Human and Clinical Nutrition
Cellular Ascorbate Depletion in Healthy Men1 ROBERT A. JACOB, FREDERICK S. PIANALTO AND ROBERT E. AGEE* USDA Western Human NutrÃ-tionResearch Center, Agricultural Research Service, Presidio of San Francisco, CA 94129 and *Department of Surgery, Urology Service, Letterman Army Medical Center, Department of the Army, Presidio of San Francisco, CA 94129 mononuclear levels correlated with plasma ascorbic acid in 41 healthy young and middle-aged adults, and Blanchard et al. (6) recently found that plasma ascorbic acid levels in women were not reliable for predicting polymorphonuclear or mononuclear leu cocyte levels of ascorbic acid within an individual. Wide variation exists in the reported levels of leu cocyte ascorbic acid, and guidelines for interpreting leucocyte ascorbic acid levels have not been stan dardized through controlled studies as for plasma ascorbic acid levels (7). We report here the levels and relationships between plasma, mononuclear leuco cyte, buccal cell and whole semen ascorbic acid for healthy adult men receiving controlled ascorbic acid intakes of 5 to 250 mg/d while residing in a metabolic unit. Reported concentrations of ascorbic acid in semen are some eight- to tenfold higher than in blood plasma (8). Low semen ascorbic acid levels in men have been linked to infertility via increased sperm agglutination and abnormal precursors, and decreased sperm via bility (8). A recent review of the subject suggests that beyond an adequate diet to prevent clinical scurvy, males above age 25 would benefit greatly with regard to sperm qualities with a higher dietary intake of ascorbic acid, especially those under stresses that in crease ascorbic acid turnover, such as smoking, toxic exposures or illness (8). We measured seminal ascorbic acid because little is known about the depletion/repletion dynamics of seminal ascorbic acid in controlled human studies. Because of the previous reports relating ascorbic acid nutriture to male fer tility (8), we also report the effect of controlled ascorbate depletion and repletion on semen and sperm qualities that are routinely determined in clinical fertility examinations.
ABSTRACT To clarify the relationship of plasma ascorbic acid to cellular ascorbic acid levels, we deter mined plasma, lymphocyte, buccal cell and semen ascorbic acid in eight healthy men consuming controlled ascorbic acid intakes of 5, 10, 20, 60 or 250 mg/d over 13 wk while living in a metabolic unit. Levels of ascorbic acid in all four specimen types were significantly lower during the three lowest intakes (5, 10, or 20 mg/d) compared with the 60 or 250 mg/d intakes, but only plasma and lymphocyte ascorbic acid levels discrimi nated between these intakes unequivocally and with no overlap. Priority for maintenance of intracellular lym phocyte ascorbic acid was indicated by rapid repletion of lymphocytes compared with plasma and semen at 60 mg/d intake. Strong correlations of plasma with lym phocyte ascorbic acid within individuals indicated that plasma levels would reliably reflect low lymphocyte levels in nutrition monitoring surveys. Buccal cell ascorbic acid may be useful as a noninvasive screening test for ascorbic acid deficiency. Semen and sperm qual ities were unchanged despite an average decline in semen ascorbic acid to 24% of baseline. Short-term ascorbic acid depletion in healthy men did not adversely affect sperm qualities related to fertility nor did mod erate supplementation improve them. J. Nutr. 122: 1111-1118, 1992. INDEXING KEY WORDS: • vitamin C •ascorbic acid •nutritional status assessment •humans
•fertility
Many of the important functions of ascorbic acid exist intracellularly; therefore it is desirable that a test of ascorbic acid status relate to cellular or tissue ascorbic acid levels in a predictable manner. Plasma ascorbic acid determination is a convenient test for assessing ascorbic acid status, yet its relation to cel lular ascorbic acid levels is not clear. In some experi mental (1-3) and population (4) studies, plasma ascorbic acid correlated well with leucocyte levels,however, Evans et al. (5) found that polymorphonuclear leucocyte ascorbic acid levels but not
'Reference to a company or product name does not imply approval or recommendation of the product by the U.S. Department of Agriculture to the exclusion of others that may be suitable.
0022-3166/92 $3.00 ©1992 American Institute of Nutrition. Received 15 July 1991. Accepted 3 December 1991. 1111 Downloaded from https://academic.oup.com/jn/article-abstract/122/5/1111/4754823 by Washington University, Law School Library user on 21 May 2018
JACOB ET AL.
1112
TABLE 1 Ascorbic
Metabolic period1234Lengthd4322828Ascorbic
acid levels of healthy men after periods
acid intakemg/d25025102010
206025060 or
of various ascorbic acid intakes*
leucocytenmol/108
cellspmol/mg
cells209
protein41
3.3a6.6 ±
9a117±
14a10 ±
0.3b5.3 ±
73b87 ±
2a9±
0.35.9 ± 0.55.5 ± 0.3b26.7 ±
995 ± 591 ± 5C201±
412± 510± 3a77±
8.864.2 ± 2217 ± 4.550.1 ± 11211± ± 7an77347224Buccal or 250a88448358Plasma\imol/L59.6 ±7.8aMononuclear
4070± 473 ± ±17
Values for ascorbic acid levels are mean ±SEMat the end of each period; n = number of subjects. Means within vertical columns not sharing the same superscript letter are significantly different by paired t test. Superscripts were derived from multiple independently run paired i tests where n > 5, i.e., 6 paired t tests for plasma and leucocyte ascorbic acid columns with P < 0.05/6 - 0.0083 considered significant. 2Subjects consumed a supplement of 250 mg ascorbic acid/d in addition to their free-living diet, for 1 to 2 wk before entering the study. 3n - 7, one value deleted due to platelet contamination.
MATERIALS AND METHODS Protocol and subjects. Twelve healthy nons moking male volunteer subjects, ages 25-43 y, were admitted to the metabolic unit of the USDA, Agricul tural Research Service Western Human Nutrition Re search Center (WHNRC), after medical and psycho logical screening. The study protocol and informed consent were approved by the Institutional Review Committee of the Letterman Army Medical Center, Department of the Army, Presidio of San Francisco, CA, and by the Human Studies Review Committee of the Agricultural Research Service, U.S. Department of Agriculture. For the duration of the 13-wk (92-d) study, subjects lived in and ate all meals in the WHNRC metabolic unit and were chaperoned at all times when outside the unit. Because of violations of the study protocol, or for personal reasons, five subjects left the study early. Three subjects left after 5 wk and one after 9 wk. Data from these four subjects were not included in this report. A fifth subject left the study after 10.3 wk, during the final repletion period. This subject was given supplemental tablets of 250 mg of ascorbic acid and was instructed to take one each day along with his regular diet. The subject remained outside the unit and came back to the unit at the end of the study when blood and semen but not buccal cells were obtained. Data from this subject were included along with the seven subjects who completed the entire study. Experimental design and diet. All subjects were fed the same ascorbic acid deficient diet throughout the study. The experimental variable was the amount of ascorbic acid supplemented into the diet, as shown Downloaded from https://academic.oup.com/jn/article-abstract/122/5/1111/4754823 by Washington University, Law School Library user on 21 May 2018
in Table 1. During Periods 3 and 4, subjects were split into two groups, receiving 10 or 20 mg ascorbic acid/d in Period 3 and 60 or 250 mg/d in Period 4. Subjects were placed in the groups so that the group averages of body weight and leucocyte ascorbic acid content were not significantly different at the start of the period. Subjects ate a 4-d rotating diet that excluded fruits, vegetables, or their juices in any form. The diet was consumed at three meals and an evening (2100 h) snack daily. The diet contained -14 oz of low fat milk daily,- beef, chicken, turkey, tuna and cheese as entrees; pasta, rice and bread as starches (no potatoes); and cookies, cakes and ice creams as desserts. As calculated from food nutrient composition tables, the diet provided 52% of energy from carbohydrates, 31% from fat (polyunsaturated/saturated fatty acid ratio of 0.33), 17% from protein, and >80% of the Recom mended Dietary Allowance (RDA) of all essential nutrients except ascorbic acid. The ascorbic acid content of the diet averaged 5.1 mg/d (range of 4.8-5.3 mg/d for the four daily menus) as calculated from food composition tables and 1.6 mg/d of ascorbic acid (reduced form) as analyzed by HPLC (range of 0.9-2.2 mg/d for four daily menus). The higher values ob tained from food composition tables are presumably due to their inclusion of the oxidized form of the vitamin, dehydroascorbic acid, which is biologically available, and to some conversion of ascorbic acid to dehydroascorbic acid during processing of the mixed diets before determination of ascorbic acid. Water was freely consumed and consistent amounts of mineral water, soda pop (noncaloric), coffee (decaffeinated) and tea with no appreciable ascorbic acid content were consumed. No aspirin was
CELLULAR ASCORBATE
given as an analgesic; however, preparations con taining ibuprofen or acetaminophen were given on an as-needed basis. The ascorbic acid intake was varied by including with each meal 100 mL of citrus-flavored soda con taining the proper amount of ascorbic acid to provide 245 [249] mg/d of ascorbic acid in Period 1, no ascorbic acid [nondetectable] in Period 2, 5 [4.4] or 15 [13.7] mg/d in Period 3, and 55 [53.2] or 245 [249] mg/ d in Period 4 (values in brackets represent analytical determinations of ascorbic acid in milligrams per 300 mL of soda as explained below). The citrus-flavored soda with added ascorbic acid was prepared the day before serving and kept at 4°Cuntil 100-mL aliquots were served at each of the three daily meals. In the same time frame and manner that aliquots were taken for serving to the subjects, separate 100-mL aliquots of the ascorbic acid-supplemented soda were com bined once during each period to provide 300 mL of soda for ascorbic acid determination by HPLC. The aliquots were added to an equal volume of a solution of cold 100 g/L metaphosphoric acid with 0.54 mmol/ L N32EDTA to preserve the ascorbic acid until analysis. The ascorbic acid used for supplementation was Ascorbic Acid USP FCC, Fine Powder, code #604565200 (Hoffmann-La Roche, Nutley, NJ). On study d 73, during the Period 4 repletion, one subject received 500 mg intravenous ascorbic acid as emer gency treatment for an apparent cardiac arrythmia that was fleeting and anomalous. The subject returned to the regular study regimen the next day and completed the study with no further complica tions. Baseline body weight for each subject (range = 63-90 kg) was established as the average weight over study d 3 to 7. Body weights throughout the study were maintained within ±2%of the baseline weight by altering portion sizes of the diet served. Specimen collections and analytical methods. Fasting blood was taken at 0700-0800 h by venipuncture 10 times throughout the study for bio chemical determinations. Semen was collected after breakfast by masturbation at the end of each of the four experimental periods. Complete 24-h urine col lections were taken throughout. Mononuclear leucocytes (mostly lymphocytes) were isolated from 5 mL of blood with LeucoPREP Cell Separation Tubes (Becton Dickinson, Lincoln Park, NJ). The cells were rinsed twice with chilled PBS solution containing 0.54 mmol/L Na2EDTA, fol lowed by centrifugation after each rinse. The final pellet was resuspended in 1 mL of PBS solution, and a portion was removed for counting of mononuclear, polymorphonuclear and platelet cells with an auto mated differential cell counter. The remaining cellPBS mixture was recentrifuged and a chilled 20 g/L metaphosphoric acid and 0.54 mmol/L Na2EDTA so lution was added to the pellet to extract and stabilize from https://academic.oup.com/jn/article-abstract/122/5/1111/4754823
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DEPLETION
1113
the ascorbic acid. The sample was vortexed and stored at -70°Cuntil analysis. Semen samples were taken within 10 min of col lection to the pathology laboratory of the Letterman Army Medical Center where the following tests were performed by standard procedures for fertility testing: semen liquefaction time, volume, pH and sperm count, motility (percent motile and activity rated 1-4) and morphology (percentage of normal forms). After fertility tests were completed, the remaining semen specimen was treated to preserve ascorbic acid. Whole semen and blood plasma samples were added to an equal volume of a 100 g/L metaphosphoric acid and 0.54 mmol/L Na2EDTA solution to stabilize ascorbic acid. The samples were centrifuged to remove precipi tated proteins and the supernatant was stored at -70°C. Buccal cells were collected with a soft toothbrush after the subjects rinsed their mouths with distilled water. The cells were removed by gently brushing 20 strokes on each side of the mouth. The toothbrush was rinsed in chilled PBS solution to remove the cells and the wash was centrifuged. The cell pellet was rewashed, and a chilled 4 g/L metaphosphoric acid, 0.54 mmol/L Na2EDTA solution was added to the final pellet to extract the ascorbic acid. The mixture was vortexed and stored at -70°C.Once thawed, the buccal cell samples were mixed well and a 20-uL portion was removed for protein analysis [a modified Lowry method (9) with comparison to bovine serum albumin]. The remaining mixture was centrifuged, filtered and analyzed for ascorbic acid without any further dilutions. Each of the four rotated daily diets was analyzed once during the study for ascorbic acid. The diets were homogenized for 2 min in 400 mL of a cold 200 g/L metaphosphoric acid, 0.54 mmol/L Na2EDTA so lution and the supernate analyzed immediately. Ascorbic acid was analyzed by HPLC separation and electrochemical detection using the method of Kutnink et al. (10). Only the reduced form of the vitamin was measured. Isoascorbic acid was added as an internal standard to all samples (except the diets) with the metaphosphoric acid/Na2EDTA preservative before freezing. Isoascorbic acid was added to the diet samples in an extra dilution just before ascorbic acid analysis. Before injection, all samples except the buccal cells were diluted 25-fold with a 1.04 mmol/L cysteine hydrochloride, 0.54 mmol/L Na2EDTA solu tion. Ascorbic acid was ion-paired and separated with a reversed-phase column and acetate mobile phase. The sampling size was 50 uL for the mononuclear leucocytes, plasma, semen, diets and supplements, and 200 uL for buccal cells. Total vitamin C (ascorbic acid plus dehydroascorbic acid) was determined in 24-h urine collections by the spectrophotometric procedure using 2,4dinitrophenylhydrazine as chromogen (11).
JACOB ET AL.
1114 Rn—
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DayFIGURE
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eighhealthy 2 Urinary excretion of vitamin C from ove92 men receiving varying intakes of ascorbic acid SEM).*,in d (values are means ±
^60Ascorbic
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-Po 100-II^ o
-8io20Q/ -B-
90% of the leucocyte cytes and platelets, but no increase in mononuclear ascorbic acid variance in halt of the subjects and leucocyte ascorbic acid levels. The implication is that the effect of ascorbic acid supplementation on blood cell ascorbic acid levels differs with the cell type and that ascorbic acid intakes above 60 mg/d may in i '00 crease intracellular ascorbic acid content of polymorAscorbic Acid 5 • u Intake, mq/d 20 A phonuclear leucocytes and platelets more effectively 60 D .85 250-1 250 O than mononuclear cells. Another interpretation is that the mononuclear cells become saturated with O o 200 ascorbic acid at a lower level of dietary intake than O) C other blood cells. In this study, ascorbic acid levels in l_ 'u T3 O 5 150mononuclear leucocytes were repleted to baseline N=10 with 60 mg/d of ascorbic acid intake for 4 wk, R2=0.970 whereas plasma ascorbic acid levels remained low 100 (Fig. 1), and the repletion of semen ascorbic acid was lowest in the one subject repleted with 60 mg/d (Fig. 4? 50 2.7 10.0 100.0 4). These results, along with the minimal urinary ascorbic acid excretion observed at 60 mg/d intake Plasma Ascorbic Acid, /¿mol/L (Fig. 2) show that the conservation and transport of FIGURE 5 Linear regression of mononuclear leucocyte ascorbic acid into lymphocytes is particularly ef ascorbic acid vs. plasma ascorbic acid (log scale) for subject fective and commands a high metabolic priority. 2.
s¡
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CELLULAR ASCORBATE DEPLETION
>75% of the leucocyte variance in seven of eight subjects. These correlations include some points at which the ascorbic acid levels were not at steady state because of changes in ascorbic acid intake from one experimental period to the next. The results show that the plasma ascorbic acid level is useful for pre dicting the mononuclear leucocyte level across a range of ascorbic acid intakes from deficient to plasma saturating. This suggests that plasma ascorbic acid levels measured in population surveys would reliably reflect low leucocyte ascorbic acid levels that resulted from deficient ascorbic acid intakes. Buccal cell ascorbate. We measured buccal cell ascorbic acid to determine whether the test might provide a noninvasive specimen for estimating cel lular ascorbic acid levels in nutrition surveys. Al though buccal cell ascorbic acid levels were signifi cantly lower in subjects receiving the low ascorbic acid intakes (5, 10 or 20 mg/d) compared with the repletion intakes (60 or 250 mg/d), there was some overlap in the ranges (3-24 vs. 17-122 pmol/mg pro tein). Buccal cell ascorbic acid did not differentiate between ascorbic acid intakes within the low range or within the repletion range. Over the entire range of ascorbic acid intakes, linear regression showed that buccal cell ascorbic acid correlated significantly (P < 0.01) with ascorbic acid levels in plasma (r = +0.616), mononuclear leucocytes (+0.605) and semen (+0.779). Buccal cell ascorbic acid distinguished between ascorbic acid-depleted and ascorbic acid-repleted in dividuals in this controlled study reasonably well, but does not seem suitable as a precise marker of ascorbic acid status among groups with similar intakes of the vitamin. The results suggest that buccal cell ascorbic acid might be useful as a noninvasive screening test for ascorbic acid deficiency, but not as a quantitative measure of ascorbic acid status over a broad range of population intakes. However, further work seems warranted on improving method sensitivity, stan dardizing the technique and assessing its validity in free-living populations. Seminal ascorbate and fertility measures. Antagglutins in male and female reproductive tissues act to prevent sperm agglutination that may limit sperm motility (18). A male antagglutin protein in seminal plasma contains active carbohydrate and sulfate residues and is active in the reduced form (18). Re ducing agents such as ascorbic acid and glutathione enhance biological antagglutin activity or restore ac tivity to oxidized antagglutins (18). In vitro studies with normal mature sperm (18, 19) and studies of scorbutic guinea pigs (20) suggest that ascorbic acid deficiency adversely affects sperm count, morphology, motility and viability. These effects have been at tributed to increased sperm agglutination in ascorbic acid-deficient semen (18, 19) and to scorbutic abnor malities of testicular and other male reproductive tissues (20). Supplementation with 1000 mg/d of Downloaded from https://academic.oup.com/jn/article-abstract/122/5/1111/4754823 by Washington University, Law School Library user on 21 May 2018
1117
ascorbic acid for 10 d, then 500 mg/d for 60 d, resulted in 100% impregnation success for 20 infertile men compared with no success with an unsupplemented control group of 10 infertile men (21). In a follow-up study, Dawson et al. (21) found that in 20 men (age 25-45 y), with sperm agglutination >25%, supplementation with 200 or 1000 mg/d of ascorbic acid produced significant increases in seminal plasma ascorbic acid levels within 1 wk, and improvements in sperm count, viability, motility, agglutination and morphology parameters within 2 wk. Therefore, changes in seminal ascorbic acid levels may affect some sperm properties such as agglutination and mo tility within weeks, even though the maturation period for sperm is approximately 90 d. Despite the average 61 and 76% declines in semen ascorbic acid in our experimental subjects during Periods 2 and 3, we found no significant changes in the sperm prop erties related to male fertility listed in Table 2, nor in semen volume, pH or liquefaction time. It should be noted that the men enrolled in the study of Dawson et al. (21) were undergoing fertility testing and were chosen to have high initial sperm agglutination (>25%), whereas our men where chosen to be healthy, were screened for normal reproductive function and showed a clinically normal semen analysis at the start of the study. Our results suggest, however, that in healthy men, short-term ascorbate depletion does not adversely affect semen and sperm qualities, nor does moderate ascorbic acid supplementation improve them.
ACKNOWLEDGMENTS The authors thank the Jones Operation and Main tenance Company (Charlotte, NC) for providing support in operating the metabolic unit, especially Edward Blonz, project manager; Louise Hoppe and staff for providing nursing services; and Esther Kwan and staff for dietary services. The authors also thank Jeffre Johnson, manager of the Bioanalytical Support Laboratory, and his staff for providing laboratory sup port; Mei Miau Wu, UCLA School of Public Health, Department of Biostatistics, for statistical analysis support; Ramona Simpson and staff of the Hematology Laboratory, Letterman Army Medical Center (Presidio of San Francisco) for performing male fer tility tests,- and Hoffmann-La Roche Inc., Roche Chemical Division (Nutley, NJ) for providing USP ascorbic acid powder for diet supplementation.
LITERATURE CITED 1. Omaye, S. T., Schaus, E. E., Kutnink, M. A. & Hawkes, W. C. (1987) Measurement of vitamin C in blood components by high-performance liquid chromatography. Implication in as-
1118
JACOB ET AL.
sessing vitamin C status. Ann. N.Y. Acad. Sci. 498: 389-401. 2. Jacob, R. A., Skala, J. H. & Omaye, S. T. (1987) Biochemical indices of human vitamin C status. Am. J. Clin. Nutr. 46: 818-826. 3. Sauberlich, H. E., Kreisch, M. J., Taylor, P. C, Johnson, H. L. & Skala, J. H. (1989) Ascorbic acid and erythorbic acid metab olism in nonpregnant women. Am. J. Clin. Nutr. 50: 1039-1049. 4. Bates, C. J., Rutishauser, I.H.E., Black, A. E. & Paul, A. A. (1977) Long term vitamin status and dietary intake of healthy elderly subjects: 2. Vitamin C. Br. J. Nutr. 42: 43-55. 5. Evans, R. M., Currie, L. & Campbell, A. (1982) The distri bution of ascorbic acid between various cellular components of blood in normal individuals, and its relation to the plasma concentration. Br. J. Nutr. 47: 473-482. 6. Blanchard, J., Conrad, K. A., Watson, R. R., Barry, P. J. & Crawley, J. D. (1990) Comparison of plasma, mononuclear and polymorphonuclear leucocyte vitamin C levels in young and elderly women during depletion and supplementation. Eur. J. Clin. Nutr. 43: 97-106. 7. Jacob, R. A. (1990) Assessment of human vitamin C status. J. Nutr. 120: 1480-1485. 8. Dawson, E. B., Harris, W. A. & Powell, L. C. (1990) Rela tionship between ascorbic acid and male fertility. In: Aspects of Some Vitamins, Minerals and Enzymes in Health and Disease (Bourne, G. H., ed.). World Rev. Nutr. Diet. 62: 1-26. 9. Peterson, G. L. (1977) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 83: 346-356. 10. Kutnink, M. A., Hawkes, W. C., Schaus, E. E. & Omaye, S. T. (1987) An internal standard method for the unattended highperformance liquid Chromatographie analysis of ascorbic acid in blood components. Anal. Biochem. 166: 424—430. 11. Omaye, S. T., Turnbull, J. D. &. Sauberlich, H. E. (1979)
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12.
13.
14.
15.
16.
17. 18. 19.
20.
21.
Selected methods for the determination of ascorbic acid in animal cells, tissues, and fluids. In: Methods in Enzymology. (McCormick, D. B. & Wright, L. D., eds.), pp. 3-11. Academic Press, New York, NY. Vanderjagt, D. J., Garry, P. J. &. Bhagavan, H. N. (1989) Ascorbate and dehydroascorbate: distribution in mononuclear cells of healthy elderly people. Am. J. Clin. Nutr. 49: 511-516. Yew, M. S. (1984) Megadose vitamin C supplementation and ascorbic acid and dehydroascorbic acid levels in plasma and lymphocytes. Nutr. Rep. Int. 30: 597-601. Ludvigsson, J., Hansson, L. O. & Stendali!, O. (1979) The effect of large doses of vitamin C on leukocyte function and some laboratory parameters. Int. J. Vit. Nutr. Res. 49: 160-165. Bergsten, P., Amitai, G., Kehrk, J., Dhariwal, K. R., Klein, H. G. & Levine, M. (1990) Millimolar concentrations of ascorbic acid in purified human mononuclear leukocytes. J. Biol. Chem. 265: 2584-2585. Washko, P., Rotrosen, D. & Levine, M. (1989) Ascorbic acid transport and accumulation in human neutrophils. J. Biol. Chem. 264: 18996-19002. Moser, U. (1987) Uptake of ascorbic acid by leukocytes. Ann. N.Y. Acad. Sci. 498: 200-215. Lindan!, P. E. (I960) Some factors influencing the biological activity of sperm agglutinins. J. Reprod. Fértil.1: 3-22. Lindahl, P. E. (1966) Sperm agglutinating and anti-agglutinating factors in normal follicular fluid from cattle. Int. J. Fértil.11: 297-305. Chinoy, N. J., Mehta, R. R., Seethalakshmi, L., Sharma, J. D. & Chinoy, M. R. (1986) Effects of vitamin C deficiency on phys iology of male reproductive organs of guinea pigs. Int. J. Fértil. 31: 232-239. Dawson, E. B., Harris, W. A., Rankin, W. E., Charpentier, L. A. & McGanity, W. J. (1987) Effect of ascorbic acid on male fertility. Ann. N.Y. Acad. Sci. 498: 312-323.
Cellular Ascorbate Depletion in Healthy Men1 ROBERT A. JACOB, FREDERICK S. PIANALTO AND ROBERT E. AGEE* USDA Western Human NutrÃ-tionResearch Center, Agricultural Research Service, Presidio of San Francisco, CA 94129 and *Department of Surgery, Urology Service, Letterman Army Medical Center, Department of the Army, Presidio of San Francisco, CA 94129 mononuclear levels correlated with plasma ascorbic acid in 41 healthy young and middle-aged adults, and Blanchard et al. (6) recently found that plasma ascorbic acid levels in women were not reliable for predicting polymorphonuclear or mononuclear leu cocyte levels of ascorbic acid within an individual. Wide variation exists in the reported levels of leu cocyte ascorbic acid, and guidelines for interpreting leucocyte ascorbic acid levels have not been stan dardized through controlled studies as for plasma ascorbic acid levels (7). We report here the levels and relationships between plasma, mononuclear leuco cyte, buccal cell and whole semen ascorbic acid for healthy adult men receiving controlled ascorbic acid intakes of 5 to 250 mg/d while residing in a metabolic unit. Reported concentrations of ascorbic acid in semen are some eight- to tenfold higher than in blood plasma (8). Low semen ascorbic acid levels in men have been linked to infertility via increased sperm agglutination and abnormal precursors, and decreased sperm via bility (8). A recent review of the subject suggests that beyond an adequate diet to prevent clinical scurvy, males above age 25 would benefit greatly with regard to sperm qualities with a higher dietary intake of ascorbic acid, especially those under stresses that in crease ascorbic acid turnover, such as smoking, toxic exposures or illness (8). We measured seminal ascorbic acid because little is known about the depletion/repletion dynamics of seminal ascorbic acid in controlled human studies. Because of the previous reports relating ascorbic acid nutriture to male fer tility (8), we also report the effect of controlled ascorbate depletion and repletion on semen and sperm qualities that are routinely determined in clinical fertility examinations.
ABSTRACT To clarify the relationship of plasma ascorbic acid to cellular ascorbic acid levels, we deter mined plasma, lymphocyte, buccal cell and semen ascorbic acid in eight healthy men consuming controlled ascorbic acid intakes of 5, 10, 20, 60 or 250 mg/d over 13 wk while living in a metabolic unit. Levels of ascorbic acid in all four specimen types were significantly lower during the three lowest intakes (5, 10, or 20 mg/d) compared with the 60 or 250 mg/d intakes, but only plasma and lymphocyte ascorbic acid levels discrimi nated between these intakes unequivocally and with no overlap. Priority for maintenance of intracellular lym phocyte ascorbic acid was indicated by rapid repletion of lymphocytes compared with plasma and semen at 60 mg/d intake. Strong correlations of plasma with lym phocyte ascorbic acid within individuals indicated that plasma levels would reliably reflect low lymphocyte levels in nutrition monitoring surveys. Buccal cell ascorbic acid may be useful as a noninvasive screening test for ascorbic acid deficiency. Semen and sperm qual ities were unchanged despite an average decline in semen ascorbic acid to 24% of baseline. Short-term ascorbic acid depletion in healthy men did not adversely affect sperm qualities related to fertility nor did mod erate supplementation improve them. J. Nutr. 122: 1111-1118, 1992. INDEXING KEY WORDS: • vitamin C •ascorbic acid •nutritional status assessment •humans
•fertility
Many of the important functions of ascorbic acid exist intracellularly; therefore it is desirable that a test of ascorbic acid status relate to cellular or tissue ascorbic acid levels in a predictable manner. Plasma ascorbic acid determination is a convenient test for assessing ascorbic acid status, yet its relation to cel lular ascorbic acid levels is not clear. In some experi mental (1-3) and population (4) studies, plasma ascorbic acid correlated well with leucocyte levels,however, Evans et al. (5) found that polymorphonuclear leucocyte ascorbic acid levels but not
'Reference to a company or product name does not imply approval or recommendation of the product by the U.S. Department of Agriculture to the exclusion of others that may be suitable.
0022-3166/92 $3.00 ©1992 American Institute of Nutrition. Received 15 July 1991. Accepted 3 December 1991. 1111 Downloaded from https://academic.oup.com/jn/article-abstract/122/5/1111/4754823 by Washington University, Law School Library user on 21 May 2018
JACOB ET AL.
1112
TABLE 1 Ascorbic
Metabolic period1234Lengthd4322828Ascorbic
acid levels of healthy men after periods
acid intakemg/d25025102010
206025060 or
of various ascorbic acid intakes*
leucocytenmol/108
cellspmol/mg
cells209
protein41
3.3a6.6 ±
9a117±
14a10 ±
0.3b5.3 ±
73b87 ±
2a9±
0.35.9 ± 0.55.5 ± 0.3b26.7 ±
995 ± 591 ± 5C201±
412± 510± 3a77±
8.864.2 ± 2217 ± 4.550.1 ± 11211± ± 7an77347224Buccal or 250a88448358Plasma\imol/L59.6 ±7.8aMononuclear
4070± 473 ± ±17
Values for ascorbic acid levels are mean ±SEMat the end of each period; n = number of subjects. Means within vertical columns not sharing the same superscript letter are significantly different by paired t test. Superscripts were derived from multiple independently run paired i tests where n > 5, i.e., 6 paired t tests for plasma and leucocyte ascorbic acid columns with P < 0.05/6 - 0.0083 considered significant. 2Subjects consumed a supplement of 250 mg ascorbic acid/d in addition to their free-living diet, for 1 to 2 wk before entering the study. 3n - 7, one value deleted due to platelet contamination.
MATERIALS AND METHODS Protocol and subjects. Twelve healthy nons moking male volunteer subjects, ages 25-43 y, were admitted to the metabolic unit of the USDA, Agricul tural Research Service Western Human Nutrition Re search Center (WHNRC), after medical and psycho logical screening. The study protocol and informed consent were approved by the Institutional Review Committee of the Letterman Army Medical Center, Department of the Army, Presidio of San Francisco, CA, and by the Human Studies Review Committee of the Agricultural Research Service, U.S. Department of Agriculture. For the duration of the 13-wk (92-d) study, subjects lived in and ate all meals in the WHNRC metabolic unit and were chaperoned at all times when outside the unit. Because of violations of the study protocol, or for personal reasons, five subjects left the study early. Three subjects left after 5 wk and one after 9 wk. Data from these four subjects were not included in this report. A fifth subject left the study after 10.3 wk, during the final repletion period. This subject was given supplemental tablets of 250 mg of ascorbic acid and was instructed to take one each day along with his regular diet. The subject remained outside the unit and came back to the unit at the end of the study when blood and semen but not buccal cells were obtained. Data from this subject were included along with the seven subjects who completed the entire study. Experimental design and diet. All subjects were fed the same ascorbic acid deficient diet throughout the study. The experimental variable was the amount of ascorbic acid supplemented into the diet, as shown Downloaded from https://academic.oup.com/jn/article-abstract/122/5/1111/4754823 by Washington University, Law School Library user on 21 May 2018
in Table 1. During Periods 3 and 4, subjects were split into two groups, receiving 10 or 20 mg ascorbic acid/d in Period 3 and 60 or 250 mg/d in Period 4. Subjects were placed in the groups so that the group averages of body weight and leucocyte ascorbic acid content were not significantly different at the start of the period. Subjects ate a 4-d rotating diet that excluded fruits, vegetables, or their juices in any form. The diet was consumed at three meals and an evening (2100 h) snack daily. The diet contained -14 oz of low fat milk daily,- beef, chicken, turkey, tuna and cheese as entrees; pasta, rice and bread as starches (no potatoes); and cookies, cakes and ice creams as desserts. As calculated from food nutrient composition tables, the diet provided 52% of energy from carbohydrates, 31% from fat (polyunsaturated/saturated fatty acid ratio of 0.33), 17% from protein, and >80% of the Recom mended Dietary Allowance (RDA) of all essential nutrients except ascorbic acid. The ascorbic acid content of the diet averaged 5.1 mg/d (range of 4.8-5.3 mg/d for the four daily menus) as calculated from food composition tables and 1.6 mg/d of ascorbic acid (reduced form) as analyzed by HPLC (range of 0.9-2.2 mg/d for four daily menus). The higher values ob tained from food composition tables are presumably due to their inclusion of the oxidized form of the vitamin, dehydroascorbic acid, which is biologically available, and to some conversion of ascorbic acid to dehydroascorbic acid during processing of the mixed diets before determination of ascorbic acid. Water was freely consumed and consistent amounts of mineral water, soda pop (noncaloric), coffee (decaffeinated) and tea with no appreciable ascorbic acid content were consumed. No aspirin was
CELLULAR ASCORBATE
given as an analgesic; however, preparations con taining ibuprofen or acetaminophen were given on an as-needed basis. The ascorbic acid intake was varied by including with each meal 100 mL of citrus-flavored soda con taining the proper amount of ascorbic acid to provide 245 [249] mg/d of ascorbic acid in Period 1, no ascorbic acid [nondetectable] in Period 2, 5 [4.4] or 15 [13.7] mg/d in Period 3, and 55 [53.2] or 245 [249] mg/ d in Period 4 (values in brackets represent analytical determinations of ascorbic acid in milligrams per 300 mL of soda as explained below). The citrus-flavored soda with added ascorbic acid was prepared the day before serving and kept at 4°Cuntil 100-mL aliquots were served at each of the three daily meals. In the same time frame and manner that aliquots were taken for serving to the subjects, separate 100-mL aliquots of the ascorbic acid-supplemented soda were com bined once during each period to provide 300 mL of soda for ascorbic acid determination by HPLC. The aliquots were added to an equal volume of a solution of cold 100 g/L metaphosphoric acid with 0.54 mmol/ L N32EDTA to preserve the ascorbic acid until analysis. The ascorbic acid used for supplementation was Ascorbic Acid USP FCC, Fine Powder, code #604565200 (Hoffmann-La Roche, Nutley, NJ). On study d 73, during the Period 4 repletion, one subject received 500 mg intravenous ascorbic acid as emer gency treatment for an apparent cardiac arrythmia that was fleeting and anomalous. The subject returned to the regular study regimen the next day and completed the study with no further complica tions. Baseline body weight for each subject (range = 63-90 kg) was established as the average weight over study d 3 to 7. Body weights throughout the study were maintained within ±2%of the baseline weight by altering portion sizes of the diet served. Specimen collections and analytical methods. Fasting blood was taken at 0700-0800 h by venipuncture 10 times throughout the study for bio chemical determinations. Semen was collected after breakfast by masturbation at the end of each of the four experimental periods. Complete 24-h urine col lections were taken throughout. Mononuclear leucocytes (mostly lymphocytes) were isolated from 5 mL of blood with LeucoPREP Cell Separation Tubes (Becton Dickinson, Lincoln Park, NJ). The cells were rinsed twice with chilled PBS solution containing 0.54 mmol/L Na2EDTA, fol lowed by centrifugation after each rinse. The final pellet was resuspended in 1 mL of PBS solution, and a portion was removed for counting of mononuclear, polymorphonuclear and platelet cells with an auto mated differential cell counter. The remaining cellPBS mixture was recentrifuged and a chilled 20 g/L metaphosphoric acid and 0.54 mmol/L Na2EDTA so lution was added to the pellet to extract and stabilize from https://academic.oup.com/jn/article-abstract/122/5/1111/4754823
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DEPLETION
1113
the ascorbic acid. The sample was vortexed and stored at -70°Cuntil analysis. Semen samples were taken within 10 min of col lection to the pathology laboratory of the Letterman Army Medical Center where the following tests were performed by standard procedures for fertility testing: semen liquefaction time, volume, pH and sperm count, motility (percent motile and activity rated 1-4) and morphology (percentage of normal forms). After fertility tests were completed, the remaining semen specimen was treated to preserve ascorbic acid. Whole semen and blood plasma samples were added to an equal volume of a 100 g/L metaphosphoric acid and 0.54 mmol/L Na2EDTA solution to stabilize ascorbic acid. The samples were centrifuged to remove precipi tated proteins and the supernatant was stored at -70°C. Buccal cells were collected with a soft toothbrush after the subjects rinsed their mouths with distilled water. The cells were removed by gently brushing 20 strokes on each side of the mouth. The toothbrush was rinsed in chilled PBS solution to remove the cells and the wash was centrifuged. The cell pellet was rewashed, and a chilled 4 g/L metaphosphoric acid, 0.54 mmol/L Na2EDTA solution was added to the final pellet to extract the ascorbic acid. The mixture was vortexed and stored at -70°C.Once thawed, the buccal cell samples were mixed well and a 20-uL portion was removed for protein analysis [a modified Lowry method (9) with comparison to bovine serum albumin]. The remaining mixture was centrifuged, filtered and analyzed for ascorbic acid without any further dilutions. Each of the four rotated daily diets was analyzed once during the study for ascorbic acid. The diets were homogenized for 2 min in 400 mL of a cold 200 g/L metaphosphoric acid, 0.54 mmol/L Na2EDTA so lution and the supernate analyzed immediately. Ascorbic acid was analyzed by HPLC separation and electrochemical detection using the method of Kutnink et al. (10). Only the reduced form of the vitamin was measured. Isoascorbic acid was added as an internal standard to all samples (except the diets) with the metaphosphoric acid/Na2EDTA preservative before freezing. Isoascorbic acid was added to the diet samples in an extra dilution just before ascorbic acid analysis. Before injection, all samples except the buccal cells were diluted 25-fold with a 1.04 mmol/L cysteine hydrochloride, 0.54 mmol/L Na2EDTA solu tion. Ascorbic acid was ion-paired and separated with a reversed-phase column and acetate mobile phase. The sampling size was 50 uL for the mononuclear leucocytes, plasma, semen, diets and supplements, and 200 uL for buccal cells. Total vitamin C (ascorbic acid plus dehydroascorbic acid) was determined in 24-h urine collections by the spectrophotometric procedure using 2,4dinitrophenylhydrazine as chromogen (11).
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90% of the leucocyte cytes and platelets, but no increase in mononuclear ascorbic acid variance in halt of the subjects and leucocyte ascorbic acid levels. The implication is that the effect of ascorbic acid supplementation on blood cell ascorbic acid levels differs with the cell type and that ascorbic acid intakes above 60 mg/d may in i '00 crease intracellular ascorbic acid content of polymorAscorbic Acid 5 • u Intake, mq/d 20 A phonuclear leucocytes and platelets more effectively 60 D .85 250-1 250 O than mononuclear cells. Another interpretation is that the mononuclear cells become saturated with O o 200 ascorbic acid at a lower level of dietary intake than O) C other blood cells. In this study, ascorbic acid levels in l_ 'u T3 O 5 150mononuclear leucocytes were repleted to baseline N=10 with 60 mg/d of ascorbic acid intake for 4 wk, R2=0.970 whereas plasma ascorbic acid levels remained low 100 (Fig. 1), and the repletion of semen ascorbic acid was lowest in the one subject repleted with 60 mg/d (Fig. 4? 50 2.7 10.0 100.0 4). These results, along with the minimal urinary ascorbic acid excretion observed at 60 mg/d intake Plasma Ascorbic Acid, /¿mol/L (Fig. 2) show that the conservation and transport of FIGURE 5 Linear regression of mononuclear leucocyte ascorbic acid into lymphocytes is particularly ef ascorbic acid vs. plasma ascorbic acid (log scale) for subject fective and commands a high metabolic priority. 2.
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CELLULAR ASCORBATE DEPLETION
>75% of the leucocyte variance in seven of eight subjects. These correlations include some points at which the ascorbic acid levels were not at steady state because of changes in ascorbic acid intake from one experimental period to the next. The results show that the plasma ascorbic acid level is useful for pre dicting the mononuclear leucocyte level across a range of ascorbic acid intakes from deficient to plasma saturating. This suggests that plasma ascorbic acid levels measured in population surveys would reliably reflect low leucocyte ascorbic acid levels that resulted from deficient ascorbic acid intakes. Buccal cell ascorbate. We measured buccal cell ascorbic acid to determine whether the test might provide a noninvasive specimen for estimating cel lular ascorbic acid levels in nutrition surveys. Al though buccal cell ascorbic acid levels were signifi cantly lower in subjects receiving the low ascorbic acid intakes (5, 10 or 20 mg/d) compared with the repletion intakes (60 or 250 mg/d), there was some overlap in the ranges (3-24 vs. 17-122 pmol/mg pro tein). Buccal cell ascorbic acid did not differentiate between ascorbic acid intakes within the low range or within the repletion range. Over the entire range of ascorbic acid intakes, linear regression showed that buccal cell ascorbic acid correlated significantly (P < 0.01) with ascorbic acid levels in plasma (r = +0.616), mononuclear leucocytes (+0.605) and semen (+0.779). Buccal cell ascorbic acid distinguished between ascorbic acid-depleted and ascorbic acid-repleted in dividuals in this controlled study reasonably well, but does not seem suitable as a precise marker of ascorbic acid status among groups with similar intakes of the vitamin. The results suggest that buccal cell ascorbic acid might be useful as a noninvasive screening test for ascorbic acid deficiency, but not as a quantitative measure of ascorbic acid status over a broad range of population intakes. However, further work seems warranted on improving method sensitivity, stan dardizing the technique and assessing its validity in free-living populations. Seminal ascorbate and fertility measures. Antagglutins in male and female reproductive tissues act to prevent sperm agglutination that may limit sperm motility (18). A male antagglutin protein in seminal plasma contains active carbohydrate and sulfate residues and is active in the reduced form (18). Re ducing agents such as ascorbic acid and glutathione enhance biological antagglutin activity or restore ac tivity to oxidized antagglutins (18). In vitro studies with normal mature sperm (18, 19) and studies of scorbutic guinea pigs (20) suggest that ascorbic acid deficiency adversely affects sperm count, morphology, motility and viability. These effects have been at tributed to increased sperm agglutination in ascorbic acid-deficient semen (18, 19) and to scorbutic abnor malities of testicular and other male reproductive tissues (20). Supplementation with 1000 mg/d of Downloaded from https://academic.oup.com/jn/article-abstract/122/5/1111/4754823 by Washington University, Law School Library user on 21 May 2018
1117
ascorbic acid for 10 d, then 500 mg/d for 60 d, resulted in 100% impregnation success for 20 infertile men compared with no success with an unsupplemented control group of 10 infertile men (21). In a follow-up study, Dawson et al. (21) found that in 20 men (age 25-45 y), with sperm agglutination >25%, supplementation with 200 or 1000 mg/d of ascorbic acid produced significant increases in seminal plasma ascorbic acid levels within 1 wk, and improvements in sperm count, viability, motility, agglutination and morphology parameters within 2 wk. Therefore, changes in seminal ascorbic acid levels may affect some sperm properties such as agglutination and mo tility within weeks, even though the maturation period for sperm is approximately 90 d. Despite the average 61 and 76% declines in semen ascorbic acid in our experimental subjects during Periods 2 and 3, we found no significant changes in the sperm prop erties related to male fertility listed in Table 2, nor in semen volume, pH or liquefaction time. It should be noted that the men enrolled in the study of Dawson et al. (21) were undergoing fertility testing and were chosen to have high initial sperm agglutination (>25%), whereas our men where chosen to be healthy, were screened for normal reproductive function and showed a clinically normal semen analysis at the start of the study. Our results suggest, however, that in healthy men, short-term ascorbate depletion does not adversely affect semen and sperm qualities, nor does moderate ascorbic acid supplementation improve them.
ACKNOWLEDGMENTS The authors thank the Jones Operation and Main tenance Company (Charlotte, NC) for providing support in operating the metabolic unit, especially Edward Blonz, project manager; Louise Hoppe and staff for providing nursing services; and Esther Kwan and staff for dietary services. The authors also thank Jeffre Johnson, manager of the Bioanalytical Support Laboratory, and his staff for providing laboratory sup port; Mei Miau Wu, UCLA School of Public Health, Department of Biostatistics, for statistical analysis support; Ramona Simpson and staff of the Hematology Laboratory, Letterman Army Medical Center (Presidio of San Francisco) for performing male fer tility tests,- and Hoffmann-La Roche Inc., Roche Chemical Division (Nutley, NJ) for providing USP ascorbic acid powder for diet supplementation.
LITERATURE CITED 1. Omaye, S. T., Schaus, E. E., Kutnink, M. A. & Hawkes, W. C. (1987) Measurement of vitamin C in blood components by high-performance liquid chromatography. Implication in as-
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JACOB ET AL.
sessing vitamin C status. Ann. N.Y. Acad. Sci. 498: 389-401. 2. Jacob, R. A., Skala, J. H. & Omaye, S. T. (1987) Biochemical indices of human vitamin C status. Am. J. Clin. Nutr. 46: 818-826. 3. Sauberlich, H. E., Kreisch, M. J., Taylor, P. C, Johnson, H. L. & Skala, J. H. (1989) Ascorbic acid and erythorbic acid metab olism in nonpregnant women. Am. J. Clin. Nutr. 50: 1039-1049. 4. Bates, C. J., Rutishauser, I.H.E., Black, A. E. & Paul, A. A. (1977) Long term vitamin status and dietary intake of healthy elderly subjects: 2. Vitamin C. Br. J. Nutr. 42: 43-55. 5. Evans, R. M., Currie, L. & Campbell, A. (1982) The distri bution of ascorbic acid between various cellular components of blood in normal individuals, and its relation to the plasma concentration. Br. J. Nutr. 47: 473-482. 6. Blanchard, J., Conrad, K. A., Watson, R. R., Barry, P. J. & Crawley, J. D. (1990) Comparison of plasma, mononuclear and polymorphonuclear leucocyte vitamin C levels in young and elderly women during depletion and supplementation. Eur. J. Clin. Nutr. 43: 97-106. 7. Jacob, R. A. (1990) Assessment of human vitamin C status. J. Nutr. 120: 1480-1485. 8. Dawson, E. B., Harris, W. A. & Powell, L. C. (1990) Rela tionship between ascorbic acid and male fertility. In: Aspects of Some Vitamins, Minerals and Enzymes in Health and Disease (Bourne, G. H., ed.). World Rev. Nutr. Diet. 62: 1-26. 9. Peterson, G. L. (1977) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 83: 346-356. 10. Kutnink, M. A., Hawkes, W. C., Schaus, E. E. & Omaye, S. T. (1987) An internal standard method for the unattended highperformance liquid Chromatographie analysis of ascorbic acid in blood components. Anal. Biochem. 166: 424—430. 11. Omaye, S. T., Turnbull, J. D. &. Sauberlich, H. E. (1979)
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