ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 10, No.

6
Copyright © 1980, Institute for Clinical Science, Inc.

Differentiation of M yoglobin and

H em oglobin in Biological Fluids
E R N E S T C. ADAMS, P h .D.

Research Products Division,

Miles Laboratories, Inc.,
Elkart, IN 46515

ABSTRACT

T h e u se o f several m ethods to d ifferen tiate m yoglobin from hem oglobin

has b e e n in v estig ated . T h e im m unochem ical m ethods, particu larly those of
h em ag g lu tin atio n in h ib itio n an d radioim m unoassay, are th e m ost useful.
T h is re p o rt sum m arizes work in th e A m es R esearch L aboratory over th e past
17 years w ith th e several m ethods.

In tro d u c tio n
M yo g lo b in , th e oxygen b in d in g p ig ­
m en t o f m u scle, a n d h em oglobin, th e oxy­
g en b in d in g p ig m e n t o f erythrocytes, are
alike, y et d ifferent. T h ey perform sim ilar
functions an d u n d erg o m any of th e sam e
reactions. T h e re are som e physical and
ch e m ic al d iffe re n c e s .3,11 D e sp ite th e ir
m any sim ilarities, m yoglobin an d hem o ­
g lo b in are im m unologically different. This
d ifferen ce, a n d som e o f th e physical and
ch em ical d ifferences, can b e u se d to dif­
feren tiate o ne from th e other. W ith the
e x c e p tio n o f th e im m u n o c h e m ic a l
m etho d s, this usu ally req u ires th e p re s­
e n c e o f re la tiv e ly la rg e q u a n titie s o f
m yoglobin an d hem oglobin.
M yoglobin is re le a se d into th e blood
plasm a as a re su lt of dam age to m uscle
tissu e. B ecause o f its sm all size an d lack of
b in d in g to hap to g lobin, it is rapidly re ­
m oved from th e blood by th e k id n ey and
e x c re te d in to th e u rin e . H e m o g lo b in ,
w h e n re le a se d from th e re d cell by in-
tra v a s c u la r h e m o ly s is , is ra p id ly a n d
t ig h tly b o u n d to h a p to g lo b in . T h e
493
0091-7370/80/1100-0493 $01.20 © Institute for Clinical Science, Inc.
h a p to g lo b in - h e m o g lo b in c o m p le x is
large, so th at it is n o t excreted in th e urin e.
T h u s, no h em oglobin appears in th e u rin e
u n til th e blo o d level o f h em o g lo b in ex­
c eed s th e hapto g lo b in b in d in g capacity.
O n c e a ll th e h a p to g lo b in is b o u n d ,
h em o g lo b in appears in the plasm a, bo th
b o u n d to a lb u m in as m eth em alb u m in an d
as free hem oglobin.
M y oglobinuria is often in fe rre d from
th e clinical sym ptom s o f m uscle w eakness
a n d p a i n ,11,20' 21 w h e n t h e r e is a
p eroxidase-like p ig m en t in th e u rin e w ith
few or no erythrocytes see n on th e m icro­
s c o p ic e x a m in a tio n . A ris e in se ru m
c re a tin e p h o sp h o k in ase (CPK) is often
u se d to b o lste r th e suspicion.21 I f th e labo­
ratory is asked to confirm this p re su m p ­
tive diagnosis, a classical m eth o d such as
so lu b ility in 80 p e rc e n t s a tu ra te d am ­
m o n iu m su lfate m ig h t b e u se d . W h en
th e re is in su fficien t p ig m en t in th e u rin e
to b e clearly visible, th ere g en erally is no
s u s p ic io n o f h e m o g lo b in u ria or m y o ­
globinuria. T h e conditions w o u ld b e d e ­
te c te d only by routinely ru n n in g occult
494
b lo o d tests. T h e d iffe re n tia tio n w o u ld
th e n h ave to b e by th e m ore sen sitiv e im ­
m un o ch em ical tests.
M e th o d s
N o n -I m m u n o l o g ic a l M e t h o d s
T h e m ore classical m ethods o f differ­
en tiatio n , su ch as am m onium sulfate sol­
u b ility , u l t r a f iltr a tio n , g e l e x c lu s io n
c h r o m a to g ra p h y , a f fin ity c h r o m a to g ­
raphy, an d spectra, are d e sc rib e d an d dis­
cu ssed in th e e a rlie r p u b licatio n .3 In some
o f th ese, th e lite ratu re m eth o d was m od­
ified b y u sin g o ccu lt blo o d tests to in­
crease th e sensitivity.
Im m u n o c h e m ic a l D if f e r e n t ia t io n
of M y o g l o b in a n d H e m o g l o b in
T h e m ost d efin itiv e d ifferen tiatio n o f
h em o g lo b in a n d m yoglobin in biological
fluids can b e ac h ie v ed by u sin g relatively
sim ple im m u n o ch em ical m ethods. T h ese
m eth o d s, w h ic h in c lu d e im m u n o d iffu ­
sion, h e m a g g lu tin a tio n in h ib itio n , a n d
im m u n o e le c tro p h o re sis, are d e p e n d e n t
u p o n th e fact th at specific a n tisera w ill
react o nly w ith its hom ologous antigen.
M oreover, th e m eth o ds w ill q u an titate the
hem o g lo b in or m yoglobin p re sen t.
Preparation o f Antisera. T h e isolation
an d pu rificatio n o f th e im m unogens, the
im m u n izatio n sch ed u les, an d th e process­
in g of th e an tisera are esse n tially th e sam e
as prev io u sly re p o rted .3 M yoglobin is iso­
lated from h u m an or rh esu s m onkey m us­
cle according to th e selectiv e p re cip ita­
tion m eth o d o f L u g in b u h l.18 H em oglobin
a n d p ro tein s o th er th a n m yoglobin are
p re cip ita te d from extracts o f m uscle at pH
8 w ith 80 p e rc e n t am m onium sulfate sat­
u ra tio n . U n d e r th e s e c o n d itio n s , th e
m yoglobin rem ain s in solution. M yoglo­
b in is p re c ip ita te d at p H 7 w ith 100 p e r­
ce n t am m onium sulfate saturation. T he
m y o g lo b in is fu rth e r p u rifie d by e le c ­
tro p h o resis on acrylam ide gel w hich re­
m oves alb u m in an d o th e r serum p rotein
contam inants. T o o btain m aterial for im ­
ADAMS
m unization, th e po rtio n o f gel containing
th e m y o g lo b in b a n d s is e le c tro e lu te d .
C o lo red e lu te d fractions are p o o led and
th e abso rb an ce in th e S oret b a n d reg io n is
m easu red . A fter th e elu tio n , th e m yoglo­
b in is in th e ferro form; at a concentration
o f one m g p e r m l, th is has an absorbance of
5.8 at 422 nm . From this value a n d the
volum e o f e lu te , th e total am o u n t o f m yo­
globin is calculated. T h e elu te, w h ich con­
tains b u ffer m atetial as w ell as m yoglobin,
is ly o p h iliz e d . F o r im m u n iz a tio n , th e
m y o g lo b in c a n b e e i t h e r h u m a n or
m onkey.
T h e h e m o g lo b in im m u n o g e n is p r e ­
p a re d from la k e d e ry th ro c y te s as d e ­
scrib ed e a rlie r3 a n d p u rifie d b y electro ­
phoresis.
T he ea rlie r im m unization sch e m e3 has
b e e n m odified in dose an d route. T h e in i­
tial im m unization in F re u n d ’s com plete
ad juvant is given into th e four footpads of
rabbits or into th e four dew claw s of goats.
T h e boosters in F re u n d ’s in co m p lete ad ­
ju v a n t are g iv en su b cu tan e o u sly or in ­
tra m u s c u la rly (ro u g h ly e q u a l am o u n ts
into th e four q u arters o f th e anim als). T he
initial dose is 45 fig p e r anim al and boos­
te r doses are 22.5 f i g p e r anim al. T he
sch e d u le o f b oosting an d b le e d in g rem ain
th e sam e. T h e an tisera are ce n trifu g ed
and p ro cessed as soon as a good clot has
fo rm e d . O n im m u n o d if f u s io n o r im ­
m u n o e le c tr o p h o r e s is , th e a n tis e r a to
m yoglobin sh o u ld give a p re c ip itin line
against a m yoglobin solution or a u rine
containing m yoglobin at levels o f 40 to 50
fig p e r m l a n d no lin e against hu m an al­
b u m in at levels o f 1000,100, an d 10 fig p er
m l or against h u m an serum . T h e antisera
to h em o g lo b in sh o u ld give a p re c ip itin
line against hem oglobin at levels o f 40 to
50 ¡jug p e r m l a n d no lines against album in
or o th er serum p ro tein s at levels of 1000,
100, an d 10 fig p e r ml.
M e th o d s f o r D e te c tin g A n tig e n -
A n tib o d y Reaction. In th is laboratory, th e
m ethods em p lo y ed for th e im m unochem ­
ical reactions o f m yoglobin an d hem oglo­
b in are im m unodiffusion, im m u n o e le c­
 M Y O G LO B IN AND H E M O G L O B IN D IF F E R E N T IA T E D IN B IO L O G IC A L FL U ID S
tro p h o resis, an d h em ag g lu tin atio n in h ib i­
tio n for u rin e , ra d io im m u n o a s s a y for
serum m yoglobin, an d latex agglutination
for special ap p lications. Im m unodiffusion
an d im m u n o electro p h o resis are th e sam e
as d esc rib ed ea rlier,3 b u t w ith m odifica­
tions o f m ed ia a n d b u ffer as in a later p a­
p er.5 P re c ip itin lin es d ev e lo p w ith in four
to 16 hours.
H e m a g g lu tin a tio n Inh ib ition . M yoglo­
b in is co u p led to form alinized sh ee p red
b lo o d cells as p rev io u sly d e s c rib e d 3 w ith
m odifications. A fter dialysis in th e carbo­
nate buffer, th e cru d e m yoglobin solution
is d ilu te d so th a t it has an absorbance of
0.79 at 410 nm . O n e h a lf ml o f this solution
is reacted w ith 6 m g pyrrole-2-carboxylic
acid azid e at p H 9.5. T h e p acked cells
from 7 ml of th e 10 p e rc e n t form alinized
sh ee p erythrocytes are re acted w ith 2 ml
b is - d ia z o b e n z id in e a t p H 3.5 . T h e
m yoglobin-pyrrole co m p o n en t is reacted
w ith th e d iazo -b en zid in e-cells at p H 7.
H em o g lo b in is co u p led to form alinized
sh ee p red blo o d ce lls.3
F o r b oth m yoglobin an d hem oglobin
conjugates, th e stab iliz ed diazonium salt,
F ast Black B, can b e su b stitu te d for the
b is-d iazo b e n zid in e.6 A solution o f 1.2 mg
F ast Black B p e r m l is e q u iv a le n t to the
p re p a re d b is-d iazo b en zid in e. T h e p re p ­
aration o f pyrrole-2 carboxylic acid azide
has b e e n d esc rib ed .6
T h e an tisera are h e a te d at 56° to destroy
c o m p le m e n t a n d a b s o r b e d w ith fo r­
m alin ized sh ee p re d blood cells as d e ­
scrib ed .3 Tw o fold d ilu tio n s o f th e anti­
sera (starting w ith 1:20) are m ade in 0.2 M,
p H 8.5 B icine bu ffer co n tain in g 1 p ercen t
n o n im m u n e ra b b it serum . T h e n u m b e r of
d ilu tio n s w ill d e p e n d on th e tite r, b u t
n in e d ilu tio n s are usually p rep ared .
M icrotiter p lates* (the tite r m ay vary
s lig h tly d e p e n d in g o n th e p a r tic u la r
b rand) are u se d for th e titration. T e n v erti­
cal row s on th e tray are m ark ed for th e
n in e a n tis e r a d ilu tio n s a n d o n e fo r
* U bottom Ames Autotray, Cooke, or Linbro.
495
p ro tein -b u ffer w ith o u t antisera. O n e ho ri­
zontal row is m arked for a neg ativ e u rin e
(titer row) one is m arked for each standard
u rin e, a n d one for each u n k n o w n u rin e. In
each w ell of th e first vertical row is p laced,
w ith a P asteu r p ip e t (0.025 ml), one drop
o f th e 1 :2 0 an tiseru m dilution; in each
w ell o f th e sec o n d v ertical row is p lace d
o ne drop o f th e 1 :4 0 dilution. T his is con­
tin u e d th ro u g h th e n in th v ertical row. In
th e te n th row is p lace d one drop o f the
protein-buffer. O ne d rop o f neg ativ e u rin e
is a d d e d to all th e w ells o f th e first h o ri­
zontal row; o n e drop o f stan d ard or u n ­
know n u rin e is a d d e d to each w ell o f th e
o th er horizontal rows. O ne drop o f th e ap ­
p ro p ria te conjugate is ad d e d to all th e
w ells.
T h e co n ten ts o f th e w ells are m ixed
e ith e r by vigorously rotating th e w hole
p la te o r b y stirrin g each w e ll w ith an
ap p licato r stick or toothpick. T h e p late is
p lace d e ith e r over a m irror or on a w h ite
b ackground for one hour. A p o sitiv e is in­
d icated b y a rin g or b u tto n o f cells in a w ell
to th e left o f th e first w ell w ith a rin g in th e
tite r row. T h e g re ater th e n u m b e r o f w ells
w ith rings, th e g re ater th e concentration
of m yoglobin or hem oglobin.
T he b e st stan d ard for m yoglobin is a
u rin e co n tain in g a large am ount o f m yo­
globin b u t no hem oglobin. T h e am o u n t o f
m y o g lo b in c a n b e d e t e r m in e d b y a
peroxidase activity m eth o d an d th e n th e
u rin e can b e d ilu te d w ith negative u rin e
to give ap p ro p riate standards. A standard
fo r m y o g lo b in c a n b e p r e p a r e d b y
chrom atography o f a m uscle extract on
Sephadex® G-100. T h e first peroxidase-,
like reactive b a n d is catalase, th e second
one is hem oglobin, a n d th e th ird is m yo­
g lo b in . T h is m y o g lo b in is n o t as im -
m unoreactive as m yoglobin ex c reted in
u rin e. I f tre a te d w ith 6 M urea, w h ich th e n
is rem o v ed on a co lu m n of S ephadex G-25,
th e im m unoreactivity is in cre ased to th at
o f ex c reted m yoglobin. T h e am o u n t o f
m yoglobin is d e te rm in e d by th e ad so rp ­
tion in th e so ret b a n d region (about 410
nm ): A (m g p e r ml) = 10.
496
H em o g lo b in stan d ards can b e p re p are d
from u rin es a n d hem olysates in a sim ilar
m anner.
S e r u m M yoglobin. B ecause o f the re la­
tively low am ounts o f m yoglobin in serum
com p ared to th a t in u rin e (nanogram s vs.
m icrogram s) an d b ecau se o f in terferen ce
by o th er serum p ro tein s, th e hem ag g lu ti­
natio n in h ib itio n m eth o d is generally no t
s u ita b le for se ru m m y o g lo b in . I f th e
serum m yoglobin co ncentration is high, as
m ay b e cau sed by ren al sh u td o w n so th at
th e seru m p ro tein s can b e d ilu te d out, the
hem agglutination inhibition m ethod may
b e used . S erum m yoglobin m ay b e d e te r­
m in ed by sep aratin g m yoglobin from m ost
o f th e s e ru m p r o te in s b y p a s s in g it
th rough an A m icon XM 50 filter and th e n
co n cen tratin g it on a UM 10 filter before
u sin g th e h e m a g g lu tin a tio n in h ib itio n
m ethod. R adioim m unoassays are b e st for
seru m m y o g lo b in . A co lu m n ra d io im ­
m unoassay w as re p o rte d previously.5 A
d o u b le an tib o d y m eth o d can b e set u p in
th e follow ing m anner.
F o r th e first an tib ody, goat anti-hum an
m y o g lo b in is d i l u te d in g o a t g am m a
g lo b u lin solution (1 m g p e r m l in 0.2 M,
p H 8.5 B icine buffer) so th a t 0.1 m l w ill
b in d 30 to 40 p e rc e n t o f the lab e lle d m yo­
globin. F o r th e seco nd antibody, th e solu­
tio n u se d is ra b b it anti-goat gam m a globu­
lin in 0.2 M, p H 8.5 B icine buffer, 0.03 M
in e t h y l e n e d ia m i n e te t r a a c e ti c a c id
(ED TA ), an d co n tain in g 2 p e rc e n t Brij 35.
L a b e lle d m y o g lo b in a n d stan d ard s are
p re p a re d in th e sam e m an n er as for the
colum n m eth o d .
D isp o sa b le tu b e s (75 x 12 m m ) are
lab elled 100 p e rc e n t blank, serum blank,
each standard, a n d each un k n o w n serum .
Into each tu b e is p lace d 0.1 m l standard or
u n k n o w n seru m as lab elled ; 0.1 ml non-
im m u ne serum is u se d for bo th th e blanks.
To each tu b e , ex cep t th e serum blank, is
ad d e d 0.1 ml o f th e first antibody. To the
serum b lan k is ad d e d 0.1 ml B icine buffer.
T h e tu b es are vigorously m ixed. A fter 15
m in u tes, 0.1 m l of la b e lle d m yoglobin is
ad d e d to each tu b e. T h e tu b es are vigor­
ADAMS
ously m ixed. A fter 30 m inutes, 0.2 m l of
th e seco n d an tib o d y is a d d e d to each tube.
T h e tu b e s are vigorously m ixed. A fter 30
m inutes, th e tu b es are again m ixed and
th e n cen trifu g ed at 3000 rpm for 15 m in ­
utes. T h e su p ern ates are d ecan ted ; the
lips o f th e tu b es (w hile in an in v erte d
position) are w ip e d w ith a tissu e. T h e
tu b es are in v e rte d on a tow el. T h e tu b es
are co u n te d in a w ell counter. T h e counts
o f th e serum b lan k are su b tracted from the
counts o f th e o th er tubes. T he n e t counts
of th e standards and unknow ns are ex­
p re sse d as percen tag es o f th e n e t co u n t of
th e 100 p e rc e n t blank. T h e p e rcen tag e of
th e standards is p lo tte d on log logit p ap e r
against th e ng o f m yoglobin. F rom this
c u rv e th e m y o g lo b in in th e u n k n o w n
serum s is calculated. A sim ilar com m er­
cial kit is available.*
T h e u s e o f th e h e m a g g lu tin a tio n
m eth o d in d e te c tin g m yoglobin in tissues
an d tissu e c u ltu re and a flu o rescen t an ti­
body te c h n iq u e for d etec tin g m yoglobin
d e p o site d in th e kid n ey was d e sc rib e d in
th e ea rlie r p u b licatio n .3 T he use of im ­
m u n o c h e m ic a l m eth o d s to d is tin g u is h
en d o g en o u s h em oglobin from m yoglobin
an d h em o g lo b in o f dietary origin was d e ­
scrib ed at th e 1973 sem inar.4
R esults a n d D iscussion
M any laboratories are d e p e n d in g on the
clinical history or th e classical am m onium
su lfate p re c ip ita tio n m eth o d for differ­
e n tia tin g h e m o g lo b in u r ia fro m m y o ­
globinuria. T h e original m ethod u sed only
th e ap p e ara n ce o f th e u rin e before and
a f te r 80 p e r c e n t s a tu ra tio n w ith a m ­
m on ium su lfa te .8 T h e sen sitiv ity is in ­
creased b y co m b in in g this w ith an occult
b lo o d te st.3 W ith this te c h n iq u e , m any
u rin es w h ic h co n tain m yo g lo b in w h e n
analyzed b y im m unochem ical m ethods,
are re p o rte d to contain only hem oglobin.
C hu e t a l 9 su g g est th a t th e am m onium su l­
fate m eth o d fails m any tim es b ecau se the
* N uclear M edical Systems.
 M Y O G LO B IN A N D H E M O G L O B IN D IF F E R E N T IA T E D IN B IO L O G IC A L F L U ID S
p H is n o t ad ju sted above 7.5. T his is not
th e co m p lete an sw e r since p H 8 has b ee n
u se d ro u tin ely by us a n d y et th ese false
positives for h em o g lo b in are obtained. In
th e u ltra filtra tio n m e th o d , m y o g lo b in
som etim es fails to pass th e filter.
B oth th e am m onium sulfate p re cip ita­
tio n an d th e u ltrafiltratio n m ethods also
fail to d e te c t a sm all am o u n t o f one o f the
p ig m e n ts in th e p r e s e n c e o f a la rg e
a m o u n t o f th e o th e r. O n ly th e im ­
m u nochem ical m ethods w ill do this.
M in im a l D e t e c t a b l e L e v e l s
In ad d itio n to b e in g m ore discrim inat­
ing, th e im m u n o chem ical tests have the
lo w est m inim al d e te c ta b le levels o f m yo­
g lo b in o r h e m o g lo b in . T h e r a d io im ­
m unoassay w ill m easu re 5 to 10 n g p e r ml
o f s e ru m .5,13,23 T h e h e m a g g lu tin a tio n
m eth o d w ill d e te c t an d m easu re from 0.3
to 1 ¡xg o f m yoglobin or h em o g lo b in p er
ml of u rin e. T h e im m unodiffusion and
im m u n o electro p h o resis system s are g en ­
erally p o sitiv e w ith 5 to 10 ¡xg p e r ml. T he
o ccu lt blood sticks w ill d e te c t from 1 to 10
fig of h em o g lo b in or m yoglobin p e r m l of
u rin e, b u t this is w ith o u t d ifferentiation of
th e tw o pig m en ts. It w o u ld b e expected
th at w h e n co m b in ed w ith th e stick test,
th e am m onium sulfate p re cip ita tio n and
th e ultrafiltratio n m ethods should b e able
to d e te c t 10 ¡xg o f h em oglobin or m yoglo­
b in p e r ml. C h u 9 h ow ever, found a m ini­
m al d etec tab le lev el o f 50 ¡xg p e r ml w ith
th e a m m o n iu m s u lf a te p r e c ip ita tio n
m ethod.
N orm al L evels
T h e n o rm al lev els o f h em o g lo b in or
m y o g lo b in in u rin e h a v e n e v e r b e e n
c le a rly d e fin e d . F ro m o u r e x p e rie n c e
w ith th e h e m a g g lu tin a tio n in h ib itio n
m ethods an d th e stick test, it was found
th at th e norm al am o u n t o f th e p igm ents in
u rin e is less th an 0.3 (xg p e r ml. W ith vig­
o rous m u sc u la r ac tiv ity , m y o g lo b in in
u rin e som etim es increases to d etec tab le
le v e ls . S a ra n c h a k a n d B e r n s te in 22 re ­
p o r te d u s in g r a d ia l im m u n o d iffu s io n
497
norm al levels to b e b elo w 5 ¡xg p e r m l and
fo llo w in g m y o c a rd ia l in fa rc tio n to b e
above 50 /xg p e r ml. B oth o f th ese n u m ­
bers are 10 tim es h ig h e r th an ours. It is
b e lie v e d this is a reflection of an erro r in
standards.
U sin g th e rad io im m u n o assay , it was
found in o u r laboratory th at th e levels of
m yoglobin in th e serum o f 10 norm al p e r­
sons ra n g ed from 0 to 69 ng p e r m l w ith a
m ean v alue of 26 an d a standard deviation
o f 26. T h is is in a g reem en t w ith values
re p o rte d b y Ju tz y 13 an d by S tone.23 T h ere
is a w id e variation in re p o rte d hem oglo­
b in levels in p lasm a d e p e n d in g on th e
care w ith w h ich th e blood is co llected an d
th e m eth o d o f assay. N a u m a n n 19 re p o rted
values o f 0.3 to 2.5 m g p e r dl w ith an aver­
age of 1.3 m g p e r dl ± 0.12 S.D.
O r ig in a lly , o u r im m u n o c h e m ic a l
m eth o d s w e re d e v e lo p e d b e c a u s e th e
q u estio n was asked, “ W hy are th e occult
b lo o d tests p ositive a n d y et no re d blood
cells are see n u n d e r m icroscopic exam ina­
tio n ?” L o n g fie ld 17 re p o rte d th at o u t of
2,700 ro u tin e hospital urines, 145 gave
po sitiv e o ccu lt blood reactions w h e n no
re d blood cell or only an occasional cell
was seen. In 1962,1 it was re p o rte d by us
th at o f 2,050 u rin e specim ens, 212 con­
ta in e d o cc u lt b lo o d a n d h ad su fficien t
volum e for fu rth e r study. By b eh a v io r of
the reactive m aterial on ultrafiltration, ion
exchange cellu lo se, co precipitation w ith
alb u m in by am m onium sulfate, and p re ­
c ip ita tio n w ith n an o n o ic a n d d ec an o ic
ac id fo llo w e d b y e x a m in a tio n o f th e ir
spectra, circu m stan tial ev id en c e o f the
p re sen ce of hem o g lo b in was obtained.
In re tro sp ect, it is now re a liz e d th at
som e of th e u rin es m ay have co n tain ed
m yoglobin. In th e p ast tw o years, 6,888
u rin e sp ecim en s w ere te ste d for occult
blood by a research laboratory at a local
hospital. O f th e s e specim ens, 1,025 gave
p o sitiv e o cc u lt b lo o d tests a n d 187 o f
th ese w ere show n b y the h em ag g lu tin a­
tion te st to contain m yoglobin.
M ost o f th e stu d ies have b e e n carried
o u t to sh o w m y o g lo b in u ria fo llo w in g
498 ADAMS
m y o c a rd ia l in fa rc tio n . In ta b le I are
show n th e p o sitiv es o b tain ed w ith th e var­
ious stu d ies. T h e stu d ies are not directly
com p arab le to each o th er since th e re a­
gents a n d th e form o f th e te st have varied
o ver th e p erio d o f tim e. T h e diagnosis of
m yocardial infarction was b a se d on clin i­
cal sym ptom s, E C G changes, an d enzym e
elevations, b u t u ltim ately on th e w illing­
n ess o f th e p artic ip a tin g cardiologist to
com m it h im self. W hen o th er conditions
know n to cause m y o globinuria are noted,
p o sitiv e tests in th e ab sen ce o f m yocardial
infarction are n eg lig ib le.
In a p rev io u s re p o rt on th e radioim ­
m unoassay for seru m m yoglobin,5 th ere
was a scattergram show ing th e d istrib u ­
tion of values o f serum s from norm al p e r­
so n s, h o s p i t a l iz e d p a tie n ts w ith o u t
m yocardial infarction, an d p atien ts w ith
d iag n o sed m yocardial infarction. N in e ­
te e n o f th e 24 p atien ts d iag n o sed as hav­
in g m yocardial infarction h ad at least one
elev a ted seru m m yoglobin. F o r tw o o f the
fiv e p a tie n ts w ith o u t e le v a te d se ru m
m y o g lo b in , th e se ru m e n z y m e s (total
CPK, SG O T, L D H ) w ere not elev ated , the
u rin e m yoglobin w as negative, and the
E C G w as q u e s tio n a b le ; h o w e v e r, th e
diagnosis was m yocardial infarction. F or
th e o th er th re e p atien ts w ith o u t e lev a ted
serum m yoglobin, it is likely th e critical
sera w ere not available. V alues as hig h as
1120 ng p e r m l w ere found w ith m ost
b ein g in th e 300 to 500 ng p e r m l range.
W hen th e tim e o f m yocardial infarction
TABLE I D u rin g th e various studies w e, or our
Myoglobinuria in Myocardial Infarction
Number Percent with
o f Cases
Institution Myoglobinuria
EH-12 44 89
EH-2 24 83
LB 7 86
B 12 33 67
P - l 15 13 85
P-2 32 84
B H 16 37 92
MM 3 100
K-l7 8 88
K-2 17 95
co u ld b e e sta b lish e d by an objective sign
such as o n se t o f ch est pain, it was show n
th at th e serum m yoglobin lev el rose in th e
first six hours, p e a k e d in ab o u t 12 hours,
an d fell to norm al w ith in 24 hours. In
som e cases th e serum m yoglobin level
re m a in e d elev a ted , in d icatin g c o n tin u ed
dam age to th e heart. In o th er cases, th e
lev el p ea k ed , fell to norm al, an d th e n later
rose again,— pro b ab ly in d icatin g a n ew at­
tack. S o m e tim e s th e re w e re e le v a te d
levels o f m yoglobin in the serum draw n
on adm ission. T h is suggests th a t th e in ­
farction h ad occurred som e tim e e a rlier
an d w as gen erally confirm ed by th e c lin i­
cal history.
T h e im portance o f th e tim ing o f th e col­
lection o f th e sp ecim en was also see n in
all th e stu d ies w ith d etec tio n o f urinary
m yoglobin. U sually, th e am o u n t of uri­
nary m yoglobin in th e first 12 hours fol­
low ing th e in su lt was greater th a n in th e
second 12 hours. F o r 15 of th e 24 p atien ts
w ith m yocardial infarction, it w as ju d g e d
th at b o th u rin e an d serum h ad b e e n col­
lecte d at th e critical tim es. T h e sera was
assa y ed b y ra d io im m u n o assay a n d th e
u rin e s by h e m ag g lu tin atio n in h ib itio n .
F o u rte e n of th e p atien ts h ad m yoglobin in
both seru m an d urin e. O ne h ad m yoglobin
in serum , b u t n o t in th e u rin e. T his m ay
reflect th e differences in sen sitiv ities o f
th e m ethods.
A variety o f conditions resulting in myo-
globinem ia and m yoglobinuria or in hem o-
g lo b in e m ia a n d h e m o g lo b in u ria w e re
liste d in ea rlie r p u b licatio n s.3' 10’14’20,21
c o o p e ra tin g in v e stig a to rs , h a v e n o te d
m y o g lo b in u r ia in c a s e s o f d e rm a to -
m yositis, trichinosis, fever an d virus in ­
f e c tio n s , a r te r ia l o c c lu s io n , c a rb o n
m o n o x id e in to x ic a tio n , c ru sh in ju rie s
(both m yoglobin an d hem oglobin), drug
o verdose, h e ro in addiction, collagen vas­
cular d isease, m yositis, alcoholic binges,
m y asth en ia gravis w ith collagen disease,
electrical shock, m alignant h y p erte n sio n ,
d iso rd er o f m uscle lip id m etabolism , an d
h ea rt an d arterial surgery. Som e o f th ese
 M Y O G LO B IN A ND H E M O G L O B IN D IF F E R E N T IA T E D IN B IO L O G IC A L F L U ID S
h av e b e e n associated w ith ren al failu re.10
In th e case o f th e p a tie n t w ith trichinosis,
en o u g h seru m m yoglobin b ecam e availa­
b le , ow ing to ren al shutdow n, th a t the
seru m co uld be d ilu te d to give a positive
in d icatio n in th e h em ag g lu tin atio n test.
T h e u rin e o f this p a tie n t w as re p o rte d to
co n tain only h em o globin b y a laboratory
u sin g th e am m o n ium sulfete so lu b ility
m eth o d . T h e im m unochem ical m ethods
sh o w ed only m yoglobin.
T h e p re se n c e of m yoglobin in kid n ey
slices from one crush injury victim was
show n by th e in d irec t flu o re scen t stain. It
w as n o t p o ssib le to fin d m yoglobin in the
u rin e from dy stro p hy p atien ts. I t m ay b e
th e se p atien ts h ad already lost too m uch
m u scle m ass and th u s w e re n o t secretin g
d e te c ta b le am ounts of m yoglobin.
T h e im m u n o ch em ical tests for hem o ­
g lo b in w ere u se d to d ifferen tiate hem o ­
g lo b in o f en d o g e n o u s o rigin from gas­
tro in testin a l b le e d in g from th e m yoglobin
a n d hem o g lo b in o f dietary origin.4
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