EXPERIMENT 8: URINALYSIS The composition of urine revealed much about body function.

The amount of urine produced is influenced by environmental temperature, fluid intake, time of day, emotional state, and many other factors. Normal urine was actually a highly complex aqueous solution of organic and inorganic substances. The majority of the constituents were either waste products of cellular metabolism or products derived directly from certain foods that were eaten. The total amount of solids in a 24-hour urine sample averages around 60 g. Of this total, 35 g were organic and 25 g were inorganic. Table 8.1 showed the physical properties including color, odor, transparency, specific gravity and pH of urine sample. The first step of any routine urine analysis was the appearance of the urine. Normal urine would vary from light straw to amber in color. The color of normal urine was due to a pigment called urochrome, which was the end-product of hemoglobin breakdown: Hemoglobin--->Hematin--->Bilirubin--->Urochrome--->Urochromogen Urine which was infected with Gram-negative organisms often had a distinctive unpleasant smell. In addition, urine infected with urea splitting organisms had an ammonical smell. If urine which had a normal odor on arrival at the laboratory develops such a small, this indicated bacterial decomposition and the specimen was unfit for most chemical analyses. Certain drugs, for example paraldehyde, impart a typical odour, as did the rare maple syrup urine disease. A fresh sample of normal urine should be transparent, but would become cloudy after standing awhile. Cloudy urine would be evidence of phosphates, urates, pus, mucus, bacteria, epithelial cells, fat, and chyle. Phosphates disappeared with the addition of dilute acetic acid and urates dissipated with heat. Other causes of turbidity could be analyzed by microscopic examination. Urine specific gravity was a measure of urine concentration. It was the weight of a substance, presented as a ratio, compared to an equal volume of water. The specific gravity of a 24-hour specimen of normal urine would be between 1.015 and 1.025. Single urine specimens would range from 1.002 to 1.030. The more solids in solution, the higher would be the specific gravity. The greater the volume of urine in a 24-hour specimen, the lower would be the specific gravity. A low specific gravity would be present in chronic nephritis and diabetes insipidis. A high specific gravity would indicate diabetes mellitus, fever, and acute nephritis. Although freshly voided urine was usually acidic (around pH 6), the normal range is between 4.8 and 7.5. The pH would vary with the time of day and diet. Twenty-four-hour specimens were less acidic than fresh specimens and would become alkaline after standing due to bacterial decomposition of urea to ammonia. High acidity was present in acidosis, fevers, and high protein diets. Excess alkalinity would be due to urine retention in the bladder, chronic cystitis, anemia, obstructing gastric ulcers, and alkaline therapy. The simplest way to determine pH was to use pH indicator paper strips. Table 8.2 presented different tests to determine the presence of inorganic and organic constituents of urine. The most important organic substances were urea, uric acid and creatinine. Urea was a product formed by the liver from ammonia and carbon dioxide. Ninety-five percent of the nitrogen content of urine was in the form of this substance. Uric acid was an end-product of the oxidation of purines in the body. By weight, there was normally about 60 times as much urea as uric acid in urine. Creatinine is a hydrated form of creatine. There would be twice as much creatinine as uric acid in the urine. The principle inorganic constituents of urine were chlorides, phosphates, sulfates and ammonia. Sodium chloride was the predominant chloride and makes up about half of the inorganic substances. Since ammonia was toxic to the body and lacking in plasma, there was very little of it normally present in fresh urine. The small amount that is present is probably secreted by nephron tubules. Urine that is allowed to stand at room temperature for 24 hours or longer may give off an odor of ammonia due to the breakdown or urea by bacterial action. Chloride was a negatively charged molecule known as an electrolyte. It works with other electrolytes, such as potassium, salt (sodium), and carbon dioxide (CO2), to help keep the proper balance of body fluids and maintain the body's acid-base balance. The normal range of chloride in the urine was 110 to 250 milliequivalents per day (mEq/day). This range depends greatly on the amount of salt and fluid consumed. The phosphate urine test measured the amount of phosphate in a sample of urine. Phosphate was a charged particle (ion) that contains the mineral phosphorus. The body needs phosphorus to build and repair bones and teeth, help nerves function, and make muscles contract. Most (about 85%) of the phosphorus contained in phosphate is found in bones. The rest of it is stored in tissues throughout the body. The kidneys help control the amount of phosphate in the body. Extra phosphate was filtered by the kidneys and passes out of the body in the urine. If there is not enough phosphate, less is found in the urine. Kidney problems could cause high or low levels of phosphate in the urine. High levels of phosphate also may be caused by eating a meal high in phosphorus, having high levels of vitamin D in your body, or by an overactive parathyroid gland. The normal value of phosphate in the urine was 0.4 1.3 grams per day. Urinary sulfate was a reflection of dietary protein intake, particularly meat, fish, and poultry, which were rich in sulfurcontaining amino acids methionine and cysteine. Urinary sulfate can be used to assess dietary protein intake for nutritional purposes. A protein-rich diet has been associated with an increased risk for stone formation, possibly due, in part, to an increase in urinary calcium excretion caused by acid production from metabolism of sulfur-containing amino acids. Indeed, urinary sulfate excretion is higher in patients who have kidney stones than in individuals who do not form stones. Thus, urinary sulfate excretion may provide an index for protein-induced calciuria. Sulfate is a major anion in the urine that has significant affinity for cations and modulates the availability of cations for reacting with other anions in the urine. It thus is an important factor of urinary supersaturation for various crystals or stones such as calcium oxalate, hydroxyapatite, and brushite. For example, a high sulfate concentration may modulate the availability of calcium for reacting with oxalate and thus affect the propensity for calcium oxalate stone or crystal formation. Urinary sulfate also has a major impact on buffering or providing hydrogen ions and as such modulates the supersaturation of uric acid. The normal value for sulphate was 7 47 mmoles per specimen. Urine urea nitrogen was a measure of protein breakdown in the body. A test can be done to measure the amount of urea in the urine. This test is mainly used to determine a person's protein balance and the amount of dietary protein needed by severely ill

patients. It is also used to determine how much protein a person takes in. Urea is excreted by the kidneys, so excretion of urea can reflect kidney function. Normal values of urea range from 12 to 20 grams per 24 hours. Low levels usually indicate malnutrition (inadequate protein in diet) and kidney problems while high levels usually indicate too much protein intake and increased protein breakdown in the body. Creatinine is a breakdown product of creatine, which is an important part of muscle. Creatinine is removed from the body entirely by the kidneys. Creatinine test can be used as a screening test to evaluate kidney function. It may also be used as part of the creatinine clearance test. It is often used to provide information on other chemicals in the urine such as albumin or protein. Urine creatinine (24-hour sample) values can range from 500 to 2000 mg/day. Table 8.3 stated the three tests namely Helloer ring test, Lange test and Benedicts test for pathologic urine. Although the large size of protein molecules normally prevents their presence in urine, certain conditions can allow them to filter through. Excessive muscular exertion, prolonged cold baths and excessive ingestion of protein may result in physiological albuminuria. Pathologic albuminuria, on the other hand, exists when albumin of the urine is due to kidney congestion, toxemia of pregnancy, febrile disease and anemias. Normal catabolism of fats produces carbon dioxide and water as final end products. When there is not an adequate amount of carbohydrate in the diet, or when there is a defect in carbohydrate metabolism, the body begins to utilize an increasing amount of fatty acids. When this increased fat metabolism reaches a certain point, fatty acid utilization becomes incomplete, and intermediary products of fat metabolism occur in the blood and urine. These intermediary substances are the three ketone bodies: acetoacetic acid (diacetic acid), acetone, and beta hydroxybutyric acid. The presence of these substances in urine is called ketonuria. Diabetes mellitus is the most common disorder in which ketonuria occurs. Progressive diabetic ketosis is the cause of diabetic acidosis, due to the increased concentration of ketoacids which can eventually lead to coma or death. It is for this reason that the detection of ketonuria in diabetics is of great significance. Only a small amount of glucose is normally present in urine (0.01 to 0.03 g/100 ml of urine). When urine contains glucose in amounts greater than this, glucosuria exists. This is usually an indication of diabetes mellitus. Lack of insulin production by the pancreas or insensitivity to insulin is the cause of the disease. Insulin is necessary to stimulate the conversion of excess glucose to glycogen in the liver and muscles. It is also essential to stimulate the oxidation of glucose by cells. A deficiency of insulin function, thus, will result in high blood concentrations of glucose. The renal threshold of glucose is around 160 mg/100 ml. Glucosuria indicates that blood concentrations of glucose exceed this amount and the kidneys are unable to accomplish 100% reabsorption of this carbohydrate. EXPERIMENT 6: PROTEINS 1. What does the reaction with litmus paper suggests? What element is supposed to be detected by the reaction of the gas produced by the protein mixture with litmus paper? Why is the gas produced by the protein mixture? The reaction with the litmus paper suggests that there is a gas (basic, ammonia) produced. Nitrogen is the element supposed to be detected by the reaction of the gas produced by the protein mixture with litmus paper. The gas is produced by the protein mixture because of the action of heat. 2. What compound is responsible for the reaction of gas produced by the protein mixture with the filter paper moistened with lead acetate? Why is this compound produced by the protein mixture? What element in protein is detected by the reaction with the lead acetate? Hydrogen sulfide is responsible for the reaction of gas produced by the protein mixture with the filter paper moistened with lead acetate. This compound is produced by the protein mixture because of the action of the 3M HCl on the mixture. Sulfur is detected by the reaction with lead acetate (in the form of lead sulfide) 3. What compound is responsible for the observed odor at the mouth of the test tube? What element in protein is suggested by this compound? Why is the compound produced by the protein mixture? Carbon dioxide is responsible for the observed odor at the mouth of the test tube. Carbon is the element in protein suggested by this compound. This compound is produced by the protein mixture because of the warming of the protein mixture after the addition of an acid, which causes decarboxylation. 4. Describe what is left in the test tube after heating. What element in protein is indicated by the presence of this residue? Why is the residue suggests the presence of such element in the protein mixture? A brown solid is left in the test tube after heating. Carbon is indicated by the presence of this residue. The residue suggests the presence of such element in the protein mixture because it is the backbone element of the protein mixture. 5. What element is implied by the substance formed near the mouth of the test tube? Why do you think the element suggested is present in the protein mixture? Clear liquid is left in the test tube after heating (water). Oxygen and hydrogen are the elements in protein indicated by the presence of the residue. The residue suggest the presence of such elements in the protein mixture because water is a by-product of neutralization reactions. 6. What is the effect of proteins? Why are the proteins affected by heat? Heat can be used to disrupt hydrogen bonds and non-polar hydrophobic interactions. This occurs because heat increases the kinetic energy and causes the molecules to vibrate so rapidly and violently that the bonds are disrupted. The proteins in eggs denature and coagulate during cooking. Other foods are cooked to denature the proteins to make it easier for enzymes to digest them. Medical supplies and instruments are sterilized by heating to denature proteins in bacteria and thus destroy the bacteria. 7. Are proteins soluble in alcohol? Why or why not?

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Proteins are soluble in water, because of the hydrogen bonding that occurs between amide groups in the secondary protein structure and also the hydrogen bonding between "side chains" occurs in tertiary protein structure in a variety of amino acid combinations. How are proteins affected by strong acids? Why are proteins affected by acid? Salt bridges result from the neutralization of an acid and amine on side chains. The final interaction is ionic between the positive ammonium group and the negative acid group. Any combination of the various acidic or amine amino acid side chains will have this effect. As might be expected, acids and bases disrupt salt bridges held together by ionic charges. A type of double replacement reaction occurs where the positive and negative ions in the salt change partners with the positive and negative ions in the new acid or base added. This reaction occurs in the digestive system, when the acidic gastric juices cause the curdling (coagulating) of milk. Explain the effect of heavy metals on proteins. Why is mercuric chloride used as component of antiseptics? What is the antidote for the ingestion of heavy metal salts? Why must vomiting be induced following the use of antidote for heavy metal poisoning? Heavy metal salts act to denature proteins in much the same manner as acids and bases. Heavy metal salts usually contain Hg+2, Pb+2, Ag+1 Tl+1, Cd+2and other metals with high atomic weights. Since salts are ionic they disrupt salt bridges in proteins. The reaction of a heavy metal salt with a protein usually leads to an insoluble metal protein salt. Mercury salts administered as Mercurochrome or Merthiolate have similar properties in preventing infections in wounds. This same reaction is used in reverse in cases of acute heavy metal poisoning. In such a situation, a person may have swallowed a significant quantity of a heavy metal salt. As an antidote, a protein such as milk or egg whites may be administered to precipitate the poisonous salt. Then an emetic is given to induce vomiting so that the precipitated metal protein is discharged from the body. Why are alkaloidal reagents so called? What is the effect of alkaloidal reagents on proteins? Why are proteins affected by alkaloidal reagents? Why is picric acid used in the treatment of burns? The alkaloid reagents owe their names to their ability of alkaloids precipitation from solutions. At pH range lower (more acidic) than the pI, each protein molecule demonstrates a positive electric charge. Protons (H+) released from dissociating acids bind to amino groups (-NH2) forming positively charged -NH3+. Protein molecule becomes a cation, which is able to interact with negatively charged alkaloid reagents. Some of them, like sulphosalicylic acid, picric acid, or ferrocyanic acid are able to precipitate cationic proteins from their solutions. How can your observations show the amphoteric property of protein? Explain explicitly. The amphoteric property of protein can be shown using the observations made from the experiment due to the changes of the colors of the indicator. The color change indicates that a reaction (neutralization reaction) has occurred. What is the purpose of Biuret test? In Biuret test, does the color in each test tube containing the sample vary? Why or why not? The biuret test is a chemical test used for detecting the presence of peptide bonds. In the presence of peptides, a copper(II) ion forms a violet-colored coordination complexes in an alkaline solution. Several variants on the test have been developed. The color in each test tube containing the sample varies because there are different concentrations of proteins in each sample. What is the purpose of Xanthoproteic test? Explain the observation for each sample in terms of principle of xanthoproteic test. The purpose of xanthoproteic test is to determine the presence of aromatic proteins. This means that only albumin and tyrosine contains an aromatic part. Which of the four samples tested reacted with Millons reagent? Which does not? Why or why not? Albumin, peptone and tyrosine reacted with Millons reagent, while gelatin does not. This is because Millons reagent tests the presence of tyrosine. Which of the four samples tested reacted with Hopkins Cole reagent? Which does not? Why or why not? Only tryptophan reacted with Hopkins Cole reagent. The rest do not. This is because only the presence of tryptophan is detected by the Hopkins Cole reagent. Which of the four samples tested reacted with reduced sulfur test? Which does not? Why or why not? Albumin and cysteine reacted with reduced sulfur test. Gelatin and glycine does not. This is because the presence of sulfurcontaining amino acids is the principle of the reduced sulfur test. This means that albumin and cysteine contains sulfur.