Retinyl Acetate Research Articles

New type of microfabricated carbon electrodes for high-performance liquid chromatography—Amperometric detection of fat-soluble vitamins and antioxidants

This study evaluates performance of thin-film carbon electrodes prepared by physical vapor deposition of the electrode material on a polyetheretherketone substrate and compares their performance with that of a standard type of glassy carbon electrodes for chromatographic detection cell of thin-layer type. Kaempferol, retinol, retinyl acetate, cholecalciferol, α-tocopherol and γ-tocopherol were selected for the study and their respective detection limits were found to be 0.63, 5.84, 5.91, 28.19, 16.80 and 16.11 pg. The calibration plots were linear for at least three orders of magnitude for all of the six analytes selected for evaluation. Hydrodynamic voltammograms are shown for both types of electrodes for the range of working electrode potentials between +0.40 and +1.40 V. Also shown and discussed are plots of signal-to-noise vs. detection potential. The working electrode potential of +1.30 V was found to be required for an improved long-term stability of detection performance and was suitable for the detection of all six analytes.

Kinetic Analysis Shows that Vitamin A Disposal Rate in Humans Is Positively Correlated with Vitamin A Stores ,

Vitamin A (VA) kinetics, storage, and disposal rate were determined in well-nourished Chinese and U.S. adults using model-based compartmental analysis. [2H8]Retinyl acetate (8.9 μmol) was orally administered to U.S. (n = 12; 59 ± 9 y; mean ± SD) and Chinese adults (n = 14; 54 ± 4 y) and serum tracer and VA concentrations were measured from 3 h to 56 d. Using the Windows version of the Simulation, Analysis and Modeling software, we determined that the average time from dosing until appearance of labeled retinol in serum was greater in U.S. subjects (40.6 ± 8.47 h) than in Chinese subjects (32.2 ± 5.84 h; P < 0.01). Model-predicted total traced mass (898 ± 637 vs. 237 ± 109 μmol), disposal rate (14.7 ± 5.87 vs. 5.58 ± 2.04 μmol/d), and system residence time (58.9 ± 28.7 vs. 42.9 ± 14.6 d) were greater in U.S. than in Chinese subjects (P < 0.05). The model-predicted VA mass and VA mass estimated by deuterated retinol dilution at 3 and 24 d did not differ. VA disposal rate was positively correlated with VA traced mass in Chinese (R2 = 0.556), U.S. (R2 = 0.579), and all subjects (R2 = 0.808). Additionally, VA disposal rate was significantly correlated with serum retinol pool size (R2 = 0.227) and retinol concentration (R2 = 0.330) in all subjects. Our results support the hypothesis that VA stores are the principle determinant of VA disposal rate in healthy, well-nourished adults.

Extraction and quantification of major carotenoids in processed foods and supplements by liquid chromatography

A simple, fast and robust method with minimum steps, small sample size and amounts of solvents was developed to determine major carotenoids contents in processed foods, tablets and gel capsules. The method involves dispersion of the sample in hot water (60 °C) with added butylated hydroxytoluene (BHT) in ethanol to minimize oxidation, followed by extraction with chloroform and analysis by liquid chromatography. Chromatographic parameters were: a C30 column protected with a C18 guard cartridge; gradient elution at the rate of 1.0 mL/min starting with 100% methanol (A) and ending with 40:60 (v/v) methanol/isopropanol (B); detection set at 450 nm for carotenoids, and 325 nm for retinol, retinyl acetate and retinol palmitate. The method exhibited: (i) high degree of repeatability (%rsd); (ii) linear calibration curves ( r 2 ⩾ 0.9998); (iii) low detection; and quantification limits. The method was validated with standard reference material 2383 for trans-β-carotene; and tested for α-, and β-carotenes, lutein, zeaxanthin, cryptoxanthin, trans-retinol in processed foods, tablets and gel supplements.

Class III Obesity and its Relationship with the Nutritional Status of Vitamin A in Pre- and Postoperative Gastric Bypass

Epidemiological findings have shown the rise of the prevalence of obesity in several segments of the world population, and more recent evidences point to a possible association with vitamin A deficiency (VAD). The aim of this study was to investigate vitamin A nutritional status in individuals with class III obesity in the preoperative period and 30 and 180 days after Roux-en-Y gastric bypass, correlating these findings with lipid profile and body mass index (BMI). The sample was composed of class III obese individuals, males and females, in the preoperative period (T1) and 30 (T2) and 180 days (T3) after bariatric surgery with 5,000 IU of supplementation of retinol acetate. Vitamin A nutritional status was assessed through biochemical indicators (retinol and beta-carotene serum levels), which were quantified by high-performance liquid chromatography with an inadequacy cutoff of <1.05 micromol/L and >or=40 microg/dL, respectively. Out of the 114 assessed patients, the mean age was 36.9+/-11.6 years and BMI was >or=40 kg/m2. The prevalence of VAD was 14%, being 37.5% in T1, 50.8% and 67.8% in T2, and 52.9% and 67% in T3, according to retinol and serum beta-carotene, respectively. A decrease of retinol and beta-carotene serum levels was observed with BMI increase in T1. An increase of very-low-density lipoprotein cholesterol (VLDLc) and triglycerides was verified with the increase of retinol, being VLDLc in T3 and triglycerides in T1 and T2. In T1, high-dense lipoprotein cholesterol presented a positive and significant correlation with beta-carotene and a negative and significant correlation in T3. High prevalence of VAD in the preoperative period and in 30 and 180 postoperative days, even during supplementation, with higher inadequacy of beta-carotene in all the three studied time periods, probably occurred because of its bioconversion to retinol due to the increased demand to which those individuals were exposed. It is suggested that the oral intake supplementation does not present the expected impact, and the need of assessing the nutritional status of vitamin A in the pre- and postoperative Roux-en-Y gastric bypass is emphasized. High prevalence of VAD in T1, with severity in T2 and T3, corroborates the utilization of the cutoff of <1.05 micromol/L as a VAD marker in the studied segment and reinforces its utilization as a tool in clinical practice to identify VAD in morbid obese patients who underwent bariatric surgery.

Simultaneous Determination of Five Fat-Soluble Vitamins in Feed by High-Performance Liquid Chromatography Following Solid-Phase Extraction

A high-performance liquid chromatography method is developed for the simultaneous determination of menadione, retinyl acetate, cholecalciferol, alpha-tocopherol, and alpha-tocopherol acetate in feed. The present study uses an enzyme to destroy the coating film, ethanol to extract free vitamins, and Oasis HLB cartridges to purify. Vitamins are separated using an Atlantis dC18 column. The mobile phase is methanol-water (98:2 v/v). Detection is performed with a UV-vis detector at 230 and 265 nm. The linearity, accuracy, and repeatability of this method are all satisfactory. Application of the method is suitable for the determination of the fat-soluble vitamins in general feed.

Expression of Stimulated by Retinoic Acid Gene 8 (Stra8) in Spermatogenic Cells Induced by Retinoic Acid: An In Vivo Study in Vitamin A-Sufficient Postnatal Murine Testes1

Vitamin A is required for male fertility and normal spermatogenesis. Retinoic acid (RA), an active metabolite of vitamin A, is necessary for spermatogonial maturation and proper entry of germ cells into meiotic prophase in the postnatal testes. The expression of Stra8, which is essential for successful meiosis in both male and female gonads and normal spermatogenesis, is directly related to the availability of RA. This study examined the developmental expression pattern of Stra8 transcript in both male and female gonads, provided specific cellular localization of STRA8 protein in the postnatal and adult testis, and investigated RA actions in adult germ cells in a vitamin A-sufficient condition. The peak of Stra8 mRNA expression coincided with the onset of meiosis in postnatal testes. STRA8 protein was detected in gonocytes as early as 5 days postpartum. The expression of STRA8 protein in the neonatal testes was not uniform among spermatogonia, perhaps heralding the asynchronous beginning of spermatogenesis. In adult testes, the highest level of Stra8 mRNA and protein was found in seminiferous epithelial stages VI-VIII. STRA8 protein was localized to some type A and B spermatogonia, preleptotene spermatocytes, and early leptotene spermatocytes. In the vitamin A-sufficient adult testes, RA but not retinol acetate stimulated Stra8 mRNA expression. STRA8 protein expression in adult spermatogonia was induced by RA stimulation, suggesting its role in spermatogonial differentiation. Retinoic acid also increased the number of preleptotene spermatocytes exhibiting 5-bromo-2-deoxyuridine incorporation, indicating a more synchronized premeiotic DNA replication.

UVA photoirradiation of anhydroretinol – formation of singlet oxygen and superoxide

Anhydroretinol is a metabolite of vitamin A (retinol) and a major photodecomposition product of retinyl palmitate and retinyl acetate. Anhydroretinol is biologically active, inducing cell death in lymphoblastoid cells, prevention of N-methyl-N-nitrosourea-induced mammary cancer, and inhibition of cell growth in lymphocytes. We have previously determined that photoirradiation of anhydroretinol in the presence of a lipid, methyl linoleate, with UVA light-induced lipid peroxidation. In the present study, electron spin resonance (ESR) spin-trap techniques were employed to explore the mechanism of lipid peroxidation initiation. Irradiation of anhydroretinol by UVA in the presence of 2,2,6,6-tetramethylpiperidine (TEMP), a specific probe for singlet oxygen, resulted in the formation of TEMPO, indicating that singlet oxygen was generated. During photoirradiation in the presence of 5,5-dimethyl N-oxide pyrroline (DMPO), a specific probe for superoxide, ESR signals for DMPO-OOH were formed, and these signals were quenched by superoxide dismutase. The involvement of singlet oxygen on the induction of lipid peroxidation was also evidenced by the observation that lipid peroxidation was inhibited by sodium azide and enhanced by deuterium oxide. Our overall results provide evidence that photoirradiation of anhydroretinol with UVA light generates reactive oxygen species, e.g. singlet oxygen and superoxide, which mediate the induction of lipid peroxidation.

Analysis of vitamins A, E and C, iron and selenium contents in infant milk-based powdered formula during full shelf-life

The stability of vitamins A, E and C, and the iron and selenium content were determined in two types of long chain-polyunsaturated fatty acid (LC-PUFA) supplemented milk-based powdered infant formulas (IF), during an 18-month storage period at 25 and 40 °C. The first type (IF-A) was supplemented with vitamin A as retinol acetate. The second type (IF-B) was supplemented with vitamin A as retinol palmitate. Both types were also supplemented with vitamin E as α-tocopherol acetate and with vitamin C as ascorbic acid. The two formulas studied had higher vitamin A (140% and 139%), vitamin E (109% and 198%) and vitamin C (167% and 118%), but lower iron (65.0% and 65.3%) and selenium (72.9% and 79.4%) than the amounts declared on the label. As expected, all the studied vitamins showed decreases during storage, and these decreases were higher in formulas stored at 40 °C. The losses of vitamin A at 40 °C after 18 months of storage were 27.5% in IF-A and 29% in IF-B, while vitamin E losses under the same conditions were 23.1% and 28.1%, and vitamin C losses under the same conditions were 28.4% and 48.6%. All these losses justify the over-fortification of the aforementioned vitamins in these LC-PUFA supplemented IFs. Iron and selenium content remained unchanged throughout storage.

Comparative studies on radical cation formation from carotenoids and retinoids

A comparative study of radical cation formation from selected polyenes, namely carotenoids (C 40) and retinoids (C 20), has been carried out by treatment with the Lewis acids BF 3 as its etherate or SbCl 3. The reaction in chloroform was monitored by vis/NIR and EPR spectroscopy at variable temperature. β,β-Carotene, β,β-caroten-4-ol, retinol (vitamin A), retinyl acetate and anhydroretinol were used as substrates. It is concluded that whereas BF 3–diethyl etherate or SbCl 3 is capable of effecting one-electron transfers to produce radical cations from the longer polyenes (carotenoids), no radical cations were obtained from the retinoids. The results of SbCl 3 treatment of the retinoids have a bearing on the current studies on the mechanism of the Carr–Price blue colour reaction previously used for quantitative analysis of vitamin A.

Bioavailability and storage stability of vitamin A fortificant (retinyl acetate) in fortified cookies

Vitamin A deficiency is one of the major nutritional deficiencies affecting population in developing regions. In Pakistan, 60% of the child deaths are due to diarrhea and respiratory infection, which are associated with vitamin A deficiency. This study was to assess the bioavailability and stability of vitamin A fortificant in cookies fortified with retinyl acetate. Cookies fortified with Vitamin A (retinyl acetate) were manufactured and physically, chemically and sensorial analyzed. Efficacy studies were carried and Serum retinol concentrations were measured to assess the bioavailability of Vitamin A (retinyl acetate), different organ weights were also measured to check the effects of Vitamin A (retinyl acetate) on organ weights and body growth. The treatment T 4 containing (257.85 μg) 45% RDA fortification level of retinyl acetate was judged best considering, physical, chemical and sensory characteristics. Baking losses were lowest in T 4 . Efficacy study revealed that Serum retinol concentrations were 125.19 μg/dl in control group and it was raised to 148.64 μg/dl in T 4 . Liver, lungs, left kidney, right kidney, heart, spleen, brain, left testicles and right testicles increased in their weights when fed with vitamin A fortified diet as compared to control. Exceptions were only brain and spleen, in spleen it remained stable and brain weight reduced as compared to control. Fortification of cookies with 257.85 μg (45% RDA) of retinol acetate improved nearly all quality attributes and through efficacy studies it was concluded that vitamin A significantly affects growth and body functioning of rats. Concludingly cookies fortified with 257.85 μg of retinol acetate can be used effectively to overcome vitamin A deficiency in the children.

Barrier Function, Epidermal Differentiation, and Human β-Defensin 2 Expression in Tinea Corporis

Barrier Function, Epidermal Differentiation, and Human β-Defensin 2 Expression in Tinea Corporis

Vitamin A: Deficiency and Food-Based Combating Strategies in Pakistan and Other Developing Countries

Vitamin A deficiency is a world wide nutritional problem especially in the developing countries that afflicts severely the health of pregnant and lactating women, infants and children. Women are the most vulnerable group affected by vitamin A deficiency during their pregnancy and lactating periods. Different strategies like food diversification, fortification, and supplementation are helpful to cope with vitamin A deficiency. Food fortification is the most cost effective, long-term approach, while supplementation is considered to be the fastidious way to uplift vitamin A status. Generally Retinyl acetate and retinyl palmitate are used in different food products as fortificants against the Vitamin A menace. In addition to these food-based strategies, educating the people regarding health and nutrition can also play an effective role.

Isolation and culture of adult neurons and neurospheres

Here we present a protocol for extraction and culture of neurons from adult rat or mouse CNS. The method proscribes an optimized protease digestion of slices, control of osmolarity and pH outside the incubator with Hibernate and density gradient separation of neurons from debris. This protocol produces yields of millions of cortical, hippocampal neurons or neurosphere progenitors from each brain. The entire process of neuron isolation and culture takes less than 4 h. With suitable growth factors, adult neuron regeneration of axons and dendrites in culture proceeds over 1-3 weeks to allow controlled studies in pharmacology, electrophysiology, development, regeneration and neurotoxicology. Adult neurospheres can be collected in 1 week as a source of neuroprogenitors ethically preferred over embryonic or fetal sources. This protocol emphasizes two differences between neuron differentiation and neurosphere proliferation: adhesion dependence and the differentiating power of retinyl acetate.

A validated liquid chromatography method for the simultaneous determination of vitamins A and E in human plasma

A fast (15 min) and simple HPLC method for determination of all- trans-retinol and α-, γ- and δ-tocopherols in human plasma has been developed. The assay utilized 200 μl of plasma to which 20 μl of internal standard solution (retinol acetate) was added followed by 200 μl of water, 400 μl of ethanol and 800 μl of hexane. The hexane layer was collected, evaporated, the residue dissolved in 200 μl of methanol and analyzed on a Zorbax Eclipse XDB-C18 column using a step gradient with a polar organic mobile phase composed of acetonitrile and methanol and variable wavelength fluorescence detection. The quantification limits for all- trans-retinol and γ-tocopherol were 20 ng/ml and for α- and δ-tocopherols 500 ng/ml and 10 ng/ml, respectively. The procedure was validated and applied to the analysis of plasma samples from the Baltimore Longitudinal Study of Aging.

Vitamin A kinetics and utilization in Chinese vs US adults

Vitamin A (VA) kinetics, storage, and utilization were compared in well-nourished Chinese (Wang Z et al., Br J Nutr; 91:–31, 2004) and US adults (Tang G et al., Am J Clin Nutr; 78:–66, 2003). [2H8]Retinyl acetate was orally administered (Chinese: n=15; 55 ± 4 y; mean ± SD and US: n=13; 60 ± 8 y), and plasma tracer and VA concentrations were measured from 3 h to 56 d. Data for plasma tracer vs time were fit to a 7-compartment model using the Simulation, Analysis and Modeling software (WinSAAM). The average time for the appearance of labeled retinol bound to retinol-binding protein in plasma was similar in both groups (~31 h). The plasma retinol pool was 3.34 ± 1.08 in Chinese vs 5.09 ± 1.11 μmol in US subjects (P<0.001); the model-predicted total VA pool was 232 ± 116 and 1866 ± 1603 μmol (P<0.002), and disposal rate was 4.74 ± 2.55 and 9.89 ± 5.15 μmol/d (P<0.0025). Based on these kinetic parameters, Chinese had 54.0 ± 26.9 d of VA stores vs 267 ± 314 d in US adults (P<0.016). Our results indicate that VA stores and disposal rates differ in healthy, well-nourished Chinese and US adults. (Chinese National Natural Science Foundation #30271121 and USDA #51000-065)

Comparing the vitamin A bioefficacy of β‐cryptoxanthin to β‐carotene from supplements and maize in Mongolian gerbils

Maize biofortified with provitamin A carotenoids maintains vitamin A (VA) status in Mongolian gerbils. β-Cryptoxanthin (βCX) contributes to VA, but the VA equivalency factor of βCX is unknown. Two studies were performed in gerbils to compare the bioefficacy of βCX to that of β-carotene (βC). Study 1 (n = 47) examined the VA value of oil supplements and study 2 (n = 57) used biofortified maize with varying levels of βCX. Gerbils were fed VA-free diets (45% white maize) for 4 wk. Seven gerbils were killed at baseline. Remaining gerbils were placed into weight-matched groups of 10. In study 1, treatments were oil control, or 35, 35, or 17.5 nmol VA (retinyl acetate), βCX, or βC, respectively. Blood and livers were collected at 3 wk. In study 2, 3 groups were fed diets with 45% maize which were mixed to contain 5.9 nmol VA/g feed assuming 100% bioefficacy. The diets had 0.30, 1.05, or 2.13 nmol βCX/g feed. Controls received either equivalent VA or oil only. Livers and serum were collected at 4 wk. In study 1, liver VA was higher in the VA (0.69 ± 0.11 μmol) and βCX (0.60 ± 0.10 μmol) groups than in the βC (0.49 ± 0.13 μmol) and control (0.37 ± 0.15 μmol) groups (P < 0.05). Study 2 will further evaluate the provitamin A value of βCX from maize. βCX contributes significant VA to the gerbil. Biofortified maize should be promoted in countries that consume maize as a staple food to enhance VA status. Supported by HarvestPlus, Hatch WIS04975 and USAID.

Maternal chronic vitamin A toxicity amplifies early fetal liver retinyl ester storage in captive Old World monkeys

Excessive preformed vitamin A (VA) intake is contraindicated during pregnancy due to teratogenic concerns. Recent studies have provided biochemical and histological evidence of chronic VA toxicity in captive Old World monkeys consuming laboratory diets containing high concentrations of retinyl acetate. This study investigated the effects of maternal chronic VA toxicity on early fetal hepatic VA storage. Monkey fetal livers (n = 19) ranging from 35 to 93 d gestational age (mid 1st to late 2nd trimester) were analyzed for VA content. Retinoic acid was quantified in a subset of livers (n = 9). Retinyl esters were identified in all fetal livers, and retinol, as a percentage, was more pronounced in younger fetuses. Hepatic VA concentration increased with gestational age (r = 0.92, P < 0.0001), ranging from 0.0011 μ mol/g in the youngest fetus (35 d) to 0.26 μ mol/g in the oldest fetus (93 d) indicating linear accumulation. Mean hepatic VA concentrations were 0.023 ± 0.008 μmol/g for the 1st trimester and 0.19 ± 0.06 μmol/g for the 2nd trimester. Hepatic VA concentrations from 2nd trimester fetuses were higher than those observed in fetal human and monkey livers from later stages of development, when growth and VA accumulation rates are assumed to be the highest. Maternal VA toxicity from excessive intake results in amplified early fetal hepatic retinyl ester storage. Supported by NIH-NIDDK RO1-61973 and 5P51 RR 000167.

Effect of a minor vitamin A deficiency on the course of infection with Ascaridia galli (Schrank, 1788) and the resistance of chickens

Abstract The effect of vitamin A deficiency was studied in chickens infected with 500 Ascaridia galli eggs and controls. Diet 1 (deficient, 500 IU vitamin A or 172 μg retinol acetate per kg diet), Diet 2 (deficient, 1000 IU vitamin A or 344 μg retinol acetate per kg diet) and Diet 3 (sufficient, 1500 IU vitamin A or 516 μg retinol acetate per kg diet) were assigned to 46 chickens each. Clinical signs, weight gains, livers’ weights, vitamin A levels, worm burdens and parasite eggs’ excretions were recorded. Infected chickens had lower weight gains than the controls fed alike. Chickens given Diet 1 stored lesser vitamin A in liver than those fed Diet 3. Although worm counts in the 3 groups did not differ significantly, chickens fed Diet 1 excreted more A. galli eggs than those fed the 2 other diets. Female worms harboured by chickens fed Diet 1 had higher fecundity at week 5 pi than those of chickens fed Diet 2. Results indicated that Vitamin A is important for poultry in the moderation of the infection with A. galli.

Modelling retinoid content in live prey: A tool for evaluating the nutritional requirements and development studies in fish larvae

This study was conducted to evaluate the accumulation of different levels of total vitamin A in live prey ( Brachionus plicatilis and Artemia nauplii) enriched with a commercial emulsion (0.15 and 0.6 g l − 1 for rotifers and Artemia nauplii, respectively) , which contained increasing levels of all- trans retinyl acetate. Emulsions used for rotifer enrichment contained 124, 138, 151, 165, 178, 192, 226, 259 and 327 μg total vitamin A l − 1 , whereas those used for Artemia nauplii contained 494, 548, 602, 629, 710, 764, 899, 1034 and 1334 μg total vitamin A l − 1 . Total vitamin A incorporation in rotifers was constant until a threshold comprised between 226 and 327 μg total vitamin A l − 1 , above which the incorporation of total vitamin A from the emulsion was maximum (806 ng total vitamin A mg DW − 1 in rotifers enriched with 327 μg total vitamin A l − 1 ). In Artemia nauplii, total vitamin A increased from 4.0 ng mg DW − 1 up to 52 ng mg DW − 1 in nauplii enriched with an emulsion containing 1334 μg total vitamin A l − 1 . Retinoid levels in live prey increased as the content of all- trans retinyl acetate augmented in the emulsion, although they did not accumulate in a dose-dependent manner because retinoid incorporation in live prey was found to be not proportional to the content in the emulsion. Rotifers exhibited a higher retinoid incorporation pattern than Artemia nauplii, which seemed to be related to species-specific differences between both live prey. Both live prey were able to absorb and metabolize the vitamin A compounds administered through the emulsion, according to the results regarding retinol and retinoic acid content although the levels were higher in the rotifers than in the nauplii. The differential pattern of total vitamin A accumulation between rotifers and Artemia nauplii should be considered when designing nutritional studies dealing with this vitamin and first feeding marine larvae reared on live prey due to the difficulty in maintaining constant levels of total vitamin A especially during the transition feeding phase from rotifers to Artemia nauplii.

Studies on the mechanism of the Carr–Price blue colour reaction

The reaction of retinoids (retinol, retinyl acetate and anhydroretinol) with Brønsted acids was studied as a model system for the Carr-Price reaction. The anhydroretinylic cation was characterised by VIS and 2D NMR spectroscopy, including an estimate of the charge distribution and region of bond inversion, observed in a mixture of identified E/Z isomers. Products obtained by quenching with NaOMe-MeOH were identified by HPLC and MS. The classical Carr-Price reaction between retinol (vitamin A) and the Lewis acid SbCl(3) in saturated chloroform solution was reinvestigated by VIS, NMR, EPR, dynamic light scattering and chemical quenching. Whereas product instability and failure to provide informative NMR spectra indicated a radical cation, EPR results excluded free-radical species. Dynamic light scattering experiments, in comparison with model systems, revealed strong aggregation for the Carr-Price complex, rationalizing the low stability, NMR problems and dimerisation observed by chemical quenching. The VIS data support structural similarity of the blue Carr-Price product with the delocalized anhydroretinylic cation, and a detailed structure of the antimony complex is evaluated.