A Comparison of Four Methods (Immunochemistry and HPLC) for Determination of 25-(OH)-Vitamin D in Postmenopausal Women.

BACKGROUND: Three immunochemical methods for the determination of 25-(OH)-vitamin D and validated HPLC method for the determination of 25-(OH)-vitamin D3 and 25-(OH)-vitamin D2 were compared. 62 patient samples from postmenopausal women were measured and the results obtained by all these methods were compared. METHODS: We used three chemiluminescent assays for determination of 25-(OH)-vitamin D. 25-(OH)-vitamin D3 and 25-(OH)-vitamin D2 were determined by HPLC with UV detection (Agilent 1200). The chemiluminescent assays were performed using the Abbott Architect i4000SR analyzer (Abbott Laboratories, Germany), the ADVIA Centaur (Siemens, USA), and the Liaison XL (DiaSorin Inc, USA). The statistical evaluation was done using GraphPad Prism 6.0. RESULTS: The data were tested by Tukey's multiple comparison test. All methods showed significant differences in comparison with the immunochemical method from DiaSorin (p < 0.001 for Abbott, p < 0.05 for Siemens, and p < 0.0001 for HPLC). The comparison of the immunochemical method from Siemens with HPLC was also significant, p < 0.05. The mean of DiaSorin measurements was 38% lower than the mean of HPLC measurements. The non-significant difference was shown by the comparison of Abbott with HPLC and also Abbott with Siemens. Means for the 25-(OH)-vitamin D methods used were: Abbott 70.2 ± 24.2 nmol/L, Siemens 67.6 ± 27.9 nmol/L, DiaSorin 53.5 ± 17.1, and HPLC 82.4 ± 40.0 nmol/L. CONCLUSIONS: The comparison of the DiaSorin immunochemical assay with other tested methods showed the greatest deviation. The mean of DiaSorin measurements was 38% lower than the mean of HPLC measurements. According to the results of the DiaSorin method, most patients treated with vitamin D would not achieve the optimal level of 25-(OH)-vitamin D and this could negatively affect the clinical decision.

Concentrations of MMP-9 and TIMP-1 in lip tissue and their impact on cleft lip surgery healing.

AIM: To compare aspects of wound healing after cleft lip surgery performed within one week of age and wound healing after surgery performed within 2 - 4 months of age, especially concentrations of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in tissue removed during surgery. METHODS: 34 tissue samples (26 boys and 8 girls) were removed during surgery within one week of age (n=19) or within 2 - 4 months of age (n=15). Tissue samples were separated into epidermis, dermis and mucous membrane. Proteins were extracted in cacodylic buffer for 24 h at a temperature 2 - 8 ºC. Total protein concentrations were examined using a modification of the Lowry method. Samples were examined using ELISA kit Amersham Biotrak Activity Assay (GE Healthcare UK) for detection of MMP-9 and TIMP-1 concentrations. RESULTS: MMP-9: early surgery - epidermis 2.168 ± 3.303 µg/g of protein (mean ± SD), dermis 1.251 ± 1.848 µg/g, 2 - 4 months surgery - epidermis 0.347 ± 0.212 µg/g, dermis 0.555 ± 0.276 µg/g. TIMP-1: early surgery - epidermis 1.762 ± 2.162 µg/g, dermis 1.628 ± 0.822 µg/g, mucous membrane 2.066 ± 1.717 µg/g, 2 - 4 months surgery - epidermis 1.881 ± 2.810 µg/g, dermis 3.117 ± 1.540 µg/g, mucous membrane 4.833 ± 6.550 µg/g. CONCLUSIONS: There were no significant differences in concentrations of protein MMP-9 in epidermis and dermis and TIMP-1 in epidermis and mucous membrane according to time of surgery. Significantly decreased levels of TIMP-1 in dermis were found in samples obtained from early surgery compared to levels in samples obtained from 2 - 4 months surgery.

Early diagnostic markers of sepsis after oesophagectomy (including thromboelastography).

BACKGROUND: Early diagnosis of sepsis and its differentiation from the noninfective SIRS is very important in order that treatment can be initiated in a timely and appropriate way. In this study we investigated standard haematological and biochemical parameters and thromboelastography (TEG) in patients who had undergone surgical resection of the oesophagus to find out if changes in any of these parameters could help in early differentiation between SIRS and sepsis development. METHODS: We enrolled 43 patients (aged 41-74 years) of whom 38 were evaluable. Blood samples were obtained on the morning of surgery and then at 24-hour intervals for the next 6 days. Samples were analysed for procalcitonin (PCT), C-reactive protein (CRP), interleukin-6 (IL- 6), aspartate transaminase (AST), alanine transaminase (ALT) , lactate, white blood count (WBC), D-dimers, antithrombin (AT), international normalised ratio (INR), activated partial thromboplastin time (APTT) and parameters of TEG. RESULTS: Significant differences between patients who developed sepsis during this period (9 patients) and SIRS were found in ALT on Day 1, in AST on Days 1-4, in PCT on Days 2-6; in CRP on Days 3-6; in IL-6 on Days 2-5; in leucocytes on Days 2, 3 and 6; and in D-dimers on Days 2 and 4. Significance values ranged from p < 0.0001 to p < 0.05. CONCLUSIONS: Sequential measurements of ALT, AST, PCT and IL-6 during the early postoperative period can be used for early differentiation of sepsis and postoperative SIRS after oesophagectomy. Among the coagulation parameters measured, only D-dimer concentrations appeared to be helpful in this process. TEG does not seem to be a useful early predictor of sepsis development; however it can be used to differentiate sepsis and SIRS from Day 5 after surgery.

Urinary transforming growth factor-ß1 in children with obstructive uropathy.

AIM: Obstructive uropathies (OU) in childhood constitute one of the major causes of chronic renal insufficiency. Transforming growth factor-ß1 (TGF-ß1) is considered to be the major fibrogenic growth factor. The aim of the present study was to investigate urinary TGF-ß1 levels in children with obstructive and non-obstructive uropathies (NOU). METHODS: This study involved 19 children with OU, 11 children with non-obstructive hydronephrosis and 21 healthy children. Urinary TGF-ß1, proteinuria, microalbuminuria and urinary α1-microglobulin were measured, and renal function was assessed. The results were statistically analyzed. RESULTS: Mean urinary TGF-ß1 concentrations in patients with OU were significantly higher than those with NOU (4.14 ± 0.67 creatinine vs 1.80 ± 0.24 pg/mmol creatinine, P < 0.05) and healthy controls (1.66 ± 0.28 pg/mmol creatinine, P < 0.05). Positive correlations of urinary TGF-ß1 concentrations with proteinuria (r = 0.87, P < 0.0001) and urinary α1-microglobulin (r = 0.82, P = 0.0002) were found in patients with OU. CONCLUSION: Children with OU have higher urinary TGF-ß1 than children with NOU. Urinary TGF-ß1 may be a useful non-invasive tool for the differential diagnosis between OU and NOU in children. A positive correlation of TGF-ß1 with markers of renal tissue damage in patients with OU was found.

Protein microarray analysis as a tool for monitoring cellular autoreactivity in type 1 diabetes patients and their relatives.

BACKGROUND: Autoreactive T cells have a crucial role in type 1 diabetes (T1D) pathogenesis. OBJECTIVES: The aim of our study was to monitor the in vitro production of cytokines by peripheral blood mononuclear cells (PBMCs) after stimulation with diabetogenic autoantigens. SUBJECTS: Ten T1D patients (tested at the time of diagnosis and 6 and 12 months later), 10 first-degree relatives of the T1D patients, and 10 controls underwent the study. METHODS: PBMCs were stimulated with glutamic acid decarboxylase 65 (GAD65) amino acids (a.a.) 247-279, 509-528, and 524-543; proinsulin a.a. 9-23; and tyrosine phosphatase (islet antigen-2)/R2 a.a. 853-872. Interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10, IL-13, interferon (IFN)-gamma, tumor necrosis factor beta, transforming growth factor beta1, and granulocyte colony-stimulating factor (GCSF) were analyzed by protein microarray. RESULTS: Differences in cytokine(s) poststimulatory and mainly in basal production were observed in all groups. The most prominent findings were in controls, the higher basal levels of IL-2, IL-4, IL-5, IL-13, and GCSF were observed when compared with relatives (p < 0.05, for all). After stimulation in controls, there was a significant decrease in IL-2, IL-13, GCSF, and IFN-gamma (p < 0.05, for all). The group of relatives was the most variable in poststimulatory production. A strong correlation between cytokines production was found but groups differed in this aspect. CONCLUSION: By multiplex analysis, it may be possible, for example, to define the risk immunological response pattern among relatives or to monitor the immune response in patients on immune modulation therapy.

Adiponectin, adipocyte fatty acid binding protein, and epidermal fatty acid binding protein: proteins newly identified in human breast milk.

BACKGROUND: Breastfeeding may protect children from developing metabolic syndrome and other diseases later in life. We investigated novel proteins in human breast milk that might play a role in this process. METHODS: We used ELISA to measure adiponectin, adipocyte and epidermal fatty acid binding proteins (AFABP, EFABP), and leptin concentrations in human breast milk obtained from 59 mothers 48 h after initiation of lactation. Using a questionnaire and medical records, we collected information about the mothers and newborns. RESULTS: Mean (SE) adiponectin concentrations in breast milk were 13.7 (0.8), range 3.9-30.4 microg/L; AFABP concentrations 26.7 (4.4), range 1.2-137.0 microg/L; EFABP concentrations 18.1 (1.4), range 0.8-47.0 microg/L; and leptin concentrations 0.50 (0.05), range 0-1.37 microg/L. We found a significant correlation between AFABP and EFABP concentrations (r = 0.593, P <0.0001). Maternal EFABP concentrations were significantly higher in mothers who delivered boys than in those who delivered girls [21.7 (2.3) vs 15.4 (1.7) microg/L, P = 0.028] and correlated with newborn birth weight (r = 0.266, P = 0.045). Maternal leptin correlated with body weight before pregnancy (r = 0.272, P = 0.043) and at delivery (r = 0.370, P = 0.005), body mass index before pregnancy (r = 0.397, P = 0.003) and at delivery (r = 0.498, P <0.0001), body weight gain during pregnancy (r = 0.267, P = 0.047), and newborn gestational age (r = 0.266, P = 0.048). Leptin was significantly lower in mothers who delivered preterm vs term babies [0.30 (0.09) vs 0.60 (0.05) ug/L, P = 0.026]. CONCLUSIONS: Concentrations of adiponectin, AFABP, and EFABP in human breast milk are related to nutritional variables of mothers and newborns and thus may play a role in the protective effects of breastfeeding.

Anti-GAD65 reactive peripheral blood mononuclear cells in the pathogenesis of cystic fibrosis related diabetes mellitus.

OBJECTIVE: A role of autoreactive T cells for type 1 diabetes pathogenesis is considered crucial. In our pilot study we addressed if autoreactive mononuclear cells are present also in peripheral blood of patients with other specific forms of diabetes as cystic fibrosis related diabetes (CFRD). METHODS: Cellular immune responses to a known beta-cell autoantigen (GAD65 and GAD65 derived peptides) were analysed by ELISPOT (IFN-gamma) and by protein microarray analysis in four patients suffering from CFRD, in four cystic fibrosis (CF) patients without diabetes, in eight type 1 diabetes patients (without CF) and in four healthy controls. RESULTS: Response to the autoantigen GAD65 (protein and peptides) was observed in 7/8 patients suffering from CF and in all type 1 diabetes patients. Post-stimulation production of Th1 cytokines (IFN-gamma, TNF-beta) was observed in 2/4 CFRD, 1/4 CF patients and in 7/8 type 1 diabetes patients. All these patients carry prodiabetogenic HLA-DQ genotype. Th2- and Th3 type of cytokine pattern was observed in 2/4 CF patients. Production of IL-8 was observed in the third CFRD as well as in the third CF patient and in 1/8 type 1 diabetes patient and borderline production of this chemokine was also observed in 2/4 healthy controls. No reaction was observed in the other 2/4 healthy controls and in the fourth CFRD patient who carried a strongly protective genotype and did not produce autoantibodies. The most potent peptide of GAD65 was amino acids 509-528. CONCLUSIONS: We consider our observations as a sign of a reaction directed against the self-antigen GAD65 that are closely connected to type 1 diabetes. In CF patients who do not develop diabetes autoreactive mechanisms are very probably efficiently suppressed by immune self-tolerance mechanisms. CFRD patients are a heterogenic group. To disclose those who may display features of autoimmune diabetes could have an impact for their therapy and prognosis.