51
Clinica Chimiea Acta, 5 8 ( 1 9 7 5 ) 5 1 - - 5 9 © E l s e v i e r S c i e n t i f i c P u b l i s h i n g C o m p a n y , A m s t e r d a m -- P r i n t e d in T h e N e t h e r l a n d s
CCA 6 8 1 4
C Y S T A T H I O N I N U R I A AND HOMOCYSTINUR[A H A R V E Y L. L E V Y * , S. H A R V E Y MADIGAN
M U D D , B. W I L L I A M U H L E N D O R F
a n d P H Y L L I S M.
Division o f Diagnostic Laboratories, Slate Laboralory Inslilule, Massachusetts D('parlmenl o f Public ~feallh, the Joseph P. Kennedy, Jr ;.~boratorics and Ncuroloj;y Service, Massachu sells General Hospital, the Department o f Neurology, llarvard Medical School, Boston, Mass. and the National lnslilute o f Menlvl lteallh, Bethcsda, Md. (U.S.A.) (Received August l, 1974)
Summary Three circumstances p r o m p t e d us to reexamine the relationship between abnormal cystathionine accumulation and possible homocystinuria resulting from this condition: (a) discovery of an infant girl with apparently alternating massive cystathioninuria and h o m o c y s t i n u r i a ; (b) the presence of homocystinuria in s(~me, b:,t not all, previously reported c~ses of cystathioninuria probably d u e to T-cystathionlne deficiency; and (c) the recent d e m o n s t r a t i o n that m a m m a l i a n cystathioninc ~-synth~se can cleave cystathionine to homocysteine. The following conclusions v,,.re reached: (a) H o m o c y s t i n e may arise as a :estdt of bacteria, c,~numunation of a urine sample initially contamint~ cystathionine, but n o t h o m o c y s t i n e . (b) After a m e t h i o n i n e load, a cystathioninuric p a t i e n t m a y excrete readily detected a m o u n t s of h o m o c y s t i n e . (c) However, h o m o c y s t i n u r i a is n o t a necessary c o n c o m i t a n t of even massive cystathioninuria. These findings and some of their implications are briefly discussed.
Introduction A urine sample from a three-week-:,Id girl, s u b m i t t e d on filter paper for r o u t i n e screening as part of the Massachusetts Metabolk" Disorders Screening Program, contained a large q u a n t i t y of cystathionine, but no homocy~tine. A follow-up liquid urine specimen sent te our laboratory from the h o m e of the patient contained h o m o c y s t i n e , but no cystathionine, and further such samples invariably contained cystathionine, or h o m o c y s t i n e , but n o t these two sulfur amino acids together. We were thus faced with the novel prob!em ef apparently alternating excessive excretions of h o m o c y s t i n e {i.e., h o m o c y s t i n u r i a ) and . f cystathionine (i.e., cystathioninuria). *
Requests for reprints should be addressed to: Harvey L. Levy, M.D., Metabolic Disorders Laboratory, State Laboratory Institute, 305 South S~reet, Jamaica Plain, Mass. 0213o, U.S.?,.
52 A search of t h e literature revealed t h a t excessive ~,' ,ary h o m o c y s t i n : has been r e p o r t e d in c o n j u n c t i o n with excessive u r i n a r y c y s t a t h i o n i n e in at least t h r e e specific i n b o r n errors o f m e t a b o l i s m [ 1 - - 5 , 1 6 - - 1 9 ] . In t w o o f these diseases i n a d e q u a t e c o n v e r s i o n of h o m o c y s t e i n e to m e t h i o n i n e leads to t h e acc u m u l a t i o n and e x c r e t i o n of ex-essive h o m o e y s t l e ) i n e [ 1 6 - - 2 0 ] . Since n o r m N a~tivities o f c y s t a t h i o n i n e '~-synthase IEC 4 • 2 . 1 . 2 2 ) are presen!: in these c o n d i :,.ons [ 1 8 , 2 0 , 2 1 ] , t h e a c c u m u l a t i o n of h o m o c y s t e i n e p r e s u m a b l y e n h a n c e s t h e rate o f c y s t a t h i o n i n e f o r m a t i o n by this e n z y m e { R e a c t i o n 1) s u f f i c i e n t l y t o lead to excessive c y s t a t h i o n i n u r i a : •
cysta thiorane
Homocysteine + serme . . . . .
fi-synthase
, cystathionine + H:O
(1)
T h e r e m a i n i n g p a t i e n t s with c o m b i n e d c y s t a t h i o n i n u r i a and h o m o c y s t i n u riaien,ia) have been ~,nown by direct e n z y m e assay [ 2 . 2 2 ] , or by i n d i r e c t genetic, ci~emical, and b i o c h e m i c a l e v i d e n c e [ 1,31, to have m a r k e d l y d e c r e a s e d activities o f - , , - c y s t a t h i o n a s e {EC' .1.4.1.I), the m a m m ~ i a n e n z y m e w h i c h cleaves c y s t a t h i o n i n e to cyst :ine and a - k e t o b u t y r a t e ( R e a c t i o n 2): Cw;tathim~ine ~- t i , O ?-cystathioaase ~'.~,'¢toino + ,a-'.->-tobutyrate + N I t ) . . . . . . . . . . . . . . . . . .
.
-.,
. . . . . . . . .
(2)
T h e p r i m a r y a b n o r m a l i t y leads to c y s t a t h i o n i n e a c c u m u l a t i o n a n d e x c r e t i o n . A possible e x p l a n a t i o n for t h e associated hon-~ocystinuria is o f f e r e d by t h e r e c e n t d e m o n s t r a t i o n that m a m m a l i a n cystathioai,r~e ,3-synthase is reversible [ 1 5 ] . T h e latter e,.~zyme is n o r m a l in p a t i e n t s w n h ~?.cystathionase d e f i c i e n c y [ 9 ] • T h u s , (_iep~pding. a m o n g o t h e r factors, u p o n :he e q u i l i b r i u m of this r e a c t i o n {Reaction I'!. a primm2, a c c u m u l a t i o n o f c y s t a t h i o n i n e m i g h t give rise to an a c c u m u !ation of sufficient h o m o c y s t e i n e to lead to h o m o c v s t i n u r i a . If this e x p l a n a t i o n i~ the correct o n e for the h o m o c y s t i , m u i a r e p o r t e d in cases of "),-cystathmnase -iefit'ie~=,cy, a d d i t i o n a l facto;'s m u s t at times play an i m p o r t a n t role since f u r t h e r p a t i e n t s s h o w n direc~iy [ 7 - - 1 ! 1 , ~"- indirectly [ 6 . 1 2 , 1 4 ] , to have .?-cystathionase deficie,.my have been r e p o r t e d ,:,,at to have associated h o m o c y s t i n u r i a . T h e investigations r e p o r t e d here were cm'ried o~tt in an a t t e m p t to u n d e r :-~,.,,..' .... c_t the relatiop.~hip . . . . . ~ t~'. ~,,.ep ,~ -,~:,' a p p a r e n t l y a l t e r n a t i n g c y s t a t h i o n i n u r i a a n d homo,.:ystinuria in our patient, a,.nct to -htrify w h a t fact:~rs m i g h t cause h o m o =_?".=tip..~sa to a c c o m p a n y prim:_u-y c y s t a t h i o n i n u r i a . An abstract covering s o r e Of "he .,•'esuits has a p p e a r e d [ 2 5 i .
=:':,r.~i~~(, acicls v.ere :=.~~.-.quantitatively s t u d i e d by two-wa'y s e q u e n t i a l elec• U'ophoresls ~.,-~'~";'~paper .::'[,.omato~raph; [241 and q u a n t i t a t e d bv i o n - e x c h a n g e ;) accordi,'~g to Efron [ 25] or ~pa,. ,, et a!. [26] T h e c o l t ' m n , -n..u.;~dog.,ui: ............. '-" ' , ' " ' ~r,,a , ",', ,_;-anide--r,i--cm,-,,;,~ ~ . , . ~ , ~~ , , . , _ , ...... est. wa~; p e r f o r m e d by pui)lished m e t h o d s e i t h e r on fil..... ~.~;paper [°"]~, o r i ~ . t t q u i d u r i n e [ 2 8 ] . For unequivoc~u i d e n t i f i c a t i o n of h o m o -.v:,th~e. the m a t e c a[ z,!utir:g in the h o m o c y s t i n e ca-ca was ~-,~.~,,,a,.,,,,,~.... by preparat>:e ion-e:,:(:hangc ,:i~:omatography {2¢[. P o r u o n s of t h e p u r i f i e d c o m p o u n d . . . . . . . . . . . . ,,:,.~e_~ ~c-, a~ ox,caL:on ~;:t, .I~erformic acid arm (b) r e d u c t i o n ~:""l~n .:-t,a-c, tnre~co. ,', ".', su.bseoueqt c o n v e r s i o n to t h e N - e t h y t m a l e i m i d e - a m i n o acid 3"
1
~
" .
.
.
.
.
53 derivative. The ninhydrin-reactive material(s} formed during each of these procedures behaved during ion-exchange chromatography [26] identically m the corresponding materials formed from authentic homocystine. N-Acetylcystathionine was identified by co-chromatography with the authentic c o m p o u n d in the following systems: (a) high-voltage electrophoresis in 8 per cent formic acid buffer (pH 1.6); (b) ascending and descending paper chromatography with butanol--acetic acid--:~'ater ( 1 2 : 3 : 5 , v/v), utilizing both ninhydrin and iodine--azide .as location reagents; (c) ,on-exchange column chromatography [25]. Further authentication was provided by the identification of t:ystathionine as one of the products of acid hydrolysis (concentrated hydrochloric acid at 120 ° for 12 hours} of the isolated compound. Plasma B, _~ concentration was measured by bioassay [29]. Urinary vitamin B6 was measured before and after acid hydrolysis [30] by bioassay [31]. Red cell glutamic-oxaloacetate transaminase was assayed according to Sass and Spear [32]. Methods for the growth of fibrotzlasts and assay of cystathionine /3-synthase activity in fibroblast extracts have been described [ 21]. UL-ine specimens from the infant pat:rot were collected by use of a U-bag (Hollister, Inc. Chicago, Ill. ). Results Bacterial conversion o f urinary eystathionine to homoeyst(e)ine Fig. 1 illustrates representative results obtained with various urine specimens from the patier.t witil apparently alternating cystathioninu~ia and homocystinuria. In addition to ab~ormal amounts of either of these two compounds, the urine invariably contained abnormally elevated amounts of N-acetylcystathionine. Urines that c:)ntained only homocystine and N-acetylcystathionine gave strongly positive ~.~actions in the cyanide---litroprusside test for disulfides, whereas uri,.~ . ~t:~ming only cystati~ionine and N-acetylc~s~athionine were negative or onl~ ¢lightly positive on testing with cyanide--nitroprusside. A number oI investigations were carried out in ~ttcmpts to define the underlying abnormality in this patient. Studies of fibroblasts cultured from a skin biopsy indicated ~hat the two known enzymatic pathways, deficient activity of which are known to result in homocy~tinuria (viz, methylation of homocysteine tG methionine and synthesi.¢ ..4. c" ~tathionine from homocyst~ine), were normal. The total plasma B, : c3n~en~,ation was normal (470 pg/m]), eliminating B, : deficiency as a -ause of the homocystinuria [33]: The findings of normai red blood cell gtutamic-oxaloacetate t r a n ~ m i n a s e activity [32] and normal urinary excrelion of free and total vitamin B6 [30] suggested there was no abnormality of Bs metabolism. Foliowing these investigations it was realized that amino acid analyses of plasma had always levealed cystathioninemia but never homocystinemia. Retrospective study showed that the urines containing homocystine also conmined bacterial ~ o w t h and had been cohected at home in container's that did not include a preservative, whereas the cystathionine-containi~,g umnes had either been collected at h o m e in containers that included a preservative (thymol or toluene) or had been obtained in the hospital and analyzed promptly, or after storage in the frozen state. Subsequent :indies outlined below showed
54
• ,,,m +
....
+-+++.
..+f
..... W+IK
t[ tprlmm °
Ats
i.:~: !
:
h,"
i : . , ~'c', i:J:',"
~,:,i>::~-.:
~}~:'+
s,,,~e:at~a!
r:ie:~ ,,',,i!c~t~:d
,.'
~)a:.)t'r+ ~'hr~,.maL ,:~-:~.]~hv. o f ~ r i n e s
',vtth
tl':;r!~+ii
,,~:.:~,.,u" ;::u:>+.,c~'~.~t- t [ , ' . , ~ u
: ~
:~'!:
~:L~',,.
; . ~t)"r
I r t ~ m the. m+a~+:t" ' : i l h
eystathi+)ninelnia
~t.b.retrllsx~o~ra~:~ c..~titailtt, d i r l d r R e d i v
. : : : : +,~:ut~g:-.~m~ ( t i f : : , ; l m d
,h~)l!l,,('v',t~n,"
JJ,.
(tarii,). increasi.d
l+~'~.")a~(I
N'+.c:,tvl-
2.'l-h u r i n e
collec-
with toluene
preser-
.
"! ,tl ,l~' J ]i:~!~ t}"',
t!~.,-~::+
Clinica Chimiea Acta, 5 8 ( 1 9 7 5 ) 5 1 - - 5 9 © E l s e v i e r S c i e n t i f i c P u b l i s h i n g C o m p a n y , A m s t e r d a m -- P r i n t e d in T h e N e t h e r l a n d s
CCA 6 8 1 4
C Y S T A T H I O N I N U R I A AND HOMOCYSTINUR[A H A R V E Y L. L E V Y * , S. H A R V E Y MADIGAN
M U D D , B. W I L L I A M U H L E N D O R F
a n d P H Y L L I S M.
Division o f Diagnostic Laboratories, Slate Laboralory Inslilule, Massachusetts D('parlmenl o f Public ~feallh, the Joseph P. Kennedy, Jr ;.~boratorics and Ncuroloj;y Service, Massachu sells General Hospital, the Department o f Neurology, llarvard Medical School, Boston, Mass. and the National lnslilute o f Menlvl lteallh, Bethcsda, Md. (U.S.A.) (Received August l, 1974)
Summary Three circumstances p r o m p t e d us to reexamine the relationship between abnormal cystathionine accumulation and possible homocystinuria resulting from this condition: (a) discovery of an infant girl with apparently alternating massive cystathioninuria and h o m o c y s t i n u r i a ; (b) the presence of homocystinuria in s(~me, b:,t not all, previously reported c~ses of cystathioninuria probably d u e to T-cystathionlne deficiency; and (c) the recent d e m o n s t r a t i o n that m a m m a l i a n cystathioninc ~-synth~se can cleave cystathionine to homocysteine. The following conclusions v,,.re reached: (a) H o m o c y s t i n e may arise as a :estdt of bacteria, c,~numunation of a urine sample initially contamint~ cystathionine, but n o t h o m o c y s t i n e . (b) After a m e t h i o n i n e load, a cystathioninuric p a t i e n t m a y excrete readily detected a m o u n t s of h o m o c y s t i n e . (c) However, h o m o c y s t i n u r i a is n o t a necessary c o n c o m i t a n t of even massive cystathioninuria. These findings and some of their implications are briefly discussed.
Introduction A urine sample from a three-week-:,Id girl, s u b m i t t e d on filter paper for r o u t i n e screening as part of the Massachusetts Metabolk" Disorders Screening Program, contained a large q u a n t i t y of cystathionine, but no homocy~tine. A follow-up liquid urine specimen sent te our laboratory from the h o m e of the patient contained h o m o c y s t i n e , but no cystathionine, and further such samples invariably contained cystathionine, or h o m o c y s t i n e , but n o t these two sulfur amino acids together. We were thus faced with the novel prob!em ef apparently alternating excessive excretions of h o m o c y s t i n e {i.e., h o m o c y s t i n u r i a ) and . f cystathionine (i.e., cystathioninuria). *
Requests for reprints should be addressed to: Harvey L. Levy, M.D., Metabolic Disorders Laboratory, State Laboratory Institute, 305 South S~reet, Jamaica Plain, Mass. 0213o, U.S.?,.
52 A search of t h e literature revealed t h a t excessive ~,' ,ary h o m o c y s t i n : has been r e p o r t e d in c o n j u n c t i o n with excessive u r i n a r y c y s t a t h i o n i n e in at least t h r e e specific i n b o r n errors o f m e t a b o l i s m [ 1 - - 5 , 1 6 - - 1 9 ] . In t w o o f these diseases i n a d e q u a t e c o n v e r s i o n of h o m o c y s t e i n e to m e t h i o n i n e leads to t h e acc u m u l a t i o n and e x c r e t i o n of ex-essive h o m o e y s t l e ) i n e [ 1 6 - - 2 0 ] . Since n o r m N a~tivities o f c y s t a t h i o n i n e '~-synthase IEC 4 • 2 . 1 . 2 2 ) are presen!: in these c o n d i :,.ons [ 1 8 , 2 0 , 2 1 ] , t h e a c c u m u l a t i o n of h o m o c y s t e i n e p r e s u m a b l y e n h a n c e s t h e rate o f c y s t a t h i o n i n e f o r m a t i o n by this e n z y m e { R e a c t i o n 1) s u f f i c i e n t l y t o lead to excessive c y s t a t h i o n i n u r i a : •
cysta thiorane
Homocysteine + serme . . . . .
fi-synthase
, cystathionine + H:O
(1)
T h e r e m a i n i n g p a t i e n t s with c o m b i n e d c y s t a t h i o n i n u r i a and h o m o c y s t i n u riaien,ia) have been ~,nown by direct e n z y m e assay [ 2 . 2 2 ] , or by i n d i r e c t genetic, ci~emical, and b i o c h e m i c a l e v i d e n c e [ 1,31, to have m a r k e d l y d e c r e a s e d activities o f - , , - c y s t a t h i o n a s e {EC' .1.4.1.I), the m a m m ~ i a n e n z y m e w h i c h cleaves c y s t a t h i o n i n e to cyst :ine and a - k e t o b u t y r a t e ( R e a c t i o n 2): Cw;tathim~ine ~- t i , O ?-cystathioaase ~'.~,'¢toino + ,a-'.->-tobutyrate + N I t ) . . . . . . . . . . . . . . . . . .
.
-.,
. . . . . . . . .
(2)
T h e p r i m a r y a b n o r m a l i t y leads to c y s t a t h i o n i n e a c c u m u l a t i o n a n d e x c r e t i o n . A possible e x p l a n a t i o n for t h e associated hon-~ocystinuria is o f f e r e d by t h e r e c e n t d e m o n s t r a t i o n that m a m m a l i a n cystathioai,r~e ,3-synthase is reversible [ 1 5 ] . T h e latter e,.~zyme is n o r m a l in p a t i e n t s w n h ~?.cystathionase d e f i c i e n c y [ 9 ] • T h u s , (_iep~pding. a m o n g o t h e r factors, u p o n :he e q u i l i b r i u m of this r e a c t i o n {Reaction I'!. a primm2, a c c u m u l a t i o n o f c y s t a t h i o n i n e m i g h t give rise to an a c c u m u !ation of sufficient h o m o c y s t e i n e to lead to h o m o c v s t i n u r i a . If this e x p l a n a t i o n i~ the correct o n e for the h o m o c y s t i , m u i a r e p o r t e d in cases of "),-cystathmnase -iefit'ie~=,cy, a d d i t i o n a l facto;'s m u s t at times play an i m p o r t a n t role since f u r t h e r p a t i e n t s s h o w n direc~iy [ 7 - - 1 ! 1 , ~"- indirectly [ 6 . 1 2 , 1 4 ] , to have .?-cystathionase deficie,.my have been r e p o r t e d ,:,,at to have associated h o m o c y s t i n u r i a . T h e investigations r e p o r t e d here were cm'ried o~tt in an a t t e m p t to u n d e r :-~,.,,..' .... c_t the relatiop.~hip . . . . . ~ t~'. ~,,.ep ,~ -,~:,' a p p a r e n t l y a l t e r n a t i n g c y s t a t h i o n i n u r i a a n d homo,.:ystinuria in our patient, a,.nct to -htrify w h a t fact:~rs m i g h t cause h o m o =_?".=tip..~sa to a c c o m p a n y prim:_u-y c y s t a t h i o n i n u r i a . An abstract covering s o r e Of "he .,•'esuits has a p p e a r e d [ 2 5 i .
=:':,r.~i~~(, acicls v.ere :=.~~.-.quantitatively s t u d i e d by two-wa'y s e q u e n t i a l elec• U'ophoresls ~.,-~'~";'~paper .::'[,.omato~raph; [241 and q u a n t i t a t e d bv i o n - e x c h a n g e ;) accordi,'~g to Efron [ 25] or ~pa,. ,, et a!. [26] T h e c o l t ' m n , -n..u.;~dog.,ui: ............. '-" ' , ' " ' ~r,,a , ",', ,_;-anide--r,i--cm,-,,;,~ ~ . , . ~ , ~~ , , . , _ , ...... est. wa~; p e r f o r m e d by pui)lished m e t h o d s e i t h e r on fil..... ~.~;paper [°"]~, o r i ~ . t t q u i d u r i n e [ 2 8 ] . For unequivoc~u i d e n t i f i c a t i o n of h o m o -.v:,th~e. the m a t e c a[ z,!utir:g in the h o m o c y s t i n e ca-ca was ~-,~.~,,,a,.,,,,,~.... by preparat>:e ion-e:,:(:hangc ,:i~:omatography {2¢[. P o r u o n s of t h e p u r i f i e d c o m p o u n d . . . . . . . . . . . . ,,:,.~e_~ ~c-, a~ ox,caL:on ~;:t, .I~erformic acid arm (b) r e d u c t i o n ~:""l~n .:-t,a-c, tnre~co. ,', ".', su.bseoueqt c o n v e r s i o n to t h e N - e t h y t m a l e i m i d e - a m i n o acid 3"
1
~
" .
.
.
.
.
53 derivative. The ninhydrin-reactive material(s} formed during each of these procedures behaved during ion-exchange chromatography [26] identically m the corresponding materials formed from authentic homocystine. N-Acetylcystathionine was identified by co-chromatography with the authentic c o m p o u n d in the following systems: (a) high-voltage electrophoresis in 8 per cent formic acid buffer (pH 1.6); (b) ascending and descending paper chromatography with butanol--acetic acid--:~'ater ( 1 2 : 3 : 5 , v/v), utilizing both ninhydrin and iodine--azide .as location reagents; (c) ,on-exchange column chromatography [25]. Further authentication was provided by the identification of t:ystathionine as one of the products of acid hydrolysis (concentrated hydrochloric acid at 120 ° for 12 hours} of the isolated compound. Plasma B, _~ concentration was measured by bioassay [29]. Urinary vitamin B6 was measured before and after acid hydrolysis [30] by bioassay [31]. Red cell glutamic-oxaloacetate transaminase was assayed according to Sass and Spear [32]. Methods for the growth of fibrotzlasts and assay of cystathionine /3-synthase activity in fibroblast extracts have been described [ 21]. UL-ine specimens from the infant pat:rot were collected by use of a U-bag (Hollister, Inc. Chicago, Ill. ). Results Bacterial conversion o f urinary eystathionine to homoeyst(e)ine Fig. 1 illustrates representative results obtained with various urine specimens from the patier.t witil apparently alternating cystathioninu~ia and homocystinuria. In addition to ab~ormal amounts of either of these two compounds, the urine invariably contained abnormally elevated amounts of N-acetylcystathionine. Urines that c:)ntained only homocystine and N-acetylcystathionine gave strongly positive ~.~actions in the cyanide---litroprusside test for disulfides, whereas uri,.~ . ~t:~ming only cystati~ionine and N-acetylc~s~athionine were negative or onl~ ¢lightly positive on testing with cyanide--nitroprusside. A number oI investigations were carried out in ~ttcmpts to define the underlying abnormality in this patient. Studies of fibroblasts cultured from a skin biopsy indicated ~hat the two known enzymatic pathways, deficient activity of which are known to result in homocy~tinuria (viz, methylation of homocysteine tG methionine and synthesi.¢ ..4. c" ~tathionine from homocyst~ine), were normal. The total plasma B, : c3n~en~,ation was normal (470 pg/m]), eliminating B, : deficiency as a -ause of the homocystinuria [33]: The findings of normai red blood cell gtutamic-oxaloacetate t r a n ~ m i n a s e activity [32] and normal urinary excrelion of free and total vitamin B6 [30] suggested there was no abnormality of Bs metabolism. Foliowing these investigations it was realized that amino acid analyses of plasma had always levealed cystathioninemia but never homocystinemia. Retrospective study showed that the urines containing homocystine also conmined bacterial ~ o w t h and had been cohected at home in container's that did not include a preservative, whereas the cystathionine-containi~,g umnes had either been collected at h o m e in containers that included a preservative (thymol or toluene) or had been obtained in the hospital and analyzed promptly, or after storage in the frozen state. Subsequent :indies outlined below showed
54
• ,,,m +
....
+-+++.
..+f
..... W+IK
t[ tprlmm °
Ats
i.:~: !
:
h,"
i : . , ~'c', i:J:',"
~,:,i>::~-.:
~}~:'+
s,,,~e:at~a!
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