CHROMATOGRAPHIC IDENTIFICATION OF
REDUCING SUGARS IN URINE*
LAWRENCE II. SOPHIAN, M.D., AND VALENTINE J. CONNOLLY, B.S.
From the United States Public Health Service, Staten Island, New York
I t is often necessary to identify the substance causing reduction by the alkaline
copper sulfate test of urine. Glucose is the most significant and frequent constituent but other sugars and chemicals may be responsible. Thus, levulosuria
may occur in association with glucose in severe cases of diabetes mellitus or as an
obscure defect of metabolism.6 Essential chronic pentosuria has been reported
as an occasional familial trait in Jews,1 and alimentary pentosuria may occur
following the ingestion of fruits rich in pentose. It is well established that lactosuria occurs in some women during the period of lactation. Galactose has been
noted in the urine of suckling children in association with gastrointestinal dysfunction.2 In addition, non-carbohydrate substances, such as urates, homogentisic
acid and ascorbic acid, will at times bring about the reduction reaction observed
when the alkaline copper test is used.
The available methods for determining nonglucose reduction depend upon
fermentation reactions, osazone formation, optical rotation and chemical color
reactions with specific reagents. For several reasons such as complexity, variety
of reagents and delay in handling, none of the above is wholly satisfactory for
employment in a routine clinical laboratory.
A recent article4 provides the basis for selecting optimum conditions for the
separation of reducing sugars by paper chromatography and stimulated us to
attempt practical application of the technic. Successful results of this type have
been reported by Odin and Werner,6 who demonstrated lactose in the urine of
5 of 10 pregnant women. Horrocks and Manning 3 applied the procedure to recover various reducing sugars added to urine.
The investigations of Jeanes, Wise and Dimler4 indicated that for the particular carbohydrates usually confused with glucose in urine, the most satisfactory
separation could be obtained employing a solvent mixture of 1-butanol, pyridine
and water in a ratio of 3:2:1.5. One hundred nil. of this solution is placed in a
glass pipet jar 15 x 30 cm. This is clone at least 30 minutes before beginning the
chromatogram, to allow for vapor saturation of the atmosphere within the vessel,
with the top firmly closed by a heavy ground-glass lid. AVe employed pure solutions of galactose, xylose, lactose, levulose and glucose in a concentration of 1.75
per cent in saturated benzoic acid. This concentration was chosen because with
the volume used in spotting the sample (0.01 ml.), a well-defined spot was produced by the development of the completed chromatogram using dinitrosalicylic
acid. This concentration is equivalent to a two-plus reaction with Benedict's
solution, a frequent range in those samples that would be of medical interest. To
provide a faster-moving'front of solvent, the paper (E & D 609) was cut in strips
* Received for publication, August 30, 1951.
41
42
SOPHIAN AND
CONNOLLY
9 x 50 cm. in such a manner that the elution was carried on along the major
axis of the paper fibers. The major axis can be determined by admitting water
slowly onto the surface of the paper to form a spot. The elliptical shape of the
#
XILOSE
m
IflTOLQSS
I2TOL0SE
"fflUJSCEE-
amass
W
•
*%£?•&
GAUCT03B
. GAIAOTOSE
m
.-^V*
..;«?L'-CTOSS
».,
F I G . 1. The heav,y line at the bottom indicates the spotting point of the specimens, lanes
A to E contain pure standard solutions in concentrations of 1.75 per cent, lane F contains a
mixture of the standard sugars each in a concentration of 1.75 per cent, and lane G, a specimen of diabetic urine.
spot marks the axis as its long diameter. The paper strips are then lightly dotted
with pencil 4 cm. from the bottom, dividing the strip into six lanes each 1.5 cm.
wide. One lane is for the unknown sample and the others for the standard solutions of the control sugar. Odin and Werner 5 pretreated the urine with absorbing
resins, but we did not find this necessary.
IDENTIFICATION
$m
O F SUGARS BY
CHROMATOGRAPHY
43
\ •
FIG. 2. Apparatus to chromatograph strips longer than height of vessel
PROCEDURE
Step 1. Adjustment of urine sample.
Each sample of urine is diluted with distilled water to approximately the concentration of the sugar standards by approximate quantitation by means of
Benedict's solution. The determination of equivalents of the various sugars
against Benedict's solution can be ignored since the concentration need be only
44
SOPHIAN AND CONNOLLY
approximate. We have found that concentrations varying greater than about
threefold (the concentration of the standard solutions) influence R/ values. If
the sample is less concentrated than the standard solutions, a greater volume of
the specimen can be added to increase the concentration, placing each drop on
the spot previously made dry.
Step 2. Spotting of sample.
A pipet of 0.2-ml. capacity with a marked volume at 0.01 ml. is used. By intermittently running out a small volume carefully and allowing a period for drying, 0.01 ml. is delivered about 4 cm. from the bottom of the strip in the 1.5-cm.
lane marked out for the purpose. These spots are allowed to dry at room temperature.
Step S. Chromatographing.
After drying, the strips are placed on the plastic holder (Fig. 2) and then into
the jars in such a manner that about 0.5 cm. of the lower edge of the paper is
immersed in solvent solution. The solvent front can be observed to move slowly
upward. In 16 hours (overnight) the solvent front travels about 42 cm., and
satisfactory resolution of spots results. The reaction is carried out at room temperature. After the solvent front has travelled the designated distance, the paper
is removed, the solvent front marked lightly with pencil and the paper kept at
room temperature, or a little warmer, to evaporate the solvent solutions.
Step 4- Developing spots.
When thoroughly dry, the paper is sprayed with a solution of 0.5 per cent
3,5-dinitrosalicylic acid in 4 per cent sodium hydroxide. After drying, the paper
is placed in a hot-air oven at 100 C. for ten minutes. The location of the sugars
is at the site of the brown spots produced and the latter are best observed by
transmitted light. Non-carbohydrate substances do not react.
Step 5. Calculating Rf value.
The center of the spot is located and the distance of travel from the original
point is measured. The distance travelled by the solvent front is measured from
the original point of spotting the specimen to the previously marked end point of
travel. The R/ (ratio of fronts) can then be calculated:
„
_
distance travelled by solute spot
distance travelled by solvent front
RESULTS
In a series of chromatograms on pure solutions, the average R F values of 10
measurements obtained with pure solutions were as follows: galactose 0.36, xylose 0.52, lactose 0.22, levulose 0.46, glucose 0.41. The values were reproducible
for the greater part, but varied slightly from run to run. For this reason we prefer
to employ a set of standards with each unknown.
IDENTIFICATION OF SUGARS BY CHROMATOGRAPHY
45
The results obtained on a sample urine obtained from a patient suffering from
diabetes are shown in Figure 1. In the column to the left of the urine sample,
we tested 0.01 ml. of a solution containing all five sugar controls, each in a concentration of 1.75 per cent. Apparently the presence of one carbohydrate interferes little with the mobility of another and satisfactory separations can be expected when more than one sugar is present. In addition we have demonstrated
galactose from a patient with galactosuria during the course of a galactose tolerance test of liver function. Lactose was detected in a number of samples of
urine from lactating mothers. Levulose was isolated from a sample of urine obtained during the performance of a levulose tolerance test.
Specific identification of some of the spots can be made by employing the reagents recommended by Horrocks and Manning. 3 With their phloroglucinol solution, pentose gives a green color, fructose an orange-brown and galactose a faint
brown. Their aniline hydrogen oxalate reaction gives a yellow color with lactose,
red with pentose, faint yellow with fructose, while glucose and galactose each
give a strong brown color. A modification of Tollen's reagent adopted by us (1
per cent phloroglucin in 20 per cent HC1) gives a violet spot with pentose, an
orange spot with fructose, and a faint yellow with galactose, when developed
at 75 C. for about fifteen minutes; while dextrose and lactose give no reaction.
In Figure 2 is illustrated the manner by which a 50 cm. strip of paper can be
chromatographed in a 30-cm. vessel, using a plastic holder to contain the strip
in a spiral S-shape fashion. The holders,* 6 cm. wide and 18 cm. long, are made
with a series of six grooves, permitting insertion of a strip of paper so that after
wetting, one fold will not be so weighted as to sag and touch the one beneath.
Two of these plastic holders may be mounted on a glass frame so that they stand
without support. This arrangement provides chromatograph strips while eliminating the large and cumbersome glass containers usually employed.
SUMMARY
Paper chromatography can be employed to identify the type of reducing sugar
in urine. The Ry values appear to be accurate and characteristic of specific
sugars.
A special apparatus is described for use with our procedure.
REFERENCES
1. BOCK, J . C : Benign meliturias. Physiol. Rev., 24: 169-176, 1944.
2. GOLDBLOOM, A., AND BRICKMAN, H . F . : Galactemia. J . Pediat., 28: 674-691, 1946.
3. HORKOCKS, R. H., AND M A N N I N G , G. B . : P a r t i t i o n chromatograph on paper; identification of reducing substances in urine. Lancet, 1: 1042-1045, 1949.
4. J E A N B S , A., W I S E , C. S., AND D I M L E R , R. J . : Improved technics in paper chromatography
of carbohydrates. Anal. Chem., 23: 415-420, 1951.
5. ODIN, L., AND W E R N E R , I.: Qualitative determination of reducing sugar in urine. Nord.
mod., 43: 470-471, 1950.
6. SILVER, S., AND R E I N E R , M . : Essential fructosuria; report of three cases with metabolic
studies. Arch. I n t . Med., 64: 412-426, 1934.
* Obtainable from Kiisch Co., Sturgis, Michigan.