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Open access

V. Manolov, B. Atanasova, V. Vasilev, K. Tzatchev and M. Velizarova

Summary

Hepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Hepcidin quantification in human blood may provide further insights for the pathogenesis of disorders of iron homeostasis and might prove a valuable tool for clinicians for the differential diagnosis of anaemia. This study describes ELISA immunoassay for hepcidin quantification in human serum. We used a sandwich ELISA method from USCN Life Science inc., that consists of ready to use, pre-coated 96-well strip plate with 2 antihepcidin-25 monoclonal antibodies. A recombinant hepcidin in 16 μg/l concentration is used as a standard; it reconstitutes with 1.0 ml standard diluent to prepare a stock solution. We correlated ELISA results of hepcidin-25 measurements in healthy population with hemodialysis patients. The sandwich ELISA was highly specific for hepcidin-25, having a low limit of quantification of 0.020 μg/l. Hepcidin- 25 concentrations were increased in hemodialysis patients (median 33.05 μg/l, range 22.31 -60.98 μg/l, n = 10) compared with healthy individuals (median 12.41 μg/l, range 6.05-18.53 μg/l, n = 40). The use of 2 monoclonal antibodies in a sandwich ELISA format provides a robust, convenient and not very expensive method for measuring concentrations of the active form of hepcidin. It should help to improve our understanding of the role of hepcidin in regulating iron metabolism.

Open access

M. P. Genova, K. Todorova-Ananieva, B. Atanasova and K. Tzatchev

Summary

The aim of the present study was to evaluate the levels of pro-insulin and pro-insulin/ insulin ratio (PIR) in pregnant with normal glucose tolerance (NGT), pregnant with gestational diabetes mellitus (GDM) and women after delivery with GDM history. Normal pregnancy is characterized by progressive insulin resistance, which is physiologically compensated by an increase in insulin secretion. The higher secretion of the insulin precursor pro-insulin has been associated with β-cell dysfunction. A total of 102 pregnant women between 24-28 gestational weeks (53 GDM pregnant, 49 with NGT) and 22 postpartum with GDM history, as assessed by a 2h oral glucose tolerance test, were included in the study. Fasting plasma insulin and pro-insulin (PI) concentrations at the basal state were measured in all women. The ratio pro-insulin/insulin was calculated. BMI was significantly higher in GDM pregnant compared to NGT weight-matched group (30.56 ± 6.9 vs. 30.56 ± 6.9; p < 0, 011) and compared to the levels after delivery (30.56 ± 6.9vs. 27.9 ± 6, 27; p < 0, 001). Significant differences in the levels of PI between NGT and GDM pregnant (3.94 ± 2.78 vs. 7.59 ± 5.27; p = 0.006), between GDM and postpartum women (7.59 ± 5.27 vs. 4.46 ± 1.14; p = 0.022) were established. No signifi cant difference in the level of PIR between two pregnant groups was observed. Separately NGT and GDM showed signifi cant difference compared to young mothers (0.41 ± 0.14 vs. 0.148 ± 0.031, p < 0.02; 0.46 ± 0.16 vs. 0.148 ± 0.031, p = 0.009). Fasting insulin was statistically higher in GDM pregnant compare to NGT and women after delivery (13.84 ± 8.43 vs. 11.35 ± 7.38, p = 0.02; 13.84 ± 8.43 vs. 10.60 ± 7.53, p < 0.01). The correlation between PIR and BMI in the three groups studied were r = 0.416; r = 0,741; r = 0,556 (with statistical significance p = 0.01 between NGT and GDM pregnancy, p = 0.02 between GDM pregnancy and postpartum, p < 0.0001 between NGT pregnancy and young mother with GDM history). In our study, comparison of PI levels between pregnant with NGT and GDM demonstrated that the OR of developing GDM was 1.194 (95% CI, 1.028-1.329, P = 0.001). Increasing the value of PI with 10 pmol/l increases the risk for development of GDM with 19.4%. According to our results, pregnant with GDM have elevated levels of pro-insulin and PIR which could serve as a markers for this condition. These results support our findings about relationship and influence of BMI on β-cell functions, established in this study with normotolerant, gestational diabetes pregnant and women postpartum with GDM history. These results demonstrate that gestational diabetics have abnormalities in pancreatic beta-cell secretion, which are likely to be important both in the etiology of gestational diabetes and non-insulin dependent diabetes.

Open access

M. P. Genova, K. Todorova-Ananieva, B. Atanasova and K. Tzatchev

Summary

The aim of the present study was to assess β-cell function using homeostasis model (HOMA-B) and disposition index (DI) in pregnant women with/without gestational diabetes, and after delivery. A total of 102 pregnant women between 24-28 gestational weeks (53 with gestational diabetes mellitus (GDM) and 49 with normal glucose tolerance (NGT) and 22 GDM postpartum women (8-12 weeks after delivery) were included in the study. All postpartum women had a history of GDM. HOMA indexes (insulin resistance - HOMA-IR and HOMA-B for assessing β-cell function) were calculated from fasting glucose and insulin concentrations. To estimate insulin secretion independent of insulin sensitivity, DI was calculated using glucose and insulin levels at 0 and 60 min during the course of a 2 h 75g oral glucose tolerance test (OGTT). In GDM pregnant women HOMA-B was significantly lower compared to NGT women (p = 0.017), but there was no significant difference compared to women after birth (NS). There was difference between NGT and postpartum women (p < 0.05). DI was significantly lower for GDM pregnant women in comparison to NGT and postpartum women (p < 0.0001; p = 0.011), between NGT and women after birth (p < 0.04). In our study, comparison of НОМА-В in NGT and GDM pregnant women demonstrated that the OR of developing GDM was 0.989 (95% CI, 0.980-0.998, P = 0.013), and comparison of DI in healthy pregnant and GDM showed that the OR of developing GDM was 0.967 (95% CI, 0.947-0.988, P = 0.002). Therefore, HOMA-B and DI appear to be protective factors in the risk of developing GDM. According to our results, assessment of β-cell function, using HOMA-B and DI, showed that they are lower in GDM than NGT group and postpartum women. It is important to note that HOMA-B did not show significant difference between GDM pregnant and women after delivery with a history for GDM. We assume that pregnant women with GDM have a pancreatic β-cell defect that remains after birth. These women are at increased risk for developing diabetes mellitus, the most frequent type 2 diabetes, in the future after birth.

Open access

I. Ivanova, B. Atanasova, A. Kostadinova, Y. Bocheva and K. Tzatchev

Summary

Copper (Cu) and zinc (Zn) are essential for life. Body Cu and Zn content depends on variety of factors - age, gender, and diet, type of drinking water, geographical location and genetic predisposition. Copper status becomes even more relevant not only in rare genetic disorders such as Wilson disease but in diseases such as cardiovascular ones, impaired glucose tolerance and neuro-degenerative and tumor diseases. The study aimed to examine the distribution of serum Cu and Zn in a representative group of the Bulgarian population and to describe factors which influence metal content. It also aimed to describe the link between serum Cu levels and the frequency of Alzheimer’s disease (AD) in Bulgarians. Cu and Zn in serum were measured in 379 individuals (172 males and 207 females) from 5 different regions in Bulgaria by flame atomic absorption using AAnalyst 400, Perkin Elmer. Statistical analyses were performed by SPSS, 19. Median and inert-quartile range (IQR) for blood Cu were 15.89 (13.87-7.89) μmol/L and for Zn - 13.00 (11.7-14.68) μmol/L in the examined group. Higher Cu levels in females than in males were found (p < 0.001). Decrease of Zn with aging was established (p > 0.05). Significant difference (p < 0.05) was found in serum Cu between young people (< 30 year old) and adults over 61 year old. Statistically significant difference in Cu and Zn was observed (p < 0.05) in respect of residences. Difference without significance was measured between serum lipids and serum Cu (p = 0.541) and Zn (p = 0.741).

Open access

I. Dimitrova Ivanova, B. Atanasova, S. Dragneva, L. Vladimirova, Z. Krastev, A. Kostadinova, A. Ivanova and K. Tzatchev

Summary

Pre-analytical factors of variation need to be carefully considered and investigated in efforts to harmonize all aspects of the total testing process. This study aimed to evaluate contamination and stability in copper (Cu) analysis of serum and urine by flame atomic absorption spectroscopy (FAAS) and to compare the stability of urine Cu in controls and in D-penicillamine (D-PA) administration. Cu was measured by AAnalyst 400, Perkin Elmer, USA. Blood was collected in BD Vacutainer®SSTTM II Advance tubes and BD Vacutainer® Trace Element tubes. Sterile polyethylene and polypropylene vessels for collection, transportation, storage and preliminary preparation of samples were used in urinalysis. Stability in serum and 24 h urine was evaluated in two temperature regimens: 15-25°C and 2-8°C, for particular time of storage. No significant differences (p = 0.20) in Cu concentration was found between the two types of tested tubes with patient`s sera. The stability of the samples (serum and urine) was better at refrigeration temperature. In urine the stability was better in D-PA administration.Standardization of Cu analysis could be achieved by assessing the aspects of pre-analytical factors of variations.