Journal of the American College of Nutrition

ISSN: 0731-5724 (Print) 1541-1087 (Online) Journal homepage: http://www.tandfonline.com/loi/uacn20

Zinc and copper status of severely burned children during TPN. J J Cunningham, M K Lydon, S E Briggs & M DeCheke To cite this article: J J Cunningham, M K Lydon, S E Briggs & M DeCheke (1991) Zinc and copper status of severely burned children during TPN., Journal of the American College of Nutrition, 10:1, 57-62, DOI: 10.1080/07315724.1991.10718127 To link to this article: http://dx.doi.org/10.1080/07315724.1991.10718127

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Zinc and Copper Status of Severely Burned Children During TPN John J. Cunningham, PhD, FACN, Martha K. Lydon, RN, Susan E. Briggs, MD, and Michael DeCheke, PhD Nutritional Support Unit and Surgical Service, Shriners Burns Institute and Massachusetts General Hospital, Boston, and Department of Nutrition and the Microanalysis Laboratory of the Graduate School, University of Massachusetts, Amherst

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Key words: zinc, copper, parenteral nutrition, burns Alterations in zinc (Zn) and copper (Cu) homeostasis have been reported during the acute recovery period following thermal injury in both children and adults. Increased urinary losses of Zn and Cu and decreased plasma concentrations of Zn, Cu, and ceruloplasmin (CP), the major copper transport protein, occur despite adequate provision of these elements in enterai feedings. We now report data for moderately to severely burned childrenreceivingtotal parenteral nutrition (TPN) supplemented to provide Zn and Cu. Hyperzincuria occurred consistently when 50 μg/kg Zn was delivered daily to older children. Similarly, when younger children received 100 μg/kg Zn daily, profound hyperzin­ curia ensued despite a reduction in total plasma Zn. Hypozincemia was accompanied by low levels of Zn in the plasma subfraction normally associated with albumin-bound Zn. The delivery of Cu via TPN was 4-12 μg/kg daily, and urinary Cu losses were not elevated. Plasma total Cu and plasma CP were invariably reduced. These findings are discussed inrelationto guidelines published for pediatrie trace element supplementation during TPN.

INTRODUCTION Zinc (Zn) and copper (Cu), which participate in wound healing, have been studied during recovery from severe bums in enterally fed humans, primarily adults [1-3]. Their urinary Zn [3,4] and Cu [1] are elevated, beginning during the hypercatabolic "flow" phase of recovery, approximately on day 3, and persisting for several weeks. Decreased plasma Zn [2] is consistent with the "acute phase response" seen in nonbum trauma [5-7]. However, the reported reductions in both plasma Cu and the major Cu transport protein ceruloplasmin (CP) [1] contrast with the "acute phase response" of Cu metabolism in trauma [8] or sepsis [9]. Continued hypozincemia, hypocupremia, and low CP during sub­ sequent recovery reflect a state of dysmetabolism for both elements [1-3]. Since absorption of enterally delivered trace elements may greatly influence the responses of Zn and Cu of thermally injured patients, we have studied the Zn and Cu status of such patients receiving our standard regimen of total parenteral nutrition (TPN) [10,11] which provides 2 mg Zn and 200 μg Cu daily. Urinary and

gastric losses of Zn and Cu were measured to determine the "apparent balance," defined here as balance not ad­ justed for wound losses. Plasma concentrations of Zn, Cu, and CP were also measured in severely burned children with ideal body weight (IBW) < 20 kg during the acute recovery period, in conjunction with urinary losses. In addition, plasma Zn was partitioned into the exchangeable fraction normally bound to albumin [12] and the remaining tightly bound fraction complexed to o^-macroglobulin [12,13].

MATERIALS AND METHODS In total, 14 children aged 2 months to 14 years were studied following admission to the acute care unit of the Shriners Bums Institute, Boston. Burn injuries in 11 cases ranged from 27 to 99% body surface area burned (BSAB), averaging 62 ± 8% BSAB, using our routine wound distribution assessment [14,15]. Our standard protocol included prompt excision and early wound closure [16,17]. Three additional children with sig­ nificant skin sloughing — two with toxic epidermal

Supported by The Shriners Hospitals for Crippled Children, Research Project 15873. Address reprint requests to John J. Cunningham, Ph.D., Nutritional Support Unit, Shiners Burns Institute, 51 Blossom Street, Boston, Massachusetts 02114.

Journal of the American College of Nutrition, Vol. 10, No. 1, 57-62 (1991) © 1991 John Wiley & Sons, Inc.

CCC 0731-5724/91/010057-06$04.00

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Zinc and Copper Status necrolysis syndrome (TENS) involving 99 and 50% BSA, and another patient who presented with lesions involving > 40% BSA secondary to meningococcal meningitis — were evaluated for Zn and Cu during TPN. Individual studies were performed during the interval between 2 and 60 days following injury while the patients were receiving TPN. The protocol was approved by the institutional human subjects review committee. Written informed consent was given by the designated parent or guardian for all research blood collections. The nutritional support goals for our patients are routinely estimated on the basis of IBW due to fluid perturbations during resuscitation. The compositions of the TPN formulations are detailed elsewhere [10,11]. TPN delivered amino acids at 2.5-3 g/kg/day and dextrose at 4-6 mg/kg min, with remaining calories sup­ plied by intravenous lipid. Infusion rates were regulated to deliver calories at approximately twice the predicted normal basal metabolic rate. Trace elements were added to only the first liter of TPN daily as 2 ml of PTE-4 (Lyphomed, Chicago, IL) to provide 2 mg Zn, 200 μg Cu, 50 μg Mn, and 2 μg Cr. In one case this supplement was added to the second daily liter of TPN to deliver up to 375 μg Cu. Data on Cr and Mn will be provided in another paper. Twenty-four-hour urinary collections were performed initially on days 2-10, either in trace metal-free plastic containers or in acid washed bottles. In some cases a follow-up collection was made 7-10 days later. In all cases 25 ml of 6 N HC1 was added. For patients of IBW > 35 kg the gastric aspirates suctioned through a nasogastric tube were also collected in similar con­ tainers. Aliquots of urine or aspirates were frozen in 50 ml plastic centrifuge tubes for later analysis. Records were reviewed to determine how many days each patient was treated with the silver (Ag) containing topical agents — silver nitrate (0.5% in saline) or silvadiene creme (l mg/g cream). Blood was collected for patients of IBW < 20 kg on days 2-19 for Zn assay {n = 7) and on days 2-20 for Cu and CP assay (n = 5). Individual collection days are depicted on the abscissa in Figures 2 and 3. One or more follow-up assays of Cu and CP were done in three cases. All blood collections were made using trace elementfree vacutainer tubes containing heparin (Fisher Scien­ tific, Medford, MA). Zn and Cu in urine or NG aspirates were measured by flame atomic absorption spectre-photometry (AAS) fol­ lowing a series of preparative steps combining wet oxidation and dry ashing. In the process, volatile chloride salts are converted to nitrates, and background contamination by HN03 is minimized. Briefly, high purity concentrated nitric acid (2.0 ml) was added to

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40-50 ml aliquots in a Pyrex beaker. Concentration was achieved by slow evaporation to 1-2 ml on a hotplate. Depending on the amount of residue, 0.5-1.0 ml of con­ centrated UNO, plus 1 ml of H202 were added and the solution was heated at 100-120°C until evaporation of the liquid phase. The temperature was then gradually raised to 300-350°C to partially char the residue. The residue was transferred to a muffle furnace and heated at 450°C overnight. Ash was wetted, dissolved in 0.5 ml HNO3, brought to a volume of 2-3 ml with water, warmed on a hotplate, and filtered. Filtered solutions were analyzed directly for Cu by AAS using background correction (Perkin-Elmer 3030 AAS), and aliquots were diluted 1:5 for Zn. Standards were added to a matrixmatched solution containing Na, Ca, Mg, Fé, K, and Mn in acid and prepared as above. Silver was analyzed in the urine samples by AAS at 328.1 nm with a detection limit of 0.01 μ^πιΐ of prepared sample. Plasma was assayed for CP by a spectrophotometric enzymatic method [18]. Samples for plasma total Cu were sent to the commercial laboratory used by Mas­ sachusetts General Hospital (Metpath, Boston, MA) for assay by flame AAS. AAS was performed at a wavelength of 324.74 nm with a detection limit of 0.05 μg/ml (personal communication). Total Zn in plasma was measured by AAS (Perkin-Elmer model 2380) fol­ lowing a 1:3 dilution with 6.7% TCAÎ12]. The subfrac­ tion of loosely bound Zn was assayed in a separate ali­ quot following precipitation of tightly bound Zn by 1:1 polyethylene glycol-6000 (EM Science), centrifugation, and removal of proteins from the supernatant by 10% TCA as detailed by Giroux [12]. The AAS method was that described by the manufacturer, namely, a single-ele­ ment Zn lamp with a wavelength of 213.9 nm and a slit of 0.7 mm. Using standards, sensitivity was 0.018 mg/L and the response was highly linear (r = 0.99). Tightly bound Zn in the o^ subfraction was calculated by sub­ traction.

RESULTS Patients with burns are separated into two subgroups for analysis and discussion because of the bimodal dis­ tribution of ages (thus IBWs) and burn severities. The older patients with IBW between 36 and 57 kg (n = 4) sustained 50-99% BSAB injuries and were studied in a pilot protocol without blood sampling. The younger children of IBW between 9.5 and 24 kg sustained generally more moderate injuries of 27-70% BSAB (n = 7). For the four bum patients with IBW > 35 kg, 24-hr urinary and nasogastric losses of Zn and Cu were

VOL. 10, NO. 1

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Fig. 1. Measured "apparent balances" for zinc (upper panel) and copper (lower panel) during a 24-hr period collected between days 2 and 17 in severely burned children of IBW 36-57 kg. Bars are the mean and standard error of the mean (n = 5) for losses through urine and nasogastric aspirates (NG) compared with intake via TPN.

Fig. 2. Plasma concentrations of copper (upper panel) and ceruloplasmin (lower panel) during TPN in severely burned children of IBW < 20 kg. The dashed line marks the lower limit of the normal range for each. The open circles display values for a 27% BSAB measured only once. The remaining connected open symbols show repeated measurements on the same child (36% BSAB squares, 60% BSAB diamonds, and 95% BSAB triangles). Filled circles depict measurements on day 6 from a patient with TENS involving 50% of the skin surface.

measured in six "apparent balance" studies between days 2 and 17. No stools were passed in these studies. Balance approximations are depicted in Figure 1. Urinary Zn los­ ses were 2160 ± 640 μg/day and NG Zn losses were 218 ± 43 μg/day. Urinary Cu was 172 ± 41 \L%làzy and NG Cu was 42 ± 11 μg/day. Net balances were -604 ± 742 for Zn and -20 ± 42 for Cu. Pronounced hyperzincuria (5070 μg/day) and negative Zn balance were also docu­ mented on day 11 for the child of IBW 50 kg with skin lesions secondary to meningococcal meningitis. For the remaining nine patients with IBW < 25 kg, including seven with burns and two with TENS, blood samples were collected in addition to the 24-hr urine

collections while on TPN. Plasma total Cu and plasma CP levels are depicted in Figure 2. Levels for CP in all 14 samples from burn patients were below normal for our institution (27-37 mg/dl), as were the 12 measure­ ments of total Cu (ni k 100 μg/dl). For one TENS patient with 50% involvement, on day 6 the plasma total Cu was 47 μg/dl with a CP of 6 mg/dl (included in Fig. 2). The corresponding urinary Cu losses for these patients were within the normal range at 31-138 μg/day. Plasma Zn was measured in six of these seven younger burn patients on days 2-19 (Fig. 3). Total Zn was 0.76 ±0.15 mg/L, with five of six patients below 0.7 mg/L. The loosely bound fraction of Zn was 0.53 ±0.13

JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION

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Zinc and Copper Status 1.8 1.6 1.4 1.2 1.0 0.8 0.6 + 0.4 0.2

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DAY 2 BSAB % 27

2 36

2 60

8 30

1 0 1 2 1 9 / 95 40 30 /

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Fig. 3. Plasma concentrations of total Zn (open bars) and loosely bound Zn (filled bars) during TPN for patients of < 20 kg IBW. Seven pairs from burned patients are arranged by day postburn and two sets of TENS patients are included thereafter. The degree of injury is noted for each patient. One set is a repeat measurement on the same patient (day 19, 30% BSAB). mg/L, comprising 66 ± 3% of total plasma Zn. The cor­ responding urinary losses were 1.4—4-fold higher than the maximal normal of 10 μg/kg, averaging 25 ± 4 μg/kg daily. Blood Zn was also determined for the two patients with major skin losses secondary to nonburn TENS (Fig. 3). Blood samples drawn on day 6 contained total plas­ ma Zn at 1.0 and 0.58 mg/L, respectively. The cor­ responding loosely bound Zn levels were 0.72 and 0.27 mg/L. Urinary Zn losses were 27.4 and 30.5 μg/kg, respectively. Silver (Ag) in concentrations of 0.03-0.24 μg/ml urine, or 17-475 μg daily, was detected in five patients. Four of these patients had low plasma Cu and CP, but were not different from the remaining patients in Figure 2, in whom urinary silver was undetectable. Analysis of five samples from PN solutions revealed no detectable Ag from this route.

DISCUSSION Four patients with severe bum injuries and IBW ex­ ceeding 35 kg received 35-55 μg/kg Zn and 3.5-5.5 μg/kg Cu via TPN daily. Two patients with 80 and 50% BSAB, studied on days 2 and 12, respectively, had posi­ tive "apparent" balances for Zn and Cu. The remaining two patients with > 95% BSAB were in profoundly negative balance for both Zn and Cu on days 4 and 10. For the latter patient, a repeat study on day 17 with 75% BSAB open wound showed improvement in total Zn balance by 1000 μg and positive Cu balance. Using max­

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imum normal adult urinary losses as appropriate indices of excretion in these patients, there was hyperzincuria (> 800 μg/day) in every case, accompanied by hypercupruria in only the two cases of > 95% BSAB. Hyperzincuria, at > 10 μg/kg [18], was always ob­ served in our younger patients who had been provided Zn in TPN at 100-120 mg/day. The magnitude, 40300% above normal, was similar to that seen in older patients. This confirms reports for adults in the literature as summarized elsewhere [3]. Low plasma total Zn levels are reported to accompany this hyperzincuria [3]. We further characterized the loosely bound fraction of plasma Zn using the method of Giroux [12]. Normal values for this assay are reported as 80% of total Zn, or 0.66 mg/L, with a standard deviation of 0.08 mg/L [12, 20]. Our patients receiving Zn in TPN averaged only 66%, or 0.53 mg/L, with most individual levels being below 0.5 mg/L. The albumin-bound Zn pool has been proposed to represent "physiologically available" Zn in healthy subjects [12]. Given the high content of free amino acids in our TPN formulation, it is possible that the loosely bound Zn fraction in plasma includes dis­ proportionately higher chelated Zn, especially cysteineZn and histidine-Zn complexes [21]. This could provide an explanation for the hyperzincuria, i.e., through en­ hanced filtration of these chelates [22]. The effect of altered speciation of plasma Zn on the physiologic availability of Zn requires further investigation. For Cu, urinary losses were normal while both plasma Cu and CP were reduced significantly. We provided 1012 μg Cu/kg daily via TPN. A potential contributor to the depression of the "acute phase" CP response might be

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Zinc and Copper Status Ag ions absorbed from the topical antimicrobial agents used in burn care [1]. The evidence from urinary Ag levels in our patients does not aid in evaluating the im­ portance of this component. It is also possible that CP is removed from circulation as a component of the physiologic response to severe trauma. Both plasma total Cu and plasma CP should be monitored in these patients so that "nonceruloplasmin Cu" [23] can be used as a clinical tool to assess the efficacy of Cu supplementa­ tion. General guidelines for the supplemental provision of Zn and Cu in TPN formulations were first issued by the American Medical Association [24]. In addition to recommendations for Cr and Mn, pediatrie targets for Zn and Cu were set at 100 and 20 μg/kg, respectively. More recently, the ASCN Subcommittee on Pediatrie Parenteral Nutrient Requirements [19] reviewed these guidelines focusing on more precise intravenous recom­ mendations for each trace element. Daily recommenda­ tions for Zn were given as 100 \ig/kg for infants and 50 μg/kg for older children to a maximum of 5000 μg daily in TPN. Additional needs secondary to the dysmetabolism of severe trauma were not considered. Our findings do not confirm the adequacy of these newly proposed [19] recommendations for Zn. Moderately to severely burned infants and young children (IBW < 20 kg) receiving Zn at 100-120 μg/kg had hypozincemia with a disproportionate reduction in the loosely bound Zn pool. This may, however, represent an exceptional trauma since a study of seven postsurgical infants reported by Shulman [25] supports the recom­ mendation for nonburn trauma. In addition, provision of 35-55 μg/kg Zn was associated with positive "apparent balance" in several of the studies of the older children. Copper supplementation in burn trauma also remains speculative, pending focused studies on the absence of an "acute phase" CP response to supplemental Cu. Our younger patients received Cu at only 10-12 μg/lcg daily consequent to our selection of multielement preparation containing Zn and Cu in a 10:1 ratio. This level of sup­ plementation was recently reported to be sufficient for balance in postsurgical infants [25], and apparent Cu balance was achieved in our older patients. However, plasma Cu and CP were not normalized, as is consistent with prior reports for burn trauma [1,2]. The absence of or masking of the acute phase CP response may reflect the severity of protein dysmetabolism in severe bum trauma.

ACKNOWLEDGMENTS This work was supported by the Shriners Hospitals for Crippled Children, Research Project 15873. Mr. Aizhong Fu of the Department of Nutrition of the Univer­ sity of Massachusetts performed plasma Zn determina­ tions.

REFERENCES 1. Boosalis MG, McCall JT, Solem LD, Ahrenholz DH, McClain CJ: Serum copper and ceruloplasmin levels and uri­ nary copper excretion in thermal injury. Am J Clin Nutr 44:899-906,1986. 2. Shewmake KB, Talbert GE, Bowser-Wallace BH, Caldwell FT, Cone JB: Alterations in plasma copper, zinc, and ceruloplasmin levels in patients with thermal trauma. J Burn Care Rehab 9:13-17, 1988. 3. Boosalis MG, Solem LD, McCall JT, Ahrenholz DH, McClain CG: Serum zinc response in thermal injury. J Am Coll Nutr 7:69-76, 1988. 4. Beisel WR, Pekarek RS, Wannemacher RW: Homeostatic mechanisms affecting plasma zinc levels in acute stress. In Prasad AS, Oberleas D (eds): 'Trace Elements in Human Health and Disease." New York: Academic, Vol 1, pp 87106, 1976. 5. Pekarek RS, Wannemacher RW Jr, Chappie FE III, Powanda MC, Beisel WR: Further characterization and species specificity of leukocytic endogenous mediator (LEM). Proc Soc Exp Biol Med 141:643-648, 1972. 6. Sobociniski PZ, Canturbury WJ Jr, Mapes CA, Dinterman RE: Involvement of hepatic metallothioneins in hypozin­ cemia associated with bacterial infection. Am J Physiol 234:Ε399-ΐ406, 1978. 7. Falchuk KH: Effect of acute disease and ACTH on serum zinc proteins. New Engl J Med 296:1129-1134, 1977. 8. Solomons NW: Biochemical, metabolic, and clinical role of copper in human nutrition. J Am Coll Nutr 4:83-105, 1985. 9. Pekarek RS, Powanda MC, Wannemacher RW Jr: The ef­ fect of leukocytic endogenous mediator (LEM) on serum copper and ceruloplasmin concentrations in the rat. Proc Soc Exp Biol Med 141:1029-1031, 1972. 10. Cunningham JJ, Harris U, Briggs SE: Nutritional support of the severely burned infant. Nutr Clin Pract 3:69-73, 1988. 11. Cunningham JJ, Anbar RA, Crawford JD: Hypomagnesemia: a multifactorial complication of treatment of severe burn trauma. JPEN 11:364-367, 1987.

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Zinc and Copper Status 12. Giroux EL: Determination of zinc distribution between al­ bumin and a2-macroglobulin in human serum. Biochem Med 12:258-266, 1975. 13. Kiilerich S, Claus C: Distribution of serum zinc between albumin and a2-macroglobulin estimated by ultracentrifugation. Clin Chim Acta 142:273-280, 1974. 14. Demling RH: Fluid resuscitation after major burns. JAMA 250:1438-1440, 1983. 15. Herrin JT, Crawford JD: The seriously burned child. In Smith CA (ed): "The Critically 111 Child," 2nd ed. Philadelphia: WB Saunders, pp 51-59, 1977. 16. Burke JF, Bondac CC: Combined bum therapy utilizing immediate skin allografting and 0.5% silver nitrate. Arch Surg 97:716-721, 1969. 17. Burke JF, Quinby WC, Bondac CC: Primary excision and prompt treatment of burns in children. Surg Clin North Am 56:477-494, 1976. 18. Ravin HA: An improved colorimetrie enzymatic assay of ceruloplasmin. J Lab Clin Med 58:161-168, 1961. 19. Greene HL, Hambidge KM, Schanler R, Tsang R: Guidelines for the use of vitamins, trace elements, cal­ cium, magnesium and phosphorous in infants and children receiving total parenteral nutrition: report of the Subcom­

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mittee on Pediatrie Issues of The American Society for Clinical Nutrition. Am J Clin Nutr 48:1324-1342,1988. Giroux E, Duriex M, Schechter PJ: A study of zinc dis­ tribution in human serum. Bioinorg Chem 5:211-218, 1976. Harris WR, Keen C: Calculations of the distribution of zinc in a computer model of human serum. J Nutr 119:1677-1682, 1989. Jeejeebhoy KN: Zinc and chromium in parenteral nutri­ tion. Bull NY Acad Med 60:118-124, 1984. Brewer GJ, Hill G, Prasad A, Dick R: The treatment of Wilson's disease with zinc. IV. Efficacy monitoring using urine and plasma copper. Proc Soc Exp Biol Med 184:446-455, 1987. Shils M, Burke AW, Greene HL, Jeejeebhoy KN, Prasad AS, Sandstead HH: Guidelines for essential trace element preparations for parenteral use. JAMA 241:2051-2054, 1979. Shulman RJ: Zinc and copper balance studies in infants receiving total parenteral nutrition. Am J Clin Nutr 49:879-883, 1989.

Received October 1989; revision accepted July 1990.

VOL. 10, NO. 1

Zinc and copper status of severely burned children during TPN.

Alterations in zinc (Zn) and copper (Cu) homeostasis have been reported during the acute recovery period following thermal injury in both children and...
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