(370) Viniegra, O., Bravo, H., Prensa Med. ?fez. 24, 73 (1959). (371) Walkenhorst, W., Staub 19,12(1959). (372) Walkley, Janet, Am. Ind. Ilyg. Assoc. J . 20, 241 (1959). (373) Wanta, R. C., Heggestad, H. E., Science 130, 103 (1959). (374) Wartburg, A. F., Brewer, A. IT: , Lodge, J. P., Jr., Division of Water and Waste Chemistry, 138th Meeting, ACS, 1960, New York, K. Y., Sept. 11-16, 1960. (375) Kasser, R. R., Am. Ind. Hyg. Assoc. J . 19, 469 (1958).
(376) Welch, -4. F., Terry, J. P., Ibid., 21, 316 (1960). (377) Rest, 1’. R., Lyles, G. L., Anal. Chim. Acta 23, 227 (1960). (378) West, P. K., Sen, R., Gibscju, N. A., ANAL.CI-IEM. 30, 1390 (1958). (379) Rest, P. IT., Sen. B.,Sant, R. R., ?bid., 31, 399 (19.59). (380) West. P. T , Weisz, H., Goeke, G. C., Jr., Lyles, G., Ihid., 32, 943 (1960). (381j Westboer, I., Statib 20, .36i (1960). (382) Whiteiey, A . R., Reed, 2. E, J . Inst. Fuel 32, 316 (1059).
(383) Vi-ilkinson, L., A-ew Zealand J . Sci 2 , 182 (1959). (384) Williams, T., Roy. SOC.Promotion Heallh J . 78, 568 (1958). (385) Wronski, M., Analyst 85,526 (1960). (386) Yaffe, I. S., Cadle, R. D., J. Phys. ’ Chem. 6 2 ; 510 (1958). (387) Yocom. J. E.. J . Air Polluti[on Contrdl Assoc. 8 , 203 ’( 1958). (388) Zewe, E., Rumler, F., Herbolshrinier, R., Wolf, G., Staub 20,37 ( 1960). (389) Zimmer, C. E., Taber, E. C . , Stern, A. C.. J . Air Pollutzon Control Assoc. 9, 136 (1959). (390) Zurlo, N., Metrico, N., Med. lavoro 51, 241 (1960). ~
Clinical Chemistry G. R. Kingsley Veterans Administration Center, 10s Angeles 25, Calif.
T
131s review of publications of significant developments in clinica! chemistry is a continuation of the author’s h s t , which ended in November 1958 and appeared in this journal
(94. REVIEWS, NEW BOOKS, AND JOURNALS
Since the interests, basic sciences, techniques, and methodology of the ,.linical chemist cover many scientific fklcis, it would be profitable for him to cover the reviews on instrumentation ‘by Muller (14.4) , polarographic theory, instrumentation, and methodology by Hume (SA), light absorption spectrometry by illellon ( I a A ) , infrared and ultrnviolet spectrophotometry by Gore ( S A ) and Hirt (511), fliJorometric analyses by White (23A), chromatography, review of fundamental developments in analysis by Strain (9ZA),gss chromatography by Nogare (18A), gas analysis by Hobbs ( G A ) , nucleonics by llIeinke (11.4) , extraction by Morrison (ISA), and statistical methods in chemistry by Np!son (ITA). Holmberg and Blom::trmd ( T A ) remewed diabetes and insulin, diseases involving metabolism of m 3 n o cicida, plasma proteins, lipides, seratonin, epinephrine, norepinep!irine, :mi urticaria pigment osa. Satelson (16iz) outlined the prosent status of m5rochtmistrp in the analuyticai laboraTory of clinical chemistry and other problenis of vital interest to the clinical chpmist. Loy and Wright (1011) reviewed the many details not ordinarily encountered in analytical chemistry in the niicrobiological assay of amino acidu, vita,mins, and antibiotics. Saito (dOA) reviewed the progress of clinical arid biochemical analysis.
A book, “Principles ani: Alet,hods of Clinical Chenuatr) fvr lifetijcal Technologists,” was +?igned ly Rice (19.4) primarily to assist the technicinn to acquire skill and cultivate a sense of awareness, which makes posible the detection of error, and to supply a basis for intelligent evaluation of laboratnry data. Wilkinson jQA; prepared a laboratory manual on “Cheirkal hIicromethods in Clinical Medicine.” Annino ( 2 A ) expanded his book, “Clinical Chenlistry P r i x i p l i s and Procedures” t o include additimal tests suci. as transaminase, iron, and protein-bound iodine. Volume I or” three volumes has been completed by R1 alytical Methods of Protein Chenl4i;try (including Polypeptides) .” The first volume of “Toxicology, Mechanisms and Analytical Methods” (two volumes) edited by Stewart and Stolman (dlAj provides accounts of the general methods of chemical analysis availabie to the toxicologist, a discussion of the principles on which they are based, snd a survey of the problems t,o which they may be applied. Volume IT {to appear early in 1961) wiil consider t h e various important groups of poisons and bring together tjhe methods awiiable for identifying the members of each group. Guidance will ais0 he give11 in the selection of tests h r neK tirugs of known cheniical composiiio~i. An important net7 journal, iinalytical Bioch,emistry, edited by ?;ason (16A) (first volume, May 1960), is intended to serve a s a central international source of information on new and improved methods in various fields of biochemistry, biophysics, and related areas of experimental biology. The new journal will publish detailed ac-
counts of fundamental contributions on analltical and preparative procedures. It will include papers on qualitative and quantitative techniques based on chemical, physical, and biological principles, methods of preparation, purification, c!iaracterization, isolation, and separati:.m of bidogical substances and related ::iLteria!s, and instrumentation. Volume Z of Journal of Lipid lilesearch. edited by Zilversmit (25A1, appearcd in October :959. An interimtiorial jciirn:tl, l’osicology and Applied Plmmacology edited by Hays (4A), began publication in 3959. APPARATUS, EQUIPMENT, INSTRUMENTATION, AND AUTOMATION
The Committee on Microchemical Apparatus, Division of Analytical Chemistry, American Chemical Soci.>ty (47B) recommended specifications for microchemiciil app:mti:s, volumetric glasswnre, and micropipets. Specific:itions %ere given for Folin-type micropipets, micro iiasliou t pipets, and density-type micro weigiiing pipets. Sanz (&B) described apparatus and equipment for quick and accurate quantitative determinations with as k t l e as 0.01 ml. of liquid together with 1,he ultramicropipets and centrifuge required. Wilkinson ( 5 0 ) described an apparatus for simple and dependable ultrafilt,mtion of 0.2 to 1.0 mi. of bociy fluids under strict,ly anaerobic conditions or p H control. A simple thermostatic device for temperature control of 1 0 . 5 ” c. in the range from 20” to 50” C. for zclls in the Beckman DU spectrophotometer was designeti by Martin (2619). Hansen and Euell i15B) developed a spectrophotometer for use between 194 and 225 mu with a constant VOL. 33, NO. 5, APRIL 1961
* 13 R
half-intensity band width of 1.5 mp. Thiers, Margoshes, and Vallee (50B) described a small, simple, portable photometer for use in the near-ultraviolet region, which is well suited for routine work. A new portable apparatus for the measurement of oxygen uptake reported by Maurath and Ruger (28B), uses the paramagnetism of oxygen to measure its concentration from 14 to 100%. The Natelson microgasometer was mechanized by O’Mai-a and Faulkner (STB). Derviz and Lazarevskil (5B) modified the Van Slyke manometric apparatus by connecting two pipets to one leveling bulb and motor, which allow one worker to conduct simultaneous analyses of venous bloods. Peirce (S9B) described a platinum polarograph for the accurate measurement of 02and C 0 2transmission through different membranes. A review by Peeters (38B), “Paper Electrophoresis: Princip!cs and Techniques,” gave an extensive critical review of one- and two-dimensional methods and also new developments in this field. An instrument for slicing gels and a photodensitometric scanner for measurement of proteins separated by electrophoresis in starch gel was described by Rubinstein, Owen, and Larscn (43%). Twelve protein components in human serum were quantitatively estimated. Strickland et al. ( 4 8 B ) constructed an agar electrophoresis apparatus for the gravimetric microdetermination of 11 serum protein components. Grunbaum and K r k (12B) designed a refined, precise microelectrophoresis apparatus by which eight 0.01- to 0.1-pl. samples were run simultaneously. An instrument was described by Natelson et al. (SIB) for the continuous separation of substances with similar but not identical electrophoretic mobilities, in which the supporting medium was moved continuously, mechanically, in a direction opposite to the migration of the ions. Application of the instrument to amino acids was described. Recht (4OB) reported the use of an electrophoresis apparatus and automatic scanner for cellulose acetate strips in routine clinical work. Audran and Reutenauer (1B)described a n apparatus consisting of a chamber in the shape of a cone enclosing a chromatographic circle for the concentration and separation of Ion concentration constituents. Ressler axd Moy ( 4 l B ) utilized a simplified fluid film method of electrophoresis which incorporated electrode compartments and running surface into a single unit and eliminated connecting filter papcr wicks. An apparatus was designed by Hannig ( I @ ) for determination of amino acids separated by ion exchange by continuously registering the extinctions of stained compounds flowing through a measuring cell b) 14 R
a
ANALYTICAL CHEMISTRY
means of a n automatic recording photometer. Foss and Andersen ( S B j described a n apparatus for automatic coloring and decoloring of electrophoresis papers. Kelley, Fisher, and Jones ( I 7B) designed a high-sensitivity, recording, wave length-scanning flame spectrophotometer which gave exceptional performance in the red spectral region. Fuwa, Thiers, and Vallee (9B) designed a burner for a cyanogen-oxygen flame spectroscopy of microliter quantities or liquid under controlled conditions. Bott (2B) described a modification of a dual-channel ultramicroflame photometer for the determinatidn of Na and K simultaneously in amounts as low as 2 ppmoles. Xatelson et al. (%B, 36B) reported the use of the x-ray in spectrochemicltl analysis in clinical chemistry, with detailed techniques for the determination of serum calcium and potassium (SZB, M B ) , chlorine and sulfur (SSB), and sulfur distribution ( 3 4 B ) in protein fractions of serum. AUTOMATION
A versatile, multijunction automatic recording photometric titrator, suitable for colorimetric, nephelometric, and chemiluminescent indicator titrimetry was assembled by blullen and Anton (SOB) from a Precision-Dow recording potentiometric titrator, a modified Beckman I3 spectropliotometer, and necessary intermediate electronic components. Thoburn, Kankowski, and Reynolds (5IB) developed a commercial automatic colorimetric titrator monitored by a light beam passing through the titration beaker, in which the change in transmittance a t the end point causes a meter relay to close and stop the delivery of reagent. A simple, rugged, compact, and highly accurate recording titrator was designed and constructed by Williams, Ruffin, and Mounter, (56B). Coulson and Cavanagh ( 4 B j developed an automatic coulometric chloride analyzer which performs rapid and accurate chloride determinatlons on small samples. Malmstadt and Winefordner (21B) applied a new, extremely sensitive and accurate precision null-point potentiometric method to the determination of chloride in 0.02 ml. of deproteinized serum or plasma. T’ecerek et al. (52B) described an apparatus for automatic serial estimations of enzymes. All pipetting and photometric measurement were fully automatic. Miller and Cummings (29B) devised an automatic recorder for serial colorimetric analysis which point-recorded a t , fixed wave length, spaced uniform points along the Y-axis of the recorder, and gave push-button control for intermittent operation. Marsh (22B) revieaed the advances
in automation of laboratory equipment used in clinical chemistry, especially measuring devices, pipets, and analytical processing machines. Gordon and Campbell (10B) reviewed the progress made in automatic recording balances. Malnistadt and Hicks (2OB) designed a n instrument to measure serum glucose by measuring accurately and automatically the rate of formation of the colored reaction product within one minute after the reaction begins, by use of the glucose oxidase method. I n the last two years most of the routine clinical chemistry methods were adapted to the AutoAnalyzer. Johnson (I6B), 11cGuckin and Power (26B, b7B), Grady and Lamar ( I I B ) , and Weller et al. (53B) contributed improvements and adaptations of the blood glucose method for use with the AutoAnalyzer. Richter and Lapointe (4ZB) adapted the urea X Chasson, Grady, and Stanley ( S B ) the creatinine, Skeggs ( 4 6 B ) and Marsters (24B) the COz, Kessler (18B) the cholesterol, Ferrari and hlacduff (7B) and Taylor and Marsh ( 4 9 B ) the amino acid, Winter (56B)the cholinesterase, Marsh, Fingerhut, and Kirsch (ZSB) the alkaline phosphatase, Schwartz, Kessler, and Bodansky (45B) the phosphohexose isomerase, Lundgren (I9B) the phosphate, Hanke and Loughead (ISB) the potassium, and Failing, Buckley, and Zak (6B) the serum protein methods for use with the AutoAnalyzer. CONTROL AND PRECISION OF CLINICAL CHEMISTRY METHODS
A Symposium on Quality Control in Clinical Chemistry moderated by Knowler (9C) was held by the rlmerican Association of Clinical Chemists in Iowa City, Iowa, a t its 10th anniversary meeting in 1958. A short review of control and accuracy in the laboratory was made by Leidler (IOC). Directions were given by Gooszen (4C) for the construction of control charts based on statistical analysis for clinical laboratories. The use of control charts in clinical chemistry in raising over-all quality of laboratory results was reported by Henry (6‘2). Tonks ( I Y C ) evaluated the accuracy of clinical chemistry determinations in 160 Canadian laboratories by the use of lyophilized samples of two serum pools. Schols (15C) described methods for construction of two general types of nomograms, using a mathematical approach for the other. For nonroutine methods of photometric calibration, Dubowski (2C) proposed the use of properly prepared and adequately monitored calibration curves and tables which in certain situations have distinct advantages over use of standards included in each series of photometric analyses-for example, \Then analytical
systems do not conform to the BeerLambert law or where appropriate standards are impractical to prepare at the time of each analysis, as in routine hemoglobinometry or in enzyme activity determinations. Annino and Relman ( I C ) studied the effect of eating on some of the clinically important chemical constituents of the blood and concluded that in apparently normal persons an average breakfast probnbly will not affect any of these constituents 45 minutes or 2 hours after the meal, except glucose and perhaps phosphorus. Reinhold, Lavefound that son, and Bayer (I.&') 100 ml. of glycerol added to 200 ml. of pooled blood on serum effectively stabilized many constituents for 55 days when kept a t 24" to 30" C. Bilirubin, phosphate, and urea were not stabilized. Since serum standards available commercially were often inadequate because of deterioration, faulty standardization, and errors in rcconstitution, Levy (2%') prepared ion-free serum, using ion exchange resins, and reconstituted with varying amounts of appropriate ionic standard solutions for use as a clinical chemistry standard. Powell, Fritz, and James (1SC) reported that cadmium acid N-hydroxyethyl (ethylenedinitrilo) N,N' - triacetate (HCdV) is a n excellent reagent for the standardization of both bases and chelating agents such as EDTA. A historical review of the evolution of certified reference materials as furnished by the National Bureau of Standards was made by Hoffman (7C). The preparation of quality control standards from urine was described by Levey and Koenig (11C) for the determination of Na, K, and Ca in urine. Free (SC) applied statistical thinking to daily routine clinical chemistry determinations. Henry (5C) pointed out that biological data frequently fall in geometric distributions, which is not well recognized by many investigators in their statistical calculations of normal range. Control microanalysis methods for capillary and venous blood were presented by Kaplan, Yuceoglu, and Seligson ( I N ) outlined Strauss (E). a system of microchemistry for the hospital laboratory designed for maintenance of a high standard of performance. Vink and Kroes (18C) made a microanalytical comparative statistical study of the reproducibility of the total protein and calcium content of venous and capillary blood of 46 individuals.
-
-
AMINO ACIDS
Bigwood et al. ( 1 0 ) reviewed quantitative amino aciduria, methods for total amino nitrogen, free and combined, and fractionation by chromatography. Opienska-Blauth (19D) also made a critical evaluation of methods used in the study of amino aciduria with par-
ticular respect to reference systems, errors involved in the determination of amino N. the necessity of desalting urine and desalting methods, identification of spots, and interpretation of chromatograms. Evered (80) separated many naturally occurring acidic and basic amino acids in fairly complex mixtures in biological fluids by lowvoltage ionophoresis on filter paper. Puranen, Puranen, and Hallman (200) developed a new high-voltage electrophoresis for the determination of plasma glutamine. A technique for two-way separation of amino acids and other ninhydrin-reacting substances by highvoltage electrophoresis on filter paper was described by Efron ( 7 0 ) . Lundquist (17'0) achieved a preliminary separation of amino acids of serum and urine into four groups by displacement chromatography on a small column of Dowex 50 ion exchange resin pretreated with isopropyl pyrazole, pyrazole, nicotinamide, and ammonia, followed by a two. dimensional paper chromatogram of each of the four fractions. Bowden ( 4 0 ) obtained a map of 63 ninhydrinreacting materials by two-dimensional chromatography using a new solvent combination of phenol-water-ammonia and ethyl methyl ketone-butanol-dicyclohexylamine-water. Zlatkis, Oro, and Kimball ( 2 6 0 ) described a method for the analysis of mixtures of amino acids that yield volatile aldehydes by direct gas chromatography. Himes and Metcalfe (11D) achieved rapid quantitative determination of amino acids of protein hydrolysates by using a solvent system of methyl ethyl ketone, propionic acid, and water (15:5:6) to develop the paper chromatogram a t 60' C. Smith ( 2 4 0 ) used silicic acid columns containing water as a stationary phase to develop two partition-type chromatographic procedures for the analysis of mixtures of adipic, glutaric, and succinic acids. Blaedel and Todd (20) made indirect polarographic determination of amino acids by utilizing their stoichiometric reaction at controlled p H with a n excess of insoluble copper phosphate. Ladik and Szekacs ( 1 4 0 ) reported a micromethod for the polarographic determination of serine based on the measurement of the decrease in the IO4 wave due to the reaction of serine. Borchers ( S D ) increased the sensitivity of the alkaline copper salt method for the spectrophotometric determination of amino acids in 0.2 ml. of blood by the use of cuprizone (biscyclohexanoneoxalyldihydrazone) for the determination of solubilized copper. Kekki ( 1 2 0 ) also used this technique for microdetermination of amino acid in plasma and urine. The method of Sakaguchi was revised by Satake and Luck ( 2 2 0 ) by use of N-bromosuccinimide or 1,3-dibromo-5,5-dimethylhy-
dantoin, in place of hypobromite for color production stable over 30 minutes. Lelievre ( 1 6 0 ) estimated cysteamine and cystamine in biological media by treatment with CH21C02H in alkaline medium for polarographic measurement. Moffat and Lytle (180) identified 20 amino acids of protein hydrolyzates on paper chromatograms by spraying with ninhydrin-cupric nitrate to characterize and identify by their characteristic color compleses after 6 hours of resolution in amounts as low as 1.2 p g . Lampson and Singher (160) determined amino acids in the presence of peptides by using a 4.0 p H ninhydrin reagent and developing the color at 60' instead of 100" C The amino acids gave the usual amount of color, while the peptides remained almost colorless. Saifer, Gerstenfeld, and Harris (220) modified the method of Yemm and Cocking for the photometric microdetermination of amino acids in biological fluids with the ninhydrin reaction. Cook and Luscombe (60) fractionated and estimated the free amino acids in serum by locating six groups with a fluorescence technique, employing o-coumaric acid in (CHs)zCO and separating into individual amino acids by descending paper chromatography with BuOH-EtOH-EtOAC-HZOAcOH(60:15:10:20:2 or 40:15:10:20: 20). Shore, Burkhalter, and Cohn (2'30) described a sensitive and specific method for the fluorometric assay of histamine in tissues, rvhich involved the evtraction of histamine into BuOH from alkalinized HClO, extracts of tissues, return to an aqueous phase, and condensation with o-phthalaldehyde to form a highly fluorescent product. A simple cnzymatic spectrophotometric method for the determination of phenylalanine in serum was described by La Du and Michael (130), based upon the measurement of the absorption of the enolborate complex of phenylpyruvic acid generated enzymatically from phenylalanine by L-amino acid ovidase of snake venom. Hess and Udenfriend ( 1 0 0 ) developed a fluorometric procedure for the measurement of tryptamine in tissues, in which tryptophan does not interfere. The method involves extraction with C s H , cyclization of the tryptamine with HCHO to produce tetrahydronorharman, and dehydrogenation of the latter by HzOZ in aqueous solution to form the highly fluorescent product norharman. A simple, new colorimetric method for the determination of tryptophan based on the chromagen production of tryptophan when treated with peracetic acid in a n anhydrous medium was proposed by Fischl ( 9 0 ) . Tompsett ( 2 5 0 ) described methods for the determination in urine of some metabolites of tryptophan, hynurenine, anthranilic acid, and 3-hydroxyanVOL. 33, NO. 5 , APRIL 1961
15 R
thranilic acid. Coppini, Benassi, and Montorsi (6D:also described a simple and rapid method for the quantitative determination of eight tryptophan (via kynurenine) metabolites. BLOOD
PRESERVATION A N D ANALYSIS
GASOMETRIC
Tullis et al. (19E) reported that when human red blood cells were processed in a closed-system Cohn centrifuge, stored in 50% glycerol solution a t -80" C. for 19 months, and then thawed, the glycerol was removed, and the cells were stored for 11 days s t 40" C. they gave no reaction9 wtcn used in transfusions. Rozhdest\ ,kaya and Mikhnoviih (16E) fourd t I l n t alcohol, sucrose, and lactose were the best agents for the prolonged preservation of blood a t temperatures between 20" and 22' C. h'ociti, Caronni, and Brambilla ( I S E ) studied the effect of storage a t 4" C. for 3 months on plasma proteins by electrophoretic and refractometric examination. The effect of angerin, the Na salt of sulfated dextran, on blood preservation was studied by Mikhnovich (IZE). Antonova and Rotf d d ( I E ) investigated the biochemical and physiological change in blood stored without preservatives. Drey (5E) described a method for preserving and transporting very small samples of biological material prior to determination of sugar. A method for the simultaneous measurement of plasma volume, red blood cell mass, and extracellular fluid volume in which radioactive iodinated serum chromate51-tagged RBC, albumin (P), and sulfur 35-tagged so4 are used, was described by Shires, Williams, and Brown ( I 7 E ) . Franks and Zizza (YE) made simultaneous measurements of plasma volume in man with T-1824 and an improved albumin method and found that the 1131 albumin used contained 2.1 to 4.8% of the 1's' in forms not bound to protein. C a m p bell, Frohman, and Reeve (SE)described a simple, rapid, and accurate method of extracting T-1824 from plasma, adapted to the routine measurement of blood volume. hfacdonald, Pomeroy, and Gardner (IOE) proposed a simple rapid method for the determination of water in serum. Robinson and Conklin (I6E) determined hydrogen dissolved in water colorimetrically by adding a n excess of alkaline permanganate to the water solution and allowing the reduction of permanganate by molecular hydrogen to proceed a t ambient temperature. A comparative study of currently available techniques of blood collection and chemical determination of the COZ content of blood was made by Still and Rodman (18E). McKay, Seligson, and Taylor ( I I E ) devised a method for measurement of COz in serum by 16R
ANALYTICAL CHEMISTRY
gas chromatography, in which COz in helium is chromatographed 011 silica gel and separated from 02,K2,and other gases. The COZ is detected by measuring the change in thermal conductivity as i t flows with the carrier gas over detecting thermistors. Ishitani (9E) used a glass electrode p H meter covered with a thin film of Teflon or polyethylene which is permeable to COz, and a solution of NaHCOa between the film and electrode for a direct electrical method for the measurement of COz tension in blood. Gambino (BE) described the use of the Severinghaus pC02 electrode for the direct measurement of arterial blood pCOz. Bartels and Reinhardt (&E) designed a simple method for measurement of Oz tension in blood with a plastic-covered platinum electrode. Feinberg, Alma, and Petzold (6E) described a quantitative method for the spectrophotometric determination of blood oxygen. Deibler et aZ. (QE)demonstrated that alkyl aryl polyether alcohol (Triton X-100) was a satisfactory hemolytic agent for the spectrophotometric measurement of blood oxygen saturation. Rappaport, Eichhorn, and Kutman (IQE) reported the adaptation of his manometric COz apparatus for the determination of oxygen in 0.1-ml. blood samples. CARBOHYDRATES
Further improvements and modifications of the glucose oxidase method for the determination of glucose, which is based on %he release of H 2 0 2 from glucose and the detection of the peroxide by a suitable indicator in the presence of peroxidase, were made by Beach and Turner ( d F ) , Solomon and Johnson (ZSF), Marks (16F), and, Cawley, Spear, and Kendall (6F). Kingsley and Getchell (16F) modified the glucose oxidaseglucose method for direct application to 0.02 ml. of plasma, omitting the preparation of proteinfree filtrates. O'Gorman, Griffith, and Bloxam (19F) reported ascorbic acid inhibition of glucose oxidase Then determining blood glucose after therapeutic doses of ascorbic acid. Washko (86F) stabilized and buffered the enzyme reagent by diluting with buffered glycerol in place of water. Momose et al. (I@) developed a new colorimetric method for the determination of sugar in blood and urine by using 3,6-dinitrophthalic acid as a color-developing agent. Torres (ZQF) measured blood glucose with a diphenylamine colorimetric reagent. Wilson (87F) adapted the aniline hydrogen phthalate method for the detection of sugars on paper chromatograms. Urinary sugars were separated and identified by means of paper electrophoresis a t high potential gradient by Iuchi and Shibata (I@').
Philippu (208') separaten ho made final measurement by ultraviolet absorption. Gerarde and Skiba (131V) determined kerosine in blood by reacting a CCld extract hith a formaldchydesulfuric acid reagent to produce a characteristic color. Curry (SW) reviewed the analysis of basic nitrogenous compounds of toucological importance. Several trchniqurs for the isolation and identification of alkaloids in biological specimens were reported by Alha and 'I'nnimincn (IT17
by infrared spectroscopy, Tewari and Tripathi (51W) by ring paper chromatography, Morgan (S8W) by iodoplatinate spray, Huang et al. (24W) by use of silicotungstic acid, and Hilf, Castano, and Lightbourn (ZIW, 22W) and Goldbaum and Williams (I5N‘) by ultraviolet spectrophotometry. Dybing (8W) described a method for paper chromatography of barbiturates using formamide and ethylene glycol as stationary phases. Goldbaum, Williams, and Koppanyi (16W) determined glutethimide in biological fluids by chloroform extraction, removal of interfering substances, and determination of its characteristic ultraviolet spectrum and rate of hydrolysis a t 236 mp. Lerner (28W) used a mixture of nitric acid, phosphoric acid, and alcohol as a new color reagent for testing for extremely small qusntities of heroin. Miithers (37W) developed a polarographic method for determining small amounts of morphine in blood and plasma. Seits and Filippova (44W) determined procaine in blood by diazotization and coupling with the disodium salt of 2-naphthy1-3,6-disulfonic acid to produce an azo dye. Sobolewski and h’adeau ( 4 S W ) described a %hour procedure to identify common sedatives, hypnotics, and ataractics in 20 ml. of urine. Reye (@W) developed a new method for serum bromide determination which depends upon oxidation of Br- to free 13r. Spectrcphotometric methods for the determination of fluoride in water and biological fluids were reported by Brownley and Howle (SW) by use of thorium-phenylfluorone lake, by Bellack and Schouboe (2W) and Korenman and Kornakova (27W) by use of zir!.onium alizarin reagent, and by Linde (3OW) by measuring the inhibitory effect of fluoride ions on the enzymatic hydrolysis of ethyl butyrate. Senkoski, Wnllish, and Shafer (&W) described a techniqiw for the determination of organically bound fluorine. Ultraviolet spectrophotometric determination of salicylate in blood was reported by Stm tison (@W) in a single extraction method with butyl ether, by Williams, Linn, and Zak (56W) by use of the differrntial characteristics of the autom:)?ic recording spectrophotometer, and by (’hirigos and Udenfriend (4W) by a fluwrmetric procedure using 310 mp l L 7 h !&vation and 400 mp for emission. syants i4SW) determined carboxyClewbglobinin blood by a method based i n the diflerences in the absorption of I?< nearest portion of the infrared specir-in by solutions of oxy- and carboxyhemoglobin. Concentrations of the latter n ere established photometrically m monochromatic light at SO0 mp. A rapid method for the determination of small amounts of CO in gas mixtures was devised by Iqsyj, Zarembo,
1,Bcyclohexanedionedioxime in the spectrophotometric determination of traces of nickel, r h i c h results in increased sensitivity. Cousins (5m’) and Watkinused 3,3’-diaminobenzidine son (54W) for the fluorometric determination of traces of selenium. Dyfverman (9TY) determined microgram amounts of thallium in biological material by wet combustion, extraction of TlC13 with C2HSOC2H5,and measurement of the absorbance of the T1 dithizonate in CHC13 at 510 mp. Holcomb and Yoe (2SW) estimated uranium spectrophotometrically with 3-(2-arsonophenylazo)3, 5-&hydroxy-2, 7-naphthalenedisulfonic acid (trisodium salt). Motojima, Yoshida, and Izawa (S9TV) employed a simple method using 8-quinolinol for the determination of uranium. Whitson and Kwasnoski (55W) determined the a-activity associated with submicrogram quantities of enriched uranium in urine. Hulcher (15677)suggested a spectrophotometric method for the determination of 0.2 to 50 pg. of vanadium in biological material, in which interference by iron was eliminated by a prc-extraction of the iron lJ8-dihydroxy-3,6-naphthalenedisulfonic complex of benzohydroxamic acid from aqueous solution into octyl alcohol a t acid, was the best indicator for the p H 8.5. microestimation of borate ion in biological solutions. Gilbert (14W) surveyed the excitation of cadmium in vaVITAMINS rious flames and found that the air-hydrogen flame is particularly advantageShimizu ( 2 2 X ) reviewed the present ous for the determination of cadmium status of physical and chemical deterat 326.1 mp with a sensitivity of 0.1 minations of vitamin A. noels and p.p.m. Halliwell @OW) reported an Trout @OX) determined carotene and alternative procedure for the colorivitamin A in human blood by precipitametic microdetermination of sexivalent tion of serum proteins with CzHjOH, chromium, using a stable colorless saponification, extraction with petrocadmium iodide-starch reagent which is leum ether, and measurement of the oxidized by chromium to the blue total carotene a t 620 mp. Mickelsen starch-iodide complex. Miller and Yoe and Yamamoto ( 1 8 X ) reviewed methods (S6W) presented a method for deterfor the determination of thiamine and mination of traces of chromium in recommended thiochrome procedures for human plasma and red cells based upon blood and urine. Malnick, D a Silva, the red-violet complex formed by the and De Angelis ( 1 6 X ) carried out paper reaction of dichromate with diphenylchromatography of thiamine in plasma carbazide. Haerdi and Monnier (ISW) and urine applied directly to the paper determined traces of lead in blood by a and developed a blue fluorescence of polarographic method. Tompsett and thiochrome by spraying with alkaline Smith (52W) modified the method of K3Fe(CN)6, which was measured with Abbott and Johnson for the determinaultraviolet light. Log ( 1 4 X ) gave detion of H g by substitution of K M n 0 4 tailed revisions of titrimetric and turfor HzOz to obtain lower blanks. bidimetric methods for the determinaHakkila and Waterbury ( 1 9 W ) seption of folic acid, nicotinic acid and arated microgram amounts of mercury amide, pantothenic acid, and ribofrom most metallic ions by extraction of flavine. the mercury-diethyl dithiocarbamate Baker et al. (SX) assayed thiamine in complex into carbon tetrachloride from biological fluids by a method based on a carbonate-buffered solution containing the protozoan Ochromonas malhamensis. (ethylenedinitri1o)tetraacetate and cyAfter a critical study of the Jansen anide to mask interfering ions. Pavlovic thiochrome method for the determinaand Asperger (41 W ) determined traces tion of thiamine, Haugen (fox) made of mercury in biological material by recommendations for simplification. A the catalytic action of mercury ions on microbiological method based on Lactothe reaction of potassium ferrocyanide bacillus casei was used by Baker et al. with nitrosobenzene. McDowell et al. ( 2 X ) for assaying folic acid activity in serum. Baker et al. ( I X ) presented ( S S W ) used xylene instead of chloromethods for the assay of pantothenic form to extract nickel(II)-4-isopropyl-
and Hanley ( S d W ) by conversion of CO to C02 by decomposed silver permanganate as a catalyst and weighing the COz in a n Ascarite tube. Dominguez et al. (7W) utilized gas-solid chromatography for the sensitive determination of CO in blood or tissue. Fleisch (11W) estimated methemoglobin and methemalbumin in blood by converting to cyanomet compounds with K C N and measuring the decrease in extinction. Leu (99W) made microdeterminations of antimony in blood and urine with crystal violet and Van Aman, Hollibaugh, and Kanzelmeyer (5SW) determined antimony in zinc compounds with rhodamine B. Smith (47W) estimated arsenic in biological specimens as small as 0.5 mg. of a single hair by activation analysis. Owens and Yoe (40W)measured extremely small quantities of beryllium with 2-phenoxyquinizarin-3,4’-disulfonic acid (dipotassium salt), which forms a stable violet complex. Silverman and Shideler (46177’) used Chrome Azurol S to determine beryllium and fluoride spectrophotometrically. Fabre and others (IOW) found t h a t Spands, B(p-sulfophenylazo)
VOL. 33, NO. 5, APRIL 1961
25 R
acid in blood, urine, and cerebrospinal fluid using Lactobacillus plantarum and Tetrahymena pyriformis. Benhamou and Amouch ( 4 X ) determined vitamin BBcomplex and its components in urine by paper electrophoresis. Five spots on the paper were obtained by spraying with diazotized p-aminoacetophenone and drying. Levine and Hansen ( 1 S X ) introduced a new color reaction for pyridoxal which reacts with thiophene in the presence of H2S04 to form a characteristic stable, brilliant, jadegreen color with absorption peak a t 615 mp. Reddy, Reynolds, and Price (19X) described a method for the fluorometric determination of 4pyridoxic acid in urine after removal of interfering fluorescent compounds by ion exchange chromatography on Dowex 1and Dowex 50. Choline was determined in serum by Wachsmuth and Van Koeckhoven (26X) by silicotungstic acid precipitation and reduction of silicotungstate to an intense blue color. Verges (24X) determined folic acid by a new method based on the color (485 mp) which develops when a HNO, solution of folic acid is boiled and treated with NHaOH. Majs and Javorkovskis (16X) determined free vitamin B I in ~ blood serum deterwith E. coli 113-2. Kato (lax) mined free and total vitamin Bl2 in human serum proteins by paper electrophoresis. A more rapid assay of vitamin Bl2 was made by Cooper ( 6 X ) by utilizing the z strain of Euglena gracilis. Hawker, Margraf, and Weischselbaum ( 1 1 X ) found that isonicotinic hydrazide gave a brilliant yellow fluorescence when sprayed with hesperetin, which could be used for the identification of groups of flavonoids. Szoke (ISX) modified the method of Roe and Keuther for the determination of ascorbic acid by dissolving the resulting osazone in 96% C2H50H instead of 85% H2SO4. Hanewald ( 9 X ) modified his vitamin D method by eliminating interfering tachysterols by the addition of maleic anhydride a t 75” C. and found the most satisfactory reagent for colorimetric determination to be a solution of 20% SbC13 in CHC13 containing 2% AcOC1. Bukin and Gakina ( 6 X ) described a quantitative spectrophotometric method for the determination of vitamin D in 5 ml. of serum. A method for the determination of dla-tocopherol esters was presented by Lushchevskaya and Savinov (16X). Epelbaum and Lushchevskaya (8X) determined vitamin E in serum by mixing with C2H50H,extracting with C6H5, and drying, purified the oil residue on a diatomite column which was eluted with C6Hs, and evaporated this extract, dissolved it in C2H50H, and determined vitamin E colorimetrically with FeClz dipyridyl solution. Duggan ( 7 X ) determined tocopherols spectrophotometri-
26 R
ANALYTICAL CHEMISTRY
cally in serum by a convenient and sensitive method for the differential determination of the free and esterified tocopherols by using a stoichiometric excess of LiAIH4 in Et20 a,nd applying ultraviolet spectrofluorimetry . Schilling and Dam ( H X ) found that 5-iminoI-thiono-l,2,4dithiazolidine (xanthane hydride) was a promising reagent for the quantitative determination of vitamin
K,. MISCELLANEOUS
Brandt (ZY) made a concise review of the informal discussion on gas chromatography which took place under sponsorship of the Division of Analytical Chemistry at the meeting of the ACS in Atlantic City, in the fall of 1959. Dressler, Mastio, and Allbritten ( S Y ) described a method for the qualitative and quantitative analysis of respiratory gases, 0 2 and C02, using the Beckman gas adsorption chromatograph. Gambino et a2. (4Y, 7 Y ) described improved techniques in the use of heparinized vacuum tubes for the collection, storing, and handling of samples of blood for the study of pH, C02 content, and oxygen saturation, compared the p H of arterial, venous, and capillary blood (6 Y ), and evaluated the routine determination of venous plasma p H in acidbase problems (6Y). Hardy ( 8 Y ) reported that blood p H can be measured electrometrically by means of aglass electrode and a calomel reference electrode, provided the blood is kept from contact with air and measurement made a t 38’ C. A technique for direct measurement of p H of the red blood cell in vivo was designed by Purcell et al. (1OY). Mendelsohn and Levin (QY) estimated total body water by nitrosating a plasma protein-free filtrate containing antipyrene and condensing the resulting nitroso compound with l-naphthylamine to form a color for colorimetric measurement and calculation of antipyrene concentration. Werbin, Chaicoff , and Imada (12Y) developed a method for the rapid, sensitive determination of H3-water in body fluids by liquid scintillation spectrometry. Reid, Balch, and Glascock (11Y ) reported that measurement of body water by tritium and antipyrene methods was in agreement with desiccation methods. Procedures for testing for several urinary constituents in urine dried on filter paper were described by Berry (1Y)-phenylpyruvic acid, sugars, protein, etc. LITERATURE CITED Reviews, New Books, and Journals
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Amino Acids
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3,43 (1658). (2E) Bartels, H., Reinhardt, W.,Arch. ges. Physiol. 271, 105 (1960). (3E) Campbell, T. J., Frohrnan, B., Reeve, E. B., J. Lab. Clin. Med. 52, 768 (1958). (4E) Deibler, G. E., Holmes, M. S.,Campbell, P. L., Gans, J., J. Appl. Physiol. 14,133 (1959). (5E) Drey, N. W., U. S.Patent 2,863,733 (1958). (6Ej- Feinberg, H., Alma, Mary, Petzold, J., J . Lab. Clin.filed. 55, 781 (1960). (7E) Franks, J. J., Zizza, F., J . Appl. Physiol. 13, 299 (1958). (8E) Gambino, S.R., Clin. Chem. 6, 389 (1960). (9E) Ishitani, K., Oyo Denki Kenkyusho Iho 11, 261 (1959). (10E) Macdonald, A., Pomeroy, J. S., Gardner, M., A m . J. Clin. Pathol. 31, 563 (1959). (11E) McKay, D. K., Seligson, D., Taylor, B. W., Clin. Chem. 5, 260 (1959). (12E) Mikhnovich, E. P , Aktuuln. Voprosy Perelivan. Krovi (Leningrad) 5, 89 (1957). (13E) Nociti, V., Caronni, E., Brambilla, G., Gazz. intern. med. e chir. 63, 1085 (1958). (14E) Rappaport, F., Eichhorn, F., Nutman, M., J . Clin.Pathol. 13, 176 (1960). (15E) Robinson, R. H., Conklin, D. B., ANAL.CHEM.31,1598 (1959). (16E) Roehdestvenskaya, M. A., Mikhnovich, E. P., Aktualn. Voprosy Perelivan. Krmi (Leningrad) 5,71 (1957). (17E) Shires, T., Williams, J., Brown, F., J. Lab. Clin. Med. 55,776 (1960). (HE) Still, G., Rodman, T., Clin. Chem. 6,388 (1960). (19E) Tullis, J. L., Ketchel, hI. M., Pyle, H. M., Pennel, R. B., Gibson, J. G., Tinch, R. J., Driscoll, S.G., J . A m . Med. Assoc. 168, 399 (1958). Carbohydrates
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~
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(14E') Jaques, 1,. B., Bell, H. J., Methods of Biochem. Anal. 7, 253 (1960). (15F) Kingsley, G. R., Getchell, G., C'lin. Chem. 6, 466 (1960). (16F) Marks, V., Clin. Chim. Acta 4, 395 (1959). (17F) Miller, G. L., Burton, A. L., ANAL. CHEM.31,1790 (1959). (18F) Momose, T., Inaba, A., Mukai, Y . , Watanabe, hi., Talanta 4, 33 (1960). (19F) O'Gorman, P., Griffith, P. D., Bloxam, H. R., Brit. Med. J . 1960, 603. (20F) Philippu, A. J., ANAL.CHEM.31, 1743 (1959). (21F) Schonenberger, M., Kellner, H., Sudhof, H., Haupt, H., 2. physiol. Chem. 309, 145 (1958). (22F) Schnwtz, V., J . Lab. Clin. Med. 56,483 (1960). (23F) Solomon, L. L., Johnson, J. E., ANAL.CHEM.31,453 (1959). (24F) Torres, J. F., Rev. assoc. bioquim. arg. 24, 102 (1959). (25F) Warren, L., Nature 186,237 (1960). (26F) Washko, M. E., Federation Proc. 19, 81 (1960). (27F) Wilson, C. M., ANAL.CHEM.31, 1199 (1959). Cations and Anions
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~__._
(20G) Goodwin, J. F., Zbid., 100, (1959). (21G) Haussler, A., Hajdu, P., Mitt. deut. pharm. Ges. 29,73 (1959). (22G) Itano, M., Williams, L. A., Zak, B., Am. J . Clin. Pathol. 32, 213 (1959). (23G) Kats, J., Golden, S. B., J. Lab. Clin. Med. 53, 658 (1959). (24G) Lai, L.-H., Yao Hsueh Hsueh Pa0 6, No. 1, 54 (1958).
28 R
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