CHEMISTRY and the MEDICAL TECHNICIAN COLETTE CORBETT Mundelein College, Chicago, Illinois
Within the past feur years scientific stuuies hane been mination of total non-prokin nitrogen, of urea, creatinine, pursued by a greater percentage of students than has and uric acid; of glucose, cholesterol, and total proteins. formerly been noted. Because women possess many of The applications of chemistry are illustrated i n functional the necessary natural abilities for laboratory work there tests, tolerance tests, and determinations of elements such has been a demand for women with background and train- as phosphorus, magnesium, celcium, iron, bromine, chloing i n science. One field in which they particularly excel rine, and hydrochloric acid in gastric contents with double is that of medical laboratory technician. indicator wed in titration. Since the human body i s a complex biochemical entity, Colloid chemistry i s represented, since the gold number chemistry plays an important r6le in diagnosis, study, and determination i s the principle of the Lange test of spinal treatment of disease. Some of the apparatus that i s com- fluid. A n example of a more recent application of monly wed in this type of work includes the calorimeters chemistry to medicine and a corresponding test to aid (both the block comparator and the depth types), the nrzscosimeter, and the Van Slyke apparatus for gas analy- i n its successful useis that of sulfonilumide. Another phase sis. Examples of substances commonly analyzed i n the of clinical laboratory work that requires chemical skill i s clinical laboratory including skeleton outlines of the bacteriologicel work. Since almost every procedure i s of a principles utilized are enumerated. Some examples are chemical nature, the position of laboratory technician the preservation and preparation of the sample, deter- offers immeasurable opportunity for the woman chemist.
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ITHIN the last few years, the percentage of students selecting the physical and natural sciences as major fields of endeavor has been steadily increasing. This increase has been noted among women students as well as men. At one time women hesitated to enter laboratory work since other studies and occupations held a greater appeal to their esthetic sense, but they have since realized that woman's natural attributes of patience, attention to detail, accuracy, and deft manipulation of materials are valuable contributions to laboratory work. However, these qualities have not been generally recognized by employers, since a woman chemist often encounters absolute hostility, or what is worse, an amused condescension. Practically the only fields in which she is tolerated are in foods, in analytical and nutritional studies, and in medical laboratories as a technician. The work of a laboratory technician is coming to be looked upon as a woman's job and more than fourfifths of such positions are held by women. The late Dr. William H. Welch of Johns Hopkins University stated that the increasing demand for women well trained in the natural or physical sciences as technicians and special workers is far in excess of the supply (1). The chemical training of the laboratory technician is very important, not only for itself, but for developing proficiency in laboratory procedures in general. Some technicians have learned the simpler routine analysis without the benefit of a formal scientific background, but it is generally conceded that the work carried on in a laboratory is of uniformly higher caliber if the technicians have bachelor's degrees in chemistry, supplemented by some courses in the biological sciences. Within a comparatively recent period, the chemical laboratory has become a valuable adjunct to the physician, whether he is engaged in research or in practice. The diagnosis depends upon the combined information obtained from the case history, the physical examination, and the laboratory reports (2). The reason for this is that the physical signs and symptoms are external manifestations of intracellular or extracellular biochemical activity, and both the internist and the surgeon consider the biochemical viewpoint as much as the medical or surgical. "The solution of many disease problems is tackled from the biochemical angle" (3). In studying the value of healing agents for bums the efficacy of the treatment may be gaged by analysis of the healed tissue for calcium deposits. In dentistry, the cause of tooth decay is considered in the light of the chemical composition of the enamel and dentine and the interaction of an acid saliva with the teeth Man's body is considered as a biochemical entity since it is an organization of atoms and a microcosm of molecules. During life the body is in constant activity composing and decomposing, secreting, and transforming matter with every breath and every act. The sum total of these chemical changes is termed metabolism, which is brought about by interdependent
and concerted action of the component cells. Deviations from the normal metabolism of cells resnlts in illness or in death according to the extent to which these disturbances have progressed. Since the cells are composed of numerous molecules of various chemical combinations and contain water, salt, minerals, alcohol, glycerin, fat, starch, albumin, amino acids, pigments, aromatic substances, and so forth, these metabolic disturbances may be due to alterations in the relative positions of component molecules of the cell brought about by some unknown cause from within the cell, or by the metabolic products elaborated by invading bacteria. These alterations in relative positions of the chemical substances may be of an electrical nature, since, if the molecules are disturbed, the number of electrons of component atoms might be increased or decreased because the atom is composed of a central nucleus carrying one or more positive electrical charges with a variable number of negative charges (electrons) traveling about the nucleus, and these are capable of lending or borrowing to keep up this balance (4). In order to maintain health it seems that the cells must maintain specific chemical equilibria and disease consists in an alteration of this balance. Some of these chemical changes may be determined qualitatively or quantitatively in the laboratory (5). Definite concentrations of various elements are required to maintain specific functions. For instance, the "minimum amount of calcium required daily to maintain calcium equilibrium and stability of the nervous system is O.Pl.5 grains and that calcium deficiency causes tetany, decay of teeth, poor healing of wounds, interference with blood coagulation, hemophilia, and a predisposition to tuberculosis while an excess resnlts in muscular hypotonia, impaired renal function, an increase in viscosity, and coagulability of the blood and acidosis" (6). It is also commonly known that the iodine balance in the body is a contributing factor in the personality and well-being of an individual. It has been pointed out that we are protected from disease in many cases by chemical defenses within the body, such as the hydrochloric acid secretions of the stomach and oily secretions of the skin which set up barriers against bacterial invasion. Dr. H. Gideon Wells of the University of Chicago claims that the attack of invading bacteria is of a chemical nature and the defense by the leucocytes of the blood and the migratory cells of the tissues is a chemical defense. Since there is no physical connection, leucocytes communicate with the invaded area by chemical means through the body fluids. The bacteria are then engulfed by the leucocytes because the surface tension of the latter a lowered by the action of the metabolic products formed by the bacteria (7). Just as we consider physical and colloidal chemistry as related fields, the processes of immunity are essentially chemical processes (8). From this evidence i t is not surprising that chemical analysis is used to a great extent in clinical laboratories.
In the laboratory, the development of the microchemical technic of analysis has been of great value in conserving time, reagents, and last, but not least, the amount of sample needed for various determinations. The use of colorimetric determinations has also provided a short cut. There are.two types in general use; one is the block comparator which is usually employed in pH determinations of blood and culture media. The unknown, with indicator added, is placed in one hole in the block and a tube containing water in back of it. On each side of the unknown are placed the color standards of known pH with the unknown fluid in front of them. Tbe block is held to the light source, and the color standard most nearly corresponding to the unknown with indicator added is obviously the pH of the unknown. Either buffered or unbuffered solutions may be used in the standards, depending upon the indicator that is used. The second type of colorirneter is better adapted to various colorimetric determinations such as cholesterol and hemoglobin. In this method, a sample of known concentration is treated with the same reagents as the unknown, in order t o obtain a standard for comparison. The two samples are placed in an instrument that is so arranged that one-half the field in the eyepiece gives the color of the standard and the other half the color of the unknown. The depth of the measuring device in the solutions is adjusted by means of a micrometer screw, which is read a t the beginning and again a t the end of a determination when the tint in both halves of the eyepiece appears uniform. The calculation and principle is based upon Beer's law, which states that light in passing through a colored medium is absorbed in direct proportion to the concentration of colored substance. Thus the intensity of the observed color is directly proportional to the concentration of the pigment in solution and inversely proportional to the depth of the observed layer. Stated mathematically
Cl (concentration) - Rz (depth) Cz (concentration) RI (depth) Therefore, knowing the concentration of one solution and the depths of each solution, we can solve for the concentration of the other. Volume factors and dilution factors can be introduced into this equation in order that the concentrations can be expressed in the proper units such as mg. per 100 cc. (9).
It is quite probable that within a short' time the electropbotometer will be used in colorimetric analysis, since it removes the subjective element in matching of tints and has greater sensitivity. The viscosimeter is another important laboratory tool used in analyzing fluids. It is used to measure the internal friction of a solution by observing the time required for i t to flow from one meniscus in a bulb to a
second meniscus. This is a measure of the relative viscosity as compared with water or some other convenient fluid. Clinically i t can be used to measure the viscosity of blood which is dependent upon the number of erythrocytes present. It is important in the study of blood pressure. In many determinations the Van Slyke methods and apparatus are utilized, especially in gas analysis such as the determination of ammonia, carbon dioxide, and oxygen in blood or any compound which will release or absorb a gas when treated with the proper reagent. Methods of gas concentration analysis are of two types-the volumetric which measures differences in volnme and the manometric which measures differences in pressure. The latter type is considered more efficient since the error in reading barometric pressure is less than in reading volnme, calculation is less complex since vapor tension and capillary attraction of mercury call be neglected; small samples may be used and accuracy is attainable over a wide range of gas concentrations (10). The solution to be analyzed is placed in a chamber over mercury and reagents added to release the desired gas from combination. The stopcock is closed and the leveling bulb lowered to form a vacuum, and the extraction of the gas is assisted by two or three minutes' shaking by a mechanical shaker operated by a small electric motor. The volume is then reduced to the original volume by readmitting the mercury, and the resultant pressure is read from the open arm manometer that is attached. The gases are either ejected or absorbed, depending upon the reagents used, and the second pressure is read with the same gas volume. The partial pressure of the gas a t that volume is then the difference between the initial and final pressures observed. A thermometer records any changes in temperature and the gas volume a t standard conditions can be calculated, and from these data the concentration can be expressed in the proper units. This method is used extensively in measuring the oxygen and carbon dioxide content and capacity of blood. Chemical analvsis in medicine falls ~ r i n c i ~ a,l into lv three or four groups--gastric, urine, blood analysis, and functional tests. Methods in chemical analysis of blood differ in expense of materials, complexity of the necessary apparatus, time required for performance of the tests, and preparation of the necessary reagents. The degree of accuracy required is the single criterion in selection of a method. In large hospitals where multiple determinations are necessary certain methods are more adaptable. In occasional clinical determinations some of the less accurate but simpler routines may be permissible (11). The laboratory findings contribute to the understanding of disease in two ways, first, by indicating sources of infection by the discovery of harmful bacteria in body fluids, and second, by measuring physical and chemical deviations from the normal by qualitative and quantitative analysis of fluids and tissues.
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Functional and tolerance tests are utilized to measure the relative efficiency of the various organs. The following are some of the applications of chemistry to hlood analysis and are hut skeleton outlines stating the principle used rather than the procedure. Just as interfering ions are removed in systematic qualitative analysis total proteins of the hlood are precipitated by tungstic acid (12) (formed by the interaction of sodium tungstate and sulfuric acid) and filtration. The filtrate contains the constituents of the blood determined by this system. Some use the precipitate for the cholesterol and lipoid phosphorous determination. Samples of hlood are protected from bacterial decomposition by the addition of toluene and xylene, and coagulation is prevented by addition of oxalate or of citrate. Gas concentrations are kept stable by keeping the samples under an oil layer until analyzed. From a portion of the filtrate non-protein nitrogen is determined by a micro Kjeldahl method using sulfuric acid and phosphoric acid as digestion mixture. The ammonia produced is distilled into standard acid and titrated using micro burets (13). Non-protein nitrogen of hlood includes the nitrogen present in urea: uric acid, creatinine, ammonia, amino acids, and other substances. The normal amount of non-protein nitrogen in hlood is twenty-five to thirty-five milligrams per hundred cubic centimeters. Higher values indicate pathological conditions such as nephritis. Urea determinations are quite important since they cover a relatively wide range of variations in diseases of the kidney. They are considered reliable for clinical purposes since they are made with a definitely known compound and not a mixture. The usual procedure is to hydrolyze the urea to ammonium carbonate by means of the enzyme urease in the presence of a buffer solution which maintains a constant pH in the mixture (14). The ammonia is distilled off and determined colorimet~icallyafter treatment with Nessler's solution (mercury bichloride, potassium iodide, and potassium hydroxide). Blood urea may also he determined by the Van Slyke manometric analysis. High hlood urea values are found in cases of lead poisoning, mercury bichloride poisoning, as well as in impaired renal function cases. Creatinine determinations are also important in diseases of the kidney. The normal amount is one to two milligrams per hundred centimeters. A blood filtrate portion is treated with an alkaline picrate solution, and the color that develops is compared with a standard in the colorimeter (15). Uric acid determinations are made by observing the blue color produced by the reducing action of uric acid upon phosphotungstic acid reagent (16). The action of 8-naphthol-quinone-sulfonicacid and alkali upon amino acids provides another colorimetric test. For glucose estimation, the protein-free hlood filtrate is heated with alkaline copper solution. The cuprous oxide formed is titrated with iodine. The micro method for hlood sugar consists in oxidiz-
ing the sugar with alkaline potassium ferricyanide and the ferrocyanide produced is converted to Prussian hlue and measured colorimetricaIly (17). Cholesterol is another chemical compound that is well adapted to colorimetric determinations. I t is extracted from whole hlood with chloroform, treated with acetic anhydride and sulfuric acid and the characteristic emerald-green color that develops is compared in the colorimeter with a freshly prepared standard at the same temperature. Cholesterol determinations have been used in basal metabolism tests of children in cases where the usual respiration analyses have failed to give dependable results. Total proteins present in serum (these include albumin and globulin) are determined colorirnetrically or by use of the refractometer. To judge the efficiency of the liver the icteric index is used. This is a comparison of the yellow tint of blood serum with a standard dilution series of potassium dichromate solutions (18). Chlorides are determined in both blood and urine according to the same principle. It is the standard method for quantitative estimation of chloride in any substance since the chlorides are precipitated by means of silver nitrate in the presence of nitric acid, and the excess of silver titrated with standard thiocyanate solution with ferric ammonium sulfate used as indicator (19). For inorganic phosphate and total acid-soluble phosphorus the protein is precipitated with trichloroacetic acid, and the filtrate is treated with molyhdic sulfuric acid reagent forming phosphomolyhdate. Stannous chloride is then added to reduce the phosphomolybdate to colloidal reduced oxides of molybdenum producing a hlue color (20) which is compared in the colorimeter. In adults normal inorganic P is 3.7 milligrams per hundred cubic centimeters. I t is found in the lecithin in the hlood and is concerned with the healing of hones and carbohydrate metabolism. Calcium is another element of importance in hone structure (21). I t is precipitated from serum by the oxalate and the latter is titrated against potassium permanganate. Sodium is also found in the blood as it plays an important part in the acid-base balance of the blood. It is precipitated as the pyroantimonate, and the autimony in the precipitate is titrated with iodine (22). Since iron is related to the oxyeen-combining power of the hlood it is important in blood chemistry. - It is detached from the hemoglobin molecule by sulfuric acid in the presence of potassium persulfate. After the removal of proteins by tungstic acid the iron is determined in the filtrate by the thiocyanate reaction (23). Blood chemistry is important, since it affords valuable diagnostic information. Urine findings are always dependent in part, at least, on the function of the kidneys and therefore do not necessarily represent the true conditions existing in the blood. By blood chemistry we can pass behind the harrier of the kidney (24).
Functional and tolerance tests are also used extensively. Functional tests for the liver and kidneys are made by injecting dyes (derivatives of phenolphthalein) into the blood stream and by a series of analyses for the intensity of the color of the excreted dye within certain time limits. A curve may be plotted wbich will indicate any deviation from normal functioning. Tolerance tests are somewhat analogous. In the case of sugar a certain amount is ingested by the patient, and the blood is analyzed a t successive time intervals. The concentration plotted against t i e will give characteristic curves indicating the relative a c i e n c y of the organs in utilizing the carbohydrate. Many of the tests for routine urine analysis are qualitative and the simpler ring tests are utilized. One ring test commonly used is the test for albumin in which a centrifuged specimen is placed in a test-tube and Robert's reagent (kieserite and nitric acid) is added and a white ring forms a t the level of contact between the two liquids. A rough estimate of the amount of albumin present is indicated on reports by a "one plus" or "two plus" according to the deiiniteness of the ring observed. Benedict's and Fehling's solutions are used in qualitative analysis for glucose; both methods can be modified to give a quantitative estimate if necessary. Acetone and diacetic acid determinations are also made in urine analysis. Bromine and iodine determinations are made by releasing from combination by sulfuric acid or nitric acid and observing their colors as they dissolve in a chloroform layer. Bile pigments are also determined qualitatively. The most common phase of gastric analysis is the determination of total and combined acids by titration against standard sodium hydroxide using double indicator. The free acid is determined k t using dimethylaminoazobenzene. At the endpoint (orange) phenolphthalein is added and the titration is continued to determine the total acid present. From the preceding data, the combined acid can be calculated. Colloid chemistry is used in clinical analysis for testmg the spinal fluid. In certain nervous disorders of bacterial origin, the organism causes changes in the
composition of the spinal fluid (25). A colloid is usually precipitated by the addition of a salt, but if there is something present to protect the colloidal gold from being reached by the salt, it will require a larger concentration of the salt to precipitate the colloid. The spinal fluid is placed in successive dilutions in 1 cc. of sodium chloride and the red colloidal gold solution added. The concentration of salt and the amount of spinal fluid which allows the colloidal gold to be precipitated (indicated by a color change from red to blue) is an indication of the protective power of the spinal fluid and hence an index to the extent to which the fluid has been changed from the normal. This test is known as the gold number and the Lange test (26). The routine in medical chemistry is by no means inflexible and many determinations have alternate procedures most of which can be modified to adapt them to particular cases. The technician is not doomed to o r d i n q routine determinations but must keep abreast of the newer developments in medical chemistry. With the advent of new therapeutic agents corresponding methods of analysis must be devised for them. For example, the doses of the new p-aminobenzenesulfonamide (sulfanilamide) that seem to be specific for bacterial infections especially the streptococci, must be carefully watched to obtain the best results. Its concentration in the blood is estimated by precipitating whole blood with ptoluenesulfonic acid and adding sodium nitrite and dimethyl-a-naphthylamine. A red color is developed which can be compared in the colorimeter with a standard of known concentration. This is one of many possible examples of adjustment to new situations. Chemical procedures are also applicable to bacteriological work in preparation of stains, culture media, buffered solutions, and pH determinations. While this brief sumey does not offer new information to those already engaged in this type of work, it may be of assistance in broadening the horizon of opportunity for the woman chemist who desires a profession that is always vitally interesting, and worthy of the skill she commands.
LITERATURE CITED
(1) B m m , "Women microbe hunters," Independent Women, 4, 379 (Dec., 1936). (2) ROWNTREE, in ''Chemistry in medicine," The Chemical Foundation Inc.. New York City, 1929, pp:41744 (3) LEWAN, "Biochemistry--the basis of medlcme," Medical Record. 143, 12. 510-12 (June 17. 1936). (4) LEV IT^, ibid. 09. cit., P. 418. (5) ROTKNTREE, op. oil.. p. 511. (6) LEVITAN, (7) WELLS,in "Chemistry in medicine," The Chemical Foundation Inc.. New York City, 1929, p. 562. (8) WELLS,ibid., p. 565. 'Tractical physiological chemistry," (9) HAWKAND BERGEIM, 10th edition, P. Blakiston's Son and Co. Inc.. Philadelphia. Penna., 1931, pq. 4 0 3 4 . ~ b z d . p. , 514. (10) HAWKa m BERGEIM, (11) HAWKAND BERGEIM, ibid., p. 400-1. (12) FOLINAND Wu, J . Biol. C h . ,38, 81 (1919).
(13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26)
HAWK~mBERGRIaa, op. cif.,p. 415. HAWKAND BERGEIM, 09. cit., p. 416. 09. c+, p. 421. HAWKAND BERGEIM, HAWKAND BERGEIM, o p . C Z ~p. . , 423. H a m AND B E R G Eop. ~ , cit., p. 434. STITT."Practical bacteriology, blood work and animal parasitology," P. Blakiston's Son and Co. Inc., Philadelphia, Penna., 8th edition, 1927. KIRKLAND, "Certain practical aspects of rend function determinations." Medical Record, 141, 184-6 (1935). op. i t . , p. 455. HAWKAND BERGEIM, HAWKAND B s R c s r ~ibid., , p. 460. ibid.., p. 463. HA- AND BERGEIM, i M . , 466. HAWKAND BERGEIM, S T I ~op. , at.. p. 695. LEMCHEN,"Laboratory diagnosis of general paralysis;' Medical Record, 143,3889 (1936). STITT,09. Cit., pp. 640-4.
