Carotenoids In Human Serum Research Articles

Simultaneous determination of fat-soluble vitamins and carotenoids in human serum using a nanostructured ionic liquid based microextraction method

The determination of fat-soluble vitamins and carotenoids in human serum provides reliable information for diagnosing malnutrition and for establishing appropriate intervention programs. Due to the complex composition of the biological samples, the efficient sample preparation is the key to the analysis. We report here a surface active ionic liquid (SAIL)-based dispersive liquid-liquid microextraction (DLLME) method coupled with a high performance liquid chromatography (HPLC) to determine four fat-soluble vitamins and six carotenoids in human serum simultaneously. Liquid crystal structures were formed in the extract phase. And the enrichment factor of the analytes treated by DLLME was 4 to 26 times of the traditional LLE method except lycopene. The limit of determination for these compounds was determined to be between 0.002 and 0.076 µg/mL. The accuracy was validated by the standard addition method with recoveries ranging from 82.4 to 114.1%. The intra-day and inter-day relative standard deviations were 2.76–12.63% and 4.01–13.54%, respectively. The proposed DLLME coupled with the HPLC method was successfully applied in the determination of fat-soluble micronutrients in human serum.

Determination of major carotenoids in human serum by liquid chromatography

A simple and rapid high performance liquid chromatographic (HPLC) method was developed to determine the major carotenoids in human serum. Lutein, β-carotene and lycopene were separated within 20 min using a C18 column and a mobile phase of methanol/methylene chloride (95:5, v/v) with sample solvent methanol/methylene chloride (45:55, v/v), detection at 476 nm and flow rate at 0.8 mL/min. A high recovery was achieved with this method, which amounted to 92, 90 and 87% for lutein, β -carotene and lycopene, respectively, while the detection limit was 5.0, 10.0 and 4.0 ng/mL. This method was applied to determine the major carotenoids in human serum samples collected from an elderly care center. A total of 62 samples were measured, and in most cases, β -carotene (17.5-1105.7 ng/mL) was present in the highest amount, followed by lutein (10.6-182.3 ng/mL) and lycopene (0-402 ng/mL).

Determination of Carotenoids in Human Serum and Breast Milk Using High Performance Liquid Chromatography Coupled with a Diode Array Detector (HPLC-DAD)

High performance liquid chromatography (HPLC) coupled with a diode array detector (HPLC-DAD) for the identification and quantification of carotenoids, namely all-trans lutein, zeaxanthin, β-cryptoxanthin, α-carotene, and β-carotene, in biological samples such as human serum and breast milk, has been developed and validated. Good chromatography separation was achieved using a binary mobile phase system on a YMC C30 column (150 × 2.1 mm, 3 µm) at 30 °C. Owing to the smaller column particle size and diameter of the column, the separation was achieved in 18 min, which is significantly reduced from the typical 30–40 min of other methods. The diode array detector (DAD) acquisition was set at a wavelength of 445 nm; 3D spectra ranging from wavelengths of 240–600 nm were also recorded. Peaks were identified by matching their retention time and spectra with those of standards. Quantification was achieved by internal standard calibration using echinenone as the internal standard. Good linearity was obtained for each compound (R2 > 0.9999). The method quantification limits (MQLs) for serum and breast milk were 10 ng/mL and 5 ng/mL, in matrix, respectively. A spike recovery study and standard reference material (SRM) from the National Institute of Standards and Technology (NIST) 968e analysis has proven that the method has a high degree of accuracy, precision, and robustness. The stability study showed that the carotenoid standard and sample extracts could be stored in a chilled autosampler at 8 °C up to 48 h without being comprised, which provides guidance on re-test time frames. The freeze/thaw process was found to be detrimental to carotenoids, and should always be avoided. Most importantly, UV standardization of the stock standard is to be performed prior to each assay, and simply taking the values on Certificate of Analysis (CoA) for calculation of the standard concentration is not recommended.

Highly sensitive and rapid profiling method for carotenoids and their epoxidized products using supercritical fluid chromatography coupled with electrospray ionization-triple quadrupole mass spectrometry

Epoxy carotenoids, which are products of carotenoid oxidation, are potential oxidative stress markers. However, it is difficult to profile epoxy carotenoids owing to their small amount and difficulty in their separation from hydroxy carotenoids. In this study, a high-performance analytical system based on supercritical fluid chromatography (SFC) coupled with tandem mass spectrometry (MS/MS) was developed for the simultaneous analysis of carotenoids and epoxy carotenoids. SFC is an effective separation technique for hydrophobic compounds, by which major carotenoids in human serum and their epoxidation products can be analyzed within 20 min. The use of MS/MS increased the sensitivity; the detection limit for each carotenoid was of the sub-fmol order. When the constructed method was applied to biological samples such as human serum and low-density lipoprotein (LDL), the precise detection of the target carotenoids was disturbed by several isomers. However, highly selective detection of epoxy carotenoids was performed by targeting product ions that were generated with a structure-specific neutral loss of 80Da. Furthermore, the sample volume needed for the analysis was only 0.1ml for the serum, indicating the efficiency of this system in performing small-scale analyses. Using the analytical system developed in this study, highly sensitive and selective analysis of epoxy carotenoids could be performed in a short time. These features show the usefulness of this system in application to screening analysis of carotenoid profiles that are easily modified by oxidative stress.

Suitability of ultra-high performance liquid chromatography for the determination of fat-soluble nutritional status (vitamins A, E, D, and individual carotenoids)

Our aim was to assess the suitability of ultra-high performance liquid chromatography (UHPLC) for the simultaneous determination of biomarkers of vitamins A (retinol, retinyl esters), E (alpha- and gamma-tocopherol), D (25-OH-vitamin D), and the major carotenoids in human serum to be used in clinical practice. UHPLC analysis was performed on HSS T3 column (2.1 x 100 mm; 1.8 microm) using gradient elution and UV-VIS detection. The system allows the simultaneous determination of retinol, retinyl palmitate, 25-OH-vitamin D, alpha- and gamma-tocopherol, lutein plus zeaxanthin, alpha-carotene, beta-carotene, alpha- and beta-cryptoxanthin and lycopene. The method showed a good linearity over the physiological range with an adequate accuracy in samples from quality control programs. Suitability of the method in clinical practice was tested by analyzing samples (n = 286) from patients. In conclusion, UHPLC constitutes a reliable approach for nutrient/biomarker profiling allowing the rapid, simultaneous and low-cost determination of vitamins A, E, and D (including vitamers and ester forms) and the major carotenoids in clinical practice.

Serum carotenoid concentrations in postmenopausal women from the United States with and without osteoporosis.

Antioxidant defenses may be compromised in osteoporotic women. Little is known about fruit and vegetable or carotenoid consumption among postmenopausal women. The primary carotenoids in human serum are alpha- and beta-carotene, lycopene, beta-cryptoxanthin, lutein, and zeaxanthin. This study investigated the interrelationships among serum carotenoid concentrations, fruit and vegetable intake, and osteoporosis in postmenopausal women (n = 59, 62.7 +/- 8.8 y). Bone density was assessed by dual energy x-ray absorptiometry and osteoporosis diagnosis was based upon T-scores. Serum samples (n = 53) and three-day diet records (n = 49) were analyzed. Logistic regression analyzed differences between carotenoids after adjusting for serum retinol; supplement usage; milk, yogurt, fruit, and vegetable intake; and body mass index (BMI). Pearson statistics correlated carotenoids with specific fruit or vegetable intake. Serum lycopene concentrations were lower in the osteoporosis group than controls (p = 0.03). Beta-cryptoxanthin intake was higher in the osteoporosis group (p = 0.0046). Total fruit and vegetable intakes were correlated with serum lycopene and beta-cryptoxanthin (p = 0.03, 0.006, respectively). Serum alpha-carotene concentration was associated with carrot intake, and zeaxanthin and beta-cryptoxanthin with lettuce intake. Carotenoids that may have beneficial skeletal effects are lower in women with osteoporosis. Research is needed to identify potential protective mechanisms or utilization of carotenoids during osteoporosis.

An improved HPLC method for determination of carotenoids in human serum

An HPLC method was developed to determine the various carotenoids in human serum. A C-30 column and a mobile phase of 100% methanol (A) and 100% methylene chloride (B) with the following gradient elution were used: 90% A and 10% B in the beginning, maintained for 5 min, decreased to 78% A at 15 min, 62% A at 30 min, 52% A at 40 min, 41% A at 50 min, 38% A at 55 min, maintained for 3 min, and returned to 100% A at 65 min. A total of 21 carotenoids, including all-trans forms of lutein, zeaxanthin, alpha-cryptoxanthin, beta-cryptoxanthin, alpha-carotene, beta-carotene and lycopene, as well as their 14 cis-isomers were resolved within 51 min at a flow rate of 1.0 mL/min and detection at 476 nm. all-trans-beta-Carotene was found to be present in highest amount (256.3-864.2 ng/mL), followed by all-trans-lycopene (64.4-569.2 ng/mL), all-trans-lutein (137.9-450.3 ng/mL), all-trans-alpha-cryptoxanthin (55.7-188.2 ng/mL), all-trans-beta-cryptoxanthin (43.1-134.5 ng/mL), all-trans-alpha-carotene (20.0-122.1 ng/mL) and all-trans-zeaxanthin (9.1-21.3 ng/mL). Similar trend was observed for cis-isomers of carotenoids.

Lycopene in serum, skin and adipose tissues after tomato-oleoresin supplementation in patients undergoing haemorrhoidectomy or peri-anal fistulotomy.

Lycopene, the main carotenoid found in tomatoes and tomato-based products, has been reported to be protective against several types of cancer. Assessment of changes in plasma concentration of carotenoids following ingestion of lycopene-rich food sources does not necessarily predict changes in lycopene concentration or distribution of its isomers in other body tissues. Our aim was to determine the relationship between concentrations of lycopene and other tomato carotenoids in human serum and body tissues after tomato-oleoresin supplementation. Tomato lycopene oleoresin (30 mg/d) or a placebo was administered for 1 to 7 weeks to seventy-five volunteers undergoing elective haemorrhoidectomy or peri-anal fistulotomy. Carotenoid concentration and isomer distribution in blood and in the surgically removed skin and adipose tissues was measured by HPLC. The serum concentration of lycopene increased after supplementation from 0.26 (SD 0.12) to 0.52 (SD 0.25) micromol/l (n 35; P<0.0001). In the placebo group (n 40), lycopene serum concentration did not change significantly. Serum lycopene concentration after treatment was 2.2-fold greater in the lycopene group than in the placebo group, a slightly higher ratio than that found in skin and adipose tissue (1.6- and 1.4-fold higher than the placebo, respectively). A significant correlation between serum and tissue concentrations was found for both beta-carotene and lycopene in the placebo group, whereas in the lycopene-supplemented group the correlation between serum and tissues remained the same for beta-carotene but for lycopene was weak. Lycopene supplementation did not significantly change the proportion of all-trans v. cis isomers in the serum and tissues, despite the fact that more than 90 % of the supplemented lycopene was in the all-trans form. These results show that tomato-oleoresin supplementation increases lycopene concentrations in serum and in adipose tissue and skin. The ability to increase lycopene levels in tissues is one of the prerequisites for using it as a food supplement with health benefits.

Amelioratory Effect of Dietary Ingestion of Lycopene and Tomato Rich in Lycopene on Learning Impairment in Senescence-Accelerated Mice (SAMP8).

We previously reported that the dietary ingestion of red bell pepper (Capsicum annuum L.) which contained a large amount of antioxidative carotenoids ameliorated the age-related phenomena of learning disorder and degradation of hair glossiness in the senescence-accelerated mouse (SAM). As lycopene is one of the most popular carotenoids in human serum and exists in abundance in tomato, we examined the effects of dietary lycopene and tomato rich in lycopene on the age-related disorders in SAMP8//Kgm, a mouse model of accelerated decline in learning and memory, and control SAMR1//Kgm mice. SAMP8 mice that received a diet containing 0.02% (w/w) lycopene or 20% (w/w) tomato powder showed much better acquisition in passive avoidance tasks than those given the control diet. Dietary lycopene and tomato had no effect on the ability of learning and memory in SAMR1 mice. Those observations indicated that the dietary ingestion of lycopene in tomato ameliorated the learning impairment in SAMP8.

Improved normal-phase and reversed-phase gradient high-performance liquid chromatography procedures for the analysis of retinoids and carotenoids in human serum, plant and animal tissues

Two high-performance liquid chromatography (HPLC) procedures, a rapid normal-phase isocratic method for the analysis primarily of retinol and retinoic acid on a 3 μ silica column, and a reversed-phase gradient method for the simultaneous analysis of retinoids and very polar to nonpolar carotenoids on a 3 μ C 18 column, are described. The normal-phase isocratic HPLC procedure is rapid (12 min), requires a sample size of 100 μl or less of serum, and is suitable for routine analysis of retinol in any serum, and of retinol and retinoic acid in serum after administration of retinoic acid. The reversed-phase gradient method is suitable for the simultaneous analysis of very polar to nonpolar carotenoids such as epoxy-xanthophylls and xanthophyll esters, along with other carotenoids and retinoids that occur normally in human serum and other plant and animal tissues. A run time of 30–70 min is necessary, depending on the presence or absence of xanthophyll esters in the sample.

The potential role of lycopene for human health.

Lycopene is one of the major carotenoids in Western diets and is found almost exclusively in tomatoes and tomato products. It accounts for about 50% of carotenoids in human serum. Among the common dietary carotenoids lycopene has the highest singlet oxygen quenching capacity in vitro. Other outstanding features are its high concentration in testes, adrenal gland and prostate. In contrast to other carotenoids its serum values are not regularly reduced by smoking or alcohol consumption but by increasing age. Remarkable inverse relationships between lycopene intake or serum values and risk have been observed in particular for cancers of the prostate, pancreas and to a certain extent of the stomach. In some of the studies lycopene was the only carotenoid associated with risk reduction. Its role in cancer risk reduction still needs to be clarified. Patients with HIV infection, inflammatory diseases and hyperlipidemia with and without lipid lowering treatment may have depleted lycopene serum concentrations. Before embarking on large-scale human trials the distribution of lycopene and its biological functions need to be further evaluated.

Liquid chromatographic determination of carotenoids in human serum using an engineered C 30 and a C 18 stationary phase

A C 30 stationary phase was specifically engineered for carotenoid separations, and carotenoid measurements using this column are compared with those obtained using a somewhat more conventional C 18 column. Both methods were used to contribute measurements for the certification of carotenoids in Standard Reference Material 968b, Fat-Soluble Vitamins and Cholesterol in Human Serum. Analytes were extracted from the serum into hexane. Measurements on the C 18 column were made using a gradient of acetonitrile, methanol, and ethyl acetate, which is described in detail elsewhere. Measurements on the C 30 column were made using a gradient of water, methanol, and methyl tert.-butyl ether.

Effect of smoking on serum nutrient concentrations in African-American women

The relationship between current cigarette smoking and serum concentrations of vitamins C, E, and A, and of five carotenoids in human serum were examined in 91 low-income, African-American women. General linear models were used to adjust geometric mean serum concentrations of micronutrients for age, dietary and supplement intakes, total energy intake, alcohol intake, medication use, body mass index, and serum concentrations of cholesterol and triglycerides. Among smokers, serum concentrations of alpha-carotene, beta-carotene, cryptoxanthin, and lycopene averaged only 71-79% of the concentrations among nonsmokers. Mean serum concentrations of vitamins C and E and lutein/zeaxanthin were only slightly lower among smokers relative to nonsmokers, and current smokers had higher serum concentrations of vitamin A. Among current smokers, mean serum concentrations of all five carotenoids decreased with an increase in the amount smoked. The negative effect of smoking on serum concentrations of antioxidant carotenoids may pose a serious health risk in low-income populations already at higher risk for many chronic diseases.

Liquid chromatographic method for the determination of carotenoids, retinoids and tocopherols in human serum and in food

A liquid chromatographic (LC) method has been developed for the quantitative measurement of the six major carotenoids in human serum (lutein, zeaxanthin, β-cryptoxanthin, lycopene, α-carotene, and β-carotene) as well as retinol, retinyl palmitate, α-tocopherol, γ-tocopherol, and δ-tocopherol. Several polar carotenoids, 2′,3′-anhydrolutein, α-cryptoxanthin, and geometric isomers of lycopene and β-carotene are also separated. Retinoids and carotenoids are monitored using a programmable ultraviolet—visible detector, while tocopherols are monitored using a fluorescence detector. The method uses a gradient containing acetonitrile, methanol, and ethyl acetate. Ammonium acetate is introduced with the methanol to minimize carotenoid losses on the LC column aggravated by the use of acetonitrile and ethyl acetate. The method is also applicable to the analysis of foods.

New simplified procedures for the extraction and simultaneous high-performance liquid chromatographic analysis of retinol, tocopherols and carotenoids in human serum

A short, simple extraction procedure and a sensitive reversed-phase high-performance liquid chromatographic assay, which utilizes isocratic elution and detection either by a photodiode-array detector or by two detectors set at 300 and 450 nm, have been developed to measure retinol, tocopherols and several carotenoids in human serum simultaneously. By relying on characteristic UV—visible spectra, seventeen carotenoids, retinol and α- and γ-tocopherols were identified in concentrated serum extracts by use of the three-dimensional data mode of a photodiode-array detector. The presence of some recently reported carotenoids in human serum has been confirmed.

Separation of beta-carotene and lycopene geometrical isomers in biological samples

The analysis of beta-carotene and lycopene, the two predominant carotenoids in human serum and tissues, was extended to the level of geometrical (cis-trans) isomers by using an improved reversed-phase HPLC methodology. We separated five geometrical isomers of beta-carotene and seven of lycopene in human serum and tissues. 13-cis-beta-Carotene was identified as the predominant cisisomer in human serum, contributing about 5% to total beta-carotene. In tissue, however, considerable amounts of 9-cis- and traces of 15-cis-beta-carotene were also detected. In contrast to beta-carotene, the lycopene isomer patterns in human serum and tissues are quite similar.

24] Extraction and analysis by high-performance liquid chromatography of carotenoids in human serum

This chapter discusses extraction and analysis by high performance liquid chromatography (HPLC) of carotenoids in human serum. The carotenoid pattern of human plasma or serum is not as complex as in vegetables and fruits. The major carotenoids occurring in human plasma are lutein, zeaxanthin, α-cryptoxanthin, β-cryptoxanthin, lycopene, α-carotene, and β-carotene. Although the trans forms predominate, cis forms of these carotenoids often occur in blood. The chapter discusses the criteria, which are important and critical in selecting the method for analysis. Continued work on the HPLC separation of carotenoids and retinoids during the past several years has made it possible to develop an extraction procedure and an isocratic HPLC procedure that enables simultaneous determination of all the major carotenoids and of retinol, retinyl esters, and the tocopherols in very small volumes of human serum. The details of the procedure are described in the chapter.

Carotenoids, retinoids and alpha-tocopherol in human serum: Identification and determination by reversed-phase HPLC

A rapid, simple and specific high performance liquid chromatographic procedure for assaying alpha- and beta-carotene is described. The method also enables the simultaneous determination of retinol and dl-alpha-tocopherol in human serum. The same chromatographic procedure can be used to assay the major carotenoids in human serum, provided analyses are replicated and the effluent is monitored at 450 nm. The conditions described also enable determination of licopene, cryptoxanthine and lutein with zeaxanthine. An aliquot of 0.5 ml serum is deproteinized with ethanol (0.5 ml) and extracted with petroleum ether (0.75 ml). The petroleum ether extract is evaporated until dry and then redissolved immediately with 0.5 ml of an eluent mixture consisting of methanol-hexane (85:15, v/v). Aliquots of 50 microl are then injected onto a 250 x 4.6 mm column packed with Spherisorb ODS-2. Owing to its good reproducibility, the procedure can be used for assays with external standards. Clinical applications are described for cases of hypercarotinemia associated with endocrine dysfunctions such as hypothyroidism and diabetes.

HPLC determination of carotenoids in human serum by gradient elution technique

Polarities of the carotenoids in human serum are very different; many nonpolar carotenoid hydrocarbons (e.g. β-carotene, lycopene) and highly polar hydroxycarotenoids (e. g. β-cryptoxanthin, zeaxanthin, lutein) can be found among them.