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Clinical Pathology

Chemical Urinalysis: Indications, Test Procedure and Results

By Dayyal Dg.Twitter Profile | Updated: Monday, 11 December 2023 14:05 UTC
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Free photo defocused female scientist holding lab substance. Freepik / @freepik

Urine serves as a diagnostic medium for detecting both physical and biochemical irregularities. This analysis aids in screening and diagnosing conditions such as urinary tract infections, kidney disorders, liver problems, diabetes, and various metabolic conditions. Prior to examination, the specimen's acceptability is assessed.

The chemical examination encompasses the analysis of the following substances in urine:

  • Proteins
  • Glucose
  • Ketones
  • Bilirubin
  • Bile salts
  • Urobilinogen
  • Blood
  • Hemoglobin
  • Myoglobin
  • Nitrite or leukocyte esterase

Proteins in Urine

Box 1: Etiologies of proteinuria
  • Glomerular proteinuria
  • Tubular proteinuria
  • Overflow proteinuria
  • Hemodynamic (functional) proteinuria
  • Post-renal proteinuria

The kidneys typically eliminate a minimal amount of protein in the urine, not exceeding 150 mg in a 24-hour period. These proteins encompass those originating from plasma, such as albumin, as well as proteins derived from the urinary tract, including Tamm-Horsfall protein, secretory IgA, and proteins originating from tubular epithelial cells, leucocytes, and other desquamated cells. Importantly, this level of proteinuria falls below the detection threshold of routine tests.

It's noteworthy that Tamm-Horsfall protein is a normal mucoprotein secreted by the ascending limb of the loop of Henle.

In adults, the term "proteinuria" denotes the excretion of protein in the urine exceeding 150 mg in a 24-hour period.

Causes of Proteinuria

Box 2: Nephrotic syndrome
  • Massive proteinuria (>3.5 gm/24 hr)
  • Hypoalbuminemia (<3.0 gm/dl)
  • Generalised edema
  • Hyperlipidemia (serum cholesterol >350 mg/dl)
  • Lipiduria

The etiologies of proteinuria can be categorized, as illustrated in Box 1.

  1. Glomerular Proteinuria: Proteinuria arising from an augmented permeability of the glomerular capillary wall is termed glomerular proteinuria. Within this category, two distinct types exist: selective and nonselective. In the initial stages of glomerular disease, there is an elevated excretion of lower molecular weight proteins such as albumin and transferrin. Selective proteinuria occurs when glomeruli can retain larger molecular weight proteins but allow passage of relatively lower molecular weight proteins. As glomerular damage progresses, selectivity is lost, resulting in the excretion of larger molecular weight proteins, including γ globulins, alongside albumin—termed nonselective proteinuria. The differentiation between selective and nonselective proteinuria can be achieved through urine protein electrophoresis. In selective proteinuria, distinct bands of albumin and transferrin are observable, while in the nonselective type, the pattern mirrors that of serum (Figure 1). Glomerular proteinuria is instigated by diseases affecting the glomerular basement membrane's permeability. The extent of proteinuria corresponds to the severity of the disease and its prognosis. Monitoring the response to treatment is facilitated by serial estimations of urinary protein. The most severe manifestation of proteinuria is observed in nephrotic syndrome (Box 2).
  2. Tubular Proteinuria: Under normal circumstances, the glomerular membrane, impermeable to high molecular weight proteins, allows the passage of low molecular weight proteins like β2-microglobulin, retinol-binding protein, lysozyme, α1-microglobulin, and free immunoglobulin light chains. These low molecular weight proteins are actively reabsorbed by proximal renal tubules. In diseases predominantly affecting the tubules, these proteins are excreted in urine while albumin excretion remains minimal. Urine electrophoresis reveals prominent α- and β-bands, representing the migration of low molecular weight proteins, and a faint albumin band (Figure 1). Tubular proteinuria is commonly observed in acute and chronic pyelonephritis, heavy metal poisoning, tuberculosis of the kidney, interstitial nephritis, cystinosis, Fanconi syndrome, and kidney transplant rejection. Purely tubular proteinuria is not detectable through reagent strip tests, sensitive to albumin. However, positive results are obtained with the heat and acetic acid test, as well as the sulphosalicylic acid test.
  3. Overflow Proteinuria: Overflow proteinuria occurs when the concentration of a low molecular weight protein rises in plasma, leading to its "overflow" into the urine. Proteins involved in this type include immunoglobulin light chains or Bence Jones proteins (associated with plasma cell dyscrasias), hemoglobin (resulting from intravascular hemolysis), myoglobin (due to skeletal muscle trauma), and lysozyme (linked to acute myeloid leukemia type M4 or M5).
  4. Hemodynamic Proteinuria: Changes in blood flow through the glomeruli cause increased protein filtration, although protein excretion is transient. This phenomenon is observed in conditions such as high fever, hypertension, heavy exercise, congestive cardiac failure, seizures, and exposure to cold. Postural (orthostatic) proteinuria occurs when the subject is standing or ambulatory, but is absent in the recumbent position. Common in adolescents (3-5%), it is likely due to a lordotic posture causing inferior vena cava compression between the liver and vertebral column. This condition usually disappears in adulthood, with proteinuria levels below 1000 mg/day. Periodic testing for proteinuria is recommended in such individuals to rule out renal disease.
  5. Post-renal Proteinuria: This type is induced by inflammatory or neoplastic conditions in the renal pelvis, ureter, bladder, prostate, or urethra.

Further reading: Methods for the Detection of Protein in Urine.

Glomerular and tubular proteinuria
Figure 1: Glomerular and tubular proteinuria. Upper figure shows normal serum protein electrophoresis pattern. Lower part shows comparison of serum and urine electrophoresis in (1) selective proteinuria, (2) non-selective proteinuria, and (3) tubular proteinuria

Glucose

Box 3: Urine glucose
  • It is advisable to assess urine glucose levels within a 2-hour timeframe following collection. This precaution is necessary due to the enzymatic breakdown of glucose by glycolysis and the presence of contaminating bacteria, which can rapidly degrade glucose.
  • The reagent strip test serves as a rapid, cost-effective, and semi-quantitative method for glucose analysis.
  • Historically utilized for at-home glucose monitoring, the reagent strip test has been supplanted by more advanced glucometers.
  • Urine glucose analysis is unsuitable for monitoring diabetes control. The variability in renal threshold among individuals, the absence of information regarding blood glucose levels below the renal threshold, and the impact of urine concentration on glucose values collectively limit its efficacy for this purpose.

The primary purpose of conducting urine glucose testing is to identify undiagnosed diabetes mellitus or to monitor known diabetic patients during follow-up.

Virtually all glucose that undergoes filtration in the glomeruli is reabsorbed by the proximal renal tubules and subsequently returned to the circulation. Under normal circumstances, only a minute quantity of glucose is excreted in the urine (typically < 500 mg/24 hours or < 15 mg/dl), a level that remains undetectable through routine tests. The presence of discernible quantities of glucose in the urine is termed glucosuria or glycosuria (Box 3). Glycosuria occurs when the filtered glucose load surpasses the reabsorptive capacity of the renal tubules, with hyperglycemia resulting from diabetes mellitus constituting the most prevalent cause.

Causes of Glycosuria

Glycosuria with hyperglycemia

  • Endocrine Diseases: Conditions encompassing diabetes mellitus, acromegaly, Cushing's syndrome, hyperthyroidism, and pancreatic disease fall within the realm of endocrine disorders.
  • Non-Endocrine Diseases: Central nervous system diseases and liver disorders are among the non-endocrine conditions associated with relevant glycosuric manifestations.
  • Drug-Induced Causes: The administration of adrenocorticotrophic hormone, corticosteroids, and thiazides represents drug-related factors contributing to glycosuria.
  • Alimentary Glycosuria (Lag-Storage Glycosuria): Following a meal, the swift intestinal absorption of glucose leads to a transient elevation of blood glucose levels beyond the renal threshold. This phenomenon may manifest in individuals with gastrectomy or gastrojejunostomy, as well as those experiencing hyperthyroidism. A glucose tolerance test reveals a peak at 1 hour exceeding the renal threshold, resulting in glycosuria, while fasting and 2-hour glucose values remain within normal limits.

Glycosuria without hyperglycemia

  • Renal Glycosuria: Constituting approximately 5% of glycosuria cases in the general population, renal glycosuria is characterized by a renal threshold—the highest blood glucose level at which glucose becomes detectable in urine through routine laboratory tests. The typical renal threshold for glucose is 180 mg/dl. Substances reaching the threshold necessitate a carrier for transport from the tubular lumen to the blood. Once the carrier becomes saturated, the threshold is attained, leading to excretion of the substance. Up to this point, glucose filtered by the glomeruli undergoes efficient reabsorption by the tubules. Renal glycosuria represents a benign condition where the renal threshold is set below 180 mg/dl, yet glucose tolerance remains normal. This disorder is inherited as an autosomal dominant trait. Other instances of glycosuria with blood glucose levels below 180 mg/dl occur in renal tubular diseases, such as Fanconi's syndrome, characterized by diminished glucose reabsorption, and in cases of toxic renal tubular damage. Pregnancy induces a reduction in the renal threshold for glucose, underscoring the importance of blood glucose estimation when the initial detection of glucose in urine occurs.

Further reading: Methods for the Detection of Glucose in Urine.

Ketones

Box 4: Urine ketones in diabetes Indications for testing
  • At the initial identification of diabetes mellitus.
  • Periodically for individuals with established diabetes and those with gestational diabetes.
  • For confirmed diabetic individuals during instances of acute illness, sustained hyperglycemia (>300 mg/dl), pregnancy, and when clinical signs of diabetic acidosis are present (such as nausea, vomiting, and abdominal pain).

The elimination of ketone bodies (specifically, acetoacetic acid, β-hydroxybutyric acid, and acetone) through urine is termed ketonuria. Ketones, derived from the breakdown of fatty acids, appearing in urine signals an elevated level of fatty acid metabolism as a source of energy.

Causes of Ketonuria

Typically, ketone bodies are undetectable in the urine of individuals in good health. When the metabolism of glucose is compromised due to issues such as defective carbohydrate metabolism, insufficient carbohydrate intake, or heightened metabolic demands, the body turns to the breakdown of fats for energy. This metabolic shift results in the production of ketone bodies, as illustrated in Figure 2.

  1. Reduced Carbohydrate Utilization:
    1. Uncontrolled Diabetes Mellitus with Ketoacidosis: In the context of diabetes, inadequate glucose utilization triggers compensatory heightened lipolysis. This process elevates the levels of free fatty acids in the plasma. The liver's degradation of these free fatty acids results in the formation of acetoacetyl CoA, subsequently giving rise to ketone bodies. These ketone bodies, potent acids, generate H⁺ ions, neutralized by bicarbonate ions. A decrease in bicarbonate levels (alkali) leads to ketoacidosis. Ketone bodies also augment plasma osmolality, inducing cellular dehydration. Individuals, particularly children and young adults with type 1 diabetes, are predisposed to ketoacidosis during acute illnesses and periods of stress. Presence of glycosuria necessitates ketone body testing. Concurrent presence of glucose and ketone bodies in urine signifies diabetes mellitus with ketoacidosis. In certain diabetes cases, blood ketone levels may rise without manifesting in urine. Detection of ketone bodies in urine can serve as a warning sign of an impending ketoacidotic coma.
    2. Glycogen Storage Disease (von Gierke’s Disease)
  2. Insufficient Carbohydrate Availability in the Diet:
    1. Starvation
    2. Persistent Vomiting in Children
    3. Weight Reduction Program (Severe Carbohydrate Restriction with Normal Fat Intake)
  3. Elevated Metabolic Demands:
    1. Fever in Children
    2. Severe Thyrotoxicosis
    3. Pregnancy
    4. Protein-Calorie Malnutrition

Further reading: Methods for the Detection of Ketones in Urine.

Formation of ketone bodies
Figure 2: Formation of ketone bodies. A small part of acetoacetate is spontaneously and irreversibly converted to acetone. Most is converted reversibly to β-hydroxybutyrate.

Bile Pigment (Bilirubin)

Bilirubin, a byproduct of hemoglobin breakdown, is typically absent in the urine of healthy individuals. The presence of bilirubin in urine is termed bilirubinuria.

Two distinct forms of bilirubin exist: conjugated and unconjugated. Following its generation from hemoglobin within the reticuloendothelial system, bilirubin circulates in the bloodstream, bound to albumin—referred to as unconjugated bilirubin. Being insoluble in water and bound to albumin, unconjugated bilirubin cannot traverse the glomeruli, and as a result, it does not manifest in the urine.

The liver plays a crucial role in processing unconjugated bilirubin. Here, it combines with glucuronic acid, forming bilirubin diglucuronide, which is categorized as conjugated bilirubin. Unlike its unconjugated counterpart, conjugated bilirubin is water-soluble, undergoes filtration by the glomeruli, and consequently, is excreted in the urine.

The identification of bilirubin in urine, coupled with the presence of urobilinogen, proves valuable in distinguishing various causes of jaundice (refer to Table 1).

Table 1: Urine bilirubin and urobilinogen in jaundice
Urine testHemolytic jaundiceHepatocellular jaundiceObstructive jaundice
Bilirubin Absent Present Present
Urobilinogen Increased Increased Absent

During acute viral hepatitis, bilirubin manifests in urine even prior to the clinical onset of jaundice. In cases of an unexplained fever, the presence of bilirubinuria suggests a potential hepatitis etiology.

The detection of bilirubin in urine signifies the presence of conjugated hyperbilirubinemia, indicative of obstructive or hepatocellular jaundice. This is attributable to the water-solubility of conjugated bilirubin. Conversely, bilirubin does not appear in urine in hemolytic jaundice due to the water-insolubility of unconjugated bilirubin.

Further reading: Methods for the Detection of Bilirubin in Urine.

Bile Salts

Bile salts comprise salts derived from four distinct types of bile acids: cholic, deoxycholic, chenodeoxycholic, and lithocholic. These bile acids combine with either glycine or taurine, forming intricate salts or acids. Transported through the bile, bile salts enter the small intestine, serving as detergents that emulsify fat and reduce surface tension on fat droplets. This action facilitates the enzymatic breakdown of fat by lipases. Following absorption in the terminal ileum, bile salts enter the bloodstream, undergo hepatic uptake, and are subsequently re-excreted in bile, constituting the enterohepatic circulation.

Further reading: Methods for the Detection of Bile Salts in Urine.

Urobilinogen

Conjugated bilirubin, excreted into the duodenum via bile, undergoes bacterial conversion to urobilinogen within the intestine. The majority of this urobilinogen is expelled through feces. A fraction is absorbed into the bloodstream, initiating recycling through the enterohepatic circulation, while a minor quantity, not reabsorbed by the liver, is excreted in urine. Initially colorless, urobilinogen transforms into urobilin upon oxidation, displaying an orange-yellow hue. Typically, 0.5-4 mg of urobilinogen is expelled in urine over 24 hours, resulting in the normal, detectable presence of a small urobilinogen quantity.

The diurnal variation of urobilinogen urinary excretion peaks in the afternoon, emphasizing the preference for a 2-hour post-meal sample for accurate assessment.

Causes of Increased Urobilinogen in Urine

  1. Hemolysis: The excessive breakdown of red blood cells results in hyperbilirubinemia, leading to an elevated production of urobilinogen in the gastrointestinal tract. Bilirubin, predominantly unconjugated in this scenario, remains absent in urine. Elevated urobilinogen in the absence of bilirubin is characteristic of hemolytic anemia. This phenomenon is also observed in megaloblastic anemia, attributed to the premature destruction of erythroid precursors within the bone marrow—a manifestation of ineffective erythropoiesis.
  2. Hemorrhage in tissues: The increased bilirubin formation results from the breakdown of red blood cells during tissue hemorrhage.

Causes of Reduced Urobilinogen in Urine

  1. Obstructive jaundice: When there's an obstruction in the biliary tract, the transport of bilirubin to the intestine is hindered, resulting in minimal or no urobilinogen formation. Consequently, this leads to pale or clay-colored stools.
  2. Reduction of intestinal bacterial flora: The decrease in the population of intestinal bacterial flora hampers the conversion of bilirubin to urobilinogen within the intestine. This phenomenon is particularly observed in neonates and following antibiotic treatment.

The analysis of urine for both bilirubin and urobilinogen proves to be valuable in assessing a case of jaundice (refer to Table 1 for details).

Further reading: Methods for the Detection of Urobilinogen in Urine.

Blood

The identification of an abnormal quantity of intact red blood cells in urine is termed hematuria. This indicates the existence of a bleeding lesion within the urinary tract. Observable bleeding in urine, either apparent to the naked eye or through macroscopic examination, is referred to as gross hematuria. In cases where bleeding is detectable only through microscopic analysis or chemical tests, it is designated as occult, microscopic, or hidden hematuria.

Causes of Hematuria

1. Diseases of urinary tract:

  • Glomerular diseases: Conditions within this category include glomerulonephritis, Berger’s disease, lupus nephritis, and Henoch-Schonlein purpura.
  • Nonglomerular diseases: This encompasses a range of conditions such as calculus, tumor, infection, tuberculosis, pyelonephritis, hydronephrosis, polycystic kidney disease, trauma, occurrences after strenuous physical exercise, and diseases of the prostate (benign hyperplasia of the prostate, carcinoma of the prostate).

2. Hematological conditions:

In the context of coagulation disorders and sickle cell disease, the presence of red cell casts, along with proteinuria and hematuria, indicates a glomerular origin of the hematuria.

Further reading: Methods for the Detection of Blood in Urine.

Hemogobin

The condition characterized by the presence of free hemoglobin in the urine is referred to as hemoglobinuria.

Causes of Hemoglobinuria

  1. Hematuria accompanied by subsequent lysis of red blood cells in urine of low specific gravity.
  2. Intravascular hemolysis: Hemoglobin becomes evident in the urine when haptoglobin, the plasma protein binding hemoglobin, is fully saturated with hemoglobin. Intravascular hemolysis manifests in various conditions, including severe falciparum malaria, clostridial infections, E. coli septicemia, trauma to red cells (such as march hemoglobinuria, extensive burns, prosthetic heart valves), glucose-6-phosphate dehydrogenase deficiency following exposure to oxidant drugs, immune hemolysis (resulting from mismatched blood transfusion, paroxysmal cold hemoglobinuria), paroxysmal nocturnal hemoglobinuria, hemolytic uremic syndrome, and disseminated intravascular coagulation.

Tests for Detection of Hemoglobinuria

Methods employed to identify hemoglobinuria include the benzidine test, ortho-toluidine test, and reagent strip test.

Hemosiderin

The occurrence of hemosiderin in urine, known as hemosiderinuria, signifies the presence of free hemoglobin in the plasma. Visualization of hemosiderin is achieved through staining urine sediment with Prussian blue stain, revealing blue granules (refer to Figure 3). These granules are situated within tubular epithelial cells or may be present independently if cellular disintegration has occurred. Hemosiderinuria is a characteristic finding in cases of intravascular hemolysis.

Staining of urine sediment with Prussian blue stain
Figure 3: Staining of urine sediment with Prussian blue stain to demonstrate hemosiderin granules (blue)

Myoglobin

Myoglobin, a protein found in striated muscles (both skeletal and cardiac), serves the function of oxygen binding. Myoglobinuria, the presence of myoglobin in urine, is associated with conditions causing injury to skeletal or cardiac muscles, such as crush injuries, myocardial infarction, dermatomyositis, severe electric shock, and thermal burns.

Chemical tests designed for the detection of blood or hemoglobin also yield a positive reaction with myoglobin, given that both hemoglobin and myoglobin exhibit peroxidase activity. The ammonium sulfate solubility test is employed as a preliminary screening test for myoglobinuria. Notably, myoglobin is soluble in an 80% saturated solution of ammonium sulfate, while hemoglobin remains insoluble and precipitates. A positive chemical test for blood conducted on the supernatant indicates the presence of myoglobinuria.

A comprehensive differentiation between hematuria, hemoglobinuria, and myoglobinuria is detailed in Table 2.

Table 2: Differentiation between hematuria, hemoglobinuria, and myoglobinuria.
ParameterHematuriaHemoglobinuriaMyoglobinuria
Urine color Normal, smoky, red, or brown Pink, red, or brown Red or brown
Plasma color Normal Pink Normal
Urine test based on peroxidase activity Positive Positive Positive
Urine microscopy Many red cells Occasional red cell Occasional red cell
Serum haptoglobin Normal Low Normal
Serum creatine kinase Normal Normal Markedly increased

Chemical Tests for Significant Bacteriuria (Indirect Tests for Urinary Tract Infection)

In addition to the direct microscopic examination of urine samples, there are commercially available chemical tests in a reagent strip form designed to detect significant bacteriuria. These tests, namely the nitrite test and leucocyte esterase test, prove valuable in settings where urine microscopy is unavailable. A positive result on these tests warrants further investigation through urine culture.

Nitrite Test: Normal urine does not contain nitrites; ingested nitrites are converted to nitrate and excreted. In the presence of gram-negative bacteria (such as E. coli, Salmonella, Proteus, Klebsiella, etc.), these bacteria, through the action of the bacterial enzyme nitrate reductase, reduce nitrates to nitrites. Reagent strip tests then detect the presence of nitrites in urine. Given that E. coli is the predominant organism causing urinary tract infections, the nitrite test serves as a useful screening tool for such infections.

Certain organisms like Staphylococci or Pseudomonas do not convert nitrate to nitrite, resulting in a negative nitrite test in these infections. It's crucial to retain urine in the bladder for a minimum of 4 hours for the conversion of nitrate to nitrite to occur; hence, a fresh early morning specimen is preferred. Adequate dietary intake of nitrate is necessary. Therefore, a negative nitrite test does not conclusively indicate the absence of a urinary tract infection, as the test detects approximately 70% of cases.

Leucocyte Esterase Test: This test identifies the esterase enzyme released in urine from the granules of leucocytes, indicating pyuria. A positive result on this test suggests the need for urine culture. The test is not sensitive to leucocytes fewer than 5 per high-power field.

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