Serum copper concentrations in sick and well preterm infants

November 1980 The Journal o f P E D I A T R I C S

795

Serum copper concentrations in sick and well preterm infants In order to define the range of serum copper concentrations in preterm infants and to determine the effect of growth upon these values, serial serum copper concentrations were measured in 26 preterm infants over their first six weeks of life. Fourteen healthy, growing preterm infants (Group I) had mean serum copper concentrations below 32 t~g/dl throughout the study. Clinical and hematologic signs of copper deficiency which responded promptly to the oral administration of copper sulfate were noted in five of these 14 infants. Twelve ill preterm infants (Group H-A), who received parenteral nutrition without supplemental copper and had slow rates of growth, had mean 'serum copper concentrations above 50 i~g/dl after the first week of life. Seven surviving infants from Group 11-A (Group 11-B) had a decrease in mean serum copper concentrations to values similar to those in Group I after two weeks of oral feedings and resumption of normal growth. Our findings suggest that preterm infants who have normal growth while receiving oral feedings are at significant risk for developing copper deficiency.

J e a n n e I. M a n s e r , M , D . , C a r o l y n S. Crawford,

M.D.,

Eileen E. Tyrala, M.D., Nancy L. Brodsky, Ph.D., and W a r r e n D. Grover, M.D.,* Philadelphia, Pa.

IMPROVED METHODS of care are resulting in the

survival of preterm infants who require various modes of alimentation. These infants have special nutritional requirements, including copper and other trace metals. At birth, normal term infants have tow total plasma copper concentrations which increase to approximately adult levels by one month of age?. '2 There are inadequate data describing serum copper concentrations in the preterm infant, s' ' Copper deficiency has been reported in infants maintained for prolonged periods of time on parenteral nutrition 5, 6 and also in the rapidly growing preterm infant?, 7 We have evaluated serial serum copper concentrations in healthy, growing preterm infants and in ill

From the Departments of Pediatrics and Neurology, Temple University School of Medicine and Sections of Neonatology and Neurology of the Department of Pediatrics and Department of Laboratories, St. Christopher's Hospital for Children and Division of Neonatology, Albert Einstein Medical Center, Northern Division. * Reprint address: Director Child Neurology, St. Christopher's Hospitalfor Children, 2600 N. Lawrence St., Philadelphia, PA 19133.

0022-3476/80/110795 + 05500.50/0 9 1980 The C. V. Mosby Co.

preterm neonates receiving parenteral nutrition in order to define the range of copper Concentrations, and to document their relationship to. rate of growth, mode of alimentation, and deficiency states. PATIENTS Twenty-six preterm infants, patients in the Neonatal Intensive Care Units at St. Christopher's Hospital for Children or Albert Einstein Medical Center, Northern Division, were studied between November, 1977, and July, 1978. Parental consent was obtained for each infant. Infants were divided into three groups according to their clinical course and mode of alimentation. Group I consisted of 14 infants receiving only oral feedings totaling 150 to 180 m l / k g / 2 4 hours by the end of the second week of life. Mean birth weight was 1.25 kg (range 1.0 to 1.64 kg) and mean gestational age was 30.5 weeks (range 28 to 32 weeks). Thirteen infants had birth weights appropriate for gestational age; one was small for gestational age. The infants who had an uncomplicated course were studied until discharge. The infants with clinical and hematologic evidence of copper deficiency were treated

Vol. 97, No. 5, pp. 795-799

796

Manser et aL

The Journal of Pediatrics November 1980

120-

100-

~.

rr hi D.. O

- -

9 GROUP I

---

9

A

280"

....... 9 G R O U P I I B

240-

80-

60"

200O9 I'--

40-

20-

o

i

~

~

~

~

A

~

WEEKS

Fig.. 1. Mean serum copper concentrations _+ SEM per week of oral feeding for Groups I and II-B and per week of life for Group II-A; *indicates significantly different from Group I (P < 0.05).

,,=,

180-

hi I:11:: eO Z

140-

A

g I00" I-'1(..9 hi

60-

:n, 20" and no longer included in the study. Formulas used were Similac, Isomil, or Similac Low Birth Weight (Ross Laboratories). No infant received more than 30 ml/kg of a blood product, either fresh-frozen plasma or packed red blood cells, during the period of study. Group II-A consisted of 12 infants nourished by intravenous alimentation for the duration of the study. The mean birth weight was 1.15 kg (range 0.7 to 2.6 kg) and mean gestational age was 30 weeks (range 26 to 35 weeks). Eleven infants had birth weights appropriate for gestational age; one was large for gestational age. Nine infants received peripheral intravenous alimentation consisting of 10% dextrose, crystalline amino acids ranging from 1.0 to 2.5 gm/kg/day, and Intralipid (Cutter Laboratories) ranging from 1.0 to 2.5 gm/kg/day. One infant received only 10% dextrose and crystalline amino acids; two infants received only intravenous dextrose, electrolytes, and water. All infants in Group II-A received multiple transfusions of fresh-frozen plasma a n d / o r packed red blood cells amounting to more than 50 ml/kg. These infants were followed until death or until oral feedings were initiated (mean six weeks), at which time they were entered into Group II-B. No infants in this group had congenital abnormalities of the bowel. Four patients developed necrotizing enterocolitis. Eight infants had congestive heart failure. All infants were suspected of having sepsis at least once during the nursery course. Group I1-B consisted of seven surviving infants from Group II-A after oral feedings were initiated. Mean age at the time of admission to this group was 6 weeks (rang# 4 to 8 weeks) and mean weight was 1.48 kg (range 0.87 to 2.93 kg). Formulas used were Simifac, Isomil, or Similac Low Birth Weight. All infants were receiving 120 to 180

hi l..IJ

O" -20-

A GROUP I 9 GROUP ITA a

GROUP II 8

-60" -I00-

WEEKS

Fig. 2. Mean weight increments _+ SEM per week of oral feeding for Groups I and II-B and per week of life for Group II-A; *indicates significantly different from Group I (P < 0.05). ml/kg/24 hours by the end of the second week after entry into Gro0.p II-B. No infants received more than 30 ml/kg of packed red blood cells during this phase of the study. All infants in this study group were asymptomatic for the duration of the study. METHOD Upon admission to the study and at five- to ten-day intervals thereafter, blood was obtained from the infants for serum copper concentrations. Increments of weight change per week were calculated from daily weights. Intakes of intravenous alimentation fluids, blood products, and oral fluids were determined and assayed for copper content. One-half milliliter of blood was obtained for serum copper determinations by heel stick and collected in Microtainer tubes (Beckton-Dickerson 5960). Blood was centrifuged and serum removed using EDTA washed Pasteur pipettes and stored in polystyrene tubes at - 2 0 ~ until time of analysis. Neither the Microtainer nor the

Volume97 Number 5

Serum copper concentrations

797

Table I. Average daily copper intake (/~g/kg _+ SEM) per week of oral feeding for Groups I and II-B and per week of life for Group II-A; ( ) indicates range of copper intake/kg

I No. infants Group 1 No. infants Group IIA No. infants Group liB

Week 14 47 +- 2 (40-71) 12 10 + 1 (1-21) 7 49 • 3 (41-75)

14 65 • 4 (20-95) 12 12 • 2 (t-30) 7 66 • 5 (65-89)

13 77 + 4 (41-120) 10 11 • 1 (8-14) 6 77 _+ 1 (76-83)

polystyrene tubes contained any detectable elutable copper. For analysis, the sera were diluted in a solution containing 140 mM NaC1, 5 mM KC1, !.8 mM magnesium acetate, 2.5 mM CaCI~, 5% glycerine, and 0.01% bovine serum albRmin? Copper analyses were performed at 324.7 nm using an Instrumentation Laboratory 351 atomic absorption spectrophotometer equipped with an air-acetylene flame. The machine was calibrated daily using the Harleco Copper Standard (7633) diluted to 50/~g/dl in the same solution as the sera. Dade Monitrol Sera, as well as previous standard solutions, were analyzed each day to verify the machine's calibration. Five readings were taken for each sample and a mean value was computed. The coefficient of variation for the Dade controls was 6.6%. In order to compare the serum copper concentrations obtained from capillary and from venous blood, simultaneous specimens were obtained by venipuncture and by fingerstick from I4 adult volunteers. No significant difference in values from serum obtained by fingerstick or by venipuncture from the same individual was noted (P = 0.55). There were no differences in the serum copper concentrations in grossly hemolyzed specimens when compared to those in normal control subjects. Data obtained from each group were compared using the unpaired Student t test and correlation coefficients. RESULTS Copper values obtained during the first week of life on all infants were similar and did not correlate with gestational age or birth weight (r = 0.13, r = 0.0059, respectively). Fig. 1 depicts the average weekly serum copper concentrations • SEM for each group. Mean serum copper concentration for Group I in the first week of life was 28 _+ 2/~g/dl. During the next four weeks, mean serum

12 76 • 3 (61-120) 10 10-+ 0.5 (7-11) 5 66 _ 6 (41-76)

12 83 • 6 (74-147) 9 9 + 0.5 (7-11) 5 66 + 6 (41-76)

10 85+7 (74-147) 8 9_+0.5 (7-11) 5 76+2 (65-79)

8 87_+9 (74-147) 5 9+_0.5 (sq0)

copper concentrations decreased slightly (20 • 2 /xg/dl) and then increased to 32 • 6/xg/dl by week six. Copper concentrations for Group II-A (30 _+ 2/~g/dl) in the first week of life were not significantly different from those in Group I (P = 0.45). Mean serum copper concentration of Group II-A increased markedly alter the first week of life to a peak value of 68 _+ 8/~g/dl by week four, then decreased slightly to 57 • 9 /~g/dl by week seven. Weekly mean copper concentrations for Group II-A were significantly higher than in Group I after the first postnatal week (P < 0.05). Mean copper concentrations for Group II-B during the first week of oral feedings (66 _+ 15/~g/dl) were similar to values in Group II-A from three to seven weeks. Serum copper concentrations in Group II-B decreased over the next five weeks of oral alimentation; after two weeks, the values were not significantly different from those of Group I. The average weekly weight increments for Group II-A were significantly less than those of Group I after the second week (Fig. 2). The weight increments per week after the first week for Group I and II-B were not significantly different. Calculation of the average daily copper intake per kilogram ( + SEM) for all three groups indicated that Group I and II-B received the largest amounts of copper by oral feeding (Table I). Transfusions in these groups contributed less than 5/,g/kg/week of copper. Group II-A received smaller amounts of copper from transfusions of packed red blood cells (mean copper content from 75 specimens was 62 + 14/,g/dl SEM), fresh-frozen plasma (84 • 19/*g/dl SEM for 16 specimens), and parenteral alimentation fluid ( < 5/,g/dl for five specimens). During the course of the study, five infants from Group I had serum copper concentrations less than 25/~g/dl and ceruloplasmin values less than 15 rag/dl. In addition, four of five infants had absolute neutrophil counts less than

798

Manser et al.

The Journal of Pediatrics November 1980

Table 11, Infants with copper deficiency before and after therapy

Infants

Gestational age (wk)/ birth weight (kg)

1

28/1.2

2 3 4 5

31/1.5 31/1.3 29/1.2 28/1.3

Diagnosis Age (days)

Serum albumin (mg/dl)

Serum Cu (l~g/dl) before~after therapy

47 36 71 67 47

3.3 2.8 3.5 3.8 3.4

15/21 23/42 15/38 20/35 19/41

Ceruloplasm (mg/ dl) before~after therapy

5/!4" 6/14t 14/-13/165

Hematologic studies (before~after therapy) Total WBC XIO ~

Absolute neutrophil count/mm 3

7.4/ 9.6 5.0/ 7.5 7.7/ 9.4 8.7/12 9.0/ 9.5

1,406/5,952 950/2,775 385/2,068 1,740/3,600 1,440/4,845

Hb gm/dl

10.1/11.9 8.7/ 9.6 8.2/ 9.9 12.2/11.6 9.4/13.8

RBCXIO 6 mm 3

3.3* /4.5 2.6t /3.0 2.7:~/5.4 4.1"~/3.7 3.1t /4.4

Reticulocyte count (%)

--/4.1/5.7 5.2/3.0 1.8/1.0 4.9w

';Three weeks of therapy. "~Twoweeks of therapy. ~One week of therapy. w transfused. 1,500/mm ~ without evidence of infection. Hemoglobin, red blood cell counts, and reticulocyte.counts are noted in Table II. Examination of the infants with low serum levels of copper revealed decrease in activity and non-pitting pedal edema in all. After one week of oral therapy with copper sulfate (1,5 mg elemental copper/day) serum copper concentrations increased by twofold or more in all infants. Hemoglobin concentrations and absolute red blood cell values increased in three of four infants (Table II). Ceruloplasmin concentrations were measured in three infants; a greater than twofold rise was noted in two patients after two weeks of copper therapy. No increase in ceruloplasmin values was noted in one patient after one week of copper therapy, increased activity and resolution of the pedal edema was noted in three patients after one to two weeks of copper therapy. DISCUSSION Copper is an essential micronutrient for the formation of many metalloproteins and cuproenzymes. The copperdeficient state may cause defective enzyme function that produces characteristic signs and symptoms. Ceruloplasmin (ferrioxidase), a glycoprotein important in oxidation of iron for hemoglobin metabolism and for copper transport, varies directly with serum copper value?, 10 Low levels of ceruloplasmin have been related to hypocupremic anemia with neutropenia responding to supplemental copper therapy. Lysyl oxidase is a cuproenzyme required for the cross linkage bonding of elastin necessary for collagen formation in blood vessels, connective tissue, and bone. Abnormal elastin tissue resulting in aortic rupture has been described in copper-deficient pigs. 11 Ascorbic acid oxidase is also a copper-containing enzyrme required for normal bone structure. States of copper insufficiency may cause defective kinetics of both the lysyl

oxidase and ascorbic acid oxidase systems, 1~ producing osteoporosis and roentgenographic signs of scurvy. The role of other cuproenzymes in the pathogenesis of the deficiency state is uncertain. Cytochrome oxidase is necessary for energy production by formation of ATP; low levels of activity have caused defective brain growth in experimental animals. TM Dopamine beta hydroxylase converts dopamine to norepinephrine, an important neurotransmitter for brain function. TM Tyrosinase (oxidation of L-tyrosine ~ L-dopa--~ melanin) and superoxide dismutase (removal of intracellular oxygen radicals) are cuproenzymes which have not been implicated in the syndromes of copper deficiency. Our data demonstrate that the serum copper concentrations of preterm infants in the first week of life are not related to gestational age or to birth weight and are similar to values reported for term infants.', 2 Healthy, growing preterm infants, receiving 150 to 180 m l / k g / d a y of standard formula, however, did not achieve the same postnatal increase in serum copper levels that is seen in the term infant. The mean serum copper concentrations of this group remained below 32/~g/dl for the first six weeks of life with an oral intake of greater than 60 ~tg/ Cu/kg/day. Despite the low copper concentrations, 64% of the infants never had hematologic evidence of copper deficiency. Factors including diminished copper stores, decreased gastrointestinal absorption and increased copper utilization by rapid growth may have contributed to the persistent hypocupremia of Group I. Preterm infants are born with decreased stores of copper TM 16 and must accumulate 70 to 90/~g of copper/kg/day in the postnatal period to approximate the fetal rate of storage during the third trimester of gestation. Dauncey et a117 demonstrated decreased absorption of copper from human milk in low birth weight infants, resulting in negative copper balance

Volume 97 Number 5

for as long as 35 postnatal days. Increased utilization of copper during rapid growth was suggested by Cordano et al TM to be a contributing factor to the hypocupremia seen in recovering marasmic infants. The higher serum copper concentrations noted in infants receiving parenteral alimentation without oral feedings (Group II-A) may have been caused by lower requirements for copper secondary to a lower growth rate, decreased biliary excretion secondary to diminished gastrointestinal motility, 19 a n d / o r greater bioavailability of copper salts contained in blood products and in parenteral alimentation fluid. After the initiation of oral feedings and the onset o f normal growth, the serum copper concentrations decreased over a period of two weeks to values comparable to those in the infants in Group I. This finding indicates that no significant increase in copper stores occurred during parenteral alimentation. The serum copper concentrations did not distinguish five of 15 infants in Group 1 with symptomatic copper deficiency from other growing preterm infants. Copper deficiency was defined by neutropenia, a serum copper concentration below 25 /tg/dl, a serum ceruloplasmin concentration less than 15 mg/dl, and resolution of the hematologic abnormality after the administration of oral copper supplementation. Several investigators have demonstrated that total serum copper values do not accurately reflect the tissue levels which cause the deficiency state?0.21 Our data indicate that a low serum copper concentration is characteristic of the rapidly growing preterm infant and that the measurement of serum copper values alone is not adequate to define the copper-deficient state. We acknowledge the assistance of Dr. Thomas Schaeffer in preparing the statistical analysis; Dr. David Smith for his critical review of the manuscript; and Dr. Phillip Zoltick for his input into the early discussions that led to this study.

S e r u m copper concentrations

4.

5. 6. 7.

8. 9.

10.

11. 12.

13.

14. 15.

16.

17.

18. 19.

REFERENCES

1. Henkin RI, Schulman JD, Schulman CB, and Bronzert DA: Changes in total, nondiffusable, and diffusable plasma zinc and copper during infancy, J PEDIATR82:831, 1973. 2. Ohtake M: Serum zinc and copper levels in healthy Japanese infants, Tohoku J Exp Med 123:265, 1977. 3. Widdowson EM: Trace elements in human development, in Barltrop D, and Burland WL, editors: Mineral metabolism

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in pediatrics, Oxford, 1969, Blackwell Scientific Publications, pp 85-98. Wilson JF, and Lahey ME: Failure to induce dietary deficiency of copper in premature infants, Pediatrics 25:40, 1960. Karpel JT, and Peden VH: Copper deficiency in long-term parenteral nutrition, J PEDIATR80:32, 1972. A1-Rashid RA, and Spangler J: Neonatal copper deficiency, N Engl J Med, 285:841, 1971, Ashkenazi A, Levin S, Djaldetti M, Fishel E, and Benvenisti D: The syndrome of neonatal copper deficiency, Pediatrics 52:525, 1973. Rosenthal RW, and Blackburn A: Higher copper concentrations in serum than plasma, Clin Chem 20:1233, 1974. Frieden E, and Osaki S: Ferrioxidases and ferrireductases: Their role in iron metabolism in protein-metal interactions, in Friedman M, editor: Advances in experimental biology, New York, 1974, Plenum Press, p 235. Holtzlnan NA, Charache P, Cordano A, and Graham GG: Distribution of serum copper in copper deficiency, Hopkins Med J 126:34, 1970. Conlson WF, and Carnes WH: Cardiovascular studies on copper deficient swine, Am J Pathol 43:945, 1963. Hambridge MK: Trace elements in pediatric nutrition, in Shulman l, editor: Advances in pediatrics, Chicago, 1977, Year Book Medical Publisher Inc., p 191. Probaska JR, and Wells WW: Copper deficiency in the developing rat brain: A possible model for Menkes' steely hair disease, J Neurochem 23:91, 1974. Fisher GL: Function and homeostasis of copper and zinc in mammals, Sci Total Environ 4:373, 1975. Widdowson EM, Chan H, Harrison GE, and Milner RDG: Accumulation of Cu, Zn, Mn, Cr and Co in the human liver before birth, Biol Neonate 20:360, 1972. Bruckmann G, and Zonder SG: Iron, copper and manganese in human organs at various ages, Biochem 33:1845, 1939. Dauncy MJ, Shaw JCL, and Urman J: The absorption and retention of magnesium, zinc and copper by low birth weight infants fed pasteurized human breast milk, Pediatr Res 11:1033, 1977. Cordano A, Baertl JM, and Graham GG: Copper deficiency in infancy, Pediatrics 34:324, 1964. Mahoney JP, Bush JA, Gubler CJ, Morety WH, Cartwright GE, and Wintrobe MM: Studies on copper metabolism, XV. The excretion of copper by animals, J Lab Clin Med 46:702, 1955. Grover WD, and Scrutton MC: Copper infusion therapy in trichopoliodystrophy, J Pediatr 86:216, 1975. Dempsey H, Cartwright GE, and Wintrobe MM: Studies on copper metabolism. XXV. Relationship between serum and liver copper, Proc Soc Exp Biol Med 98:520, 1958.