Urinalysis of four urine samples

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Urinalysis Practical

Background:

As you have learned, the urinary system performs many vital functions in the body including:

  1. Regulating blood volume and pressure by regulating water excretion,
  2. Regulating plasma ion/solute concentrations by adjusting urine composition,
  3. Assisting blood pH stabilisation,
  4. Removing nitrogenous waste,
  5. Conserving water and important nutrients and
  6. Assisting the liver in detoxifying poisons.

Therefore, analysing a sample of urine from a person can provide important information on the health of that person. Urinalysis can reveal diseases such as diabetes mellitus, urinary tract infections and renal (kidney) infections such as glomeronephritis and kidney stones (renal calculi).

A medical professional may perform a urinalysis for several reasons:

  1. As a general health check-up,
  2. Diagnosing metabolic or systemic diseases that affect renal function (heart failure will lead to decreased blood flow to the kidneys, pre-eclampsia during pregnancy will lead to increased protein in the urine),
  3. Diagnosis of endocrine disorders e.g. infertility (low levels of FSH and/or LH),
  4. Diagnosis of urinary system disease,
  5. Monitoring of glucose levels in patients with diabetes,
  6. Testing for pregnancy (hCG levels secreted by the embryo),
  7. Screening for drug use.
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Urinalysis is a technique involving physical, chemical and microscopic analyses of a sample of urine.

  1. Physical parameters: Normal urine is a clear yellow colour due to the presence of uribilin. Abnormal urine may be dark orange, red or brown and cloudy in appearance. This can be due to the presence of red and/or white blood cells or pigments and may indicate a urinary tract or renal infection or disease, liver or gall bladder disease. Normal urine has a specific gravity of between 1.002 – 1.028 (this is a measure of the number of particles/solutes in the urine, its concentration). A urine sample that has an elevated specific gravity can indicate dehydration, diarrhea/vomiting, glucosuria, inappropriate ADH secretion. A diminished specific gravity may indicate such diseases as renal failure or pyelonephritis.
  1. Chemical parameters: The chemical analysis of urine is routinely performed using an inexpensive and relatively accurate dipstick test (Uristix from Bayer or other brands). The test uses a reagent-coated plastic stick that is placed or dipped into the urine sample. The reagent areas change colour according to the presence of glucose and/or protein.

(a)G10_42b.jpg

Figure 1. Colour chart (a) for determination of glucose and/or protein.

The glucose test on the dipstick is based on a double sequential enzyme reaction. One enzyme, glucose oxidase, catalyses the formation of gluconic acid and hydrogen peroxide from the oxidation of glucose (if present in the urine). A second enzyme, peroxidase, catalyses the reaction of hydrogen peroxide with a potassium iodide chromogen to oxidise the chromogen to colours ranging from green to brown. Normal urine has less than 0.1% glucose concentration.

The protein test on the dipstick is based on the protein-error-of-indicators principle. At a constant pH, the development of any green colour is due to the presence of protein. Colours range from yellow for ‘negative’ through yellow-green and green to green-blue for ‘positive’ reactions. Normal urine has a protein concentration of less than 100 µg/ml. Although the dipstick test is semi-quantitative, significantly more accurate levels of glucose and protein can be determined by other means. In this practical you will use a BCA Assay (discussed later) to quantify the amount of protein present in a sample of urine.

Urinalysis may also include assaying for levels of ketones (an indicator of diabetic ketosis, fasting or starvation), blood cells (indicating infection or kidney stones), bilirubin (liver or gall bladder disease), drugs and many other substances.

  1. Microscopic parameters: The urine sample can also be analysed by a microscope, often after staining to reveal any pathogens such as bacteria, urine crystals, cells and/or mucous. The presence of any of these may indicate infection or disease and further medical investigation will provide a thorough diagnosis.
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Aim:

The aim of this practical is to perform glucose and protein urinalysis techniques on five samples of ‘urine’ provided by five ‘patients’ and use this information to provide an initial diagnosis for each patient.

Part One: Using Dipsticks To Provide A Qualitative Measure of Protein And/Or Glucose.

Materials:

  1. 5 samples of urine labelled A – E (these will be required for Parts One and Two),
  2. 5 Uristix dipsticks.

Method:

  1. Perform a basic physical analysis of the urine samples noting the colour and cloudiness of each sample:

Urine A

Urine B

Urine C

Urine D

Urine E

Colour

Cloudiness

  1. Immerse a dipstick into each of the samples, wait 60 seconds and record your results using the colour chart in Figure 1 to determine if the sample contains glucose and/or protein or neither substance:

Urine A

Urine B

Urine C

Urine D

Urine E

Glucose

Protein

Ketones

Negative

Negative

Negative

Negative

Strongly positive

Blood

Negative

Negative

Trace

Negative

Negative

Part Two: Using A Commercial BCA Assay To Provide A Quantitative Measure of Protein.

Background:

The BCA Protein Assay exploits the chemical reduction of Cu2+ to Cu1+ by protein in an alkaline medium with the selective colorimetric detection of the cuprous cation (Cu1+) by bicinchoninic acid (BCA). The first step is the chelation of copper with protein in an alkaline environment to form a blue coloured complex. In this reaction, known as the biuret reaction, peptides containing three or more amino acid residues form a coloured chelate complex with cupric ions in an alkaline environment containing sodium potassium tartrate. Single amino acids and dipeptides do not give the biuret reaction, but tripeptides and larger polypeptides or proteins will react to produce the light blue to violet complex that absorbs light at 540 nm.

In the second step of the colour development reaction, BCA, a highly sensitive and selective colorimetric detection reagent reacts with the Cu1+ that was formed in step 1. The purple-coloured reaction product is formed by the chelation of two molecules of BCA with one Cu1+. The BCA/Cu complex is water-soluble and exhibits a strong linear absorbance at 562 nm with increasing protein concentrations.

The rate of BCA colour formation is dependent on the incubation temperature, the types of protein present in the sample and the relative amounts of reactive amino acids contained in the proteins.

BCAFig1.jpg

Figure 2. Reaction diagram for the bicinchoninic acid (BCA) protein assay.

Materials:

  1. The 2 samples of urine from Part One that were positive for protein,
  2. Protein stock standard (BSA, bovine serum albumin) at 1mg/ml,
  3. 0.9% Saline (diluent)
  4. BCA (bicinchoninic acid) Working Reagent (labelled BCA WR),
  5. 6 Tubes for dilutions for the standard curve,
  6. 96 Well microtitre plate,
  7. P100, P200 & P1000 pipettes & tips,
  8. Marker pen,
  9. 37ËšC Incubator,
  10. Microtitre plate reader set to read at a wavelength of 562 nm.
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Method:

  1. Set up the dilutions for your standard curve,
    1. Label your tubes as 1,2,3,4,5 & 6,
    2. Prepare your standards according to the table below:

Dilution tube #

Volume of diluent (µl)

Volume of BSA or from tube # (µl)

Final BSA (protein) concentration (µg/ml)

1

0 µl

300 µl BSA

1000

2

250 µl

250 µl bsa

500

3

250 µl

250 µl Tube 2

250

4

250 µl

250 µl Tube 3

125

5

800 µl

200 µl Tube 4

25

6 Blank

250 µl

0

0

  1. Label your microtitre plate so that you know which wells hold your standards and which contain your samples (perform in triplicate),
  2. Pipette 25 µl of each standard (in triplicate) and sample (in triplicate) to each well,
  3. Add 200 µl of the Working Reagent to each standard or sample and shake for 30 seconds,
  4. Cover the plate and incubate at 37ËšC for 30 minutes,
  5. Cool the plate to room temperature,
  6. Measure the absorbances at 562 nm on a microtitre plate reader, insert the average values in the table below:

Dilution tube #

Average absorbance at 562 nm (add 3 values & divide by 3)

Subtract blank

(Tube 6) from

value in previous column

Final BSA (protein) concentration (µg/ml)

1

1000

2

500

3

250

4

125

5

25

6 Blank

0

Sample 1

Sample 2

  1. Prepare your standard curve: use the corrected absorbance readings for standards 1 – 6 (in column 3 in the previous table) and plot them against the amount of BSA in each tube,
  2. Once you have plotted your standard curve, you can determine the protein concentration in your samples, enter this value into the table above.

The patient scenarios are outlined below. You now need to match the urine samples with their corresponding patient scenarios and include justification for your decisions in your practical report:

Patient Scenarios:

  1. Kidney Stones: Nida is a 17 year old student. She arrives at her GP feeling nauseous, feverish & with acute pain in her lower back. She is also passing large amounts of blood in her urine.
  2. Glucose Drink: Thomas has just started a new job but is feeling quite stressed & has lost weight. He arrives to see his GP but has had to skip lunch & so drinks a litre of cola to maintain his energy levels.
  3. Diabetic: Jenny is studying for her A levels. Recently she has been losing weight although she is eating much more than usual & is always hungry.
  4. Athlete: Dave is a professional athlete & requires a blood & urine test before competing in his next event. His test results are negative for drugs but are positive for another substance.

Nephrotic syndrome: Keely is a 20 year old student. She has been feeling very unwell for some time with general fatigue, listlessness, weight loss & puffiness around her eyes & ankles. Her urine is very sparse & very dark in colour