Taurine in Fresh Seafishes and Processed Seafishes
Pornpimol Muangthai2015, Asian journal of natural and applied sciences
Sign up for access to the world's latest research
checkGet notified about relevant papers
checkSave papers to use in your research
checkJoin the discussion with peers
checkTrack your impact
Abstract
The aim of this work was to study the effects of processed on taurine content in fishes samples from Gulf of Thailand. Taurine in samples were qualitative tested by thin layer chromatography (TLC) and quantitatively analysed by Ultraviolet –Visible Spectrophotometry technique. The method in the analysis was also validated. The results revealed that the rapid qualitative test by TLC showed all fresh fish samples contain taurine in their.The quantitative analysis of taurine was performed with high sensitivity and selectivity with LOD and LOQ as 6.27 μM amd 189.99 μM,respectively. Taurine in Russel’s snapper contained the highest taurine level approximately as 5548.68 ± 7.78 mg/100g. Processed method such as frying , grilling and steaming effect on taurine content in fish samples.The experiment showed the effect on taurine content depend on processing method.
Related papers
Inventory and Extraction of Taurine from Deep Sea Fishes: Escolar (<em>Lepidocybium flavobrunneum</em>) and Lobster
Advance Journal of Food Science and Technology, 2014
Today the utilization of deep sea fishes for consumption as food supplements and pharmaceuticals is being developed. Escolar (Lepidocybium flavobrunneum) and deep sea lobster, originating from South Sea of Java-Indonesia can be used for product development as a source of taurine. The purpose of this study was to determine the characteristics of escolar and lobster as raw materials and to determine the best extraction treatment to produce crude taurine powder. Analysis of taurine level in fresh samples indicated that taurine level of escolar flesh was higher than the lobster. Escolar and lobster had yield of flesh at 59.81 and 20.47%. The yield of escolar and lobster viscera were 11.41 and 4.62%, respectively. Protein level which was calculated as the sum of free nitrogen in escolar was higher than the lobster. Highest amino acid content in escolar flesh was glutamic acid (2.04%) for nonessential amino acid and lysine (1.21%) for essential amino acid. Highest taurine level in escolar crude taurine powder produced from the extraction treatment at 30°C for 60 min with taurine levels of 83.71 mg/100 g.
The effect of dietary taurine on growth performance and liver histopathology in Persian sturgeon, Acipenser persicus (Borodin, 1897) fed plant-based diet
This experiment was conducted to illustrate the effect of dietary taurine levels on Persian sturgeon, Acipenser persicus (mean weight of ~35 g) fed plant-based diet. A basal diet containing low level of fishmeal (19%) was formulated containing 0.05% endogenous taurine (control diet). To this diet were added five levels of exogenous taurine: 0.25 (0.25T), 0.5 (0.5T), 0.8 (0.8T), 1.2 (1.2T) and 1.6% (1.6T). These diets were fed to the experimental fish for 42 days. Results showed that growth performance decreased and food conversion ratio increased as dietary taurine increased, although no significant difference was observed between the control and 0.25T treatments. Increased dietary taurine led to increased carcass moisture and decreased carcass lipid percentages. Feed intake was significantly and negatively correlated with dietary taurine. Carcass taurine content significantly increased with dietary taurine elevation up to 1.2% and then decreased. Hepatic tau-rine content showed a significant increase in 0.25T treatment and then decreased along with dietary taurine elevation and reached the control values at 1.6% taurine level. Dietary taurine elevation led to more hepatic damages compared to the control. In conclusion, although within the range reported for other studied fish, the tested taurine levels in this study might be supra-optimal causing adverse effects in Persian sturgeon and further study with lower taurine levels is encouraged.
Taurine supplementation in diet for olive flounder at low water temperature
Fisheries and Aquatic Sciences, 2017
The objective of this study was to examine the effect of dietary supplementation of taurine for juvenile olive flounder (Paralichthys olivaceus) at low water temperature (16.4 ± 0.36°C). Fish meal (FM)-based diet was used as the control diet. Four other experimental diets were prepared by adding taurine to FM-based diet at 0.25, 0.50, 1.00, and 1. 50% (T1, T2, T3, and T4, respectively). Each experimental diet was fed to triplicate groups of fish (initial mean body weight, 19.5 g) for 10 weeks. At the end of the feeding trial, growth performance and feed utilization, hematological parameters, non-specific immune responses, whole-body proximate composition, and liver mRNA expression of insulin-like growth factor-1 (IGF-1) were investigated. Feed conversion ratio was significantly reduced while protein efficiency ratio was significantly increased in taurine-supplemented groups. Hematocrit and hemoglobin were also significantly increased while plasma cholesterol levels were decreased in taurine-supplemented groups than those in the control group. Nitroblue-tetrazolium, myeloperoxidase and lysozyme activities, and plasma immunoglobulin level were significantly increased by taurine supplementation. These results suggest that dietary taurine supplementation is effective in improving growth performances, feed utilization, and innate immunity of olive flounder in low water temperature season.
Effect of Taurine Supplementation on Skin Thickness and Scale Detachability in Red Sea Bream Pagrus major
2012
Taurine supplementation, to compensate for the reduction of fish meal (FM) in fish diets, has been the subject of numerous investigations. However, the taurine contents of FM-based diets are still lower than in diets of raw fish, even in diets where FM is the only protein source. In this study, the effect of taurine supplementation to commercial feed (containing 57% FM) on skin thickness and scale detachability in red sea bream Pagrus major was investigated. Three different levels of taurine were used: 0% taurine (control), 1% taurine (Tau-1%), and 2% taurine (Tau-2%). Red sea bream (average body weight, 107 g) were fed these diets for 60 days. No effects of taurine supplementation on growth and feeding efficiency were observed. However, skin thicknesses of fish from the Tau-1% and Tau-2% groups were significantly higher than for the control group, and scale detachability of the control group was significantly higher than for the Tau-1% and Tau-2% groups. These results suggested tha...
Taurine improves the growth performance, health status and liver histopathology of meagre (Argyrosomus regius) fed a reduced fish meal diet
Research has focused on alternative plant materials or additives that can be used instead of fish meal due to limited stocks and increased feed prices, although it is important for carnivorous fish species. Taurine is a functional amino acid supplemented to low fish meal diets to improve the growth performance of fish. In this study, eight experimental diets with 0%, 0.5%, 1% and 2% ratios of taurine supplemented (T0, T0.5, T1 and T2) high (HFM) and low fish meal (LFM) diets were prepared (defined as HFMT0, HFMT0.5, HFMT1, HFMT2, LFMT0, LFMT0.5, LFMT1 and LFMT2). The experiment was carried out in twenty-four 400 L rectangular fiberglass tanks conducted in a marine water system, and fish (initial mean weight of 23.5 g) were stocked in each tank. The nutrition trial was carried out for 90 days with three replicates. Meagre fed with the HFMT1 diet had a higher final mean weight (FMW) and specific growth rate (SGR) than fish fed with HFMT0 and all LFM diets. Including taurine in all LFM...
High taurine supplementation in plant protein-based diets improves growth and organoleptic characteristics of European seabass (Dicentrarchus labrax)
Scientific Reports
Plant-based proteins are increasingly being used in aquafeeds, but one of the limitations is taurine deficiency that can be especially detrimental for carnivorous fish. In this study, taurine supplementation in high plant protein diets (low fish meal, 15%) was investigated on the growth performance and fillet organoleptic characteristics of European seabass (Dicentrarchus labrax), juveniles (78 ± 0.4 g). Five diets were formulated to contain 0 (C−), 5 (T5), 10 (T10) or 20 (T20) added taurine (g/kg), while a control diet (C+) included two-fold higher amount of fishmeal (30%) with no taurine supplementation. Seabass fed the T20 or C+ diet showed similar growth, which was significantly higher compared to those in the C-treatment. Histological examination of the muscle, liver and intestine were similar among treatments. No effect on spoilage bacterial growth or production of total volatile basic nitrogen was observed. Taurine supplementation significantly reduced fillet drip loss, while...
Protective Effect of Dietary Taurine from ROS Production in European Seabass under Conditions of Forced Swimming
Animals
Taurine (Tau) is an amino sulfonic acid, which is widely distributed in animal tissues, whereas it is almost lacking in plants with the exception of certain algae, seaweeds, and few others. In the aquafeed industry, Tau is mainly used as a feed additive to promote growth in marine fish species with limited cysteine sulfinate decarboxylase activity. In particular, Tau supplementation is required in feeds in which fishmeal (FM) is substituted with high percentages of plant-derived protein sources such as soybean meals (SBM) that have much lower levels of Tau than FM. In addition to being a growth promoter, Tau exert powerful antioxidant properties being a scavenger of the reactive oxygen species (ROS). Under sustained swimming conditions, an intracellular increase in ROS production can occur in fish red muscle where the abundance of mitochondria (the main site of ROS formation) is high. Accordingly, this study aimed at investigating the effects of dietary Tau on European seabass (Dice...
Effects of Dietary Taurine on Growth, Body Composition, Blood Parameters, and Enzyme Activities of Juvenile Sterlet (Acipenser ruthenus)
Aquaculture Nutrition
To evaluate the effects of dietary taurine on Acipenser ruthenus, a total of 180 fish with an initial weight of 14.13 ± 0.10 g, were stocked into 15 (300 liters) tanks (12 fish/replicate) for 70 days. Five experimental diets (38.60% crude protein and 18.78% crude lipid) supplemented with 0 (T0), 5 (T5), 15 (T15), 30 (T30), and 50 (T50) g/kg taurine were used. The experimental fish were fed to visual satiation four times daily. At the end of the experimental period, weight gain, thermal growth coefficient, and survival in fish fed T30 diet was higher ( P < 0.05 ) than other experimental fish. Food conversion ratio in all treatments containing taurine was significantly lower than T0 treatment ( P < 0.05 ). The highest and the lowest total essential amino acids of muscle were found in T50 and T0 treatments, respectively. The highest serum cholesterol and low-density lipoprotein levels were observed in the T30 treatment ( P < 0.05 ). An increasing trend was observed in the val...
Assessment of dietary taurine essentiality on the physiological state of juvenileTotoaba macdonaldi
Aquaculture Research, 2017
Information on specific nutrients like taurine is important to support a nutritionally balanced diet for marine species such as totoaba Totoaba macdonaldi under culture conditions. Eight isoproteic (50%) and isolipidic (12%) experimental diets were formulated to contain graded levels of taurine (0.23%, 0.45%, 0.91%, 1.28%, 1.76%, 2.20%, 2.72%, 3.01% as-is) using ethanol-washed fishmeal (FM) as primary protein source. Green liver, low gallbladder-somatic index (GBSI), low apparent digestibility coefficient (ADC) of lipid, low erythrocyte turnover, low plasma cholesterol and triglycerides, as well as low visceral fat, were detected in the basal diet (T-0.23) after 10 weeks. Thermal-unit growth coefficient (TGC) was best modelled by a fiveparameter saturation kinetic model (5-SKM, p < .001) with marginal differences (R 2 = .13) of juvenile totoaba (initial weight 10.0 AE 1.0 g). Low GBSI (0.095%) was accompanied with low ADC of lipid (78.9%) in the low-taurine diet (T-0.23). GBSI and ADC of lipid were best modelled by four-parameter SKM (4-SKM, R 2 =.72 and .87, respectively, p < .001). Plasma cholesterol increased linearly with dietary taurine (R 2 = .75, p < .001), while plasma triglycerides increased quadratically (R 2 = .53, p < .001), suggesting modulations of lipid metabolism in totoaba. Plasma bilirubin levels increased linearly, and the lowest concentration was found in the basal diet (T-0.23); however, haematocrit and haemoglobin levels were changed slightly between the treatments. This study demonstrates that supplementation with a low level of taurine (0.45% as-is) in a washed FM-based diet can normalize the physiological condition of juvenile totoaba. Green liver, GBSI, ADC of lipid, plasma cholesterol and triglycerides may be informative diagnostic tools for taurine deficiency in totoaba.
Development of an LC–MS–MS method for the quantification of taurine derivatives in marine invertebrates
Analytical Biochemistry, 2004
Sulfur amino acids, such as taurine, hypotaurine, and thiotaurine, were found in high quantities in tissues of marine symbiotic organisms (e.g., bivalves, tubeworms) living close to hydrothermal vent sites. Therefore, they are assumed to play a key role in the S-oxidizing base metabolism or sulfide detoxification. We propose here a specific, rapid, and original analytical procedure for the direct determination of sulfur amino acids at the level of a few parts per billion in biological samples, avoiding the classical low specific post-column orthophthaldialdehyde derivatization step required by non-ultraviolet-absorbing molecules. Indeed, by coupling liquid chromatography on a porous graphitic stationary phase under isocratic conditions (10 mM ammonium acetate buffer adjusted to pH 9.3) to tandem mass spectrometry (ionization process by pneumatically assisted electrospray in negative ion mode), it is possible to perform specific quantification of these metabolites in less than 10 min directly in biological matrices without any derivatization step or other tedious sample treatments. Thus, taurine, hypotaurine, and thiotaurine have been identified and assayed in several deep sea organisms, showing that the developed method is well suited for this kind of application.
Related topics
Related papers
Natural Taurine Extraction of Escolar (Lepidocybium flavobrunneum) As Deep Sea Fish from Southern Java Ocean
2014
This study aimed to determine chemical composition and natural taurine content of escolar (Lepidocybium flavobrunneum). The proximate analysis was carried out using methods of AOAC. Results showed that escolar’s flesh and viscera contained protein at 16,40% and 12,11%, moisture at 63,38% and 76,78%, ash content at 0,59% and 2,52%, and fat content at 18,34% and 7,51%. Extraction of escolar using boiling and steaming method was conducted to produce crude taurine extract. Analysis of taurine content using HPLC method showed that fresh escolar’s flesh and viscera contained 44,201 mg/100g and 43,915 mg/100 g taurine. The concentration of taurine obtained by steaming treatment of escolar’s flesh and viscera was 112,203 and 128,918 mg/100 g. The concentration of taurine obtained by boiling treatment of escolar’s flesh and viscera was 103,324 mg/100 g and 105,230 mg/100 g. Taurine content of crude taurine extract which obtained by steaming method was higher than boiling method. Based on the...
Effects of taurine supplementation in low fish meal diets for red seabream (Pagrus major) in low water temperature season
Fisheries and Aquatic Sciences, 2019
BackgroundTaurine is a conditional essential amino acid for fish. A study was conducted to investigate the compensating effect of supplemental taurine in diets for red seabream (Pagrus major) on impaired growth performance by fish meal (FM) replacement with soybean meal (SM) at low water temperature (14.15 ± 1.95 °C).MethodsA FM-based diet was considered as a high FM diet and three other experimental diets were formulated to replace FM with SM by 20, 35, or 50% (HFM, SM20, SM35, or SM50, respectively) without taurine and other four diets were formulated by adding 1% taurine to the diets (HFM-T, SM20-T, SM35-T, or SM50-T, respectively). Triplicate groups of fish (108.9 ± 1.58 g/fish) were distributed into 24 polyvinyl circular tanks (215 L) with 20 fish per tank and fed one of the diets to satiation for 20 weeks.ResultsGrowth performance and feed utilization of red seabream were significantly improved by the dietary taurine supplementation. SM20-T and SM35-T diets increased fish grow...
Dietary taurine improved growth performance, nutrient utilization, and antioxidant enzyme activities in pangasius Pangasianodon hypophthalmus
Journal of the World Aquaculture Society, 2021
Taurine is a β sulphonic amino acid and is conditionally essential for many fish species. In the present study, the effect of dietary taurine (tau) on Pangasianodon hypophthalmus fry (initial mean weight 0.8 ± 0.6 g) was evaluated by feeding six isonitrogenous (370 g/kg crude protein) and isolipidic (100 g/kg crude lipid) casein-based purified diets with graded levels of tau at 0 (basal diet), 5, 10, 15, 20, and 25 g/kg feed. Fish were randomly stocked in triplicate groups in 100 L plastic tubs and fed to apparent satiation over two feedings at 10.00 and 16.00 hr daily for 45 days. Final weight, weight gain, specific growth rate, and protein efficiency ratio improved significantly (p < .05) with increasing dietary tau up to 15 g/kg feed and plateaued thereafter. Whole-body protein content did not change beyond tau 15 g/kg feed, but carcass lipid content significantly (p < .05) decreased. The group of fish fed with tau 25 g/kg feed exhibited significantly higher (p < .05) superoxide dismutase, catalase, and glutathione s-transferase activity. According to the findings of this study, the optimum dietary tau content in P. hypophthalmus fry feed was estimated to be 15.05-16.31 g/kg feed as per the broken-line regression
Taurine concentrations in animal feed ingredients; cooking influences taurine content
Journal of Animal Physiology and Animal Nutrition, 2003
The aim of this study was to determine the taurine content in a variety of animal feeds. There is very little information on the taurine content of ingredients used in homeprepared diets for dogs and cats, and foods fed to wild animals in captivity. This study reports the taurine content of both common and alternative feed ingredients, and compares taurine loss as a result of different methods of food preparation. Foods were selected based on their use in commercial and home-prepared diets. Animal muscle tissue, particularly marine, contained high taurine concentrations. Plant products contained either low or undetectable amounts of taurine. The amount of taurine that remained in a feed ingredient after cooking depended upon the method of food preparation. When an ingredient was constantly surrounded by water during the cooking process, such as in boiling or basting, more taurine was lost. Food preparation methods that minimized water loss, such as baking or frying, had higher rates of taurine retention.
Taurine modulates hepatic oxidative status and gut inflammatory markers of European seabass (Dicentrarchus labrax) fed plant feedstuffs-based diets
Amino Acids, 2019
This study aimed to evaluate the effect of taurine (tau) supplementation to low fishmeal (FM) diets on growth performance, oxidative status, and immune response of European seabass juveniles. Four isoproteic (46% crude protein) and isolipidic (19% crude lipid) diets were formulated to contain either 25 or 12.5% FM and a mixture of plant feedstuffs, supplemented or not with 1% tau. Twelve groups of 20 fish (IBW = 9.4 g) were fed each diet for 9 weeks. Reduction of dietary FM from 25 to 12.5% impaired growth performance, feed efficiency, and protein efficiency ratio but had no effect on nitrogen retention (% N intake). Independently of FM level, dietary tau supplementation improved growth performance and nitrogen retention without affecting feed efficiency. Dietary FM level reduction increased liver G6PDH activity, but did not affect lipid peroxidation or activities of redox key enzymes. Contrarily, dietary tau supplementation decreased hepatic G6PDH and GPX activities and lipid peroxidation. Gene expression COX-2 was not affected either by FM or tau levels but TNF-α increased with the reduction of FM level but not with the tau level. Dietary tau supplementation decreased Casp3 and Casp9 expression regardless of dietary FM level. Overall, this study evidenced that dietary tau supplementation improved growth performance and antioxidant response and reduced intestine inflammatory and apoptosis processes.
Evaluation of dietary taurine concentrations in microparticulate diets provided to larval California yellowtail (Seriola dorsalis) postlarvae
Aquaculture Nutrition, 2017
Taurineisanimportantaminoacidderivativeformarineandfreshwaterfish, especially duringearlydevelopment.Weinvestigatedtherangeoftaurineconcentrationsthat influencethegrowthandsurvivalratesofCaliforniayellowtail(CYT;Seriola dorsalis) during transition from live feeds to microparticulate diets, as well as the extent to whichnutrientleachingfromthemicroparticulatedietsaffectstheseranges.Wetested particle-assistedrotationallyagglomerated(PARA)particleswithfourlevelsoftaurine: 4 (low taurine; LT), 45 (medium taurine; MT), 93 (high taurine; HT) and 122 g/kg (veryhightaurine;VHT).OurresultsshowedthatCYTpostlarvaehadnosignificant differencesingrowth,survivalandfeedconsumptionratesbetweentheMT,HTand VHT treatments. However, it should be noted that the PARA particles containing 122g/kg(VHT)taurinewereespeciallypronetoleachingandmayhavehadtaurine concentrations as low as 34.9g/kg before they settled on the bottom of the tank. Therefore,theactualdietarytaurineconcentrationsexperiencedbythelarvaewere likelylowerthantheinitialdietaryconcentrations.Moreresearchisneededtoevaluate the potential nutrient toxicity of elevated dietary taurine concentrations for marine fishlarvaeandjuveniles.
Utilisation of fish industry residuals: Screening the taurine concentration and angiotensin converting enzyme inhibition potential in cod and salmon
Journal of Food Composition and Analysis, 2009
Is a taurine supplement necessary in fishmeal-based feeds for juvenile European sea bass (Dicentrarchus labrax)?
Aquaculture International, 2019
The effect of taurine supplementation of fishmeal-based feeds for juvenile European sea bass (Dicentrarchus labrax) was investigated. Fish (initial weight ca. 5 g) were reared for 90 days in 12 concrete tanks (1 m 3), with 50 fish in each tank. Four feeds (44% crude protein (CP) and 17% lipids (L)) containing 0 (control (CTRL)), 0.4 (T4), 0.7 (T7) and 1.0 (T10) % taurine were prepared and were fed to apparent visual satiation to triplicate groups of fish. The effects on fish growth, feed utilization, proximate chemical composition, haematology, immune biomarkers and muscle morphometry were examined. An increase in the dietary taurine incorporation resulted in progressive increases in the growth metrics, with the highest values being observed for fish receiving the T10 feed (weight gain 22 vs 18 g/fish in CTRL; protein productive value 31 vs 28% in CTRL). Taurine addition at all levels significantly increased the fish protein percentage, but the percentage lipid was reduced significantly in T4 fish only (7.8% compared with 9.4% in CTRL fish). Taurine supplementation resulted in elevated blood lymphocyte and monocyte counts and increased serum phagocytic and lysozyme activities. Taurine addition at 1% (T10 feed), the suggested level to boost growth, altered the dorsal muscle cellularity and myofibril ultrastructure, suggesting enhanced muscle function and firmness in comparison with that of the fish given the CTRL feed.
Effect of dietary taurine and methionine supplementation on growth performance, body composition, taurine retention and lipid status of Persian sturgeon,<i>Acipenser persicus</i>(Borodin, 1897), fed with plant-based diet
Aquaculture Nutrition, 2017
The aim of this study was to investigate the effects of dietary taurine and methionine on Persian sturgeon (Acipenser persicus) fed with plant-based diets. To this purpose, sturgeons were fed with either of methionine + taurine-supplemented (MT), without taurine supplementation (M) or without methionine supplementation (T) diets. Growth performance, body composition, tissue amino acids and serum glucose and lipids were studied at the end of the experiment. Results showed that the treatments T and MT had similar growth performance and feed efficiency, which were significantly higher than the treatment M. Dietary taurine and methionine significantly altered carcass and liver taurine and methionine contents. The highest serum glucose was observed in the treatment M and the lowest in treatment MT. Serum cholesterol and triglyceride levels in the treatments T and MT were similar and significantly higher than those of the treatment M. This study showed that Persian sturgeon needs taurine supplementation when fed with plant-based diets, but the supplemented level seems to be lower than those of the other species. Taurine has hypoglycaemic and lipidogenesis effects on Persian sturgeon. However, further studies are required to illustrate taurine and methionine metabolism in Persian sturgeon.
Effect of Dietary Taurine Supplementation on Growth Performance and Body Composition of Snapper, Lutjanus colorado Juvenile
Turkish Journal of Fisheries and Aquatic Sciences, 2018
This study was conducted to evaluate the effect of dietary taurine supplementation on growth, immunity and resistant to dry stress of rice field eel (Monopterus albus) fed low fish meal diets. Six isonitrogenous and isolipid diets (32% fish meal) supplemented with six taurine concentrations (0, 0.3, 0.6, 0.9, 1.2 and 1.5 g/kg; designated as T 0 , T 0.03 , T 0.06 , T 0.09 , T 0.12 and T 0.15 groups, respectively) were prepared. A diet including 42% fish meal (FM group) was also included as a reference. The results showed that specific growth rate (SGR) in FM group was significantly higher than that in lower fish meal treatments. SGR significantly increased and slowly decreased with the increase in taurine supplementation level. Lipase activity value in intestine of M. albus fed FM diet was maximum, and with the increase in taurine supplementation level, lipase activity significantly increased and slowly decreased. The FM group had relative higher total antioxidant capacity (T-AOC) content, catalase (CAT), total superoxide dismutase (T-SOD), and lyzozyme (LZM) activities in serum than the other groups. With the increase in dietary taurine supplementation level, the CAT, T-SOD, T-AOC and LZM activities in serum significantly increased and then decreased. In the dry stress experience, the adrenaline (AD), cortisol (COR), glucose (GLU), total cholesterol (CHOL), and malondialdehyde (MDA) concentrations, T-AOC content, CAT and T-SOD activities in serum of M. albus in the four groups first increased and reached the peak at 2 hr, and then decreased under air-exposure stress. Compared to the FM group, T 0.
Loading Preview
Sorry, preview is currently unavailable. You can download the paper by clicking the button above.