Mohd Hairi Bin A.Hamid Bsc.

Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)

Introduction of pancreatic function

Elongated gland organ in the digestive and endocrine that lies in the back of the mid-abdomen Weight 75 125 g Length 10 20 cm Width 3 5 cm Thickest in the head (1.5 3.5 cm) & thinnest in the tail (0.8 2.5 cm) Functions: Endocrine functions o Origin is Islet of Langerhans (, , cells) o Production of insulin, glucagon, and gastrin Exocrine functions o The islet cells surrounded by acinar cell which involved in exocrine functions. o Secretion of digestive enzymes in pancreatic juice (Eg: Amylase, Lipase) o Help in the breakdown of carbohydrates, protein, and fat in the chime Pancreatitis

An inflammation of the pancreas which pancreatic enzymes escaped into the surrounding tissues either high or low level. Types: Acute pancreatitis and Chronic Pancreatitis

Page | 1

Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)

Acute pancreatitis Definition Lab Findings Ransons Score @ Ransons prognostics signs Inflammation of pancreas that occurs suddenly as one attack and can be recurrent with resolutions. Nausea, vomiting, diarrhea Fever Muscle spasm with epigastric pain Tachycardia Jaundice Restless and dehydration Idiopathic Autoimmune disease (Eg: Systemic Lupus Erythematosus, SLE) Alcohol consumption (Eg: Ethanol) Radiation Trauma Steroids Hyperlipidemia, Hypercalcemia, Hypothermia Serum-Urine Amylase Significantly increased (2 12 Hours) Significantly increased (12 72 Hours) Lipase Normal/ Slightly increase (2 12 Hours) Normal/ Significantly increase (12 72 Hours) Hepatic Function Studies Transaminase levels Function: To estimate severity of acute pancreatitis Includes: Age > 55 years Estimated sequestration > 6000 ml Hematocrit 10% AST > 250 IU/L LDH > 350 IU/L Calcium < 8 mg/dL Arterial pCO2 < 60 mmHg Blood glucose > 200 mg/dL Base deficit > 4 mmol/L BUN > 5 mg/dL Occur within 2 weeks of onset of pain. 2 pancreatic infections common cause of death Necrosis, organ failure and includes: Cardiovascular system Pulmonary system Renal systems

Signs and Symptoms

Aetiology

Complications

Page | 2

Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)

Treatment:

Surgery No oral alimentation Analgesics Release pain IVF and colloids Maintain normal intravascular volume Nasogastric suction Decrease gastrin release from the stomach Prevent gastric contents from entering the duodenum

Chronic Pancreatitis Definition Lab Findings A continual inflammation and destruction of the pancreas, that change its normal structure and functions Thin Epigastric tenderness Abdominal pain (Steatorrhoea) Severe nausea Pale Weight loss Skin pigmentation over abdomen & back Idiopathic Chronic Alcohol consumption Chronic Steroids used Autoimmune diseases Cystic Fibrosis (Common) Severe protein-energy malnutrition (Common) Serum-Urine Amylase Normal/ Slightly increased (2 12 Hours) Normal/ Slightly increased (12 72 Hours) Lipase Normal/ Slightly increased (2 12 Hours) Normal/ Slightly increased (12 72 Hours) Peptic ulcer Malabsorption of fat and protein Chronic calcifies pancreatitis (70%) Diabetes (30%) Therapeutic endoscopy Surgery Insulin therapy for diabetes Analgesics Release pain

Signs and symptoms

Aetiology

Complications

Treatment

Page | 3

Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)

Diagnosing of acute pancreatitis and chronic pancreatitis

2 12 Hours

ACUTE PANCREATITIS Amylase Lipase

ACUTE PANCREATITIS Amylase Normal Lipase

CHRONIC PANCREATITIS N/slight Amylase N/slight Lipase

12 72+ Hours

Amylase serum, urine Lipase

Amylase serum, urine Normal Lipase

N/ slight Amylase serum, urine N/ slight Lipase

Patient sign & symptoms shows acute pancreatitis Radiology graphs diagnostics

Patient sign & symptoms shows acute pancreatitis Radiology graphs NOT diagnostics INVESTIGATE SECONDARY CAUSES

ISOENZYME ANALYSIS RANSONS PROGNOSTIC SIGNS 1) Age > 55 years 2) Estimated sequestration > 6000 ml 3) Hematocrit 10% 4) AST > 250 IU/L 5) LDH > 350 IU/L 6) Calcium < 8 mg/dL 7) Arterial pCO2 < 60 mmHg 8) Blood glucose > 200 mg/dL 9) Base deficit > 4 mmol/L 10) BUN > 5 mg/dL S type and P type SECONDARY CAUSES Biliary tract disease Intra-abdominal disease Alcoholism Surgery Drugs Pancreas & kidney transplant Sepsis Diabetic Ketoacidosis Burns & Shock Renal Failure Hyperlipidemia Macroamylasemia

MORTALITY PREDICTION < 3 Signs = 1% 3 4 Signs = 16% 5 6 Signs = 40% > 6 Signs = 100%

Amylase Creatinine Clearance Ratio

Page | 4

Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)

Introduction of amylase Amylase is the enzyme that split complex carbohydrate constituted of -Dglucose units linked Has 2 types: -amylase = plant and bacterial -amylase = human (optimum pH 6.9-7.0 assayed at 37C) = small, can pass via glomeruli (kidney) = the only plasma enzyme normally found in urine Source = Greatest concentration in Pancreas (P type) = Present also in Salivary glands (S type) = in serum and urine usually (P type) -amylase

Macroamylase: complex of amylase (usually S type) with IgA, IgG or other abnormal high molecular weight plasma proteins Not filtered, retained in plasma No clinical symptoms, detectable after investigation of abdominal pain Clinical significance

Amylase in serum and urine Diagnose of pancreatic disease and to investigate pancreatic function Increase amylase in pancreatitis = urine amylase > serum amylase ACCR% (Amylase creatine clearance ratio) Useful in diagnosis Calculated from amylase activity and creatinine concentration determined from the same urine and from a single serum specimen obtained at time of urine collection ACCR% = urine amylase (U/L) x Serum creatinine (mg/L) Serum amylase (U/L) x urine creatinine (mg/L) Method dependant Reference level (2-5%) Random or short (2-4hours) collection adequate ACCR% also increase in burns, ketoacidosis etc The specificity of -amylase for pancreatic disorders is not very high. Therefore, measurement of lipase (confirmatory) should be performed Increase Decrease # Acute pancreatitis Pancreatic neoplasm # Abscess Intestinal infarction Appendicitis Salivary gland inflammation # Diabetic ketoacidosis

# Chronic pancreatitis # Hepatic necrosis

Page | 5

Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)

Method of amylase determination Saccharogenic Rarely used, enzyme activity followed by measuring amount of reducing materials formed Amylase Starch Glucose + Maltose Reduced substrate

Amyloclastic assays Enzyme activity evaluated by following decrease in starch substrate concentration rather than measuring product formed Rate of blue complex is proportional to amylase activity Can measured turbidimetrically
Amylase

Starch + I2

Blue complex

Red complex

Chromogenic assay Dye labelled enzyme substrate Substrate (amylose/amylopectin) linked to various dyes and indicator group Dye labelled substrates in buffer and enzyme attack substrate Small fragments of dye containing substrate Separate fragments from unreacted substrate Measure small fragment photometrically Amylase Dye-labelled starch small dye-labelled fragment Separation step Measure photometrically

Defined substrate method couple enzyme assay uses auxillary and indicator enzyme -amylase Starch maltose + maltotriose + dextrins -glucosidase Maltose + maltoriose Glucose oxidase Glucose + 1.5 O2 H2O2 + d-glucunolactone glucose

Rate of glucose produced determined by glucosidase Decrease in pO2 measured by O2 electrode 1 mole of glucose oxidized = 0.5 mole O2 consumed Therefore, rate of O2 consumed directly proportional to amylase activity in the sample
Page | 6

Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)

Determination and differentiation of amylase isoenzymes in lab There are several methods includes: Electrophoresis Ion-exchange chromatography Ion selective electrode (ISE) Selective inhibition (wheat germ inhibitor) or immunoassay The most reliable method by using electrophoresis method Used primarily to detect acute pancreatitis Electrophoresis of Isoenzyme amylase Patient preparation: No special preparation Samples: saliva, urine, serum amylase Storage: 4C, unstable in acidic urine Reference range: 45% - 70% S type Medium: Agarose agar Buffer: Tris-sodium barbital-barbital buffer (pH 8.8) Voltage: 50 400 volts Temperature: 2 25C Time preferred for separation: < 3 Hours Band separated: P1, P1b, P2, P3,P4, S1, S2, S3, S4, S5 bands Band result : S1, P3, S2, P4, S3, S4, and S5 bands anode (anodal) : P1 and P1b bands cathode (cathodal) : P2 band remains close to the point of application

P3

P1

P1B

P2

S1

P3

S2

P4

S3

S4

S5

+
P3

Cathodal

Anodal

Migration of amylase isoenzymes in electrophoresis

After completed Stain the gels using chemical system (eg: Phadebas amylase tablets) System reaction: produce colour when amylase present.

Page | 7

Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)

Picture of electrophoresis pattern of isoenzyme amylase

Amylase isoenzymes in serum of patients with mumps and acute pancreatitis

Isoenzyme detected in urine (A) Normal person (B) Patient with acute pancreatitis

Amylase isoenzymes in pancreatic, parotid glands, saliva in serum of patient with acute pancreatitis, mumps, and pancreatectomy Page | 8

Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)

References 1. Makoto Otsuki et.al (October 09, 1975) Electrophoretic Pattern of Amylase Isoenzyme in Serum and Urine of Normal Person.pdf, Kobe University School of Medicine, Kobe, Japan 2. Vicky L.Royse et.al (March,1983) Agarose gel electrophoresis technique for the determination of amylase isoenzymes.pdf, US Patent Issues, Paternstorm retrieved on March 19th,2010 from websites http://www.patentstorm.us 3. Craig A.Lehmann (1998), Saunders Manual of Clinical Laboratory Science (1st Edition): Pancreas and Gastrointestinal Functions, W.B Saunders Company. 4. Acute Pancreatitis (February 10, 2010), retrieved on February 10, 2010 from http://en.wikipedia.org/wiki/Acute_pancreatitis 5. Chronic Pancreatitis (February 10, 2010), retrieved on February 10, 2010 from http://en.wikipedia.org/wiki/Chronic_pancreatitis 6. Amylase (February 10, 2010), retrieved on February 10, 2010 from http://en.wikipedia.org/wiki/Amylase

Page | 9