i Analybai Chemistry intheanquest&
.
r4 l 1
I A B E T
a relative or absolute defiDiabetes mellitus is one of the 4154 diseases that ciency of the hormone insulin. This results in an elafflict mankind throughnalytical chemistry has had a key evation of blood sugar and out the world, according to the medical subject headthe excretion of sugar in role in the conquest of diabetes the urine. There are two ings of “Index Medicus.” since very early times. The contriAnalytical chemistry has forms of diabetes mellitus. butions of analytical chemistry to underplayed and is continuing One of these occurs frequently 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 At the present time the Type I, or insulin-depenthan any other disorder of mankind. Alnumber of analytical dent diabetes mellitus. chemical measurements This form of diabetes is though dramatic progress has been made, made with relation to diasusceptible to ketoacimany challenges yet remain. As the techbetes each year rivals the dosis, and if not treated nology of analytical chemistry becomes with insulin usually renumber of analytical more sophisticated it is reasonable to envichemical measurements sults in death within two made on all of the 4153 reyears. The other form is sion that this area of chemical science will maining diseases together. called noninsulin-depenmake major contributions to the conquest These measurements indent diabetes mellitus, of other infectious, metabolic, and degenvolve urine sugar measureType 11,or maturity onset ments, blood sugar meadiabetes. Ketoacidosis erative diseases. surements, tests on urine does not occur in this form and blood for ketone hodof diabetes. Both forms ies. serum insulin assays. manifest a comnarahle hemoglobin A,, estimations, glucagon pend to a great extent on analytical number of secondary complications if assays, and numerous other blood, chemical measurements in their efcareful treatment is not provided. urine, and tissue analyses. These meaforts to unlock the final doors to the These complications affect the eyes, surements are primarily made to prototal conquest of the worldwide diskidneys, nerves, and blood vessels. vide information of aid in diagnosis ease, diabetes. Diabetes mellitus occurs in all parts and to provide guidelines for monitorDiabetes is a metabolic disease of the world and is identified as the ing or controlling the treatment of the characterized by abnormal endocrine number-three killer in the US.The disease. In addition, researchers defunction of the pancreas that involves secondary complications of diabetes
A
__
664A
* ANALYTICAL CHEMISTRY. VOL. 56, NO. 6. MAY 1984
0003-2700/84/0351-664A$O1 Sol0
0 1984 American Chemical Society
Report Helen M. Free Alfred H. Free Ames Division Miles Laboratories, Inc.
P.O. Box 70 Elkhart, Ind. 46515
S
ted to Diabet
300 000 p e r s ~ l die s of diabetes each year
Approximately 5% of the pcpulation has diabetes Half of the patknts with Insulin&pendent diabetes die of chronic renal failure
I
! 8
present very serious problems. Some of the facts related to diabetes, its secondary complications,and its dehilitating effects are provided in Table I.
Early History Diabetes was discovered independently in several parts of the world prior to the time there were physicians or chemists. The discovery was made by observing that insects were attracted to the urine of certain persons with wasting disease, by what was later appreciated as the sweet taste of the urine. Such independent discovery occurred in China, India, and Egypt more than ZOO0 years ago. In India it was appreciated that there were two forms of the disease, one form being characterized by emaciation, dehydration. and uolvuria (tvuical of ” w e I diabetes)-and the other typified by obesity and excessive food intake (typical of Type I1 diabetes). The early Egyptians (ca 1500 BC.) mote of a disorder (presumably diabetes) in which the passing offrequent and large quantities of urine occurred. At the time of the Roman Empire, 2000 years ago, Aretaeus of Cappadocia gave the disease the name diabetes, which means siphons or “liquid
-
Diabetes is the leading cause of new blindness Diabetics are 25 thnes 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 Maethan80%ofmajwa ns occw in diabetics 10% of patients seen by ice physicians have diabetes 600 000 new cases of diabetes appear annually The likelihood of having diabetes doubles with each 20% excess weiw As the American pgruiah grows older it is predicted that there will be 20 000 000 p e r m wlth diabetes by 1990 Diabetics are 20 times more likely to develop gangene than nondiabetics Diabetics are 17 times nmre likely to develop kidney disease than nondiabetics 75% of patients wlth noninsuiindapendent diabetes die of aUwoscierosis 14% of patients with diabetes are bedridden for an average of six weeks per year One in 300-400 white children in the US. will have insulin-dependentdabete the time they are 18 years old Patients with diabetes have a greater dependence on enaiytical chemistry for appropriate care than patients with any other diseesa
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 Paracelsus made an analytical error and mistakenly identified crystals obtained from the evaporation of urine from a diabetic patient as salt rather
I
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 association of glycosuria as an important diagnostic feature of diabetes was gradually accepted. And the test for
ANALYTICAL CHEMISTRY. VOL. 56. NO. 6. MAY 1984
865A
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 failureand so h e 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. 666A
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 evevy hour and the reagent was getting paler, paler. (Indicatinga 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, hut 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 he 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 centurybecause the injected animals did not die of diabetic ketoacidosis but rather of hypoglycemia, because the insulin content of the extracts was prohahly 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 Patho-
ANALYTICAL CHEMISTRY. VOL. 56, NO. 6 . MAY 19PA
from 0.2% to 0.12% to a normal 0.09 %! This was the most exciting moment of Banting's lifeor my own. Life became a blurred nightmare of work. . . dogs had to be injected, blood had to be drawn for testing, urine collected. lt 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 Gsneral Hospital was 74-year-old Leonard Thompson. After two years of diabetes he was down to 65pounds. By the usual criteria he would have, at the most, only a few weeks left. . . Banting and l rolled up our sleeves. l 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 73 years and died in 1935of pneumonia following a motorcycle accident.
logical Chemistry Laboratory a t the New York Postgraduate Medical School and Hospital in New York City. Today such a laboratory would be called a clinical chemistry lahoratory. 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-
111
vent
epect, the concept of pre~ y i destruction m of insulin
duct ligation was not the only critical factor in the Banting and Best discovety
Banting was lost in a trans-Atlantic flight during World War il. Best had illustrious career-he finished mediI school, was appointed professor of iene and public health at the Univerof Toronto School of Medicine in 27 and in 1929 was appointed pro. or and chairman of the department physiology at the University of Toron. He held this post until his retirement 1965. During his tenure as departnt chairman at the Universityof TOto he continued an active research ogram and with Norman Taylor at+ thored one of the maior textbooks of
extract
versity, was the director of the laboratory. Diabetes mellitua was one of the disorders that received attention from the small staffof three or four persons. One of the first research activities that Myers carried out was to create a practical method for measuring blood sugar. This method, published 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 analytical 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 recognized and as a result Myers was invited by the American Medical Association to provide demonstrations and information about the laboratory a t several of the association's annual meetings. 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 amlyticai chemi5 by in the studies, particularly the information obtained from the Myers-Bailey blood sylar values. The dramatic blood sugar decreases in a diabetic dog following 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 diabetics in all parts of the world. These studies are described in great detail in the initial publication of Banting and Best (8). nd Macleod received the Prize in Medicine. Best was .There was very great critiIS decision so that as a result nting shared half of his prize with t. and Macleod shared half of his
their patients. Today, practically every hospital in the world has a laboratory where analyses are carried out on body fluids of patients in the hospital as well as those coming to the outpatient clinics. Evolving from the initial Pathological Chemical Laboratory a t the Postgraduate Hospital with three or four employees,there are now more than 100 OOO such laboratories throughout the world with a total personnel of approximately a million persons.
The blood sugar method described by Myers and Bailey involved precipitation 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 protein-free filtrate, which was mixed with saturated sodium carbonate and heated in a boiling water bath for 15 minutes. This resulted in the reduction of the picrate anion to picramate by the glucose of the blood. The picramate ion has an orange-browncolor, which was read in a visual colorimeter. This MyereBailey 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 discovery 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 single 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 recognizing 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 sample that was sugar-free was taken as an indication that the amount of insulin administered was adequate; if the urine contained excreted glucose it would give a positive Benedict's test, indicating that insulin dosage was inadequate or had not been injected.
ANALYTICAL CHEMISTRY, VOL. 56, NO. 6, MAY 1884
.
607A
was defined on an analytical chemical hasis relating to the carbon dioxide content of the blood plasma. Patients with COn content of less than 20 volumes per cent were considered to have diabetic acidosis or diabetic coma; those with COz 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 COn content, and serum electrolytes in addition to chloride. 18981914
19141922
19221925
Pre-Insulin Era
1 9 s 1929
1930- 19371936 1944
19451960
1960 1966
Insulin Era
Figure 2. Deaths due to diabetic ketoacidosislwm as a percent of all diabetic patients Data ot 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 hy laboratories throughout the world as a means of recognizing diabetes and monitoring its course of treatment. inHiai Analytical Measurements in MonHoring of insulin Therapy The Joslin Clinic in Boston was founded prior to the discovery of insulin and became an outstanding diahetic treatment center. At present it still remains a world-famous center for t b study and treatment of diabetes. One of the fundamental activities used hy the clinic in the treatment of diabetes was the emphasis on teaching patients how to carlr out the analyti-cal chemical procedure of testing their own urine for the presence of glucose. It was further emphasized that insulin dosace be adjusted so that the urine be kept s u g a h e e as indicated by four testa at different times each dav. 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 hy the major. ity of the expert diabetologists of the time, it was appreciated that a great M
A
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 a t 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. A t 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 he 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 a t 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 wart for m a w i g erd colw reanimranges from navy blue (negative) Io oraw-ted (2%
ANALYTICAL CHEMISTRY. VOL. .56, NO. 6, MAY 1984
OT
more)
LC-MS is here. For the scientist that demands the best.
/ ,
The VG 12-250 from VG I P Q + ~ ~
A
/
A
A
I
I
, . ,.. :
.-
. ., i
I
/
..
...
1 -VG INSTRUMENTS /G m-\ = 7
Instruments Inc 300 Broad Street Stamford, CT 06901 (203) 322-4546
Circle Reada Service Card NO. 220 for literawe.
_Ju
UI
Clrcle Reader SBrYiuI Card No. 221 fa Sales Repesentative 10 call you.
ANALYTICAL CHEMISTRY, VOL. 56, NO, 6. MAY 1984
669A
NMR and Macromolecules
, Dynamic, snd Domain Structure
Chrompack Introduces the New Style in HPLC columns: GLASS! The new Chrompack cartridge system consists of a glass cartridge and a stainless steel holder with cartridge compressor that offers many advantages Over other systems.
!
GLASS CONSTRUCTION
It's glass, so the inner wall is smooth and inert, and the packing material can be checked visually. FLEXIBLE
Because the Chrompack system has 1/16 capillary end connectors the cartridge holder can be mounted in any HPLC syst6m. HIGH PERFORMANCE
It guarantees theoretical plate. counts of 45.000 (55,000 with modern equipment) per meter thanks to the unique CP'" Spher packing materials. ECONOMICAL Each cartridge costs only $75.00. To order a Chrompack HPLC cartridge system. or for help with any of your chromatography needs, call us at 800-526-3687', You'll reach Chrompack specialists in chromatography and chromatcgraphic products forover 15 years. *In N.J. call 201-722-8930
I
chromatography specialists
Chrompack, Inc. PO Box 6795 Bridgewater, NJ 08807 800-526-3687 timcu 670 A
33 Lm n -
s E R V K 2 CARD
ANALYTICAL CKMISTRY.
voc.
56, NO. 6. MAY 1984
cess was achieved by using exceptionally dry ingredients and then carrying out the mixing and tabletting operation in an extremely low humidity environment. 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 hicarbonate to provide an effervescence, which is important in the mixing effect 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 cuprous ions mix to form various shades of green and brown depending on the amount of glucose in the urine being tested. The color chart used for quantitation is also shown. The total reaction 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 treab ment of diabetes was very real and its use gradually appeared as a recommendation in texts devoted to diahetes. The test gradually replaced the Benedict test as the standard and popular means of recognizing glucose in urine. Furthermore, the concept expanded to considering effective treatment as the maintenance of sugar-free urine shown by as many &s four tests per day. The number of diabetic patients 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 analysis used by both physicians and pa. tienta in health care. In 1947 the method of choice for recognizing impending diabetic acidaxis or actual ketoacidosis was to demonstrate the presence of ketone bodies in urine. Our group a t the Miles-Ames Research Laboratory initiated research to develop a conveniencetest for recognizing 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 1956dip-and-read colorimetric enzymatic tests were introduced as new methods for recognizing glucose in urine (10).Two independent organizations 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 Division of Miles Laboratories and TesTape was a product of Eli Lilly and Company. The chemical reaction systems of the two reagent compositions are similar. Glucose oxidase and peroxidase provide a double sequential enzyme system in which glucose oxidase catalyzes the oxidation of glucose in the urine with oxygen of the air to form gluconic acid and hydrogen peroxide. 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 specimen of urine or to moisten it in the urine stream. The reactions involved are:
-
+
glucose (C6H1206) oxygen (of the air) glucose hydrogen peroxide (HzOz) gluconic acid (C~HI~O,) orthotolidine (C12H16N2) H20z
+
+
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 diabetes. Different chromogens have been used in various reagent systems, but the glucose oxidase-peroxidase oxidation mechanism for glucose color reactions 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 CIRCLE 19 ON READER SERVICE CARD
ANALYTICAL CHEMISTRY, VOL. 56,
NO. 6, MAY 1984
671A
FTIR Only Nic this much problem New levels of FT-IR simplicity, eIBciency, productivity. Getting started with Nicolet FT-IR is surprisingly simple. Our new menu-driven software package gives you convenient access to the full power of FT-IR. All analytical functions, from data collection to spectral identification and quantitative analysis, are easily selected from displayed menus. You initiate most menu selections and analytical procedures with single keystrokes. Our interactive software provides unparalleled versatility and streamlined operating efficiency for the experienced FT-IR user as well as for the novice. Macro programming allows you to easily access all system functions, including data collection, analysis, display, storage, and plotting. You can select from a library of Nicolet pre-programmed Macro
experiments and operations, or construct your own Macros to fit your unique needs - you then perform an entire experiment or complex system function at the touch of a single soft-key. Nicolet FT-IR combjnes elegant operational simplicty with sophisticated analytical power. Our extensive software estate gives you unmatched analytical problem-solving versatility. The new NICOS operating sJstem in UNIT tike ih strucfure, but gives you enhanced real-time capabilities for your most demanding laboratory work. Fast, accurate sample ideo tillcation. With Nicolet FT-IR you don't need to manually page through volumes of reference spectra to identify your compound. You have instant access to the largest on-line data base of high-quality FT-IR spectra.
"Y
'' wbile uIewin8 mulls.
Automatic Spectral Search
and Identitication A
Our easv-to-use search orograms perfdrm rapid and aScurate spectral library searching, inter-pretation, and identification. You can also create you own libraries tailored to your needs - with up to 160 Mbytes of storage, extensive on-line data storage is no problem. NIC-QUANT:a powerful and versatile tool for quantitative analysis. With the NICQUANT software package, you have the flexibility and precision to analyze virtually any type of sample. This convenient menudriven program guides you through setup and calibration once parameters are defined, highly accurate analyses can be pedormed by even a novic operator.
53
Lp
FO
n
0
MRIN M
2.
C p u m
3. M T r I p 4.
EDIT PmfrmVa
I
5 . RINT-P E. STaR - P
7. W T
Baseline adjustment gives you versatility for 'Jine-tuning
Nicolet GC-IR separation and identi5cation simple, rapid, automatic. Designed for ' the chromatographer, our GCIR package provides superb sensitivity and streamlined operation, I from sample injection to component identification. The software performs real-time data collection, chromatogram reconstruction, peak location and spectral computation for each chromatographic peak, and spectral search
~
I
i
,
1
and identification for e ponent in the GC-IR ru
-
Advanced capabilities for your special applications. For demanding dynamic studies such as chemical kinetics and catalysis, our rapid-scan (up to 50 scans/ second) and real-time analysis capablities are without peer. Our special micro-beam capabilities allow analysis of small samples, micro-ATR, micro-pellets, and
1
spatial mapping. Tell us ab your demanding applic tough analysis problems, so we can tell you how Nicolet FT-IR can solve them. In addition to our proven performance benefits, we provide a one year full warranty on all of our FT-IR systems, as well as the most responsive support in the industry. Please call or write to find our more about the advantages of Nicolet FT-IR.
Automatic search, rem'eual,and identification of components.
Real-time GC-IR letsyou observe peaks during elution.
I
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 products provided ease of carrying the tests in a pocket or purse. Dip-andread urine glucose tests were not only accepted and used by diabetic patients, they also became standard practice in the physician's office and in the hospital laboratory. The evolution of additional dipand-read tests followed quite promptly. Alhustix, 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 provided by Ames was Ketostix, a rapid colorimetric 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 diabetic 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 reagent 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, recording the results so that their physicians could look a t 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 con674A
quest of diabetes by analytical chemistry. 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 reaction as the dip-and-read urine tests. But the reagent is coated with a semipermeable 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 reagent and reacts to give a blue color. Large molecules, such as hemoglobin and the cells of the blwd, are too large to pass through the semipermeable membrane and remain on the surface to be washed away. This system prevents the red hemoglobin of the blood from masking the developing color. Many laboratory analysts and physicians 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 followed his dietary and therapeutic regime (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 w88 not until 1969 with the introduction of the Ames Reflectance meter that blood glucose monitoring 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 ohvious that reading numbers from a dial face gave a great deal of confidence to users of Dextrostix reagent strips, who were accustomed to seeing results in incremental color block values. The original meter has been improved through seveial models and today is available as the Glucometer Reflectance Photometer, a small, battery-operated, lightweight instrument that automatically times the one-minUte waiting period and signals that the strip should he washed and placed in the instrument for reading. The instrument is shown in Figure 4. There
ANALYTICAL CHEMISTRY, VOL. 56. NO. 6. MAY 1984
BUY YOUR EM SCIENCE REAGENTS FROM THESE NATIONAL AND REGIONAL
THE WORLD OF EM SCIENCE Introduces the
New Standard in Purity; Products &L Packaging EM SCIENCE has combined the interna. tional resources of E. Merck, Darmstadt, Germany, the foremost source of reagent chemicals in the world, with the proven expertise of MCB's past 40 years of service. We have done this t o provide you with the highest standard available today for purity, products and packaging. This unique combination will provide you with* BASIC SOURCE CAPABILITY-The assurance of consistent lot-to-lot purity, based on large scale production facilities dedicated to producing high purity reagents.
I
STATE-OF.THE.SCIENCE PRODUCTSOver 2,000 researchers worldwide are dedicated to providing new, innovative roducts for the advancement of the scientitc communityand the betterment of mankind.
.
.
KNOWLEDGE-With our New UNIKRSAL PRODUCT INFORMATIONSYSTEM, concise information about each of our product's purity, proper storage, safety and health precautions, is clearly h*.
; 1;;;gM
EM
SCIENCE-The finest products, the highest ouritv. convenient packagesand thecom. plete knowmgeto provide ou with the best VArUE in Reagent Chemicals.
For additional information about EM SCIENCES NEW STANDARDS. contact one of our distributors listedon the left or call or write:
I
480 Democrat Road Gibbstown, New Jersey 08027 (609) 423-6300 Arwrialeof
E. Mer&
Darmssdl. &many
CIRCLE 65 ON READER SERVICE CARD
ANALYTICAL CHEMISTRY, VOL. 56. NO. 6. MAY 1984
676A
Sulfur -
The Dohrmann" System 702 X-ray Fluorescence Sulfur Analyzer has: -A non-radioactive source, X-ray tube is safe, requires no license. -A 19-samplecarousel with builtin check standard for rapid, unattended analyses. Single pushbutton operationnothing could be simpler. -Automatic calibration, and statistical analysis of data. -Widest range of concentrations and sample types, from 0.01 % to 100% sulfur. Multi-point calibrations, multiple curves. For more details on the world's easiest-to-operate and most productive sulfur analyzer, circle reader service number or contact:
-
are more than 250 000 of these instruments now used throughout the world in hospital laboratories, physician office laboratories, and by diabetic patients themselves. In the past few years, other instruments and reagents have been introduced, for example the Chemstrip bG and the instrument AccuChek, available in the US.from Bio-Dynamics Division of Boehringer Mannheim. Other instruments in Europe and Japan have been designed for use with Dextrostix or Chemstrip hG or with reagent strips made by other companies. The availability of easy-to-do analytical blood sugar methods and lowpriced instruments to facilitate reading the color reactions created interest in the practice of self-testing by diabetic patients. The practice was initiated first in Europe, particularly by diabetologists taking care of pregnant patients. The results were dramatically successful and accordingly American diabetologists recommended selftesting to their patients. C. M. Peterson of Rockefeller Hospital in New York was one of the first to report results in the US.with pregnant diabetic patients who were taught to monitor their own blood sugar ( 1 1 ) . When several blood sugar measurements were made each day by the pregnant diabetic it was possible, with very few exceptions, to keep the blood sugar within normal limits throughout the day and night. The net effect of such a normalization process was the birth of healthy, normal babies. This was in contrast to what had been the usual outcome of pregnancy in the diabetic, which involved miscarriages, fetal
I I
Circle WSB-Please send Iltershrre. Circle W57-I need a dsmonstratlon.
676A
ure 4. Dextrostix and GI one drop of fingertip blood
ANALYTICAL CHEMISTRY, VOL. 56. NO. 6, MAY 1984
deaths, and many malformed babies. It is now generally agreed among experts that with careful self-testing and normalization of hlocd sugar achieved with careful control of food intake and insulin dosage, the diabetic patient can anticipate a normal pregnancy in the same way a nondiabetic would. The practice of self-testing of blocd sugar has been adopted by a very large number of diabetologists and diabetic patients as a result of the success achieved by the pregnant diabetic. Patients with both Type I and Type I1 diabetes have become zealous advocates of self-testing. It is pertinent to point out that monitoring the treatment of diabetes with urine sugar tests provides the opportunity to avoid glycosuria indicative of high blood sugar values but does not give the opportunity to recognize hypoglycemia, which is one of the major problems in diabetes. Although most patients on blood sugar self-testing regimes prefer to use instruments, improved blood sugar reagents are currently available for visual use. Visidex I1 from Ames and Chemstrip bG from Bio-Dynamicsare labeled with color charts that make it practical to get good results without the need for an instrument. Blood sugar values up to 800 mg/dL can be measured with these analytical chemical systems. Immunoassay of Insulin With the discovery and widespread use of insulin, great interest evolved in the measurement of insulin in hlocd. Initially this information was surmised from the level of blood sugar. Thus, if blood sugar is normal, it indi-
In microscopy, as in language, clear understanding depends on clear definition. That’s why man scientists depend on the Ni on Opti hot microscope. The starting clarity, remarkable resolutionand crisp contrast of its unique CF optics reveal a precise, undistorted picture of the specimen. For fast, sure documentation,
I
the optional FX photomicrographic systems provide microprocessor-controlled exposure accuracy. Add whatever accessories you require and the Optiphot can easily keep pace with your curiosity. See how your investigations could benefit from a more definitive view. Ask to look into an Optiphot. For
Circle #?49 far
m e intormation.
full information and a reproduction of this photomicrograph, write: Nikon Inc., .27-. Instrument Division. .. , 6 . Stewart Avenue, Garden 2220200. City, NY ll5M (Y6) ~
lvikon
Extending Man‘s Vision
Circle #I50 for a demonsbation
The new generation
l O O q 0 1 Glucose Orally
!r
Hours
@re 5. Serum insulin levels obtained after a glucose challenge in healthy rbjects and in adult onset diabetics (Type 11) both of whom produce insulin, and in veniie (Type I)diabetics who produce little cf no insulin ~ t e an s optimal concentration of in-
blood sugar is below normal, it iggesta an excess of insulin; and if le blood sugar is above normal, it reecta an inadeauate or below-normal mount of insuiin. Since many factors ther than insulin cause blood sugar hmges, thii method is at best crude. ubsequently, a biological assay for isulin was developed that related to le effect of insulin on rat epididymal din; if
TheCAMAGTLC SCANNER II is capable of: scanning thinlayer chromatograms by absorbance or by fluorescence . scanning electropherograms or similar objects in transmined light . scanning armlar/anticircular chromatograms . automatic recording of spectra dualwavelength scanning
tissue. This procedure also was difficult, i n a m a t e , and time consuming. Rosalyn Yalow and Solomon Berson developed a radioimmunoassay that introduced a new Drincinle of analvtical chemistry to &e field of diabet;?s (12).This m a y was based on cornpetitive binding of antibody (guinea pig antibeef insulin serum) to human insulin (in the serum being assayed) and to tracer beef insulin (iodinated with
Ask for our 12-page fullcolour product bulletin rCAMAG TLC SCANNER Ilr
0 - 4 1 3 2 MuttenzlSwii2erland For further informationmntaR
APPLIED ANALYTICAL INDUSTRIES
6
Weeks of Treatment CAMAG has agents on alm05t all countrier cn u 5 A pinfly represented by A N
:IRCLE 34 ON READER SERVlCE CAR0
878A
:Igure 6. Illustrationof the value of glycosylated =rum protein measurements in he evaluation of short-term treatment of diabetes and the value of glycosyiated hemoglobin measurements in the evaluation of long-term treatment
ANALYTKAL CHEMISTRY, VOL. 56, NO. 6, MAY 1984
explore the advantage of the world's most sophisticated x-ray diffraction software.
Discover it at Philips. The Philips computerized APD x-ray diffractionsystem represents the solution to an unparalleled range of analytical needs. It provides optimum results, yet requires little more than the touch of a button. The Philips APD is totally user oriented. By combining a flexible hardware configuration with the most comprehensive software library available, it offers ease of operation and maximum system capability Interactivegraphic operation guides the user tep by step. In routine studies, a batch program automatically collects the data, then provides final analysis and a printout of identified phases or a calculation of concentrations. Unequaled accuracy in identification of phases is accomplished by a fast, automated searchlmatch routine. A wide choice of algorithms provides for quantitative and profileanalyses. For more sophisticatedapplications, the APD software package includes programs for indexing powder patterns,isostructural searches aiding in solid solutions analysis, preferred orientation alculations. automated texture analvsis. ,~ ~.incomoration user-generated patterns into a data base and calculatioi f lattice parameters to provide ultra-accurate'd' spacings. Over 80 years of x-ray experience on an international scale has resulted in the largest user base and a global network of sales, training and service personnel that meet our customers' needs worldwide. The Philips computerized APD x-ray diffraction system is a prime example of our unique ability to anticipate the technology of tomorrow. For an even greater discovery of the capabilities of the APD or informationon an upgrade of your present system, talk with Philips today Contact: N.V. Philips Analytical X-ray, Lelyweg 1, 7602 EA Almelo, The Netherlands.Tel. (31) 5490-18291. :In U.S.A.) Philips Electronic Instruments. Inc.. Analytical X-ray Group, 85 McKee Drive, Mahwah, NJ 07430. Tel. (201) 529-3800.
\
I
I
InstrumentsSA o h m a complete line of ICP Spectrometersdesigned especially for waste wate analysis. Callor urrite today for a brochure and application note. 173 Essex Avo., Metuchen, NJ 08840 2OIl494-8660,In Eumpe, 16-18Rue du Canal 91160Lmgjumeau. France, Tel. 909.34.93.
JOBIN YVEl
I
InStNlllMltS SA, IW. J-Y OpllUl sp.un*0l"l.lO"
CIRCLE 109 ON READER SERVICE CARD
First of all. Bio-Rad's new Model 1770 Differential Refractometer is highly compact, measuring only 150 mm wide and 100 mm high. It's also the most sensitive differential refractometer for HPLC, thanks in Dart to
I
1
- . - -. - .- -
which reduces fiow effect. Heated cell orovides constant *B.U iemperature operation to assure a stable baseline and a Datented Dhoiodeiector'assures an extremely accurate out u t signal. Convenience Peatures such as t h e bar readout (which visually indicates the presence of eluting peaks) make this t h e easiest of all refractometers to use. For more details, contact us for Bulletin 1158.
I
.,.C.**yd,.l. 2. C.*hldr..
~
0
I
I
Bio-Rad Laboratories Chemical Division 2200 Wright Ava Richmond, CA 94804 2
4
(415) 234-4130
"n"
I
Sem us at The Amorlcan Soclely ot Blologlcal Chernl8tslAAl Booth S-3 680 A
CIRCLE 16 ON READER SERVICE CARD ANALYTICAL CHEMISTRY, VOL. 56. NO. 6. MAY 1984
,S1I). Insulin bound to antibody was ieparated by paper chromatography after mixtures had been incubated a t L "C for 4 days. Free insulin remained it the point of origin and insulin 3ound to the antibody migrated with b e globulins. The ratio of "bound" to 'free" tracer insulin with no human nsulin added decreased proportionaly with addition of increasing amounts If serum containing insulin. One of the most significant facts reiulting from the study of the actual imounts of insulin in the blood of iealthy persons and in the blood of Jersons with diabetes is that people with noninsulin-dependent or maturi,y onset or nonketosis-prone diabetes :currently designated Type I1 diabe2 s ) have blood insulin levels compa.able to those of healthy persons. On ;he other hand, insulin-dependent or iuvenile, or ketosis-prone diabetics :currently designated Type I diabetes) have very subnormal levels of insulin. It also becomes evident that ind i n is released from the pancreas with the ingestion of glucose or a meal :ontaining carbohydrate. Figure 5 ihows typical serum insulin response ,f healthy subjects, patients with maturity onset, and patients with juvenile diabetes. In the 1982 "Clinical Laboratory Reference" (8th Edition) published by Medical Economics, Inc., there are eight manufacturers listed who produce kits for immunoassay of insulin. The importance of the work of Yalow and Berson in developing a radioimmunoassay for insulin-an important clinical analytical assay-is evidenced by the fact that Rosalyn Yalow received the Nobel Prize in Medicine in 1977. Glycosylated Hemoglobin Electrophoretic study of blood from patients with poorly controlled diabetes has revealed the presence of a modified form of hemoglobin. Further study indicated that this hemoglobin is firmly conjugated with a molecule of glucose. This glycosylation involves about 5% of total hemoglobin in healthy individuals and about 10-14% in patients with poorly controlled diabetes. Hemoglobin is a component of the red blood cell that has a life of about 120 days. After the red cell breaks down, its hemoglobin is converted to bilirubin. Thus the half-life of glycosylated hemoglobin is about 60 days (the same as the half-life of the red cell's hemoglobin). This is why glycosylated hemoglobin concentration indicates the blood glucose levels over the 60-day or so period preceding the determination. Analytical procedures for the measurement of glycosylated hemoglobin have established its determination as a means of evaluating the effectiveness of blood sugar
The revolution in micraration.
/
3 Recover
1. Fill
All-new centrifugal filtration system. Less than 15 microlbers hold-up volume. B B B
Maximum recovery Disposable 20 pl to 5 ml
0 0 0
For aqueous or organicSampies Color-coded for easy identification Pre-assembled, pre-sterilized
Contact S&S for technical data and a selection guide.
SChkkhWSl Innovators in Separation SCEence
Schleicher 81Schuell, Inc., 543 Washington Street, Keene N.W. 03431. Phone: (603)55248lO Schleicher 6 Schuell GmbH, D33y -el, Mi G e ~ n ~ nSchlrichef y. 6Schwll AG, C " U h l d b . c h M . Xhleicher 6Xhuell NsderlandBV, 's-ktogenbMlch. The Nethnrhnda CIRCLE 196 ON R F A D t R StRVlCf CARD
Swkerland
,
control over the period of the preceding six to eight weeks. Careful regulation of blood glucose within the normal range during a six- to eight-week period results in normal concentrations of glycosylatedhemoglobin, or hemoglobin AI,, as it is also known. The measurement of HbA1, has become established as a frequently used monitor in evaluating the compliance of a patient to a recommended diet and insulin dosage. A shortcoming of this analytical chemistry monitoring system is that the frequency or extent of potentially harmful hypoglycemia is not indicated by the results of the measurement. Recognition of the glycosylation of hemoglobin has led to investigation of the same type of reaction involving other plasma proteins. The albumin of blood plasma is among the glycosylated proteins that provide information relating to effectiveness of diabetes control. The half-life of plasma alhumin and other proteins is much shorter than that of hemoglobin, and therefore the analytical determination of glycosylated albumin or other plasma protein is indicative of blood glucose values during the preceding one or two weeks. Belfiore's review of recent diabetic congresses indicated a 37% mean fall in glycosylatedserum proteins after one week's treatment of poorly controlled diabetics compared to an 8% fall in HbAlc during the same period (13).These differences are shown graphically in Figure 6 and indicate the advantage of measuring glycosylated proteins for short-term treatment evaluation and the advantage of glycosylated hemoglobin measurement in the evaluation of long-term treatment. There is a considerable amount of evidence to indicate that many of the secondary complications of diabetes are related to the glycosylation in tissue proteins that occurs with continued elevation of blood sugar.
Research Studies In Diabetes Analytical chemistry plays a very critical role in research studies being (182A
carried out to better understand and unravel the mysteries of diabetes. One of the interesting instruments contributing to further research activities is the Biostator Glucose Controlled Insulin Infusion System (14).This instrument involves the use of a glucose sensor placed in the vein of the patient being studied. The sensor, attached to a recording pen, provides a continuous measurement and continually records the blood sugar level. When the blood glucose falls below a certain level, the instrument responds by injecting glucose intravenously; when the blood sugar exceeds a given level, the instrument automatically injects insulin. A picture of the Biostator functioning in a hospital environment is shown in Figure 7,in which a diabetic patient is attached to the instrument but is nonetheless involved in a game of chess with his son. This quite complex individual instrument has led the way for the evolution of easier-to-use, less-expensive, continuous insulin pump systems.
Vistas of the Future The ultimate objective in the conquest of any disease is its elimination as a cause of illness and death. This objective is a very real one in the minds and activities of many diabetes researchers. Two of the efforts that provide encouragement involve analytical chemistry. The hope of providing tissue implants of viable pancreatic islet tissue has received much study and in some experimental animals has
.
achieved modest success. Blood sugar and urine sugar measurements define the effectiveness of the implant (15). The biggest difficulty is that of immune rejection of the implant islet tissue. The application of modern immunosuppressive drugs and the monitoring of the level of immunosuppression gives an indication of the effectiveness of providing an environment that will allow the transplant to survive and provide a regulated output of insulin. A second interesting approach relative to the possible elimination of diabetes is concerned with efforts to identify the causative agent responsible for the disorder. There is some evidence that this is a virus that infects cells of the pancreas, the cells in turn being destroyed by the body's own immune system. If such a virus is found and isolated, it is then possible that a vaccine can be prepared that would be effective in preventing the initial infection. In the meantime, there is an increasing amount of evidence that Type I1 diabetes, which has a genetic background, can he made much less severe or possibly may be completely avoided by a program of careful weight control and exercise (16). Cahill has suggested that the application of molecular biology to effect cell modification may in the future allow the creation of new cells within the diabetic patient that will have the capability of synthesizing insulin and releasing it in response to appropriate stimuli (17). (continued on p . 684 A )
.. .._-._--._. ..._...._. ...--...._l..I....s.. dtion and glucose levels
,_, ,..LI of a diabetic under haspitalizatm
Though thn patient is "hooked up'' to thn instrument. which constantly reguiatesinsulin d w g e and giucose administmtion by computer. he is able to remain nonnaliy ache.. photographcartesy of AMI. photogapher Marvin Silver
ANALYTICAL CHEMISTRY, VOL. 56, NO. 6, MAY 1984
Complete thermal analysis in cold print. Only from Mettler, The TA3000 ThermalAnalysisSystem The proof is in this printout of a glass transition (DSC) test. Only Mettler gives you such complete results,without an external computer or storage, for $22,620. From starting tests to finished evaluations, the TA3000 does it all. That's true whether you need differential scanning calorimetry (DSC), thermogravimetry (TG) or thermomechanicalanalysis (TMA),in research and development or quality control. The processor, which is the heart of the system, prompts the operator in setting up a test, then performs it unattended. Results appear as hard
copy on the TA3OOO's printer/plotter. You end up with a complete record of the test parameters, digital results, and an easy-to-read plot of the curve.
Smart and compact The TA3000 uses state-of-the-art microprocessor technology to increase the "smartness" of thermal analysis while reducing the size of the hardware. The TA3000 conserves lab space, stores your methods, is selfcalibrating and has complete diagnostics built in. Economical and versatile In addition to its moderate price, the CIRCLE 147 ON READER SERVICE CARD
.....I
I .
IthTlLER A , =.
S"B,E*
...
I.."..
TA3000 is extremely versatile. For the basic price, you can get a complete TA3000 system for DSC from 25°C to 6OO0C,including all programs and data evaluations. Low-temperature DSC, TG, and TMA can be readily added.
On-site demonstration Because the Mettler TA3000 system is very compact, we can arrange a demonstration in your lab with no trouble at all. Write to set one up. Mettler Instrument Corporation, Box 71, Hightstown, NJ 08520. Or call: (609) 448-3000.
References
AI h/Applied HI ksolvents in Safetv Coated Containiirs ...
Because we’re concerned about lab safety. W W W W
Safety Coated Bottles Distllled in Glass l0Oo/o Nitrogen Blanketed Color Coded Labels
Distilled in glass, high purity solvents packaged in color coded, easy-to-handle, hard-to-break, safety coated amber glass containers for your added protection.
All solvents are placed under nitrogen blankets to avoid contamination and final filtered through 0.2 micron TFE membranes. Supplied with NFPA code, U.V. absorbence, GC assay, evaporation residue water content, safet and first aid data.
A
I
d
11) Dodson. M. Med. Obs. Soe. Phvs. London 1776;298,316. 12) Bouchardat, A. “A la Glycosurie ou Diabete Sucre; son Traitment Hygienique”; Germer-Baillere: Paris. France. 1875. :3)von Mering, J.; Minkowski, 0. Arch. Exp. Pathol. Phormakol. 1889-90.26, 371. 14) Myers, V.C.; Bailey, C. V. J. Bid. Chem. 1916,24,14741. (5) Benedict, S.R. J. Bid. Chem. 1908-4. 5,485487. 16) Folin, 0 Wu, H. J. Biol. Chem. 1920. 41,367-74. 17) Jukes,T. M.J. Nutr. 1980,110,19-21. 18) Banting, F. G.;Best, C. H. J. Lob. Clin. Med. 1922.7.25146. :9)Joslin, E. P.; Root, H. F.; White, P.; Marble, A. “Treatment of Diabetes Mellitus”: 7th ed.: Lea & Febieer: - Philadelphia, Pa., 1940. (10) Free, A. H.; Adams, E. C.; Kereher, M. L.;Free, H. M.; Caok, M. H.Clin. Chem. 1957,3,163-I. 111) Peterson, C. M., Ed. “Diabetes Management in the 808:The Role of Home Blood Glucose Monitorin,. and New Insulin Delivery Systems”; Praeger Publishers: New York, N.Y., 1982. (12) Yalow, R.; Berson, S. J. Clin. Inuest. 1960,39,1157-75. (13)Belfiore, F. “Frontiers in Diabetes”; Karger: New York, N.Y., 1981;Vol. I, pp. 68-71. (14)Fogt, E. J.; Dodd, L. M.; Jenning, E. M.; Clemens, A. H.; Clin. Chem. 1978, 24,1366-72. (15)Lacy, P. E. In “World Book of Diabetes in Practice”; Krall, L., Ed.; Excerpta Medica: Amsterdam, The Netherlands, 1982;pp. 207-8. (16)Bonar, J. R. “Diabetes, A Clinical Guide”; Medical Examination Publishing Co.: Garden City, N.Y., 1980;pp. 9d032Q
(17)Cabill, G . F. In “Diabetes Mellitus’:; Rifkin, R.; Raskm, P., Eds.; Robert Brady Co.: Bowie, Md., 1981;Vol. V, pp. 375-8.
Helen and A1 Free are a husband and wife team with a combined total of over 80 years in the profession of chemistry with emphasis on diagnostics and medicine. They are avid attendees of scientific meetings all over the world where one or both are always involved with program presentations, and avid proponents of good health via low-cholesterol-low-salt diets with moderate exercise (usually tennis). CIRCLE 1 ON READER SERVICE CARD
684A
ANALYTICAL CHEMISTRY, VOL. 56. NO. 6, MAY 1984
