Determination of a Serum 25-Hydroxyvitamin D Reference Ranges in Japanese Adults Using Fully Automated Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Despite an increasing interest in vitamin D status, a reference range of the nutrient has not been fully established. This is partly due to a paucity of standardized measuring systems with high throughput. In addition, the range may vary by populations and may change with modernization of lifestyles. OBJECTIVES: This study aims to calculate the current reference concentration of 25-hydroxyvitamin D (25(OH)D) among healthy people living in an urban area in Japan. METHODS: A newly developed fully automated liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) system was used to measure serum 25(OH)D concentrations. Reproducibility was assessed by measuring standardized samples. Accuracy was validated by comparing with commercially available immunoassays. Then, mass screening was conducted targeting participants who received medical checkups in Tokyo from April 2019 to March 2020, and the reference ranges were calculated. RESULTS: The coefficients of variations of interoperator and interday reproducibility were 4.1%-8.5% and 3.7%-8.0% for 25-hydroxyvitamin D2 (25(OH)D2) and 4.7%-7.0% and 4.0%-6.9% for 25-hydroxyvitamine D3, respectively. The measured total 25(OH)D concentrations correlated well with those measured by immunoassays. In total, 5518 participants were measured for 25(OH)D concentrations, among whom 98% showed inadequate concentrations (<30 ng/mL). The reference ranges of total 25(OH)D for female, male, and total participants were 7-30 ng/mL, 5-27 ng/mL, and 6-29 ng/mL, respectively. After excluding those with abnormal renal and liver function, the range was 6-30 ng/mL. CONCLUSIONS: The high prevalence of vitamin D insufficiency among seemingly healthy population may be attributed to lifestyle characteristics of people living in urban areas of Japan, including spending less time outdoors and lower intake of traditional foods. Longitudinal follow-up and mass screenings targeting different population will help elucidate reasons for discrepancies between official guidelines and the observed concentrations, to which the well-validated measurement system is essential.

[Clinical verification of serum procalcitonin measurement in sepsis].

To evaluate the clinical application of measuring procalcitonin (PCT) level for diagnosis of bacterial sepsis in patients with and without systemic inflammatory response syndrome(SIRS), we studied the relationship between blood culture (BC) and serum PCT level in clinical 207 cases. In addition, we evaluated the time courses of PCT and other inflammatory markers: tumor necrosis factor-alpha (TNF-alpha), interleukin 6 (IL-6), E-selectin, WBC count and C-reactive protein (CRP) in 5 bacterial septic patients with SIRS. Serum PCT showed sensitivity of 41% and specificity of 61%, while BC showed specificity of 88%. In 27 BC-positive cases, serum PCT was significantly elevated in gram-negative bacterial sepsis. We observed 11 cases with BC(+) and serum PCT below 0.5 ng/ml. Major causes of these discrepancies were probably due to gram-positive bacterial infection, local bacterial infection or pretreatment with broad-spectrum antibiotics. In contrast, 10 cases with BC(-) and serum PCT over 10 ng/ml were presumably due to some cytokine elevation caused by virus infection or collagen diseases. In 5 cases studied for inflammatory markers, TNF-alpha level elevated earlier than the others and followed by PCT, IL-6, WBC, CRP, and E-selectin. It was suggested that the measurement of serum procalcitonin in septic patients is clinically useful marker to diagnose gram-negative bacterial septic patients with SIRS.

[Relationship between glycated albumin (GA) and glycated hemoglobin (A1c) in 255 patients with liver diseases using cross-sectional laboratory data].

To investigate how liver disease alter the serum glycated proteins as markers of diabetic control, we studied serum GA, A1c and especially GA/A1c ratio in 255 patients having over 35IU/L in ALT(transaminase) compared with those of 829 type 2 diabetes mellitus (DM) in cross sectional manner. 255 patients with liver diseases were divided into 69 patients with biopsy proven liver cirrhosis (LC), 66 patients with chronic hepatitis(CH) and 120 patients with fatty liver(FL) diagnosed by abdominal echography. The mean GA/A1c ratio (+/-SD) was significantly higher (p<0.0001) in LC group(3.71+/-1.03) than the other groups (3.03+/-0.45 for CH, 3.05+/-0.42 for DM), while the mean GA/A1c ratio in FL group was significantly lower(2.74+/-0.31) (p<0.0001)) than that of DM groups. In LC group the GA/A1c ratio increased significantly depending upon serum albumin and/or platelet reductions. The GA/A1c ratio was significantly correlated with the other laboratory data such as serum albumin, cholinesterase, total cholesterol levels and weakly correlated with serum hemoglobin level. We also followed the serum levels of GA and A1c and the GA/A1c ratio during about 13 months (5 times blood collections) in 18 patients enrolled in this study. Resultantly the coefficient of variation of GA/A1c ratio was the smaller than the others(GA, A1c). The ROC curve of GA/A1c ratio for LC versus FL group was the most reliable between four groups and the cut-off value for LC versus FL was 2.94. Theses results suggest that GA/A1c ratio could be an useful marker for different diagnosis when facing patients with abnormal serum ALT level in a clinical setting.

Changes in the albumin glycation site, plasma pentosidine and esRAGE concentrations before and after intensive diabetic treatment in patients with abnormally high glycated albumin levels.

Background We have reported that the blood glucose normalization treatment reduced the albumin glycation sites and the intensity of albumin AGE fluorescence in patients with abnormally high glycated albumin levels. To clarify the relationship between glycaemic control status and levels of glycated proteins and related markers, we studied the change of the markers of the DM patients with and without fatty liver, liver cirrhosis and dialysis before and after the intensive diabetic treatment. Methods Eight diabetic patients with abnormally high glycated albumin levels (no complications: 2, fatty liver: 3, liver cirrhosis: 2, dialysis: 1) were recruited. In the hypoglycaemic treatment for these patients, the HbA1c, glycated albumin, albumin AGE fluorescence, pentosidine, endogenous secretory receptors for AGE (esRAGE) and glycation sites of albumin were determined. Results Glycated albumin and HbA1c levels dropped after the treatment. Albumin glycation sites decreased in almost the same pattern, irrespective of the type of complications. The fluorescence intensity and pentosidine concentrations decreased significantly. However, post-treatment pentosidine concentrations were higher than the reference interval in all cases. Average esRAGE concentrations did not change and were lower than the reference interval. Conclusions Hypoglycaemic treatment reduced the glycated albumin levels, glycation sites of albumin and AGE concentrations but not esRAGE concentrations in diabetic patients with or without fatty liver, liver cirrhosis, and dialysis. Checking and maintaining low glycated albumin levels would prevent the formation of AGE and may be useful to prevent the onset or progression of diabetes complications.

Structural and glycation site changes of albumin in diabetic patient with very high glycated albumin.

BACKGROUND: Glycated albumin (GA) has been utilized to monitor mid-term glycemic control, and reflects the status of blood glucose more rapidly and effectively than hemoglobin A(1c) (HbA(1c)). To examine the relationship between GA level and structural changes or glycation sites of albumin, we analyzed pre- and post-treatment samples from a diabetic patient with extraordinary increase of GA. METHOD: A female diabetic patient with poor glycemic control had a GA >94% and was treated with intensive insulin therapy to decrease blood glucose. We analyzed changes in fluorescence derived from tryptophan (Trp) and advanced glycation end product (AGE) of albumin isolated/purified from pre- and post-treatment samples. To determine the sites of glycation of albumin, samples were carboxymethylated and digested by Glu-C endoprotease, and peptides were analyzed using liquid chromatography/mass spectrometry. RESULTS: GA level decreased almost linearly and reflected the improved glycemic state well. Trp-related fluorescence of pre- and post-treated samples did not change while AGE-related fluorescence increased depending on GA level. Ten major glycation sites were detected in the pre-treatment sample, while 3 major glycation sites were detected in post-treated samples. CONCLUSIONS: GA level reflects the status of blood glucose more rapidly than HbA(1c). Since GA level was related to AGE-related fluorescence and number of glycation sites, it might be a good marker for not only glycemic control of diabetic patients but also structural and functional changes of albumin.