Pergamon Camp. Biochem. Physiol. Vol. 1 IOA, No. 4. pp. 367-373. 1995

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Maturational differences in coronary flow and interstitial transudate adenosine during alteration of perfusate oxygenation in isolated rabbit hearts

Gerard Holmes,* M. L. Epstein?_ and G. Paul MatherneJ: *Department of Pediatrics (Cardiology), University of New Mexico School of Medicine, Albuquerque, New Mexico, U.S.A.; TDivision of Cardiology, Children’s Hospital of Michigan, Detroit, Michigan, U.S.A.; and SDepartments of Pediatrics and Physiology, University of Virginia College of Medicine, Charlottesville, Virginia, U.S.A.

This study was designed to investigate effects of perfusate oxygenation and maturation on coronary flow (CF), interstitial transudate adenosine (ITA) and coronary effluent adenosine (CEA) in isolated rabbit hearts. Hearts were paced at a fixed rate and were perfused under constant pressure at two different levels of perfusate oxygenation: baseline (B) (PO*= 408 +_ 7 mmHg, O2 content = 1.28 +_ 0.03 ml OJdl) and a lower level (L) (pOz=189+4 mmHg, O2 content =0.59f0.02 ml O,/dl). CF was higher in immature (I, age 5 weeks) compared with mature (M, age 12 weeks) hearts at both levels of perfusate oxygenation (9.7 f 0.4 vs. 7.7 t 0.3 and 12.9 ) 0.4 vs. 9.2 + 0.3 ml/min/g). I hearts had correspondingly higher values for myocardial oxygen consumption (MVOS (53 _+ 3 vs. 49 &- 2 and 39 + 3 vs. 35 + 2 PL OJmin/g), but similar values for venous oxygen tensions (~02) (240 f 9 vs. 219 + 10 and 74 _+ 4 vs. 62 + 3 mmHg), compared with M hearts at B and L. Although interstitial transudate adenosine (ITA) concentration was similar in I and M hearts at B (409) 75 vs. 254 + 39 nM), it was lower in I than M hearts at L (2500+ 770 vs. 4210& 1000 nM). Comparing responses to reduction of perfusate oxygenation, a similar decrement in v02 (175+ 10 vs. 166+ 12 mmHg) was found to correspond to a lesser increment in ITA (2090 +_ 110 vs. 3950 +_480), but a greater increment in CF (3.2 +_ 0.2 vs. 1.5 _+ 0.4 ml/min/g) in I than in M hearts. CEA concentration was, in general, higher at L than at B. but was similar in I and M hearts for both experimental conditions of perfusate oxygenation. These results suggest that: (1) CF was higher in I than M hearts due to greater metabolic demand; (2) CF was equally effective in maintaining myocardial oxygenation in both groups; (3) adenosine may help mediate hypoxia-induced coronary vasodilation in isolated hearts from both age groups; and (4) the relationships between myocardial oxygenation and interstitial adenosine concentration as well as between interstitial adenosine concentration and coronary flow are altered as a consequence of maturation in isolated rabbit hearts.

Key words: Maturation; Coronary flow; Adenosine; Oxygenation.

Comp. Biochem. Physiol. IlOA, 367-373, 1995.

Correspondence to: Gerard Holmes M.D., Ph.D., Dept. Introduction Pediatrics, UNM. School of Medicine, ACC 3 West, Albuquerque, NM 87131-5311, U.S.A. Although coronary blood flow is affected by

Received I5 April 1994; revised 17 August 1994: accepted 15 September 1994

a variety of determinants, including hydro-

Abbreoiarions: CF: coronary flow; ITA: interstitial transu- static forces, autonomic tone, and anatomic

date adenosine; CEA: coronary effluent adenosine; factors, it is primarily regulated in accordance

MVOI: myocardial oxygen consumption; RH: reactive with myocardial metabolic need. Adenosine. hyperemia. a naturally occurring vasodilatory cardiac

367

368 G. Holmes er al

nucleoside, is thought to be an important medi- ator of this process (Feigl, 1983, Belloni 1979; Klocke and Ellis, 1980). The mechanism through which myocardial metabolism and adenosine release are linked remains to be completely elucidated; however, changes in the ratio of oxygen supply to myocardial demand and the cytosolic phosphorylation potential have been implicated (Berne, 1980; Sparks and Barden- heuer, 1986; Bardenheuer and Schrader, 1986).

Vascular density is higher in immature than mature myocardium (Tomanek and Hovanec, 198 1, Tomanek et al., 1982). This has been shown to impart to immature myocardium a compara- tively greater capacity for maximal perfusion (Toma et al., 1989; Holmes and Epstein, 1993) and may also reasonably be expected to result in differences in the way coronary flow and cardiac adenosine levels are regulated with respect to myocardial oxygen balance (Toma et al., 1988). Such differences might also be expected to arise as a consequence of changes in myocardial energy metabolism occurring during growth and maturation (Matherne et al., 1990).

In support of maturational differences in the way coronary flow and cardiac adenosine are regulated, Toma et al. (1988) found that equiv- alent levels of myocardial metabolic stimulation with norepinephrine produced greater coronary flow, higher myocardial oxygen delivery and lower coronary effluent adenosine concentration in immature compared with mature isolated guinea-pig hearts. Similarly, Matherne et al. (1990) found that an equivalent reduction of perfusate oxygen availability resulted in lower coronary resistance in immature than mature isolated rabbit hearts. However, in contrast to the findings of Toma et al (1988), they found interstitial transudate adenosine levels to be similar in immature and mature hearts at the lower perfusate oxygen tension.

Matherne et al. (1990) perfused hearts at constant flow and may have failed to find maturational differences in interstitial transudate adenosine concentration at the lower level of perfusate oxygenation because of excessive limitation to myocardial oxygen delivery. Accordingly, this study was designed to investigate potential maturational differences in the effects of reduction in perfusate oxygenation on coronary flow and interstitial transudate adenosine levels in a preparation in which coronary flow and myocardial oxygen delivery were allowed to be physiologically regulated.

Materials and Methods Animals and preparation

Twenty-one immature (age 5 weeks), and 15 mature (age 12 weeks) rabbits of either sex

were anesthetized with ketamine and xylazine (50 mg kg and 5 mg/kg IM, respectively). Following tracheostomy and initiation of mechanical ventilation, a thoracotomy was performed. The right and left superior vena cava and the inferior vena cava were ligated and the hearts rapidly excised. The aorta was cannulated and retrograde perfusion was instituted in a non-recirculating Langendorff mode. Total ischemic time between excision and onset of perfusion was less than 30 sec. Hearts were perfused at a constant inflow pressure (70 mmHg) with oxygenated modified Krebs-Henseleit bicarbonate buffer containing (mmol/l): NaCl 122, KC1 4.7, CaC& 2.5, MgS04 1.2, NaHC03 22, KH,P04 1.2 and glucose 11.

After onset of perfusion, the pulmonary artery was cannulated to allow collection of the venous effluent, to vent the right ventricle to atmospheric pressure, and to prevent leakage of coronary effluent onto the cardiac surface. A left ventricular vent was placed through the cardiac apex to drain the thebesian return away from the cardiac surface. Pacing electrodes were placed on the right ventricular epicardial surface and recording electrodes were placed on the posterior left ventricular epicardial surface. Hearts were paced at 350 beats/min.

Hemodynamic monitoring

Coronary inflow pressure was monitored by a fluid filled line attached to the perfusion cannula and connected to a Statham P23 ID pressure transducer (Gould Inc., Cleveland, Ohio). Coronary flow was measured using a cannulating (2 mm ID) Biotronex electromagnetic flowmeter. Surface electrograms were monitored via the left ventricular recording electrodes. Pressure, flow and electrographic signals were recorded with a Gould ES 1000 recorder. Samples of perfusate and coronary venous effluent were obtained in gas-tight syringes. Gas tensions and pH were measured directly using a Corning 168 pH/Blood gas analyzer. Myocardial oxygen consumption (MV02) was calculated as:

MVO: (37 ~1 OJmin/g) = (P‘%OZ - P,O?)

x coronary flow (c/760).

where P,Oz and P,O, refer to perfusate and venous effluent PO? (mmHg). respectively. and c=O.O239 (Bunsen solubility coefficient of oxygen dissolved in perfusate at 37 C, ml OJatm/ml).

Experimental protocol

The Krebs-Henseleit buffer was initially equilibrated with a gas mixture containing 95% O?, 5% CO? at 37 C. This produced pO? 408+_7 mmHg, pC0, 30+ 1 mmHg and pH 7.41 + 0.01 with an O? content of 1.28 + 0.03 ml

Maturation and interstitial adenosine 369

02/dl. This condition of perfusate oxygenation will henceforth be referred to as the baseline condition (B). Hearts were equilibrated for a 30 min period under baseline conditions to allow recovery from the anoxic effects of isolation. During this time, period coronary flow was observed to stabilize to a steady state. Following this equilibration period, perfusate and coronary venous effluent were sampled for measurement of gas pressures and pH. Sampling of the interstitial transudate and the coronary venous effluent was also done to allow measurement of their respective concentrations of adenosine. Coron- ary flow, coronary inflow pressure and heart rate were recorded. Myocardial oxygen consumption was calculated for this sampling period (baseline or B) as indicated above.

Perfusion of hearts at a lower level of oxygenation was then initiated by equilibrating perfusate with a gas mixture containing 30% 02, 5% CO, and 65% N,. This produced pOz 189+4mmHg,pCOz29+1,andpH7.39~0.01 with an O2 content of 0.59 + 0.02 ml OJdl. This condition of perfusion will hereafter be referred to as one of lower perfusate oxygenation (L). After a 5 min equilibration period, during which coronary flow was observed to stabilize to a steady state, repeat samplings and measurements of hemodynamic parameters were done.

Prior to termination of experiments, the peak reactive hyperemic response (RH) to 90 set total coronary inflow occlusion was measured for the purposes of determining maximum myocardial flow rates and coronary flow reserve.

Measurement of interstitial transudate and coronary efluent adenosine

Methods for measurement of ITA were as previously described (Matherne et al., 1990). Briefly, epicardial discs (6 mm diameter) were cut from sheets of porous hydrophilic Micro-Sep Magna Nylon 66 Membrane Filters (Micron Separation, Inc. Westboro, MA) with pore size 0.45 pm. Discs were weighed before soaking in Krebs-Henseleit solution, and for each sampling period, two wetted discs were applied to the anterior epicardial surface of the left ventricle and left in place for 2 min to allow equilibration of disc fluid with the interstitial transudate fluid. The discs were then removed, reweighed to determine sample volume, and stored in vials at

- 80°C until analyzed for adenosine content by HPLC.

Discs were thawed and placed in 200400 ~1 of distilled water to elute the contents over 60 min. The diluted samples were filtered (0.2 pm) in Spin-X tubes (Costar INC., Cambridge MA) with the aid of centrifugation and then placed in HPLC tubes. One hundred to 200 ,~l of this sample or filtered undiluted venous effluent were injected onto a C-18 reverse phase column (Su-plelco LC 18s; Supelco, Inc., Bellefonte, PA) and then eluted using a liner 30 min buffer gradient (100 mM KH?PO+ 1% methanol, pH 5.53 at time zero to 100 mM KH2P04, 25% methanol, pH 5.58 at 23 min) at 1.3 ml/min. Absorbance was continuously monitored at 254 nM with a Kratos model 773 variable wavelength detector (Kratos Analytical Instru- ments, Ramsey, NJ). Peaks were quantitated by comparison of peak areas with those from standards. Standards were routinely run with each set of samples. Preliminary studies showed that recovery of known amounts of adenosine applied to discs exceeded 94%.

Statistics

Values were expressed as means + SEM, unless otherwise indicated. Statistical comparisons among maturational stages and experimental conditions were made using a two way analysis of variance (ANOVA) and the Newman-Keuls range test. Comparisons between maturational stages for data expressed as the change from baseline were made using Student’s unpaired t-test. A P value of 10.05 was considered statistically significant in all instances.

Results

Values for body weight, wet heart weight, dry heart weight, and percent myocardial water content are given in Table 1. Immature and mature hearts had similar water content; thus, while results are given below in terms of wet heart weight, similar results are obtained when values are expressed in terms of dry heart weight.

Values for coronary flow, oxygen delivery, oxygen extraction, oxygen consumption, venous oxygen tension, venous carbon dioxide, and venous pH are given in Table 2. Coronary flow was higher in immature compared with mature

Table 1. Body weight, wet and dry heart weight, percent water content in heart (% water), and hematocrit percent in immmature and mature rabbits

Immature Mature

Body wt

(grams)

886+28 2193_f58*

Heart wt Dry heart wt Hct (grams) (grams) % water (%)

2.302 *0.071 0.438 * 0.022 81k1 41fl 4.940f0.138* 1.005 * 0.047* so*1 43+1

*Mature differs from immature, P~0.05

370 G. Holmes et al.

Table 2. Hemodynamic parameters measured in isolated hearts of immature and mature rabbits at baseline and at the

lower level of uerfusate oxvgenation

CF

(mhminig)

Oxygen del.

(Nminig) a-v oxygen

(mmHg) Oxygen cons.

Wminig) Venous 02

(mmHg) Venous CO2

(mmHg) Venous pH

Lower Hyperemia

Baseline Lower

Baseline Lower

Baseline Lower

Baseline Lower

Baseline Lower

Baseline

9.7_+0.4* 12.9*0.4*,t 19.0+0.7*,$ 128_+6* 79 f 3*.t

186+9 122*4t 53+3* 49 + 2*,t

240+9 74*4t 40*1 38_+1

7.29+0.01

7.7kO.3 9.2+0.3* 12.5~0.5f

94_+5 52f3T

169+11 119_+5t 39*3 35+2t

219_+ 10 62+3t 40*1 40&l

7.30+0.01 Lower 7.27+0.01 7.29+0.01

*Immature differs from mature, PGO.05. tLower (L) differs from baseline (B), P < 0.05. $Differs from baseline (B) and lower perfusate oxygenate(C),

PSO.05.

hearts under both conditions of perfusate oxygenation and also at a peak response to 90 set of inflow occlusion. Immature hearts increased their coronary flow (Fig. 2) to a comparatively greater extent in going from baseline to conditions of lower perfusate oxygenation than did mature hearts (3.2 + 0.2 vs. 1.5 + 0.4 ml/min/ g; P~0.05). Despite this, immature hearts used less of their maximal coronary flow rates, as determined by measured peak reactive hyper- emit flow responses to inflow occlusion, than did mature hearts under both experimental con- ditions of perfusate oxygenation (51 f 2 vs. 68 + 2% at baseline and 62 f 2 vs. 74 f 1% at the lower level of oxygenation, respectively; PIO.O5%).

‘r

VOZ (mm W

Fig. 1. Interstitial transudate adenosine (ITA) versus venous oxygen tension (001) in isolated hearts of immature (-) and mature (--3 rabbits under conditions of baseline and lower perfusate oxygenation (mean_+ SEM). *Similar decrements in 002 corresponded to a greater increment in ITA in mature compared with immature hearts in response to reduction in

perfusate oxygenation (PsO.05).

14

7-

Fig. 2. Coronary Aow versus interstitial transudate adenosine concentration in immature (-) and mature (---) isolated rabbit hearts at baseline and at the lower level of perfusate oxygenation (meanf SEM). *A greater increment in ITA corresponded to a lesser increment in coronary flow in mature compared to immature hearts in response to reduction in

perfusate oxygenation (P 5 0.05).

The increased coronary flow seen at the lower level of perfusate oxygen availability did not fully compensate for the reduced oxygen content in our preparation, resulting in myocardial oxygen delivery being lower in both immature and mature hearts under this condition than at baseline (38 +2 vs. 45 f 2% of baseline, respectively; P = non-significant). Oxygen deliv- ery was, however, higher in immature than in mature hearts under both experimental con- ditions, reflecting the higher coronary flow rates seen in this group.

Immature and mature hearts both extracted less oxygen at the lower level of perfusate oxygenation compared with baseline. There were no statistically significant maturational differ- ences in oxygen extraction at either level of perfusate oxygenation.

Myocardial oxygen consumption was de- pressed to a similar extent relative to baseline values in immature and mature hearts (8 + 1% vs. 10 + 1% of baseline, respectively; P= non- significant). Immature hearts, however, had higher oxygen consumption than did mature hearts under both conditions of perfusate oxygenation.

Coronary venous oxygen tension (Fig. 1) was decreased to a similar extent from baseline in going to the lower level of perfusate oxygenation in both immature and mature hearts (175 f 10 vs. 166 +_ 12 mmHg; P = non-significant). Immature and mature hearts had similar coronary venous oxygen tensions under both experimental conditions of perfusate oxygenation. Venous carbon dioxide tension and venous pH values were similar with respect to experimental condition and maturational stage.

Maturation and interstitial adenosine 371

Table 3. Adenosine concentration in interstitial transudate (ITA) and coronary effluent (CEA) at baseline and at the lower level of perfusate oxygenation in isolated hearts of

immature and mature rabbits

Immature Mature

ITA Baseline 409 f 75 254+39

(nM) Lower 2500 f 773t 4210+ lOOO*,t CEA Baseline 142+30 125,24

(nM) Lower 656 + 63t 588,917

*Mature differs from immature, PGO.05. tLower differs from baseline, P~0.05.

Values for interstitial transudate adenosine and coronary effluent adenosine concentrations are given in Table 3. Interstitial transudate adenosine concentration (ITA) was similar in immature and mature hearts at the baseline level of perfusate oxygenation, but was approximately twice as high in mature than in immature hearts at the lower level of perfusate oxygenation. Although ITA was increased at the lower level of perfusate oxygenation compared with baseline in both experimental groups, this increase was greater in mature compared with immature hearts (3950 + 480 vs. 2090 f 110 nM, respect- ively; P_C 0.02).

Coronary effluent adenosine concentration was increased in both immature and mature hearts in response to lowering oxygen availability (5 14 + 69 vs. 463 f 44 nM, respectively; P = non- significant). Coronary effluent adenosine con- centration did not differ between immature and mature hearts at either level of perfusate oxygen availability.

Discussion

The higher values for coronary flow and myocardial oxygen delivery seen in immature compared with mature hearts in this study could have been either a response to higher myocardial metabolic need or a result of an increased vascular capacity for myocardial perfusion. Because myocardial oxygen consumption was higher, while venous oxygen tension was similar under both conditions of perfusate oxygenation in immature compared with mature hearts, we conclude that the higher coronary flow seen in immature hearts was attributable to greater metabolic demand. Inasmuch as peak reactive hyperemic responses to transient inflow occlu- sion (90 sets) were also higher in immature compared with mature hearts, we also conclude that the immature hearts had a comparatively greater maximum capacity for myocardial perfusion than mature rabbit hearts. Indeed, immature hearts used less of their maximal coronary flow rates than did mature hearts under both levels of perfusate oxygenation studied, demonstrating that they had a higher coronary

reserve under these conditions. The present findings regarding maturational differences in maximal coronary flow rates corroborate our previous findings in rabbits (Holmes and Epstein, 1993) and are consistent with those of others in other species (Toma et al., 1984).

It is unclear why both immature and mature hearts had apparently significant coronary flow reserve as determined by their peak reactive hyperemic responses to coronary inflow occlu- sion, yet did not increase coronary flow sufficiently to maintain myocardial oxygen consumption during reduction of perfusate oxygenation to the lower level. At least two possibilities, however, must be considered. First, it is possible that, during reperfusion, there was flow dependent release of a vasoactive compound such as nitric oxide which was not released during reduction in perfusate oxygenation. Second, it is likely that inflow occlusion caused a build up of additional metabolites which did not build up during reduction of perfusate oxygenation and that these metabolites either augmented the vasodilatory effects of other compounds like adenosine or stimulated ad- ditional vasodilation directly.

Venous oxygen tension did not differ in immature and mature hearts under either condition of perfusate oxygenation studied, suggesting that these hearts were equivalent with regard to the degree to which coronary flow maintained myocardial oxygenation. A similar decrease in ~0~ (166+ 12 vs. 175+ 10; P=non- significant) produced a greater increase in ITA concentration (3950 + 480 vs. 2090 &_ 110 ]nM, PsO.05) in mature compared with immature hearts (Fig. 1), suggesting that immature hearts produced less adenosine in response to a similar decrement in myocardial oxygen tension than mature hearts. Such a maturational difference in the relationships of myocardial oxygenation and adenosine concentration might seem contrary to what would be expected, given the probable link between cardiac adenosine production and cytosolic phosphorylation potential (Sparks and Bardenheuer, 1986; Bardenheuer and Schrader, 1986) and considering that the efficiency with which ATP is utilized is thought to be lower in immature than mature myocardium (Mahony, 1988; Kissling and Rupp, 1986; Nakanishi et al., 1986). However, these differences may be accounted for inasmuch as immature hearts are thought to have a greater capacity for anaerobic production of ATP than mature hearts (Jar- makani et al., 1978), and may, thus, be better able to maintain cytosolic phosphorylation potential than mature hearts under conditions of low oxygen availability. It is also important, in this regard, to recognize that, because venous oxygen tension is an imperfect measure of myocardial

372 G. Holmes et al

oxygenation, inferences regarding maturational differences in the relationships between myocar- dial oxygenation and adenosine production using venous oxygen tension should be made cautiously.

The concentrations of interstitial transudate and coronary effluent adenosine obtained in immature and mature hearts (Table 3) are of similar magnitude as have been previously reported in isolated heart preparations (Toma et al., 1988; Matherne et al., 1990; Decking ef al., 1988; Fenton et al., 1990; Heller and Mohrman, 1988). Interstitial transudate and coronary sinus adenosine concentrations were increased at the lower level of perfusate oxygenation compared with the baseline in both immature and mature hearts. Inasmuch as interstitial transudate adenosine levels have been reported to be a close approximation of interstitial adenosine levels (Decking et al., 1988; Fenton et al., 1990; Heller and Mohrman, 1988) these data are consistent with a role for adenosine in mediating the coronary vasodilation seen during reduction of perfusate oxygenation in these groups.

Contrary to the findings of Matherne et al (1990) interstitial transudate adenosine concen- tration was found to be similar in immature and mature hearts at the baseline, but lower in immature compared with mature hearts at the lower level of perfusate oxygenation. Although several potential causes need to be considered, we speculate that the most likely cause for these differences is preparation-dependent differences in myocardial oxygenation as Matherne et al. (1990) fixed coronary flow and myocardial oxygen delivery in isovolumetric working hearts, whereas in this study, non-working hearts were allowed to physiologically regulate coronary flow and oxygen delivery.

A greater change in ITA concentration (3950 _+ 480 vs. 2090 _+ 110 nM) corresponded to a lesser change in coronary flow (1.5 +0.4 vs. 3.2 kO.2 ml/min/g) in mature compared with immature hearts (Fig. 2). These data suggest increased coronary sensitivity to interstitial adenosine in immature compared with mature hearts. Although increased coronary sensitivity to interstitial adenosine in immature compared with mature rabbit hearts is consistent with the findings of Matherne et al. (1990) it is in apparent opposition to the findings of Buss et al. (1987). These later workers reported reduced coronary responsiveness to extrinsically infused adenosine in hearts of neonatal com- pared with adult rabbits. However, these apparent differences in study findings may be explained by considering that interstitial adenosine may act at a different receptor level (smooth muscle) than extrinsic adenosine (endothelial) (Bellardinelli et al., 1989) and

that these sites may have different sensitivities to adenosine. Alternatively, it is also possible that there are differences in adenosine receptor sensitivity between the neonatal hearts studied by Buss et al. (1987) and the 5-week old hearts used in this study. Finally, it should also be pointed out that the present data may also be explained by postulating that adenosine is a relatively more important mediator of hypoxia- related coronary vasodilation in mature than immature hearts. For example, it is possible that a mediator other than adenosine was also released in response to reduction in perfusate oxygenation in immature hearts lessening the relative stimulus or need for adenosine release and resulting in a comparatively lower ITA concentration.

Because isolated, unloaded, paced hearts were used in the present study, higher myocardial oxygen consumption in immature compared with mature hearts may be interpreted as indicating that either the intrinsic work of contracting at a given heart rate was higher in immature than mature hearts or that the efficiency (mechanical or chemical) of contract- ing at a given rate was lower in immature compared with mature hearts. Although pre- vious experimental data exists to support either of these potential causes for higher oxygen consumption in immature hearts (Matherne et al., 1992; Mahony, 1988; Kissling and Rupp, 1986; Nakanishi et al., 1986) the present data is insufficient to determine which of these factors was responsible.

Ackno,vledgements-This study was supported by the American Heart Association-Florida Affiliate (II A 92-95) and by funds from the Children’s Miracle Network Telethon. The authors wish to thank Mr David Coverston and Mr Dan Peters for technical support in raising the animals, in performing the experiments. and in the preparation of illustrations. We also wish to acknowledge MS Deborah Floyd in preparing this manuscript.

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