Nutritional characterization of Mucuna pruriens: 4. Does replacing soybean meal with Mucuna pruriens in lamb diets affect ruminal,blood and tissue l-dopa concentrations?

The harmful effects of the 3,4-dihydroxy-l-phenylalanine (l-dopa) in Mucuna pruriens (Velvet bean) seeds have limited its use as a protein supplement for monogastrics and humans. Little is known about the extent of metabolism of Mucuna l-dopa in ruminants or its accumulation in ruminant tissues consumed as food by humans. This study aimed to determine if replacing soybean meal (SB) with Mucuna increases concentrations of l-dopa in rumen fluid, blood and muscle tissue of lambs. Twenty-seven RM lambs (RM; initial body weight, BW = 33.8 ± 5.44 kg) and 12 Florida Native (FN; initial BW = 24.9 ± 8.63 kg) lambs were assigned to four treatments and fed a basal diet of coastal bermudagrass hay, corn grain, and liquid molasses for 42 (FN) or 49 days (RM) and then slaughtered. Dietary supplements were formulated by substituting 0 (SB), 330 (Lo), 670 (Med) or at least 1000 (Hi) g/kg of SB with rolled Mucuna seeds (l-dopa, 24 g/kg dry matter, DM). Body weight was measured weekly and carcass characteristics and concentrations of l-dopa in rumen fluid, blood and the sterno-mandibularis muscle were measured at slaughter when concentrations of blood urea N, glucose, haptoglobin and cerruloplasmin were also measured. Lambs fed SB had higher (P<0.05) average daily gain than those fed Mucuna (0.20 versus 0.15 kg/day for RM; 0.21 versus 0.14 kg/day for FN). However, concentrate protein source did not affect dressing and concentrations of blood urea N, or blood glucose. Feeding the Hi diet versus the SB diet did not increase concentrations of blood cerruloplasmin, or l-dopa or concentrations of l-dopa metabolites in blood. No l-dopa was found in the ruminal fluid of the lambs and l-dopa concentrations in the sterno-mandibularis muscle were low (i.e., <5 ng l-dopa/g), and unaffected (P>0.05) by diet. Ingested Mucuna l-dopa was extensively metabolized in lambs, and did not accumulate to toxic levels in muscle tissues.     

Nutritional characterization of Mucuna pruiriens: 2. In vitro ruminal fluid fermentability of Mucuna pruriens,Mucunal-dopa and soybean meal incubated with or without l-dopa

Three in vitro experiments were conducted to determine the rumen fermentability of Mucuna (M) pruriens (24 g 3,4-dihydroxy-l-phenylalanine (l-dopa)/kg dry matter (DM) and soybean meal treated with (SBD) or without (SB) 138 g l-dopa/kg DM). Additional objectives were to determine if l-dopa inhibits rumen fermentation, and if ruminal microbes can adapt to l-dopa or M. In Experiment 1, ground (1 mm) substrates were incubated in triplicate at 38 °C in 9 ml nutrient media and 1 ml rumen fluid in a series of six, 48 h, consecutive batch cultures. The first culture was inoculated with rumen fluid from two donor cows. Subsequent cultures were inoculated with fluid (1 ml) from the previous culture. The DM digestibility (DMD, 616 g/kg vs. 540 g/kg; P<0.01) and gas production (51.7 ml/g vs. 44.2 ml/g DM; P<0.05) were higher from fermentation of M versus SB but similar for SB and SBD (540 g/kg vs. 554 g/kg and 44.2 ml/g DM vs. 43.5 ml/g DM, respectively). The slopes of the relationships between DMD (g/kg) or gas production (ml/g DM) and fermentation period were not reduced by fermenting M (−0.014 DMD slope; 2.28 gas production slope) or SBD (−0.014 DMD slope; 0.459 gas production slope), instead of SB (−0.002 DMD slope; 1.039 gas production slope), indicating microbial adaptation to M and SBD. Total volatile fatty acid concentration (VFA; 53.7, 54.9 and 54.9 mmol/l) and molar proportions of VFA were similar among substrates. Gas production kinetics of M versus SB (Experiment 2), and SB versus SBD (Experiment 3) were also measured after substrates were incubated in triplicate in buffered rumen fluid for 24 h using a non-linear exponential model to fit the data. Residual l-dopa was measured after separate fermentation of substrates in triplicate for 0, 4, 8, 16 and 24 h. Fermentation of M versus SB produced more (P<0.05) gas (250 ml/g vs. 100 ml/g DM) and total VFA (203 mmol/l vs. 180 mmol/l) and a lower (P<0.05) acetate:propionate ratio (1.35 vs. 1.87; P<0.05). Adding l-dopa to SB increased (P<0.01) gas production (92 ml/g DM vs. 200 ml/g DM), and total VFA concentration (132 mmol/l vs. 188 mmol/l), but reduced (P<0.05) gas production rate (0.08 ml/h vs. 0.05 ml/h). The concentration of l-dopa in fermented M and SBD decreased by 53 and 47%, respectively during fermentation. In conclusion, M was more fermented than SB and degradation of l-dopa during ruminal fermentation and microbial adaptation to l-dopa were confirmed. Adding l-dopa to SB did not impair ruminal fermentation.     

Tyrosinase inactivation in its action on dopa

Under aerobic or anaerobic conditions, tyrosinase undergoes a process of irreversible inactivation induced by its physiological substrate l-dopa. Under aerobic conditions, this inactivation occurs through a process of suicide inactivation involving the form oxy-tyrosinase. Under anaerobic conditions, both the met- and deoxy-tyrosinase forms undergo irreversible inactivation. Suicide inactivation in aerobic conditions is slower than the irreversible inactivation under anaerobic conditions. The enzyme has less affinity for the isomer d-dopa than for l-dopa but the velocity of inactivation is the same. We propose mechanisms to explain these processes.     

Aryl boronic acid inhibition of synthetic melanin polymerization

Inhibitors of melanin formation are sought after for a range of applications. Boronophenylalanine is known to inhibit melanogenesis via boronic acid–catechol interactions. A spectroscopic assay was developed to study the polymerization of l-dopa to synthetic melanin in the presence of para-substituted aryl boronic acids. The best inhibition was observed for aryl boronic acids with electron-withdrawing substituents. The IC₅₀ values exhibit a correlation with the Hammett σ_p parameter (ρ = 0.97, r² = 0.92).     

Effect of L-leucine on p-aminohippurate accumulation by mammalian renal cortical slices

The effect of several amino acids (L-glutamate, L-phenylalanine, L-leucine, glycine, L-tryptophan, L-histidine, L-valine) on p-aminohippurate accumulation was evaluated in rat, mouse and rabbit kidneys. Only leucine was found to enhance p-aminohippurate accumulation in rat and mouse renal cortical slices but not in rabbit slices. Leucine had no effect on tetraethylammonium accumulation. In rat renal cortical slices, leucine increased the uptake and decreased the runout of p-aminohippurate, each effect contributing to the increase in p-aminohippurate accumulation. The apparent K_m of p-aminohippurate uptake was decreased by leucine with no change in the apparent V. Aminooxyacetate (an inhibitor of transamination of leucine) partially depressed the stimulating effect of leucine on p-aminohippurate accumulation, whereas α-ketoisocaproic acid (a metabolite of leucine formed by transamination) enhanced p-aminohippurate accumulation, suggesting that the metabolism of leucine in kidney slices may be necessary for the stimulating effect on p-aminohippurate transport.     

Interference of carbidopa and other catechols with reactions catalyzed by peroxidases

Background

A number of compounds, including ascorbic acid, catecholamines, flavonoids, p-diphenols and hydrazine derivatives have been reported to interfere with peroxidase-based medical diagnostic tests (Trinder reaction) but the mechanisms of these effects have not been fully elucidated.

Design,synthesis and biological evaluation of peptide derivatives of l-dopa as anti-parkinsonian agents

A series of dipeptide derivatives of l-dopa were synthesized and investigated for their pharmacological activity using the unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rat as an experimental model of Parkinson’s disease. Among them, (S)-isopropyl 2-(2-amino-2-methylpropanamido)-3-(3,4-dihydroxyphenyl)propanoate (4g) was found to be the most active compound, with 106% AUC activity and 149% peak activity of l-dopa after oral administration.     

A kinetic characterization of the rabbit plasmin isozymes.

Steady-state and presteady-state kinetic parameters for plasmins derived from the two rabbit plasminogen isozymes have been determined. Steady-state kinetic experiments with N-α-tosyl-l-arginine methyl ester indicate that each isozyme has a similar V. Plasmin isozyme 2 has a higher K_m value for this substrate as well as a higher K_i, for the competitive inhibitor, benzamidine-HCl. Presteady-state kinetic experiments, using the p-nitrophenyl esters of p-(methylethylsulfoniummethyl)benzoate, p-(pyridiniummethyl) benzoate, p-(thiouroniummethyl)benzoate and p-(guanidinium)benzoate, indicate that each plasmin has similar rate constants of acylation (k₂). However, values of the dissociation constant (K_S) indicate that plasmin isozyme 1 has a greater binding affinity for these substrates than does isozyme 2. The magnitude of this difference varies with the substrate and is the greatest for those containing analogs of the guanidino moiety of l-arginine.     

Kinetic study of the pathway of melanizationn between l-dopa and dopachrome

The first part of the melanization pathway from l-dopa to dopachrome has been studied as a system of various chemical reactions coupled by an enzymatic reaction. A theoretical and experimental kinetic approach is proposed for such a system. Rate constants for the implicated chemical steps at different pH and temperature values can be evaluated from measurement of the lag period arising from the accumulation of dopachrome that takes place when l-Dopa was oxidized at acid pH. The thermodynamic parameters of the chemical steps, the deprotonation of dopaquinone-H⁺ into dopaquinone and the internal cyclization of dopaquinone into leukodopachrome, have been obtained. From the results presented, an alternative series of chemical reactions to the Raper-Mason scheme are proposed and discussed.     

Simultaneous removal of 2-chlorophenol, phenol, p-cresol and p-hydroxybenzaldehyde under nitrifying conditions: kinetic study

The kinetic behavior of a stable nitrifying consortium exposed to 2-chlorophenol (2-CP), phenol, p-cresol and p-hydroxybenzaldehyde (p-OHB) was evaluated in batch assays. Phenolic compounds were evaluated either individually or in mixture. In individual assays, 2-CP inhibited stronger the nitrification, diminishing the ammonium consumption efficiency (16%) and the nitrate production rate (at 91%). Nonetheless, the consumption efficiencies for all phenolics were of 100%. On the other hand, in mixture, the inhibitory effect of 2-CP diminished significantly, since ammonium consumption efficiency and nitrate production rate were improved. Consumption efficiencies for most of the phenolic compounds were high. Furthermore, the kinetic of 2-CP oxidation was 2.4-fold-faster than the individual assays. Finally, the experimental results showed the potential of nitrifying consortium for removing 2-CP, phenol, p-cresol and p-OHB. This is the first work showing the simultaneous removal of these pollutants and also this information might be useful for treating wastewaters of chemical complexity.     

Folic acid deficiency and methyl group metabolism in rat brain: effects of L-dopa

Rats deficient in folic acid were found to have decreased concentrations of S-adenosylmethione in brain, kidney, and liver. They also showed decreased concentrations of methionine in serum, but not in brain. Administration of l-dopa (a methyl acceptor) in doses comparable to those used in the treatment of Parkinson's disease caused significant reductions in the concentrations of brain methionine in rats deficient in folic acid (45%, 45 min after administration), but failed to alter methionine concentrations in control animals. The changes in brain methionine brought about by l-dopa were not paralleled by similar changes in serum methionine, which decreased by only 20%. These observations suggest that de novo methyl group synthesis contributes significantly to the maintenance of brain methionine concentrations. The possibility is raised that the daily requirements for folic acid and for vitamin B₁₂ may be increased in human patients treated chronically with large doses of l-dopa.     

Specific spectrophotometric assays for cathepsin B1.

Cathepsin B₁ from bovine spleen was partially purified by acetone fractionation and by chromatography on Sephadex G-150 and DEAE Sephadex A-50. The enzyme was shown to catalyze the hydrolysis of p-nitrophenyl benzyloxycarbonylglycinate and p-nitrophenyl α-N-benzyloxycarbonyl-l-lysinate. Under the assay conditions, cathepsin B₁ is the major enzyme present in bovine spleen homogenates hydrolyzing these substrates. The kinetic parameters for the hydrolysis of p-nitrophenyl benzyloxycarbonylglycinate and p-nitrophenyl α-N-benzyloxycarbonyl-l-lysinate were measured and compared with those obtained for other cathepsin B₁ substrates. These results form the basis of an improved spectrophotometric assay for this enzyme in which the liberation of p-nitrophenol from either the N-benzyloxycarbonyl glycine or lysine p-nitrophenyl ester is monitored continuously at 326 nm.     

Partial characterization of peroxidase isoenzymes from rust-affected wheat leaves

Four anodic peroxidase isoenzymes from wheat leaves were purified by column chromatography and their kinetic behavior with common substrates were examined. One isoenzyme is more active in wheat resistant to stem rust fungi and differed from the others in carbohydrate content and also by a specific activity 2–4-fold higher with non-physiological electron donors. As a substrate, eugenol exhibited kinetic behavior different from p-phenylenediamine, guaiacol or o-dianisidine with all isoenzymes. All four isoenzymes showed similar pH and temperature optima and kinetic behavior and apparent K_m values for both H₂O₂ and non-physiological electron donors.     

Effects of acute and sub-chronic l-dopa therapy on striatal l-dopa methylation and dopamine oxidation in an MPTP mouse model of Parkinsons disease

Aims

The molecular mechanisms for the loss of 3,4-dihydroxyphenylalanine (l-dopa) efficacy during the treatment of Parkinson's disease (PD) are unknown. Modifications related to catecholamine metabolism such as changes in l-dopa and dopamine (DA) metabolism, the modulation of catecholamine enzymes and the production of interfering metabolites are the primary concerns of this study.

Main methods

Normal (saline) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) pre-treated mice were primed with 100 mg/kg of l-dopa twice a day for 14 days, and a matching group remained l-dopa naïve. l-dopa naive and primed mice received a challenge dose of 100 mg/kg of l-dopa and were sacrificed 30 min later. Striatal catecholamine levels and the expression and activity of catechol-O-methyltransferase (COMT) were determined.

Key findings

Normal and MPTP pre-treated animals metabolize l-dopa and DA similarly during l-dopa therapy. Administration of a challenge dose of l-dopa increased l-dopa and DA metabolism in l-dopa naïve animals, and this effect was enhanced in l-dopa primed mice. The levels of 3-OMD in MPTP pre-treated animals were almost identical to those in normal mice, which we found are likely due to increased COMT activity in MPTP pre-treated mice.

Significance

The results of this comparative study provide evidence that sub-chronic administration of l-dopa decreases the ability of the striatum to accumulate l-dopa and DA, due to increased metabolism via methylation and oxidation. This data supports evidence for the metabolic adaptation of the catecholamine pathway during long-term treatment with l-dopa, which may explain the causes for the loss of l-dopa efficacy.     

ATP-dependent calcium transport and its correlation with Ca2+-ATPase activity in basolateral plasma membranes of rat duodenum

Isolated basolateral plasmamembrane vesicles from rat duodenum epithelial cells exhibit ATP-dependent calcium-accumulation and Ca²⁺-dependent ATPase activity. Calcium accumulation stimulated by ATP is prevented by the calcium ionophore A23187, inhibited 80% by 0.1 mM orthovanadate but is not effected by oligomycin. Calcium accumulation is not observed with the substrate β-γ-(CH₂)-ATP, ADP and p-nitrophenyl phosphate. Kinetic studies reveal an apparent K_m of 0.2 μM Ca²⁺ and a V_max of 5.3 nmol Ca²⁺/min per mg protein for the ATP-dependent calcium-uptake system. Calmodulin and phenothiazines have no effect on calcium accumulation in freshly prepared membranes, but small effects are inducable after a wash with a 5 mM EGTA. The kinetic parameters of Ca²⁺-ATPase are: K_m = 0.25 μM Ca²⁺ and V_max = 19.2 nmol P_i/min per mg protein. Three techniques, osmotic shock, treatment with Triton X-100 or the channel-forming peptide alamethacin, reveal that about 40% of the vesicles are resealed. Assuming that half of the resealed vesicles have an inside-out orientation, the V_max of ATP-dependent calcium uptake amounts to 25 nmol Ca²⁺/min per mg protein and of the Ca²⁺-ATPase to 23 nmol P_i/min per mg protein. The close correlation between kinetic parameters of Ca²⁺-ATPase and ATP-dependent calcium-transport strongly suggests that both systems are expressions of a Ca²⁺-pump located in duodenal basolateral plasma membranes.     

A continuously monitored spectrophotometric assay of glycosidases with nitrophenyl glycosides

A general method for a continuously monitored spectrophotometric assay of glycosidases at all values of pH using p-nitrophenyl glycosides is presented. The method is demonstrated specifically by the development of a routine assay for α-galactosidase from fig and Mortierella vinacea using p-nitrophenyl galactopyranoside (NPG) at pH 3.9 and 5.8, respectively, and also for jack bean meal β-N-acetylhexosaminidase using p-nitrophenyl-β-2-acetamido-2-deoxy-d-glucopyranoside (NPADG) at pH 5.0. A number of different wavelengths may be used for the assay depending upon the criterion of the user; maximum sensitivity at a selected pH, determination of enzyme pH optima with a pH-independent difference extinction coefficient, or the reduction of background absorbance for kinetic studies at high substrate concentrations.     

Intracellular accumulation of bilirubin as a defense mechanism against increased oxidative stress

Antioxidant, anti-inflammatory and anti-atherogenic effects have been associated with elevations of unconjugated bilirubin (UCB) in serum and with the induction of heme oxygenase-1 (HO-1), the rate-limiting enzyme in UCB synthesis. The aim of this study was to investigate the intracellular metabolism and antioxidant properties of UCB in human hepatoblastoma HepG2 cells and tissues of Wistar rats exposed to oxidative stressors and lipopolysaccharide (LPS), respectively. Intracellular UCB concentrations in HepG2 cells correlated with its levels in culture media (p < 0.001) and diminished lipid peroxidation in a dose-dependent manner (p < 0.001). Moreover, induction of HO-1 with sodium arsenite led to 2.4-fold (p = 0.01) accumulation of intracellular UCB over basal level while sodium azide-derived oxidative stress resulted in a 60% drop (p < 0.001). This decrease was ameliorated by UCB elevation in media or by simultaneous induction of HO-1. In addition, hyperbilirubinemia and liver HO-1 induction in LPS-treated rats resulted in a 2-fold accumulation of tissue UCB (p = 0.01) associated with enhanced protection against lipid peroxidation (p = 0.02). In conclusion, hyperbilirubinemia and HO-1 induction associated with inflammation and oxidative stress increase intracellular concentrations of UCB, thus enhancing the protection of cellular lipids against peroxidation. Therefore, the previously reported protective effects of hyperbilirubinemia and HO-1 induction are at least in part due to intracellular accumulation of UCB.     

Phloem loading of sucrose: involvement of membrane ATPase and proton transport

p-Chloromercuribenzenesulfonic acid markedly inhibited sucrose accumulation into sugar beet source leaves without inhibiting hexose accumulation. The site of inhibition is proposed to be the plasmalemma ATPase, since the ATPase-mediated H⁺ efflux was completely inhibited by p-chloromercuribenzenesulfonic acid under conditions where intracellular metabolism, as measured by photosynthesis and hexose accumulation, was unaffected. Fusicoccin, a potent activator of active H⁺/K⁺ exchange, stimulated both active sucrose accumulation and proton efflux in the sugar beet leaf tissue. These data provide strong evidence for the phloem loading of sucrose being coupled to a proton transport mechanism driven by a vectorial plasmalemma ATPase.     

Identification de la 4-O-méthyl dopamine dans les tissus de rat par chromatographie liquide en phase inverse

Identification of 4-O-methyldopamine in rat tissues by reversed-phase liquid chromatography4-O-Methyldopamine was identified and assayed in tissues from l-dopa treated rats by reversed-phase high-performance liquid chromatography. The initial steps in the separation of catecholamines were performed by alumine, a weak cation-exchange resin, and thin-layer chromatographic techniques.After L-[³H] dopa administration, the radiochromatogram was superimposed on the fluorochromatogram obtained with authentic marker 4-O-methyldopamine. This metabolite was detected in kidney but not in brain.The 4-O-methyldopamine:3-O-methyldopamine ratio was 0.032 in kidney. The influence of various treatments on this ratio was investigated. A 160% increase was found after l-dopa administration. This effect was potentiated by nialamide pretreatment (550% increase).     

Phylogenetic analysis and in-depth characterization of functionally and structurally diverse CE5 cutinases

Cutinases are esterases that release fatty acids from the apoplastic layer in plants. As they accept bulky and hydrophobic substrates, cutinases could be used in many applications, ranging from valorization of bark-rich side streams to plastic recycling. Advancement of these applications, however, requires deeper knowledge of cutinases’ biodiversity and structure–function relationships. Here, we mined over 3000 members from carbohydrate esterase family 5 for putative cutinases and condensed it to 151 genes from known or putative lignocellulose-targeting organisms. The 151 genes were subjected to a phylogenetic analysis, which showed that cutinases with available crystal structures were phylogenetically closely related. We then selected nine phylogenic diverse cutinases for recombinant production and characterized their kinetic activity against para-nitrophenol substrates esterified with consecutively longer alkyl chains (pNP-C₂ to C₁₆). Each investigated cutinase had a unique activity fingerprint against the tested pNP substrates. The five enzymes with the highest activity on pNP-C₁₂ and C₁₆, indicative of activity on bulky hydrophobic compounds, were selected for in-depth kinetic and structure–function analysis. All five enzymes showed a decrease in k_cat values with increasing substrate chain length, whereas K_M values and binding energies (calculated from in silico docking analysis) improved. Two cutinases from Fusarium solani and Cryptococcus sp. exhibited outstandingly low K_M values, resulting in high catalytic efficiencies toward pNP-C₁₆. Docking analysis suggested that different clades of the phylogenetic tree may harbor enzymes with different modes of substrate interaction, involving a solvent-exposed catalytic triad, a lipase-like lid, or a clamshell-like active site possibly formed by flexible loops.