Development of a high-performance liquid chromatographic glycated albumin assay using finger-prick blood samples.

BACKGROUND: Home blood glucose monitoring can be effective for the self-management of diabetic patients. Hemoglobin A1c (HbA1c) is a widely used marker that reflects the average blood glucose within 1-2 months but does not sensitively respond to behavioral changes. Self-monitoring of blood glucose, continuous glucose monitoring, and flush glucose monitoring are sensitive; however, the cost and invasiveness of these tests prevent their widespread use. We focused on glycated albumin (GA), which reflects the average blood glucose levels over 1-2 weeks, and established a GA measurement method for self-sampling, finger-prick blood, which may be submitted for testing through postal service to receive weekly results. METHODS: A high-performance liquid chromatography assay was established to measure GA levels in finger-prick blood samples from 103 diabetic patients and the results were compared with venous blood measurements using an enzymatic method. Furthermore, conditions for sending blood samples by mail were evaluated. Under these conditions, samples from 27 healthy and 32 patient volunteers sent through postal service were compared with samples stored in the laboratory. RESULTS: GA levels were measured in samples containing > 20 µg albumin, which resulted in a CV less than 0.3%. The correlation between the GA levels of finger-prick blood measured using HPLC and the GA levels of venous blood measured using the enzymatic method was R2 = 0.988 with the slope âˆ¼ 1.0, suggesting that the two were nearly equivalent. GA levels were stable for four days at 30 °C and two days at 37 °C. Mail-delivered samples exhibited a high correlation with samples that were not sent (R2 > 0.99). CONCLUSIONS: We established a method to measure GA levels in self-sampled, finger-prick blood sent through postal service in Japan. The method is applicable for weekly feedback of GA levels, which is potentially useful for motivating behavioral changes. In addition to markers such as HbA1c and blood glucose, GA can be used as a marker for assessing dietary and physical activities. This study highlighted the importance of GA monitoring by developing a suitable measurement method for weekly monitoring of GA levels.

Redox state as assessed using the measurement of human non-mercaptalbumin in embryo culture media is associated with successful embryo development in human in vitro fertilization.

The role of oxidative stress in the pathogenesis of various diseases has been attracting attention. We speculated as to whether the redox state of treatment solutions used for various diseases may play a role in treatment success. In the current study, we focused on the human embryo culture medium used for in vitro fertilization (IVF). A total of 173 oocytes from a total of 91 patients treated with IVF were enrolled. The redox state was assessed by measuring the levels of human non-mercaptalbumin (HNA). We analyzed factors related to blastocyst formation on day 5 or 6 after insemination. We also developed a random forest (RF) model for the prediction of blastocyst formation. The variable importance in the predictive model was assessed using the mean decrease in the Gini impurity. Blastocyst formation was observed in 41.04% (71/173) of the oocytes and was associated with a lower %HNA in the culture medium, a younger patient age, and the fertilization method (standard IVF or intracytoplasmic sperm injection). The RF model developed using these factors and 70% of the samples (training set, n = 121) was validated in the remaining testing set (n = 52) and produced an area under the curve of 0.761, where the %HNA in the culture medium was the most important variable for predicting blastocyst formation. In conclusion, lower levels of oxidative stress in embryo culture media were associated with the success of IVF treatment. The redox state of treatment solutions should be considered to support treatment success.

A simple colorimetric assay to determine the concentration and proportion of human mercaptalbumin.

Objectives: Human serum albumin can take on two forms, mercaptalbumin (HMA) or non-mercaptalbumin (HNA), depending on the redox status of its Cys34. The ratio of HMA and HNA is considered to be a novel biomarker of oxidative stress. While HPLC and mass spectrometry are established methods to measure HMA and HNA, a simple colorimetric assay was applied to measure this biomarker. Design and methods: Michler's Hydrol (4,4'-Bis(dimethylamino)benzhydrol) is a blue dye with a maximum absorption at 612 nm, and its absorption decreases when it reacts with a thiol group. Concentrations of HMA in serum samples from 36 healthy subjects were measured based on absorption changes of Michler's Hydrol. The proportion of HMA (HMA%) in total albumin was also obtained by dividing the HMA concentration by total albumin concentration, which was obtained by a bromocresol purple (BCP) assay. The proportion of HNA (HNA%) was obtained by subtracting HMA% from 100%. Results: HMA concentrations obtained by Michler's Hydrol assay were highly correlated (r2 = 0.97) with reference values obtained by HPLC (HMA%) and BCP assay (total albumin). The HNA% obtained by Michler's Hydrol and BCP assays combined also gave a good correlation (r2 = 0.96) and a small deviation (average 2.4%) with respect to HPLC as a reference method. Conclusions: A colorimetric assay using Michler's Hydrol was optimized for a 96-well plate format so that it can be easily performed in a standard laboratory setting. This assay gives HMA concentrations and HNA proportions comparable to HPLC.

Non-mercaptalbumin is significantly associated with the coronary plaque burden and the severity of coronary artery disease.

Human non-mercaptalbumin (HNA), oxidized form of serum albumin, has been reported as a useful marker in oxidative stress-related diseases; however, few reports have examined the association between HNA and the severity of coronary artery disease (CAD). The present study evaluated whether the HNA fraction is correlated with coronary artery stenosis in 140 patients considered to have a high risk of CAD or who were suspected of having acute coronary syndrome. The severity of CAD was defined by the number of stenotic coronary vessels and a severity score system (the Gensini score). HNA measurements were performed using our newly established high-performance liquid chromatography methodology. The results had shown that HNA was significantly increased in patients with three-vessel disease, compared with those without CAD or with single-vessel disease (p = 0.025), and was positively correlated with the Gensini score (ρ = 0.421, p < 0.001). A multivariate analysis showed that the number of stenotic vessels was an independent and significant factor associated with HNA (ρ = 1.246, p = 0.012). A logistic regression analysis showed that HNA was a strong predictor of multivessel CAD (odds ratio, 1.12; 95% confidence interval, 1.020-1.229; p = 0.017). These findings indicate that the measurement of HNA could be clinically practical for predicting the severity of coronary artery stenosis.

Oxidized albumin in blood reflects the severity of multiple vascular complications in diabetes mellitus.

Albumin has an oxidized form, known as non-mercaptalbumin (HNA), which reflects systemic oxidative stress. The association between serum HNA levels and diabetic complications are yet to be reported. In this cross-sectional study, we investigated 164 diabetic subjects to assess the correlation between HNA% (the proportion in the total albumin) and various clinical parameters. HNA% was significantly associated with the severity of multiple complications including neuropathy (23.3 ± 4.1% vs 26.2 ± 5.1%) and nephropathy (24.1 ± 3.9%, 24.6 ± 4.2%, 28.5 ± 6.1%, 31.3 ± 5.7%, 37.8 ± 2.9%, stage 1/2/3/4/5, respectively). These findings highlight the universal importance of oxidative stress, indicating HNA% potential as a versatile marker of the severity of diabetic complications.

Nonmercaptalbumin as an oxidative stress marker in Parkinson's and PARK2 disease.

OBJECTIVE: To investigate the oxidized albumin ratio, which is the redox ratio of human nonmercaptalbumin (HNA) to serum albumin (%HNA), as a biomarker in idiopathic Parkinson's disease (iPD) and related neurodegenerative disorders. METHODS: This prospective study enrolled 216 iPD patients, 15 patients with autosomal recessive familial PD due to parkin mutations (PARK2), 30 multiple system atrophy (MSA) patients, 32 progressive nuclear palsy (PSP) patients, and 143 healthy controls. HNA was analyzed using modified high-performance liquid chromatography and was evaluated alongside other parameters. RESULTS: iPD and PARK2 patients had a higher %HNA than controls (iPD vs. controls: odds ratio (OR) 1.325, P < 0.001; PARK2 vs. controls: OR 1.712, P < 0.001). Even iPD patients at an early Hoehn & Yahr stage (I and II) showed a higher %HNA than controls. iPD patients had a higher %HNA than MSA and PSP patients (iPD vs. MSA: OR 1.249, P < 0.001, iPD vs. PSP: OR 1.288, P < 0.05). When discriminating iPD patients from controls, %HNA corrected by age achieved an AUC of 0.750; when discriminating iPD patients from MSA and PSP patients, an AUC of 0.747 was achieved. Furthermore, uric acid, an antioxidant compound, was decreased in iPD patients, similar to the change in %HNA. INTERPRETATION: %HNA was significantly increased in iPD and PARK2 patients compared with controls, regardless of disease course and severity. Oxidative stress might be increased from the early stages of iPD and PARK2 and play an important role in their pathomechanisms.

Clinical usefulness of human serum nonmercaptalbumin to mercaptalbumin ratio as a biomarker for diabetic complications and disability in activities of daily living in elderly patients with diabetes.

BACKGROUND: Oxidative stress may play an important role in the development of diabetic complications. The ratio of human nonmercaptalbumin (HNA; oxidized form) to human mercaptalbumin (HMA; reduced form) has attracted attention as an indicator for systemic redox states. In this study, we measured the ratio in elderly patients with diabetes and evaluated its association with diabetic complications and disability in activities of daily living (ADL disability). METHODS: One hundred twenty-six elderly patients with diabetes, aged 70 years and older, under medical care at Yukuhashi Central Hospital from April 2018 to June 2018, were continuously recruited. HNA%, defined as HNA / (HNA + HMA) × 100, was measured by a high-performance liquid chromatography method. First, multivariate regression analysis was performed to evaluate which variables were significant determinants for HNA%. Next, to evaluate the association of HNA% with ADL disability, logistic regression analysis in various models was performed. Then we plotted the receiver operating characteristic (ROC) curve and calculated the under area the curve (AUC), sensitivity, and specificity in each model. RESULTS: In elderly patients with diabetes, multiple regression analysis showed that serum bilirubin levels and albumin levels, both of which are major endogenous anti-oxidants, and chronic renal failure (or proliferative nephropathy) were significantly associated with HNA%, suggesting that HNA% may be a good biomarker for oxidative stress in those patients. We then evaluated the association of HNA% with ADL disability in various logistic regression models. Model using only HNA% showed that it was a significant determinant for ADL disability (OR 1.158, 95% CI 1.077-1.244, P < 0.001). Model using HNA% and age showed that both variables were significant determinants for ADL disability (OR 1.160, 95% CI 1.069-1.258, P < 0.001; OR 1.258, 95% CI 1.110-1.427, P < 0.001, respectively). ROC analysis showed that the AUC of HNA% alone was 0.765. The AUC of model using HNA% and age was further increased to 0.866. CONCLUSIONS: HNA% was significantly associated with diabetic complications and ADL disability, thereby may be clinically useful as an oxidative stress marker in elderly patients with diabetes.

Establishment of a stable sampling method to assay mercaptoalbumin/non-mercaptoalbumin and reference ranges.

Oxidative stress is reportedly associated with many diseases such as cancer, arteriosclerosis, diabetes and aging, but no practical biomarkers are currently available in actual clinical practice. Human mercaptoalbumin (HMA) and human non-mercaptoalbumin (HNA) are expected to become markers of oxidative stress, but the stability of HMA/HNA has been problematic. We investigated the conditions for stabilizing HMA/HNA and found that HMA/HNA was stable at room temperature for 25 h if whole blood samples were mixed with a citrate buffer so that the citric acid concentration after mixing was 70 mM or higher and the pH of the added buffer was less than pH 6.0. Whole blood samples were then collected under the above conditions, and the reference range for HNA was set at 21.8% ±â€¯7.4% (HMA, 78.2% ±â€¯7.4%) based on samples from 65 volunteers (28 males and 37 females; average age, 55.0 ±â€¯13.8 years). The clinical usefulness of HMA/HNA as an oxidative stress marker should be clarified for specific pathological conditions using the previously reported, highly accurate measurement method under the conditions required for HMA/HNA stability.

Redox state of albumin affects its lipid mediator binding characteristics.

Depending on its redox status, albumin is known to exist as two forms: reduced albumin or human mercaptalbumin (HMA); and oxidised albumin or human nonmercaptalbumin (HNA). The ratio of HNA to HMA is reportedly elevated in several diseases. Since lipid mediators, such as eicosanoids and lysophospholipids, are typically bound to albumin, we examined the possible preferences of lipid mediators for HNA or HMA. We observed that DHA-derived and EPA-derived eicosanoids preferred to be bound to HMA, while the levels of lysophospholipid mediators, such as lysophosphatidic acids and sphingosine 1-phosphate, were higher in the HNA fraction. Considering the bioactivities reported in previous basic studies, these results suggest that proatherosclerotic lipid mediators might generally prefer HNA, while antiatherosclerotic ones might prefer HMA. Oxidative stress affects the redox status of albumin, which might modulate the dynamism of lipid mediators. This pathway might be partly involved in the association between oxidation and atherosclerosis.

A rapid method for measuring serum oxidized albumin in a rat model of proteinuria and hypertension.

Oxidative stress is a risk for and cause of various disease, however, measurements of oxidative stress are either time-consuming or non-specific. Here, we established a rapid method of using high performance liquid chromatography (HPLC) to measure serum oxidized albumin in a rat model. We optimized HPLC conditions for rat oxidized albumin. To validate our method, three-week-old male Sprague-Dawley rats were uninephrectomized and treated normal diet, high salt diet or high salt diet with Tempol, a superoxide dismutase (SOD) mimetic. After 4 weeks of treatment, we analyzed serum oxidized albumin. The main findings are listed as below. (i) Our method of oxidized albumin measurement only takes 16 minutes, with an intra-day and inter-day deviation within 1% and a detection limit concentration of 6.4 mg/ml. (ii) Oxidized albumin levels were significantly higher in the high salt diet group than in the normal salt diet group, and this effect was reversed by Tempol. (iii) Oxidized albumin levels also correlated with urinary protein and 8-isoprostane levels. In conclusion, we have established a simple method for evaluating rat serum oxidized albumin using HPLC. Our method is rapid and has an advantage over conventional methods and may be useful for animal models of oxidative stress.

Non-mercaptalbumin, Oxidized Form of Serum Albumin, Significantly Associated with Renal Function and Anemia in Chronic Kidney Disease Patients.

Oxidative stress plays a major role in development of cardiovascular disease in patients with chronic kidney disease (CKD). Human mercaptalbumin (HMA), a reduced form of serum albumin, and non-mercaptalbumin (HNA), an oxidized form of serum albumin, are known as indicators for evaluating oxidative stress in systemic circulation, including end-stage renal disease cases. We investigated factors associated with fraction of HNA [f(HNA)] in 112 pre-dialysis CKD patients (63.6 ± 14.0 years old; 59 males, 53 females) using a newly established anion-exchange column packed with hydrophilic polyvinyl alcohol gel as well as high performance liquid chromatography. Mean f(HNA) in our CKD patients was 30.0 ± 6.1%, higher than that previously reported for healthy subjects. In multiple regression analysis, age (ß = 0.200, p = 0.014), eGFR (ß = -0.238, p = 0.009), hemoglobin (ß = -0.346, p < 0.001), and ferritin (ß = 0.200, p = 0.019) were significantly and independently associated with f(HNA) (R2 = 0.356, p < 0.001). In addition, factors related to CKD-mineral and bone disorder (CKD-MBD), including intact-PTH (ß = 0.218, p = 0.049) and 1,25-dihydroxyvitamin D (1,25(OH)2D) (ß = -0.178, p = 0.040), were significantly and independently associated with serum f(HNA) (R2 = 0.339, p < 0.001), whereas fibroblast growth factor-23 was not. These findings indicate the importance of management of hemoglobin and ferritin levels, as well as appropriate control of CKD-MBD factors for a better redox state of serum albumin in CKD patients.

A simple, rapid and validated high-performance liquid chromatography method suitable for clinical measurements of human mercaptalbumin and non-mercaptalbumin.

Background Human mercaptalbumin and human non-mercaptalbumin have been reported as markers for various pathological conditions, such as kidney and liver diseases. These markers play important roles in redox regulations throughout the body. Despite the recognition of these markers in various pathophysiologic conditions, the measurements of human mercaptalbumin and non-mercaptalbumin have not been popular because of the technical complexity and long measurement time of conventional methods. Methods Based on previous reports, we explored the optimal analytical conditions for a high-performance liquid chromatography method using an anion-exchange column packed with a hydrophilic polyvinyl alcohol gel. The method was then validated using performance tests as well as measurements of various patients' serum samples. Results We successfully established a reliable high-performance liquid chromatography method with an analytical time of only 12 min per test. The repeatability (within-day variability) and reproducibility (day-to-day variability) were 0.30% and 0.27% (CV), respectively. A very good correlation was obtained with the results of the conventional method. Conclusions A practical method for the clinical measurement of human mercaptalbumin and non-mercaptalbumin was established. This high-performance liquid chromatography method is expected to be a powerful tool enabling the expansion of clinical usefulness and ensuring the elucidation of the roles of albumin in redox reactions throughout the human body.

Evaluation of human nonmercaptalbumin as a marker for oxidative stress and its association with various parameters in blood.

Oxidative status of albumin was not a useful biomarker for oxidative stress in practical use due to time-consuming measuring method. We evaluated oxidized, human nonmercaptalbumin measured more quickly than ever by a novel method using anion-exchange HPLC. In 60 subjects taking a general health examination, mean serum human nonmercaptalbumin level was 25.1 ± 3.0% with no gender difference but positive correlation with age. There were no links between human nonmercaptalbumin and C-reactive protein, γ-glutamyltransferase or iron, reportedly associated with oxidative stress. Human nonmercaptalbumin correlated with systolic blood pressure, pulse pressure and body mass index among physical findings. Positive correlations were observed between human nonmercaptalbumin and AST, LDH, BUN, or creatinine, suggesting that oxidative stress may link with liver injury and renal function. Human nonmercaptalbumin correlated with uric acid in female but not in male, suggesting that higher uric acid levels may be associated with increased oxidative stress only in female. As another gender difference, white blood cell counts correlated with human nonmercaptalbumin in female, while the parameters for red blood cells correlated with human nonmercaptalbumin in male. In conclusion, serum human nonmercaptalbumin level in healthy subjects was approximately 25% as previously reported. Oxidative stress may be closely associated with hypertension, obesity, liver injury, renal function, and anemia.

Committee on Diabetes Mellitus Indices of the Japan Society of Clinical Chemistry-recommended reference measurement procedure and reference materials for glycated albumin determination.

BACKGROUND: Glycated albumin is an intermediate glycaemic control marker for which there are several measurement procedures with entirely different reference intervals. We have developed a reference measurement procedure for the purpose of standardizing glycated albumin measurements. METHODS: The isotope dilution liquid chromatography/tandem mass spectrometry method was developed as a reference measurement procedure for glycated albumin. The stable isotopes of lysine and fructosyl-lysine, which serve as an internal standard, were added to albumin isolated from serum, followed by hydrogenation. After hydrolysis of albumin with hot hydrochloric acid, the liberated lysine and fructosyl-lysine were measured by liquid chromatography/tandem mass spectrometry, and their concentrations were determined from each isotope ratio. The reference materials (JCCRM611) for determining of glycated albumin were prepared from pooled patient blood samples. RESULTS: The isotope dilution-tandem mass spectrometry calibration curve of fructosyl-lysine and lysine showed good linearity (r = 0.999). The inter-assay and intra-assay coefficient of variation values of glycated albumin measurement were 1.2 and 1.4%, respectively. The glycated albumin values of serum in patients with diabetes assessed through the use of this method showed a good relationship with routine measurement procedures (r = 0.997). The relationship of glycated albumin values of the reference material (JCCRM611) between these two methods was the same as the relationship with the patient serum samples. CONCLUSION: The Committee on Diabetes Mellitus Indices of the Japan Society of Clinical Chemistry recommends the isotope dilution liquid chromatography/tandem mass spectrometry method as a reference measurement procedure, and JCCRM611 as a certified reference material for glycated albumin measurement. In addition, we recommend the traceability system for glycated albumin measurement.