Protein glycation in diabetes mellitus.

Diabetes mellitus is the ninth leading cause of mortality worldwide. It is a complex disease that manifests as chronic hyperglycemia. Glucose exposure causes biochemical changes at the proteome level as reflected in accumulation of glycated proteins. A prominent example is hemoglobin A1c (HbA1c), a glycated protein widely accepted as a diabetic indicator. Another emerging biomarker is glycated albumin which has demonstrated utility in situations where HbA1c cannot be used. Other proteins undergo glycation as well thus impacting cellular function, transport and immune response. Accordingly, these glycated counterparts may serve as predictors for diabetic complications and thus warrant further inquiry. Fortunately, modern proteomics has provided unique analytic capability to enable improved and more comprehensive exploration of glycating agents and glycated proteins. This review broadly covers topics from epidemiology of diabetes to modern analytical tools such as mass spectrometry to facilitate a better understanding of diabetes pathophysiology. This serves as an attempt to connect clinically relevant questions with findings of recent proteomic studies to suggest future avenues of diabetes research.

Comprehensive profiling and kinetic studies of glycated lysine residues in human serum albumin.

Lysine residues of proteins slowly react with glucose forming Amadori products. In hyperglycemic conditions, such as diabetes mellitus, this non-enzymatic glycation becomes more pervasive causing severe medical complications. The structure and conformation of a protein predisposes lysine sites to differing reactivity influenced by their steric availability and amino acid microenvironment. The goal of our study was to identify these sites in albumin and measure glycation affinities of lysine residues. We applied a bottom-up approach utilizing a combination of three LC-MS instruments: timsTOF, Orbitrap, and QTRAP. To prove applicability to samples of varying glycemic status, we compared in vitro glycated and non-glycated HSA, as well as diabetic and non-diabetic individual samples. The analysis of lysine glycation affinities based on peptide intensities provide a semi-quantitative approach, as the results depend on the mass spectrometry platform used. We found that glycation levels based on multiple reaction monitoring (MRM) quantitation better reflect individual glycemic status and that the glycation percentage for each site is in linear relation to all other sites. To develop an approach which more accurately reflects glycation affinity, we developed a kinetics model which uses results from stable isotope dilution HPLC-MRM methodology. Through glycation of albumin at different glucose concentrations, we determine the rate constants of glycation for every lysine residue by simultaneous comparative analysis.

Higher degree of glycation of hemoglobin S compared to hemoglobin A measured by mass spectrometry: Potential impact on HbA1c testing.

BACKGROUND: Glycated hemoglobin (GHb), reported as HbA1c, is used as marker of long-term glycemia for diabetic patients. HbA1c results from boronate affinity methods are generally considered to be unaffected by most hemoglobin variants; this assumes comparable glycation of variant and non-variant (HbAA) hemoglobins. In this report, glycation of HbA beta chain (ßA) and HbS beta chain (ßS) for the most common Hb variant trait (HbAS) are examined. METHODS: We analyzed 41 blood samples from subjects with HbAS, both with and without diabetes. Using LC-MS, ratios of glycated HbS to glycated HbA were determined by comparison of areas under the curves from extracted ion chromatograms. RESULTS: Glycation of ßS chains was significantly higher (p<0.001) than ßA chains; this difference was consistent across subjects. Total (α+ß) glycated HbAS was theoretically estimated to be ~5% higher than glycated HbAA. CONCLUSION: This novel mass-spectrometric approach described allows for relative quantification of glycated forms of ßS and ßA. Although ßS glycation was significantly higher than that of ßA, the difference in total glycation of HbAS versus HbAA was smaller and unlikely to impact clinical interpretation of boronate affinity HbA1c results. These data support the continued use of boronate affinity to measure HbA1c in patients with HbAS.

Two-step ion-exchange chromatographic purification combined with reversed-phase chromatography to isolate C-peptide for mass spectrometric analysis.

A liquid chromatography with mass spectrometry on-line platform that includes the orthogonal techniques of ion exchange and reversed phase chromatography is applied for C-peptide analysis. Additional improvement is achieved by the subsequent application of cation- and anion-exchange purification steps that allow for isolating components that have their isoelectric points in a narrow pH range before final reversed-phase mass spectrometry analysis. The utility of this approach for isolating fractions in the desired "pI window" for profiling complex mixtures is discussed.

Isotope dilution assay in peptide quantification: the challenge of microheterogeneity of internal standard.

Isotope dilution analysis allows quantitation of elements and different compounds in complex mixtures. The quantitation is based on a known amount of reference material (internal standard, IS) added to a sample that makes the result critically dependent on the value assigned to the standard. In the case of peptides, IS concentration is determined by nitrogen and amino acid analysis while purity is normally assessed by methods such as chromatography or electrophoresis that might not be able to detect many possible amino acid modifications, either naturally occurring or chemically induced. Microheterogeneity of the IS, if it is not accounted for when assigning a reference value to the standard, results in highly overestimated values in target analyte quantitation. In this viewpoint article, we illustrate the problem of internal standard microheterogeneity by analyzing synthetic human C-peptide labeled analogs.

Measurement of Hba(1C) in patients with chronic renal failure.

BACKGROUND: Carbamylated hemoglobin (carbHb) is reported to interfere with measurement and interpretation of HbA(1c) in diabetic patients with chronic renal failure (CRF). There is also concern that HbA1c may give low results in these patients due to shortened erythrocyte survival. METHODS: We evaluated the effect of carbHb on HbA(1c) measurements and compared HbA(1c) with glycated albumin (GA) in patients with and without renal disease to test if CRF causes clinically significant bias in HbA(1c) results by using 11 assay methods. Subjects included those with and without renal failure and diabetes. Each subject's estimated glomerular filtration rate (eGFR) was used to determine the presence and degree of the renal disease. A multiple regression model was used to determine if the relationship between HbA(1c) results obtained from each test method and the comparative method was significantly (p<0.05) affected by eGFR. These methods were further evaluated for clinical significance by using the difference between the eGRF quartiles of >7% at 6 or 9% HbA(1c). The relationship between HbA(1c) and glycated albumin (GA) in patients with and without renal failure was also compared. RESULTS: Some methods showed small but statistically significant effects of eGFR; none of these differences were clinically significant. If GA is assumed to better reflect glycemic control, then HbA(1c) was approximately 1.5% HbA(1c) lower in patients with renal failure. CONCLUSIONS: Although most methods can measure HbA(1c) accurately in patients with renal failure, healthcare providers must interpret these test results cautiously in these patients due to the propensity for shortened erythrocyte survival in renal failure.

Human C-peptide Quantitation by LC-MS Isotope-Dilution Assay in Serum or Urine Samples.

In this communication we report a simple and efficient approach to C-peptide quantitation using isotope dilution mass-spectrometry analysis. The method facilitates quantitation of C-peptide levels at least one order of magnitude lower compared to concentration levels achieved with an IDA method reported previously. The improvement was due to more intensive sample preparation procedure that, in turn, makes it possible to increase the sample load without a corresponding increase in matrix effects. We also show the results of a comparison study with a second laboratory using a similar previously reported method for C-peptide quantitation.

Use of cation exchange chromatography for human C-peptide isotope dilution - mass spectrometric assay.

An application of ion exchange chromatography for C-peptide analysis is described here. At the stage of C-peptide isolation, a strong cation exchanger (SP HP or MonoS) was used to purify the analyte from ballast proteins and peptides. The conditions of ion-exchange chromatographic separations were optimized using theoretical modeling of the net surface electric charge of the peptide as a function of pH. The purified and concentrated sample was further subjected to LC-MS/MS. In order to improve the reliability of analysis, two fragment ions were monitored simultaneously both for native C-peptide and internal standard, isotopically labeled C-peptides analogues (fragments with m/z of 927.7 and 147.2). Using ion-exchange chromatography, it became possible to process larger sample volumes, important for testing patients with very low C peptide levels, compared to currently used solid phase extraction methods.

Effects of hemoglobin (Hb) E and HbD traits on measurements of glycated Hb (HbA1c) by 23 methods.

BACKGROUND: Glycohemoglobin (GHB), reported as hemoglobin (Hb) A(1c), is a marker of long-term glycemic control in patients with diabetes and is directly related to risk for diabetic complications. HbE and HbD are the second and fourth most common Hb variants worldwide. We investigated the accuracy of HbA(1c) measurement in the presence of HbE and/or HbD traits. METHODS: We evaluated 23 HbA(1c) methods; 9 were immunoassay methods, 10 were ion-exchange HPLC methods, and 4 were capillary electrophoresis, affinity chromatography, or enzymatic methods. An overall test of coincidence of 2 least-squares linear regression lines was performed to determine whether the presence of HbE or HbD traits caused a statistically significant difference from HbAA results relative to the boronate affinity HPLC comparative method. Deming regression analysis was performed to determine whether the presence of these traits produced a clinically significant effect on HbA(1c) results with the use of +/-10% relative bias at 6% and 9% HbA(1c) as evaluation limits. RESULTS: Statistically significant differences were found in more than half of the methods tested. Only 22% and 13% showed clinically significant interference for HbE and HbD traits, respectively. CONCLUSIONS: Some current HbA(1c) methods show clinically significant interferences with samples containing HbE or HbD traits. To avoid reporting of inaccurate results, ion-exchange chromatograms must be carefully examined to identify possible interference from these Hb variants. For some methods, manufacturers' instructions do not provide adequate information for making correct decisions about reporting results.

The effect of elevated fetal hemoglobin on hemoglobin A1c results: five common hemoglobin A1c methods compared with the IFCC reference method.

Hemoglobin A1c (HbA1c) is an important indicator of risk for complications in patients with diabetes mellitus. Elevated fetal hemoglobin (HbF) levels have been reported to interfere with results of some HbA1c methods, but it has generally been assumed that HbA1c results from boronate-affinity methods are not affected by elevated HbF levels. None of the previous studies used the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference method as the comparative HbA1c method. We, therefore, measured HbA1c in samples with normal and elevated HbF levels by several common assay methods and compared the results with those of the IFCC reference method.HbF levels of more than 20% artificially lowered HbA1c results from the Primus CLC 330/385 (Primus Diagnostics, Kansas City, MO), Siemens DCA2000 (Siemens Healthcare Diagnostics, Tarrytown, NY), and Tosoh 2.2+ (Tosoh Bioscience, South San Francisco, CA), but not the Bio-Rad Variant II (Bio-Rad Laboratories, Hercules, CA) and Tosoh G7. Physicians and laboratory professionals need to be aware of potential interference from elevated HbF levels that could affect HbA1c results, including those from boronate-affinity methods.

Effects of sample storage conditions on glycated hemoglobin measurement: evaluation of five different high performance liquid chromatography methods.

BACKGROUND: Glycated hemoglobin, reported as hemoglobin A1c (HbA1c), is widely used as a measure of long-term glycemic control in patients with diabetes. The accuracy of measurements depends in part on proper storage of the sample prior to analysis. METHODS: Three whole blood (WB) samples at three HbA1c levels were collected and stored at -70 degrees C, -20 degrees C, 4 degrees C, room temperature (17-23 degrees C), and 37 degrees C. One aliquot from each temperature was analyzed by each method on days 1, 2, 3, 6, 7, 10, 14, 21, 28, and 57. RESULTS: The Primus CLC (385 and 330) (Primus Corp., Kansas City, MO) showed stability of WB at -20 degrees C and 4 degrees C for 57 days, room temperature for 14 days, and 37 degrees C for 1 day. The Tosoh 2.2 Plus (Tosoh Bioscience, Inc., South San Francisco, CA) showed stability at -20 degrees C for 3 days, 4 degrees C for 14 days, room temperature for 3 days, and 37 degrees C for less than 24 h. With the Tosoh G7, results were acceptable at -20 degrees C for 10 days, 4 degrees C for 57 days, room temperature for 7 days, and 37 degrees C for less than 24 h. The Bio-Rad Variant (Bio-Rad Laboratories, Hercules, CA) showed stability at -20 degrees C for 6 days, 4 degrees C for 14 days, room temperature for 3 days, and 37 degrees C for less than 24 h. The Bio-Rad Variant II showed stability at -20 degrees C for 28 days, 4 degrees C for 57 days, room temperature for 7 days, and 37 degrees C for less than 24 h. CONCLUSIONS: All methods either met or exceeded manufacturers' claims for stability. The CLC 385/330, Tosoh G7, and Bio-Rad Variant II high performance liquid chromatography methods showed better stability than the Tosoh 2.2 Plus and Bio-Rad Variant.