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Role of Doxycycline in Prostate Cancer With Microarray

Bioinformatics analysis of the role of doxycycline in prostate cancer with microarray
Running title: Roles of doxycycline in prostate cancer
High lights:
Significant pathways involved in the progression are analyzed.
PPI network was constructed and total 392 common DEGs were identified.
BUB1, MYC, IGF-1, CCNE2, CYP2E1 and ALDH3B2 were identified as key genes.
Purpose: We aim to identify the key genes and pathways in prostate cancer (PC)with treatment of doxycycline for different time.
Methods: The microarray expression profile of GSE24261 was obtained from gene expression omnibus (GEO) database, including doxycycline treated samples and control samples at day 1 and day 2. Differentially expressed genes (DEGs) in doxycycline samples at different time point were identified using the limma package in R language. Gene ontology (GO) Term and kyoto encyclopedia of genes and genomes (KEGG) pathway analysis of DEGs were performed using the DAVID and protein-protein interaction (PPI) network was constructed using the Cytoscape. Moreover, the common DEGs in day1 and day2 doxycycline case samples were selected and analyzed.
Results: Totally, 460 DEGs in day1 samples and 1336 DEGs in day2 samples were screened. The up-regulated DEGs were mainly enriched in metabolism of xenobiotics by cytochrome P450 and drug metabolism-cytochrome P450, while the down-regulated genes were mainly enriched in cell cycle and DNA replication. The common DEGs, aldehyde dehydrogenase 3 family, member B2 (ALDH3B2), cytochrome P450, family 2, subfamily E, polypeptide 1 (CYP2E1), v-mycavian myelocytomatosis viral oncogene homolog (MYC) insulin-like growth factor 1 (IGF–1), cyclin E2 (CCNE2), were identified as key genes in response to doxycycline, which were involved in some significant pathways including cell cycle.
Conclusion: Doxycycline inhibits the expression of BUB1, MYC, IGF-1 and CCNE2 and increase the expression of CYP2E1 and ALDH3B2, leading to the inhibition of tumoregenesis.
Keywords: prostate cancer; doxycycline; pathway analysis; differentially expressed genes.
Prostate cancer (PC) is a cancer develops in prostate in the male reproductive system, which is predicted to account for approximately 240000 new cases in America and is the leading cause of cancer mortality [1]. The morbidity of invasive PC increases depending on patients` ages, with the risk that rises from 1 in 37 males between 40 to 59 of age to 1 in 8 males of 70 or older [2]. Radical prostatectomy combined with chemotherapeutic is the main therapy for PC, yet at least a third of the patients will have a recurrence and the cancer cells may spread from the prostate to other parts of human body, such as bones and lymph nodes [4]. However, it is little known on the efficient inhibition therapy of prostate, thus the research on the roles of some valid drugs, such as doxycycline, is of vital significance.
Doxycycline is a member of tetracycline family which has been used as antibiotics effectively for decades [5]. Doxycycline blocks shedding of MHC class I polypeptide-related sequences from a panel of human tumor cells and acted to increase their expression and cell surface translocation [6]. Besides, doxycycline also selectively enhanced the replication of oncolytic vaccinia in various tumor cell lines, which leads to increased sensitivity to these therapies [6]. In recent years, doxycycline has been used for treatment of various human cancers, such as breast cancer, in which doxycycline is highly effective in the inhibition of matrix metalloproteinases, the important enzymes in tumor cell invasion and metastatic ability [7]. Besides, the doxycycline has also been used for PC treatment [7]. However, the definite mechanism of doxycycline in PC cell has not been fully clarified.
Sanjeev K et al [8] take treatment of doxycycline on PC cells to analyze the mRNA expression in response to RunX2, an osteoblast master transcription factor and Gillian H et al [9] has identified RunX2-regulated genes in PC cells with treatment of doxycycline. Both two researched above make use of microarray of GSE24261, which was used in the present study. In the present study, we identified differentially expressed genes (DEGs) in PC cells using the microarray GSE24261, which was obtained based on PC cell samples treated with or without doxycycline at different time points.. Besides, pathways and protein-protein interaction (PPI) network were analyzed and common DEGs were identified to clarify the role of doxycycline in PC.
Materials and methods
Microarray data
The gene expression profile of GSE24261 was downloaded from Gene Expression Omnibus (GEO) database in National Center for Biotechnology Information (NCBI) ( based on the platform of GPL6883 (Illumina HumanRef-8 v3.0 expression beadchip). The study contains a total of 16 samples, including 4 day1 doxycycline case samples, 4 day1 control samples, 4 day2 doxycycline case samples and 4 day2 control samples.
Data preprocessing and DEGs analysis
By using Limma package [10] in Bioconductor and Illumina microarray probe annotation profile from Brain Array Lab, the probe-level data was converted into expression measures, during which Background Correction, Quantile Normalization and Probe Summarization were performed and the significantly DEGs in doxycycline case samples of PC cells were identified. A combination of FDR 2 was used as the threshold.
Gene ontology (GO) and pathway enrichment analysis
GO analysis has become a widely used approach for the studies of large-scale genomic or transcriptomic data in function [12]. Kyoto encyclopedia of genes and genomes (KEGG) is a widely used collection of online database which deals with genomes, enzymatic pathways, and biological chemicals. [13] In this study, the functions and pathways of the screened DEGs in both day1 and day2 doxycycline case samples were analyzed using the DAVID from the GO and KEGG pathway database with the p-value < 0.05, respectively.
Functional annotation of DEGs
Functional annotation of DEGs was performed for the detection of oncogenes and tumor suppressor genes. Two databases, tumor suppressor genes (TSGs) database [14] and tumor associated genes (TAG) database [15] were used for screening tumor suppressor genes and oncogenes.
PPI network and sub-network construction

The Search Tool for the Retrieval of Interacting Genes (STRING) database [16] is a database of known and predicted protein interactions, including both experimental and predicted interaction information. Cytoscape [17] is an open source bioinformatics software platform, which is used for the visualization of molecular interaction networks and integrating with gene expression profiles and other state data. In this study, STRING was used to predict the interactions of selected DEGs [18] with Required Confidence (combined score) > 0.9 and Cytoscape was used to visualize the PPI network. [19]. Besides, sub-network was also constructed using ClusterOne [20] in Cytoscape and pathways were enriched on sub-network with p-value < 0.05.
Selection of common DEGs in samples at two time points
To analyze the effects of doxycycline works for different time, the common DEGs in both day1 and in day2 samples were identified. Afterwards, the functions and pathways of the screened common DEGs were analyzed using the DAVID from the KEGG pathway database with the p-value < 0.05.
DEGs selection
Totally, 460 DEGs (324 up-regulated and 136 down-regulated) in day 1 samples, and 1336 DEGs (707 up-regulated and 629 down-regulated) in day 2 samples were screened. The heat map of both day1 and day2 samples were showed in Figure 1A and Figure 1B, respectively.
GO and KEGG enrichment analysis of DEGs
GO and KEGG enrichment were performed with p-value < 0.01 for the functional and pathway analysis of DEGs, respectively. The main KEGG pathways of DEGs screened in day1 doxycycline case samples were listed in Table 1. The up-regulated genes were involved in metabolism of xenobiotics by cytochrome P450 and drug metabolism-cytochrome P450 (Table 1A), while the down-regulated genes participated in the cell cycle, TGF-beta signaling pathway and PC (Table 1B). In addition, the GO terms of the DEGs were also showed in Table 1. We can see from the results that the up-regulated genes were mainly enriched in the signal transduction and signaling (Table 1C), while the down-regulate genes were involved in negative regulation of binding and positive regulation of cellular component organization (Table 1D).
The main KEGG pathways of DEGs identified in day2 doxycycline case samples were listed in Table 2. The up-regulated genes were involved in metabolism of xenobiotics by cytochrome P450 and drug metabolism-cytochrome P450 (Table 2A), while the down-regulated genes participated in the DNA replication and cell cycle (Table 2B). In addition, the GO terms of the up-regulated genes were mainly enriched in the metabolic process (Table 2C), while the down-regulate genes were involved in cell cycle (Table 2D).
Functional annotation of DEGs
Tumor suppressor genes (TSG) and oncogenes were screened. According to the results (Table 3), in the day1 doxycycline case samples, 4 up-regulated oncogenes and 2 down regulated oncogenes, and 27 up-regulated TSGs and 4 down-regulated TSGs were screened. In day2 doxycycline case samples, totally 15 oncogenes and 44 TSGs in up-regulated genes, and 17 oncogenes and 26 TSGs were screened, which is more than that in day1 doxycycline case samples.
PPI network construction
The network constructed of all DEGs in both day1 doxycycline case samples and day2 doxycycline case samples was shown in Figure 2. In the PPI network of day1 doxycycline case sample (Figure 2A), 102 nodes and 152 protein pairs were obtained. In these nodes, vascular endothelial growth factor A (VEGFA), cytochrome P450, family 2, subfamily E, polypeptide 1 (CYP2E1), glutathione S-transferase mu 1 (GSTM1), glutathione S-transferase alpha 5 (GSTA5), epoxide hydrolase 1, microsomal (EPHX1), glutathione S-transferase alpha 1 (GSTA1), cell division cycle 6 (CDC6), glutathione S-transferase mu 2 (GSTM2) and glutathione S-transferase alpha 4 (GSTA4) were identified as key genes.
In the PPI network of day2 doxycycline case samples, 548 nodes and 2501 protein pairs were obtained. Two sub-network modules, module A and module B, were constructed using ClusterOne and were shown in Figure 2B. BUB1 mitotic checkpoint serine/threonine kinase (BUB1), polo-like kinase 1 (PLK1), cyclin A2 (CCNA2), cell division cycle 20 (CDC20), baculoviral IAP repeat containing 5 (BIRC5) and cyclin B1 (CCNB1) were identified key nodes in the network. For further analysis of the modules, KEGG and GO term were performed on module A and module B, respectively (Table 4). According to the results, the main pathways those nodes enriched in were pathways related to cell cycle and mitotic, such as cell cycle, oocyte meiosis and p53 signaling pathway in module A, and DNA replication, mismatch repair and nucleotide excision repair in module B
Analysis on common DEGs
Totally, 392common DEGs were obtained, including 283 up-regulated genes and 109 down-regulated genes, and the heat map of these genes was shown in Figure 1C According to the pathways analysis (Table 5), up-regulated genes, such as aldehyde dehydrogenase 3 family, member B2 (ALDH3B2), cytochrome P450, family 2, subfamily E, polypeptide 1 (CYP2E1) and glutathione S-transferase alpha 1 (GSTA1), were mainly enriched in metabolism of xenobiotics by cytochrome P450 and Leukocyte transendothelial migration, while down-regulated genes, such as cyclin E2 (CCNE2), v-mycavian myelocytomatosis viral oncogene homolog (MYC) and insulin-like growth factor 1 (IGF-1), were mainly enriched in cell cycle, prostate cancer and p53 signaling pathway.
PC is the most common cancer found in men in America, which has a high rate of reccurence in spite of primary therapy [21]. As a widely used antibiotic in inflammation, doxycycline is also used in treatment of PC, but quite little is known about the mechanism of doxycycline in PC. In this study, PC cell samples with or without treatment of doxycycline at different time point have been used for the identification of key genes in PC in response to doxycycline. Our study showed that BUB1, IGF-1, MYC, ALDH3B2, CYP2E1, and CCNE2 were identified as key genes, which were involved in metabolism of xenobiotics by cytochrome P450, cell cycle, prostate cancer and p53 signaling pathway.
In the PPI network of day2 doxycycline case sample, we found that BUB1 was a down-regulated key gene enriched in cell cycle pathway. BUB1 is a member of BUB family, which encode proteins that are part of a large multi-protein kinetochore complex and believed to be key components of the checkpoint regulatory pathway [22]. Known as a serine/threonine-protein kinase, BUB1 plays a central role in mitosis and functions in part by phosphorylating members of the mitotic checkpoint complex and activating the spindle checkpoint [23]. This kinase accumulates at unattached kinetochores where it mediates the recruitment of mitotic arrest deficient (Mad) dimers [24] and prevents the premature separation of sister chromatids until all chromosomes are properly attached to kinetochores to make sure chromosome segregation is correct [25]. Its defects may cause the instability of chromosome or aneuploidy in human cancer cell lines and underexpression of BUB1 in colon cancer [26] and wilms tumor [27] has been reported. As aneuploidy is an important prognostic factor in PC, BUB1 plays a major role in PC tumorigenesis. Our data showed that with treatment of doxycycline for 2 days, BUB1 expressed down-regulated and was enriched in cell cycle, indicating that doxycycline inhibit the tumorigenesis and tumor growth by controlling the expression of genes associated with mitotic checkpoint, such as BUB1.
In this study, with treatment of doxycycline, the expression of MYC was down-regulated, which was enriched in cell cycle, TGF-beta signaling pathway, small cell lung cancer and bladder cancer. MYC is a proto-oncogene implicated in PC development, which is present on human chromosome 8q24 [28]. The MYC onco-protein is a transcription factor that regulates cellular processes including cell proliferation, metabolism, protein synthesis, mitochondrial function and stem cell renewal [29]. Associated with a partner protein MAX, MYC is able to alternatively associate with other proteins and bind DNA to influence transcription [30]. In PC cells, a region encompassing the MYC locus is somatically amplified at low levels in a subset of patients, which correlates with both worse prognosis and high historical grade. Based on our study and published reportd, overexpression of MYC associated with somatic genetic alterations including translocations and gene amplification lead to the tumorigenesis and growth of PC. In our findings, MYC was down-regulated, leading to the inhibition of main pathways related to cell cycle and TGF-beta signaling pathway, which is important for cytokines secretion and cellular proliferation and migration, and therefore, the growth of PC cells are decreased.
In the common genes, IGF-1 was expressed down-regulated with treatment of doxycycline and mainly enriched in PC, oocyte meiosis and pathway in cancer. IGF-1(encoded byIGF-1) is a member of insulin-like growth factor family and plays a role in prostate development and carcinogenesis [31]. IGF-1 stimulates mitogenic and antiapoptotic activities of prostate epithelial cells in both prostate development and tumorigenesis [31]. General view is that IGF-1 binds to its binding protein IGFBP-1 or IGFBP-3, which modulate the bioavailability of IGF-1 and regulated by insulin, promoting the proliferation of epithelial cells [32]. Doxycycline treatment causes the expression of IGF-1 down-regulated, which decreases the stimulation of cell proliferation and resistance to apoptosis and consequently inhibits the occurrence of PC.
In the present study, CCNE2, encoded by the down-regulated gene CCNE2, was enriched in significant pathways including cell cycle, prostate cancer, oocyte meiosis and p53 signaling pathway. CCNE family members are important regulators of S phage entry concurrent with DNA replication in cell cycle and they are frequently deregulated in some human cancers [33]. They drive the transition from G1 to S phase in mitotisis cycle through assembly of pre-replication complexes and activation of CDK kinases, which leads to the initiation of DNA synthesis [34]. Because of the fundamental role in promoting proliferation, CCNEs have been identified as oncogenes in various cancers [35]. As for CCNE2, it functions as a regulator of CDK2 and specifically interacts with CDK2 inhibitors [36], playing a key role in cell cycle G1/S transition. Our study may drawn a conclusion that doxycycline down-regulates the exhibition of CCNE2, leading to the restrain of G1/S transition in cell cycle, thereby the growth of tumor is inhibited.
Cytochrome P450 enzymes are a family of heme-containing enzymes involved in xenobiotics metabolism [37] and specific isoenzymes of the family have been identified in tumors [38]. CYP2E1 is a key enzyme in the metabolic activation of procarcinogens [39], which is up-regulated with doxycycline treatment in this study and enriched in drug metabolism-cytochrome P450 and metabolism of xenobiotics by cytochrome P450. CYP2E1 metabolizes some small molecules such as acetaminophen, ethanol and pro-carcinogenes like azo compounds and nitrosamines [40], during which toxic intermediates and excessive amounts of reactive oxygen species (ROS) are generated [41]. High ROS levels induces autophagy pathway and triggers accumulation of autophagy-regulated genes [42], thereby stimulating the formation of autophagosome in cancer [43]. In the present study, the express level of CYP2E1 was high, which increases ROS generation and inhibits the migration of the highly invasive cancer cells [38].
In the doxycycline case samples, another key gene enriched in drug metabolism-cytochrome P450 and metabolism of xenobiotics by cytochrome P450, ALDH3B2, was also expressed up-regulated. Aldehyde dehydrogenase (ALDH) is a family of cytosolic isoenzymes that are responsible for oxidizing intracellular aldehydes and contributing to the oxidation of retinol to retinoic acid in early stem cell differentiation [33]. Some member, such as ALDH1 enzymes, has been identified as being responsible for the resistance of progenitor cells to chemotherapeutic agents [35]. ALDH3 is induced by chlorinated compounds and polycyclic aromatic hydrocarbons and in hepatoma and lung cancer cell lines it is directly correlated with the degree of deciation [34]. ALDH3B2 encodes a member of ALDH family, which is a isozymes play a role in the detoxification of aldehydes generated by alcohol metbollism and peroxidation of lipid. The role of ALDH3B2 is similar with CYP2E1 in response to doxycycline and this gene is a target of the drug, whose up-regulated expression decreases the tumorigenesis.
In conclusion, our study identified key genes in prostate cancer cells with treatment of doxycycline at different time point, including BUB1, MYC, IGF-1, ALDH3B2, CYP2E1 and CCNE2. Doxycycline inhibits the expression of BUB1, MYC, IGF-1 and CCNE2 while increases the expression of CYP2E1 and ALDH3B2, leading to the inhibition of tumoregenesis. However, the sample size is relatively less in this study and no experiments have been performed to confirm our conclusion, which is a limitation. Therefore, more analysis and experiments should be performed for further research.

Urea Recycling in Ruminants

Animals have a certain state of protein metabolism, varying from negative to positive protein balances. This balance level is influenced e.g. by the efficiency of nitrogen (N) utilization in animals. A simple strategy to increase the efficiency of N utilization is by reducing the N content in the feed converted to urea, for which a correlation of about r2=0.77 was found. However, this was mainly based on studies with mature or slow growing, small ruminants in which most of the absorbed N is converted to urea to maintain the N balance of the whole body close to zero (Lapierre and Lobley, 2001). More recent and extensive data show much weaker correlations between N intake and urea production for growing sheep (r2=0.33) and cattle (r2=0.58). Moreover, this strategy is not always realizable due to minimal absolute N requirements in animal feed, especially for growing animals.
In addition to N intake, the protein balance level is influenced by the efficiency of N recycling in animals, especially in ruminants. Nitrogen recycling takes place between blood and the digestive tract in the form of endogenous protein-N, secreted-N (e.g. enzymes in saliva) and urea-N (Reynolds and Kristensen, 2008).
In this chapter, the recycling of urea-N is explained. Amino acids and ammonia, which are absorbed from the digestive tract, are converted to urea in the liver. Urea (re)enters the digestive tract, mainly through the rumen wall, where it can be absorbed again or be (re)used for microbial protein synthesis and finally anabolic purposes.
Amino acids and ammonia are absorbed into the portal bloodstream and converted into urea in the liver (ureagenesis). Urea can reenter the rumen, where it can be absorbed (again) or be used for microbial protein synthesis.
Absorption of amino acids and ammonia Urea is the mammalian end-product of the amino acid metabolism. In the rumen, proteins are degraded into amino acids and finally into ammonia (NH3) by means of rumen fermentation (Shingu et al., 2007). Then, absorption of both amino acids and NH3 through the rumen wall and entrance into the portal circulation to the liver can take place (figure 3.1). The NH3 absorption depends on the pH and the ratio of NH3 to NH4 in the rumen (Siddons et al., 1985).
Ureagenesis In the liver, detoxification of NH3 takes place, because urea is synthesized from the nitrogen (N) compound of both NH3 and amino acids (which appear in the portal circulation due to absorption from the intestine into the blood) (Obitsu and Taniguchi, 2009). The synthesis of urea, called ureagenesis, takes place by means of the urea or ornithine cycle. This cycle of biochemical reactions occurs in many animals that produce urea ((NH2)2CO) from ammonia (NH3), mainly in the liver and to a lesser extent in the kidney. The key compound is ornithine, which acts as a carrier on which the urea molecule is built up. At the end of the reaction sequence, urea is released by the hydrolysis of arginine, yielding ornithine to start the cycle again (Bender, 2008). Mitochondrial ammonia and cytosolic aspartate are precursors for the ornithine cycle (Van den Borne et al., 2006). The presence of arginine is needed to produce ornithine in the body, so higher levels of this amino acid should increase ornithine production. Furthermore, ornithine, citrulline and arginine (all components of the ornithine cycle) seem to stimulate urea synthesis, with a concurrent decrease in plasma ammonia.
Temporarily high ammonia fluxes seem to stimulate amino acid utilization for ureagenesis (Milano and Lobley, 2001). Urea is produced in the liver in greater amounts than which is eliminated in the urine. This is because urea from the liver is released to the blood circulation and then, next to excretion in the urine also is reabsorbed in the distal renal tubules, where it maintains an osmotic gradient for the reabsorption of water (Bender, 2008). Furthermore, urea from the blood can re-enter the digestive tract via saliva, secretions or directly across the rumen wall in the form of endogenous proteins or urea respectively (Lapierre and Lobley, 2001; Shingu et al., 2007; Obitsu and Taniguchi, 2009). Thus not all urea is secreted directly into the urine after entering the bloodstream.
Entry into digestive tract Entry of urea into the digestive tract is, until certain concentrations (sheep: 6 mM (= 84 mg/L); cattle: 4 mM (= 56 mg/L) (Harmeyer and Martens, 1980; cattle: 80 mg/L (Kennedy and Milligan, 1978)) partly affected by plasma urea concentrations (Harmeyer and Martens, 1980). Above these concentrations, boundary layer effects with NH3 inhibit the urea entry into the digestive tract (Lapierre and Lobey, 2001). Urease activity is lower with increased NH3 concentrations and N intake (Marini et al., 2004). This inhibits the entry of urea into the digestive tract (Kennedy and Milligan, 1978). Thus high ammonia concentrations in the rumen result in a lower gut entry rate (Kennedy and Milligan, 1978; Bunting et al., 1989a).
Urea, which flows from the blood into the rumen and enters the digestive tract, is hydrolyzed by bacterial urease to carbon dioxide (CO2) and ammonia (NH3) (figure 3.1). NH3 can be either reabsorbed into the blood or be used as N source for microbial protein synthesis or microbial growth (Sarraseca et al., 1998; Shingu et al., 2007). This latter process may provide a mechanism for the salvage of urea-N into bacterial protein which can be digested and yields amino acids to the animal when they are absorbed in the lower parts of the digestive tract. Thus, urea nitrogen incorporated in microbial protein and possibly absorbed in the gut gets ‘a second chance’ for absorption and deposition/anabolic purposes. Therefore, urea recycling can be regarded as a mechanism with positive effects at the protein balance of ruminants.
Gut entry location and gut entry rate (GER) The gut entry rate (GER) of urea is simply the amount of urea N recycled into the digestive tract. The amount of urea which entered the digestive tract that can be used for anabolic purposes depends e.g. on the gut entry location (Lapierre and Lobley, 2001). Urea appears to enter all parts of the digestive tract, including via saliva and pancreatic juice, but with different rates. The GER could be influenced by the concentration gradient of urea between the plasma and the fluids in the digestive tract (Harmeyer and Martens, 1980). The concentration gradient is again dependent on the activity of ureolytic bacteria and could therefore be influenced by diverse bacteria-influencing compounds in the feed. Also, the presence of carrier mediated, facilitative urea transport mechanisms have been reported in the ovine colon and rumen epithelia (Ritzhaupt et al., 1997). The carrier mediated, facilitative urea transporters in the ovine colon and rumen epithelia permit bi-directional flows (Ritzhaupt et al., 1997), and thus may the total gut entry rate (GER) be underestimated if urea molecules are reabsorbed without being metabolized (Lapierre and Lobley, 2001).
Post-stomach tissues can greatly influence the (GER) (up to 70%), but their contribution to potential anabolic salvage of N is not certain.
The majority of conversions of urea into anabolic compounds occur in the fore-stomach, mainly the rumen (Kennedy and Milligan, 1980). As summarized by Lapierre and Lobley (2001), in sheep, the part of the total gut urea entry (GER) transferred to the rumen varies from 27 to 60% (Kennedy and Milligan, 1978) and 27 to 54% (Siddons et al., 1985) depending on type of diet. This proportion seems to increase when animals get high levels of rumen-degradable energy in feed (Lapierre and Lobley, 2001; Theuer et al., 2002).
Also saliva contributes to the total urea entry into the rumen, depending on the type of diet ingested. E.g. this proportion varies extensively from 15 (Kennedy and Milligan, 1978) to almost 100% (Norton et al., 1978) in sheep. It has been found in growing beef steers that forage diets, e.g. alfalfa hay, result in higher proportions of saliva entering the gut (36% of GER) (Taniguchi et al., 1995) compared to high concentrate diets (17% of GER) (Guerino et al., 1991). Thus the fore-stomachs are important for the anabolic salvage of N, however, this depends on the type of feed ingested (and animal species).
Small intestine
Also the small intestine contributes to the anabolic salvage of N. It has been found in sheep that 37 and 48% of the total GER of urea entered the small intestine in case of grass silage and dried grass, respectively (Siddons et al., 1985). However, the quantities of anabolic N formed may by small, e.g. because ammonia production seems to exceed urea entry across the small intestine, although this depends on the type of feed ingested (Lapierre and Lobley, 2001).
Likely most microbial protein synthesized from urea that enters the hindgut is excreted. All the evidence so far would suggest that hindgut usage of urea involves only catabolic fates, at least in terms of amino acids supply to the animal (Lapierre and Lobley, 2001).
Fate of urea that enters the digestive tract Urea that enters the gut by means of saliva or flowing through the gut wall can be used for anabolic purposes or is transformed into ammonia and returned to the liver (Lapierre and Lobley, 2001). Much of the NH3 in the GI tract is reabsorbed and used in the liver for the synthesis of glutamate and glutamine, and then a variety of other nitrogenous compounds (Bender, 2008).
Urea-N that entered the gut contributed for 33% of the rumen ammonia flux in sheep offered dried grass, while this percentage was lower in case of grass silage (Siddons et al., 1985). Lapierre and Lobley (2001), based on several references, summarized that sheep, dairy cows and growing steers have a efficient reuse of N because urea-N atoms can return to the gut on more than one occasion. This increases the overall probability of appropriation towards an anabolic fate. This multiple-recycling process can result in improvements of 22 to 49% of GER used for anabolic purposes in both cattle and sheep (Lapierre and Lobley, 2001). A substantial proportion of urea that enters the digestive tract is returned to the body as ammonia in both sheep (32 to 52%; Sarreseca et al., 1998) and cattle (26 to 41%; Archibeque et al., 2001). This means that a large proportion of net ammonia absorption across the PDV is due to recycled N, rather than arising directly from ingested N. These anabolic and catabolic fates of urea then explain why net appearances of amino acid-N and ammonia across the PDV can equal or exceed apparent digestible-N (Lapierre and Lobley, 2001). The net result of all these N transactions is that the apparent conversion of digestible N into net absorbed amino acid N can be high, with individual values of 27 to 279% calculated for both cattle and sheep. These ‘efficiencies’ are lower (24 to 58%) when other inputs are considered, mainly the urea-N inflow into the rumen. Apparent digestible N represents the net available N to the animal and thus the amino acid absorption cannot normally exceed this unless other N sources like amino acids obtained due to catabolism (released on a net basis during submaintenance intake) or urea recycling. N recycling via the digestive tract increases the opportunity for catabolism N to be reconverted to an anabolic product. This recycling can be considered analogous to the synthesis and breakdown of proteins within tissues, where the dynamic flow maintains metabolic fluidity with minimum loss (see figure …; Lapierre and Lobley, 2001).
Thus, urea-N kinetics can, as an approximation, be considered as a mechanism, where hepatic synthesis is similar to digested N, with one-third lost via the kidneys into urine, while the remaining two-thirds is returned to the digestive tract. Half of this is then reconverted to anabolic N (mainly amino acids) that can be reabsorbed and used for productive purposes. Most of the remaining half of GER is reabsorbed as ammonia that is reconverted to urea and can be further re-partitioned between urinary loss and GER (see figure…). The process thus allows conversion of a catabolic products (urea-N) into anabolic forms, contains these for longer within the body, and provides the animal with increased opportunities to utilize products derived from dietary N (Lapierre and Lobley, 2001).
Figure… Urea recycling: values in circles represent the fraction of hepatic ureagenesis destined either for urinary output or to gut entry rate (GER); values in rectangles represent the fractions of gut entry rate lost in feces, returned as ammonia to the hepatic ornithine cycle or converted to anabolic products (mainly amino acid N). Thus, on average, 33% of hepatic urea-N flux is eliminated in urine while 67% enters the various sites of the digestive tract. Of this latter N, 10% is lost in feces, 40% is reabsorbed directly as ammonia, while the remaining 50% is reabsorbed as anabolic-N sources (mainly AA’s). Data are simplified means for steers, dairy cows and sheep (from Archibeque et al., 2001; Sarraseca et al., 1998; summarized by Lapierre and Lobley, 2001)
Efficiency of N utilization In both cattle and sheep, the inefficient use of intake-N is associated with large ammonia absorption representing on average 0.46 and 0.47 of N available from the lumen of the gut (digestible N plus urea-N entry across the PDV) (Lapierre and Lobley, 2001). As mentioned earlier, one strategy is to reduce the amount of N directed towards ammonia absorption and hepatic ureagenesis, but the situation is more complex than that. The target of reduction of ammonia absorption has to be integrated in a wider context where this decrease would result 1) from a smaller degradation of dietary N into the rumen or 2) from an increased utilization of rumen ammonia for microbial protein synthesis. Lowered N degradation can result from diet manipulation. Lapierre and Lobley (2001) summarized from several studies that cattle fed concentrate-based diets had decreased ammonia absorption, both in absolute amounts and relative to digested N, compared with forage rations. Increased utilization of N for bacterial synthesis can also be influenced by dietary manipulation, particularly provision of additional energy. From several studies, it can be concluded that supplements of rumen fermentable energy sources increase the transfer of urea into the rumen, and therefore the capture of dietary N and GER into anabolic products, mainly amino acids. However, there appear to be upper limits to the overall efficiency of the process (Lapierre and Lobley, 2001). The limited data available suggest that a maximum of 50 to 60% of dietary N, or 70 to 90% of apparently digested N, will be converted into amino acids released into the portal vein. Energy sources may also improve utilization of dietary and urea-N by less direct means, e.g. by energy-sparing effects within the cells of the gut tissues rather than alteration of rumen fermentation (Lapierre and Lobley, 2001).
Intrarumen recycling Recycling of N can also occur within the rumen, due to the presence of proteolytic bacterial and protozoa. These ‘graze’ and digest the rumen bacteria, increasing ammonia content and release within the rumen, and reducing microbial N outflow within the rumen because of increased recycling of bacteria (Lapierre and Lobley, 2001). Thus changes in the microbial population of the rumen can have substantial effect on anabolic N flow. Such modifications of the rumen microflora may contribute to the differences in N recycling and conversion to amino acids that occur between diets and animal species (Lapierre and Lobley, 2001).
Amino acid supply
In many circumstances, inefficiencies for conversion of feed N to animal protein may not be a feature of total amino acid supply, but rather depend more on the profile of absorbed amino acids. Hereby you can think of e.g. limiting essential amino acids.
In ‘short’ the definition of urea recycling is: the flow of urea from the blood into the digestive tract so that urea nitrogen salvage could happen.
Figure … Use of [15N15N] urea and isotopomer analysis of urinary [15N15N], [14N15N] and [14N14N] urea to quantify flows and fates of urea that enters the digestive tract. Part of the infused [15N15N]urea enters the digestive tract were it can be excreted in the faeces or is hydrolyzed to [15N]ammonia. This latter is either used by the microbial population to synthesize bacterial proteins ([15N]) or it is absorbed directly as [15N]ammonia. [15N]ammonia is removed by the liver were [15N14N]urea is formed. The ratio of [14N15N]:[14N14N]urea in the urine reflects the proportion of urea flux that is converted to ammonia in the digestive tract and returned directly to the hepatic ornithine cycle (Lapierre and Lobley, 2001).
The utilities of urea recycling Both ruminants and non-ruminants, including omnivores, have a mechanism in which urea produced by the liver can enter the intestinal tract and where it is used for microbial protein production or urea production. However, the amount of urea recycled in ruminants is in much larger proportions compared to non-ruminants, which emphasizes the importance of urea recycling in ruminants (Lapierre and Lobley, 2001). Next to reducing feed costs (due to the lower dietary N contents required), there are three important reasons to obtain a good and efficient urea recycling in ruminants (Huntington and Archibeque, 1999):
Maximization of the microbial functioning in the rumen;
Optimization of the amino acid supply to the host ruminant – improvements of adaptation;
Minimization of the negative effects of nitrogen excretion into the environment.
Maximization of microbial functioning In ruminants, synthesis of urea by the liver can exceed apparent digestible N. This would result in a negative N balance (even at high intakes) if no salvage mechanism existed to recover some of this N (Lapierre and Lobley, 2001). Recycling of urea synthesized in the liver can provide a substantial contribution to available N for the gut. Lapierre and Lobley (2001) summarized that this can increase the digestible N inflow from 43 to 85% in growing steers, 50 to 60% in dairy cows and 86 to 130% in growing sheep. Moreover, in veal calves shifts the major origin of absorbable amino acids in the small intestine after weaning from milk protein to microbial protein (Obitsu and Taniguchi, 2009). With this, it is important to realize that a higher level of urea recycling results in a higher production of microbial protein. This protein source will be largely used for anabolic uses and performance which will result, on the long term, in improved production efficiency (Lapierre and Lobley, 2001). What urea-N recycling does is to increase N transfers through the body to convert more of the N into anabolic form and thus acts as a conservation mechanism. Therefore, the combined inflows of dietary N and urea GER can be considered analogous to protein turnover within the body, where the anabolic and catabolic processes of synthesis and degradation greatly exceed inputs (intake) and outputs (oxidation and gain). This is believed to provide an overall plasticity to allow rapid response to any challenges or changes in metabolic status.
Optimization of amino acid supply – adaptation As a consequence of the salvage mechanism to recover some N, nitrogen and urea recycling in ruminants are important regarding the adaptation to different environmental (living) circumstances but mainly to nutritional conditions. Examples are periods of dietary protein deficiency or an asynchronous supply of carbohydrates and proteins (Reynolds and Kristensen, 2008). Ammonia and microbial protein produced in the gut and urea synthesized in the liver are major components in N-recycling transactions (Obitsu and Taniguchi, 2009). An increase in the total urea flux, caused by the return to the ornithine cycle from the gut entry, is considered to serve as a labile N pool in the whole body to permit metabolic plasticity under a variety of physiological (productive), environmental and nutritional conditions (Obitsu and Taniguchi, 2009; Lapierre and Lobley, 2001). Therefore, ruminant species have different characteristics of their urea recycling due to different living conditions varying from tropical conditions with poor quality feed to intensive systems in temperate/cold conditions with high quality feed. High ambient temperatures seem to increase urea production but reduce urea gut entry (Obitsu and Taniguchi, 2009).
Minimization of N excretion into the environment Finally, a more efficient urea recycling in ruminants results in a less urea-N excretion in the urine. This is will minimize the negative effects of nitrogen excretion into the environment (Huntington and Archibeque, 1999).