APPENDIX III

ANALYTICAL METHODS

1. Determination of Vitamin C in whole blood or plasma¹

Reagents

1. Trichloroacetic acid, 6% (W/V) solution

2. 2, 4-Dinitrophenylhydrazine reagent:

   Dissolve 2 g of the solid in 100 ml of approximately 9N-sulphuric acid

3. Acid washed Norit:

   Place 200 g Norit in a large flask, add 1 litre of 10% HCL heat to boiling and filter with suction. Wash three times with distilled water to remove ferricions. Dry overnight at 110° to 120° C.

4. Thiourea solution, 10% aqueous solution.

Procedure

1. Add 1.0 ml whole blood or plasma, dropwise with stirring to 3.0 ml 6% trichloroacetic acid
2. Stand for five minutes: centrifuge
3. Pour supernatant into another test tube.
4. Add 150 mg acid washed Norit to supernatant and shake vigorously
5. Centrifuge
6. Take 1 ml of filtrate and add 1 drop 10% thiourea solution and 0.2 ml 2, 4-DNPH reagent
7. Incubate at 37%C for exactly 3 hours
8. Remove and place in ice water bath
9. Add drop wire 1.25 ml 85% H₂ SO₄ with stirring.

Blank

6a. Place bank tube containing 1 ml Norit filtrate and 1 drop thiourea into ice-water bath
7a. Add drop wise 1.25 ml 85% H₂ SO₄ with stirring
8a. Add 0.25 ml 2.4-DNPH reagent.

10. Remove tubes from ice bath and stand for 30 minutes.
11. Read at 540 mm (set for 100% transmission) with blank tube.

Standards

Test 1 ml aliquots containing 0.25–15.0 mcg ascorbic ascorbic acid/ml through procedures 1–11. From curve estimate unknown and multiply dilution factor to obtain ascorbic acid of sample.

¹ Adapted from: H. Varley (1969) Practical Clinical Biochemistry, 4th Edition, William Heinemann Medical Books, London

2. Glutamate-Pyruvate Transaminase, GPT²

Glutamate-pyruvate transaminase, GPT, is found in the following tissues: liver, kidney, heart, skeletal muscle, pancreas, spleen, lung, and serum.

Principle

The colorimetric method is based on the following net reaction³.

L-alanine + 2-oxoglutarate

GPT
pyruvate + L-glutamate

The chromogen used, 2, 4-dinitrophenyl hydrazine, reacts with both the pyruvate and the residual 2-oxoglutarate to form the respective hydrazones. However, when measurements are made between 500 and 550 mu, the contribution of 2-oxoglutarate hydrazone to optical extinction is low.

Conditions for measurement

Relatively low 2-oxoglutarate concentrations are used (usually not higher than 1.7 mM).

Reagents required

1. Potassium dihydrogen phosphate, KH₂PO₄

2. Dipotassium hydrogen phosphate, K₂HPO₄

3. DL-Alanine

4. 2-Oxoglutaric acid

5. Sodium pyruvate, 98–100% pure

6. 2, 4-Dinitrophenylhydrazine

7. Hydrochloric acid, IN

8. Sodium hydroxide, analytical grade

² Adapted from Sidney P. Colowick, et. al., editors (1979) Methods of Enzymology. Academic Press, New York.
³ Full reaction: (i) 2-oxoglutarate + pyridoxamine pyridoxal + glutamate
pyridoxal + alanine
pyruvate + pyridoxamine

Preparation of solutions (for about 50 determinations)

Prepare all solutions fresh, with doubly distilled water.

1. I. Buffer/substrate solution (0.1 M phosphate buffer, pH 7.4; 0.2M dl-alanine; 2 mM 2-oxoglutarate).

   Dissolve	1.5 g K2HPO4
   	0.2 g KH2 PO4
   	0.030 g 2-oxoglutarate
   	1.78 g dl-alanine

   in distilled water make up to 100 ml. Check pH if necessary, adjust with 0.4M NaOH.

2. 2, 4-Dinitrophenylhydrazine (1 mM solution) Dissolve: 20 mg DNPH in 1 N HC1 and make up to 100 ml

3. Sodium hydroxide (0.4 N solution) Dissolve: 16 g NaOH pellets in distilled water make up to 1 000 ml.

4. Pyruvate solution (2 mM) Dissolve: 22.0 mg sodium pyruvate in distilled water and make up to 100 ml.

Stability of solutions

Solutions II and III are stable indefinitely.

Store solutions I and IV at about 4°C in the absence of bacterial growth (by addition of a few drops of chloroform) these are stable for more than two months.

Procedure

Obtain blood without venestasis using anticoagulants such as oxalate (1 mg/ml), citrate (1 mg/ml), fluoride (2 mg/ml), heparin (0.2 mg/ml) or EDTA (1 mg/ml) centrifuge for 10 min. at about 3000 rpm to obtain plasma or serum use only fresh blood free from haemolysis.

GPT activity in serum decreases by about 15% in 24 h at room temperature, by about 11% at 4°C and by about 10% at - 20°C. No further loss in activity is thought to occur after 72 h at 4°C.

1. Pipet into test tube:
   1.00 ml buffer/substrate solution I
   0.20 ml plasma (or serum)

2. Mix; incubate for exactly 30 min at 37°C in water bath

3. Pipet 1.00 ml chromogen solution II

4. Mix and allow to stand for 20 min. at room temperature

5. Add in 10.00 ml sodium hydroxide solution III

6. Mix and read OD after 5 min at 546 mu against blank

7. Compute volume activity (units/litre, or, umoles pyruvate formed/litre plasma/minute)

3. Transketolase, TK⁴

Transketolase, TK, is an enzyme of the pentose phosphate cycle (hexose monophosphate shunt). It is present in liver, kidney, spleen, intestine, lung, brain, heart, skeletal muscle and enthrocytes. It contains thiamine pyrophosphate (TPP) and required Mg ions for activity. In thiamine deficiency, TK activity in erythrocytes is significantly reduced and the addition of TPP in assay systems involving thiamine deficient animals will produce what is known as the ‘TPP effect’. This effect is manifest by a larger percentage increase in TK activity after the addition of TPP to the assay mixture.

Principle

Assay of TK activity is based on

1. decrease of pentose phosphates

2. formation of S-7-P

3. formation of GAP

4. formation of hexose

Conditions of measurement

There is relatively little accumulation of GAP and S-7-P since these partially react further.

On the other hand there are progressively larger quantities of hexoses accumulated, and despite their breakdown in glycolysis the build-up of hexoses is at a much faster rate.

⁴ Adapted from: Sidney P. Colowick, et. al., editors (1979) Methods of Enzymology, Academic Press, New York

Reagents required

1. For incubation

   1. Sodium chloride
   2. Potassium chloride
   3. Dipotassium hydrogen phosphate
   4. Hydrochloric acid
   5. Magnesium sulphate
   6. Thiamine pyrophosphate cocarboxylase, TPP. 4H₂O
   7. Ribose 5-phosphate, R-5-P, monobarium salt
   8. Trichloroacetic acid
   9. Sodium sulphate

   10. Potassium hydroxide

2. For Anthrone method

   11. D-Glucose
   12. Anthrone
   13. Thiourea

   14. Sulphuric acid, 66% (w/w)

3. For Orcinol method

   15. D-Ribose
   16. Orcinol
   17. Ferric chloride, FeCL₃. 6H₂O

   18. Hydrochloric acid, 30%

Preparation of Solutions

For Incubation

1. Buffer (4 mM Na⁺: 115 mM K⁺; 20 mM PO^3-₄; 5 mM Mg^#; pH 7.4)

   mix 40 ml	0.9% NaC1
   1030 ml	1.15% KC1
   200 ml	1.75% K2H PO4 (pH 7.4 with HCl)
   10 ml	3.82% MgSO4 7H2O

   
   

2. Thiamine pyrophosphate solution (0.26 mM)
   Dissolve 29 mg TPP. 4H₂O in 24 ml Buffer (I)
   Dilute 1 ml of this with 8 ml of Buffer (I)

3. Ribose-5-phosphate (47 mM)
   Dissolve 3.24 g R-5-P-Ba in 8.5 ml in HCl
   Dilute to 45 ml with distilled water
   Slowly add 8 ml saturated sodium sulphate solution
   Centrifuge off precipitate
   Adjust the filtrate to pH 7.4 with 5 N KOH
   Determine ribose content (using orcinol reaction)
   Adjust to exactly 7 mg ribose/ml (47 mM)

4. Trichloroacetic acid (7.5% W/V, about 0.45 M)

   Dissolve 75 g trichloroacetic acid in distilled water and make up to 1000 ml.

For Anthrone method

5. Hexose standard solution (0.55 mM)

   Dissolved 25 mg d-glucose in 25 ml distilled water Dilute 5 ml of this to 50 ml with TCA (IV) Standard contains 100 ug glucose/ml.

6. Anthrone reagent (2.5 mM anthrone 0.13 M thiourea)

   Dissolve 0.5 g anthrone and 10 g thiourea in about 200 ml 66% H₂SO₄ by warming to 60–70°C Cool, dilute to 1000 ml with 66% H₂SO₄ Stand for at least 24 h in cold before use.

For Orcinol method

7. Pentose standard solution (67 mM):

   Dissolve 25 mg d-ribose in 25 ml distilled water Dilute 1 ml of this to 100 ml with TCA (IV) Standard contains 10 ug ribose/ml

8. Orcinol reagent (14 mM orcinol; 0.62 mM Fecl₃)

   Dissolve 4.0 g orcinol and 335 mg FeCl₃6H₂O in distilled water Make up to 100 ml Dilute to 2000 with 30% HCl

Stability of solutions

Store solutions I, IV, VI and VIII at about 4°C
Store solutions II, III, V and VII at - 20°C
Solution III is stable for three months.
Other solutions stable indefinitely.

Procedure

Blood should be treated with anticoagulants, centrifuged for 10 minutes at 2000 rpm. After removal of plasma, transfer erythrocytes to test-tube containing equal volumes of water. Freeze overnight before assay.

For organ tissues, homogenize organs (fresh or frozen) in buffer I. Use 50-fold volume buffer for liver and kidney and 10-fold volume for other tissues.

TK activity in the frozen tissue is stable for at least two months.

Fresh blood can be stored for up to three days in a refrigerator before haemolysis, while the haemolyzate can be stored for about 3 months.

Incubation system⁵

Pentose Assay

620 mu
Room temperature
Read against water blank
Standardization with solution VII

Hexose Assay

620 mu
Room temperature
Read against water blank
Standardize with solution V