Olestra is the common name for a mixture of hexa-, hepta- and octaesters of sucrose formed from long-chain fatty acids prepared from edible fats and oils. Olestra (Olean, Procter & Gamble, Cincinnati, OH) is not hydrolyzed by gastric or pancreatic enzymes (Mattson and Volpenhein 1972) and it is not absorbed intact from the gastrointestinal (GI)⁴ tract (Miller et al. 1995). As a result, it contributes no calories to the diet. Olestra has physical properties similar to those of a triglyceride with the same constituent fatty acids (Jandacek and Webb 1978) and organoleptic and thermal properties similar to those of traditional fats (Kester 1993). Because of these unique properties, olestra can serve as a zero-calorie replacement for conventional dietary fat. Olestra is an approved food additive for use in the preparation of savory snacks such as potato and corn chips and crackers.

As observed for other lipophilic nonabsorbed substances (Javert and Marci 1941, Matschiner et al. 1967), olestra can reduce the absorption of lipophilic nutrients such as the fat-soluble vitamins. This happens because a portion of lipophilic nutrients eaten at or near the same time as olestra partitions into the olestra in the GI tract and thus become unavailable to the intestinal micelles for transport to absorptive sites (Jandacek 1982).

The effects of olestra on the status of vitamins A, D and E have been determined in pig (Cooper et al. 1997b) and human studies (Schlagheck et al. 1997b). The study in the pig was conducted under conditions that modeled the situation in which olestra would be present in all foods, not in selected food forms. These studies showed that water-soluble nutrients are unaffected by olestra. These and other studies (Cooper et al. 1997c, Jones et al. 1991, Koonsvitsky et al. 1997, Schlagheck et al. 1997a) showed that the status of vitamin K, as assessed by functional tests, is also unaffected by olestra.

It has also been demonstrated in both pigs (Cooper et al. 1997a) and humans (Schlagheck et al. 1997a) that the effects of olestra on the status of vitamins A, D and E can be offset by adding extra amounts of the vitamins to the diet. Further, it has been demonstrated in the pig that the amount of additional vitamin required to maintain tissue or blood concentrations of the vitamin at control concentrations (i.e., the restoration level) is essentially a linear function of the dietary concentration of olestra over a range of intakes encompassing and exceeding the expected human intake of olestra (Webb et al. 1997).

In 12-wk studies in the pig, which defined the magnitude and nature of the response of the status of vitamins A, D and E to dietary concentrations of olestra and of the vitamins (Cooper et al. 1997a and 1997b), the pigs were fed daily amounts of olestra that exceeded by severalfold the estimated human intake of olestra from savory snacks, its initial intended use. The studies covered the period of the pigs' most rapid growth, which occurs at about 8 wk of age (Martin and Crenshaw 1989), and therefore the period of maximum nutrient demand. At the end of the studies, the pigs were 18-19 wk old and had not passed through major developmental phases such as sexual maturity, which generally occurs around 28 wk of age (Zimmerman et al. 1981). Although 12 wk is a sufficient period for tissue pools to respond to the presence of olestra and fat-soluble vitamins in the diet, it may not be long enough for nutrients such as calcium and vitamin A to reach equilibrium. Therefore data collected over periods >12 wk would provide assurance that the results from the 12-wk studies represented the full effect of olestra on the status of such nutrients.

The present studies were conducted to obtain data over periods during which the pigs reach sexual maturity and accumulate most of their nutrients stores, while eating olestra at intakes typical of human chronic intake from savory snacks. In addition, the studies were long enough to allow body stores of nutrients with slow turnover times to reach equilibrium. The primary purposes of the 26-wk study were as follows: 1 ) to determine the amounts of vitamins A and E required to restore tissue concentrations of these vitamins to control concentrations at olestra intakes, 5-10 g/d, typical of expected chronic 90th-percentile human intake from savory snacks (Webb et al. 1997); and 2 ) to confirm that the consumption of olestra during the period when most of the body's nutrient stores are accumulated does not produce effects different than those seen in the 12-wk studies (Cooper et al. 1997a and 1997b). The primary purposes of the 39-wk study were as follows: 1 ) to determine the effects of an olestra intake typical of the mean chronic human intake, 2.0-4.4 g/d depending on age or 3.1 g/d for the total population, on selected nutrients over a period that encompasses the major growth and developmental phases of the pig, including sexual maturity; and 2 ) to confirm that the responses of tissue concentrations of vitamins A and E to added dietary amounts of the vitamins, in the presence of olestra, are the same as those observed in shorter-term studies. Both studies were designed to provide data on the effect of olestra on dietary vitamin D₂ in the absence of cutaneously synthesized vitamin D₃ and on vitamin K function at low dietary vitamin K intakes, i.e., about 20% of the NRC's requirements for 5- to 10-kg swine (NRC 1988). The two studies were conducted concurrently.

MATERIALS AND METHODS

Table 1. Treatment groups and target amounts of olestra and vitamins A and E fed to pigs for 26 or 39 wk [View Table]

In the 26-wk study, five groups were fed purified diet (ICN Biomedicals, Cleveland, OH) to which had been added graded concentrations of olestra (OA) ranging from 0.25 to 5.5% (wt/wt). The purified basal diet provided the NRC requirements for nutrients for 5- to 10-kg swine (NRC 1988). This was essentially the same basal diet as fed in other pig studies (Cooper et al. 1997a-c, Daher et al. 1997a). These groups were used to determine the dose-response effects of olestra on vitamins A, D, E and K, and selected water-soluble nutrients, over a range of olestra intakes that encompassed the estimated human intake of olestra.

Three groups were fed purified diet containing 0.25% olestra (wt/wt) and 682, 727 or 816 µg retinol equivalents (RE) of vitamin A/kg diet, added directly to the diet, and 1.5, 3.1 or 4.6 mg d--tocopheryl acetate/g olestra, added in the olestra. Tocopheryl acetate was added to the olestra rather than directly to the diet because that is the way it will be done for olestra-containing snacks. These concentrations are identified as low vitamin (LV), medium vitamin (MV) and high vitamin (HV), respectively. The amounts of vitamin A added were 43, 88 and 177 RE/kg diet over and above the concentration in the basal diet (Table 1). The amounts of vitamin E added were 4, 7 and 11 mg -tocopherol equivalents (-TE)/kg diet greater than the concentration in the basal diet. These groups were used to determine the amounts of vitamins A and E required to restore tissue concentrations of the vitamins to control concentrations at an olestra intake typical of expected human intake.

One group was fed purified diet containing 5.5% (wt/wt) olestra, 955 RE/kg and 71 -TE/kg over and above the concentrations in the basal diet (Table 1); these amounts are the same, on a per gram olestra basis, as the amounts in the 0.25% LV diet. This group was included to allow any potential effects on iron, calcium and vitamin B₁₂ status that might result from poor vitamin A or vitamin E status to be distinguished from direct effects of olestra on those nutrients. Secondary effects of poor vitamin A and vitamin E status were observed in the 12-wk dose-response study (Cooper et al. 1997b). This group was exposed to 1-2 min of UV light daily to stimulate endogenous synthesis of vitamin D₃. A control group was fed the basal purified diet with no olestra or added vitamins A and E.

In the 39-wk study, two groups of pigs were fed purified diet containing 0.25% (wt/wt) olestra with and without low levels (LV) of additional vitamin A (45 RE/kg) and vitamin E (4 -TE/kg); a control group was fed the basal diet. These groups were used to confirm that tissue concentrations of vitamins A and E respond in the same way when pigs are fed a daily olestra intake typical of human intake over a period encompassing major growth and developmental phases, including sexual maturation, as when they are fed for shorter times.

The purified diet fed in the studies provided the NRC requirements for nutrients for 5- to 10-kg swine, with the following two exceptions: ergocalciferol was added to the diet fed in the 26-wk study at twice the NRC requirement, and phylloquinone was added to the diet fed in the 39-wk study at one fifth the NRC requirement. The amount of ergocalciferol in the diet for the 26-wk study was increased, relative to that fed in other pig studies, to ensure that serum 25-hydroxyergocalciferol [25(OH)D₂] would be great enough to allow any effect of olestra to be measured. In 12-wk studies in the pig in which ergocalciferol was present in the diet at the NRC requirement, serum 25(OH)D₂ concentrations were 10 nmol/L (Cooper et al. 1997a and 1997b). The amount of phylloquinone in the diet for the 39-wk study was decreased to confirm that the lack of an effect on vitamin K status observed in the other pig studies was not due to the vitamin being present in excessive amounts. In both studies, a control group was fed the purified basal diet with no olestra or added vitamins A and E.

The dietary concentrations of olestra were selected on the basis of the results from the 12-wk studies (Cooper et al. 1997a and 1997b). From findings in those studies, it was estimated that 0.25% olestra would provide the pigs with 1-2 g/d olestra at the beginning of the study and 4-5 g/d at the end; these intakes encompass the estimated average chronic human intake (Webb et al. 1997). In the 12-wk dose-response study, the effects of olestra on the fat-soluble vitamins did not increase significantly as dietary concentrations of olestra were increased above 5.5%. Thus 5.5% was selected as the highest dietary concentration of olestra fed in the 26-wk study. This amount of olestra would provide the pigs with a daily intake more than 10 times the estimated 90th-percentile chronic intake, 10 g/d, of the heaviest consumers of savory snacks, 13- to 17-y-old adolescents (Webb et al. 1997).

In both studies, the amounts of vitamin A and vitamin E (LV) added to the diets containing 0.25% olestra were chosen to restore tissue concentrations of the vitamins to control concentrations, on the basis of the results of the previous 12-wk study (Cooper et al. 1997a).

The olestra was prepared by the method of Rizzi and Taylor (1978). The olestra was of the same composition as that used in previous pig studies and was heated in a similar manner before being added to the diets.

With the exception of the diets to which additional vitamins A and E were added, all olestra-containing diets delivered the same nutrients per megajoule of digestible energy as the basal diet. The concentrations of nutrients and olestra and the homogeneity of the diets were confirmed by analysis. The stability of olestra in the diet was determined as described by Cooper et al. (1997c). Analysis of the stored samples for periods exceeding the length of the studies showed that olestra was stable (data not shown). The compositions and digestible energy of the diets are shown in Table A in the Appendix.

Blood was collected from the cranial vena cava after an overnight fast at wk 0, 4, 8, 12, 16, 20, 24 and 26 in the 26-wk study and at wk 0, 12, 16 and 39 in the 39-wk study. The serum or plasma samples were stored at 20°C until analyzed. A complete battery of clinical chemistry and hematological measurements was made, including total iron, total iron-binding capacity (TIBC), and concentrations of zinc, calcium and inorganic phosphorus.

Serum was analyzed for retinol, -tocopherol, 25-hydroxyergocalciferol [25(OH)D₂ ], 25-hydroxycholecalciferol [25(OH)D₃ ], and 1,25-dihydroxyvitamin D [1,25(OH)₂D], as in previous studies (Cooper et al. 1997a and 1997b). Serum parathyroid hormone (PTH) concentration was measured as an indicator of calcium status. PTH is a principal regulator of calcium homeostasis (Allen and Wood 1994) and has been shown to increase when calcium intake decreases (Silver 1992).

Plasma was analyzed for folate, and prothrombin time (PT) was measured. In the 26-wk study, blood samples also were taken for PT measurements at wk 14, 18 and 22 from the group fed 5.5% olestra with low concentrations of added vitamins A and E. In the 39-wk study, blood samples were taken for PT measurements from all groups at wk 2, 4, 6, 8, 10, 20, 24, 28, 32 and 36.

Liver, bone and adipose tissue were collected as described previously (Cooper et al. 1997b). Liver was analyzed for retinyl esters, -tocopherol, vitamin B₁₂, iron and zinc. Bone was analyzed for ash content and for zinc, calcium and phosphorus concentrations. Adipose tissue was analyzed for -tocopherol.

Methods used to measure tissue nutrient concentration, with the exception of PTH, have been described previously (Cooper et al. 1997b). The serum concentration of PTH was measured by a noncompetitive immunoradiometric assay using a commercial kit (INCSTAR, Stillwater, MN) based on principles described originally by Miles and Hales (1968). The assay employs two different polyclonal antibodies, specific for two different regions of the PTH molecule. One antibody, bound to polystyrene beads, is specific for PTH 39-84, a portion that includes the C-terminal of the molecule. The second, labeled with ¹²⁵I, is specific for PTH 1-34, the portion that includes the N-terminal. Thus the two antibodies bind only intact PTH.

After incubating the serum samples with both antibodies, the amount of bound ¹²⁵I- labeled antibody was determined by gamma counting. Quantitation was from calibration curves established with human serum samples. PTH concentration was expressed in terms of human equivalents (pmol he/L). This method was validated for swine by using high PTH serum samples collected from pigs fed a diet low in vitamin D and calcium until they displayed signs of rickets.

Repeated-measures ANOVA was conducted to evaluate the effect of olestra on hematology and clinical chemistry parameters; gender, group and gender by group were used as classification variables (Steel and Torrie 1960). To facilitate interpretation of the results of the repeated-measures ANOVA, two-way ANOVA was performed for all time points, with group and gender as class variables. Comparisons among groups were based on the protected least significant difference (LSD) test (Carmer and Swanson 1973, Welsch 1977). No significant gender-by-group interactions existed; therefore, data for males and females were not analyzed separately.

All comparisons were conducted at the two-tailed 0.05 significance level with SAS Version 6.07 software (SAS Institute, Cary, NC).

RESULTS

Table 2. Daily olestra consumption for pigs fed olestra for 26 and 39 wk1 [View Table]

Olestra or added vitamins A and E had no significant effect on the cumulative amount of feed consumed, the cumulative weight gained or the feed efficiency (Table 3). Weight gain expressed in relation to wk-0 body weight or wk-0 body surface area (Kleiber 1975) also did not differ significantly among the groups (data not shown).

Table 3. Cumulative energy consumption, cumulative weight gain and feed efficiency for pigs fed olestra for 26 and 39 wk1 [View Table]

No significant differences in growth were found among any of the groups in the 26-wk study, although pigs fed 5.5% olestra without added vitamins A and E were growing at a slower rate by the end of the study (Table 3). Growth slowed slightly in all groups after about wk 20 of both studies.

In both studies, the female pigs reached sexual maturity as evidenced by swollen vulvae and vaginal discharges. These signs were observed in animals distributed randomly among all groups.

No olestra-related deaths occurred in either study. One female pig in the 3.3% OA group in the 26-wk study died during wk 12 from stress triggered by phlebotomy. Three pigs in the 26-wk study were killed before the end of the study because of unresolving leg or joint disabilities resulting from long-term housing on concrete. A male in the 0.25% MV group was killed during wk 10, a male in the 5.5% OA group was killed during wk 23, and a female in the 0.25% OA group was killed during wk 25. Three pigs also were killed before the end of the 39-wk study: a female in the control group was killed during wk 23 because of a congenital defect in a hind leg that grew progressively worse, a male in the 0.25% OA group was killed during wk 10 because of acute respiratory distress, and a female in the 0.25% OA group was killed during wk 13 because of trauma induced by falling.

Vitamin A. The liver concentration of vitamin A decreased with increasing dietary concentrations of olestra for the five groups of pigs fed increasing amounts of olestra and no added vitamins A or E (Table 4) for 26 wk. The liver vitamin A concentration was 55% of control for pigs fed 0.25% olestra for 26 wk and 50% of control for pigs fed 0.25% olestra for 39 wk. Liver vitamin A for the control pigs was 71% greater than the base-line value in the 26-wk study and 153% greater in the 39-wk study.

Table 4. Liver vitamin A concentration for pigs fed olestra for 26 or 39 wk1 [View Table]

The addition of graded amounts of vitamin A to the diet containing 0.25% olestra in the 26-wk study produced a dose-responsive increase in liver vitamin A concentration. The concentration for the group fed 5.5% olestra with added vitamin A (5.5% LV) was not significantly different than control. Addition of vitamin A to the diet containing 0.25% olestra in the 39-wk study increased liver vitamin A concentration but did not restore it to control concentration.

The liver vitamin A concentrations for pigs fed 0.25% olestra with low, medium or high levels of vitamin A for 26 wk encompassed the control value, indicated by the dashed line (Fig. 1). Regression of the liver vitamin A concentrations measured in the individual pigs against the above-basal concentrations of vitamin A in the diet produced the following relationship between liver concentration of vitamin A and the amount of added vitamin A: where C_LA is the liver concentration of vitamin A in nmol/g liver and V_A is the amount of vitamin A added to the basal diet in RE/kg of diet. This relationship is shown by the solid line in Figure 1.

The vitamin A restoration level, obtained by inserting the mean control liver vitamin A concentration (112 nmol/g liver) into the above equation for C_LA , is calculated to be 114 RE/kg of diet or 93 µg retinyl palmitate/g olestra. This is the amount of additional vitamin A required to restore liver vitamin A concentration for pigs fed 0.25% olestra to the mean control concentration.

Olestra reduced serum vitamin A concentration in both studies. Values measured at wk 0, 12, 26 and 39 are shown in Table E in the Appendix. Addition of vitamin A to the diet slightly increased serum vitamin A concentration. Serum concentrations measured at wk 4, 8, 16, 20 and 24 (not shown) showed similar trends.

Vitamin E. Liver and serum concentrations of vitamin E for pigs fed olestra for 26 wk decreased in a dose-responsive manner with increasing dietary concentrations of olestra (Table 5). A similar response was observed at wk 12 (Table F in the Appendix). The effect of 0.25% olestra on tissue concentrations of vitamin E was essentially the same in the two studies, and the effects on liver and serum concentrations were essentially equal. In the 26-wk study, the serum vitamin E concentration for the group fed 0.25% was 75% of control, compared with 74% of control for the 39-wk study. For these groups, the liver concentration of vitamin E was 74 and 78% of control, respectively. Liver and serum concentrations for the group fed 1.1% olestra for 26 wk were 47 and 49% of control, respectively; for the group fed 5.5% olestra, the values were 25 and 22% of control. Comparison of the data from the groups fed 0.25% olestra in the two studies shows that the effect of olestra did not change after 26 wk.

Table 5. Liver and serum vitamin E concentrations for pigs fed olestra for 26 or 39 wk1 [View Table]

In the 26-wk study, the addition of vitamin E to the basal diet produced increases in the liver and serum concentrations of vitamin E (Fig. 2, 3). The resultant concentrations encompassed the mean concentrations for the control groups, indicated by the dashed horizontal lines in the figures.

Regression of the liver vitamin E concentrations for the individual pigs against the amount of vitamin E added to the basal diet produced the relationships shown by the solid line in Figure 2. This relationship is described by the following equation: where C_LE is the liver concentration of vitamin E in nmol/g liver and V_E is the above-basal amount of vitamin E in -TE/kg diet.

The equation describing the relationship produced by regression of the individual serum vitamin E concentrations, shown by the solid line in Figure 3, is as follows: where C_SE is the serum concentration of vitamin E in µmol/L and V_E is as described above.

Solving these equations as described above for vitamin A produces a restoration level for vitamin E of 4.2 -TE/kg diet from the liver data, and a value of 4.4 -TE/kg diet from the serum data.

Olestra produced a dose-responsive decrease in the concentration of vitamin E in adipose tissue (Table G in the Appendix). The magnitude of the effect was essentially the same as that for liver and serum vitamin E concentrations. The mean concentration of vitamin A in adipose tissue for pigs fed 1.1% olestra for 26 wk was 49% of control; for pigs fed 5.5% olestra, it was 26% of control. Derivation of the amount of additional vitamin E required to offset the effect of 0.25% olestra on the adipose vitamin E concentration, as described above for liver and serum, produced a value of 3.7 -TE/kg diet.

Liver, serum, and adipose vitamin E concentrations for the 0.25% LV group were not significantly different from control values at the end of the 39-wk study. These findings showed that the addition of 4 -TE/kg diet offset the olestra effect, in agreement with the above calculations (Table 5 and Table G in the Appendix). Liver, serum and adipose vitamin E concentrations for the 5.5% LV group were not significantly different than control values at the end of the 26-wk study; again, these results showed that the same amount of added vitamin E, on a mg/g olestra basis, essentially offset the effect of olestra.

Vitamin D. In the 26-wk study, only 5.5% olestra produced a significant decrease in serum 25(OH)D₂ (Table 6). The mean concentration for that group of pigs was 82% of control at wk 26. In the 39-wk study, 0.25% olestra reduced serum 25(OH)D₂ concentration about 14%, on average, across wk 12, 26 and 39. Adding vitamins A and E to the diet had no effect on serum 25(OH)D₂ concentration.

Table 6. Serum 25-hydroxyergocalciferol [25(OH)D2] concentration for pigs fed olestra for 26 or 39 wk1 [View Table]

The serum 25(OH)D₂ concentration for the group fed 5.5% olestra and exposed to UV light was lower than that for the group fed 5.5% with no exposure to UV light. This apparent depression of serum 25(OH)D₂ concentration by production of vitamin D₃ was observed in the 12-wk studies in the pig (Cooper et al. 1997a and 1997b).

The serum concentrations of 25(OH)D₃ were essentially at the detection limit of 4.4 nmol/L in all groups except the 5.5% LV group in the 26-wk study group that received UV exposure (Table 7). The highly variable value of serum 25(OH)D₃ at wk 12 for the 5.5% OA group and the high and variable values for the 0.25% LV and 0.25% HV groups probably resulted from inadvertent and variable exposure to UV light. These observations are not olestra-related because they did not occur at other time points or for other groups.

Table 7. Serum 25-hydroxycholecalciferol [25(OH)D3], total 25-hydroxyvitamin D [25(OH)D] and 1,25-dihydroxyvitamin D [1,25(OH)2D] concentrations for pigs fed olestra for 26 or 39 wk1 [View Table]

No olestra dose response was observed on serum 25(OH)D₃ , total 25-hydroxyvitamin D [25(OH)D] or 1,25(OH)₂D in either study (Table 7). The serum concentration of 1,25(OH)₂D declined with age for all groups in both studies.

Vitamin K. There was no effect of olestra on PT in either study; values measured at wks 0, 12, 26 and 39 are shown in Table 8. PT values at other time points were similar (not shown).

Table 8. Plasma prothrombin time (PT) for pigs fed olestra for 26 or 39 wk1 [View Table]

Olestra did not affect the measures of status of any of the water-soluble micronutrients. No effect of olestra was observed on plasma folate concentration (Table 9) or liver vitamin B₁₂ concentration (Table 10). Liver concentration of iron was unaffected by olestra (Table 11), as were serum total iron concentration and TIBC (Tables H and I in the Appendix). Bone, liver (Table 12) or serum (Table J in the appendix) concentration of zinc was not affected by olestra.

Table 9. Plasma folate concentration for pigs fed olestra for 26 or 39 wk1 [View Table]

Table 10. Liver vitamin B12 concentration for pigs fed olestra for 26 or 39 wk1 [View Table]

Table 11. Liver iron, serum iron and serum total iron-binding capacity for pigs fed olestra for 26 and 39 wk1 [View Table]

Table 12. Liver, bone and serum zinc concentrations for pigs fed olestra for 26 or 39 wk1 [View Table]

Olestra had no effect on bone ash content or bone concentrations of phosphorus and calcium (Table 13), serum concentration of PTH (Table 14), or serum concentrations of calcium or inorganic phosphorus (Table 15 and Tables K and L in the Appendix).

Table 13. Bone concentrations of ash, phosphorus and calcium for pigs fed olestra for 26 or 39 wk1 [View Table]

Table 14. Serum parathyroid hormone (PTH) concentration for pigs fed olestra for 26 or 39 wk1 [View Table]

Table 15. Serum calcium and inorganic phosphorus concentrations for pigs fed olestra for 26 or 39 wk1 [View Table]

DISCUSSION

The daily amounts of olestra eaten by the pigs fed 0.25% olestra in these studies were typical of the estimated average chronic human intake from savory snacks, 3.1 g/d. The daily intake for the pigs fed 0.5% olestra approximated the estimated 90th-percentile chronic human intake, 6.9 g/d. The daily intake for the pigs fed 5.5% olestra in the 26-wk study exceeded the average chronic human intake by a factor of about 44 and exceeded the 90th-percentile chronic human intake by a factor of about 20. These daily intakes of olestra had no adverse effects on the pigs. No olestra-related clinical signs of ill health were observed. In addition, hematology and clinical chemistry measurements showed no adverse effects of olestra.

Because protein, carbohydrate and fat are the major dietary energy sources and provide certain essential nutrients such as amino acids and fatty acids, an effect of olestra on the absorption or digestion of these macronutrients would be reflected in reduced growth or reduced feed efficiency during this high need period. Weight gain and digestible feed efficiency did not differ among the groups in either study, an indication that olestra had no effect on the availability or utilization of macronutrients. This finding is consistent with observations in other studies in the pig (Cooper et al. 1997a-c, Daher et al. 1997a).

For the first 20 wk of the studies, the pigs grew at a rate comparable to that reported for these crossbred pigs fed standard swine feed (Martin and Crenshaw 1989) and consistent with growth rates observed in the previous 12-wk olestra feeding studies. After this, the growth rate declined for all groups in both studies, regardless of diet. Over the 26-wk study, the rate decreased from ~0.23 to 0.03 kg gain/(kg body weight·wk), a decline of about 87%. This is similar to the 78% decline in the growth rate in humans from childhood to young adulthood (NRC 1989).

The slower but not significantly different growth rate for the pigs fed 5.5% olestra for 26 wk was probably a reflection of their poor vitamin A and vitamin E status. Pigs in the group fed 5.5% olestra with added vitamins A and E (5.5% LV group) did not show a decline in growth but rather had the fastest growth rate of any group in the study.

The slower growth of the pigs fed 0.25% olestra with added vitamin A and E after about wk 15 in the 39-wk study apparently represents random biological variation. There was no evidence that it was related to olestra intake. Cumulative weight gain, digestible feed efficiency and body weight adjusted for wk-0 weight or wk-0 body surface area for this group were not significantly different from control values. In addition, the group fed 0.25% olestra without added vitamins A and E grew at the same rate as the control group.

The length of these studies was such that olestra had the opportunity to affect the status of nutrients that are present in the body in large pools and whose status thus changes slowly in response to changes in intake. Such nutrients include vitamin A and calcium. More than 90% of the body stores of vitamin A are in the liver (Olson 1984). For the pigs in these studies, most of the final liver store of vitamin A was accumulated while they were eating olestra. To illustrate, at the end of the 26-wk study, the pigs weighed ~170 kg, of which about 2% is the liver (Filer et al. 1966). The liver vitamin A concentration for the control group at wk 26 was ~112 nmol/g liver, which corresponds to ~380,800 nmol of vitamin A. At the start of the study, the pigs weighed ~20 kg and had a liver vitamin A concentration of ~65 nmol/g liver, or a total liver pool of vitamin A of ~26,000 nmol. The 354,800 nmol of vitamin A liver stores accumulated during the study, about 93% of the final amount, were acquired while the pigs ate olestra at each meal. This situation provided ample opportunity for the effect of olestra of vitamin A availability to be fully manifested.

Serum vitamin A concentrations remained above the value of 0.18 µmol/L associated with signs of vitamin A deficiency in pigs, even in the groups fed the greatest amounts of olestra (Hentges et al. 1952). In pigs, signs of vitamin A deficiency occur at serum concentrations of ~0.35 µmol/L, about one half of the concentration associated with impaired dark adaptation, night blindness and ocular lesions in humans (Gibson 1990). At the start of these studies, the serum vitamin A concentrations (0.84-0.98 µmol/L) of the pigs were slightly greater than the concentrations (0.63-0.86 µmol/L) measured in the two 12-wk studies (Cooper et al. 1997a and 1997b.). Factors that might account for these differences include the age of the pigs at the start of the studies, the composition of the diet fed during acclimation and differences in vitamin A status at receipt resulting from differences in vitamin A intake during suckling and weaning.

The effect of olestra on vitamin A absorption measured in the 26-wk study represents the complete effect of olestra on that vitamin, as evidenced by the comparison of the effect with that measured in the 12-wk dose-response study (Cooper et al. 1997b). In both the 12-wk study and the 26-wk study, liver vitamin A concentrations were 65 and 12%, respectively, of control for pigs fed 1.1 or 3.3% olestra.

Olestra did not affect the measures of calcium status even though the pigs acquired most of their calcium pools while eating olestra. About 99% of the total calcium pool is found in bone (Peo 1991). On the basis of the percentage of body weight coming from bone (~9% in young pigs and ~6% in mature pigs) and the bone concentration of calcium at the beginning and the end of the 26-wk study, the pigs acquired more than 80% of their calcium pools while being fed the olestra-containing diets.

These studies provided two other important confirmatory findings with respect to the effect of olestra on the status of fat-soluble vitamins. First, the 26-wk study provided data confirming the effect of olestra on the availability of dietary vitamin D, as reflected by measurements of serum 25(OH)D₂ , in the absence of any potential interference from vitamin D₃. In the 12-wk studies, serum 25(OH)D₂ concentration apparently was suppressed by competition between vitamin D₂ and vitamin D₃ for the liver 25--hydroxylase. In pigs, vitamin D₃ is the preferred substrate for liver 25--hydroxylase (Horst et al. 1982). In the present studies, with the exception of one group in the 26-wk study, the pigs were not exposed to UV light, and 25(OH)D₃ contributed <20% to overall vitamin D status. Under these conditions, 5.5% olestra reduced serum 25(OH)D₂ concentration to about 73% of control, when averaged over the measurements made from wk 12 through 26. In the 12-wk study in which the pigs were exposed to UV light daily, 25(OH)D₃ contributed almost 90% of total vitamin D status. In that study, serum 25(OH)D₂ concentration for pigs fed 5.5% olestra, averaged across the measurements made at wk 8 and 12, was ~67% of control (Cooper et al. 1997b). The similarity of the effects shows that the competition between the two forms of the vitamin for the liver hydroxylase does not prevent the measurement of the olestra effect on the availability of dietary vitamin D.

The 26-wk study provided additional data consistent with the competitive sensitivity of liver 25--hydroxylase in the pig. For the group of pigs fed 5.5% olestra and exposed to UV light daily (5.5% LV), 25(OH)D₃ contributed about 66% of total vitamin D status. In contrast, 25(OH)D₃ contributed about 14% of total status for the group fed 5.5% olestra but not exposed to UV light (5.5% OA). The serum 25(OH)D₂ concentration for the group with the higher 25(OH)D₃ contribution was about 39% less than for the group with the lower 25(OH)D₃ contribution.

The decrease in serum 1,25(OH)D₂ concentration with time, which occurred in both studies, was also observed in the 12-wk pig studies. It is consistent with an age effect observed in pigs by other researchers (Horst and Littledike 1982).

The second important confirmatory finding from these studies with respect to the fat-soluble vitamins was that olestra did not affect vitamin K status, as measured by PT. The 12-wk studies showed that PT was unaffected by olestra when vitamin K intake was near the NRC requirement for 5- to 10-kg swine. In the 39-wk study, a daily olestra intake of >1.5 times the estimated average chronic human intake from savory snacks, 3.1 g/d, did not affect PT, and the pigs were eating only about one fifth of the NRC requirement of vitamin K for 5- to 10-kg swine. This finding provides assurance that an effect of olestra on PT did not occurr in the previous pig studies and was masked by excessive vitamin K intakes.

Prothrombin time is a rather insensitive measure of vitamin K status (Suttie 1992). However, measurements of vitamin K status in humans using sensitive functional tests confirmed that olestra does not affect vitamin K functional status. Measurements of the urinary excretion of -carboxyglutamic acid and the plasma concentration of des--carboxyprothrombin in subjects consuming up to 32 g/d of olestra for 8 wk showed no effects on vitamin K function (Schlagheck et al. 1997a and 1997b). In addition, measurement of the circulating concentration of fully -carboxylated prothrombin in free-living subjects consuming 20 g/d olestra for 6 wk (Jones et al. 1991) or 18 g/d for 16 wk (Koonsvitsky et al. 1997) showed no effect of olestra on vitamin K status.

The 12-wk dose-response study showed a steep, hyperbolic dose response of olestra on liver vitamin A concentration (Cooper et al. 1997b). The present 26-wk study further defined the shape of the response. The effects of 0.25 and 0.5% were about three times greater than the magnitudes that would have been predicted by interpolation of the data from the 12-wk study. The extreme hyperbolic nature of the dose response at low olestra intakes reflects the fact that the availability of retinyl palmitate and of -carotene is affected differently by olestra. Data from human studies illustrate the different effects of olestra on the availability of the two dietary sources of vitamin A. Serum -carotene concentration was reduced by 50% in human subjects given 8 g/d olestra for 4 wk (Schlagheck et al. 1997b). In contrast, 8 or 20 g of olestra in a meal had no effect on the absorption of retinyl palmitate from that meal (Daher et al. 1997b).

The two dietary sources of vitamin A are affected differently by olestra because they differ in lipophilicity, a primary factor controlling olestra's effect on nutrient availability (Jandacek 1982). The difference in lipophilicities of the two dietary sources of vitamin A is illustrated by the octanol-water partition coefficients (log₁₀ p_c) of -carotene and of retinol (retinol because retinyl palmitate is hydrolyzed rapidly in the gut). The log₁₀ p_c value for retinol is 7.6 and for -carotene is 17.6. The coefficients were calculated by the method developed by Meylan and Howard (1995). These coefficients indicate that -carotene is about 10 orders of magnitude more lipophilic than retinol.

Because of the difference in the olestra effect on the two dietary sources of vitamin A, low olestra dietary concentrations (e.g., 0.25 and 0.5%) would be expected to substantially reduce that portion of liver vitamin A stores that comes from -carotene and to have essentially no effect on the portion that comes from preformed vitamin A. Further, an incremental increase in the dietary olestra concentration at low olestra intakes will produce a larger effect on liver vitamin A stores than the same incremental increase at higher olestra intakes. This is the case because that portion of the stores that comes from -carotene has already been reduced greatly at higher olestra intakes; therefore the additional effect is due primarily to the effect on retinyl palmitate, which is affected less per gram or per percentage of olestra in the diet.

Because olestra has different effects on the availability of the two sources of vitamin A stores, the restoration level of vitamin A determined in the present 26-wk study, 114 RE/kg diet, is expected to be greater than the restoration level calculated from the relationship derived in the 12-wk study in which pigs were fed 1.1-7.7% olestra (Cooper et al. 1997b). The restoration level determined from pigs fed 0.25% olestra, as in the 26-wk study, reflects almost totally the effect of olestra on -carotene absorption because that dietary concentration of olestra has little (if any) effect on retinyl palmitate absorption. In contrast, the restoration level derived from pigs fed 1.1-7.7% olestra in the 12-wk study reflects the effect of olestra on the absorption of both -carotene and retinyl palmitate. At estimated human olestra intakes, even at the 90th percentile, 6.1 g/d, the only effect of olestra on vitamin A stores is the effect on -carotene absorption.

The responses of tissue stores to the added amounts of vitamin A and vitamin E were the same in the two studies, an indication that restoration efficiency does not change as the pigs age. However, the above-basal amounts of vitamin A added to the 0.25% olestra diet were different in the two studies. In the 26-wk study, the amount of vitamin A in the 0.25% LV diet was 44 RE/kg diet above the basal concentration. This amount restored liver vitamin A concentration from 55 to 70% of control. In the 39-wk study, the 0.25% LV diet contained a lower amount of vitamin A, 27 RE/kg diet above basal. On the basis of the results from the 26-wk study, 27 RE/kg of added vitamin A would be predicted to restore the liver concentration from the 50% of control value measured for the 0.25% OA group to 59% of control. Actual restoration reached 57% of control.

The responses of tissue concentrations of vitamin E obtained in the 39-wk study by the added vitamin E were also consistent with the responses noted in the 26-wk study. The measured above-basal amount of vitamin E in the 0.25% LV diet fed in both studies was 3.6 -TE/kg. In the 26-wk study, this added vitamin E restored liver, serum and adipose concentrations to 98, 100 (averaged over wk 12 and 26) and 103% of control, respectively. In the 39-wk study, liver, serum, and adipose concentrations were restored to 88, 103 (averaged over wk 26 and 39) and 92% of control, respectively.

The similarity of the results of olestra consumption on liver and serum concentrations of vitamin E, measured after different periods of ingestion, provides assurance that the effect of olestra on vitamin E status has been measured fully. The similarity of the effect of olestra on the two tissue pools provides assurance that serum vitamin E concentration can be used as a reliable indicator of vitamin E body stores. This finding increases confidence in the measurement of an olestra effect on vitamin E status by measuring serum vitamin E concentration in humans.

The restoration level of vitamin E determined from liver and serum measurements in the pig agrees well with the value determined from serum measurements in humans. The restoration levels for vitamin E determined in the present 26-wk study, 4.2 -TE/kg diet determined from liver data or 4.4 -TE/kg diet determined from serum data, translate to 2.1 and 2.2 mg d--tocopheryl acetate/g olestra. A restoration level of 2.1 mg d--tocopheryl acetate/g olestra was derived from serum measurements in humans (Schlagheck et al. 1997a). These results also substantiate the appropriateness of the pig as a model in which to assess the nutritional effects of olestra and in which to determine restoration levels of fat-soluble vitamins. This consideration is important, given the difficulties in determining a vitamin A restoration level in humans.

The restoration level of vitamin A derived in the present 26-wk study from pigs fed 0.25% olestra and a 3:1 ratio of retinyl palmitate and -carotene, 114 RE/kg diet, is appropriate to the human situation because the dietary source of vitamin A modeled the average found in the U.S. diet and because the grams per day intake of olestra was typical of expected human consumption.

ACKNOWLEDGMENTS