Relationship between selected micronutrient deficiencies and oxidative stress biomarkers in diabetes mellitus patients with foot ulcers in Ibadan, Nigeria

Background/aim: Micronutrients are indispensable in the prevention of diseases and maintenance of good health. Their deficiencies have been implicated in several diseases associated with oxidative stress (OS). This study aimed at investigating the levels of some vitamins and minerals in association with OS markers in diabetic foot ulcer (DFU) patients in Ibadan, Oyo State, Nigeria. Materials and methods: Seventy DFU patients and 50 apparently healthy volunteers (controls) were recruited for the study. Blood samples of 10 mL were collected after a 10-h overnight fast from each participant after obtaining their consent. Levels of oxidative stress biomarkers such as lipid peroxide (LPO), 8-hydroxyl-2?-deoxyguanosine (8-OHdG), total antioxidant status (TAS), superoxide dismutase (SOD), and glutathione peroxidase (GPx) and micronutrients such as vitamin C, vitamin E, copper, selenium, and zinc were determined. Results: Significant increases in the levels of LPO and 8-OHdG and GPx activity were found in DFU patients compared to controls (P < 0.001). Significant decreases in vitamin C (P = 0.003), selenium, vitamin E, and TAS concentrations were detected between DFU patients and controls (P < 0.001). However, nonsignificant decreases in SOD activity and copper and zinc levels were observed when DFU patients and controls were compared (P > 0.05). Vitamin C was significantly positively correlated with GPx and selenium was significantly negatively correlated with 8-OHdG in the DFU group. However, nonsignificant correlations were observed between other micronutrients and oxidative stress biomarkers of both the DFU and control groups. Conclusion: Diabetes mellitus patients with foot ulcers may require micronutrient supplementation for proper control and maintenance of oxidant/antioxidant homeostasis.

Calcium - Magnesium imbalance implicated in benign prostatic hyperplasia and restoration by a phytotherapeutic drug - Croton membranaceus Müll.Arg.

BACKGROUND: Calcium (Ca)- magnesium (Mg) imbalance is implicated in prostate cancer. Ca/Mg ratio increases or decreases with proliferation or apoptosis, respectively. The study examined whether this Ca/Mg imbalance exists in BPH patients and the effect of a phytotherapeutic drug on the Ca/Mg ratio. METHODS: Thirty (30) BPH patients who used the ethanolic root extract of Croton membranaceus (60 mg/day) for 3 months were examined for serum Ca, Mg, phosphate, parathyroid hormone (PTH), vitamin D, prostate specific antigen (PSA) levels and renal function tests (RFT) before (BT) and after treatment (AT) alongside thirty (30) controls. Twenty (20) trace element including Mg and Ca were determined in the drug by neutron activation analysis (NAA). RESULTS: RFT, PTH and vitamin D for BT, AT and controls (C) were normal. Mean PSA was 1.0 ± 0.64 (C), 27.9 ± 19.0 (BT) and 16.2 ± 11.8 ng/mL (AT) (p = 0.002). Mg, Ca/Mg ratio BT, AT and control were significantly different (p = 0.0001, respectively). After treatment, Mg and Ca/Mg ratio were not different from controls. The prevalence of Ca/Mg imbalance was 80% (BT), 13.3% (AT) and 3.3% (control group). CONCLUSION: Ca/Mg ratio imbalance is associated with BPH. This has previously not been demonstrated. The imbalance was significantly corrected after treatment with the phytotherapeutic drug.

Effects of exogenous antioxidants on dietary iron overload.

In dietary iron overload, excess hepatic iron promotes liver damage. The aim was to attenuate free radical-induced liver damage using vitamins. Four groups of 60 Wistar rats were studied: group 1 (control) was fed normal diet, group 2 (Fe) 2.5% pentacarbonyl iron (CI) followed by 0.5% Ferrocene, group 3 (Fe + V gp) CI, Ferrocene, plus vitamins A and E (42x and 10x RDA, respectively), group 4 (Fe - V gp) CI, Ferrocene diet, minus vitamins A and E. At 20 months, glutathione peroxidase (GPx), superoxide dismutase (SOD), Oxygen Radical Absorbance Capacity (ORAC), Ames mutagenicity test, AST, ALT and 4-hydroxynonenal (4-HNE) immunohistochemistry were measured. 8OHdG levels of the Fe + V and Fe - V groups were 346 +/- 117 and 455 +/- 151, ng/g w.wt, respectively. Fe + V and Fe - V differences were significant (p<0.005). A positive correlation between DNA damage and mutagenesis existed (p<0.005) within the iron-fed gps. AST levels for Fe + V and Fe - V groups were 134.6 +/- 48.6 IU and 202.2 +/- 50.5 IU, respectively. Similarly, ALT levels were 234.6 +/- 48.3 IU and 329.0 +/- 48.6 IU, respectively. However, Fe - V and Fe + V groups transaminases were statistically insignificant. 4-HNE was detected in Fe + V and Fe - V gp livers. Vitamins A and E could not prevent hepatic damage.

Antioxidant and oxidative stress status in type 2 diabetes and diabetic foot ulcer.

OBJECTIVE: Oxidative stress (OS) has been implicated in the aetiology and progression of diabetic complications including diabetic foot ulcer. In this study, the levels of lipid peroxides (LPO) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) as well as the enzymatic antioxidant activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) in type 2 diabetes mellitus and diabetic foot ulcer subjects were assessed and compared with apparently healthy normal subjects to understand the involvement of OS in the subjects. METHOD: The above-mentioned OS markers were measured in 50 subjects for each of the following groups: type 2 diabetes mellitus (DM), diabetic foot ulcer (DF) and non-diabetic control (NC). RESULTS: Significant elevated values of LPO (39.86%) and 8-OHdG (45.53%) were found in DM subjects compared with the NC subjects. This increase in both parameters was greater for DF subjects: 80.23% and 53.91% respectively. SOD activities were significantly reduced in DM (14.82%) and DF (4.09%) subjects in contrast with elevated activities of GPx observed in DM (21.87%) and DF (20.94%) subjects. Glycated haemoglobin/fasting plasma glucose (HbA1c/FPG) correlated positively with LPO, 8-OHdG and GPx, whereas a negative correlation was observed for SOD. CONCLUSION: Increased oxidation subsequent to diabetic conditions induces an over-expression of GPx activity suggesting a compensatory mechanism by the body to prevent further tissue damage in the subjects.

Hepatocellular carcinoma caused by iron overload: a possible mechanism of direct hepatocarcinogenicity.

BACKGROUND/AIMS: Excess hepatic iron may be both directly and indirectly carcinogenic. The aim of this study was to determine if generation of reactive oxygen species and the resulting oxidative damage induced by free hepatic iron is directly hepatocarcinogenic. METHODS: Sixty male Wistar albino rats were iron-loaded by ferrocene supplementation of their diet. Biochemical parameters of oxidative damage and lipid peroxidation, DNA unwinding and strand breaks, and the Ames Mutagenesis Test were measured at 4 monthly intervals and correlated with the degree of hepatic iron overload, the presence of iron-free preneoplastic foci in the liver, and the development of hepatocellular carcinoma in comparison with 60 control rats. RESULTS: Levels of lipid hydroperoxides, malonaldehyde, 8-isoprostane and 8-hydroxy-2'-deoxyguanosine increased, reaching peak concentrations at 20-24 months, and correlating with an increase in the rate of DNA unwinding, strand breaks, and positive Ames Tests. Iron-free neoplastic foci became evident at 16 months and thereafter increased in number. Preneoplastic foci were present in five of eight rats remaining at 32 months and HCC had developed in one of the five. CONCLUSIONS: Our findings are compatible with the hypothesis that the direct hepatocarcinogenic effect of free iron is mediated by the generation of oxygen reactive species and oxidative damage that are mutagenic and carcinogenic.

Iron-free neoplastic nodules and hepatocellular carcinoma without cirrhosis in Wistar rats fed a diet high in iron.

Although excess hepatic iron in hereditary haemochromatosis and dietary iron overload in the African causes hepatocellular carcinoma, it usually does so in the presence of cirrhosis. A direct hepatocarcinogenic effect of iron has not been proved. Moreover, an animal model of hepatocellular carcinoma induced by iron overload has not been available. The aim of this study was to develop such a model and to use it to ascertain whether excess hepatic iron is directly hepatocarcinogenic. Sixty Wistar albino rats were fed a chow diet and 60 the same diet supplemented initially with 2% carbonyl iron for 12 months, followed by 0.5% ferrocene for 20 months. Five rats from each group were sacrificed every 4 months for 24 months for histological and biochemical monitoring. By 16 months, hepatocytes in all the rats receiving the iron-supplemented diet showed grade 4 iron overload, comparable in degree with that seen in patients with advanced hereditary haemochromatosis and dietary iron overload. Altered hepatic foci and pre-neoplastic nodules were first seen at 16 months. These increased in size and number with time, were iron-free, stained positively with placental glutathione sulphydryl transferase, and showed the same histological features as the iron-free foci described in patients with hepatocellular carcinoma complicating hereditary haemochromatosis. At 32 months the eight surviving rats in the iron overloaded group were sacrificed. The livers of five of these rats contained pre-neoplastic nodules and one showed, in addition, an iron-free, well-differentiated hepatocellular carcinoma. The tumour stained positively for placental glutathione sulphydryl transferase. Neither cirrhosis nor portal fibrosis was present in this or any iron-loaded animal. We conclude that hepatocellular carcinoma may complicate dietary hepatic iron overload in Wistar albino rats in the absence of fibrosis or cirrhosis, confirming an aetiological association between dietary iron overload and the tumour and suggesting that iron may be directly hepatocarcinogenic.

Inhibitory effects of the extracts of Sutherlandia frutescens (L.) R. Br. and Harpagophytum procumbens DC. on phorbol ester-induced COX-2 expression in mouse skin: AP-1 and CREB as potential upstream targets.

Numerous anti-inflammatory agents have been shown to exert chemopreventive activity by targeting cyclooxygenase (COX)-2, a rate-limiting enzyme involved in the inflammatory process. Sutherlandia frutescens (L.) R. Br. and Harpagophytum procumbens DC., which are commonly known as Cancer bush (CB) and Devil's claw (DC), respectively, have long been used in South Africa for the management of pain and inflammation. In the present study, we investigated the effects of methanolic extracts of CB and DC on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 expression in mouse skin. Topical application of both extracts inhibited TPA-induced COX-2 expression. As an underlying mechanism of COX-2 inhibition, these extracts diminished TPA-stimulated catalytic activity of extracellular signal-regulated protein kinase (ERK), which is known to regulate the activation of eukaryotic transcription factors mediating COX-2 induction. While TPA-induced activation of nuclear factor-(kappa)B remained unaffected by both extracts, they inhibited TPA-induced activation of activator protein-1 (AP-1) and attenuated the expression of its key component c-Fos. In another study, topical application of TPA induced DNA binding of cyclic AMP response element binding (CREB) protein in mouse skin in vivo, which was abrogated by pretreatment with either CB or DC.

Inhibition of phorbol ester-induced COX-2 expression by some edible African plants.

Cancer bush (CB, Sutherlandia frutescens), Devil's claw (DEV, Harpagophytum procumbens), Rooibos tea (RT, Aspalathus linearis), and Bambara groundnut (BB, Vignea subterranean) have been used to treat some malignancies and inflammatory disorders in Africa. However, biochemical basis for chemopreventive effects of these medicinal plants remains unclear. An abnormally elevated expression of cyclooxygenase-2 (COX-2) has been implicated in pathogenesis and progression of carcinogenesis. In the present study, we found that the methanol extracts of CB, DEV, RT, and BB inhibited, to a different extent, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 expression in human breast epithelial (MCF10A) cells and in mouse skin in vivo. To determine the molecular mechanism of COX-2 inhibition by the above medicinal plants, we examined their effects on activation of NF-kappaB which is one of the major transcription factors responsible for regulating COX-2 expression. Methanol extracts of both CB and BB inhibited the DNA binding of NF-kappaB activated by TPA in MCF10A cells in a dose-dependent manner. Based on above findings, CB and BB are likely to inhibit TPA-induced COX-2 expression through suppression of DNA binding of NF-kappaB, which may contribute to the chemopreventive or chemoprotective activity of these African plants.