Method for determination of hemoglobins

Details Method for determination of hemoglobins Method for determination of hemoglobins

2001-02-07

2002-08-06

Therkorn, Ernest G. (Department: 1723)

Liquid purification or separation

Processes

Liquid/liquid solvent or colloidal extraction or diffusing...

C210S656000, C210S198200, C436S066000, C436S161000, C530S385000, C530S413000, C530S416000, C530S417000

Reexamination Certificate

active

06428704

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a method for determining hemoglobins, specifically directed to determine stable hemoglobin A
1c
by cation exchange liquid chromatography.
DESCRIPTION OF PRIOR ART
Hemoglobin A
1c
(hereinafter abbreviated as HbA
1c
) has been frequently listed as a test item in a screening test of diabetes mellitus or as a test item for grasping how well the blood glucose level of diabetics is controlled, because its ratio by composition to all hemoglobins reflects an average blood glucose level (blood glucose concentration) over the preceding 1-2 months.
HbA
1c
is glycated hemoglobin (hereinafter abbreviated as GHb) produced via a reaction between glucose and hemoglobin A (hereinafter abbreviated as HbA) present in the blood. HbA
1c
, if produced via a reversible reaction therebetween, is called labile HbA
1c
and, if produced via an irreversible reaction involving the labile HbA
1c
, is called stable HbA
1c
.
Normally, hemoglobin is a tetrameric protein composed of two pairs of two different subunits. HbA has &agr;-chain and &bgr;-chain subunits. Binding of glucose to N-terminal amino acid(s) of this/these &bgr;-chain results in HbA
1c
. The stable HbA
1c
better reflects an average blood glucose level over the preceding 1-2 months. In the field of clinical testing, it has been demanded to develop a method whereby the stable HbA
1c
level (%) can be measured with high precision.
Conventional HbA
1c
determination methods mostly employ liquid chromatography (hereinafter abbreviated as LC) which, according to a cation exchange technique, separates hemoglobins present in a sample prepared by hemolytically eluting a blood specimen on the basis of difference in positively charged state between hemoglobins (for example, Japanese Patent Publication No. Hei 8-7198).
The separation of hemoglobins present in the hemolyzed sample by means of cation exchange LC, if performed over a sufficiently long period of time, generally results in the sequential elution of hemoglobin A
1a
(hereinafter abbreviated as HbA
1a
) and hemoglobin A
1b
(hereinafter abbreviated as HbA
1b
), hemoglobin F (hereinafter abbreviated as HbF), labile HbA
1c
, stable HbA
1c
and hemoglobin A
0
(hereinafter abbreviated as HbA
0
). HbA
1a
, HbA
1b
and HbA
1c
each is GHb in the form of glycated HbA. HbF is fetal hemoglobin composed of &agr; and &ggr; chains. HbA
0
consists of a group of hemoglobin components, includes HbA as its primary component and is retained more strongly to a column than HbA
1c
.
The prior techniques have suffered from the following deficiencies: The separation of labile HbA
1c
from stable HbA
1c
is insufficient; and “modified hemoglonins”, such as acetylated hemoglobin (hereinafter abbreviated as AHb) and carbamylated hemoglobin (hereinafter abbreviated as CHb), are eluted together with stable HbA
1c
.
That is, in the determination of hemoglobins present in a blood sample, primarily purposed to measure a stable HbA
1c
level (%) by cation exchange LC, it has been difficult to separate labile HbA
1c
, AHb and CHb peaks from a stable HbA
1c
without affecting measurement of stable HbA
1c
level, since their elution behaviors resemble each other.
Hemoglobin S (hereinafter abbreviated as HbS) and hemoglobin C (hereinafter abbreviated as HbC) are known as “abnormal hemoglobins”. HbS and HbC result from substitution of glutamic acid located in a sixth position from an N-terminal of the &bgr; chain of HbA for valine and lysine, respectively.
Hemoglobin A
2
(hereinafter abbreviated as HbA
2
) is composed of &agr; and &dgr; chains and, like HbF, its elevated level is interpreted as evidence of Mediterranean anemia (thalassemia).
In the normal determination of hemoglobins by cation exchange LC, they are eluted in the sequence of HbA
0
, HbA
2
, HbS and HbC.
In the determination of stable HbA
1c
present in a specimen which also contains HbS, HbC or other abnormal hemoglobins, it is required that peaks of these hemoglonins be separated from a HbA
0
peak and that the stable HbA
1c
level (%) be determined by calculating a ratio of a peak area of stable HbA
1c
to a total peak area of hemoglobin components exclusive of abnormal hemoglobins.
When the simultaneous examination of Mediterranean anemia is desired, an elustion condition is established that allows separation of HbA
2
from HbA
0
. In this case, the ratio in level of HbF and HbA
2
to all hemoglobins is calculated to provide measurement result.
In the normal separation of hemoglobins by means of cation exchange LC, the hemoglobin components included in the HbA
0
peak (hereinafter abbreviated as HbA
0
components) are classified into the following two cases depending upon the measurement conditions used. Under the measurement condition where a blood specimen containing HbA
2
, abnormal hemoglobins or the like which tend to become retained more strongly by packing materials than HbA is subjected to a single-step elution, a resulting peak includes not only HbA but also those hemoglobin components. On the other hand, under the measurement condition that effects separation of HbA
2
, abnormal hemoglobins and the like, a peak primarily of HbA results.
A typical LC used in the determination of Hb's is cation exchange LC (for example, Japanese Patent Publication No. Hei 8-7198).
Examples of packing materials known to be useful for determination of hemoglobins include those made via a reaction of inorganic or organic polymer particles with a compound having an ion exchange group, and those made via polymerization of a monomer having an ion exchange group with a crosslinking monomer.
One of important factors that determine performances of such cation-exchange packing materials is the ion exchange capacity. Conventional cation-exchange packing materials have ion exchange capacities in the approximate range of several meq-several tens meq/g, 0.2-0.3 meq/g at the lowest. The ion exchange capacity depends not only upon the amount of the ion exchange group-containing compound to be reacted and the condition under which it is reacted, but also upon the particle size, specific surface area, pore size, pore volume and the like of the packing material used. It is thus considered very important to achieve simultaneous optimization of ion exchange capacity, pore size, specific surface area, pore volume and the like for the sake of precise separation.
Japanese Patent Laying-Open No. Hei 7-27754 discloses reacting porous particles having pore diameters in the range of 20-2,000 angstroms and specific surface areas of 0.1-100 m
2
/g with ion exchange group-containing compounds to thereby obtain packing materials having ion exchange capacities in the range of 0.5-3.0 meq/g.
The packing materials described in this reference are obtained by reacting polymeric particles and the like with ion exchange group-containing compounds. Accordingly, it has been difficult to introduce a controlled amount of ion exchange group-containing compounds into the polymeric particles and the like. The problem of poor reproducibility thus remains (Yoshimawari, Hosoya, Kimata and Tanaka; Chromatography, vol.16(1), pp. 7-12 (1995)).
Also, in the case where the polymeric particles are silica particles, the following problems arise: a pH range of an eluent is limited; resolution is reduced by non-specific adsorption; and service lives of columns are shortened. In the case of natural polymeric particles which show the increased tendency to swell and shrink, a problem of low pressure resistance arises.
On the other hand, the packing materials made via polymerization of an ion exchange group-containing monomer with a crosslinking monomer appear to be favored over the above-described packing materials since their use increases reproducibility, eases manufacture and extends service lives of columns. However, packing materials are not yet reported which result from polymerization of an ion exchange group-containing monomer with a crosslinking monomers and have optimized pore size, specific surface area, pore volume, ion exchange capacity and the like.
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