Analytical Chemistry in the Conquest of
D I Α Β Ε Τ a relative or absolute defi Diabetes mellitus is one ciency of the hormone in of the 4154 diseases that sulin. This results in an el afflict mankind through nalytical chemistry has had a key evation of blood sugar and out the world, according to the excretion of sugar in the medical subject head role In the conquest of diabetes the urine. There are two ings of "Index Medicus." since very early times. The contri forms of diabetes mellitus. Analytical chemistry has butions of analytical chemistry to under One of these occurs fre played and is continuing quently in children and to play a major role in the standing and providing effective treatment may be called juvenile, conquest of this disease. of diabetes are greater with this disease Type I, or insulin-depen At the present time the than any other disorder of mankind. Al dent diabetes mellitus. number of analytical This form of diabetes is chemical measurements though dramatic progress has been made, susceptible to ketoaci made with relation to dia many challenges yet remain. As the tech dosis, and if not treated betes each year rivals the nology of analytical chemistry becomes with insulin usually re number of analytical sults in death within two chemical measurements more sophisticated it is reasonable to envi years. The other form is made on all of the 4153 re sion that this area of chemical science will called noninsulin-depenmaining diseases together. make major contributions to the conquest dent diabetes mellitus, These measurements in Type II, or maturity onset volve urine sugar measure of other infectious, metabolic, and degen diabetes. Ketoacidosis ments, blood sugar mea erative diseases. does not occur in this form surements, tests on urine ——— of diabetes. Both forms and blood for ketone bod^—^ manifest a comparable ies, serum insulin assays, number of secondary complications if hemoglobin A l c estimations, glucagon pend to a great extent on analytical careful treatment is not provided. assays, and numerous other blood, chemical measurements in their ef These complications affect the eyes, urine, and tissue analyses. These mea forts to unlock the final doors to the kidneys, nerves, and blood vessels. surements are primarily made to pro total conquest of the worldwide dis vide information of aid in diagnosis ease, diabetes. Diabetes mellitus occurs in all parts and to provide guidelines for monitor of the world and is identified as the Diabetes is a metabolic disease ing or controlling the treatment of the number-three killer in the U.S. The characterized by abnormal endocrine disease. In addition, researchers desecondary complications of diabetes function of the pancreas that involves
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664 A · ANALYTICAL CHEMISTRY, VOL. 56, NO. 6, MAY 1984
0003-2700/84/0351 -664A$01.50/0 © 1984 American Chemical Society
Report Helen M. Free Alfred H. Free Ames Division Miles Laboratories, Inc. P.O. Box 70 Elkhart, Ind. 46515
Table I.
Ε S
Facts Related to Diabetes Mellitus in the U.S.
Diabetes is the third leading cause of death 300 000 persons die of diabetes each year Approximately 5 % of the population has diabetes Half of the patients with insulin-dependent diabetes die of chronic renal failure Diabetes is the leading cause of new blindness Diabetics are 25 times more likely to become blind than are nondiabetics The likelihood of having diabetes doubles with each decade of life Diabetics are twice as likely to develop heart disease as nondiabetics
present very serious problems. Some of the facts related to diabetes, its sec ondary complications, and its debili tating effects are provided in Table I.
More than 8 0 % of major amputations occur in diabetics
Early History Diabetes was discovered indepen dently in several parts of the world prior to the time there were physicians or chemists. The discovery was made by observing that insects were attract ed to the urine of certain persons with wasting disease, by what was later ap preciated as the sweet taste of the urine. Such independent discovery oc curred in China, India, and Egypt more than 2000 years ago. In India it was appreciated that there were two forms of the disease, one form being characterized by emaciation, dehydra tion, and polyuria (typical of Type I diabetes) and the other typified by obesity and excessive food intake (typical of Type II diabetes). The early Egyptians (ca. 1500 B.C.) wrote of a disorder (presumably dia betes) in which the passing of frequent and large quantities of urine occurred. At the time of the Roman Empire, 2000 years ago, Aretaeus of Cappadocia gave the disease the name diabe tes, which means siphons or "liquid
The likelihood of having diabetes doubles with each 2 0 % excess weight
1 0 % of patients seen by family practice physicians have diabetes 600 000 new cases of diabetes appear annually
As the American population grows older it is predicted that there will be 20 000 000 persons with diabetes by 1990 Diabetics are 20 times more likely to develop gangrene than nondiabetics Diabetics are 17 times more likely to develop kidney disease than nondiabetics 7 5 % of patients with noninsulin-dependent diabetes die of atherosclerosis 1 4 % of patients with diabetes are bedridden for an average of six weeks per year One in 3 0 0 - 4 0 0 white children in the U.S. will have insulin-dependent diabetes by the time they are 18 years old • Patients with diabetes have a greater dependence on analytical chemistry for appropriate care than patients with any other disease
runs through," and indeed this is one of the dominant aspects of the disease. Many times the person with diabetes observes a very unquenchable thirst and an increase in the frequency and amount of urine excreted. During the middle ages the famous chemist Para celsus made an analytical error and mistakenly identified crystals ob tained from the evaporation of urine from a diabetic patient as salt rather
than sugar. In the same year that the Declaration of Independence of the American colonies was signed, 1776, Mathew Dodson described analytical chemical studies in which he provided evidence that the sweetness of urine and blood in diabetes is due to sugar (i). From this point in history the as sociation of glycosuria as an important diagnostic feature of diabetes was gradually accepted. And the test for
ANALYTICAL CHEMISTRY, VOL. 56, NO. 6,
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Discovery of Insulin by Banting and Best In 1921 a young Canadian physician who had been a surgeon in the Canadian Army during World War I arrived at the department of physiology of the University of Toronto. This 29-year-old physician was Fredrick G. Banting. He had set up a medical practice in London, Ontario, following his discharge from the army. This endeavor was a total failure and so he had sold his books, office furniture, and medical instruments in order to go to Toronto to seek a cure for diabetes. At this time it was known that the removal of the pancreas from a dog would cause the animal to develop a characteristic diabetes with polydipsia, polyuria, marked elevation of the blood sugar concentration, and the excretion of large quantities of sugar in the urine. Within a short time the animal would die. There was also a considerable amount of evidence to indicate that the islet cells in the pancreas were associated with a function that prevented diabetes. Another important fact was that if the ducts that carry digestive secretion from the pancreas to the small intestine were ligated or tied off in an experimental animal, the secretory cells that produce the digestive enzymes would undergo deterioration, but the islet cells would not undergo any apparent change and the animal would not develop diabetes. The hypothesis Banting proposed to Professor J.J.R. Macleod, chairman of the physiology department, was that
glycosuria or glucose in urine was one of the first clinical analytical chemical diagnostic procedures. It was at this time that the word mellitus was added to diabetes in order to distinguish this disorder from diabetes insipidus, a completely different disease in which there is a greatly increased urine volume but no sugar. This disease is completely unrelated to diabetes mellitus. Bouchardat, a French physician, studied the blood and urine of his patients with diabetes and provided further evidence that the sweetness of blood and urine in diabetes was due to the sugar glucose. He used a fermentation test, a polariscope, and copper reduction tests in his identification tests for glucose (2). Oscar von Mering and Joseph Minkowski of the Halle Medical Polyclinic carried out critical studies with dogs demonstrating that the removal of the pancreas gave rise to a condition resembling human diabetes with glucosuria, polydipsia (excessive water ingestion), polyuria (excessive urine excretion), and death within a few days. 666 A · ANALYTICAL CHEMISTRY, VOL.
earlier attempts to obtain active extracts of the pancreas that would have antidiabetic effects had failed because the enzymes of the pancreas had destroyed the active antidiabetic material during the preparation. Banting's plan was to ligate the pancreatic ducts of dogs and allow degeneration of the enzyme-producing cells. After this had occurred, the remaining organ, which should contain intact islet cells, would be used to prepare an extract to be tested for antidiabetic activity in dogs that had been made diabetic by complete removal of the pancreas. Professor Macleod agreed that the hypothesis appeared logical and consented to the details of Banting's request. This involved providing a small working space, 10 dogs, and an assistant who knew chemistry and physiology. Banting proposed that the study should take approximately eight weeks. The total cost of the project was not more than $100. Charles H. Best, a 22-year-old medical student, was the volunteer assistant "who knew chemistry and physiology" assigned to work with Banting. Together they started work on May 16, 1921, while Professor Macleod went to Scotland for the summer. The first successful experiment was carried out July 27, 1921 and was described by Best (7) some 43 years later. There was a new test every hour and the reagent was getting paler, paler. (Indicating a drop in the blood sugar.) Blood sugar was going down . . .
These scientists made extracts of the pancreases of healthy animals and injected the extract into the depancreatized animals, but these animals did not survive. At that time there was no available analytical procedure to measure the blood sugar concentration of either normal persons or diabetic persons or for such measurements to be carried out on the experimental animals of von Mering and Minkowski (3). If there had been such methods and they had been used, insulin might have been discovered in the 19th century rather than in the 20th century— because the injected animals did not die of diabetic ketoacidosis but rather of hypoglycemia, because the insulin content of the extracts was probably sufficient to cause the blood sugar to fall to lethal levels. The First Two Decades of the Twentieth Century
The most significant event in the conquest of diabetes during the first two decades of the twentieth century was the establishment of the PathoNO. 6, MAY 1984
from 0.2% to 0.12% to a normal 0.09%! This was the most exciting moment of Banting's life or my own. Life became a blurred nightmare of work... dogs had to be injected, blood had to be drawn for testing, urine collected. It was an hourly, round the clock schedule. We stretched out on lab benches to get what sleep we could. Other experiments confirmed the first result and within six months the process of preparing an extract of pancreas was worked out with the invaluable assistance of J. B. Collip; the process no longer depended on ligating the ducts of experimental animals, but rather involved the acid extraction of pancreas from carcasses of animals being slaughtered for food. Best described the first human clinical trial: Across the street in Toronto General Hospital was 14-year-old Leonard Thompson. After two years of diabetes he was down to 65 pounds. By the usual criteria he would have, at the most, only a few weeks left. . . Banting and I rolled up our sleeves. I injected him with our extract and he injected me—next day we had slightly sore arms, that was all. So in January 1922 the dying boy was injected. Blood sugar dropped—dramatically. Leonard began to eat normal meals. Leonard lived another 13 years and died in 1935 of pneumonia following a motorcycle accident.
logical Chemistry Laboratory at the New York Postgraduate Medical School and Hospital in New York City. Today such a laboratory would be called a clinical chemistry laboratory. The function of this first-of-itskind-in-the-world laboratory was to provide an organized and continuing service of analytical chemical measurements on blood, urine, and fecal specimens from the patients. Prior to this time hospitals did not have laboratories and the very meager amount of worldwide study of this type was carried out by scientists in universities or by physicians as an avocation or hobby. The few analytical procedures that had evolved by this time were cumbersome and time consuming and the quality of the information created ranged from very bad to very good. Furthermore, physicians in general did not know how to use the information created by analytical measurements in either diagnosing or monitoring the disease. Victor C. Myers, a young PhD biochemist who had studied at Yale Uni-
In retrospect, the concept of pre venting enzyme destruction of insulin during its extraction from pancreas by
duct ligation was not the only critical factor in the Banting and Best discovery of insulin. Much more important was
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Figure 1. Blood sugar changes in a diabetic dog following injection of pancreatic extract The first Banting and Best experiment. Yellow · denotes day of removal of pancreas. Arrows denote in jection of 5 mL of pancreatic extract from another dog
versity, was the director of the labora tory. Diabetes mellitus was one of the disorders that received attention from the small staff of three or four per sons. One of the first research activi ties that Myers carried out was to create a practical method for measur ing blood sugar. This method, pub lished by Myers and Bailey (4), was used to create information on diabetic patients. Obviously the initiation and use of this laboratory was a significant milestone in the involvement of ana lytical chemistry not only in diabetes, but also in the process of health care delivery relative to all diseases. The importance of this laboratory function in a hospital was soon recog nized and as a result Myers was invit ed by the American Medical Associa tion to provide demonstrations and in formation about the laboratory at sev eral of the association's annual meet ings. These demonstrations attracted much attention and as a result other hospitals throughout the country created similar laboratories to provide analytical chemical information on
the extensive use of analytical chemis try in the studies, particularly the infor mation obtained from the Myers-Bailey blood sugar values. The dramatic blood sugar decreases in a diabetic dog fol lowing injection of pancreatic extract in the first Banting and Best experiment are shown in Figure 1. This relatively simple and crude experiment was a life-saving event for millions of diabet ics in all parts of the world. These studies are described in great detail in the initial publication of Banting and Best (8). Banting and Macleod received the 1923 Nobel Prize in Medicine. Best was not included. There was very great criti cism of this decision so that as a result Banting shared half of his prize with Best, and Macleod shared half of his prize with Collip. Banting was lost in a trans-Atlantic air flight during World War II. Best had an illustrious career—he finished medi cal school, was appointed professor of hygiene and public health at the Univer sity of Toronto School of Medicine in 1927 and in 1929 was appointed pro fessor and chairman of the department of physiology at the University of Toron to. He held this post until his retirement in 1965. During his tenure as depart ment chairman at the University of To ronto he continued an active research program and with Norman Taylor au thored one of the major textbooks of physiology, "The Physiological Basis of Medical Practice."
their patients. Today, practically every hospital in the world has a labo ratory where analyses are carried out on body fluids of patients in the hospi tal as well as those coming to the out patient clinics. Evolving from the ini tial Pathological Chemical Laboratory at the Postgraduate Hospital with three or four employees, there are now more than 100 000 such laboratories throughout the world with a total per sonnel of approximately a million per sons. The blood sugar method described by Myers and Bailey involved precipi tation of the protein in 2 mL of whole blood (diluted with water) using solid dry picric acid. The mixture was then filtered or centrifuged to obtain a pro tein-free filtrate, which was mixed with saturated sodium carbonate and heated in a boiling water bath for 15 minutes. This resulted in the reduc tion of the picrate anion to picramate by the glucose of the blood. The picra mate ion has an orange-brown color, which was read in a visual colorimeter. This Myers-Bailey method had a very
special role in the conquest of diabetes because it was the method used a few years later by Banting and Best in their studies which led to the discov ery of insulin (see above). A second very significant analytical procedure created during this period was the urine sugar test described by Stanley Benedict (5). This was a sin gle copper reagent that had distinct advantages over the then-used Fehling solution for recognizing the presence of reducing sugar in urine. Benedict's solution became the method of choice for testing urine as an aid in recogniz ing and diagnosing diabetes. This test enjoyed great popularity following the discovery of insulin, and at that time it was used as a means of determining whether or not the dosage of insulin being used was adequate. A urine sam ple that was sugar-free was taken as an indication that the amount of insu lin administered was adequate; if the urine contained excreted glucose it would give a positive Benedict's test, indicating that insulin dosage was in adequate or had not been injected.
ANALYTICAL CHEMISTRY, VOL. 56, NO. 6,
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was defined on an analytical chemical basis relating to the carbon dioxide content of the blood plasma. Patients with CO2 content of less than 20 volumes per cent were considered to have diabetic acidosis or diabetic coma; those with CO2 content values above 20 volumes per cent were considered not to have this complication of diabetes. Figure 2 indicates the changes in mortality from diabetic acidosis, but the advancement in using analytical chemical methods to aid in proper therapy also played a key role. Among these analytical methods were those used to measure serum ketones, serum chloride, plasma CO2 content, and serum electrolytes in addition to chloride. 1945- 19601960 1966 Pre-lnsulin Era
Insulin Era
Figure 2. Deaths due to diabetic ketoacidosis/coma as a percent of all diabetic patients Data of the Joslin Clinic
This type of usage of analytical chemical tests is currently spoken of as monitoring. In 1920 Professor Otto Folin of the Harvard Medical School, in collaboration with Hsein Wu, devised a copper reduction method for measuring blood sugar (6). This method also received widespread use by laboratories throughout the world as a means of recognizing diabetes and monitoring its course of treatment. Initial Analytical Measurements in Monitoring of Insulin Therapy
The Joslin Clinic in Boston was founded prior to the discovery of insulin and became an outstanding diabetic treatment center. At present it still remains a world-famous center for the study and treatment of diabetes. One of the fundamental activities used by the clinic in the treatment of diabetes was the emphasis on teaching patients how to carry out the analytical chemical procedure of testing their own urine for the presence of glucose. It was further emphasized that insulin dosage be adjusted so that the urine be kept sugar-free as indicated by four tests at different times each day. In 1940 Joslin (9) continuously emphasized to his patients, and also in his writing for the benefit of other physicians, the dictum that "without urinary examinations for sugar a diabetic patient cannot secure the best results." Although the importance of frequent testing of urine was well recognized and emphasized by the majority of the expert diabetologists of the time, it was appreciated that a great
number of physicians and an even greater proportion of patients did not heed these teachings and ignored the testing of urine. In retrospect this failure to test urine in the monitoring of insulin dosage was partly because Benedict's test, the method of choice at that time, was a cumbersome procedure that only a few of the more conscientious patients used on a continuing basis. During the two decades following the discovery of insulin a major effort was directed to preventing diabetic coma or diabetic ketoacidosis and to effectively treating it when it did occur. At the Joslin Clinic diabetic acidosis and diabetic coma were used as comparable terms, and the condition
Convenience Analytical Chemistry Tests in Diabetes
In 1941 a new concept was introduced in the use of analytical chemistry tests for diagnosis and the monitoring of insulin administration in diabetes. This was a single reagent composition in the form of a tablet that could be added to a very small specimen of diluted urine in a test tube. This tablet provided the reagents employed in the by-then-classic Benedict test. The tablet also was self-heating and caused the solution to boil so that the reducing effect of glucose in urine was to alter the blue color of the cupric sulfate to the orange-red color of cuprous ion. This tablet reagent, which was called Clinitest, was devised by Walter A. Compton, a recent graduate of Harvard Medical School, and Maurice Treneer, the chief chemist at Miles Laboratories. Treneer was an expert tablet maker and was able to mix solid sodium hydroxide, solid citric acid, and sodium bicarbonate along with anhydrous cupric sulfate into a stable tablet. This whole pro-
Figure 3. Various stages during boiling reaction of Clinitest Chart for matching end color reaction ranges from navy blue (negative) to orange-red (2 % or more)
668 A · ANALYTICAL CHEMISTRY, VOL. 56, NO. 6, MAY 1984
NMR and Macromolecules Sequence, Dynamic, and Domain Structure
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He*»' J a m e s C . R a n d a l l , Jr., Editor Phillips Petroleum Company Reports on recent innovations and developments in the use of nuclear magnetic resonance spectroscopy for characterizing polymers. Includes in troductory information for those be coming familiar with NMR of polymers as well as gives detailed descriptions of how this analytical technique pro vides useful structural data. Covers NMR studies of both solid and liquid polymers. Serves as a useful refer ence book and as a guide for those in terested in polymer characterization. CONTENTS NMR and Macromolecules Overview · Intro duction to NMR Spectroscopy of Solid Sam ples · Molecular Motion in Glassy Polysty renes · Solid State 2H NMR Studies of Molecular Motion · Spin Relaxation and Local Motion in Dissolved Aromatic Polyformal · Characterization of Molecular Motion in Solid Polymers by Variable Temperature Magic An gle Spinning ,3C NMR · New NMR Experi ments in Liquids · Application of INEPT Method to 13C NMR Spectral Assignments in Low Density Polyethylene and Ethylene-Pro pylene Copolymers · 13C NMR in Polymer Quantitative Analysis · Synthesis of Novel Regioregular Polyvinylfluorides and Their Char acterization by High-Resolution NMR · Com position and Sequence Distribution of Dichlorocarbene-Modified Polybutadiene by ,3 C NMR · NMR Spectra of Styrene Oligo mers and Polymers · 75MHz ,3C NMR Stud ies on Polystyrene and Epimerized Isotactic Polystyrenes · Stereospecific Polymerization of α-Olefins · Structural Characterization13of Naturally Occurring c/s-Polyisoprenes · C NMR Study of Radiation-Induced Structural Changes in Polyethylene Based on a symposium sponsored by the Divi sion of Polymeric Materials: Science and Engi neering of the American Chemical Society ACS Symposium Series No. 247 181 pages (1984) Clothbound LC 84-366 ISBN 0-8412-0829-8 US & Canada $34.95 Export $41.95
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cess was achieved by using exception ally dry ingredients and then carrying out the mixing and tabletting opera tion in an extremely low humidity en vironment. The source of the heat is a combination of the heat of solution of the sodium hydroxide and the heat of neutralization of the citric acid by the strong alkali. Some of the citric acid also interacts with the sodium bicar bonate to provide an effervescence, which is important in the mixing ef fect needed for good reaction. Figure 3 pictures a sequential series of tubes showing the boiling reaction of Clinitest. The blue of the unreduced cupric ions and the orange-red of reduced cu prous ions mix to form various shades of green and Thrown depending on the amount of glucose in the urine being tested. The color chart used for quan titation is also shown. The total reac tion time for performing an analytical measurement on urine is less than one minute and all the equipment needed is a small test tube, dropper, rack, and the convenience reagent tablet. The effect of Clinitest on the treat ment of diabetes was very real and its use gradually appeared as a recom mendation in texts devoted to diabe tes. The test gradually replaced the Benedict test as the standard and popular means of recognizing glucose in urine. Furthermore, the concept ex panded to considering effective treat ment as the maintenance of sugar-free urine shown by as many as four tests per day. The number of diabetic pa tients who began to test their urine for sugar showed a constant increase. Clinitest established the important contribution that convenience can make to an analytical chemical analy sis used by both physicians and pa tients in health care. In 1947 the method of choice for recognizing im pending diabetic acidosis or actual ke toacidosis was to demonstrate the presence of ketone bodies in urine. Our group at the Miles-Ames Re search Laboratory initiated research to develop a convenience test for rec ognizing ketones. This resulted in an
easy-to-do analytical test for ketones in urine using the same nitroprusside compound used in the liquid test for ketone bodies. This test reagent was also a tablet, Acetest; carrying out a test simply involved placing a drop of urine on the surface of the tablet and observing for the development of a purple color indicating ketone bodies. In addition to sodium nitroprusside, the tablet contains glycine and trisodium phosphate. Dip-and-Read Tests In 1956 dip-and-read colorimetric enzymatic tests were introduced as new methods for recognizing glucose in urine (10). Two independent orga nizations provided test systems that had somewhat different forms; one was a unitized reagent strip and the other a reagent-impregnated tape. Clinistix was a product of Ames Divi sion of Miles Laboratories and TesTape was a product of Eli Lilly and Company. The chemical reaction sys tems of the two reagent compositions are similar. Glucose oxidase and per oxidase provide a double sequential enzyme system in which glucose oxi dase catalyzes the oxidation of glucose in the urine with oxygen of the air to form gluconic acid and hydrogen per oxide. In turn, the second enzyme, peroxidase, catalyzes the interaction of hydrogen peroxide and the chromogen orthotolidine to generate a blue color as an indicator of the presence of glucose in the urine. The procedure for using either Clinistix or TesTape is to dip the test composition in a speci men of urine or to moisten it in the urine stream. The reactions involved are:
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glucose (C6H1206) + oxygen (of the air) glucose hydrogen peroxide (H2O2) oxidase + gluconic acid (C6H12O7) orthotolidine (Ci 2 H 16 N 2 ) + H 2 0 2 peroxidase
*• oxidized orthotolidine (blue color)
This basic chemical reaction sequence is still employed in the billions of urine glucose systems that are used throughout the world each year. It is also the basis of practically all of the rapid blood-sugar-measuring systems that have become critical items in the recognition and monitoring of diabe tes. Different chromogens have been used in various reagent systems, but the glucose oxidase-peroxidase oxida tion mechanism for glucose color reac tions is prevalent. The introduction of glucose oxidase ready-to-react systems provided a new dimension to the analytical chemistry testing of urine by the diabetic. The convenience of either TesTape or Clinistix was unexcelled. One simply
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 6, MAY 1984 · 671 A
dipped the test strip in a specimen of urine and compared the reacted color to a color block chart one minute later. The compact nature of the test prod ucts provided ease of carrying the tests in a pocket or purse. Dip-andread urine glucose tests were not only accepted and used by diabetic pa tients, they also became standard practice in the physician's office and in the hospital laboratory. The evolution of additional dipand-read tests followed quite prompt ly. Albustix, a colorimetric dip-andread test for protein in urine, provides a sensitive test for recognizing early kidney damage, one of the prominent secondary complications of diabetes. A third dip-and-read test soon provid ed by Ames was Ketostix, a rapid col orimetric test for recognizing ketones in the urine. Ketoacidosis is preceded and accompanied by the excretion of ketone bodies in the urine. The use of a urine self-test for ketones by the dia betic patient provides a warning signal of impending ketoacidosis at a time when preventive treatment steps by the patient and his physician can be carried out effectively. Keto-Diastix, a somewhat later combination dip-andread test provides both a glucose and a ketone measurement in a single re agent strip. During the almost three decades that dip-and-read urine tests have been available, a number of products with minor variations have been produced by several companies in different parts of the world. Blood Sugar and Its Use in Self-Testing For many years, diabetics faithfully followed their physicians' advice and checked their urine for glucose two or three times a day and for ketones whenever glucose was positive, record ing the results so that their physicians could look at the records during scheduled appointments. But they had to wait for scheduled visits to physicians or to hospital clinics to find out their blood sugar levels. In 1963 another battle was won in the con
quest of diabetes by analytical chem istry. This was the development and marketing of Dextrostix, a product for measuring blood sugar that required less than two minutes from start to finish. This novel test is based on the same double sequential enzyme reac tion as the dip-and-read urine tests. But the reagent is coated with a semi permeable membrane, and its range of reaction (sensitivity) is adjusted so that it reacts with about 20-500 mg/ dL, whereas urine glucose tests must function in the range of 0-5000 mg/ dL. The procedure for blood glucose determination is to place a large drop of blood (easily obtained from a finger puncture) on the reagent strip, allow it to remain for exactly one minute, wash off the blood and compare the color of the reaction with a color block chart. During the one-minute waiting time, glucose diffuses through the semipermeable membrane into the re agent and reacts to give a blue color. Large molecules, such as hemoglobin and the cells of the blood, are too large to pass through the semipermeable membrane and remain on the surface to be washed away. This system pre vents the red hemoglobin of the blood from masking the developing color. Many laboratory analysts and physi cians saw the advantages of the new test, which would allow them to obtain a blood sugar while the patient was sitting on the opposite side of the desk. And the patient could see that his blood sugar was normal, showing that for the past few hours he had fol lowed his dietary and therapeutic re gime (or that it was abnormally high and that changes had to be made in his treatment). Some physicians even taught their brittle diabetic patients to use the test and thus gave them better control over day-to-day coping with the disease. However, it was not until 1969 with the introduction of the Ames Reflec tance meter that blood glucose moni toring was really put on its way. This small instrument was designed to measure the reflectance of light from a reacted Dextrostix and translate the signal onto a dial face calibrated to show the results in mg/dL. It was ob vious that reading numbers from a dial face gave a great deal of confi dence to users of Dextrostix reagent strips, who were accustomed to seeing results in incremental color block val ues. The original meter has been im proved through several models and today is available as the Glucometer Reflectance Photometer, a small, bat tery-operated, lightweight instrument that automatically times the one-min ute waiting period and signals that the strip should be washed and placed in the instrument for reading. The in strument is shown in Figure 4. There
674 A · ANALYTICAL CHEMISTRY, VOL. 56, NO. 6, MAY 1984
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Curtin Matheson Scientific, Inc. Marietta, GA Orlando, FL W o b u r n , MA Beltsville, MD Wayne. NJ VWR Scientific Inc. Atlanta, GA Baltimore. MD S Plainfield. NJ Boston. MA Philadelphia. PA Miami, FL Rochester, NY Thomas Scientific Philadelphia. PA Ace Scientific Supply Co. East Brunswick, NJ AWC Alabama, Inc. Mobile, AL Bodman Chemicals Media, PA Doravilte. GA Borden & Remington Div of Tillotson Corp. Everett, MA Brinkmann Instruments, Inc. Westbury, NY Caplree Chemical Corp. OldBethpage.NY Washington, DC Delray Chemical Co. Delray Beach. FL Doe 4 Ingalls. Inc. Medford, MA Eastern Scientific Providence. Rl Educational Modules Rochester, NY General Laboratory Supply Co. Wayne, Ν J Jeffery Chemical Co., Inc. Wilmington, MA Kern Chemical Corp. Mt. Vernon, NY Krackeler Scientific, Inc. Albany, NY MacAlasler Bicknell Co. Millville. NJ New Haven. CT Syracuse, NY Morgan Scientific Corp. North-Strong, Div. Rockville, MD New England Scientific Supply Bndgewater, CT Para Scientific Fairless Hills. PA Preiser Scientific Silver Spring. MD Preiser/Mineco St. Albans, WV Reagents, Inc. Charlotte, NC Sargent Welch Scientific Co. Springfield. NJ Birmingham, AL SGA Scientific. Inc. Bloomfield, NJ Tennessee Reagents Nashville, TN Wilshire Chemical Company Gardena. CA
WESTERN DISTRIBUTORS
CENTRAL DISTRIBUTORS Curtin Matheson Scientific, Inc. Kansas City. M 0 Detroit, Ml Houston, TX Cleveland, OH Tulsa, OK Cincinnati. OH Dallas, TX Minneapolis, MN Elk Grove Village, IL Harahan. LA Maryland Heights, M 0 VWR Scientific Inc. Pittsburgh, PA Bellwood, IL Midland. Ml Detroit. Ml Columbus • OH New Orleans, LA Kansas City. M 0 Minneapolis, MN St. Louis. M 0 Allometrics Baton Rouge, LA AWC Baton Rouge, LA Thomas Scientific Philadelphia, PA Anspec Ann Arbor. Ml Warrensville, (Chicago) IL Central Scientific Co. Franklin Park, (Chicago) IL Frey Scientific Company Mansfield, OH Hawkins Chemical Company Minneapolis, MN
Curtin Matheson Scientific, Inc. Dallas. Tx Brisbane. CA Denver. CO Seattle, WA Brea, CA Houston, TX Tukwila, WA Tulsa, OK VWR Scientific Inc. Seattle, WA Los Alamos, NM Anchorage. AK Phoenix, AZ Irving, TX Portland, OR Denver, Co San Diego, CA Honolulu, HI San Francisco, CA Houston, TX Salt Lake City, UT Los Angeles, CA Thomas Scientific Philadelphia, PA Alameda Chemical and Scientific Escondido, (San Diego) CA Oakland. CA Alaskan Prospectors Anchorage, AK Fairbanks, AK Albuchemisl, Inc. Albuquerque. NM American Scientific & Chemical Seattle, WA Atlas Mfg. Chemical Company San Diego, CA Bryant Labs Berkeley. (San Francisco) CA Capitol Chemical Little Rock, AR Chemical Sales Denver, CO Chemonics Scientific Houston. TX Phoenix, AZ Portland, OR Delmon Craft Company Deer Park. TX General Laboratory Supply Co Pasadena, TX Industrial Chemical 4 Supply Houston, TX MRC Scientific Tucson. AZ Northwest Scientific, Inc. Billings, MT Nurnberg Thermometer Portland. OR Berkeley, CA Orange County Chemical Co. Santa Ana. CA Escondido, CA Refinery Supply Company Tulsa, OK Sargent Welch Scientific Anaheim. CA Denver, CO Sherman's Inc. Alameda, NM Spectrum Chemical Inc. Gardena, CA Technical Support Lab Orange, TX Universal Scientific Arizona Tempe, AZ Whittaker General Scientific Tampa, FL
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A D i v i s i o n of E M Industries, Inc. 480 Democrat Road G i b b s t o w n , N e w Jersey 08027-9899 (609)423-6300
EM S C I E N C E Assiiciate ut F. Merck. ()drmt
