Blood methemoglobin analysis

Details Blood methemoglobin analysis Blood methemoglobin analysis

1999-01-22

2001-02-27

Wallenhorst, Maureen M. (Department: 1743)

Chemistry: analytical and immunological testing

Hemoglobin, myoglobin, or occult blood

C436S008000, C436S017000, C436S018000, C436S164000, C436S166000, C436S176000, C252S408100

Reexamination Certificate

active

06194218

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to the field of blood analysis, specifically the testing of blood for methemoglobin content.
BACKGROUND OF THE INVENTION
Normally, the hemoglobin in circulating blood is usually more than 99% in the ferrous (Fe
2+
) form. The ferrous form transports oxygen. Another form of hemoglobin, known as methemoglobin, contains the ferric ion (Fe
3+
). Methemoglobin is unable to transport oxygen. In healthy individuals, the methemoglobin concentration in circulating blood is usually less than one percent. The iron in hemoglobin is kept in the ferrous valence state by the activity of a special methemoglobin reductase system. See Metzler,
Biochemistry
, Academic Press, New York, N.Y., 1997, pp. 564-565.
Methemoglobin is monitored to evaluate biological exposure to biotoxins and possible carcinogenic compounds which contain primary aromatic amino-, nitro-, nitroso-, azo- and/or certain phenolic moieties. These residues have been directly correlated to methemoglobin production both in vivo and in vitro. An elevated methemoglobin concentration can be indicative of exposure to these and/or other toxicants.
The normal methemoglobin concentration is less than 1% in normal, living persons. Shortly after blood is drawn from a living person, or after a person dies, the methemoglobin enzymatic reduction systems become disrupted and the hemoglobin iron starts moving towards a new equilibrium between the ferrous and the ferric state, i.e., from normal Fe
2+
hemoglobin toward Fe
3+
methemoglobin. In less than one hour after blood collection, the methemoglobin concentration can become spontaneously elevated to a level which is above the biological exposure limit of 1.5%, resulting in an erroneous interpretation of excessive toxicant exposure. In forensic cases, spontaneous bacterial or other thanatologic causes can not only produce apparently “toxic” levels of methemoglobin, but can also result in lowering causally significant levels of methemoglobin, thus rendering those levels misinterpretable.
The ability to accurately measure methemoglobin is presently limited to a narrow time window after sample collection via venipuncture or other collection methods, due to the spontaneous oxidation of hemoglobin to methemoglobin. What is needed is a quantitative methemoglobin analysis method wherein the time from blood collection to analysis may be extended without the risk of spontaneous methemoglobin production in the blood sample, which would otherwise undesirably increase the actual methemoglobin concentration present at the time of collection.
ABBREVIATIONS AND DEFINITION
By “hemoglobin” is meant the oxygen-carrying heme protein found in red blood cells consisting of two pairs of polypeptide chains and an iron-containing heme group. Unless indicated otherwise, “hemoglobin” shall mean normal hemoglobin, that is, the form of the protein which contains iron in the ferrous, that is, the Fe
2+
, valence state.
By “methemoglobin” is meant the form of hemoglobin in which the contained iron is oxidized to the ferric state, that is, the Fe
3+
, valence state.
By “oxyhemoglobin” is meant hemoglobin which contains bound oxygen, reversible bound to the iron in the heme group.
By “carboxyhemoglobin” is meant hemoglobin which contains bound carbon monoxide in lieu of oxygen. Carboxyhemoglobin may be generated by contacting hemoglobin with carbon monoxide.
By “cyanmethemoglobin” is meant methemoglobin which contains bound cyanide ions.
By “deoxyhemoglobin” is meant hemoglobin which does not contain bound oxygen. It is also known as “reduced hemoglobin”.
By “erythrocytolysis agent” is meant an agent capable of lysing erythrocytes.
By “total hemoglobin”, unless indicated otherwise, is meant the total of all forms of hemoglobin in a blood sample, comprising all of the above forms, and any other hemoglobin forms which may be present in a sample.
By “substantially convert” with respect to the conversion of one hemoglobin form present in a sample to another hemoglobin form is meant that at least about 90% of the initial form in the sample is converted to the other form. The degree of conversion is more preferably at least about 95%, most preferably at least about 99%.
By “fully oxygenated blood” is meant whole blood which has been purged with oxygen to the extent that essentially all of the hemoglobin contained therein is converted to oxyhemoglobin.
SUMMARY OF THE INVENTION
According to the present invention, a buffer composition is provided for stabilizing blood samples for methemoglobin determination. The buffer composition comprises:
(a) carbon monoxide-containing water;
(b) a tetraborate salt selected from the group consisting of sodium tetraborate, potassium tetraborate and combinations thereof; and
(c) a cyanide compound selected from the group consisting of KCN, NaCN and combinations thereof. The buffer composition is added to a blood sample comprising lysed erythrocytes. Lysis releases the hemoglobin contained in the erythrocytes for analysis according to the present invention.
According to another embodiment of the invention, a method for preparing a buffer composition for stabilizing blood samples for methemoglobin determination is provided comprising dissolving in carbon monoxide-containing water the aforesaid tetraborate salt and cyanide compound. The carbon monoxide-containing water is preferably carbon monoxide-saturated water.
According to another embodiment, the invention comprises, in combination, a resealable spectrophotometer cuvette suitable for conducting spectrophotometric analysis over the wavelength range 500 nm to 650 nm, and a volume of the buffer composition contained in the cuvette.
According to another embodiment of the invention, a buffered blood preparation is provided comprising a blood sample and an amount of the buffer composition sufficient to substantially convert methemoglobin in the blood sample to cyanmethemoglobin. The solution contains a tetraborate salt selected from the group consisting of sodium tetraborate, potassium tetraborate and combinations thereof.
In yet another embodiment, the invention is a method of stabilizing blood for determination of methemoglobin content. The blood is treated with a carbon monoxide-saturated water solution providing cyanide ions in an amount sufficient to substantially convert methemoglobin in the blood sample to cyanmethemoglobin.
In another embodiment, the invention is a method for determining the percent methemoglobin content of a blood sample comprising:
(a) substantially converting oxyhemoglobin and deoxyhemoglobin in the sample to carboxyhemoglobin, and substantially converting methemoglobin in the sample to cyanmethemoglobin;
(b) measuring the spectrophotometric absorbance of the sample at wavelengths 528.3 nm (A
528.3
) and 557.8 nm (A
557.8
);
(c) determining the amount of total hemoglobin in the sample; and
(d) computing the percent methemoglobin according to the relationship:
PercentMethemoglobin
=
(
A
528.3
⁢
 
-
A
557.8
TotalHemoglobin
)
⁢
(
1000
)
Oxyhemoglobin and deoxyhemoglobin in the blood sample are substantially converted to carboxyhemoglobin, and methemoglobin in the sample is substantially converted to cyanmethemoglobin. This may be achieved by dissolving the sample in a carbon monoxide-containing water buffer solution providing cyanide ions in an amount sufficient to substantially convert the methemoglobin in the blood sample to cyanmethemoglobin.
These and other embodiments of the invention are apparent from consideration of the following detailed description.


REFERENCES:
patent: 4013417 (1977-03-01), Raffaele
patent: 4436820 (1984-03-01), Reiter
patent: 5051353 (1991-09-01), Stratton et al.
patent: 5352773 (1994-10-01), Kandler et al.
patent: 5476764 (1995-12-01), Bitensky
patent: 5692503 (1997-12-01), Kuenstner
patent: 5858794 (1999-01-01), Malin
patent: 5961469 (1999-10-01), Roizen et al.
patent: WO 97/39027 (1997-10-01), None
Sakata et al.Clinical Chemistry,vol. 28, No. 3, pp. 508-511, 1982.
Rossi-Bernardi et al.Clinical Chemistry,vol

Affiliated with

Also associated with

Rating

Blood methemoglobin analysis does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Blood methemoglobin analysis, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Blood methemoglobin analysis will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2613899