BIOCHEMISTRY - Laboratory Practical - NAU 2015

MODULE V

HORMONAL REGULATION OF METABOLISM

Laboratory Work 13

DETECTION OF HORMONES IN MODEL SOLUTIONS

Objective: to confirm the presence of Hormones with various chemical structures in model solutions.

Basic Theoretical Background

Hormones are BIOLOGICALLY ACTIVE SUBSTANCES produced by living organisms that regulate metabolic processes and physiological Functions at extremely low concentrations. Hormones are classified into true (or classical) hormones, which are secreted into the bloodstream and affect target Organs at a distance, and tissue hormones (or histohormones), which are secreted in and affect the same Tissues where they are produced. Chemically, true hormones are divided into protein-Peptide Hormones, Amino Acid Derivatives, and Steroid Hormones. Most tissue hormones are derivatives of arachidonic (eicosatetraenoic) acid, as well as amino acid derivatives.

THYROID HORMONES—derivatives of the Amino Acids Phenylalanine and Tyrosine, namely triiodothyronine (T₃) and tetraiodothyronine or thyroxine (T₄)—are synthesized by The Thyroid Gland. Their Structure incorporates three or four iodine atoms, respectively. These hormones affect METABOLISM/26.html">Energy Metabolism (acting as uncouplers of Respiration and phosphorylation), and activate Protein Biosynthesis and morphogenesis (stimulating the formation and development of organs).

Hormones of The adrenal medulla—adrenaline (epinephrine) and noradrenaline (norepinephrine)—are also derivatives of phenylalanine and tyrosine. Adrenaline increases Heart rate and force, raises Blood pressure, and affects the Muscle tone of the gastrointestinal tract, eyes, etc. Noradrenaline functions primarily as a neurotransmitter.

Insulin is a protein hormone produced by the Pancreas. It consists of 51 amino acid residues and contains 2 polypeptide chains: chain A with 21 amino acid residues and chain B with 30 amino acid residues. The chains are interconnected by two Disulfide Bonds, and an additional disulfide bond is located within chain A. The primary Biological Role of insulin is to lower blood glucose levels.

Steroid hormones are derivatives of sterane (cyclopentanoperhydrophenanthrene); Cholesterol serves as the precursor for their synthesis in the body:

 sterane     cholesterol

Steroid hormones include corticosteroids (Adrenal Cortex Hormones—mineralocorticoids and glucocorticoids), Sex Hormones (estrogens, androgens), and calcitriol (a derivative of vitamin D₃, a hormone-like compound involved in calcium Metabolism regulation). Steroid hormones differ in their substituents at the 10th, 13th, and 17th carbon atoms. Corticosteroids contain methyl radicals at the 10th and 13th atoms, and an ethyl radical at the 17th atom (a total of 21 carbon atoms, referred to as C₂₁-Steroids). Androgens lack the ethyl radical at the 17th atom (C₁₉-steroids). Estrogens have only a single methyl radical at the 13th atom (C₁₈-steroids). Steroid hormones contain hydroxyl and keto groups at various carbon positions. For example, androsterone and estrone (folliculin) have a keto group at position 17 and a hydroxyl group at position 3. Steroid hormones are excreted from the body in the urine as 17-ketosteroids, at a rate of 1–25 mg per day, depending on AGE AND SEX, with maximum excretion observed at age 25. Under stress conditions, the levels of 17-ketosteroids in blood and urine increase.

Equipment: test tube rack, pipettes, Glass stirring rods, dropper bottles, Water bath, fluorometer.

13.1. Qualitative reactions for Thyroid Hormones

Materials and Reagents: thyreoidin, concentrated HNO₃,
10% potassium iodate solution KIO₃, chloroform, 10% potassium bicarbonate solution KHCO₃, 1% starch solution, 10% sulfuric acid solution.

Procedure

During the acid Hydrolysis of thyroid hormones, hydriodic acid is formed, which reacts with potassium iodate to release free iodine:

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Iodine in chloroform exhibits a violet coloration. Place a few crystals of thyreoidin into a test tube, add 10 drops of concentrated nitric acid, and heat in a water bath for 3–5 minutes. Then add 20 drops of 10% potassium iodate solution. Mix the contents and allow to cool.

Add 15 drops of chloroform to the test tube, shake, and observe The Development of coloration.

During the alkaline hydrolysis of thyroid hormones (boiling with KHCO₃), KI is formed. The reaction between iodide and iodate is a redox process: potassium iodide (KI) acts as a reducing agent, while potassium iodate (KIO₃) acts as an oxidizing agent. The released iodine is detected using starch (resulting in a blue coloration) in an acidic medium:

10 tablets of thyroidin are placed in a mortar and thoroughly ground. The ground mass is transferred to a hydrolysis flask, and 20 ml of 10% sodium bicarbonate solution is added. The flask, equipped with a reflux condenser, is placed on an asbestos wire gauze, and the contents are boiled for 15 minutes (from the moment of boiling) under moderate heating.

25 drops of the cooled hydrolyzate are measured into a test tube, 3 drops of 1% starch solution are added, followed by 4 drops of 10% potassium iodate solution and approximately 10—15 drops of 10% sulfuric acid until discoloration occurs, and further until the appearance of a blue color resulting from the reaction of the released free iodine with starch.

13.2. Color Reactions for Insulin

Materials and Reagents: insulin solution (insulin preparation in ampoules), 0.1 M ninhydrin solution, concentrated nitric acid, ammonia, Folin's reagent, Millon's reagent, concentrated sodium hydroxide solution, 10% sodium (or potassium) hydroxide solution,
1% copper sulfate solution, 20% sulfosalicylic acid (SSA) solution.

Procedure

5 drops of injectable insulin are added to each of five test tubes, and the Folin, Millon, ninhydrin, xanthoproteic, and biuret reactions are carried out (see Laboratory Works 1, 2).

The protein precipitation reaction using SSA is highly sensitive. 1 drop of SSA solution is added to a test tube containing the insulin solution (10—20 drops), and The formation of a white precipitate is observed.

13.3. Color Reactions for Adrenaline

Materials and Reagents: 0.1% adrenaline solution (adrenaline preparation in ampoules), 1% iron(III) chloride solution, 25% ammonia solution, 1% sulfanilic acid solution, 5% sodium nitrite solution, 10% sodium carbonate solution, 0.2% potassium hexacyanoferrate(III) solution, crystalline ascorbic acid,
10% sodium hydroxide solution, 10% potassium iodate solution KIO₃, 10% acetic or phosphoric acid solution, 5% Hydrochloric acid solution, concentrated hydrochloric acid, nitrite-molybdate reagent (5 g of sodium nitrite and 5 g of sodium molybdate dissolved in 50 ml of distilled water).

Procedure

Reaction for adrenaline with iron(III) chloride

Adrenaline readily undergoes oxidation-reduction reactions. The hormone and its oxidation products can be detected using color reactions. With iron(III) chloride solution, adrenaline and noradrenaline form an emerald-green complex compound. Upon The addition of a drop of ammonia solution, the color turns cherry-red, and then orange-red.

10 drops of 0.1% adrenaline solution and 1 drop of 1% iron(III) chloride solution are placed in a test tube. Then, 1 drop of ammonia is added. The appearance and change of color are observed.

Reaction for adrenaline with diazobenzenesulfonic acid

The method is based on the ability of adrenaline to form a red compound with diazobenzenesulfonic acid.

To prepare diazobenzenesulfonic acid, 3 drops of 1% sulfanilic acid solution and 3 drops of 5% sodium nitrite solution are measured into a test tube and mixed by shaking. Then, 5 drops of 0.1% adrenaline solution and 3 drops of 10% sodium carbonate solution are added to the test tube. The contents are mixed by shaking, and the color development is observed.

Reaction for adrenaline with potassium iodate

In an acidic medium, adrenaline reacts with potassium iodate to form a red-violet compound.

1 drop of 0.1% adrenaline solution and 5 ml of water are placed in a test tube. 0.5 ml of the diluted adrenaline solution is transferred to another test tube, 1 ml of KIO₃ solution and 10 drops of acetic or phosphoric acid solution are added, and the mixture is heated to 60 — 65 °C. The appearance of color is observed.

Reaction for adrenaline with nitrite-molybdate reagent

In an acidic medium, adrenaline interacts with the nitrite-molybdate reagent to form a yellow-orange compound. Upon the Addition of an alkali, the color changes to raspberry-red, which turns lemon-yellow in the presence of hydrochloric acid.

5 drops of 0.1% adrenaline solution are placed in a test tube, followed by 5 drops of 5% hydrochloric acid solution and 5 drops of the nitrite-molybdate reagent. The mixture is mixed, and the appearance of color is observed. Then, 1 drop of 10% sodium hydroxide solution is added, mixed, and 1—2 drops of concentrated hydrochloric acid are added, observing the color changes at each step of the experiment.

Determination of adrenaline via fluorescence of its oxidation products

Adrenaline is oxidized under The Influence of alkalis to form compounds characterized by green fluorescence.

Add 0.5 ml of distilled water, 5 drops of 10% sodium hydroxide solution, and 5 drops of 0.1% adrenaline solution to a test tube, and mix. Observe the appearance of fluorescence using a fluorometer.

Determination of adrenaline via the formation of its fluorescent oxidation product, adrenolutin

Adrenaline is oxidized by potassium hexacyanoferrate(III) into adrenochrome, which in an alkaline medium forms adrenolutin, exhibiting a yellow-green fluorescence:

adrenaline    adrenochrome  adrenolutin

Dispense 1 drop of 0.1% adrenaline solution into each of two test tubes, then add 5 ml of water to the first and 10 ml to the second, and mix. Transfer 2 ml of these diluted adrenaline solutions into two separate test tubes, add 0.3 ml of 0.2% potassium hexacyanoferrate(III) solution to each, and let stand for 5 min (during which adrenaline is oxidized to adrenochrome). To each test tube, add a spatula tip of crystalline ascorbic acid and 2 ml of 10% sodium hydroxide solution (ascorbic acid prevents further oxidation of adrenochrome, while sodium hydroxide promotes its conversion to adrenolutin). Place the test tubes in the fluorimeter rack and compare the fluorescence intensity of the samples.

13.4. Formation of Foliculin Phenolate

Materials and reagents: 1% alcoholic solution of foliculin,
30% NaOH solution.

Procedure

Estrone (foliculin) contains a phenolic hydroxyl group. In an alkaline medium, it forms water-soluble foliculin phenolate.

Pour 0.5 ml of alcoholic foliculin solution into each of two dry test tubes. Add 1 ml of water to the first and observe the formation of a foliculin emulsion (the solution becomes turbid). Add 1 ml of 30% NaOH solution to the second test tube — no turbidity appears because water-soluble foliculin phenolate is formed in the alkaline medium.

13.5. Qualitative test for 11-dehydro-17-oxycorticosterone (cortisone)

Materials and reagents: 1% alcoholic solution of cortisone (cortisone acetate), Fehling's reagent (see Laboratory Work 2).

Procedure

Cortisone can reduce Cu ⁺² to Cu ⁺¹, which allows its detection via the reaction with Fehling's reagent.

Add 1 ml of Fehling's reagent to 1 ml of 1% alcoholic cortisone solution (cortisone acetate) and heat in a water bath (80 —
90 °C). Observe the formation of a brick-red copper(I) oxide precipitate.

13.6. Qualitative tests for 17-ketosteroids

Materials and reagents: 2% alcoholic solution of m-dinitrobenzene, 30% sodium hydroxide solution, 1% alcoholic solution of estrone, 1% alcoholic solution of androstane, urine.

Procedure

In an alkaline medium, 17-ketosteroids react with m-dinitrobenzene to form violet-pink to cherry-red Condensation products with a Light absorption maximum at a wavelength of 530 nm. The color intensity is proportional to The amount of 17-ketosteroids in the urine.

Add 5 drops of 1% alcoholic foliculin solution to the first test tube, 5 drops of 1% alcoholic androstane solution to the second, and 5 drops of urine to the third. Add 5 drops of 30% sodium hydroxide solution and 5 drops of 2% alcoholic m-dinitrobenzene solution to each test tube. Shake the tubes and observe the development of color after 2–3 min.

Processing of experimental data

Construct a table listing the hormones known to you; indicate their biological role, chemical nature, and the endocrine gland that produces them.

Draw a Conclusion regarding the presence of specific amino acid residues in the insulin molecule. Compare the Methods for detecting adrenaline.

Selection/5.html">Control Questions and tasks

1. How are hormones classified according to their chemical structure? Give Examples.

2. Which hormones are referred to as true hormones? Which Endocrine glands secrete them?

3. What is the MECHANISM OF ACTION of lipid-derived and thyroid hormones?

4. What is The Mechanism of Action of Protein, peptide, and amino acid-derived hormones?

5. Outline the cascade mechanism of hormonal enzyme activation involving cyclic AMP (using Glycogen phosphorylase as an example).

6. What are tissue hormones (histohormones)? What is their chemical nature?

7. Name the iodine-containing thyroid hormones. Write the formula of The amino acid from which they are synthesized.

8. Define enolic hydroxyl and phenolic hydroxyl. Write the reaction of phenol with FeCl₃. Which hormone contains a phenolic hydroxyl group?

9. Explain THE PRINCIPLE OF qualitative reactions for adrenaline.

10. What are 17-ketosteroids? How can they be detected in biological fluids?

11. How can protein hormones be detected in solutions?

12. Write the structural formulas of sterane and cholesterol.

13. How do corticosteroids and sex hormones differ structurally?

14. What Structural Features of folliculin allow it to be detected via qualitative reactions?

References: [1; 4—7].