Efficient Quantification of Milk Metabolites from 1H NMR Spectra Using the Signature Mapping (SigMa) Approach: Chemical Shift Library Development for Cows' Milk and Colostrum.

Cow milk contains essential nutrients for humans, and its bulk composition is usually analyzed using Fourier transform infrared spectroscopy. The higher sensitivity of nuclear magnetic resonance (NMR) spectroscopy can augment the extractible qualitative and quantitative information from milk to nearly 60 compounds, enabling us to monitor the health of cows and milk quality. Proton (1H) NMR spectroscopy produces complex spectra that require expert knowledge for identifying and quantifying metabolites. Therefore, an efficient and reproducible methodology is required to transform complex milk 1H NMR spectra into annotated and quantified milk metabolome data. In this study, standard operating procedures for screening the milk metabolome using 1H NMR spectra are developed. A chemical shift library of 63 milk metabolites was established and implemented in the open-access Signature Mapping (SigMa) software. SigMa is a spectral analysis tool that transforms 1H NMR spectra into a quantitative metabolite table. The applicability of the proposed methodology to whole milk, skim milk, and ultrafiltered milk is demonstrated, and the method is tested on ultrafiltered colostrum samples from dairy cows (n = 88) to evaluate whether metabolic changes in colostrum may reflect the metabolic status of cows.

Net absorption and liver metabolism of amino acids and heat production of portal-drained viscera and liver in multiparous sows during transition and lactation.

BACKGROUND: Determination of nutrient requirements in the late gestating and lactating sows is essential to optimize sow productivity. The objectives of the present study were to quantify amino acid (AA) fluxes and heat production across portal-drained viscera (PDV) and liver in multiparous sows during transition and lactation. METHODS: Eight second parity sows were fitted with indwelling catheters in the femoral artery and in the mesenteric, portal and hepatic veins. Eight hourly sets of blood samples were taken starting 0.5 h before feeding at - 10, - 3, + 3, and + 17 d in milk (DIM). Blood gases, plasma metabolites and apparent total tract digestibility (ATTD) of nutrients were measured. RESULTS: Feed intake, the ATTD of DM, energy, nitrogen, fat and crude fiber changed with DIM (P < 0.001). Except for Glu, O2, and urea, all net portal fluxes were positive, and all were affected by DIM (P < 0.05) and by sampling time (P < 0.01). Compared with pre partum levels, net portal uptake of AA was 3-63% lower at + 3 DIM but 40-100% higher at + 17 DIM. Net portal fluxes of AA peaked at 1.5 to 2.5 h after feeding except for Glu, and they were positively correlated with changes in sow feed intake across DIM. The net portal recovery was low for Met (49%), Thr (54%), and His (54%) and high for the remaining essential AA (63-69%) and none of them differed across DIM. Net hepatic uptake (i.e. hepatic oxidation) of Lys, Thr, Ile, Leu and Phe peaked at 0.5 to 2.5 h after feeding, whereas uptake of Trp, Val, and His was constant, while that of Met was close to zero. CONCLUSION: The net portal recovery was substantially lower for Met, Thr, and His than the remaining essential AA. Hepatic AA oxidation peaks 0.5 to 2.5 h after feeding. The heat production in PDV and liver was approximately two-fold higher at peak lactation compared to other stages. The study suggests that lysine was the limiting AA in peak lactation but not in early lactation.

Net Absorption and Metabolism of ß-Hydroxy- ß-Methyl Butyrate during Late Gestation in a Pig Model.

The leucine metabolite, ß-hydroxy-ß-methyl butyrate (HMB), is widely used in human nutrition and animal production as a nutritional supplement. Although the HMB usage during late gestation has been demonstrated to have a positive effect on fetal development, knowledge on net absorption and metabolism of HMB and impact of HMB on branched chain amino acids (BCAAs) metabolism is lacking. To address this, we conducted a study using pigs during the perinatal period as a model organism. Eight-second parity sows were fitted with indwelling catheters in the femoral artery and in the portal, hepatic, femoral, and mesenteric veins. Eight hourly sets of blood samples were taken starting 30 min before the morning meal on day -10 and day -3 relative to parturition. Four control (CON) sows were fed a standard lactation diet from day -15 and throughout the experiment, and 4 HMB sows were fed the control diet supplemented with 15 mg Ca(HMB)2/kg body weight mixed in one third of the morning meal from day -10 until parturition. Blood gases, plasma metabolites, milk compositions, and apparent total tract digestibility of nutrients were measured. Arterial plasma concentrations of HMB (p < 0.001), Cys (p < 0.001), and Lys (p < 0.10) were increased in HMB supplemented sows, while arterial plasma triglycerides concentration was decreased (p < 0.05). The net portal recovery of Ala and Asp were increased in HMB sows (p < 0.05). Sows fed HMB had increased hepatic vein flow and net hepatic fluxes of Met, Asn, and Gln (p < 0.05). In contrast, the femoral extraction rates of Ala and Ser were decreased by dietary HMB supplementation (p < 0.05). Dietary HMB treatment and sampling time relative to feeding had an interaction on arterial concentrations, net portal fluxes, and femoral extraction rates of BCAAs. The net portal recovery of HMB was 88%, while 14% of supplemented HMB was excreted through urine and 4% through feces. Moreover, the gastrointestinal tract metabolized 8% while the liver metabolized 12%. Finally, 26% of the daily intake of HMB was secreted via colostrum at the day of farrowing. This study demonstrated that dietary HMB supplementation increased net uptake of amino acids and increased fatty acid oxidation through improving blood flow and insulin sensitivity during the late gestation. Most importantly, oral HMB administration could maintain a stable postprandial absorption and altered metabolism in BCAAs. Net portal flux of HMB at 5.5 to 6.5 h after feeding approached zero, indicating that HMB ideally should be administrated two or three times, daily.

Simultaneous quantification of purine and pyrimidine bases, nucleosides and their degradation products in bovine blood plasma by high performance liquid chromatography tandem mass spectrometry.

Improved nitrogen utilization in cattle is important in order to secure a sustainable cattle production. As purines and pyrimidines (PP) constitute an appreciable part of rumen nitrogen, an improved understanding of the absorption and intermediary metabolism of PP is essential. The present work describes the development and validation of a sensitive and specific method for simultaneous determination of 20 purines (adenine, guanine, guanosine, inosine, 2'-deoxyguanosine, 2'-deoxyinosine, xanthine, hypoxanthine), pyrimidines (cytosine, thymine, uracil, cytidine, uridine, thymidine, 2'-deoxyuridine), and their degradation products (uric acid, allantoin, ß-alanine, ß-ureidopropionic acid, ß-aminoisobutyric acid) in blood plasma of dairy cows. The high performance liquid chromatography-based technique coupled to electrospray ionization tandem mass spectrometry (LC-MS/MS) was combined with individual matrix-matched calibration standards and stable isotopically labelled reference compounds. The quantitative analysis was preceded by a novel pre-treatment procedure consisting of ethanol precipitation, filtration, evaporation and reconstitution. Parameters for separation and detection during the LC-MS/MS analysis were investigated. It was confirmed that using a log-calibration model rather than a linear calibration model resulted in lower CV% and a lack of fit test demonstrated a satisfying linear regression. The method covers concentration ranges for each metabolite according to that in actual samples, e.g. guanine: 0.10-5.0 µmol/L, and allantoin: 120-500 µmol/L. The CV% for the chosen quantification ranges were below 25%. The method has good repeatability (CV%≤25%) and intermediate precision (CV%≤25%) and excellent recoveries (91-107%). All metabolites demonstrated good long-term stability and good stability within-runs (CV%≤10%). Different degrees of absolute matrix effects were observed in plasma, urine and milk. The determination of relative matrix effects revealed that the method was suitable for almost all examined PP metabolites in plasma drawn from an artery and the portal hepatic, hepatic and gastrosplenic veins and, with a few exceptions, also for other species such as chicken, pig, mink, human and rat.

Validation of 99mTechnetium-labeled mebrofenin hepatic extraction method to quantify meal-induced splanchnic blood flow responses using a porcine model.

The aim of this study was to evaluate the measurement of the total splanchnic blood flow (SBF) using a clinical diagnostic method based on Fick's principle and hepatic extraction of 99mTc-mebrofenin (99mTc-MBF) compared with a paraaminohippuric acid (pAH) dilution method in a porcine model. Another aim was to investigate whether enterohepatic cycling of 99mTc-MBF affected the SBF measurement. Five indwelling catheters were placed in each pig (n = 15) in the portal, mesenteric, and hepatic veins, as well as in the aorta and the vena cava. The SBF was measured using both methods. The portal blood flow; the intestinal and hepatic oxygen uptake; the net fluxes of oxygen, lactate, and glucose; and the extraction fraction (EF) of 99mTc-MBF were measured before and for 70 min after feeding. The mean baseline SBF was 2,961 ml/min vs. 2,762 ml/min measured by pAH and 99mTc-MBF, respectively, and increased significantly to 3,977 ml/min and 3,981 ml/min postprandially. The hepatic EF of 99mTc-MBF decreased from 40% at the start of the investigation to 16% 70 min after feeding. The arterial-portal difference in 99mTc-MBF concentration was 0.21% (P = 0.48), indicating no intestinal extraction or metabolism. The clinical method for measuring the SBF based on hepatic 99mTc-MBF extraction is robust compared with the indicator dilution method, despite the decrease seen in hepatic extraction of 99mTc-MBF. Because there was no difference in the content of 99mTc-MBF between the arterial and portal vein plasma, the SBF can be calculated from an arterial and a hepatic vein sample.

Effect of dietary nitrogen content on the urine metabolite profile of dairy cows assessed by nuclear magnetic resonance (NMR)-based metabolomics.

NMR-based metabolomics was applied on urine samples from 32 cows that were fed four levels of crude protein (124, 135, 151, and 166 g/kg DM, respectively) in a crossover design with the aim of identifying urinary metabolites related to nitrogen intake and nitrogen efficiency. Principal component analysis (PCA) on selected regions of the obtained (1)H NMR spectra revealed an effect of crude protein intake on NMR signals in the 0.5-3.0 and 5.0-10.0 ppm regions. Partial least-squares (PLS) regressions confirmed a correlation between the NMR metabolite profile and both nitrogen intake and efficiency. The NMR signals that correlated with nitrogen intake and efficiency included urea, hippurate, phenylacetylglutamine, and p-cresol sulfate, which all contributed to the prediction of nitrogen intake and efficiency. Thus, it was not possible to identify a single metabolite that could be used as a marker to predict nitrogen efficiency, and it can be concluded that a wide-ranging urinary metabolite profile is needed to evaluate nitrogen efficiency in ruminants.

Proton nuclear magnetic resonance spectroscopy based investigation on propylene glycol toxicosis in a Holstein cow.

BACKGROUND: It is unknown which metabolites are responsible for propylene glycol (PG)-induced toxicosis, and a better understanding of the underlying mechanisms explaining incidences of abnormal behaviour of dairy cows fed PG is therefore needed. METHODS: The study included three cows of which one developed PG toxicosis. In order to investigate how the metabolism of PG differed in the cow developing toxicosis, proton nuclear magnetic resonance (NMR) spectroscopy was applied on ruminal fluids and blood plasma samples obtained before and after feeding with PG. RESULTS: PG toxicosis was characterized by dyspnea and ruminal atony upon intake of concentrate containing PG. The oxygen saturation of arterial blood haemoglobin and the oxygen pressure in arterial blood decreased along with the appearance of the clinical symptoms. NMR revealed differences in plasma and ruminal content of several metabolites between the cow responding abnormally to PG and the two control cows. CONCLUSION: It is concluded that PG-toxicosis is likely caused by pulmonary vasoconstriction, but no unusual metabolites directly related to induction of this condition could be detected in the plasma or the ruminal fluid.

Effects of supplemental dietary fatty acids on milk yield and fatty acid composition in high and medium yielding cows.

The present study tested the hypothesis that supplemental dietary fatty acids (FA) affect the energy corrected milk yield in proportion to the milk production level of dairy cows, and increase both long chain FA proportion of milk FA and milk fat globule diameter. Sixteen Danish Holstein cows were divided into four 4x4 Latin squares with two squares of medium yielding cows (32.2 kg energy corrected milk (ECM)/d; 158 days in milk (DIM)) and two squares of high yielding cows (40.0 kg ECM/d; 74 DIM). Experimental length was 12 weeks, with three weeks for each of the four periods. The four treatments were no supplementation (17 g FA/kg dry matter (DM)) and three diets with supplemented FA (29, 40, and 52 g total FA/kg DM, respectively) obtained by substituting barley with Palm Fatty Acid Distillate (PFAD) fat. Diets were offered as total mixed rations with 63% grass/clover silage (DM basis). Dry matter intake decreased with increasing FA supplementation, but net energy intake was not affected. The general linear responses to 10 g/kg DM increase in FA level were 1.1 kg ECM (P<0.0001), 0.061 kg milk fat (P<0.0001), 0.012 kg milk protein (P=0.09) and 0.052 kg lactose (P=0.0002) per day, and linear responses in milk composition were 0.39 g fat (P=0.07), -0.71 g protein (P<0.0001) and 0.05 g lactose (P=0.3) per kg milk, and 0.092 microm (P<0.0001) in milk fat average globule diameter. Fatty acid supplementation decreased short- and medium-chain FA and C16:0 and increased C18:1 proportions of total FA in milk. Supplemental dietary FA increased ECM yield but not in proportion to production level as anticipated, and increased average FA chain length and milk fat globule diameter.