However, Fisher’s tcst did not give any significant difference (11). LATVRY et al. ( 7 ) . The results of total serum cholesterol by the present method were also compared with those of Lawry et al. Venous blood from 300 normal persons of different ages and sexes was studied. To avoid personal deviation, onr technician was responsible for all determinations. The results obtained by the present method were similar to those of Lawry el al. (Table 111). The data for men between 50 and 59 and women between 60 and 69 were higher than those of Lawry et a/., probably because of the sampling difference. Males consistently had higher values than females until the sixth decade. The level rose with age in both sexes. The male reached the maximum a t 50 to 60, but the female continued to rise. LITERATURE CITED
( 1 ) Block, J. R., Uurrum, E. L., Zweig, G.. .‘Manual of Paper Chromatography and Paper Electrophoresis,’’ p. 576, -4cademic Press, Xew York, 1958. ( 2 ) Bloor, W. R., J . Biol. Chem. 24, 227 (1916). ( 3 ) Iliirchard, H., Chem. Zentr. 61 ( l ) , 25 I 1890).
Table 111.
Total Serum Cholesterol Contents at Various Ages
(Mg. %) Our Results
s o . of
ilge, Years Day old 1-9 10-19 20-29 30-39 40-49 50-59 60-69 83
Subjects 13 13 4 4 6 5 13 19 14 30 15 28 11 22 17 10 1
91
* 10
Lawry’s Data 11 F
F
M .
73 f 10
180 f 23 120 f 27
~~.
~
150 5 49 147 f 33 211 f 49
212 201 f 41
236 f 62 236
* 50
222
51
* 59
290 240
230 A 44
273 A 55 237
198 230 225 244
247 f 19
252 235
335 f 22
263
135
( 4 ) Ferro, P. V., Ham, A. B., Tech. Bull. Registry Med. Technologists 30, 71 (1960). (5) Hawk, P. B., Oser, B. L., Summerson,
W.H., “Practical Physiological Chem-
istry,” 13th ed., p. 23, McGraw-Hill, Yew York, 1958. (6) Klungsoyr, L., Haukenes, E., Closs, K., Clin. Chim. Acta 3, 514 (1960). (7) Lawry, E. Y., Mann, G. V., Peterson, A., Kysocki, A . P., Connell, R. O., Stare, F. J., Am. J . M e d . 22, 605 (1957).
(8) Liebermann, C., Ber. deut. Chem. Ges. 18, 1803 (1885). (9) Pearson, S., Stern, S., McGavack, T. H., ANAL.CHEM.25, 813 (1953). (10) Schoenheimer, D., Sperry, W. W., J . Biol. Chem. 106,745 (1934). (11) Snedeyr, G. W., “Statistical
Methods, Iowa State College Press, Ames, Iowa, 1946.
RECEIVEDfor review January 16, 1961. Accepted June 14, 1961.
Spec t ro photo metric Dete rmi natio n of Seve ra I Bile Acids as Conjugates Extraction with Ethyl Acetate SAMUEL J. LEVIN, C. G. JOHNSTON,I and A. J. BOYLE Department o f Chemistry and Surgery, Wayne State University, Detroit 2,
b Because of the recent evidence indicating a radical change in the state of conjugation of bile acids in liver disease as well as the continued interest in these compounds because they are the main cholesterol metabolites, the determination of the bile acids has assumed great importance. Most analytical methods currently used are not suitable because they require a hydrolysis step. The process causes gross losses and makes it impossible to obtain information concerning the state of conjugation. This paper presents a procedure for the rapid separation of the glycine and taurine conjugates of cholic and deoxycholic acids, and their spectrophotometric determination. Representative specimens of animal and human gall bladder bile have been analyzed and values for the four conjugates are presented.
Mich.
T
HE bile acids, 24-carbon carboxylic acids of the cholane series, are found conjugated with glycine and taurine in the bile of most vertebrates and all mammals (6). The commonly occurring bile acids are the conjugates of cholic (3,7,12-trihydroxy-), deoxycholic (3,12-dihydroxy-), and chenodeoxycholic acids (3,7-dihydroxycholanic acid). Recent studies have shown a drastic change in the proportions of conjugates in bile from patients suffering from liver and biliary disease
(15).
Most procedures for the estimation of these compounds in bile have in common a hydrolysis step. This has been necessary because of the difficulties involved in the extraction and subsequent separation of the conjugates owing t o the great hydrophilic properties of these compounds. It has been repeatedly recog-
nized that hydrolysis caused a structural alteration of the bile acid molecule, which results in large errors in the subsequent spectrophotometric results (1, 9). The hydrolytic step, however, does. obviate the exacting and tedious countercurrent (1) and chromatographic (19) methods, which are unsuitable for rapid clinical procedures. This paper describes a relatively rapid process for the separation and subsequent spectrophotometric estimation of the conjugates of cholic and deoxycholic acids in bile. METHODS AND MATERIALS
Purification and Separation of Conjugates. REAGENTS.Reagent grade
absolute ethyl alcohol, ether, hexane, Deceased June 3, 1960. VOL. 33, NO. 10, SEPTEMBER 1961
1407
ethyl acetate, hydrochloric acid, ammonium hydroxide, and n-butyl alcohol. Barium Hydroxide Solution. Dissolve 6.0 grams of Ba(OH)2.8Hz0 in 100 ml. of distilled water (freshly boiled). Filter quickly through glass wool and store in a tightly stoppered polyethylene bottle. Zinc Sulfate Solution. Dissolve 10 grams of ZnSOd.7H20 in 100 ml. of distilled water, and.store in a glassstoppered bottle. Ethyl Scetate Solvent. To approximately 500 ml. of reagent grade ethyl acetate in a separatory funnel, add 2 drops of concentrated ammonium hydroxide and 20 ml. of distilled water, shake well, allow the layers to separate, and discard the aqueous layer. Wash .the organic layer well with two 20-ml. portions of water and two portions of 0.001N hydrochloric acid solution. The second acidic wash should turn Congo red paper blue. If this does not occur, continue washing with the 0.001N hydrochloric acid solution. The amount of organic solvent remaining is enough to extract 10 samples. Prepare this solution just before use and discard any excess. Butyl Alcohol. Saturate approximately 400 ml. of reagent grade n-butyl alcohol with 1% hydrochloric acid solution, adding the acidic solution until two layers are formed. This solvent is relatively stable and may be stored in a cool place for several weeks. PROCEDURE FOR SEPARATION (see Figure 1). Add 1 to 2 ml. of filtered bile to approximately 10 ml. of absolute alcohol in a 25-ml. volumetric flask. Add 1.O ml. of barium hydroxide solution and boil the solution for a few seconds in a hot water bath. To this hot solution add 0.5 ml. of zinc sulfate solution. Wash down the sides of the flask with ethyl alcohol and after cooling to room temperature, make the solution up to volume with ethyl alcohol. After tration (Whatman 541) transfer a 20ml. aliquot to a 50-ml. centrifuge tube (T 16) and evaporate by a n air jet in a warm water bath. To the cooled residue add 5 ml. of distilled water, and extract the aqueous solution with two 15-ml. portions of diethyl ether, discarding the organic layer. Then perform a third extraction with 15 ml. of hexane, which is also discarded. Add a small square of Congo red paper, and acidify the aqueous solution with 1% hydrochloric acid solution until the paper turns blue. Extract the solution with two 15-ml. portions of hexane, which are discarded. Then extract the aqueous solution with three 15-ml. portions of freshly prepared ethyl acetate solvent, which is centrifuged to separate the layers completely. Remove the organic layer by aspiration into a 20 X 200 mm. test tube and wash the ethyl acetate solution once with approximately 0.5 ml. of 0.001N hydrochloric acid solution which is forcibly injected into the solution with a dropper. Extract the acid wash from the bottom of the container with a dropper and add it to the aqueous solution in the centrifuge tube. Transfer the ethyl acetate
a-
I
1408
ANALYTICAL CHEMISTRY
I
Figure 1 .
IOtHCl
Flowsheet
solution to 100-ml. beakers and evaporate by means of an air jet a t a temperature not exceeding 45" C. Make the residue up to volume in a 10-ml. volumetric flask with ethyl alcohol. It contains the glycine conjugates (G extract). To the remaining aqueous solution add 1 drop of concentrated hydrochloric acid and extract the solution with three 10-ml. portions of the butyl alcohol solvent as before. No washing is necessary for this extract. Add 2 drops of concentrated ammonium hydroxide to the extract and evaporate the solution in a warm water bath (65' to 75" C.) with the aid of an air jet. Make the resulting residue up to volume in a 10ml. volumetric flask with ethyl alcohol. It contains the taurine conjugates (T extract). Spectrophotometric Determinations of Cholates. REAGENTS. Standard solutions of the conjugated bile acids containing 0.05 to 0.20 mg. per ml. of ethyl alcohol are prepared from synthesized compounds. The conjugates were synthesized by the methods of Norman (If) and Cortese et al. (2, 3, 4). They were demonstrated to be chromatographically pure (IS), and the infrared spectra and other characteristic physical properties also indicated authenticity. 0.5% Furfural Solution. Dissolve 0.5 ml. of freshly distilled furfuraldehyde in 100 ml. of distilled water and refrigerate. PROCEDURE. Evaporate suitable aliquots of the respective conjugated cholate standard solutions and dilutions of the G and T extracts in 19 X 150 mm. test tubes in a boiling water bath. After cooling, add a tube for the blank, and 6.0 ml. of 16N sulfuric acid solution to each tube. Then add 2.0 ml. of the furfural solution and stir the contents of each tube well with a glass rod. Transfer the tubes simultaneously to a 65" C. water bath and incubate for 13 minutes. At the end of this period, cool the tubes in a water bath and add 5.0 ml. of glacial acetic acid. After stirring well, transfer the contents of each tube to matched colorimeter tubes and determine absorbance on a Coleman
Junior spectrophotometer, Model 6A, with the blank set a t zero. The optimum wave length for this determination is 620 mp. Calculate the content of cholate conjugate with the aid of the readings of the standards and the dilution factor. Spectrophotometric Determination of Deoxycholates. PROCEDURE. Prepare a special reagent by adding slowly and with cooling 600 ml. of concentrated sulfuric acid to 400 ml. of 85% orthophosphoric acid and 100 ml. of distilled water. Refrigerate in a glass-stoppered bottle. After evaporation of suitable aliquots of the respective conjugate standards and dilution of the G and T extracts, add a blank tube. Add 4.0 ml. of a freshly prepared 1% solution of redistilled benzaldehyde in the special reagent to each tube, starting a timer a t the first addition. Allow the tubes to stand in a refrigerator (3" to 5" C.) for 120 minutes. Mix the contents of each tube by swirling two or three times during the first 15 minutes. At the end of this initial color development period, add exactly 4.0 ml. of cold ethyl acetate slowly to each tube, so that two layers form. Place the rack holding the tubes in cool running water, and as a timer is started, stir the contents of each tube well with a glass rod. Then transfer the solutions to matched colorimeter tubes. Read absorbances a t the end of 25 minutes in a Coleman Junior spectrophotometer, wave length being 660 mp. Calculate the concentrations of conjugated deoxycholate from the readings and dilution factors. DISCUSSION
'
The barium hydroxide-zinc sulfate deproteinization is a modification of the Somogyi method for blood sugar (16). It adequately removes the bile protein which sometimes reaches high concentrations in pathological bile specimens, as well as precipitating most of the bile pigments as their sparingly soluble barium salts. The series of ether and hexane extractions removes all of the cholesterol and free fatty acids, as well as a t least 98% of the phospholipide present, as demonstrated by commonly used colorimetric and titrimetric methods. In the search for a rapid but accurate method for the separation of the two types of conjugates, the chromatographic and countercurrent solvent systems of Sjovall (IS) and Ahrens and Craig ( I ) , respectively, were investigated. These systems were not adequate for a single-stage extraction procedure. Ether extraction of aqueous solutions of various acidities resulted in some degree of separation, but glycocholic acid could not be extracted because of its higher polarity. While attempting the extraction with several solvents, it was found that ethyl acetate separated the glycine
'1
I
;O
16
j,
ic
50
4
ea
+
Minutes Figure 2. Effect time period
of
B
9p
m
I~OLZO
dt 3%.
variation of initial
conjugates from the taurine conjugates quickly and completely. Small amounts of acetic acid normally present in the solvent were found to affect the distribution of the bile acids profoundly. For this reason the washing procedure was instituted. Evaporation of the G extract could not proceed a t temperatures higher than 45' C. because of the formation of a gummy residue which resisted re-solution. For the acidification of the aqueous solution, several mineral acids were investigated. Sulfuric, nitric, and phosphoric acids were found to cause destruction of the bile acid present. Hydrochloric acid, when used in the amounts specified, caused no loss, probably because the excess is easily volatilized with a n air stream. The distribution coefficients for the bile acids were determined from colorimetric data obtained from both the aqueous and organic phases. For the ethyl acetate solvent, the coefficients are 2.7, 35.8, < 0.05, and < 0.02 for glycocholic acid, glycodeoxycholic, taurocholic, and taurodeoxycholic acids, respectively. The distribution coefficients for taurocholate and taurodeoxycholate in this solvent are > 15 and > 60, respectively. While the coefficient for glycocholic acid in ethyl acetate does not seem very large, less than 0.3% of the original concentration will remain in the aqueous layer if the amounts of solvents specified are used. Since the properties of the chenodeoxycholic acid derivatives are so similar to those of the deoxycholates, it is assumed that its respective conjugates are separated in a similar manner; indeed, chromatographic studies indicate t h a t such is the case. The spectrophotometric methoc cholates is essentially the Irvin, J mston, and Kopala procedure (8). Minor modifications in the amounts and concentrations of reagents have been made for convenience. However, the final furfural, sulfuric, and acetic acid concentrations are the same.
Several procedures are available for the colorimetric determination of the deoxycholic acid content of bile. Most use some strongly acidic reagent of a n aromatic aldehyde for color formation. Szalkowski and Mader used salicylaldehyde in 67% sulfuric acid and subsequent dilution with acetic acid (17). The reaction must be timed with stopwatch precision. A deeply colored blank is obtained and only a small number of samples may be run simultaneously. The method also yields results approximately 8 and 12% high, respectively, when equal amounts of cholates and chenodeoxycholates are present. Vanillin-phosphoric acid reagents, used by some investigators (IO, I d ) , are not specific for deoxycholates and therefore yield high results when other bile acids are present. While in the process of reinvestigating most of these colorimetric procedures, the reaction described in this paper was discovered. I n a mixed solvent of sulfuric and phosphoric acids and water, and in the presence of benzaldehyde, a yellowish orange color is formed by all the bile acids. However, on the addition of ethyl acetate, a blue-green color is formed with the deoxycholates but not with the other common bile acids. The factors of this reaction with the deoxycholates in bile acid mixtures were examined and the optimum conditions for quantitation are described below. The criterion for most of the studies on optimum conditions was the minimization of the per cent of error caused by the presence of the corresponding chenodeoxycholate conjugate. The composition of the reagent is critical, in both acid content and benzaldehyde concentration. A reagent composition with a ratio of 1:4:6 water-phosphoric-sulfuric acids yielded minimum error when comparing absorbances derived from like amounts of deoxycholates and chenodeoxycholates. Increasing the acid concentration of the reagent, especially sulfuric acid, causes a great increase in sensitivity; however, the error due to the other conjugates also increases, so as to make the method useless for the analysis of mixtures. The benzaldehyde concentration also was found to exert a profound influence, the best results being obtained when the concentration was 1%. The time and temperature of the initial color development are also important factors. Investigations a t room temperature, 40°, and 3' to 5' C. for varying periods indicated that for least error the refrigerator temperature was best. Figure 2 shows the effect of variation of the incubation period on the per cent error caused by glycochenodeoxycholic acid. The volume of ethyl acetate added
WMIength (nyr) Figure 3.
Spectra of conjugates
0 Glycodeoxycholic acid, 0.1 0 mg.
_ _ - _Sodium tourodeoxycholate, 0.1 0 mg. 0 Glycocholic acid, 1.0 mg. - Sodium taurocholate, 1 .O mg.
X Glycochenodeoxycholic acid, 1 .O mg. 0000 Sodium taurochenodeoxycholate, 1 .O mg.
for final color development is also critical. If one half the necessary volume of ethyl acetate is added (2 ml.), a n intense red color develops, but since the other bile acids also absorb in this spectral region, the color could not be utiliied. When the volume of ethyl acetate added equals the volume of the benzaldehyde reagent (4 ml,), the other conjugates interfere the least. Addition of large volumes of the ester increases the error and lowers the sensitivity by diluting the blue-green color formed. The 25-minute period after stirring in the ethyl acetate gives adequate time to mix thoroughly and transfer the contents of each tube to matched colorimeter tubes. The 22- to 30-minute interval after initial stirring represents the optimum time for reading the absorbance, since the error resulting from the presence of other bile acids is minimal during this period. The reagent blank, used to set the instrument to zero absorbance, has a constant absorbance during the 15- to 35-minute period and therefore may be used to set the instrument during the entire sample reading interval. Figure 3 shows the spectra obtained from solutions of the conjugated bile acids with the deoxycholate procedure. The spectra were determined with a Coleman Junior spectrophotometer, Model 6A, in 19-mm. matched tubes. The absorption peak for the deoxycholates were examined more fully with a Beckman DU spectrophotometer and found to be a t 660 mp. Several determinations on successive days indicated that the molar absorptivities of the two deoxycholate conjugates are the same and appear to be 900 to 975 liter cm-l. mole-'. For the two conjugates, Beer's law is obeyed throughout the 0.05- to 0.50-mg. region. Some VOL 33, NO. 10, SEPTEMBER 1961
1409
deviation from linearity occurs below the 0.05-mg. level but is easily compensated for with a calibration curve. Investigation of the interferences caused by simultaneous presence of bile acids other than those determined yields the data shown in Table I. The interferences are slight for the naturally occurring compounds.
Table 1.
Interferences in Colorimetric Procedures
Ratio of Molar Absorptivities
x- 1ooa
Compound Cholic acid Glycocholic acid Sodium taurocholate Deoxycholic acid Glycodeoxycholic acid Sodium taurodeoxycholate Chenodeoxycholate acid G1ycochenodeoxycholic acid Sodium taurochenodeoxycholate Cholanic acid Dehydrocholic acid Lithocholic acid Hyodeoxycholic acid Palmitic acid Cholesterol
DeoxyCholate cholate procedure procedure 97 100 92
0.6 1 2 1.3
1
39
2
100
2
100
0 4
2 9
2
2.2
4 0 0 0 0
2.1 0 0 0 0.8
0 1
0 2.0
a Glycocholic and glycodeoxycholic acids assigned a value of 100 in their respective procedures.
Table II.
Figure 4.
GC. Glycocholic acid GD. Giycodeoxycholic acid GGD. Glycochenodeoxycholic acid TC. Sodium taurocholate TD. Taurodeoxychalate TCD. Taurochenodeoxycholate Left. Separation of glycine Conjugates Right. Separation of taurine conjugates
RESULTS
Figure 4 illustrates the chromatograms obtained when the G and T extracts of a human bile sample were examined by paper partition chromatography (1.9). Glycine conjugates were separated (descending, 18 hours) with 60: 40 isopropyl ether-heptane/ 70% acetic acid. Taurine conjugates were separated (descending, 17 hours) with n-butyl alcohol saturated with 1% acetic acid. The locating reagent was 10% phosphomolybdic acid in ethyl alcohol. It is apparent that no glycine
Recovery of Conjugates in Protein Solution
Conjugate Added, Mg. Conjugate Recovered, Mg. GC TC G D T D G C D T C D GC TC GD TD 10 10 10 10 10 5 10 5
6 6
3
6
6 3
6 6
6 6
3
3
6 3
S
3
10 10 10 5 6 6 6 3
3
3
6
3
Table 111.
Conjugate Added, Mg. GC TC GD TD 3..
1.5 3 3 1.5
14 10
3.. 1.5 3 1.5 3
3.1.5 1.5 3 1.5
Chromatograms
3'1.5 1.5 1.5 3
10.5 10.8 5.85 5... 8- . 9 2.94 5.77
9 . 7 6 10.8 10.7 4 . 7 4 10.6 5.45 5.86 6 . 2 9 5 . 8 8
2 .. 9.7 -
6 36
3 02
5.90 5.78
3.16
3.05
i:i4 8:SS
Per Cent Recovery GC TC GD TD 97.6 108 107 94.7 106 109 9 7 . 7 105 9 8 . 1 9 9 . 0 106 101 9S:i 98.3 io5 -98.1 9 6 . 3 9 6 . 3 105 102
105 108 97.5 98 2
Recovery of Conjugates Added to Bile
Conjugate Determined, Mg. GC TC GD T D 8:29 6.94 8.34 8.55 7.12
ANALYTICAL CHEMISTRY
3.69 2.07 3.53 2.04 3.51
3:29 1.77 1.87 3.38 1.84
GC
Per Cent Recovery TC GD TD
3 . 0 2 98:3 104 99.3 9 7 . 8 102 97.7 9 6 . 8 1 . 5 4 100 101 1.60 9 8 . 8 99.2 103 101 1.58 101 102 99.3 97.2 98.7 101 3 . 0 0 103
conjugates originate from the 1' estract and no taurine conjugates trail the spots derived from the compounds in the G extract. Infrared spectra derived from the two extracts mere compared with those obtained from mixtures of the authentic conjugates combined in the proportions determined by the colorimetric methods outlined in this paper. The spectra were practically coincident throughout the 2- to 15-micron range, demonstrating the authenticity of the compounds present in the extracts. It may therefore be concluded that this method adequately separates the glycine conjugates from the taurine derivatives, as shown by chromatography and infrared spectra. Table I1 describes the reroveries of bile acid conjugates in protein Solution. The specified amount of conjugate mas placed in a 25-ml. volumetric flask by evaporation of standard solution aliquots and 2 ml. of 0.5% cgg albumen solution in physiologival saline, p H adjusted to 7.5 with 0.1.V NaOH, was then added to the bile acid residue. The resulting solution was carried through the procedure as for bile. The range of recoveries of the conjugates is seen to be from 95 to 109%. The average recovery is 101% (24 determinations). Table I11 shows the recoveries of conjugates added to a specimen of human T-tube bile from a patient on whom a choledochostomy had been performed. In this case, aliquots of the conjugate standard solutions were evaporated in 25-ml. flasks, 2 to 3 drops 0.01N NaOH were added to neutralize the free acids, and 1.0 nil. of bile was pipetted into the flask with swirling to dissolve the bile salts. The resulting solution was then processed according to the procedure. The range of recoveries was 96.8 to 104%. The average recovery for the 20 determinations was 100.0% with a standard deviation of 2.2%. Reproducibility of the analysis was studied by determining the conjugates six times in aliquots of the same normal human bile specimen. The results show that the relative standard deviations for glycocholic acid and sodium taurocholate were 1.7 and 0.9%, respectively, and 3.7 and 3.8% for glycodeoxycholic acid and sodium taurodeoxycholate, respectively. Table IV gives the values for the four conjugates obtained by analysis of certain animal samples. All specimens were obtained from the gall bladder, except where noted. The second to fifth columns indicate the actual concentrations found in milligrams per milliliter. The last four columns show the ratios between the conjugates. As indicated by Sjovall ( l j ) , the ratios between the various bile acids are often
more revealing than the actual cuncentrations present. Several investigations (7, 14) have revealed that dog bile contains only taurine conjugates of cholic and deoxycholic acids, the cholate predominating. The present study demonstrates that this is so. In the hepatic dog bile analyzed, taurocholate exceeds taurodeoxycholate by ratios of 2:l to 24:1, the average being about 9 3 . Although it has been stated that no glycine conjugates exist in dog bile, results in Table IV show the presence of such bile acids. On examination of the composition of the G extracts of these specimens by chromatographic and infrared methods, it was readily apparent that the chromogenic materials present were traces of taurocholate and taurodeoxycholate. However, this glycine conjugate content was rarely found t o constitute more than 1 to 2% of the total amount of conjugates determined and was not considered in the calculation of the ratios between the conj ugates. Table V shows results obtained on analysis of human gall bladder bile taken froin patients during operation for other than hepatic or biliary ailments. The ratios between the respective glycine and taurine conjugates of cholic and deoxycholic acids are constant, approximately 70y0 of each acid being conjugated with glycine. It also appears that cholates predominate in each fraction. Further study with more bile specimens will be necessary, however, before normal values for the conjugates are established. REFERENCES
(1) Ahrens, E. H., Craig, L. C., J.
Table IV.
Sample cow
ox
Cebus Rhesus, hepatic . Dog, gall bladder GBB 8AV 9AV 13AV Dog, hepatic 1-1 1-2 4a
GC 27.4 39.9
Analysis of Representative Animal Bile
Mg Conjugate/Ml. Bile GD TC TD
GC/ GD/ GC/ TC TD GD
TC’TD
0.7
3.4 4.3
2.7 4.0
0 9 0 7
8.5 9.3
29.3 54.6
5.5 1.53 4.38 0.1
2 87 1.17 2.50 0.06
Q.6 7.16 24.2 56.8
1.34 6.09 5.12
0.1 0.2 0.2 ...
0.3 0.9 0.4
1.9 1.3 1.8
...
...
5.I 5.8 4.0 11.1
3.6
0.42
3.73
0.56
0.9
0.6
7.0
5.1
4.6 0.78 0.41 0.65
1.0 0.62 0.53 0.71
149 83.6 86.9 122
... ... ... ...
...
...
... ...
... ... ...
3.4 5.0 7.8 2.3
0.66 0.69 0.34
0.32 0.16 0.20
33:6 47.9 46.3
4.85 2.00 4.91
...
...
...
...
... ...
0.13 1.44
0.12 0.87
31.0 74.3
5.11
... ...
6.9 23.9 9.4 19.3 5.9 22.6 43 22 6.1 9.1
11.2
13.7 8.44
43.3 16.7 11.2 63.7
...
0.8
...
...
,..
5a .~
2b
5b
2a 6b
6c 7BH
8.22
...
...
...
...
V. Analysis of Normal Human Gall Bladder Bile Mg. Conjugate/Ml. Bile GC GD TC T D GC/TC GD/TD GC/GD TC/TD 29.9 6.7 10.8 1.57 2.9 4.3 4.5 6’.9 7.66 1.55 1.83 0.41 4.2 3.8 4.9 4.5 1.8 7.2 8.5 9.25 1.28 6.13 0.72 1.5 16.3 1.22 8.13 1.13 2.0 1.0 13.3 7.2 8.1 2.81 5.34 1.43 1.5 2.0 2.9 3.7 21.6 1.80 13.7 1.06 1.6 1.7 15.7 12.9 16.1 8.0 4.17 0.52 3.3 1.6 13.7 0.85 2.6 3.2 3.33 3.1 3.8 32.5 12.7 10.6 3.3 4.2 3.53 2.2 2.8 32.3 9.73 14.7 17.0 15.3 1.74 1.7 1.5 45.8 2.69 26.7 Av. 2 . 4 2.4 8.1 7.4
Table
Sample NGBB 24 31 34 NG 46 52 54 55 59
Biol.
Chem. 195, 763 (1952). (2) Cortese. F.. Bashour. J . T.. Ibid.. .
I
119. 177 (1937). (3) Cortese; F., Bauman, L., J . Am. C h a . SOC.57, 1393 (1935). (4) Cortese, F., Bauman, L., J. Biol. Chem. 113, 779 (1936). (5) Haslewood. G. A. D.. Biochem. J. (6) Haslewood, G. -4.D., Physiol. Rev. 35, 178 (1955). (7) Haslewood, G. A. D., Wootton, I. D. P., Biochem. J . 47,584 (1950). (8) Irvin, J. L., Johnston, C. G., Kopala, J., J . Biol. Chem. 153,439 (1944). (9) Josephson, B., Jungner, G., Biochem. J . 30, 1953 (1936)
(10) Murakami, E., J. Biochem. (Tokyo) 39, 17 (1952). (11) Norman, A., Arkiv Kemi 8, 331 (1955). (12) Pratt, E. L., Corbitt, H. B., ANAL. CHEM.24,166 (1952). (13) Sjovall, J., Acta C h a . S a n d . 8 , 339 (1954). (14)Sjovall; J., Biochem. J . 57, 126 (1954). (15) Sjovall, J., Clin. Chim. -4cta 4, 652 (1959). (16) Somogyi, M., J. Biol. Chem. 160, 69 (1945).
(17) Szalkowski, C. R., Mader, W. J., ANAL.CHEM.24, 1602 (1952). RECEIVEDfor review April 17, 1961. Accepted June 19, 1961. Abstracted from a thesis submitted by Samuel J. Levin to the Graduate School of Wayne State University, April 1961, in partial fulfillment of the requirements for the degree of doctor of philosophy. Work supported in part by Parke, Davis & Co., Research Corp. of Receiving Hospital, and National Institutes of Health grants A-699 and A-224.
VOL 33, NO. 10, SEPTEMBER 1961
1411
