Effects of Freeze-Thaw Cycles and Assessment of Short-Term Storage Stability on Serum Iron, Ferritin, and Transferrin.

BACKGROUND: Most errors in the laboratory occur due to processes related to the pre-analytical phase. The purpose of this study was to investigate the effects of multiple freeze-thaw cycles on serum iron, transferrin, and ferritin, as well as the impact of short-term storage on the stability of these analytes. METHODS: Serum samples from ten volunteers were submitted to three consecutive freeze-thaw cycles at -20°C and -80°C. Serum aliquots were also kept at 4°C, -20°C, and -80°C for 28 days. Iron, ferritin, and transferrin were measured after 1, 7, 14, 21, or 28 days of storage. RESULTS: Serum ferritin and transferrin showed variations from -2.6 to 2.6% and -1.7 to 2.4%, respectively, in their concentrations during the three freeze-thaw cycles of -20°C and -80°C. However, the variations were statistically significant only at -20°C. No significant changes were found for iron at both temperatures. The storage at temperatures of 4°C, -20°C, and -80°C for up to 4 weeks significantly affected the serum concentrations of iron, ferritin, and transferrin (p < 0.01 for all). CONCLUSIONS: Serum iron, ferritin, and transferrin were affected by the storage of samples at temperatures of 4°C, -20°C and -80°C for up to 4 weeks, and the freeze-thaw cycles at -20°C influenced the measurement of serum ferritin and transferrin.

Low frataxin mRNA expression is associated with inflammation and oxidative stress in patients with type 2 diabetes.

BACKGROUND: The mitochondrial protein frataxin is involved in iron metabolism, as well as regulation of oxidative stress. To elucidate the association of frataxin with the pathophysiology of diabetes, we evaluated the mRNA levels of frataxin in leukocytes of patients with type 2 diabetes (T2D). In addition, we investigated the relation between frataxin mRNA levels, inflammatory cytokines, and oxidative stress biomarkers. METHODS: A study including 150 subjects (115 patients with T2D and 35 healthy subjects) was performed to evaluate the frataxin mRNA levels in leukocytes. We assessed the relation between frataxin and interleukin (IL)-6, IL-1, tumour necrosis factor-alpha (TNF-α), total oxidation status (TOS), total antioxidant capacity (TAC), and serum iron. RESULTS: The frataxin mRNA levels in the T2D group were significantly lower than those in healthy subjects. It was also demonstrated that T2D patients with frataxin mRNA levels in the lowest quartile had significantly elevated levels of serum iron, TOS, and inflammatory cytokines, such as TNF-α, IL-1, and IL-6, while TAC levels were significantly lower in this quartile when compared with the upper quartile. CONCLUSIONS: Our findings showed that T2D patients with low frataxin mRNA levels showed a high degree of inflammation and oxidative stress. It is speculated that frataxin deficiency in T2D patients can contribute to the imbalance in mitochondrial iron homeostasis leading to the acceleration of oxidative stress and inflammation.

Sarcopenia and Its Association with Vertebral Fractures in People Living with HIV.

The prevalence of chronic diseases is increasing in people living with HIV (PLHIV) in the post ART era. Sarcopenia is prevalent in the elderly and is associated with many chronic diseases. Our study aimed to evaluate the frequency of sarcopenia in PLHIV and its association with bone mineral density and fracture. A cross-sectional study was carried out at Santa Maria, South Brazil. It included PLHV age ≥ 50 years and registered to receive antiretroviral therapy. A structured questionnaire was applied, blood samples collected, muscle strength evaluated, body composition measured, and vertebral morphometry performed. Sarcopenia and presarcopenia were defined according to the European Working Group on Sarcopenia in Older People. Of the 101 patients recruited, 83 underwent DXA and muscle strength measurements. The prevalence of sarcopenia and presarcopenia in the individuals studied was 12% and 16.9%, respectively. 66.7% of sarcopenic individuals had morphometric vertebral fractures and there was a tendency towards a higher frequency of multiple vertebral fractures when compared with non-sarcopenic subjects (44.4% vs. 16.2%, p = 0.066). BMI and total hip BMD were significantly lower in sarcopenic than non-sarcopenic individuals (p ≥ 0.035 and 0.032 respectively). In multiple regression analysis, sarcopenia was associated with age and multiple vertebral fractures. Sarcopenia was present in 12% of this population of PLHIV age ≥ 50 years and was associated with lower hip BMD and a high prevalence of vertebral fractures.

Association between Urinary Levels of Interleukin-6, Interleukin-10 and Tumor Necrosis Factor-Alpha with Glomerular and Tubular Damage Indicators in Patients with Type 2 Diabetes.

BACKGROUND: This study investigated the association between urinary levels of interleukin (IL)-6, IL-10, and tumor necrosis factor-alpha (TNF-α) with estimated glomerular filtration rate (eGFR), urinary albumin/creatinine ratio (uACR), and urinary neutrophil gelatinase-associated lipocalin (uNGAL) in patients with type 2 diabetes (T2D). METHODS: Urinary concentrations of IL-6, IL-10, TNF-α, ACR, and NGAL were measured in 121 patients with T2D. RESULTS: Urinary IL-6 and TNF-α increased 45.5% and 49.4% in the highest uACR quartile compared to lowest quartile. Urinary IL-10 levels decreased 40.9% in the highest uACR quartile compared to the lowest quartile. Urinary IL-6 and TNF-α were 75.3% and 81.6%, higher in the highest uNGAL quartile compared to the lowest quartile. Urinary IL-10 concentration was 69.8% lower in patients from the highest uNGAL quartile compared to lowest quartile. CONCLUSIONS: Urinary IL-6, IL-10, and TNF-α were associated with indicators of glomerular and tubular injuries in patients with T2D.

The yerba mate intake has a neutral effect on bone: A case-control study in postmenopausal women.

Nutritional factors have been associated with osteoporosis and fractures. The intake of coffee may increase the risk of fracture whereas the intake of black and green tea is associated with its reduction. Recently, consumption of yerba mate was associated with increased bone mineral density in postmenopausal women. Nonetheless, its influence on fracture is not known. The aim of this study was to evaluate the effect of yerba mate tea intake on fractures, bone markers, calcium homeostasis, and oxidative stress in postmenopausal women. A case-control study was carried out in South Brazil, 46 women with fractures and 49 controls completed the study. There was no significant difference between the frequency of fractures in women who drank mate tea and women who did not (48.3% vs. 48.5%, p = .99). Moreover, there was no significant difference concerning the serum levels of total calcium, phosphorus, PTH, vitamin D, P1NP, and CTX in the subjects with the history of yerba mate use when compared to controls. Higher serum levels of NOx were found in women who drank the yerba mate infusion. In conclusion, the yerba mate intake is not associated with fracture, and it appears to have a neutral effect on the bone metabolism.

Melaleuca alternifolia essential oil nanoparticles ameliorate the hepatic antioxidant/oxidant status of silver catfish experimentally infected with Pseudomonas aeruginosa.

Oxidative stress has been recognized as a conjoint pathological mechanism that contributes to initiation and progression of liver injury, such as that caused by bacterial diseases. Natural antioxidants are considered a rational curative strategy to prevent and cure hepatic diseases involved with oxidative stress. Thus, the aim of this study was to evaluate, for the first time, whether treatment with bactericidal Melaleuca alternifolia essential oil (TTO) nanoparticles prevents or reduces the hepatic damage in silver catfish (Rhamdia quelen) experimentally infected with Pseudomonas aeruginosa (PAO1). Liver samples from fish infected with P. aeruginosa showed increased thiobarbituric acid reactive substances (TBARS), protein carbonylation and advanced oxidation protein product (AOPP) levels, while catalase (CAT) activity was reduced compared to uninfected animals. The prophylactic treatment with nanoencapsulated TTO prevented these alterations. Based on this evidence, we concluded that P. aeruginosa infection causes hepatic damage, evidenced by increased TBARS, protein carbonylation and AOPP levels, which inhibits the antioxidant defense system, contributing to disease pathophysiology. Thus, this treatment may be considered an important approach for the prevention of hepatic oxidative damage caused by P. aeruginosa infection in fish.

Fowl typhoid in laying hens cause hepatic oxidative stress.

The aim of this study was to analyses nitric oxide, antioxidant status, and oxidative profile in the liver of laying hens naturally infected by Salmonella enterica subsp enterica serovar Gallinarum (S. Gallinarum). The nitrite/nitrate (NOx), reactive oxygen species (ROS), thiobarbituric acid-reactive substances (TBARS), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione S-transferase (GST) activities were measured in liver samples, as well the biomarkers of hepatic function (alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), total protein and albumin levels measured in serum. NOx levels and CAT activity were reduced in hepatic tissue of infected hens. On the other hand, TBARS and ROS levels, GR, GPx and GST activities were higher in infected animals. On biomarkers of tissue damage, ALT, AST, GGT and total protein levels were higher in serum of infected hens, and showed decreased albumin levels. In summary, ROS and TBARS production lead to damage on the membrane lipids that alter activities of antioxidant enzymes CAT, GR, GPx and GSH, an adaptive response against S. Gallinarum infection, contributing to the pathophysiology and clinical signs of the disease.

Achyrocline satureioides essential oil-loaded in nanocapsules reduces cytotoxic damage in liver of rats infected by Trypanosoma evansi.

The aim of this study was to evaluate whether the treatment with Achyrocline satureioides essential oil-loaded in nanocapsules (AS-NC) is able to protect the hepatic tissue against cytotoxic damage caused by Trypanosoma evansi. Thus, the rats were divided into four groups (n = 6 per group): uninfected/saline, uninfected/AS-NC, infected/saline, and infected/AS-NC. At day 4 post-infection (PI), the animals were euthanized and liver and sera samples were collected to perform the hepatic cell viability assay, and to determine seric levels of reactive oxygen species (ROS) and nitric oxide metabolites (NOx). Cell viability decreased (p < 0.05) in the infected/saline group compared to uninfected/saline group, while the treatment with AS-NC avoided this alteration in infected rats. Seric ROS and NOx levels increased (p < 0.05) in the infected/saline group compared to uninfected/saline group, while the treatment with AS-NC avoided this effect on ROS levels of infected rats. In summary, the treatment with AS-NC was able to protect the liver tissue against the cytotoxic effect caused by the parasite by avoiding exacerbated production of ROS.

Nanoparticle formulation increases Syzygium cumini antioxidant activity in Candida albicans-infected diabetic rats.

CONTEXT: Syzygium cumini (L.) Skeels (Myrtaceae) is a medicinal plant widely used in folk medicine for the treatment of diabetes mellitus (DM). However, studies on the use of this plant and of nanoparticle formulations against DM-related fungal infections are scarce. OBJECTIVE: To evaluate the effect of the treatments with aqueous seed extract of S. cumini (ASc) and ASc-loaded polymeric nanoparticles (NPASc) on biochemical parameters in Candida albicans-infected diabetic rats. MATERIALS AND METHODS: Male Wistar rats were divided into eight groups: Control, DM, C. albicans, C. albicans + ASc, C. albicans + NPASc, DM + C. albicans, DM + C. albicans + ASc and DM + C. albicans + NPASc. Rats were daily treated with ASc or NPASc (100 mg/kg) for 21 days. Biochemical parameters in serum and urine, advanced oxidation protein product (AOPP) and TBARS levels in the serum, kidney, liver and pancreas and N-acetyl-ß-d-glucosaminidase (NAG) activities in kidney and urine were evaluated. RESULTS: Biochemical and oxidative stress parameters increased in rats with DM and/or candidiasis. NPASc was more effective than ASc in decreasing glucose (56%), cholesterol (33%) and creatinine (51%) levels; serum (16%) and pancreatic (46%) AOPP and renal (48%) TBARS levels when compared with DM + C. albicans group. In C. albicans group, both treatments decreased NAG activity but did not decrease creatinine levels. CONCLUSIONS: These data suggest that the use of nanotechnology is able to improve plant extract properties such as antioxidant activity that may be useful in diabetes-related complications.

Iron metabolism and oxidative profile of dogs naturally infected by Ehrlichia canis: Acute and subclinical disease.

The aim of this study was to evaluate the oxidant profile and iron metabolism in serum of dogs infected by Ehrlichia canis. Banked sera samples of dogs were divided into two groups: negative control (n = 17) and infected by E. canis on acute (n = 24), and subclinical (n = 18) phases of the disease. The eritrogram, leucogram, and platelet counts were evaluate as well as iron, ferritin, and transferrin levels, latent iron binding capacity (LIBC), and transferrin saturation index (TSI) concentration. In addition, the advanced oxidation protein products (AOPP) and ferric reducing ability of plasma (FRAP) in sera were also analyzed. Blood samples were examined for the presence of E. canis by PCR techniques. History and clinical signals were recorded for each dog. During the acute phase of the disease, infected animals showed thrombocytopenia and anemia when compared to healthy animals (P < 0.05) as a consequence of lower iron levels. Ferritin and transferrin levels were higher in both phases (acute and subclinical) of the disease. The AOPP and FRAP levels increased in infected animals on the acute phase; however, the opposite occurred in the subclinical phase. We concluded that dogs naturally infected by E. canis showed changes in the iron metabolism and developed an oxidant status in consequence of disease pathophysiology.

Synergistic effects of resveratrol (free and inclusion complex) and sulfamethoxazole-trimetropim treatment on pathology, oxidant/antioxidant status and behavior of mice infected with Toxoplasma gondii.

This study aimed to investigate the synergistic effects of resveratrol and sulfamethoxazole-trimethoprim (ST) on the treatment of mice experimentally infected by Toxoplasma gondii during the chronic phase of the disease considering infection, behavior, and oxidative/antioxidants profile aspects. For the study, 60 mice were initially divided into two groups: uninfected (n = 24) and infected by T. gondii (n = 36). These two groups were later subdivided into other groups and treated with resveratrol (free and inclusion complex containing resveratrol) alone and co-administered with ST: groups A to D were composed by healthy mice and groups E to J were consisted of animals infected by T. gondii (VEG strain). Treatments began 20 days post-infection for 10 consecutive days with oral doses of 0.5 mg kg(-1) of ST (groups B and F), 100 mg kg(-1) of free resveratrol (groups C and G) and inclusion complex of resveratrol (nanoparticles containing resveratrol) (groups D and H), and lastly an co-administration of both drugs (groups I and J). Behavioral tests (memory, anxiety and locomotion) were performed after treatment. Liver and brain fragments were collected to evaluate pathological changes, brain cysts counts, as well as oxidant and antioxidant levels. A reduction on the number of cysts in the brain of animals treated with both drugs combined was observed; there was also reduced number of lesions on both organs. This drug combined effect was also able to reduce oxidative and increase antioxidant levels in infected mice, which might be interpreted as a resveratrol protective effect. In addition, the combination of ST and resveratrol was able to prevent behavioral changes in infected mice. Therefore, the use of co-administration drugs enhances the therapeutic effect acting on a synergic way, reducing the oxidizing effects of the chemical treatment for toxoplasmosis. In addition, resveratrol in inclusion complex when co-administered with ST showed an improved therapeutic effect of ST reducing oxidative damage, liver damage and the number of cysts in the brain of T. gondii infected mice.

Evaluation of the In Vitro Interference of Hemoglobin, Lipids, and Bilirubin on the Measurement of Plasma Advanced Oxidation Protein Products.

BACKGROUND: The pre-analytical phase is the most vulnerable and contributes to the majority of errors in laboratory assays. Therefore, the aim of this study was to evaluate in vitro interference of hemoglobin, lipids, and bilirubin on measurement of plasma advanced oxidation protein products (AOPPs). METHODS: AOPPs were measured in a sample spiked with increasing concentrations of hemoglobin, Intralipid® 20% or bilirubin. Then, the relative deviation of the result from the nonspiked baseline value was calculated. RESULTS: We found that all interferents analyzed influenced AOPPs baseline value significantly. Hemoglobin produced a non-linear negative bias, with a maximum decrease of 21.1%. Otherwise, lipids and bilirubin produced a positive bias, ranging from 20.0 to 90.9%, and 12.1 to 33.2%, respectively. CONCLUSIONS: The addition of interferents produced a significant bias in the measurement of AOPPs and in different degrees at the majority of concentrations added.

Relation between iron metabolism and antioxidants enzymes and δ-ALA-D activity in rats experimentally infected by Fasciola hepatica.

The aim of this study was to evaluate the iron metabolism in serum, as well as antioxidant enzymes, in addition to the Delta-Aminolevulinic Acid Dehydratase (δ-ALA-D) activity in the liver of rats experimentally infected by Fasciola hepatica. Thirty male adult rats (Wistar) specific pathogen free were divided into four groups: two uninfected group (CTRL 1 and CTRL 2) with five animals each and two infected groups (INF 1 and INF 2) with 10 animals each. Infection was performed orally with 20 metacercariae at day 1. On day 15 (CTRL 1 and INF 1 groups) and 87 PI (CTRL 2 and INF 2 groups) blood and bone marrow were collected and the animals were subsequently euthanized for liver sampling. Blood was allocated in tubes without anticoagulant for serum acquisition to measure iron, transferrin and unsaturated iron binding capacity (UIBC). δ-ALA-D, superoxide dismutase (SOD), and catalase (CAT) activities were measured in the liver. A decrease in iron, transferrin and UIBC levels was observed in all infected animals compared to control groups (P < 0.05). Furthermore, iron accumulation was observed in bone marrow of infected mice. Infected animals showed an increase in δ-ALA-D activity at 87 post-infection (PI) (INF 2) as well as in SOD activity at days 15 (INF 1) and 87 PI (INF 2). On the other hand, CAT activity was reduced in rats infected by F. hepatica during acute and chronic phase of fasciolosis (INF 1 and INF 2 groups), when moderate (acute) and severe necrosis in the liver histopathology were observed. These results may suggest that oxidative damage to tissues along with antioxidant mechanisms might have taken part in fasciolosis pathogenesis and are also involved in iron deficiency associated to changes in δ-ALA-D activity during chronic phase of disease.