Immunohistochemical detection of betaine-homocysteine S-methyltransferase in human, pig, and rat liver and kidney.

Betaine-homocysteine S-methyltransferase (BHMT) has been shown to be expressed at high levels in the livers of all vertebrate species tested. It has also been shown to be abundant in primate and pig kidney but notably very low in rat kidney and essentially absent from the other major organs of monogastric animals. We recently showed by enzyme activity and Western analysis that pig kidney BHMT was only expressed in the cortex and was absent from the medulla. Using immunohistochemical detection, we report here that in human, pig, and rat kidney, BHMT is expressed in the proximal tubules of the cortex. Immunohistochemical staining for BHMT in human, pig, and rat liver indicate high expression in hepatocytes. The staining patterns are consistent with cytosolic expression in both organs.

Association of betaine-homocysteine S-methyltransferase with microtubules.

In mammals, betaine of the mitochondrial matrix is used in the cytosol by betaine-homocysteine S-methyltransferase for methionine synthesis. The resulting dimethylglycine is shuttled back into the mitochondrial matrix for further degradation. Nanospray tandem mass spectrometry and N-terminal amino acid sequencing of microtubule-associated proteins from rat liver tubulin revealed that betaine-homocysteine S-methyltransferase is microtubule associated. This was confirmed by confocal laser scanning microscopy of HepG2 cells labeled with betaine-homocysteine S-methyltransferase- and alpha-tubulin-specific monoclonal antibodies. The association of betaine-homocysteine S-methyltransferase with the cytoskeleton may functionally integrate the mitochondrial and cytoplasmic compartments of choline degradation.

Isolation and characterization of a mouse betaine-homocysteine S-methyltransferase gene and pseudogene.

Betaine-homocysteine S-methyltransferase (BHMT) is one of the enzymes involved in the branch point metabolism of homocysteine. Elevated levels of plasma homocysteine may be a risk factor for the development of vascular disease; however, whether BHMT has a significant role in the regulation of plasma levels of homocysteine remains to be determined. As a prelude to creating a mouse strain deficient in BHMT activity, we screened a lambda library containing mouse SvJ 129 genomic DNA for the mouse BHMT gene using random probes made from the human cDNA. One genomic isolate was completely sequenced and found to encode an intronless BHMT pseudogene (mBHMT-ps). mBHMT-ps was then used as a template for the generation of random probes that were used to screen a BAC library containing mouse 129 Sv/Ev genomic DNA. In order to discriminate between pseudogenes and the authentic BHMT gene, a secondary PCR-based screen was employed which used primers designed from the pseudogene sequence that would predictably amplify across introns. Using this strategy, we isolated six mouse genomic clones that tested positive for the presence of all seven introns characteristic of the human gene, and the BHMT gene of one clone was completely sequenced. Like the human BHMT gene, the mouse gene spans 21kb and is encoded by eight exons interrupted by seven introns. The structure of the mouse BHMT gene is described herein as well as the 5'-flanking region of the gene adjacent to exon 1, which we demonstrate is capable of conferring basal promoter activity in Chinese Hamster Ovary cells.

Recombinant human liver betaine-homocysteine S-methyltransferase: identification of three cysteine residues critical for zinc binding.

Betaine-homocysteine S-methyltransferase (BHMT; EC 2.1.1.5) catalyzes the transfer of an N-methyl group from betaine to homocysteine to produce dimethylglycine and methionine, respectively. The enzyme is found in the pathway of choline oxidation and is abundantly expressed in liver and kidney. We have recently shown that human BHMT is a zinc metalloenzyme [Millian, N. S., and Garrow, T. A. (1998) Arch. Biochem. Biophys. 356, 93-98]. To facilitate the rapid purification of human BHMT for further physical and mechanistic studies, including characterizing its metal binding properties, we have overexpressed the enzyme in E. coli as a fusion construct which facilitated its subsequent purification by a self-cleavable affinity tag system (IMPACT T7). Using this expression and purification system in conjunction with site-directed mutagenesis, we have identified Cys217, Cys299, and Cys300 as zinc ligands. Mutating any of these Cys residues to Ala results in the complete loss of activity and a significant reduction in the ability of the protein to bind zinc. Comparing the regions of BHMT amino acid sequence surrounding these Cys residues with similar amino acid sequences retrievable from protein databases, we have identified the following motif: G[ILV]NCX(20,100)[ALV]X(2)[ILV]GGCCX(3)PX(2)I, which we propose to be a signature for a family of zinc-dependent methyltransferases that utilize thiols or selenols as methyl acceptors. Some of the members of this family include the vitamin B(12)-dependent methionine synthases, E. coli S-methylmethionine-S-homocysteine methyltransferase, and A. bisulcatus S-methylmethionine-selenocysteine methyltransferase.

Homocysteine metabolism in cardiovascular cells and tissues: implications for hyperhomocysteinemia and cardiovascular disease.

We have determined the activity and protein levels of CBS in a number of cardiovascular cells and tissues by direct enzyme assay and Western blot analysis, respectively. We have also determined the activity of BHMT in these same tissues and cells and have come to the conclusion that neither enzyme is expressed. This results suggests that in the human cardiovascular system homocysteine metabolism is limited to the remethylation pathway catalyzed by MS. Thus, hyperhomocysteinemia in conjunction with a limited metabolic capacity for homocysteine in the cardiovascular system could result in cellular dysfunction.

Apolipoprotein B mRNA and lipoprotein secretion are increased in McArdle RH-7777 cells by expression of betaine-homocysteine S-methyltransferase.

The cDNA encoding rat betaine-homocysteine S-methyltransferase (BHMT) was isolated through production of monoclonal antibodies against protein fractions enriched with apolipoprotein B (apo B)-mRNA-editing complexes. BHMT mRNA was expressed predominantly in liver, and also in kidney, but not in small intestine. In stable McArdle RH-7777 (McA) cell lines expressing differing levels of BHMT, the editing efficiency of apo B mRNA was unchanged. Evaluation of apo B-mRNA expression revealed that steady-state levels were increased significantly and in parallel with BHMT protein expression. The highest levels of BHMT mRNA and BHMT enzyme activity expressed in stably transfected McA cells were comparable with those found in rat hepatocytes. In contrast to the changes in apo B-mRNA abundance, levels of other apolipoprotein-encoding mRNAs and several liver-specific and ubiquitously expressed mRNAs were unchanged by BHMT expression. In the cell line expressing the highest level of BHMT, apo B-containing lipoprotein secretion was increased, indicating utilization of increased endogenous message. Results suggest that apo B-mRNA abundance in McA cells is related to the expression of BHMT, an enzyme important in homocysteine metabolism.

Interaction between dietary methionine and methyl donor intake on rat liver betaine-homocysteine methyltransferase gene expression and organization of the human gene.

We previously showed that rat liver betaine-homocysteine methyltransferase (BHMT) mRNA content and activity increased 4-fold when rats were fed a methionine-deficient diet containing adequate choline, compared with rats fed the same diet with control levels of methionine (Park, E. I., Renduchintala, M. S., and Garrow, T. A. (1997) J. Nutr. Biochem. 8, 541-545). A further 2-fold increase was observed in rats fed the methionine-deficient diet with supplemental betaine. The nutrition studies reported here were designed to determine whether other methyl donors would induce rat liver BHMT gene expression when added to a methionine-deficient diet and to define the relationship between the degree of methionine restriction and level of methyl donor intake on BHMT expression. Therefore, rats were fed amino acid-defined diets varying in methionine and methyl donor composition. The effect of diet on BHMT expression was evaluated using Northern, Western, and enzyme activity analyses. Similar to when betaine was added to a methionine-deficient diet, choline or sulfonium analogs of betaine induced BHMT expression. The diet-induced induction of hepatic BHMT activity was mediated by increases in the steady-state level of its mRNA and immunodetectable protein. Using methyl donor-free diets, we found that methionine restriction was required but alone not sufficient for the high induction of BHMT expression. Concomitant with methionine restriction, dietary methyl groups were required for high levels of BHMT induction, and a dose-dependent relationship was observed between methyl donor intake and BHMT induction. Furthermore, the severity of methionine restriction influenced the magnitude of BHMT induction. To study the molecular mechanisms that regulate the expression of BHMT, we have cloned the human BHMT gene. This gene spans about 20 kilobases of DNA and contains 8 exons and 7 introns. Using RNA isolated from human liver and hepatoma cells, a major transcriptional start site has been mapped using the 5' rapid amplification of cDNA ends technique, and this start site is 26 nucleotides downstream from a putative TATA box.

Betaine-homocysteine methyltransferase is a developmentally regulated enzyme crystallin in rhesus monkey lens.

We describe herein the characterization of a major 45-kDa protein from the soluble betaH-crystallin fraction of rhesus monkey (Macaca mulatta) lens. Based on partial peptide sequence, immunoreactivity, and enzymatic activity, this protein has been identified as betaine-homocysteine S-methyltransferase (BHMT: EC 2.1.1.5), an enzyme that catalyzes the methylation of homocysteine using either betaine or thetins as methyl donors. This protein was found to be expressed abundantly in the nuclear region of the monkey lens, reaching approximately 10% of the total nuclear protein, but was barely detectable in the epithelium and cortex regions of the lens. Because the nucleus represents the early embryonic and fetal stages of lens development, we infer that BHMT expression in the lens of the eye is developmentally regulated. By virtue of its high abundance, BHMT can be considered an enzyme crystallin (psi-crystallin). This is the first enzyme crystallin to be found in primate lenses.

Human betaine-homocysteine methyltransferase is a zinc metalloenzyme.

We have overexpressed recombinant human liver betaine-homocysteine methyltransferase (BHMT; EC 2.1.1.5) in Escherichia coli and have purified the enzyme to homogeneity. The Michaelis constants for betaine and l-homocysteine are 2.2 mM and 4 microM, respectively. Analysis of the pure protein for metals by inductively coupled plasma emission spectrometry indicate that the recombinant enzyme contains zinc. Extensive dialysis in buffer containing high levels of EDTA could not strip the protein of zinc. However, dialysis against buffer containing EDTA and methyl methanethiosulfonate, followed by buffer containing EDTA and dithiothreitol, could remove zinc from the enzyme with concomitant loss of activity. Dialyzing the zinc-depleted enzyme against buffer containing 1 M urea and 2 mM zinc, followed by dialysis with buffer alone, completely restored BHMT activity and zinc content. BHMT was also partially purified from human liver. The purest BHMT-containing fractions also contained zinc and the enzyme was kinetically indistinguishable from the recombinant enzyme. As with the recombinant enzyme, the partially purified human liver enzyme could be inactivated by treatment with methyl methanethiosulfonate, EDTA, and dithiothreitol. Reconstitution of the zinc-depleted enzyme completely restored activity. We conclude that BHMT is a major zinc metalloenzyme in liver and that cysteineresidues are likely involved in zinc binding.

Hepatic and renal betaine-homocysteine methyltransferase activity in pigs as affected by dietary intakes of sulfur amino acids, choline, and betaine.

In Exp. 1, young pigs were fed a basal diet containing .17% methionine (Met) (.14% digestible Met), and .48% cystine (.38% digestible cystine) for 14 d (34 to 48 d of age). Treatment additions were .25% DL-Met, .34% betaine, .30% choline, or .25% DL-Met and .34% betaine. Methionine, but not betaine or choline supplementation, increased (P < .05) weight gain and feed efficiency. Hepatic betaine-homocysteine methyltransferase (BHMT) activity was increased (P < .05) by betaine and choline supplementation but was not affected by Met deficiency. Renal BHMT activity was increased (P < .05) by Met deficiency and was further increased (P < .05) by betaine supplementation. In Exp. 2, 10-kg pigs were fed the basal diet from Exp. 1 supplemented with enough DL-Met to bring the total basal Met to .24% (.20% digestible Met). Treatment additions consisted of .20% DL-Met or .34% betaine, and diets were fed for 16 d (34 to 50 d of age). Feed efficiency increased (P < .05) in response to Met, but not to betaine, supplementation. Hepatic BHMT activity increased (P < .05) in response to betaine and Met, but no changes in renal BHMT activity occurred. Although statistically significant changes in hepatic and renal BHMT activity occurred in both experiments, the magnitude of the responses was probably not physiologically important. Therefore, in contrast to previous findings with rats and chicks, it does not seem that hepatic and renal BHMT activity in pigs is influenced substantially by Met deficiency, or by surfeit levels of choline or betaine.

Betaine-homocysteine methyltransferase expression in porcine and human tissues and chromosomal localization of the human gene.

We have prepared antibodies against porcine liver betaine-homocysteine methyltransferase (BHMT; EC 2.1.1.5) and recently cloned cDNAs encoding the porcine and human liver enzymes. Porcine tissues were evaluated for BHMT expression by measuring catalytic activity and Western analysis. Liver and kidney were the only organs tested that had immunodetectable levels of BHMT, and these organs expressed high levels of enzyme activity. BHMT was expressed in the kidney cortex and not the medulla. Porcine pancreas, brain, heart, lung, and spleen were devoid of BHMT activity and immunodetectable protein. Human tissues were tested for BHMT expression by Northern analysis. Human liver and kidney were the only organs tested that expressed BHMT mRNA. Human pancreas, brain, heart, skeletal muscle, spleen, and placenta were devoid of BHMT mRNA. The human BHMT gene has been mapped to chromosome 5q13.1-q15.

Development of an experimental diet for determining bioavailable choline concentration and its application in studies with soybean lecithin.

Attempts to determine choline bioavailability have encountered criticism of experimental diets and protocol. Our objectives were to develop a choline-deficient soy isolate diet to quantify bioavailable choline concentration of soybean lecithin and to compare results to those obtained with a purified diet. In Assay 1, weight gain of chicks fed a choline-free crystalline amino acid diet responded linearly (P < .01) to graded doses of choline chloride, fluid lecithin (FL), or deoiled lecithin (DL). Multiple linear regression analysis indicated a bioavailable choline content of 2.3 and 3.7% for FL and DL, respectively. In Assay 2, a choline-deficient soy isolate diet was supplemented with 2-amino-2-methyl-1-propanol (AMP) to inhibit choline biosynthesis. Weight gain, feed intake, and feed efficiency increased (P < .05) markedly with the addition of choline chloride, but supplemental methionine or betaine had no effect (P > .10). Addition of 10% soybean meal to the diet severely deficient in choline per se produced a growth response (P < .05), whereas the same addition to the diet made adequate in choline did not elicit a growth response. In Assay 3, addition of graded levels of choline chloride, FL, or DL to the choline-deficient soy isolate diet containing AMP resulted in a linear (P < .01) increase in weight gain and feed intake. Multiple linear regression analysis indicated a bioavailable choline content of 2.0 and 3.5% for FL and DL, respectively. The AMP-containing soy isolate diet seems well suited for determination of bioavailable choline content in products containing secondary nutrients. Bioavailable choline content of FL and DL was similar to estimates of total choline content, suggesting that the choline in these products was fully available.

Purification, kinetic properties, and cDNA cloning of mammalian betaine-homocysteine methyltransferase.

Porcine liver betaine-homocysteine methyltransferase (BHMT; EC) was purified to homogeneity, and the Michaelis constants for betaine, dimethylacetothetin, and L-homocysteine are 23, 155, and 32 microM, respectively. The maximum rate of catalysis is 47-fold greater using dimethylacetothetin as a methyl donor compared with betaine. Partial amino acid sequence of porcine BHMT was obtained, and inosine-containing redundant oligonucleotide primers were used to amplify an 815-base pair sequence of the porcine cDNA by polymerase chain reaction (PCR). Nondegenerate oligonucleotide primers based on the porcine cDNA were synthesized and used to isolate a 463-base pair fragment of the human cDNA by PCR. The human PCR DNA product was then used to screen a cDNA library by plaque hybridization, and cDNAs encoding human BHMT were isolated. The primary structure of the human cDNA is reported here, and the open reading frame encodes a 406-residue protein of Mr 44,969. The deduced amino acid sequence of human BHMT shows limited homology to bacterial vitamin B12-dependent methionine synthases (EC). A plasmid containing the human BHMT cDNA fused in frame to the N terminus of beta-galactosidase was transformed into Escherichia coli, and transformants expressed BHMT activity, an activity that is absent from wild type E. coli.

Hepatic betaine-homocysteine methyltransferase activity in the chicken is influenced by dietary intake of sulfur amino acids, choline and betaine.

There is much interest in the metabolism of homocysteine, because elevated plasma homocysteine [hyperhomocyst(e)inemia] is an independent risk factor for the development of cardiovascular disease. Four chick assays were conducted to determine the effects of varying dietary sulfur amino acids, choline and betaine on the activity of hepatic betaine-homocysteine methyltransferase (BHMT), an enzyme likely to be important in modulating plasma homocysteine. In Experiment 1, chicks were fed a purified crystalline amino acid diet containing adequate sulfur amino acids and choline. Excess dietary methionine, or the combination of excess cystine with choline or betaine, caused a small increase (P < 0.05) in BHMT activity. In Experiment 2, use of a methionine-deficient purified diet resulted in a threefold increase (P < 0.05) in BHMT activity, and addition of choline or betaine further increased (P < 0.05) BHMT activity. In Experiment 3, use of a methionine-deficient corn-peanut meal diet increased BHMT (P < 0.05) relative to that of chicks supplemented with adequate methionine, and addition of surfeit choline to the methionine-deficient basal diet caused a further increase (P < 0.05). In Experiment 4, addition of both surfeit choline and surfeit betaine to the methionine-deficient corn-peanut meal diet caused an increase (P < 0.05) in BHMT activity relative to that observed in chicks fed the methionine-deficient basal diet. These assays show that large increases in BHMT activity can be produced under methionine-deficient conditions, especially in the presence of excess choline or betaine.

Purification and properties of human cytosolic folylpoly-gamma-glutamate synthetase and organization, localization, and differential splicing of its gene.

Human cytosolic folylpolyglutamate synthetase (FPGS) was expressed in Escherichia coli and purified to homogeneity. Tetrahydrofolate and dihydrofolate were the most effective substrates, while 5-substituted folates were poor substrates. Most pteroyldiglutamates were better substrates than monoglutamates. The human FPGS gene spans 12 kilobases and contains 15 exons and 14 introns. A single FPGS gene was located to chromosome region 9q34.1. Four exon 1 variants were identified, each of which was spliced to exon 2. The exon 1 variant corresponding to the isolated cDNA contains two ATG codons and multiple transcription start sites in this region generates mitochondrial and cytosolic FPGS (Freemantle, S. J., Taylor, S. M., Krystal, G., and Moran, R. G. (1995) J. Biol. Chem. 270, 9579-9584). Exons 1B and 1C, generated by alternate splicing in intron 1, and exon 1A, which is 5' to exon 1 and may encode an additional mitochondrial isoform, are preceded by a number of potential promoter sites. Chinese hamster ovary cell transfectants expressing FPGS activity in the mitochondria contained normal mitochondrial and low cytosolic folylpolyglutamate pools. Mitochondrial FPGS activity is required for mitochondrial folate accumulation, while cytosolic FPGS activity is needed for establishment of normal cytosolic folate pools. The reconstructed FPGS gene restored normal cytosolic and mitochondrial folate metabolism in hamster cells.

Rapid decline in folylpolyglutamate synthetase activity and gene expression during maturation of HL-60 cells. Nature of the effect, impact on folate compound polyglutamate pools, and evidence for programmed down-regulation during maturation.

These studies in HL-60 cells examined the regulation of folylpolyglutamate synthetase (FPGS) activity at the level of gene expression during terminal maturation. Following addition of 210 mM Me2SO to cultures of HL-60 cells at a concentration that induces maturation of 85-90% of the cells, FPGS activity, but not folylpolyglutamate hydrolase (FPGH) activity, was reduced 2-7-fold within 1-5 days. The initial decline in FPGS activity preceded any effect of Me2SO on rate of growth and the increase in appearance of nitro blue tetrazolium-positive cells, a marker of cellular maturation, and the decrease after 5 days of exposure to Me2SO was solely accounted for by a 7-fold decrease in value for Vmax. The same time and concentration dependence for Me2SO was shown for the decline in FPGS activity, increase in nitro blue tetrazolium-positive cells, and decline in the level of a 2.1-kilobase FPGS mRNA during exposure to this inducer. This decline in FPGS mRNA was reversible when Me2SO was removed from the culture medium but only until that time when an appreciable number of cells were committed to terminal maturation. Following growth of HL-60 cells with [3H]MTX, used as a model folate compound, a large reduction in its intracellular polyglutamate pools was shown during maturation which quantitatively reflected the decline in FPGS activity as well as folate transport inward (Sirotnak, F.M., Jacobson, D.M., and Yang, C-H. (1986) J. Biol. Chem. 261, 11150-11156). Other data showed that folate status or obviation of the folate requirement during growth of these cells strongly influenced the rapidity of the onset of maturation following exposure to inducer. Overall, these results show that FPGS activity in HL-60 cells is a marker for proliferative capacity and that the underlying basis for the decline in FPGS activity during maturation is altered cognate gene expression which is manifested as early reversible and late irreversible phases. They also suggest that the coordinate reduction observed in folate transport, FPGS activity, dihydrofolate reductase, and probably other folate related enzymes by limiting macromolecular biosynthesis may be early programmed events in the maturation process that influence the switch from proliferation to senescence in these cells.

Cloning of human cDNAs encoding mitochondrial and cytosolic serine hydroxymethyltransferases and chromosomal localization.

Human cDNAs for cytosolic and mitochondrial serine hydroxymethyltransferase (SHMT) were cloned by functional complementation of an Escherichia coli glyA mutant with a human cDNA library. The cDNA for the cytosolic enzyme encodes a 483-residue protein of M(r) 53,020. The cDNA for the mitochondrial enzyme encodes a mature protein of 474 residues of M(r) 52,400. The deduced protein sequences share a high degree of sequence identity to each other (63%), and the individual isozymes are highly homologous to the analogous rabbit liver cytosolic (92% identity) and mitochondrial (97% identity) SHMT isozymes (Martini, F., Angelaccio, S., Pascarella, S., Barra, D., Bossa, F., and Schirch, V. (1987) J. Biol. Chem. 262, 5499-5509; Martini, F., Maras, B., Tanci, P., Angelaccio, S., Pascarella, S., Barra, D., Bossa, F., and Schirch, V. (1989) J. Biol. Chem. 264, 8509-8519). SHMT is a highly conserved protein with the human isozymes retaining about 43% sequence identity with the E. coli protein. The human cytosolic and mitochondrial SHMT genes were localized to chromosome regions 17p11.2 and 12q13, respectively. The high degree of nucleotide sequence identity between the two isozymes, and the presence of keratin genes in both chromosomal regions, is consistent with these regions of chromosome 12 and 17 arising by a duplication event.

Expression cloning of a human cDNA encoding folylpoly(gamma-glutamate) synthetase and determination of its primary structure.

A human cDNA for folypoly(gamma-glutamate) synthetase [FPGS; tetrahydrofolate:L-glutamate gamma-ligase (ADP forming), EC 6.3.2.17] has been cloned by functional complementation of an Escherichia coli folC mutant. The cDNA encodes a 545-residue protein of M(r) 60,128. The deduced sequence has regions that are highly homologous to peptide sequences obtained from purified pig liver FPGS and shows limited homology to the E. coli and Lactobacillus casei FPGSs. Expression of the cDNA in E. coli results in elevated expression of an enzyme with characteristics of mammalian FPGS. Expression of the cDNA in AUXB1, a mammalian cell lacking FPGS activity, overcomes the cell's requirement for thymidine and purines but does not overcome the cell's glycine auxotrophy, consistent with expression of the protein in the cytosol but not the mitochondria.

Influence of hypertension and dietary copper on indexes of copper status in rats.

The Dahl salt-sensitive rat was used to investigate the effect of hypertension on indexes of copper status and to determine the extent to which dietary manipulation of copper attenuated, or exacerbated, the rate of sodium chloride-induced hypertension. Weanling salt-sensitive rats were fed, in a 2 x 3 factorial design, one of six diets that contained one of three levels of copper (2.0 micrograms/g marginal, 12 micrograms/g adequate, or 50 micrograms/g supplemental) and either control (0.4%) or high (4%) levels of sodium. Diets were fed to the rats for 11 weeks. Rats fed the high sodium diets were characterized by high plasma copper concentrations and ceruloplasmin activities compared with their respective control sodium rats. The magnitude of the sodium-induced rise in plasma copper and ceruloplasmin was affected by dietary copper intake; however, dietary copper intake had no effect on the development of hypertension in the high sodium groups. These results suggest that altered copper metabolism is secondary, rather than primary, to the development of sodium chloride-induced hypertension in the salt-sensitive rat. Red blood cell superoxide dismutase activity was reduced in rats fed the low copper diets compared with the adequate and supplemented copper groups. At the lower levels of copper intake, sodium chloride-induced hypertension increased red blood cell superoxide dismutase activity in a manner consistent with the plasma copper and ceruloplasmin changes observed. However, at adequate or supplemental levels of dietary copper, red blood cell superoxide dismutase activity plateaued, suggesting possible saturation of copper at sites of hematopoeisis.