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P work from this laboratoryl-S has demonstrated the heterogeneity of crystalline trypsin by paper and starch column electrophoresis. This article presents an electrophoretic method of greater resolution. Using this technique it was possible to demonstrate in a sample of crystalline trypsin the occurrence of fractions showing different ratios of proteolytic to amidasic activity. In the experiments recorded here a buffer of pyridine acetate of pH 4·9 (0,48 : 0·56 : 98·96 v/v of acetic acid: pyridine: water) was used for electrophoresis in a Durrumtype apparatus. To a strip of Whatman 3 filter paper of 12 x 50 cm, 0·12 ml. of an 8 per cent trypsin solution in 0·001 N hydrochloric acid were applied. An electrical tension of 450 V was used and the experiment was allowed to run for 5 h in the cold room. In these conditions at the end of the experiment the temperature of the buffer was 9° C. The strip, while still wet, was cut in 1-cm wide transversal segments and transferred individually to test-tubes containing 3ml. of a 0·25 M pyridine formate buffer of pH 2·7. This extraction was allowed to continue for about 20 h in the cold room. The extract of each tube was then analysed for protein content, proteolytic activity against azo casein and amidasic activity towards cebenzoyl-L-arginine amide (BAA). The protein content was determined by the FolinLowry4 method as applied to the 'auto analyseI" system·. Proteolytic activity against azo casein was measured by the method of Charney and Tomarelli". The hydrolysis of BAA was followed colorimetrically in the auto analyser'. Chromatography of trypsin in carboxy methyl cellulose was carried out according to the method of Liener8• The trypsin samples used in this work were purchased from the 'Worthington Biochemical Corporation ('Cryst. Trypsin, lyophilized, lot TL 747-58'), Freehold, New Jersey, and from Novo Industri ('Cryst. Trypsin Novo, batch 114-3'), Copenhagen, Denmark. The results obtained by the fractionation of the Worthn~on trypsin are shown in Fig. 1. The presence of three

THE application of the principle of electrophoretic separation on paper to steroids is limited by their relatively low polarity, so that they do not readily migrate under the influence of an electric current, and also by their sparing solubility in aqueous media. By converting the steroids into derivatives with polar groupings, some separation has been achieved (Voigt and Beckmann1, 1953, using the hemisuccinates; McKinley2, 1955, using the mono- and di-phenylhydrazone derivatives). Our early experiments, begun before the publication of these authors' results, were along similar lines, and with somewhat similar results. It did not seem likely, however, that electro-phoretic separation would be so convenient a method as chromatography, which has been adapted by Lembart and Schneider3 for the separation on paper of mixtures of pure steroid conjugates. The natural occurrence of more polar steroid derivatives, the sulphates and glucuronides in urine, suggested to us that these might undergo electrophoretic separation on paper. Although the individual steroids could not be resolved, the two groups of conjugates were sharply separated.

MANNOSIDOSTREPTOMYCIN in some streptomycin preparations is conveniently detected by the paper chromatographic procedure of Winston and Eigen1. In the presence of high concentrations of salts such as in ion-exchange column eluates, or in fermentation broths, this procedure is not applicable: attempts to desalt such solutions, by mixed-bed ion-exchange, electrolytic de-ionization or solvent extraction, have proved unsuccessful. We have found it possible to separate streptomycin, mannosidostreptomycin and allied substances by paper electrophoresis, having developed (independently) a technique similar to that used for new antibiotic broths by Hosoya et al. 2, and King and Doery3.

IN work concerned with the composition and estimation of neurohypophysial hormones in the posterior pituitary, the hypothalamus and body fluids, it would be desirable to have a chromatographic procedure which permits the identification in and recovery of small amounts of the active peptides from impure extracts. It would also be useful to know whether the substituted synthetic oxytocins which have recently been prepared1 can be distinguished by a simple chromatographic method. Taylor2 separated oxytocin, and arginine- and lysine- vasopressin by ion-exchange chromatography on resin columns. He used du Vigneaud's highly purified peptides, and the smallest amounts applied were equivalent to 0.6 mgm. of this material. Benfey3 purified Stehle and Fraser's4 preparations of ox posterior pituitary hormones (200 U./mgm. pressor activity and 250 U./mgm. oxytocic activity) by paper chromatography using ninhydrin as the colour reagent. The smallest amounts of the hormone preparations used were 1 mgm. However, since only terminal amino-groups react with ninhydrin, it is obviously disadvantageous to use this reagent when attempting to visualize very small quantities of the posterior pituitary peptides. Rydon and Smith5 have shown that micrograms of peptides of all kinds, including cyclopeptides, can be detected by chlorination followed by spraying with a starch–potassium iodide solution. Reindel and Hoppe6 improved this procedure in two ways. First, before exposure to chlorine, they moistened the paper with a mixture of watery ethanol and acetone, which prevents background staining. Secondly, they treated the N-chloropeptides with a mixture of potassium iodide and either benzidine or o-tolidine in acetic acid and thereby increased the sensitivity of the method.

FOR Chromatographic identification of iodide and iodinated protein derivatives, it is often necessary to locate inorganic and organic iodide tracers on the same chromatogram. In paper chromatography a number of methods have been used for the detection of iodide. These include spraying with silver nitrate1 ; potassium chlorate, acid and starch2 ; 1 per cent aqueous palladium chloride3 ; a mixture of ferric nitrate and hydrogen peroxide4 ; the ceric-arsenite reagent5; and a mixture of FeCl3.6H2O, K3Fe(CN)6 and NaAsO2 (FFCA reaction)6. Most of these tests for iodide interfere with those commonly used for the identification of iodinated amino-acids, namely, ninhydrin7, diazotized sulphanilic acid3, or its diethyl amide8. The ceric-arsenite and the FFCA reagents are the most sensitive (0.01 µm. I−) and can moreover be used for the detection of inorganic iodide, together with iodinated amino-acids on the same chromatogram6,9. The FFCA reagent, however, is not specific for iodine but also gives positive reactions with certain reducing substances6. The ceric-arsenite reaction has several disadvantages and is generally not used in the spraying technique9.

IT has been shown by paper electrophoresis that xanthine dehydrogenase activity is bound to the globulin fractions of rat serum1. Using differential centrifugation, it was demonstrated that xanthine oxidase and xanthine dehydrogenase activities are present in the supernatant fluid corresponding to the ‘cell sap’ containing the soluble proteins of the rat liver2.

THE separation of phospholipids by paper chromatography has generally been performed on modified papers. Marinetti and Stotz1 and Lea et al.2 report the use of silicic acid-impregnated paper, and Brown et al.3 have used silicic acid-impregnated glass-fibre ‘paper’. Rouser et al.4 investigated the possibility of using paper impregnated with mineral oil, rubber, silicone, and formamide, and paper oxidized by periodic acid, but found all these to be unsuitable. Recently, however, in this laboratory5 a method has been evolved for the separation and differential staining of phospholipids on untreated paper. During work on this method difficulty was encountered with the separation of lecithin from phosphatidyl ethanol-amine, and a procedure for separating them on paper impregnated with zinc salts is described here.

INORGANIC chloride is commonly present in mixtures of biological origin and, if not removed, may interfere with the identification of the organic constituents. A silver nitrate reagent has been developed which will detect as little as 1 µgm. of chloride ion on a paper chromatogram.

PHOSPHATE esters separated on paper chromatograms are usually detected by degrading them on the paper with acid to give ortho-phosphoric acid and detecting this by the reaction with molybdate and a reducing reagent. The procedure of Hanes and Isherwood1 is based upon this and is widely used. This technique suffers from two disadvantages. In the first place, the prolonged initial ‘digestion’ necessary to break down the more resistant esters frequently leaves the paper in a very fragile state; and secondly, further analyses cannot be carried out on the spot after treatment. In order to avoid these disadvantages the following technique was developed. It depends upon the fixation of ferric ions by the esters and the reaction of the free ferric ion with salicylsulphonic acid. If the paper is not strongly buffered it is sprayed with 0.1 per cent FeCl3.6H2O in 80 per cent alcohol, dried in air at room temperature and then sprayed with 1 per cent salicylsulphonic acid in 80 per cent alcohol. Upon drying, the phosphates appear as white spots on a pale mauve background, ortho-phosphate having a band of deeper mauve surrounding it. The colour formation occurs only when the pH of the residual moisture in the paper is about 1.5–2.5. This can be checked conveniently with thymol blue, since the colour formation occurs most satisfactorily when the paper just turns this indicator red. Further indications of unsatisfactory pH are that below this range no colour is formed, whereas above the range the colour is orange-yellow and definition of the spots is very poor.

WHEN ethyl pyruvate and 4,5-diaminopyrimidine were refluxed in water, between pH 1 and 3, a solid of molecular weight about 320 was formed which was seen as two spots1 after dissolution in 0.1 N sodium hydroxide and chromatography on a sheet of Whatman No. 1 paper in water at pH 5.5 or 8.5. The spots (Fig. 1A) had RF values of 0.55 and 0.65 respectively and appeared identical when viewed in ultra-violet light. When eluted, and rechromatographed, each spot generated only one spot, which corresponded in RF to the spot from which it was eluted (Fig. 1B and C).

THE observation and analysis of isolated cell fragments gives much information on the way in which the more complex structures are put together. The usual method used for separating cell fragments is differential centrifugation, in which the difference in size and particle density is the determining factor. Albertsson1 has studied the separation of cells and cell fragments using column chromatography. This is a method of interest since it separates the cell fragments on the basis of the physical and chemical properties of the surface of the particles. This communication points out that the well-known technique of paper chromatography can be extended from the separation of molecules to the separation of cell fragments of very large molecular weight.

ELECTROPHORETIC fractionation of pancreatic juice components in a dog has been described in the literature1–4. Some of the authors2,3 isolated part of the fractions and studied their enzymatic activities. All authors used the free electrophoresis method with the exception of Delcourt and Delcourt4, who employed paper electrophoresis for the separation of the different fractions.

THE identification of sulphonamides in biological fluids is usually attempted by microscopical examination of crystals and by the use of various, rather non-specific, colour reactions1. Since it is common for only small quantities of fluid to be available, it was decided to investigate the possibilities of paper partition chromatography.

IN 1955, Scott1,2 described a method for the preparative separation of polysaccharides containing different amounts or types of anionic residues, based on the formation of insoluble complexes with cetyl pyridinium chloride (CPC) or similar long-chain cations. The resulting complexes can be selectively dissociated by varying concentrations of electrolytes (sodium sulphate, sodium chloride, magnesium chloride)3,4.

IN recent studies, normal adults ingested 120–400 gm. of fat (as 40 per cent butterfat cream) and, after 3–4½ hr., they received 100 mgm. of heparin intravenously. Serum samples taken 30 min. later were compared with their fasting and pre-heparin samples by electropapergram techniques. The post-heparin samples consistently showed a number of changes which are listed below and indicated by Roman numerals.

THE method described below was developed with the view of detecting and estimating the small amounts of steroids present in the blood and tissues of mammals. It has proved very useful for studying the output of the adrenal gland, and is sufficiently sensitive to be used on small samples of human blood.

THE most sensitive chemical method for determining epinephrine in solution is the reaction with ethylenediamine. The reaction yields a number of products, several of which are fluorescent. These substances can be used for quantitative measurement. Nor-epinephrine produces a fluorescence of different colour so that mixtures of the two compounds can be determined by the selection of appropriate filters in measuring the fluorescent light1.

THE Burton, Zaffaroni and Keutman1 method for the paper chromatographic analysis of the adrenal cortical steroids has been found to be suitable for the non-steroid œstrogens of the hexœstrol series (hexœstrol, stilbœstrol, dienœstrol, trienœstrol2) and ethinyl œstradiol. It can also be used for the natural œstrogens (private communication from H. Grundy). Development is carried out using the toluene – propylene glycol system, and the detecting reagent, a mixture of 1 per cent ferric chloride and 1 per cent potassium ferricyanide as used by Barton, Evans and Gardner3, gave a blue spot with less than 2 µgm./sq. cm. of the run synthetic œstrogen. In addition, stilbœstrol, dienœstrol, trienœstrol but not hexœstrol gave a strong absorption at 2300 A., and dienœstrol a blue and trienœstrol a green fluorescence at 3600 A.

WE have already described a fortuitous combination of events which led to the discovery that American newspapers and other paper products contain a potent analogue of the juvenile hormone of the European bug, Pyrrhocoris apterus L. (Heteroptera ; Pyrrhocoridae)1. When reared in contact with ‘active paper’ or when exposed to surfaces impregnated with extracts of the ‘paper factor’ (PF), fifth-stage Pyrrhocoris larvae undergo one or more supernumerary larval moults and finally die without completing metamorphosis or attaining sexual maturity. This same result is observed when 1 µg of partially purified PF is topically applied to young fifth instar larvae1,2.

IN the first report1,2, on ‘crossing paper electrophoresis’, a direct demonstration of enzyme-substrate complex has been described. The enzymes tested included crystallized pure trypsin, chymotrypsin and ribonuclease.

R. P. LINSTEAD et al.1 have described the separation by means of paper Chromatography of aluminium, gallium, indium and zinc from solutions of their chlorides in n-butyl alcohol containing hydrochloric acid as a solvent. According to their work, aluminium scarcely moves at all from the original position, while the other elements move varying distances down the paper. We have recently followed their technique in an attempt to separate aluminium and beryllium, but instead of developing the aluminium with ‘Aluminon', we have incorporated the technique of development by fluorescence first proposed by Pollard et al.2. After separation on the paper, the positions of the aluminium and beryllium spots are shown by spraying with an alcoholic solution of 8-hydroxyquinoline. Both the aluminium and the beryllium then fluoresce under ultra-violet light and can, indeed, be observed in strong sunlight. The fluorescence is intensified on drying the paper and exposing to ammoniacal vapours.

ACCORDING to Miyazaki, Omachi and Kamata1, antibiotics can be grouped according to their salting-out chromatograms. Pure antibiotics and materials containing antibiotics were examined by the aid of ascending paper chromatography. As irrigation solvents in each case and with each antibiotic, distilled water and rising concentrations of ammonium chloride (0.5, 1, 2, 3, 5, 10, 20 per cent and saturated) were used. Location of the spots of the antibiotics, that is, the R F values, were ascertained bio-autographically.

BY means of filter-paper partition chromatography, Fink, Dent and Fink1 located several different radioiodine fractions in hydrolysates prepared from thyroids of rats that had been injected 24–28 hours earlier with iodine-131. Surprisingly, little or none of the radioactivity was found as thyroxine. Since this is not in accord with previous results from this laboratory2, we examined the method of filter-paper chromatography as applied to thyroid iodine.

PROTEINS of the lens tissue of the eye have been extensively examined because of their relationship with cataract formation. In all forms of cataract a decrease was found in the total amount of lens proteins as compared with normal lens of the same age1. Changes in the proportion of the soluble proteins of the lens measured by electrophoresis have been reported in different kinds of cataract; but, according to van Heyningen2, no consistent patterns have emerged from these investigations. The work recorded here was carried out to determine the behaviour on paper electrophoresis of soluble lens proteins of the rat, as well as the possible changes in the proportion of the different fractions during lactose feeding.

Paper Chromatography of Muscle Tissue Constituents: A Phosphate-absorbing Protein from Muscular Tissue

ALTHOUGH publications concerning measurement of vasopressin and oxytocin in blood and urine are legion, the lack of specificity of technique for concentration and bioassay of the trace amounts presumed to be present in biological fluids has led to wide variation in results and frank scepticism concerning their physiological significance1. Since bioassay techniques, although extremely sensitive, are inherently subject to wide error, a reliable quantitative method applicable to biological fluids demands a specific and sensitive technique for isolation and concentration of these polypeptides from blood or urine prior to bioassay.

EXTRACTION methods are usually used for the isolation and purification of antibiotics soluble in organic solvents which are immiscible with water. This method can be applied if the partition coefficient of the antibiotic between the solvent and water is sufficiently large. The partition coefficient often depends on the pH. value, particularly for acidic or basic compounds.

VARIOUS colour-producing spraying reagents have been developed for the detection of di- and tri-hydroxy phenols on paper chromatograms. Excellent results are obtainable with the diazotized sulphanilic acid of Evans, Parr and Evans1 and the phospho-molybdic acid reagent of Riley2. Bate-Smith3,4 applied Partridge's5 ammoniacal silver nitrate reagent for furnishing diagnostic evidence regarding the position and number of hydroxy groups and also used ultra-violet light, both in the presence and absence of ammonia vapour, to obtain useful information. The majority of the above class of C6 compounds give characteristic coloured spots with a 2 per cent aqueous ferric chloride spray3,4, although meta- and para-hydroxy phenols (for example, orcinol, phloro-glucinol, resorcinol and hydroquinone) show ill-defined greyish spots.

Paper Chromatography of Muscle Tissue Constituents: Paper Chromatography of Adenosine Phosphates

POLYCHLOROBIPHENYLS (PCB) cause chloracne and liver damage in man and induce oestradiol degradation1. They are widely distributed in the ecosystem in birds, fish and shellfish1–4. The miserable episode of “Yusho”, a mass food poisoning involving more than 1,000 people which was caused by chronic ingestion of rice oil contaminated with ‘Kanechlor-400’, a commercial brand of PCB, occurred in western Japan in 19685,6.

THE acid phosphatase test has proved its usefulness in seminal stain examinations and is now in widespread use1–4. The desirability of preparing a stable form of the reagent was obvious, more especially for ‘scene-of-crime’ searching, post-mortem examinations and for scientists and clinicians without laboratory facilities.

THE electrophoretic analysis by the ordinary Tiselius apparatus of the soluble proteins isolated from rat liver has already been described1–3; but the constituent components reported by these authors differed one from the other.

LITTLE is known of the metabolism (in man and experimental animals) of the alkaloids of Rauwolfia serpentina. We are particularly interested in the reported antifertility effects of reserpine in rats1 and the mechanisms of sedative action2 of Rauwolfia serpentina alkaloids in schizophrenic patients. Reserpine is known3 to be hydrolysed in vivo to methyl reserpate and presumably trimethoxybenzoic acid. The latter compound has been shown4 to influence certain enzyme systems in vitro which are of importance in relation to the mode of action of œstrogens on the uterus. Using a fertility performance test, we have studied the effects of the administration to male and female rats of daily oral doses of 80–100 mgm. of sodium trimethoxybenzoate given over a period of ten weeks. Since no alteration in fertility was observed, we conclude that reserpine itself or some polycyclic metabolite is responsible for the observations of previous workers1.

THE numerous paper chromatographic methods1–3 so far presented for the separation of corticosteroids all have limitations; our results may therefore be of interest. We have found that by impregnating the papers with a 90 per cent methanol solution we could separate the corticosteroids rapidly at room temperature (16–20°C.). The method also works for prednisone and prednisolone (Table 1). Furthermore, it is suitable for quantitative and semi-quantitative determinations.

WE have examined recently blood containing fibrinolysins in high titre. The blood was taken from a man who was later diagnosed as suffering from prostatic malignancy. The presence of high-titre fibrinolysins, raised formal acid phosphatase, and greatly reduced fibrinogen content, were all carried out by routine, accepted biochemical methods, and were fully diagnostic. It was then considered that there could be present a circulating auto-immunological factor which we might be able to demonstrate.

METABOLITES of numerous biologically active endogenous and exogenous organic compounds are excreted in bile and urine as glucuronic acid conjugates. In the course of studying the mechanism of action of carcinogenic polycyclic hydrocarbons, we wished to identify the metabolites of certain of these agents. For this purpose, 14C-3,4-benzpyrene was injected into rats, and bile was collected by an indwelling cannula, utilizing a technique already described1. The bile fractions were collected in an automatic fraction collector, and enzymatic hydrolysis with β-glucuronidase was undertaken. Characterization of all the metabolites by this technique proved to be impossible because of the extreme instability of some of the hydrolysis products. We therefore attempted to determine whether the extent of oxidation and the class of conjugate formed could be established by direct analysis of the conjugates.

THE issue of the first volume of this work in the year 1880 was the beginning of the valuable series of reprints of mathematical and physical papers for which we are indebted to the Cambridge Press. It was felt at the time that no more auspicious beginning could have been made, and the publication was widely appreciated; but a gradual and increasing sense of disappointment supervened when, after the second volume, the continuation seemed to be suspended indefinitely. A third instalment has however now appeared, after the lapse of eighteen years, and although the regrets we have referred to cannot be altogether appeased, the contents of the volume will assure it of as hearty a welcome as was accorded to its predecessors. Mathematical and Physical Papers, vol. iii. By Sir G. G. Stokes. Pp. viii + 451. (Cambridge University Press, 1901.) Price 15s.

WE have found that, when a mixture containing barbiturate, salicylate and acetophenetidin is subjected to chromatography on Whatman No. DE20 cellulosic ion-exchange paper in dilute ammonia solution, a partial resolution of the components is possible. Salicylate is clearly separated from the other two compounds, which move together with only partial resolution (Fig. 1).

THE presence of acylating compounds on paper is frequently deduced from reaction with hydroxylamine and subsequent treatment with ferric chloride to reveal the hydroxamates so formed. This method has been used for the detection of nucleotide peptides and similar ‘activated’ complexes1–8.

TWENTY-SEVEN authors contribute to this symposium, which will be welcomed by all who have to deal with any aspect of cancer. The papers, some of which are highly technical, are reprinted from many journals and form four main groups: three of general clinical interest; four on radiation therapy; eight on calculations and physical measurements of radiation; eighteen on experimental research, and a review of the world literature 1938–1939 on chemical compounds as carcinogenic agents. The latter paper, by Profs. Cook and Kennaway, follows an earlier review on the same subject and is a masterly piece of work, including a bibliography of some five hundred new references. About half the volume is concerned with the synthesis and testing of chemical carcinogens and growth inhibitors. It is not yet certain that any of these substances are directly related to human cancer other than industrial cancer. A careful perusal of the results shows that many new facts could not have been established by studying only the earliest and best-known carcinogenic hydrocarbons. To the reviewer, the most interesting new experiments are those dealing with growth-inhibiting hydrocarbons that are not necessarily carcinogenic; with carcinogens that act on remote organs as well as locally; with those that show the need for more than one kind of biological test before a substance can be regarded as non-carcinogenic; and, particularly, with the extraction from human livers (not necessarily from cancer subjects) of unidentified carcinogens. Selected Papers from the Royal Cancer Hospital (Free) and the Chester Beatty Research Institute Published by order of the Governors of the Royal Cancer Hospital (Free), London. Vol. 2. Pp. viii + 414. (London: Royal Cancer Hospital (Free), 1943.) 16s.

AN easy solution of the paper-band puzzle described by Prof. C. V. Boys in NATURE of June 9, p. 774, is obtained as follows: Hold the hand with thumb up and palm towards you; place the paper band over the index finger, letting the ends hang down. Observe which way the original four half-twists were applied. Treat the nearest of these to the index finger on the palm side of the hand as if it were that of an ordinary single half-twist band; which complete, by looping up one-half of the band over the finger (the other twists being pushed out of the way into the remaining half). Then apply the surfaces one upon another at the finger; and turn the other half of the band inside out so as to get rid of two of the twists. It will be found to fit exactly upon the first half, as required.

IN two previous communications, we1,2 have shown that a mixture of phenobarbitone, acetophenetidin, salicylate and p-acetylaminophenol can be separated in 60–75 min. by a combined technique of chromatography and ionophoresis on Whatman ion-exchange cellulose papers. We have now been able to effect the resolution of a mixture of basic and neutral drugs, namely, promazine, quinine, sulphacetamide and acetophenetidin.

IN a previous communication, we1 showed that a mixture of salicylate, barbiturate, and acetophenetidin could be separated by a combination of chromatography and ionophoresis on a sheet of cellulosic anion-exchanger.

Simple oligonucleotide maps can be obtained by two-dimensional paper electrophoresis from enzyme digests of 5S RNA from KB carcinoma ribosomes. These maps can be distinguished from those obtained from 5S RNA derived from E. coli and from RNA components of low molecular weight derived from KB cells with and without adenovirus infection.

THE application of paper electrophoresis to the study of new antibiotics1 has been in use at these laboratories for some time. Our technique consists of applying spots of culture fluid, or a concentrate of it, to paper strips soaked in buffer, and subjecting the strips to electrophoresis by the technique of Kunkel and Tiselius2. Spots of chloromycetin are applied to the paper on each side of the spot of unknown antibiotic as a control for electro-osmosis. After migration, the position of the antibiotic is revealed by the microbiological technique as usual in paper chromatography of antibiotics3. Thus it is possible to determine whether unknown antibiotics in culture fluids are acidic, basic or amphoteric, and to obtain an approximate measure of their isoelectric point as a guide to subsequent purification procedures. The migration of an unknown antibiotic 179, relative to chloromycetin, to the cathode at pH 3 and pH 8, with lessened mobility at pH 8, indicates a basic substance with an isoelectric point greater than pH 8, as illustrated in the accompanying photograph.

SEVERAL cases of moderately severe hypochromic haemolytic anaemia have recently been observed in Burgas, Bulgaria1. The affected individuals were unrelated white adult men and women. They showed splenomegaly, slight icterus, decreased osmotic fragility of the red cells and 40–90 per cent target cells. Paper and starch-gel electrophoresis2 at pH 8.6 of their haemoglobin showed complete absence of normal adult haemoglobin (Hb A) and presence of a slow-moving abnormal haemoglobin. The electrophoretic mobility of this abnormal haemoglobin was similar in paper electrophoresis to that of haemoglobin C (Hb C). In starch-gel electrophoresis the abnormal haemoglobin moves slightly ahead of Hb C. In agar-gel electrophoresis at pH 6.03 it moved between Hb A and Hb C, in the position of haemoglobin S (Fig. 1).

THE antigenic properties of insulin are now well recognized, but no simple method has yet been described for the routine detection and assay of insulin antibodies. The method described here is based on the classical observation by Berson, Yalow, Bauman, Rothschild and Newerly1. They showed that during chromato-electrophoresis on filter paper, 131I-labelled insulin bound by insulin antibodies in human serum moves with the β-γ-globulins, while free insulin remains fixed at the point of application of the serum-insulin mixture on the filter paper. When an excess of unlabelled insulin mixed with a trace of labelled hormone is added to the anti-insulin serum, the excess insulin is adsorbed from solution by cellulose and the amount bound by the antibodies is measured in the supernatant solution. We have used this assay system for serum obtained from guinea-pigs treated with bovine insulin2 and capable of neutralizing insulin at rates ranging from 0.1 to 3.5 units per ml. serum. It can, however, be adapted for the detection and assay of antibody concentrations lower than this; it has not yet been used for anti-insulin sera of other animals or of man.

ANTIMONY pentachloride has been used as an agent for the detection of steroids in paper chromatography by a number of investigators1–4. It has been found that antimony pentachloride is a useful agent for detecting certain digitalis compounds (digoxin, digitoxin and digitoxigenin) on paper chromatograms.