Basic Examination of Urine

Red Urine

The most common abnormal color for urine is red or red-brown. When seen in females, menstrual flow contamination should be considered. Hematuria (presence of red blood cells [RBCs]), hemoglobinuria, and myoglobinuria may produce pink, red, or red-brown coloration. All three of these conditions are easily detectable on reagent strip testing; however, further evaluation is necessary for absolute differentiation (see the Blood, Hemoglobin, Hemosiderin, and Myoglobin in Urine section later in the chapter).

In the porphyrias, urine coloration is variable. It is usually red in congenital erythropoietic porphyria and porphyria cutanea tarda; however, in lead porphyrinuria, the urine color is generally normal. In acute intermittent hepatic porphyria, it is normal but darkens on standing. Red urine also may be associated with the use of drugs and dyes in diagnostic tests—for example, phenolsulfonphthalein, which is sometimes used in assessing renal function, will cause a red color in alkaline urine. Patients with an unstable hemoglobin may produce urine with red-brown color that does not give a positive indication of hemoglobin or bilirubin. The pigment is probably a dipyrrole or bilifuscin. An innocuous red urine associated with ingestion of beets is seen in genetically susceptible persons.

Yellow-Brown or Green-Brown Urine

Yellow-brown or green-brown urine is generally associated with bile pigments, chiefly bilirubin. On shaking the urine specimen, a yellow foam may be seen, which distinguishes bilirubin from a normal, dark, concentrated urine, which will have white foam. In severe obstructive jaundice, the urine may be dark green.

Orange-Red or Orange-Brown Urine

Excreted urobilinogen is colorless but is converted in the presence of light and low pH to urobilin, which is dark yellow to orange. Urobilin will not color the foam on shaking; in this way, it may be confused with a concentrated normal urine. Reagent strip testing would be confirmatory in this situation.

Dark Brown or Black Urine

Acid urine containing hemoglobin will darken on standing because of the formation of methemoglobin. “Cola-colored” urine may be seen with rhabdomyolysis ( ) and in some patients taking l -dopa. Rarer causes of dark-brown urine are homogentisic acid (alkaptonuria) and melanin. Urine-containing homogentisic acid will darken more rapidly when alkaline.

Blue, Green, or Blue-Green Urine

A blue, green, or blue-green discoloration of the urine is most commonly due to food dyes or additives, some foods, or medications, including amitriptyline, doxorubicin, cimetidine, flutamide, indomethacin, methocarbamol, mitoxantrone, phenylbutazone, phenergan, propofol, promethazine, triamterene, rinsapin, and sildenafil ( ; ; ). Rare causes of blue or green urine include Pseudomonas infection and some inherited diseases, such as Hartnup disease, indicanemia, indicanuria, and familial hypercalcemia. In cancer patients receiving chemotherapy, the methylene blue included in several dysuria medications, including Prosed DS, Trac Tabs, Urised, and Uroblue, may cause green or bluish-green urine discoloration ( ; ).

Purple Urine

Urine with a bright purple color has been reported in association with long-term urinary catheterization with coexistent urinary tract infection ( ).

Chyluria

This is a rare condition in which the urine contains lymph. It is associated with obstruction to lymph flow and rupture of lymphatic vessels into the renal pelvis, ureters, bladder, or urethra ( ). Although parasitic infection with Wuchereria bancrofti (filariasis) is the prevailing cause ( ), abdominal lymph node enlargement, tumors, scoliosis surgery, and pregnancy have also been associated with chyluria. Even with filariasis, this condition is rare.

The appearance of the urine varies with the amount of lymph present, ranging from clear to opalescent or milky. Clots may form and, if sufficient lymph is present, the urine may layer with the chylomicrons on top and fibrin and cells beneath. Chylomicrons may not be apparent microscopically unless they have coalesced as microglobules. This fatty material can be extracted from urine using an equal volume of ether or chloroform. Urine phosphates, in contradistinction, will not clear with this method. Pseudochyluria occurs with the use of paraffin-based vaginal creams for the treatment of Candida infection.

Lipiduria

Fat globules appear in the urine most often with nephrotic syndrome; these consist of neutral fats (triglycerides) and cholesterol (Streather et al., 1993). Lipiduria can also be present in patients who have sustained skeletal trauma with fractures to major long bones or the pelvis. Presumably, the source of lipid is exposed fatty marrow. Keep in mind that in addition to these endogenous lipids, oily contaminants such as paraffin may float on the urine surface. Microscopic examination of the urine may be required to classify fatty materials as Oil Red O–positive droplets or cholesterol esters with polarization.

Defective Hormonal Regulation of Volume Homeostasis

Diabetes insipidus can be due to a deficiency (central/pituitary variety) of, or renal unresponsiveness (nephrogenic) to, antidiuretic hormone. In either situation, excessive thirst and water intake occur, together with marked polyuria and nocturia. Up to 15 L of urine per day may be produced.

Defective Renal Salt/Water Absorption

This can be due to the administration of diuretic agents or an abnormality of the renal tubules, resulting in sodium wasting or impairment of the countercurrent mechanism. In progressive chronic renal failure, functioning renal tissue is diminished and the ability to concentrate urine is gradually lost. To excrete the daily renal water and solute load, an increase in urine volume per residual nephron results and the urine eventually becomes isoosmotic with the plasma ultrafiltrate.

Osmotic Diuresis

In diabetes mellitus with hyperglycemia, an excessive amount of glucose is excreted, causing a solute diuresis.

Prerenal

Loss of intravascular volume may result from hemorrhage or from dehydration associated with prolonged diarrhea, vomiting, excess sweating, or severe burns. So-called third spacing is the shifting of intravascular fluids to extracellular spaces. Additionally, conditions such as congestive heart failure, sepsis, anaphylaxis, or renal artery embolic occlusion may result in a decrease in renal blood flow.

Postrenal

Bilateral hydronephrosis, resulting from high-grade or long-standing obstruction of the urinary tract, may be associated with a marked decrease in urine flow and even anuria. This can occur with prostatic hyperplasia and carcinoma. Bilateral ureteral obstruction due to stones, clots, and sloughed tissue, and urethral obstruction due to stricture or valves, are other forms of obstruction. The anuria associated with sulfonamide therapy and dehydration is due to obstruction caused by the precipitation of crystals in the renal tubules when the urinary pH is acidic.

Renal Parenchymal Disease

This should be considered after other prerenal and postrenal causes of oliguria have been ruled out. The list of conditions is extensive and includes various vascular disorders, glomerulonephritis, interstitial nephritis, and ATN. A common cause of ATN is renal ischemia due to heart failure or hypotension. Numerous nephrotoxic agents may produce ATN, including several antibiotics, mercury, cadmium, carbon tetrachloride, and glycerol. Other causes include hemoglobinuria and myoglobinuria, associated with hemolysis and muscle damage, respectively, as well as excessive quantities of intratubular proteins or crystals.

Chronic renal failure, a progressive and irreversible loss of renal function, results from several disease entities. These include hypertensive and diabetes-associated nephrosclerosis, chronic glomerulonephritis, polycystic kidney disease, and other urologic disorders. Urinary specific gravity is low and proteinuria, casts, and renal cells may be evident. Pyelonephritis or interstitial nephritis will cause predominantly tubular dysfunction with polyuria early in the disease, but later oliguria of chronic renal failure supervenes.

Methods

Several methods are available to measure specific gravity—reagent strip, refractometer, urinometer, and the falling drop method.

Reagent Strip

This is an indirect method for measuring specific gravity. The reagent area has three main ingredients present: polyelectrolyte, indicator substance, and buffer. The principle of this method is based on the pKa change of pretreated polyelectrolytes in relation to the ionic concentration of the urine. When the ionic concentration is high, the pKa (acid dissociation constant) is decreased, as is the pH. The indicator substance then changes color relative to ionic concentration, which is translated to specific gravity values. The results obtained by this method should be used with caution, since it is not affected by high amounts of glucose, protein, or radiographic contrast material, all of which tend to elevate the specific gravity readings obtained from refractometers and urinometers, described in the following sections. The reagent strip assay for urine pH must be performed carefully to avoid runover from adjacent test areas, which can cause false readings.

Refractometer (see Chapter 4)

This is also an indirect method. The refractive index of a solution is related to the content of dissolved solids present. The index is the ratio of the velocity of light in air to the velocity of light in a solution. It varies directly with the proportion of particles in solution and, therefore, with the specific gravity.

The optical analog handheld clinical refractometer is a device that requires only a few drops of urine (unlike the 15 mL of urine necessary with the urinometer). Although the refractometer measures the refractive index of a solution, the scale used is valid only for urine and cannot be used to indicate the specific gravity of salt or sugar solutions. This should be kept in mind if salt solutions are to be used for calibration. Special graphs or tables are required to convert refractive index scale numbers to solute concentrations in aqueous solutions if this should be required (American Optical Catalog Number 10403). The specific gravity reading on the refractometer is generally slightly lower than a urinometer reading on the same urine specimen by about 0.002. Digital refractometers are now available for human clinical applications.

Procedure

A temperature-compensated hand model is widely used. The instrument is temperature compensated between 60°F and 100°F (15°C to 38°C). It is damaged by heat above 150°F (66°C) and by immersion of the eyepiece and focusing ring in water. It should read zero with distilled water; the zero reading can be reset if necessary by breaking the seal over the setscrew, turning it with a small screwdriver, and resealing. Check calibration daily. Copper sulfate solutions can be adjusted to monitor a high specific gravity level as an additional check.

To make a specific gravity determination of urine, clean the surfaces of the cover and prism with a drop of distilled water and a damp cloth, and allow them to dry. Close the cover. Hold horizontally and apply a drop of urine at the notched bottom of the cover so that it flows over the prism surface by capillary action. Point the instrument toward a light source at an angle that gives optimal contrast. Rotate the eyepiece until the scale is in focus. Read directly on the specific gravity scale the sharp dividing line between light and dark contrast. The entire procedure should be repeated with a second drop of urine from the same sample.

Urinometer

This is a hydrometer that is adapted to directly measure the specific gravity of urine at room temperature. It should be checked each day by measuring the specific gravity of distilled water. If the urinometer does not give a reading of 1.000, an appropriate correction must be applied to all readings taken with that urinometer. The accuracy of a urinometer may be further checked with solutions of known specific gravity. An automated urinometer using a capacitive sensor has been described ( ; ).

Because temperature influences the specific gravity, urine samples should be allowed to come to room temperature before a reading is made or a correction of 0.001 should be made for each 3°C above or below the calibration temperature indicated on the urinometer. Corrections must also be made for protein or glucose present; subtract 0.003 for every 1 g/dL of protein and 0.004 for every 1 g/dL of glucose.

Procedure

The urinometer vessel is filled three-fourths full with urine (minimum volume required is about 15 mL). The urinometer is inserted with a spinning motion to make sure that it is floating freely. (When reading the urinometer, be sure that it is not touching the sides or the bottom of the cylinder. Avoid surface bubbles, which obscure the meniscus.) Read the bottom of the meniscus.

Falling Drop Method

This is a direct method for measuring specific gravity. It is more accurate than the refractometer and is more precise than the urinometer. This method utilizes a specially designed column filled with water-immiscible oil. A measured drop of urine is introduced into the column; as this drop falls, it encounters two beams of light. Breaking the first beam starts a timer and breaking the second turns it off. The falling time is measured electronically and is expressed as a specific gravity ( ). Despite its accuracy and use of small specimen volume, this method is not widely practiced.

Methods

The freezing-point depression method is commonly employed. A solution containing 1 osmol or 1000 mOsm/kg water depresses the freezing point 1.86°C below the freezing point of water. For methods, see Chapter 4 .

Reagent Strip

Indicators methyl red and bromothymol blue give a range of orange, green, and blue colors as the pH rises, permitting estimation of pH values to within half a unit within the range of 5 to 9. It should be read immediately, but time is not critical. Care should be taken not to have excessively wet strips where acid buffer from the protein patch runs into the pH patch, causing it to become orange.

Measurement of urine pH and acidity must always be made on freshly voided specimens. If precise measurements are required, the container should be filled to minimize the amount of dead space, and the urine covered tightly. The container should be kept cold, preferably on ice, but not frozen ( ). On standing, the pH tends to rise because of loss of carbon dioxide and because bacterial growth produces ammonia from urea.

pH Electrode

Although the estimate of the pH obtainable by indicator strip is usually sufficient, more accurate measurement with a pH meter and glass electrode may be indicated in some clinical circumstances, such as the diagnosis and treatment of patients with disturbances of acid-base balance or monitoring urine alkalinization in patients receiving high-dose methotrexate therapy or undergoing treatment for nephrolithiasis ( ; ).

Because the pH meter may tend to drift, it must be standardized with three buffers of known pH immediately before use. After standardization, spray the electrodes with distilled water, clean, and dry with a tissue. Immerse the electrode in the urine sample and report the pH of urine at the temperature of measurement.

Titratable Acidity of Urine

The pH of the urine is largely dependent on the amounts of monobasic and dibasic phosphate present. Titratable acidity is measured by titrating an aliquot of 24-hour urine (collected on ice) with 0.1 N NaOH, with pH 7.4 as an end point. Measurement may be used, together with urinary ammonia determination, in patients with chronic acidosis of obscure origin. Normal titratable acidity is in the range of 200 to 500 mL 0.1 N NaOH (or 6 mL 0.1 N NaOH per kg of body weight) or 20 to 40 mEq/24 hours. This procedure is described in previous editions of this textbook.

Heavy Proteinuria (>4 g/day)

Heavy protein loss is characteristically seen with nephrotic syndrome. Classically, a low serum albumin level, generalized edema, and increased serum lipids (cholesterol, triglycerides, and phosphatides) accompany this disorder. Lipoproteins, low-density and very-low-density, are increased in serum, whereas high-density lipoprotein, a smaller molecule, has been demonstrated in the urine ( ). It has been suggested that loss of lipoprotein lipase in urine contributes to the rise in serum lipid levels. γ-Globulin is also lost in the urine, which may contribute to susceptibility to bacterial infections commonly found in nephrotic patients. When lipid is lost in urine, many granular casts, fatty casts, and fat-filled renal tubular epithelial cells (oval fat bodies) are found in the sediment. Cholesterol ester droplets may be demonstrable by polarization.

Nephrotic syndrome is principally associated with glomerular dysfunction/damage due to (1) primary renal diseases, including idiopathic disease, and (2) systemic diseases with renal involvement ( ). Transient or mechanical causes include severe congestive heart failure, constrictive pericarditis, and renal vein thrombosis. The last can be a consequence of nephrotic syndrome because of losses of anticlotting factors in urine and elevation of serum fibrinogen. In children, a common cause of nephrotic syndrome is minimal change disease (also known as nil lesion ), a steroid-responsive glomerular disorder. Acute, rapidly progressive, and chronic types of glomerulonephritis are causes of heavy proteinuria and may be accompanied by urinary erythrocytes or erythrocyte casts. Diabetes mellitus and lupus erythematosus are systemic diseases that frequently cause glomerular injury and heavy proteinuria. Urine sediment may be “telescoped,” that is, may display all types of cells and casts in lupus nephritis or with a hypersensitivity reaction ( ). Malaria, malignant hypertension, toxemia of pregnancy, heavy metals (gold, mercury), drugs (penicillamine), neoplasia in general, amyloidosis, sickle cell disease, renal transplant rejection, and, rarely, primary antiphospholipid syndrome ( ) are additional causes of heavy proteinuria.

Moderate Proteinuria (1.0–4.0 g/day)

Moderate proteinuria may be found in the vast majority of renal diseases, including those mentioned previously, as well as nephrosclerosis, multiple myeloma, and toxic nephropathies. Also included are degenerative, malignant, and inflammatory conditions of the lower urinary tract, including irritative conditions such as the presence of calculi.

Minimal Proteinuria (<1.0 g/day)

Minimal proteinuria may be noted in chronic pyelonephritis, in which case it may be intermittent, and in relatively inactive phases of glomerular diseases. It is also seen with nephrosclerosis, chronic interstitial nephritis, congenital diseases such as polycystic disease and medullary cystic disease, and renal tubular diseases. In tubular diseases, the urinary sediment usually is not abnormal, but erythrocytes, leukocytes, and tubular cells may be seen with interstitial nephritis. However, significant sediment findings may sometimes accompany trace protein results. Minimal proteinuria is also present in postural proteinurias and transient proteinuria.

Glomerular Pattern

Glomerular disease causes proteinuria, which may be heavy (>3–4 g/day). Loss or reduction of the fixed negative charge on the glomerular basement membrane allows albumin to permeate into the Bowman space in large quantities, more than can be reabsorbed by the proximal tubular cells. When serum albumin is lost in urine, other proteins of similar size or charge are also lost (e.g., antithrombin, transferrin, prealbumin, α ₁ -acid glycoprotein, α ₁ -antitrypsin). Because tubular function may still be normal, very small plasma proteins are largely reabsorbed. Large proteins, in contradistinction, are not seen in urine while the glomerulus is still selective (e.g., α ₂ -macroglobulin, β-lipoprotein). As larger proteins appear, the proteinuria is less selective, indicating greater damage to the glomerulus (e.g., with membranous nephropathy and proliferative glomerulonephritis).

Mechanisms of proteinuria in diabetic kidney disease with specific attention to glomerular damage were recently reviewed, with special recognition that CKD is actually a multifactorial, complex disease involving a combination of both glomerular and tubulointerstitial scarring ( ; ; ; ; ).

Tubular Pattern

This is associated with loss of a small amount of urinary protein that would otherwise be largely reabsorbed. These proteins most often are of low molecular weight (e.g., α ₁ -microglobulin, N-acetyl-β-D-glucosaminidase [NAG], β-globulins such as β ₂ -microglobulin, light-chain immunoglobulins, cystatin C, and lysozyme), usually without a clear predilection for albumin-sized molecules. By radioimmunoassay, β ₂ -microglobulin excretion has been measured in microgram amounts in urine as an indication of tubular damage; its normal excretion is about 100 μg/day. A tubular pattern proteinuria occurs with renal tubular diseases such as Fanconi syndrome, cystinosis, Wilson disease, and pyelonephritis, and with renal transplantation rejection. The amount of proteinuria is typically lower than that seen with glomerular disease, at about 1 to 2 g/day. Tubular proteinuria may be missed by the reagent strip test because of the absence or very low amounts of albumin, but it may be detected by an acid precipitation method ( ). In addition, urinary tubular indexes comparing tubular proteins to albumin, particularly the ratios of α-1-m to albumin and NAG to albumin, reportedly have a high sensitivity and specificity in the differentiation between primary tubulointerstitial diseases and primary glomerular diseases ( ). There is also interest in the utilization of specific molecules released into the urine during tubular injury as monitors of renal insufficiency in patients with diabetic nephropathy. The biomarkers under study include neutrophil-gelatinase–associated lipocalin (NGAL), kidney injury molecule 1 (KIM-1), liver–fatty-acid–binding protein (L-FABP), calprotectin, inflammatory cytokines, and others ( ; ; ; ; ).

Overflow Proteinuria

Overflow proteinuria is due to the overflow of excess levels of a protein in the circulation, and can be seen with hemoglobin, myoglobin, or immunoglobulin loss into the urine. These proteins are not initially associated with glomerular or tubular diseases but may themselves cause renal damage. Myoglobin may cause ATN (see the Blood, Hemoglobin, Hemosiderin, and Myoglobin in Urine section, and case 4 in Chapter 9). Hemoglobin in low amounts is not thought to be toxic unless hypovolemia is present.

Bence Jones Proteinuria

Bence Jones protein is the light chain from a monoclonal immunoglobulin that is filtered in the kidney. Bence Jones proteinuria is associated with multiple myeloma, macroglobulinemia, primary (AL) amyloidosis, and malignant lymphomas. The incidence of Bence Jones proteinuria in multiple myeloma has been estimated as 50% to 80%; however, its demonstration depends greatly on the technique used. Bence Jones protein may be missed altogether if only a reagent strip test for protein is used. Electrophoresis and immunofixation electrophoresis methods (see Chapter 20 ) are the best detection and quantification methods, along with the immunoassay measurement of free light chains ( ) (see Chapter 45 ).

Excretion of Bence Jones protein in large amounts, sometimes several grams in 24 hours, causes the tubular cells to deteriorate because of the high levels of protein reabsorbed. Inclusions may form in the cells, and desquamated cells may form casts in the tubular lumen. Casts also form from immunoglobulin and Tamm-Horsfall protein mixtures. With renal failure, less protein is reabsorbed and more Bence Jones protein and other proteins appear in the urine. The damaged kidney is sometimes called a myeloma kidney , and the nephrotic syndrome may follow.

Microalbuminuria

Microalbuminuria is the presence of albumin in urine above the normal level but below the detectable range of conventional urine dipstick methods. Several authors have suggested that these lower urine albumin levels ranging from 20 to 200 mg/L (or an approximate rate of excretion of 20 to 200 μg/min) are an indicator of early and possibly reversible glomerular damage ( ; ). In diabetic patients, microalbuminuria is associated with a fourfold to sixfold increase in cardiovascular mortality and is an independent risk factor for renal mortality ( ; ; ). It is also more prevalent in hypertensive subjects and may be an indicator of subclinical renal and extrarenal organ damage ( ; ). Various analytic methods have been introduced, including immunologic test systems and dye-binding chemical test strips, both of which are discussed in the next section.

Reagent Strip

This method takes advantage of the protein error of pH indicators. Because proteins carry a charge at physiologic pH, their presence will elicit a pH change. The reagent strip is impregnated with tetrabromophenol blue buffered to an acid pH of 3, or tetrachlorophenol-tetrabromosulfophthalein. In the absence of protein, the strip is yellow; 30 to 60 seconds following urine application, variable shades of green develop depending on the type and concentration of protein present. Results may be read in a “plus” system as negative, trace, and 1+ to 4+. Most methods will detect 5 to 20 mg of albumin per deciliter.

As stated previously, reagent strips tend to be more sensitive to albumin than to globulins, Bence Jones protein, or mucoprotein. “Trace” results may be seen with physiologic normal excretion of protein in concentrated urine specimens from healthy individuals. High salt levels will lower results. Exceptionally alkaline and/or highly buffered urine samples may give positive results in the absence of significant proteinuria (e.g., with a patient on alkaline medication or with bacterial contamination). False-positive results can occur with highly pigmented urine, quaternary ammonium compounds, amidoamines in fabric softeners, chlorhexidine, and excessive leaching of the acid buffer of the test strip by excessive wetting. The method is unaffected by urine turbidity, radiographic media, and most drugs or their metabolites.

Sulfosalicylic Acid Method—Qualitative

This method depends on formation of a precipitate for determination of the presence of protein.

Procedure

Specimens should be centrifuged and a clear supernatant used. To approximately 3 mL of supernatant urine in a clean test tube, aliquot an equal amount of 3% SSA. Invert to mix. Let stand exactly 10 minutes. Invert again twice. Using ordinary room light (not a lamp), observe the degree of turbidity and/or precipitation, and grade the results according to the following descriptions:

• Negative—no turbidity (≈5 mg/dL or less)

• Trace—perceptible turbidity (≈20 mg/dL)

• 1+ —Distinct turbidity, but no discrete granulation (≈50 mg/dL)

• 2+ —Turbidity with granulation, but no flocculation (≈200 mg/dL)

• 3+ —Turbidity with granulation and flocculation (≈500 mg/dL)

• 4+ —Clumps of precipitated protein or solid precipitate (≈1.0 g/dL or more)

This method will detect about 5 to 10 mg/dL. Albumin, globulins, glycoproteins, and Bence Jones proteins are all detected ( ). High levels of detergents may decrease the result. When radiographic dye is present, SSA precipitate will increase on standing, and typical crystals are seen on microscopic examination of the precipitate. In this situation, another urine specimen from the patient should be assayed. However, the effects of the radiographic media may persist for up to 3 days. A reagent strip test may be substituted or heat and the acetic acid method may be used. In the acetic acid method, radiographic contrast media will clear with heat, whereas protein will increase turbidity.

Quantitative Protein Determinations and Confirmatory Methods

Quantitative measurements of urine protein are typically adaptations of one of the various precipitation methods or are colorimetric in nature. SSA and trichloroacetic acid (TCA) are commonly used as precipitants; the resultant turbidity can be measured by a photometer or a nephelometer. If a visual interpretation is performed, a set of gelled commercial standards that correspond to 10, 20, 30, 40, 50, 75, and 100 mg/dL may be used, with results reported in milligrams per deciliter as opposed to the “plus” method of screening precipitation tests. With SSA, the turbidity produced with albumin is 2.4 times that produced with globulin; polypeptides, glycoproteins, and Bence Jones proteins are also precipitated with this method. Of historic note, Exton reagent contains SSA, sodium sulfate, and an indicator—bromphenol blue. TCA, in contradistinction, will cause γ-globulin to be precipitated with greater turbidity than albumin; however, the difference is not marked.

More precise measurements suitable for smaller amounts of protein are available. In these methods, a TCA precipitate is dissolved in sodium hydroxide and measured by use of the biuret reaction. The quantitative TCA-biuret method is tedious but gives good precision. A color correction blank is used. For a comparison of biuret methods with the SSA turbidity method, see Lizana and colleagues ( ).

Several dye-binding colorimetric methods are available to quantitate urine protein. These include Coomassie blue, Ponceau S, and benzethonium chloride turbidity methods ( ). Pyrogallol Red-Molybdate will also react with protein to form a blueish-purple complex that absorbs at 600 nm.

Methods used to quantitate urinary protein have not been satisfactory. Participants in the College of American Pathologists proficiency testing surveys will be aware that the mean values reported vary twofold between methods, with the SSA method producing high values. Precision is poor, with the SSA turbidimetric method showing the poorest coefficient of variation. The TCA-biuret, Coomassie blue, and TCA turbidity methods show closer agreement and about half the coefficient of variation of the SSA method. Problems arise from nonstandardized methods. With turbidity methods, these include different acid concentrations and timing, along with variation in the protein standard.

Microalbuminuria Determination Methods

Very small amounts of proteins, such as albumin and β ₂ -microglobulin, are measured by immunologic means using antibodies to the proteins, nephelometric methods, immunoassay, protein strip electrophoresis, high-performance liquid chromatography (HPLC), or other means ( ). The Micral II test strip (Boehringer Mannheim, Indianapolis, IN) is an immunologic test system that gives an almost immediate, reliable semiquantitative determination of low urine albumin concentrations ( ). Oxytetracycline may interfere with this method, causing higher readings. There is no interference with pH. A newer method, Clinitek microalbumin (Bayer Diagnostics, Tarrytown, NY), is a highly sensitive dye-binding method ( ; ). It has the further advantage of an additional test pad for simultaneous measurement of creatinine concentration. This method is not absolutely specific for albumin, for the dye compound also reacts with Tamm-Horsfall mucoprotein. HPLC is a highly sensitive method for the early detection of microalbuminuria but is not widely utilized at this time ( ).

Bence Jones Proteinuria Determination Methods

Methods for detection of Bence Jones protein in urine include protein electrophoresis, immunofixation electrophoresis, capillary zone electrophoresis, and immunoassay for free light chains ( ) (see Chapter 47 ). The traditional electrophoretic procedure employs the Amido black stain on a 200-fold concentrated urine. Newer methods, performed on less concentrated urine, including a modified Coomassie brilliant blue stain, are comparably sensitive and specific ( ; ). The presence of Bence Jones globulin or clonal production of immunoglobulin is indicated by a single sharp peak in the globulin region on protein electrophoresis. Bence Jones globulin represents either the κ or the λ immunoglobulin light chain.

Bence Jones protein precipitates at temperatures between 40°C and 60°C and redissolves at near 100°C. Other methods depend on precipitation in the cold with salts, ammonium sulfate, and acids. In the presence of marked Bence Jones proteinuria, most methods yield positive results. When only a small amount of Bence Jones protein is present or when other globulins are present, results may be doubtful. False-positive reactions are seen when other globulins are precipitated by acetic acid in the heat precipitation method. A false-negative reaction may occur if the Bence Jones protein is too concentrated and the precipitate does not redissolve on boiling.

Diabetes Mellitus

Although hyperglycemia alone is not necessarily indicative of diabetes mellitus, the appearance of glucose in the urine necessitates further workup. When glycosuria is present, it is typically accompanied by polyuria and thirst. Inadequate carbohydrate utilization in these patients results in elevated ketone levels in the blood and urine due to increased fat metabolism ( ).

For diabetic individuals, the advantage of a urine method over a blood test for glucose is that it is painless and inexpensive. Urine glucose measurements are most useful for well-controlled diabetic individuals who do not have to make frequent adjustments in their insulin/hypoglycemic agents. In insulin-dependent diabetes, a negative urine measurement could correspond to a wide range of serum glucose levels; this is attributed to the great variation in renal threshold for glucose in diabetic patients. Therefore, urine measurements may be misleading, and home blood glucose monitoring is preferred.

Monitoring for glycosuria in diabetic patients is not without problems. Reagent strips may be difficult to interpret at the 1-g/dL (1%) and 2-g/dL (2%) glucose levels. Copper reduction tests or newer, more sensitive reagent strips may be more efficacious. With the Clinitest tablet method, diabetic patients are able to estimate reducing substance levels in urine to about 10 g/dL, using one drop of specimen rather than two or five drops. In some clinics, the 24-hour urine glucose measurement is found to be useful for monitoring patients. It represents a defined longer time period, and, with blood levels of glycated hemoglobin, it contributes to the regular overall long-term management of the disease.

Several studies have looked at the usefulness of urine dipstick testing for glycosuria as a screening method for diabetes, and the results have been mixed. concentrated on patients over 50 years of age in a general practice setting and found this method to be practical and effective, whereas came to an opposing conclusion. The latter authors suggest that if diabetes screening is carried out in general practice, blood glucose measurements should be utilized for patients in selected risk groups. The primary role of blood glucose and HbA _1c measurements in the diagnosis and management of diabetes mellitus was later endorsed by the American Association for Clinical Chemistry and other organizations ( ). However, found that both blood glucose and urine glucose self-monitoring resulted in improved HbA _1c levels at 18 months in newly diagnosed patients with type 2 diabetes if structured education was provided. Routine dipstick glucose analysis can identify gravidas at increased risk for gestational diabetes ( ).

Other Causes of Glycosuria

Glycosuria with concomitant hyperglycemia is seen in several endocrine disorders. These include pituitary and adrenal disorders such as acromegaly, Cushing syndrome, hyperadrenocorticism, functioning α- or β-cell pancreatic tumors, hyperthyroidism, and pheochromocytoma. Pancreatic disease with loss of functioning islet cells is also associated with glycosuria—for example, carcinoma, pancreatitis, and cystic fibrosis.

Numerous other causes of glycosuria with hyperglycemia have been recognized, among them being central nervous system disorders, including brain tumor or hemorrhage, hypothalamic disease, and asphyxia. Disturbances of metabolism associated with burns, infection, fractures, myocardial infarction, and uremia, as well as liver disease, glycogen storage disease, obesity, and feeding after starvation, may all be associated with glycosuria, as are certain drugs (e.g., thiazides, corticosteroids, adrenocorticotropic hormone, and birth control pills).

In pregnancy, an increase in glomerular filtration rate occurs and all of the filtered glucose may not be reabsorbed. In this situation, glycosuria may appear at relatively low blood glucose levels. Persistent or greater than trace amounts of glycosuria should be investigated. In some patients, diabetes occurs only during pregnancy. Glycosuria after a glucose challenge has been used during pregnancy, but its predictive value for gestational diabetes, preeclampsia, and low birth weight has been questioned ( ). Glucose tolerance may also be decreased in older adults, especially when patients have a poor intake of carbohydrates, but this is not necessarily accompanied by glycosuria.

Glycosuria without hyperglycemia is usually associated with renal tubular dysfunction. True inherited renal glycosuria is uncommon and is associated with reduced glucose reabsorption. In renal tubular transport diseases, glycosuria may be accompanied by impaired reabsorption of water, amino acid, bicarbonate, phosphate, and sodium—a pattern seen in Fanconi syndrome. Galactosemia, cystinosis, lead poisoning, and myeloma are additional examples of conditions associated with renal tubular dysfunction and possible glycosuria.

Fructose

Fructose appears in urine in association with inherited enzyme deficiencies that cause benign essential fructosuria and serious fructose intolerance associated with severe vomiting and liver and kidney disease ( ). Fructosuria may also be seen with parenteral feedings that include fructose. Urinary fructose has been used as a marker of sucrose intake in dietary intervention studies ( ).

Galactose

Galactose is found in the urine in genetic disorders of galactose metabolism associated with a deficiency of galactose-1-phosphate uridyl transferase or galactokinase ( ). In these diseases, galactose derived from dietary lactose is not converted to glucose. Early detection followed by dietary restriction may control the disease.

Lactose

Lactose may appear in the urine late in normal pregnancy or during lactation. In lactose intolerance, high levels of sugars accumulate in the gut; lactose will be absorbed and excreted unchanged in the urine.

Pentose

Pentosuria may follow the ingestion of large amounts of fruit, causing the excretion of l -xylulose and l -arabinose in amounts up to 0.1 g/day. It may also be seen with certain drug therapies and with benign essential pentosuria.

Sucrose

Sucrose may appear in the urine after the ingestion of very large amounts of sucrose. Sucrase deficiency is associated with intestinal diseases such as sprue in the same manner as lactase deficiency. Sucrose intolerance is an inherited disorder associated with sucrase and α-dextrinase (isomaltase) deficiencies. Symptoms are similar to those seen with lactase deficiency and occur in the first few weeks of life when sweetened food is ingested. Tolerance may develop, but sucrose may have to be avoided permanently. Factitious sucrosuria may create a high specific-gravity urine with negative glucose oxidase and negative copper reduction tests.

Reagent Strip

This method is based on a specific glucose oxidase and peroxidase method, a double sequential enzyme reaction; reagent strips differ only in the chromogen used. The method is specific for glucose—no reaction is seen with lactose, galactose, fructose, or reducing metabolites of drugs. The reagent strips may be used for semiquantitative results, which should be reported as approximate grams per deciliter. Combination glucose and ketone reagent strips detect not only ketonuria but also suppression of the glucose reaction by ketones seen with some reagent strips.

False-positive readings may be produced by strongly oxidizing cleaning agents in the urine container. Low specific gravity may falsely elevate results. Sodium fluoride used as a preservative will cause false-negative readings, as can high specific gravity and occasionally ascorbic acid. Glycolytic enzymes from cells and bacteria will reduce glucose levels in urine on standing; prompt refrigeration or testing is essential.

Chemistry .

Copper sulfate, sodium hydroxide, sodium carbonate, and citric acid are incorporated into each Clinitest tablet. Copper sulfate reacts with reducing substances in the urine, converting cupric sulfate to cuprous oxide. Based on Benedict’s copper reduction reaction,

Heat is caused by the reaction of sodium hydroxide with water and citric acid.

Procedure

Clinitest reagent tablets will detect 250 mg of reducing substance per deciliter of urine. Both five-drop and two-drop Clinitest methods can be used; corresponding color charts are available ( ). The two-drop method was developed in response to a so-called pass-through phenomenon that may occur if more than 2 g/dL of sugar is present in the urine. In the pass-through phenomenon, the solution that results after addition of the Clinitest tablet goes through the entire range of colors and back to a dark-greenish brown. This final color does not compare with any section of the color chart; however, it corresponds most closely to a significantly lower result. It is important to observe the entire reaction and to continue to observe for 15 seconds after boiling inside the tube has stopped so that reversion to a different color is not missed and a falsely low result reported.

Five-Drop Method

Place five drops of urine in a dry test tube and add 10 drops of water. Add one Clinitest tablet by easing it into the tube without touching it—it contains strong alkali. Watch while boiling takes place, but do not shake or touch the bottom of the tube—it is hot. Wait for 15 seconds after boiling stops, then shake the tube gently and immediately compare the color of the solution with the color scale. Results correspond to the following approximate concentrations: Negative, 0.25 g/dL, 0.5 g/dL, 0.75 g/dL, 1.0 g/dL, 2.0 g/dL, and pass-through. It is important to watch the solution carefully while it is boiling. If the solution passes through orange to a dark shade of greenish-brown, this indicates that more than 2 g/dL sugar is present. This should be recorded as greater than 2 g/dL without reference to the color scale. Urine samples showing this pass-through phenomenon should be retested with the two-drop method.

Two-Drop Method

Place two drops of urine in a test tube and add 10 drops of water. Add one Clinitest tablet. Watch while boiling takes place, but do not shake. Wait 15 seconds after the boiling stops, then shake the tube gently. Compare the color of the solution with the color scale supplied for the two-drop method. The pass-through phenomenon may also occur with the two-drop method with large concentrations of sugar—over 5 g/dL. Report results as 1 g/dL, 2 g/dL, 3 g/dL, 5 g/dL, and more than 5 g/dL if a pass-through reaction occurs. With negative or low-level results, the five-drop method should be performed.

Precautions .

Observe the precautions in the literature supplied with the Clinitest tablets. The bottle must be kept tightly closed at all times to prevent absorption of moisture and kept away from direct heat and sunlight in a cool, dry place. The tablets normally have a spotted bluish white color. If not stored properly, they will absorb moisture or deteriorate from heat, turning dark blue or brown. In this condition, they will not give reliable results. They are also available individually packaged in aluminum foil to help prevent this absorption of moisture. Although more expensive, such packaging is useful when a limited number of measurements are performed.