Search for the Majorana Nature of Neutrinos in the Inverted Mass Ordering Region with KamLAND-Zen.

The KamLAND-Zen experiment has provided stringent constraints on the neutrinoless double-beta (0νßß) decay half-life in ^{136}Xe using a xenon-loaded liquid scintillator. We report an improved search using an upgraded detector with almost double the amount of xenon and an ultralow radioactivity container, corresponding to an exposure of 970 kg yr of ^{136}Xe. These new data provide valuable insight into backgrounds, especially from cosmic muon spallation of xenon, and have required the use of novel background rejection techniques. We obtain a lower limit for the 0νßß decay half-life of T_{1/2}^{0ν}>2.3×10^{26} yr at 90% C.L., corresponding to upper limits on the effective Majorana neutrino mass of 36-156 meV using commonly adopted nuclear matrix element calculations.

Colchicine protects against cartilage degeneration by inhibiting MMP13 expression via PLC-γ1 phosphorylation.

OBJECTIVE: Low molecular weight compounds that reduce the expression of MMP13 at the mRNA level might serve as disease-modifying osteoarthritis (OA) drugs (DMOADs). The objective of this study was to identify a candidate DMOAD that targets MMP13 expression. DESIGN: High-throughput screening was performed to identify compounds that suppress inflammatory cytokine-induced MMP13 expression. Ingenuity pathway analysis (IPA) using isobaric tags for relative and absolute quantification (iTRAQ)-based proteomic analysis was conducted to identify signaling pathways related to cytokines. MMP13 expression in chondrocytes was evaluated through RT-qPCR and western blotting analyses. Additionally, 10-week-old mice were subjected to destabilization of the medial meniscus (DMM) surgery to induce OA and were sacrificed 12 weeks post-surgery for pathological examination. OA was evaluated using the OARSI scoring system. RESULTS: Colchicine was identified as a DMOAD candidate as it inhibited inflammatory cytokine-induced MMP13 expression in vitro, and the colchicine-administered mice with DMM presented significantly lower OARSI scores (adjusted P: 0.0242, mean difference: 1.6, 95% confidence interval (CI) of difference: 0.1651-3.035) and significantly lower synovial membrane inflammation scores (adjusted P: 0.0243, mean difference: 0.6, 95% CI of difference: 0.06158-1.138) than mice with DMM. IPA further revealed that components of the Rho signaling pathways are regulated by cytokines and colchicine. IL-1ß and TNF-α activate RAC1 and SRC signals, respectively, leading to the phosphorylation of PLC-γ1 and synergistic induction of MMP13 expression. Most notably, colchicine abrogates inflammatory cytokine-induced phosphorylation of PLC-γ1, leading to the induction of MMP13 expression. CONCLUSIONS: Colchicine is a potential DMOAD candidate that inhibits MMP13 expression and consequent cartilage degradation by disrupting the SRC/RAC1-phospho-PLCγ1-Ca2+ signaling pathway.

Change in sense of food safety associated with social connectedness and information sources-A 10-year longitudinal survey before and after the Fukushima nuclear power plant accident.

OBJECTIVES: This study analyzes the change in the sense of food safety over time, from 2 years before to 8 years after the Fukushima accident in 2011, and its association with social connectedness using cohort data, taking into account regional differences in Fukushima Prefecture. STUDY DESIGN: Repeated cross-sectional data from Fukushima Prefectural Government from 2009 to 2018 were used. METHODS: We randomly selected 1300 people every fiscal year (FY). The survey gathered data on age, gender, occupation, residential region, and the explanatory variables 'sense of social connectedness' and 'recovery-related information source' (information source). The prefecture was divided into three regions for the survey-Hamadori region, where the nuclear power plant is located, Nakadori region, where the air dose rate after the earthquake was high, and in Aizu, far from the nuclear power station but has suffered from harmful rumors. RESULTS: Focusing on FY 2014, when the sense of safety first showed recovery, we performed a binominal logistic regression analysis with 'sense of safety' as the outcome and 'sense of social connectedness' and 'information source' as the explanatory variables. The sense of safety significantly decreased in all regions in 2011 relative to earlier years. The sense of food safety decreased markedly in Hamadori and Nakadori but started to improve 3 years after the earthquake and reached the pre-earthquake level in 2018. The effect sizes were larger in the Hamadori region and in Nakadori than in Aizu. In FY 2014, multivariate analysis found that a sense of food safety was significantly positively associated with a sense of social connectedness, whereas the use of information from newspapers and TV and word of mouth was negatively associated. CONCLUSION: Although the recovery of a sense of food safety may take some time, a focus on social connectedness during recovery and scrutiny of information sources may facilitate recovery. Health communication has an important role when the provider sends information intelligibly and the recipient can distinguish good news from bad and link it to self-determination. It is necessary to improve literacy.

Predictive performance of a genetic risk score using 11 susceptibility alleles for the incidence of Type 2 diabetes in a general Japanese population: a nested case-control study.

AIMS: To assess the predictive ability of a genetic risk score for the incidence of Type 2 diabetes in a general Japanese population. METHODS: This prospective case-control study, nested within a Japan Public Health Centre-based prospective study, included 466 participants with incident Type 2 diabetes over a 5-year period (cases) and 1361 control participants, as well as 1463 participants with existing diabetes and 1463 control participants. Eleven susceptibility single nucleotide polymorphisms, identified through genome-wide association studies and replicated in Japanese populations, were analysed. RESULTS: Most single nucleotide polymorphism loci showed directionally consistent associations with diabetes. From the combined samples, one single nucleotide polymorphism (rs2206734 at CDKAL1) reached a genome-wide significance level (odds ratio 1.28, 95% CI 1.18-1.40; P = 1.8 × 10-8 ). Three single nucleotide polymorphisms (rs2206734 in CDKAL1, rs2383208 in CDKN2A/B, and rs2237892 in KCNQ1) were nominally significantly associated with incident diabetes. Compared with the lowest quintile of the total number of risk alleles, the highest quintile had a higher odds of incident diabetes (odds ratio 2.34, 95% CI 1.59-3.46) after adjusting for conventional risk factors such as age, sex and BMI. The addition to the conventional risk factor-based model of a genetic risk score using the 11 single nucleotide polymorphisms significantly improved predictive performance; the c-statistic increased by 0.021, net reclassification improved by 6.2%, and integrated discrimination improved by 0.003. CONCLUSIONS: Our prospective findings suggest that the addition of a genetic risk score may provide modest but significant incremental predictive performance beyond that of the conventional risk factor-based model without biochemical markers.

A cluster randomized trial on the effect of a multifaceted intervention improved the technical quality of diabetes care by primary care physicians: The Japan Diabetes Outcome Intervention Trial-2 (J-DOIT2).

AIMS: To evaluate the effect of multifaceted interventions using the Achievable Benchmark of Care (ABC) method for improving the technical quality of diabetes care in primary care settings. METHODS: We conducted a 1-year cluster randomized controlled trial in 22 regions divided into an intervention group (IG) or control group (CG). Physicians in the IG received a monthly report of their care quality, with the top 10% quality of diabetes care scores for all physicians being the achievable benchmark. The change in quality-of-care scores between the IG and CG during follow-up was analysed using a generalized linear model considering clustering. RESULTS: A total of 2199 patients were included. Their mean (sd) age was 56.5 ± 5.9 years and the mean (sd) HbA1c level was 56.4 ± 13.3 mmol/mol (7.4 ± 1.2%). The quality-of-care score in the CG changed from 50.2%-point at baseline to 51%-point at 12 months, whereas the IG score changed from 49.9%-point to 69.6%-point, with statistically significant differences between the two groups during follow-up [the effect of intervention was 19.0%-point (95% confidence interval 16.7%- to 21.3%-point; P < 0.001)]. CONCLUSIONS: Multifaceted intervention, measuring quality-of-care indicators and providing feedback regarding the quality of diabetes care to physicians with ABC, was effective for improving the technical quality of care in patients with Type 2 diabetes in primary care settings. ( TRIAL REGISTRATION: umin.ac.jp/ctr as UMIN000002186).

Characterization of ion-irradiated poly-L-lactic acid using nano-cutting.

Effects on the mechanical strength of poly-L-lactic acid (PLLA) upon irradiation with 150 keV He(+) ion were studied. Changes in the irradiated surface were investigated using a surface texture and contour measuring instrument and an atomic force microscope. Observations made with the atomic force microscope revealed that the irradiated surface subsided significantly as the fluence increased. In order to investigate the dependence on fluence of the depth of the Bragg peak for the ion implantation, the cutting strength, Σ, was analysed [F. Saito, I. Nishiyama and T. Hyodo, Mater. Lett., 2012, 66, 144-146]; this value is an indicator of the strength of a material against cutting, and is obtained from the cutting resistance. The averaged ion projected range increased from about 1.1 µm for a fluence of 1 × 10(15) He(+)/cm(2) to about 4 µm for a fluence of 1 × 10(16) He(+)/cm(2). The density of the region following irradiation was estimated using a combination of cutting resistance measurements and positron annihilation γ ray Doppler broadening measurements made with an energy-variable positron beam. The density decreased from the value of 1.27 g cm(-3) to about 0.6 g cm(-3) after irradiation with a fluence of 3 × 10(15) He(+)/cm(2). By considering the decrease in the density and the subsidence of the surface, it is concluded that only 30% of the original weight remained in the irradiated region after exposure to the He(+) ions. Anisotropic change in the cutting resistance suggests that mechanical strength in the direction normal to the surface increased while that in the lateral direction decreased.

Time course of the antibody response in humans compared with rats experimentally infected with hepatic alveolar echinococcosis.

Human alveolar echinococcosis (AE) is caused by the accidental ingestion of Echinococcus multilocularis eggs. Early detection is essential as surgical resection is the only treatment for a complete cure. However, details are unclear about changes in the antibody response during the initial stages of infection, yet such information is useful for early serodiagnosis. Therefore, a long-term investigation was performed into the time course of the antibody response before 'positive' detection. Patient sera were used for enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB) analysis using crude antigens extracted from E. multilocularis protoscoleces. Rats were experimentally infected with AE and similarly analysed by ELISA and WB. Among the markers for diagnoses, the 18 kDa band (main marker) appeared last in the preoperative stages and disappeared first after operation in a WB test. Although the 18 kDa antigen can be useful as a marker for AE diagnosis, it cannot contribute to the detection of some patients before the 18 kDa band appearance. To avoid misdiagnosis, different diagnostic antigens such as the 26-28 and 7-8 kDa bands should also be considered. These bands tend to appear earlier than the 18 kDa band and thus offer the potential for early detection of AE. We first observed changes in the antibody response in a relatively early stage after infection in human AE cases. Notably, changes in the antibody response of two intermediate species were similar. These findings provide valuable information for the early detection of human AE cases in the future.

Time-varying living arrangements and suicide death in the general population sample: 14-year causal survival analysis via pooled logistic regression.

AIMS: While past research suggested that living arrangements are associated with suicide death, no study has examined the impact of sustained living arrangements and the change in living arrangements. Also, previous survival analysis studies only reported a single hazard ratio (HR), whereas the actual HR may change over time. We aimed to address these limitations using causal inference approaches. METHODS: Multi-point data from a general Japanese population sample were used. Participants reported their living arrangements twice within a 5-year time interval. After that, suicide death, non-suicide death and all-cause mortality were evaluated over 14 years. We used inverse probability weighted pooled logistic regression and cumulative incidence curve, evaluating the association of time-varying living arrangements with suicide death. We also studied non-suicide death and all-cause mortality to contextualize the association. Missing data for covariates were handled using random forest imputation. RESULTS: A total of 86,749 participants were analysed, with a mean age (standard deviation) of 51.7 (7.90) at baseline. Of these, 306 died by suicide during the 14-year follow-up. Persistently living alone was associated with an increased risk of suicide death (risk difference [RD]: 1.1%, 95% confidence interval [CI]: 0.3-2.5%; risk ratio [RR]: 4.00, 95% CI: 1.83-7.41), non-suicide death (RD: 7.8%, 95% CI: 5.2-10.5%; RR: 1.56, 95% CI: 1.38-1.74) and all-cause mortality (RD: 8.7%, 95% CI: 6.2-11.3%; RR: 1.60, 95% CI: 1.42-1.79) at the end of the follow-up. The cumulative incidence curve showed that these associations were consistent throughout the follow-up. Across all types of mortality, the increased risk was smaller for those who started to live with someone and those who transitioned to living alone. The results remained robust in sensitivity analyses. CONCLUSIONS: Individuals who persistently live alone have an increased risk of suicide death as well as non-suicide death and all-cause mortality, whereas this impact is weaker for those who change their living arrangements.

Barriers and motivators to blood donation among university students in Japan: development of a measurement tool.

BACKGROUND AND OBJECTIVES: Despite growing demand for transfusion, the number of voluntary young blood donors has steadily decreased over recent years in Japan. This study aimed to develop an easy-to-use survey tool to assess barriers and motivators to blood donation among Japanese university students. MATERIALS AND METHODS: We conducted cross-sectional studies at two universities in Fukushima Prefecture, Japan, in December 2011 (Stage 1) and February 2012 (Stage 2) using self-administered questionnaires. A short list of motivators and barriers to blood donation was developed from the open-ended questions asked of 50 students in Stage 1. In the Stage 2, we asked 105 students how important these items were when they decided whether or not to donate blood. Items showing a significant difference between donors and non-donors were kept in the final list. RESULTS: Overall, 56% of the 100 participants analysed in Stage 2 were men, and ages ranged from 19 to 24 with a median of 20 years. Comparison of motivators and barriers between donors and non-donors revealed that only barrier item 8 ('Frightened by blood donation') showed a significant difference (P = 0·0006) in an expected direction and with a consistency between two universities. CONCLUSIONS: This study identified fear as being the most significant barrier to blood donation among Japanese university students, which could be used as a single convenient indicator to assess their readiness to donate. More academic and clinical efforts are needed to understand and address students' fear towards blood donation in order to increase the donor pool in Japan.

Mapping a gene for combined hyperlipidaemia in a mutant mouse strain.

Familial combined hyperlipidaemia (FCHL) is a common, multifactorial disorder associated with elevated levels of plasma triglyceride, cholesterol, or both. A characteristic feature is increased secretion of very low density lipoproteins (VLDL) and apolipoprotein B (apoB). Although FCHL is the most common cause of premature coronary artery disease (CAD), accounting for over 10% of cases, its aetiology remains largely unknown. One powerful approach to the dissection of complex genetic traits involves the use of animal models. We have identified a mouse strain, HcB-19/Dem (HcB-19), which exhibits hypertriglyceridaemia, hypercholesterolaemia and elevated levels of plasma apoB. Like FCHL patients, HcB-19 mice also exhibit increased secretion of triglyceride-rich lipoproteins, and their hyperlipidaemia becomes progressively more severe with age. It is likely that the hyperlipidaemia results from a mutation of a novel gene that arose during development of strain HcB-19. We mapped the hyperlipidaemia gene (Hyplip1) to the distal portion of mouse chromosome 3. This region is syntenic to human chromosome 1q21-q23, which has recently been shown to harbour a gene associated with FCHL in families from a Finnish isolate.

Ethical and societal aspects of radiological protection for offspring and next generations.

PURPOSE: Over the last decade or so, ethical and societal aspects of radiological protection have received increasing attention. This is also reflected in the publications of the International Commission on Radiological Protection (ICRP). The current paper aims at identifying relevant ethical and societal topics which should receive attention in the context of radiological protection for offspring and next generations. MATERIALS AND METHODS: We present a non-comprehensive review of the subject, based on presentation made at an ICRP workshop in Budapest in 2022. We first discuss the ethical values promoted by ICRP, and the application of these values in cases of (potential) pre-conceptual and prenatal radiation exposures. We then consider experience gained after the Fukushima accident indicating particular societal concerns about the health effects of such exposures. RESULTS AND CONCLUSIONS: Beneficence/non-maleficence, prudence, justice and dignity, the "core values" of the system of radiological protection have special roles to play when heritable and/or in utero effects are to be considered. Prudence, in particular, must be taken account of in view of the fact that solid scientific data in humans are largely lacking in this area, and it is necessary to rely on insights from animal experiments as well as theoretical considerations. As regards societal considerations, the perception of risk among (potentially) affected populations needs to be taken seriously. Accountability, transparency, and inclusivity, the "procedural values" promoted by ICRP for the practical implementation of the system of radiological protection play a central role in overcoming skepticism and creating trust. Stakeholder involvement should emphasize cooperation and dialogue, which allows for the joint evaluation of an exposure situation by experts and affected people.

Comparison of the effects of two commercially available prescription diet regimens on the fecal microbiomes of client-owned healthy pet dogs.

In the present study, we used next-generation sequencing to investigate the impacts of two commercially available prescription diet regimens on the fecal microbiomes of eleven client-owned healthy pet dogs. We tested an anallergenic diet on 6 dogs and a low-fat diet on 5 dogs. Before starting the study, each dog was fed a different commercial diet over 5 weeks. After collecting pre-diet fecal samples, the anallergenic or low-fat diet was administered for 5 weeks. We then collected fecal samples and compared the pre- and post-diet fecal microbiomes. In the dogs on the anallergenic diet, we found significantly decreased proportions of Bacteroides, Ruminococcaceae, and Fusobacteriaceae, belonging to the phyla Bacteroidetes, Firmicutes, and Fusobacteria, respectively. The proportion of the genus Streptococcus belonging to the phylum Firmicutes was significantly increased upon administering the anallergenic diet. In the dogs on the low-fat diet, although the phyla Actinobacteria and Bacteroidetes tended to increase (p=0.116) and decrease (p=0.147) relative to the pre-diet levels, respectively, there were no significant differences in the proportions of any phylum between the pre- and post-diet fecal microbiomes. The anallergenic diet induced a significantly lower diversity index value than that found in the pre-diet period. Principal coordinate analysis based on unweighted UniFrac distance matrices revealed separation between the pre- and post-diet microbiomes in the dogs on the anallergenic diet. These results suggest that, even in pet dogs kept indoors in different living environments, unification of the diet induces apparent changes in the fecal microbiome.

Tongue Muscle for the Analysis of Head Muscle Regeneration Dynamics.

Tongue muscle damage impairs speaking and eating, thereby degrading overall health and quality of life. Skeletal muscles of the body are diverse in embryonic origin, anatomic location, and gene expression profiles. Responses to disease, atrophy, aging, or drugs vary among different muscles. Currently, most muscle studies are focused on limb muscles and the tongue is neglected. The regenerative ability of tongue muscle remains unknown, and thus there is need for tongue muscle research models. Here, we present a comprehensive characterization of the spatiotemporal dynamics in a mouse model of tongue muscle regeneration and establish a method for the isolation of primary tongue-derived satellite cells. We compare and contrast our observations with the tibialis anterior (TA) limb muscle. Acute injury was induced by intramuscular injection of cardiotoxin, a cytolytic agent, and examined at multiple timepoints. Initially, necrotic myofibers with fragmented sarcoplasm became infiltrated with inflammatory cells. Concomitantly, satellite cells expanded rapidly. Seven days postinjury, regenerated myofibers with centralized nuclei appeared. Full regeneration, as well as an absence of fibrosis, was evident 21 d postinjury. Primary tongue-derived satellite cells were isolated by enzymatic separation of tongue epithelium from mesenchyme followed by magnetic-activated cell sorting. We observed that tongue displays an efficient regenerative response similar to TA but with slightly faster kinetics. In vitro, tongue-derived satellite cells differentiated robustly into mature myotubes with spontaneous contractile behavior and myogenic marker expression. Comparison of gene expression signatures between tongue and TA-derived satellite cells revealed differences in the expression of positional-identity genes, including the HOX family. In conclusion, we have established a model for tongue regeneration useful for investigations of orofacial muscle biology. Furthermore, we showed that tongue is a viable source of satellite cells with unique properties and inherited positional memory.

Formation of a fluvial non-parasitic population of Lethenteron camtschaticum as the first step in petromyzontid speciation.

To elucidate the petromyzontid speciation process, the genetic independence of the fluvial non-parasitic populations within the anadromous parasitic Lethenteron camtschaticum was estimated by using polymorphic microsatellite loci. Abundant gene flow was revealed in multitemporal scales between potentially sympatric populations, suggesting ongoing gene flow resulting from imperfect size-assortative mating between them and plastic determination of life histories. On the contrary, landlocked fluvial non-parasitic populations in the upper region of dams were genetically divergent from anadromous parasitic populations. The temporal heterogeneity of gene flow, i.e. contemporary little gene flow but significant gene flow over the long-term between the landlocked fluvial non-parasitic and anadromous parasitic populations was elucidated. In addition, the divergence time of isolation of the landlocked populations from the ancestral anadromous parasitic population was estimated to have occurred 17.9-428.2 years ago, which includes the construction times of an initial dam c. 90 years ago. These instances indicate that the landlocked populations should have very recently been established, and subsequent accumulation of divergence and development of reproductive isolation are predicted. The present landlocked fluvial non-parasitic populations should be analogous to the founder populations in terms of petromyzontid speciation. The data also strongly support the hypothesis of multitemporal and multispatial speciation in the petromyzontid stem-satellite species complex.

Feedback assessment from the audience as part of health literacy training for health professionals: a case from Fukushima after the nuclear accident.

Following the accident at Fukushima Daiichi nuclear power plant in 2011, a series of health literacy training workshops for local health workers was developed and implemented. This study aimed to analyse who among the intended audience gained the greatest benefit from the training. Nine health workers attended a workshop consisting of classroom training and a follow-up feedback assessment of materials they created in the workshop. The materials were assessed by a total of 131 intended readers. Using Sakai's tool, those items asking readers to rate the accessibility of written information were used to compare the materials before and after revision. The total score for Sakai's measure showed a significant improvement, and the elderly and those without regular doctor visits were more likely to notice improvements after revision. Such health literacy training could serve as a model programme to prevent inequity in access to health information in the face of a regional health crisis.

Development of an application tool to support returnees in Fukushima.

To promote radiation protection and health promotion among returning residents (returnees) in coastal areas of Fukushima, eHealth principles were used to develop a new application tool (app) that can record radiation exposure and health status while providing comprehensive support to returnees. Intended users are returnees and health and welfare workers. After assessing their needs, a flowchart and prototype for operational logic were created using commercially available software tools. Professional developers will focus on improving the user interface and ensuring data security. The finished app will be compatible with mobile telephones and tablets. Utility and ease of use are paramount to serve returnees of all ages effectively.

Peripartum changes in serum activities of three major alkaline phosphatase isoenzymes in Holstein dairy cows.

This study investigated changes in serum levels of hepatic, bone, and intestinal alkaline phosphatase (ALP) isoenzymes (ALP2, ALP3, and ALP5, respectively) in Holstein cows around parturition. Tartrate-resistant acid phosphatase 5b (TRAP5b) activity and calcium (Ca) concen-trations were also measured. We analyzed blood samples from 11 late-pregnant heifers (primipa-rous group) and 13 multiparous (2-4 lactations; multiparous group) cows at 3 weeks (18-24 days prepartum; -3 weeks), 2 weeks (17-11 days prepartum; -2 weeks), and 1 week (10-4 days prepar-tum; -1 weeks) before parturition; the day of calving (within 12 h post-calving; day 0); and 5 days postpartum (5 days). ALP3 activity was significantly higher in the primiparous group than in the multiparous group, whereas the activities decreased significantly in both groups after 5 days. ALP2 and ALP5 activities did not change, whereas ALP2 activity was significantly higher in the primiparous group than in the multiparous group. TRAP5b activity was significantly higher in the primiparous group than in the multiparous group and showed a transient significant increase at day 0. Ca concentration significantly decreased at day 0 in both groups; the Ca level at day 0 was significantly higher in the primiparous group than in the multiparous group. These data show that ALP3 activity in serum may indicate a change in osteoblastic bone forma-tion after calving, but further study is needed to determine the clinical application for measuring ALP isoenzymes in bovine medicine.

Agarose gel electrophoresis pattern of serum alkaline phosphatase isoenzymes in Holstein cows during lactation.

A recent study found that an agarose gel electrophoresis (AGE) method yielded two distinct major bands corresponding to the hepatic and bone ALP isoenzymes (ALP2 and ALP3, respec-tively) in bovine serum treated with protease and neuraminidase (PN-treatment), although there were concerns that the intestinal ALP isoenzyme (ALP5) often overlapped with ALP3 in human serum treated with neuraminidase. Because ALP5 was separated from ALP3 in bovine serum treated with protease alone (P-treatment), we used a modified method employing both P- and PN-treated bovine sera to measure the activities of the three ALP isoenzymes in 53 lacta-ting Holstein cows: 24 primiparous and 29 multiparous. Upon electrophoresis, 51 of 53 samples (96.2%) subjected to P-treatment yielded a distinct fraction corresponding to ALP5, as did the control serum. All PN-treated sera yielded a definite ALP2 fraction. The ALP3 fraction was calculated as the remainder after excluding ALP2 and ALP5. The activities of total ALP (t-ALP) and ALP3 in primiparous cows were higher than those in multiparous cows (p ⟨ 0.001) at early-to-peak [10-110 days in milk (DIM)] and mid (111-220 DIM) lactation. In the multi-parous cows, the ALP3 activity at late lactation (221-477 DIM) was significantly higher than that at early-to-peak lactation. Thus, the modified AGE method described here is able to discrimi-nate three fractions of ALP isoenzymes in the sera of lactating cows. The AGE pattern of circu-lating ALP isoenzymes will contribute to the understanding of the physiological bone metabolism status in lactating cows.