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Lesson 2
Chemical Measures


The specific gravity of urine indicates the relative proportions of dissolved solid components to the total volume of the specimen. It reflects the relative degree of concentration or dilution of the specimen. Knowledge of the specific gravity is needed in interpreting the results of most tests performed in routine urinalysis. Under appropriate and standardized conditions of fluid restriction or increased fluid intake, specific gravity measures the concentrating and diluting abilities of the kidney.

a. Expected Values. Specific gravity of urine may range from 1.003 to 1.030, but usually remains between 1.010 and 1.025. Specific gravity is highest in the first morning specimen, which is usually greater than 1.020. A specimen gravity of 1.025 or above in any random urine specimen indicates normal concentrating ability.

b. Clinical Significance.

(1) Low specific gravity.

(a) Diabetes insipidus, a disease caused by impaired functioning of the antidiuretic hormone (ADH), is the most obvious and severe example of the loss of effective concentrating ability. This disease is characterized by large volumes of urine with low specific gravity. Specific gravity in such cases usually ranges between 1.001 and 1.003.

(b) Low specific gravity may also occur in patients with glomerulonephritis, pyelonephritis, and various renal anomalies. In these cases, the kidney has lost its ability to concentrate the urine because of tubular damage.

(2) High specific gravity. Specific gravity is high in patients with adrenal insufficiency, hepatic disease, and congestive cardiac failure. It is elevated whenever there has been excessive loss of water, as with sweating, fever, vomiting, and diarrhea.

(3) Fixed specific gravity. Urine with a fixed low specific gravity (approximately 1.010) which varies little from specimen to specimen is known as isosthenuric. This condition is indicative of severe renal damage with disturbance of both the concentrating and diluting abilities of the kidney.

c. Determination.

(1) Specific gravity is a measurement that indicates the density of the urine. It is a number derived from the ratio of the weight of a given volume of urine to the weight of the same volume of water, under standardization conditions.

Specific Gravity = Weight of Urine
Weight of Water

(2) Water has a specific gravity of 1.000. Since urine is a solution of minerals, salts, and organic compounds in water, the specific gravity is greater than 1.1. Specific gravity is a measure of the total solids in urine.

2-2. pH

The kidneys and the lungs are the two major organs that regulate the acid-base balance of the body. The lungs excrete carbon dioxide while the kidneys regulate excretion of the nonvolatile acids produced by the normal metabolic processes of the tissues. The acidity of urine is due primarily to acid phosphates, with only a minor portion contributed by organic acids such as pyruvic, lactic, and citric acids. These acids are excreted in the urine as salts, primarily sodium, potassium, calcium, and ammonium salts. The kidney regulates the selective excretion of the various cations in order to maintain normal acid-base balance. This is accomplished primarily through the reabsorption of a variable amount of sodium ion by the tubules and the tubular secretion of hydrogen and ammonium ions in exchange. Urine becomes increasingly acid as the amount of sodium retained by the body increases.

a. Expected Values. The pH of urine is a measure of its hydrogen ion concentration. A pH below 7 indicates acid urine. A pH above 7 indicates alkaline urine. Normal kidneys are capable of producing urine that can vary from a pH of 4.5 to slightly higher than 8.0. Freshly voided urine from patients not on special diets is acid and has a pH of about 6.0.

b. Clinical Significance.

(1) Acid urine. Acid urine with pH lower than 6.0 may be excreted by patients on high protein diets. Certain medications, such as ammonium chloride and mandelic acid, may also produce acid urines. Patients with acidosis and/or uncontrolled diabetes mellitus excrete urine containing large amounts of acid.

(2) Alkaline urine. Alkaline urine is frequently excreted after meals as a normal response to the secretion of HCl in the gastric juice. It also occurs in individuals consuming diets high in vegetables, milk, and other dairy products. Renal tubular acidosis is a specific disease of the kidneys in which the renal tubules are unable to adequately excrete hydrogen ions although severe systemic acidosis is present within the body. The urine pH of these patients usually remains approximately neutral and never falls below pH 6.0. Highly alkaline urines may represent either urinary tract infection or possible bacterial contamination of an old specimen with urea-splitting organisms.

(3) Renal stones. Renal stone formation significantly depends on the pH of urine. Phosphate and calcium carbonate stones develop in alkaline urine. Uric acid, cystine, and calcium oxalate stones precipitate in acid urine.

c. Determination. For routine analysis, urinary pH may be measured with reagent strips and a color chart. When more exact determinations are needed, a pH meter is used. The reagent strip is dipped into the urine specimen and the color change is compared to a standardized color chart on the bottle label that shows pH values 5 through 8.5.


Glucose is the sugar most commonly found in urine, although other sugars (such as lactose, fructose, galactose, and pentose) may also be found under certain conditions.

a. Clinical Significance.

(1) The presence of detectable amounts of glucose in urine is known as glycosuria. Glycosuria occurs whenever the blood glucose level exceeds the reabsorption capacity of the renal tubules (renal threshold); that is, when the glomerular filtrate contains more glucose than the tubules are able to reabsorb. The condition may be either benign or pathological, and the physician must distinguish between the two types.

(2) Diabetes mellitus, a pathological state, is the chief cause of glycosuria. This condition is associated with a marked elevation of blood glucose and usually an increase in urine volume.

b. Determination. There are various tests for glucose that can be applied to urine. Those most frequently used are of two types listed below.

(1) Enzymatic tests based on the action of glucose oxidase on glucose.

(2) Reduction tests based on the reduction of certain metal ions by glucose.

c. Enzymatic Tests.

(1) The enzymatic glucose oxidase tests for glucose, as applied to urine, are specified for glucose. In these tests, glucose oxidase catalyzes the oxidation of glucose to gluconic acid and hydrogen peroxide. The peroxide, in the presence of peroxidase, oxidizes an indicator that produces a color change. Other sugars, such as lactose, fructose, galactose, and pentose, are not substrates for glucose oxidase and, therefore, do not react with this test.

(2) CLINISTIX® Reagent Strip is another glucose test strip using the glucose oxidase principle. The results of this test are qualitative. Quantitation is only approximate because of the variable effect different urines may have on the color development. The glucose test strips will distinguish urines containing glucose only.

d. Reduction Tests.

(1) Metalic ions. The reduction of metallic ions such as Cu++ is nonspecific for glucose. This is because the reaction may be brought about by any reducing substance that may be present in the urine, such as creatinine, uric acid, ascorbic acid, or some other reducing sugar. The nonspecificity of the copper reduction test can be an advantage in that it will detect sugar other than glucose. It has a disadvantage in that it will detect reducing substances other than sugars.

(2) CLINITEST® Reagent Tablets. The copper reduction test has been greatly simplified by CLINITEST Reagent Tablets. When the tablet is added to a small test tube containing 10 drops of water and 5 drops of urine, it dissolves and produces carbon dioxide and heat. In the process, if a reducing substance such as glucose is present, the color changes from blue to orange, depending on the amount of sugar present. By comparing the color with a reference color chart, the amount of reducing substance in the urine can be estimated.

e. Non-Glucose Reducing Sugars (Galactose).

(1) Galactose is found in the urine of infants afflicted with galactosemia. These children are deficient in the enzyme necessary for converting galactose into glucose. This is a severe condition, which can be treated by eliminating lactose and other sources of galactose from the diet. If not treated properly, the infant will rapidly deteriorate physically and mentally and early death will result. Galactose can be detected with CLINITEST.

(2) Some pediatricians will screen infants to detect for galactosemia by using CLINITEST and CLINISTIX® to detect the presence of non-glucose reducing substances (positive CLINITEST, negative CLINISTIX).


Normally, the body completely metabolizes fats to carbon dioxide and water. Whenever there is inadequate carbohydrate in the diet or a defect in carbohydrate metabolism or absorption, the body metabolizes increasing amounts of fatty acids. When this increase is large, fatty acid utilization is incomplete. Intermediary products of fat metabolism appear in the blood and are excreted in the urine. These intermediary products are the three ketone bodies: acetoacetic acid (which is also called diacetic acid), acetone, and beta-hydroxybutyric acid. They are derived from acetoacetic acid. All three ketone bodies are present in the urine of patients with ketonuria in the relative proportions of 20 percent acetoacetic acid, 2 percent acetone, and 78 percent beta- hydroxybutyric acid.

a. Expected Values. Normally there is no detectable amount of ketones in the urine.

b. Clinical Significance.

(1) Diabetes mellitus is the most important disorder in which ketonuria occurs. Diabetes mellitus is a disorder of glucose metabolism. In insulin-deficient diabetes, glucose metabolism is sufficiently impaired that fatty acids are utilized to meet the body’s energy requirements. When this type of diabetes is untreated or inadequately treated, excessive amounts of fatty acids are metabolized. This results in the accumulation of ketone bodies in the blood (ketosis) that are excreted in urine (ketonuria). Progressive diabetic ketosis is the cause of diabetic acidosis, which can eventually lead to coma and even death. The term ketoacidosis is frequently used to designate the combined ketosis and acidosis of diabetes.

(2) Ketonuria also accompanies the restricted carbohydrate intake that occurs in association with fevers, anorexia, gastrointestinal disturbances, fasting, starvation, cyclic vomiting, pernicious vomiting of pregnancy, and cachexia.

c. Determination. In ketonuria, acetoacetic acid, acetone, and beta- hydroxybutyric acid are all excreted in the urine. Consequently, a general test procedure that indicates the presence of one of these components is usually satisfactory for the diagnosis of ketonuria.

(1) Nitroprusside reactions. The reagent strip method is the simplest technique for determination of ketonuria. The strip is dipped into fresh urine, tapped to remove excess urine, and compared to the color chart after exactly 15 seconds. The chart has six color blocks indicating negative, trace (5 mg/dL), small (15 mg/dL), moderate (40 mg/dL), or large (80 mg/dL) and (160 mg/dL) concentrations of ketone, and ranging in color from buff to lavender and maroon. The test is sensitive to acetoacetic acid. It does not react with beta-hydroxybutyric acid or acetone.

(2) ACETEST® reaction tablet. To detect acetone and acetoacetic acid:

(a) Place tablet on a clean surface, preferably a piece of white paper.

(b) Put one drop of urine (or serum, plasma, or whole blood) on the tablet.

(c) Compare urine ketone test results to color chart at 30 seconds.


Approximately one-third of normal urinary protein is albumin. This albumin appears to be identical to serum albumin. The majority of normal proteins in the urine are globulins. A high molecular weight mucoprotein, the Tamm-Horsfall protein, occurs in normal urine quantities up to 2.5 mg/dL. In nephrosis, it may occur in higher concentrations. It is not found in plasma and is thought to originate in the kidneys.

a. Expected Values. Normally, between 40 and 80 mg of protein are excreted daily, but as much as 100 to 150 mg per day may be considered within normal limits. Since the average daily urine volume may range from 1,000 to 1,500 mL, the average normal concentration of protein in the urine varies from 2 to 8 mg/dL. This wide range of normal values is the result of biological variations and differences in the methods used for the determination of protein.

b. Clinical Significance.

(1) Proteinuria refers to an increased amount of protein in the urine and is one of the most important indicators of renal disease.

(2) Albumin constitutes between 60 percent and 90 percent of protein excreted in most disease states. The urine of patients with multiple myeloma contains increased amounts of a low molecular weight globulin (Bence Jones protein).

(3) Proteinuria depends on the precise nature of the clinical and pathological disorder and upon the severity of the specific disease. Proteinuria may be intermittent or continuous. Transient, intermittent proteinuria is usually caused by physiologic or functional conditions rather than by renal disorders.


a. Proteinuria.

(1) Marked proteinuria is characterized by the excretion of more than 4 gm per day. It is typical of the nephrotic syndrome, but also occurs in severe cases of glomerulonephritis, nephrosclerosis, amyloid disease, systemic lupus erythematosus, and severe venous congestion of the kidney produced by renal vain thrombosis, congestive heart failure, or constrictive pericarditis.

(2) Moderate proteinuria refers to the daily excretion of between 0.5 and 4 gm of protein. It is found in the vast majority of renal diseases, as well as all of the disorders listed above. It is also found in chronic glomerulonephritis, diabetic nephropathy, multiple myeloma, toxic nephropathy, preeclampsia, and inflammatory, malignant, degenerative, and irritative conditions of the lower urinary tract, including the presence of calculi.

(3) Minimal proteinuria is the excretion of less than 0.5 gm of protein per day. It is associated with chronic glomerulonephritis polycystic disease of the kidneys, renal tubular disorders, the healing phase of acute glomerulonephritis, latent or inactive stages of gloemerulonephritis, and various disorders of the lower urinary tract.

b. Determination. A number of simple, semiquantitative tests and more complex quantitative tests are available for the determination of all proteins in urine.

(1) Colorimetric reagent strip test.

(a) The colorimetric reagent strip test is based on the ability of proteins to alter the color of some acid-base indicators without altering the pH. In the presence of protein, the color will change to green and then to blue with increasing protein concentrations.

(b) Most multiple reagent strips contain an area for protein determination, along with test areas for other urinary constituents. Protein is determined simply by dipping the strip into well-mixed uncentrifuged urine and comparing the resultant color with the chart provided on the reagent strip bottle.

(2) Turbidimetric or precipitation test. The sulfosalicylic acid method is a simple method for semiquantitating protein concentration in terms of trace through four “plus” precipitation. The precipitation is read and interpreted as follows:

(a) Negative. No tubidity, or no increase in turbidity (approximately 5 mg/dL or less).

(b) Trace. Perceptible turbidity (approximately 20 mg/dL).

(c) 1+. Distinct turbidity, but no discrete granulation (approximately 50 mg/dL)

(d) 2+. Turbidity with granulation, but no flocculation (approximately 200 mg/dL).

(e) 3+. Turbidity with granulation and flocculation (approximately 500 mg/dL)

(f) 4+. Clumps of precipitated protein, or solid precipitate (approximately 1 g/dL or more).

c. Sulfosalicylic Acid Method.

(1) Place 3 mL centrifuged urine in a test tube.

(2) Add 3 mL of 3 percent sulfosalicylic acid.

(3) Mix thoroughly and estimate the amount of turbidity 10 minutes later.

2-7. BLOOD

Although protein in urine is the most important indication of renal dysfunction, the presence of blood in urine is also an indication of damage to the kidney or urinary tract. Blood may appear as intact red cells or as free hemoglobin. Usually, the presence of free hemoglobin indicates that the cells have ruptured because of the traumatic passage through the kidney and urinary tract to the bladder or because the cells have been exposed to dilute urine in the bladder, which has caused them to hemolyze. Free hemoglobin is excreted from the blood into the urine in special cases only, such as transfusion reactions.

NOTE: Hematuria is defined as the presence of red blood cells in urine. In contrast, hemoglobinuria is defined as the presence of free hemoglobin.

a. Expected Values. Normally, there is no detectable amount of occult blood present in urine, even with very sensitive chemical methods.

b. Clinical Significance. The presence of blood in urine, as indicated by a positive test for occult blood, most likely indicates bleeding in the urinary tract. This may occur in a variety of renal disorders, infectious disease, neoplasms, or trauma affecting any part of the urinary tract. Free hemoglobin is likely to be found in any of the above disorders. Free hemoglobin may also indicate transfusion reaction, hemolytic anemia, or paroxysmal hemoglobinuria. It may also appear in various poisonings or following severe burns. A positive chemical test without the presence of red cells may indicate myoglobinuria as a result of traumatic muscle injury.

c. Determination.

(1) The reagent strip method is the simplest and most direct test for the presence of blood in urine.

(2) The color of the strip is compared with a color chart 60 seconds after the strip is dipped into the urine.


Bilirubin in the urine indicates the presence of hepatocellular disease or the presence of intrahepatic or extrahepatic biliary obstruction. It is an early sign of these disorders and, therefore, a useful diagnostic tool. Bilirubin is formed by the breakdown of hemoglobin in the reticuloendothelial cells of the spleen and bone marrow. It is linked to albumin in the bloodstream and transported to the liver. This albumin-bound form, which is also known as indirect bilirubin, is insoluble in water and does not appear in the urine. In the liver cells, it is separated from the albumin and conjugates with glucuronic acid to form water-soluble conjugated bilirubin, also know as direct bilirubin. The liver cells that form the conjugated bilirubin excrete it into the bile and it is then excreted into the intestinal tract through the bile duct. This conjugated bilirubin in the intestinal tract is converted by bacterial action to urobilinogen. Being water soluble, conjugated bilirubin can be excreted by the kidneys, although normally its level in the blood is not high enough to cause significant amounts to appear in the urine.

a. Expected Values. Biliruben present in urine is approximately 0.02 mg/dL, reflecting the normally low blood levels of conjugated bilirubin. This amount is not detected by routine qualitative or semiquantitative techniques.

b. Clinical Significance.

(1) Bilirubin excretion in the urine will reach significant levels in any disease process that increases the amount of conjugated bilirubin in the bloodstream. In some liver diseases due to infectious or hepatotoxic agents, liver cells are unable to excrete all of the conjugated bilirubin into the bile. Therefore, sufficient amounts are returned to the blood to elevate blood levels and cause significant bilirubinuria.

(2) In obstructive biliary tract disease, biliary stasis interferes with the normal excretion of conjugated bilirubin via the intestinal tract. This causes a buildup in the bloodstream with resulting bilirubinura. Since bilirubin may often appear in the urine before other signs of liver dysfunction (jaundice, clinical illness) are apparent, bilirubinuria is an important diagnostice sign of liver disease and a bilirubin test should be part of every routine urinalysis.

c. Determination.

(1) The bilirubin reagent area on multiple strips is the simplest test for the determination of bilirubin. The reagent strip is dipped into fresh, uncentrifuged urine, tapped to remove excess urine, and, after a 30-second wait, compared to the color chart on the reagent strip bottle.

(2) Reagent tablets and special absorbent test mats are a highly sensitive and convenient method for the determination of bilirubinuria. The procedure is:

(a) Place 10 drops of urine on one special test mat. If bilirubin is present in the specimen, it will be absorbed onto the mat surface.

(b) Place an ICTOTEST Reagent Tablet on the moistened area of the mat.

(c) Flow two drops of water over the tablet.

(d) When elevated amounts of biliruben are present in the urine specimen, a blue to purple color forms on the mat within 60 seconds. The rapidity of the formation of the color and the intensity of the color development are proportional to the amount of bilirubin in the urine.


Bacterial action in the intestinal tract converts the bilirubin to a group of compounds known as urobilinogen. It is estimated that as much as 50 percent of the urobilinogen formed in the intestines is reabsorbed into the portal circulation and re- excreted by the liver. Small amounts are normally excreted in the urine, but the major excretion is in the feces.

a. Expected Values. Normally, between 1 and 4 mg (1 to 4 Ehrlich units) of urobilinogen is excreted in urine in a 24-hour period. The concentration of urobilinogen in a random normal urine is 0.1 to 1.0 Ehrlich unit/dL (1 EU/dL ~ 1 mg/dL).

b. Clinical Significance.

(1) Urinary urobilinogen is increased by any condition that causes an increase in the production of bilirubin and by any disease that prevents the liver from normally removing the reabsorbed urobilinogen from the portal circulation. Urobilinogen is increased whenever there is excessive destruction of red blood cells as in hemolytic anemias, pernicious anemia, and malaria. It is increased also in infectious hepatitis, toxic hepatitis, portal cirrhosis, or congestive heart failure. Determination of urinary urobilinogen is a useful procedure in routine urinalysis since it serves as a guide in detecting and differentiating liver disease, hemolytic disease, and biliary obstruction. Sequential determination also assists in evaluating progress of the disease and response to management.

(2) Urinary urobilinogen is decreased or absent when normal amounts of biliruben are not excreted into the intestinal tract. This usually indicates partial or complete obstruction of the bile ducts such as may occur in cholelithiasis, severe inflammatory disease, or neoplastic disease. Also, during antibiotic therapy, suppression of normal intestinal flora may prevent conversion of bilirubin to urobilinogen, leading to an absence of urobilinogen in urine.

c. Determination (Urobilinogen Reagent Area). The strip is dipped into fresh uncentrifuged urine collected without preservatives. It is then removed and, after exactly 60 seconds, the color reaction is compared to the color chart.


The finding of significant numbers of bacteria by culture methods is considered indicative of a urinary tract infection, especially if the specimen is a clean-voided midstream sample collected under aseptic conditions in a sterile container that is immediately closed with a sterile cap. However, a positive test for nitrite on any random urine specimen always indicates bacteriuria.

a. Clinical Significance.

(1) Bacteriuria is considered significant when microbiological laboratory findings show the presence of 100,000 (105) or more bacteria per mL of three separate urine specimens.

(2) Significant urinary tract infections may be present in patients who have experienced no symptoms. Despite an absence of symptoms, these infections are serious because they have the potential for causing severe kidney damage before the patient is aware of them. This condition is known as significant asymptomatic bacteriuria.

b. Determination (Nitrite Test).

(1) The nitrite test is fast and inexpensive and it provides an indirect method for early detection of significant bacteriuria.

(2) The nitrite area of the reagent strips has to produce a pink color. Thus, a positive result from a nitrite test is an indication of significant bacteriuria. However, a negative test result should never be interpreted as indicating an absence an absence of bacteriuria.


a. This test detects the esterase released from the white blood cells (neutrophils) in the urine. Usually the presence of a significant number of white blood cells (leukocytes) in the urine indicates bacteriuria or a urinary tract infection.

b. The detection of leukocyte esterase as an indication of bacteriuria is an indirect test for infection. Pyuria (the presence of white blood cells in urine in significant numbers) has long been an indication of the possibility of urinary tract infection.

David L. Heiserman, Editor

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Revised: June 06, 2015