January, 19581 PAUL 37 Determination of the Major Constituents of Small Amounts of Tissue (H.E.H. T . l‘isszre Czrltuve Laboratory, Department of Biochemistry, The Univevsity, Glasgow, W.2) BY JOHN PAUL A fractionation procedure that permits the determination of lipid, carbo- hydrate, protein nitrogen, ribonucleic acid and deoxyribonucleic acid in single samples of tissue of about 1 mg dry weight is described. Lipids are extracted with organic solvents and determined by oxidation with chromic acid. Acid-soluble substances are extracted with cold N sulphuric acid and carbo- hydrate is determined in this extract by the anthrone method. Nucleic acids are extracted, together with the remaining carbohydrate, with hot N perchloric acid. The carbohydrate is determined by the anthrone method and the nucleic acids are determined by ultra-violet absorption. Deoxy- ribonucleic acid is determined by reaction with indole and ribonucleic acid is found by difference.The method is straightforward and reliable. Protein nitrogen is determined in the residue, IN previous studies the fractionation procedure of Schmidt and Thannhauserl has been applied to small amounts of tissue, such as tissue cultures, for the determination of ribo- nucleic acid and deoxyribonucleic acid.2 Other components of tissue have been dete~mined,~~~ but this has usually involved the analysis of replicate specimens, owing to the very small amounts of material available in tissue culture studies, and the necessity to prepare the samples for analysis in different ways. The method to be described, which is applicable to a very wide variety of tissues, was evolved to overcome this difficulty.It is based on Schneider’s procedure5 for the determination of nucleic acids. EXPERIMENTAL Before fractionation is commenced, it is necessary to remove any medium or serum adhering to the tissue without removing cell constituents. It was found that this could be done by washing the tissue rapidly with a physiological salt solution while the cells were still alive. DETERMINATION OF LIPID- A modification of Bloor’s method6 for determining total lipid by the colorimetric deter- mination of the lipid-soluble material that can be oxidised by chromic acid was used, the intensity of the coloured solution being measured at 620 mp. It was found that prior extrac- tion of the tissue with the usual acids interfered with the reaction, since trichloroacetic acid appeared in the solvents and perchloric acid caused oxidation of the lipid material on evapora- tion of the solvents.By performing the extraction with fat solvents first, as in Ogur and Rosen’s procedure,’ these difficulties were avoided. An ethanol - ether mixture was used as the first solvent in order to minimise the risk of extracting protein. A carbon tetra- chloride-ether mixture was used as the second solvent, as the more usual chloroform interfered in the colorimetric reaction. Subsequent extraction of the lipid solvents with 1 per cent. sodium hydrogen carbonate solution gave slightly lower results for lipid material, presumably owing to the removal of small acid radicles. The mean of six determinations of lipid in embryonic chick liver by Bloor’s method without extraction with 1 per cent.sodium hydrogen carbonate was 685 pg, with a standard deviation of 15 pg, and the mean of six determinations after extraction with 1 per cent. sodium hydrogen carbonate was 642 pg, with a standard deviation of &15pg. DETERMINATION OF NUCLEIC ACIDS- The procedure of Ceriotti* was modified. Excellent results were obtained for some tissues by the original fractionation, but it was found that with others, especially embryonic chick tissues, a constant loss of ribonucleic acid occurred during treatment with cold per- chloric acid. It was observed that pure solutions of ribonucleic acid were much more stable to treatment with trichloroacetic acid or sulphuric acid than they were to perchloric acid and, when lipid extraction of the tissue was performed as the first stage, N sulphuric acid38 [Vol.83 could be used successfully to remove acid-soluble material. Thereafter, measurement of the ultra-violet absorption and determination of the deoxyribonucleic acid by the indole methods in a warm perchloric acid extract, as described by Ceriotti,8 gave results for ribo- nucleic acid and deoxyribonucleic acid in good agreement with those obtained by the Schmidt and Thannhauser procedure (see Table I). Trichloroacetic acid was not used for the extraction of the acid-soluble substances, since its ulta-violet absorption interfered with the subsequent determination of nucleic acids. PAUL : DETEKMINATION O F THE: MAJOR CONSITUENTS OF TABLE I COMPARISON OF AMOUNTS OF RIBONUCLEIC ACID AND DEOXYRIBONUCLEIC ACID DETERMINED BY THE SCHMIDT AND THANNHAUSER PROCEDURE AND BY THE Determinations were performed on at.least six aliquots of each tissue PROPOSED FRACTIONATION SCHEME Schmidt and Thannhauser rnethod A I 7 Deoxyribo- Ribonucleic nucleic phosphorus phosphorus Ratio of acid acid ( X I , CLg (Y), CLg -y to y Ehrlich mouse ascites 80.5 42.2 1.91 Mouse liver (ascitic) . . 67.7 15.85 4-26 Embryonic chick liver. . 62.4 22.3 2.80 tumour . . . . k3.03 2 0.354 - + 5.36 20.771 k 1.76 1.04 Proposed scheme Deoxyribo- r Ribonucleic nucleic phosphorus phosphorus Ratio of 82.5 43.9 1.88 64.9 17.6 3.69 62.4 23.15 2.69 A \ acid acid ( X ) , CLg ( Y ) , pg X t o y 2 1.36 k0.171 & 1.19 - -b 0.165 k 2.36 & 1.26 DETERMINATION OF CARBOHYDRATES- factory without modification.The anthrone method, as described by Trevelyan and Harri~on,~ was found to be satis- DETERMINATION OF PROTEIN NITROGEN- It was expected that protein nitrogen could be determined in the residue after the fractionation procedure had been completed, since, as shown by the following results, there was no loss of protein nitrogen at any stage during this procedure. The results for protein nitrogen in aliquots of horse serum determined in the residue after (a) extraction with lipid solvents, (b) extraction with lipid solvents and three extractions with cold N sulphuric acid and (c) extraction with lipid solvents, three extractions with cold N sulphuric acid and two extractions of 30 minutes each with N perchloric acid at 70" C were 1200 22 pg, 1236 However, in some tissues a considerable deficit was found between the protein nitrogen determined in this way and the total nitrogen that could be precipitated by trichloroacetic acid.In order to exclude the possibility that protein nitrogen might be lost by hydrolysis in the warm perchloric acid, the distribution of nitrogen in the different fractions was investi- gated (see Table 11). Although a considerable amount of nitrogen was found in all fractions, the amount found in the perchloric acid fraction was not more than could be accounted for by nucleic acids, which indicates that hydrolysis of protein at this stage is probably negligible. 53 pg and 1200 & 45 pg, respectively.TABLE I1 DISTRIBUTION OF NITROGEN I N EHRLICH MOUSE ASCITES TISSUE Nitrogen Pi3 Material tested found, Lipid fraction . . .. .. .. 337 Sulphuric acid fraction . . .. . . 225 Perchloric acid fraction . . .. .. 150 Trichloroacetic acid wash . . . . 12 Residual fraction .. .. .. 2050 Part of total nitrogen, 12-15 8-11 5.41 0.43 % 73.9 If perchloric acid is not removed before protein is digested, the results for nitrogen may be low, owing to decomposition of ammonium perchlorate' and therefore a single wash with trichloroacetic acid was introduced before carrying out the digestion.January, 19581 SMALL AMOUNTS OF TISSUE METHOD REAGENTS- 39 All reagents should be of recognised analytical grade. Balanced salt solution-Prepared by dissolving 80 g of sodium chloride, 4 g of potassium chloride, 1.4 g of calcium chloride and 2 g of magnesium sulphate, MgS0,.7H20, in 1 litre of distilled water. For use, dilute the solution 1 + 9 with distilled water.The solution must not contain glucose or phosphate. Ethanol - ether mixture (3 + 1 v / v ) . Ether - carbon tetrachloride mixture (3 + 1 v / v ) . Sulphuric acid, N and 10 N. Perchloric acid, N and 4 N. Trichloroacetic acid, N. Sodium hydrogen carbonate solution, 1 per cent. w/v. Ammonium molybdate solution, 2.5 per cent. A minonaphtholsulphonic acid reagent-Prepared by dissolving 1 g of 1-amino-2-napht hol- 4-sulphonic acid, 59.5 g of sodium bisulphite and 2 g of anhydrous sodium sulphite in 1 litre of distilled water. Chromic acid-Prepared by dissolving 12.5 g of silver dichromate in 500 ml of concen- trated sulphuric acid.(As a precipitate may be formed when analytical-reagent grade sulphuric acid is used, it may be necessary to use the ordinary grade.) Anthrone solution-Prepared by dissolving 200mg of anthrone in 100 ml of 25 N sulphuric acid. Indole solution, 0-04 per cent. Chloroform. Selenium dioxide solution-A solution containing 1 per cent. of selenium dioxide in 50 per cent. v/v sulphuric acid. Nessler reagent (modi$ed)-Prepared by adding a solution of 3.5 g of gum acacia in 750 ml of water to a solution of 4 g of potassium iodide and 4 g of mercuric iodide in 25 ml of water and adjusting the volume to 1 litre. FRACTIONATION PROCEDURE- The tissue is collected in a centrifuge tube and, after it has been spun in a centrifuge, the medium is discarded.Then 2 ml of the balanced salt solution are added and the tissue is stirred with a platinum wire and quickly spun in a centrifuge, the supernatant liquid being discarded. A 2-ml portion of ethanol - ether mixture is added and the resulting mixture is shaken and then set aside for 15 to 20 minutes at room temperature; it is then spun in a centrifuge and the supernatant liquid is collected. This procedure is repeated, first with 2 ml of the ether - carbon tetrachloride mixture and then with a further 2 ml of the ethanol - ether mixture, the supernatant liquid from each being combined to form the lipid fraction. To the residue in the centrifuge tube are added 2 ml of ice-cold N sulphuric acid. The contents of the tube are mixed and left at 0" C for 10 minutes, with occasional stirring, and then spun in a refrigerated centrifuge, the supernatant liquid being collected.This procedure is repeated twice, the supernatant liquid from each being combined to form the sulphzcric acid fraction. A 2-ml portion of N perchloric acid is added to the material remaining in the tube and the contents are stirred as before. The tube is then immersed in a water bath a t 70" C and left for 20 minutes. The tissue is again separated by centrifugation and the supernatant liquid is collected. This procedure is repeated once, the supernatant liquid from each being combined to form the perchloric acid fraction. The residue in the centrifuge tube is washed once with 2 ml of N trichloroacetic acid and the washing is discarded, PROCEDURE FOR THE LIPID FRACTION- The lipid fraction is shaken with 2ml of 1 per cent.w/v sodium hydrogen carbonate solution and, in order to facilitate the separation of the phases, 2 ml of ether may be added. When the layers have been separated, the sodium hydrogen carbonate layer is washed twice with 2 ml of ether each time and these washings are added to the ethanol - ether - carbon tetrachloride layer. This layer is then adjusted to a measured volume and aliquots are taken During this period the contents are stirred occasionally. The residual fraction remains.4a PAUL: DETERMINATION OF THE MAJOR CONSTITUENTS OF [Vol. 83 for total lipid, lipid phosphorus or other determinations. The aliquots, preferably in short wide tubes, are evaporated to dryness in a hot-air bath and the lipid phosphorus and total lipid are determined as follows.Lipid Phosphorm-To the dry residue are added 0-5ml of 1 0 N sulphuric acid and 0.5 ml of 4 N perchloric acid. This digestion mixture is concentrated by evaporation on a sand-bath at 160" C and digestion is completed over an open flame. It is necessary to add an anti-bump rod, made by indenting the end of a 6-inch length of &inch diameter glass rod with a copper wire. The phosphorus is then determined by the method of Griswold, Humoller and McIntyre.lO To the digested sample is added 0.5 ml of 2.5 per cent. ammonium molybdate solution and 0.5 ml of aminonaphtholsulphonic acid reagent and the volume is adjusted to 5 ml with water. After the mixture has been heated for 10 minutes in a boiling-water bath, the blue colour is measured in a suitable spectrophotometer (a Unicam SP600 was used) at 820mp or with a red filter.This method gives very accurate results in the range 0.2 to 2p.g of phosphorus. Note that the final concentration of acid in the reagents must not be less than N , otherwise artificially high results will be obtained. Total lipid (BZoor's method6)-To the sample to be analysed are added 3 ml of the silver dichromate reagent and then the sample tube is placed in a boiling-water bath for 15 minutes. The tube is removed and cooled, 3 ml of water are added and then the green colour is measured in a suitable absorptiometer at 620 mp. Lard dissolved in carbon tetrachloride can be used as a standard. Note that errors are most likely to be caused by inadequate removal of the solvents.This test is suitable for 50 to 500 pg of fat. PROCEDURE FOR THE SULPHURIC ACID FRACTION- Aliquots are taken for the determination of carbohydrate and phosphorus. Determina- tion of phosphorous is performed in exactly the same way as for the phosphorus in the lipid fraction after 0.5 ml of 10 N sulphuric acid and 0-5 ml of 4 N perchloric acid have been added to the aliquot and the volume has been reduced as before. Carbohydrate is determined by the anthrone method, as described by Trevelyan and Harri~on.~ To each of a number of tubes, 5 ml of the anthrone solution are added and 1 ml of the solution to be tested is carefully inserted so as to form a layer on top of the reagent. At zero time the two layers are intimately mixed by means of a glass rod with a flattened end, which is plunged up and down once or twice in the mixture.The tubes are dealt with in strict rotation and are placed in a boiling-water bath immediately after mixing. After exactly 10 minutes (timed with a stop-watch), they are transferred in the same order to a bath of iced water. When the solutions are cool, the green colours are measured at 620 rnp. The useful range of the reaction is from 10 to 150 pg of glucose or its equivalent. PROCEDURE FOR THE PERCHLORIC ACID FRACTION- Total nucleic acids are determined by measuring the ultra-violet absorption at 268 mp.8 Carbohydrate is then determined by the anthrone method as before in a 1-ml aliquot of the fraction. A 2-ml aliquot of the fraction is taken for the determination of deoxyribonucleic acid by the method of Ceriotti.ll The 2-ml aliquot of the fraction is placed by pipette in a boiling-tube, and 1 ml of indole solution and 1 ml of concentrated hydrochloric acid are added.The tube is placed in a boiling-water bath for 10 minutes and then removed and cooled. The reaction mixture is extracted three times with 4-ml portions of chloroform and the chloroform extracts are dis- carded. The tube is then spun in a centrifuge for a short time at low speed in order to remove any emulsion that may have formed and the yellow colour is measured at 490'mp. The useful range of the reaction is from 5 to 30 pg of deoxyribonucleic acid. PROCEDURE FOR THE RESIDUAL FRACTION- The residue consists almost entirely of protein and the protein nitrogen is determined as follows.To the residue is added 0.5 ml of the selenium dioxide solution. Water is removed by heating on a sand-bath and the mixture is then digested over an open flame until clear. Protein nitrogen is thus converted to ammonium sulphate and the ammonia can be determined by means of a modified Nessler' reagent.January, 19583 SMALL AMOUNTS OF TISSUE 41 The digestion mixture is diluted with distilled water to contain about 10 pg of nitrogen per ml. To 2 ml of this test solution are added 2 ml of the modified Nessler reagent and 3 ml of 2 N sodium hydroxide. After 16 minutes the yellow colour is measured at 490 mp. RESULTS AND CONCLUSIONS Table I11 shows that the recoveries of all substances were satisfactory.TABLE I11 RECOVERY OF MATERIALS ADDED TO THE VARIOUS FRACTIONS Amount of material Amount of Amount of Material originally material material Fraction added present, added, found, tG P.tg PLg Lipid . . . . Fat 44 1 250 699, 673, 699,699 Sulphuric acid . . Glucose 82.5 225 318.1, 318.1 Perchloric acid . . Glucose 28.2 150 190, 196 Perchloric acid . . Deoxyribonucleic 18.2 7-75 26.5 acid* 15.5 31-7, 33.2 Perchloric acid . , Deoxyribonucleic 4.44 1.83 6.27, 6.3, acid t 6.4 Recovery, % 101, 97.5, 101,101 103.5, 103.5 106, 110 102 94, 98-5 100, 100.5. 102 * Determined by indole method. t Determined by ultra-violet absorption method. Table IV shows the results of the analyses of a variety of tissues by this procedure. For all except embryonic chick heart, at least six samples were analysed so that the standard deviation of the determinations could be found.The analyses of embryonic chick heart indicate the degree of reproducibility between samples of about 1 mg dry weight. TABLE IV RESULTS OF THE FRACTIONATION AND ANALYSIS OF A VARIETY OF TISSUES BY THE METHOD DESCRIBED Aliquots of mouse and embryonic chick liver were obtained from aqueous homogenates, of Ehrlich mouse ascites tumour and strain L cells from cell suspensions, and of embryonic chick heart from approximately equal amounts of tissue fragments Tissue Ehrlich mouse ascites tumour . . Mouse liver (ascitic) . . Embryonic chick liver . . .. Embryonic chick A heart R C Strain L mouse fibroblast * Total lipid, mg 2-368 & 0.099 1-428 10.062 2.570 2 0.062 0.094 0-072 0.072 0-610 &0.014 sulphuric acid fraction, Pg 111-6 * 3.3 759.6 159.3 & 0.78 75 53.4 64-2 0 & 48 perchloric acid fraction, Clg 50 & 1.35 563-6 49.2 44-5 43 53.5 22.7 f 0.8 f 50 & 3-33 total, Clg 161-6 1313-2 208.5 119.5 96-4 117.7 22.7 Carbohydrate (as glucose) in- Protein nitrogen, mg. 2.855 f 0.15 1.619 & 0-072 1.483 * 0.033 0.107 0.0914 0.112 0.268 f 0.004 Ribo- nucleic acid phosphorus, PLg 82.5 1-36 64-9 62-4 - + 2.36 3.57 3.54 3.73 17-2 0-7 & 1-19 Deoxyribo- nucleic acid phosphorus, Pg 43.9 20.171 17.6 3-0.165 23-15 1.26 1.836 1.63 1.796 24-52 1.75 * Stored for some months in a deep-freeze cabinet before testing.The results in Table IV are in agreement with other figures published for such tissues. I t may be noted that the general scheme that has been described is capable of considerable flexibility.Since an accurate determination of deoxyribonucleic acid is possible by omitting a11 the steps in the fractionation except extraction with hot perchloric acid,12 it is possible42 RILEY : SIMULTANEOUS DETERMINATION OF WATER AND [Vol. 83 to omit various stages when only limited information is required. Also, when it is particu- larly desired to separate the different phosphoru!; fractions (usually for the determination of the incorporation of phosphorus-32), we have found it convenient to employ a Schmidt and Thannhauser separation after the sulphuric acid stage. Instead of proceeding to the extraction with hot perchloric acid, 0 4 m l of N sodium hydroxide is added to the precipitate and it is incubated overnight (18 hours) a t 37" C.After aliquots have been removed for determinations of carbohydrate and protein nitrogen, the digest is cooled to 0" C and 0.2 ml of 2.5 N hydrochloric acid and 0.3 ml of 4 N perchloric acid are added. Deoxyribonucleic acid is thereby precipitated and, after standing for 10 minutes at 0" C to allow the precipitate to flocculate, it can be separated by centrifugation in a refrigerated centrifuge. I t is necessary to wash the precipitate twice with 0.5 ml of N perchloric acid each time. Ribonucleic acid phosphorus can be determined in the super- natant liquid and deoxyribonucleic acid phosphorus in the precipitate by digesting and continuing with the procedure described for lipid phosphorus. This work was supported by grants from the Scottish Hospitals Endowments Research Trust. I thank Professor J. N. Davidson for the provision of facilities and Miss E. Pearson and Miss I. Withers for their assistance. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. REFERENCES Schmidt, G., and Thannhauser, S. J., J . B i d . Chem., 1948, 161, 83. Davidson, J. N., Leslie, I., and Waymouth, C., Biochem. J., 1949,44, 5. Davidson, J. N., and Leslie, I., Exp. Cell Res., 1951, 11, 366. Cailleau, R., Moss, S., and Siegel, B. V., J . Nut. Cancer Igzst., 1956, 16, 1011. Schneider, W. C., J . Biol. Chem., 1945, 161, 293. Bloor, W. R., Ibid., 1947, 170, 671. Ogur, M., and Rosen, G., Arch. Biochem., 1950, 25, 262. Ceriotti, G., J . Biol. Chem., 1955, 214, 59. Trevelyan, W. E., and Harrison, J. S., Biochem. J., 1962, 50, 299. Griswold, B. L., Humoller, F. L., and McIntyre, A. R., Anal. Chem., 1951,23, 192. Ceriotti, G., J . B i d . Chem., 1952, 198, 297. Paul, J., J . Biophys. Biochem. Cytol., 1966, 2, 5'97. Received November 30th, 1966