COPPER
DEFICIENCY
IN
INFANCY
Angel Cordano, M.D., Juan M.
Baertl,
M.D., and George G. Graham, M.D.Departtnent of
Research,
British American Hospital, Lima, Peru, and Pan American Health Organization(Submitted December 23, 1963; accepted for publication May 7, 1964.)
This investigation was supported by grants Nos. AM-04635 and 05935 from the National Institutes
of Health, U. S. Public Health Service.
ADDRESS: (G.G.G.) British American Hospital, Apartado 2713, Lima. Peru.
PEDIATRICs, September 1964
324
-I_’
LETARY copper deficiency in animals.LJ
and the role of Cu in erythropoesisand bone metabolism have been
exten-sively 2 hi 1931 Josephs3
re-ported the probable existence of
simultane-ous Fe and Cu deficiencies in milk-fed
in-fants. Subsequently this was questioned
and modern textbooks state that dietary Cu
deficiency has never been documented in
humans.
A series
of
reports
exist
of
in-fants with anemia, hypoproteinemia,
hypo-ferremia, and hypocupremia which were
felt to be of dietary origin.8#{176} Recent work
suggests that most of these belong in the
category of exudative enteropathy.h1
Cart-wright mentions two patients with
non-tropical sprue who had anemia and
hypo-cupremia, but he was not able to make a
proper therapeutic trial.12
We have observed 4 infants, recovering
from marasmus on exclusive milk diets,
de-velop Cu deficiency, with many of the
characteristics of that produced
experi-mentally in pigs.13 It is probable that they
had long-standing intestinal losses due to
chronic diarrhea and intestinal
malabsorp-tion as well as poor intakes and that, when
growth was rapidly accelerated on a diet
of low Cu content, they developed overt
signs of deficiency.
Case 1
AC. is a mestizo male, born at term, weight
unknown, who was breast fed for 15 days and
then supposedly given evaporated cow’s milk until
1 month of age, when he developed vomiting and
diarrhea, was given acid skim milk and then again
evaporated milk, in very small quantities. At 7
months he started to receive broth and occasional
small amounts of potato and squash. Diarrhea was
almost continuously present from age 1 month,
anorexia from 6 months. At age 8 months, two
days prior to admission, he became obviously
fe-brile and vomited all feedings; the following day
he developed severe diarrhea and was noted to
be flaccid and stuporous. He was severely
under-nourished, subcutaneous fat almost absent, muscle
mass much decreased, and there were signs of
dehydration with decreased muscle tone and deep
tendon reflexes. Weight was 4.780 kg, length 62.3
cm. Shortly after admission flaccid paralysis of
the left leg and some of the back muscles was
noted; the subsequent course suggested that this acute illness was poliomyelitis. On the 171st
hos-pital day, he suffered a pathological fracture of
the right femur.
DuET: By the 3rd day he tolerated modified cow’s milk and by the 5th was getting 2 gm protein and
75 calories per kilogram of body weight per day
(cal/kg/day) from this preparation.#{176} For 6 months
protein intake was maintained at the same level,
recalculated daily. By the 11th day caloric intake
was increased to 100/kg/day by adding cane
sugar and cottonseed out to the milk and by the
47th day to 175 cal/kg/day. On the 99th day it
was reduced to 150 and on the 121st to 125/kg/
day. On the 182nd day protein intake was
in-creased to 3 gm/kg/day, keeping caloric intake
at 125/kg/day.
Although the milk contained added Fe, the
relatively small amounts given yielded only 1.4
mg elemental Fe and 28 tg Cu/kg/day during
the first 182 days and 2.1 mg Fe and 42 tsg Cu/ kg/day thereafter. An additional 90 mg Fe/days were given from the 17th to the 49th day, when
it was decreased to 45 and on the 199th day to
15 mg/day. On that day and the next he was
given 50 mg Fe intramuscularly. On the 207th
day he was started on 10 drops daily of 1% CuSO4
solution yielding 2.5 mg Cu/day, which added to
Similac with Iron, supplied by Ross Labora-tories, Columbus, Ohio.
I
Supplied by Anderson, Clayton & Co., Lima, Peru.t
Fer-in-sol, supplied by Mead JohnsonIn-ternational, Evansville, Indiana.
Imferon, supplied by Fisons Overseas, Ltd.,
ARTICLES
325the amount in the milk gave a total of 362 pig!
kg/day. On the 216th day it was decreased to 104
and on the 225th it was increased to 167 g/k
day.
During 103 days, he received only the vitamins
in the milk, which in the amounts used should
yield 25 to 30 mg of vitamin C, 1,300 units of
vitamin A, 220 units of vitamin D, 0.35 mg of
vitamin B1 and 0.55 mg of riboflavin daily. On
the 103rd day a supplement of 50 mg/day of
ascorbic acid was added. On the 109th day this
was replaced by 1.2 ml of a multivitamin prepar-ation,#{176}0.6 ml of which yield 50 mg ascorbic acid,
5,000 U vitamin A, 1,000 U vitamin D, 1.0 mg
thiamine HC1, 1.2 mg riboflavin, 1.0 mg
pyridox-me HCI, 5 mg pantothenic acid, and 10 mg of
nicotinamide. On the 127th day this supplement
was halved. From the 168th to the 177th day he
was given 50 mg/day of ascorbic acid
intramuscu-laxly. On the 141st day 10 mg/day of folic acidf
were added and on the 162nd day this was
re-duced to 5 mg daily.
LABORATORY STUDIES (Fig. 1): Body weight,
polymorphonuclear (PMN) count, hematocrit (Hct),
reticulocytes (Ret), and pertinent hematinic treat-ment have been plotted against age and hospital
days. On the 45th day PMN count had dropped to
350/mm3 and total white blood cell (WBC) count was 5,050/mm3. During the next 168 days WBC
varied between 3,500 and 5,150 and PMN
be-tween 70 and 650/mm3, with the exception of a
single determination on the 193rd day of 6,500
WBC and 2,665 PMN associated with an acute
infection. On the 213th day, 6 days after
supple-mental Cu was started, WBC rose to 6,300 and
PMN to 900 (17%); thereafter there was a rapid
rise to normal values of more than 6,000 WBC’s
and 2,000 PMN’s/mm. On the 222nd day, 5 days
after decreasing the intake of Cu, WBC was 5,600
with 30% PMN (1,680/mm3); on the 225th day,
6,000 with 12% PMN (720) and on the 228th day,
4,500 with 10% PMN (450). On the 232nd day,
7 days after doubling the supplemental Cu, WBC
rose to 5,800 with 34% PMN (1,970/mm3) and
thereafter remained at normal levels.
On admission, Hct was 36% and Hgb 10.65 gm %. On the 85th day they started to drop rapidly
to low values of 16% and 4.75 gm by the 117th
day, 14 days after increasing the intake of
as-corbic acid and 8 days after adding the
multi-vitamin preparation, from which point they rose
slowly to a value of 20% and 6.5 gm on the 140th
day. After the addition of folic acid, Hct and
Hgb rose steadily to 30% and 8.91 gm on the
163rd to again fall slowly to 21% and 7.0 gm on
#{176}Abdecol, supplied by Parke, Davis & Co., Lima, Peru.
f
Folvite, supplied by Lederle Laboratories,Pearl River, N.Y.
A#{149}g#{149}
:
PMN1O3
Hct. 1.
Ret.’!. -
-100
Fe mg .
CUPWk
_________________________
9- 0 -.-.-.-.-.-m1/fI/III/I/A
Vit.Crng.125T
rA.mg. 10T
,,,,-Agemonths
?
, 1 ,9
1 Hosp- days 0 60 120 180 240FIG. 1 (Case 1). Evolution of body weight (Wt),
polymorphonuclear neutrophils (PMN), hematocrit (Hct), and reticulocytes (Ret) during recovery from
marasmus on a high-calorie, low-copper diet.
In-take of iron (Fe) in mg/day, copper (Cu) in jsg/
kg/day, vitamin C in mg/day and folic acid (F.A.)
in mg/day are also shown in relation to the
chronologic age in months and the number of days after admission.
the 207th day, when Cu supplementation was
started. In the next 13 days they rose to 33% and
10 gm and from that day until the 262nd were
never below 33% and 10.0 gm or above 35% and
11.0 gm. He was then changed to a mixed diet
and Hct and Hgb rose further to stable values of
38% and 12 gm. At its lowest on the 120th day
the red blood cell (RBC) count was 1.34 million/
mm3; on the 140th day it had risen to 2.14
mil-lion. Following treatment with folic acid it rose
to over 3 million but by the 207th day had again
fallen to 2.67 million. Fifteen days after Cu was started it had risen to 4.0 million.
Microscopic examination of the blood for 103
days showed slight hypochromia and anisocytosis.
Thirteen days after increasing ascorbic acid
in-take, there was no hypochromia, some
macrocyto-sis, and moderate anisocytosis. Immediately
poly-chromatophilia; 49 days later there was very slight hypochromia and anisocytosis. Nine days
after the addition of Cu, there was very slight
hypochromia and anisocytosis; 6 days later the
smear was normal. Platelets were adequate at all
times.
Reticulocytes were below 1.4% until 5 days
after ascorbic acid was increased to 75 mg/day, when there was a rise to 3.9%. Two days after folic
acid was first given they again rose to 4.1% and
stayed near that level for about 1 week, then fell
below 1% until 3 days after Cu was started, when
they rose to 2.1%. On the 213th day they were
4.9% and on the 217th day, 4.2%. After this they fell to less than 0.5%.
Biopsy of bone marrow on the 116th day
re-vealed some diminution of erythroid elements,
conservation of normal maturation proportions and
a preponderance of orthochromatic normoblasts
over more juvenile elements. The granulocytic
series revealed a quantitative diminution of its
elements with a predominance of mature
ele-ments; there was an increase in the proportion of
lymphocytes. Repeat biopsy on the 247th day
(40 days after Cu was given) revealed marked
regenerative hyperplasia. Red cells had elements in all stages of development, with a preponderance of orthochromatic normoblasts. The myeloid series, equally hyperplastic, had a preponderance of meta-myelocytes and non-segmented neutrophils.
Mega-karyocytes were normal.
On the 207th day serum Cu was 68 pig/100
ml; on the 288th day it was 168 g/100 mi.#{176}
BONE: Radiologic appearance was normal until
the 161st day when widening and irregularity of
the left distal ulnar metaphysis were noted, with bony spurs on the medial and lateral aspects (Fig.
2a). The right wrist was similar. The knees
re-vealed loss of density and pattern of the
can-cellous bone of the femora and tibiae, with a
“ground-glass” appearance. The cortex of the
epiph-yses was very thin and more obvious than
usual, giving a ring-like appearance. On the 171st
day x-ray of the right femur (Fig. 2b) revealed
an incomplete anterior fracture 1 cm above the
distal metaphysis. Three bony spurs were also
noted on the posterior aspect of the femoral
me-taphysis. The left wrist on the 193rd day showed
widening of the ulnar metaphysis due to the
ex-ternal spur, persistent irregularity, and also loss of density in the medial aspect of the radial
me-taphysis. The right femur and knee on the 203rd day (Fig. 2c) revealed consolidation of the fracture
with hard callus and periosteal reaction extending
#{176}Serum copper determinations were kindly
per-formed by Miss Louise Gray, U.S. Plant, Soil and
Animal Nutrition Laboratories, Ithaca, N.Y.
Av-erage normal adult values in her laboratory are
109 ± 17 pig/100 ml.
to the mid-shaft. On the 215th, 8 days after Cu was begun, there was increased density and osse-ous trabeculation in the femoral epiphysis. On the
218th (Fig. 2d) there was bony union between
the ulnar metaphysis and the spur. Irregularity of
the ulnar metaphysis and loss of density of the
radial metaphysis persisted. On the 245th the
ossification centers of the knee were much more
clearly delineated, the fracture better consolidated
and there was some degree of osteoscierosis of
the metaphysis and neighboring areas of the
diaph-ysis of the left femur. Both tibiae revealed
growth arrest lines. The ulnar metaphysis lacked
lineal demarcation and had a hazy appearance.
On the 274th day there were no structural or
morphologic alterations of either knee.
SUMMARY: A.C. was rehabilitated on a diet of
milk with added sugar and oil. For the first 182
days this yielded 28 g and for the next 25 days,
42 g Cu/kg/day. Weight gain was satisfactory
and in accord with his caloric intake.14
By the 45th day moderate leukopenia, with
marked neutropenia, was evident and persisted
until the 213th day with the exception of one
brief neutrophilic response to an active infection. Six days after dietary Cu was raised to 362 pig/kg/
day there was evident a rapid increase in
neu-trophils. When this was reduced to 104 pig there
was a drop in neutrophils and when it was raised
to 167 g, they again increased to normal values.
The bone marrow changed from moderate
ma-turation arrest and hypoplasia to pronounced hy-perplasia.
Despite early administration of Fe, after the
85th day a severe, predominantly normocytic and
normochromic anemia developed. There was a
fair reticulocytic and hematologic response to
as-corbic acid and a further, incomplete response
to folic acid. When Cu intake was increased,
there was a prompt but moderate reticulocyte response and a rapid climb to nearly normal values
in Hgb, Hct, and RBC. When he was given a full
diet there was a further increase.
Five months after admission marked osteoporosis
of bone and changes reminiscent of scurvy
be-came apparent by x-ray. Shortly thereafter he
suffered a pathological fracture which healed with callus formation. Promptly after the administration of Cu, increased density of bone and healing of
the abnormal changes became evident. During
the period of Cu deficiency there was practically
no advance in radiologic “bone age”; after treat-ment there was a rapid advance.
Case 2
M.M. is a mestizo female, one of twins, who
was breast fed for a few days and then changed
to very dilute evaporated milk. At 6 months she
started to receive clear broth and occasional
a
b
fs
‘9
d
r
1
FR;. 2 (Case 1). (a) X-ray’ of left wrist 161 (lays after admission showing widening and irregularity of the ulnar motaphysis with bony spurs on the medial and lateral aspects. (b) X-ray of the right knee 171
days after admission showing incomplete fracture above the distal femoral metaphvsis as well as bony spurs on its posterior aspect. Also “ground-glass” appearanCe of long bones and “ring-like” epiphyses. (c) Right knee on the 203rd day shows consolidation of fracture with hard callus and periosteal reaction;
no change in the epiphyses. (d) Left wrist on 218th day reveals marked improvement.
hospital with a weight of 3 kg, after a 3-week
history of diarrhea and vomiting. She was given
acid whole milk with added casein, FeSO,, and
multivitamins. She had positive Kahn and
Maz-zini reactions; x-ravs of the long bones were
re-portedlv normal. She reached 4.75 kg by the time
she was transferred to our hospital.
On admission, age 14h months, she was alert,
had a protuberant abdomen, could not sit, and was grossly undernourished, with very scanty sub-cutaneous fat and muscle mass. She developed gradually increasing pallor and some increased pigmentation and roughness of the skin overlying
the malar areas of the face. On the 124th hospital day she received a transfusion of 120 ml of
whole blood. Because her serological tests for
syphilis were positive on admission, she was given penicillin for 9 days. Because of a positive
tuber-culin test and x-ray evidence of primary
pulmo-nary tuberculosis she was given isoniazid, 75 mg/
day orally, from the 6th to the 89th day, when it was discontinued because of unexplained low-grade fever and progressive anemia.
DIET: On admission she was placed on modified
cow’s milk yielding 2 gm protein and 75 cal!
4+
PMN.103
2-caloric intake was gradually raised to 175 cal/
kg/day by the 38th day. On the 73rd day it was
reduced to 150 cal/kg/day. Protein intake
re-mained at 2 gm/kg/day until the 123rd day when
it was increased to 3 gm by the addition of
casein.#{176} On the 141st day the caloric intake was
reduced to 100 cal/kg/day. On the 190th day it
was raised to 125 cal/kg/day and maintained at
this level, with 3 gui protein/kg/day, until the
289th day (age 2 yr) when she weighed 8.54 kg
and had a length of 70 cm.
She received an estimated 1.4 mg Fe and 28
g Cu/kg/day from the milk. On the 50th day
90 mg/day of Fe were added; on the 64th day
this was reduced to 45 mg/day. On the 214th day
she received 40 mg and on the 215th, 50 mg of
Fe intramuscularly. On the 229th day a daily
sup-plement of 2.4 mg Cu was started, bringing her
intake to 378 jig/kg/day. Ten days later it was
reduced to 168 pig/kg/day.
Until the 94th day she received the vitamins
present in the milk (cf. Case 1). She was then
given 25 mg/day of pyridoxine HC1 for 10 days,
100 mg/day for 12 days and 20 mg/day for 27
days. On the 108th day she was started on 10
mg/day of folic acid, on the 142nd it was
de-creased to 5, on the 181st to 2.5 and on the 279th to 0.3 mg/day. After the 117th day she received 75 mg or more of ascorbic acid daily.
LBORATORY Snmis (Fig. 3): By the 94th
day, PMN’s had dropped to 376/mm3 and until
the 229th day, when Cu was started, varied
be-tween 43 and 675/mm3. On the 232nd day there
were 448/mm3 (7% of 6,400); on the 235th there were 2,263/mm3 (31% of 7,300). After that, PMN’s
were never below 1,728/mm3 and WBC’s never below 6,000/mm3.
On admission, Hct was 32% and Hgb 9.6 gm;
rose to 38 and 10.65 on the 27th; 34 and 9.6 on
the 64th and then dropped to 12% and 3.48 gin by the 12.4th day, when a transfusion was given. The
following day they were 27% and 7.94 gin; rose to
30% and 9.6 gin in a few days, and then fell slowly to 20% and 7 gin by the 228th day. Seven days after Cu was started, they had risen to 32.5% and 9.0 gm
and then remained between 31 and 10.0 and 35%
and 11.0 gin. On a varied diet, Hct was between
35 and 37%. At its lowest, on the 124th day, RBC
was 1 million/mm. Ten days after transfusion it
was 3 million, but by the 228th day had fallen to
2.2 million. On the 3rd day of Cu supplementation,
it was 2.5 million; in another 3 days it was 2.7 mil-lion and in another 5 days, 3.3 million. It then rose gradually to approximately 4 million.
Blood smear was normal the 72nd day, when
moderate hypochromia and anisocytosis were
evi-dent. From the 106th to the 123rd days
anisocy-o Casec, supplied by Mead Johnson
Interna-tional, Evansville, Indiana.
::. ‘:i
Hct- ‘I.
Ret.’!.
Fe mg -
‘F
277M77777I h72772____]300
Cu,isqfkg .
Vit.Cmg. ‘25T
F.A.mg. 10T 77fl)rmv,- 1
Agemonths p1.5 , 17 1 21 2,3
Hosp. dzys 50 110 170 230 290
Fic. 3 (Case 2). Evolution of body weight (Wt),
polymorphonuclears (PMN), hematocrit (Hct) and reticulocytes (Ret)) during recovery from marasmus on a high-calorie, low-copper diet. Intake of iron
(Fe) in mg/kg/day, copper (Cu) in pig/kg/day,
vitamin C in mg/day and folic acid (F.A.) in
mg/day are also shown in relation to age in
months and hospital days. The arrow indicates
transfusion of 120 ml of blood.
tosis became more marked and there was moderate
macrocytosis; hypochromia became severe. After
transfusion red cells were normal until the 204th
day, when there was moderate hypochromia and
some anisocytosis. On the 232nd day there was
pronounced hypochromia, some
polychromato-philia, and moderate anisocytosis and macrocyto-sis, this last probably related to reticulocytosis. Five days later there was only slight anisocytosis and thereafter there were no abnormalities. Plate-lets were adequate at all times.
Reticulocytes were below 1.3% until the 232nd
day, 3 days after Cu was started, when they rose
to 3.9%, with 7% nucleated RBC’s. On day 235
reticulocytes were 6.2%, nucleated RBC 3%.
Bone marrow on the 94th day revealed
eryth-roid hypoplasia with questionable early megalo.. blastic changes and some cytoplasmic vacuolation, maturation arrest of the myeloid series with some
giant metamyelocytes and marked lymphocytic
re-ARTICLES
vealed a normal myeloid series and some
eryth-roid hyperplasia. On the 260th day, 31 days
after Cu was begun, there was marked
hyper-plasia, particularly myeloid, with cells in all stages
of maturation, predominantly PMN’s; slight
in-crease in eosinophilic PMN’s, and normal
mega-karyocytes. Erythroid elements were hyperplastic,
with a predominance of orthochromatic
normo-blasts.
On the 95th day serum Cu was 119 pig/100
ml; on the 222nd day it was 72 ig/100 ml; on
the 296th day it was 170 pig/100 ml.
BONE: The day after admission x-rays revealed,
in the right tibia, hyperostoses, particularly on the internal surface of the diaphysis, as well as small, rounded, osteolytic images in its proximal third.
There was another elongated osteolytic image on
the external border of the previously mentioned
area of hyperostoses. These findings were felt to
be due to congenital lues. On the 178th day
there was osteoporosis and ring-like distal femoral epiphyses as well as several lines of growth arrest.
On the 204th day there was irregularity of the
left distal metaphysis. On the 233rd day there
was very slight irregularity within the radial
metaphysis and a normal ulnar metaphysis and
ulnar and radial diaphyses. On the 241st day
there was bony trabeculation apparent within the
femoral epiphysis but its density was still
sub-normal. On the 260th day the bony density and
pattern of the femoral epiphyses, ulnar and radial metaphyses were normal.
SUMMARY: M.M. was rehabilitated on a diet of
milk with sugar and oil which yielded 28 g of
Cu/kg/day. Gain in weight was satisfactory and
gain in length was slow. Marked neutropenia and
severe anemia developed by the 94th day.
Trans-fusion on the 124th day temporarily corrected
the anemia but not the neutropenia. Iron,
pyri-doxine, folic acid, ascorbic acid, and a
multivita-mm preparation were of no benefit; increasing
the Cu intake to 378 pig/kg/day produced a
prompt and dramatic response in neutrophils and
erythrocytes. Bone changes were present by the
178th day and returned to normal within 31 days
after Cu supplementation.
Case 3
CM. is the twin of Case 2, with identical past
and feeding histories. When admitted to another
hospital at the age of 12% months she weighed
3.625 kg. She also received acid whole milk and
1 month later weighed 4.24 kg. Her serological
tests for syphilis were positive.
At the time of transfer, at age 14% months, she weighed 4.34 kg and was 60 cm long. Her clinical
conditions and course were similar to those of
her twin, including pallor and brawny
pigmenta-tion of the skin and low-grade fever from the
86th to the 289th days. She did not receive a
blood transfusion. She was treated with penicillin for 9 days because of positive serological tests for
syphilis and with isoniazid, 75 mg/day orally,
from the 5th to the 89th hospital days.
DWT: She was given modified cow’s milk
yield-ing 2 gin protein and 75 cal/kg/day. On the 5th
day the caloric intake was raised to 100, and by
the 38th day to 175 cal/kg/day. On the 129th
day it was reduced to 150/kg/day and the protein
intake raised to 3 gm/kg/day by the addition
of casein. On the 141st day calories were reduced
to 100/kg/day protein intake remaining at 3
gm/kg/day. On the 158th the caloric intake was
increased to 125 and on the 190th day to 150
cal/kg/day. On the 258th day it was reduced to
125 cal/kg/day. On the 289th day, at age 2 years,
she weighed 9.86 kg and had a length of 70.5 cm.
Until the 289th day she received no more than
1.4 mg of Fe and 28 pig Cu/kg/day from the
milk. On the 41st day an additional 90 mg/day of Fe were added; on the 79th day this was reduced
to 45 and on the 214th to 15 mg/day. On the
115th day she received 100 mg of Fe
intramus-cularly. On the 214th, 215th, and 216th days she
received 25, 25, and 50 mg of Fe intramuscularly.
From the 263rd to the 272nd days she received
a daily supplement of 2.5 mg Cu, bringing her
intake to just over 300 pig/kg/day. On the 273rd
day this was reduced to 135 pig/kg/day.
For 94 days she received only the vitamins in
the milk (cf. Cases 1 and 2). She was then given
25 mg pyridoxine HC1 daily for 14 days, then
20 mg/day for 47 days. On the 124th day she
was started on a daily supplement of 1.2 ml of
the same multivitamins used in the other cases,
raising intake of ascorbic acid from 25 to 125
mg/day. On the 142nd day it was reduced to
0.6 nil/day. From the 173rd to the 177th day she
received daily intramuscularly 1 ml of a
multi-vitamin preparation containing 5 pig of
cyano-cobalamin and 100 mg ascorbic acid. On the
156th day she started receiving 10 mg/day of
folk acid; on the 177th day this was reduced to
5 mg/day. From the 229th to the 233rd days she
received 50 pig/day of vitamin B12 intramuscularly. LABORATORY STUDIES (Fig. 4): By the 94th day,
PMN’s had dropped to 960/mm3 and by the 115th
to 275. Thereafter they varied between 157 and
546 until the 239th day. There was one isolated
rise to 7,500 WBC, with 10% PMN, on the 193rd
day, associated with a mild diarrheal episode. On
the 239th day, 5 days after completing the 5-day
course of B12, PMN’s rose to 600 (17% of 3,500); on the 2,46th day to 650; on the 251st to 728;
on the 257th to 1,155 and on the 263rd day to
1,350/mm3 (27% of 5,000). On the 267th day,
4 days after beginning Cu, there was a spurt to
2,016 PMN’s, and thereafter a prompt rise to
normal values: 40 to 50% of 6 to 7,000 WBC’s.
Cu 1lkg.
j
125
Vit.Cmg. 0
some macrocytosis possibly due to reticulocytosis;
after the 173rd day slight hypochromia, marked
anisocytosis, some macrocytosis, and discreet poi-kiocytosis; on the 187th day slight hypochromia, moderate anisocytosis, and slight poikilocytosis.
The morphology then remained unchanged until
the 242nd day when it was judged normal.
Plate-lets were adequate.
Reticulocytes were below 1.2% until the 134th
day, 10 days after being started on multivitamins, when there was a rise to 3.3%, sustained between
1.6 and 4.7% until the 193rd day. There was a
very slight rise from 1.3 to 2.2% when B12 was
given. After Cu, reticulocytes remained above 1%
for 11 days and then dropped to 0.5% or less.
Bone marrow on the 94th day showed
matura-_______________________________ tion arrest of the myeloid series, with marked
lymphocytic infiltration. The erythroid series was
.. . - --J quantitatively decreased, with cytoplasmic
vacuo-lation. The same marrow was interpreted by
an---
other observer as showing questionable earlymegaloblastic changes. Bone marrow on the 108th
showed erythroid hyperplasia and little change in
myeloid elements. On the 135th day, 6 days after
increasing protein intake, the bone marrow was
_________________________________ hyperplastic, erythroblastic, with a preponderance
of orthochromatic normoblasts. The myeloid series _________________________________ was less cellular, with inversion of
myeloid/eryth-roid ration. There was a preponderance of
mye-locytes and to a lesser degree of metamyelocytes. Mature elements were scarce. Biopsy after B12 and before Cu revealed active production of cellular
elements in both series. The myeloid had cells in
all phases of development, with a preponderance
of immature myelocytes, suggesting maturation
arrest. On the 277th day, 14 days after beginning
of Cu, the bone marrow revealed marked
hyper-plasia, predominantly neutrophilic, with very large numbers of mature, segmented neutrophils.
On the 95th day serum Cu was 72 pig/100 ml;
on the 222nd day it was 69 pig/nil; on the 296th
day it was 160 pig/100 ml.
BONE: On the 172nd day, radiologic appearance
of the wrist (Fig. 5a) was normal. On the 206th
day there was some irregularity of the distal
borders of the radial and ulnar metaphyses, with
minimal spur formation. On the 242nd day (Fig.
Sb) there was widening, irregularity, and fraying of the distal metaphyseal border of the ulna, with
lateral bony spurs; also an irregular lineal shadow
of calcification 1 to 2 mm below the medial half of the radial metaphysis. On the 261st day (Fig. 5c) there was some haziness of the ulnar and radial
metaphyses and some filling in of the space
be-tween the irregular area of calcification and the distal metaphysis of the radius. Part of the bony spur on the lateral aspect of the ulnar metaphysis was still present. On the 292nd day there still was
some haziness of the ulnar metaphysis alone. On
the 303rd day (Fig. 5d) the bony density and
C.
M.Wt. kg. 10
PMN,10’
‘I.
Ret. ‘I. ____.__
-lOOy
Fe mg. - t
FA.mg. 10i 977)
B’2’ig 50i - I _1
Age months 15 17 19 #{149}21
Hosp. days 0 50 110 170 230 29C
Fic. 4 (Case 3). Evolution of body weight (Wt),
polymorphonuclears (PMN), hematocrit (Hct), and
reticulocytes (Ret) during recovery from marasmus on a high-calorie, low-copper diet. Intake of iron
(Fe) in mg/day, copper (Cu) in pig/kg/day,
vita-mm C in mg/day, folic acid (F.A.) in mg/day and
vitamin B12 in pig/day are also shown in relation to age and hospital days.
on the 94th day, Hct fell to 27 and Hgb to 8.9
and then continued to fall steadily to a low of
14% and 4.5 gm on the 131st day, one week after the addition of multivitamins. From that point
they climbed slowly to 24% and 8.0 gm. Two days
after completing the 5 days of B12, Hct rose to
27 and Hgb to 8.6; then to 30% and 9.25 gm by
the 246th day; they remained near this level until
Cu supplementation after which they rose to
39% and 11.0 gm. RBC’s fell from 3.0 million to
1.45 million by the 134th day; rose slowly to
over 2.0 million by the 170th and to just under
3.0 million by the 233rd day. They then
in-creased to over 3.5 million after B12 and to 4.0 million after Cu.
Blood smear on admission was normal; by the
102nd day there was moderate hypochromia and
anisocytosis, some macrocytosis, and an occasional target cell; on the 118th day less hypochromia and
anisocytosis and fewer macrocytes; on the 134th
FIG. 5 (Case 3). (a) X-ray of the left wrist on the 172nd day was still essentially normal. (b) On the 242nd day there is definite widening, irregularity, fraying, and lateral bony spurs of the ulnar
metaph-ysis; also an irregular, lineal shadow of calcification just below the medial half of the radial metaphysis. (c) On the 261st day, one month after B12 treatment, there is still some haziness of the metaphyses.
(d) On the 303rd day, 2 weeks after Cu supplementation, the appearance is alniost normal.
structure of the ulnar and radial rnetaphyses were normal.
SUMMARY: CM. was rehabilitated on a diet
yielding no more than 28 g Cu/kg/day. Because
of intentionally planned additional caloric intake, she gained in weight and length at a faster rate than her twin. Marked neutropenia was evident by the 94th day and anemia became evident at the
same time, becoming most severe by the 131st day. There was an incomplete erythroid response, with reticulocytosis, to a multivitamin preparation, probably related to its ascorbic acid content. There was no clear response to folic acid and to small doses of B12. There was additional incomplete
erythroid response and fair myeloid response to
larger doses of B2, with little additional reticulocy-tosis.Cu produced a further erythroid and myeloid
response. Bone lesions became evident on the 206th day and improved with the B12 and Cu therapy.
Case
4
MG. is a mestizo male who weighed 2.3 kg at
birth and was breast fed for 2 months. At that age he started subsisting on rice water, oat water, and various herb teas. lie supposedly received 100 ml
2
0
“vomiting” until the age of 15 months, when he
was admitted because of undernutrition, weighing 3.24 kg and measuring 55.5 cm. He was not able to raise his head and sucked his hand continuously.
Physical examination revealed extreme
undemutri-tion and an increase in body hair but was other-wise unremarkable. His course was complicated by the fact that he was a classic “ruminator”; this was finally overcome after 3 months of consider-able personal attention. Pitting edema of the lower
extremities and hypoalbuminemia were apparent
from the 8th to the 63rd hospital days. He was
found to harbor giardia lamblia in the stool and
was treated with
chloro-methoxy-acridil-amino-diethyl-aminopropanol dichiorhydrate from the
42nd to the 46th days.
DIET: He received 2 gui protein/kg/day until
the 119th day, when it was raised to 3 gm/kg/day, all from the milk. Caloric intake was 50/kg/day until day 9; 75 kg/day until day 12; 100/kg/day
until the 26th day and 150/kg/day until the 83rd
day, when it was raised to 175/kg/day. On the
141st day it was decreased to 150 and on the 147th day to 125 cal/kg/day, at which level it remained
until the 222nd day, when he weighed 8.275 kg
and measured 64.5 cm.
For 118 days he received an estimated 1.4 mg
Fe and 28 g Cu/kg/day from the milk.
There-after he received approximately 2.1 mg Fe and 42
pig Cu/kg/day. From the 27th to the 179th days he received a supplement of 15 mg of Fe daily. On the 140th and again on the 141st days he received
50 mg of Fe intramuscularly. From the 189 to the
201st days Cu intake was 384 pig/kg/day and from the 202nd to the 222nd, 173 pig/kg/day.
For 64 days he received only the vitamins in the milk. He then received 0.6 ml daily of the same
multivitamins used in the previous 3 cases. From
the 65th to the 102nd days he received 10 mg a
day of folic acid and from the 103rd to the 222nd, 5 mg daily. From the 148th to the 152nd days he received 50 g daily of vitamin B intramuscu-larly.
LABORATORY STUDIES (Fig. 6): By the 75th day
PMN’s had dropped to 828/mm’ (18% of 4,600
WBC) and then remained between 45 and 440
until the 163rd day, 15 days after B12 was first
given, when they rose to 738. On the 166th day
they were 584; on the 169th, 1,428; on the 177th,
1,231; and on the 188th day, 1,743/nun’ (21% of
8,300). On the 191st day, 2 days after Cu was
started, they were 2,070 (30% of 6,900); on the
196th day, 3,108 (37% of 8,400); thereafter they
were always above 3,500/mm’.
During the first 60 days Hct was between 34
and 38% and Hgb above 10.0 gm. On the 65th day
Hct had fallen to 23%, Hgb to 7.32 gm. After folic
acid and the additional vitamins, Hct rose rapidly to 30% on the 69th day and then ranged between
28 and 30, with a Hgb of 8.91 until the 104th,
MG.
wt. kg.
PMNo1O3
Hct. ‘!.
Ret. ‘I.
F. mg
300
Cu Ag/kg. o L
Vi t. C mg. ‘
FA.mg. T 777Th-J . I
B12g. 50T i
Age months 15 17 19 21 23
I- , ‘ , ‘ . ‘ I
Hosp.days 30 90 150 210
FIG. 6 (Case 4). Body weight (Wt),
polymorpho-nuclears (PMN), hematocrit (Hct), and
reticulo-cytes (Ret) during recovery from marasmus on a
high-calorie, low-copper diet. Daily intake of iron (Fe) in mg, copper (Cu) in pig/kg, vitamin C in
mg, folic acid (F.A.) in mg and vitamin B12 in
pig are also shown in relation to age and hospital
days.
when it fell to 26, reaching a low of 16% with a
Hgb of 5 gin on the 146th day. After the adminis-tration of 1312 they rose steadily to 34% and 10.0 gm.
After Cu was added, Hct ranged between 34 and
36% and Hgb rose to 11.0 gm. On a full diet, Hct
rose further to 40% and Hgb to 12.1 gm. RBC’s
were 2.4 million on the 64th day. With folic acid and multivitamins they rose to over 3.0 million on the 75th day, only to fall again to a low of 1.7 mil-lion on the 148th. After B12 they rose steadily to 3.5, and with the further addition of Cu, to be-tween 3.7 and 4.0 million.
Red blood cells revealed moderate hypochromia, slight anisocytosis and polychromatophilia by the
70th day. Four days later the appearance was
ARTICLES
333after B12 was given, there was moderate anisocy-tosis, slight hypochromia, and discreet
polychroma-tophilia and macrocytosis. After this, there was
only slight anisocytosis; after Cu was given, the
morphology was normal. Platelets were always
ade-quate.
With the exception of a single value of 2.7% on the 54th day, reticulocytes were below 1.0% during the first 64 days; 2 days after folic acid was added
there was a rise to 2.5% on day 67, 3.2% on day
69, 4.3% on day 70 and 3.0% on day 75. Values
between 1.2 and 2.2% persisted until the 133rd day. After the administration of B2 there was a second sharp rise to 4.6% by the 161st day. They then
re-mained between 1 and 2% until 18 days after Cu
was started, when they fell to below 1% and
re-mained low.
Biopsy on admission revealed a hypoplastic bone
marrow with a relative scarcity of cellular
ele-ments. Myeloid elements were diminished and
pri-manly polymorphonuclear. The erythroid series
was markedly decreased with only occasional
ortho-chromatic normoblasts. Megakaryocytes were also decreased. On the 188th day, 23 days after B12 was
first given and immediately before Cu was
in-creased, the myeloid series had elements in all
stages of development, with a predominance of
immature cells, especially myelocytes, suggesting
some degree of maturation arrest. On the 203rd
day, 14 days after Cu was started, the bone
mar-row revealed marked hyperplasia, particularly of
the myeloid series, which was represented by huge numbers of mature PMN’s.
On the 148th day serum Cu was 43 pig/100 ml;
on the 222nd day, 183 pig/100 ml.
BONE: Radiologic appearance was normal until the 138th day, when there was some irregularity of
the distal ulnar metaphysis with a small lateral
bony spur and loss of density and pattern within
the distal femoral and proximal tibial epiphyses,
which had a “ring-like” appearance. Fifteen days
later there was little change. On the 159th day
there was evidence of bone formation between the
previously mentioned spur and the ulnar epiphysis. On the 188th day there was normal bone structure apparent within the femoral and tibial epiphyses as well as an increase in size.
SUMMARY: M.C. was rehabilitated on a diet
yielding 28 pig Cu/kg/day for 118 days and 42
pig/kg/day thereafter. Marked neutropenia became
evident on the 75th day, anemia by the 65th day,
and bone lesions by the 138th day. Folic acid and/
or ascorbic acid produced a good reticulocyte
re-sponse and a partial erythroid remission but had
no effect on the myeloid series. Vitamin B12
pro-duced a slow but almost complete erythroid and
myeloid response as well as healing of the bone
lesions. Supplemental Cu produced a total myeloid
remission and further increased the erythroid
re-sponse; it also led to progress in bone age.
COMMENT
These 4 cases had important features in
common; very short periods of breast
feed-ing followed by semi-starvation diets and
repeated episodes of diarrhea or vomiting.
In 3 of them, significant infections
pre-ceded admission. All were rehabilitated on
milk diets modified by the addition of fat
and carbohydrate which resulted in marked
acceleration of weight gain and thus
cre-ated a relative deficiency of nutrients which
would otherwise have been present in
ade-quate amounts in the milk used.
Fe deficiency was anticipated but they
developed severe anemia 64 to 108 days
after admission, which was hypochromic to
normochromic; microcytes were not
prom-inent, and minimal macrocytosis was
pres-ent in all. Cases 1 and 3 had reticulocyte
responses and temporary erythroid
remis-sions following increase in ascorbic acid
in-take. One of these (Case 1) had an
addi-tional, similar response to folic acid. Case
4 had a similar response but the ascorbic
acid and folic acid had been increased
al-most simultaneously; Case 2 had received
a previous blood transfusion and had no
response to either.
In Case 4 there was a short period of
hypoalbuminemia; serum proteins were
otherwise within normal limits.
All 4 had been receiving only 28 p.g
Cu/kg/day during most of their stay and
developed significantly low serum Cu
values. Two of them (1 and 4) received
42 g Cu/kg/day for brief periods after
the anemia had developed, but without any
noticeable improvement. Cases 1 and 2 had
prompt and dramatic erythroid responses
to supplemental Cu, going up to nearly
normal values in a few days. Cases 3 and 4
had slower but good responses to vitamin
B12, and then had further responses to Cu.
All had slight but additional further
re-sponses, particularly in 11gb, when given
varied diets, suggesting that perhaps some
other nutrient was deficient. Subsequent
experience with other infants has shown
that a complete mineral
mixture15
givenre-suits in normal values for Hct, 11gb, and
RBC, suggesting that the additionally
de-ficient nutrient was one of the minerals.
Perhaps the most striking finding was a
marked neutropenia, which became
ap-parent with, or even before, the anemia.
Cases 1 and 3 were able to respond with
moderate leucocytosis and some increase in
neutrophils to intercurrent infections. There
was no improvement in the neutropenia
from ascorbic or folic acid, and transfusion
in Case 2 also failed to produce any
im-provement. Cases 1 and 2 had prompt and
complete responses to Cu; in Case 1 it was
possible to produce a temporary
neutro-penic relapse by decreasing the Cu intake
prematurely. Cases 3 and 4 had much
slower but good responses to vitamin B22
and subsequent complete responses to Cu.
After B12 the myeloid elements in the bone
marrow were hyperplastic but
predom-inantly immature; after Cu was added,
ma-ture PMN’s were evident in enormous
amounts.
Experimental work in pigs12 has
charac-terized the anemia of Cu deficiency as due
to a limitation in the rate of synthesis of red
cells by the bone marrow in the face of a
significantly decreased survival time. The
very slight but persistent reticulocytosis
during the entire period of Cu deficiency
in all 4 cases argues for the same
mech-anism in man. The additional
reticulocyto-sis, but with only transient and incomplete
hematologic response which was obtained
in 3 cases with ascorbic and folic acids,
suggests that these agents favored the
pro-duction of red cells by the marrow without
prolonging their life span. On the basis of
its action on different stages of Cu
metab-olism, it is possible that ascorbic acid may
have some other mode of action.16 Only in
the work of Lahey et al.13 have we been
able to find evidence of significant
leuko-penia and particularly neutropenia in Cu
deficient animals. There is a report of
neu-tropenia as a complication of penicillamine
therapy in Wilson’s Disease17 but in none
of the supposed cases of Cu deficiency that
we have been able to find in the literature
has this manifestation been reported. Dr.
Harold E. Harrison has suggested to us
that the neutropenia of the cases of
hyper-glycinemia reported by Childs, Nyhan et
al.18 might be due to the ability of glycine
to chelate Cu.
The good, though incomplete, remissions
obtained in 2 of our cases with vitamin B,2
might be explained in a variety of ways:
1. Vitamin
B12
may have increased theintestinal absorption cf Cu or mobilized it
from other sites. Unfortunately, we do not
have serum Cu values after B12 and before
Cu supplementation.
2. It may have further stimulated the
production of red cells and also of white
blood cells by the marrow. The
hyper-plastic response observed lends support to
this possibility.
3. It may have increased the life span
of red cells produced, and, if we assume
that the neutropenia is also the result of
decreased survival, it may also have
in-creased the life span of neutrophils.
On the basis of the skimpy evidence
available, we cannot say if any or all 3
of these mechanisms were operant. We are
not aware of any experimental work
relat-ing vitamin B12 to Cu metabolism.
Case 1 developed the most severe
osse-ous lesions, and although a pathological
fracture occurred, it healed with good
cal-lus formation. All developed, in varying
degrees, osteoporosis, metaphyseal
irregu-larity, and spur formation. These lesions
were suggestive of scurvy but gave no
clinical indication of their existence.
Strik-ing apparent retardation in bone ag&9 was
present in all cases (Fig.
7).
This is inmarked contrast to the normal maturation
of bone reported in Cu-deficient dogs.2#{176}
In the first 2 there was prompt
improve-ment in the appearance of bone after Cu
supplementation. In the last 2, after B12
administration there was definite
improve-ment which continued after the addition of
Cu. Advance in radiologic bone age was
more prompt in the first 2 and quite slow
in the 4th case. These lesions are not
un-like those reported in dogs20 and pigs2’ who
were made Cu deficient.
exam-Is
15
12
E
Ld9 0 4
‘Ii
z 6
0
03
3
o--o
pies of growth “imbalance”-Cu deficiency
resulting from growth acceleration
pro-duced by a high-calorie, low-Cu intake in
infants whose stores were probably low.
We can only speculate as to the possible
role of isoniazid in Cases 2 and 3, as this
product has Cu-binding properties.22
Bush4 suggested that the human infant’s
Cu requirement was on the order of 80
g/kg/day, but Wilson and Lahey23
thought that it might be as low as 15
g/kg/day, as at this level they were
un-able, in 60 days, to produce any indication
of Cu deficiency in premature infants. It
must be remembered, however, that the
newborn has very ample stores in the liver
and that this length of time was probably
not enough to deplete them, particularly at
an age when utilization is probably very
low, despite the lower liver Cu stores of the
premature. Our cases, assuming that their
stores were low because of poor intake and
chronic intestinal losses, took 64 to 108 days
to show clear signs of deficiency. The fact
that 28 were not enough to prevent
de-ficiency and that 42 tg/kg/day produced
no improvement, suggests that their
mini-mum daily requirement was above this
second figure. In Case 1, 362 tg/kg/day
produced a prompt remission, but after 10
days on this dose, 104 tg/kday were
in-adequate and 167 &g/kday were
ade-quate to maintain it.
In Case 2, 158 .g, in Case 3, 135 tg, and
in Case 4, 173 tg/kg/day were adequate to
maintain remission. Consequently, we
might assume that the minimal daily
re-quirement for a rapidly growing infant
with inadequate stores is over 42 but below
135 pg/kg/day. This is in striking accord
with the requirements for pigs estimated by
Teague and Carpenter2’ of 77 to 110 g/kg.
The diet on which Cu deficiency was
in-advertently produced in these 4 infants is
not likely to occur by chance in everyday
practice and was probably complicated by
marginal or deficient intakes of other
nu-trients. We have, however, seen
hypocu-premia and the same hematologic picture
develop and respond promptly to Cu in one
other infant receiving the same diet
supple-‘3
2p/
,...‘,
I
I
I1.1
74
/p/.,_.
:
r
..P-;g::,:::g
I
8i1i0’
9 12 15 15 21 26 27
AGE mos.
Fic. 7. Relation of radiologic bone age in months to chronologic age in 4 cases of marasmus recover-ing on a high-calorie, low-copper diet. The arrows indicate the beginning of copper supplementation.
Case 3 shows rapid advancement in bone age
be-fore copper, possibly under the influence of
vita-mm B12.
mented with folic and ascorbic acids.
In-fants receiving high caloric intakes often
have undiluted milk as the sole food and
probably receive barely adequate amounts
of Cu. The commonly used supplements to
the diet of infants, such as cereals, fruits,
and vegetables supply ample Cu. Also, the
commonly used utensils probably
contami-nate food with sufficient Cu. In our kitchen
the diets were mixed in glass containers
and placed in plastic nursing bottles,#{176} thus
inadvertently decreasing the possibility of
contamination with Cu. The casein added
to the diets of Cases 2 and 3 contains only
2 ppm of Cu and the amounts in the cane
sugar and oil are probably negligible. We
would agree with the contention that Cu
deficiency cannot occur with any frequency
in human beings. It must, however, be
taken into account in milk-fed infants who
become anemic, particularly if they have a
history of increased stool losses and
de-creased absorption.
SUMMARY
In four severely malnourished infants
who were rehabilitated on high-calorie,
low-Cu diets, we have observed the
ap-pearance of severe anemia, marked
neutro-penia, scurvy-like bone changes, and
hypo-cupremia. In 2 cases there was a prompt
and dramatic response to Cu
supplementa-tion. In the other 2, slower but good
re-sponses were obtained with vitamin B,,;
supplemental Cu produced further
re-sponses. The Cu requirement of rapidly
growing infants with poor stores is
esti-mated at between 42 and 135 tg/kg/day.
REFERENCES
1. Marston, H. R. : Cobalt, copper and
molyb-denum in the nutrition of animals and
plants. Physiol. Rev., 32:66, 1952.
2. Scheinberg, I. H. : Copper metabolism, a
re-view. In Wilson’s Disease, Some Current
Concepts. Edited by Walshe, J. M., and
Cummings, J. N. Oxford: Blackwell, 1961,
pp. 4-17.
3. Josephs, H. W.: Treatment of anemia in
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4. Bush, J. A.: The role of trace elements in
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5. Moore, C. V.: The essential trace elements. In
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6. Wintrobe, ‘M. M.: Clinical Hematology. 5th
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19. Greulich, W. W., and Pyle, S. I.: Radiographic
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