A simple dot-blot-Sirius red-based assay for collagen quantification.

The assessment of collagen content in tissues is important in biomedical research, since this protein is altered in numerous diseases. Hydroxyproline and Sirius red based assays are the most common methods for collagen quantification. However, these procedures have some pitfalls, such as the requirement of oxygen-free medium or expensive equipment and large sample size or being unsuitable for hydrolyzed collagen, respectively. Our objective was to develop a specific, versatile, and user-friendly quantitative method applicable to small tissue samples and extracts obtained from elastin purification, therefore, suitable for simultaneous quantification of elastin. This method is based on the binding of Sirius red to collagen present in a sample immobilized on a PVDF membrane, as in the dot-blot technique, and quantified by a scanner and image analysis software. Sample loading, Sirius red concentration, temperature and incubation time, type of standard substance, albumin interference, and quantification time are optimized. The method enabled the quantification of (1) intact collagen in several rat tissue homogenates, including small resistance-sized arteries, (2) partially hydrolyzed collagen obtained from NaOH extracts, compatible with elastin purification, and (3) for the detection of differences in collagen content between hypertensive and normotensive rats. We conclude that the developed technique can be widely used since it is versatile (quantifies intact and hydrolyzed collagen), requires small sample volumes, is user-friendly (low-cost, easy to use, minimum toxic materials, and reduced time of test), and is specific (minimal interference with serum albumin).

Liver growth factor treatment restores cell-extracellular matrix balance in resistance arteries and improves left ventricular hypertrophy in SHR.

Liver growth factor (LGF) is an endogenous albumin-bilirubin complex with antihypertensive effects in spontaneously hypertensive rats (SHR). We assessed the actions of LGF treatment on SHR mesenteric resistance and intramyocardial arteries (MRA and IMA, respectively), heart, and vascular smooth muscle cells (VSMC). SHR and Wistar-Kyoto (WKY) rats treated with vehicle or LGF (4.5 µg LGF/rat, 4 ip injections over 12 days) were used. Intra-arterial blood pressure was measured in anesthetized rats. The heart was weighted and paraffin-embedded. Proliferation, ploidy, and fibronectin deposition were studied in carotid artery-derived VSMC by immunocytochemistry. In MRA, we assessed: 1) geometry and mechanics by pressure myography; 2) function by wire myography; 3) collagen by sirius red staining and polarized light microscopy, and 4) elastin, cell density, nitric oxide (NO), and superoxide anion by confocal microscopy. Heart sections were used to assess cell density and collagen content in IMA. Left ventricular hypertrophy (LVH) regression was assessed by echocardiography. LGF reduced blood pressure only in SHR. LGF in vitro or as treatment normalized the alterations in proliferation and fibronectin in SHR-derived VSMC with no effect on WKY cells. In MRA, LGF treatment normalized collagen, elastin, and VSMC content and passive mechanical properties. In addition, it improved NO availability through reduction of superoxide anion. In IMA, LGF treatment normalized perivascular collagen and VSMC density, improving the wall-to-lumen ratio. Paired experiments demonstrated a partial regression of SHR LVH by LGF treatment. The effective cardiovascular antifibrotic and regenerative actions of LGF support its potential in the treatment of hypertension and its complications.

Short-term treatment of spontaneously hypertensive rats with liver growth factor reduces carotid artery fibrosis, improves vascular function, and lowers blood pressure.

OBJECTIVE: Liver growth factor (LGF), a mitogen for liver cells, reduces fibrosis in a rat model of cirrhosis. The present study assesses the possible vascular antifibrotic and antihypertensive effects of LGF treatment on spontaneously hypertensive rats (SHR). METHODS: Six-month-old male SHR and normotensive Wistar Kyoto rats (WKY) were treated with LGF (4.5 microg LGF/rat i.p. twice a week for 2 weeks). Haemodynamic parameters were measured in anaesthetized rats. Vascular structure and function were studied in carotid arteries using optical and confocal microscopy, radioimmunoassay for desmosine, and isometric tension recording. RESULTS: LGF reduced systolic and diastolic blood pressure only in SHR. When compared to those of untreated SHR, carotid arteries from LGF-treated SHR showed: 1) a 50% reduction in collagen area and an increase in vascular smooth muscle cell number in the media, 2) no difference in total elastin content, but an increase in size of fenestrae in the internal elastic lamina, and 3) enhanced relaxation to acetylcholine, sodium nitroprusside, and forskolin. These effects were specific for SHR, since no changes were observed in LGF-treated WKY. CONCLUSION: Short-term treatment with a low dose of LGF induced a large improvement in vascular structure and function and significantly reduced blood pressure in a rat model of essential hypertension. The present results could open future research to explore the vascular effects of this endogenous factor in order to determine its potential as an antifibrotic and antihypertensive agent in humans.

Modulatory role of the adventitia on noradrenaline and angiotensin II responses role of endothelium and AT2 receptors.

OBJECTIVE: We have studied the modulatory role of the adventitia on vascular tone and nitric oxide (NO) availability in response to noradrenaline (NA) and angiotensin II (Ang II). METHODS: Changes in isometric tension were determined in carotid arteries from 3-month-old Sprague-Dawley rats denuded from adventitia (-A) and compared to intact rings (+A). NO availability was assessed by the fluorescent NO indicator, 4,5-diaminofluorescein diacetate (DAF-2). RESULTS: Responses to NA (10(-10) to 10(-6) M) were: (i) significantly lower in -A compared to +A rings; (ii) equally enhanced in +A and -A rings without endothelium; and (iii) reduced in +A and -A rings incubated with superoxide dismutase (SOD; 15 U/ml). Responses to Ang II (10(-10) to 10(-7) M) were: (i) similar between +A and -A segments; (ii) equally reduced in both groups by SOD; and (iii) increased by endothelial denudation in both +A and -A arteries. Blockade of AT2 receptors with PD 123,319 (10(-7) M) significantly increased Ang II-induced contractions in +A rings. In segments preincubated with losartan (10(-5) M) and precontracted with NA (10(-7) M), Ang II elicited a relaxation that was abolished by l-NAME (10(-4) M), PD 123,319 (10(-7) M), and endothelium or adventitial removal. NO availability was increased in carotid rings stimulated with Ang II, but not with NA. This NO release was blocked by PD 123,319 (10(-7) M) and endothelium denudation. CONCLUSIONS: These results suggest that the adventitia differently modulates responses to vasoconstrictors and that it is a key layer in Ang II-induced contractions, mediating NO release from the endothelium via AT2 receptors. This increase in NO counterbalances basal superoxide release.

A novel, micro, rapid and direct assay to assess total antioxidant capacity of solid foods.

A novel, micro, rapid and direct procedure to measure the total antioxidant capacity of solid foods using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) (mR-QUENCHER-DPPH) was developed and validated. The mR-QUENCHER-DPPH assay was performed in semi-aqueous medium (methanol-Tris buffer) using very small sample amounts (below 3.6 µg), as estimated by a Bradford reagent-based chemical predictor, and it was completed in 10 min at room temperature. The total antioxidant capacity (TAC) of solid foods was expressed as scavenging capacity index (SCI, mmol DPPH scavenged per kg sample), a theoretical and stoichiometric parameter deduced in this study. SCI values measured by mR-QUENCHER-DPPH assay for cereals cous-cous (7.20±0.35), amaranth (7.99±0.35) and buckwheat (194.2±6.72); Goji fruit (91.27±3.98); lotus root (2402±168); and spices turmeric (3767±355), ginger (2493±283), and cinnamon (10461±2133) were further validated using Folin-Ciocalteau assay. Bland-Altman analysis showed that there were not statistically significant differences in TAC values as measured by both assays. In the same way, TAC values measured by mR-QUENCHER-DPPH were correlated with free (r=0.8088, P=0.0151), bound (r=0.9668, P<0.0001) and total (r=0.9067, P=0.0019) reducing capacity of extracts from solid foods as assessed by Folin-Ciocalteau assay. The mR-QUENCHER-DPPH assay allows to measure TAC values using micro-gram amounts in solid food samples with a wide content range of antioxidants (low, high and very high), and omitting the time-consuming dilution cellulose-step commonly employed in the traditional QUENCHER procedures.

Physical features, phenolic compounds, betalains and total antioxidant capacity of coloured quinoa seeds (Chenopodium quinoa Willd.) from Peruvian Altiplano.

Physical features, bioactive compounds and total antioxidant capacity (TAC) of coloured quinoa varieties (Chenopodium quinoa Willd.) from Peruvian Altiplano were studied. Quinoa seeds did not show a pure red colour, but a mixture which corresponded to different fractal colour values (51.0-71.8), and they varied from small to large size. Regarding bioactive compounds, total phenolic (1.23-3.24mg gallic acid equivalents/g) and flavonol contents (0.47-2.55mg quercetin equivalents/g) were highly correlated (r=0.910). Betalains content (0.15-6.10mg/100g) was correlated with L colour parameter (r=-0.569), total phenolics (r=0.703) and flavonols content (r=0.718). Ratio of betaxanthins to betacyanins (0.0-1.41) was negatively correlated with L value (r=-0.744). Whereas, high TAC values (119.8-335.9mmol Trolox equivalents/kg) were negatively correlated with L value (r=-0.779), but positively with betalains (r=0.730), as well as with free (r=0.639), bound (r=0.558) and total phenolic compounds (r=0.676). Unexploited coloured quinoa seeds are proposed as a valuable natural source of phenolics and betalains with high antioxidant capacity.

Rapid high-throughput assay to assess scavenging capacity index using DPPH.

A new microplate-adapted DPPH rapid assay was developed to assess the antioxidant capacity of pure compounds and foods. The assay was carried out in buffered medium (methanol: 10mmol/l Tris buffer pH 7.5, 1:1 v/v) and reaction was completed at 10min. The scavenging capacity index (SCI), a theoretical antioxidant parameter directly related to the antioxidant capacity of samples, was calculated. SCI for pure compounds: gallic acid (6.76±0.08), quercetin (7.89±0.24), catechin (6.05±0.23), trolox (2.32±0.03), ascorbic acid (2.52±0.15) and gluthatione (1.08±0.08) and foods (µmol DPPH scavenged/100ml): tropical juice (655.62±12.18), mediterraneo juice (702.87±11.13), apple juice (212.52±17.22), pomegranate juice (319.83±9.45), red grape nectar (1093.05±18.69), Don Simon orange juice (632.94±17.22) and date syrup (15992.34±250.7) were comparable to those in previous reports using the classic DPPH assay. The relative standard deviation (RSD) for the SCI on the same and different days was less than 8.12% in all cases.

Liver growth factor treatment reverses vascular and plasmatic oxidative stress in spontaneously hypertensive rats.

BACKGROUND: Liver growth factor (LGF) is an albumin-bilirubin complex with antioxidant actions in vitro. In spontaneously hypertensive rats (SHRs), short LGF treatment exerts antihypertensive and antifibrotic effects. METHOD: We aimed to determine if LGF treatment (4 i.p. injections, 4.5 µg/rat over 12 days) reduces oxidative stress in SHRs using Wistar-Kyoto (WKY) as control strain. We assessed the following: plasma oxidative stress biomarkers [protein-bound malondialdehyde (MDA); protein carbonyls and advanced glycation end products (AGEs)]; superoxide anion basal production in carotid artery-derived vascular smooth muscle cells (VSMCs) detected by dihydroethidium and confocal microscopy; and expression (western blot) and activities (spectroscopic methods) of NADPH and xanthine oxidases, CuZn, Mn and extracellular superoxide dismutases (SODs) and catalase in carotid arteries. RESULTS: LGF treatment had the following effects: reversed the increase in plasma MDA and protein carbonyls and VSMC superoxide anion levels observed in SHRs, without any effect on WKY strain; reversed the alterations in SHR vascular p22phox expression as well as NADPH oxidase, xanthine oxidase and catalase activities; had no effect on vascular CuZn-SOD and Mn-SOD expression or total SOD activity; and reversed the elevation in SHR vascular glycated/free extracellular-SOD expression ratio and plasma glucose without changes in plasma AGEs. CONCLUSION: LGF treatment of SHRs normalizes the level of plasma oxidative stress biomarkers through a reduction of vascular superoxide anion produced by NADPH and xanthine oxidases. These effects might be linked to the cardiovascular regenerative actions of LGF.

The antioxidant activity and thermal stability of lemon verbena (Aloysia triphylla) infusion.

Because of its good sensorial attributes, lemon verbena is used as a primary ingredient in infusions and nonalcoholic drinks. The present study was designed to assess the antioxidant activity (AA) of lemon verbena infusion (LVI) as well as the thermal stability of its AA and the content of polyphenolic compounds. The values reflecting the AA of LVI, including AA index, fast scavenging rate against 2,2-diphenyl-1-picrylhydrazyl, Trolox equivalent antioxidant capacity, and hydroxyl radical scavenging, are higher than those of many herbal infusions and antioxidant drinks estimated from reported data. In addition, the slope lag time and specific oxyradical antioxidant capacity values of LVI are comparable to those of a commercial antioxidant drink based on green tea. Hence, LVI is a source of bifunctional antioxidants, and thus in vivo studies of the antioxidant capacity of LVI would be useful to evaluate its potential as an ingredient in antioxidant drinks.

Antioxidant activity of liver growth factor, a bilirubin covalently bound to albumin.

We previously reported that treatment of spontaneously hypertensive rats (SHR) with liver growth factor (LGF), an albumin-bilirubin complex with a covalent bond, reduces blood pressure, improves nitric oxide (NO)-dependent vasodilatation, and exerts vascular antifibrotic actions. Because bilirubin, albumin, and albumin-bound bilirubins have antioxidant properties, we hypothesize that LGF might exert its cardiovascular actions through an antioxidant mechanism. We have tested in vitro the capacity of LGF to scavenge ABTS cation and peroxyl and hydroxyl radicals and to protect vascular NO from degradation by superoxide anion. We have also compared the antioxidant capacity of LGF with that of its molecular components albumin and bilirubin and the reference antioxidant trolox. LGF exhibited antioxidant capacity against all free radicals tested at lower concentrations than albumin, bilirubin, and trolox. LGF, bilirubin, and albumin were also able to protect endothelial NO from superoxide anion degradation in a fashion similar to that of superoxide dismutase or tiron, but at much lower concentrations. These data, together with our previous results in SHR, suggest that LGF might exert its cardiovascular regenerative actions, at least in part, through an antioxidant mechanism and that LGF could be a relevant circulating antioxidant in situations of oxidative stress.