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, 3,
, and
* Departments of Biochemistry, Ophthalmology, and Radiation Oncology, Medical College of Wisconsin, Milwaukee, WI 53226
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
LITERATURE CITED
ABSTRACT 
Previous studies showed a higher percentage of neutrophils from vitamin A deficient rats are hypersegmented and contain lower levels of cathepsin G than the neutrophils from control rats. In this study chemotaxis, phagocytosis and oxidant generation were studied using either isolated neutrophils or neutrophils in whole blood from four dietary groups of rats: 1) vitamin A deficient rats; 2) vitamin A deficient rats that received vitamin A for 16, 8, 4 or 2 d prior to killing; 3) weight-matched rats pair-fed a vitamin A-complete diet; and 4) rats fed nonrestricted, vitamin A complete diet. Chemotaxis towards P. aeruginosa conditioned medium and formylated methinyl leucinyl phenylalanine was significantly lower for neutrophils from vitamin A-deficient rats than for neutrophils from weight-matched pair-fed rats, nonrestricted vitamin A sufficient rats and vitamin A deficient rats that received vitamin A for 16 d prior to killing. No differences in chemotaxis towards activated rat serum were noted among the neutrophils from the four groups of rats. Adhesion of P. aeruginosa organisms, phagocytosis of these organisms and generation of active oxidative molecules were significantly lower in the neutrophils from the vitamin A-deficient rats relative to these functions in the neutrophils from the vitamin A deficient rats that received vitamin A for 16 d, weight-matched rats pair-fed a vitamin A complete diet; and rats fed nonrestricted, vitamin A-complete diet. Eight days after vitamin A administration to vitamin A deficient rats, the ability of the neutrophils to phagocytose P. aeruginosa organisms and to generate active oxidant molecules was restored to the levels observed for weight-matched, pair-fed rats and rats fed nonrestricted, vitamin A complete diet. The elucidated alterations in neutrophil function in vitamin A deficient rats probably contribute to the altered ability of vitamin A deficient rats to fight infections.
Key words: vitamin A deficiency, neutrophils, phagocytosis, chemotaxis, rats, oxidant generation.INTRODUCTION
Vitamin A deficient people and rats are prone to more severe infections and have a higher mortality than vitamin A sufficient people and rats (Beaton et al. 1992
, Fawzi et al. 1994, Twining et al. 1996b). This increase is observed even in the early stages of the deficiency (Semba et al. 1993, Twining et al. 1996b). Numbers of Pseudomonas aeruginosa organisms that are subinfectious in corneas of normal and pair-fed, weight matched rats are capable of inducing keratitis in corneas of vitamin A deficient rats (Twining et al. 1996b).
The first line of defense against infections, including corneal infections, is the neutrophil (Smith 1994). This cell is the first to respond to infections and targets bacteria (Ferrante et al. 1993, Twining et al. 1996b), fungi, (Roilides et al. 1993), protozoa (Ferrante et al. 1989), viruses (Klebanoff and Coombs 1992, Ratcliffe et al. 1988), virus-infected cells (Ratcliffe et al. 1988) and tumor cells (Weitzman and Gordon 1990). The neutrophil phagocytoses the target organism or cell and then uses an arsenal of active oxygen species and other microcidal molecules to kill its target (Rosen et al. 1995, Smith 1994). The importance of the neutrophil is observed in animals with neutropenia or a deficiency of one or more key neutrophil enzymes (Edwards 1994). In these animals, infections which normally are mild can be life threatening.
The neutrophil differentiates and matures in the bone marrow. Differentiation from the promyelocyte to the mature neutrophil requires the oxidized form of retinol, retinoic acid (Robertson et al. 1992, Tsai and Collins 1993). Recently, we showed that in severely vitamin A deficient rats, the concentration of retinol is not less in the bone marrow as we hypothesized, but is elevated (Twining et al. 1996a). This sequestration of retinol indicates the importance of bone marrow cells for the survival of the animals.
Vitamin A deficiency does not alter the distribution of myeloid derived cells in the bone marrow with the exception of a significantly greater occurrence of hypersegmented neutrophils (six or more lobes) in vitamin A deficient rats (2.1%) relative to the control rats (
0.1%). The blood of the vitamin A deficient rats contained significantly higher numbers (P < 0.01) of hypersegmented neutrophils (67%) relative to those in the control rats (2-7%). The hypersegmentation of the neutrophils in vitamin A deficient rats was not due to a concurrent deficiency of vitamin B-12 or folate (Twining et al. 1996a).
Incubation of neutrophils with retinoids can influence their function. At a concentration of 25 µmol/L, all trans-retinal and retinol can stimulate the release of superoxide (Badwey et al. 1989). Retinoic acid in combination with other neutrophil activators such as phorbol esters or formylated methinyl leucinyl phenylalanine (f-Met-Leu-Phe),4 inhibit superoxide production (Fumarulo et al. 1991, Sharma et al. 1990, Varani et al. 1991), chemiluminescence (Fumarulo et al. 1991), degranulation (Fumarulo et al. 1991, Perkins et al. 1991) and endothelial cell injury (Varani et al. 1991).
Very little is known about polymorphonuclear leukocyte function in vitamin A deficient animals. Using a rat model, Ongsakul et al. (1985) reported fewer numbers of active phagocytic polymorphonuclear leukocytes in deficient animals. The number of bacteria ingested per active phagocytic cell was similar. The purpose of this study was to determine the effects of vitamin A deficiency on the neutrophil functions: chemotaxis, adhesion of organisms, phagocytosis and killing.
MATERIALS AND METHODS
,7Dichlorofluorescein diacetate (DCFH-DA) and hydroethidine were from Molecular Probes (Eugene, OR). The Coulter Diagnostics Clone Immuno-Lyse and Clone Fixer (Hialeah, FL) were used. Unless otherwise stated chemicals and enzymes were obtained from Sigma Chemical Company (St. Louis, MO). Myeloperoxidase was from Athens Research Technologies (Athens, GA). Polycarbonate chemotaxis membranes were obtained from Neuropore (Cabin John, MD). RPMI medium was purchased from GIBCO-BRL (Grand Island, NY). A FITC-labeled rabbit anti-rat PMN antibody was obtained from Accurate (Westbury, NY).
A (vitamin A deficient), +A (weight-matched pair-fed), R (vitamin A deficient rats refed vitamin A for 16 d) and N (nonrestricted, vitamin A complete diet) rats with time of incubation. Whole blood, pretreated with hydroethidine, was incubated with FITC-labeled killed P. aeruginosa, strain 107, at 37°C for increasing times. The reaction was stopped at 2, 7, 15 and 30 min by the addition of Immuno-Lyse followed by Clone Fixer. Flow cytometry was used to quantify phagocytosis and superoxide generation by measuring the increase in florescence at 510-530 nm due to the association of FITC P. aeruginosa organisms with the cells (LFL1) and the increase in fluorescence at 600 nm from the oxidation of hydroethidine by superoxide (LFL3), respectively. The neutrophils were selected by electronic gating. Fluorescence values are given as log fluorescence (LFL) in these representative plots.
Vitamin A deficient and control rats. WAG/Rij/MCW rats were derived from animals established in 1970 at Yale University from rats supplied by H. R. Reinhold (Radiological Institute, TNO, Rijswyk, The Netherlands). These rats were raised in a defined microflora environment, and vitamin A deficient and control rats were prepared as previously reported (Twining et al. 1996a
). Briefly, nursing females were fed an autoclavable casein-based vitamin A deficient diet5 beginning 16 d postpartum. The pups were weaned on d 21. The male pups were divided into four dietary groups: 1) Sixteen rats (A) were fed a casein-based retinoid deficient diet. 2) Twenty-six rats (R) were fed the retinoid deficient diet. On d 74 (16 d before killing), 14 rats were fed a bolus of 500 µg retinyl palmitate in 200 µL safflower oil and then given free access for 16 d to the retinyl palmitate-supplemented control diet (vitamin A deficient diet supplemented with retinyl palmitate at 15 mg/kg). On d 82, 86 and 88 (8, 4 and 2 d before killing), groups of four vitamin A deficient rats were fed a bolus of retinyl palmitate (500 µg retinyl palmitate in 200 µL safflower oil). These rats were then given free access to the retinyl palmitate-supplemented control diet. 3) Fourteen rats (+A) were restricted in their intake of the retinyl palmitate-supplemented diet so that their weight gain matched that of the A rats. 4) Fourteen nonrestricted (N) rats were given free access to the retinyl palmitate-supplemented diet. Because P. aeruginosa was not present in the known microflora environment, the rats were immunized with killed P. aeruginosa, strain 107, on d 42, 49 and 56 (Twining et al. 1996b). Because the rats used in these experiments were from multiple litters born on different days, the experiments were staggered. Each of the experiments described was carried out as 3 or 4 independent trials.
80°C until extracted for retinoids. All procedures were carried out under yellow lights to preserve the retinoids. Samples were stored in either foil or yellow tubes for protection from light.
). The entire liver was weighed, homogenized, saponified and extracted using hexane in the presence of 80 mmol pyrogallol/L (Nauss et al. 1985, Thompson et al. 1971). Retinyl acetate was added as an internal standard to all samples prior to extraction. The samples were solubilized in ethanol and separated by HPLC (Dionex, Sunnyvale, CA) on a Whatman (Hillsboro, OR) EQC 125A C18 column (4.6 mm i.d. × 108 mm) using a linear gradient of methanol/H2O (90:10, v/v) and ethyl acetate/isopropanol (90:10, v/v) at a flow rate of 1.0 mL/min (Rudy et al. 1992).
Neutrophil chemotaxis.
Neutrophils were isolated from heparinized blood using dextran sedimentation followed by separation on a Histopaque density gradient (Twining et al. 1996). Preparations containing at least 95% neutrophils were used for chemotaxis experiments. Activated rat serum was prepared by incubating plasma from N rats with zymosan for 1 h at 37°C followed by heat inactivation at 56°C for 30 min (Metcalf et al. 1986). RPMI medium was conditioned by Pseudomonas aeruginosa strain 107 for 48 h at 37°C. The organisms were removed by centrifugation followed by filtration through a 0.2 µm filter.
) using Neuro Probe 48-well micro chemotaxis chamber (Neuro Probe Inc., Cabin John, MD) and gelatin coated 5 µm pore membranes. Neutrophils (2 × 107), in RPMI medium, were placed in the bottom wells, and the chemoattractants, 10% activated rat serum, f-Met-Leu-Phe (10
10 to 105 mol/L) or 0-16 mL/100 mL P. aeruginosa conditioned medium were placed in the top wells. For 4 × 4 checkerboard assays (to test for chemotaxis rather than chemokinesis), chemoattractants were placed in both the upper and lower wells at graded concentrations (0, 4, 8 and 12 mL/100 mL for the P. aeruginosa conditioned medium and 1010 to 106 mol/L for f-Met-Leu Phe). The chamber was inverted and incubated at 37°C in a humidified 5% CO2 environment for 1 h. The contents of the upper wells (containing the cells which chemotaxed) were mixed with hexadecyltrimethylammonium bromide to a final concentration of 5 g/L. Chemotaxis was quantified by determination of the myeloperoxidase content of these samples relative to the myeloperoxidase content of isolated neutrophils (Williams et al. 1983).
) and labeled with fluorescein isothiocyanate (Twining 1984). Whole blood was incubated with hydroethidine (added in dimethylformamide) at a final concentration of 5 µmol/L for 10 min at 37°C prior to initiation of experiments. Hydroethidine is permeable to membranes. Inside neutrophils, hydroethidine is oxidized to the membrane impermeable molecule, ethidium, mainly by superoxide during the respiratory burst (Rothe and Valet 1990). Upon oxidation, the fluorescence of this molecule undergoes a red shift. In some experiments, DCFH-DA (in ethanol) was also added at a final concentration of 5 µmol/L (Rothe and Valet 1990). DCFH-DA is a nonfluorescent, membrane permeable compound which upon oxidation becomes fluorescent and membrane impermeable (Rothe and Valet 1990). This reagent follows mainly the myeloperoxidase catalyzed formation of hypohalous compounds. The specificities of the oxidants for hydroethidine and DCFH-DA, however, is not absolute.
|
Table 1. Final weights, plasma retinol and total liver retinol concentrations of vitamin A-deficient and control rats1 |
) using a Coulter Epics V Flow Cytometer. A 488 nm argon laser with a 470-500 nm bandpass filter was used for excitation. A 510-530 band pass filter was used for emission wavelength selection when either FITC-labeled P. aeruginosa organisms or DCFH-DA was used. When hydroethidine was used, a 600 nm longpass filter was used for emission wavelength selection. For each sample, the fluorescence of 5000 to 10,000 cells was measured. The neutrophils were gated using the forward angle scattering vs. 90° light scattering. The position of the neutrophils was determined using cells labeled with an FITC-labeled antibody specific for rat neutrophils. The data were analyzed using a Cytometer Elite software package (Coulter Diagnostics, Hialeah, FL).
) was used to determine whether an overall difference existed among the A, R, +A and N groups of data. The Kruskal-Wallis analysis of variance was used when variances were unequal between groups or the data did not pass the normality test. Multiple comparisons were carried out using Tukey's Comparison Method (Tukey 1949) except where the data did not pass the normality test. In this later case, Dunn's multiple comparison of ranks (Dunn 1961) was used.
RESULTS
A and +A rats were significantly different from those of the R16 and N rats (P < 0.05; Table 1). No significant differences in the final average weights were observed among the A, +A, R2, R4 and R8 groups. The plasma and liver retinol concentrations at the time of killing confirmed that the A rats were vitamin A deficient and that the R rats had received vitamin A (Table 1). The plasma retinol concentrations of the A rats were significantly different from those of the +A, R2, R4, R8, R16 and N rats (P < 0.05). The liver retinol concentrations of the A rats were significantly different from those of the +A, R16 and N rats (P < 0.05) but not those of the R2, R4 or R8 rats (P < 0.05).
A (Vitamin A deficient), +A (Weight-matched pair-fed), R16 (Vitamin A deficient rats refed vitamin A for 16 days) and N (Nonrestricted, vitamin A complete diet) rats. Isolated neutrophils from vitamin A deficient and control rats were placed in modified Boydon Chambers and allowed to migrate towards 10% activated rat serum, 12% P. aeruginosa strain 107 conditioned RPMI medium and 1 nmol f-Met-Leu-Phe/L for one hour under 5% CO2 and a humidified atmosphere. Chemotaxis was quantified by assaying the cells that migrated through the filter into the well containing the chemoattractant for myeloperoxidase. Values were corrected for the myeloperoxidase content of the neutrophils isolated from the same rat. Chemotaxis of the cells toward activated rat serum was used as the control. Values are given as percent migration of the neutrophils towards either P. aeruginosa, strain 107 conditioned medium or f-Met-Leu-Phe relative to the migration of the neutrophils towards 10% activated rat serum (ARS). The bars represent the means, and the error bars represent the SD, n = 10. An overall significant difference in chemotaxis was determined by ANOVA among the neutrophils from A, R, +A and N rats. P < 0.05 for a vs. b and c vs. d based on Tukey's Comparison Method (Tukey 1949).
DISCUSSION A, R, +A and N rats was studied using activated rat serum (ARS, control), P. aeruginosa conditioned medium and f-Met-Leu-Phe. In dose response experiments, maximal chemotaxis towards the P. aeruginosa conditioned medium was observed at concentrations of 14 to 16 mL/100 mL and towards f-Met-Leu-Phe at concentrations of 1 to 10 nmol/L for the A, R16, +A and N rat neutrophils. Checkerboard analyses (4 × 4) for both P. aeruginosa conditioned medium and f-Met-Leu-Phe revealed migration in the direction of the gradient and confirmed the assays measured chemotaxis and not chemokinesis. Chemotaxis towards ARS varied among experiments. However, within a given experiment, there were no significant differences in the chemotaxis activity of A PMN relative to +A, R16 and N PMN towards this chemoattractant. Chemotaxis towards P. aeruginosa conditioned medium and 10 nmol f-Met-Leu-Phe/L relative to ARS was significantly less (P < 0.05) for the neutrophils from the A rats when compared to the neutrophils from the +A, R and N rats (Fig. 1).
Fig. 3.
Effect of vitamin A deficiency and refeeding of vitamin A to vitamin A deficient rats on neutrophil phagocytosis (A, C) and superoxide generation (B, D). Neutrophils in whole rat blood from vitamin A deficient rats (A), vitamin A deficient rats refed vitamin A for 2, 4 or 8 d (R2, R4, R8) and non-restricted, vitamin A complete diet rats (N) were assayed as given in Figure 2. A: Comparison of FITC-P. aeruginosa fluorescence associated with neutrophils from A, +A, and N rats. n = 8 per group. B: Comparison of superoxide oxidation of hydroethidine by neutrophils from A, +A, and N rats. n = 8 per group. C: Effect of refeeding vitamin A to vitamin A deficient rats for 2, 4 or 8 d on FITC-P. aeruginosa fluorescence associated with neutrophils. n = 4 per group. D: Effect of refeeding vitamin A to vitamin A deficient rats for 2, 4 or 8 d on superoxide oxidation of hydroethidine by neutrophils. n = 4 per group. The points represent the average of the mean fluorescence channel, and the error bars represent the SD. An overall significant difference in phagocytosis for the data in A plus C and in killing for the data in B plus D was determined by ANOVA among the neutrophils from A, +A, R2, R4, R8 and N rats at 7, 15 and 30 min for both phagocytosis and killing and, in addition, at 2 min for phagocytosis. P < 0.01 for a vs. b in A, and c vs. d in B; P < 0.05 for a (in A) vs. e (in C) and for c (in B) vs. f (in D) based on Tukey's Comparison Method (Tukey 1949).
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A samples (32 ± 3%) at 7 min relative to the R16 (61 ± 5%), +A (58 ± 3%) and N (56 ± 4%) samples (n = 8 for each group). By 15 and 30 min, the differences in distribution of cells among the groups were insignificant. The average of the mean cell fluorescence channel for both green fluorescence, due to FITC-labeled P. aeruginosa organisms (Fig. 3A), and red fluorescence, due to hydroethidine oxidation (Fig. 3B), was significantly (P < 0.01) lower for the total A neutrophil population than the total +A and the total N neutrophil populations at 7, 15 and 30 min. The number of organisms phagocytosed by the neutrophils reached a plateau by 10 min (Fig. 3A), however, the oxidation of hydroethidine was still linearly increasing at 30 min (Fig. 3B). In neutrophils obtained from rats refed vitamin A two (R2) d and four (R4) days before killing, the averages of the fluorescence channel means representing the phagocytosis of FITC-P. aeruginosa organisms (Fig. 3C) and the oxidation of hydroethidine (Fig. 3D) were higher at all time points. No significant differences were observed between the values for neutrophils from the R2 d and R4 d rats and those for the neutrophils from the A rats. Neutrophils, obtained from vitamin A deficient rats 8 d after vitamin A was given, had significantly greater numbers of phagocytosed FITC-organisms (Fig. 3C) and oxidized hydroethidine (Fig. 3D) (P < 0.05) than that for the neutrophils from A rats. Similar values were observed at 16 days (not shown). No significant differences in the amounts of phagocytosed FITC-organisms and oxidized hydroethidine were observed among the neutrophils from the vitamin A-deficient rats refed vitamin A for 8 and 16 d, the +A and N rats.
A, R, +A and N rats. The generation of hypochlorous acid was significantly less (P < 0.05), as quantified by the amount of DCFH-DA oxidized at 2, 7 and 15 min, in the neutrophils from the A rats in comparison to the neutrophils from the R16, +A and N rats (Fig. 4). By 30 min, no significant differences were noted among the neutrophils from the A, R, +A and R rats.
Fig. 4.
The effect of vitamin A-deficiency on myeloperoxidase oxidation of 27-dichlorofluorescein diacetate (DCFH-DA) determined by comparing activated neutrophils from A (vitamin A deficient), +A (weight-matched pair-fed), R16 (vitamin A deficient rats refed vitamin A for 16 d) and N (nonrestricted, vitamin A complete diet) rats. The experiment was carried out in the same manner as given in Figure 2 with the exceptions that unlabeled P. aeruginosa and DCFH-DA were used. n = 8 for A; n = 4 for +A, R16 and N. Values are the average of the mean fluorescence channel and the error bars represent SD. An overall significant difference in DCFH oxidation was determined by ANOVA among the neutrophils from A, +A, R16 and N rats at 2, 7 and 15 min. P < 0.05 for a vs. b at each time point based on Tukey's Comparison Method (Tukey 1949).
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A rats at 2 and 15 min was significantly less (P < 0.05) than that seen with the neutrophils from the R, +A or N rats (Fig. 5). At 30 min, only the A and R neutrophils had significantly different numbers of organisms associated. No significant differences were observed among the neutrophils from the A, +A and N rats.
Fig. 5.
Comparison of adhesion of P. aeruginosa to neutrophils from A (vitamin A deficient), +A (weight-matched pair-fed), and R16 (vitamin A deficient rats refed vitamin A for 16 d) rats and N (nonrestricted, vitamin A complete diet) rats. The experiment was carried out in the same manner as given in Figure 2 with the exception that the reaction was carried out at 4°C. n = 8 for A, n = 4 for +A, R and N. Values are the average of the mean fluorescence channel and the error bars represent the SD. An overall significant difference in adhesion of P. aeruginosa was determined by ANOVA among the neutrophils from A, +A, R16 and N rats at 2, 15 and 30 min. P < 0.05 for a vs. b at each time point based on Tukey's Comparison Method (Tukey 1949).
[View Larger Version of this Image (22K GIF file)]
A rats relative to neutrophils from +A, R and N rats. Chemotaxis of the neutrophils from the A rats towards activated rat serum was not altered by the deficiency. In vivo, the differences in neutrophil chemotaxis towards bacterial products did not affect the total number of neutrophils that responded within 24 h to a corneal P. aeruginosa infection at high numbers of organisms topically applied to scratched corneas (107-108) (Twining et al. 1996b). This may be due to the fact that, in vivo, multiple chemotactic factors are generated by the host in response to the infective organism.
A rats was lower than that of the neutrophils from the R, +A and N rats. This is probably related to a lower initial rate of adhesion of the organisms to the A neutrophils. The initial number of cells which have undergone the respiratory burst (indicated by a large increase in red fluorescence) was less in the neutrophil population of A rats. This may also reflect differences among the cells in the rate of adhesion of the organisms or it may also be related to a lower rate of the respiratory burst. The differences in kinetics in the generation of oxidized hydroethidine and DCFH-DA may reflect the partitioning of superoxide for the oxidation of hydroethidine and the generation of hydrogen peroxidase. Hydrogen peroxide is needed as a substrate for myeloperoxidase and subsequent oxidation of DCFH-DA. A difference in the amount of DCFH-DA oxidized, but not in the amount of hydroethidine oxidized, was noted by Rothe and Valet (1990) when the two fluorescent molecules were present during phagocytosis of bacteria.
) or to very low retinol concentrations in blood. The bone marrow of the vitamin A deficient rats sequesters retinol with fourfold higher levels than in control rats (Twining et al. 1996a). The low retinol levels in the blood could play a role in the adhesion, phagocytosis and killing experiments reported here because they were carried out using whole blood from the respective rats.
A, +A, R and N rats, only the A rat corneas became infected within 24 h (Twining et al. 1996b). These differences in neutrophil functions probably contribute to the lessened ability of vitamin A deficient animals to fight infections of various types.
). The severity of infections is affected more than the incidence of infections by the vitamin A status. Because neutrophils are the first cell that responds to an infection, they play a role in limiting the scope of the infection by reducing the number of organisms present (Ferrante et al. 1993, Ratcliffe et al. 1988, Twining et al. 1996b). The reduced ability of the neutrophils from the vitamin A deficient rats to chemotax, phagocytose and kill P. aeruginosa probably is related to the increased severity of infections in vitamin A deficient animals.
, Pastatiempo et al. 1990, and 1994, Rothe and Valet 1990), lower numbers of natural killer cells (Zhao et al. 1994), abnormal T-cell proliferation (Cantorna et al. 1994, Friedman and Sklan 1989), and increased interferon
secretion by T-cells (Carman and Hayes 1991). Thus, changes in neutrophil, T and B cell and natural killer cell function probably contribute to the decreased ability of vitamin A deficient animals to fight infections.
FOOTNOTES
A, vitamin A deficient rats; +A, weight-matched pair-fed control rats; DCFH-DA, 27-dichlorofluorescein diacetate; FITC, fluorescein isothiocyanate; f-Met-Leu-Phe, formylated methinyl-leucinyl-phenylalanine; LFL, log fluorescence; N, nonrestricted vitamin A-complete diet rats; PMN, polymorphonuclear neutrophil; R, vitamin A-deficient rats subsequently receiving vitamin A for 2, 4, 8 or 16 d (R2, R4, R8, R16).
-tocopherol, 160; vitamin B-12, 45.
Manuscript received 29 August 1996. Initial reviews completed 28 October 1996. Revision accepted 20 December 1996.
LITERATURE CITED
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