Urine Sugar Testing- State of the Art

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1 V TEST ESIRE twswsm w"» w* 1 USE BALL POINT PEN "'"SSFiXSJSS?'" PRESS FIRMLY ijr?sl!!?! <(( JL «V- M, a ^ ' J.i, I The price of progress is continuous change. This applies to the state of the art in urine sugar testing as it does to all other areas of modern laboratory science. Testing for urine sugar is one of the oldest of laboratory activities and it has accordingly undergone and is still undergoing more changes than many other laboratory testing areas. Tests for urine sugar are of significance in screening, in diagnosing, in monitoring, and in exploration or research. B«I«W. wi MTUV.TO e-nn ww*i*«rows -W»HCT -HIP. wnra wen Urine Sugar Testing- State of the Art by Alfred H. Free, Ph.., and Helen M. Free History Urine sugar testing was established as a clinical laboratory procedure during the 19th Century. The reducing action of glucose in urine in a hot alkaline solution of blue divalent copper was the basis of the first tests which were described. Trommer proposed a procedure which was followed by the classic test of Fehling. This involved the use of two separate stable solutions which were mixed prior to testing. Stanley Benedict, during the time he was a college student at the University of Cincinnati, proposed a test for urine sugar (utilizing a single stable reagent) which has come to be called Benedict's qualitative reagent. 1 This test involves heating a portion of the copper-containing reagent with a few drops of urine. Shortly thereafter, the quantitative procedure of Benedict appeared. This also involves a liquid reagent utilizing a titration procedure with hot reagent and urine. The first commercial test for sugar in the urine appeared in the early 1940's during World War II. This procedure utilizes a tablet called Clinitest (Ames Company) which, on dissolving, provides an alkaline copper reagent and contributes its own source of heat. The tablets are manufactured in a very dry environment so that the ingredients do not interact when stored properly. Alfred H. Free, Ph.., is Vice President, Technical Services and Scientific Relations, Ames Company, Elkhart, Indiana. Mrs. Helen M. Free is Senior New Product Manager, Ames Company. In 1956, two enzyme tests for recognition of glucose in urine appeared almost simultaneously. These were Clinistix (Ames) and Tes-Tape (Eli Lilly & Co., Indianapolis). Each of these tests is based on the use of the enzyme, glucose oxidase. Two years later, the first of a series of combination tests for urine constituents, including glucose, were made available as Uristix (Ames). Subsequently, other combination tests including urine glucose have become available. iastix is an enzyme dip-and-read test for urine glucose which provides good quantitation. It was introduced by Ames to the urine testing series in The first automated procedure for routine urinalysis appeared in 1972 as Clinilab (Ames). This system involves a test for urine glucose similar to Clinistix. LABORATORY MEICINE VOL. 6, NO. 2, FEBRUARY ownloaded from

2 The most recent test for urine sugar is a specific enzymatic test for urine galactose Galactostix (Ames) which became available in Methods Laboratory Methods Since the basic methods of Benedict for qualitative and quantitative measurements of reducing substances during the first decade of the present century, there have been several laboratory procedures described. But none has achieved any significant usage. Benedict's qualitative test has to be done in a uniform standardized way if consistent useful results are to be achieved. Neither the dinitrosalicylic acid reduction method nor the anthrone procedure has been successfully adapted for urine sugar determination. In addition, liquid enzymatic methods ordinarily require prior treatment of the urine. Benedict's tests, therefore, remain the only significant laboratory methods. Commercial Methods uring the past three decades there has been an impressive increasing usage of commercial tests for urine sugar testing. The first of these, Clinitest, has achieved widespread usage and is employed extensively at the present time. Enzyme tests specific for glucose provide a very sensitive and specific test system for urine glucose. These have greatly expanded the volume of testing for urine sugar because of their convenience, either as single tests for urine glucose only or as a part of a multiple test system for routine urinalysis. Procedures Benedict's Qualitative Test. This time-honored test is based on the reduction of blue cupric ions to orange cuprous ions by sugar in the presence of heat and alkali. Five ml. of Benedict's qualitative reagent and eight drops of urine are mixed in a test tube and heated in a boiling water bath for five minutes. The mixture is cooled, the precipitate is resuspended, and the colors are graded. Cook et a/. 2 have proposed the following scheme: negative (blue and turbidity slight enough to read newsprint through the tube), trace (green and more turbidity), 1+ (yellow-green, green predominating), 2+ (green-yellow, yellow predominating), 3+ (orange or brown), and 4+ (brick red). Clinitest. This standarized semi-quantitative copper reduction tablet test was invented by Compton and Treneer. 3 The same principle applies as for Benedict's qualitative test. However, the tablet method is unique in that no external source of heat is required. The tablet ingredients include citric acid and sodium hydroxide in addition to cupric LABORATORY MEICINE VOL. 6, NO. 2, FEBRUARY 1975 ownloaded from sulfate and sodium carbonate. When a tablet is added to 5 drops of urine and 10 drops of water in a small tube, the heat of solution of the sodium hydroxide and its interaction with citric acid provide the necessary heat for reduction of cupric ions by the sugar present in the urine. The color of the final reaction is matched to a color chart ranging from blue through green and brown to orange with color blocks showing typical reaction for 0, VA, VI, 3 A, 1, and 2% reducing sugars. Close observation is important while the reaction boils since a phenomenon called the "pass-through" may occur with large concentrations of sugar. A concentration of more than 2% causes a fleeting orange color which fades back to brown. This color change may be missed, and the final reaction may erroneously be matched to the 1% color block. A special color chart is available which is used with 2 drops of urine instead of 5 drops. The special 2-drop color chart has color blocks over the same blue to orange range which represent color formation with 0, trace (approximately 14%), Vi, 1, 2, 3, and 5% sugar. Its use with specimens containing large amounts of sugar avoids most of the "passthrough" reactions. 4 Galatest. This product is based on the reduction of bismuth salt to bismuth oxide by urine sugar. A mound of powder which contains sodium carbonate is moistened with a drop of urine and observed for a color change indicating reduction of white bismuth salt to brown or black bismuth oxide. Benedict's Quantitative Method. In the hand of an experienced analyst, this copper reduction method provides quantitation of the amount of reducing sugars present. Benedict's quantitative reagent is carefully made to contain cupric ions in each 25 ml. of reagent equivalent to the reducing activity of 50 mg. glucose. Urine is added from a burette while the reagent with added sodium carbonate is kept boiling. Titration is continued to the endpoint, which is the disappearance of the last trace of blue from the solution and the appearance of insoluble cuprous thiocyanate. This shows the reduction of blue cupric ions to cuprous ions which react with the thiocyanate in the reagent. Calculation is then made of the urine sugar level. Clinistix. This dip-and-read test is based on the enzyme glucose oxidase. Therefore, unlike the others based on reduction of metallic salts, it is specific for glucose. The dry, solid-state reagent also contains, in addition to glucose oxidase, another enzyme, peroxidase, and the chromogen, orthotolidine. When the strip is dipped into a urine specimen, any glucose present reacts with oxygen in the (Continued on page 27) For free information circle 8 on Reader Service Card

3 Good Control *, Fig. 1. State of the Art. (Continued from page 24) presence of glucose oxidase to form gluconolactone strip Uristix (for glucose and protein) to Multistix, and hydrogen peroxide. 5 In turn, the hydrogen a seven-way strip which tests for glucose, protein, peroxide in the presence of peroxidase oxidizes the ph, ketones, occult blood, bilirubin, and urobilinogen, chromogen to give blue oxidized orthotolidine. A red background dye is added to give an easily Tes-Tape. This test is based on the same principle matched negative color, and three positive color as Clinistix, and the active ingredients are the same, blocks are light, medium and dark purple. ]t > too > is specific since it is based on the enzyme, glucose oxidase. The product form is a tape in a This same glucose oxidase composition is a part dispenser. A portion of the tape is torn off and of all multiple reagent strips from the two-test dipped into the urine specimen. The darkest area For free information circle 8 on Reader Service Card LABORATORY MEICINE VOL. 6, NO. 2, FEBRUARY 1975 ownloaded from

4 Reagent Care Specimen Care Test Procedure Recording Results Clinical Interpretation Personnel training Proficiency & Controls S Table I 25-point Check List, Optimal Utilization Urine Sugar Testing Reagent storage protected from heat and moisture Bottle cap replacement tightly after each use Collection clean, disposable container used Identification-from collection to receipt of results Fresh urine specimen promptly brought to laboratory Prompt testing efficient laboratory handling Refrigeration or preservation used when necessary irections always carefully followed Repeat procedure whenever results are questionable Prompt recording immediately after results are obtained Recording and reporting efficient procedure used All results given careful consideration Repeat specimens & tests ordered when appropriate Follow-up laboratory study performed when appropriate Understanding results continuing education Interfering substances possibilities known and considered All personnel involved in urinalysis testing appropriately trained in: Regular use of controls Participation in proficiency programs reagent handling specimen handling testing procedures recording and reporting results I known reference specimens blind controls corrective action based on results C results considered / corrective action taken of the moistened tape is matched to a color chart at one minute. Color blocks are given for 0, 1/10, 14, Vi, and 2% glucose. The two higher concentrations of glucose require an extra minute for color development. The background dye in this reagent is yellow, so positive results give various shades of green. 6 iastix. The third test composition based on the exzyme glucose oxidase is iastix. It uses potassium iodide as the chromogen, and it contains a light blue background dye. A blue color is negative, and color blocks with shades of green through brown represent 1/10, 14, VI, 1, and 2% glucose. Color reactions are matched at 30 seconds. Galactostix. The newest dip-and-read urine sugar test is one specific for galactose. It is similar to the enzymatic glucose tests except that the enzyme catalyzing the first part of the chemical reaction previously described is galactose oxidase instead of glucose oxidase. The products of this reaction are galactohexodialdose and hydrogen peroxide. The reaction of hydrogen peroxide with the chromogen produces a blue color in one minute as a positive test for galactose in urine. The reaction is not quantitative, but there is no need for it to be since the presence of qualitatively detectable amounts of galactose in urine is abnormal. 28 LABORATORY MEICINE VOL. 6, NO. 2, FEBRUARY 1975 ownloaded from

5 Clinical Utility Urine sugar tests have established themselves as having great clinical utility as screening tests for recognizing diabetes and other abnormalities of carbohydrate metabolism that result in the excretion of sugar in the urine. It should be well recognized that some of the conditions which give rise to urinary reducing substances or glucose are benign, whereas others can be quite serious and merit early detection. Reducing sugars that may be present, other than glucose, include pentoses, lactose, galactose and fructose. The great majority of positive urine sugar tests are due to glucose; however, the recognition of rare, inborn errors of metabolism, such as galactosemia, can be quite important. The clinical utility of a test for sugar in urine is such that this test is a part of essentially every "routine urinalysis." Urine sugar tests are used as an aid in diagnosing diabetes in conjunction with blood sugar measurements and carbohydrate challenges or glucose tolerance tests. Ordinarily in the diagnosis of diabetes, there is a tendency to give more weight to information gained in studying the blood sugar than to information relating to the excretion of glucose in urine. However, there are few clinicians who ignore or disregard results obtained from urine tests in diagnosing diabetes. Urine sugar tests also play an important role in the differential diagnosis of certain acute problems. The time-honored example, which is taught to every medical and paramedical student, is the contribution a positive or negative test for urine sugar can make in the differential diagnosis of comatose conditions where either ketoacidosis or insulin shock is suspected. In one case, the urine is likely to contain large quantities of glucose; and in the other, it is likely that the urine will be free of sugar. Important diagnostic information is contributed by urine sugar tests not only in known diabetics, but in any situation involving comatose or semicomatose patients. Another of the very great usages of urine sugar tests is in the monitoring of diabetes. r. Eliot P. Joslin of the Joslin Clinic in Boston and r. Russell Wilder of the Mayo Clinic in Rochester, Minnesota both played prominent roles in establishing the utility and popularizing the practice of diabetics testing their own urine. Specific recommendations of diabetic patients vary depending on the severity of the disease. Semiquantitative tests give patients and their physicians a means of obtaining an index of the degree of imbalance. Most patients benefit from use of Keto- iastix, the combination ketone-glucose test when ketonuria appears to be a possibility. Free and Free 7 have devised a test using baker's yeast and finger cots which allows blind diabetics to test their own urine using a "touch" technique. Figure 1 presents a series of photographs representing several aspects regarding the state of the art of urine sugar testing. This group includes illustrations of Benedict's qualitiative and quantitative tests, the use of single commercial tests for urine sugar Tes-Tape, iastix, Clinistix, Clinitest, and 2- drop Clinitest, the use of glucose tests as a component of multiple tests, such as Uristix, for screening in OB/GYN offices, or Multistix or the automated Clinilab for routine urinalysis, galactose testing directly from the diaper, and the use of the yeast fermentation test for blind diabetics. It also stresses the attention required for good results. This includes training of laboratory personnel, use of controls and proficiency testing, protection of reagents from heat and moisture, and familiarization with interfering substances that might be encountered. It also illustrates good instruction to the diabetic patient, testing by patients with strict attention to the directions for testing, and literature reference. Advantages The advantage of using specific enzyme tests for glucose or galactose lies in their specificity and in the clinical application of this quality. For example, in diabetes detection drives, a specific test for glucose is of great advantage in eliminating other urine constitutents which might possess reducing activity and be unrelated to the disease being sought. For screening infants, the specific test for galactose is appropriate. The enzyme tests are more sensitive than the reducing tests and, therefore, an enzyme test may give a positive result on the same urine which gives a negative reducing test. It is important to realize that neither test is "wrong." This type of reaction indicates that about 0.1% sugar is present; it does not indicate a "false positive" enzyme test. There is no substance excreted in urine which has been found to give false positive enzyme test. Limitations The reducing sugar tests lack specificity and, therefore, can be erroneously interpreted if this fact is not kept in mind. The sensitivity of Clinitest has been adjusted to avoid trace amounts of nonspecific reducing substances. However, several drugs are excreted in the urine as glucuronides which are (Continued on page 45) ownloaded from LABORATORY MEICINE VOL. 6, NO. 2, FEBRUARY