AN ABSTRACT OF THE THESIS OF Xu Wang for the degree of Doctor of Philosophy in Nutrition and Food Management presented on May 3. 1995. Title: Effect of Controlled Vitamin B-6 Intake on in vitro Lymphocyte Proliferation and Interleukin 2 Production in Young Women ■__ Abstract approved: ; Lorraine T. Miller In two studies we tested the effect of vitamin B-6 (B-6) intake on in vitro lymphocyte, proliferation and IL-2 production in healthy young women. In Study I, 6 women were fed a constant diet containing 0.84 mg (4.96 jimols) of B-6 for 12 d, and 1.24 mg (7.33 jimols) and 2.44 mg (14.42 nmols) of B-6 during two subsequent 10-d periods. Lymphocyte proliferation in response to the mitogens concanavalin A (Con A) and pokeweed mitogen (PWM) was significantly higher (p < 0.05) at 2.44 mg than at 0.84 mg intake and the prestudy value. In Study II, 10 women who consumed their self- selected diets were randomly divided into a PN and a placebo group of five each. Following a 5-d baseline period, the PN group received a daily supplement of 1.5 mg (7.29 jimols) and 50 mg (243 nmols) pyridoxine.HC1 (PN.HC1) for 7 and 6 d, respectively. This was followed by a 28-d washout period during which no supplement or placebo was administered. After daily 1.5 mg supplementary PN.HC1 for seven days, lymphocyte proliferation in response to phytohemagglutinin (PHA), Con A and PWM, and IL-2 production were significantly higher than the baseline (p < 0.05) and that of the placebo group (p < 0.05). The 50 mg PN.HC1 supplement increased IL-2 production, but not lymphocyte proliferation as compared with the 1.5 mg PN.HC1 supplement period. Lymphocyte proliferation and IL-2 production were significantly correlated with lymphocyte pyridoxal 5'phosphate concentrations (p < 0.01). We conclude that a B-6 intake of 1.5 to 2 times the RDA improves lymphocyte proliferation and IL-2 production in healthy young women and this effect of B-6 on immunocompetence is transitory. To explore the basis for the effect of B-6 on immune function, putrescine was added to the culture medium (2 - 200 pimol/L) . In vitro lymphocyte proliferation and IL-2 production were not changed by the addition of putrescine under our experimental conditions. Effect of Controlled Vitamin B-6 Intake on in vitro Lymphocyte Proliferation and Interleukin 2 Production in Young Women by Xu Wang A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Completed May 3, 1995 Commencement June, 1995 Doctor of Philosophy thesis of Xu Wang presented on May 3. 1995 APPROVED: Major Professor, representing Nutrition and Food Management Head of Department of Nutrition and Food Management Dean of Gradiiate School I understand that my thesis will become part of the permanent collection of Oregon State University library. My signature below authorizes release of my thesis to any reader upon request. Xu Wang, Author TABLE OF CONTENTS Page INTRODUCTION 1 REVIEW OF LITERATURE 3 A. Vitamin B-6 Metabolism 3 B. The Safety of PN.HCl Supplementation 9 C. Recommended Dietary Allowance for Vitamin B-6 10 D. Assessment of Vitamin B-6 Status 11 E. Vitamin B-6 and Immunocompetence 15 1. Animal Studies 2. Human Studies F. Proposed Roles of Vitamin B-6 in Immune Function 1. 2. 3. 4. One-carbon Unit Metabolism Modulation of Hormone Receptors Polyamine Synthesis Vitamin B-6 and Polyamines MATERIALS AND METHODS 15 18 23 24 25 27 30 32 A. Subjects 32 B. Experimental Designs 33 C. Blood Drawing 38 D. Isolation of Mononuclear Cells 38 E. Cell Viability Tests and Counts 39 F. Lymphocyte Proliferation 39 G. IL-2 Production (Study II) 40 H. Effect of Putrescine on Lymphocyte Proliferation and IL-2 Production (Study II) 41 I. PLP Determination 42 TABLE OF CONTENTS (Continued) Page J. Erythrocyte Aspartate Aminotransferase Activity Coefficient (EAST AC) (Study II) 43 K. Collection of Dietary Information (Study II) 43 L. Statistics 44 RESULTS A. Study I 1. Plasma PLP Concentration 2. Lymphocyte Proliferation B. Study II 1. Subjects' Dietary Vitamin B-6 Intake and Nutritional Status 2. Plasma and Lymphocyte PLP Concentrations 3. Erythrocyte Aspartate Aminotransferase Activity Coefficient (EAST AC) 4. Lymphocyte Proliferation 5. IL-2 Production 6. Effect of Putrescine on Lymphocyte Proliferation and IL-2 Production 45 45 45 45 49 49 52 57 58 66 69 DISCUSSION 75 SUMMARY AND CONCLUSIONS 92 BIBLIOGRAPHY 96 LIST OF FIGURES Figure 1. Formulae of B-6 vitamers, metabolites and PN-glucoside Page 4 2. The experimental protocols 37 3. Vitamin B-6 intake and the changes of lymphocyte proliferation in response to Con A and PWM stimulation (Study I) 47 4. Changes of plasma and lymphocyte PLP concentrations in the PN and placebo groups (Study II) 56 5. Lymphocyte proliferation in response to PHA (A), Con A (B) and PWM (C) stimulation in the PN and placebo groups (Study II) 62 6. Correlation between lymphocyte proliferation, IL-2 production and lymphocyte PLP concentrations (Study II) 64 7. IL-2 production in response to PHA stimulation in the PN and placebo groups (Study II) 68 8. Changes of lymphocyte proliferation and IL-2 production in response to putrescine concentrations in culture medium (Study II) 74 LIST Of TABLES Table Page 1. Descriptive data on subjects in Study I 34 2. Descriptive data on subjects in Study II 36 3. Vitamin B-6 intake and plasma PLP concentration in six healthy young women consuming a constant diet with controlled vitamin B-6 intake (Study I) 46 4. Vitamin B-6 intake and mitogen response, in cpm, of lymphocytes in six healthy young women consuming a constant diet with controlled vitamin B-6 intake (Study I) 48 5. Average daily dietary intakes of energy, vitamin B-6 and protein by the PN and placebo groups during baseline, supplement and washout periods (Study II) 50 6. Average weekly hemoglobin (Hb) concentration and hematocrit (Hct) value of the PN and placebo groups (Study II) 51 7. Plasma PLP concentrations of the PN and placebo groups (Study II) 54 8. Lymphocyte PLP concentrations of the PN and placebo groups (Study II) 55 9. Erythrocyte aspartate aminotransferase activity coefficient (EAST AC) of the PN and placebo groups (Study II) 57 10. PN.HCl supplementation and mitogen response, in cpm, of lymphocytes to PHA in the PN and placebo groups (Study II) 59 11. PN.HCl supplementation and mitogen response, in cpm, of lymphocytes to Con A in the PN and placebo groups (Study II) 60 LIST OF TABLES (Continued) Table Page 12. PN.HCl supplementation and mitogen response, in cpm, of lymphocytes to PWM in the PN and placebo groups (Study II) 61 13. PN.HCl supplementation and IL-2 production (U/L) of lymphocytes in the PN and placebo groups (Study II) 67 14. Effect of PN.HCl supplementation and putrescine concentrations in the culture medium on mitogen response, in cpm, of lymphocytes to PHA in the PN and placebo groups (Study II) 70 15. Effect of PN.HCl supplementation and putrescine concentrations in the culture medium on mitogen response, in cpm, of lymphocytes to Con A in the PN and placebo groups (Study II) 71 16. Effect of PN.HCl supplementation and putrescine concentrations in the culture medium on mitogen response, in cpm, of lymphocytes to PWM in the PN and placebo groups (Study II) 72 17. Effect of PN.HCl supplementation and putrescine concentrations in the culture medium on IL-2 production (U/L) of lymphocytes in the PN and placebo groups (Study II) 73 EFFECT OF CONTROLLED VITAMIN B-6 INTAKE ON IN VITRO LYMPHOCYTE PROLIFERATION AND INTERLEUKIN 2 PRODUCTION IN YOUNG WOMEN INTRODUCTION Vitamin B-6 is necessary for the development and maintenance of normal immune function (1). Although the effect of vitamin B-6 deficiency on immune function has been examined widely in animals (2-5), relatively few studies have been conducted in humans. In a vitamin B-6 depletion- repletion study in elderly people, vitamin B-6 deficiency significantly decreased the percentage and total number of lymphocytes, in vitro proliferation responses of peripheral blood lymphocytes to T- and B-cell mitogens and interleukin 2 (IL-2) production (6). These decreased parameters for immune function were not restored completely by a vitamin B6 intake of 2.88 mg/d by men and 1.90 mg/d by women, which is slightly higher than the current recommended dietary allowance (RDA) for vitamin B-6. In independently living elderly having an adequate vitamin B-6 intake, a pharmacological level of vitamin B-6 supplementation (50 mg pyridoxine hydrochloride per day) significantly increased immunocompetence as evaluated by lymphocyte subpopulation analysis and in vitro lymphocyte proliferation in response to mitogen stimulation (7). Also in independently living elderly having an adequate vitamin B-6 intake, a supplement of 3 mg vitamin B-6 per day, together with other micronutrients, for one year significantly increased IL-2 production, IL-2 receptor expression (8), delayed hypersensitivity skin test (DHST) (9) and the resistance to infectious diseases (8) of the subjects. On the other hand, there has been reported that short-term marginal vitamin B-6 deficiency did not affect immune response in health young men (10). There have been no data to evaluate the influence of vitamin B-6 intake on immune function in young women. The research presented in this thesis was designed to test the effect of different vitamin B-6 intakes on in vitro lymphocyte proliferation and IL-2 production of healthy young women in both controlled and self-selected dietary conditions. We determined the relationship between vitamin B-6 intake, plasma and lymphocyte pyridoxal 5'phosphate concentrations and in vitro peripheral blood lymphocyte proliferation in response to mitogen stimulation and IL-2 production. The duration of the effect of vitamin B-6 supplement on lymphocyte proliferation and IL-2 production was also determined. An attempt was made to explore a possible mechanism of the effect of vitamin B-6 on immune function. REVIEW OF LITERATURE A. Vitamin B-6 Metabolism Vitamin B-6 was first defined and delineated as a distinct entity in the vitamin B-2 complex by Gyorgy sixty years ago (11). Eight years later, Snell and his colleagues (12) provided evidence that vitamin B-6 is a group of related compounds. Vitamin B-6 is recommended as the generic descriptor for all 3-hydroxy-2-methylpyridine derivatives exhibiting qualitatively the biological activity of pyridoxine in rats (13). The six forms of vitamin B-6 include the free forms pyridoxine (PN), pyridoxal (PL) and pyridoxamine (PM); and their phosphorylated forms, pyridoxine 5'-phosphate (PNP), pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP) (Fig. 1). The predominate urinary metabolite of vitamin B-6 is 4-pyridoxic acid (4-PA) (Fig. 1), which has no vitamin B-6 activity. Vitamin B-6 occurs in a wide variety of foods. In general, PN and PNP are the predominant forms of vitamin B-6 in plant-derived foods, while PL and PM and their phosphorylated forms are found chiefly in animal-derived foods (14). In addition, glycosylated pyridoxine (pyridoxine 5'-fi-D-glucoside) (Fig. 1) has been identified and comprises from 5% to more than 80% of the total vitamin B-6 in rice bran, wheat, orange juice, and other plant CH, -R2 HoC Rl (PN) Pyridoxine (PM) Pyridoxamine Pyridoxal (PL) (PNP) Pyridoxine 5'-phosphate Pyridoxamine 5•-phosphate (PMP) (PLP) Pyridoxal 5'-phosphate (4-PA) 4-pyridoxic acid Pyridoxine 5'-B-D-glucosidee (PN-glucoside) -CH2OH -CH2NH3+ -CHO -CHjOH -CH2NH3+ -CHO -COO" -CH2OH R2 -OH -OH -OH -OP03= -OP03= -OPO3-OH -B-D-glucose Fig. 1. Formulae of B-6 vitamers, metabolites and PN-glucoside. foods (15-17). Glycosylated vitamin B-6 is less bioavailable than the free and phosphorylated forms of vitamin B-6. Studies in human subjects using an orally administered, purified deuterium-labeled PN-glucoside indicated that the bioavailability of this form of vitamin B-6 is 50-70% relative to PN (18). In the intestinal lumen, PLP and PMP are dephosphorylated by intestinal phosphatases. The three free forms are absorbed by the jejunum through a nonsaturable, passive process (19). The B-6 vitamers cross the basolateral membrane to enter circulation, mainly in the nonphosphorylated forms (19,20). After transport to the liver, the free forms of vitamin B-6 enter hepatocytes by diffusion followed by metabolic trapping (21,22). Liver is the primary organ responsible for vitamin B-6 metabolism. In human hepatocytes, PL, PN and PM are phosphorylated by a single kinase to their respective phosphorylated forms. The activity of pyridoxal kinase (EC 2.7.1.35) appears to be zinc- and ATP-dependent (23). Since the activity of pyridoxal kinase predominates over hepatic phosphatase, phosphorylated forms of vitamin B-6 are the major forms of the vitamin B-6 in liver. PNP and PMP are oxidized to PLP by the FMN-dependent oxidase, pyridoxine (pyridoxamine) 5'-phosphate oxidase (EC 1.4.3.5) (24). PLP is bound to apoenzymes and other proteins or released into plasma attached to albumin. When PLP is synthesized in excess of the binding capacity of hepatic proteins, the phosphate ester binding PLP is hydrolyzed by alkaline phosphatases (25). Finally, excess PL is oxidized by aldehyde oxidase (EC 1.2.3.1) to 4-PA, the dead-end catabolite and the major urinary vitamin B-6 excretory product (23). Plasma PLP bound to albumin is the major transport form of vitamin B-6. Protein binding protects PLP from dephosphorylation by phosphatases. cross cell membranes. PLP does not readily PL, formed when PLP is dephosphorylated by a membrane phosphatase, is the primary form crossing cell membranes. Within the cells PL can be rephosphorylated to PLP by the action of pyridoxal kinase, which exists in most tissues (26) . In contrast, the distribution of PNP (PMP) oxidase has been identified only in the liver (23) and erythrocytes in humans (27). The absence of PNP (PMP) oxidase in skeletal muscle is important because this tissue represents the major storage site of vitamin B-6 in the body. with In an isolated hind-limb perfused 3 H-PN, there was a large accumulation of labeled PNP, but virtually no PLP (28) . This means that the liver, via plasma PLP, provides PL which other tissues take up from circulation and convert to PLP for their own usage. The erythrocytes also have an important role in metabolizing and transporting vitamin B-6. are rapidly taken up by red cells (29). Both PL and PN Since both pyridoxal kinase and PNP (PMP) oxidase exist in human erythrocytes (27,30), PL and PN are readily phosphorylated and PNP is converted to PLP. PLP, the biologically active form of vitamin B-6, is utilized as a coenzyme by over 100 enzymes (31). For example, PLP (or PMP) plays an essential role as a coenzyme for aminotransferases in the interconversion of amino acids and their respective a-ketoacids (e.g., aspartateoxaloacetate aminotransferase, EC 2.6.1.1; alanine-pyruvate aminotransferase, EC 2.6.1.2; etc.). Transamination is central to the biosynthesis and catabolism of essential and non-essential amino acids. In a number of cases, transamination provides a simple link between amino acids and intermediates of the glycolysis pathway and the tricarboxylic acid cycle. Reactions of PLP-dependent decarboxylases, another example, include the synthesis of various amines such as polyamines, serotonin, tyramine and x-amino butyric acid by the decarboxylation of precursor amino acids. Withers et al. (32) reported a structural role of PLP in glycogen phosphorylase (EC 2.4.1.1) by demonstrating conformational changes in the enzyme due to ionic interactions of the phosphate group of PLP with the active site in the enzyme. Glycogen phosphorylase is the major binding protein for PLP in muscle (33) and this enzyme accounts for as much as 5% of the soluble protein in skeletal muscle, which is approximately 40% of the lean body mass in humans (34). Krebs and Fisher (35) proposed that muscle glycogen phosphorylase is a storage site for vitamin B-6 in the body. Black et al. (33) reported that high intakes of vitamin B-6 increased muscle glycogen phosphorylase activity, but it was the starvation rather than a vitamin B-6 deficient diet that decreased the activity of muscle glycogen phosphorylase in rats (36). A deficiency of vitamin B-6 in rats resulted in decreased activity of liver glycogen phosphorylase (37). Plasma PLP concentration increases during and immediately following a strenuous exercise (38). In a recent study, Crozier et al. (39) reported that this rapid increase in plasma PLP and 4-PA concentrations after the onset of exercise was not affected by exercise intensity. They proposed that the elevated plasma PLP concentration immediately after exercise came from the redistribution of PLP from extravascular spaces to the plasma pool along with albumin and other proteins shifted from interstitial space into blood during exercise. This increased plasma PLP was oxidized to 4-PA and was destined for urinary excretion rather than for utilization by liver to facilitate gluconeogenesis during exercise. B. The Safety of PN Supplementation The acute toxicity of pyridoxine is very low. A single dose in the range of 3 to 4 g per kg body weight produces convulsions and death in animals (40). In mice and rats, the LD5Q of an oral dose is 4 - 6 g per kg body weight (41). Such extremely large doses of vitamin B-6 intake does not occur naturally at any place but toxicological laboratories. Chronic toxicity of pyridoxine is also low. Schaumburg et al. (42) reported that in humans who had used PN for self-medication, toxic effects occurred after 2 to 6 g of pyridoxine intake daily for 2-40 months. Clinical manifestations of pyridoxine toxicity included peripheral sensory neuropathy with atactic gait disorders, absence of limb reflexes and impairment of the sensation of touch, vibration, temperature, pinprick and the joint position. Anatomically, the peripheral sensitive nerves undergo nonspecific axonal degeneration of large and small myelinated fibers (42). Considerable and even complete improvement occurred within 6 months of withdrawing pyridoxine. Rudman et al. (43) speculated that the toxic side effects of pyridoxine were due to the basic pyridine structure of vitamin B-6 (Fig. 1). Rudman et al. also suggested that high pyridoxine concentrations may competitively inhibit the binding of PLP to apoenzymes, leading to a deficiency of active enzymes in the peripheral 10 nervous system. Possibly, toxic impurities in vitamin B-6 preparations may be significant in high-dose intakes. Human data on the safety of pyridoxine have shown that doses of PN less than 500 mg per day appear to produce no detectable adverse effects for periods ranging from 6 months to 6 years (44-46). C. Recommended Dietary Allowance for Vitamin B-6 The crucial role of vitamin B-6 in metabolism clearly points to the importance of an adequate intake of this nutrient. The 1989 edition of RDA sets the recommended allowance of vitamin B-6 as 2 mg for adult men and 1.6 mg for adult women per day (47). Since the requirement for vitamin B-6 is related to protein intake, this recommendation is based on the ratio of 0.016 mg of vitamin B-6 to one gram of protein intake. It was calculated from the reported average daily protein intakes of approximately 100 g by adult females and 125 g by adult males. The present RDA for vitamin B-6 is lower than the one in 19 86, which set the recommendation for vitamin B-6 as 2.2 mg for men and 2 mg for women per day (47). These numbers were based on the ratio of 0.02 mg of vitamin B-6 to one g of protein intake. Justification for reducing the RDA for vitamin B-6 in 1989 included human studies and survey data of protein intake. 11 Studies showed acceptable biochemical measurements of vitamin B-6 status in subjects who were receiving less than the 1986 RDA for vitamin B-6. Survey data also indicated that the average daily protein intakes of women and men were 60 g and 100 g, respectively. The use of dietary intake data assumes that the survey sample represents all populations of different age and socioeconomic groups, the nutrient databases are accurate and the populations studied are healthy. Very few studies have aimed to assess human nutrient requirements in terms of maximizing bodily functions. Based on the measurements of immunocompetence in the elderly receiving controlled intakes of vitamin B-6, Meydani et al. (6) suggested that the elderly need a higher intake of vitamin B-6 than the present RDA to maintain normal immune function. D. Assessment of Vitamin B-6 Status The indices for assessing vitamin B-6 nutriture can be divided into three categories: direct and indirect biochemical measures of vitamin B-6 status and dietary vitamin B-6 intake. Direct measurement of the concentrations of vitamin B-6 and its metabolites in blood and urine is widely used to assess vitamin B-6 status. Since PLP is the active form of 12 vitamin B-6 and the level of PLP in plasma reflects tissue levels of vitamin B-6 (48), plasma PLP concentration is considered a good indicator of vitamin B-6 status (49). Plasma PLP levels respond to changes in vitamin B-6 intake (50,51). Leklem (52) recommended that the normal value for plasma PLP concentration is > 30 nmol/L. Protein (53,54) and riboflavin (55,56) intake influence plasma PLP levels. Other factors affecting plasma PLP concentration include plasma volume and physical activity (37), smoking (57), age (58), and use of oral contraceptive agents (59). Plasma PL is also an important indicator of vitamin B-6 status. Fifteen minutes after an orally administrated physiological dose of 3 H-labeled pyridoxine (60) or pyridoxamine (61) to mice, more than 50% of the B-6 vitamers in plasma was PL. Barnard et al. 3 H-labeled (62) reported that in pregnant women the plasma PLP concentration was 50% lower than in nonpregnant controls, but plasma PLP and PL together only showed a slightly lower value in pregnant than in nonpregnant women. Another direct measure of vitamin B-6 status is the urinary excretion of 4-PA, the dead-end catabolite and the major urinary excretory product of vitamin B-6 (54). Urinary excretion of 4-PA normally accounts for about 40 - 60% of the dietary intake of vitamin B-6 (50,54). Given individual doses of 100 mg PL, PM or PN, human subjects excreted 90% of PL and PM, and 70% of PN in urine 13 as 4-PA within 3 6 hours (63). A short-term indicator of vitamin B-6 status, urinary 4-PA excretion parallels plasma PLP concentrations in vitamin B-6 depletion-repletion studies (64,65). The liver is thought to be the major site of 4-PA formation (23,66). Indirect measurements of vitamin B-6 status include activity of erythrocyte aminotransferases, tryptophan load test and methionine load test. A commonly used measure of vitamin B-6 status is the activity of erythrocyte alanine aminotransferase (EALT) and aspartate aminotransferase (EAST). For assessing vitamin B-6 status, the basal activity and the activity of these enzymes stimulated with exogenous PLP added in vitro are measured. When vitamin B-6 status is inadequate, less endogenous PLP is available to bind to the apoenzyme, resulting in lower basal activity and higher PLP-stimulated activity. The ratio of stimulated to basal aminotransferase activity, known as the activity coefficient (AC), indicates vitamin B-6 status (67). Leklem (52) suggests that an activity coefficient of < 1.25 and < 1.80 for EALT and EAST, respectively, reflects adequate vitamin B-6 intake. Vitamin B-6 deficiency results in higher activity coefficients for both aminotransferases. Since erythrocyte has a long life span (120 days), erythrocyte aminotransferase activity is a long term index of vitamin B-6 status. Tryptophan metabolism requires PLP for the conversion 14 of 3-hydroxykynurenine to 3-hydroxyanthranilic acid (68). In vitamin B-6 deficiency, 3-hydroxykynurenine accumulates and is converted to xanthurenic acid, which is another PLP-dependent step. Measurement of urinary tryptophan metabolites, particularly xanthurenic acid, following an oral load (2 - 5 g) of L-tryptophan was at one time used to assess vitamin B-6 status in humans (69). Activity of enzymes in the tryptophan-niacin pathway is also affected by dietary protein intake (53), specific hormones and use of oral contraceptive agents (70). These factors complicate the interpretation of tryptophan load test as an index of vitamin B-6 status. In addition, in 19 89 Food and Drug Administration (FDA) banned L-tryptophan from the market following the determination that a contaminated lot of this product had contributed to public health problems (71). The methionine load test is also an indirect measure of vitamin B-6 status (72). In the metabolism of methionine, conversion of cystathionine to a-ketobutyrate and cysteine is PLP-dependent (73). Following a 3-g oral load of L-methionine, increased urinary excretion of cystathionine indicates inadequate vitamin B-6 status. Like tryptophan load test, the level of protein influences methionine metabolism (72). Dietary vitamin B-6 intake alone (i.e., without using biochemical test of vitamin B-6 status) is not sufficient to assess vitamin B-6 status in humans. The inherent problems 15 in collecting dietary intake data include difficulties of obtaining accurate food intakes (74) and incomplete nutrient databases used to determine vitamin B-6 content of foods (75). Additionally, nutrient databases do not include information on the glycosylated form of this vitamin in plant-derived foods which may affect bioavailability of vitamin B-6 (76). However, collecting dietary intake data is relatively easy, cheap and quick (77). The expected improvement of nutrient databases and data collecting methodology make dietary intake data still valuable in estimating intake and identifying vulnerable populations who may be susceptible to vitamin B-6 deficiency, especially along with other biochemical or functional measurements of vitamin B-6 status. Several studies have shown that the majority of those who had evidence of biochemical vitamin B6 deficiency also had low intakes of this vitamin (78-80). E. Vitamin B-6 and Immunocompetence l. Animal Studies Since there are several recent reviews on the effect of vitamin B-6 deficiency on immune function in animals (81-83), this subject will be reviewed only briefly here. Vitamin B-6 plays a critical role in the development and maintenance of normal immune function in experimental animals. As early as in 1944, Stoerk and Zucher (84) showed 16 that vitamin B-6 deficiency produced a severe atrophy of the thymus in rats. Thymuses from vitamin B-6 deficient rats were virtually depleted of lymphocytes and consisted almost exclusively of epithelial cells and stroma. Stoerk and Zucher also observed atrophied lymph nodes and impaired antibody responses in these vitamin B-6 deficient rats. Splenic hypoplasia was reported in fetuses of pregnant rats fed a vitamin B-6 deficient diet or given the vitamin B-6 antagonist, deoxypyridoxine (85). Pups of vitamin B-6 deficient dams had diminished numbers of circulating blood lymphocytes, fewer splenic plaque-forming cells, and diminished vitamin B-6 content of splenic and thymic tissues (86) . Vitamin B-6 deficiency produces profound effects on cell-mediated immunity. Cell-mediated immune function, measured by delayed dermal hypersensitivity, allograft response and mixed lymphocyte cultures, was decreased in the vitamin B-6 deficient rats (87), mice (88), and guinea pigs (89). Thoracic duct lymphocytes from vitamin B-6 deficient female rats and their three-month old offspring showed decreased ability to respond in mixed lymphocyte cultures and to incorporate tritiated uridine (90). Axelrod et al. (91) reported that vitamin B-6 deficient guinea pigs inoculated with Mycobacterium tuberculosis had depressed delayed type hypersensitivity skin response to this antigen. Axelrod et al. (92) also reported that in rats immune 17 responses to antigens were inhibited and viability of skin homografts was increased by vitamin B-6 deficiency. al. Ha et (93) found that in mice challenged with Moloney sarcoma virus, tumor incidence was higher and regression time was longer in animals fed diets containing 0 or 0.1 mg of PN/kg than in those fed 0.5 - 1.0 mg of PN/kg diet. Gridley et al. (94) observed that the proliferation response of peripheral blood and splenic lymphocytes to T-cell mitogens was higher in mice fed diets containing 7.7 and 74.3 mg of PN/kg than in those fed 0.2 and 1.2 mg of PN/kg diets. Ha et al. (95) investigated different levels of vitamin B-6 on cell-mediated immunity in mice. In female C57BL/6 mice, they observed that primary and secondary splenic and peritoneal T-cell mediated cytotoxicity was significantly reduced in animals receiving 10 and 0% of their required vitamin B-6 compared with animals receiving 700 and 100% of their vitamin B-6 requirement. Increasing the vitamin B-6 intake to as much as seven times the requirement did not further improve immunocompetence. These results suggest that high doses of vitamin B-6 may not produce benefits beyond those observed with moderate supplementation of the vitamin in mice. Vitamin B-6 deficiency impairs humoral immunity. Stoerk and coworkers (96) observed that the formation of circulating antibodies to sheep red blood cells was suppressed in rats fed a vitamin B-6 deficient diet. 18 Subsequent studies confirmed this original work and demonstrated further that vitamin B-6 deficiency significantly inhibited antibody production in response to influenza virus (97), B. typhosus (98), Salmonella pullorum (99), or a synthetic antigen poly Glu-Lys-Tyr (100). Additionally, antibodies produced by vitamin B-6 deficient animals have decreased in vitro binding affinity for antigens (5). 2. Human Studies In contrast to the numerous animal studies, studies exploring the effect of vitamin B-6 status on immune function in human beings are few. An early study by Hodges et al. (101) investigated the effect of vitamin B-6 deficiency on antibody production in response to immunization with tetanus and typhoid antigens in six healthy young men. The subjects were made vitamin B-6 deficient by feeding them a vitamin B-6 deficient diet with and without deoxypyridoxine, a vitamin B-6 antagonist. Compared with the controls who took 2 mg PN daily, the vitamin B-6 deficient subjects had decreased antibody responses to tetanus and typhoid antigens. Hodges and coworkers also observed that the combined deficiencies of vitamin B-6 and pantothenic acid resulted in a further decline of antibody titer in response to tetanus and typhoid antigen immunization (102). 19 Van den Berg et al. (10) investigated the effect of marginal vitamin B-6 status on immunological indices in 24 young males. During the first two weeks, all subjects received the basal diet, which provided 0.68 mg of vitamin B-6, plus a multinutrient supplement containing 4 mg of vitamin B-6. During the following 11 week period, 12 subjects were maintained on this regimen, while 12 other subjects were given the same diet without vitamin B-6 supplementation. At the end of the 11 weeks, the subjects receiving no vitamin B-6 supplement were marginally depleted as evaluated by decreased plasma PLP concentrations and erythrocyte aminotransferase activities. The percentage of T-helper cells and the Ig D concentration were slightly lower in the vitamin B-6 deficient group than in the vitamin B-6 supplemented group. Other indices (total lymphocytes, total T-cells, total B-cells, IL-2 receptors, other immunoglobulins and complement concentrations) were not significantly different between the two groups. The authors concluded that short-term marginal vitamin B-6 deficiency did not affect immune function in young men. However, the subjects in this study received twice the RDA for all other vitamins and minerals. Many of those vitamins and minerals, such as vitamins A (103), C (104) and E (105), and minerals Se (106) and Zn (107) may improve immune function of the subjects. Additionally, what the authors measured is actually a static status of the immune cells rather than the 20 dynamic response of immune system to a stimulation. It is possible that the marginal vitamin B-6 nutriture could maintain apparently normal cell number without challenge, but the immune system may not have the optimal competence upon a stimulation. Talbott et al. (7) examined the effect of PN supplementation on lymphocyte function in an independently living elderly population. The mean dietary vitamin B-6 intake in this study group was 1.51 mg/d or 0.022 mg vitamin B-6/g protein, close to the current RDA. Eleven subjects received 50 mg PN.HC1 per day for 2 months and 4 subjects received a placebo. The results showed clearly that PN supplementation significantly increased lymphocyte proliferation response to both T- and B-cell mitogens in the elderly. The percentage of CD3+ and CD4+ cells increased significantly in subjects receiving the PN supplement. Moreover, when the PN-treated subjects were divided into two groups according to their pre-study plasma PLP concentrations, the effect of PN supplementation on lymphocyte proliferation was much greater in the subjects who had a low pre-study plasma PLP concentration ( z 30 nmol/L) than in those with normal level ( > 30 nmol/L). More recently, Meydani et al. (6) reported that in elderly persons who had been depleted in vitamin B-6, lymphocyte proliferation in response to mitogen stimulation and IL-2 production increased with vitamin B-6 repletion. 21 The decline in IL-2 production and lymphocyte proliferation induced by vitamin B-6 depletion, however, was not completely restored to the baseline level by a vitamin B-6 intake of 2.88 mg/d by men and 1.9 0 mg/d by women. On the other hand, in response to 50 mg PN.HCl/day for 4 days, lymphocyte proliferation and IL-2 production increased above the baseline level. Meydani et al. suggested that for maintenance of normal immune function, older adults may require more vitamin B-6 than the current RDA. Vitamin B-6 status indicators (tryptophan load test, plasma PLP level, urinary 4-PA excretion, and erythrocyte aspartate aminotransferase activity coefficient) measured in the same subjects during vitamin B-6 depletion and repletion also supported this conclusion (108). It is not certain, however, whether this higher vitamin B-6 requirement for maintaining optimal immune function is specific to the elderly or is a general effect regardless of age. From the studies summarized above, we see that an intake of vitamin B-6 above the RDA improves in vitro immune responses in the elderly. Although less certain, the study by Van den Berg et al. suggested that young subjects may also have an increased immune response with the increased intake of vitamin B-6. However, in these studies the number of subjects was small and the duration of vitamin B-6 was relatively short and follow-up on the subjects' health was not done. 22 There are only two relatively large scale field studies of long duration on the effect of micronutrients including vitamin B-6 supplementation on immune function. In a double-blind controlled investigation reported by Chandra (8), 96 healthy Canadians over 65 years of age and in the upper middle to high socioeconomic status were divided into a supplemented group and a placebo group of 48 each. Daily for one year, the supplemented group received a multivitamin and mineral supplement containing 3 mg of vitamin B-6 and approximately 1-2 times of the RDA for other vitamins and essential trace elements. The percentage of the subjects with plasma PLP concentrations lower than 50 nmol/L in supplemented group was decreased from 16.7% at the beginning of the study to 4.4% after taking the supplement for one year. This supplement of physiological amounts of vitamins and minerals resulted in a significant improvement in immune indices, including percentage of CD4+ cells, natural killer (NK) cells, IL-2 production and IL-2 receptors, and in vitro lymphocyte proliferation in response to PHA stimulation. The supplemented group also had a significantly decreased incidence of infectious diseases compared with the placebo group. Although this study did not clarify the role of individual vitamins or minerals, it confirmed both the effect of physiological amounts of supplementing vitamins and minerals on laboratory tests of immune response and improvement in health. 23 Recently, Bogden et al. (9) administered daily for one year an over the counter micronutrient supplement to 29 independently living healthy elderly subjects, aged 59 - 85 years, whose vitamin B-6 intake was adequate (1.55 - 2 mg of vitamin B-6). This over the counter supplement included 3 mg of vitamin B-6 and other vitamins and minerals in the range of 20 - 450% of the RDA. Daily intake of this nutrient supplement for one year resulted in a significant increase of DHST in response to a panel of seven recall antigens. The authors concluded that a modest daily supplement of micronutrients enhanced cellular immunity, and suggested that the current RDAs of micronutrients including vitamin B-6 are too low to support optimal immunity in older individuals. Again, since the subjects had increased intake of other vitamins and minerals, the role of individual vitamin or mineral in immune response could not be clarified in this investigation. F. Proposed Roles of Vitamin B-6 in Immune Function Although the importance of vitamin B-6 in developing and maintaining normal immune function has been recognized for a long time (5,80,101), the mechanism of the effect of vitamin B-6 on immune system is still unclear. Discussed here are the three proposed mechanisms which include the role of vitamin B-6 in one-carbon unit metabolism, hormone 24 receptor modulation and polyamine synthesis. 1. One-carbon Unit Metabolism PLP is the coenzyme of serine hydroxymethyltransferase (EC 2.1.2.1), which catalyzes a step in one-carbon unit metabolism. In 1964, Axelrod and Trakatellis (5) proposed this as a role of vitamin B-6 in immune function. This enzyme catalyzes the transfer of a hydroxymethyl group from serine to tetrahydrofolate to form N5,N10methylenetetrahydrofolate (109). The methylene group from N5,N10-methylenetetrahydrofolate participates in the biosynthesis of thymidylic acid from deoxyuridine 5'phosphate and of purine bases from glycine amide ribotide. DNA and RNA synthesis precedes all cell proliferation. In response to PHA stimulation, mRNA synthesis in cultured human T-cells increased significantly within ten minutes (110). Trakatellis et al. reported that vitamin B-6 deficient rats had less DNA in splenic cells (111) and less ribosomal RNA in liver and splenic cells (112) than the corresponding control animals. Axelrod and Trakatellis (5) reported that vitamin B-6 deficiency resulted in a decreased incorporation of C rat tissue. from serine-3-C14 into DNA and RNA in Vitamin B-6 deficiency, therefore, may suppress immune function by compromising DNA and RNA synthesis when immune cells are activated. To the knowledge of this reviewer, there is no recent 25 research on this role of vitamin B-6 on one-carbon unit metabolism. Many authors (4,6,82,83,113) cite this as the role of vitamin B-6 in immune function. 2. Modulation of Hormone Receptors An active biomolecule, PLP interacts with many proteins forming a Schiff base with the e-amino group of the lysine residue of the protein. Nishigori et al. (114) were the first to report that PLP inhibits the binding of avian progesterone receptors to ATP-sepharose. After PLP treatment, this inhibition could be made irreversible by reduction with NaBH4 which fixes the formed Schiff base. Following binding with specific receptors, transporting the glucocorticoid-receptor complex from cytoplasm to the nucleus and binding the complex to DNA is considered to be the first step in glucocorticoid function in cells (115) . Cidlowski et al. (116) demonstrated that PLP inhibited the binding of the receptor-hormone complex to the nuclei of rat thymic lymphocytes, which contain specific, high-affinity glucocorticoid receptors. Their study also showed that only PLP effectively inhibited the binding of the receptorhormone complex to nuclei. Neither PL, PN, PMP nor 5- deoxypyridoxal (a nonphysiological analog of PL) fulfilled this role. In in vitro studies, PLP inhibited the binding of estrogen (117) and androgen (118) receptors to DNA and nuclei. Compared with control animals, vitamin B-6 26 deficient ones accumulated more radioisotope-labeled estradiol in uterine tissue (117), while vitamin B-6 sufficient animals exhibited a decreased nuclear localization of uterine estrogen receptors (119). Although the mechanism of the action is unclear, the immunosuppressive effect of glucocorticoid hormones has been recognized for a long time (120,121). Allgood and Cidlowski (122) proposed that the effect of vitamin B-6 on immune function may be mediated by compromising the immunosuppressive effect of glucocorticoid hormones. In human lymphocytes treated in vitro, glucocorticoids inhibited production of a variety of cytokines (123) and the expression of Fc receptors (12 0). Glucocorticoid treatment induced specific restriction endonuclease degradation of DNA in rat thymocytes (124). The morphologic characteristics of apoptosis have been observed widely in glucocorticoidinduced atrophy of lymph tissues (125,126). Recently, Reeve et al. (127) reported that vitamin B-6 intake equivalent to four times the RDA for mice protected these animals from suppression of contact hypersensitivity induced by ultraviolet B (UVB) radiation (280 - 320 nm) or cis-urocanic acid. Reeve et al. proposed that vitamin B-6 modulates the binding of cis-urocanic acid to H-2 histamine receptors and compromises the immunosuppressive effect induced by cis-urocanic acid or UVB radiation. Trans- urocanic acid, a metabolite of histidine, is a natural 27 epidermal constituent. UVB radiation photoisomerizes trans - urocanic acid to the cis-form, which suppresses immune response in mice (128). Cis-urocanic acid shares the imidazole structure of histamine. Both cis-urocanic acid and histamine activate T-suppressor cell function by binding to H-2 histamine receptors on surface of T-lymphocytes (129) . 3. Polyamine Synthesis Polyamines are aliphatic amines associated with nucleic acids and are synthesized in all cells. The most common ones are putrescine, spermidine and spermine. The first and rate limiting step of polyamine biosynthesis is the conversion of ornithine, an intermediate in the urea cycle, to putrescine by ornithine decarboxylase (ODC, EC 4.1.1.17), a PLP-dependent enzyme (130). Putrescine is subsequently converted to spermidine and spermine by the action of spermidine synthase (EC 2.5.1.16) and spermine synthase (EC 2.5.1.22), respectively (131). ODC, the key regulatory enzyme in this pathway, has a short half-life (11 - 90 minutes depending on source of tissue and experimental conditions) (132). Rapidly growing cells have higher ODC activities and higher concentrations of polyamines than non-proliferating cells (133). Growth stimulation results in a parallel rise in the levels of polyamines, DNA and RNA (134). Although 28 the exact role of polyamines in cell growth and development is not clear, polyamines are important in cellular macromolecule (e.g., DNA, RNA and protein) synthesis. Polyamines affect replication, transcription and translation possibly by influencing the conformation of templates and their products (135). At a physiological pH, polyamines function as polyvalent cations which interact with nucleic acids and nucleic acid-containing structures such as ribosomes (13 6). Polyamines are required for isolated nuclei from herpes simplex virus infected cells to synthesize viral DNA (137). Blocking polyamine synthesis by adding the ODC inhibitor, difluoromethylornithine (DFMO), resulted in loss of malarial DNA polymerase (EC 2.7.7.7) activity. The addition of putrescine to DFMO-treated cultures led to increased DNA polymerase activity (13 8). Physiological concentrations of polyamines stimulated transcription of both bacterial and eukaryotic RNA polymerases (EC 2.7.7.6) in vitro (136). Polyamines affect a number of steps of protein synthesis: low concentrations of spermidine stimulate aminoacyl tRNA synthetase activity (139); and polyamines facilitate the movement of the ribosomes along mRNA, enabling them to surmount obstacles (140). Atkins et al. (141) demonstrated that the addition of polyamines resulted in a qualitative difference in the polypeptides synthesized in a cell-free system. They showed that when the system was supplemented with polyamines, 29 polypeptides synthesized in vitro were larger and resembled the in vivo products more closely. In absence of polyamines, the cell-free polypeptide synthesis system produced a greater amount of incomplete translation products (142). Abraham et al. (142) and Jelenc and Kurland (143) suggested that polyamines contribute to the specificity of the codon anti-codon interaction and that the high fidelity of translation is partly due to the presence of polyamines. It is well documented that the conversion of many types of mammalian cells from resting to the rapidly growing and dividing state is accompanied by a marked increase in the ODC activity and subsequent polyamine content of cells (130,144-146). Upon antigenic or mitogenic stimulation, lymphocytes are quickly transformed into a rapidly proliferative state. Fillingame et al. methylglyoxal bis(guanylhydrazone) (147) reported that (MGBG), an anti-leukemic agent and a potent inhibitor of polyamine synthesis, inhibited DNA synthesis in bovine lymphocytes activated by Con A by 60% compared with the control. Addition of exogenous spermidine or spermine 24 hours after Con A stimulation produced complete reversal of inhibition of DNA synthesis by MGBG. Moreover, when MGBG was added more than 40 hours after Con A stimulation, the length of time lymphocytes need to accumulate polyamines after stimulation, no inhibition of DNA synthesis was observed. More recently, Singh et al. (148) tested the effect of DFMO and exogenous 30 putrescine on normal immune responses and malignant cell growth. Inhibition of ODC by DFMO suppressed lymphocyte proliferation in response to Con A and lipopolysaccharide. DFMO-mediated inhibition of cell proliferation correlated with depletion of intracellular polyamines. Repletion with putrescine reversed the inhibitory effects of DFMO. Scott et al. (149) showed that ODC activity and polyamine biosynthesis were rapidly increased by lectin or lymphokine stimulated human T-lymphocytes. Putrescine also reversed the growth inhibitory effects of DFMO on four tumor cell lines (148) . After activation, lymphocyte proliferation is regulated by the production of IL-2 by T-helper cells and the induction of specific membrane receptors for IL-2 (150,151). Responding to stimulation, T-helper cells also produce IL-3 (152). IL-3, which is species-specific, stimulates stem cell growth and fosters a dose-dependent rise in peripheral blood lymphocyte counts (153). Depletion of polyamines by DFMO reduces both IL-2 and IL-3 dependent proliferation of cell lines. The addition of exogenous putrescine reversed this decreased response to lymphokines (154). Intracellular polyamine biosynthesis is also required for IL-2 responsiveness during lymphocyte mitogenesis (155) . 4. Vitamin B-6 and Polyamines Since PLP is the coenzyme of ODC, the relationship 31 between vitamin B-6 status and polyamine biosynthesis has been explored. Pegg (156) reported that polyamine concentrations in the liver, kidney and brain of adult vitamin B-6 deficient rats were significantly lower than in the control group. ODC activity (measured without adding PLP in vitro) in hepatic extracts from the vitamin B-6deficient rats was significantly lower than that of the controls. After adding PLP to the hepatic extracts, ODC activity increased markedly and was higher than that of the controls. Pegg suggested that more ODC apoenzyme had been synthesized in the vitamin B-6 deficient rats and that the reduced tissue polyamine concentration was due to the decreased availability of PLP for ODC. Recently, Sampson et al. (157) reported that ODC apoprotein, measured as total ODC activity (with PLP added in vitro). was elevated 2.7-, 5.8-, and 8.5-fold, respectively, in the kidney, liver, and intestine, respectively, in vitamin B-6 deficient rats compared with control animals. This is consistent with the results reported by Pegg (156) . On the other hand, Sampson et al. found that ODC holoenzyme activity (measured without adding PLP in vitro) also increased in the kidney, liver and intestine of vitamin B-6-deficient rats. They did not measure tissue polyamine concentrations and gave no explanation for the increased ODC holoenzyme in vitamin B-6 deficient rats. 32 MATERIALS AND METHODS Two studies were conducted in young women. In Study I, the effect of vitamin B-6 intake ranging from 0.84 mg to 2.44 mg vitamin B-6 on lymphocyte proliferation in vitro was investigated. This study was designed primarily to determine the effect of varying physiological levels of vitamin B-6 intake on the metabolism of this nutrient in young women. Results on vitamin B-6 metabolism from this investigation will be reported elsewhere. Study II was designed to test the effect of physiological and pharmacological doses of supplementary PN and its removal on in vitro lymphocyte proliferation and IL-2 production in young women. Unless mentioned otherwise, all reagents used in this investigation were obtained from Sigma Chemical Company, St. Louis, MO. A. Subjects The subjects were recruited by advertisements placed on bulletin boards on Oregon State University (OSU) campus and in the city of Corvallis, Oregon. The volunteers completed a questionnaire to provide information on their health, menstrual cycle, eating habits, medical history, use of drugs including alcoholic beverages and nutritional 33 supplements, and physical activity. Written informed consent was obtained from each subject. These two investigations were approved by the OSU Committee for Protection of Human Subjects. In both studies, all subjects were women between the ages of 2 0 - 35 years, and, as determined by a questionnaire, were in good health; had no acute or chronic disease; were non-smokers; were not pregnant; were not taking an oral contraceptive agent (except one subject in Study II) or any other medications such as hydrazine derivatives, cycloserine, L-Dopa, and penicillamine which could affect the metabolism of vitamin B-6 (158); and were not taking vitamin supplements. In addition, the subjects were not taking drugs that are known to affect immune function, such as corticosteroids, cyclosporine, azathioprine, and cyclophosphamide, or receiving any immunostimulative agent such as lymphokines (159) . B. Experimental Designs Study I was a repeated measures design. In this 32-day investigation, the six subjects (Table 1) received a constant basal diet which was adequate in all nutrients (except vitamin B-6 during the first 12-day period) and was composed of natural foods. The basal diet contained 13.6 g N (85 g crude protein) and 0.84 mg (4.96 jimols) of vitamin 34 B-6. To lower the subjects' body stores of vitamin B-6, the subjects received the basal diet without a PN supplement during the first 12 days of the study. This preliminary 12- day period was followed by two consecutive 10-day periods. The subjects' basal diet was supplemented daily with 0.4 mg PN (as PN.HCl) during the first 10-day period, and with 1.6 mg PN (as PN.HCl) during the second 10-day period. The subjects' total daily vitamin B-6 intake was 0.84 mg (4.96 jimols) , 1.24 mg (7.33 ^mols) and 2.44 mg (14.42 nmols) , respectively, during the 3 consecutive periods (Fig. 2A). The composition of the basal diet, as well as preparation and administration of the PN supplements are reported elsewhere. Table l. Descriptive data on subjects in Study I Age Height (y) (cm) beginning end of study 1 2 3 4 5 6 28 28 27 33 28 25 162 185 162 183 170 173 68.2 64.5 94.1 68.8 62.2 57.7 67.7 63.2 95.0 68.4 61.8 57.7 Mean ±SD 28.2 2.6 172.5 9.9 69.3 12.8 69.0 13.4 Subject Weight (kg) 35 The experimental protocol for Study II is illustrated in Fig. 2B. This was a completely randomized design study. Ten female subjects, described in Table 2, were randomly assigned to two groups of five each: one group received a PN.HCl supplement (PN group), the other received a placebo (placebo group). The subjects consumed self-selected diets throughout the 46-day investigation. Following a 5-day preliminary period, the PN group received 1.5 mg of supplementary PN.HCl (7.29 iimols) daily for 7 days, followed by a supplement of 50 mg PN.HCl (243 umols) daily during the subsequent 6-day period. The control group received a placebo during these 13 days. The subjects were not aware of which preparation they were receiving. Compliance was verified by providing the subjects their PN supplement or placebo every morning during this period. This "treatment" period was followed by a 28-day "washout" period during which the subjects received no PN supplement or placebo. The subjects provided their 3-day dietary records between days 1 and 5, 6 and 12, and 3 6 and 46 of the study. 36 Table 2. Descriptive data on subjects in Study II Sub- Group ject Age Height Weight (kg) (y) (cm) 34 36 20 24 25 25 34 22 24 27 164 160 175 160 162 179 168 171 164 161 62.2 53.1 68.1 56.3 53.3 72.2 61.3 63.1 52.7 54.9 61.7 53.1 68.1 55.4 52.7 72.6 59.9 64.0 52.9 52.9 Means ±SD 25. 8 6. 3 165.4 7.1 59.1 6.3 58.7 6.9 Placebo Means ±SD 28. 4 5. 1 167.4 6.8 60.3 8.0 60.0 8.1 1 2 3 4 5 6 7* 8 9 10 placebo PN PN PN placebo Placebo placebo PN placebo PN beginning end of study Group PN * Took an oral contraceptive agent during the experiment. The vitamin B-6 supplement was prepared by mixing PN.HCl with dextrose at ratios of 1:400 and 1:12 for the 1.5 mg PH.HC1 and 50 mg PN.HCl supplements, respectively. Pyridoxine hydrochloride and dextrose were mixed thoroughly and sieved 7 times. Six hundred milligrams of this PN.HC1- sugar mixture were placed in a gelatin capsule. Six hundred milligrams of dextrose were placed in a gelatin capsule for the placebo. The concentration of the PN.HCl in capsules was verified spectrophotometrically in 0.1 N HC1 using an extinction coefficient of 422 at 291 run (160). 37 (A) blood draws 1 I I 0.84mg B-6/day 1.24mg B-6/day 2.44mg B-6/day day 12 1 22 32 (B) 3day dietary record blood draws I +50mg +1.5mg washout PN/day (no PN or placebo) PN/day or placebo day 12 15 18 22 26 36 46 Fig. 2. The experimental protocols. (A). Study I. During the first period, days 1 through 12, the subjects received 0.84 mg (4.96 jimols) of vitamin B-6 daily, and during days 13 through 22, and days 23 through 32, they received a total of 1.24 mg (7.33 jimols) and 2.44 mg (14.42 jimols) of vitamin B-6 daily, respectively. Blood was drawn from fasting subjects in the morning of day 1, and in the morning following days 12, 22, and 32. (B). Study II. The subjects consumed their self-selected diets throughout this investigation. Following a 5-day preliminary period, the PN group received a supplement of 1.5 mg PN.HC1 (7.29 jimols) daily for seven days, followed by a supplement of 50 mg PN.HC1 (243 nmols) daily during the subsequent six day. The placebo group received a placebo during these 13 days. This "treatment" period was followed by a 28-day "washout" period of no supplement or placebo. The subjects provided their 3day dietary records between days 1 and 5, 6 and 12, and 3 6 and 46. 38 C. Blood Drawing Between 7 and 9 am on specified days for both studies (Fig. 2A and 2B), blood was obtained from the antecubital vein of fasting subjects by a registered medical technologist. The blood was collected in evacuated tubes containing heparin and processed immediately. 25 ml of blood were drawn each time. Approximately Of this volume, approximately 8 ml of blood were used in both studies to separate mononuclear cells for lymphocyte proliferation test (both studies) and for measuring interleukin 2 production and lymphocyte PLP concentration (Study II only). The plasma and erythrocytes (washed with normal saline) from the remaining blood were stored at -20 0 C for measuring plasma PLP concentration (both studies) and erythrocyte aspartate aminotransferase activity later (Study II only). D. Isolation of Mononuclear Cells Immediately after it was obtained, whole blood was diluted 1:1 with phosphate buffered saline. Diluted blood was carefully layered with a Pasteur pipette on top of 8 10 ml of lymphocyte separation reagent (Histopaque-1077) in a 50 ml centrifuge tube. The this tube was centrifuged at 400 x g for 3 0 minutes at room temperature. Mononuclear cells at the interface were removed with a Pasteur pipette, 39 placed in a 15 ml centrifuge tube, and washed twice with RPMI 1640. E. Lymphocyte Viability Tests and Counts Lymphocyte viability was determined by trypan blue exclusion. The percentage of viable cells in the cell suspension was greater than 95%. For further experiments, the cells were then resuspended in appropriate amount of RPMI 1640 with 10% fetal bovine serum (FBS) (10% RPMI) to obtain a concentration of 1X109 viable cells/L. F. Lymphocyte Proliferation Lymphocyte proliferation was measured by 3H-thymidine incorporation following culture with the T-cell mitogens, PHA and Con A, and the T-cell-dependent B-cell mitogen, PWM. Appropriate dilutions of each mitogen were made in 10% RPMI. PHA was diluted to concentrations of 10, 2.5, and 0.625 jig/ml; Con A was diluted to 40, 10, and 2.5 ng/ml ; and PWM was diluted to 4, l, and 0.25 ng/ml. These concentrations of each mitogen were plated in triplicate, 0.1 ml per well, into a 96-well flat-bottomed microtiter plate. One tenth milliliter of cell suspension of IxlO9 cells/L was plated into each well. All plates were incubated 96 hours at 370C in an atmosphere of 5% CO2 (Napco 40 CO2 incubator, Model 304; Napco Inc., Minneapolis, MN). The microtiter plates were wrapped with plastic film to prevent water evaporation from the medium. After 72 hours, 0.5 jiCi 3 H-thymidine (6.7 Ci/M; ICN Biomedical, Inc. Irvine, CA) was added to each well in a volume of 20 jil. At the end of 96 hours, the plates were frozen at -700C until harvested. When thawed, the cells were harvested onto microfiber paper using a semi-automatic cell harvest system (Type 11019, Skatron Instrument Inc., Sterling, VA). Filter disks were dried at 350C and placed in pre-labeled minivials. After adding 2 ml of liquid scintillation fluid to each vial, the vials were counted in a S-scintillation counter (Beckman Model LS5000TD, Beckman Instruments, Inc., Fulleton, CA). Data were recorded as counts per minute (cpm) using a tritium channel. The cpm of the unstimulated, control culture was subtracted from each subject's mitogen responses. G. IL-2 Production (Study II) Lymphocytes (IxlO9 cells/L) in 10% RPMI were cultured in 24-well, flat-bottomed plates with PHA (2.5 mg/L) for 48 hours. The cell-free supernatant was stored at -700C for measuring IL-2. IL-2 activity was measured by the microassay method by Coligan et al. (161) using CTLL-2 cell line (ATCC TIB 214). Recombinant human IL-2 was used as the 41 standard. IL-2 standard and samples were diluted with 10% RPMI. One tenth milliliter of standard or sample was plated in triplicate into a 96-well flat-bottomed microtiter plate. CTLL-2 cells were counted and cell concentration was adjusted to SxlO7 cells/L in 10% RPMI. One tenth milliliter of the cell suspension was added to each well of the microtiter plates containing either an IL-2 standard or sample. A negative control with no standard or sample was included in each assay. The plates were wrapped with plastic film and incubated for a total of 2 8 hours at 3 70C and 5% CO2. After 20 hours of incubation, 0.5 nCi 3 H- thymidine was added to each well in a volume of 20 jil. At the end of 28 hours, the plates were frozen and stored at -700C until harvested. Cells were harvested and 3H- thymidine incorporation was measured as described for the lymphocyte proliferation assay given above. IL-2 activity was calculated using probit analysis (161). H. Effect of Putrescine on Lymphocyte Proliferation and IL-2 Production (Study II) To examine the effect of putrescine on the proliferation of lymphocytes, putrescine was added in culture medium at concentrations of 0, 2, 20 and 200 pimol/L when testing the lymphocyte proliferation in response 42 to PHA, Con A and PWM stimulation. putrescine on IL-2 production, To test the effect of of 0, 20 and 200 nmol/L putrescine were added in the culture medium. The procedures for testing lymphocyte proliferation and IL-2 production were then followed as described above. I. PLP Determination Plasma PLP was determined by measuring the 14 C02 evolved during the decarboxylation of tyrosine-l-14C by tyrosine decarboxylase apoenzyme. The method used was proposed by Chabner and Livingston (162) and modified by Leklem (163). A minimum of 1 x 105 lymphocytes were used to measure PLP concentration in the cells. The lymphocytes were broken by freezing and thawing, followed by sonification (Sonifier Model 200, Branson Sonic Power Company, Danbury, CT) for 30 seconds. Complete breakage of cells was verified under a light microscope. Lymphocytes and FBS PLP concentrations were measured the same as plasma as described above. The coefficient of variation (CV) of a control plasma sample analyzed with each assay was 4.2% (n = 11). The percent recovery of PLP added to the subjects' plasma samples was 91.7% ± 5.7% (mean ± SD). 43 J. Erythrocyte Aspartate Aminotransferase Activity Coefficient (EAST AC) (Study II) The kinetic method described by Beutler (164) was used to measure erythrocyte aspartate aminotransferase activity with and without in vitro PLP stimulation. Results are expressed as erythrocyte aspartate aminotransferase activity coefficient (activity with PLP stimulation/activity without PLP stimulation). K. Collection of Dietary Information (Study II) Between the days 1 and 5, 6 and 12 and 3 6 and 46 of Study II, each subject completed three three-day dietary records. Each three-day dietary record included two week days and one weekend day. Foods were coded for computer analysis for general nutrient intake with special emphasis on vitamin B-6 and protein intake. The nutrient database used was The Food Processor Plus (Nutrition & Fitness Software, Version 5.0, ESHA Research, Salem, OR). This nutrient database is based on data from USDA and information from manufacturers for over 5000 food items. 44 L. Statistics The data were analyzed for statistical significance by using the software packages Statgraphics (Statistical Graphics System, Version 5.0, STSC, Inc., Rockville, MD) and SAS (Version 6.04, SAS Institute Inc., Gary, NC). Comparisons between means were made with ANOVA, multiple range test and Student's t test. Correlation coefficients were calculated to determine relationships between immune parameters and plasma PLP concentration and lymphocyte PLP concentration. p 4 0.05 Statistical significance was based on 45 RESULTS A. Study I 1. Plasma PLP Concentration The six subjects' plasma PLP concentrations are given in Table 3. Their initial (baseline) vitamin B-6 status was adequate according to Leklem (52). After a daily vitamin B- 6 intake of 0.84 mg for 12 days, the subjects' mean plasma PLP concentration dropped more than 40 %, from 47.4 nmol/L at baseline to 26.5 nmol/L. An intake of 1.24 mg of vitamin B-6 per day for 10 days did not restore the subjects' plasma PLP to the baseline level. After ten days of 2.44 mg vitamin B-6 daily intake, the subjects' mean plasma PLP level increased to 58.9 nmol/L, almost 25 % higher than the baseline level. 2. Lymphocyte Proliferation When the subjects received 0.84 mg of vitamin B-6, the lymphocyte proliferation response to the T-cell mitogen, Con A, decreased slightly, while the response to the T-dependent B-cell mitogen, PWM, did not change from the baseline response (Table 4 and Fig. 3). An intake of 1.24 mg of vitamin B-6 increased their lymphocyte proliferation response to both mitogens, but only the response to Con A was significantly higher (p < 0.05) than that at 0.84 mg of 46 vitamin B-6 intake. Increasing the subjects' vitamin B-6 intake to 2.44 mg per day resulted in a significantly higher (p < 0.05) lymphocyte response to both mitogens than at baseline and when the subjects received 0.84 mg of vitamin B-6. Table 3. Vitamin B-6 intake and plasma PLP concentrations in six healthy young women consuming a constant diet with controlled vitamin B-6 intake (Study I)* B-6 intake (mg/d) Baselinefl 0.84§ 1.24§ 2.44§ Plasma PLP (nmol/L) 47.4 26.5 29.4 58.9 ± ± ± ± 17.9 12.4 12.5 22.3 * Mean ± SD; plasma PLP was measured by C. Hanson; details are described elsewhere. II First day of the study; reflects previous self-selected diet. § Blood drawn on days 13, 23, and 33, after subjects had consumed 0.84 mg, 1.24 mg, and 2.44 mg of vitamin B-6 per day for 12, 10, and 10 days, respectively. 47 Con A H PWM 150 120 E Q. O 9 ^ I2 3«= I 2 o gb a o Baseline 0.84 1.24 B-6 intake (mg) 2.44 Fig. 3. Vitamin B-6 intake and lymphocyte proliferation in response to Con A and PWM stimulation (Study I). Baseline reflects the pre-study value (self-selected diet); 0.84, 1.24, and 2.44 mg B-6 indicate the subjects' vitamin B-6 intake. (*) shows significant difference (p < 0.05) compared with the baseline value and (#) significant difference (p < 0.05) compared with the value during 0.84 mg vitamin B-6 intake period. y 48 Table 4. Vitamin B-6 intake and mitogen response, in cpm, of lymphocytes in six healthy young women consuming a constant diet with controlled vitamin B-6 intake (Study I)* Mitogens B-6 intake (mg/d) Baselinefl 0.84§ 1.24§ 2.44§ Con A 73154 67306 91130 107447 ± 11604 ± 4425 ± 10325B ± 5697^ PWM 24644 27058 35876 48792 ± 5983 ± 5458 ± 7960 ± 10704AB * Mean ± SEM. fl First day of the study; values reflect subjects' previous self-selected diet. § Blood drawn on days 13, 23 and 33, after subjects consumed 0.84 mg, 1.24 mg and 2.44 mg of vitamin B-6 per day for 12, 10 and 10 days, respectively. A Significant difference (p < 0.05) compared with baseline value. B Significant difference (p < 0.05) compared with 0.84 mg vitamin B-6 intake period. 49 B. Study II 1. Subjects' Dietary Vitamin B-6 Intake and Nutritional Status The subjects' dietary intakes were adequate in protein, vitamin B-6 and energy (Table 5) as well as other nutrients (data not reported here). There were no significant differences between the PN and placebo groups in their dietary intakes of vitamin B-6 and protein. Their mean vitamin B-6 intake and mean ratio of mg of vitamin B-6 to g of protein were above the RDA for women (47). There were no significant differences between the PN and placebo groups in their hemoglobin concentrations and hematocrit values (Table 6). The hemoglobin and hematocrit values of all 10 subjects were within the normal ranges for women (165) . 50 Table 5. Average daily dietary intakes of energy, vitamin B-6, and protein by the PN and placebo groups during baseline, supplement and washout periods (Study II)*1I Periods Baseline Supplement! Washout 1642 ± 136 1872 + 431 1694 ± 490 1822 ± 259 Vitamin B-6 (mg) PN 1.75 ± 0.23 Placebo 1.75 ± 0.40 1.80 ± 0.23 1.81 ± 0.73 1.83 ± 0.37 2.33 ±- 0.80 Protein (g) PN Placebo 70.6 ± 16.0 68.9 ± 9.2 65.4 ± 27.5 65.5 ± 15.6 0.026 ± 0.007 0.027 ± 0.013 0.032 ± 0.020 0.035 ± 0.005 Energy (kcal) PN Placebo 1733 ± 286 1831 ± 668 66.0 ± 13.4 61.8 ± 19.6 B-6/Protein (mg/g) PN 0.028 ± 0.009 Placebo 0.031 ± 0.011 * Mean ± SD; n = 5 per group. There were no significant differences in dietary intakes between the PN and placebo groups. II The subjects consumed their self-selected diet throughout this investigation. They recorded their three-day dietary intakes between days 1 and 5, 6 and 12 and 36 and 46. § Vitamin B-6 intake of PN group during the days 6 to 12 does not include the 1.5 mg PN.HC1 supplement. 51 Table 6. Average weekly hemoglobin (Hb) concentration and hematocrit (Hct) value of the PN and placebo groups (Study II)* Time Baseline PN Placebo Hb(g/L) Hct(l) 127 ± 8 0.41 ± 0.01 128 ± 8 0.41 ± 0.01 Supplement period +1.5mg PN.HCl/d or placebo day 7 Hb(g/L) Hct(l) 128 ± 8 0.41 ± 0.02 132 ± 7 0.41 ± 0.01 +50mg PN.HCl/d or placebo day 6 Hb(g/L) Hct(l) 133 ± 13 0.41 ± 0.03 134 ± 8 0.40 ± 0.01 Washout period day 4 Hb(g/L) Hct(l) 132 ±7 0.41 ± 0.01 133 ± 6 0.40 ± 0.01 day 8 Hb(g/L) Hct(l) 128 ± 9 0.40 ± 0.02 131 ± 10 0.40 ± 0.02 day 18 Hb(g/L) Hct(l) 131 ± 10 0.40 ± 0.02 127 ± 4 0.40 ± 0.01 day 28 Hb(g/L) Hct(l) 131 ± 9 0.41 ± 0.02 130 ± 4 0.41 ± 0.02 * Mean ± SD; n = 5 per group. The subjects consumed their self-selected diet throughout this investigation. There were no significant differences in Hb and Hct between the PN and placebo groups. 52 2. Plasma and Lymphocyte PLP concentrations The mean plasma PLP concentrations at baseline of the two groups were normal (Table 7, Fig. 4A) (52). Although the range of plasma PLP concentrations within the placebo group was large and the mean was higher than that of the PN group, there was no significant difference between the PN and placebo groups in their baseline plasma PLP concentrations. Following PN.HCl supplementation, the PN group had higher plasma PLP concentration than the placebo group. The PN-treated subjects' mean plasma PLP concentration increased from 35.6 nmol/L at baseline to 54.3 nmol/L after a daily 1.5 mg PN.HCl supplement for seven days. After having received 50 mg of PN.HCl for three days, mean plasma PLP concentration increased to 248 nmol/L, seven times higher than the baseline level. The mean plasma PLP concentration plateaued at approximately 250 nmol/L in response to 50 mg of PN.HCl supplement. During the washout period, the plasma PLP concentrations in the PN group dropped rapidly. After 18 days of no PN.HCl supplementation, the PN group's mean plasma PLP concentration dropped to the baseline level. The placebo group's plasma PLP level remained relatively stable during the 46 days of study. In general, the changes in lymphocyte PLP concentration 53 in the PN group during the supplement and washout period paralleled those in plasma PLP concentration (Table 8, Fig. 4B). The initial mean lymphocyte PLP concentrations in two groups were similar. In the PN group, 1.5 mg PN.HC1 supplement increased mean lymphocyte PLP concentration from 0.27 to 0.55 pmol/10° cells. After six days of supplement with 50 mg PN.HC1 per day, this value increased to 1.68 pmol/106 cells. During the washout period, the PN group's lymphocyte PLP concentration dropped more slowly compared with the decline in plasma PLP concentration. After 18 days of PN.HC1 withdrawal, their lymphocyte PLP concentration dropped to the baseline level. Lymphocyte PLP concentration in the placebo group did not change during the 46 days of this study. Note in Table 8 that sufficient sample size was not always available for the lymphocyte PLP measurement. 54 Table 7. Plasma PLP concentrations groups (Study II)* (nmol/L) of the PN and placebo PN Placebo Baseline 35.6 ± 7.4 54.8 ± 38.8 Supplement period +1.5mg PN.HCl/d or placebo day 7 54.3 ± 8.8J 44.3 ± 30.9 +50mg PN.HCl/d or placebo day 3 day 6 Washout period day 4 day 8 day 18 day 28 248.0 ± 32.3 AB 254.8 ± 48.1 AB 100.6 70.2 47.1 39.7 ± ± ± ± 17.1 AB 19.5^ 6.9 7.0 60.8 ± 39.4 46.7 ± 37.2 48.2 42.7 50.5 54.6 ± ± ± ± 30.3 33.6 28.7 43.1 * Mean ± SD; n = 5 per group. The subjects consumed their self-selected diet throughout this investigation. A Significant difference (p < 0.05) compared with baseline value. B Significant difference (p < 0.05) compared with control group during same period. 55 Table 8. Lymphocyte PLP concentrations (pmol/106 cells) of the PN and placebo groups (Study II)* PN (n=5§) Placebo (n§) Baseline 0.27 ± 0.04 0.26 ± 0.04(4) Supplement period +1.5mg PN.HCl/d or placebo day 7 0.55 ± 0.30 0.32 ± 0.13(5) 1.28 ± 0-21^ 1.68 + 0.45^ 0.57 ± 0.25(3) 0.52 ± 0.33(2) 1.17 1.10 0.40 0.50 0.37 0.35 0.54 0.52 +5 0mg PN.HCl/d or placebo day 3 day 6 Washout period day 4 day 8 day 18 day 2 8 ± 0-35^ ± 0>49AB ± 0.12 ± 0.30 ± ± + ± 0.21(2) 0.26(2) 0.02(2) 0.33(3) * Mean ± SD. The subjects consumed their self-selected diet throughout this investigation. § Number of subjects. Sufficient blood was not always available to measure lymphocyte PLP in all of the subjects in the placebo group. A Significant difference (p < 0.05) compared with baseline value. B Significant difference (p < 0.05) compared with control group during same period. 56 -+- PN — O— Placebo 300i 200 a E V) a 100:. a. Ilia lllb lllc Hid a. -i a. u nid Fig. 4. Changes of plasma and lymphocyte PLP concentrations in the PN and placebo groups (Study II). A: Plasma PLP concentration; B: Lymphocyte PLP concentration. Periods are: (B) baseline value; (I) a daily supplement of 1.5 mg PN.HCl or placebo for 7 days; (Ila) a daily supplement of 50 mg PN.HCl or placebo for 3 days and (lib) for 6 days; (Ilia) indicates discontinuation of PN supplement or placebo for 4 days, (lllb) for 8 days, (IIIc) for 18 days and (Hid) for 28 days. (*) shows significant difference (p < 0.05) compared with the baseline value and (#) significant difference (p < 0.05) compared with the placebo group. 57 3. Erythrocyte Aspartate Aminotransferase Activity Coefficient (EAST AC) Although the PN group's EAST AC decreased slightly during the supplement period, there were no significant differences in EAST AC between the two groups throughout this investigation (Table 9). Table 9. Erythrocyte aspartate aminotransferase activity coefficient (EAST AC) of the PN and placebo groups (Study II)* PN Placebo Baseline 1.67 ± 0.22 1.77 ± 0.18 Supplement period +1.5mg PN.HCl/d or placebo day 7 1.55 ± 0.12 1.60 ± 0.18 +50mg PN.HCl/d or placebo day 3 day 6 1.43 ± 0.28 1.56 ± 0.23 1.60 ± 0.34 1.90 ± 0.24 1.56 1.59 1.47 1.59 1.78 1.64 1.76 1.57 Washout period day 4 day 8 day 18 day 28 ± ± ± ± 0.29 0.29 0.26 0.28 ± ± ± ± 0.18 0.22 0.36 0.20 * Mean ± SD; n = 5 per group. The subjects consumed their self-selected diet. There were no significant differences at EAST AC between the PN and the placebo groups throughout this investigation. 58 4. Lymphocyte Proliferation The lymphocyte proliferation in response to PHA (Table 10, Fig. 5A), Con A (Table 11, Fig. 5B) and PWM (Table 12, Fig. 5C), significantly increased following a daily supplement of 1.5 mg PN.HC1 for 7 days. Increasing the PN.HC1 supplement to 50 mg, however, did not further increase lymphocyte proliferation in response to PHA and PWM stimulation and enhanced the lymphocyte proliferation response to Con A only slightly. The increased lymphocyte proliferation in response to all three mitogens in PN group during the supplement period were statistically significant when compared with that in the placebo group. During the washout period, the elevated lymphocyte proliferation in response to three mitogens in the PN group gradually decreased. However, three weeks after removed the PN.HC1 supplement, the lymphocyte proliferation in response to all three mitogens remained elevated and significantly higher than baseline and placebo group values. By the end of the fourth week, the proliferation response of lymphocytes to all mitogens from the PN group dropped to the pre-supplement level and were not significantly different from the placebo group. In the placebo group, the lymphocyte proliferation in response to mitogen stimulation showed only minor fluctuations throughout this 46-day study. 59 Table 10. PN supplementation and mitogen response, in cpm, of lymphocytes to PHA in the PN and placebo groups (Study II)* Period Baseline PN Placebo 84679 ± 3893 82117 ± 2838 Supplement period +1.5mg PN.HCl/d or placebo day 7 106120 ± 5042 AB 91892 ± 2326 +50mg PN.HCl/d or placebo day 3 day 6 106983 ± 2443^ 108262 ± 3690^ 93069 ± 2812 84821 ± 3143 108329 107945 97654 85645 82249 85483 75681 86194 Washout period day 4 day 8 day 18 day 28 ± ± ± ± 4757^ 6424^? 3017AB 5866 ± ± ± ± 3449 6316 3150 3067 * Mean ± SEM; n = 5 per group. The subjects consumed their self-selected diet throughout this investigation. A Significant difference (p < 0.05) compared with baseline value. B Significant difference (p < 0.05) compared with control group during same period. 60 Table 11. PN supplementation and mitogen response, in cpm, of lymphocytes to Con A in the PN and placebo groups (Study II)* Period PN Placebo Baseline 69473 ± 1974 Supplement period +1.5mg PN.HCl/d or placebo day 7 81287 ± 3031 AB 73073 ± 3588 +50mg PN.HCl/d or placebo day 3 day 6 92626 ± 2705^ 83351 ± 941 AB 80555 ± 3255 69325 ± 5648 Washout period day 4 day 8 day 18 day 28 88676 83546 79451 74239 ± ± ± ± 1400 AB 2946"AB 5224^ 4425 69813 ± 3928 68136 73925 67581 68544 ± ± ± ± 6123 5598 4148 3418 * Mean ± SEM; n = 5 per group. The subjects consumed their self-selected diet throughout this investigation. A Significant difference (p < 0.05) compared with baseline value. B Significant difference (p < 0.05) compared with control group during same period. 61 Table 12. PN supplementation and mitogen response, in cpm, of lymphocytes to PWM in the PN and placebo groups* Period PN Placebo Baseline 19335 ± 3007 Supplement period +1.5mg PN.HCl/d or placebo day 7 36219 ± 5796AB 22273 ± 1084 +50mg PN.HCl/d or placebo day 3 day 6 38682 ± 4158^ 34240 ± 5658^ 27421 ± 5482 25110 ± 3665 43478 33077 37805 24011 23196 ± 5288 30492 ± 4737A 28595 ± 3224A 28203 ± 5945 Washout period day 4 day 8 day 18 day 28 ± ± ± ± 4156^ 5947A 2366^ 5839 19641 ± 2521 * Mean ± SEM; n = 5 per group. The subjects consumed their self-selected diet throughout this investigation. A Significant difference (p < 0.05) compared with baseline value. B Significant difference (p < 0.05) compared with control group during same period. 62 PN mM Placebo ISO' PHA 12<H 90- 60 30 I B lla Mb Ilia lllb lllc Hid ISO 120 B I lla Mb Ilia lllb lllc Hid 150 PWM Fig. 5. Lymphocyte proliferation in response to PHA (A), Con A (B) and PWM (C) stimulation in the PN and placebo groups (Study II). Periods, * and # are the same as in Fig. 4. 63 To explore further the relationship between lymphocyte PLP concentration and the proliferation response of lymphocytes to mitogen stimulation, correlation analyses were conducted. The lymphocyte proliferation in response to each of the three mitogens was positively and significantly (p < 0.01) correlated to lymphocyte PLP concentrations (Fig. 6A-C). The correlation coefficient for lymphocyte proliferation in response to PHA was 0.44 (p < 0.01), to Con A was 0.47 (p < 0.01) , and to PWM was 0.45 (p < 0.01) . The best-fit correlation lines in Figure 6A-C could be roughly separated into two parts at a PLP concentration of 1.0 pmol/10° cells. Changes in proliferation response were greater at the lower concentrations of lymphocyte PLP ( < 1.0 pmol/106 cells) than those at the higher lymphocyte PLP concentrations ( ^ 1.0 pmol/106 cells). 64 Fig. 6. Correlation between lymphocyte proliferation, IL-2 production and lymphocyte PLP concentrations (Study II). Data are from both PN and placebo groups. A: lymphocyte proliferation in response to PHA stimulation; B: lymphocyte proliferation in response to Con A stimulation; C: lymphocyte proliferation in response to PWM stimulation; D: IL-2 production. The correlations are positive and statistically significant (p < 0.01). 65 (X 1000) (X 1000) Con fii PHA r=0.47,P<0.01 r=0.44,P<0.01 104 146 94 8 o 126 84 a a c o a. n e o a 74 106 L C a a o 64 86 54 44 66 J 1 1 1 1 ' 0 0.4 0.8 1.2 1.6 2 2.4 J ' ' 0 0.4 0.8 L. 1.2 1 1 1.6 2 L 2.4 <X 10000) PUfl r = 0.45,P<0.01 IL-2 r=0.63,P<0.01 3 a a c 0 a. m a L C 0 a o J L 0 0.4 0.8 J 1 1 L 1. 2 1.6 2 2.4 J 1 1 L J L 0 0.4 0.8 1.2 1.6 2 Lymphocyte PLP concentration (pmol/106 cells) 2.4 66 5. IL-2 Production There was no significant difference in IL-2 production between PN and placebo groups at baseline (Table 13, Fig. 7) . In the PN group, a daily supplement of 1.5 mg PN.HC1 for 7 days increased the mean IL-2 production by more than 60%. When the PN.HC1 supplement was increased to 50 mg per day, mean 11-2 production in the PN group increased to 3470 U/L of culture medium after 3 days and 433 6 U/L of culture medium after 6 days. Withdrawing the PN.HC1 supplement resulted in decreased IL-2 production, which was still higher than the baseline level and the placebo group during the first three weeks of the washout period. At the end of fourth week of the washout period, the IL-2 production in PN group was still higher than the baseline level but showed no significant difference between the PN and placebo groups. In the placebo group, IL-2 production fluctuated slightly from time to time during the 46-day study. IL-2 production in the PN group was significantly (r = 0.63, p < 0.01) correlated to lymphocyte PLP concentration (Fig. 7D). 67 Table 13 PN supplementation and IL-2 production (U/L) of lymphocytes in the PN and placebo groups (Study II)* Period PN (n=5) Baseline 988 ± 62 Placebo (n=3) 1137 ± 192 Supplement period +1.5mg PN.HCl/d or placebo day 7 1602 ± 122^ 1207 ± 212 +50mg PN.HCl/d or placebo day 3 day 6 3474 ± 866^ 4336 ± 676AB 1880 ± 700 1710 ± 416 3622 3032 2032 1220 1300 1460 1390 1063 Washout period day 4 day 8 day 18 day 28 ± ± ± ± 740^ 925^^ 213^^ 120A ± 72 ± 292 ± 10 ± 144 * Mean ± SEM. The subjects consumed their self-selected diet throughout this investigation. A Significant difference (p < 0.05) comparing with baseline value; B Significant difference comparing with control group during same period. 68 -+- RN -o- Placebo SOOOn 4000 c o o 3 •o o Q. 3000" 2000- CVJ I _l 1000^ = Hid Fig. 7. IL-2 production in response to PHA stimulation in the PN and placebo groups (Study II). Periods, * and # are the same as in Fig. 4. 69 6. Effect of Putrescine on Lymphocyte Proliferation and IL-2 Production Effect of putrescine added in vitro on proliferation of lymphocytes from subjects in the PN and placebo groups was also tested in Study II (Table 14-16). Lymphocyte proliferation in response to each mitogen was at its peak after the PN group subjects had taken the 50 mg PN.HC1 supplement for 3 days. Figure 8A shows the effect of 0, 2, 20, and 200 nmol/L of putrescine concentrations on lymphocyte proliferation at this peak response to mitogen stimulation. For all three mitogens, PHA, Con A and PWM, the lymphocyte responses to different putrescine concentrations in both PN and placebo groups were flat lines (Fig. 8A), indicating that the putrescine concentrations used did not affect the lymphocyte response to mitogen stimulation under the experimental conditions used. Putrescine was also added in culture medium at concentrations of 0, 20, 200 nmol/L to test the effect of this polyamine on IL-2 production by lymphocytes from the PN and placebo groups (Table 17). Figure 8B shows the IL-2 production at the different added putrescine concentrations by the placebo group and PN group who had received 50 mg PN.HC1 for 6 days. The putrescine added to culture medium did not significantly affect the IL-2 production in the experiment. 70 Table 14. Effect of PN supplementation and putrescine concentrations in the culture medium on mitogen response, in cpm, of lymphocytes to PHA in the PN and placebo groups (Study II)* Putrescine Cone. (iAmol/L) PN placebo Baseline 0 2 20 200 84679 80882 82506 75976 ± ± ± ± 3893 4388 5200 4037 82117 78918 78514 74994 ± ± ± ± 2838 4046 2803 3814 Supplement period +1.5mg PN.HCl/d or placebo day 7 0 2 20 200 106120 115314 114154 111747 ± ± ± ± 5042 5033 5271 8565 91892 92357 86750 81596 ± ± ± ± 2326 2685 1396 2798 +50mg PN. HCl/d or placebo day 3 0 2 20 200 106983 104838 102916 96315 ± ± ± ± 2443 3193 4485 3554 93069 90340 91230 89535 ± ± ± ± 2812 3290 3899 2940 0 2 20 200 108262 107006 105234 97484 ± ± ± ± 3690 1968 2152 3903 84821 81700 81088 81687 ± ± ± ± 3143 2655 2738 3984 0 2 20 200 108329 111065 111116 103560 ± ± ± ± 4757 6704 6553 3850 82249 78000 77036 71675 ± ± ± ± 3449 4328 4551 6812 day 8 0 2 20 200 107945 108677 110053 101969 ± ± ± ± 6424 6023 4344 4710 85483 82662 85232 86172 ± 6316 ± 5964 ± 6125 ± 803 day 18 0 2 20 200 97654 97154 98069 88131 ± ± ± ± 3017 3761 3390 6310 75681 71318 72045 71379 ± ± ± ± 3150 2124 2024 2409 day 28 0 2 20 200 85645 91908 91426 86991 ± ± ± ± 5866 5873 7073 5381 86194 81450 80993 78811 ± ± ± ± 3067 4978 4490 3472 day 6 Washout period day 4 * Mean ± SEM; n = 5 per group. The subjects consumed their self-selected diet throughout this investigation. 71 Table 15. Effect of PN supplementation and putrescine concentrations in the culture medium on mitogen response, in cpm, of lymphocytes to Con A in PN and placebo groups (Study II)* Putrescine Cone. (jimol/L) PN placebo Baseline 0 2 20 200 69473 66231 65376 65458 ± ± ± ± 1974 2051 1814 2764 69813 63514 64984 61064 ± ± ± ± 3928 3149 2313 3770 Supplement period +1.5mg PN.HCl/d or placebo day 7 0 2 20 200 81287 73925 74014 70809 ± ± ± ± 3031 2983 2559 3841 73073 71146 68801 72102 ± ± ± ± 3588 3922 3459 3168 +50mg PN. HCl/d or placebo day 3 0 2 20 200 92626 89198 88305 89014 ± ± ± ± 2705 2114 2220 4353 80555 78544 79949 79011 ± ± ± ± 3255 2565 5365 4447 0 2 20 200 83351 77742 77010 77160 ± 941 ± 1796 ± 1931 ± 2378 69325 67832 65651 68134 ± ± ± ± 5648 7423 7433 6522 0 2 20 200 88676 85820 85318 87581 ± ± ± ± 1400 2777 2399 2291 68136 64603 67167 61749 ± ± ± ± 6123 5925 5297 8717 day 8 0 2 20 200 83546 78335 79205 77648 ± ± ± ± 2946 3656 3830 1641 73925 68585 68950 71144 ± ± ± ± 5598 4132 5554 2987 day 18 0 2 20 200 79451 68480 67254 72983 ± ± ± ± 5224 4605 3953 4806 67581 64984 65357 66149 ± ± ± ± 4148 3953 4090 4013 day 2 8 0 2 20 200 74239 66954 64499 65219 ± ± ± ± 4425 6882 7317 5640 68544 68061 67842 68499 ± ± ± ± 3418 1906 1126 2709 day 6 Washout period day 4 * Mean ± SEM; n = 5 per group. The subjects consumed their self-selected diet throughout this investigation. 72 Table 16. Effect of PN supplementation and putrescine concentrations in the culture medium on mitogen response, in cpm, of lymphocytes to PWM in the PN and placebo groups (Study II)* Putrescine Cone.(nmol/L) eline PN placebo 0 2 20 200 19335 19129 18343 15965 ± ± ± ± 3007 2923 1531 2495 19641 19765 20953 16095 ± ± ± ± 2521 3202 3523 2388 Supplement period +1.5mg PN.HCl/d or placebo day 7 0 2 20 200 36219 35976 33444 23896 ± ± ± ± 5796 5378 5580 5273 22273 23570 22051 25272 ± ± ± ± 1084 1379 1648 6880 +50mg PN. HCl/d or placebo day 3 0 2 20 200 38682 39286 38882 33329 ± ± ± ± 4158 3816 3802 4131 27421 27444 29325 24422 ± ± ± ± 5482 6473 6429 6257 0 2 20 200 34240 35479 33460 28168 ± ± ± ± 5658 5554 5329 6100 25110 28124 27662 21810 ± ± ± ± 3665 5282 5079 4375 0 2 20 200 43478 44352 44530 38996 ± ± ± ± 4156 3987 4156 6113 23196 24755 24830 21167 ± ± ± ± 5288 6210 6162 6233 day 8 0 2 20 200 33077 32213 34059 28227 ± ± ± ± 5947 5682 6171 7605 30492 28174 32726 29978 ± ± ± + 4737 2416 5640 5640 day 18 0 2 20 200 37805 35664 37130 32047 ± ± ± ± 2366 2218 2115 4621 28595 31555 30671 26565 ± ± ± ± 3224 2733 3217 3312 day 2 8 0 2 20 200 24011 25509 25181 21563 ± ± ± ± 5839 6260 5736 5998 28203 31382 31063 24628 ± ± ± ± 5945 5829 5538 4764 day 6 'ashout peiriod day 4 * Mean ± SEM; n = 5 per group. The subjects consumed their self-selected diet throughout this investigation. 73 Table 17. Effect of PN supplementation and putrescine concentrations in the culture medium on IL-2 production (U/L) of lymphocytes in the PN and placebo groups (Study II)* Putrescine Cone, (iimol) Placebo (n=3i 62 75 44 1137 ± 192 1173 ± 186 1070 ± 55 1602 ± 122 1406 ± 137 1440 ± 346 1207 ± 213 1240 ± 171 1057 ± 243 0 20 200 3474 ± 866 4304 ± 813 2820 ± 160 1880 ± 700 1325 ± 345 1030 ± 90 0 20 200 4336 ± 676 4492 ± 598 3464 ± 937 1710 ± 416 1287 ± 272 1197 ± 200 0 20 200 3622 + 740 3634 ± 568 2512 ± 338 1300 ± 72 1267 ± 123 1093 ± 47 day 8 0 20 200 3032 ± 925 3210 ± 723 2608 ± 716 1460 ± 292 1380 ± 355 957 ± 109 day 18 0 20 200 2032 ± 213 2082 ± 232 1886 ± 48 1390 ± 10 1505 ± 25 1430 ± 330 day 28 0 20 200 1220 ± 120 1506 ± 112 1234 ± 131 1063 ± 144 1423 ± 160 1250 ± 190 Baseline 0 20 200 PN (n=5) Supplement period 0 +1.5mg PN.HCl/d 20 200 or placebo day 7 +50mg PN.HCl/d or placebo day 3 day 6 Washout pe:riod day 4 988 ± 1116 ± 1020 ± * Mean ± SEM. The subjects consumed their self-selected diet throughout this investigation. 74 - + - pN — O— placebo 200 E Q. S 3 +— 100 S O— £ o '—+ CO -i +putrescine 50001 1 2 1— 20 200 umol/L B 4000 3000 a. 2000 1000- +putr9»cino h-- ~r20 -2 200 umol/L Putroscino in medium Fig. 8. Changes of lymphocyte proliferation and IL-2 production in response to putrescine concentrations in culture medium (Study II). A: lymphocyte proliferation after a daily supplement of 50 mg PN.HCl or placebo for 3 days; B: IL-2 production after a daily supplement of 50 mg PN.HCl or placebo for 6 days. 75 DISCUSSION While the damaging effect of vitamin B-6 deficiency on cellular and humoral immunity in animals is well documented (1,2,3,4,5), in limited human studies the effect of vitamin B-6 on immune function is not as clear as shown in animal studies. In addition to the inherent limitations of human studies, such as not existing in real life of single vitamin B-6 difference and unethical to induce a severe B-6 deficient status in controlled studies, the different experimental designs and the short experiment duration all contribute to the uncertainty of the reported human study results. The two studies reported here show the effect of controlled vitamin B-6 intake on in vitro lymphocyte proliferation and IL-2 production in young women. The subjects who participated in these studies were apparently healthy and free of any known disorder. any illness throughout each study. They were free of The subjects also had adequate intakes of protein, vitamin B-6 and energy (Table 5) as well as other nutrients. After taking 2.44 mg vitamin B-6 per day (about 1.5 times of the RDA) for 10 days, the subjects in Study I had a significantly increased lymphocyte proliferation in response to T-cell mitogen, Con A, and T-dependent B-cell mitogen, PWM, stimulation, compared with the baseline and the vitamin 76 B-6 intake of 0.84 mg per day (Table 4). In Study II, addition of daily supplement of 1.5 mg PN.HC1 to their selfselected diet (Table 5) brought the total vitamin B-6 intake of the PN group to about 2 times of the RDA. With this intake of vitamin B-6, there was a significant (p < 0.05) increase in the subjects' lymphocyte proliferation in response to PHA, Con A and PWM, and IL-2 production compared with the placebo group and the PN group's response at the baseline. The results of these two studies reported here clearly show that a vitamin B-6 intake above the RDA increased in vitro lymphocyte proliferation and IL-2 production in healthy young women as had been shown in elderly (6,7). Lymphocyte proliferation in response to mitogen stimulation and IL-2 production were used to assess the effect of vitamin B-6 intake on immunocompetence in healthy young women in our studies. In vitro proliferation of peripheral blood lymphocytes in response to mitogen stimulation is a widely used method for assessing immunocompetence in humans (166) and is thought to mimic the events occurring in vivo following challenge by specific antigens. Lymphocyte activation by either antigens or mitogens results in similar intracellular changes and subsequent proliferation. The difference is that a specific antigen stimulates a specific cell clone, while mitogens (such as those used in this investigation) are multiclonal 77 stimulators (167). IL-2, formerly called T-cell growth factor, is one of the key factors in immune response. Stimulated T-cells produce IL-2, which is essential to initiate the proliferation of activated T-cells and NK cells (168,169) . High affinity receptors for IL-2, which are absent on resting T-cells, appear within hours of activation. The binding of IL-2 to these receptors induces the clonal expansion of the T-cells activated by the specific antigen (168). Activated B-cells also express receptors for IL-2, which can act on those B-cells as a potent growth factor (167). Hodges et al. (101,102) found slightly impaired antibody response to tetanus and typhoid antigens in young men maintained on a vitamin B-6 deficient diet for five weeks. In an independently living elderly population with a normal vitamin B-6 intake according to the current RDA levels of B-6 intake and the ratio of the intakes of vitamin B-6 and protein, a 50 mg PN.HC1 supplement per day for 2 months resulted in a significant increase of lymphocyte proliferation response to both T- and B-cell mitogen stimulation, compared with the pre-supplemental level and the control group. When the PN-treated subjects were divided into two groups according to their pre-study plasma PLP concentration, the effect of PN supplementation on lymphocyte proliferation was much greater in the subjects who had a low pre-study plasma PLP concentration (i 30 78 nmol/L) than in those with normal level (> 30 nmol/L). Although the total lymphocyte count did not change with the PN supplement, the number of CD3+ and CD4+ cells increased significantly (7). In a vitamin B-6 depletion-repletion study, the depletion of vitamin B-6 from elderly people resulted in a significant decrease of lymphocyte proliferation response to both T- and B-cell mitogen stimulation and a decline in IL-2 production. An intake of 2.88 mg vitamin B-6 per day for elderly men and 1.90 mg per day ^for elderly women, which was slightly higher than the current vitamin B-6 RDA for men and women, respectively, partly restored lymphocyte proliferation and IL-2 production. A supplement of 50 mg PN.HC1 per day further increased lymphocyte proliferation and IL-2 production in these elderly subjects (6). There are no reported studies to evaluate the effect of vitamin B-6 intake on immune function in young women. It has been known that a decline in immunocompetence is one of the general physiological changes associated with aging (170-173). It is possible that a high vitamin B-6 intake in the elderly specifically stimulated the decreased immune function accompanying aging through a not yet clearly defined nonnutritional effect. It is important, therefore, to study the effect of vitamin B-6 intake, especially in a nutritional level, on immune function in young adults. Based on a 13 weeks study of 24 young males, Van 79 den Berg et al. (10) concluded that short term vitamin B-6 marginal deficiency did not affect immune function as evaluated by total lymphocytes, total T- and B-cells, IL-2 receptor expression, immunoglobulin and complement concentrations. However, these subjects, whether or not they received vitamin B-6 supplement, received twice the RDA for all other vitamins and minerals. Many of these vitamins and minerals, such as vitamins A (103), C (104) and E (105), and minerals Se (106) and Zn (106) , may improve immune function of the subjects. Second, what the authors measured is actually a static status of the immune cells, rather than dynamic response of immune system to a stimulation. It is possible that the marginal vitamin B-6 status could maintain apparently normal immune indices without challenge, but the immune system may not have the optimal competence upon a stimulation. Additionally, sexual difference of the subjects may also be a contributing factor in the discrepancy between their conclusion and our results. The American Medical Association (AMA) stated that, in general, the nutritional supplementary intake of vitamins is 0.5 - 1.5 times that of the RDA, while 2 - 10 times RDA of supplementary vitamins are therapeutic amounts for certain nonnutritional or pharmacological purposes (174). (The amount suggested by the AMA is unrealistic in view of the levels of vitamin B-6 administered in some studies (6,7,175) and amount available over the counter.) In the two studies 80 reported here, the vitamin B-6 intakes included both nutritional and pharmacological levels. The results of our studies demonstrated that in young healthy women, a vitamin B-6 intake 1.5-2 times of the current RDA increased the lymphocyte function evaluated by in vitro proliferation in response to mitogen stimulation and IL-2 production. In Study II, a daily supplement of 50 mg PN.HC1 in PN group brought their total vitamin B-6 intake to about 25 times the RDA of this vitamin for women. This amount of vitamin B-6 intake did not further increase lymphocyte proliferation in response to mitogen stimulation (Table 10-12, Fig. 5) compared with the period of lower vitamin B-6 intake (2 times of the RDA). IL-2 production was increased following 50 ml of PN.HC1 supplement (Table 13, Fig.7). We speculate that the effect of vitamin B-6 on immune function is by virtue of providing PLP, the coenzyme for some enzymes which in turn support the activation of immune cells. Conceivably, there are apoenzymes in lymphocytes that are inactive under normal conditions; in response to a higher intake of vitamin B-6, these apoenzymes are activated by PLP and support a rapid proliferation of lymphocytes in response to mitogen stimulation. In our study, lymphocyte proliferation in response to mitogen stimulation could be roughly separated into two phases (Fig. 6): changes in proliferation were greater at the lower lymphocyte PLP concentrations (roughly < 1.0 pmol/106 cells) than those at 81 the higher lymphocyte PLP concentrations {z. 1.0 pmol/106 cells). Talbott et al. (7) also reported that the effect of PN supplementation on lymphocyte proliferation was much greater in the subjects who had low pre-study plasma PLP concentrations U 30 nmol/L) than in those with normal levels (> 30 nmol/L). These results suggest that at lower lymphocyte and plasma PLP concentrations, there are more apoenzymes in lymphocytes that are activated by increased PLP concentration and support lymphocyte proliferation. It makes sense that a high level of supplementary vitamin B-6 (i.e., 50 mg PN.HC1) would not have a proportionate effect on immunocompetence, and that at higher lymphocyte and plasma PLP levels the effect of PN supplementation on lymphocyte proliferation was smaller than at lower PLP levels, since the available amount of PLP-dependent enzymes in the cells would be a limiting factor. The results of Talbott's and our studies support the view that the effect of vitamin B-6 on immune function is by virtue of a nutritional or physiological role rather than a nonnutritional or pharmacological one. To our knowledge, the persistence of improved immunocompetence after withdrawing a micronutrient supplement has not been reported elsewhere. The study reported here showed that when vitamin B-6 supplementation ended, the increased lymphocyte proliferation in response to mitogen stimulation and IL-2 production 82 gradually decreased, paralleling the decrease in plasma and lymphocyte PLP concentrations (Fig. 4,5,7). It is therefore demonstrated that the effect of a high vitamin B-6 intake on immunocompetence is transitory. These results support our speculation that the increased intake of vitamin B-6 provides the coenzyme for PLP-dependent enzymes of lymphocytes, which, in turn, facilitate the rapid proliferation in response to stimulation. After cessation of the higher intake of vitamin B-6, the plasma and lymphocyte PLP concentration gradually decreased to a lower level. The improved laboratory values for immune function during PN supplementation decreased since the higher plasma and lymphocyte PLP concentrations to support the increased lymphocyte proliferation and IL-2 production were no longer available. If a higher intake of vitamin B-6 improves immunocompetence as indicated by in vitro lymphocyte proliferation and IL-2 production, does this mean the subjects have improved in vivo immune function? Long-term population studies are needed to clarify this question. Two studies which tested the effect of supplementary multiple micronutrients including vitamin B-6 on immune function in humans have been reported. elderly populations. Both studies were carried out in Bogden et al. (9) gave 29 independently living, healthy elderly subjects a daily multiple micronutrient supplement including 3 mg of vitamin 83 B-6 for one year. At the end of one year, the subjects receiving this daily supplement had a significantly increased DHST in response to a panel of seven recall antigens compared with the controls who took a placebo. Bogden et al. concluded that a modest supplement of micronutrients including vitamin B-6 improved cell-mediated immunity in these elderly subjects. Chandra (8) gave 4 8 independently living healthy elderly persons a daily multinutrient supplement which also included 3 mg of vitamin B-6 for one year. At the end of the year, the subjects had a significant improvement in immune indices including percentage of CD4+ cells, NK cells, IL-2 production, IL-2 receptor expression and lymphocyte proliferation in response to PHA stimulation. Additionally, Chandra observed a significantly (p < 0.05) decreased incidence of infectious diseases among the subjects receiving the micronutrient supplement compared with the control group who received a placebo. This in vivo experiment by Chandra suggests that the elderly subjects benefitted from the improved immunocompetence resulting from increased intake of micronutrients including vitamin B-6 by having a lower incidence of infectious diseases. It is recognized that the elderly subjects who took the supplement received vitamins and minerals at the level of approximately 1-2 times of the RDA. Many of these micronutrients such as vitamins A (103), C (104), E (105) and minerals Se (106), Zn (107) and 84 S-carotene (176) have been reported to improve laboratory tests of immune function. Therefore, the improvement of immunocompetence reported in these investigations can not be attributed to any individual nutrient or combination. Population studies with long-term, low dose (i.e., 2-3 mg) vitamin B-6 supplement only are needed to confirm the beneficial effects, such as a lower incidence of infections and/or a higher titer of the antibody in response to immunizations, of a higher vitamin B-6 intake on immune function in humans. Nutrient requirements are currently defined as the amounts of nutrients needed to maintain normal body functions (177). The indices of nutritional status, therefore, are designed to measure if intake of a nutrient equaled output, or if body functions dependent on that nutrient remained "normal". Along with our improved understanding of nutrient functions, a new concept on nutrient requirements has gradually formed: that it is not only desirable to maintain "normal" function but also to maximize body functions. There is a shortage of sophisticated methods to assess nutritional status in terms of maximal function. Potential measurements of maximal function include reproduction, growth and development, behavior and cognitive function and immunocompetence. these measurements, immune function is the most viable direction. Among 85 The immune system is extremely important to health and normal function of the whole body. The immune system is a large component of the body's nonmuscle lean mass. Lymphocytes alone account for approximately 2 per cent of total body weight and 8 per cent of fat-free solids (177). The short life and active metabolism of most immune cells make the immune system very sensitive to variations in nutritional status. The results of the two studies reported here indicate that in healthy young women whose vitamin B-6 intake is adequate according to the RDA and plasma PLP concentration is normal (52), the addition of 1.5 - 2.5 mg vitamin B-6 per day can significantly (p < 0.05) increase the in vitro lymphocyte proliferation in response to mitogen stimulation and IL-2 production, the same as had been reported in elderly (6,7). This suggests that the current RDA for vitamin B-6 may not be the best level for maximizing immune function. Our results also suggest that the indices of immunocompetence may be sensitive measurements of vitamin B-6 and/or other nutrient status. Since many factors affect immune function (103-107,159,166-169), much more work needs to be done before we can practically use immune parameters as indices of nutritional status. The results of our studies and the investigations by Meydani et al. Chandra (8), Bogden et al. (9), Talbott et al. (6), (7) and Miller and Kerkvliet (178) provided an encouraging perspective for using immune function to assess nutritional 86 status. The subjects' mean baseline lymphocyte PLP concentration measured in Study II (Table 8, Fig. 4B) was 0.27 pmol/106 cells. To our knowledge, there has been no report on PLP concentrations in lymphocytes. Early reports revealed that the mean leukocyte PLP concentration in adults (179), children (179) and mice (180) was 0.6, 1.0 and 1.2 pmol/10 cells, respectively, which is in the same order as our results. In response to supplementary PN, lymphocyte PLP concentrations increased in a much flatter pattern than did plasma PLP levels (Table 7,8, Fig. 4). Since PLP can not cross cell membranes directly, its transmembrane movement is mediated through dephosphorylation by a membrane-bound phosphatase followed by rephosphorylation by a cellular pyridoxal kinase (181). When a high intake of vitamin B-6 dramatically increases plasma PLP concentration, lymphocyte PLP concentrations are enhanced. PLP must be in lymphocytes to perform its physiological role in immune response. Lymphocyte PLP concentrations were well correlated with the changes of lymphocyte proliferation in response to mitogen stimulation (to PHA, r = 0.44, p < 0.01); to Con A, r = 0.47, p < 0.01; to PWM, r = 0.45, p < 0.01) and IL-2 production (r = 0.63, p < 0.01) (Fig. 6). Lymphocyte PLP, therefore, could be a good measure for vitamin B-6 nutriture. To measure the in vitro lymphocyte proliferation in 87 response to mitogen stimulation and IL-2 production, the subjects' lymphocytes were put in the culture medium for 48 - 96 hours. It is plausible that components in the culture medium influence the response of lymphocytes to mitogen stimulation. In RPMI 1640, which was used in these experiments, the form of vitamin B-6 is PN (182). However, PNP (PMP) oxidase, an enzyme converting PN to PLP, has been identified only in liver and erythrocytes in humans (i.e., no one has measured this enzyme in lymphocytes) (23,27). If there is no PNP (PMP) oxidase in lymphocytes, then they can not directly use PN as a source of PLP without first converting PN to PLP in the liver or erythrocytes. In fact, the omission of pyridoxine from the culture medium does not significantly alter the response of lymphocytes in the culture medium (183). Ha et al. (93) also found that in a culture medium containing the major part of vitamin B-6 as PN and small amount of PLP, secondary T-cell responses generated in vitro did not recover the impaired cytotoxic response of the T-cells from vitamin B-6 deficient mice. However, Sergeev et al. (184) reported that when PLP was added to the medium, the impaired function of cytotoxic Tcells from vitamin B-6 deficient mice was partially restored. The results of these studies support the view that lymphocytes can not convert PN into PLP in vitro. Short-term mitogen activation and initial growth of activated lymphocytes over a period of 5 days results in 88 about three cell divisions (185). If the lymphocytes have a reserve of PLP, a PLP-free culture medium may still support optimal growth. Therefore, the concentration of PLP in lymphocytes when they are isolated from the subjects is an important factor influencing lymphocyte proliferation in response to mitogen stimulation. A small amount of PLP in our culture medium was from added 10% of FBS. The FBS used in our studies contained 4.8 nmol/L of PLP, which is very low compared with the average PLP concentration in human plasma (normally > 30 nmol/L). It is unlikely that this small amount of PLP in the culture medium could provide enough PLP to affect the difference of PLP in lymphocytes from the subjects of different vitamin B-6 status. However, a complete elimination of PLP and control of all components in the culture medium is the best way of studying the effect of vitamin B-6 on lymphocyte proliferation in in vitro experiments. Serum-free cell culture may provide an ideal tool for further investigation in this area (186,187). PLP is a highly active biomolecule. In in vitro studies, PLP, in concentrations of 0.2 - 0.5 mmol/L, which is much higher than the physiological concentration of plasma PLP, has been reported to inactivate human hepatitis B virus and human immunodeficiency virus (HIV) (189,190). The maximum plasma PLP concentration that could be achieved in response to different amounts of supplementary vitamin B-6 in humans and the possible pharmacological effects of 89 elevated plasma PLP are interesting research topics and worthy of further studies. Talbott et al. (7) reported that elderly people given 50 mg PN.HCl per day for one month, had a plateau of plasma PLP concentration at around 200 nmol/L as long as the subjects continued taking the PN supplement. Speitling et al. (188) found that young healthy males given daily supplement of 40 mg PN.HCl for one day increased their mean plasma PLP concentration from 78 nmol/L to 298 nmol/L. A steady state of about 500 nmol/L of plasma PLP occurred after 4 days of receiving the daily supplement of 40 mg PN.HCl. Coburn et al. (175) reported that in healthy young men a daily supplement of 200 mg PN.HCl for six weeks increased the subjects' mean plasma PLP concentration to 455 nmol/L. In our study, a daily supplement of 50 mg PN.HCl for 3 days increased young women's mean plasma PLP concentration to around 250 nmol/L and remained at this level after 6 days of 50 mg PN.HCl supplement (Fig. 4A). EAST AC did not change significantly in response to two weeks' PN supplement in Study II. EAST AC is considered an indirect long-term indicator of vitamin B-6 nutriture because of the long life span of erythrocyte. Our samples had been stored at -200C for 8 weeks and were thawed and refrozen once due to a freezer malfunction. The nature of EAST AC as a long-term indicator of vitamin B-6 status, long storage time and thawing and refreezing of our samples may all have led to errors in measuring EAST AC in our study. 90 Westrick and Smolen (191) recently reported that EAST and EALT activity in young erythrocytes separated by fractionated centrifugation are better indicators of recent vitamin B-6 status. Polyamines are important for DNA, RNA, and protein synthesis (144). Activation of mammalian cells from a resting to a rapidly dividing state is accompanied by a marked increase in the polyamine content (135). ODC, a PLP- dependent enzyme, is the first and rate-limiting enzyme in polyamine biosynthesis (131). ODC (11 - 90 minutes) The very short half-life of (132) makes this enzyme sensitive to regulatory factors and availability of its coenzyme, PLP. Fillingame et al. observed that inhibiting ODC activity suppressed lymphocyte proliferation in response to Con A and lipopolysaccharide, and, furthermore, this inhibition was correlated with the depletion of intracellular polyamines (147). Adding exogenous polyamines to the culture medium reversed the inhibition of DNA synthesis in Con A stimulated cells by the ODC inhibitor (147). Vitamin B-6 deficient rats had decreased tissue ODC activity and a lower tissue polyamine concentration compared with normal vitamin B-6 status rats (156). In a culture medium, putrescine at concentrations of 2 0 - 25 jimols/L had the strongest stimulatory effect on human (192) and mouse (193) lymphocytes. Higher putrescine concentrations (100 - 200 nmol/L), however, inhibited proliferation of cultured 91 lymphocytes (154,192). Based on these experimental results, we hypothesized that a higher vitamin B-6 intake improves immunocompetence by increasing intracellular PLP concentration, which, in turn, increases ODC activity and polyamine synthesis. Increased concentration of polyamine would optimize lymphocyte proliferation in response to stimulations. If this was true, exogenous polyamines added to the culture medium would compromise the effect of different vitamin B-6 status on the lymphocyte proliferation in response to stimulation. In our experiment, however, the addition of polyamine to the culture medium (0 - 200 nmol/L) had no effect on lymphocyte proliferation in response to mitogen stimulation in both PN and placebo groups (Fig. 8). Recently, Sampson et al. (157) reported that in vitamin B-6 deficient rats, ODC activity in liver, kidney and small intestine tissues actually increased. Further studies such as measuring ODC activity and polyamine concentration in lymphocytes with different B-6 status are needed to clarify our hypothesis. 92 SUMMARY AND CONCLUSIONS The effect of vitamin B-6 on in vitro lymphocyte proliferation and IL-2 production in healthy women (20 - 35 years old) was examined in two studies. The subjects of both studies were healthy and in adequate nutritional status as evaluated by dietary intake, health questionnaire, hemoglobin and hematocrit values. In Study I, a repeated measure design, six subjects received a constant diet supplying o.84 mg (4.96 jimols) vitamin B-6 per day for 12 days, followed by 1.24 (7.33 nmols) and 2.44 mg (14.42 nmols) vitamin B-6 daily during the following two 10-day periods, respectively. Lymphocyte proliferation in response to mitogens Con A and PWM was higher when the subjects received 1.24 mg of vitamin B-6 than when their intake was 0.84 mg of vitamin B-6. But only the response to Con A increased significantly (p < 0.05) . Increasing vitamin B-6 intake to 2.44 mg, which is about 50 % higher than the RDA, significantly (p < 0.05) increased lymphocyte proliferation in response to both mitogens, compared with the response at baseline and 0.84 mg vitamin B-6 intake. In study II, ten subjects, who consumed their selfselected diets throughout this 46-day investigation, were randomly assigned to a PN group and a placebo group (5 subjects each). Their daily mean vitamin B-6 intakes 93 calculated from dietary records were 1.80 mg (10.64 jimols) and 1.96 mg (11.58 nmols) for the PN and placebo groups, respectively. After a 5-day baseline period (no treatment), the PN group received a supplement of 1.5 mg PN.HCl (1.23 mg PN) daily for 7 days, followed by a supplement of 50 mg PN.HCl (41.12 mg PN) daily for the subsequent six days. The daily total mean vitamin B-6 intakes (supplement plus intake from diets) for the PN group during the two time periods of PN supplementation were 3.03 mg (17.91 jimols) and 42.92 mg (253.60 jimols) , respectively. The placebo group received a capsule containing no PN during this period. This supplementary period was followed by a 28-day washout period without a PN supplement or placebo. In the PN group, lymphocyte proliferation in response to mitogens PHA, Con A and PWM, and IL-2 production significantly increased following a daily 1.5 mg PN.HCl supplement compared with the baseline (p < 0.05). Increasing PN.HCl supplementation to 50 mg daily did not further enhance lymphocyte proliferation in response to PHA and PWM. However, when the PN group received a daily supplementation of 50 mg PN.HCl, lymphocyte response to Con A stimulation peaked after 3 days and IL-2 production maximized after 6 days. During the PN supplementation period, the PN group's lymphocyte proliferation and IL-2 production were significantly higher (p < 0.05) than placebo group's. After withdrawing the PN supplement, lymphocyte proliferation in response to mitogen 94 stimulation and IL-2 production gradually decreased. At the end of the 4-week washout period, lymphocyte proliferation in PN group dropped to the baseline level and was not significantly different from the placebo group. IL-2 production in the PN group was still higher at the end of the 28-day washout period compared with the baseline but not significantly different from the placebo group. The 1.5 mg and 50 mg supplement of PN.HC1 significantly (p < 0.05) increased both plasma and lymphocyte PLP concentrations in the PN group. Lymphocyte PLP concentration changed in a flatter pattern in response to PN.HC1 supplement and subsequent withdrawal compared with changes in plasma PLP. Lymphocyte PLP concentrations were significantly correlated with the lymphocyte proliferation and IL-2 production (p < 0.01). The changes of lymphocyte proliferation and IL-2 production were greater at lower lymphocyte PLP concentrations than at higher lymphocyte PLP concentrations. To explore a possible mechanism of the effect of vitamin B-6 on immune function, 2 to 200 nmol/L of putrescine were added to culture medium to assess its effect on lymphocyte proliferation and IL-2 production. However, putrescine added in the culture medium in these concentrations did not affect lymphocyte proliferation and IL-2 production. Further investigation is needed to clarify the relationship between vitamin B-6 status, 95 polyamine metabolism and immune function. The results of the studies reported here demonstrate that a vitamin B-6 intake 1.5-2 times the RDA significantly (p < 0.05) increased in vitro lymphocyte proliferation in response to mitogen stimulation and IL-2 production in healthy young women as had been shown in elderly. The pharmacological level of vitamin B-6 intake (i.e., 50 mg PN.HC1) did not improve these laboratory tests of immune function proportionately. Since lymphocyte PLP concentrations were significantly (p < 0.01) correlated with the lymphocyte proliferation and IL-2 production, this suggests that lymphocyte PLP concentration may be a good indicator of vitamin B-6 status. During the washout period, decreased lymphocyte proliferation in response to mitogen stimulation and IL-2 production paralleled the drop of plasma and lymphocyte PLP concentration. The stimulatory effect of an increased intke of vitamin B-6 on immune function was transitory. 96 BIBLIOGRAPHY 1. Bendich A, and Cohen M. 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