V O L U M E 2 7 , NO. 4, A P R I L 1 9 5 5 (103) ROSS,J. H., ANAL.C I I E J I . , 25, 1285 (1953). (104) Russian Pharmacopoeia, 7th ed., ;\loscow, 1952. (105) Sale, J. W., et al., d r n . Pe,:fuiner Essent. Oil. Rec., 61, 4 7 i (1952); J . Assoc. O$lc. Agr. Chemists. 36, 112 (1952). (106) Salomaa, P., Suomen Kemistilehii. 27B, S o . 2, 12 (1954). (107) Schenck, G., and Fromniing, K. H., - ~ a t u r ~ i s s e n s c h a e40, n, 276 (1953). (108) Schirm, hI., De7~t.Bpoth.-Zig., 93,273 (1953). (109) Schoniger, W., Mikrochirri. Acta, 1953, 434. (110) Schults, 0. E . , and Gmelin, R., Z . .l-atiLrforsch., Bb, 151 (1953). (111) Ibid., 9b, 27-9 (1954). (112) Schults, 0. E . , Gmelin, R., and Iceller, d.,Ibid., Bb, 14 (1953). (113) Siggia, Sidney, “Quantitative Organic -1nalysis Via Functional Groups,” 2nd ed.. p. 227, Wiles, S e w Tork, 1954. (114) Siggia, Sidney, and Segal. E.. .IsaL. CHEX, 25, 610-2 (1953). (115) Ihid., pp. 830-1. (116) Sjostrom, E . , Stensk Kern. Tidskr.. 64, 301 (1952). (117) Snell, F. D . , and Snell, C . T.. “Colorimetric Methods of Analysis. Tol. I. Organic 3Iethods,” p. 614, Van Sostrand, S e w York, 1953. (118) Society for Analytical Chemistry, Analytical Methods Committee, Analyst, 79, 397 (1954). (119) Souchay, P., and Graizon, AI.,Chin7. anal., 36, 85 (1954).
617 (120) Stahl, E., Mikrochemie Fer. Mikrochim. dcta, 40, 367 (1953). , 86-90 (1954). (121) Sully, B. D., A t ~ a l y s t 79, (122) Sutherland, RI. D., Perfumery Essent. Oil Record, 43, 453 (1952). (123) Sgkora, I-.,and Prochlska, Z., Chem. Lis&, 47, 1674 (1953). (124) Syndicat Kational des Fabricants et Importateurs d’Huiles Essentielles e t Produits Aromatiques Saturels de Grasse. “Nethodes et constantes analytiques des huiles essentielles,” Grasse (Var), France, 1954. (125) Terent’ev, -1.P., and Zabrodina, K. S., Doklady Akad. S a u k S.S.S.R., 95, 85 (1954). (126) Torres, J. C., Inform. guim. anal., 6, 145 (1952). (127) Torres, J. C., and de Diego, F. B. P., Farmacognosia Madrid, 11, 419 (1952). (128) Van Duin, H . , Rec. trav. chim.,73, 68-77 (1954). (129) Van Duuren, A. J., Ibid., 72, 889-92 (1953). (130) Voge, C. I. B., and Sinclair, F. E., Intern. Perfumer, 2, KO. 7 , 70 (1952). (131) Wegner, E., Pharm. Zentralhalle, 93, 139 (1954). (132) Wojahn, H . , Ibid., 91, 326 (1952). (133) Xavier, 31. L., T-erghese, J., and Yeddanapalli, L. AI., Current Sci., 22, 112 (1953). (134) Yarborough, Y. .A,, -%SAL. CHEX, 25, 1914-16 (1953). (135) Zeehuisen, J. J., J . Sci. Research Indonesia, 2, 24 (1953).
Clinical Chemistry
I
R. M. ARCHIBALD Hospital of the Rockefeller lnstitote for M e d i c a l Research, N e w York 21,
T
THIS ievien does not attempt to eo-rer all of the many contributioiTq to quanritstive clinical chemistry which have appeared during the past 2 years, up to September 30, 1954 Many of those not mentioned here ma! prove eventuallj to be exceptionally significant contributions. However, in t h e light of the author’s present knonledge, and in the opinion of the colleagues with TI hom he has conferred and to whom he is thus indebted, the contribution> mentioned are among the most important. S T A h D 4 R D TIETHODS
T h e moqt significant advance for clinical biochenii2trj during the past 2-pear period has been the appearance of t h e first volunie of “Standard Methods of Clinical Chemistry” (48) dltliougli this publication presents no new methods or concepts, it is the result of the effort of clinical chemists to arrive a t an understanding as to M hich of several available chemical methods is the best for routine measurement of a given suhstance or p r o p a t ) of clinical importance. Thus an a t t e x p t has been made to provide for clinical chemists a compilation of standard procedure. equivalent to that ’i\ hich the Association of Official Agricultural Chemists provides in its official p.!hlication (6) foi agricultural chemists. It is obvious t h a t many ieviewers feel t h a t the work leaves much to be desired: that the selection of methods presented is not t h e best (33),t h a t purit>-of ieagents to be employed should be specified (IZ),t h a t stability of reagents should he indicated (8), t h a t t h e allowable percentage deviation of duplicates for each method should be specified (8), and that qualitative as ne11 as quantitative tests should be included ( 3 6 ) . I t is important, however, t h a t the start has been made. It is inevitable t h a t the choice of method. is not acceptable to all. Tn the present volunie each selection was made on the basis of the judgment of a relativelv small nuinhei of clinical chemists. Subsequent volumes nill gain in value and recognition as an
N. Y.
accepted compilation of standard methods, to t h e extent to which other clinical chemists effectively voice their constructive criticism regarding the selection of methods and manner of presentation, and t h e editors and contributors weigh and act on the suggestions of their colleagues, voiced either in personal communications 01 in the book reviews which follow the appearance of each volume. As the eyperience of a n ever-increasing number of clinical chemists is brought to bear on t h e subject, the work nill acquire greater prestige as a standard compilation until it beconies regarded lyith an esteem comparable to t h a t in which the AOAC publication is held. Wherever the contributors and editorial board c h o x e not to follow suggeitions made by a n appreciable number of readers, it would seem desirable to state in subsequent editions the Ieasons nhy, in the judgment of the board, it is deemed unnise to comply. T h r reading public will then be able to neigh the choices in the light of the reasons given, and to comment furthei if necessary. T n e evolution of such a collection of standard methods n oi t h y of almost universal acceptance 1% ill be a n arduous and continuing but n-orth-n-hile task. It lvould appear to this reviener t h a t it nould be helpful t o have in such a manual not only the directions for t h e procedures to be followed, but also numerous notes, in small print, to indicate the probable source2 and causes of trouble and means of preventing or overcoming difficulties when thece arise X good evample of this may be the type of notes in small print in -A. A. Soyes’s s j stem of quantitative t of t h e American hssociation of Clinical Chemists is t h e launching of its official journal, Clznzcal Chenizstry. A moie coniprehensive coverage of methods of clinical chemistrj ha. been presented clearly and concisely by Hiller ( 2 7 ) for the guidance of technicians. Hepler’s book (26) gives more attention to the nonchemical procedures of the clinical laboratory.
ANALYTICAL CHEMISTRY
678 The last edition of “Practical Physiological Chemistry” (25) contains a selection of micromethods for clinical chemists. VITAMINS AND HORMONES
A new method for the determination of ascorbic acid has proved useful for the measurement of this vitamin in food and pharmaceut,ical preparations (53). It has a high degree of specificity attributable to the reaction of the enediol grouping of ascorbic acid with diazotized Pmethoxy-2-nitroaniline. Dehydroascorbic acid does not interfere. The procedure has not yet been adapted to the measurement of the small amounts ordinarily present in blood, urine, and small samples of tissue, but as the recovery of relatively large added amounts is quantitative (54), there is reasonable hope t h a t this reaction may prove useful to the clinical chemist as well as to biochemical investigators. A modification of the more conventional method has been described by Kingsley and Schaffert (32). Recent developments in the field of quantitative determination of steroids have been reviewed extensively (49,47). The methods described, with few exceptions, are not suitable for use in the clinical laboratory. Simpler and more specific methods will need to be developed before i t will be practical to assay many steroids in the central laboratory of most hospitals. Some of the methods appear to be well adapted for use in investigational work. It has been widely assumed t h a t the adrenal cortical hormones and their metabolites are either reducing steroids or Porter-Silber chromogens. However, only a small percentage of the administered hormone can be accounted for by steroids which either have appreciable reducing properties or react with PorterSilber reagent. Fukushima et al. (20) have found t h a t in neutral ether a t least 30% of the metabolites of hydrocortisone were in the form of four new sterols which neither reduce nor react as Porter-Silber chromogens. The failure of t h e present classical methods to effect sharp fractionation of steroids is indicated by the fact t h a t because of relatively high water solubility some of these new steroids could be detected in the phenolic fraction despite the fact t h a t they are not acidic. The Porter-Silber reaction is reported to yield abnormally high values ( 4 2 ) after administration of chloral hydrate, paraldehyde, iodide, or iodine. The chromogens obtained after iodine or iodide yield an absorption curve identical with t h a t derived from hydrocortisone. The routine determination of chloride has been simplified by Kingsley and Dowdell (28) by elimination of the use of thiosulfate. The simpler method for measurement of cholesterol (SO), mentioned in the previous review ( 4 ) ,is proving to be useful and more widely accepted. Improved methods for measurement of creatine and creatinine have recently appeared (48, page 55; 64). Sunderman (61, 62) and Somogyi (59) have improved reagents used for the measurement of blood sugar. The method outlined by Reinhold (49, page 6 5 ) is a modification of the earlier method of Nelson. Somogyi’s studies ( 5 8 ) on arteriovenous differences in blood sugar (concentration in fingertip blood-Le., capillary blood-less concentration in peripheral venous blood) may prove useful in clinical studies. Whether or not Binkley’s simple method for the determination of glutathione and its degradation products (10) will prove useful in the study of diabetes remains to be seen. The measurement of adrenogenic amines in shock or for the diagnosis of pheochromocytoma or as aids in the differentiation of conditions involving hypertension has been facilitated by development of methods for ephedrine and norephedrine (41, 65). Other methods have been developed for the extraction of noradrenaline and adrenaline and hydroxytyramine from urine ( 1 7 ) . Perhaps the best and probably t h e most widely used method for protein-bound iodine ( 7 ) is still giving unsatisfactory results in the hands of many operators. Figures for protein-bound iodine, when the procedure is working well, are of considerable clinical significance. However, i t is hoped t h a t simpler and more readily reproducible laboratory methods will be developed
shortly t o yield clinical information of equivalent or greater value. Flame photometric measurements of sodium, potassium, and cdcium have been made more sensitive by the use of photomultiplier tube attachments arid a high concentration of acetone in the solutions to be atomized (29, $1, 66, 68). The 2- to 20fold elevation of serum glutamic oxaloacetic transaminase activity observed 12 to 24 hours after myocardial infarction is of interest (58). After further investigation measurement of this enzyme might prove to be a useful index of acute tissue damage. Those eoncerned with forensic chemistry will be interested in recent methods for the measurement of alcohol (40, 51;) and mercury (67) in blood or urine. The ultraviolet absorption procedures of Goldbaum (21) have facilitated differentiation and determination of b:trbituratep (1-3).
Tissue respiration studies form a part of the basic research underlying the development of mew drugs and new chemical techniques
Several modifications of the bromosulfaline liver test have appeared. The improvements which they introduce are small compared with the inherent limitations of the procedure, results of which are determined not only by liver function but also by such factors as blood flow in the liver and renal excretion of dye. Apparently improved methods have appeared for the measurement of serum cholinesterase ( I S , 49), plasma heparin (19), cystine and cysteine ( 1 4 ) , choline (44, page 265), and nucleic acids (44, page 287). Considerable progress is being made in the fractionation of protein mixtures ( 6 7 )and in the specific determination of the various proteins so separated (50752). It seems reasonable to expect that the detection and identification of abnormal hemoglobins may become a matter of considerable clinical significance, now that the work of Itano and his colleagues ( 2 4 )has facilitated such detection. NEW TECHNIQUES
The location of the boundary between the realm of relatively simple, straightforward methods which are generally regarded as capable of yielding results of clinical importance, and on the other hand, the methods which are useful primarily in investigative work, will vary somewhat from one laboratory to another,
V O L U M E 27, NO. 4, A P R I L 1 9 5 5 depending upon the competence and interest of the technical staff and the interests and demands of the clinical investigators. Methods which are generally regarded as too complicated or exacting for a general clinical laboratory, or methods which yield results of as yet unproved clinical significance, will probably be conducted by t h e general clinical laboratory staff in locations where the clinicians are interested in pursuing a related investigation. Although many of thebe will be dropped when lark of clinical significance of results is recognized, nevertheless from this group will come new procedures which will yield information of very definite clinical significance, and will replace some of the standard methods now generally employed. It appears mobt likely t h a t these new and promising techniques will use some of t h e very potent tools of fractionation Jvhich have been exploited to excellent adrantage during the past several years (34). Countercurrent fractionation has made possible improved methods of measuring estrogens in biological material (16, 46). Starch block (44,page 141) and paper (15, 22) zone electrophoresis ( 1 8 ) have permitted the separation and identification of complex lipoproteins ( 3 7 ) , enzymes (-$), and other piotein components of mixtures ( l b , 56,39), and separation of amino acids ( 15, 22) and iodine-containing derivatives, precursors, or metabolites of thyroxin (15). Paper chromatography continues to be evploited to good advantage for fractionation of barbiturates ( 1 , 2), hemoglobins ( 2 4 ) , uiine wgars (44, page 205; 6 0 ) ) thyroid (25,44,page 2431. and adrenal hormones (11, 44, page 171). These methods have heen improved by the use of more or less specific stains for gixvm types of compounds-e g., Sudan black for lipides (37, 6Y), peliodate followed by Schiff’s reagent for carbohydrates (96).and bromophenol blue for proteinP. Tests Jvhich employ radioactive isotopic labels v, ere once considered as scarcely falling n-it’iin the scope of the work of the clinical laboratory. However, in the not too distant future, use of these is likely to become sufficiently n-idespread and helpful to justify training t h e technical help of the clinical laboratory to handle a t least the biological material from patients subjected to tracer studies, if not the undiluted solutions of radioactive isotopes. T h e clinicians responsible for the ordering and administration of the tests should be aell versed in the limitations of the tests, the possible fallacies of the interpretation, and the need to avoid exposure of the patient and personnel to hazardous doses of radiation. Good preparations of iodine-131-labeled albumin and gamma-globulin are no\\ available and are admirably adapted to the determination of plaqnia volume ( 9 , 5 5 ) .
LITERATURE CITED
(1) Algeri, E. J., and lIcBay, -4.J., A m . J . Clin. Pathol., 23, 654
(1953). (2) -4lgeri E. J., and Walker, J. T., Ibid., 22, 37 (1952). ( 3 ) AllgBn, L. G., Svensk Farm. Tidskr., 57, 188 (1953). (4) drchibald, R. bI., ASAL.CHEW,25,2 (1953).
(5) Xrchibald, R. lI.,unpublished manuscript. (6) Association of Official dgricultural Chemists, Washington, D. C., “Official Methods of Analysis,” 7th ed., 1950. (7) Barker, S. B., Humphrey, 31. J., and Soley, 31. H., J . Clin. Invest., 30, 55 (1951). (8) Belk, TTT. P., Am. J . Clin. Pathol., 24,209 (1954). (9) Berson, S. A, and Yalow, R. S., J . Clin. Invest., 31, 572 (1952). (10) Binkley, F., Fujii , and Kimmel, J. R., J . Biol.Chem., 186,159 (1950). (11) Bush, I. E., Brit. Med. Bull., 10, 229 (1954). (12) Clarke, B. L., Sci. Monthly, 78,393 (1954). (13) de la Huerga, J., Yesinick, C., and Popper, H., Am. J . Clin. Pathol., 22, 1126 (1952). (14) Dent, C. E., Senior, B., and Walshe, J. >I., J . Clin. Invest., 33, 1216 (1954). (15) Durrum, E. L., J . Am. Chem. Soc., 72,2943 (1950). 116) Engel, L. L., Slaunwhite, IT. R., J r . , Carter, P., Olmsted, P. C., and Sathanson, I. T., Ciba Foundation, Colloquia on Endocrinology, Vol. 2, p. 104, Blakiston, Philadelphia, 1952.
679 Euler, U. 9. von, and Hellner, S., Acta physiol. Scand., 22, 161 (1951). Fisher, B., Am. J . Clin. Pathol., 23, 246 (1953). Freeman, L., Engelberg, H., and Dudley, d.,Ibid., 24, 599 (1954). Fukushima, D. K., Leeds, N. S., Bradlow, H. L., Kritchevsky, T. H., Stoken, 11. B.. and Gallagher, T. F., J . Biol. Chem., in press. Goldbaum, L. R., ASAL. CHEM.,24,1604 (1952). Grassmann, W.,and Hannig, K., SaturwissenschaftPn, 37, 397 (1950). Gross, J., Brit.Med. Bull., 10, 218 (1954). Hal-inga, E., and Itano, H. A , Proc. Satl. Acad. Sci., 39, 65 (1953). Hawk, P. B., Oser, B. L., and Summeraon, W. H., “Practical Physiological Chemistry,” 13th ed., p. 662, Blakiston, New York, 1954. Hepler, 0. E., “3Ianual of Clinical Laboratory Methods,” 4th ed., C. C Thomas, Springfield, Ill., 1953. Hiller. d.. “Practical Clinical Chemistry,” C. C Thomas, Springfield, Ill., 1953. Kingsley, G. R., and Dowdell, L. d.,J . Lab. Clin. &fed., 35, 637 (1950). Kingsley, G. R., and Schaffert, R. R., AXAL.CHEM.,25, 1738 (1953). Kingsley, G. R., and Schaffert, R. R., J . B i d . Chem., 180, 315 (1949). Ibid., 206,807 (1954). Ibid., in press. Kirk, P. L., . ~ R ’ A L .CHEX.,26, KO.3 . 30-1 (1954). Kirk. P. L., and Duggan, E. L., I b i d . , 26, 163 (1954). Kleiner, I. S., Arch. Biochem. Biophys., 51, 553 (1954). Kiiiw, E., and Gronwall, A , , Scand. J . Clin. & Lab. Incest., 4, 244 (1952). Kunkel, H. G., and Slater, R. J., J . Clin.Inuest., 31, 677 (1952). LaDue, J . S., Wroblewski, F., and Karmen, d.,Science, 120, 497 (1954). Lerin, B., and Oberholaer, V. G., Am. J . Clin. Pathol., 23, 205 (1953). McConnell, IT. B., Ibid., 22, 1223 (1952). Manger, W.51., et al., Proc. Sta$ Meetings M a y o Clinic, 28, 526 (1953). Marks, L. J . , and Leftin, J. H., J . Clim. Endocrinol. & Metabolism, 14, 1263 (1954). Mem. Soc. Endocrinol. (Gt. Brit.), No. 2 (1953). Methods ofBiochem. Analysis, 1 (1954). LIigeon, C. J., J . Clin. Endocrinol. & Metabolism, 13, 674 (1953) Soyes, A. d.,Bray, W.C., and Spear, E. B., J . Am. Chem. Soc., 30, 481 (1908). Recent Progr. Hormone Research, 9 (1954). Reiner, AI., ed., “Standard Methods of Clinical Chemistry,” Tal. 1, Academic Press, N e w York, 1953. Reinhold, J. G., Tourigny, L. G., and Yonan, V. L., Arn. J . Clin. Pathol., 23, 645 (1953). Saifer, A , , Am. J . M e d . , 13,730 (1952). Saifer, A, and Zymaris, 11.C., J . Clin.Invest., 31, 1 (1952). Saifer, A , Zymaris, M . C., and Berger, H., Ibid., 31, 12 (1952). Schmall, XI., Pifer, C. W., and Wollish, E. G., - 4 s . k ~ .CHEY.,25, 1486 (1953). Schmall, AI., Pifer, C. W., Wollish, E. G., Duschinsky, R.. and Gainer, H., Ibid., 26, 1521 (1954). Schults, -4.L., Hammarsten, J. F., Heller, B. I., and Ebert, R. V., J . Clin. Invest., 32, 107 (1953). Shupe, L. l I . , and Dubowski, K. 11..A m . J . Clin. Pathol., 22, 901 (1952). (57) Simonsen, D. G., Ibid., 23,789 (1953). (58) Somogyi, SI.,J . Biol. Chem., 174, 189, 597 (1948); 179, 217, 1289 (1949). (59) Ibid., 195, 19 (1952). (60) Sophian, L. H., and Connolly, V. J., Am. J . Clin. Pathol., 22, 41 (1952). (61) Sunderman, F. W., Ibid., 23, 193 (1953). (62) Sunderman, F. TV., and Fuller, J. B., Ibid.. 21, 1077 (1951). (63) Swahn, B., Scand. J . Clin. &Lab. Invest., 5, Suppl. 9 (1953). (64) Taussky, H. H., J . Biol. Chem., 208, 853 (1954). (65) Weil-Malherbe, H., and Bone, A. D., Lancet, 1, 974 (1953). (66) Winer, A. D., and Kuhns, D. M., Am. J . Clin. Pathol., 23, 1259 (1953). (67) Wolfson, W.Q., and Cohn, C., “Rapid Chemical Micromethods for Analytic Fractionation of Serum Proteins,” Overbeck Co., -4nn Arbor, Mich., 1951. (68) Zak, B., Mosher, R. E., and Boyle, A. J., Am. J . Clin. Pathol., 23, 60 (1953).
