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From www.bloodjournal.org by guest on April 29, 2017. For personal use only. BRIEF REPORT Effect of Vitamin Deficiencies B12 and on Neutrophil By Sandra S. Kaplan and Morphological and quantitative neutrophil abnormalities are common in the megaloblastic anemias of vitamin B,2 and folic acid deficiency. Little is known, however, about the role of these vitamins in normal leukocyte function. Seven patients with megaloblastic bone marrows, four with vitamin B,2 deficiency and three with folic acid deficiency, were studied to determine the effect, if any, of these deficiencies on leukocyte function. Phagocytosis of staphylococci, hexose monophosphate shunt activation with phagocytosis, and microbicidal capacity against Staphylococcus aureus were determined prior to the institution of specific therapy. In two instances, these studies were repeated T HE MEGALOBLASTIC ficiencies developing of ficiencies are and usually Function following treatment. There was no impairment of phagocytosis per se, and resting metabolism was not significantly decreased. With phagocytosis, however, metabolic activation was decreased to 35%-36% of control values in the leukocytes of patients with vitamin B,2 deficiency but not in the leukocytes of patients with folic acid deficiency. Bacterial killing was slightly decreased in vitamin B,2 but not in folic acid deficiency. These abnormalities of function were reversed after specific therapy. These findings suggested a specific role for vitamin B,2 in the production of intermediates necessary for normal cell function. of vitamin with qualitative polymorphonuclear associated with Acid R. E. Basford ANEMIAS associated mature are Folic acid de- and quantitative abnormalities (PMN) leukocytes. Such de- macrocytic B,2 PMN and precursors folic and reduced numbers of circulating PM N leukocytes with hypersegmented nuclei.’ Relatively little research has been done on the role ofvitamin B,2 acid in leukocyte hypersegmentation to impaired the production states, selves metabolism of PMN DNA of leukocyte suggest ordered synthesis.2 succinyl the has Vitamin B,2 coenzyme CoA from methylmalonyl propionate that leukocyte morphology impairment qualitative or function, but leukocytes generally oxidation function ofdeveloping of leukocyte platelet defect and function in in vitamin macrocytosis been believed also folic and nuclear to be related serves as a cofactor for CoA and, in deficiency is reduced.3’4 These may be impaired. mature and facts do However, leukocytes, as well protein-calorie B,2 deficiency7’8 malnutrition provoked not the as the 5,6 this themdis- known and the investi- gation. The four cific leukocyte anemia. associated From instances, of Pathology patients, three was evaluated studies were with folic efficiency of phagocytosis, monophosphate shunt and Biochemistry. acid deficiency prior to the institution repeated after reversal the (HMPS) and of speof the phagocytosisand micro- University of Pittsburgh Medical School. University of Pittsburgh Medical School. Pa. Submitted Blood, several the Departments A ddress Pittsburgh. of seven B,2 deficiency, This evaluation included activation of the hexose Pittsburgh, © function with vitamin therapy. In for August 21. 1975; reprint requests: Pa. accepted Sandra November S. Kaplan. 27, 1975. M.D.. /526/. 1976 by Grune Vol. 47, No. & Stratton. 5 (May). Inc. 1976 801 From www.bloodjournal.org by guest on April 29, 2017. For personal use only. 802 KAPLAN 1. Table Hexose Monophosphate Shunt Resting B12 deficiency Vitamin 4.5 4.4 39 10 R.R. 0.6 125 45 136 9.1 p 26 12 ± 37 <0.001 C.W. 6.6 295 161 EM. 6.5 302 170 N.J. 2.8 185 5.3 ± p 1.25 249 ± 23 pro duced 93 141 ± >0.5 9.9 ± 1.3 n <0.005 38.15 ± 261 <0.3 values 14C02 57 102 ± <0.2 Mean *Nanomoles 36 108 4.6 ± 2 Normal Staph D.C. E.H. deficiency Associated Latex Ci. Mean Folic acid BASFORD Activity* Phagocytosis Conditions AND <0.5 12.9 ± 104 23 fro m 1- ‘4C glucose per i07 leukocytes 24.34 8.4 18 per 30 mm; mean ± SE. bicidal capacity against Staphylococcus aureus. Our results indicate that, although leukocytes appeared to be quantitatively and morphologically similar in vitamin B,2 and folic acid deficiencies, their functional integrity, in terms of the parameters studied, is decidedly dissimilar with impaired function in vitamm B,2 deficiency and essentially normal function in folic acid deficiency. MATERIALS Segmented patients done neutrophils were from counts Wright’s including crest were folic acid red deficiency in two lost. All B,2 had and of had tests. value of 57 basis of prior was pg/ml. assessment grown Bacteria for activity was either These basis was of not patients, resulting in had who Ime lower of of E.H. the at the and lack of a markedly posterior superior iliac patients with three initial diagnosis. and limits of for the The patients B,2 values R.R., were acid with vitamin B,2 One Ci. vitamin abnormal but and was therapy and diagnosis, leukocytes, labora- patient folic anemia. vitamin B,2 absorption. deficient diet while the third to were polymorpho- of normal of his All showed vitamin time were blood the responded serum anemic of specimen patients low Complete the polymorphonuclear of phagocytosis 1- ‘4C counts the addition in two and trypticase from shunt glucose from he had vitamin deficient of had a B,2 on the the patients, characteristic as of a suitably of bacteria. The were and heat smears rest and of 4C02 described.9 0.01-mI cell were stained at evolution previously diluted activity washed prepared diluted killed at 60’C prepared 30 from to an for 30 and/or an 18-hr appropriate mm. 120 Phago- mm after per cell. activity the broth, studies Wright’s or ten staphylococci determined microbicidal soy use in metabolic evaluated monophosphate particles was of (R.R.) the leukocytes. marrow original improvement the these diabetic aureus introducing colony of surgery below levels. the vitamin-deficient preparation Each bone The hypersegmented S. concentration. ence Two bowel were ng/ml). with four for of Hexose marrow, levels B,2 on at from WBC from patients. anemia the PMN aspirates megaloblastic (<2.5 reticulocytosis of the marrow and of anemia. Bacteria culture acid diagnosed One bone 85%-90% macrocytic vitamin bone showed a frankly patients normal a juvenile pernicious cytic had controls counts vitamin-deficient a marked Folic were megaloblastic the normal Differential and and of all three a characteristic Schilling after the three deficiency D.C., all hypersegmentation. tory with indices in METHODS from quantitated. smears cell examined leukocytes) and stained thrombocytopenic, nuclear (PMN prepared9 AND ratio aliquot of these during uptake after 30 Microbicidal taken at of mm of staphylococci incubation activity time leukocyte-bacteria 0 and was at 30, suspensions in or latex the pres- determined 60, and was 1:2. from 120 mm From www.bloodjournal.org by guest on April 29, 2017. For personal use only. NEUTROPHIL 803 FUNCTION l0 -0- 06 Fig. 1 . The mean microbicidal activity of 20 normal controls at 30, 60, and 120 mm is shown by the solid line. The shaded area indicates 2 SD above and below the mean. The microbicidal activity of leukocytes from three of the four patients with vitamin B,2 deficiency are shown by dashed killed lines. fewer The mean After 2 hr of incubation, staphylococci than microbicidal activity three patients by the dotted of differences acid of leukocytes between deficiencies had expected. with folic acid deficiency line. All were within normal Significance B,2 and folic all normally from is shown limits. normal 30 120 MIN. control was determined 60 with and the the Student’s results t from patients with vitamin test. RESULTS The effects associated 1 and Fig. lower than of vitamin activation I. Resting normal. B,2 and folic acid deficiencies on the phagocytosis- ofthe HMPS and on bacterial killing are shown in Table metabolism tended to be low, but it was not significantly With phagocytosis either of latex or of staphylococci, how- ever, there was a statistically and 35% ofcontrol in the regardless of the cause of significant impairment ofHMPS activation to 36% leukocytes of patients with vitamin B,2 deficiency, the deficiency. Microbicidal activity at 2 hr after inoculation was reduced by more than 2 SD from the mean control level three of the four vitamin B,2-deficient patients where this was measured. patient with true pernicious anemia (C.J.) showed the greatest impairment in all The of microbicidal activity. Two of these patients, R.R. and E.H., were restudied after receiving vitamin B,2. Of these, E.H. demonstrated a marked improvement of phagocytosis-associated HMPS activation by the tenth posttreatment day. Initially, with metabolism the tenth posttreatment The other patient, leukocyte function The patients with HMPS phagocytosis of latex and staphylococci, her leukocytes’ was 43% and 54% of the mean control value (Table I). At day, this metabolic activity was 100% of the control. at 25 days posttreatment, also showed improvement of from approximately 40% of control to about 60% of control. folic acid deficiency did not show a decrease either of phagocytosis-associated HMPS activation or of microbicidal activity. DISCUSSION The primary role of the PMN leukocyte in host defense against bacterial invaders is well recognized. It has been suggested that deficiencies of any one or more of the B-complex vitamins may impair the metabolic activities associated with phagocytosis.’#{176} Thiamin, riboflavin, and pyridoxine deficiencies all have been associated with decreased activities of enzymes involved in HMPS activity. From www.bloodjournal.org by guest on April 29, 2017. For personal use only. 804 KAPLAN In addition, the leukocytes have decreased metabolic of children with and microbicidal severe protein-calorie activities,5 which decreased activity of granule-bound NADPH oxidase.6 demonstrated quantitatively impaired NBT reduction and kinase activity with normal phagocytosis in the leukocytes refractory Leukocyte anemias of unspecified function, in terms types.’1 of metabolic the course of vitamin B,2 and folic investigated. However, two previous mm B,2 deficiency have shown that 4 and using work an that the newborn increased suspected studies, tabolism deficiency. decrease susceptibility with caused multiple betic. to fatal malnutrition be related can to Another study has decreased pyruvate of patients with microbicidal activity acid deficiencies, has not been studies on leukocyte metabolism 3-’4C propionate oxidation to of methylmalonyl fed diets deficient BASFORD during specifically in vitaCO2 is im- CoA mutase is decreased.3 Other in folic acid has demonstrated infections,’2 and low tissue folate levels are of being a predisposing factor in the bacteriuria of ‘#{176} Our however, have shown no impairment of phagocytosis-associated meor microbicidal activity in the leukocytes of patients with folic acid Only in the four patients with vitamin B,2 deficiency did a marked in the phagocytosis-associated HMPS activation occur. These patients also have shown bicidal activity. able activity animals and AND a slight Although specific therapy, by vitamin deficiencies Only one to moderate both of these it is not impairment defects of possible B,2 deficiencies. Two due to malabsorption, of the patients had of been to of PMN intraleukocytic function assume that they are microcorrect- are entirely these patients may have possessed and one of the patients also is dia- vitamin B,2 deficient on the basis of a deficiency of intrinsic factor. It is not unreasonable to suppose that, in view of the impaired DNA and protein synthesis in vitamin B,2 deficiency, one or more of the enzymes involved in HMPS activation are reduced in the cell. Since hexokinase and NADP are rate-limiting for HMPS activity,’3 quantitation of these factors or the substances necessary for their formation would seem to be the most reduced potentially productive PMN reactivity are not avenues for future investigation. necessarily incompatible with Our findings the infrequency of oflife-threatening infection in vitamin B,2 deficiency. Metabolic activity is reduced by approximately 40% and microbicidal activity only by 5%-lO%. Much more profound reductions probably are necessary before serious compromise of PMN functional integrity occurs. The fact that this defect occurs with vitamin B,2 deficiency, vitamin B,2 in the tabolism and but not with folic acid deficiency, suggests a specific production of intermediates necessary for normal role for cell me- function. ACKNOWLEDGMENT We thank tion Michael Nardi of the manuscript. for We expert would also technical like assistance to thank Dr. and Mary Ralph Klayner Schmeltz for for help in prepara- allowing us to study in Am his patient. REFERENCES 1. Herbert New 2. V: York, Grune Nixon PF, The Megaloblastic & Stratton, Bertino JR: Anemias. 1959 Interrelationships of vitamin B,2 48:555-561, 3. Contrearas and folate man. J Med 1970 E, Giorgio Al: Leukocyte From www.bloodjournal.org by guest on April 29, 2017. For personal use only. NEUTROPHIL 805 FUNCTION methylmalonyl-CoA deficiency. mutase Am J Clin Nutr 4. Seashore MR. Hsia SL, Rosenberg LE: Defective tion in leukocytes In vitro I. YE, Am B,2 Scully K, Nutr 9. Durant and Selvaraj RI, Seetharam Med 26:873-875, activities protein-calorie 25:166-174, 6. Selvaraj cytosis and calorie Bhat K: of malnutrition. Am J Clin in Nutr 1972 Ri, leukocyte Bhat K: enzymes malnutrition. Biochem in J Phago- J Clin 11. 12. natal and 7. Stefanini bocytopathy Lancet M, in patients 1:400-402, Levine severe Karoca PH: vitamin M: with Acquired throm- pernicious A deficiency. platelet defect Beck Response, mal Paul Jr: in and 232:271-283, Biol health JB, 1:37-38, to WS: and J, Troube Shigella Nutr late of leukocytes. re- and in the 1972 control pathway I neo- growth infection 25:992-996, oxidation 1958 in Effect on Occurrence leukemic disease. 1973 KC: deprivation J Clin Strauss 1974 Haltalin acid Am B, Bactericidal P, Boucherot Lancet phosphogluconate qualitative B,2 13. of Exp abnormalities susceptibility pig. anemia. 1966 Ri, GW 27:629-637, anemia. guinea Soc Mitchell J, Boivin folic the impairment Selvaraj phagocytes Nelson on 1970 Nutr Hakim 1973 Studies Proc Blood-granulocyte fractory protein- 127:255-259, 1972 in Am SC: function. AA, of activities vitamin 78:533-539, chloramphenicol Al, iacobs H: Seetharam Sbarra after Med Finch 134:287-290, RR, Metabolic leukocytes SS, of thrombosis Intern leukocyte 10. bactericidal 8. KapLan human pigs: 1973 5. and Ann mechanism oxida- B2-deficient J Clin hyperresponse, B,2 therapy. 1972 propionate of vitamin correction. Vitamin 25:695-702, of the in Biol norChem From www.bloodjournal.org by guest on April 29, 2017. For personal use only. 1976 47: 801-805 Effect of vitamin B12 and folic acid deficiencies on neutrophil function SS Kaplan and RE Basford Updated information and services can be found at: http://www.bloodjournal.org/content/47/5/801.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.