CCHM LAB MIDTERM: Enzyme determination

Use only clear, unhemolyzed serum • Serum must be separated from clot within two hours after collection. (ACP activity is labile at RT) • Treated serum samples will remain stable for 7 days if kept in ref temp (2-8 oC)

ACID PHOSPHATASE

• L-tartrate reagent • Acetate Buffer: 5M, pH 5.0 o 20ul of buffer per 1mL of serum

ACP Reagent

Serum (should be free of hemolysis) • Heparinized plasma (*Heparin is the only suitable anticoagulant)

ALKALINE PHOSPHATASE

Reagent 1: Diethanolamine buffer, pH 10.2 Magnesium Chloride 0.1% Sodium Azide (preservative)

ALP R1

p-nitrophenylphosphate pH of working reagent: 9.8 Avoid exposure to direct light

ALP R2

Composition: • Alpha-ketoglutarate • NADH • Stabilizers • Preservatives (Sodium Azide) • Distilled Water

ALT Reagent (Enzyme) - R2

This enzymatic procedure is based on the modification of Wallenfels

AMYLASE DETERMINATION

hydrolyzes p-Nitrophenyl-Dmaltohepatoside (PNPG7) to p-Nitrophenylmaltostriose (PNPG3) and maltotetraose. Glucoamylase hydrolyzes PNPG3 to pNitrophenylglycoside (PNPG1) and glucose. Then is hydrolyzed by glucosidase to glucose and p-Nitrophenol

AMYLASE DETERMINATION

Reagent Stability: Stable until the expiration date

AST ALT

Composition: L-Aspartate MDH Tris Buffer, pH 7.5 Stabilizers Preservatives (Sodium Azide)

AST Reagent (Buffer) -- R1

Composition: 2-Oxoglutarate NADH Stabilizers Preservatives (Sodium Azide) Distilled Water

AST Reagent (Enzyme) - R2

Reference Value for lipase determination

Adults 10 - 150 U/L age more than 60 old: 18 - 180 U/L

Composition when reconstituted: o p- Nitrophenyl D- Maltohepatoside (PNPG7) o Glucosidase (microbial source) o Glucoamylase (microbial source) o Sodium chloride o Calcium chloride

Amylase Reagent (powder)

Inhibits up to 2000 u/L of CK-MM at 37 degrees Celsius

Anti-human CK-M antibody (mouse)

• ADP • G6PDH • Creatinine phosphate • NAD • Hexokinase (yeast) • D-glucose • Anti-human CK-M antibody (mouse)

CK-MB Reagent (powder):

is a substance that speeds up or slows down the rate of reaction.

Catalyst

SPECIMEN OF CHOICE FOR CK-MB DETERMINATION

Clear unhemolyzed serum is the recommended specimen sample. No special additives or preservatives are required

They function as biochemical or biological catalysts

Enzymes

are mostly protein in their nature and if there is an exception to this rule, it's very rare or seldom.

Enzymes

are enzymes that catalyze the same reaction but differ in enzyme form/structure.

Isoenzymes or Isozymes a

: • L-lithium Lactate • 2-Methyl-2-Amino-1-Propanol; pH 8.8

LDH Buffer (R1)

specifically catalyzes the oxidation of lactate to pyruvate with the subsequent reduction of NAD to NADH. The rate at which NADH form is proportional to LDH activity. The method described determines NADH absorbance increase per minute at 340 nm

LDH DETERMINATION

• NAD Reagent

LDH Enzyme (R2):

Buffer: Tris Buffer 69 mM, Sodium deoxycholate 10mM, pH 9.0

Lipase determination

Reagent Preparation: 1. Add lipase buffer to a 50mL Erlenmeyer flask. Add 25 mL distilled water and swirl to dissolve 2. Pipette 1mL of well-mixed lipase substrate into buffer solution

Lipase determination

Substrate: 0.8% (w/v) olive oil in alcohol

Lipase determination

adapted from TECO diagnostics.

Lipase determination

U/L = ΔA/minute x 860

NON-PROSTATIC ACP

The value is obtained by subtracting the result of the non prostatic acid phosphatase assay (B) from the total acid phosphatase assay (A)

PROSTATIC ACP

other specimen which can be used for amylase determination

Plasma from heparin tubes Urine but the pH should be adjusted to 7.0 and should be kept refrigerated

Preparation and Stability LDH DET

Prepare Working Reagent in the ratio of 5 parts Buffer (R1) to 1 part Enzyme(R2) (i.e., 25 mL Buffer and 5 mL Enzyme)

Triglyceride + H2o ---------> mono + di-glycerides + fatty acids

Quantitative Turbidimetric Determination of Pancreatic Lipase in Human Serum

Wavelength: 340nm Temperature: 37 degree Celsius

- LDH DETERMINATION - CREATINE KINASE - MB DETERMINATION - ASPARTATE AMINOTRANSFERASE DETERMINATION - ALANINE AMINOTRANSFERASE DETERMINATION

REF VALUE FOR CK-MB DETERMINATION (30˚C)

0 - 14 U/L

REF VALUE FOR CK-MB DETERMINATION (37˚C)

0 - 24 U/L

Reference Value for ALT (30°C)

0-26 U/L

Reference Value for AST (30°C)

0-28 U/L

REF VALUE FOR Prostatic Acid Phosphatase:

0-3 U/L

Reference Value for ALT (37°C)

0-38 U/L

Reference Value for AST (37°C)

0-40 U/L

REF VALUE FOR Total Acid Phosphatase:

0-9 U/L

Lipase activity in serum is stable at room temperature for _______

1 week

ALP REF VALUE IN ADULT 37˚C (U/L)

100 - 290

REF VALUE LDH DET FEMALES (37˚C)

103 - 227 U/L

ALP REF VALUE IN CHILDREN 25˚C (U/L)

110 - 720

Reference Value for ALT (NEWBORNS)

12.0 U/L

PROCEDURE FOR NON-PROSTATIC ACP 1. Add 1.0 mL of reagent to appropriately labelled tubes. 2. Add 10 µl (0.01 mL) of L-Tartrate Reagent and mix. 3. Zero spectrophotometer with water at 405 nm. Set cuvette temperature at 37˚C 4. After incubation, read and record absorbance every minute for 5 minutes to determine 5. Add 100µL (0.10mL) of sample to respective tube and allow incubating at 37˚C for 5 minutes. (ΔA/minute) 6.Values U/L are obtained by multiplying (ΔA/minute) by the factor 7. Repeat procedure for each sample

1235476

ALT PROCEDURE 1. Prepare ALT Working Reagent according to instructions. 2. For each sample, add 1.0 mL Working Reagent to test tube and warm to 37°C. 3. Zero spectrophotometer at 340 nm with Distilled water. 4. Add 100 uL serum to its respective tube and mix gently. Incubate for 1 minute at 37°C. 5. Read and record decrease in absorbance at 1 minute. Continue incubating at 37°C and record absorbance again at 2 and 3 minutes to get the change in absorbance per minute (ΔA/minute). Rate should be constant.

12435

PROCEDURE FOR LDH DET 1. For each sample add 1.0 mL working reagent into a cuvette/test tube. Warm for 37˚C for 3 minutes. 2. Determine average abs. per min. (∆ A/min), multiply by factor 3367 for results in U/L 3. Add 0.050 mL (50µL) serum to its respective tube and mix gently. Incubate for 1 minute at 37˚C. 4. Read and record absorbance at 1 minute. Continue incubating at 37 degrees Celsius and record absorbance again at 2 and 3 minutes. Rate should be constant.

1342

AMYLASE DETERMINATION 1. For each sample add 1.0 ml reconstituted reagent to cuvette or test tube and pre warm at 37°C for at least 3 minutes. 2. Determine the mean absorbance difference per minute (ΔAbs/min). 3. Zero spectrophotometer with water at 405 nm. 4. Multiply the Δabs/min by 4824 to obtain the result in U/L. 5. Add 0.025 ml (25μL) serum to its respective tube and read immediately 6. Record increase in absorbance at 30 second intervals for 2 minutes. - You must have 5 absorbances at the end. 1 for initial reading and 4 for the follow-up reading (2 mins with 30 sec interval)

135624

ALP REF VALUE IN CHILDREN 30˚C (U/L)

145 - 950

ALP REF VALUE IN CHILDREN 37˚C (U/L)

180 - 2000

ALP REAGENT STABLITY FOR 7 DAYS AT

2 - 8˚C

ALP REAGENT STABLITY FOR 24 HOURS AT

20 - 25˚C

PROCEDURE FOR TOTAL ACP 1. Pipet 1.0mL of reagent in all tubes 2. Label tubes as "control" and "patient" 3. Add 100µL (0.10mL) of sample to respective tube and allow incubating at 37˚C for 5 minutes. 4. Zero spectrophotometer with water at 405 nm. Set cuvette temperature at 37˚C 5. Repeat procedure for each sample 6. After incubation, read and record absorbance every minute for 5 minutes to determine (ΔA/minute) 7. Values U/L are obtained by multiplying (ΔA/minute) by the factor

2143657

Reference Value for amylase determination

Serum: 25 - 125 U/L Urine: 1 - 17 U/Hour

modified the method proposed by Voget et. al and eliminated some interferences.

Shihabi et. al.

U/L = ΔA/minute x 853

TOTAL ACP

what type of specimen is used in lipase determination

fresh serum

sample should NOT be contaminated with bacteria because bacterial contamination can result in ___________ LPS activity.

increase

Serum lipase hydrolyses the __________________ and the decrease in turbidity at 400 nm is proportional to the lipase activity in the specimen

olive oil emulsion

Serum lipase hydrolyses the olive oil emulsion and the decrease in turbidity at 400 nm is _________ to the lipase activity in the specimen

proportional

the rate of increase in absorbance is measured at 405 nm is __________ to the amylase activity in the sample

proportional

lipase sera may be stored for 3 weeks in the ___________ and for several months if frozen.

refrigerator (4-8˚C)

Don't use plasma (Anticoagulants can inhibit ACP activity which may cause ________)

turbidity

Amylase hydrolyzes p-Nitrophenyl-Dmaltohepatoside (PNPG7) to p-Nitrophenylmaltostriose (PNPG3) and maltotetraose. Glucoamylase hydrolyzes PNPG3 to pNitrophenylglycoside (PNPG1) and glucose. Then is hydrolyzed by glucosidase to glucose and p-Nitrophenol, which produce a ______

yellow color

1. Read and record absorbance of blank and place back in heating bath. 2. Reconstitute lipase reagent according to instruction 3. Using timed intervals, add 0.1 mL (100µL) of sample of each tube, mix, and read initial absorbance. 4. Label test tubes "blank", "control", "patient", etc. - Prepare three (3) test tubes for this 5. Return each tube to heating bath after initial reading. 6. Pipette 3.0 mL of reagent into appropriate (3) test tubes and pre-warm at 37˚C for at least 5 minutes. 7. Exactly 5 minutes after the initial absorbance reading, using the same timed intervals, remove each tube from heating bath and mix each tube. Read the absorbance of the blank and each sample tube against the distilled water - You must have 2 absorbances at the end. 1 for initial and 1 from the final reading (5 mins after initial) 8. Zero spectrophotometer with distilled water at 400nm (390-420)

24681357

Working Reagent Storage and Stability: AST & ALT

4 weeks at 2-8°Celsius. 5 days at room temperature (20- 22°C)

Serum lipase hydrolyses the olive oil emulsion and the decrease in turbidity at ____ is proportional to the lipase activity in the specimen

400 nm

wavelength used in amylase determination

405 nm

REF VALUE LDH DET MALES (30˚C)

50 - 166 U/L

AST PROCEDURE 1. Read and record decrease in absorbance at 1 minute. Continue incubating at 37°C and record absorbance again at 2 and 3 minutes to get the change in absorbance per minute (ΔA/minute). Rate should be constant. 2. For each sample, add 1.0 mL Working Reagent to test tube and warm to 37°C. 3. Add 100 uL serum to its respective tube and mix gently. Incubate for 1 minute at 37°C. 4. Zero spectrophotometer at 340 nm with Distilled water. 5. Prepare AST Working Reagent according to instructions.

52341

MANUAL PROCEDURE FOR CK-MB DETERMINATION 1. Record the increase in absorbance at 60 seconds intervals for the next 2 minutes (ΔA/minute). The rate of change should be constant. 2. After 5 minutes has elapsed, read and record the absorbance 3. Set the wavelength of the instrument at 340 nm. Zero the instrument with distilled water. 4. Add 50µL (0.050 mL) of sample to its respective tube, mix well and incubate for 5 minutes at 37˚C. 5. For each sample add 1.0 mL CK - MB working reagent into a cuvette / test tube and warm for approximately 5 minutes at 37˚C. (only one test tube is needed here)

54321

Amylase Reagent (powder) buffer pH:

6.9 ± 0.1

REF VALUE LDH DET FEMALES(30˚C)

60 - 132 U/L

ALP REF VALUE IN ADULT 25˚C (U/L)

60 - 170

ALP REF VALUE IN ADULT 30˚C (U/L)

80 - 220

REF VALUE LDH DET MALES (37˚C)

80 - 285 U/L

REF VALUE FOR CK-MB DETERMINATION % CK- MB

< 5.5%

specimen of choice for amylase determination

Unhemolyzed serum

proposed the use of olive oil emulsion in measuring the change of rate in turbidity over a specific unit of time.

Voget et. al.

REAGENT STORAGE AND STABILITY: LDH DET

Working Reagent is stable for 2 weeks at 2-8°C or 1 day at room temperature (15-30°C).

Spectrophotometer proper sequences A—J A. Clean the outside of the cuvette and put it inside the cuvette holder B. Open the compartment C. Turn the spectrophotometer on and allow for a warm-up time of 15-20 mins. D. Select the desired wavelength by pressing the wavelength increase / decrease button E. Select the transmittance or absorbance mode by pressing the MODE button F. Get the cuvette containing blank, standard and sample solution G. Connect the spectrophotometer to an electrical outlet. H. Close the compartment lid I. Read the % Transmittance /Absorbance of the remaining solution by moving the cuvette holder. J. With the blank solution on the first compartment, set the reading of the spectrophotometer to zero ( blank) by pressing the Abs/100\% transmittance a. abcdefghij b. bdegjacfhi c. gcedbfahji d. hejadbcgfi

c

Pipette proper sequences A—J A. Make sure that the tip is firmly attached to the tip cone. B. Place the tip just under the surface of the liquid (2-3 mm) and smoothly release the operating button . C. Prepare all the needed materials D. Liquid is dispensed by gently depressing the thumb button to the first stop . E. Depress the operating button to the first stop. F. Make sure that the liquid and container vessel are clean and that the pipettor, tips and the liquid are also clean and at the same temperature. G. Carefully withdraw the tip from the liquid , touching against the edge of the container to remove excess H. Hold the pipettor vertically during aspiration. I. After a short delay continue to depress the thumb button to the second stop , this procedure will empty the tips and ensure accurate delivery J. After dispensing , remove the pipette tip by pressing on the ejector button .

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