Journal of Functional Foods 77 (2021) 104344

Contents lists available at ScienceDirect

Journal of Functional Foods

journal homepage: www.elsevier.com/locate/jff

Evaluation of the ameliorative effect of Spirulina (Arthrospira platensis)

supplementation on parameters relating to lead poisoning and obesity in
C57BL/6J mice
Hokuto Nakata a, Shouta M.M. Nakayama a, Andrew Kataba a, b, Yared Beyene Yohannes a, c,
Yoshinori Ikenaka a, d, Mayumi Ishizuka a, *
a
Laboratory of Toxicology, Department of Environmental Veterinary Sciences, Faculty of Veterinary Medicine, Hokkaido University, Kita 18 Nishi 9, Kita-ku, Sapporo
060-0818, Japan
b
The University of Zambia, School of Veterinary Medicine, P.O. Box 32379, Lusaka, Zambia
c
Department of Chemistry, College of Natural and Computational Science, University of Gondar, Ethiopia
d
Water Research Group, School of Environmental Sciences and Development, North-West University, South Africa

A R T I C L E I N F O A B S T R A C T

Keywords: The current study aimed to validate the possible ameliorative effects of Spirulina (Arthrospira platensis) on lead
Lead poisoning poisoning and obesity using C57BL/6J mice. After a treatment period, we performed metal analyses, as well as
Obesity prevention hematocrit and plasma biochemical parameter measurements, and assayed oxidative stress markers and eryth­
Spirulina
rocyte δ-aminolevulinic acid dehydratase (ALAD) activity. Our results highlighted the effectiveness of Spirulina
Cost-effective
C57BL/6J
in improving anemia status with the normalization of hematocrit levels and ALAD activity ratios, even in mice
that exhibited obesity in addition to lead poisoning. Spirulina treatment also decreased epididymal white adipose
tissue weight and increased plasma high-density lipoprotein levels, which are normally reduced after lead
exposure. However, most of the studied plasma biochemical parameters and oxidative stress markers did not
show large changes after treatment, likely because of the short duration of treatment. Further studies with
longer-term exposures are required to validate the usefulness of Spirulina suggested in the present study.

1. Introduction
Lead (Pb) is a non-essential element that occurs ubiquitously in the
environment, although anthropogenic Pb sources generally cause Pb
pollution. The World Health Organization (WHO) reported that Pb ac­
counts for 0.6% of the global disease burden, which is highest in
developing countries (WHO, 2009). Elevation of blood Pb levels has
been reported in humans from several developing countries (Dooyema
et al., 2012; Nakata et al., 2020; Parnia et al., 2018). Additionally, low-
income communities tend to be more susceptible to pollution (Miranda,
Edwards, Keating, & Paul, 2011). Lead has been implicated in many
adverse health outcomes, such as kidney disorders (Sabath and Robles-
Osorio, 2012) and anemia, as Pb inhibits δ-aminolevulinic acid dehy­
dratase (ALAD) (Gonick, 2011). One of the important mechanisms un­
derlying Pb toxicity is oxidative-stress induction resulting from the
generation of reactive species and/or depletion of the antioxidant
defensive system (Matović, Buha, Ðukić-Ćosić, & Bulat, 2015).
Being overweight or obese are also common health issues and are
major risk factors for chronic non-communicable diseases (NCDs),
including cardiovascular diseases and type 2 diabetes (WHO, 2011).
Nearly 80% of NCD deaths occur in low- and middle-income countries
(WHO, 2011). Drewnowski and Specter (2004) reported that the highest
rates of obesity occur among population groups with the highest poverty
rates. They also stated in linear programming models that a reduction in
diet costs leads to energy-dense diets, which consist of refined grains or
fats and are similar in composition to the diets of people with low in­
comes. Similarly, economic deprivation leads people to choose cheaper
foods, namely, food high in fats and carbohydrates, rather than foods
high in protein (Hruschka, 2012). Hence, the poor in the developing
world generally take more calories from fats and carbohydrates instead
of protein and are likely to suffer from both Pb poisoning and obesity.
Spirulina (Arthrospira platensis) is the general name of a filamentous
cyanobacterium (blue-green algae). Spirulina exists in tropical and sub-
tropical lakes in Africa, Asia, and South and Central America. Although

* Corresponding author.
E-mail address: ishizum@vetmed.hokudai.ac.jp (M. Ishizuka).

https://doi.org/10.1016/j.jff.2020.104344
Received 5 September 2020; Received in revised form 3 December 2020; Accepted 14 December 2020
Available online 8 January 2021
1756-4646/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
H. Nakata et al.
it has been used as a traditional medicine and as a food source in some
areas, Spirulina has recently attracted greater attention because of its
potential use in nutritional and medical applications (FAO/IOC 2011). It
has high protein content, approximately 50%–70% of its dry weight
(Hoseini, Khosravi-Darani, & Mozafari, 2013), and is rich in essential
minerals, including iron (Fe), which results in significantly higher he­
moglobin (Hb) content (Campanella, Crescentini, & Avino, 1999).
Further, A. platensis provides antioxidant protection due to the antioxi­
dant activity of the vitamins it contains, such as β-carotene and to­
copherols, as well as phycocyanin (Estrada, Bescós, & Del Fresno, 2001;
Miranda, Cintra, Barros, & Mancini-Filho, 1998). Upasani and Balara­
man (2003) reported that the administration of A. platensis to Pb-
exposed rats significantly inhibited lipid peroxidation (LPO) and
restored the levels of endogenous antioxidants, including superoxide
dismutase (SOD), catalase, and glutathione (GSH), in various organs. It
was indicated that co-administration of A. platensis and Pb acetate
minimized the increased levels of LPO and other oxidative stress
markers (Khalil, Elhady, Elewa, Abd El-Hameed, & Ali, 2018). The anti-
oxidative property of A. platensis was also demonstrated in sub-
chronically Pb-exposed rabbits (Aladaileh et al., 2020). Moreover,
A. platensis has a protective effect against obesity-related metabolic
disorders (Yousefi, Mottaghi, & Saidpour, 2018) and anemia (Selmi
et al., 2011). Zhao et al. (2019) recently reported that the protein hy­
drolysate of A. platensis exhibited good anti-obesity effects with modu­
lation of gene expression.
On the basis of this available data, we hypothesized that Spirulina
(A. platensis) supplementation may play a role in ameliorating Pb
poisoning and obesity. Because both Pb poisoning and obesity are
common health concerns in developing countries, people in these re­
gions may be considered doubly affected. However, to the best of our
knowledge, there have been no studies examining the possible mitiga­
tive effects of Spirulina on this combination of conditions. To investigate
this possibility, we designed a laboratory exposure experiment using
mice. We assessed hematotoxicity-related parameters including eryth­
rocyte ALAD activity, which can be induced by Pb exposure. As kidney
damage is also a common clinical effect of Pb, we assayed several plasma
markers including clusterin, cystatin C, and neutrophil gelatinase-
associated lipocalin (NGAL) which have recently attracted attention
because of their sensitivity and their characteristic of being unaffected
by other factors (Vinken et al., 2012; Zhang, Lu, Sheng, & Jin, 2011).
Moreover, two markers in liver were measured to assess the oxidative
stress status of the mice. To assess obesity, we measured epididymal
white adipose tissue (eWAT) weight, as well as plasma parameters. As
Spirulina is cultivated and exists naturally worldwide, Spirulina sup­
plementation has great potential as a cost-effective solution for Pb
poisoning and obesity.
2. Materials and methods
2.1. Animals and experimental design
All animal experiments were performed with the approval and su­
pervision of the Institutional Animal Care and Use Committee of Hok­
kaido University (approval number 19–0119), Japan. Forty-two male
C57BL/6J mice (Mus musculus, 7 weeks old) were purchased from San­
kyo Labo Service Corporation, Inc. (Tokyo, Japan) and Hokudo Co., Ltd.
(Hokkaido, Japan). Food (rodent chow, Labo MR Stock; Nosan Corpo­
ration, Yokohama, Japan) and distilled water were provided ad libitum
during the one-week acclimation period at the Faculty of Veterinary
Medicine, Hokkaido University. After acclimation, the mice (8 weeks of
age) were divided into seven groups (n = 6 for each group) and treated
for ten days as follows: 1) given no treatment (control, NC), 2) given
special diet (SD), 3) given 1,000 mg/L lead acetate (Wako Pure Chemical
Industries, Osaka, Japan) in water (PB), 4) given Spirulina powder
(1,000 mg/kg body weight/day) (SP), (5) given special diet and Spir­
ulina powder (SD-SP), (6) given lead acetate water and Spirulina powder
2
Journal of Functional Foods 77 (2021) 104344
(PB-SP), and (7) given a special diet, lead acetate water, and Spirulina
powder (SD-PB-SP). A control diet (D12450H, Research Diets, Inc., NJ,
USA) was given to the groups, which were not given the special diet
during the treatment period. The special diet (modified D12451,
Research Diets, Inc.) contained more fats and carbohydrates, as well as
lower protein compared to the control diet, with a calorie-based
formulation under the premise that mice eat for calories and adjust
their overall intake in food weight to achieve a certain caloric intake
(Bullen et al., 2004). The detailed blending ratios of ingredients are
shown in Supplementary Table S1. Purified Spirulina (A. platensis)
powder (DIC Lifetec Co., Ltd., Tokyo, Japan) was mixed with distilled
water, then the suspension was given orally to mice everyday using
sonde. The suspension was prepared at time of use for each animal to
ensure the same concentration. The compositions of the Spirulina
product were reported by the previous study (Okamoto et al., 2019).
Both control and special diets were given ad libitum. Prepared lead ac­
etate as given in the drinking water ad libitum. The mice in the Pb-
exposed group had an average of 5.0 mL/day/animal of water to
drink, indicating the animals ingested 5.0 mg/day/animal of lead ace­
tate in average. The mice were housed under a 12 h light–12 h dark cycle
throughout the acclimation and treatment periods.
2.2. Sample collection
After the treatment period, mice were euthanized by carbon dioxide
(CO2) gas inhalation. Heparinized whole blood was then collected from
the postcava using syringe and needle, as well as feces, liver, kidneys,
and eWAT. The collected eWAT was immediately weighed. Plasma was
collected after centrifugation (10 min at 2,000 × g at room temperature)
of heparinized whole blood for further analyses. The collected samples
were stored at − 20 ◦ C prior to the analyses.
2.3. Sample digestion and metal extraction
Sample digestion and metal extraction were performed as previously
described (Nakata et al., 2016) with minor modifications. Fifty micro­
liters of whole blood were placed in pre-washed digestion vessels, fol­
lowed by acid digestion using 5 mL of two-fold diluted nitric acid
(atomic absorption spectrometry grade, 60%; Kanto Chemical Co.,
Tokyo, Japan) and 1 mL of hydrogen peroxide (Cica reagent, 30%; Kanto
Chemical Co.). Similarly, approximately 0.1 g of dried liver and kidney,
as well as 50 mg of dried feces, were treated using the same volume of
nitric acid and hydrogen peroxide as the blood sample. The digestion
and metal extraction were conducted using a microwave digestion sys­
tem (Speed Wave MWS-2; Berghof, Eningen, Germany) following the
manufacturer’s instruction. The operating conditions of the microwave
system are given in Supplementary Table S2. After cooling, the sample
solutions were transferred into 15 mL polypropylene tubes and diluted
to a final volume of 10 mL with double distilled and deionized water
(Milli-Q; Millipore, Bedford, MA, USA).
2.4. Metal analysis
The concentrations of the metals (Pb and Fe) were determined using
an inductively coupled plasma-mass spectrometer (ICP-MS) (7700 Se­
ries; Agilent Technologies, Tokyo, Japan). Detailed operating conditions
are shown in Supplementary Table S3. Analytical quality control was
performed using the following certified reference materials: Seronorm™
Trace Elements Whole Blood L-2 (Sero, Billingstad, Norway), DORM-3
(fish protein; National Research Council of Canada, Ottawa, Canada),
and DOLT-4 (dogfish liver; National Research Council of Canada). A
replicate analysis of these reference materials showed good accuracy (a
relative standard deviation of less than 3%) and recoveries (95%–
105%). The instrument detection limit was 0.001 µg/L.
H. Nakata et al.
2.5. Hct and plasma biomarker analyses
Hct values were measured using an Hct centrifuge (Kubota 3220;
Kubota Corp., Osaka, Japan). A conventional blood biochemical
analyzer (Spotchem EZ SP-4430; Arkray Inc., Kyoto, Japan) was used to
analyze the levels of plasma high-density lipoprotein (HDL-C), triglyc­
eride (TG), glucose (Glu), and total cholesterol (T-Cho). Another con­
ventional blood biochemical analyzer (Fuji Drichem 7000 V; Fujifilm
Corporation, Tokyo, Japan) was used to analyze plasma total protein
(TP), albumin (Alb), blood urea nitrogen (BUN), uric acid (UA), and
creatinine (Cre) levels. The analyses were done following the manu­
facturer’s instructions.
A Milliplex MAP Mouse Kidney Injury Magnetic Bead Panel 2 Kit
(EMD, Millipore Corporation, Billerca, MA, USA) was used with a Bio-
Plex 200 System (Bio-Rad, Hercules, CA, USA) and Bio-Plex Manager
software version 6.0 (Bio-Rad) to measure clusterin, cystatin C, and
NGAL. Five mice were selected at random from each group for the
Milliplex MAP assay, unlike in the other experiments.
2.6. Oxidative-stress marker analysis
Genomic DNA was extracted from the liver with the sodium iodide
method using a DNA Extractor WB Kit (Fujifilm Wako Pure Chemical
Corporation, Tokyo, Japan) according to the manufacturer’s manual.
The DNA concentration was measured spectrophotometrically using a
NanoDrop spectrophotometer (ND-1000; Thermo Scientific, Newark,
DE) and diluted to 100–200 ng/µL. The EpiQuik One-Step DNA Hydro­
lysis Kit (Epigentek Group Inc., NY, USA) was used to hydrolyze DNA
according to the manufacturer’s protocol. Afterward, the 8-hydroxy-2′ -
deoxyguanosine (8-OHdG) assay was done using the highly sensitive 8-
OHdG Check ELISA Kit (KOG-HS10/E, Japan Institute for the Control of
Aging, Nikken Seil Co., Ltd., Shizuoka, Japan).
The extraction of protein carbonyls from the liver was performed as
described previously (Augustyniak et al., 2015) with minor modifica­
tions. Approximately 0.4 g of liver tissue was homogenized in ice-cold
phosphate-buffered saline (PBS; pH 7.4) at 2:1 PBS/tissue ratio using a
rotor stator homogenizer. Phenylmethylsulphonyl fluoride (Nacalai
Tesque, Kyoto, Japan) was added as a protease inhibitor at a final
concentration of 1 mM immediately before homogenization. The ho­
mogenate was then centrifuged (600 × g, 5 min at 4 ◦ C). The collected
supernatant was re-centrifuged (3,000 × g, 20 min at 4 ◦ C). The super­
natant was collected again and re-centrifuged using an ultracentrifuge
(100,000 × g, 4 h at 4 ◦ C), after which the supernatant containing sol­
uble proteins was collected. Protein concentration in the collected su­
pernatant was measured using a Pierce BCA Protein Assay Kit (Thermo
Scientific, IL, USA) following the manufacturer’s instructions. Finally,
the protein carbonyl assay was carried out with 250 µg of protein using a
Protein Carbonyl ELISA Kit (ALX-850-312-KI01; Enzo Life Sciences, NY,
USA).
2.7. ALAD activity assay
Erythrocyte enzymatic activity and the ratio between non-activated
and enzymes activated in vitro were measured as described in a previous
study (Nakata et al., 2020), which was slightly modified from the orig­
inal method (Espín, Martínez-López, Jiménez, María-Mojica, & García-
Fernández, 2015; Scheuhammer, 1987). Three aliquots (20 µL each) of
erythrocytes from each mouse were separated to measure non-activated
ALAD activity, reactivated ALAD activity, and the activity of the matrix.
For the non-activated enzyme activity assay, 80 µL of 0.1% Triton X-100
(Sigma–Aldrich, MO, USA) was added to the erythrocytes to form a
lysate. Subsequently, 100 µL of 0.5 M PBS (pH 6.8), 50 µL of 60 mM 5-
aminolevulinic acid (ALA) hydrochloride (Sigma–Aldrich) solution in
PBS, and 50 µL of distilled water were added. For the reactivated-
activity assay, 0.1% Triton X-100, 0.5 M PBS, and 60 mM ALA hydro­
chloride were added to the sample with the same volume of each
3
Journal of Functional Foods 77 (2021) 104344
solution as used in the non-activated enzyme activity assay. Subse­
quently, 25 µL of 0.8 mM zinc acetate (Kishida Chemical Co., Ltd.,
Osaka, Japan) and 25 µL of 1 M dithiothreitol (Fujifilm Wako Pure
Chemical Corporation) were added. For the matrix blank assay, 80 µL of
Triton X-100, 150 µL of PBS, and 50 µL of distilled water were added,
followed by 200 µL of 0.4 M trichloroacetic acid (Fujifilm Wako Pure
Chemical Corporation)/60 mM mercury chloride (HgCl2) (Kanto
Chemical Co.) as a stop solution. After 60 min incubation and termina­
tion of the reaction by HgCl2 in both the non-activated and reactivated
activity assays, all samples were centrifuged at 10,000 rpm for 5 min.
Supernatants were transferred to new tubes and mixed with 750 µL of
modified Ehrlich’s reagent, which consisted of dimethylamino­
benzaldehyde (Nacalai Tesque) in acetic acid (glacial; 99.7%, Thermo
Fisher Scientific, Lancashire, UK) and perchloric acid (Kanto Chemical
Co.). After 10 min, the absorbance was read at 555 nm against the
appropriate blank using a UV spectrophotometer (Shimadzu UV-2600;
Shimadzu Inc., Kyoto, Japan). The activity was expressed as µmol por­
phobilinogen/h/L red blood cells using the equation provided by
Scheuhammer (1987). The ratio between the non-activated and the
in vitro reactivated enzymes was then calculated.
2.8. Statistical analysis
IBM SPSS Statistics 26 (IBM Corporation, Armonk, NY, USA) was
used to evaluate significant differences in the data in all statistical an­
alyses. The data were log-transformed and fitted with a normal distri­
bution. The Tukey–Kramer test was used to compare food and water
consumption, body and eWAT weight, Hct values, blood biochemical
parameters, renal function parameters, oxidative stress markers, and
ALAD activity among groups. All statistical analyses were performed
with a significance level of 0.05 (p < 0.05).
3. Results
3.1. Metal concentrations in mice tissues and feces
The sample size for the SP and PB-SP groups decreased by one in each
because of deaths during the treatment period attributed to Spirulina
powder aspiration. The remainder of the samples (n = 5 for SP and PB-
SP, n = 6 for other groups) were used in the laboratory experiments.
There were no significant differences in body weight or in food and
water consumption rates among groups throughout the experiment
period.
The Pb and Fe levels in the blood, liver, kidney, and feces of the mice
were analyzed and are summarized in Table 1 and Table 2, respectively.
The blood and liver Pb levels of the Pb-administered groups (PB, PB-SP,
and SD-PB-SP) were within the range of 11.74–22.51 µg/dL and
1.23–4.22 mg/kg, respectively, significantly higher than those of groups
that were not given Pb. The kidney and feces Pb concentrations of the
PB, PB-SP, and SD-PB-SP groups similarly showed significant elevation
compared to those of other groups. However, the kidney Pb level of the
SD-PB-SP group was significantly lower than that of the PB group. The
SD-PB-SP group also showed a significant reduction of Pb concentration
in their feces compared with the PB-SP group.
In contrast to Pb level, blood and liver Fe levels did not differ sta­
tistically among groups, although the SD-SP group showed a significant
increase in kidney Fe concentration compared with the NC group. The
ranges of blood and liver Fe for all groups were 311.90–524.04 µg/mL
and 202.88–797.50 mg/kg, respectively. The excreted Fe levels in the
feces of the SD, SD-SP, and PB-SP groups were also greater than those of
the NC group.
3.2. Adipose weight, Hct, and blood biochemical parameters
The values of eWAT weight and Hct, as well as plasma HDL-C, TG,
Glu, and T-Cho, were measured (Fig. 1, Table 3). A significant increase
H. Nakata et al. Journal of Functional Foods 77 (2021) 104344

Table 1
Pb levels in blood, liver, kidney, and feces samples by group (control group and treated groups with sole or combination of special diet, lead acetate and spirulina
powder).
Pb level Blood (µg/dL) Liver (mg/kg) Kidney (mg/kg) Feces (mg/kg)

NC 0.69 ± 0.14 a 0.01 ± 0.00 a 0.01 ± 0.00 a 0.04 ± 0.01 a

(0.52–0.88) (0.01–0.01) (0.01–0.02) (0.03–0.05)
SD 0.71 ± 0.12 a 0.01 ± 0.00 a 0.01 ± 0.00 a 0.11 ± 0.04 a
(0.56–0.89) (0.01–0.01) (0.01–0.01) (0.06–0.19)
PB 19.25 ± 2.51 b 2.06 ± 0.62 b 13.20 ± 1.86 c 705.83 ± 252.26 bc
(14.98–22.06) (1.23–3.16) (11.11–15.54) (360.34–1008.95)
SP 0.55 ± 0.18 a 0.01 ± 0.00 a 0.01 ± 0.00 a 0.17 ± 0.07 a
(0.44–0.87) (0.01–0.01) (0.01–0.01) (0.08–0.27)
SD-SP 0.55 ± 0.08 a 0.01 ± 0.00 a 0.01 ± 0.00 a 0.20 ± 0.16 a
(0.43–0.66) (0.00–0.02) (0.01–0.02) (0.07–0.49)
PB-SP 16.94 ± 3.05 b 1.96 ± 0.43 b 11.42 ± 1.60 bc 948.41 ± 494.33 c
(12.57–21.07) (1.30–2.40) (9.77–13.93) (481.76–1654.53)
SD-PB-SP 16.48 ± 3.48 b 2.90 ± 1.14 b 10.62 ± 2.15 b 431.37 ± 85.14 b
(11.74–22.51) (1.31–4.22) (7.83–13.96) (277.06–520.25)

Note: Mean ± standard deviation in the upper row, and range in the lower row.
Note: Different small letters (a, b and c) between columns indicate significant differences among groups.

Table 2
Fe levels in blood, liver, kidney, and feces samples by group (control group and treated groups with sole or combination of special diet, lead acetate and spirulina
powder).
Fe level Blood (µg/mL) Liver (mg/kg) Kidney (mg/kg) Feces (mg/kg)

NC (N = 6) 472.29 ± 36.50 a 246.41 ± 40.76 a 337.45 ± 40.60 b 224.65 ± 57.57 b

(414.46–521.30) (208.49–298.00) (297.07–385.82) (159.53–294.09)
SD (N = 6) 490.97 ± 11.87 a 281.56 ± 50.31 a 373.83 ± 54.00 ab 441.75 ± 97.31 a
(479.78–507.28) (213.59–336.73) (291.79–434.19) (371.04–633.12)
PB (N = 6) 484.86 ± 16.62 a 268.07 ± 40.03 a 361.53 ± 34.04 ab 335.68 ± 77.74 ab
(466.96–511.18) (202.88–324.50) (335.54–414.48) (214.44–418.90)
SP (N = 5) 468.25 ± 30.68 a 301.78 ± 40.37 a 397.30 ± 25.34 ab 341.47 ± 51.55 ab
(424.65–493.66) (262.26–366.62) (361.86–422.15) (271.46–389.67)
SD-SP (N = 6) 429.91 ± 59.49 a 417.73 ± 156.22 a 426.44 ± 36.23 a 455.54 ± 161.21 a
(311.90–477.18) (275.30–627.99) (379.61–470.19) (230.04–729.87)
PB-SP (N = 5) 422.96 ± 15.02 a 394.28 ± 228.07 a 398.94 ± 32.93 ab 445.43 ± 194.22 a
(402.66–441.69) (243.50–797.50) (362.29–446.30) (289.33–749.04)
SD-PB-SP (N = 6) 449.34 ± 49.80 a 348.55 ± 61.07 a 387.47 ± 18.75 ab 380.88 ± 85.07 ab
(389.03–524.04) (278.88–457.22) (359.12–415.16) (287.49–484.82)

Note: Mean ± standard deviation in the upper row, and range in the lower row.
Note: Different small letters (a and b) between columns indicate significant differences among groups.

55% 0.6
A a B
a
a a a 0.5
52%
ab ab
b b b
eWAT weight (g)

b 0.4
49% b b
Hct (%)

b
0.3
46%
0.2

43%
0.1

40% 0.0
C

P
PB
SD

SP

P
P
C

P
P
PB
SD

SP

-S

S
-S
S
-S

-S

N
N

B-
B-

SD

PB
PB
SD

-P
-P

SD
SD

Fig. 1. Means and standard deviation values (error bars) of blood Hct (A) and eWAT weight (B) by groups. Different small letters (a and b) indicate significant
differences among groups. NC = control without any treatment, SD = given special diet, PB = given 1,000 mg/L Pb acetate, SP = given Spirulina powder (1,000 mg/
kg body weight/day), SD-SP = given special diet and Spirulina powder, PB-SP = given Pb acetate and Spirulina powder, SD-PB-SP = given special diet, Pb acetate and
Spirulina powder.

in eWAT weight was recorded in the SD group, with a mean increase and than that of the NC, SD, SP, and SD-SP groups. The SD group showed
standard deviation of 0.44 ± 0.07 g over the eWAT weight of other significantly elevated HDL-C compared with the PB-SP and SD-PB-SP
groups. The Hct of the PB group (46.0 ± 2.2%) was significantly lower groups, and there was no significant difference in TG, Glu, or T-Cho

4
H. Nakata et al. Journal of Functional Foods 77 (2021) 104344

Table 3
Values of plasma biochemical parameters relating to obesity assessment by group (control group and treated groups with sole or combination of special diet, lead
acetate and spirulina powder).
HDL-C (mg/dL) TG (mg/dL) Glu (mg/dL) T-Cho (mg/dL)

NC 51.2 ± 9.4 ab 103.3 ± 17.4 a 271.5 ± 66.8 a 104.5 ± 19.4 a

(40–66) (90–137) (193–367) (71–127)
SD 61.5 ± 6.2 a 110.0 ± 14.8 a 324.3 ± 51.9 a 114.0 ± 7.3 a
(56–71) (85–130) (249–384) (102–124)
PB 48.7 ± 2.6 ab 118.3 ± 23.0 a 324.3 ± 44.1 a 116.3 ± 9.1 a
(44–51) (96–147) (275–396) (109–128)
SP 49.6 ± 9.7 ab 116.6 ± 12.4 a 356.4 ± 52.1 a 114.4 ± 4.6 a
(40–66) (100–134) (289–407) (110–122)
SD-SP 54.0 ± 5.2 ab 111.2 ± 6.2 a 353.8 ± 17.1 a 116.5 ± 8.4 a
(48–62) (103–118) (335–380) (105–125)
PB-SP 38.0 ± 14.1 b 121.8 ± 11.5 a 344.4 ± 39.5 a 107.2 ± 15.5 a
(17–54) (112–139) (298–387) (85–124)
SD-PB-SP 38.8 ± 10.9 b 113.0 ± 13.7 a 352.2 ± 37.7 a 106.7 ± 14.0 a
(21–51) (92–127) (290–401) (81–119)

Note: Mean ± standard deviation in the upper row, and range in the lower row.
Note: Different small letters (a and b) between columns indicate significant differences among groups.

among groups.
3.3. Renal function parameters
The analyzed data for renal function parameters, including TP, Alb,
BUN, UA, Cre, clusterin, cystatin C, and NGAL, are summarized in
Table 4. Significant elevations of TP and Alb for the SD group as
compared with the NC, PB, SP, and SD-PB-SP groups were recorded. The
SD, SP, SD-SP, and SD-PB-SP groups showed significantly BUN lower
than those of the NC group. A significant increase in UA was observed for
the Spirulina-administrated groups (SP, SD-SP, PB-SP, and SD-PB-SP) as
compared with the NC group. No significant differences were found for
the other four target plasma parameters.
3.4. Oxidative-stress parameters
The 8-OHdG value ranged from 0.05 to 0.24 ng/mL, and the protein
carbonyl level ranged from 0.12 to 0.84 nmol/mg for all groups. No
statistically significant differences were observed among groups for
either 8-OHdG or protein carbonyl (Table 5).
3.5. ALAD activity
The SP group showed the highest ALAD activity ratio with statistical
significance, followed by the SD, SD-SP, and NC groups (Fig. 2, Sup­
plementary Table S4). The ALAD activity ratio of the PB group was
significantly reduced compared with the other groups. There were no
significant differences in assays other than the non-activated assay.
4. Discussion
In the current study, a Spirulina solution was administrated to mice
that had been exposed to either Pb acetate, a special diet high in fats and
carbohydrates, or a combination of both to elucidate the potential
ameliorative effect of Spirulina on Pb poisoning and obesity. However,
because of the unfortunate deaths of two mice during the experimental
period, we had to discontinue administration after 10 days, which is a
major limitation of this study.
The Pb concentrations in all of the studied tissues and feces of Pb-
exposed groups (PB, PB-SP, and SD-PB-SP) were significantly elevated
relative to those of the other groups (NC, SP, SD, and SD-SP). Spirulina
administration did not significantly change Pb levels in the blood, liver,
and feces, but the kidney Pb level in the SD-PB-SP group was signifi­
cantly lower than that of the PB group. Although the reason for the
reduction in the renal Pb concentration in the SD-PB-SP group was un­
clear, these results suggest that Spirulina supplementation does not
affect Pb deposition in tissues or excretion through the feces. This is in
agreement with a previous laboratory-exposure study that used Wistar
rats (Upasani and Balaraman, 2003). On the other hand, Khalil, Elhady,
Elewa, Abd El-Hameed, & Ali. (2018) found the significant reduction of
Pb accumulation levels in blood and liver by Spirulina supplementation
in their exposure study albeit the longer treatment period (30 days) and
lower dose of the intraperitoneally administrated Pb acetate (50 mg/kg
body weight).
The previous study administered Spirulina to healthy Wistar rats for
30 days and did not record a change in serum Fe concentration (Nasir­
ian, Dadkhah, Moradi-kor, & Obeidavi, 2018), supporting our results
showing no change in blood Fe levels after Spirulina treatment. Ac­
cording to Simsek, Karadeniz, & Karaca (2007), erythrocyte formation
and Hb synthesis were stimulated by Spirulina administration in rats.
Moreover, in a previous study on humans, Spirulina supplementation
increased the mean corpuscular volume, mean corpuscular Hb, and
mean corpuscular Hb concentration (Selmi et al., 2011). In our study, a
protective effect of Spirulina supplementation was observed in two in­
dicators of Pb hematotoxicity. The Hct value dropped significantly in the
PB group compared with the groups not exposed to Pb, however the Hct
value was restored by Spirulina treatment. Similarly, the reduction of
the ALAD activity ratio observed in the PB group was significantly
ameliorated in the PB-SP and SD-PB-SP groups. Furthermore, the SP
group exhibited the highest ALAD activity ratio among groups, indi­
cating a promotive effect of Spirulina on ALAD activity. Simsek, Kar­
adeniz, Kalkan, Keles, & Unal (2009) also reported that Spirulina
treatments in rats improved red blood cell counts and Hb concentra­
tions, as well as improved Hct values reduced by Pb administration. The
results in the current and previous studies support Spirulina having an
ameliorative effect on anemia. It should also be emphasized that the
same extent of restoration in the Hct value and ALAD activity ratio were
seen in the SD-PB-SP group as in the PB-SP group; this suggests that
Spirulina supplementation improves anemia status even in mice fed a
high-fat, low-protein diet.
Regarding the renal injury assessment, the three plasma proteins
(clusterin, cystatin C, and NGAL), which are preferred for detecting
changes in renal function at an early stage over traditional parameters
(Vinken et al., 2012; Zhang et al., 2011), did not differ among the groups
in our study, contrary to our expectations. Although Poręba, Gać, Por­
ęba, Antonowicz-Juchniewicz, & Andrzejak (2011) reported a signifi­
cant association between blood Pb and serum cystatin C levels in
humans, the short duration of exposure period in our study could have
resulted in the lack of observed kidney injury. On the other hand, for
some traditional parameters, significant differences among the groups
were recorded. The increase in Alb in the SD group is especially inter­
esting because the special diet contains less protein than the control diet.

5
H. Nakata et al. Journal of Functional Foods 77 (2021) 104344

Table 5

a
Values of oxidative-stress markers by group (control group and treated groups
with sole or combination of special diet, lead acetate and spirulina powder).

NGAL (µg/mL)
8-OHdG (ng/mL) Protein carbonyl (nmol/mg)

(0.14–0.21)

(0.21–0.60)

(0.12–0.16)

(0.15–0.41)

(0.14–0.28)

(0.22–3.58)

(0.15–6.06)
0.17 ± 0.03

0.33 ± 0.17

0.14 ± 0.02

0.26 ± 0.10

0.22 ± 0.05

0.98 ± 1.46

1.38 ± 2.62
NC 0.11 ± 0.02 a 0.18 ± 0.05 a
Values of plasma biochemical parameters relating to kidney injury assessment by group (control group and treated groups with sole or combination of special diet, lead acetate and spirulina powder).

(0.09–0.13) (0.13–0.27)
SD 0.12 ± 0.02 a 0.26 ± 0.09 a
(0.09–0.14) (0.12–0.40)
PB 0.08 ± 0.03 a 0.17 ± 0.05 a
(0.05–0.13) (0.12–0.25)
a

a
SP 0.10 ± 0.03 a 0.18 ± 0.04 a
(0.06–0.12) (0.12–0.24)
Cystatin C (µg/mL)

SD-SP 0.12 ± 0.04 a 0.34 ± 0.27 a

(0.08–0.18) (0.14–0.84)
PB-SP 0.12 ± 0.07 a 0.29 ± 0.15 a
(0.36–0.53)

(0.41–0.58)

(0.36–0.53)

(0.40–0.72)

(0.45–0.64)

(0.51–0.61)

(0.50–0.61)
0.45 ± 0.07

0.49 ± 0.07

0.44 ± 0.06

0.53 ± 0.12

0.55 ± 0.08

0.56 ± 0.04

0.54 ± 0.04
(0.08–0.24) (0.15–0.55)
SD-PB-SP 0.12 ± 0.03 a 0.40 ± 0.20 a
(0.08–0.15) (0.22–0.74)

Note: Mean ± standard deviation in the upper row, and range in the lower row.
Note: One small letter of a indicates no significant difference among groups.
a

a
Clusterin (µg/mL)

a
(67.94–105.44)

(59.66–133.35)

(64.18–107.57)

(80.41–151.99)

(71.76–107.79)
106.85 ± 29.68
(59.50–78.50)

(64.62–79.17)
80.49 ± 15.93

93.38 ± 26.96

94.90 ± 17.69

93.41 ± 13.45
72.32 ± 7.80

74.31 ± 5.64

0.800

b b b
0.600
ALAD activity ratio
a

c c
Cre (mg/dL)

0.400
(0.24–0.31)

(0.19–0.29)

(0.22–0.29)

(0.24–0.30)

(0.21–0.26)

(0.25–0.27)

(0.24–0.27)
0.27 ± 0.03

0.25 ± 0.04

0.25 ± 0.03

0.26 ± 0.03

0.24 ± 0.02

0.26 ± 0.01

0.25 ± 0.01

d
0.200
abc

ab

bc

bc

Note: Different small letters (a, b and c) between columns indicate significant differences among groups.
a

0.000
UA (mg/dL)

P
P

P
PB
SD

SP
(0.3–1.3)

(0.9–2.2)

(1.1–1.7)

(1.5–2.7)

(1.6–2.5)

(1.1–2.9)

(1.6–2.9)
0.9 ± 0.4

1.6 ± 0.4

1.4 ± 0.2

2.0 ± 0.5

2.0 ± 0.3

2.3 ± 0.7

2.2 ± 0.5

S
-S
-S
N

B-
PB
SD

-P
SD
Fig. 2. Means and standard deviation values (error bars) of erythrocyte
δ-aminolevulinic acid dehydratase activity ratio by groups. Different small
ab

ab
bc
a

Note: Mean ± standard deviation in the upper row, and range in the lower row.

letters (a, b, c and d) indicate significant differences among groups.

NC = control without any treatment, SD = given special diet, PB = given
1,000 mg/L Pb acetate, SP = given Spirulina powder (1,000 mg/kg body
BUN (mg/dL)

weight/day), SD-SP = given special diet and Spirulina powder, PB-SP = given
(7.4–9.1)

(4.2–5.4)

(6.4–8.8)

(4.2–6.9)

(4.3–5.5)

(5.1–8.7)

(3.1–5.9)
8.0 ± 0.6

4.8 ± 0.4

7.6 ± 0.8

6.1 ± 1.1

4.7 ± 0.4

7.3 ± 1.4

4.5 ± 1.0

Pb acetate and Spirulina powder, SD-PB-SP = given special diet, Pb acetate and
Spirulina powder.

Although to the best of our knowledge, there have been no studies

ab

ab
b

reporting Alb increases due to a low protein diet, it could be hypothe­

a

sized that more lipid was used for energy metabolism instead of Alb
because of the high-fat diet; this results in the temporal plasma Alb in­
Alb (g/dL)

(0.4–0.8)

(0.8–1.3)

(0.6–0.8)

(0.6–0.9)

(0.4–1.3)

(0.6–1.0)

(0.6–1.1)
0.6 ± 0.2

1.1 ± 0.2

0.7 ± 0.1

0.7 ± 0.1

0.8 ± 0.3

0.8 ± 0.1

0.8 ± 0.2

crease on day 10 of treatment. In our study, feeding mice the special

diet, which had low levels of protein, also caused the decline of plasma
BUN. This phenomenon is well-known and can be explained by the
theory that low protein intake suppresses protein metabolism, resulting
ab

ab
b

in low BUN synthesis in the liver (Boulay, Scott, Conly, Stevenson, &
a

Koski, 1998). Another interesting finding is the increase in UA in mice

fed Spirulina. Considering the fact that high UA is usually caused by a
TP (g/dL)

(1.7–2.5)

(2.2–2.9)

(1.9–2.3)

(2.0–2.3)

(2.1–3.0)

(2.0–2.5)

(1.9–2.6)
2.0 ± 0.3

2.6 ± 0.3

2.1 ± 0.1

2.1 ± 0.1

2.5 ± 0.3

2.2 ± 0.2

2.2 ± 0.3

high intake of dietary purines, the purine-rich alkaloids in Spirulina

(Kata et al., 2018) would have enhanced purine metabolism in liver and
increased the UA metabolite. It is also generally known that changes in
these indices can result from renal dysfunction; however, the absence of
SD-PB-SP

changes in parameters such as clusterin, cystatin C, and NGAL suggests

Table 4

SD-SP

PB-SP

that renal disorders did not occur in the present study.

NC

SD

PB

SP

6
H. Nakata et al.
The plasma HDL-C, TG, Glu, and T-Cho levels were assessed to
evaluate obesity status. A previous study showed that type 2 diabetes
mellitus patients who took Spirulina supplements for two months had
decreased blood TG, Glu, and T-Cho, coupled with a marginal increase in
HDL-C (Parikh, Mani, & Iyer, 2001). Likewise, a study using CD-1 mice
with experimental diabetes demonstrated the elevation of HDL-C after
four weeks of Spirulina administration (Rodrıguez-Hernández, Ble-
Castillo, Juarez-Oropeza, & Dıaz-Zagoya, 2001). Serban et al. (2016)
concluded that Spirulina supplementation reduced plasma concentra­
tions of TG and T-Cho and elevated those of HDL-C. In our study,
however, such phenomenon was not observed; the plasma parameters
did not differ greatly among groups. The meta-analysis study explained
this unexpected result with the finding of significant associations be­
tween Spirulina supplementation duration and plasma HDL-C, TG, and
T-Cho concentration changes (Serban et al., 2016). In contrast to the
other parameters, HDL-C in the SD group was significantly greater than
that of the PB-SP and SD-PB-SP groups. The observed increase in HDL-C
in C57BL/6J mice due to the high-fat diet was in agreement with a
previous study (Yang, Du, Hosokawa, & Miyashita, 2020). A former
exposure study on lead acetate in rats revealed a significant reduction in
blood HDL-C in, the Pb-exposed group (Abdel-Moneim et al., 2015).
Similarly, decreases in serum and liver HDL-C levels in Pb-exposed rats
have also been reported (Newairy and Abdou, 2009). Taken together,
these suggest the HDL-C decline in the PB-SP and SD-PB-SP groups was
caused by the Pb administration. In fact, HDL-C in the PB group also
marginally decreased relative to the NC group. Additionally, the com­
parison of the PB group with the PB-SP and SD-PB-SP groups supports
the hypothesis that Spirulina treatment induces the reductive effect Pb
has on plasma HDL-C.
The eWAT weight results, which are widely used as an indicator of
obesity status in mice (Pham et al., 2019; Yang, Du, Hosokawa, &
Miyashita, 2020), showed a clear trend. The significant increase in
eWAT weight in the SD group was observed to be similar to that in a
previous study on C57BL/6J mice on a 21-weeks high-fat diet (Duval
et al., 2010). In the comparison of the SD group with the SD-SP and SD-
PB-SP groups, the increase of eWAT weight due to the high-fat diet was
significantly decreased upon Spirulina supplementation, indicating the
mitigative effect Spirulina has on eWAT accumulation. Moreover, the SP
group showed the lowest mean eWAT weight, followed by PB-SP,
although the difference between these two groups was not statistically
significant. A similarly significant reduction of total white adipose tissue
(WAT) and mesenteric WAT weights of C57BL6/J mice by Spirulina has
been reported, albeit there was no significant difference in eWAT weight
(Yang, Du, Hosokawa, & Miyashita, 2020). But in contrast to our study,
Pham et al. (2019) treated C57BL6/J mice with a high-fat/high-sucrose
diet (34%/38%, w/w) for 20 weeks and found that dietary Spirulina
supplementation (2%, w/w) did not affect eWAT weight. Considering
the fact that our study used a diet containing a relatively low weight
ratio of fat and sucrose (24%/20%) compared with the study by Pham
et al. (2019), it can be hypothesized that Spirulina supplementation is
not effective in reducing WAT weight after fat and sucrose intakes
exceed a certain level.
Unlike the other parameters that showed significant changes with
treatment, there were no significant differences between the groups in
terms of oxidative-stress status on either of the two studied indices.
Given that increases of reactive oxygen species, antioxidant capacity,
and reactive oxygen metabolites due to Pb exposure are commonly
known (Matović et al., 2015), the most likely explanation is the short
exposure period, similar to the reason behind the absence of renal
damage. Thus, further studies with longer treatment periods are
considered necessary.
5. Conclusion
We examined the potential mitigative effects of Spirulina on Pb
poisoning and obesity using a mouse model. While Spirulina treatment
7
Journal of Functional Foods 77 (2021) 104344
did not greatly affect the Pb and Fe accumulation patterns, probably
because of the short duration of the experimental period, the anemia
caused by Pb administration was improved by Spirulina, as shown
through the Hct values and ALAD activity ratios, even for mice fed a
high-fat low-protein diet. Regarding obesity-related indicators, Spir­
ulina supplementation aided in decreasing eWAT weight, although the
plasma biochemical parameters for obesity assessment did not greatly
change in mice fed a high-fat diet. However, Spirulina treatment may
have normalized the plasma HDL-C, which otherwise declines after Pb
administration. No changes in any of the studied oxidative-stress-related
indices were recorded in the current study. In summary, our study
provided some positive insights into the efficacy of Spirulina; however,
further validations are needed.
Ethics statements
All animal experiments were performed with the approval and su­
pervision of the Institutional Animal Care and Use Committee of Hok­
kaido University (approval number 19-0119), Japan. The institute of
Faculty of Veterinary Medicine, Hokkaido University has been accredi­
ted by The European Association of Establishments for Veterinary Ed­
ucation (EAEVE) and regulated under the EU Directive 2010/63/EU,
and all animal experiments are reviewed by AAALAC International.
CRediT authorship contribution statement
Hokuto Nakata: Conceptualization, Data curation, Formal analysis,
Funding acquisition, Investigation, Methodology, Validation, Visuali­
zation, Writing - original draft. Shouta M.M. Nakayama: Funding
acquisition, Investigation, Methodology, Supervision, Writing - review
& editing. Andrew Kataba: Investigation, Methodology, Validation,
Writing - review & editing. Yared Beyene Yohannes: Investigation,
Methodology, Validation, Writing - review & editing. Yoshinori Ike­
naka: Funding acquisition, Resources, Supervision, Writing - review &
editing. Mayumi Ishizuka: Funding acquisition, Project administration,
Supervision, Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was supported by Grants-in-Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science, and Technol­
ogy of Japan awarded to M. Ishizuka (No. 16H01779, 18KK0287), Y.
Ikenaka (No. 17K20038, 18H04132), S.M.M. Nakayama (No.
17KK0009, 20K20633), and H. Nakata (No. 19K20472) and the foun­
dation of JSPS Bilateral Open Partnership Joint Research Projects
(JPJSBP120209902), and the Environment Research and Technology
Development Fund (SII-1/3-2, 4RF-1802/18949907) of the Environ­
mental Restoration and Conservation Agency of Japan. We also
acknowledge financial support from The Soroptimist Japan Foundation,
The Nakajima Foundation, The Sumitomo foundation, The Nihon Seimei
Foundation, The Japan Prize Foundation, The Akiyama Life Science
Foundation, and Hokkaido University SOUSEI Support Program for
Young Researchers in FY2020 (SMMN). This research was also sup­
ported by JST/JICA, SATREPS (Science and Technology Research
Partnership for Sustainable Development; No. JPMJSA1501), JST aXis
(Accelerating Social Implementation for SDGs Achievement; No.
JPMJAS2001) and Program for supporting introduction of the new
sharing system (JPMXS0420100619). Our appreciation is extended to
Sayako Yamamoto, Yumi Miyabe and Ami Matsuno for their assistance,
as well as Takahiro Ichise and Nagisa Hirano for their technical support.
H. Nakata et al.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jff.2020.104344.
References
Abdel-Moneim, A. M., El-Toweissy, M. Y., Ali, A. M., Allah, A. A. M. A., Darwish, H. S., &
Sadek, I. A. (2015). Curcumin ameliorates lead (Pb 2+)-induced hemato-
biochemical alterations and renal oxidative damage in a rat model. Biological trace
element research, 168(1), 206-220. PMID: 25947936, https://doi.org/10.1007/
s12011-015-0360-1.
Aladaileh, S. H., Khafaga, A. F., Abd El-Hack, M. E., Al-Gabri, N. A., Abukhalil, M. H.,
Alfwuaires, M. A., … Abdelnour, S. (2020). Spirulina platensis ameliorates the sub
chronic toxicities of lead in rabbits via anti-oxidative, anti-inflammatory, and
immune stimulatory properties. Science of The Total Environment, 701, 134879.
PMID: 31734488, https://doi.org/10.1016/j.scitotenv.2019.134879.
Augustyniak, E., Adam, A., Wojdyla, K., Rogowska-Wrzesinska, A., Willetts, R., Korkmaz,
A., …, Griffiths, H. R. (2015). Validation of protein carbonyl measurement: a multi-
centre study. Redox Biology, 4, 149-157. PMID: 25560243, https://doi.org/
10.1016/j.redox.2014.12.014.
Boulay, M., Scott, M. E., Conly, S. L., Stevenson, M. M., & Koski, K. G. (1998). Dietary
protein and zinc restrictions independently modify a Heligmosomoides polygyrus
(Nematoda) infection in mice. Parasitology, 116(5), 449-462. PMID: 9614328,
https://doi.org/10.1017/s0031182098002431.
Bullen Jr, J. W., Ziotopoulou, M., Ungsunan, L., Misra, J., Alevizos, I., Kokkotou, E., …,
Mantzoros, C. S. (2004). Short-term resistance to diet-induced obesity in A/J mice is
not associated with regulation of hypothalamic neuropeptides. American Journal of
Physiology-Endocrinology and Metabolism, 287(4), E662-E670. PMID: 15361355,
https://doi.org/10.1152/ajpendo.00114.2004.
Campanella, L., Crescentini, G., & Avino, P. (1999). Chemical composition and
nutritional evaluation of some natural and commercial food products based on
Spirulina. Analusis, 27(6), 533–540. https://doi.org/10.1051/analusis:1999130.
Dooyema, C. A., Neri, A., Lo, Y. C., Durant, J., Dargan, P. I., Swarthout, T., …
Brown, M. J. (2012). Outbreak of fatal childhood lead poisoning related to artisanal
gold mining in northwestern Nigeria, 2010. Environmental Health Perspectives, 120
(4), 601–607. https://doi.org/10.1289/ehp.1103965.
Drewnowski, A., & Specter, S. E. (2004). Poverty and obesity: the role of energy density
and energy costs. The American Journal of Clinical Nutrition, 79(1), 6-16. PMID:
14684391, https://doi.org/10.1093/ajcn/79.1.6.
Duval, C., Thissen, U., Keshtkar, S., Accart, B., Stienstra, R., Boekschoten, M. V.,
Roskams, T., Kersten, S., & Müller, M. (2010). Adipose tissue dysfunction signals
progression of hepatic steatosis towards nonalcoholic steatohepatitis in C57BL/6
mice. Diabetes, 59(12), 3181-3191. PMID: 20858684, https://doi.org/10.2337/
db10-0224.
Estrada, J. P., Bescós, P. B., & Del Fresno, A. V. (2001). Antioxidant activity of different
fractions of Spirulina platensis protean extract. Il farmaco, 56(5-7), 497-500. PMID:
11482785, https://doi.org/10.1016/s0014-827x(01)01084-9.
Espín, S., Martínez-López, E., Jiménez, P., María-Mojica, P., & García-Fernández, A. J.
(2015). Delta-aminolevulinic acid dehydratase (δALAD) activity in four free-living
bird species exposed to different levels of lead under natural conditions.
Environmental research, 137, 185-198. PMID: 25569843, https://doi.org/10.1016/
j.envres.2014.12.017.
FAO and IOC (2011). Spirulina: a livehood and a business venture. Available: http://
www.fao.org/3/a-az386e.pdf [accessed 20 April 2020].
Gonick, H. C. (2011). Lead-binding proteins: a review. Journal of Toxicology, 2011.
PMID: 21941540, https://doi.org/10.1155/2011/686050.
Hoseini, S. M., Khosravi-Darani, K., & Mozafari, M. R. (2013). Nutritional and medical
applications of spirulina microalgae. Mini Reviews in Medicinal Chemistry, 13(8),
1231-1237. PMID: 23544470, https://doi.org/10.2174/1389557511313080009.
Hruschka, D. J. (2012). Do economic constraints on food choice make people fat? A
critical review of two hypotheses for the poverty–obesity paradox. American Journal
of Human Biology, 24(3), 277-285. PMID: 22345082, https://doi.org/10.1002/
ajhb.22231.
Kata, F. S., Athbi, A. M., Manwar, E. Q., Al-Ashoor, A., Abdel-Daim, M. M., & Aleya, L.
(2018). Therapeutic effect of the alkaloid extract of the cyanobacterium Spirulina
platensis on the lipid profile of hypercholesterolemic male rabbits. Environmental
Science and Pollution Research, 25(20), 19635-19642. PMID: 29736642, https://
doi.org/10.1007/s11356-018-2170-4.
Khalil, S. R., Elhady, W. M., Elewa, Y. H., Abd El-Hameed, N. E., & Ali, S. A. (2018).
Possible role of Arthrospira platensis in reversing oxidative stress-mediated liver
damage in rats exposed to lead. Biomedicine & Pharmacotherapy, 97, 1259-1268.
PMID: 29145152, https://doi.org/10.1016/j.biopha.2017.11.045.
Matović, V., Buha, A., Ðukić-Ćosić, D., & Bulat, Z. (2015). Insight into the oxidative
stress induced by lead and/or cadmium in blood, liver and kidneys. Food and
Chemical Toxicology, 78, 130-140. PMID: 25681546, https://doi.org/10.1016/j.
fct.2015.02.011.
Miranda, M. S., Cintra, R. G., Barros, S. B. D. M., & Mancini-Filho, J. (1998). Antioxidant
activity of the microalga Spirulina maxima. Brazilian Journal of Medical and
biological research, 31(8), 1075-1079. PMID: 9777014, https://doi.org/10.1590/
s0100-879x1998000800007.
Miranda, M. L., Edwards, S. E., Keating, M. H., & Paul, C. J. (2011). Making the
environmental justice grade: the relative burden of air pollution exposure in the
8
Journal of Functional Foods 77 (2021) 104344
United States. International Journal of Environmental Research and Public Health, 8
(6), 1755-1771. PMID: 21776200, https://doi.org/10.3390/ijerph8061755.
Nakata, H., Nakayama, S. M., Yabe, J., Liazambi, A., Mizukawa, H., Darwish, W. S.,
Ikenaka, Y., & Ishizuka, M. (2016). Reliability of stable Pb isotopes to identify Pb
sources and verifying biological fractionation of Pb isotopes in goats and chickens.
Environmental Pollution, 208, 395-403. PMID: 26549754, https://doi.org/10.1016/
j.envpol.2015.10.006.
Nakata, H., Nakayama, S. M., Yabe, J., Muzandu, K., Toyomaki, H., Yohannes, Y. B., …
Ishizuka, M. (2020). Clinical biochemical parameters associated with the exposure to
multiple environmental metals in residents from Kabwe, Zambia. Chemosphere,
127788. https://doi.org/10.1016/j.chemosphere.2020.127788.
Nasirian, F., Dadkhah, M., Moradi-kor, N., & Obeidavi, Z. (2018). Effects of Spirulina
platensis microalgae on antioxidant and anti-inflammatory factors in diabetic rats.
Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy, 11, 375-380.
PMID: 30104892, https://doi.org/10.2147/DMSO.S172104.
Newairy, A. S. A., & Abdou, H. M. (2009). Protective role of flax lignans against lead
acetate induced oxidative damage and hyperlipidemia in rats. Food and Chemical
Toxicology, 47(4), 813-818. PMID: 19271316, https://doi.org/10.1016/j.
fct.2009.01.012.
Okamoto, T., Kawashima, H., Osada, H., Toda, E., Homma, K., Nagai, N., …, Ozawa, Y.
(2019). Dietary spirulina supplementation protects visual function from photostress
by suppressing retinal neurodegeneration in mice. Translational Vision Science &
Technology, 8(6), 20-20. PMID: 31788349, https://doi.org/10.1167/tvst.8.6.20.
Parikh, P., Mani, U., & Iyer, U. (2001). Role of Spirulina in the control of glycemia and
lipidemia in type 2 diabetes mellitus. Journal of Medicinal Food, 4(4), 193-199.
PMID: 12639401, https://doi.org/10.1089/10966200152744463.
Parnia, A., Chakravartty, D., Wiseman, C. L., Archbold, J., Copes, R., Zawar, N., …, Cole,
D. C. (2018). Environmental factors associated with blood lead among newcomer
women from South and East Asia in the Greater Toronto Area. Science of the Total
Environment, 624, 558-566. PMID: 29268227, https://doi.org/10.1016/j.
scitotenv.2017.11.336.
Pham, T. X., Lee, Y., Bae, M., Hu, S., Kang, H., Kim, M. B., …, Lee, J. Y. (2019). Spirulina
supplementation in a mouse model of diet-induced liver fibrosis reduced the pro-
inflammatory response of splenocytes. British Journal of Nutrition, 121(7), 748-755.
PMID: 30806344, https://doi.org/10.1017/S0007114519000126.
Poręba, R., Gać, P., Poręba, M., Antonowicz-Juchniewicz, J., & Andrzejak, R. (2011).
Relation between occupational exposure to lead, cadmium, arsenic and
concentration of cystatin C. Toxicology, 283(2-3), 88-95. PMID: 21356263, https://
doi.org/10.1016/j.tox.2011.02.008.
Rodrıguez-Hernández, A., Ble-Castillo, J. L., Juarez-Oropeza, M. A., & Dıaz-Zagoya, J. C.
(2001). Spirulina maxima prevents fatty liver formation in CD-1 male and female
mice with experimental diabetes. Life Sciences, 69(9), 1029-1037. PMID: 11508645,
https://doi.org/10.1016/s0024-3205(01)01185-7.
Sabath, E., & Robles-Osorio, M. L. (2012). Renal health and the environment: heavy
metal nephrotoxicity. Nefrología (English Edition), 32(3), 279-286. PMID:
22508139, https://doi.org/10.3265/Nefrologia.pre2012.Jan.10928.
Scheuhammer, A. M. (1987). Erythrocyte δ-aminolevulinic acid dehydratase in birds. I.
The effects of lead and other metals in vitro. Toxicology, 45(2), 155-163. PMID:
3603581, https://doi.org/10.1016/0300-483X(87)90101-6.
Selmi, C., Leung, P. S., Fischer, L., German, B., Yang, C. Y., Kenny, T. P., …, Gershwin, M.
E. (2011). The effects of Spirulina on anemia and immune function in senior citizens.
Cellular & Molecular Immunology, 8(3), 248. PMID: 21278762, https://doi.org/
10.1038/cmi.2010.76.
Serban, M. C., Sahebkar, A., Dragan, S., Stoichescu-Hogea, G., Ursoniu, S., Andrica, F., &
Banach, M. (2016). A systematic review and meta-analysis of the impact of Spirulina
supplementation on plasma lipid concentrations. Clinical Nutrition, 35(4), 842-851.
PMID: 26433766, https://doi.org/10.1016/j.clnu.2015.09.007.
Simsek, N., Karadeniz, A., & Karaca, T. (2007). Effects of the Spirulina platensis and
Panax ginseng oral supplementation on peripheral. Revue Méd. Vét, 158(10),
483–488.
Simsek, N., Karadeniz, A., Kalkan, Y., Keles, O. N., & Unal, B. (2009). Spirulina platensis
feeding inhibited the anemia-and leucopenia-induced lead and cadmium in rats.
Journal of Hazardous Materials, 164(2-3), 1304-1309. PMID: 18976856, https://doi.
org/10.1016/j.jhazmat.2008.09.041.
Upasani, C. D., & Balaraman, R. (2003). Protective effect of Spirulina on lead induced
deleterious changes in the lipid peroxidation and endogenous antioxidants in rats.
Phytotherapy Research: An International Journal Devoted to Pharmacological and
Toxicological Evaluation of Natural Product Derivatives, 17(4), 330-334. PMID:
12722134, https://doi.org/10.1002/ptr.1135.
Vinken, P., Starckx, S., Barale-Thomas, E., Looszova, A., Sonee, M., Goeminne, N., …,
Lampo, A. (2012). Tissue Kim-1 and urinary clusterin as early indicators of cisplatin-
induced acute kidney injury in rats. Toxicologic Pathology, 40(7), 1049-1062. PMID:
22581811, https://doi.org/10.1177/0192623312444765.
World Health Organization (WHO), 2009. Global health risks: mortality and burden of
disease attributable to selected major risks. Available: http://whqlibdoc.who.int/
publications/2009/9789241563871_eng.pdf [accessed 24 May 2020].
World Health Organization, 2011. Global status report on noncommunicable diseases
2010. Available: https://apps.who.int/iris/bitstream/handle/10665/44579/
9789240686458_eng.pdf;jsessionid=8C88471C98B5D9640B654D832C983F8C?
sequence=1 [accessed 20 April 2020].
Yang, Y., Du, L., Hosokawa, M., & Miyashita, K. (2020). Effect of Spirulina lipids on high-
fat and high-sucrose diet induced obesity and hepatic lipid accumulation in C57BL/
6J mice. Journal of Functional Foods, 65, Article 103741. https://doi.org/10.1016/j.
jff.2019.103741.
Yousefi, R., Mottaghi, A., & Saidpour, A. (2018). Spirulina platensis effectively
ameliorates anthropometric measurements and obesity-related metabolic disorders
H. Nakata et al.
in obese or overweight healthy individuals: A randomized controlled trial.
Complementary Therapies in Medicine, 40, 106-112. PMID: 30219433, https://doi.
org/10.1016/j.ctim.2018.08.003.
Zhang, Z., Lu, B., Sheng, X., & Jin, N. (2011). Cystatin C in prediction of acute kidney
injury: a systemic review and meta-analysis. American Journal of Kidney Diseases,
58(3), 356-365. PMID: 21601330, https://doi.org/10.1053/j.ajkd.2011.02.389.
Journal of Functional Foods 77 (2021) 104344
Zhao, B., Cui, Y., Fan, X., Qi, P., Liu, C., Zhou, X., & Zhang, X. (2019). Anti-obesity effects
of Spirulina platensis protein hydrolysate by modulating brain-liver axis in high-fat
diet fed mice. PloS One, 14(6), e0218543. PMID: 31220177, https://doi.org/
10.1371/journal.pone.0218543.