Analysis of ATP and Its Breakdown Products in Beef by Reversed-Phase HPLC

Analytical conditions for the quantitative determination of adenosine 5′-triphosphate(ATP), adenosine 5′-diphosphate(ADP), inosine 5′-monophosphate(IMP), adenosine 5′-monophosphate(AMP), hypoxanthine(Hyp), xanthine(Xan), inosine(Ino) and adenosine(Ado) in meat extracts by reversed-phase high-performance liquid chromatography (HPLC) were examined. A commercial ODS column with a 5-μm particle diameter was used, and expeimental parameters affecting the separation were discussed. Peaks in chromatograms of meat extracts were identified by retention time, co-injection with standards, absorbance ratios and the enzymatic peak shift method. The procedure proposed was adaptable as an indication of freshness of meat.     

A Simple Method for Following Postmortem ATP Depletion in Lamb Muscle

Relationship between adenosine 5′-triphosphate (ATP) concentration and R-value, defined as the absorption ratio of the degradation derivatives inosine 5′-monophosphate (IMP), inosine and hypoxanthine (E₂₅₀) to adenosine nucleotides (E₂₆₀), was studied in postmortem lamb Longissimus dorsi, until onset of rigor mortis, at different temperatures of muscle storage and lamb carcass weight. Results showed a high overall correlation coefficient (r =?0.960), nondependent of storage temperature or carcass weight. Thus, lamb postmortem ATP concentrations can be indirectly calculated through a simple, rapid, economic and reliable method such as R-value measurements.     

Early Postmortem Detection of Exudative Pork Meat Based on Nucleotide Content

The content of nucleotide metabolites in the muscle L. dorsi was analyzed for the detection of pale, soft and exudative (PSE) pork meats at just 2 h postmortem. PSE meat was characterized by significantly lower (p < 0.05) amounts of adenosine 5'-triphosphate (ATP) and higher adenosine 5'-monophosphate (AMP), inosine 5'-monophosphate (IMP), inosine (ino) and hypoxanthine (hyp) than normal meat. IMP and ino classified all the samples from both groups with statistical significance (p < 0.05). The K₀ value, R' value and IMP/ATP ratios were also useful indicators for full distinction of PSE meats. Thus, the assay of any of these nucleotide metabolites may allow a good detection of PSE meats at very short, only 2 h, postmortem time.     

Concentration of Creatine Phosphate, Adenine Nucleotides and Their Derivatives in Electrically Stimulated and Nonstimulated Beef Muscle

The concentration of creatine phosphate (CP), adenosine tri-, di- and monophosphate (ATP, ADP and AMP, respectively), inosine monophosphate (IMP) and inosine in longissimus muscle removed from electrically stimulated (ES) and nonstimulated (NS) beef sides was enzymatically determined. After aging the carcass 7 days, steaks were removed for flavor evaluation. R values (absorbance ratios) were obtained from muscle samples removed over time from both sides. Data indicate more rapid catabolism of CP, ATP, and ADP in ES samples, with subsequent fluctuations in IMP and inosine concentration. At 12 and 24 hr post-stimulation, ES samples had more inosine; however, this difference did not exist after aging. Flavor differences were not observed after the 7-day aging period. R values parallel the degradation of adenine nucleotides and indicate rapid onset of rigor mortis in ES muscle.     

Hydrophilic interaction chromatographic determination of adenosine triphosphate and its metabolites

A new HPLC method based on hydrophilic interaction chromatography (HILIC) has been developed for the simultaneous analysis of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), inosine monophosphate (IMP), inosine (Ino), hypoxanthine (Hx) and nicotinamide adenine dinucleotide (NAD⁺) in pork loin muscle. The chromatographic analysis has been developed using a zwitterionic polymeric column and it has been optimised in terms of linearity, repeatability, reproducibility and recovery. Detection limit values of 0.10, 0.15, 0.05, 0.16, 0.16, 1.72 and 0.15 μg/mL for ATP, ADP, AMP, IMP, Ino, Hx and NAD⁺ respectively were achieved. A comparison between the pork meat concentrations of the compounds analysed by hydrophilic interaction chromatography (HILIC), using a ZIC®-pHILIC column, and ion-pair reversed-phase chromatography (IP-RP-HPLC), using a Zorbax Eclipse XDB-C18 column, has been done, concluding that the new chromatographic methodology constitutes a valid and reliable alternative to the existing methods. Finally, this method has also proved to be very effective in the isolation of guanosine, guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), creatinine (Cn) and uric acid (UA) from a standards mixture.     

Extractive Components of the Adductor Muscle of Japanese Baking Scallop and Changes During Refrigerated Storage

The changes in concentration of adenosine triphosphate (ATP) and related compounds were examined as well as free amino acids, organic acids, betaines, glycogen and glycolytic metabolites in the Japanese baking scallop (Pecten albicans) during storage at 0° and 5°C. High amounts of free amino acids and glycine betaine were found. Adenosine monophosphate, inosine and hypoxanthine accumulated during storage, but inosine monophosphate was not detected. Hypoxanthine concentration was considered more appropriate as a freshness index than K value. Taurine, glycine and arginine were the main free amino acids and accounted for 91% of the total immediately after death. D-lactate increased, but no substantial change of L-lactate was detected during storage.     

不同宰前断水时间对肉鸭食用品质及能量代谢的影响

本研究以38日龄肉鸭的胸肉为研究对象,研究了不同宰前断水时间(0 h、1 h、3 h、5 h)对肉鸭食用品质及能量代谢相关限速酶活力与产物的影响,包括p Hi(初始p H值)、p Hu(最终p H值)、色泽、蒸煮损失率、加压失水率、剪切力、肌糖原、乳酸、ATP、ADP、AMP、IMP、己糖激酶、丙酮酸激酶、肌酸激酶、乳酸脱氢酶等,并对结果进行相关性分析。研究结果表明,断水处理过的肉鸭,其鸭胸肉的丙酮酸激酶活力与ATP含量呈显著负相关;己糖激酶活力与肌糖原含量有良好的正相关性;5 h断水组的IMP水平最低,ATP含量也维持在较高水准,肌糖原含量最高;经过5 h断水后的肉鸭,其鸭胸肉的p Hi显著低于3 h内断水组,使其肉质下降。综上可得,对肉鸭进行不超过5 h的短时间宰前断水处理,其食用品质、能量代谢指标处于相对较好的水平,易最大程度缓解应激,并保持肉质最佳。     

Effects of pithing on pH, creatine phosphate and ATP-related compounds of beef Psoas major and Longissimus dorsi muscles

The effects of pithing at slaughter on meat quality were studied in M. longissimus dorsi (LD muscle) and M. psoas major (PM muscle) from steers. Ten steers, half of them pithed and the others not pithed at slaughter, were used for determining pH values and concentrations of creatine phosphate (CP), adenosine triphosphate (ATP) and its breakdown products in the muscles at 2·0 h post mortem. CP was not detected in any PM muscles from pithed steers and no significant differences were observed between pithed and non-pithed steers on the mean values of pH and any compounds, but significant differences were observed in the variance of pH value, ATP, (inosine monophosphate IMP) and inosine levels in PM muscles and of (adenosine diphosphate ADP) and IMP levels in LD muscles. When two steers with excitable temperament were excluded from the pithed group significant differences of variance between the treatments were observed only in PM muscles. The pithing procedure thus affects the levels of pH and of some of the ATP-related compounds in the PM but not in LD muscles.     

Effects of meat quality and storage on the breakdown of adenosine triphosphate in muscle from swine

The quality of pork is dependent on animal genotype, pre-slaughter handling, processing, maturation, and storage. We investigated the pattern of adenosine triphosphate (ATP) breakdown as it related to these factors. Samples of the thoracis portion of the longissimus dorsi muscle were obtained from 19 randomly selected German Landrace-Pietrain crossbreed swine. Based on their 40 min post-mortem pH and electrical conductances, three groups were classified: (1) PSE meat, pH ≤ 5·6 and conductance >10 mS (n = 7); (2) intermediate quality, pH 5·6-6·0 and conductance between 4 and 10 mS (n = 5); and (3) normal quality, pH ≥ 6·0 and conductance < mS (n = 7). Hence, the animals investigated included those susceptible to malignant hyperthermia (porcine stress-syndrome). Twenty-four hours post mortem samples were evaluated for the following parameters: ATP metabolism, pH values, electrical conductance, meat colour, water binding capacity, shear force and general composition (i.e. total protein, fat, mineral and water contents). Muscle composition was the same in each group, but for the other parameters there were clear differences. Following different storage periods and conditions (1 or 5 days at 4°C and 27 days at -18°C), the degree of ATP metabolism as well as general meat quality (i.e. including sensory evaluation) were reassessed. Samples from the pre-selected groups became less discernible following prolonged storage. In all animals, the pattern of ATP breakdown was similar, the major metabolites including inosine monophosphate, hypoxanthine, adenosine monophosphate, and inosine. The degree of breakdown was dependent on the duration and temperature of storage, but not on animal type. The muscular samples for the intermediate and normal muscle groups, which were stored for 27 days at -18°C, were given the highest sensory evaluation scores. The simple HPLC measurement of ATP metabolism was considered as a useful means to assess appropriate storage.     

Patterns of Nucleotide Catabolism as Freshness Indicators in Flatfish from the Gulf of Alaska

Hypoxanthine was the major catabolite of adenosine triphosphate (ATP) in rock, yellowfin, flathead and Dover soles examined from the Gulf of Alaska. Inosine monophosphate (IMP)/hypoxanthine (Hx) ratios were as effective as K₁ values for assessing freshness. In contrast, inosine (HxR) was the end catabolite in rex sole and hypoxanthine was barely detectable. The gradual accumulation of hypoxanthine in rock and yellowfin soles made this catabolite a useful freshness indicator for these species.     

冷藏过程中三疣梭子蟹的营养物质变化

为了解冷藏条件下三疣梭子蟹的营养物质变化规律,采用基于核磁共振（nuclear magnetic resonance,NMR）的代谢组学技术对冷藏过程中的梭子蟹肌肉和肝胰腺的营养物质进行分析。结果表明,NMR技术共检测到梭子蟹肌肉中的30?种物质和肝胰腺中的27?种物质。鲜活梭子蟹在4?℃海水中冷藏保存,肌肉的营养物质变化主要发生在前2?d,而肝胰腺的营养物质变化主要发生在第1天。冷藏可导致肌肉谷氨酸、谷氨酰胺、精氨酸、牛磺酸和三磷酸腺苷水平的显著降低,乳酸、三甲胺-N-氧化物、肌苷、肌苷酸和单磷酸腺苷水平的显著升高;同时导致肝胰腺中多种氨基酸、甜菜碱、牛磺酸、葡萄糖、三甲胺-N-氧化物、胆碱-O-硫酸和2-吡啶甲醇等水平的显著降低,而乳酸水平显著升高。这些物质的变化显著改变了梭子蟹的口味和营养价值。     

Effects of freshness on ATP-related compounds in retorted chub mackerel Scomber japonicus

To examine the usefulness of measurement of adenosine triphosphate (ATP)-related compounds for estimation of the quality of canned and/or retorted fishes, we investigated the ATP-related compounds, in sashimi (for raw-eating)-grade and for-cooking-grade chub mackerel Scomber japonicus before and after retort process (121 °C for 30 min) by HPLC method. By the deterioration (storage time) and retort process, inosine monophosphate (IMP), an umami-taste compound, was decreased and inosine (HxR), hypoxantine (Hx) and the K-value, an index of fish freshness defined as the ratio of the sum of HxR and Hx to the sum of ATP-related compounds, were increased. By the retorting, the K-values of sashimi-grade, for-cooking-grade and unacceptable-grade meats were increased from 9.9%, 26.6% and 70% to 36%, 46% and 81%, respectively. These results suggest that the measurement of ATP-related compounds in retorted fish products may estimate the freshness in raw materials, the product quality and/or the good manufacture practices.     

Sapid Components in Carrot

SUMMARY: The relationship between various substances present in carrot to its taste was studied. Nucleic acid derivatives found in hot water extracts of carrot were adenine, adenosine, inosine, hypoxanthine, 5'-AMP, 5'-UMP, UDP, but 5'-IMP and 5'-GMP were absent. The contents of these derivatives were extremely small. Silica gel chromatography showed the presence of small quantities of succinic acid, α-ketoglutaric acid, lactic acid, pyroglutamic acid, citric acid and glycolic acid.
Amino acids in hot water extracts of carrot were detected by two-dimensional thinlayer chromatography and automatic amino acid analyzer. Identified were glutamic acid, valine, leucine, aspartic acid, lysine, and serine etc. Glutamic acid content was relatively large. Sucrose, maltose and glucose were detected in carrot and these carbohydrates were responsible for the sweetness of carrot. The taste of carrot was due mainly to the presence of glutamic acid and the buffer action of various amino acids.     

POSTMORTEM CHANGES IN QUALITY INDICES OF ICE-STORED FLOUNDER (PARALICHTHYS PATAGONICUS)

The characteristics of flounder (Paralichthys patagonicus) were evaluated during a 12‐day period of storage in ice by monitoring the biochemical, microbiological and sensory parameters. The adenosine‐5′‐triphosphate and adenosine‐5′‐diphosphate were not detected, and the initial content of adenosine‐5′‐monophosphate (AMP) rapidly decreased to zero. Inosine‐5′‐monophosphate (IMP) but not adenosine (Ado) was present in muscle extracts. The AMP deaminase activity was found in the muscle. Conversely, the Ado deaminase activity was not detected. A 55% decrease in IMP concentration occurred during the first 2 days of storage. The inosine content was barely detectable (<0.1 μmol/g), probably because of the high nucleoside phosphorylase activity in the muscle. Hypoxanthine significantly (P < 0.01) increased up to the seventh day and then remained approximately constant. The muscle pH fluctuated between 6.6 and 7.0. The iced flounder was microbiologically acceptable for up to 7 days with aerobic plate counts reaching <10⁷ cfu/g. The sensory score, determined by the Quality Index Method, linearly increased (r² = 0.96) with the keeping time. All the parameters, including the K‐value, indicated that the shelf life of iced flounder was 7 days.     

Monitoring of physical–chemical and microbiological changes in fresh pork meat under cold storage by means of a potentiometric electronic tongue

This work describes the correlation found along 10 days between potentiometric measurements obtained by using an electronic tongue and the variation in certain physicochemical, microbial and biochemical parameters measured on a whole piece of pork loin stored under refrigeration. The electronic tongue consists of a set of six electrodes made of Au, Ag, Cu, Pb, Zn and C, and a reference electrode. Through the use of various multivariate analysis techniques, such as: PCA and two types of artificial neural networks (i.e. multilayer perceptron (MLP) and fuzzy ARTMAP) it was found that it is possible to determine the time elapsed in relation to the degradation of the loin by using simple potentiometric measurements. Additionally, in the same pork sample used to measure redox potentials with the electronic tongue, the following parameters were also determined; pH, microbial count, concentrations of inosine 5′-monophosphate (IMP), inosine (Ino) and hypoxanthine (Hx). Through the use of PLS analysis, it was found a rather good correlation between pH and the potentiometric data. Also a remarkable correlation was observed between the measures carried out with the electronic tongue and the so-called K-index that simultaneously measures the variation in the adenosine triphosphate (ATP) degradation products. These results suggest that this simple, or a similar electronic tongue, could be useful for the undemanding qualitative or semi-quantitative evaluation of freshness in meat samples in a wide range of situations.     

Studies on nucleotides and related compounds in plants. II. An adenyl-cleaving enzyme system in potato tubers

Adenyl-cleaving activity was demonstrated in clarified potato juice. A stock freezedried enzyme preparation, obtained after (NH₄)₂SO₄ fractionation, hydrolysed adenosine and deoxyadenosine but had no effect on other possible nucleoside substrates including cytidine, deoxycytidine, guanosine, deoxyguanosine, inosine, hypoxanthosine, uridine and thymidine. The rate of adenine release for adenosine was some 60% of that for deoxyadenosine under the conditions of the digest. AMP, ADP, ATP and dAMP were all susceptible to hydrolysis releasing adenine. Evidence is produced to show that dephosphorylation is a necessary prerequisite to adenine release. The adenine nucleosidase has an optimum pH 4.1–4.6 and an apparent temperature optimum of 50 °C.     

古尼拟青霉小孢变种核苷类成分分析

以已知核苷为标样,并以冬虫夏草生药作对比,利用反相高效液相色谱法对古尼拟青霉小孢变种RCEF0864菌丝体和发酵液中核苷类化学成分进行分析,结果表明古尼拟青霉小孢变种RCEF0864菌丝体中主要含有胞苷、尿苷、鸟苷、肌苷、环磷腺苷和腺苷等6种,其中腺苷和尿苷的含量均为冬虫夏草生药的3倍左右,肌苷和鸟苷的含量则分别是冬虫夏草生药的7倍和10倍左右,尿苷的含量也高于对照品冬虫夏草生药。古尼拟青霉小孢变种RCEF0864发酵液中主要含有尿苷、胞苷、胸苷、肌苷、环磷腺苷和腺苷等6种核苷类物质,其中,腺苷和胞苷的含量均高于对照品冬虫夏草生药。     

苯酚提取-超高效液相色谱法测定烟草中的磷酸腺苷

摘 要: 建立了苯酚提取超高效液相色谱法（UPLC）同时测定新鲜烟叶中三磷酸腺苷（ATP）、二磷酸腺苷（ADP）和单磷酸腺苷（AMP）3种磷酸腺苷的分析方法。苯酚提取样品溶液中的ATP、ADP和AMP较稳定,不易水解,样品在2 h以内各磷酸腺苷变化率小于10 %。采用6 ‰三乙胺（pH=6.6）和甲醇作为流动相梯度洗脱,3种磷酸腺苷在10分钟内实现了较好的分离,线性范围（5～200）μg/mL内,磷酸腺苷浓度与峰面积具有很好的相关系数（r>0.9999）,3种磷酸腺苷检测限分别为0.5 μg/mL、0.4 μg/mL、0.3 μg/mL,回收率在91.68 %～110.79 %之间,相对标准偏差RSD在1.53 %～3.67 %之间。该方法可快速、准确检测烟草样品中ATP含量,进而为研究烟草能量代谢尤其不育系的产生机理,提供重要的检测指标。      

NUCLEOTIDE CATABOLISM IN COLD STORED ADDUCTOR MUSCLE OF SCALLOP (ZYGOCHLAMYS PATAGONICA)

The postmortem catabolism of adenosine triphosphate (ATP) in cold stored scallop adductor muscles was examined. The change In the pH of stored muscles was also investigated. The ATP content increased for a short time after death and afterwards decreased up to 24 h of storage. Thereafter, the nucleotide level remained unchanged up to 120 h of storage. The ADP content slightly decreased up to 48 h and after that remained unchanged. The AMP slowly accumulated to around 15% of the total nucleotide concentration when the ATP decreased. Small amounts of IMP were detected in all samples. Conversely, adenosine (Ado) was not detected. Inosine (HxR) slightly increased after 48 h of storage and hypoxanthine (Hx.) significantly increased after 24 h. The 260/250‐absorbance ratio of muscle extracts and the pH of stored muscles fell sharply up to 24 h and then decreased slowly. The Hx contents were positively correlated (P < 0.01) with both the Hx/AMP ratios and the K values.     

草鱼冷藏期间ATP关联物含量及新鲜度变化

目的：分析草鱼冷藏期间腺嘌呤核苷三磷酸关联物含量的变化,研究草鱼肉中各核苷酸的含量与其新鲜度的相关性。方法：利用强碱将不稳定的核苷酸关联物反应生成其钠盐形式,通过高效液相色谱法检测核苷酸含量,并通过总挥发性盐基氮含量和感官性状的变化进行鲜度评价。结果：生成钠盐形式的腺嘌呤核苷三磷酸关联物能通过色谱很好地分离。新鲜鱼肉中含有高含量的肌苷酸,达504.34 mg/kg。冷藏2 d内草鱼腺嘌呤核苷三磷酸、肌苷酸含量快速下降,次黄嘌呤含量则迅速增加。冷藏后腺嘌呤核苷三磷酸未检出,肌苷酸下降幅度达92.77%,次黄嘌呤增加幅度达89.82%。冷藏期间挥发性盐基氮含量一直上升,冷藏6 d后超出最大值限度,冷藏草鱼的贮藏期限为6 d。