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Article

The Food Matrix of Spinach Is a Limiting Factor in Determining the Bioavailability of ß-Carotene and to a Lesser Extent of Lutein in Humans

^a Division of Human Nutrition and Epidemiology and ^b Food Science Group, Department of Food Technology and Nutritional Sciences, Wageningen Agricultural University, 6700 EV Wageningen, The Netherlands and ^c Unilever Research Vlaardingen, 3130 AC Vlaardingen, The Netherlands

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Carotenoid bioavailability depends, amongst other factors, on the food matrix and on the type and extent of processing. To examine the effect of variously processed spinach products and of dietary fiber on serum carotenoid concentrations, subjects received, over a 3-wk period, a control diet (n = 10) or a control diet supplemented with carotenoids or one of four spinach products (n = 12 per group): whole leaf spinach with an almost intact food matrix, minced spinach with the matrix partially disrupted, enzymatically liquefied spinach in which the matrix was further disrupted and the liquefied spinach to which dietary fiber (10 g/kg wet weight) was added. Consumption of spinach significantly increased serum concentrations of all-trans-ß–carotene, cis-ß–carotene, (and consequently total ß-carotene), lutein, -carotene and retinol and decreased the serum concentration of lycopene. Serum total ß-carotene responses (changes in serum concentrations from the start to the end of the intervention period) differed significantly between the whole leaf and liquefied spinach groups and between the minced and liquefied spinach groups. The lutein response did not differ among spinach groups. Addition of dietary fiber to the liquefied spinach had no effect on serum carotenoid responses. The relative bioavailability as compared to bioavailability of the carotenoid supplement for whole leaf, minced, liquefied and liquefied spinach plus added dietary fiber for ß-carotene was 5.1, 6.4, 9.5 and 9.3%, respectively, and for lutein 45, 52, 55 and 54%, respectively. We conclude that the bioavailability of lutein from spinach was higher than that of ß-carotene and that enzymatic disruption of the matrix (cell wall structure) enhanced the bioavailability of ß-carotene from whole leaf and minced spinach, but had no effect on lutein bioavailability.

KEY WORDS: • carotenoids • bioavailability • processing spinach • dietary fiber • humans

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Carotenoids have many functions in the human body, including being precursors of retinol and retinoids, and acting as antioxidants (Krinsky 1993 ). They are found in dark-green, leafy vegetables; in yellow and orange colored fruits and vegetables; and, to a lesser extent, in animal products. Carotenoids are found in the chloroplasts of all green plant tissues, where they occur in the photosynthetic pigment-protein complexes, and in the chromoplasts of fruits. Some carotenoids, including ß-carotene, lycopene, and some oxycarotenoids such as lutein and zeaxanthin, exert antioxidant activity in lipid phases by quenching oxygen or free radicals (Sies and Stahl 1995 ). This may explain why a higher dietary intake of carotenoids has been found to be associated with a lower risk for age-related macular degeneration (Seddon et al. 1994 ) and some forms of cancer and cardiovascular disease (VAN Poppel 1996 ).

Bioavailability is defined as the fraction of an ingested nutrient that is available to the body for utilization in normal physiological functions or for storage (Jackson 1997 ). We have developed the mnemonic SLAMENGHI, which includes all factors that could affect bioavailability, especially bioavailability of ß-carotene and other carotenoids (Castenmiller and West 1998 , de Pee and West 1996 ). The SLAMENGHI factors are as follows: Species of carotenoids, molecular Linkage, Amount of carotenoids consumed in a meal, Matrix in which the carotenoid is incorporated, Effectors of absorption and bioconversion, Nutrient status of the host, Genetic factors, Host-related factors, and Interactions. Current information on carotenoid bioavailability is limited, fragmentary, and often conflicting. Apart from the many factors that determine bioavailability, the lack of adequate indicators has made it difficult to establish the bioavailability of carotenoids in food.

Carotenes dissolved in oil are more readily absorbed than when they are incorporated in foods such as fruits and vegetables. The plasma response to ingestion of ß-carotene dissolved in oil was found to be about five times the response to a similar amount of ß-carotene in carrots (Brown et al. 1989 , Micozzi et al. 1992 ). Recent studies in Indonesia have shown that feeding ß-carotene from dark-green, leafy vegetables produced a lower plasma response than similar quantities of ß-carotene in a fat matrix (de Pee et al. 1995 ). The serum response to ß-carotene from fruits was four times higher than that from vegetables (de Pee et al. 1998 ). Cooking or fine grinding of foods could increase the bioavailability of carotenes by disrupting or softening plant cell walls and disrupting carotenoid-protein complexes (Hussein and El-Tohamy 1990 , VAN Zeben and Hendriks 1948 ). The greater ease with which carotenoids in thermally treated foods can be extracted during analysis may imply that they are also biologically more available.

We describe a controlled dietary intervention study with differently processed spinach products to examine the effect of the food matrix on carotenoid concentrations in human serum. Different processing techniques were used as a means of testing the role of cellular structure. One of the spinach products was treated with a mixture of enzymes, which causes disruption of the cell wall structure as well as depolymerization of the cell wall components, reducing the dietary fiber content of the spinach product. Dietary pectin added to meals reduced plasma responses to ß-carotene in humans (Rock and Swendseid 1992 ). Thus, by adding sugar beet fiber, we also examined the effect of restoring fiber to the enzyme-treated spinach on serum carotenoid concentrations. Although the fiber added replaced that lost during enzyme treatment, it did not restore cell wall structure or cellular integrity.

	MATERIALS AND METHODS

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Subjects.

The subjects were 72 healthy, non-smoking, normolipidemic volunteers: 42 women and 30 men, aged 18–58 y. The subjects, students of Wageningen Agricultural University and other residents of the Wageningen area, were recruited through local advertisements and all gave their written informed consent. None of the subjects were taking oral medication, apart from oral contraceptives, nor supplements of any kind during the last 3 mo before the study started and during the study period. Subjects were screened for elevated fasting glucose and protein levels in urine and for low hemoglobin and abnormal hematology. All subjects completed a medical and a general questionnaire and food frequency questionnaires to estimate their habitual intakes of energy, carotenoids and vitamin A. All subjects had normal body mass indices (18–28 kg/m²), fasting serum cholesterol concentrations < 6.5 mmol/L and fasting triacylglycerol concentrations < 2.8 mmol/L. During the study, two male volunteers withdrew from the study for personal reasons. Characteristics of the subjects are presented in Table 1 . The protocol for this study was approved by the Medical-Ethical Committee of the Division of Human Nutrition and Epidemiology of Wageningen Agricultural University.

The study started with a 3-wk run-in period followed by a dietary intervention period of 3 wk. During the intervention period, subjects were allocated to one of six experimental groups. Blood was taken by venipuncture from fasting subjects between 7.15 and 10.00 h at the start of the run-in period and at days 0, 1, 8, 15, 21 and 22 of the dietary intervention period. Blood samples, to which no anticoagulant was added, were left to clot and were centrifuged within 1 h after being drawn. The serum was separated and stored at -80^°C until analysis.

During the first 3 wk of the study (run-in period), subjects chose their own diets but were instructed to avoid foods rich in carotenoids and vitamin A. During the dietary intervention period, subjects were supplied with total diets, except for a limited choice of free products (approximately 10 energy%), which did not contain carotenoids or retinol. Subjects received a hot meal at the Division of Human Nutrition and Epidemiology and foods for their other meals and snacks were packed to be taken home. A maximum of two alcoholic consumptions per day was allowed but were not permitted to be consumed together with the hot meal. Duplicate portions of diets were collected for chemical analysis. The daily selection of free choice foods was recorded in a diary, and the nutrient content was calculated (Stichting NEVO 1995 ). Individual body weights throughout the study were maintained ± 2 kg.

Diet.

All subjects in the six treatment groups were fed the same control diet throughout the study, and the menu was changed daily on a weekly cycle. The control diet consisted of foods, other than fruits and vegetables with moderate or high amounts of carotenoids, and met the requirements of Dutch Recommended Daily Allowances (Netherlands Food and Nutrition Council 1992 ). Four groups received a spinach product, one control group received no additional source of carotenoids and one group received a carotenoid supplement. The carotenoid supplement was a suspension in vegetable oil of microcrystalline ß-carotene (40 g/kg; Hoffmann-La Roche, Basel, Switzerland) and crystalline lutein and zeaxanthin derived from marigold flowers (60 g/kg and 3 g/kg, respectively; FloraGLO, Kemin Industries, Des Moines, IA), added to salad dressing. The energy content of the diets of the control group and carotenoid supplement group was adjusted to that of the spinach groups by providing extra amounts of appropriate foods. The spinach groups received 20 g whole leaf spinach, minced spinach, liquefied spinach or liquefied spinach to which dietary fiber was added/MJ. All spinach products originated from one batch and were prepared and provided by Langnese-Iglo in Wunstorf (Germany) for Unilever Research Vlaardingen (The Netherlands). Four different spinach products were prepared: whole leaf spinach with an almost intact food matrix; minced spinach in which the matrix is partially disrupted; enzymatically liquefied spinach in which the matrix is disrupted; and the enzymatically liquefied spinach to which dietary fiber was added. The whole leaf spinach was washed and subsequently blanched for 90 s and cooled down quickly. The minced spinach was minced to 5 mm after blanching. The liquefied spinach was prepared by treating minced spinach with an enzymatic preparation with pectinase, hemicellulase and cellulase activities (Rapidase LIQ plus, Gist-brocades, Seclin, France) during 2.5 h at 35^°C. After the enzyme treatment, the spinach was boiled for 5–10 min to inactivate the enzymes. The spinach products were frozen immediately after processing. Liquefaction resulted in a breakdown of cell wall material. Therefore, the fourth group received the liquefied spinach plus fiber prepared from sugar beet (10 g/kg wet weight, Fibrex 600, TEFCO Food Ingredients b.v., Bodegraven, The Netherlands) to compensate for the loss of dietary fiber. This fiber product contains per 100 g of product, 73 g of dietary fiber of which one third is soluble and 22 g is pectin. All frozen spinach was thawed and heated by microwave before consumption. The spinach products contained no measurable nitrite, and the content of nitrate was less than 1000 mg/kg, thus ensuring that the nitrate intake of the subjects was below the Acceptable Daily Intake for nitrate (FAO/WHO 1995 ). Microbiological counts showed normal values and confirmed that the spinach products were safe for human consumption.

Serum measurements.

Serum carotenoids and retinol were measured by high performance liquid chromatography (HPLC) (Craft and Wise 1992 ). To avoid day-to-day analytic variations, all samples from an individual were analyzed sequentially as a set. After precipitation with ethanol, extraction followed with hexane twice; samples were evaporated under nitrogen and injected into the HPLC system described below. Serum cholesterol and triacylglycerol concentrations were analyzed with the Abbott Spectrum high performance diagnostic system following a standard procedure (Siedel et al. 1983 , Sullivan et al. 1985 ).

Food measurements.

The duplicate portions of the daily food intake of one subject collected throughout the study were mixed thoroughly as weekly portions, and subsequently pooled samples were stored at -20^°C until analysis. The moisture level and the ash content in each weekly portion were determined (Osborne and Voogt 1978 ) using a vacuum oven at 85^°C and a muffle furnace at 550^°C. The protein concentration was determined by the Kjeldahl method using a conversion factor of 6.25. The Folch method (Folch et al. 1957 ) was used to extract fat. Digestible carbohydrate was calculated by difference. Dietary fiber was analysed according to the AOAC Official Method 992.16 for total dietary fiber (AOAC 1996 ). The dietary fiber content of the various spinach products was determined as methanol-ether insoluble solids (MEIS)⁴and the amount of pectin (polygalacturonic acid) in the spinach products was measured by hydrolyzing pectic substances to water-soluble galacturonic acid using an enzyme preparation (Katan and van de Bovenkamp 1981 ). Carotenoids and retinol were determined in the pooled samples and in spinach products and salad dressings containing the carotenoid supplement. Carotenoids and retinol were extracted from wet material after homogenisation, using tetrahydrofuran (THF) and redissolved in THF/methanol (1:1 v/v) and injected into the HPLC system described below.

Chemicals and instrumentation for carotenoid and retinol analysis.

Pure grades of all-trans-–carotene, all-trans-ß–carotene and lycopene were obtained from Sigma Chemical Co. (St Louis, MO) and all-trans-lutein, all-trans-zeaxanthin, and all-trans-ß-cryptoxanthin from Hoffmann-La Roche Ltd (Basel, Switzerland). The carotenoid concentrations of the standards were measured in a Zeiss M4 QIII spectrophotometer (Carl Zeiss, Oberkochen/Württemberg, Germany). The HPLC system, manufactured by Thermo Separation Products (San Jose, CA), was equipped with a pump (P4000), a solvent degasser (SCM1000), a temperature controlled autosampler (AS3000), a UV-visible forward optical scanning detector (Spectra Focus UV3000), interface (SN4000), and control and integration software (PC1000, version 3.0). A reversed phase Vydac 218 TP 54 column containing silica polymerically modified with C18 from The Separations Group (Hesperia, CA) was used, and the metal frits in the column were replaced by a PAT (Peek Alloyed with Teflon) frits from the same manufacturer to minimize carotenoid degradation on the column. The mobile phase consisted of a mixture of methanol and THF (98:2 v/v). For each series of analyses of serum samples, a control serum sample was analysed. The coefficients of variation (CV) within runs for serum analysis of -carotene, ß-carotene, lutein, zeaxanthin, ß-cryptoxanthin, lycopene and retinol in control pools averaged 7.4, 3.9, 3.6, 8.7, 4.5, 10.4 and 1.6%, respectively. For each series of analyses of food samples, a control sample (homogenized baby food) was extracted in duplicate and injected into the HPLC system for monitoring the stability of the analytical procedure over time. The CV within runs for food analysis of -carotene, ß-carotene and lutein in control pools averaged 5.7, 6.8 and 8.8%, respectively. All sample preparations and extractions were carried out in duplicate and under subdued yellow light with minimal exposure to oxygen (Hulshof et al. 1997 ).

Statistical analysis.

Serum concentrations were averaged for d 0 and 1 and for d 21 and 22 for each subject. For each person, the response to treatment was calculated as the change in serum concentrations from the start to the end of the intervention period. Two-tailed t-tests for independent samples were performed to evaluate differences in serum responses between the control and treatment groups and between the carotenoid supplement and pooled spinach groups. To compare differences in responses among the groups receiving various spinach products, after significant F-tests (ANOVA) the Tukey method for multiple comparisons was used (Godfrey 1985 ). Analyses were carried out using general linear models to compare differences in response among intervention groups, controlling for several factors and covariables, including sex, vegetarian diet, age, body mass index and change in cholesterol and triacylglycerol concentrations. Spearman correlation coefficients for the entire study group were calculated when relevant. Differences associated with P < 0.05 were regarded as significant (SPSS/PC 7.5, 1997, SPSS Inc., Chicago, IL).

The relative bioavailability of ß-carotene and lutein was calculated by dividing the serum response (µmol/L) relative to the intake of the respective carotenoid in each spinach group (mg/MJ) by the serum response to the carotenoid in the supplement group relative to the intake of carotenoid in the carotenoid supplement (mg/MJ). The serum carotenoid responses were adjusted for serum responses in the control group, and the amount of carotenoid consumed (mg/MJ) by the control group was subtracted from the amount consumed by each spinach group.

	RESULTS

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Carotenoids.

The composition of the diets is given in Table 2 . Consumption of spinach significantly increased the serum concentrations of all-trans-ß–carotene, cis-ß–carotene (and consequently total ß-carotene), lutein (Table 3 , Fig. 1and Fig. 2), -carotene and retinol (P < 0.05); decreased the serum concentration of lycopene as compared to the control group (P < 0.05); and had no effect on serum concentrations of zeaxanthin and ß-cryptoxanthin. The serum responses (changes in serum concentrations from the start to the end of the intervention period) of total ß-carotene and lutein were significantly related (r_s = 0.51; P < 0.001). The -carotene response was significantly correlated to the total ß-carotene response (r_s = 0.69; P < 0.001), and the all-trans-ß-carotene and cis-ß-carotene responses were significantly correlated (r_s = 0.79; P < 0.001).

View larger version (18K): [in this window] [in a new window]   Figure 1. Serum ß-carotene concentrations in groups of healthy subjects fed a control diet or a control diet with a carotenoid supplement or with various spinach products. Values are expressed as means and as means ± SD for the carotenoid supplement group; n = 12, for control group: n = 10. Serum concentrations were averaged for d 0 and 1 (d 0) and for d 21 and 22 (d 21) for each subject. For significant differences among the study groups, see Table 3 .

View larger version (20K): [in this window] [in a new window]   Figure 2. Serum lutein concentrations in groups of healthy subjects fed a control diet or a control diet with a carotenoid supplement or with various spinach products. Values are expressed as means and as means ± SD for the control group; n = 12, for control group: n = 10. Serum concentrations were averaged for d 0 and 1 (d 0) and for d 21 and 22 (d 21) for each subject. For significant differences among the study groups, see Table 3 .

The carotenoid supplement group had significantly higher responses of serum concentrations of all-trans-ß–carotene, cis-ß–carotene, (and consequently total ß-carotene), lutein (Table 3) , zeaxanthin and -carotene than the control group, and significantly higher responses of all-trans-ß–carotene, cis-ß–carotene, (and consequently total ß-carotene) (Table 3) , zeaxanthin, -carotene, lycopene and ß-cryptoxanthin than the pooled spinach groups. Thus, the food matrix (cellular structure) played an important role in the uptake of ß-carotene from spinach. Processing spinach in various ways did not affect lutein bioavailability.

Dietary fiber.

The all-trans-ß-carotene, cis-ß-carotene, total ß-carotene and lutein serum responses in the liquefied spinach group were not different from the responses in the liquefied spinach plus added dietary fiber group. Adding 10 g of sugar beet fiber per kg wet weight of spinach had no effect on serum carotenoid response. The enzyme-treated spinach contained 13% less MEIS (methanol-ether insoluble solids) and 17% less polygalacturonic acid than the whole leaf spinach, which contained per 100 g dry matter 71 g MEIS and 11 g polygalacturonic acid, respectively. The composition of the sugar beet fiber added to spinach was comparable to the fiber present in the whole leaf spinach, but had a higher amount of pectin.

Retinol.

The decrease in serum concentration of retinol was significantly less (P = 0.04) in the pooled spinach groups than in the control group. Serum retinol concentrations decreased in the control group (7.6%), in the minced spinach group (5.1%) and liquefied spinach plus dietary fiber group (2.9%), but increased in the carotenoid supplement group (2.9%), whole leaf spinach group (1.4%) and liquefied spinach group (0.4%).

Cholesterol and triacylglycerol.

The changes in serum cholesterol and triacylglycerol concentrations after 3 wk of dietary intervention (serum cholesterol and triacylglycerol responses) of the carotenoid supplement group and pooled spinach groups were not significantly different from the response in the control group. Normalized serum concentrations of total ß-carotene and lutein were calculated as follows: carotenoid concentration/(cholesterol + triacylglycerol concentration). Adjustment of serum carotenoid concentrations for concentrations of cholesterol and other lipids may provide a better reflection of dietary intake of carotenoids (Willett et al. 1983 ). Analysis of the normalized serum carotenoid concentrations of total ß-carotene and lutein showed significant differences of the normalized ß-carotene responses between the whole leaf spinach and liquefied spinach group (P = 0.05) and between the whole leaf spinach group and liquefied spinach plus added dietary fiber group (P < 0.02).

Diets.

The chemical analysis of the diets supplied showed consistent data for the 3 wk and among the treatment groups. Surprisingly, the enzyme-treated spinach products, which were treated for 2.5 h at 35^°C, did not have a higher concentration of cis-ß–carotene than the whole leaf spinach. Chemical analysis of the salad dressings showed that the carotenoid supplement should provide daily per 11 MJ energy intake: ß-carotene, 10.9 mg (all-trans-ß–carotene, 10.5 mg;cis-ß–carotene, 0.4 mg); lutein, 12.2 mg; and zeaxanthin, 1.0 mg. However, from the analysis of the duplicate portions of ~11 MJ of the carotenoid supplement group, the daily intake of lutein was 46% lower (6.6 ± 0.2 mg) than was to be expected from the lutein concentrations in the salad dressings, whereas the daily intake of ß-carotene in the duplicate food portions was 10% lower than expected based on the amounts analysed in the salad dressing (Table 2) . Prepared spinach products, as consumed during the dietary intervention period, contained 15–18% of the ß-carotene as cisisomers. The amounts of total ß-carotene and lutein in the duplicate food portions were 0–9% and 31–43%, respectively, lower than those expected to be present in the daily food from analysis of the spinach products. The amount of carotenoids measured in the duplicate portions was used to calculate relative bioavailability.

Estimate of relative bioavailability.

Based on the analysis of the duplicate portions, the bioavailability of ß-carotene from spinach as compared to the carotenoid supplement was 5.1, 6.4, 9.5 and 9.3% for whole leaf, minced, liquefied and liquefied spinach plus added dietary fiber, respectively. The bioavailability of ß-carotene from whole leaf spinach was calculated as follows:

The relative bioavailability of lutein from spinach was 45, 52, 55 and 54% for whole leaf, minced, liquefied and liquefied spinach plus added dietary fiber, respectively. Thus, enzymatic treatment increased the relative bioavailability of ß-carotene in spinach by about half. The relative bioavailability of lutein in spinach was more than five times higher than that of ß-carotene and was not affected by the enzymatic treatment.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Consumption of processed spinach products significantly increased serum concentrations of all-trans-ß–carotene, cis-ß–carotene, (and consequently total ß-carotene), lutein, -carotene and retinol and decreased serum concentrations of lycopene. For ß-carotene, but not for lutein, there was a significant effect of processing: liquefied spinach, in which the vegetable food matrix is disrupted, produced a higher serum response than whole leaf spinach, where the food matrix is still intact, and minced spinach. Addition of dietary fiber to liquefied spinach does not restore the cellular structure, but did compensate for the fiber that was broken down and had no effect on serum responses of carotenoids as compared to serum responses after consumption of liquefied spinach.

There are three possible explanations for the low bioavailability of ß-carotene (5.1–9.5%) from spinach compared to the carotenoid supplement. First is the food matrix in which the carotenoids are embedded. Other investigators also found a low bioavailability of ß-carotene from vegetables as compared to pure ß-carotene: stir-fried vegetables, 7% (de Pee et al. 1995 ); carrots, 18–26% (Brown et al. 1989 , Micozzi et al. 1992 , Törrönen et al. 1996 ). Rock et al. (1998) found a trend for a greater percentage increase in plasma concentrations of total ß-carotene in the period when thermally processed and pureed carrots and spinach were fed to healthy women as compared to the period when these women were fed raw carrots and spinach.

Second is the isomeric form of ß-carotene in spinach. The carotenoid supplement dissolved in oil contained mainly all-trans-ß–carotene (approximately 96%), whereas in the spinach products 15–18% of the ß-carotene was present as cisisomers. Several investigators have shown that all-trans-ß–carotene is more readily absorbed than its cis isomers (Ben-Amotz and Levy 1996 , Gaziano et al. 1995 , Jensen et al. 1987 , Tamia et al. 1995 ). Rock et al. (1998) recently found that feeding subjects thermally processed and pureed spinach that provided an increased proportion of cis-ß–carotene was not associated with a significant increase in the plasma cis-ß–carotene concentration after 4 wk. They concluded that isomerization of ß-carotene in foods by heat treatment does not negate the enhanced ß-carotene uptake associated with consuming processed vegetables compared with raw vegetables. In our study, a significant increase in the serum concentrations of the cis-ß-carotene was observed in the treatment groups at the end of the dietary intervention period. The relative bioavailability of all-trans-ß–carotene for whole leaf, minced, liquefied and liquefied spinach plus added dietary fiber was 6.2, 6.8, 10.1 and 9.9% and of cis-ß–carotene was 4.1, 4.2, 6.6 and 6.2%, respectively. These results provide some support for more ready absorption of all-trans-ß–carotene than cis-ß–carotene. However, the issue needs further research. The ratio of serum all-trans- tocis-ß–carotene was strongly correlated in our study. It is not known whether this is due to an artefact produced by the chemical analysis because measuring cis-ß–carotene isomers in chlorophyl-containing foods is extremely difficult or whether ß-carotene isomers are in some sort of equilibrium in human serum.

Third, there may be an effect of other carotenoids on the bioavailability of ß-carotene. Kostic et al. (1995) found that when ß-carotene and lutein were given to subjects in the same dose, ß-carotene significantly reduced the serum area-under-the-curve values for lutein. Also Micozzi et al. (1992) found that oral doses of ß-carotene (12 or 30 mg daily for 6 wk) lowered serum lutein concentrations in men. In contrast, a preferential increase in chylomicron concentrations of lutein compared to ß-carotene in the presence of high amounts of ß-carotene and small amounts of lutein was described by Gärtner et al. (1996) . Although our study was not designed to examine the interaction between carotenoids, we found no evidence to conclude that ß-carotene and lutein interact with each other.

The relative bioavailability of lutein from spinach products ranged from 45 to 55%. Thus, we conclude that the relative bioavailability of lutein from spinach is greater than that of ß-carotene and less affected by the food matrix. Lutein, which is a dihydroxycarotenoid, is about 0.1% as lipophylic as ß-carotene. This may explain why the matrix in which lutein is embedded in spinach does not reduce lutein absorption as it does the absorption of ß-carotene. The amount of lutein found in the duplicate portions was much lower than was to be expected from addition of the results of the separate analyses of the control diet and either the carotenoid supplement or the spinach products. Lutein from spinach products and from the carotenoid supplement was apparently lost when added to a complete, daily diet. The composition of the carotenoid supplement was based on analysis of the various spinach products and experiments to examine the preferred preparation of the spinach for this study. Unexpectedly, at the time of the intervention study, the lutein content of the spinach products was found to be higher than those analyzed several months before the study, which partly explains why the carotenoid supplement group received less lutein than the spinach groups.

One of the effects of dietary fiber on lipid metabolism centres upon its interactions with bile acids, resulting in their increased loss by faecal excretion, disturbance of micelle formation and thus a decreased absorption of fats and fat soluble substances (Olson 1994 ). Addition of dietary fiber to liquefied spinach had no effect on the serum ß-carotene responses. Rock and Swendseid (1992) reported earlier that adding 12 g dietary citrus pectin to controlled meals with 25 mg synthetic ß-carotene (0.48 g pectin/mg ß-carotene) reduced the increase in plasma ß-carotene. In our study, where the proportion of dietary fiber added (0.23 g fiber/mg ß-carotene) was half that used earlier (Rock and Swendseid 1992 ), we were not able to demonstrate an effect of dietary fiber.

In all groups, intake of carotenoids other than ß-carotene and lutein was rather low. We found that the -carotene response was significantly correlated to the ß-carotene response, which is in line with the finding of others that ß-carotene supplementation increases concentrations of -carotene (Castenmiller and West 1998 ).

In conclusion, this study confirmed that the bioavailability of ß-carotene, and to a lesser extent of lutein, was affected by the food matrix and that processing spinach had an effect on the matrix (disruption of cell wall structure and loss of cellular integrity), and thus on the bioavailability of ß-carotene from spinach. The bioavailability of ß-carotene from liquefied spinach was higher than from whole leaf or minced spinach. We could not demonstrate an effect on serum ß-carotene or lutein responses from the addition of dietary fiber to liquefied spinach. This suggests that once the cell wall components are broken down, addition of dietary fiber in amounts previously present in the food, has no effect on bioavailability of carotenoids. A limitation of the present study may be that subjects in the control group were fed low-carotenoid diets. The effects of feeding carotenoids in this type of study may result in greater apparent increases and differences in serum carotenoid responses. To obtain estimates of absolute bioavailability and to understand the mechanisms involved, it will be necessary to carry out studies using other experimental approaches including those involving isotopically-labelled carotenoids.

	ACKNOWLEDGMENTS

 
We thank all participants for their interest, enthusiasm and perseverance to complete the trial. A number of other persons are acknowledged for their invaluable contribution to the study: Hanneke Reitsma for pilot studies on spinach liquefaction; Jörg Kramer (Langnese-Iglo GmbH, Wunstorf, Germany) for the production of the spinach products; Saskia Meyboom, Karin Roosemalen, Els Siebelink and Jeanne de Vries for work on dietary aspects of the study; Joke Barendse, Peter van de Bovenkamp, Jan Harryvan, Robert Hovenier, Paul Hulshof, Truus Kosmeijer, Frans Schouten, Marga van der Steen, Pieter Versloot, and Johan de Wolf for drawing blood and chemical analyses of blood and food samples. Ingrid Bakker, Tiny Hoekstra, Wanda Vos, Aviva van Campen and Goverdien Klerk worked on the project as students in the framework of their training programmes. The advice of Jan Burema on statistical matters was much appreciated.

	FOOTNOTES

 
¹ To whom correspondence should be addressed.

¹ Supported in part by Unilever Research Vlaardingen, The Netherlands and by the Commission of the European Communities, Agriculture and Fisheries (FAIR) specific RTD programme CT95–0158, Improving the quality and nutritional value of processed foods by optimal use of food antioxidants (Project Leader: B. Sandström, Copenhagen, Denmark). This paper does not necessarily reflect the Commission's views and in no way anticipates its future policy in this area.

² The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ''advertisement'' in accordance with 18 USC section 1734 solely to indicate this fact.

³ Abbreviations used: CV, coefficients of variation; MEIS, methanol-ether insoluble solids; PAT, Peek Alloyed with Teflon; THF, tetrahydrofuran.

Manuscript received August 25, 1998. Initial review completed September 16, 1998. Revision accepted November 4, 1998.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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