Background: The first step in iron absorption requires the reduction of ferric iron to ferrous iron, a change that is catalyzed by duodenal ferric reductase. Iron deficiency is associated with high iron absorption, high ferric reductase activity, and high duodenal ascorbate concentrations in experimental animals, but it is not known whether a relation between reductase and ascorbate is evident in humans. Objective: The objective of the study was to assess the relation between ferric reductase activity in human duodenal biopsy specimens and ascorbate concentrations in iron-replete and iron-deficient subjects. Design: Patients and control subjects were overnight-fasted adults presenting sequentially for upper gastrointestinal endoscopic investigation. Ferric reductase activity in duodenal biopsy specimens was assayed by using nitroblue tetrazolium. Ascorbate was assayed in duodenal biopsy specimens and plasma. Results: Iron-deficient patients had significantly higher reductase activity (n = 6–9;P < 0.05) and duodenal (n = 20;P < 0.001) and plasma (n = 6;P < 0.001) ascorbate concentrations than did control subjects. Incubation of biopsy specimens with dehydroascorbate (to boost cellular ascorbate) increased reductase activity in the tissues that initially had normal activity (n = 9;P < 0.01) but inhibited reductase activity in the tissues that already had high reductase activity (n = 13;P < 0.001). Conclusions: Iron deficiency in humans is associated with increased duodenal ascorbate concentrations. This finding suggests that increased reductase activity is partly due to an increase in this substrate for duodenal cytochrome b reductase 1.

Ascorbate has long been thought to play an important role in intestinal iron absorption. The recent identification of a possible ascorbate-dependent duodenal ferric reductase suggests a role for intracellular ascorbate in the control of iron absorption. We set out to determine whether duodenal ascorbate concentrations are altered by treatments known to alter the rate of iron absorption and whether ascorbate levels affect duodenal reductase activity. Duodenal ascorbate was extracted and assayed by HPLC and/or a chemical assay. Ferric reductase was assayed in vitro with ferric nitrilotriacetate or nitroblue tetrazolium as substrates. Duodenal ascorbate concentrations were increased by iron deficiency, genetic hypotransferrinemia, and hypoxia. Parenteral iron overload increased iron stores but did not affect duodenal ascorbate concentrations. Hemolytic anemia induced in mice by phenylhydrazine injection also did not affect duodenal ascorbate concentrations. In vitro studies with incubated duodenum showed that decreased tissue ascorbate was associated with decreased mucosal ferric reductase activity, whereas incubation with dehydroascorbate prevented both the decrease in ascorbate concentration and reductase activity. Mouse duodenum ascorbate concentrations changed in response to treatments that altered iron absorption rates; in particular, ascorbate levels generally increased when iron absorption was increased by iron deficiency, hypoxia, or genetic hypotransferrinemia. We conclude that changes in ascorbate levels are associated with changes in ferric reductase activity. These findings are consistent with the proposal that duodenal ascorbate plays a role in intestinal iron absorption.

Hepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Hepcidin quantification in human serum provides new insights for the pathogenesis of disorders of iron homeostasis. This study describes an ELISA immunoassay for hepcidin quantification in human serum and reference ranges for Bulgarian population. 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. We correlated ELISA results of hepcidin-25 measurements in healthy population to ferritin, hemoglobin concentration in reticulocytes, transferrin, and iron levels. The sandwich ELISA was highly specific for hepcidin-25. We found that serum hepcidin levels for Bulgarian population are 3.052 μg/L - 37.750 μg/L, which is quite similar to that established by WCX-TOF MS from the Laboratory of Genetic, Endocrine and Metabolic Diseases; Dept. of Laboratory Medicine, Radbound University Medical Centre; Nijmegen, The Netherlands. Ferritin levels and hemoglobin concentration in reticulocytes correlated significantly to serum hepcidin levels (0.3 < r < 0.5, p < 0.010). Transferrin levels showed negative and no significant correlation to hepcidin in serum (r = -0.111). The use of two monoclonal antibodies in a sandwich ELISA format provides a reliable, reproducible, and not very expensive method for measuring serum concentrations of the bioactive form of hepcidin in Bulgarian laboratory practice.

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.

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.