Clinical Chemistry 46: 1144-1149, 2000;
(Clinical Chemistry. 2000;46:1144-1149.)
© 2000 American Association for Clinical Chemistry, Inc.
Glyceraldehyde Preserves Glucose Concentrations in Whole Blood Specimens
Michael Landt1
1
Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, and St. Louis Children’s Hospital, One Children’s Place, St. Louis, MO 63110.
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Abstract
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Background: Glucose concentrations decrease in blood specimens
during transport/processing, primarily because of continuing metabolism
(glycolysis) by erythrocytes. Several means to reduce the loss of
glucose in blood specimens have been developed, but all have major
drawbacks. Glyceraldehyde, which has antiglycolytic activity, was
assessed for potential in preserving glucose in blood specimens.
Methods: Heparinized blood from volunteers was treated with
glyceraldehyde and other agents. After incubation for various times,
plasma concentrations of glucose and other common analytes were
determined with prevalent commercial analyzers.
Results: The racemic mixture of glyceraldehyde
(D,L-GA) preserved glucose concentrations for up to
8 h at room temperature. Half-maximal effect was attained with 0.9
mmol/L D,L-GA. Trials of the D and
L stereoisomers individually indicated that the
L isomer (L-GA) was responsible for all or most
of the antiglycolytic activity of the racemic mixture. Other related
compounds were ineffective. Measurements of most common clinical
laboratory analytes were unaffected by the presence of
D,L-GA or L-GA.
Conclusions: Glyceraldehyde (D,L-GA or
L-GA) effectively preserves glucose concentrations in whole
blood specimens for up to 8 h. Specimens collected with
D,L-GA or L-GA are suitable for analysis of
many analytes commonly comeasured with glucose.
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Introduction
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Glucose is one of the most commonly measured components of blood
because of its central role in metabolism and the high prevalence of
diseases of glucose homeostasis. A continuing problem in the accurate
measurement of glucose is the loss of glucose from specimens because of
glycolysis by erythrocytes during transport and processing
(1). In recent years this phenomenon has been more evident
as laboratory services have consolidated and many more specimens are
transported to distant laboratories for analysis. Several approaches
have been proposed to minimize glucose loss, including
centrifugation/decantation of plasma immediately after specimen
collection (1); refrigeration/cooling on ice during
transport (2); addition of antiglycolytic agents such as
iodoacetate (3), fluoride (4), or mannose
(5) to the collection tubes; and the use of glucose
analyzers designed for near-patient testing, at the bedside
(6). All of these approaches are in current use, and the use
of fluoride in blood collection tubes is prevalent in circumstances
where substantial delay between collection and analysis is anticipated;
however, all have substantial limitations [for review, see Ref.
(7)]. To various degrees, these approaches are limited in
efficacy by incomplete inhibition of glycolysis, interference in
testing for co-analytes (e.g., electrolytes, creatinine, and urea),
disturbance of cellular integrity (e.g., hemolysis), or promotion of
leakage of intracellular potassium.
The ideal approach to eliminating glucose loss would provide reasonably
stable glucose concentrations for the period needed for transport to a
centralized laboratory, avoid costly near-patient analysis, and yield a
specimen that was suitable for analysis of many other common analytes
so that separate collection of specimen for those analytes was not
necessary. From a practical standpoint, the best way to achieve this
goal is discovery of an antiglycolytic agent that could be added to
collection tubes but did not alter cellular integrity or interfere in
common analytical methodologies. Such an agent should also be effective
at low concentrations (minimizing volume addition to avoid dilution
errors), dissolve rapidly during the collection process, be nontoxic,
be stable in the room-temperature storage environment of blood
collection devices, and be inexpensive. Here I present studies of the
glucose preservation properties of glyceraldehyde, which appears to
have potential as an additive in blood collection devices.
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Materials and Methods
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Subjects
Whole blood specimens collected in tubes containing sodium heparin
as anticoagulant were obtained from healthy adult volunteers or from
adult diabetic patients visiting an outpatient clinic, after informed
consent was obtained. Specimens were used within 1 h of
collection. In a few experiments, heparinized whole blood specimens
arriving at the clinical laboratory for analysis for glucose and other
common analytes were intercepted, and aliquots were removed for
experimental purposes. These studies were conducted in accordance with
a protocol approved by the Human Studies Committee of Washington
University.
Experimental design
Aliquots of heparinized whole blood (0.96 mL) were pipetted into
microcentrifuge tubes containing 0.040 mL of saline or antiglycolytic
agent in saline. Antiglycolytic agents were prepared as 250 mmol/L
stock solutions within 1 h of use. At various times, plasma was
prepared from incubations by centrifugation at 8000g for 2
min; plasma was decanted into 12 x 75 mm tubes and stored frozen
until analysis. All incubations of whole blood were conducted at room
temperature (23 °C) on a rotating table shaker, which kept the cells
in dispersed suspension. D,L-Glyceraldehyde
(D,L-GA), methylglyoxal,
D-glyceraldehyde (D-GA),
glycolaldehyde, and dihydroxyacetone were obtained from Sigma.
L-Glyceraldehyde (L-GA) was
obtained from Fisher Scientific.
Analytical procedures
Glucose analyses were routinely performed on a Cobas MIRA analyzer
(Roche Diagnostic Systems), using reagents based on glucose oxidase,
manufactured by Sigma Diagnostics. In some experiments, glucose and
many other common laboratory analyses were performed on a Vitros 250
Analyzer (Ortho-Clinical Diagnostics), a Hitachi 747 Analyzer
(Boehringer-Mannheim) and a RxL Analyzer (Dade Behring).
Statistical analysis
All results are stated as means ± 1 SD, unless otherwise
stated. Correlative studies are reported graphically, after calculation
of a least-squares linear regression relationship. Statistical
significance was assessed with the Student t-test, with
P values <0.05 considered significant.
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Results
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Stabilization of blood glucose concentrations by d,l-GA
Whole heparinized blood was incubated for 0–8 h in the presence
or absence of 10 mmol/L D,L-GA, and the glycolytic loss of
glucose was followed over time (Fig. 1
). A linear decrease in glucose concentration was evident in
incubations without additive, with 38% of the initial glucose
concentration lost in 8 h (0.23 mmol/L per hour). In incubations
containing D,L-GA, glucose concentrations remained nearly
constant throughout the 8-h period, with the loss at 8 h only 2%.
Thus D,L-GA appeared to be highly effective in preventing
loss of glucose in whole blood for up to 8 h. The presence of
D,L-GA did not promote visible hemolysis.
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Figure 1. Time course of glucose disappearance in the absence ( )
and presence (•) of 10 mmol/L D,L-GA.
Error bars are shown for triplicate replicates where the
error exceeds the size of the symbols used.
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Dose-dependent stabilization of glucose concentrations by d,l-GA
Whole heparinized blood was incubated for 8 h in the presence
of 0–10 mmol/L D,L-GA (Fig. 2
). Similar to preceding experiments, glucose concentrations
declined from 4.8 ± 0.1 mmol/L to 2.6 ± 0.2 mmol/L (46%
decline) when no D,L-GA was present, but increasing
concentrations of D,L-GA effectively preserved glucose
concentrations so that at 5 mmol/L D,L-GA, decreases in
glucose were nearly completely eliminated (residual glucose, 4.6
± 0.3 mmol/L at 8 h). The concentration of D,L-GA
that reduced glycolytic loss by one-half was ~0.9 mmol/L. The
presence of D,L-GA did not appear to interfere in the
glucose assay at any of the concentrations examined (Fig. 2
).
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Figure 2. Concentration dependence of the glucose preservation
action of D,L-GA.
Two curves are presented. One curve presents data at various
D,L-GA concentrations at the start of the experiment
(0 hours; •), and the other curve presents data after
8 h of incubation at room temperature (8 hours;
). Error bars, when error exceeds the size of the
symbols used, are shown for triplicate replicates.
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Efficacy of d,l-GA in patient specimens
Specimens arriving at the clinical laboratory for glucose and
other common laboratory analyses were split into three aliquots: one
was analyzed immediately to establish the initial glucose concentration
in each specimen; one was incubated for 8 h without additive; and
the third aliquot was incubated for 8 h in the presence of 10
mmol/L D,L-GA. Results are presented by plotting initial
glucose concentration against glucose concentrations in either the
presence or absence of D,L-GA (Fig. 3
). The line generated with incubations without additive had a
slope of 1.01 and an intercept of -2.2 mmol/L, indicating that 8
h of incubation reduced glucose concentrations an average of 2.2 mmol/L
regardless of starting concentration (0.28 mmol/L per hour). The
relationship of initial glucose to glucose concentration after 8 h
of incubation in the presence of D,L-GA also yielded a
slope of 1.01, but the intercept was only 0.3 mmol/L, indicating that
D,L-GA reduced the extent of glucose loss more than
sevenfold compared with the absence of D,L-GA. Furthermore,
D,L-GA appeared to be equally effective regardless of the
starting glucose concentration, which ranged from 3.9 to 13.4 mmol/L.
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Figure 3. Glycolytic loss in a range of patient specimens.
Glucose concentration at the start of the experiment is plotted on the
x axis, and glucose concentration after 8 h of
incubation on the y axis. Incubations containing either
10 mmol/L D,L-GA () or no additives (•) are plotted on
separate curves.
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Effect of d,l-GA on erythrocyte potassium and lactate homeostasis
Some antiglycolytic measures, such as storage on ice, so
effectively inhibit erythrocyte metabolism that the cells are unable to
maintain the potassium gradient between plasma and intracellular fluid,
leading to significant increases in plasma potassium concentrations.
The time course of potassium concentration changes was followed for
8 h in whole blood incubations under three conditions:
(a) incubation at room temperature (without additions);
(b) incubation at room temperature with 10 mmol/L
D,L-GA; and (c) incubation at 4 °C
(without additions). In room temperature incubations without additive,
potassium decreased modestly from 4.0 ± 0 mmol/L at 0 h to
3.5 ± 0.1 mmol/L at 8 h. Incubation for 8 h in the
presence of D,L-GA produced an increase of
similar magnitude (to 4.5 ± 0 mmol/L). In contrast, incubation at
4 °C, which has been advocated as an effective way to prevent
glucose loss for short periods (2), produced an increase to
6.0 ± 0.1 mmol/L.
An analogous experiment examining the time course of lactate
concentration changes found that the time-dependent increase in plasma
lactic acid was not prevented by D,L-GA, whereas incubation
on ice preserved initial lactate concentrations (data not shown).
Glucose preservation activity of related compounds
Several compounds related biochemically or structurally to
D,L-GA were tested for their ability to preserve specimen
glucose; incubations without inhibitor and with D,L-GA were
included for comparison (Table 1
). All compounds were tested at a final concentration of 10
mmol/L, and incubations were conducted at 0 and 8 h for each
compound. Only the D and L stereoisomers of
glyceraldehyde (D-GA and L-GA) prevented
glucose loss. The inhibition by D-GA was only partly
effective, reducing glucose loss from 41% (no additions) to 17%. Loss
of glucose was completely prevented by L-GA. The partial
inhibition by D-GA could have been attributable to
small contamination of the D-GA preparation with the
L isomer, and the efficacy of D,L-GA could be
attributable entirely to the action of the L isomer in this
preparation. To test this possibility, an experiment varying the
concentration of L-GA was performed in analogy with the
earlier experiment varying the D,L-GA concentration (see
above). If L-GA is the active portion of the
D,L-GA mixture (and D-GA is inert), then the
concentration of L-GA needed to inhibit glycolysis will be
one-half that of D,L-GA. The concentration that reduced
glucose loss by 50% was ~0.65 mmol/L, which was close to one-half of
the D,L-GA concentration needed (see above). Furthermore,
2.5 mmol/L L-GA was nearly completely effective in
eliminating glycolytic loss during an 8-h incubation (data not shown),
whereas twice that concentration of D,L-GA was needed for
comparable influence. These results were interpreted to suggest that
the antiglycolytic action of D,L-GA was attributable to the
L-GA component of the racemic mixture.
Effect of d,l-GA and l-GA on common clinical chemical tests
The potential for D,L-GA and L-GA to
interfere in testing for commonly ordered co-analytes was investigated
by drawing blood from six diabetic patients in a clinic setting and
adding to aliquots 10 mmol/L D,L-GA, 5 mmol/L
L-GA, or the same volume of saline. The aliquots were
centrifuged immediately, and plasma was decanted for analysis on three
common laboratory automated systems: Vitros 250 (Ortho-Clinical
Diagnostics), RxL (Dade Behring), and Hitachi 747 analyzers. The
analytes tested were those in the standard "basic metabolic
panel/comprehensive metabolic panel" (Table 2
). Glucose results were similar on all three analyzers, which
suggested that L-GA and D,L-GA do not effect
either glucose oxidase methods (Vitros 250 and Hitachi 747) or methods
based on hexokinase (RxL). The other analytes examined were also free
of significant interference from either L-GA or
D,L-GA, except creatinine. Creatinine analyses on the
Vitros 250 and Hitachi 747 (both enzymatic methods) were not influenced
by the presence of either D,L-GA or L-GA, but
the alkaline picrate method of the Dade Behring RxL was highly
susceptible to positive interference by either form of GA (Table 2
).
Aspartate aminotransferase results were modestly depressed in
the presence of GA on all three analyzers, with greater decreases
observed on the Dade Behring RxL and lesser effects in specimens
treated with L-GA, but these decreases were too small to
reach statistical significance (Table 2
).
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Discussion
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Considerable effort has been expended in the past to find a highly
effective preservative of glucose for blood collection that does not
interfere in other common clinical chemical tests, does not cause
hemolysis or other loss of cellular integrity, is nontoxic, is stable
for storage at room temperature, and is inexpensive. GA, and
specifically L-GA, seems to meet these criteria better than
any currently available alternative. Specimens containing GA were
suitable for most common clinical chemical determinations, although
careful evaluation will be needed in individual laboratories to
determine the potential for interference in such methodologies as the
alkaline picrate method for creatinine. Unlike the most prevalently
used glucose preservation agent, fluoride ion, GA does not cause
hemolysis, and potassium concentrations in plasma from GA-treated
specimens remain suitable for assessment of potassium homeostasis for
up to 8 h. From the standpoint of stability and solubility, GA
seems ideal as an additive because it is highly soluble (30 g/L)
(8) and, according to the source, Sigma Chemical,
D,L-GA can be stored at room temperature in the
crystalline form. Because L-GA is fully effective
at concentrations as low as 2.5 mmol/L, the actual amount of
L-GA needed for the standard evacuated 7-mL
collection tube is 1.58 mg. This small dose limits the expense of use
of GA and eliminates the potential that volume dilution of the specimen
by the additive might decrease glucose and other determinations, in
contrast to the equivalent fluoride/oxalate 7-mL tube, which contains
>31 mg of additive. Volume dilution of specimen by additive becomes
more important when collection tubes are only partially filled.
Prevalent approaches to minimizing glycolytic loss of glucose have
considerable limitations. The use of fluoride or iodoacetate to inhibit
cellular glycolytic enzymes is only partially effective; although
significantly decreased, glycolysis continues in the presence of either
agent, particularly in the first few hours after collection
(9)(10). Iodoacetate interferes in at least some
methods of glucose analysis (11). Specimens collected in
standard fluoride/oxalate tubes are invariably hemolyzed, which makes
them unsuitable for analysis of other important analytes that are
frequently requested in conjunction with glucose, such as potassium.
The use of mannose as an antiglycolytic agent has been hampered by
reports of interference of mannose in several glucose methods
(12)(13). Mannose was also judged unsuitable for
preservation of specimens for electrolyte analysis because of
appearance of cellular potassium in the plasma phase during storage
(14). Immediate cooling of specimens and transportation on
ice effectively preserves glucose concentrations (2), but it
imposes additional costs and burden on the transportation process.
Because metabolism is reduced by cooling, cellular potassium rapidly
diffuses into the plasma phase of whole blood specimens, which
significantly increased plasma potassium concentrations after 1 h
(2). The use of near-patient analytical devices to measure
glucose immediately after specimen collection effectively eliminates
glucose loss, but these analytical devices typically are expensive to
operate and place a considerable burden on patient care staff. Glucose
meters are a prevalent and relatively inexpensive example of this
approach, but they offer only glucose determinations (7).
The antiglycolytic effect of L-GA has been noted
previously, but L-GA has never been tested as an additive
for preservation of blood specimens for glucose analysis [for review,
see Ref. (15)]. The ability of L-GA
to inhibit the formation of glucose from D-GA in
slices of rat kidney cortex-mix was documented in 1966 (16).
Thornalley and Stern (17) noted L-GA
inhibition of lactate/pyruvate formation from radioactive glucose in
erythrocytes but did not measure glucose concentrations in blood or
plasma in their experiments. Glyceraldehyde has been investigated as an
agent to promote insulin secretion from isolated pancreatic islets
(15); both D-GA and
L-GA were effective in promoting secretion. The
action of D- and L-GA was
related to autooxidation to methylglyoxal, which is a highly reactive
inhibitor of many cellular functions.
The mechanism of L-GA inhibition of glycolysis in
erythrocytes has not been fully established. L-GA is poorly
metabolized (15)(16), through conversion to
glycerol (17). Hexokinase has been proposed as the site of
inhibition by L-GA (17), through
condensation with dihydroxyacetone phosphate to form
sorbose-1-phosphate, which is an inhibitor of hexokinase
(18). On the basis of this hypothesis, I anticipated that
L-sorbose might mimic the antiglycolytic activity
of L-GA, through cellular conversion to
sorbose-1-phosphate, but sorbose was not effective at concentrations
similar to those of D,L-GA that were effective in
preserving glucose concentrations. It is also possible that
L-GA inhibits glycolysis through autooxidation to
methylglyoxal or pyruvaldehyde, which are known to form covalent
adducts with proteins and to inhibit many cellular processes
(15). However, results from my study argue against this
mechanism. Both D- and L-GA
readily autooxidize, but only L-GA seemed to be
the active agent in my experiments. Methylglyoxal and related compounds
were ineffective as glucose preservation agents when incubated with
erythrocytes at the same concentration that was effective for
L-GA. Further studies are needed to determine the
metabolic fate of L-GA in erythrocytes and to
determine the site of inhibition of glycolysis by
L-GA or a metabolic product.
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Acknowledgments
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This work was supported in part by the General Clinical Research
Center (RR-00036) and the Diabetes Research and Training Center
(DK-20579) of Washington University. Michael Morris and Joy Brothers
provided excellent technical assistance for this study, and Barbara
Hartman was invaluable in preparing the manuscript.
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Footnotes
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Address correspondence to: Department of Pediatrics, Washington
University School of Medicine, One Children’s Place, Room 2N-68, St.
Louis, MO 63110. Fax 314-454-2274; e-mail landt{at}kids.wustl.edu
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Abstract
Introduction
