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Background: Oxidative enzymes targeting lignocellulosic substrates are presently classified into various auxiliary activity (AA) families within the carbohydrate-active enzyme (CAZy) database. Among these, the fungal AA3 glucose-methanol-choline (GMC) oxidoreductases with varying auxiliary activities are attractive sustainable biocatalysts and important for biological function. CAZy AA3 enzymes are further subdivided into four subfamilies, with the large AA3_2 subfamily displaying diverse substrate specificities. However, limited numbers of enzymes in the AA3_2 subfamily are currently biochemically characterized, which limits the homology-based mining of new AA3_2 oxidoreductases. Importantly, novel enzyme activities may be discovered from the uncharacterized parts of this large subfamily., Results: In this study, phylogenetic analyses employing a sequence similarity network (SSN) and maximum likelihood trees were used to cluster AA3_2 sequences. A total of 27 AA3_2 proteins representing different clusters were selected for recombinant production. Among them, seven new AA3_2 oxidoreductases were successfully produced, purified, and characterized. These enzymes included two glucose dehydrogenases (TaGdhA and McGdhA), one glucose oxidase (ApGoxA), one aryl alcohol oxidase (PsAaoA), two aryl alcohol dehydrogenases (AsAadhA and AsAadhB), and one novel oligosaccharide (gentiobiose) dehydrogenase (KiOdhA). Notably, two dehydrogenases (TaGdhA and KiOdhA) were found with the ability to utilize phenoxy radicals as an electron acceptor. Interestingly, phenoxy radicals were found to compete with molecular oxygen in aerobic environments when serving as an electron acceptor for two oxidases (ApGoxA and PsAaoA), which sheds light on their versatility. Furthermore, the molecular determinants governing their diverse enzymatic functions were discussed based on the homology model generated by AlphaFold., Conclusions: The phylogenetic analyses and biochemical characterization of AA3_2s provide valuable guidance for future investigation of AA3_2 sequences and proteins. A clear correlation between enzymatic function and SSN clustering was observed. The discovery and biochemical characterization of these new AA3_2 oxidoreductases brings exciting prospects for biotechnological applications and broadens our understanding of their biological functions., (© 2024. The Author(s).)

Background: The Carbohydrate-Active enZymes (CAZy) auxiliary activity family 3 (AA3) comprises flavin adenine dinucleotide-dependent (FAD) oxidoreductases from the glucose-methanol-choline (GMC) family, which play auxiliary roles in lignocellulose conversion. The AA3 subfamily 1 predominantly consists of cellobiose dehydrogenases (CDHs) that typically comprise a dehydrogenase domain, a cytochrome domain, and a carbohydrate-binding module from family 1 (CBM1)., Results: In this work, an AA3_1 gene from T. myriococcoides CBS 398.93 encoding only a GMC dehydrogenase domain was expressed in Aspergillus niger. Like previously characterized CDHs, this enzyme (TmXdhA) predominantly accepts linear saccharides with β-(1 → 4) linkage and targets the hydroxyl on the reducing anomeric carbon. TmXdhA was distinguished, however, by its preferential activity towards xylooligosaccharides over cellooligosaccharides. Amino acid sequence analysis showed that TmXdhA possesses a glutamine at the substrate-binding site rather than a threonine or serine that occupies this position in previously characterized CDHs, and structural models suggest the glutamine in TmXdhA could facilitate binding to pentose sugars., Conclusions: The biochemical analysis of TmXdhA revealed a catalytic preference for xylooligosaccharide substrates. The modeled structure of TmXdhA provides a reference for the screening of oxidoreductases targeting xylooligosaccharides. We anticipate TmXdhA to be a good candidate for the conversion of xylooligosaccharides to added-value chemicals by its exceptional catalytic ability., (© 2022. The Author(s).)

Pyranose dehydrogenases (PDHs; EC 1.1.99.29; AA3_2) demonstrate ability to oxidize diverse carbohydrates. Previous studies of these enzymes have also uncovered substrate-dependent regioselectivity, along with potential to introduce more than one carbonyl into carbohydrate substrates. Enzymatic oxidation of carbohydrates facilitates their further derivatization or polymerization into bio-based chemicals and materials with higher value; accordingly, PDHs that show activity on xylooligosaccharides could offer a viable approach to extract higher value from hemicelluloses that are typically fragmented during biomass processing. In this study, AbPDH1 from Agaricus bisporus and AmPDH1 from Leucoagaricus meleagris were tested using linear xylooligosaccharides, along with xylooligosaccharides substituted with either arabinofuranosyl or 4- O -(methyl)glucopyranosyluronic acid residues with degree of polymerization of two to five. Reaction products were characterized by HPAEC-PAD to follow substrate depletion, UPLC-MS-ELSD to quantify the multiple oxidation products, and ESI-MS n to reveal oxidized positions. A versatile method based on product reduction using sodium borodeuteride, and applicable to carbohydrate oxidoreductases in general, was established to facilitate the identification and quantification of oxidized products. AbPDH1 activity toward the tested xylooligosaccharides was generally higher than that measured for AmPDH1. In both cases, activity values decreased with increasing length of the xylooligosaccharide and when using acidic rather than neutral substrates; however, AbPDH1 fully oxidized all linear xylooligosaccharides, and 60-100% of all substituted xylooligosaccharides, after 24 h under the tested reaction conditions. Oxidation of linear xylooligosaccharides mostly led to double oxidized products, whereas single oxidized products dominated in reactions containing substituted xylooligosaccharides. Notably, oxidation of specific secondary hydroxyls vs. the reducing end C-1 depended on both the enzyme and the substrate. For all substrates, however, oxidation by both AbPDH1 and AmPDH1 was clearly restricted to the reducing and non-reducing xylopyranosyl residues, where increasing the length of the xylooligosaccharide did not lead to detectable oxidation of internal xylopyranosyl substituents. This detailed analysis of AbPDH1 and AmPDH1 action on diverse xylooligosaccharides reveals an opportunity to synthesize bifunctional molecules directly from hemicellulose fragments, and to enrich for specific products through appropriate PDH selection., (Copyright © 2020 Karppi, Zhao, Chong, Koistinen, Tenkanen and Master.)

Plant-derived carbohydrates are an abundant renewable resource. Transformation of carbohydrates into new products, including amine-functionalized building blocks for biomaterials applications, can lower reliance on fossil resources. Herein, biocatalytic production routes to amino carbohydrates, including oligosaccharides, are demonstrated. In each case, two-step biocatalysis was performed to functionalize d-galactose-containing carbohydrates by employing the galactose oxidase from Fusarium graminearum or a pyranose dehydrogenase from Agaricus bisporus followed by the ω-transaminase from Chromobacterium violaceum (Cvi-ω-TA). Formation of 6-amino-6-deoxy-d-galactose, 2-amino-2-deoxy-d-galactose, and 2-amino-2-deoxy-6-aldo-d-galactose was confirmed by mass spectrometry. The activity of Cvi-ω-TA was highest towards 6-aldo-d-galactose, for which the highest yield of 6-amino-6-deoxy-d-galactose (67 %) was achieved in reactions permitting simultaneous oxidation of d-galactose and transamination of the resulting 6-aldo-d-galactose., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Aim: Low-density lipoprotein cholesterol (LDL-c) and high-density lipoprotein cholesterol (HDL-c), which are components of total cholesterol, have each been suggested to be linked to the risk of sudden cardiac death (SCD). However, the relationship between LDL-c/HDL-c ratio and the risk of SCD has not been previously investigated. We aimed to assess the associations of LDL-c, HDL-c, and the ratio of LDL-c/HDL-c with the risk of SCD., Methods: Serum lipoprotein concentrations were assessed at baseline in the Finnish Kuopio Ischemic Heart Disease prospective cohort study of 2,616 men aged 42-61 years at recruitment. Hazard ratios (HRs) (95% confidence intervals [CI]) were assessed., Results: During a median follow-up of 23.0 years, a total of 228 SCDs occurred. There was no significant evidence of an association of LDL-c or HDL-c with the risk of SCD. In analyses adjusted for age, examination year, body mass index, systolic blood pressure, smoking, alcohol consumption, physical activity, years of education, diabetes, previous myocardial infarction, family history of coronary heart disease, and serum high sensitivity C-reactive protein, there was approximately a two-fold increase in the risk of SCD (HR 1.94, 95% CI 1.21-3.11; p=0.006), comparing the top (＞4.22) versus bottom (≤2.30) quintile of serum LDL-c/HDL-c ratio., Conclusion: In this middle-aged male population, LDL-c or HDL-c was not associated with the risk of SCD. However, a high serum LDL-c/HDL-c ratio was found to be independently associated with an increased risk of SCD. Further research is warranted to understand the mechanistic pathways underlying this association.

There is limited knowledge on the relationship between kidney function and incidence of atrial fibrillation. Thus, this prospective study was designed to evaluate whether various biomarkers of kidney function are associated to the risk of atrial fibrillation. The study population consisted of 1840 subjects (615 women and 1225 men) aged 61-82 years. Cystatin C- and creatinine-based estimation of glomerular filtration rate (eGFRcys and eGRFcreat , respectively) and urinary albumin/creatinine ratio (ACR) were assessed to investigate their relationship with the risk of atrial fibrillation. During a median follow-up of 3.7 years, a total of 159 incident atrial fibrillation cases occurred. After adjustment for potential confounders, the risk of atrial fibrillation was increased (hazard ratio 2.74, 95% confidence interval (CI) 1.56-4.81, P < 0.001) in subjects with reduced kidney function (eGFRcys , 15-59 mL/min per 1.73 m(2) ) compared to subjects with normal kidney function (≥90 mL/min per 1.73 m(2) ). Similar results were also found when comparing the respective groups of subjects defined by their eGRFcreat levels (hazard ratio 2.41, CI 1.09-5.30, P = 0.029). Consistently, subjects with ACR ≥300 mg/g had an increased risk of incident atrial fibrillation (hazard ratio 2.16, CI 1.35-2.82, P < 0.001) compared to those with ACR <30 mg/g. Reduced eGFR and albuminuria were associated with an increased risk of atrial fibrillation., (© 2016 Asian Pacific Society of Nephrology.)

Background: Left ventricular (LV) mass ascertained using echocardiography may enhance risk stratification for sudden cardiac death. The objective of this study was to assess the association between left ventricular mass and the risk of sudden cardiac death in a population-based cohort and determine its incremental value beyond conventional risk predictors., Methods and Results: Assessment of LV mass was based on echocardiography in a sample of 905 middle-aged men representative of the general population (aged 42 to 61 years). During the follow-up period of 20 years, there were a total of 63 sudden cardiac deaths. In a comparison of the top versus the bottom quartile of LV mass adjusted by body surface area (>120 versus <89 g/m(2)), the multivariable adjusted hazard ratio was 2.57 (95% CI 1.24 to 5.31, P=0.010). Further adjustment for LV function only modestly attenuated the risk of sudden cardiac death among men with LV mass of >120 g/m(2) (hazard ratio 2.29, 95% CI 1.10 to 4.74, P=0.026). Addition of LV mass adjusted by body surface area to a conventional risk factor model for sudden cardiac death improved the integrated discrimination index by 0.033 (95% CI 0.009 to 0.057, P=0.007) and the category-free net reclassification index by 0.501 (95% CI 0.092 to 0.911, P=0.016)., Conclusions: Indexed LV mass by body surface area is an independent predictor of sudden cardiac death and may help improve the risk prediction of sudden cardiac death beyond conventional cardiovascular risk factors., (© 2014 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Background: Fruit and vegetable intake has been associated with lower risk for cardiovascular diseases, but data on congestive heart failure (CHF) are inconsistent. The association of serum carotenoids, biomarkers reflecting fruit and vegetable intake, with the risk of CHF has not been well documented in previous studies. We therefore examined the association between carotenoid levels and the risk of CHF., Methods: Data were available for 1031 males aged 46 to 65 years participating in the Kuopio Ischaemic Heart Disease Risk Factor Study (Finland). Baseline data for the present study were collected between 1991 and 1993. The association between serum concentrations of carotenoids and the risk of CHF was examined by using Cox proportional hazard models., Results: During the median of 17.8 follow-up years, CHF occurred in 72 patients. Age and examination year adjusted risk (hazard ratio, HR) for CHF among men within the lowest quartile of serum β-carotene was 4.08 (95% CI, 1.90-8.78, p<0.001) as compared to men in the highest quartile of serum β-carotene. After further adjustment for many potential confounders, men with the lowest quartile of β-carotene had almost 3-fold increased risk of CHF (HR=2.78, 95% CI, 1.23-6.25, p=0.014). However, serum concentrations of lycopene and α-carotene were not related to the risk of CHF., Conclusions: The present study suggests that low concentrations of serum β-carotene may be associated with an increased risk of CHF., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Background: Several previous epidemiologic studies have shown that high blood levels of carotenoids may be protective against early atherosclerosis, but results have been inconsistent. We assessed the association between atherosclerotic progression, measured by intima-media thickness of the common carotid artery wall, and serum levels of carotenoids., Methods: We studied the effect of carotenoids on progression of early atherosclerosis in a population-based study. The association between concentrations of serum carotenoids, and intima-media thickness of the common carotid artery wall was explored in 840 middle-aged men (aged 46-65 years) from Eastern Finland. Ultrasonography of the common carotid arteries were performed at baseline and 7-year follow-up. Serum levels of carotenoids were analyzed at baseline. Changes in mean and maximum intima media thickness of carotid artery wall were related to baseline serum carotenoid levels in covariance analyses adjusted for covariates., Results: In a covariance analysis with adjustment for age, ultrasound sonographer, maximum intima media thickness, examination year, body mass index, systolic blood pressure, smoking, physical activity, serum LDL cholesterol, family history of coronary heart disease, antihypertensive medication and serum high sensitivity C-reactive protein, 7-year change in maximum intima media thickness was inversely associated with lycopene (p = 0.005), α-carotene (p = 0.002) and β-carotene (p = 0.019), respectively., Conclusions: The present study shows that high serum concentrations of carotenoids may be protective against early atherosclerosis.

Objective: The aim of the study was to determine whether impaired fasting plasma glucose (FPG) and type 2 diabetes may be risk factors for sudden cardiac death (SCD)., Research Design and Methods: This prospective study was based on 2,641 middle-aged men 42-60 years of age at baseline. Impaired FPG level (≥5.6 mmol/L) among nondiabetic subjects (501 men) was defined according to the established guidelines, and the group with type 2 diabetes included subjects (159 men) who were treated with oral hypoglycemic agents, insulin therapy, and/or diet., Results: During the 19-year follow-up, a total of 190 SCDs occurred. The relative risk (RR) for SCD was 1.51-fold (95% CI 1.07-2.14, P = 0.020) for nondiabetic men with impaired FPG and 2.86-fold (1.87-4.38, P < 0.001) for men with type 2 diabetes as compared with men with normal FPG levels, after adjustment for age, BMI, systolic blood pressure, serum LDL cholesterol, smoking, prevalent coronary heart disease (CHD), and family history of CHD. The respective RRs for out-of-hospital SCDs (157 deaths) were 1.79-fold (1.24-2.58, P = 0.001) for nondiabetic men with impaired FPG and 2.26-fold (1.34-3.77, P < 0.001) for men with type 2 diabetes. Impaired FPG and type 2 diabetes were associated with the risk of all-cause death. As a continuous variable, a 1 mmol/L increment in FPG was related to an increase of 10% in the risk of SCD (1.10 [1.04-1.20], P = 0.001)., Conclusions: Impaired FPG and type 2 diabetes represent risk factors for SCD.

Systemic inflammation, oxidative stress, and atrial fibrosis are identified as the main mechanisms for atrial fibrillation (AF). Antioxidant micronutrients may have antiarrhythmic benefit in humans. We studied whether plasma carotenoids are related to the risk of AF in elderly subjects. The study population consisted of 1,847 Finnish subjects (620 women and 1,227 men) aged 61-82 years from the Kuopio Ischaemic Heart Disease Risk Factor study cohort. Plasma concentrations of carotenoids were measured by high-performance liquid chromatography. The multivariable-adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) of plasma carotenoids were analyzed using the Cox proportional hazard's model. During a median follow-up of 2.8 years a total of 105 subjects developed incident AF. The HRs of AF among subjects with levels in the highest through the lowest tertile after adjustment for age, examination year, gender, systolic blood pressure, smoking, alcohol consumption, education, diabetes, use of antihypertensive medication, congestive heart failure, recurrent AF, prevalent coronary heart disease and baseline prevalence of myocardial infarction were 1.70 (95% CI: 1.01-2.86, p = 0.047) for lutein, 1.99 (95% CI: 1.14-3.48, p = 0.016) for zeaxanthin. Risk of AF was borderline significant for β-carotene (HR = 1.69, 95 % CI: 1.00-2.87, p = 0.050). Results remained unchanged, when season was added to the multivariable model. Lycopene, β-cryptoxanthin, α-carotene and total carotenoids were not associated with the risk of AF. Results of the present study suggest that low plasma concentrations of lutein and zeaxanthin were associated with an increased risk of AF in elderly population.

Objectives: To examine whether serum concentrations of carotenoids are related to the risk of sudden cardiac death (SCD) in middle-aged men., Methods: The study population consisted of 1031 Finnish men aged 46-65 years of the Kuopio Ischemic Heart Disease Risk Factor (KIHD) cohort. Serum concentrations of carotenoids were measured by high-performance liquid chromatography. The hazard ratios (HR) of serum β-carotene, lycopene and α-carotene were estimated by using the Cox proportional hazard model after adjusting for age and other potential confounding factors., Results: During a median follow-up of 15.9 years a total of 59 incidents of SCD occurred. After controlling for age, systolic blood pressure, waist circumference, smoking, alcohol consumption, years of education, serum LDL cholesterol, serum hs-CRP, diabetes, prevalent coronary heart disease (CHD) and congestive heart failure (CHF), men in the lowest tertile of serum concentrations of β-carotene had a 2-fold increased risk of SCD (HR=2.15, 95% CI: 1.02-4.51; p=0.044) as compared to those in the highest tertile. The risk of SCD was borderline significant for lycopene. In addition, low serum β-carotene concentrations increased the risk of cardiovascular disease (CVD) and total mortality. Lycopene and α-carotene were not related to the risk of SCD., Conclusions: Our findings suggest that low serum β-carotene concentrations may increase the risk of SCD in middle-aged Finnish men. Furthermore, low serum β-carotene concentrations may be related to the risk of CVD and total mortality., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Objective: Previous studies have shown that high intake or concentrations of serum carotenoids may protect against acute myocardial infarction (AMI). The role of carotenoids on the risk of AMI remains inconsistent. The aim of the present study was to examine if serum concentrations of major carotenoids are related to AMI in men., Methods: The study population consisted of 1031 Finnish men aged 46-65 years in the Kuopio Ischaemic Heart Disease Risk Factor (KIHD) cohort. Serum concentrations of carotenoids, retinol and α-tocopherol were measured by high-performance liquid chromatography. The association between the serum concentrations of lycopene α-carotene and β-carotene and the risk of AMI was studied by using the Cox proportional hazard models., Results: A total of 194 incident AMI cases occurred during an average of 11.5 follow-up years. After adjusting for potential confounders, the risk of AMI for men in the lowest tertile of serum concentrations compared with men in the highest tertile was 1.55 (95% CI 1.05- 2.30; P = 0.028) for lycopene and 1.60 (95% CI 1.09-2.35; P = 0.017) for β-carotene., Conclusions: This cross-sectional study shows that low serum lycopene and β-carotene concentrations may increase the risk of AMI in men.

Oxidative stress plays an important role in cataractogenesis. Previous studies have shown that long-term dietary intake of antioxidants (lutein and zeaxanthin) may decrease the risk of age-related cataracts. The aim of the present study was to examine whether plasma concentrations of lutein and zeaxanthin are related to age-related nuclear cataract in the elderly population. Subjects were participants in the Kuopio Ischaemic Heart Disease Risk Factor Study and they were classified into tertiles according to plasma concentrations of lutein and zeaxanthin. The association of plasma lutein and zeaxanthin concentrations with age-related nuclear cataract in 1689 elderly subjects (aged 61-80 years) was investigated in the present cross-sectional study by using the Cox proportional hazards model. A total of 113 cases of incident age-related cataracts were confirmed, of which 108 cases were nuclear cataracts. After adjustment for age, examination year, sex, BMI, smoking, alcohol consumption, serum LDL-cholesterol, serum HDL-cholesterol, years of education, use of oral corticosteroids, history of diabetes and history of hypertension with current use of antihypertensive medication, subjects in the highest tertiles of plasma concentrations of lutein and zeaxanthin had 42 and 41 % lower risks of nuclear cataract, respectively, compared with those in the lowest tertiles (relative risk (RR) = 0·58, 95 % CI 0·35, 0·98; P = 0·041 for lutein and RR = 0·59, 95 % CI 0·35, 0·99; P = 0·046 for zeaxanthin). In conclusion, we suggest that high plasma concentrations of lutein and zeaxanthin were associated with a decreased risk of age-related nuclear cataract in the elderly population.

Results from epidemiologic studies on the association between circulating carotenoid concentrations and the risk of prostate cancer are still inconsistent. We studied whether serum concentrations of carotenoids were associated with the risk of developing prostate cancer. The study population consisted of 997 middle-aged Finnish men (56.1 ± 6.6 yr) in the Kuopio Ischaemic Heart Disease Risk Factor (KIHD) cohort. Serum concentrations of carotenoids were measured by high-performance liquid chromatography. Subjects were classified into tertiles according to their serum concentrations of antioxidants. Relative risks (RRs) were estimated by using the Cox proportional hazard models. During the mean follow-up time of 15 yr, a total of 68 prostate cancer cases occurred. After adjusting for age, examination yr, family history of cancer, BMI, pack-yr of smoking, alcohol consumption, education, physical activity, serum total cholesterol, and serum α-linolenic acid, men in the highest tertile of serum concentrations of β-carotene had 2.3-fold higher risk of prostate cancer as compared to those in the lowest tertile (RR = 2.29, 95% CI: 1.12-4.66; P = 0.023). α-Tocopherol and retinol were not associated with prostate cancer. This prospective study suggests that high-serum β-carotene concentrations may increase the risk of prostate cancer in middle-aged men.

Oxidative modification of low-density lipoprotein (LDL) in the vascular endothelium is considered to be important in the development of early atherosclerosis. The aim of this study was to investigate the main determinants of serum LDL conjugated dienes in women (n=124) and men (n=225). We focused on the influence of fat-soluble vitamins and carotenoids on the concentration of conjugated dienes in LDL. In multivariate linear regression models, including age, body mass index, diastolic blood pressure, symptomatic ischaemic heart disease (IHD) or IHD history, statin medication, leukocytes and serum triglycerides as covariates, plasma lycopene (standardized beta=-0.33; P=0.002) and lutein (standardized beta=-0.22; P=0.027) concentrations were the strongest determinants of serum LDL conjugated dienes in women, whereas plasma beta-carotene (standardized beta=-0.23; P=0.002) was the most important factor in men. Furthermore, statin medication, diastolic blood pressure, age and serum triglycerides were significant determinants of LDL conjugated dienes. The regression model with lycopene contributed to 29% in women and 15% in men with beta-carotene of the variation of serum LDL conjugated dienes. Results of the present study suggest that plasma lycopene, lutein and beta-carotene are the most powerful antioxidants for explaining the content of in vivo oxidatively modified LDL in serum., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)

Purpose: Lycopene is thought to decrease the risk of cancers, although previous epidemiologic studies have produced inconsistent results. The aim of the present study was to evaluate the protective effect of lycopene against the risk of cancer., Methods: The study population consisted of 997 middle-aged Finnish men in the Kuopio Ischaemic Heart Disease Risk Factor (KIHD) cohort. During the mean follow-up time of 12.6 years, a total of 141 cancer cases appeared, of which 55 were prostate cancers. The association between the serum concentrations of lycopene and the risk of cancer was studied using the Cox proportional hazard models., Results: An inverse association was observed between serum lycopene and overall cancer incidence. The adjusted risk ratio (RR) in the highest tertile of serum lycopene was 0.55 (95% confidence interval [CI], 0.34-0.89; p=0.015) compared with the lowest serum lycopene group. No association was observed between the lycopene concentrations and a prostate cancer risk. RR for other cancers was 0.43 (95% CI, 0.23-0.79; p=0.007)., Conclusions: These findings suggest that in middle-aged men, the higher circulating concentrations of lycopene may contribute to the lower risk of cancer, with the exception of prostate cancer.

The present study aimed to evaluate the suitability of Smopex-102 cation-exchange fiber for the separation of acidic and basic model drugs from biological fluids (e.g. serum) prior to chromatographic analysis. In addition, the interactions of the drugs with the fiber were studied. The study found that basic antidepressant model drugs bound to a considerably greater extent than acidic drugs to poly(acrylic acid) (PAA) grafted Smopex-102 cation-exchange fiber from 25 mM HEPES buffer (pH 7.0) and spiked serum. Drug binding from serum decreased except for acidic drugs due to drug distribution between serum proteins and cation-exchange fiber. Electrostatic interactions were possibly the most important factors affecting drug binding to the fiber. Basic drugs were released most effectively from the fiber by using acetic acid (mean released amount 123.7 +/- 36.3% and mean absolute recovery 95.4 +/- 23.8%). Results demonstrated that the cation-exchange fiber evaluated might be a potential material for separating basic drugs from protein-free and proteinaceous (e.g. serum) liquid solutions for subsequent monitoring and evaluation. However, the drug release solution and release time must be optimized more precisely in order to validate described sample preparation method for each basic drug.

A simple and sensitive isocratic reversed-phase high-performance liquid chromatography (HPLC) method for simultaneous determination of retinol, alpha-tocopherol and six carotenoids in human plasma was described. Sample preparation of the earlier published method was further developed by addition of ultrapure water, which enabled aqueous layer to freeze facilitating phase separation without pipetting thus also improving precision of the method. Developed method appeared to be less laborious and time consuming compared to the traditional extraction methods, which require removal of organic layer by pipetting. The recoveries (absolute and relative) were between 80% and 103%. The intra-assay CVs were 1.1-4.0% (normal level) and 3.3-9.0% (low level). Inter-assay CVs were 5.3-8.8%. Reference method for all these analytes was not available, but a comparison with another published method was carried out. The results of the comparison matched satisfactorily. The method is used routinely in our laboratory in a large population-based study.

Isolation of acidic and basic model drugs by using pH sensitive poly(acrylic acid) grafted poly(vinylidene fluoride) (PAA-PVDF) cation-exchange membrane from biological fluids was reported. Effects of drug charge and lipophilicity on adsorption were also investigated. In the present study, basic model drugs adsorbed to a considerably greater extent onto the membrane than acidic drugs. Albumin was not adsorbed onto the membrane. Results of our study exposed, that electrostatic interactions between positively charged basic drug and negatively charged PVDF-PAA membrane were the most important factor affecting drug adsorption onto the membrane. Adsorption of acidic and basic drugs onto the PVDF-PAA membrane was not related to drug lipophilicity. The results of present study demonstrated that basic drugs adsorbed extensively onto the membrane, but albumin did not, proposing that PAA-PVDF membrane may be suitable for isolating basic drugs from proteinaceous biological fluids (i.e. serum) for subsequent monitoring and evaluation.

The influence of charge and lipophilicity of acidic and basic model drugs on their adsorption onto poly(N,N-dimethyl aminoethyl methacrylic acid) grafted poly(vinylidene fluoride) (DMAEMA-PVDF) membranes was evaluated. The effect of serum proteins (albumin, IgG) and hormones (cortisol, free thyroxine (T(4)F) and thyrotropin (TSH)) on drug adsorption was also studied. Acidic model drugs (antiepileptics and benzodiazepies) adsorbed to a greater extent onto the membrane from Hepes buffer at ionic strength of 25mM and pH 7.0 than basic drugs (antidepressants) did. Adsorption of acidic model drugs was based on electrostatic interactions between positively charged tertiary amino groups of DMAEMA side-chain and acidic negatively charged drug. Albumin diminished the adsorption of drugs from serum onto the membrane. Lipophilicity was related to the adsorption of acidic model drugs from serum onto the membrane. The degree of grafting had the greatest effect on adsorption of lipophilic drugs, but no influence was observed on adsorption of hydrophilic drugs. The present results showed that acidic drugs and albumin adsorbed onto the membrane, which suggests that the PVDF-DMAEMA membrane may be suitable for separating acidic drugs from protein-free substances for subsequent monitoring and evaluation.

Astaxanthin, the main carotenoid pigment in aquatic animals, has greater antioxidant activity in vitro (protecting against lipid peroxidation) and a more polar configuration than other carotenoids. We investigated the effect of three-month astaxanthin supplementation on lipid peroxidation in healthy non-smoking Finnish men, aged 19-33 years by using a randomized double-blind study design. Also absorption of astaxanthin from capsules into bloodstream and its safety were evaluated. The intervention group received two 4-mg astaxanthin (Astaxin) capsules daily, and the control group two identical-looking placebo capsules. Astaxanthin supplementation elevated plasma astaxanthin levels to 0.032 pmol/L (p < 0.001 for the change compared with the placebo group). We observed that levels of plasma 12- and 15-hydroxy fatty acids were reduced statistically significantly in the astaxanthin group (p = 0.048 and p = 0.047 respectively) during supplementation, but not in the placebo group and the change of 15-hydroxy fatty acid was almost significantly greater (p = 0.056) in the astaxanthin group, as compared with the placebo group. The present study suggests that intestinal absorption of astaxanthin delivered as capsules is adequate, and well tolerated. Supplementation with astaxanthin may decrease in vivo oxidation of fatty acids in healthy men.

Oxidation of LDL generates biologically active platelet-activating factor (PAF)-like phospholipid derivatives, which have potent proinflammatory activity. These products are inactivated by lipoprotein-associated phospholipase A2 (Lp-PLA2), an enzyme capable of hydrolyzing PAF-like phospholipids. In this study, we generated an adenovirus (Ad) encoding human Lp-PLA2 and injected 10(8), 10(9), and 10(10) plaque-forming unit doses of Adlp-PLA2 and control AdlacZ intra-arterially into rabbits to achieve overexpression of Lp-PLA2 in liver and in vivo production of Lp-PLA2-enriched LDL. As a result, LDL particles with 3-fold increased Lp-PLA2 activity were produced with the highest virus dose. Increased Lp-PLA2 activity in LDL particles decreased the degradation rate in RAW 264 macrophages after standard in vitro oxidation to 60-80% compared with LDL isolated from LacZ-transduced control rabbits. The decrease was proportional to the virus dose and Lp-PLA2 activity. Lipid accumulation and foam cell formation in RAW 264 macrophages were also decreased when incubated with oxidized LDL containing the highest Lp-PLA2 activity. Inhibition of the Lp-PLA2 activity in the LDL particles led to an increase in lipid accumulation and foam cell formation. It is concluded that increased Lp-PLA2 activity in LDL attenuates foam cell formation and decreases LDL oxidation and subsequent degradation in macrophages., (Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.)

In this study, we present significant changes occurring in serum drug concentrations while using blood collection tubes that contain a barrier gel. This report also contains results with antidepressant drugs, which have not been studied before with human samples. The drug concentrations were measured either with high performance liquid chromatography (HPLC) or fluorescence polarization immunoassay (FPIA). The results show that gel tubes are suitable for blood collection for antiepileptic, antibiotic, asthma and cardioactive drug measurements, since only slight adsorption was seen (0-5%). However, the studied tubes are not suitable for blood collection of antidepressants nor benzodiazepines, because the adsorption can be 5-30%. The adsorption was even higher (up to 40%) when samples were stored for 24 h after centrifugation in gel tubes. When the centrifugation step was performed after storage the effect of the barrier gel was lower (only 0-13%). Antidepressant drug measurements performed from patient specimens collected in the studied gel tubes and stored for 3 h showed <10% adsorption of the studied drugs. After 24 h storage time, concentrations of all analysed drugs decreased even more: adsorbed amount of drugs were about 5-20%. The studied gel tubes are proposed to be satisfactory for blood collection for antidepressant drug measurements if separation step is performed within 3 h after blood clotting. With the spiked samples the adsorption to barrier gel was higher, so it seems that adsorption is faster when drugs are not highly bound to serum proteins.

The influence of pH, ionic strength and the concentration of albumin in the adsorption medium as well as the charge and lipophilicity of a model drug on their adsorption onto poly(acrylic acid) grafted poly(vinylidene fluoride) (PAA-PVDF) membranes was evaluated. The PAA-PVDF membrane is a responsive porous polymer membrane that we have studied for controlled drug delivery. Sodium salicylate (anionic), flunitrazepam (neutral), primidone (neutral), desipramine (cationic) and thioridazine (cationic) were used as model drugs. The extent of drug adsorption was dependent on pH. Drug adsorption was enhanced by the dissociation of the grafted PAA chains and by a positive charge and a high lipophilicity of the drug. Increasing the ionic strength of the medium retarded the adsorption of the cationic drugs. Interestingly, the present results showing that drugs are adsorbed onto the membrane while albumin is not adsorbed onto the membrane suggest that the PAA-PVDF membrane may be suitable for separating drugs from proteinaceous substances for subsequent monitoring and evaluation.