Association of lysophosphatidic acid molecules with liver fibrosis: different roles indicated.

Lysophosphatidic acid is composed of lysophosphatidic acid (LPA) molecules with varied chemical forms. The present cross-sectional study was conducted to investigate the associations of various LPA molecules with liver fibrosis. Forty-six patients affected by various types of liver disease who underwent an ultrasound-guided liver biopsy were recruited for this study. Liver fibrosis was evaluated using histological grading, as well as shear wave velocity (Vs) and serum level of type IV collagen 7S (T4c7s). Serum levels of LPA molecules were determined using liquid-chromatography tandem mass-spectrometry (LC-MSMS). Total LPA showed a significant positive association with fibrosis severity evaluated based on histological grading, Vs, and T4c7s used as parameters, following adjustment for other confounding factors, including disease type, age, gender, body mass index, and high-sensitivity C-reactive protein. This association was replicated when 16:0-LPA was substituted for total LPA. In contrast, when 20:4-LPA was substituted for total LPA, no significant association with liver fibrosis was observed. In conclusion, the degree of association varied among the different LPA molecule chemical forms, suggesting different pathophysiological roles of individual LPA molecules, although total LPA concentration was shown to be associated with liver fibrosis.

Neither Trimethylamine-N-Oxide nor Trimethyllysine Is Associated with Atherosclerosis: A Cross-Sectional Study in Older Japanese Adults.

Recent evidence suggests that trimethylamine-N-oxide (TMAO), a metabolite of L-carnitine and choline, is linked to atherosclerosis and cardiovascular diseases. As TMAO content is very high in fish, we raised the following question: why do Japanese people, who consume lots of fish, show a low risk of atherosclerosis? To address this question, we investigated the effects of TMAO and other L-carnitine-related metabolites on carotid intima-media thickness (IMT). Participants were recruited from a small island and a mountainous region. Plasma L-carnitine, γ-butyrobetaine (γBB), TMAO, trimethyllysine (TML), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) levels were measured using liquid or gas chromatography-mass spectrometry. Plasma L-carnitine concentration was higher in men than in women. TMAO and TML were significantly higher in the residents of the island than in the mountainous people. In multiple linear regression analyses in all participants, TML showed a significant inverse association with max-IMT and plaque score (PS), whereas TMAO did not show any associations. In women, L-carnitine was positively associated with max-IMT and PS. TMAO was correlated with both EPA and DHA levels, implying that fish is a major dietary source of TMAO in Japanese people. Our study found that plasma TMAO was not an apparent risk factor for atherosclerosis in elderly Japanese people, whereas a low level of TML might be a potential risk. L-carnitine may be a marker for atherosclerosis in women.

A modulatory effect of L-arginine supplementation on anticancer effects of chemoimmunotherapy in colon cancer-bearing aged mice.

Myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) are increased in cancer-bearing aged hosts. Arginase-I in MDSCs degrades L-arginine, an amino acid required for T cell activation and proliferation. In this study, we compared the therapeutic efficacy of 5-fluorouracil (5-FU)/oxaliplatin (L-OHP) and cyclophosphamide (CP) between young and aged colon cancer-bearing mice. Therapy with 5-FU/L-OHP and CP significantly suppressed the in vivo growth of CT26 and MC38 colon carcinomas in syngeneic young mice, whereas this effect was attenuated in aged mice. L-arginine monotherapy showed no effect in aged mice. However, additional therapy with anti-programmed cell death (PD)-1 antibody and L-arginine supplementation boosted the effect of chemoimmunotherapy in aged mice, and some mice were cured. During all combination therapy, tumor-specific cytotoxic T lymphocytes (CTLs) were generated from mice with non-progressing tumor, but not from those with progressing tumor. Plasma L-arginine levels were lower in aged than young mice, and chemotherapy tended to decrease the plasma L-arginine levels in aged mice. Compared to young mice, CT26-bearing aged mice decreased arginase activity, arginase-I expression, and the proportion of monocytic MDSCs in tumor tissues, whereas contrasting results were observed in MC38-bearing aged mice. Importantly, the induction of tumor-specific CTLs was impaired at lower doses of L-arginine in vitro, and the infiltration of CTLs into CT26 tissues after chemoimmunotherapy was promoted by L-arginine administration in vivo. These results indicate that chemoimmunotherapy was less effective in cancer-bearing aged mice, but that L-arginine supplementation can modulate its therapeutic efficacy via its effect on tumor-specific CTLs.

A Simple Flow Injection Analysis-Tandem Mass Spectrometry Method to Reduce False Positives of C5-Acylcarnitines Due to Pivaloylcarnitine Using Reference Ions.

Flow injection analysis−tandem mass spectrometry (FIA-TMS) has been applied in a first-tier test of newborn screening (NBS). Although isovalerylcarnitine (i-C5), which is a diagnostic indicator of isovaleric acidemia (IVA), is isobaric with pivaloylcarnitine (p-C5), 2-methylbutyrylcarnitine, and n-valerylcarnitine, these isomers cannot be distinguished by the FIA-TMS. There are many reports of false positives derived from p-C5 due to the use of pivalate-conjugated antibiotics. In this study, we developed a new FIA-TMS method to distinguish i-C5 and p-C5. We found that the intensity ratio of product ions for i-C5 and p-C5 was different in a certain range even under the same analytical conditions. The product ions with the most distinct differences in ionic intensity between the isomers and the collision energies that produce them were determined to be m/z 246.2 > 187.1 and −15 V, respectively. In addition to the quantification ion, a reference ion was defined, and the similarity of the i-C5 and p-C5 reference ion ratios (i-C5 score and p-C5 score, respectively) were used to estimate which isomer (i-C5 and p-C5) was responsible for elevated C5 acylcarnitine in dried blood spots (DBSs). As a result of analyses of 11 DBS samples derived from pivalate-conjugated antibiotics and four DBS samples from IVA patients using our method, it was found that our method was able to correctly determine the type of C5-acylcarnitine (i-C5 or p-C5) in the DBS samples. Implementation of this new FIA-TMS method into the current NBS protocol will allow for a reduction in false positives in IVA.

Milk Fermented with Mushrooms Prevents Stroke in the Stroke-Prone Spontaneously Hypertensive Rats Independently of Blood Pressure.

OBJECTIVES: In a previous study, a mushroom was shown to digest milk protein to a mixture of oligopeptides and free amino acids. The aim of this study was to examine effects of this mixture, i.e., mushroom-fermented milk, on blood pressure and stroke susceptibility in the stroke-prone spontaneously hypertensive rats (SHRSP). MATERIALS AND METHODS: Rats were fed mushroom-fermented milk with or without 1 % salt water. Blood pressure was monitored either by the tail-cuff method or the telemetry system. Symptoms of stroke were examined every day to determine the stroke latency. RESULTS: Mushroom-fermented milk at 120 mg/Kg BW/day (estimated as a peptides/amino acids content) did not ameliorate hypertension in SHRSP. In contrast, mushroom-fermented milk significantly improved stroke susceptibility under salt-loading. The effects were replicated using milk fermented with three different mushrooms. To elucidate the effective components in mushroom-fermented milk, spermidine (3 mM), one of major components of mushroom-fermented milk, and a mixture of amino acids (0.8 g/L) was examined, both of which showed no significant effects on stroke susceptibility. Intake of mushroom-fermented milk did not affect sodium content significantly either in feces or in urine of the rats given 1% salt water. This observation indicated sodium absorption by the digestive system was not inhibited by intake of mushroom-fermented milk. CONCLUSION: Despite that the mechanisms were not elucidated, intake of mushroom-fermented milk effectively prevented stroke in SHRSP. Mushroom-fermented milk would be a new candidate for a supplemental nutrient supporting the cardiovascular health.

Supplementation of l-arginine boosts the therapeutic efficacy of anticancer chemoimmunotherapy.

Myeloid-derived suppressor cells (MDSCs) play a crucial role in immunosuppression in tumor-bearing hosts. MDSCs express arginase-I and indoleamine 2,3-dioxygenase; they suppress T-cell function by reducing the levels of l-arginine and l-tryptophan, respectively. We examined the anticancer effects of supplementation of these amino acids in CT26 colon carcinoma-bearing mice. Oral supplementation of l-arginine or l-tryptophan (30 mg/mouse) did not affect tumor growth, whereas oral supplementation of d-arginine was lethal. Supplementation of l-arginine showed a tendency to augment the efficacy of cyclophosphamide (CP). CP reduced the proportions of granulocytic MDSCs and increased the proportions of monocytic MDSCs in the spleen and tumor tissues of CT26-bearing mice. l-Arginine supplementation alone did not affect the MDSC subsets. CP treatment tended to reduce the plasma levels of l-arginine in CT26-bearing mice and significantly increased the number of tumor-infiltrating CD8+ T cells. In addition, l-arginine supplementation significantly increased the proportions of tumor peptide-specific CD8+ T cells in draining lymph nodes. Importantly, additional supplementation of l-arginine significantly increased the number of cured mice that were treated with CP and anti-PD-1 antibody. Totally, l-arginine supplementation shows promise for boosting the therapeutic efficacy of chemoimmunotherapy.

Validation of Liquid Chromatography-Tandem Mass Spectrometry-Based 5-Plex Assay for Mucopolysaccharidoses.

Mucopolysaccharidoses (MPSs) are rare lysosomal storage diseases caused by the accumulation of undegraded glycosaminoglycans in cells and tissues. The effectiveness of early intervention for MPS has been reported. Multiple-assay formats using tandem mass spectrometry have been developed. Here, we developed a method for simultaneous preparation and better measurement of the activities of five enzymes involved in MPSs, i.e., MPS I, MPS II, MPS IIIB, MPS IVA, and MPS VI, which were validated using 672 dried blood spot samples obtained from healthy newborns and 23 patients with MPS. The mean values of the enzyme activities and standard deviations in controls were as follows: α-iduronidase (IDUA), 4.19 ± 1.53 µM/h; iduronate-2-sulfatase (I2S), 8.39 ± 2.82 µM/h; N-acetyl-α-glucosaminidase (NAGLU), 1.96 ± 0.57 µM/h; N-acetylgalactosamine-6-sulfatase (GALNS), 0.50 ± 0.20 µM/h; and N-acetylgalactosamine-4-sulfatase (ARSB), 2.64 ± 1.01 µM/h. All patients displayed absent or low enzyme activity. In MPS I, IIIB, and VI, each patient group was clearly separated from controls, whereas there was some overlap between the control and patient groups in MPS II and IVA, suggesting the occurrence of pseudo-deficiencies. Thus, we established a multiplex assay for newborn screening using liquid chromatography tandem mass spectrometry, allowing simultaneous pretreatment and measurement of five enzymes relevant to MPSs.

Association of High-Density Lipoprotein Subclasses with Carotid Intima-Media Thickness: Shimane CoHRE Study.

AIMS: Recent studies suggested that subclasses of high-density lipoprotein (HDL) may be a better biomarker to predict the risk of atherosclerotic disorders. We aimed to examine the association of HDL2- and HDL3-cholesterol (HDL2-C and HDL3-C) with carotid intima-media thickness (IMT) using a new method to quantify the HDL-C subclasses. METHODS: Participants were 657 Japanese subjects (434 women) who received a health examination (mean age: 73 years). Serum samples were analyzed by the homogenous assay for HDL-C and HDL3-C. HDL2-C was calculated indirectly by subtracting HDL3-C from HDL-C. HDL3-C measured by this assay was well correlated with that measured by ultracentrifugation (r=0.898, p＜0.001). The maximum IMT (max-IMT) and plaque score (PS) were evaluated by ultrasonography following the standard protocol. RESULTS: HDL3-C was associated with age both in men (r=－0.322, p＜0.0001) and women (r=－0.315, p＜0.0001). In a simple regression analysis, max-IMT showed an inverse association with HDL3-C, whereas no significant association was observed with HDL2-C. A multiple linear regression analysis indicated, however, that the association between HDL3-C and max-IMT was not significant in both aged and younger populations when age was included in the analysis. Further, not only HDL2-C but also HDL3-C was not a significant predictor of 'atherosclerotic arteries' defined as the max-IMT ≥1.5 mm. Similar results were observed in the analysis on PS. CONCLUSIONS: Neither HDL3-C nor HDL2-C was significantly associated with carotid atherosclerosis in the Japanese population in this study.

Plasma arginine/ADMA ratio as a sensitive risk marker for atherosclerosis: Shimane CoHRE study.

BACKGROUND: Asymmetric dimethylarginine (ADMA), which acts an endogenous inhibitor of nitric oxide synthase (NOS), is involved in the pathogenesis of cardiovascular disease. Arginine (Arg) may regulate vascular endothelial function, since Arg is the substrate of NO competing with ADMA. In our previous study, low Arg/ADMA ratio is an independent risk for microangiopathy-related cerebral damage. PURPOSE: Here, we performed a cross-sectional study to evaluate the association between the Arg/ADMA ratio and the maximal intima-media thickness (IMT) in the carotid artery. SUBJECTS AND METHODS: Participants were 785 community-dwelling Japanese people without any severe disorders. Plasma concentration of Arg and ADMA in fasting blood sample was determined using HPLC. IMT was measured in the bilateral carotid artery by ultrasonography. RESULTS: Among quartiles stratified by the Arg/ADMA ratio, ANOVA showed a significant difference in IMT and the IMT in Q1 (the lowest quartile) was significantly higher than that in Q4 (the highest quartile). In multiple linear regression analysis, age, the male gender, lower BMI, the presence of hypertension and lower Arg/ADMA ratio were independently correlated with IMT, while IMT was not correlated with Arg or ADMA alone. In addition, the Arg/ADMA ratio was associated with IMT independent of age, sex, BMI and the presence of hypertension with odds ratio 0.21 (95%CI: 0.05-0.88) in multiple logistic regression analysis for IMT 1.5 mm or more. CONCLUSION: Imbalance of Arg and ADMA is independently involved in the progression of atherosclerosis, and the Arg/ADMA ratio may be a sensitive marker for atherosclerosis.

Asymmetric dimethylarginine as a risk factor for cardiovascular disease in Japanese patients with type 2 diabetes mellitus.

AIMS: Although asymmetric dimethylarginine (ADMA) is known to be involved in the developing process of cardiovascular diseases (CVD), little is known about the effects of ADMA on atherosclerosis in Asian patients with diabetes, who have the racial feature of lower body mass index (BMI) and decreased capacity of insulin secretion and sensitivity. METHODS: We employed 55 Japanese patients with type 2 diabetes mellitus (mean age, 64·2 years; 56% men) in a 6-month-longitudinal study and 450 patients (mean age, 62·7 years; 56% men) in a cross-sectional study and examined the association of serum ADMA with atherosclerosis parameters [intima-media thickness (IMT) and brachial-ankle pulse wave velocity (baPWV)] as well as with the presence of CVD. RESULTS: In the longitudinal study, multiple regression analysis showed that basal serum ADMA level had a significantly positive association with changes in IMT (ß=0·35, P=0·03) independently of age, duration of diabetes, BMI, blood pressure, low-density lipoprotein and high-density lipoprotein (LDL and HDL) cholesterol, HbA(1c) , and renal function. In the cross-sectional study, the serum ADMA level was significantly and positively associated with the presence of CVD (odds ratio=7·22, 95% confidence interval 1·29-40·40, P=0·02, by logistic analysis) and with baPWV (ß=0·14, P <0·01, by multiple regression analysis). In contrast, serum symmetric dimethylarginine level, a structural isomer of ADMA, was associated neither with parameters for atherosclerosis nor with the presence of CVD in both studies. CONCLUSIONS: Serum ADMA is a predictor of atherosclerosis and associated with the presence of CVD in Japanese patients with type 2 diabetes mellitus.

Relationships between dimethylarginine and the presence of vertebral fractures in type 2 diabetes mellitus.

OBJECTIVE: Although previous studies suggested that nitric oxide (NO) could affect bone metabolism, little is known about whether asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthase, is associated with the risk of osteoporotic fractures. METHODS: A cross-sectional study was carried out to examine the associations of serum dimethylarginines with bone mineral density (BMD), bone turnover markers, and the presence of vertebral fractures in 226 men and 152 postmenopausal women with type 2 diabetes. RESULTS: In subjects with vertebral fractures, there was significantly higher serum ADMA in men (0.61 ± 0.20 [mean ± SD] vs 0.55 ± 0.19 µm, P = 0.030) and symmetric dimethylarginine (SDMA) in postmenopausal women (0.72 ± 0.37 vs 0.56 ± 0.16 µm, P < 0.001) than in those without fractures. In men with vertebral fractures, serum ADMA was negatively correlated with Z-score at one-third radius (multiple regression ß = -0.28, p = 0.016), and SDMA was positively correlated with serum osteocalcin (ß = 0.38, P = 0.044) and urinary N-terminal cross-linked telopeptide of type-I collagen (ß = 0.40, P = 0.027). In addition, serum ADMA in men and SDMA in postmenopausal women were positively associated with the presence of vertebral fractures after adjusting for age, duration of diabetes, and lumbar BMD (multiple logistic regression odds ratio [OR] = 1.36, P = 0.044 and OR = 1.78, P = 0 .006, respectively). CONCLUSIONS: We report, for the first time, that serum dimethylarginines may be independent risk factors for prevalent vertebral fractures in patients with type 2 diabetes.

Efficacy of Corrected Rapid Turnover Protein Increment Index (CRII) for Early Detection of Improvement of Nutrition Status in Patients with Malnutrition.

Serum prealbumin level is useful for assessment of changes in nutritional status but it is markedly affected by the inflammation. In this study, we examined the efficacy of the corrected rapid turnover protein increment index (CRII) for prealbumin, which is calculated as [prealbumin level/C-reactive protein (CRP) level on the assessment day]/[prealbumin level/CRP level on the day of starting nutritional care], for prediction of improvement of nutritional status in patients with malnutrition. The subjects were 50 hospitalized patients with low albuminemia, who were receiving nutritional care. Serum concentrations of albumin, prealbumin and CRP were measured every week for 5 weeks. We defined patients whose serum albumin level was elevated by more than 0.2 g/dl after 5 weeks as those showing improved nutritional status. There was a significant difference in the prealbumin level between improved and unimproved patients at 5 weeks after the start of nutritional support. On the other hand, the prealbumin CRII value showed a significant difference between the groups at 1 and 2 weeks after the start of nutritional support. In conclusion, assessment of prealbumin CRII is useful for early prediction of improved nutritional status in patients with malnutrition.

[Specificity of homogenous assays for the LDL-cholesterol measurement].

The plasma level of LDL cholesterol (LDL-C) is an independent risk factor for atherosclerosis. Recently, homogenous assays were developed to measure the level of LDL-C directly. In some cases, however, these assays give inconsistent results probably due to their difference in measurement principles and reactivity. In this study, we therefore evaluated the specificity of three homogeneous assay reagents currently available commercially. In the experiment, measures of the homogeneous assays were compared with the cholesterol levels in the fractions separated by ultracentrifugation. We found that the reactivity to IDL and VLDL differed substantially among the assays studied.

Evaluation of asymmetric dimethylarginine and homocysteine in microangiopathy-related cerebral damage.

BACKGROUND: Microangiopathy-related cerebral damage (MARCD) is an entity of cerebrovascular disease based on arteriosclerosis in deep white matter, which includes lacunar infarction and white matter hyperintensity (WMH). As asymmetric dimethylarginine (ADMA), an endogenous inhibitor of the nitric oxide (NO) synthases, and homocysteine are both potential risk factors for arteriosclerosis, the plasma levels of these two substances were evaluated in individuals with MARCD. METHODS: Consecutive participants of a health examination (401 males and 311 females) were recruited for this cross-sectional study. All participants received an magnetic resonance imaging examination, and those with either lacunar infarction or WMH (grade > or =2) were classified into MARCD (+) (N = 146). The plasma ADMA concentration was measured with high performance liquid chromatography. The total homocysteine (tHcy) concentration was measured using a commercial kit. RESULTS: The ADMA level (P < 0.001), symmetric dimethylarginine (SDMA) level (P < 0.05) and L-arginine (Arg)/ADMA ratio (P < 0.01) differed significantly between MARCD (+) and (-) according to nonparametric Wilcoxon test, while the tHcy level did not (P = 0.37). Classic risk factors such as age, blood pressure, and the presence of hypertension differed significantly between the two groups as well. In the logistic analysis, the association of Arg/ADMA with MARCD remained significant (odds ratio and 95% confidence interval, 0.19 (0.05, 0.73), P < 0.05) even after adjusting for the effects of age and hypertension. CONCLUSIONS: ADMA and tHcy levels were studied in 712 subjects with or without MARCD. The Arg/ADMA ratio was suggested to be an independent risk factor for MARCD. A large-scale prospective study is warranted to confirm the causal relationship between Arg/ADMA and MARCD.

Involvement of cystatin C in pathophysiology of CNS diseases.

Cystatin C Leu68Gln variant is known to induce amyloid deposition in cerebral arterioles, resulting in Icelandic type cerebral amyloid angiopathy (CAA). Wild-type cystatin C is also observed in solitary CAA involving amyloid beta protein (Abeta), and accelerates the amyloidogenicity of Abeta in vitro. In neurological inflammatory diseases and leptomeningeal metastasis, low cystatin C levels are accompanied with high activities of cathepsins in the cerebrospinal fluid. Among the cells in CNS, astrocytes appear to secrete cystatin C in response to various proteases and cytokines. Co-localization of Abeta and cystatin C in the brains of Alzheimer's disease (AD) led to the hypothesis that cystatin C is involved in the disease process. We demonstrated that cystatin C microinjection into rat hippocampus induced neuronal cell death in dentate gyrus. Furthermore, apoptotic cell death was observed in neuronal cells treated with cystatin C in vitro. Up-regulation of cystatin C was observed in glial cells with neuronal cell death in vivo. These findings indicate the involvement of cystatin C in the process of neuronal cell death.

Sympathetic regulation of the renal functions in rats reciprocally congenic for chromosome 1 blood pressure quantitative trait locus.

The role of the chromosome 1 blood pressure quantitative trait locus (QTL) on the sympathorenal interaction was studied using congenic strains. The two reciprocal congenic strains, WKYpch1.0 and SHRSPwch1.0, were respectively constructed by introgressing the stroke-prone spontaneously hypertensive rat (SHRSP)-derived fragment for the QTL into a Wistar-Kyoto rat (WKY) and vice versa. The role of the sympathetic nervous system in the kidney was evaluated by comparing the renal functions between denervated and sham-operated kidneys under anesthesia. The denervation was performed by stripping the adventitia off and applying 10% phenol to the blood vessels at the left renal hilus. Polyfructosan was continuously injected intravenously to determine the renal plasma flow and the glomerular filtration rate. A reciprocal and significant alteration in the renal norepinephrine (NE) content was observed between WKY and WKYpch1.0 and between SHRSP and SHRSPwch1.0. Concomitantly, the renal vascular resistance differed significantly between the congenic and the background parental strains. By contrast, no significant difference was observed in the fractional excretion of sodium, an index of the tubular function. While the denervation elicited a significant decrease of the renal NE content in all of the four strains studied, the significant effects of the denervation on the renal functions were observed only in SHRSP and WKYpch1.0, both of which harbored the SHRSP-derived QTL fragment. These results indicated that the chromosome 1 blood pressure QTL modulated the renal functions through the sympathetic nerve activity in the kidney.

[HOMA-IR and related clinical parameters].

Metabolic syndrome (MS) is based on visceral obesity and insulin resistance (IR) and, shows abnormalities in fat and glucose metabolism as well as hypertension. We investigated the relationship between IR and various risk factors for atherosclerosis in 378 male subjects (averaged age: 63.8 +/- 15.2 years) and 509 female subjects (averaged age: 67.1 +/- 13.5 years) recruited from a health check examination. Homeostasis model assessment (HOMA-IR) was used as an indicator of IR. Body mass index (BMI), triglyceride (TG), remnant-like particle cholesterol (RLP-C) and C-reactive protein (CRP) showed a good correlation (r > 0.2) with HOMA-IR. LDL cholesterol(LDL-C), small dense LDL(sdLDL) and HbA1c showed less stringent (r < 0.2) but still significant correlation with HOMA-IR. In contrast, oxidized LDL (oxLDL) and systolic blood pressure did not show any correlation. A multiple regression analysis indicated that BMI, RLP-C, HDL-C, HbA1c and CRP were independently correlated with HOMA-IR. These observations suggested that, among the three new lipid parameters (i.e., RLP-C, sdLDL and oxLDL), RLP-C was the most closely related to the IR status.