The effect of a nucleotide-nucleoside solution on hepatic regeneration after partial hepatectomy in rats

BASIC NUTRITIONAL INVESTIGATION

Nutn’tion Vol. 12, Nos. 11112. 1996

The Effect of a Nucleotide-Nucleoside Solution on Hepatic Regeneration After Partial Hepatectomy in Rats MAKOTO USAMI, MD, PHD, KOUJI FURUCHI, MD, PHD, MITSUTOSHI OGINO, HIROSHI KASAHARA, MD, PHD, TAICHI KANAMARU, MD, PHD, YOICHI SAITOH, MD, PHD, FACS, HIROOMI YOKOYAMA, AND SEIICHIRO KANO* From the First Department of Surgery, Kobe University School of Medicine, *Nutrition Research Otsuka Pharmaceutical Factory, Inc., Naruto-city, Tokushima, Japan

Date accepted:

Laboratory,

1 May 1996

ABSTRACT After hepatectomy, purine and pyrimidine metabolism is a key process in the synthesis of DNA and RNA and maintaining cellular energy metabolism. The purpose of this study is to evaluate changes in blood purine and pyrimidine levels after partial hepatectomy and the effect of purine and pyrimidine nucleoside solution injection on hepatic regeneration under the hypothesis that the rat after partial hepatectomy requires substrates for salvage nucleotide synthesis and changes blood nucleoside and nucleobase levels. Blood levels of nucleotides, nucleosides, and nucleobase by high-performance liquid chromatography method and liver ATP level by enzymatic analysis, and the effect of preoperative injection of nucleoside solution (OG-VI) on hepatic regeneration ratio and hepatocytes DNA synthesis, were assessed in rats after 70% partial hepatectomy. Decreased liver adenosine triphosphate and increased plasma xanthine and hypoxanthine after partial hepatectomy indicated an increase in catabolism of purine nucleotides in regenerating liver. Plasma thymidine and cytidine levels increased, then returned to the prevalue, suggesting that the thymidine and cytidine pool was enlarged. OG-VI increased labeling indices of hepatocytes at postoperative d 1 (POD) and hepatic regeneration ratio at POD 14. Blood purine nucleobase and pyrimidine nucleoside levels change after partial hepatectomy and preoperative supply of nucleoside solution OEZsevier is effective for increasing hepatocytes DNA synthesis and hepatic regeneration after partial hepatectomy. Science Inc. 1996 Nutrition 1996; 12:797 - 803 Key words: plasma pyrimidine nucleoside, liver purine nucleotide, plasma nucleobase, partial hepatectomy, hepatic regeneration, hepatocyte DNA synthesis

INTRODUCTION Regeneration of rat liver after 70% partial hepatectomy has been widely used as an experimental model for rapid growth of normal cells.‘,’ The metabolic effect of operative stress itself is considered to be much less significant than the remarkable changes that occur in hepatic metabolism in the regenerating liver in rats.3 Nucleotides and nucleosides play an important role in the synthesis of DNA and RNA, and in the energy metabolism: an increased uptake of erotic acid, uridine, and thymidine into the intracellular acid soluble fraction at 0.5-l h,4 an increase in the purine nucleotide pool at 3 h,5 and an increase in the uridine level in the remnant liver at 6 h after

the partial hepatectomy.6 These observations indicate purine and pyrimidine pool changes during hepatic regeneration. Recently, a solution containing a mixture of nucleosides and a nucleotide (OG-VI) was developed as a nutritional supplement.’ Addition of OG-VI in an optimal concentration enhanced DNA and RNA syntheses in primary-cultured rat hepatocytes.’ Intravenous administration of OG-VI with total parenteral nutrition (TPN) solution after partial hepatectomy increased protein synthesis and turnover rates, and improved nitrogen balance in the rat model. However, no improvement of hepatic regeneration was mentioned.7,9 Continuous intravenous infusion of OG-VI after hepatectomy without an energy source to rabbits resulted in a suppressed hepatic adenylate energy

Correspondence to: Makoto Usami, MD, PhD, First Department of Surgery, Kobe University School of Medicine. 7-5-2 Kusunoki-cho, Chuo-ku, Kobe, Japan 650.

Nutrition 12:797-803, 1996 OElsevier Science Inc. 1996 Printed in the USA. All rights reserved.

ELSEVIER

0899-9007/96/$15.00 PII: SO899-9007(96100292-4

798

HEPATIC REGENERATION

charge and mitochondrial phosphorylation rate, which was lower than the subjects that received only physiologic saline infusion.” Changes in the purine and pyrimidine metabolism occur at 1-6 h after partial hepatectomy.4-6 In this study, the effect of preoperative treatment with OG-VI under normal feeding conditions on hepatic regeneration in rats was evaluated under the hypothesis that preoperative administration of OG-VI in combination with sufficient nutrition is an important factor in increasing purine and pyrimidine pool size and salvage nucleotide synthesis, then enhancing hepatic regeneration after partial hepatectomy. We hypothesized that changes in blood purine and pyrimidine levels reflect the changes of purine and pyrimidine metabolism in the regenerating liver. Blood purine and pyrimidine levels were evaluated in rats after partial hepatectomy. This is because plasma nucleoside is thought to originate from the nucleoside pool in the acid-soluble fraction of various tissues and high levels of plasma uridine and cytidine reflect larger salvage pathway of synthesis.” Culture cells secrete nucleosides in the medium.” MATERIALS AND METHODS Hepatectomy

Studies were approved by the Animal Experiment Committee of Kobe University School of Medicine. Male Wistar rats weighing 250 g were used in the experiments (SLC, Shizuoka, Japan). A total of 10 mL *kg-’ * d-’ of OG-VI (Table I) was administered intraperitoneally twice, 24 h and 2 h prior to hepatectomy in the OG group. Controls (S) were administered the same volume of normal saline. OG-VI is a mixture of inosine, 5 ‘-GMP, cytidine, uridine, and thymidine (Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima, Japan). All rats were fed standard food (CE2, Nihon Crea, Japan) ad libitum during the experiment. Surgery was performed under light open ether anesthesia between 9 and 12 A.M. The method of Higgins and Anderson2 for a 70% hepatectomy was followed. Five to seven rats from each group were sacrificed under general anesthesia before and 2 h after the second drug administration, and 1, 2, 3, 7, and 14 d after the operation. Sampling

Liver specimens were freeze-clamped in situ and blood samples were obtained from the inferior caval vein. Body weight and wet weight of the remnant liver were measured at sacrifice. The hepatic regeneration ratio was calculated as the percent

TABLE I. OG-VI SOLUTION

Inosine Cytidine 5’-GMP 2Na Uridine Thymidine Total concentration of components Total nitrogen Na

contents (wthol %)

mmol/L

Molar ratio

0.80 0.73 1.22 0.55 0.18

29.83 30.01 29.96 22.52 7.43

4 4 4 3 1

3.35 0.59 6

AND A NUCLEOTIDE-NUCLEOSIDE

SOLUTION

ratio of regenerating remnant liver weight on sacrifice to the resected liver weight. Assessment

of DNA Synthesis

of the Liver

Twenty mg/kg of 5-bromo-deoxyuridine (BrdU, Sigma Chemical Co., St. Louis, MO, USA), which is a thymidine analogue and taken up by cells in DNA synthesis, was injected intraperitoneally 1 h before sacrifice. Histoimmunochemical staining of BrdU was performed using an anti-BrdU monoclonal antibody (Sigma) and the antiperoxidase method on the liver specimens following Morystin’s I3method. DNA synthesis of liver cells was evaluated by the labeling index (LI) of regenerating livers hepatocytes at 24 h after 70% hepatectomy. LI was calculated as the percent ratio of BrdU positive cells seen against 1500 hepatocytes on one tissue slide with 400-fold magnification in a blinded fashion. Analysis

of Purine and Pyrimidine

Whole blood diluted with an equal volume of phosphatebuffered saline or plasma was mixed in cold 20% perchloric acid and centrifuged to obtain an acid-soluble fraction. The solution from the blood was neutralized with 0.7 M KzC03. Processed blood, plasma, and urine samples were then stored at -80°C. Nucleotides in blood, and nucleosides and nucleic bases in plasma were measured according to the method developed by Stocchi et ali4 and Yamamoto et al.” using highperformance liquid chromatography.‘4,‘5 A CLC-ODS-M column (Shimazu, Tokyo, Japan) for nucleotide measurement and a TSK gel ODS-80TM column (Shimazu) for nucleoside and nucleobase measurement were used in the LCdAD HPLC system (Shimazu). The ratio of xanthine to hypoxanthine was calculated using plasma xanthine and hypoxanthine levels. Adenosine triphosphate (ATP) in the liver was measured using enzymatic analysis following the method of Jaworek et alI6 The level under the measurable limit was assigned to zero in a time course after the operation. Statistical

Analysis

All data are expressed as mean t SD and Student’s t test is used for statistical evaluation. RESULTS Distribution Treatment

of Purine and Pyrimidine

in the Blood before

Purine nucleotides in the blood cells formed a majority in blood purine and pyrimidine in rats (Table II). ATP formed about 80% of purine nucleotides, while adenosine diphosphate and guanosine triphosphate formed about 8- 10% each. Purine nucleotides, such as adenosine monophosphate and guanosine monophosphate, and purine nucleosides, such as inosine, adenosine, and guanosine, which were intermediate catabolic products of purine nucleotides, were not detected in this assay. The plasma levels of pyrimidine nucleosides were less than 2% of blood cells purine nucleotide levels in normal rats. Cytidine and uridine levels were higher than thymidine. Levels of xanthine and hypoxanthine were under the lower limit of measurement, which was 0.25 nmol/mL. Liver ATP Level after the Partial Hepatectomy

The ATP level in the liver decreased at postoperative days l-3 (POD) and recovered gradually at POD 14, but was still lower than the preoperative level (P < 0.05-0.01, Fig. 1).

HEPATIC

REGENERATION

AND

A NUCLEOTIDE-NUCLEOSIDE TABLE

SOLUTION

799

II.

EFFECT OF OG-VI ADMINISTRATION ON PURINE AND PYRIMIDINE LEVELS IN THE BLOOD BEFORE PARTIAL HEPATECTOMY IN RATS (nmol/mL) Untreated Blood nucleotide ATP ADP GTP GDP Plasma nucleosides Cytidine Uridine Thymidine Uric acid

669.5 70.1 80.9 12.6

2 5 z +

3.77 1.47 0.77 53.75

t + + t

OG Group

106.8 8.1

S Group

0.9

696.3 70.2 86.7 12.6

+ + t t

89.7 11.7 9.8 1.0

624.5 61.7 71.6 10.7

f f 2 2

68.3 6.9 8.7 1.0

0.65 0.50 0.09 12.16

90.50 4.09 3.36 79.69

z 2 2 i-

34.15* 1.23* 1.04* 50.58

9.65 1.46 1.08 51.03

2 + + t-

2.06 0.83 0.06 23.77

11.4

OG group and S group received 10 mL/kg of OG-VI or normal saline, respectively, before partial hepatectomy. Values are mean 2 SD of 3-6 rats. * P < 0.01 versus untreated or S group, Student’s t test. Hypoxanthine and xanthine levels are under the measurable limit (0.25 nmol/mL). OG-VI,

mixture

triphosphate;

of nucleosides

GDP, guanosine

Blood Purine Nucleobases Hepatectomy

and nucleotide;

S, controls;

ATP, adenosine

and Uric Acid Levels After Partial

Whole blood levels of purine nucleotides were not changed (Fig. 1). Plasma xanthine levels increased abruptly at POD 1 and decreased gradually to preoperative value by POD 14 (P < 0.01) . Plasma hypoxanthine levels increased following hepatectomy and remained at a higher level until POD 14 (P < 0.01, Fig. 2 ). The plasma xanthine/hypoxanthine ratio showed the same changes as plasma xanthine levels with a sharp peak at POD 1 (Fig. 2). There was no normal value in the plasma xanthine/hypoxanthine ratio nor statistically significant differences between the data after hepatectomy and preoperative levels, because plasma hypoxanthine levels were under the measurable unit before operation. The plasma uric acid levels slightly increased at POD 1 and gradually decreased thereafter to be significantly lower at POD 14 than the preoperative level (P < 0.01, Fig. 2). There were no statistically significant differences between the OG and S groups in blood purine levels. Blood Pyrimidine Hepatectomy

triphosphate;

ADP, adenosine

diphosphate;

GTP. guanosine

diphosphate.

Nucleoside

Levels After Partial

The plasma cytidine level increased in both the S and OG groups after hepatectomy (P < 0.01). The peak value was observed at POD 2-3 and decreased gradually thereafter until POD 14 (Fig. 3). The level at POD 3 in the OG group was less than that in the S group without a statistically significant difference. The plasma uridine level at POD 1 was within the normal range, but decreased until POD 3 and decreased more thereafter (P < 0.01) The plasma thymidine level in the OG group increased after hepatectomy, remained at a high level until POD 7 and then decreased gradually, but remain higher than the preoperative level (P < 0.01, Fig. 3 ) . The thymidine level at POD 7 in the S group was lower than that in the OG group but recovered at POD 14 (P < 0.05 ). Effect of OG-VI Administration on Hepatic Regeneration on Purine and Pyrimidine Level

and

The effect of preoperative administration of OG-VI on blood purine and pyrimidine levels is shown in Table II. No differences were noted in blood purine nucleotide levels between the

OG and the S groups. However, plasma pyrimidine nucleosides levels were significantly higher in the OG group (P < 0.01). The plasma uric acid level was also insignificantly higher in

the OG group. After partial hepatectomy, liver ATP, blood purine nucleotides, and plasma purine nucleobase levels changed in the OG group in exactly the same fashion as in the S group (Figs. l-3). Plasma pyrimidine nucleosides in the OG group showed almost the same changes as in the S group. The effect of OG-VI on hepatic regeneration is shown in Table III. The LI of the hepatocytes on POD 1, which was calculated from BrdU-stained specimen (Fig. 4), was significantly higher in the OG group indicating increased DNA synthesis (P < 0.01, Table III). The hepatic regeneration

ratio on POD 14 also

showed augmented hepatic regeneration under OG-VI administration (P < 0.025). No differences in body weight changes or survival rates ( 100%) were observed between the two experimental groups. DISCUSSION This is the first report to indicate change in blood nucleosides and nucleobases after partial hepatectomy in rats and that administration of a nucleoside solution is effective for improvement of liver regeneration. In the first study, we examined changes in both blood purine and pyrimidine levels and in liver ATP levels after hepatic surgery. Distributions of purines and pyrimidines in rat blood have rarely been reported. However, results in this rat experiment were comparable to those in man.‘7-‘9 Urine and pyrimidine nucleosides and nucleobases were detected in the plasma in 0.25-100

nmol/mL ranges but not detected in blood cells. The reason is considered to be that nucleotides cannot pass through cell membrane; for example ATP in red blood cells passes through the cell membrane as adenosine, and the intermediate metabolic product is not detected in cells due to its rapid metabolism.z0 After partial hepatectomy, liver tissue ATP levels decreased and blood levels of the catabolic products of purine nucleotide, plasma hypoxanthine, xanthine, and uric acid reached a peak at POD 1. The increase in the plasma xanthine/hypoxanthine ratio was also obvious. These changes in purine nucleotides and their catabolic products suggest that ATP in the remnant

HEPATIC

800

REGENERATION

AND

A NUCLEOTIDE-NUCLEOSIDE

SOLUTION

Liver tissue .4TP content

0 1

2

3

I

14 (days)

Blood purine nucleotide content i @ a! 2,

1

%

BB

500

ii

P

1 0

I 1

I 2

I 3

I 7

I 14 (days)

Time after partial hepatectomy FIG. 1. Changes in liver ATP content and whole blood purine nucleotide level after partial hepatectomy in rats. Bars correspond to If: SD, *P < 0.01 and tP < 0.05, versus untreated preoperative value. no differences between the two groups was observed using Student’s I test. Shadow indicates, untreated preoperative value (mean t SD).

liver decreases due to the acceleration of ATP-utilizing reaction, in response to metabolic overload for hepatic regeneration3 Catabolism from purine nucleotides to nucleosides and nucleobases increases. An increase in the xanthinefhypoxanthine ratio is thought to reflect either increased xanthine oxidase activity or a decreased hypoxanthine level. Increases in both xanthine and uric acid levels indicate increased xanthine oxidase activity. However, Kurokawa et al.‘s*’ study shows that xanthine oxidase activity change is minimum in normal rats after 70% hepatectomy. From these observations, utilization of hypoxanthine by the salvage purine nucleotide synthetic pathway is highly suspect. And, higher hypoxanthine and lower uric acid levels on POD14 suggest that changes in purine catabolism and/or salvage synthesis do not fully recover until POD 14 after surgery. As for blood pyrimidine changes after partial hepatectomy, plasma pyrimidine nucleosides showed an increase in cytidine and thymidine levels, and a decrease in uridine level at POD 2-3 during hepatic regeneration. Recently, Nagaoka” reported an increase in plasma cytidine and adenosine at 3 -6 h posthepatectomy in rats, the same as our results. An increased plasma cytidine level is considered to reflect increased pyrimidine syn-

thesis and its pool size in the regenerating liver.’ An increase in salvage and de novo synthesis of pyrimidine nucleotides after partial hepatectomy increases pyrimidine nucleotide pool size and also increases catabolic products of nucleosides in liver and blood.5,6,1’A decrease in plasma uridine is related to higher uridine phosphorylase activity, a plasma catabolic enzyme for uridine, compared to the absence of cytidine deaminase, a plasma catabolic enzyme for cytidine, in rats.” These data suggest that uridine clearance increases after hepatectomy in rats in relation to hepatic regeneration. In summary, changes in blood purine nucleobases and pyrimidine nucleosides after partial hepatectomy in rats were indicated. Increased utilization and catabolism of purine and pyrimidine nucleotide in the regenerating liver are suspected to be the reason for blood level changes. Also, the addition of substrate as a requirement for salvage nucleotide synthesis is suggested after partial hepatectomy. In the second study, we examined whether a substrate for nucleotide synthesis, exogenously administered to well-nourished rats, promotes hepatic regeneration. The result that the LI and regenerating ratio of the remnant liver in the OG group were higher than those in the S group supports the enhancement

HEPATlC

AND A NUCLEOTIDE-NUCLEOSIDE

REGENERATION

801

SOLUTION

XanthinelHypoxanthine Ratio

Xanthine

FIG.2.Changes in plasma purine nucleobase (xanthine, hypoxanthine, and uric acid) levels after partial hepatectomy in rats. Bars correspond to 2 SD, *P < 0.01 and t P < 0.05, versus untreated preoperative value, no differences between the two groups was observed using Student’s r test. Shadow indicates untreated preoperative value (mean t SD).

Cytidine

1

Thymidine T

I

2.0

= I.5 1 3 y

1.0

I

2

7

3

Time

after pulid

heptectomy

FIG. 3. Changes in plasma pyrirnidine nucleoside (cytidine, uridine, and thymidine) level after partial hepatectomy in rats. Bars correspond to -C SD, *P < 0.01, versus untreated preoperative value, tP < 0.05, versus S group using Student’s t test. Shadow indicates untreated preoperative value (mean -C SD).

802

HEPATIC REGENERATION

AND A NUCLEOTIDE-NUCLEOSIDE

SOLUTION

TABLE III. EFFECT OF PREOPERATIVE OG-VI ADMINISTRATION ON HEPATIC REGENERATION AFTER PARTIAL HEPATECTOMY IN RATS (%) OG Group Hepatocyte labeling POD 1 Hepatic regeneration POD 14

S Group

index at 5

36.0 + 3.8*

4

25.5 2 3.5

7

160 2 13

ratio at 7

185 ?22t

OG group and S group received 10 mL/kg of OG-VI or normal saline, respectively, before partial hepatectomy. Value is number of rats. Values are mean 2 SD, * P < 0.01, t P < 0.025 versus S group. Student’s t test. OG-VI, mixture of nucleosides and nucleotide; S, controls; POD, postoperative day. FIG. 4. Histoimmunochemical staining of BrdU-positive nuclei in regenerating liver in rats at 24 h after partial hepatectomy. Left side in the OG group and right side in the S group, 200-fold magnification, antiperoxidase staining using DAB against BrdU monoclonal antibody. BrdU, 5-bromodeoxyuridine.

effect of preoperative OG-VI administration in liver regeneration after partial hepatectomy.’ DNA synthesis of the regenerating liver could be evaluated using radio-labeled thymidine uptake. However, the method was not selected in this study after preliminary evaluation, because thymidine incorporation is influenced by the injected nonlabeled thymidine in OG-VI solution itself and a suitable calculation and comparison method between the S group has not been established. Also, hepatocyte LI is the better method for evaluating DNA synthesis in hepatocytes themselves by excluding other nonparenchymal cellular components of the liver under microscopy. Concerning the mechanism of facilitation of hepatic regeneration, increased levels of plasma pyrimidine nucleosides at 2 h after the second administration of OG-VI, which occurred immediately before the hepatectomy, indicate increased pyrimidine nucleoside pools without a corresponding increase in puke nucleotide, nucleoside, and nucleobase in the OG group. Although no difference in blood purine and pyrimidine levels was observed between the two groups after hepatectomy, preoperative administration of OG-VI was responsible for improvement in hepatic regeneration. But timing and times of OG-VI administration is not sufficient for changing their blood levels after surgery.

to our result, Ogoshi et al? and Yamaguchi et In contrast al.” reported that postoperative administration of OG-VI had no beneficial effects on hepatic regeneration or oxidative phosphorylation. These differences suggest that postoperative supplementation of OG-VI is not sufficient for increasing puke and pyrimidine pool size under abrupt metabolic changes occurring at an early stage of hepatic regeneration.3-6 Yamaguchi et al.” reported that the administration of OG-VI in the absence of a nutritional supplement causes less augmentation on oxidative phosphorylation reaction in hepatectomized rabbits. However, purine and pyrimidine metabolism tends to fluctuate depending on the nutritional status.4.5,‘9,22 Administration of OG-VI with adequate nutrition by either TPN or normal diet is therefore important for improvement of hepatic regeneration. The results of a plasma level 2 h after OG-VI administration suggests differences between puke and pyrimidine in utilization and metabolic rate in the liver cells.8 Increased pyrimidine levels, without a corresponding change in the purine nucleoside level, may have resulted from a shortage in the purine component in this OG-VI solution. In conclusion, preoperative administration of OG-VI to partially hepatectomized rats enhances hepatic DNA synthesis in a way similar to the in vitro study.8 In order to create the best nutritional management for hepatectomized patients, more detailed studies, including an analysis of the timing and route of OG-VI administration, and reexamination of solute composition in nucleoside solution are necessary.

REFERENCES 1. Harkness RD. Regeneration of liver. Br Med Bull 1957; 13:87 2. Higgins DM, Anderson RM. Experimental pathology of the liver: 1. Restoration of the liver of the white rat following partial surgical removal. Arch Path01 1931; 12:186 3. 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:G.585 4. Ord MG, Stocken LA. Uptake of amino acids and nucleic acid precursors by regenerating rat liver. Biochem J 1972; 129:175 5. Bucher NLR, Swaffield MN. Ribonucleic acid synthesis in relation to precursor pools in regenerating rat liver. Biochem Biophys Acta 1969; 174:491 6. 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:l 7. Ogoshi S, Iwasa M, Yonezawa T, et al. Effect of nucleotide and nucleoside mixture on rats given total parenteral nutrition after 70% hepatectomy. J Parenter Enteral Nutr 1985;9:339

8. 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 Parenter Enteral Nutr 1989; 13:51 9. Ogoshi S. Iwasa M, Mizobuchi S, et al. Effect of nucleoside and nucleotide mixture on protein metabolism in rats given total parenteral nutrition after 70% hepatectomy. In: Tanaka T, Okada A, eds. Nutritional support in organ failure. Amsterdam: Elsevier Science Publishers, 1990:309 10. Yamaguchi T, Takeda Y, Shimahara Y, et al. A mixture of nucleosides and nucleotide alters hepatic energy metabolism 34 hours after hepatectomy in rabbits. J Nutr 1992; 122:340 R. Salvage of circulating 1 l. Moyer JD, Oliver JT, Handschumacher pyrimidine nucleosides in the rat. Cancer Research 1981;41:3010 12. Uziel M, Selkirk JK. Pyrimidine nucleoside, pseudouridine, and modified nucleoside excretion by growing and resting fibroblasts. J Cell Physiol 1979;99:217 13. Morystin G, Hsu SM, Kinsella T, et al. Bromodeoxyuridine in

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14.

15.

16.

17.

REGENERATION

AND A NUCLEOTIDE-NUCLEOSIDE

tumors and chromosomes detected with a monoclonal antibody. J Clin Invest 1983;72:1844 Stocchi V, Cucchiarine L, Canestrari F, et al. A very fast ionpair reversed-phase HPLC method for the separation of the most significant nucleotides and their degradation products in human red blood cells. Anal Biochem 1987; 167:181 Yamamoto T, Moriwaki Y, Takahashi S, et al. Separation of hypoxanthine and xanthine from pyrazinamide and its metabolites in plasma and urine by high-performance liquid chromatography. J Chromatogr 1986;382:270 Jaworek D, Gruber W, Bergmeyer HU. Adenosine-5’triphosphate: determination with 3-phosphoglycerate kinase. In: Bergmeyer HU. ed. Methods of enzymatic analysis. New York; Academic Press, 1974:2097 Nagaoka T. Changes in plasma nucleoside levels during hepatic

. . .

18. 19. 20. 21. 22.

SOLUTION regeneration in partial hepatectomized and Ccl, damaged rats. Acta Hepatologica Japonica 1991;32:633 (in Japanese) Furuchi K, Usami M, Kasahara H, et al. Blood and urine levels of nucleic acid after surgery. Jap J Surg Metab Nutr 1993;27:11 (in Japanese) Yamamoto T, Yokoyama H, Moriwaki Y, et al. The effect of completely purine-free diet of low sodium content on purine intermediates and end-product. Eur J Clin Nutr 1990;44:659 Sato F, Ito T, Akiyoshi H, et al. Metabolism of adenine nucleotides in the rat. Studies in vivo by intravenous and intraduodenal administration, and in vitro using red and fat cells. J Biochem 1978;83:1 Kurokawa T, Nonami T, Sugiyama S, et al. Effect of long acting superoxide dismutase on 1ive;metabolism after major hepatic resection in rats with cirrhosis. Eur Surg Res 1991;23:65 Tseug J, Barelkovski J, Gurpide E Rates of formation of bloodborne uridine and cytidine in dogs. Am J Physiol 1971;222:869

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