Labile glycated haemoglobin and carbamylated haemoglobin are still critical points for HbA1c measurement.

INTRODUCTION: Haemoglobin A1c (HbA1c) is a key analyte for the monitoring of glycemic balance in diabetic patients and is used for diabetes diagnosis in many countries. The potential interference of carbamylated haemoglobin (cHb) and labile glycated haemoglobin (LA1c) on HbA1c assays must remain a matter of vigilance. Such a situation has occurred in our laboratory with a kit replacement on the Bio-Rad Variant™ II testing system, a cation-exchange high performance liquid chromatography (HPLC) system. With this method, LA1c and cHb coeluted in a same peak which may have different consequences on HbA1c values. MATERIALS AND METHODS: The influence of increasing LA1c and cHb values on HbA1c results was studied with in vitro glycation and carbamylation of samples. Samples from patients with high and normal blood urea concentrations were assayed by HPLC and immunological assay. RESULTS: We observed that the degree of interference greatly varied depending on the nature of the interfering Hb fractions found under the so-called "LA1c peak". Thus, we have decided to apply a decision tree using "LA1c" thresholds depending on: (i) the retention time, (ii) the shape of the peak, (iii) other analytes, like urea. If the peak recognized as "LA1c" is mainly formed by LA1c, we consider that there is no interference until 4%. If the peak is mainly formed by cHb, we consider an interference threshold equal to 2%. CONCLUSIONS: This situation reminds that cHb and LA1c remain critical issues in chromatography-based HbA1c assays and that adapted criteria must be set up for result interpretation.

Homocitrulline as marker of protein carbamylation in hemodialyzed patients.

BACKGROUND: Homocitrulline (HCit) is a carbamylation-derived product (CDP) that has been identified as a valuable biomarker of morbidity and mortality in patients with chronic kidney disease (CKD). The aim of this study was to determine whether initiation of hemodialysis therapy (HD) could induce variations of HCit concentrations in CKD patients. METHODS: Serum HCit concentrations were determined by LC-MS/MS in CKD patients (n=108) just before (M0) and six months (M6) after the initiation of HD therapy. RESULTS: Mean HCit concentrations reached 1000µmol/mol Lysine before initiation of HD therapy and decreased by 50% within 6months after HD onset. HCit concentrations remained stable over time as assessed during a 24-months follow-up period. HCit was mostly found in its protein-bound form in HD patients. HCit concentrations obtained at M0 were positively correlated with urea (r=0.58) and carbamylated hemoglobin (r=0.41), and are likely to be promising predictive markers of mortality. However, no correlations were found between HCit concentrations and Kt/V values, suggesting that HCit is not a marker of HD efficiency. CONCLUSION: HCit concentrations reflect the intensity of protein carbamylation and are stable over time during HD treatment, making HCit a reliable biomarker in the follow-up of CKD patients.

Simple accurate mathematical models of blood HbO2 and HbCO2 dissociation curves at varied physiological conditions: evaluation and comparison with other models.

PURPOSE: Equations for blood oxyhemoglobin (HbO2) and carbaminohemoglobin (HbCO2) dissociation curves that incorporate nonlinear biochemical interactions of oxygen and carbon dioxide with hemoglobin (Hb), covering a wide range of physiological conditions, are crucial for a number of practical applications. These include the development of physiologically-based computational models of alveolar-blood and blood-tissue O2­CO2 transport, exchange, and metabolism, and the analysis of clinical and in vitro data. METHODS AND RESULTS: To this end, we have revisited, simplified, and extended our previous models of blood HbO2 and HbCO2 dissociation curves (Dash and Bassingthwaighte, Ann Biomed Eng 38:1683­1701, 2010), validated wherever possible by available experimental data, so that the models now accurately fit the low HbO2 saturation (SHbO2) range over a wide range of values of PCO2, pH, 2,3-DPG, and temperature. Our new equations incorporate a novel PO2-dependent variable cooperativity hypothesis for the binding of O2 to Hb, and a new equation for P50 of O2 that provides accurate shifts in the HbO2 and HbCO2 dissociation curves over a wide range of physiological conditions. The accuracy and efficiency of these equations in computing PO2 and PCO2 from the SHbO2 and SHbCO2 levels using simple iterative numerical schemes that give rapid convergence is a significant advantage over alternative SHbO2 and SHbCO2 models. CONCLUSION: The new SHbO2 and SHbCO2 models have significant computational modeling implications as they provide high accuracy under non-physiological conditions, such as ischemia and reperfusion, extremes in gas concentrations, high altitudes, and extreme temperatures.

Impact of carbamylation on three different methods, HPLC, capillary electrophoresis and TINIA of measuring HbA1c levels in patients with kidney disease.

AIMS: Aim of this study was to investigate whether established IFCC traceable routine measurements of HbA1c levels in patients with kidney diseases result in comparable and valid results. Additionally, the influence of carbamylation as a marker of uremia on the measurement methods was assessed. METHODS: We compared three different measurement methods (high-performance liquid chromatography (HPLC), capillary electrophoresis and turbidimetric inhibition immunoassay (TINIA)) based on 407 nephrology samples for HbA1c determination with specific analysis of the threshold areas where different HbA1c measurements could result in different diagnoses (diabetes mellitus vs prediabetes and prediabetes vs non-diabetes). Indirectly, a potential effect of carbamylated hemoglobin was assessed based on determination of BUN levels in serum. RESULTS: In nephrological samples, we were able to show that three different measurement methods provide similar results regarding HbA1c in routine diagnostics. Our results show that with BUN concentrations <80 mg/dl and ≥80 mg/dl, similar HbA1c levels were determined, independent of measurement method. CONCLUSION/DISCUSSION: While routine measurements follow IFCC standards, many interfering factors remain which can influence HbA1c determination, e.g. carbamylation of proteins. As kidney disease is the most common complication in patients with diabetes mellitus, interference in the measurement of HbA1c due to carbamylated proteins as markers of uremia must be recognized and if necessary corrected. We found not only a weak influence of carbamylation on all methods of HbA1c determination, but also that, in nephrological samples, the three different measurement methods provided similar results regarding HbA1c in routine diagnostics.

Evaluation of the In2it analyzer for HbA1c determination.

AIM: The prominent role of HbA(1c) in the follow-up of glycaemic balance in patients with diabetes mellitus necessitates the use of robust and reliable methods of assay. The purpose of this study was to evaluate the In2it analyzer, a new device allowing HbA(1c) evaluation within 10 min, using 10 microL of blood, in the laboratory or clinical unit, using an affinity-based method. METHODS: The analytical performance of the In2it analyzer was tested for precision, interference and linearity, and correlated with two other analyzers - the high-performance liquid chromatography (HPLC)-based Variant II analyzer in a laboratory, and the immunology-based DCA 2000 analyzer in a clinical unit - for practicability and its compliance with good laboratory practices. RESULTS: HbA(1c) assay is linear from 4 to 14%, with coefficients of variations ranging from 2.4 to 3.9%. In2it correlation was satisfactory with both the HPLC Variant II (r(2)=0.974, P<0.001) and DCA 2000 (r(2)=0.794, P<0.001) analyzers although, with the latter, unpredictable differences were randomly observed. However, the method is free of interference from common haemoglobin variants, labile glycated haemoglobin and carbamylated haemoglobin, hyperbilirubinaemia (<520 micromol/L) and hypertriglyceridaemia (<6 mmol/L). The practicability of the analyzer is good. However, software specifications need to be upgraded, especially for quality-control management, traceability of results and data safety. CONCLUSION: The In2it analyzer is suitable for HbA(1c) assay in small laboratory series and for point-of-care testing, and its analytical performance is satisfactory overall. However, several issues related to software need to be improved for optimal application. Also, special attention should be paid concerning the possibility of underestimation of results in cases of high hypertriglyceridaemia.

Solution 1H NMR study of the active site molecular structure and magnetic properties of the cyanomet complex of the isolated alpha-chain from human hemoglobin A.

The solution electronic and molecular structure for the heme pocket of the cyanomet complex of the isolated alpha-chain of human adult hemoglobin (HbA) has been investigated by homonuclear two-dimensional 1H NMR in order to establish an assignment protocol for the dimeric chain that will guide similar assignments in the intact, heterotetrameric HbA complex, and to compare the structures of the alpha-chain with its subunit in HbA. The target residues are those that exhibit significant (>0.2 ppm) dipolar shifts, as predicted by a "preliminary" set of magnetic axes determined from a small set of easily assigned active site residues. All 97 target residues (approximately 70% of total) were assigned by taking advantage of the temperature dependence predicted by the "preliminary" magnetic axes for the polypeptide backbone; they include all residues proposed to play a significant role in modulating the ligand affinity in the tetramer HbA. Left unassigned are the A-helix, the end of the G-helix and the beginning of the H-helix where dipolar shifts are less than 0.2 ppm. The complete assignments allow the determination of a robust set of orientation and anisotropies of the paramagnetic susceptibility tensor that leads to quantitative interpretation of the dipolar shifts of the alpha-chain in terms of the crystal coordinates of the alpha-subunit in ligated HbA which, in turn, confirms a largely conserved molecular structure of the isolated alpha-chain relative to that in the intact HbA. The major magnetic axis, which is correlated with the tilt of the Fe-CN unit, is tilted approximately 10 degrees from the heme normal so that the Fe-CN unit is tilted toward the beta-meso-H in a fashion remarkably similar to the Fe-CO tilt in HbACO. It is concluded that a set of "preliminary" magnetic axes and the use of variable temperature two-dimensional NMR spectra are crucial to effective assignments in the cyanomet alpha-chain and that this approach should be similarly effective in HbA.

The role of carbamylated hemoglobin in identifying acute and chronic renal failure.

Carbamylated hemoglobin (CarbHb) levels expressed as valipe hydantoin (VH) were measured by high performance liquid chromatography (HPLC) in patients with acute renal failure (ARF, n=35) and chronic renal failure (CRF, n=39). CarbHb levels in CRF patients were approximately 2.5 times of those in ARF ones (121.2 +/- 8 vs 54.8 +/- 6 microgVH/gHb, p<0.01). CarbHb levels of 80 microgVH/gHb provided the best statistical values (sensitivity of 89% and specificity of 82%). CarbHb/BUN and Carb/Cr ratios were also effective determinants in differentiation between ARF and CRF. CarbHb/BUN ratio of 1.5 and CarbHb/Cr ratio of 20 were the best statistical cut off points. As such, measurement of CarbHb levels could be a reliable non-invasive method in identifying ARF from CRF patients.

Use of capillary electrophoresis to quantitate carbamylated hemoglobin concentrations in dogs with renal failure.

OBJECTIVE: To evaluate quantification of the amount of carbamylated hemoglobin (CarbHb), using capillary electrophoresis (CE) and a new dynamic capillary coating system to separate hemoglobin derivatives, and to assess the use of CarbHb amounts to evaluate long-term urea exposure and differential diagnoses of azotemia in dogs. ANIMALS: 8 dogs with renal failure, 2 dogs with diabetes mellitus, and 7 control dogs. PROCEDURE: Optimal analytic conditions for separation of CarbHb and other hemoglobin derivatives in blood samples obtained from dogs were determined, using a commercial analysis system developed for the detection of glycohemoglobin Hb A1c (GlycHb) in human blood samples. Relative content of hemoglobin derivatives in blood from 10 dogs with renal failure or endocrine diseases were compared with values for 7 dogs without renal or endocrine diseases. RESULTS: Satisfactory resolution of hemoglobin derivatives was obtained, which permitted identification and quantitation of the amount of CarbHb as a percentage of the total amount of hemoglobin. Normal or increased amounts of GlycHb did not interfere with CarbHb analysis. Dogs with chronic renal failure had considerably higher peak amounts of CarbHb than dogs with acute renal failure, a dog with chronic renal failure that was treated by use of hemodialysis, or dogs without renal disease. CONCLUSIONS AND CLINICAL RELEVANCE: Amounts of CarbHb in blood samples obtained from dogs can be readily quantified by use of capillary electrophoresis. Assessment of the amount of CarbHb can be used to facilitate evaluation of the cause of azotemia in dogs.

Carbamylated hemoglobin and carbamylated plasma protein in hemodialyzed patients.

The carbamylation reaction in vivo involves the nonenzymatic, covalent attachment of isocyanic acid, the spontaneous dissociation product of urea, to proteins. Carbamylated proteins have been proposed as markers of uremia and indicators of uremic control. However, the utility of measuring carbamylated proteins has not been investigated adequately. Therefore, this study was done to determine the relationship between the carbamylation of long-lived protein (hemoglobin) with that of short-lived proteins (plasma proteins) in hemodialyzed patients. Significantly higher carbamylated hemoglobin (CHb; 157 +/- 40 microg valine hydantoin/g Hb) and carbamylated protein (CTP; 0.117 +/- 0.011 absorbance/mg protein) concentrations were found in hemodialyzed patients (N = 13) as compared to normal individuals (N = 9, 53 +/- 20 microg valine hydantoin/g Hb and 0.08 +/- 0.01 absorbance/mg protein, respectively). A high correlation was found between CHb and CTP concentrations (r = 0.87, P < 0.0001), demonstrating a strong relationship between these two different half-lived proteins. A six-month longitudinal study of seven hemodialyzed patients showed that the between subject correlations were significant for CHb versus CTP as well as CHb versus pre-dialysis urea. Correlations were not significant for CTP versus pre-dialysis urea or Kt/V, nor CHb versus Kt/V. Carbamylated hemoglobin fluctuated the most over this time period (30.1% +/- 20.2%), pre-dialysis urea and CTP varied less (18.3% +/- 13.4% and 14.9% +/- 7.5%, respectively), and Kt/V varied the least (6.3% +/- 3.3%). Within subject correlations were not significant between any two tests. It is unclear whether the lack of correlations found is real or a function of the small sample size. However, these data do show that CHb and CTP are positively associated and reflect the degree of urea exposure in the blood, but their usefulness for patients on maintenance hemodialysis is not clear.

Use of carbamylated hemoglobin concentration to differentiate acute from chronic renal failure in dogs.

OBJECTIVE: To determine usefulness of carbamylated hemoglobin (CarHb) concentration for differentiation of acute renal failure (ARF) from chronic renal failure (CRF) in dogs. SAMPLE POPULATION: Samples from dogs with ARF or CRF and from nonazotemic control dogs. PROCEDURE: CarHb concentration was determined in heparinized blood samples by measuring the micrograms of valine hydantoin (VH) per gram of hemoglobin (Hb), using a high-performance liquid chromatography assay, in which carbamyl valine is converted to VH via acid hydrolysis. RESULTS: CarHb concentration was significantly higher in dogs with ARF and CRF, compared with values in control dogs (ARF vs control, P < 0.05; CRF vs control, P < 0.001). Furthermore, CarHb concentration was significantly (P < 0.001) higher in dogs with CRF, compared with that in dogs with ARF. Carbamylated hemoglobin concentration did not correlate with serum urea nitrogen or creatinine concentration. Using a cutoff value of 100 micrograms of VH/g of Hb, the sensitivity and specificity of CarHb concentration for differentiating ARF from CRF was 96.1 and 84.2%, respectively. CONCLUSIONS: CarHb concentration was useful in the differentiation of ARF from CRF in the dogs of this study. CLINICAL RELEVANCE: CarHb concentration may be used to increase the accuracy of identifying ARF, so that early, aggressive management can be instituted, thereby increasing the chance of recovery.

Carbamylated hemoglobin in hemodialysis patients.

We have previously found that carbamylated hemoglobin (carHb) levels are increased in chronic renal failure and correlate positively with blood urea nitrogen (BUN) levels and with the duration of exposure to urea. In a fashion analogous to glycosylated hemoglobin in diabetic patients, it is possible that carHb may better reflect BUN levels before hemodialysis (preBUN) and also between hemodialysis sessions. We therefore tested the hypothesis that carHb could be a better index of adequacy of hemodialysis than the urea reduction ratio (URR). Fifty hemodialysis patients had carHb measured every 2 months for 14 months; the carHb level was compared with URR and preBUN levels, as assessed by changes in absolute numbers and trends of the BUN levels between hemodialyses. Mean URR was above 61% throughout the 14 months. Mean carHb levels did not change significantly during the study and were only weakly correlated with URR. However, there was a much better correlation between predialysis BUN and carHb, suggesting that carHb levels reflect more accurately the changes in BUN between hemodialysis sessions. To further test this hypothesis, we subdivided the patients arbitrarily, depending on the change in preBUN between two consecutive carHb measurements. We found significantly lower carHb levels when BUN decreased or remained stable than when it increased or was persistently high. In patients with decreasing or stable BUN, carHb was significantly lower than in patients with persistently high or increasing BUN (carHb 81.5 +/- 3.6 microg valine hydantoin [VH]/g Hb v 123.7 +/- 11.7 microg VH/g Hb, respectively; P < 0.001). URR was not different between groups. In addition to changes in BUN levels, carHb was correlated by multiple regression analysis with the presence of diabetes, weight, and plasma HCO3. The relationship between diabetic patients and carHb levels was complex because such patients tend to have higher preBUN levels, higher protein catabolic rate, and lower HCO3 levels. These results demonstrate that carHb reflects the changes between dialysis BUN and may serve as a more accurate index of uremia control. Clinically, it appears that well-dialyzed patients have carHb levels lower than 100 microg VH/g Hb.

Temporal changes and reversibility of carbamylated hemoglobin in renal failure.

The detection of carbamylated hemoglobin (CarHb) is known to be useful in determination of the chronicity of uremia. However, the time course of the in vivo reaction between isocyanic acid and terminal valine residues of the hemoglobin chain is not clearly defined. To assess the temporal relationship and reversibility of carbamylation, we prospectively measured CarHb as micrograms of valine hydantoin per gram of hemoglobin (microg VH/g Hb) by high-performance liquid chromatography in 37 patients with acute renal failure (ARF), 53 patients with chronic renal failure (CRF), and six patients with successful kidney transplant. Patients with ARF had a lower median CarHb concentration (53.2 microg VH/g Hb; range, 24.6 to 97.1 microg VH/g Hb) than those with CRF (115.0 microg VH/g Hb; range, 34.6 to 286.5 microg VH/g Hb; P < 0.01), but had a higher value (53.2 microg VH/g Hb; range, 24.6 to 97.1 microg VH/g Hb) than 31 normal controls (36.6 microg VH/g Hb; range, 19.9 to 62.9 microg VH/g Hb; P < 0.05). In patients with ARF, the CarHb concentration positively correlated with the number of days of illness (r = 0.74; P < 0.01). The patients with ARF of 10 or more days' duration had a higher CarHb concentration (68.7 microg VH/g Hb; range, 36.0 to 93.9 microg VH/g Hb) than those with a shorter duration of ARF (33.7 microg VH/g Hb; range, 24.6 to 55.8 microg VH/g Hb; P < 0.01) despite similar blood urea nitrogen and serum creatinine values. However, they had a lower concentration of CarHb (68.7 microg VH/g Hb; range, 36.0 to 93.9 microg VH/g Hb) than CRF patients with comparable serum creatinine values (112.5 microg VH/g Hb; range, 34.6 to 286.5 microg VH/g Hb; P < 0.01). In patients with a kidney transplant, CarHb concentration declined by 19.7% (range, 12.3% to 35.6%) within 2 to 3 weeks after receiving the graft, while the level of hemoglobin increased by 25% (range, 4.0% to 46.6%) during the same period. Therefore, the total blood CarHb (CarHb x hemoglobin concentration) was not significantly changed. We concluded that the in vivo reaction of carbamylation of hemoglobin progressed during the period of uremia, and there might exist some irreversible preformed CarHb in advanced stages of CRF.

Carbamylated hemoglobin: a potential marker for the adequacy of hemodialysis therapy in end-stage renal failure.

Urea can dissociate in vivo to form isocyanic acid which can react with hemoglobin to form carbamylated hemoglobin. Previous work has shown that formation of carbamylated hemoglobin depends upon both the severity and the duration of renal failure. To determine whether carbamylated hemoglobin can be used as an assessment of the adequacy of hemodialysis treatment, we prospectively studied 55 stable patients who regularly attended our hospital dialysis program. Carbamylated hemoglobin was greater in those patients with a Kt/V of < or = 1.1 compared to those with a Kt/V of > 1.1 (120 +/- 8 micrograms VH/gHb versus 99 +/- 7, P < 0.01), and there was a negative correlation with Kt/V (r = -0.37, P = 0.007). There were positive correlations between carbamylated hemoglobin and the time-averaged urea concentration (r = 0.4, P = 0.004), and a negative correlation with the urea reduction ratio (r = -0.37, P = 0.01). Carbamylated hemoglobin may therefore be a useful marker of the degree of uremia, just as glycosylated hemoglobin is used in the assessment of patients with diabetes mellitus.

Specificity of hemoglobin A1c measurement by cation exchange liquid chromatography. Evaluation of a Mono S column method.

A method was developed for evaluating the specificity of the ion exchange chromatographic HbA1c method by Pharmacia which uses a Mono S column. To investigate hemoglobin fractions potentially interfering in HbA1c analysis, the chromatographic resolution was enhanced and a peak integration program was used which enables the quantitation of overlapping and even shoulder-like peaks. Hemoglobin was incubated with glucose in vitro and the chromatograms were analyzed before and after incubation. Five minor peaks were detected, close to hemoglobin A1c, which could not be conclusively identified as hemoglobins previously described. Four of these peaks, the stable ones, were included in the fraction of the chromatogram to be measured as HbA1c by the routine method. In practice, these peaks comprise 20%-35% of the routine HbA1c result in diabetic patients and 30%-45% in non-diabetic subjects. Three of these minor peaks were non-dependent on glucose. The chromatography and the peak integration method described can also be used to study hemoglobin adducts other than glycohemoglobin.