Clinical Interpretations of the M easurem ent of F olic Acid and Vitamin B12 in Neuromuscular Disease

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1 ANNALS O F C L IN IC A L A ND LABORATORY SCIEN C E, Vol. 6, No. 2 Copyright 1976, Institute for Clinical Science Clinical Interpretations of the M easurem ent of F olic Acid and Vitamin B12 in Neuromuscular Disease ED W A RD E. M ORSE, M.D. AND BARBARA MAXW ELL, M.D. University o f Connecticut Health Center, Farmington, CT ABSTRACT Fifty-six patients w ere studied in an attem pt to define neurologic com plications associated w ith folic acid and vitam in B12 deficiency. Two patien ts had abnorm al levels o f vitam in B12. O n e o f th e se show ed d ecreased vibratory sense in th e legs. T en of 15 patients w ith serum folate less than 4.0 n g p er m l an d 16 o f 23 patien ts w ith seru m folate less th an 6.0 ng p er m l by radioassay h ad neurologic abnorm alities. T h e control group dem on strated 17/31 w ith neu ro lo gic abnorm alities, a figure not significantly differen t from th e d eficien t group. It is su g g ested th a t m ore p atien ts w ith pure folate d eficien cy m u st b e analyzed to e lim in ate th e p o ssib le role of toxic effects o f alcohol and o th er diseases o f th e nervous system in patien ts w ith low serum folate and neurologic problem s. Introduction 137 It is w ell e sta b lish e d th at vitam in B12 d e fic ie n c y is a s s o c ia te d w ith v ario u s n e u ro lo g ic a l c o m p lic a tio n s in c lu d in g p e rip h e ra l n e u ro p a th y, s u b a c u te co m b in e d system s d isease and, less com m o n ly, n e u r o p s y c h ia trie m a n ife s ta tio n s.4,6 F olic acid d eficiency w as not reco g n iz e d as b e in g a sso c ia te d w ith any neu ro lo gical d isease u n til recen t years. In fact, folic acid w as th o u g h t to reverse th e m egaloblastic b o n e m arrow changes o f B12 d e fic ie n c y, w h ile a llo w in g th e n e u ro lo g ic c o m p lic a tio n o f B 12 d e f i ciency.14 F olic acid deficiency is com m only associated w ith a poor d ie t and c h ro n ic alco h o lism, n e u ro lo g ic a b n o r m alities in these circum stances are usually thought to be related to the m ore direct effects of alcohol on th e nervous syste m.2 In 1965 G ran t e t al3 p o in te d to folic acid d eficien cy as th e p o ten tial reason for neurologic problem s in a group of nutritionally deprived individuals. A dditionally, a n u m b e r o f p atien ts b e in g tre a te d w ith d ip h e n y lh y d a n to in for e p ile p s y w ere o b serv ed to d ev elo p m egaloblastic anem ia an d n eu ro p ath y un resp o n siv e to v ita m in B 12 b u t r e s p o n s iv e to fo lic a c id.1,2,12 M any o f th e se p a tie n ts w ere discov ered to have low levels o f both vitam in B12 an d folate in th e serum. P roof th at iso lated d eficien cy o f folic acid can produce neurologic dam age is lacking. Strachan and H enderson in 1967 d e scribed tw o patients w ith advanced dem en tia an d m egaloblastic anem ia d u e to folate d e fic ie n c y.10 F o late th erap y p ro

2 138 MORSE AND MAXWELL d u c e d a m a rk e d im p ro v e m e n t in th e m e n ta l state. R e c e n tly, R e y n o ld s an d c o w o rk e rs9 re o p e n e d th e q u e s tio n o f neu ro lo g ic disease associated w ith folate deficiency. T w enty -four patients w ith folate levels less th an 3 n g p e r m l w ere stu d ie d. F o r co n tro ls, p a tie n ts c h o se n w ere m atch ed for age a n d sex w ith folic acid levels over 6.9 ng per ml. A statistically significant in crease in organic brain syndrom e w as d isco v ered as w as positive B abinski resp o n se in th e folate d eficient p atien ts. In th e ir series, 17/24 p atien ts w ith low folate lev els h ad evid en ce o f organic b ra in syndrom e. Also 5/24 folate d eficien t p atien ts h ad a B abinski reflex w h ile n o n e o f th e p atien ts w ith norm al folate h ad a p o sitive B abinski response. T h e s e in v e s tig a to r s s h o w e d th a t p e rip h e ra l n e u ro p a th y w as m ore com m on in th e alcoholic patients in both folate d eficien t an d n o n -d eficient patien ts, bu t that evidence of organic brain syndrom e was m ore frequently seen in patients w ith low folate and did not correlate w ith a history of excessive intake of alcohol. It was concluded th at consideration m u st b e given to th e possibility th at n u tritio n al d eficien cy o f folate m ay re su lt in neurological com plications. T o review th e p o ssib le role of folate and vitam in B12 in neurom uscular disease, th e p re se n t authors have stu d ied a series of anem ic patients in whom vitam in B12 and folate in th e serum was m easured. M ethods Fifty-six anem ic p atien ts in w hom vitam in B12 and folate lev els w ere m easured u p o n ad m issio n into th e h o sp ital w ere studied to determ ine th e presence or absence of neu ro lo gic findings. A nem ia w as d efin ed as h ct < 35 p erc e n t or hb < 12 g per dl. R eview o f th e m edical history and physical findings w as carried o u t by one investigator, (B.M.) w ithout know ledge o f th e seru m levels of vitam in B12 and folic acid. V itam in B12 and folic acid w ere m eas u re d b y ra d io a ssa y.7,8' S eru m w as fro z e n im m e d ia te ly a fte r c o lle c tio n. N orm al controls w ere serum s from R ed C ross b lo o d donors. A pool of 20 serum s w as u se d for daily quality control and to determ ine replication of th e assays. A fter p atie n t data h ad b e e n co llected an d th e assays h ad b e e n com p leted, an a tte m p t to fin d c o rre la tio n s b e tw e e n n euro lo g ic abnorm alities an d vitam in B 12 an d folic acid lev els w as m ade b y th e other investigator. (E.M.) R esults F ifty norm al sera w ere exam ined in d u p licate and p ro d u ced a m ean norm al value for folic acid o f 12 ± 4 ng p e r m l an d for vitam in B 12 o f 650 ± 175 pg p er m l. T h irty rep licate d eterm in atio n s on th e sam e pool of norm al sera p ro d u ced a coefficient of variation o f 13 p ercen t for vitam in B12 and 17 p e rc e n t for folic acid. T he low er lim it of norm al for vitam in B 12 w as tak en as 300 pg p e r m l w ith a norm al range to 1000 p g p er ml. F o r folic acid, th e low er lim it was 4.0 ng p e r m l w ith a norm al range to 20 ng p e r ml. T h e p atien ts ran g ed in age from 7 to 91 w ith a m ean o f 47 years, th e m ale fem ale ratio was 38/18. O f the 56 anem ic patients review ed, 15 had folic acid levels less than 4.0 n g p e r m l and tw o h ad vitam in B12 lev els less th an 300 pg p er m l. T h ere w ere eight patients w ith folic acid levels b e tw e e n 4.0 an d 6.0 ng p er m l w h ich w ere an aly zed separately. T h e re w e re a p p ro x im a te ly tw ic e as m any m en as w o m en in each o f th e th re e gro u p s d e te rm in e d b y th e ir folic acid le v e ls. B o th m ale a n d fe m a le g ro u p s w ere fo u n d to b e eq u ally d istrib u te d b e tw e e n th ose w ith neu ro lo gical findings and those w ithout. No statistically significant difference o f age or sex distributio n o ccu rred in any o f th e groups. O f th e tw o patients w ith low vitam in B12 levels,

3 VITAMIN B12 IN NEUROMUSCULAR DISEASE 139 n e i th e r h a d p e r ip h e r a l b lo o d m acrocytosis and bone m arrow exam inations w ere n o t perform ed. O n e h ad a norm al n eu ro lo g ic exam ination. T h e o th er had loss of vibratory sen se in th e lo w er leg. T h ese patients are not con sid ered furth er. O f 15 p a tie n ts w ith lo w fo lic a c id le v e ls, 11 h a d p e r ip h e r a l b lo o d m acrocytosis (seven w ith m ean corpuscular v o lu m e (M CV ) g re a te r th a n 100, fo u r w ith m acro cytes d e s c rib e d on sm ear). T w o h a d b o n e m a rro w e x a m in a tio n s, b o th o f w h ic h s h o w e d m o d e ra te to m ark ed m egaloblastic changes. O f eig h t p atien ts w ith in term ed iate values o f folic a c id, six h a d p e r ip h e r a l b lo o d m acrocytosis (four w ith M CV g re a te r th an 1 0 0, tw o w ith m acrocytes d esc rib e d on sm ear). In th e g ro u p o f p a tie n ts w ith n o rm al lev els o f v itam in B12 an d folic acid, 14 show ed p e rip h eral b lo o d macrocytosis (nine w ith M CV g reater th an 1 0 0, five w ith m acro cytes d e s c rib e on sm ear). S even b o n e m arrow exam inations w ere d o n e an d th re e sh o w ed slig h t to m oderate m egaloblastic changes. F o u r of th e fiv e p a tie n ts w ith m e g a lo b la s tic c h a n g e s in th e b o n e m a rro w h a d p e rip h eral blood m acrocytosis, b u t th re e o f th e four patien ts w ho had no evid en ce o f m agaloblastic changes in th e m arrow also h ad p erip h eral blood m acrocytosis. O n ly s e v e n o f 29 p a tie n ts w h o h a d p erip h e ra l blood m acrocytosis h ad b o n e m arrow exam inations perform ed. T h ese results w ere statistically insignificant. E x am in atio n o f th e re la tio n s h ip b e tw e e n folic acid lev els an d n eu ro lo g ic d is e a s e r e v e a le d n e u ro lo g ic a b n o r m alities in 10 out of 15 patients (67 perc e n t) w ith lo w fo lic a c id. H o w e v e r, n eu ro lo gic abnorm alities w ere also found in 23 o f 39 patien ts w ho h ad folic acid levels greater th an 4.0 ng p e r m l (59 p e r cent) as show n in tab le I. T h e re w ere e ig h t p a tie n ts w h o se folic a c id le v e ls w ere betw een 4.0 and 6.0 ng per ml, a TABLE I Neurologic Disease in Folic Acid Deficiency Folic Acid Neurologic No Evidence of ng per ml Disease Neurologic Disease > < lev el w h ich w as co n sid ered by us su sp icious for folic acid deficiency. Six o f th e eig h t had n eu ro lo gic abnorm alities. N one of th ese in terrelatio n sh ip s p ro v ed to be statistically significant using X2 m ethod. E valuation of th e relatio n sh ip b e tw e e n th e ty p e s o f n e u ro lo g ic a b n o rm a litie s found and th e folic acid lev el revealed m ore instances in each category in th e p a tie n ts w ith n o rm al le v e ls o f folic acid th an in th o se w ith low folic acid (table II). N eurologic abnorm alities w ere significantly co rrelated w ith m acrocytosis p < 0.02 (table III). D iscussio n In th is series o f p a tien ts, correlatio n b e tw e e n serum lev els o f folic acid and vitam in B12 and n eu ro lo gic findings w ere difficult to dem o nstrate. O f o u r series o f anem ic p atien ts, only tw o (less th an four p ercent) d em o n strated low v itam in B 12 w h ile 15 (27 percent) d em o n strated low folic acid. T h e n u m b e r o f folate d eficien t patien ts increases to 23 (41 p ercen t) if th e bo rd erlin e group w ith folic acid m easurem ents b e tw e e n 4.0 and 6.0 n g p e r m l is included w ith th e abnorm als. T h e se fin d in g s strongly su g g est th at w hile nutritional deficiency in this po pulation of patients m ay account for a substantial p art o f th e b lood and neu ro lo gic abnorm alities seen, d eficien cy o f vitam in B12 or folic acid as d e te rm in e d by serum level does not provide the entire explanation. A lthough R eynolds e t al9 sh o w ed a hig h er in cid e n c e o f chronic b rain syndrom e and extrapyram idal signs in their

4 140 MORSE AND MAXWELL TABLE II Types of Neurologic Findings Folic Acid Decreased Vibration Sense Decreased Deep Paresthesis Hallucination ng per ml Seizures or Proprioception Tendon Reflexes Mental Confusion >4.0 <4.0 folic acid d e fic ie n t group, th a t fin d in g was u n ab le to b e confirm ed b y us ev en w h e n in c lu d in g p a tie n ts w ith fo la te lev els b e tw e e n 4.0 an d 6.0 ng p e r m l. It w o uld b e en lig h te n in g to know w h at th e neurologic status w as in R eynolds patien ts w ith folate levels b e tw e e n 3 ng p er m l an d 6.9 ng p er ml. On th e other hand, the choice of controls m atch ed only by age and sex in th e Yale stu dy m ay have in tro d u ced a bias by selecting hem atologically norm al in d i v id u a ls w h o w o u ld h a v e m u c h le s s c h an ce o f h av in g n e u ro lo g ical d ise a se from o th er n u tritio n al deficiencies. W hile th e p atien ts w ere analyzed for a history of alcoholism, m anifestations o f anem ia or o th er nutritio n al abnorm alities to m atch th e folate d eficien t p atien ts w ith th e controls w ere not included. O ur study suggests a strong relations h ip b e tw e e n m a c ro c y to s is in th e p erip h eral blood and neurological d isease. W h e th e r th e m acrocytosis sh o u ld b e in te rp re te d as a m anifestation o f folate deficiency, w h ich could n o t b e m easu red by serum levels, or a m anifestation of TABLE III Correlation of Neurologic Abnormalities With Macrocytosis Neurologic Macrocytosis (MCV* >100) Disease Present Absent Present Absent *Mean corpuscular volume. X =6.166 P<0.02 o th e r n u tr itio n a l d e fic ie n c y, also a s sociated w ith n eu ro lo g ic abn orm alities, is a m oot point. A stu d y o f tissu e (red blood cell) folate w o uld b e o f in te re st a n d w ill b e u n d ertak en, since seru m folate levels fluctuate m ore readily th an tissue folate lev els d o.5 In a p o p u latio n w h ere alcoholism and o th e r n e u ro lo g ic p r o b le m s m ay b e su p erim p o sed on nutritio n al deficiency, it is difficult to differen tiate th e effects ow ing to folate deficiency. U ntil larger n u m b ers o f p atien ts have b e e n stu d ied w ithout these interfering factors, the association o f neu ro lo gic d isease w ith p u re folic acid deficiency rem ains b u t an intrig u in g specu lation. R eferen ces 1. A n an d, M. P.: Iatrogenic m egaloblastic anem ia w ith neurological com plications. Scott M ed. J. 9: , C h a n a r in, I.: T h e M egaloblastic A nem ias. Blackwell Scientific Publications. Oxford, E n gland, pp , G r a n t, H. C., H o f f b r a n d, A. V. and W e lls, D. G.: Folate deficiency and neurological disease. Lancet ii: , H a n se n, H. A., N o r d q u is t, P., an d S o u ra n - d e r, P.: M egaloblastic anem ia and neurologic disturbance com bined w ith folic acid deficiency. Acta Med. Scand. 176: , HERBERT, V.: Folic acid deficiency in man. V itam ins and H orm ones, Vol. 26 A cadem ic Press, N ew York pp , H u n t e r, R., J o n e s, M., J o n e s, T. G., and M a tth e w s, D. M.: Serum B i2 and folate concentrations in m ental patients, Brit. J. Psychiat. 113: , L au, K. S., G o t t l i e b, C., W a sse rm a n, L. R., and H e r b e r t, V.: M easurem ent o f serum vitam in B12 level using radioisotope d ilution an d coated charcoal. Blood 26: , 1965.

5 VITAMIN B12 IN NEUROMUSCULAR DISEASE M o llin, D. L. and W a te r s, A. H.: T he Study o f V itam in B12 A b so rp tio n U sing L a b e lle d C o b alam in s. M ed ical M o nograph 6, W hite F riors P ress L td. A m ersham, G reat B ritain, R e y n o ld s, E. H., R o t h f i e l d, P. andpin C U s,j. H.: N eurological disease associated w ith folate deficiency. Brit. M ed. J. 2: , S t r a c h a n, R. W. and H e n d e r s o n, J. G.: D e m entia and folate deficiency. Quart. J. M ed. 36: , T a ju d d in, M. and GARDYNA, H. A.: Radioassay o f serum folate w ith use o f a serum blank and n o n -d ia ly z e d m ilk as fo late b in d e r. C lin. C hem. 19: , U n g a r, B. and C o w lin g, D. C.: M egaloblastic an em ia associated w ith an tico n v u lsan t drug therapy. M ed. J. Aust. : , W axm an, S., S c h r e ib e r, C., and H e r b e r t, V.: R adioisotopic assay for m easu rem en t of serum folate levels. Blood 38: , W illia m s, W. J., B e u t l e r, E., E r s l e v, A. J., and R u n d le S, R. W.: H em atology. M cgraw- H ill Co., New York, 1972.