Potato Research 35 (1992) 5 5 - 5 8 SHORT COMMUNICATION

A microplate reader assay for rapid enzymatic quantification

of sugars in potato tubers

R. VIOLA and H. V. DAVIES

Department of Cellular and Environmental Physiology, Scottish Crop Research Institute,

Dundee DD2 5DA, United Kingdom

Accepted for publication: 19 July 1991

Additional keywords: glucose, fructose, sucrose, Solanum tuberosum L.

Summary
A sensitive, rapid and inexpensive method has been developed for quantifying the glucose,
fructose and sucrose content of potato tubers. The method, based on selective enzyme-coupled
reaction systems and the reduction of NAD to NADH, uses a microplate reader fitted with a
340 nm filter. In one microtiter plate 96 samples can be analysed for all three sugars in less than
two hours. Several plates can be processed in parallel.

Introduction

Analysis of potato tuber reducing sugar and sucrose content is important to our
understanding of sugar metabolism and hexose accumulation in storage. The accu-
mulation of glucose and fructose beyond certain limits affects, adversely, the process-
ing quality of tubers due to a typical Maillard reaction with amino groups at high
temperature. The inherent variability in sugar composition between tubers can be
substantial and adequate numbers of samples must be processed to obtain an accurate
assessement of sugar balance e.g. when examining the effects of cultural practice and
environmental factors. Furthermore, breeding programmes designed to improve the
storage potential of tubers also require the analysis of large numbers of samples to
identify promising clones. There are many methods currently available for quantify-
ing soluble sugars. Many are potentially non-specific, relatively insensitive colouri-
metric tests, often using toxic or otherwise hazardous chemicals. A list would include
alkaline ferricyanide (Baijal & Van Vliet, 1966), anthrone (Cerning-Beroard, 1975)
and Somogyi's copper sulphate-based formulation (Cronin & Smith, 1979). Analysis
of individual sugars commonly involves the use of H P L C , gas chromatography and
enzymatic (autoanalyser) methods (Davies, 1988 and references therein). The present
paper reports the use of a microplate reader for the determination of glucose, fructose
and sucrose in potato tubers.

Materials and melhods

Instrumentation. The microplate reader used was a Dynatech MR700 equipped with
a 340 nm filter. Flat bottomed microtiter plates were also obtained from Dynatech
(Billingshurst, Sussex).

Potato Research 35 (1992) 55

R. VIOLA A N D H,V. DAVIES

Reagents. The reagent for glucose determination was obtained from Technicon
Diagnostics (product number T 11-1832). This is commonly used to determine glucose
with an autoanalyser. It contains, in a dry form, yeast hexokinase (EC 2.7.1.1),
glucose-6-phosphate dehydrogenase (EC 1.1.1.49), ATP, NAD, Mg 2+, buffer and
stabiliser. For the microplate assay 1 g of reagent was dissolved in 8 ml of distilled
water.
Phosphoglucoseisomerase [PGI] (cat. no. 128139; EC 5.3.1.9) and invertase (cat.
no. 104914; EC 3.2.1.26) were obtained from Boehringer Mannheim. Care must be
taken in selecting a suitable source of invertase as some preparations are contami-
nated with glucose. The glucose reagent and protocol outlined below can be used to
identify suitable batches.

Protocol. In our laboratory transverse sections (3 mm thick) are taken through the
mid-region of the tuber, rapidly frozen and freeze-dried. Adjacent sections are taken
for fry tests. Freeze-dried samples are finely ground and 100 mg extracted in a sealed
glass bottle with 5 ml of 80 ~ technical grade alcohol. The bottles are immersed in
a shaking water bath at 70 ~ for 2 h during the extraction process. Particles are then
allowed to sediment under gravity and the ethanol solution diluted, if necessary, with
water prior to analysis. For any batch of tubers a suitable dilution has to be deter-
mined by trial and error. As a guideline, for tubers with a sugar content of ca. 0.25 %
of the fresh weight (2.5 mg g-1 FW) the ethanol extract can be used undiluted,
analysing volumes between 30 and 50/A. When the sugar content is high (around
1.5 % of the fresh weight [15 mg g- ~FW]) it is advisable to dilute samples x 5 or x 10
and use volumes in the region of 50 #1. The requirement for dilution will depend on
the degree of accuracy of the pipetting system used and the competence of the
operator. It is important that the total sugar content (glucose + fructose + sucrose) of
the sample falls within the linear range of the microplate reader (see Fig. 1). The
protocol outlined recovers >95 % of soluble sugars. Preliminary experiments in
which tuber samples were spiked with 14C sucrose showed that < 5 % of the sucrose
was degraded during the extraction procedure (data not shown).
Up to 100/zl of extract or standard solution is added to a microplate well and the
volume made up to 220/A with distilled water. The starting absorbance of each well
is then recorded at 340 nm (AI). Glucose reagent (30 tzl) is added (to give 1.5 units
hexokinase, 1.2 units glucose-6-phosphate dehydrogenase, 2/~mol ATP, 1.2 p.mol
NAD and 15 ~mol Mg 2 + per well) and the absorbance at 340 nm recorded again once
the reaction is complete at room temperature (A2). A 2 - A 1 gives the increase in
absorbance due to the conversion of glucose to 6-phosphogluconate and the corre-
sponding reduction of NAD to NADH. PGI is then added to all wells (10/zl = 0.2
units; enzyme diluted with water) and the increase in absorbance at 340 nm allowed
to proceed to completion, again at room temperature (A3). A3 - A2 gives the increase
in absorbance due to fructose. Invertase (10 t~l = 10 units; enzyme prepared in 0.1 M
acetate buffer, pH 5) is added to all wells and the increase in absorbance at 340 nm
determined (A4). A 4 - A 3 gives the increase in absorbance due to the glucose and
fructose released from sucrose inversion. The standard solution contains 60 Izg m l -
each of glucose, fructose and sucrose and 50 izl is assayed per well (3 replicates).

56 Potato Research 35 (1992)

 A RAPID ENZYMATIC METHOD FOR QUANTIFYING SUGARS

Results and discussion

When standard solutions of glucose, fructose and sucrose were analysed individually
by the microplate method the increase in absorbance at 340 nm was directly propor-
tional to the quantity of sugar in each well. The detection limit was ca. 1 ~g and the
upper limit ca. 12 t~g (Fig. 1). Upper and lower limits will be determined to some
extent by the microplate reader used. Standards should allways be analysed in parallel
with samples to ensure that the method operates consistently.
When measuring glucose and fructose each reaction is complete within 30 min at
18~ (Fig. 2). The measurement of sucrose can take longer (up to 1 h) as the pH
optimum for invertase differs substantially from the optima for the coupling enzymes

/
A

1.2

E
C
0.9
0
0'3

%
0.6.
c
..E)

0.3
..,0

Fig. 1. Relation between absorbance at

i i i i 340 nm and the quantity of glucose (e),
fructose (,x) or sucrose ( 9 in wells of mi-
3 6 9 12
crotiter plates. Bar indicates maximum stand-
pg sugar per well ard error (n = 5 for each measurement).

1.6

E
C
1.2
0
("3

u
0.8
c

O3
x~ 0.4
Fig. 2. Reaction times for quantifying glu-
cose, fructose and sucrose in the same sample.
A
Microtiter plate wells contained 4 ~g each of
i i i i
all three sugars. A) addition of glucose reagent,
0 15 30 45 60 B) addition of 0.05 units of PGI, C) addition
min of 10 units of invertase.

Potato Research 35 (1992) 57

R, VIOLA A N D H.V. DAVIES

used to metabolise the hexoses. Since the plate reader scans 96 wells in 1.5 min it is
relatively easy to determine the timing of reaction end-points. It is essential that well
lids are replaced when reactions are taking place to prevent evaporation and drifting
of absorbance.
With the technique described it is possible to process hundreds of samples in one
day for glucose, fructose and sucrose. The method is much faster than others
currently available e.g. H P L C (ca. 10 rain analysis time per sample for the three
sugars) and autoanalyser (40 samples per hour for glucose alone). The use of enzyme-
coupled reactions also offers the specificity that many other methods do not. If a
microplate reader is available then the major limitation to sugar analysis is now the
speed of sample preparation. The microplate reader has also been used in our
laboratory for quantifying starch (glucose released after amyloglucosidase treat-
ment), amino acids, a m m o n i u m ions, and inorganic phosphate and for rapid localisa-
tion of enzymes eluted from ion exchange columns etc. during protein purification.
The latter includes hexokinases, pyrophosphatase, invertase and sucrose synthase. It
also has a proven potential in our laboratory for examining enzyme kinetic character-
istics.

Acknowledgements

This work forms part of a programme supported by EEC grant ST2 0424, which the
authors duly acknowledge.

References
Baijal, B. D. & W. F. van Vliet, 1966. The chemical composition in different parts of the potato
tuber during storage. European Potato Journal 9: 179- 190.
Cerning-Beroard, M., 1975. A note on sugar determination by the anthrone method. Cereal
Chemistry 52:875 - 860.
Cronin, D.A. & S. Smith, 1979. A simple and rapid procedure for the analysis of reducing,
total and individual sugars in potatoes. Potato Research 22: 9 9 - 105.
Davies, H.V., 1988. Rapid determination of glucose, fructose and sucrose in potato tubers by
capillary gas chromatography. Potato Research 31: 569- 572.

58 Potato Research 35 (1992)