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The effect of a nucleotide-nucleosidesolution on hepatic regeneration in rats after partial hepatectomy and in primary monolayer culture of hepatocytes
SYMPOSIUM
Nutrition Vol. 13, No. 4, 1997
PROCEEDING
The Effect of a Nucleotide-Nucleoside Solution on Hepatic Regeneration in Rats After Partial Hepatectomy and in Primary Monolayer Culture of Hepatocytes MAKOTO
USAMI, MD, PHD, AND YOICHI SAITOH, MD, PHD, FACS
From the First Department of Surgery, Kobe University School of Medicine, Kobe, Japan ABSTRACT Purine and pyrimidine metabolism is a key process after hepatic surgery. To evaluate the effect of purine and pyrimidine supplementation on hepatic regeneration, the following clinical in vitro and in vivo experiments were performed. Changes in blood nucleotides, nucleosides and nucleobase were analyzed by high performance liquid chromatography in patients and rats after partial hepatectomy. The effect of supplementation of nucleotide-nucleoside solution (OG-VI) or their components on nucleic acids syntheses in primary monolayer cultured hepatocytes and preoperative intraperitoneal injection of OG-VI on hepatic regeneration in the partially hepatectomized rats were evaluated. Blood purine and pyrimidine levels in patients change after hepatectomy and their changes indicate increased salvage synthesis of pufine and pyrimidine in the regenerating liver. Addition of appropriate amounts of inosine, GMP, uridine, or thymidine, the substrates for salvage purine and pyrimidine syntheses, to primary cultures of hepatocytes enhanced both DNA and RNA syntheses by the salvage and de novo pathways. The OG-VI mixture also enhanced the syntheses of DNA and RNA. Preoperative administration of OG-VI to partially-hepatectomizedrats enhances hepatic DNA synthesis in a way similar to the in vitro study. Nutrition 1997; 13:365-368. ©Elsevier Science Inc. 1997
INTRODUCTION Purine and pyrimidine metabofism is a key process after hepatic surgery and it plays an important role in the syntheses of DNA and RNA and in energy metabolism. The pool changes of purine and pyrimidine during hepatic regeneration are reported in rats 1-3 and the effect of their supplementation on hepatic regeneration has, consequently, been expected. A solution containing a mixture of nucleosides and nucleotide (OG-VI) has recently been developed as a nutritional supplement.4 Intravenous administration of OG-VI with total parenteral nutrition (TPN) solution after partial hepatectomy increases protein synthesis and turnover rates, and improves nitrogen balance in the rat model.4'5 However, the improvement of hepatic regeneration by nucleoside supplementation has not been reported. Since hepatocellular carcinoma is seen in cirrhotic rivers, and the mortality and morbidity after cirrhotic river resection are high, it is important to evaluate the effect of a nucleosides-nucleotide mixture on a cirrhosis model in an effort to control the metabolic derangement after hepatectomy. The purpose of this study is to evaluate (1) liver ATP content and DNA synthesis in rats after partial hepatectomy and blood purine and pyrimidine levels in hepatectomized patients
to assess their pool changes; (2) the effect of nucleoside and nucleotide supplementation on DNA and RNA syntheses in primary-cultured rat hepatocytes; (3) the effect of preoperative OG-VI administration on hepatic ATP level and hepatic regeneration in normal rats after partial hepatectomy; and (4) the effect of continuous postoperative administration of OG-VI with TPN on liver nucleic acid content, protein fractional synthesis rate, and nitrogen balance in cirrhotic rats after 70% partial hepatectomy. MATERIALS AND METHODS
Analysis of Purine and Pyrimidine After Partial Hepatectomy6 Blood and urine levels of purine and pyrimidine nucleotides, nucleosides, and nucleobases were measured in 15 hepatectomized patients, 7 cases with and 8 cases without cirrhotic liver, in comparison with 19 nonhepatectomized patients, and in male Wistar rats after 70% partial hepatectomy by Higgins and Anderson's method using high-performance liquid chromatography. 6 ATP in the rat liver was assessed by enzymatic analysis.
Correspondence to: Makoto Usami, MD, First Department of Surgery, Kobe University School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe, 650 Japan.
Nutrition 13:365-368, 1997 ©Elsevier Science Inc. •997 Printed in the USA. All rights reserved.
ELSEVIER
0899-9007/97/$17.00 PII S0899-9007(96)00374-7
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THE EFFECT OF NUCLEOTIDE-NUCLEOSIDE SOLUTION ON HEPATIC REGENERATION TABLE I.
EFFECTS OF NUCLEOSIDES AND A NUCLEOTIDE ON DNA AND RNA SYNTHESES BY THE SALVAGE PATHWAY IN PRIMARY MONOLAYER CULTURES OF HEPATOCYTES
Inosine GMP Cytidine Uridine Thymidine OG-VI
DNA Synthesis
RNA Synthesis
3-30 ¢zM --* 30 #M I" 3-30 #M ~ 23 #M "f 7.4/zM "t 3.35 mg/dL ?
300 #M "~ 300/zM 1' 3 #M $ 230 #M "~ 740 #M t 33.5 mg/dL 1"
t, increase; ~, No change; $, decrease. GMP, guanosine 5'monophosphate; OG-VI, nucleosides/nucleotidemixture.
Primary Monolayer Cultures of Hepatocytes z
plasma was <2% of blood purine nucleotide levels in patients and rats. Plasma xanthine and xanthine/hypoxanthine levels of cirrhotic patients were higher before operation (P < 0.01 ).
Blood Purine, Pyrimidine, and Uric Acid Levels After the Partial Hepatectomy The levels of plasma xanthine, hypoxanthine, and uric acid, catabolic products of purine nucleosides, decreased on POD 1 and recovered by POD 3 - 7 in the hepatectomized group (P < 0.05). The ratio of xanthine to hypoxanthine in the plasma increased on POD 1, decreased to its preoperative level by POD 3, and then maintained a constant level in the hepatectomized group, but the ratio did not change in the nonhepatectomized group (P < 0.05-0.01). Urinary excretion of xanthine and hypoxanthine in the hepatectomized group was less. The plasma uridine level in the hepatectomized group decreased more than that in the nonhepatectomized group (P < 0.05). Rats after partial hepatectomy showed the same changes in the plasma xanthine/hypoxanthine ratio as hepatectomized patients.
Inoculum of 3 × 10 4 hepatocytes, cm -z. mL -1 was introduced into tissue culture dishes containing Williams E medium with 5% fetal calf serum and hepatotrophic factors; 10 -9 M insulin, 10 -6 M dexamethasone, and 20 ng/mL of epidermal growth factor. Inosine, guanosine 5'-monophosphate (GMP), cytidine, uridine, thymidine, or their mixture (4:4:4:3:1, OGVI) was added at various concentrations. DNA and RNA syntheses by the de novo pathway were measured by 3H orotic acid incorporation into DNA and RNA fraction and those by the salvage pathway were measured by 3H thymidine and 3H uridine incorporation, respectively. Uptake of nucleotide and nucleosides in the medium was evaluated by measuring their concentrations before and after cultivation.7
DNA and RNA Syntheses in Cultured Hepatocytes
Partial Hepatectomy and Assessment of Energy Metabolism, Hepatic Regeneration, and Protein Synthesis 8'9
Plasma levels of pyrimidine nucleosides were significantly higher in the OG group (P < 0.01 ) but no difference was noted in blood purine nucleotide levels at 2 h after administration of OG-VI. Plasma pyrimidine nucleoside levels after partial hepatectomy in the OG group showed almost the same changes as in the control group. Hepatic ATP content at 5 h after hepatectomy in the OG group showed a higher level than in the control group. The LI of the hepatocytes on POD 1 was significantly higher in the OG group, indicating increased DNA synthesis (P < 0.01, Table II). The hepatic regeneration ratio on POD 14 also
OG-VI (10 mL-kg - l . d -1) or normal saline was administered intraperitoneally two times, 24 h and 2 h prior to hepatectomy in the OG and the S groups who were fed chow ad libitum. Liver ATP content and hepatic regeneration ratio was measured. Hepatocyte DNA synthesis was assessed by the labeling index (LI) of histoimmunochemical staining of 5bromo-deoxyuridine in the remnant liver nuclei 24 h after 70% hepatectomy. Liver cirrhosis was induced by repeated intraperitoneal injection of thioacetamide in rats.l° Postoperative continuous administration of 0.35 mL. kg -~. d -1 of OG-VI with standard TPN consisting of hypertonic glucose plus an amino acid mixture was performed for 7 d in cirrhotic rats after 70% partial hepatectomy in the metabolic cage. 9 Liver nucleic acid content, protein fractional synthesis rate by 24 h infusion of 15N-glycine, and nitrogen balance were measured.
Addition of an appropriate amount of inosine, GMP, uridine, or thymidine enhanced both DNA and RNA syntheses by the salvage and the de novo pathways. Cytidine appeared to have lower optimal concentration for enhancing their syntheses. The OG-VI mixture also enhanced hepatocytes DNA and RNA syntheses (Table I). Uptake of GMP, inosine, uridine, and thymidine in the medium was observed, but cytidine was not. Most of the purine, inosine, and GMP were metabolized during a 24-h culture; hypoxanthine and xanthine were then produced.
Effect of OG-VI Administration on Hepatic Regeneration and on Purine and Pyrimidine Level
TABLE II. EFFECT OF PREOPERATIVE OG-VI ADMINISTRATION ON HEPATIC REGENERATION AFTER PARTIAL HEPATECTOMY IN RATS (%)
RESULTS
Liver DNA Synthesis and ATP Level After the Partial Hepatectomy Hepatic DNA synthesis showed a sharp peak at postoperative day (POD) 1, the LI was about 30%. The ATP level in the liver decreased at PODs 1-3 (P < 0.05-0.01) and recovered gradually at POD 14.
Distribution of Purine and Pyrimidine in the Blood Before Treatment Purine nucleotides in the blood cells formed a majority in blood purine and pyrimidine nucleotides, nucleosides, and nucleobases. Total amount of pyrimidine nucleosides in the
Hepatic ATP level at 5 h Hepatocyte labeling index at POD 1 Hepatic regeneration ratio at POD 14
n
OG Group
n
S Group
5
36.0 +_ 3.8*
4
25.5 _+ 3.5
7
185 + 22?
7
160 ___13
OG group and S group received 10 mL/kg of OG-VI or normal saline, respectively, before partial hepatectomy. Values are number of rats and mean _+ SD, * P < 0.01; t P < 0.025 versus S group; Student's t test; OG, OG-VI, nucleosides/nucleotidemixture; POD, postoperative day; S, normal saline.
THE EFFECT OF NUCLEOTIDE-NUCLEOSIDE SOLUTION ON HEPATIC REGENERATION showed augmented hepatic regeneration under OG-VI administration (P < 0.025). Liver RNA content and protein fractional synthesis rate in the OG group were higher than in the control group, but the DNA level was the same. Nitrogen balance in the control group was negative until POD 3, but in the OG group showed a positive value at POD 2 with statistical significance. DISCUSSION Increased DNA synthesis and decreased ATP content of the rat liver after 70% partial hepatectomy, the standard experimental procedure for liver regeneration, indicate the importance of nucleotide in the syntheses of nucleic acids and in energy metabolism.l-3 Decreased blood ATP level and increased ratio of plasma xanthine to hypoxanthine on POD 1 in patients also indicate rapid changes in ATP metabolism after hepatectomy in response to various surgical stresses and metabolic overload, including hepatic regeneration, the operative procedure itself, tissue damage, ischemia, reperfusion, and blood transfusion. 8'm 12 An increase in the xanthine/hypoxanthine ratio is thought to reflect either increased xanthine oxidase activity or decreased hypoxanthine level. Increases in both xanthine and uric acid level suspect increased xanthine oxidase activity. However, Kurokawa et al.'s 11 study shows that there is minimal change in xanthine oxidase activity in normal rats after 70% hepatectomy. From these results, utilization of plasma hypoxanthine by the salvage synthetic pathway of purine nucleotides is highly suspected. These changes of purines and pyrimidines of the liver and blood after partial hepatectomy in patients and rats indicate augmented utilization and catabolism of purine and pyrimidine nucleotides in the regenerating liver and requirement of substrate for salvage nucleotide synthesis. Studies of radio-labeled pyrimidine nucleoside uptake show similar results: an increased uptake of orotic acid, uridine, and thymidine into the intracellular acid soluble fraction at 0.5-1 hi; an increase in the purine nucleotide pool at 3 h2; and an increase in the uridine level in the remnant liver at 6 h after partial hepatectomy.3 These observations indicate the possibility of nucleotide and nucleoside supplementation to increase the size of the purine and pyrimidine pool to improve hepatic regeneration after partial hepatectomy, m3 Primary hepatocyte culture in the in vitro system reproduces in vivo hepatic regeneration without various responses in extrahepatic organs after operation. However, de novo nucleotide synthesis without nucleotides, nucleosides, and their catabolites is the mechanism of cellular proliferation in a usual cell culture system. Purine and pyrimidine supplementation in the medium has been studied in combination with cancer chemotherapy using 5 ' fluorouracil, the fluorinated analogue of uracil, and cancer cells 14 but has not been reported in primary hepatocyte culture. In this study, uptake of a nucleotide and nucleosides in the medium and excretion of hypoxanthine and xanthine clarified that GMP, inosine, uridine, and thymidine were metabolized by regenerating hepatocytes themselves] Enhancement of both DNA and RNA syntheses by the salvage pathway as a result of the addition of a nucleotide and nucleosides and better result by a mixture of the five compounds, OG-VI, indicates augmentation of synthesis of DNA and RNA with the optimal
367
concentration in the range of 10 6-10 7 M. The results of this synthesis suggest the effectiveness of a nucleotide and nucleosides in enhancing nucleic acid metabolism when they are present in suitable concentrations. Surprisingly, the profile of DNA and RNA syntheses by the de novo pathways showed a similar augmentation to those by the salvage pathway. It is suggested that nucleotide and nucleoside supplementation have the unknown pharmacologic effect of enhancing DNA synthesis by the de novo synthesis pathway in addition to the supplementation of substrates for nucleotide syntheses. In the third study, we examined whether a substrate for nucleotide synthesis, exogenously administered to well-nourished rats, promotes hepatic regeneration) Improvement of LI and regenerating ratio of the remnant liver in the OG group indicates the enhancement effect of preoperative OG-VI administration in liver regeneration after partial hepatectomy in the same way the in vitro studies d o ) Indicated mechanisms for facilitation of hepatic regeneration are as follows: increased levels of plasma pyrimidine nucleosides before hepatectomy indicate increased pyrimidine nucleoside pool size, and an increased ATP level in the remnant liver shortly after operation improved energy metabolism. These metabolic effects augmented pleiotypic responses of hepatic regeneration in combination with the direct effects on nucleic acid syntheses, then improved DNA synthesis and hepatic regeneration. In comparison with other hepatotrophic factors, OG-VI does not increase liver ATP level nor DNA synthesis without partial hepatectomy. This is in agreement with Yamaguchi et al.'s 15 report showing that liver ATP level is not increased without nutrients with OG-VI suggesting the complex mechanism involved in improving energy metabolism. Depressed energy metabolism and depressed DNA synthesis in cirrhotic patients after hepatic surgery is a well-established observation and our experimental cirrhosis rats indicate the same change. 1° Increased hepatic RNA content facilitates protein synthesis and improved nitrogen balance in cirrhotic rats after partial hepatectomy under intravenous administration of OG-VI with TPN. These results in cirrhotic rats are comparable to Ogoshi et al.'s 4'5 report using normal rats. The OG-VI solution is an effective nutritional supplement to the metabolism of cirrhotic rats after partial hepatectomy. CONCLUSION Blood purine and pyrimidine levels in patients change after hepatectomy and these changes indicate increased salvage synthesis of purine and pyfimidine in the regenerating liver. A significant decrease in hypoxanthine and an increase in the ratio of xanthine/ hypoxanthine indicate increased requirement of nucleotide after hepatectomy. Addition of an appropriate amount of inosine, GMP, uridine, or thymidine to primary cultures of hepatocytes enhanced both DNA and RNA syntheses by the salvage and de novo pathways. The OG-VI mixture also enhanced the syntheses of DNA and RNA. Preoperative administration of OG-VI to partially hepatectomized rats enhances hepatic DNA synthesis in a way similar to the in vitro study. Postoperative intravenous administration with TPN in cirrhotic rats increased hepatic RNA levels and improved protein metabolism.
REFERENCES
1. Ord MG, Stocken LA. Uptake of amino acids and nucleic acid precursors by regenerating rat liver. Biochem J 1972; 129:175 2. Bucher NLR, Swaffield MN. Ribonucleic acid synthesis in relation to precursor pools in regenerating rat liver. Biochem Biophys Acta 1969; 174:49t 3. Yngner T, Engelbrecht C, Lewan L, et al. Anabolism versus catabolism of (5-3H) Uridine and its relationship to ribonucleic acid
labeling in mouse liver after partial hepatectomy. Biochem J 1979; 178:1 4. Ogoshi S, Iwasa M, Yonezawa T, et al. Effect of nucleotide and nucleoside mixture on rats given total parenteral nutrition after 70% hepatectomy. J Paren Ent Nutr 1985;9:339 5. Ogoshi S, Iwasa M, Mizobuchi S, et al. Effect of nucleoside and nucleotide mixture on protein metabolism in rats given total paren-
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6.
7.
8. 9.
T H E E F F E C T OF N U C L E O T I D E - N U C L E O S I D E teral nutrition after 70% hepatectomy. In: Tanaka T, Okada A, eds. Nutritional support in organ failure. Amsterdam: Elsevier Science, 1990:309 Usami M, Furuchi K, Kasahara H, et al. The effect of partial hepatectomy on blood purine levels in rats and patients. In: Sahota A, Taylor M, eds. Purine and pyrimidine metabolism in man VIII. New York: Plenum Press, 1995:11 Ohyanagi H, Nishimatsu S, Kambara Y, et al. Effect of nucleosides and a nucleotide on DNA and RNA syntheses by the salvage and de novo pathway in primary monolayer cultures of hepatocytes and hepatoma cells. J Paren Ent Nutr 1989; 13:51 Usami M, Furuchi K, Ogino M, et al. The effect of a nucleotidenucleoside solution on hepatic regeneration after partial hepatectomy in rats. Nutrition 1996;12:797 Usami M, Iso A, Kasahara H, et al. The effect of a parenteral nucleoside-nucleotide mixture on hepatic metabolism in partially hepatectomized cirrhotic rats. Nutrition 1996; 12:436
S O L U T I O N ON H E P A T I C R E G E N E R A T I O N
10. Usami M, Fumchi K, Shiroiwa H, et al. Effect of repeated portaltriad cross-clamping during hepatectomy on hepatic regeneration in normal and cirrhotic rats. J Surg Res 1994;57:541 11. Kurokawa T, Nonami T, Sugiyama S, et al. Effect of long acting superoxide dismutase on liver metabolism after major hepatic resection in rats with cirrhosis. Eur Surg Res 199i;23:65 12. Itakura M, Maeda N, Tsuchiya M, et al. Increased rate of de novo purine synthesis and its mechanism in regenerating liver. Am J Physiol 1986;251:G585 13. Moyer JD, Oliver JT, Handschumacher R. Salvage of circulating pyrimidine nucleosides in the rat. Cancer Research 1981;41:3010 14. Wang J, Usami M, Yasuda I, et al. Effect of nucleosides and a nucleotide mixture on proliferation of human gastric cancer cells (KATO Ill). Kobe J Med Science 1994;40:65 15. Yamaguchi T, Takeda Y, Shimahara Y, et al. A mixture of nucleosides and a nucleotide alters hepatic energy metabolism 34 hours after hepatectomy in rabbits. J Nutr 1992; 122:340
ANNOUNCEMENT The 34th Annual Congress of the Japanese Society for Surgical Metabolism and Nutrition July 9-11, 1997 The Westin Tokyo, Tokyo, Japan International Symposium: "Advances in Metabolic and Nutritional Support for Trauma and Sepsis"
Official Language: English Free Papers and Educational Lectures
Official Language: Japanese~English Contact: Masaharu Hasebe, MD Congress Secretariat Trauma and Critical Care Center Teikyo University School of Medicine 2-11-1 Kaga, Itabashi-ku, Tokyo 173, Japan Tel: +81 3 3964-1211 ext. 3319 Fax: +81 3 5375-3120 E-maih dangan @ppp.bekkoame.or.jp
Nutrition Vol. 13, No. 4, 1997
PROCEEDING
The Effect of a Nucleotide-Nucleoside Solution on Hepatic Regeneration in Rats After Partial Hepatectomy and in Primary Monolayer Culture of Hepatocytes MAKOTO
USAMI, MD, PHD, AND YOICHI SAITOH, MD, PHD, FACS
From the First Department of Surgery, Kobe University School of Medicine, Kobe, Japan ABSTRACT Purine and pyrimidine metabolism is a key process after hepatic surgery. To evaluate the effect of purine and pyrimidine supplementation on hepatic regeneration, the following clinical in vitro and in vivo experiments were performed. Changes in blood nucleotides, nucleosides and nucleobase were analyzed by high performance liquid chromatography in patients and rats after partial hepatectomy. The effect of supplementation of nucleotide-nucleoside solution (OG-VI) or their components on nucleic acids syntheses in primary monolayer cultured hepatocytes and preoperative intraperitoneal injection of OG-VI on hepatic regeneration in the partially hepatectomized rats were evaluated. Blood purine and pyrimidine levels in patients change after hepatectomy and their changes indicate increased salvage synthesis of pufine and pyrimidine in the regenerating liver. Addition of appropriate amounts of inosine, GMP, uridine, or thymidine, the substrates for salvage purine and pyrimidine syntheses, to primary cultures of hepatocytes enhanced both DNA and RNA syntheses by the salvage and de novo pathways. The OG-VI mixture also enhanced the syntheses of DNA and RNA. Preoperative administration of OG-VI to partially-hepatectomizedrats enhances hepatic DNA synthesis in a way similar to the in vitro study. Nutrition 1997; 13:365-368. ©Elsevier Science Inc. 1997
INTRODUCTION Purine and pyrimidine metabofism is a key process after hepatic surgery and it plays an important role in the syntheses of DNA and RNA and in energy metabolism. The pool changes of purine and pyrimidine during hepatic regeneration are reported in rats 1-3 and the effect of their supplementation on hepatic regeneration has, consequently, been expected. A solution containing a mixture of nucleosides and nucleotide (OG-VI) has recently been developed as a nutritional supplement.4 Intravenous administration of OG-VI with total parenteral nutrition (TPN) solution after partial hepatectomy increases protein synthesis and turnover rates, and improves nitrogen balance in the rat model.4'5 However, the improvement of hepatic regeneration by nucleoside supplementation has not been reported. Since hepatocellular carcinoma is seen in cirrhotic rivers, and the mortality and morbidity after cirrhotic river resection are high, it is important to evaluate the effect of a nucleosides-nucleotide mixture on a cirrhosis model in an effort to control the metabolic derangement after hepatectomy. The purpose of this study is to evaluate (1) liver ATP content and DNA synthesis in rats after partial hepatectomy and blood purine and pyrimidine levels in hepatectomized patients
to assess their pool changes; (2) the effect of nucleoside and nucleotide supplementation on DNA and RNA syntheses in primary-cultured rat hepatocytes; (3) the effect of preoperative OG-VI administration on hepatic ATP level and hepatic regeneration in normal rats after partial hepatectomy; and (4) the effect of continuous postoperative administration of OG-VI with TPN on liver nucleic acid content, protein fractional synthesis rate, and nitrogen balance in cirrhotic rats after 70% partial hepatectomy. MATERIALS AND METHODS
Analysis of Purine and Pyrimidine After Partial Hepatectomy6 Blood and urine levels of purine and pyrimidine nucleotides, nucleosides, and nucleobases were measured in 15 hepatectomized patients, 7 cases with and 8 cases without cirrhotic liver, in comparison with 19 nonhepatectomized patients, and in male Wistar rats after 70% partial hepatectomy by Higgins and Anderson's method using high-performance liquid chromatography. 6 ATP in the rat liver was assessed by enzymatic analysis.
Correspondence to: Makoto Usami, MD, First Department of Surgery, Kobe University School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe, 650 Japan.
Nutrition 13:365-368, 1997 ©Elsevier Science Inc. •997 Printed in the USA. All rights reserved.
ELSEVIER
0899-9007/97/$17.00 PII S0899-9007(96)00374-7
366
THE EFFECT OF NUCLEOTIDE-NUCLEOSIDE SOLUTION ON HEPATIC REGENERATION TABLE I.
EFFECTS OF NUCLEOSIDES AND A NUCLEOTIDE ON DNA AND RNA SYNTHESES BY THE SALVAGE PATHWAY IN PRIMARY MONOLAYER CULTURES OF HEPATOCYTES
Inosine GMP Cytidine Uridine Thymidine OG-VI
DNA Synthesis
RNA Synthesis
3-30 ¢zM --* 30 #M I" 3-30 #M ~ 23 #M "f 7.4/zM "t 3.35 mg/dL ?
300 #M "~ 300/zM 1' 3 #M $ 230 #M "~ 740 #M t 33.5 mg/dL 1"
t, increase; ~, No change; $, decrease. GMP, guanosine 5'monophosphate; OG-VI, nucleosides/nucleotidemixture.
Primary Monolayer Cultures of Hepatocytes z
plasma was <2% of blood purine nucleotide levels in patients and rats. Plasma xanthine and xanthine/hypoxanthine levels of cirrhotic patients were higher before operation (P < 0.01 ).
Blood Purine, Pyrimidine, and Uric Acid Levels After the Partial Hepatectomy The levels of plasma xanthine, hypoxanthine, and uric acid, catabolic products of purine nucleosides, decreased on POD 1 and recovered by POD 3 - 7 in the hepatectomized group (P < 0.05). The ratio of xanthine to hypoxanthine in the plasma increased on POD 1, decreased to its preoperative level by POD 3, and then maintained a constant level in the hepatectomized group, but the ratio did not change in the nonhepatectomized group (P < 0.05-0.01). Urinary excretion of xanthine and hypoxanthine in the hepatectomized group was less. The plasma uridine level in the hepatectomized group decreased more than that in the nonhepatectomized group (P < 0.05). Rats after partial hepatectomy showed the same changes in the plasma xanthine/hypoxanthine ratio as hepatectomized patients.
Inoculum of 3 × 10 4 hepatocytes, cm -z. mL -1 was introduced into tissue culture dishes containing Williams E medium with 5% fetal calf serum and hepatotrophic factors; 10 -9 M insulin, 10 -6 M dexamethasone, and 20 ng/mL of epidermal growth factor. Inosine, guanosine 5'-monophosphate (GMP), cytidine, uridine, thymidine, or their mixture (4:4:4:3:1, OGVI) was added at various concentrations. DNA and RNA syntheses by the de novo pathway were measured by 3H orotic acid incorporation into DNA and RNA fraction and those by the salvage pathway were measured by 3H thymidine and 3H uridine incorporation, respectively. Uptake of nucleotide and nucleosides in the medium was evaluated by measuring their concentrations before and after cultivation.7
DNA and RNA Syntheses in Cultured Hepatocytes
Partial Hepatectomy and Assessment of Energy Metabolism, Hepatic Regeneration, and Protein Synthesis 8'9
Plasma levels of pyrimidine nucleosides were significantly higher in the OG group (P < 0.01 ) but no difference was noted in blood purine nucleotide levels at 2 h after administration of OG-VI. Plasma pyrimidine nucleoside levels after partial hepatectomy in the OG group showed almost the same changes as in the control group. Hepatic ATP content at 5 h after hepatectomy in the OG group showed a higher level than in the control group. The LI of the hepatocytes on POD 1 was significantly higher in the OG group, indicating increased DNA synthesis (P < 0.01, Table II). The hepatic regeneration ratio on POD 14 also
OG-VI (10 mL-kg - l . d -1) or normal saline was administered intraperitoneally two times, 24 h and 2 h prior to hepatectomy in the OG and the S groups who were fed chow ad libitum. Liver ATP content and hepatic regeneration ratio was measured. Hepatocyte DNA synthesis was assessed by the labeling index (LI) of histoimmunochemical staining of 5bromo-deoxyuridine in the remnant liver nuclei 24 h after 70% hepatectomy. Liver cirrhosis was induced by repeated intraperitoneal injection of thioacetamide in rats.l° Postoperative continuous administration of 0.35 mL. kg -~. d -1 of OG-VI with standard TPN consisting of hypertonic glucose plus an amino acid mixture was performed for 7 d in cirrhotic rats after 70% partial hepatectomy in the metabolic cage. 9 Liver nucleic acid content, protein fractional synthesis rate by 24 h infusion of 15N-glycine, and nitrogen balance were measured.
Addition of an appropriate amount of inosine, GMP, uridine, or thymidine enhanced both DNA and RNA syntheses by the salvage and the de novo pathways. Cytidine appeared to have lower optimal concentration for enhancing their syntheses. The OG-VI mixture also enhanced hepatocytes DNA and RNA syntheses (Table I). Uptake of GMP, inosine, uridine, and thymidine in the medium was observed, but cytidine was not. Most of the purine, inosine, and GMP were metabolized during a 24-h culture; hypoxanthine and xanthine were then produced.
Effect of OG-VI Administration on Hepatic Regeneration and on Purine and Pyrimidine Level
TABLE II. EFFECT OF PREOPERATIVE OG-VI ADMINISTRATION ON HEPATIC REGENERATION AFTER PARTIAL HEPATECTOMY IN RATS (%)
RESULTS
Liver DNA Synthesis and ATP Level After the Partial Hepatectomy Hepatic DNA synthesis showed a sharp peak at postoperative day (POD) 1, the LI was about 30%. The ATP level in the liver decreased at PODs 1-3 (P < 0.05-0.01) and recovered gradually at POD 14.
Distribution of Purine and Pyrimidine in the Blood Before Treatment Purine nucleotides in the blood cells formed a majority in blood purine and pyrimidine nucleotides, nucleosides, and nucleobases. Total amount of pyrimidine nucleosides in the
Hepatic ATP level at 5 h Hepatocyte labeling index at POD 1 Hepatic regeneration ratio at POD 14
n
OG Group
n
S Group
5
36.0 +_ 3.8*
4
25.5 _+ 3.5
7
185 + 22?
7
160 ___13
OG group and S group received 10 mL/kg of OG-VI or normal saline, respectively, before partial hepatectomy. Values are number of rats and mean _+ SD, * P < 0.01; t P < 0.025 versus S group; Student's t test; OG, OG-VI, nucleosides/nucleotidemixture; POD, postoperative day; S, normal saline.
THE EFFECT OF NUCLEOTIDE-NUCLEOSIDE SOLUTION ON HEPATIC REGENERATION showed augmented hepatic regeneration under OG-VI administration (P < 0.025). Liver RNA content and protein fractional synthesis rate in the OG group were higher than in the control group, but the DNA level was the same. Nitrogen balance in the control group was negative until POD 3, but in the OG group showed a positive value at POD 2 with statistical significance. DISCUSSION Increased DNA synthesis and decreased ATP content of the rat liver after 70% partial hepatectomy, the standard experimental procedure for liver regeneration, indicate the importance of nucleotide in the syntheses of nucleic acids and in energy metabolism.l-3 Decreased blood ATP level and increased ratio of plasma xanthine to hypoxanthine on POD 1 in patients also indicate rapid changes in ATP metabolism after hepatectomy in response to various surgical stresses and metabolic overload, including hepatic regeneration, the operative procedure itself, tissue damage, ischemia, reperfusion, and blood transfusion. 8'm 12 An increase in the xanthine/hypoxanthine ratio is thought to reflect either increased xanthine oxidase activity or decreased hypoxanthine level. Increases in both xanthine and uric acid level suspect increased xanthine oxidase activity. However, Kurokawa et al.'s 11 study shows that there is minimal change in xanthine oxidase activity in normal rats after 70% hepatectomy. From these results, utilization of plasma hypoxanthine by the salvage synthetic pathway of purine nucleotides is highly suspected. These changes of purines and pyrimidines of the liver and blood after partial hepatectomy in patients and rats indicate augmented utilization and catabolism of purine and pyrimidine nucleotides in the regenerating liver and requirement of substrate for salvage nucleotide synthesis. Studies of radio-labeled pyrimidine nucleoside uptake show similar results: an increased uptake of orotic acid, uridine, and thymidine into the intracellular acid soluble fraction at 0.5-1 hi; an increase in the purine nucleotide pool at 3 h2; and an increase in the uridine level in the remnant liver at 6 h after partial hepatectomy.3 These observations indicate the possibility of nucleotide and nucleoside supplementation to increase the size of the purine and pyrimidine pool to improve hepatic regeneration after partial hepatectomy, m3 Primary hepatocyte culture in the in vitro system reproduces in vivo hepatic regeneration without various responses in extrahepatic organs after operation. However, de novo nucleotide synthesis without nucleotides, nucleosides, and their catabolites is the mechanism of cellular proliferation in a usual cell culture system. Purine and pyrimidine supplementation in the medium has been studied in combination with cancer chemotherapy using 5 ' fluorouracil, the fluorinated analogue of uracil, and cancer cells 14 but has not been reported in primary hepatocyte culture. In this study, uptake of a nucleotide and nucleosides in the medium and excretion of hypoxanthine and xanthine clarified that GMP, inosine, uridine, and thymidine were metabolized by regenerating hepatocytes themselves] Enhancement of both DNA and RNA syntheses by the salvage pathway as a result of the addition of a nucleotide and nucleosides and better result by a mixture of the five compounds, OG-VI, indicates augmentation of synthesis of DNA and RNA with the optimal
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concentration in the range of 10 6-10 7 M. The results of this synthesis suggest the effectiveness of a nucleotide and nucleosides in enhancing nucleic acid metabolism when they are present in suitable concentrations. Surprisingly, the profile of DNA and RNA syntheses by the de novo pathways showed a similar augmentation to those by the salvage pathway. It is suggested that nucleotide and nucleoside supplementation have the unknown pharmacologic effect of enhancing DNA synthesis by the de novo synthesis pathway in addition to the supplementation of substrates for nucleotide syntheses. In the third study, we examined whether a substrate for nucleotide synthesis, exogenously administered to well-nourished rats, promotes hepatic regeneration) Improvement of LI and regenerating ratio of the remnant liver in the OG group indicates the enhancement effect of preoperative OG-VI administration in liver regeneration after partial hepatectomy in the same way the in vitro studies d o ) Indicated mechanisms for facilitation of hepatic regeneration are as follows: increased levels of plasma pyrimidine nucleosides before hepatectomy indicate increased pyrimidine nucleoside pool size, and an increased ATP level in the remnant liver shortly after operation improved energy metabolism. These metabolic effects augmented pleiotypic responses of hepatic regeneration in combination with the direct effects on nucleic acid syntheses, then improved DNA synthesis and hepatic regeneration. In comparison with other hepatotrophic factors, OG-VI does not increase liver ATP level nor DNA synthesis without partial hepatectomy. This is in agreement with Yamaguchi et al.'s 15 report showing that liver ATP level is not increased without nutrients with OG-VI suggesting the complex mechanism involved in improving energy metabolism. Depressed energy metabolism and depressed DNA synthesis in cirrhotic patients after hepatic surgery is a well-established observation and our experimental cirrhosis rats indicate the same change. 1° Increased hepatic RNA content facilitates protein synthesis and improved nitrogen balance in cirrhotic rats after partial hepatectomy under intravenous administration of OG-VI with TPN. These results in cirrhotic rats are comparable to Ogoshi et al.'s 4'5 report using normal rats. The OG-VI solution is an effective nutritional supplement to the metabolism of cirrhotic rats after partial hepatectomy. CONCLUSION Blood purine and pyrimidine levels in patients change after hepatectomy and these changes indicate increased salvage synthesis of purine and pyfimidine in the regenerating liver. A significant decrease in hypoxanthine and an increase in the ratio of xanthine/ hypoxanthine indicate increased requirement of nucleotide after hepatectomy. Addition of an appropriate amount of inosine, GMP, uridine, or thymidine to primary cultures of hepatocytes enhanced both DNA and RNA syntheses by the salvage and de novo pathways. The OG-VI mixture also enhanced the syntheses of DNA and RNA. Preoperative administration of OG-VI to partially hepatectomized rats enhances hepatic DNA synthesis in a way similar to the in vitro study. Postoperative intravenous administration with TPN in cirrhotic rats increased hepatic RNA levels and improved protein metabolism.
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10. Usami M, Fumchi K, Shiroiwa H, et al. Effect of repeated portaltriad cross-clamping during hepatectomy on hepatic regeneration in normal and cirrhotic rats. J Surg Res 1994;57:541 11. Kurokawa T, Nonami T, Sugiyama S, et al. Effect of long acting superoxide dismutase on liver metabolism after major hepatic resection in rats with cirrhosis. Eur Surg Res 199i;23:65 12. Itakura M, Maeda N, Tsuchiya M, et al. Increased rate of de novo purine synthesis and its mechanism in regenerating liver. Am J Physiol 1986;251:G585 13. Moyer JD, Oliver JT, Handschumacher R. Salvage of circulating pyrimidine nucleosides in the rat. Cancer Research 1981;41:3010 14. Wang J, Usami M, Yasuda I, et al. Effect of nucleosides and a nucleotide mixture on proliferation of human gastric cancer cells (KATO Ill). Kobe J Med Science 1994;40:65 15. Yamaguchi T, Takeda Y, Shimahara Y, et al. A mixture of nucleosides and a nucleotide alters hepatic energy metabolism 34 hours after hepatectomy in rabbits. J Nutr 1992; 122:340
ANNOUNCEMENT The 34th Annual Congress of the Japanese Society for Surgical Metabolism and Nutrition July 9-11, 1997 The Westin Tokyo, Tokyo, Japan International Symposium: "Advances in Metabolic and Nutritional Support for Trauma and Sepsis"
Official Language: English Free Papers and Educational Lectures
Official Language: Japanese~English Contact: Masaharu Hasebe, MD Congress Secretariat Trauma and Critical Care Center Teikyo University School of Medicine 2-11-1 Kaga, Itabashi-ku, Tokyo 173, Japan Tel: +81 3 3964-1211 ext. 3319 Fax: +81 3 5375-3120 E-maih dangan @ppp.bekkoame.or.jp