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Chromium picolinate, [Cr(pic)3], is the second most popular nutritional supplement after calcium supplements. However, the supplement, unlike simple i...
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FEBRUARY 2002 VOLUME 15, NUMBER 2 © Copyright 2002 by the American Chemical Society

Articles In Vivo Distribution of Chromium from Chromium Picolinate in Rats and Implications for the Safety of the Dietary Supplement Dion D. D. Hepburn and John B. Vincent* Department of Chemistry and Coalition for Biomolecular Products, The University of Alabama, Tuscaloosa, Alabama 35487-0336 Received May 14, 2001

Chromium picolinate, [Cr(pic)3], is the second most popular nutritional supplement after calcium supplements. However, the supplement, unlike simple inorganic Cr(III) salts, has been shown in the presence of biological reducing agents in vitro to catalytically generate appreciable quantities of hydroxyl radicals, resulting in DNA damage. The complex has also been shown to be remarkably stable in vitro at neutral, basic, or weakly acidic pHs. Thus, the significance of this ability to generate hydroxyl radicals depends on whether the complex is absorbed by cells intact along with the stability and concentration of the complex in cells. Consequently, male Sprague Dawley rats have been injected with 51Cr- and 3H-labeled [Cr(pic)3]. The tissue distribution, urinary and fecal loss, and subcellular hepatocyte distribution and concentration of the labels suggest that [Cr(pic)3] has a lifetime of less than 1 day in vivo, minimizing the potential threat from the supplement itself.

Introduction In the past decade, chromium(III) picolinate, [Cr(pic)3],1 has become a very popular nutritional supplement; products containing [Cr(pic)3] generated nearly one-half billion dollars in sales in 2000, with the supplement being second only to calcium supplements (1). The complex is available over-the-counter in numerous forms including pills, chewing gums, sports drinks, and nutrition bars and is advertised as a weight loss agent and an agent for enhancing lean body mass. [Cr(pic)3] is a relatively wellabsorbed form of chromium (2-5% efficiency compared to dietary chromium which is only absorbed with ap1

Abbreviations: [Cr(pic)3], chromium picolinate.

proximately 0.5% efficiency) (2, 3). This degree of absorption or “bioavailability” versus dietary chromium or inorganic chromic salts is not unique and is shared by other organic ligand-chromium(III) complexes (2, 3). The study of the effects of chromium picolinate on mammals has been extremely contentious (4). In 1999, Lukaski reviewed in detail the effects of supplemental chromium picolinate on humans (5). This extensive survey suggested that [Cr(pic)3] “per se does not promote beneficial changes in body composition in humans”. The Federal Trade Commission came to the same conclusion in 1997 (6), as did Vincent in a 2001 review article examining effects on both rats and humans (7). In 1995, the first questions arose regarding the safety of [Cr(pic)3] as a dietary supplement when Wetterhahn

10.1021/tx010091t CCC: $22.00 © 2002 American Chemical Society Published on Web 01/09/2002

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and co-workers showed that the compound caused clastogenic damage in a Chinese hamster ovary (CHO) cell model (8). Unfortunately, these studies used high, nonphysiological levels of chromium in the culture media that cast doubt on the significance of these findings. Stohs and co-workers have subsequently observed DNA fragmentation in cultured macrophages treated with [Cr(pic)3], although the Cr concentrations were also nonphysiological (9). Wetterhahn and co-workers have suggested that taking [Cr(pic)3] supplements for 5 years could result in liver tissue concentrations of 13 µM (10). In recent in vitro investigations (11, 12), physiologically relevant concentrations of chromium as [Cr(pic)3] (e.g., 120 nM, more than 100 fold less than the estimate above) and of biological reductants, such as ascorbic acid and thiols (5 mM, the approximate ascorbate concentration of most cells), resulted in catalytic production of hydroxyl radicals, which cleaved DNA. These findings were consistent with earlier results that showed mutagenic forms of chromium(III) possessed chelating ligands containing pyridine-type nitrogens coordinated to the metal (13). Recently, [Cr(pic)3] has been found in vivo to result in increased levels of peroxidized lipids in the liver and kidneys of rats (Hepburn and Vincent, submitted for publication); thus, the compound can give rise to oxidative damage in vivo. Isolated incidents of deleterious effects of [Cr(pic)3] supplementation have been reported: weight loss, anemia, thrombocytopenia, liver dysfunction, and renal failure (14); renal failure (15); rhaddomyolysis (16); dermatitis (17); acute, short-lasting cognitive, perceptial and motor changes (18); and exanthematous pustulosis (19). The significance of these incidents is difficult to ascertain. No effect on 5-hydroxymethyl uracil levels was observed in 10 obese women given 400 µg of [Cr(pic)3]/ day for 8 weeks (20); however, this is not surprising given the expected level of accumulation of [Cr(pic)3] in tissue in this short time at this level of intake. The same criticism holds for a preliminary report of a study in which rats were given an oral dose of [Cr(pic)3] up to 2000 mg of complex/kg of body mass; chromosomes from bone marrow cells of femurs removed 18 or 42 h after the dose showed no increase in damage versus controls (21). Longterm studies of the effects of chromium picolinate supplementation are required to determine the significance of this chemistry, especially potential DNA damage. Recent in vitro studies have also shown that [Cr(pic)3] is remarkably stable in buffered aqueous solution (11, 22) and in synthetic gastric fluid and passes unhindered through the jejunum (23). Consequently, absorbed [Cr(pic)3] probably enters cells intact, i.e., in the potentially harmful form. In vitro [Cr(pic)3] does not release its chromium efficiently to biological chromium-binding species such as apotransferrin or apochromodulin unless the metal is reduced to the chromous level (12). Recently Kelley and co-workers have reported that hepatocyte microsome extracts can catalytically modify the picolinate ligands, resulting in Cr release (24). Consequently, while Cr from [Cr(pic)3] supplements can accumulate in cells, the form of this trivalent Cr is not known. Herein are reported studies to determine whether [Cr(pic)3] is stable in vivo and to what concentrations Cr as [Cr(pic)3] can accumulate in cells in order to aid in elucidating the potential for the supplement to have deleterious effects on humans.

Hepburn and Vincent

Experimental Procedures Materials. 51CrCl3 in 0.5 M HCl was obtained from ICN; [3H(G)]picolinic acid was obtained from Moravek Biochemicals. Labeled derivatives of [Cr(pic)3] were made by the method of Press et al. (25) except that either a tracer amount of 51CrCl3 (1-5 mCi) was mixed with the initial aqueous solution of CrCl3 or a tracer amount of [3H(G)]picolinic acid (0.5 mCi) was mixed with picolinic acid. The product from this synthesis has been characterized by a variety of spectroscopic techniques (22, 26, and references therein). Male Sprague-Dawley rats weighed between 550 and 650 g. All procedures were performed using doubly distilled deionized water unless otherwise noted and performed in plasticware whenever possible. The University of Alabama Institutional Animal Use and Care Committee approved the procedures involving the use of rats. In Vivo Experiments. The male Sprague Dawley rats were maintained for 15 days in metabolic cages. During the first 14 days, the rats were injected in the tail vein each morning with 150 µL of an aqueous saturated solution of a radiolabeled derivative of [Cr(pic)3] (∼600 µM Cr). Urine and feces were collected every 12 h. On the morning of day 15, the rats were sacrificed by carbon dioxide asphyxiation, and tissue samples were harvested and weighed. 51Cr-label experiments were performed with five rats except where otherwise noted. Because of the previous 51Cr results and the expense of [3H(G)]picolinic acid, only one rat was used in studies with 3H-labeled [Cr(pic)3]. Contents of the blood were calculated assuming the blood comprised 6% of the body mass. Hepatocyte Subcellular Fractionation and Chromatography. Subcellular liver fractions were obtained by differential centrifugation according to established literature procedures (27). Components of the urine and soluble hepatocyte fraction were separated by G-15 column chromatography using 50 mM NH4OAc buffer, pH 6.5. For scintillation counting, feces and tissues were homogenized with a Waring blender. Aquasol-2 (Packard) was used as the scintillation fluid. As a result of a technical difficulty with the differential centrifugation of one sample, results for hepatocyte subcellular distribution of 51Cr are based on only four rats. Instrumentation. Ultraviolet-visible measurements were made on a Hewlett-Packard 8453 spectrophotometer. Gamma counting was performed on a Packard Cobra II auto-gamma counter. Scintillation counting was performed on a Packard 1900 TR scintillation counter. NMR spectra were obtained using a Bruker AM-360 spectrophotometer.

Results and Discussion 51 Cr Studies. To determine the stability and distribution of [Cr(pic)3], rats were treated daily for 2 weeks with 51Cr- or 3H-labeled complex, allowing the fate of the complex, the chromium, and the organic component to be followed. [Cr(pic)3] at the concentration in cells cannot be followed by paramagnetic NMR (26) or ultravioletvisible spectroscopy (22). The complex was administered by injection to allow the amount entering the bloodstream to be known and avoiding complications associated with absorption from the gastrointestinal tract. The amount of the complex injected daily corresponds to 5 µg of chromium. Nutritional supplements generally provide 200-600 µg of Cr/day. Assuming an average human body mass of 75 kg, this is equivalent to 1.3 to 3.9 µg of Cr/ day for a 0.5 kg rat. Thus, 5 µg of Cr is slightly more than rats would receive if given a dose comparable to humans. Because of 5% or less of Cr from [Cr(pic)3] is actually absorbed when given orally, the injection given the rats actually represents a g25-fold excess of Cr compared to humans taking the supplement orally. If rats can degrade this quantity of [Cr(pic)3], they should be

In Vivo Distribution of Chromium Picolinate

Figure 1. Representative urinary and fecal 51Cr loss during the 2-week period of treatment with 51Cr-labeled [Cr(pic)3]. Time zero represents the time of the first injection of an aliquot of a solution of the labeled complex.

able to degrade quantities of [Cr(pic)3] equivalent to normal human diet supplementation. Anderson and coworkers have fed rats diets containing up to 100 mg of Cr/kg diet as [Cr(pic)3] for 20 weeks without observing any acute toxic effects (28). Thus, assuming the rats consumed a normal quantity of food in the study of Anderson and co-workers, the dosage of Cr administered daily for only 2 weeks in the current work was unlikely to have any acute toxic effects. No ill effects were observed from the injections; all tissues appeared normal when harvested. Giving [Cr(pic)3] intravenously in the current study is essential. As [Cr(pic)3] given orally is only absorbed with an efficiency of 2-5%, insufficient quantities of [Cr(pic)3] could be introduced to allow detection of [Cr(pic)3] (if present intact) in the tissues (vide infra). While Cr from [Cr(pic)3] accumulates in the tissues (28), the half-life of 51Cr (27.7 days) becomes an issue in long-term studies; the chemical form of the accumulated chromium is also unknown and may not be [Cr(pic)3]. Thus, if [Cr(pic)3] does not remain intact in vivo when administered intravenously in the quantity used in the current study, then the quantities of [Cr(pic)3] which could enter the bloodstream and tissues after oral ingestion will certainly be degraded. Urine samples collected during the 2 weeks of treatment with 51Cr-labeled [Cr(pic)3] reveal a distinct pattern of Cr excretion (Figure 1). Loss of chromium is maximal during the first 12 h after injection and significantly reduced the next 12 h. The pattern of chromium loss in the feces is similar the second week of injections; however, samples taken 12-24 h after injection contain more label than samples taken the first 12 h. The pattern of fecal Cr loss the first week is irregular. Urinary chro-

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Figure 2. Elution profile from G-15 size exclusion column of rat urine sample from day 8 (0-12 h after injection). The solid line and circles represent the absorbance at 270 nm; the dotted line and open circles represent the cpm of 51Cr (scaled by dividing by 4000).

mium loss throughout exceeds fecal Cr loss severalfold. Total daily urinary and fecal Cr loss is rather small, starting at ∼10% of the daily quantity of injected Cr lost during days 1 and 2 and increasing to ∼20% by days 13 and 14. This change is accompanied by an increase from day 1 to day 14 in the amount of Cr lost 12-24 h after injection. Previously, a three-component model has been proposed to explain the kinetics of Cr exchange and distribution in studies with rats and humans (29-31). Plasma Cr was proposed to be in equilibrium with three pools of Cr: a small pool with rapid exchange (t1/2 < 1 day), a medium pool with a medium rate of exchange (days), and a large, slowly exchanging pool (months). Chromium lost within 12 h of injection primarily then reflects this small pool in rapid equilibrium; the slow increase in Cr levels 12-24 h after injection may reflect Cr exchanging with the medium pool that undergoes exchange of a period of days. To determine the form(s) of Cr lost in the urine, urine from a few of the 12-h periods was subjected to G-15 size exclusion chromatography (Figure 2); regardless of the time samples collected, the elution profiles were almost identical. 51Cr eluted in a single band, eluting just ahead of the major components that absorb ultraviolet light at 270 nm. This band does not correlate to the molecular weight of [Cr(pic)3]; this band elutes at the same position as the oligopeptide chromodulin (∼1500 molecular weight). This is consistent with other studies that have demonstrated that Cr from other sources including Cr2-transferrin is ultimately lost in the urine bound to chromodulin

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Figure 3. 51Cr content of tissues after two weeks of treatment with 51Cr-labeled [Cr(pic)3].

(27, 32-33). Thus, all the urinary 51Cr represents chromium picolinate that has been degraded in vivo. Fecal Cr loss probably represents Cr lost in the bile. Previous work has shown that only minor amounts of Cr are lost in the bile (34, 35). In bile, chromic ions occur as part of a low-molecular-weight organic complex (36), although the molecular weight was not estimated. The authors did postulate that this complex might be responsible for passage of Cr from the liver to the bile. In the current work, the low concentration of fecal Cr prevented further studies on the form of the Cr. Studies are needed on bile duct-cannulated rats given labeled Cr sources to determine the nature of bile Cr. After the two-week period of treatment with [51Cr(pic)3], the distribution of the radiolabel in the tissues was examined (Figure 3). Of the tissues examined, the greatest amount was located in the liver, followed by the kidneys. This distribution reproduces that observed in previous studies in which rats were given [Cr(pic)3] (28, 37). Anderson and co-workers have shown that Cr accumulation in rat kidneys and liver from the supplement increases linearly with the amount of the supplement given (28). The amounts of chromium retained by rats and in their blood 4 or 24 h after consuming a diet supplemented with CrCl3 or [Cr(pic)3] are similar; however, the tissue concentrations of chromium are about 5-6 times greater using [Cr(pic)3] (37). CrCl3 in the blood readily serves as a source of Cr for transferrin (27, 34), which binds Cr(III) tightly at neutral or slightly basic pHs (12). Transferrin releases its chromic ions in cells (after entering by endocytosis) where the Cr subsequently is eliminated in the urine, probably as the oligopeptide chromodulin (27). The different tissue concentrations but similar blood concentrations of Cr when using the two supplements would be consistent with Cr(pic)3 remaining intact and entering cells by a different mechanism (probably passive diffusion). If after entering the cells, the [Cr(pic)3] would readily degrade releasing chromic

Figure 4. Subcellular distribution of 51Cr in hepatocytes after 2 weeks of treatment with 51Cr-labeled [Cr(pic)3]. Error bars indicate standard deviation.

ions, then the cells could handle the chromium in a similar fashion, resulting eventually in similar blood Cr levels. The subcellular distribution of 51Cr from [Cr(pic)3] in rat hepatocytes is rather distinct with ∼75% of the Cr in the cytosol. Very little chromium comparatively is in the nucleus or mitochondria, lowering the risk of DNA damage from any generated hydroxyl radicals. The distributions of Cr from injected CrCl3 and Cr2-transferrin are nearly identical to one another (as would be expected by transferrin binding available chromic ions in the blood) with the nearly half the Cr being found in the nucleus (27, 36). The difference in subcellular Cr from injected [Cr(pic)3] and injected CrCl3 or Cr2-transferrin strongly suggests that the [Cr(pic)3] maintains its integrity in the blood and diffuses into cells intact, otherwise the Cr from [Cr(pic)3] available in the blood would bind to transferrin and subsequently be handled in a similar fashion. In time (shorter times for the lower doses given in oral studies), the Cr released from [Cr(pic)3] in cells should be in equilibrium in the cells, resulting in a similar retention and distribution of the Cr as compared to the other sources. To determine the form of Cr in the hepatocyte cytosol, the cytosol fraction was applied to a G-15 column. Cytosol from five different rats treated with the labeled [Cr(pic)3] was applied separately to the column. In all but one case, the 51Cr migrated with the solvent front, indicating that the Cr-containing species all had molecular weights well in excess of 1500 or in a shoulder just behind the front. No Cr eluted in fractions corresponding to the position that [Cr(pic)3] elutes from the column. In the one instance (Figure 5), a small, broad band of radiolabel eluted where [Cr(pic)3] would have been expected to elute. Given the broadness of the band, all that can be said is that the Cr

In Vivo Distribution of Chromium Picolinate

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Figure 6. Representative urinary and fecal 3H loss during 2-week period of treatment with 3H-labeled [Cr(pic)3]. Time zero represents the time of the first injection of an aliquot of a solution of the labeled complex. Figure 5. Elution profile of the soluble fraction of hepatocytes from 51[Cr(pic)3]-treated rat from G-15 column. Inset: enlargement of the region where a low-molecular-weight Cr-containing species elutes.

is part of some complex(es) with the approximate molecular weight of [Cr(pic)3]. The Cr in this band corresponds to a ∼1 µM concentration in the cytosol in vivo and is nearly 10 times higher than the 120 nM [Cr(pic)3] previously observed to result in DNA damage in vitro (11). Therefore, it is possible in individual rats that cellular concentrations of [Cr(pic)3] could be in the range that hydroxyl radical generation can be significant. One obvious conclusion from these studies is that [Cr(pic)3] is degraded rather rapidly in cells, i.e., within 24 h of injection. The concentration of chromium in cells is not equivalent to the concentration of [Cr(pic)3]. Thus, while liver and kidney appreciably accumulate chromium from [Cr(pic)3] when given as a diet supplement (28), the vast majority, if not all, of the Cr is not in the form of [Cr(pic)3] after 24 h or less. Consequently, the prediction of Wetterhahn et al. (10) that [Cr(pic)3] could accumulate to levels of 13 µM should be interpreted as the levels of chromium that could be accumulated. However, a steadystate concentration of [Cr(pic)3] in cells of under 1 µM would not be possible to detect using the techniques in this work; the ability to detect a low-molecular-weight Cr species in only one of five rats may suggest that such a species exists in all three rats but varies from just at detection limits to somewhat lower concentrations. While this decomposition minimizes the risk of damage from [Cr(pic)3] itself in that the vast majority of the complex degrades within 24 h, it does not eliminate the need for long-term studies to examine whether [Cr(pic)3] leads to increased oxidative damage in cells or whether products of the biotransformation of the complex might have derogatory effects.

These results (that when large quantities of Cr(pic)3 enter the bloodstream the complex is degraded rapidly and does not accumulate in cells intact) also indicate that orally administered Cr(pic)3 (which cannot reasonably achieve comparably high concentrations in the bloodstream) should readily be degraded within 24 h. This also suggests that following the fate of orally administered Cr(pic)3 from the gastrointestinal tract into the bloodstream to determine whether the complex remains intact will require more sensitive detection techniques. 3H Studies. The original intention of the 3H-labeled picolinate studies was to determine which tissues and fluids should be emphasized based on a similar content of both 51Cr and 3H (suggesting that the [Cr(pic)3] might exist intact in these samples) in the respective studies. However, the 51Cr studies which reveal that [Cr(pic)3] dissociates in 24 h to levels below detection limits suggested the tritium studies would provide little if any additional information in this regard. Given the scale of the tritium experiments, only general comments will be presented. Treatment of a rat with 3H-labeled [Cr(pic)3] results in a much greater portion of the label appearing in the feces and urine (Figure 6). As with urine chromium, 3H appears in the urine primarily in the first 12 h after injection. Fecal loss is irregular during the first 2 days but afterward follows a regular pattern with maximal loss 12-24 h after injection. Urinary loss is up to more than 20-fold greater than fecal loss. Fecal and urinary loss of the label during day 1 corresponded to 100% of the label (within experimental error). After day one, retention increases and after day six becomes stable at approximately 30%. Thus, throughout the 2-week study, the vast majority of the 3H is lost in these waste products. Tritium in the urine is localized to one tight band (with a small shoulder on the lead edge) upon G-15 size

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Figure 8. 3H content of tissues after 2 weeks of treatment with 3H-labeled [Cr(pic) ]. 3

Figure 7. Elution profile from G-15 column of rat urine sample from day 4 (0-12 h after injection). The solid line represents the absorbance at 270 nm; the dotted line represents the cpm of 3H (scaled by dividing by 1250).

exclusion chromatography (Figure 7). This band is distinct from the major feature containing chromium, which elutes ahead of the position of the 3H. The band elutes at a position that suggests a molecular weight much greater than picolinic acid or N-1-methylpicotinamide. The latter was identified as the product of degradation of [Cr(pic)3] by hepatocyte microsomal proteins (24). Thus, the cells appear to process the picolinic acid derivative further. Fractions containing 3H were combined and freeze-dried, and the residue was dissolved in D2O. The 1 H NMR spectrum of the residue contained several features in the region 7.5-8.5 ppm. Resonances from the pyridine ring of picolinate, methylpicolinamide, or other derivatives could not be unambiguously identified. Among tissues examined, the liver retains the largest portion of the label. The epididymal fat contains a substantial quantity of the tritium from the picolinate ligands as well (Figure 8). The distribution of the label is distinct from that of chromium from the complex. This reflects the difference between the handling of chromic ions and picolinate by the body. Picolinate is cleared rapidly while absorbed Cr3+ is largely retained in the slow and medium exchanging pools. The distribution of the 3H in hepatocytes is also distinctly different than that of chromium (Figure 9), with most of the tritium in the cytosol and lysosomes. Because ∼70-100% of the label is excreted daily, insufficient quantities of the tritium remains in the organs, including the liver and thus in the hepatocyte subcellular fractions, to attempt to sepa-

rate 3H-containing species by size exclusion chromatography. Implications for Toxicity of Cr(pic)3. The potential DNA-damaging ability of [Cr(pic)3] comes from the combination of chromic ions and picolinate; the redox potential of the chromium is altered by the pyridinebased ligands such that it is susceptible to biologically relevant reducing agents such as thiols and ascorbate (11). Oxidation by oxygen or other species then can produce high-valent chromium complexes that generate hydroxyl radicals. Dillion et al. have proposed that chromic complexes with similar ligands may also be enzymatically oxidized to Cr(V) with the Cr(V) producing oxidative damage (38, 39). However, rapid enzymatic degradation of the complex would tend to minimize the potentially deleterious effects of the complex by either mechanism. The question then arises as to the safety of the products from the break down of [Cr(pic)3]. Chromic ions when bound to the type of ligands normally found in biological systems are not susceptible to the same types of redox chemistry (40); in fact, concentrations of chromic ions in cells required to make the ion unsafe are so large that determining suggested upper levels for human intake has been difficult to date (41, 42). Picolinic acid is a naturally occurring tryptophan catabolite, generated as an end product in the kynurenine pathway (43). However, safety concerns regarding picolinic acid have arisen several times (44-48), resulting in suggestions that picolinic acid by itself should not be used as a dietary supplement. This raises concerns about chromium picolinate in vivo, which could potentially release 3 equiv of picolinate/molecule of the complex. Yet, picolinic acid, at least when attached to chromium, appears to be enzymatically modified which

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of oxidative damage from long-term supplementation are required to firmly establish the risk or safety of [Cr(pic)3] diet supplementation.

Acknowledgment. Funding was provided by the American Diabetes Association (J. B. V.).

References

Figure 9. Subcellular distribution of 3H in hepatocytes after 2 weeks of treatment with 3H-labeled [Cr(pic)3].

may prevent the generation of significant quantities of the acid. However, the potential for the products of this activity to have deleterious effects needs to be investigated, especially since molecules generated along the kynurenine pathway tend to have neurological effects (49).

Conclusions The tissue distribution, pattern of urinary and fecal loss, and subcellular hepatocyte distribution of 51Cr and 3H from labeled [Cr(pic) ] injected daily into male 3 Sprague-Dawley rats daily for 2 weeks suggest that [Cr(pic)3] has a lifetime less than 24 h in vivo. Cr from [Cr(pic)3] was largely retained in cells over the period of investigation, while picolinate derivatives are readily expelled from the body in the urine and feces. As cell concentrations of Cr are not equivalent to [Cr(pic)3] concentrations and cellular Cr is found primarily in the cytosol rather than the nucleus, the potential for DNA damage from the catalytic production of hydroxyl radicals is significantly less than previous literature suggestions. However, the potential existence of low steady-state concentrations of [Cr(pic)3] in cells cannot be eliminated. Given that the compound was administered intravenously to achieve large concentrations of [Cr(pic)3], the compound when taken orally (when such high concentrations in the bloodstream cannot readily be achieved) should readily be degraded in cells. Studies of the extent

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