B i o c h e m i s t r y P r a c t i c a l N o t e s

Authors:Ida Fakla Péter Ferdinandy Janos Fischer Gyorgyi Jakab —.. ^Margit Keresztes Zsuzsanna Kiss ' Im re Ocsovszki Janos Rohan Marianna Török Erno Zador ^

Editors:Laszlo Dux Zsuzsanna Kiss

Reviewer:Dr. György Falkay associate professor

Albert Szent-Györgyi Medical University Department of Biochemistry 1995

C o n t e n t s

Safety regulations..................................................................................Substrate specificity and temperature optimum of Amylase enzyme

activity..................................................................................................Determination of albumin in serum.......................................................Determination of total protein in serum................................................Assay of the activity of Alkaline Phosphatase........................................

Practical enzyme kinetics.......................................................................Determination of Glucose-6-phosphatase activity in rat liver..... .........Enzymatic-colorimetric assay of urea in human serum.........................Detection of the oxygen consumption of isolated mitochondria by

methylene blue reduction....................................................................Serum protein electrophoresis..............................................................Determination of orosomucoid concentration in human serum with

immunonephelomctry..........................................................................Determination of uric acid concentration in serum...............................Determination of triglycerides in serum................................................Principle of dry chemistry diagnostic tests............................................Determination of serum bilirubin...........................................................Determination of enzyme activity..........................................................

Fixed-time (Two-point) assay of the a-Amylase enzyme..............Kinetic assay (Multipoint, Continuous Monitoring) of Aspartate

Aminotransferase and Alanine Aminotransferase enzymes.....Determination of serum calcium, potassium, and chloride ion

concentration.......................................................................................Enzymatic determination of blood glucose level...................................Assay of glycosylated haemoglobin........................................................

Safety regulations

1. It is compulsory to wear labcoats during practices. Coats and bags etc. are to be kept in the wardrobe on the corridor during practices.

2. Laboratory equipment, chemicals and samples must be used as described in the protocol.

3. Spilling or splashing any chemicals or samples is prohibited. Contact of the chemicals/samples with the skin (inhalation, contact with eyes or swallowing) should be prevented.

4. Harmful chemicals (concentrated acids, oc. bases, organic solvents and toxic materials) are to be handled with special care, following the safety instructions. When such materials are in liquid form, only automatic pipettes or glass syringe dispensers are allowed.

Before sampling or dispensing, proper operation of the pipette must be checked (no dripping is allowed).Should the above chemicals get into contact with the skin, it has to be removed immediately with plentiful water. When spilled on clothes or onto lab equipment it has to be removed immediately with towels or tissue paper (while wearing gloves). Upon contact of the chemicals with the eye, the eye must be rinsed using a neutr alizing solution and/or plentiful water, and the supervisor must be notified.

5. Extreme care must be taken when handling human samples because of the potential risk of infections. In addition to the above general rules, all manipulations must be done using gloves and the prescribed disinfecting handwashes are compulsory.

6. Laboratory glassware is fragile and might cause injuries. Should any injuries occur, the supervisor must be notified in order to obtain appropriate treatment of the injury.

7. Smoking, eating and pipetting by mouth is strictly forbidden in

the laboratory. Fire precautions

1.After using Bunsen-burners (to prepare boiling water-bath) gas valves both under the fume hood and the mains must be returned to the closed position.

2. Use of electric equipment in improper condition is forbidden. Upon finishing the work in the lab, electric switches (on the benches etc.) must be turned off.

3. Students should be familiar with the location and the mode of the operation of the fire-extinguisher.

4. Electric fires must not be "put out" with water. Proper fire-extinguishers are to be used or the air supply to the fire should be blocked, if possible (e.g. using textile) or else.

5. Routes in the lab as well as exits must be kept free, unblocked!

Substrate specificity and temperature optimum of Amylase enzymeactivity

Amylase (1,4-a-glucosidase) is responsible for the enzymatic hydrolysis of alimentary starch. In the hydrolytic reaction disaccharide units (maltose) are removed from the unbranched glucose chain termini of starch. Amylase is also capable of hydrolyzing glycogen, though this polysaccharide is not considered to be an important nutrient (glycogen, while present as a minor component of white muscle cells, is rapidly decomposed during food processing).

In the alimentary tract, the breakdown of polysaccharides is initiated by the amylase of the saliva. This is followed by a more effective and complete pancreatic amylase hydrolysis in the small intestine. Amylase, in low concentration, can be traced in blood serum. Due to its molecular mass (Mw: 50 000), amylase readily appears in kidney filtrate and urine. Elevated activity of amylase in serum and urine indicates diseases of the pancreas (acute pancreas necrosis) or the salivary glands.

In this practical demonstration we use amylase as a model enzyme to show the influence of temperature on catalytic activity. We also compare the efficiency and specificity of enzymatic versus acidic hydrolysis of starch.

Principle

Amylase activity can be detected by thc Fehling reaction, which is based on the measurement of reducing sugar released by enzymatic hydrolysis. Undegraded starch is traceable by iodine colour reaction.

Materials

- Amylase solution: is obtained by extraction from germinating barley seeds

- Iodine reagent: 2% KI and 1% I2 in aqueous solution- Fehling I reagent: 4% CUSO4 solution- Fehling II: 20% K-Na-tartarate + 15% NaOH solution- 1% starch solution- phosphate-citrate buffer solution, pH 6.8 (0.2 M Na2HPC>4

and 0.1 M citricacid in 7.7:2.3 volume ratio)

- 10% H2S04 solution- 20% NaOH solution- 1% sucrose solution- 20% sulfosalicylic acid

The influence of temperature on the enzymatic and acidic hydrolysis

of starch

Procedure

Dispense the following amounts of reagents into 6 test tubes in the order given below.

Table 1.t e s t t u b e s

reagents (ml) 1 2 3 4 5 6

starch 2 2 2 2 2 2

buffer 5 5 5 - - -

< ;£Kfmin. preincubation (°C)

0 37 100 0 37 100

amylase 1 1 1 - - -

10% h2so4- - - 6 6 6

15 min. incubation (°C)

0 37 100 0 37 100

20% sulfosalicylic acid 0.5 0.5 0.5 n - -

Incubation time is counted from the point of the addition of the enzyme solution or H2SO4; the enzymatic reaction is stopped by addition of 20% sulfosalicylic acid.

Divide the content of each test tube into two equal portions.To one part add Fehling I and Fehling II reagents. Since the

reaction requires alkaline conditions, 20% (w/v) NaOH solution should be added to adjust the pH of H2SO4 -containing (4 to 6) tubes (till the colour of the solution turns blue; approx. 3 ml NaOH is required).

Table 2.t e s t t u b e s

reagents (ml) 1/A 2/A 3/A 4/A 5/A 6/A

Fehling I 1 1 1 1 1 1

Fehling II 1 1 1 1 1 1

20% NaOH - - - 3 3 3

5 min. incubation at 100°C

(boiling waterbath)

Compare the intensity of the colour reactions. (It is proportional to the extent of starch hydrolysis).

To the other part of the reaction mixture 1 to 6 add iodine reagent as follows:

Table 3.t e s t t u b e s

reagents (ml) 1/B 2/B 3/B 4/B 5/B 6/B

iodine 1 1 1 1 1 1

Mix well and compare the intensity of the colour reactions (this is proportional to the amount of starch present).

Evaluation

By comparing the results obtained with the Fehling and iodine reactions determine the optimal conditions of enzymatic versus acidic hydrolysis. Write down the possible explanation of the results.

Analysis of Ihc substrate specificity of the enzyme

Procedure

Dispense the following reagents in 4 test tubes in the order given below: Table 4.

t e s t t u b e s

reagents (ml) 1 2 3 4buffer 5 5 - -

starch 2 - 2 -

sucrose - 2 - 2

10 min. preincubation (°C)

37 37 100 100

amylase 1 1 - -

10% H2SO4- -

6 615 min. incubation (°C)

37 37 100 100

20% sulfosalicylic acid 0.5 0.5 - -

Divide the content of each tube evenly as done earlier. With half of the reaction mixture perform the Fehling reaction. Table 5.

t e s t t u b e s

reagents (ml) 1 2 3 4

Fehling I 1 1 1 1

Fehling II 1 1 1 1

20% NaOH - - 3 3

5 min. incubation at 100 °C

Evaluation

Compare the efficiency of enzymatic (amylase) and acidic hydrolysis of starch and sucrose . Interpret the results in terms of specificity.- y -

- 1 0 -

Dctcrmination of albumin in serum

Introduction

Albumin makes up about 60% of the loial plasma proteins. It has two important biological functions: It is responsible for 75-80 % of colloid osmotic or oncotic pr essure of the plasma, and it is the most important carrier protein.

Because of diminished synthesis, plasma albumin levels fall in any chronic illness. Very low levels arc often found in patients with advanced hepatocellular disease and in patients with conditions in which diminished synthesis is accompanied by loss of large amounts of albumin into the urine (nephrosis) or into the gastrointestinal tract (protein-losing enteropathies).

Principle

Bromcresol green binds to albumin specifically forming a green colored complex in the presence of Brij 35.

Color reagent:

Dist. waterGlycine solution (1 M) HCI (1 N) Brij 35 (30%)Bromcresol green stock solution

1600 ml 189 mlI 1 ml8 ml 6 ml

Procedure

Dispense in the test tubes the following amounts of solutions:

t e s t t u b e s

reagents 1sample

2standard

3blank

color reagent(ml) 5 5 5

serum(pl) 50 - -

standaid(pl) - 50 -

dist. water(jil) - - 50

15 min. incubation at 25 °C

Measure the absorbance of

sample and standard at 625 nm.

Evaluation-^sample

x ^--standard = ^serum (S^O^-standard

A = absorban c

_

Introduction- 12-

ce C = concentration

me>

r<£ S-pocî cc CQ^OJ (hOi^xhc§51'oUL cUoa^

yiOl- <oidu p o k lu** (jKUu^ i u O K d r fi ' SioU CUClal^

QjbunX^^

Determination of total protein in serum (Lowry method)

Plasma proteins serve a number of different functions in the organism. Not only the relative amount of plasma protein fractions but the total protein content can undergo a change depending on the different disease states. A large number of methods are available for determination of plasma proteins and they are different in their sensitivity and specificity. The commonly used spectrophotometric methods are based on the change of absorbance resulting from the interaction of the reagents with peptide bounds or certain amino acid residues. Some examples are as follows:- Biuret reaction - Substances containing peptide linkages give a violet coloration with the reagent. The method is of low sensitivity, but it is not affected by free amino acids.- Lowry method - The intensity of color formed is due to the reduction of phosphomolybdate by Tip and Tyr present in the proteins. The method is fairly sensitive, but free amino acids inteifere with the reaction.- Bradford reaction - The color reagent binds to basic (especially Arg) and aromatic 1 amino acid residues giving a blue color complex. A reliable method of high sensitivity and specificity.

Principle

The method is based on the color reaction of the Folin reagent (phosphotungstomolybdic acid) with Trp and Tyr side chain of proteins.

Materials

Sol."A": 4% Na2C03 in 0.2N NaOH

Introduction- 14-

Sol."B": 1.1% CUSO4 x 5H20, 2.2% K-Na tartarate in 1:1 ratio Sol."C": "A" and "B" Sol. in 50:1 ratioFolin reagent (dilute with dist. water in 1:1 ratio before use)distilled waterprotein standard: 1 mg/mldiluted serumphosphate buffer (pH:7.2)

Procedure

Dilute the Folin reagent with distilled water in 1:1 ratio

Prepare Sol."C" by mixing Sol."A"

and Sol."B" in 50:1 ratio Dispense in

the test tubes the following amounts

of solutions

t e s t t u b e s

reagents 1 2 3 4 5 6

standard(jil) 10 50 100 150 200 -

dil. serum(pl) - - - - - 200

phosphate buffer(pl) - - - - - -

dist. water(jjl) 590 550 500 450 400 400

Sol. "C" ml) 3 3 3 3 3 3

10 min. preincubation at 25 °C

dil. Folin reagent(fjl) 300 300 300 300 300 300

30 min. incubation at 25 °C

Measure the absorbance of the samples at 710 nm

Evaluation

Make a calibration curve using the values obtained with standards (1-5. test tubes), (abscissa: protein standard concentration fpg/mlj, ordinate: measured absorbance).

Use this curve to determine protein concentration of the diluted serum (6. test tubes).

Introduction- 1 6 -

Assay of the activity of Alkaline Phosphatase

Phosphatases represent a group of enzymes, which hydrolyze phosphate esters

»

(phosphatides, carbohydrate phosphoric acid esters, glycerophosphoric acid, nucleotides). According to their pH-optima phosphatases are classified as alkaline or acidic phosphatases. They are activated by and Mn2+ ions, while enzymeactivity is hindered by Zn2+, Bc2+, PO43", ASO43", CN~, oxalate, citrate ions, and thiol compounds. Both groups have a fairly broad substrate specificity, thus they are capable of hydrolyzing a variety of physiological and nonphysiological substrates, like glycerol phosphate and p-nitrophenyl phosphate, which are widely used in in vitro enzyme assays.

Phosphatases are involved in bone matrix formation, polysaccharide and protein biosynthesis of the connective tissue, in nucleic acid metabolism, in regulation of glycogen metabolism and signalization processes taking place in membranes. Serum alkaline phosphatase activity is the result of different isoenzymes of intestinal , placental, liver, kidney, and osteoblast origin. A physiological increase of enzyme activity occurs in pregnancy, while both severe starvation and protein-rich diet result in decreased activity. Certain pathologic conditions, like certain bone and liver t diseases (especially those involving biliary obstruction) and recovery from bone fractures are associated with increased phosphatase activity. Enzyme activity of the serum is decreased in hypothyreosis and during osteoblast damage.

In this practical, the dependence of enzyme activity on substrate concentration is studied by using the alkaline phosphatase (ALP) as a model system.

Normal value (in human serum) adult: 13-45 U/linfant (2 to 12 months): 40-160 U/l

(One unit [U] of alkaline phosphatase activity is, defined as the activity of enzyme which produces reaction in 1 pmole of substance in 1 minute under defined conditions. The values of enzyme activity are referred to 1 liter serum.)

- 17 -

Principle

p-Nitrophenyl phosphate is hydrolyzed by alkaline phosphatase. The resulting p- -nitrophenol, which is yellow in alkaline medium , can be quantified by spec troph otometry.

Materials

- alkaline buffer solution (pH 10.5; 220 mM Tris(hydroxymethyl)aminomethane, 5.5 mM MgCl2)- substrate, 55 mM p-nitrophenyl phosphate (Mw. 371.1) stock solution- 0.2 M NaOH solution- blood serum

Procedure

We will make a bisecting dilution series from the stock substrate solution as follows: Pipette 1 ml of the stock solution (55 mM p-nitrophenyl phosphate) into the first tube (1/a). Then pipette 0.5 ml of distilled water into the tubes marked 2/a, 3/a, 4/a and 5/a, respectively. Then take out 0.5 ml of the stock solution from the first tube (1/a) and put it into the second tube (2/a). After thorough mixing the p- nitrophenyl phosphate concentration in this tube is the 1/2 of the original one, namely 27.5 mM. Pipette 0.5 ml of the diluted solution from the second tube (2/a) into the third tube (3/a). As a result, the concentration of the mixed solution is the 1/4 of the original concentration, 13.75 mM. Prepare 1/8 and 1/16 dilutions (6.87 mM; 3.43 mM) in the same way. This substrate dilution series is used in alkaline phosphatase enzyme assay.

- 18 -

t e s t t u b e s

reagents (ml) 1 2 3 4 5 6(blank)

buffer 0.7 0.7 0.7 0.7 0.7 0.7

corresponding diluted substrate

0,1 0.1 0.1 0.1 0.1 -

dist. water - - - - 0.1

serum 0.2 0.2 0.2 0.2 0.2 -

15 min. incubation at 37°C

0.2M NaOH 2 2 2 2 2 2

serum - - - - - 0.2

Mix the samples (tubes 1-5) again and determine absorbances (extinctions) at 400 nm. Use control sample (tube 6) as a reference.

Evaluation

Absorbance values are proportional to the quantity of p-nitrophenol and the rate of the enzyme reaction. Plot the results on graph paper indicating the measured absorbances (A) as a function of concentrations of the substrate according to Michaelis-Menten. (All substrate were diluted lOx, thus the concentrations are the follows: 0.343; 0.687; 1.37; 2.75 and 5.5 mM.) Evaluate the experimental results on the basis of Lineweaver-Bnrk and Direct Linear Methods for comparison as well. (Theoretical bases and practical applications can be found in the section of Practical Enzyme Kinetics:)

Practical enzyme kinetics

The Michaelis-Mcnten equation

According to Michaelis and Menten, the kinetics of enzyme catalysis can bedescribed with the following equation:

kx k2E + S ES -------------> E + T

k3if and only the conditions numbered 1-5 are fulfilled.

- 1 9 -

where E — enzyme S = substrateES = enzyme-substrate complex P = productkj, k2, = reaction rate constants

1.There has to be an ES temporary complex during the reaction.2. The product P is not converted back to ES (therefore there is no k4 reaction

constant). It can be supposed because P is converted further at once in most reactions.

3. We observe the reaction only with one substrate (first order reaction) and consider only one ES complex and one product to be formed.

The velocity of the reaction equals with the velocity of the product formation:V = k2 [ES]

4. We suppose the reaction to be at steady state. This means that the [ES] is not hanging because the speed of its formation and its breakdown are equal. (The situation is different at the beginning stage of the reaction where more ES is formed than broken down.)

ES complex formed at Vf velocity and is broken down at V^ velocity:

Vf=ki [E] [S]Vf = ki [E] [S]Vb = k2 [ES] + k3 [ES] = (k2+k3) [ES]

Steady state means Vf = Vb

ki [E] [S] = (k2 + kg) [ES]

by rearranging the equation we get: [E] [S] k2 + k3

[ES] ki

this is a new constant, called the Michaelis constant (Kjyj)

k2 + k3k m = -------------

kl

this includes the 3 reaction constants, [E] [S]------------ =KM

[ES]

where [E] = free enzyme concentration: [E] = [Et] - [ES] [EJ = total enzyme concentration[S] = the actual substrate concentration equals with total substrate

concentration because 5. We suppose that free substrate concentration supersede many times that of the enzyme linked one, so since enzyme concentration is relatively low compared to substrate concentration, we always have surplus substrate.

To calculate enzymatic velocity (V = k2 [ES]), we have to determine the actual ES concentration. On substituting [E] for [EJ - [ES]

( [Et] - [ES] ) [S]------------------------------ =KM

[ES]

([EJ - [ES]) [S] = KM [ES] [Ed

[S] - [ES] [S] = KM [ES] [EJ

[S]=KM [ES] + [ES] [S] [Et]

[S] = [ES] ( KM + [S] ) [Et]

[S][ES] = ----------------

KM + [S]

Now on substituting this into V = k2 [ES] we get

[Et] [S] V = k2 -------------------------

KM + [S]

Here only [S] is a variable.

Now let us examine when the maximal rate Vmax is attained. This is the case when the enzyme active sites are saturated with substrate; in other words [S] is much greater than Km» thus

- 2 1 -

[S]= 1 (approaches 1)

[S] + Km

Since Vmax = k2 [Et],

Vmax lEt[ [S]substituting k2 = ----------- into the equation V = k2 ----------------

lEt] [S] + KM

we get

- 22 -

[S]^ = ^max

[Si + Km

This is the Michaelis-Menten equation which with two constants accounts for therelation of the rate of enzyme catalysis to the concentration of substrate.

The Michaelis-Menten equation describes a hyperbole where Vmax is theasymptote of the curve, while Km is the concentration of substrate [S], having the

half of the maximal value of the velocity. This is evident, if we substitute Vmax/2into the Michaelis-Menten equation:

[S]^ max/2 = ^max

[S] + KM

by rearranging the equation

1=2[S]/(KM + [S]) KM + [S] = 2[S][S] = KM (when V = Vmax/2)

That is, Km is numerically equal to the substrate concentration, at which reaction rate is half of its maximal value.

The Km value is a characteristic constant of the particular enzyme (isoenzyme). The lower Km reflects to a lower substrate concentration, at which Vmax can be attained.

The conversion of the Michaelis-Menten equation to a Straight Line Plot

It is difficult to estimate Km and Vmax values from the hyperbolic Michaelis curve graphically, since the asymptote can not correctly be determined. However, for determination of Km the correct asymptote value is needed.

Exact graphical representation is possible, if we linear ize the original equation. There are many different methods for doing this but we present only one here,

the one called the Lineweaver and Burk linearization. The method is based on the reciprocal transformation of the sides of the Michaelis-Menten equation that

gives a straight line plot.1/V = (KM + [S])/

v max [S] 1/V = KM/Vmax [S] + 1/Vmax

- 23 -

Graphical representation means plotting 1/V as a function of 1/[S]. If our enzyme follows the Michaelis-Menten kinetics, we get a straight line.

The slope is : i\M/Vmax

The intercept on the y axis : 1/Vmax

The intercept on the x axis : - 1/Km

Because of the nature of this linearization Vmax can be determined more correctly than Kjyj-

Direct Linear Method

The other method to determine Km and Vmax correctly is the so-called "direct linearization method". Here we use the original Michaelis-Menten curve to generate a constant in the following way.

We draw parallel lines with the x and y axis which run through the original determined points, thus we get a series of rectangles. We draw the diagonal line from the lower right to the upper left corner and extend it. All diagonal lines will intercept Vmax at the -Km- To prove this let us take up a point on the x axis and let this point be at Km-

Fig. 1. Graphical Determination of Km Using the Direct Linear Method

- 24 -

abc and ABC triangles are equal because B = Vmax/2 = b, so A = K^ Hence, this line will intercept the Vmax asymptote at -Kj^j-

We can similarly prove it for any other point on the Michaelis curve. Let us now prove this at the substrate concentration = 2Km» so

2 K MV = V

max2 KM + KM

V = 2/3 Vmax

The relation of triangles is:

(l/3)/(2/3) = a/(2KM)

1/2 = a/2 KM a= KM

Therefore, these diagonal lines intercept the Vmax asymptote at -Kj^j-

KM 2KM

max

Fig. 2. Determination of K^ at 2 Kj^ Concentration of the Substrate

b/B = a/A

Determination of GIucose-6-phosphatase activity in rat liver

Glucose-6-phosphatase is an integral membrane protein of the endoplasmic reticulum (ER), found mostly in liver, also in kidney, and in intestinal mucosa. The enzyme present in liver is functioning by catalyzing the hydrolysis of glucose-6- phosphate originated from glycogen catabolism and through this by maintaining constant blood glucose level. The glucose-6-phosphatase system involves also 3 translocases apart from the enzyme, which allow the transport of glucose-6-phosphate to the ER, or the efflux of inorganic phosphate or glucose from the ER, respectively.

Physiologically, damaged or no activity of the enzyme can be shown in the plasma; in cases of liver damage (hepatitis, cirrhosis) enzyme activity is increased in the plasma, while reduced in liver. In type I/a glycogen storage disease (Gierke s disease) low blood glucose level is found (fasting hypoglycemia) caused by the lack of the enzyme. Similar" symptoms are seen in type I/b glycogen storage disease where glucose-6-phosphate translocase is missing. During long fasting increased synthesis of glucocorticoid hormones bring about increased synthesis of glucose-6-phosphatase.

Principle

The enzyme present in liver homogenate cleaves inorganic phosphate from glucose-6-phosphate; the amount of inorganic phosphate is determined by a turbidimetric assay. Phosphate reacting with ammonium molybdate forms a phosphomolybdate complex; adding Triton X-100 (a detergent) to the solution, turbidity will develop, the degree of which is related to the concentration of the complex. EDTA and NaF (in the reaction mixture) inhibit the activity of acidic and alkaline phosphatases.

Materials

- liver homogenate (rat liver homogenized in 3 vol buffer solution containing 10 mM Tris, 2 mM MgCl2, 30 mM NaCl, pH 7.3); used after a 5-fold dilution- malate buffer solution (75 mM malate, 12.3 mM EDTA, 12.3 mM NaF pH 6.0)- glucose-6-phosphate (diNa salt) (200 mM)

Introduction - 2 5 -

- 26 -

- 12% trichloracetic acid- 1% Triton X-100- 2.5% ammonium molybdate x 4 H20 in a solution containing 16.6% (v/v) conc.

sulfuric acid.

Be careful! Strong

acid! Procedure

Take 2 test tubes: 1 for the enzyme reaction (sample tube) and 1 for the determination of inorganic phosphate present in the glucose-6-phosphate solution and in the liver homogenate (control). Fill 0.55 ml malate buffer in all test tubes. Pipette 50-50 pi (5 x diluted) liver homogenate to the sample and to the sample control tubes. (Before pipetting shake the liver homogenate well!). Incubate all tubes for 5 minutes at 37 °C. Then add 2 ml 12% tr ichloroacetic acid to the control tube and 0.1-0.1 ml glucose-6-phosphate solution to the sample and to the control tubes. Incubate for 15 minutes at 37 °C. Stop the reaction by adding 2 ml cold 12% trichloroacetic acid to the sample tube. Then filter the contents of the test tubes into 2 labelled short tubes.

Three long test tubes are labelled for the determination of inorganic phosphate (1 sample, 1 control, 1 blank). Deliver 0.4 ml filtrate to the appropriate tube, then add 5- 5 ml dist. water to each. Prepare the blank one by adding 5.4 ml dist. water to a tube. Fill 60-60 pi 1% Triton X-100 in all tubes and mix well. Then add 0.6-0.6 ml ammonium molybdate solution to all tubes and mix them again. Read absorbance 4 (against blank) at 620 nm 20 minutes later.

Table 1.t e s t t u b e s

reagents 1sample

2control

malate buffer(ml) 0.55 0.55

liver homogenate dil.(pl)

50 50

5 min. preincubation at 37 °C

12% TCA(ml) - 2

glucose-6-phosphate(pl) 100 100

- 27 -

15 min. incubation at 37 °C

12% TCA(ml) 2 -

filtration + +

Table 2.t e s t t u b e s

reagents 1sample

2control

3blank

filtrate(ml) 0.4 0.4 -

dist. water(ml) 5 5 5.4

Triton X-lOO(jil) 60 60 60

ammonium-molybdate(ml)

0.6 0.6 0.6

reading absorbance at 620 nm 20 minutes later

= i - ^ ^Calculation

Take the average of the absorbance values of the 2 samples, 2 substrate controls and 2 sample controls, respectively. Read the inorganic phosphate (Pj) amounts (in nmol) corresponding to these absorbance values from the Pj calibration curve. Subtract the P^ values of the substrate plus sample controls from the P^ value of the sample, thus we get ?\ liberated by the enzyme.

Enzyme activity calculated for 1 ml diluted liver homogenate (nmol Pi / ml sample x min):

Pi (nmol) x 6.75

diluted 0.05 ml homogenate x 15 min

Pi (nmol)calculated for 1 hour: -------------------------------------------- x 60

diluted ml homogenate x min

calculated for the undiluted liver homogenate (diluted 5x):

Pi (nmol) x 5 —>

- 28 -

ml homogenate x h

volume factor:final volume of reaction mixture 2.7ml ---------------- .= = 6.75

volume of filtrate used for Pi determination 0.4ml

If protein concentration of the liver homogenate is known, specific activity corresponding to mg protein can be calculated: enzyme activity (nmol Pi / mg protein x min):

Pi (nmol) x 6.75

protein quantity in 50 ul (in mg) x 15 min

This value can be calculated with, as described above; units in nmol Pj can be converted to higher units.

¿Ul^lM A Staple : (So*- C f S r & ^ y - Z ^ l i ^ t ^

Enzymatic-colorimetric assay of urea in human serum

Due to the degradation of nitrogen containing compounds, 80-90% of the total excreted nitrogen gets into the urine as urea. Urea is synthesized in the liver in the "urea cycle" consuming NH3, CO2, the amino nitrogen of aspartate, and ATP. This process detoxicates the highly toxic ammonia formed by the metabolism of nitrogen compounds, and by the enteral bacteria. In case of severe insufficiency of the urea cycle due to hereditary enzyme deficiency or hepatic cirrhosis, the extrahepatic detoxication of the accumulated ammonia may lead to metabolic disorders causing mainly neurological symptoms. High ammonia concentration and low urea concentration in the plasma have great diagnostic value in this case. Elevated urea concentration may be observed as a result of protein-rich diet, or enhanced protein degradation that occurs in starvation or due to overproduction of glucocorticoid hormones. Insufficiency of kidney function also leads to high urea concentration.

Principle

Urease cleaves urea producing carbon dioxide and ammonia in alkaline pH.

urea + H20------> 2NH3 + C02

Ammonia is immediately converted to ammonium ion in aqueous solution. Ammonium forms a green compound with salicylate and Na-hypochlorite in the presence of the catalyst Na-nitroprusside. The intensity of the color measured at 600 nm wave length is proportional to the concentration of urea in the range of 0 -2 5 mmol/1.

Materials

Reagent 1.: urease powderReagent 2.: (phosphate buffer):

Na-salicylate (60 mmol/1) Na-nitroprusside (3.5 mmol/1) EDTA (1.31 mmol/1)

Reagent 3.: Na-hypochlorite (45 mmol/1)NaOH (1 mmol/1) Standard: urea

(8.325 mmol/1)

Introduction - 2 9 -

- 30 -

Sample: serum, or heparinized plasmaurine (100 times dilution)

Procedure

t e s t t u b e s

reagents 1sample

2standard

3blank

1.+2. reagent(ml) 1 1 1

dist. water(jil) - - 10

standard(jil) - 10 -

sample(pl) 10 - -

5 min. incubation at 25 C

3. reagent(ml) 1 1 1

15 min. incubation, then measurement of absorbance

at 600 nm

Calculation

[urea] (mmol/1) = (A sample / A standard) x [standard] (mmol/1)

- 28 -

Reagent 3.: Na-hypochlorite (45 mmol/1)NaOH (1 mmol/1)

Standard: urea (8.325 mmol/1)Sample: serum, or heparinized plasma

urine (100 times dilution)

Procedure

t e s t t u b e s

reagents 1sample

2standard

3blank

1.+2. reagent(ml) 1 1 1

dist. water(jil) - - 10

standard(pl) - 10 -

sample(pl) 10 - -

5 min. incubation at 25 C

3. reagent(ml) 1 1 1

15 min. incubation, then measurement of absorbance

at 600 nm

[urea] (mmol/1) = (A sample / A standard) x [standard] (mmol/1) »

- 29 -

Calculation

- 30 -

Detection of the oxygen consumption of isolated mitochondria bymethylene blue reduction

Mitochondria - as the organelles of intracellular respiration - produce high amount of reduced coenzymes ( NADH+H+, FADH2 ) in mitosol during the breakdown of acetyl-CoA by the enzymes of the citric acid cycle. The elections from these reduced coenzymes are transported to molecular 02 by the enzymes of the electron transport chain localized in the inner mitochondrial membrane. Under normal conditions this process is strictly connected with ATP production which is regulated by oxygen as a substrate and even by the ATP/ADP ratio.

The velocity of the intracellular respiration depend therefore on the actual 02

supply and the energy saturation of the cell.Certain drugs or chemicals interfere with this regulation mechanism, either

destroying the proton (pH) gradient which exists between the matrix and the intramembranous space, or blocking the transport of electrons which in turn is responsible for the generation and maintenance of the proton gradient. Such chemicals are the so called uncoupling agents (2,4-dinitrophenol, dinitrocresol, pentachlorphenol) arid enzyme inhibitors (barbiturate, atractyloside, rotenone, antimycin, oligomycin, KCN).

Principles

Mitochondria are easily isolated from soft tissues (i.e. liver) and under physiological conditions they remain functionally active for a longer period of time. The aim of this practice is to demonstrate some functional aspects of intact mitochondria.

The oxygen consumption will be followed therefore suspension containing mitochondria and substrates has to be overlayered by paraffin-oil to exclude further 02 supply. In this case the available 02 content of the suspension equals to the amount of 02 which has been dissolved into the medium from the air to its partial pressure at room temperature.

We will also add redox-indicator molecules to the suspension. Elections flowing through the electron transport system of the inner mitochondrial membrane will reduce the dye molecules into a colorless (leukomethylene blue) product, if molecular oxygen is no more available.

Materials

- mitochondria suspension (prepared from rat liver in isotonic solution)

- 31 -

- incubation solution: 20mM TRIS buffer pH 7.2 containing 8mM KC1, ImM EDTA, lOmM KH2P04, 0.01 M Na-succinate- solutions of lOmM 2,4-dinitrophenol, 0.1M KCN, 0.3M ADP- 0.5% methylene blue

Proceduret e s t t u b e s

reagents (ml) 1 2 3 4 Remarks

incubation solution 4 4 4 4

mitochondrium susp. 1 1 1 1 Shake well before use!

ADP sol. - 0.2 - -

dinitrophenol - - 0.2 - Toxic!

KCN sol. - - - 0.2 Toxic!

dist. water 0.2 - - -

After shaking, 2 min. preincubation 37 C at

methylen blue 0.1 0.1 0.1 0.1 Put the meth. blue first

into the 4. tube. Shake well and overlay with

paraffin-oil carefully!

Shake the mixture!

paraffin-oil 1 1 1 1

incubation at 37 C

Check the tubes every 30 seconds and compare their

colors! Evaluation

Bleaching of methylene blue refers to the loss of dissolved 02 from the reaction mixture which in turn is an indicator of an intact electron transport.

Serum protein electrophoresis

Protein molecules migrate in electric field. The migration is determined by their structure and the pH of the solvent. The majority of proteins has isoelectric point (when the molecule is neutral) around or under pH 7. In a moderate basic solvent (pH 8-9) the molecule dissociates protons, it becomes negatively charged and migrates toward the positive electrode (anode). Protein molecules with isoelectric point at basic pH are less frequent and move toward the negative electrode (cathode), if the solvent has moderate basic pH. On a suitable carrier matrix (filter paper, agarose, acrylamide, cellulose acetate) this feature can be utilized for electric separation - for the electrophoresis.

The electrophoresis has been used in biological, medical research and in clinical diagnosis for nearly thirty years. It is a basic method that has been developed into more sophisticated ones. Here we introduce the basic principles of some of these methods.

Native electrophoresisProtein isoforms occurring in a small quantity but having catalytic activity can

be identified by this method. After separation resulting in a relatively low resolution, enzymes can be stained histochemically. Special gels like starch-agarose or cellulose acetate membrane are used to preserve enzyme activity.

SDS-gel electrophoresisMolecular weight of protein subun stermined by electrophoresis. After

boiling with sodium-dodecilsulfate (SDS) and DTT that breaks the disulphide bounds, proteins dissociate into subunits, become negatively charged and move toward the anode. The speed of migration is inversely proportional to the molecular weight. The resolution can be improved by repeating the electrophoresis among altered conditions (pH, gel concentration) into an other direction.

Isoelectric focusing

Protein molecules are separated according to their isoelectric point on a gel carrying pH gradient. Proteins migrate to the pH zone of their isoelectric point and stop there.

ImmunelectrophoresisThe sample is applied to gel made with the antibody or the antibody is

electrophoresed toward the sample. At the zone where the proteins and the antibody form complexes precipitation arches are formed. The place and size of these arches are evaluated quantitatively and qualitatively.

Electrophoresis of nucleic acidsThe separation of nucleic acid fragments is also made by electrophoresis in

molecular biology. Nucleic acids are macromolecules therefore must be digested by restriction enzymes before electrophoresis. The length of the restriction fragments will characterize the nucleic acid macromolecule or the chromosomal DNA. The linearized double stranded DNA fragments migrate toward the anode at basic pH in 1% agarose . Single stranded nucleic acid molecules have to be denatured for the electrophoresis. The electrophoretic mobility is in inverse proportion with the size. The size of the fragments can be determined by standards.

Gel blottingProtein molecules or nucleic acid fragments can be transferred to nitro-

cellulose/nylon membrane from a gel after denaturation. Protein molecules are transferred by electric field, while the nucleic acid fragments mostly by capillary * diffusion. The transferred molecules are fixed to the membrane by heat, vacuum or UV irradiation. The membrane has the advantage of keeping its size during the wetting and drying procedure. Proteins on the membrane can be hybridized with antibodies - this is the Western blot. Blots made from nucleic acid gels are hybridized with a nucleic acid fragment labelled with radioactive nucleotide. If the DNA fragments are on the membrane that is the Southern analysis. If RNA fragments are on the membrane that is the Northern analysis.

Protocol for the practice

This practical demonstrates the electrophoresis of serum proteins on a special ready made agarose gel. Under these circumstances the protein molecules migrate according to the electric charge and molecular size. The

Introduction - 3 3 -

serum protein bands, in the order of their migration: the albumin, the ct[ and ct2, the P-globulin and the y- -globulin - has clinical significance. Except the albumin, each band contains multiple proteins. The ratio of these proteins might change in diseases. Certain additional proteins might also accumulate in the serum in acute infections.

- 35 -

Use plastic gloves!

1.Open protective bag and blot the buffer from the gel.2. Place template on gel, remove air bubbles and apply 5 pi of the diluted serum.

Wait 5 minutes until sample infiltrates the gel.3. Remove template, center gel over bridge, squeeze and locate on paragon.4. Pour 45 ml buffer into each cell reservoir, connect the chamber into the electric

circuit and electrophorese at 100 V for 25 minutes.5. Place gel in frame and the frame into frame holder.

Keep the gel in:6. Acetic acid-methanol for 3 minutes.7. Drying oven (maximum 90 °C), dry gel completely, aprox. 30 minutes.8. Blue stain solution for 3 minutes.9. Acetic acid for 2 minutes.10.Acetic acid-alcohol solution for 2 minutes.11.Acetic acid solution for 2 minutes12.Dry gel completely (maximum at 90 °C).

Gel can be scanned by densitometer at 600 nm. The ratio of serum proteins: albumin: 58.8-69,6%; globulins: cq: 1.8-3.8%; a2: 3.7-13.1%; P: 8,9-13,6%; y: 8,4- 18,3%; A/G ratio 1.39-2.23%. Ratios might be different in pathological samples.

- 36 -

Determination of orosomucoid concentration in human serum withimmunonephclometry

The major portion of plasma seromucoids has been recognized as orosomucoid (acid a j-glycoprotein). Orosomucoid is an acute phase protein. Increased orosomucoid serum can be observed in less than 24 hours after the tissue damage. Increases occur also in rheumatoid arthitis, systemic lupus erythematosus, malignant neoplasms and in myocardial infarction. Decreases occur in malnutrition, severe hepatic damage and severe protein-losing gastroenteropathies.

Measurement range: ca. 0.2-3.0 g/1Expected values: 0.3-1.1 g/1

Principle

Following dilution of serum, anti-orosomucoid antibody is added. The intensity of the antigen - antibody reaction can be measured by nephelometry. This method is based on the light-scattering properties of immun-complexes (precipitates) and the measured values correlate with the amount of these complexes.

Light Source

Recorder

Immunoprecipitate----------- of

Analyte

- 37 -

Materials

- sample: serum- buffer for the antibody solution- blank buffer- antibody solution- calibrator solution

- 0.9 % NaCl solution (physiological salt

sol.) Procedure

Human serum is used for the determination of orosomucoid, therefore the use of surgical gloves is required!1.Antiserum buffer solution: pipette 500 pi antiserum reagent into the 30 ml

buffer. Mix gently. Solution is stable for 12 weeks when stored at +2-+8 °C.2. Blank-buffer: ready to use.3. Calibrator: dilute 1:51 (10pl+500pl) with 0.9% NaCl.4. Sample: dilute 1:51 (10pl+500pl) with 0.9% NaCl.5. Pipette into the cuvettes the following solutions (prepare the calibrator as a

duplicate):

calibrator

blankcalibrator test sample

blank

sample

test

dil. sample(jjJL) - - 25 25

dil. calibrator 25 25 - -

blank buffer (HO

500 - 500 -

dil. antiserum <H0

- 500 - 500

- 38 -

Mix by shaking gently. Let stand for 30+5 minutes at room temperature. Note: Should small bubbles develop during incubation, they will disappear when the cuvette is shaken gently.

Measurement

1.Mix each cuvette gently before measuring.2. Measure the calibrator and the samples according to the instructions for

the instrument.3. Read the cuvettes in the following order:

Calibrator blank Calibrator test 1 Calibrator test 2

Sample 1 blank Sample ltest

Sample 2 blank Sample 2 test

Determination of uric acid concentration in serum

Uric acid formed during the catabolism of purine bases cannot be further used in the "salvage" reactions; it is produced from hypoxantine (in 2 steps) upon the action of xanthine oxidase. (In a healthy individual, 1 g is formed daily; most of which /cca. 600 mg/ is excreted in the urine by secretion of kidney tubules /active transport/.) While most mammals further catabolize uric acid to the highly water-soluble allantoin, in humans lacking uricase, uric acid is the end product of the degradation. At the normal pH value of the blood plasma, uric acid occurs primarily in the form of Na urate (solubility: 380 jimol/1 = 6.4 mg%). In the urine, below pH 5, mostly undissociated uric acid can be found which is poorly water-soluble (solubility is 20 times less, than that of Na urate).

Elevated uric acid level may occur physiologically, following strenuous physical exercise. Pathological, primar y hipexuricemia results either from reduced excretion of uric acid in urine or from enhanced biosynthesis (e. g. in Lesch-Nyhan

- 39 -

syndrome). Secondary hyperuricemia may occur a./ as a consequence of increased cell death in leukemia and in the case of other malignant tumors (especially following cytostatic or irradiation treatment), in addition, in hemolytic anemia, in heart attack and in severe pneumonia; b./ as a result of reduced excretion in kidney failure, in metabolic and respiratoric acidosis.

Prolonged hyperuricemia leads to accumulation of uric acid in the organism, the consequence of which is the condition of gout. The symptoms are due to the appearance of Na urate and uric acid crystals in acidic milieu, primarily in the joints of foot and hand and/or less often, in the kidney.

Normal valuemale : 120-400 pmol/1 (2.1-6.7 mg%)

female: 110-380 pmol/1 (2.0-6.4 rng%)

Principle

Uric acid is converted to allantoin and hydrogen peroxide by uricase. Hydrogen peroxide - in the presence of peroxidase enzyme - oxidizes the colour-forming molecule (3.5-dichloro-2-hydroxybenzenesulfonic acid /3.5-DCHBS/) to a red- coloured compound, the absorbance of which is detected at 520 nm.

uricaseuric acid + 02 + 2H202 --------------> allantoin + C02 + H202

peroxidase2H202 + 3.5-DCHBS + 4-aminophenazone -----------------> ACSB + HC1 + 4 H20

(coloured)

Materials

reagent solution: (phosphate buffer /pH 7/ 50 mM; 4-aminophenazone 0.3 mM; 3.5- DCHBS 4 mM; uricase 200 UA; peroxidase 1000 U/l; stabilizers, activators)standard solution: 10 mg% =-595 pmol/1 uric acid

. Hfrserum sample

Procedure

t e s t t u b e s

- 40 -

components 1sample

2standard

3blank

reagent(ml) 1 1 1

serum(pl) 76 - -

standard(pl) - 20 -

dist. water(pl) - - 20

10 min. incubation at 25 °C,

reading absorbance at nm (in 15 min.)

Calculation, evaluation

-y>5"V A sample uric acid concentration (pmol/I) = 595 x -------------------

A standard A

sampleuric acid concentration (mg%) = 10 x-------------

A standar dDetcrmination of triglycerides in serum (by

enzymatic-colorimetric method)

Triglycerides are both ingested with the food (exogenous triglycerides) or synthesized in the body (endogenous triglycerides). They are transported in the blood in the form of lipoproteins. The mucosal cells in the small intestine convert the exogenous triglycerides into chylomicrons, which are the lipoproteins that contain the largest amount of triglycrides. The liver esterifies free fatty acids, coming for the greater part from the adipose tissue, into endogenous triglycerides. Part of these is released into the blood in the protein-bound form, this fraction consisting of pre- (3 lipoproteins. Like hypercholesterolemia, a rised serum triglyceride level is a major risk factor for atherosclerosis and myocardial infarction.

- 41 -

Principle

LPLtriglyceride + 3 H2O ----------> glycerol + 3 RCOOH

GKglycerol + ATP -----------> glycerol-3-phosphate +ADP

GDHgIycerol-3-phosphate + NAD ----------> dihydroxyacetone

phosphate + NADH+ H+

diaphoraseindicator + NADH+H+ ------------------> indicator + NAD(colorless) (reddish)

LPL : lipoprotein lipase GK : glycerokinase

GDH : glycerophosphate

dehydrogenase Materials1.Buffer solution pH 7,92. Reagent mixture (LPL, GK, GDH, diaphorase, ATP, indicator, NAD)3. Standard solution: 2,29 mmol/14. Sample (serum)

Working solution preparation: dissolve the reagent mixture in buffer solution. (Solution is stable for 5 days when stored at +2 - +8 °C for 1 day when stored at +20 - +25 °C)

Procedure

Pipette into the test tubes the following solution:

t e s t t u b e s

components Isample

2standard

3blank

working sol.(ml) 1 1 1

serum(jjl) 10 - -

- 42 -

standard(pl) - 10 -

dist. water(pl) - - 10

20 min. incubation at 25 °C, reading absorbance at 520 nm

JS vi u_

Calculation

ASConcentr ations = ------------ x 2.29 (mmol/1)

ASt

S : Sample St: Standard A : Absorbance

- 43 -

Principle of dry chemistry diagnostic tests (measurement by Reflotron system)

After cleaning the measuring chamber and subsequently check the performance of the optical system:

1.Determination of cholesterol content in plasma2. Determination of HDL cholesterol content in plasma3. Determination of triglyceride content in plasma4. Calculation of LDL cholesterol from the values of cholesterol,triglycerides

and HDL cholesterol on the basis of the Friedewald formula (F3 key)5. Risk calculation according to -FRAMINGHAM- (F2 key)

In the -FRAMINGHAM- study performed in the USA, the incidence of myocardial infarction in dependence of defined risk factors was ascertained in 30- to 74-year old women and men.

Evaluation

The example shows an incidence of 12.5% (incidence of infarction in percent over 6 years). The minimum and maximum incidences (range) of the sex-related age group are listed for better risk assessment The individual risk is elevated by the factor 5.5 over the minimum incidence of the sex-related age group.

Test principle

Determination of cholesterol

cholesterolcholesterol ester ---------------------> cholesterol + RCOOH

esterase

cholesterolcholesterol + 02----------------------> cholestenon + H202

oxidase

94 4

PODINCID. RANGE MULT.12 .5% 2 .5%-26% 5 . 5

indicator (colorless) + H202 -------------> indicator (blue) + H20

- 45 -

Determination of triglyceride

esterasetriglyceride + 3 H20 --------------> glycerol + 3 RCOOH

GKglycerol + ATP -------------> L- a -glycerol phosphate + ADP

GPOL- a -glycerol phosphate + 02 --------------> hydroxyacetone phosphate + H202

PODindicator (colorless) + H202 ---------------> indicator (blue) + H20

GK:glycerol kinase GPO:glycerol-3-phosphate oxidase POD:peroxidase

Determination of HDL cholesterol

Precipitation of chylomicrons, VLDL and LDL by means of magnesium ions / dextran sulfate and subsequent determination of HDL cholesterol.

94 6

Reflotron optical system

Measurement of the reflectance, i.e. of the diffusely reflected light intensity, is performed by the Ulbricht sphere. Strictly selected light-emitting diodes (LEDs) with wavelengths centered on 567, 642 and 951 nm serve as light sources. Measurements can thus be taken at the wavelength that is most appropriate for the individual test. Two symmmetrical-positioned photodiodes serve as light detectors: a reference photodiode (Dp) and a measuring photodiode (D).

Diagrammatic illustration of the structure of Ulbricht sphere

Structure of Reflotron reagent carriers

Determination of serum bilirubin

The partial degradation of hem in human body starts with cytochrome P450 heme oxygenase in the RES cells. Oxidation of porphyrin ring results in biliverdin. In birds and reptiles this green coloured substance is the final product of hem degradation. In spite of the very poor solubility of bilirubin, mammals have been utilizing its advatage being a very effective antioxidant. Humans secrete bilirubin- diglucuronide into the bile. Further transformation of bilirubin occurs in the gut by bacteria.

In normal blood serum, bilirubin can be found in a low concentration. According to the reactivity in the "van den Bergh reaction" "direct" and "indirect" bilirubin levels can be determined. Direct bilirubin gives a direct colour reaction with diazo reagent, while indirect bilirubin reacts only in the presence of accelerators, such as caffeine or ethanol. Direct bilirubin is predominantly bilirubin-diglucuronide, a conjugate produced by the liver. Indirect bilirubin is the serum albumin bound form. In normal serum, bilirubin is found almost exclusively in the indirect form. One albumin molecule can bind and transport 2 molecules of bilirubin. The liver cells take up this bilirubin using a high capacity carrier system. Bilirubin level of newborns is physiologically high in the first two weeks following birth. In that case the yellow color of blood serum is originated almost exclusively from serum bilirubin, and directly measurable by spectrophotometer at 455 nm.

In the case of adults, high serum bilirubin level may indicate occlusion of the bile duct (direct bilirubin), haemolytic processes (indirect bilirubin) or liver damage (both direct and indirect forms).

Normal values in human serum

total bilirubin : 8.6-17 uM (0.5-1 mg%) direct bilirubin: 0.8-4.3 uM (0.05-0.3 mg%)

Principle

In the "van den Bergh reaction" bilirubin reacts with diazo-benzoyl sulfanilic acid forming an azo-compound (azorubin) which is red at neutral pH. Direct

Introduction - 4 7 -

- 48 -

bilirubin is determined without, while total bilirubin with the addition of caffeine to the reaction mixture.

Materials

- human serum (is to be kept in dark before the test. Best results are obtained with fresh serum. Hemolysed serum can not be used.)- sulfanilic acid solution (29 mM in 0.17 M HC1)- sodium nitrite solution (25 mM NaN02)- Na-benzoate + caffeine solution (0.52 M and 0.26 M, respectively)- K-Na-tartarate solution (0.93 M in 1.9 M NaOH)- physiological NaCl solution (0.85%)

Procedure

Transfer 0.2 ml aliquots of sulfanyl reagent to four test tubes (2 for samples + 2 for blank). Add 0.05-0.05 ml NaN02 solution to the two sample tubes.

A. Total bilirubin

Add 1 ml aliquots of caffeine reagent, then 0.2 ml aliquots of serum to a sample and a blank tube. Stir the contents thoroughly and incubate the tubes for 10 min at room temperature. Then add 1 ml tartarate solution to both tubes, mix well again, continue incubation for five more minutes. Measure absorbance of the sample at 580 nm using the blank as reference.

B. Direct bilirubin

Add 2 ml aliquots of physiological NaCl solution, then 0.2 ml aliquots of serum to the two remaining (sample and blank) tubes. Stir their content thoroughly and incubate them for 5 min at room temperature. Measure absorbance, using blank as reference, at 540 nm.

t e s t t u b e s

total bilirubin direct bilirubin

components (ml) sample blank sample blank

sulfanilic acid 0.2 0.2 0.2 0.2

NaN02 0.05 - 0.05 -

Na-benzoate + caffeine 1 1 - -

- 49 -

phys. NaCl - - 2 2

serum 0.2 0.2 0.2 0.2

incubation at 25 °C 10 min. 5 min.

tartarate 1 1 - -

incubation at 25 °C 5 min. -

reading absorbance 580 nm 540 nm

- 6 7 O t o m

Calculation

total bilirubin concentration (uM): 185xA58q (mg%): 10.8 x A580

direct bilirubin concentration (uM) : 246 x A540(mg%): 14.4 x A540 (9 Q( <{ db^kXUX CJ>

Determination of enzyme activity I.Fixed-time (Two-point) assay of the a-Amylase enzyme

Hydrated starch and glycogen are attacked by the endosaccharidase a-amylase, which is present in saliva and pancreatic juice. Amylase has specificity for internal a(l-4)-glucosidic bonds; a(l-6) bonds are not attacked, nor are a(l-4) bonds of glucose units that serve as branch points. The pancreatic isoenzyme is secreted in a large excess relative to starch intake and is more important than the salivary enzyme from a digestive point of view. The products of the digestion by a-amylase are mainly maltose (glucose-a( l-4)-glucose), maltotriose (glucose-a( l-4)-glucose-a(l- 4)-glucose) and a-limit dextrins containing on an average eight glucose units with one or more a( l-6)-glucosidic bonds.

Aim:Quantitative assay of a-amylase in serum by using Phadebas Amylase Test.

Clinical Use:Acute pancreatis is often characterized by high serum and urinary a-

amylase levels.Elevated levels may also occur in mumps, hepatic disorders, pancreatic

pseudocyst, impaired renal function (serum only), penetrating or perforating ulcer, peritonitis, intestinal obstruction, pancreatic carcinoma, after abdominal operations, and following opiate therapy.

In pancreatic insufficiency decreased levels may be found.

- 50 -

Principle of the procedure

This enzyme test use a fixed time for the reaction and expressed the enzyme activity as the amount of substrate transformed by a specified volume of serum under the particular conditions of the test. The substrate is a water-insoluble cross-linked starch polymer carrying a blue dye. It is hydrolyzed by a-amylase to form water- soluble blue fragments. The absorbance of the blue solution is a function of the a- amylase activity in the sample.

Caution: Use rubber gloves, please! 1. Pipette into two centrifuge tubes the

following solutions:

t e s t t u b e s

components 1blank

2sample

scrum(fil) - 200

dist. water(j.il) 200 -

dist. water(ml) 4 4

5 min. incubation at 37 °C

2. Pre-incubate the tubes for 5 minutes at 37 °C in water bath.3. Add one tablet to each tube - use forceps - immediately vortex for 10 seconds

and replace in water bath.4. Incubate by standing the tubes in a well stirred water bath at 37°C for

exactly 15 minutes.5. Stop the reaction exactly 15 minutes after tablet addition by adding 1.0 ml of

0.5 M sodium hydroxide into each tube. Vortex immediately.6. Centrifuge at > 1500 g for 5 minutes . Pipette the blue supernatant into a

cuvette.7. Measure the absorbance of the supernatant at 620 nm against distilled water

using a cuvette with 1 cm light path.

Calculation

Subtract the absorbance value of the blank from that of the sample and read thea-amylase activity in U/l from the standard curve. This gives directly the ct-

amylaseactivity in serum samples.

Normal valuesi

- 51 -

Reference values in serum: 70-300 U/l. (Linearity of the method: 35-

1000 U/l.)

- 52 -

0.4 0.5 0.S o v a n 0.91 2 3 4 5 S 7 H 9 10 20 3 4 5 8 7 » » 100 150(jh«UlPhadebas® Amylase Test

Absorbance 620 nm

Detcrmination of enzyme activity II.Kinetic assay (Multipoint, Continuous Monitoring) of Aspartate

Aminotransferase (ASAT, GOT) and Alanine Aminotransferase(ALAT, GPT) enzymes

Aspartate Aminotransferase (ASAT) is found in practically every tissue of the body, including red blood cells. It is in particularly high concentration in cardiac muscle and liver, intermediate in skeletal muscle and kidney, and in much lower concentrations in the other tissues.

The measurement of the serum ASAT level is helpful for the diagnosis and following of cases of myocardial infarction, hepatocellular- disease, and skeletal muscle disorders.

The concentration of Alanine Aminotransferase (ALAT) in tissues is not nearly as great as for ASAT. It is present in moderately high concentration in liver, but is low in cardiac and skeletal muscles and other tissues. Its use for clinical purposes is primarily for the diagnosis liver disease.

Aim:Quantitative assay of ASAT (GOT) [Aspartate Aminotransferase] or ALAT (GPT)

[Alanine Aminotransferase] in serum by using LJV kinetic method.

Principle of the procedure

Kinetic Assay: the rate of the enzyme reaction as a function of time is measured by incubating serum under specific reaction conditions and measuring the rate of change in substrate , cofactor, or product concentration. The assay of ASAT/ALAT is based on the following series of reactions:

ASATL-Aspartate + a-ketoglutarate —*------> Oxaloacetate + L-Glutamate

MDHOxaloacetate + NADH + H+ ----------> L-Malate + NAD+

or

ALATL-Alanine + a-ketoglutarate -----------> Pyruvate + L-Glutamate

- 54 -

- 51 - LDHPyruvate + NADH + H+ -----------> Lactate + NAD+

NDH: malate dehydrogenaseLDH: lactate dehydrogenase

The enzyme, aspartate aminotransferase (ASAT), reversibly transfers an amino group from aspartate to a-ketoglutarate and forms oxaloacetate in the process. This reaction is coupled with that of malate dehydrogenase, in which the oxaloacetate is reduced to malate as NADH is simultaneously oxidized to NAD+.

The enzyme that transfers an amino group from the amino acid alanine to a- ketoglutarate is alanine aminotransferase (ALAT).

The resulting decrease in absorbance is followed spectrophotometrically and is directly proportional to the activity of ASAT (or ALAT) in serum.

Reagent composition: Tris buffer, L-aspartate or L-alanine, a-ketoglutarate, MDH, LDH, NADH, stabilizers, non-reactive vehicle substances.

Test procedure^

Caution: Use rubber gloves, please!

Parameters of the method: Wavelength: 340 nm Light path: 1 cm Temperature: 25°C

Procedure

1. Pipette 1000 pi reagent solution and 100 pi serum into a cuvette.2. Mix, wait for 1 minute and measure the absorbance.3. Measure it again exactly 1, 2 and 3 minutes later and determine 5A/min.

Calculation

8A/min x 1745 = U/l ASAT (or ALAT) activity

Normal values: ALAT:male:

up to 22 U/l at 25 °C

- 5 2 -female: up to 17 U/l at 25°C

- 56 -

ASAT:male: up to 18 U/l at 25°Cfemale: up to 15 U/l at 25°C

- 57 -

Determination of serum calcium, potassium, and chloride ion

concentration

It is apparent that the concentration of inorganic ions (Na+, K+, Ca++, Mg++, CI", PO^" etc.) is regulated within very narrow limits in order maintain physiological osmotic

- 58 -

pressure, resting membrane potential, and the excitability of the cells. In the clinical laboratory inorganic ions are usually determined by using either flame photometry, colorimetry, refractometry, ion selective electrodes, or atomic absorption spectrophotometry. The subject of the practical is the determination of K+,

- 59 -

Ca++, and CI" by using refractometry, and colorimetry.

Hypokalemia (decreased K+

concentration in the plasma) develops mostly as a consequence of diuretic and insulin treatment, excess salt loss due to sweating or diarrhoea, Cushing-syndrome, Conn syndrome, and metabolic alkalosis. The most frequently occurring symptoms of

- 60 -

hypokalemia are muscle weakness, cardiac arrhythmias, cardiac arrest, hypotension. Decreased mineralocorticoid activity due to Addison's disease or spironolactone treatment, metabolic acidosis, chronic renal failure etc. lead to an increase in extracellular K+ concentration i.e. hyperkalemia. The most characteristic

- 61 -

symptoms of hyperkalemia are cardiac arrhythmias, smooth muscle spasm.

Pathological changes in the plasma chloridc concentration usually occur with parallel changes in Na+

content in severe salt or water loss/loading.

The calcium in the plasma is present in three different foxms. Approximately 41% of the total

- 62 -

calcium is combined with the plasma proteins, and in this form is undiffusible through the capillary membranes. About 9% of the calcium is diffusible, however, it is combined with phosphate, citrate, oxalate etc. in such a manner that it is not ionized. The remaining 50% of the calcium is both diffusible through the capillary membranes and ionized, therefore this

- 63 -

fraction is considered as active calcium. Hypocalcemia occurs in alkalosis, deteriorated vitamin-D metabolism, insufficient parathyroid hormone activity, excess oxalate intake, and steatoxThoea. Hypocalcemia results in tetanic muscle contractions, insufficient cardiac functions, hypotension, and obstipation. A 50% decrease in plasma Ca++ level is life

- 64 -

threatening. Hypercalcemia may develop as a result of D-hypervitaminosis, increased level of parathyroid and growth hormones, decreased activity of calcitonin. The symptoms of hypercalcemia are characterized by mental depression, decxeased reflex activity, cardiac arrhythmias. A two fold increase in plasma Ca++ content may lead to

- 65 -

precipitation of calcium phosphate throughout the body.

Determination of K+ by

refractometry ("Reflotron"

system) Test principlePotassium ion binds to the K+

selective ionophore valinomycin, than

- 66 -

the complex binds to the anion of an acid-base indicator. The anion of the dissociated indicator is a colourful compound.

K+ + valinomycin + ind-H = [valinomycin-K]+ [indj" + H+

No

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rmal value

- 68 -

s: plasma:

- 69 -

3.5-4.6 mm

- 70 -

ol/1 serum

- 71 -

:3.6-5.0 m

- 72 -

mol/1The plasma or the serum should

be separated from the cells within one hour to avoid the release of intracellular K+

- 73 -

Determination of Ca++ by colorimetry

Test principle

The calcium ion forms a deep purple complex with o-cresolphthaleine in alkaline pH. Magnesium ions (up to 4 mmol/1) which may interfere with the reaction

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are bond with 8-hydroxy-quinoline. The linearity of the assay extends up to 3.25 mmol/1 Ca++

Reagents

1.reagent: 360 mmol/1 diethylamine

2. reagent: 0.15 mmol/1 phthalein purple, 17.2

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mmol/1 8-hydroxy-quinoline Standard: 2.5 mmol/1 Ca++

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Procedure

Ten pi of distilled water, standard, or sample is added to 1 ml of reagent mixture (1:1), respectively. Absorbance is measured in 1 cm narrow cuvette using 570 nmlighL S

reagent control standard sample

1ml reagent

lOpl dist water

1 ml reagent

lOpl standard

1 ml reagent

lOpl sample

[Ca++sample] - (Asampie/Astanciarcj) x [Ca++

Stancjar{j] Normal value: serum, plasma:

2.2-2.55 mmol/1

Determination of CI" by colorimetry

Test principle

Chloride releases rhodanide from mercury-rhodanide, and forms mercury- chloride. Rhodanide forms a red complex with Fe^+.

2 CI" + Hg(SCN)2---------> HgCl2 + 2 SCN"

Fe3+ + 3 SCN"---------> Fe(SCN)3

Normal value: 95-105 mmol/I

Procedure

blank standard sample

2ml reagent

20pl dist. water

2ml reagent

20pl standard

2ml reagent

20pl sample

Ten minutes of incubation period (20-25 °C) is needed for the complete reaction. Subsequently, absorbance is measured using 1 cm cuvette and 457 nm light.

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[crsampleJ ~ (^sample^standard) x l^l

standard J (standard: 100 mM CI")Enzymatic determination of blood glucose level

Carbohydrates are primarily functioning in metabolism as a fuel to be oxidized and provide energy for other metabolic processes. In this role, carbohydrates are utilized by cells mainly in form of glucose. Maintenance of constant glucose level in blood is controlled by neurohormonal action.

A number of methods using colored product with glucose have been described (iodometrical method, with o-toluidine). Enzymatic determination is the most specific method in clinical use nowadays because other reducing hexoses can not alter the results.

Principles

Glucose oxidase catalyses the oxidation of nonphosphorylated glucose to glucono-l,5-lactone with the formation of H2O2. In the presence of peroxidase H2O2 is converted to oxygen free radicals and these produce a color compound (4-p- Benzoquinone-monoimino-phenazone) with phenol and 4-amino antipyrine.

glucose-oxidaseglucose + O2 + H2O -------------------------> glucono-l,5-lactone + H2O2

peroxidase

2H2O2 + phenol + 4-amino antipyrine---------------> color compound + 4H2O

Normal values:

Serum:

3.9-5.8 mmol/1 (70-105 mg/dl; 0.7-1.05 g/1)Liquor:

2.8-3.9 mmol/1 (50-70mg/l)

Materials

Trinder reagent (diluted 1+2 in distilled water)Standard solution

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Sample (serum or liquor)Distilled water

Procedure

Components(ml) test tubes

1 2 3

Blank Sample Standard

Trinder reagent 2 2 2

Dist. water 20 - -

Standard - - 20

Sample - 20 -

Mix and incubate for 15 min. at 37aC.

Measure the absorbance at 520 nm against blank. Reaction dye is stable for 1

h. Calculation^sample

concentration sample (mmol/1) = ----------------- x concentration standard^standard

Assay of glycosylated haemoglobin by DCA 2000™ HbA|c system

The term "glycosylated haemoglobin" refers collectively to a series of stable compounds that are formed between haemoglobin and sugars. Their concentrations are increased within erythrocytes of patients with diabetes mellitus. The aim of therapy by clinicians is to maintain their patients' glucose levels at constant normal or near normal levels in blood. Measurement of serum or plasma glucose levels by laboratory or home monitoring techniques, gives the measure of glucose regulation over very short time period. It has been found, however, that a measure of long term blood glucose control can be achieved by measuring the glycosylated haemoglobin fractions of erythrocytes.

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Principle

The DCA 2000™ System relies on an immunochemical technique for the measurement of HbA|c. A monoclonal antibody has been developed which reacts specifically with certain amino acid sequences following the N-terminal valine on b- chain of HbAic. The antibody doesn't binds the haemoglobin glycated at other positions, because both glucose and specific acid sequences must be present for binding. An agglutinator present as one of reagents causes agglutination of specific anti-HbA|c coated latex particles. This is measured as an increase in turbidity by the spectrophotometer. HbAjc present in the patient's sample causes an inhibition of the agglutination in proportion to its concentration in the blood.

The simplified reaction is described as follows:

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Assay Principle Inhibition Of Latex Agglutination

(Antibody-

Latex) <>

(Agglutinat

or) £r

— H i g h

Scattering (Increased Absortiance)

« A I »

Hemoglobin A1c- - -< ► in Patient's Blood yJJ --------------------------—L o w Scattering

O Inhibition k,C>T (Decreased Absortance)

Agglutination Inhibited

DCA 2000™ HbA1c Reagent

Cartridge

•eraAgglutination

- 81 -

BufferSolution Tray with Foil Seal (600 ¿xL)

[P Agglutinator

Antibody Latex

Optical Window

1 fiL Blood Sample

Cartridge Removal Tab

Absorbant(Picks up all liquid at test end)

Capillary Holder

Pull-Tab(Pull to release buffer from tray)

Oxidant

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The assay sequence is shown as follows.

2.

Capillary holder inserted into cartridge.

Cartridge inserted into instrument. Pull-tab pulled and discarded. Buffer

solution flows to read area of the cartridge where a "blank" reading is obtained.

5 .

1. Sample collected in capillary.

Optical window

o

-

C

\J.M.

Cartridge rotates to mixantibody- latex and agglutinator.

4. Cartridge rotates to mix blood

sample and

ferricyanide.

7. Cartridge rotates to read position where HbAic

reading is obtained.

Cartridge rotates to read position where total hemoglobin reading is taken.