values are:

Serum amylase: 60 – 160 SU/100 ml

Urinary amylase: 35 – 260 SU/ hr60, 61

CARAWAY METHOD

In this method the serum amylase value is expressed in Caraway Units (CU) per 100

ml. The caraway unit is approximately equal to Somogyi Unit. The normal value of serum

amylase is 9532 CU/100 ml62.


Review of literature

HUGGIN AND RUSSEL METHOD63

In this method serum amylase is expressed in units / 100 ml. Normal value of serum

amylase is 9 – 35 U.

HENRY AND CHIAMORI METHOD64

In this method serum amylase values are expressed in units/100ml. The normal

values are

Serum amylase is males : 38 to 118 U/100ml

Serum amylase in females : 46 to 141 U/100 ml

On the whole serum amylase in adults is 40 – 140 U/100 ml

Urinary amylase: 66 – 870 units / 100 ml or 43 – 245 Units / 24hrs.

AMYLOCHROME PROCEDURE65

According to this procedure

Serum amylase: 45 to 200 AMYLOCHROME UNITS / 100 ml

Urinary amylase: 40 to 330 AMYLOCHROME UNITS / hour


Review of literature

ENZYME COUPLED METHOD

Serum amylase-5 to 21 IU/L.

HYPERAMYLASEMIA AND HYPERAMYLASURIA

Any increase in the serum or urine amylase from the above mentioned normal value is

known as hyperamylasemia and hyperamylasuria respectively. This may be due to :

1. Pancreatic disease

2. Non Pancreatic diseases

3. Salivary gland disorders and

4. Disorders of other complex origin.

I. PANCREATIC DISEASES

A. Pancreatitis

1) Common causes

 Alcoholism

 Gall bladder disease

2) Uncommon causes

 Vasculitis

 Uraemia
Review of literature

3. Drug induced

(eg) Chlorthalidone

Tetracycline

Thiazide diuretics

Oral contraceptive steroids

4. Post endoscopy

5. Post-operative

6. Renal transplantation

B. Pancreatic carcinoma

C. Pancreatic trauma

II. NON PANCREATIC DISEASES

1. Renal insufficiency

2. Tumor hyperamylasemia

III. SALIVARY GLAND DISORDERS

1. Mumps

2. Calculi

3. Sialadenitis

4. Maxillofacial surgery

5. Drugs – Oxyphen butazone, phenyl butazone

6. Parotitis

7. Trauma
Review of literature

IV. DISORDERS OF OTHER COMPLEX ORIGIN

1. Biliary tract disease

2. Intra-abdominal causes other than pancreatitis.

a. Perforated gastric ulcer

b. Intestinal obstruction

c. Ruptured ectopic pregnancy

d. Mesentric infarction

e. Afferent loop syndromes

f. Aortic aneurysm with dissection.

g. Peritonitis

h. Acute appendicitis

i. Salphingitis

3. Cerebral trauma

4. Burns and traumatic shock

5. Post-operative hyperamylasemia

6. Diabetic ketoacidosis

7. Renal transplantation

8. Pneumonia

9. Acquired Bis albuminemia

10. Prostatic disease

11. Drugs – anticoagulants, salicylates, corticosteroids.

12. Pancreatic pleural effusion.

13. Mediastinal pseudocyst

14. Pregnancy
15. Review of literature

EVALUATION OF PANCREATITIS

Pancreatic amylasemia
With or without
lipasemia

Symptoms No symptoms

Pancreatic Previous pancreatitis Occasional


Aspecific or other pancreatitis finding
diseases

Illness Illness Familial Drugs


Difficult Post-Surgery
discharge of Post-ERCP
pancreatic juice Dyslipidemia
Pancreatitis Macroenzymemia
Abdominal or Viral hepatitis
(Acute, systematic
recurrent, Systemic diseases
diseases SO Malignancy
chronic
dysfunction,
Wirsung’s duct
stenosis
LIPASE

Lipase is also one of the pancreatic enzymes which are elevated in pancreatitis.

Lipase is the enzyme that catalyzes the hydrolysis of lipids.

Serum lipase is more specific and sensitive indicator to diagnose acute pancreatitis as

the serum lipase is mostly of pancreatic origin66, 67.

Serum lipase increases in the serum usually on the first day of clinical illness like that

of amylase68, 69. It also remains elevated for a longer period of about 10 days70, 71.
Review of literature

Many lipases are found in Gastro intestinal tract like lingual lipase, gastric lipase,

pancreatic lipase, phospholipase A2 and pancreatic nonspecific lipase. Apart from this

adipose tissue also contains lipoprotein lipase, liver contains hepatic lipase and endothelium

synthesizes endothelial lipase72. But the extent to which these nonpancreatic lipases

contribute to serum lipase levels has not been elucidated.

Phospholipase A2 splits the fatty acid of lecithin to form lysolecithin.

This lysolecithin damages the cell membrane. In acute pancreatitis phospholipase A2 is

activated in the pancreatic duct to form lyolecithin which can disrupt the pancreatic tissue and

also necrotize the surrounding fat.

Pancreas is the major source of serum lipase whose molecular weight is


73
approximately 50,000 . It occurs in several molecular forms. Although a more exact

description of the isoenzymes are needed, at least 2 forms are known to exist that differ in

molecular size, ionic strength requirements, co-lipase requirements, thermal stabilities etc74.

As their molecular properties are better characterized, the usefulness of measuring the lipase

associated with acute pancreatitis may improve75.

MECHANISM OF ACTION

Lipase hydrolyzes emulsified triglycerides of long chain fatty acids76. The site of

action of lipase is the interface between the oil drops and the aqueous phase77, so that the

degree of emulsification plays an important part in establishing the active substrate

concentration.
Review of literature

This distinguishes lipase from esterase which reacts with water soluble substrates. Pancreatic

lipase acts in the presence of colipase and bileacids78 at a pH range of 6 – 8, with a rapid fall

in activity below pH 579.

Lipase preferentially hydrolyses the outer 1 and 3 fatty acids. Its action on 2-

monoglyceride is weak and is probably advantageous in providing a mechanism for the

entry of glycerol into the cell, where it is required for re-esterification.

CH2 O C R1
CH2 OH R1 COOH
O
Lipase CH OH + COOH
CH2 O C R2 + 3H2O R2

O CH2 OH R3 COOH
CH2 O C R3
Glycerol Fatty Acid
O

Tri Acyl Glycerol

RENAL HANDLING OF PANCREATIC LIPASE

As lipase is completely absorbed by the proximal convoluted tubules after filtration

by the glomerulus, lipase is not found in urine of healthy people80. Therefore there is no

lipolytic activity in urine.


Review of literature

NORMAL VALUES OF SERUM LIPASE

Cherry and Crandall unit: 0.01 to 1 conventional unit. One conventional unit represent

50 micro mole of fatty acid split off in 180 minutes reaction run or 500/180 = 0.2778

micromole per minute.

The conditions in which there is hyperlipasemia81,82 :

1. Acute pancreatitis

2. Renal insufficiency

3. Dyslipidemia

4. Hyperlipidemia

5. Hepatobiliary disorders

6. Other intra-abdominal conditions like Irritable bowel syndrome

In acute pancreatitis, the serum lipase values increase like that of amylase because of

its absorption into general circulation from pancreatic bed. Even when the serum amylase

level has reached normal, the serum lipase in acute pancreatitis is one of the valuable aids to

the diagnosis of the same.

Serum amylase level is increased in many other conditions. The

estimation of serum lipase in those conditions will exclude the diagnosis of pancreatitis in

those cases. But in abdominal conditions the serum lipase levels are also increased making

the diagnosis difficult.

Patients with hypertriglyceridemia are prone to develop acute pancreatitis. This is because the

Review of literature
Review of literature

lipase in the pancreatic capillaries hydrolyze the triglycerides to release free fatty acids. This

in turn activates the trypsinogen to cause pancreatic capillary damage. In such cases even if

the serum amylase is normal , hyperlipasemia will be present83.

SERUM CALCIUM IN PANCREATITIS

Most of the patients with pancreatitis have hypocalcaemia. This may be due to

hypoalbuminemia which accompanies acute pancreatitis or sometimes patients suffering

from pancreatitis also have decreased free calcium ion irrespective of hypoalbuminemia.

If this type of hypocalcaemia occurs the prognosis is poor.

SERUM CALCIUM AND PHOSPHORUS IN CRF

Impaired kidney function is associated with variations in electrolytes

concentration. This may be due to bone disorders which occur in CRF. The main

pathophysiological changes which occur in bone disorders are increased PTH which leads to

abnormal mineral metabolism. CRF leads to decreased phosphate excretion causing hyper

phosphatemia. Increased levels of phosphorus in the blood acts on the 1-hydroxylase enzyme

and decreases its activity leading to decreased synthesis of 1, 25-Dihydroxy

cholecalciferol/calcitriol , which is the active form of vitamin D.

When the synthesis of calcitriol is decreased, the calcium absorption from the

gastro intestinal tract is also decreased. This causes hypocalcaemia. Thus hyperphosphatemia

can cause hypocalcaemia by decreased synthesis of calcitriol.

Hypocalcaemia and hyperphosphatemia together leads to increased synthesis

of PTH and increased proliferation of parathyroid cells, which ultimately leads to secondary

hyper parathyroidism.
Review of literature

Thus increased PTH stimulates the osteoblasts of the bone to cause a high bone

turnover. Low bone turnover diseases occur in osteomalacia. Disorders which occur due to

decreased bone turnover are accompanied with decreased number of osteoclasts and

osteoblasts and reduced activity of osteoblasts also.

Demineralized bone matrix is seen in osteomalacia. Therefore serum levels of

calcium and phosphorus should be estimated in the early stages of CRF to assess the

electrolyte and bone metabolism.

LABORATORY FINDINGS IN ACUTE PANCREATITIS

1. Pancreatic Enzymes in Body Fluids

Serum amylase : Elevated particularly P – isoamylase

Urinary amylase : Elevated

Cam / Ccr % : Elevated

Serum lipase : Increased

Pleural and Peritoneal fluid : Elevated amylase levels are found

Other Enzymes : Trypsin, phospholipase A2, elastase are

found to beelevated in acute pancreatitis.

Methaemoglobin : Increased

Serum triglycerides : Increased

2. STANDARD BLOOD TESTS

Increase in serum bilirubin, alkaline phosphatase and aspartate transaminase is more

in biliary tract disease than in alcoholic pancreatitis.


Review of literature

Blood glucose – Elevated because of increased glucagon.

Serum calcium – Lowered because of loss of albumin into the retroperitoneum,

partially because of calcium soap formation and possibly because of effects of paratharmone.

In case of acute renal failure occurring as a complication, blood urea nitrogen and

serum creatinine may be elevated.

Circulatory may be elevated failure leading to lactic acidosis and hence increased

lactate Dehydrogenase.

3. Tests for Anatomic Derangement

X-ray, ultrasonography, CT scan

As mentioned earlier, P2, S2 Pattern is normally seen,

when serum is electrophoresed. In acute pancreatitis, the P – type isoamylase is more than S

– type. Even when the serum amylase reaches normal in acute pancreatitis, the serum does

contain an elevated level of P-type isoamylase. In acute pancreatitis, the isoenzymes pattern

is P2, P3, S2, P3 which is normally undetectable in normal serum81. The origin of P3 in acute

pancreatitis remains unclear. A comparison with acquired Bis albuminemia may provide a

possible explanation of its origin. In acquired Bis albuminemia, electrophoresis of serum

proteins show a double band for albumin, one is corresponding to the normal albumin, the

other an abnormal band migrating more anodally. The latter is thought to be a product of

hydrolysis of normal albumin by pancreatic peptidase. Such a process may involve some

pancreatic proteins and P2 isoamylase be hydrolyzed by this process to give the faster enzyme

P3.Post translational modifications (deamidation or deglycosidation) were described by


Review of literature

Merritt and Karn84, 85. Such modifications of P2 induced by pancreatitis could also account

for P3. Evaluation of the Kinetics of P3 concentration in sera shows a peak of P3 appearing

latter than that of P2. The delay of the peak value of P3 is possibly explained by the time

required for it to be produced from P2.

ACUTE PANCREATITIS IN RENAL FAILURE

Serum amylase, P-isoamylase and lipase levels are increased in renal failure patients

both in chronic and also in acute renal failure86, 87.

THE REASON FOR INCREASED AMYLASE IN RENAL FAILURE

Renal Failure

1. Only approximately 24% of amylase is handled by the kidney88.

2. P- isoamylase is filtered 80% faster than salivary isoamylase.

Because of reduced renal function, the P – isoamylase is not filtered by the Kidney.

Hence P – isoamylase and total amylase levels increase in renal failure patients.

Even though the total amylase is elevated in renal failure patients, it is never more

than 2 fold. This is because only 24% is handled by the kidney and the rest of the amylase is

handled by the reticuloendothelial system89.

The interpretation of hyperamylasemia in CRF patients with pain abdomen becomes

difficult. The renal clearance ratio for amylase and creatinine has been suggested as a useful

aid in such a situation.


Review of literature

Previous reports have stated that the ratio of amylase clearance to creatinine clearance

remains normal in renal insufficiency and is increased in the presence of pancreatitis 53, 90.

But it has been proved that even in renal failure patients the Cam / Ccr ratio is elevated91, 92.

The increase in serum amylase is less than corresponding rise in serum creatinine. The

increased Cam / Ccr ratio must also be related to greater rise in serum creatinine than in

serum amylase. A decrease in the renal function does not reduce amylase clearance and

creatinine clearance proportionately. In severe renal failure, the Ccr is reduced out of

proportion to Cam thereby increasing the ratio93.

As mentioned above the presence of significant P3 activity has been associated almost

exclusively with acute or chronic pancreatitis, with pancreatic pseudocyst and sometimes

with renal failure because of decreased clearance of the enzyme. When P3 is detected in renal

failure, without clinical pancreatitis, diagnosis confusion can be reportedly minimized by

measuring the fractional percentage of the pancreatic isoamylase. As explanation, it has been

suggested that impaired renal excretion of plasma amylase facilitates post-translational

modification of the pancreatic P2 form within the circulation with the formation of the P3

isofoms94.

SERUM LIPASE IN RENAL FAILURE

Even though this also is one of the diagnostic aids in acute pancreatitis, this has been

proved to be elevated in renal failure patients95, 96.

THE REASON FOR ELEVATED LIPASE LEVELS

Lipase is removed from the serum mainly by glomerular filtration. Reabsorption of


lipase is almost complete in contrast to that of amylase97. The difference in handling of both

enzymes is due to their differing affinities for hydrophobic and hydrophilic surfaces. Serum

lipase activity appears to increase proportionately to the degree of renal failure, up to a

maximum of approximately 4 times of normal value87,98.

The patients with end stage renal disease and who were on maintenance hemodialysis

also have elevated serum amylase, P-isoamylase and serum lipase values. There is no

significant difference in pancreatic enzymes between pre and post hemodialysis sample99.
MATERIALS AND METHODS
Materials and methods

The present study was carried out at the Nephrology unit of Sree Mookambika

Institute of Medical Sciences, Kulasekaram from August 2012 to May 2013 for a time period

of 10 months. This cross sectional study was approved by the institutional Human Ethical

Committee. Voluntary informed consent was taken from all the subjects of the study.

SOURCE OF DATA

Fifty patients suffering from Chronic Renal Failure were selected from the

Nephrology ward as well as Nephrology outpatient department of Sree Mookambika Institute

of Medical Sciences, Kulasekaram. The history of the patient was taken by audit

questionnaire.

STUDY GROUP

Based on the serum urea and serum creatinine values the CRF patients were categorized in to

three groups for the study

GROUP 1: Mild CRF (Serum Urea 40-80 mg% and serum creatinine 0 to 3mg %)

GROUP 2: Moderate CRF (Serum Urea 81-100 mg% and serum creatinine 3.1 to 4.5 mg %)

GROUP 3: Severe CRF (Serum Urea >100 mg% and serum creatinine 4.6 to 8 mg %)

TYPE OF STUDY: Cross-Sectional Study

INCLUSION CRITERIA

Fifty patients with CRF from Nephrology Department of Sree Mookambika Institute

of Medical Sciences between the age group from thirty to eighty years were included in the

study.

EXCLUSION CRITERIA

1) Chronic alcoholics with portal hypertension.

2) Known case of previous pancreatitis


Materials and methods

Parameters to be studied

Serum Urea, Serum Creatinine, Serum Amylase, Serum Lipase, Serum Calcium,

Serum Phosphorus, Uninary Amylase and Urinary Creatinine.

Sample collection

In the diseased patients studied, a sample of timed 4 hours urine was collected in

sterile containers during which period about 5 ml blood was collected in a red capped

Vaccutainer. Blood samples were then centrifuged at 3000 rpm for 10 minutes and the serum

was separated for further tests.

ESTIMATION OF SERUM AMYLASE

METHOD (CNPG3 Kinetic Method)

Historically various methods have been used, but all have relied on either an ill-

defined substrate (starch) or the use of helper enzymes with defined substrates. Here we

describe the direct colorimetric assay for  – amylase in which a defined substrate is used
that is predominantly cleaved directly by  – amylase to yield free chromophore. The

reaction is α Amylase

5CNPG3 3CNP + 2 CNPG3 + 2 Glucose + 3 Maltotriose

Rate of increase in absorbance due to formation of CNP is measured at 405 nm and is

proportional to the  -amylase activity in the sample.


Materials and methods

REAGENTS

Amylase Mono Reagent (R1)

R1 MES buffer pH 6.0 90 mmol/L

Sodium Chloride 500 mmol/L

Pottasium Sulpha cyanide 0.60 mol/L

Calcium Acetate 7.2 mmol/L

CNPG3 2.7 mmol/L

PREPARATION OF THE REAGENT:

Peracetylation of maltotriose with acetic anhydride was done in the presence of

sodium acetate which acts as catalyst ,at 120 to 140 degree centigradefor 1 to 2 hours. This

results in formation of a peracetate with beta configuration at the anomeric carbon. This is

then heated with 3 to 10 equivalents of phosphorus pentachloride along with carbon tetra

chloride or chloroform (supporting solvent).This results in formation of a melt which is then

further heated at 70 to 90 degree centigrade for 3 to 8 hours to give 1-beta-chloro-2-trichloro

acetyl derivative which is then de-trichloro acetylated to 1- beta-chloro-2-hydroxy derivative

by treating with saturated ammonia in diethyl ether for 10 to 60 minutes at 0 to 10 degree

centigrade. This is then treated with a secondary amine in an aromatic hydrocarbon like

benzene or toluene at 15 to 30 degree centigrade for 8 to 24 hours which results in formation


Materials and methods

of an alpha-anhydride. This anhydride reacts specifically with the hydroxyl group of the

chromogen in an aromatic solvent like toluene to yield alpha-maltotriosides. Equimolar

concentration of chromogen and the anhydride are mixed and refluxed for 4 to 24 hours in

toluene or benzene. When the alpha maltotrioside is deacetylated, a less side product is

formed which is usually associated with the reactions at the glycosidic bond. The compound

is reacted with a mixture of concentrated hydro chloric acid, methanol and chloroform in the

ratio 1:10:4 respectively at 20 to 25 degree centigrade for 2 to 4 days to give deacetylated

alpha-maltotriosides which was then isolated by neutralizing the acid, removing the residual

organic solvents and freeze drying.

Specimen

Unhemolysed serum should be used EDTA or citrate should not be used as

anticoagulant as they decrease the activity of the enzyme by 15%.

Procedure

The semiautoanlyzer was programmed as per the assay parameters.

BLANK SAMPLE

Working reagent 1000µl 1000µl

Distilled water 25 µl -

Sample - 25 µl
Materials and methods

1000  L of the reagent and 25  L of serum was taken in a test tube and mixed well.

Reading was taken in the semi auto analyzer.

LINEARITY

The reaction is linear up to a concentration of 1000 units per litre. Samples with values more

than 1000 U/L must be diluted with normal saline.

Reference Range

Serum  -amylase activity is 35– 115 IU/L.

ESTIMATION OF URINARY AMYLASE

The procedure is the same except that the sample was diluted with purified water in

the ratio 1:5. Instead of serum, diluted urine was added and all the steps were followed as for

serum amylase determination.

Reference range

Urinary  -amylase activity is up to 1360 IU/24 hours.

ESTIMATION OF SERUM LIPASE

Ready to use two liquid reagent systems was used.

Principle

The Chromogenic lipase substrate 1,2-0- dilauryl – rac – glycerol -3-glutaric acid

ester (6 – methyl resorufin) is cleaved by catalytic activity of lipase to form 1,2-0- dilauryl –

rac – glycerol and an unstable intermediate glutaric acid ester. This decomposes

spontaneously in alkaline medium to form glutarate and methyl resorufin. The lipase activity
Materials and methods

in the serum is proportional to the rate of formation of methyl resorufin and can be

determined photo metrically at 578 nm.

1,2, - O-Dilauryl – rac – glycerol – 3 – glutaric acid – ester


(6 methyl resorufin)

Lipase / Colipase

1,2, - O-Dilauryl – rac – glycerol +glutaric acid


(6 methyl resorufin) ester

Lipase / Colipase
Alkaline medium

glutaric acid + methyl resorufin

COMPONENTS

Reagents Concentration

R1 Bicin buffer (pH 8) 50 mmol/lt

Colipase  1 mg/lt

Sodium deoxycholate 1.6 mmol/lt

Calcium chloride 10 mmol/lt

R2 Tartarate buffer (PH4) 10 mmol /lt

Tauro deoxy cholate 8.8 mmol/lt

1,2 – o-Dilaury – rac – Glycero – 3 glutaric 0.27 mmol/lt

acid – 6 Methyl / resorufin


Materials and methods

Specimen Collection

Serum is preferred. Lipase activity in serum is stable for 5 days at 2-8oC or for 24

hours at 20-25oC.

Procedure

Reagent blank Calibrator Serum/Plasma

R1 0.8 ml 0.8 ml 0.8 ml

Distilled H2O 0.01 ml 0.08 ml 0.8 ml

Mix and incubate for five minutes at 37o C

R2 0.2 ml 0.2 ml 0.2 ml

800  l of R1 and 200 of R2 was taken and mixed well. It was incubated at room

temperature for 5 minutes. Then 10  l serum was added and mixed well. Then the value

was read in the Semi atuto analyzer.

Reference Interval

Serum lipase activity is 13-60 IU/L

If lipase activity exceeds 300 IU/L, dilute the specimen with normal saline and repeat

the assay. That result should be multiplied with dilution factor to obtain the correct lipase

activity.
Materials and methods

Estimation of serum urea

Serum urea was determined by kinetic UV method using Gesan kit.

Principle

Urea is hydrolysed in the presence of water in urease to produce Co2. The ammonia

formed in the first reaction combines with  - Ketoglutarate and NADH which in the

presence of glutamate dehydrogenase form glutamate and NAD+. The decrease in

absorbance at 340 nm due to depletion of NADH is proportional to the urea concentration in

the sample

Reagents

Concentration

R1: Goods buffer pH 7.6 130 mmol/l

ADP 1.2 mmol/l

Urease  8000 u/l

GLDH  1500 u/l


R2 Goods buffer pH 10.2 100 mmol /l

 - ketoglutarate 65 mmol/l

NADH 1.2 mmol/l

Reagents are liquid and ready to use. About using as mono reagent pour the content of R2

vial into the R1 vial and let stand for 6.15 minutes at least. For minority use add 1 ml of R2
Materials and methods

reagent to every 4 ml of R1 reagent. Keep the reagents out of the refrigerator only for

use.

Store the kit at 2-8oc. After opening the vials, R1 and R2 are stable for 90 days if

recapped, immediately and is protected from contamination, evaporation, direct light and is

stored at the appropriate temperature. The working solution (R1+R2) is stable for 20 days at

2-8oC.

Specimen collection

Specimens used are serum or plasma. Urine specimen is diluted to 1:20 ratio. Do not

use hemolysed samples. Do not use ammonia – heparinate and fluorides as anticoagulants.

Urea in the serum is stable upto 3 days if stored at 2-8o c or for three months at – 20oC.

Procedure

The procedure is done at wavelength 340 nm and the working temperature was 37oC.

The reagents were brought at 15oC – 25oC before using them.

Monoreagent procedure “Sample Starter”

Blank Standard Sample

Working reagent 1000  l 1000  l 1000  l

Distilled water 10  l - -

Sample - - 10  l

Standard - 10  l -
Materials and methods

All the reagents were prepared as above and incubated at 37oC. After 30 minutes of

addition of the sample the absorbance values of first reading was taken as E1C and standard

was taken as E1 Std. After 60 minutes the absorbance value of second reading was taken as

E2 C and that of standard was taken as E2 STD.

Calculation

S. Urea is calculated as,

E2C  E1 C 
S.Urea (mg/dl) = xConc. Std
E2 Std  E1 std 
For diluted urine – Multiply the result with dilution factor.

Conversion factor

Urea (mg/dl) x 0.1665 = Urea (mmol/l)

Reference values

Serum Urea = 10 – 50 mg/dl (OR) 1.67 – 8.32 mmol/l

Urea in Urine = 20-35g/24 hour (OR) 3330583 mmol/24hour

Linearity

The reaction is linear upto a concentration of 300 mg/dl (49.95 mmol/l) with a range

of 4.9-300 mg/dl (0.81 – 49.95 mmol/l). Samples with values exceeding 300 mg/dl must be

diluted with saline solution. Then multiply the result for dilution factor.

ESTIMATION OF SERUM CREATININE

The method used for estimation of creatinine in serum or urine was Jaffe’s

calorimetric method without deproteinization.


Materials and methods

Principle

Creatinine reacts with picrate in alkaline environment to give a coloured compound

whose intensity is proportional to the creatinine concentration in the sample.

Components

Reagents Concentration

R1 : Lithium hydroxide 120 mmol/l

Boric acid 80 mmol/l

R2 : Picric acid 67 mmol /l

Reagents are liquid and ready to use. For using as monoreagent (sample starter

procedure) the reagents R1 and R2 were mixed in equal parts. After opening the vials R1 and

R2 are stable for 90 days if recapped immediately and protected form contamination,

evaporation, direct light and stored at correct temperature.

Specimen collection

Serum or plasma and urine. Do not use hemolysed samples. The creatinine is stable

in the samples up to 24 hours at 2-8oC.

Procedure: (Mono reagent ) Procedure “Sample starter”

Wavelength 510 nm

Working temperature 37oC

Reaction – fixed time


Materials and methods

Blank Standard Sample

Working reagent 1000  l 1000  l 1000  l

Distilled water 50  l - -

Sample - - 50  l

Standard - 50  l -

The reagents were mixed as above and incubated at 37oC. After 30 minutes the

absorbance of sample (E1C) and standard (E1 std) was read against the reagent blank. After

another one minute the second reading was taken as E2C and E2 std.

Calculation

E2C  E1 C 
S. Creatinine (mg/dl) = xConc. Std
E2 Std  E1 std 
Conversion factor

S.Creatinine (mg/dl) x88.4 = S.Creatinine (  mol/l)

Reference intervals

S.Creatinine in males is 0.9 – 1.3 mg/dl (80-115  mol/l)

In females 0.6-1.1 mg/dl (53-97  mol/l)

ESTIMATION OF URINE CREATININE

The same procedure was followed but the sample urine (100  l) was diluted with
Materials and methods

900  l distilled water. The same procedure was done using 500  l of creatinine reagent

and 50  l of diluted urine.

Reference interval

Normal urine creatinine is 60-130 mg/dl.

ESTIMATION OF SERUM CALCIUM

Serum calcium was estimated using calcium Bio systems kit.

Principle

Calcium in the sample reacts with arsenazo III forming a coloured complex which can

be measured by spectrophotometry.

Contents

A.Reagent - Arsenazo III 0.2 mmol /l

- Imidazole 75 mmol/l

S.Standard - Calcium / magnesium standard

Calcium 10mg/dl, Magnesium 2mg/dl

Aqueous primary standard.

Sample: Serum

Calcium in Serum / plasma is stable for 10 days at 2-8oC. Anticoagulants other than

heparin should not be used.


Materials and methods

Procedure

Blank Standard Sample

Calcium Standard (S) - 15 µL -

Sample - - 15 µL

Reagent (A) 1000 µL 1000 µL 1000 µL

The reagent was brought to room temperature 500  l of the reagent was taken and

10  l serum was added to it and mixed well and then incubated at room temperature for 5

mts and the value was taken in a semiautoanalyzer.

Reference Range

Serum calcium is 8.6 – 10.3 mg/dl = 2.15 – 2.58 mmol/lt

ESTIMATION OF SERUM PHOSPHORUS

Method used was molybdate /UV method

Principle: End point Analysis. Inorganic phosphorous reacts with Ammonium molybdate in

acid environment to form phospho molybdate complex. The increase in absorbance is

proportional to the inorganic phosphorous in the sample.

Constituents of the reagent

Reagent Concentration

Hydrochloric acid 1.0 mmol/L

Ammonium Molybdate 0.70 mmol/L

Surface active agents Anionic and poly anionic


Materials and methods

Specimen: Serum/ Plasma

Procedure

Reaction type - End point

Wave length - 340 nm

Temperature - 37oC

Incubation - 5 minutes at room temperature

Working reagent was prepared 250  L of reagent 1 and 250  l of reagent 2 were

mixed well. (This reagent is stable for 2 days at 2-8oC).

BLANK STD SAMPLE

Working reagent 1000  L 1000  L 1000  L

Distilled Water 10  L - -

Sample - - 10  L

Standard - 10  L -

Mix, then incubate for one minute at 37o C. Measure the absorbance of the sample (EC)

and standard (ESTD) against the reagent blank.

Reference interval

Children 4 - 7 mg/dl

Adults 2.5 – 5 mg/dl


COLOUR PLATES
C.P 1Collection of Venous blood sample

C.P 2 Various blood collection tubes


C.P 3 Table top centrifuge

C.P 4 Separation of serum


C.P 5 Semi auto analyzer

C.P 6 Structure of alpha amylase


C.P 7 Action of alpha amylase

C.P 8 Mechanism of action of lipase


RESULTS AND OBSERVATIONS
Results and observations

RESULTS AND STATISTICAL ANALYSIS


The results of the various biochemical parameters obtained on analysing the blood and urine

samples of 50 cases of chronic renal failure selected for the study were tabulated in three

different tables.

Data was entered in excel spread sheet and was analysed using statistical software Epi

Info Version3.5.3.Analysis was done by simple proportion,mean±SD and Chisquare test.

The mean and standard deviation were calculated for each of the biochemical

parameters pertaining to renal and pancreatic functions namely serum urea, serum creatinine,

urine creatinine, creatinine clearance, serum amylase, urinary amylase and serum lipase in all

the three tables giving the results of mild, moderate and severe renal failure individuals. In

addition to the above parameters which would give detailed analysis of renal and pancreatic

functions, the other biochemical parameters which are involved in renal diseases like Serum

Calcium and phosphorous were also assessed to complete the study and tabulated in the

above tables.

For mild chronic renal failure individuals the mean serum urea was 58.69±9.65mg/dl

and the mean of serum and urea Creatinine was 2.52±0.26 mg/dl and 64.16±11.81mg/dl

respectively. The mean serum amylase was 158.36±31.45U/dl whereas the urinary amylase

was 101.15±25.45U/dl. The mean Creatinine clearance and amylase clearance calculated

from the above values were 57.24±14.40ml/min and 1.44±0.41ml/min respectively. The

mean ratio of amylase clearance to that of Creatinine clearance was 2.62±0.86±%.The mean

serum lipase was153.32±73.81U/L. The other parameters like serum calcium and serum

phosphorus for this group were 8.4±0.65mg/dl and 4.88±0.71mg/dl respectively.

For moderate chronic renal failure the mean serum urea was 87.09±6.02 mg/dl. The

serum Creatinine and urine Creatinine were 3.68±0.75 mg/dl and 74.59±17 mg/dl
respectively. The mean serum amylase and urinary amylase were 185.72±71.28 U/dl and

83.94±27.85U/dl respectively. The mean Ccr and Cam were 26.44±5.6 ml/min and

0.62±0.25ml/min respectively. Cam/Ccr ratio was found to be 2.39±0.92%. The mean serum

lipase was 166.53±99.64U/L. The mean serum calcium and phosphorous were

8.19±0.46mg/dl and 5.07±0.62mg/dl respectively.

The results of severe chronic renal failure patients are as follows. The mean serum

urea, serum and urinary Creatinine were 142.02±38.56mg/dl, 7.39±2.56mg/dl and

61.27±15.22mg/dl respectively. The mean values of serum and urinary amylase were

196.21±44.39U/dl and 86.10±17.51U/dl respectively. The Ccr and Cam calculated from the

above values were 7.11±4.01ml/min and 0.34±0.13ml/min respectively. The Cam/Ccr ratio

was found to be 5.67±2.22%. The mean value of serum lipase was 168.17±71.68U/L. The

mean serum calcium and phosphorous were 8.18±0.29mg/dl and 5.41±0.5.1mg/dl

respectively.

To understand the significance of assessing the serum and urinary pancreatic enzymes

and the ratio of amylase to Creatinine clearance in renal failure, these parameters as well as

the other parameters assessed for renal failure(serum calcium and phosphorous) are

compared between various grades of renal failure in the following tables.


Table 1 Comparison between mild and moderate renal failure

Serum Urinary Amyla 𝑪𝒂𝒎 Serum Serum Serum


%
Amylase amylase se 𝑪𝒄𝒓 Lipase Calciu phosphor
U/100ml U/100ml clearan U/L m ous
ce mg% mg%
(Cam)
ml/min
Mild
158.36±31 101.15±25 1.44±0. 2.62±0. 153.32±73 8.4±0.6
renal 4.88±0.71
.45 .45 41 86 .81 5
failure
Moderat
185.72±71 83.94±27. 0.62±0. 2.39±0. 166.53±99 8.19±0.
e renal 5.07±0.62
.28 85 25 92 .64 46
failure
T-value 2.08 3.44 47.05 0.56 0.19 1.17 0.68
P-value 0.16 0.07 0.00 0.46 0.67 0.29 0.42
Significa
NS NS S NS NS NS NS
nce

Table 1 shows the comparison of parameters between mild and moderate renal failure

Here we found out that the T value for serum amylase was 2.08, for urinary amylase

was 3.44, for serum lipase was 0.67, for serum calcium was 0.29, for serum phosphorous was

𝑪𝒂𝒎
0.68 and for % was 0.56. Their P values were >0.05 and hence were not of significance.
𝑪𝒄𝒓

But for amylase clearance T value was 47.05 and the P value was 0.000(<0.01) which

showed a significant variation.


Table 2Comparison between moderate and severe renal failure

Serum Urinary Amylas 𝑪𝒂𝒎 Serum Serum Serum


%
Amylase amylase e 𝑪𝒄𝒓 Lipase Calciu phosphor
U/100ml U/100ml clearan U/L m ous
ce mg% mg%
(Cam)
ml/min
Moderat
185.72±71 83.94±27. 0.62±0. 2.39±0. 166.53±99 8.19±0.
e renal 5.07±0.62
.28 86 25 92 .64 46
failure
Severe
196.21±44 86.1±17.5 0.34±0. 5.67±2. 168.17±71 8.18±0.
renal 5.40±0.51
.39 1 13 22 .68 29
failure
T-value 0.26 0.07 17.19 30.58 0.003 0.002 2.93
P-value 0.61 0.79 0.00 0.00 0.96 0.97 0.1
Significa NS NS S S NS NS NS
nce

Table 2 shows the comparison of biochemical parameters between moderate and severe renal

failure. Here the T values for all the parameters compared were serum amylase – 0.26,

urinary amylase – 0.07, serum lipase – 0.003, serum calcium – 0.002 and serum phosphorous

– 2.93. Their P values were >0.05 and hence were not significant. But the T values for

𝑪𝒂𝒎
amylase clearance was 17.192 and for % was 30.58 whose P values were 0.00(> 0.01)
𝑪𝒄𝒓

which showed a significant variation.


Table 3 Comparison between mild and severe renal failure

Serum Urinary Amyla 𝑪𝒂𝒎 Serum Serum Serum


%
Amylase amylase se 𝑪𝒄𝒓 Lipase Calciu phosphor
U/100ml U/100ml clearan U/L m ous
ce mg% mg%
(Cam)
ml/min
Mild
158.36±31 101.15±25 1.44±0. 2.62±0. 153.32±73 8.4±0.6
renal 4.88±0.71
.45 .45 41 86 .8 5
failure
Severe 8.18±0. 5.41±0.51
196.21±44 86.1±17.5 0.34±0. 5.67±2. 168.16±71
renal 29
.39 0 13 22 .68
failure
T-value 8.23 4.04 112.19 27.86 0.35 1.60 6.21
P-value 0.01 0.05 0.000 0.000 0.56 0.22 0.02
Significa S NS S S NS NS S
nce

Table 3 shows the comparison of all the parameters between mild and severe renal failure.

Here we found out that T values for urinary amylase was 4.04., for serum lipase it was 0.35

and for serum calcium it was 1.6. Their P values were>0.5 and showed no significance.

But the T value for serum amylase was 8.23 and the P value was 0.007(<0.05) which

showed significant variation. Similarly the T value for amylase clearance was 112.19 and

𝑪𝒂𝒎
% was 27. 86 and their P values were 0.000(<0.01) which showed significant variation.
𝑪𝒄𝒓

The T value for serum phosphorous was 6.21 and its P value was 0.02(<0.05) which also

showed significant variation.


Table 4 Correlation of serum amylase and serum urea in various study groups

Mild renal Moderate Severe renal


failure renal failure failure
Serum Urea(mg%) 58.69±9.65 87.09±6.02 142.02±38.56
Serum
158.36±31.45 185.72±71.28 196.21±44.39
Amylase(U/100ml)
Pearson correlation
0.33 -0.11 -0.03
Coefficient
p-Value 0.20 0.69 0.92
Significance NS NS NS

To find out the correlation level of each of the pancreatic enzymes with that of serum

urea or serum Creatinine which are the two most important parameters of renal function,

Pearson correlation coefficient was obtained by comparing their level in various study groups

like mild, moderate & severe renal failure & the significance of the same was obtained.

In Table 4 Correlation between serum urea and serum amylase has been made in

various study groups mild, moderate and severe renal failure respectively. The P value

derived from the Pearson correlation coefficient on comparison of the parameters in various

study groups showed that serum amylase had no significant correlation P (>0.05), with

serum urea in all the study groups.


Table 5 Correlation of serum lipase and serum urea in various study groups

Mild renal Moderate renal Severe renal


failure failure failure
SERUM
58.69±9.65 87.09±6.02 142.02±38.56
UREA(mg%)
Serum
153.32±73.81 166.53±99.64 168.17±71.68
Lipase(U/L)
Pearson
correlation 0.12 -0.27 0.09
Coefficient
P-Value 0.66 0.31 0.72
Significance NS NS NS

In Table 5 a comparison has been made between serum lipase and serum urea in various

study groups mild, moderate & severe renal failure respectively. The P value derived from

the Pearson correlation coefficient on comparison of the parameters in various study groups

showed that serum lipase had no significant correlation ( P>0.05) with serum urea in various

study groups.
Table 6 Correlation of serum creatinine and serum amylase in various study groups

Mild renal Moderate renal Severe renal


failure failure failure
Serum
Creatinine 2.52±0.26 3.68±0.63 7.39±2.56
(mg%)
Serum
Amylase(U/100 158.36±31.45 185.72±71.28 196.21±44.39
ml)
Pearson
correlation 0.37 0.65 0.25
Coefficient
P-Value 0.14 0.01 0.34
Significance NS HS NS

Table gives a comparison of serum amylase with serum Creatinine in various study groups

mild, moderate & severe renal failure. The P value was derived from Pearson correlation

coefficient by comparing the parameters in various study groups, The P vale was >0.05 when

serum amylase was compared serum Creatinine in mild & severe renal failure, but the P value

was 0.01 in moderate renal failure which showed significance.


Table 7 Correlation of serum creatinine and serum lipase in various study groups

MILD RENAL MODERATE SEVERE


FAILURE RENAL RENAL
FAILURE FAILURE
Serum Creatinine
2.52±0.26 3.68±0.631 7.39±2.56
(mg%)
Serum Lipase (U/L) 153.37±73.81 166.53±99.64 168.17±71.68
Pearson correlation
0.05 0.45 0.21
Coefficient
P-Value 0.84 0.08 0.43
Significance NS NS NS

Table 7 gives a comparison of serum lipase with serum Creatinine in various study groups

mild, moderate & severe. The P value was derived from Pearson Correlation coefficient by

comparing these parameters in all the study groups. The p value was not significant (>0.05)

when serum lipase was compared with serum Creatinine in all the study groups.
Table 8 Correlation of serum creatinine and amylase clearance in various study groups

MILD RENAL MODERATE SEVERE


FAILURE RENAL RENAL
FAILURE FAILURE
Serum Creatinine
2.52±0.26 3.68±0.63 7.39±2.56
(mg%)
Amylase clearance
1.44±0.41 0.62±0.25 0.34±0.13
(ml/min)
Pearson correlation
-0.06 -0.54 -0.64
Coefficient
P-Value 0.82 0.03 0.01
Significance NS S HS

Table 8 shows the correlation of serum Creatinine & amylase clearance in various

study groups – mild, moderate & severe renal failure respectively. The P value was derived

from Pearson correlation coefficient by comparing the parameters in various study groups.

The P value was >0.05 when serum Creatinine was compared to amylase clearance in mild

RF, but the P value was <0.05 in moderate RF & <0.01 in severe RF which showed two

tailed significance.
𝑪𝒂𝒎
Table 9 Correlation of serum creatinine and ratio in various study groups
𝑪𝒄𝒓

Mild renal Moderate renal Severe renal


failure failure failure
Serum Creatinine
2.52±0.26 3.68±0.63 7.39±2.56
(mg%)
𝑪𝒂𝒎
% 2.62±0.86 2.39±0.92 5.6741±2.22
𝑪𝒄𝒓
Pearson correlation
0.45 -0.41 0.56
Coefficient
P-Value 0.07 0.12 0.02
Significance NS NS S

Table 9 Shows a correlation between serum Creatinine & Cam ration in various study

groups –mild, Moderate & severe renal failures respectively. The P value was derived from

Pearson correlation coefficient by comparing these parameters in all the study groups. The P

value was found to be >0.05 in mild & moderate renal failure which showed no significance.

The P value was <0.05 in severe renal failure which showed significance.
Table 10 Correlation of serum creatinine and serum calcium in various study groups

Mild renal Moderate renal Severe renal


failure failure failure
Serum Creatinine
2.52±0.26 3.68±0.63 7.39±2.56
(mg %)
Serum calcium
8.4±0.65 8.19±0.46 8.18±0.29
(mg %)
Pearson correlation
0.04 -0.42 0.12
Coefficient
P-Value 0.87 0.11 0.65
Significance NS NS NS

Table 10 shows the correlation between serum Creatinine & serum phosphorous in various

study groups mild, moderate & severe renal failure. The P value was derived from Pearson

correlation coefficient by comparing the parameters in various study groups. The P value was

>0.05 when serum Creatinine was compared with serum phosphorous in mild& severe renal

failure which showed no significance, but in moderate renal failure the P value was <0.01

which showed two tailed significance.


Table 11 Correlation of serum creatinine and serum phosphorus in various study groups

Mild renal Moderate renal Severe renal


failure failure failure
Serum Creatinine
2.52±0.26 3.68±0.63 7.39±2.56
(mg %)
Serum Phosphorus
4.88±0.71 5.07±0.62 5.41±0.51
(mg %)
Pearson correlation
0.14 0.74 0.05
Coefficient
P-Value 0.59 0.00 0.86
Significance NS HS NS

Table 11 shows the correlation between serum Creatinine & serum calcium in various study

groups- mild, moderate & renal failure respectively. The P value was derived from the

Pearson correlation coefficient. The P value was found to be >0.05 in all the study groups

which showed no significance.


Table 12 Correlation of serum calcium and serum phosphorus in various study groups

Mild renal Moderate renal Severe renal


failure failure failure
Serum Calcium (mg
8.4±0.65 8.19±0.46 8.18±0.29
%)
Serum Phosphorus
4.88±0.71 5.07±0.62 5.41±0.51
(mg %)
Pearson correlation
-0.57 -0.45 -0.23
Coefficient
P-Value 0.02 0.07 0.37
Significance S NS NS

Table 12 shows the correlation of serum calcium & serum phosphorous in various study

groups – mild, moderate & severe renal failure respectively. The P value was derived from

the Pearson correlation coefficient. The P value was <0.05 in mild renal failure which showed

significance, but the P value was >0.05 in moderate & severe renal failure which showed no

significance.
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