Medical Engineering & Physics 26 (2004) 473481

www.elsevier.com/locate/medengphy

On the suitability of refractometry for the analysis of glucose

in blood-derived uids5
K. Zirk a,
, H. Poetzschke b
a
SES-Entwicklung GmbH, Emil-Figge-Strasse 80, D-44227 Dortmund, Germany
b
Bereich Klinische Physiologie, Private Universitaet Witten/Herdecke, D-58448 Witten (Ruhr), Germany

Received 8 October 2003; received in revised form 27 February 2004; accepted 19 March 2004

Abstract

Background: Refractometry is the determination of the optical refractive index of a substance or a mixture of substances. It is a
very sensitive method for the detection and quantication of dissolved analytes, but it is incapable of distinguishing between dif-
ferent analytes. The aim of this investigation was to determine the principle suitability of refractometry for the quantication of
glucose (blood sugar) in blood and various blood uids which can readily be obtained for medical diagnosis, in particular blood
plasma, blood serum, and their ultraltrates.
Methods and results: After the oral intake of freshly dissolved a-glucose, the in vivo blood contents of the a and b anomers of
glucose were found to be in an at least approximate equilibrium at all times. This observation is a prerequisite for a refractome-
trical determination of glucose due to the fact that both molecule forms have dierent refractive index increments. An assessment
of the glucose content in untreated blood uids was not possible, since no suitable relationship to the refractive index was found,
most probably due to the inuence of the many other substances present in blood on this parameter. However, after removal of
certain macromolecules by ultraltration, value pairs showed a high level of correlation, providing the nominal molecular weight
limit (cut-o) of the ultralter used possessed a maximum of 300 kDa. Besides macromolecules, the osmolality of the uids
undergoing measurement also proved to be a considerable interfering factor, particularly when values were outside the normal
physiological range between 285 and 293 mmol/L.
Conclusions: If a clinical application of this method is to be contemplated it is imperative (1) that blood cells are separated and
removed, (2) that macromolecules present in plasma or serum are removed, e.g. by ultraltration, and (3) that beyond the results
presented the inuence of all small molecules other than glucose on the overall refractive index be determined and included in the
calculation of analysis results.
# 2004 IPEM. Published by Elsevier Ltd. All rights reserved.

Keywords: Blood glucose; Glucose analysis; Refractometry; Blood uids; In vivo specicity

1. Introduction metrical determinations are substance unspecic, which

means that they are suitable for analytical purposes
Refractometry is the determination of the optical only if the analyte is known [4]. Technical applications
refractive index (n) of transparent substances or mix-
of refractometry are therefore usually employed in two
tures of substances. The refractive index of a solution
component systems, e.g. in solutions of an analyte in a
is the sum of the refractive index of the solvent and the
solvent, or in multi-component systems in which only
content-dependent refractive index contributions of the
the content of one component changes. Such systems
dissolved components [13]. Refractometry allows a
very sensitive detection (down to Dn 108 ) and can be found in the media resulting from substance-
quantication of dissolved substances [3], but refracto- separating analysis processes, e.g. the elutes of chroma-
tographic columns. So, refractometry is often applied
in the analysis, process-steering and quality control of
5
This work contains parts of the doctoral thesis of K. Zirk.

chemically or technically produced mixtures of sub-
Corresponding author. Tel.: +49-231-9742-523; fax: +49-231-
9742-524. stances, e.g. chemical or pharmaceutical intermediate
E-mail address: zirk@ses-entwicklung.de (K. Zirk). and nal products, solutions of salts and sugars,
1350-4533/$ - see front matter # 2004 IPEM. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.medengphy.2004.03.008
474 K. Zirk, H. Poetzschke / Medical Engineering & Physics 26 (2004) 473481
preparations of detergents and antifreeze, essences and
aromatic substances [2,3,5].
Providing a sucient specicity for D-glucose (or
dextrose, or, within the body, blood sugar, hereafter
more briey referred to as glucose) could be obtained,
refractometry as a purely physical measurement
method could be clinically applicable both, for the in
vitro determination of blood glucose levels in blood
samples, and within the frame-work of an insertable or
implantable technical in vivo probe for glucose. The
subcutaneous connective and fatty tissue appears to be
a suitable candidate for probe insertion or implan-
tation, whereby its interstitial uid (which corresponds
to the molecular composition of blood uid [6]) is the
substance undergoing investigation [7,8].
The aim of this study was, in principle, to determine
the suitability of refractometry for quantication of the
glucose content in several blood uids which are easily
obtainable for medical diagnosis (full blood (especially
venous and capillary), as well as its derivates blood
plasma, blood serum, and ultraltrates of plasma and
serum). Of special interest was the possible extent of
the (real or in vivo) specicity for glucose in these
uids. This paper contains a description of basic
experiments on the suitability of the refractive indices
of body uids for the analysis of glucose contained in
such uids, as well as a consideration of the disturb-
ance of the measurement due to other components in
the investigated samples.
2. Problems and solving attempts
A prerequisite for a refractometrical determination
of glucose in body uids is the technical ability to
exactly account for changes in the measuring quan-
titythe refractive index ncaused by changes in the
glucose concentration. Currently available precision
refractometers are able to determine the expected
refractive index changes (about 14  106 per 0.1 g/L
(10 mg/dL) change in glucose concentration) su-
ciently [9].
In aqueous solutions glucose predominantly exists in
two isomeric (anomeric) forms: a- and b-glucose (both
are pyranoses: a-glucopyranose and b-glucopyranose).
Each anomer can be transformed into the other through
molecular conversionthe so-called mutarotation [2]:
a-D-glucose  D-glucose aldose  b-D-glucose
A variety of catalytic mutarotase activities accelerate
the attainment of a temperature-dependent, steady state
equilibrium between these anomeric forms, e.g. at body
temperature the equilibrium is between 36.4%
a-glucose and 63.6% b-glucose, with only traces of the
intermediate ring-opened aldehyde form [10].
The physiological co-existence of two dierent
molecular forms of glucose in the body prompts an
examination of the possibility of the determination of
the overall content of dissolved glucose in blood using
refractometry. A refractometrical determination relies
on an equilibrium of the glucose anomers since the
total refractive index is determined, which includes the
contributions of both glucose anomers with their dier-
ent respective refractive indices, 147:6  106 L=g for
a-glucose, and 149:0  106 L=g for b-glucose [11].
Moreover, the question of whether a sucient equi-
librium of the anomers exists at all times in the frame-
work of this work was linked to the necessity to nd a
simple, but nevertheless reliable reference method for
the determination of glucose contents. There are a num-
ber of analytical methods available for the analysis of
glucose based on an enzymatically catalysed chemical
reaction. Very often, those analytical methods speci-
cally use only one anomer of the dissolved glucose as a
substrate. With technical and clinical analytical meth-
ods, the enzymes glucose oxidase (b-D-glucose: oxygen
1-oxidoreductase) and glucose dehydrogenase (b-D-
glucose: NAD(P)+ 1-oxidoreductase), which are highly
specic for b-glucose, are frequently used [12,13].
Glucose oxidase catalyses the reaction
b-D-glucose O2 H2 O  D-gluconic acid H2 O2 ;
and glucose dehydrogenase catalyses the reaction
b-D-glucose NAD  D-gluconolactone NADH
H :
In several commercially available analytical glucose tests
the only real analyte is b-glucose, because the analysis
reaction time (often much less than one minute) is far
too short to encompass the analysis of a succeeding com-
plete mutarotation of the a-glucose present in the
material to be analysed. So, only if the anomers of glu-
cose are in a steady state equilibrium during calibration
and during measurement, the measuring results obtained
with those enzymatic methods are correct.
The prerequisite of a glucose anomers equilibrium for
analysis is most certainly fullled in the body uids
during phases where a stable glucose level and a constant
body temperature prevail. However, at times when
glucose levels change, such an equilibrium may not be
present, and could thus present a measurement problem.
No information could be found in the available
literature to clarify the existence of an equilibrium
between a- and b-glucose in the human body at all
times. Imbalances may occur, particularly during pha-
ses of postprandial absorption (uptake of substances in
the gastrointestinal tract following a meal) or during
hormonally or neurally stimulated mobilisation of
glucose from glycogen, the storage form of glucose in
the body, consisting of polymers of covalently com-
bined a-glucose [10].
 K. Zirk, H. Poetzschke / Medical Engineering & Physics 26 (2004) 473481
An attempt was therefore made to shift the equilib-
rium between the two anomers through oral ingestion
of a substantial amount of a-glucose, and to prove this
shifting with the help of a detection method specic for
one anomer.
Refractometry is not substance-specic, and many
substances present in the blood contribute to the
bloods refractive index. The contents of these sub-
stances therefore need to be considered as disturbing
quantities if the blood glucose level is to be determined
using refractometry. In order to identify interfering
substances or groups of such substances a number of
approaches are possible.
One is to remove supposed disturbing substances by
means of a substance-separating process. Under the
assumption that these components (present in varying
concentrations) are of high molecular size (lipopro-
teins, proteins, etc.), a suitable method for their
removal is the ltration of the material to be analysed
through ultralters [14,15].
A further approach to the identication of disturbing
substances is a global evaluation of the inuence of all
dissolved components through an in vivo dilution or
concentration of the body uids. When a large amount
of water with a low salt content is ingested, an acute
dilution of the extracellular uid can be achieved. Cor-
respondingly, a restriction of water intake should lead
to a concentration of these uids. During both phases
the blood content of glucose is normally held constant
by hormone-mediated endogenous regulation.
3. Experimental
3.1. Materials and preparative methods
Crystalline a-D-(+)-glucose (dextrose) was obtained
from Roth (Karlsruhe, D; product numbers: M 198,
17 or 6780 (a-D-(+)-glucose monohydrate), purity:
99.5%, Ph.Eur., DAB).
Blood samples were obtained from healthy (non-
diabetic) women and men of between 23 and 42 years
of age, who participated voluntarily. Prior to each
measurement, venous blood samples were obtained by
aspiration puncture of a slightly congested cubital vein,
and capillary blood by piercing a nger tip with a lan-
cet following hyperaemerisation. 20 IU (international
units) of heparin sodium salt (4 lL Heparin-Natrium-
25000-Ratiopharm from Ratiopharm, Ulm, D) were
added to each millilitre of venous blood to prevent
coagulation.
Blood plasma was obtained from this heparinised
full blood by centrifugation (15; 000  9:81 m=s2 for 10
min), and blood serum by separating the plasma from
the cellular components of native full blood using cen-
trifugation (10; 000  9:81 m=s2 for 2 min), incubation
475
v
of the plasma for 1 h at 37 C (in order to obtain a
complete coagulation) and removal of the brin clots
by centrifugation (15; 000  9:81 m=s2 for 10 min).
Ultraltrates of blood plasma and serum were pro-
duced with ultraltration stirring cells of dierent
Nominal Molecular Weight Limits (NMWL, referring
to the separation capabilities, the values describe the
molecular masses (in Dalton or g/mol) of globular pro-
teins which are withheld in a standardised ultraltra-
tion process to approximately 90%). Technical grade
nitrogen served as the propellant (alternatively, oxygen
gave the same results). The stated in vivo values for the
glucose contents and the refractive indices are stable
plateau values obtained in the course of an ultraltra-
tion process.
3.2. Analytic methods and measuring devices
Two identical commercially available test strip analy-
sers served for the determination of glucose content in
body uids (Accutrend Sensor, Boehringer Mannheim,
Mannheim, D). They are based on the oxidation of
b-D-glucose (enzymatically catalysed by means of
glucose dehydrogenase), and the reaction rate is quan-
tied amperometrically.
A calibration of the measurement results from the
two devices was carried out by the construction of cali-
bration curves for all used media. Gravimetrically mea-
sured amounts of the analyte glucose (as a-glucose)
served as references and were added to the matrix of
full blood, plasma, serum, and their ultraltrates, as
well as to water, and mixed suciently (in accordance
with our results concerning the kinetics of all media in
terms of reaching a steady state equilibrium of mutaro-
tation between a- and b-glucose). The calibration
curves were generally linear, and readings showed a
relative imprecision of (usually) less than 5% (regarding
ve single measurements at each glucose content), cor-
responding well with the values of Wahl [16].
Two dierent commercially available devices were
used for the determination of optical refractive indices.
The rst was a simple manually held refractometer
(Eclipse from Bellingham & Stanley, Turnbridge
Wells, UK; reading accuracy: 5  104 ) of which the
display was calibrated to the value of distilled water
v
(n 1:333) at room temperature (22 C). The second
was a precision refractometer DUR by Schmidt &
Haensch (Berlin, D), with which all measurements were
v
carried out at a temperature of 30.0 C (set with a cir-
culating thermostat), with the display being accordingly
adjusted to the value of the refractive index of distilled
v
water at 30.0 C (1.33192). The imprecision of this
refractometer is 105.
Osmolalities (the sum of the particular molal con-
tents of all dissolved components) were determined
476 K. Zirk, H. Poetzschke / Medical Engineering & Physics 26 (2004) 473481
with an osmometer (Halbmikro-Osmometer Typ M
from Knauer, Berlin, D), the measuring principle of
which is the determination of the decrease in the freez-
ing point. The device was calibrated with a NaCl cali-
bration solution (12.687 g NaCl/kg H2O, Knauer).
According to the manufacturers operating instructions
the inaccuracy of the measurements is
2%. decreasing pore diameter, and a comparison was made
Multiple measurements of each sample were per-
formed with both the precision refractometer and the
osmometer. Measurement values are presented as arith-
metic means, and the standard deviation (SD) serves as
a measure of scatter.
3.3. Protocols
The kinetics of anomeric conversion of freshly
dissolved a-glucose until a substance equilibrium with
b-glucose is reached were determined in preliminary
experiments. Venous blood of a fasting non-diabetic
male (H.P.) was used for this purpose, from which
heparinised full blood, plasma and serum were
obtained. After addition of a-glucose to these derivates
in vitro, as well as to water, an incubation of the uids
v v
(with stirring) at 22 or 37 C ( 1 C in both cases) fol-
lowed, and glucose content was determined over time.
The existence of an equilibrium of the mutarotation
of a- and b-glucose in the human body (in vivo) was
examined, especially during a phase where a large
inux of a-glucose into the blood was occurring (i.e. an
increase in the blood level). This inow was attained
by oral ingestion of a-glucose. Within 2 min, the same
volunteer drank a freshly prepared solution of 75 g
a-glucose in 0.2 L water (this is the glucose quantity
used for a diagnostic oral glucose tolerance (OGT) test
in Germany).
At once, and after 10, 15, 25, 30, 37 and 40 min,
capillary blood was used to obtain an in vivo blood
sugar prole, in which the glucose contents (cmGluAS)
were determined with the help of the blood sugar mea-
suring device Accutrend Sensor (AS), which exclusively
detects b-glucose. In order to detect a possible imbal-
ance in the mutarotation of the glucose anomers in cir-
culating blood, at 3, 7 and 16 min after intake of the
glucose solution venous blood samples were obtained,
v
heparinised and incubated at 37 C for up to 20 min,
and were used to determine time-related changes (in
vitro) in the measurable glucose content (cmGluAS). In
the chosen time period the inuence of the glucose con-
sumption by the erythrocytes is smaller than the scat-
tering interval of the values of the analysers, and can
therefore be ignored [17].
For an assessment of the inter-individual dependence
of the refractive index of blood plasma on the mass
concentration of glucose, glucose contents and refrac-
tive indices of heparinised plasma samples from 11
non-diabetics (ve female and six male, all non-fasting)
were determined (using the manually held refrac-
tometer Eclipse).
To explore possible improvements in the correlation
of measured refractive indices and glucose contents, the
refractive indices of the ultraltrates of plasma samples
were obtained, using a number of ultralters with
between ultraltrated and non-ltrated plasmas from
dierent volunteers: Heparinised plasma samples,
obtained from the blood of 6 of the above-mentioned,
non-fasting volunteers (three female, three male) were
ltered through ultraltration membranes of dierent
NMWLs (10, 30, 100, 300, and 1000 kDa). The refrac-
tive indices (determined using the manually held refrac-
tometer Eclipse) and the glucose concentrations were
determined in both the plasma samples and the corre-
sponding ultraltrates.
For the analogue but more precise determination of
the inter-individual dierences of the dependence of the
refractive index on the glucose concentration in ultra-
ltrates of blood serum, blood sera (from blood
obtained from four non-fasting, non-diabetic volun-
teers; three male, one female) were ltered through
ultraltration membranes with a NMWL of 3 kDa.
The ultraltrated serum of one male volunteer was div-
ided into three parts and dierent amounts of a-glucose
were added to two of them. These two aliquots were
v
then incubated for 15 minutes at 37 C in order to
achieve a mutarotation equilibrium between a- and
b-glucose. Two of the male volunteers (H.P. and K.Z.)
additionally drank a freshly prepared solution of 75 g
a-glucose in 0.2 L water within 2 min. At the approxi-
mate time of the maximum value for the venous blood
glucose content (see Fig. 2), a further blood sample
was taken, and the refractive indices (using the precise
refractometer DUR) and glucose concentrations of the
ultraltrates of these sera were determined.
The inuence of the osmolality of the serum solutes
and its possible in vivo extent on the refractive index of
a serum ultraltrate was examined, as well as the
(refractometrically) indirectly determinable (apparent)
glucose concentration. A non-fasting, non-diabetic
male (H.P.) drank 2.5 L water within 15 min (in order
to decrease serum osmolality), and venous blood sam-
ples were taken 12 and 60 min thereafter in order to
obtain serum. The refractive indices (precise refrac-
tometer DUR), the glucose concentrations, as well as
the osmolalities were determined from 3 kDa ultra-
ltrates of the respective serum.
Afterwards, the volunteer underwent a phase of
approximately 26 h uid starvation (in order to
increase the osmolality) followed by analogue determi-
nation of the mentioned quantities in the serum ultra-
ltrate.
 K. Zirk, H. Poetzschke / Medical Engineering & Physics 26 (2004) 473481 477

4. Results

To ensure correct determination of glucose contents

using the analysing device Accutrend Sensor, the times
required for freshly dissolved a-glucose to reach a
mutarotation equilibrium with b-glucose were determ-
ined. This was especially important for the construc-
tion of valid calibration curves for the dierent media
(full blood, blood plasma, blood serum and its ultra-
ltrates, as well as aqueous glucose solutions).
Fig. 1a shows an exemplary progression of the
measurable glucose content in blood plasma (as mea-
sured with the analysing device Accutrend Sensor) at
v
37 C, after an additional 3.00 g a-glucose per litre
were dissolved in it. After about 15 to 20 min, a stable
nal value was reached. The time required for the
actual value to reach 90% of the dierence between the
start and nal valuethe so-called 90% setting time
(s90)more precisely characterises the graphs course
and was found to be 12.8 min as shown in Fig. 1a.

Fig. 2. The time course of the determined mass concentrations of

glucose cmGluAS (each data point indicates the mean of three mea-
surements) of a (male) fasting non-diabetic after an oral intake of
75 g a-glucose, (a) in vivo from capillary blood, and (b) in vitro in
venous blood samples, obtained 3 (tq), 7(_&) and 16 (uv) min after
ingestion of the glucose drink. The lled and open symbols mark the
individual measurements of one blood sugar detecting device, and the
value ranges in (b) indicate the 68% area (x SD) of a normal distri-
bution of the measurement values.

An outline of the 90% setting times in several media

Fig. 1. (a) An example of the dependence of the mass concentration
of glucose cmGluAS determined by an enzymatic method (AS, specic
for b-glucose) as a function of time after addition of a-glucose to
v
blood plasma at 37 C. cmGluAS was assessed using two measuring
devices (u and v, single measurements). s90 is the time necessary for
the measurement value to reach 90% of the mutarotation equilibrium
value. (b) The 90% setting-time s90 as a function of glucose concen-
tration (nal detected values) to show the dependence of the kinetics
of the murarotation between a- and b-glucose on the dissolving
media, the amount of added a-glucose, and the temperature. Dissolv-
ing media were water (u), blood plasma (t and ), blood serum
(_&) and decoagulated full blood ( ). The temperatures were 37 C
v
v
(tu _) or 22 C (&).
(blood, plasma, serum, aqueous solutions), partially
with additionally varied temperature and varied added
amounts of a-glucose is shown in Fig. 1b. Under the
chosen in vitro conditions, the equilibrium in full
v
blood at 37 C has the fastest 90% setting time with
only 7 min compared to plasma and serum with 12 and
13 min, respectively, and last of all water with 32 min.
Additionally, the setting times for plasma in the
exemplary examined case were found to be inde-
pendent of the amount of added glucose.
In the attempt to examine whether a- and b-glucose
in the human body are (at least predominantly) in an
equilibrium of mutarotation even during phases of a
supposed inux of a-glucose into the blood, the follow-
ing results were achieved: After oral ingestion of 75 g
a-glucose (through rapid ingestion of a freshly pre-
pared aqueous solution), glucose content was measured
with the analysator Accutrend Sensor, which is only
sensitive for b-glucose. The blood samples (taken at 3,
478 K. Zirk, H. Poetzschke / Medical Engineering & Physics 26 (2004) 473481
7, and 16 min) showed constant levels for glucose con-
tent. Fig. 2a depicts the time course of the (measur-
able) glucose content in the volunteers blood (in vivo)
and Fig. 2b presents the course of the measured values
(in vitro) for the three blood samples obtained.
For orientation, Fig. 3 shows a graph in which the
refractive index of native blood plasma (the refractive
index of full blood is not determinable with the refrac-
tometer used) is plotted against the glucose contents of
these plasma samples. A useful, recognisable corre-
lation is not evident, with the measurement value
pairsrepresented by dots in the diagramappearing
as a cloud.
Fig. 4 shows a comparable diagram of the refractive
indices obtained in blood plasma ultraltrates plotted
against glucose contents. It is evident that ultraltrates
gained using lters with a nominal molecular weight
cut-o lower than 1000 kDa (300 kDa and smaller)
possess a much smaller refractive index than non-ultra-
ltrated plasmas and ultraltrates gained using 1000
kDa ultralters.
Fig. 3. The refractive index n of the blood plasma of 11 non-
diabetics (ve females and six males) as a function of the glucose
concentration cmGlu. Each point represents a mean of ve
measurements.
Fig. 4. Dependence of the refractive index n of human native (unl-
trated) and ultraltrated blood plasma (taken from three male and
three female non-diabetics) on the glucose concentration cmGlu. The
numbers on the graph symbols of ultra-ltrated plasma indicate the
nominal molecular weight limit (NMWL in kDa) of the ultra-lters
used. The dashed line separates the areas representing ultraltrated
and unltrated plasma. Identical symbols mark values from the same
volunteer, and each symbol represents a mean of ve measurements.
For all further tests only blood serum was used. The
use of blood serum instead of blood plasma eliminates
the possible inuence of variable amounts of the non-
endogenous macromolecular decoagulant heparin sul-
phate as well as of brinogen. Since the size of the lter
pores has only a slight inuence on the processing time
(ltration time), an ultralter with the smallest pore
size can be used, if an ultraltration is required at all.
Ultraltrates of the serum were therefore obtained only
using ultralters with a nominal molecular weight limit
of 3 kDa. In addition, ultralters with a small pore
size are able to hold back further disturbing serum
components. Because of the expected very small chan-
ges in the determining quantity, the refractive index n,
a precise refractometer was applied.
The refractive index of aqueous solutions of glucose
in the examined glucose concentration range are well
known, exhibiting an approximately linear relation [5].
Fig. 5 shows the refractive index of ultraltrates of
blood serum obtained from four volunteers together
with their respective glucose contents. In some cases,
these contents were increased by the ingestion of
glucose solutions (in vivo), or by mixing crystalline
glucose into these serum samples (in vitro).
The values correlate very well, with a correlation
coecient R of 0.973. The greatest single dierence
between the measured glucose content and the value of
the regression line is 0.1 g/L, which corresponds to a
relative error of about 9% in this particular case.
The dependence of the refractive index of the ultra-
ltrates (obtained using lters with a nominal molecu-
lar weight limit of 3 kDa) of human blood serum on
their glucose contents, as shown in Fig. 5, can serve as
a calibration curve to determine glucose content by
refractive index measurement.
To test the extent of the inuence of glucose content
on the accuracy of such determinations, global changes
Fig. 5. Dependence of the refractive index n of human ultraltrated
(NMWL 3 kDa) blood serum from four non-diabetics (one female
and three male), on the glucose concentration cmGlu (mean of four
measurements each). Identical symbols mark the values from the
same volunteers. Increased blood sugar levels were seen in vivo in
two volunteers (shown on the right: +a-glucose; _ and u) following
oral ingestion of a-glucose. The open circles (v) mark the values
measured after in vitro addition of a-glucose to a serum sample of
one volunteer ().
 K. Zirk, H. Poetzschke / Medical Engineering & Physics 26 (2004) 473481
Fig. 6. The mass concentration of glucose cmGlu in human ultra-
ltrated (NMWL 3 kDa) blood serum from a non-diabetic (male)
as a function of the osmolality cnOsm (data points show means of at
least ve measurements). The lled symbols (u) indicate the measure-
ments of the glucose concentration (glucose analysator Accutrend
Sensor), the open symbols (v) indicate values obtained using the
refractometry calibration function as shown in Fig. 5. The two verti-
cal dashed lines mark the boundaries of the physiological area (68%)
for the osmolality [15].
in the contents of all substances, which are potentially
disturbing factors, were achieved by dilution or con-
centration of the body uids. It became evident that a
dilution of the extracellular uid could easily be
obtained by the ingestion of a substantial amount of
water with low salt content. Restriction of water intake
however resulted in only a minimal concentration of
the body uids.
Fig. 6 shows the refractometrically determined glu-
cose contents determined using the reference method
(Accutrend Sensor) over the osmolality which is
dened as the sum of all molal contents of all osmoti-
cally eective, dissolved components, i.e. dissolved non-
electrolyte molecules and ions [6,9]. The osmolality
showed a clear inuence on the accuracy of the refrac-
tometrical determinations of the glucose contents. In
both the diluted (at an osmolality of 273 mmol/kg)
and the slightly concentrated (at an osmolality of 296
mmol/kg) extracellular uid, the relative deviations
were more than 50%.
5. Discussion
The starting conditions for the determination of
mutarotation kinetics encompassed only the predomi-
nance of a-glucose, primarily because this is the usual
crystalline form of glucose (resulting from the dehy-
dration of aqueous solutions), and secondly, since the
bodys endogenous store of glucose (glycogen in the
liver and muscles) consists of polymerised a-glucose
[10]. The momentary anomer equilibrium in the blood
may be shifted following an oral supply as well as with
an endogenous release of glucose. In both cases the
equilibrium will be inuenced by an increase in the
fraction of a-glucose.
479
A prerequisite for a refractometrical determination
of glucose in body uids which are accessible to
medical diagnosis, is at least an approximate mutaro-
tation equilibrium between glucose anomers in the
body. In order to examine the permanent existence of
such a mutarotation equilibrium, measurements using a
method specic for only one glucose anomer were
performed.
The results obtained show that, for the investigated
conditions, the anomers of the glucose in blood (in
vivo) are in a sucient mutarotation equilibrium at all
times. After ingestion of a-glucose, a- and b-glucose
were found to be in a substance equilibrium each time
a blood sample was taken, despite the fact that at these
times the total content of glucose in the blood was rap-
idly increasing.
There is no reason to assume that there was not an
equilibrium at any other moment, so the measured in
vivo time course correctly reects the glucose content
course in the volunteers blood.
Furthermore, it can be concluded that in vivo glu-
cose contents determined with the Accutrend Sensor
are correct, so that an indication of the determination
quantity as a method-dependent quantity (and thus an
index AS as initially used as a precaution in Figs. 1
and 2) is not necessary. The method is therefore suit-
able as a practical reference.
Since the rst blood sample was taken only 3 min
after rapid ingestion of the freshly prepared glucose
solution, it is probable that, during the gastrointestial
absorption of glucose into the mucous membrane and
its subsequent passage into the portal vein blood, an
overall greater mutarotation activity occurs in compari-
son with full blood. Known mutarotase catalysts
besides temperatureare unspecic proton activity
extremes (high and low pH values) as well as the enzy-
matic activities of certain proteins [2]. These factors
occur in the digestive juices, the latter perhaps also in
the cells which glucose has to pass through.
A hormonally (for example by glucagon or adrena-
line) or neurally (by activation of the sympathetic ner-
vous system) induced endogenous glucose release from
the bodys glycogen stores should predominantly result
in a-glucose (with the intermediates a-glucose 1-phos-
phate and a-glucose 6-phosphate) [10]. An interesting
question is whether such a release can occur more rap-
idly than the absorption by the gastrointestinal tract,
and whether a measurable imbalance of the glucose
anomers results.
A further necessity for determining glucose by
refractometrical analysis is that the concentrations of
other substances contained within the body uids show
only small variation ranges as compared to glucose,
since only then would their contribution to variations
of the total refractive index be small. If one regards the
composition of an adults blood, it consists of 42%
480 K. Zirk, H. Poetzschke / Medical Engineering & Physics 26 (2004) 473481
blood cells and 58% blood plasma. Plasma in turn con-
tains 90% water, approximately 7% proteins, 0.40.7%
lipids, 0.070.1% glucose, 0.030.07% amino acids,
0.020.05% carbamide, 0.0080.02% lactic acid and
approximately 0.5% inorganic salts [6,15].
Besides water, protein dominates in the liquid phase
of blood and thus a refractometrical protein determi-
nation is possible, as was already shown at the begin-
ning of the 20th century [9,18]. Additionally, as shown
here the glucose content of untreated blood plasma is
not determinable refractometrically. The individual
variation of the protein level and its contribution to the
total refractive index exceeds the contribution of glu-
cose many times.
After removing high molecular substances by means
of ultraltration, the inuence of these substances on
the refractive index was reduced clearly. The necessary
separation limit (NMWL) of the ultralters of approxi-
mately 300 kDa found seems to be indicative of plasma
proteins rather than large lipoprotein complexes (chy-
lomicrons, chylomicron-remains, and the other lipopro-
teins VLDL, IDL, LDL and HDL) [13], since the latter
can be eliminated through ultraltraters with separ-
ation limits far greater than 300 kDa.
Besides high molecular substances, low molecular
solutes can also have an inuence on the total refractive
index and are thus able to reduce the specicity of a
refractometrical glucose determination. The smallest
ultralter used had a nominal molecular weight limit of
3 kDa. The remaining matrix components were there-
fore salts (in particular sodium and chloride ions, etc.),
amino acids and other low molecular organic substances
(carbamide, lactate, etc.) [6,15], all of which generally
show very similar specic refractive index increments
(Dn/Dc) of approximately 140 200  106 L=g (Ref. [4],
or determined by ourselves). However, the absolute con-
centration (or content) of a disturbing substances is not
the decisive factor, but rather its uctuations and there-
fore its variable contribution to the total refractive
index.
By dilution or concentration of the body uids,
many possible interfering quantities may change simul-
taneouslywhereas the content of the analyte glucose
should be held constant by endogenous mechanisms.
Such changes in the bodys hydration state are
assumed to occur frequently, especially dehydration
caused by physical training, in warmer climates, and in
elderly persons. We found that an acute experimental
dilution of the extracellular uids was easily possible
through ingestion of a large amount of water (with a
low salt content), while restriction of water input
hardly resulted in a concentration of these uids, pre-
sumably because the kidneys act to markedly reduce
water excretion (anti-diuresis) [19].
Nevertheless, through the achieved variation of the
plasma osmolality it could be shown that the concen-
tration of the dissolved components has a clear inu-
ence on the true accuracy of the refractometrical
determination of the glucose content. The relative
deviations already amount to more than 50%, both in
the diluted (at a osmolality of 273 mmol/kg) and the
slightly more concentrated (at a osmolality of 296
mmol/kg) extracellular uid.
In summary, refractometry is a suitable method for
the determination of the glucose content of blood
(using blood plasma or serum ultraltrates as the
measurement object) of non-diabetics within a small
physiological hydration range. However, when this
hydration range limit is exceeded, there are distinct
deviations of determined glucose concentrations from
the true values, so that a refractometrical method alone
appears unsuitable for a clinical application.
Even so, the latter problem can presumably be
solved since the hydration state of a person can be
determined inexpensively by a technically simple
measurement of the electrical conductivity of extra-
cellular body uids, including blood-derived uids. If a
universal interindividual dependency (with sucient
accuracy) exists between the body uids osmolalities
(at constant glucose levels) and these uids optical
refractive index values, then the glucose analysis cali-
bration functions for various hydration states can be
determined. By means of mathematical interpolations it
should then be possible to calculate a calibration func-
tion valid for any actual hydration state. Further
research on this possibility of eliminating the disturbing
and biasing eects of the salts and other solutes con-
centrations is planned.
Acknowledgements
We express our sincere gratitude to Dr. Debra
Kelleher for her careful and critical assistance in pre-
paring this manuscript.
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