Clinical Chemistry (CHE 221)

Experiment # 11 Glucose, Glycosylated Hemoglobin, and the Michaelis-Menten Constant (KM) of Hexokinase

Name ______________________________

Date Performed______________________

Date Submitted______________________

Partners Name(s)_____________________

Partners Name(s)_____________________

Experiment title: Glucose, Glycosylated Hemoglobin, and the Michaelis-Menten Constant (KM) of Hexokinase Reading assignment: Bishop 6th edition pages 283-285, and the attached protocols. Pre-lab questions None

Protocol(s) Part 1. Serum glucose determination by hexokinase (Pointe Scientific protocol attached) Part 2. Hb A1c determination by ion-exchange purification (Pointe Scientific protocol attached)

Part 3. Determination of Michaelis-Menten Constant (KM) of Hexokinase. Your instructor will demonstrate how to collect the kinetic data required for this part of the experiment on the Evolution 60 Spectrophotometer. The instrument will collect absorbance values every two seconds at 340 nm for a total time of one minute.

1) Prepare the first solution shown in Table 3. Add the glucose portion last. Take care to pipette gently so the solutions have a minimum of bubbles. After adding the glucose portion be prepared to work quickly through the next two steps.

2) Place the lid on the cuvette and gently invert it so as not to create bubbles. Do not shake the tube!

3) Collect kinetic data for one minute on the sample. 4) Record the rate calculated for the first 60 seconds in Table 3. 5) Pour the reaction mixture into a waste bottle and clean and dry the cuvette gently

with Kimwipes. 6) Repeat steps 1-5 for each of the reaction mixtures in the Table 3. 7) Enter the concentration and rate data in Table 3 into Microsoft Excel. 8) Calculate a new column which expresses the rates in units of mM/min rather than

absorbance units/min by dividing the absorbance values by b. Note the molar absorptivity of NADH is 3.22 x104 M-1cm-1.

9) Prepare a Michaelis-Menten graph by plotting rate in units of mM/min on the y-axis versus Glucose Concentration in units of mM on the x-axis.

10) Calculate two new columns in Excel: 1/Rate in units of min/mM, and 1/Glucose Concentration in units of mM-1.

11) Prepare a Lineweaver-Burk plot by graphing 1/Rate on the y-axis and 1/Glucose Concentration on the x-axis.

12) Determine the best fit line for the data by right clicking on the data points, selecting Add Trend Line, then choosing Linear and checking the box to Display Equation on chart.

+ 20 L level I standard

2000 Lhexokinase

reagent

Standard

Glucose Flowchart

+ 20 L patient serum

2000 Lhexokinase

reagent

Blank

2000 Lhexokinase

reagent

Patient

+ 20 Lwater

1) Zero the instrument with water

2) Incubate at 37 C for 3 minutes3) Record the absorbance of all tubes

including the blank

Liquid Glucose (Hexokinase) Reagent Set

Phone: 734-487-8300 • Toll Free: 800-445-9853 • Fax: 734-483-1592 • www.pointescientific.com

Intended Use For the quantitative determination of glucose in serum. For in vitro diagnostic use only. Clinical Significance Determination of glucose in serum is most commonly performed for the diagnosis and treatment of diabetes mellitus. Test Summary There are a large number of methods in existence for the measurement of glucose in biological fluids. Early methods such as the Folin-Wu1 and Somogyi-Nelson2 depended on the reduction of heavy metals by the aldehyde group of glucose. These methods are subject to interference by carbohydrates other than glucose. The ortho-toluidine method, introduced in 19593 and later modified 4,5 to react directly with serum, is specific for aldoses but uses a strong, noxious, corrosive acid requiring incubation at elevated temperatures. Enzymatic methods were first described in the 1940’s6 with varied modifications described to date.7,8 The present hexokinase method is based on a modification of Slein9, using hexokinase and glucose-6-phosphate-dehydrogenase to catalyze the reaction. The method is also based on the reference method proposed by the FDA for measuring glucose. Principle

HK Glucose + ATP ------------------- G6P + ADP

G6PDH G6P + NAD ----------------------- 6-Phosphogluconate + NADH + H+ Glucose is phosphorylated with adenosine triphosphate (ATP) in the reaction catalyzed by hexokinase (HK). The product, glucose–6-phosphate (G6P) is then oxidized with the concomitant reduction of nicotinamide adenine dinucleotide (NAD) to NADH in the reaction catalyzed by glucose-6-phosphate-dehydrogenase (G6PDH). The formation of NADH causes and increase in absorbance at 340nm. The increase is directly proportional to the amount of glucose in the sample. Reagent Composition Liquid Glucose (Hexokinase) Reagent: Hexokinase (yeast) ≥ 2000U/L, G6PDH (Leuconostoc mesenteroides) ≥ 1500U/L, ATP ≥ 1.5mM, NAD ≥ 2.0mM, Buffer pH 7.5 ± 0.1. Nonreactive stabilizers and sodium azide (0.095%) as preservative. Reagent Preparation The reagent is ready to use. Reagent Storage and Stability The reagent set is stored at 2-8°C. Once opened the reagent will remain stable at least 30 days when properly stored and handled. Precautions 1. This reagent is for in vitro diagnostic use only. 2. Reagent contains Sodium Azide as a preservative. Sodium Azide may

form explosive compounds in metal drain lines. When disposing of reagents through plumbing fixtures, flush with copious amounts of

water. For further information, refer to “Decontamination of Laboratory Sink Drains to remove Azide Salts,” in the Manual Guide-Safety Management No. CSC-22 issued by the Centers for Disease Control, Atlanta, Georgia.

3. Do not use the reagent if the reagent blank has an absorbance greater than 0.500 against water at 340 nm, or contains obvious microbial growth.

4. Reagent should not be used if it fails to recover stated values in control sera. 5. All specimens and controls should be handled in accordance with good

laboratory practices using appropriate precautions as described in the CDC/NIH Manual, “Biosafety in Microbiological and Biomedical Laboratories,” 2nd ed., 1988, HHS Publication No. (CDC) 88-8395.

Specimen Collection and Storage 1. Serum: Use fresh, unhemolyzed serum. 2. Plasma: Unhemolyzed samples from tubes containing oxalate, citrate,

EDTA, fluoride or heparin may be used. 3. Specimen collection should be carried out in accordance with NCCLS M29-

T2.10 No method can offer complete assurance that human blood samples will not transmit infection. Therefore, all blood samples should be considered potentially infectious.

4. Serum and plasma must be separated from the red cells promptly to prevent glycolysis. Glucose will decrease approximately 7% per hour when left in contact with red cells.11 The addition of sodium fluoride to the specimen may prevent glycolysis.

5. Glucose in serum or plasma is stable for 8 hours at room temperature and 24 hours refrigerated at 2-8°C.

6. Specimen collection should be carried out in accordance with NCCLS M29-T2.10 No method can offer complete assurance that human blood samples will not transmit infection. Therefore, all blood samples should be considered potentially infectious.

Interferences 1. Young, et al12 has published a comprehensive list of drugs and substances

that may affect glucose values. 2. Bilirubin to the level of 20 mg/dl has been found to exhibit negligible

interference (≤5%) in this assay. 3. Hemoglobin to the level of 400 mg/dl has been found to exhibit negligible

interference (≤5%) in this assay. NOTE: Glucose level was 177 mg/dl for the Bilirubin study and 181 mg/dl for the Hemoglobin study. Materials Provided Glucose (Hexokinase) Reagent. Materials Required but not Provided 1. Accurate pipetting devices. (10ul and 1.0ml) 2. Timer. (For five minutes) 3. Test tubes/rack 4. Spectrophotometer with ability to read 340 nm. 5. Heating block or water bath (37°C). Procedure (Automated-General) Wavelength: 340 nm Assay Type: Endpoint Sample/Reagent Ratio: 1:101 Reaction Direction: Increasing Temperature: 37°C Incubation Time: 180 seconds Low Normal: 70 mg/dl High Normal: 110 mg/dl

Glycohemoglobin Reagent Set

Phone: 734-487-8300 • Toll Free: 800-445-9853 • Fax: 734-483-1592 • www.pointescientific.com

Intended Use For the quantitative determination of Glycohemoglobin (HbA1) in blood by cation exchange resin. The test is to be used to monitor long-term glucose control in diabetes mellitus. Summary and Explanation of Test Throughout the circulatory life of the red cell, glycohemoglobin is formed continuously by the adduction of glucose to the N-terminal of the hemoglobin beta chain. This process, which is non-enzymatic, reflects the average exposure of hemoglobin to glucose over an extended period. In a classical study, Trivelli et al1 showed glycohemoglobin in diabetic subjects to be elevated 2-3 fold over the levels found in normal individuals. Several investigators have recommended that glycohemoglobin serve as an indicator of metabolic control of the diabetic, since glycohemoglobin levels approach normal values for diabetics in metabolic control.2,3,4 Glycohemoglobin has been defined operationally as the “fast fraction” hemoglobins (HbA1a, A1b, A1c) that elute first during column chromatography with cation-exchange resins. The non-glycosylated hemoglobin, which consists of the bulk of the hemoglobin has been designated HbA0. The present glycohemoglobin procedure employs a weak binding cation-exchange resin for the rapid separation of glycohemoglobin (fast fraction) from non-glycosylated hemoglobin. Over 80% of the labile fraction of glycohemoglobin is removed during the separation step in this procedure due to the inclusion of the borate buffer system.5 Principle A hemolyzed preparation of the whole blood is mixed continuously for five minutes with a weak binding cation-exchange resin. During this time, HbA0 binds to the resin. After the mixing period, a filter is used to separate the supernatant containing the glycohemoglobin from the resin. (Note: This binding is temperature dependent. Therefore, a standard should be included in each run.) The percent glycohemoglobin is determined by measuring the absorbance at 415 nm (405-420 nm acceptable) of the glycohemoglobin fraction and the total hemoglobin fraction. The ratio of the two absorbances gives the percent glycohemoglobin. Reagents 40 Test Kit containing: 1 x 120ml Bottle 8mg/ml Cation-exchange Resin in a borate buffer, pH 6.9. 1 x 30ml Bottle Glycohemoglobin Lysing Resin, 10mM Potassium Cyanide, surfactant added. 40 serum separators. Reagent Storage Store reagent at room temperature (21-26°C). Expiration Dating All reagents are stable to expiration date stated on the labels. Do not use the reagents past their expiration date. Reagent Deterioration Alterations in the physical appearance of the reagents or values of control materials outside of the manufacturer’s acceptable range may be an indication of reagent instability. Instruments Use a spectrophotometer able to read at 415nm with linearity to at least 1.5 O.D. units. (405-420 nm is acceptable.)

Precautions 1. This reagent is for in vitro diagnostic use only. 2. Not for internal or external use in humans or animals. 3. Lysing reagent contains cyanide (poison). Do not ingest. Do not mix with

acid or HCN gas may be released. Specimen Collection and Preparation Special preparation of the patient is unnecessary. Fasting specimens are not required. No special additives or preservatives other than anticoagulants are required. Collect venous blood with EDTA using aseptic technique. All human specimens should be regarded as potentially biohazardous. Therefore, universal precautions should be used in specimen handling (gloves, lab garments, avoid aerosol production, etc.). Storage Glycohemoglobin in whole blood collected with EDTA is stable for one week at 2-8°C. Interferences Samples that are severely lipemic may cause elevated results. It has been reported that bilirubinemia may interfere with ion-exchange methods.6 Fetal Hemoglobin (HbF) may interfere in this assay. Blood samples with total hemoglobin greater than 18 g/dl should be diluted x 2 with deionized water before assay. Materials Provided Refer to “Reagents” Materials Required but not Provided 1. 20 ul and 100 ul Micropipettors 2. 500 ul, 3 ml and 5 ml Pipettes or Dispensers 3. 13x100 mm Glass Tubes 4. Glass or plastic Test Tubes to hold 0.6 ml and 5 ml 5. Rocker or Rotator (e.g. Miles Inc., Diagnostics Division 4651) 6. Glycohemoglobin standard (Cat. No. G7540-STD) 7. Glycohemoglobin controls (Cat. No. G7540-2) 8. Deionized water Procedure a. Hemolysate Preparation

1. Dispense 500ul Lysing Reagent into tubes labeled: Standard, Control, etc. Note: Plastic or glass tubes of appropriate size are acceptable.

2. Place 100ul of the well-mixed blood sample, standard or control into the appropriately labeled lysing reagent tube. Mix.

3. Allow to stand for 5 minutes or until complete lysis is evident. b. Glycohemoglobin Preparation

1. Dispense 3.0ml of Glycohemoglobin Cation-exchange Resin into 13 x 100 mm glass tubes labeled: Standard, Control, etc. Note: Before use, mix the resin by inverting at least 10 times. Swirl the bottle after addition to each 5 tubes.

2. Add 100 ul of the hemolysate (from step A3) to resin reagent. 3. Position the filter separators in the tubes so that the rubber sleeve is

approximately 1cm above the liquid level. 4. Place the tubes on the rocker or rotator and mix continuously for 5

minutes. 5. Remove the tubes from the rocker or rotator. 6. Push the filter separators into the tubes until the resin is firmly packed. 7. The supernatant may be poured into another tube or directly into a

cuvette for absorbance measurement.

Glycohemoglobin Reagent Set

8. Zero the instrument at 415 nm (405-420nm acceptable) with deionized water as the blank.

9. Read and record the absorbance values for Standard, Control, etc. These readings are for glycohemoglobin.

c. Total Hemoglobin Fraction 1. Dispense 5.0ml deionized water into plastic, or glass tubes

labeled: Standard, Control, etc. 2. Place 20 ul of the hemolysate (from step A3) into the

appropriately labeled tube of total hemoglobin diluent. Mix. 3. Adjust the instrument to zero absorbance at 415nm (405-420nm

acceptable) with deionized water as the blank. 4. Read and record the absorbance values for Standard, Control etc.

These readings are for total hemoglobin. NOTE: This glycohemoglobin assay should be performed at room temperature, 21-26°C. The final reaction products for glycohemoglobin and total hemoglobin appear quite stable. However, the test samples should be read within an hour before evaporation becomes significant. Limitations 1. This assay should not be used for the diagnosis of diabetes mellitus. 2. This method can be influenced by temperature. Patient specimens

should always be assayed with a calibrator included in the run to eliminate temperature influences.

3. Glycosylated HbS and HbC bind more tightly than HbA, and produce lower values. Other hemoglobinopathies (e.g., betathalassemia and hemolytic anemia also produce lowered results.)

4. Results may be inconsistent in patients who have the following conditions: opiate addiction, lead-poisoning, uremia (carbamylated Hb), alcoholism, ingest large doses of aspirin.7,8,9,10

Quality Control The reliability of test results should be monitored whenever patient samples are assayed using a standard and quality control materials analyzed in the same manner employed for the unknowns. We suggest the use of commercially available glycohemoglobin controls with an assayed range. If controls do not fall into the assayed range patient values from that run should not be reported. The run should be repeated, making sure that all mixing and handling instructions are strictly followed. Linearity of the assay should be verified with a commercial linearity check set, or dilutions of a high specimen, at least every six months. Calculations Results for the unknowns and controls are calculated as follows: For each sample, calculate the ratio (R) of the glycohemoglobin absorbance to the total hemoglobin absorbance. Use the following equation to determine unknown concentrations: Unknown (%) = R (Unk) x Std Conc. (%) R (Std) Example: A standard containing 10.0% glycohemoglobin had Abs. = 0.490 for the glycohemoglobin fraction and Abs. = 0.560 for the total hemoglobin fraction. An unknown sample had glycohemoglobin Abs. = 0.750 and total hemoglobin Abs. = 0.625. The glycohemoglobin concentration of the unknown is: Standard R = 0.490 = 0.875 0.560 Unknown R = 0.750 = 1.200 0.625

Unknown % = 1.200 x 10.0% = 13.7% 0.875 Expected Values Normal: 6.0 – 8.3% The normal range represents the 95% confidence interval for 75 subjects with normal glucose values and no history of diabetes. Each laboratory should establish its own expected values. In using glycated Hb to monitor diabetic patients, results should be interpreted individually. That is, the patient should be monitored against him or herself. There is a 3-4 week time lag before % glycohemoglobin reflects changes in blood glucose level. Performance 1. Linearity: The glycohemoglobin assay shows linearity for glycohemoglobin

levels in the range of 4.0%-20%. 2. Comparison: A study on normal and abnormal human specimens between

this glycohemoglobin procedure and a widely used column procedure yielded a correlation coefficient of 0.970 and a linear regression equation of y=1.10x-3.00. (n=36, range=5.9-14.2%)

3. Precision: Within Run: The intra assay precision was established by assaying blood with normal and elevated glycohemoglobin levels twenty times each. Level Mean Std. Dev. % C.V. Normal 7.7 0.24 3.1 Elevated 12.8 0.21 1.6

Run To Run: The inter run precision was established by assaying blood with normal and elevated glycohemoglobin levels for ten runs conducted over a five day period. Level Mean Std. Dev. % C.V. Normal 8.0 0.32 4.0 Elevated 14.8 0.45 3.0

4. Sensitivity: This glycohemoglobin procedure has a sensitivity of 0.02% glycohemoglobin per 0.001 units of absorbance.

References 1. Trivelli, L.A., Ranney, H.M., and Lai, H.T., New Eng. J. Med. 284,353 (1971). 2. Gonen, B., and Rubenstein, A.H., Diabetologia 15, 1 (1978). 3. Gabbay, K.H., Hasty, K., Breslow, J.L., Ellison, R.C., Bunn, H.F., and Gallop,

P.M., J. Clin. Endocrinol. Metab. 44, 859 (1977). 4. Bates, H.M., Lab. Mang., Vol 16 (Jan. 1978). 5. PSI Records (8/84). 6. Eissler, S.M., Diabetes 29, p. 467-474 (1980). 7. Corielo, A., et al, Diabetologia 22, p. 379 (1962). 8. Goldstein, D.E., et al, Clin. Chem. 32, pp. 364-370 (1986). 9. Fluckiger, R., et al, Med Intelligence 304 p. 823-827 (1981). 10. Nathan, D.M., et al, Clin. Chem. 29, p. 466-469 (1983). Manufactured for Pointe Scientific, Inc.

5449 Research Drive, Canton, MI 48188 European Authorized Representative: Obelis s.a. Boulevard Général Wahis 53 1030 Brussels, BELGIUM Tel: (32)2.732.59.54 Fax:(32)2.732.60.03 email: mail@obelis.net

Rev 12/09 P803-G7540-01

Table 1. Absorbance data for glucose determination.

Trial Absorbance

at 340 nm

Standard

Patient

Level I Standard Glucose Concentration _________ mg/dL

Table 2. Absorbance data for Hb A1c determination.

Trial Purified A1c Absorbanceat 415 nm

Hemolysate Absorbance at

415 nm

Standard

Patient

Standard A1c percentage _________

Table 3. Rate of phosphorylation of glucose standards in the presence of hexokinase.

Trial

Volume

Water

(µL)

Volume

Hexokinase

Stock (µL)

Volume of 55

mM Glucose

Stock (µL)

Glucose

Concentration

(mM)

Rate (Au/min)

1 499 2000 1 0.0222

2 498 2000 2 0.0444

3 497 2000 3 0.0667

4 495 2000 5 0.111

5 490 2000 10 0.222

6 475 2000 25 0.556

7 450 2000 50 1.11

8 400 2000 100 2.22

9 300 2000 200 4.44

10 0 2000 500 11.1

Calculations

Post-lab questions

1) In a Lineweaver-Burk plot one over the y-intercept is known as Vmax. It is the maximum rate the reaction proceeds and occurs when there is a large enough excess of substrate that the enzyme is essentially always bound to substrate catalyzing the reaction. Determine the value of Vmax from your Lineweaver-Burk plot. Show your work on the graph and/or below.

2) If part 3 of this experiment was performed with a higher concentration of enzyme would you expect Vmax to be larger?

3) In a Lineweaver-Burk plot one over the x-intercept is known as the Michaelis-Menten constant (KM). If the formation of the product from the enzyme-substrate complex:

Enzyme-Substrate Complex Product + Enzyme

is the rate-determining step then KM is approximately equal to the equilibrium constant for the enzyme and substrate to bind together:

Enzyme + Substrate Enzyme-Substrate Complex

Determine the value of KM from your Lineweaver-Burk plot. Show your work on the graph and/or below.

4) If part 3 of this experiment was performed with a higher concentration of enzyme would you expect KM to be larger?