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Upshots of Anti-Diuretic Hormone

A Laboratory Experiment
Presented to
Ms. Annlyn B. Sanchez, RMT, MLS (ASCPi)

by

Alegria, Neil Jenkin M.

Casañas, Dennielle G.
De Jesus, Earl James Leonard P.
Lopez, Katrina May E.
Lee, Kristine Joy G.
Rabe, Yasmin Clarice C.
July 2018
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INTRODUCTION

Water is considered as one of the fundamental necessity that man needs

in order to survive. It is the solvent for all processes in the human body. It

transport nutrient to cells, determines cell volume by its transport into and out

of the cells, removes waste products by ways of urine, and acts as the body’s

coolant by ways of sweating (Bishop, Fody, & Schoeff, 2010). Furthermore, the

distribution of water in both intracellular and extracellular compartment makes

it convenient in managing the body’s electrolyte concentration and blood

pressure and promoting homeostasis by regulation of the antidiuretic hormone.

Arginine vasopressin hormone (AVP), formerly called antidiuretic

hormone (ADH) is synthesized in the neurons of the supraoptic nuclei of the

hypothalamus and stored and secreted by the posterior pituitary gland. The

primary function of AVP in the body is to regulate extracellular fluid volume. It

acts on the renal collecting ducts to increase water permeability which leads to

decreased urine formation. An increase of 1% to 2% in osmolality causes a

fourfold increase in the circulating concentration of AVP, and a 1% to 2%

decrease in the osmolality shuts off AVP production (Bishop, Fody, & Schoeff,

2010). The second function of the AVP is vasoconstriction. It binds to V 1 receptor

on the vascular smooth muscle to cause vasoconstriction through the inositol

triphosphate signal transduction pathway which increases arterial pressure

(Ouden, DT, & and Meinders, 2005).

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OBJECTIVES

The students seek to accomplish the following objectives:

1. To determine the blood urea nitrogen of the patient

2. To know the patient’s serum electrolytes, specifically sodium,

potassium and chloride.

3. To be able to provide the urine electrolytes, specifically sodium,

potassium and chloride of the patient

4. To identify the serum osmolality of the sample

5. To be able to provide an accurate urine glucose and specific gravity

of the patient’s sample

6. To make a comparison between the patient’s values and the

reference range

7. To correlate the results of the tests done with the fluctuation, or

lack thereof, of ADH in the body

8. To determine any discrepancies that may arise in the patient

sample
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METHODOLOGY

I. Blood Urea Nitrogen

1. Pipette 1.5 ml of BUN Enzyme Reagent into labelled test tubes. Allow to
equilibrate to room temperature.

2. Add 0.010 ml of each sample to tis respective tube. Mix gently.

3. Incubate for five minutes at 37oC

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4. Add 1.5 ml BUN Color Developer. Mix gently.

5. Incubate for 5 minutes at 37oC.

6. Zero Spectrophotometer with the reagent blank at 580nm - 630nm.

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7. Read and record absorbance of samples of all tubes.

II. Blood Na, K, Cl/ Urine Na, K, Cl

1. Prepare 5ml of blood and place it in a non-coagulant tube.

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2. Dilute the sample with the ratio 1:10 for urine, while for blood let the
serum be the sample.

3. Place the sample under the tube in the electrolyte machine.

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4. Read the result.

Serum:

Urine:
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III. SERUM OSMOLALITY

Computation of serum osmolality by using the values obtained from procedure A

and B

IV. URINE GLUCOSE, SPECIFIC GRAVITY

1. Collect urine sample to a sterile container

2. Transfer urine sample to a test tube

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3. Dip totally but briefly the reagent strip to the urine. Blot the reagent strip
to a tissue to absorb the excess urine to prevent discrepancies to the
results

4. Compare the results from the chart attached to the container of the
reagent strip
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RESULTS AND DISCUSSION

I. Blood urea nitrogen

Urea is a nonprotein nitrogen (NPN) compound present in highest

concentrations in the blood and contributes to the blood’s osmolality (Bishop,

Fody, & Schoeff, 2013). For this experiment, it is determined by using the BUN

colorimetric method using the Berthelot reaction. In this reaction, urease

hydrloyzes urea converting it to ammonium ion then reacts with salicylate,

sodium nitroprusside, and hypochlorite that yields a blue-green complex with

intensity that is proportional to urea concentration in the sample (Pointe

Scientific, Inc.)

The absorbance of the unknown was divided by the absorbance of

the standard then it is multiplied by the concentration of the standard which was

20mg/dL. The resulting urea-nitrogen concentration of the patient was below

the normal range which was (indicate value), (indicate normal range). The low

concentration of BUN may be due to the patient’s diet in which there are not

enough nutrients in the food he’s taking or maybe due to overhydration or

excess fluid intake.

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II. Blood Na, K, Cl/ Urine Na, K, Cl

In our body, water makes up most of our body mass. Water serves

as an essential carrier for different metabolites and nutrients. Fluid balance in

our body are maintained and regulated by hormones and electrolytes,

specifically sodium, potassium and chloride.

Sodium is the one responsible for the homeostasis of the

intravascular and interstitial volumes in our body, and also influences

extracellular fluid volume. Secretion of this electrolyte can be seen through

urine, but may also be possible through sweat and feces. In this experiment the

male patient resulted to have a serum sodium of 142.5 which drops in the

normal range of serum sodium which is 135-145 milliequivalents per liter, but its

urine sodium is quite low which is 34.2 mEq/L per day which may be due to the

preparation of the tube inserted at the easy lite. Chloride is the major anion of

the extracellular fluid. It usually parallels that of sodium, but external losses and

excretion can occur independently, mainly in equilibrium with bicarbonate

status. The chloride of the patient serum was 98.5 (96-106 mEq/L normal value)

and 25.6 (25-40 mEq/L normal value), which indicates that the patient may have

a normal dietary intake of chloride in its daily living. Lastly, potassium is the

major intracellular cation, and the pool that correlates well with the lean body

mass. In this experiment, 4.55 mEq/L of potassium was determined which lies

down the normal value which is 3.5-5.5mEq/L and its urine potassium 16.7 also
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is said to be below normal (<20mEq/mL low potassium level) and suggests that

the patient has a poor dietary intake of food rich in potassium (Balci, 2013).

III. Serum osmolality

Serum osmolality is an essential laboratory parameter to

understand several clinical disorders such as electrolyte disturbances, exogenous

intoxication and hydration status. The measurement of serum osmolality is an

important evaluation and decision-making laboratory tool in cases of

hydroelectrolytic imbalance. Osmolality is the measure of the number of

osmoles of solute per kilogram of water. Usually, osmolal concentration is

expressed in milli-osmoles per kilogram of water (mOsm/KgH2O). The osmolality

of a solution does not depend on the nature of the particle, but on the number

of the dispersed particles (Faria 2017). In this experiment the following formula

was followed to determine the serum osmolality of the patient:

Serum osmolality = 279.45

With the provided computations and results, the patient’s serum

osmolality is said to be normal, since the normal value was 285-329 mmol/L.
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IV. Urine Glucose and Specific Gravity

The patient’s urine showed negative in glucose and a favorable

urine specific gravity upon testing using the reagent strip.

The glucose test is designed to detect the presence of glucose in

urine by color changes of reagent-treated strip (King M.D. & Hainline Jr. Ph.D.) It

utilizes the principle of a double sequential enzyme reaction which is produced

when the testing area is impregnated with the mixture of glucose oxidase,

peroxidase, chromogen, and a buffer. Glucose oxidase serves as a catalyst in the

reaction between glucose and oxygen. The reaction produces gluconic acid and

peroxide. The peroxidase acts on the reaction between peroxide and chromogen

that yields an oxidized colored chromogen that is directly proportional to the

concentration of glucose in the urine. (Strasinger & Di Lorenzo, 2014). For the

urine tested in the experiment, it did not react with the test for glucose on the

reagent strip. No color change occurred indicating that there were no

concentrations of glucose in the urine.

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Specific gravity is also part of the test in the reagent strip. Its

principle is based on the change in the dissociation constant (pKa) of a

polyelectrolyte in an alkaline medium. An increase in specific gravity changes the

indicator blue (1.000 [alkaline]) to shades of green to yellow (1.030 [acid])

(Strasinger & Di Lorenzo, 2014).

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BIBLIOGRAPHY

(n.d.). Retrieved July 17, 2018, from Pointe Scientific, Inc.:

http://www.pointescientific.com/uploads/inserts/B7551-01-891.pdf

Balci, A. (2013). Characteristics of patients with electrolyte imbalance. Retrieved

from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4129840/

Bishop, M. L., Fody, E. P., & Schoeff, L. E. (2013). Clinical Chemistry Principles,

Techniques, and Correlations. Philadelphia: Lippincott Williams & Wilkins.

Faria, D. (2017). Measurement of serum osmolality. Retrieved from

http://www.scielo.br/pdf/jbpml/v53n1/1676-2444-jbpml-53-01-0038.pdf

King M.D., J. W., & Hainline Jr. Ph.D., A. (n.d.). COMMERCIAL GLUCOSE OXIDASE

PREPARATIONS.

Strasinger, S. K., & Di Lorenzo, m. S. (2014). Urinalysis and Body Fluids.

Philadelphia: F.A. DAVIS COMPANY.