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J. Anim. Sci. 2002. 80:3307-3314
© 2002 American Society of Animal Science

Binding affinity and capacities for ytterbium(3+) and hafnium(4+) by chemical entities of plant tissue fragments

R. Worley1,2, A. Clearfield3 and W. C. Ellis2,4

Texas A&M University, College Station 77843

4 Correspondence:
Department of Animal Science, 2471 TAMU (phone: 979-845-5063; fax: 979-845-5292; E-mail:
w-ellis{at}tamu.edu).


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
The binding affinity of ytterbium (Yb3+) and hafnium (Hf 4+) to ligands of chemical entities of fragments of bermudagrass tissues and their resistance to exchanging Yb with other ligands and to displacement by protons were investigated. Chemical entities of acid resistant NDF (ARNDF), 0.1 N acid detergent fiber (0.1 N ADF), and permanganate cellulose (CELL) were prepared from fragments of bermudagrass hay (Cynodon dactylon [L.] Pers.) obtained by grinding to pass a 2-mm sieve. 175Ytterbium and Yb, as YbCl3, were initially bound to each preparation by soaking for 12 h in pH 5.5 borate buffer to obtain Yb bound onto ligands having affinity constants for Yb equal to or greater than that for the weakly stable borate ligand, Yb >= borate. The fraction of Yb >= borate was measured and fragments then sequentially exposed to acetate, citrate, nitrotriacetate (NTA), and EDTA ions to allow exchange of Yb from Yb >= borate with ligands having affinity constants for Yb equal to or greater than acetate (Yb >= acetate), citrate (Yb >= citrate), NTA (Yb >= NTA), and EDTA (Yb >= EDTA) ions. Binding of Yb >= borate indicated the existence of two species of ligands: strong ligands binding essentially 100% of added Yb at levels of 1 to 1,300 ppm (0.1 N ADF) and at 1 to 7,000 ppm (ARNDF); and weaker ligands binding 4 and 8% of the Yb, respectively, at levels of added Yb greater than 1,300 ppm and 7,000 ppm. Ytterbium >= acetate of ARNDF, but not 0.1 N ADF, was as resistant to exchange as Yb >= citrate. Ytterbium >= borate was exchanged extensively (85% or greater) with soluble ligands having affinity constants >= NTA. Ytterbium resistance to proton displacement at pH of 1.5 increased with Yb >= EDTA > Yb >= NTA > Yb >= citrate > Yb >= acetate. Very efficient binding of Yb to CELL suggested that such chemical preparations are not representative of native cellulose. Hafnium (4+) was strongly bound to plant tissues rendering both Hf and Hf-bound DM insoluble at a pH of 1.5 and insoluble in a modified NDF solvent without EDTA. It is concluded that Yb specifically applied as Yb >= acetate and Hf4+ are indelible markers for estimating sojourn time of undigested plant tissues at the normal pH of the rumen. Because of its resistance to proton displacement, Hf4+ would be an indelible marker for estimating sojourn time in more acidic postgastric segments of the gastrointestinal tract.

Key Words: Ytterbium • Plant Tissues • Markers


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
Rare earth elements form very stable complexes with various subsistent groups (ligands) of chemical entities of plant tissues and have, therefore, been proposed as flow markers for undigested residues of plant tissues during their ruminal sojourn. The affinity (or formation) constant of a Yb•ligand-X complex is the molar proportion of bound Yb/unbound Yb+++ at equilibrium (Martell and Calvin, 1952). The Yb•ligand-X complex will remain intact unless 1) Yb is exchanged with a metal cation having a greater affinity constant for ligand -X than Yb, 2) Yb is exchanged with another ligand having a greater affinity constant for Yb than ligand-X, or 3) proton concentrations became sufficiently large (low pH) that proton displacement of Yb occurs by mass action or change the ligand’s structure such that its affinity for Yb is lost. It was postulated that an array of ligands exists in plant tissues (ligand-X1, ligand-X2, ligand-X3, ..., Xn) of different affinity constants and binding capacities for Yb+++. To test this hypothesis, chemically defined entities of plant tissues were exposed to varied concentrations of Yb+++ to determine the distribution of ligands of different affinity constants and their binding capacities for Yb+++ (Yb•ligand-X1, Yb•ligand-X2, Yb•ligand-X3, ..., Yb•Xn). Affinity of Yb bound to ligands of these insoluble chemical entities of plant tissues was then evaluated by exposure to soluble ligands of progressively greater and known affinity constants (ligand-S1, ligand-S2, Yb•ligand-S3, ..., Yb•ligand-Sn). By this means, Yb was specifically bound to plant tissue ligands having affinity constants >= ligand-S1, >= ligand-S2, >= ligand-S3, ..., >= ligand-Sn and their resistance to proton displacement of Yb was then evaluated at a pH simulating the rumen and the duodenum. Lastly, the indelibility of hafnium to fragments of plant tissues was investigated.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Implications
 Literature Cited
 
These experiments were conducted following an Animal Use Protocal reviewed and approved by the Institutional Agricultural Animal Care and Use Committee of Texas A&M University.

Yb Experiment

The objectives of this experiment were to evaluate effects of Yb concentration and of soluble competitive ligands of known affinity constants upon binding level and resistance of bound Yb to proton displacement at a pH typical of the rumen (5.5) and the duodenum (1.5). Three chemical entities of bermudagrass plant tissues were used; NDF, 0.1 N acid detergent insoluble fiber (0.1 N ADF) and permanganate cellulose (CELL). To avoid confounding effects of proton displacement of Yb from bound DM as compared to solubilization of DM bound Yb, acid insoluble residues of NDF (ARNDF) of ground bermudagrass hay (< 2 mm sieve) were used. The ARNDF was prepared as the NDF residue that was insoluble after soaking for 2 h in pH 1.5 HCl. To avoid problems of residual EDTA in the NDF, EDTA was omitted from the NDF solvent (Goering and Van Soest, 1970) used to prepare the NDF. The 0.1 N ADF was prepared as the residues that were insoluble after a 1-h reflux of the bermuda fragments in an acid detergent solvent as specified by Goering and Van Soest (1970) with the substitution of 0.1 N sulfuric acid for the 1 N sulfuric acid. Acid permanganate cellulose was prepared as specified by Goering and Van Soest (1970). Each tissue residue was washed with distilled water until the effluent had a pH of 6 to 7 and then used for initial binding with Yb.

Initial Binding of Yb. Approximately 3-g portions of each tissue preparation were initially labeled in 60 mL of 0.1 M borate buffer (pH 5.5) containing 10,000 dpm of 175YbCl3•8H2O (specific activity of approximately 10,000 dpm per microgram of Yb) and providing approximately 1 ppm Yb. Additional increments of YbCl3•8H2O were subsequently added to each sample to provide a range of 1 to 100,000 ppm Yb/3 g of tissue preparation. Tissue preparations were soaked with various ppm Yb in borate buffer for 12 h to allow for achieving equilibrium in binding of Yb to ligands having binding affinities for Yb concentrations greater than that of Yb for the soluble ligand borate, Yb >= borate. Excess borate buffer and unbound Yb were removed by filtration with subsequent rinsing with distilled water through a 40-µm porosity sintered glass crucible and dried. The fraction of residual DM was determined by weighing, and Yb bound to sites having an affinity for Yb >= borate was measured as residual 175Yb >= borate/(initial 175Yb•specific activity of initial Yb). 175Ytterbium was assayed as emissions between 470 and 494 keV as detected by counting the filter crucible in the well of a sodium iodide crystal with emissions captured by a pulse height analyzer. The number of disintegrations per minute was determined for Yb dispersed on bound residues vs the 0.1-mL of initial dose of 175Yb placed in the center of a volume of cellulose powder equal to that of the bound residues.

Specifically Bound Yb. Tissue preparations containing Yb >= borate were then soaked successively in 60 mL of pH 5.5 buffers of 0.1 M acetate, citrate, nitrilotriacetic acid, or EDTA. After soaking for 12 h, excess buffer and soluble ligand-bound Yb were removed by filtrations and washing with the respective buffer-ligand. The preparations of Yb specifically bound to plant tissue ligands having binding affinities >= the soluble ligands, Yb >= acetate, Yb >= citrate, Yb >= NTA, and Yb >= EDTA, were then dried, and bound 175Yb was determined to evaluate resistance of bound Yb to proton displacement.

Acid-Resistant Bound Yb. Tissue preparations Yb >= borate, Yb >= acetate, Yb >= citrate, Yb >= NTA, and Yb >= EDTA resulting from initial exposure to 100,000 ppm Yb were soaked in pH 1.5 HCl for 6 h. Acid-resistant residues were rinsed with distilled water until the filtrate was > pH 6, dried, and assayed for DM and 175Yb as above.

Hafnium Experiments

The objective of these experiments was to evaluate effects of initial binding conditions upon the subsequent resistance of Hf to removal by acid and by varied DM solvents.

Preparation of Ammonium Hafnyl Carbonate. Initially, a 0.5 M solution of ammonium hafnyl carbonate having a pH of 9.5 was obtained by custom synthesis (Oremet-Wah Chang, Albany, OR). Subsequently, this source became unavailable and a 0.25 M solution of ammonium hafnyl carbonate was prepared as follows.

Distilled water (250 mL) was placed in a 500-mL beaker and cooled to near 0°C in a hood. Wearing eye protection, gloves, and lab coat, 50 g of HfCl4 was cautiously added a spoonful at a time to the cold water with constant stirring. Appropriate precautions were taken against the large volume of HCl gas evolved. Ice was added to the bath as needed to absorb the evolved heat of solution. The reacting solution was continuously stirred after all HfCl4 had been added and allowed to warm to near room temperature. A second solution was prepared by dissolving 47.9 g of ammonium carbonate, (NH4)2CO3, or 56.92 g of (NH4)2CO3•H2O in 250 mL of distilled water with stirring. Under a hood, 37 mL of concentrated reagent grade ammonium hydroxide was added with all precaution to avoid breathing the ammonia solution to the carbonate solution. Then, 10 mL of the ammonium chloride solution was added drop-wise with stirring to the ammonium carbonate solution. After slow addition of 10 mL of the hafnium chloride solution, the remaining volume of the hafnium solution was added to the ammonium carbonate solution, and the graduate cylinder was rinsed with 10 mL of distilled water and added to the beaker. After adding the remainder of the HfCl4 solution, the pH was adjusted to 8.2 using dilute NH4OH or HCl as required. Distilled water was then added to give a final volume of 678 mL (0.25 M).

Experiment 2. Ammonium 181hafnyl carbonate was prepared by irradiation of a portion of the above solution with slow neutrons in the Texas A&M University Nuclear Reactor. Approximately 3-g samples of plant tissue fragments of bermudagrass were soaked for 12 h in 60 mL of 0.1 M ammonium 181hafynyl carbonate (approximately 100,000 dpm) in one of four buffered solutions: 1) ammonium carbonate, pH 9.5, 2) ammonium acetate, pH 9.5, 3) ammonium acetate, pH 7, or 4) ammonium EDTA, pH 9.5. After filtration and washing with distilled water until the effluent was pH 6 to 7, residual DM and bound 181Hf were measured. Resistance of bound 181Hf to displacement was determined subsequent to sequential 1) 4-h soak in pH 4 sodium acetate, 2) refluxing with modified neutral detergent solvent (without EDTA), and 3) a 4-h soak in pH 1.4 HCl.

Experiment 3. Residues were prepared by extracting bermudagrass tissues for 1 h with 1) distilled water, 2) pH 7 sodium phosphate buffer with 3% sodium lauryl sulfate followed by 6 h at pH 1.5, 3) pH 7 buffered sodium phosphate solution, 4) pH 7 buffered sodium phosphate solution with 3% sodium lauryl sulfate, 5) pH 1.5 HCl, or 6) 0.1 N HCl. Two grams of each extracted tissue (EBG) were individually soaked in 60 mL of 0.1 M ammonium 181hafynyl carbonate buffered with either 0.1 M ammonium acetate, pH 7, or 0.1 M ammonium carbonate, pH 9.5, for 8 or 96 h. After filtration and washing with distilled water until the effluent was pH 6 to 7, residual DM and bound 181Hf were measured and resistance of each to displacement was determined subsequent to a 4-h soak in pH 4 sodium acetate and either refluxing with modified neutral detergent solvent (without EDTA) or a 4-h soak in pH 1.4 HCl.

Statistics. Procedure REG was used to estimate the regression coefficient for linear relations between bound Yb vs added Yb. Procedure GLM of SAS (SAS Inst., Inc., Cary, NC) was used to evaluate statistical significance of differences in the dependent variable (usually radioisotope bound) with chemical entity/fragments and binding conditions as independent variables. When differences existed due to treatment, the SNK procedure was used to detect differences among treatment means.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Implications
 Literature Cited
 
It should be emphasized that the affinity constants as measured here is intended to reflect the affinity of the metal•ligand complex at equilibrium and not the kinetics of formation and exchange reactions. The time allowed for reaction was used after affirming that further reaction times of 96 h resulted in increased binding of less than 2% of that achieved by 12 h.

Being of unknown chemical composition, the molar affinity constants of plant tissue ligands could not be determined but are referred to here in terms of their resistance to displacement by soluble ligands of known molar affinity. Thus, the term resistance to displacement is used to express the inferred affinity constant of the plant tissue ligand(s) for Yb relative to the known affinity constant of the soluble ligand and Yb. Resistance to displacement of Yb by protons as measured here does not distinguish effects of pH on solubilization of the ligand bound Yb or in altering the affinity constant of the insoluble ligand.

Yb Experiments

Binding Capacities and Stabilities. The affinity and binding capacities for various chemical entities of plant tissue for Yb were evaluated by expressing the accumulative percent Yb bound vs the accumulative Yb added (Figure 1Go). In the presence of the borate buffer, Yb appeared bound to acid-resistant NDF and to 0.1 N ADF as two species of ligands. More stable species of ligands formed at the lower concentration range of added Yb with less stable species forming when the binding capacities of the more stable species were exceeded. These groups of ligands will be referred to as the stronger and weaker species of ligands, respectively. Data presented in Figure 1Go indicate that the binding capacity of the stronger species of Yb >= borate and Yb >= acetate occurred in the order of 1,300 and 7,000 ppm added Yb to 0.1 N ADF and ARNDF, respectively. In the case of 0.1 N ADF in borate buffer, linear regression of the accumulative percent bound as Yb >= borate vs accumulative ppm Yb added yielded binding efficiencies of 103.5 ± 2.5% for the stronger species over the range of 1 to 1,300 ppm of added Yb. In contrast, the weaker species of ligands yielded binding efficiencies of 8.2 ± 1.2% over this range. Binding efficiencies greater than 100% appeared to be the result of failure of the 175Yb standard to exactly duplicate the counting geometry of bound tissues samples. The 175Yb standard was placed in the approximate geometrical center of an equal volume of each preparation of forage tissue fragments and may not have equaled the counting geometry of the uniformly dispersed 175Yb of the test samples of tissue fragments.



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  		Figure 1. Accumulative percent of added Yb bound (ppm) to 0.1 N ADF, acid-resistant NDF (ARNDF), or cellulose (CELL) in a borate buffer (Borate) and resistant to subsequently displacement by acetate, citrate, nitrilotriacetic acid (NTA) or ethylenediaminetetraacetic acid (EDTA) ions.

 
A biphase distribution of binding efficiencies also occurred for acid-resistant NDF. Linear regression of accumulative percent bound as Yb >= acetate vs accumulative Yb added yielded binding efficiencies of 98.5 ± 1.8% over the range of 1 to 7,000 ppm and 4 ± 1.2% over the range of 7,000 to 100,000 ppm ARNDF. Thus, within the precision of the procedures used here, Yb was nearly completely bound as Yb >= borate and Yb >= acetate within binding capacities of 1,300 and 7,000 ppm added Yb to 0.1 N ADF and ARNDF, respectively. When these binding capacities were exceeded, Yb was only weakly bound.

The observed binding efficiencies in borate (for 0.1 N ADF) or acetate (for ARNDF) represent interactions among Yb bound to ligands of the plant tissue preparations and the soluble ligands of the buffer (borate or acetate ions). Thus, the observed binding efficiencies are for ligands of the plant tissue preparations having affinity constants for Yb greater than the affinity constants for borate (Yb >= borate) or acetate (Yb >= acetate). Subsequent exposure of this weakly bound Yb (Yb >= borate for 0.1 N ADF or Yb >= acetate for ARNDF) to soluble ligands having stronger affinity constants will result in exchange of Yb from the ligand of weaker affinity constant to the ligand of stronger affinity constant. Thus, the subsequent distribution of bound Yb in the presence of soluble ligands of stronger affinity constants (Figure 1Go) was used to evaluate the distribution of ligands having affinity constants greater than or equal to the soluble ligands of acetate, citrate, NTA, and EDTA (Yb >= acetate, Yb >= citrate, Yb >= NTA, and Yb >= EDTA, respectively).

The mean proportion of strongly bound ligands of 0.01 ADF having affinity constants for Yb >= acetate, Yb >= citrate, Yb >= NTA, and Yb >= EDTA did not differ (P > 0.05) over the range of 1 to 1300 ppm added Yb and represented mean proportions of 0.95 ± 0.5, 0.11 ± 0.022, 0.10 ± 0.035, and 0.02 ± 0.009, respectively, of that for Yb >= borate (Figure 1Go, 0.1 ADF). In general, these proportions prevailed for Yb bound to the weakly bound species of ligands of 0.1 N ADF. In contrast to 0.1 N ADF, ligands of Yb >= acetate on ARNDF were completely resistant to exchange with the stronger citrate ligand over the range of both the stronger and weaker bound species of ligands. However, like 0.1 N ADF, ligands of ARNDF extensively exchange with NTA and EDTA.

When exposed to permanganate cellulose, Yb exhibited a positive concentration dependent profile in the presence of all competitive soluble ligands (Figure 1Go CELL). The unique nature of the results for CELL suggest that the oxidative procedures used to prepare CELL materially altered the nature of ligands relative to the milder effects of solubility and hydrolysis used in preparation of ARNDF and 0.01 N ADF.

Binding Capacities for Yb. Based on the binding profiles exhibited in Figure 1Go, the total binding capacity for Yb onto acid-resistant NDF and 0.1 N ADF was approached at 100,000 ppm added Yb. The levels of Yb bound in the presence of 100,000 ppm added Yb and in the presence of various competitive soluble ligands is summarized in Table 2Go. Greater levels of Yb were bound to acid-resistant NDF than to 0.01 N ADF, and levels of Yb bound to ligands of Yb >= acetate, Yb >= citrate, Yb >= NTA, Yb >= acetate, and Yb >= EDTA decreased progressively for each chemical entity (Table 2Go). In contrast, near total binding of Yb occurred for permanganate cellulose.


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  		Table 2. Effect of binding conditions on levels of Hf bound to acid-resistant NDF, ARNDF, and to hay particles, HAY, and fractional resistance of bound Hf to displacement by acid solubilization by a modified neutral detergent solvent (NDS) without ethylenediaminetetraacetic acid (EDTA)
 
Resistance to Proton Displacement. When bound to acid-resistant NDF at initial concentrations of 100,000 ppm added Yb, fractional resistance to proton displacement at pH 1.5 increased progressively for Yb >= borate, Yb >= acetate, Yb >= citrate, Yb >= NTA, or Yb >= EDTA (Table 1Go). When bound to 0.1 ADF, detectable fractional resistance to proton displacement did not increase beyond Yb >= citrate. In contrast, more than 90% of Yb bound to permanganate cellulose was resistant to proton displacement without regard to the competitive soluble ligand.


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  		Table 1. Levels of Yb bound (ppm) when various entities of bermudagrass (acid-resistant NDF, 0.1 N ADF, and cellulose) were exposed to 100,000 ppm of Yb in the presence of various soluble competitive ligands at pH 5.5 and fraction of bound Yb resistant to displacement during a 2 h exposure to pH 1.5
 
Hf Experiments

Experiment 2. When bound in the presence of the strong ligands of EDTA, the level of Hf bound was smaller than when bound in the presence of the weak ligands of acetate or carbonate (Table 2Go) and especially so for acid-resistant NDF vs total plant tissue (HAY). The fraction of Hf bound in the presence of EDTA at pH 9.5 was also less resistant to proton displacement at pH 6 and 1.5 and to solubilization by the modified NDS solvent. With these exceptions, the level of binding of Hf onto both acid-resistant NDF and HAY did not differ (P < 0.05).

Additional measurements of DM removed (data not shown), suggested that the modified NDF solvent removed DM without concomitant removal of Hf from acid-resistant NDF. Thus, the specific activity of the residual tissue DM, 181Hf/DM, after pH 1.5 or NDS, appeared constant regardless of the fraction of DM removed. Experiment 3 was conducted to more extensively compare Hf binding to plant tissues and to residues of plant tissues subsequent to extraction with various nonhydrolytic solvents in addition to the hydrolytic solvents used in Exp. 2.

Experiment 3. As shown in Table 2Go, binding conditions of pH 9.5 ammonium carbonate or pH 9.5 and pH 7.0 acetate had no effects upon level of binding or resistance to displacement for HAY and acid-resistant NDF. These same binding conditions were used in Exp. 3 involving HAY or residues of HAY after extraction with various nonhydrolytic solvents (SEHAY). Therefore, means for recovery of DM and DPM in Exp. 2 for acid-resistant NDF and for HAY are combined with results for SEHAY used in Exp. 3 and summarized in Table 3Go. The recovery of 181Hf subsequent to exposure to pH 1.5 HCl did not differ among unextracted bermudagrass (HAY), nonhydrolyzing solvent extracted bermudagrass preparations (SEHAY), or acid-resistant residues of bermudagrass (ARNDF). However, 0.78 to 0.79% of the DM of HAY and SEHAY was solubilized by pH 1.5 HCl. Significant loss of DM without concomitant loss of 181Hf suggests that Hf-bound DM was resistant to the mild hydrolysis associated with pH 1.5 HCl. Similiarly, Hf bound to acid-resistant NDF was essentially resistant to solubilization by the NDF solvent (without the strong chelating agent, EDTA) even though the NDF solvent removed considerable DM from the acid-resistant NDF subsequent to its 1 h exposure to pH 1.5.


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  		Table 3. Mean (N) fractional recovery of 181Hf (dpm) and DM from bermudagrass (HAY), solvent extracted HAY (SEHAY) and acid-resistant fiber (ARNDF) when Hf was bound in pH 7.0 acetate or pH 9.5 carbonate solutions and then exposed for 2 h at a lower pH or a 1 h reflux with neutral detergent solvent without NDS
 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Implications
 Literature Cited
 
These data suggest that a complex array of ligands for Yb exist in plant tissues and that these ligands have varying stabilities for binding of Yb. Ligands having a strong affinity for Yb (Huston et al., 1968; Pond et al., 1989) have been suggested to be associated with polyphenols of lignified tissues (Tetter et al., 1984; Owens and Hanson, 1992), soluble pectins (Allen, 1982; Allen et al., 1985) and other chemical entities (McBurney et al., 1986). The current results indicate an abundance of binding sites having affinities for Yb equal to or greater than acetate (Yb >= acetate), the principal nonplant tissue and competitive ligand of rumen fluid. Sixty percent of the Yb >= borate remained bound to sites of Yb >= acetate (14,181 ppm/23,635 ppm, Table 1Go) and were resistant to proton displacement at pH 5.5, well within the usual pH of the ruminal digesta of foraging ruminants. Thus, if Yb is specifically bound to ligands having affinity constants >= acetate, then no exchange of Yb should occur at ruminal digesta pH above 5.5 (Beever and Ellis, 1986). Postruminal flow of digesta solids and solutes is homogenous (Wylie et al., 2000), so any post-ruminal exchange of Yb due to proton displacement would be inconsequential in estimating sojourn time of plant tissues in the rumen. Thus, if no more stable ligands than acetate exist in the rumen, then flow to the feces of Yb specifically bound to feed sites of Yb >= acetate should be valid for estimating rumen sojourn time. Wylie et al. (1986), Ellis et al. (2002), and Faichney et al. (1989) have provided more direct evidence for specifically applied rare earth elements as valid flow markers for estimating ruminal sojourn time of the undigested residues of such marked feeds.

The affinity constants are similar for all rare earth elements (NIST, 1998), so the results obtained here for Yb should apply to all rare earth elements. A protocol has been suggested for specifically binding rare earth elements to plant tissues (Ellis et al., 1994). The protocol essentially involves applying Yb by soaking the plant tissue in pH 5 to 6 acetic acid solution. Preference should be given to the use of rare earth acetates (Siddon et al., 1985) because of the dominant levels of acetate in rumen fluid.

In view of the current indications that Yb bound DM affects DM solubility (Table 3Go) and previous observations that bound cations in the order of 40 mg/g DM increases specific gravity and retards ruminal sojourn time in the rumen (Ehle, 1984), additions of cations to marked plant tissue should be minimal (< 40 mg/g DM) to avoid altering specific gravity and sojourn time. Further, in view of the important role of ruminal flow-paths (Ellis et al., 2000) and timing within a meal (Pond et al., 1985) in determining their sojourn time, it is increasingly apparent that the marked tissues should be as large a portion of the meal as possible in order to minimize effects due to time and quantity of dose. Thus, levels of cation binding well below their capacities are preferred and are consistent with specific binding onto the less abundant, more stable cation-ligand complexes. It is the size of dosed meal and the analytical method available to the investigator for the cation that determines the required amount of marked meal to administer.

Evidence that bound rare earth elements migrate from the feed fragments other than those to which they are initially added appear most likely due to 1) additions of rare earth elements in excess of their binding capacities, 2) failure to remove unbound cations, and 3) assumptions concerning application and recovery of cations to specified size of fragments (Hartnell and Satter, 1979; Crooker et al., 1982; see discussion of these items by Owens and Hanson, 1992).

It seems improbable that complexes of rare earth elements would be "digested" in view of: 1) such binding of Yb would likely interfere with binding by a hydrolysase (Van Soest, 1994), 2) most likely ligands for Yb binding involve relatively indigestible entities associated with refractory compounds, 3) depressive effects upon dry matter digestibility of relatively large levels of Yb (Tetter et al., 1984), 4) effects of Hf observed here on solubility of DM by neutral detergent solution, and 5) the positive effects on indigestibility of more extreme effects of "mordanting" with rare earths and other group four elements. However, if rare earths are bound at the low levels indicated here, effects on digestibility and specific gravity would be negligible.

The most direct test for usefulness of rare earth markers for estimating ruminal sojourn time in vivo is from comparisons of rumen sojourn time for specifically applied rare earth elements and for indigestible entities. Similar estimates have been obtained from a number of different laboratories (Wylie et al., 1986; Faichney et al., 1989; Huhtanen et al., 1995; Huhtanen and Vanhatalo, 1997; Rinne et al., 1997; Ellis et al., 2002).

Hafnium as ammonium hafnyl carbonate proved to bind very tenaciously to plant tissues. Affinity constants are much greater for the group four (Hf) vs the group three rare earth elements. For example, the affinity constant for Hf4+ is in the order of 1.6-fold greater than for Yb3+ (NIST, 1998). No critical affinity constants for Hf-acetate were reported due to difficulty in measurements of the group four elements and the variability in their estimated affinity constants (NIST, 1998).

The results suggest that Hf-bound DM is resistant to mild hydrolysis (pH 1.5 at 20°C for 6 h), conditions simulating the most extreme expected in the digestive tract of mammals. In contrast to the rare earths, Hf was observed to have a high binding level and affinity for cereal grains (Hill, 1991). Thus, Hf4+ is recommended if resistance to proton displacement is required and(or) an indelible marker for undigested grain residues is desired.

Allen et al. (1985) reported that the group four elements Hf and Zr possessed strong binding affinities for forage tissues. However, they reported relatively slow binding of Hf and Zr under the unspecified conditions they used. In contrast, Hf from ammonium hafynyl carbonate bound relatively rapidly in our experiments. Of the Hf bound after 96 h, 0.8% was bound by 8 h in Exp. 4 and possessed equivalent binding attributes.

The strong binding of tetravalent elements Hf4+ and Zr4+ and the resistance of Hf-bound DM to solubilization are similar to observations concerning the "indigestible" attributes of "mordants" produced by more extreme treatments of feed residues with other tetravalent elements (Cr4+ and Mo4+; Uden et al., 1980). Binding of Hf4+, or expectantly Zr4+, as a slightly alkaline complex offers a simpler alternative to "mordanting." Hafnium was preferable, in our case, due to its extreme sensitivity for its analysis via neutron activation analysis. A limitation of Hf is the general unavailability of an ionizable source of Hf4+ and hence the need for custom synthesis. Possible use of the soluble HfCl4 was not investigated. However, use of this tetrachloride compound presents associated health problems to the user. The ionic nature of ammonium hafnyl carbonate as used here is maintained by surrounding Hf4+ within a basic ammonium complex. This complex slowly decomposes to insoluble hafnyl hydroxides over months even under refrigeration. This synthesis route for ammonium hafnyl carbonate is based on the synthesis of ammonium Zr4+ carbonate starting with Hf tetrachloride (Clearfield, 1969).

Tissues from different plants and different parts of the plant appear to differ in the ruminal and gastric sojourn time and, therefore, require different specifically applied markers. Elements of the rare earth series and Hf and Zr of the group four elements all have large affinity constants. These results and those reported by Wylie et al. (1986) and Ellis et al. (2002) suggest that any of the rare earth elements specifically applied to binding sites resistant to pH 4 acetic acid will simulate ruminal sojourn time in ruminants whose ruminal pH does not fall below the binding pH. Thus, any of the rare earth elements together with Hf and Zr that are amenable to available analytical methodology of sufficient sensitivity may be used where differential flow is expected.


   Implications
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
Literature Cited
 
Ytterbium specifically bound to plant tissue sites having binding affinities for Yb greater than that of acetate has suitable attributes as flow markers for undigested residues. Hafnium from ammonium hafnyl carbonate firmly binds to plant tissues, and DM of Hf-bound ligands is also resistant to mild acid hydrolysis and solubilization. Thus, specifically bound Hf would be a suitable marker for flow of undigested residues through more acidic segments of gastrointestinal digesta.


   Footnotes
 
1 Deceased. Back

2 Department of Animal Science. Back

3 Department of Chemistry. Back

Received for publication November 15, 2001. Accepted for publication May 30, 2002.


   Literature Cited
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 


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