Source: CLEMSON UNIVERSITY submitted to NRP
THE EFFECT OF DILUTION RATE AND PH ON THE CONVERSION OF STABLE ISOTOPICALLY LABELED FATTY ACIDS TO TRANS MONOENES BY RUMINAL BACTERIA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0194028
Grant No.
2003-35206-12835
Cumulative Award Amt.
(N/A)
Proposal No.
2002-03642
Multistate No.
(N/A)
Project Start Date
Dec 1, 2002
Project End Date
Nov 30, 2005
Grant Year
2003
Program Code
[42.0]- (N/A)
Recipient Organization
CLEMSON UNIVERSITY
(N/A)
CLEMSON,SC 29634
Performing Department
ANIMAL & VETERINARY SCIENCES
Non Technical Summary
Trans fatty acids have surfaced as potent regulators of lipid synthesis causing a decline in the percentage of fat in both body tissues and milk across a wide variety of animal species. Therefore, some trans fatty acid isomers are important as metabolic regulators to control nutrient partitioning and mammary function. Despite the importance of these trans fatty acids as inhibitors of lipid synthesis, and their confirmed presence in ruminal contents of cattle, the pathways for their formation are unknown. Therefore, this study will examine how manipulation of environmental conditions in the rumen affect the movement of carbons from 13C labeled fatty acids when they are added to cultures of ruminal microbes. It is the goal of this project to determine what conditions must exist in the rumen to enhance the production of trans isomers that are the most active inhibitors of fat synthesis. The ultimate beneficiary of this work will be the consumer who has been demanding meat and milk choices with lower fat content that conform to current medical recommendations for a healthy diet. A The long-term goal of this proposal is to reduce the fat content of meat and milk by manipulating the gut microbial population of cattle to increase their production of selected trans fatty acid isomers known to be potent inhibitors of fat synthesis. B This study will examine how manipulation of environmental conditions in the rumen (interactions of pH and dilution rates) affect the movement of carbons from 13C labeled fatty acids when they are added to cultures of ruminal microbes
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3023310100010%
3023310101020%
3023410100020%
3023410101050%
Knowledge Area
302 - Nutrient Utilization in Animals;

Subject Of Investigation
3310 - Beef cattle, live animal; 3410 - Dairy cattle, live animal;

Field Of Science
1010 - Nutrition and metabolism; 1000 - Biochemistry and biophysics;
Goals / Objectives
Determine how changes in dilution rate (0.05 and 0.10 h-1) and pH (5.5 and 6.5) and their interactions affect the movement of carbons from 13C labeled cis-9 octadecenoic acid (oleic) or trans-9 octadecenoic acid (elaidic acid) to isomers of trans monenes in cultures of mixed ruminal microbes.
Project Methods
Two experiments will be conducted with 13C labeled cis-9 octadecenoic acid (oleic) examined in experiment 1 and 13C labeled trans-9 octadecenoic acid (elaidic acid) examined in experiment 2. Four treatments in each experiment will consist of A) dilution rate 0.10 h-1 and pH 6.5 B) dilution rate 0.10 h-1 and pH 5.5 C) dilution rate 0.05 h-1 and pH 6.5 and D) dilution rate 0.05 h-1 and pH 5.5. Each of the four treatments will be evaluated in each of four continuous culture fermenters in a 4 x 4 Latin square design. Whole ruminal contents will be taken from a ruminally-fistulated cow and filtered through double-layered cheesecloth prior to incubation in the fermentors. Approximately 700 ml of the strained ruminal fluid were transferred into each of the four fermentors. Culture vessels used in this study were an all glass, closed system type with a continuous independent flow of liquid and particulate matter. Air leak into the fermentor is avoided by 1) rubber seals and 2) a continuous flow of CO2 (20 ml/min) that maintains anaerobic conditions as well as a positive internal pressure. Artificial saliva will be delivered using a precision pump set to achieve the desired flow rates. The artifical saliva will be titrated with sodium hydroxide to achieve the desired pH treatment. The temperature of ruminal cultures will be maintained at 39oC by a circulating water bath. Fatty acids will be added to the cultures at 40 mg/g feed each day introduced into the fermentors in gelatin capsules at each feeding. Unlabelled oleic acid will be placed into the fermenters via gelatin capsules on d 1-9 of each period. On d 10, the capsule for the morning feeding will contain 0.75 g unlabeled oleic acid plus 0.25 g 13C -oleic labeled at either carbon 1 or carbon 18. Samples from the fermentors will be converted to methyl esters (FAME), and then separated into saturated, trans monoene, cis monoene, and diene fractions using an Isolute SCX-2 (International Sorbent Technology, Mid Glamorgan, UK) solid phase extraction column (500 mg, 10 ml reservoir). The FAME in the trans and cis fractions will be incubated with dimethyl disulfide (DMDS) to form methyl thio adducts at the location of the double bond. Analysis of the FAME in the saturated and diene fractions, and the DMDS derivatives in the trans and cis fractions will be analyzed by gas chromatography-mass spectroscopy to determine 13C enrichment. The DMDS derivatives of FAME produce two distinctive spectral fragments that are indicative of the double bond position when analyzed by mass spectroscopy. The F fragment is the methyl thio adduct of the methyl end of the FAME. The G fragment is the methyl thio adduct of the carboxyl end of the FAME. The atom percent excess (APE) will be calculated from the mass abundance of the F and F + 1 fragments using the equation APE = (F + 1)/[F + (F + 1)]. Enrichment of the fatty acid with 13C was calculated as (APE labeled - average APE unlabeled)*100.

Progress 12/01/02 to 11/30/05

Outputs
In a previous in vitro study, mixed ruminal microorganisms converted oleic acid to a variety of trans monenes when grown in batch cultures under constant environmental conditions. To determine if a similar conversion occurs under environmental conditions more typical of the rumen, conversion of 13C-labeled oleic acid to biohydrogenation intermediates was determined in ruminal microorganisms grown in continuous culture at two pH (5.5 and 6.5) and liquid dilution rates (0.05 and 0.10 h-1) arranged factorially. After each morning feeding of the dual-flow continuous cultures, 250 mg oleic acid in 5 ml of ethanol was injected into each culture. On d 10, 250 mg of oleic-1-13C replaced the unlabelled oleic acid in ethanol. Trans fatty acids were isolated from culture samples by solid phase extraction, and 13C enrichment and identity of double bond position determined by gas chromatography-mass spectroscopy. At 6.5 pH and 0.10 h-1 dilution rate, 13C enrichment was detected in all trans-C18:1 isomers having double bond positions from C6 through C16 in the acyl chain. However, when pH or dilution rate in fermentors was lowered, no 13C enrichment was detected in any trans isomer with a double bond position beyond C10. The results of this study confirm that ruminal microorganisms are capable of converting oleic acid to a wide variety of trans-C18:1 positional isomers when ruminal conditions are favorable (such as the pH 6.5 and 0.10 h-1 dilution rate treatment). However, at low pH and dilution rate, the conversion of oleic acid to trans-C18:1 still occurs, but positional isomers produced are restricted to to double bond positions from C6 to C10. The formation of hydroxystearic acid (HSA) and ketostearic acid (KSA) from oleic acid transformation has been documented in a variety of microbial species, including several isolated from the rumen of domesticated ruminant species. However, their ruminal production rates have not been established as influenced by fatty acid source. Dosing continuous cultures of mixed ruminal microorganisms with 1-13C-oleic acid led to increased 13C enrichment of both HSA and KSA at 24 hours post-dosing, and showed that the majority (96 and 85%, respectively) of the HSA and KSA present in the 24 h samples originated from oleic acid. Several experiments using batch cultures of ruminal microorganisms showed that production of HSA and KSA was directly related to oleic acid input but not affected by elaidic acid input, and that HSA was further metabolized to KSA but not metabolized to other fatty acids. When continuous cultures of ruminal microorganisms were supplemented with soybean oil or canola oil, production of 10-HSA + 10-KSA was related to oleic acid input but not to linoleic acid input. Daily production of 10-HSA + 10-KSA across treatments was 14.4 umol per 100 umol oleic acid input into the cultures or 31.1 umol per 100 umol oleic acid net loss. The results of this study quantify the formation of 10-HSA and 10-KSA from oleic acid transformation by ruminal microorganisms, and show that their accumulation in ruminal contents is directly related to the extent of oleic acid input and biotransformation by the rumen microbiota.

Impacts
Oleic acid is a major unsaturated fatty acid found in many fat sources used as feed ingredients for dairy cattle. Microorganisms in the rumen of dairy cattle transform oleic acid to stearic acid and a multitude of trans-C18:1 isomers. This study showed that low pH or fractional dilution rate in ruminal contents prevented the conversion of oleic acid to some of these trans-C18:1 isomers, specifically those where the double bond was positioned beyond C10 in the fatty acyl chain. The results also detail the formation of an alternative pathway of oleic acid transformation that involves its hydration to hydroxy stearic acid followed by subsequent oxidation of the hydroyl group to a keto group. These results will aid in modeling the production of bioactive trans fatty acids that originate from oleic acid biohydrogenation by ruminal microorganisms.

Publications

  • Jenkins, T. C., A. A. AbuGhazaleh, S. Freeman, and E. J. Thies. 2006. The production of 10-hydroxystearic acid and 10-ketostearic acids is an alternate route of oleic acid transformation by the ruminal microbiota in cattle. J. Nutr. 176: In Press.
  • AbuGhazaleh, A. A., M. B. Riley, E. E. Thies, and T. C. Jenkins. 2005. Dilution rate and pH effects on the conversion of oleic acid to trans C18:1 positional isomers in continuous culture. J. Dairy Sci. 88:4334-4341.
  • Jenkins, T. C., A. A. AbuGhazaleh, E. J. Thies, and M. B. Riley. 2005. Conversion of oleic acid to 10-hydroxy and 10-keto stearic acids in vitro and their accumulation in milk of cows fed added fat. J. Dairy Sci. (Suppl. 1):371.
  • AbuGhazaleh, A. A., M. B. Riley, and T. C. Jenkins. 2004. The effect of dilution rate and pH on the conversion of stable isotopically labelled oleic acid to trans monoenes in continuous culture. J. Dairy Sci. 87 (Suppl. 1):337.


Progress 01/01/04 to 12/31/04

Outputs
In a previous in vitro study, mixed ruminal microorganisms converted oleic acid to a variety of trans monenes when grown in batch cultures under constant environmental conditions. To determine if a similar conversion occurs under environmental conditions more typical of the rumen, conversion of 13C-labeled oleic acid to biohydrogenation intermediates was determined in ruminal microorganisms grown in continuous culture at two pH (5.5 and 6.5) and liquid dilution rates (0.05 and 0.10 h-1) arranged factorially. At 6.5 pH and 0.10 h-1 dilution rate, 13C enrichment was detected in all trans monoenes by GC-MS. However, when pH or dilution rate in fermentors was lowered, no 13C enrichment was detected in any trans monoene having a double bond position between carbons 11 to 16 of the acyl chain. The 13C enrichment of trans-10 was reduced under low pH and dilution rate conditions. The 13C enrichment also was found in two additional peaks identified as 10-hydroxy stearic acid and 10-keto stearic acid. The results of this study show that ruminal microorganisms are capable of converting oleic acid to trans monoenes, and 10-hydroxy and 10-keto stearic acids and that conversion was reduced under low pH and dilution rate conditions. Additional batch cultures of mixed ruminal microorganisms were prepared to examine possible pathways for oleic acid conversion to 10-hydroxy and 10-keto stearic acids. In the first experiment, 10, 20, or 30 mg of oleic acid was added to cultures resulting in linear net increases in both stearic acid derivatives. When equal quatnties of trans-9 octadecenoic acid was added to the cultures, neither 10-hydroxy or 10-keto stearic acids were seen, indicating that oleic acid is directly converted to these stearic acid derivatives. In another experiment, 17 mg of 12-hydroxy stearic acid was added to cultures. After 24 hours of incubation, there was a 4.24 mg loss of the 12-hydroxy stearic acid and a 5.93 mg increase in 10-keto stearic acid without any increases in trans-octadecenoic acid or stearic acid. These results show that hydroxy stearic acid arises from oleic acid but not trans monoenes or stearic acid, and that 10-hydroxy stearic acid is converted by ruminal anaerobes to 10-keto stearic acid over time.

Impacts
This study shows that dietary factors that maintain high ruminal pH or dilution rate will maximize conversion of oleic acid to trans monoenes at the expense of stearic acid, thus having the potential to enhance trans-10 isomers associated with inhibition of fat synthesis. Also, hydroxy and keto derivatives of stearic acid are produced directly from oleic acid, which have been shown previously to act as inhibitors of cell proliferation.

Publications

  • AbuGhazaleh, A. A., M. B. Riley, and T. C. Jenkins. 2004. The effect of dilution rate and pH on the conversion of stable isotopically labelled oleic acid to trans monoenes in continuous culture. J. Dairy Sci. 87 (Suppl. 1):337.


Progress 01/01/03 to 12/31/03

Outputs
A previous in vitro study using batch cultures of mixed ruminal microbes showed that oleic acid was converted to trans monoene isomers. This study was conducted to determine if a similar conversion can occur under different rumen environmental conditions. Four dual-flow continuous fermenters were used to determine the effects of pH and liquid dilution rates on microbial biohydrogenation of 13C-labeled oleic acid to trans monoenes. A 4 ? 4 Latin square design with a factorial arrangement of treatments was used, with four 11-d consecutive periods. Treatments were 1) pH 6.5 and 0.10 h-1 dilution rate, 2) pH 6.5 and 0.05 h-1 dilution rate, 3) pH 5.5 and 0.10 h-1 dilution rate, and 4) pH 5.5 and 0.05 h-1 dilution rate. Fermenters were fed twice daily (22g/d) a TMR diet consisting of 55% alfalfa, and 45% concentrate mix (DM basis). The 13C-labeled oleic acid (250 mg) dissolved in 5 ml of 95% ethanol was added into fermenters after the morning feeding on d-10 of each period. Samples of digesta were taken at 0 h (before adding 13C-labeled oleic acid) and at 24 h after adding 13C-labeled oleic acid. Both pH and dilution rate affected trans monoene formation from oleic acid. At pH 5.5, 13C enrichment was not detected on the trans double bonds between carbons 11 and 16 compared with pH 6.5. Similarly, at 0.05 h -1 dilution rate 13C enrichment was not detected on the trans double bonds between carbons 12 and 16 compared with 0.10 h -1 dilution rate. The percentage of trans-9 and 10 produced from oleic acid was not affected by pH (P > 0.05). The percentage of trans-6, 9, 10, and 11 produced from oleic acid was higher (P < 0.05) at 0.10 h -1 dilution rate compared with 0.05 h -1 dilution rate. The percentage of stearic acid produced from oleic acid was not affected by dilution rate (P > 0.05) and it was higher (P < 0.05) at pH 5.5 (72%) compared with pH 6.5 (26%). The results of this study show that conversion of oleic acid to trans monoenes was reduced under low dilution rate and pH conditions.

Impacts
This study shows that changing conditions in the rumen of cattle significantly affects the degree of fatty acid isomerization. Thus, the formation of specific trans monoene isomers can be affected by changes in ruminal pH and dilution rate that accompany dietary manipulation. Dietary manipulation has the potential to enhance trans-10 isomers associated with inhibition of fat synthesis.

Publications

  • AbuGhazaleh, A. A., M. B. Riley, and T. C. Jenkins. 2004. The effect of dilution rate and pH on the conversion of stable isotopically labelled oleic acid to trans monoenes in continuous cultures. J. Dairy Sci.(Suppl. 1): Submitted.