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Institute of Nutritional Sciences, University of Technology of Munich, 85350 Freising-Weihenstephan, Germany.
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • vitamin B-12 deficiency • hyperhomocysteinemia • high trace element diets • pigs
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)
and animals (Kennedy et al. 1994
, Stangl et al. 2000
, Van der Westhuyzen et al. 1985
,
Young et al. 1997
) and a derangement of folate
metabolism similar to that caused by a primary folate deficiency
(Bässler 1997
).
Recent results from a few studies demonstrated that experimentally
induced vitamin B-12 deficiency and hyperhomocysteinemia can change the
metabolism of the trace elements iron, copper and nickel. These changes
include elevated tissue iron stores in rats and pigs (Brown and Strain 1990
, Stangl et al. 2000
, Young et al. 1997
), depressed body copper concentrations in rats
(Brown and Strain 1990
) and an accumulation of liver
nickel in cattle (Stangl et al. 1999
). These
observations suggest that vitamin B-12 and homocysteine may alter
processes relating to trace element mobilization and transport. A
further conjecture is that trace elements might also have some
influence on vitamin B-12–dependent processes. This hypothesis is
supported by a few observations in rat studies. Nielsen et al. (1993)
and Uthus and Poellot (1996)
suggested that nickel has a biological
function in a metabolic pathway in which vitamin B-12 is important.
Tamura et al. (1999)
reported that copper is necessary for maximum
activity of methionine synthetase. If trace elements are involved in
vitamin B-12–dependent processes, we suggest that trace element
supplementation during vitamin B-12 deficiency could influence the
outcome of deficiency symptoms including hyperhomocysteinemia. The
specific objectives of this experiment were to obtain further clues as
to the locus of the biochemical action of iron, copper, nickel and
cobalt in vitamin B-12 metabolism. Pigs were used as the model, and the
degree of vitamin B-12 deficiency was monitored by analysis of serum
and liver concentrations of vitamin B-12 and folate, by analysis of
blood cell counts and by measurement of the concentration of
circulating homocysteine.
Moreover, homocysteine, which occupies a branch point in the
transsulfuration pathway of vitamin B-12–dependent methionine
metabolism, is of special interest because of its claimed link with
endothelial cell dysfunction, an early event in the progression of
atherothrombotic disease (Outinen et al. 1999
). The
ability of excessive homocysteine to generate hydrogen peroxide has
been implicated as a potential mechanism leading to endothelial
dysfunction, which was observed in subjects with hyperhomocysteinemia
(Kang et al. 1986
). This study therefore also focused on
the measurement of variables indicative of imbalances in oxidative and
antioxidative status. These included the concentrations of circulating
serum tocopherols and thiols, liver concentrations of oxidized
glutathione (GSSG) and the antioxidant enzymes glutathione peroxidase
(GSHPx, cofactors selenium, copper, EC 1.11.1.9) and catalase (EC
1.11.1.6).
Experimental vitamin B-12 deficiency can be produced in two different
ways, either nutritionally or by exposure to the Co (I) vitamin
B-12–inactivating agent nitrous oxide, which has often been used as a
model for vitamin B-12 deficiency responses in pigs (e.g., Scott et al. 1994
, Weir et al. 1992
). However,
metabolism of nitrous oxide could produce toxic intermediates and could
affect cytochrome C oxidase and Ca2+-ATPase, side
effects that are unrelated to cobalamin inactivation
(Einarsdottir and Caughey 1988
, Hong et al. 1980
, Horn et al. 1999
). Because of these
side effects, and with a view to simulating situations as they occur
with completely vegetarian diets or trophic changes at vitamin B-12
absorption sites, we decided to induce vitamin B-12 deficiency by
dietary factors alone. This was done by prolonged feeding of a vitamin
B-12-free diet without additional folate for 166 d, combined with
a relatively high dietary methionine concentration because
methionine-loading has been shown to exacerbate
hyperhomocysteinemia in miniature pigs and rabbits
(Jourdheuil-Rahmani et al. 1995
, Koyama 1995
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this experiment, 48 piglets (German Landrace x
Pietrain) from multiple litters of commercial crossbred sows
with a body weight of 8.61 ± 0.14 (mean ± SEM)
kg were assigned to one of six treatment groups. Their allocation to
dietary treatments was random from blocks based on litter, sex and
initial weight. Each group comprised 8 piglets with 4 male castrates
and 4 females. The dietary treatments were as follows: one control
diet, in which pigs were fed a vitamin B-12-adequate and
folate-supplemented diet with normal trace element concentrations
(B12-C), or one of five vitamin B-12–free, folate nonsupplemented
diets, with either normal trace element concentrations (B12-D), high
iron concentrations (B12-D + Fe), high copper concentrations
(B12-D + Cu), high cobalt concentrations (B12-D + Co) or high nickel
concentrations (B12-D + Ni). The experiment lasted 166 d. Each
group received the same basal diet to limit observed differences in the
actions of vitamin B-12 and the trace elements with the diets alone.
From d 1 to 42 all pigs received basal diet 1 that was formulated to
meet the nutrient requirements of piglets (5–25 kg;
Gesellschaft für Ernährungsphysiologie 1987
,
National Research Council 1998
). From d 43 to 166, a
grower diet 2 was administered for all groups whose nutrient
composition was designed to satisfy the mean requirement of pigs
weighing 25–90 kg (Gesellschaft für Ernährungsphysiologie 1987
, National Research Council 1998
). The diet and nutrient compositions are shown in
Table 1
. The basal nonsupplemented diet that was offered from d 1 to 42
contained (per kg dry matter,
DM3) no vitamin B-12 and 0.35 mg folate, 100 mg iron, 6.0 mg copper, 0.13
mg cobalt and 1.01 mg nickel. The basal nonsupplemented grower diet
that was offered from d 43 to 166 contained (per kg DM) no vitamin B-12
and 0.37 mg folate, 86 mg iron, 6.4 mg copper, 0.14 mg cobalt and 1.27
mg nickel. All vitamin B-12–deficient diets were formulated to contain
no vitamin B-12 and no additional folate. The high iron diet was
supplemented with FeSO4 x 7
H2O to contain 300 mg/kg DM iron, the high copper
diet was supplemented with CuSO4 x 5
H2O to contain 30 mg/kg DM copper, the high
cobalt diet was supplemented with CoSO4 x 7
H2O to contain 1 mg/kg DM cobalt and the high
nickel diet was supplemented with NiSO4 x 6
H2O to contain 6 mg/kg DM nickel. For the vitamin
B-12– and folate-adequate control diet, the basal diets fed from d
1 to 42 and d 43 to 166 were fortified with 30 µg/kg DM
vitamin B-12 and 150 and 130 µg/kg DM folate,
respectively, to contain 0.5 mg/kg DM folate (National Research Council 1998
). Dietary methionine was fed in relatively high
concentrations to exacerbate the hyperhomocysteinemia. Therefore, the
basal diets that were offered to the vitamin B-12–adequate group and
the vitamin B-12–deficient groups were supplemented with
DL-methionine to contain 12 g/kg DM and 7.5 g/kg DM
methionine, respectively. The energy density was 13.1 MJ metabolizable
energy/kg feed for all diets. Except for vitamin B-12 and folate, all
diets were fortified with recommended amounts of vitamins and minerals
(Gesellschaft für Ernährungsphysiologie 1987
, National Research Council 1998
).
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Analyses.
Five days before slaughter, on d 161 of the experiment, 12 h after the last feeding, venous blood samples were collected by puncture of the jugular vein for determination of the blood cell count and measurement of serum concentrations of vitamin B-12, folate, homocysteine, clinico-chemical variables, enzymes and tocopherols. Blood cell counts and hematological variables were determined in EDTA-treated blood. Blood for determination of the serum metabolites was collected into untreated tubes. On d 166, 18 h after the last feeding, all pigs were slaughtered and livers excised for analysis of vitamin B-12, folate and trace elements. Serum and liver samples for measurements were stored at -80°C pending analysis.
Serum and liver concentrations of vitamin B-12 and folate were
determined using a competitive binding RIA kit (ICN, Costa Mesa, CA)
that worked with an extracting reagent (containing 1 mol/L sodium
hydroxide and an organic extracting enhancer) to release vitamin B-12
from transcobalamines. The RIA test kit used in this study served to
remove the nonspecific vitamin B-12–binding R-protein by affinity
chromatography. Prior to the RIA quantification, vitamin B-12 and
folate were released from liver binders by papain proteolysis using the
double extraction method of Van Tonder et al. (1975)
.
Serum concentrations of total homocysteine (free, bound to proteins or
as mixed disulphide) were determined by HPLC (Cornwell et al. 1993
). Serum samples were prepared for derivatization according
to the method of Ubbink et al. (1991)
using
7-fluorbenzo-2-oxa-1,3-diazole-4-sulfonamide as derivatization reagent.
Homocysteine was separated using a reverse-phase column (Nucleosil
120–5 C18; 250 x 4.6 mm internal diameter, 5-µm-thick film;
Machery & Nagel, Düren, Germany). The fluorescence
spectrophotometer was operated at an excitation wavelength of 385 nm
and an emission wavelength of 515 nm. The mobile phase, pumped at 1.5
mL/min, consisted of 0.1 mol/L potassium dehydrogenphosphate (adjusted
to pH 2.1 with orthophosphoric acid, containing 2.4 mol/L
acetonitrile).
Hematological variables such as blood cell counts, hemoglobin concentration, hematocrit and mean corpuscular volume (MCV) were determined with a Coulter counter and a hemoglobinometer (Coulter Electronics GmbH, Krefeld, Germany). Total protein, albumin, creatinine, urea, glucose and the lipids cholesterol, triacylglycerol and phosphatidylcholine in serum were determined by standardized procedures using an auto analyzer (model 704; Hitachi, Tokyo, Japan) and Boehringer kit reagents (Boehringer, Mannheim, Germany).
For trace element analysis of the liver, 10-g aliquots were dry-ashed for 3 d at 480°C. The dry-ashed samples were then dissolved in 0.6 mol/L hydrochloric acid. One blank was included for each dry-ashing stage. The cobalt and nickel concentrations of the samples were then determined by absorbance at 240.7 and 232.0 nm, respectively, after the specimens were loaded into a pyrolytically coated graphite tube of an atomic absorption spectrophotometer (model 5100, HGA-600 Graphite Furnace; Perkin–Elmer, Überlingen, Germany). Iron and copper concentrations of the liver were measured directly by their absorbance at 248.3 and 327.4 nm by aspirating the dry-ashed samples dissolved in hydrochloric acid into the flame of the atomic absorption spectrophotometer. All specimens were analyzed in duplicate. In the analysis of iron and copper, the CV for duplicate analyses were typically <2%, and in the analysis of cobalt and nickel, the CV for duplicate analyses was <5%. All trace element concentrations were expressed on a fresh weight basis.
Liver concentration of oxidized GSSG was determined by HPLC
(Asensi et al. 1994
). To 0.5 g of freeze-dried
liver 3 mL of 1.2 mol/L ice-cold perchloric acid containing 40
mmol/L N-ethylmaleimide and 2 mmol/L
bathophenanthrolinedisulfonic acid was added.
N-ethylmaleimide was used to prevent GSH oxidation
during sample preparation. Then samples were centrifuged for 5 min at
15,000 x g (4°C), and supernatant was used for
derivatization. 1-Fluoro-2,4-dinitrobenzene was used as derivatization
reagent (Asensi et al. 1994
). GSSG was separated using a
Spherisorb aminopropyl column (250 x 4.6 mm internal diameter;
5-µm-thick film; Sigma Chemical, St. Louis, MO). The UV
spectrophotometer was operated at a wavelength of 365 nm. The flow rate
was 1.0 mL/min during the procedure. The mobile phases and the gradient
were used as described by Aseni et al. (1994)
.
For determination of hepatic catalase (EC 1.11.1.6), liver homogenates
(250 g/L) prepared in 0.25 mmol/L sucrose (containing 20 mmol/L
ethanol) were centrifuged at 700 x g for 10 min to
remove nuclei, unbroken cells and cell debris. Then supernatant was
used for the determination of the catalase activity. Total catalase
activity was measured spectrophotometrically (Aebi 1970
), after pretreatment of the enzyme source with Triton
X-100 to a final concentration of 1% to disrupt the peroxisomal
membranes. The determination of the enzyme activity was based on the
measurement of the rate of converting hydrogen peroxide at 240 nm and a
temperature of 25°C in the presence of the enzyme. Protein in 700
x g supernatant used for enzyme determination was
measured by a method of Smith and co-workers (1975) using
bicinchoninic acid and bovine serum albumin as a standard. The activity
of GSHPx (EC 1.11.1.9) in serum was determined by a spectrophotometric
method (Paglia and Valentine 1967
). The determination of
total thiol groups in serum (from protein and glutathione) was done by
a spectrophotometric method of Hu (1994)
. Prior to measuring the thiols
the serum was analyzed for its protein concentration by a standardized
procedure using an auto analyzer (model 704; Hitachi) and Boehringer
kit reagents.
Concentrations of individual tocopherols in serum were determined by
HPLC (Balz et al. 1993
). Serum samples (100 µL) were
mixed with 1 mL of a 80 mmol/L pyrogallol solution (in ethanol,
absolute) and 150 µL saturated sodium hydroxide solution. This
mixture was heated for 30 min at 70°C, and tocopherols were extracted
with n-hexane. Individual tocopherols of the extracts were
separated isocratically using a mixture of n-hexane and 1,4-dioxane
(96:4, v/v) as mobile phase and a LiChrosorb Si 60 column (5-µm
particle size, 250-mm length, 4-mm internal diameter; Merck, Darmstadt,
Germany) and detected by fluorescence (excitation wavelength,
295 nm; emission wavelength, 320 nm).
Statistical analysis.
Single classification ANOVA was used for analysis of serum and liver concentrations of vitamin B-12 and folate, blood cell counts, the concentration of circulating homocysteine, the response of the antioxidant system and the concentrations of trace elements in liver. Data were subjected to logarithmic transformation where necessary to achieve homogeneity of variances. Differences between dietary treatment groups were compared by the Student-Newman-Keul’s (SNK) test. Significant difference was assigned at P < 0.05. Data are presented as mean ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The removal of vitamin B-12 from the diets generally resulted in a
significant reduction in serum and liver vitamin B-12 concentrations
compared with concentrations found in pigs receiving the corresponding
vitamin B-12–adequate diet (Table 2
). However, the magnitude of the decrease was different among the
vitamin B-12–deficient groups. Among pigs receiving no vitamin B-12,
the group fed the high nickel diet had significantly higher serum and
liver concentrations of vitamin B-12 than pigs fed normal amounts of
trace elements or diets fortified with iron, copper and cobalt. As was
the case with vitamin B-12, the concentration of folate in liver was
lower in pigs fed the vitamin B-12-deficient diets than in pigs fed the
vitamin B-12–adequate control diet. The serum concentration of folate
was significantly reduced only in the vitamin B-12–deficient group fed
normal amounts of trace elements compared with all the other groups.
Vitamin B-12–deficient pigs fed the iron-, copper-, cobalt- and
nickel-fortified diets had serum folate concentrations not
different from those observed in the vitamin B-12–adequate group.
|
). The highest homocysteine concentrations, which differed significantly
from those of the vitamin B-12–adequate pigs, were found in serum of
vitamin B-12–deficient pigs fed normal trace element amounts and the
iron- and copper-fortified diets. Intermediate values were observed
with the vitamin B-12–free high cobalt or high nickel diets. Vitamin
B-12–deficient pigs fed the high cobalt or nickel diets had about 40%
lower (P > 0.05) serum concentrations of homocysteine
than the corresponding vitamin B-12-deficient pigs fed the normal trace
element amounts or the iron- and copper-fortified diets.
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. MCV
values were significantly influenced by the dietary treatments. Pigs
fed the vitamin B-12–deficient, high-iron diet had higher MCV
values than the vitamin B-12–adequate control group. MCV values of the
other vitamin B-12–deficient groups did not significantly differ from
those of the control group. White blood cell counts (B12-C: 18.0
± 0.6, B12-D: 18.5 ± 1.9, B12-D + Fe: 15.1 ± 1.7,
B12-D + Cu: 15.4 ± 1.7, B12-D + Co: 17.6 ± 1.2 and B12-D +
Ni: 17.7 ± 1.0 109/L), hemoglobin
concentration (B12-C: 130 ± 8, B12-D: 113 ± 7, B12-D + Fe:
128 ± 7, B12-D + Cu: 131 ± 6, B12-D + Co: 130 ± 4 and
B12-D + Ni: 121 ± 8 g/L) and hematocrit (B12-C: 0.423
± 0.017, B12-D: 0.365 ± 0.017, B12-D + Fe: 0.404 ± 0.014, B12-D + Cu: 0.416 ± 0.014, B12-D + Co: 0.411
± 0.007 and B12-D + Ni: 0.388 ± 0.019) did not differ among
the groups.
Iron concentration in liver was twice as high in pigs fed the vitamin
B-12–deficient diets with normal or high amounts of iron and copper as
in the vitamin B-12–adequate group (Table 3
). Intermediate values were observed for the vitamin B-12–deficient
group fed the high cobalt diet. The iron concentration in livers of
vitamin B-12–deficient pigs fed the high nickel diet reached values
not different from those in the vitamin B-12–adequate group. The
copper concentration in liver was highest in the vitamin
B-12–deficient group fed the high copper diet. Additionally, the
vitamin B-12–deficient group fed the high cobalt diet also had
significantly higher copper concentrations in liver than the control
group. Copper concentration in livers of pigs fed the vitamin
B-12–free diet with normal trace element amounts or excessive iron or
nickel was not significantly different from that of the vitamin
B-12–adequate pigs. Vitamin B-12–adequate pigs and vitamin
B-12–deficient pigs fed the high cobalt diet had much higher liver
concentrations of cobalt than the other groups. The dietary treatments
had no significant influence on the nickel concentration of those
livers.
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.
The serum concentrations of 
-tocopherol (B12-C: 2.68
± 0.16, B12-D: 2.87 ± 0.19, B12-D + Fe: 2.77 ± 0.31, B12-D + Cu: 2.88 ± 0.18, B12-D + Co: 2.78 ± 0.16 and
B12-D + Ni: 2.68 ± 0.19 µmol/L) and thiols (B12-C:
58.4 ± 6.6, B12-D: 74.3 ± 9.7, B12-D + Fe: 73.5 ± 5.1, B12-D + Cu: 83.5 ± 6.3, B12-D + Co: 68.4 ± 4.3 and
B12-D + Ni: 68.8 ± 7.6 µmol/L) were not influenced
by the dietary treatment. The serum concentration of
-tocopherol was diminished in all groups fed the vitamin
B-12–deficient diets compared with the vitamin B-12–adequate control
group (P < 0.05). The dietary treatment had
significant effects on the activities of serum GSHPx and liver
catalase. Vitamin B-12–adequate pigs and vitamin B-12–deficient pigs
fed excessive iron, copper, cobalt and nickel had similar GSHPx
activities in serum. The vitamin B-12–deficient pigs fed normal
amounts of trace elements had the lowest activity of GSHPx, although
this difference was only significant in comparison with pigs fed the
high cobalt diet (Table 3)
. Vitamin B-12–deficient pigs fed normal
trace element amounts had a greater activity of liver catalase than the
control group. Catalase activities in the other groups did not differ
from that of the control group. The liver concentrations of GSSG did
not differ among the groups (B12-C: 1.18 ± 0.18, B12-D: 1.16
± 0.24, B12-D + Fe: 1.23 ± 0.20, B12-D + Cu: 1.04 ± 0.10, B12-D + Co: 1.12 ± 0.17 and B12-D + Ni: 0.87 ± 0.12
mmol/kg). Serum concentrations of the clinico-chemical variables (including total protein, albumin, creatinine, urea, and glucose) were not influenced by the dietary treatments (data not shown). The serum concentrations of cholesterol, triacylglycerols and phosphatidylcholine also did not differ among the groups (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). However, the magnitude of changes differed among the
vitamin B-12–deficient groups. The data demonstrate clearly that
cobalt and nickel influence the response of the pig to vitamin B-12
deficiency when labile methyl group metabolism is compromised. The
findings further indicate that there is a collaborative relationship
between the vitamins B-12 and folate and the trace elements nickel and
cobalt, although the responses of nickel and cobalt to vitamin B-12
deficiency differed in some respects. Nickel was more effective than
cobalt at improving vitamin B-12 deficiency outcomes. The main effects
of nickel were an improvement of the vitamin B-12 status and a decline
of the homocysteine concentration in serum, although these variables
did not reach the values observed in the control group, and a complete
normalization of the iron concentration in liver. In contrast, cobalt
supplementation only improved homocysteine accretion in serum and
partly diminished abnormal iron accretion in liver, whereas the vitamin
B-12 status remained completely unchanged.
First, the interaction between nickel and vitamin B-12 could be the
result of nickel being used to synthesize vitamin B-12 by bacteria in
the animals’ gut, which would result in a greater intake of vitamin
B-12 through coprophagy. Nickel is an essential trace element for many
microorganisms, and bacteria require nickel for the synthesis of
enzymes such as urease, carbon monoxide dehydrogenase, many
hydrogenases and methyl-coenzyme-M reductase (Ankel-Fuchs and Thauer 1988
). Enterobacteriaceae, which have been proved to be
capable of synthesizing vitamin B-12 (Vorob’eva et al. 1987
), require the activity of nickel-containing
hydrogenase for their anaerobic growth (Ankel-Fuchs and Thauer 1988
). Because nickel is known to be poorly absorbed from
ordinary diets and is excreted mostly in the feces, the high nickel
diet used in this study might have contributed to improved bacterial
growth and vitamin B-12 synthesis in the gut. The fecal bacterial flora
is a major source of vitamins for coprophagic animals. Coprophagy
is a phenomenon mainly known from rodents, but not so much from pigs,
although it cannot be excluded that pigs also may coprophagy in states
of nutritional imbalance. However, the efforts that were made to remove
feces from cages to prevent coprophagy together with failure to
demonstrate any effect of cobalt supplementation on vitamin B-12
status, although cobalt is an integral compound of vitamin B-12 and
could be used for vitamin B-12 synthesis by bacteria, indicate that
mechanisms other than intestinal synthesis and coprophagy must be
responsible for the attenuation of vitamin B-12 deficiency in nickel-
and cobalt-supplemented pigs.
In any event, there is experimental evidence of a metabolic interaction
between vitamin B-12 and trace elements. As early as 1952 Lecoq
reported that nickel acted in synergy with vitamin B-12 in stimulating
hematopoiesis (Lecoq et al. 1952
). It was subsequently
suggested that nickel has a physiologic function related to vitamin
B-12 metabolism, possibly one-carbon metabolism (Nielsen et al. 1989
, Poellot et al. 1990
, Uthus and Poellot 1996
). The present study shows that vitamin B-12–free
diets with nickel concentrations of 6 mg/kg can improve several
clinical outcomes of vitamin B-12 deficiency compared with vitamin
B-12–deficient diets containing 1 mg nickel/kg diet, a nickel
concentration that has been considered ample for animal growth
(Schnegg and Kirchgessner 1975
). In addition to the
statement of Nielsen and co-workers (1993) to the effect that
vitamin B-12 is necessary for the optimal expression of the biological
role of nickel, we maintain that nickel has a vitamin B-12–sparing
effect in animals deprived of vitamin B-12. Moreover, cobalt was found
to act in a manner similar to that of nickel on the serum concentration
of homocysteine, without influencing vitamin B-12. The reason for this
phenomenon is unclear, but previous studies have established that
cobalt ions induce a series of metabolic changes in experimental
animals such as inhibition of cytochrome P-450 metabolism (Zhang et al. 1998
), greatly reduced zinc output in urine
(Rosenberg and Kappas 1989
), stimulation of hepatic
concentrations of reduced glutathione (Sasame and Boyd 1978
) and regulation of renal tubular reabsorption processes
(Goncharevskaia et al. 1985
). Although this study does
not show any specific site of action of cobalt in these processes, it
is conceivable, for example, that cobalt may lower serum homocysteine
concentration via the promotion of the renal clearance of this amino
acid. Whether cobalt and nickel will interfere the
betaine-dependent reaction, mediated by the zinc-containing
betaine-homocysteine methyltransferase (EC 2.1.1.5; Millian and Garrow 1998
), which supplements to a small extent the activity
of the methylcobalamin-dependent methionine synthetase when the
latter is impaired in severe vitamin B-12 deficiency (Maree et al. 1990
), remains speculative.
Additionally, an interesting observation may be made from the serum
concentration of folate in the vitamin B-12–deficient groups. The
vitamin B-12–deficient groups fed either iron, copper, cobalt or
nickel, like the vitamin B-12–adequate group, had higher serum
concentrations of folate than the vitamin B-12–deficient group fed
normal amounts of trace elements. At present, we do not have a
mechanistic explanation for why high trace element diets lead to
different responses in serum concentration of folate. The liver exerts
substantial regulatory effect in folate homeostasis because of its
large and rapid folate turnover, the large flux through the
enterohepatic cycle (Steinberg 1984
) and also the
concentration of folate binders in tissues may play an important role
in folate turnover (Lakshmaiah and Bamji 1981
). However,
there is no evidence to date that trace elements may interfere in these
processes, and we think that there is an obvious need for more
information about the mode of action of metals on folate metabolism.
In this study we failed to demonstrate that homocysteine exhibits a
distinct prooxidative potential. A reduction of the
-tocopherol concentration in serum was the only
indication for diminished antioxidant status caused by vitamin B-12
deficiency, although this observation is of minor importance because of
the low -tocopherol concentration relative to
-tocopherol. In contrast, other studies have
unequivocally demonstrated that hyperhomocysteinemia compromised plasma
redox thiol status (Ueland et al. 1996
) and led to
increased lipid peroxidation (Brown and Strain 1990
,
Young et al. 1997
). However, there are also in vivo
studies that have failed to demonstrate marked changes in the
oxidative/antioxidative balance through hyperhomocysteinemia
(Dudman et al. 1993
, Mele and Meucci 1996
, Stangl et al. 2000
). The ability of
homocysteine to trigger oxidative procedure in vivo is therefore a
subject of some controversy. Preibisch et al. (1993)
were able to
demonstrate in vitro that homocysteine exhibits a significant
prooxidant potential only in the presence of either copper or iron as
transition-metal ions. Our results indicate clearly that the
combined effect of vitamin B-12–deficiency–induced
hyperhomocysteinemia and administration of high cationic concentrations
did not induce a greater loss of antioxidant potential than
hyperhomocysteinemia alone.
It can be concluded from these observations that vitamin B-12–deficiency symptoms, including an accumulation of serum homocysteine, can be attenuated by nickel and cobalt, although the mode of action of these elements seems to differ. If the effects of nickel and cobalt are indeed mediated by metabolic processes, then the relevance to human nutrition is quite plausible.
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FOOTNOTES |
|---|
3 Abbreviations used: B12-C, vitamin B-12–adequate
control group; B12-D, vitamin B-12–deficient group fed normal
concentrations of iron, copper, cobalt and nickel; B12-D+Fe, vitamin
B-12–deficient, high-iron group; B12-D+Cu, vitamin
B-12–deficient, high-copper group; B12-D+Co, vitamin
B-12–deficient, high-cobalt group; B12-D+Ni, vitamin
B-12–deficient, high-nickel group; DM, dry matter; GSHPx,
glutathione peroxidase; GSSG, oxidized glutathione; MCV, mean
corpuscular volume; RBC, red blood cells; Tcp, tocopherols; WBC, white
blood cells.
Manuscript received May 19, 2000. Initial review completed June 13, 2000. Revision accepted August 11, 2000.
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REFERENCES |
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|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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