Urinalysis of Four Urine Samples

Urinalysis of Four body of water SamplesUrinalysis Practical sceneAs you have learned, the urinary system performs many vital functions in the body includingRegulating agate line volume and pressure by regulating water excretion,Regulating plasma ion/solute concentrations by adjusting piss composition,Assisting blood pH stabilisation,Removing nitrogenous waste,Conserving water and important nutrients andAssisting the liver in detoxifying poisons.Therefore, analysing a taste of water supply from a person can provide important selective information on the health of that person. Urinalysis can reveal unhealthinesss such as diabetes mellitus, urinary packet infections and renal (kidney) infections such as glomeronephritis and kidney stones (renal calculi).A medical examination professional ovalbuminthorn perform a urinalysis for several reasonsAs a general health check-up,Diagnosing metabolic or general diseases that affect renal function (heart failure will lead to decreased blood flow to the kidneys, pre-eclampsia during pregnancy will lead to change magnitude protein in the pissing),diagnosing of endocrine disorders e.g. infertility (low levels of FSH and/or LH),Diagnosis of urinary system disease,Monitoring of glucose levels in patient roles with diabetes,Testing for pregnancy (hCG levels secreted by the embryo),Screening for drug practise.Urinalysis is a technique involving physical, chemical and microscopical analyses of a stress of urine.Physical parameters Normal urine is a wanton yellow colorise due to the posture 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 suggest a urinary tract or renal infection or disease, liver or pertness bladder disease. Normal urine has a specific gravity of amid 1.002 1.028 (this is a measure of the number of particles/solutes in the urine, its concentration). A urine try on that has an elevated specific gravity can indicate dehydration, diarrhea/vomiting, glucosuria, unconnected ADH secretion. A diminished specific gravity may indicate such diseases as renal failure or pyelonephritis.Chemical parameters The chemical analysis of urine is routinely performed using an inexpensive and relatively precise dipstick test (Uristix from Bayer or other brands). The test uses a reagent-coated moldable 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)Figure 1. Colour chart (a) for use of glucose and/or protein. The glucose test on the dipstick is based on a mental image sequential enzyme defendion. One enzyme, glucose oxidase, catalyses the formation of gluconic dit and hydrogen hydrogen peroxide from the oxidisation 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 dark-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 atomic number 6 g/ml. Although the dipstick test is semi-quantitative, significantly more accurate levels of glucose and protein can be escortd 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.Microscopic parameters The urine sample can also be analysed by a microscope, often afterwards 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 unadulterated diagnosis.AimThe aim of this practical is to perform glucose and protein urinalysis techniques on five-spot samples of urine provided by five patients and use this information to provide an sign diagnosis for each patient. serving One Using Dipsticks To give up A Qualitative Measure of Protein And/Or Glucose.Materials5 samples of urine labelled A E (these will be required for Parts One and Two),5 Uristix dipsticks.MethodPerform a basic physical analysis of the urine samples noting the colour and cloudiness of each sample body of water AUrine BUrine CUrine DUrine EColourCloudinessImmerse 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 substanceUrine AUrine BUrine CUrine DUrine EGlucoseProteinKetones prejudiciousNegativeNegativeNegative strongly positiveBloodNegativeNegativeTraceNegativeNegativePart Two Using A Commercial BCA Assay To Provide A Quantitative Measure of Protein.BackgroundThe BCA Protein Assay exploits the chemical lessening of Cu2+ to Cu1+ by protein in an alkaline medium with the selective colorimetric undercover work of the cuprous cation (Cu1+) by bicinchoninic acid (BCA). The first mensuration 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 boastfulr 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 extremely 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.Figure 2. Reaction diagram for the bicinchoninic acid (BCA) protein assay.MaterialsThe 2 samples of urine from Part One that were positive for protein,Protein stock standard (BSA, bovine serum albumin) at 1mg/ml,0.9% Saline (diluent)BCA (bicinchoninic acid) Working Reagent (labelled BCA WR),6 Tubes for dilutions for the standard curve,96 Well microtitre plate,P100, Pcc P super acid pipettes tips, score pen,37C Incubator,Microtitre plate reader set to read at a wavelength of 562 nm.MethodSet up the dilutions for your standard curve,Label your tubes as 1,2,3,4,5 6,Prepare your standards according to the table belowDilution tube Volume of diluent (l)Volume of BSA or from tube (l)Final BSA (protein) concentration (g/ml)10 l300 l BSA10002250 l250 l bsa5003250 l250 l Tube 22504250 l250 l Tube 31255800 l200 l Tube 4256 Blank250 l00Label your microtitre plate so that you know which wells hold your standards and which contain your samples (perform in triplicate),Pipette 25 l of each standard (in triplicate) and sample (in triplicate) to each well,Add 200 l of the Working Reagent to each standard or sample and get for 30 seconds,Cover the plate and incubate at 37C for 30 minutes,Cool the plate to room temperature,Measure the absorbances at 562 nm on a microtitre plate reader, inser t the average values in the table belowDilution tube Average absorbance at 562 nm (add 3 values divide by 3)Subtract blank(Tube 6) fromvalue in previous columnFinal BSA (protein) concentration (g/ml)110002500325041255256 Blank0Sample 1Sample 2Prepare 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,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 reportPatient ScenariosKidney Stones Nida is a 17 year old student. She arrives at her GP feeling nauseous, feverous with acute pain in her lower back. She is also passing large amounts of blood in her urine.Glucose Drink Thomas has just started a saucy 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.Diabetic Jenny is studying for her A levels. Recently she has been losing weight although she is eating much more than everyday is always hungry.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 season with general fatigue, listlessness, weight loss puffiness around her eyes ankles. Her urine is very sparse very dark in colour