labelled in this way have been used for the determination of the total circulating

Transcription

1 382 J. Physiol. (1251) II4, A CRITICAL ASSESSMENT OF THE USE OF BLOOD CELLS LABELLED WITH RADIOACTIVE PHOSPHORUS FOR THE QUANTITATIVE STUDY OF SKIN BLOOD CONTENT BY R. W. E. WATTS From the Department of Physiology, St Bartholomew's Hospital Medical College, London (Received 4 January 1951) No completely satisfactory technique is at present available for the comparison ofthe blood content ofindividual tissues under different physiological conditions. Blood cells which have been labelled with radioactive phosphorus (32p) can readily be detected after they have been injected into the circulation. Cells labelled in this way have been used for the determination of the total circulating red blood cell volume by an isotope dilution technique (Hevesy, 1948; Nieset, Porter, Trautman, Bell, Parson, Lyons & Mayerson, 1948). The experimental work described below was undertaken to see if the radioactivity of the skin which results from the presence of such labelled corpuscles can be used to measure the skin blood content. When this work was undertaken it was hoped that it might prove practicable to apply the method to the study of the distribution of blood in other tissues including tumours. Reiss, Badrick, Halkerston & White (1949) described a method for the continuous graphic recording of the concentration of radioactive materials in superficial structures. An attempt was made to apply the technique described by these workers to the detection of changes in the distribution of 32P-labelled cells in the skin of anaesthetized rats. The window of an end-window Geiger- Muller counter was screened to a diameter of 1 0 cm. The screened counter was placed in contact with the animal's skin and the count rate recorded. The intravenous injection of as much as 50 ug. adrenaline, to produce intense cutaneous vaso-constriction, did not alter the count rate significantly. It was concluded, therefore, that the count rate recorded under these circumstances was mainly conditioned by the amount of radioactive material in the tissues deep to the skin, and that this technique was not suitable for the measurement of skin blood content. The technique finally adopted for the determination of skin blood content depended on the measurement of the radioactivity of the phosphorus present

2 RADIO ACTIVE PHOSPHORUS AND SKIN BLOOD CONTENT 383 in samples of skin and separated from the acid hydrolysed skin as magnesium ammonium phosphate. The skin blood content before and after the injection of adrenaline was compared in fourteen animals. METHODS Preparation of cells for injection Freshly drawn heparinized blood was used. The corpuscles were separated from the plasma and were washed twice with an equal volume of 0-9% NaCl. Approximately 200 1Lc. carrier-free inorganically combined radioactive phosphorus were dissolved in 0-03 ml. of 0-75 M-NaHCO3 saturated with CO2 and the solution diluted to 0-6 ml. with distilled water. This buffered solution is isotonic with mammalian blood corpuscles and has a ph of approximately 7-5. The ph of the solution was checked immediately before use and a final adjustment to ph made by adding one or two drops of u-hci. 1-0 ml. of washed packed blood cells was added to 0-6 ml. of the isotonic radioactive phosphorus solution in a clean dry centrifuge tube. The centrifuge tube was attached to the stem of an electric stirrer and rotated as slowly as possible in a water-bath at 370 C. for 2 hr. The labelled cells were then separated by centrifuging, and were washed twice with an equal volume of non-radioactive rat plasma. Eaeh washing lasted 5 min. Removal of skin specimens The rat ( g. in weight) was anaesthetized with pentobarbitone ('nembutal'), and 0-1 ml. freshly labelled cells was injected intravenously. After 5 min., an area of skin about i in. square was removed from the anterior aspect of the left side of the animal's thorax. The skin specimen was divided into two similar-sized portions which were transferred to separate weighed 'Pyrex' boiling tubes. Dissecting instruments and operating gloves were changed at this stage of the experiment. Adrenaline (50 jg.) was injected intravenously, and a similar area of skin removed from the right side of the anterior thoracic wall during the period of apnoea following the injection of adrenaline. This specimen was divided into two approximately equal portions, and each was transferred to a weighed 'Pyrex' boiling tube. No attempt was made to depilate the skin either before or after its removal. Major blood vessels were avoided during the dissection. The specimens were carefully inspected to ensure that they were free from attached subcutaneous adipose tissue and that they were not obviously contaminated with extraneous blood. A strip of skin was left attached to the subcutaneous tissue between the two denuded areas in order to diminish the risk of the second skin specimen becoming contaminated by radioactive blood or tissue fluid liberated from the site of the first dissection. Chemical manipulations The specimens of skin were weighed, dried in an air oven at C. for 24 hr., cooled in a vacuum desiccator, reweighed, and heated with a mixture of conc. HNO3, copper wire and 10% NaClI3 solution as described by Boursnell, Francis & Wormall (1946). The residue from this procedure was dissolved in 25-0 ml. N-HCI, and the solution was boiled gently and cooled. Saturated NaH2PO4 (0-3 ml.) was added to provide 'carrier' phosphate. 'Magnesia mixture' (25-0 ml.) (Vogel, 1948) was then added, and magnesium ammonium phosphate precipitated by the addition of excess concentrated ammonia (sp.gr ) as described by Vogel (1948). The precipitate was allowed to settle overnight, filtered and washed with 5% ammonia solution until the filtrate was no longer blue, i.e. until free from the cuprammonium compound formed during the final stage of the precipitation. The precipitate was dissolved in N-HCI and the solution made up to exactly 15 ml.

3 384 R. W. E. WATTS Radioactivity measurements The activity of 10 0 ml. of the final solution was measured using a liquid counter (Veall, 1948). The number of counts recorded in each determination was sufficient to give a probable error of ±2-5% (Hevesy, 1948). 'Recovery' experiment Accurately measured volumes of a solution of radioactive phosphorus were added to specimens of skin similar to those used in the injection experiments. The specimens were dried and the phosphorus was extracted, precipitated and 'counted' as described above. 100% recovery was obtained in each experiment. RESULTS The values obtained for the radioactivity of the specimens of skin are shown in Table 1. TABLE 1. The radioactivity of samples of rat skin after the intravenous injection of blood cells labelled with 32p Radioactivity (counts/min./g. dry skin) Exp. (a)* (b)* Mean 1 Before adrenaline Before adrenaline Before adrenaline Before adrenaline Before adrenaline Before adrenaline Before adrenaline Before adrenaline Before adrenaline Before adrenaline Before adrenaline Before adrenaline Before adrenaline Before adrenaline * (a) and (b) give values obtained with different portions of the same sample of skin. In each experiment the mean value for the 32p content of the skin is lower after the injection of adrenaline than before administration of the drug. The values for the duplicate determinations of radioactivity upon which each of the mean figures is based show considerable disagreement. Dr J. 0. Irwin, of the Medical Research Council's Statistical Research Unit, submitted these results

4 RADIO ACTIVE PHOSPHORUS AND SKIN BLOOD CONTENT 385 to a statistical analysis and found that the difference between the mean values for radioactivity before and after the injection of adrenaline was significant at the level of 01 %. The discrepancies between pairs of duplicate determinations were shown to be equivalent to a coefficient of variation of 'counts' under comparable conditions of about 25%. DISCUSSION It is necessary to seek an explanation for the discrepancies between pairs of duplicate determinations which were found in these experiments. Previous workers, quoted by Hevesy (1948), have shown that the 32p which migrates into blood corpuscles during labelling in vitro is sufficiently firmly bound foi there to be no appreciable 10s8 of 32p from the cells during the first half hour following their re-introduction into the circulation of a living animal of the same species. Nieset et al. (1948) reported a variable loss of 32p not exceeding 5% during the same period of time. The results of experiments performed by the present writer have confirmed these findings. All the skin specimens were removed between 5 and 10 min. after the injection of labelled cells. It would appear, therefore, that the large discrepancies observed cannot be ascribed to 32p becoming disseminated throughout the animal's tissues during the experiment. Hevesy, Koster, Sorensen, Warburg & Zerahn (1944) and Nylin & Hedlund (1947) have shown that intravenously injected blood cells are uniformly distributed among the recipient's corpuscles 3 min. after injection. Hence, in the experiments described, the injected cells must have been uniformly distributed in the animal's blood stream by the time the first specimen of skin was removed. A different animal was used for each experiment, and although the skin specimens were removed from the same anatomical region in each case, it is possible that the minutiae of the capillary blood supply to a given region differ from animal to animal, and between adjacent areas of skin in the same animal. The dissection technique described under 'methods' was evolved in order to avoid the transfer of extraneous radioactive blood to the skin specimens. The weight of highly radioactive material necessary to affect the final 'count' in these experiments significantly would be minute, and it is considered that invisible contamination of the skin specimens with small numbers of radioactive blood cells was responsible for the discrepancies which occurred. Any further modification of the technique used for the removal of the skin specimens in order to make contamination with amounts of blood of this order impossible, would not be practicable. It is concluded that the technique described, although it might have a qualitative value, is not suitable for the quantitative comparison of skin blood content under different physiological conditions.

5 386 R. W. E. WATTS SUMMARY 1. The work described was undertaken in order to determine whether blood corpuscles labelled with 32p could be used as a means of comparing the blood content of the skin under different physiological conditions. 2. The mean value for the radioactivity (counts/min./g. dry weight) of the skin removed from rats previously injected with 32P-labelled blood cells was consistently lower after the injection of adrenaline than it was before the administration of the drug. 3. The radioactivity values obtained for different portions of the same sample of skin usually showed marked differences. Possible reasons for this finding are discussed, and it is concluded that these discrepancies seriously limit the accuracy of this method of measuring skin blood content. I am indebted to Prof. A. Wormall for making supplies of radioactive phosphorus available for this work and for his continued encouragement and advice. I am grateful to Prof. K. J. Franklin for his advice and criticism. My colleague Mr R. Tupper collaborated with me in some of the initial work, and I am indebted to Dr J. 0. Irwin for his statistical report on the results. The expenses of this work were defrayed by a departmental grant from the British Empire Cancer Campaign. REFERENCES Boursnell, J. C., Francis, G. E. & Wormall, A. (1946). Biochem. J. 40, 743. Hevesy, G. (1948). Radioactive Indicators. London: Interscience Publishers. Hevesy, G., Koster, K. J., Sorensen, G., Warburg, E. & Zerahn, K. (1944). Acta med. Scand. 116, 561. Nieset, R. T., Porter, B., Trautman, W. V., Bell, R. M., Parson, W., Lyons, C. & Mayerson, H. S. (1948). Amer. J. Physiol. 155, 226. Nylin, G. & Hedlund, S. (1947). Amer. Heart J. 33, 770. Reiss, M., Badrick, F. E., Halkerston, J. M. & White, J. H. (1949). Biochem. J. 44, 254. Veall, N. (1948). Brit. J. Radiol. 21, 347. Vogel, A. I. (1948). A Textbook of Quantitative Inorganic Analysis. London: Longmans, Green and Co.