Rapid Screening And Evaluation Of Diabetes And Prediabetes By Glycated Hemoglobin Mass Spectrometry

Abstract

The method describes rapid screening of whole blood samples, pin prick and blood spot cards, subjected to MALDI-ToF Mass spectrometry. The spectra is generated and compared to those from normal healthy controls. Characteristic spectra are indicative of the presence of a hemoglobinopathy and the method can be used to screen/diagnose all sickle cell diseases, alpha and beta Thalassemias.

Claims

1. A method of detecting prediabetes or diabetes comprising subjecting a blood sample obtained from a subject to direct mass spectral analysis and determining the proportion of glycated hemoglobin (Hb) present in the sample.

2. The method according to claim 1 wherein determining the proportion of glycated hemoglobin present comprises measuring (i) the levels of glycated α globin and unglycated α globin and/or (ii) the levels of glycated β globin and unglycated β globin.

3. The method according to claim 1, wherein the blood sample is lysed prior to said direct mass spectral analysis.

4. The method according to claim 1, wherein the blood sample is diluted prior to direct mass spectral analysis.

5. The method according to claim 1, wherein the sample subjected to direct mass spectral analysis is not processed other than lysis and/or dilution.

6. The method according to claim 1, wherein the spectra obtained from the direct mass spectral analysis is normalized.

7. The method according to claim 1, wherein the proportion of glycated hemoglobin is calculated as the percentage glycated hemoglobin.

8. The method according to claim 1 wherein the spectra obtained from the direct mass spectral analysis is examined in the range 7,000 to 16,500 m/z.

9. The method according to claim 1, wherein determining the proportion of glycated hemoglobin comprises measuring the levels of the singly charged hemoglobin or globin molecules.

10. The method according to claim 9, wherein the spectra obtained from the direct mass spectral analysis is examined in the range 15,000 to 16,200 m/z.

11. The method according to claim 1, wherein determining the proportion of glycated hemoglobin comprises measuring the levels of the doubly charged hemoglobin or globin molecules.

12. The A method according to claim 11, wherein the spectra obtained from the direct mass spectral analysis is examined in the range 6,000 to 8,100 m/z.

13. The method according to claim 1, wherein the amount of glycated Hb and unglycated Hb is measured using the total area under the curve of the mass spectra obtained from the direct mass spectral analysis.

14. The method according to claim 1, wherein the amount of glycated Hb and unglycated Hb is measured using the relative peak height of the mass spectra obtained from the direct mass spectral analysis.

15. The method according to claim 1, wherein a percentage of glycated α-globin between 3-4% and/or a percentage of glycated β-globin between 3-6% is indicative of pre-diabetes.

16. The method according to claim 1, wherein a percentage glycated α globin >4% and/or a percentage glycated β-globin >6% is indicative of diabetes.

17. The method according to claim 1, wherein the mass spectral analysis carried out is matrix-assisted laser desorption/ionization spectrometry (MALDI).

18. The method according to claim 17, wherein the MALDI spectrometry is time-of-flight mass spectrometry (MALD-ToF MS).

Description

[0056] The application will now be described in the examples below which refer to the following figures:

[0057] FIG. 1 shows a spectra of normal blood in a 12 year old male

[0058] FIG. 2 shows a spectra of normal blood in an obese 52 year old male

[0059] FIG. 3 shows a spectra of blood in a patient with sickle cell disease HbSC

[0060] FIG. 4 shows an overlay of the 12 year old boy and 52 year old male blood spectra.

[0061] FIG. 5—A table of calculated relative amounts of glycated α-globin and β-globin and other globin molecules in 9 adults and 7 patients with various hemoglobinopathies

EXAMPLE 1

[0062] Method

[0063] Sample Processing

[0064] The optimal dilution for whole blood or dried blood spot is between 1/1000 and 1/2000 in either ddH.sub.2O or 0.1% TFA in ddH.sub.2O after an initial lysis of sample with ddH.sub.2O (1:1 v/v). This dilutional step effectively purifies the Hb from other components of blood for mass spectral analysis as Hb is the most abundant protein. In addition the dilution in ddH.sub.2O (of 0.1% TFA/ddH.sub.2O) dissociates the constituent globin proteins for resolved analysis by MALDI-ToF Mass spectrometry.

[0065] Dilutions higher than 1/8000 results in progressively weaker mass spectral signal.

[0066] MALDI ToF Mass Spectral Analysis

[0067] The optimal matrices are sinnapinic acid (SA), ferulic acid (FA) and alpha 4-cyano hydroxycinnamic acid (CHCA). Sinapinic acid being the preferred matrix mixed or as pre-coating layer to a mixed drop of 1/1000 to 1/8000 diluted sample (optimal 1/2000).

[0068] Steel MALDI plates (384 wells) were prepared by pipetting 0.5 μl of matrix solution (sinapinic acid—20 mg/ml dissolved in 50/50 v/v acetonitrile (ACN)/ddH.sub.2O and 0.1% trifluoacetic acid (TFA)) and allowed to dry. 0.5 μl of sample, was mixed with SA and spotted on the dry matrix. This was allowed to dry at room temperature for 1 hour before MALDI TOF MS analysis.

[0069] The mass spectrometric analysis was carried out using a Shimadzu Axima plus MALDI mass spectrometer: the pulse nitrogen laser (λ.sub.max=337 nm), was fired at 75 to 80% arbitrary units of power. The ions were accelerated by a 20 kV electrical field down a 1.2 m linear tube and detected by a micro-channel plate detector at a sampling rate of 500 MHz. Spectra were generated by summing 20-30 laser shots. A positive linear mode with delayed extraction was used in order to acquire the spectra.

[0070] The instrument was internally calibrated whereby a 1/1000 diluted sample of blood was spiked with 10 pmoles/ul Cytochrome C (1:2, v/v) The two points calibration generated was at [M+H]+=12 361 m/z and [M+2H]2+=6181 m/z

[0071] A mass spectral region of between 6,000 and 17,000 m/z was collected and analyzed and in particular the range of 7500 to 8200 m/z examined for doubly charged globin proteins.

[0072] These are characterized both in respect to centroid mass assignment and relative peak intensity either as comparative normalized peak height or normalized peak area in the spectral range examined.

[0073] Results

[0074] FIG. 1 spectra of blood globins from a boy of 12 years.

[0075] A blood sample from a normal male child of 12 years demonstrated a spectra with clearly evident peaks corresponding to α and β globin and corresponding matrix (sinnapinic acid—SA) adducts (see FIG. 1 and table 1). Other globin adducts and other globins (δ, Gγ, Aγ and ε) are barely detected (see table 1). Furthermore glycated α and β-globin orthologs were barely visible (indicated in FIG. 1) at <1% of the parent globin.

[0076] FIG. 2 spectra of blood globins from a 52 year old man.

[0077] A normal but obese adult blood sample reveals peaks for α-globin and β-globin along with their associated SA adducts. In addition elevated δ-globin is noted along with other α and β-globin adducts, whilst other globins (Gγ, Aγ and ε) are barely detected (see FIG. 2 and table 1). The blood sample revealed glycated α-globin and β-globin peaks representing 3% and 5% of the parent globins.

[0078] FIG. 3 spectra of blood globins from a patient with Hb SC—Sickle cell diseases.

[0079] Blood sample from a patient with sickle cell disease (HbSC) FIG. 3 revealed normal and glycated α-globin peaks and a peak for .sup.Sβ at 7920 m/z clearly resolved from β-globin at 7934 m/z and .sup.Cβ approx. 7933 m/z. Baseline elevation of δ and Gγ globins at 7965 and 7996 m/z was evident as was a new peak at 8023 m/z. The blood sample revealed normal and glycated α-globin peaks and the % comparison of intensity was 1.8% whilst the glycated β-globin was 6.3% of the parent β-globin.

[0080] FIG. 4 Comparison of blood globins of a 52 year old male and a 12 year old boy.

[0081] Superimposing the 52 year old adult male blood spectra over the spectra from the 12 year old boy's, illustrates the elevation in circulating glycated α and β globins in the adult sample (FIG. 4).

[0082] FIG. 5 Tabulated comparison of 9 adult spectra peak intensities with that from 7 assorted hemoglobinopathy patients.

[0083] Glycated α-chain was detected in all samples and was clearly resolved in phenotypically normal samples and those with hemoglobinopathies, including sickle cell disease. Glycated β-globin was clearly distinguishable in all normal samples, but compromised by fetal globin expression which was generally elevated in all hemoglobinopathies (FIG. 5). Expressed as a percentage of the α-globin intensity, the glycated ortholog represented between 0.5 and 3% (mean 1.78%, SD 0.79%) for phenotypic normal adults; and between 1 and 2.7% (mean 2.1, SD 0.57%) for those with hemoglobinopathies, including carriers. Expressed as a percentage of the β-globin intensity, the glycated ortholog represented between 2 and 5.2% (mean 3.15%, SD 1.07%) for phenotypic normal adults; and between 0 and 9.1% (mean 5.1%, SD 2.78%) for those with hemoglobinopathies including carriers (see FIG. 5).

[0084] Discussion

[0085] Elevated Glycation of Hemoglobin is associated with poor regulation of blood glucose. HbA1c was first identified as a chromatographic fraction of Hemoglobin in 1971 and characterized as a measure of the beta-N-1-deoxy fructosyl component of hemoglobin. Normal levels of glucose produce a normal amount of glycated hemoglobin. As the average amount of plasma glucose increases, the fraction of glycated hemoglobin increases in a predictable way. This serves as a marker for average blood glucose levels over the previous 3 months prior to the measurement as this is the half-life of red blood cells.

[0086] In diabetes mellitus, higher amounts of glycated hemoglobin, indicating poorer control of blood glucose levels, have been associated with cardiovascular disease, nephropathy, and retinopathy. Monitoring HbA1c in type 1 diabetic patients has been adopted as a measure to improve outcomes. Several methods to measure “HbA1c” including HPLC, immunoassay and capillary electrophoresis are in clinical use. Largely regarded as a measure of β-globin glycation, its measurement is compromised by fetal β-like globin expression and with β-globin gene mutations such as in sickle cell disease and beta-thalassemia, and carriers of such mutation. However, elevated glycation has recently been shown to be associated with such hemoglobinopathies and may reflect an increased chemical susceptibility to glycation in such affected blood cells.

[0087] The present method clearly resolves the glycated forms of α- and β-globin from each other and their respective non-glycated parents. This gives a finer discrimination of the glycation of hemoglobin and is not compromised by the presence of a mutation or aberrant globin gene expression that adversely affects other methods to measure Hb glycation.

[0088] Conclusion

[0089] MALDI-Tof MS spectral analysis of drop or dried spot whole blood, following 1/2000 dilution in ddH2O reveals resolved globin proteins and clear resolution of glycated orthologo of α and β globins. Comparison of signal intensity of the glycated α-globin peak to the parent α-globin peak represents a rapid and economic screening and monitoring testing method for diabetes and pre-diabetes.

TABLE-US-00001 TABLE 1 Identification of Globins - The best resolution was achieved in the m/z range 7500 to 8100 corresponding to [M = 2H].sup.2+ ions. Peak assignment to Globin chains M/Z of [M = 2H].sup.2+ A 7564 m/z, ±5 m/z Acetyl and Carbonyl adducts of α-globin 7594 m/z, ±5 m/z Glycated α 7645 m/z, ±5 m/z Matrix (SA) adduct of α-globin 7671 m/z, ±5 m/z .sup.sβ 7921 m/z B 7936 m/z, ±5 m/z Δ 7965 m/z, ±5 m/z Gγ 7996 m/z, ±5 m/z Aγ 8005 m/z, ±5 m/z Glycated β 8017 m/z, ±5 m/z Matrix (SA) adduct of β-globin 8039 m/z ±5 m/z Marker 8088 m/z possibly ε-globin 8088 m/z ±5 m/z