- Email: [email protected]
RAPID DETERMINATION OF SOME PLASMA AMINOACIDS IN SUBCLINICAL KWASHIORKOR
250
isoleucine, and valine) and methionine are particularly affected, but glycine, serine, glutamine, and taurine remain within the normal range. The screening test
TABLE II-EFFECTS ON CONSCIOUSNESS
could be attracted in a sustained way through questioning by the examiner. The aphasic, but otherwise conscious patient could, in our experience, readily be distinguished from the patient who had lost consciousness. The injection on the side showing dominance for speech resulted, in most patients, in loss of consciousness, while injection on the side not dominant for speech led to loss of consciousness far less often. When loss of consciousness did follow injection of the non-dominant side its duration was shorter than that after injection of the other side; and, further, in no case did unconsciousness follow injection of the minor hemisphere alone. Also, those patients with apparent bilateral representation of speech showed no difference between the two sides in terms of consciousness, whether injection of both sides or neither side resulted in aphasia. CONCLUSION
The
finding that consciousness tends to be lost when the speech-dominant hemisphere rather than the non-dominant is injected with amylobarbitone has not, as far as we are aware, been reported before. It has been suggested that there is a close interaction in the function of speech between the relevant parts of the dominant hemisphere and the upper brain-stem.4 It seems possible that the speech-dominant hemisphere may, by virtue of this interaction, also acquire a dominant role in the matter of consciousness, which, in man, is heavily dependent on symbolic processes. We are grateful for the help given in this investigation by Mr. M. A. Falconer and the staff of the Guy’s-Maudsley Neurosurgical Unit. One of us (E.A.S.) was in receipt of a grant from Guy’s Hospital Medical School.
E. A. SERAFETINIDES M.D. Athens., D.P.M. Research Fellow
The Guy’s-Maudsley Neurosurgical Unit and the Department of Experimental Neurology (Institute of The
Psychiatry), Maudsley Hospital, London, S.E.5
R. D. HOARE M.B. Lond., D.M.R.D. Consultant
Radiologist
M. V. DRIVER
described in this paper is based on the estimation of these aminoacids in two groups, and the calculation of an " imbalance " ratio. Aminoacids are imbalanced not only in severe cases of kwashiorkor but also in children who, although living on a poor diet, show few or none of the clinical signs, except a small size for age; the imbalance is therefore the earliest biochemical abnormality so far discovered. Full details of the correlations of the ratio with the signs are being published elsewhere," but a preliminary communication to the International Congress of Nutrition in Edinburgh last August has resulted in requests for details of the method for use as a potential diagnostic test. In the developing countries where kwashiorkor is most prevalent, skilled biochemical assistance is not usually available, and the procedure is here described in full for those who may not be familiar with routine quantitative paper
chromatography. MATERIALS
ml. of demineralised water added
to 900 Ethanol, 90% v/v.-100 ml. absolute alcohol. Chromatography solvent.-120 ml. n-butanol, 30 ml. glacial acetic acid, and 50 ml. water. In tropical countries the solvent should be prepared afresh for each batch of estimations, and used once only. Ninhydrin, 0-2% w/v in acetone.-l g. ninhydrin (indane-l:2:3trione hydrate) in 500 ml. acetone. About 50 ml. is needed for each batch. It should be used once only: if it is used repeatedly, a strong background colour will develop. Ethanolic copper nitrate.-l ml. saturated copper nitrate in 100 ml, absolute ethanol plus 0-2 ml. 10% v/v nitric acid. Methanol (reagent grade). Isopropanol (isopropyl alcohol), 10% v/v.-100 ml. added to 900ml. distilled water. Heparin, B.P.-5000 units per ml.
APPARATUS
Heparinised capillary tubes.-Thin-walled lymph tubes such as those used for vaccines, 90 mm. long and 1-5 mm. in internal diameter, are filled with the heparin solution, drained, and dried in of air. Burner.-For sealing the tubes, a small cylinder of butane gas attached to a microburner has been found very satisfactory. It is a current
easily portable. Small centrifuge.-A clinical centrifuge that will take 10 ml. centrifuge tubes packed with the lumph tubes. Capillary tube rack.-Before they are sealed, the filled capillary tubes must be kept horizontal; and after sealing they must be held vertically, or on a slope, so that the blood does not run up into the hot end and become ha:molysed. The rack in fig. 1 serves both purposes.
Centrifuge tubes, conical, 10 ml. Glass specimen tubes, 50 x 25 mm., with flat bottoms.
M.B., PH.D. Lond. Senior Lecturer
Water bath
RAPID DETERMINATION OF SOME PLASMA AMINOACIDS IN
SUBCLINICAL KWASHIORKOR ABNORMALITIES in the amounts of some aminoacids free plasma in advanced kwashiorkor have been reported from several countries.5-1o In general, the essential or in
are reduced in concentration whilst the non-essential or dispensable ones remain The branch-chain aminoacids (leucine, unaltered.
indispensable aminoacids 4. 5. 6. 7. 8. 9. 10.
Penfield, W., Roberts, L. Speech and Brain Mechanisms. Princeton, 1959. Westall, R. G., Roitman, E., de la Pena, C., Rasmussen, H., Cravioto, J., Gomez, F., Holt, L. E. Arch. Dis. Childh. 1958, 33, 499. Edozien, J. C., Phillips, E. J., Collis, W. R. F. Lancet, 1960, i, 615. Arroyave, G., Wilson, D., de Funes, C., Behar, M. Amer. J. clin. Nutr. 1962, 11, 517. Vis, H. L. in Aspects et mécanismes des hyperamino-aciduries de l’enfance; p. 89. Bruxelles, 1963. Snyderman, S. E, Holt, L. E., Norton, P. M., Roitman, E., Finch, J. Amer. J. clin. Nutr. 1963, 12, 333. Holt, L. E., Snyderman, S. E., Norton, P., Roitman, E., Finch, J. Lancet 1963, ii, 1343.
or
boiler with
a
metal
cover.
20 gives the best separation, also be used. A convenient size is 31.5x
Chromatography paper.-Whatman no. but Whatman 33-5 cm. or
no.
1
can
Chromatography apparatus.-Any container designed for ascending descending chromatography may be employed. We have mostly
used
a
13 in. Universal Sheet Chromatotank but the 8 in. Frame
Chromatotank, with the squares of paper specially supplied already perforated, has been found adequate. Good results were also obtained with descending chromatography in a 50 cm. Chromatojar. A dip tray (shallow and gently concave) enables papers to be passed through a small amount of solution. All this chromatography apparatus is made by the Shandon Scientific Co. Ltd., 65, Pound Lane, London, N.W.10. METHOD
Collection The finger, after being cleaned with 80% ethanol, is pricked, and 100-200 1. blood is collected in two of the heparinised tubes. The tubes are sealed at one end and after cooling in the rack (fig. 1) are centrifuged. If the plasma cannot be analysed 11. Whitehead, R. G., Dean, R. F. A. Amer. J. clin. Nutr. (in the press).
251 chain aminoacids (leucine plus isoleucine, and valine) are easily identified as the two major spots in the upper half of the chromatogram. The valine spot contains a small quantity of methionine. The spot used for a representative sample of the dispensable aminoacids is less well separated from its neighbours but is easily identified because it is the largest spot on the chromatogram. Also, its colour is distinctive; it is red-purple, with a deep purple fringe. The spot contains glycine, serine, glutamine and taurine. The chromatogram is then dipped in the alcoholic copper nitrate solution, after which it is air-dried. The colours turn from purple to salmon-red. In kwashiorkor an abnormal ninhydrin-positive compound sometimes travels on the chromatogram to a position just below the valine-methionine spot. The method is not affected, since after the dip in the copper-nitrate solution the spot does not change to salmonred but remains blue-purple. It cannot therefore be mistaken for the valine-methionine spot. The compound is being
investigated.
Fig. I-In the foreground is the wooden rack that holds the capillary tubes horizontally before sealing, and vertically afterwards. Behind, solutions of aminoacids in ethanol are being evaporated in the flat-bottom specimen tubes on the water-bath, in a current of air from the fan.
.
immediately the tubes are sealed at the open end and stored upright at 5°C. In specimens so stored, the ratio of the groups of aminoacids does not change for at least 14 days. The specimens can be preserved for several months if the tubes are snapped at the cell-plasma boundary and the part containing the plasma is kept in a deep=freeze cabinet. Repeated estimations of deep-frozen serum have shown no alteration in periods of up
to
6 months,
Analysis The plasma separated from the cells is blown out of the capillary tube into 4 ml. of the 90% ethanol in a centrifuge tube. The mixture is vigorously stirred with a glass rod and then allowed to stand for 10 minutes. The protein precipitate is separated by centrifugation. The clear supernatant is poured off into a flat-bottomed specimen tube and evaporated to dryness over a water bath (fig. 1). If air from an electric fan is blown over the tubes the evaporation is complete within 30 minutes, although the temperature does not rise above 60°C. The residue is then dissolved in 200 pd. 10% isopropanol. The residue is fatty and the last remnants must be scraped off with a glass rod. The extract is applied to the chromatography in amounts of about 25 nl. as a streak 2 cm. Ions. A shorter streak will not give good results. An exact quantitative transfer is not necessary since the method estimates the ratio of the groups of aminoacids, not the
paDer
absolute concentrations. The chromatogram is run in the solvent overnight. The temperature need not be closely controlled and a variation of 10° (from 20° to 30°C) in room temperature has no adverse effects. A satisfactory separation of the am-inoacids is achieved when the solvent front has risen about 20 cm. The papers are removed from the tank and dried in air with the help of the electric fan. When they no longer smell of acetic acid, they are dipped through the ninhydrin solution, drained, and air-dried for 5 minutes, then placed for 5 minutes in an oven at 110°C. It is important to ensure that the temperature does not fall below 90’C: at a lower temperature, or if the period of heating is too short, colour development is incomplete. After the chromatograms are removed from the oven they are allowed to stand at room temperature for5 minutes. In this period the colour of the spots changes from red-purple to blue-purple. The compounds selected for analysis are now ringed with pencil as shown on the left in fig. 2. The branch-
The spots containing leucine and isoleucine (Rf 0-67: see fig. 2) and valine and methionine (Rf 0-51) are cut out and cut into small strips and placed in a suitable boiling tube. The glycine-containing spot (Rf 0-13) is similarly treated. To each tube is added 4 ml. methanol and the salmon-red colour is eluted for an hour with occasional gentle shaking. The extinction of the coloured methanol is measured at 509 m[J. in a spectrophotometer such as the Unicam sp.500, using methanol as the blank. The imbalance ratio is derived from the extinction of the two solutions, that for the glycme-containing solution being the numerator. For the two examples shown in fig. 2, the calculations were: before-treatment sample-0-36/0-08; ratio= 4-5. After-treatment sample-0-45/0-30; ratio =1-5. (The. extinctions, obtained in a Unicam spectrophotometer, were rounded off to the nearest 0-01.) The colour is stable for at least 4 hours in daylight. An expensive spectrophotometer is not essential. When the colour values of thirteen samples were read in a Unicam spectrophotometer and again in an EEL colorimeter, a difference of ratio of more than 0-2 was found in only one case. With experience, the ratio can be judged with fair accuracy by inspection of the chromatograms. The accuracy could probably be enhanced by the use of a simple comparator consisting of a set of standard colours covering the usual range of concentrations. The variation of the ratios obtained from aliquots of a single blood sample, each taken through the whole procedure, is less than 5%, largely because of the simplified method of calculation. The variation between plasma from blood taken bv
Fig. 2-Left: aminoacids separated from plasma of a child before and after treatment for kwashiorkor. Right: Schematic representation of the aminoacid spots. Those used for the calculation of the ratio are shaded. The others are PH phenylalanine, TY tyrosine, PR proline, AL alanine, GL A glutamic acid, and TH threonine, L etc. Lysine, cystine, cysteine, &c.
252
finger-prick, and serum from blood taken almost simultaneously by venepuncture, is of the
same
order.
USE OF THE METHOD
In Uganda, the ratio for healthy European children and African children successfully treated for kwashiorkor with a milk-protein diet is less than 2-0, with an average of 1-5. Children living on a diet of the type known to produce kwashiorkor, but not showing the clinical signs typical of the disease in more than the slightest degree, have values of 2-0 or more, and, in general, the size of the ratio is related to the clinical assessment of the degree of malnutrition in the child. Children with ratios above 3-5 are classed as cases of potential kwashiorkor. Before treatment, children with obvious signs nearly always have ratios in the range from 4-0 to 10-0. In this range, the correlation with clinical condition is not exact and the highest ratios (15-0 and 21-0 have been found) were not obtained from the children who were the most severely ill. The method is very easy and a single African technician can estimate 25-35 samples a day. The cost for all materials (in Kampala) for 35 estimations (each in duplicate) is about 10 English shillings (140 U.S. cents): the most expensive items are the lymph tubes and the heparin, which together account for .two-thirds of the total cost. Ninhydrin is expensive but the amounts used very small. The usefulness of the method is not confined to the diagnosis of potential kwashiorkor. For instance, it offers a rapid screening test for the analysis of the serum aminoacids in disease, and to complement information obtained from chromatograms of the urine; and it might be used to investigate many conditions in which protein synthesis is disturbed, such as virus infections, vaccination, and inoculation. We are using it in an attempt to compare the efficacy of various therapeutic diets. Reports of the use of the method will be welcomed.
are
Medical Research Council Infantile Malnutrition Research Unit, Mulago Hospital, P.O. Box 351, Kampala, Uganda, E. Africa
R. G. WHITEHEAD PH.D.
Leeds
CULTIVATION IN VITRO OF HUMAN LYMPHOBLASTS FROM BURKITT’S MALIGNANT LYMPHOMA SINCE Burkittfirst recognised that various unusual sarcomas of children in Africa represented different manifestations of a single bizarre multifocal tumour syndrome, the condition has been classified as a malignant lymphomacomposer of poorly differentiated lymphoid cells and varying numbers of non-malignant histiocytes.34 The syndrome is common, and is of special significance because of its peculiar geographical distribution 1and the factors which seem to determine this. 61 A comprehensive review of the subject and its implications has
recently appeared.8 Laboratory investigations into Burkitt’s lymphoma have included attempts to grow the tumour cells in vitro, using several culture methods on biopsies flown to London from Uganda; this communication reports the successful cultivation of such cells from one of the 26 samples provided so far. 1. 2. 3. 4. 5. 6. 7. 8.
Burkitt, D. Brit.J. Surg. 1958, 46, 218. O’Conor, G. T. Cancer, 1961, 14, 270. Wright, D. H. Brit. J. Cancer, 1963, 17, 50. Clift, R. A., Wright, D. H., Clifford, P. Blood, 1963, 22, 243. Burkitt, D., O’Conor, G T. Cancer, 1961, 14, 258. Burkitt, D. Brit. med. J. 1962, ii, 1019. Burkitt, D. Nature, Lond. 1962, 194, 232. Burkitt, D. in International Review of Experimental Pathology; vol. n, p. 67 (edited by G. W. Richter and M. A. Epstein). New York and London, 1963.
Fig. 1-Leishman-stained smear of cultured lymphoma cells growing individually or as doublets; the lower right-hand doublet contains a prophase mitosis. Many cell " ghosts " can be seen. Photomicrograph x 187. Fig. 2-Detail of fig. 1 showing a cell in mitosis on the right, promi. nent nucleoli in the left-hand cell, and small clear cytoplasmic inclusions in both. Photomicrograph x 1750. METHODS
Biopsy.-Biopsy material was taken from a child with a typical maxillary lymphoma, and 1 mm. cube fragments were placed in a bijou bottle with 2 ml. of 50% guineapig serum in Hanks B.s.s., to protect the cells in transit. The specimen travelled at aircraft-cabin temperature and reached the laboratory in London within 24 hours of operation. Preparation of cultures.-On 2rrival, the suspending fluid was turbid and contained swarms of single round cells shaken free from the biopsy fragments. After settling out, the cells and fragments were distributed into six hard glass 12 ml. insulin bottles (Hodges & Co., London, E.C.1), and 2 ml. of Eagle’s Basal medium 9 with 10% human serum was added to each. Maintenance of cultures.-Incubation has been at 37’C. Bottles were at first kept upright on their flat bottoms; so that the cells settled on a small surface as close together as possible, but were later rolled at 5° to the horizontal at 12 r.p.h. When stationary, the cells were fed by removing 0’5 ml. of medium from near the surface and replacing with fresh; for roller cultures the cells were allowed to settle first. The cultures were divided by doubling their volume with fresh medium, suspending the cells evenly in the increased fluid, and then transferring half of this to a new bottle. OBSERVATIONS AND DISCUSSION
After 16
days’ stationary incubation, a pH fall occurred bottle and a wet film of the culture fluid showed single sparse, uniform, clear round cells between dead cells and debris. The culture was fed and reincubated as before. Turbidity and a further pH drop developed by the 26th day and the culture was divided. Feeding and division has been continued whenever indicated by pH change and turbidity. Between the 42nd and 44th days of culture, bottles were transferred individually to the roller apparatus.
in
a
9. W.H.O. Scientific Group on Human Director General, 1962, p. 32.
Diploid Cells: Report
to
the
isoleucine, and valine) and methionine are particularly affected, but glycine, serine, glutamine, and taurine remain within the normal range. The screening test
TABLE II-EFFECTS ON CONSCIOUSNESS
could be attracted in a sustained way through questioning by the examiner. The aphasic, but otherwise conscious patient could, in our experience, readily be distinguished from the patient who had lost consciousness. The injection on the side showing dominance for speech resulted, in most patients, in loss of consciousness, while injection on the side not dominant for speech led to loss of consciousness far less often. When loss of consciousness did follow injection of the non-dominant side its duration was shorter than that after injection of the other side; and, further, in no case did unconsciousness follow injection of the minor hemisphere alone. Also, those patients with apparent bilateral representation of speech showed no difference between the two sides in terms of consciousness, whether injection of both sides or neither side resulted in aphasia. CONCLUSION
The
finding that consciousness tends to be lost when the speech-dominant hemisphere rather than the non-dominant is injected with amylobarbitone has not, as far as we are aware, been reported before. It has been suggested that there is a close interaction in the function of speech between the relevant parts of the dominant hemisphere and the upper brain-stem.4 It seems possible that the speech-dominant hemisphere may, by virtue of this interaction, also acquire a dominant role in the matter of consciousness, which, in man, is heavily dependent on symbolic processes. We are grateful for the help given in this investigation by Mr. M. A. Falconer and the staff of the Guy’s-Maudsley Neurosurgical Unit. One of us (E.A.S.) was in receipt of a grant from Guy’s Hospital Medical School.
E. A. SERAFETINIDES M.D. Athens., D.P.M. Research Fellow
The Guy’s-Maudsley Neurosurgical Unit and the Department of Experimental Neurology (Institute of The
Psychiatry), Maudsley Hospital, London, S.E.5
R. D. HOARE M.B. Lond., D.M.R.D. Consultant
Radiologist
M. V. DRIVER
described in this paper is based on the estimation of these aminoacids in two groups, and the calculation of an " imbalance " ratio. Aminoacids are imbalanced not only in severe cases of kwashiorkor but also in children who, although living on a poor diet, show few or none of the clinical signs, except a small size for age; the imbalance is therefore the earliest biochemical abnormality so far discovered. Full details of the correlations of the ratio with the signs are being published elsewhere," but a preliminary communication to the International Congress of Nutrition in Edinburgh last August has resulted in requests for details of the method for use as a potential diagnostic test. In the developing countries where kwashiorkor is most prevalent, skilled biochemical assistance is not usually available, and the procedure is here described in full for those who may not be familiar with routine quantitative paper
chromatography. MATERIALS
ml. of demineralised water added
to 900 Ethanol, 90% v/v.-100 ml. absolute alcohol. Chromatography solvent.-120 ml. n-butanol, 30 ml. glacial acetic acid, and 50 ml. water. In tropical countries the solvent should be prepared afresh for each batch of estimations, and used once only. Ninhydrin, 0-2% w/v in acetone.-l g. ninhydrin (indane-l:2:3trione hydrate) in 500 ml. acetone. About 50 ml. is needed for each batch. It should be used once only: if it is used repeatedly, a strong background colour will develop. Ethanolic copper nitrate.-l ml. saturated copper nitrate in 100 ml, absolute ethanol plus 0-2 ml. 10% v/v nitric acid. Methanol (reagent grade). Isopropanol (isopropyl alcohol), 10% v/v.-100 ml. added to 900ml. distilled water. Heparin, B.P.-5000 units per ml.
APPARATUS
Heparinised capillary tubes.-Thin-walled lymph tubes such as those used for vaccines, 90 mm. long and 1-5 mm. in internal diameter, are filled with the heparin solution, drained, and dried in of air. Burner.-For sealing the tubes, a small cylinder of butane gas attached to a microburner has been found very satisfactory. It is a current
easily portable. Small centrifuge.-A clinical centrifuge that will take 10 ml. centrifuge tubes packed with the lumph tubes. Capillary tube rack.-Before they are sealed, the filled capillary tubes must be kept horizontal; and after sealing they must be held vertically, or on a slope, so that the blood does not run up into the hot end and become ha:molysed. The rack in fig. 1 serves both purposes.
Centrifuge tubes, conical, 10 ml. Glass specimen tubes, 50 x 25 mm., with flat bottoms.
M.B., PH.D. Lond. Senior Lecturer
Water bath
RAPID DETERMINATION OF SOME PLASMA AMINOACIDS IN
SUBCLINICAL KWASHIORKOR ABNORMALITIES in the amounts of some aminoacids free plasma in advanced kwashiorkor have been reported from several countries.5-1o In general, the essential or in
are reduced in concentration whilst the non-essential or dispensable ones remain The branch-chain aminoacids (leucine, unaltered.
indispensable aminoacids 4. 5. 6. 7. 8. 9. 10.
Penfield, W., Roberts, L. Speech and Brain Mechanisms. Princeton, 1959. Westall, R. G., Roitman, E., de la Pena, C., Rasmussen, H., Cravioto, J., Gomez, F., Holt, L. E. Arch. Dis. Childh. 1958, 33, 499. Edozien, J. C., Phillips, E. J., Collis, W. R. F. Lancet, 1960, i, 615. Arroyave, G., Wilson, D., de Funes, C., Behar, M. Amer. J. clin. Nutr. 1962, 11, 517. Vis, H. L. in Aspects et mécanismes des hyperamino-aciduries de l’enfance; p. 89. Bruxelles, 1963. Snyderman, S. E, Holt, L. E., Norton, P. M., Roitman, E., Finch, J. Amer. J. clin. Nutr. 1963, 12, 333. Holt, L. E., Snyderman, S. E., Norton, P., Roitman, E., Finch, J. Lancet 1963, ii, 1343.
or
boiler with
a
metal
cover.
20 gives the best separation, also be used. A convenient size is 31.5x
Chromatography paper.-Whatman no. but Whatman 33-5 cm. or
no.
1
can
Chromatography apparatus.-Any container designed for ascending descending chromatography may be employed. We have mostly
used
a
13 in. Universal Sheet Chromatotank but the 8 in. Frame
Chromatotank, with the squares of paper specially supplied already perforated, has been found adequate. Good results were also obtained with descending chromatography in a 50 cm. Chromatojar. A dip tray (shallow and gently concave) enables papers to be passed through a small amount of solution. All this chromatography apparatus is made by the Shandon Scientific Co. Ltd., 65, Pound Lane, London, N.W.10. METHOD
Collection The finger, after being cleaned with 80% ethanol, is pricked, and 100-200 1. blood is collected in two of the heparinised tubes. The tubes are sealed at one end and after cooling in the rack (fig. 1) are centrifuged. If the plasma cannot be analysed 11. Whitehead, R. G., Dean, R. F. A. Amer. J. clin. Nutr. (in the press).
251 chain aminoacids (leucine plus isoleucine, and valine) are easily identified as the two major spots in the upper half of the chromatogram. The valine spot contains a small quantity of methionine. The spot used for a representative sample of the dispensable aminoacids is less well separated from its neighbours but is easily identified because it is the largest spot on the chromatogram. Also, its colour is distinctive; it is red-purple, with a deep purple fringe. The spot contains glycine, serine, glutamine and taurine. The chromatogram is then dipped in the alcoholic copper nitrate solution, after which it is air-dried. The colours turn from purple to salmon-red. In kwashiorkor an abnormal ninhydrin-positive compound sometimes travels on the chromatogram to a position just below the valine-methionine spot. The method is not affected, since after the dip in the copper-nitrate solution the spot does not change to salmonred but remains blue-purple. It cannot therefore be mistaken for the valine-methionine spot. The compound is being
investigated.
Fig. I-In the foreground is the wooden rack that holds the capillary tubes horizontally before sealing, and vertically afterwards. Behind, solutions of aminoacids in ethanol are being evaporated in the flat-bottom specimen tubes on the water-bath, in a current of air from the fan.
.
immediately the tubes are sealed at the open end and stored upright at 5°C. In specimens so stored, the ratio of the groups of aminoacids does not change for at least 14 days. The specimens can be preserved for several months if the tubes are snapped at the cell-plasma boundary and the part containing the plasma is kept in a deep=freeze cabinet. Repeated estimations of deep-frozen serum have shown no alteration in periods of up
to
6 months,
Analysis The plasma separated from the cells is blown out of the capillary tube into 4 ml. of the 90% ethanol in a centrifuge tube. The mixture is vigorously stirred with a glass rod and then allowed to stand for 10 minutes. The protein precipitate is separated by centrifugation. The clear supernatant is poured off into a flat-bottomed specimen tube and evaporated to dryness over a water bath (fig. 1). If air from an electric fan is blown over the tubes the evaporation is complete within 30 minutes, although the temperature does not rise above 60°C. The residue is then dissolved in 200 pd. 10% isopropanol. The residue is fatty and the last remnants must be scraped off with a glass rod. The extract is applied to the chromatography in amounts of about 25 nl. as a streak 2 cm. Ions. A shorter streak will not give good results. An exact quantitative transfer is not necessary since the method estimates the ratio of the groups of aminoacids, not the
paDer
absolute concentrations. The chromatogram is run in the solvent overnight. The temperature need not be closely controlled and a variation of 10° (from 20° to 30°C) in room temperature has no adverse effects. A satisfactory separation of the am-inoacids is achieved when the solvent front has risen about 20 cm. The papers are removed from the tank and dried in air with the help of the electric fan. When they no longer smell of acetic acid, they are dipped through the ninhydrin solution, drained, and air-dried for 5 minutes, then placed for 5 minutes in an oven at 110°C. It is important to ensure that the temperature does not fall below 90’C: at a lower temperature, or if the period of heating is too short, colour development is incomplete. After the chromatograms are removed from the oven they are allowed to stand at room temperature for5 minutes. In this period the colour of the spots changes from red-purple to blue-purple. The compounds selected for analysis are now ringed with pencil as shown on the left in fig. 2. The branch-
The spots containing leucine and isoleucine (Rf 0-67: see fig. 2) and valine and methionine (Rf 0-51) are cut out and cut into small strips and placed in a suitable boiling tube. The glycine-containing spot (Rf 0-13) is similarly treated. To each tube is added 4 ml. methanol and the salmon-red colour is eluted for an hour with occasional gentle shaking. The extinction of the coloured methanol is measured at 509 m[J. in a spectrophotometer such as the Unicam sp.500, using methanol as the blank. The imbalance ratio is derived from the extinction of the two solutions, that for the glycme-containing solution being the numerator. For the two examples shown in fig. 2, the calculations were: before-treatment sample-0-36/0-08; ratio= 4-5. After-treatment sample-0-45/0-30; ratio =1-5. (The. extinctions, obtained in a Unicam spectrophotometer, were rounded off to the nearest 0-01.) The colour is stable for at least 4 hours in daylight. An expensive spectrophotometer is not essential. When the colour values of thirteen samples were read in a Unicam spectrophotometer and again in an EEL colorimeter, a difference of ratio of more than 0-2 was found in only one case. With experience, the ratio can be judged with fair accuracy by inspection of the chromatograms. The accuracy could probably be enhanced by the use of a simple comparator consisting of a set of standard colours covering the usual range of concentrations. The variation of the ratios obtained from aliquots of a single blood sample, each taken through the whole procedure, is less than 5%, largely because of the simplified method of calculation. The variation between plasma from blood taken bv
Fig. 2-Left: aminoacids separated from plasma of a child before and after treatment for kwashiorkor. Right: Schematic representation of the aminoacid spots. Those used for the calculation of the ratio are shaded. The others are PH phenylalanine, TY tyrosine, PR proline, AL alanine, GL A glutamic acid, and TH threonine, L etc. Lysine, cystine, cysteine, &c.
252
finger-prick, and serum from blood taken almost simultaneously by venepuncture, is of the
same
order.
USE OF THE METHOD
In Uganda, the ratio for healthy European children and African children successfully treated for kwashiorkor with a milk-protein diet is less than 2-0, with an average of 1-5. Children living on a diet of the type known to produce kwashiorkor, but not showing the clinical signs typical of the disease in more than the slightest degree, have values of 2-0 or more, and, in general, the size of the ratio is related to the clinical assessment of the degree of malnutrition in the child. Children with ratios above 3-5 are classed as cases of potential kwashiorkor. Before treatment, children with obvious signs nearly always have ratios in the range from 4-0 to 10-0. In this range, the correlation with clinical condition is not exact and the highest ratios (15-0 and 21-0 have been found) were not obtained from the children who were the most severely ill. The method is very easy and a single African technician can estimate 25-35 samples a day. The cost for all materials (in Kampala) for 35 estimations (each in duplicate) is about 10 English shillings (140 U.S. cents): the most expensive items are the lymph tubes and the heparin, which together account for .two-thirds of the total cost. Ninhydrin is expensive but the amounts used very small. The usefulness of the method is not confined to the diagnosis of potential kwashiorkor. For instance, it offers a rapid screening test for the analysis of the serum aminoacids in disease, and to complement information obtained from chromatograms of the urine; and it might be used to investigate many conditions in which protein synthesis is disturbed, such as virus infections, vaccination, and inoculation. We are using it in an attempt to compare the efficacy of various therapeutic diets. Reports of the use of the method will be welcomed.
are
Medical Research Council Infantile Malnutrition Research Unit, Mulago Hospital, P.O. Box 351, Kampala, Uganda, E. Africa
R. G. WHITEHEAD PH.D.
Leeds
CULTIVATION IN VITRO OF HUMAN LYMPHOBLASTS FROM BURKITT’S MALIGNANT LYMPHOMA SINCE Burkittfirst recognised that various unusual sarcomas of children in Africa represented different manifestations of a single bizarre multifocal tumour syndrome, the condition has been classified as a malignant lymphomacomposer of poorly differentiated lymphoid cells and varying numbers of non-malignant histiocytes.34 The syndrome is common, and is of special significance because of its peculiar geographical distribution 1and the factors which seem to determine this. 61 A comprehensive review of the subject and its implications has
recently appeared.8 Laboratory investigations into Burkitt’s lymphoma have included attempts to grow the tumour cells in vitro, using several culture methods on biopsies flown to London from Uganda; this communication reports the successful cultivation of such cells from one of the 26 samples provided so far. 1. 2. 3. 4. 5. 6. 7. 8.
Burkitt, D. Brit.J. Surg. 1958, 46, 218. O’Conor, G. T. Cancer, 1961, 14, 270. Wright, D. H. Brit. J. Cancer, 1963, 17, 50. Clift, R. A., Wright, D. H., Clifford, P. Blood, 1963, 22, 243. Burkitt, D., O’Conor, G T. Cancer, 1961, 14, 258. Burkitt, D. Brit. med. J. 1962, ii, 1019. Burkitt, D. Nature, Lond. 1962, 194, 232. Burkitt, D. in International Review of Experimental Pathology; vol. n, p. 67 (edited by G. W. Richter and M. A. Epstein). New York and London, 1963.
Fig. 1-Leishman-stained smear of cultured lymphoma cells growing individually or as doublets; the lower right-hand doublet contains a prophase mitosis. Many cell " ghosts " can be seen. Photomicrograph x 187. Fig. 2-Detail of fig. 1 showing a cell in mitosis on the right, promi. nent nucleoli in the left-hand cell, and small clear cytoplasmic inclusions in both. Photomicrograph x 1750. METHODS
Biopsy.-Biopsy material was taken from a child with a typical maxillary lymphoma, and 1 mm. cube fragments were placed in a bijou bottle with 2 ml. of 50% guineapig serum in Hanks B.s.s., to protect the cells in transit. The specimen travelled at aircraft-cabin temperature and reached the laboratory in London within 24 hours of operation. Preparation of cultures.-On 2rrival, the suspending fluid was turbid and contained swarms of single round cells shaken free from the biopsy fragments. After settling out, the cells and fragments were distributed into six hard glass 12 ml. insulin bottles (Hodges & Co., London, E.C.1), and 2 ml. of Eagle’s Basal medium 9 with 10% human serum was added to each. Maintenance of cultures.-Incubation has been at 37’C. Bottles were at first kept upright on their flat bottoms; so that the cells settled on a small surface as close together as possible, but were later rolled at 5° to the horizontal at 12 r.p.h. When stationary, the cells were fed by removing 0’5 ml. of medium from near the surface and replacing with fresh; for roller cultures the cells were allowed to settle first. The cultures were divided by doubling their volume with fresh medium, suspending the cells evenly in the increased fluid, and then transferring half of this to a new bottle. OBSERVATIONS AND DISCUSSION
After 16
days’ stationary incubation, a pH fall occurred bottle and a wet film of the culture fluid showed single sparse, uniform, clear round cells between dead cells and debris. The culture was fed and reincubated as before. Turbidity and a further pH drop developed by the 26th day and the culture was divided. Feeding and division has been continued whenever indicated by pH change and turbidity. Between the 42nd and 44th days of culture, bottles were transferred individually to the roller apparatus.
in
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9. W.H.O. Scientific Group on Human Director General, 1962, p. 32.
Diploid Cells: Report
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