Dyeing Method for Quantitative Deter minuti on of Lipides and Lipoproteins Directly on Filter Paper KATHERINE F. TALLUTO, RUTH R. BENERITO, and W. S. SINGLETON Southern Utilization Research and Development Division,
b A quantitative dyeing technique was required to measure accurately and rapidly the changes in serum lipide fractions after ,the administration of intravenous fat emulsions. The use of a 50% aqueous solution of diacetin saturated with Oil Red 0 dye at 25" C. prevents the loss of lipides from the paper strips into the dye bath, as usually occurs in alcoholic solutions of the dye a t 25" C., and results in a linear relationship between dye uptake and lipide concentration for various classes of lipides. Direct scanning of the dyed strips b y the method of transmission densitometry results in a linear relationship between integrated area and the concentration of a given lipide. Due to the differences in solubilities of the oil-soluble dye in various classes of lipides, there are differences in the partition coefficients of the dye between the dye bath and the various classes of lipides. However, within a given class, the variation is, the with alkyl group is small-that dye uptake of tristearin, for instance, varies little from the dye uptake of an equal quantity of triolein.
F
AS ISVESTIGATION by paper electrophoresis of the changes in animal sera following the in vivo and in vitro addition of fat emulsions, a quantitative dyeing technique was neccssary to locate and measure accurately and rapidly the amount of lipide associated with each serum protein fraction. The literature contains many references to the techniques of staining lipides by various oil-soluble dyes. However, there are many conflicting statements on the following: OR
The proportionality of dye uptake to lipide concentration for a given lipide The variation of dye uptake with the class of lipide a t a given concentration The validity of Beer's law in the case of absorbances obtained by direct ..canning of the paper strips stained for lipides I n general, the dye bath used in most methods for staining lipides is an alcohol-water solution (usually 50 to GOYo ethyl alcohol) which is saturated with one of the oil-soluble dyes. The majority of investigators interested in the study of serum lipides fractionated
U. S.
Deparfment of Agriculture, New Orleans, l a .
11ypaper electrophoresis use the method of S m h n (8, 9). I n this method the dye bath is approximately a 55y0 aqueous solution of ethyl alcohol which has been saturated with Sudan Black B, and the rinsing solution is also a *55y0 ethyl alcohol solution. I n his early work Swahn (8) found that the lipides in 10 ~ 1 of. serum could be dyed nnd scanned directly without elution. In later work he did not scan the dyed lipides directly, but eluted the dyed lipides n ith 2OY0 acetic acid in absolute ethyl alcohol and determined the extinction coefficients spectrophotometlieally a t 590 mp (9). I n the latter investigation he found that triolein, :in impure lecithin, corn oil, and serum lipides had the same color intensity, :ind all his experimental points fell dong the same straight line when the extinction coefficients were plotted against the concentrations of total lipides in milligrams per 100 ml. The concentration of lipides in this series cJf experiments covered a range from ,500 to 5000 mg. per 100 ml. of solution, hut the main lipide component in wery sample was a triglyceride. One of the few investigators who -canned the dyed lipide paper strips directly was Roboz (7), who also used the method of Swahn for lipide staining. In the investigation by Roboz et al., a linear relationship between absorbance expressed as scanned area and total lipide concentration in milligrams per 100 ml. was found on direct scanning. These strips were not treated t o minimize scattering of light, and the concentration range covered was rather =mall. The dye uptake was the same for serum lipides as for olive oil a t a given lipide concentration. However, the concentration range covered was within the range where the lipides are bound tightly to the filter paper strips. Fasoli (3) stained his electrophoreograms with Sudan 111. However, Durrum et al. (9) found Oil Red 0 dye wperior to Sudan I11 for lipide stainings. These latter investigators immersed the paper strips in a saturated solution of Oil Red 0 in 60% ethyl alcohol for 16 hours. KO data were given in evidence of the reproducibility of the dye uptake or the variation of dye uptake with the type of lipide. I n later
~ o r kJencks, Durrum, and Jetton (6) immersed the paper strips for 18 hours in a 60% ethyl alcohol solution saturated v i t h Oil Red 0 a t 30" C. and reported that the increase in dye uptake with increasing lipoprotein concentration is not strictly linear, that a portion of the lipides from serum lipoproteins dissolves off the papers into 60% ethyl alcohol solution in the absence of the dye, and that their method dyed triglycerides and lecithin but not cholesterols, oleic acid, palmitic acid, or ethyl esters of saturated fatty acids 17-ith 8 t o 16 carbon atoms. Adlersberg el al. (1) were unable to obtain reproducible results on staining paper strips from serum lipoproteins iT-ith Sudan Black because of the variations in uptake of the dye. These same investigators found that the uptake of Sudan I V by serum lipides v a s only about half of that obtained with Oil Red 0 in 60% ethyl alcohol. The densitometric readings with Oil Red 0 checked closely with results by elution and colorimetry. However, these investigators did not look into the mriation of dye uptake with different classes of lipides. I n the present investigation, quantitatively reproducible results were not obtained with any of the previously published dyeing techniques for the estimation of various classes of lipides on paper strips. Therefore, the technique described here was developed for the dyeing of serum lipoproteins as well as other lipides present in serum after the addition of a fat emulsion. The use of a 50% aqueous solution of diacetin saturated with Oil Red 0 at 25" C. prevented the loss of lipides from the paper strips into the dye bath and resulted in a linear relationship between dye uptake and lipide concentration within a given class of lipides. EQUIPMENT
The Junior Ionograph Model, manufactured by the Precision Scientific Co., was used to prepare the paper electrophoreograms. The dyed electrophoreograms or dyed papers which had been stripped with various kinds of lipides were scanned in a balanced-beam type of recording photometer called the Analytrol, manufactured by the Spinco Co. This instruVOL. 30, NO. 6, JUNE 1958
1059
ment has automatic scanning, integrating, and recording mechanisms which measure the dye uptake as an integrated area in square centimeters x 10-1. Accurately measured volumes of all lipide samples were applied to the paper strips by means of the Spinco stripper, a device consisting of parallel wires, approximately 1.5 cm. long and 0.1 cm. apart, cemented into a stainless steel holder.
scattering of light by the strips on direct scanning: Saturated solution of sucrose in water a t 25" C. A solution consisting of 200 ml. of xylene (Baker), 1 liter of paraffin oil (white, viscosity 125/135), 500 ml. of a-bromonaphthalene (Fisher), and 17 grams of Span 80 (Atlas Chemical Co. commercial emulsifier), a t 25" C. PROCEDURE
REAGENTS
Lipide Samples. T h e various types of lipides used were laboratory preparations of mono-olein, monostearin, diolein, distearin, triolein, tristearin, oleic acid, and stearic acid; a refined, bleached, winterized, and deodorized cottonseed oil; a clarified virgin olive oil; cholesterol; pooled normal human serum; and dog serum from blood withdrawn a t the height of chylomicra count after the oral administration of fat emulsions. Emulsions. Tn-o fat emulsions were used in a n oral f a t diet to produce lipemic dog serum. Oil-free fractionated soybean phosphatides, a sample of which was furnished by The Upjohn Co., and Pluronic F68 constituted the emuliifying system in one emulsion, I n the other the emulsifying agents were Drumulse, T E N , and Pluronic F68. Drumulse, a fatty acid ester of polyglycerol; T E N , an acetylated tartaric acid ester of a monoglyceride; and Pluronic F68, a polyoxyethylenepropylene, were commercial products obtained from E. F. Drew and Co., Hachmeister, Inc., and Wyandotte Chemical Corp., respectively. Both einulsions contained 15% by weight of a refined, bleached, winterized, and deodorized cottonseed oil, 5% by weight of dextrose in distilled water, and the indicated emulsifiers. The emulsions were prepared in a two-stage Cherry Burrell Superhomo homogenizer a t 3500 p.s.i.g. and contained only occasional particles as large as 1 micron in diameter. The general particle diameter was less than 0.7 micron after sterilization a t 120' C. for 15 minutes at 15 p.s.i.g. The emulsifiers previously mentioned were also applied individually to paper strips and their ability to retain fat dyes mas noted. Dye Baths. The dye baths were saturated solutions of Oil Red 0, (Sational Aniline Division, Allied Chemical and Dye Corp.), in t h e follou ing : 1. 60% ethyl alcohol in water a t 25" C. 2. 80% ethyl alcohol in water at 4" C. 3. 50'70 diacetin in water at 25' C. 4. Solution 2 diluted fourfold with 60% ethyl alcohol at 4' C. 5. Solution 1diluted fourfold with SOY0 ethyl alcohol a t 25" C.
Solution to Minimize Light Scattering by Strips. T h e following two solutions were found t o be most satisfactory in reducing to a minimum t h e 1060
ANALYTICAL CHEMISTRY
Preliminary experiments with Whatman 3 A131 filter paper, which was used in the electrophoresis experiments, indicated that the use of Oil Red 0 was preferred to Sudan I V or Sudan Black B because the Sudan dyes gave more intense background colors. Each lipide sample, with the exception of the serum lipides, was applied to the strip as a 1% by weight solutioii in chloroform. The serum lipides were applied as such and not in solvent solution. I n all experiments each lipide sample was applied as a film across the wires of the Spinco stripper from a graduated micropipet, and thence transferred to the paper strips. Concentrations of each sample ranging from 10 t o 50 pl. were applied to several paper strips. These strips, containing known concentrations of the various lipides, then n-ere immersed in the different dye baths, rinsed, dried, treated with a solution to minimize light scattering, and scanned in the automatic photometer and integrator. Paper electrophoresis of serum was carried out according t o the procedure given in detail previously (4). RESULTS A N D DISCUSSION
K h e n the most generally adopted procedure of preparing a dye bath by saturating 60% ethyl alcohol a t 25" C. with Oil Red 0 was followed, the stearic acid, oleic acid, monostearin, mono-olein, and Pluronic F68 samples were removed completely from the papers. The diglycerides and triglycerides 'ivere removed only partially. I n the case of the triglycerides, a maximum adherence of sample to paper was reached when 20 pl. of solution were applied. The amount of dye uptake leveled off and was the same for samples of 20, 30, 40, and 50 pl., within experimental error. Only in the case of the lipoproteins was the dye uptake proportional to the concentration of sampIe applied to the strip. Data in Table I are typical of the results obtained rvith Oil Red 0 in 607, ethyl alcohol a t 25" C. Under these conditions the partition coefficients for the dye between dye bath and lipide samples varied greatly. For those samples which did not dissolve off the papers, the average values for IO-pl. samples of 1% solutions expressed as integration units (sq. cm. X 10-1) were as follows: soybean phosphatides, 3; cholesterol, 5 ; TEX, 8;
distertrin, 15; Drumulse', 16; diolein, 20; tristearin, 18; olive &I, 28; and cottxlnaeed oil, 31. In order to determine if the loss of certain types of lipides from the strips could be minimized by lowering the temperature and dye concentration, two dye baths rrere used. I n one, an aqueous solution of 60% e t h r l alcohol at 4" C. was saturated with Oil Red 0 and kept in a cold room a t 4' C. The other mas made by diluting a 60% ethyl alcohol solution, which previously had been saturated with Oil Red 0 a t 4" C., fourfold with a 60% solution of ethyl alcohol at 4" C. Some of these results also are included in Table I. Figure 1 shows the variation of dye uptake by triglycerides with time, temperature, and concentration of the dye. The graph indicates that a t least 16 hours were required t o reach equilibrium. Curves in Figure 1 and data in Table I show that at 4" C. the solubility of the Oil Red 0 was too small even in the triglycerides to give large enough areas when the strips were scanned directly. A t this low temperature the larger samples of triglycerides did not dissolve off the paper as they did a t 25" C., as data in Table I show that the scanned area was proportional to the concentration of a given triglyceride throughout the concentration range investigated at 4' C. However, the monoglycerides and fatty acids were soluble in the 60% ethyl alcohol even at the lower temperature. Figure 1 and the data in Table I show that use of ethyl alcohol solutions of Oil Red 0 did not give sufficiently quantitative results for the intended use a t 25" C. The solubility of the dye in a given lipide was proportional to the amount of lipide applied to the strip only when the dye was absorbed from a saturated solution of Oil Red 0 in 50% diacetin at 25" C. for a period of a t least 16 hours. I n the diacetin dye bath there was much greater dye uptake by the pure triglycerides than by the lipoproteins, Drumulse, distearin, or soybean phosphatides. I n the instances of olive oil and cottonseed oil, the average areas were 23 f 1.1 and 24 =t2.4 integration units per 10 pl, of the 1% solutions, respectively. These n-ere the averages of 22 samples covering a concentration range of 10 to 50 pl, of solutions applied to the strips, dyed from 18 to 24 hours in the diacetin dye bath, washed, sprayed with a-bromonaphthalene solution, and scanned in the automatic scanner and integrator. The niem values for 10 pl. of 1% solutions of soybean phosphatides, cholesterol, T E N , distearin, diolein, Drumulse, and tristearin under similar conditions nere 1, 2, 3, 6, 7 , 3, and 6 sq. cm. X lo-*, respectively.
Table I.
Dye Uptake after 18 Hours in Dye Baths" 10
X
Sample Lipemic dog serumh 1 tristearin lyc olive oil 15%Wesson oil
I
a
18 28
31
-
Lipemic dog serum* 1c,c tristearin 1% olive oil IC,%Wesson oil
1
1
'
B
12
IS
TIME
IN
20
24
a
'3
11
12
28
DYE B A T H , HOURS
Figure 1. Variation in dye uptake with concentration and temperature of d y e bath
0 3
Wesson oil Olive oil Dye baths contoin saturated solution of Oil Red 0 in: A. 60% ethyl alcohol a t 25' C. E. Solution A diluted fourfold with 6OY0 ethyl alcohol a t 25' C. C. 60% ethyl alcohol a t 4 ' C. D. Solution C diluted fourfold with 60% ethyl alcohol at 4 " C. E . 50% diacetin a t 25' C.
111 the determination of lipide fractions in all electrophoreogran~sof animal cera, the 50% aqueous solution of diacetin saturated with Oil Red 0 n a s used as the dye bath because the diglycerides and triglycerides did not nash off the paper strips during the 16-hour immersion period at 25' C. The electrophoreograms dyed for lipide fractions were always sprayed IT ith an a-bromonaphthalene solution in order to fill the interstices of the papel. with a solution possessing the same indrx of refraction as that of the cellulose ( n = 1.55). This spraying technique, which minimized the aiiiount of light scattering when thc strips n u e scanned in the Analytrol, prevented the spreading of the fat spots 11 hich occurred during immersion of the dyed strips in various solutions possessing the proper indices of refraction. T h r ~only other technique found to iiiiniiiiize the amount of light scattering nitliout the spreading of the lipides u a q the immersion of the dyed strips in a saturated solution of sucrosc, and the scanning of the strips immediately after drying in an oven a t 110' C. If the strips were completely dry n hen they were scanned, the interstices n ere filled with sucrose of approsinlately the same index of refraction as the cellulose. However, these sugar-coated strips became slightly dainp a i d limp due to the absorption of moisture and n ere no longer as transparent as the dr? strips. Therefore, the method of
-
1% . - olive oil 152 n'esson oil
Area, Sq. Cm. X 10-1) S o h ._____ -4 10 15 20 25 34 42 39 40 60 60 61 GO 57 GO 58 60 Area, Sq. Cm. X Soln. B ~ _ _ G 10 12 15 12 18 25 30 17 26 33 18 27 36 Area, Sq. Cm. X 10-l) Soln. C __ ~~~~22 2J 24 25 25 24 24 26 Area, Sq. Cm. X 10-l) Soln. D 10 1!5 2 0 25 10 1G 20 24 ~
8
G
IC; olive oil lC& \Yesson oil
1
Volume of Lipide Sample or Strip, p l . __ 20 30 40 50
~~
5 J
Area, Sq. Cm. X 10-l, Soln. E ~ _ _ _ _ -I Pooled normal human serumc 14 20 28 34 Lipemic dog serumb 6 13 19 25 38 15%tristearin ii 12 20 24 30 157, olive oil 2 :3 46 6;; 94 113 lYc \Yesson oil 25 54 79 109 125 1'3; distearin 6 12 19 26 31 15; soybean phosphatides 1 2 3 4 0 1% Drumulse 3 5 9 13 15 a Saturated solutions of Oil Red 0 in: A, 60% ethyl alcohol at 25" C.: B, 60% ethyl alcohol at 4" C.: C, A diluted fourfold TTith 60% ethyl alcohol at 25" C.; D, B dilut,ed fourfold with 60% ethyl alcohol at 4' C.; E, 507, diacetin at 25' C. Contained 540 mg. of lipides and 105 mg. of cholesterol per 100 ml. Contained GO0 mg. of lipides and 230 mg. of cholesterol per 100 ml. Table
II. Variation in Scanned Areas after Treatment to Minimize Light Scattering
Volume of 1:; Treatment before Scanning Sone
Dye Batha A A B
Dipped in saturated aqueous solution of sucrose and dried Spra!-edRithsolutionof a-bromonaphthalene in paraffin oil
-4 -k B -4
Lipide Sample Cottonseed oi 1
10
Lipide Solutions Applied
to Strips, pl.
25 30 35 40 45 50 60 Area, Sq. Cm. X 10-' __ 8 18 20 25 YO
olive oil 5 9 14 20 Cottonseed 28 40 48 42 43 50 56 44 oil Cottonseed 8 18 27 36 oil Olive oil 10 20 30 42 Cottonseed 27 54 65 80 95 110 oil Cottonseed 8 17 23 27 32 37 40 45
68
54
oil
.4 B
Olive oil 8 17 Cottonseed 23 46
26 6i
33
94 113 oil -111 strips immersed 22 hours in dye bath of saturated solution of Oil Red 0 in: A, GOC, ethyl alcohol at 4 ' C.; B, 50yGdiacetin at 25" C. (1
spraying with the a-bromonaphthaleiie solution was adopted. Data in Table I1 emphasizc the iniportance of treating the dyed paper strips before scanning with a substance which minimizes the scattering of light. The areas scanned on the untreated strips do not vary linearly with the concentrations of lipidrs. Howerer, after these same strips are treated n-ith sucrose or with oil of n = 1.55, a linear variation is obtained. When the electrophoreograiiis of pooled normal human serum were
dyed from 16 to 24 hours in a 50% aqueous solution of diacetin saturated with OilRed 0, two bands were observed. One band, the a-lipoprotein, always occurred between the cy1- and arglobulins; its scanned area represented 25y0 of the stainable lipides. The other band, the p-lipoprotein fraction, almays occurred with the 0-globulins and represented approximately 70yo of the stainable lipides. The electrophoreograms of dog serum showed a distribution of fat different from that observed in human serum. VOL. 30, NO. 6 , JUNE 1958
1061
Quite frequently, the P-lipoprotein fraction was absent in the electrophoreograms of a fasting dog serum. I n the electrophoreograms of the dog serum obtained at the height of chylomicra count after the oral administration of fat emulsions, the relative percentage of a-lipoprotein was greater than that of the p-lipoprotein. This difference in distribution of lipoproteins in dog and human serum has been observed by other investigators. h'ikkila (6) observed that the greatest reduction on delipidation of dog serum occurs in the a-globulin portion and not in the pglobulin portion, whereas the greatest reduction in human serum on delipidation occurs in the beta fraction. With a given serum from either humans or dogs, the scanned areas varied linearly with the quantity of serum applied to the strip when the electro-
phoreograms were stained for at least 16 hours in a 50% aqueous solution of diacetin saturated with Oil Red 0 a t 25' C. All electrophoreograms obtained on a given serum and stained for lipides gave quantitatively reproducible a- and P-lipoprotein areas n-hen scanned in the Analytrol. ACKNOWLEDGMENT
This investigation was supported in part by funds from the Office of the Surgeon General, and was carried out in one of the laboratories of the Southern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture. LITERATURE CITED
(1) Adlersberg, D., Bossak, E. T., Sher, I. H., Sobotka, H., Clin. Chem. 1, 18 (1955).
Durrum, E. L., Paul, 11, H., Smith, E. R. B., Science 116, 428 (1952). Fasoli, .\.,'Lancet 1, 106 (1952). Formusa, Katherine AI.,, Benerito, Ruth R., Singleton, JJ-. S., White, J. L., *\XAL. CHEDI. 29, 1816 ( 1 9 5 7 ~ Jencks, 13'. P., Durrum, E. L., Jetton, R., J . Clin.Incest. 34, 1437 (1955:. Xkkila, E., Scand. J . Clin. K. Lab. Inaest. 5 : S U P D 8. ~ . 5 (1953). Roboz, E.,. Heis; IT: C', Forster, F. 11.,Temple, D. AI,, A . X . A Arch. Seurol. Pszichicit. 72. 154 (1954). (8) Swahn, B., Scand. J . C l i n . ~k Lab. Incest. 4, 98 (1952). (9) Ibid., 5 ; Suppl. 9, 5 (1953). '
RECEIVEDfor review June 26, 1957. Accepted January 16, 1958. Trade names have been used only t o identify equipment or materials actually used in conducting this work. Their use does not imply endorsement or recommendation by the U. S. Department of Agriculture over other 6rms or similar products not mentioned,
Determination of Zirconium in Titanium AIIoys Using p-Bromo- or p-Chloromandelic Acid ROLAND A. PAPUCCI
F. C. Broeman and Co., Cincinnati, Ohio JOSEPH J. KLINGENBERG Xavier University, Cincinnati, Ohio
b Zirconium has found application as an alloying element in titanium. Zirconium in amounts from 0.1 to 10.0% can b e successfully determined in commercial titanium alloys by the halomandelate procedure using p bromo- or p-chloromandelic acid as reagent.
T
use of p-chloro- and p-bromomandelic acid in the determination of zirconium in steels, aluminum alloys, and magnesium alloys has been reported (1, 2, 3). This paper deals with the use of these reagents for the determination of zirconium in titanium alloys. A somewhat similar determination using mandelic acid has been proposed ( 5 ) . The halomandelate method is accurate in the range from 0.1 to 2.0% zirconium and in the range from 2.0 to 10.0% if the zirconium is reprecipitated. Successful determinations were carried out in the presence of known amounts of aluminum, chromium, iron, manganese, and tungsten as alloying elements. Unanalyzed trace amounts of beryllium, boron, calcium, cobalt, copper, lead, magnesium, molybdenum, nickel, niobium, silicon, silver, tantalum, tin, HE
1062
ANALYTICAL CHEMISTRY
Table I.
Sominal Composition of Alloy RC-130B bIn-4,OO -41-4.00 0-0.30 K-0.05 WFO. 04 C-0 .06
Determination of Zirconium in Titanium
Zirconium Added,
Z i r c o h m Found, % p-Bromomandelic acid p-Chloromandelic wid Wthout Reprecipitation 0.100 0.10 0.09 0.09 0 08
%
0.09
0.09
0.09 0.09 0.10
0.10 0.09
0.i0 0.10 0.10
0. ii
0.10
0.10 0.096
Av. 0.093 0.500
0.50 0.53 0.49 0.52
1.00
0.51 0.53 0.50 0.50 0.50 Av. 0.509 1.02 1.02 1.02
1.04 1.03 1.00 1.01 1.00
Av. 1.02
0.51 0.50 0.49 0.51 0.53 0.52 0.52
0.54 0.50 0.513
1.02 1.00 1.01 1.01 1.01 1.02 1.02 1.04 1.01 1.02
