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Review for the pharmacist…Diagnostic aids
review for the pharmacist •••
diagnostic aids by Paul J. Wurdack *
A
diagnostic aid is an agent used in the detection of some abnormal condition of the body. Community and hospital pharmacists have an increasing number of these products added to their inventories each year. The pharmacist cannot assist the physician and the patient intelligently in the use of these aids without knowing how they are applied in conditions for which they are intended. At the present time, most diagnostic aids sold in community pharmacies are used to detect one or more of the following conditions l ,21. Glycosu ria-the presence of glucose in the urine; most commonly associated with diabetes mellitus. 2. Glucose in the blood-most commonly associated with diabetes mellitus. 3. Cervical glucose-the presence of glucose in the cervical mucus; most commonly associated with determinations of the period of fertility. 4. Proteinuria-the presence of protein bodies in the urine; most commonly associated with kidney impairment. 5. Ketonuria or ketosis-the presence of ketone bodies in the urine; most commonly associated with an altered carbohydrate metabolism. 6. Bilirubinuria-the presence of bilirubin in the urine; most commonly associated with liver disorders or common bile duct obstruction and subsequent jaundice. 7. Occult blood-the presence of hidden or visually undetectable blood in the urine or fecal matter; most commonly associated with neoplasms of the colon, gastric or duodenal ulcers, kidney stones, acute appendicitis, nephritis, acute liver atrophy, hemorrhoids and vitamin C and K deficiencies. 8. Phenylketonuria-presence of phenylketone bodies (principally phenylpyruvic acid) in the urine; most commonly associated with phenylpyruvic oligophrenia-a preventable mental deficiency in infants. 9. pH-the determination of the acid-base balance of urine or other secretions-most commonly associated with the patient's metabolic picture.
In addition to these conditions, the
* Presented
to tl;1e section on pharmaceutical technology a t the 1962 APHA annual meeting in Las Vegas .
598
hospital pharmacist frequently dispenses diagnostic aids used for the following diagnostic procedures 3- various x-ray procedures of the alimentary tract, heart and blood vessels, hollow viscera, sinus and fistula tracts, intradural tumors, urologic disorders as well as blood volume and blood circulation time, gall bladder and liver functions, gastrointestinal anacidity agents, myxedema, hypertension and ophthalmic involvements to name a few. An increasing number of radioactive substances such as arsenic (As74) , bismuth (Bi 205,206), calcium (Ca 45,47), cobalt (C 0 60), Gold (Au I98 ), iodine (P31), phosphorus (P32), potassium (K42), rubidium (Rb86) and sulfur (8 35) are being employed. The community pharmacist normally handles diagnostic aids which are either powders, solutions, tablets or chemical and enzyme impregnated paper strips.
urine glucose (glycosuria) The most commonly dispensed diagnostic aids are those used for the detection of urine glucose (glycosuria) by the diabetic patient. 4- 9 This urine glucose may be detected using products which are either tablets, solutions, enzyme impregnated paper strips or a powder. The solution and tablet products involve a metallic oxide reduction using copper sulfate. 10 The cupric ion in the presence of a reducing sugar, heat and alkali is reduced to cuprous oxide with a resulting color change. This color change can be compared to a standard color chart provided with the diagnostic aid. The chart indicates either percent glucose or is referred to a negative, or reaction. Commercially available diagnostic aids using this reaction are Benedict's solution, Clinitest® and Sheftel Kit®. Enzyme impregnated paper strips involve two enzymes, glucose oxidase and peroxidase, and a dye substrate orthotolidine.1l,I2 In the presence of glucose the enzyme glucose oxidase reacts to form hydrogen peroxide. The hydrogen peroxide reacts with the enzyme peroxidase and oxygen is liberated. The dye orthotolidine is oxidized with resulting color formations which may be compared with a color chart provided with the diagnostic aid . Depending on the particular product, the chart is calibrated as to percent glu-
+++
++++
Journal of the AMERICAN PHARMACEUTICAL ASSOCIATION
+, ++,
Newly appointed assistant professor at the University of Pittsburgh school of pharmacy, Paul J. Wurdack is interested in diagnostic aids in pharmacy and trade marked dosage forms. These interests have brought him speaking and teaching engagements at seminars at the University of Pittsburgh. He has authored several articles on the subjects. A Pittsburgh graduate, he went on to earn his MS and PhD at his alma mater and served as assistant professor for two years. He is returning to Pittsburgh after serving a year at the University of North Carolina as an assistant professor. He is a member of APhA, Sigma Xi, Rho Chi and Kappa Psi and has taught pharmacology to nursing students at the Pittsburgh hospital.
cose or negative, light, medium or dark. Commercially available products utilizing the enzyme reactions are Clinistix® and Tes-Tape®. A positive test for glucose using improved Clinistix®gives a shade of purple while Tes-Tape® gives a shade varying from green to blue. Because both of these products are designed to be specific for glucose, there are few possible errors involved with other reducing sugars as contaminants. A third reaction involving the detection of glucose is that using a sodium hydroxide-bismuth oxychloride-sodium silicate powder combination. I3 In the presence of a reducing sugar the normally white powder is turned gray or black by the formation of partly colloidal, metallic bismuth. A color chart for comparison is provided with
the diagnostic aid and is cali brated both as percent and negative, or reaction . This product is marketed as Sugar T est , Denco® (Galatest®). Dip-and-read paper strips used in the detection of severa l conditions have been marketed . A water-im pervious band separates each area to prevent the colors from runnin g together. One portion of the paper strip is used to detect urine glucose; a second area is used in the detection of proteins and in one of the products a th ird area is used in the measurement of pH. Uristi x® is used to detect urine glucose and proteins while Comhistix® is used to detect both of these conditions and in addition measure pH .
+++
++++
+, ++,
glucose in the blood Glucose in the blood may be determined using a diagnostic aid which first precipitates blood proteins and a fter these are removed by filtration , the clear filtrate is used for the detection of glucose. A two- tablet product utilizing these ste ps is marketed as Dextrotest®. One tablet designated as the " P " tablet contains sulfosalicylic acid which is used to precipitate insoluble blood proteins. After fi ltration, the second tablet designated as the "S" tablet is used to test the clear filtrate for the presence of glucose. The second tablet uses the principle of metallic-oxide reduction as previously cited for the detection of glucose in the urine.
over-the-counter diagnostic aids 1. 2. 3. 4. 5. 6. 7. 8.
trade name Acetest Acetone Test Albumin Test Solution Albustix Albutest Benedict's Qualitative Solution Bumintest Clinilab
9. Clinistix
10. Clinitest 11. Combistix
14. 15. 16. 17.
Dextrotest Ferric Chloride Solution Fertility Tape Galatest (Suga r Test) Hematest Hemoccult
18. 19. 20. 21.
Icotest Ketostix Litmus Paper Nitrazine Paper
22. 23.
Occultest Phenistix
24. 25 .
Hydrion Paper Rhythm Meter and Non-Medicated Paper
26. 27.
Sheftel Kit Tes-Tape
28.
Uristix
12.
13.
cervical glucose In the past several years at least two companies have marketed devices which, used in conjunction with certain enzyme impregnated paper strips, are capable of detecting glucose in the cervical mucus. 14 , 15 Cervical glucose is at its highest concentration in these fluids just before or on the day of ovulation . By use of these devices and paper strips, it is possible to determine on which days of the month the cervical glucose is at its highest concentration. Thus, the time of ovulation and the period of fertility can be accurately determined. The products and paper strips used for this determination are either medicated or non-medicated. The plastic device itself serves as a means to hold ar:d insert the paper strip which must come in contact with the cervix. Nonmedicated papers such as those which come with the Rhythm Meter® must be touched to a medicated paper such as Clinistix®, Fertility Tape® or Tes-Tape® when the device and paper are withdrawn from the vagina. Because the Food and Drug Administration 16-18 on numerous occasions has cited the dan gers of using orthotolidine dye-impregnated paper strips directly against the cervix, the products Clinistix® and Tes-Tape®
test for ketone bodies in the urine ketone bodies in the urine proteins in the urine
manufacturer Ames Company Denver Chemical Co. Endo La boratories
proteins in the urin e proteins in the urine glucose in the urine
Ames Company Ames Company various companies
proteins in the urine bilirubin , ketone bodies, occult blood, proteins, glucose in the urine, pH, others possible dependin g on how wish to stock glucose in the urin e or in conjunction with nonmedicated paper in cervical glucose glucose in the urine pH, proteins and glucose in the urine glucose in the blood phenylketone bodies in th e urine cervical glucose gl ucose in the urin e occult blood (fecal m atter) occult bloo d (urine or fecal m atter) bilirubin in the urin e ketone bodies in th e urine pH pH of th e urine or other fluids occult blood (urine) phenyl ketone bodi es in the urine pH cervical gl ucose in conjunction with Tes-Tape , Clinistix or Fertility Tape glucose in the urin e glucose in the urine or in conjunction with nonmedicated paper in cervical glucose proteins a nd glucose in the urin e
Am es Company Ames Company
should not be used in these devices. They may be utilized, however, by touching a non-medicated paper strip to them after its removal from the vagina. The product Fertility Tape® does not use the orthotolidine dye and thus may be used directly against the cervix in the device marketed as the Fertility Testor®.
proteinuria The presence of proteins in the urine generally is a sign of kidney impairment. 19 The tests for these protein bodies are based on either color or turbidity formations. The "protein error of indicators" is utilized as a colorimetric test using the indicator tetrabromphenol. In the absence of proteins, this indicator has a yellow color at a pH of three. If proteins are present, the color becomes green to blue, Albustix® reagent strips and Albutest® reagent tablets utilize this same principle. One area of each of the diagnostic aids Combistix® and Uristix®
Ames Company
Am es Company Ames Company Am es Company vari ous companies Weston Laboratorie s Denver Chemical Co . Am es Compa ny Schiefflin and Company Ames Co mpany Am es Company various companies E.R . Squibb and Sons Compa ny Am es Company Ames Company various companies G and M Laboratories
Eli Lilly and Company Eli Lill y a nd Company
Ames Company
incorporates this same reaction in the detection of proteins. A second test for proteins in the urine utilizes the degree of turbidity which results when proteins are precipitated from solutions such as urine. Sulfosalicylic acid will precipitate these proteins and the degree of turbidity may be estimated quantitatively. Bumintest® reagent tablets and Albumin Test Solution, Endo Laboratories, both utilize this principle. The "P" tablet of the product Dextrotest® incorporates this same principle for the precipitation of blood proteins.
keton u ria-ketosis Ketone bodies, principally acetone and acetoacetic acid , may accompany any disorder of the body characterized by an altered carbohydrate metabolism. 20 Diabe tic patients, particularly those who are pregnant and those using oral hypoglycemic agents, are frequently (con tinued on page 602) Vol. NS2, No . 10, October 1962
599
diagnostic aids color-tests urine-glucose Benedict's Solution Clinistix (old style) Clinistix (improved) Clinitest Combistix (glucose portion) Galatest (Su gar Test-Denco) Sheftel Kit Tes-Tape U ristix (gl ucose portion)
positive Green to Orange Blue to Blue-gree n Purple Green to Oran ge Lavender to Purple Gray to Black Green to Oran ge Green to Blue Lavender to Purple
negative Blue White (no change) Pink Blue Pink White Blue Yellow Pink
blood glucose Dextrotest
Gree n to Oran ge-brown
Blue
cervical glucose Clinistix (old style) Blu e to Blue-green Clinistix (improved) Purple Fertility Tape Blue Rhythm Meter (use either Clinistix or Tes-Tape in conjunction paper) Tes-Tape Green to Blue
Yellow
proteins in urine Albumin Test Solution-Endo Albustix Albutest Bumintest Combistix (protein portion) Uristix (protein portion)
Turbidity degree Green to Blue- gree n Blue- green Turbidity degree Yellow-green to Gre en Yellow-green to Green
No turbidity Yellow No color change No turbidity Yellow Yellow
ketone bodies in urine Acetest
Lavender to Purple
No color change or cream color White No color change or very light pink
Acetone Test (Denco) Ketostix
Pink to Purple Purple
phenylketone bodies in urine Ferric Chloride Solution 10% Phenistix
Gree n Gray to Blue-gray
Yellow Orange (no color change)
bilirubin in urine Icotest
Blue to Purple
No color cnange or slight pi n k to red
occult blood Hematest Hemoccult Occultest
Blue Blue Rin g Blue
No color change No color chan ge No color change or appearance
pH of urine Combistix Litmus Paper Nitrazine Hydrion
Oran ge, lowest pH 5 to blue, highest pH 9 Red is aci d ; blue is alkaline Orange, lowest pH 4.5 to blue , highest pH 7.5 Various colors depending on range of pH
diagnostic aids (continu ed from page 599)
requested to test their urine using these diagnostic aids. The detection of these ketone bodies may indicate the need for further medical treatments to prevent ketoacidosis and diabetic coma. Diagnostic aids used in the detection of ketonuria are based on a color complex with nitroprusside. A pink to lavender color is formed when acetoacetic acid and acetone react with nitroprusside. There are three products which utilize this reaction. They are Acetest®, a tablet diagnostic aid ; Acetone Test®, a powder diagnostic aid, and Ketostix®, a paper strip diagnostic aid. 60Z
White (no change) Pink Pink with non-medicated
bilirubinuria Bilirubin is a breakdown product of hemoglobin and is formed in the bone marrow and the spleen. Its presence in the urine (bilirubinuria) is often the first sign of liver disorders and common bile duct obstruction. 21 This is usually followed by jaundice. The test for bilirubin involves the chemical coupling of bilirubin to a diazo compound with a resulting blue to purple color. The product Icotest® reagent tablets uses p-nitrobenzene diazonium p-toluene sulfonate - termed "bilazo" by the manufacturer.
occult blood Occult or hidden blood is that which is
Journal of the AMERICAN PHARMACEUTICAL ASSOCIATION
hidden from the naked eye and may occur in excreted fecal matter or the urine. 22 Tests for occult blood are based on the orthotolidine color reaction . A positive reaction is associated with the peroxidase properties of hemoglobin. H ydrogen peroxide is formed in these products from a reaction between tartaric acid, calcium acetate and strontium peroxide. The hemoglobin of the blood cataly tically decomposes the hydrogen peroxide to li berate oxygen. The oxygen combines with the orthotolidine dye to produce a blue color, a positive reaction for this test. The products which utilize this reaction are Hematest®, used primarily with fecal matter specimens, and Occultest®, specifically designed to be used with urine specimens. A second t est for the presence of occult blood utili zes a guaiac impregnated paper strip and a hydroge n peroxide developer. In the presence of occult blood a blue rin g is formed when the paper strip is developed. The product is designed to detect both fecal matter and urine specimens containing occult blood. This product is mar keted as I-Iemoccult®.
phenylketonuria or PKU Phenylketonuria results from the a bnormal metabolism of the essential amino acid , pheny lalanine. 23 . 24 This condition must be detected early in in fan cy before permanent a nd irreversible damage to the brain occurs with resul tant mental retarda tion. Several different products utilizing ferric ions are used for the detection of this condition. A fresh ly prepared 10 percent ferri c chloride solution a pplied to the urinated diaper of an infant suffering from this condition will turn green. A paper strip25 impregnated with ferric ammonium sulfa te , magnesium sulfate and cyclohexylsulfamic acid is marketed as a colorimetric dip-andread test under the trade name of Phenistix®. A positive reaction using this paper is a gray to gray-green color. A new test using blood from the susceptible infants has recently been reported. This test is based on bacterial growth of micro-organisms if phenylalanine is present in the blood of the infants. This product is being developed for laboratory and hospital use at the present time. The infant suffering from this condition is treated by being placed on a low phenylalanine diet using such products as Ketonil® and Lofenalac®.
pH The pH of the urine and other body fluid s is an important consideration in many medical diagnoses. The normal pH of the urine is approximately six. An upper limit of pH 8 and a lower limit of pH 4.5, indicates that the bod y has
more capacity to handle acidic substances than basic ones. The tests for pH utilize a series of dyes which are sensitive to certain hydrogen ion concentrations. Such dyes as methyl red and bromthymol blue are frequently utilized . There are several brands and types of paper strips which can be used to measure pH . Litmus paper will only indicate acid or alkaline pH. Medical quality Hydrion® papers are available for the determination of urine pH. The product Nitrazine® papers (Phenapthazine-Squibb) is supplied in l5-foot rolls and can be used to make approximately 180 tests. The product has seven color gradations in a pH range from 4.5 to 7.5. When using this paper stri p for pH determinations of the urine, a pH of 0.2 should be subtracted from the indica ted readin g after comparing with the sup plied color chart. The urine used in makin g these determina tions should not be diluted with water or other fluids . Both the Hydrion® and Nitrazine® papers a re supplied with a color chart on the package . The multiple diagnostic aid product Combistix® contains one section of the paper strip for pH measurements from five to nine. Thus with this one dip-
Announcing
and-read paper strip patients may determine the presence or absence of glucose and proteins and also measure the pH of their urine. A color chart for each of these determinations is attached to the container. At the present time, these are the diagnostic aids most frequently sold by pharmacists to physicians and patients. The community and hospital pharmacist should be aware of the various trade products and their uses, so that they are able to give professional advice when requested. The charts on pages 599 and li02 will provide a summary of their uses. color form ations and manufacturers.
9. 10.
11.
12.
What Is the R ole of the Enzymatic Uriue
13 .
Glucose Te st , Ames Co. Su gar Te st, Den co, Denver C hemical Co., Ne w York , N. Y.
14 . [.') .
ttl. 17.
18 . 19 .
references 1.
2. 3.
4. ;,) , 6.
7. 8.
Drugs of Choice, C. V. Mosby Compan y , Phi lade lp hi a, Pa. 1960-6 1, p. 795. Ph ysicians' Desk R eference, 1962, p. 224. Franck e, Don E. , H ospital Formulary of Selected Drugs, H amilton P ress, Hami lton, 111. , 1954, p. 235. "The Neglected Art of Ur ine T'estin g," Pictacl inic, Ames Co., Elkhart, Ind. Guidebook /0 1' the N ew Diabeti c P aticlll , ibid. A Few W ords About Urine - Sugar Te j lin g, ibid. The Where, Why, and H ow of a S eparate Diabetic Department for Your Phal'1l'tG cy, ibid. "Urine
Diagnosis
of
'Inborn
Errors ,'"
Physician s' Bulletin, 26 ( May 1961).
A P ocket Ref erence for the Diabetic, E li Lilly and Co., Indi anapoli s . Ind . "Compari son of T es-Tape a nd Copper Red uction Methods fo r the Det ermina ti on of U rin e Gl ucose," J . Tex. State Soc. Amer. Me d. Techu%gists, 3 (Dec. 1956). Adams, E.C., JL, Mast , R.L ., and Free, A.H. , "Specificity of Glucose Oxidase, " Arch. of Biochem. and Biophysics, 91 (D ec. 1960).
20 21 . 22 .
2;).
2-1. 2;"i.
Birnberg, C. H. , Kurzrok, R ., and Laufer, A" "S im ple T est for Determining Ovulation Time ," lAMA, 166 (Ma r . 8,1958). Doy le , J osep h B ., Ewers, Frank J. Jr., a nd Sapit , Donald , "The New Fertility Testing T ape, A Predictive Test for the Fertile P eriod ," l AMA, 172, 1744(A pril 16, 1960) . American Druggist l\1agazine (N ov. 16 ,1959). THI S JO U RNAL (Pract. Ed.), 20, 733(Dec. 1051)). Drug Topics (Nov. 23, 1959). " H ome-T esting with Colo rimetric Paper Strips, " l AMA , 173, 14 78(J ul y 30, 1960). Stanl ey, Ph y llis, " The Detecti o n o f Aceto n e in the Urine ," Amer. J . 0/ Jv12d. Technol ogy , 9 (Jan . 1943) . Hepatic Tests , W yeth Laboratories, Philadelphia , Pa . Paul , W .O. , a nd H am ilto n , H.E., "The Importance of Occult B lood in th e S tool ," Amer. l . of Dig. Diceu"" IS, 23(Jan . 1948) . Baird , H . W ., III , J. Pe diat ., 52, 7 15 (Jun e 19!i8). Ph enylketonu ria, Mead John son and Co., Evansvill e, Ind . "Simple Paper Test for Detection o f Phen y lketonur ia," Amer. Pra ct. Dig. Treat., 136 (Feb . 1960).
SEVEN RESEARCH ACHIEVEMENT AWARDS
for 1963
by American Pharmaceutical Association Foundation The American Pharmaceutical Association Foundation through the Committee on Awards and Prizes A.Ph.A. Scientific Section, invites nominations before January 1, 1963, for the second annual presentation of the seven research achievement awards ma de for the first time at the annual meeting of the Association in Las Vegas. The seven areas include the followin g : 1. Drug Standards and Assays 3. Pharmacodynamics 5. Physical Pharmacy 2. Advancement of Pharmacy 4. Natural Products 6. Stimulation of Research 7. Pharmaceutical and Medicinal Chemistry
Each award carries with it an honorarium of $1000 (one thousand dollars ) . In addition to the $1000 for each award, travel expenses up to $300 are included to enable attendance at the annual meeting of the Association at which the award will be presented. Awards are intended to recognize and encourage outstanding individuals for meritorious contributions in their chosen field of pharmaceutical research, either theoretical and basic or applied and t echnological. To be eligible, nominees need not be members of the Association but should be active in a field or pursuit closely r elated to pharmacy or pharmaceuticals, such as pharmacy schools, industrial pharmaceutical concerns, or a department of a private institution or government agency. Nominations may be submitted by any member of the A.Ph.A. They must be in prior to January 1, 1963 to the Research Ach ievement Awards, A.Ph.A. Foundation 2215 Constitution Avenue, N.W., Washington 7, D.C.
Previous nominations must be renominated for reconsideration . Refer ence can be made to pr evious nomination and supporting file and only new information, data, reprints, etc. need be submitted with the new nomination. Further detai ls and a booklet on Research Achievement Awards is available from the American Pharmaceutical Association Foundation office at the above address .
VOl . NS2. No . 10, October 1962
603
diagnostic aids by Paul J. Wurdack *
A
diagnostic aid is an agent used in the detection of some abnormal condition of the body. Community and hospital pharmacists have an increasing number of these products added to their inventories each year. The pharmacist cannot assist the physician and the patient intelligently in the use of these aids without knowing how they are applied in conditions for which they are intended. At the present time, most diagnostic aids sold in community pharmacies are used to detect one or more of the following conditions l ,21. Glycosu ria-the presence of glucose in the urine; most commonly associated with diabetes mellitus. 2. Glucose in the blood-most commonly associated with diabetes mellitus. 3. Cervical glucose-the presence of glucose in the cervical mucus; most commonly associated with determinations of the period of fertility. 4. Proteinuria-the presence of protein bodies in the urine; most commonly associated with kidney impairment. 5. Ketonuria or ketosis-the presence of ketone bodies in the urine; most commonly associated with an altered carbohydrate metabolism. 6. Bilirubinuria-the presence of bilirubin in the urine; most commonly associated with liver disorders or common bile duct obstruction and subsequent jaundice. 7. Occult blood-the presence of hidden or visually undetectable blood in the urine or fecal matter; most commonly associated with neoplasms of the colon, gastric or duodenal ulcers, kidney stones, acute appendicitis, nephritis, acute liver atrophy, hemorrhoids and vitamin C and K deficiencies. 8. Phenylketonuria-presence of phenylketone bodies (principally phenylpyruvic acid) in the urine; most commonly associated with phenylpyruvic oligophrenia-a preventable mental deficiency in infants. 9. pH-the determination of the acid-base balance of urine or other secretions-most commonly associated with the patient's metabolic picture.
In addition to these conditions, the
* Presented
to tl;1e section on pharmaceutical technology a t the 1962 APHA annual meeting in Las Vegas .
598
hospital pharmacist frequently dispenses diagnostic aids used for the following diagnostic procedures 3- various x-ray procedures of the alimentary tract, heart and blood vessels, hollow viscera, sinus and fistula tracts, intradural tumors, urologic disorders as well as blood volume and blood circulation time, gall bladder and liver functions, gastrointestinal anacidity agents, myxedema, hypertension and ophthalmic involvements to name a few. An increasing number of radioactive substances such as arsenic (As74) , bismuth (Bi 205,206), calcium (Ca 45,47), cobalt (C 0 60), Gold (Au I98 ), iodine (P31), phosphorus (P32), potassium (K42), rubidium (Rb86) and sulfur (8 35) are being employed. The community pharmacist normally handles diagnostic aids which are either powders, solutions, tablets or chemical and enzyme impregnated paper strips.
urine glucose (glycosuria) The most commonly dispensed diagnostic aids are those used for the detection of urine glucose (glycosuria) by the diabetic patient. 4- 9 This urine glucose may be detected using products which are either tablets, solutions, enzyme impregnated paper strips or a powder. The solution and tablet products involve a metallic oxide reduction using copper sulfate. 10 The cupric ion in the presence of a reducing sugar, heat and alkali is reduced to cuprous oxide with a resulting color change. This color change can be compared to a standard color chart provided with the diagnostic aid. The chart indicates either percent glucose or is referred to a negative, or reaction. Commercially available diagnostic aids using this reaction are Benedict's solution, Clinitest® and Sheftel Kit®. Enzyme impregnated paper strips involve two enzymes, glucose oxidase and peroxidase, and a dye substrate orthotolidine.1l,I2 In the presence of glucose the enzyme glucose oxidase reacts to form hydrogen peroxide. The hydrogen peroxide reacts with the enzyme peroxidase and oxygen is liberated. The dye orthotolidine is oxidized with resulting color formations which may be compared with a color chart provided with the diagnostic aid . Depending on the particular product, the chart is calibrated as to percent glu-
+++
++++
Journal of the AMERICAN PHARMACEUTICAL ASSOCIATION
+, ++,
Newly appointed assistant professor at the University of Pittsburgh school of pharmacy, Paul J. Wurdack is interested in diagnostic aids in pharmacy and trade marked dosage forms. These interests have brought him speaking and teaching engagements at seminars at the University of Pittsburgh. He has authored several articles on the subjects. A Pittsburgh graduate, he went on to earn his MS and PhD at his alma mater and served as assistant professor for two years. He is returning to Pittsburgh after serving a year at the University of North Carolina as an assistant professor. He is a member of APhA, Sigma Xi, Rho Chi and Kappa Psi and has taught pharmacology to nursing students at the Pittsburgh hospital.
cose or negative, light, medium or dark. Commercially available products utilizing the enzyme reactions are Clinistix® and Tes-Tape®. A positive test for glucose using improved Clinistix®gives a shade of purple while Tes-Tape® gives a shade varying from green to blue. Because both of these products are designed to be specific for glucose, there are few possible errors involved with other reducing sugars as contaminants. A third reaction involving the detection of glucose is that using a sodium hydroxide-bismuth oxychloride-sodium silicate powder combination. I3 In the presence of a reducing sugar the normally white powder is turned gray or black by the formation of partly colloidal, metallic bismuth. A color chart for comparison is provided with
the diagnostic aid and is cali brated both as percent and negative, or reaction . This product is marketed as Sugar T est , Denco® (Galatest®). Dip-and-read paper strips used in the detection of severa l conditions have been marketed . A water-im pervious band separates each area to prevent the colors from runnin g together. One portion of the paper strip is used to detect urine glucose; a second area is used in the detection of proteins and in one of the products a th ird area is used in the measurement of pH. Uristi x® is used to detect urine glucose and proteins while Comhistix® is used to detect both of these conditions and in addition measure pH .
+++
++++
+, ++,
glucose in the blood Glucose in the blood may be determined using a diagnostic aid which first precipitates blood proteins and a fter these are removed by filtration , the clear filtrate is used for the detection of glucose. A two- tablet product utilizing these ste ps is marketed as Dextrotest®. One tablet designated as the " P " tablet contains sulfosalicylic acid which is used to precipitate insoluble blood proteins. After fi ltration, the second tablet designated as the "S" tablet is used to test the clear filtrate for the presence of glucose. The second tablet uses the principle of metallic-oxide reduction as previously cited for the detection of glucose in the urine.
over-the-counter diagnostic aids 1. 2. 3. 4. 5. 6. 7. 8.
trade name Acetest Acetone Test Albumin Test Solution Albustix Albutest Benedict's Qualitative Solution Bumintest Clinilab
9. Clinistix
10. Clinitest 11. Combistix
14. 15. 16. 17.
Dextrotest Ferric Chloride Solution Fertility Tape Galatest (Suga r Test) Hematest Hemoccult
18. 19. 20. 21.
Icotest Ketostix Litmus Paper Nitrazine Paper
22. 23.
Occultest Phenistix
24. 25 .
Hydrion Paper Rhythm Meter and Non-Medicated Paper
26. 27.
Sheftel Kit Tes-Tape
28.
Uristix
12.
13.
cervical glucose In the past several years at least two companies have marketed devices which, used in conjunction with certain enzyme impregnated paper strips, are capable of detecting glucose in the cervical mucus. 14 , 15 Cervical glucose is at its highest concentration in these fluids just before or on the day of ovulation . By use of these devices and paper strips, it is possible to determine on which days of the month the cervical glucose is at its highest concentration. Thus, the time of ovulation and the period of fertility can be accurately determined. The products and paper strips used for this determination are either medicated or non-medicated. The plastic device itself serves as a means to hold ar:d insert the paper strip which must come in contact with the cervix. Nonmedicated papers such as those which come with the Rhythm Meter® must be touched to a medicated paper such as Clinistix®, Fertility Tape® or Tes-Tape® when the device and paper are withdrawn from the vagina. Because the Food and Drug Administration 16-18 on numerous occasions has cited the dan gers of using orthotolidine dye-impregnated paper strips directly against the cervix, the products Clinistix® and Tes-Tape®
test for ketone bodies in the urine ketone bodies in the urine proteins in the urine
manufacturer Ames Company Denver Chemical Co. Endo La boratories
proteins in the urin e proteins in the urine glucose in the urine
Ames Company Ames Company various companies
proteins in the urine bilirubin , ketone bodies, occult blood, proteins, glucose in the urine, pH, others possible dependin g on how wish to stock glucose in the urin e or in conjunction with nonmedicated paper in cervical glucose glucose in the urine pH, proteins and glucose in the urine glucose in the blood phenylketone bodies in th e urine cervical glucose gl ucose in the urin e occult blood (fecal m atter) occult bloo d (urine or fecal m atter) bilirubin in the urin e ketone bodies in th e urine pH pH of th e urine or other fluids occult blood (urine) phenyl ketone bodi es in the urine pH cervical gl ucose in conjunction with Tes-Tape , Clinistix or Fertility Tape glucose in the urin e glucose in the urine or in conjunction with nonmedicated paper in cervical glucose proteins a nd glucose in the urin e
Am es Company Ames Company
should not be used in these devices. They may be utilized, however, by touching a non-medicated paper strip to them after its removal from the vagina. The product Fertility Tape® does not use the orthotolidine dye and thus may be used directly against the cervix in the device marketed as the Fertility Testor®.
proteinuria The presence of proteins in the urine generally is a sign of kidney impairment. 19 The tests for these protein bodies are based on either color or turbidity formations. The "protein error of indicators" is utilized as a colorimetric test using the indicator tetrabromphenol. In the absence of proteins, this indicator has a yellow color at a pH of three. If proteins are present, the color becomes green to blue, Albustix® reagent strips and Albutest® reagent tablets utilize this same principle. One area of each of the diagnostic aids Combistix® and Uristix®
Ames Company
Am es Company Ames Company Am es Company vari ous companies Weston Laboratorie s Denver Chemical Co . Am es Compa ny Schiefflin and Company Ames Co mpany Am es Company various companies E.R . Squibb and Sons Compa ny Am es Company Ames Company various companies G and M Laboratories
Eli Lilly and Company Eli Lill y a nd Company
Ames Company
incorporates this same reaction in the detection of proteins. A second test for proteins in the urine utilizes the degree of turbidity which results when proteins are precipitated from solutions such as urine. Sulfosalicylic acid will precipitate these proteins and the degree of turbidity may be estimated quantitatively. Bumintest® reagent tablets and Albumin Test Solution, Endo Laboratories, both utilize this principle. The "P" tablet of the product Dextrotest® incorporates this same principle for the precipitation of blood proteins.
keton u ria-ketosis Ketone bodies, principally acetone and acetoacetic acid , may accompany any disorder of the body characterized by an altered carbohydrate metabolism. 20 Diabe tic patients, particularly those who are pregnant and those using oral hypoglycemic agents, are frequently (con tinued on page 602) Vol. NS2, No . 10, October 1962
599
diagnostic aids color-tests urine-glucose Benedict's Solution Clinistix (old style) Clinistix (improved) Clinitest Combistix (glucose portion) Galatest (Su gar Test-Denco) Sheftel Kit Tes-Tape U ristix (gl ucose portion)
positive Green to Orange Blue to Blue-gree n Purple Green to Oran ge Lavender to Purple Gray to Black Green to Oran ge Green to Blue Lavender to Purple
negative Blue White (no change) Pink Blue Pink White Blue Yellow Pink
blood glucose Dextrotest
Gree n to Oran ge-brown
Blue
cervical glucose Clinistix (old style) Blu e to Blue-green Clinistix (improved) Purple Fertility Tape Blue Rhythm Meter (use either Clinistix or Tes-Tape in conjunction paper) Tes-Tape Green to Blue
Yellow
proteins in urine Albumin Test Solution-Endo Albustix Albutest Bumintest Combistix (protein portion) Uristix (protein portion)
Turbidity degree Green to Blue- gree n Blue- green Turbidity degree Yellow-green to Gre en Yellow-green to Green
No turbidity Yellow No color change No turbidity Yellow Yellow
ketone bodies in urine Acetest
Lavender to Purple
No color change or cream color White No color change or very light pink
Acetone Test (Denco) Ketostix
Pink to Purple Purple
phenylketone bodies in urine Ferric Chloride Solution 10% Phenistix
Gree n Gray to Blue-gray
Yellow Orange (no color change)
bilirubin in urine Icotest
Blue to Purple
No color cnange or slight pi n k to red
occult blood Hematest Hemoccult Occultest
Blue Blue Rin g Blue
No color change No color chan ge No color change or appearance
pH of urine Combistix Litmus Paper Nitrazine Hydrion
Oran ge, lowest pH 5 to blue, highest pH 9 Red is aci d ; blue is alkaline Orange, lowest pH 4.5 to blue , highest pH 7.5 Various colors depending on range of pH
diagnostic aids (continu ed from page 599)
requested to test their urine using these diagnostic aids. The detection of these ketone bodies may indicate the need for further medical treatments to prevent ketoacidosis and diabetic coma. Diagnostic aids used in the detection of ketonuria are based on a color complex with nitroprusside. A pink to lavender color is formed when acetoacetic acid and acetone react with nitroprusside. There are three products which utilize this reaction. They are Acetest®, a tablet diagnostic aid ; Acetone Test®, a powder diagnostic aid, and Ketostix®, a paper strip diagnostic aid. 60Z
White (no change) Pink Pink with non-medicated
bilirubinuria Bilirubin is a breakdown product of hemoglobin and is formed in the bone marrow and the spleen. Its presence in the urine (bilirubinuria) is often the first sign of liver disorders and common bile duct obstruction. 21 This is usually followed by jaundice. The test for bilirubin involves the chemical coupling of bilirubin to a diazo compound with a resulting blue to purple color. The product Icotest® reagent tablets uses p-nitrobenzene diazonium p-toluene sulfonate - termed "bilazo" by the manufacturer.
occult blood Occult or hidden blood is that which is
Journal of the AMERICAN PHARMACEUTICAL ASSOCIATION
hidden from the naked eye and may occur in excreted fecal matter or the urine. 22 Tests for occult blood are based on the orthotolidine color reaction . A positive reaction is associated with the peroxidase properties of hemoglobin. H ydrogen peroxide is formed in these products from a reaction between tartaric acid, calcium acetate and strontium peroxide. The hemoglobin of the blood cataly tically decomposes the hydrogen peroxide to li berate oxygen. The oxygen combines with the orthotolidine dye to produce a blue color, a positive reaction for this test. The products which utilize this reaction are Hematest®, used primarily with fecal matter specimens, and Occultest®, specifically designed to be used with urine specimens. A second t est for the presence of occult blood utili zes a guaiac impregnated paper strip and a hydroge n peroxide developer. In the presence of occult blood a blue rin g is formed when the paper strip is developed. The product is designed to detect both fecal matter and urine specimens containing occult blood. This product is mar keted as I-Iemoccult®.
phenylketonuria or PKU Phenylketonuria results from the a bnormal metabolism of the essential amino acid , pheny lalanine. 23 . 24 This condition must be detected early in in fan cy before permanent a nd irreversible damage to the brain occurs with resul tant mental retarda tion. Several different products utilizing ferric ions are used for the detection of this condition. A fresh ly prepared 10 percent ferri c chloride solution a pplied to the urinated diaper of an infant suffering from this condition will turn green. A paper strip25 impregnated with ferric ammonium sulfa te , magnesium sulfate and cyclohexylsulfamic acid is marketed as a colorimetric dip-andread test under the trade name of Phenistix®. A positive reaction using this paper is a gray to gray-green color. A new test using blood from the susceptible infants has recently been reported. This test is based on bacterial growth of micro-organisms if phenylalanine is present in the blood of the infants. This product is being developed for laboratory and hospital use at the present time. The infant suffering from this condition is treated by being placed on a low phenylalanine diet using such products as Ketonil® and Lofenalac®.
pH The pH of the urine and other body fluid s is an important consideration in many medical diagnoses. The normal pH of the urine is approximately six. An upper limit of pH 8 and a lower limit of pH 4.5, indicates that the bod y has
more capacity to handle acidic substances than basic ones. The tests for pH utilize a series of dyes which are sensitive to certain hydrogen ion concentrations. Such dyes as methyl red and bromthymol blue are frequently utilized . There are several brands and types of paper strips which can be used to measure pH . Litmus paper will only indicate acid or alkaline pH. Medical quality Hydrion® papers are available for the determination of urine pH. The product Nitrazine® papers (Phenapthazine-Squibb) is supplied in l5-foot rolls and can be used to make approximately 180 tests. The product has seven color gradations in a pH range from 4.5 to 7.5. When using this paper stri p for pH determinations of the urine, a pH of 0.2 should be subtracted from the indica ted readin g after comparing with the sup plied color chart. The urine used in makin g these determina tions should not be diluted with water or other fluids . Both the Hydrion® and Nitrazine® papers a re supplied with a color chart on the package . The multiple diagnostic aid product Combistix® contains one section of the paper strip for pH measurements from five to nine. Thus with this one dip-
Announcing
and-read paper strip patients may determine the presence or absence of glucose and proteins and also measure the pH of their urine. A color chart for each of these determinations is attached to the container. At the present time, these are the diagnostic aids most frequently sold by pharmacists to physicians and patients. The community and hospital pharmacist should be aware of the various trade products and their uses, so that they are able to give professional advice when requested. The charts on pages 599 and li02 will provide a summary of their uses. color form ations and manufacturers.
9. 10.
11.
12.
What Is the R ole of the Enzymatic Uriue
13 .
Glucose Te st , Ames Co. Su gar Te st, Den co, Denver C hemical Co., Ne w York , N. Y.
14 . [.') .
ttl. 17.
18 . 19 .
references 1.
2. 3.
4. ;,) , 6.
7. 8.
Drugs of Choice, C. V. Mosby Compan y , Phi lade lp hi a, Pa. 1960-6 1, p. 795. Ph ysicians' Desk R eference, 1962, p. 224. Franck e, Don E. , H ospital Formulary of Selected Drugs, H amilton P ress, Hami lton, 111. , 1954, p. 235. "The Neglected Art of Ur ine T'estin g," Pictacl inic, Ames Co., Elkhart, Ind. Guidebook /0 1' the N ew Diabeti c P aticlll , ibid. A Few W ords About Urine - Sugar Te j lin g, ibid. The Where, Why, and H ow of a S eparate Diabetic Department for Your Phal'1l'tG cy, ibid. "Urine
Diagnosis
of
'Inborn
Errors ,'"
Physician s' Bulletin, 26 ( May 1961).
A P ocket Ref erence for the Diabetic, E li Lilly and Co., Indi anapoli s . Ind . "Compari son of T es-Tape a nd Copper Red uction Methods fo r the Det ermina ti on of U rin e Gl ucose," J . Tex. State Soc. Amer. Me d. Techu%gists, 3 (Dec. 1956). Adams, E.C., JL, Mast , R.L ., and Free, A.H. , "Specificity of Glucose Oxidase, " Arch. of Biochem. and Biophysics, 91 (D ec. 1960).
20 21 . 22 .
2;).
2-1. 2;"i.
Birnberg, C. H. , Kurzrok, R ., and Laufer, A" "S im ple T est for Determining Ovulation Time ," lAMA, 166 (Ma r . 8,1958). Doy le , J osep h B ., Ewers, Frank J. Jr., a nd Sapit , Donald , "The New Fertility Testing T ape, A Predictive Test for the Fertile P eriod ," l AMA, 172, 1744(A pril 16, 1960) . American Druggist l\1agazine (N ov. 16 ,1959). THI S JO U RNAL (Pract. Ed.), 20, 733(Dec. 1051)). Drug Topics (Nov. 23, 1959). " H ome-T esting with Colo rimetric Paper Strips, " l AMA , 173, 14 78(J ul y 30, 1960). Stanl ey, Ph y llis, " The Detecti o n o f Aceto n e in the Urine ," Amer. J . 0/ Jv12d. Technol ogy , 9 (Jan . 1943) . Hepatic Tests , W yeth Laboratories, Philadelphia , Pa . Paul , W .O. , a nd H am ilto n , H.E., "The Importance of Occult B lood in th e S tool ," Amer. l . of Dig. Diceu"" IS, 23(Jan . 1948) . Baird , H . W ., III , J. Pe diat ., 52, 7 15 (Jun e 19!i8). Ph enylketonu ria, Mead John son and Co., Evansvill e, Ind . "Simple Paper Test for Detection o f Phen y lketonur ia," Amer. Pra ct. Dig. Treat., 136 (Feb . 1960).
SEVEN RESEARCH ACHIEVEMENT AWARDS
for 1963
by American Pharmaceutical Association Foundation The American Pharmaceutical Association Foundation through the Committee on Awards and Prizes A.Ph.A. Scientific Section, invites nominations before January 1, 1963, for the second annual presentation of the seven research achievement awards ma de for the first time at the annual meeting of the Association in Las Vegas. The seven areas include the followin g : 1. Drug Standards and Assays 3. Pharmacodynamics 5. Physical Pharmacy 2. Advancement of Pharmacy 4. Natural Products 6. Stimulation of Research 7. Pharmaceutical and Medicinal Chemistry
Each award carries with it an honorarium of $1000 (one thousand dollars ) . In addition to the $1000 for each award, travel expenses up to $300 are included to enable attendance at the annual meeting of the Association at which the award will be presented. Awards are intended to recognize and encourage outstanding individuals for meritorious contributions in their chosen field of pharmaceutical research, either theoretical and basic or applied and t echnological. To be eligible, nominees need not be members of the Association but should be active in a field or pursuit closely r elated to pharmacy or pharmaceuticals, such as pharmacy schools, industrial pharmaceutical concerns, or a department of a private institution or government agency. Nominations may be submitted by any member of the A.Ph.A. They must be in prior to January 1, 1963 to the Research Ach ievement Awards, A.Ph.A. Foundation 2215 Constitution Avenue, N.W., Washington 7, D.C.
Previous nominations must be renominated for reconsideration . Refer ence can be made to pr evious nomination and supporting file and only new information, data, reprints, etc. need be submitted with the new nomination. Further detai ls and a booklet on Research Achievement Awards is available from the American Pharmaceutical Association Foundation office at the above address .
VOl . NS2. No . 10, October 1962
603