Hydroxyapatite-coated titanium oxide ameliorates dextran sulphate sodium-induced colitis by attenuating both innate and acquired immune reaction.

Introduction: Titanium oxide (TiO2) is a widely used oxidizer for environmental management. The power of TiO2 has been demonstrated by its photocatalytic activity. Hydroxyapatite (HA)-coated TiO2 (HA-TiO2) was used to test the in vivo effect on dextran sulphate sodium (DSS)-induced colitis in mice. Material and methods: Mice were monitored for body weight and then sacrificed on the seventh day, and the colon length was measured. Their faeces were analysed for intestinal microbiota distribution, and colon tissue was subjected to histological examination and immunohistochemical analysis. Results: Weight loss was significantly lower in HA-TiO2-fed mice than in mice without HA-TiO2. The colon length in the DSS colitis-induced mice was shortened, but HA-TiO2 feeding lessened this effect. Histological and immunohistochemical analyses of the colon revealed that macrophages and CD4+CD8+ T cells were observed in the colitis-occurring site, indicating the involvement of innate and acquired immunity in determining the degree of DSS-induced colitis. Intestinal microbiota analysis in faeces revealed changes in the distribution of multiple bacterial species after DSS colitis induction, and the increase/decrease of 2 Clostridium (sub)clusters moved in response to the colitis phenomenon. All the described effects of HA-TiO2 were photocatalytic activity-dependent because mice that were kept in the dark showed similar results to those treated with DSS alone without HA-TiO2. Conclusions: HA-coated TiO2 ameliorated DSS-induced colitis through photocatalytic activity, while HA-TiO2 diminished the changes in intestinal microbiota and immune reactions caused by DSS.

Reflux-related Extraesophageal Symptoms Until Proven Otherwise: A Direct Measurement of Abnormal Proximal Exposure Based on Hypopharyngeal Multichannel Intraluminal Impedance as a Reliable Indicator for Successful Treatment Outcomes.

BACKGROUND/AIMS: The Lyon Consensus defined parameters based on upper endoscopy and 24-hour combined multichannel intraluminal impedance-pH (MII-pH), that conclusively establish the presence of gastroesophageal reflux disease (GERD). However, the true role of upper endoscopy and MII-pH to evaluate patients with extraesophageal symptoms (EES) has not been well established. Hypopharyngeal MII (HMII), which directly measures laryngopharyngeal reflux (LPR) events, has been utilized to evaluate patients with EES suggestive of LPR. METHODS: This was a retrospective study involving patients with EES for > 12 weeks despite proton pump inhibitor therapy, and had no endoscopic confirmatory evidence for GERD and negative MII-pH. All patients were subsequently referred for further evaluation of EES with "unknown" etiology and underwent laryngoscopy and HMII. Based on HMII, abnormal proximal exposure (APE) was defined as LPR ≥ 1/day and/or full column reflux (reflux 2 cm distal to the upper esophageal sphincter) > 4/day. Patients with APE were offered antireflux surgery (ARS) and the outcome of ARS was objectively assessed using Reflux Symptom Index. RESULTS: Of 21 patients with EES which was thought to be GERD-unrelated based on endoscopy and MII-pH, 17 patients (81%) had APE. Eight patients with APE who had undergone ARS had significant symptomatic improvement in the Reflux Symptom Index score (19.6 ± 4.9 pre-ARS to 5.8 ± 1.4 post-ARS, P = 0.008). CONCLUSIONS: A conventional diagnostic approach using endoscopy and MII-pH may not be sufficient to evaluate patients with EES suggestive of LPR. HMII is essential to evaluate patients with EES, and APE could be a reliable indicator for successful treatment outcomes.

"Gas" laryngopharyngeal reflux cause unexplained chronic cough.

Hypopharyngeal multichannel intraluminal impedance (HMII) that can measure laryngopharyngeal reflux (LPR) events has supported the causal relationship between chronic cough (CC) and LPR containing liquid. However the role of "gas" LPR associated with CC has been poorly understood. We present two cases of patients with CC who had negative LPR containing liquid but had multiple episodes of "gas" LPR on HMII. The majority of "gas" LPR events had a minor pH drop at hypopharynx. Since any etiology of CC was excluded and medical therapy failed, both patients underwent laparoscopic antireflux surgery (LARS). Both of the patients had complete resolution of cough postoperatively. The present cases demonstrated successful outcome of LARS to treat the patients with CC who had documented "gas" LPR on HMII, thus suggesting the causal relationship between CC and "gas" LPR. The number of "gas" LPR events may need to be considered as an important diagnostic parameter.

Effect of modified ultrafiltration on cytokines and hemoconcentration in dogs undergoing cardiopulmonary bypass.

Cardiac surgery using cardiopulmonary bypass (CPB) generates severe inflammatory reactions secondary to hemodilution and surgical stress. This study was conducted to evaluate whether modified ultrafiltration (MUF) could be performed safely and to clarify its effects during mitral valve repair in dogs in terms of hemodilution and the status of inflammatory cytokines. We retrospectively studied 38 dogs with mitral valve disease who underwent MUF immediately after mitral valve repair under CPB. To determine the effect of MUF, we measured the pre- and post-MUF blood dilution and blood cytokine levels. The levels of red blood cells, hematocrit (HCT), and albumin were significantly increased after MUF, whereas interleukin (IL)-6 levels were significantly increased from 24.3 (range 9.6-54.6) to 32.3 (15.9-65.1) pg/ml. The levels of IL-8 and IL-10 declined significantly after MUF, from 368.2 (246.1-669.4) and 45.4 (28.6-76.1) to 272.2 (174.1-414.4) and 28.8 (18.8-44.5) pg/ml, respectively. Our results demonstrated that MUF can be applied in dogs undergoing CPB and is effective in achieving hemoconcentration. Moreover, MUF may be useful for the removal of cytokines. Further studies are needed to validate these findings and clarify the effects of inflammatory cytokines after CPB.

CD8+CD122+CD49dlow regulatory T cells maintain T-cell homeostasis by killing activated T cells via Fas/FasL-mediated cytotoxicity.

The Fas/FasL (CD95/CD178) system is required for immune regulation; however, it is unclear in which cells, when, and where Fas/FasL molecules act in the immune system. We found that CD8(+)CD122(+) cells, which are mostly composed of memory T cells in comparison with naïve cells in the CD8(+)CD122(-) population, were previously shown to include cells with regulatory activity and could be separated into CD49d(low) cells and CD49d(high) cells. We established in vitro and in vivo experimental systems to evaluate the regulatory activity of CD122(+) cells. Regulatory activity was observed in CD8(+)CD122(+)CD49d(low) but not in CD8(+)CD122(+)CD49d(high) cells, indicating that the regulatory cells in the CD8(+)CD122(+) population could be narrowed down to CD49d(low) cells. CD8(+)CD122(-) cells taken from lymphoproliferation (lpr) mice were resistant to regulation by normal CD122(+) Tregs. CD122(+) Tregs taken from generalized lymphoproliferative disease (gld) mice did not regulate wild-type CD8(+)CD122(-) cells, indicating that the regulation by CD122(+) Tregs is Fas/FasL-dependent. CD122(+) Tregs taken from IL-10-deficient mice could regulate CD8(+)CD122(-) cells as equally as wild-type CD122(+) Tregs both in vitro and in vivo. MHC class I-missing T cells were not regulated by CD122(+) Tregs in vitro. CD122(+) Tregs also regulated CD4(+) cells in a Fas/FasL-dependent manner in vitro. These results suggest an essential role of Fas/FasL as a terminal effector of the CD122(+) Tregs that kill activated T cells to maintain immune homeostasis.

TCR affinity for thymoproteasome-dependent positively selecting peptides conditions antigen responsiveness in CD8(+) T cells.

In the thymus, low-affinity T cell antigen receptor (TCR) engagement facilitates positive selection of a useful T cell repertoire. Here we report that TCR responsiveness of mature CD8(+) T cells is fine tuned by their affinity for positively selecting peptides in the thymus and that optimal TCR responsiveness requires positive selection on major histocompatibility complex class I-associated peptides produced by the thymoproteasome, which is specifically expressed in the thymic cortical epithelium. Thymoproteasome-independent positive selection of monoclonal CD8(+) T cells results in aberrant TCR responsiveness, homeostatic maintenance and immune responses to infection. These results demonstrate a novel aspect of positive selection, in which TCR affinity for positively selecting peptides produced by thymic epithelium determines the subsequent antigen responsiveness of mature CD8(+) T cells in the periphery.

Acceleration of ileal pacemaker activity in mice lacking interleukin 10.

BACKGROUND: Epidemiological studies indicate the genetic association between irritable bowel syndrome and inflammatory bowel disease, including genetic mutations related with interleukin 10 (IL-10), serotonin, and so on. On the other hand, it becomes clearer that interstitial cells of Cajal (ICC) play a major role in gut motility by coordinating the electric activity of cellular members and generating pacemaker potentials. METHODS: Ileal musculatures containing the myenteric plexus and ICC were isolated from wild-type (WT) and IL-10-deficient mice. A microelectrode array system was used to simultaneously measure 8 × 8 field potentials over a ∼1 mm area. Nifedipine and tetrodotoxin were applied to predominantly evaluate ICC electric activity. Histological changes were also assessed by immunohistochemistry. RESULTS: Potential mapping revealed that spontaneous electric activity was synchronized throughout the recording area in ileal musculature preparations of both WT and IL-10-deficient mice, but rapid propagation was observed in the latter. The spectral power in the frequency range of 9.4 to 30.0 cpm (Pw9.4-30.0) did not differ between these preparations, but the oscillation frequency estimated using autocorrelation analysis was significantly higher in IL-10-deficient mice than in WT mice (22.16 ± 4.10 versus 15.72 ± 1.61 cpm). In immunohistochemistry, no significant changes were observed in ICC, macrophages, and enteric neurons in the ileum of WT and IL-10-deficient mice. CONCLUSIONS: This study provides evidence for accelerated pacemaker activity in the ileum of IL-10-deficient mice, not accompanied by any significant histological changes. This could be accounted, as an example, by a genetic cross-link between inflammatory bowel disease and irritable bowel syndrome.

CD8+ CD122+ regulatory T cells contain clonally expanded cells with identical CDR3 sequences of the T-cell receptor ß-chain.

We identified CD8(+)  CD122(+) regulatory T cells (CD8(+)  CD122(+) Treg cells) and reported their importance in maintaining immune homeostasis. The absence of CD8(+)  CD122(+) Treg cells has been shown to lead to severe systemic autoimmunity in several mouse models, including inflammatory bowel diseases and experimental autoimmune encephalomyelitis. The T-cell receptors (TCRs) expressed on CD8(+)  CD122(+) Treg cells recognize the target cells to be regulated. To aid in the identification of the target antigen(s) recognized by TCRs of CD8(+)  CD122(+) Treg cells, we compared the TCR diversity of CD8(+)  CD122(+) T cells with that of conventional, naive T cells in mice. We analysed the use of TCR-Vß in the interleukin 10-producing population of CD8(+)  CD122(+) T cells marked by high levels of CD49d expression, and found the significantly increased use of Vß13 in these cells. Immunoscope analysis of the complementarity-determining region 3 (CDR3) of the TCR ß-chain revealed remarkable skewing in a pair of Vß regions, suggesting the existence of clonally expanded cells in CD8(+)  CD122(+) T cells. Clonal expansion in Vß13(+) cells was confirmed by determining the DNA sequences of the CDR3s. The characteristic TCR found in this study is an important building block for further studies to identify the target antigen recognized by CD8(+)  CD122(+) Treg cells.

Autophagic activity in thymus and liver during aging.

Impaired or deficient autophagy is believed to cause or contribute to aging, as well as several age-related pathologies. Thymic epithelial cells had a high constitutive level of autophagy. The autophagic process may play a supporting role or even a crucial role in the presentation of self-Ags in the thymus to shape the T-cell repertoires. Autophagic activity in the liver is important for the balance of energy and nutrients for basic cell functions. The abundance of autophagic structure in both cortical and medullary thymic epithelial cells and liver with mouse age has not been examined in detail. Here, we demonstrated that the architecture of mouse thymus and liver markedly changed with age. We found that the expression of LC3 detected by immunofluorescence and Western blot analysis was greatly decreased in thymus and liver of 12-month-old mice. The same level of reduction was observed in thymus and liver of 24-month-old mice. Ultrastructure analysis by an electron microscope revealed that the number of autophagic structure/vacuole in total thymic epithelial cells and hepatocytes decrease with age. The age-related decrease of autophagic structure in thymic epithelial cells may cause the reduction of immunocompetent T-cell pool in aged mice. The age-related decrease of autophagy in liver may induce accumulation of cellular materials in liver of aged mice.

Cladribine enhances apoptotic cell death in lung carcinoma cells over-expressing DNase γ.

Worldwide, lung cancer is the most common form of cancer and often has a poor prognosis. Establishment of effective therapies for lung cancer is a major concern in clinical cancer research. We compared the cytotoxic effects of chemotherapeutic agents including cisplatin, 5-fluorouracil, vinorelbine and cladribine, on a human lung cancer cell line, A549, and its derivative transfected with the DNase γ gene. We observed selective cytotoxicity of cladribine on the DNase γ-expressing sub-cell line of A549. Cladribine induces selective apoptosis in DNase γ-expressing A549 cells, which depends on activation of caspases. These results suggest that a combination therapy that includes cladribine along with the introduction of DNase γ has potential as a new therapeutic strategy for lung cancer.

Establishment of induced pluripotent stem cells from aged mice using bone marrow-derived myeloid cells.

If induced pluripotent stem (iPS) cells are to be used to treat damaged tissues or repair organs in elderly patients, it will be necessary to establish iPS cells from their tissues. To determine the feasibility of using this technology with elderly patients, we asked if it was indeed possible to establish iPS cells from the bone marrow (BM) of aged mice. BM cells from aged C57BL/6 mice carrying the green fluorescence protein (GFP) gene were cultured with granulocyte macrophage-colony stimulating factor (GM-CSF) for 4 days. Four factors (Oct3/4, Sox2, Klf4 and c-Myc) were introduced into the BM-derived myeloid (BM-M) cells. The efficiency of generating iPS cells from aged BM cultured in GM-CSF was low. However, we succeeded in obtaining BM-M-iPS cells from aged C57BL/6 mice, which carried GFP. Our BM-M-iPS cells expressed SSEA-1 and Pou5f1 and were positive for alkaline phosphatase staining. The iPS cells did make teratoma with three germ layers following injection into syngeneic C57BL/6 mice, and can be differentiated to three germ layers in vitro. By co-culturing with OP9, the BM-M-iPS cells can be differentiated to the myeloid lineage. The differentiated BM-M-iPS cells proliferated well in the presence of GM-CSF, and lost expression of Nanog and Pou5f1, at least in part, due to methylation of their promoters. On the contrary, Tnf and Il1b gene expression was upregulated and their promoters were hypomethylated.

Up-regulation of Gr1+CD11b+ population in spleen of dextran sulfate sodium administered mice works to repair colitis.

Dextran sulfate sodium (DSS) is commonly used in rodent IBD models to chemically induce acute intestinal inflammation. The acute course of colitis includes colon tissue damages and recovery from wounded tissues. As skin wound repair was delayed by splenectomy, we asked whether splenectomy would induce the delay of colonic wound healing. In splenectomized mice, body weight recovery, disease score and colon length were delayed. Surprisingly we found a great increase of Gr1+CD11b+ cells in spleen and bone marrow of DSS-administered mice. Anti-Gr-1 antibody treatment worsened the DSS- administered colitis. These results indicate that Gr1+CD11b+ cells induced by DSS worked to repair colon wound healing and repair colitis.

CD8+CD122+ regulatory T cells (Tregs) and CD4+ Tregs cooperatively prevent and cure CD4+ cell-induced colitis.

We identified CD8(+)CD122(+) regulatory T cells (Tregs) and demonstrated their importance in the maintenance of immune homeostasis and in the recovery from experimental autoimmune encephalomyelitis. In this paper, we show that CD8(+)CD122(+) Tregs effectively prevent and cure colitis in a mouse model. In our experiments, colitis was induced in lymphocyte-deficient RAG-2(-/-) mice by transferring CD4(+)CD45RB(high) cells that were excluded with CD4(+) Tregs. Cotransfer of CD8(+)CD122(+) cells clearly suppressed the development of colitis, and this suppressive effect was similar to that of CD4(+)CD45RB(low) cells that were mostly CD4(+) Tregs. CD8(+)CD122(+) cells obtained from IL-10(-/-) mice were unable to suppress colitis, indicating that IL-10 is an important effect-transmitting factor in the suppression of colitis. CD8(+)CD122(+) cells showed a suppressive effect when they were transferred 4 wk after CD4(+)CD45RB(high) cells, indicating the therapeutic potential of CD8(+)CD122(+) cells. A mixture of CD8(+)CD122(+) cells and CD4(+)CD45RB(low) cells was far more effective than single Tregs, indicating the synergistic effect of these Tregs. These overall findings demonstrate the potential role of CD8(+) Tregs, and possibly together with CD4(+) Tregs, in the medical care of inflammatory bowel disease patients.

Differentiation of induced pluripotent stem cells to thymic epithelial cells by phenotype.

Thymic epithelial cells (TECs) are present in both cortical and medullary thymic areas, and have crucial roles in functional T-cell development. In this study, we studied the differentiation of induced pluripotent stem cells (iPSCs) to TEC. When iPSC were cultured for 4 days in collagen IV-coated dishes in the presence of both activin A and lithium chloride (LiCl), the cells differentiated to definitive endoderm through mesendoderm. Further treatment with Fgf8 followed by Fgf7, Fgf10 and BMP4 differentiated iPSC to thymic epithelial progenitor cells (TEPCs) by phenotype. Gene expression of Hoxa3, Pax1 and Pax9 was observed and cell surface proteins EpCAM1 and MTS24 were detected at day 14 of iPSC differentiation. TEPCs differentiated to medullary TECs (mTECs) by phenotype following the addition of receptor activator nuclear factor B ligand with LiCl. Thus, we successfully induced efficient differentiation from mouse iPSC to TEPCs and mTEC by phenotype using chemically defined conditions.

GADD34 suppresses wound healing by upregulating expression of myosin IIA.

Wound healing consists of sequential steps of tissue repair, and cell migration is particularly important. In order to analyze the potential function of growth arrest and DNA damage inducible protein 34 (GADD34) in tissue repair, we performed in vitro and in vivo wound healing experiments. In an in vitro scratch assay, GADD34 knockout (KO) mouse embryonic fibroblasts (MEFs) had higher migration rates than did wild type (WT) MEFs. Furthermore, the rate of wound closure was faster in GADD34 KO MEFs than in WT MEFs. Using in vivo punch biopsy assays, GADD34 KO mice had accelerated wound healing compared to WT mice. WT mice expressed higher amounts of myosin IIA in migrating macrophages and myofibroblasts than did GADD34 KO mice. These results indicate that GADD34 negatively regulates cell migration in wound healing via expression of myosin IIA.

Toxic effects of D-galactose on thymus and spleen that resemble aging.

Continuous low-dose injection of d-galactose induces changes in mice that resemble accelerated aging. As such, these mice have been used as models to study mechanisms of aging. Here, we examined whether repeated (daily, for 60 days) subcutaneous injections (at 50 mg D-galactose/kg) into young adult (i.e., 2-month-old) mice induced changes in key immune system organs that were on par with those associated with aging. The results showed that galactose-treated mice develop histologic changes in their thymic cortical and medullary regions; immunohistochemical analysis revealed unorganized distributions of keratin-5 and keratin-8 proteins in the thymus of these hosts. These histological changes in the thymus of D-galactose-treated mice were also observed in the organs of aged (i.e., 24-month-old control mice); however, in this latter group, these changes were accompanied by a strong infiltration of adipose cells. Galactose-treated mice also evinced alterations within their splenic white and red pulp. Further, ultrastructural analyses of the thymus and spleen of the treated mice revealed increases in irregularly shaped lymphocytes bearing visible pyknosis. It was also seen that levels of autophagy within thymic epithelial cells were greatly decreased in the tissues of the galactose-treated mice, an outcome also seen in aged mice. Lastly, the level of memory T-lymphocytes and percentage of IgM-B220-B-lymphocytes in spleens of the galactose-treated mice were both increased (albeit insignificantly so) relative to values among splenocytes of age-matched control; however, these levels were not clearly as elevated as would be expected in "elderly" mice. Taken together, our results strongly suggest that d-galactose treatment can induce structural changes in the thymus and spleen, and some changes in organ-associated cell phenotypes, that are similar to several effects seen with aging. However, the fact that many endpoints do not appear to be truly reflective of what should be seen in immune system organs/cells of "elderly" mice now calls into question the appropriateness of the use of d-galactose (i.e., is it histologically/immunotoxicologically-proper?) to create age-mimicry in mice.

Intra-bone marrow bone marrow transplantation rejuvenates the B-cell lineage in aged mice.

Age-related reductions in the frequency and absolute number of early B lineage precursors in the bone marrow of aged mice have been reported. Reversal of B-cell lineage senescence has not been achieved. Age-related impairment of the B-cell lineage is caused by the decreasing functionality of hematopoietic and B lineage precursors, and reduced efficacy of bone marrow stromal cells that constitute the bone marrow microenvironment. To induce rejuvenation of aged B cells, we injected whole bone marrow from young donors to irradiated aged recipients through the tibia and analyzed B-cell development and immune responsiveness. In aged mice, we found significant reductions in the frequencies and absolute numbers of pro-B cells (B220(+)CD43(+)CD24(+)BP-1(-) and B220(+)CD43(+)CD24(int)BP-1(+)) and pre-B cells (B220(+)CD43(+)CD24(high)BP-1(+) and B220(+)CD43(-)IgM(-)IgD(-)). Intra-bone marrow bone marrow transplantation (IBM-BMT) of young marrow cells including both hematopoietic stem cells and bone marrow stromal cells reversed the reduction of pro-B cells and pre-B cells. In the periphery, the frequency and absolute number of marginal zone-B cell were not significantly different between young, old and IBM-BMT group. The frequency of follicular-B cells in the IBM-BMT group was significantly increased compared to old group. The frequency of B1a B cells in the peritoneal cavity was significantly decreased in the IBM-BMT group. Antibody production against T-independent antigens was not different among the young, the aged and IBM-BMT groups.

Human CD8+CXCR3+ T cells have the same function as murine CD8+CD122+ Treg.

The importance of CD8(+)CD122(+) Treg in the maintenance of immune homeostasis has been previously demonstrated in mice. Because the expression pattern of CD8 and CD122 in humans is different from that in mice, human CD8(+) Treg that correspond to the murine CD8(+)CD122(+) Treg have not been identified. In this study, we performed DNA microarray analyses to compare the gene expression profiles of CD8(+)CD122(+) cells and CD8(+)CD122(-) cells in mice and found that CXCR3 was preferentially expressed in CD8(+)CD122(+) cells. When we analyzed the expression of CD122 and CXCR3 in murine CD8(+) cells, we observed a definite population of CD122(+)CXCR3(+) cells. CD8(+)CXCR3(+) cells in mice showed similar regulatory activities to CD8(+)CD122(+) cells by in vivo and in vitro assays. While CD8(+)CD122(+)CXCR3(+) cells are present in mice, CD8(+)CXCR3(+) cells, but not CD8(+)CD122(+) cells, are present in humans. In the in vitro assay, human CD8(+)CXCR3(+) cells showed the regulatory activity of producing IL-10 and suppressing IFN-gamma production from CD8(+)CXCR3(-) cells. These results suggest that human CD8(+)CXCR3(+) T cells are the counterparts of murine CD8(+)CD122(+) Treg.

Antibodies to wounded tissue enhance cutaneous wound healing.

The wound repair process is a highly ordered sequence of events that encompasses haemostasis, inflammatory cell infiltration, tissue regrowth and remodelling. Wound healing follows tissue destruction so we hypothesized that antibodies might bind to wounded tissues, which would facilitate the engulfment of damaged tissues by macrophages. Here, we show that B cells, which produce antibodies to damaged tissues, are engaged in the process of wound healing. Splenectomy delayed wound healing, and transfer of spleen cells into splenectomized mice recovered the delay in wound healing. Furthermore, wound healing in splenectomized nude mice was also delayed. Transfer of enriched B220(+) cells by magnetic beads accelerated wound healing in splenectomized mice. We detected immunoglobulin G1 (IgG1) binding to wounded tissues by using fluorescein isothiocyanate-labelled anti-IgG1 6-24 hr after wounding. Splenectomy reduced the amount of IgG1 binding to wounded tissues. Immunoblotting studies revealed several bands, which were reduced by splenectomy. Using immunoprecipitation with anti-IgG bound to protein G we found that the intensity of several bands was lower in the serum from splenectomized mice than in that from sham-operated mice. These bands were matched to myosin IIA, carbamoyl-phosphate synthase, argininosuccinate synthase, actin and alpha-actinin-4 by liquid chromatography tandem mass spectrometry analysis.

Increased bone resorption may play a crucial role in the occurrence of osteopenia in patients with type 2 diabetes: Possible involvement of accelerated polyol pathway in its pathogenesis.

In order to investigate the underlying mechanism of alterations in bone mineral metabolism in patients with type 2 diabetes, we determined circulating levels of bone functional markers along with urinary excretion of sorbitol (SOR) and bone mineral density (BMD), and also examined their mutual interrelationship. A total of 151 male type 2 diabetic patients were examined in this study. Forty-eight age-matched male healthy subjects were also studied as the controls. A significant reduction of serum intact osteocalcin (i-OC) was found in the diabetic groups (p<0.01). On the other hand, circulating levels of tartrate resistant acid phosphatase (TRAP) in the diabetic patients were significantly higher than those in the controls (p<0.01). Interestingly, a significantly negative relationship was observed between BMD and serum TRAP (p<0.01), although no significant relationship was noted between BMD and serum i-OC in diabetic patients. Urinary excretion of SOR was significantly elevated in the diabetic patients when compared with the controls (p<0.01). In addition, a significantly positive correlation was observed between serum TRAP and urinary SOR (p<0.01), but not between serum i-OC and urinary SOR. Elevated serum TRAP in diabetes was reduced after the administration of aldose reductase inhibitor (p<0.05). It seems most likely that the increase in osteoclastic function probably due to accelerated polyol pathway plays a crucial role in the pathogenesis of decreased bone mineral content in male patients with type 2 diabetes.