Loss-of-function variants in SAT1 cause X-linked childhood-onset systemic lupus erythematosus.

OBJECTIVES: Families that contain multiple siblings affected with childhood onset of systemic lupus erythematosus (SLE) likely have strong genetic predispositions. We performed whole exome sequencing (WES) to identify familial rare risk variants and to assess their effects in lupus. METHODS: Sanger sequencing validated the two ultra-rare, predicted pathogenic risk variants discovered by WES and identified additional variants in 562 additional patients with SLE. Effects of a splice site variant and a frameshift variant were assessed using a Minigene assay and CRISPR/Cas9-mediated knock-in (KI) mice, respectively. RESULTS: The two familial ultra-rare, predicted loss-of-function (LOF) SAT1 variants exhibited X-linked recessive Mendelian inheritance in two unrelated African-American families. Each LOF variant was transmitted from the heterozygous unaffected mother to her two sons with childhood-onset SLE. The p.Asp40Tyr variant affected a splice donor site causing deleterious transcripts. The young hemizygous male and homozygous female Sat1 p.Glu92Leufs*6 KI mice spontaneously developed splenomegaly, enlarged glomeruli with leucocyte infiltration, proteinuria and elevated expression of type I interferon-inducible genes. SAT1 is highly expressed in neutrophils and encodes spermidine/spermine-N1-acetyltransferase 1 (SSAT1), a rate-limiting enzyme in polyamine catabolism. Young male KI mice exhibited neutrophil defects and decreased proportions of Foxp3 +CD4+ T-cell subsets. Circulating neutrophil counts and proportions of Foxp3 +CD4+ T cells correlated with decreased plasma levels of spermine in treatment-naive, incipient SLE patients. CONCLUSIONS: We identified two novel SAT1 LOF variants, showed the ability of the frameshift variant to confer murine lupus, highlighted the pathogenic role of dysregulated polyamine catabolism and identified SAT1 LOF variants as new monogenic causes for SLE.

Polyamine-Rich Diet Elevates Blood Spermine Levels and Inhibits Pro-Inflammatory Status: An Interventional Study.

The Japanese diet and the Mediterranean diet are rich in polyamines (spermidine and spermine). Increased polyamine intake elevated blood spermine levels, inhibited aging-associated pro-inflammatory status (increases in lymphocyte function-associated antigen-1 (LFA-1) on immune cells), suppressed aberrant gene methylation and extended the lifespan of mice. To test the effects of increased polyamine intake by humans, 30 healthy male volunteers were asked to eat polyamine-rich and ready-to-eat traditional Japanese food (natto) for 12 months. Natto with high polyamine content was used. Another 27 male volunteers were asked not to change their dietary pattern as a control group. The volunteers' age of intervention and control groups ranged from 40 to 69 years (median 48.9 ± 7.9). Two subjects in the control group subsequently dropped out of the study. The estimated increases in spermidine and spermine intakes were 96.63 ± 47.70 and 22.00 ± 9.56 µmol per day in the intervention group, while no changes were observed in the control group. The mean blood spermine level in the intervention group gradually rose to 1.12 ± 0.29 times the pre-intervention level after 12 months, and were significantly higher (p = 0.019) than those in the control group. Blood spermidine did not increase in either group. LFA-1 on monocytes decreased gradually in the intervention group, and there was an inverse association between changes in spermine concentrations relative to spermidine and changes in LFA-1 levels. Contingency table analysis revealed that the odds ratio to decrease LFA-1 by increased polyamine intake was 3.927 (95% CI 1.116-13.715) (p = 0.032) when the effect of acute inflammation was excluded. The results in the study were similar to those of our animal experiments. Since methylation changes of the entire genome are associated with aging-associated pathologies and our previous studies showed that spermine-induced LFA-1 suppression was associated with the inhibition of aberrant gene methylation, the results suggest that dietary polyamine contributes to human health and longevity.

Polyamine biomarkers as indicators of human disease.

The significant increase of periodontitis, chronic kidney disease (CKD), Alzheimer's disease and cancer can be attributed to an ageing population. Each disease produces a range of biomarkers that can be indicative of disease onset and progression. Biomarkers are defined as cellular (intra/extracellular components and whole cells), biochemical (metabolites, ions and toxins) or molecular (nucleic acids, proteins and lipids) alterations which are measurable in biological media such as human tissues, cells or fluids. An interesting group of biomarkers that merit further investigation are the polyamines. Polyamines are a group of molecules consisting of cadaverine, putrescine, spermine and spermidine and have been implicated in the development of a range of systemic diseases, in part due to their production in periodontitis. Cadaverine and putrescine within the periodontal environment have demonstrated cell signalling interfering abilities, by way of leukocyte migration disruption. The polyamines spermine and spermidine in tumour cells have been shown to inhibit cellular apoptosis, effectively prolonging tumorigenesis and continuation of cancer within the host. Polyamine degradation products such as acrolein have been shown to exacerbate renal damage in CKD patients. Thus, the use of such molecules has merit to be utilized in the early indication of such diseases in patients.

Solid-Supported Amplification of Aggregation Emission: A Tetraphenylethylene-Cucurbit[6]uril@Hydroxyapatite-Based Supramolecular Sensing Assembly for the Detection of Spermine and Spermidine in Human Urine and Blood.

The development of sensitive and selective tools for the detection and quantification of biomarkers is important in the diagnosis and treatment of clinical diseases. Spermine (SP) and spermidine (SPD) act as biomarkers for early-stage diagnosis of cancer in humans as their increased levels in urine are indicative of abnormal biological processes associated with this fatal disease. In this study, we introduced a strategy for solid-supported amplification of the effective aggregation-induced-emission (AIE) effect of a water-soluble tetraphenylethylene (TPE)-based probe in developing a supramolecular sensing platform for the rapid, sensitive, and selective detection of SP and SPD in water. The nonemissive TPE derivative (TPEHP) forms a less emissive conjugate with hydroxyl cucurbit[6]uril (CB[6]OH) in water, which undergoes several-fold enhancement of effective emission upon electrostatic interaction with the solid surface of hydroxyapatite nanoparticles (HAp NPs), dispersed in the aqueous media. The corresponding three-component supramolecular assembly disrupts by the intrusion of SP and SPD in the CB[6] portal because of the stronger binding ability with CB[6], resulting in a turn-off fluorescence sensor for SP and SPD with enhanced sensitivity. The assembly-disassembly-based sensing mechanism was thoroughly demonstrated by carrying out isothermal titration calorimetry (ITC), spectroscopic, and microscopic experiments. The sensing system showed low limits of detection (LODs) of 1.4 × 10-8 and 3.6 × 10-8 M for SP and SPD, respectively, which are well below the required range for the early diagnosis of cancer. Besides, a good linear relationship was obtained for both SP and SPD. Nominal interference from various metal ions, anions, common chemicals, amino acids, and other biogenic amines makes this sensing platform suitable for the real-time, low-level measurement of spermine (and spermidine) in human urinary and blood samples.

Digestive involvement in a severe form of Snyder-Robinson syndrome: Possible expansion of the phenotype.

Snyder-Robinson syndrome (OMIM #309583) is a rare X-linked condition, caused by mutation in the SMS gene (MIM *300105), characterized by a wide spectrum of clinical signs including developmental delay, epilepsy, asthenic habitus, dysmorphism, osteopenia, and renal or genital anomalies. Here we describe two maternal half-brothers who both presented with severe neurodevelopmental delay, seizures, hearing loss, facial dysmorphism, renal and ophthalmologic anomalies, failure to thrive and premature death. A novel p.(Gly203Asp) variant was found at the hemizygous state in the two boys, and an elevated Spermidine/Spermine ratio confirmed the diagnosis of Snyder-Robinson syndrome. One of the brothers presented with gastrointestinal symptoms, with jejunal stenosis, enteral feeding intolerance, failure to thrive due to a dysfunctional gastrointestinal system, cholestasis and exocrine pancreatic insufficiency. Although more studies will be needed to understand its mechanisms, this observation lends further support to the possibility of severe digestive involvement in Snyder Robinson syndrome.

N8-Acetylspermidine: A Polyamine Biomarker in Ischemic Cardiomyopathy With Reduced Ejection Fraction.

Background Patients with ischemic cardiomyopathy (ICM) have worse outcomes than those with coronary artery disease alone and those with non-ICM. N8-acetylspermidine (N8AS) is a polyamine that regulates ischemic cardiac apoptosis and resultant cardiac dysfunction. We hypothesized that N8AS is a mechanistic biomarker of adverse outcomes in patients with ICM. Methods and Results High-resolution plasma metabolomics profiling and mass spectrometry were used to quantitate N8AS levels in a discovery cohort of 474 patients with coronary artery disease (age: 68±11 years, 12% black, 26% women): 154 with ICM, and 320 without ICM; and in an external validation cohort of 85 patients with ICM (age: 60±12 years, 37% black, 19% women). Patients without heart failure (HF) at baseline were followed for incident HF. The association between N8AS (log2-transformed, standardized) and outcomes of all-cause mortality and incident HF were examined using Cox regression. N8AS was higher (10.39 [interquartile range, 7.21-17.75] versus 8.29 nmol/L [interquartile range, 5.91-11.42]; P<0.001) in patients with ICM compared with patients who had coronary artery disease without ICM. Higher N8AS levels were associated with higher mortality in patients with ICM (hazard ratio [HR], 1.48; 95% CI, 1.19-1.85 per SD increase [P=0.001]), independent of B-type natriuretic peptide, high-sensitivity troponin I, and high-sensitivity C-reactive protein. Findings were validated in the independent cohort. Moreover, higher N8AS level was associated with incident HF in patients without HF at baseline (HR, 4.16; 95% CI, 1.41-12.25 per SD increase [P=0.01]). Conclusions Independent of traditional HF measures, higher N8AS levels are associated with higher mortality in patients with ICM and incident HF in those who have coronary artery disease without HF. N8AS is a novel mechanistic biomarker in ICM.

Association Between Plasma Diacetylspermine and Tumor Spermine Synthase With Outcome in Triple-Negative Breast Cancer.

BACKGROUND: MYC is an oncogenic driver of development and progression in triple-negative breast cancer (TNBC). Ornithine decarboxylase, the rate-limiting enzyme in polyamine metabolism, is a transcriptional target of MYC. We therefore hypothesized that a plasma polyamine signature may be predictive of TNBC development and progression. METHODS: Using liquid chromatography mass spectrometry, polyamine levels were determined in plasma samples from newly diagnosed patients with TNBC (n = 87) and cancer-free controls (n = 115). Findings were validated in plasma samples from an independent prospective cohort of 54 TNBC, 55 estrogen receptor negative (ER-) and progesterone receptor negative (PR-) and HER2 positive (HER2+), and 73 ER+ case patients, and 30 cancer-free control subjects. Gene expression data and clinical data for 921 and 2359 breast cancer tumors were obtained from The Cancer Genome Atlas repository and the Oncomine database, respectively. Relationships between plasma diacetylspermine (DAS) and tumor spermine synthase (SMS) mRNA expression with metastasis-free survival and overall survival were determined using Cox proportional hazard models; Fisher exact tests were used to assess risk of distant metastasis in relation to tumor SMS mRNA expression. RESULTS: An increase in plasma DAS, a catabolic product of spermine mediated through SMS, was observed in the TNBC subtype of breast cancer. Plasma levels of DAS in TNBC associated with increased risk of metastasis (plasma DAS value ≥ 1.16, hazard ratio = 3.06, 95% confidence interval [CI] = 1.15 to 8.13, two-sided P = .03). SMS mRNA expression in TNBC tumor tissue was also found to be predictive of poor overall survival (top 25th percentile hazard ratio = 2.06, 95% CI = 1.04 to 4.08, one-sided P = .04) and increased risk of distant metastasis in TNBC (comparison of lowest SMS quartile [reference] to highest SMS quartile relative risk = 1.90, 95% CI = 0.97 to 4.06, one-sided Fisher exact test P=.03). CONCLUSIONS: Metabolomic profiling identified plasma DAS as a predictive marker for TNBC progression and metastasis.

Gender-Related Differences on Polyamine Metabolome in Liquid Biopsies by a Simple and Sensitive Two-Step Liquid-Liquid Extraction and LC-MS/MS.

Polyamines are involved in the regulation of many cellular functions and are promising biomarkers of numerous physiological conditions. Since the concentrations of these compounds in biological fluids are low, sample extraction is one of the most critical steps of their analysis. Here, we developed a comprehensive, sensitive, robust, and high-throughput LC-MS/MS stable-isotope dilution method for the simultaneous determination of 19 metabolites related to polyamine metabolism, including polyamines, acetylated and diacetylated polyamines, precursors, and catabolites from liquid biopsies. The sample extraction was optimized to remove interfering compounds and to reduce matrix effects, thus being useful for large clinical studies. The method consists of two-step liquid-liquid extraction with a Folch extraction and ethyl acetate partitioning combined with dansyl chloride derivatization. The developed method was applied to a small gender-related trial concerning human serum and urine samples from 40 obese subjects. Sex differences were found for cadaverine, putrescine, 1,3-diaminopropane, γ-aminobutyric acid, N8-acetylspermidine, and N-acetylcadaverine in urine; N1-acetylspermine in serum; and spermine in both serum and urine. The results demonstrate that the developed method can be used to analyze biological samples for the study of polyamine metabolism and its association with human diseases.

Decreased plasmatic spermidine and increased spermine in mild cognitive impairment and Alzheimer's disease patients

Abstract Background Current evidence suggests that upregulation of polyamines system plays a role both in cognitive deficit and synaptic loss observed in Alzheimer's disease (AD). Objective The aim of this study was to determine the plasmatic concentration of polyamines in mild cognitive impairment (MCI) and AD patients in comparison with healthy controls (HC). Methods Plasmatic polyamines were quantified using the AbsoluteIDQ® p180 and liquid chromatography coupled to tandem mass spectrometry (LC/MS-MS). Results The study group comprised 34 AD patients, 20 MCI and 25 HC. All individuals were followed for 4 years. During this period 8 amnestic MCI patients (40% of the MCI sample at baseline) converted to AD. Spermidine level was lower in both patient groups (AD; MCI) compared to HC (p = 0.007). Plasma levels of spermine were higher in the MCI group (p < 0.001), but decreased in the sub-sample of MCI patients who converted to AD (p = 0.043). No statistically significant differences were found in ornithine and putrescine levels (p = 0.056 and p = 0.126, respectively). Discussion Our results suggest dynamic changes in the expression of polyamines in the MCI-AD continuum.

Gene polymorphisms of SFTPB rs7316, rs9752 and PAOX rs1046175 affect the diagnostic value of plasma Pro-SFTPB and DAS in Chinese Han non-small-cell lung cancer patients.

Plasma pro-surfactant protein B (pro-SFTPB) and N1,N12-diacetylspermine (DAS) can be used as markers for the diagnosis of non-small-cell lung carcinoma (NSCLC). Whether the genetic diversity affects the application value of Pro-SFTPB and DAS as a diagnostic marker for NSCLC is still unknown. This study aims to explore the relationship between SFTPB rs7316, rs9752 and PAOX rs1046175 gene polymorphisms and the diagnostic value of plasma Pro-SFTPB and DAS in patients with Chinese Han lung cancer. SFTPB rs7316, rs9752 and PAOX rs1046175 genotypes were analyzed by direct sequencing in 425 patients with NSCLC and 425 controls, and the levels of Pro-SFTPB and DAS in plasma were determined by enzyme-linked immunosorbent assay (ELISA). The area under the curve (AUC) of the SFTPB rs7316 locus TT genotype for the diagnosis of NSCLC was 0.758, and the AUC of the TC/CC genotype for the diagnosis of NSCLC was 0.872. The AUC of the SFTPB rs9752 locus GG genotype for the diagnosis of NSCLC was 0.935, and the AUC of the GC/CC genotype for the diagnosis of NSCLC was 0.648. The AUC of the PAOX rs1046175 locus GG for the diagnosis of NSCLC was 0.669, and the AUC of the GC/CC genotype for the diagnosis of NSCLC was 0.749. In conclusion, SFTPB rs7316, rs9752, and PAOX rs1046175 gene polymorphisms affect the diagnostic value of plasma Pro-SFTPB and DAS in patients with Chinese Han NSCLC.

Serum polyamine metabolic profile in autoimmune thyroid disease patients.

OBJECTIVE: Polyamines are indispensable polycations and play important physiological roles in living cells. Some polyamine metabolites have been associated with autoimmune disorders. The aims of this study were to profile polyamine metabolites in autoimmune thyroid disease (AITD) and predict whether polyamine metabolites are associated with thyroid hormone, thyroid autoantibodies or disease progression. DESIGN, PATIENTS AND MEASUREMENTS: A total of 136 participants were recruited, including Graves' disease (GD) (n = 36), Hashimoto's thyroiditis (HT) (n = 33) and thyroid autoantibody-positive (pTAb) (n = 29) patients and 38 age- and sex-matched healthy controls (HCs). Fourteen polyamine metabolites, including polyamine precursors, polyamines and polyamine catabolite, were measured by UFLC-MS/MS RESULTS: Both GD and HT patients had higher L-arginine, L-ornithine, lysine and agmatine levels and lower putrescine, 1,3-diaminopropane, spermine, N-acetylputrescine levels than HCs. Some polyamine metabolite levels were different only in GD or HT patients compared to HCs: GD patients had significantly higher spermidine, N-acetylspermidine and γ-aminobutyric acid and lower cadaverine, whereas HT patients had significantly decreased N-acetylspermine. Only spermine and N-acetylspermine were significantly lower in pTAb than HCs. The spermine:spermidine ratio was significantly reduced in all AITD patients. In addition, spermine was negatively correlated with thyroid-specific antibodies grade. N-acetylspermidine might be a risk factor for pTAb progression to overt hypothyroidism. CONCLUSIONS: Compared with the HCs, most metabolites of GD and HT showed similar patterns, suggesting the possibility of a common pathophysiological basis or metabolic pathway. Moreover, pTAb progression to overt hypothyroidism may be related to high N-acetylspermidine. Thyroid autoimmunity was associated with low spermine.

A Metabolomics Profiling of Glaucoma Points to Mitochondrial Dysfunction, Senescence, and Polyamines Deficiency.

Purpose: To determine the plasma metabolomic signature of primary open-angle glaucoma (POAG). Methods: We compared the metabolomic profiles of plasma from individuals with POAG (n = 36) with age- and sex-matched controls with cataract (n = 27). A targeted metabolomics study was performed using the standardized p180 Biocrates Absolute IDQ p180 kit with a QTRAP 5500 mass spectrometer. Multivariate analyses were performed using principal component analysis (PCA) and the least absolute shrinkage and selection operator (LASSO) method. Results: Among the 151 metabolites accurately measured, combined univariate and multivariate analyses revealed 18 discriminant metabolites belonging to the carbohydrate, acyl-carnitine, phosphatidylcholine, amino acids, and polyamine families. The metabolomic signature of POAG points to three closely interdependent pathophysiologic conditions; that is, defective mitochondrial oxidation of energetic substrates, altered metabolism resembling that observed in senescence, and a deficiency in spermidine and spermine, both polyamines being involved in the protection of retinal ganglion cells. Conclusions: Our results highlight a systemic and age-related mitochondrial defect in the pathogenesis of POAG.

Ionic Self-Assembled Platform of Perylenediimide-Sodium Dodecylsulfate for Detection of Spermine in Clinical Samples.

The detection and quantification of spermine in clinical practice is important for early diagnosis of many diseases. Chromatographic and immunoassay-based methods are commonly used. However, a fluorescence-based assay could provide real-time detection. Herein, the synthesis and aggregation properties of a dicationic perylene probe (N1 -dodecyl-N3 -(4-phenyl)benzimidazolium-functionalized perylenediimide (DAB-PDI)) used to develop a fluorescent "turn-on" ensemble for the detection of spermine are discussed. The fluorescence of DAB-PDI (10 µm, Φ=0.55) is efficiently quenched by negatively charged sodium dodecylsulfate (SDS) through the formation of ionic self-assembled aggregates (charge ratio of negative (N) in SDS to positive (P) in DAB-PDI (N/P)=9). This negatively charged ionic self-assembly between DAB-PDI and SDS has been characterized by using photophysical, microscopic, dynamic light scattering, isothermal titration calorimetry, and HRMS techniques. The addition of spermine to this ensemble solution results in the breakdown of the DAB-PDI-SDS ensemble owing to strong binding of spermine with SDS and, as a result, the fluorescence of DAB-PDI is recovered. This ensemble exhibits high sensitivity and selectivity for spermine detection in water, urine, and blood serum. The lowest limit of detection is 27.5 nm, which is at least about 36 times lower than that required for early diagnosis of cancer (1 to 10 µm for urinary spermine).

Microwave assisted synthesis of tyrosine protected gold nanoparticles for dual (colorimetric and fluorimetric) detection of spermine and spermidine in biological samples.

In this work, tyrosine-protected gold nanoparticles (Tyr-Au NPs) were fabricated by one-step reduction of Au3+ ion using Tyr as a reducing and capping agent under microwave irradiation. The Tyr-Au NPs were successfully used as a dual probe for colorimetric and fluorescence turn-on assays of spermine and spermidine in biological samples. Upon addition of spermine and spermidine, the characteristic surface plasmon resonance (SPR) band of Tyr-Au NPs was red-shifted to 596 and 616nm and the emission peak (Tyr) at 410nm was gradually increased with increasing concentration of both analytes, confirming the aggregation of Tyr-Au NPs induced by spermine and spermidine, which results to restore fluorescence of Tyr on the surfaces of Au NPs. In addition, it shows high selectivity for sensitive detection of prostatic cancer biomarkers spermine and spermidine in real clinical applications with reduced sample preparations.

Development and validation of a rapid and sensitive UPLC-MS/MS method for quantification of kukoamine B in human plasma: Application to a clinical pharmacokinetic study.

A rapid, accurate and robust method was firstly developed using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay to quantify kukoamine B, which is a novel drug under clinical development for the treatment of sepsis, in human plasma. Solid-phase extraction (SPE) was used to extract kukoamine B from human plasma. The extracts were separated on a Waters Acquity HSS T3 column (2.1×50mm i.d., 1.8µm) with a gradient elution method, using mobile phases of A (formic acid-water (1:1000, v/v)) and B(formic acid-methanol (1:1000, v/v)). Kukoamine B and internal standard (5-deuterated isotope kukoamine B) were detected under the multiple-reaction monitoring mode by an API 5500 triple quadrupole mass spectrometer with electrospray ionization. The method showed good linearity from 0.100 to 50.0ng/mL according to 1/x2 weighted linear regression analysis. Inter- and intra-batch precision of kukoamine B were less than 15% and the accuracy was within 85-115%. The extraction recoveries and matrix effect of kukoamine B at three concentration levels were consistent. The sensitivity, specificity and stabilities under various conditions were validated. In conclusion, the validation results showed that this method was rapid, accurate, robust and can successfully fulfill the requirement of clinical pharmacokinetic study of kukoamine B mesylate in Chinese healthy subjects.

Uremic toxins: some thoughts on acrolein and spermine.

Chronic kidney disease (CKD) is characterized by the progressive reduction of glomerular filtration rate and subsequent retention of organic waste compounds called uremic toxins. While patients with CKD are at a higher risk of premature death due to cardiovascular complications, this increased risk cannot be completely explained by classical cardiovascular risk factors such as hypertension, diabetes mellitus, and obesity. Instead, recent research suggests that uremic toxins may play a key role in explaining this marked increase in cardiovascular mortality in patients with CKD. While spermine, a tetra-amine, has previously been hypothesized to act as an uremic toxin, the following review presents a summary of recent literature that casts doubt on this assertion. Instead, acrolein, an oxidative product of spermine and the triamine spermidine, is likely responsible for the toxic effects previously attributed to spermine.

Identification of arginine and its "Downstream" molecules as potential markers of breast cancer.

Breast cancer (BC) is the most commonly diagnosed cancer in women worldwide. Arginine is a semiessential amino acid in humans and is essential for several biological pathways in malignant and normal cells, such as ornithine and N1, N12-diacetylspermine (DiAcSpm). This study aimed to determine the role of arginine and these downstream molecules in BC. Plasma arginine, ornithine, and arginine-to-ornithine ratio (AOR) were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Urine samples were measured by the colloid gold aggregation to test determination of urinary diAcSpm. A principal component analysis was performed to evaluate the results observed between breast tumor and control characteristics. Differences in individual metabolite concentrations between BC patients and controls were tested by receiver operating characteristics (ROC) analyses. Student's t tests were used to detect the differences between two groups of normally distributed variables, and Wilcoxon sign rank tests were performed for asymmetrically distributed variables. As we analyzed, BC patients had lower plasma arginine and arginine/ornithine level, and higher plasma ornithine and urinary DiAcSpm concentrations as compared with control patients (P = 0.028, 0.020, 0.002, and 0.011, respectively). And the ROC curve was drawn and the area under the curve of the metabolites was calculated to be 0.659 (P = 0.028), 0.645 (P = 0.045), 0.7233 (P = 0.002), 0.683 (P = 0.011), respectively. In addition, our analysis showed that arginine concentrations and AOR had a positive correlation with ER status, while ornithine had a negative correlation with T stage (P = 0.042, 0.023, respectively).In conclusion, arginine and these downstream molecules were biomarkers for BC. More studies are needed to highlight the theoretical strengths. © 2016 IUBMB Life, 68(10):817-822, 2016.

Polyamines, myosin heavy chains, and collagen specific amino acids after a repeated bout of eccentric exercise.

We investigated alternatives to commonly used biomarkers of exercise-induced tissue damage. Over 5 days following two bouts of 100 drop-to-vertical jumps (inter-bout rest period of 3 weeks), myosin heavy chain 1, hydroxylysine (HYL), hydroxyproline (HYP), spermine (SPM) and spermine synthase (SMS) were measured in the serum of 10 participants. HYL significantly increased from 5.92 ± 1.49 ng/mL to 6.48 ± 1.47 ng/mL at 24 h. A similar trend was observed for bout 2, but without reaching significance. SPM significantly increased only after bout 1 from 0.96 ± 0.19 ng/mL at pretest to a peak level of 1.12 ± 0.26 ng/mL at 24 h, while B2 increments remained non-significant. Myosin heavy chain 1, HYP and SMS values remained below the detection limit of the applied enzyme-linked immunosorbent assay (ELISA) kit. Though HYL and SM increased after the intervention, both markers showed a large standard deviation (SD) combined with small increments. Therefore, none of the investigated biomarkers provides a meaningful alternative to commonly used damage markers.

Ultra performance liquid chromatography tandem mass spectrometry assay for determination of kukoamine B in human blood and urine.

In this paper, we report a sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method which is capable of quantifying kukoamine B (KB) levels in human blood and urine. Following solid phase extraction and direct dilution process, the analyte and its internal standard (D5-KB) run on an Acquity UPLC(®) HSS T3 column (2.1×50mm i.d., 1.8µm) by using a gradient elution method (run time was 1.5min). The mass spectrometric analysis was performed by using an API-5500 mass spectrometer coupled with an electro-spray ionization source. The MRM transitions of m/z 531.3(+)→222.1(+) and 536.3(+)→222.1(+) were used to quantify KB and D5-KB respectively. This assay method has been fully validated in terms of selectivity, linearity, lower limit of quantification, precision, accuracy, stability, recovery and matrix effect. The concentration range of this method is 10.0-2000.0ngmL(-1) in blood and 0.5-500.0ngmL(-1) in urine. Linearity (R(2)) of calibration curves were 0.9964±0.0022 and 0.9935±0.0053 for blood and urine, respectively (regression equation: y=ax+b). The precision (RSD%) of quality control samples is less than 10.3% for blood and less than 10.5% for urine. The accuracy (RE%) is within -4.0-11.3% and -11.7-12.5% for blood and urine respectively. KB was stable after 4h in ice-water bath, 1 freeze/thaw cycles and 180days at -80°C for blood samples; and was stable after 3h at room temperature, 3 freeze/thaw cycles and 180days at -80°C for urine samples. Recoveries of KB were 4.7±0.9% in blood and 96.5±1.3% in urine, respectively. Additionally, the applicability of this method has been proved by analyzing clinical samples from pharmacokinetic study of KB in human.

[POLYAMINE CONCENTRATION IN ERYTHROCYTES IN THE BLOOD OF PREGNANT WOMEN WITH INTRAUTERINE GROWTH RETARDATION].

The aim of the study was to determine polyamine concentration in erythrocytes in the blood of pregnant women with intrauterine growth retardation of different severity. The study included 100 pregnant women (from 23 to 40 weeks of gestation). The main group consisted of 80 pregnant women with intrauterine growth retardation. The control group consisted of 20 women with physiological course of pregnancy. The patients of the main group were divided into three clinical groups regarding intrauterine growth retardation staging. Group I included 38 pregnant women with stage I IUGR, 22 pregnant women with stage II IUGR were in group II and 20 pregnant women with stage III IUGR - in group III.Polyamine concentration in erythrocytes in the blood of pregnant women with intrauterine growth retardation was determined by using Agilent 1200 series (USA) high performance liquid chromatography [4]. The standards of polyamine hydrochlorides were obtained from Sigma-Aldrich Company (USA). The variational methods were used to make the statistical analysis of outcomes by standard licensed computer programs: STATISTICA 6.0, Microsoft Excel, ANOVA «Statistica¼. The study results were presented in the form of M±m and differences were considered reliable at р<0,05 by Student's t-criterion. The conducted study has revealed that polyamine concentration in erythrocytes in the blood of pregnant women with intrauterine growth retardation is drastically lower if compared with pregnant women with physiological course of pregnancy. At the same time the putrescine concentration is higher, andspermidineandspermine concentrations are significantly reduced in the pregnant women with intrauterine growth retardation in comparison with the control group.According to the obtained results the polyamine exchange proves to be disturbed in pregnant women with intrauterine growth retardation. The progression of polyamine imbalance depends on the severity of fetal growth retardation in pregnant women.