We previously reported that type 2 diabetes risk, early impaired glucose tolerance and insulin resistance can be predicted by measuring the fasting levels of certain biomarkers. Here we validated these findings in randomly recruited healthy volunteers (n = 101) based on biomarker expression as well as various non-invasive indices. Weight, body mass index, waist circumference and visceral fat differed between individuals with impaired fasting glucose and/or impaired glucose tolerance, and normal subjects. Fasting plasma levels of glycated hemoglobin, leptin, pro-insulin and retinol binding protein 4 differed between impaired fasting glucose/impaired glucose tolerance and normal subjects group and between newly detected diabetes and normal subjects group. Insulin resistance was correlated with fasting levels of insulin and leptin/adiponectin (r = 0.913); of insulin, retinol binding protein 4 and leptin/adiponectin (r = 0.903); and of insulin, glycated albumin, and leptin/adiponectin (r = 0.913). Type 2 diabetes risk, early impaired glucose tolerance and insulin resistance were predicted with >98% specificity and sensitivity by comparing fasting glucose levels to the estimated Matsuda Index based on fasting levels of insulin, adiponectin and leptin with or without oxidative lineolate metabolites. Non-invasive indices are slightly correlated with glucose tolerance and insulin resistance but do not increase the accuracy of predicting type 2 diabetes risk.

We have previously found that fasting plasma levels of totally assessed 10- and 12-(Z,E)-hydroxyoctadecadienoic acid (HODE) correlated well with levels of glycated hemoglobin (HbA1c) and glucose during oral glucose tolerance tests (OGTT); these levels were determined via liquid chromatography-mass spectrometry after reduction and saponification. However, 10- and 12-(Z,E)-HODE alone cannot perfectly detect early impaired glucose tolerance (IGT) and/or insulin resistance, which ultimately lead to diabetes. In this study, we randomly recruited healthy volunteers (n = 57) who had no known history of any diseases, and who were evaluated using the OGTT, the HODE biomarkers, and several additional proposed biomarkers, including retinol binding protein 4 (RBP4), adiponectin, leptin, insulin, glycoalbumin, and high sensitivity-C-reactive protein. The OGTT revealed that our volunteers included normal individuals (n = 44; Group N), "high-normal" individuals (fasting plasma glucose 100-109 mg/dL) with IGT (n = 11; Group HN+IGT), and diabetic individuals (n = 2; Group D). We then used these groups to evaluate the potential biomarkers for the early detection of type 2 diabetes. Plasma levels of RBP4 and glycoalbumin were higher in Group HN+IGT, compared to those in Group N, and fasting levels of 10- and 12-(Z,E)-HODE/linoleic acids were significantly correlated with levels of RBP4 (p = 0.003, r = 0.380) and glycoalbumin (p = 0.006, r = 0.316). Furthermore, we developed a stepwise multiple linear regression models to predict the individuals' insulin resistance index (the Matsuda Index 3). Fasting plasma levels of 10- and 12-(Z,E)-HODE/linoleic acids, glucose, insulin, and leptin/adiponectin were selected as the explanatory variables for the models. The risks of type 2 diabetes, early IGT, and insulin resistance were perfectly predicted by comparing fasting glucose levels to the estimated Matsuda Index 3 (fasting levels of 10- and 12-(Z,E)-HODE/linoleic acids, insulin, and leptin/adiponectin).

Current diagnostic tests such as glycemic indicators have limitations for early detection of impaired glucose tolerance (IGT), which leads to diabetes. Oxidative stress induced by various oxidants in a random and destructive manner is considered to play an important role in the pathophysiology of a number of human disorders and diseases such as impaired glucose tolerance. We have developed an improved method for the measurement of in vivo lipid peroxidation, where the presence of 8-iso-prostaglandin F-2 alpha (8-iso-PGF(2 alpha)), hydroxyoctadecadienoic acids (HODEs), hydroxyeicosatetraenoic acids (HETEs), and 7-hydroxycholesterol (7-OHCh), as well as their parent molecules, linoleic acid (LA) and cholesterol (Ch), was determined by performing LC-MS/MS (for 8-iso-PGF(2 alpha), HODE, and HETE) and GC-MS (for 7-OHCh, LA, and Ch) after reduction with triphenyl phosphine and saponification by potassium hydroxide. We then applied this method to volunteers (n = 57), including normal type (n = 43), "high-normal'' (fasting plasma glucose, 100-109 mg/dL, n = 7), pre-diabetic type (IGT, n = 5), and diabetic type (n = 2) subjects who are diagnosed by performing oral glucose tolerance tests (OGTTs). Several biomarkers in plasma, such as insulin, leptin, adiponectin, interleukin-6, tumor necrosis factor-a, high sensitivity-C-reactive protein, HbA1c, and glucose levels were measured during OGTT. We found that the fasting levels of (10- and 12-(Z,E)-HODE)/LA increased significantly with increasing levels of HbA1c and glucose during OGTT and with insulin secretion and resistance index. In conclusion, 10-and 12-(Z,E)-HODE may be prominent biomarkers for the early detection of IGT and "high-normal'' type without OGTT.

Sandwich enzyme-linked immunosorbant assay (ELISA) using a 96-well plate is frequently employed for clinical diagnosis, but is time-and sample-consuming. To overcome these drawbacks, we performed a sandwich ELISA on a microchip. The microchip was made of cyclic olefin copolymer with 4 straight microchannels. For the construction of the sandwich ELISA for interleukin-6 (IL-6) or tumor necrosis factor-α (TNF-α), we used a piezoelectric inkjet printing system for the deposition and fixation of the 1st anti-IL-6 antibody or 1st anti-TNF-α antibody on the surface of the each microchannel. After the infusion of 2 µl of sample to the microchannel and a 20 min incubation, 2 µl of biotinylated 2nd antibody for either antigen was infused and a 10 min incubation. Then 2 µl of avidin-horseradish peroxidase was infused; and after a 5 min incubation, the substrate for peroxidase was infused, and the luminescence intensity was measured. Calibration curves were obtained between the concentration and luminescence intensity over the range of 0 to 32 pg/ml (IL-6: R(2) = 0.9994, TNF-α: R(2) = 0.9977), and the detection limit for each protein was 0.28 pg/ml and 0.46 pg/ml, respectively. Blood IL-6 and TNF-α concentrations of 5 subjects estimated from the microchip data were compared with results obtained by the conventional method, good correlations were observed between the methods according to linear regression analysis (IL-6: R(2) = 0.9954, TNF-α: R(2) = 0.9928). The reproducibility of the presented assay for the determination of the blood IL-6 and TNF-α concentration was comparable to that obtained with the 96-well plate. Simultaneous detection of blood IL-6 and TNF-α was possible by the deposition and fixation of each 1st antibody on the surface of a separate microchannel. This assay enabled us to determine simultaneously blood IL-6 and TNF-α with accuracy, satisfactory sensitivity, time saving ability, and low consumption of sample and reagents, and will be applicable to clinic diagnosis.

Several types of microchips have been developed for application in clinical diagnosis. A microchip made of cyclic olefin copolymer with straight microchannels (300 microm width and 100 microm depth) was employed for sandwich ELISA for the determination of serum type I C-peptide (PICP), a biomarker of osteoporosis. This assay enabled us to determine PICP with accuracy and high sensitivity, reducing the time for the immunoassay to 1/6, and the consumption of samples and reagents to 1/50 compared with the conventional method. Furthermore, cell microarray chips with 20,944 microchambers (105 microm width and 50 microm depth), made of polystyrene, were employed for malaria diagnosis and the detection of carcinoma cells among the leukocytes. Around 100 erythrocytes or leukocytes were accommodated in each microchamber with the formation of a monolayer. For malaria diagnosis, it offered 10-100 times higher sensitivity in the detection of malaria infected erythrocytes than conventional light microscopy, and easy operation within 15 min. By double staining for epithelial cells on the cell microarray chip, one carcinoma cell could be detected among 1,800,000 leukocytes. These results indicate the potential of microchips for clinic diagnosis.

Muscle-type carnitine palmitoyltransferase I (M-CPTI) is the key enzyme for fatty acid beta-oxidation in heart and skeletal muscles and in adipose tissue. So far, M-CPTI mRNA has been detected in white adipocytes from epididymal fat pads of rats and humans, but not in mouse adipocytes. To characterize the gene expression of M-CPTI in mice, we isolated the genomic DNA encoding mouse M-CPTI and determined its transcription initiation site. As a result, the mouse M-CPTI gene seemed to have multiple initiation sites, as in the case of the rat and human genes. Furthermore, the conserved nucleotide sequence of the response element for peroxisome proliferators was shown to exist in the upstream of the mouse gene as in that of the rat and human genes. From these observations, we suggest that the anomalous expression of M-CPTI in mouse adipocytes reported previously may be regulated by factors other than peroxisome proliferators. Previously, we reported that there were transcripts containing regions of both CK/EK-beta and M-CPTI genes in humans. In this study, we found that such transcripts also exist in rodents and that the amounts of the transcripts containing regions of both of these genes did not depend on the expression level of CK/EK-beta.