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Chromatography of sugars in body fluids
Chromatography II. Paper
of Sugars
in Body
Chromatographic Pattern in Five Solvents1 VLADIMIR
VITEK
of Urinary
AND KVl?TA
Fluids Sugars
VITEK
Research Center, Rockland State Hospital, Orangeburg, New York, and Center fos the Study of Trauma, University of Mary,?und, School of Medicine, Baltimore, Maryland 21201
ReceivedJune
25,
1970
By our analysis the pattern of neutral sugars in normal human urine is more constant and complex ( 1) than has been reported in other chromatographic and electrophoretic studies (2-5). A lo-20-minute aliquot of urine always contains at least 23 sugars plus a varying number of sugars occurring occasionally ( 1) . Th is regular sugar pattern includes some rare saccharides such as allulose identified by Strecker et al. (S), allose and alloheptulose, both of which were tentatively identified by us (1) and have yet to be found in other mammalian material, two unknown aldoses, and three unknown ketoses. In the course of analyzing metabolic changes of %-labeled sugars in healthy men (‘7, 8) we compared the W-labeled neutral metabolites with sugars normally present in the urine. Since the mixture of labeled and unlabeled metabolites was very complex and displayed large differences in the concentration of individual components, a separation procedure with a reliable reproducibility and high sensitivity was required. The properties of five solvent systems selected or designed to fulfill this task were compared. In order to maintain actual working conditions urine samples from healthy individuals as well as artificial mixture of standards were utilized. MATERIALS
AND
PROCEDURES
Urine specimens were collected from 43 healthy volunteers. After overnight fasting, the initial voiding was discarded and 2- or S-hour specimens were collected and kept at -20” until analyzed. ‘Supported in part by the U. S. Army Research and Development Department of the Army, under Research Contract DA 49-193-2229 and Institutes of Health, Division of General Medical Sciences, under Contract
282
Command, the National GM 15700.
CHROMATOGRAPHY
OF
URINARY
SUGARS
283
The solvents, at least of ACS grade, were supplied by various companies and used without further purification. Liquid phenol (A931), chromatographic grade, was supplied by Fisher Scientific Company. Preparation of Specimens. The urine samples were filtered through dry filter paper prewashed with water and deionized by successive passage through columns of Amberlite IR 120 (H) and Amberlite IRA 410 (acetate) in a modified procedure (9) of White and Hess (2). The pooled effluents and washings were evaporated on a rotary flash evaporator at 38’ until dry or lyophilized. Chromatography. The dry, sometimes syrupy, residue was dissolved in water and a portion corresponding to a IO-2O-minute aliquot of diuresis was streaked on a 36mm line on a sheet of Whatman No. 17 paper (46 X 57 cm). The application line was drawn parallel to the shorter side of the sheet, 5 cm from the edge. Aldo-and keto-standards were applied in separate groups alongside the urine. Ascending chromatography was carried out in square jars with outside dimensions of 31 X 62 cm (Fisher Scientific Company). The paper sheet was arched and the edges stitched to an additional 30-cm wide strip carrying other urinary samples and standards. The solvent filled the bottom submerging the paper about 10-15 mm. Chromatography was started without preliminary exposure to the vapors. During repeated runs, the solvent was refilled to the original level. Testing of Soluents. The suitability of the solvent systems was tested with a mixture of standard sugars similar to those occurring in the urine and later with the urine itself. After each run, the sugars were located to find the optimal schedule for the separation. Between individual irrigations and after the final run, the chromatograms were dried at room temperature. The drying period between individual runs containing n-butanol or more volatile components was 2 hours, for less volatile it was extended accordingly. Chromatograms from phenolic solvents were allowed to dry overnight. Detection. After the odor of the solvents completely disappeared, the paper chromatograms were viewed in short-wave UV light. The UVabsorbing spots were marked with a pencil, serving as an indicator of the regularity of the flow. Then a line was drawn in the middle of the urine sample. After cutting the paper along the line, the part of the chromatogram containing half of the urinary spots with adjoining standards of aldoses was sprayed with aniline citrate according to White and Hess (2) with a modification (10) which ensures stepwise detection of different classes of aldoses and avoids nonspecific reactions of some ketoses. The remaining part of the chromatogram was sprayed with orcinol-trichloroacetic acid reagent to reveal ketoses ( 11). Stained sugar spots were
284
V&K
AND
ViTEK
also observed in the fluorescent bulb light of a vic’w box for rocntgenograms and their fluorescence recorded under the UV lamps ~lincrallight-UVS-11 (260 rnp main wavelength) and Black Ray-UVL-21 (360 mp), both made by Ultraviolet Products, Inc., San Gabriel, California. The position and size of the spots in actual dimensions were recorded on graphic cross-section paper. The part detected with orcinol was matched to that revealed with aniline citrate. In the presence of both fluorescence and color, the size of the spot was recorded according to the more sensitive fluorescence which provided larger spots than the color itself. For the preparation of chromatographic maps illustrated in Fig. 1, the urine of five persons was subjected to chromatography in duplicate samples. One of them was chromatographed with the addition of 100 pg quantities of d-allose, d-glycero-d-alloheptulose, maltose, galactose, and 200 pg of N-acetylglucosamine. Quantitative Determinations. Total reducing sugars in the desalted urine were determined by Nelson’s modification of Somogyi’s method ( 12), glucose with glucose oxidase preparation Glucostat of Worthington Biochemical Corp., Freehold, N. J., and galactose by galactose dehydrogenase method supplied by Boehringer Mannheim Corp., New York, N. Y. RESULTS
In a search for acidic, basic, neutral, phenolic, and ethylacetate type of solvent systems suitable to separate neutral sugars, we tested 47 solvent combinations. These included various combinations of ethanol, n-propanol, isopropanol, n-butanol, isobutanol, sec. butanol, amylacohol, terc. amylalcohol, aceton, methyl ethyl ketone, benzylalcohol, phenol, the formic, acetic and boric acids, pyridine, and ammonia. Among them were also some well-known systems such as n-butanol-acetic acid-water (4 : 1: 5)) ethyl acetate-acetic acid-water (3: 1: 3)) n-butanol-ethanol acid-water (5: 1: 4)) ethyl acetate-pyridine-water (2: 1: 2)) n-propanolacetic acid-water (7: 1:2), and phenol saturated with water ( 13, 14). The most frequent reasons for unsatisfactory performance were: (a) no separation, (b) resolution with streaking, particularly of the fastest sugars or of the slowest moving ones, (c) diffise spots, either generally or in the fastest migrating fraction, (d) a too fast flow resulting in crowding of monosaccharides in the upper part of the chromatogram, (c) unsufEcient ascent of the solvent because of large viscosity, and (f) unsufficient stability of the solvent in the conditions of multiple chromatography. The search resulted in the selection of five systems: Solvent A: n-butanol-pyridine-benzene-w ater ( S: 3 : 1: 3 )-not miscible ( 1.5) : B :
CHROMATOGRAPHY
OF
URINARY
285
SUGARS
GM SOLVEWT
FROM /
------_-_
50
--------
15 40 35 30 25 20 15 IO 5 A
K
A
K
0 n-Bul-Py-or-w 5:3 :I .3
Isobul-AcA-W 4 I ‘I
Ethylac- AcA-W ‘I 3 ‘I
sec. But-Ethylac7 2
W 2
n-But-Phe-AcA-W 5 5 : 2
:10
FIG. 1. Distribution pattern of urinary sugars in five proposed solvent systems. The schema was drawn on the basis of the results of chromatography in two duplicates of two urinary specimens obtained from five healthy subjects after overnight fasting using the schedule and conditions described in Procedures and in Table 1. It includes the spots of all sugars forming the constant spectrum of human urine, and some sugars revealed occasionally, i.e., maltose, sucrose, and glucuronolactone. The dimensions in the direction of the flow corresponded to the actual size of spots. Aldoses (A) detected with aniline citrate reagent (2, 10): 1, lactose; 2, maltose; 3, unknown red spot, tentatively identified as a glucosylxylose; 4, galactose; 5, glucose; 6, allose; 7, mannose; 8, arabinose; 9, N-acetylghrcosamine; 10, xylose; II, fucose; 12, ribose; 13, unknown yellow orange spot; 14, glucuronolactone; 15, unknown pink spot, later light brown. Ketoses ( K) detected with orcinol-trichloroacetic acid reagent ( 11) : 1, sucrose; 2, mannoheptulose; 3, sedoheptulose; 4, fructose; 5, alloheptulose; 6, unknown beige to beige-pink spot; 7, allulose; 8, xylulose; 9, ribulose; 10, unknown pale yellow; 11, unknown yellow. Ketoses 10 and 11 may be identical with aldose spots 13 and 15, respectively.
isobutanol-acetic
acid-water
water
II:
( 4: 1: 1) (3); C: ethyl acetate-acetic acidbutanol-ethyl acctatt,-\~,atcr ( 7 ::! :2 ) ; I< : n.-butanol-phenol-acetic acid-water (5: 5: 2: IO)--not miscible ( 16). The solvents A-E were employed according to the schcdulc given in Table 1. It applies to Whatman No. 17. (3:l:l);
X‘C.
TSBLE SCHEDULE
OF MUL'ITIJI~E:
__System
1
A B C 1) E
18 7 18 24.6 24.5
__~-2 21 18 18 24.5 94.5
1
ASCENI)ING CHROMATOGRAPHY Fiw SoLvewr SYSTEMS~
:3 24 21 21.5 x2.5 26.5
Number of developments ~. ..-I .i ti 26 22.5 26 33 26.-i
30
38.5
34 31
36.5
OF I~RINAHY
.__-7 38.5
SU(:.~RS
s 41.5
IN
Total hours 89.5 217
a The composition of individual solvent system is described under procedures. The duration of runs is given in hours. The schedule is proposed for Whatman No. 17 filt,er paper.
Figure 1 shows the separation of 23 regularly present sugars and 3 occasionally occurring ones (sucrose, glucuronolactone, and maltose). The illustration also indicates (1) the degree of overlapping between neighboring spots of aldoses and ketoses, (2) the relative size of the spot in the flow direction, and (3) the actual position of the front after the last development. The aniline-positive spots 13 and 15 and ketoses 6, 10, and 11 are constant, as yet not identified, constituents of the urine. As the temperature fluctuated only between 23-26’, the positions of spots were very well reproducible in all systems but the best results were obtained in isobutanol solvent (system B) where the positions of individual spots usually could be predicted with a greater accuracy than 10 mm providing that the schedule was strictly observed. The compactness of the spots decreased in the order: solvent B, A, D, C, E. Solvents A, D, and C showed only little differences in the quality of the spots. System E displayed the largest diffusion but other tested phenolic systems were similar or often worse. The background of chromatograms is very good in systems B, C, and D. System A is slightly inferior, particularly in the front zone, where occasionally unidentified aniline-positive yellow spots appear. The possibility of interference with the detection is more serious in the system F which gives a colored background of various intensity and a considerabIy broader solvent front. With the exception of glucuronolactone and N-acetylhexosamine all
CJSROMATOCRAPHY
OF
URINARY
SUGARS
287
nonphenolic systems separate simple sugars in the same order but the quality of separation of certain pairs or larger groups differs significantly as evident from Fig. 1. For easier orientation, the characteristic separator-y properties of the proposed systems are summarized in Table 2. The stability of urinary sugars in the selected systems is good, except glucuronolactone, which in the neutral system D and especially in the basic A decomposes and a distinct streak links the spot of the liberated glucuronic acid with the original lactone. We did not observe this in the normal urine probably because of the very low concentration in the latter. In all systems standards of ribulose, obtained from three different sources, formed two spots of the same staining and fluorescenting properties as marked in Fig. 1 (keto-spot No. 9)) the smaller one usually partially masking the spot of allulose (spot No. 7). We did not notice this phenomenon upon analyzing the urine. For many experimental or diagnostic purposes, where the detection of the smallest components, such as allose, alloheptulose, mannoheptulose, N-acetylglucosamine, and the fast migrating aniline-positive spot No. 15, are not a necessity, a much faster monodimensional descending chromatography was developed using Whatman paper No. 3 MM and the solvent n-butanol-pyridine-water (6: 4 : 3) and/or ethyl acetateacetic acid-water (3: 1: 1). The schedule for individual runs is given in Table 3. The quality of separation is good providing that not more than a 5-10 minute aliquot of diuresis is employed and that the glucose content is within the normal range, or only moderately increased, i.e., less than 150 mg/24 hr. DISCUSSION
The complexity as well as the constancy of composition of the urinary sugar spectrum have not been realized in previous reports. White and Hess (2) reported in 1956 the occurrence of 11 aldoses and 8 ketoses in the normal urine. Kopecky and Kellen (3) confirmed the findings of White and Hess (2) and the presence of mannose, mentioned earlier ( 17). Moreover, they described vaguely a few additional unidentified spots. A later report by Fleury and Eberhard ( 18) mentioned only nine sugars in the normal urine, one of them identified as maltose. According to these authors (2, 3, 17, 18) the majority of sugars occurs in the urine only occasionally. We have found, however, that the number of sugars in the urine is much less erratic and depends mainly on numerous technical factors, such as the quality of deionization, applied volume, quality of resolution, sensitivity and specificity of the detection methods. Diet plays also an important part since it may considerably influence the levels of certain sugars such as glucose, fructose, and especially sucrose, lactose, and galactose, and according to Date (19) also of pentoses. Our
2 PROPOSED
Ethyl acetate-acetic (3:l:l)
acid-water
SOLVENT
Unknown aldose (31-galactose (4) xylose (lo)-fucose (ll)-ribose (121 unknown aldose C13 )-gltlc:rironolact.one i 14 ) allohept\dose (5)-unknown ketose 16 1 alluloae (7 I-xylulose (8) unknown ketose (6i~allulose (71
ITnknowll aldose CB)-galactose (4 I allose (6j-mannose (7) mannoheptulose (‘L)-sedoheptllloae (3) ribose (121-unknown aldose (131
acid-water
(1 I
separation
THE
Isobutanol-acetic 14:l:l)
good
(1 )-maltose (2’1 (4)-glucose (5 1 (8)-xylose (9) (Cribulose (9) aldose (3)~sucrose
to very
0~
TABLE
Lactose galactose arabinose xylulose unknown
Fair
CHARACTERISTICS
Butanol-pyridine-benzene-water (5:3:1:3)
System
SEPAR.4TlON
Poor
or no separation
Glucose i.i I-allose (6 i unknown aldose (Is)-galactose il sedoheptulose (X-fructose (4) xylose (lo)-fltcose ill I-ribose 112’1 mannose (7)-fructose (411 mannose (7i-arahinose (8, alloheptulose (.i I-~~tnknown ketosr /6 frllctose 14 1 -nlloheptlllose (3 i
SYSTEMS"
CHROMATOGRAPHY
OF
URINARY
SUGARS
2239
290
n-Butanol-pyridine-water (6:4:3) Ethyl acetate-acetic acid-water (3:l:l)
ViTEk’
AND
ViTEE
6
x
5.0
6.0
1% 6 .5
0 The duration of individual runs is given in hours. The schedule What.man No. 3 MM filter paper.
14 7.5
40 26
is proposed
for
experience, however, shows that with the exception of sucrose and POSsibly an unknown ketose (spot No. 6) all sugars (Fig. 1) occur in the urine even under complete caloric starvation. The urinary sugars belong to various classes. Among them simple aldoses and ketoses are most common (Fig. 1). They form groups of isomers, such as galactose-glucose-allose-mannose; arabinose-xyloseribose; fructose-allulose; mannoheptulose-sedoheptulose-alloheptulose; xylulose-ribulose. Some groups of isomers, e.g., fructose-allulose, arabinose-xylose-ribose or glucose-mannose, may be separated relatively easily in a single run, particularly in an overflow arrangement. In order to achieve the best separation of all groups on a single chromatogram we employed multiple chromatography. Its mechanism and conditions of separation were extensively studied by Jeanes et al. (20) and later by Thoma (21). Using simple mixtures of sugars, they proved (20, 21) that the sluggishness of the procedure is highly compensated by the quality of resolution. Figure 1 demonstrates, however, that a perfect resolution of such a complex mixture as urinary sugars cannot be achieved even by multiple ascending chromatography where the ascent of solvent is an additional factor favoring further concentration of spots. An overlapping of some sugar spots in varying degree occurs in any of the proposed systems (Fig. 1). It is unavoidable even in two-dimensional chromatography (2, 18) which is less convenient for serial analyses and for a comparison of the positions of aldoses and ketoses. Therefore a sensitive and highly specific detection method is of importance not only in successful differentiation between aldoses and ketoses but also in revealing compounds which occur in very low concentrations. These compounds need more attention especially when their RF values are very close to other sugars which are present in several times higher concentrations. The most illustrative example is the group galactose-glucoseallose.
sugar
74.0 43.8 95.8 56.8 29.4 70.0 56.0 123.0 103.0 163.0 81.4 40.3
A.J. B.R. H.M. T.A. R.T. B.L. A.K. H.J. V.V. W.E. Mean 3~ SD
u The
edml
Name
-
content
61.20 73.73 59.18 57.89 54.29 58,90 62.64 57.02 96.48 89.79 67.11 14.75
w/24 hr
GLUCOSE,
was estimated
0.425 0.512 0.411 0.402 0.377 0.409 0.435 0.396 0.670 0.693 0.473 0.116
w/lo min
Glucose
FREE
specimens
6.34 2.78 4.32 1.65 8.35 4.42 10.46 12.48 3.74 7.20 6.17 3.47
in urine
0.036 0.033 0.019 0.012 0.107 0.026 0.081 0.040 0.024 0.031 0.041 0.030
&ml
collected
5.24 4.18 2.66 1.66 15.42 3.71 11.69 5.79 3.50 4.41 5.87 4.31 from
healthy
340 225 340 275 125 255 260 550 360 493 327 127
dml
Total
individuals
2.239 2.628 1.459 1.944 1.604 1.489 2.018 1.771 2.340 2.093 1.959 0.384
mg/lO min
after
322.42 378.43 210.70 279.94 230.48 214.42 290.59 255.02 336.96 301.39 287.02 52.30
w/24 hr
(Somogyi)
IN NORMAL
red . sugars
TABLE 4 REDUCING SUGARS
w/24 hr
TOTAL
Galactose
AND
mg/lO min
GA~ACTOSE, OF
fasting.
1:11.7 15.7 22.2 35.0 3. 5 15.9 5.4 9.9 27.6 22.7 16.9 9.5
27.47 21.55 22.36 28.63 33.11 24.34 4.70
22.93
18.98 19.48 28.17 20.68
yi (:lucose of total sugars
ADULTS"
Galactose glucose
overnight
URINE
1.63 1.24 1.27 0.59 6.68 1.73 4.02 2.27 1.04 1.46 2.19 1.83
y. Galactose of total reducing sugars
$ 54
2
il & 4
%
$
s F: ?
292
ViTEK
ALND
ViTEK
Glucose is the strongest component in the entire sugar spectrum of the normal urine, representing on average 35% of the total reducing powri of the desalted urine (Table 4). Allose, and frequently also galactose (Table 4), is present in very small amounts. If glucose is markedly elevated its spot becomes very large, tends to streak and absorb or at least to blur the neighboring weak spots of galactose and allosc. If it is increased even more, it may ruin the separation of the entirc specimen. This problem has been solved to a great extent by using Whatman No. 17 chromatographic paper, which unlike other papers has the capacity not only to carry the heavy load of the sample but also to separate sufficiently galactose and allose even if the glucose concentration is increased up to 400 mg/24 hr. Comparing the performance of the proposed systems and some solvents very popular in the carbohydrate chemistry we found that the systems A, B, and C gave almost identical results with n-butanol-pyridine-water ( 6: 4 : 3)) n-butanol-acetic acid-water ( 4 : 1: 5 ) , and ethyl acetate-acetic acid-water (3: 1: 3), respectively. For this reason the resolution pattern shown in Fig. 1 may be helpful as a guide in determining the relative positions and the separation quality of urinary sugars in other solvents of similar composition. In addition it may serve as a reliable base for any specialized investigational task. During these studies with sugar chromatography we have observed, contrary to the generally accepted view, that neutral sugars are not capable of ionization in the absence of complex forming agents, that the quality of separation of two sugars seems to be dependent on the basicity or acidity of the medium, In all acidic nonphenolic solvents, regardless whether the secondary component is an aliphatic alcohol or ethyl acetate, resolution of certain isomeric pairs such as galactose-glucose, arabinosexylose, xylulose-ribulose and less distinctly mannoheptulose-sedoheptulose is worse than in basic or even neutral systems. It seems to be of minor importance what produces the basicity since the effects of pyridine and ammonia are very similar. This phenomenon is also evident from a comparison of the results obtained in the systems B and C in contrary to those achieved in solvents A and D. Although the mechanism of this behavior is not elucidated we assume that heavy hydration of the molecules of isomeric sugars, which occurs during migration (23). is not equally affected by the profound change of the hydrogen ion concentration of the medium. SUMMARY
A method for chromatographic separation on Whatman No. 17 and 3 MM filter papers is described by means of which at least 21 identified
CHROMATOGRAPHY
OF
URINARY
SUGARS
293
and 5 unknown neutral sugars regularly present in the normal human urine may be revealed. The pattern of their resolution is compared in five solvent systems of different nature. The procedure uses Whatman No. 17 in ascending multiple modification for urine specimens corresponding to a lO-20-minute diuresis. For smaller aliquots of urine Whatman No. 3 MM and descending repeated developments are proposed. The former modification has an advantage of revealing better the minor constituents and possessing superior reproducibility. ACKNOWLEDGMENTS Our thanks are due to Dr. Nelson X. Richtmeyer of the National Institutes of Health, Bethesda, Maryland, for generous samples of &&se, sedoheptulose, mannoheptulose, and d-glycero-d-alloheptulose; to Dr. Gilbert Ashwell of the same Institute for samples of xylulose and ribulose and also to Dr. R. J. Suhadolnik of the Albert Einstein Center, Philadelphia, Pennsylvania, for a gift of d-allulose and psicofuranine. Appreciation is also expressed to Dr. Harriet L. Frush of the National Bureau of Standards for suggesting the Whatman No, 17 filter paper. We also are grateful to Mrs. Hsi-Chiang Lin, MS., for the aid in analysis. ADDENDUM Our further investigations proved that isomaltose is also a constant constituent of human urine. The approximate range in healthy persons is 2-f mg/24 hrs. In the proposed five systems isomaltose spot has the RF value identical or very similar to that of lactose. REFERENCES 1. V~TEK, V., AND V~TEK, K., Clin. Res. 16, 556 (1968). 2. WHITE, A. A., AND HESS, W. C., Arch. Biochem. Biophys. 64, 57 ( 1956). 3. KOPECKP, A., AND KELLEN, J,, “Papierchromatographie der Zucker in der Klinik,” pp. 58, 40. Edition, Leipzig, 1963. 4. LATO, M., BRUNELLI, B., CIUFFINI, G., AND MEZZELLI, T., J. Chromutogr. 34, 26 (1968). 5. JOLLXY, R. L., AND FREEMAN, M. L., Clin. Chem. 14, 538 (1968). 6. STRECKER, G., G~UBEL, B., AND MONTREUIL, J., Compt. Rend. Acad. Sci. 260, 999 (1965). 7. V~TEK, V., V~TFX, K., AND SACKS, W., Fed. Proc. Fed. Amer. Sot. Exp. Biol. 27, 254 (1968). 8. V~TEK, V., V~TEK, K., AND SACKS, W., Fed. PTOC. Fed. Amer. Sot. Exp. Biol. 28, 906 (1969). 9. %EK, v., AND ViTEK, K., In preparation. 10. V~TEK, V., AND V~TEK, K., In preparation. 11. KLEVSTIUND, R., AND NORDAL, A., Acta Chem. Scud 4, 1320 (1950). 12. SOMOGYI, M., j. Biol. Chem. 195, 19 (1952). 13. MACEK, K., in “Paper Chromatography” (I. M. Hais and K. Macek, ea.), p. 303. . Publishing House of the Czechoslovak Academy of Sciences, Prague, 1963. 14. BAILEY, R, W., AND PFUDHAM, J. B., in “Chromatographic Reviews,” Vol. 4. Elsevier Press, Amsterdam, 1962.
294
ViTEh:
AND
ViTEE;
IS. ALBON, N. II., AND Gnossq D., AnuZtJst 75, 454 (19.50). 16. KRISHNAILIUIITY, Ii., AND SWAhlINATHAN, M., J. %i. kd. Res. 14c, 310 (1955). 17. MONTREUIL, J., ASI> BOWLANGER, P., Compt. Rend. Acad. Sci. 236, 337 (1953). 18. FLEURY, P., AND E~EIIHAR~, H., Ann. Biol. C.&n. Puris 23, 1175 (1965). 19. DATE, J. W., Scaud. j. Chr. Lub. Inwst. 10, 115 (1958). 20. JEANES, A., WISE, C. S., AND DIMLER, R. J., Anal. Chem. 23, 415 (1951). 21. THOMA, J. A., Anal. Chem. 35, 214 (1963). 22. TOWER, D. B., PETERS, E. L., AND POGORELSIUN, M. A., Neurology 6, 37 (1956). 23. ISHERWOOD, F. A., Brit. Med. Bull. 10, 202 (1954).
of Sugars
in Body
Chromatographic Pattern in Five Solvents1 VLADIMIR
VITEK
of Urinary
AND KVl?TA
Fluids Sugars
VITEK
Research Center, Rockland State Hospital, Orangeburg, New York, and Center fos the Study of Trauma, University of Mary,?und, School of Medicine, Baltimore, Maryland 21201
ReceivedJune
25,
1970
By our analysis the pattern of neutral sugars in normal human urine is more constant and complex ( 1) than has been reported in other chromatographic and electrophoretic studies (2-5). A lo-20-minute aliquot of urine always contains at least 23 sugars plus a varying number of sugars occurring occasionally ( 1) . Th is regular sugar pattern includes some rare saccharides such as allulose identified by Strecker et al. (S), allose and alloheptulose, both of which were tentatively identified by us (1) and have yet to be found in other mammalian material, two unknown aldoses, and three unknown ketoses. In the course of analyzing metabolic changes of %-labeled sugars in healthy men (‘7, 8) we compared the W-labeled neutral metabolites with sugars normally present in the urine. Since the mixture of labeled and unlabeled metabolites was very complex and displayed large differences in the concentration of individual components, a separation procedure with a reliable reproducibility and high sensitivity was required. The properties of five solvent systems selected or designed to fulfill this task were compared. In order to maintain actual working conditions urine samples from healthy individuals as well as artificial mixture of standards were utilized. MATERIALS
AND
PROCEDURES
Urine specimens were collected from 43 healthy volunteers. After overnight fasting, the initial voiding was discarded and 2- or S-hour specimens were collected and kept at -20” until analyzed. ‘Supported in part by the U. S. Army Research and Development Department of the Army, under Research Contract DA 49-193-2229 and Institutes of Health, Division of General Medical Sciences, under Contract
282
Command, the National GM 15700.
CHROMATOGRAPHY
OF
URINARY
SUGARS
283
The solvents, at least of ACS grade, were supplied by various companies and used without further purification. Liquid phenol (A931), chromatographic grade, was supplied by Fisher Scientific Company. Preparation of Specimens. The urine samples were filtered through dry filter paper prewashed with water and deionized by successive passage through columns of Amberlite IR 120 (H) and Amberlite IRA 410 (acetate) in a modified procedure (9) of White and Hess (2). The pooled effluents and washings were evaporated on a rotary flash evaporator at 38’ until dry or lyophilized. Chromatography. The dry, sometimes syrupy, residue was dissolved in water and a portion corresponding to a IO-2O-minute aliquot of diuresis was streaked on a 36mm line on a sheet of Whatman No. 17 paper (46 X 57 cm). The application line was drawn parallel to the shorter side of the sheet, 5 cm from the edge. Aldo-and keto-standards were applied in separate groups alongside the urine. Ascending chromatography was carried out in square jars with outside dimensions of 31 X 62 cm (Fisher Scientific Company). The paper sheet was arched and the edges stitched to an additional 30-cm wide strip carrying other urinary samples and standards. The solvent filled the bottom submerging the paper about 10-15 mm. Chromatography was started without preliminary exposure to the vapors. During repeated runs, the solvent was refilled to the original level. Testing of Soluents. The suitability of the solvent systems was tested with a mixture of standard sugars similar to those occurring in the urine and later with the urine itself. After each run, the sugars were located to find the optimal schedule for the separation. Between individual irrigations and after the final run, the chromatograms were dried at room temperature. The drying period between individual runs containing n-butanol or more volatile components was 2 hours, for less volatile it was extended accordingly. Chromatograms from phenolic solvents were allowed to dry overnight. Detection. After the odor of the solvents completely disappeared, the paper chromatograms were viewed in short-wave UV light. The UVabsorbing spots were marked with a pencil, serving as an indicator of the regularity of the flow. Then a line was drawn in the middle of the urine sample. After cutting the paper along the line, the part of the chromatogram containing half of the urinary spots with adjoining standards of aldoses was sprayed with aniline citrate according to White and Hess (2) with a modification (10) which ensures stepwise detection of different classes of aldoses and avoids nonspecific reactions of some ketoses. The remaining part of the chromatogram was sprayed with orcinol-trichloroacetic acid reagent to reveal ketoses ( 11). Stained sugar spots were
284
V&K
AND
ViTEK
also observed in the fluorescent bulb light of a vic’w box for rocntgenograms and their fluorescence recorded under the UV lamps ~lincrallight-UVS-11 (260 rnp main wavelength) and Black Ray-UVL-21 (360 mp), both made by Ultraviolet Products, Inc., San Gabriel, California. The position and size of the spots in actual dimensions were recorded on graphic cross-section paper. The part detected with orcinol was matched to that revealed with aniline citrate. In the presence of both fluorescence and color, the size of the spot was recorded according to the more sensitive fluorescence which provided larger spots than the color itself. For the preparation of chromatographic maps illustrated in Fig. 1, the urine of five persons was subjected to chromatography in duplicate samples. One of them was chromatographed with the addition of 100 pg quantities of d-allose, d-glycero-d-alloheptulose, maltose, galactose, and 200 pg of N-acetylglucosamine. Quantitative Determinations. Total reducing sugars in the desalted urine were determined by Nelson’s modification of Somogyi’s method ( 12), glucose with glucose oxidase preparation Glucostat of Worthington Biochemical Corp., Freehold, N. J., and galactose by galactose dehydrogenase method supplied by Boehringer Mannheim Corp., New York, N. Y. RESULTS
In a search for acidic, basic, neutral, phenolic, and ethylacetate type of solvent systems suitable to separate neutral sugars, we tested 47 solvent combinations. These included various combinations of ethanol, n-propanol, isopropanol, n-butanol, isobutanol, sec. butanol, amylacohol, terc. amylalcohol, aceton, methyl ethyl ketone, benzylalcohol, phenol, the formic, acetic and boric acids, pyridine, and ammonia. Among them were also some well-known systems such as n-butanol-acetic acid-water (4 : 1: 5)) ethyl acetate-acetic acid-water (3: 1: 3)) n-butanol-ethanol acid-water (5: 1: 4)) ethyl acetate-pyridine-water (2: 1: 2)) n-propanolacetic acid-water (7: 1:2), and phenol saturated with water ( 13, 14). The most frequent reasons for unsatisfactory performance were: (a) no separation, (b) resolution with streaking, particularly of the fastest sugars or of the slowest moving ones, (c) diffise spots, either generally or in the fastest migrating fraction, (d) a too fast flow resulting in crowding of monosaccharides in the upper part of the chromatogram, (c) unsufEcient ascent of the solvent because of large viscosity, and (f) unsufficient stability of the solvent in the conditions of multiple chromatography. The search resulted in the selection of five systems: Solvent A: n-butanol-pyridine-benzene-w ater ( S: 3 : 1: 3 )-not miscible ( 1.5) : B :
CHROMATOGRAPHY
OF
URINARY
285
SUGARS
GM SOLVEWT
FROM /
------_-_
50
--------
15 40 35 30 25 20 15 IO 5 A
K
A
K
0 n-Bul-Py-or-w 5:3 :I .3
Isobul-AcA-W 4 I ‘I
Ethylac- AcA-W ‘I 3 ‘I
sec. But-Ethylac7 2
W 2
n-But-Phe-AcA-W 5 5 : 2
:10
FIG. 1. Distribution pattern of urinary sugars in five proposed solvent systems. The schema was drawn on the basis of the results of chromatography in two duplicates of two urinary specimens obtained from five healthy subjects after overnight fasting using the schedule and conditions described in Procedures and in Table 1. It includes the spots of all sugars forming the constant spectrum of human urine, and some sugars revealed occasionally, i.e., maltose, sucrose, and glucuronolactone. The dimensions in the direction of the flow corresponded to the actual size of spots. Aldoses (A) detected with aniline citrate reagent (2, 10): 1, lactose; 2, maltose; 3, unknown red spot, tentatively identified as a glucosylxylose; 4, galactose; 5, glucose; 6, allose; 7, mannose; 8, arabinose; 9, N-acetylghrcosamine; 10, xylose; II, fucose; 12, ribose; 13, unknown yellow orange spot; 14, glucuronolactone; 15, unknown pink spot, later light brown. Ketoses ( K) detected with orcinol-trichloroacetic acid reagent ( 11) : 1, sucrose; 2, mannoheptulose; 3, sedoheptulose; 4, fructose; 5, alloheptulose; 6, unknown beige to beige-pink spot; 7, allulose; 8, xylulose; 9, ribulose; 10, unknown pale yellow; 11, unknown yellow. Ketoses 10 and 11 may be identical with aldose spots 13 and 15, respectively.
isobutanol-acetic
acid-water
water
II:
( 4: 1: 1) (3); C: ethyl acetate-acetic acidbutanol-ethyl acctatt,-\~,atcr ( 7 ::! :2 ) ; I< : n.-butanol-phenol-acetic acid-water (5: 5: 2: IO)--not miscible ( 16). The solvents A-E were employed according to the schcdulc given in Table 1. It applies to Whatman No. 17. (3:l:l);
X‘C.
TSBLE SCHEDULE
OF MUL'ITIJI~E:
__System
1
A B C 1) E
18 7 18 24.6 24.5
__~-2 21 18 18 24.5 94.5
1
ASCENI)ING CHROMATOGRAPHY Fiw SoLvewr SYSTEMS~
:3 24 21 21.5 x2.5 26.5
Number of developments ~. ..-I .i ti 26 22.5 26 33 26.-i
30
38.5
34 31
36.5
OF I~RINAHY
.__-7 38.5
SU(:.~RS
s 41.5
IN
Total hours 89.5 217
a The composition of individual solvent system is described under procedures. The duration of runs is given in hours. The schedule is proposed for Whatman No. 17 filt,er paper.
Figure 1 shows the separation of 23 regularly present sugars and 3 occasionally occurring ones (sucrose, glucuronolactone, and maltose). The illustration also indicates (1) the degree of overlapping between neighboring spots of aldoses and ketoses, (2) the relative size of the spot in the flow direction, and (3) the actual position of the front after the last development. The aniline-positive spots 13 and 15 and ketoses 6, 10, and 11 are constant, as yet not identified, constituents of the urine. As the temperature fluctuated only between 23-26’, the positions of spots were very well reproducible in all systems but the best results were obtained in isobutanol solvent (system B) where the positions of individual spots usually could be predicted with a greater accuracy than 10 mm providing that the schedule was strictly observed. The compactness of the spots decreased in the order: solvent B, A, D, C, E. Solvents A, D, and C showed only little differences in the quality of the spots. System E displayed the largest diffusion but other tested phenolic systems were similar or often worse. The background of chromatograms is very good in systems B, C, and D. System A is slightly inferior, particularly in the front zone, where occasionally unidentified aniline-positive yellow spots appear. The possibility of interference with the detection is more serious in the system F which gives a colored background of various intensity and a considerabIy broader solvent front. With the exception of glucuronolactone and N-acetylhexosamine all
CJSROMATOCRAPHY
OF
URINARY
SUGARS
287
nonphenolic systems separate simple sugars in the same order but the quality of separation of certain pairs or larger groups differs significantly as evident from Fig. 1. For easier orientation, the characteristic separator-y properties of the proposed systems are summarized in Table 2. The stability of urinary sugars in the selected systems is good, except glucuronolactone, which in the neutral system D and especially in the basic A decomposes and a distinct streak links the spot of the liberated glucuronic acid with the original lactone. We did not observe this in the normal urine probably because of the very low concentration in the latter. In all systems standards of ribulose, obtained from three different sources, formed two spots of the same staining and fluorescenting properties as marked in Fig. 1 (keto-spot No. 9)) the smaller one usually partially masking the spot of allulose (spot No. 7). We did not notice this phenomenon upon analyzing the urine. For many experimental or diagnostic purposes, where the detection of the smallest components, such as allose, alloheptulose, mannoheptulose, N-acetylglucosamine, and the fast migrating aniline-positive spot No. 15, are not a necessity, a much faster monodimensional descending chromatography was developed using Whatman paper No. 3 MM and the solvent n-butanol-pyridine-water (6: 4 : 3) and/or ethyl acetateacetic acid-water (3: 1: 1). The schedule for individual runs is given in Table 3. The quality of separation is good providing that not more than a 5-10 minute aliquot of diuresis is employed and that the glucose content is within the normal range, or only moderately increased, i.e., less than 150 mg/24 hr. DISCUSSION
The complexity as well as the constancy of composition of the urinary sugar spectrum have not been realized in previous reports. White and Hess (2) reported in 1956 the occurrence of 11 aldoses and 8 ketoses in the normal urine. Kopecky and Kellen (3) confirmed the findings of White and Hess (2) and the presence of mannose, mentioned earlier ( 17). Moreover, they described vaguely a few additional unidentified spots. A later report by Fleury and Eberhard ( 18) mentioned only nine sugars in the normal urine, one of them identified as maltose. According to these authors (2, 3, 17, 18) the majority of sugars occurs in the urine only occasionally. We have found, however, that the number of sugars in the urine is much less erratic and depends mainly on numerous technical factors, such as the quality of deionization, applied volume, quality of resolution, sensitivity and specificity of the detection methods. Diet plays also an important part since it may considerably influence the levels of certain sugars such as glucose, fructose, and especially sucrose, lactose, and galactose, and according to Date (19) also of pentoses. Our
2 PROPOSED
Ethyl acetate-acetic (3:l:l)
acid-water
SOLVENT
Unknown aldose (31-galactose (4) xylose (lo)-fucose (ll)-ribose (121 unknown aldose C13 )-gltlc:rironolact.one i 14 ) allohept\dose (5)-unknown ketose 16 1 alluloae (7 I-xylulose (8) unknown ketose (6i~allulose (71
ITnknowll aldose CB)-galactose (4 I allose (6j-mannose (7) mannoheptulose (‘L)-sedoheptllloae (3) ribose (121-unknown aldose (131
acid-water
(1 I
separation
THE
Isobutanol-acetic 14:l:l)
good
(1 )-maltose (2’1 (4)-glucose (5 1 (8)-xylose (9) (Cribulose (9) aldose (3)~sucrose
to very
0~
TABLE
Lactose galactose arabinose xylulose unknown
Fair
CHARACTERISTICS
Butanol-pyridine-benzene-water (5:3:1:3)
System
SEPAR.4TlON
Poor
or no separation
Glucose i.i I-allose (6 i unknown aldose (Is)-galactose il sedoheptulose (X-fructose (4) xylose (lo)-fltcose ill I-ribose 112’1 mannose (7)-fructose (411 mannose (7i-arahinose (8, alloheptulose (.i I-~~tnknown ketosr /6 frllctose 14 1 -nlloheptlllose (3 i
SYSTEMS"
CHROMATOGRAPHY
OF
URINARY
SUGARS
2239
290
n-Butanol-pyridine-water (6:4:3) Ethyl acetate-acetic acid-water (3:l:l)
ViTEk’
AND
ViTEE
6
x
5.0
6.0
1% 6 .5
0 The duration of individual runs is given in hours. The schedule What.man No. 3 MM filter paper.
14 7.5
40 26
is proposed
for
experience, however, shows that with the exception of sucrose and POSsibly an unknown ketose (spot No. 6) all sugars (Fig. 1) occur in the urine even under complete caloric starvation. The urinary sugars belong to various classes. Among them simple aldoses and ketoses are most common (Fig. 1). They form groups of isomers, such as galactose-glucose-allose-mannose; arabinose-xyloseribose; fructose-allulose; mannoheptulose-sedoheptulose-alloheptulose; xylulose-ribulose. Some groups of isomers, e.g., fructose-allulose, arabinose-xylose-ribose or glucose-mannose, may be separated relatively easily in a single run, particularly in an overflow arrangement. In order to achieve the best separation of all groups on a single chromatogram we employed multiple chromatography. Its mechanism and conditions of separation were extensively studied by Jeanes et al. (20) and later by Thoma (21). Using simple mixtures of sugars, they proved (20, 21) that the sluggishness of the procedure is highly compensated by the quality of resolution. Figure 1 demonstrates, however, that a perfect resolution of such a complex mixture as urinary sugars cannot be achieved even by multiple ascending chromatography where the ascent of solvent is an additional factor favoring further concentration of spots. An overlapping of some sugar spots in varying degree occurs in any of the proposed systems (Fig. 1). It is unavoidable even in two-dimensional chromatography (2, 18) which is less convenient for serial analyses and for a comparison of the positions of aldoses and ketoses. Therefore a sensitive and highly specific detection method is of importance not only in successful differentiation between aldoses and ketoses but also in revealing compounds which occur in very low concentrations. These compounds need more attention especially when their RF values are very close to other sugars which are present in several times higher concentrations. The most illustrative example is the group galactose-glucoseallose.
sugar
74.0 43.8 95.8 56.8 29.4 70.0 56.0 123.0 103.0 163.0 81.4 40.3
A.J. B.R. H.M. T.A. R.T. B.L. A.K. H.J. V.V. W.E. Mean 3~ SD
u The
edml
Name
-
content
61.20 73.73 59.18 57.89 54.29 58,90 62.64 57.02 96.48 89.79 67.11 14.75
w/24 hr
GLUCOSE,
was estimated
0.425 0.512 0.411 0.402 0.377 0.409 0.435 0.396 0.670 0.693 0.473 0.116
w/lo min
Glucose
FREE
specimens
6.34 2.78 4.32 1.65 8.35 4.42 10.46 12.48 3.74 7.20 6.17 3.47
in urine
0.036 0.033 0.019 0.012 0.107 0.026 0.081 0.040 0.024 0.031 0.041 0.030
&ml
collected
5.24 4.18 2.66 1.66 15.42 3.71 11.69 5.79 3.50 4.41 5.87 4.31 from
healthy
340 225 340 275 125 255 260 550 360 493 327 127
dml
Total
individuals
2.239 2.628 1.459 1.944 1.604 1.489 2.018 1.771 2.340 2.093 1.959 0.384
mg/lO min
after
322.42 378.43 210.70 279.94 230.48 214.42 290.59 255.02 336.96 301.39 287.02 52.30
w/24 hr
(Somogyi)
IN NORMAL
red . sugars
TABLE 4 REDUCING SUGARS
w/24 hr
TOTAL
Galactose
AND
mg/lO min
GA~ACTOSE, OF
fasting.
1:11.7 15.7 22.2 35.0 3. 5 15.9 5.4 9.9 27.6 22.7 16.9 9.5
27.47 21.55 22.36 28.63 33.11 24.34 4.70
22.93
18.98 19.48 28.17 20.68
yi (:lucose of total sugars
ADULTS"
Galactose glucose
overnight
URINE
1.63 1.24 1.27 0.59 6.68 1.73 4.02 2.27 1.04 1.46 2.19 1.83
y. Galactose of total reducing sugars
$ 54
2
il & 4
%
$
s F: ?
292
ViTEK
ALND
ViTEK
Glucose is the strongest component in the entire sugar spectrum of the normal urine, representing on average 35% of the total reducing powri of the desalted urine (Table 4). Allose, and frequently also galactose (Table 4), is present in very small amounts. If glucose is markedly elevated its spot becomes very large, tends to streak and absorb or at least to blur the neighboring weak spots of galactose and allosc. If it is increased even more, it may ruin the separation of the entirc specimen. This problem has been solved to a great extent by using Whatman No. 17 chromatographic paper, which unlike other papers has the capacity not only to carry the heavy load of the sample but also to separate sufficiently galactose and allose even if the glucose concentration is increased up to 400 mg/24 hr. Comparing the performance of the proposed systems and some solvents very popular in the carbohydrate chemistry we found that the systems A, B, and C gave almost identical results with n-butanol-pyridine-water ( 6: 4 : 3)) n-butanol-acetic acid-water ( 4 : 1: 5 ) , and ethyl acetate-acetic acid-water (3: 1: 3), respectively. For this reason the resolution pattern shown in Fig. 1 may be helpful as a guide in determining the relative positions and the separation quality of urinary sugars in other solvents of similar composition. In addition it may serve as a reliable base for any specialized investigational task. During these studies with sugar chromatography we have observed, contrary to the generally accepted view, that neutral sugars are not capable of ionization in the absence of complex forming agents, that the quality of separation of two sugars seems to be dependent on the basicity or acidity of the medium, In all acidic nonphenolic solvents, regardless whether the secondary component is an aliphatic alcohol or ethyl acetate, resolution of certain isomeric pairs such as galactose-glucose, arabinosexylose, xylulose-ribulose and less distinctly mannoheptulose-sedoheptulose is worse than in basic or even neutral systems. It seems to be of minor importance what produces the basicity since the effects of pyridine and ammonia are very similar. This phenomenon is also evident from a comparison of the results obtained in the systems B and C in contrary to those achieved in solvents A and D. Although the mechanism of this behavior is not elucidated we assume that heavy hydration of the molecules of isomeric sugars, which occurs during migration (23). is not equally affected by the profound change of the hydrogen ion concentration of the medium. SUMMARY
A method for chromatographic separation on Whatman No. 17 and 3 MM filter papers is described by means of which at least 21 identified
CHROMATOGRAPHY
OF
URINARY
SUGARS
293
and 5 unknown neutral sugars regularly present in the normal human urine may be revealed. The pattern of their resolution is compared in five solvent systems of different nature. The procedure uses Whatman No. 17 in ascending multiple modification for urine specimens corresponding to a lO-20-minute diuresis. For smaller aliquots of urine Whatman No. 3 MM and descending repeated developments are proposed. The former modification has an advantage of revealing better the minor constituents and possessing superior reproducibility. ACKNOWLEDGMENTS Our thanks are due to Dr. Nelson X. Richtmeyer of the National Institutes of Health, Bethesda, Maryland, for generous samples of &&se, sedoheptulose, mannoheptulose, and d-glycero-d-alloheptulose; to Dr. Gilbert Ashwell of the same Institute for samples of xylulose and ribulose and also to Dr. R. J. Suhadolnik of the Albert Einstein Center, Philadelphia, Pennsylvania, for a gift of d-allulose and psicofuranine. Appreciation is also expressed to Dr. Harriet L. Frush of the National Bureau of Standards for suggesting the Whatman No, 17 filter paper. We also are grateful to Mrs. Hsi-Chiang Lin, MS., for the aid in analysis. ADDENDUM Our further investigations proved that isomaltose is also a constant constituent of human urine. The approximate range in healthy persons is 2-f mg/24 hrs. In the proposed five systems isomaltose spot has the RF value identical or very similar to that of lactose. REFERENCES 1. V~TEK, V., AND V~TEK, K., Clin. Res. 16, 556 (1968). 2. WHITE, A. A., AND HESS, W. C., Arch. Biochem. Biophys. 64, 57 ( 1956). 3. KOPECKP, A., AND KELLEN, J,, “Papierchromatographie der Zucker in der Klinik,” pp. 58, 40. Edition, Leipzig, 1963. 4. LATO, M., BRUNELLI, B., CIUFFINI, G., AND MEZZELLI, T., J. Chromutogr. 34, 26 (1968). 5. JOLLXY, R. L., AND FREEMAN, M. L., Clin. Chem. 14, 538 (1968). 6. STRECKER, G., G~UBEL, B., AND MONTREUIL, J., Compt. Rend. Acad. Sci. 260, 999 (1965). 7. V~TEK, V., V~TFX, K., AND SACKS, W., Fed. Proc. Fed. Amer. Sot. Exp. Biol. 27, 254 (1968). 8. V~TEK, V., V~TEK, K., AND SACKS, W., Fed. PTOC. Fed. Amer. Sot. Exp. Biol. 28, 906 (1969). 9. %EK, v., AND ViTEK, K., In preparation. 10. V~TEK, V., AND V~TEK, K., In preparation. 11. KLEVSTIUND, R., AND NORDAL, A., Acta Chem. Scud 4, 1320 (1950). 12. SOMOGYI, M., j. Biol. Chem. 195, 19 (1952). 13. MACEK, K., in “Paper Chromatography” (I. M. Hais and K. Macek, ea.), p. 303. . Publishing House of the Czechoslovak Academy of Sciences, Prague, 1963. 14. BAILEY, R, W., AND PFUDHAM, J. B., in “Chromatographic Reviews,” Vol. 4. Elsevier Press, Amsterdam, 1962.
294
ViTEh:
AND
ViTEE;
IS. ALBON, N. II., AND Gnossq D., AnuZtJst 75, 454 (19.50). 16. KRISHNAILIUIITY, Ii., AND SWAhlINATHAN, M., J. %i. kd. Res. 14c, 310 (1955). 17. MONTREUIL, J., ASI> BOWLANGER, P., Compt. Rend. Acad. Sci. 236, 337 (1953). 18. FLEURY, P., AND E~EIIHAR~, H., Ann. Biol. C.&n. Puris 23, 1175 (1965). 19. DATE, J. W., Scaud. j. Chr. Lub. Inwst. 10, 115 (1958). 20. JEANES, A., WISE, C. S., AND DIMLER, R. J., Anal. Chem. 23, 415 (1951). 21. THOMA, J. A., Anal. Chem. 35, 214 (1963). 22. TOWER, D. B., PETERS, E. L., AND POGORELSIUN, M. A., Neurology 6, 37 (1956). 23. ISHERWOOD, F. A., Brit. Med. Bull. 10, 202 (1954).