Influence of the dark/light rhythm on the effects of UV radiation in the eyestalk of the crab Neohelice granulata

Crustaceans are interesting models to study the effects of ultraviolet (UV) radiation, and many species may be used as biomarkers for aquatic contamination of UV radiation reaching the surface of the Earth. Here, we investigated cell damage in the visual system of crabs Neohelice granulata that were acclimated to either 12L:12D, constant light, or constant dark, and were exposed to UVA or UVB at 12:00 h (noon). The production of reactive oxygen species (ROS), antioxidant capacity against peroxyl radicals (ACAP), lipid peroxidation (LPO) damage, catalase activity, and pigment dispersion in the eye were evaluated. No significant differences from the three groups of controls (animals acclimated to 12L:12D, or in constant light, or not exposed to UV radiation) were observed in animals acclimated to 12L:12D, however, crabs acclimated to constant light and exposed to UV radiation for 30 min showed a significant increase in ROS concentration, catalase activity, and LPO damage, but a decrease in ACAP compared with the controls. Crabs acclimated to constant darkness and exposed to UV for 30 min showed a significantly increased ROS concentration and LPO damage, but the ACAP and catalase activity did not differ from the controls (animals kept in the dark while the experimental group was being exposed to UV radiation). Pigment dispersion in the pigment cells of eyes of animals acclimated to constant light was also observed. The results indicate that UVA and UVB alter specific oxidative parameters; however, the cell damage is more evident in animals deviated from the normal dark/light rhythm.     

Role of vitamin E as a lipid-soluble peroxyl radical scavenger: in vitro and in vivo evidence

Multiple reactive oxygen/nitrogen species induce oxidative stress. Mammals have evolved with an elaborate defense network against oxidative stress, in which multiple antioxidant compounds and enzymes with different functions exert their respective roles. Radical scavenging is one of the essential roles of antioxidants and vitamin E is the most abundant and important lipophilic radical-scavenging antioxidant in vivo. The kinetic data and physiological molar ratio of vitamin E to substrates show that the peroxyl radicals are the only radicals that vitamin E can scavenge to break chain propagation efficiently and that vitamin E is unable to act as a potent scavenger of hydroxyl, alkoxyl, nitrogen dioxide, and thiyl radicals in vivo. The preventive effect of vitamin E against the oxidation mediated by nonradical oxidants such as hypochlorite, singlet oxygen, ozone, and enzymes may be limited in vivo. The synergistic interaction of vitamin E and vitamin C is effective for enhancing the antioxidant capacity of vitamin E. The in vitro and in vivo evidence of the function of vitamin E as a peroxyl radical-scavenging antioxidant and inhibitor of lipid peroxidation is presented.     

Reactive oxygen species in pregnant rats: effects of exercise and thermal stress

With the aim of evaluating the effect of interaction between physical training or exercise only during pregnancy and thermal stress on oxidative stress, and antioxidant mechanism sedentary pregnant rats (PS), exercised pregnant rats only during pregnancy (PE) and trained rats submitted to also exercise during pregnancy (PT) were compared (N=63). Exercise sessions consisted of swimming at 80% of maximal work load supported into water at 28 degrees C (hypothermia, PS 28, PE28, PT28) or 35 degrees C (thermal neutrality, PS35, PE35, PT35) or 39 degrees C (hyperthermia, PS39, PE39, PT39), for 30 min. The initial body weight in all groups of rats was from 177 to 207 g. On the 20th day of pregnancy, 24 h after the last immersion or swimming session venous blood was collected to determine oxidative stress. Plasma concentrations of means malondialdehyde (MDA) values measured as thiobarbituric acid reactive substances (TBARS); total glutathione (GSH) and vitamin E were determined. The oxidative stress index was calculated from the ratio TBARS/GSH and TBARS/Vitamin E. TBARS did not change on the group PE at different temperatures of water; TBARS were higher for PS28 than PS35 and PS39; PT35 had higher values than PT28 and PT39. For GSH, PS39 was lower than PS35; PE28 was higher than PE35 and PE39 and PT35 were lower than PT28 and PT39. Plasma concentration of vitamin E did not present any difference for sedentary rats at different water temperatures, but for PE28, the values were lower than for PE35 and PE39, whereas PT39 was lower than PT35 and PT28. In relation to TBARS/GSH, it was verified an increase in oxidative stress for PS28 (in relation to PS35 and PS39), PE35, and PT35 (in relation to PE28 and PE39 or PT28 and PT39); regarding the ratio TBARS/vitamin E, the highest values were obtained at 35 degrees C for PS and PT groups and at 39 for PE group. These results have shown the great complexity of the interaction between physical training, thermal stress and pregnancy. Apparently, hypothermia produces large index of oxidative stress only in sedentary rats, but this index was greater at 35 degrees C in relation to extreme temperatures for trained rats. These results have suggested that physical training allows a more efficient activation of antioxidant mechanisms under thermal stress.     

Dietary caloric restriction improves the redox status at the onset of diabetes in hepatocytes of streptozotocin-induced diabetic rats

Enhanced production of free radicals and oxidative stress induced by hyperglycemia play a central role in the pathogenesis of diabetes and its complications. This study assessed the attenuation by dietary caloric restriction on the oxidative and lipid peroxidative effects of diabetes in the liver through reduction in body and organ weights and concomitant metabolic changes. Three-month-old male Wistar rats were subjected to ad libitum feeding and 30% caloric restriction for 9 weeks before induction of diabetes by intraperitoneal injection of 35 mg/kg body weight streptozotocin. The animals were sacrificed 2 weeks after streptozotocin treatment depicting the onset of diabetes. Caloric restriction significantly reduced the organ weights (p<0.01), malondialdehyde (p<0.01) and catalase activity (p<0.01), but significantly increased glutathione reductase activity (p<0.01), and GSH/GSSG ratios (p<0.05). Caloric restriction also non-significantly reduced reactive oxygen species, superoxide dismutase and oxidized glutathione but increased glutathione peroxidase activity and reduced glutathione levels in the diabetic rats. Our data indicate a decrease in lipid peroxidation, improvement in the antioxidant defense systems and restoration of the redox status in the liver by caloric restriction. Therefore, this could provide a non-invasive antioxidant therapy early in diabetes to prevent the development of the complications associated with the disease.     

Effect of indole acetic acid administration on the neutrophil functions and oxidative stress from neutrophil, mesenteric lymph node and liver

This study was done to investigate the effect of the in vivo administration of indole acetic acid (IAA) on the neutrophil function, the activities of antioxidants enzymes in neutrophils, the mesenteric lymph node and on the oxidative stress in liver and plasma. The animals received subcutaneous administration of IAA in a phosphate-buffered saline (the control group received only the phosphate-buffered saline). The other groups received IAA at concentrations of 1 mg (T1), 2 mg (T2) and 18 mg (T3) per kg of body mass per day. Administration of IAA in both treatments T2 and T3 promoted a significant rise in the phagocytic capacity of neutrophils (by 51%), in comparison with the control. Another alteration was observed in antioxidant enzyme activities of the neutrophil and lymph node. But in the liver, the treatments imposed a significant decrease in the activity of catalase of 19% and 30% for T2 and T3, respectively, in comparison with the control. A similar effect was observed in the activity of hepatic glutathione peroxidase for T3 where a significant decrease of 31%, compared with the control, was obtained. The IAA did not show another significant alteration of the activities of superoxide dismutase and glutathione reductase activities in liver. The hepatic lipid peroxidation level, available by reactive products with thiobarbituric acid, has shown a significant decrease of 27% and 29% with T1 and T3 respectively, in comparison with the control. IAA treatment did not show a significant alteration in reduced glutathione contents in comparison with the liver and plasma controls. In conclusion, the IAA administration has a good potential animal utilization for increasing the phagocytic capacity with no prooxidant effect.     

Antioxidant responses and lipid peroxidation in gills and erythrocytes of fish (Rhabdosarga sarba) upon exposure to Chattonella marina and hydrogen peroxide: Implications on the cause of fish kills

Chattonella marina, a red tide or harmful algal bloom species, has caused mass fish kills and serious economic loss worldwide, and yet its toxic actions remain highly controversial. Previous studies have shown that this species is able to produce reactive oxygen species (ROS), and therefore postulated that ROS are the causative agents of fish kills. The present study investigates antioxidant responses and lipid peroxidation in gills and erythrocytes of fish (Rhabdosarga sarba) upon exposure to C. marina, compared with responses exposed to equivalent and higher levels of ROS exposure. Even though C. marina can produce a high level of ROS, gills and erythrocytes of sea bream exposed to C. marina for 1 to 6 h showed neither significant induction of antioxidant enzymes nor lipid peroxidation. Antioxidant responses and oxidative damage did not occur as fish mortality began to occur, yet could be induced upon exposure to artificially supplied ROS levels an order of magnitude higher. The result of this study implies that ROS produced by C. marina is not the principal cause of fish kills.     

The effects of dietary restriction on oxidative stress in rodents

Oxidative stress is observed during aging and in numerous age-related diseases. Dietary restriction (DR) is a regimen that protects against disease and extends life span in multiple species. However, it is unknown how DR mediates its protective effects. One prominent and consistent effect of DR in a number of systems is the ability to reduce oxidative stress and damage. The purpose of this review is to comprehensively examine the hypothesis that dietary restriction reduces oxidative stress in rodents by decreasing reactive oxygen species (ROS) production and increasing antioxidant enzyme activity, leading to an overall reduction of oxidative damage to macromolecules. The literature reveals that the effects of DR on oxidative stress are complex and likely influenced by a variety of factors, including sex, species, tissue examined, types of ROS and antioxidant enzymes examined, and duration of DR. Here we present a comprehensive review of the existing literature on the effect of DR on mitochondrial ROS generation, antioxidant enzymes, and oxidative damage. In a majority of studies, dietary restriction had little effect on mitochondrial ROS production or antioxidant activity. On the other hand, DR decreased oxidative damage in the majority of cases. Although the effects of DR on endogenous antioxidants are mixed, we find that glutathione levels are the most likely antioxidant to be increased by dietary restriction, which supports the emerging redox-stress hypothesis of aging.     

Mitochondrial free radical overproduction due to respiratory chain impairment in the brain of a mouse model of Rett syndrome: protective effect of CNF1

Rett syndrome (RTT) is a pervasive neurodevelopmental disorder mainly caused by mutations in the X-linked MECP2 gene associated with severe intellectual disability, movement disorders, and autistic-like behaviors. Its pathogenesis remains mostly not understood and no effective therapy is available. High circulating levels of oxidative stress markers in patients and the occurrence of oxidative brain damage in MeCP2-deficient mouse models suggest the involvement of oxidative stress in RTT pathogenesis. However, the molecular mechanism and the origin of the oxidative stress have not been elucidated. Here we demonstrate that a redox imbalance arises from aberrant mitochondrial functionality in the brain of MeCP2-308 heterozygous female mice, a condition that more closely recapitulates that of RTT patients. The marked increase in the rate of hydrogen peroxide generation in the brain of RTT mice seems mainly produced by the dysfunctional complex II of the mitochondrial respiratory chain. In addition, both membrane potential generation and mitochondrial ATP synthesis are decreased in RTT mouse brains when succinate, the complex II respiratory substrate, is used as an energy source. Respiratory chain impairment is brain area specific, owing to a decrease in either cAMP-dependent phosphorylation or protein levels of specific complex subunits. Further, we investigated whether the treatment of RTT mice with the bacterial protein CNF1, previously reported to ameliorate the neurobehavioral phenotype and brain bioenergetic markers in an RTT mouse model, exerts specific effects on brain mitochondrial function and consequently on hydrogen peroxide production. In RTT brains treated with CNF1, we observed the reactivation of respiratory chain complexes, the rescue of mitochondrial functionality, and the prevention of brain hydrogen peroxide overproduction. These results provide definitive evidence of mitochondrial reactive oxygen species overproduction in RTT mouse brain and highlight CNF1 efficacy in counteracting RTT-related mitochondrial defects.     

Effects of linuron and dimethenamid on antioxidant systems in weeds associated with soybean

Changes in antioxidant systems in soybean and associated weeds (Ambrosia artemisiifolia L., Chenopodium album L., Convolvulus arvensis L and Sinapis arvensis L.) were studied in relation to treatment with herbicides linuron and dimethenamid in the field experiment. Differences in the total superoxide dismutase (SOD) and catalase (Cat) activities were observed in plants after application of herbicide formulation. Quantities of superoxide (O₂.-) and hydroxyl (·OH) radicals and malonyldialdehyde (MDA), reduced glutathione (GSH) and total polyphenols content were also determined. In addition to this, potential antioxidant activity of the plant ethanolic extracts were assessed based on the scavenging activity of stable DPPH free radicals. Results obtained suggest that plants investigated 1) expressed different antioxidant systems in response to herbicide treatment; 2) enzymatic and non-enzymatic protective mechanisms were complementary; 3) some weed species showed distinctive and combined activity of several biochemical parameters, such as Ambrosia artemisiifolia.     

The role of reactive oxygen species and oxidative stress in carbon monoxide toxicity: An in-depth analysis

Abstract

The underlying mechanism of the central nervous system (CNS) injury after acute carbon monoxide (CO) poisoning is interlaced with multiple factors including apoptosis, abnormal inflammatory responses, hypoxia, and ischemia/reperfusion-like problems. One of the current hypotheses with regard to the molecular mechanism of CO poisoning is the oxidative injury induced by reactive oxygen species, free radicals, and neuronal nitric oxide. Up to now, the relevant mechanism of this injury remains poorly understood. The weakening of antioxidant systems and the increase of lipid peroxidation in the CNS have been implicated, however. Accordingly, in this review, we will highlight the relationship between oxidative stress and CO poisoning from the perspective of forensic toxicology and molecular toxicology.     

The effects of reactive oxygen species on amphibian aging

To clarify the role of reactive oxygen species (ROS) in the aging process of amphibians, antioxidant enzyme activity and indexes of ROS damage were investigated biochemically using the livers of 3- and 10-year-old Rana nigromaculata frog males and females. Findings revealed no significant difference in survival rate between males and females. Antioxidant enzyme activity displayed an age-related decline. Superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) activity in 10-year-old liver decreased 40-80% from 3-year-old liver levels. In contrast, urate oxidase activity in the 10-year-old liver increased more than 200% from 3-year-old liver levels. At the same time levels of ROS damage, including the concentration of inorganic peroxide and thiobarbituric acid reactive substances (TBARS), greatly increased with age. Liver catalase from 10-year-old frogs proved to be more susceptible to aminotriazole and urea, losing approximately 80% of its original activity after 30 min of treatment. It seems likely that liver catalase in older frogs has diverged from liver catalase in younger frogs through oxidative modification. These findings suggest that a decrease in the activity of antioxidant enzymes over time results in increased levels of ROS damage in the livers of older frogs.     

The complex interplay of iron metabolism,reactive oxygen species,and reactive nitrogen species: Insights into the potential of various iron therapies to induce oxidative and nitrosative stress

Production of minute concentrations of superoxide (O₂⁻) and nitrogen monoxide (nitric oxide, NO) plays important roles in several aspects of cellular signaling and metabolic regulation. However, in an inflammatory environment, the concentrations of these radicals can drastically increase and the antioxidant defenses may become overwhelmed. Thus, biological damage may occur owing to redox imbalance—a condition called oxidative and/or nitrosative stress. A complex interplay exists between iron metabolism, O₂⁻, hydrogen peroxide (H₂O₂), and NO. Iron is involved in both the formation and the scavenging of these species. Iron deficiency (anemia) (ID(A)) is associated with oxidative stress, but its role in the induction of nitrosative stress is largely unclear. Moreover, oral as well as intravenous (iv) iron preparations used for the treatment of ID(A) may also induce oxidative and/or nitrosative stress. Oral administration of ferrous salts may lead to high transferrin saturation levels and, thus, formation of non-transferrin-bound iron, a potentially toxic form of iron with a propensity to induce oxidative stress. One of the factors that determine the likelihood of oxidative and nitrosative stress induced upon administration of an iv iron complex is the amount of labile (or weakly-bound) iron present in the complex. Stable dextran-based iron complexes used for iv therapy, although they contain only negligible amounts of labile iron, can induce oxidative and/or nitrosative stress through so far unknown mechanisms. In this review, after summarizing the main features of iron metabolism and its complex interplay with O₂⁻, H₂O₂, NO, and other more reactive compounds derived from these species, the potential of various iron therapies to induce oxidative and nitrosative stress is discussed and possible underlying mechanisms are proposed. Understanding the mechanisms, by which various iron formulations may induce oxidative and nitrosative stress, will help us develop better tolerated and more efficient therapies for various dysfunctions of iron metabolism.     

Fasting induces cyanide-resistant respiration and oxidative stress in the amoeba Chaos carolinensis: implications for the cubic structural transition in mitochondrial membranes

Summary. Large free-living amoeba (Chaos carolinensis) can survive in spring water without food intake for several weeks. Starvation is associated with a dramatic change in mitochondrial cristae from random tubular to ordered (paracrystalline) cubic morphology. Whole-cell polarography was used to monitor changes in respiratory activity during fasting. Basal respiration per cell decreased progressively during starvation, while the cyanide-resistant fraction increased. Spectrofluorometric assay of H₂O₂ and reactive oxygen species (ROS) in cell lysates (using the dye 2′,7′-dichlorofluorescein diacetate) indicates greater H₂O₂ and ROS generation in starved than in fed cells. Fluorescence microscopy of intact cells incubated with the same dye demonstrates that H₂O₂ and ROS tend to accumulate in vacuoles. A remarkable generation of O₂ observed with starved cells after addition of KCN may be explained by release of H₂O₂ from these compartments into the cytosol, where it can react with catalase. Together, these observations suggest that fasting increases oxidative stress in the amoeba and that this organism has several protective mechanisms to deal with it, including activation of a plantlike alternative oxidase. The hypothesis is forwarded that the cubic structural transition of the mitochondrial inner membrane represents another protective mechanism, reducing oxidative damage by enhancing the efflux of H₂O₂ and ROS and by reducing the susceptibility of membrane lipids to the oxidants. Received September 1, 2001 Accepted January 7, 2002     

Exogenous insulin can reverse the effects of caloric restriction on mitochondria

It has been proposed that part of the anti-aging mechanism of caloric restriction (CR) involves a reduction in both the generation rate of reactive oxygen species (ROS) by mitochondria, and a reduction in peroxidizability of mitochondrial membranes. It was hypothesized that these effects may be due to upstream changes in hormonal status, since certain hormones (such as insulin) are stimulatory for ROS production, effect fatty acid composition, and are lowered by CR. To investigate this hypothesis, young male Brown-Norway rats on 55% CR (4 months duration) were subjected to insulin replacement by use of mini-osmotic pumps. ROS and free radical-induced malondialdehdye production were significantly lower in mitochondria from CR animals compared to those from fully fed, and these effects were reversed by insulin. It is concluded that the beneficial changes induced by CR, as seen at the mitochondrion, may in part be downstream effects of alterations in hormonal signalling.     

Studies on the protective effects of betaine against oxidative damage during experimentally induced restraint stress in Wistar albino rats

Stress can be defined as physical and psychological modifications that disrupt the homeostasis and the balance of organisms. Stress is known as one of the most important reasons of several diseases. In the present study, the anti-stress effect of betaine was evaluated with reference to its antioxidant property. Wistar albino rats were divided into four groups such as control, betaine, restraint stress (6 h/day for 30 days), and betaine + restraint stress. The oxidative damage was assessed by measuring the protein and corticosterone in plasma, lipid peroxidation, non-enzymic (reduced glutathione), and enzymic antioxidants (glutathione peroxidase, glutathione-S-transferase, catalase, and superoxide dismutase) in the lymphoid organs of thymus and spleen. Followed by the induction of restraint stress, the non-enzymic and enzymic antioxidants were significantly decreased with concomitant increase observed in the levels of corticosterone and lipid peroxidation. Oral pretreatment with betaine (250 mg/kg body weight daily for a period of 30 days) significantly (P < 0.001) prevented the restraint stress-induced alterations in the levels of protein and corticosterone in plasma of experimental groups of rats. It counteracted the restraint stress-induced lipid peroxidation and maintained the antioxidant defense system in the lymphoid tissues at near normal. The findings suggest that betaine possesses significant anti-stress activity, which may be due to its antioxidant property.     

The quality of the antioxidant defence system in term and preterm twin neonates

Abstract

Objective: Multiple pregnancy is associated with an enhanced metabolism and demand for O2, which may lead to the overproduction of reactive oxygen species and the development of oxidative stress. The degree of oxidative damage depends on the level of the antioxidant protection system of the foetus. The objective of the study was to identify the relationship between the state of the maturity and the antioxidant status of twin neonates. Investigations of the umbilical cord blood were carried out to detect differences in the antioxidant defence system between mature and premature twin neonates.

Methods: The activities of the superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) enzymes, the levels of reduced glutathione (GSH), protein carbonyls and oxidized lipids and the total antioxidant capacity of the plasma were determined.

Results: The level of lipid peroxidation was significantly higher in the premature neonates. An increase in the total antioxidant capacity was accompanied by a decrease in the damaged protein concentration. Significantly elevated activities of GPx alone were observed in the premature twins, though the GSH content too tended to be increased. The activity of SOD was decreased in the premature neonates.

Discussion: The antioxidant status of twin neonates are mainly influenced by maturity. We suggest that the level of lipid peroxidation might be of clinical value as a marker of pre- and perinatal distress in twins.     

Influence of vitamin C and vitamin E on redox signaling: Implications for exercise adaptations

The exogenous antioxidants vitamin C (ascorbate) and vitamin E (α-tocopherol) often blunt favorable cell signaling responses to exercise, suggesting that redox signaling contributes to exercise adaptations. Current theories posit that this antioxidant paradigm interferes with redox signaling by attenuating exercise-induced reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation. The well-documented in vitro antioxidant actions of ascorbate and α-tocopherol and characterization of the type and source of the ROS/RNS produced during exercise theoretically enable identification of redox-dependent mechanisms responsible for the blunting of favorable cell signaling responses to exercise. This review aimed to apply this reasoning to determine how the aforementioned antioxidants might attenuate exercise-induced ROS/RNS production. The principal outcomes of this analysis are (1) neither antioxidant is likely to attenuate nitric oxide signaling either directly (reaction with nitric oxide) or indirectly (reaction with derivatives, e.g., peroxynitrite); (2) neither antioxidant reacts appreciably with hydrogen peroxide, a key effector of redox signaling; (3) ascorbate but not α-tocopherol has the capacity to attenuate exercise-induced superoxide generation; and (4) alternate mechanisms, namely pro-oxidant side reactions and/or reduction of bioactive oxidized macromolecule adducts, are unlikely to interfere with exercise-induced redox signaling. Out of all the possibilities considered, ascorbate-mediated suppression of superoxide generation with attendant implications for hydrogen peroxide signaling is arguably the most cogent explanation for blunting of favorable cell signaling responses to exercise. However, this mechanism is dependent on ascorbate accumulating at sites rich in NADPH oxidases, principal contributors to contraction-mediated superoxide generation, and outcompeting nitric oxide and superoxide dismutase isoforms. The major conclusions of this review are: (1) direct evidence for interference of ascorbate and α-tocopherol with exercise-induced ROS/RNS production is lacking; (2) theoretical analysis reveals that both antioxidants are unlikely to have a major impact on exercise-induced redox signaling; and (3) it is worth considering alternate redox-independent mechanisms.     

Determination of urinary malondialdehyde by isotope dilution LC-MS/MS with automated solid-phase extraction: a cautionary note on derivatization optimization

A highly sensitive quantitative LC-MS/MS method was developed for measuring urinary malondialdehyde (MDA). With the use of an isotope internal standard and online solid-phase extraction, urine samples can be directly analyzed within 10 min after 2,4-dinitrophenylhydrazine (DNPH) derivatization. The detection limit was estimated as 0.08 pmol. This method was further applied to assess the optimal addition of DNPH for derivatization and to measure urinary MDA in 80 coke oven emission (COE)-exposed and 67 nonexposed workers. Derivatization optimization revealed that to achieve complete derivatization reaction, an excess of DNPH is required (DNPH/MDA molar ratio: 893-8929) for urine samples that is about 100 times higher than that of MDA standard solutions (molar ratio: 10-80). Meanwhile, the mean urinary concentrations of MDA in COE-exposed workers were significantly higher than those in nonexposed workers (0.23±0.17 vs 0.14±0.05 μmol/mmol creatinine, P<0.005). Urinary MDA concentrations were also significantly associated with the COE (P<0.005) and smoking exposure (P<0.05). Taken together, this method is capable of routine high-throughput analysis and accurate quantification of MDA and would be useful for assessing the whole-body burden of oxidative stress. Our findings, however, raise the issue that derivatization optimization should be performed before it is put into routine biological analysis.