The Kinetics of Urea in the Body after Liver Resection in the Experiment

Материал и методы. Опыты проведены на 45 белых крысах (самках) массой 180—220 г. Резекцию пече ни (РП) проводили под эфирным наркозом, удаляя 15—20% от массы органа. Исследовали содержание мо чевины в биологических жидкостях (артериальная кровь, кровь v. porta, v. hepatica, v. renalis, желчь холедо ха, моча) и тканях висцеральных органов (щитовидная железа, легкие, сердце, печень, почки, селезенка, желудок, кишечник) на 3 и, 7 е и 14 е сутки после РП.


Introduction
One of the end products of protein metabolism in mammals is ammonia.The increase in its concen tration has a toxic effect on cells [1,2] and the whole organism [3,4].One of the main ways of neutraliza tion of ammonia in mammals is its irreversible bind ing through synthesis of urea [5] that occurs in hepa tocytes of the periportal zone of liver lobules [6], where a complete set of enzymes of Krebs Henseleit ornithine cycle [1], including arginase, is found.This enzyme catalyzes the last reaction of the cycle: the hydrolytic cleavage of arginine synthesized therein accompanied with ornithine re synthesis and pro duction of urea [5].Since arginase was detected not only in the liver, but also in other mammalian organs and tissues [7], one can speak of «extra hepatic» syn thesis of urea wherein ammonia neutralization does not take place.Different arginase activity in tissues of mammals [7] allows expecting a different contri bution of each organ to the formation of physiologi cal concentration of urea in the blood.However, it is currently unknown how it happens in reality in the healthy and sick body.The aim of the present study was to investigate the kinetics of urea in the body after liver resection in the experiment.

Materials and Methods
The experiments were performed on 45 albino female rats weighing 180-220 g.Liver resection was performed under ether anesthesia by removing, with an electrotherm, a part of the left lobe that was 15-20% by weight of organ.The animals were divided into 4 series of experiments: se ries 1 -intact animals (norm), series 2, 3 and 4 -the ani mals investigated, respectively, on the 3 rd , 7 th , and 14 th day after LR.The objects of the study were: thyroid, lungs, heart, left and middle lobe of the liver, spleen, stomach, duodenum, colon, kidneys, arterial blood (aorta), venous blood (v.porta, v.hepatica, v.renalis), bile, and urine.Blood sampling was performed in animals previ ously anes Urea content was studied in biological fluids (arterial blood, venous -v.porta, v.hepatica, v.renalis -blood, chole dochal bile, urine), and tissues of visceral organs (the thyroid gland, lungs, heart, liver, kidneys, spleen, stomach, intestine) on days 3, 7, and 14 after LR.
Results.LR, while reducing the urea content in the v. hepatica blood, does not lead to similar changes in the arterial blood.This is accompanied by increased urea re absorption in kidneys and higher v.porta blood urea con tent, which, depending on the postoperative time, results either from reduced urea excretion into the small intes tine lumen or from its greater production by enterocytes followed by metabolite intake into the portal blood flow.The urea intake from hepatocytes into the hepatic bile ducts did not change on day 3 after LR; however, it increased on day 7 and slowed down on day 14.LR caused no changes in the gastric tissues urea content; never theless, it led to its increased content in the duodenal and colonic tissues.Without affecting the cardiac muscle urea content, LR entailed its increase in the lungs and thyroid gland on postoperative days 3, 7, and 14.At the background of absence of similar changes in the arterial blood data indicates promotion of urea production by the cells of these organs or metabolite retention therein.

Conclusions.
LR not only changes urea kinetics in the portal system organs, but also activates extrahepatic mechanisms aimed at preventing development of the arterial blood urea deficit because of its abnormal intake from the resected organ into the central blood flow.
To receive urine, the animal was placed for 2-4 hours in a cage container, and 0.1 ml of 60% trichloroacetic acid was previously added into tubes designated for this pur pose, in order to suppress urease activity of urine.A urine sample for determination of urea was diluted 100 times that was taken into account when calculating the resulting figure.The content of urea in the blood, tissues, bile, and urine were determined by the diacetylation method [8].The content of urea was expressed in mmol/kg wet tissue for tissue, and in mmol/L for biological fluids (blood, bile, urine).The results were processed statistically by Student's t test and Wilcoxon Mann Whitney test.Statistical analysis was performed using a personal com puter with the help of programs «Staistica 5.5» и «Microsoft Exel XP».The differences in the series of experiments were considered significant at P<0.05.

Results and Discussion
As can be seen from table 1, the normal content of urea in the v.hepatica blood significantly exceeds the same indicator in the arterial blood and v.porta blood, respectively, by 25% and 58%.So, hAVD and PVD for urea are negative values, indicating urea incretion by the intact liver into the bloodstream.This is consistent with contemporary ideas of liver as the main organ the synthesis of urea in mammals [1].Unlike v.hepatica blood, the concentration of urea in the common duct bile did not differ significantly from the similar index in v. porta blood, but com pared to arterial and v. hepatica blood it was reduced, respectively, by 19% and 35% (table 1).Taking into account the existence in mammals of the enterohep atic circulation of urea [9] and its ability to tran scend the biological membranes [10], comparison of the results suggests a different nature of urea in v.hepatica the blood and common duct bile.This is indicated by a significant difference between the urea content in the bile of the common bile duct and arterial blood found in healthy rats (table 1).On this basis, we can assume that mainly urea produced by hepatocytes in the Krebs cycle ornithine will be sup plied to v.hepatica blood, whereas mainly urea deliv ночно кишечного кругооборота мочевины [9] и ее способности легко преодолевать биологические мембраны [10], можно говорить о различной при роде мочевины в крови v. hepatica и желчи холедо ха.На это указывает и достоверное различие меж ду содержанием мочевины в желчи холедоха и артериальной крови, выявленное у здоровых крыс (табл.1).Исходя из этого, можно полагать, что в кровь v. hepatica будет поступать преимуществен но мочевина, образованная гепатоцитами в орни тиновом цикле Кребса Хенселяйта, тогда как в желчные капилляры преимущественно выделять ся мочевина, доставленная с кровью v. porta.
As can be seen from the table.1, the concentra tion of urea in the v.porta v and.renalis blood was, respectively, 21% and 23% lower than in the arterial blood, so APD and rAVD of urea were positive val ues.The first reason would be secretion of a part of 'arterial' urea into the gastrointestinal lumen.The second reason is urea excretion from the body with urine, where the concentration of this metabolite, due to the concentrating ability of the renal tubules [11], is 10 times higher than the same indicator in the arterial blood (table 1).
As can be seen from the table 2, in healthy ani mals' tissues of visceral organs studied, the maximum urea concentration was noted in the kidneys, and the minimal -in the thyroid gland.In turn, urea con centration in the tissue of lungs did not reliably dif fered from that of the cardiac tissue: no difference was found in the urea concentration in the healthy animals' liver lobes studied lobes (table 2).In the intestine, urea concentration of in the colon tissue was reliably lower than its content in the walls of the stomach and duodenum, respectively, by 20% and 18% (table 2).Comparison of the results obtained allows speaking about the original difference of urea ki netics in the organs of mammals, which is associ ated not only with different speed of its generation therein, but, most likely, with different permeability of the histohematical barrier of their tis sues for this metabolite.
Если в артериальной крови содержание мо чевины после резекции печени существенно не изменялось, то в крови v. porta она превышала норму на 3 и, 7 е и 14 е сутки послеоперационно го периода, соответственно на 45, 25 и 41% (табл.1).Благодаря этому ПВР по мочевине на 3 и и 14 е сутки исследования становилась положительной величиной (табл.1).С учетом появления у опери рованных животных преобладания концентрации мочевины в v. porta над аналогичным показателем в желчи, можно говорить о ретенции «порталь blood remained was not changing (table 1).Short term normalization of urea concen tration on the 7 th day of the postoperative period was accompanied by its 19% increase in the ar terial blood (table 1).Despite this, hАVD for urea remained unreliable at all observation times (table 1).This indicates dis turbed incretion of urea from the operated liver into the blood flow.In the common duct bile, the urea concentration on day 3 after LR remained within the normal range without reliable difference from the similar indicator in the arterial blood and v. hepatica blood (table 1).A comparison of the results shows that on the 3rd postoperative day, decreased urea incretion from the liver into the blood flow takes place against the background of its intact release by hepatocytes into bile capillaries.On the 7 th day liver resection, flow of urea from its cells into the bile cap illaries increased as evidenced by increase of its con tent in the bile of the common bile duct by 15% (table 1).This was, probably, due to «portal» urea, which is supported by a negative correlation (r=0.88,P<0.05) between the urea content in the bile of the common bile duct and v. porta blood.Meanwhile, a (12%) decline of urea concentration of in the bile by day 14 after liver resection against the background of its increased inflow to the operated organ with v. porta blood (table1) suggests inhibition of urea inflow from hepatocytes into bile capillar ies.
Whereas the arterial blood urea content after liver resection did not significantly changed, it was above normal in v. porta on days 3, 7, and 14 after liver resection, respectively, by 45%, 25%, and 41% (table 1).Due to this, PVD for urea on the 3rd and 14 th days of the study was a positive value (table 1).Taking into account the occurrence in the operated animals of predomi nance of urea concentration in v. porta blood vs. similar figure in the bile, we can speak about 'portal' urea retention in the liver part remain ing after resection.This, along with decreased incre tion of urea from the operated liver into the blood, must lead to its accumulation in the organ part remaining after resection.However, on the 3 rd day after liver resection, the content of urea in the left and middle lobes of the liver was reduced, respec tively, by 11% and 15%, whereas on the 7 th and 14 th days of the study it was within the normal range (table 2).This discrepancy can be ex plained by two factors: firstly, by urea synthesis dysfunction of hepa tocytes, which was detected after liver resection [12]; secondly, by urea drop from the free into the bound state.This process is one of the mechanisms of cell adaptation to the action of an extreme irritant [13] and repre sents urea interaction both with lipoproteins of subcellular organelles' membranes [14] and with enzymes [15], resulting in alteration of the catalytic properties of the latter.
Since liver resection promotes arginase activity of splenocytes [16], a change in the splenic urea con centration had been reasonably expected.However, it remained within the normal limits on postopera tive days 3 and 14 (table 2), which suggests either its increased inflow into v. porta blood, or its transition from the free into the bound state.The latter explains the lack of urea ac cumulation by spleno cytes on the 7th day after liver resection, when its increased content in arte rial blood was noted.Since urea diffusing through a tissue barrier easily alter nates between the free and bound state [17], involve ment of this mechanism in stabilizing urea concen tration in op erated body's splenocytes within the normal range, irrespectively of its content in the arterial blood, cannot be excluded.
Как видно из табл.2, резекция печени не вы зывала достоверных изменений содержания мо чевины в сердечной мышце.В легочной ткани ее концентрация на 3 и, 7 е и 14 е сутки послеопера ционного периода превышала норму, соответст increase the v. renalis blood.This is indica tive of increased reabsorption of the metabolite from renal tubules during the said observation period.On the 7th postoperative day, the rate of urea reabsorption in the kidneys became appar ently normal.This is indicated by the recovery of urea concentration in urine (table 1).Mean while, on the 7th day after liver resection, showed an increase of urea concen tration in arterial blood and v. renalis blood by 19% and 46%, respectively, was established (table 1).This discrep ancy can be explained by promotion of urea production by renal tubules' cells and its further in cretion into the blood flow.It can be reasonably assumed that this mechanism is one of the rea sons for delayed accumulation of urea in kidneys 14 days postoperatively (table 2).In kidneys, mRNA that encodes generation of carbamoyltransferase -I [18] responsible for the involvement of ammonia in the urea synthesis was not found [5].Therefore, despite the presence of arginase in the cells of renal tubules [19], it is possible to speak about urea production of by nephrocytes, which is not accompanied with ammonia neutralization.
As can be seen from table 2, LR did not cause reliable changes in the urea concentration in the heart muscle.In the lung tissue, its concentration on the 3rd, 7th, and 14th postoperative day was above normal by 30%, 29%, and 40%, respectively (table 2).Given the presence of lung tis sue's arginase activity [7], we can speak about stimulation of urea produc tion therein after liver resection.In turn, mapping the dynamics of changes in the concentration of urea in the lungs and arterial blood after liver resection indicates its retention latency in the lung tissue.
Without exerting a significant influence on the concentration of urea in arterial blood (table 1), LR caused its increase in thyroid tissue on the 3rd, 7th, and 14th postoperative day, by 62%, 55%, and 28%, respectively (table 2).The lack in the available sci entific literature of data about the presence of arginase activity in thyrocytes makes one think about distortion of urea diffusion from blood thyro cytes after liver resection as the cause of its accumu lation by the thyroid tissue.It cannot be excluded that urea accumulation in the thyroid gland, along with activation of am monia neutralization reactions therein in the conditions of postoperative hyperam monemia [20], is one of the reasons for reduced hor monal activity of thyrocytes after liver resection that has been detected earlier.

Заключение
Таким образом, резекция здоровой печени изменяет кинетику мочевины в организме.С од synthesis by hepatocytes and reduced inflow of urea from the liver part remaining after resection into the central blood flow.On the other hand, these are func tional and metabolic changes that taking place in organs during adaptation of the body to operative aggression.As a result, not only their permeability of blood tissue interface for urea alters, but the kinetics and production of this metabolite therein, too.This explains the mismatch between the changes of urea concentration in visceral organs and the changes of its arterial blood content discovered in the postoper ative period.
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