One of the numerous benefits from eating “Paleo” or switching to a contemporary Paleo Diet if you eat in the standard American way is that you and your spouse/partner/ will greatly improve your chances for successful conception, pregnancy and a healthy baby. Here’s the information you may want to become acquainted with, particularly if you have avoided meats and fish and have previously focused upon whole grain cereals, legumes and processed foods in your diet.
If you fail to regularly eat animal foods (eggs, meat, fish and seafood) you will become deficient in vitamin B12 because plant food sources contain no B12. Further plant foods do not contain the specific types of vitamin B6 which are readily absorbable in the human gut. Accordingly, diets devoid of or lacking in animal proteins, will invariably be deficient in vitamin B12 and vitamin B6. Deficiencies or insufficiencies in these vitamins ultimately increase the level of homocysteine in our blood.
Elevated blood concentrations of homocysteine result primarily from too little vitamin B12. B6 and folate in our diets. When adequate stores of these B vitamins are present from nutritious foods in our diet (e.g. meats, fresh fruits and veggies), then our cells can defuse the poisonous effects of homocysteine and convert it into less toxic compounds. However, when B12 is lacking or deficient, as it almost always is in vegetarian and vegan diets, then homocysteine builds up in our bloodstream and literally infiltrates nearly every cell in our bodies.
Healthy egg cells in women and healthy sperm cells in men are absolutely essential requirements for getting pregnant, staying pregnant and producing normal embryos, vigorous infants and healthy children. Vitamin B12 deficiencies can elevate blood levels of homocysteine and cause numerous adverse health problems in pregnant women, their unborn fetuses and nursing infants. In addition to these unfavorable effects, a diet deficient or marginal in vitamins B12, B6 and folate can severely reduce your chances for successful fertilization and conception.
Infertility is a huge problem in both the U.S. and Europe and affects at least 6 million people in the U.S. or more importantly about 7.4 % of the reproductive age population. Many environmental and genetic factors may be involved. However, one thing is certain, as a couple, if you or your partner’s blood levels of vitamin B12, B6 and/or folate are low and your homocysteine is elevated, your chances for normal conception will be significantly reduced.
It is becoming increasingly evident that the low vitamin B12, B6 and folate status responsible for elevated homocysteine is toxic to both sperm and egg cells and may represent a major, previously un-recognized risk factor for infertility. More than 30 years ago, at least one group of researchers pointed out that Indian vegetarian men maintained lower vitamin B12 concentrations in their sperm than non-vegetarians and attributed these values to their vegetarian diet. Additionally, a number of these earlier studies hinted that vitamin B12 supplementation could improve sperm function and vigor and even boost male fertility.
If we fast forward to the 21st century, in the past five years similar nutritional patterns have been discovered in western populations. In a recent (2009) study of 172 men and 223 women who were unable to conceive, 36 % of men and 23 % of women had vitamin B12 deficiencies. Almost 40 % of the infertile men had abnormal semen that was directly related to their vitamin B12 deficiencies. Other recent studies in men show that low dietary folate and vitamin B12 are associated with high blood concentrations of homocysteine that likely underlie abnormal sperm function. On the flip side of the equation, women with compromised dietary B12 and folate intakes frequently have elevated blood levels of homocysteine which prevent them from becoming pregnant. We are not completely sure how these blood chemistry changes impede successful pregnancies in women, but tissue studies suggest that egg cells infiltrated by homocysteine and deficient in vitamin B12 and folate make them fragile and unable to continue with a normal pregnancy once fertilized.
Menstrual Problems caused by Vegetarian and Low Meat Diets
In addition to B vitamin deficiencies and elevated blood concentrations of homocysteine, vegetarian/low meat diets are frequently associated with menstrual problems known to affect fertility. A total of five studies have compared the incidence of menstrual irregularities between vegetarians and meat eaters. Four out of these five studies demonstrated significantly higher rates of menstrual complications in vegetarians. Let’s take a look at the only randomized controlled trial investigating vegetarian diets on menstrual health.
Dr. Pirke and researchers at the University of Trier in Germany randomly divided 18 young women with normal menstrual periods into either vegetarian or non-vegetarian diet groups. After six weeks, 7 of the 9 women assigned to the vegetarian diet stopped ovulating, whereas only a single woman in the meat eating group experienced this problem. The results of this experiment are shocking – within only six weeks of consuming a vegetarian diet, 78% of healthy, normally cycling women ceased ovulating. The take home message is this: if you are trying to get pregnant, one of your best strategies is to avoid vegetarian diets. While you’re at it, make sure your husband or partner does the same.
Zinc Deficiencies Impair Sperm Function
One of the most frequent nutritional shortcomings of vegetarian and vegan diets is that they fall short of recommended intakes for zinc. In the largest epidemiological study ever of vegetarians (The EPIC-Oxford Study) Dr. Davey and colleagues noted that vegans had “. . . the lowest intakes of retinol [vitamin A], vitamin B12, vitamin D, calcium and zinc. . .” when compared to meat and fish eaters. More importantly, with zinc it’s not just how much is present in your food, but how much is actually absorbed in your body. Although dietary zinc intakes in vegetarian diets sometimes appear to be adequate on paper – in the body they actually result in deficiencies because most of plant based zinc is bound to phytate and therefore unavailable for absorption. Phytate is an antinutrient found in whole grains, beans, soy and other legumes that prevents normal assimilation of many minerals. Laboratory experiments show that vegetarians only absorb about half as much zinc as meat eaters because zinc from animal food is much better assimilated than from plant foods.
Based upon this information, you might expect blood concentrations of zinc to be lower in vegetarians than meat eaters. Sometimes scientists have found this to be the case, but not always. The problem here has to do with where zinc ends up in our bodies after we ingest it. Most zinc finds its way into the interior of cells and does not accumulate in the liquid portion (plasma) of blood. Consequently, unless scientists examine zinc concentrations within cells, readings obtained in blood plasma frequently do not accurately reflect body stores of this essential mineral. In virtually every study of vegetarians which measured zinc levels inside various cells (red blood cells, hair cells and skin cells in saliva), plant based diets caused zinc deficiencies. In one study, 12 meat eating women were put on a lacto-ovo vegetarian diet, and after only 22 days Dr. Freeland-Graves and co-workers reported that zinc concentrations in the women’s salivary cells plunged by 27%. Similar results were described by Dr. Srikumar and colleagues from a longer term experiment in which 20 meat eating men and women adopted a lactovegetarian diet for an entire year. In this study, both hair cells and blood levels of zinc sharply declined and remained low throughout the 12 month experiment.
So, I’ve set the stage for zinc deficiencies and infertility problems. Because of their low zinc content and bioavailability, long term vegetarian diets almost always cause zinc deficiencies. Numerous epidemiological studies have shown that infertile men had poor sperm counts that were associated with reduced zinc levels in their semen. Virtually every well controlled experimental study ever conducted shows that men put on zinc deficient diets ended up with reduced sperm counts, impaired sperm health and often depressed blood testosterone levels. The good news is that these deleterious changes in male reproductive function can be reversed if zinc rich diets (e.g. The Paleo Diet) are consumed, or if zinc pills are supplemented. Dr. Steegers-Theunissen’s research group in the Netherlands showed dramatic improvements in the reproductive health of 103 sub-fertile men when zinc and folic acid were supplemented. Following the six month supplementation program, sperm counts increased significantly in the sub-fertile men while sperm abnormalities declined by 4 %. A similar study of 14 infertile men from India also indicated that zinc supplementation increased sperm health, sperm counts and shortly thereafter resulted in three successful conceptions by these men’s wives.
Whether you are a man or woman, if you want to sidestep infertility problems, the best advice I can give you is to abandon vegetarian/low meat diets and adopt the nutritional patterns that have sustained our hunter-gatherer ancestors for the past 2.6 million years. There are no known risks to adopting The Paleo Diet, and in fact, regular consumption of meat, seafood and fresh fruit and vegetables at the expense of cereals, dairy and processed foods will prevent vitamin B12 and folate deficiencies. In turn these essential vitamins will ensure that your blood levels of homocysteine will return to normal – effectively reducing your risk for cardiovascular, neurological, bone and reproductive diseases.
Loren Cordain, Ph.D., Professor Emeritus
1. Alexander D, Ball MJ, Mann J. Nutrient intake and haematological status of vegetarians and age-sex matched omnivores. Eur J Clin Nutr. 1994 Aug;48(8):538-46.
2. Appleby P, Roddam A, Allen N, Key T. Comparative fracture risk in vegetarians and nonvegetarians in EPIC-Oxford. Eur J Clin Nutr. 2007 Dec;61(12):1400-6.
3. Bhushan S, Pandey RC, Singh SP, Pandey DN, Seth P. Some observations on human semen analysis. Indian J Physiol Pharmacol. 1978 Oct-Dec;22(4):393-
4. Bennett M. Vitamin B12 deficiency, infertility and recurrent fetal loss. J Reprod Med. 2001 Mar;46(3):209-12.
5. Berker B, Kaya C, Aytac R, Satiroglu H. Homocysteine concentrations in follicular fluid are associated with poor oocyte and embryo qualities in polycystic ovary syndrome patients undergoing assisted reproduction. Hum Reprod. 2009 Sep;24(9):2293-302
6. Bissoli L, Di Francesco V, Ballarin A, Mandragona R, Trespidi R, Brocco G, Caruso B, Bosello O, Zamboni M. Effect of vegetarian diet on homocysteine levels. Ann Nutr Metab. 2002;46(2):73-9.
7. Boivin J, Bunting L, Collins JA, Nygren KG. International estimates of infertility prevalence and treatment-seeking: potential need and demand for infertility medical care. Hum Reprod. 2007 Jun;22(6):1506-12.
8. Boxmeer JC, Smit M, Weber RF, Lindemans J, Romijn JC, Eijkemans MJ, Macklon NS, Steegers-Theunissen RP. Seminal plasma cobalamin significantly correlates with sperm concentration in men undergoing IVF or ICSI procedures. J Androl. 2007 Jul-Aug;28(4):521-7
9. Boxmeer JC, Brouns RM, Lindemans J, Steegers EA, Martini E, Macklon NS, Steegers-Theunissen RP. Preconception folic acid treatment affects the microenvironment of the maturing oocyte in humans. Fertil Steril. 2008 Jun;89(6):1766-70.
10. Boxmeer JC, Smit M, Utomo E, Romijn JC, Eijkemans MJ, Lindemans J, Laven JS, Macklon NS, Steegers EA, Steegers-Theunissen RP. Low folate in seminal plasma is associated with increased sperm DNA damage. Fertil Steril. 2009 Aug;92(2):548-56.
11. Brown KH, Peerson JM, Baker SK, Hess SY. Preventive zinc supplementation among infants, preschoolers, and older prepubertal children. Food Nutr Bull. 2009 Mar;30(1 Suppl):S12-40.
12. Bucciarelli P, Martini G, Martinelli I, Ceccarelli E, Gennari L, Bader R, Valenti R, Franci B, Nuti R, Mannucci PM. The relationship between plasma homocysteine levels and bone mineral density in post-menopausal women. Eur J Intern Med. 2010 Aug;21(4):301-5
13. Campbell-Brown M, Ward RJ, Haines AP, North WR, Abraham R, McFadyen IR, Turnlund JR, King JC. Zinc and copper in Asian pregnancies–is there evidence for a nutritional deficiency? Br J Obstet Gynaecol. 1985 Sep;92(9):875-85
14. Cappuccio FP, Bell R, Perry IJ, Gilg J, Ueland PM, Refsum H, Sagnella GA, Jeffery S, Cook DG. Homocysteine levels in men and women of different ethnic and cultural background living in England. Atherosclerosis. 2002 Sep;164(1):95-102.
15. Clarke R, Sherliker P, Hin H, Nexo E, Hvas AM, Schneede J, Birks J, Ueland PM,Emmens K, Scott JM, Molloy AM, Evans JG. Detection of vitamin B12 deficiency in older people by measuring vitamin B12 or the active fraction of vitamin B12, holotranscobalamin. Clin Chem. 2007 May;53(5):963-70
16. Davey GK, Spencer EA, Appleby PN, Allen NE, Knox KH, Key TJ. EPIC-Oxford: lifestyle characteristics and nutrient intakes in a cohort of 33 883 meat-eaters and 31 546 non meat-eaters in the UK. Public Health Nutr. 2003 May;6(3):259-69.
17. de Bortoli MC, Cozzolino SM. Zinc and selenium nutritional status in vegetarians. Biol Trace Elem Res. 2009 Mar;127(3):228-33.
18. Dhonukshe-Rutten RA, van Dusseldorp M, Schneede J, de Groot LC, van Staveren WA. Low bone mineral density and bone mineral content are associated with low cobalamin status in adolescents. Eur J Nutr. 2005 Sep;44(6):341-7.
19. Dror DK, Allen LH. Effect of vitamin B12 deficiency on neurodevelopment in infants: current knowledge and possible mechanisms. Nutr Rev. 2008 May;66(5):250-5.
20. Ebisch IM, Peters WH, Thomas CM, Wetzels AM, Peer PG, Steegers-Theunissen RP. Homocysteine, glutathione and related thiols affect fertility parameters in the (sub)fertile couple. Hum Reprod. 2006 Jul;21(7):1725-33.
21. Ebisch IM, Pierik FH, DE Jong FH, Thomas CM, Steegers-Theunissen RP. Does folic acid and zinc sulphate intervention affect endocrine parameters and sperm characteristics in men? Int J Androl. 2006 Apr;29(2):339-45.
22. Elmadfa I, Singer I.Vitamin B-12 and homocysteine status among vegetarians: a global perspective. Am J Clin Nutr. 2009 May;89(5):1693S-1698S.
23. Fischer Walker CL, Ezzati M, Black RE. Global and regional child mortality and burden of disease attributable to zinc deficiency. Eur J Clin Nutr. 2009 May;63(5):591-7.
24. Freeland-Graves JH, Ebangit ML, Hendrikson PJ. Alterations in zinc absorption and salivary sediment zinc after a lacto-ovo-vegetarian diet. Am J Clin Nutr. 1980 Aug;33(8):1757-66.
25. Freeland-Graves JH, Bodzy PW, Eppright MA. Zinc status of vegetarians. J Am Diet Assoc. 1980 Dec;77(6):655-61
26. Gilsing AM, Crowe FL, Lloyd-Wright Z, Sanders TA, Appleby PN, Allen NE, Key TJ. Serum concentrations of vitamin B12 and folate in British male omnivores, vegetarians and vegans: results from a cross-sectional analysis of the EPIC-Oxford cohort study. Eur J Clin Nutr. 2010 Sep;64(9):933-9
27. Hansen CM, Leklem JE, Miller LT. Vitamin B-6 status indicators decrease in women consuming a diet high in pyridoxine glucoside. J Nutr. 1996 Oct;126(10):2512-8
28. Herbert V. Staging vitamin B-12 (cobalamin) status in vegetarians. Am J Clin Nutr. 1994 May;59(5 Suppl):1213S-1222S
29. Herrmann W, Obeid R, Schorr H, Geisel J. Functional vitamin B12 deficiency and determination of holotranscobalamin in populations at risk. Clin Chem Lab Med. 2003 Nov;41(11):1478-88.
30. Herrmann M, Widmann T, Colaianni G, Colucci S, Zallone A, Herrmann W.Increased osteoclast activity in the presence of increased homocysteine concentrations. Clin Chem. 2005 Dec;51(12):2348-53
31. Herrmann W, Schorr H, Obeid R, Geisel J. Vitamin B-12 status, particularly holotranscobalamin II and methylmalonic acid concentrations, and hyperhomocysteinemia in vegetarians. Am J Clin Nutr. 2003 Jul;78(1):131-6.
32. Herrmann M, Peter Schmidt J, Umanskaya N, Wagner A, Taban-Shomal O, Widmann T, Colaianni G, Wildemann B, Herrmann W. The role of hyperhomocysteinemia as well as folate, vitamin B(6) and B(12) deficiencies in osteoporosis: a systematic review. Clin Chem Lab Med. 2007;45(12):1621-32
33. Herrmann W, Obeid R, Schorr H, Hübner U, Geisel J, Sand-Hill M, Ali N, Herrmann M. Enhanced bone metabolism in vegetarians–the role of vitamin B12 deficiency. Clin Chem Lab Med. 2009;47(11):1381-7.
34. Hirwe R, Jathar VS, Desai S, Satoskar RS. Vitamin B12 and potential fertility in male lactovegetarians. J Biosoc Sci. 1976 Jul;8(3):221-7
35. Huang YC, Chang SJ, Chiu YT, Chang HH, Cheng CH.The status of plasma homocysteine and related B-vitamins in healthy young vegetarians and nonvegetarians. Eur J Nutr. 2003 Apr;42(2):84-90.
36. Humphrey LL, Fu R, Rogers K, Freeman M, Helfand M. Homocysteine level and coronary heart disease incidence: a systematic review and meta-analysis. Mayo Clin Proc. 2008 Nov;83(11):1203-12.
37. Hunt JR, Matthys LA, Johnson LK. Zinc absorption, mineral balance, and blood lipids in women consuming controlled lactoovovegetarian and omnivorous diets for 8 wk. Am J Clin Nutr. 1998 Mar;67(3):421-30.
38. Hunt JR, Roughead ZK. Nonheme-iron absorption, fecal ferritin excretion, and blood indexes of iron status in women consuming controlled lactoovovegetarian diets for 8 wk. Am J Clin Nutr. 1999 May;69(5):944-52
39. Jathar VS, Hirwe R, Desai S, Satoskar RS. Dietetic habits and quality of semen in Indian subjects. Andrologia. 1976;8(4):355-8.
40. Katre P, Bhat D, Lubree H, Otiv S, Joshi S, Joglekar C, Rush E, Yajnik C. Vitamin B12 and folic acid supplementation and plasma total homocysteine concentrations in pregnant Indian women with low B12 and high folate status. Asia Pac J Clin Nutr. 2010;19(3):335-43.
41. Koebnick C, Hoffmann I, Dagnelie PC, Heins UA, Wickramasinghe SN, Ratnayaka ID, Gruendel S, Lindemans J, Leitzmann C. Long-term ovo-lacto vegetarian diet impairs vitamin B-12 status in pregnant women. J Nutr. 2004 Dec;134(12):3319-26.
42. Krivosíková Z, Krajcovicová-Kudlácková M, Spustová V, Stefíková K, Valachovicová M, Blazícek P, Nĕmcová T. The association between high plasma homocysteine levels and lower bone mineral density in Slovak women: the impact of vegetarian diet. Eur J Nutr. 2010 Apr;49(3):147-53
43. Kumar J, Garg G, Sundaramoorthy E, Prasad PV, Karthikeyan G, Ramakrishnan L, Ghosh S, Sengupta S. Vitamin B12 deficiency is associated with coronary artery disease in an Indian population. Clin Chem Lab Med. 2009;47(3):334-8.
44. Mezzano D, Kosiel K, Martínez C, Cuevas A, Panes O, Aranda E, Strobel P, Pérez DD, 45. Pereira J, Rozowski J, Leighton F. Cardiovascular risk factors in vegetarians. Normalization of hyperhomocysteinemia with vitamin B(12) and reduction of platelet aggregation with n-3 fatty acids. Thromb Res. 2000 Nov 1;100(3):153-60.
45. Molloy AM, Kirke PN, Brody LC, Scott JM, Mills JL. Effects of folate and vitamin B12 deficiencies during pregnancy on fetal, infant, and child development. Food Nutr Bull. 2008 Jun;29(2 Suppl):S101-11
46. Molloy AM, Kirke PN, Troendle JF, Burke H, Sutton M, Brody LC, Scott JM, Mills JL. Maternal vitamin B12 status and risk of neural tube defects in a population with high neural tube defect prevalence and no folic Acid fortification. Pediatrics. 2009 Mar;123(3):917-23.
47. Michie CA, Chambers J, Abramsky L, Kooner JS. Folate deficiency, neural tube defects, and cardiac disease in UK Indians and Pakistanis. Lancet. 1998 Apr 11;351(9109):1105.
48. Misra A, Vikram NK, Pandey RM, Dwivedi M, Ahmad FU, Luthra K, Jain K, Khanna N, Devi JR, Sharma R, Guleria R. Hyperhomocysteinemia, and low intakes of folic acid and vitamin B12 in urban North India. Eur J Nutr. 2002 Apr;41(2):68-77.
49. Pront R, Margalioth EJ, Green R, Eldar-Geva T, Maimoni Z, Zimran A, Elstein D. Prevalence of low serum cobalamin in infertile couples. Andrologia. 2009 Feb;41(1):46-50.
50. Refsum H, Yajnik CS, Gadkari M, Schneede J, Vollset SE, Orning L, Guttormsen AB, Joglekar A, Sayyad MG, Ulvik A, Ueland PM. Hyperhomocysteinemia and elevated methylmalonic acid indicate a high prevalence of cobalamin deficiency in Asian Indians. Am J Clin Nutr. 2001 Aug;74(2):233-41.
51. Reynolds RD: Bioavailability of vitamin B-6 from plant foods. Am J Clin Nutr 1988;48:863-67.
52. Roed C, Skovby F, Lund AM. Severe vitamin B12 deficiency in infants breastfed by vegans].Ugeskr Laeger. 2009 Oct 19;171(43):3099-101
53. Rush EC, Chhichhia P, Hinckson E, Nabiryo C. Dietary patterns and vitamin B(12) status of migrant Indian preadolescent girls. Eur J Clin Nutr. 2009 Apr;63(4):585-7. Epub 2007 Dec 19.
54. Selhub J, Morris MS, Jacques PF. In vitamin B12 deficiency, higher serum folate is associated with increased total homocysteine and methylmalonic acid concentrations. Proc Natl Acad Sci U S A. 2007 Dec 11;104(50):19995-20000.
55. Singh K, Singh SK, Sah R, Singh I, Raman R. Mutation C677T in the methylenetetrahydrofolate reductase gene is associated with male infertility in an Indian population. Int J Androl. 2005 Apr;28(2):115-9.
56. Srikumar TS, Johansson GK, Ockerman PA, Gustafsson JA, Akesson B.Trace element status in healthy subjects switching from a mixed to a lactovegetarian diet for 12 mo. Am J Clin Nutr. 1992 Apr;55(4):885-90.
57. Stabler SP, Allen RH. Vitamin B12 deficiency as a worldwide problem. Annu Rev Nutr. 2004;24:299-326
58. Stephen EH, Chandra A. Declining estimates of infertility in the United States: 1982-2002. Fertil Steril. 2006 Sep;86(3):516-23.
59. Taneja S, Bhandari N, Strand TA, Sommerfelt H, Refsum H, Ueland PM, Schneede J, Bahl R, Bhan MK. Cobalamin and folate status in infants and young children in a low-to-middle income community in India. Am J Clin Nutr. 2007 Nov;86(5):1302-9.
60. te Velde E, Burdorf A, Nieschlag E, Eijkemans R, Kremer JA, Roeleveld N, Habbema D.
Is human fecundity declining in Western countries? Hum Reprod. 2010 Jun;25(6):1348-53.
61. Tikkiwal M, Ajmera RL, Mathur NK. Effect of zinc administration on seminal zinc and fertility of oligospermic males. Indian J Physiol Pharmacol. 1987 Jan-Mar;31(1):30-4.
62. van Mil NH, Oosterbaan AM, Steegers-Theunissen RP. Teratogenicity and underlying mechanisms of homocysteine in animal models: a review. Reprod Toxicol. 2010 Dec;30(4):520-64. Vegetarianism in American. Vegetarian Times Magazine, 2008. http://www.vegetariantimes.com/features/archive_of_editorial/667
63. Verkleij-Hagoort AC, Verlinde M, Ursem NT, Lindemans J, Helbing WA, Ottenkamp J, Siebel FM, Gittenberger-de Groot AC, de Jonge R, Bartelings MM, Steegers EA, Steegers-Theunissen RP. Maternal hyperhomocysteinaemia is a risk factor for congenital heart disease. BJOG. 2006 Dec;113(12):1412-8.
64. Vogel T, Dali-Youcef N, Kaltenbach G, Andrès E. Homocysteine, vitamin B12, folate and cognitive functions: a systematic and critical review of the literature. Int J Clin Pract. 2009 Jul;63(7):1061-7
65. Wald DS, Law M, Morris JK. Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis. BMJ. 2002 Nov 23;325(7374):1202.
66. Waldmann A, Dörr B, Koschizke JW, Leitzmann C, Hahn A. Dietary intake of vitamin B6 and concentration of vitamin B6 in blood samples of German vegans. Public Health Nutr. 2006 Sep;9(6):779-84.
67. Wong WY, Merkus HM, Thomas CM, Menkveld R, Zielhuis GA, Steegers-Theunissen RP. Effects of folic acid and zinc sulfate on male factor subfertility: a double-blind, randomized, placebo-controlled trial. Fertil Steril. 2002 Mar;77(3):491-8.