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A method is described for rapid analysis of phenylalanine and tyrosine using eluates of whole blood that has been impregnated into filter paper. This method employs semi-automated ion-exchange column chromatography (amino acid analyzer) and will yield reasonably accurate and consistent quantitative values for phenylalanine and tyrosine concentrations within two hours. The technique has significant value in rapidly confirming the presence of increased phenylalanine with or without a concomitant increase in tyrosine concentration in autoclaved or unautoclaved blood filter paper specimens. Thus phenylketonuria can be differentiated from hyperphenylalaninemia due to tyrosinemia within hours after the initial identification of an elevated phenylalanine concentration by a screening method such as the Guthrie test. In addition, further evaluations of phenylalanine and tyrosine concentrations can be facilitated in infants on whom only blood filter paper specimens are obtainable.

Paper chromatography is very helpful in the identification and comparison of antibiotics, but very few results from the application of this technique have been published so far. The awakening of interest in the search for antibiotics which are active against fungi pathogenic to plants and animals makes the investigator aware of the scant information that is available to help characterize such agents. A knowledge of the Rf values of the known antifungal agents would help advance the search for such therapeutic materials. Often these values, in conjunction with the biological inhibitory activities, are the only criteria by which one can determine whether the unknown agent appears to be a new material. When the Rf data are obtained for shake cultures, one can eliminate many previously described antibiotic-producing organisms from further study, thus allowing more research to be applied to antibiotics which are new and different. The studies reported in this paper should serve as a guide to the identification of some of the known antifungal antibiotics.

A paper chromatographic system was developed for the resolution of mixtures of sulfated mucopolysaccharides. The effect of varying the ammonium formate buffer/isopropanol ratio in the developing solution on the RF values of heparin and chondroitin sulfate is presented. A complete separation of heparin from chondroitin sulfate was obtained with three solvent mixtures containing different proportions of buffer and isopropanol. One of these solvent mixtures, ammonium formate buffer-isopropanol (65:35) v/v, resolved commercial beef heparin into two principal components, on “fresh” paper. This system was used to compare beef heparin with mouse mast cell tumor heparin, rat heparin, sheep lung heparin, chondroitin sulfate, and β-heparin. A common metachromatic component with an RF of approximately 0.5 was0found in all of the heparin preparations chromatographed with this solvent mixture. “Aging” of the filter paper resulted in the loss of the ability of the buffer-isopropanol mixture (65:35) v/v to resolve the beef heparin. Increase of the buffer-isopropanol ratio to (70:30) v/v restored the resolution of the beef heparin on “aged” paper. Heparitin sulfate and α-heparin monosulfate were compared to beef heparin using the latter system.

Publisher Summary This chapter discusses the principles of paper chromatography of antibiotics. It includes all steps of analysis beginning with the preparation of samples and papers, development techniques, detection methods, and means of documentation. Paper chromatography of the best-known groups of antibiotics is also presented in the chapter. General theoretical aspects of paper chromatography are not dealt with except for those that directly emerged from studies of antibiotics. The chapter concludes with a discussion of paper chromatographic classification and systematic analysis of antibiotics.

1.1. Filter paper electrophoretic analysis of the serum and urine proteins in thirty-five cases of multiple myeloma has been carried out. In all thirty-five cases characteristic protein abnormalities were observed; seventeen cases showing homogeneous peaks in both the serum and urine, eleven cases in serum only, seven cases in urine only. 2.2. All urine samples showing a positive Bence-Jones protein reaction also displayed a characteristic electrophoretic abnormality. Eight of the twenty-four electrophoretically positive urine samples were Bence-Jones protein negative by the usual tests. Because of the relative simplicity of urine electrophoresis by the filter paper technic, this test is considered to be a practical clinical procedure. 3.3. Two cases are presented in detail because certain atypical clinicopathologic features of their disease patterns possibly assist in an understanding of the relationship of the plasma cell dyscrasias to chronic lymphatic leukemia and the lymphosarcomas. A third case, which demonstrates the value of serial electrophoretic studies in myeloma, is also presented. 4.4. Consideration is given to some of the physicochemical properties of the myeloma serum and urine proteins, and to the possible nature of their interrelationship.

The results obtained with freshly prepared solutions indicate that ferric ions may be adsorbed by Whatman No. 1 filter paper by a process of ion exchange. However, on standing the system becomes complicated by the diverse nature of the hydrolysis products formed. Increased adsorption takes place, apparently through the retention of hydroxy compounds of lower net charge.

A systematic analysis of keto acids and other carbonyl compounds in biological material was based on the following procedures: (1) their conversion to DNPH derivatives under mild conditions, (2) preliminary chromatographic separation into acidic and non-acidic DNPH derivatives, (3) isolation of each component by paper chromatography and (4) identification by means of rechromatography and catalytic reduction to the corresponding amino derivative. The method is quantitative for keto acids and its accuracy is between 10–20%.

Two phase solvent mixtures are described in which di-substituted steroid ketones and esters have partition coefficients such that they may be readily separated by partition chromatography on paper. 80 steroids and derivatives have been characterised in these solvent systems and their RF values are presented. Notable features include the resolution of otherwise similar and ostane and pregnane derivatives, 3-hydroxy-17-ketone from 17-hydroxy-3-ketones in the androstane series, aetiocholanolone from its -9: ii - unsaturated analogue and substantial inversion of general order of separation as compared with the order observed in procedures for unesterified steroids. Up to 0.5 mg of steroid or derivative per cm2 may be chromatographed from each origin.

Filter paper electrophoretic analyses of serum were carried out in a series of eighteen proved cases of multiple myeloma, twenty-three normal controls and 130 patients with non-myelomatous diseases, and compared with the results of classical Tiselius electrophoresis and chemical fractionation. Abnormal electrophoretic patterns were found in all of the cases of multiple myeloma studied, and tall narrow spikes representing abnormal proteins with varying mobilities in the globulin range were demonstrated in 78 per cent. The latter were considered diagnostic of myelomatosis since similar patterns have not been described in any other disease and since none were observed in any of the present controls. In six of the eighteen cases the diagnosis was first established on the basis of these electrophoretic patterns and subsequently confirmed by bone marrow biopsy. In eight others they were merely confirmatory in previously diagnosed cases. Of special interest were the diagnostic patterns found in four patients in whom myelomatosis was not suspected. There were significant quantitative differences between the results obtained by filter paper and classical electrophoresis, and the results of both differed from those of chemical fractionation. However, the diagnostic peaks of myelomatosis were equally well demonstrated by both electrophoretic methods. It is concluded (1) that the electrophoretic patterns of serum obtained by both the filter paper and Tiselius methods exhibit highly characteristic peaks in a high proportion of individuals with multiple myeloma, (2) that such patterns are of great diagnostic value and (3) that the filter paper method offers the advantages of simplicity, low cost and speed, without any loss in sensitivity or specificity, which make it especially valuable for screening large numbers of specimens in patients suspected of having myelomatosis.

CHANG (1955) reported the lowering of the blood hemoglobin of chicks due to free gossypol feeding and postulated that free gossypol in the diet leads to interference with the synthesis of the proteins of the blood in these chicks. In view of the useful information that has been obtained in recent years using the electrophoresis technique in the study of normal and pathological sera, an experiment was planned to study the electrophoretic patterns of serum samples obtained from birds fed a normal diet, as well as from birds fed free gossypol in the diet. An electrophoresis cell using filter paper strips, as originally designed by Durrum (1950) and marketed by Spinco, was employed. Later it was discovered that the resolution of the globulins of chick serum is very poor on filter paper. More serum samples were obtained from birds used in a study of the effect of protein level on …

A method for the estimation of unconjugated cortisol concentration in human urine is described. Using highly sensitive fluorimetric assay after paper chromatographic purification of cortisol, it is possible to quantitate the amount of endogenous cortisol excreted in 24 hours urine, after steroid suppression and corticotrophin stimulation (ACTH). According to this method, normal values (30 males and 30 females aged between 18–50 years) are 35.8–120 μg/24 hours; fluorimetric recoveries of cortisol from urine are 82.5–95.4% and radioactive recoveries are 74.8–88.6%. A trained person can perform 12 determinations in 5 working days.

A simple paper chromatographic method was found to be effective for the study of phenylketonuria and tyrosyluria. It proved to be much more reliable than conventional tests for the detection of abnormal amounts of ketoacids in urine and was suitable and convenient for following the effects of dietary variations on the excretion of these compounds. Information obtained about ketones other than those of primary importance to the conditions studied included confirmation of the excretion of p-hydroxyphenylpyruvic acid in phenylketonuria. A modification of the method applicable to histidinaemia was devised.

A method for the determination of urinary normetadrenaline and metadrenaline is described whereby these 3-O-methoxy metabolites of noradrenaline and adrenaline are separated by paper chromatography before estimation by Pisano's method. Results in 80 normal subjects are given.

The technique of paper chromatography has been adapted to the identification of gluconic, 2-ketogluconic, and α-ketoglutaric acids. Combinations of methyl and ethyl alcohol were found to be the most suitable solvents and ammoniacal silver nitrate was found to give the most satisfactory reaction. When grown under normal physiological conditions where glucose was metabolized to carbon dioxide and water, Pseudomonas aeruginosa 9027 was shown to have oxidized glucose by way of gluconic and 2-ketogluconic acids. Since a strong system for oxidizing both gluconic and 2-ketogluconic acids was demonstrated, the presence of these acids over at least an eight hour period of growth is taken as evidence of their importance as intermediates in the oxidation of glucose by this organism.

Various isomers are always present in commercial samples of R-, G- and Schaeffer acids.According to our experiments, high-voltage paper electrophoresis seems to provide the best separation and determination of these isomers on the micro scale.The procedure was as follows.Known quantities of samples (ca. 100g) were submitted to paper electrophoresis by using borate buffer (pH 10) as an electrolyte, in the condition of 75V/cm.As soon as 10 minutes of separation time elapsed, the separated zones were cut off wet, extracted seperately with borate buffer solution (pH 10) as rapidly as possible on the water bath, the quantities of dyes in extracted fractions were fluorophotometrically determined in comprison with standard naphthol-sulfonic acids.Commercial samples were examined according to this method. The results are shown in Table 5.

The formation and stability of Te(VI) compounds during chlorination of acidic, neutral and alkaline solutions of Te(IV) compounds were followed by a simple paperchromatographic method. It was found that about 2 μg of Te(VI) could be detected in the presence of considerable amounts of Te(IV) and that less than 1 μg of Te(IV) could be detected in the presence of large quantities of Te(VI). Chromatography was carried out at 20° on Schleicher and Schull 2043A filter paper by the ascending technique, using n-butanol saturated with 3N HCl as developing agent. The papers were sprayed with stannous chloride solution. The RF value of Te(VI) was found to be 0.08–0.15 and that of Te(IV) compounds, depending on the basicity of the spotted solution, 0.51–0.61, which decreases with increasing basicity.

In few conditions is the electrophorogram of serum proteins capable alone of deciphering the diagnosis. In conjunction with the assembled clinical data and relevant applied pathological tests the definition of the disease is often clarified. The patterns as observed by the applied pathologist are serially discussed and the importance of the variations in different fractions presented. In several instances, exemplified by liver disease, the assistance rendered toward prognosis has been considerable. Perhaps the most clearcut diagnosis is possible in the agammaglobulinaemia syndrome which is immediately evident by the technique of paper electrophoresis. Current advances are mainly in the direction of estimating the protein-bound carbohydrate and lipid constituents which are believed to reflect the abnormalities in several infections and metabolic diseases. Utilisation of non-ionic detergents has also allowed separation of subfractions of the globulins into numerous new components, including lipo-proteins, some of which had been previously observed during the application of improved buffer systems designed to restrict interference of heavy metals and to facilitate accentuation of fractions having intermediate mobility.

Separation of hypoglycemic agents as tolbutamide or similar compounds is carried out on paper chromatograms using different developers. Detection of the spot contents has been done by new colouring agents which proved to be good tools for resoltion of the spot. Among these reagnets, ninhydrin proved to be a useful tool for determination of hypoglycemic agents under investigation. Radioactivation of the dried developed chromatograms, using I131 has been applied as a tool for measuring the Rt-values of the same hypoglycemic agents. Invetigation on S35-labelled hypoglycemic agents prepared in our laboratories proved that paper chromatography accompanied by radioscanning or autoradiography of the dry chromatogram could be used for the separation, detection, and determination of these agents.

An indirect immunofluorescence test using small discs (0.4 cm in diameter) cut from blood smears on filter paper is described. Discs plus three drops of phosphate-buffered saline are laid on antigenic areas on slides, which are then incubated. During incubation, eluted antibodies react with antigen. The method was tested with 10 eluates from healthy persons and with 30 eluates from three groups of 10 patients with Chagas’ disease, schistosomiasis Mansoni and South American blastomycosis, respectively. Sera from the same blood samples were tested also by conventional indirect immunofluorescence tests. Both tests gave similar results. The method proposed is useful as a rapid qualitative screening test when dealing with a large number of samples.

By the method of paper electrophoresis a comparative quantitative study was made of the sundanophilic serum lipids of two groups of persons; one group consisted of twenty-one patients who had had previous myocardial infarction, the other of eighteen women with no clinically important arteriosclerosis. The electrolipograms in all cases revealed two zones, the larger probably corresponding to β-lipoproteins, the smaller to α-lipoproteins. In the atherosclerotic group the major lipid band constituted a larger fraction of the total lipid than in the control group. The major lipid band of one-third of the atherosclerotic persons extended farther with respect to the β-globulin.

A rapid filter paper disk assay for cyclic AMP based on the activation of a cyclic AMP dependent protein kinase from rabbit skeletal muscle is described. This procedure permits determination of as little as 0.2 picomoles of cyclic AMP. In addition this procedure employs ATP-γ-33P instead of ATP- γ-32P.

Summary A qualitative method of paper chromatography for phenolic compounds is discussed. Whatman No. 7 paper was impregnated with a saturated solution of boric acid. The solvent used was the organic phase of n -butanol saturated with 2% NH 4 OH. The time of development was 15 hours. Beside the classical methods of spot detection, two new processes were employed: (a) ultra-violet examination of the chromatogram after Maule's test; (b) spraying with a solution of diazotized sulphanilic acid, exposure to ammonia vapours followed by spraying with a solution of diazotized p -nitroaniline. A table of R F values of 34 pure phenolic compounds is given, together with methods for their identification.

An investigation of the electrophoretic data from patients with various malignant diseases shows that the possibilities of paper electrophoresis as a diagnostic tool are limited. The relative inaccuracy of the method may cover up important details; but more physiological information can scarcely be expected from data concerning only one physical property of a protein molecule. Quantitative evaluation of the data still gives rather qualitative results. With more material it may be possible to differentiate between various groups of diseases. The use of the “entropy” of a serum protein mixture does not give greater differentiation. Only in a limited number of cases can it be used to distinguish pathological and normal sera. The high correlation in the normal group between the albumin contribution to the total “entropy” and the total “entropy” provides a criterion for the detection of pathological serum protein mixtures in most cases.

The electrophoresis and immunodiffusion steps for an open, atmospheric-un-equilibrated system in dilute agar gel on vegetable parchment paper has been described. Studies were carried out on resolution and precipitin identification. Figures showing the attempt to identify individual components of serum were presented.

Mean R F and R M values have been obtained for a number of steroids and dyes in Bush aqueous methanol systems. Scrutiny of the Δ R M values verifies the stability of the condition achieved.

A RADIOIMMUNOASSAY FOR HUMAN PITUITARY LUTEINISING HORMONE USING PAPER CHROMATO-ELECTROPHORESIS

Summary The electrophoretic pattern of serum proteins has been studied in 30 infants suffering from toxicosis. During the stage of acute dehydration the main changes were a moderate or marked increase of alpha globulin, particularly of the α2 fraction and a decrease of albumin. In some cases there was also an increase of the gamma globulin. During recovery the alpha globulin decreases while albumin increases. However, the return to normal of the electrophoretic pattern is not always achieved when clinical recovery occurs. The absolute amounts of total serum proteins have also been estimated and their significance has been discussed. Electrophorese sur papier des proteines du serum au cours de la toxicose. L'aspect electrophoretique des proteines du serum a eteetudie chez 30 ehfants presentant une toxicose. Au cours de la periode de deshydratation aigue, on a pu noter une augmentation nette des alphaglobulines (en particulier la fraction alpha 2), et une diminution de l'albumine. Dans quelques cas, on a pu noter aussi une augmentation des gammaglobulines. La diminution des alphaglobulines, et l'augmentation de l'albumine est survenue avec la guerison. Neanmoins, le retour a la normale de la courbe d'electrophorese n'est pas toujours acheve au moment de la guerison elinique. Papierelektrophorese von Serum-Protein bei Sauglingstoxikose. Die Elektrophoresebefunde von Serumproteinen sind bei 30 an Toxikose leidenden Sauglingen untersucht worden. Wahrend des Stadiums der akuten Dehydration waren die Hauptveranderungen eine massige oder ausgepragte Zunahme des Alpha-Globulins, insbesondere der α2-Fraktion und eine Abnahme des Albumins. In einigen Fallen be-stand auch eine Zunahme des Gamma-Globulins. Wahrend der Genesung nimmt das Alpha-Globulin ab, wahrend das Albumin ansteigt. Jedoch ist der Ruckgang zum nor-malen elektrophoretischen Bild nicht immer vollendet wenn die klinische Genesung eintritt. Electroforesis en papel de las proteinas sericas en la toxicosis. Se estudiaron las imagenes electroforeticas de las proteinas sericas en 30 lactantes afectos de toxicosis. Durante el periodo de deshidratacion aguda, los cambios mas notables fueron un moderado o marcado aumento de la alfa globulina, especialmente de alfa 2. Durante la convalecencia, la fraecion alfa globulina disminuyo mientras au-mentaba la albumina. Sin embargo, la vuelta a la imagen electroforetica normal no siempre es completa al ocurrir, la mejoria clinica.

Metabolism of the antioxidant, 2, 6-di-tert-butyl-p-cresol (BHT), was studied in a rabbit. After administration of 0.5g./kg. of BHT by mouth, 37.5% of it was excreted as glucuronides, 16.7% as ethereal sulfates, and 6.8% as free phenols. α-Hydroxy-2, 6-di-tert-butyl-p-cresol, 3, 5-di-tert-butyl-4-hydroxybenzoic acid and 4, 4'-ethylenebis (2, 6-di-tert-butylphenol) were identified by paper chromatography from the rabbit urine 24 hours after the administration of 0.5g./kg. of BHT. Unchanged BHT was isolated from the feces, but not from the urine.

A PAPER Chromatographie study of urinary aminoacids has been carried out in 69 affected and 565 unaffected relatives from 64 families with various types of muscular dystrophy. Only a small proportion of subjects had either generalized or specific hyperaminoaciduria.

Maleic hydrazide added to solutions of partially purified plant extracts can be determined semiquantitatively by a paper chromatographic method in which the area of the maleic hydrazide spot, detected by spraying with a mixture of ferric chloride and potassium ferricyanide, is measured. By this method as little as 0.2 micrograms of maleic hydrazide can be detected with an error of about 35%. This method is not applicable to plants treated with maleic hydrazide because of the presence of interfering substances.

The phosphatides of the erythrocytes of several species of mammals have been analysed chromatographically. The cells of the true ruminants contained substantial amounts of cephalin and sphingomyelin but usually no lecithin. The cells of dog and guinea-pig contained lecithin but relatively small amounts of sphingomyelin. The biological implications of the lack of lecithin in ruminant cells have been discussed.

Various birch-pollen and dog-epithelium allergen extracts were chromatographed using Sephadex G-75 and Sephadex G-200 columns. The chromatographic tractions were then tested by an in vitro paper disc radioallergosorbent test, using sera from allergen-sensitive patients. Calibrated columns were used to approximate molecular weights of some birch-pollen and dog-epithelium allergenic active components.

Paper chromatography has proved to be suitable for the separation of both Degranol and Mannitol-Myleran. Experiments made in this connection showed that Degranol was more stable than the other agent.

Birch pollen and dog epithelium allergen extracts were studied by ultracentrifugation in linear sucrose gradients. The various sucrose-gradient fractions were then tested for allergenic activity by the paper disc radioallergosorbent test, using sera from allergen sensitive patients.

Summary In 21 premature infants, the birth weights of whom ranged between 1500 and 2400 g and the ages between 1 and 56 days, the total serum protein values have been determined and the serum protein components have been electrophoretieally differentiated. All infants were fed on human milk but, in 10 infants additional protein was administered (Casein preparation Secoosan). Total protein values were found to be lower in premature infants than in full-term infants. During the first eight weeks of life total protein values decreased, from 5.63 g per cent to 4.03 g per cent. The decrease in total protein is caused by a decrease in the amount of albumin (from a little below 3 g per cent in the first week of life to a little above 2 g per cent in the eighth week) and of gamma-globulin (from about 1 g per cent to about 0.5 g per cent) while the alpha1-, alpha2-and beta-globulin values remain rather constant. If casein is added to the diet the total serum protein values and the serum albumin values will range at higher levels, but the decrease with increasing age will not be prevented. On the contrary, additional casein to the diet will cause a further reduction in the gamma-globulin concentrations (Fig. 1). Taux des proteines dans le serum des prematures. Des determinations du taux des proteines totales ainsi que des taux particuliers des differentes proteines ont ete effectuees par electrophorese chez 21 bebes nes avant terme. Tous ces enfants etaient nourris au lait maternel et 10 d'entre eux recurent des supplements de proteines. Il est apparu que le taux des proteines totales etait plus faible chez les prematures que chez les enfants nes a terme. Au cours des huit premieres semaines qui suivirent la naissance, le taux des proteines totales descendit de 5,63 g%a 4,03 g%. Cet abaissenient du taux des proteines totales provenait de la diminution de la concentration en alburnine (qui d'abord legerement inferieure a 3 g % au cours de la premiere semaine etait descendue a un niveau legerement superieur a 2 g % au bout de huit semaines) et en gamma-globuline (qui, etant d'environ 1 g %etait descendue a environ 0,5 g %), tandis que les taux des globulines alpha1, alpha2 et beta etaient restes relativement constants. L'addition de caseine a la nourriture fit monter les taux des proteines totales et de l'albumine a des niveaux plus eleves, mais ces concentrations diminuerent egalement avec le temps. D'autre part, l'addition de caseine a la nourriture entraina une diminution plus importante du taux des gamma-globulines. Serumproteinwerte bei fruhgeborenen Kmdern. Bei 21 fruhgeborenen Kindern wurden die gesamten Serumproteinwerte bestimmt und die Serumproteinbestandteile elektrophoretisch differenziert. Alle Kinder wurden nut Frauenmilch ernahrt, aber 10 Kinder erhielten einen Eiweisszusatz. Die gesamten Proteinwerte erwiesen sich bei den fruhgeborenen Kindern als niedriger als in vollreif geborenen. Wahrend der ersten 8 Lebenswochen fielen die totalen Proteinwerte von 5,63 % auf 4,03 % ab. Der Fall im Gesamtproteingehalt ist durch einen Abfall im Albumingehalt bedingt (von ein wenig unter 3 g % in der ersten Lebenswoche auf ein wenig uber 2 g % in der achten Woche) und Gammaglobulingehalt (von ca. 1 g % auf ca. 0,5 g%), wahrend die Alpha1-, Alpha2-und Betaglobulinwerte ziemlich konstant bleiben. Wenn Kasein zur Diat zugefugt wird, werd. en die gesamten Serumproteinund Serumalbuminwerte ein hoheres Niveau erreichen, aber der mit dem Alter einhergehende Abfall wird nicht verhindert werden. Im Gegenteil, der Kaseinzusatz zur Diat bewirkt eine weitere Herabsetzung der Gammaglobulinkonzentrationen. Valores seroproteicos en los prematuros. Han sido determinados, en 21 prematures, los valores seroproteicos totales y diferenciados electroforeticamente los componentes seroproteicos. Todos los ninos fueron alimentados con leche humana, pero a 10 de ellos se les administraron proteinas adicionales. Hallose que los valores proteicos totales eran inferiores en los prematuros a los de los ninos nacidos a termino. Durante las ocho primeras semanas de la vida, los valores proteicos totales disminuyeron. de 5.63 g % a 4.03 g %. La disminucion del total proteico esta causada por una disminueion de la cantidad de albumina (desde un poco menos de 3 g % en la primera semana de la vida hasta un poco mas de 2 g % en la octava semana) y de gammaglobulina (desde aproximadamente 1 g % a aproximadarnente 0.5 g %) mientras los valores globulinicos alfa1, alfa2 y beta, permanecen bastante constantes. Si se anade caseina a la dieta, los valores seroproteicos totales y los valores seroalbuminosos alcanzaran niveles mas elevados, pero la disminueion, al aumentar la edad, no podra evitarse. La caseina, contraria, adioionada a la dieta, causara todavia mas reduccion en las concentraciones gammaglobulinicas.

The author describes a method, for the determination of bacterial L-histidine, L-tyrosine, L-tryptophan, L-arginine, L-lysine, L-ornithine, L-serine, L-phenyl-alanine, L-glutamic acid and L-aspartic acid decarboxylase by means of high voltage paper electrophoresis, using electrolytes of low ionic strength. For the group of test microorganisms (Enterobacteriaceae, Achromobacteriaceae andPseudomonas), L-histidine, L-arginine, L-lysine, L-ornithine and L-glutamic acid decarboxylases were found to be of the greatest practical significance. The optimal conditions for maximum formation of these decarboxylases by some of the test strains ofEscherichia coli, Hafnia andProteus are discussed.

A method for the determination of insulin potency using circular chromatography is described. The eluted spots are measured with respect to a standard which is run simultaneously with the unknown. The results of this determination do not differ significantly from those obtained by biological assay, at least for preparations with a potency higher than 19 I.U./mg on a moisture-free basis. For the determination of less pure samples the use of a standard insulin of corresponding purity is recommended. The precision of the method compares with that of a biological assay with 250 rabbits. Protamine and excess of zinc do not interfere with the determination.

Urine from normal subjects and from patients with a variety of metabolic disturbances was examined for organic acids by ion exchange purification followed by two-dimensional paper chromatography. Two patients showed slight variations from the normal pattern. One adult male with acromegaly showed an increased citric acid excretion, and one young girl with hepatitis showed an increased α-oxoglutaric acid excretion. Patients with a variety of renal tubular disorders, metabolic bone diseases, and other metabolic disturbances showed no variation from the normal urinary pattern of organic acid excretion. One patient with Lowe's syndrome of “organic aciduria” had a generalized aminoaciduria but no detectable abnormality of urinary organic acid excretion.