New Smoothened ligands based on the purine scaffold as potential agents for treating pancreatic cancer.

Aberrant activation of the Hedgehog (Hh) signalling pathway has been associated with the development and progression of pancreatic cancer. For this reason, blockade of Hh pathway by inhibitors targeting the G protein-coupled receptor Smoothened (SMO) has been considered as a therapeutic target for the treatment of this cancer. In our previous work, we obtained a new SMO ligand based on a purine scaffold (compound I), which showed interesting antitumor activity in several cancer cell lines. In this work, we report the design and synthesis of 17 new purine derivatives, some of which showed high cytotoxic effect on Mia-PaCa-2 (Hh-dependent pancreatic cancer cell lines) and low toxicity on non-neoplastic HEK-293 cells compared with gemcitabine, such as 8f, 8g and 8h (IC50 = 4.56, 4.11 and 3.08 µM, respectively). Two of these purines also showed their ability to bind to SMO through NanoBRET assays (pKi = 5.17 for 8f and 5.01 for 8h), with higher affinities to compound I (pKi = 1.51). In addition, docking studies provided insight the purine substitution pattern is related to the affinity on SMO. Finally, studies of Hh inhibition for selected purines, using a transcriptional functional assay based on luciferase activity in NIH3T3 Shh-Light II cells, demonstrated that 8g reduced GLI activity with a IC50 = 6.4 µM as well as diminished the expression of Hh target genes in two specific Hh-dependent cell models, Med1 cells and Ptch1-/- mouse embryonic fibroblasts. Therefore, our results provide a platform for the design of SMO ligands that could be potential selective cytotoxic agents for the treatment of pancreatic cancer.

New Inhibitors of Bcr-Abl Based on 2,6,9-Trisubstituted Purine Scaffold Elicit Cytotoxicity in Chronic Myeloid Leukemia-Derived Cell Lines Sensitive and Resistant to TKIs.

Bcr-Abl is an oncoprotein with aberrant tyrosine kinase activity involved in the progression of chronic myeloid leukemia (CML) and has been targeted by inhibitors such as imatinib and nilotinib. However, despite their efficacy in the treatment of CML, a mechanism of resistance to these drugs associated with mutations in the kinase region has emerged. Therefore, in this work, we report the synthesis of 14 new 2,6,9-trisubstituted purines designed from our previous Bcr-Abl inhibitors. Here, we highlight 11b, which showed higher potency against Bcr-Abl (IC50 = 0.015 µM) than imatinib and nilotinib and exerted the most potent antiproliferative properties on three CML cells harboring the Bcr-Abl rearrangement (GI50 = 0.7-1.3 µM). In addition, these purines were able to inhibit the growth of KCL22 cell lines expressing Bcr-AblT315I, Bcr-AblE255K, and Bcr-AblY253H point mutants in micromolar concentrations. Imatinib and nilotinib were ineffective in inhibiting the growth of KCL22 cells expressing Bcr-AblT315I (GI50 > 20 µM) compared to 11b-f (GI50 = 6.4-11.5 µM). Molecular docking studies explained the structure-activity relationship of these purines in Bcr-AblWT and Bcr-AblT315I. Finally, cell cycle cytometry assays and immunodetection showed that 11b arrested the cells in G1 phase, and that 11b downregulated the protein levels downstream of Bcr-Abl in these cells.

Design, Synthesis, In Silico Studies and Inhibitory Activity towards Bcr-Abl, BTK and FLT3-ITD of New 2,6,9-Trisubstituted Purine Derivatives as Potential Agents for the Treatment of Leukaemia.

We report 31 new compounds designed, synthesized and evaluated on Bcr-Abl, BTK and FLT3-ITD as part of our program to develop 2,6,9-trisubstituted purine derivatives as inhibitors of oncogenic kinases. The design was inspired by the chemical structures of well-known kinase inhibitors and our previously developed purine derivatives. The synthesis of these purines was simple and used a microwave reactor for the final step. Kinase assays showed three inhibitors with high selectivity for each protein that were identified: 4f (IC50 = 70 nM for Bcr-Abl), 5j (IC50 = 0.41 µM for BTK) and 5b (IC50 = 0.38 µM for FLT-ITD). The 3D-QSAR analysis and molecular docking studies suggested that two fragments are potent and selective inhibitors of these three kinases: a substitution at the 6-phenylamino ring and the length and volume of the alkyl group at N-9. The N-7 and the N-methyl-piperazine moiety linked to the aminophenyl ring at C-2 are also requirements for obtaining the activity. Furthermore, most of these purine derivatives were shown to have a significant inhibitory effect in vitro on the proliferation of leukaemia and lymphoma cells (HL60, MV4-11, CEM, K562 and Ramos) at low concentrations. Finally, we show that the selected purines (4i, 5b and 5j) inhibit the downstream signalling of the respective kinases in cell models. Thus, this study provides new evidence regarding how certain chemical modifications of purine ring substituents provide novel inhibitors of target kinases as potential anti-leukaemia drugs.

A New Smoothened Antagonist Bearing the Purine Scaffold Shows Antitumour Activity In Vitro and In Vivo.

The Smoothened (SMO) receptor is the most druggable target in the Hedgehog (HH) pathway for anticancer compounds. However, SMO antagonists such as vismodegib rapidly develop drug resistance. In this study, new SMO antagonists having the versatile purine ring as a scaffold were designed, synthesised, and biologically tested to provide an insight to their mechanism of action. Compound 4s was the most active and the best inhibitor of cell growth and selectively cytotoxic to cancer cells. 4s induced cell cycle arrest, apoptosis, a reduction in colony formation and downregulation of PTCH and GLI1 expression. BODIPY-cyclopamine displacement assays confirmed 4s is a SMO antagonist. In vivo, 4s strongly inhibited tumour relapse and metastasis of melanoma cells in mice. In vitro, 4s was more efficient than vismodegib to induce apoptosis in human cancer cells and that might be attributed to its dual ability to function as a SMO antagonist and apoptosis inducer.

Evaluation of methodologies of urine collection to estimate microbial synthesis in bovines diets containing lipids or not / Avaliação de metodologias de coleta de urina para estimar a síntese microbiana em dietas de bovinos contendo ou não lipídios

This study investigated the effectiveness of the urine sample collection method in predicting the volume urinary and synthesis of microbial nitrogen. Eight fistulated steers were used with accessible rumens and kept in individual stalls. Their diets consisted of corn silage; corn silage + concentrate; corn silage + concentrate with addition of lipids in the form of soybean oil; and corn silage + concentrate with addition of lipids in the form of soybean grains. Estimates of microbial protein synthesis were obtained based on the urinary excretion of purine derivatives. There was no effect of diets on daily creatinine excretion (P>0.05). There were differences (P<0.05) between the urinary volume and microbial synthesis values determined by the total urine collection and those estimated from the urine spot samples and equations proposed by different authors. The estimation of microbial synthesis based on the urine excretion of purine derivatives should be performed from the total collection of the urine for a period of 24 hours.

Este estudo investigou a eficácia do método de coleta de amostras de urina sobre a predição do volume urinário e síntese de nitrogênio microbiano. Oito novilhos fistulados foram utilizados com rúmen acessível e mantidos em baias individuais. Suas dietas consistiram de silagem de milho; silagem de milho + concentrado; silagem de milho + concentrado com adição de lipídios na forma de óleo de soja; e silagem de milho + concentrado com adição de lipídios na forma de grãos de soja. Estimativas de síntese de proteína microbiana foram obtidas com base na excreção urinária de derivados de purina. Não houve efeito de dietas na excreção diária de creatinina (P> 0,05). Houve diferenças (P <0,05) entre o volume urinário e os valores de síntese microbiana determinados pela coleta total de urina e os estimados a partir das amostras de manchas de urina e equações propostas por diferentes autores. A estimativa da síntese microbiana baseada na excreção de urina dos derivados de purina deve ser realizada a partir da coleta total da urina por um período de 24 horas.

New 2,6,9-trisubstituted purine derivatives as Bcr-Abl and Btk inhibitors and as promising agents against leukemia.

Bcr-Abl and Btk kinases are among the targets that have been considered for the treatment of leukemia. Therefore, several strategies have focused on the use of inhibitors as chemotherapeutic tools to treat these types of leukemia, such as imatinib (for Bcr-Abl) or ibrutinib (for Btk). However, the efficacy of these drugs has been reduced due to resistance mechanisms, which have motivated the development of new and more effective compounds. In this study, we designed, synthesized and evaluated 2,6,9-trisubstituted purine derivatives as novel Bcr-Abl and Btk inhibitors. We identified 5c and 5d as potent inhibitors of both kinases (IC50 values of 40 nM and 0.58/0.66 µM for Abl and Btk, respectively). From docking and QSAR analyses, we concluded that fluorination of the arylpiperazine system is detrimental to the activity against two kinases, and we also validated our hypothesis that the substitution on the 6-phenylamino ring is important for the inhibition of both kinases. In addition, our studies indicated that most compounds could suppress the proliferation of leukemia and lymphoma cells (HL60, MV4-11, CEM, K562 and Ramos cells) at low micromolar concentrations in vitro. Finally, we preliminarily demonstrated that 5c inhibited the downstream signaling of both kinases in the respective cell models. Therefore, 5c or 5d possessed potency to be further optimized as anti-leukemia drugs by simultaneously inhibiting the Bcr-Abl and Btk kinases.

Promising 2,6,9-Trisubstituted Purine Derivatives for Anticancer Compounds: Synthesis, 3D-QSAR, and Preliminary Biological Assays.

We designed, synthesized, and evaluated novel 2,6,9-trisubstituted purine derivatives for their prospective role as antitumor compounds. Using simple and efficient methodologies, 31 compounds were obtained. We tested these compounds in vitro to draw conclusions about their cell toxicity on seven cancer cells lines and one non-neoplastic cell line. Structural requirements for antitumor activity on two different cancer cell lines were analyzed with SAR and 3D-QSAR. The 3D-QSAR models showed that steric properties could better explain the cytotoxicity of compounds than electronic properties (70% and 30% of contribution, respectively). From this analysis, we concluded that an arylpiperazinyl system connected at position 6 of the purine ring is beneficial for cytotoxic activity, while the use of bulky systems at position C-2 of the purine is not favorable. Compound 7h was found to be an effective potential agent when compared with a currently marketed drug, cisplatin, in four out of the seven cancer cell lines tested. Compound 7h showed the highest potency, unprecedented selectivity, and complied with all the Lipinski rules. Finally, it was demonstrated that 7h induced apoptosis and caused cell cycle arrest at the S-phase on HL-60 cells. Our study suggests that substitution in the purine core by arylpiperidine moiety is essential to obtain derivatives with potential anticancer activity.

Nitrogen metabolism in lambs fed diets containing peach palm meal.

The objective of this study was to evaluate the nitrogen metabolism of lambs fed diets containing peach palm meal substituting maize (0, 10, 40, 60, and 85 % DM). Thirty Santa Inês uncastrated lambs with an average initial body weight of 21.6 ± 0.87 kg were distributed in a completely randomized design with five diets and six replicates. The nitrogen (N) intake (g/day, %BW) and the fecal N decreased linearly as the peach palm meal was added to the concentrate. The urinary N, however, responded quadratically, with maxima at the level of 29.97 and 40.44 % of substitution of maize for peach palm meal when expressed in g/day and %BW, respectively. The substitution of maize for peach palm meal reduced the retention of nitrogen and the microbial protein synthesis in the lambs.

Nitrogen metabolism in lactating goats fed with diets containing different protein sources.

This study aimed to evaluate urea excretion, nitrogen balance and microbial protein synthesis in lactating goats fed with diets containing different protein sources in the concentrate (soybean meal, cottonseed meal, aerial part of cassava hay and leucaena hay). Four Alpine goats whose mean body weight was 42.6±6.1 kg at the beginning of the experiment, a mean lactation period of 94.0±9.0 days and a production of 1.7±0.4 kg of milk were distributed in a 4×4 Latin square with four periods of 15 days. Diets were formulated to be isonitrogenous, containing 103.0 g/kg of CP, 400 g/kg of Tifton 85 hay and 600 g/kg of concentrate. Diet containing cottonseed meal provided (p<0.05) increased excretion of urea and urea nitrogen in the urine (g/d and mg/kg of BW) when compared with leucaena hay. The diets affected the concentrations of urea nitrogen in plasma (p<0.05) and excretion of urea nitrogen in milk, being that soybean meal and cottonseed meal showed (p<0.05) higher than the average aerial part of the cassava hay. The use of diets with cottonseed meal as protein source in the concentrate in feeding of lactating goats provides greater nitrogen excretion in urine and negative nitrogen balance, while the concentrate with leucaena hay as a source of protein, provides greater ruminal microbial protein synthesis.

Microbial protein synthesis and nitrogen metabolism in cows bred on tropical pasture and fed on cassava root and corn / Síntese de proteína microbiana e metabolismo de nitrogênio em vacas suplementadas com raiz de mandioca e milho em pastagem tropical

Current experiment evaluated the effect of replacement of full corn meal by dehydrated ground cassava roots at levels 0%, 25%, 50%, 75% and 100% in experimental supplements for lactating cows grazing on irrigated and fertilized tropical pastures. Ten Holstein cows were divided into two 5 x 5 Latin squares, with average initial 150 days of lactation, milk production 22±3.30 kg day-1 at the beginning of experiment and initial body weight of 603±65 kg. Cows were maintained on pasture consisting of elephant grass (Pennisetum purpureum, Schum) cultivar Pioneiro, intercropped with Tifton 85 (Cynodon nlemfuensis), fertilized with 600 kg nitrogen per hectare year-1. There was no significant difference (p > 0.05) between the substitution levels of corn meal by ground and dehydrated cassava root in the concentrate on the synthesis of microbial protein with an estimated average of 1,288.49 g day-1 and efficiency in the synthesis of microbial protein per kilogram of TDN with estimated average of 91.30 g kg-1 TDN. Nitrogen equilibrium showed an estimated average of 218.79 g day-1 of retained nitrogen. The microbial protein synthesis and nitrogen balance were not affected by treatments.(AU)

Síntese de proteína microbiana e metabolismo de nitrogênio em vacas suplementadas com raiz de mandioca e milho em pastagem tropical. O experimento foi realizado para avaliar o efeito da substituição do milho moído por raiz integral de mandioca moída e desidratada nos níveis 0, 25, 50, 75 e 100% nos suplementos experimentais, para vacas lactantes mantidas em pastagens tropicais irrigadas e adubadas. Foram utilizadas dez vacas da raça Holandesa, divididas em dois quadrados latinos 5 x 5, com média inicial de 150 dias de lactação, produção leiteira inicial de lactação de 22±3,3 kg dia-1 e peso corporal inicial médio de 603±65 kg, mantidos em pastagem formada por capim-elefante (Pennisetum purpureum, SCHUM) cultivar Pioneiro, consorciado com capim Tifton 85 (Cynodon nlemfuensis), adubada com 600 kgde nitrogênio por hectare ano-1. Não foram encontradas diferenças significativas (p > 0,05) entre os níveis de substituição do milho moído por raiz de mandioca moída e desidratada no concentrado, sobre a síntese de proteína microbiana, que apresentou média estimada de 1.288,49 g dia-1 e, eficiência na síntese de proteína microbiana por quilograma de NDT com média estimada de 91,30 g kg-1 NDT, o balanço de nitrogênio apresentou média estimada de 218,79 g dia-1 de nitrogênio retido (p > 0,05). A síntese de proteína microbiana e balanço de compostos nitrogenados não sofreram efeitos dos tratamentos.(AU)

Microbial protein synthesis and nitrogen metabolism in cows bred on tropical pasture and fed on cassava root and corn / Síntese de proteína microbiana e metabolismo de nitrogênio em vacas suplementadas com raiz de mandioca e milho em pastagem tropical

Current experiment evaluated the effect of replacement of full corn meal by dehydrated ground cassava roots at levels 0%, 25%, 50%, 75% and 100% in experimental supplements for lactating cows grazing on irrigated and fertilized tropical pastures. Ten Holstein cows were divided into two 5 x 5 Latin squares, with average initial 150 days of lactation, milk production 22±3.30 kg day-1 at the beginning of experiment and initial body weight of 603±65 kg. Cows were maintained on pasture consisting of elephant grass (Pennisetum purpureum, Schum) cultivar Pioneiro, intercropped with Tifton 85 (Cynodon nlemfuensis), fertilized with 600 kg nitrogen per hectare year-1. There was no significant difference (p > 0.05) between the substitution levels of corn meal by ground and dehydrated cassava root in the concentrate on the synthesis of microbial protein with an estimated average of 1,288.49 g day-1 and efficiency in the synthesis of microbial protein per kilogram of TDN with estimated average of 91.30 g kg-1 TDN. Nitrogen equilibrium showed an estimated average of 218.79 g day-1 of retained nitrogen. The microbial protein synthesis and nitrogen balance were not affected by treatments.

Síntese de proteína microbiana e metabolismo de nitrogênio em vacas suplementadas com raiz de mandioca e milho em pastagem tropical. O experimento foi realizado para avaliar o efeito da substituição do milho moído por raiz integral de mandioca moída e desidratada nos níveis 0, 25, 50, 75 e 100% nos suplementos experimentais, para vacas lactantes mantidas em pastagens tropicais irrigadas e adubadas. Foram utilizadas dez vacas da raça Holandesa, divididas em dois quadrados latinos 5 x 5, com média inicial de 150 dias de lactação, produção leiteira inicial de lactação de 22±3,3 kg dia-1 e peso corporal inicial médio de 603±65 kg, mantidos em pastagem formada por capim-elefante (Pennisetum purpureum, SCHUM) cultivar Pioneiro, consorciado com capim Tifton 85 (Cynodon nlemfuensis), adubada com 600 kgde nitrogênio por hectare ano-1. Não foram encontradas diferenças significativas (p > 0,05) entre os níveis de substituição do milho moído por raiz de mandioca moída e desidratada no concentrado, sobre a síntese de proteína microbiana, que apresentou média estimada de 1.288,49 g dia-1 e, eficiência na síntese de proteína microbiana por quilograma de NDT com média estimada de 91,30 g kg-1 NDT, o balanço de nitrogênio apresentou média estimada de 218,79 g dia-1 de nitrogênio retido (p > 0,05). A síntese de proteína microbiana e balanço de compostos nitrogenados não sofreram efeitos dos tratamentos.

Nitrogenous compounds balance and microbial protein synthesis in steers supplemented with sunflower crushed in partial replacement of soybean meal / Balanço de compostos nitrogenados e síntese de proteína microbiana em novilhos suplementados com torta de girassol em substituição parcial ao farelo de soja

Four steers in individual paddocks with Marandu grass (B. Brizantha) in 4x4 square design were used to evaluate sunflower crushed supplementation in pasture-grazing animals on nitrogen balance and microbial protein synthesis. Supplements at 6 g kg <->1 body weight comprised corn, soybean meal, and mineral and soybean meal substituted at proportions 0, 20, 40 and 60%. Diet contained averages 6.79, 6.96, 7.10 and 6.87% nitrogen respectively for substitution levels 0, 20, 40 and 60%. The inclusion of sunflower crushed (SC) increased nitrogen intake and fecal excretion of nitrogen while providing a positive balance. Animals plasma urea concentration supplemented with SC was 28.13% lower than that of supplemented animals without SC. SC inclusion did not change allantoin concentration, purine derivatives, microbial nitrogen, crude microbial protein and microbial efficiency microbial, with mean rates totaling 150.98 mmol day <->1; 158.06 mmol day <->1, 112.35 g day <->1, 702.18 g day <->1; 146.41 crude protein (CP) microbial kg <->1 of TDN. Partial replacement of soybean meal by sunflower crushed improves nitrogen balance without altering microbial protein synthesis and excretion of urea and creatinine.(AU)

Para se avaliar a suplementação de torta de girassol em novilhos mantidos a pasto, sobre o balanço de nitrogênio e a síntese de proteína microbiana, foram utilizados quatro animais em piquetes com capim Marandu (B. Brizantha), em quadrado latino 4x4. Os suplementos foram fornecidos na quantidade de 6 g kg <->1 de peso vivo dia <->1; constituídos de milho, farelo de soja e mineral, sendo o farelo de soja substituído nas proporções de 0, 20, 40, e 60%. A dieta disponível apresentava em média 6,79; 6,96; 7,10 e 6,87% de nitrogênio, para os níveis de substituição de 0, 20, 40 e 60%. A inclusão da torta de girassol (TG) elevou o N ingerido e o N fecal, proporcionando balanço positivo. A concentração plasmática de ureia dos animais suplementados com TG foi 28,13% inferior aos animais suplementados sem TG. A inclusão de TG não alterou a concentração de alantoína, os derivados de purina, o nitrogênio microbiano, proteína bruta microbiana e eficiência microbiana, apresentando valores médios de 150,98 mmol dia <->1; 158,06 mmol dia <->1; 112,35 g dia <->1; 702,18 g dia <->1; 146,41 proteína bruta microbiana kg <->1 NDT. A substituição parcial do farelo de soja pela torta de girassol melhorou o balanço de nitrogênio, sem alterar a síntese de proteína microbiana e a excreção de ureia e creatinina.(AU)

Nitrogenous compounds balance and microbial protein synthesis in steers supplemented with sunflower crushed in partial replacement of soybean meal / Balanço de compostos nitrogenados e síntese de proteína microbiana em novilhos suplementados com torta de girassol em substituição parcial ao farelo de soja

Four steers in individual paddocks with Marandu grass (B. Brizantha) in 4x4 square design were used to evaluate sunflower crushed supplementation in pasture-grazing animals on nitrogen balance and microbial protein synthesis. Supplements at 6 g kg

Para se avaliar a suplementação de torta de girassol em novilhos mantidos a pasto, sobre o balanço de nitrogênio e a síntese de proteína microbiana, foram utilizados quatro animais em piquetes com capim Marandu (B. Brizantha), em quadrado latino 4x4. Os suplementos foram fornecidos na quantidade de 6 g kg

Substituição de uréia por cloreto de amônio em dietas de bovinos: digestibilidade, síntese de proteína microbiana, parâmetros ruminais e sanguíneos / Replacing urea with ammonium chloride in cattle diets: digestibility, synthesis of microbial protein, and rumen and plasma

Foram utilizados cinco bovinos machos, castrados, da raça Holandesa Preta e Branca, com 450 kg de peso vivo em um delineamento quadrado latino 5 x 5, sendo os tratamentos cinco níveis de substituição, 0, 25, 50, 75 e 100% de ureia por cloreto de amônio, como fontes de nitrogênio não-proteico da dieta. Houve redução linear (p < 0,05) na ingestão e no fluxo fecal, além de aumento na digestibilidade da MS, MO, PB, FDN e EE com a inclusão do cloreto de amônio na dieta. A inclusão de cloreto de amônio na dieta influenciou de forma quadrática (p < 0,05) o pH da urina, aumentou (p < 0,05) o volume urinário assim como a excreção de ácido úrico. Os tratamentos não influenciaram (p > 0,05) a excreção de alantoína e de derivados de purinas na urina, purinas absorvidas, síntese de compostos nitrogenados microbianos e eficiência de síntese de proteína microbiana. Houve queda linear (p < 0,05) no pH do líquido ruminal e N ureico plasmático com a substituição da ureia pelo cloreto de amônio, mas os tratamentos não influenciaram (p > 0,05) a concentração de amônia no rúmen. O cloreto de amônio pode ser utilizado como fonte de NNP para bovinos em níveis de até 1,4% da matéria seca total da dieta.(AU)

Five Holstein steers weighting 450 kg were used in a 5 x 5 Latin square statistical design, where treatments consisted of five replacement levels: 0, 25, 50, 75 and 100% of urea by ammonium chloride, as non-protein nitrogen in the diet. There was a linear decrease (p < 0.05) on intake and fecal flow, as well as a linear increase in apparent digestibility of DM, OM, CP, NDF and EE as result of ammonium chloride inclusion in the diet. There was a quadratic effect (p < 0.05) on urine pH and a linear increase of urine volume and uric acid (p < 0.05) as a result of ammonium chloride use. Treatments did not influence (p > 0.05) daily excretion of allantoin, purine derivatives, absorbed purines, as well as microbial nitrogen compounds and microbial efficiency synthesis. Rumen pH and plasma urea nitrogen decreased linearly (p < 0.05) but treatments did not influence (p > 0.05) ruminal ammonia concentration as urea was replaced by ammonium chloride. Ammonium chloride can be used as a nonprotein nitrogen source in ruminant diets up to level of 1.4% of diet dry matter.(AU)

Substituição de uréia por cloreto de amônio em dietas de bovinos: digestibilidade, síntese de proteína microbiana, parâmetros ruminais e sanguíneos / Replacing urea with ammonium chloride in cattle diets: digestibility, synthesis of microbial protein, and rumen and plasma

Foram utilizados cinco bovinos machos, castrados, da raça Holandesa Preta e Branca, com 450 kg de peso vivo em um delineamento quadrado latino 5 x 5, sendo os tratamentos cinco níveis de substituição, 0, 25, 50, 75 e 100% de ureia por cloreto de amônio, como fontes de nitrogênio não-proteico da dieta. Houve redução linear (p 0,05) a excreção de alantoína e de derivados de purinas na urina, purinas absorvidas, síntese de compostos nitrogenados microbianos e eficiência de síntese de proteína microbiana. Houve queda linear (p 0,05) a concentração de amônia no rúmen. O cloreto de amônio pode ser utilizado como fonte de NNP para bovinos em níveis de até 1,4% da matéria seca total da dieta.

Five Holstein steers weighting 450 kg were used in a 5 x 5 Latin square statistical design, where treatments consisted of five replacement levels: 0, 25, 50, 75 and 100% of urea by ammonium chloride, as non-protein nitrogen in the diet. There was a linear decrease (p 0.05) daily excretion of allantoin, purine derivatives, absorbed purines, as well as microbial nitrogen compounds and microbial efficiency synthesis. Rumen pH and plasma urea nitrogen decreased linearly (p 0.05) ruminal ammonia concentration as urea was replaced by ammonium chloride. Ammonium chloride can be used as a nonprotein nitrogen source in ruminant diets up to level of 1.4% of diet dry matter.