- Email: [email protected]
Porphyrin metabolism V. The metabolism of purines in experimental porphyria
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
80,
416-457
(1959)
Porphyrin Metabolism V. The Metabolism of Purines in Experimental Porphyria’ Ellen L. Talman, Robert F. Labbe,” Robert A. Aldrich” and David Sears3 Frott~
the Departments Oregon
of Riochenristr!/ Medical School,
Received
and Pediatrics, Portlnnd, Oreyon
C:niwrsitg
of
June 27, 1058
INTRODCCTION
A previous report from t,his laboratory (1) described t’he experimental induction of hepatic porphyria in chick embryos by inject,ing nllylisopropylacet,ylcarbamide (Sedormidm) into their yolk sacs. Anatomical abnormslit,ies, retarded growth, and difficulty in hatching were found to constitute parts of the syndrome in this species. The possibility of deranged purine metabolism in experimental porphyria was also ment,ioned. Investigation of this possibilit’y by quuntitation of uric acid output established a marked reduct,ion in the excretion of this compound by porphyric embryos (2). This finding could reflect impaired purine synthesis, catabolism, or excretion. The latt,er two possibilities have now been eliminated by demonst,rating t,hat exogenous adenine is excreted as uric acid by porphyric embryos even more efficient,ly than by their controls. Furthermow, t,racer studies utilizing glycine-2-Cl4 have shown t,hat, porphyric embryos synthesize purines at, a decreased rate. Supplementary adenine also reduced the porphyrin excretion and improved t,he growt,h of Sedormid-t’reated embryos. The latter observations led t,o st,udies showing that, ndeuine improves the growt,h and reduces t,he incidence of congenital anomalies in Sedormid-t’reated chicks. A soluble non-hypnot,ic Sedormid derivative, allylisopropy1acetamide (AM), has largely replaced t’he former compound as a porphyria-producing 1 Supported by a special research grant of the Eli Lilly Company; the Helen Hay Whitney Foundation; A.;C4.A. Council on Pharmacy and Chemistry, William Volker Grant No. 7; National Institute of Arthrit,is and Metabolic Diseases, National Inst,itutes of Health, U. 8. Public Health Service ((irant No. A-752). 2 Present address: Departzment of Pediatrics. IYniversity of Washington School of Medicine, Seattle 5, Washington. 8 With the technical assistance of Margaret S. Shafer. 446
PORPHYRIN METABOLISM. V
447
agent (3-5). Injecting the soluble adenine derivative, adenosine, with AIA produced results qualitatively similar to those obtained with Sedormid and adenine. The findings reported here support the previous suggestion (2, 6) that, in hepatic porphyria, both porphyrin and purine metaholism are deranged. EXPERIMENTAL
Eggs. See Ref. (5) Preparation of Materials
for Injection
Concentrations of all materials were adjusted so that the dose given was contained in a volume of 0.50 ml. Suspensions of Sedormid (20 mg./ml.), adenine (10 mg./ml.), and Sedormid plus adenine (20 and 10 mg./ml., respectively) in isotonic glycerol were prepared and sterilized as described previously (1). AIA (10 mg./ml.) and adenosine (20 mg./ml.) were dissolved in 0.6y0 NaCl, and AIA plus adenosine (10 and 20 mg./ml., respectively) in 0.45’% NaCl. Glycine-2-C14 (2.0 mg./ml., 13.6 pc./mg.) was dissolved in 0.9% NaCl. These solutions were sterilized in the autoclave.
Procerhre Except for glvcine-2-CL4, the compounds and combinations of compounds under investigation were injected into the yolk sacs of S-day embryonated eggs. In studies utilizing Sedormid to induce porphyria, one group of embryos received 10.0 my. Sedormid, a second 10.0 mg. Sedormid plus 5.0 mg. adenine (or 5.6 mg. guanine or .uanthine), a third 5.0 mg. adenine (or 5.6 mg. guanine or xant#hine), and the control group received 0.50 ml. of sterile suspension medium. When ndenine was used, six embryos from each group were sacrificed at 24.hr. int#ervals for a period of 6 days following injection, and their allantoic fluids were aspirated and pooled. After analyzing these pools quantitatively for coproporphyrin, uroporphyrin, and uric acid, the average amount of each compound contained in the allantoic fluid of a single egg was calculated (7). Embryos were then separated from their extraembryonic t,issues, blotted on absorbent paper, and weighed. When guanine and xanthine were used, a similar procedure was carried out hut only on the second and fourth days after t.re:ttment. In experiments utilizing AIA to induce porphyria, one group of emhryos received 5.0 mg. AIA, a second 5.0 mg. AIA plus 10.0 mg. adenosine, a third 10.0 my. adenosine, and the controls received 0.50 ml. of sterile 0.9% NaCl solution. At intervals of 2, 4, and 6 days after treatment, eight or nine eggs from each group were sacrificed, their allantoic fluids pooled for analysis, and the embryos weighed as before. Tracer studies were carried out 6 days after injecting the porphyria-producing drug. At that time, 0.25 ml. (6.8 PC.) of the glycine-2-C14 solution (2.0 mg./ml.) was injected intravenously (S), and the eggs were returned to the incubator. After a designated time had elapsed, the allantoic fluids from each group were collected for the isolation of uric acid (9), and the embryos were removed and quickly frozen in dry ice-acetone. The purines were isolated (10, 11) from 50% homogenates of each group of embryos, and adenine and guanine were then separated from each other chromatographically (12) prior to determination of their Cl4 act,ivities.
TALMAN, LABBE, ALDRICH AND SEARS
448
Analytical
Methods
The techniques of Schwartz and associates for determining coproporphyrin uroporphyrin, as slightly modified in this laboratory (13), were used. Uric acid was determined by the method of Buchanan, Block, and Christman Incubation with uricase was found to be unnecessary for allantoic fluid.
and (14).
Studies with Hatched Chicks An experiment was carried out in which &day embryonated eggs were treated as described above with Sedormid and adenine, then allowed to hatch. At the end of the 21st day of incubation, chicks unable to hat,ch alone were assisted in leaving t,he shell. Shortly after hatching, all chicks were examined for gross anatomical abnormalities and tagged with wing bands, and the development of individual birds was followed for 30 days. During fed a commercial st.art,er mash.
this t,ime the c-hicks were housed in box brooders
and
RESULTS
Porphyria
I,nduced by Sedorm.id
I:ric Acid Excretion. These dnta, summnrized
in Table I, are presented in terms of totnl uric ncid per egg for the following reasons. Kot. only does the volume of nllnntoic fluid in the embryonnted egg vary wit,h the stage TABLli:
I
ITric .lcirE Excretion of Control and Sedormid-Treated Chick Embryos .Ldrninistration of Exogenous Pltrines via the Yolk Sac Total Days after treatment
Purine
1. Control
uric
acid per embryo.
.i
Adkd purine
p - c‘”
3.12
1.45 2.56 2.33 1 .83 3.00 2.31 0.10 -0.30 0.29 -0.25
j
Following
mi:
.+.
1u mg Sedormid
,--. A&nine* ma.)
(5
(;uaninec (5.6 mg.) Santhinec (5.6 mg.)
1 2 3 4 5 6 2 4 2 4
1.67 3.00 4.08 5.73 7.14 10.84 2.36 5.00 2.36 5.00
-
5.65 6.41 7.66 10.14 13.15 2.46 4.70 2.65 4.75
i / : i ’
1.ot I .04 2.07 3.94 5.06 6.46 1.68 3.35 1.68 3.35
2.44 4.70 5.i1 8.1; 8.G 11.16 1.81 3.98
1 / j j / /
1.~1 i
3.23
1.43 2 76 2.74 4.23 3.61 4.70 0.13 0.68 0.1:Jl -0.12
L
a I’ - C = purine minus control; S + I’ - S = Sedormid + purine minus Sedormid. Represents additional uric acid formed from exogenous purines. * Data derived from five separate experiments utilizing the pooled allantoic fluids from six eggs for each sample. c Data from one experiment utilizing the pooled allantoic fluids from six eggs fol each sample.
PORPHYRIN
METABOLISM.
V
419
of incubation, but also the allantoic fluid volume of porphyric embryos is somewhat greater than that of control embryos during the later stages of these experiments (7). Therefore, comparison of the outputs of any excretory product by the various groups of eggs has more meaning when these outputs are evaluated in terms of the total amount of the product present in the allantoic fluid of an average egg at a given time than when they are expressed simply as concentrations in the allantoic fluid. Examination of the figures on uric acid concentration in allantoic fluid revealed several noteworthy facts, however: (a) With any one lot of eggs, the concentration of uric acid in the allantoic fluid of control embryos was always greater than that in the nllantoic fluids of porphyric embryos and only rarely was there any overlap in the values obtained in different experiments. (b) The same relationships existed with regard to uric acid concentration in the allantoic fluids from adenine-treated embryos as compared to control embryos. (c) Adenine-treated embryos always exhibited higher concentrations of uric acid in their nllantoic fluids than porphyric embryos, with no overlap between the two groups in the values obt’ained in different experiments. Results of statist,ical analysis of the concentration figures for uric acid in allantoic fluid in experiments utilizing adenine are outlined in Table II, from which it can be seen that the differences in uric acid output presented in ~01s. 3 and 6 of Table I represent significant differences. Comparison of ~01s. 3 and 6 in Table I shows that on the first day after treatment the increments in uric acid excretion of normal and porphyric embryos receiving ndenine were almost identical. However, beginning on the second and continuing through the sixth day after treatment, porphyric embryos displayed somewhat greater increments in uric acid output than did the controls, t’he difference being especially noticeable at 4 and 6 days. This phenomenon will be discussed further below. Neither guanine nor xanthine produced significant changes in the uric acid out,put of chick embryos, whether normal or porphyric. Porphyrin Excretion. The data on porphyrin excretion in experiments utilizing adenine, illustrated in Figs. 1 and 2, are also presented as total porphyrin per egg for the reasons given above. Results of statistical analysis of the concentrat,ion figures for porphyrins in allantoic fluid may be summarized as follows: (a) Neither the uroporphyrin nor coproporphyrin outputs of adenine-treat,ed embryos differed significanbly (p > 0.05) from those of the controls at any time. (b) The coproporphyrin excretions of porphyric embryos were significantly higher (p < 0.01) than those of control embryos at all stages of the experiment. (c) The uroporphyrin outputs of embryos receiving Sedormid and Sedormid + adenine were not significantly different from those of the controls after 1 day. After 2 days, the uroporphyrin excret’ions of embryos receiving Sedormid +
450
TALMAN,
L~~BBE, ALDRIC~H TABLE
Statistical
.wn SEARS
II
dnalgsis of Concentration Figures .for Vric :lcid in the dllantoic L’ontrol and Sedormid-Treated Chick Embryos Following Treatment with Exogenous ddenine via the Iyolk Saca
Fluid
of
-
2
Control vs. adenine Control vs. Srdormid Cont,rol vs. Hedormid + :tdeninc Adenintt vs. Sedormid .\denine vs. Sedormid + :rdenine vs. Sedormid Sedormid + adenine
p < 0.02 I
~
p < 0.02
p
p < 0.01 p < 0.01 j p < 0.01
p < 0.0’2
p < 0.01
p < 0.01
p < 0.01
p < 0.01
p < 0.05
p < 0.05
p > 0.05
p > 0.0.5
p > 0.05
p < 0.01
,’ < 0.01
p < 0.01
p < 0.01
p < 0.01
p > 0.05
p > 0.05
p < 0.05 / p < 0.05
p > 0.05
p < 0.01
p < 0.01
p < 0.01
/I < 0.02 !
p < 0.05 1
I
‘I Sedormid dosage = IO mg.; ndenine dosage = 5 mg. IData analyed obtained from five separate experiments utilizing the pooled nlhmtoic fluids from six eggs for inch samplr.
adrnine still did not differ significunt,ly from those of control embryos t)ut, were significantly lower (p < 0.01) than those of embryos receiving Sedormid alone. Uroporphyrin excretion by the latt,er group was significant,ly higher (p < 0.01) than that of the controls. (d) Differences in uroporphyrin and coproporphyrin excret,ion between all other possible combinations of groups at, all ot,her times were highly significant, (p < 0.01). ,& may be seen from Figs. 1 and 2, administration of adenine simultaneously with Sedormid reduced both uroporphyrin and coproporphyrin excretion. It should he not,ed that, adenine-treat,ed porphyric embryos excret’ed porphyrins very slowly between the third and fourth and fifth and sisth days after treatment but quite rapidly between the fourth and fifth days. Comparison of these findings with the increases in uric acid output resulting from adenine treatment shows that the increment in uric acid excretion displayed by porphyric embryos was more t,han twice as great as that of the controls on the fourth and sixt,h days but, only about 20 % greut,er on the fifth day. Thus, the active met,abolism of supplementary adenine is accompanied by a markedly reduced rate of porphyrin excretion. Exogenous gunnine and xant’hine not only failed to increase uric acid out,put, hut also had no effect on the porphyrin excretions of porphyric embryos.
6.0 r
4.0 t
I 9
2 3 Days After IO - II Embryonic
4 5 6 Treatment 12 13 14 Age (Days)
content per egg FIG. 1. Effect of exogenous adenine on the total “uroporphyrin” in allantoic fluid in normal and porphyric embryos. 0, control; X, adenine only; A, 10 mg. Sedormid; n , 10 mg. Sedormid + 5 mg. adenine.
I
2
3
4
5
6
FIG. 2. Effect of exogenous adenine on the total coproporphyrin
in allantoic fluid in normal and porphyric embryos. 0, control; X, sdenine only; A, 10 mg. Sedormid; adenine. 451
n , 10 mg.
content per egg Sedormid + 5 mg.
452
TALMAS,
LABBE,
dL1)ILICH
B,c’D SE$BS
Embryonic Growth. The multiple-range test developed by Duncan (15) was used for statistical analysis of t.hese data. Statistically significant differences (95 % confidence level) in embryonic weight were not observed until the fourth day aft,er treatment. At that time, Sedormid-treated embryos (mean 5.22 g.) were significantly smaller than the controls (mean 5.86 g.) and those receiving Sedormid plus adenine (mean 5.82 g.), hub not significantly smaller than embryos receiving adenine only (mean 5.39 g.). However, adenine-trea,ted embryos failed to differ significantly from the controls or the adenine-treated porphyric embryos. On the fifth and sisth days, the Sedormid-treated group (means 6.9$ and 9.44 g., respectively) was significantly smaller than the control (means 8.36 and 11.15 g.) and adenine-treated (means 7.93 and 10.17 g.) groups, hut not, significantly smaller than the group receiving Sedormid plus adenine (means 7.45 and 10.29 g.). The lat,ter group, however, failed to differ significantly from the cont,rol and adenine-treated groups. Thus, supplementary adenine significantly improved the growth of Sedormid-t’reated embryos 4 days after treat,ment. Although this clear-cut difference failed to persist through the fift.h and sixth days, growth continued to be improved sufficiently to prevent embryos receiving ndenine with Sedormid from becoming significantly smaller than the control or adenine-t,reat,ed groups. Wit’h a larger number of observations or wit,h experiments extended a day or t’wo longer, t,he difference between the porphyric and ndenine-treatsect porphyric groups might again prow to bc significant. Obserrations
on Hatched Chicks
Hatchabilit!/. Hatchability of the control and adenine-treated groups WIS 81 and 87 %, respectively, and the proport,ions of these groups requiring assist’ancc in leaving the shell were 21 and 33 %. Hatchability of porphyric chicks was reduced to 69 %. However, 48 % of t,hose receiving Sedormid alone required assistance in leaving the shell, while only 31 % of t,hose receiving ndcnine as well as Sedormid required such assistance. (‘ongenital dno~~~alies. Table III sumn~ariaes the occurrence of gross abnormnlit~ies. The incidence of congenital anomalies in the Sedormidt,rent,ed group n-as significantly higher (~2 test, 95 % confidence level) t.han in the group receiving ndenine wit,h Sedormid. Differences in the incidence of anomalies in the control, adenine, and Sedormid plus ndeninc groups were not significant’ statistically. Only obviously abnormal chicks were list’ed as deformed. Persons more familiar with the anatomy and physiology of t’he chick might have been able to detect, more subtle physiological and morphological defect,s. Nearly all afflicted chicks displ:lycd curled toes, the condit,ion varying from a simple bending of the toes (most often the long toe, and usually medially) to a curling of t.he whole foot,,
PORPHYRIN
Effect
METBBOLISM.
TABLE III Adenine on the Incidence in Sedormid-Treated Chic&
of Supplementary Anomalies
of Congenital
Per cent exhibiting congenital anomalies
Group
Control Adenine Sedormid Sedormid + adenine a All materials administered
453
V
9 6 53 19
to S-day embryonated
eggs via the yolk sac.
so that, if the chick were able to walk at all, it walked on its hocks. Signs of neurologicnl involvement were often present. These signs included paresis in the legs with the legs spread and practically useless, tremors in the legs, and occasionally tremors in the wings. Growth after Hatching. Since the embryo draws the yolk sac into its abdominal cavity during the last 2448 hr. in the egg, hatching weights were not significantly different. From the second day after hatching, however, the control chicks and those receiving adenine only were significantly larger than Sedormid-treated chicks (Student’s t test, 95 % confidence level). Chicks receiving adenine in addition to Sedormid were significantly larger than those receiving only Sedormid from t’he second through the twelfth day after hatching. After that time their growth rate gradually declined until it became almost identical with that of Sedormid-treated chicks from the nineteenth day on. Porphyria
Induced by Allylisopropylacetamide
To demonstrate the effect, of adenine supplementation on experimental porphyria induced by injecting AIA, which is soluble, it was found necessary to use a soluble adenine derivative, adenosine. liric Acid Excretion. Results were similar to those obtained using Sedormid and ndenine, i.e., injection of supplementary adenosine with AIA resulted in considerably greater increments in uric acid output in porphyric embryos than in their controls after the early stages of the experiment. Porphyrin Excretion. Exogenous adenosine reduced the porphyrin outputs of AIA-treated embryos, although this reduction was not so marked as that observed in Sedormid-treated embryos receiving adenine. The decreased efficacy of adenosine in this respect may arise from the less efficient, utilization of the nucleoside for nucleic acid synthesis as compared to the free purine base (18). significant differences in Embryonic Growth. Here again, statistically embryonic
weight
were not observed
until
4 days after
treatment.
From
454
TALMAN,
LABBE,
.~LDRICH
TABLE
Irccorporation of Glycirwf-(
-
SESKS
IV
“14into Pwines by Normal und Porphyric Chick Embryosa
T
Adenine Incubation
INI)
Guanine
Control”
Controlh
Porphyrkh
Uric acid
I
time Porphyric’
ControlC
1 Porphpric’
---,--min.
1.6 3 7 2. 7
20
60 180
-
0 .6 3.s 8.4
8.0 00 1.5
X.8 3.5 9.6
i
7220 6620
i
s;~o 4100
-
u Cilycine-S-C’“, 6.8 pc., was given intravenously I,orl,hvrin-producirlR drug via the yolk sac. * Counts/mm./pmole at mfinite thinness. c Total counts/min. in uric acid per egg.
6 days after
administr:ttion
of
that time on, AIA-treated embryos were significnntly smaller than the contxols, and exogenous ndenosine failed to improve their growth. This failure of adenosine to enhance gro&h, again, probably reflects the less efficient, ut,ilizat,ion of the nucleoside. Incorporation of G’lycinr-2-P into Purincs. These data are summarized in Table IV. Results obtained after 20 min. incubation indicate that the rate of purine synt,hesis in control embryos is at least t,wice that of the porphyrics. The obserxxtion that t,he specific ncti&ies of the tissue purincs from porphyric embryos ntt,nined higher values than the controls after 180 min. suggests t,hat (a) their rate of turnover is much reduced, and (b) the newly formed purines are probably diluted somewh:~t less by enclogenous purines. The latt,er possibility is supported by the observation that homogennt~es of porphyric embryos alw:~ys yielded smaller cluantities of purint than equal volumes of homogenates of control embryos t#hroughout t.he isolation procedure. I’reliminury ohservat~ions on t,he incorporation of glyciuc-2-C”’ ;md adenine-8-C~14 mto . liver purines in porphyric rats support, these conclusiolxl
The dat.u reported here indicate that chick embryos, like rat.s (I?), readily ut’ilize preformed adenine, but metabolize preformed guanine und xant’hine only slightly, if nt all. This agrees also with the findings of I,u and Winnick (18) who have reported good utilizat,ion of adenine-(2’4 and very poor utJilizntion of guanine-C I4 for nucleic acid synthesis in tissue cultures of embryonic chick hewt. N&her, Snell, and Cravens (In), on t,he ot,hcr hand, found guanine as well as adenine to be met~nholicnlly active, when used in conjunction with thymidine, in reversing nminopterin in-
PORPHYRIN
METABOLISM.
V
455
hibition of chick embryos. Xanthine, again, was ineffect,ive. A difference in procedure, however, may account for the divergent results. The more efficient oxidation and excretion of exogenous adenine by porphyric embryos as compared to the controls leave little doubt that purine catabolism and excretion are unimpaired and suggest that purine biosynthesis is defective in experimental porphyria. This suggestion is supported by the observation that the rate of incorporation of glycine-2-P into nucleic acid purines is reduced approximately 50% in porphyric embryos. A plausible explanat,ion for the smaller increment in uric acid output by control embryos following treatment with supplementary adenine is found in the results of earlier work on ndenine-treated mice and rats (20). Those studies demonstrated t#hat, exogenous adenine, administered in quantities greater than a certain threshold amount, is oxidized to 2,8dioxyadenine, and the latter is t,hen deposited in the renal tubules as crystalline occlusions, leading to a secondary uremia. It may well be t,hat, a similar impairment of renal function occurs in normal chick embryos receiving adenine. Porphyric embryos, however, in which purine biosynt,hrsis seems to be impaired, may be expected to ut,ilize preformed adenine more efficiently than normal emhryos. Thus, the t.hreshold quantity of adenine producing such sequelae may he elevat,ed in experiment’al porphyria . Since the decreased uric acid output of porphyric chick embryos apparently reflects a defect in purine biosynthesis rat#her than in purine catabolism, the question arises as to t,he nnt,ure of a defect causing simultaneous impairment in purine biosynthesis and enhancement of porphyrin biosynthesis. The work of Shemin and associates (21, 22) showing that through a common porphyrins and purines are related metabolically precursor, &nminolevulinic acid, makes it reasonable to postulate that’ an error in the met~abolism of the lat,ter may he responsible for such a phenomenon. Accordingly, this group advanced t’he following hypot’hexis to account, for its findings in experimental porphyria: A block in Shemin’s succinate-glycine cycle retarding the entry of the d-carbon at,om of b-nminolevulinic acid into the C1 pool, and thence into purines, results in accumulat,ion of this common precursor which is t,hen disposed of via the porphyrin pathway. This hypot,hesis adequately explains the findings reported here, hut studies designed t,o test this hypothesis furt’her, which are described in the accompanying paper, indicate t,hat, t,he metabolic block in experimental porphyria is prohahly located at some other point. However, the possibility that, some error in t,he metabolism of &aminolevulinic acid may be involved in experimental porphyria gains support from the observation of h-euherger and associates (23, 24) that the activit,y of the enzyme, &aminolevulinic acid dehydrase is increased twofold hy Sedormid treatment. This
45G
TALMAN, LABBE, ALDRICH ASD SEARS
latter effect might reflect a primary pot,entisting action of the drug on the enzyme system or it might arise secondary bo a block in another metabolic pathway normally open to ALA. Thus, the data presented here provide evidence that purine catabolism is unimpaired but a decreased rat’e of purine biosynthesis exists in experimental porphyria. Reduced porphyrin outputs of porphyric embryos following treatment with exogenous adenine likely reflect a sparing action of the preformed purine. The organism may respond to a purine deficit by elaborating more ALA in a vain attempt to overcome the metabolic block by mass action. When adenine is supplied preformed, however, some of the stimulus for ALA formation is removed and porphyrin formation is decreased. Supplementation of one of the metabolites in short supply with preformed material also probably accounts for t,he improved growt,h of Sedormid-treated embryos receiving adenine. SUMMARY
In order t,o assess the efficiency of purine catabolism in experimental porphyria, t.he ability of chick embryos to oxidize and excrete purines was tested by administering preformed adenine, guanine, and xant,hine. Both normal and porphyric embryos were found t’o convert adenine, but not, guanine or xant’hine, to uric acid, with the porphyric embryos apparently cntuholizing exogenous adenine even more efficiently than t,he controls. Furthermore, adenine supplementat,ion reduced t,he porphyrin output and improved the growt’h of Sedormid-t’rrated embryos. Although adenosine was somewhat less effective than adenine, it’s use with allylisopropylacet.amide gave qu:llit,atively similar results. Results of tracer studies using glycine-NY support the hypothesis of impaired purinc biosynt,hesis in experimental porphyria.
1. TALMAN, 12. I,., CARE, J. II., NEVE, II. A.. T,ABBE, R. F., ANI) A~,arcw, II. A., d. Rid. (%em. 212, 663 (1955). 2. I,ABBE, R. F., TAI.MAS, I<. I,., AND AI,URI(-H, II. A., Hiwhim. et Bioph!/s. .-trtt~ 16, 500 (195-l). 3. (GOLDBERG, .4., Riochem. Sot. S!/wposia (Cambridge, Engl.) 12, 27 (195-&). 1. (~OLI~BERG, A., AND RIMINGTON, C., Proc. Roil. Sot. (London) B143, 257 (1955). 5. TALMAN. l<. I,., I,ABBE, R. F., ANI) AI,DRI(-H, It. A., .lwh. Biochem. Bioph!/s. 66, 289 (1957). 6. I,ABBE, R. F., TALMAN, E. I,., AND ALDRIVH, It. A., Frrleratio~~ Pror. 14,241 (1955). 7. TALMAX, 15. I,., HUTCHENS, ‘I’. T., ANI) ALDRICII, R. A., Prof.. 80~. Expt/. Rio/. Med. 96, 130 (1957). 8 BEVERIISE, W. I. B., AND BL-RNET, F. M., “The Cnlt,ivntion of Virnses 2nd
Rirkettsiae in t,he Chick lXmt,ryo.” Series No. 256, T,ondon, 1946.
IbIedic:d Research Council Special Report
PORPHYRIN
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 2-I.
METABOLISM.
V
457
ST. JOHN, J. L., AND JOHNSON, O., J. Biol. Chem. Sa, 41 (1931). SCHNEIDER, W. C., J. Biol. Chem. 161, 293 (1945). HITCHINGS, G. H., J. Biol. C’hem. 139, 843 (1941). ABRAMS, R., Arch. Biochem. 30, 44 (1951). TALMAN, E. I,., i,n. “Standard Methods of Clinical Chemistry” (Sefigson, I>.), Vol. II, pp. 137-X. Academic Press, New York, 1958. BCCHANAN, 0. H., BLOCK, W. D., AND CHRI~TMAN, A. A., J. Biol. C’hem. 16’7, 181 (1945). DUNCAN, D. B., Bion~etrics 11, 1 (1955). BROWN, G. B., AND ROI,L, I’. M., in “The Nucleic Acids” (Chargaff, E., and Davidson, J. N., eds.), Vol. 2, p. 383. Academic Press, New York, 1955. BROWN, G. B., AND ROLL, I’. M., in “The Nucleic Acids” (Chargaff, E., and Davidson, J. N., eds.), Vol. 2, p. 344. Academic Press, New York, 1955. Lu, K. H. AND WINKICK, T., Ezptl. Cell Research 6, 345 (1954). NABER, E. C., SXELL, E. E., AND CRAVENS, W. W., Proc. Sot. Exptl. Biol. Med. 81, 20 (1952). PHILIPS, F. S., THIERSCH, J. B., AND BENDICH, A., J. Pharmacol. Exptl. l’herap. 104, 20 (1952). SHEMIN, D., AND RUSSELL, C. S., J. .4m. Chenz. Sot. 76, 4853 (1953). SHEMIN, D., RUSSELL, C. S., ANU ABRAMYKY, T., J. Biol. Chent. 216, 613 (1955). GIBSON, K. D., NEUBERGER, A., AND SCOTT, J. J., Biochem. J. 61, 618 (1955). XEUBERGER, A., Proc. 3rd. Intern. Congr. Biochem., 1966, p. 204.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
80,
416-457
(1959)
Porphyrin Metabolism V. The Metabolism of Purines in Experimental Porphyria’ Ellen L. Talman, Robert F. Labbe,” Robert A. Aldrich” and David Sears3 Frott~
the Departments Oregon
of Riochenristr!/ Medical School,
Received
and Pediatrics, Portlnnd, Oreyon
C:niwrsitg
of
June 27, 1058
INTRODCCTION
A previous report from t,his laboratory (1) described t’he experimental induction of hepatic porphyria in chick embryos by inject,ing nllylisopropylacet,ylcarbamide (Sedormidm) into their yolk sacs. Anatomical abnormslit,ies, retarded growth, and difficulty in hatching were found to constitute parts of the syndrome in this species. The possibility of deranged purine metabolism in experimental porphyria was also ment,ioned. Investigation of this possibilit’y by quuntitation of uric acid output established a marked reduct,ion in the excretion of this compound by porphyric embryos (2). This finding could reflect impaired purine synthesis, catabolism, or excretion. The latt,er two possibilities have now been eliminated by demonst,rating t,hat exogenous adenine is excreted as uric acid by porphyric embryos even more efficient,ly than by their controls. Furthermow, t,racer studies utilizing glycine-2-Cl4 have shown t,hat, porphyric embryos synthesize purines at, a decreased rate. Supplementary adenine also reduced the porphyrin excretion and improved t,he growt,h of Sedormid-t’reated embryos. The latter observations led t,o st,udies showing that, ndeuine improves the growt,h and reduces t,he incidence of congenital anomalies in Sedormid-t’reated chicks. A soluble non-hypnot,ic Sedormid derivative, allylisopropy1acetamide (AM), has largely replaced t’he former compound as a porphyria-producing 1 Supported by a special research grant of the Eli Lilly Company; the Helen Hay Whitney Foundation; A.;C4.A. Council on Pharmacy and Chemistry, William Volker Grant No. 7; National Institute of Arthrit,is and Metabolic Diseases, National Inst,itutes of Health, U. 8. Public Health Service ((irant No. A-752). 2 Present address: Departzment of Pediatrics. IYniversity of Washington School of Medicine, Seattle 5, Washington. 8 With the technical assistance of Margaret S. Shafer. 446
PORPHYRIN METABOLISM. V
447
agent (3-5). Injecting the soluble adenine derivative, adenosine, with AIA produced results qualitatively similar to those obtained with Sedormid and adenine. The findings reported here support the previous suggestion (2, 6) that, in hepatic porphyria, both porphyrin and purine metaholism are deranged. EXPERIMENTAL
Eggs. See Ref. (5) Preparation of Materials
for Injection
Concentrations of all materials were adjusted so that the dose given was contained in a volume of 0.50 ml. Suspensions of Sedormid (20 mg./ml.), adenine (10 mg./ml.), and Sedormid plus adenine (20 and 10 mg./ml., respectively) in isotonic glycerol were prepared and sterilized as described previously (1). AIA (10 mg./ml.) and adenosine (20 mg./ml.) were dissolved in 0.6y0 NaCl, and AIA plus adenosine (10 and 20 mg./ml., respectively) in 0.45’% NaCl. Glycine-2-C14 (2.0 mg./ml., 13.6 pc./mg.) was dissolved in 0.9% NaCl. These solutions were sterilized in the autoclave.
Procerhre Except for glvcine-2-CL4, the compounds and combinations of compounds under investigation were injected into the yolk sacs of S-day embryonated eggs. In studies utilizing Sedormid to induce porphyria, one group of embryos received 10.0 my. Sedormid, a second 10.0 mg. Sedormid plus 5.0 mg. adenine (or 5.6 mg. guanine or .uanthine), a third 5.0 mg. adenine (or 5.6 mg. guanine or xant#hine), and the control group received 0.50 ml. of sterile suspension medium. When ndenine was used, six embryos from each group were sacrificed at 24.hr. int#ervals for a period of 6 days following injection, and their allantoic fluids were aspirated and pooled. After analyzing these pools quantitatively for coproporphyrin, uroporphyrin, and uric acid, the average amount of each compound contained in the allantoic fluid of a single egg was calculated (7). Embryos were then separated from their extraembryonic t,issues, blotted on absorbent paper, and weighed. When guanine and xanthine were used, a similar procedure was carried out hut only on the second and fourth days after t.re:ttment. In experiments utilizing AIA to induce porphyria, one group of emhryos received 5.0 mg. AIA, a second 5.0 mg. AIA plus 10.0 mg. adenosine, a third 10.0 my. adenosine, and the controls received 0.50 ml. of sterile 0.9% NaCl solution. At intervals of 2, 4, and 6 days after treatment, eight or nine eggs from each group were sacrificed, their allantoic fluids pooled for analysis, and the embryos weighed as before. Tracer studies were carried out 6 days after injecting the porphyria-producing drug. At that time, 0.25 ml. (6.8 PC.) of the glycine-2-C14 solution (2.0 mg./ml.) was injected intravenously (S), and the eggs were returned to the incubator. After a designated time had elapsed, the allantoic fluids from each group were collected for the isolation of uric acid (9), and the embryos were removed and quickly frozen in dry ice-acetone. The purines were isolated (10, 11) from 50% homogenates of each group of embryos, and adenine and guanine were then separated from each other chromatographically (12) prior to determination of their Cl4 act,ivities.
TALMAN, LABBE, ALDRICH AND SEARS
448
Analytical
Methods
The techniques of Schwartz and associates for determining coproporphyrin uroporphyrin, as slightly modified in this laboratory (13), were used. Uric acid was determined by the method of Buchanan, Block, and Christman Incubation with uricase was found to be unnecessary for allantoic fluid.
and (14).
Studies with Hatched Chicks An experiment was carried out in which &day embryonated eggs were treated as described above with Sedormid and adenine, then allowed to hatch. At the end of the 21st day of incubation, chicks unable to hat,ch alone were assisted in leaving t,he shell. Shortly after hatching, all chicks were examined for gross anatomical abnormalities and tagged with wing bands, and the development of individual birds was followed for 30 days. During fed a commercial st.art,er mash.
this t,ime the c-hicks were housed in box brooders
and
RESULTS
Porphyria
I,nduced by Sedorm.id
I:ric Acid Excretion. These dnta, summnrized
in Table I, are presented in terms of totnl uric ncid per egg for the following reasons. Kot. only does the volume of nllnntoic fluid in the embryonnted egg vary wit,h the stage TABLli:
I
ITric .lcirE Excretion of Control and Sedormid-Treated Chick Embryos .Ldrninistration of Exogenous Pltrines via the Yolk Sac Total Days after treatment
Purine
1. Control
uric
acid per embryo.
.i
Adkd purine
p - c‘”
3.12
1.45 2.56 2.33 1 .83 3.00 2.31 0.10 -0.30 0.29 -0.25
j
Following
mi:
.+.
1u mg Sedormid
,--. A&nine* ma.)
(5
(;uaninec (5.6 mg.) Santhinec (5.6 mg.)
1 2 3 4 5 6 2 4 2 4
1.67 3.00 4.08 5.73 7.14 10.84 2.36 5.00 2.36 5.00
-
5.65 6.41 7.66 10.14 13.15 2.46 4.70 2.65 4.75
i / : i ’
1.ot I .04 2.07 3.94 5.06 6.46 1.68 3.35 1.68 3.35
2.44 4.70 5.i1 8.1; 8.G 11.16 1.81 3.98
1 / j j / /
1.~1 i
3.23
1.43 2 76 2.74 4.23 3.61 4.70 0.13 0.68 0.1:Jl -0.12
L
a I’ - C = purine minus control; S + I’ - S = Sedormid + purine minus Sedormid. Represents additional uric acid formed from exogenous purines. * Data derived from five separate experiments utilizing the pooled allantoic fluids from six eggs for each sample. c Data from one experiment utilizing the pooled allantoic fluids from six eggs fol each sample.
PORPHYRIN
METABOLISM.
V
419
of incubation, but also the allantoic fluid volume of porphyric embryos is somewhat greater than that of control embryos during the later stages of these experiments (7). Therefore, comparison of the outputs of any excretory product by the various groups of eggs has more meaning when these outputs are evaluated in terms of the total amount of the product present in the allantoic fluid of an average egg at a given time than when they are expressed simply as concentrations in the allantoic fluid. Examination of the figures on uric acid concentration in allantoic fluid revealed several noteworthy facts, however: (a) With any one lot of eggs, the concentration of uric acid in the allantoic fluid of control embryos was always greater than that in the nllantoic fluids of porphyric embryos and only rarely was there any overlap in the values obtained in different experiments. (b) The same relationships existed with regard to uric acid concentration in the allantoic fluids from adenine-treated embryos as compared to control embryos. (c) Adenine-treated embryos always exhibited higher concentrations of uric acid in their nllantoic fluids than porphyric embryos, with no overlap between the two groups in the values obt’ained in different experiments. Results of statist,ical analysis of the concentration figures for uric acid in allantoic fluid in experiments utilizing adenine are outlined in Table II, from which it can be seen that the differences in uric acid output presented in ~01s. 3 and 6 of Table I represent significant differences. Comparison of ~01s. 3 and 6 in Table I shows that on the first day after treatment the increments in uric acid excretion of normal and porphyric embryos receiving ndenine were almost identical. However, beginning on the second and continuing through the sixth day after treatment, porphyric embryos displayed somewhat greater increments in uric acid output than did the controls, t’he difference being especially noticeable at 4 and 6 days. This phenomenon will be discussed further below. Neither guanine nor xanthine produced significant changes in the uric acid out,put of chick embryos, whether normal or porphyric. Porphyrin Excretion. The data on porphyrin excretion in experiments utilizing adenine, illustrated in Figs. 1 and 2, are also presented as total porphyrin per egg for the reasons given above. Results of statistical analysis of the concentrat,ion figures for porphyrins in allantoic fluid may be summarized as follows: (a) Neither the uroporphyrin nor coproporphyrin outputs of adenine-treat,ed embryos differed significanbly (p > 0.05) from those of the controls at any time. (b) The coproporphyrin excretions of porphyric embryos were significantly higher (p < 0.01) than those of control embryos at all stages of the experiment. (c) The uroporphyrin outputs of embryos receiving Sedormid and Sedormid + adenine were not significantly different from those of the controls after 1 day. After 2 days, the uroporphyrin excret’ions of embryos receiving Sedormid +
450
TALMAN,
L~~BBE, ALDRIC~H TABLE
Statistical
.wn SEARS
II
dnalgsis of Concentration Figures .for Vric :lcid in the dllantoic L’ontrol and Sedormid-Treated Chick Embryos Following Treatment with Exogenous ddenine via the Iyolk Saca
Fluid
of
-
2
Control vs. adenine Control vs. Srdormid Cont,rol vs. Hedormid + :tdeninc Adenintt vs. Sedormid .\denine vs. Sedormid + :rdenine vs. Sedormid Sedormid + adenine
p < 0.02 I
~
p < 0.02
p
p < 0.01 p < 0.01 j p < 0.01
p < 0.0’2
p < 0.01
p < 0.01
p < 0.01
p < 0.01
p < 0.05
p < 0.05
p > 0.05
p > 0.0.5
p > 0.05
p < 0.01
,’ < 0.01
p < 0.01
p < 0.01
p < 0.01
p > 0.05
p > 0.05
p < 0.05 / p < 0.05
p > 0.05
p < 0.01
p < 0.01
p < 0.01
/I < 0.02 !
p < 0.05 1
I
‘I Sedormid dosage = IO mg.; ndenine dosage = 5 mg. IData analyed obtained from five separate experiments utilizing the pooled nlhmtoic fluids from six eggs for inch samplr.
adrnine still did not differ significunt,ly from those of control embryos t)ut, were significantly lower (p < 0.01) than those of embryos receiving Sedormid alone. Uroporphyrin excretion by the latt,er group was significant,ly higher (p < 0.01) than that of the controls. (d) Differences in uroporphyrin and coproporphyrin excret,ion between all other possible combinations of groups at, all ot,her times were highly significant, (p < 0.01). ,& may be seen from Figs. 1 and 2, administration of adenine simultaneously with Sedormid reduced both uroporphyrin and coproporphyrin excretion. It should he not,ed that, adenine-treat,ed porphyric embryos excret’ed porphyrins very slowly between the third and fourth and fifth and sisth days after treatment but quite rapidly between the fourth and fifth days. Comparison of these findings with the increases in uric acid output resulting from adenine treatment shows that the increment in uric acid excretion displayed by porphyric embryos was more t,han twice as great as that of the controls on the fourth and sixt,h days but, only about 20 % greut,er on the fifth day. Thus, the active met,abolism of supplementary adenine is accompanied by a markedly reduced rate of porphyrin excretion. Exogenous gunnine and xant’hine not only failed to increase uric acid out,put, hut also had no effect on the porphyrin excretions of porphyric embryos.
6.0 r
4.0 t
I 9
2 3 Days After IO - II Embryonic
4 5 6 Treatment 12 13 14 Age (Days)
content per egg FIG. 1. Effect of exogenous adenine on the total “uroporphyrin” in allantoic fluid in normal and porphyric embryos. 0, control; X, adenine only; A, 10 mg. Sedormid; n , 10 mg. Sedormid + 5 mg. adenine.
I
2
3
4
5
6
FIG. 2. Effect of exogenous adenine on the total coproporphyrin
in allantoic fluid in normal and porphyric embryos. 0, control; X, sdenine only; A, 10 mg. Sedormid; adenine. 451
n , 10 mg.
content per egg Sedormid + 5 mg.
452
TALMAS,
LABBE,
dL1)ILICH
B,c’D SE$BS
Embryonic Growth. The multiple-range test developed by Duncan (15) was used for statistical analysis of t.hese data. Statistically significant differences (95 % confidence level) in embryonic weight were not observed until the fourth day aft,er treatment. At that time, Sedormid-treated embryos (mean 5.22 g.) were significantly smaller than the controls (mean 5.86 g.) and those receiving Sedormid plus adenine (mean 5.82 g.), hub not significantly smaller than embryos receiving adenine only (mean 5.39 g.). However, adenine-trea,ted embryos failed to differ significantly from the controls or the adenine-treated porphyric embryos. On the fifth and sisth days, the Sedormid-treated group (means 6.9$ and 9.44 g., respectively) was significantly smaller than the control (means 8.36 and 11.15 g.) and adenine-treated (means 7.93 and 10.17 g.) groups, hut not, significantly smaller than the group receiving Sedormid plus adenine (means 7.45 and 10.29 g.). The lat,ter group, however, failed to differ significantly from the cont,rol and adenine-treated groups. Thus, supplementary adenine significantly improved the growth of Sedormid-t’reated embryos 4 days after treat,ment. Although this clear-cut difference failed to persist through the fift.h and sixth days, growth continued to be improved sufficiently to prevent embryos receiving ndenine with Sedormid from becoming significantly smaller than the control or adenine-t,reat,ed groups. Wit’h a larger number of observations or wit,h experiments extended a day or t’wo longer, t,he difference between the porphyric and ndenine-treatsect porphyric groups might again prow to bc significant. Obserrations
on Hatched Chicks
Hatchabilit!/. Hatchability of the control and adenine-treated groups WIS 81 and 87 %, respectively, and the proport,ions of these groups requiring assist’ancc in leaving the shell were 21 and 33 %. Hatchability of porphyric chicks was reduced to 69 %. However, 48 % of t,hose receiving Sedormid alone required assistance in leaving the shell, while only 31 % of t,hose receiving ndcnine as well as Sedormid required such assistance. (‘ongenital dno~~~alies. Table III sumn~ariaes the occurrence of gross abnormnlit~ies. The incidence of congenital anomalies in the Sedormidt,rent,ed group n-as significantly higher (~2 test, 95 % confidence level) t.han in the group receiving ndenine wit,h Sedormid. Differences in the incidence of anomalies in the control, adenine, and Sedormid plus ndeninc groups were not significant’ statistically. Only obviously abnormal chicks were list’ed as deformed. Persons more familiar with the anatomy and physiology of t’he chick might have been able to detect, more subtle physiological and morphological defect,s. Nearly all afflicted chicks displ:lycd curled toes, the condit,ion varying from a simple bending of the toes (most often the long toe, and usually medially) to a curling of t.he whole foot,,
PORPHYRIN
Effect
METBBOLISM.
TABLE III Adenine on the Incidence in Sedormid-Treated Chic&
of Supplementary Anomalies
of Congenital
Per cent exhibiting congenital anomalies
Group
Control Adenine Sedormid Sedormid + adenine a All materials administered
453
V
9 6 53 19
to S-day embryonated
eggs via the yolk sac.
so that, if the chick were able to walk at all, it walked on its hocks. Signs of neurologicnl involvement were often present. These signs included paresis in the legs with the legs spread and practically useless, tremors in the legs, and occasionally tremors in the wings. Growth after Hatching. Since the embryo draws the yolk sac into its abdominal cavity during the last 2448 hr. in the egg, hatching weights were not significantly different. From the second day after hatching, however, the control chicks and those receiving adenine only were significantly larger than Sedormid-treated chicks (Student’s t test, 95 % confidence level). Chicks receiving adenine in addition to Sedormid were significantly larger than those receiving only Sedormid from t’he second through the twelfth day after hatching. After that time their growth rate gradually declined until it became almost identical with that of Sedormid-treated chicks from the nineteenth day on. Porphyria
Induced by Allylisopropylacetamide
To demonstrate the effect, of adenine supplementation on experimental porphyria induced by injecting AIA, which is soluble, it was found necessary to use a soluble adenine derivative, adenosine. liric Acid Excretion. Results were similar to those obtained using Sedormid and ndenine, i.e., injection of supplementary adenosine with AIA resulted in considerably greater increments in uric acid output in porphyric embryos than in their controls after the early stages of the experiment. Porphyrin Excretion. Exogenous adenosine reduced the porphyrin outputs of AIA-treated embryos, although this reduction was not so marked as that observed in Sedormid-treated embryos receiving adenine. The decreased efficacy of adenosine in this respect may arise from the less efficient, utilization of the nucleoside for nucleic acid synthesis as compared to the free purine base (18). significant differences in Embryonic Growth. Here again, statistically embryonic
weight
were not observed
until
4 days after
treatment.
From
454
TALMAN,
LABBE,
.~LDRICH
TABLE
Irccorporation of Glycirwf-(
-
SESKS
IV
“14into Pwines by Normal und Porphyric Chick Embryosa
T
Adenine Incubation
INI)
Guanine
Control”
Controlh
Porphyrkh
Uric acid
I
time Porphyric’
ControlC
1 Porphpric’
---,--min.
1.6 3 7 2. 7
20
60 180
-
0 .6 3.s 8.4
8.0 00 1.5
X.8 3.5 9.6
i
7220 6620
i
s;~o 4100
-
u Cilycine-S-C’“, 6.8 pc., was given intravenously I,orl,hvrin-producirlR drug via the yolk sac. * Counts/mm./pmole at mfinite thinness. c Total counts/min. in uric acid per egg.
6 days after
administr:ttion
of
that time on, AIA-treated embryos were significnntly smaller than the contxols, and exogenous ndenosine failed to improve their growth. This failure of adenosine to enhance gro&h, again, probably reflects the less efficient, ut,ilizat,ion of the nucleoside. Incorporation of G’lycinr-2-P into Purincs. These data are summarized in Table IV. Results obtained after 20 min. incubation indicate that the rate of purine synt,hesis in control embryos is at least t,wice that of the porphyrics. The obserxxtion that t,he specific ncti&ies of the tissue purincs from porphyric embryos ntt,nined higher values than the controls after 180 min. suggests t,hat (a) their rate of turnover is much reduced, and (b) the newly formed purines are probably diluted somewh:~t less by enclogenous purines. The latt,er possibility is supported by the observation that homogennt~es of porphyric embryos alw:~ys yielded smaller cluantities of purint than equal volumes of homogenates of control embryos t#hroughout t.he isolation procedure. I’reliminury ohservat~ions on t,he incorporation of glyciuc-2-C”’ ;md adenine-8-C~14 mto . liver purines in porphyric rats support, these conclusiolxl
The dat.u reported here indicate that chick embryos, like rat.s (I?), readily ut’ilize preformed adenine, but metabolize preformed guanine und xant’hine only slightly, if nt all. This agrees also with the findings of I,u and Winnick (18) who have reported good utilizat,ion of adenine-(2’4 and very poor utJilizntion of guanine-C I4 for nucleic acid synthesis in tissue cultures of embryonic chick hewt. N&her, Snell, and Cravens (In), on t,he ot,hcr hand, found guanine as well as adenine to be met~nholicnlly active, when used in conjunction with thymidine, in reversing nminopterin in-
PORPHYRIN
METABOLISM.
V
455
hibition of chick embryos. Xanthine, again, was ineffect,ive. A difference in procedure, however, may account for the divergent results. The more efficient oxidation and excretion of exogenous adenine by porphyric embryos as compared to the controls leave little doubt that purine catabolism and excretion are unimpaired and suggest that purine biosynthesis is defective in experimental porphyria. This suggestion is supported by the observation that the rate of incorporation of glycine-2-P into nucleic acid purines is reduced approximately 50% in porphyric embryos. A plausible explanat,ion for the smaller increment in uric acid output by control embryos following treatment with supplementary adenine is found in the results of earlier work on ndenine-treated mice and rats (20). Those studies demonstrated t#hat, exogenous adenine, administered in quantities greater than a certain threshold amount, is oxidized to 2,8dioxyadenine, and the latter is t,hen deposited in the renal tubules as crystalline occlusions, leading to a secondary uremia. It may well be t,hat, a similar impairment of renal function occurs in normal chick embryos receiving adenine. Porphyric embryos, however, in which purine biosynt,hrsis seems to be impaired, may be expected to ut,ilize preformed adenine more efficiently than normal emhryos. Thus, the t.hreshold quantity of adenine producing such sequelae may he elevat,ed in experiment’al porphyria . Since the decreased uric acid output of porphyric chick embryos apparently reflects a defect in purine biosynthesis rat#her than in purine catabolism, the question arises as to t,he nnt,ure of a defect causing simultaneous impairment in purine biosynthesis and enhancement of porphyrin biosynthesis. The work of Shemin and associates (21, 22) showing that through a common porphyrins and purines are related metabolically precursor, &nminolevulinic acid, makes it reasonable to postulate that’ an error in the met~abolism of the lat,ter may he responsible for such a phenomenon. Accordingly, this group advanced t’he following hypot’hexis to account, for its findings in experimental porphyria: A block in Shemin’s succinate-glycine cycle retarding the entry of the d-carbon at,om of b-nminolevulinic acid into the C1 pool, and thence into purines, results in accumulat,ion of this common precursor which is t,hen disposed of via the porphyrin pathway. This hypot,hesis adequately explains the findings reported here, hut studies designed t,o test this hypothesis furt’her, which are described in the accompanying paper, indicate t,hat, t,he metabolic block in experimental porphyria is prohahly located at some other point. However, the possibility that, some error in t,he metabolism of &aminolevulinic acid may be involved in experimental porphyria gains support from the observation of h-euherger and associates (23, 24) that the activit,y of the enzyme, &aminolevulinic acid dehydrase is increased twofold hy Sedormid treatment. This
45G
TALMAN, LABBE, ALDRICH ASD SEARS
latter effect might reflect a primary pot,entisting action of the drug on the enzyme system or it might arise secondary bo a block in another metabolic pathway normally open to ALA. Thus, the data presented here provide evidence that purine catabolism is unimpaired but a decreased rat’e of purine biosynthesis exists in experimental porphyria. Reduced porphyrin outputs of porphyric embryos following treatment with exogenous adenine likely reflect a sparing action of the preformed purine. The organism may respond to a purine deficit by elaborating more ALA in a vain attempt to overcome the metabolic block by mass action. When adenine is supplied preformed, however, some of the stimulus for ALA formation is removed and porphyrin formation is decreased. Supplementation of one of the metabolites in short supply with preformed material also probably accounts for t,he improved growt,h of Sedormid-treated embryos receiving adenine. SUMMARY
In order t,o assess the efficiency of purine catabolism in experimental porphyria, t.he ability of chick embryos to oxidize and excrete purines was tested by administering preformed adenine, guanine, and xant,hine. Both normal and porphyric embryos were found t’o convert adenine, but not, guanine or xant’hine, to uric acid, with the porphyric embryos apparently cntuholizing exogenous adenine even more efficiently than t,he controls. Furthermore, adenine supplementat,ion reduced t,he porphyrin output and improved the growt’h of Sedormid-t’rrated embryos. Although adenosine was somewhat less effective than adenine, it’s use with allylisopropylacet.amide gave qu:llit,atively similar results. Results of tracer studies using glycine-NY support the hypothesis of impaired purinc biosynt,hesis in experimental porphyria.
1. TALMAN, 12. I,., CARE, J. II., NEVE, II. A.. T,ABBE, R. F., ANI) A~,arcw, II. A., d. Rid. (%em. 212, 663 (1955). 2. I,ABBE, R. F., TAI.MAS, I<. I,., AND AI,URI(-H, II. A., Hiwhim. et Bioph!/s. .-trtt~ 16, 500 (195-l). 3. (GOLDBERG, .4., Riochem. Sot. S!/wposia (Cambridge, Engl.) 12, 27 (195-&). 1. (~OLI~BERG, A., AND RIMINGTON, C., Proc. Roil. Sot. (London) B143, 257 (1955). 5. TALMAN. l<. I,., I,ABBE, R. F., ANI) AI,DRI(-H, It. A., .lwh. Biochem. Bioph!/s. 66, 289 (1957). 6. I,ABBE, R. F., TALMAN, E. I,., AND ALDRIVH, It. A., Frrleratio~~ Pror. 14,241 (1955). 7. TALMAX, 15. I,., HUTCHENS, ‘I’. T., ANI) ALDRICII, R. A., Prof.. 80~. Expt/. Rio/. Med. 96, 130 (1957). 8 BEVERIISE, W. I. B., AND BL-RNET, F. M., “The Cnlt,ivntion of Virnses 2nd
Rirkettsiae in t,he Chick lXmt,ryo.” Series No. 256, T,ondon, 1946.
IbIedic:d Research Council Special Report
PORPHYRIN
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 2-I.
METABOLISM.
V
457
ST. JOHN, J. L., AND JOHNSON, O., J. Biol. Chem. Sa, 41 (1931). SCHNEIDER, W. C., J. Biol. Chem. 161, 293 (1945). HITCHINGS, G. H., J. Biol. C’hem. 139, 843 (1941). ABRAMS, R., Arch. Biochem. 30, 44 (1951). TALMAN, E. I,., i,n. “Standard Methods of Clinical Chemistry” (Sefigson, I>.), Vol. II, pp. 137-X. Academic Press, New York, 1958. BCCHANAN, 0. H., BLOCK, W. D., AND CHRI~TMAN, A. A., J. Biol. C’hem. 16’7, 181 (1945). DUNCAN, D. B., Bion~etrics 11, 1 (1955). BROWN, G. B., AND ROI,L, I’. M., in “The Nucleic Acids” (Chargaff, E., and Davidson, J. N., eds.), Vol. 2, p. 383. Academic Press, New York, 1955. BROWN, G. B., AND ROLL, I’. M., in “The Nucleic Acids” (Chargaff, E., and Davidson, J. N., eds.), Vol. 2, p. 344. Academic Press, New York, 1955. Lu, K. H. AND WINKICK, T., Ezptl. Cell Research 6, 345 (1954). NABER, E. C., SXELL, E. E., AND CRAVENS, W. W., Proc. Sot. Exptl. Biol. Med. 81, 20 (1952). PHILIPS, F. S., THIERSCH, J. B., AND BENDICH, A., J. Pharmacol. Exptl. l’herap. 104, 20 (1952). SHEMIN, D., AND RUSSELL, C. S., J. .4m. Chenz. Sot. 76, 4853 (1953). SHEMIN, D., RUSSELL, C. S., ANU ABRAMYKY, T., J. Biol. Chent. 216, 613 (1955). GIBSON, K. D., NEUBERGER, A., AND SCOTT, J. J., Biochem. J. 61, 618 (1955). XEUBERGER, A., Proc. 3rd. Intern. Congr. Biochem., 1966, p. 204.