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.

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.

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.

Functional properties of the glycosylated minor hemoglobins A1a-1,A1a-2 and A1b. EPR evidence for increased stability of the low affinity quaternary structure and decreased susceptibility to organic phosphate.

Electron paramagnetic resonance (EPR) spectra of the glycosylated minor hemoglobins A1a-1, A1a-2, A1b and A1c and the major hemoglobin A0 in the nitrosyl form have been obtained in the absence and presence of inositol hexaphosphate. In the absence of inositol hexaphosphate, nitrosyl hemoglobins A1a-1, A1a-2 and A1b exhibited a triplet hyperfine structure centered at g = 2.009 which has been shown to be diagnostic of the low affinity (T) quaternary structure. Addition of inositol hexaphosphate to nitrosyl hemoglobins A0, A1c, A1b and A1a-2 developed a triplet hyperfine structure of the EPR spectra but the magnitude of the hyperfine was decreased in the order of hemoglobins A0, A1c, A1b and A1a-2. However, inositol hexaphosphate had essentially no effect on the EPR spectrum of nitrosyl hemoglobin A1a-1. The present results account qualitatively for the oxygen binding properties of these glycosylated minor hemoglobins in the framework of a two-state allosteric model.

A rapid chemical means for removing labile glycohemoglobin.

We have devised a rapid and convenient method of eliminating the contribution of labile glycosylated hemoglobin in the hemoglobin A1 assay. The procedure requires a 30-min incubation of erythrocytes in 30 mM semicarbazide and 12 mM aniline (pH 5, 38 degrees C). This chemical means of eliminating the labile fraction is as effective as a 14-h incubation in isotonic saline as measured with high-pressure liquid chromatography or electrophoresis methods. The removal of the labile glycosylated hemoglobin fraction preserves the assay as a reliable index of chronic metabolic control.

Bimodality of glycosylated hemoglobin distribution in Pima Indians: relationship to fasting hyperglycemia.

Glycosylated hemoglobin (HbA1) concentrations were determined in 300 Pima Indians aged 15 yr and older. Frequency distributions of HbA1 were unimodal in the 15--24-yr-old age group, but were bimodal in those aged 25 yr and over. The bimodality indicated that the subpopulation with diabetes could be identified by the presence of elevated HbA1 levels. This group was comprised primarily of subjects who also had fasting plasma glucose levels of less than or equal to 140 mg/dl, but subjects with impaired glucose tolerance without fasting hyperglycemia had HbA1 levels that were not significantly higher than those with normal glucose tolerance. The prevalence of diabetic retinopathy was much higher in the subgroup with elevated HbA1 levels and increased with increasing HbA1 level. HbA1 levels and triglyceride concentrations showed only a modest association. HbA1 determinations provided no advantage over fasting or post challenge glucose levels in the diagnosis of diabetes.

Critical factors in the chromatographic measurement of glycohemoglobin (HbA1).

Measurement of glycohemoglobin has been proposed as a criterion for the management of diabetes mellitus. We evaluated various conditions critical to the accuracy and precision of the cation-exchange method. Tolerance limits for each variable were defined as follows: phosphate-eluding buffer (0.06 +/- 0.005 mol/L pH 6.70--6.72), column temperature (19--21 degrees C), and resin equilibration (to phosphate buffer, 0.07 mol/l, pH 6.70 +/- 0.01). Hemoglobin absorbance measured in the Sorét region (approximately 416 nm) of the first chromatographic fraction divided by that of the total hemolysate provided the most accurate and precise result. Overall between-run precision expressed as coefficient of variation (CV, in percent) of normal and diabetic pools was 4.8% and 5.1%, respectively. When purified HbA1c was added to hemolysates, recovery was 90--95%. Results were linear to at least 18% glycohemoglobin. Hemoglobin F (HbF) interfered with the method, whereas HbC and HbS did not. Red cells could be stored frozen for at least 6 days, thus easing transport of outpatient samples. A reference range of 6.0--8.8% glycohemoglobin was established from 85 nondiabetic adults (ages 23--65 yr). In a clinical study, only 4 of 13 treated diabetic patients believed to be in good control showed glycohemoglobin results within the normal range. All of the 19 treated diabetics in fair or poor control showed glycohemoglobin results greater than 10% of total Hb, ie., well above the normal range.

Comparison of a colorimetric assay for glycosylated hemoglobin with ion-exchange chromatography.

Because levels of glycosylated hemoglobin (GHb) are increased in diabetes and reflect the previous metabolic control, clinicians and clinical investigators are finding increasing applications for measurements of GHb in diabetic patients. We report the characterization of a colorimetric assay procedure for GHb and compare its performance with that of a commonly used assay by ion-exchange chromatography. Although results of GHb determination by both methods correlate highly (r = 0.946, P less than 0.001), the two procedures estimate different glycosylated fractions. The colorimetric procedure is nonstoichiometric, requiring careful standardization of assay conditions, including the concentration of total hemoglobin in the assayed aliquot, to achieve precision and permit comparison of results. We characterized the effect of storage of hemolysates or packed erythrocytes on the subsequent determination of GHb by both methods. Determinations of GHb by the colorimetric method, but not by column chromatography, are reproducible on hemolysates or packed erythrocytes on the subsequent determination of GHb by both methods. Determinations of GHb by the colorimetric method, but not by column chromatography, are reproducible on hemolysates or packed erythrocytes stored frozen for at least 5 mo. A unique advantage of the colorimetric procedure is the capability to estimate GHb levels when variant hemoglobins, including fetal and sickle hemoglobins, are present.

HbA1 in subjects with abnormal glucose tolerance but normal fasting plasma glucose.

HbA1(a+b+c)(HbA1) was determined chromatographically in 107 subjects with normal fasting plasma glucose (FPG) and 112 patients with overt diabetes. Subjects with normal FPG were divided into two groups based on their response to two oral glucose tolerance tests (OGTTs), at an interval of 2 mo. In 40 subjects with normal OGTT (group I), HbA1 ranged from 5.2% to 7.2%, while in 67 subjects with abnormal OGTT (group II), it ranged from 6.3% to 9.6%. HbA1 levels were significantly higher in group II than in group I (7.7 +/- 0.09% versus 6.4 +/- 0.08%, mean +/- SEM, P less than 0.0005), but 14 subjects of group II had HbA1 levels less than 7.2%. No correlation was found between HbA1 and FPG, OGTT peak, and curve area in either group. However, the correlation became significant in all 107 subjects with normal FPG (groups I + II). In patients with overt diabetes, HbA1 ranged from 6.3% to 18% (11.9 +/- 0.22%) and correlated with FPG (r = 0.78, P less than 0.0005). The traditional OGTT seems more sensitive than the HbA1 measurement in detecting subjects with reduced carbohydrate tolerance. HbA1 level, on the other hand, is known to be more specific indicator of structural abnormalities following long-term hyperglycemia. Thus HbA1 determination might be a helpful test along with OGTT to improve both selection and follow-up subjects with true borderline diabetes.

Clinical trial of an aldose reductase inhibitor in diabetic neuropathy.

A single-blind, nonrandomized, placebo crossover clinical trial of an aldose reductase inhibitor, Alrestatin (AY 22, 284) was performed over a 4-mo period in nine patients with diabetic peripheral neuropathy. Most patients had subjective benefit, but objective measures of conduction were essentially unchanged. Substantial toxicity was evident, particularly photosensitive skin rash.

Serum glycosidase activity in diabetes mellitus.

The activity of three glycosidic enzymes, B-glucuronidase, N-acetylglucosaminidase, and B-galactosidase were measured in plasma samples from 163 diabetic subjects and 72 normal controls. No age- or sex-based variation in concentration was noted in controls. Plasma activity of B-glucuronidase and N-acetylglucosaminidase in diabetics correlated directly with the overall level of glycemia as measured by HbA1c levels. B-galactosidase activity was consistently normal in diabetics. A significant age-based variation was noted in the diabetic group for B-glucuronidase and N-acetylglucosaminidase. Prior to age 12 B-glucuronidase and N-acetylglucosaminidase were normal in diabetics, but activity increased significantly after the age of 12, a change that appeared to coincide with the development of puberty.

Changes in nerve conduction velocity after six weeks of glucoregulation with portable insulin infusion pumps.

Near normal glucoregulation was maintained in 10 patients with insulin-dependent (type I) diabetes mellitus for 6 wk with preprogrammed continuous subcutaneous insulin infusion using a portable battery-powered infusion pump (CSII). This form of therapy resulted in a statistically significant increase in motor nerve conduction velocity in the median and peroneal nerves compared with baseline values. There was no significant change in the motor nerve conduction velocity in the ulnar nerve or in the sensory nerve conduction studies. No changes occurred in five additional patients studied in similar fashion while on a conventional insulin regimen. These results suggest that the prevention of sustained hyperglycemia with CSII could theoretically result in the prevention of diabetic neuropathy. However, only long-term studies of CSII will provide the information necessary to determine the clinical relevance of the findings.

Nonenzymatic glucosylation of serum proteins and hemoglobin: response to changes in blood glucose levels in diabetic rats.

The relationship between concentrations of blood glucose and nonenzymatically glucosylated serum proteins was studied in rats with alloxan-induced diabetes of varying severity. Fasting serum glucose correlated strongly with both glucosylated albumin (r = 0.91, P less than 0.001) and glucosylated serum protein (r = 0.93, P less than 0.001). The relative rates of response of serum protein and hemoglobin glycosylation to changes in blood glucose were also compared. Following withdrawal of insulin from diabetic rats, the half-times to reach new steady state levels of blood glucose, glucosylated serum proteins, and glycohemoglobins were about 2, 3, and 8 days, respectively. Similarly, on reinstitution of insulin therapy, the half-times for these same indices to return to baseline values were 2, 3.5, and 15 days, respectively. Changes in glucosylated albumin were more sensitive than glycohemoglobins to changes in serum glucose, consistent with the observation that albumin was glucosylated at about 10 times the rate for hemoglobin in incubations in vitro. These data indicate that glucosylated serum protein measurements can serve as a sensitive, short-term integrator of blood glucose homeostasis in diabetes.

Glycohemoglobin. Its use in the follow-up of diabetes and diagnosis of glucose intolerance.

In 140 persons with a normal glucose tolerance test (GTT), the median glycohemoglobin (GHb) level was 8.1% (95% limits, 6.5% to 9.0%). The GHb level was not significantly different in 30 patients with impaired GTT (8.6%). In all 14 patients with diabetic GTT and fasting plasma glucose (PG) level above 140 mg/dL, GHb level was above 10% (median, 11.3%). Of 25 patients with diabetic GTT but without fasting hyperglycemia, only two had GHb levels 10% or above. The GHb level correlated with the mean daily PG level in both 176 insulin-dependent (rs, +.530) and 107 non-insulin-dependent (rs, +.734) diabetics. Correlation was almost zero between GHb level and mean one-day PG level in 14 most brittle insulin-dependent diabetics. However, in them, there was a strong correlation (rs, +.763) between GHb level and the mean of 20 or more PG values for the preceding month. Follow-up of 100 diabetics showed strong correlation (rs, +.845) between the changes in GHb level and in the mea PG level. Decrease of the mean PG level of 100 mg/dL was accompanied by a drop of the GHb level of about 2%.

Glycosylated hemoglobin in diabetes mellitus: correlations with fasting plasma glucose, serum lipids, and glycosuria.

The levels of glycosylated hemoglobin (GlHb), fasting plasma glucose (FPG), urine glucose, serum triglycerides, cholesterol, and HDL-cholesterol were monitored in several hundred patients attending an adult diabetic clinic. Thirty-five percent of the patients were being treated with insulin, 30% with diet alone, and 20% with sulfonylurea. Therapy was changed during the study in the remaining 15%. The levels of GlHb and FPG were significantly lower in the patients treated with diet alone than in the other groups. The overall coefficient of correlation (r) between FPG and GlHb was 0.64, with the highest r (0.70) for the diet group and the lowest r (0.55) for the insulin group. A follow-up study showed that in about 50% of the patients GlHb and FPG levels changed in different directions from one visit to the next. The absence of glycosuria was noted in two-thirds of the patients, while in 78% of the patients GlHb values were above the normal range. A weak but positive correlation was found between GlHb and serum triglycerides and cholesterol. There was no significant correlation between GlHb and HDL-cholesterol. The HDL-cholesterol levels were significantly lower in male than in female diabetic subjects, but no significant difference was found between the diabetic and nondiabetic subjects of the same sex. The results of this study suggest that (1) although there is a positive correlation between GlHb and FPG, the two tests cannot be used interchangeably in the evaluation of diabetic control; and (2) in the majority of the patients the absence of glycosuria tends to create the false assurance of satisfactory blood glucose control.

Changes in basement membrane thickening and pulse volume concomitant with improved glucose control and exercise in patients with insulin-dependent diabetes mellitus.

Ten patients with type I diabetes mellitus were enrolled in a program of exercise and carbohydrate "control" using self-monitored glucose determinations and self-adjusted insulin. Glucose control was improved for the group, although normoglycemia was not uniformly achieved. Pulse volume measurements performed at the onset and after 8-10 mo documented a drop in systolic arm blood pressure with increases in the ankle-arm index (P < 0.001). Quadriceps biopsy was successfully performed at the beginning and after 8-10 mo for analysis of basement membrane thickening (BMT) in seven patients. Six patients showed a decrease in basement thickening on rebiopsy (P = 0.02). One patient showed increased BMT. This was the only patient to maintain a mean hemoglobin A1c of greater than 10% for the duration of the study. These observations indicate that abnormalities of BMT and pulse volume recordings may be more labile measurements than previously thought and may be amenable to therapeutic intervention. The relationship of these variables to the severe micro- and macrovascular sequelae of diabetes mellitus remains to be established.

Hemoglobin A1c levels in children and adolescents with diabetes mellitus.

Hemoglobin A1c (HbA1c) was measured as an indicator of glucose control in 180 children and adolescents with diabetes mellitus who received two daily injections of insulin as part of a highly structured treatment program. A total of 426 HbA1c determinations was made in the group of 180 patients. HbA1c values were elevated in most patients despite the aggressive treatment. The HbA1c level was very elevated at diagnosis, fell to near normal after 60-90 days of insulin therapy, increased gradually, and reached a plateau after approximately 4 yr duration (at about twice the level in normal subjects) (mean +/- SEM, 10.0 +/- 0.2% and 5.34 +/- 0.07%, respectively). Mean insulin dose (U/kg/24 h) paralleled both HbA1c and duration of diabetes. The relationship between endogenous insulin secretion and glucose control was examined in those patients with diabetes for longer than 5 yr. Patients were separated into three groups based on HbA1c levels: those with HbA1c less than 9% (N = 22), between 9 and 11% (N = 26), and greater than 11% (N = 28). Serum C-peptide and glucose concentrations were measured 2 h after a standard breakfast in those patients in the "low" and "high" HbA1c groups (mean HbA1c values 8.2% and 12.7%, respectively). C-peptide was detectable in all patients and the mean C-peptide levels did not differ significantly in the two groups, although postprandial glucose concentrations were significantly lower in the "low" HbA1c group (means +/- SEM, 96 +/- 11 and 211 +/- 21 mg/dl, respectively; P less than 0.001).