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A densitometric method for the quantitative determination of protein fractions after agar gel electrophoresis
397
SHORT COMMUNICATIONS
by phosphorus analysis each time, were also in good agreement, as indicated in Table I. Applying successive increments of each phospholipid, from 5 to 20 mg, to plates and then analyzing by the above procedures revealed a linear relationship of concentration to both densitometry recording and phosphorus analysis. This method is advantageous in that it can be used with large plates in preparative thin-layer chromatography, fractions can be collected and eluted for subsequent fatty acid or other analysis, and the percentage composition can be determined from the border of the same plate by densitometry. In addition, a permanent record of the separation can be retained for later reference. Departments
R. J.
of Pathology, Harbor General Hospital, Torrance, California
and the U.C.L.A.
School of Medicine,
MORIN
Los Angeles, Calif. (U.S.A.)
I H. K. ~UANGOLD AND R. KAMMERECK, J. Am. Oil Chem. Sot., 3g (1962) 201. 2 M. 1~. BLANK, J. A. SCHMIT AND 0. S. PRIVETT,J. Am. OilChem. Sot., 41 (1964) 371. 3 0. S. PRIVETT,M. L. BLANK, D. W. CODDING AND E. C.MICKELL, J. Am. OilChem. Sot.,
(1965) 381. 4 D. B. ZILVERSMIT AND A. K. DAVIS, J. Lab. C&n. Med.,
35 (1950)
4z
155.
Received October roth, 1965 Clin. Chim. Acta,
A densitometric
13 (1966)
method for the quantitative determination
395-397
of
protein fractions after agar gel electrophoresis The difficulties concerning the densitometry of amidoblack-stained protein fractions in serum or cerebrospinal fluid after agar gel electrophoresis have been discussed by Wiemel. Special attention is drawn to the difference in dye-binding capacity of the various fractions. Van der Helm2 proposed a method for quantitative determination by elution of the fraction and estimating the amount of dye bound by simple calorimetry. In this paper a densitometric procedure is described, that gives values which can be compared with Van der Helm’s method. Moreover it is less complicated and by carrying out both methods we found them to be equally accurate (Table I). TABLE
I MIXTURES
ANALYSESOFALBUMIN-y-GLOBULIN MEAN
VALUES
Mixture 1
FROM
IO ANALYSES
___~__~~
MIXTURE
Densitometry
El&ion
65%
53% 37%
35% Mixture II
PER
albumin globulin S.D. 2.4%
750/balbumin 250/bglobulin S.D. 2%
method
S.D. 1.2:~ 75% 25% S.D. 39; ~____ Clin. Chim.
Acia,
13 (1966)
397-399
SHORT COMMUNICATIONS
398 Electro$horesis
The agar used is the commercially available Difco-Noble product, which contains several impurities3. A gel is made by dissolving 9 g of agar in 500 ml of boiling water. The hot solution is poured out in Petri dishes and after cooling the gel is cut into small cubes of about I cm size. Purification is then performed by leaving the cut agar during 3 h in 500 ml of 50% ethyl alcohol. The flask is occasionally shaken and the same washing is repeated once more. After the second alcohol treatment the latter is removed by swirling the agar gently with 500 ml of water. The gel is then dissolved in 500 ml of buffer pH 8.4 containing 17 g of sodium diethyl barbiturate and 23 ml of I N HCl per IOOOml. After cooling, z-mm thick slides are prepared in the usual way. A 4-mm long slit is then made with a razor blade and filled instantaneously with 0.5~1 of serum containing about 30 ,ug of protein. The slit is not dried with filter paper as usual and the application of the serum in the slit is carried out with a very thin capillary glass micro pipette, which is specially constructed and calibrated for this purpose. Electrophoresis is done in a Vitatron apparatus (slightly modified Wieme apparatus) at a constant voltage of 150 V per slide at 20’ during 25 min. The slides are fixed for I 11 in a mixture containing acetic acid, ethyl alcohol and water in a ratio of I : 16: 4 by volume. Next staining of the proteins is performed in an amido-black-IoB solution for 30 min, because we found that reproducibility of results is rather unsatisfactory after staining for a short time only. The color bath contains 0.5 g of amidoblack-roR and 5 g of mercuric chloride in IOO ml of acetic acid 57;. Densitonzetry
The electropherograms are scanned with a Vitatron recording densitometer using a slit of 4x2 mm. The lamp is a mercury vapor discharge tube, the light of which is used without a filter. Discussion
and resdts
With mixtures of known composition of albumin and ;,-globulin we studied the relation between the real amount of protein present in its region on the slide and the values obtained by densitometry. A possible correction factor for calculating the amount of protein from densitometry data is related, a.o., to the dye-binding capacity of each kind of protein and also to the dimensions of the used slit in the apparatus, the kind of light used and the type of stain used. In the procedure described here, this factor does not differ from unity. TABLE
II
REPRODUCIBILITY
OF THE
METHOD
~-~
Mean values of 17 normal sera in percent of total protein S.D. of the mean S.D. in percent of total protein for of the same serum
15
analyses
Alb.
m,
aa
8,
82
Y
59.0
4.3
12.5
7.5
3.4
12.9
3.8
1.0
2.2
1.6
0.7
2.6
I.9
1.0
I.5
1.0
0.6
1.0
__-
C&z.
Chim.
Ada,
rj
(1966)
397-399
399
SHORT COMMUNICATIONS
The reproducibility of the method is checked by performing 15 separate analyses from the same serum (Table II). Normal values for human serum are determined from the analyses of the sera of 17 normal persons; standard deviations are calculated (Table II). A. H. J. GIJZEN E. M. M. A. VAN NUNEN P. E. VERHEESEN
Clinicd Laboratory of St. Joseph Hospital*, Heerlec (The Netherlands) in H. PEETERS (Ed.), Protides of the Biological Elsevier, Amsterdam, 1964. p. 397. 2 H. J. VAN DER HELM, Cliw. Chim. Acta, IO (1964) 483. 3 H. H. KREUTZER, C&z. Chim. Acta, g (1964) 404. I
R. J.WIEME,
Fluids.
11th
Coil.
Bruges,
1963.
Received October zoth, 1965 ____ * Head: P. J. Brombacher Clin.
The fatty acid composition of muscular dystrophy
of infiltrating
Chim.
Acta,
13 (1966)
397-399
fat in muscle from a case
One of the characteristic features of human muscular dystrophy is the infiltration of the diseased muscle by fat cells. The extent of infiltration varies with the site of the muscle and the stage of the disease. In the later stages some muscles may show a virtually complete replacement of fibres by fat cells. In general, fatty filtration is most prominent in the Duchenne (childhood) type of muscular dystrophy, and less so in the adult forms. It is less evident in other muscle diseases such as polymyositis and neurogenic muscular atrophies. The origin of the fat cells has not been established. Histologically they are similar to those of normal adipose tissue. No detailed data on the composition of the fat in dystrophic muscles is available. It was of interest, therefore, to determine the nature of the constituent fatty acids, which could then be compared with those of normal adipose tissue. Information on the latter has been provided by a fewworkers1-3. Samples were obtained from three different muscles at autopsy on a seventeenyear-old boy with advanced Duchenne-type muscular dystrophy. In each case the fibres had been almost completely replaced by fat. The tissues were stored at --zoo for several days and then extracted with 15 ~01s. of chloroform-methanol (z : I).Thinlayer chromatography on silica gel indicated that the lipids were almost entirely (99.9%) triglyceride, with traces of phospholipid, diglyceride and free fatty acids. The triglycerides were eluted from the plates and methylated by refluxing in methanol-benzene-H,SO, (150: 75 :7.5) (3 ml/zo mg lipid) for 90 min. Gas-liquid chromatography was carried out with an 8 ft. long 4-mm column of polyethylene glycol succinate (13%) on Celite (IOO-IZO mesh) at ISO’ and N, flow rate of 40 ml/min, using a flame ionization detector. The fatty acids found and the proportion of each Clin. Chim.
Acta,
13 (1966)
399-400
SHORT COMMUNICATIONS
by phosphorus analysis each time, were also in good agreement, as indicated in Table I. Applying successive increments of each phospholipid, from 5 to 20 mg, to plates and then analyzing by the above procedures revealed a linear relationship of concentration to both densitometry recording and phosphorus analysis. This method is advantageous in that it can be used with large plates in preparative thin-layer chromatography, fractions can be collected and eluted for subsequent fatty acid or other analysis, and the percentage composition can be determined from the border of the same plate by densitometry. In addition, a permanent record of the separation can be retained for later reference. Departments
R. J.
of Pathology, Harbor General Hospital, Torrance, California
and the U.C.L.A.
School of Medicine,
MORIN
Los Angeles, Calif. (U.S.A.)
I H. K. ~UANGOLD AND R. KAMMERECK, J. Am. Oil Chem. Sot., 3g (1962) 201. 2 M. 1~. BLANK, J. A. SCHMIT AND 0. S. PRIVETT,J. Am. OilChem. Sot., 41 (1964) 371. 3 0. S. PRIVETT,M. L. BLANK, D. W. CODDING AND E. C.MICKELL, J. Am. OilChem. Sot.,
(1965) 381. 4 D. B. ZILVERSMIT AND A. K. DAVIS, J. Lab. C&n. Med.,
35 (1950)
4z
155.
Received October roth, 1965 Clin. Chim. Acta,
A densitometric
13 (1966)
method for the quantitative determination
395-397
of
protein fractions after agar gel electrophoresis The difficulties concerning the densitometry of amidoblack-stained protein fractions in serum or cerebrospinal fluid after agar gel electrophoresis have been discussed by Wiemel. Special attention is drawn to the difference in dye-binding capacity of the various fractions. Van der Helm2 proposed a method for quantitative determination by elution of the fraction and estimating the amount of dye bound by simple calorimetry. In this paper a densitometric procedure is described, that gives values which can be compared with Van der Helm’s method. Moreover it is less complicated and by carrying out both methods we found them to be equally accurate (Table I). TABLE
I MIXTURES
ANALYSESOFALBUMIN-y-GLOBULIN MEAN
VALUES
Mixture 1
FROM
IO ANALYSES
___~__~~
MIXTURE
Densitometry
El&ion
65%
53% 37%
35% Mixture II
PER
albumin globulin S.D. 2.4%
750/balbumin 250/bglobulin S.D. 2%
method
S.D. 1.2:~ 75% 25% S.D. 39; ~____ Clin. Chim.
Acia,
13 (1966)
397-399
SHORT COMMUNICATIONS
398 Electro$horesis
The agar used is the commercially available Difco-Noble product, which contains several impurities3. A gel is made by dissolving 9 g of agar in 500 ml of boiling water. The hot solution is poured out in Petri dishes and after cooling the gel is cut into small cubes of about I cm size. Purification is then performed by leaving the cut agar during 3 h in 500 ml of 50% ethyl alcohol. The flask is occasionally shaken and the same washing is repeated once more. After the second alcohol treatment the latter is removed by swirling the agar gently with 500 ml of water. The gel is then dissolved in 500 ml of buffer pH 8.4 containing 17 g of sodium diethyl barbiturate and 23 ml of I N HCl per IOOOml. After cooling, z-mm thick slides are prepared in the usual way. A 4-mm long slit is then made with a razor blade and filled instantaneously with 0.5~1 of serum containing about 30 ,ug of protein. The slit is not dried with filter paper as usual and the application of the serum in the slit is carried out with a very thin capillary glass micro pipette, which is specially constructed and calibrated for this purpose. Electrophoresis is done in a Vitatron apparatus (slightly modified Wieme apparatus) at a constant voltage of 150 V per slide at 20’ during 25 min. The slides are fixed for I 11 in a mixture containing acetic acid, ethyl alcohol and water in a ratio of I : 16: 4 by volume. Next staining of the proteins is performed in an amido-black-IoB solution for 30 min, because we found that reproducibility of results is rather unsatisfactory after staining for a short time only. The color bath contains 0.5 g of amidoblack-roR and 5 g of mercuric chloride in IOO ml of acetic acid 57;. Densitonzetry
The electropherograms are scanned with a Vitatron recording densitometer using a slit of 4x2 mm. The lamp is a mercury vapor discharge tube, the light of which is used without a filter. Discussion
and resdts
With mixtures of known composition of albumin and ;,-globulin we studied the relation between the real amount of protein present in its region on the slide and the values obtained by densitometry. A possible correction factor for calculating the amount of protein from densitometry data is related, a.o., to the dye-binding capacity of each kind of protein and also to the dimensions of the used slit in the apparatus, the kind of light used and the type of stain used. In the procedure described here, this factor does not differ from unity. TABLE
II
REPRODUCIBILITY
OF THE
METHOD
~-~
Mean values of 17 normal sera in percent of total protein S.D. of the mean S.D. in percent of total protein for of the same serum
15
analyses
Alb.
m,
aa
8,
82
Y
59.0
4.3
12.5
7.5
3.4
12.9
3.8
1.0
2.2
1.6
0.7
2.6
I.9
1.0
I.5
1.0
0.6
1.0
__-
C&z.
Chim.
Ada,
rj
(1966)
397-399
399
SHORT COMMUNICATIONS
The reproducibility of the method is checked by performing 15 separate analyses from the same serum (Table II). Normal values for human serum are determined from the analyses of the sera of 17 normal persons; standard deviations are calculated (Table II). A. H. J. GIJZEN E. M. M. A. VAN NUNEN P. E. VERHEESEN
Clinicd Laboratory of St. Joseph Hospital*, Heerlec (The Netherlands) in H. PEETERS (Ed.), Protides of the Biological Elsevier, Amsterdam, 1964. p. 397. 2 H. J. VAN DER HELM, Cliw. Chim. Acta, IO (1964) 483. 3 H. H. KREUTZER, C&z. Chim. Acta, g (1964) 404. I
R. J.WIEME,
Fluids.
11th
Coil.
Bruges,
1963.
Received October zoth, 1965 ____ * Head: P. J. Brombacher Clin.
The fatty acid composition of muscular dystrophy
of infiltrating
Chim.
Acta,
13 (1966)
397-399
fat in muscle from a case
One of the characteristic features of human muscular dystrophy is the infiltration of the diseased muscle by fat cells. The extent of infiltration varies with the site of the muscle and the stage of the disease. In the later stages some muscles may show a virtually complete replacement of fibres by fat cells. In general, fatty filtration is most prominent in the Duchenne (childhood) type of muscular dystrophy, and less so in the adult forms. It is less evident in other muscle diseases such as polymyositis and neurogenic muscular atrophies. The origin of the fat cells has not been established. Histologically they are similar to those of normal adipose tissue. No detailed data on the composition of the fat in dystrophic muscles is available. It was of interest, therefore, to determine the nature of the constituent fatty acids, which could then be compared with those of normal adipose tissue. Information on the latter has been provided by a fewworkers1-3. Samples were obtained from three different muscles at autopsy on a seventeenyear-old boy with advanced Duchenne-type muscular dystrophy. In each case the fibres had been almost completely replaced by fat. The tissues were stored at --zoo for several days and then extracted with 15 ~01s. of chloroform-methanol (z : I).Thinlayer chromatography on silica gel indicated that the lipids were almost entirely (99.9%) triglyceride, with traces of phospholipid, diglyceride and free fatty acids. The triglycerides were eluted from the plates and methylated by refluxing in methanol-benzene-H,SO, (150: 75 :7.5) (3 ml/zo mg lipid) for 90 min. Gas-liquid chromatography was carried out with an 8 ft. long 4-mm column of polyethylene glycol succinate (13%) on Celite (IOO-IZO mesh) at ISO’ and N, flow rate of 40 ml/min, using a flame ionization detector. The fatty acids found and the proportion of each Clin. Chim.
Acta,
13 (1966)
399-400