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Articles |
1
Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom.
a Author for correspondence. Fax 44-1603-255237; e-mail
tony.wright{at}bbsrc.ac.uk
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Abstract |
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Methods: The conventional method of erythrocyte lysis was modified to include saponin, a known effective hemolyzing agent. The influence of saponin was determined at various lysate pHs, using the microbiological (Lactobacillus rhamnosus) folate assay. The effect of saponin during lysate preparation was subsequently compared with either the effect of 30 s of sonication or a single 1-h freeze-thaw cycle.
Results: Saponin addition was found to increase assayable RCF up to ninefold, depending on lysate pH. Sonication of lysates had no effect, and freezing-thawing lysates once did not always guarantee complete hemolysis. Lysates created with 10 g/L ascorbic acid (a historically widely used diluent) without pH adjustment produced assayable folate concentrations significantly lower than optimal.
Conclusions: A lysing agent should be incorporated into RCF
assays to guarantee complete hemolysis. Ten-fold dilution of blood with
10 g/L ascorbic acid, without pH adjustment, produces lysates with pHs
(pH 4.0) below the point (pH 4.7) at which hemoglobin can denature
irreversibly. The optimum pH for hemolysates is 
5.0.
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Introduction |
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-fetoprotein estimation or fetal
ultrasound examination) has led to a reduction in the number of
children born with NTDs, through termination of pregnancy
(1)(2), it cannot prevent the development of
these defects or the associated parental distress. There now is firm evidence that an adequate intake of periconceptional folate (a B-group vitamin found especially in leafy green vegetables, some other vegetables and fruits, and whole grain cereals) can prevent a significant proportion of NTDs. Numerous studies (case-control and cohort studies and nonrandomized and randomized controlled trials) have shown that increased intake of either dietary folates or supplemental folic acid reduces the risk of NTDs (3). It is not surprising, therefore, that there is renewed interest in the analysis of red cell folate (RCF) as an indicator of folate deficiency risk in women of child-bearing age. Moreover, inverse associations between RCF concentration and the risk of cardiovascular disease (4) and various cancers (5)(6) have generated additional interest in the analysis of RCF as an indicator of risk of folate deficiency in the general population.
Interlaboratory comparison studies of analytical methods for folate (7)(8)(9) not only demonstrated large interassay variation in whole-blood folate analysis, but also large intraassay variation. There are discussions in the literature about method comparisons (8)(10)(11) and possible sources of variability (9)(12)(13)(14)(15)(16). Recently, we suggested that the assumptions underpinning RCF analysis are flawed (17). The first assumption in erythrocyte folate analysis is that dilution of whole blood with hypotonic diluents produces complete hemolysis and, hence, the release of all folate; historically, 10-fold dilution of whole blood with ascorbic acid (AA) has been widely used to create lysates (7)(10)(18)(19)(20). However, this assumption has not been tested within the range of conditions currently used in RCF analysis, although it is known that elements within the RCF assay could influence the degree of hemolysis. It is also known that erythrocyte folate is electrostatically associated with deoxyhemoglobin, within the hollow central cavity of the quaternary structure of the hemoglobin (Hb) molecule (21)(22)(23)(24). After hypotonic lysis of erythrocytes, transitory holes appear in the erythrocyte membrane that are large enough to allow the escape of Hb. However, these holes can reseal without complete release of Hb (25). To maximize the release of erythrocyte folate, and thus ensure appropriate measurement of folate status, it may be necessary to use a hemolyzing agent within the RCF assay to ensure complete release of Hb. Because pH is a major determinant of the rate of the resealing process (26)(27), characterization of the interaction of lysate pH with the selected hemolyzing agent is essential.
Saponin was chosen as the hemolyzing agent in the current studies not just because of its historical use for the hemolysis of erythrocytes, but because it was anticipated that the thick peptidoglycan outer cell wall of the gram-positive bacterium Lactobacillus rhamnosus (previously known as L. casei), used in the classical microbiological assay of folate, would prevent the saponin molecule from reaching the outer bacterial membrane.
Saponins (sapogenin glycosides) consist of an aglycone unit (sapogenin;
a sterol or triterpene) linked to a sugar unit (glycone; one or more
carbohydrate chains); they possess surface-active or detergent
properties because the carbohydrate portion of the molecule is
water-soluble whereas the sapogenin is fat-soluble (28). It
is thought that the release of Hb from erythrocytes is the direct
result of membranolytic saponins combining irreversibly with membrane
cholesterol to form permeable micelle-like aggregates, with a central
pore 80 Å (8 nm) in diameter, within the plane of the membrane
(29). Transmission electron microscopy has revealed
irreversible destruction of the erythrocyte lipid bilayer by saponin
(30). Thus, saponin-lysed erythrocytes do not reseal.
Preexperimental work indicated that the microbiological growth response of L. rhamnosus to a folic acid calibration series was not perturbed even at a saponin concentration fivefold higher than would be encountered in diluted hemolysates in the current studies. Furthermore, under pH conditions where the use of saponin generated significantly higher RCF values, preexperimental work also indicated that the use of saponin at either twice or one-half the concentration actually used in the current studies gave RCF values that were not significantly different. Therefore, the saponin concentration used in the experiments described here neither bordered on being too low nor affected the growth of L. rhamnosus.
This report describes two studies designed to determine (a) the effect of the addition of a hemolyzing agent (saponin) on the RCF assay at a range of lysate pHs, and (b) the effectiveness of freezing-thawing or sonication of erythrocytes in comparison with the addition of saponin. Results obtained are discussed in relation to conditions currently used in RCF analysis and possible modification of the assay.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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stock saponin solution
Saponin (11.111 g, containing 90 mg/g sapogenins, "white
suitable for hemolysis"; cat. no. 43650-2L; BDH-MERCK) was
diluted to 100 mL with H2O to create a stock
solution containing 10 g/L saponin.
blood diluents
Freshly prepared 10 g/L (57 mmol/L) solutions of AA were adjusted,
as appropriate, with 1 mol/L NaOH through a range of pHs from
4.0 to 6.0. In addition, if appropriate, 1 mL of stock saponin solution
was added to 99 mL of diluent (producing a final working saponin
concentration of 100 mg/L) before the pH was adjusted. This working
saponin concentration has recently been shown to produce 100%
hemolysis, 50% hemolysis being achieved at a concentration of 10
mg/L (30).
study i
Study I was divided into two parts:
Study I(a).
Study I(a) involved an investigation of assayable
RCF concentration, comparing the use of AA at pH 2.7 (the historically
used diluent) with pH-adjusted AA or water, and concomitant
determination of the effect of added saponin as a hemolyzing agent.
Fresh anticoagulated whole blood was diluted 10-fold with 10 g/L AA (pH
2.7); 10 g/L AA adjusted with 1 mol/L NaOH to pH 4.0, 4.25, 4.5,
4.75, 5.0, and 6.0; or water (analytical reagent grade), each with or
without added saponin; the pHs of the lysates were 4.0, 4.7, 5.2, 5.5,
6.0, 6.3, 7.0, and 7.4, respectively. Six independent lysates were made
using each diluent.
Study I(b).
Study I(b) investigated, in the absence of added
saponin, whether incomplete hemolysis (seen with increasing
diluent/lysate pH) is a function of pH per se or whether it is
attributable to the decreased hypotonicity of diluents to which NaOH
was added during pH adjustment. Fresh anticoagulated whole blood was
diluted 10-fold with 10 g/L AA adjusted with 1 mol/L NaOH to pH
4.5, 4.75, and 5.0, and with 10 g/L AA first adjusted with 1 mol/L NaOH
to pH 6.0 and then readjusted immediately with 1 mol/L HCl back to pH
4.5 (each diluent without added saponin). Four independent lysates were
made using each diluent.
study ii
Study II was an investigation into the effects of freezing-thawing
and saponin addition on lysis with water, and the effect of
freezing-thawing, sonication, and saponin addition on lysis with AA, pH
5.0 (lysate, pH 6.3). Fresh anticoagulated whole blood was diluted
10-fold with water (analytical reagent grade), (a) without
saponin, (b) without saponin again, and (c) with
saponin. Eighteen separate lysates were prepared using each condition.
The second set of 18 lysates without saponin was frozen immediately in
solid CO2 for 1 h and then thawed.
Additionally, fresh anticoagulated whole blood was also diluted 10-fold
with 10 g/L AA, pH 5.0, (a) without saponin, (b)
without saponin again, (c) without saponin for a third time,
and (d) with saponin. Eighteen separate lysates were
prepared using each condition. The second set of 18 lysates without
saponin was frozen immediately in solid CO2 for
1 h and then thawed. The third set of 18 lysates was sonicated for
30 s in an ultrasonic water bath.
creation of lysates
After gentle mixing, 100 µL of anticoagulated whole blood was
reverse-pipetted into 900 µL of the appropriate diluent in 2-mL
plastic screw-cap microcentrifuge tubes and mixed gently by multiple
inversions.
folate polyglutamate deconjugation
All lysates (including those that may have undergone preliminary
freezing-thawing) were incubated for 2 h at 37 °C so that any
released red cell folyl-polyglutamates could undergo deconjugation,
with naturally occurring plasma folate conjugase (
-Glu-X
carboxypeptidase; EC 3.4.19.9), to the monoglutamate form, which is the
appropriate form for assay. The pHs of lysates/hemolysates, once
cooled, were monitored with the use of a micro pH probe (pH Boy KS501;
CAMLAB Limited) that had been calibrated immediately before use.
postdeconjugation treatment
Deconjugated samples were remixed gently by multiple inversions
and diluted 20-fold by reverse-pipetting 100 µL into a 2-mL screw-cap
microcentrifuge tube containing 1.9 mL of 0.1 mol/L sodium
phosphate buffer, pH 6.2 (so that samples could be added to the
microbiological assay at the same pH as the assay medium). The tubes
were then capped, and the samples were remixed gently by multiple
inversions and centrifuged in a microcentrifuge at 16 000g
for 1 min to pellet partially hemolyzed or nonhemolyzed erythrocytes.
Four 50-µL volumes of each supernatant were then transferred
aseptically, by reverse-pipetting, to 96-well Cell Culture Cluster
plates (cat. no. 3596; Corning-Costar®) for the
microbiological assay.
preparation of growth media
Difco Lactobacilli Agar AOAC (cat. no. 0900-15; Becton Dickinson
UK) was used to prepare agar for the maintenance of the microorganism
as "stab" cultures. Hot liquid agar (3 mL) was dispensed into 5-mL
screw-cap bottles, which were subsequently securely capped, autoclaved
for 15 min at 121 °C (103 x 103 Pa), and
stored at 2–8 °C.
Medium for the revitalization of either lyophilized or stab cultures was prepared with Difco Lactobacilli Broth AOAC (cat. no. 0901-15; Becton Dickinson UK). Aliquots (10 mL) were dispensed into thick-walled glass test tubes, capped with Clark Fincaps (Clark Scientific), autoclaved for 15 min at 121 °C (103 x 103 Pa), and stored at 2–8 °C.
Medium for the preparation of cultures in log-phase growth and for the main folate assay was prepared with Difco Folic Acid Casei Medium (cat. no. 0822-15; Becton Dickinson UK). Dehydrated medium (47 g) was gently brought to the boil for 1–2 min in 900 mL of H2O and then cooled to room temperature. At pH >6.2, there can be a divergence in growth response to folic acid (assay calibrant) and 5-methyltetrahydrofolic acid from deconjugated erythrocyte folate samples (12) because of a peculiarity of L. rhamnosus’ metabolism of the monoglutamate form of 5-methyltetrahydrofolate not shared with mammalian metabolism (13). For this reason, 1.0 g of L-AA was then added to the cooled medium, and if necessary, the pH was further adjusted with either 1 mol/L acetic acid or 1 mol/L NaOH to pH 6.2 before the addition of H2O to a final volume of 1 L. Aliquots (200 mL) were distributed into four or five 250-mL wide-mouthed screw-cap bottles, autoclaved at 121 °C (103 x 103 Pa) for 5 min (to minimize caramelization of the medium), and stored at 2–8 °C. One bottle was enough for nine 96-well assay plates. Initially, and thereafter occasionally, 200 mL of medium was distributed in 10-mL aliquots into thick-walled glass test tubes, capped with Clark Fincaps (Clark Scientific), autoclaved for 5 min at 121 °C (103 x 103 Pa), and stored at 2–8 °C. On the day of assay, 1 ng of folic acid would be added to one of these tubes to create an "inoculum" tube.
microorganism
Lyophilized cultures of L. rhamnosus NCIMB 6375 (also
known as ATCC 7469) were obtained from the National Collection of
Industrial and Marine Bacteria. L. rhamnosus was previously
known as L. casei, subsp. rhamnosus.
preparation of culture
Cultures were initiated by transfer of freeze-dried cells to 10 mL
of Lactobacilli Broth AOAC and overnight incubation at 37 °C. A
subsample (100 µL) of this broth was then recultured in fresh
Lactobacilli Broth AOAC overnight. After loop transfer, six stabs (four
"weekly" stabs, a "monthly maintenance" stab, and a "spare"
stab) were incubated for 2 days at 37 °C and stored at 2–8 °C.
L. rhamnosus was maintained long-term as stab
cultures and refreshed every 4th week from the monthly
maintenance stab.
preparation of folic acid stock solution
Precisely 20 mg of folic acid (cat. no. F 7876; Sigma-Aldrich) was
dissolved with 200 µL of 1 mol/L NaOH. Approximately 50 mL of
H2O was added, and the solution was neutralized
to pH 7.0 with 1 mol/L HCl. After the addition of 20 mL of ethanol, the
solution was finally made up to 100 mL with H2O,
transferred to an aluminum foil-covered brown glass volumetric flask,
and stored at 2–8 °C. This folic acid stock solution had a nominal
concentration of 200 mg/L.
estimation of true concentration of folic acid stock solution
It is critically important to note that not only do commercial
supplies of folic acid contain water, but that opened supplies also
pick up water rapidly. Hence, it should be considered mandatory that
the absolute concentrations of folic acid stock solutions be determined
using the specific absorptivity for a 10 g/L solution in 0.1
mol/L alkali at 365 nm, the absorption maximum (E365nm1%,1 cm = 195)
(31). In the present studies, the real concentration of the
stock solution was actually 155.9 mg/L. In practice, it was easier to
use the nominal folic acid concentrations (200 mg/L) of the stock
solution and any subsequent dilutions throughout all experimental
calculations and to correct final tabulated answers only; in our case,
we corrected by multiplying by 155.9/200.
preparation of folic acid calibration solutions (of nominal values)
On the day of assay, the stock solution was diluted. For solution
A (200 µg/L), 100 µL of stock solution was diluted to 100 mL with
water. For solution B (4 µg/L), 2.0 mL of solution A was diluted to
100 mL with water (this solution was used to prepare the inoculum
tube). For solution C (4 µg/L), 2.0 mL of solution A was diluted to
100 mL with 0.1 mol/L sodium phosphate buffer, pH 6.2 (this solution
was used to prepare the folic acid calibration series). Solution D was
0.1 mol/L sodium phosphate buffer, pH 6.2. This solution was used to
dilute deconjugated samples before plating for assay, and to dilute the
folic acid calibration solution (solution C).
preparation of calibration series
For the calibration series, 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, and 1.0 mL of folic acid solution C (4 µg/L) were added to 2-mL
plastic screw-cap microcentrifuge tubes already containing 2.0, 1.9,
1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, and 1.0 mL, respectively, of
solution D (0.1 mol/L sodium phosphate buffer, pH 6.2) to form a
10-point calibration series of 0–2000 ng/L such that a 50-µL aliquot
from each solution contained 0, 20, 30, 40, 50, 60, 70, 80, and 100 pg,
respectively. Aliquots (50 µL) of each calibrator were added
aseptically (sterile laminar-flow cabinet) to four wells of every
sterile, flat-bottomed 96-well Cell Culture Cluster plate that was to
be used.
preparation of inoculum and combined inoculum/folic acid casei
medium, and timetable of assay
On the day before the assay, bacteria were transferred from one of
the stab cultures to a tube of Lactobacilli broth and incubated
overnight at 37 °C to reinitiate active growth. Early on the day of
the assay, folic acid solutions A, B, and C (see above) were
constructed from the stock folic acid solution. Solution B (250 µL,
containing 1 ng of folic acid in H2O) was added
to a tube containing 10 mL of Folic Acid Casei Medium, together with
100 µL of bacterium from the Lactobacilli broth grown overnight, to
create an inoculum tube, which was incubated 6 h at 37 °C to
generate organisms in the logarithmic phase of growth. During this
period of time, folic acid calibration samples were prepared together
with blood samples for the assay.
Using sterile pipette tips, we added four 50-µL aliquots of the calibration samples (every plate) and the experimental treated/diluted blood samples to sterile 96-well plates in a sterile microbiological laminar-flow cabinet. After the inoculum had grown for 6 h, 2 mL was transferred to one bottle containing 200 mL of Folic Acid Casei Medium and mixed; 200 µL of the combined growth medium/inoculum was then added, by reverse-pipetting, to all plate wells with an eight-lane multitip pipette. Plates were subsequently placed in a sealable plastic box lined with wetted absorbent paper; assay plates were stacked on top of two unused plates to keep them well above the wetted absorbent paper lining the box bottom. Before the box was sealed with a lid and incubated at 37 °C, a small container of water was also placed inside to ensure that humidity was maintained and that there was no volume change in the wells of the assay plates during incubation.
After overnight (18 h) incubation, we measured the bacterial growth
in the sample wells by reading the absorbance at 630 nm with an
automatic microplate reader. Before the absorbance in the sample wells
was measured, the absorbance for the four 0 pg folic acid calibration
wells in each plate was reset to an approximate zero absorbance
reading.
calculation of results
For each separate plate, the mean absorbance for each group of
four calibration or sample replicates was calculated, and any residual
mean absorbance for the 0 pg/well calibrant was subtracted (thus
ensuring a zero absorbance for the 0 pg/well calibrant and
allowing the absolute growth on different plates, both within
and between assays, to be compared). A spreadsheet
(MicroSoft EXCEL®) was used to plot (pg/well on
the y axis, and absorbance on the x axis) the
calibration for each plate and to calculate a polynomial (third degree)
equation: y = C + ax +
bx2 +
cx3. The resulting equation for each
plate was used to convert the absorbances of experimental samples into
pg of folate/well. Folate concentrations (in ng of folic acid/mL of
whole blood = µg/L) were generated by multiplying pg of
folate/well (50 µL of lysate) by an overall factor of 4 (i.e., x 20
for conversion to pg/mL diluted lysate; x 20 for dilution of lysate;
x 10 for original dilution of whole blood; divided by 1000 to convert
from pg to ng).
statistical analysis
Study I(a).
The effect of added saponin at any one diluent pH
was analyzed by the Student unpaired t-test. The effect of
pH, separately for with or without added saponin, on assayable RCF
compared with 10 g/L AA, pH 2.7 (lysate, pH 4.0), was assessed by
parametric one-way ANOVA and the least significant difference
test.
Study I(b).
The effect of pH on assayable RCF was assessed by
parametric one-way ANOVA and the least significant difference test.
Study II.
With water as the diluent, the effect on assayable
RCF (a) without saponin, (b) without saponin +
freezing-thawing, and (c) with saponin was assessed by
parametric one-way ANOVA and the least significant difference test.
Because of nonhomogeneous variances with 10 g/L AA, pH 5.0, as the
diluent, the effect on assayable RCF (a) without saponin,
(b) without saponin + freezing-thawing, (c)
without saponin + sonication, and (d) with saponin was
assessed by nonparametric Kruskal-Wallis one-way ANOVA by ranks and the
Mann–Whitney U-test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The addition of saponin to the 10 g/L AA diluent, pH 2.7, 4.0,
and 4.25 (whole blood lysates, pH 4.0, 4.7, and 5.2, respectively), did
not produce significantly different assayable RCF concentrations. In
contrast, addition of saponin to the 10 g/L AA diluent, pH 4.5, 4.75,
5.0, and 6.0, and water (lysates, pH 5.5, 6.0, 6.3, 7.0, and 7.4,
respectively) produced significantly higher (P <0.05)
assayable RCF concentrations: a 27% increase in the case of water
(lysate, pH 7.4), and a ninefold increase in the case of 10
g/L AA diluent, pH 6.0 (lysate, pH 7.0). Even with added saponin,
the use of 10 g/L AA diluents more neutral than the historically widely
used diluent (10 g/L AA, pH 2.7) produced significantly higher
(14%) assayable RCF concentrations.
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The results (µg/L folate) for study I(b) are given in Table 2
. On the basis of results from study I(a), it would be expected
that assayable RCF for a diluent of 10 g/L AA, pH 6.0, would be very
poor and even lower than that for 10 g/L AA, pH 5.0. However, results
from this study indicated that the immediate readjustment of 10 g/L AA,
pH 6.0, diluent back to pH 4.5 produced assayable RCF concentrations
considerably higher than expected: 85% of the values observed when 10
g/L AA adjusted directly to pH 4.5 was used as diluent.
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The results for study II are given in Table 3
. When water was used as the diluent (lysate, pH 7.4), the
assayable RCF concentration when saponin was not added was 81.7% of
the value measured when saponin was added. This value rose to 90.1%
with one cycle of freezing-thawing before RCF deconjugation. The
failure of freezing-thawing to completely hemolyze water lysates was
confirmed by visual observation. After deconjugation and subsequent
lysate dilution before microbiological assay, centrifugation of samples
revealed the presence of small red pellets in the freeze-thawed samples
that were not seen in saponin-treated samples. When 10 g/L AA, pH 5.0,
was used as the diluent (lysate, pH 6.3), the assayable folate when
saponin not added was 15.1% of the value measured when saponin was
added. This value increased to 15.7% with 30 s of sonication in
an ultrasonic water bath (a nonsignificant response). In contrast, the
comparative value of assayable folate when saponin was not added
(15.1%) rose to 98.9% with one cycle of freezing-thawing before RCF
deconjugation, a value indistinguishable from 100% because there was
no statistically significant difference in the RCF values of the
freeze-thawed samples compared with the saponin-treated samples (see
Table 3
).
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Discussion |
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Recently, we suggested that several assumptions underpinning RCF analysis are deeply flawed (17). The studies presented here addressed the first of these assumptions, that the dilution of whole blood with hypotonic diluents always produces red cell lysis and, hence, the release of all erythrocyte folate. Most lysates are prepared initially by diluting whole blood 10- to 21-fold with AA solutions (10 to 0.5 g/L), although it has been reported (14)(20) that some laboratories use sodium ascorbate solutions or sodium ascorbate adjusted to pH 4.5 or 6.1. These variations in diluent type (AA vs ascorbate), concentration, and dilution factor will produce lysates with different pHs. The studies presented here assessed the effect of lysate pH, using a historically widely used diluent (10 g/L AA) and dilution factor (10-fold).
Results from study I(a) demonstrated that RCF concentrations can be up to ninefold higher in the presence of added saponin, depending on diluent/lysate pH. We consider these results indicative of incomplete hemolysis. Unexpectedly, when lysis was induced with plain water, the ultimate hypotonic diluent, the presence of saponin increased the RCF concentration by 27%, which also was indicative of incomplete hemolysis. Study I(a) showed that an ascorbate diluent at pH 6.0 produced very poor hemolysis and, hence, low assayable RCF concentrations. It is arguable that this was a direct consequence of the decrease in diluent hypotonicity produced by the ions added when NaOH was used to adjust the 10 g/L AA diluent to pH 6.0. However, study I(b) showed that if diluent adjusted to pH 6.0 was immediately readjusted back to pH 4.5 with 1 mol/L HCl before use (the added ions decreasing its hypotonicity even further), assayable RCF essentially reverted to the much higher concentration seen when 10 g/L AA was adjusted directly to pH 4.5. This result implies that the substantially lower assayable RCF concentration obtained with increasing diluent pH is mainly a function of lysate pH per se and is not attributable to an inherent decrease in the hypotonicity of diluents caused by the addition of increasing amounts of NaOH as a pH modifier.
Study I(a) also demonstrated, surprisingly, that 10-fold dilution of whole blood with unadjusted 10 g/L AA (pH 2.7), historically a widely used diluent and dilution factor that produces a lysate pH of 4.0, yields a RCF concentration significantly lower than that achieved at the higher lysate pHs of 4.7, 5.2, or 5.5. This result was unexpected and raises a question concerning hemolysis conditions that have been accepted as a "gold standard" for many decades. An empirical observation made during the course of the current studies was that hemolysates generated with 10 g/L AA changed from a dark red/purple to a dark brown color within minutes, which suggested changes in the structure of Hb. It previously has been shown that Hb denatures irreversibly in solution at pH <4.7, with the degree of denaturation being directly related to increasing acidity and time (33). Because RCF associates with Hb (21)(22)(23)(24), it is perhaps not surprising that the use of 10 g/L AA, pH 2.7 (lysate, pH 4.0), leads to lower RCF concentrations. Under such conditions, RCF would be expected to coprecipitate with denatured Hb. This may explain problems encountered in a field study investigating the potential for screening blood folate concentrations by the collection of small finger-prick samples stored on filter paper as dried blood spots, where a generally decreased stability of blood folate concentrations in lysates or dried blood spots in the presence of AA was reported (34).
Results from study II, which used a diluent of 10 g/L AA, pH 5.0
(lysate, pH 6.3), demonstrated that although sonication for 30 s
in an ultrasonic water bath had no significant impact on RCF
concentration and, by implication, the degree of hemolysis, subjecting
samples to one freeze-thaw cycle before RCF deconjugation increased the
assayable folate concentration from 15% to 100% of the comparative
values seen in lysates with added saponin. Although there have been
reports that freezing and thawing lysates once
(14)(35), but not repeatedly (35),
increases assayable folate concentrations, we have yet to find a
currently used commercial RCF assay with blood preparation procedures
that suggests that freezing and thawing may induce higher assayable
values or that requires a single freeze-thaw cycle of lysates before
RCF deconjugation. However, in contrast to the effect seen when 10 g/L
AA, pH 5.0, was used as the diluent, results from study II demonstrated
that subjecting samples to one freeze-thaw cycle before RCF
deconjugation of whole blood previously diluted 10-fold with water
still did not produce complete hemolysis, a failure confirmed by visual
observation. Thus, freezing and thawing lysates once may not always
guarantee complete hemolysis.
The results from studies I and II demonstrate that it should not be assumed that 10-fold dilution of whole blood with hypotonic solutions containing 10 g/L AA/ascorbate will always produce complete hemolysis. The results for water diluent suggest that the assumption of complete hemolysis should not be made unless lysing diluents are accompanied by appropriate addition of a hemolyzing agent. Of course, any hemolyzing agent that interferes with terminal folate analysis, microbiological assays, or any of the various commercially available competitive folate binding assays should not be used.
Once complete hemolysis has been achieved (in the absence of Hb
denaturation), the next important step before assay is the complete
enzymatic deconjugation of erythrocyte polyglutamyl folates through the
judicious choice of pH, time, and temperature. The most obvious optimum
pH is 5.0 (4.7–5.2), an acidic pH close to the optimum for folate
deconjugases, whose maximum activity is at pH 4.5 (36), but
not low enough to induce Hb denaturation. Although deconjugase activity
at pH 6.0 should be 40% of the maximum exhibited at pH 4.5
(36), incomplete deconjugation has been reported in pH 6.0
lysates incubated for as long as 90 min at 37 °C
(20). However, it may be important to reevaluate this report
because the authors initially diluted blood 10-fold with 10
g/L sodium ascorbate before adjusting the resulting lysate to pH
6.0. We have observed (unreported observations) that a diluent
containing 10 g/L sodium ascorbate (pH 8.0) produces a lysate with a pH
of 7.1, incomplete hemolysis, and a very low assayable RCF
concentration that is indistinguishable from the concentration obtained
when 10 g/L AA, pH 6.0, is used as the diluent (lysate, pH 7.0). In any
future work (on completely hemolyzed solutions) into the efficiency of
deconjugation at any chosen hemolysate pH and temperature, it may be
informative to attempt to distinguish between incomplete deconjugation
attributable to low deconjugase activity per se and that attributable
to binding of RCF to Hb. This could be accomplished by assessing both
the distribution of folate polyglutamate forms in subsamples of
hemolysates by HPLC (20) and the distribution of folate
polyglutamate forms in the filtrate of subsamples centrifuged through a
molecular size-exclusion membrane that would prevent the passage of Hb.
Samples showing incomplete folate deconjugation in hemolysates, but
complete deconjugation in filtrates, would indicate that potentially
effective deconjugase activity is being hindered by the binding of
erythrocyte folate to Hb, perhaps in a manner ("oxy-hemoglobin
folate-trap hypothesis") that we have previously proposed
(17). Dimerization of Hb would negate any potential physical
"trapping" of folate within the hollow central cavity of the
quaternary structure of the Hb molecule. The choice of an acidic
hemolysate pH that maximizes folate deconjugase activity, short of
inducing denaturation of Hb (i.e., pH 5.0) will also tend to
maximize Hb tetramer-dimer dissociation, which has been shown to occur
extensively at pH <6.0 (37).
The degree of hemolysis and/or the completeness of deconjugation will obviously influence the estimation of erythrocyte folate concentrations, but may affect the accuracy and stability of the classical biological method (microbiological assay) and the now widely used competitive folate-binding protein assays (radioassays and immunoassays) differently. The microbiological assay is more tolerant of the presence of polyglutamate RCF forms and will tend to underestimate total folate where hemolysis is complete but deconjugation is not. In contrast, under similar conditions, folate-binding protein assays may bind polyglutamate forms with a greater affinity than monoglutamate and thus overestimate total folate (38).
In conclusion, the inaccuracy and incomparability of erythrocyte folate measurements are hindering evaluation of the effects of national folic acid supplementation programs and our emerging understanding of the relationships between erythrocyte folate concentrations (folate status) and the risk of NTDs, vascular disease, and cancer. Erythrocyte folate assays vary in the strength of the AA/ascorbate diluent and the dilution factor used for preparing lysates. This produces different lysate pHs, some of which may not fully induce either complete hemolysis and/or complete RCF deconjugation, and some which may irreversibly denature Hb, thus potentially coprecipitating any RCF electrostatically held within its central cavity (17)(21)(22)(23)(24). This report concludes that:
5.0, an acidic pH that does
not induce Hb denaturation but would maximize both plasma folate
deconjugase activity and Hb dimerization.
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