Redistribution of labile plasma zinc during mild surgical stress in the rat

Redistribution of Labile Plasma Zinc During Mild Surgical Stressin the Rat

Edward Kelly, MD, Jeffrey Mathew, MD, Jonathan E. Kohler, MD, Amy L. Blass, BS, andDavid I. Soybel, MDDepartment of Surgery, Brigham and Women's Hospital and Harvard Medical School, Boston,Massachusetts

AbstractZinc is an essential trace element and co-factor for many cellular processes. Uptake of Zn2+ inperipheral tissues depends on its total content in the circulation, and on mechanisms facilitatingdelivery to tissues in its labile form. Understanding mechanisms of Zn2+ delivery has beenhindered by the absence of techniques to detect labile Zn2+ in the circulation. In this study, wereport the use of the fluorescent zinc-binding dye, ZnAF-2, to detect changes in labile Zn2+ in thecirculating plasma of the rat under standardized conditions, including exogenous infusions to raiseplasma Zn2+, and infusion of the chelator, citrate, to lower labile Zn2+ in the plasma withoutaltering total Zn2+ content. In a model of mild surgical stress (unilateral femoral arterial ligation),plasma levels of total and labile Zn2+ decreased significantly 24 hours following operation.Ultrafiltration of plasma into high and low molecular weight macromolecule fractionationsindicated that binding capacity of zinc in the high molecular weight fraction is impaired for theentire 24 hour interval following induction of mild surgical stress. Affinity of the filtrate fractionwas rapidly and reversibly responsive to anesthesia alone, decreasing significantly at 4 hours andrecovering at 24 hours; in animals subjected to moderate surgical stress this responsiveness waslost. These are the first reported measurements of labile Zn2+ in the circulation in any form of mildsystemic stress. Zinc undergoes substantial redistribution in the plasma, response to surgical stress,leading to increased availability in lower molecular weight fractions and in its labile form.

IntroductionZinc is an essential trace element for all living cells and is a co-factor for many cellularprocesses including protein synthesis, energy metabolism, nucleic acid synthesis, genetranscription, and programmed cell death (1,2). A wealth of experimental (3,4,5,6) andclinical (7,8,9) data indicate that total levels of Zn2+ in the circulation are decreased in avariety of chronic and acute conditions associated with impaired immune response. Thegroundbreaking work of Prasad, beginning in the 1960s demonstrated the biologicalsyndrome of chronic zinc deficiency in humans (10,11,12), and clinical response to zincsupplementation (13,14). Subsequent research has also demonstrated the value of zinc

© 2010 Mosby, Inc. All rights reserved.Corresponding Author: David I. Soybel, M.D., Department of Surgery, Brigham and Women’s Hospital, Division of General andGastrointestinal Surgery, 75 Francis St, Boston, MA 02118, Tel: 617 732 7930, FAX: 617 232 9576, [email protected]'s Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to ourcustomers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review ofthe resulting proof before it is published in its final citable form. Please note that during the production process errors may bediscovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

NIH Public AccessAuthor ManuscriptTransl Res. Author manuscript; available in PMC 2012 March 1.

Published in final edited form as:Transl Res. 2011 March ; 157(3): 139–149. doi:10.1016/j.trsl.2010.12.004.

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supplementation in other zinc-avid conditions such as bone marrow transplantation (15),uremia (16), and neurodegenerative disease (17).

To date most zinc assays used in biological research have reported total zinc content oftissue or fluids. The total content of Zn2+ in the plasma reflects components that are boundto plasma proteins such as albumin and alpha-2 macroglobulin (18), constituting more than99% of the total content in the plasma. The component that is loosely bound or free isdesignated as “labile” Zn2+. It is this component that is accessible to meet the requirementsfor key signaling and phagocytic functions (19,20,21) of cells in the circulation (22,23),including leukocytes (24) and endothelium (2). These considerations indicate that ongoingavailability of labile Zn2+ in the circulation and its delivery to peripheral tissues is a vitalfactor in the cellular response to shock, injury and infection. As with calcium, a divalentcation with parallel activities in biology, the availability of free zinc may not correlatedirectly to the total circulating concentration.

Investigation of the mechanisms of delivery has been impeded by the absence of a method todetect free Zn2+ in the circulation. This report summarizes our experiments using ZnAF-2, afluorescent dye that binds zinc selectively, as a reporter for nanomolar concentrations of freezinc concentration in the plasma. The present study was performed to investigate theconcentration of circulating total and labile Zn2+, in a rat model without prior zincdeprivation under conditions of moderate surgical stress. Elimination of malnutrition andzinc deficiency from the model enables investigation of labile zinc delivery to tissues inresponse to stress alone, in order to study the alteration of zinc availability that may bephysiologically adaptive, as opposed to the effects of zinc depletion. To our knowledgethese are the first reported efforts to obtain measurements of changes in plasma labile Zn2+

in such a clinical model.

MATERIALS AND METHODSReagents and solutions

Unless otherwise noted, all reagents were from Sigma-Aldrich (St. Louis, MO). ZnAF-2 waspurchased from Axxora (San Diego, CA). Fluozin-3 was purchased from Invitrogen/Molecular Probes (Carlsbad, CA). Ringer’s solutions used for preliminary screening studiescontained NaCl 145mM, KH2PO4 2.5mM, MgSO4 1mM, HEPES 10mM, EGTA 0.3mM,pH 7.4, with Ca2+ and Zn2+ added to maintain Ca2+ at 1mM and Zn2+ at desiredconcentrations. Free concentrations for Ca2+ and Zn2+ in Ringer’s solutions containingchelators such as EGTA or citrate were calculated based on the internet-accessibleWEBMAXCHELATOR (http://www.stanford.edu/~cpatton/webmaxc/webmaxcS.htm).

Experimental Animals and Surgical ProceduresMale Sprague Dawley rats weighing 300–350g were used for all experiments (Charles RiverLabs, Waltham, MA). For some studies, animals were purchased with cannulae(microrenethane MRE-040, external diameter 0.04 inches, internal diameter 0.025 inches)pre-placed in the femoral artery or vein several days prior to blood draw, in order tominimize acute stress. Rats were housed using standard animal care procedures (12:12 hourlight-dark cycle, food and water ad libitum). All animal care and experimental proceduresused were consistent with National Institutes of Health Animal Care and Use Guidelines andwere approved by the Institutional Animal Care and Use Committee at Harvard MedicalSchool. Rats were maintained under general anesthesia using pentobarbital intraperitonealinjection. Warming pads were used to maintain temperature.

For vascular access, the lower abdomen and groins were shaved and the femoral vesselswere surgically exposed. Cannulation of artery or vein was performed with 24-gauge IV

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http://www.stanford.edu/~cpatton/webmaxc/webmaxcS.htm

catheters (Angiocath, BD Medical, Sandy, Utah), accompanied by distal ligation using finesilk ligatures. The arterial catheter was used for invasive blood pressure measurement usinga Spacelabs multichannel monitor (Spacelabs Healthcare, Issaquah, Washington). Thecatheter was flushed with normal saline to maintain patency and provide fluid boluses, andwas also used for withdrawal of blood samples (1.5 ml at each time point) from the mostproximal port (~10 cm from the catheter) to minimize dead space.

Screening of conditions and candidate reporters for measuring free Zn2+ in plasmaPreliminary studies were performed to identify optimal conditions for assay, and includedexploration of the influence of simple salt solutions (100uM NaCl) containing different pH-buffers (10mM HEPES, Glycine, N-methyl-D-glucamine, or mono-/bi-sodium phosphate)on responsiveness of those reporters to Zn2+. The resulting solutions were plated in 96-wellplates and fluorescence was measured in a Synergy 2 microplate reader (Biotek Inc,Winooski, VT). Since content of labile Zn2+ in the plasma has been predicted to reside in thenanomolar range or lower (25,26,27), Fluozin-3 and ZnAF-2 (50nM each) were selected forinvestigation based on reports of their high selectivity for zinc and dissociation constants inthe low nanomolar range in aqueous solution (28,29,30). Among the buffers, HEPES wasthe only one that did not interfere with measurements of reporter fluorescence in simpleaqueous solution. In contrast to anticoagulants that are also chelators (EDTA, citrate) ofdivalent cations, we observed that heparin does not interfere with measurements in HEPES-buffered Ringer’s if placed in dilutions of 1:50 or less (data not shown).

Standardization of conditions for plasma assay of labile Zn2+ and fractionation of samplesPlasma samples were collected and measured immediately, as preliminary experimentsdemonstrated increased variability of measurements in samples stored on ice. Bloodspecimens were collected from the femoral artery catheter in 1.5 mL aliquots andcentrifuged at 10,000 ×g for 10 minutes. The plasma fraction was immediately assayed forlabile zinc. Total zinc, total protein and albumin were assayed in batches at the end of theexperiment.

In a subset of experiments, a 500 µl aliquot of plasma from each sample was filtered througha 10 kDa plasma filter (Amicon Ultra, Millipore, Billerica, MA) at 14,000 ×g for 15 minutesat 4°C. The filtrates were retained and concentrates were resuspended in zinc-free 18 MΩMilli-Q water. For measurements of labile Zn2+, whole or fractionated samples of plasma(50µl/sample) were plated in duplicate or triplicate on black 96-well plates (NUNC, ThermoScientific, Roskilde, Denmark). After an initial baseline fluorescence reading (ex. 485, em.528), ZnAF-2 (1.5µl of a 1 µM solution in DMSO) was added to each well, for a finalconcentration of 30 nM. Samples were mixed by gentle shaking for 10 seconds, andfluorescence was re-read.

Total Plasma Zinc assayPlasma total zinc was determined using the Quantichrom Zinc Assay kit (BioAssay Systems,Hayward CA), according to the manufacturer’s instructions. This zinc assay is a colorimetricassay based on zinc binding to a chromogen that reports at 425 nm. Results were read on themicroplate reader.

Plasma Albumin assayPlasma albumin was assayed using the BCP Albumin Assay Kit (BioAssay Systems,Hayward, CA) according to the manufacturer’s instructions, and results were read at 610 nmon the plate reader.

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StatisticsData were analyzed using standard statistical software (SigmaStat v3.5, Systat Inc, Chicago,IL). Continuous variables are expressed as mean plus or minus standard error. One-wayANOVA was used for statistical comparison of multiple groups, and Student’s t-test wasused for before and after comparison within groups where appropriate.

RESULTSEvaluation of fluorescent Zn2+ reporters for plasma assay

In order to assess the validity of using vital dyes for determination of zinc concentration insolution, we evaluated two candidate reporters, based on their known binding affinities forzinc in the nanomolar range: Fluozin-3 and ZnAF-2 (both in their free acid forms). Bothreporters excite in the green component region of the spectrum (485nm), with emissionsmeasured at 528 nm. When studied in simple HEPES-buffered Ringer’s solutions, goodtitration curves were obtained for Fluozin-3, confirming published reports that the Kd forZn2+ (31,32) lies in the low nanomolar range. When evaluated in Ringer’s solutions (Figure1A), ZnAF-2 provided a reliable range of fluorescence responses (from Fmin to Fmax) inresponse to increases in [Zn2+] from ~0nM (Ringer’s + 500µM EGTA) to 32nM. Whenevaluated in plasma, the response curve maintained its dynamic range, that is, the ratio ofthe fluorescence minimum when all labile Zn2+ is chelated with excess EGTA, compared tothe fluorescence maximum in the presence of excess Zn2+ (33) In HEPES-Ringer’s andplasma, this ratio is about 1:8. In addition, the curve shifted markedly to the right (Figure1B), indicating that the dye is capable of monitoring changes in labile Zn2+ despite thepresence of high affinity binding within the plasma. These observations accord withpublished estimates of labile zinc concentration in the plasma in the range of 2–10 nM.

Of note, excitation of plasma at 485nm does yield background fluorescence. We furtherfound that background fluorescence increases with Zn2+ content, increasing from about 10%of signal under baseline conditions to about 20% of signal when 100µM of Zn2+ is added toa plasma sample. As a result, all measurements require correction for background for eachexperimental condition, and this was done in the studies that are reported below. Of note,Figure 1A shows that the background fluorescence of dye in Ringers solution is about 4000units, which was constant throughout our experiments. On the other hand, in plasma (Figure1B) the difference between background fluorescence of dye in plasma and fluorescence inresponse to added zinc is much less. Because of the difficulty of independently calibratingthe dye in a complex fluid such as plasma under different circumstances, it seemed mostappropriate to summarize results by comparison with baseline measurements (normalizationto a baseline level of 1.0). Extrapolations to quantitative measurements are provided in thediscussion, below.

Sequential measurements in plasma during exogenous infusions of Zn2+ and citrateWe next determined whether the ZnAF-2 assay is capable of detecting controlled changes inthe labile Zn2+ content of plasma. Rats underwent cannulation of both femoral veins and onefemoral artery and were divided into three groups (n=4 each). In the first (control) group,only normal saline was infused in each of the venous catheters (1cc over 10 min). In thesecond group (Zn2+-infusion), a brief bolus infusion of ZnCl2 was performed through one ofthe venous cannulae (0.4 mg/kg, 1cc over 10 min) while saline was administered through theother (1cc over 10 min). In order to demonstrate detection of transient changes in labile Zn2+

in plasma, a third group of animals received infusion of a Zn2+ bolus infusion through onecannula while receiving infusion of sodium citrate (30mg/kg in 1cc, over 10 min) throughthe other. Citrate is a moderate affinity chelator for divalent cations including Zn2+ (Kd for

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Zn2+ 18µM). Arterial blood samples were drawn before (time 0) and 5, 15, 30, and 60minutes after infusion) for measurements of total and labile Zn2+ in plasma.

Measurements of total Zn2+ content in plasma, using a commercial colorimetric assay, areshown in Figure 2 and those of labile Zn2+ are shown in Figure 3. Among control animals,neither total nor labile Zn2+ levels were changed significantly over baseline. Among theanimals who received infusion of Zn2+ alone, levels of total Zn2+ were elevated immediatelyfollowing the infusion and remained elevated for the duration of study period, slowlyreturning toward baseline from a peak at 5 minutes. In these animals, increases in levels oflabile Zn2+ were also detected, peaking at 40% to 50% above baseline at 10 to 20 minutesand then slowly declining thereafter. Among the animals receiving both Zn2+ and thedivalent cation chelator sodium citrate, each infused through separate catheters, there was asimilar rise and time course of elevation in levels of total Zn2+. In contrast to animalsundergoing infusion of Zn2+ alone, initial increases in labile levels were observed before asharp, transient downturn was detected when the metal and the chelator had time to intermixin the circulation.

Alterations in plasma Zn2+ levels during moderate surgical stressTo evaluate disturbances in circulating levels of labile Zn2+, we performed studies in a ratmodel of moderate surgical stress. Rats underwent pentobarbital anesthesia, skin incision,tissue dissection, unilateral femoral artery ligation and cannulation, and blood sampling attime points A (time 0), B (one hour following cannulation), and C (five hours followingcannulation). The femoral artery was then decannulated and ligated, and rats were allowedto awaken. Twenty-four hours following the start of the initial surgical procedure, rats werere-anesthetized and the femoral artery was cannulated on the contralateral side for collectionof a final blood sample (time point D). Rats were then euthanized by pentobarbital overdoseand exsanguination. Rats that had been pre-cannulated in the femoral artery served ascontrols and were placed under anesthesia only using an identical protocol. Measurementswere obtained in both groups (n=6 for each group).

Summarized in Figure 4 are changes in levels of total Zn2+ in plasma, in pre-cannulatedcontrol rats undergoing anesthesia alone (Figure 4A) and in rats acutely, but mildly stressedby anesthesia and femoral artery cannulation (Figure 4B). While some variations wereobserved over the 24 hours of study in the control group, there was a significant decline intotal plasma content of Zn2+, in the stressed group at the 24 hour time point. Summarized inFigure 5 are changes in labile Zn2+. In control animals these were remarkably constant(Figure 5A) over the period of study, while in the stress group (Figure 5B) they decreasedsignificantly in period D. In this 24 hr interval following initiation of study, significantalterations in plasma albumin -- a key serum binding protein for Zn2+-- were not observed incontrol or stressed groups (Figure 6). Thus, in period D, the ratios of total (Figure 7) Zn2+ toserum albumin were markedly decreased (p<0.05). These findings suggest that the decreasein total Zn2+ was not due to alterations in plasma concentration of binding proteins; rather,the decline in total Zn2+ appears to reflect unloading of Zn2+ due to changes in plasmaprotein binding capacity.

We then explored potential alterations in distribution of Zn2+ among serum proteinfractions. Plasma proteins were separated by filtration of whole plasma samples (0.5ml), intohigh (>10kDa) and low (<10kDa) fractions. Volume of each fraction was adjusted to 0.5ml,using 18 MΩ water, then assayed for total Zn2+ content (200µl aliquots). Shown in Figure8A are measurements of total Zn2+ in plasma fractions filtrate (F, <10kDa) and concentrate(C, >10kDa) obtained from a group of animals (n=4) subjected to moderate surgical stress.Summarized in Figure 8B are calculations of the ratio of total Zn2+ in the F vs. C fractions,used as an index of redistribution between the two. The results indicate that changes in total

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Zn2+ content of plasma (Figure 4) largely reflect those in the large molecular weightfraction. In period D (24 hours), it appears that there is a transfer of Zn2+ content from thehigh molecular weight fraction to the low molecular weight fraction.

We then tested the hypothesis that surgical stress had altered the affinity of the larger andsmaller protein fractions for Zn2+. Aliquots of reconstituted plasma proteins (50µl) weremixed with 1.5µl ZnAF-2, and then fluorescence (Ex 485nm/Em 528nm) was measured atbaseline and following addition of 3.5µl aliquots of Zn2+ that increased total Zn2+ content inthe sample by an increment of 8µM. Shown in Figure 9 are summaries of studies in controls(n=6) and animals subjected to mild surgical stress (n=6). Labile zinc signal is shown bothbefore and after addition of ZnCl2.

A first observation is that there is considerable affinity for Zn2+ in both the high molecularweight (concentrate or C) and low molecular weight (Filtrate or F) fractions. In the Cfractions from control animals (Figure 9A), ZnAF-2 fluorescence was not altered followingaddition of 8 µM exogenous Zn2+, indicating a very high capacity for binding of Zn2+.Fluorescence in the F fraction (Figure 9B) was also much attenuated with addition of 8µMZn2+, a level that would be expected to saturate the reporter. A second observation is that inperiods B and C there was variability in the affinity for Zn2+ in the F fraction in controlanimals (Figure 9B), restored to baseline levels (period A) by the time measurements wereobtained in period D.

Of principal note were the responses in samples taken from animals subjected to stress. Asshown in Figure 9C, even mild surgical stress led to significant impairment of binding in theC fraction, in all periods following induction of stress. Conversely, stress seemed toeliminate the rapid responsiveness observed in control animals and, if anything, to enhanceZn2+ binding in the F fraction. Taken together, these observations are consistent with ourhypothesis that even moderate surgical stress leads to significant redistribution of Zn2+ fromthe larger- to smaller-sized proteins. In addition, these findings suggest that concentrationsof labile Zn2+ in the circulation remain remarkably constant due, in part, to suchredistribution and alterations in affinity of plasma proteins.

DISCUSSIONPrior reports of assays for free Zn2+ in extracellular fluids have focused on use of dialysates(27,32). In an early report (25) concentrations of Zn2+ were determined in equine plasma,utilizing an enzymatic bioassay, which required both time for equilibration, larger volumesand manipulation of conditions to exclude interference by Mg2+. In a more recent reports(34,35), a fluorometric assay based on an early generation reporter for Zn2+, zinquin, wasused to measure labile Zn2+ in the micromolar range in samples of plasma and other fluids,including cell-conditioned media. A key contribution of that report was the recognition that“lability” of the metal divalent cation is defined in large part by the selectivity, affinity andconcentration of the reporter itself. Such studies were conducted on samples obtained fromexperimental subjects that were not acutely stressed. To our knowledge, measurements oflabile Zn2+ in plasma samples have not been reported in patients who are acutely ill oranimals that are under experimental stress.

In plasma, background fluorescence with excitation in the green range is not ideal, butmanageable for semi-quantitative measurements. Optimal conditions for assay includeimmediate processing and use of minimal volume additions (<3%). In addition the reporter(~30nM) should be used in final concentrations that minimize, respectively, dilution ofsample and significant chelation by the reporter itself. In the studies reported here,

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utilization of ZnAF-2 was guided by its selectivity for Zn2+ and high fluorescence yield, aswell as the preservation of its dynamic range even in a complex fluid such as plasma.

If the reporter maintains its dissociation constant (Kd) for Zn2+ in a complex fluid such asplasma, then it is possible to utilize ZnAF-2 fluorescence to calculate approximateconcentrations of free Zn2+. Utilizing the Kd (5nM) obtained in Ringer’s solutions, whichaccords well with published values, and the data obtained in Figure 1B, we may infer a freeconcentration of Zn2+ of 1nM to 3nM, under baseline conditions (see arrow in Figure 1A).These calculations agree reasonably with predictions and experimental observationssuggesting that unbound Zn2+ levels in plasma are likely to be in the nanomolar range (26,27, 28).

An important caveat of such calculations is that changes from baseline fluorescence levelsare not linearly proportional to the changes in concentrations of labile Zn2+. In the middle ofthe dynamic range of the dye (about 5nM), they may be so, but not toward the margins ofthe response curve. Thus, when baseline concentrations rise or fall by 40%, as shown inFigures 2 and 5, there may be corresponding changes in actual concentration by a factor of 2or 3. Such disturbances may seem small, because they occur in the nanomolar range. It mustbe remembered, however, that such changes in this range can initiate or terminatephysiologic or pathologic processes, including initiation of pathways of apoptosis (36, 37)and degradation of extracellular matrix (38). The small scales on which such changes mayoccur should not obscure their potential importance in regulating activities of circulatingcells and peripheral tissues. Nevertheless, the feasibility of fully quantitative measurementsof labile Zn2+ in the circulation awaits development of dyes that do not require laboriouscorrections and normalizations against background fluorescence.

With this caveat, our studies with infusions of zinc with or without the nonspecific chelator,sodium citrate, confirm responsiveness of ZnAF-2 to changes in labile zinc in samples takenfrom the circulation. With infusions at 0.4 mg/kg (0.12mg for a 300g rat = 0.12mg/66mg/mmol=1.8uM), and given an initial volume of distribution limited to plasma (~0.2L/kg or6ml for a 300g rat), we would estimate initial rise in total plasma Zn2+ content of 300µM/L.Such expected increases in initial plasma Zn2+ content would be detected at the upper limitof the colorimetric assay for total Zn2+, as was observed (Figure 2A). The observed rise inZnAF-2 fluorescence accords well with that expected from an exogenous addition of Zn2+ tothe highly buffered plasma (Figure 1B). Moreover, we show that this reporting system canbe used to monitor changes in plasma Zn2+ when high capacity chelators, such as citrate, aresimultaneously introduced into the circulation.

Our studies also demonstrate that ZnAF-2 can be used to interrogate the affinity of plasma,and its different protein fractions, for free Zn2+. Using ZnAF-2, we confirm that normalplasma has marked binding capacity for zinc, and that the binding capacity largely—but notexclusively—reflects binding by large molecular weight macromolecules (Figure 9) such asalbumin and alpha-2 macroglobulin (18). We also provide evidence for binding in lowermolecular weight (<10kD) fractions, which becomes proportionately more important duringacute stress. Our studies indicate that changes in affinity can be monitored in large and smallmolecular weight fractions in samples taken directly from the circulation.

With characterization of the assay and its limitations, we have been able to investigate labilezinc distribution in a model of mild surgical stress. The model itself involves dissection,cannulation, and ligation of the femoral vessels in the rat hind limb. This degree of surgicalstress has been characterized in previous studies as mild and is not associated with elevationin circulating markers of injury (39). In our model we show that plasma albumin, and acutephase reactant, is unchanged throughout the experiment. This finding confirms that

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alterations in total and free levels of circulating Zn2+ are not easily attributed to changes inlevels of important binding proteins, but also confirming the mild nature of the surgicalstress in this model. In this study, we find that even mild surgical stress leads to decreases incirculating Zn2+ overall and causes alterations in distribution of Zn2+ among differentfractions of plasma proteins. The decline in total zinc levels was not attributable toconcomitant decline in plasma albumin concentration, which did not change significantly in24 hours following induction of stress. The subsequent studies using plasma fractionsindicate that changes in zinc affinity of plasma proteins is the likely cause of the overalldecline in total Zn2+ levels, and may also function to regulate concentrations of labile Zn2+

in the plasma that are observed throughout the study. The results of our experiments extendand clarify very early clinical reports that zinc in the plasma is unloaded to peripheral tissuesduring some forms of acute stress (40).

Our studies suggest that Zn2+ content of plasma can shift between high affinity, highcapacity pools in high molecular-weight fractions to low affinity or labile pools duringsurgical stress. Much like the ionized calcium pool in acute calcium deficient conditions(41,42), this redistribution of Zn2+ may occur so that it can more easily be transferred tomeet demand in peripheral tissues. Our observations also emphasize that anesthesia andmoderate surgical stresses can elicit subtle, acute responses in delivery and clearance ofmetal ions from the circulation. Modification of plasma albumin metal ion(Cobalt) bindingaffinity has been shown to occur in response to organ ischemia (43,44). Our modelcharacterizes the responses of both the total circulating zinc pool and the small labilefraction in acute stress; our results indicate that total zinc and labile zinc both decrease inmild stress, and that zinc binding increases in the lower molecular weight plasma fraction.Our results and other reports (43,44) suggests that metal ion binding has a dynamic role inthe metabolic response to stress, and can be studied independently of other markers. Inaddition, chronic zinc deficiency and zinc responsive conditions such as chronic steroid usemay be studied in terms of labile zinc affinity.

Acute changes in zinc availability have not been recognized until now as having clinicalsignificance. Chronic Zn2+ deficiency is well recognized as a risk factor for infection andpoor healing. Increasingly, however, it has become clear that demand of peripheral tissuesand parenchymal cells for Zn2+ may occur in response to endocrine stimulation (45), normalphysiologic activity (46), as well as oxidative stress (47,48) and systemic sepsis (24).Rapidly deployable assays for free Zn2+ in extracellular fluids such as the one describedhere are crucial for development of studies to understand how such acute demand for Zn2+ issatisfied and to identify experimental conditions and clinical circumstances in which thecapacity of the circulation to deliver Zn2+ is insufficient to meet the demand. Clinicalconditions previously identified as zinc deficiency may be need to re-defined in the contextof zinc utilization and distribution.

AcknowledgmentsThe authors gratefully acknowledge Christopher J. Frederickson PhD and Leonard L. Giblin PhD for insightfulreview and commentary on this series of experiments.

Grant Support: American College of Surgeons Resident Research Fellowship (JEK), T32 DK007754 (JEK, JM),intramural support from the Department of Surgery, Brigham and Women’s Hospital (EK) and RO1 DK069929,(DIS).

Abbreviations

Zn2+ ionized divalent zinc

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EGTA Ethylene glycine tetra acetate

Kd Dissociation constant

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Figure 1.Titration curves for ZnAF-2 fluorescence measured with excitation 485nm and emission at528nm. In Ringer’s solutions (A), the dye is responsive to changes in free concentration ofZn2+, over the range 0 nM to 32 nM, confirming the profound buffering capacity of theplasma for Zn2+. In rat plasma (B), the same range responsiveness is observed in rat plasmawith all Zn2+ chelated [POINT X] or in samples supplemented with high concentrations ofexogenous ZnCl2, at levels up to 16 mM, indicating that responsiveness of the dye ispreserved in plasma. In panel B the vertical dashed line indicates the fluorescence ofuntreated plasma minus background. This value, about 1400 RFU, corresponds with aconcentration of about 1 nM of labile zinc as predicted by the Grynkiewicz fluorescenceequation (33). The arrow in panel A marks the corresponding concentration, 1 nM protein-free Ringers solution. All data are summarized as mean relative fluorescence units (RFU) ±SD, N = 3 for all data points.



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Figure 2.Total zinc measurements in whole plasma samples from rats undergoing infusions of saline(5ml over 20 min, symbol ), exogenous zinc sulfate (0.4 mg/kg in 5 ml over 20 min,symbol ), or exogenous zinc sulfate (0.4 mg/kg) and sodium citrate (30 mg/kg) throughseparate infusion catheters (symbol ). Data points are shown as mean ± SEM. * = p < 0.05vs. control, N =4.



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Figure 3.Labile Zn2+ measurements in whole plasma samples, using ZnAF2 fluorescence, duringinfusions of saline, zinc sulfate or zinc sulfate and citrate (same animal groups as in Figure2). Rats underwent infusions of saline (5ml over 20 min, symbol ), exogenous zinc sulfate(0.4 mg/kg in 5ml over 20 min, symbol ), or exogenous zinc sulfate (0.4 mg/kg) andsodium citrate (30 mg/kg) through separate infusion catheters (symbol ). Labile zinc risessharply in the earliest time points after infusion, buffered transiently with the concurrentinfusion of the chelator, citrate. Data points are shown as mean ± SEM. * = p < 0.05 vs.control, N =4.



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Figure 4.Total zinc measurements in whole plasma samples from animals pre-cannulated to avoidacute surgical stress, that were subjected to anesthesia alone or animals subjected toanesthesia and a mild surgical stress. In control animals (A), there were not significantalterations throughout the 24 hour experimental period. In stressed animals (B), levelsdecreased progressively, becoming statistically different from baseline at 24 hours (TimePoint D). Data points are shown as mean ± SEM. * = p < 0.05 vs. Time Point A, N = 6.



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Figure 5.Labile Zn2+ in whole plasma samples from animal groups undergoing anesthesia alone ormild surgical stress (same animal groups as in Figure 4). In control animals (A), nosignificant changes were observed during the 24 hr period of observation. Stressed animals(B) exhibited significant decrease; mean 40% below baseline at 24 hrs (Time Point D). Datapoints are shown as mean ± SEM. * = p < 0.05 vs. Time Point A, N = 6.



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Figure 6.Plasma albumin levels measured in plasma samples of animals undergoing anesthesia alone(A) or mild surgical stress (B); same animal groups as in Figure 4. No significant differenceswere observed over time in either group, confirming that stress in both groups was mild. Thestable level of albumin concentration is consistent with mild stress, without significant acutephase response. Data points are shown as mean ± SEM. N = 6.



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Figure 7.Whole plasma total zinc:albumin ratio calculated from measurements reported in Figures 4and 5. In the group subjected to mild surgical stress, there is a more than 50% decrease inthe total zinc to albumin ratio in the Stress group at 24 hours (Time Point D). Data points areshown as mean ± SEM. * = p < 0.05 vs. Time Point A, N = 6.



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Figure 8.A. Total zinc content in high and low molecular weight fractions obtained from plasma ofanimals undergoing mildly surgical stress. Plasma was separated by centrifugal filtrationacross a 10 kDa filter into concentrate (> 10 kDa, grey bars) and filtrate (< 10 kDa, blackbars) fractions at each time point. B: Calculations for the ratio of Zn2+ content in theconcentrate (>10kD fraction) to that in the low molecular weight fraction (<10kD fraction).The data indicate that, over the 24 hour time course of the experiment, even mild surgicalstress induces a shift of Zn2+ content from the high- to the low-molecular weight fraction.Data points are shown as mean ± SEM. p = NS versus Time Point A. N = 4.



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Figure 9.Measurements of buffering capacity for Zn2+ within high- and low-molecular weightfractions of plasma proteins taken from control animals and animals undergoing mildsurgical stress. Buffering capacity was assessed by mixing samples with ZnAF2 andmeasuring fluorescence before and after addition of a standard amount (8µM) of ZnCl2.Responses of higher magnitude to the addition of exogenous Zn2+ indicate lower bufferingcapacity. Panels A and B provide information about buffering for samples (high-molecularweight concentrates and low-molecular weight filtrates) from control animals, while PanelsC and D provide information for samples from animals undergoing mild surgical stress. A:in control animals subjected only to anesthesia, a very high level of binding capacity isobserved within the high molecular weight fraction, demonstrated as nearly identical levelsof labile zinc with () or without () added zinc. B: in control animals, a transient increasein binding capacity of the lower molecular weight fraction is observed and restored at 24hrs. C: following anesthesia and mild surgical stress, significant decreases in bindingcapacity in the high molecular weigh fraction are observed and are not fully restored at 24hours. D: in the stressed animals, the rapid responsiveness is lost that was observed undercontrol conditions (Panel B). Data points are shown as mean ± SEM. * = p < 0.05 vs.Control, N = 6.



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Redistribution of labile plasma zinc during mild surgical stress in the rat