Carotenoid measurement

EPR studies of host-guest complexes of carotenoids Measuring distances between carotenoid radicals and distant metals in matrices by using ESEEM methods and pulsed EPR relaxation techniques EPR studies of radical cations on activated alumina and silica-alumina... 

Muller L, Frohlich K, Bohm V. Comparative antioxidant activities of carotenoids measured by ferric reducing antioxidant power (FRAP), ABTS bleaching assay (aTEAC), DPPH assay and peroxyl radical scavenging assay. Food Chemistry. 2011 129 139-148... 

F4.2 Extraction of Photosynthetic Tissues Chlorophylls and Carotenoids F4.3 Chlorophylls and Carotenoids Measurement and Characterization by UV-VIS Spectroscopy... 

This example illustrates several important lessons. First, this application takes advantage of Raman signal enhancements to measure concentrations normally considered too low for Raman spectroscopy. Extremely strong Raman signal is a hallmark of carotenoid measurements. Traditionally, this enhancement has been considered a resonance effect but others believe it is due to coupling between 7r-electrons and phonons.37 It can be beneficial to use such enhancements whenever possible. 

More specific methods involve chromatographic separation of the retinoids and carotenoids followed by an appropriate detection method. This subject has been reviewed (57). Typically, hplc techniques are used and are coupled with detection by uv. For the retinoids, fluorescent detection is possible and picogram quantities of retinol in plasma have been measured (58—62). These techniques are particularly powerful for the separation of isomers. Owing to the thermal lability of these compounds, gc methods have also been used but to a lesser extent. Recently, the utiUty of cool-on-column injection methods for these materials has been demonstrated (63). 

The antioxidant activities of carotenoids and other phytochemicals in the human body can be measured, or at least estimated, by a variety of techniques, in vitro, in vivo or ex vivo (Krinsky, 2001). Many studies describe the use of ex vivo methods to measure the oxidisability of low-density lipoprotein (LDL) particles after dietary intervention with carotene-rich foods. However, the difficulty with this approach is that complex plant foods usually also contain other carotenoids, ascorbate, flavonoids, and other compounds that have antioxidant activity, and it is difficult to attribute the results to any particular class of compounds. One study, in which subjects were given additional fruits and vegetables, demonstrated an increase in the resistance of LDL to oxidation (Hininger et al., 1997), but two other showed no effect (Chopra et al, 1996 van het Hof et al., 1999). These differing outcomes may have been due to systematic differences in the experimental protocols or in the populations studied (Krinsky, 2001), but the results do indicate the complexity of the problem, and the hazards of generalising too readily about the putative benefits of dietary antioxidants. 

The absorption and transport processes of many of the phytochemicals present in food are complex and not fully understood, and prediction of their bioavailability is problematic. This is particularly true of the lipid-soluble phytochemicals. In this chapter the measurement of carotenoid bioavailability will be discussed. The carotenoids serve as an excellent example of where too little understanding of food structure, the complexity of their behaviour in foods and human tissues, and the nature and cause of widely different individual response to similar intakes, can lead to misinterpretation of study results and confusion in our understanding of the relevance of these (and other) compounds to human health. 

The measurement of carotenoid absorption is fraught with difficulties and riddled with assumptions, and it is therefore a complex matter. Methods may rely on plasma concentration changes provoked by acute or chronic doses, oral-faecal mass balance method variants and compartmental modelling. 

Classically, to measure absolute absorption the plasma area imder the curve from an intravenous dose would be compared to that caused by the feeding of an oral dose. However, the carotenoids are lipid-soluble and are normally incorporated in chylomicrons synthesised in the enterocytes, a situation that cannot be replicated and applied to studies in humans because an intravenous preparation that would behave naturally is not possible. 

HART J D and SCOTT K J (1995) Development and evaluation of an HPLC method for the analysis of carotenoids in foods and the measurement of carotenoid content of vegetables and fruits commonly consumed in the UK. Food Chem. 54(1) 101-111. 

STAHL w, HEINRICH u, JUNGMANN H, TRONNIER H and SIES H (2000) Carotenoids in hiunan skin non-invasive measurement and identification of dermal carotenoids and carotenoid esters. Methods Enzymol 319 494-502. 

With investigations of phytochemicals and functional foods, the outcome measure is generally going to be a biomarker of disease, such as serum cholesterol level as a marker of heart disease risk, or indicators of bone turnover as markers of osteoporosis risk. Alternatively, markers of exposure may also indicate the benefit from a functional food by demonstrating bioavailability, such as increased serum levels of vitamins or carotenoids. Some components will be measurable in both ways. For instance, effects of a folic acid-fortified food could be measured via decrease in plasma homocysteine levels, or increase in red blood cell folate. 

In the traditional balance method, the apparent absorption of a carotenoid or any other pigment is determined by the difference between its intake (inpnt) and its excretion (ontpnt). This techniqne requires carefnl measurements of all inpnt and ontpnt of the compound of interest. The approach has the advantage of being non-invasive and thns able to be used directly on hnman subjects. 

Experimental evidence in humans is based upon intervention studies with diets enriched in carotenoids or carotenoid-contaiifing foods. Oxidative stress biomarkers are measured in plasma or urine. The inhibition of low density lipoprotein (LDL) oxidation has been posmlated as one mechanism by which antioxidants may prevent the development of atherosclerosis. Since carotenoids are transported mainly via LDL in blood, testing the susceptibility of carotenoid-loaded LDL to oxidation is a common method of evaluating the antioxidant activities of carotenoids in vivo. This type of smdy is more precisely of the ex vivo type because LDLs are extracted from plasma in order to be tested in vitro for oxidative sensitivity after the subjects are given a special diet. 

Comparative studies of the widely employed spectrophotometric readings at the Soret and Q bands (405 and 630 nm, respectively) and the elemental analysis of copper and nitrogen showed that the spectrophotometric assay based only on the Soret band can overestimate the purity of a preparation. Erroneous data were attributed to an increase in absorptivity at the Soret band when other colored compounds like metal-free analogs and carotenoids are present. Indeed, copper-free chlorin e6 exhibits a specific absorbance 3.6 times greater than that of its coppered counterpart. Therefore, measurements at the Q band (630 mn) and the establishment of the S Q ratio are preferred. 

Solvent — The transition energy responsible for the main absorption band is dependent on the refractive index of the solvent, the transition energy being lower as the refractive index of the solvent increases. In other words, the values are similar in petroleum ether, hexane, and diethyl ether and much higher in benzene, toluene, and chlorinated solvents. Therefore, for comparison of the UV-Vis spectrum features, the same solvent should be used to obtain all carotenoid data. In addition, because of this solvent effect, special care should be taken when information about a chromophore is taken from a UV-Vis spectrum measured online by a PDA detector during HPLC analysis. 

It must be underlined that independently of the MS equipment characteristics, no information about stereo-chemistry can be obtained. In fact, cis and trans isomers of the corresponding carotenoid showed identical mass spectra, as did carotenoids with epoxide groups at 5,6 and 5,8 positions. In addition, special care should be taken in assigning carotenoid molecular masses to avoid confusion due to the various ions that may be formed depending on measurement conditions. 

However, several hours of measurement are typically needed to achieve good signal-to-noise and high resolution, especially for 2D techniques. Other 2D techniques were used for structure elucidation of carotenoids from guava and annatto seeds. ... 

The determination of the absolute configuration of a carotenoid is only possible by circular dichroism (CD) measurement. The spectrum interpretation can only be done by comparison with reference or model compounds with known chiralities. The sample requirement is as low as 5 to 50 pg, but CD facilities are not so commonly available. Buchecker and Noack reported experimental aspects and discussion of the relationships of carotenoid structures and CD spectra. 

Eor estimation of the total carotenoid content of a sample, generally the absorbance is measured in petroleum ether at 450 nm and a mean value of 2500 forAj is used. In case a known carotenoid is present in the sample, the absorbance measured at the of that carotenoid and its corresponding Aj can be used for quantifi-... 

The accuracy and precision of carotenoid quantification by HPLC depend on the standard purity and measurement of the peak areas thus quantification of overlapping peaks can cause high variation of peak areas. In addition, preparation and dilution of standard and sample solutions are among the main causes of error in quantitative analysis. For example, the absorbance levels at of lutein in concentrations up to 10 mM have a linear relationship between concentration and absorbance in hexane and MeOH on the other hand, the absorbance of P-carotene in hexane increased linearly with increasing concentration, whereas in MeOH, its absorbance increased linearly up to 5 mM but non-linearly at increasingly higher concentrations. In other words, when a stock solution of carotenoids is prepared, care should be taken to ensure that the compounds are fuUy soluble at the desired concentrations in a particular solvent. 

The lag-phase measurement at 234 nm of the development of conjugated dienes on copper-stimulated LDL oxidation is used to define the oxidation resistance of different LDL samples (Esterbauer et al., 1992). During the lag phase, the antioxidants in LDL (vitamin E, carotenoids, ubiquinol-10) are consumed in a distinct sequence with a-tocopherol as the first followed by 7-tocopherol, thereafter the carotenoids cryptoxanthin, lycopene and finally /3-carotene. a-Tocopherol is the most prominent antioxidant of LDL (6.4 1.8 mol/mol LDL), whereas the concentration of the others 7-tocopherol, /3-carotene, lycopene, cryptoxanthin, zea-xanthin, lutein and phytofluene is only 1/10 to 1/300 of a-tocopherol. Since the tocopherols reside in the outer layer of the LDL molecule, protecting the monolayer of phospholipids and the carotenoids are in the inner core protecting the cholesterylesters, and the progression of oxidation is likely to occur from the aqueous interface inwards, it seems reasonable to assign to a-tocopherol the rank of the front-line antioxidant. In vivo, the LDL will also interact with the plasma water-soluble antioxidants in the circulation, not in the artery wall, as mentioned above. 

At least some of the antioxidant nutrients are essential to human health, and others (such as carotenoids) may be highly beneficial, particularly in preventing cancer (e.g. Block et /., 1992). However, we do not yet know what dietary intakes are optimal. In principle, this could be investigated by varying the dietary intake of antioxidants and measuring free-radical damage in the human body. This is one of our current research directions. 

---