Abtlg Allgemeine Piidiatrie

Gerd Liebezeit

Gfr Kiel

Gisela Georgi

Klaus Dirner

Roger Dawson

Universitits-Kinderklinik Kiel

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The clinical applicability of a new
automatic sugar analyser
Klaus Dirner,* Gisela Georgi,
Universitits-Kinderklinik, Kiel, Abtlg. Allgemeine Piidiatrie, Kiel, GFR.
and Gerd Liebezeit, Roger Dawson,
Institut fiir Meereskunde, Universitdt Kiel, Kiel, GFR.
Introduction
The clinical laboratory is regularly faced with the task of
establishing the causes of non-diabetic melituria. These causes
may be: physiological for neonates ], caused by hereditary
defects of carbohydrate metabolism [2], brought about by
congenital or acquired deficiencies of disaccharidases.[3], or
an abnormal sugar excretion may be found with enteritis
[4]. The necessary analysis is often approached qualitatively
by using thinlayer chromatography [5, 6, 7] ;this gives a quick
result which is often sufficient. Ion exchange chromatography
allows the quantitative determination of a range of carbohy-
drates in urine whereas enzymatic methods only measure
single compounds.
Column chromatographic methods previously described for
use on clinical materials [8, 9, 10] suffer from disadvantages;
long analytical run-times are required and the corrosive
orcinol-conc, sulphuric acid reagent is used to detect any non-
reducing sugars. Recently a method 11 usesthe non-corrosive
Nanochrom II reagent, and is investigated here for its applic-
ability to routine clinical analysis.
Material and methods
Instruments
The sugar analyser (Fluorimetric Sugar Module, Breda Scien-
tific, Breda, The Netherlands) was used packed with DA X-4
0/.t (Durrum Chemical Co., Palo Alto, USA)in the borate
form treated according to Mopper [12]. Pressure measured
at the pump head was below 30 bar (3MPa). A single channel
flowthrough filter fluorimeter was used as the fluorimetric
detector with a combined controller-integrator-recorder unit
(Figure 1). The UV absorption was measured with a UV spec-
trophotometer UV-24 (Beckman, Munich, Germany). This
company also supplied the glucose analyser for glucose and
disaccharide determinations.
* correspondence to this author
6
7 10
12
1) Reogent/Buffer 5) Injection port 9) Integrator/Con-
reservoir 6) Column troller termlnel
2) Precolumn 7) Reector 10) mY-Recorder
3} Bubble trop 8) Fluorimeter 11} Flow meter
4} High pressure pump 12) Woste
Figure 1. Block diagram of the sugar analyser.
Ultrafiltration of samples was performed with a micro-
filtration apparatus 16538 (Sartorius, GSttingen, Germany)
with a nitrogen overpressure of 10 bar (1MPa).
Chemicals
All chemicals (Merck, Darmstadt, Germany) were of the
highest purity grade. Enzymes and the test kit for galactose
were from Boehringer, Mannheim, Germany (Catalogue No.
124273). Xylose was determined using the test kit "Determin-
ation of pentoses in urine" (Hoffmann-La-Roche, Grenzach,
Germany Catalogue No. 0180). Nanochrom II (ethylenedia-
mine, EDA) was obtained from Breda Scientific, Breda, The
Netherlands. Standard solutions contained 0.7 mmoles/1
glucose, xylose, galactose, fructose, mannose and sucrose
respectively, and 0.18 mmoles/1 of lactose.
Sugar solutions for addition experiments contained 50g
sugar/1.
Methods
The borate complexes of the carbohydrates were ’separated
isocratically using a 0.7 M boric acid eluting buffer adjusted
to pH 8.90 + 0.01 with 8 N NaOH. The Column temperature
was set to 80 8 IC and a flow rate of 48 ml/h was used.
Carbohydrates are detected by the formation of a fluoro-
phore in alkaline medium with EDA according to Honda et al
[13]. As the reaction proceeds sufficiently rapidly only at
elevated temperatures, it was possible to add the reagent
(Nanochrom II) with a final concentration of 500/A/1 to the
eluting buffer. After separation the sugars were reacted with
EDA at 155 C for approximately 8 minutes. The absorbance
of the resulting fluorophore was measured at two wavelengths,
345 and 450 nm. Details of the reaction conditions (effects
of buffer molarity, pH, reaction temperature and time) are
scheduled to be published elsewhere 11 ]. To eliminate possible
effects of changes in the buffer]reagent composition, 50/al of
the standard solution was injected each day before analysis
of samples. Quantification and peak identification was
achieved by comparison of peaks and retention times with
those of standard mixtures. The volume of the ultrafiltered
samples (occasionally diluted 10 with doubly distilled water)
was kept constant at 50 /al. For urine samples the running
time was extended to 3 hours in order to elute uronic acids
which, having longer retention times than glucose (90 min),
would otherwise appear as a continuous base line increase
during the next runs.
Quantitative evaluation of chromatograms was carried out
by comparing peak heights multiplied by width at half peak
height, because matrix effects present on the baseline posed
difficulties for electronic integration (see later).
The enzymic determinations ofglucose, xylose, and galactose
were carried out according to the kit instructions, lactose and
sucrose were determined according to D6rner 14].
Bacterial activity in urine was suppressed by the addition
of 1% (v/v) thymol in isopropanol (10% w/v). For recovery
Volume 3 No.1 Januaxy 1981 27
D6rner et al New automatic sugar analyser
experiments 9 ml of urine was made up to 10 ml with stand-
ard solution and doubly distilled water. Serum and spinal
fluid were ultrafiltered and either analysed immediately or
stored at -18 C.
Results
Choice of sugars investigated
Several urine samples from newly born infants and healthy
adults were analysed to establish the "normal" pattern and to
decide which sugars to investigate. A typical chromatogram
with pure sugars is shown in Figure 2, and from neonatal urine
in Figure 3. Lactose, xylose, galactose, and glucose were
routinely found to be present with occassional occurence of
mannose, fructose, and sucrose. With the exception ofmannose
and fructose chemical methods already existed in the authors’
clinics and could be used for comparative purposes.
Samples of serum and spinal fluid from subjects without
metabolic disorders contained glucose with only traces of
other sugars as shown in Figure 4.
Linearity and sensitivity
The chromatographic method was tested with glucose in
standard addition experiments as described above and found
to be linear up to 2.5 mmoles/1 (Figure 5). Higher concen-
trations were not tested. The reference methods were tested
similarly and their linearity found to be satisfactory.
The detection limit is dependent on fluorimeter and re-
corder/integrator settings. For purposes of this study the
smallest peak area that could be evaluated was considered to
be 0.87 cm2 corresponding to an integrator value of 100. For
the reference methods, when an extinction difference of
0.005E was taken as being measurable, the detection limits
were as shown in Table 1.
Precision
Analytical precision was determined with 6 standard runs on
two separate days. Results are given in Table 2. Coefficients
of variation for sugars with long retention times are markedly
smaller for the manual evaluation than for electronic integra-
tion. Repeated analysis (n 6) of a newborn’s urine gave co-
efficients of variation for lactose and galactose of 2.8% and
liN bilirubinlinterference
could be
observed.
II 1
111111111/,, .llllll111111 III1 I1111111111111111111 I11 II1.,11, !1 3.4% respectively. The retention times showed good repro-
_1 ]l[ll[llllfr 171TINlF177 I1]111 llllIlll IIlllllllll !lllll]ll lllllllll ill Accuracy and recovery_I d[ll[[l] $ 115 [IIIt;IIIIlllNll111NI ’N NIl [ An indication of accuracy was given on one sample by extra-polation of the recovery curve to y 0 (Figure 5). The inter
_, NINI N,, }l lllllllllll]lllllllllllll],lll].lllllll,l,l ,l!lll cept concentration of 0.78 mmoles/1 was in good agreementwith the value of 0.68 mmoles/1 determined on the initialIi[, I1[1 g I, 11, 1[ sample without addition. Nine recovery experiments weretli]t ILIIILIJ&LIII]II]I lIll II l[]llIttlll Ill carried out employing 4 different urines. Table 3 liststhecon-Ill[ [lii l. Ir]]ll[]l[ lll.. centration ranges together with recoveries found from chro-_, Illl Ii 1 Ill Illlll lllll llllllll t]llll,l II I,II [1 I1/[1311] matographic and conventional methods.
]1 [l IllI gliillll[ll[111 Ill [II I111!!II Interference by preservatives and drugs, etc.-I ]lllll!I [ l[]r[lm ll!ll Ill I]1111 Addition of thymol in isopropanol was found to have no
-’," 3lllt[11 lllll Illlllllll]llllllllllllilltl ill II !I]-1 quenching effect on the fluorimetric signals.
llllll, Il[l llltl tli111 lll lllllll lll lllllllll[ l,IllIll,[ [ll ll[111 Addition of sodium, salicylate, sulfanilamide, ascorbic acid
-l lllt liltllll lll ][]!l]llllll l][l[][]ll, and aldactone showed no quenching effect but rather aslight
-:I. 1111[ tllll llIftllllllllllIlll If! IM ll[llllll tll[l Illlllll{[ llllll increase of the fluorimetric signal within the range of the
-I [I [11 Illl Illl Ill lllll 111I standard deviation (cf. Table 3).
Figure 2. Chromatogram ofpure sugars; flow rate 52 ml
h-linsteadf48mlh-lasemplOyedtherwise’thus
re__suiting in s_______horter retention times ]i iiiiiiiiiiiiiiiiiiiiiiiiillliiiiiil iiiiiiiiiiiillliiiiiiiiiiiiiiiilliiiiiiiiiil iiiiii
_Jl""il1’l"l’[ lll]lll II II[l lllllll Ili llllll[lllllI[l l[ll[ll lll
._.tl LIIIIIIIIIIIIIilIIIWTTiiiiiiI!!IlTNI-
i
-i 1111I lil ,
/I ll
_ll,,,.,Jllli I11111 I,. 1111 llll t111 llll]l IIIII[I]I!J_L].I, ill]il[i
7l"’11111 Iii1117111 l,llll..lid:lllll]Illlllll I11111IIII!ttl 1II!t ._1, ]lllit lIll !111 I,I/1 tllllll_ll_llI [1! lllll]lJ]lll
I N 1 11111 111!1 IIIIlI!III-lilil]NIItlI11111IIII!I1111 N I1!i11!II ,l l[Iltlll llt<Il I111 llllll Jllllllt[lilllll
m
7 "1 i’""" ,f,,ll -, 7tiilil’flt,t 1.11 !Ill!mllllilIll l,l7Lmii
’-1I t’ll
/ 11I!11,I _7II,11., t _+l_" +".’,".,. ""l_li 1, ll!!lll !i,li,lI,II 111 11 Ill ,IIl!ll L
iiii]7"__llill,i,l.-tlll ]11 Itl ill!l.,i 11!it!!!1 7[ ]_/ ilil ltll 11111]1II11 lI!lll!ll[lll!!lIlll
ql II ,,i
1_, _. Illll Itllllt I II11 I1,1111, ll,l!ili [ __,l .,,ll,l.llfl],l il I1, lllllillllll1111111 1111111 lll ll ill 11111 l]llHl
._][ i]illl Ill IN]Ii[I[ [lllll lllllil!llll l[ll!l!i ii[!iiU ; llmlrl’lli Ill lit lllltilllllllllllllllllllll 111 I111 tll 111
_]l ;d&Lt] IIIIIiLLililll I!! tllitlllilllll ]] Figure 4. Chromatogram from serum of an infant withi lTill] Ill/-I lit I11il llitl IIIllllllllllll Ill! 1111111 baiary cirrhosis. The following concentrations were found."
Figure 3. Chromatogram of urinary sugars from a healthy 3.84 mmoles/1 glucose, 0.0618 mmoles/l mannose, and
newborn infant collected on the 4th day after birth; same O. 0255 mmoles/l lactose. Sensitivity setting was higher
running conditions as in Figure 2. than usual and the running conditions were as in Figure 2.
28 Journal of Automatic Chemistry
Irner et al New automatic sugar analyser
Table 1. Detection limits of the methods used
Sugar
Sucros
Lactos
Galactose
Xylos
Glucos
Chromatography
(mmoles/l)
0.38
0.02
0.07
0.17
0.17
Reference
(mmoles/l)
0.56
0.56
0.05
0.43
0.28
Table 2. Coefficients of variation of the peak areas(n 6)
and retention times
Sugar
Sucrose
Lactose
Galactose
Xylose
Glucose
retention t,’m)e [min]mean maximum mlmmum
27.6 27.7 27.4
36.2 36.3 36.0
61.0 62.1 60.9
72.1 72.9 71.8
88.9 89.6 88.6
coefficient of variation
eldctro’nic manual
9.9 5.4
4.9 4.4
5.2 3.9
10.7 3.1
11.3 1.2
Discussion
The analysis of complex mixtures of carbohydrates in physio-
logical fluids has been carried out in the past mainly by paper
15 or thin layer chromatography 5, 6, 7 ]. For quantitative
approaches ion exchange chromatography has certain ad-
vantages 8, 9, 10, 16, 17 ]. Long running times, and the use
of corrosive reagents have been largely overcome by recently
developed techniques. Special resin treatment 12] and the
use of the non-corrosive reagent Nanochrom II [11] have
made it possible to carry out rapid determinations of carbohy-
drates. This reagent has the advantage over the non-corr0sive
Cu(II)-sodium bicinchionate-aspartic acid 18] in that non-
reducing sugars are also detected. Since fluorimetric methods
are sometimes sensitive to interferences from various external
agents it is important to ascertain that the matrix (urine for
example) does not influence the determination. No signif-
icant interference by sodium salicylate, ascorbic acid sulfanil-
amide, and aldactone could be established. As shown in Table
3 recoveries are generally good and superior to conventional
methods. The ability of the built-in integrator to followvarying
baselines and changing backgrounds encountered with urine
samples was not entirely satisfactory with the basic integration
program and a more complicated baseline tracking program is
Icm
20-
15-
10-
5"
1
-0.1 0:.25 0:5 b I:5 2’.0 215 .addled lucos
ImmorAl
Figure 5. Test of linearity with increasing addition of
glucose standard to urine.
Table 3. Results of recovery after the simultaneous addition
of sucrose, lactose, galactose, xylose, and glucose
concentration range mean recoveries %]
Sugar [mmoles/l] chromatography conventional
Sucros
Lactos
Galactos
Xylos
Glucos
3.65- 5.84
1.70- 2.38
4.16- 9.71
3.44 12.6
5.73- 12.49
96.2
89.6
83.4
90.4
99.2
method
81.2
81.1
85.4
121.7
79.3
required. Since the peaks were symmetrical it was possible
to use a manual method for peak area evaluation with good
precision [Table 2 ]. The retention times showed little variance
between runs. A slight shift towards shorter retention times
was observed for urine samples due to their high salt content.
This effect may be overcome by using a desalting step prior
to analysis. This also serves to remove unwanted matrix sub-
stances.
Galactose/fructose and maltose]lactose were not separated
with 0.7 molar buffer used. If fructosuria or maltosuria are
suspected or if unsymmetrical peaks are detected, a second
analysis should be carried out using a buffer oflower molarity.
In the samples employed for this study these two compounds
were not established to be present.
Uronic acids have retention times longer than glucose (90
min) and appear as extremely broad peaks. Theirdetermination
within two hours is possible by changing running parameters
(higher buffer molarity, lower column temperature and add-
ition of 0.03 to 0.06 mmoles]l sodium chloride).
Samples of urine, serum, and spinal fluid need only be
ultrafiltered, no desalting or concentration was necessary.
A decrease in separating ability with the analysis of physio-
logical samples could not be detected. The observed yellow
discolouration of the resin at the top of the column was
probably due to protein and polysaccharide material of low
molecular weight and had no/narked effect on the separations.
The detection limits of the chromatographic system were
good and can be decreased if necessary. Settings of the fluori-
meter and recorder/integrator were adjusted to analyse normal
urines without dilution.
REFERENCES
Bickel, H., (1960) Med. Probl. Paediat. 6, 313
[2] Stanbury, J.B., Wyngaarden, J.B. and Frederickson, D.S. (1978),
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[3] Erdin, H.P., Schweiz. (1978) Rundschau Med. (PRAXIS)67,
334
[4] Lebenthal, E. (1975),Peal. Clin. North America 22, 757
[5] Young, D.S. and Jackson, A.J. (1970), Clin. Chem. 16, 954
[6] Kraffczyk, F., Helger, R. and Bremer, H.J. (1972), Clin. Chim.
Acta 42, 303
[7] Menzies, I.S., Mount, J.N. and Sheeler, M.J. (1978), Ann. Clin.
Bioehem. 15, 65
[8] Jolley, R.L. and Freeman, M.L. (1968), Clin. Chem. 14, 538
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10] Jolley, R.L., Warren,K.S., Scott, Ch.D., Jainehill, J.L., Freeman,
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[11] Mopper, K., Dawson, R., Liebezeit, G., Hansen, H.P. (1980).
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12] Mopper, K., (1978) Anal. Biochem. 87, 162
[13] Honda, S., Kakimoto, K., Sudo, K., Kakehi, K. and Takiura, K.
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[14] Dtirner, K. (1977),Europ. J. Ped. 126, 45
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[17] Kennedy, J.F. (1974),Biochem. Soc. Trans. 2, 54
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Volume 3 No.1 January 1981 29
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The clinical applicability of a new automatic sugar analyser

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The clinical applicability of a new automatic sugar analyser

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