Gary R. Beecher

Separation, identification, and quantification of carotenoids in fruits, vegetables and human plasma by high performance liquid chromatography

Separation, identification, and quantitation by high performance liquid chromatography of prominent carotenoids and carotenol fatty acid esters in various fruits and vegetables are reviewed. Examples of transformations of carotenoids as a result of food preparation are discussed. Recent developments in separation and identification of several carotenoids in human plasma are presented. The micronutrients considered in this text will be largely limited to carotenoids, but the analysis of vitamin A and two forms of vitamin E (y- and a-tocopherol) in human plasma will be briefly discussed. Carotenoids are among the most abundant naturally occumng pigments that are found in plants and plant foods. The present number of naturally occumng carotenoids isolated from various sources is in excess of 560 (ref. 6). Current food composition tables (ref. 7 & 8) lack detailed analytical information in that they only provide data on vitamin A activity which is largely contributed by about four carotenoids in plant foods (ref. 9). In the past decade, with the implementation of high performance liquid chromatography (HFLC), more attention has been focused on the separation of all the carotenoid constituents of foods. In the following section, the separation of various classes of carotenoids by HPLC is described.

Separation and quantitation of carotenoids in foods

Publisher Summary This chapter describes high-performance liquid chromatography (HPLC) procedures that allow effective separation and accurate quantitation of individual carotenoids in commonly consumed foods. Carotenoids are one of the most common dietary agents that have been studied as cancer preventive agents. Although the number of naturally occurring carotenoids isolated from various sources is in excess of 600, the number of carotenoids that are abundant in common foods is less than 50. HPLC has been shown to be the most efficient technique for the analysis of sensitive compounds in complex food extracts. Numerous HPLC conditions have been developed to separate the carotenoids in extracts of natural products. Such HPLC procedures can be highly advantageous and provide valuable information on the identity and the levels of these compounds in their natural state in foods. However, HPLC procedures that can be universally used for the separation and quantification of carotenoids in extracts of foods are lacking. The HPLC profiles of extracts from fruits and vegetables are illustrated in chapter.

Separation and quantification of carotenoids in human plasma

Publisher Summary This chapter discusses separation and quantification of carotenoids in human plasma. A number of studies have reported an inverse relationship between the high consumption of certain fruits and vegetables and a lower incidence of several types of cancer. The first successful separation of carotenoids from an extract of human serum was performed by using nonaqueous reversed-phase chromatographic conditions on a Zorbax ODS column, with a mixture of acetonitrile, dichloromethane, and methanol as eluent. Under these conditions, six carotenoids were separated and identified in an extract from human serum: lutein, zeaxanthin, β cryptoxanthin, lycopene, α -carotene, and β -carotene. This chapter describes the methodologies for detailed separation and quantitation of 18 carotenoids along with vitamin A and 2 forms of vitamin E in human plasma by high-performance liquid chromatographic (HPLC) on reversed-phase and silica-based nitrilebonded columns.

Relation between sodium balance and menstrual cycle symptoms in normal women.

Women often have cyclical physical symptoms of bloating, swelling, and breast tenderness. During the luteal phase of the menstrual cycle [1, 2] and pregnancy [3], osmoregulation changes significantly [1, 2] and sodium-retaining hormone secretion [4-7] and salt preference [8] increase. The concurrence of these cyclical changes has indicated that water and sodium retention may cause physical symptoms during the luteal phase [9, 10]. If sodium is retained during the menstrual cycle, the mechanisms involved might include increased salt intake or urinary sodium retention, or both, related to the luteal phase. We hypothesized that sodium balance, a product of total sodium intake and sodium excretion, affects expression of somatic symptoms and inferred that a lower sodium intake or sodium balance should alleviate these symptoms. We therefore explored the effects of a decrease in sodium intake on the expression and severity of menstrual symptoms in women studied during three consecutive menstrual cycles. Methods Participants Thirteen healthy menstruant women without the premenstrual syndrome [10-12] (age, 21 to 35 years; weight, 50 to 80 kg; height, 160 to 180 cm) were recruited by a newspaper advertisement that requested volunteers for a diet and hormone study. Participants were taught how to complete food frequency questionnaires, collect 24-hour urine samples, and complete visual analogue scale questionnaires about the severity of menstrual symptoms [11, 12]. Study Design Diet Baseline sodium chloride intake averaged 115 23.5 mmol/d (6.7 1.3 g/d) or 2.6 1.0 g of sodium ion (Na+) per day (from food frequency records analysis, University of Minnesota Nutrition Coordinating Center, Minneapolis, Minnesota). Intake of dietary salt was then reduced by 30% (to 73.0 12.2 mmol/d [4.3 0.6 g/d]; Na+ intake, 1.6 0.2 g/d) during the next two menstrual cycles to create a moderate salt-deprivation stimulus [13]. To determine whether the use of salt depended on cycle phase, women were allowed to add salt to their food during the second month of this diet (salt-access cycle). All meals were prepared by the metabolic kitchen at the Beltsville Human Nutrition Research Center (U.S. Department of Agriculture, Beltsville, Maryland); the same menu was used every week. The use of added salt was determined from the difference in weight of packaged salt before and after each meal (each packet weighed 10 to 13 mmol [0.57 to 0.74 g]). Cycle Periods Each cycle was divided into five time periods: I = early follicular phase, days 1 to 4 (menses); II = mid-follicular phase, days 5 to 8; III = late follicular phase (luteinizing hormone surge 2 days); IV = mid-luteal phase (days 20 to 25; 7 days after the luteinizing hormone surge); and V = late luteal phase (4 days before menses). Blood specimens to determine plasma renin activity, plasma sodium levels, atrial natriuretic peptide levels, and aldosterone levels (during the 2 months of the salt-restricted diet) and two consecutive 24-hour urine samples to determine total volume, creatinine, and sodium excretion were obtained in the mid-follicular, late-follicular, and mid-luteal phases (time periods II, III, and IV, respectively) of the cycle. Luteinizing hormone levels were measured from day 11 until the luteinizing hormone surge occurred, and progesterone levels were measured 1 week after the luteinizing hormone surge. Levels of all hormones and peptides were measured by established radioimmunoassays. All samples except those of luteinizing hormone, estradiol, and progesterone were run in the same assay to avoid interassay variability. Sodium levels were measured by ion-selective electrodes. Fasting weights were obtained daily during the last 2 months of the study. Documentation of Menstrual Symptoms The visual analogue questionnaire was completed by 11 women at the same time every day for 3 months [10, 14] and was used to record ratings on questions assessing somatic symptoms and sensory cravings. Statistical Analysis A mean value was generated from the questionnaire results, body weights, and amounts of added salt (during the salt-access cycle) for the days encompassing each of the five time periods. Data were analyzed using repeated-measures analysis of variance and paired t-tests. Significance was set at a P value less than 0.05 for quantitative data and a P value less than 0.01 for symptom questionnaire scores (using the Bonferroni adjustment for interdependent questions). All expressions of variability represent the 95% CI. Data are presented as means one half of the 95% CI. Results Effects of Salt Condition Decreasing dietary sodium intake significantly altered extracellular fluid volume and sodium balance. During the first month of the salt-restricted diet (salt-restriction cycle), urinary sodium excretion decreased by 40.3 18 mmol/d from a baseline value of 115 20 mmol/d (P = 0.001) (Figure 1, top). Plasma sodium levels decreased by 0.9 0.9 mmol/L from a baseline value of 139.1 0.9 mmol/L (P = 0.018) (Figure 1, upper middle), and plasma renin activity increased by 0.14 0.08 ng/(Ls) from a baseline value of 0.25 0.08 ng/(Ls) (P = 0.008) (Figure 1, lower middle) during the 2 months of the salt-restricted diet. Levels of atrial natriuretic peptide decreased during period III of the salt-restriction cycle by 6.1 8.1 pg/mL from a mean value of 58.2 13 pg/mL in period III of the baseline cycle (P = 0.024). Figure 1. Variables measured during the baseline cycle, the salt-restriction cycle, and the salt-access cycle; during the latter, women were allowed to add salt to their food. P P Top. Bottom. P P Effects of Time of Cycle All women had ovulatory cycles during the study and equivalent estrogen and progesterone levels during each cycle. Body weight did not change significantly between or within cycles (data not shown). Plasma sodium levels were significantly lower in the luteal phase than in the follicular phase; they decreased by 1.8 1.3 mmol/L (from 140.0 0.9 mmol/L) in the baseline cycle and by 1.23 0.5 mmol/L (from 138.8 0.8 mmol/L) in the salt-restriction cycle (Figure 1, upper middle). Retention of urinary sodium was not seen in any cycle period. An increase in urinary sodium excretion of 27 16 mmol/d (from 65 10 mmol/d) was seen in period IV (mid-luteal phase) of the salt-restriction cycle (Figure 1, top). Plasma renin activity increased in the luteal phase of the baseline cycle by 0.17 0.08 ng/(mLs) from a mean value in the follicular phase of 0.19 0.08 ng/(mLs), in the luteal phase of the salt-restriction cycle by 0.25 0.17 ng/(mLs) from a mean value in the follicular phase of 0.28 0.08 ng/(mLs), and in the luteal phase of the salt-access cycle by 0.19 0.14 ng/(mLs) from a mean value in the follicular phase of 0.31 0.17 ng/(mLs) (P < 0.001) (Figure 1, lower middle). Aldosterone levels doubled in all cycles during the luteal phase (data not shown). Breast tenderness and bloating were phase dependent during all cycles studied (Figure 1, bottom). Severity ratings of swelling or bloating were higher in period I than in period II by 25 12 points (period II value, 15 11 points) in the baseline cycle, 31 15 points (period II value, 27 15 points) in the salt-restriction cycle, and 40 11 points (period II value, 15 9) in the salt-access cycle (P < 0.001) (Figure 1, bottom). These symptoms were also affected by the salt condition: Peak severity ratings were 16 19 points higher during the salt-restricted diet cycles (salt-restriction cycle and salt-access cycle) than during the baseline cycle (peak severity rating, 38 11 points; P < 0.001). Breast tenderness was most severe 4 days before menses, during which time ratings increased from the nadir of the follicular phase by 42 16 points (from 5.0 2.2 points) during the baseline cycle, by 35.0 11 points (from 10.0 11.1 points) during the first salt-restriction cycle, and by 39 6 points (from 6.0 4.4 points) during the salt-access cycle (P < 0.001). Breast tenderness was not affected by sodium balance. The highest physical comfort ratings coincided with the lowest severity ratings for breast discomfort and bloating (periods II and III). Compared with nadir ratings in the follicular phase, ratings of thirst and cravings for salt and sweets increased and peaked during the late luteal phase (time period V) (P = 0.006 for the baseline cycle, P < 0.001 for the salt-restriction cycle, and P = 0.002 for the salt-access cycle) (data not shown). In the salt-access cycle (second salt-restricted diet cycle), use of added salt was less than 10 mmol/d and did not vary with phase of the cycle (P > 0.2; data not shown). Dietary salt condition and time of cycle did not affect appetite. Discussion To study the relation between menstrual cycle symptoms and sodium balance in normal women, we changed sodium balance by decreasing intake of sodium by 30% for 2 months. This modest change in sodium balance did not decrease the severity or the cycle-dependent expression of somatic symptoms during the luteal phase or menses. We found no evidence of urinary sodium retention, and, paradoxically, sodium loss was seen during the luteal phase in the first month of the sodium-restricted diet (salt-restriction cycle), despite biochemical evidence of extracellular fluid volume contraction [13]. Although the information was not documented, we believe that natriuresis occurred during the luteal phase of the other two cycles (baseline cycle and salt-access cycle), because levels of urinary sodium excretion remained high despite elevated renin activity and aldosterone levels. Progesterone is a competitive antagonist of aldosterone [15], and high salt intake can reverse the progesterone-related increase in renin and aldosterone levels [16, 17]. These findings, together with our data, suggest that progesterone enhances natriuresis during the luteal phase in normal women and that the changes in the renin-aldosterone system during this time are probably secondary to primary natriuresis. Use

[12] Gas-phase reaction mass spectrometric analysis of carotenoids

Publisher Summary The coupling of sample introduction with a direct exposure probe together with gas-phase reaction chemistry within the mass spectrometer ionizing chamber provides several powerful techniques for examining the structure of carotenoids and determination of their molecular weights. During isolation procedures, electron-capture negative-ion (ECNI) mass spectrometry provides a useful method for the detection of carotenoid compounds in the presence of non-electron-capturing contaminants. Increased sensitivity for molecular weight determinations is realized with the application of chemical ionization techniques, and electron-capture negative ion spectrometry provides even greater sensitivity. ECNI sensitivity is usually in excess of 100-fold that of conventional electron ionization using a direct exposure probe. The use of deutero-containing reagent and buffer gases allows determining the number of exchangeable hydrogens by either chemical ionization or the more sensitive ECNI process. Ions indicative of the acyl groups of fatty acyl esters of carotenols are readily determined by chemical ionization methods and with even greater sensitivity by ECNI techniques. These gas-phase reaction techniques provide complementary and confirmatory evidence of structure. The coupling of an high-performance liquid chromatographic (HPLC) equipped with a diode array detector to a mass spectrometer provides chromatographic retention data and ultraviolet (UV) or visible (VIS) spectra and mass spectra, and, in many cases, is sufficient for on-line identification of unknown carotenoids.