Effect of short-chain fructooligosaccharides on intestinal calcium absorption and calcium status in postmenopausal women: a stable-isotope study

¹ From the Centre de Recherche en Nutrition Humaine d’Auvergne, Unité Maladies Métaboliques et Micronutriments, Centre de Recherche INRA Clermont-Ferrand/Theix, Saint Genès Champanelle, France (MT, JCT, CF-C, YR, and CC); the Laboratoire de Biologie de Stress Oxydant, Faculté de Pharmacie, Domaine de la Merci, La Tronche, France (JA, VD, and AMR); Nutri-Health, Rueil-Malmaison, France (FRJB); the Laboratoire de Nutrition Humaine, Centre de Recherche en Nutrition Humaine d’Auvergne, Clermont-Ferrand, France (CB-D and MB); the Laboratoire de Contrôle des Eaux, Institut Louise Blanquet, Faculté de Médecine et Pharmacie, Clermont-Ferrand, France (DP); Eridania Béghin-Say, Vilvoorde, Belgium (FB); and the Nutrition and Toxicology Research Institute, Maastricht University, Maastricht, Netherlands (FB).

² Supported by The French Ministry of National Education and Scientific Research and Technology (98 G-0069).

³ Address reprint requests to C Coudray, Centre de Recherche en Nutrition Humaine d’Auvergne, Unité Maladies Métaboliques et Micronutriments, Centre de Recherche INRA Clermont-Ferrand/Theix, 63122 Saint Genès Champanelle, France. E-mail: coudray{at}clermont.inra.fr.

ABSTRACT  
Background: An enhancing effect of short-chain fructooligosaccharides (scFOSs) on intestinal calcium absorption has been shown in animals and in some short-term human studies. However, the long-term effect of scFOSs on calcium absorption in humans is still unknown.

Objective: We investigated the long-term effect of a moderate daily dose (10 g) of scFOSs on intestinal calcium absorption in postmenopausal women.

Design: In a randomized, double-blind crossover protocol, 12 healthy, postmenopausal women received scFOSs or placebo for 5 wk. The treatments were separated by a 3-wk washout period. Subjects orally received ⁴⁴Ca (stable isotope) and a fecal marker. Feces were collected after the isotope intake for 5–7 d to measure unabsorbed isotope. Calcium-status indexes, calciotropic hormones, and bone turnover were also assessed.

Results: Mean (±SD) intestinal calcium absorption with scFOS treatment was not significantly different from that with placebo treatment (35.63 ± 9.40% and 36.55 ± 8.48%, respectively). However, a tendency for calcium absorption to be higher with scFOS treatment than with placebo treatment was observed in women who had been going through menopause for >6 y.

Conclusions: scFOSs do not modify intestinal calcium absorption in postmenopausal women who do not receive hormonal replacement therapy. The results from a subgroup of women who had been going through menopause for >6 y (n = 6) suggest that scFOSs may influence calcium absorption in the late postmenopausal phase. The small number of subjects and the related P value warrant verification and further investigation with women in late menopause only.

Key Words: Short-chain fructooligosaccharides • fermentation • intestinal absorption • calcium • stable isotopes • postmenopausal women • bone turnover • ⁴⁴Ca

INTRODUCTION  
Complex carbohydrates may have important implications for human health, eg, for bowel function, carbohydrate and lipid metabolism, and cancer prevalence. Since our early work in 1977 (1), important findings have accumulated from animal studies showing that fermentable complex carbohydrates, potato-resistant starch, inulin, and oligosaccharides, in particular fructooligosaccharides (FOSs), may enhance intestinal absorption of calcium and magnesium and probably that of some trace elements such as iron and zinc (2–7).

One of the physiologic changes characterizing aging and menopause is decreased intestinal calcium absorption. This may be due to decreased renal formation of 1,25-dihydroxyvitamin D (8), combined with reduced intestinal 1,25-dihydroxyvitamin D receptors (9–11) and receptor responsiveness (12). In animal studies, calcium deficiency was associated with decreased bone growth, increased bone resorption, and bone fragility (13–16). Impaired calcium status and bone mineral content have often been reported in postmenopausal, osteoporotic women. Prince et al (17) showed that calcium supplementation increases bone density in postmenopausal women. Thus, increasing not only the dietary intake of calcium but also its bioavailability might help postmenopausal women avoid bone loss and prevent osteoporosis (18).

During the past 5 y, a few studies were carried out in different human groups (adolescents and adults) to investigate the potential beneficial effect of FOSs on the intestinal absorption of minerals, particularly that of calcium. Two studies found a beneficial effect of FOSs on intestinal calcium absorption in humans (19, 20), although one study showed no effect (21). More studies are therefore required to assess the potential beneficial effect of FOSs on intestinal calcium absorption, particularly in postmenopausal women. In the present study, we investigated the effect of ingesting a moderate daily dose (10 g) of short-chain fructooligosaccharides (scFOSs) on intestinal calcium absorption in postmenopausal women who were not receiving hormonal replacement therapy.

SUBJECTS AND METHODS  
Reagents and equipment
Enriched ⁴⁴Ca (96.5% by wt; stable isotope), in the form of carbonate, was obtained from Chemgas (Paris). Suprapure nitric acid, suprapure hydrogen peroxide, suprapure hydrochloric acid, lanthanum oxide, and a standard solution of calcium (1 g/L) were obtained from Merck (Darmstadt, Germany). All other chemicals were of the highest quality available, and demineralized water was used throughout the study.

Isotope ratio measurements were performed by using an inductively coupled plasma mass spectrometer (Plasmaquad-II system; Fisons Instruments, Manchester, United Kingdom) fitted with a Meinhard nebulizer. A Perkin-Elmer 560 atomic absorption spectrometer (Perkin-Elmer, St-Quentin-en-Yvelines, France) was used for total calcium measurements.

Subjects
Fourteen healthy, postmenopausal women were recruited, and 12 of them completed the study. Subjects were included in the study if they were 50–70 y of age, had been going through menopause for >2 y, were not receiving hormonal replacement therapy, and had a body mass index (in kg/m²) of 20–27. Subjects were excluded from the study if they had digestive, hepatic, renal, or inflammatory diseases; consumed vegetarian, lactovegetarian, or ovolactovegetarian diets; had undergone any surgery in the 3 mo before the study; or took medication that might modify intestinal mineral absorption. Subjects did not take nutritional supplements (vitamins, minerals, polyols, fiber, or FOSs) during the study or during the 3 mo before the study.

The subjects’ health state was assessed by medical history, physical examination, and routine clinical laboratory tests. The volunteers were fully informed of the aims and purposes of the study and gave their written informed consent. The protocol of this study, registered under the reference AU260, was approved by the local ethics committee of the Human Nutrition Research Center at Clermont-Ferrand, France (20 August 1998).

Preparation of stable-isotope solutions
The carbonate form of ⁴⁴Ca (96.5%) was moistened with demineralized water, and concentrated hydrochloric acid was slowly added to convert the carbonate form into the chloride form. The pH was adjusted to 2–3 with a 1-mol NaOH/L solution. The concentration of total calcium (3.00 mg Ca/mL) and the isotope enrichment of the solution were checked before use. The physical and chemical characteristics of the stable-isotope solution were determined, and a toxicity test was performed on mice by a specialized company (CERB, Baugy, France). The pharmacist of Clermont-Ferrand Hospital certified the pharmaceutic conformity of this solution for human use.

Study design
The subjects were treated for 5 wk with scFOSs and a placebo (sucrose) according to a randomized, double-blind crossover design. The treatments were separated by a washout period of 3 wk. Both treatments were supplied to the volunteers as packets containing 5 g powder. The scFOSs or placebo was administered progressively to obtain the best tolerance. Subjects first received 5 g scFOSs/d for 4 d at their midday meal and then received 10 g scFOSs/d (5 g at the midday meal and 5 g at the evening meal). During the first 23 d, the subjects were asked to maintain their normal food intake and not to consume either FOS-containing food products or mineral or vitamin supplements. After this first part of each period, the subjects received a controlled diet in the metabolic unit that consisted of 2100 kcal/d, of which 15% was protein, 35% was fat, and 50% was carbohydrate (Table 1). This standardized diet contained 900 mg Ca/d, 250 mg Mg/d, and 12 g dietary fiber/d. The subjects continued to consume FOSs or placebo until days 33–35. On day 28, urine, fecal, and blood samples were collected in the morning to determine calcium indexes and basal isotope ratios before stable-isotope intake. During their midday meal on day 29, the subjects received the isotope dose (30.01 mg ⁴⁴Ca) and 40 radiopaque pellets as a fecal marker. Ten milliliters of the ⁴⁴Ca solution was diluted with 100 mL aromatized water (Volvic, Paris), and the subjects then drank the resulting solution. The receptacle was then rinsed twice with 50 mL aromatized water (Volvic), and these rinsings were also drunk by the subjects. Blood was sampled 24 h after ⁴⁴Ca isotope intake to determine ⁴⁴Ca plasma isotope enrichment. Fecal collections to assess intestinal mineral absorption started immediately after stable-isotope intake and lasted 5–7 d. Feces were collected individually every 24 h, frozen, and X-rayed to count radiopaque markers. Urine was also collected individually every 24 h for 2 d after stable-isotope intake (from the midday meal on day 29 to the midday meal on day 31) to determine urinary ⁴⁴Ca isotope enrichment and excretion.

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TABLE 1 . Composition of the diets  
Assessment of food intake
Before the start of the study, the food consumption of the subjects was assessed by interview to determine dietary patterns and estimate mineral and fiber intakes. At the middle of each experimental period, the subjects were also asked to complete a food-intake questionnaire for 4 d. They reported the type and amount of each food they consumed during this period. Daily mineral and fiber intakes were determined by using composition tables (22–24). For the 5–7 d after the stable-isotope intake, standard meals were prepared and any unconsumed food was collected to estimate its calcium content. This content was then subtracted from the mineral content of the meals to estimate the real mineral intake of the subjects. This also enabled us to determine apparent intestinal absorption and the conventional balance of total calcium (input minus output).

Stable-isotope analysis
Total fecal collections per subject and per treatment were weighed, pooled, and thoroughly homogenized. They were then freeze-dried, and the dry weight was measured. Subsamples (0.25 g) were heated at 500°C for 10 h to produce ash. The ash was dissolved in 0.5 mL of a 14-mol HNO₃/L solution and in 0.2 mL H₂O₂ (30% by vol) and heated at 110°C for 2 h. The temperature was then increased to 130°C until all the acid had evaporated, and 10 mL of a 0.14-mol HNO₃/L solution was added to all the samples. The calcium was concentrated by precipitation of calcium with saturated ammonium oxalate, and the calcium oxalate formed was then dissolved in a 0.14-mol HNO₃/L solution (25).

Calcium concentrations in the calcium oxalate extracts were measured by atomic absorption spectrometry, and the concentrations were then adjusted to 2–3 mg Ca/L by appropriate dilution in a 0.14-mol HNO₃/L solution before calcium isotope ratio analysis. The ratios of ⁴⁴Ca to ⁴³Ca in basal and 5–7-d fecal samples were determined by inductively coupled plasma mass spectrometry. The mass spectrometer settings and plasma conditions were optimized with a solution of 10 µg indium/L, and the instrument operating conditions were as follows: radio frequency generator, 27.12 MHz; forward radio frequency power, 1350 W; reflected radio frequency power, <3 W; outer argon flow rate, 14 L/min; intermediate argon flow rate, 0.7 L/min; nebulizer argon flow rate, 0.76 L/min; mass resolution (at 10% of peak height), 0.9 Da. Data-collection variables were as follows: total replicates per integration, 5; signal integration time per replicate, 40 s; dwell time per sweep, 20.4 s; scanning mode, peak hopping (5 points/peak); sample uptake rate, 0.6 mL/min.

Calculations
Intestinal calcium absorption (%) was calculated as

RESULTS  
The main characteristics of the subjects were as follows: age, 59.8 ± 6.2 y; length of menopause, 8.3 ± 7.1 y; weight, 62.1 ± 7.5 kg; body mass index, 25.2 ± 1.9. Compliance and tolerance to treatments (placebo or scFOSs), as evaluated by returned treatment packets and questionnaires, were considered very good: the subjects consumed 9.78 ± 0.19 g placebo/d and 9.83 ± 0.19 g scFOSs/d.

Food-intake assessment
At inclusion into the study, the subjects had a calcium intake of 1171 mg/d and were instructed to decrease their consumption of dairy products. Their calcium intake was thus 900 mg/d at the middle and the end of each period, which is close to the recommended dietary allowance for this population. Daily fiber intake was 16.7 g at inclusion into the study and 14.3 and 17.2 g at the middle of the placebo and scFOS treatments, respectively. This intake was slightly lower in the controlled diet periods and averaged 12 g/d. Daily vitamin D intake was 1.89 µg at inclusion into the study and ranged between 1.73 and 2.35 µg at the middle of the treatment periods. The intake of vitamin D during the controlled diet periods was 1.25 or 1.28 µg/d. These intakes were substantially lower than the recommended dietary allowance for this vitamin (5 µg/d). There was no significant difference in vitamin D intake between the 2 experimental periods. However, dietary intake of vitamin D does not take into account endogenous synthesis, which is closely linked to sun exposure.

Intestinal calcium absorption and calcium-status indexes
"True" intestinal calcium absorption values, determined with ⁴⁴Ca stable isotope, were not significantly different between the scFOS and placebo treatment periods (Table 2). True intestinal calcium absorption was 35.63 ± 9.40% during placebo treatment and 36.55 ± 8.48% during the scFOS treatment. When the subjects were divided into 2 subgroups according to their menopause duration (n = 6/group), intestinal calcium absorption tended to be higher with scFOS treatment than with placebo treatment in the women who were in late menopause (P = 0.097) but not in the women who were in early menopause (P = 0.370). Apparent intestinal calcium absorption was also determined from the dietary intake and the fecal excretion of total calcium. As shown in Table 3, total daily calcium intake, fecal calcium excretion, and calcium absorption (expressed as mg/d or as %) were not significantly different between the 2 treatments.

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TABLE 2 . True intestinal ⁴⁴Ca absorption by menopause duration in postmenopausal women who received placebo or short-chain fructooligosaccharides (scFOSs)¹  

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TABLE 3 . Daily calcium intake, fecal calcium excretion, and apparent intestinal calcium absorption in postmenopausal women who received placebo or short-chain fructooligosaccharides (scFOSs)¹  
Total calcium plasma concentrations remained unchanged during the scFOS and placebo treatments, and ⁴⁴Ca plasma enrichment did not differ significantly between the 2 treatments (Table 4). Daily total urinary calcium excretion and urinary ⁴⁴Ca enrichment and excretion also did not differ significantly between the 2 treatments (Table 5). These urine results agree with the results obtained for intestinal calcium absorption, which was unmodified by the treatment. As shown in Figure 1, these results clearly show that intestinal ⁴⁴Ca absorption is well correlated with plasma Ca⁴⁴ enrichment (r = 0.563, P = 0.0042). Intestinal ⁴⁴Ca absorption was also closely correlated with urinary ⁴⁴Ca excretion in both urine collections (r = 0.83 and 0.84 for the first and second 24-h urine samples collected after ⁴⁴Ca intake, respectively, P < 0.0001). Intestinal ⁴⁴Ca absorption was not correlated with daily vitamin D intake (data not shown).

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TABLE 4 . Plasma calcium-status indexes in postmenopausal women who received placebo or short-chain fructooligosaccharides (scFOSs)¹  

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TABLE 5 . Urinary calcium indexes in postmenopausal women who received placebo or short-chain fructooligosaccharides (scFOSs)¹  

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FIGURE 1. . Correlations between intestinal ⁴⁴Ca absorption and both plasma ⁴⁴Ca enrichment and urinary ⁴⁴Ca excretion in postmenopausal women.

 
Calciotropic hormones and bone turnover biomarkers
Plasma PTH and 1,25-dihydroxyvitamin D concentrations did not change significantly from the start to the end of the experimental periods (Table 6). Nevertheless, when all the subjects were taken together, 1,25-dihydroxyvitamin D concentrations showed a tendency to decrease during scFOS treatment (P = 0.065). However, when the subjects were divided into 2 subgroups [early menopause (2–6 y) and late menopause (>6 y)], plasma 1,25-dihydroxyvitamin D concentrations in the early menopause subgroup significantly decreased during the scFOS period (P = 0.045) (Figure 2).

View this table:
TABLE 6 . Plasma calciotropic hormone concentrations and bone turnover markers in postmenopausal women who received placebo or short-chain fructooligosaccharides (scFOSs)¹  

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FIGURE 2. . Plasma 1,25-dihydroxyvitamin D [1,25(OH)₂D] concentrations in women who had been going through menopause for 2-6 y (early menopause; n = 6) or >6 y (late menopause; n = 6) and who received separate treatment with a placebo (n = 12) and with short-chain fructooligosaccharides (scFOSs; n = 12).

 
Bone turnover was assessed by measuring plasma osteocalcin concentrations and urinary deoxypyridinolidine excretion as markers of bone formation and bone resorption, respectively. There were no significant differences between the 2 treatments in plasma osteocalcin concentrations, changes in plasma osteocalcin concentrations, or urinary deoxypyridinolidine excretion (Table 6).

DISCUSSION  
The aim of this study was to investigate the effect of ingesting a moderate daily dose of short-chain FOSs (10 g) for 5 wk on intestinal calcium absorption and calcium-status indexes in postmenopausal women who were not receiving hormonal replacement therapy. The results obtained showed that under our conditions, the consumption of scFOSs for 5 wk had no effect on overall intestinal calcium absorption. The unchanged apparent and true absorption percentages were confirmed by the lack of change in total calcium status and in ⁴⁴Ca enrichment assessed in plasma and urine after oral intake of the stable isotope ⁴⁴Ca. The use of ⁴⁴Ca enabled us to measure the true intestinal absorption of calcium, whereas the conventional balance (measurement of the ingested and the excreted amounts of total calcium) gave us the apparent intestinal absorption of calcium. True intestinal absorption is known to be generally higher than apparent intestinal absorption (26), thus explaining the high values of true intestinal calcium absorption in the present study. In postmenopausal women, true intestinal calcium absorption values between 28% and 32% have been reported (28). The values of apparent intestinal calcium absorption obtained in our subjects were very close to those reported elsewhere (19).

During the past 3 decades, the potential beneficial effects of fermentable carbohydrates, including FOSs, on intestinal absorption of calcium and magnesium were extensively investigated by our group (1, 29–31) and by others (6–8, 32, 33). Animal studies clearly showed a beneficial effect of FOSs on intestinal calcium absorption, although this effect is generally less marked for calcium than for magnesium and depends on the experimental conditions (experiment duration, age of animal, FOS doses, amount of calcium in the diet, etc). Several hypotheses have been advanced to explain the potential positive effect of fermentable carbohydrates on intestinal mineral absorption in animals. The fermentation of FOSs is accompanied by a strong production of short-chain fatty acids (31), resulting in a decrease in luminal pH and an increase in the concentration of ionized minerals (30). Consequently, mineral solubility is increased, and the active and passive diffusion of minerals may thus be enhanced (34, 35). Also, short-chain fatty acids may directly influence intestinal mineral absorption by modifying various electrolyte exchanges (calcium-hydrogen, magnesium-hydrogen), and calcium or magnesium may pass through the cell membrane more readily in the form of a lower-charge complex with short-chain fatty acids than in the ionic form (35–38). Carbohydrate fermentation in the animal’s cecum is also accompanied by cecum enlargement, which may increase the intestinal absorption exchange area. Thus, these fermentation-induced changes would theoretically improve the intestinal absorption of nearly all minerals. In a previous paper, we reported that scFOSs significantly increase intestinal magnesium absorption in postmenopausal women (39). However, intestinal calcium absorption was unchanged in the experimental conditions of the present study. The mechanism and sites of intestinal calcium absorption are different from those of intestinal magnesium absorption. This may explain the different effect that FOSs may have on the intestinal absorption of these 2 major minerals, both for the entire intestinal length and for various segments of the intestine.

Intestinal calcium absorption involves 2 mechanisms. The main absorptive pathway is a saturable transcellular process, which is regulated by vitamin D via calcium binding protein. This is a highly regulated process, occurring mainly in the duodenum. The second process is a nonsaturable, paracellular transport that occurs throughout the length of the intestine (40). If FOSs increase intestinal calcium absorption, this effect would take place in the large intestine, most likely via both passive, paracellular and active, transcellular pathways (fermentation effects). The mechanisms involved in intestinal magnesium absorption are a saturable process (facilitated diffusion or active absorption) and a passive diffusion. The portion of intestinal magnesium absorption that occurs via passive diffusion from the distal part of the intestine is very important. Intestinal magnesium absorption is only weakly controlled, if at all.

A possible explanation of the lack of effect of scFOSs on intestinal calcium absorption in the present study is down-regulation of the active pathway of calcium absorption in the small intestine after several weeks of scFOS feeding (41, 42). We speculate that scFOS feeding does increase intestinal calcium absorption in the lower parts of the intestine, as is also the case for magnesium. Over several weeks, this may result in a feedback effect that decreases calcium absorption in the proximal segment of the intestine to maintain serum calcium concentrations in a narrow, finely regulated range. Therefore, a potential positive effect of scFOSs on colonic calcium absorption might be masked when overall intestinal calcium absorption is considered. This adaptation may be mediated through changes in the amount of calcium binding protein in the intestine in response to a decreased serum concentration of 1,25-dihydroxyvitamin D, the main regulator of intestinal calcium absorption (43). In the present study, a nonsignificant decrease in the concentration of this metabolite was observed after scFOS treatment but not after placebo treatment (P = 0.06). However, this adaptation is apparently very active during early menopause, because 1,25-dihydroxyvitamin D concentrations in the women who were in early menopause (2–6 y) significantly (P = 0.045) decreased after scFOS treatment, despite the lack of any significant effect of scFOSs on intestinal calcium absorption in these women (P = 0.37). Experimental data obtained from studies of rats support this assumption of adaptation. Chonan et al (44) noted that galactooligosaccharide supplementation initially (9 d) increases intestinal calcium absorption in ovariectomized rats but that the effect disappears at 28 d. Other investigators reported a reduction in the calbindin-D9k concentration in the small intestine in rats after scFOS or lactose feeding, supporting the occurrence of such adaptation (43, 45). In human studies, scFOSs and lactulose increased intestinal calcium absorption in adolescent and postmenopausal women when the supplementation lasted only 9 d (20, 28), whereas other studies conducted in adults did not show any effect after a 21-d supplementation (21; D Barclay, P Kastenmayer, F Couzy, et al, personal communication, 2000).

Another factor that may explain why scFOSs did not have a significant effect on overall calcium absorption in the present study is the type of subjects. All the subjects had been menopausal for at least the previous 2 y. Their menopause duration ranged widely (2–22 y). Thus, the subjects in this study were quite heterogeneous with respect to this variable. It is well known that the first 5 y after the onset of menopause are marked predominantly by hormonal disturbances, and it is apparently difficult to modulate calcium metabolism variables during this period. Dawson-Hugues et al (46) reported that calcium supplements were more effective in preventing bone loss in late menopause (>6 y after the onset of menopause) than in early menopause (2–6 y after the onset of menopause). We thus divided the subjects of our study into 2 subgroups according to menopause duration: those who had been menopausal for 2–6 y (n = 6) and those who had been menopausal for >6 y (n = 6). Our results showed that in the subjects who were in early menopause, there was no significant effect of scFOSs on intestinal calcium absorption (P = 0.35). In contrast, in the late menopause subgroup, we observed a tendency for intestinal calcium absorption to increase with scFOS treatment (P = 0.097). Also, measurements of 1,25-dihydroxyvitamin D concentrations in the 12 subjects showed a downward trend between the start and the end of the scFOS period. Interestingly, plasma 1,25-dihydroxyvitamin D concentrations in the subjects who were menopausal for 2–6 y significantly decreased (P = 0.0457) after scFOS treatment, whereas no variation was observed for the subjects in late menopause (P = 0.37). Therefore, feedback adaptation of calcium absorption seems to occur more effectively in early menopause than in late menopause. Thus, scFOSs may be more beneficial to calcium absorption in women who are in late menopause. This observation is consistent with the results obtained by Van den Heuvel et al (28), who observed a positive effect of lactulose in women who had been going through menopause for >5 y. scFOSs may also be beneficial to calcium absorption in populations with high calcium needs (adolescents) or impaired active intestinal calcium absorption, such as elderly people, in whom down-regulation of calcium absorption is less likely.

In conclusion, under the conditions of the present study, scFOS feeding did not significantly affect intestinal calcium absorption in postmenopausal women. The results from a subgroup of subjects who had been going through menopause for >6 y (n = 6) suggest that scFOSs may influence calcium absorption in the late postmenopausal phase. The small number of subjects and the related P value warrant verification and further investigation with women in late menopause only.

ACKNOWLEDGMENTS  
We are grateful for the use of the human metabolic facilities in the Center de Recherche en Nutrition Humaine d’Auvergne. We also gratefully acknowledge the cooperation of the subjects, who so conscientiously and cheerfully suffered the restrictions imposed on them by this study. Special appreciation goes to Liliane Morin and Paulette Rousset for nursing help and to Guy Manhiot for kitchen assistance. We also thank Jean Vernet for statistical help and Marie Jeanne Davicco and Elyett Gueux for excellent technical assistance.

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