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North Carolina State
University Return to main menu Plasma Diamine Oxidase: a Biomarker of Copper Deficiency in the Bovine L. R. Legleiter and J. W.
Spears Summary Liver copper (Cu), plasma
Cu, and plasma ceruloplasmin activity indicated that cattle receiving diets
designed to induce Cu deficiency did become Cu deficient. In addition to these traditional indices
of Cu status, plasma diamine oxidase (DAO) activity, a relatively simple
assay, was an equally effective biomarker of Cu deficiency in the bovine. Introduction Copper deficiency in the
bovine, a widespread problem in many areas of North America, may result in
decreased growth, anemia, weak bones, cardiac failure, depigmentation of
hair, and reduced reproductive efficiency (NRC, 1996). Currently there are several ways to
diagnose Cu deficiency; however, each poses unique challenges. For example, the enzymatic activity assay
used to measure plasma ceruloplasmin is relatively simple; however,
ceruloplasmin is an acute phase protein that is increased during inflammation
and may therefore erroneously over-predict Cu status during stress and
infection (DiSilvestro,1990).
Further, liver Cu is an accurate predictor of Cu status, but requires
rather involved means of sample collection, and like plasma Cu, requires
advanced analytical equipment to quantitatively measure Cu
concentrations. It would be
advantageous to have an indicator of Cu status that is both easy to acquire
and simple to analyze with few confounding factors. Plasma DAO is a
copper-containing enzyme responsible for the oxidative deamination of
diamines (cadaverine and putrescine), their derivatives, and histamine
(Wolvekamp and DeBruin, 1994). A
relatively simple and sensitive colorimetric assay for plasma DAO was
developed by Takagi et al. (1994) and slightly modified by Kehoe et al.
(2000). Further, recent research in
rodent models has indicated that plasma DAO may serve as a sensitive
biomarker of Cu deficiency (Kehoe et al., 2000). Thus, the objective of this study was to determine the efficacy
of plasma diamine oxidase activity as a biomarker for Cu deficiency in the
bovine. Materials
and Methods Twenty-eight Angus steers
(n = 11) and heifers (n = 17) were used to determine the efficacy of plasma
diamine oxidase activity as a biomarker of copper (Cu) deficiency in the
bovine. The steers and heifers were
born to dams assigned to one of the following treatments: 1) control, 2) Cu
deficient (-Cu), and 3) Cu deficient plus high dietary manganese (-Cu+Mn; Table 1).
Thiomolybdates, a three-way interaction between copper, molybdenum
(Mo), and sulfur, are strong Cu antagonists that form in the rumen. Thus, by providing supplemental molybdenum
and sulphur, treatments -Cu and -Cu+Mn were designed to induce Cu deficiency
via the formation of thiomolybdates.
These animals were on a concurrent study evaluating the relationships
between Cu, Mn, and brain prion proteins; thus the need for treatment
-Cu+Mn. Following weaning (7 months
of age) calves were maintained on the same treatments (Table
1) through 15 months of age and were individually fed via Calan
gates. Cattle received a corn
silage-based diet during the growing phase and a corn-based diet during the
finishing phase. Samples were collected
from all animals at 13 months of age to assess Cu status and the
effectiveness of plasma DAO as a biomarker of Cu deficiency. Blood samples, used for the determination
of plasma Cu, ceruloplasmin activity, and plasma DAO were collected via jugular
venipuncture. Liver biopsies were obtained
for the analysis of liver Cu concentrations. The DAO assay was
conducted at 37˚C in 96-well plates.
Thirty microliters of plasma or standard was added to 20 ul PIPES
dilution buffer and incubated with 130 ul cadaverine substrate solution (30
mM cadaverine in 25 mM PIPES buffer containing 0.5% Triton X-100, pH 7.2) for
30 minutes. Next, 150 ul of color
solution containing the chromagen DA-67 (Wako Chemical, Osaka, Japan),
ascorbate oxidase, and peroxidase type X (Sigma Chemical, St. Louis, MO) was
added. Methylene blue was allowed to
develop for 15 minutes at which point the absorbance was read at 668 nm using
a microplate spectrophotometer.
Diamine oxidase activity (U/ml) was quantified using a standard curve
prepared from diamine oxidase (Sigma Chemical, St. Louis, MO). Results and
Discussion Using supplemental
molybdenum and sulphur to form ruminal thiomolybdates to induce Cu deficiency
was successful as suggested by all indices of Cu status (Table
2). Plasma Cu was significantly
decreased (P < 0.01) in cattle
receiving -Cu and -Cu+Mn treatments compared to the control. High dietary Mn (-Cu+Mn) further depressed
(P < 0.01) plasma Cu compared to
the -Cu diet alone. Liver Cu stores
and ceruloplasmin activity were significantly reduced (P < 0.01) in Cu deficient cattle compared to the
controls. Plasma diamine oxidase
activity was decreased (P <
0.01) in Cu deficient animals compared to controls and tended (P = 0.19) to be further depressed in
animals receiving high Mn. These data
demonstrate that plasma diamine oxidase activity can be used as a biomarker
of Cu deficiency in the bovine. Plasma diamine oxidase as
a biomarker of Cu deficiency appears to have several advantages over
conventional measures of Cu adequacy.
First, it is a relatively simple and quick assay that only requires
basic lab ware and a spectrophotometer to complete. To measure liver and plasma Cu requires the use of strong acids
to digest the samples and an atomic absorption spectrophotometer to
quantitate the Cu. Secondly, to
measure DAO only requires the collection of a blood sample versus a liver
biopsy for liver Cu. Finally, DOA is
not an acute-phase protein like ceruloplasmin that is affected by stress and
sickness, which can erroneously over-predict Cu status. It is well known that Mo
acts as a strong Cu antagonist through the formation of thiomolybdates;
however, this study also indicates that high levels of Mn may further
decrease Cu bioavailability when present in conjunction with Mo. Manganese homeostasis is tightly
controlled in the body and excess concentrations may alter the relative
absorption and/or bioavailability of other minerals such as Cu. Implications Plasma diamine oxidase
activity may serve as a relatively simple and rapid diagnostic test for Cu
deficiency in the bovine. This
biomarker may also prove useful in nutritional research related to Cu
absorption and metabolism. References DiSilvestro,
R. A. 1990. Influence of dietary copper, copper injections and inflammation
on rat serum ceruloplasmin activity levels.
Nutr. Res. 10:355-358. Kehoe, C.
A., M. S. Faughnan, W. S. Gilmore, J. S. Coulter, A. N. Howard, and J. J.
Strain. 2000. Plasma diamine oxidase activity is greater
in copper-adequate than copper-marginal or copper-deficient rats. J. Nutr.
130:30-33. NRC. 1996.
Nutrient Requirements of Domestic Animals. Nutrient Requirements of Beef Cattle. 7th Ed. Natl. Acad. Press, Washington, D. C. Takagi, K.,
M. Nakao, Y. Ogura, T. Nabeshima, and A. Kunii. 1994. Sensitive
colorimetric assay of serum diamine oxidase.
Clinica Chimica Acta 226:67-75. Wolvekamp,
M. C. and R. W. DeBruin. 1994. Diamine oxidase: an overview of historical, biochemical and functional aspects. Dig. Dis.
12:2-14. Table 1. Minerals supplemented to the basal diet to form
three treatments Table 2. Copper indices of cattle fed control, Cu deficient
and Cu deficient plus high Mn diets |
