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The automated dithionite test for the detection of hemoglobin S has been modified for use with AutoAnalyzers having a filter paper read-out. This method was compared with results of hemoglobin electrophoresis in 425 individuals, of whom 58 were positive for hemoglobin S. The two methods were in complete agreement. The automated filter paper dithionite test is a rapid, economical, accurate method for detection of hemoglobin S and could be easily incorporated into currently available mass screening equipment.

The separations of steroid oestrogens achieved by paper chromatography have been reviewed with special reference to studied involving epimeric oestrogens and to oestrogens which have been isolated recently. Methods of detection of these compounds on paper chromatograms have also been discussed. The mobilities of thirty-four different oestrogens and their derivatives in many solvent systems have been tabulated. It is hoped that this review will be found useful in providing a collection of data from which separations of mixtures of oestrogens can be planned.

1. 1. A method for the detection of Jaffe-positive substances in human blood serum using the technique of paper partition chromatography is described. 2. 2. A partial solution of the problem of apparent creatinine has been furnished by identification of pyruvate as a second Jaffe-positive compound beside creatinine in a water-soluble fraction of acetone-filtrate of sera; this finding was confirmed polarographically as well as spectrophotometrically . Up till now the quantitative significance of this substance has not been considered in connection with this problem. 3. 3. After a few preliminary experiments, paper chromatography of the insoluble residue of acetone-filtrate of serum has been left open for further investigation. It seems likely that the final solution of the problem of apparent creatinine will cover both possibilities which were discussed in the introduction, irrespectively of whether the chromatographic investigation of the insoluble fraction might show the presence of some new unknown Jaffe-positive component.

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.

A technique has been developed by which complex mixtures of amines can be sepated by column chromatography on the ion exchange resins Amberlite CG-50 and Amberlite CG-120. Identification of amines is made on the basis of the speed of their elution from the resin column, as well as by subsequent paper chromatography of effluent fractions. The method can be applied to amines derived from various physiological fluids, and offers the possibility of isolating unidentified compounds for chemical characterization. Using the combined technique of ion exchange column chromatography followed by paper chromatography, 26 amines were identified in human urine. these were: methylamine, dimethylamine, ethylamine, ethanolamine, β-hydroxypropylamine, pyrrolidine, piperidine, putrescine, cadaverine, 2,2'-dithiobis(ethylamine), histamine, N-acetylhistamine, i -methylhistamine, p -hydroxybenzylamine, 3-methoxy-4-hydroxybenzylamine, p -tyramine, m -tyramine, octopamine, synephrine, 3-methoxytyramine, normetanephrine, metanephrine, tryptamine, serotonin, bufotenin, and kynuramine. An additional 20 unidentified bases, presumably amines, were found to be regularly excreted in urine. Evidence is presented supporting the identification of kynuramine, β-hydroxy-propylamine, putrescine, cadaverine, and 2,2'-dithiobis-(ethylamine). These 5 amines have not previously been considered to be constituents of normal human urine. It is suggested that study of the urinary excretion of amines may provide useful clues as to the mechanisms involved in various metabolic disorders, including those characterized by mental dysfunction.

A survey of various buffers as agents for the paper ionophoresis of hexose phosphate esters and inorganic phosphate has revealed 0.2 M borate buffer at pH 9.5 as the most suitable for separation of the phosphate compounds into four distinct groups. Variations in molarity and pH of the borate can be utilized to separate the three reducing aldohexoses not resolved by the above conditions. These procedures have been used to identify tentatively a previously unknown compound in potatoes as fructose 1-phosphate. The advantages of paper electrophoresis as well as correlation between movement and relative charge density are discussed.

1. Low voltage paper electrophoresis in 0.05 M citrate buffer of pH 4.8, at a voltage of 200 V, for 312 hours, is a suitable method for determining adenine nucleotides in erythrocytes. 2. Before applying the sample to the paper it is necessary to remove the excess of deproteinisation agent. 3. By means of the method described 42.6 ± 14.5 mg% ATP and 10.7 ± 9.5 mg % ADP were determined in the erythrocytes.

Paper chromatography with new solvents applied to some commercial samples of thiazine stains, especially azures, resulted in improved resolution which gave more spots than had been obtained with previously reported solvents. To a Whatman No. 1 filter paper strip, 50 cm in length, 0.08 ml of a 0.1% solution in water was spotted 18 cm from the edge of the strip. The solvent used in most cases was a mixture of t-butylalcohol, methylcellosolve and a 1% NH4Cl solution in distilled water (60:20:20, v/v) with the ascending technique. Several batches of thionin were found to have a high degree of purity (concerning their content of coloured components). One sample of thionin (Chroma) contained only one component (violet). The thionin-SO2 reagent showed a chromatogram with more spots than the thionin sample from which the reagent was prepared. Qualitatively a sample of azure A appeared to have practically the same composition as a sample of azure C. At least six spots were visible in the chromatogram of these azur...

Summary and conclusions1. Serum from 6 patients who had received radioiodide or I131-labeled thyroxine was studied by zone electrophoresis in filter paper, at pH 8.6. Most of the I131 had a mobility intermediate between the alpha1- and alph2-glabulins, and a small proportion had the mobility of albumin. No differences were detected between thyroxine added to serum (in vivo) and the thyroid hormone secreted by a normal thyroid gland, by a Graves' disease thyroid gland, and by a functioning thyroid carcinoma. 2. Two patients had received very large doses of radio-iodide and their serum contained large amounts of butanol-insoluble I131, whereas in the other patients the serum I131 was largely butanol soluble, and behaved like thyroxine on paper chromatography. These two forms of serum I131 had similar mobility on zone electrophoresis, but could be differentiated by fractional precipitation of the serum proteins with phosphate buffer. The radioiodine, therefore, was probably attached, in each instance, to a d...

A silicic acid paper chromatographic method for the separation of four major classes of cholesterol esters, namely saturated cholesterol esters, cholesterol oleate, cholesterol linoleate and cholesterol linolenate plus cholesterol arachidonate is described. This method uses a benzene-hexane solvent. The use of combined column and paper chromatography as well as gas chromatographic analysis is presented for definitive identification of the various spots. The predominance of cholesterol linoleate in human plasma, and cholesterol arachidonate in rat plasma is confirmed.

A paper Chromatographic method for the analysis of phenylalanine in serum is described. The amino acids, including phenylalanine, are isolated from the deproteinised serum by means of a cation exchanger. The phenylalanine is then separated by paper chromatography at elevated temperature and estimated colorimetrically. Normal values are given. The excretion of phenylalanine and phenylpyruvic acid is correlated with the serum phenylalanine level. The method is specific and the reproducibility is good. Its application in a case of phenylketonuria is reported.

Paper-electrophoretic separation of intrinsic factor-related “primary” and “secondary” B12 binders in pooled normal human gastric juice was achieved by using Teorell-Stenhagen buffers of pH 4.0–5.0, at which pH the “primary” binder moves to the cathode, while the “secondary” one travels to the anode. The isoelectric point of the “primary” binder is about pH 5.5, while that of the “secondary” one is pH 2.5–3.0. The electrophoretic mobility of none of the major carbohydrate-, proteinor peptide-nondialyzable components of gastric juice correlates with that of the “primary” or “secondary” B12 binders when run on electrophoresis in the pH range of from 2.5 to 10.0. This supports previous conclusions that none of the major components of gastric juice resolved by paper electrophoresis represents the intrinsic factor-related B12 binders.

The possible degradation of tryptophan and seven of its metabolites during and after paper chromatography with n-butanol-acetic acid-water has been investigated. It has been found that extensive degradation results from developing and drying the chromatograms at temperatures over 27° and storing them in room light for extended periods before the second development. Lesser amounts of degradation were found after limited exposure of compounds to ultraviolet light between developments and occasionally with the use of unwashed paper. Procedures to eliminate degradation have been described.

Benzene (pregnanediol) or chloroform (pregnanetriol) extracts of urine after enzymatic hydrolysis were chromatographed by an ascending technique on potassium- silicate impregnated glass fiber paper strips in cyclohexane: acetone. Chromatography was completed within 50–60 minutes. Steroids were eluted quantitatively with ethyl acetate by a descending elution technic, and measured spectrophotometrically in sulfuric acid. Experimental data are presented which demonstrate the accuracy, specificity, reproducibility and clinical usefulness of the method. The simplicity and rapidity of glass fiber paper chromatography make the method highly suitable for routine determinations.

A simple method for the extraction and purification of human growth hormone has been developed, by which, after the initial alkaline extraction of the acetone-dried pituitary powder, the bulk of the impurities is precipitated at pH 4.8 and the crude hormone subsequently precipitated with alcohol at 25% v/v concentration. The crude hormone is then purified by a single Sephadex G-100 gel nitration, 75% of the activity of the crude being obtained as virtually pure monomer and the remaining 25% in the form of two fractions containing two separate polymers. A simple and rapid paper chromatographic assay method has also been developed for following the extraction and purification stages which gives good agreement with the corresponding radioimmunoassays.

A simplified procedure for filter paper electrophoresis is described in which disturbing factors such as evaporation, heating, buffer concentration gradients, and pH changes in the electrode vessels were reduced to a minimum. Artificial mixtures of highly purified proteins could be separated and the components isolated. The application of the method to a variety of studies on serum proteins is demonstrated. Protein concentration in paper segments was determined by two different methods of protein estimation. Curves were obtained showing the same five major peaks for normal serum as found by the classical methods of free electrophoresis. Comparisons were made of the areas of the various components under the curves obtained with the different methods. Two dimensional electrophoresis was applied to serum and serum components. It proved of value in demonstrating the heterogeneity of fractions such as the γ-globulin of serum. The polysaccharide dextran was used as an index of the extent of electro-osmotic flow during the course of the various experiments. The ratio of the distance of electroosmotic flow and the distance of protein migration was shown to be constant for a given type of paper. For serum albumin on Munktell 20 paper this ratio was 0.35. A formula for mobilities applicable to liquid in a highly porous supporting medium is presented. Mobility values for human serum albumin at various pH levels on paper showed approximate agreement with those obtained in free solution giving a similar isoelectric point.

A simple quantitative chromatographic method is described for the separation of epitetracycline and tetracycline in pharmaceutical preparations. The method is applicable qualitatively to human serum and quantitatively to the biological determination of tetracycline in urine. Whatman's modified cellulose phosphate cation-exchange paper is developed by the descending technique using 0·1 per cent w/v ammonium chloride solution. The spots are located as yellow fluorescent spots under ultra-violet light and ammonia vapour, eluted in 10 per cent w/v ammonium chloride, and the extinction determined at 356 mμ. The method is not suitable for the separation of chlortetracycline or oxytetracycline from tetracycline.

The mammalian respiratory tract produces a secretion in which the major macromolecular components are mucin-type glycoproteins. High-voltage paper electrophoresis has proved a valuable research tool in studies of the formation of these glycoproteins. Separations of hexoses, amino sugars, oligosaccharides, sugar nucleotides and mucin glycoproteins are rapidly and easily obtained by paper electrophoresis at voltages of 50 to 100 V/cm. The use of high-voltage paper electrophoresis to follow amino sugars interconversions and the transfer of glycosyl units to mucin acceptors is described.

SummaryUsing the paper chromatographic technic significant changes in the protein patterns have been demonstrated in patients with multiple sclerosis. We are unable to state at present the relationship of these changes to the activity of the disease, but our evidence would seem to indicate that they may occur concomitantly with, or immediately following the onset of exacerbation. Furthermore the changes in protein patterns revert to normal during remissions.

Recoveries of metanephrines from urine by three methods were compared. Ion-exchange gave higher recoveries than did solvent extraction. Best results were obtained using Zeo Karb 226 which yielded extracts in which the amines could be estimated after separation by paper chromatography.

Construction and use of a simple scanning device are described for locating small amounts of radioactivity on large uncut filter papers used for chromatography and electrophoresis. With this device the use of autoradiographs in eliminated from the original method for determining urinary hydroxyproline on the principle of isotope dilution. The modified method is described, and eight results are reported for the recovery of added nonradioactive dipeptide. Percentage of recovery varied from 84.5 to 111% with an average of 98.4%.

A paper electrophoresis method is described in which four serum lipoprotein components are separated without the use of albumin in the buffer. Tris-EDTA-boric acid buffer, Oil Red O staining solution prepared in 52.8% instead of the usual 60% alcohol, and solvent-extracted paper strips are the distinguishing features of this procedure. The system was effective not only in separating chylomicron, β- and α-lipoproteins into well-defined bands, but also in separating very low density or pre-β-lipoprotein distinctly in some samples and partially in others. The advantages of this procedure are low background staining and, in comparison to the procedure using albumin in the buffer, a sharper alpha band. This sharpened α-band gives a sharper peak in densitometric scanning. Electrophoresis could be performed for 16 hr or 4 hr. The 4-hr electrophoresis run produced electrophoregrams with even denser and sharper bands than the 16-hr run.