1210
ANALYTICAL CHEMISTRY
Table I.
Tryptophan C o n t e n t of Proteins Using Light a n d S o d i u m Nitrite to Develop Color Tryptophan Content' Sodium nitrite
Protein
Light
% Casein @-Lactoglobulin Ovalbumin, denatured Conalbumin, denatured CS-54R ( 1 )
APPARATUS, MATERIALS, AYD M h T H D D S Difference
%
%
1.75 2.50
* 0.01(3)* * O.Ol(3)
1.70 2.52
2.9 0.8
1.55
*
1.46
6.2
0.02(3)
oxidation could be used in an analytical procedure with a reasonable degree of accuracy if use of sodium nitrite were not feasible.
3.01 12.3 3 . 3 8 * 0.04(3) 0.991 0.907 * 0.013(3) 8.5 0.561 CS-56R( I ) 0.524 * 0.005(2) 6.6 Same protein samples used for analyses using light and sodium nitrite. Results are on ash- and water-free ,basis and va!ues were corrected as described in ($). Values obtained using sodium nitrite were determined by Procedure 0 and were taken from Table XI11 ( 8 ) . b Numbers in parentheses show number of determinations made on each protein.
The relative color intensities obtained on 20 minutes' illumination of tryptophan, casein, ovalbumin, and a cottonseed allergenic polysaccharidic protein CS-54R ( 1 ) were 97, 99, 100, and 99%, respectively, of the color intensity obtainable by a 60minute period of illumination of each substance. These data indicate that the photochemical development of color, reaction 11, is slightly more rapid for proteins than for free tryptophan and for greatest accuracy in an analytical method the exact time of maximum color development for tryptophan and each protein should be determined as was done previously for r e action I1 with sodium nitrite ( 2 , S). In the present work, however, a 20-minute pertod of illumination was adopted as sufficiently accurate to illustrate the method. Results of tryptophan analysis of several representative proteins using light to develop the color are compared in Table I with values obtained using sodium nitrite. The difference between values varied from a minimum of 0.8% with p-lacte globulin to a maximum of 12.3% with conalbumin, and the average difference for six proteins was 6.2%. The precision using photochemical oxidation was * 1%, while that using sodium nitrite was *0.88%. These results indicate that photochemical
Apparatus, materials, and general procedures have been described in detail (2-4). Reaction I for free tryptophan and for proteins can bc carried out by any appropriate procedure. The apparatus and procedure for illumination of. test solutions have been described ( 4 ) C, Figure 1, is the same as described in Figure 4 of ( 2 ) . 1, and .Y were obtained as described below using Procedure E ( 2 )for reaction I.
Procedure S. To 1.003 mg. of tryptophan and 250.8 mg. of p-dimethylnminobenzaldehyde (30 mg. per 10 ml.) in a 125ml. glass-stoppered Erlenmeyer flask were added 83.6 ml. of 19 N sulfuric acid at 25", solution A. In another flask 100 ml. of 19 N acid were added to 300 mg. of p-dimethylaminobenzaldehyde, solution B. Required volumes of solutions A and B were mixed immediately in 25-ml. glass-stoppered flasks to give test solutions containing 10 to 120 micrograms of tryptophan and 30 mg. of p-dimethylaminobenzaldehyde in a volume of 10 ml. The solutions were reserved in the dark at 25" for 19 hours. Each solution was then illuminated for 20 minutes a t 25" and transmittancies were read using a blank solution similar to the test solution, except that tryptophan waa omitted. The lowest transmittancy obtained over the wave length ranging from 580 to 620 mp was used for calculation of results. Results are plotted as L. To each solution was then added 0 1 ml. of 0.04% sodium nitrite solution and after standing for 30 minutes t r a n s mittancies were read. Results are lotted as N . For the proteins, Procedure 0 ($was used with approximately individual optimum times for reaction I (5). Solutions were illuminated for 20 minutes. Transmittancies were converted to weight of tryptophan from L,Figure 1. LITERATURE CITED
Spies, J. R., Bernton, H. S., and Stevens, H., J. Am. Chem. Soc., 63,2163 (1941). (2) Spies, J. R., and Chambers,D. C., ANAL.CHEM., 20,30(1948). (3) Ibid., 21,1249-66 (1949). (4) Spies, J. R.,and Chambers, D. C., J . Am. Chem. SOC.,70, 1682 (1948).
(1)
RECEIVED November 2, 1949. Paper V in series entitled "Chemical Determination of Tryptophan." For previous paper see ($).
Determination of Cholesterol Adaptation of Schoenheimer-Sperry Method to Photoelectric Instruments FRANCIS F. FOLDES A N D B. CRAIG WILSON D e p a r t m e n t of Anesthesia, Mercy Hospital, Pittsburgh, Po. HIS paper describes a modification of the SchoenheimerT Sperry method (I), simple enough for clinical w e and accurate enough for research purposes. A similar adaptation,
0.500, and 0.750 mg. were prepared with each series of determinstions. APPARATUS
published by Sperry (6),has limited distribution and is a t present out of print. The authors have tried to eliminate some of the difficulties of the method which in the past have been made ita introduction into the routine of any laboratory a tedious and timeconsuming procedure.
The a paratus was that described by S erry ( 4 ) , except that 15-ml. gfrtss-stoppered, heavy-duty borosi6aate glass centrifuge tubes (obtained from MacAllaster-Bicknell, Cambridge, Mass.) were substituted for the stirring rods and preserving jars.
REAGENTS
EXPERIMENTAL
With three exceptions, the reagents used are the m e aa described by Sperry (4). Instead of redistilled alcohol and acetone, C.P. acetone and absolute alcohol are entirely satisfactory. The 0.4yoaqueous digitonin solution has been replaced by an 0 . 5 7 digitonin solution in 50% alcohol, as recommended by Sobel and Mayer ( 2 ) . The use of alcoholic di i t o i h solution results in an evenly dispersed, fine precipitate. $he precipitate is not sticky, and the washed precipitate easily dissolves ill glacial acetic acid without the development of the sediment referred to by Sperry ( 4 ) . The stork standard used contained 2 mg. of cholesterol per ml., and three different working standards containing 0.250,
Ih attempting to adapt the method to the spectmphotometer and photoelectric colorimeter it soon became evident that, the development of the color and the determination of its intensity are the most sensitive points of the reaction. The time relationship of the color development and the relationship between the color ,intensity and the Beer-Lambert law seemed to be the moat important in this respect. In his earlier publications Sperry recommended that if the color is developed a t 25' C. it should be read a t 30 minutes ( 9 . 4 ) . In his
1211
V O L U M E 22, NO. 9, S E P T E M B E R 1 9 5 0 Table I. Sample
Optical Density (-log T) of Samples Containing 0.5 Mg. of Cholesterol 30 Min. 3 0 . 5 Min. 31 Min. 3 1 . 5 hlin. 32 Min. 33 Min. Determined with Spectrophotometer
2 3
48.9 49.0 49.0
48.9 49.1 49.1
5 6
..
..
1
4
..
48.9 49.1 49.1 49.3
48.9 49.1 49.0
..
..
..
48.9 49.1 49.0 49.3 49.8
49:9
Determined kitti Photoelectric Colorimeter 1 2 3 4 5 6
48.8 48.5 48.4
48 8 48.5 48.4
.. .. ..
.. .. ..
40 Min.
4 0 . 5 Min.
48.8 48 5 18.5 48.7
.. ...
49.1 48.5 48.7
..
41 Min. . 4 1 . 5 Min.
48.9 48.6 48.; 48., 48.5
... 48'5
42 >fin. 43 Min.
Determined with Spectrophotometer 48.5 48.9 49.0
..
48.3 48.5 48 S 48.t
..
..
7 8 9 10 11 12
48.7 49.0 49.0
7 8 9 10 11 12
49.8 49.7 49.7
49.8 49.6 49.7
..
..
.. , .
..
..
48.3 48.4 48.7
..
.. ..
48.1
..
..
47:5
48.2 48.5 48.2 48.2
..
..
Determined with Photoelectric Colorimeter
..
..
49.7 49.7 49.7 49.7
49.7 49.6 49.7
..
49.7 49.6 49.t 49.3 49.4
49:3
latest publication (6) he specified 40 minutes as the optinial time for the determination of the color intensity.
To clarify this point, color was developed a t 25" C. in a seriej of standards containing 0.5 mg. of cholesterol dissolved in glacial acetic acid, by the addition of the acetic anhydride-sulfuric acid reagent. R'hen the color intensity was to be determined in the photoelectric colorimeter (Fisher Scientific Company, Pittsburgh, Pa.) 0.5 my. of cholesterol was dissolved in 1 ml. of glacial acetic acid to which 2 ml. of acetic anhydride-sulfuric acid reagent were added, and the color was read in a microcuvette of approsimately IO-mm. diameter, using a 650 mp filter. When a spectrophotometer (Coleman Junior Model 6a, Maywood, Ill.) was used, 0.5 mg. of cholesterol was dissolved in 2 ml. of glacial acetic acid, 4 ml. of acetie anhydride-reagent were used, and the reading was made in a cuvette of 19-mm. diameter a t a wave length of 625 mp. Each series consisted of six samples, and the acetic anhydride reagent was added a t 3-minute intervals. The color intensity in the first and second series was determined in the spectrophotometer and in the third and fourth series in the photoelectric colorimeter. In three samples of the first and third series the color was read a t 30 minutes, then the samples were left in the cuvette and the color was read again a t 30.5, 31, 31.5, and 32 minutes. Another sample was left in the water bath a minute longer and the color was read a t 31 and 32 minutes, and in two other samples the color was read a t 32 and 33 minutes, respectively. .4similar procedure was carried out v ith the second and fourth series; the color was read a t 40, 41, 42, and 43 minutes. Findings are presented in Table I.
It can be seen from the table that there is little or no difference in the color,intensity read a t or around 30 minutes, or a t or around 40 minutes. At 30 minutes there is a slight tendency to a further increase of the color intensity with time, and a t 40 minutes a similar tendency to a decrease of the color intensity, but for practical purposes the color can be considered stable, and its intensity can be determined at any time between 30 and 40 minutes. To study the relationship between the color intensity and the Beer-Lambert Ian, color was developed in samples containing 0.125, 0.250, 0.500, 0.750, and 1.000 mg. of cholesterol. When the determinations were made on the spectrophotometer. the above quantities of cholesterol were dissolved in 2 ml. of glacial acetic acid. to which 4 ml. of acetic acid anhydride reagent were added. When photoelectric colorimeter was used the cholesterol w s s dissolved in 1 ml. of glacial acetic acid and 2 ml. of acetic acid anhydride were used. The results are presented in Table 11.
The color intensity as determined with either instrument follows the Beer-Lambert law fairly closely. The parallelism is closer when the color intensity (or optical density) is determined with the spectrophotometer than when it is determined by the photoelectric colorimeter. The spectrophotometer in higher concentrations tends to give slightly higher values. The results obtained with the photoelectric colorimeter are somewhat higher in low concentrations, and considerably lower in higher concentrations than would follow from the Beer-Lambert law. The error caused by this discrepancy can be largely eliminated by the use of several standards of different concentrations and comparison of the unknown with the standard nearest to it. This point is brought out more clearly in connection with the results of the recovery tests PROCEDURE FOR ANALYSIS O F CHOLESTEROL IN BLOOD OR PLASMA
Extraction. Add 5 ml. of heparinized plasma (or whole blood) rapidly, in a fine stream, to 75 to 80 ml. of 1 to 1 alcohol-acetone mixture in a 100-ml.volumetric flask. Bring to boil, and keep a t boiling point for 30 seconds in a water bath, rotating to avoid bumping. After cooling to room temperature, make up t o volume with 1 to 1 alcohol-acetone, mis, and filter through fat-free filter paper into a glass-stoppered bottle.
'Table 11. Optical Density of Cholesterol Solutions Read 40 Minutes after Development of Color Cholesterol Content of Sample, Mg. 0.125 0.250 0.500
0.750 1.000
Photoelectric Coloriineter 14.3 14.. 28.. 28., 49.1 50.1 67.1 6 7 .I
Spectrophotometer 11.3
Determination of Total Cholesterol. 1. Place 2 drops of 50% pbtassium hydroside in a 15ml. glassstoppered centrifuge tube, add 5 ml. of extract, and shake vigorously. 2. Place the centrifuge tube in a water bath a t 40" C. for 30 minutes. Do not allow the temperature of the bath to fall below 37" c . 3. Remove centrifuge tube from water bath, cool to room temperature, add 1 drop of phenolphthalein solution, and titrate with 10% acetic acid solution. Add 1 drop in excess. 4. Add 3 ml. of 0.50/, alcoholic digitonin solution to the slightly acid extract in centrifuge tube, stopper, mix thoroughly, and keep a t room temperature overnight. 5. Remove stopper, centrifuge a t 2500 t o 2800 r.p.m. fw 15 minutes, and decant supernatant fluid. Blow in 2 ml. of 1 t o 2 acetone-ether mixture in a rapid stream t o break up sediment and wash down the wall of centrifuge tube with another 2 ml. 6. Centrifuge again for 10 minutes, decant, and wash with 4 ml. of ether. 7. Centrifuge again for 10 minutes, decant, and evaporate ether with a stream of filtered air for 1 minute. 8. Heat a 3- t o 4-cm. layer of sand in a pan to 110" to ll5OC. in an oven. Replace the stopper loosely in the centrifuge tube (preferably by placing a fine string about halfway down the stop per), place the centrifuge tube in hot sand, and keep in oven for 30 minutes. 9. Take the centrifuge tube from the sand brtth, remove the stopper, and allow 2 ml. of glacial acetic acid to run down its wall. Replace stopper, shake vigorously, and put back into sand bath for 2 to 3 minutes. Determination of Free Cholesterol. Put 10 ml. of extract into a glass-stoppered centrifuge tube. Omit steps 2 and 3 and carry out steps 4 to 9, inclusive, ss described for total cholesterol, but do step 6 twice. Development of Color. Remove centrifuge tubes from tba oven and cool in a 25' C. water bath. Prepare standards containing 0.25,0.50, and 0.75 mg. of chfesterol in 2 ml. of glacial acetic acid and a blank containing 2nd. of glacial acetic acid.
A N R L Y TICAL C H E M I S T R Y
1212
Prepare acetic anhydTide-sulfuric acid (20 to 1) reagent aiid keep in a glsss-stoppered bottle in an ice-cold water bath. Add a t regular intervals ( 1 minute is suitable after some practice) 4 ml. of ice-cold anhydride reagent to blank, each unknown, and standards, mix well, and place in 25" C. water bath kept in a dark cabinet. Adjus the instrument to zero with blank in place. A t an evact time (any time between 30 and 40 minutes may be chosen) from the addition of the anhydride reagent, and in the same order the reagent wm added, read the color. If the photoelectric colorimeter is used, dissolve both unknowns and standards in 1 ml. of acetic acid and use only 2 ml. of anhydride reagent. This rhangd is made necessary by the fact that with this type of instrument the color is read in a 10-mm. cuvette and the decreased thickness of the solution must be compensated for by increased concentration. It was also necessary to use a cushion in the cuvette holder to raise the cuvette and therkby bring the small quantity of solution into the path of the light of the instrument. CALCULATION
Recovery of Cholesterol from Acetone-Alcohol Solutions of Known Concentration Photoelectric Colorimeter, Mg. % ' Calculated Nearest Single standard standardU 49.0 55.0 50.0 56.0 99.0 111.0 99.0 111.0 197.0 197.0 198.0 198.0 300,O 265.0 300.0 265.0
Cholesterol Concentration, IIg. %
50 100 200 300 a
=
Table V.
reading of unknown reading of standard
To obtain results in milligrams per cent, the cholesterol content of the sample must he multiplied by the dilution factor of the sample. If 5 ml. of plasma (whole blood) are extracted in 100 ml. (dilution 1 to 20) and 5 ml. of the extract are used in a sample, the factor converting to milligrams per cent will be 20 X 20 = 400. If 10 ml. of extract are used (as in the determination of free cholesterol), the factor will be 10 X 20 = 200. Simpler formulas can be obtained if the milligrams of cholesterol in the sample are multiplied by the dilution factor used. For convenience the factors to be used are presented in Table 111. With the use of the above combined factors Mg. % of cholesterol in sample =
reading of unknown XF' reading of standard
Table 111. Dilution Factors Cholesterol in Standard, Mg.
5 ml. of extract
Factor F 10 ml. of extract 50 100 150
0.25 0.50 0.75
100 200 300
CONTROL OF METHOD BY RECOVERY OF KNOWN QUAVTITIES OF CHOLESTEROL
The cholesterol concentration was determined as total cholesterol in 50, 100, 200, and 300 mg. % acetone-alcohol solutions. The total and free plasma cholesterol content was determined in two pregnant women, with and without the addition of 0.125 mg of cholesterol to the samples. It follows from the above calculations that the addition of 0.125 mg. of cholesterol to a sample should produce a 50 nlg. % increase in the total cholesterol concentration and a 25 mg. % increase in the free cholesterol concentration. The results are presented in Tables IV and V. Table I V shows good agreement between the expected and determined cholesterol concentrations, when the calculations were made with the nearest standard. The maximum difference was 3%. When the results were calculated with a single standard, the maximum difference between. the calculated and determined cholesterol concentration waa as high as 12%.
Single standard'' 45.0 46.0 97.0 96.0 199.0 200.0 298.0 300.0
Recovery of Cholesterol Added to Plasma Extracts
With added cholesterol
Total Cholesterol, Mg. % Detd. CaIcd.
Free Cholesterol.
hfg. % ___Detd. CaIcd.
271. 272
I'lasnia 59 Without added cholesterol With added cholesterol
mg. of cholesterol in standard X
Spectrophotometer, Mg. % Calculated Nearest standard 49.0 50.0 98.0 97.0 199.0 200.0 299.0 300.0
Standard used contained 0.5 mg. of cholesterol rr 200 mg. %.
Sample
For accurate results calculations should be made with the standard that gave the nearest reading to the unknown in quwtion. The readings are made 6n the optical density (-log Z') scale of the instruments. Mg. of cholesterol in sample
Table IV.
255.0 257.0 308.0 306.n
... 306.0
..
77,5 77.0 103.5 103.0
IO? 8
Table V shows that good recovery was obtained when 0.125 mg. of cholesterol was added t'o samples of plasma extracts in which total and free cholesterol was determined. DISCUSSION
If a given color reaction is to be considered adaptable to analysis with photoelectric instruments, the optical density-Le., the concentration of the sample-must be within the sensitive range of the instruments. Pre1iminai.y experiments indicated that accurate results could be expected if the cholesterol content of the samples was between 0.125 and 0.750 mg. Thus, when a 5-ml. blood or plasma aliquot is extracted with 100 ml. of 1 to 1 alcohol acetone and 5 and 10 ml. of the extract are used for the determination of total and free cholesterol, respectively, the cholesterol content of the samples will be between 0.125 and 0.750 mg., provided that the total cholesterol concentration is between 50 and 300 mg. %, and the free cholesterol concentration is between 25 and 150 mg. %. If lower or higher concentrations are encountered the determinations should be repeated using more, or less, extract. The use of glass-stoppered test tubes offers several advantages. The extract can be mixed with the potassium hydroxide used in the total cholesterol determination more satisfactorily. Frequently, in the last washing of cholesterol digitonide a small quantity of ether is trapped under the centrifuged precipitate. When the tubes are placed in the oven for drying, the trapped ether evaporates rapidly. This may scatter the precipitate and lead to a loss of material. This can be prei-ented by looselv stoppering the centrifuge tube. Furthermore, in the course of the repeated washing of the precipitate some of it unavoidably sticks to the wall of the centrifuge tube. This precipitate can be dissolved more completely with the relatively small quantity (1 to 2 ml.) of acetic acid used if thecentrifuge tube can be stoppered and its contents well shaken. Another advantage of the stoppered test tube is that it will prevent absorption of atmospheric moisture by the hygroscopic glacial acetic acid and acetic acid anhydride, thereby eliminating the interference with the color development caused by the presence of water. Close agreement was found (especially with the use of the spectrophotometer) with the Beer-Lambert law when the color was developed in glacial acetic acid solutions of cholesterol. Re
1213
V O L U M E 22, NO. 9, S E P T E M B E R 1 9 5 0 covery tests on acetone-alcohol solutions of cholesterol, however (Table IV), gave better results when the samples were compared to standards containing approximately the same quantity of cholesterol. The discrepancy resulting from the use of a single standard was greater when the photoelectric colorimeter was used. It is therefore suggested that three different standards containing 0.250, 0.500, and 0.750 mg. be prepared for each series of photoelectric determination of plasma (or blood) cholestcsrol. It was necessary for the authors’ purposes to extract 5 ml. of plasma (or whole blood), but total cholesterol can be determined in 0.25 ml. and free cholesterol in 0.50 ml. of plasma. In this case the extraction should be done as recommended by Sotiel and hlaycr (b),and from then on the procedure continued as described here. SUIMLMMARY
The Schoenheimer-Sperrv method of cholesterol determination has been adapted to photoelectric instruments. The iiitroduc-
tion of glass-stoppered centrifuge tubes obviates the use of stirring rods and preserving jars recommended in the original method. Cholesterol may be determined accurately by photoelectric methods in samples containing 0.125 to 0.750 mg. of cholesterol. The accuracy of the Schoenheimer-Sperry method may be. enhanced by the use of serial standards; calculations are based on the comparison of the unknown t o the standard of approximating concentration. LITERATURE CITED
(1) Schoenheimer, R.,and Sperry, W. M., J . B i d . Chetn., 32, 745 (1934). (2) Sobel, A. E., and Mayer, A. M., Ibid., 157,255 (1945). (3) Sperry, W.M.,,fim. J. Clin. Path., Tech. SuppZ., 2, 91 (1938). (4) Sperry, W.M., Schoenheimer-Sperry Method for Determination of Fholesterol,” New York, 1945. (5) Sperry, W.M.,“Technical Manual for Laboratory Technicians,” War Dept.. TM-8-227, r-. S.Government Printing Office, 1946. R E C E I V E Dneceiiiber 6 , 1949.
Dropping Mercury Electrode Apparatus W. CHARLES COOPER’
AND
M. M. WRIGHT*
Princeton llniversity, Princeton, N .
4RIOUS dropping mercury electrode assemblies have been Vduggested (1-7). In the majority of these, rubber tubing with its inherent disadvantages is used to some extent. For polarographic analysis based on standardized diffusion current constants, the apparatus of Lingane ( 4 )with the stop-clock circuit for the rapid, automatic determination of the rate of flow of mercury is probably the most advantageous. The apparatus drscribed in this paper is particularly well suited for routine polarographic analysis by comparative methods. Designed entirely of glass and having ground-glass connections, there is n? contact between mercury and rubber and hence no possibility of the mercury being fouled from this source. The three-piece assembly is compact, is more readily disassembled and cleaned than the glass-rubber or all-glass apparatus currently in use, and is not considered by the authors aa extremely complicated or fragile. These advantages offset the initial expense. Figure 1 illustrates the entire apparatus, an enlarged view of one section of the stand tube, and a hand-drawn capillary.
J.
capillaries have been discussed by Novak (8). The major disadvantage in their use is the fact that they are easily breakable. Figure 1, right, shows a hand-drawn capillary for which this disadvantage has been overcome. Such a capillary has been used
C
The apparatus is made up of three pieces: A , the capillary; B , the mercury reservoir vessel; and C, the stand tube with adjoining stopcock and blood pressure bulb and valve. In A a 5.5-cm. length of Corning marine barometer tubing is joined to 6-mm. soft- lass tubing have a 7/25 J female joint. Electrical contact to &e mercury in B is made by rpeans of the tungsten contact and mercury well, D. The details of the construction of the stand tube, C, in the region of the ground-glass joint, E, 12/30 J , are shown in Figure 1, center. The operation of the apparatus is simple. By means of the blood pressure bulb and valve, F , mercury can be raised to any desired height in the stand tube. The air forced in when the bulb is squeezed (valve is closed and stopcock is open during this operation) enters the ieservoir vessel through a hole, G , close to the lower inner seal. As a result of the pressure which is built up in the reservoir vessel, the mercury proceeds up tube H. The stopcock is closed when the mercury has reached the desired level. When the polarographic analysis has been completed, the capillary is washed carefully with a stream of distilled water and immersed in either distilled water or ure mercury. Then the mercury column is lowered by opening t i e stopcock and the valve, whereupon the pressure in the vessel is returned to atmospheric.
I
$7125
P B
$12130
In polarographic work hand-drawn capillaries can be used as well as marine barometer tubing. Various features of such 1 Present address, Department of Chemistry, University of Wisconsin, Madison, Wis. 1 Preaent address, Chemistry Division, National Research Council, Ottawa, Canada.
Figure 1. Dropping Mercury Electrode Apparatus Left.
Entire apparatus Enlar ed view of section of s t a n d tube Hand-&awn capillary w i t h protective shield
Center.
Right.
