Urine pH Measurement by Dipstrip

The test pad on the dipstrip is impregnated with various combinations of reagents that produce color gradations depending on the urinary pH of 5–9. Most test pads include a mixture of methyl red and bromothymol blue resulting in shades of orange, yellow, green, and blue. The lowest pH (5.0) generates orange color followed by yellow and green for intermediate pH values, and the highest pH (8.5) generates blue [4, 8, 33]. The color that develops on the reagent pad is compared visually to a color chart provided by the dipstrip manufacturer immediately following the application of urine to the dipstrip. The pH that is recorded is the one that most closely matches the color block on the chart and is estimated to the nearest 0.5 or 1.0 pH unit depending on the manufacturer of the dipstrip. The measurement of pH by dipstrip is considered accurate to within 1 pH unit (±1 unit) when compared to pH meter [8, 26], a high level of discordance that is not generally appreciated in veterinary medicine. Reporting urinary pH to the nearest 0.5 pH unit can lead to the false impression that the dipstrip pH measurement provides a high level of accuracy and precision (Heather Wamsley personal communication 2019). For this reason, some urinary dipstrips are manufactured that provide pH to the nearest whole unit pH value instead (e.g. 6, 7, and 8).

Historically, urine pH by dipstrip has been considered useful in providing an approximation of urine pH during routine urinalysis [34, 35]. Unfortunately, the estimation of urine pH by dipstrip shows only poor‐to‐moderate agreement with pH measured by meter. The measurement of urine pH by dipstrip has been described as inherently inaccurate and its continued use is one of convenience instead of accuracy [36]. Consequently, urine pH by dipstrip often is not accurate enough to make informed clinical decisions in many disorders. Urine pH by dipstrip in spot urine samples was compared to pH measured by an electrode in 24‐hour urine samples from humans. Greater inaccuracy was found in urine samples with a spot urine pH > 6.5, but pH by dipstrip was associated with a large range in pH by electrode in this study. Based on these findings, urine pH by dipstrip from spot urine was considered unacceptable in the monitoring and management of human patients with urolithiasis [37].

The inaccurate determination of urine pH by dipstrip could also result in faulty treatment strategies to alter urinary pH to prevent urolithiasis. For example, the dipstick method tended to overestimate pH in one study of dogs suggesting the presence of alkaline urine when the urine was mildly acidic by pH meter [35]. Urinary pH by dipstrip reported acidic urine in cats when readings by pH meter indicated a neutral to mildly alkaline urine in some cats [34]. The clinical interpretation of inaccurate urinary pH readings by dipstrip is most likely to occur when pH values are near neutral [38]. Urine pH values more removed from neutral are likely to align in the same bin when measured by meter (high–high or low–low) despite differing actual values.

When measured by dipstrip, visible blood contamination lowered the score assigned to urinary pH in both dogs and cats of one study, though the magnitude of change was considered small. Urinary pH was significantly lower in red urine samples from dogs and cats, and in combined red (light pink, dark pink, and red) samples from cats [12].

Extremes in urinary pH have the potential to alter color development on other reagent pads on the dipstrip, as can occur with a high urine pH and an increased reaction for protein in urine that is highly concentrated. Also, contamination of the pH pad with acid buffer flowing from the protein reagent pad during poor technique is a consideration for a falsely low urinary pH [12, 39].

Automated readers are available to read dipstrip results and may provide more consistent and more accurate readings of urinary pH. Pooled urine samples from dogs that were acidic, neutral, or alkaline were measured by dipstrip for pH by visual and by automated methods to detect color development. Compared to urinary pH by meter as the gold standard, accuracy for pH readings was higher with the use of automated readings compared to that recorded following visual analysis assessed by multiple operators for all the samples [22]. In another study, there was good agreement between urinary pH determined by visual assessment and an automated reader in urine from dogs of one study (a pH meter was not used for comparison) [20]. Similarly, correlation between urinary pH by visual reading and those by an automated reader was considered excellent in another study of urine from dogs and cats [21].

Urine pH Measurement by a pH Meter

The gold standard for the measurement of urinary pH is provided by either benchtop or portable pH meters that have been properly maintained and calibrated [14, 30,34–36]. If accurate measurement of urine pH is important in clinical decision‐making, a pH meter should be used [26,34–36, 38]. Relatively inexpensive portable pH meters can be used in‐house, or a refrigerated urine sample can be shipped to a reference laboratory within 24 hours of collection to obtain an accurate urinary pH [30, 35].

Comparisons of Dipstrip and pH Meter Methodology

There can be significant discordance between urinary pH measured by dipstrip and pH meter. A pH meter can be used to confirm or determine more accurate results, particularly when pigmenturia obscures or alters test pad color [32].

The degree of agreement for urinary pH was compared between a benchtop pH meter, two portable pH meters, urinary dipstrip, and pH paper in urine from normal cats. The expiration of the electrodes on one of the portable pH meters accounted for a negative bias on the pH readings. The pH paper had poor agreement, and dipstrip pH readings had intermediate agreement with pH measured by the benchtop analyzer. The pH measured by dipstrip had an average negative bias of −0.12 pH units compared to the benchtop analyzer, but displayed much greater discordance at lower pH values on the dipstrip. The difference in urine pH measured by the benchtop analyzer after 24 hours of refrigeration (mean increase of 0.04) compared to time 0 was not considered clinically relevant. Electrode maintenance and daily calibration of portable pH meters is important to ensure the accuracy of test results [34]. In another study of urine from hospitalized dogs, urinary pH was compared between a benchtop pH meter, four portable pH meters, urinary dipstrip, and pH paper. Three of the four portable pH meters provided nearly identical results to that from the benchtop meter. However, there was moderate‐to‐poor agreement for urinary pH results determined by dipstrip or pH paper compared to the benchtop meter [35]. Mean urinary pH measured by dipstrip was significantly higher (7.11 ± 0.12) than when measured by pH meter (6.83 ± 0.12) in urine from dogs; urinary pH did not change when analyzed before or after centrifugation [40].

In cats, urinary pH by dipstrip was consistently lower compared to measurement by pH meter; a consistent error in direction of pH was not found in dogs of the same study. The authors of this study suggested that pH by dipstrip should be within ±0.25 pH units of that measured by pH meter in order to be clinically useful, but this only occurred in 40% of the samples analyzed. A dipstrip pH of 7 corresponded to a pH meter pH of 6.2–8.2 [36]. In another study in cats, a dipstrip urinary pH of 6 corresponded to a meter pH of 5.81–7.18 [34].

Expected Urine pH in Health and Disease

The normal urinary pH of dogs and cats is generally considered to be from 5.0 to 7.5 [41, 42] or from 6.0 to 7.5 [3, 43] depending on age, nutrients in the diet, food intake (ad libitum or meal fed), and timing of the urine sample collection. The mean urinary pH of 5.1 ± 0.3 in one day old Great Dane puppies significantly increased on day 5 of age to 5.3 ± 0.6. The initially low urinary pH was attributed to a combination of respiratory and metabolic acidosis in newborn pups [44].

Bone growth of the young is associated with the production of calcium apatite and protons that are excreted into urine, which decreases urinary pH. When bone growth is finished, urinary pH tends to increase [36, 45]. The possibility for the elaboration of slightly more acid urine following the sleeping hours and mild respiratory acidosis [42] has not been critically evaluated in dogs or cats.

The amount and type of protein, acidifying or alkalinizing minerals [46], and organic anions and cations in the diet contribute to urinary pH. Decreased urinary pH is expected when eating higher concentration of proteins, especially those with sulfur‐containing amino acids, which generate acid products following metabolism (Table 5.3). The consumption of organic cations usually increases the urinary pH (Table 5.4). The ingestion of diets based on cereal or vegetables may generate alkaline urine due to the excretion of alkaline end products following metabolism. Urinary pH is lower in young cats compared to adult cats eating the same diet. Urinary pH tends toward neutral to slightly acidic during fasting of normal animals, as demonstrated by the finding of a urinary pH by meter of 6.5–7 regardless of the baseline pH when normal adult cats were fasted for three days [45].

An increased urinary pH of varying magnitude following food intake is sometimes observed in dogs and cats. This transient increase in urinary pH has been attributed to the so‐called postprandial alkaline tide as a response following gastric acid secretion [42], but this explanation appears too simplistic [47]. A fall in urinary acid excretion was still seen following food intake despite blockade of gastric acid secretion in one study of humans, so the mechanism for the generation of this alkaline urine remains unclear. Also, a decrease in urinary acid excretion was seen after the feeding of high compared to lower concentrations of dietary protein, fasting, or ingestion of water alone [47]. The postprandial alkaline urine effect can be protracted, and it may require 8–12 hours fasting after feeding for more acidic urine to return. For these reasons, the timing of the urine sample collection in relationship to eating or fasting should be factored into the interpretation of neutral to alkaline urinary pH.

An abnormal urine pH (>7.5 pH) was detected in 17.4% of samples from dogs and in 8.2% of samples from cats at a veterinary teaching hospital. Of these abnormal pH samples, 52.3% from the dogs and 74.4% from the cats were subsequently found to be associated with some microscopic abnormality [41]. A wide range of urinary pH was encountered in dogs evaluated at a teaching hospital, from 5.0 to 9.0 as determined by visual inspection of the color reactions. Urinary pH was 8–9 in 40% of these samples and 5.0 in 24%. A pH of 6.5–7.0 was encountered in 22% of the samples [48].

Urinary pH is not a reliable indicator for the precise assessment of acid–base status, as there are many instances where urinary pH is discordant from systemic acidemia or alkalemia. Urinary pH is influenced by acid–base balance, but this is not a simple function of the blood pH, as this can be affected by systemic disease, renal tubular health (e.g. CKD and AKI), effective circulating blood volume, and concurrent electrolyte disorders. In general, metabolic and respiratory acidosis result in aciduria, whereas metabolic and respiratory alkalosis result in alkaline urine following compensatory mechanisms. The optimal interpretation of urinary pH is made when blood gases are available to fully characterize the nature of the acid–base disturbance.

Paradoxical aciduria occurs in some dogs and cats that have metabolic alkalosis in association with the contraction of circulating blood volume, along with the depletion of sodium, potassium, and chloride as can occur during gastrointestinal (GI) obstruction. In these instances, alkalinuria develops as a compensatory response to restore normal acid–base balance following fluid therapy to restore circulating volume and chloride and potassium repletion. Paradoxical alkalinuria potentially develops in states characterized predominantly by hyperkalemia in which the translocation of potassium into and hydrogen ions out of cells occurs. Uncommonly, distal renal tubular acidosis may be responsible for alkaline urine or an inability to maximally acidify urine. Because of these variations, evaluation of urinary pH by itself can be misleading and lead to the wrong characterization of an acid–base disturbance. Intermittent alkaline urine was documented in a cat that was fed an acidifying diet, likely due to stress‐induced respiratory alkalosis during visits to the veterinary hospital [49].

Drugs may affect urine pH, and the drug history of the animal should be considered when evaluating urine pH. Urinary acidifiers (e.g. ascorbic acid, d,l‐methionine, and ammonium chloride) result in the production of acidic urine. The administration of alkalinizing agents, such as sodium bicarbonate or citrate, and carbonic anhydrase inhibitor diuretics (e.g. acetazolamide) causes the formation of alkaline urine.

Urinary pH often aligns with certain types of crystals in urinary sediment. For example, urate, cystine, and calcium oxalate crystals are more apt to form in acid urine, whereas struvite tends to form in alkaline urine. The prediction of the urolith type in the absence of quantitative stone analysis is facilitated by knowing the urinary pH in addition to breed, age, sex, and radiodensity of the stone. Manipulation of urinary pH is often attempted to try to prevent the recurrence of urolithiasis based on crystal type and solubility characteristics.

Urinary pH may not indicate how much acid is being excreted by the kidneys in some instances, if the urine is changed after renal processing [50]. Urinary tract infection (UTI) with urease‐producing bacteria (Proteus spp, Staphylococcus aureus, Mycoplasma spp) can be associated with persistently alkaline urine, often with urinary pH ≥ 7.5 and as high as 9.0 in some dogs. Many bacterial UTIs, however, do not result in alkaline urine and exist in neutral or acid urine [51]. Alkaline urine may also develop if a sample of normal urine is contaminated with urease‐producing bacteria. If this urine is allowed to stand at room temperature, bacteria proliferate and urea can be hydrolyzed, resulting in alkaline urine the same as happens with a real urinary infection.

Methodology

Increasing protein concentrations in the urine cause the chemical indicator in the reagent pad (tetrabromophenol blue or tetrachlorophenol‐tetrabromosulfonphthalein) to change color [1, 4, 5, 32]. A positive reaction on the reagent pad does not identify a particular protein, but the indicator is much more sensitive to albumin than to globulins, Bence Jones protein, mucoproteins, hemoglobin, myoglobin, and other nonalbumin proteins [4, 8, 32, 33, 50, 52]. The color change on the reagent pad is based on the ability of free amino groups of proteins in the urine to bind to the color indicator, which varies by specific protein. This test pad detects albumin most readily since albumin has an abundance of free amino groups compared to other proteins in urine and so develops the strongest color. Nonalbumin urinary proteins can be detected if they have enough free amino groups and the protein concentration is high enough. Consequently, a negative reagent pad reaction for protein does not automatically exclude the presence of these nonalbumin proteins [1, 5, 32, 38, 53]. Specific bins for the degree of color development on the reagent pad and the protein concentration reported vary by manufacturer, especially at the trace and maximal amount of protein that is reported.

Albumin in the urine is the protein of greatest interest in most clinical cases, as it is the predominant urinary protein in disease and in health [1, 32, 38,54–58]. The gradations in color intensity for each block/bin of protein reagent pad on the dipstrip are subtle making it difficult at times to assign the proper block during visual inspection. It appears likely that the assignment of the proper color block for urinary protein has the most errors of any pad on the dipstrip when assessed manually using visual inspection. Any abnormal color of the urine (hemorrhage bilirubin) also makes it difficult to impossible to properly read the color that develops on the reagent pad [3].

The lower limit of sensitivity for detection of proteinuria using conventional dipstrips is approximately 10–20 mg/dL for a trace reading on the reagent pad, depending on the specific manufacturer. The ability to detect protein in urine (sensitivity) depends on the type and proportion of urinary proteins [4]. Maximal intensity color reaction occurs at protein concentrations ≥2000 mg/dL, ≥1000 mg/dL, ≥600 mg/dL, 500 mg/dL, or 300 mg/dL depending on the specific dipstrip manufacturer. In general, the protein concentration of urine measured by dipstrip is reported qualitatively as negative (<10 or 15 mg/dL), trace (10–20 mg/dL), 1⁺ (30 mg/dL), 2⁺ (100 mg/dL), 3⁺ (300 mg/dL), or 4⁺ (1000 mg/dL). 1⁺ and 2⁺ reactions are the most consistent reagent pad determinations among different manufacturers. Due to difficulty in the interpretation of trace protein reactions, the reporting of trace values can be suppressed by some laboratories [1].

Interferences

Visibly bloody urine can result in the reporting of false‐positive protein results from discoloration of the reagent pad [8, 12]. A repeat dipstrip measurement using urine supernatant after RBC have settled by gravity or after centrifugation can lessen the discoloration of the reagent pad. The dipstrip reagent pad for protein did not develop a positive reaction (100 mg/dL) until the hemoglobin concentration was >5 mg/dL in one study from dogs. Less than maximal reactions for hemoglobin on the dipstrip at the same time that the protein dipstrip is positive indicates that the protein being detected on the reagent pad cannot be attributed to hemoglobin [52]. According to one package insert, false‐positive reactions are also noted to occur when reagent pads are exposed to strongly alkaline urine (>8.0 pH), following treatment with some intravenous (IV) blood substitutes, and when the urine sample has been contaminated with disinfectants (quaternary ammonium or chlorhexidine) that activate color development on the reagent pad (Table 5.5) [33]. False‐negative results for reagent pad protein can occur in urine that is highly acidic (pH < 6.0) or urine that is minimally concentrated or dilute (1.001–1.017 USG). Results on the reagent pad do not appear to be affected by turbidity, radiographic contrast agents [59], most medications, and preservatives that are in the urine sample.

Interpretation

Small amounts of protein are excreted into urine by the normal dog and cat, which often are read as trace to 1⁺ on the reagent pad [32, 54, 55, 57, 58]. Highly concentrated urine magnifies the concentration of small amounts of protein normally found in the urine [3, 43]. The protein reagent pad was negative in 21%, trace in 27%, and positive at ≥1+ in 52% of 97 apparently healthy senior and geriatric dogs [27]. Mean dipstrip proteinuria ≥30 mg/dL was not detected in any voided urine sample from healthy Great Dane puppies of 0–28 days of age. Trace reactions for protein were detected in 65.5% of these urine samples at 10 days of age [44].

Most of the protein in urine from normal and diseased animals is albumin [32, 38]; small amounts of globulins from the urethra and genital tract may be detected in samples collected by voiding from normal animals. Randomly collected, voided urine samples from normal dogs contained 0–56 mg/dL of protein as measured by a urinary dipstrip or heat precipitation [60]. In another study of 31 normal dogs, positive urinary dipstrip reactions for protein recorded as trace (15 mg/dL) or 1+ (30 mg/dL) were common. This resulted in the recommendation that trace to 1+ reactions for protein on the dipstrip be considered within the reference range for dogs and not automatically be considered a false positive. By benchtop methods, the urinary total protein ranged from 3.0 to 39.0 mg/dL with a mean of 20.4 mg/dL, median of 15.0 mg/dL, and with the highest UPC at 0.21 [61]. When the dipstrip for urinary protein is ≥2+ regardless of USG, the UPC will usually be >0.4 for the cat or >0.5 for the dog. In minimally concentrated or dilute urine, a trace or 1+ dipstrip reaction for protein may indicate clinically relevant proteinuria when further assessed by UPC or microalbumin (MA). In samples with isosthenuria or very dilute urine (1.001–1.017), the dipstrip may register as negative or trace despite the presence of clinically relevant proteinuria due to the lower of limit of sensitivity (10–20 mg/dL) of the reagent pad.

The accurate interpretation of proteinuria necessitates integration of findings from the history, physical examination, urinary sediment activity, degree of urine concentration, and method of urine collection (Table 5.6). Postrenal proteinuria (UTI, stones, neoplasia) is most commonly encountered, renal origin is second in frequency, and prerenal proteinuria is uncommon in dogs and cats with clinical disorders. The origin of proteinuria as prerenal, postrenal, genital, glomerular, or tubular cannot be determined by the magnitude of the proteinuria in isolation on the dipstrip reagent pad. Persistent proteinuria associated with a normal urinary sediment (inactive sediment with few WBC, RBC, nonsquamous epithelial cells, no bacteria) or a sediment with excessive numbers of hyaline and granular casts is the hallmark of glomerular disease (e.g. glomerulonephritis, glomerular amyloidosis, glomerulosclerosis). Proteinuria associated with hematuria or pyuria does not allow anatomical localization, as inflammation or hemorrhage could occur anywhere along the urinary or genital tracts, allowing the entry of plasma proteins into the urine. Marked hematuria can be associated with the detection of proteinuria on the reagent pad, while disorders causing substantial pyuria can result in concomitant mild‐to‐moderate proteinuria by reagent pad. If the sediment is active and proteinuria is mild to moderate, consider inflammatory renal disease or disease of the lower urinary or genital tract to account for the proteinuria [62, 63].

Urine protein results should be integrated with the degree of urine concentration (USG) for the interpretation of whether the proteinuria is likely to be pathological or not. Higher protein concentrations in mg/dL are expected from urine samples with a high USG, and lower protein concentrations are expected from those with minimal urine concentration. For example, when the same amount of protein is excreted into urine, the protein may be measured by dipstrip at 30 mg/dL when the USG was 1.015 and at 300 mg/dL when the USG was 1.055. In another example, a trace or 1+ proteinuria (30 mg/dL) in a urine sample with a USG of 1.007 may be clinically relevant. The same 1+ proteinuria via reagent pad in a urine sample with a USG of 1.065 is less likely to be clinically relevant as a consequence of the concentration of urinary proteins following the removal of water by the kidneys in such a highly concentrated urine sample. Any positive protein pad color reaction in patients with minimally concentrated or dilute urine (1.001–1.020) may be clinically relevant and should be further investigated with UPC [1, 5, 32, 43, 53].

Trace and 1⁺ dipstrip reactions are the most problematic, regardless of the degree of urine concentration, to know if they indicate pathological proteinuria or not without confirmatory testing (UPC or MA). Dipstrip protein ≥2⁺ are usually associated with pathologic proteinuria and albuminuria. In some commercial veterinary laboratories, a trace or 1⁺ positive color reaction on the dipstrip is confirmed with sulfosalicylic acid (SSA) precipitation testing. If both are positive, then the proteinuria is deemed to be “real.” Other laboratories do not confirm the dipstrip reaction with SSA, but rather recommend a UPC to verify the significance of the positive pad reaction [38]. Quantitative methods for protein determination using the UPC are recommended to confirm proteinuria detected by dipstrip reagent pads. Urinary electrophoresis can be helpful to identify globulins entering urine, especially in cases with multiple myeloma. Since urinary protein excretion can display biological variation [4], it should be determined if findings from reagent pad positive reactions for protein are repeatable on subsequent urine samples. Alternate methods to measure protein as well as causes and interpretation of proteinuria are discussed in greater detail in Chapter 7.