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Introduction to discussion of Heinz body anemias—Unstable hemoglobinopathies
Exp. Eye Rcs. (1971 ) 1 l, 3 6 5 - 3 7 5
I n t r o d u c t i o n to D i s c u s s i o n o f H e i n z B o d y A n e m i a s H cmoglobinopathies VIP.GII, F.
Unstable
FAIRBANKS
,q,'cHo~: ¢f Clinical Pathology, 3layo Clinic, I?,~clmster, 3Iimwsota, 56901, U.S.A. l)urin,.: the past IS years, tmmerous eases of "congenital Heinz body anemia" have been I'Ol)~Wl('~I. It is now eh.ar thaL tmstablt:, therumlabile hemoglobins are presenL in most~ or all such ( ,~...,-.~. I.;nstnlflt: ht'nto~lol)ins Ztirieh. tI. i'~oln. I lasharort and L Ferrara, have been rel~)rt,.d ~n more than otu: family. Most. of th(" unsta})lo hemoglobins m a y be a t t r i b u t e d Ix) point Inut:).tions in the L'~ell¢'ti(."code. suc]| as a change from guanine to ndenino. NEarly 30 such :tn.'~tal)lo h(,mo,_,lol)inOlmthtes haw., been characterized f.rolll tile standpoint, of LhE nmlet'u!.~r pathology--that, is. the chan~e in structure of the globilt chains of hemoglobin. 'l'ho Ina)in:.r by which tht.so, moh, t.uhtr changt-s cause instability of the hemoglobin molecule st,.ins to b~: wtriablo, but very liktdy t tie effect ill most. is to loosen t,ho bond between heine nl)(I .L,Iol)m. fi)llowe¢t t,y fi)rlnnt ion of mixed tlisulti(lez~ anti denaturation and i)reeipitalbion of glottal. F.rylhro('ytos that contain unstable hemoglobhm may exhibiL ~Lceelera.ted glucose ('ol)sumpth-~. low ,~,ltltnthioltl: vo~wetltration, decreased glutathione sbability, and low (:-o)~('o)ltra t I()llS Of a(lmmsilm t ril)hOs |)hate. The rat e of methemoglobin formation anti of in v i t r o llll|O}lOlllO]X'~i.'~, Ill.fly })e lilt'teased, Oxygen dissociation is abnormal ill l!lolnE o f these cli~orxleru. Tile inheritance of th(.se disortiers is co~ntnoilly autosomal dominant;. HEmolysis m a y bo mild to sevcr~; it may occur only after sult'onamide ingestion or m a y no(; be demonstrable at all. Npldnectomy has been beneficial in a few cases of severe hemolytio anemia due to Illtstabiu hemoglobinopathy. I n 1890, t l c i n z describe(1 s m a l l , s p h e r i c a l , r e f l ' a c t i l e b o d i e s in t h e e r y t b r o c y t e s o f e x p e r i m e n t a l a n i m a l s e x p o s e d t o p h e n y l h y d r a z i n e a n d its d e r i v a t i v e s . ~k l a r g e n u m b e r o f c h e m i c a l s a r e n o w k n o w n t o be c a p a b l e o f i n d u c i n g s u c h I-teinz b o d y f o n . ~ a t i o n i n h u m a n e r v d t r o c v t e s . T h e s e i n c l u d e botll m e d i c i n a l s , s u c h a s - s u l f o n a m l d e s , a n d s u b s t a n c e s e n c o u n t e r e d hi a c c i d e n t a l o r occ:upationa.1 p o i s o n i n g , s u c h as n a p h t h a l e n e . S o m e c h a r a c t e r i s t i c s o f t h e H e i n z b o d y a r e t h a t it is s e e n in s a f i n e s u s p e n s i o n s o f c r y t h r o c y t e s , t h a t it s t a i n s w i t h a n u m b e r o f s u p r a v i t a l s t a i n s suci~ as c r y s t a l v i o l e t , a n d that. it. is n o t s e e n in W r i g h t - s t a i n e d b l o o d fibns. T h i s is p r o b a b l y d u e t o t h e s o l u b i l i t y o f ~he t-[einz b o d y in a l c o h o l f i x a t i v e s ( F e r t m a n a n d F e r t m a n , 1955). T h e H e h ~ z b o d y is b e l i e x e d to be c o m p o s e d p r i m a r i l y o f p r e c i p i t a t e d g l o b i n , b u t i t a l s o c o n t a i n s s m a l l q u a n t i t i e s o f p o r p h y r i n s , f e r r i t i n , a n d n u c l e i c acids. I n ] 952, C a t h i e o f L o n d o n , I g n g l a n d , r e p o r t . e d t h e e a s e oi" a y o u n g b o y w i t h c o n g e n i t a l h e m o l y t i c a n e m i a who, a f t e r s p l e n e c t o m y , m a n i f e s t e d l a r g e n u m b e r s o f i n t r a e r y t h r o c y t i c i n c l u s i o n s t l m t w e r e t i n c t o r i a l l y s i m i l a r t o t~eh~z b o d i e s . [ T h i s c a s e w a s s u b s e q u e n t l y r e s t u d i e d , a n d t h e p r e s e n c e o f an u n s t t t b l e h e m o g l o b i n ( h e m o g l o b i n B r i s t o l , a2fl.2'~Tasp) was c o n f i r m e d ( S t e a d m a n , ~:'ates a n d t-~uehns, 1970).] T h e r e f o l l o w e d t e l ) e r r s f , ' o m s e v e r a l c o u n t r i e s o f " c o n g e n i t a l [{einz b o d y " a n e m i a s ( A n d r 6 , D r e y f u s a n d L e B o l l o c h - C o m b r i s s o n , 1964; B a c h m ~ m n a n d M a r t i , 1962; B e r e t t a , P r a t e , G a l l o \ a n d L e h m a n n , 1968; C a r r e l l , L e h m a n n , Lorki,1, l ~ a i k a n d .l-Itmter, 1967; D a e i e eL al., ] 96,t.; ] ) a c i e , S h i n t o n , G a f f n e y , C a r r e l l a n d L e h m a n n , 1967 ; .De Vries, J o s h u a , L e h m a n n , H i l l a n d F e l l o w s , 1963; F a i r b a n k s , O p f e l l a n d BurgerI;, 1969; F r i c k , I-Iitzig a n d Sl~auffer, 1961; G o u d e m a n d , B i s e r t e , H a b a y a n d Voisin, 1964; G r i m e s , M e i s l e r a n d D a c i e , i 9 6 4 ; L e l o n g e t el., 1961; M o z z i c o n a c c i , A t t a l , : P h a m , 5 : [ a l a s s e n e t a n d 365
~6
Y. F. FA1/~13AN/(S Txnt,g I
Un,~table hem(×jlobins l~siduo no.
SubstAtutio~
Refcrenct-
Clinical effect
Design a t i o n
,~-Chain v a r i a ~ t ~ 43* ,t7
phe~'-vn! n.~p-~ g l y
Torino I3eilinson (L F c c r a r a )
Sul fonnmidc-hemo]5's~a Hemolysis
V r i e s el al. ( 1 9 6 3 ) ; ~'N'a~cl o t a l . i'lgO.~)
47 47
; t a p - - his ~ p - - ~ his
l-I a ~ h a r o n Sinai. ~ e a l y
~'one
80
lcu---~ ar~
A~;n A r b o r
~ot~e
Ite, l h r c c h ; vt aL (1967) Chn.r~ehe a n d , M o n d ~ e (1967): Ch-',rachc t:t al. {1969J Osteri.ag a m | Smith( t 96S),,~rhncider e t M. { tgf, s ~, I{uckrmgc, l (l~-r.c,mal
84 112 136 ~
.~e.r -~-a r g his--gtu Ieu---~.pro
Etobicokc~ Dakar Bibba
~N*one ltemolymn }temolysi.~
Cr~oksi~m ct at. (1911c,l) l¢o~a eL ;~1. [ 19118) K l e i h a u e r et hi. (t96~;1
Hemolysis
59 heine 88
B e r e t t n eL al. I t968) B i a ~ c o et al. (lfl6:|): l.)c
~
l r l I I l I i | t i (',l~t t4 iI 1 }
/3.Chain v a r i a n t s 6 14 23
gtu d e l e t e d ]eu--*arg va| deleted
24 2,8
gty--~ a rg leu--~pro
givemale-Bronx ('.~ ~2rl O g a,
~N'ot k n o ~ ~ Nm~c Hemolysis and mcth. emogh~binemi~ tt emo~yain Hernolyaia
30
arg-~ser
Tacoma
None
35 4 .°*
tyr--~pho pbe->~er
Philly Hammeramitb
Hemolyaia ]:I emolysi~
63*
his--~arg
Ziiricb
Su I f o n a m i d e . h e m o t y a i s
67*
val--~ a.~p
Bristol
H-emolysi~
6"1"
val--> al~
Sidney
:Hemoly~is
7,t 76
gly---~azp ala-~glu
Shepherd's Bush Seattle
88*
] eu---~pro
Santa Ana
tKomoJysia
88" 91"
leu~r, rg leu--->pro
Borha Sabine
Not}_e }:[emoJysis
93--98*
deletion
G u n ]:till
Hemolysia
Leiden Sogn Freiburg
Homol)"~i~ "l!temolysia
heine
I)c J o u g e~ al. (196~) MoJm eb al. (19~8) J o n e ~ et al. ( I 9 6 6 ) lCan2~cy et at. (1965) Sanzo~m e t al. (1967}; S a n ~ o n e a n d P i k (1965) ]3aur and Motulsky (1965); B r l m h a l l e t al. (t969) R i e d ~ r e t a } . (} 9l~t)) D a c i v et M. ( t 9 6 4 , 1967); Grlmc~ e t al, (1964) Bachmatm and Marti (1962); ,Friok o~ al. (1961); IXie~!er e t at. (1965); R i g a s el, at. (1956) C a t h i e (1952); ,Sta~,aclman e t ~.i. (1970) C~rr~ll et al. (1967) ; ~ a i k et. a~. (1967} Whitx~ e t al. ( I 9 7 0 ) t l u e h n a (1965); H u e h n s e t al. (1970); Mot, u l s k y
(196~) l~'ttirba~l_k~ e£ al. ( 1 0 6 9 ) ; Opfell e t al. (1968) H o l l a n d e r e t al, (1969) MjII~ eg al. (1968); S c h n e i d e r o t a l . (1969) 13radIoy a n d Risxtec (1965); : B r a d l e y et al. (1967); L o r k l a e t al. (1970)~ l'~iedar a n d B r ~ l e y (1968)
HEINZ
367
1301)Y A N E M I A S "I'An L r I ( r o n t i n a e d )
9S*
124 130 14:1
val-~n)et
]
Hemolysis
pro-~arg t vr~asp h i s - ~ a r:~
Khart<,um ~Vien Ohnsted
Not, k n o w n Hemolysis Hemolysis
C a r r e l ] e t a l . (1966, 1967); ] I u t , chi~on e t a l . (19{~i) : J a c k s o n e t al. ( 1 9 6 q ) ; J o n e s ct, a l . ( 1 9 6 7 ) ; _Pribilla e t al. (1965) C l c g g e t al. (1969) : P i e t s e h m a n n e t el. (1969) F a i r b a n k s e t el. ( 1 9 6 9 ) ; L o r k i n e t el. (1970)
O t h e r vaxiant.s flj
1!
Ifemolysis
J o n e s e t e l . (1959) ; FCigas e~ el. (1956)
* Amino acid poMtion~ immc
Bc.~.qis, 1961 : Muller and Kingma, 1961 ; R a i k , H u n t e r and Lindsay, 1967; :Rieder, Z i n k h a m and t [ o l t z m a n , 1965: Rigas, Koler and Osgood, 1956; Schmid, Brecher and Clemens. 1959 ; Scott, t l a u t . C a r t w r i g h t and Wintrobe, 1960 ; Sheehy, 1964 ; Shibata, Iuchi, Miyaji, Ueda and Takeda, 1963; S t e a d m a n et el., 1970; White, Brahl, Lorkin, L e h m a n n and Smith, 1970; \Vorms, Bernard, Bessis and Malassenet, 1961). ) l o s t of the,no cases had the following features in c o m m o n : a n e m i a persisting from infancy, the appearance of e r y t h r o c y t i e inclusions after splenectomy, a n d an tmusually d a r k urine c o n t a i n i n g excessive a m o u n t s of poorly characterized, p r o b a b l y pyrrolic, pigments. An autosomal d o m i n a n t i n h e r i t a n c e of the " H e i n z b o d y a n e m i a " was Txm.E
II
lb,latit'e electrophoretiv Tos'~im~s of u.~stable hemoglobins* Standard
Unstable l[ Seattle S h e p h e r d ' s ]3ush Dakar
Tacoma IIammcrsmith, Genova~, ~ristol, Philly, Wien, Torino, Olmmted
A
S
A2
{
BorAs Santa Ana, Sidney K61n Freiburg, Z ( i r i c h
Etobicoke, Sogn, Leiden, Bibba, Hasharon (Sinai), Riverdale-Bronx, .Ann A r b o r Sabine Gun Hill
Khartoum,
L Fcrrara
C F r c e a chains:~ Origin * A t p i t 8.6 on s t a r c h g e l ; r e l a t i v e p o s i t i o n s a r e a p p r o x i m a t i o n s b a s e d o n p u b l i s h e d d a t a . Htts also been reported to have mobility similar to that of hemoglobin S (Sansone, Carrell and : L e h m a n n , 1967). :]: A f a i n t b a n d m a y b e s e e n in t h i s p o s i t i o n in m a n y o f t h e u n s t a b l e h e m o g l o b i n s in w h i c h t h e f l . c h a i n is a n o m a l o u s ; this b a n d is d u o t o f r e e ,,-chains.
36S
V.F.
FAIRBANKS
clearly discerned in m a n y families, t-Iemoglobin elcctrophoresis showed in m o s t cases a broad trail e x t m u l i n g c a t h o d a l l v from the h e m o g l o b i n ..k I han, l. ; \ u t n h e m o [ ) ' s i s was t y p e I in m o s t cases studied. Frick et ft. (196I) a p p e a r to have linen the first to r e p o r t t,hcrn~ohtbility of h e m o g l o b i n (hemoglobin Ziirich) in this ~roup of tlisc"~rders. Daoie et al. (1964) showed that, t h e i r cases of H e i n z b o d y a n e m i a also had a,n easily demons t r a t e d themnolabile h e m o g l o b i n later described as hemoglolfin .t[:mlmorsmitl,. neff, a~ser (Dacie et al.. 1967). S u b s e q u e n t l y . t h e r m o l a l f i l i t y of hemoglo~,tn has been demonst.rated in n e a r l y all cases of I t e i n z b o d y a n e m i a , a n d it is for this reason t l m t we now regard these disorders as being due I,rimarily to the presence of an u n s t a h l e hemo~,lobin A t least. '> _5" u n s t a b l e h e m o g l o b i n s h a v e been identified a n d characterized it,, considerable d e t a i l w i t h respect to their clinical effects, e l e c t r o p h o r e t i e p'ttterns, a n d molecular a b n o r m a l i t i e s (Tables I a n d 1I). Of t.hese, h e m o g l o b i n s K61n, H a n d H a s h a r o n seem to h a v e been recognized in a g r e a t e r numl)er of cases t h a n a u v of the o t h e r s . . . 8 o m e of t h e u n s t a b l e hemoglobix~s h',ve been ,'cported i n d e p e n d o n l l y t,y d_ifferent g-roups of i n v e s t i g a t o r s , t h e r e b y i n t r o d u c i n g more t h a n one d e s i g n a t i o n for t h e s a m e m o l e c u l a r v a r i a n t . '['hus. h e m o g l o b i n s Beilinson a n d [~ F e r r a r a a p p e a r to ~,~be t h e same v a r i a n t . ~imflarls', t h e hmnoglobin v a r i a n t ¢~.a4;~'lsfl~ has been d e m o n s t r a t e d in p a t i e n t s in Israel, Texas. M a r y l a n d , and Michigan a n d has been called h e m o g l o b i n H a s h a r o n , Seal)'. Sinai. and :Michigan. A t least six fi~nilies h a v e been identified in which t h i s h e m o g l o b i n o p a t h y has occurred, all of A s h k e n a z i e J e w i s h a n c e s t r y b u t o t h e r w i s e n o t k n o w n to be related. I n the Israeli and r e x a s cases the p a t i e n t s were not anemic, a n d h e m o g l o b i n ]-Iasharon (Scaly) is believed not to be t h e r m o l a b i l e ( L e h m a n n , personal c o m m u n i c a t i o n ) . H o w e v e r , in the ot.her t~wo ins t a n c e s ( h e m o g l o b i n "'Sinai" a n d . " M i c h i g a n " ) t,he propositi had Jnild h e m o l y t i c a n e m i a s a n d t h e r m o l a b i l e hemog!obins. I n fact., the criteria for d e s i g n a t i o n of a h e m o g l o b i n as b e i n g unsta ble seem nol~ to h a v e been u n i f o r m l y applied, F o r e,cample, hemoglobin ]';tobieoke was not t h e r m o labile in fresh h e m o l y s a t e s but, was so a f t e r prolonged storage in t h e frozen, state. H e m o g l o b i n H o p k i n s - I I , w h e n purified, is t h e r m o l a b i l e b u t occurs in such low conc e n t r a t i o n in h e m o l y s a t e s t h a t turbidit, v is n o t observed on hea~;ing whole h e m o l y s a t e s (Charache, personal c o m m u n i c a t i o n ) . ]-liemoglobin K a n s a s h a s been described rep e a t e d l y as b e i n g u n s t a b l e , a l t h o u g h t h e basis for t h i s is n o t elea~ from p u b l i s h e d d a t a . H e m o g l o b i n Iro,ao~ e (~8~_ ~"-~Lu) (Rosa et, el., 1969) also ba-s been r e g a r d e d b y some as b e i n g u n s t a b l e , a l t h o u g h p u b l i s h e d d a t a do no~, p r o v i d e e v i d e n c e t h a t this is so. A few other h e m o g l o b i n s h a v e been r e p o r t e d as being u n s t a b l e , b u t t h e s t r u c t u r e of t h e s e is n o t k n o w n as of t h i s w r i t i n g ( S a g h i a p a l a n a n d l~obinson, .t968; S c h w a r t z , Schreiber, G o l d m a n a n d lVagelman, 1968). To date, m o s t of t h e u n s t a b l e h e m o g l o b i n o p a t h i e s h a v e been r e p o r t e d o n l y in single families; some u n d o u b t e d l y r e p r e s e n t n e w m u t a t i o n s because t h e y h a v e not~ been d e t e c t e d in o t h e r f a m i l y m e m b e r s . T h e i n h e r i t a n c e a p p e a r s to h a v e been autos o m a / d o m i n a n t in those families in w h i c h several m e m b e r s were affected. T h e st,r u c t u r a l c h a n g e s in t h e h e m o g l o b i n molecule for t h e m o s t p a r t m a y be e x p l a i n e d on t h e basis of c h a n g e s of single nueleotides in t h e . g e n e t i c code, as f r o m g u a n i n e to a d e n i n e . T w o disorders ( F r e i b u r g a n d L e i d e n ) are explicable as d e l e t i o n s of single c o d o n s (nueleotide t r i p l e t s in t h e g e n e t i c code). I:[emoglobin G u n Hill ~nust represent, d e l e t i o n of a 15-nucIeotide seq~mnce, l:[emog[obin [£ is believed to be d u e to demreased ¢,.-ehain s y n t h e s i s as a r e s u l t of d o u b l e h e t e r o z y g o s i t y for t h r e e a.-thalassemia genes ( L e h m a n n , 1970). -
HEINZ
369
I3OI)Y ANEMIAS
Several of ~he unst~able hemoglobins have substitutions of polar (hydrophilic) for non-polar (hydrophobic) amino acids in the pocket of the globin chain t h a t contains the heine componen~ (Figs 1 and 2). The presence of hydroptfilie radicals in this "greas}, cleft" facilitates t,he entry of a water molecule, and tlfis in tm'n leads to methemogiobin for.ination and ultimately to hemoglobin denaturation. This t y p e of alteration occurs in hemoglobins ~,Vien, Barbs, Sogn, Ann ~krbor and Shepherd's Bush. These and at,her substitutions also m a y cause distortion of spatial re]ationships t h a t nomrm,lly, exist between globin and heine, thereby weakening the Van der Weals forces and causing dissociation of heine from its globin chain. In hemoglobin Khba, for example; there is substitution of methionine for the slightly smaller reline in a posit, ion.., contig,~ous to he,mi.~l?l~is,,,, slight, change in size of amino acids in a critical area. results in r~n increased tend~'.nc) of heine and globin to dissociate and denature (Jacob, Brain and Daeie, 1967; kIae6b, Brain, Daeie, Carrel/and Lehmann, 1968;
156 B
~Torino
43
'~L. Ferrara 47 Hashoron Sinai
80AnnA r b o r ~ ~
F i e . I. Sites o f c h a n g e s in c¢ chain responsible for u n s t a b l e h e m o g l o b i n o p a t h i e s . Of t h e f o u r s u b s t i t u tions indicated, t,wo (residues 4q a n d 47) are in the h y d r o p h o b i a p o c k e t tha~ c o n t a i n s t h e heine m o i e t y , a n d residue 43 is a eriticM heine c o n t a c t . This position is analogolm t o position 4-2 in t h e fl ohain. I { e m o globin L F c r r a r a (Beilinson) a n d h e m o g l o b i n ~ I a s h a r o n (Sinai, Scaly, ~'[iehigan) b o t h invobze s u b s t i t u tion in a m i n o acid residue 47. I n a d d i t i o n to the u n s t a b l e h e m o g l o b i n s indieaeed in t h i s ~Jlust,r a t i o n , t h r e e o t h e r s h a v e been a t t r i b u t e d to subst, i t u t i o n in t h e ~-ehain b u t details of t h e s e . a r e n e f f c o m p l e t e . Gun Hill 9 3 - 9 8
88San,eBores Ana.//L 14 Sogn/
~~Freiburg23 \ ~'.~
Geneva 2 8
" Zurich 63
""-~S heph~ir~;e; 2sTh74 Seattle 76 :Fro. 2. Sites of c h a n g e s in/3 c h a i n leading to h e m o g l o b i n insCability. Bfos~ o f t.hese cluster abou¢ elefl~ wherein heine m o i e t y is lodged, a n d some are a t eri~ieM heine eontao~ p o i n t s such as a m i n o acid p o s i t i o n s 98, 42 trod 63. T h e c h a n g e in h e m o g l o b i n O l m s t e d in the /~ c h a i n i s a n a l o g o u s to t h a t in h e m o g l o b i n ]31bbo, in ghe ~z chain.
370
V.F.
FA!RBANKS
J a c o b and "Winterhaltcr, 1970). Analogous to this are the changes in henaoglobins Torino, Sidney. H a m m e r s m i t h , Bristol, Gun Hill and Shepherd's Bush. In flint, in hemoglobin Gun }Jill the/9 chains appear to have lost their he,he groups. The introduction of proline (as in hemoglobins Genova, S a n t a :kna, l~ibba, and Sabine) into a n o r m a l l y helical segment of the globin chain prevent~s helix formation, thu.~ m a r k e d l y altering the confi~lration a n d stability of the hemoglolfin molecule. In henaoglobin Ziirich, the histidine residue t h a t normally binds heine to globin isreplaecd by arginine. a change t h a t would be expected to result in a greater tendency of hezne to dissociate from globin. I n sex'eral of the unstable hemoglobins, the substitution al)p(:ars in an e x t e r n a l l y located amino acid a t a site distant from the heine moiety, and it is uncertain in w h a t m a n n e r these alterations m a y lead to instability of the hemo.,_,lobin molecule. I n the unstable hemoglobinopathies, hemolysis ramges from severe {he,noglobins S a n t a Ana. Bristol, Sabine. H a m m e r s m i t h and Olmsted) to none (hemoglobin.s H a s h a r o n , Tacoma, Sogn and Bor'as). Some unstable hemoglobins, such as Gun Hill and Freiburg, are associated with mild hemolysis in spite of the severity of the molecular lesion. Hemoglobin Ziirich is associated with hemolytic (tisemse only after its bearer ingests sulfonamides. Sulfonamide-induced hemolysis is also characteristic of hemoglobin Torino. W h e t h e r drug ingc:stion nmy prec.il)itate hemolysis in other of these disorders is not y e t known. I t seems prudent, however, to advise such patients against indiscriminate d r u g use, and speckficall),, sulfonamides should not be used ~o t r e a t infections in these patients. Some of the unstable hemoglobins have been associated with abnormal oxygen dissociation (K61n, Freiburg, Gun Hill), b u t m a n y have not as yet been studied from this standpoint. Abnormalities of e r y t h r o c y t e metabolism h a v e been reported in m a n y patient,-.' with unstable hemoglobinopathies (Table I I I ) . Low concentratim).s of adenosine t r i p h o s p h a t e (ATP) (Grimes et el., 1964; Mills, Lcvil~ a n d Alperin, 1968; R a i k et el., 1967; Schneider, Ueda, _Mperin, Brimhall and Jones, 1969) and of glutathione have been reported ( F a i r b a n k s et el., 1969; Grimes et al., 1964). Glut~tthionc stability m a y be" decreased (Jones, Grimes, CarrelJ, and L e h m a n n , 1967; R a i k et al., 196"7). There m a y be an enhanced t e n d e n c y toward methemoglobin formation (Berett~ et al., 1968 ; H u t c h i s o n et al.', 1964 ; J o n e s et al., 1967 ; K l e i h a u e r et el., 1968 ; Mozziconacci et el., 1961; Schneider et aI., 1969; S t e a d m a n et al., 1970). Glucose consumption m a y be accelerated, particularly by the hexose m o n o p h o s p h a t e " s h u n t " p a t h w a y (Jacob et al., 1967). The relationship of these metabolic aberrations to abnormalities of hemoglobin structure would seem to merit f u r t h e r investigation. T A B L E III
Metabolic abnormalities reported in some un.stabIe hemoglobinopathies Abnormal
hemoglobin
K61n Santa Ana Sabine Hammersmith
Glutathionc
,JIethemoglobin
ATP
Low to normal Low to normal Normal Low
Righ }tigh High High
Low -Low Low
Glucose consumption --High. High
W h e n there is little or no hemolysis or a n e m i a is mild, there is little reason for concern a b o u t the p a t i e n t ' s well-being, his activities need not be restricted, a n d no
IIEINZ
.BODY ANEMIAS
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t r e a t ~ n e n b is mlvise¢t. ( ) u r e x p e r i e n c e w i t h p a t i e n t s w i t h h e m o g l o b i n K61n d i s e a s e is t h a t t h i s is a r e l a t i v e l y b e n i g n d i s o r d e r . M a n y o t h e r u n s t a b l e h e m o g l o b i n o p a t h i e s a r e s i m i l a r l y b e n i g n . \Ve a d v i s e againsl~ a d m i n i s t r a t i o n of drugs such as sulfonamides, whicI~ M~.ould t)e e x p e c t e d t o a c c e l e r a t e h e m o l y s i s . S o m e p a t i e n t s w i t h m o r e s e v e r e a n e m i a h a v e b e e n b e n e f i t e d m a r k e d l y b y s p l e n e c t o m y . T h i s h a s b e e n t r u e in p a t i e n t s wit.h Itenm,..,Iobin S a n t a A n a o r I t a n d in a f e w w i t h h e m o g l o b i n K 6 1 n d i s e a s e . S p l e n t , ( . t o m v sl~(>uhl I)o s e r i o u M y c o n s i d e r e d in p a t i e n t s w}~o h a v e m o d e r a t e o r s e v e r e am.'~nia. T i m rt, s u h , s o f s p I c a e ( ; t o m y , b o w e v e r , c a n n o t b e p r e d i c t e d w i t h c o n f i d e n c e , a n d s u ~ m p a t i e n t . ~ c o n t i n u e to h a v e s e v e r e a n e m i a . [ t is h o p e d t h a t e l u c i d a t i o n o f t h e :noleculnr and biochemical bases of this group of diseases may lead ultimately to a rationul 'rod .successful thcral)y for such patients. l:{ E F E I ~,E N C E S Andr,'., It., Dr%vltzs. l;. and l.cltolloch-Combrlsson. A. (19ti4). Bull. ;litre. 8oe. '.lied. I-lo19. Pari~ 115, 475. l~achmann, 1". a n d .Marl.i, It. 1{. (1902). 131cod 20, "~"~ Baur, E. ~,V. a u d Motulsk.v. :\. G. (1965). llu~J~t~ule,letilc I, 13~21. l~,ert~t.ta, :\., I'rato. V.. italic. E. anti l.,ehnltum, It. (I.9ti8). N a l u r e (Lo?Mo~)217, 1016. Bronco, I.. Modia,m. (.;., l{ottini, E. a n d Lucci, I.{. (1963). N a l u r e (London) 198, 395. |gradh~y. T. B.. J r . and I{iedcr, I~. F. (1965). Blood 28, 975. i{radh,y, T. It., J r., \VoM. 1¢,. C. a n d l{.ieder, i{. F. (1967). ~cie?rce, :V. Y . 157, 1581. I{rm'thall, 13., J o n e s . l~. "1'.. ltaur, E. \V. and 5Iotulsky, t\. G. (1969). 13ioch~emi.stry 8, 2125. Carrcll. II. \V.. l ~ h m a n n , H. a n d l h l t c h i s o n , it. E. (1966). Nalure. (Londo?~) 210, 915. (.:arrell, 1<. \V., l,¢q~m,um. 11.. l~)rkin. P . . \ . , l{ai'k. E. and l-[ttnter, E. (1967). N a t u r e ( I ~ t o ~ ) 215, 626. Carrell, l<. \V., I,eil,nann. It. and PribiHa, \V. (1967). Kli,~. Wsch?'. 45, 1189. Cathie, i. ;\. B. (195:2). (;re¢1l Orpnotul S t J . J u n e , pp. 43-48. Chart, che, S. a n d Mondzac, A. M. (1967). Blood 30,879. Ch:trache, S., Mon(tzac, .-\. 31. a a d Go.,kqner. U. (1969). J . Cli;z. lnvest. 48, 8::14. Clcgg, J . 13.. \Veatherall, D..J., Boon, \V. 1-I. a n d Mustafa. D. (19139). N a t u r e (Lolulon) 222, 379. Crookston, J . H., F a r q u h a r s o n , 11. A.. 13eale, D. a n d l.,clmmm~, 1-I. (1969). Canad. J . ]3iocl~en~. 47, 1-13. l)acic, J. V., Gri,ncs. 4.. ,1.. 31eisler, A., Stcingold, L., Hcmst, ed, E. H., 13eavcn, O. H., a n d \Vhite, J. C. (1964). B r i t . . 1 . llaen~olol. 10, 388. l)acic, J . V., Shinton. N. K., ('laffney, P. J., ,Jr.. Carrell, R. \V. and L e h m a n n , H. (1967). ~Yalure (Lo~uion) 216, (163. De J o n g . DJ. ~\r. V~q. ~Vmat, l,. N. artd 13ernini, L. F. (1968). N a t u r e (1london,) 220, 788. De Vries, A., J o s h u a , tI., l~chnaann, H., Hill, 1~. L. a n d l~'ellows, IR. E. (1963). 2-3rit. J . H a e~atol. 9, 484. Fairbanks, V. I?., Ol)fcll, 1{. \V. a n d B u r g e r t , E. O., ,Jr. (1969). A mer. J . 2~[ed. 46, 344. l,'ertman. 31. It., a n d F e r t m a n , hi. B. (1955). J/'ed/cine (Balti~ore) 34, 131. Frick, 1". G., l"Iitzig, \V. H. a n d Stauffer, U. (1961). Schweiz. 'lied. lVochenschr. 91, 1203. G o u d e n m n d , hlt., Biserte, 0., t[~tbay, D. n n d Voisin. D. (1964). N o u v . Rev. I,'r. £lernato.l: 4, 487. Grimes, A. ,j., ~Ieisler. A. a n d Daeie, J. V. (1964)..Brit. J. Haematol. 10, 281. I t a l b r e c h t , I., lsaacs, \V. -k., Lehma~m, H. and/3on-:Porat, F. (1967). Israel J . '.Med. 8ci. 3, 827. Heinz, II. (1890). Virchows Arch. Pathol. A n a t . Phy8.1ol. K(. .~Ied. 122, 112. Hollcndcr, A., I m r k i n . P. A., L e h n m m L H. a n d Svensson, ]3. (1969). N a t u r e (Lo?u/on) 222, 953. H u e h n s , E. 12.. (1965). Prov. Roy. ,.~oc. ~lIed. 58, 514. I-[tmhns, E. 1%., H e e h t , I0.. Yoshida, A., Stamat, oyannopoulos, G., 1-Iartman, J. a n d l~To~ulsky, A. G. (1970). -Blood 36, 209. H u t e h i s o n , H. E., P i n k e r t o n , P. tt., W a t e r s , P., Douglas, A. S., L e h m a n n , tI. a n d :Beale, D. (1964)..Brit..,lied. J. ii, 1099. Jackson, J. hi., \Vay, B. ,j. and Woodliff. H. J. (1967). Brit. J. Haematol. (Suppl) 13, 474. Jacob, H. S., Brain, ~I. C. and Daeie, ,j. V. (1967). J. Clin. lnvest. 46, 1073. J a c o b , H. S., Bruin, h L C., Dacie, J. V., Carroll, 1~. W . ~nd Lehraann, H. (1968). N a t u r e (London) 218, 1214.
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Jacob, 11. and \Vinicrhalier, K. (1970). ]'roe..\'~.t. A~d. ~ci. U.S...t. 65, 697. J o n e s , If. '1".. 13rimhaU..13., t t u i s m a n . '1". 1t. O., l~leihauer, 12. a n d B c t k c , K. (1966). Science, ?v'. Y. 154, 1024. J o n e s , 11. T., Schroeder, "~V. :\., 13alog, J . 1~]. a n d Vinograd, J. 1l. (1959). J . . - l i n e r . Chem. Soc. 81, 316I. J o n e s , 11. V., Grimes, A. J., Can-ell. R. \V. a n d L e h m a u n , t i . (1967). ltrit, d. llr~em~ltol. 13, 394. 1,21eihauer. E. 1;., Ile3nlolds, C. A., Dozy, A. 31., \Vilson. d. 1~., .Moores, 11. 11., lSercz,~tu*, 31. 1'., "~Vright, C.-S., a n d H u i s m a n , T. t t . J . (1968). Bio,:him. JJiophys. ,.tct~, 154, 220. L e h m a m l , t l . (1970). LaTtcet ii, 78. I_~long. 3i., I.'leury, J . , Alagille, D.. ~[alassenet, 1~., L o r t h o l a r y , P. a n d P a r s , M. (191.;1). A'ouc. Re','..Fr. Hematol. 1, 819. Ix~rkin, P. A., I . ~ h m a n n . t t . , F a i r b a n k s , X'. 10, / l e r g l u n d . G. a n d L e o n h a r d t , T. (19;0). Proc. Biochem. Soc. J u l y S. 51ills, (4. C.. Levin. \V. C. a n d :\lperin, J. ]3. (196S). Bh~c,d 32, 15. 5lonn. E., Gait'hey, P. J . , J r . a n d I . e h m a n n , 1t. (196S). b'cand, d. ]laem~dol. 5, 333. 3 i o t u l s k y , A. G. (1969). Cited in L e h m a n n , 1t.. a n d Carrell. If. \V. B r i t . . U c d . Bull. 25, 14. 5Iozzieo:mcei. P., A t t a l . C.. l~ham. It.-T.. Malassenet, II. a n d 13essis. 51. (1961). :Yo~,,. ]~'et'. ~'r. Hematol. 1, $32. 51uller, C. J . a n d K i n g m a , S. (1961). Bicwhlm. B i o p h b s . . . l o i s 50,/59~5. Nagel, I~. L.. ]~Ianney. 1t. 31.. B r a d l e y , T. 13.. J a c o b s , :\. a n d Udem. I,. (1969). l~lood 34, 157. Opfell, R. \V., I~orkin. P. A. a n d L e h m a n n . }I. (1968). ,1. 3led. Gvnet. 5, 292. Ostertag. ~\'. a n d S m i t h , E. \V. (196S). Humamye,tetik 6, ;377. P i e t s c h m a m l . 1t., ~[.,orkin, P. A., Le.hmann, It. and ]~raunsteiner, 1|. (19119). Cited in l x q m l a n n , H. a n d Can-el!. II. \V. Brit. 31ed. Bull. 2S, 14. Pribilla, \V., I~lesse. P., Betke. K.. I . e h m a n n , t1. a n d l~eale. D. (19t;5). K i l n . l|'ochenscltr. 43, I(M9. R a i k , E.. ]~untx~r, E. G. a n d L i n d s a v , D. A. (1967). ~lled. J . Australia !, 955. l l a n n e y , t{. _M., ,Jaeobs. A. S., U d e m , L. a n d Z a l u s k y , 1t. (1968). Bio,'l~em. Biophy.~. Rcs. Commull. 33, 1004. l t i e d e r , ]t. F. a n d Bradley. T. 13., J r . (1968).)31cod 32, 35.5. llieder, l/. F., Oski. F. A. a n d Clegg. J. B. (1969). J . Clin. Inve.~t. 48, 1627. l:lieder, 11. F., Zin "tdmm, \V. II. a n d t l o l t z m a n , N. 2\. (1965). Amer. J . _;led. 39, 4. tligas, D. A.. l,Loler, R. D. a n d Osgood, E. E. ([956). J . Le~b. Clin. 3Ied. 47, 51. Rosa. J . , Labie. D.. \ V a j c m a n , It., Boigne. J . 5[.. Cabannes. 11., Bicrme. 1:~. a n d lluftlo..J. (1969). N a t u r e (London) 223, 190. l l o s a . J . , O u d a r t . J . L.. Pagnier. J . . B e l k h o d j a . If., ]3oigne. J . M. a n d Labia, D. (19GS). 12lh Intermr~tional Society of Hematology. p. 72. New York. Sansone, G., Carrell, r~. \ v . a n d L e h m a n n , I1. (1967). Nature (London) 214, 877. Sansone, G. a n d Pik, C. (1965). Brit. J . Haenmlol. 11, 511. S a t h i a p a l a n , R.. a n d R o b i n s o n . M. 0 . (1968)..Brit. J . lIaemalol. 15,579. S e h m i d , R., Breeher, C. a n d Clemens, T. (1959). ]Mood 14, 991. Schneider, 1R. O., Ued~, S., Alperin, J. B., Brimhall, 13. a n d J o n e s , R. T. (1968)..elmer. J. H u m a n Ge~et. 20, 151. Schneider, R. G., U e d a , S., Alperin, J . :B., Brimhall: B. a n d J o n e s , I),. 2:'. (1969). N e w Emil. J. :Ved. 280~ 739. S c h w a r t z , J . M., Sehreiber, Z., G o l d m a n , B. a n d l " a g e h n a n , K . (1968). 12th I n t e r n a t i o n a l Society of Hematology, p. 73, N e w ~_-ork. Scott, J. L., 1-Iaut, A., Cart~right, G. E. and ~,Vintrobe, M..~I. (1960). Blood 16, 1239. S h e e h y , T. ~,V. (1964). Arch. I n t . 2led. 114, 83. Shibat~, S., Iuchi, I., ~[iyaji, T., Ueda, S. a n d T a k e d ~ , I. (1963). A c t s Hae~natol. J a p . 26, 164. S t e ~ d m a n . J . 1~., Y a t e s , A. a n d J-Iuehr~s, E. II.. (1970). Br;A. J . llae~t.tol. 18, 435. W h i t e , J . M., B r a i n , hi. C., L o r k i n , P. A., L e h m a n n , J~. a n d S m i t h . M. (1970). Nature (London) 225, 939. Worms, I~., Bernard, J., Bessis, l~I. and ~lalassenet, 1%. (1961). N o s y . . R e v . F t . Hermztol. 1,805.
DISCUSSION :DR BEu-I'~ER. I think t h a t one of the things t h a t both Dr¢ K~-osmTA and I hoped ~ ould come oueoof this very lucid description of the unstable hemoglobins is the realization on the part of the lens contingent t h a t there m u s t be crystallinopathies, which should be the analog of hemoglobinopathics. These disorders, I would think, would be dominantly t r a n s m i t t e d cat~ractm These c~taracts ought to be always
HEINZ
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ANEMIAS
373
b i l a t e r a l . '['h.ey m i g h t I)e i,,e~ent at b i r t h , b u t also c o u l d o c c u r l a t e r in life. [ t h i n k t h i s is r e a l l y o n e o f t h e ~no.~t f r u i t f u l ~ r e a s :,f~nah,~y, a n d ] a m e s p e c i a l l y l o o k i n g f o r w a r d to "DR SI'EC~'OI~.:s d e s c r i p t i o n o f c r y s t a l l i n c h e m i s t r y , w h i c h i t h i n k p r o b a b l y is n o t q u i t e as f~r a l o n g as h e m o g l o b i n c h e m i s t r y , a t t h i s p o i n t . T h e r e is o n e q u e s t i o n t h a t I w o u l d like t o d i r e c t t o DR J A c o n . l i e i n d i c a t e d t h a t t h e h e x o s e m o n o t~ho~t)hate s h u n t a c t i v i t y was r e g u l a t e d b y t h e level o f G S I [ . I t s e e m s t o m e m u c h m o r e l i k e l y t h a t tile a c t i v i t y o f t h e s h u n t is r e g u l a t e d by t h e level o f G S S G or o t h e r s u b s t r a t e s for g l u t a t h i o n e r e d u c t a s e . As I)R S~t~v,~sT,~v~ i n d i c a t e d y e s t e r d a y , t h e h e m o g l o b i n o f G S l [ m i x e d disulfide, w h i c h Dr¢ JACOB h a s s h o w n o c c u r s in tit(.st, u n s t a b l e hcnm~:lobiu s y n d r o m e s , is a s u b s t r a t a for t h i s e n z y m e . T h e r e f o r e , m y g u e s s w o u l d I)c t h a t t h e increasc(I h e x o s e m o n o p h o s l ) h a t e s h u n t a c t i v i t y r e p r e s e n t s a v a i l a b i l i t y o f t h i s s u b st r a t e for -,.h)tat h irene n.<~uetast,. l ) a J.~c:)l,. I t.hink t h a t is a v e r y a t t r a c t i v e h y p o t h e s i s . I d i d n ' t m e a n to i n d i c a t e that, t h e level of g l u t a thion(, a h ) n c u t i m u l a t e s th(: s h u n t . I t h i n k y o u k n o w o f o u r p u b l i c a t i o n i n t h e Jour~,l of.Biological (.'he~ni,vtr!/. T h e sin~zle b e s t c o r r e l a t e o f hexose m o n o l ) h o s p h a t e s h u n t a c t i v i t y is t h e r a t i o of G S S G to G S i t . A n y t h i n g ~hat w o u l d incrca.sa G S S G w o u h l i n c r e a s e t h e s h u n t ; a n d a n y t h i n g w i t h a g i v e n a m o u n t o f (;.%~G th-~t wo,~ld d e c r c , ~ e G S t [ w o u h l i n c r e a s e t h e s h u n t . W e s i m p l y s u g g e s t e d t h a t t h e r e d u c t i o n i n ( ; S I t b y b i n d i n g t h r o u g h the d e n a t u r i n g h e m o g l o b i n is a s t i m u l u s . I t h i n k y o u r i d e a o f g l u t a t h i o n c r e d u c ta.~e a c t i n g o n th,~" m i x e d d i s u l l l d e is also valid. ])l'. I<[)'rHSTEIN. ~[ was vt.ry f a s c i n a t e d b y 131~J A c o n ' s d e m o n s t r a t i o n t h a t t h e H e i n z b o d i e s a r c b o u n d to he mere b r a h e t h r o u g h d isu}~i,le b o n d s , a n d f u r t h e r , t h a t t h i s r e s u l t s i n t h e c h a n g e i n p o t a s s i u m p e r m e a b i l i t y , l a m l m r t i e u l a r l y i n u ' r e s t e 4 i~ t h e f a c t t h a t in s o m e o f t h e e l e c t r o n m i c r o g r a p h s , i t ' s q u i t e clear t h a t the~o Hei,~z b o d i e s r e a c t w i t h o n l y a s m a l l f r a c t i o n o f t h e m e m b r a n e ; y o u k n o w , w i t h o n e o r t w o s p o t s a r o u n ( I . ])o y o u hnv,~ a n y i n f o r m a l i o n o n h o w m a n y - S } [ g r o u p s i n t e r a c t w i t h t h e H e i n z b o d y in p r o d u e i n g t h i s eff(.ct.',' I t w o u h l seem t hat, it m u s t be a r a t h e r s m a l l n u m b e r a n d t h a t w o u l d be v e r y i n t e r e s t ins. 1)I: J.xc,.~l:. 1 d o n ' t h a v e a n a n s w e r in t e r m s o f n u m b e r s . I w o u l d like to s u g g e s t to y o u , t h o u g h , t h a t o n e o f t h c thin~zs we sug~zested in t h e p a p e r we a r e a b o u t to p u b l i s h is t h a t w h a t o n e is seeing i n t h e e l e c t r o n m i c r o g r a p h s (Jf R i f k i n a n d l ) a n o n is t h e e n d st~lge d e n a t u r a t i o n t h a t ' s t h e p r e c i p i t a t e d h e m o g l o b i n , w h i c h is (,bvi()us. I t ' s cluite c l e a r to u s t h a t t h e s o l u b l e i n t e r m e d i a t e c o m p o u n d , or t h e a n a l o g i n t h e case o f t h e ~ e n e t i c a l l y u n s t a b l e h e m o g l o b i n s h a v i n g fl c h a i n a b n o r m a l i t i e s , is still s o l u b l e , a n d h a s c t m r m o u ~ a f f i n i t y fc.r b o t h g l u t a t h i o n e a n d for m e m b r a n e s . ~Vc c o u l d n ' t see t h i s , but, p r e s u m a b l y t h i s s o l u b l e m a t e r i a l is b i n d i n g -¢~}{ g r o u p s o f t h e cell m e m b r a n e s also. I ' m n o t s u r e we c a n d e m o n s t r a t e t h i s m o r p h o l o g i c a l l y an(l [ c e r t a i n l y d o n ' t h a v e a n y c h e m i c a l s t u d i e s to s h o w h o w m a n y - S I { g r o u p s a r e i n v o l v e d in this. [ a m t r y i n g to s ~ i m u l a t e s o m e o f t h e lens p e o p l e . H a s t h e m e m b r a n e , w h a t e v e r t h a t is, o f t h e l e n s been s h o w n to ]utve a l t e r e d p 2 r m e a b i l i t y t o c a t i o n s w h e n aflticted w i t h c a t a r a c t ? T h e a n s w e r to this, to me, m a y be t h e a n a l o g t h a t 1)it Bb:U~'t,F.I~ is l o o k i n g f o r - - o f p e r h a p s d 0 n a t u r i n g p r o t e i l l or a l t e r e d p r o t e i n a t t a c h i n g to t h e m e m b r a n e a n d , i n so d o i n g , altering its c h a r a c t e r i s t i c s . I s t h e r e a n y i n f o r m a t i o n a b o u t it? D a Kx.~osI[ITa. I n galacLose c a t a r a c t s we find t h a t o n e o f t h e e a r l i e s t c h a n g e s is o s m o t i c s w e l l h l g t h a t s e e m s t o l e a d t o a n i n c r e a s e in c a t i o n flux as m e , ~ u r e d b y p o t a s s i u m e x c h a n g e . I s u s p e c t t h a t m a n y o f t h e s e e x p e r i ' n c n t a l c a t a r a c t s i n v o l v i n g c h a n g e s in t h e c o r t e x d o first i n v o l v e c h a n g e s i n p e r m e a b i l i t y to o a t i o n s . D r I w a t a from O s a k a U n i v e r s i t y h a s b r o u g h t t o t h i s c o u n t r y a s t r a i n o f ' m i c e t h a t d e v e l o p h e r e d i t a r y c a t a r a c t s . O n e o f t h e e a r l i e s t c h a n g e s we see in t h e s e h e r e d i t a r y c a t a r a c t s is a m a r k e d c h a n g e in e l e c t r o l y t e m e t a b o l i s m . DR Elcxsa" 11. J ~ F F ~ . Does a n y o n e else h a v e a n 5, c o m m e n t s ? I w o u l d like to a s k :DR JACOB a q u e s t i o n a b o u t t h e 5-t0-2S0 r a t i o that~ he h a s u s e d to d e m o n s t r a t e t h e h o m e - d e p l e t e d g l o b i n c h a n g e . I s t h e r e an5" p o s s i b i l i t y [ h a t p a r t o f this is a c t u a l l y m e t h e n m g l o b i n f o r m a t i o n b e c a u s e t h e 5 4 0 - 2 S 0 r a t i o o f m e t b e m o g l o b i n w o u l d be lower t h a n t h a t of o x y h e m o g l o b i n ? D~t JACO;~. ~ ' o w t k a t ' s v e r y p e r c e p t i v e . [ s u s p e c t t h a t h e t h o u g h t t h i s o n e t h r o u g h b e f o r e t h e s e e x p e r i m e n t s . T w o t h i n g s were d o n e to c o n t r o l t h a t : (1) i n e u b a t i o l ~ s were s h o r t - l i v e d ; a n d (2) t h e a m o u n t o f m e t h c m o g l o b i n was a b o u t 10~,. Thus, one could m a k e calculations t h a t would reduce the ratio b y a b o u t 0.02, w h i c h is i n s i g n i f i c a n t as c o m p a r e d w i t h t h e r a t h e r m a r k e d d r o p s t h a t were o c c u r r i n g . C y a n i d e w a s a d d e d ns well, w h i c h h e l p s t h e s i t u a t i o n d r a m a t i c a l l y i n t e r m s o f t h e 5 4 0 - 2 8 0 r a t i o . T h e s e w e r e p o i n t s o f c r i t i c i s m leveled a t D a ~,~'INTERtL~,LTER'Sa n d m y p a p e r a n d were a n s w e r e d b y D[¢ WI'~,'TERtI,~.LTER q u i t e well in a n u p c o m i n g a r t i c l e . DR SRrVASTAV,~. D i d y o u e v e r t r y to b l o c k t.he --StI g r o u p s b y A r - e t h y l m a l e i m i d o m a i n l y a t t h e 93 p o s i t i o n o f f l - e h a i n s a n d t h e n s u b j e c t i n g t h e m t o o x i d a t i v e s t r e s s for H e i n z b o d y f o r m a t i o n ?
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DR J . ~ c o s . I n o u r first s t u d i e s wc f o u n d that. N E b l - t r c a t e d r e d eell.~ i n t h e s e p a t i e n t s c o m p l e t e l y b l o c k e d a n y u n b o u n d g l u t a t h i o n o ; t h e s e cells w e r e m o r e susct, p t i b l c t o h e a t p r e c i p i t a t i o n . T h a t in, t h e h e m o l y ~ t e s d e r i v e d t h e r e f r o m , so t h a t t h i s is a n a d d i t i v e effect. I n a d d i t i o n . H g b - A t h a t h a s it,s fl-93 s u l f h y d r T I s i n h i b i t e d also b e c o m e s a h e a t - p r e c i l n t a b l e h e t a o g l o b i n . I t m i m i c s m a r k c ~ l l y t he u n s t a b l e h e m o g l o h i t a . ])r~ SRtVAST.~V.~. 1 h a v e a n o t h e r q u e s t i o n . ~X'_~ultI y o u e l a b o r a t e f u r t h e r o n h o w t i m m i x e d d i s u l f i d e o f ]-lgb--S-S-C, a t t a c h e s t o t h e m e m b r a n e . T h e s e q u e n c e t h a t y o u h a v e p o i n t e d o u t is that. llgb--~S-.'q- (; r e a c t s w i t h t h e m e m b r a n e s u l f h y d r y l . N o w in t h a t ease. otto w o u l d expect, t h a t t h e G S I I w o u l d be relea,~cd f r o m t h e h e m o g l o b i n . D i d y o u e v e r t r y t o see o r h a v e y o u e v e r m e a s u r e d t h e a m o u n t o f (~.%_'1[ t h a t m released during the attachment of the Heinz bodies to the membrane? :DR J.~COB. T h a t w o u l d be a n i c e e x l ~ c r i m e n t . W e h a v e n ' t d o n e t h a t . I t h i n k w h a t y o u a r e s u g g e s t i n ~ is m a y b e t o m i x d i s u l f i d e w i t h r a d i o a c t i v e g l u t a t h i o n e , w h i c h c a n b e d o n e , a n d t h e n use t h a t m a t e r i a l w i t h g h o s t t o see if y o u get. a r e l e a s e o f r a d i o a c t i v i t y . 1 t h i n k t h a t w o u l d be ver b, p r e t t y . ~Ve h a v e n ' t b<.eu s m a r t e n o u g h t o h a v e t h o u g h t o f t h a t a h e a d o f t h i s m e e t i n g . I also w o u l d s u g g e s t t o y o u . t h o u g h , t h a t i t p r o b a b l y is n o t j u s t fl-93 s u l f h y d r y l s t h a t a r e involvc~l in t h e b i n d i n g o f t h e red cell gho~t~. 1 t h i n k t h e f u r t h e r s t e p h e r e is, as J a n d l a n d A l l e n h a v e showt~, t h a t t h e c h e m i c a l d a m a g e is t h e u n f o l d i n g . N o w o n e is a b l e t o t i t r a t e m a n y m o r e - S H g r o u p s t h a n t h e o r i g i n a l l y free o n e s o f fl-93. "l'h~tt is. ot~e ii~ at a n d a n o t h e r i n t h e fl c h a i n . The-refers, f o u r m o r e b e c o m e a v a i l a b l e as t h i s s p h e r i c a l m o l e c u l e s t a r t s t o o p e n u p i n i t s p r o - p r e c i p i t a t i o n . So t h o s e - S H g r o u p s are. I s u s t ~ c t , a l s o n o w a v a i l a b l e for m e m b r a n e b i n d i n g . I d o n ' t h a v e a n y real e v i d e n c e of t h a t . Dl~ SRIVAST.~VA. O n e l a s t q u e s t i o n , D ~ J A ¢ o I u :Did y o u e v e r t r y t o i n c u b a t e c e r t a i n u n s t a b l e h e m o g l o b i n s w i t h T P N H a n d g l u t a t h m n e r t ~ l u c t a s e a n d t r y zo s p l i t off (~SII t o s h o w t h a t t h e t r t p e p t i d e is b o u n d to t h e h e m o g l o b i n ? W h a t is t h e e v i d e n c e t h a ~ s u c h h e m o g l o b i n is in c o m b i n a t i o n w i t h G S S ( I o r GSH ? D n J A c o B . V~re h s r v e n ' t d o n e w h a t y o u ' v e aske~l. T h e e v i d e n c e h a s boa's t h a t o n e c a n s p h t g l u t ~ , t h i o n e f r o m t h e s e m a t e r i a l s w i t h m e r c s p t o c t h a n o l . 1 t h i n k t h a t ' s t h e m a j o r e v i d e n c e . T h e r e is e v i d e n c e t h a t i t is t h e f l - c h a i n t h a t b i n d s t h e r a d i o a c t i v e g l u t a t h i o n o . O n e c a n r~,split t h a t a n d c h r o m a t o g r a p h tt, a n t i f i n d t h i s o n t h e f l - c h a i n . T h i s is r e a l l y j u s t a p r e s u m p t i o n t h a t i t ' s t h e fl-93, b u t t h a t ' s t h e o n l y o n e t h a t is f r e e l y t i t r a t a b l e . W e k n o w i t ' s f l - c h a i n a n d w e a r e p r e s u m i n g t h a t i t ' s o n t h e 93. W e h a v e n o c v i d t . n c e of that at the moment. :DR EFSTEI.~'. I t h i n k o n e o f t h e purpos~_~ o f t h i s m e e t i n g is t o h e l p u s c x a n i i n e o u r o w n b i a s e s a n d prec o n c e i v e d n o t i o n s i n l i g h t o f e v i d e n c e i n o t h e r fields, b l y o w n b i a s , a s y o u h e a r d y e s t e r d a y , is t h a t e a r l y c a t a r a c t f o r m a t i o n is p r i m a r i l y a m e m b r a n e p h e n o m e n o n , a n d t h a t c h a n g e s i n t h e l e n s p r o t e i n c r y s t a d l i n a r e s e c o n d a r y t o c h a n g e s i n m e m b r a n e . 1 t b i n l t t h a t t h i s o c c u r s i n d i a b e t i c c a t a r a c t s , w h i c h I)R CttrLAC~ will d i s c u s s . I t is b e l i e v e d t h a t c h a n g e s i n t h e m e m b r a n e a r e s e c o n d a r y t o s w e l l i n g o f t h e l e n s cells c a u s e d b y t h e a c c u m u l a t i o n o f p o l y o l . H o w e v e r , it, w o u l d be v e r y i n t e r e s t i n g t o a p p r o a c h t h i s f r o m tJ~c o t h e r viewTpoint, n a m e l y , t h a t t h e r e is s o m e p r i m a r y d e f e c t i n t h e l e n s c r y s t a l l i n s a n d t h a t t h i s d e f e c t affects t h e m e m b r a n e s . I t c o u l d b e s a i d t h a t i t is o f a d v a n t a g e t o t h e w h o l e o r g a n i s m t h a t r e d ceils a r e u n a b l e t o c a r .ry o x y g e n i f t h e r e is d a m a g e t o h e m o g l o b i n . ' £ h i s is a m e c h a n i s m w h e r e b y t h e effect oat h e m o g l o b i n can relate to the membrane and by changing membrane -Sit groups and affecting membrane functions, t h e b o d y c a n r i d i t s e l f o f d e f e c t i v e r e d cells. S i m i l a r l y , t h e r e m a y b e d a m a g e t o t h e p r o t e i n in t h e l e n s cells w i t h a s e c o n d a r y e f f e c t o n t h e m e m b r a n e s b e c a u s e t h e l e n s c a n n o t rid i t s e l f o f t h e s e cells. As y o u k n o w , n a t u r e u s u a l l y d o e s n ' t c o n f o r m t o o u r b i a s e s , a n d i t w o u l d b e v e r y i n t e r e s t i n g t o t r y to i n v e s t i g a t e s o m e o f :DR JACOB'S ~,~,eories i n t h e l e n s . :DR ~ L ~ . I h o p e l ' m n o t g o i n g t o l e t t h e l e n i n v e s t i g a t o r s d o w n b y a g r e e i n g w i t h Dlt J a c o b , b u t w e h a v e n ' t g o t h a l f as f a r w i t h t h e s t u d y o f t h e c h e m i s t r y a n d c h a n g e s o f t h e e r y s t ~ l l i n s a s h e h a s , a n d o t h e r p e o p l e h a v e w i t h h e m o g l o b i n . I n o u r s t u d i e s o f c h a n g e s i n p r o t e i n s i n c a ~ r a c t , f o r m a t i o n s , in c a t a r a c t f o r m a t i o n in h u m a n beings, w e ' v e used u r e a i n s o l u b i l i t y as a sign of - ~ - S - f o r m a t i o n , a n d I w o n d e r w h e t h e r t h i s h a s b e e n u s e d a t all b y a n y b o d y i n t h e s t u d y o f h e m o g l o b i n s . A l s o , w e h a v e f o u n d t h a t b y a n a e r o b i c e x t r a c t i o n o f c e r t a i n c a t a r a c t s t h a t w o u l d , i f e x t r a c t e d a e r o b i c a l l y , g i v e us a l o t o f u r e a i n s o l u b l e p r o t e i n h e l d i n s o l u b l e b y - S - S - forms. I f we e x t r a c t a n a e r o b i c a l l y , t h e n we do n o t got this - S - S - b o n d f o r m a t i o n . T h e t h e o r y is, t h a t t h e f i r s t c h a n g e i n t h e d i a b e t i c h u m a n c a t a r a c t , w h i c h is a p r e t t y s e v e r e n u c l e a r c a t a r a c t , is a k i n d o f u n f o l d i n g o f t h e p r o t e i n a l l o w i n g - S H f o r m s t o b e a v a i l a b l e f o r a e r o b i c o x i d a t i o n w h e n t h e w h o l e s t r u c t u r e is g r o u n d u p a n d e x t r a c t e d . T h e q u e s t i o n I a s k is h a v e you studied the hemoglobins and the urea solubility or insolubility under certain and different circum. stances? :DR JACOB. I ' m s u r e t h a t ' s b e e n d o n e . I c a n ' t r e m e m b e r . I l o o k e d o v e r a t E r n i e b e c a u s e i t s e e m s t o m e t h a t h e r e p o r t e d s o m e t h i n g l i k e t h a t i n o n e o f h i s e a r l y p a p e r s . I k n o w J i m J a n d l a n d :Dave A l l e n h a v e
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look~l a t urea* solubility of p h e n y l h y d r ~ z i n c . t r e a t c d hemoglobins, or m a y b e someone knows w h a t the results arc. 1)a CJl ~'t.,U'K. 1 ] usu have a couple of c o m m e n t s on Da EI'~TEI2~'S remarks. We studied the expcriment~tl sugar c~taract, the diabetic cataract, and wc found t h a t the accumulation of sorbitol causes the lens liter to swell and Clltion flux to increase. This a p p c a r s to be an increased cation exchange prior to an absolute clmngc in the level of cations. A b o u t 3 ¢htys after the flux incrcascs, opacification develops, and then there is a sudden change in the absolute level of cations. This was interpreted to be an indication t h a t as m e m b r a n e i~,rmeability increa.~,d, p u m p a c t i v i t y incrcased and compensated for this, b u t then its the p u m p a~.'tion was exceeded, there was a sort of influx of sodium and a m a r k e d change in the absolute level of eatmns. Now this is an e.
I n t r o d u c t i o n to D i s c u s s i o n o f H e i n z B o d y A n e m i a s H cmoglobinopathies VIP.GII, F.
Unstable
FAIRBANKS
,q,'cHo~: ¢f Clinical Pathology, 3layo Clinic, I?,~clmster, 3Iimwsota, 56901, U.S.A. l)urin,.: the past IS years, tmmerous eases of "congenital Heinz body anemia" have been I'Ol)~Wl('~I. It is now eh.ar thaL tmstablt:, therumlabile hemoglobins are presenL in most~ or all such ( ,~...,-.~. I.;nstnlflt: ht'nto~lol)ins Ztirieh. tI. i'~oln. I lasharort and L Ferrara, have been rel~)rt,.d ~n more than otu: family. Most. of th(" unsta})lo hemoglobins m a y be a t t r i b u t e d Ix) point Inut:).tions in the L'~ell¢'ti(."code. suc]| as a change from guanine to ndenino. NEarly 30 such :tn.'~tal)lo h(,mo,_,lol)inOlmthtes haw., been characterized f.rolll tile standpoint, of LhE nmlet'u!.~r pathology--that, is. the chan~e in structure of the globilt chains of hemoglobin. 'l'ho Ina)in:.r by which tht.so, moh, t.uhtr changt-s cause instability of the hemoglobin molecule st,.ins to b~: wtriablo, but very liktdy t tie effect ill most. is to loosen t,ho bond between heine nl)(I .L,Iol)m. fi)llowe¢t t,y fi)rlnnt ion of mixed tlisulti(lez~ anti denaturation and i)reeipitalbion of glottal. F.rylhro('ytos that contain unstable hemoglobhm may exhibiL ~Lceelera.ted glucose ('ol)sumpth-~. low ,~,ltltnthioltl: vo~wetltration, decreased glutathione sbability, and low (:-o)~('o)ltra t I()llS Of a(lmmsilm t ril)hOs |)hate. The rat e of methemoglobin formation anti of in v i t r o llll|O}lOlllO]X'~i.'~, Ill.fly })e lilt'teased, Oxygen dissociation is abnormal ill l!lolnE o f these cli~orxleru. Tile inheritance of th(.se disortiers is co~ntnoilly autosomal dominant;. HEmolysis m a y bo mild to sevcr~; it may occur only after sult'onamide ingestion or m a y no(; be demonstrable at all. Npldnectomy has been beneficial in a few cases of severe hemolytio anemia due to Illtstabiu hemoglobinopathy. I n 1890, t l c i n z describe(1 s m a l l , s p h e r i c a l , r e f l ' a c t i l e b o d i e s in t h e e r y t b r o c y t e s o f e x p e r i m e n t a l a n i m a l s e x p o s e d t o p h e n y l h y d r a z i n e a n d its d e r i v a t i v e s . ~k l a r g e n u m b e r o f c h e m i c a l s a r e n o w k n o w n t o be c a p a b l e o f i n d u c i n g s u c h I-teinz b o d y f o n . ~ a t i o n i n h u m a n e r v d t r o c v t e s . T h e s e i n c l u d e botll m e d i c i n a l s , s u c h a s - s u l f o n a m l d e s , a n d s u b s t a n c e s e n c o u n t e r e d hi a c c i d e n t a l o r occ:upationa.1 p o i s o n i n g , s u c h as n a p h t h a l e n e . S o m e c h a r a c t e r i s t i c s o f t h e H e i n z b o d y a r e t h a t it is s e e n in s a f i n e s u s p e n s i o n s o f c r y t h r o c y t e s , t h a t it s t a i n s w i t h a n u m b e r o f s u p r a v i t a l s t a i n s suci~ as c r y s t a l v i o l e t , a n d that. it. is n o t s e e n in W r i g h t - s t a i n e d b l o o d fibns. T h i s is p r o b a b l y d u e t o t h e s o l u b i l i t y o f ~he t-[einz b o d y in a l c o h o l f i x a t i v e s ( F e r t m a n a n d F e r t m a n , 1955). T h e H e h ~ z b o d y is b e l i e x e d to be c o m p o s e d p r i m a r i l y o f p r e c i p i t a t e d g l o b i n , b u t i t a l s o c o n t a i n s s m a l l q u a n t i t i e s o f p o r p h y r i n s , f e r r i t i n , a n d n u c l e i c acids. I n ] 952, C a t h i e o f L o n d o n , I g n g l a n d , r e p o r t . e d t h e e a s e oi" a y o u n g b o y w i t h c o n g e n i t a l h e m o l y t i c a n e m i a who, a f t e r s p l e n e c t o m y , m a n i f e s t e d l a r g e n u m b e r s o f i n t r a e r y t h r o c y t i c i n c l u s i o n s t l m t w e r e t i n c t o r i a l l y s i m i l a r t o t~eh~z b o d i e s . [ T h i s c a s e w a s s u b s e q u e n t l y r e s t u d i e d , a n d t h e p r e s e n c e o f an u n s t t t b l e h e m o g l o b i n ( h e m o g l o b i n B r i s t o l , a2fl.2'~Tasp) was c o n f i r m e d ( S t e a d m a n , ~:'ates a n d t-~uehns, 1970).] T h e r e f o l l o w e d t e l ) e r r s f , ' o m s e v e r a l c o u n t r i e s o f " c o n g e n i t a l [{einz b o d y " a n e m i a s ( A n d r 6 , D r e y f u s a n d L e B o l l o c h - C o m b r i s s o n , 1964; B a c h m ~ m n a n d M a r t i , 1962; B e r e t t a , P r a t e , G a l l o \ a n d L e h m a n n , 1968; C a r r e l l , L e h m a n n , Lorki,1, l ~ a i k a n d .l-Itmter, 1967; D a e i e eL al., ] 96,t.; ] ) a c i e , S h i n t o n , G a f f n e y , C a r r e l l a n d L e h m a n n , 1967 ; .De Vries, J o s h u a , L e h m a n n , H i l l a n d F e l l o w s , 1963; F a i r b a n k s , O p f e l l a n d BurgerI;, 1969; F r i c k , I-Iitzig a n d Sl~auffer, 1961; G o u d e m a n d , B i s e r t e , H a b a y a n d Voisin, 1964; G r i m e s , M e i s l e r a n d D a c i e , i 9 6 4 ; L e l o n g e t el., 1961; M o z z i c o n a c c i , A t t a l , : P h a m , 5 : [ a l a s s e n e t a n d 365
~6
Y. F. FA1/~13AN/(S Txnt,g I
Un,~table hem(×jlobins l~siduo no.
SubstAtutio~
Refcrenct-
Clinical effect
Design a t i o n
,~-Chain v a r i a ~ t ~ 43* ,t7
phe~'-vn! n.~p-~ g l y
Torino I3eilinson (L F c c r a r a )
Sul fonnmidc-hemo]5's~a Hemolysis
V r i e s el al. ( 1 9 6 3 ) ; ~'N'a~cl o t a l . i'lgO.~)
47 47
; t a p - - his ~ p - - ~ his
l-I a ~ h a r o n Sinai. ~ e a l y
~'one
80
lcu---~ ar~
A~;n A r b o r
~ot~e
Ite, l h r c c h ; vt aL (1967) Chn.r~ehe a n d , M o n d ~ e (1967): Ch-',rachc t:t al. {1969J Osteri.ag a m | Smith( t 96S),,~rhncider e t M. { tgf, s ~, I{uckrmgc, l (l~-r.c,mal
84 112 136 ~
.~e.r -~-a r g his--gtu Ieu---~.pro
Etobicokc~ Dakar Bibba
~N*one ltemolymn }temolysi.~
Cr~oksi~m ct at. (1911c,l) l¢o~a eL ;~1. [ 19118) K l e i h a u e r et hi. (t96~;1
Hemolysis
59 heine 88
B e r e t t n eL al. I t968) B i a ~ c o et al. (lfl6:|): l.)c
~
l r l I I l I i | t i (',l~t t4 iI 1 }
/3.Chain v a r i a n t s 6 14 23
gtu d e l e t e d ]eu--*arg va| deleted
24 2,8
gty--~ a rg leu--~pro
givemale-Bronx ('.~ ~2rl O g a,
~N'ot k n o ~ ~ Nm~c Hemolysis and mcth. emogh~binemi~ tt emo~yain Hernolyaia
30
arg-~ser
Tacoma
None
35 4 .°*
tyr--~pho pbe->~er
Philly Hammeramitb
Hemolyaia ]:I emolysi~
63*
his--~arg
Ziiricb
Su I f o n a m i d e . h e m o t y a i s
67*
val--~ a.~p
Bristol
H-emolysi~
6"1"
val--> al~
Sidney
:Hemoly~is
7,t 76
gly---~azp ala-~glu
Shepherd's Bush Seattle
88*
] eu---~pro
Santa Ana
tKomoJysia
88" 91"
leu~r, rg leu--->pro
Borha Sabine
Not}_e }:[emoJysis
93--98*
deletion
G u n ]:till
Hemolysia
Leiden Sogn Freiburg
Homol)"~i~ "l!temolysia
heine
I)c J o u g e~ al. (196~) MoJm eb al. (19~8) J o n e ~ et al. ( I 9 6 6 ) lCan2~cy et at. (1965) Sanzo~m e t al. (1967}; S a n ~ o n e a n d P i k (1965) ]3aur and Motulsky (1965); B r l m h a l l e t al. (t969) R i e d ~ r e t a } . (} 9l~t)) D a c i v et M. ( t 9 6 4 , 1967); Grlmc~ e t al, (1964) Bachmatm and Marti (1962); ,Friok o~ al. (1961); IXie~!er e t at. (1965); R i g a s el, at. (1956) C a t h i e (1952); ,Sta~,aclman e t ~.i. (1970) C~rr~ll et al. (1967) ; ~ a i k et. a~. (1967} Whitx~ e t al. ( I 9 7 0 ) t l u e h n a (1965); H u e h n s e t al. (1970); Mot, u l s k y
(196~) l~'ttirba~l_k~ e£ al. ( 1 0 6 9 ) ; Opfell e t al. (1968) H o l l a n d e r e t al, (1969) MjII~ eg al. (1968); S c h n e i d e r o t a l . (1969) 13radIoy a n d Risxtec (1965); : B r a d l e y et al. (1967); L o r k l a e t al. (1970)~ l'~iedar a n d B r ~ l e y (1968)
HEINZ
367
1301)Y A N E M I A S "I'An L r I ( r o n t i n a e d )
9S*
124 130 14:1
val-~n)et
]
Hemolysis
pro-~arg t vr~asp h i s - ~ a r:~
Khart<,um ~Vien Ohnsted
Not, k n o w n Hemolysis Hemolysis
C a r r e l ] e t a l . (1966, 1967); ] I u t , chi~on e t a l . (19{~i) : J a c k s o n e t al. ( 1 9 6 q ) ; J o n e s ct, a l . ( 1 9 6 7 ) ; _Pribilla e t al. (1965) C l c g g e t al. (1969) : P i e t s e h m a n n e t el. (1969) F a i r b a n k s e t el. ( 1 9 6 9 ) ; L o r k i n e t el. (1970)
O t h e r vaxiant.s flj
1!
Ifemolysis
J o n e s e t e l . (1959) ; FCigas e~ el. (1956)
* Amino acid poMtion~ immc
Bc.~.qis, 1961 : Muller and Kingma, 1961 ; R a i k , H u n t e r and Lindsay, 1967; :Rieder, Z i n k h a m and t [ o l t z m a n , 1965: Rigas, Koler and Osgood, 1956; Schmid, Brecher and Clemens. 1959 ; Scott, t l a u t . C a r t w r i g h t and Wintrobe, 1960 ; Sheehy, 1964 ; Shibata, Iuchi, Miyaji, Ueda and Takeda, 1963; S t e a d m a n et el., 1970; White, Brahl, Lorkin, L e h m a n n and Smith, 1970; \Vorms, Bernard, Bessis and Malassenet, 1961). ) l o s t of the,no cases had the following features in c o m m o n : a n e m i a persisting from infancy, the appearance of e r y t h r o c y t i e inclusions after splenectomy, a n d an tmusually d a r k urine c o n t a i n i n g excessive a m o u n t s of poorly characterized, p r o b a b l y pyrrolic, pigments. An autosomal d o m i n a n t i n h e r i t a n c e of the " H e i n z b o d y a n e m i a " was Txm.E
II
lb,latit'e electrophoretiv Tos'~im~s of u.~stable hemoglobins* Standard
Unstable l[ Seattle S h e p h e r d ' s ]3ush Dakar
Tacoma IIammcrsmith, Genova~, ~ristol, Philly, Wien, Torino, Olmmted
A
S
A2
{
BorAs Santa Ana, Sidney K61n Freiburg, Z ( i r i c h
Etobicoke, Sogn, Leiden, Bibba, Hasharon (Sinai), Riverdale-Bronx, .Ann A r b o r Sabine Gun Hill
Khartoum,
L Fcrrara
C F r c e a chains:~ Origin * A t p i t 8.6 on s t a r c h g e l ; r e l a t i v e p o s i t i o n s a r e a p p r o x i m a t i o n s b a s e d o n p u b l i s h e d d a t a . Htts also been reported to have mobility similar to that of hemoglobin S (Sansone, Carrell and : L e h m a n n , 1967). :]: A f a i n t b a n d m a y b e s e e n in t h i s p o s i t i o n in m a n y o f t h e u n s t a b l e h e m o g l o b i n s in w h i c h t h e f l . c h a i n is a n o m a l o u s ; this b a n d is d u o t o f r e e ,,-chains.
36S
V.F.
FAIRBANKS
clearly discerned in m a n y families, t-Iemoglobin elcctrophoresis showed in m o s t cases a broad trail e x t m u l i n g c a t h o d a l l v from the h e m o g l o b i n ..k I han, l. ; \ u t n h e m o [ ) ' s i s was t y p e I in m o s t cases studied. Frick et ft. (196I) a p p e a r to have linen the first to r e p o r t t,hcrn~ohtbility of h e m o g l o b i n (hemoglobin Ziirich) in this ~roup of tlisc"~rders. Daoie et al. (1964) showed that, t h e i r cases of H e i n z b o d y a n e m i a also had a,n easily demons t r a t e d themnolabile h e m o g l o b i n later described as hemoglolfin .t[:mlmorsmitl,. neff, a~ser (Dacie et al.. 1967). S u b s e q u e n t l y . t h e r m o l a l f i l i t y of hemoglo~,tn has been demonst.rated in n e a r l y all cases of I t e i n z b o d y a n e m i a , a n d it is for this reason t l m t we now regard these disorders as being due I,rimarily to the presence of an u n s t a h l e hemo~,lobin A t least. '> _5" u n s t a b l e h e m o g l o b i n s h a v e been identified a n d characterized it,, considerable d e t a i l w i t h respect to their clinical effects, e l e c t r o p h o r e t i e p'ttterns, a n d molecular a b n o r m a l i t i e s (Tables I a n d 1I). Of t.hese, h e m o g l o b i n s K61n, H a n d H a s h a r o n seem to h a v e been recognized in a g r e a t e r numl)er of cases t h a n a u v of the o t h e r s . . . 8 o m e of t h e u n s t a b l e hemoglobix~s h',ve been ,'cported i n d e p e n d o n l l y t,y d_ifferent g-roups of i n v e s t i g a t o r s , t h e r e b y i n t r o d u c i n g more t h a n one d e s i g n a t i o n for t h e s a m e m o l e c u l a r v a r i a n t . '['hus. h e m o g l o b i n s Beilinson a n d [~ F e r r a r a a p p e a r to ~,~be t h e same v a r i a n t . ~imflarls', t h e hmnoglobin v a r i a n t ¢~.a4;~'lsfl~ has been d e m o n s t r a t e d in p a t i e n t s in Israel, Texas. M a r y l a n d , and Michigan a n d has been called h e m o g l o b i n H a s h a r o n , Seal)'. Sinai. and :Michigan. A t least six fi~nilies h a v e been identified in which t h i s h e m o g l o b i n o p a t h y has occurred, all of A s h k e n a z i e J e w i s h a n c e s t r y b u t o t h e r w i s e n o t k n o w n to be related. I n the Israeli and r e x a s cases the p a t i e n t s were not anemic, a n d h e m o g l o b i n ]-Iasharon (Scaly) is believed not to be t h e r m o l a b i l e ( L e h m a n n , personal c o m m u n i c a t i o n ) . H o w e v e r , in the ot.her t~wo ins t a n c e s ( h e m o g l o b i n "'Sinai" a n d . " M i c h i g a n " ) t,he propositi had Jnild h e m o l y t i c a n e m i a s a n d t h e r m o l a b i l e hemog!obins. I n fact., the criteria for d e s i g n a t i o n of a h e m o g l o b i n as b e i n g unsta ble seem nol~ to h a v e been u n i f o r m l y applied, F o r e,cample, hemoglobin ]';tobieoke was not t h e r m o labile in fresh h e m o l y s a t e s but, was so a f t e r prolonged storage in t h e frozen, state. H e m o g l o b i n H o p k i n s - I I , w h e n purified, is t h e r m o l a b i l e b u t occurs in such low conc e n t r a t i o n in h e m o l y s a t e s t h a t turbidit, v is n o t observed on hea~;ing whole h e m o l y s a t e s (Charache, personal c o m m u n i c a t i o n ) . ]-liemoglobin K a n s a s h a s been described rep e a t e d l y as b e i n g u n s t a b l e , a l t h o u g h t h e basis for t h i s is n o t elea~ from p u b l i s h e d d a t a . H e m o g l o b i n Iro,ao~ e (~8~_ ~"-~Lu) (Rosa et, el., 1969) also ba-s been r e g a r d e d b y some as b e i n g u n s t a b l e , a l t h o u g h p u b l i s h e d d a t a do no~, p r o v i d e e v i d e n c e t h a t this is so. A few other h e m o g l o b i n s h a v e been r e p o r t e d as being u n s t a b l e , b u t t h e s t r u c t u r e of t h e s e is n o t k n o w n as of t h i s w r i t i n g ( S a g h i a p a l a n a n d l~obinson, .t968; S c h w a r t z , Schreiber, G o l d m a n a n d lVagelman, 1968). To date, m o s t of t h e u n s t a b l e h e m o g l o b i n o p a t h i e s h a v e been r e p o r t e d o n l y in single families; some u n d o u b t e d l y r e p r e s e n t n e w m u t a t i o n s because t h e y h a v e not~ been d e t e c t e d in o t h e r f a m i l y m e m b e r s . T h e i n h e r i t a n c e a p p e a r s to h a v e been autos o m a / d o m i n a n t in those families in w h i c h several m e m b e r s were affected. T h e st,r u c t u r a l c h a n g e s in t h e h e m o g l o b i n molecule for t h e m o s t p a r t m a y be e x p l a i n e d on t h e basis of c h a n g e s of single nueleotides in t h e . g e n e t i c code, as f r o m g u a n i n e to a d e n i n e . T w o disorders ( F r e i b u r g a n d L e i d e n ) are explicable as d e l e t i o n s of single c o d o n s (nueleotide t r i p l e t s in t h e g e n e t i c code). I:[emoglobin G u n Hill ~nust represent, d e l e t i o n of a 15-nucIeotide seq~mnce, l:[emog[obin [£ is believed to be d u e to demreased ¢,.-ehain s y n t h e s i s as a r e s u l t of d o u b l e h e t e r o z y g o s i t y for t h r e e a.-thalassemia genes ( L e h m a n n , 1970). -
HEINZ
369
I3OI)Y ANEMIAS
Several of ~he unst~able hemoglobins have substitutions of polar (hydrophilic) for non-polar (hydrophobic) amino acids in the pocket of the globin chain t h a t contains the heine componen~ (Figs 1 and 2). The presence of hydroptfilie radicals in this "greas}, cleft" facilitates t,he entry of a water molecule, and tlfis in tm'n leads to methemogiobin for.ination and ultimately to hemoglobin denaturation. This t y p e of alteration occurs in hemoglobins ~,Vien, Barbs, Sogn, Ann ~krbor and Shepherd's Bush. These and at,her substitutions also m a y cause distortion of spatial re]ationships t h a t nomrm,lly, exist between globin and heine, thereby weakening the Van der Weals forces and causing dissociation of heine from its globin chain. In hemoglobin Khba, for example; there is substitution of methionine for the slightly smaller reline in a posit, ion.., contig,~ous to he,mi.~l?l~is,,,, slight, change in size of amino acids in a critical area. results in r~n increased tend~'.nc) of heine and globin to dissociate and denature (Jacob, Brain and Daeie, 1967; kIae6b, Brain, Daeie, Carrel/and Lehmann, 1968;
156 B
~Torino
43
'~L. Ferrara 47 Hashoron Sinai
80AnnA r b o r ~ ~
F i e . I. Sites o f c h a n g e s in c¢ chain responsible for u n s t a b l e h e m o g l o b i n o p a t h i e s . Of t h e f o u r s u b s t i t u tions indicated, t,wo (residues 4q a n d 47) are in the h y d r o p h o b i a p o c k e t tha~ c o n t a i n s t h e heine m o i e t y , a n d residue 43 is a eriticM heine c o n t a c t . This position is analogolm t o position 4-2 in t h e fl ohain. I { e m o globin L F c r r a r a (Beilinson) a n d h e m o g l o b i n ~ I a s h a r o n (Sinai, Scaly, ~'[iehigan) b o t h invobze s u b s t i t u tion in a m i n o acid residue 47. I n a d d i t i o n to the u n s t a b l e h e m o g l o b i n s indieaeed in t h i s ~Jlust,r a t i o n , t h r e e o t h e r s h a v e been a t t r i b u t e d to subst, i t u t i o n in t h e ~-ehain b u t details of t h e s e . a r e n e f f c o m p l e t e . Gun Hill 9 3 - 9 8
88San,eBores Ana.//L 14 Sogn/
~~Freiburg23 \ ~'.~
Geneva 2 8
" Zurich 63
""-~S heph~ir~;e; 2sTh74 Seattle 76 :Fro. 2. Sites of c h a n g e s in/3 c h a i n leading to h e m o g l o b i n insCability. Bfos~ o f t.hese cluster abou¢ elefl~ wherein heine m o i e t y is lodged, a n d some are a t eri~ieM heine eontao~ p o i n t s such as a m i n o acid p o s i t i o n s 98, 42 trod 63. T h e c h a n g e in h e m o g l o b i n O l m s t e d in the /~ c h a i n i s a n a l o g o u s to t h a t in h e m o g l o b i n ]31bbo, in ghe ~z chain.
370
V.F.
FA!RBANKS
J a c o b and "Winterhaltcr, 1970). Analogous to this are the changes in henaoglobins Torino, Sidney. H a m m e r s m i t h , Bristol, Gun Hill and Shepherd's Bush. In flint, in hemoglobin Gun }Jill the/9 chains appear to have lost their he,he groups. The introduction of proline (as in hemoglobins Genova, S a n t a :kna, l~ibba, and Sabine) into a n o r m a l l y helical segment of the globin chain prevent~s helix formation, thu.~ m a r k e d l y altering the confi~lration a n d stability of the hemoglolfin molecule. In henaoglobin Ziirich, the histidine residue t h a t normally binds heine to globin isreplaecd by arginine. a change t h a t would be expected to result in a greater tendency of hezne to dissociate from globin. I n sex'eral of the unstable hemoglobins, the substitution al)p(:ars in an e x t e r n a l l y located amino acid a t a site distant from the heine moiety, and it is uncertain in w h a t m a n n e r these alterations m a y lead to instability of the hemo.,_,lobin molecule. I n the unstable hemoglobinopathies, hemolysis ramges from severe {he,noglobins S a n t a Ana. Bristol, Sabine. H a m m e r s m i t h and Olmsted) to none (hemoglobin.s H a s h a r o n , Tacoma, Sogn and Bor'as). Some unstable hemoglobins, such as Gun Hill and Freiburg, are associated with mild hemolysis in spite of the severity of the molecular lesion. Hemoglobin Ziirich is associated with hemolytic (tisemse only after its bearer ingests sulfonamides. Sulfonamide-induced hemolysis is also characteristic of hemoglobin Torino. W h e t h e r drug ingc:stion nmy prec.il)itate hemolysis in other of these disorders is not y e t known. I t seems prudent, however, to advise such patients against indiscriminate d r u g use, and speckficall),, sulfonamides should not be used ~o t r e a t infections in these patients. Some of the unstable hemoglobins have been associated with abnormal oxygen dissociation (K61n, Freiburg, Gun Hill), b u t m a n y have not as yet been studied from this standpoint. Abnormalities of e r y t h r o c y t e metabolism h a v e been reported in m a n y patient,-.' with unstable hemoglobinopathies (Table I I I ) . Low concentratim).s of adenosine t r i p h o s p h a t e (ATP) (Grimes et el., 1964; Mills, Lcvil~ a n d Alperin, 1968; R a i k et el., 1967; Schneider, Ueda, _Mperin, Brimhall and Jones, 1969) and of glutathione have been reported ( F a i r b a n k s et el., 1969; Grimes et al., 1964). Glut~tthionc stability m a y be" decreased (Jones, Grimes, CarrelJ, and L e h m a n n , 1967; R a i k et al., 196"7). There m a y be an enhanced t e n d e n c y toward methemoglobin formation (Berett~ et al., 1968 ; H u t c h i s o n et al.', 1964 ; J o n e s et al., 1967 ; K l e i h a u e r et el., 1968 ; Mozziconacci et el., 1961; Schneider et aI., 1969; S t e a d m a n et al., 1970). Glucose consumption m a y be accelerated, particularly by the hexose m o n o p h o s p h a t e " s h u n t " p a t h w a y (Jacob et al., 1967). The relationship of these metabolic aberrations to abnormalities of hemoglobin structure would seem to merit f u r t h e r investigation. T A B L E III
Metabolic abnormalities reported in some un.stabIe hemoglobinopathies Abnormal
hemoglobin
K61n Santa Ana Sabine Hammersmith
Glutathionc
,JIethemoglobin
ATP
Low to normal Low to normal Normal Low
Righ }tigh High High
Low -Low Low
Glucose consumption --High. High
W h e n there is little or no hemolysis or a n e m i a is mild, there is little reason for concern a b o u t the p a t i e n t ' s well-being, his activities need not be restricted, a n d no
IIEINZ
.BODY ANEMIAS
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t r e a t ~ n e n b is mlvise¢t. ( ) u r e x p e r i e n c e w i t h p a t i e n t s w i t h h e m o g l o b i n K61n d i s e a s e is t h a t t h i s is a r e l a t i v e l y b e n i g n d i s o r d e r . M a n y o t h e r u n s t a b l e h e m o g l o b i n o p a t h i e s a r e s i m i l a r l y b e n i g n . \Ve a d v i s e againsl~ a d m i n i s t r a t i o n of drugs such as sulfonamides, whicI~ M~.ould t)e e x p e c t e d t o a c c e l e r a t e h e m o l y s i s . S o m e p a t i e n t s w i t h m o r e s e v e r e a n e m i a h a v e b e e n b e n e f i t e d m a r k e d l y b y s p l e n e c t o m y . T h i s h a s b e e n t r u e in p a t i e n t s wit.h Itenm,..,Iobin S a n t a A n a o r I t a n d in a f e w w i t h h e m o g l o b i n K 6 1 n d i s e a s e . S p l e n t , ( . t o m v sl~(>uhl I)o s e r i o u M y c o n s i d e r e d in p a t i e n t s w}~o h a v e m o d e r a t e o r s e v e r e am.'~nia. T i m rt, s u h , s o f s p I c a e ( ; t o m y , b o w e v e r , c a n n o t b e p r e d i c t e d w i t h c o n f i d e n c e , a n d s u ~ m p a t i e n t . ~ c o n t i n u e to h a v e s e v e r e a n e m i a . [ t is h o p e d t h a t e l u c i d a t i o n o f t h e :noleculnr and biochemical bases of this group of diseases may lead ultimately to a rationul 'rod .successful thcral)y for such patients. l:{ E F E I ~,E N C E S Andr,'., It., Dr%vltzs. l;. and l.cltolloch-Combrlsson. A. (19ti4). Bull. ;litre. 8oe. '.lied. I-lo19. Pari~ 115, 475. l~achmann, 1". a n d .Marl.i, It. 1{. (1902). 131cod 20, "~"~ Baur, E. ~,V. a u d Motulsk.v. :\. G. (1965). llu~J~t~ule,letilc I, 13~21. l~,ert~t.ta, :\., I'rato. V.. italic. E. anti l.,ehnltum, It. (I.9ti8). N a l u r e (Lo?Mo~)217, 1016. Bronco, I.. Modia,m. (.;., l{ottini, E. a n d Lucci, I.{. (1963). N a l u r e (London) 198, 395. |gradh~y. T. B.. J r . and I{iedcr, I~. F. (1965). Blood 28, 975. i{radh,y, T. It., J r., \VoM. 1¢,. C. a n d l{.ieder, i{. F. (1967). ~cie?rce, :V. Y . 157, 1581. I{rm'thall, 13., J o n e s . l~. "1'.. ltaur, E. \V. and 5Iotulsky, t\. G. (1969). 13ioch~emi.stry 8, 2125. Carrcll. II. \V.. l ~ h m a n n , H. a n d l h l t c h i s o n , it. E. (1966). Nalure. (Londo?~) 210, 915. (.:arrell, 1<. \V., l,¢q~m,um. 11.. l~)rkin. P . . \ . , l{ai'k. E. and l-[ttnter, E. (1967). N a t u r e ( I ~ t o ~ ) 215, 626. Carrell, l<. \V., I,eil,nann. It. and PribiHa, \V. (1967). Kli,~. Wsch?'. 45, 1189. Cathie, i. ;\. B. (195:2). (;re¢1l Orpnotul S t J . J u n e , pp. 43-48. Chart, che, S. a n d Mondzac, A. M. (1967). Blood 30,879. Ch:trache, S., Mon(tzac, .-\. 31. a a d Go.,kqner. U. (1969). J . Cli;z. lnvest. 48, 8::14. Clcgg, J . 13.. \Veatherall, D..J., Boon, \V. 1-I. a n d Mustafa. D. (19139). N a t u r e (Lolulon) 222, 379. Crookston, J . H., F a r q u h a r s o n , 11. A.. 13eale, D. a n d l.,clmmm~, 1-I. (1969). Canad. J . ]3iocl~en~. 47, 1-13. l)acic, J. V., Gri,ncs. 4.. ,1.. 31eisler, A., Stcingold, L., Hcmst, ed, E. H., 13eavcn, O. H., a n d \Vhite, J. C. (1964). B r i t . . 1 . llaen~olol. 10, 388. l)acic, J . V., Shinton. N. K., ('laffney, P. J., ,Jr.. Carrell, R. \V. and L e h m a n n , H. (1967). ~Yalure (Lo~uion) 216, (163. De J o n g . DJ. ~\r. V~q. ~Vmat, l,. N. artd 13ernini, L. F. (1968). N a t u r e (1london,) 220, 788. De Vries, A., J o s h u a , tI., l~chnaann, H., Hill, 1~. L. a n d l~'ellows, IR. E. (1963). 2-3rit. J . H a e~atol. 9, 484. Fairbanks, V. I?., Ol)fcll, 1{. \V. a n d B u r g e r t , E. O., ,Jr. (1969). A mer. J . 2~[ed. 46, 344. l,'ertman. 31. It., a n d F e r t m a n , hi. B. (1955). J/'ed/cine (Balti~ore) 34, 131. Frick, 1". G., l"Iitzig, \V. H. a n d Stauffer, U. (1961). Schweiz. 'lied. lVochenschr. 91, 1203. G o u d e n m n d , hlt., Biserte, 0., t[~tbay, D. n n d Voisin. D. (1964). N o u v . Rev. I,'r. £lernato.l: 4, 487. Grimes, A. ,j., ~Ieisler. A. a n d Daeie, J. V. (1964)..Brit. J. Haematol. 10, 281. I t a l b r e c h t , I., lsaacs, \V. -k., Lehma~m, H. and/3on-:Porat, F. (1967). Israel J . '.Med. 8ci. 3, 827. Heinz, II. (1890). Virchows Arch. Pathol. A n a t . Phy8.1ol. K(. .~Ied. 122, 112. Hollcndcr, A., I m r k i n . P. A., L e h n m m L H. a n d Svensson, ]3. (1969). N a t u r e (Lo?u/on) 222, 953. H u e h n s , E. 12.. (1965). Prov. Roy. ,.~oc. ~lIed. 58, 514. I-[tmhns, E. 1%., H e e h t , I0.. Yoshida, A., Stamat, oyannopoulos, G., 1-Iartman, J. a n d l~To~ulsky, A. G. (1970). -Blood 36, 209. H u t e h i s o n , H. E., P i n k e r t o n , P. tt., W a t e r s , P., Douglas, A. S., L e h m a n n , tI. a n d :Beale, D. (1964)..Brit..,lied. J. ii, 1099. Jackson, J. hi., \Vay, B. ,j. and Woodliff. H. J. (1967). Brit. J. Haematol. (Suppl) 13, 474. Jacob, H. S., Brain, ~I. C. and Daeie, ,j. V. (1967). J. Clin. lnvest. 46, 1073. J a c o b , H. S., Bruin, h L C., Dacie, J. V., Carroll, 1~. W . ~nd Lehraann, H. (1968). N a t u r e (London) 218, 1214.
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Jacob, 11. and \Vinicrhalier, K. (1970). ]'roe..\'~.t. A~d. ~ci. U.S...t. 65, 697. J o n e s , If. '1".. 13rimhaU..13., t t u i s m a n . '1". 1t. O., l~leihauer, 12. a n d B c t k c , K. (1966). Science, ?v'. Y. 154, 1024. J o n e s , 11. T., Schroeder, "~V. :\., 13alog, J . 1~]. a n d Vinograd, J. 1l. (1959). J . . - l i n e r . Chem. Soc. 81, 316I. J o n e s , 11. V., Grimes, A. J., Can-ell. R. \V. a n d L e h m a u n , t i . (1967). ltrit, d. llr~em~ltol. 13, 394. 1,21eihauer. E. 1;., Ile3nlolds, C. A., Dozy, A. 31., \Vilson. d. 1~., .Moores, 11. 11., lSercz,~tu*, 31. 1'., "~Vright, C.-S., a n d H u i s m a n , T. t t . J . (1968). Bio,:him. JJiophys. ,.tct~, 154, 220. L e h m a m l , t l . (1970). LaTtcet ii, 78. I_~long. 3i., I.'leury, J . , Alagille, D.. ~[alassenet, 1~., L o r t h o l a r y , P. a n d P a r s , M. (191.;1). A'ouc. Re','..Fr. Hematol. 1, 819. Ix~rkin, P. A., I . ~ h m a n n . t t . , F a i r b a n k s , X'. 10, / l e r g l u n d . G. a n d L e o n h a r d t , T. (19;0). Proc. Biochem. Soc. J u l y S. 51ills, (4. C.. Levin. \V. C. a n d :\lperin, J. ]3. (196S). Bh~c,d 32, 15. 5lonn. E., Gait'hey, P. J . , J r . a n d I . e h m a n n , 1t. (196S). b'cand, d. ]laem~dol. 5, 333. 3 i o t u l s k y , A. G. (1969). Cited in L e h m a n n , 1t.. a n d Carrell. If. \V. B r i t . . U c d . Bull. 25, 14. 5Iozzieo:mcei. P., A t t a l . C.. l~ham. It.-T.. Malassenet, II. a n d 13essis. 51. (1961). :Yo~,,. ]~'et'. ~'r. Hematol. 1, $32. 51uller, C. J . a n d K i n g m a , S. (1961). Bicwhlm. B i o p h b s . . . l o i s 50,/59~5. Nagel, I~. L.. ]~Ianney. 1t. 31.. B r a d l e y , T. 13.. J a c o b s , :\. a n d Udem. I,. (1969). l~lood 34, 157. Opfell, R. \V., I~orkin. P. A. a n d L e h m a n n . }I. (1968). ,1. 3led. Gvnet. 5, 292. Ostertag. ~\'. a n d S m i t h , E. \V. (196S). Humamye,tetik 6, ;377. P i e t s c h m a m l . 1t., ~[.,orkin, P. A., Le.hmann, It. and ]~raunsteiner, 1|. (19119). Cited in l x q m l a n n , H. a n d Can-el!. II. \V. Brit. 31ed. Bull. 2S, 14. Pribilla, \V., I~lesse. P., Betke. K.. I . e h m a n n , t1. a n d l~eale. D. (19t;5). K i l n . l|'ochenscltr. 43, I(M9. R a i k , E.. ]~untx~r, E. G. a n d L i n d s a v , D. A. (1967). ~lled. J . Australia !, 955. l l a n n e y , t{. _M., ,Jaeobs. A. S., U d e m , L. a n d Z a l u s k y , 1t. (1968). Bio,'l~em. Biophy.~. Rcs. Commull. 33, 1004. l t i e d e r , ]t. F. a n d Bradley. T. 13., J r . (1968).)31cod 32, 35.5. llieder, l/. F., Oski. F. A. a n d Clegg. J. B. (1969). J . Clin. Inve.~t. 48, 1627. l:lieder, 11. F., Zin "tdmm, \V. II. a n d t l o l t z m a n , N. 2\. (1965). Amer. J . _;led. 39, 4. tligas, D. A.. l,Loler, R. D. a n d Osgood, E. E. ([956). J . Le~b. Clin. 3Ied. 47, 51. Rosa. J . , Labie. D.. \ V a j c m a n , It., Boigne. J . 5[.. Cabannes. 11., Bicrme. 1:~. a n d lluftlo..J. (1969). N a t u r e (London) 223, 190. l l o s a . J . , O u d a r t . J . L.. Pagnier. J . . B e l k h o d j a . If., ]3oigne. J . M. a n d Labia, D. (19GS). 12lh Intermr~tional Society of Hematology. p. 72. New York. Sansone, G., Carrell, r~. \ v . a n d L e h m a n n , I1. (1967). Nature (London) 214, 877. Sansone, G. a n d Pik, C. (1965). Brit. J . Haenmlol. 11, 511. S a t h i a p a l a n , R.. a n d R o b i n s o n . M. 0 . (1968)..Brit. J . lIaemalol. 15,579. S e h m i d , R., Breeher, C. a n d Clemens, T. (1959). ]Mood 14, 991. Schneider, 1R. O., Ued~, S., Alperin, J. B., Brimhall, 13. a n d J o n e s , R. T. (1968)..elmer. J. H u m a n Ge~et. 20, 151. Schneider, R. G., U e d a , S., Alperin, J . :B., Brimhall: B. a n d J o n e s , I),. 2:'. (1969). N e w Emil. J. :Ved. 280~ 739. S c h w a r t z , J . M., Sehreiber, Z., G o l d m a n , B. a n d l " a g e h n a n , K . (1968). 12th I n t e r n a t i o n a l Society of Hematology, p. 73, N e w ~_-ork. Scott, J. L., 1-Iaut, A., Cart~right, G. E. and ~,Vintrobe, M..~I. (1960). Blood 16, 1239. S h e e h y , T. ~,V. (1964). Arch. I n t . 2led. 114, 83. Shibat~, S., Iuchi, I., ~[iyaji, T., Ueda, S. a n d T a k e d ~ , I. (1963). A c t s Hae~natol. J a p . 26, 164. S t e ~ d m a n . J . 1~., Y a t e s , A. a n d J-Iuehr~s, E. II.. (1970). Br;A. J . llae~t.tol. 18, 435. W h i t e , J . M., B r a i n , hi. C., L o r k i n , P. A., L e h m a n n , J~. a n d S m i t h . M. (1970). Nature (London) 225, 939. Worms, I~., Bernard, J., Bessis, l~I. and ~lalassenet, 1%. (1961). N o s y . . R e v . F t . Hermztol. 1,805.
DISCUSSION :DR BEu-I'~ER. I think t h a t one of the things t h a t both Dr¢ K~-osmTA and I hoped ~ ould come oueoof this very lucid description of the unstable hemoglobins is the realization on the part of the lens contingent t h a t there m u s t be crystallinopathies, which should be the analog of hemoglobinopathics. These disorders, I would think, would be dominantly t r a n s m i t t e d cat~ractm These c~taracts ought to be always
HEINZ
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ANEMIAS
373
b i l a t e r a l . '['h.ey m i g h t I)e i,,e~ent at b i r t h , b u t also c o u l d o c c u r l a t e r in life. [ t h i n k t h i s is r e a l l y o n e o f t h e ~no.~t f r u i t f u l ~ r e a s :,f~nah,~y, a n d ] a m e s p e c i a l l y l o o k i n g f o r w a r d to "DR SI'EC~'OI~.:s d e s c r i p t i o n o f c r y s t a l l i n c h e m i s t r y , w h i c h i t h i n k p r o b a b l y is n o t q u i t e as f~r a l o n g as h e m o g l o b i n c h e m i s t r y , a t t h i s p o i n t . T h e r e is o n e q u e s t i o n t h a t I w o u l d like t o d i r e c t t o DR J A c o n . l i e i n d i c a t e d t h a t t h e h e x o s e m o n o t~ho~t)hate s h u n t a c t i v i t y was r e g u l a t e d b y t h e level o f G S I [ . I t s e e m s t o m e m u c h m o r e l i k e l y t h a t tile a c t i v i t y o f t h e s h u n t is r e g u l a t e d by t h e level o f G S S G or o t h e r s u b s t r a t e s for g l u t a t h i o n e r e d u c t a s e . As I)R S~t~v,~sT,~v~ i n d i c a t e d y e s t e r d a y , t h e h e m o g l o b i n o f G S l [ m i x e d disulfide, w h i c h Dr¢ JACOB h a s s h o w n o c c u r s in tit(.st, u n s t a b l e hcnm~:lobiu s y n d r o m e s , is a s u b s t r a t a for t h i s e n z y m e . T h e r e f o r e , m y g u e s s w o u l d I)c t h a t t h e increasc(I h e x o s e m o n o p h o s l ) h a t e s h u n t a c t i v i t y r e p r e s e n t s a v a i l a b i l i t y o f t h i s s u b st r a t e for -,.h)tat h irene n.<~uetast,. l ) a J.~c:)l,. I t.hink t h a t is a v e r y a t t r a c t i v e h y p o t h e s i s . I d i d n ' t m e a n to i n d i c a t e that, t h e level of g l u t a thion(, a h ) n c u t i m u l a t e s th(: s h u n t . I t h i n k y o u k n o w o f o u r p u b l i c a t i o n i n t h e Jour~,l of.Biological (.'he~ni,vtr!/. T h e sin~zle b e s t c o r r e l a t e o f hexose m o n o l ) h o s p h a t e s h u n t a c t i v i t y is t h e r a t i o of G S S G to G S i t . A n y t h i n g ~hat w o u l d incrca.sa G S S G w o u h l i n c r e a s e t h e s h u n t ; a n d a n y t h i n g w i t h a g i v e n a m o u n t o f (;.%~G th-~t wo,~ld d e c r c , ~ e G S t [ w o u h l i n c r e a s e t h e s h u n t . W e s i m p l y s u g g e s t e d t h a t t h e r e d u c t i o n i n ( ; S I t b y b i n d i n g t h r o u g h the d e n a t u r i n g h e m o g l o b i n is a s t i m u l u s . I t h i n k y o u r i d e a o f g l u t a t h i o n c r e d u c ta.~e a c t i n g o n th,~" m i x e d d i s u l l l d e is also valid. ])l'. I<[)'rHSTEIN. ~[ was vt.ry f a s c i n a t e d b y 131~J A c o n ' s d e m o n s t r a t i o n t h a t t h e H e i n z b o d i e s a r c b o u n d to he mere b r a h e t h r o u g h d isu}~i,le b o n d s , a n d f u r t h e r , t h a t t h i s r e s u l t s i n t h e c h a n g e i n p o t a s s i u m p e r m e a b i l i t y , l a m l m r t i e u l a r l y i n u ' r e s t e 4 i~ t h e f a c t t h a t in s o m e o f t h e e l e c t r o n m i c r o g r a p h s , i t ' s q u i t e clear t h a t the~o Hei,~z b o d i e s r e a c t w i t h o n l y a s m a l l f r a c t i o n o f t h e m e m b r a n e ; y o u k n o w , w i t h o n e o r t w o s p o t s a r o u n ( I . ])o y o u hnv,~ a n y i n f o r m a l i o n o n h o w m a n y - S } [ g r o u p s i n t e r a c t w i t h t h e H e i n z b o d y in p r o d u e i n g t h i s eff(.ct.',' I t w o u h l seem t hat, it m u s t be a r a t h e r s m a l l n u m b e r a n d t h a t w o u l d be v e r y i n t e r e s t ins. 1)I: J.xc,.~l:. 1 d o n ' t h a v e a n a n s w e r in t e r m s o f n u m b e r s . I w o u l d like to s u g g e s t to y o u , t h o u g h , t h a t o n e o f t h c thin~zs we sug~zested in t h e p a p e r we a r e a b o u t to p u b l i s h is t h a t w h a t o n e is seeing i n t h e e l e c t r o n m i c r o g r a p h s (Jf R i f k i n a n d l ) a n o n is t h e e n d st~lge d e n a t u r a t i o n t h a t ' s t h e p r e c i p i t a t e d h e m o g l o b i n , w h i c h is (,bvi()us. I t ' s cluite c l e a r to u s t h a t t h e s o l u b l e i n t e r m e d i a t e c o m p o u n d , or t h e a n a l o g i n t h e case o f t h e ~ e n e t i c a l l y u n s t a b l e h e m o g l o b i n s h a v i n g fl c h a i n a b n o r m a l i t i e s , is still s o l u b l e , a n d h a s c t m r m o u ~ a f f i n i t y fc.r b o t h g l u t a t h i o n e a n d for m e m b r a n e s . ~Vc c o u l d n ' t see t h i s , but, p r e s u m a b l y t h i s s o l u b l e m a t e r i a l is b i n d i n g -¢~}{ g r o u p s o f t h e cell m e m b r a n e s also. I ' m n o t s u r e we c a n d e m o n s t r a t e t h i s m o r p h o l o g i c a l l y an(l [ c e r t a i n l y d o n ' t h a v e a n y c h e m i c a l s t u d i e s to s h o w h o w m a n y - S I { g r o u p s a r e i n v o l v e d in this. [ a m t r y i n g to s ~ i m u l a t e s o m e o f t h e lens p e o p l e . H a s t h e m e m b r a n e , w h a t e v e r t h a t is, o f t h e l e n s been s h o w n to ]utve a l t e r e d p 2 r m e a b i l i t y t o c a t i o n s w h e n aflticted w i t h c a t a r a c t ? T h e a n s w e r to this, to me, m a y be t h e a n a l o g t h a t 1)it Bb:U~'t,F.I~ is l o o k i n g f o r - - o f p e r h a p s d 0 n a t u r i n g p r o t e i l l or a l t e r e d p r o t e i n a t t a c h i n g to t h e m e m b r a n e a n d , i n so d o i n g , altering its c h a r a c t e r i s t i c s . I s t h e r e a n y i n f o r m a t i o n a b o u t it? D a Kx.~osI[ITa. I n galacLose c a t a r a c t s we find t h a t o n e o f t h e e a r l i e s t c h a n g e s is o s m o t i c s w e l l h l g t h a t s e e m s t o l e a d t o a n i n c r e a s e in c a t i o n flux as m e , ~ u r e d b y p o t a s s i u m e x c h a n g e . I s u s p e c t t h a t m a n y o f t h e s e e x p e r i ' n c n t a l c a t a r a c t s i n v o l v i n g c h a n g e s in t h e c o r t e x d o first i n v o l v e c h a n g e s i n p e r m e a b i l i t y to o a t i o n s . D r I w a t a from O s a k a U n i v e r s i t y h a s b r o u g h t t o t h i s c o u n t r y a s t r a i n o f ' m i c e t h a t d e v e l o p h e r e d i t a r y c a t a r a c t s . O n e o f t h e e a r l i e s t c h a n g e s we see in t h e s e h e r e d i t a r y c a t a r a c t s is a m a r k e d c h a n g e in e l e c t r o l y t e m e t a b o l i s m . DR Elcxsa" 11. J ~ F F ~ . Does a n y o n e else h a v e a n 5, c o m m e n t s ? I w o u l d like to a s k :DR JACOB a q u e s t i o n a b o u t t h e 5-t0-2S0 r a t i o that~ he h a s u s e d to d e m o n s t r a t e t h e h o m e - d e p l e t e d g l o b i n c h a n g e . I s t h e r e an5" p o s s i b i l i t y [ h a t p a r t o f this is a c t u a l l y m e t h e n m g l o b i n f o r m a t i o n b e c a u s e t h e 5 4 0 - 2 S 0 r a t i o o f m e t b e m o g l o b i n w o u l d be lower t h a n t h a t of o x y h e m o g l o b i n ? D~t JACO;~. ~ ' o w t k a t ' s v e r y p e r c e p t i v e . [ s u s p e c t t h a t h e t h o u g h t t h i s o n e t h r o u g h b e f o r e t h e s e e x p e r i m e n t s . T w o t h i n g s were d o n e to c o n t r o l t h a t : (1) i n e u b a t i o l ~ s were s h o r t - l i v e d ; a n d (2) t h e a m o u n t o f m e t h c m o g l o b i n was a b o u t 10~,. Thus, one could m a k e calculations t h a t would reduce the ratio b y a b o u t 0.02, w h i c h is i n s i g n i f i c a n t as c o m p a r e d w i t h t h e r a t h e r m a r k e d d r o p s t h a t were o c c u r r i n g . C y a n i d e w a s a d d e d ns well, w h i c h h e l p s t h e s i t u a t i o n d r a m a t i c a l l y i n t e r m s o f t h e 5 4 0 - 2 8 0 r a t i o . T h e s e w e r e p o i n t s o f c r i t i c i s m leveled a t D a ~,~'INTERtL~,LTER'Sa n d m y p a p e r a n d were a n s w e r e d b y D[¢ WI'~,'TERtI,~.LTER q u i t e well in a n u p c o m i n g a r t i c l e . DR SRrVASTAV,~. D i d y o u e v e r t r y to b l o c k t.he --StI g r o u p s b y A r - e t h y l m a l e i m i d o m a i n l y a t t h e 93 p o s i t i o n o f f l - e h a i n s a n d t h e n s u b j e c t i n g t h e m t o o x i d a t i v e s t r e s s for H e i n z b o d y f o r m a t i o n ?
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DR J . ~ c o s . I n o u r first s t u d i e s wc f o u n d that. N E b l - t r c a t e d r e d eell.~ i n t h e s e p a t i e n t s c o m p l e t e l y b l o c k e d a n y u n b o u n d g l u t a t h i o n o ; t h e s e cells w e r e m o r e susct, p t i b l c t o h e a t p r e c i p i t a t i o n . T h a t in, t h e h e m o l y ~ t e s d e r i v e d t h e r e f r o m , so t h a t t h i s is a n a d d i t i v e effect. I n a d d i t i o n . H g b - A t h a t h a s it,s fl-93 s u l f h y d r T I s i n h i b i t e d also b e c o m e s a h e a t - p r e c i l n t a b l e h e t a o g l o b i n . I t m i m i c s m a r k c ~ l l y t he u n s t a b l e h e m o g l o h i t a . ])r~ SRtVAST.~V.~. 1 h a v e a n o t h e r q u e s t i o n . ~X'_~ultI y o u e l a b o r a t e f u r t h e r o n h o w t i m m i x e d d i s u l f i d e o f ]-lgb--S-S-C, a t t a c h e s t o t h e m e m b r a n e . T h e s e q u e n c e t h a t y o u h a v e p o i n t e d o u t is that. llgb--~S-.'q- (; r e a c t s w i t h t h e m e m b r a n e s u l f h y d r y l . N o w in t h a t ease. otto w o u l d expect, t h a t t h e G S I I w o u l d be relea,~cd f r o m t h e h e m o g l o b i n . D i d y o u e v e r t r y t o see o r h a v e y o u e v e r m e a s u r e d t h e a m o u n t o f (~.%_'1[ t h a t m released during the attachment of the Heinz bodies to the membrane? :DR J.~COB. T h a t w o u l d be a n i c e e x l ~ c r i m e n t . W e h a v e n ' t d o n e t h a t . I t h i n k w h a t y o u a r e s u g g e s t i n ~ is m a y b e t o m i x d i s u l f i d e w i t h r a d i o a c t i v e g l u t a t h i o n e , w h i c h c a n b e d o n e , a n d t h e n use t h a t m a t e r i a l w i t h g h o s t t o see if y o u get. a r e l e a s e o f r a d i o a c t i v i t y . 1 t h i n k t h a t w o u l d be ver b, p r e t t y . ~Ve h a v e n ' t b<.eu s m a r t e n o u g h t o h a v e t h o u g h t o f t h a t a h e a d o f t h i s m e e t i n g . I also w o u l d s u g g e s t t o y o u . t h o u g h , t h a t i t p r o b a b l y is n o t j u s t fl-93 s u l f h y d r y l s t h a t a r e involvc~l in t h e b i n d i n g o f t h e red cell gho~t~. 1 t h i n k t h e f u r t h e r s t e p h e r e is, as J a n d l a n d A l l e n h a v e showt~, t h a t t h e c h e m i c a l d a m a g e is t h e u n f o l d i n g . N o w o n e is a b l e t o t i t r a t e m a n y m o r e - S H g r o u p s t h a n t h e o r i g i n a l l y free o n e s o f fl-93. "l'h~tt is. ot~e ii~ at a n d a n o t h e r i n t h e fl c h a i n . The-refers, f o u r m o r e b e c o m e a v a i l a b l e as t h i s s p h e r i c a l m o l e c u l e s t a r t s t o o p e n u p i n i t s p r o - p r e c i p i t a t i o n . So t h o s e - S H g r o u p s are. I s u s t ~ c t , a l s o n o w a v a i l a b l e for m e m b r a n e b i n d i n g . I d o n ' t h a v e a n y real e v i d e n c e of t h a t . Dl~ SRIVAST.~VA. O n e l a s t q u e s t i o n , D ~ J A ¢ o I u :Did y o u e v e r t r y t o i n c u b a t e c e r t a i n u n s t a b l e h e m o g l o b i n s w i t h T P N H a n d g l u t a t h m n e r t ~ l u c t a s e a n d t r y zo s p l i t off (~SII t o s h o w t h a t t h e t r t p e p t i d e is b o u n d to t h e h e m o g l o b i n ? W h a t is t h e e v i d e n c e t h a ~ s u c h h e m o g l o b i n is in c o m b i n a t i o n w i t h G S S ( I o r GSH ? D n J A c o B . V~re h s r v e n ' t d o n e w h a t y o u ' v e aske~l. T h e e v i d e n c e h a s boa's t h a t o n e c a n s p h t g l u t ~ , t h i o n e f r o m t h e s e m a t e r i a l s w i t h m e r c s p t o c t h a n o l . 1 t h i n k t h a t ' s t h e m a j o r e v i d e n c e . T h e r e is e v i d e n c e t h a t i t is t h e f l - c h a i n t h a t b i n d s t h e r a d i o a c t i v e g l u t a t h i o n o . O n e c a n r~,split t h a t a n d c h r o m a t o g r a p h tt, a n t i f i n d t h i s o n t h e f l - c h a i n . T h i s is r e a l l y j u s t a p r e s u m p t i o n t h a t i t ' s t h e fl-93, b u t t h a t ' s t h e o n l y o n e t h a t is f r e e l y t i t r a t a b l e . W e k n o w i t ' s f l - c h a i n a n d w e a r e p r e s u m i n g t h a t i t ' s o n t h e 93. W e h a v e n o c v i d t . n c e of that at the moment. :DR EFSTEI.~'. I t h i n k o n e o f t h e purpos~_~ o f t h i s m e e t i n g is t o h e l p u s c x a n i i n e o u r o w n b i a s e s a n d prec o n c e i v e d n o t i o n s i n l i g h t o f e v i d e n c e i n o t h e r fields, b l y o w n b i a s , a s y o u h e a r d y e s t e r d a y , is t h a t e a r l y c a t a r a c t f o r m a t i o n is p r i m a r i l y a m e m b r a n e p h e n o m e n o n , a n d t h a t c h a n g e s i n t h e l e n s p r o t e i n c r y s t a d l i n a r e s e c o n d a r y t o c h a n g e s i n m e m b r a n e . 1 t b i n l t t h a t t h i s o c c u r s i n d i a b e t i c c a t a r a c t s , w h i c h I)R CttrLAC~ will d i s c u s s . I t is b e l i e v e d t h a t c h a n g e s i n t h e m e m b r a n e a r e s e c o n d a r y t o s w e l l i n g o f t h e l e n s cells c a u s e d b y t h e a c c u m u l a t i o n o f p o l y o l . H o w e v e r , it, w o u l d be v e r y i n t e r e s t i n g t o a p p r o a c h t h i s f r o m tJ~c o t h e r viewTpoint, n a m e l y , t h a t t h e r e is s o m e p r i m a r y d e f e c t i n t h e l e n s c r y s t a l l i n s a n d t h a t t h i s d e f e c t affects t h e m e m b r a n e s . I t c o u l d b e s a i d t h a t i t is o f a d v a n t a g e t o t h e w h o l e o r g a n i s m t h a t r e d ceils a r e u n a b l e t o c a r .ry o x y g e n i f t h e r e is d a m a g e t o h e m o g l o b i n . ' £ h i s is a m e c h a n i s m w h e r e b y t h e effect oat h e m o g l o b i n can relate to the membrane and by changing membrane -Sit groups and affecting membrane functions, t h e b o d y c a n r i d i t s e l f o f d e f e c t i v e r e d cells. S i m i l a r l y , t h e r e m a y b e d a m a g e t o t h e p r o t e i n in t h e l e n s cells w i t h a s e c o n d a r y e f f e c t o n t h e m e m b r a n e s b e c a u s e t h e l e n s c a n n o t rid i t s e l f o f t h e s e cells. As y o u k n o w , n a t u r e u s u a l l y d o e s n ' t c o n f o r m t o o u r b i a s e s , a n d i t w o u l d b e v e r y i n t e r e s t i n g t o t r y to i n v e s t i g a t e s o m e o f :DR JACOB'S ~,~,eories i n t h e l e n s . :DR ~ L ~ . I h o p e l ' m n o t g o i n g t o l e t t h e l e n i n v e s t i g a t o r s d o w n b y a g r e e i n g w i t h Dlt J a c o b , b u t w e h a v e n ' t g o t h a l f as f a r w i t h t h e s t u d y o f t h e c h e m i s t r y a n d c h a n g e s o f t h e e r y s t ~ l l i n s a s h e h a s , a n d o t h e r p e o p l e h a v e w i t h h e m o g l o b i n . I n o u r s t u d i e s o f c h a n g e s i n p r o t e i n s i n c a ~ r a c t , f o r m a t i o n s , in c a t a r a c t f o r m a t i o n in h u m a n beings, w e ' v e used u r e a i n s o l u b i l i t y as a sign of - ~ - S - f o r m a t i o n , a n d I w o n d e r w h e t h e r t h i s h a s b e e n u s e d a t all b y a n y b o d y i n t h e s t u d y o f h e m o g l o b i n s . A l s o , w e h a v e f o u n d t h a t b y a n a e r o b i c e x t r a c t i o n o f c e r t a i n c a t a r a c t s t h a t w o u l d , i f e x t r a c t e d a e r o b i c a l l y , g i v e us a l o t o f u r e a i n s o l u b l e p r o t e i n h e l d i n s o l u b l e b y - S - S - forms. I f we e x t r a c t a n a e r o b i c a l l y , t h e n we do n o t got this - S - S - b o n d f o r m a t i o n . T h e t h e o r y is, t h a t t h e f i r s t c h a n g e i n t h e d i a b e t i c h u m a n c a t a r a c t , w h i c h is a p r e t t y s e v e r e n u c l e a r c a t a r a c t , is a k i n d o f u n f o l d i n g o f t h e p r o t e i n a l l o w i n g - S H f o r m s t o b e a v a i l a b l e f o r a e r o b i c o x i d a t i o n w h e n t h e w h o l e s t r u c t u r e is g r o u n d u p a n d e x t r a c t e d . T h e q u e s t i o n I a s k is h a v e you studied the hemoglobins and the urea solubility or insolubility under certain and different circum. stances? :DR JACOB. I ' m s u r e t h a t ' s b e e n d o n e . I c a n ' t r e m e m b e r . I l o o k e d o v e r a t E r n i e b e c a u s e i t s e e m s t o m e t h a t h e r e p o r t e d s o m e t h i n g l i k e t h a t i n o n e o f h i s e a r l y p a p e r s . I k n o w J i m J a n d l a n d :Dave A l l e n h a v e
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look~l a t urea* solubility of p h e n y l h y d r ~ z i n c . t r e a t c d hemoglobins, or m a y b e someone knows w h a t the results arc. 1)a CJl ~'t.,U'K. 1 ] usu have a couple of c o m m e n t s on Da EI'~TEI2~'S remarks. We studied the expcriment~tl sugar c~taract, the diabetic cataract, and wc found t h a t the accumulation of sorbitol causes the lens liter to swell and Clltion flux to increase. This a p p c a r s to be an increased cation exchange prior to an absolute clmngc in the level of cations. A b o u t 3 ¢htys after the flux incrcascs, opacification develops, and then there is a sudden change in the absolute level of cations. This was interpreted to be an indication t h a t as m e m b r a n e i~,rmeability increa.~,d, p u m p a c t i v i t y incrcased and compensated for this, b u t then its the p u m p a~.'tion was exceeded, there was a sort of influx of sodium and a m a r k e d change in the absolute level of eatmns. Now this is an e.