Proton nuclear magnetic resonance spectroscopy based investigation on propylene glycol toxicosis in a Holstein cow
© Bertram et al., licensee BioMed Central Ltd. 2009
Received: 19 March 2009
Accepted: 13 June 2009
Published: 13 June 2009
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.
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.
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.
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.
Propylene glycol (PG) has been used as a glucogenic feed supplement for ruminants for decades . Metabolism of PG in ruminants involves microbial metabolism in the rumen and hepatic metabolism of products of ruminal fermentation (propanol, propanal, and propionate) as well as PG absorbed to the portal blood . Various application forms of PG are in use: oral drench, oro-ruminal infusion devices, top dressed on feed, mixed into pelleted feeds, and mixed into total mixed rations. Numerous studies report beneficial effects of PG on glucose and fat homeostasis in periparturient dairy cows, for review see . However, reports from practice and sparse reports in the literature describe abnormal behaviour involving observation of shallow breathing, ataxia, salivation, somnolence and depression when adding PG to the feed of dairy cows . In a field trial involving 7 dairy herds, cows were fed either 0, 150, 300 or 450 g PG/d from 20 to 14 d antepartum . In 3 out of the 7 herds approximately 36% of the cows reacted during the first few days of application by signs described as hyperventilation and somnolence.
The present study is based on a test-feeding trial with a pelleted concentrate containing PG, which is under development for use in very early lactation. Feeding this concentrate induced unexpectedly a condition in one out of three cows resembling PG toxicosis and the present study aimed to investigate the metabolites responsible for PG-induced toxicosis using proton nuclear magnetic resonance (NMR) spectroscopy. Since it remains unknown which metabolites are responsible for a PG-induced toxicosis, a non-selective analytical method that detects as many metabolites as possible would be attractive. Proton NMR spectroscopy, which in principle enables detection of all hydrogen-containing molecules, has turned into a commonly applied technique for metabolic profiling of biofluids, among other to identify biochemical changes in response to disease in mammals . In the present study proton NMR spectroscopy was applied on ruminal and blood plasma samples from the cows used in the study.
Materials and methods
Animals and feeding
Composition of pelleted concentrate1
Inclusion, % of concentrate (as mixed)
Sugar beet pulp
The study complied with the Danish Ministry of Justice Law no. 382 (June 10, 1987), Act no. 726 (September 9, 1993) concerning experiments with animals and care of experimental animals.
Each cow was sampled for one day after being fed the experimental diet for 14 d. On sampling days, continuous infusion of p-aminohippuric acid (pAH; 29 ± 1 mmol/h) into the mesenteric vein was initiated at 05:30. The pAH infusate was a sterilized 250 mM solution of pAH (4-aminohippuric acid 99%, Acros, Geel, Belgium) adjusted to pH 7.4. Ten sets of ruminal and blood samples were obtained 0.5 h before feeding, and 0.5, 1.5, 2.5, 3.5, 5.0, 6.5, 8.0, 9.5, and 11 h after feeding. Blood was sampled by simultaneously drawing blood from the artery and hepatic portal vein into 20 mL syringes and was immediately transferred to heparin vacuettes (#455051; Greiner Bio-One GmbH, Kremsmuenster, Austria). Plasma was harvested by centrifugation at 3000 g for 20 min and stored at -20°C until analysis. Separate blood samples were obtained in 1 mL heparinized syringes for blood gas measurements just before collection of the main blood samples. One extra arterial 1 mL sample was obtained from the cows that reacted to feeding 1 h after feeding. Ruminal fluid was sampled from the ventral ruminal sac using a suction strainer (#RT extended version, Bar Diamond, Parma, ID) and a 50 mL syringe. Ruminal fluid pH was measured immediately after sampling (IQ 150 pH meter; IQ Scientific Instruments Inc., Carlsbad, CA), and a subsample of ruminal fluid was stabilized with 5% meta phosphoric acid and frozen at -20°C.
Blood sampled in 1 mL syringes was immediately taken for blood gas and oximetry analysis (ABL 520, Radiometer A/S, Copenhagen, Denmark).
The NMR measurements were performed on a Bruker 800 spectrometer, operating at a 1H frequency of 799.40 MHz, and equipped with a 5-mm 1H observe TXI cryoprobe (Bruker BioSpin, Rheinstetten, Germany). For both plasma and ruminal samples the NMR measurements were carried out on samples collected 0.5 h before feeding, and 0.5, 1.5, 2.5, 3.5, 5.0 and 8.0 h after feeding. On plasma samples the NMR measurements were carried out at 310 K, while measurements on ruminal fluid samples were carried out at 298 K. Prior to NMR measurements, the samples were thawed, and 500 μl aliquots were mixed with 100 μl D2O. Sodium trimethylsilyl- [2,2,3,3-2H4]-1-propionate (TSP) was added as an internal chemical shift standard (0.10% w/w). The NMR measurements were essentially carried out as described previously described . For ruminal fluid samples 1H NMR spectra were obtained using a standard single 90° pulse experiment, while for plasma samples two 1H NMR spectra were obtained on each sample; i) a standard one-dimensional spectrum acquired using single 90° pulse experiment, and ii) a one-dimensional spectrum acquired with a Carr-Purcell-Meiboom-Gill (CPMG) delay of 50 ms added in order to attenuate broad signals from high-molecular-weight components. On plasma samples 64 scans were acquired in the CPMG experiment, 32 scans were acquired in the standard spectrum, while 64 scans were acquired on ruminal fluid samples. In all NMR experiments water suppression was achieved by irradiating the water peak during the relaxation delay of 5 s and 16 K data points spanning a spectral width of 13.03 ppm were collected. An exponential line-broadening function of 0.3 Hz was applied to the free induction decay (FID) prior to Fourier transformation (FT). All spectra were referenced to the TSP signal at 0 ppm.
To aid spectral assignment 2D 1H-1H correlation (DQFCOSY) and 2D 1H-13C HSQC spectra were recorded on selected ruminal fluid samples using water suppression. The DQFCOSY spectra were acquired with a spectral width of 10000 Hz in both dimensions, 4096 data points, 512 increments with 64 transients per increment and zero filled in the F1 dimension. The HSCQ spectra were acquired with a spectral width of 10000 Hz in the F2 dimension and 30153 Hz in the F1 dimension, a data matrix with a size of 2048 × 512 data points and 32 transients per increment, and the spectra were zero filled in both dimensions.
Post-processing and multivariate data analysis
Principal component analysis (PCA) was applied to explore any clustering behaviour of the samples using the Unscrambler software version 9.2 (Camo, Oslo, Norway). PCA is an unbiased mathematical algorithm that lowers data dimensionality whilst retaining variation in a large dataset. By identifying directions (principal components) in which variation are at maximum, samples can be explained by a relatively low number of components instead of thousands of variables. Following analysis of the components plots can then be used to identify similarities and differences between samples . The NMR spectra were subdivided into 0.002 ppm integral regions and integrated, and for ruminal fluid spectra the regions 0.5–4.6–10.0 ppm and for plasma spectra the regions 0.5–4.5 and 5.1–10.0 were included in the PCA.
Cows were fed the experimental diet for 14 d prior to sampling and no signs of lack of tolerance to the ration were noticed. On the sampling day, which was designated as the sampling day, two of the cows consumed the entire amount of offered concentrate within 15 min whereas the third cow had consumed approximately half and stopped eating. Three min later the remaining amount of the concentrate was introduced into the rumen via the ruminal cannula. Twelve min after feeding the concentrate directly into the rumen (30 min after feeding) and immediately before starting the second blood sampling the third cow developed severe dyspnea and ruminal atony. The cow remained standing although she was severely affected by the incidence. Two h after feeding the symptoms had completely disappeared and it was observed that the cow started eating hay. By 2.5 h after feeding she was observed drinking water and had apparently completely recovered.
Examples of PG-induced toxicosis have been reported in ruminants  and other animals [10–13]. However, these animals have rarely been further examined, and the molecular mechanisms causing the abnormal response are unknown. In the present study PG was applied in a pelleted concentrate and the effects of PG cannot be separated from the effects of other ingredients as such. However, the observed clinical signs of the cow that responded badly to the ration are in good agreement with the reports from farmers, extension personnel, and veterinarians on reaction to introduction of PG containing concentrates in dairy herds. Accordingly, evidence exists that the incidence under investigation is PG toxicosis; however, it cannot be ruled out at present if other dietary components contributed to the incidence.
The oximetry data together with the clinical picture suggests that the hypoxia of the affected cow was caused by decreased gas exchange between the pulmonary alveoli and the blood and not caused by changes in oxygen affinity of haemoglobin (both oxygen tension and saturation decreased in parallel) and the breathing of the cow appeared to be both of high frequency and with full depth. The condition of the cow could be caused by pulmonary vasoconstriction (brisket disease) similar to the response of cattle seen at high altitude . However it is unlikely that PG itself induced the pulmonary vasoconstriction because high plasma concentrations of PG have been attained in previous studies without any apparent effects on the cows [2, 15].
The present investigation is the first to report the use of 1H NMR-based metabolic profiling in the study of PG metabolism and toxicity. High-resolution 1H NMR spectra could be obtained in both ruminal fluid and plasma samples, enabling the detection of several metabolites. PCA on the serial metabolite profiles revealed differences between the abnormal cow and the two control cows both in ruminal fluid, arterial and portal plasma samples. Accordingly, data indicated that the PG-induced toxicosis was associated with a different metabolic response to the feeding. Further analysis of the metabolite profiles of the ruminal samples revealed that this abnormal response was reflected in lower contents of isopropanol, isobutyrate, lactate, acetate, acetone, citrate and some unidentified, smaller esters and alcohols in the ruminal fluid shortly (0.5 h) after feeding. However, the lower concentrations as compared with the control cows could indicate a general decrease in microbial fermentation in the rumen. Later in the course corresponding to 1.5–2.5 h after feeding, the 1H NMR spectra of ruminal fluid from the cow responding abnormally to the ration were characterized by considerably higher intensities of signals ascribed to propanol. However, also propanol has previously been observed in high ruminal and plasma concentrations without affecting the cows .
1H NMR spectroscopy of the plasma samples revealed that the cow that responded abnormally to PG was characterized by a lower content of isopropanol, isobutyrate, β-hydroxybuturate, acetate, acetone and acetoacetate shortly after feeding (0.5 h) compared with the respective arterial and portal plasma samples from the two control cows. However, the lower concentrations as compared with the control cows is likely caused by reduced fermentation activity in the rumen in combination with reduced absorption because of ruminal atony.
In addition to these differences in the concentration of low-molecular-weight metabolites, the 1H NMR spectra also revealed a higher lipid content in plasma of the cow responding abnormally to PG compared with the control cows. This is very unlikely an effect of PG, as it was also present in the samples obtained before feeding. In contrast, the higher lipid content in plasma of the cow responding abnormally to PG probably reflects a natural variation. It remains unknown if the higher plasma lipid content is associated with a higher susceptibility for development of PG toxicosis.
Beside lower concentrations of common metabolites/fermentation products in ruminal fluid and plasma samples immediately after feeding, the NMR spectra could not reveal the presence of any "extraordinary" or unusual metabolites in the biofluids of the cow developing toxicosis. It has recently been suggested that sulphur-containing compounds produced during fermentation of PG could be the cause of side effects . We observed no indications of the presence of sulphur-containing compounds in the NMR spectra. Plausible explanations for the lack of detection in the NMR spectra exist, as the compounds are volatile or present in concentrations below the detection limit of NMR. It has recently been established that H2S is an important signalling substance in hypoxic vasoconstriction in vertebrates including cattle . Therefore sulphur compounds or specifically H2S appear as promising candidates for explaining the link between PG application to the rumen and the dyspnea of the cow.
The present study showed that the symptoms of PG-toxicosis are likely to be caused by pulmonary vasoconstriction, however, it was not possible to identify the metabolites inducing the response by use of 1H NMR spectroscopy.
The Danish Technology and Production Research Council (FTP; NMR-based metabonomics on tissues and biofluids #274-05-339) and the Danish Cattle Federation are acknowledged for financial support of the study. The 800 MHz spectra were obtained using the Bruker 800 spectrometer of the Danish Instrument Center for NMR Spectroscopy of Biological Macromolecules. The Weipass and MetaSmart were provided from Raiffeisen Hauptgenossenschaft Nord AG, Kiel, Germany, and Adisseo, Antony, France, respectively.
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