Clin. Biochem. 9, ( l ) 22-23 (1976)

The Effect of Diphenylhydantoin on the Microbial Assay

of Serum Folate N O R M A N F O S T E R and B R U C E C A M P B E L L , J r .

D e p a r t m e n t o f C h e m i s t r y , M a c M u r r a y Col lege , J a c k s o n v i l l e , I l l i no i s 62650, U S A

( A c c e p t e d A u g u s t 25, 1975)

CLBIA 9, (1) 22-23 (1976) Clin. Biochem.

Foster, Norman and Campbell Jr., Bruce

Department of Chemistry, MacMurray College, Jack- sonville, Illinois 62650 USA.

THE E F F E C T OF D I P H E N Y L H Y D A N T O I N ON THE MICROBIAL ASSAY OF SERUM FOLATE.

1. Two modifications of the usual method of assay for foiic acid in solutions are reported which ap- pear to improve this procedure. These are a reduction of incubation time and dilution with 95% ethanol.

2. Experimental results are reported which indicate that Dilantin (5,5-diphenylhydantoin) does not interfere with the microbiological assay for folic acid.

SINCE I~ANNHEIMER FIRST DESCRIBED TWO CASES OF

MEGALOBLASTIC ANEMIA associated with the use of anticonvulsant drugs '') there have been numerous re- ports of other cases of megaloblastic anemia and ma- crocytosis connected with such drug use These symp- toms are related as both may be caused by impairment of B,~ and/or folic acid metabolism. Indeed the admi- nistration of folic acid and/or vitamin therapy as well as withdrawal of the drugs have been used to bring about remissions. 's-,~ It is of obvious importance to monitor the serum folate level when using such drugs. The typical procedure involves the incubation of L. casei which is folic acid dependent and turbi- dimetric analysis of total growth. Other workers '~* using radioactive triglutamic folic acid found results which seemed inconsistent with one interpretation of data from growth assay and they suggest that this may be due to inherent limitation in the usual micro- biological for folic acid.

It then seemed useful to study directly the L. casei assay with known levels of folic acid and Dilantin (sodium diphenylhydantoin). As a consequence the common microbiological assay has been modified with apparent improvement and assays of folic acid in the presence of Di lant in have been carr ied out wi th this modif ied method.

METHODS AND MATERIALS

The original method of assay followed that described by Herbert '9~ and by Cooperman"°k The method consists

Correspondence: Bruce Campbell, Jr., Department of Chemistry, SUNY College, Postdam, N.Y. 13676, U.S.A.

essentially of combining the solution to be tested with media containing what is needed to support the growth of L. casei except folic acid and inoculating with a culture of L. easel. The samples are incubated at 37 ° along with suitable blanks and/or standards, originally, for 24 hrs or more. Additional details are available from the authors. The media, Lactobacilli Agar, Lactobacilli Broth, and Folic Acid Casei Medium and the culture Lactobacillus easel, AATC 7469, were obtained front Difco. One major modification was to shorten the incuba- tion time between the addition of the inoculum and the reading. Although it appeared that growth was about complete after 24 hrs there was a linear relationship of growth and folic acid concentration at shorter times. Thus readings were taken after 15 to 18 hrs of incubation. Two other difficulties were met by an additional modifi- cation. The difficulties were too heavy growth in the range of concentration (0.1 to 1.0 l~g/ml) of folic acid that was generally employed and some increase of the readings with time was observed af ter removal from the incubation bath especially if not all the folic acid had been completely utilized. A number of procedures were tried to stop the reaction and dilute it before reading. These included water, 1% lysol, 1 ~ NaOH, 95% ethanol and autoclaving. The best approach seemed to be 1:1 dilu- tion with 95% ethanol before reading.

In the final experiment 6 sets of assays were run using the optimized procedure. They were all identical in media, inoculum and total volume and parallel in folic acid concentration. For 2 sets the concentration of 5,5- diphenylhydantoin (DPH) was 5 y.g/ml; for 2 it was 50 ,xg/ml; while in the final 2 there was none. The concen- tration of folic acid in the members of each set were 0, .1, .2, .5, and 1 pg/ml. The incubation was at 37 ° for 15 hours.

RESULTS AND DISCUSSION

The modi f ica t ions of the usual procedure seemed to have s~me very s i g n i f i c a n t advantages . The t ime requi red for this assay can be reduced by about 9 hours. This would appear qui te useful in a cl inical se t t ing. I f ex t remes of folic acid concent ra t ion are ex- pected it may be well to include some t r ia l s where incubat ion periods a re 8-12 hrs and 24-36 hrs to de te rmine if the g rowth is l inear wi th t ime and if modi f ica t ion in the folic acid concent ra t ions of the s tandards should be made. S tandards should be run wi th each set of assays because of the possible va r i a - tions. The pr inciple of cont inuous g rowth cul tures sugges ts tha t a t su f f i c i en t ly low concent ra t ions of g rowth fac to r there is a reg ion in which g rowth ra te is direct ly propor t ional to the concent ra t ion of g rowth fac to r . " This would appear to be the case in the s tudies repor ted here. This would obvia te the neces- s i ty of wa i t i ng for the t e rmina t ion of g rowth to make this de te rmina t ion .

ASSAY FOR FOLIC ACID 23

The d i lu t ion wi th 95% ethanol was qui te advan- tageous . By s topp ing g rowth i t f a c i l i t a t ed m a k i n g d e t e r m i n a t i o n be fo re the t e r m i n a t i o n of g rowth f rom exhaus t ion of the folic acid. The r e a d i n g s were more cen te red on the scale and seem more rel iable. Re- p l ica te runs were usual ly cons i s t en t wi th in -- 0 .5%T. However i t was judged t ha t d i f f e r ences mus t be 3 % T or g r e a t e r to be cons idered s ign i f i can t . The r ead ings were qui te cons i s t en t w i th t i m e when s tab i l ized wi th ethanol . F o r t u i t o u s l y the e thanol also seemed to im- prove the qua l i ty of the suspension, which shaken or mixed before r ead ing , was f i ne r and showed l i t t le or no tendency to clump.

The f ina l expe r imen t was des igned to s tudy the e f fec t of 5 ,5 -d iphenylhydanto in ( D P H ) on the a s say for folic acid. The lower concen t ra t ion of D P H was p red ica t ed on a reasonable ped i a t r i c dosage "2) and the sugges t ion of Ki ips t e in (7~ as to a r easonab le r ange of D P H plasma levels. The h i g h e r was de t e rmined f rom cons ide ra t ion of the mean le thal dosage in adul ts . ''2) I f i n t e r f e r ence is to be observed i t should be observed wi th such concen t ra t ions f rom 2 to 10 t imes g r e a t e r than expected p lasma levels. The resu l t s of each p a i r of sets were p lot ted wi th absorbance vs concen t r a t i on of folic acid in /xg/.ml. The r e su l t i ng curves were a lmos t ident ica l p a r t i c u l a r l y in slope. Where dev ia t ions froin the s t r a i g h t l ine occurred they were a lmost complete ly paral le l . There was no

evidence fo r the i n t e r f e r ence of fol ic dependent g rowth by D P H in th i s s tudy. The quest ion of d i f - fe rences in s tud ies made us ing d i f f e r e n t assays , then, is s t i l l open.

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