Anal. Chem. 1982, 5 4 , 2079-2082
are observed for treatment with NaHC03, the sample is not affecte to as great a depth as for treatment with H2S04. L. NaOH Treatment. Treatment with NaOH had the most dramatic effect on surface of Haynes alloy. Before sputtering Cr and W concentrations ares decreased and Co is increased as is C contamination without significant change in the Ni concentration1 (Figure 7) relative to the "as-received samples. Even after sputtering the concentrations of the metals on a surface treated with NaOIl are not within the control limits established by the "as-received" samples (Figure 8). AES depth profiling indicates that the depth of the layer affected by NaOH treatment is on the order of 30-35 A. However, the oxide layer thickness calculated from ESCA data is approximately 10 A. Layer thicknesses calculated from AES and ESCA data for the samples treated iin hot NaOH are not in agreement as they were for the other treatments. It appears that the species on the surface are changed dramaticaly by the hot NaOH treatment. Therefore, the ESCA escape depths and peak area ratios used to calculate the ESCA layer thickness are not applicable to the samples which were treated with hot NaOH.
C(3NCLUSIONS The major result of the Haynes alloy studies is that the surface is inert to most acid and base treatments, probably because of the chromium passivatiug layer on the surface. Treatments which affect the surface (H2S04,NaHCO,, and NaOH) are all solutiona in which Cr203is at least slightly soluble. Another important aspect of this study is the complementary nature of the data obtqined from the surface analysis
2079
techniques. AES data gave elemental concentrations and information about thickness of the topmost layer and showed that only short sputtering times were required to reach bulk concentrations after most treatments. SIMS showed similar information about the increased Cr concentration of the surface of Haynes alloy; but because of the limitations of the quadrupole, SIMS gave little information about the tungsten. ISS data agreed well with AES about the increased Cr and W concentrations on the alloy surface. ESCA data give information about the composition of the Haynes alloy surfaces including concentrations of the elements present, information about the oxides present, and an approximate thickness of the oxide layer. The latter is in good agreement with the layer thickness calculated from AES data. By use of a combination of surface analysis techniques, a reasonably complete description of the surface of Haynes alloy can be obtained, including elemental composition, oxidation states of the metals present, and thickness of the surfqce layer.
LITERATURE CITED (1) "Handbood of Auger Electron Spectroscopy"; Physical Electronics, Inc.; Minnesota (1972). (2) Wagner, C. D. Anal. Chem. 1972, 4 4 , 1050-1053. (3) Weast, R. C., Ed. "CRC Handbook of Chemistry and Physics", 51st ed.; Chemical Rubber co.: Cleveland, OH, 1970. (4) Seah, M. P. J . Catal. 1979, 57, 450-457. (5) Penn, D. R. J. Electron Spectrosc. Relat. Phenom. 1976, 9 , 29-40. (6) Storp, S.; Berreshelm, K.; Wilmers, M. SJA Surf. Interface Anal. 1979, I , 96-99. (7) Graham, S . W. Ph.D. Thesis, University of Pittsburgh, 1981.
RECEIVED for review February 16, 1982. Accepted June 25, 1982. This work was supported, in part, by Grant No. CHE-81-08495 from the National Science Foundation.
Determination of Bile Acid Monomers in Micellar Solutions Helmut V. Ammon" and L. Greg Walter Gastroenterology Section, \/A Medical Center, 5000 West National Avenue, Wood, Wisconsin 53 193
A simple method for the! determinatlon 01 monomer concentrations of conjugated blile aclds in mlcellar and mixed mlcellar solutlons Is described. I t utilizes the small exclusion llmits of Sephadex G-10 beads. Only bile acids in the monomer phase are able to enter the bemds. When small amounts of these beads are incubated in mixed micellar solutlons contalnlng 14Clabeledbile aclds and 3H-labeled rafflnose or sucrose, the monomer concentration cf blle aclds can be determined from the known concentratloin of the blle aclds In the incubation mlxture and the ratio of tlhe markers In the Incubation medium and in the beads. Monaimer concentrations of taurocholate and taurodeoxycholate were determlned for solutlons contalnlng 0.01-50 mM blle aclds. Phosphatidylcholinereduced the monomer concentratllon of 5 mM taurodeoxycholate. The results are consistent wltb the mlcellar properties of these solutlons and with predictions about the monomer concentration from thermodynamic calculations.
Determination of monomer concentration of bile acids in mixed micellar solutions is important for an understanding of their interaction with biological membranes (1)and of the
mechanisms of micelle formation (2),biliary secretion (3), and dissolution of gallstones ( 4 ) . So far no satisfactory method has been available. Equilibrium dialysis has the disadvantage that it cannot distinguish between monomers and dimers in the intermicellar phase (5). Ultrafiltration has similar shortcomings (6). A recently described membrane electrode (7) produces data for the critical micellar concentrations of bile acids inconsistent with previously published data (8). Here we describe a simple method which utilizes Sephadex G-10 beads to separate monomers of bile acids from dimers and mixed micelles without disturbing the dynamic micellemonomer equilibrium.
EXPERIMENTAL SECTION Materials. [14C]Taurodeoxycholateand ['4C]taurocholatewere purchased from California Bionuclear Corporation, Sun Valley, CA, [3H]raffinose,[3H]sucrose,and [14C]polyethyleneglycol-4OOO (PEG-4000) from New England Nuclear, Boston, MA, taurocholate and taurodeoxycholate from Calbiochem, La Jolla, CA, raffinose from J. T. Baker Chemical Co., Fhillipsburg, NJ, and sucrose and egg phosphatidylcholine from Sigma Chemical Co., St. Louis, MO. Egg phosphatidylcholinewas further purified by column chromatography (9). Procedures. The method uses the fact that monomers of conjugated bile acids (molecular weight 450-516) can enter
0003-2700/82/0354-2079$01.25/00 1982 American Chemlcal Society
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 12, OCTOBER 1982
Sephadex G-10 beads (exclusionlimits 700 daltons), while dimers are too large. When Sephadex G-10 beads are incubated in a micellar solution of bile acids, the bile acid concentration within the beads, therefore, will represent the monomer concentration in the incubation medium. By using 14C-labeledbile acids and tritium labeled raffinose or sucrose as aqueous phase markers, one can therefore determine the monomer concentration of a given bile acid in a micellar solution without disturbing the phase equilibrium. A 200-mg portion of Sephadex G-10 beads was incubated in 10 mL of a buffered solution of bile acids. In some validation experiments Sephadex G-15, G-25, and G-50 were also used. The incubation mixture consisted of 20 mM sodium phosphate buffer, pH 7.4, known amounts of bile acids, and lipids under investigation, with NaCl added to a final Na concentration of 156 mM. In addition, the mixture contained the corresponding 14C-labeled bile acids (0.3 pCi/lO mL) and 3.0 pCi/lO mL of [3H]sucroseor [3H]raffinosewith a final sugar concentration of 10 ymol/L. The incubation mixture was shaken at 190 oscillations/min for 48 h at room temperature. The beads were then separated from the mother liquor by filtration through a 450-nm Millipore filter (Millipore Corp., Bedford, MA). To remove any label adherent to the surface of the beads, we rinsed them once with the buffer solution under rigidly controlled conditions. A 5-mL portion of the buffer solution was injected into the Millipore filtration cell (25 mm id.) which was then rapidly pressurized with 10 1b/ins2 nitrogen by the turn of a three-way stopcock. The rinsed beads were divided between two scintillation vials, 1mL of buffer was added, and, after the mixture was shaken for 48 h, 10 mL of scintillation fluid (Ready Solv HP, Beckman Instruments, Fullerton, CA) was added and the radioactivity determined. The time for the initial incubation of the beads with the buffer solution and for the washout of the marker was chosen arbitrarily based on the studies by Duane (IO). An extension of these time periods to 72 h did not change the results. Shorter incubation periods might be sufficient. The concentration of a bile acid in the monomer phase [BA], was calculated from the known concentration of the bile acid in the incubation mixture [BA]i and the ratio of 14C(bile acid) and 3H (aqueous phase marker) in the incubation medium (i) and in the beads (b) as follows: [BA], = r[BAIi
(1)
where
Under ideal conditions, r should equal 1 below the critical micellar concentration (CMC). Because bile acids interact with the beads (see below) and because the effective molecular radius of the aqueous phase marker differs from that of the bile acids, marker and bile acid will be washed out at different rates during the rinsing. If r for a given bile acid is constant over a range of concentrations below the CMC, it can be used as a correction factor (r,) to calculate monomer concentrations above the CMC provided that r, is determined with each experiment. The final calculation, therefore, is
(3) r, was determined for each run as the mean of the values obtained for 0.01, 0.1, and 1 mM of the bile acid under study. To detect any interaction between the beads, the aqueous phase markers, and the bile acids, we determined the elution volume (VJ and the partition coefficient (Kav)(11)for trace amounts of these compounds between the gel phase and the aqueous phase by chromatography on Sephadex G-10. The elution buffer was identical with that used in the incubation experiments and contained 0, 10, or 50 mM bile acids. The column used for the determination of K,, of the sugars was 28.5 cm long with a 1.5 cm internal diameter. For the determination of K, of the bile acids the column was 25 cm long with an internal diameter of 0.4 cm. The operating pressure was 50 cm of water. The void volume (V,) was determined with 14CPEG-4000, the total volume of the
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53
(mM)
Monomer concentration of taurocholate (n = 3) and taurodeoxycholate (n = 6) in relationship to increasing bile acid concentration. The data are means f standard deviation. The aqueous phase marker was raffinose. Flgure 1.
,.
IE
” rn 15 4 3
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u
+
10
o
10
20
30
40
50
BULK CONCENTRATION (mM)
Flgwe 2. Uptake of taurdeoxycholate by Sephadex beads of varying size in relation of bile salt concentration in the bulk phase. Data are means f standard deviation (n = 4 for G-50,G-25,and G-15 and n = 6 for G10). All data for G15 above 2 mM taurodeoxycholate were statlstically significantly different from those for G10 (P < 0.01). The aqueous phase marker was raffinose. The standard deviations for the data points below 10 mM taurdeoxycholate were too small for graphic display.
gel bed (V,) with tritiated water. All volumes were measured at the peak of the elution curves.
RESULTS AND DISCUSSION Figure 1shows the monomer concentrations of taurocholate (n = 3) and taurodeoxycholate (n = 6) over a range of bile acid concentrations from 0.01 to 50 mM with raffinose as the aqueous phase marker. The monomer concentration for 10 mM taurocholate measured in nine separate determinations was 6.7 f 0.55 mM. The small standard deviations indicate an excellent precision of the method. The monomer concentration of taurodeoxycholate rises linearly with the concentration in the bulk phase up to the critical micellar concentration (CMC). I t shows only a minimal increase thereafter. As judged by the changes in monomer concentration, the CMC of taurodeoxycholate is about 1.8 mM. In contrast, the monomer concentration of taurocholate continues to rise significantly with the bile acid concentration in the bulk phase above the CMC, and it would be difficult to define a CMC from the monomer data. When sucrose was used, a virtually identical curve was obtained for taurodeoxycholate; the data obtained for taurocholate, however, showed too great a scatter to be useful. Therefore, raffinose is the preferred aqueous phase marker. T o test our assumption that the measured uptake of bile acids by the Sephadex G-10 beads is a function of the exclusion radius, we measured bile acid uptake by Sephadex beads with larger exclusion radii (Figure 2). Bile acid concentration inside the beads increased with increasing exclusion radius. In the larger beads it also increased with increasing bile salt concentration in the bulk phase. This is consistent with the concept that micelles of different sizes coexist in micellar solutions (2). Although the exclusion radius of Sephadex beads has been calibrated with dextran molecules, Sephadex G-10 beads seem to exclude dimers of conjugated bile salts effectively for the
ANALYTICAL CHEMISTRY, VOL. 54, NO. 12, OCTOBER 1982
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Table I. Partition Coefficients (K,) of Sugars and Bile Acids during Column Chromatography on Sephadex G-10 %VU
a
aqueous phase
raffinose
sucrose
taurodeoxycholate
taurocholate
phosphate buffer taurodeoxycliolate 10 mM taurodeoxycholate 50 mM taurocholate 1.0 mM taurocholate 50 mM
0.19 i 0.00 0.24 f 0.01 0.21 i 0.01 0.20 i. 0.00 0.20 f 0.00
0.34 i. 0.00 0.37 f 0.04 0.37 i 0.00 0.35 f 0.00 0.36 f 0.00
6.33 i: 0.09 1.06 i 0.02
1.85 i. 0.05 1.31 i. 0.01
The data are means +. standard deviation from three determinations.
Table 11. Correction Factor r, in Relationship to Bile Acid Concentration and Aqueous Phase Marker rea at different bile acid concentrations bile acid/aqueous phase marker 0.01 mM 0.1 mM 1mM taurodeoxyeholate/raffinose taurodeoxycholate/sucrose
taurocholate/raffinose tau roch ol ate /sucrose
32.3 t 2.9 22.5 i 0.7 10.1 i 0.3 6.8 i. 0.8
32.0 i. 3.3 21.2 i 2.4 9.6 i 0.3 6.9 i 0.8
33.4 i 1.7 21.8 i 1.7 9.7 i 0.2 5.9 i 1.6
no. of determns 6 3 3 7
7 he data are means i standard deviation. following reasons: (1)a significant entiry of dimers into the beads would result in an overestimation of the monomer concentration and of tihe CMC. The CMC data obtained for taurodeoxycholate, however, were very close to those obtained by other methods (8). (2) The intermicellar bile salt concentration for taurocholate is about half of that obtained by equilibrium dialysis, which cannot distinguish between monomer and dimers in the intermicellar phase (5). (3) The increase in the exclusion radius from 700 daltons (G-10) to 1500 daltons (G-15) resulted in a significant increase in bile acid uptake. Since G- 15 is far too small to admit tetramers (aggregation weight 1998.6), this increase in uptake can only be attributed to the uptake of dimers by the (3-15 beads. If dimers entered G-10 bmds, the curves obtained for G-10 and G-15 beads should be identical. Since no reliable direct measurements of monomer concentrations of bile acids in micellar solutions are available, the validity of the results can only be judged by comparison with data from studies of micelle formation. The CMC for taurodeoxycholate is close to results obtained by totally different methods (8). The rise of the monomer concentration of taurocholate above the CMC is consistent with thermodynamic calculations ( I ) . It is also conisistent with the observation by Duane that the intermicellar concentration of taurocholate continued to rise above the CMC in mixtures of taurocholate and phosphatidylcholintr (10). The absence of a distinct break of the monomer concentration curve a t the CMC of taurocholate is also consistent with the observation that the CMC of cholate cannot be well-defined by the solubilization of naphthalene (12). Our data show signikantly lower intermicellar concentrations for taurocholate than data obtained by equilibrium dialysis (10). This can be explained by the fact that equilibrium dialysis cannot eliminate dimers from the measurement of intermicellar bile salt concentration ( 5 ) . The principle of the method is based on the assumption that there is no interaction of the aqueous phase marker with other constituents in the solution or with the Sephadex beads. This assumption is supported by the chromatographic studies (Table I). The partition coefficients, K,,, for raffinose and sucrose were less than 1,K,, for sucrose was greater than that for raffinose, and the vallues were only minimally altered by either taurocholate or taurodeoxycholate. This indicates that there was no significant interaction between the gel and the sugars or between the bile acid micelles and the sugars and that they are therefore suitable aqueous phase markers in this
Table III. Effect of Phosphatidylcholine on Monomer Concentration of 5 inM Taurodeoxycholate monomer concn,' mM control phosphatidylcholine, 1.25 mM phosphatidylcholine, 2.5 mM phosphatidylcholine, 5 mM
2.05 i 0.08 1.45 i 0.06 1.11i 0.04 1.08 f 0.08
a The data are means i standard deviation from three determinations. The aqueous phase marker was sucrose.
system. In contrast, K,, for trace amounts of bile acids was greater than 1indicating an interaction between the bile acids and the beads. Consequently, K,, did not drop to 0 when micellar solutions of bile acids were used in the elution buffer. The reduction of K,, for T C and TCD in the presence of micellar solution is due to the incorporation of the labeled bile acids into micelles and not due to saturation of binding sites because r, did not change when bile acid concentration rose over 100-fold (Table 11). The fact that r, remained constant below the CMC (Table 11) makes it possible to use it as correction factor for the calculation of the monomer concentrations of the bile acids from the isotope data. The method can be used to measure monomer concentrations of bile acids in mixed micellar solutions. We studied the effects of phosphatidylcholine on monomer concentrations of taurodeoxycholate because phosphatidylcholine blocks the effects of dihydroxy bile acids on electrolyte and water transport in the jejunum (13)and the gallbladder (14) (Table 111). Increasing concentrations of phosphatidylcholine reduced the monomer concentrations of 5 mM taurodeoxycholate in a nonlinear fashion, where 2.5 mM phosphatidylcholine reduced monomer concentration almost by 50%, while 5 mM phosphatidylcholine had little additional effect. This reduction in monomer concentration is consistent with thermodynamic calculations which predict a reduction of the monomer concentration of a detergent by the addition of another hydrophobic or amphiphilic compound to the solution (15). The method allows the determination of the monomer concentration of only one bile acid at a time. This is, however, no shortcoming, since micellar behavior of bile acids is usually studied in model solutions using individual bile acids. Moreover, the different biological and biophysical properties
Anal. Chem. 1982, 5 4 , 2082-2085
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of individual bile acids, such as their ability to induce intestinal fluid secretion (13) or to solubilize cholesterol (161,render the determination of “monomer concentration” of a mixture of bile acids in biological samples meaningless.
LITERATURE CITED (1) Helenius, A.; Slmons, K. Eiochim. Biophys. Acta 1975, 415, 29-79. (2) Mazer, N. A.; Benedek, G. B.; Carey, M. C. Biochemistry 1980, 19, 60 1-6 15. (3) O’Maille, E. R. L. J . fhysiol. (London) 1980, 302, 107-120. (4) Kwan, K. H.; Higuchi, W. I.; Hofmann, A. F. J . fharm. Sci. 1978, 6 7 , 1 17 1- 17 14. (5) Duane, W. C. 6ioch;m. Biophys. Acta 1975, 398, 275-286. (6) Shiau, Y.; Levine, G. M. Am. J . fhysiol. 1980, 2339,G177-Gl82. (7) Gilligan, T. J.; Cussler, E. L.; Evans, D. F. Blochim. Biophvs. Acta 1877, 497, 627-630. (8) Carey, M. C.; Small, D. M. J. Coilold Interface Sci. 1969, 31, 362-396.
(9) Singleton, W. S.; Gray, M. S.; Brown, M. L.; White, J. L. J . Am. Oil. Chem. SOC. 1965, 4 2 , 53-56. Duane, w, c, Blochem, Blophys, Res, Commun, 1977, 74 223-229. ( 1 1 ) Laurent, T. C.; Killander, J. J . Chromafogr. 1984, 1 4 , 317-330. (12) Mukerjee, P.; Cardinal, J. R. J . fharm. Sci. 1976, 6 5 , 882-886. (13) Wingate, D. L.; Phillips, S . F.; Hofmann, A. F. J . Clin. Invest. 1973, 52, 1230-1236. (14) Ammon, H. V . Gastroenterology 1979, 76, 778-783. (15) Tanford, C. “The Hydrophobic Effect: Forrnatlon of Micelles and Biological Membranes”; Wiley: New York, 1973; Chapter 10. (16) Armstrong, M. J.; Carey, M. C. J . LipidRes. 1982, 23, 70-80.
RECEIVED for review September 8, 1981. Resubmitted June 17, 1982. Accepted July 1,1982. This research was supported by the Research Service Of the Veterans Administration and NIH Grant AM 17941.
Quench Correction in Liquid Scintillation Counting by a Combined Internal Standard-Samples Channels Ratio Technique Erik Dahlberg Karolinska Instltutet, Department of Medical Nutrltion, Research Center, F 69, Huddinge University-Hospital, S- 14 1 86 Huddinge, Sweden
A well-known problem in liquid sclntlllation counting (LSC) Is that radioactlvlty cannot be measured with 100% efflclency, e.g., due to ”quenching”, which thus needs be corrected for. Three methods (vlz., those of internal standard (IS),samples channels ratio (SCR), and external standard channels ratio (ESCR)) are In common use to accomplish quench correction. None of these methods is ideal. This paper shows that a comblnatlon of the IS and SCR methods (IS-SCR) ameliorates the malor dlsadvantages of both techniques and also offers some advantages over the ESCR method. Thus, the dependence on accurate pipetting in the IS technique and the disadvantage of the SCR technlque at low count rates have been eliminated In the IS-SCR method, which also has a low volume dependence compared to the IS and ESCR methods. The IS-SCR method is not affected by tlme-dependent dlffusion of solutes and solvents Into the walls of plastic counting vials, whlch Is a major drawback of the ESCR technique. Used wlth a simple llnear regression technique, the IS-SCR quench curves may be llnearized over wide ranges of efficlencles. I n view of the wide-spread appllcation of LSC, the IS-SCR technique Is therefore llkely to be useful to many investlgat ors.
LSC is commonly applied to determine the radioactive decay from a number of isotopes often used in research, such as the p emitters 3Hand 14C (1-10). A well-known problem is that radioactivity cannot then be measured with 100% efficiency, partly due to ”quenching” (11-13), which needs to be corrected for. Three methods are in common use to accomplish this (12,13), all of which have their advantages and disadvantages (14-18). Counting efficiency is volume dependent in LSC (15, 17, 19-21), and the volume of sample and scintillator must be kept constant at an optimum. Of the three common quench-cor-
rection methods, particularly that of ESCR is affected by volume and geometry of samples (15, 17). Several authors have found the SCR method superior to the ESCR method (14-18,22,23). A major problem of the latter technique is the time-dependent changes in ESCR values due to diffusion of solvents and solutes into the walls of the high-density polyethylene vials most often used (7,16,20, 24,251. Background counting rate also increases (16). By contrast, SCR values are unaffected (16). The SCR method is not applicable for samples having low count rates (12,17,18). The fact that the ESCR technique is the least accurate method is especially true for highly quenched samples (18). The method using an IS is the most reliable quench-correction technique, but it is expensive, time-consuming, and highly dependent upon accurate pipetting (18). It would seem theoretically possible to avoid the drawback of t h e SCR method at low count rates by adding an IS to low-radioactivity samples. By then using SCR values for quench correction, instead of calculating the efficiency from the contribution of the known amount of radioactivity added (”classical”IS procedure), one could perhaps at the same time eliminate the dependence of the IS technique on accurate pipetting. The data in this paper show that both assumptions are correct. The combined qethod (IS-SCR) is a useful improvement of quench correction.
EXPERIMENTAL SECTION Apparatus. The liquid-scintillationcounter used was a WalIac 1215 RackBeta (LKB, Stockholm, Sweden),equipped with an ES using 226Raas the y source. Instrument performance was checked regularly as outlined by Patterson et al. (15). Standard 20-mL high-density polyethylene counting vials (28 X 60 mm) were purchased from Packard Instrument Co. Inc. (Downers Grove, IL). Reagents. The scintillator fluids used were Insta-Gel and Scintillator 299 (both from Packard). The former is a well-known solgel scintillator, equal or superior to many other fluors in most respects (e.g.,ref 26-30). The latter is a new scintillator containing
0003-2700/82/0354-2082$01.25/0 0 1982 American Chemical Society
