Sulfuric acid

Antioxidant (DPPH and ABTS radical scavenging, reducing power (CUPRAC and FRAP), phosphomolybdenum, and metal chelating (ferrozine method)) and enzyme inhibitory activities [cholinesterase (ChE) Elmann’s method], tyrosinase (dopachrome method), α-amylase (iodine/potassium iodide method), and α -glucosidase (chromogenic PNPG method)) were determined using the methods previously described by Zengin et al. (2014) (link) and Dezsi et al. (2015) (link).
For the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay: Sample solution (1 mg/mL; 1 mL) was added to 4 mL of a 0.004% methanol solution of DPPH. The sample absorbance was read at 517 nm after a 30 min incubation at room temperature in the dark. DPPH radical scavenging activity was expressed as millimoles of trolox equivalents (mg TE/g extract).
For ABTS (2,2′-azino-bis(3-ethylbenzothiazoline) 6-sulfonic acid) radical scavenging assay: Briefly, ABTS+ was produced directly by reacting 7 mM ABTS solution with 2.45 mM potassium persulfate and allowing the mixture to stand for 12–16 in the dark at room temperature. Prior to beginning the assay, ABTS solution was diluted with methanol to an absorbance of 0.700 ± 0.02 at 734 nm. Sample solution (1 mg/mL; 1 mL) was added to ABTS solution (2 mL) and mixed. The sample absorbance was read at 734 nm after a 30 min incubation at room temperature. The ABTS radical scavenging activity was expressed as millimoles of trolox equivalents (mmol TE/g extract) (Mocan et al., 2016a (link)).
For CUPRAC (cupric ion reducing activity) activity assay: Sample solution (1 mg/mL; 0.5 mL) was added to premixed reaction mixture containing CuCl₂ (1 mL, 10 mM), neocuproine (1 mL, 7.5 mM) and NH₄Ac buffer (1 mL, 1 M, pH 7.0). Similarly, a blank was prepared by adding sample solution (0.5 mL) to premixed reaction mixture (3 mL) without CuCl₂. Then, the sample and blank absorbances were read at 450 nm after a 30 min incubation at room temperature. The absorbance of the blank was subtracted from that of the sample. CUPRAC activity was expressed as milligrams of trolox equivalents (mg TE/g extract).
For FRAP (ferric reducing antioxidant power) activity assay: Sample solution (1 mg/mL; 0.1 mL) was added to premixed FRAP reagent (2 mL) containing acetate buffer (0.3 M, pH 3.6), 2,4,6-tris(2-pyridyl)-S-triazine (TPTZ) (10 mM) in 40 mM HCl and ferric chloride (20 mM) in a ratio of 10:1:1 (v/v/v). Then, the sample absorbance was read at 593 nm after a 30 min incubation at room temperature. FRAP activity was expressed as milligrams of trolox equivalents (mg TE/g extract).
For phosphomolybdenum method: Sample solution (1 mg/mL; 0.3 mL) was combined with 3 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The sample absorbance was read at 695 nm after a 90 min incubation at 95°C. The total antioxidant capacity was expressed as millimoles of trolox equivalents (mmol TE/g extract) (Mocan et al., 2016c (link)).
For metal chelating activity assay: Briefly, sample solution (1 mg/mL; 2 mL) was added to FeCl₂ solution (0.05 mL, 2 mM). The reaction was initiated by the addition of 5 mM ferrozine (0.2 mL). Similarly, a blank was prepared by adding sample solution (2 mL) to FeCl₂ solution (0.05 mL, 2 mM) and water (0.2 mL) without ferrozine. Then, the sample and blank absorbances were read at 562 nm after 10 min incubation at room temperature. The absorbance of the blank was sub-tracted from that of the sample. The metal chelating activity was expressed as milligrams of EDTA (disodium edetate) equivalents (mg EDTAE/g extract).
For ChE inhibitory activity assay: Sample solution (1 mg/mL; 50 μL) was mixed with DTNB (5,5-dithio-bis(2-nitrobenzoic) acid, Sigma, St. Louis, MO, United States) (125 μL) and AChE [acetylcholines-terase (Electric ell AChE, Type-VI-S, EC 3.1.1.7, Sigma)], or BChE [BChE (horse serum BChE, EC 3.1.1.8, Sigma)] solution (25 μL) in Tris–HCl buffer (pH 8.0) in a 96-well microplate and incubated for 15 min at 25°C. The reaction was then initiated with the addition of acetylthiocholine iodide (ATCI, Sigma) or butyrylthiocholine chloride (BTCl, Sigma) (25 μL). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (AChE or BChE) solution. The sample and blank absorbances were read at 405 nm after 10 min incubation at 25°C. The absorbance of the blank was subtracted from that of the sample and the cholinesterase inhibitory activity was expressed as galanthamine equivalents (mgGALAE/g extract) (Mocan et al., 2016b (link)).
For Tyrosinase inhibitory activity assay: Sample solution (1 mg/mL; 25 μL) was mixed with tyrosinase solution (40 μL, Sigma) and phosphate buffer (100 μL, pH 6.8) in a 96-well microplate and incubated for 15 min at 25°C. The reaction was then initiated with the addition of L-DOPA (40 μL, Sigma). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (tyrosinase) solution. The sample and blank absorbances were read at 492 nm after a 10 min incubation at 25°C. The absorbance of the blank was subtracted from that of the sample and the tyrosinase inhibitory activity was expressed as kojic acid equivalents (mgKAE/g extract) (Mocan et al., 2017 (link)).
For α-amylase inhibitory activity assay: Sample solution (1 mg/mL; 25 μL) was mixed with α-amylase solution (ex-porcine pancreas, EC 3.2.1.1, Sigma) (50 μL) in phosphate buffer (pH 6.9 with 6 mM sodium chloride) in a 96-well microplate and incubated for 10 min at 37°C. After pre-incubation, the reaction was initiated with the addition of starch solution (50 μL, 0.05%). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (α-amylase) solution. The reaction mixture was incubated 10 min at 37°C. The reaction was then stopped with the addition of HCl (25 μL, 1 M). This was followed by addition of the iodine-potassium iodide solution (100 μL). The sample and blank absorbances were read at 630 nm. The absorbance of the blank was subtracted from that of the sample and the α-amylase inhibitory activity was expressed as acarbose equivalents (mmol ACE/g extract) (Savran et al., 2016 (link)).
For α-glucosidase inhibitory activity assay: Sample solution (1 mg/mL; 50 μL) was mixed with glutathione (50 μL), α-glucosidase solution (from Saccharomyces cerevisiae, EC 3.2.1.20, Sigma) (50 μL) in phosphate buffer (pH 6.8) and PNPG (4-N-trophenyl-α-D-glucopyranoside, Sigma) (50 μL) in a 96-well microplate and incubated for 15 min at 37°C. Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (α-glucosidase) solution. The reaction was then stopped with the addition of sodium carbonate (50 μL, 0.2 M). The sample and blank absorbances were read at 400 nm. The absorbance of the blank was subtracted from that of the sample and the α-glucosidase inhibitory activity was expressed as acarbose equivalents (mmol ACE/g extract) (Llorent-Martínez et al., 2016 (link)).
All the assays were carried out in triplicate. The results are expressed as mean values and standard deviation (SD). The differences between the different extracts were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference post hoc test with α = 0.05. This treatment was carried out using SPSS v. 14.0 program.

Negative dog sera - Negative sera were from 16 dogs living in the city of La Paz, where no Chagas transmission exists. Dogs were born in the city and never went out in an endemic Chagas region. Negativity was checked by the Chagas STAT-PAK rapid test which is an accurate test for Chagas diagnosis in dogs (Nieto et al. 2009 (link)), and by polymerase chain reaction (PCR) targeting the kDNA of T. cruzi following Fernandes et al. (2001) (link), slightly modified by one of us (Aliaga et al. 2011 (link)). The 16 negative sera were included as negative controls in each of the processed titer-plates.
Positive dog sera - 10 positive dog sera were obtained from dogs originated from the same region of the field sample (see below) and diagnosed positive both by PCR using the same protocol as above, and by the Chagas STAT-PAK rapid test following the manufacturer’s instructions. Then, in each ELISA plate, five-10 of them were included as positive controls to allow the computation of a cut-off value with formula F₃ (Table I).
TABLE I

Formula	a	f	Computation	Comment
F1	2	0	2 x MEAN of negative controls	-
F2	3	0	3 x MEAN of negative controls	-
F3	1	0	MEAN of negative controls + 0.13 x MEAN of positive controls	Pan et al. (1992) formula
F4a	1		MEAN + f x SD, with f = 2.197	Frey et al. (1998) formula. Confidence level (1-α) for t computation: 97.5%
F5a	1		MEAN + f x SD, with f = 3.848	Frey et al. (1998) formula. Confidence level (1-α) for t computation: 99.9 %
F6	1	3	(MEAN + 3 x SD) of negative controls	Classen et al. (1987)

a: for the computation of F₄ and F_5,j is the number of negative controls used in the plate (16 in the present study) and t is the (1-α)^th percentile of the one-tailed Student t-distribution with (j-1) degrees of freedom. Because 16 negative controls were used in the study, and taking into account the confidence level for the computation of the Student t, the f values were 2.197 and 3.848 for F₄ and F₅ respectively.

Sera of field sample - A field sample of 231 dog sera was obtained from four Bolivian populations. Villages of dog’s origin were Eje Pampa (Lat -18.54º Long -65.17º) (47 individuals) and Lagar Pampa (Lat -18.45º Long -64.99º) (26 individuals) in the dry inter-Andean valleys, and La Brecha (Lat -19.51º Long -62.56º) (72 individuals) and Palmarito (Lat -19.49º Long -63.46º) (78 individuals) in the Chaco region. For each dog, 10 mL of blood was taken from the cephalic vein. 5 mL were put in 6 M Guanidine Hydrochlorid/EDTA 0.2 M for DNA analysis (for T. cruzi identification) and 5 mL in EDTA vacuum tubes for the ELISAs. At the field site, blood samples were allowed to clot and were kept at 4ºC.
IgG-ELISA protocol to detect antibodies against T. cruzi - In the laboratory, tubes containing blood samples of dogs were centrifuged at 3000 rpm for 10 min for plasma separation. The ELISA protocol was from Lauricella et al. (1998) (link) which is routinely used for Chagas diagnosis in dogs (Enriquez et al. 2013 (link)). It was slightly modified as follow: ELISAs were carried out in 96-well micro-titer plates (NUNC Maxisorp, flat bottom) coated with a homogenate of T. cruzi epimastigote culture. The homogenate was prepared as follow: 1 mL of pure culture of epimastigotes (forms cultured at 28ºC in LIT liquid medium) was centrifuged in a 5 mL Eppendorff tube at 4000 rpm at 4ºC for 10 min. The supernatant was discarded, 1 mL of phosphate buffer saline (PBS) at pH7.2 was added and the tube vortexed. This washing operation was realised three times. Then, 1 mL of PBS was added and the tube vortexed. A dilution of 1/1000 of the solution was realised in PBS in carbonate buffer (100 µL of “parasites” in PBS + 9900 µL of carbonate buffer), vortexed, and 100 µL of the solution was then added in each well. The plate was sealed with adhesive plastic sheet and incubated overnight at 4ºC. The following day, the content was discarded by inversion. The plate was washed three times with 120 µL/well of washing buffer (PBS - 0.01% Tween 20). Then each well was loaded with 100 µL of blocking buffer (PBS - 3% skimmed milk REGILAIT, France) and incubated 1 h at 37ºC. Then, the plate was washed three times with 120 µL/well of washing buffer. Dog sera were diluted at 1/100 in dilution buffer (PBS - 1% skimmed milk) in 1.5 mL Eppendorff tubes, vortexed and kept at 4ºC until loaded in the plate. Diluted sera were loaded in duplicate at 50 µL/well and incubated 1 h at 37ºC. The plate was then emptied by inversion and washed three times with 120 µL/well of washing buffer. Anti-dog IgG were diluted at 1/1200 in dilution buffer. Each well was loaded with 50 µL of peroxidase conjugated antibodies anti-IgG and incubated 1 h at 37ºC. Then the plate was emptied by inversion and washed three times with 120 µL/well of washing buffer. Then 50 µL of TMB (3, 3’, 5, 5’ - Tetramethylbenzidine, SIGMA) was added in each well and the plate was incubated for 5 min at room temperature. Then, 50 μL/well of sulfuric acid 1 N were added to stop the reaction and absorbance values were obtained at 450 nm in a microwell plate reader (Multiskan). The mean absorbance of each pair of duplicate sera was calculated. When the difference between both values was more than 30%, the sample was retested (Lauricella et al. 1998 (link)). In total, the 231 dog sera and the controls were processed in seven titer plates.
Cut-off formulas (Table I) - For each of the seven titer-plates analysed, cut-off values were computed using six usual formulas (F_i, i = 1 to 6). The value of the f coefficient in formulas F4 and F5 was 2.197 and 3.848 respectively, according to Frey et al. (1998) (link).
Change-point analysis - The whole set of sera was also analysed by change-point analysis which does not need the presence of known positive or negative sera (blind analysis). Change-point analysis is aimed at identifying points in a series where the statistical properties change. In particular, such analysis can be used to detect abrupt steps in the mean level of a series. In the case of ELISA, if absorbance values of a micro-titer plate are ordered in ascending order, negative samples are supposed to be the lower ones in the series while positive ones (if they exist) would be the higher. However, values are not supposed to increase regularly if positive samples exist in the series. Indeed, as positive controls are supposed to be “different” from negative ones, a step, even small, should appear in the series, separating the negative from the positive values. Therefore, change-point algorithms might be used to detect such a change and locate the value where in the series this change occurs. The detected value is therefore a kind of specific cut-off proxy that discriminates between positive and negative samples.
For each of the seven processed titer-plates, absorbance values were first arranged in ascending order and each series was analysed using the R package “changepoint” (Killick & Eckley 2014 ) which detects a change-point if it exists and locates it in the series. In this package, the Pruned Exact Linear Time (PELT) algorithm was selected (Killick et al. 2012) with the CUSUM method as detection option (Page 1954) . The PELT algorithm divides iteratively the series of absorbance values in sub-groups of increasing size. In each, it calculates the minimum of a “cost” function that takes into account the method of detection (Killick et al. 2012) . Minima indicate where the change-points are located within the series. The PELT algorithm can therefore rapidly detect various change-points in a series. The CUSUM method is based on cumulative sums and operates as follow: The absorbance values x are ordered in ascending values (x₁,…x_n) and sums (S) are computed sequentially as S₀ = 0, S_i+1 = max (0, S_i+ x_i - L_i), where L_i is the likelihood function. When the value of S exceeds a threshold, a change-point has been detected.
Approval for the study was granted by the WHO’s Research Ethics Review Committee (ERC), project #A90281 and by the Comisión de Ética de la Investigación del Comité Nacional de Bioética (CEI-CNB) of Bolivia (letters 3 august 2010 and 21 august 2012).

Using the Seaman approach with some modifications [23 (link),26 (link)], an aliquot (0.45 mL) of sulfuric acid (72%, w/w) was added to aliquots (5 mg) of the HMW fractions and kept at room temperature for 2 h, respectively. Afterwards, the solution was diluted with water (5.8 mL) and heated for 2 h and 8 h at 100 °C, respectively. The reaction mixtures were cooled to room temperature and neutralized with aqueous sodium hydroxide solution (1 mol/L). For purification, the samples were washed with water (15 mL) using a water-conditioned C18 SPE-cartridge (1000 mg, Chromabond^® C₁₈ ec, Machery-Nagel, Düren, Germany). The effluent was filled up with water (25 mL) and concentrated to 2.5 mL for quantitative measurements. The purification procedure was repeated for the native HMW fractions (5 mg in 2 mL water). The whole procedure was performed in triplicates for all HMW fractions.

EPS concentrations were determined using ethanol precipitation [23 ]. The cell liberated supernatants were collected by centrifugation at 14,240 ×g at 4°C for 5 min. The EPS was precipitated from the supernatant with three quantities of cold ethanol (95% purity) at 4°C overnight, and gathered by centrifugation at 14,240 ×g for 20 min. The EPS pellets were dissolved in distilled water. Quantitative analysis of EPS yield was performed using the phenol-sulfuric acid method [24 (link)]. First, 500 μl of considered samples were added with 500 μl of 4% phenol afterward the addition of 2.5 ml of sulfuric acid (96% purity) into all the tubes. The solutions were analyzed at 490 nm using a UV spectrophotometer, with distilled water as the blank and glucose as the standard. The absorbance values of the measured samples were adjusted with distilled water below 1.0. Finally, the EPS concentrations present in the samples were determined based on a graph plotting the absorbance against the EPS calibration standards.