Incorporation of N from intravenously administered 15N labelled urea into the bacterial protein in the sheep.

The experiment carried out on two wethers demonstrated that nitrogen of intravenously injected urea, labelled with 15N was incorporated into total and bacterial nitrogen fraction of the digesta flowing through the rumen and duodenum. The amount of 15N in the bacterial fraction flowing throught the rumen and duodenum was relatively low in comparison with the amount of 15N in the total nitrogen (14,8% and 8,1% in the rumen and 6,6% and 7,9% in the duodenum. The ratio of the amount of bacterial-N to total-N in the rumen content (12,7 and 7,5%) was only slightly lower than the ratio of bacterial 15N to total 15N. In the duodenum this ratio was a little higher (8,7 and 10,0%). Blood urea nitrogen was utilized only partly in biosynthesis of bacterial protein. The results showed that only a small amount of blood urea nitrogen retained in the organism was utilized for microbial protein synthesis and the majority in some different way.     

Effect of propionate on the utilization of nitrogen from 15NH4Cl for urea synthesis in hepatocytes isolated from sheep liver

1. The effect of ornithine (2.0 mM) and propionate (5.0 mM) on the utilization of N from 15NH4Cl (5.0 mM) for urea synthesis in hepatocytes isolated from sheep liver was investigated. 2. The capacity of sheep hepatocytes to utilize [15N]ammonia in the absence of the other exogenous substrates was very low and amounted 132 +/- 37.3 mumol/hr per 1 g dry wt. 3. Ornithine failed to affect the total [15N]ammonia uptake and total urea synthesis, but at the same time it markedly increased the utilization of [15N]ammonia for ureagenesis and diminished the rate of urea synthesis from endogenous sources. 4. Propionate markedly increased total [15N]ammonia utilization and total urea formation; this increase resulted from the rise of ammonia utilization for urea synthesis and it was similar in the presence or absence of ornithine. 5. The capacity of sheep liver cells to utilize ammonia in the presence of propionate (in the presence or absence of ornithine) amounted to 256 mumol/hr per 1 g dry wt, thus being similar to the values in vivo. 6. It is concluded that in sheep hepatocytes both ornithine and propionate stimulate the utilization of ammonia for urea synthesis and these effects take place independently and occur by different mechanisms.     

Regulation of urea synthesis by agmatine in the perfused liver: studies with 15N

Administration of arginine or a high-protein diet increases the hepatic content of N-acetylglutamate (NAG) and the synthesis of urea. However, the underlying mechanism is unknown. We have explored the hypothesis that agmatine, a metabolite of arginine, may stimulate NAG synthesis and, thereby, urea synthesis. We tested this hypothesis in a liver perfusion system to determine 1) the metabolism of l-[guanidino-15N2]arginine to either agmatine, nitric oxide (NO), and/or urea; 2) hepatic uptake of perfusate agmatine and its action on hepatic N metabolism; and 3) the role of arginine, agmatine, or NO in regulating NAG synthesis and ureagenesis in livers perfused with 15N-labeled glutamine and unlabeled ammonia or 15NH4Cl and unlabeled glutamine. Our principal findings are 1) [guanidino-15N2]agmatine is formed in the liver from perfusate l-[guanidino-15N2]arginine ( approximately 90% of hepatic agmatine is derived from perfusate arginine); 2) perfusions with agmatine significantly stimulated the synthesis of 15N-labeled NAG and [15N]urea from 15N-labeled ammonia or glutamine; and 3) the increased levels of hepatic agmatine are strongly correlated with increased levels and synthesis of 15N-labeled NAG and [15N]urea. These data suggest a possible therapeutic strategy encompassing the use of agmatine for the treatment of disturbed ureagenesis, whether secondary to inborn errors of metabolism or to liver disease.     

Passage of the intravenously administered 15N urea into the digestive tract and its excretion in the sheep.

The experiments performed on two wethers provided with simple rumen cannulas and reentrant cannulas, inserted into the proximal duodenum and ileum, showed a passage of 15N from labelled urea, injected intravenously, from the blood to the digestive tract. The amount of the 15N in the digesta was the highest in duodenum, slightly lower in the rumen and slightly lower in ileum. Approximately 50% of the injected 15N was excreted in urine. The amount of the 15N eliminated with feces was very small; 0.6 to 2.8% of the dose injected per day. About 73--84% of the 15N which passed the duodenum was absorbed in further parts of the digestive tract. It can be concluded that all parts of the digestive tract take part in utilization of the endogenous urea.     

Utilization of nitrogen from 15NH4Cl and [15N]alanine for urea synthesis in hepatocytes from fed and starved rats

Utilization of N from 15NH4Cl and [15N]alanine for urea synthesis in hepatocytes isolated from fed and 24 hr starved rats was investigated. In hepatocytes isolated from fed rats, 54 and 65% of the added [15N]ammonia was utilized for urea synthesis in the presence of 0.5 and 2.0 mM NH4Cl, respectively. This utilization of [15N]ammonia in hepatocytes from starved rats was 2-fold lower. The amount of urea synthetized from endogenous sources was, in the presence of 0.5 and 2.0 mM NH4Cl, about 44 and 60% higher than in the control conditions (without NH4Cl). The considerable amount of added ammonia (30-44%) was utilized in processes other than urea synthesis. Alanine markedly diminished the utilization of 15N from NH4Cl in hepatocytes from both fed and starved rats. In these conditions (NH4Cl present), alanine significantly increased the urea formation in hepatocytes from starved rats and failed to affect the urea production in hepatocytes from fed rats. On the basis of 15N determination, it was concluded that both NH4Cl and alanine caused an increase in the utilization of nitrogen from endogenous sources in rat hepatocytes. This conclusion is in contrast with the results based only on the changes in ammonia and urea concentrations.     

Relative concentrations of N15 in urinary ammonia N and urea N after feeding N15-labeled compounds

Available data on the isotopic ratio See PDF for Equation of ammonia (r_a) and that of urea (r_u) after a single feeding of glycine, aspartic acid, and ammonium citrate are analyzed. From this analysis the following conclusions are drawn. 1. The isotopic ratio See PDF for Equation of ammonia (r_a) is always higher than that of urea (r_u) in the initial period after a single feeding of isotopic glycine or aspartic acid, but the relation is reversed later. A similar relation probably holds after feeding isotopic ammonia. 2. It is pointed out that the ratio of average r_a to average r_u depends on the time interval for which urine is collected, on the schedule of feeding, and probably also on the amount taken at each feeding. When the amount fed and the feeding schedule are unknown, theoretical interpretation of the ratio of average r_u to average r_u is impossible. 3. At the point of maximum isotopic ratio of urea, it is very probably equal to the isotopic ratio of ammonia. A possible explanation is suggested.     

Nucleic acid synthesis in preimplantation mouse embryos

Summary Mouse embryos were collected at the 2-cell stage, cultured in vitro in the presence of³H deoxyuridine or uridine for 6 or 4 h and autoradiographed.Deoxyuridine is actively incorporated into the DNA of cleaving mouse embryos indicating the existence of thymidylate synthetase activity at least at the 4-cell stage and presumably already before this.RNAase treatment of embryos squashed on slides shows a weak but obvious incorporation of uridine into DNA of cleaving mouse embryos, from the 4-cell stage onwards; this incorporation is totally inhibited by hydroxyurea. The reduction of ribonucleotides to deoxyribonucleotides is a metabolic pathway already required for cleavage, as shown by hydroxyurea experiments.The second polar pody, known to incorporate thymidine, is unable to incorporate either deoxyuridine or uridine.     

Appearance of intraportally infused [15N]urea in blood and urine of domestic fowl

The time course of the occurrence of intraportally infused [15N]urea in blood and urine was investigated in chickens. The infused urea appeared in ureteral urine, mostly in the form of urea, as early as 30 min after the start of infusion and the excretion further increased up to the end of 2 hr infusion. Blood urea concentration rapidly increased and reached about three times the initial level at the end of the experiment (P < 0.05 after 20 min), but no significant effects were observed on uric acid, ammonia and glutamine concentrations. Fifty-seven percent of blood urea N and 3% of blood glutamine-amide N and 1% of blood ammonia N, which were determined at the end of experiment, were derived from the infused urea N. It is concluded that urea, which is rapidly increased in blood and urine after feeding urea to chickens, is mostly derived from dietary urea.     

Nucleic acid synthesis in cancerous cells under the effect of gnidilatimonoein from Daphne mucronata

Cytotoxicity evaluation of gnidilatimonoein, the most active isolated diterpene ester from Daphne mucronata [Sadeghi H, Mianabadi M, Yazdanparast R, (2002) Journal of Tropical. Medicinal Plant1 3: 169-173], revealed the strong antiproliferative activity among several different human cancer cell lines (K562, CCRF-CEM, HL-60 and MOLT-4 leukemia cell lines, LNCaP-FGC-10 a prostate cancer cell line) and a mouse BALB/C fibrosarcoma cell line (WEHI-164). Using flow cytometry technique, it was found that treatment of the most responsive cells (K562) with gnidilatimonoein inhibited the progression of cells through G1 phase by almost 15% compared to the untreated cells. The population of the treated cells in the S and G2 phases also reduced by 8.3% and 5.4%, respectively. Based on the extent of [3H]-thymidine and [3H]-uridine incorporation into DNA and RNA, respectively, the major metabolic effects of gnidilatimonoein were found to be mainly on DNA and to a less extent on RNA synthesis. Additionally, the activity of inosine-5'-monophosphate dehydrogenase (IMPDH), under the effects of genidilatimonoein, was reduced in the treated cells by 44%. These data strongly suggest that the purine biosynthetic pathway is significantly affected by gnidilatimonoein.