Acute effect of tea, wine, beer, and polyphenols on ecto-alkaline phosphatase activity in human vascular smooth muscle cells

Article abstract of DOI:10.1021/jf060505u

Alkaline phosphatase (ALP) is an ecto-enzyme widely distributed across species. It modulates a series of transmembranar transport systems, has an important role in bone mineralization, and can also be involved in vascular calcification. Polyphenol-rich di

Full text of DOI:10.1021/jf060505u

4982 J. Agric. Food Chem. 2006, 54, 4982−4988
Acute Effect of Tea, Wine, Beer, and Polyphenols on
ecto-Alkaline Phosphatase Activity in Human Vascular Smooth
Muscle Cells
MARIA R. NEGRA˜ O,*,† ELISA KEATING,† ANA FARIA,†,‡ ISABEL AZEVEDO,† AND
†
MARIA J. MARTINS
Department of Biochemistry, Faculty of Medicine (U38-FCT), University of Porto,
4200-319 Porto, Portugal, and Department Porto Chemistry Investigation Centre,
Department of Chemistry, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal
Alkaline phosphatase (ALP) is an ecto-enzyme widely distributed across species. It modulates a
series of transmembranar transport systems, has an important role in bone mineralization, and can
also be involved in vascular calcification. Polyphenol-rich diets seem to have protective effects on
human health, namely, in the prevention of cardiovascular diseases. We aimed to investigate the
effects of polyphenols and polyphenol-rich beverages upon membranar alkaline phosphatase (ecto-
ALP) activity in intact human vascular smooth muscle cells (AALTR). The ecto-ALP activity was
determined at pH 7.8, with p-nitrophenyl phosphate as the substrate, by absorbance spectrophotometry
at 410 nm. Cell viability was assessed by the lactate dehydrogenase (LDH) method, and the polyphenol
content of beverages was assessed using the Folin-Ciocalteu reagent. All polyphenols tested inhibited
ecto-ALP activity, in a concentration-dependent way. Teas, wines, and beers also inhibited ecto-
ALP activity, largely according to their polyphenol content. All tested compounds and beverages
improved or did not change AALTR cell viability. Stout beer was an exception to the described
behavior. Although more studies must be done, the inhibition of AALTR ecto-ALP activity by
polyphenolic compounds and polyphenol-containing beverages may contribute to their cardiovascular
protective effects.
KEYWORDS: Alkaline phosphatase; smooth muscle cells; polyphenols; wine; tea; beer
INTRODUCTION
zyme consisting of a group of isoenzymes, all glycoproteins.
Human ALPs are encoded by four different gene loci, which
express the tissue nonspecific (TNALP), intestinal (IALP),
placental (PALP), and germ-cell (GALP) isoenzymes. Because
kidney, bone, and liver express relatively high amounts of the
same ALP protein (isoform), it is usually denominated TNALP.
Liver, bone, and kidney ALP isoforms are formed through
posttranslational modifications of the tissue nonspecific gene
product. IALP, PALP, and GALP are expressed in high amounts
in intestine, placenta, and germ cells, respectively, and are
denominated tissue-specific ALPs (5). Despite its wide distribu-
tion in nature, ALP-specific functions remain unclear. ALP
cellular location mainly in the plasma membranes of exchange
surfaces, where extensive transport takes place, is suggestive
of a biological role related to transport processes (5, 6). It has
been suggested that ALP is involved in the renal transport of
phosphate (5), in the modulation of hepatocyte P-glycoprotein
transport activity (7, 8), in the transport of organic cations in
the blood-brain barrier (9), in taurocholate uptake by rat
hepatocytes (10), and in the intestinal transport of lipids (5).
IALP is present in surfactant-like particles (SLPs) that surround
fat droplets (11). SLPs are formed inside enterocytes and then
Atherosclerosis is widely prevalent in western populations.
It is a complex process, associated with high low-density
lipoprotein (LDL) levels in serum and inflammation of injured
vascular tissue, frequently resulting in dystrophic calcification.
It begins much earlier in life than initially thought, a reason
why its prevention should begin in adolescence (1). The
migration of vascular smooth muscle cells toward the intima
and the calcification of the extracellular matrix initiated, at least
in part, by these cells, constitute also important mechanisms in
the establishment of atherosclerosis. Atherosclerotic calcification
as well as medial arterial calcification, processes highly
characteristic of diabetes and end-stage renal disease, resemble
bone formation (2).
Alkaline phosphatase (ALP) (EC 3.1.3.1.) has been investi-
gated for more than 70 years (3). Its position as a front-line
chemical test in hepatobiliary and bone diseases justifies that
interest (4). In eukaryotes, it is a membrane-bound metalloen-
* To whom correspondence should be addressed. Tel/Fax: 351 225513624.
E-mail: ritabsn@med.up.pt.
† Department of Biochemistry, University of Porto.
‡ Department of Chemistry, University of Porto.
10.1021/jf060505u CCC: $33.50
© 2006 American Chemical Society
Published on Web 06/10/2006

ecto-Alkaline Phosphatase Activity in Human Muscle
J. Agric. Food Chem., Vol. 54, No. 14, 2006 4983
were maintained in a humidified atmosphere of 5% CO2/95% air and
grown in DMEM supplemented with 4.5 g L-1 glucose, 3.7 g L-1
sodium hydrogencarbonate, 10% fetal bovine serum, 100 units mL-1
penicillin, 100 µg mL-1 streptomycin, and 0.25 µg mL-1 amphotericin
B. The culture medium was changed every 2-3 days, and the cell
culture was split every 7 days. For subculturing, cells were washed
with phosphate-buffered saline solution, enzymatically removed with
0.25% trypsin-0.02% EDTA (3 min, 37 °C), split 1:4, and subcultured
in plastic culture dishes (21 cm2; Ø 60 mm; Corning Coastar, Corning,
NY). For enzymatic assays, the cells were seeded, after splitting, on
24 well plastic cell culture clusters (2 cm2; Ø 16 mm; Corning Coastar).
Membranar alkaline phosphatase (ecto-ALP) activity studies were
generally performed 5 days after seeding when cells formed a
monolayer, between passage numbers 18-24.
Assay of ecto-ALP in Intact Cells. ecto-ALP assays were performed
in intact cells. On the experiment day, the culture medium was removed
and cell monolayers were washed once with buffer A [pH 7.8, with
the following composition (mM): 5.5 glucose, 136 NaCl, 2.6 KCl, 20
Hepes, and 10 tricine] at 37 °C. Then, cell monolayers were preincu-
bated at 37 °C for 30 min in buffer A. The ecto-ALP reaction was
carried out in 0.3 mL of buffer A containing 5 mM pNPP and 1 mM
MgCl2. After 60 min of incubation, 0.2 mL of the supernatant solution
was transferred into hemolyse tubes containing 0.02 M NaOH at 4 °C;
the amount of pNP released from pNPP, reflecting ecto-ALP activity,
was measured at 410 nm [adapted from Anagnostou (35)]. In experi-
ments performed to evaluate the effect of polyphenols or polyphenol-
rich beverages on ecto-ALP activity, polyphenols (10-200 µM) and
polyphenol-rich beverages (200 µL/mL buffer A) were present during
both the preincubation and the incubation periods. EGCG was dissolved
in water. Catechin, epicatechin, and xanthohumol were dissolved in
100% ethanol. Resveratrol, quercetin, myricetin, rutin, and chrysin were
dissolved in DMSO. The final concentration of the solvents used, in
the incubation medium, was 0.1%. Controls of these drugs and
beverages (12.0% ethanol for both red and white wines, 5.6% ethanol
for alcohol-containing beers, and water for alcohol-free beverages and
teas) were run in the presence of the corresponding solvents. The results
were expressed as ecto-ALP specific activity (nmol pNP released/min/
mg cell protein) or as percentage of activity recovered in the presence
of a given compound or beverage, when compared with the corre-
sponding control.
Determination of Cell Viability. Cell viability was assessed by
measuring the lactate dehydrogenase (LDH) activity, as described by
Bergmeyer and Bernt (36). The release of the intracellular LDH into
the media was used to calculate cell viability, since released LDH is a
stable enzymatic marker correlating linearly with cell death. The LDH
activity was derived by measuring NADH oxidation at 340 nm during
the reduction of pyruvate to lactate. Optical density values were
recorded for 2 min, and the rate of reduction was calculated. Cells from
control cultures were solubilized with 0.3 mL of 0.1% (v/v) Triton
X-100 (in 5 mM Tris-HCl, pH 7.4) and placed for 30 min at 37 °C.
This cell-associated LDH activity was added to the LDH activity
released from control cultures, and the total activity was considered to
represent 100% cell death. Then, for each treatment [polyphenols (200
µM) or polyphenol-rich beverages (200 µL/mL buffer A)], the amount
of LDH present in the medium was calculated as a percentage of total
LDH, which reflects the percent cell death in the sample.
Total Polyphenol Content. The total polyphenol content of the
beverages was determined following the Folin-Ciocalteu method
adjusted to a microscale (37). In an Eppendorf tube, 790 µL of distilled
water, 10 µL of sample, and 50 µL of Folin-Ciocalteu reagent were
mixed. After 1 min, 150 µL of aqueous 20% (w/v) Na2CO3 was added,
and the mixture was mixed and allowed to stand at room temperature
in the dark for 120 min. The absorbance was read at 750 nm, and the
total polyphenol concentration was calculated from a calibration curve,
using catechin as the standard. The results were expressed as mg/L
catechin equivalents.
secreted into either the intestinal lumen or the blood stream (12).
IALP gene expression is regulated by ZBP-89 and seems to
have an important role in retarding the rate of fat absorption
(13, 14). The most well-established function of TNALP is its
role in bone mineralization, as clearly shown by the clinical
situation of hypophosphatasia (15) and confirmed by studies
with knockout mice models (16, 17). ALP may also contribute
to pathological mineralization in other tissues, including the
vascular endothelium (18, 19), a biological process sharing
similarities with bone formation, for example, in the presence
of bone regulatory factors (2, 20, 21).
Polyphenols occurring in fruits, vegetables, grains, and
beverages, such as tea, beer, or red wine, have been extensively
studied in the last few years due to the emerging possibility of
their disease-preventive properties on cancer, coronary heart
disease, and osteoporosis suggested by several epidemiological
studies (22-28).
Tea and red wine intakes have been epidemiologically shown
to be inversely related to coronary heart disease. Tea and red
wine are very rich in polyphenols. Their properties as powerful
antioxidant beverages and as protectors of the cardiovascular
system have been extensively studied (22, 25-28). Recent
studies have identified compounds with antioxidant and estro-
genic characteristics (29-33) with protective effects against
cardiovascular diseases in hops and beer (29, 32, 33).
Our aim was to investigate the putative direct modulation of
ALP activity in vascular smooth muscle cells by polyphenols
and some beverages rich in these compounds (such as wine,
tea, and beer). We hypothesize that the modulation of this
enzyme activity may be another process that explains polyphenol
protection against cardiovascular diseases.
MATERIALS AND METHODS
Materials. p-Nitrophenyl phosphate (pNPP), p-nitrophenol (pNP),
levamisole, sodium orthovanadate, (+)-catechin hydrate, (-)-epicat-
echin, (-)-epigallocatechin-3-gallate (EGCG), trans-resveratrol, quer-
cetin dihydrate, myricetin, rutin hydrate, chrysin, coomassie brilliant
blue G, albumin, tris-HCl [tris(hydroxymethyl)aminomethane hydro-
chloride], Hepes, Dulbecco’s modified Eagle’s medium (DMEM) cell
culture medium, fetal bovine serum, penicillin, streptomycin, ampho-
tericin B, and tricine were purchased from Sigma (Sigma Alcobendas,
Madrid, Spain). Dimethyl sulfoxide (DMSO), ethanol, Folin-Ciocalteu
reagent, D-glucose, Triton X-100, sodium hydrogencarbonate, sodium
chloride, potassium chloride, and sodium hydroxide were purchased
from Merck (Darmstadt, Germany). All other reagents were of the
highest quality and purity available. Xanthohumol was kindly supplied
by Hopsteiner (Mainburg, Germany) through iBeSa, Beverage and
Health Institute (Porto, Portugal). Lager type beer (SuperBock), stout
type beer (SuperBock Stout), and alcohol-free beer (Cheers) were
purchased from the local market (Portuguese beers produced by
UNICER, Portugal). Green and black teas (both from Tetley) were
purchased from the local market. Red wine (Terrac¸os do Douro, 1999,
Sandeman) and white wine (Terrac¸os do Douro, 2000, Sandeman) were
produced in the Douro region (North of Portugal) and were also
purchased from the local market. Alcohol-free red and white wines
were prepared by extracting ethanol from the corresponding intact wines
(kindly prepared and supplied by Prof. Paula Guedes de Pinho from
ESBUC, College of Biotechnology, Portuguese Catholic University,
Porto, Portugal).
Beverages Preparation. All beverages were used at pH 7.8. The
gas was removed from the beers before use. Green and black teas were
prepared, according to the recommendations of the supplier, by infusing
one tea bag (green tea, 1.75 g for 5 min; black tea, 1.5 g for 2 min) in
250 mL of boiling water.
Cells and Culture Conditions. The adult aortic smooth muscle cell
line (AALTR) was generously provided by Dr. McDougall (Fred
Hutchinson Cancer Research Center, United States) (34). AALTR cells
Protein Determination. The protein content of cell monolayers was
determined as described by Bradford (38), with human serum albumin
as the standard.
Calculations and Statistics. Km and Vmax values for ALP activity
were calculated from nonlinear regression analysis of the saturation

4984 J. Agric. Food Chem., Vol. 54, No. 14, 2006
Negra˜o et al.
beverages, intact cells were incubated with 10.0 mM pNPP to
ensure that the enzyme was working under saturated conditions.
Enzymatic assays were performed at pH 7.8 since viability of
AALTR cells was not affected by this pH value (results not
shown) and ecto-enzyme activity was higher.
Ecto-ALP activity modulation by levamisole and orthovana-
date was investigated in order to characterize the ALP isoen-
zyme present in the AALTR cell line. A 500 µM concentration
of levamisole and 100 µM orthovanadate significantly inhibited
ecto-ALP activity from AALTR cells (57.94 ( 6.02 and 51.04
( 3.95% of recovered activity, respectively).
Effect of Polyphenols on ecto-ALP Activity and AALTR
Cells Viability. The effect of various polyphenols on ecto-ALP
activity in AALTR cell line was studied. All of the polyphenols
tested (10-200 µM) (the stilbene resveratrol; the prenylated
chalcone xanthohumol; the flavanols EGCG, catechin, and
epicatechin; the flavonols quercetin, myricetin, and rutin; and
the flavone chrysin) produced a significant concentration-
dependent decrease of ecto-ALP activity in AALTR cells. The
maximal reductions found and the IC50 values that could be
determined were summarized in Figure 2A and Table 1,
respectively. As can be seen (Table 1), EGCG, xanthohumol,
and myricetin were the most potent inhibitory polyphenols with
very similar IC50 values: 51.91, 56.22, and 54.40 µM, respec-
tively, followed by quercetin and chrysin. None of the com-
pounds studied diminished cell viability in the conditions tested,
when compared with the respective controls; on the contrary,
EGCG and myricetin appeared to have a beneficial effect on
AALTR viability (Figure 2B).
Figure 1. pNP formation dependence on time (A) and substrate (pNPP)
concentration (B) in AALTR cells. Cells were incubated at 37
°C with 5
mM (A) or increasing (0.125 10.0 mM) concentrations of pNPP (B) at
−
pH 7.8. Data represent arithmetic means
each performed in triplicate.
± SEM of 3−6 experiments,
Effect of Polyphenol-Rich Beverages on ecto-ALP Activity
and AALTR Cells Viability. Tea, wine, and beer are currently
described as polyphenol-rich beverages. We quantified the total
polyphenol content of all tested beverages, summarized in Table
2. The effect of these beverages on ecto-ALP activity was also
assessed.
curves using the Graphpad Prism statistics software package (39). The
activity of an enzyme is defined by the rate constant Kenzyme (Kenzyme
max/Km), indicating that the higher the Vmax and the lower the Km are
the higher is the activity of the enzyme (40).
)
V
For IC50 calculation, Hill equation parameters for multisite inhibition
(41) were fitted to the experimental data by a nonlinear regression
analysis, using a computer-assisted method (39). The IC50 values were
given as geometric means (42) with 95% confidence limits.
The ecto-ALP activity, cell viability, and polyphenol content results
were expressed as arithmetic means ( standard error of the mean
(SEM). Statistical significance of difference between various groups
was evaluated by one-way analysis of variance followed by the
Bonferroni test. For comparison between two groups, Student’s t-test
was used. Differences were considered to be significant when p < 0.05.
All tested beverages inhibited ecto-ALP activity in intact
AALTR cells: Teas and red wine were more potent than white
wines and beers. Green and black teas inhibited enzyme activity
almost completely. There was no difference between the two
teas (green tea, 7.29 ( 3.07%; black tea, 13.89 ( 2.96%)
although green tea tended to have a stronger effect, in agreement
with its higher polyphenol content (Figure 3A and Table 2).
Green tea significantly increased the viability of AALTR cells,
and black tea showed the same tendency (Figure 3B).
The effect of red wine was similar to that obtained for teas
(14.01 ( 5.16%; Figure 4A), in accordance with the fact that
these beverages showed the highest values for total polyphenol
content (Table 2). White wine also reduced the ecto-ALP
activity (44.67 ( 3.02%), albeit with less potency than red wine.
These effects are again correlated with red and white wine
polyphenol contents (Table 2). There were no differences
between the effects obtained for wines and the corresponding
alcohol-free beverages (Figure 4A) (alcohol-free red wine, 1.44
( 1.02%; alcohol-free white wine, 42.18 ( 3.81%). Neverthe-
less, alcohol-free red wine seems to have a stronger effect on
ecto-ALP activity (Figure 4A), in agreement with its polyphenol
content (Table 2). Cell viability was improved by all tested
wines. Red wine seems to be more protective in what concerns
AALTR cell viability than white wine (Figure 4B).
RESULTS
ecto-ALP Activity in AALTR Cells. The time course of pNP
formation was determined using pNPP as the substrate in a final
concentration of 5.0 mM. pNP formation increased in a time-
dependent way up to 90 min of incubation time (Figure 1A).
So, in subsequent determinations of ecto-ALP activity, an
incubation period of 60 min was used.
To determine the kinetic parameters of ecto-ALP activity in
AALTR cells, intact cells were incubated with increasing
concentrations of pNPP (0.125-10.0 mM). As can be seen in
Figure 1B, the ecto-ALP activity appeared to be saturable and
dependent on the concentration of pNPP used. The kinetic
parameters obtained by nonlinear analysis of the saturation
curves were as follows: Km ) 389.3 ( 46.3 µM pNPP, Vmax
)
2.18 ( 0.05 nmol pNP/min/mg protein, and Kenzyme ) 0.005
nmol pNP/min/mg protein/µM pNPP.
In subsequent experiments, performed to evaluate the modu-
lation of ecto-ALP activity in AALTR cells by levamisole,
orthovanadate, polyphenolic compounds, and polyphenol-rich
Beers also reduced the ecto-ALP activity as shown in Figure
5A. Lager type beer was more potent than stout type beer in
decreasing ecto-ALP activity (26.99 ( 4.09 vs 47.23 ( 5.10%).
Once again, there were no differences between lager type beer
and alcohol-free beer (39.51 ( 3.88%; Figure 5A). None of

ecto-Alkaline Phosphatase Activity in Human Muscle
J. Agric. Food Chem., Vol. 54, No. 14, 2006 4985
Figure 2. Effect of various polyphenols on ecto-ALP activity (A) and in cell viability (B) in AALTR cell line. Cells were preincubated for 30 min and
incubated for 60 min at 37 C with 10.0 mM pNPP, in the absence (water, H2O; ethanol, Et; and DMSO controls) or in the presence of different
polyphenols (200 M): resveratrol (Resv), xantohumol (Xant), EGCG, catechin (Cat), epicatechin (Epi), quercetin (Querc), myricetin (Myr), rutin (Rut),
°
µ
and chrysin (Chry). AALTR cells viability was then assessed by the LDH method. Each value represents the arithmetic mean
different from the control; p < 0.05.
± SEM. *Significantly
Table 1. Effect of Polyphenols on ecto-ALP Activity in AALTR Cellsa
AALTR ecto-ALP inhibition
polyphenol
IC50
(µ
M)
95%
n
xantohumol
EGCG
quercetin
myricetin
chrysin
56.22
51.91
65.43
54.40
93.18
16.65
−
−
−
−
−
189.8
5−8
4−8
6−8
5−6
5−6
39.24
38.36
26.33
23.13
68.67
111.6
112.4
375.5
a IC50 values are geometric means with 95% confidence intervals.
Table 2. Total Polyphenol Content of Studied Beveragesa
total polyphenol content
beverage
green tea
black tea
red wine
alcohol-free red wine
white wine
alcohol-free white wine
lager type beer
alcohol-free beer
stout beer
(mg/L catechin equivalents)
937.4
653.5
1672.6
2102.9
221.7
225.6
445.6
249.2
987.4
±
±
±
±
±
±
±
±
±
41.9 a
5.3 b
186.8 c
124.1 d
0.5 e
10.8 e
10.7 f
18.2 f
15.3 g
Figure 3. Effect of teas on ecto-ALP activity (A) and in cell viability (B)
in AALTR cells: green tea (GT) and black tea (BT). Cells were
preincubated for 30 min and incubated for 60 min at 37
pNPP in the absence (water control, C) or in the presence of teas (200
L/mL buffer). AALTR cells viability was then assessed by the LDH
°C with 10.0 mM
µ
method. Each value represents the arithmetic mean
different from the control; p < 0.05.
± SEM. *Significantly
with different ethanol concentrations. There was no alteration
in ecto-ALP activity (results not shown).
a Values represent means
± SEM of triplicate determinations (n ) 3). The
concentration is expressed as mg/L catechin equivalents. The mean values followed
by different letters, in each group of beverages, are significantly different at p <
0.05.
DISCUSSION
the beers diminished cell viability with the exception of stout
beer (Figure 5B).
Finally, to ensure that ethanol, by itself, did not alter the
enzyme activity, AALTR cells were incubated with solutions
ALP has been characterized as an ecto-phosphatase enzyme
in eukaryotes, being anchored to the plasma membrane via a
covalent linkage to glycosylphosphatidylinositol. This ecto-
phosphatase activity is maintained during vesicles formation in

4986 J. Agric. Food Chem., Vol. 54, No. 14, 2006
Negra˜o et al.
in several cell lines (35, 43, 44). McComb et al. (6) defined an
alkaline pH optimal for ALP in their studies with the purified
enzyme.
The kinetic parameters obtained in this work show that km
and Vmax values for ALP in AALTR cell lines were in agreement
with those obtained by other authors also for TNALP (48).
In AALTR cells, ecto-ALP inhibition by levamisole was far
from complete (57.94 ( 6.02%). Taking into consideration the
cell line origin, we expected to obtain a higher degree of
inhibition since levamisole is known to be a specific inhibitor
of TNALP (35). In agreement with our results, there are some
reports describing differences in the behavior of TNALP
isoforms activity when levamisole and other compounds are
tested, probably due to different glycosylation patterns associated
with the isoform source (49). A 100 µM concentration of
orthovanadate also inhibited the enzyme activity in AALTR cells
(51.04 ( 3.95%). Vanadate is a phosphotyrosyl protein phos-
phatase inhibitor (50), and so, the inhibition obtained with
vanadate may indicate that ALP works, at least in part, as an
ecto-tyrosine phosphatase in AALTR cells.
Figure 4. Effect of wines on ecto-ALP activity (A) and in cell viability (B)
in AALTR cells: red (RW) and white wine (WW), alcohol-free red
(AF-RW), and alcohol-free white wine (AF-WW). Cells were preincubated
Teas, wines, and beers were used in this experimental work
because of their extensive worldwide consumption and described
health benefits in preventing cardiovascular diseases (22-28,
32, 33). The evolution of these pathologies seems to be
exacerbated in estrogen-deficient women. Health benefits of
these beverages have been attributed mainly to their polyphenol
contents, and the classification of many polyphenols as phy-
toestrogens (ligands for estrogen receptors) led the scientific
community to consider this estrogenic effect as a possible mode
of action (51, 52). Nevertheless, it has been demonstrated
that polyphenols also inhibited the oxidation of LDL, increased
high-density lipoprotein cholesterol, induced nitric oxide-
dependent vasorelaxation, and inhibited platelet aggregation,
inflammation, and proliferation of vascular smooth muscle cells
(23, 27) mechanisms that contribute to their cardiovascular
protection.
for 30 min and incubated for 60 min at 37
absence (control-ethanol 12%, C) or in the presence of wines (200
L/mL buffer). AALTR cells viability was then assessed by the LDH
°C with 10.0 mM pNPP in the
µ
method. Each value represents the arithmetic mean
different from the control; p < 0.05.
± SEM. *Significantly
Studying ALP activity using direct modulation of polyphenols
and polyphenolic-rich beverages, we are trying to understand
the relation between the above-mentioned compounds and their
cardiovascular protection as well as the involvement of ALP in
the process. All polyphenols tested inhibited ecto-ALP activity
in a concentration-dependent way. Chrysin is not known to exist
in these beverages, but it is present in many plants and is part
of human nutrition (23).
Published results of long-term effect of polyphenols on ALP
activity are inconclusive and almost all refer to osteoblasts or
osteoblast-like cells. Yamaguchi and Jie (53) referred to an
increase in ALP activity and in calcium tissue content with 100
µM genistein, a reduction in ALP activity with 100 µM EGCG,
and no alterations in ALP activity with 100 µM catechin. Choi
et al. (54) described an increase in ALP activity with 10-100
µM catechin. The precise mechanisms by which polyphenols
interact with ALP in long-term treatments are not yet known.
None of the tested polyphenols diminished cell viability; on
the contrary, myricetin and EGCG improved the viability of
AALTR cells. Similar results in osteoblasts have been described
for 10-100 µM catechin (54).
Figure 5. Effect of beers on ecto-ALP activity (A) and in cell viability (B)
in AALTR cells: lager type beer (LB), alcohol-free beer (AF-B), and stout
type beer (SB). Cells were preincubated for 30 min and incubated for 60
min at 37
C) or in the presence of beers (200
was then assessed by the LDH method. Each value represents the
arithmetic mean SEM. *Significantly different from the control; p < 0.05.
°
C with 10.0 mM pNPP in the absence (control-ethanol 5.6%,
µ
L/mL buffer). AALTR cells viability
±
bone and is important for mineralization (5, 15-17, 35,
43-47) as well as for the regulation of lipid absorption (11,
12).
The phosphatase activity studied in this work can be
characterized as an ecto-phosphatase activity: The substrate used
was only present in the external medium since it does not
penetrate plasma membranes (45); cell viability did not diminish,
under the described conditions; substrate hydrolysis was linear
with time, confirming that cells were intact during the incubation
period; pNPPase activity was not detected in the external
medium (data not shown), which excludes enzymatic action of
contaminants or leakage of intracellular enzymes.
As far as we know, this is the first study of the direct effect
of these beverages on ecto-ALP activity in this cell line. The
results of experiments with polyphenol-rich beverages are in
accordance with the results for individual polyphenols. All tested
beverages significantly inhibited ecto-ALP activity in AALTR
cells. This inhibition is tightly related to the polyphenol content
for each type of beverage.
As previously mentioned, we chose pH 7.8 to study ALP in
AALTR intact cells. Other authors have already described the
physiological pH value as optimal for the ALP enzyme activity

ecto-Alkaline Phosphatase Activity in Human Muscle
J. Agric. Food Chem., Vol. 54, No. 14, 2006 4987
There were no differences in ecto-ALP activity inhibition
between green and black teas, although green tea seems to have
a stronger effect, in agreement with its polyphenol contents.
Other authors have found a different variety of polyphenols but
the same total polyphenol contents for green and black teas (55,
56). The effects of green tea ingestion have been intensively
studied, and some authors defend that only green tea has health
benefits. Others attribute to black tea the same potentialities.
Red wine reduction of ecto-ALP activity was similar to that of
teas, despite its higher polyphenol contents. These results as
well as the results obtained for alcohol-free red wine can be
explained by the very high inhibition of ecto-ALP activity,
already observed for teas. On the other hand, red wine ALP
inhibition was higher than that obtained with white wine, in
accordance with the respective polyphenol contents. Concerning
beers, lager type beer, unexpectedly, had a stronger inhibitory
effect on ALP activity than stout type beer. Theoretically, stout
type beer has a higher antioxidant content than lager type beer
(57). We confirmed that the total polyphenol content of the stout
beer used in the study was higher than that of lager type beer.
Therefore, the beer polyphenol content does not seem to be
determinant of these beverages effects on ecto-ALP activity.
In this context, it must be stressed that polyphenolic compounds
present in beers are generally very different from polyphenols
of other beverages. None of the beverages tested diminished
cell viability, with the exception of stout beer.
cells (AALTR) to Dr. McDougall from Fred Hutchinson Cancer
Research Center (United States).
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ABBREVIATIONS USED
ALP, alkaline phosphatase; DMSO, dimethyl sulfoxide; ecto-
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Received for review February 20, 2006. Revised manuscript received
May 10, 2006. Accepted May 12, 2006. This research was supported
by FCT, POCTI, and FEDER (Programa Cieˆncia, Tecnologia e
Inovac¸a˜o do Quadro Comunita´rio de Apoio) and iBeSa, Beverage and
Health Institute, Porto, Portugal (Grant P09-03).
JF060505U