Only few benefits from propylene glycol drench in early lactation for cows identified as physiologically imbalanced based on milk spectra analyses.

The main objective of this study was to test the efficiency of a management system combining metabolic clustering of cows based on Fourier-transform mid-infrared (FT-MIR) spectra of milk and targeted treatment of metabolically imbalanced cows with propylene glycol drench. We hypothesized that cows identified in a metabolically imbalanced status during early lactation were associated with subsequent impaired health, reproduction, and production, and that treatment with propylene glycol treatment would improve health, reproduction, and production relatively more in these cows than in control cows. We completed a prospective, randomized controlled trial with 356 early-lactation cows in 2 private dairy herds in Denmark from December 2017 to April 2018. Milk samples of cows were collected before treatment, from 4 to 9 d in milk, and after treatment, from 22 to 27 d in milk. Milk samples were analyzed using FT-MIR spectroscopy. We also measured 4 milk metabolites (ß-hydroxybutyrate, isocitrate, malate, and glutamate) and fat and protein contents. Based on FT-MIR spectra and cluster analyses, cows were clustered into groups of metabolically imbalanced and healthy cows. Within each group, cows were allocated randomly to treatment with propylene glycol (500 mL for 5 d) or no treatment. We analyzed the effect of the treatment on cow-level variables: metabolic cluster, milk metabolites, fat and protein contents, and fat-to-protein ratio at a milk sampling after the treatment. Furthermore, we analyzed daily milk yield, calving to first service interval, and disease occurrence. Results showed only a few effects of propylene glycol treatment and few interactions between treatment and metabolic clusters. We found no significant main effects of propylene glycol treatment in any of these analyses. A negative effect of the imbalanced metabolic cluster was found for the outcome of calving to first service interval for multiparous cows. In conclusion, we found a longer calving to first service interval in metabolically imbalanced cows, but we were not able to demonstrate overall benefits from the applied detection of cows in imbalanced metabolic status in early lactation and follow-up by treatment with propylene glycol.

Metabolomic profile of diabetic retinopathy: a GC-TOFMS-based approach using vitreous and aqueous humor.

AIM: To identify the potential metabolite markers in diabetic retinopathy (DR) by using gas chromatography coupled with time-of-flight mass spectrometry (GC-TOFMS). METHODS: GC-TOFMS spectra were acquired from vitreous and aqueous humor (AH) samples of patients with DR and non-diabetic participants. Comparative analysis was used to elucidate the distinct metabolites of DR. Metabolic pathway was employed to explicate the metabolic reprogramming pathways involved in DR. Logistic regression and receiver-operating characteristic analyses were carried out to select and validate the biomarker metabolites and establish a therapeutic model. RESULTS: Comparative analysis showed a clear separation between disease and control groups. Eight differentiating metabolites from AH and 15 differentiating metabolites from vitreous were highlighted. Out of these 23 metabolites, 11 novel metabolites have not been detected previously. Pathway analysis identified nine pathways (three in AH and six in vitreous) as the major disturbed pathways associated with DR. The abnormal of gluconeogenesis, ascorbate-aldarate metabolism, valine-leucine-isoleucine biosynthesis, and arginine-proline metabolism might weigh the most in the development of DR. The AUC of the logistic regression model established by D-2,3-Dihydroxypropanoic acid, isocitric acid, fructose 6-phosphate, and L-Lactic acid in AH was 0.965. The AUC established by pyroglutamic acid and pyruvic acid in vitreous was 0.951. CONCLUSIONS: These findings have expanded our understanding of identified metabolites and revealed for the first time some novel metabolites in DR. These results may provide useful information to explore the mechanism and may eventually allow the development of metabolic biomarkers for prognosis and novel therapeutic strategies for the management of DR.

Effect of postpartum propylene glycol allocation to over-conditioned Holstein cows on concentrations of milk metabolites.

The objective of the study was to investigate the effect of propylene glycol (PG) allocation on concentrations of milk metabolites with potential use as indicators of glucogenic status in high yielding postpartum dairy cows. At time of calving, nine ruminally cannulated Holstein cows were randomly assigned to ruminal dosing of 500 g/d tap water (CON, n = 4) or 500 g/d PG (PPG, n = 5). The PG was given with the morning feeding week 1-4 postpartum (treatment period) and cows were further followed during week 5-8 postpartum (follow-up period). All cows were fed the same postpartum diet. Milk samples were obtained at each milking (3 times/d) in the treatment period, and at morning milking during the follow-up period. Weekly blood samples were obtained from -4 to +8 weeks relative to calving and daily blood samples from -7 until +7 d relative to calving. The main effect of PG allocation was an increased glucogenic status, e.g. visualised by a prompt marked increase in blood fructosamine. During the treatment period, milk concentration of free glucose tended to be greater, whereas milk concentrations of isocitrate and BHBA were lower for PPG compared with CON. It is proposed that the ratio between free glucose and isocitrate in milk may be a potential biomarker for glucogenic status in the vulnerable early postpartum period. We will pursue this issue in the future.

Fluorometric determination of free and total isocitrate in bovine milk.

Isocitrate is an intermediate metabolite in the citric acid cycle found both inside the mitochondria as well as outside in the cytosolic shunt. Oxidation of isocitrate is believed to deliver large fractions of energy [i.e., reducing equivalents (NADPH) in the bovine udder] used for fatty acid and cholesterol synthesis. This study describes a new analytical method for determination of free and total isocitrate in bovine milk where time-consuming pretreatment of the sample is not necessary. Methods for estimation of both total isocitrate and free isocitrate are described, the difference being the esterified or even lactonized isocitrate. On average, 20% (6-27%) of cow milk isocitrate was esterified and free isocitrate correlated significantly with total isocitrate (r=0.98). The present fluorometric determination correlated well with the traditional spectrophotometric determination of isocitrate. Milk samples from Danish Holstein cows (984) contained significantly less isocitrate than milk from Danish Jersey cows (760; i.e., 0.134 vs. 0.211 mmol/L). Isocitrate in milk is correlated to milk protein, fat, and citrate, and it is speculated, based on biochemistry, former studies, and the present, that isocitrate may reflect the energy situation in the mammary gland. The use of isocitrate as a biomarker of the energy status in the dairy cow is warranted.

Effects of granulation on organic acid metabolism and its relation to mineral elements in Citrus grandis juice sacs.

We investigated the effects of granulation on organic acid metabolism and its relation to mineral elements in 'Guanximiyou' pummelo (Citrus grandis) juice sacs. Granulated juice sacs had decreased concentrations of citrate and isocitrate, thus lowering juice sac acidity. By contrast, malate concentration was higher in granulated juice sacs than in normal ones. The reduction in citrate concentration might be caused by increased degradation, as indicated by enhanced aconitase activity, whilst the increase in malate concentration might be caused by increased biosynthesis, as indicated by enhanced phosphoenolpyruvate carboxylase (PEPC). Real time quantitative reverse transcription PCR (qRT-PCR) analysis showed that the activities of most acid-metabolizing enzymes were regulated at the transcriptional level, whilst post-translational modifications might influence the PEPC activity. Granulation led to increased accumulation of mineral elements (especially phosphorus, magnesium, sulphur, zinc and copper) in juice sacs, which might be involved in the incidence of granulation in pummelo fruits.

Determination of α-hydroxy acids and their enantiomers in fruit juices by ligand exchange CE with a dual central metal ion system.

The content of α-hydroxy acids and their enantiomers can be used to distinguish authentic and adulterated fruit juices. Here, we investigated the use of ligand exchange CE with two kinds of central metal ion in a BGE for the simultaneous determination of enantiomers of dl-malic, dl-tartaric and dl-isocitric acids, and citric acid. Ligand exchange CE with 100 mM d-quinic acid as a chiral selector ligand and 10 mM Cu(II) ion as a central metal ion could enantioseparate dl-tartaric acid but not dl-malic acid or dl-isocitric acid. Addition of 1.8 mM Sc(III) ion to the BGE with 10 mM Cu(II) ion to create a dual central metal ion system permitted the simultaneous determination of these α-hydroxy acid enantiomers and citric acid. The proposed ligand exchange CE was thus well suited for detecting adulteration of fruit juices.

(-) Epicatechin attenuates mitochondrial damage by enhancing mitochondrial multi-marker enzymes, adenosine triphosphate and lowering calcium in isoproterenol induced myocardial infarcted rats.

Cardiac mitochondrial damage plays an important role in the pathology of myocardial infarction. The protective effects of (-) epicatechin on cardiac mitochondrial damage in isoproterenol induced myocardial infarction were evaluated in rats. Rats were pretreated with (-) epicatechin (20 mg/kg body weight) daily for a period of 21 days. After the pretreatment period, isoproterenol (100 mg/kg body weight) was injected subcutaneously into rats twice at an interval of 24 h to induce myocardial infarction. Isoproterenol induced myocardial infarcted rats showed a significant increase in the levels of cardiac diagnostic markers, heart mitochondrial lipid peroxidation, calcium, and a significant decrease in the activities/levels of heart mitochondrial glutathione peroxidase, glutathione reductase, reduced glutathione, isocitrate, succinate, malate, α-ketoglutarate and NADH-dehydrogenases, cytochrome-C-oxidase and adenosine triphosphate. (-) Epicatechin pretreatment showed significant protective effects on all the biochemical parameters evaluated. The in vitro study revealed the superoxide and hydroxyl radical scavenging activity of (-) epicatechin. The possible mechanisms for the beneficial effects of (-) epicatechin on cardiac mitochondria could be attributed to scavenging of free radicals, decreasing calcium, increasing multi-enzymes (antioxidant, tricarboxylic acid cycle and respiratory chain enzymes), reduced glutathione and adenosine triphosphate. Thus, (-) epicatechin attenuated mitochondrial damage in isoproterenol induced myocardial infarcted rats.

Enantioseparation of DL-isocitric acid by a chiral ligand exchange CE with Ni(II)-D-quinic acid system.

The ratio of citric acid to D-isocitric acid can be used to distinguish authentic and adulterated fruit juices. To separate DL-isocitric acid enantiomers, we used ligand exchange CE. D-Quinic acid was used as a chiral selector ligand and Mn(II), Fe(III), Co(II), Ni(II), Cu(II), and Zn(II) ions were used as the central ions of the chiral selector in the BGE. DL-Isocitric acid was found to be enantioseparated with the above metal ions except for Mn(II) ion. The optimum running conditions for the analysis of D- and L-isocitric acids along with citric acid, an isomer of isocitric acid, were found to be a BGE (pH 5.0) containing 30% ACN, 20 mM acetic acid, 20 mM NiSO(4), and 80 mM D-quinic acid. Under these conditions, DL-isocitric and citric acids in fruit juices were analyzed successfully.

Sugar and nonvolatile acid composition of blackberries.

Sugar and nonvolatile acid analyses were conducted on 52 samples of blackberries (Rubus spp), the objective being to develop a compositional database for evaluating authenticity and quality. Brix ranged from 6.88 to 16.83, with a mean of 10.82. Titratable acidity ranged from 0.52 to 2.24 g citric acid/100 mL, with a mean of 1.35. Sucrose levels (range, 0-12.9%; mean, 4.6%) were highly variable. The overall glucose:fructose ratio ranged from 0.81 to 1.17, with a mean of 1.01. Malic, isocitric, lactoisocitric, citric, shikimic, and fumaric acids were identified, with succinic acid being present in some samples. Malic acid ranged from 5.2 to 35.3% of total acids (87.5-603 mg/100 g), with a mean of 16.4% (280 mg/100 g). Isocitric acid ranged from 4.7 to 71.6%, with a mean of 34.7% (599 mg/100 g), and lactoisocitric acid ranged from 3.4 to 32.6% with a mean of 17.3% (293 mg/100 g). Citric acid ranged from 1.3 to 80.2%, with a mean of 31.6 (572 mg/100 g). Shikimic, fumaric, and succinic were present in trace quantities. Two patterns of nonvolatile acid compositions were evident. Ten commercial blackberry juice samples were analyzed, and it was possible to determine whether they were Marion, Evergreen, or a mixture of the two from their acid profiles.

Impact of coenzyme regeneration on the performance of an enzyme-based optical biosensor: a computational study.

A mathematical model of a reagent-less optical sensing scheme composed of an enzymatic reaction coupled to light-controlled photochemical coenzyme regeneration is described. The model is based on previous experimental work describing the regeneration of NADPH from NADP(+) by excited state thionine coupled to the oxidation of isocitrate by isocitrate dehydrogenase. The system is capable of repeated isocitrate measurements without the addition of exogenous coenzyme. The model is simulated using numerical integration to determine the effect of regeneration on the sensor sensitivity, response time and maximum sample throughput rate. Prediction of these effects without a model is difficult due to activation and inhibition of the dehydrogenase by both forms of the coenzyme. The regeneration parameters, including thionine concentration and thionine excitation pattern, are varied to determine optimal sensor conditions to maximize performance. A periodic regeneration approach is found to be superior to a continuous regeneration approach as the former maximizes sensitivity and minimizes response time in most cases. In addition periodic regeneration results in a maximum sample throughput frequency that is achieved at a single optimal thionine level and is independent of the analyte concentration. In contrast the optimal thionine concentration during continuous regeneration varies with the sample analyte concentration. These findings highlight the importance of designing controllable regeneration for dehydrogenase-based optical biosensors.

Using capillary isotachophoresis for the determination of biogenic amines and D-isocitric acid in food products.

This paper presents results achieved in the determination of biogenic amines in various food products and quality evaluation of some commercial orange juices by capillary isotachophoresis. The highest amount of histamine was determined in frankfurters (146.75 mg x kg(-1)); the content of tyramine was either under detection limit of method or it was present in very low concentrations. The highest amount of cadaverine was determined in Karicka cheese (666.36 mg x kg(-1)). The limit of determination was 1.59 mg x kg(-1) for histamine and 1.25 mg x kg(-1) for tyramine. The recovery of method ranged from 95.4 to 104.9%. Surprisingly it was found that many organge juice samples did not reach required values according to the Code of Practice. The lowest ratio of citric to D-isocitric acids was found in Happy day juice (44.1). Limit of determination was 1.62 mg x dm(-3) for citric acid and 2.00 mg x dm(-3) for D-isocitric acid. The recovery of the method ranged from 89 to 96.5%.

[Diagnostic significance of analysis the energy metabolism substrates in biological fluids]. / Diagnosticheskaia znachimost' opredeleniia substratov énergeticheskogo obmena v biologicheskikh zhidkostiakh.

Changes in energy metabolism substrates in biological fluids (venous and umbilical blood, urine, and amniotic fluid) were studied. Lactate concentrations were 2-4--fold increased in venous blood and 2-fold in umbilical blood. Urinary excretion of energy metabolism substrata was increased in women with gestosis. An increase of lactate concentration above 0.110 mmol/liter and of isocitrate concentration above 0.238 mmol/liter indicated fetal hypoxia.

[Determination method of isocitric acid in food additive citric acid].

A simple and rapid method using HPLC was developed for the determination of isocitric acid in food additive citric acid. One gram of sample was dissolved in 100 mL of water. HPLC separation was performed on an Inertsil ODS-3 column (4.6 mm i.d. x 250 mm) using 0.1% phosphoric acid as the mobile phase at a flow rate of 1 mL/min. Isocitric acid was detected at 210 nm. The calibration graph was rectilinear from 5 to 100 micrograms/mL. The recoveries of isocitric acid from sample at the levels of 0.1% and 0.4% were 98% and 99%, respectively, and the determination limit was 0.05%.

Capillary electrophoresis for evaluating orange juice authenticity: a study on Spanish oranges.

Fruit juices have very distinct organic acid profiles that can be used as fingerprints for establishing possible adulteration. Recently, our group developed and validated a capillary electrophoresis method using UV detection for determining citric, isocitric, tartaric, and malic acids in natural and commercial orange juices. Sample treatment consisted of only dilution and centrifugation or filtration. This method has been applied to evaluate these acids and their ratios in 63 samples of Navelina, the most common variety of Spanish oranges, over a three month period. This evaluation has been conducted to establish ranges of acid concentrations and to compare them with those found in commercial juices. The more reliable parameter, because of the lower variability in fresh samples, was found to be the citrate/isocitrate ratio with a value of 113 (RSD = 10%). Only one of nine ramdonly selected commercial juices presented values within the range of those of the population of just-pressed Navelina orange juice. Moreover, three of them had measurable tartrate values, which is not a natural component of orange juice, showing mixtures with cheaper fruits.

Development and validation of a capillary electrophoresis method for direct measurement of isocitric, citric, tartaric and malic acids as adulteration markers in orange juice.

Fruit juices each have very distinct organic acids profiles that can be used as fingerprints for establishing authenticity. A method has been developed, optimised and validated for measuring by capillary electrophoresis citric, isocitric, malic and tartaric acids as authenticity markers in orange juices, without any sample treatment other than dilution and filtration. Final conditions were phosphate buffer 200 mM, pH 7.50, -14 kV as applied potential, and 57 cm length neutral capillary. Detection was direct UV at 200 nm. Different kinds and marks of orange juice, chosen from the great variety existent in the market, were analysed and clear differences could be found between them and just pressed orange juice.

Gas chromatographic determination of organic acids from fruit juices by combined resin mediated methylation and extraction in supercritical carbon dioxide.

A procedure in which anionic analytes, trapped on ion exchange resin, are simultaneously methylated and released using methyl iodide in either supercritical carbon dioxide or acetonitrile has been extended to polyfunctional organic acids. The combined SFE methylation of fruit juice acids trapped onto ion exchange resin proceeds in good yield producing the methyl esters of fumaric, succinic, malic, tartaric, isocitric and citric acids which are readily separated by GC. Using this procedure low concentrations of one acid can be detected and quantitated in the presence of very high concentrations of another. This new method detects tartaric acid at levels of 10 ppm in juices containing 10,000 ppm citric acid. Quantitation was performed either by using GC-FID with triethyl citrate or diethyl tartrate as internal standards or with the element specific calibration capability of the GC-AED. A simple new technique for the determination of citric/isocitric acid ratio is now available. Also, in contrast to HPLC methods, the identity of an analyte is readily confirmed by GC-MS.

Isocitric and citric acid in human prostatic and seminal fluid: implications for prostatic metabolism and secretion.

Human prostatic secretion is remarkably rich in citric acid but the mechanisms to account for this accumulation are not well understood. One factor may be the extent of citrate oxidation to isocitrate, catalyzed by aconitase. The citrate-to-isocitrate ratio will help characterize the relative significance of this reaction in prostatic production and secretion of citrate. Isocitric acid and citric acid were measured in samples of seminal fluid and expressed prostatic secretion (EPS). A constant ratio between citrate and isocitrate of about 33:1 was found (r = 0.93, P < 0.0001) despite the wide variation in concentrations. Citrate ranged from 1 to 180 mM in EPS and from 13 to 50 mM in seminal fluid while isocitrate varied between 0 to 4.8 mM in EPS and from 0.4 to 1.5 mM in seminal fluid. Isocitrate is present in EPS and semen at much higher levels than found in most other animal or plant tissues or fluids and may be actively secreted by the same mechanism as citrate. The high citrate to isocitrate ratio of about 33:1, compared to the expected value of about 10:1, supports suggestions that citrate to isocitrate oxidation by aconitase is a rate limiting step in prostatic citrate metabolism. A low aconitase activity will therefore play a significant role in enabling accumulation of high citrate levels in prostatic epithelia and acini.

Automated docking in crystallography: analysis of the substrates of aconitase.

Automated docking of substrates to proteins of known structure aids the process of crystallographic analysis in two ways. First, automated docking can be used to generate a small number of starting models for substrates using only protein coordinates from an early stage of refinement. Second, automated docking provides a method for exploring aspects of catalysis that are inaccessible to crystallography by postulating binding modes of catalytic intermediates. This paper describes the use of automated docking to explore the binding of substrates to aconitase. The technique starts with a substrate molecule in an arbitrary configuration and position and finds favorable docked configurations in a (static) protein active site based on a molecular mechanics type force field. Using protein coordinates from an early stage of refinement of an aconitase-isocitrate complex, we successfully predicted the binding configuration of isocitrate. Four configurations were found, the energetically most favorable of which fit the observed electron density well and was used as a starting model for further refinement. Two configurations were found in citrate docking experiments, the second of which approximates the mode of substrate binding in an aconitase-nitrocitrate complex. We were also able to propose two binding modes of the catalytic intermediate cis-aconitate. These correspond closely to the isocitrate and the citrate binding modes. The relation of these new results to the proposed reaction mechanism is discussed.

The onset of mammary secretion of medium-chain-length triglyceride fatty acids in the cow milked pre-partum.

Six Friesian cows were milked daily, and arterial and mammary venous blood samples were taken between day 266 of gestation and day 6 after parturition. Samples of blood plasma were analysed for their content of acetate and progesterone, and mammary secretion for potassium, isocitrate, 2-oxoglutarate and triglyceride fatty acids. The onset of secretion of medium-chain triglyceride fatty acids was preceded by, or coincided with, increases of potassium and isocitrate concentration in the secretion. The onset of fatty acid secretion was not accompanied by any change in the gland's extraction of acetate from the circulation, and did not occur at a consistent time relative to parturition or changes in plasma progesterone concentration.

Enzymatic determination of isocitrate by amperometric monitoring of the rate of oxygen consumption.

A coupled enzymatic method elaborated for NAD+-dependent dehydrogenases has been adapted for NADP+-dependent isocitrate dehydrogenase, in combination with amperometric measurements. The isocitrate dehydrogenase activity dependent linearly on the isocitrate concentration in the range 0-2 X 10(-4) M. Application of this method affords a sensitive estimation of isocitrate even in turbid liquids such as fermentation broths.