Cholesterol sulfate has been found to be a significant component of stratum corneum lipids and plays an important role in keratinocyte differentiation and development of the barrier, but we did not understand how it was made. In 2001, we found cholesterol sulfotransferase (SULT2B1b) which sulfonates cholesterol with universal sulfate donor (PAPS) and expressed in skin and the primary culture of human keratinocyte cell. SULT2B1b also sulfonates several oxysterols including cytotoxic 7-ketocholesterol, and cholesterol sulfate is completely nontoxic to cells. Recently, it has been reported that relationships between sulfonation of oxysterols and nuclear receptors which were involved in the regulation of genes engaged in lipid metabolism. These reports suggested that sulfonation of oxysterols might regulate lipid metabolism.

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We review the characterization of lactate dehydrogenase (LD)-immunoglobulins complex and the mechanism of LD inhibition by abnormal immunoglobulin. In Bence Jones protein (BJP) which combines with LD, it has been considered that LD binds the abnormal immunoglobulin which has an aromatic amino acid at the first portion of N-terminal, such as tyrosine and abnormal three-dimensional structure, such as longer β-sheet structure of N-terminal. However, the mechanism that is responsible for formation of immunoglobulins with LD inhibition is unclear. We recently discovered a patient with low LD activity and an abnormal LD isozyme pattern. The LD activity of the patient's serum was inhibited more strongly at 4° than at 37°. The immunoglobulin responsible for LD inhibition was an IgAl- λ. The LD inhibition by the patient's IgAl was blocked by reduction and alkylation and by NADH. Polymerization of the patient's IgAl might play an important role in its interaction with LD. Moreover, the possibility exists that part of the patient's IgAl gets into a pocket of LD in the absence of NADH, and that LD recognizes the patient's IgAl at the NAD⁺-binding site of LD, since that part of the patient's IgAl may appear similar in structure to NAD⁺.

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Based on confirmation of lysine 525 (Lys⁵²⁵) in albumin as the primary glycation site in a study of glycation sites on glycated albumin (GA), we determined levels of peptides containing glycated and non-glycated Lys⁵²⁵ in albumin, and developed a GA measurement method in which the ratio of these peptides was used as an indicator of blood glucose kinetics. Substances for measurement were defined as peptides containing glycated Lys⁵²⁵ (G-525Lys, Lys⁵²⁵- N^ε-(1-deoxy-D-fructos-1-y1)-undecapeptide) and those containing non-glycated Lys⁵²⁵ (NG-525Lys, Lys⁵²⁵-undecapeptide) obtained from enzymatic hydrolysis of plasma or serum using endoproteinase Glu-C. Quantification was performed by liquid chromatography/mass spectrometry (LC/MS) using the respective synthetic peptides as calibrators. GA level was expressed as [G-525Lys/(G-525Lys + NG-525Lys)] in milimoles/mole.Within-run and day-to-day CVs were< 1.0%. Correlation coefficients between the present method and common measurement methods were > 0.97. The present GA measurement method enables GA level to be expressed in SI units.

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Measurement of glycated albumin (GA) has been introduced into clinical laboratory medicine as a new marker for diabetes, and it is now widely used as a routine test in Japan. However, due to absence of a reference measurement method, it was necessary to promptly establish a standardized procedure. Since 2002, the following activities have been conducted as part of the GA standardization project: 1) a survey of the actual conditions to understand the current status of GA measurement; 2) a basic study to define GA; 3) preparation of reference materials and establishment of a reference method; and 4) determination of reference intervals.
The basic study to define GA involved peptide mapping, since albumin has multiple glycation sites and it was necessary to determine suitable glycation sites exist in GA. Based on these results, in the present study, GA was defined as “albumin containing lysine residues bound to glucose (N^E(1-deoxy-D- fructos- 1-yl)-L-lysine: DOF-Lys) ” A method based on isotope dilution mass spectrometry (ID-MS) is proposed as the reference measurement procedure to be used as a traceable tool of GA standardization. Herein, we present the ID-MS procedure and its performance results.
Methods: After isolating albumin from serum, stable isotopes of DOF-Lys and lysine (Lys) were added to albumin fractions as internal standards and glycation sites were subject to hydrogenation followed by hydrolysis. All freed DOF-Lys and Lys residues were recovered by reduced pressure drying, and their isotopic ratios were determined using liquid chromatography-mass spectrometry. GA levels were expressed in terms of molar ratios (unit: mmol/mol) of all liberated DOF-Lys residues and albumin calculated from all liberated Lys residues.
Results: The measurement precision of the present method was CV 1.2% for within-run reproducibility (n=10) and CV 1.4% for day-to-day reproducibility (n=15); favorable compatibility with routine tests (r=0.996) was demonstrated.
Conclusion: The present ID-MS method of GA. measurement is a valid reference measurement procedure for GA.

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