Induction of aminolevulinic acid synthase gene expression and enhancement of metabolite, protoporphyrin IX, excretion by organic germanium

Article abstract of DOI:10.1016/j.ejphar.2010.12.002

Poly-trans-[(2-carboxyethyl) germasesquioxane], Ge-132 is a water-soluble organic germanium compound. Oral intake of dietary Ge-132 changes fecal color and we attempted to identify the fecal red pigment, which increased by the intake of dietary Ge-132. Sprague Dawley rats were given diets containing Ge-132 from 0 to 0.5% concentration. Fecal red pigment was extracted and purified for optical and structural studies. We examined the fecal red pigment content by high performance liquid chromatography (HPLC), and hepatic gene expressions relating to heme synthesis by reverse transcription polymerase chain reaction (RT-PCR). The purified red pigment had particular optical characteristics on the ultraviolet (UV)-visible spectrum (Soret band absorbance at 400 nm) and fluorescence emission at 600 nm by 400 nm excitation, and was identified as protoporphyrin IX by LC-MS analysis. Protoporphyrin IX significantly (P < 0.05) increased 2.4-fold in the feces by the intake of a 0.5% Ge-132 diet. Gene expression analysis of the liver explained the increase of protoporphyrin IX by dietary Ge-132 as it enhanced (P < 0.05) aminolevulinic acid synthase 1 (Alas1), a rate-limiting enzyme of heme synthesis, expression 1.8-fold, but decreased ferrochelatase (Fech) expression 0.6-fold (P < 0.05). The results show that the intake of dietary Ge-132 is related to heme metabolism. Because protoporphyrin IX is used to treat chronic hepatitis, Ge-132 may be a beneficial substance to increase protoporphyrin IX in the liver.

Full text of DOI:10.1016/j.ejphar.2010.12.002

European Journal of Pharmacology 653 (2011) 75–81
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Pulmonary, Gastrointestinal and Urogenital Pharmacology
Induction of aminolevulinic acid synthase gene expression and enhancement of
metabolite, protoporphyrin IX, excretion by organic germanium
a,
a
a
b
b
c
Takashi Nakamura ⁎, Miki Saito , Yasuhiro Shimada , Haruhiko Fukaya , Yasuo Shida , Yoshihiko Tokuji
a
Asai Germanium Research Institute Co., Ltd., Hakodate, Hokkaido, Japan
Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan
Department of Agricultural and Life Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 October 2010
Received in revised form 3 December 2010
Accepted 7 December 2010
Available online 15 December 2010
Poly-trans-[(2-carboxyethyl) germasesquioxane], Ge-132 is a water-soluble organic germanium compound.
Oral intake of dietary Ge-132 changes fecal color and we attempted to identify the fecal red pigment, which
increased by the intake of dietary Ge-132. Sprague Dawley rats were given diets containing Ge-132 from 0 to
0.5% concentration. Fecal red pigment was extracted and purified for optical and structural studies. We
examined the fecal red pigment content by high performance liquid chromatography (HPLC), and hepatic
gene expressions relating to heme synthesis by reverse transcription polymerase chain reaction (RT-PCR). The
purified red pigment had particular optical characteristics on the ultraviolet (UV)–visible spectrum (Soret
band absorbance at 400 nm) and fluorescence emission at 600 nm by 400 nm excitation, and was identified as
protoporphyrin IX by LC-MS analysis. Protoporphyrin IX significantly (Pb0.05) increased 2.4-fold in the feces
by the intake of a 0.5% Ge-132 diet. Gene expression analysis of the liver explained the increase of
protoporphyrin IX by dietary Ge-132 as it enhanced (Pb0.05) aminolevulinic acid synthase 1 (Alas1), a rate-
limiting enzyme of heme synthesis, expression 1.8-fold, but decreased ferrochelatase (Fech) expression 0.6-
fold (Pb0.05). The results show that the intake of dietary Ge-132 is related to heme metabolism. Because
protoporphyrin IX is used to treat chronic hepatitis, Ge-132 may be a beneficial substance to increase
protoporphyrin IX in the liver.
Keywords:
Organic germanium
Ge-132
Hepatic function
Protoporphyrin
Alas1
Chronic hepatitis
©
2010 Elsevier B.V. All rights reserved.
1
. Introduction
the germanium compoundis veryimportant for safe use. In the past, fatal
accidents occurred by the intake of health foods containing germanium
compounds. In these cases, they were sold as organic germanium unless
they contained inorganic germanium; therefore, it is necessary to avoid
contamination with inorganic germanium.
Poly-trans-[(2-carboxyethyl) germasesquioxane], Ge-132, is the
most common water-soluble organogermanium compound (Fig. 1); it
has very low toxicity. Ge-132 was first synthesized by Tsutsui et al.
(
1976), and its immunomodulation activities have been revealed (Aso
The intake of Ge-132 changes the fecal color of mammals to yellow.
Recently, we reported (Nakamura et al., 2010) that this color change
was the increase of the fecal pigment stercobilin, a metabolite of
bilirubin. Mammalian feces contain many porphyrin metabolic pig-
ments, such as stercobilin, urobilinogen, bilirubin, protoporphyrin IX
(PpIX) and so on. Urobilinoids are metabolites of bilirubin by clostridia
(Vitek et al., 2006). We have demonstrated the anti-oxidation activity of
urobilinogen previously (Nakamura et al., 2006). Dietary Ge-132
increased various antioxidants related to bilirubin and urobilinoids.
We also showed the increase of a red pigment in the feces by Ge-132
intake and considered the red pigment to be related to PpIX (Nakamura
et al., 2010). PpIX is synthesized in many tissues, mainly bone marrow
(Surinya et al., 1997), and is secreted into the feces in bile (Beukeveld
et al., 1994). On the other hand, there are diseases such as porphyria;
erythropoietic protoporphyria is a disease caused by excess PpIX as a
result of ferrochelatase deficiency (Lecha et al., 2009). Meanwhile,
protoporphyrin disodium is used to treat chronic hepatitis in Japan. This
suggests that PpIX in the intestine affects the suppression of hepatic
et al., 1989; Ebina et al., 2004; Aso et al., 1985). In particular, the
activation of natural killer cells and macrophages (Aso et al., 1985) has
been reported; therefore, interferon gamma is induced in the serum of
humans (Miyao and Tanaka, 1988) and mice (Brutkiewicz and Suzuki,
1
987) after the administration of Ge-132. Many physiological
functions of Ge-132 have been reported: anti-rheumatoid (Arimori
et al., 1990), anti-osteoporosis (Fujii et al., 1993), anti-inflammatory
(
functional foods and in cosmetics in Japan and the U.S.A.
On the other hand, an inorganic germanium dioxide, GeO
toxicity. The difference in the intake of GeO
Sanai et al. (1991). GeO is accumulated in the kidney and causes renal
failure, whereas Ge-132 is not accumulated in any tissue. The structure of
Furusawa et al., 1987) and so on. Recently, Ge-132 has been used in
2
, has fatal
2
and Ge-132 was reported by
2
⁎
Corresponding author. Asai Germanium Research Institute Co.,Ltd. 3-131, Suzuranoka,
Hakodate, Hokkaido 042-0958, Japan. Tel.: +81 138 32 0032; fax: +81 138 31 0132.
E-mail address: nakamura@asai-ge.co.jp (T. Nakamura).
0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2010.12.002

76
T. Nakamura et al. / European Journal of Pharmacology 653 (2011) 75–81
Fig. 1. Structure of poly-trans-[(2-carboxyethyl) germasesquioxane], Ge-132, and its hydrolyzed compound, trihydroxy germyl propanic acid. The 3D structure model shows
germanium in green, oxygen in red and carbon in gray, respectively. Hydrogen is omitted.
inflammation. Nagai et al. (1992) studied the anti-inflammation effect
of zinc protoporphyrin and described the anti-inflammation effect of
PpIX.
In this report we investigated the effect of oral Ge-132 intake on
fecal pigmentation in rats. The increasing red pigment was purified
from feces and identified as PpIX by TOF-MS analyses. The PpIX
secretion in the feces affected by the dietary Ge-132 was then
quantified by HPLC. Moreover, hepatic gene expressions were
analyzed for heme synthesis and breakdown to bile pigment.
2.3. Characteristic studies and quantification of fecal red pigment
Sixteen male Sprague Dawley rats (four weeks old; Clea Japan Inc.,
Tokyo Japan) were housed in a divided cage and kept under the same
conditions as described above. They were acclimated with the basal
diet for a week and then randomly divided into two groups. One group
was given the control diet and the other group was given the 0.5% Ge-
132 diet for two weeks. Water was available ad libitum. The
composition of each diet based on AIN 76 is shown in Table 1. On
the final day, the animals were anesthetized with pentobarbituric acid
and killed by bleeding from the abdominal aorta; total blood was
collected into a heparin-treated tube, and the liver was removed and
used for analysis. Each blood sample was centrifuged immediately to
divide the serum. During the feeding period, the feces were collected
for pigment analyses and kept at −80 °C.
2
. Materials and methods
2
.1. Chemicals
Ge-132 (Lot No. 006316A) was synthesized at Asai Germanium
Research Institute Co., Ltd (Hakodate, Japan). Its purity was over
9.99%, and it was used for all experiments. Protoporphyrin IX and
protoporphyrin IX disodium salt were obtained from Sigma Chemical
Co. (St. Louis, MO, USA). CD OD for nuclear magnetic resonance
NMR) analysis was obtained from Merck KGaA (Darmstadt,
2
2
.4. Extraction of fecal pigments
9
.4.1. HPLC analysis
Fecal pigments were extracted with organic solvent as follows: 4-fold
3
(
ethyl acetate/acetic acid (4:1, vol/vol) was added to the feces and each
sample mixture was homogenized with a polytron homogenizer. The
homogenates were centrifuged and supernatants were collected. The
pellets underwent extraction twice. The supernatant was mixed and
filtrated, and then evaporated and resolved in aliquots with chloroform/
methanol (2:1, vol/vol).
Germany). All organic solvents for extraction and HPLC were obtained
from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
2
.2. Fecal color change by the intake of different amounts of Ge-132
Four male Sprague Dawley rats (four weeks old; Clea Japan Inc.,
Tokyo Japan) were housed in a divided cage. They were kept in an air-
conditioned room (temperature 23–25 °C) on a 12 h light–dark cycle
and maintained on a basal purified diet for a week. They were then
given diets containing different amounts of Ge-132 for a week. The
amount of Ge-132 in each diet was 0, 0.005, 0.5 and 0.5%, respectively
2.4.2. TOF MS analysis
Twenty grams of fecal sample was homogenized and then 100 ml
chloroform/methanol (2:1) and 5 ml acetic acid were added. The
sample was stirred for 2 h and filtered, and then 100 ml chloroform/
methanol (2:1) was added to the filtered sample. The extraction was
repeated 2 times. The total filtrate was mixed and evaporated, and
then the pigments were purified by chromatography.
(
Table 1). We then observed the fecal color changes.
Table 1
2.5. Separation of fecal red pigment
Composition of diets for Ge-132 administering in different concentration.
Control
0.005%Ge-132
0.05%Ge-132
0.5%Ge-132
The total extract of fecal pigments was placed in a silicic acid
column (Silicagel 60; Merck, Germany) for chromatography. Several
solvents, chloroform, acetone and methanol, were used for elution.
The eluted solutions were spotted on a silica gel thin layer
chromatograph, TLC (Silicagel 60; Merck, Germany) and separated
for confirmation by the developing solvent of chloroform/methanol/
water (65:25:4, vol/vol). Fluorescence was detected by irradiating the
TLC plate at 365 nm with a trans-illuminator (UVP; LLC, Upland CA,
USA). The red pigment was eluted with the acetone fraction and then
treated and attached to an OASIS sample extraction product, Oasis
MAX Plus (Waters Co., Milford, MA, USA) ion exchange resin. The red
pigment was eluted with 2% formic acid in acetone. The fraction
containing red pigment obtained from ion exchange chromatography
(
Weight %)
Casein
Corn starch
Corn oil
DL-methionine
Vitamin mixture
Mineral mixture
Cellulose
Choline
Ge-132
Total
23
61.5
5
0.3
1
4
5
0.2
23
61.495
5
0.3
1
23
61.45
5
0.3
1
23
61
5
0.3
1
4
5
4
5
4
5
0.2
0.005
100
0.2
0.05
100
0.2
0.5
100
-
100
HPLC analysis was carried using control and 0.5%Ge-132 groups.
Gene expression was studied using control and 0.05%Ge-132 groups.

T. Nakamura et al. / European Journal of Pharmacology 653 (2011) 75–81
77
was placed on a TLC plate to purify. The TLC was developed with
chloroform/methanol/water (65:25:4, vol/vol). The separated fraction
of red pigment was scraped from the TLC plate for extraction. The red
pigment attaching to silica gel was extracted twice with 1 ml
methanol each time, and the extract was filtered by DISMIC
2.11. Gene expression analysis of enzymes relating to heme metabolism
Fourteen male Sprague Dawley rats (four weeks old; Clea Japan
Inc.) were housed in a divided cage and maintained under the same
conditions as described above. They were acclimated with the basal
diet for a week and then randomly divided into two groups. One group
was given the control diet and the other group was given the 0.05%
Ge-132 diet for two weeks. Water was available ad libitum. The
composition of each diet based on AIN 76 is shown in Table 1. On the
final day, the animals were anesthetized with diethyl ether and killed
by bleeding from the abdominal aorta; their liver was removed and
they were dipped in RNAlater (Applied Biosystems, Carlsbad, CA, USA)
for gene expression analysis. They were kept at −30 °C.
Total RNA was extracted from approximately 50 mg liver tissue.
The tissue was homogenized with 1 ml RNAiso Plus (Takara Bio, Shiga
Japan). Total RNA was extracted according to the usual phenol–
chloroform extraction method. The DNA of the extracted sample was
digested with TURBO DNA-free (Applied Biosystems), and then the
sample was checked for quality and quantified. Two micrograms of
total RNA sample was used in reverse transcription to synthesize
template cDNA with oligo deoxythymidine primer and Super Script III
reverse transcriptional enzyme (Invitrogen). The reaction was
performed at 50 °C for 60 min.
(
0.2 μm; ADVANTEC, Tokyo Japan). The obtained pigments were
analyzed by an optical spectrometer and LC/MS.
2
.6. Preparation of protoporphyrin IX methyl ester
ProtoporphyrinIX(SigmaChemicalCo.) 10 mgwasdissolved in 4 ml
of 5% HCl in methanol. The mixture was then esterified at −20 °C for an
hour, after which 8 ml chloroform and 3 ml water were added to the
reaction mixture and mixed well. The lower phase was evaporated and
dissolved in chloroform. The solution was added to a silicic acid column
and eluted with chloroform/methanol (8:2, vol/vol) to isolate the two
major red pigments. The obtained fractions containing red pigment
were purified with silica gel TLC, as described above. Two red pigments
possessing different Rf values on the TLC were obtained and named red
pigment A and B. The obtained pigments were used for TOF/ MS and
NMR analyses.
2
.7. Structural analysis of synthesized protoporphyrin IX methyl ester
The synthesized cDNA was diluted 5-fold with PCR Easy Dilution
(Takara Bio) and then used for quantitative real-time polymerase chain
Two purified methyl esters of PpIX (PPME), named A and B, were
reaction (PCR). The expressions of genes relating to heme metabolism
were measured as the expression ratio to beta actin as a housekeeping
gene. The primers were as follows: Alas1 (Genbank accession No.
NM_024484.2) forward 5′-TGT GGC CCA TGA GTT TGG AG-3′, Alas1
reverse 5′-GAA CGC TTT ACC GAG TGT TCC AG-3′, Alas2 (Genbank
accession No. NM_013197.1) forward 5′-TGC GCC AGC TAC TAA TGG ACA
G-3′, Alas2 reverse 5′-CGA GGC ACA GTT GGG TAG TTG A-3′, Fech
identified by TOF/MS and NMR analyses. Both pigments were
dissolved in 50% methanol for TOF/MS analysis. TOF mass spectra
were recorded on an ESI: LCT (Waters Co.) mass spectrometer.
Samples (approximately 10pmol) with 50% acetonitrile were injected
and measured.
3
Purified PPME A and B were dissolved in CD OD and then analyzed
1
by H NMR using a Gemini 300 (Varian Technologies Japan Ltd., Tokyo
Japan). Measurements were performed at 25 °C probe temperature.
PPME A could be analyzed easily but PPME B did not show data with
separate sharp peaks.
(Genbank accession No. NM_ 001108434.1) forward 5′-ATT TCC AAG
AAC AGT GCC ACC AG-3′, Fech reverse 5′-TTC AAG GCT ATC AAA CAC GGT
CAA-3′, Hmox1 (Genbank accession No. NM_012580.2) forward 5′-AGG
TGC ACA TCC GTG CAG AG-3′, Hmox1 reverse 5′-TCC AGG GCC GTA TAG
ATA TGG TAC A-3′, Actb (Genbank accession No. NM_031144.2) forward
2
.8. Analyses of separated synthesized or fecal red pigments
5′-GGA GAT TAC TFC CCT GGC TCC TA-3′ and Actb reverse 5′-GAC TCA
TCG TAC TCC TGC TTG CTG-3′. The cDNA was amplified using SYBR
Premix Ex Taq II (Takara Bio) on an Opticon 2 (Bio-Rad Laboratories,
Hercules, CA, USA), which was programmed for 95 °C for 30 s, followed
by 45 cycles of denaturation (95 °C for 5 s), annealing and extension
The spectra of purified red fecal pigment were recorded with a UV–
visible (vis) spectrophotometer, UV2200 (Shimadzu, Kyoto Japan) and a
fluorescence spectrophotometer, FP777 (Jasco, Tokyo Japan). The
maximum absorption was confirmed by the UV–vis spectrophotometer.
The pigment sample was then excited at the obtained maximum
absorption wavelength and the fluorescence spectrum scanned using a
fluorescence spectrophotometer.
(60 °C for 30 s).
2.12. Peripheral blood ferric ion
The serum sample was used for the quantitative study of ferric ion.
2
.9. Mass analysis of purified fecal red pigment
The unsaturated iron binding capacity was measured using a UIBC test
Wako kit (Wako Pure Chemical Industries, Ltd.). The measurement
was performed according to the manufacturer's manual with a UV–vis
spectrometer.
The LC/TOF mass spectra of purified fecal red pigment were recorded
on an ESI: LCT (Waters Co.) mass spectrometer. Samples (approximately
0 pmol) with 50% acetonitrile were injected and separated by ODS-3
1.5×150 mm; GL Science Inc., Tokyo Japan) using a semi-microcolumn.
1
(
2
.13. Ethics of animal experiments
Elution was carried with 50% MeOH at a flow rate of 0.2 ml/ min. The
separated peak at 10.8 min was analyzed for mass study.
All animal experiments were conducted according to the guide-
lines of the ethics committee of experimental care at Asai Germanium
Research Institute Co., Ltd., which are based on public guidelines set
by the Japanese Ministry of Education, Culture, Sports, Science and
Technology.
2
.10. HPLC analysis of rat fecal protoporphyrin
For HPLC analysis, the pigments were methylated by the same
methods described above. They were then dissolved in 1 ml chloroform,
and a 10 μl sample was injected onto an HPLC column. The pigments
were quantified by HPLC equipped with a silicic acid column
Chromosorb Silica 100 (Merck). The mobile phase was a mixture of n-
hexane/ethyl acetate (1:1, vol/vol), and the flow rate was 2.5 ml/ min.
Detection was carried out using a fluorescent spectrophotometric
detector, FP821 (Jasco) at EX: 405 nm, Em: 630 nm.
2.14. Statistical analysis
The results of the experiments are presented as the mean± standard
error of the means (S.E.M.). Significant differences among two groups
were analyzed using Student's t test. The statistical significance of all
experiments was defined as Pb0.05.

78
T. Nakamura et al. / European Journal of Pharmacology 653 (2011) 75–81
3
. Results
3.1. Characteristic studies of fecal red pigment enhanced by Ge-132
administration
Ge-132 is a water-soluble organogermanium compound with a
polymer structure that is hydrolyzed to 3-trihydroxy germyl propanoic
acid in water. Its structure is shown in Fig. 1. Dietary Ge-132 changes
mammalian fecal color and we administrated different amounts of Ge-
1
32 orally to rats; their feces changed color clearly. The control feces
were dark, and feces after administration of 0.005% Ge-132 diet were
not so different; however, administration of the 0.05% Ge-132 diet made
the fecal color lighter than the control, whereas the 0.5% Ge-132 diet
made the feces slightly redder than 0.05% Ge-132. The extracted fecal
pigments were separated on a silica gel TLC plate, as shown in Fig. 2A.
The red pigment increased in a dose-dependent manner.
The rat fecal pigments after ingesting both diet compositions,
shown in Table 1, were extracted by organic solvent. The TLC pattern
of extracted fecal pigments is shown in Fig. 2B. Several spots of fecal
pigments were found and the content of dietary Ge-132 affected the
amount of red pigment, with an Rf value at 0.8. The concentration of
red pigment in the feces after Ge-132 intake was higher than the
Fig. 2. Fecal color change by Ge-132 diets. Feces samples were collected from rats given 0,
.005, 0.05 and 0.5% Ge-132 diets. The TLC pattern of extracted pigments is shown in (A).
Extracts from the same amount of feces were spotted and developed by chloroform–
0
methanol (95:12, v/v). Lanes are: 1: control (0% Ge-132), 2: 0.005% Ge-132, 3: 0.05% Ge-132,
4: 0.5% Ge-132, respectively. Lane ste. was spotted with commercial stercobilin as a
standard. Each pigment extract from 5 g feces was dissolved to 1 ml. TLC separation of
extracted pigment is shown in (B). Twenty microliters of extract was spotted in each lane.
Commercial stercobilin was spotted between two groups as a reference. Developing solvent,
C-M-W (65:25:4, v/v), was used. Lower image of the TLC plate was irradiated by ultraviolet
light at 365 nm.
Fig. 3. Final identification of protoporphyrin IX. Assignment chemical shifts of synthesized protoporphyrin IX dimethyl ester by NMR are shown in (A). Purified red pigment from rat feces
was dissolved in methanol. UV–vis spectrum was measured from 190 to 800 nm and is shown in (B). Fluorescent spectrum was measured using 400 nm for excitation and scanned
emission from 420 to 820 nm, as shown in (C). TOF mass spectrum was used to measure the purified fecal red pigment, which increased with dietary Ge-132, as shown in (D). The red
pigment was identified as protoporphyrin IX. The sample was dissolved in methanol, injected onto LC-MS, and separated by 50% methanol eluent. The peak detected at 10.8 min was
analyzed by TOF/MS.

T. Nakamura et al. / European Journal of Pharmacology 653 (2011) 75–81
79
control. On the other hand, UV irradiation showed strong red
fluorescence of the red pigment, which was increased by dietary
Ge-132. We purified this red pigment using silicic column separation.
The pigment was purified by ion exchange chromatography and then
used for optical characteristic and structural studies.
3
.2. Structural studies of synthesized protoporphyrin methyl ester
We synthesized the methyl ester of protoporphyrin (PPME) for
standard HPLC analysis. Two different pigments were obtained by
chromatography separation and were named A and B. Synthesized
PPME A and B were purified using a silicic column and Silicagel TLC,
and analyzed by TOF/MS and NMR analyses. PPME A was identified as
a protoporphyrin dimethyl ester, PPDM, by TOF/MS analysis. Distinct
peaks accorded with the fecal red pigment and were mainly observed
Fig. 4. Effect of Ge-132 administration on protoporphyrin amount in rat feces. Fecal
protoporphyrin of rats was measured by HPLC. Extracted protoporphyrin was methylated
and identified as protoporphyrin dimethylester. Pigments were quantified by HPLC equipped
with a silicic acid column, Chromosorb Silica 100 (Merck, Germany). The mobile phase was a
mixture of n-hexane/ethyl acetate (1:1, vol/vol), and the flow rate was 2.5 ml/min. Detection
was carried out using a fluorescent spectrophotometric detector, FP821 (JASCO, Japan) at EX:
405 nm, Em: 630 nm. The intake of 0.5% Ge-132 diet increased fecal protoporphyrin. Data are
presented as the mean. Error bars are presented as S.E.M. Value with asterisk is significantly
different from the control group (Pb0.05).
+
at m/z 591 [M ], 517, 503, 459, 445 and 431. The fragment ion peaks
suggested that the sample was protoporphyrin IX dimethyl ester,
PPDM. In addition, proton NMR analysis data showed that all the
peaks originating from the twelve groups bonding to the skeleton of
porphyrin were PPDM. The assigned chemical shifts of each radical of
synthetic PPDM are shown in Fig. 3A. On the other hand, PPME B was
identified as a protoporphyrin monomethyl ester, PPMM, by TOF/MS
analysis. The parental peak was detected at m/z 577. PPDM was used
as the standard in optical analyses, TLC and HPLC analysis.
to 1.15) by dietary Ge-132, suggesting that porphyrin synthesis in the
liver is enhanced by Ge-132 administration, while Fech, which is also a
rate-limiting enzyme, was significantly decreased about 0.6-fold (1.29
to 0.79). This enzyme regulated the insertion of ferric ion to PpIX.
Hmox 1 catalyzed the breakdown of hemin to biliverdin and was
increased 1.7-fold (0.96 to1.63), with no significance.
3
.3. Optical and structural characteristics of purified fecal red pigment
The observations of purified fecal red pigment with a UV–vis
spectrometer and a fluorescent spectrometer are shown in Fig. 3B and
C. The red pigment extracted from feces had several absorption
maxima at 405, 505, 540 and 575 nm. Optic absorption was highest at
3.6. Ferric ion
4
05 nm, which is specific to porphyrin products. Strong absorption at
about 400 nm, so-called Soret band absorption, and excitation at this
wavelength highlights strong red fluorescence of about 600 nm
We evaluated the concentration of peripheral blood ferric ion by
the intake of Ge-132. The unsaturated iron binding capacity of the
control and 0.05% Ge-132 was 4.93 and 5.02 μg/ml, respectively. No
change was found by the intake of Ge-132.
(
Fig. 3C.). The characteristics of these optical analyses were very
similar to fecal PpIX, synthesized PPDM and PPMM.
The red pigment separated from feces was analyzed by a TOF mass
spectrometer equipped with LC. The red pigment was separated with LC
with 50% MeOH as the elution solvent. The peak of the red pigment was
identified at 10.8 min. The TOF mass spectrum of the peak is shown in
4
. Discussion
The effects of dietary Ge-132 on fecal color change were evaluated.
Fig. 3D. The parental peak in the mass spectrum was observed at m/z
+
We previously reported the fecal color change by Ge-132 intake
Nakamura et al., 2010) and mainly described tetra pyrols, metabolites
5
63 [M+H ]; therefore, the red pigment was identified as PpIX.
(
of heme; however, we found unknown red spots in silicagel TLC analysis
of fecal pigments. In this study, we purified the red pigment, which
was increased by dietary Ge-132, showed its optical characteristics and
3
.4. HPLC analysis of rat fecal protoporphyrin IX
Fecal PpIX in the extracted sample was methylated and then
identified by HPLC equipped with a silicic column. The content of PpIX
in the fecal extract from rat feces is shown in Fig. 4. The amount of
PpIX in the control group was 9.3 and in the Ge-132 group was 21.9
(
μg/g wet feces), which was about two-fold significance (Pb0.05). On
the other hand, the cecal content was not different between groups.
Feces were strongly concentrated from the cecal content, and the
absorption of PpIX might have differed in the rectum. One reason for
the increase of PpIX excretion in feces is considered to be the
progression of synthesis in the bone marrow or the liver.
3
.5. Gene expression of enzymes relating to heme metabolism in liver
The gene expressions of aminolevulinic acid synthase (Alas) 1, Alas 2,
ferrochelatase (Fech) and heme oxigenase 1 (Hmox1) in rat liver were
evaluated. 5-Aminolevulinic acid (ALA) is made from glycine and
succinyl-CoA catalyzed by Alas. This reaction is the first step in
porphyrin synthesis as it limits the rate of the whole synthesis of heme.
The expressions of Alas 1, Alas 2, Fech and Hmox 1 in the liver are
shown in Fig. 5. No change in Alas2 was found in either group. On the
other hand, the expression of Alas1 was increased about 1.8-fold (0.65
Fig. 5. Effect on gene expression of Alas1 in liver by Ge-132 administration. Hepatic
gene expressions of Alas1, Alas2, Fech and Hmox1 against Actb were measured by qPCR.
The diets, Ge-132 free (Control) and 0.05% Ge-132 (containing 0.05% Ge-132), were
given to the rats for two weeks. Samples of total RNA were extracted from the liver of
rats. Value with asterisk is significantly different from the control group (Pb0.05).

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T. Nakamura et al. / European Journal of Pharmacology 653 (2011) 75–81
identified the structure. Moreover, an increased excretion amount was
identified by HPLC.
We confirmed the increasing tendency of Hmox1 gene expression in
the liver by the intake of Ge-132. This change is in agreement with our
previous report on bile pigment secretion (Nakamura et al., 2010). Hmox1
is inducible by stimulation with hemin, many non-heme substances (for
example, heavy metals) and oxidative stress, and this enzyme is a stress
protein. Ishikawa et al. (1983) reported the induction of Hmox1 by
exogenous factors, showing the high induction of Hmox1 from 4.8- to
25.5-fold by hemin and cadmium. In this study we also found increased
Hmox1 but it was milder than other inducing factors. Hmox1 prevented
inflammation by carbon monoxide, a reaction product (Sass et al., 2003);
therefore, the intake of dietary Ge-132 may act as a mild inducer of anti-
inflammation by carbon monoxide from heme degradation.
PpIX is used to treat chronic hepatitis. The absorption and
excretion of PpIX were revealed by Yokoyama et al. (1973) The
pharmacological functions of PpIX are the regulation of hepatic
function (Sato et al., 1986), increase of oxygen used by tissues
(Yamamoto et al., 1966), acceleration of synthesize hepatic proteins
(Takahashi et al., 1984) and anti-inflammation (Nagai et al., 1992).
Additionally, hepatic protection effects have been reported (Takeuchi
et al., 1980; Ueno et al., 1978; Kawakami et al., 1978). These
properties of PpIX are similar to the physiological actions of Ge-132
previously reported (Sasaki et al., 1984); for example, on the one
hand, PpIX is used to treat hepatic inflammatory disease as a
pharmaceutical, whereas Ge-132 was reported to have a preventive
effect against the elevation GOT and GTP activity in the serum of
carbon tetrachloride-induced acute hepatitis (Sasaki et al., 1984).
Many protective effects of Ge-132 on hepatic function have been
shown (unpublished data).
Dietary Ge-132 increases PpIX in the intestine, which may protect
hepatic function. It is suggested that some physiological functions of
dietary Ge-132 are expressed through the role of induced PpIX.
Dietary Ge-132 increases heme metabolites in the intestine as bile
secretion. Pigments bilirubin, urobilinogen and PpIX are beneficial for
health; namely, bilirubin and urobilinogen act as antioxidants
(Nakamura et al., 2010) and PpIX acts as a protective compound for
hepatic function. Meanwhile, Ge-132 has an immune regulatory
function and its safety is well known, so daily intake of this compound
as a functional food will maintain health. In this study, we evaluated
the genes relating to heme metabolism in the liver. It would be
interesting to study the effect of Ge-132 on erythroid in the bone
marrow because heme is synthesized mainly in progenitor cells of
erythroid cells, not in adult RBCs. Moreover, it should be studied how
to regulate the upstream of the gene expression of heme metabolism
by Ge-132. Further studies are expected.
The administration of 0.5% Ge-132 diet to rats changed the fecal color
clearly as a result of the red pigment (Fig. 2), which had specific optical
characteristics (Fig. 3). Absorption maxima were found at 405, 505, 540
and 575 nm, and were Soret band absorption (Rimington, 1960), and
alpha, beta and gamma band absorption, respectively, suggesting that it
was a porphyrin product. In addition, with excitation at 400 nm, the
pigment had strong fluorescence emission at 600 nm. These data also
reflect the characteristics of porphyrin (shown in Fig. 3).
Analysis of the data peak of TOF-MS confirmed that the red
compound was PpIX (Fig. 3). Rimington studied the spectral
absorption coefficients of some porphyrins in the Soret band region
(
Rimington, 1960), and found that the Soret bands of protoporphyrin
dimethyl ester and protoporphyrin were at 407.5 nm and 408 nm,
respectively. These are almost identical, and we found the same
pattern with synthesized PPDM and PpIX purified from fecal extract in
this study. It is known that PpIX is a minor fecal component. The
porphyrins are synthesized mainly in bone marrow (Surinya et al.,
1
997) and PpIX is the last precursor of hemin before insertion of a
ferric ion (Beukeveld et al., 1994); therefore, PpIX is one of the most
important compounds for mammalian respiration. Heme, which is
synthesized in different organs, is used in different ways in the bone
marrow and liver. The former is used as a component of hemoglobin in
red blood cells (Surinya et al., 1997), and the latter in cytochrome
enzymes (Sato et al., 1986). The increase of PpIX in the feces by dietary
Ge-132 was confirmed by HPLC analysis (Fig. 4), and a two-fold
increase was detected. We described the secretion of bilirubin in a
recent study (Nakamura et al., 2010). Bilirubin is a metabolite of heme,
therefore shortening heme, although the hematocrit value was not
changed by Ge-132 intake; hence, supplying the heme system must
keep the hematocrit value constant, suggesting that heme synthesis
must occur at the same time to maintain homeostasis. Excess
synthesized porphyrin, increased by dietary Ge-132, is excreted in
feces as fecal protoporphyrin. We evaluated the PpIX increase with a
0
.5% Ge-132 diet, which is too high for daily intake. The content was
defined to identify the significant statistical difference. We confirmed
the PpIX increase with 0.05% Ge-132, as shown in Fig. 2; therefore, the
gene expression was studied using the 0.05% Ge-132 diet.
We confirmed that PpIX synthesis in the liver was increased. In
Fig. 5, the gene expression of Alas1 was increased, while Fech was
decreased in the liver by Ge-132 intake. In heme synthesis by two
enzymes, Alas1 and Alas2, which catalyze the first step, and Fech,
which catalyzes the last step, both are rate-limiting enzymes;
therefore, the increase of Alas1 expression and decrease of Fech
expression can upregulate the PpIX pool level in the liver. Hemin is
produced by Fech, an enzyme inserting ferric ion into PpIX. The PpIX
level is regulated according to the amount of hemoglobin and
unsaturated iron. Surplus PpIX may be excreted into the intestine
when dietary Ge-132 is ingested, and excessed PpIX in blood can
cause protoporphyria (Lecha et al., 2009); however, we could not find
any indication of protoporphyria symptoms in animals or humans
administrated dietary Ge-132 (data not shown). Increased fecal PpIX
suggests its increased secretion via bile. Rapid secretion of PpIX by
dietary Ge-132 may protect against disease from excessive PpIX.
Tephly et al. revealed that Alas was induced by various drugs in the rat
liver (Tephly et al., 1971) and they discussed the results relating to the
induction of cytochrome P-450. Cytochrome P-450 enzyme catalyzes
many drugs and endogenous compounds by oxidation or reduction,
and reduces the toxicity (phase I reaction) to the liver from harmful
compounds (Boron and Boulpaep, 2009). Ge-132 may also induce
cytochrome P-450 by Alas in detoxification. These results of a gene
expression study suggest an increasing supply of PpIX and excretion
of excessive porphyrin as PpIX and bile pigments. Fech may be
controlled by the need for cytochrome P-450. The peripheral free
ferric ion was not changed as a result of only increasing PpIX.
5
. Conclusion
We evaluated the function of a water-soluble organic germanium
compound, Ge-132, in the induction of a red pigment, which was
identified as PpIX. Ge-132 affected the regulation of the gene expression
of rate-limiting enzymes of heme metabolism. PpIX is using to treat
chronic hepatitis; therefore, the beneficial effect of Ge-132 for chronic
hepatitis is expected.
Acknowledgements
We sincerely thank Mitsuo Akiba Ph.D. for his beneficial advice and
support of this study.
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