Glyoxylate reductase/hydroxypyruvate reductase regulates the free d-aspartate level in mammalian cells.

Multiple d-amino acids are present in mammalian cells, and these compounds have distinctive physiological functions. Among the free d-amino acids identified in mammals, d-aspartate plays critical roles in the neuroendocrine and endocrine systems, as well as in the central nervous system. Mammalian cells have the molecular apparatus necessary to take up, degrade, synthesize, and release d-aspartate. In particular, d-aspartate is degraded by d-aspartate oxidase (DDO), a peroxisome-localized enzyme that catalyzes the oxidative deamination of d-aspartate to generate oxaloacetate, hydrogen peroxide, and ammonia. However, little is known about the molecular mechanisms underlying d-aspartate homeostasis in cells. In this study, we established a cell line that overexpresses cytoplasm-localized DDO; this cell line cannot survive in the presence of high concentrations of d-aspartate, presumably because high levels of toxic hydrogen peroxide are produced by metabolism of abundant d-aspartate by DDO in the cytoplasm, where hydrogen peroxide cannot be removed due to the absence of catalase. Next, we transfected these cells with a complementary DNA library derived from the human brain and screened for clones that affected d-aspartate metabolism and improved cell survival, even when the cells were challenged with high concentrations of d-aspartate. The screen identified a clone of glyoxylate reductase/hydroxypyruvate reductase (GRHPR). Moreover, the GRHPR metabolites glyoxylate and hydroxypyruvate inhibited the enzymatic activity of DDO. Furthermore, we evaluated the effects of GRHPR and peroxisome-localized DDO on d- and l-aspartate levels in cultured mammalian cells. Our findings show that GRHPR contributes to the homeostasis of these amino acids in mammalian cells.

Structure and kinetic properties of human d-aspartate oxidase, the enzyme-controlling d-aspartate levels in brain.

d-Amino acids are the "wrong" enantiomers of amino acids as they are not used in proteins synthesis but evolved in selected functions. On this side, d-aspartate (d-Asp) plays several significant roles in mammals, especially as an agonist of N-methyl-d-aspartate receptors (NMDAR), and is involved in relevant diseases, such as schizophrenia and Alzheimer's disease. In vivo modulation of d-Asp levels represents an intriguing task to cope with such pathological states. As little is known about d-Asp synthesis, the only option for modulating the levels is via degradation, which is due to the flavoenzyme d-aspartate oxidase (DASPO). Here we present the first three-dimensional structure of a DASPO enzyme (from human) which belongs to the d-amino acid oxidase family. Notably, human DASPO differs from human d-amino acid oxidase (attributed to d-serine degradation, the main coagonist of NMDAR) showing peculiar structural features (a specific active site charge distribution), oligomeric state and kinetic mechanism, and a higher FAD affinity and activity. These results provide useful insights into the structure-function relationships of human DASPO: modulating its activity represents now a feasible novel therapeutic target.

Prenatal expression of D-aspartate oxidase causes early cerebral D-aspartate depletion and influences brain morphology and cognitive functions at adulthood.

The free D-amino acid, D-aspartate, is abundant in the embryonic brain but significantly decreases after birth. Besides its intracellular occurrence, D-aspartate is also present at extracellular level and acts as an endogenous agonist for NMDA and mGlu5 receptors. These findings suggest that D-aspartate is a candidate signaling molecule involved in neural development, influencing brain morphology and behaviors at adulthood. To address this issue, we generated a knockin mouse model in which the enzyme regulating D-aspartate catabolism, D-aspartate oxidase (DDO), is expressed starting from the zygotic stage, to enable the removal of D-aspartate in prenatal and postnatal life. In line with our strategy, we found a severe depletion of cerebral D-aspartate levels (up to 95%), since the early stages of mouse prenatal life. Despite the loss of D-aspartate content, Ddo knockin mice are viable, fertile, and show normal gross brain morphology at adulthood. Interestingly, early D-aspartate depletion is associated with a selective increase in the number of parvalbumin-positive interneurons in the prefrontal cortex and also with improved memory performance in Ddo knockin mice. In conclusion, the present data indicate for the first time a biological significance of precocious D-aspartate in regulating mouse brain formation and function at adulthood.

D-Aspartate oxidase: distribution, functions, properties, and biotechnological applications.

Recently, substantial levels of acidic D-amino acids, such as D-aspartate and D-glutamate, have been identified in many organisms, from bacteria to mammals, suggesting that acidic D-amino acids have multiple physiological significances. Although acidic D-amino acids found in animals primarily originate from foodstuffs and/or bacteria, the D-aspartate-synthesizing enzyme aspartate racemase is identified in various animals. In eukaryotic organisms, acidic D-amino acids are primarily degraded by the flavoenzyme D-aspartate oxidase (DDO). DDO is found in multiple eukaryotic organisms and may play important roles in acidic D-amino acid utilization, elimination, and intracellular level regulation. Moreover, owing to its perfect enantioselectivity and stereoselectivity, DDO may be a valuable tool in several biotechnological applications, including the identification and quantification of acidic D-amino acids. In this mini-review, previous DDO reports are summarized and the potential bioengineering and biotechnological applications of DDO are discussed. Key Points ・Occurrence and distribution ofd-aspartate oxidase. ・Fundamental properties of d -aspartate oxidase of various eukaryotic organisms. ・Biotechnological applications and potential engineering ofd-aspartate oxidase.

New Evidence on the Role of D-Aspartate Metabolism in Regulating Brain and Endocrine System Physiology: From Preclinical Observations to Clinical Applications.

The endogenous amino acids serine and aspartate occur at high concentrations in free D-form in mammalian organs, including the central nervous system and endocrine glands. D-serine (D-Ser) is largely localized in the forebrain structures throughout pre and postnatal life. Pharmacologically, D-Ser plays a functional role by acting as an endogenous coagonist at N-methyl-D-aspartate receptors (NMDARs). Less is known about the role of free D-aspartate (D-Asp) in mammals. Notably, D-Asp has a specific temporal pattern of occurrence. In fact, free D-Asp is abundant during prenatal life and decreases greatly after birth in concomitance with the postnatal onset of D-Asp oxidase expression, which is the only enzyme known to control endogenous levels of this molecule. Conversely, in the endocrine system, D-Asp concentrations enhance after birth during its functional development, thereby suggesting an involvement of the amino acid in the regulation of hormone biosynthesis. The substantial binding affinity for the NMDAR glutamate site has led us to investigate the in vivo implications of D-Asp on NMDAR-mediated responses. Herein we review the physiological function of free D-Asp and of its metabolizing enzyme in regulating the functions of the brain and of the neuroendocrine system based on recent genetic and pharmacological human and animal studies.

Characterization and improvement of substrate-binding affinity of D-aspartate oxidase of the thermophilic fungus Thermomyces dupontii.

D-Aspartate oxidase (DDO) is a valuable enzyme that can be utilized in the determination of acidic D-amino acids and the optical resolution of a racemic mixture of acidic amino acids, which require its higher stability, higher catalytic activity, and higher substrate-binding affinity. In the present study, we identified DDO gene (TdDDO) of a thermophilic fungus, Thermomyces dupontii, and characterized the recombinant enzyme expressed in Escherichia coli. In addition, we generated a variant that has a higher substrate-binding affinity. The recombinant TdDDO expressed in E. coli exhibited oxidase activity toward acidic D-amino acids and a neutral D-amino acid, D-Gln, with the highest activity toward D-Glu. The Km and kcat values for D-Glu were 2.16 mM and 217 s-1, respectively. The enzyme had an optimum pH and temperature 8.0 and 60 °C, respectively, and was stable between pH 5.0 and 10.0, with a T50 of ca. 51 °C, which was much higher than that in DDOs from other origins. Enzyme stability decreased following a decrease in protein concentration, and externally added FAD could not repress the destabilization. The mutation of Phe248, potentially located in the active site of TdDDO, to Tyr residue, conserved in DDOs and D-amino acid oxidases, markedly increased substrate-binding affinity. The results showed the great potential of TdDDO and the variant for practical applications.

Rat d-aspartate oxidase is more similar to the human enzyme than the mouse enzyme.

d-Aspartate oxidase (DDO) is a degradative enzyme that is stereospecific for the acidic amino acid d-aspartate, an endogenous agonist of the N-methyl-d-aspartate (NMDA) receptor. Dysregulation of NMDA receptor-mediated neurotransmission has been implicated in the onset of various neuropsychiatric disorders including schizophrenia, as well as chronic pain. Thus, appropriate regulation of d-aspartate is believed to be important for maintaining proper neural activity in the nervous system. Accordingly, much attention has been paid to the role(s) of DDO in the metabolism of d-aspartate in vivo, and the physiological functions of DDO have been actively investigated using experimental rats and mice. However, detailed characterisation of rat DDO has not yet been performed, and little is known about species-specific differences in the properties of mammalian DDOs. In this study, the structural and enzymatic properties of purified recombinant rat, mouse and human DDOs were examined and compared. The results showed that rat DDO is more similar to human DDO than to mouse DDO. This work provides useful insight into the use of rats as an experimental model for investigating the biological significance of human DDO and/or d-aspartate. This article is part of a Special Issue entitled: d-Amino acids: biology in the mirror, edited by Dr. Loredano Pollegioni, Dr. Jean-Pierre Mothet and Dr. Molla Gianluca.

Atopic asthma after rhinovirus-induced wheezing is associated with DNA methylation change in the SMAD3 gene promoter.

Children with rhinovirus-induced severe early wheezing have an increased risk of developing asthma later in life. The exact molecular mechanisms for this association are still mostly unknown. To identify potential changes in the transcriptional and epigenetic regulation in rhinovirus-associated atopic or nonatopic asthma, we analyzed a cohort of 5-year-old children (n = 45) according to the virus etiology of the first severe wheezing episode at the mean age of 13 months and to 5-year asthma outcome. The development of atopic asthma in children with early rhinovirus-induced wheezing was associated with DNA methylation changes at several genomic sites in chromosomal regions previously linked to asthma. The strongest changes in atopic asthma were detected in the promoter region of SMAD3 gene at chr 15q22.33 and introns of DDO/METTL24 genes at 6q21. These changes were validated to be present also at the average age of 8 years.

Age-Related Changes in D-Aspartate Oxidase Promoter Methylation Control Extracellular D-Aspartate Levels and Prevent Precocious Cell Death during Brain Aging.

The endogenous NMDA receptor (NMDAR) agonist D-aspartate occurs transiently in the mammalian brain because it is abundant during embryonic and perinatal phases before drastically decreasing during adulthood. It is well established that postnatal reduction of cerebral D-aspartate levels is due to the concomitant onset of D-aspartate oxidase (DDO) activity, a flavoenzyme that selectively degrades bicarboxylic D-amino acids. In the present work, we show that d-aspartate content in the mouse brain drastically decreases after birth, whereas Ddo mRNA levels concomitantly increase. Interestingly, postnatal Ddo gene expression is paralleled by progressive demethylation within its putative promoter region. Consistent with an epigenetic control on Ddo expression, treatment with the DNA-demethylating agent, azacitidine, causes increased mRNA levels in embryonic cortical neurons. To indirectly evaluate the effect of a putative persistent Ddo gene hypermethylation in the brain, we used Ddo knock-out mice (Ddo(-/-)), which show constitutively suppressed Ddo expression. In these mice, we found for the first time substantially increased extracellular content of d-aspartate in the brain. In line with detrimental effects produced by NMDAR overstimulation, persistent elevation of D-aspartate levels in Ddo(-/-) brains is associated with appearance of dystrophic microglia, precocious caspase-3 activation, and cell death in cortical pyramidal neurons and dopaminergic neurons of the substantia nigra pars compacta. This evidence, along with the early accumulation of lipufuscin granules in Ddo(-/-) brains, highlights an unexpected importance of Ddo demethylation in preventing neurodegenerative processes produced by nonphysiological extracellular levels of free D-aspartate.

Structure-function relationships in human d-aspartate oxidase: characterisation of variants corresponding to known single nucleotide polymorphisms.

d-Aspartate oxidase (DDO) is a degradative enzyme that is stereospecific for the acidic amino acid d-aspartate, an endogenous agonist of the N-methyl-d-aspartate (NMDA) receptor. Dysregulation of NMDA receptor-mediated neurotransmission has been implicated in the onset of various neuropsychiatric disorders including schizophrenia and in chronic pain. Thus, appropriate regulation of the amount of d-aspartate is believed to be important for maintaining proper neural activity in the nervous system. Herein, the effects of the non-synonymous single nucleotide polymorphisms (SNPs) R216Q and S308N on several properties of human DDO were examined. Analysis of the purified recombinant enzyme showed that the R216Q and S308N substitutions reduce enzyme activity towards acidic d-amino acids, decrease the binding affinity for the coenzyme flavin adenine dinucleotide and decrease the temperature stability. Consistent with these findings, further experiments using cultured mammalian cells revealed elevated d-aspartate in cultures of R216Q and S308N cells compared with cells expressing wild-type DDO. Furthermore, accumulation of several amino acids other than d-aspartate also differed between these cultures. Thus, expression of DDO genes carrying the R216Q or S308N SNP substitutions may increase the d-aspartate content in humans and alter homeostasis of several other amino acids. This work may aid in understanding the correlation between DDO activity and the risk of onset of NMDA receptor-related diseases.

ampliMethProfiler: a pipeline for the analysis of CpG methylation profiles of targeted deep bisulfite sequenced amplicons.

BACKGROUND: CpG sites in an individual molecule may exist in a binary state (methylated or unmethylated) and each individual DNA molecule, containing a certain number of CpGs, is a combination of these states defining an epihaplotype. Classic quantification based approaches to study DNA methylation are intrinsically unable to fully represent the complexity of the underlying methylation substrate. Epihaplotype based approaches, on the other hand, allow methylation profiles of cell populations to be studied at the single molecule level. For such investigations, next-generation sequencing techniques can be used, both for quantitative and for epihaplotype analysis. Currently available tools for methylation analysis lack output formats that explicitly report CpG methylation profiles at the single molecule level and that have suited statistical tools for their interpretation. RESULTS: Here we present ampliMethProfiler, a python-based pipeline for the extraction and statistical epihaplotype analysis of amplicons from targeted deep bisulfite sequencing of multiple DNA regions. CONCLUSIONS: ampliMethProfiler tool provides an easy and user friendly way to extract and analyze the epihaplotype composition of reads from targeted bisulfite sequencing experiments. ampliMethProfiler is written in python language and requires a local installation of BLAST and (optionally) QIIME tools. It can be run on Linux and OS X platforms. The software is open source and freely available at http://amplimethprofiler.sourceforge.net .

Biosynthesis of D-aspartate in mammals: the rat and human homologs of mouse aspartate racemase are not responsible for the biosynthesis of D-aspartate.

D-Aspartate (D-Asp) has important physiological functions, and recent studies have shown that substantial amounts of free D-Asp are present in a wide variety of mammalian tissues and cells. Biosynthesis of D-Asp has been observed in several cultured rat cell lines, and a murine gene (glutamate-oxaloacetate transaminase 1-like 1, Got1l1) that encodes Asp racemase, a synthetic enzyme that produces D-Asp from L-Asp, was proposed recently. The product of this gene is homologous to mammalian glutamate-oxaloacetate transaminase (GOT). Here, we tested the hypothesis that rat and human homologs of mouse GOT1L1 are involved in Asp synthesis. The following two approaches were applied, since the numbers of attempts were unsuccessful to prepare soluble GOT1L1 recombinant proteins. First, the relationship between the D-Asp content and the expression levels of the mRNAs encoding GOT1L1 and D-Asp oxidase, a primary degradative enzyme of D-Asp, was examined in several rat and human cell lines. Second, the effect of knockdown of the Got1l1 gene on D-Asp biosynthesis during culture of the cells was determined. The results presented here suggest that the rat and human homologs of mouse GOT1L1 are not involved in D-Asp biosynthesis. Therefore, D-Asp biosynthetic pathway in mammals is still an urgent issue to be resolved.

Characterization of the enzymatic and structural properties of human D-aspartate oxidase and comparison with those of the rat and mouse enzymes.

D-Aspartate (D-Asp), a free D-amino acid found in mammals, plays crucial roles in the neuroendocrine, endocrine, and central nervous systems. Recent studies have implicated D-Asp in the pathophysiology of infertility and N-methyl-D-Asp receptor-related diseases. D-Asp oxidase (DDO), a degradative enzyme that is stereospecific for acidic D-amino acids, is the sole catabolic enzyme acting on D-Asp in mammals. Human DDO is considered an attractive therapeutic target, and DDO inhibitors may be potential lead compounds for the development of new drugs against the aforementioned diseases. However, human DDO has not been characterized in detail and, although preclinical studies using experimental rodents are prerequisites for evaluating the in vivo effects of potential inhibitors, the existence of species-specific differences in the properties of human and rodent DDOs is still unclear. Here, the enzymatic activity and characteristics of purified recombinant human DDO were analyzed in detail. The kinetic and inhibitor-binding properties of this enzyme were also compared with those of purified recombinant rat and mouse DDOs. In addition, structural models of human, rat, and mouse DDOs were generated and compared. It was found that the differences among these DDO proteins occur in regions that appear involved in migration of the substrate/product in and out of the active site. In summary, detailed characterization of human DDO was performed and provides useful insights into the use of rats and mice as experimental models for evaluating the in vivo effects of DDO inhibitors.

Current knowledge of D-aspartate in glandular tissues.

Free D-aspartate (D-Asp) occurs in substantial amounts in glandular tissues. This paper reviews the existing work on D-Asp in vertebrate exocrine and endocrine glands, with emphasis on functional roles. Endogenous D-Asp was detected in salivary glands. High D-Asp levels in the parotid gland during development suggest an involvement of the amino acid in the regulation of early developmental phases and/or differentiation processes. D-Asp has a prominent role in the Harderian gland, where it elicits exocrine secretion through activation of the ERK1/2 pathway. Interestingly, the increase in NOS activity associated with D-Asp administration in the Harderian gland suggests a potential capability of D-Asp to induce vasodilatation. In mammals, an increase in local concentrations of D-Asp facilitates the secretion of anterior pituitary hormones, i.e., PRL, LH and GH, whereas it inhibits the secretion of POMC/α-MSH from the intermediate pituitary and of oxytocin from the posterior pituitary. D-Asp also acts as a negative regulator for melatonin synthesis in the pineal gland. Further, D-Asp can stereo-specifically modulate the production of sex steroids, thus taking part in the endocrine control of reproductive activity. Although D-Asp receptors remain to be characterized, gene expression of NR1 and NR2 subunits of NMDAr responds to D-Asp in the testis.

On the regulation of human D-aspartate oxidase.

The human flavoenzyme D-aspartate oxidase (hDASPO) controls the level of D-aspartate in the brain, a molecule acting as an agonist of NMDA receptors and modulator of AMPA and mGlu5 receptors. hDASPO-induced D-aspartate degradation prevents age-dependent deterioration of brain functions and is related to psychiatric disorders such as schizophrenia and autism. Notwithstanding this crucial role, less is known about hDASPO regulation. Here, we report that hDASPO is nitrosylated in vitro, while no evidence of sulfhydration and phosphorylation is apparent: nitrosylation affects the activity of the human flavoenzyme to a limited extent. Furthermore, hDASPO interacts with the primate-specific protein pLG72 (a well-known negative chaperone of D-amino acid oxidase, the enzyme deputed to D-serine degradation in the human brain), yielding a ~114 kDa complex, with a micromolar dissociation constant, promoting the flavoenzyme inactivation. At the cellular level, pLG72 and hDASPO generate a cytosolic complex: the expression of pLG72 negatively affects the hDASPO level by reducing its half-life. We propose that pLG72 binding may represent a protective mechanism aimed at avoiding cytotoxicity due to H2 O2 produced by the hDASPO enzymatic degradation of D-aspartate, especially before the final targeting to peroxisomes.

3-Chloropropane-1,2-diol exposure adversely influenced the bio-accessibility signatures of digested infant foods by suppressing the destabilization of α-lactalbumin and d-aspartate oxidase in a dose-dependent manner.

The potential mechanisms about the health risks of endogenous 3-MCPD remain elusive. Here, we researched the influences of 3-MCPD on the metabolic landscape of digested goat infant formulas via integrative UHPLC-Q-Orbitrap HRMS-MS/MS-based peptidomics and metabolomics (%RSDs ≤ 7.35 %, LOQ 2.99-58.77 µg kg-1). Digested goat infant formulas under 3-MCPD-interference caused metabolic perturbation by down-regulating levels of peptides VGINYWLAHK (5.98-0.72 mg kg-1) and HLMCLSWQ (3.25-0.72 mg kg-1) pertained to health-promoting bioactive components, and accelerated the down-regulation of non-essential amino acids (AAs, l-tyrosine 0.88-0.39 mg kg-1, glutamic acid 8.83-0.88 µg kg-1, and d-aspartic acid 2.93-0.43 µg kg-1), semi-essential AA (l-arginine 13.06-8.12 µg kg-1) and essential AAs (l-phenylalanine 0.49-0.05 mg kg-1) that provide nutritional value. Peptidomics and metabolomics interactions elucidated that 3-MCPD altered the stability of α-lactalbumin and d-aspartate oxidase in a dose-dependent manner, and affected the flavor perception of goat infant formulas, leading to a decline of nutritional value of goat infant formulas.

New insights on the role of free D-aspartate in the mammalian brain.

Free D-aspartate (D-Asp) occurs in substantial amounts in the brain at the embryonic phase and in the first few postnatal days, and strongly decreases in adulthood. Temporal reduction of D-Asp levels depends on the postnatal onset of D-aspartate oxidase (DDO) activity, the only enzyme able to selectively degrade this D-amino acid. Several results indicate that D-Asp binds and activates N-methyl-D-aspartate receptors (NMDARs). Accordingly, recent studies have demonstrated that deregulated, higher levels of D-Asp, in knockout mice for Ddo gene and in D-Asp-treated mice, modulate hippocampal NMDAR-dependent long-term potentiation (LTP) and spatial memory. Moreover, similarly to D-serine, administration of D-Asp to old mice is able to rescue the physiological age-related decay of hippocampal LTP. In agreement with a neuromodulatory action of D-Asp on NMDARs, increased levels of this D-amino acid completely suppress long-term depression at corticostriatal synapses and attenuate the prepulse inhibition deficits produced in mice by the psychotomimetic drugs, amphetamine and MK-801. Based on the evidence which points to the ability of D-Asp to act as an endogenous agonist on NMDARs and considering the abundance of D-Asp during prenatal and early life, future studies will be crucial to address the effect of this molecule in the developmental processes of the brain controlled by the activation of NMDARs.

D-Aspartate acts as a signaling molecule in nervous and neuroendocrine systems.

D-Aspartate (D-Asp) is an endogenous amino acid in the central nervous and reproductive systems of vertebrates and invertebrates. High concentrations of D-Asp are found in distinct anatomical locations, suggesting that it has specific physiological roles in animals. Many of the characteristics of D-Asp have been documented, including its tissue and cellular distribution, formation and degradation, as well as the responses elicited by D-Asp application. D-Asp performs important roles related to nervous system development and hormone regulation; in addition, it appears to act as a cell-to-cell signaling molecule. Recent studies have shown that D-Asp fulfills many, if not all, of the definitions of a classical neurotransmitter-that the molecule's biosynthesis, degradation, uptake, and release take place within the presynaptic neuron, and that it triggers a response in the postsynaptic neuron after its release. Accumulating evidence suggests that these criteria are met by a heterogeneous distribution of enzymes for D-Asp's biosynthesis and degradation, an appropriate uptake mechanism, localization within synaptic vesicles, and a postsynaptic response via an ionotropic receptor. Although D-Asp receptors remain to be characterized, the postsynaptic response of D-Asp has been studied and several L-glutamate receptors are known to respond to D-Asp. In this review, we discuss the current status of research on D-Asp in neuronal and neuroendocrine systems, and highlight results that support D-Asp's role as a signaling molecule.

Crystallization and preliminary crystallographic analysis of D-aspartate oxidase from porcine kidney.

D-Aspartate oxidase (DDO) from porcine kidney was crystallized by the sitting-drop vapour-diffusion method using PEG 8000 as a precipitant. The crystal belonged to space group P2(1), with unit-cell parameters a = 79.38, b = 144.0, c = 80.46 Å, ß = 101.1°, and diffracted to 1.80 Å resolution. Molecular-replacement trials using the structure of human D-amino-acid oxidase, which is 42% identical in sequence to DDO, as a search model provided a satisfactory solution.

Enzymatic assay for D-aspartic acid using D-aspartate oxidase and oxaloacetate decarboxylase.

An enzymatic assay system for D-Asp was established using D-aspartate oxidase and oxaloacetate decarboxylase. In this system, D-Asp is converted to pyruvate, which is determined fluorometrically with 1,2-diamino-4,5-methylenedioxybenzene. This method makes possible D-Asp measurement at the micromolar level. The D-Asp contents of an edible brown alga, Hijika fusiforme, a lactic acid bacteria beverage, and pig testis were determined by the method.