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Orotic acid-induced fatty liver
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Orotic Metabolic
From the Laboratory
13-19 (1965)
Acid-Induced
Studies
H. G. WISDMUELLER,
109,
Fatty
in Conventional
Liver
and Germ-Free
E. G. McDAXIEL,
AND
ALBERT
Rats SPAETH
of AVutrition and Endocrinology, National Institute of Arthritis Diseases, AVational Institutes of Health, Bethesda, Maryland Received
September
and Metabolic
25, 1901
Germ-free rats fed l?,i erotic acid in their diet absorb G6Y0 of the compound from their intestinal tracts and continuously excrete the remaining one third in their feces. In conventional rats fed erotic acid, the intestinal microflora adapts rapidly to catabolize all nonabsorbed erotic acid, which disappears from the feces within 5 days after feeding is begun. Orotic acid-fed, germ-free rats develop a fatty liver which can be prevented by adenine administration, as is the case with conventional rats. Of the erotic acid absorbed by germ-free rats, 3Oc/;, is excreted in urine and the remainder is rapidly decarboxylated and apparently converted to uridylic acid despite high steady-state concentrations of uridine nucleotides in the liver. The addition of adenine to the diet does not influence intestinal absorption but increases urinary excretion and decreases somewhat the amount decarboxylated. The magnitude of the adenine effect seems inadequate to account for the protective action of adenine against erotic acid-induced fatty liver or to explain the ability of adenine to depress rapidly the steady-state concentrations of uridine nucleotides. ilddition of ly& erotic acid to the diet does not influence the excretion of pseudouridine but doubles the amount, of uracil appearing in the urine.
The precursor role of erotic acid (OA) in obje&ives the following: (1) to determine how much OA is absorbed from the gaskopyrimidine biosynthesis is well established (1). However, recently OA has been the intestinal tract, how much is converted t,o subject of renewed attention following the UIJII’ by the animal, and how much is eliminated in urine; (2) to determine the report (2) that it produces grossly fatt,y effect’ of adenine 011 the above processes, in livers when fed t’o rat’s as 1% of their diet. Livers from OA-fed rats contain high levels an effort t,o explain the protective capacity of triglycerides (3), cholest,erol (4), and of t’his compound in OA-fed rats; and (3) acid-soluble uridine nucleotides, and de- through the use of germ-free rats, to assess the role of the intestinal microflora in the pressed levels of adenine and pyridinc metabolism and fatty liver-producing action nucleotides (5). Furthermore, in such livers of OA. there is an inhibition of pyridine nuclcotidc synthesis (6, 7) and of lipoprot,ein secretion EXPERIRlE?;TAL (8, 9), t)he lat’ter being an apparent, cause of Rats and diets. RIale rats (GO-150 gm) of the hepatic fat accumulation. These effects of strain were maintained OA can all be prevent,ed or rapidly reversed NIH Sprague-Hawley individually in wire-bottomed cages and were by the addition of small amounts of adcnine allowed free access to food and water unless otherto the diet (7, 10). wise specified. The basal diets varied with the reThere are no quantit’ativc dat,a available quirements of the rxperiment and are described relating to the ut’ilizat’ion and metabolism of below: R-l, a complete synthetic diet containing large amounts of dietary OA by the rat. 18% casrin, 5% corn oil, and GS.570 glucose monoTherclforc the present siudies had as t,heir hydrate (11); R-1.FF, same as 11-1, with the corn 13
oil replaced by an cquivnlcnt amount of glucose (8); W-B (used for tube-feeding after being mixed same as with 75 or 100~~ by weight of water), II-I-FF with the cascxin replaced by ctnzymically hydrolyzed casein (Nutritional Biochcmicals Corp.); GF-1 (sterilized by autoclaviltg), similar to R-1, with 20${, casein, 5(:‘, corn oil, and 717,;) corn starch as the carbohydrate source. IXats that had been fed one of the above diets supplcmcnt,ed with 15;: OA or l’{;, OA plus O.25(:; Sd,S04 for 5 days or more will be referred to as “0.4-adapted” and rats fed only the basal diets as “unadapted.” Conversion of 0.1 to widine-5’.phosphute. The recovery of respiratory Cl402 after administration of OA-7.Cl4 was used as the index of uridinc-5’. phosphate (UPIIP) formation (12). Rats wcrc placed in glass metabolism cages which were COIItinuously flushed with dry, COS-free air. Expired COZ was trapped in 3-5 IV KOH, of which one portion was titrated with dilute HCL aftrr addition of an excess of BaC12. To d&ermine C”Os, another portion was transferred to the main compartment of a large Warburg-type flask (13), and CO, was released by addition of HzS04 from the side arm. Carbon dioxide was trapped in t,he center well, which was made large enough to reccivc a 2Q-ml liquid scintillat,ion counting vial containing 2 ml of phenethylamine-methanol (1: 1, v/v) (14). The flasks, sealed with ground-glass stoppers coated with glycerol, were shaken gent,ly for 2 hours to effect a quantitative transfer of CO: to the counting vials. Radioactivity was determined after addition of 10 ml toluene scintillation solution containing 0.5’;lc PPO (2,5-dipherlyloxazole) and O.l%O POPOP [1,4-his-2.(5.phenylo~ozolyl). benzene]. Orotic acid determinations. Feces and the gastrointestinal tract plus its contents were extracted with 0.02-0.05 S LiOH by homogenization and high-speed centrifugation. The choice of LiOH as extractant was based on the relatively high solubility of lithium orotate. IJrinc, collected under toluene in metabolism cages, was mixed with 0.05 X LiOH and centrifuged. Portions of the supernatant extracts were assayed for OA by the enzymic method of Rosenbloom and Sccgmiller (15). By using this procedure, recovery of OA added to fecal homogenates was quantitative. Feces were collected from beneath raised wire cages or, in some experiments, by use of tail cups. The Cl4 content of the LiOH extracts was determined (where appropriate) by liquid scintillation counting in Bray’s solution (l(i). Ger?n;free rats. (berm-free rats were maintained in isolators by using standard techniques. For collection of COZ, the rats were transferred into sterile metabolism cages constructed from battery jars and were int,roduccd aseptically into the iso-
later. The jars wcrc scsalcd in the isolator., csccpt for air inHow and outflow ports, which w-erc ~IYtccted with t):tctc~riologic:~1 filt.crs. The jars wc1rc t hcrr rcsmovcd from the isolator and continuously flushed wit.h COt-free air in the usual \vay. The germ-free state was verified by periodically culturing frcal cxtrncts antl, at the end of each csp(Jrimerit., thr cccal cont,cnts of each germ-frecx rat used in thcsc st,udics. 13lMt-LTS
Following the intragastric administration of OA-7-Cl4 to (IA-adapted conventional rats, 90-95 % of tjhc CL4 was recovered as C140, wit bin 2-l hours (Fig. 1). The remaining 5-10 ‘2 of the radioactivitIy was recovered in urine, while feces cottt,ained only traces of radioact’ivity. The over-all recovery of Cl4 was 98-103 ‘X . Addit,ion of adenitte sulfate (Ad.SO.,) to the diet had little or no effect on the distribution of radioactivity. When unadapted rat’s were similarly tubefed a meal containing OX-7-C14, recovery of radioact,ivity in <:140~ was slower and accounted for only 71 ‘X of t)he total after -18 hours (Fig. 1). Fecal recovery of Cl4 averaged 23 %a(6-33 “: ) of the dose, urinary recovery averaged 3 ‘,G (2-h S), and over-all recovery was 97 ‘X (92-99 72). Of the Cl4 recovered in the feces, 66 “4 was present as OA, as determined by independent enzymic assay. Evidence t’hat, t’he apparent adapt,at,ion to OA involved t’hc ittt,estinal microflora was obtained from the experiment. described in Fig. 2. The recovery of fecal OA in convetttional rats was initially low and fell to zero in all animals aft’cr 3-5 days of OA ingestion. On the other hand, germ-free rats continuously excreted large amounts of OA in theit feces. i2ft,er being ittt,ragastricalIy inoculated wit’h a fecal extract’ frotn OA-ingesting conventional rats, gerrtikfree rats (ex-germfree) exhibited the adaptive response t,o OA, and after 3 days of OA-feeding, none of the compound could be detected in their feces. It is clear, therefore, that the intestinal microflora adapts rapidly and is soon capable of catabolizing large quantities of OA. Thus, much of the CY402 recovered following the intragastric administraton of OA-7-Cl4 in conventional rats (Fig. 1) could represent microbial rather than t’issue activity.
METABOLISM
OF EXOGENOUS
OROTIC
ACID
15
100 90
HOURS
1. Recovery of Cl402 from rats given OA-7-Cl4 by stomach tube. Weanling rats were fed basal diet R-1.FF for 7-10 days. Then the diet of some was supplemented with OA or OA + Ad.S04 for (i days (adaptation). Finally, all were tube fed 5 ml of diet W-G + 1% OA-7-C14 (based on dry weight), containing 2 PC C’4, with or without the addition of Ad.SOa , and COz was collected for 2738 hours. FIG.
Legend
No. rats
A--A
6 2 2
o--o o--o
Supplement to diet RlFF during adaptation (6 days)
Supplement to diet W-6 for tube-feedin:
None 1%) Oh
I’;, OA-7-V 1%; OA-7-C’” I’,,; OA-7-C”
170 0.-1 + 0.25% Ad .SOd
It is noteworthy that in the germ-free rats fed 1% OA for 5 days or more (with basal diet GF-I), liver triglyceride and cholesterol levels were elevated, and plasma triglycerides, cholesterol, and phospholipids were greatly depressed, the situation being qualitatively and quantitatively identical to that previously found in conventional rats ingest’ing similar diets (8). And, as with conventional rats, the addition of 0.25 % diet,aryAd.S04preventedall of thesechanges URIDINE-5’-PHOSPHATE GERM-FREE
FORMATION
IN
RUTS
When microbial activity is excluded, the production of Cl402 following the administration of OA-7-C14 is a measure of the conversion of OA to UMP (12). Figure 3 shows rats recovery of C1402 from germfree adapted to diets containing 1% OA, with and without addition of Ad.SOd, and tube fed a meal containing OA-7-C14. In contrast with conventional rats to experiments (Fig. l), less than 25 % of t’he radioactivity was recovered in respired COz. The presence
+ 0.257;, Ad.SO1
of Ad. SO4 in the diet (and in the tube-fed meal) further reduced recovery in COz by 25 70, mostly after 12 hours. As will be indicated in subsequent tables, the remainder of the Cl4 was recovered in feces plus intestinal contents (53-6.5%; avg. 59%) and in urine (6-11% ; avg. 9 %). Over-all recovery was 89 %’ (83-92 %). EXCRETION
OF OROTIC
PYRrnfmrms AND
ACID IN
AND
OTHER
URINE
FECES
Recoveries of OA in the feces and urine during continuous administration of the compound in the diet, after administration of a tube-fed meal containing 1% OA-7-Cl4 and after intraperitoneal injection of OA-7-04, are given in Tables I and II. When OA-7-Cl4 was administered, recovery is expressed in terms of recovery of CY4.To det,ermine if t,he Cl4 in these samples was OA-7-C14, the following test was applied. An aliquot, of t,he LiOH extract of the sample was incubated in a closed vessel (large, Warburg-type) for 144u hours with crude orotidylic acid pyro-
16
WINDMUELLER,
MCDANIEL, AND HPAETH
24,
phosphorylase and orotidylic acid decarboxylase prepared from yeast (15), with and without addition of ?phosphoribosyl-lpyrophosphate (PRPP). The react’ion was stopped by addit’ion of HaSOd, and all COa liberated was trapped in a mixture of phenethylamine and methanol and counted (see E.rperz&ental). The counts obtained were compared to those obtained by counting directly a similar aliquot of the LiOH extract’ (see flzpe~in~ental). If all the counts in t,he extract were recovered as C1402 when PRPP was added t’o the incubation and less than 13 when it was not, then extract radioactivity was assumed to be OA-7-Cl4. This was found to be t,he case for all urine samples and for fecal and intestinal samples from gern-free rats. When 1% OA was continuously supplied in t,he diet, conventional rats, once they were adapted, excret)ed none in feces but 20 9%in urine (Tables I and II). Germ-free rats excreted one t’hird of their intake in feces and about 10 % in urine. The addition of dietary Ad.SO, did not influence fecal excretion but, did increase by 50 ‘i; urinary excretion in both conventional and germ-free animals. When OA-7-C14 was tube fed, less of the dose was absorbed as indicated by the greater fecal recovery (feces plus int’estinal contents) in the germfree rats and the generally reduced urinary recovery in both convent,ional and germ-free rats when compared with results of ad Zibitum OA administration. Adaptation did not significantly change the amount excreted in urine by conven-
I
_. i 56r
Germ/ree (91
16 8 0 OAI DAY AFTER OA FEEDING WAS BEGUN
Flc. 2. Fecal excretion of erotic acid by conventional and germ-free rats. Rats (120-150 gm) were fed basal diet GF-1 for 10 days. Then 1% OA was added as a supplement (indicated by arrow). Fecal collections (24.hour) were made 1 day before and for 9 days after the addition of OA. E&germfree rats were removed from a sterile isolator on day -1, inoculated intragastrically with 1 ml of an homogenate of feces from rats fed for several months on a diet containing 1’;; OA, and thereafter handled as conventional rats. Numbers in parentheses indicate the number of animals, and vertical lines represent one standard error of the mean. TABLE FEV.IL
EXCRETION
OF EXCIGENOIJS
ORWIC
are expressed as mean (j& of administered number of rats. All rats were OA-adapted
Data indicate
Method of 0.4 administration
Ad lib.feeding
as 1% of diet (It-1 or GF-1) OA-7-C’” tube-fed as lyO of diet
(W-6)"
I
ACID
IS
CUNVENTIONAL
.\KD
GERM-VKEE
dose (OA or Cl”) & SE. Numbers (see Ezpe~?mental).
R.\Ts
in parentheses
Germ-free
Conventional -Ada
+4d
0.0
0.0
(7) 0.4 zk 0.2
(2) 0.6 zk 0.3
(2)
(2)
-Ad
33.5 f 3.6 (JP 57.2 f 2.3c (3)
o Ad denotes addltlon of O.25,0 (” Ad.804 to the diet. h Mean yc recovery from 4 rats over a ‘i-day period. c 27?;; in feces and 30% in the gastrointestinal tract (48 hours after tube-feeding). d 217; in feces and 41y0 in the gastrointestinal tract (48 hours after tube-feeding). e For more detail, see legend to Figs. 1 and 2.
+Ad
33.3 zk 2.3 (I)* 62.3 It 2.5d
(2)
METABOLISM 26
I
I
4
8
OF EXOGENOUS I
OROTIC
17
ACID
I
I
I
I
I
28 24 HOURS
32
36
40
44
I
24 22 20
4
0 0
12
16
20
4t
of CPO2 from germ-free rats given OA-7-C’” by stomach tube. Weanling germ-free FIG. 3. Recovery rats were fed basal diet GF-1 for 7-20 days. Then the diet was supplemented for 7-10 days with OA or OA + Ad.SO, Finally, all were tube fed 4 ml of diet W-6 + 1% OA-7-P (based on dry weight), containing 2 PC Cr4, with or without the addition of Ad.SOa , and COZ was collected for 47 hours. The germ-free state was maintained throughout, as described under Ezperimental. Vertical bars represent one standard error of the mean. Legend
No.
rats
o--o
4
o--o
3
Supplement to diet GF-1 during adaptation (7-10 days)
Supplement
I?& 08 1R 08
1% OA-7-P 1% OA-7-C’”
+ 0.25y0 Ad.SOa
tional rats, nor did dietary Ad.SO, appear to alter consistently urinary or fecal elimination in any of the rats. However, when OA-7-Cl4 (tracer dose) was injected intraperitoneally, without fasting the rats, the presence of dietary Ad. SO, did consistently increase urinary elimination of the labeled compound. The urinary elimination of uracil appears to be increased by feeding 1% OA, and the increase is largely prevented by Ad. SO, (Table III). The excretion of pseudouridine in urine was altered little, if any, by the diet modifications.
to diet
W-6 for tube-feeding
+ 0.25% Ad.SOd
DISCUSSION
The intestinal microflora of conventional rats rapidly adapts to catabolize all OA which is not absorbed from the gut. Such microbial activity is not related, however, to the effects of dietary OA on hepatic lipid and nucleotide metabolism, as is evident from the similar way in which conventional and germ-free rats respond to to dietary OA. The products of OA fermentat’ion by a common intestinal bacterium, ZymobacteGwn oroticum, grown in liquid culture, have been identified as ethanol,
18
WINDMUELLER,
MCDANIEL, TABLE
AND
SPAETH
II
~JRIN.~RY EXCRETION OB ADMINISTERED OHOTIC ACID IN CONVENTION.IL .IND GERM-FREE Ii.1~ Data are expressed as mean “i, of administered dose (OA or Cl*) + SE. Numbers in parentheses indicate number of rats. Conventional Method of OA administration
Ohadapted
Unadapted -Ada
Ad lib. feeding as 1% of diet (P-1 or GF-1) OA-7-C14 tube-fed as 1:; of diet (W-6)* OA-7-C’4 injected i.p. (tracer dosejc
-Ad
+Ad
2.5 (1)
3.1 * 0.4 (5) 13.2 f 6.0 (3)
19.8 zt 2.3 (5Y 4.4 f 0.6
Germ-free +Ad
29.6 + 1.2 (5P 7.3 r!z 0.5
(2)
(2)
12.6 + 2.2
40.1 zt 5.3 (5)
(6)
(I Ad denotes addition of 0.25(/;, Sd.SOa to the diet. b For more detail see legend to Figs. 1 and 2. c IJrine was collected for 24 hours after injection of 2 kc (0.24 rmoles d Urinary OA was determined for each rat for at least 4 days. TABLE
III
EFFECT OF FEEDING OROTIC ACXD AND ADENINE ON THE URINARY EXCRETION OF URACIL AND PSEUDOURIDINE~
Additions
to basal (RlFF)
Excreted (m,n/Z+
die@
Uracil
None lYO OA lyO OA + 0.25% Ad.SO4
in Urine hours)’ Pseudouridine
0.30 f- 0.021.41 0.72 + 0.061.07 0.47 zt 0.081.20
zk 0.06 i 0.04 i 0.15
a The authors are very grateful to Dr. S. Weissman for performing these analyses (17). B Diets were fed 10 days preceding and also during the urine collection. c Each number is the mean of 4 values from 2 rats + SE. acetic acid, COZ, and KH3.l The present studies emphasize the large role which the intestinal microflora may play in the metabolism of compounds administered intragastrically, and they also indicate that. a substantial portion of the total COz output. of conventional rats may be of microbial origin. The average hourly CO2 production of the conventional rats in these studies was 20.7 f 0.7 (SE) mmoles/lOOgm body weight compared with 15.4 f 0.8 mmoles/lOO gm body weight for the germ-free rats. Van ’ W. E. C. Moore,
personal
communication.
-Ad
10.3 & 0.9 (3Y 10.0 * 0.8 (4)
(OA-adapted) +Ad
15.2 + 1.2 (3P 6.9 Ik 0.5 (3)
of OA-7-C14).
Euler et al. (5) reported highly variable fecal recoveries after tube feeding OA-6-C14, and they also found a higher recovery of C1402 when labeled OA was administered to rats fed OA for 10 days as compared to 1 day. Bot’h these findings are now explicable in terms of microbial activity in the gut,. Under the conditions in which dietary O-4 alters hepatic lipid and nucleotide concentrations, only 25-56 % of the compound is metabolized. The higher figure, obtained when rats had free access t’o diet, is probably more nearly correct. The lower estimate was obt,ained from tube-feeding experiments and reflects the decreased absorption of OA when administered in t’his way. Thus, lOO-gm rat ingesting 12 gm of diet is converting, daily, about 430 pmoles of exogenous OA to UMP. That, this conversion occurs largely in the liver is indicated by the st’udies of Hurlbert and Potter (18) and Rabkin et al. (12). This conversion continues despite hepatic accumulation of UMP (5), which, in soluble preparations from rat liver, conpetitively inhibits orotidylic acid decarboxylase (19). Thus our in viva experiments do not provide evidence for the suggestred (19) feedback regulating role of UMP in controlling pyrimidine nucleotide biosynthesis. Liver slices from OA-fed rats also show 110 diminished UMP formation from OA (lo), although their UR4P cont,ent, is elevated.
METABOLISM
OF EXOGENOUS
The presence of adenine in t,he diet does not, influence the amount of OA absorbed from the int,estinal tract. It does, however, iucrease
somewhat
the amount
excreted
in
urine and decrease the amount convert,ed to UJII’. The increase in urinary excretion is very large when the OA is administered intraperitoneally. These in vivo eff ect.s of adenine are compatible with the finding that in soluble preparations from rat liver, adenine can reduce UNIP formation from OA when available PRPP is limiting (5). It is tempting to invoke such a reduct,ion t’o explain the rapid disappearance of the elevated hepatic acid-soluble uridine level when OA-fed rats are supplemented with adenine (8). However, small effects of adenine on the relatively URlP format,ion from OA in t)hc whole animal
make
it unlikely
that’
competition
for PRPP is the only fact,or involved. Even though the presence of adenine in the diet diminishes
only
slight’ly
the amount
of 31
met,abolized by the rat, it, prevents almost completely the accumulation of high steadyst,ate concentrations of U?tIP and other acidsoluble uridine derivatives in the liver (5, 7). This action of adenine, as well as it,s ability
to e1evat.e rapidly
hepatic
adenine
nucleotide levels which have been depressed by feeding OA, suggests that, adenine counkrs the effect, of OA by augmenting the endogenous acid-soluble hepatic adenine pool, which is diminished in size by OA in an as yet, unknown way. The failure of dietary OA, with or without adenine, to produce increased pseudo-uridine excretion (Table III) indicates that 110 accelerat.ed turnover of soluble volved (17).
ribouucleic
acid
is in-
OROTIC
ACID
19
ACKNOWLEDGMENT
We gratefully acknowledge the assistance of Mr. Edward Barron in the care of the germ-free rats. REFERENCES 1. REICHARD, P., Advan. Enzymol.
21, 263 (1959). 2. STANDERFEK, S. B., AND HANDLEK, P., Proc. Sot. Erptl. Biol. Med. 20,270 (1955). L., and HAND3. CREASEY, W. A., HANKIN, SCHUMACHER, R. E., J. Biol. Chem. 236, 2064 (1961). 4. RAJALAKSHMI, S., SARMA, D. S. R., AND SARMA, P. S., Biochem. J. 80,375 (1961). 5. VON EULER, L. H., RUBIN, R. J., AND HANDSCHUMACHER, R. E., J. Biol. Chem. 236, 2464 (1963). 6. RAJALAKSHMI, S., SAKMA, D. S. R., AND SARMA, P. S., Znd. J. Ezptl. Biol. 1,63 (1963). 7. WINDMUELLER, H. G., submitted to J. h’utr. 8. WINDMUELLER, H. G., J. Biol. Chem. 239, 530 (1964). 9. WINDMUELLER, H. G., Biochem. Biophys. Res. Commun. 11, 496 (1963). 10. HANDSCHUMACHEII, R. E., CREASEY, W. A., JAFFE, J. J., PASTERNAK, C. A., AND HANKIN, I,., Proc. K’atl. Acad. Sci. U. S. 46, 178 (1960). 11. WILLIAMS, J. N., JR., J. Kutr. 73,199 (1961). 12. RABKIN, M. T., FREDERICK, E. W., LOTZ, &I., AND SMITH, L. H., JR., J. Clin. Invest. 41, 8il (1962). 13. EISENBERG, F., in “Liquid Scintillation
Counting,” p. 123. Macmillan (Pergamon), New I7ork (1958). 14. WOELLER, F. H., Anal. Biochem. 2, 580 (1961). 15. ROSENBLOOM, F. hr., AND SEEGMILLER, J. E., J. Lab. Clin. Med. 63, 492 (1964). 16. BRAY, G. A., Anal. Biochem. 1,279 (1960). 17. WEISSMAN, S., EISEN, A. Z., LEWIS, Al., AND KARON, i\l., J. Lab. Clin. Med. 60,40 (1962). 18. HURLBERT, K. B., AND POTTER, 1.. It., J. Biol. Chem. 196, 257 (1952). 19. BLAIR, I). G. R., AND POTTER, I-. R., J. Biol. Chem. 236, 2503 (1961).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Orotic Metabolic
From the Laboratory
13-19 (1965)
Acid-Induced
Studies
H. G. WISDMUELLER,
109,
Fatty
in Conventional
Liver
and Germ-Free
E. G. McDAXIEL,
AND
ALBERT
Rats SPAETH
of AVutrition and Endocrinology, National Institute of Arthritis Diseases, AVational Institutes of Health, Bethesda, Maryland Received
September
and Metabolic
25, 1901
Germ-free rats fed l?,i erotic acid in their diet absorb G6Y0 of the compound from their intestinal tracts and continuously excrete the remaining one third in their feces. In conventional rats fed erotic acid, the intestinal microflora adapts rapidly to catabolize all nonabsorbed erotic acid, which disappears from the feces within 5 days after feeding is begun. Orotic acid-fed, germ-free rats develop a fatty liver which can be prevented by adenine administration, as is the case with conventional rats. Of the erotic acid absorbed by germ-free rats, 3Oc/;, is excreted in urine and the remainder is rapidly decarboxylated and apparently converted to uridylic acid despite high steady-state concentrations of uridine nucleotides in the liver. The addition of adenine to the diet does not influence intestinal absorption but increases urinary excretion and decreases somewhat the amount decarboxylated. The magnitude of the adenine effect seems inadequate to account for the protective action of adenine against erotic acid-induced fatty liver or to explain the ability of adenine to depress rapidly the steady-state concentrations of uridine nucleotides. ilddition of ly& erotic acid to the diet does not influence the excretion of pseudouridine but doubles the amount, of uracil appearing in the urine.
The precursor role of erotic acid (OA) in obje&ives the following: (1) to determine how much OA is absorbed from the gaskopyrimidine biosynthesis is well established (1). However, recently OA has been the intestinal tract, how much is converted t,o subject of renewed attention following the UIJII’ by the animal, and how much is eliminated in urine; (2) to determine the report (2) that it produces grossly fatt,y effect’ of adenine 011 the above processes, in livers when fed t’o rat’s as 1% of their diet. Livers from OA-fed rats contain high levels an effort t,o explain the protective capacity of triglycerides (3), cholest,erol (4), and of t’his compound in OA-fed rats; and (3) acid-soluble uridine nucleotides, and de- through the use of germ-free rats, to assess the role of the intestinal microflora in the pressed levels of adenine and pyridinc metabolism and fatty liver-producing action nucleotides (5). Furthermore, in such livers of OA. there is an inhibition of pyridine nuclcotidc synthesis (6, 7) and of lipoprot,ein secretion EXPERIRlE?;TAL (8, 9), t)he lat’ter being an apparent, cause of Rats and diets. RIale rats (GO-150 gm) of the hepatic fat accumulation. These effects of strain were maintained OA can all be prevent,ed or rapidly reversed NIH Sprague-Hawley individually in wire-bottomed cages and were by the addition of small amounts of adcnine allowed free access to food and water unless otherto the diet (7, 10). wise specified. The basal diets varied with the reThere are no quantit’ativc dat,a available quirements of the rxperiment and are described relating to the ut’ilizat’ion and metabolism of below: R-l, a complete synthetic diet containing large amounts of dietary OA by the rat. 18% casrin, 5% corn oil, and GS.570 glucose monoTherclforc the present siudies had as t,heir hydrate (11); R-1.FF, same as 11-1, with the corn 13
oil replaced by an cquivnlcnt amount of glucose (8); W-B (used for tube-feeding after being mixed same as with 75 or 100~~ by weight of water), II-I-FF with the cascxin replaced by ctnzymically hydrolyzed casein (Nutritional Biochcmicals Corp.); GF-1 (sterilized by autoclaviltg), similar to R-1, with 20${, casein, 5(:‘, corn oil, and 717,;) corn starch as the carbohydrate source. IXats that had been fed one of the above diets supplcmcnt,ed with 15;: OA or l’{;, OA plus O.25(:; Sd,S04 for 5 days or more will be referred to as “0.4-adapted” and rats fed only the basal diets as “unadapted.” Conversion of 0.1 to widine-5’.phosphute. The recovery of respiratory Cl402 after administration of OA-7.Cl4 was used as the index of uridinc-5’. phosphate (UPIIP) formation (12). Rats wcrc placed in glass metabolism cages which were COIItinuously flushed with dry, COS-free air. Expired COZ was trapped in 3-5 IV KOH, of which one portion was titrated with dilute HCL aftrr addition of an excess of BaC12. To d&ermine C”Os, another portion was transferred to the main compartment of a large Warburg-type flask (13), and CO, was released by addition of HzS04 from the side arm. Carbon dioxide was trapped in t,he center well, which was made large enough to reccivc a 2Q-ml liquid scintillat,ion counting vial containing 2 ml of phenethylamine-methanol (1: 1, v/v) (14). The flasks, sealed with ground-glass stoppers coated with glycerol, were shaken gent,ly for 2 hours to effect a quantitative transfer of CO: to the counting vials. Radioactivity was determined after addition of 10 ml toluene scintillation solution containing 0.5’;lc PPO (2,5-dipherlyloxazole) and O.l%O POPOP [1,4-his-2.(5.phenylo~ozolyl). benzene]. Orotic acid determinations. Feces and the gastrointestinal tract plus its contents were extracted with 0.02-0.05 S LiOH by homogenization and high-speed centrifugation. The choice of LiOH as extractant was based on the relatively high solubility of lithium orotate. IJrinc, collected under toluene in metabolism cages, was mixed with 0.05 X LiOH and centrifuged. Portions of the supernatant extracts were assayed for OA by the enzymic method of Rosenbloom and Sccgmiller (15). By using this procedure, recovery of OA added to fecal homogenates was quantitative. Feces were collected from beneath raised wire cages or, in some experiments, by use of tail cups. The Cl4 content of the LiOH extracts was determined (where appropriate) by liquid scintillation counting in Bray’s solution (l(i). Ger?n;free rats. (berm-free rats were maintained in isolators by using standard techniques. For collection of COZ, the rats were transferred into sterile metabolism cages constructed from battery jars and were int,roduccd aseptically into the iso-
later. The jars wcrc scsalcd in the isolator., csccpt for air inHow and outflow ports, which w-erc ~IYtccted with t):tctc~riologic:~1 filt.crs. The jars wc1rc t hcrr rcsmovcd from the isolator and continuously flushed wit.h COt-free air in the usual \vay. The germ-free state was verified by periodically culturing frcal cxtrncts antl, at the end of each csp(Jrimerit., thr cccal cont,cnts of each germ-frecx rat used in thcsc st,udics. 13lMt-LTS
Following the intragastric administration of OA-7-Cl4 to (IA-adapted conventional rats, 90-95 % of tjhc CL4 was recovered as C140, wit bin 2-l hours (Fig. 1). The remaining 5-10 ‘2 of the radioactivitIy was recovered in urine, while feces cottt,ained only traces of radioact’ivity. The over-all recovery of Cl4 was 98-103 ‘X . Addit,ion of adenitte sulfate (Ad.SO.,) to the diet had little or no effect on the distribution of radioactivity. When unadapted rat’s were similarly tubefed a meal containing OX-7-C14, recovery of radioact,ivity in <:140~ was slower and accounted for only 71 ‘X of t)he total after -18 hours (Fig. 1). Fecal recovery of Cl4 averaged 23 %a(6-33 “: ) of the dose, urinary recovery averaged 3 ‘,G (2-h S), and over-all recovery was 97 ‘X (92-99 72). Of the Cl4 recovered in the feces, 66 “4 was present as OA, as determined by independent enzymic assay. Evidence t’hat, t’he apparent adapt,at,ion to OA involved t’hc ittt,estinal microflora was obtained from the experiment. described in Fig. 2. The recovery of fecal OA in convetttional rats was initially low and fell to zero in all animals aft’cr 3-5 days of OA ingestion. On the other hand, germ-free rats continuously excreted large amounts of OA in theit feces. i2ft,er being ittt,ragastricalIy inoculated wit’h a fecal extract’ frotn OA-ingesting conventional rats, gerrtikfree rats (ex-germfree) exhibited the adaptive response t,o OA, and after 3 days of OA-feeding, none of the compound could be detected in their feces. It is clear, therefore, that the intestinal microflora adapts rapidly and is soon capable of catabolizing large quantities of OA. Thus, much of the CY402 recovered following the intragastric administraton of OA-7-Cl4 in conventional rats (Fig. 1) could represent microbial rather than t’issue activity.
METABOLISM
OF EXOGENOUS
OROTIC
ACID
15
100 90
HOURS
1. Recovery of Cl402 from rats given OA-7-Cl4 by stomach tube. Weanling rats were fed basal diet R-1.FF for 7-10 days. Then the diet of some was supplemented with OA or OA + Ad.S04 for (i days (adaptation). Finally, all were tube fed 5 ml of diet W-G + 1% OA-7-C14 (based on dry weight), containing 2 PC C’4, with or without the addition of Ad.SOa , and COz was collected for 2738 hours. FIG.
Legend
No. rats
A--A
6 2 2
o--o o--o
Supplement to diet RlFF during adaptation (6 days)
Supplement to diet W-6 for tube-feedin:
None 1%) Oh
I’;, OA-7-V 1%; OA-7-C’” I’,,; OA-7-C”
170 0.-1 + 0.25% Ad .SOd
It is noteworthy that in the germ-free rats fed 1% OA for 5 days or more (with basal diet GF-I), liver triglyceride and cholesterol levels were elevated, and plasma triglycerides, cholesterol, and phospholipids were greatly depressed, the situation being qualitatively and quantitatively identical to that previously found in conventional rats ingest’ing similar diets (8). And, as with conventional rats, the addition of 0.25 % diet,aryAd.S04preventedall of thesechanges URIDINE-5’-PHOSPHATE GERM-FREE
FORMATION
IN
RUTS
When microbial activity is excluded, the production of Cl402 following the administration of OA-7-C14 is a measure of the conversion of OA to UMP (12). Figure 3 shows rats recovery of C1402 from germfree adapted to diets containing 1% OA, with and without addition of Ad.SOd, and tube fed a meal containing OA-7-C14. In contrast with conventional rats to experiments (Fig. l), less than 25 % of t’he radioactivity was recovered in respired COz. The presence
+ 0.257;, Ad.SO1
of Ad. SO4 in the diet (and in the tube-fed meal) further reduced recovery in COz by 25 70, mostly after 12 hours. As will be indicated in subsequent tables, the remainder of the Cl4 was recovered in feces plus intestinal contents (53-6.5%; avg. 59%) and in urine (6-11% ; avg. 9 %). Over-all recovery was 89 %’ (83-92 %). EXCRETION
OF OROTIC
PYRrnfmrms AND
ACID IN
AND
OTHER
URINE
FECES
Recoveries of OA in the feces and urine during continuous administration of the compound in the diet, after administration of a tube-fed meal containing 1% OA-7-Cl4 and after intraperitoneal injection of OA-7-04, are given in Tables I and II. When OA-7-Cl4 was administered, recovery is expressed in terms of recovery of CY4.To det,ermine if t,he Cl4 in these samples was OA-7-C14, the following test was applied. An aliquot, of t,he LiOH extract of the sample was incubated in a closed vessel (large, Warburg-type) for 144u hours with crude orotidylic acid pyro-
16
WINDMUELLER,
MCDANIEL, AND HPAETH
24,
phosphorylase and orotidylic acid decarboxylase prepared from yeast (15), with and without addition of ?phosphoribosyl-lpyrophosphate (PRPP). The react’ion was stopped by addit’ion of HaSOd, and all COa liberated was trapped in a mixture of phenethylamine and methanol and counted (see E.rperz&ental). The counts obtained were compared to those obtained by counting directly a similar aliquot of the LiOH extract’ (see flzpe~in~ental). If all the counts in t,he extract were recovered as C1402 when PRPP was added t’o the incubation and less than 13 when it was not, then extract radioactivity was assumed to be OA-7-Cl4. This was found to be t,he case for all urine samples and for fecal and intestinal samples from gern-free rats. When 1% OA was continuously supplied in t,he diet, conventional rats, once they were adapted, excret)ed none in feces but 20 9%in urine (Tables I and II). Germ-free rats excreted one t’hird of their intake in feces and about 10 % in urine. The addition of dietary Ad.SO, did not influence fecal excretion but, did increase by 50 ‘i; urinary excretion in both conventional and germ-free animals. When OA-7-C14 was tube fed, less of the dose was absorbed as indicated by the greater fecal recovery (feces plus int’estinal contents) in the germfree rats and the generally reduced urinary recovery in both convent,ional and germ-free rats when compared with results of ad Zibitum OA administration. Adaptation did not significantly change the amount excreted in urine by conven-
I
_. i 56r
Germ/ree (91
16 8 0 OAI DAY AFTER OA FEEDING WAS BEGUN
Flc. 2. Fecal excretion of erotic acid by conventional and germ-free rats. Rats (120-150 gm) were fed basal diet GF-1 for 10 days. Then 1% OA was added as a supplement (indicated by arrow). Fecal collections (24.hour) were made 1 day before and for 9 days after the addition of OA. E&germfree rats were removed from a sterile isolator on day -1, inoculated intragastrically with 1 ml of an homogenate of feces from rats fed for several months on a diet containing 1’;; OA, and thereafter handled as conventional rats. Numbers in parentheses indicate the number of animals, and vertical lines represent one standard error of the mean. TABLE FEV.IL
EXCRETION
OF EXCIGENOIJS
ORWIC
are expressed as mean (j& of administered number of rats. All rats were OA-adapted
Data indicate
Method of 0.4 administration
Ad lib.feeding
as 1% of diet (It-1 or GF-1) OA-7-C’” tube-fed as lyO of diet
(W-6)"
I
ACID
IS
CUNVENTIONAL
.\KD
GERM-VKEE
dose (OA or Cl”) & SE. Numbers (see Ezpe~?mental).
R.\Ts
in parentheses
Germ-free
Conventional -Ada
+4d
0.0
0.0
(7) 0.4 zk 0.2
(2) 0.6 zk 0.3
(2)
(2)
-Ad
33.5 f 3.6 (JP 57.2 f 2.3c (3)
o Ad denotes addltlon of O.25,0 (” Ad.804 to the diet. h Mean yc recovery from 4 rats over a ‘i-day period. c 27?;; in feces and 30% in the gastrointestinal tract (48 hours after tube-feeding). d 217; in feces and 41y0 in the gastrointestinal tract (48 hours after tube-feeding). e For more detail, see legend to Figs. 1 and 2.
+Ad
33.3 zk 2.3 (I)* 62.3 It 2.5d
(2)
METABOLISM 26
I
I
4
8
OF EXOGENOUS I
OROTIC
17
ACID
I
I
I
I
I
28 24 HOURS
32
36
40
44
I
24 22 20
4
0 0
12
16
20
4t
of CPO2 from germ-free rats given OA-7-C’” by stomach tube. Weanling germ-free FIG. 3. Recovery rats were fed basal diet GF-1 for 7-20 days. Then the diet was supplemented for 7-10 days with OA or OA + Ad.SO, Finally, all were tube fed 4 ml of diet W-6 + 1% OA-7-P (based on dry weight), containing 2 PC Cr4, with or without the addition of Ad.SOa , and COZ was collected for 47 hours. The germ-free state was maintained throughout, as described under Ezperimental. Vertical bars represent one standard error of the mean. Legend
No.
rats
o--o
4
o--o
3
Supplement to diet GF-1 during adaptation (7-10 days)
Supplement
I?& 08 1R 08
1% OA-7-P 1% OA-7-C’”
+ 0.25y0 Ad.SOa
tional rats, nor did dietary Ad.SO, appear to alter consistently urinary or fecal elimination in any of the rats. However, when OA-7-Cl4 (tracer dose) was injected intraperitoneally, without fasting the rats, the presence of dietary Ad. SO, did consistently increase urinary elimination of the labeled compound. The urinary elimination of uracil appears to be increased by feeding 1% OA, and the increase is largely prevented by Ad. SO, (Table III). The excretion of pseudouridine in urine was altered little, if any, by the diet modifications.
to diet
W-6 for tube-feeding
+ 0.25% Ad.SOd
DISCUSSION
The intestinal microflora of conventional rats rapidly adapts to catabolize all OA which is not absorbed from the gut. Such microbial activity is not related, however, to the effects of dietary OA on hepatic lipid and nucleotide metabolism, as is evident from the similar way in which conventional and germ-free rats respond to to dietary OA. The products of OA fermentat’ion by a common intestinal bacterium, ZymobacteGwn oroticum, grown in liquid culture, have been identified as ethanol,
18
WINDMUELLER,
MCDANIEL, TABLE
AND
SPAETH
II
~JRIN.~RY EXCRETION OB ADMINISTERED OHOTIC ACID IN CONVENTION.IL .IND GERM-FREE Ii.1~ Data are expressed as mean “i, of administered dose (OA or Cl*) + SE. Numbers in parentheses indicate number of rats. Conventional Method of OA administration
Ohadapted
Unadapted -Ada
Ad lib. feeding as 1% of diet (P-1 or GF-1) OA-7-C14 tube-fed as 1:; of diet (W-6)* OA-7-C’4 injected i.p. (tracer dosejc
-Ad
+Ad
2.5 (1)
3.1 * 0.4 (5) 13.2 f 6.0 (3)
19.8 zt 2.3 (5Y 4.4 f 0.6
Germ-free +Ad
29.6 + 1.2 (5P 7.3 r!z 0.5
(2)
(2)
12.6 + 2.2
40.1 zt 5.3 (5)
(6)
(I Ad denotes addition of 0.25(/;, Sd.SOa to the diet. b For more detail see legend to Figs. 1 and 2. c IJrine was collected for 24 hours after injection of 2 kc (0.24 rmoles d Urinary OA was determined for each rat for at least 4 days. TABLE
III
EFFECT OF FEEDING OROTIC ACXD AND ADENINE ON THE URINARY EXCRETION OF URACIL AND PSEUDOURIDINE~
Additions
to basal (RlFF)
Excreted (m,n/Z+
die@
Uracil
None lYO OA lyO OA + 0.25% Ad.SO4
in Urine hours)’ Pseudouridine
0.30 f- 0.021.41 0.72 + 0.061.07 0.47 zt 0.081.20
zk 0.06 i 0.04 i 0.15
a The authors are very grateful to Dr. S. Weissman for performing these analyses (17). B Diets were fed 10 days preceding and also during the urine collection. c Each number is the mean of 4 values from 2 rats + SE. acetic acid, COZ, and KH3.l The present studies emphasize the large role which the intestinal microflora may play in the metabolism of compounds administered intragastrically, and they also indicate that. a substantial portion of the total COz output. of conventional rats may be of microbial origin. The average hourly CO2 production of the conventional rats in these studies was 20.7 f 0.7 (SE) mmoles/lOOgm body weight compared with 15.4 f 0.8 mmoles/lOO gm body weight for the germ-free rats. Van ’ W. E. C. Moore,
personal
communication.
-Ad
10.3 & 0.9 (3Y 10.0 * 0.8 (4)
(OA-adapted) +Ad
15.2 + 1.2 (3P 6.9 Ik 0.5 (3)
of OA-7-C14).
Euler et al. (5) reported highly variable fecal recoveries after tube feeding OA-6-C14, and they also found a higher recovery of C1402 when labeled OA was administered to rats fed OA for 10 days as compared to 1 day. Bot’h these findings are now explicable in terms of microbial activity in the gut,. Under the conditions in which dietary O-4 alters hepatic lipid and nucleotide concentrations, only 25-56 % of the compound is metabolized. The higher figure, obtained when rats had free access t’o diet, is probably more nearly correct. The lower estimate was obt,ained from tube-feeding experiments and reflects the decreased absorption of OA when administered in t’his way. Thus, lOO-gm rat ingesting 12 gm of diet is converting, daily, about 430 pmoles of exogenous OA to UMP. That, this conversion occurs largely in the liver is indicated by the st’udies of Hurlbert and Potter (18) and Rabkin et al. (12). This conversion continues despite hepatic accumulation of UMP (5), which, in soluble preparations from rat liver, conpetitively inhibits orotidylic acid decarboxylase (19). Thus our in viva experiments do not provide evidence for the suggestred (19) feedback regulating role of UMP in controlling pyrimidine nucleotide biosynthesis. Liver slices from OA-fed rats also show 110 diminished UMP formation from OA (lo), although their UR4P cont,ent, is elevated.
METABOLISM
OF EXOGENOUS
The presence of adenine in t,he diet does not, influence the amount of OA absorbed from the int,estinal tract. It does, however, iucrease
somewhat
the amount
excreted
in
urine and decrease the amount convert,ed to UJII’. The increase in urinary excretion is very large when the OA is administered intraperitoneally. These in vivo eff ect.s of adenine are compatible with the finding that in soluble preparations from rat liver, adenine can reduce UNIP formation from OA when available PRPP is limiting (5). It is tempting to invoke such a reduct,ion t’o explain the rapid disappearance of the elevated hepatic acid-soluble uridine level when OA-fed rats are supplemented with adenine (8). However, small effects of adenine on the relatively URlP format,ion from OA in t)hc whole animal
make
it unlikely
that’
competition
for PRPP is the only fact,or involved. Even though the presence of adenine in the diet diminishes
only
slight’ly
the amount
of 31
met,abolized by the rat, it, prevents almost completely the accumulation of high steadyst,ate concentrations of U?tIP and other acidsoluble uridine derivatives in the liver (5, 7). This action of adenine, as well as it,s ability
to e1evat.e rapidly
hepatic
adenine
nucleotide levels which have been depressed by feeding OA, suggests that, adenine counkrs the effect, of OA by augmenting the endogenous acid-soluble hepatic adenine pool, which is diminished in size by OA in an as yet, unknown way. The failure of dietary OA, with or without adenine, to produce increased pseudo-uridine excretion (Table III) indicates that 110 accelerat.ed turnover of soluble volved (17).
ribouucleic
acid
is in-
OROTIC
ACID
19
ACKNOWLEDGMENT
We gratefully acknowledge the assistance of Mr. Edward Barron in the care of the germ-free rats. REFERENCES 1. REICHARD, P., Advan. Enzymol.
21, 263 (1959). 2. STANDERFEK, S. B., AND HANDLEK, P., Proc. Sot. Erptl. Biol. Med. 20,270 (1955). L., and HAND3. CREASEY, W. A., HANKIN, SCHUMACHER, R. E., J. Biol. Chem. 236, 2064 (1961). 4. RAJALAKSHMI, S., SARMA, D. S. R., AND SARMA, P. S., Biochem. J. 80,375 (1961). 5. VON EULER, L. H., RUBIN, R. J., AND HANDSCHUMACHER, R. E., J. Biol. Chem. 236, 2464 (1963). 6. RAJALAKSHMI, S., SAKMA, D. S. R., AND SARMA, P. S., Znd. J. Ezptl. Biol. 1,63 (1963). 7. WINDMUELLER, H. G., submitted to J. h’utr. 8. WINDMUELLER, H. G., J. Biol. Chem. 239, 530 (1964). 9. WINDMUELLER, H. G., Biochem. Biophys. Res. Commun. 11, 496 (1963). 10. HANDSCHUMACHEII, R. E., CREASEY, W. A., JAFFE, J. J., PASTERNAK, C. A., AND HANKIN, I,., Proc. K’atl. Acad. Sci. U. S. 46, 178 (1960). 11. WILLIAMS, J. N., JR., J. Kutr. 73,199 (1961). 12. RABKIN, M. T., FREDERICK, E. W., LOTZ, &I., AND SMITH, L. H., JR., J. Clin. Invest. 41, 8il (1962). 13. EISENBERG, F., in “Liquid Scintillation
Counting,” p. 123. Macmillan (Pergamon), New I7ork (1958). 14. WOELLER, F. H., Anal. Biochem. 2, 580 (1961). 15. ROSENBLOOM, F. hr., AND SEEGMILLER, J. E., J. Lab. Clin. Med. 63, 492 (1964). 16. BRAY, G. A., Anal. Biochem. 1,279 (1960). 17. WEISSMAN, S., EISEN, A. Z., LEWIS, Al., AND KARON, i\l., J. Lab. Clin. Med. 60,40 (1962). 18. HURLBERT, K. B., AND POTTER, 1.. It., J. Biol. Chem. 196, 257 (1952). 19. BLAIR, I). G. R., AND POTTER, I-. R., J. Biol. Chem. 236, 2503 (1961).