- Email: [email protected]
The excretion of formiminoglutamic acid by the rat. Influence of dietary ethionine and fat
ARCHIVES
OF BIOCHEMISTRY
The
AND
87, 306-311
(1960)
Excretion of Formiminoglutamic Influence of Dietary Ethionine
M. SILVERMAN, From
BIOPHYSICS
the NationalInstitute Public Health
Service,
R. C. GARDINER
of Arthritis and Metabolic Department of Health, Received
AND
Diseases, Education,
October
Acid by the and Fat
Rat.
H. A. BAKERMAN National Institutes of Health, United and Welfare, Bethesda, Maryland
States
26, 1959
Dietary ethionine blocks the metabolism of histidine in the rat at the level of urocanic acid and induces the urinary excretion of this metabolite. High concentrations of dietary fat tend to lower the folic acid and vitamin B12 concentrations of the liver; consequently, the excretion of formiminoglutamic acid is increased. INTRODUCTION
MATERIALS
The quantity of formimino-L-glutamic acid (FGA) excreted in urine by the rat depends upon the availability of histidine (the precursor of FGA) (l), and the degree to which the rat is depleted of folic acid (2, 3) and vitamin Biz (4). After elevated excretion of FGA has been induced, it can then be greatly diminished by suitable concentrations of dietary methionine (4). Thus, it was anticipated that dietary ethionine, a methionine antagonist (5), would tend to increase the urinary excretion of the formimino compound. Instead, it has been found that ethionine (a) reduced the urocanase activity of liver, (b) blocked the metabolism of histidine at the level of urocanic acid, and (c) consequently diminished or abolished the output of FGA. Dietary fat is another factor which indirectly influences the excretion of the formimino compound. It has been found that rats fed high levels of fat tend to excrete increased amounts of FGA. This phenomenon appears to be related to reduced amounts of folic acid and vitamin Blz in the livers of these rats. The present communication describes the observations made on (a) the influence of ethionine on urocanic acid and FGA excretions, (b) the influence of dietary fat on the excretion of FGA, and (c) an attempt to relate the excretion of FGA by the rat to the folic acid and vitamin B12 concentrations in its liver
The major sisted of 9% oil, and 67% vitamin B,, adequate with
AND
METHODS
components of the basal diet concasein, 20% hydrogenated vegetable sucrose. Except for folic acid and which were omitted, the diet was respect to the salts and vitamins
(4). Month-old male Sprague-Dawley rats weighing between 40 and 45 g. were used. Folic acid in liver was determined by the procedure of Chang (6) modified by the addition of 8 mg. ascorbic acid/ml. digest. Essentially the same values were obtained by assay with Streptococcus jaecalis and Pediococcus cereuisiae (Leuconostoc citrovorum, 8081), indicating that all of the folic acid activity released after autolysis was in the form of Nh-formyltetrahydrofolic acid. Vitamin Bi2 was determined by the U.H.P. procedure, one-tenth the recommended concentration of sodium metabisulfite (7) being used. Total nitrogen in liver homogenates was determined by a modified Kjeldahl procedure (8). Urocanase activities of liver extracts were determined by the method of Tabor and Mehler (9). ESTIMATION
OF
UROCANIC
ACID
AND
FGA
In the technique employed for the detection of EGA, crude dialyzed extracts of chicken liver were used as the source of the enzyme complex which (a) generates tetrahydrofolic acid and (b) converts the formimino carbon to a formyl group linked to the Nro position of tetrahydrofolic acid. The latter, after conversion to NS-formyltetrahydrofolic acid is measured microbiologically. In this test system, urocanic acid is readily converted to FGA and thus is an active formylating agent (IO, 11). However, after being autoclaved in dilute alkali, the imidazole compound retains it,s activity, 306
EXCRETION
OF
FORMIMINOGLUTAMIC
whereas FGA is degraded to inactive compounds. For the determination of FGA and urocanate in urine, the total formylating activity in the untreated sample was determined. An aliquot (1 ml. + 1 drop 0.5 N KOH) was autoclaved at 10 lb. steam pressure for 10 min. and neutralized (1 drop of 0.5 N HCl) (12). The formylating activity in the sample so treated was due to urocanic acid. FGA was represented by the difference in activity between the unheated and heated samples. From 90 to 100% of urocanic acid or FGA added to the digests was recovered. All of the formylating activity of the urine samples studied could be accounted for as FGA and urocanic acid. This was demonstrated by the resolution of the activity of urine into two fractions by adsorption on Dowex 50 H+ and selective elution. One fraction eorresponded in stability and spectrophotometric and elution properties to urocanic acid. The other contained a heatand alkali-labile compound which gave rise to glut,amic acid on inactivation by alkali and which W.&S eluted from Dowex 50 H+ in the FGA fraction, and in the assay system gave rise to N1o-formyltetrahydrofolic acid. The total formylating activity of urine resided in these two fractions. Urocanic acid, when present in sufficiently high concentrations in rat urine may be determined spectrophotometrically. Rat urine corresponding in volume to one-sixth of a day’s output was passed through 3 ml. Dowex 50 H+ packed in a 25-ml. buret (10 mm. diameter). The resin was washed with 25 ml. water, followed by 40 ml. of 1.1 N H&SO4 , and the urocanic acid was eluted with 60 ml. of 1.95 N HzS04 . Then 0.4 ml. of the eluate was diluted to 3.0 ml. with water, the absorbancy at 268 rnh was determined, and the concentration of urocanate was calculated using the molar extinction coefficient 19,600. When determined by this procedure, values for urocanic acid corresponding to 2,.~moles/day or less are of little significance. Values in this order of magnitude have been found in samples of urine from normal rats which show no urocanate when tested by the procedure employing chick liver extracts. As seen below, both of the above methods when applied to the rat urines under study gave results which were in reasonable agreement. Values shown are in micromoles/day. smple Spectrophotometric Biologically as myltetrahydrofolic
A B _______ N6-foracid
C
D
E
18.013.727.418.525.2 15.8 12.824.4 19.9 24.4
When high concentrations of FGA were present, the spectrophotometric procedure was employed.
307
ACID ISOLATION
OF
UROCANIC
ACID
Two groups of six rats each were fed diets containing 0.05% nL-ethionine and 1% L-histidine. One group was fed the basal diet and another the basal diet plus folic acid and vitamin B12 together with the supplements indicated above. Urine from these animals was collected for 3 days. Bioassay indicated a total excretion of 332 mg. urocanic acid. The urine, total volume of 500 ml., was passed through a column of Dowex 50 H+ (3 cm. diameter, 30 cm. long). The resin containing the urocanic acid was washed with 5 1. water and 1 1. of 1.1 N HxS04 . The active material was eluted with 1.95 N H,SO, . Fractions varying in volume from 75 to 200 ml. were collected. At this stage, the active fractions selected were those which in 0.26 N H&O4 showed an absorption peak at 268 rnp, characteristic of urocanic acid. The most active fractions were combined and contained 258 mg. urocanic acid. The combined fractions, total volume 1.34 I., were freed of sulfuric acid with solid BaC03 added in 16 portions. After the addition of each portion, the suspension was stirred until the evolution of CO* ceased, and the BaS04 formed was separated by filtration and washed with water. The wash and filtrate were combined and concentrated to a convenient volume. This process was repeated until all the sulfate had been removed. The solution was concentrated (30 mm. Hg, 40°C.) to 25 ml., and a white crystalline product separated. The product (160 mg.) was recovered by filtration and dried for 2 hr. at room temperature in zxxcuo (0.1 mm. Hg). The mother liquor was concentrated t6 5-6 ml., and a second crop of crystals (63 mg.) was obtained. Both crystalline products possessed the ultraviolet absorption spectrum of urocanic acid, and by direct comparison with a synthetic sample’ were SO”]O pure. The first product was recrystallized from water and dried as above. Elemental analysis2 showed it contained 52.26% C, 4.39% H, and 20.09’% N. Theoretical values for CsNtHaOz are 52.17y0 C, 4.38oja H, and 20.28% N. The melting points of the isolated compound and an authentic sample of urocanic acid were in agreement; isolated compound 229-230’; synthetic 228-229”; mixed 229-230”. The melting point values are uncorrected and were determined on a Fisher-Johns block. In butanol-water-acetic acid (40:50: 10) both the isolated and synthetic compounds migrated at the same rate (I?, = 0.56). Spectra in the ult,raviolet were consistent with those of urocanic acid: in 0.01 N HCl, maximum absorption 1 Kindly 2 Carried Alford.
supplied by Dr. Howard out under the direction
Bond. of Dr.
W. C.
308
SILVERMAN,
GARDINER
occurred at 267 rnM; in 0.01 N KOH, at 278 rnb; in 0.01 M PO4 pH 7.2, at 277 rnp, and in 6 N KOH, at 308 rnp (13). In 0.01 M PO& pH 7.2, E27~ = 18,400; under these conditions, Mehler and Tabor (13) have reported E271 = 18,800. It was concluded the substance isolated was urocanic acid. RESULTS
Animals fed diets containing ethionine and given a test dose of histidine generally excreted lessFGA than did the control animals. This was true whether or not the diets were supplemented with folic acid or vitamin Blz (Table I). The animals fed ethionine excreted large amounts of a stable substance capable of yielding an active formyl group which condensed with tetrahydrofolic acid. This substance in the urine has been characteriied as urocanic acid (see Materials and Methods). The accumulation of urocanic acid in the urine suggestedthat the dietary ethio&e interfered with the activity of urocanase. Data concerned with the nature of the lesion (reduced urocanase activity) induced by ethionine and the reversibility of this lesion are shown in Table II. Two groups of animals were employed for this purpose: one had been maintained on the basal diet to which both vitamin Blz and folic acid had been added and another maintained on the basal diet alone. Six animals in each group were fed diets containing ethionine. After the injection of a test dose of histidine, those animals maintained on the ethionine ,diets excreted significant amounts of urocanic acid. After 6 weeks the initial diets I TABLE EFFECTS
OF ETHIONINE
ON
THE
URINARY
AND
BAKERMAN
of selected animals were changed. The new diets were fed for a period of 4 days, samples of urine were collected, and the animals were sacrificed. The results obtained are shown in Table II. The control animals (Nos. 58-59) excreted insignificant’ amounts of urocanic acid and their livers contained high concentrations of urocanase activity. Animals Nos. 61 and 62 continued to excrete the same amounts of urocanic acid, and extracts of their livers were almost devoid of urocanase activity. The addition of methionine (Nos. 63-64) or the removal of ethionine (Nos. 6546) resulted in (a) reduction in the excretion of urocanic acid and (b) the apparent regeneration of urocanase activity of liver. Removal of the ethionine appeared to be more effective than addition of methionine in elevating the urocanase activity. Roughly the same results were obtained with animals fed the basal diet devoid of vitamin B12 and folic acid. However, with respect to reversal of the urocanase activity the results were less dramatic. This may be due in part to the requirement for folic acid in the synthesis of urocanase (14). Of interest is the observation that upon the removal of ethionine from the diet (Nos. 77-78) the animals regained the ability to convert histidine to FGA. No evidence could be found for the presence of an inhibitor of urocanase in the inactive extracts. In three trials employing mixtures of equal volumes of inactive and active extracts, no loss of urocanase activity occurred. I EXCRETION
OF FGA
AND
UROCANIC
ACID
Values shown are for the metabolites excreted in 24 hr. after the intraperitoneal injection of 48~moles L-histidine. HCl. Average values are given for each group of five animals; ranges are indicated in parentheses. The rats were fed the indicated diets for 5 weeks with the following average weight gains: Group 1,33.4 g.; Group 2, 4.8 g.; Group 3, 24.6 g.; and, Group 4, 18.8 g. Addition
to basal diet
FGA
Urocanic
pmoles
None (2) 0.05% nL-ethionine (3) Vitamin @lzO + folic acidb (4) Vitamin B~z + folic acid + 0.05q7, nL-ethionine (1)
0 One hundred micrograms/kg. 6 Twenty milligrams/kg.
14.4 3.0 4.0 0.2
(10.2-19.5) (0.0-7.9) (1.0-7.0) (0.0-0.9)
acid
$moles
3.0 17.3 1.6 12.0
(2.9-3.4) (10.4-24.9) (0.2-4..0) (6.1-15.6)
EXCRETION
OF
FORMIMINOGLUTAMIC
TABLE REVERSAL
OF THE
309
ACID
II
EFFECTS
OF ETHIONINE
Values shown are for themetabolites excreted in 24 hr. after theintraperitoneal injectionof 48rmoles L-histidine.HCl. The animals were fed the initial diets for 6 weeks and the final diets for 4 davs. Rat No.
Initial
diet
FGA
pmoles
58 59 61 62 63 64 65 66
-I- 0.05’% nt-ethionine + + + + +
Do. Do. Do. Do. Do.
71
14.3
72 73 74 75 76 77 78
6.1 4.5 0.3 7.9 15.8 0.0 2.3
+ + + + +
Do. Do.. Do. Do. Do.
Final
diet
(ch;,m;y
from initial
f.mzoles
6.1
Basal None None 14.4 None 18.8 None 24.9 1% L-methionine 3.1 1.1
17.8 18.0 10.4
FGA
&mwles
Basal + vitamin Bz+ folic acidb 0.9 0.5 None 3.8 1.6 None 0.3 11.6 None 0.9 13.0 None 1.2 3.1 1% r,-methionine added 0.0 15.6 0.0 13.6 Ethionine omitted 0.0
f 0.05% nL-ethionine
U yca$c
Ethionine
omitted
added
0.5 6.9 0.2
1.8 0.7 0.2 6.6 4.8
23.4 16.7 9.7 8.5 3.0 16.2 18.5 26.5
Urocanic acid
Units of urocana?e/ mg. N m homogenate
&wtkoles 0.5 1.7 12.1 11.2 0.5 0.8 2.3 2.3
24.8 33.2 2.8 2.1 13.4
4.1 3.1
28.2 20.1 4.5 3.3 5.1 8.3 11.5 7.2
11.8 14.2 7.0 4.8 6.3 7.4
9.1 17.2 34.1
a One hundred micrograms/kg. a Twenty milligrams/kg.
In a second trial employing as many animals, essentially the same results as those shown in Table II were obtained. INFLUENCE
OF
FAT
In an attempt to investigate the influence of dietary fak on the excretion of FGA by the rat, four groups of animals were fed diets containing 0, 5, 20, and 30 % fat (hydrogenated vegetable oil, Crisco) in the basal diet. At the end of 5 weeks, the excretion of FGA by each of the rats was determined, the animals were sacrificed, and the concentrations of folic acid and vitamin Blz in the livers were determined. In order to facilitate treatment of the data, the animals receiving 20 and 30 ‘% fat were treated as one group and divided into two subgroups, low and high excretors of FGA. Examination of Table III suggeststhat animals receiving the higher levels of dietary fat excreted greater amounts of FGA. In turn, this may be correlated with reduced concentrations of folic
acid and vitamin B12in the liver. In general, if the patterns for the individual animals are examined, it is found that elevated excretion of FGA is associated with a decreased concentration of folic acid or vitamin B1z in the liver. The converse situation is demonstrated by the findings with animal No. 118. This rat, in the group fed 20-30% fat, had the lowest FGA excretion and the highest vitamin content in liver. The results shown in Table IV are an attempt to relate FGA excretion and the folic acid and vitamin Blz concentrations in the livers of rats fed the two B vitamins. The rats employed were fed the indicated diets for 17 weeks prior to sacrifice. The two animals on the vitamin B1z-deficient diet (Nos. 83-84) appeared to be recovering spontaneously from the metabolic defect associated with the excretion of FGA. In any case, it is evident that despite elevated vitamin B1z levels in the livers of animals Nos. 79 and 80, significant amounts of FGA appeared in
TABLE
III
INFLTJENCE OF DIETARY FAT ox FGA Rat No.
Per cent fat in diet
101 102 103 104 105 106 107 108 109 110
0 0 0 0 0 5 5 5 5 5
1.6 5.0 5.6 1.7 1.4 2.3 2.2 1.1 5.9 6.0
111 113 118 119 120
20 20 30 30 30
4.5 5.8 3.3 8.2 6.7
112 114 115 116 117
20 20 20 30 30
12.4 17.8 13.0 27.1 10.9
FGAj24 hr.
Folic acid in liver
Average
rmoles
2.72 1.24 1.05 1.46 1.46 2.09 1.18
3.1
(k&b
0.96 1.04 1.50 0.78 0.92
71 64 96 52 45
1.04 (2.88)b
1.34 0.50 0.57 0.48 0.22
Q In all diets the fat was replaced by an equal weight present. The animals were fed the above diets for 5 weeks 6 ( ) Fisher’s t value. c Significant at 1% level. Per cent fat in diet Gain in weight g. range 37.4 (27-44) 0 5 38.8 (31-42) 20 41.0 (31-52) 32.2 (20-46) 30
The values for this group
(?16,
66 (0.59)”
35 32 39 25 34
0.62 (3.39)bz”
of sucrose. No vitamin prior to sacrifice.
TABLE IV AND VITAMIN Blz
33 (3.58)be”
Biz or folic
FGA
Vitamin Bu
acid
was
Weight of liver g. rLl?@v 3.93 (3.29-4.36) 3.81 (3.32-4.42) 4.74 (4.06-5.06) 3.33 (2.90-4.40)
IN LIVER
AND FGA
EXCHETION
in parentheses are those obtained from another group of rats. The EGA were not obtained. Rats were 21 (12) weeks of age at time of sacrifice.
Additions to basal diet and rat NO.
b
excretors
16.2 (3.91)bfG
ACID
1.69 (1.93)b
75
excretors
5.7 (1.53)b
FOLK
w4.ln. 61 68 60 117 69 76 53 83 78 58
1.96
2.56 1.30 1.32
High
OF
AVerape
#R./R.
Low
CONCENTRATIONS
EXCRETION”
excretion
rates
Folic acid
pg./g. liwer
g.
6.
None 86 Folio acid” 83 84 Vitamin Brzb 79 80 Folic acid + 81 82 0 Diet 6 Diet
vitamin
contained contained
81.0
40
(81) (43)
0.68
(1.11) (1.22)
176
7.66
13.2 5.4
44 (67) 66 (35)
9.15 9.50
(8.53) (7.13)
219 178
6.49 6.45
31.2 49.2
138 (178) 136 (174)
1.26 .92
(1.24) (1.06)
184 217
6.88 9.06
4.0 2.8
145 (127) 120 (155)
(13.47) (9.70)
234 224
7.76 6.92
Brz
20 mg. folio acid/kg. 100 pg. (20 pg.) vitamin
Blz/kg. 310
11.10 12.70
EXCRETION
OF
FORMIMINOGLUTAMIC
the urine. This was associated with low folic acid concentrations in the liver. Moderate amounts of FGA (13.2 pmoles/day) occurred in the urine of rat No. 83 whose liver contained 44 mpg. vitamin B1z and, as to be expected, a -normal concentration of folic acid (6). On the other hand, the companion rat (No. 84) whose excret.ion of the formimino compound was almost within normal limits, contained in his liver 66 mwg. vitamin BnJg., an amount apparently sufficient to satisfy the requirements for FGA metabolism. No consistent pattern emerges which relates dietary vitamin Blz to folic acid concentrations in liver.
ACID
311
fat in the basal diet reduces the concentrations of folic acid and vitamin Bn! in the livers of the rats. To the extent that this is true, increased FGA excretion is a consequence of the decreased vitamin content. Under the conditions employed, FGA excretion is induced in the rat when the folic acid or vitamin Blz concentrations in the liver are in t’he region of or below 1.0 pg. or 50 mpg./g., respectively (Tables III and IV). No attempt has been made to establish the mechanism through which fat exerts its effects; however, Spivey, Fox, Ortiz, and Briggs (15) have shown that high dietary fat increases the vitamin B12 requirement for growth of the chick.
DISCUSSION
In the rat the metabolism of histidine via the urocanic acid pathway (histidine -+ urocanic acid -+ imidazolonepropionic acid -+ FGA -+ “formyl” + glutamic acid) can be obstructed at two stages. The absence of dietary folic acid or vitamin Blz imposes a block at the FGA level. The inclusion of dietary ethionine induces a block at the urocanic acid level. Both inhibitions are indicat,ed by increased excretion of these metabolites in urine. The nature of the block at the urocanic acid level is not altogether clear. If one considers the group of animals on the complete diet (Table II, Nos. 58-66), it appears likely that inhibition of urocanase synthesis by ethionine induces the excretion of urocanic acid. However, this interpretation is complicated by the observations that in multiple deficiencies (Table II, Nos. 71-78) the magnitude of the changes in urocanase activities after addition of methionine or removal of ethionine are not consistently of the same order observed for changes in urocanic acid excretion. Complications in interpreting these result’s arise from the fact that (a) methionine in itself tends to reduce the excretion of FGA (4), and (b) the blocks imposed are not 100 % effective. Despite these limitations on the interpretations, it is possible to select individuals from a group of rats fed ethionine and the doubly deficient diet in which the metabolism of histidine is partially obstructed at both the urocanic acid and FGA stages. The data suggest that the presence of 20 %
ACKNOWLEDGMENTS We are grateful to ~Mrs. Marjorie K. Romine for performing many of the microbiological assays and to Miss Paula R. Silverman for assistance in the preparation of diets. REFERENCES 1. TABOR, H., SILVERMAN, M., MEHLER, A. H., DAFT, F. S., AND BAUER, H., J. Am. Chem. Sue. 76, 756 (1953). 2. BAKERMAN, H. A., SILVERMAN, M., AND DAFT, F. S., J. Biol. C&em. 188, 117 (1951). 3. SILVERMAN, M., GARDINER, R. C., AND BAKERMAN, H. A., J. Biol. Chem. 134, 815 (1952). 4. SILVERMAN, M., AND PITNEY, A. J., J. Biol. Chem. 233, 1179 (1958). 5. DYER, H. M., J. Bid. Chem. 124, 519 (1938). 6. CHANG, S. C., J. Bid. Chem. 200, 827 (1953). 7. “The Pharmacopeia of the United States of America,” 15th ed., p. 885. Mack Printing Co., Easton, Pa., 1955. 8. HILLER, A., PLAZIN, J., AND VAN SLYKE, D. D., J. Biol. Chem. 176, 1401 (1948). 9. TABOR, H., ANU MEHLER, A. H., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. II, p. 228. Academic Press, New York, 1955. 10. MILLER, A., ANU WAELSCH, H., Biochim. et Biophys. Acta 17, 278 (1955). 11. SILVERMAN, M., GARDINER, R. C., AND CONDIT, P. T., J. Natl. Cancer Inst. 20,71 (1958). 12. TABOR, H., AND WYNGARDEN, L., J. Clin. Invest. 37, 824 (1958). 13. MEHLER, A. H., AND TABOR, H., J. Bid. Chem. 201, 775 (1953). 14. BALDRIDGE, R. C., 1. Biol. Chem. 231, 207 (1958). 15. SPIVEY Fox, M. R., ORTIZ, L. O., AND BRIQGR~, G. M., Proc. Sot. Ezptl. Biol. Med. 93, 501 (1956).
OF BIOCHEMISTRY
The
AND
87, 306-311
(1960)
Excretion of Formiminoglutamic Influence of Dietary Ethionine
M. SILVERMAN, From
BIOPHYSICS
the NationalInstitute Public Health
Service,
R. C. GARDINER
of Arthritis and Metabolic Department of Health, Received
AND
Diseases, Education,
October
Acid by the and Fat
Rat.
H. A. BAKERMAN National Institutes of Health, United and Welfare, Bethesda, Maryland
States
26, 1959
Dietary ethionine blocks the metabolism of histidine in the rat at the level of urocanic acid and induces the urinary excretion of this metabolite. High concentrations of dietary fat tend to lower the folic acid and vitamin B12 concentrations of the liver; consequently, the excretion of formiminoglutamic acid is increased. INTRODUCTION
MATERIALS
The quantity of formimino-L-glutamic acid (FGA) excreted in urine by the rat depends upon the availability of histidine (the precursor of FGA) (l), and the degree to which the rat is depleted of folic acid (2, 3) and vitamin Biz (4). After elevated excretion of FGA has been induced, it can then be greatly diminished by suitable concentrations of dietary methionine (4). Thus, it was anticipated that dietary ethionine, a methionine antagonist (5), would tend to increase the urinary excretion of the formimino compound. Instead, it has been found that ethionine (a) reduced the urocanase activity of liver, (b) blocked the metabolism of histidine at the level of urocanic acid, and (c) consequently diminished or abolished the output of FGA. Dietary fat is another factor which indirectly influences the excretion of the formimino compound. It has been found that rats fed high levels of fat tend to excrete increased amounts of FGA. This phenomenon appears to be related to reduced amounts of folic acid and vitamin Blz in the livers of these rats. The present communication describes the observations made on (a) the influence of ethionine on urocanic acid and FGA excretions, (b) the influence of dietary fat on the excretion of FGA, and (c) an attempt to relate the excretion of FGA by the rat to the folic acid and vitamin B12 concentrations in its liver
The major sisted of 9% oil, and 67% vitamin B,, adequate with
AND
METHODS
components of the basal diet concasein, 20% hydrogenated vegetable sucrose. Except for folic acid and which were omitted, the diet was respect to the salts and vitamins
(4). Month-old male Sprague-Dawley rats weighing between 40 and 45 g. were used. Folic acid in liver was determined by the procedure of Chang (6) modified by the addition of 8 mg. ascorbic acid/ml. digest. Essentially the same values were obtained by assay with Streptococcus jaecalis and Pediococcus cereuisiae (Leuconostoc citrovorum, 8081), indicating that all of the folic acid activity released after autolysis was in the form of Nh-formyltetrahydrofolic acid. Vitamin Bi2 was determined by the U.H.P. procedure, one-tenth the recommended concentration of sodium metabisulfite (7) being used. Total nitrogen in liver homogenates was determined by a modified Kjeldahl procedure (8). Urocanase activities of liver extracts were determined by the method of Tabor and Mehler (9). ESTIMATION
OF
UROCANIC
ACID
AND
FGA
In the technique employed for the detection of EGA, crude dialyzed extracts of chicken liver were used as the source of the enzyme complex which (a) generates tetrahydrofolic acid and (b) converts the formimino carbon to a formyl group linked to the Nro position of tetrahydrofolic acid. The latter, after conversion to NS-formyltetrahydrofolic acid is measured microbiologically. In this test system, urocanic acid is readily converted to FGA and thus is an active formylating agent (IO, 11). However, after being autoclaved in dilute alkali, the imidazole compound retains it,s activity, 306
EXCRETION
OF
FORMIMINOGLUTAMIC
whereas FGA is degraded to inactive compounds. For the determination of FGA and urocanate in urine, the total formylating activity in the untreated sample was determined. An aliquot (1 ml. + 1 drop 0.5 N KOH) was autoclaved at 10 lb. steam pressure for 10 min. and neutralized (1 drop of 0.5 N HCl) (12). The formylating activity in the sample so treated was due to urocanic acid. FGA was represented by the difference in activity between the unheated and heated samples. From 90 to 100% of urocanic acid or FGA added to the digests was recovered. All of the formylating activity of the urine samples studied could be accounted for as FGA and urocanic acid. This was demonstrated by the resolution of the activity of urine into two fractions by adsorption on Dowex 50 H+ and selective elution. One fraction eorresponded in stability and spectrophotometric and elution properties to urocanic acid. The other contained a heatand alkali-labile compound which gave rise to glut,amic acid on inactivation by alkali and which W.&S eluted from Dowex 50 H+ in the FGA fraction, and in the assay system gave rise to N1o-formyltetrahydrofolic acid. The total formylating activity of urine resided in these two fractions. Urocanic acid, when present in sufficiently high concentrations in rat urine may be determined spectrophotometrically. Rat urine corresponding in volume to one-sixth of a day’s output was passed through 3 ml. Dowex 50 H+ packed in a 25-ml. buret (10 mm. diameter). The resin was washed with 25 ml. water, followed by 40 ml. of 1.1 N H&SO4 , and the urocanic acid was eluted with 60 ml. of 1.95 N HzS04 . Then 0.4 ml. of the eluate was diluted to 3.0 ml. with water, the absorbancy at 268 rnh was determined, and the concentration of urocanate was calculated using the molar extinction coefficient 19,600. When determined by this procedure, values for urocanic acid corresponding to 2,.~moles/day or less are of little significance. Values in this order of magnitude have been found in samples of urine from normal rats which show no urocanate when tested by the procedure employing chick liver extracts. As seen below, both of the above methods when applied to the rat urines under study gave results which were in reasonable agreement. Values shown are in micromoles/day. smple Spectrophotometric Biologically as myltetrahydrofolic
A B _______ N6-foracid
C
D
E
18.013.727.418.525.2 15.8 12.824.4 19.9 24.4
When high concentrations of FGA were present, the spectrophotometric procedure was employed.
307
ACID ISOLATION
OF
UROCANIC
ACID
Two groups of six rats each were fed diets containing 0.05% nL-ethionine and 1% L-histidine. One group was fed the basal diet and another the basal diet plus folic acid and vitamin B12 together with the supplements indicated above. Urine from these animals was collected for 3 days. Bioassay indicated a total excretion of 332 mg. urocanic acid. The urine, total volume of 500 ml., was passed through a column of Dowex 50 H+ (3 cm. diameter, 30 cm. long). The resin containing the urocanic acid was washed with 5 1. water and 1 1. of 1.1 N HxS04 . The active material was eluted with 1.95 N H,SO, . Fractions varying in volume from 75 to 200 ml. were collected. At this stage, the active fractions selected were those which in 0.26 N H&O4 showed an absorption peak at 268 rnp, characteristic of urocanic acid. The most active fractions were combined and contained 258 mg. urocanic acid. The combined fractions, total volume 1.34 I., were freed of sulfuric acid with solid BaC03 added in 16 portions. After the addition of each portion, the suspension was stirred until the evolution of CO* ceased, and the BaS04 formed was separated by filtration and washed with water. The wash and filtrate were combined and concentrated to a convenient volume. This process was repeated until all the sulfate had been removed. The solution was concentrated (30 mm. Hg, 40°C.) to 25 ml., and a white crystalline product separated. The product (160 mg.) was recovered by filtration and dried for 2 hr. at room temperature in zxxcuo (0.1 mm. Hg). The mother liquor was concentrated t6 5-6 ml., and a second crop of crystals (63 mg.) was obtained. Both crystalline products possessed the ultraviolet absorption spectrum of urocanic acid, and by direct comparison with a synthetic sample’ were SO”]O pure. The first product was recrystallized from water and dried as above. Elemental analysis2 showed it contained 52.26% C, 4.39% H, and 20.09’% N. Theoretical values for CsNtHaOz are 52.17y0 C, 4.38oja H, and 20.28% N. The melting points of the isolated compound and an authentic sample of urocanic acid were in agreement; isolated compound 229-230’; synthetic 228-229”; mixed 229-230”. The melting point values are uncorrected and were determined on a Fisher-Johns block. In butanol-water-acetic acid (40:50: 10) both the isolated and synthetic compounds migrated at the same rate (I?, = 0.56). Spectra in the ult,raviolet were consistent with those of urocanic acid: in 0.01 N HCl, maximum absorption 1 Kindly 2 Carried Alford.
supplied by Dr. Howard out under the direction
Bond. of Dr.
W. C.
308
SILVERMAN,
GARDINER
occurred at 267 rnM; in 0.01 N KOH, at 278 rnb; in 0.01 M PO4 pH 7.2, at 277 rnp, and in 6 N KOH, at 308 rnp (13). In 0.01 M PO& pH 7.2, E27~ = 18,400; under these conditions, Mehler and Tabor (13) have reported E271 = 18,800. It was concluded the substance isolated was urocanic acid. RESULTS
Animals fed diets containing ethionine and given a test dose of histidine generally excreted lessFGA than did the control animals. This was true whether or not the diets were supplemented with folic acid or vitamin Blz (Table I). The animals fed ethionine excreted large amounts of a stable substance capable of yielding an active formyl group which condensed with tetrahydrofolic acid. This substance in the urine has been characteriied as urocanic acid (see Materials and Methods). The accumulation of urocanic acid in the urine suggestedthat the dietary ethio&e interfered with the activity of urocanase. Data concerned with the nature of the lesion (reduced urocanase activity) induced by ethionine and the reversibility of this lesion are shown in Table II. Two groups of animals were employed for this purpose: one had been maintained on the basal diet to which both vitamin Blz and folic acid had been added and another maintained on the basal diet alone. Six animals in each group were fed diets containing ethionine. After the injection of a test dose of histidine, those animals maintained on the ethionine ,diets excreted significant amounts of urocanic acid. After 6 weeks the initial diets I TABLE EFFECTS
OF ETHIONINE
ON
THE
URINARY
AND
BAKERMAN
of selected animals were changed. The new diets were fed for a period of 4 days, samples of urine were collected, and the animals were sacrificed. The results obtained are shown in Table II. The control animals (Nos. 58-59) excreted insignificant’ amounts of urocanic acid and their livers contained high concentrations of urocanase activity. Animals Nos. 61 and 62 continued to excrete the same amounts of urocanic acid, and extracts of their livers were almost devoid of urocanase activity. The addition of methionine (Nos. 63-64) or the removal of ethionine (Nos. 6546) resulted in (a) reduction in the excretion of urocanic acid and (b) the apparent regeneration of urocanase activity of liver. Removal of the ethionine appeared to be more effective than addition of methionine in elevating the urocanase activity. Roughly the same results were obtained with animals fed the basal diet devoid of vitamin B12 and folic acid. However, with respect to reversal of the urocanase activity the results were less dramatic. This may be due in part to the requirement for folic acid in the synthesis of urocanase (14). Of interest is the observation that upon the removal of ethionine from the diet (Nos. 77-78) the animals regained the ability to convert histidine to FGA. No evidence could be found for the presence of an inhibitor of urocanase in the inactive extracts. In three trials employing mixtures of equal volumes of inactive and active extracts, no loss of urocanase activity occurred. I EXCRETION
OF FGA
AND
UROCANIC
ACID
Values shown are for the metabolites excreted in 24 hr. after the intraperitoneal injection of 48~moles L-histidine. HCl. Average values are given for each group of five animals; ranges are indicated in parentheses. The rats were fed the indicated diets for 5 weeks with the following average weight gains: Group 1,33.4 g.; Group 2, 4.8 g.; Group 3, 24.6 g.; and, Group 4, 18.8 g. Addition
to basal diet
FGA
Urocanic
pmoles
None (2) 0.05% nL-ethionine (3) Vitamin @lzO + folic acidb (4) Vitamin B~z + folic acid + 0.05q7, nL-ethionine (1)
0 One hundred micrograms/kg. 6 Twenty milligrams/kg.
14.4 3.0 4.0 0.2
(10.2-19.5) (0.0-7.9) (1.0-7.0) (0.0-0.9)
acid
$moles
3.0 17.3 1.6 12.0
(2.9-3.4) (10.4-24.9) (0.2-4..0) (6.1-15.6)
EXCRETION
OF
FORMIMINOGLUTAMIC
TABLE REVERSAL
OF THE
309
ACID
II
EFFECTS
OF ETHIONINE
Values shown are for themetabolites excreted in 24 hr. after theintraperitoneal injectionof 48rmoles L-histidine.HCl. The animals were fed the initial diets for 6 weeks and the final diets for 4 davs. Rat No.
Initial
diet
FGA
pmoles
58 59 61 62 63 64 65 66
-I- 0.05’% nt-ethionine + + + + +
Do. Do. Do. Do. Do.
71
14.3
72 73 74 75 76 77 78
6.1 4.5 0.3 7.9 15.8 0.0 2.3
+ + + + +
Do. Do.. Do. Do. Do.
Final
diet
(ch;,m;y
from initial
f.mzoles
6.1
Basal None None 14.4 None 18.8 None 24.9 1% L-methionine 3.1 1.1
17.8 18.0 10.4
FGA
&mwles
Basal + vitamin Bz+ folic acidb 0.9 0.5 None 3.8 1.6 None 0.3 11.6 None 0.9 13.0 None 1.2 3.1 1% r,-methionine added 0.0 15.6 0.0 13.6 Ethionine omitted 0.0
f 0.05% nL-ethionine
U yca$c
Ethionine
omitted
added
0.5 6.9 0.2
1.8 0.7 0.2 6.6 4.8
23.4 16.7 9.7 8.5 3.0 16.2 18.5 26.5
Urocanic acid
Units of urocana?e/ mg. N m homogenate
&wtkoles 0.5 1.7 12.1 11.2 0.5 0.8 2.3 2.3
24.8 33.2 2.8 2.1 13.4
4.1 3.1
28.2 20.1 4.5 3.3 5.1 8.3 11.5 7.2
11.8 14.2 7.0 4.8 6.3 7.4
9.1 17.2 34.1
a One hundred micrograms/kg. a Twenty milligrams/kg.
In a second trial employing as many animals, essentially the same results as those shown in Table II were obtained. INFLUENCE
OF
FAT
In an attempt to investigate the influence of dietary fak on the excretion of FGA by the rat, four groups of animals were fed diets containing 0, 5, 20, and 30 % fat (hydrogenated vegetable oil, Crisco) in the basal diet. At the end of 5 weeks, the excretion of FGA by each of the rats was determined, the animals were sacrificed, and the concentrations of folic acid and vitamin Blz in the livers were determined. In order to facilitate treatment of the data, the animals receiving 20 and 30 ‘% fat were treated as one group and divided into two subgroups, low and high excretors of FGA. Examination of Table III suggeststhat animals receiving the higher levels of dietary fat excreted greater amounts of FGA. In turn, this may be correlated with reduced concentrations of folic
acid and vitamin B12in the liver. In general, if the patterns for the individual animals are examined, it is found that elevated excretion of FGA is associated with a decreased concentration of folic acid or vitamin B1z in the liver. The converse situation is demonstrated by the findings with animal No. 118. This rat, in the group fed 20-30% fat, had the lowest FGA excretion and the highest vitamin content in liver. The results shown in Table IV are an attempt to relate FGA excretion and the folic acid and vitamin Blz concentrations in the livers of rats fed the two B vitamins. The rats employed were fed the indicated diets for 17 weeks prior to sacrifice. The two animals on the vitamin B1z-deficient diet (Nos. 83-84) appeared to be recovering spontaneously from the metabolic defect associated with the excretion of FGA. In any case, it is evident that despite elevated vitamin B1z levels in the livers of animals Nos. 79 and 80, significant amounts of FGA appeared in
TABLE
III
INFLTJENCE OF DIETARY FAT ox FGA Rat No.
Per cent fat in diet
101 102 103 104 105 106 107 108 109 110
0 0 0 0 0 5 5 5 5 5
1.6 5.0 5.6 1.7 1.4 2.3 2.2 1.1 5.9 6.0
111 113 118 119 120
20 20 30 30 30
4.5 5.8 3.3 8.2 6.7
112 114 115 116 117
20 20 20 30 30
12.4 17.8 13.0 27.1 10.9
FGAj24 hr.
Folic acid in liver
Average
rmoles
2.72 1.24 1.05 1.46 1.46 2.09 1.18
3.1
(k&b
0.96 1.04 1.50 0.78 0.92
71 64 96 52 45
1.04 (2.88)b
1.34 0.50 0.57 0.48 0.22
Q In all diets the fat was replaced by an equal weight present. The animals were fed the above diets for 5 weeks 6 ( ) Fisher’s t value. c Significant at 1% level. Per cent fat in diet Gain in weight g. range 37.4 (27-44) 0 5 38.8 (31-42) 20 41.0 (31-52) 32.2 (20-46) 30
The values for this group
(?16,
66 (0.59)”
35 32 39 25 34
0.62 (3.39)bz”
of sucrose. No vitamin prior to sacrifice.
TABLE IV AND VITAMIN Blz
33 (3.58)be”
Biz or folic
FGA
Vitamin Bu
acid
was
Weight of liver g. rLl?@v 3.93 (3.29-4.36) 3.81 (3.32-4.42) 4.74 (4.06-5.06) 3.33 (2.90-4.40)
IN LIVER
AND FGA
EXCHETION
in parentheses are those obtained from another group of rats. The EGA were not obtained. Rats were 21 (12) weeks of age at time of sacrifice.
Additions to basal diet and rat NO.
b
excretors
16.2 (3.91)bfG
ACID
1.69 (1.93)b
75
excretors
5.7 (1.53)b
FOLK
w4.ln. 61 68 60 117 69 76 53 83 78 58
1.96
2.56 1.30 1.32
High
OF
AVerape
#R./R.
Low
CONCENTRATIONS
EXCRETION”
excretion
rates
Folic acid
pg./g. liwer
g.
6.
None 86 Folio acid” 83 84 Vitamin Brzb 79 80 Folic acid + 81 82 0 Diet 6 Diet
vitamin
contained contained
81.0
40
(81) (43)
0.68
(1.11) (1.22)
176
7.66
13.2 5.4
44 (67) 66 (35)
9.15 9.50
(8.53) (7.13)
219 178
6.49 6.45
31.2 49.2
138 (178) 136 (174)
1.26 .92
(1.24) (1.06)
184 217
6.88 9.06
4.0 2.8
145 (127) 120 (155)
(13.47) (9.70)
234 224
7.76 6.92
Brz
20 mg. folio acid/kg. 100 pg. (20 pg.) vitamin
Blz/kg. 310
11.10 12.70
EXCRETION
OF
FORMIMINOGLUTAMIC
the urine. This was associated with low folic acid concentrations in the liver. Moderate amounts of FGA (13.2 pmoles/day) occurred in the urine of rat No. 83 whose liver contained 44 mpg. vitamin B1z and, as to be expected, a -normal concentration of folic acid (6). On the other hand, the companion rat (No. 84) whose excret.ion of the formimino compound was almost within normal limits, contained in his liver 66 mwg. vitamin BnJg., an amount apparently sufficient to satisfy the requirements for FGA metabolism. No consistent pattern emerges which relates dietary vitamin Blz to folic acid concentrations in liver.
ACID
311
fat in the basal diet reduces the concentrations of folic acid and vitamin Bn! in the livers of the rats. To the extent that this is true, increased FGA excretion is a consequence of the decreased vitamin content. Under the conditions employed, FGA excretion is induced in the rat when the folic acid or vitamin Blz concentrations in the liver are in t’he region of or below 1.0 pg. or 50 mpg./g., respectively (Tables III and IV). No attempt has been made to establish the mechanism through which fat exerts its effects; however, Spivey, Fox, Ortiz, and Briggs (15) have shown that high dietary fat increases the vitamin B12 requirement for growth of the chick.
DISCUSSION
In the rat the metabolism of histidine via the urocanic acid pathway (histidine -+ urocanic acid -+ imidazolonepropionic acid -+ FGA -+ “formyl” + glutamic acid) can be obstructed at two stages. The absence of dietary folic acid or vitamin Blz imposes a block at the FGA level. The inclusion of dietary ethionine induces a block at the urocanic acid level. Both inhibitions are indicat,ed by increased excretion of these metabolites in urine. The nature of the block at the urocanic acid level is not altogether clear. If one considers the group of animals on the complete diet (Table II, Nos. 58-66), it appears likely that inhibition of urocanase synthesis by ethionine induces the excretion of urocanic acid. However, this interpretation is complicated by the observations that in multiple deficiencies (Table II, Nos. 71-78) the magnitude of the changes in urocanase activities after addition of methionine or removal of ethionine are not consistently of the same order observed for changes in urocanic acid excretion. Complications in interpreting these result’s arise from the fact that (a) methionine in itself tends to reduce the excretion of FGA (4), and (b) the blocks imposed are not 100 % effective. Despite these limitations on the interpretations, it is possible to select individuals from a group of rats fed ethionine and the doubly deficient diet in which the metabolism of histidine is partially obstructed at both the urocanic acid and FGA stages. The data suggest that the presence of 20 %
ACKNOWLEDGMENTS We are grateful to ~Mrs. Marjorie K. Romine for performing many of the microbiological assays and to Miss Paula R. Silverman for assistance in the preparation of diets. REFERENCES 1. TABOR, H., SILVERMAN, M., MEHLER, A. H., DAFT, F. S., AND BAUER, H., J. Am. Chem. Sue. 76, 756 (1953). 2. BAKERMAN, H. A., SILVERMAN, M., AND DAFT, F. S., J. Biol. C&em. 188, 117 (1951). 3. SILVERMAN, M., GARDINER, R. C., AND BAKERMAN, H. A., J. Biol. Chem. 134, 815 (1952). 4. SILVERMAN, M., AND PITNEY, A. J., J. Biol. Chem. 233, 1179 (1958). 5. DYER, H. M., J. Bid. Chem. 124, 519 (1938). 6. CHANG, S. C., J. Bid. Chem. 200, 827 (1953). 7. “The Pharmacopeia of the United States of America,” 15th ed., p. 885. Mack Printing Co., Easton, Pa., 1955. 8. HILLER, A., PLAZIN, J., AND VAN SLYKE, D. D., J. Biol. Chem. 176, 1401 (1948). 9. TABOR, H., ANU MEHLER, A. H., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. II, p. 228. Academic Press, New York, 1955. 10. MILLER, A., ANU WAELSCH, H., Biochim. et Biophys. Acta 17, 278 (1955). 11. SILVERMAN, M., GARDINER, R. C., AND CONDIT, P. T., J. Natl. Cancer Inst. 20,71 (1958). 12. TABOR, H., AND WYNGARDEN, L., J. Clin. Invest. 37, 824 (1958). 13. MEHLER, A. H., AND TABOR, H., J. Bid. Chem. 201, 775 (1953). 14. BALDRIDGE, R. C., 1. Biol. Chem. 231, 207 (1958). 15. SPIVEY Fox, M. R., ORTIZ, L. O., AND BRIQGR~, G. M., Proc. Sot. Ezptl. Biol. Med. 93, 501 (1956).