Analytical Problems in Biology and Medicine

REPORT FOR ANALYTICAL

CHEMISTS

Analytical Problems in Biology and Medicine by William B. Mason, School of Medicine and Dentistry, of Rochester, Rochester, Ν. Υ.

The

University

Although a great diversity of disciplines has contributed to progress in biology and medicine, analytical chemistry, in one form or another, may justly be said to constitute the bedrock underlying today's knowledge in these fields. Many measurements that seemed impossible one or two decades ago are now commonplace, or will become so within a few years. Even so, an almost endless array of challenging analytical problems re mains essentially unsolved. Some of these problems are challenging be cause the constituents sought occur at extremely low concentrations. Some are challenging because of the extremely complex nature of bio logical materials; others, because of the small samples available for analysis. Most real problems are combinations of these. /"Venerally speaking, analytical problems in biology and medicine are attacked with the same tools used by investigators working in other fields. Thus spectro photometers (ultraviolet, visible, and infrared), fluorometers, spectrographs (optical and mass), radioactivity detectors, gas chromatographs, flame photometers, and polarographs, all find application. Even so, many of the analytical problems in biology and medicine differ from the usual problems encountered in industry. Represen tative samples are often difficult to obtain because bio logical materials are extremely unhomogeneous, constit uents are usually complex, and quantities available for examination may be very small. This report focuses attention upon a few analytical problems that are rep resentative of those encountered in biology and medicine.

CYTOCHEMISTRY Chemical study of individual cells, as opposed to cells organized into organs, is termed "cytochemistry." Most cells contain numerous small structures, some of which

can be seen with the ordinary light microscope. Others —for example, the ciliary filaments shown in Figure 1— are not visible without higher magnification. Determi nation of the composition and function of these sub cellular components is one of the most fascinating and difficult analytical problems in biology and medicine. In general, there have been two approaches. In some instances it is possible to disrupt the cells and, by appli cation of techniques such as differential centrifugation, prepare suspensions composed largely of identical com ponents. These suspensions are then analyzed in the usual fashion. Alternatively, microprocedures may be applied to the components within individual cells. These procedures usually involve treatment ("staining") with reagents that are more or less specific, followed by visual or spectrophotometric measurements. An indication of the complex subcellular organ ization and function existing in cells is provided by Figure 1, a cross section through the tail of a rat spermatozoon. The entire sperm weighs about 10~* μg. Approximately half of this is concentrated into a compact head. The tail is a long filamentous struc ture having two well-defined parts: a thicker mid-piece VOL. 34, NO. 3, MARCH 1962

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REPORT FOR ANALYTICAL CHEMISTS

that is attached to the head by a short neck, and a thinner main-piece which extends beyond the mid-piece. In rat spermatozoa, the mid-piece (shown in cross sec tion in Figure 1) extends for about 65 microns and includes 40% of the total tail length. Spermatozoa are highly motile, normally traveling many times their body length per minute, and remain active for hours. Motility results from rapid movements of the tail. Very little is known about the chemical reac tions providing energy for motility, or the means by which energy is converted into mechanical move ment. Likewise, little is known about the complex sequence of chemical events that begins upon fertilization of an ovum by a sperm head and leads ultimately to a ma ture embryo. TRACE O R G A N I C CONSTITUENTS

Many organic constituents occuring in trace concentrations are extremely important in the regula tion of physiological processes. As in cytochemistry, however, investi gations have often been hampered by lack of analytical methods hav ing sufficient specificity and sen sitivity. This has been the case with aldosterone. 24 A

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ANALYTICAL CHEMISTRY

This steriod (Figure 2) is ex tremely important in the regulation of sodium levels in human body fluids. Even so, relatively little is known about normal levels of aldosterone, or their fluctuation in disease. Chemical determination has been difficult because of the small amount of aldosterone in nor mal human urine (about 10 μ-g. per 24-hour specimen) and the simul taneous occurrence of several score other steroids, many in much larger concentration. Recently Kliman and Peterson (2) developed a double isotope derivative method for aldosterone which promises to be useful. In their procedure, the residue from a crude urinary extract is treated with tritium-labeled acetic anhy dride, which converts aldosterone quantitatively to the tritiumlabeled diacetate. A measured amount of authentic aldosterone diacetate-C 14 is then added, and the doubly labeled steroid is purified by paper chromatography. Three separate chromatographic proce dures are employed, with chromic acid oxidation between the second and third steps. Although the final product is not pure aldosterone, the sequence is adequate to separate aldosterone from other tritiumlabeled materials, and aldosterone is estimated reliably by measuring tritium and carbon-14 activities.

Using this technique, it has been possible to assay aliquots of urine containing about 0.1 /xg. of aldos terone with an uncertainty of ±15%. METABOLIC PATHWAYS

Another difficult and important problem has been elucidation of steps in the metabolism of food stuffs, from the time they enter the body until they become parts of body structures, or until their meta bolic end products are ultimately excreted in the urine, feces, breath, or perspiration. Much of the prog ress in determining these metabolic pathways can be attributed to studies using isotopically labeled materials. The analytical problems in such studies lie in identifying the reaction products and determin ing the proportion of reactant being converted to each product. Be cause of the small amounts of ma terials converted, and the complex ity of the medium, these analyses would have been virtually impos sible without the aid of isotopic labels. Stable and radioactive iso topes have both been used, and many metabolic sequences now seem well established for both ani mals and plants. Many of the en zymes and cofactors necessary for individual steps have also been iso lated and characterized.


^

Figure 1

Electron micrograph of a tail of rat spermatozoon sectioned transversely through the mid-piece. (The oval ap pearance results mainly from compres sion during sectioning, rather than from an oblique cut.) The outermost en velope is the cell membrane. Within this is a layer of mitochondria whose complex system of membranes is clearly visible. These elongated mito chondria form four parallel spirals of many turns, one turn being included in this micrograph. Ciliary filaments ex tend from the sperm head to the tip of the tail. They are seen in the center of the section and consist of nine double filaments arranged in a circle around a pair of central filaments. One member of each doublet is hollow in

these preparations while the other con tains moderately dense material. Both members of the central pair are hollow. If the micrograph is rotated in a clock wise direction, the hollow member is on the leading side of each doublet. This general organization of nine-by-two fila ments is common to many cilia regard less of source and is presumably as sociated with ciliary motility. Between the ciliary filaments and the mito chondria in the sperm tail are nine rather large, homogeneous elements which run through the mid-piece and appear to be associated with the nine ciliary doublets. Their function and composition is unknown. (Photograph and description kindly supplied by Dr. M. L. Watson, University of Rochester School of Medicine and Dentistry and Atomic Energy Project.)

METABOLISM O F DRUGS Tracer techniques have also g r e a t l y simplified investigation of drug metabolism. I n the case of nicotine (Figure 3 ) , for example, which has been studied extensively, i t h a s been shown t h a t dogs receiv i n g intravenous injections of r a n domly labeled nicotine excrete 80 t o •90% of t h e C 1 4 a c t i v i t y in their u r i n e within 1 t o 2 d a y s . Some of t h e excreted m a t e r i a l is unchanged nicotine, b u t most of t h e C 1 4 a c tivity resides in d e g r a d a t i o n p r o d ucts (3). In separate experi m e n t s using nicotine containing C 1 4 only in t h e m e t h y l group, it w a s shown (using r a t s ) t h a t a b o u t 10% of the C 1 4 a p p e a r e d in t h e ex p i r e d air within 24 hours (4) · I t t h u s seems possible t o account •quantitatively for n e a r l y all t h e m e t a b o l i c products from nicotine. T h i s is unusual, a n d equally com p l e t e d a t a are available for only a few drugs. P r e p a r a t i o n of labeled m a t e r i a l s m a y p r e s e n t a difficult problem in organic synthesis. Sometimes, as in t h e case of nicotine, it is possible t o utilize a biologic process. I n this i n s t a n c e tobacco w a s grown in a special greenhouse h a v i n g a n a t m o s phere containing C 1 4 0 2 . Labeled nicotine w a s t h e n isolated from t h e tobacco.

Dr. William Β. Mason is associate professor of bio chemistry, medicine, and pathology (clinical chemistry) at the School of Medicine and Dentistry, University of Rochester. He was born in Warren, Ohio, in 1920. He obtained his B.S. in chemistry with high distinction from the University of Rochester (1942). He then went to Princeton University where he served as a teaching assistant ( 1 9 4 2 - 4 4 ) and obtained his M,A. in analytical chemistry (1944). From 1944 to 1946 he worked on the Manhattan District Project at Prince ton and received his Ph.D. in analytical chemistry (1946) working with Prof. Ν. Η. Furman. He then went to the University of Rochester where he worked on the atomic energy project (1946—58). During this period he studied medicine and received his M.D. with honor ( 1 9 5 0 ) . Since 1950 he has served at the University of Rochester School of Medicine and Dentistry in several capacities: intern in pathology ( 1 9 5 0 - 5 1 ) , assistant resident in pathology and part time instructor in biochemistry ( 1 9 5 1 - 5 2 ) , scientist in clinical chemistry and instructor in biochemistry (1951—58), as sistant professor of biochemistry and medicine (1957—59), and associate pro fessor of biochemistry and medicine since 1959. In 1 9 6 1 his appointment was extended to include pathology. During the academic year 1961—62 he is on sabbatical leave as a Fellow in Clinical Pathology at the National Institutes of Health, Bethesda, M d . He is a member of the Phi Beta Kappa, Sigma Xi, American Chemical So ciety, American Association for Advancement of Science, Rochester Academy of Medicine, American Association of Clinical Chemists, The Coblentz Society, Association of American Medical Colleges, (Individual Member), New York Academy of Sciences, and the Society for Applied Spectroscopy. Dr. Mason has published a number of papers in the area of analysis of radioactive materials, biological effects of radiation, and analytical procedures in clinical chemistry, particularly with respect to spectrophotometric techniques in the micro and ultramicro range.

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Figure 4 Differentiation of human hemoglobins by starch-gel zone electrophoresis at pH 9.05 in 0.030M borate buffer. Migration rates for normal adult hemoglobin and sickle-cell hemoglobin are dis tinctly different, and both types are clearly evident in blood from patients having sickle-cell trait (mild form of sickle-cell anemia). In hemoglobin-C sickle cell disease, normal adult hemoglobin is absent; the hemoglobin is partly sickle cell hemoglobin and partly the very slow-moving variety termed hemoglobin-C. For additional details, see reference 1.

MOLECULAR DISEASES

The phrase "molecular disease" has been used (5) to designate dis eases that "result from the manu facture by the patient of abnormal molecules in place of normal mole cules which are manufactured by normal individuals." In a broader sense, all diseases are probably mo lecular diseases. Those falling out side the above definition most likely involve either the presence of mole cules that do not occur normally— viruses, bacterial toxins, abnormal metabolites, for example—or ab normal quantities of one or more molecules necessary for specific physiological processes. Diabetes, as is well known, is a disease result ing primarily from inadequate pro duction of insulin. Clear demonstration that mo lecular diseases do indeed exist re sulted from the careful application of analytical techniques to hemo globin obtained from patients hav ing an unusual form of anemia. This anemia, characterized by the tendency of the red blood cells to change from their normal discoid shapes into crescents when the blood cells are exposed to low par tial pressures of oxygen, had been recognized as a clinical entity about 1910. During the following three

decades it was shown that this socalled "sickle-cell anemia" ex hibited clear genetic transmission through family lines, and that the disease existed in mild and severe forms. It was postulated that the severe form resulted when a pair of genes controlling hemoglobin syn thesis were abnormal, and that the milder disease occurred when only one gene was abnormal. In 1946, H. A. Itano, a physician embarking upon graduate study in chemistry with Linus Pauling at the California Institute of Technology, began sys tematic studies that led within a few years to chemical differentia tion between normal and sickle-cell hemoglobin (6). Electrophoresis proved extremely useful, and with this technique it was possible to show that all the hemoglobin from patients having the severe form of sickle-cell anemia was abnormal. By contrast, less than half of the hemoglobin from patients having the milder disease was of the sicklecell type. The existence of hemoglobins having different electrophoretic characteristics spurred protein chemists to investigate the chemical basis of the difference. I t now seems established that the amino acid compositions of normal adult hemoglobin and sickle-cell hemo-


globin are identical except for a single amino acid per half-molecule —i.e., one amino acid in approximately 300. It is not yet known how extensive a change this may produce in the configuration of the hemoglobin molecule. Zone electrophoresis, as in Figure 4, simplified study of hemoglobins, and contributed greatly to the rapid recognition of many abnormal forms other than that associated with sickle-cell anemia. Today more than a score of abnormal hemoglobins have been identified. Some of these occur in the absence of recognized disease. Advances in the chemical characterization of hemoglobin have piwided important new tools for geneticists and anthropologists.

CLINICAL

CHEMISTRY

Chemical procedures useful in detecting or diagnosing disease, or in evaluating therapy, are collectively referred to as "clinical chemistry." These procedures range from simple wet tests, as for glucose and other reducing substances in urine, to complex instrumental methods involving equipment such as the ultracentrifuge. The past 20 years have witnessed tremendous growth in clinical chemistry, with regard to both numbers of determinations and complexity of procedures. It has been estimated that nearly a million clinical chemical determinations are currently being done each day in the United States, and that the work load is increasing 10 to 15% per year. Improvements in analytical techniques have contributed substantially to this growth. Sodium and potassium determinations, for example, now constitute about 10% of the total clinical chemistry work load. Both measurements were made extremely infrequently prior to the commercial introduction of relatively simple flame photometers during the 1940's. Calcium, by contrast, although equally important, is determined about one fourth as frequently, and magnesium, which is essential in many biological reactions, is seldom determined

except for research purposes. The apparent discrepancy between the biological significance of these elements and the relative infrequency of their determination reflects the absence of good analytical procedures applicable to biological samples. Many physicians have predicted that a great increase in magnesium and calcium measurements will occur as soon as more satisfactory analytical methods become generally available. Much of the growth in clinical chemistry has stemmed from new biochemical knowledge. A decade ago the measurement of enzymes in blood, for example, was limited to infrequent estimation of less than a half-dozen enzymes. Today, procedures for determining more than a score are available, and the measurement of blood enzymes constitutes about 10% of the total clinical chemistry work load. Especially interesting are recent observations that several enzymes apparently occur in different molecular forms,

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and that both the absolute and relative concentrations of these isozymes vary from disease to disease. Determination of individual isozymes poses a more complex problem than determination of total enzyme activity, but may provide considerable additional information having clinical value. Perhaps the most important single factor in the growth of clinical chemistry is the growing realization that analytical measurements are among the most promising of the few techniques capable of supplying new kinds of information about sick people. It seems doubtful that much new information can be obtained by more refined clinical examination of patients, or by more searching questions regarding the development of their illness, or by better x-ray studies. Laboratory tests offer the greatest hope for the future, and of these, analytical chemistry presents the clearest potential and one of the greatest challenges. Consider, for example, the

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many hundreds of unrecognized or unmeasured constituents that probably exist in low concentration in urine and in expired air, both of which are easily available in large quantities. In addition to a growing utilization of clinical chemistry in evaluating sick patients, it seems likely, within a few years, that clinical chemistry examinations will be made periodically upon thousands of well persons in an effort to detect the earliest stages of cancer and other diseases. As in other experiments, many of the data collected in these studies will perhaps appear to have no immediate value. Nevertheless, if continued systematically, this approach may increase our understanding of many illnesses and, by facilitating early recognition of disease, lead to early corrective therapy and reduced mortality. Many of the clinical facilities required for such long-range studies already exist, but analytical facilities must be expanded and developed.

CLINICAL CHEMISTRY METHODOLOGY

Until recently, only the simplest analytical methods found general application in clinical chemistry. This is well illustrated by electrophoresis. The use of moving boundary electrophoresis for determining the types of proteins present in human blood plasma was already well established by 1939. Nearly 20 years elapsed, however, before electrophoresis came into general use in clinical chemistry; then only after development of the much simpler techniques of zone electrophoresis. Paper electrophoresis of serum proteins is now routine in many hospitals and has stimulated extensive study of a diverse group of diseases, formerly considered to be rare, which have been termed "paraproteinemias." Several factors are responsible for the introduction of more complex analytical methods in clinical chemistry. It is becoming apparent that many important constituents cannot be measured by simple procedures. In addition, with increasing concern for problems of health,

REPORT

funds are being made available to support more complex analytical procedures. Most importantly, professional groups, both among physicians and among chemists, have become actively engaged in educational programs aimed at im proving clinical chemistry. Use of automatic and semiauto matic equipment has become com monplace. It is interesting to note that the first fully automatic sys tem of colorimetric analysis was developed for use in clinical chem istry (7). This equipment, com mercially available from the Technicon Co., Chauncey, Ν. Υ., has also found wide application in in dustrial laboratories. Micromethods have great practi cal importance, especially when de terminations must be made on very small samples, as from infants. Utilization of micromethods, how ever, is not yet widespread, al though several manufacturers have made available complete micro systems which include reagents as well as the specialized equipment required. NEED FOR BETTER METHODS

Investigators working in biology and medicine have great need for better analytical methods. Several forms of improvement over existing procedures are required. Most pressing is the need for methods that combine very high sensitivity with good specificity. Methods are required which will permit identify ing and measuring relatively simple organic compounds (80 atoms or less per molecule) occurring in amounts no larger than 10 -4 μg•! preferably no larger than 10~5 μg. These small amounts of material usually occur as trace constituents in mixtures containing hundreds of other organic compounds, some of which may be closely similar to the constituent sought; hence the need for good specificity. Procedures employing formation of radioactive derivatives, as in the determination of aldosterone men tioned earlier in this report, are among the most sensitive and spe cific at present available. Fluores cence techniques, utilizing either fluorescence which is inherent to the material being measured, or

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Figure 5 Separation of Amino Acid Derivatives by Gas Chromatography. The combination of Golay columns and programmed temperature operation gives excellent resolution in short analysis time. This run was made with a 150 ft. by 0.010 in. Golay column coated with QF-10056 fluorinated alkyl silicone. Column temperature was programmed from an initial isothermal temperature of 125° C. at a rate of 25° C. per minute up to 200° C , and then maintained at that temperature. A 2 μ\. sample was used, with 1/1000 of the effluent passing into a flame ionization detector. Temperature of the injection block was 300° C.

that resulting from derivative for mation, also provide good sensi tivity and specificity. For the most part, however, existing procedures based upon fluorescence or radio activity measurements fall several orders of magnitude below the de sired sensitivity. They are also tedious and time-consuming. Gas chromatography offers considerable promise, with regard to both sensi tivity and ease of measurement, but considerable advancement will be required beyond present commer cial instruments for many applica tions. There is also need for methods capable of detecting very small dif ferences in large molecules, such as proteins. An example of the speci ficity required has been provided by the work on hemoglobin already cited. Techniques utilizing highly specific immune reactions are es pecially promising. By combining precipitin formation with gel elec trophoresis ("Immunoelectropho resis"), it has been possible, for ex ample, to demonstrate the existence of a great many different proteins in normal human serum. Remarkable separations of serum proteins have also been obtained by using poly mers (acrylamide) as the support in zone electrophoresis. Even so,

much finer resolution of complex molecules is required. Finally, there is great need for methods that are faster and less laborious than those at present in use. Determination of the amino acid sequence in proteins, for ex ample, was greatly facilitated by the development of automatic col umn chromatographic procedures. Even so, many hours are required for analysis of a single amino acid mixture by this technique. By gas chromatography the time required for this analysis has now been re duced to a few minutes (Figure 5). Similar improvements are required in many determinations. References 1. Engle, R. L., Jr., Markey, Ann, Pert, J. H., Woods, K. R., Clin. Chim. Acta 6, 136 (1961). 2. Kliman, B., Peterson, R. E., / . Biol. Chem. 235, 1639 (1960). 3. McKennis, Herbert, Jr., Bowman, E. R., Turnbull, L. B., Proc. Soc. Exptl. Med. 107, 145 (1961). 4. McKennis, Herbert, Jr., Wada, E., Bowman, E. R., Turnbull, L. B., Nature 190, 910 (1961). 5. Pauling, Linus, Harvey Lectures 49, 236 (1954). 6. Pauling, Linus, Itano, Η . Α., Singer, S. J., Wells, I. C , Science 110, 543 (1949). 7. Skeggs, L. T., Am. J. Clin. Pathol. 28, 311 (1957).

Analytical Problems in Biology and Medicine

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