The Folic Acid Content of Milk: Revised Figures Based on an Improved Assay Method

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PEDIATRIcs, August 1964

Experience

and Reason

Briefly

Recorded

‘ ..

“Iii \I((li(iiic’ Omit mutmst u1y attentiomi not to I)huttmsit)lc theorizing i)umt to expcricmice amid r(ason togcthier. . . . I agree that theorizing is to lId’ approved, i)rovidle(l that it is based on facts, and

sstematicall make its deductions from-mi what is observed. . . . But conclusiomis c!rawmi from

UIiaidld’dl reason can hardly be serviceai)le; only thd)se drawn from-mi observed fact.” Hippocrates: Pr(cepts.

S

#{149}

(This heading (10(1 text seem appropriate for a section of short cmnmunications requiring onit, rapid editorial ret;iew (111(1 little, if iiiy, reVision. It is hoped that soc/i factual material may thus I)(’ pul)li.shed (llfllO.St (1.5 pioinptltj (it the opinions, coinmeiits, (111(1 criticisms tehicli will continue to

(1J)/)C(JT as Letters to time Editor.)

The

Folic

Acid

Content

of

Milk:

Revised

Figures

Based

on

an

Improved

Assay

Method

Previously published data imiolicate that milk

has

a low

folio

acid

comitent.

The

figcmres

of

1-2 tg -‘b of milk were obtaimied by

micro-biologic assay usimlg Streptococcus fecalis as

the

assay

organism.

Iii these

assas’s

ascorbic

acid

was mlc)t added to) the incubation medium to

Prtect

the

labile

folates

from

oxidative

de-structiomi.

Niore recemit sttmdliesi jimchicate that the

or-ganisrn Lac’to1,cilliis caso’i exhibits a niuch

greater growth response thami does

Strepto-coccus fecali.s to) the folic acid-active material

Presemit ill hummiiami serum and that this activity

correlates well with the status d)f fohic acid

nutrition iii these patiemits. It seems likely,

therefore, that the L. casei folate activity of

milk and other foodstuffs would give a

closer

approximatiomi of the content of fohic acid-active material that is of significance to human miutritiomi.

Because milk is the most important and

often the sole source of nutrition for infants

in

the

first

few

mouths of life it was

felt

that

the folate cd)ntemit of milk should be

re-evahm-ated

usin g Iresent assay methoc!s.

METHODS

Folate activity was determimied

by

the

pro-cedure as o)tmtlined by

Herbert

using

the

aseptic

addition

niethod

and

an

ascorbic

acid

concemitration of

1 gm100

ml

in the assay

mec!ium.1

Because final readimigs are based on ti-me

timrbk!ity d)f the niixtures, the folbowimig steps

were

taken

to

correct

possible

inaccuracies

in

readimigs due to) the turbidity imparted by the

milk alone to the cultum’e media.

1. \Vith each flask containing milk and tb-me

assay

orgamiism a “mi-milk blank” was used

in

which no bacterial imimiocimlum was added.

Subtractimig this reading from that obtaimied

with the inmioculum gave a nieasure of tb-me

turl)idity

clue

to

the

milk

itself.

2. After imicubation,

the

flask

were

centrifuged at 3,000 r.p.m. for 10 mi-minutes,

the supernatamit aspirated amid ti-me sedimemit

(

consisting of bacterial growth) was

recoil-stituted to the origimab volumne with fmeshi basal mei!ium prior to reaclimg.

Samples of cow’s milk, goat’s milk,

evapo-rated milk, and 13 proprietar milk

prepara-tions

were

assayed

by

ti-me foregoimig

nethod.

Each assay was performeol on a differemt can or bottle of the milk product. The samples

were freshly’ opemied before each test. Iii the case of the proprietary milks the contents

were reCo)mlStitJited with water according to

the recommendations of the mnamiufactumer.

Ahiquots of the same milk formula were

as-sayed! i)efore amid after atmtoclaving (210#{176} F. amci 20 lb presstmre for 30 minutes) iii the Milk Laboratory oif the Chik!ren’s Hospital.

All milks vhose folate contemit was less than

100 g

/

1 were

assayed

on at beast

five

separate

occasions uSimig different samples.

i\4ilks

whose

folate content exceeded 100 .g I were

as-sayed at least twice for purposes of

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TABLE I

. _I _roauct

Number of

kSamj)les’ issayea

(in

duplicate)

Po!ats (‘on/eat (ig/l)

.1. Before auk’elarmng . B. 4fter auloclari rig

Mean Range . Ascorbic . Acid (‘onlent (g/qt)5 .1 B -1 B

W’lmole (Ow’s niilk*

Evaporated mnilkt (;mmt’s niilkt Similac (powder) SMA (liquid) Lactuni (powder) i1odihmic (hiqumid) Breniil (powder)

Bakers Mo(lifie(1 (powder) Enfamiiil (liquid)

Meat Base (liquid)

Muhisoy (liquid) Sobee (powder) Sovalac (powder) Nutramigen (powder) Lofenalac (powder) 7 6 6 5 6 7 .5 5 2 5 5 4 2 2

89.5 51 .4

84 .7 47.8

10.7 18.7

61 46.6

18.8 15.8

63.5 31 .5

> lot) > 100

42 .‘2 40. 7

100 100

49.’2 35.5

50.4 41 .1

> 100 > 100 > 100 > 100 > 1(M) > 100 > 100 > 100

> 100 > 100

62100 21-100 65-100 8-86 7.-13 12--30.5 36-88 24-58 9-29 9.6-t3 31-1(5) 13-54 mnme 30-70 31-60 same 35-68 27-45 ‘2R-64 20.5-58 same same same same sumac 0 0 0 .50 50 0) 45 50 .50 50 40 30 50 0 30 30

EXPERIENCE

AND

REASON-BRIEFLY

RECORDED

275

, 1100(1 Dairy Co., Bostomi, Massachusetts.

t

Carnation Milk Conipamiy, Los Angeles, California. I)asvn Dairy Imic., Castleton, New York (pasteurized).

RESULTS

The

folate

comtent

of the

various

milk

prep-aratiolls is given imi

Table

I. To

facilitate

in-terpretatiom

of

the

effect

of

autoclaving

in

relation to ascorbic acid contemit of the milks, published values for the latter are also listed.

COMMENT

The

“folate

content”

of

milk

determined

by the presemit assay represents the amount

of fohic acid-active material that preferentially supports growth of the organism L. casei.

Studies of humam sertmm suggest that the

major

source

of

this

activity

is

the

mono-glutamate,

N2-methyl

tetrahydrofolate.6

Whether the same compound comprises the

fohic

acid-active

material

of

milk

is

not

yet

known.

However,

in

view

of

the

close

corre-lation of L. casei

folate

activity

of serum

with

the

status

of fohic

acid

nutritiomi,

it seems likely

that

the

activity

present

in milk

is o)f biological

significance im the htmman. Umtil more is

known about the absorption and utilization

of ti-me folate compounds in milk (am-md other

foodstuffs) by the body, no statememit can

be

made

comiceniing

the

relationship

of

L.

casei

folate

activity

to

the

true

total

folate

comitemit

of milk biologically available to the himmami. This study has revealed the folate content

of

milk to be much higher than the earlier

published

figtmres

of 1-2

&g/l.

The

meami values

for

the

milk

preparations

in

the

present

study

varied

from

10.7 .tg/l

to

over

100 sg/l.

A real

variation

was

found

among

different

samples of the same milk. This may be

re-lated

to

factors

such

as

the

folio

acid

content

of the cows

feed

or

the

methods

of

milk

processing am-md storage. No significant

corre-lation

was

found

between

the

lability

of folate

activity

with

autoclavimig

of

the

milk

amid

its

ascorbic acid content.

That

the

values

for

the

folate

content

of

milk obtaimied in the present study are of clinical significamice is suggested by the

fob-lowing

observatiomis:

1. Megaloblastic

amiemia

is

a

well-recog-nized

consequence

of

excessive

comisumption

of

goat’s

milk,

am-md the

folate

content

of

the

latter

(mean

10.7 p.g/1)

was

the

lowest

of all

the milks tested.

2. Children who develop iromi-deficiency

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276

INTESTINAL

BIOPSY

milk (with a poor imitake of solid foods) rarely show evidence of folic acid deficiemcy,7

sug-gesting

that

the

relatively

high

com-mtem-mtof

folate iii cow’s milk (89.5 ig 1) is sufficient to

meet needs of such children. (Studies of

otherss have confirmed the relatively high folate comitemit of cow’s milk.)

Until information is available concermiimg

the

minimal

folio

acid

reqtmirernent

of

the

normal infant, no further statememt can be

made

regarding

the

adequacy

of these

variotms

milks

irm satisfying

this

requiremiient.

Such

in-formation woulc! be of especial imnportance

during the first few momths of life heri milk

constitutes the major source of nutrition for

imifants. In this regard, Shojamiia amid Gross

found lower levels of sertmm folate activity iii prcmiiature imifants compared to full-term in-fants at 1-2 months of age.

No

significant

differemice imi hemoglobin level was found

be-bveei-m the tWo groups of infants. The full

significamice of this iii relatiom to the folate

content

of

milk

must

await

further

observa-tioms concermiimig the absorption, utilizatiom,

amd meec!s O)f folic acid b such infants.

SUMMARY

Using

au

iml)rovedl

assay

technique

with

ascorbate-protecteci L. casei, the folate

con-temit of variotms milk preparatiomis was

deter-mined. There was much variation among the

differemit milk preparations,

but

im all

cases

the vakmes were muchi higher than those

previ-ousby reported. Problems in the interpretation

of these data arid their clinical significance

are

discussed.

J.

LAWIIENCE NAIMAN, M.D.

FRANK A. Osmu,

\I.D.

The Department of Pediatrics, Harvard Medical School am-md ti-me Children’s Hospital Medical Center,

Boston, Massachusetts

.300 Longwood

Avenue

Boston 02115

REFERENCES

1. llodson, A. Z. : The microbiological assay of

“fohic acid!” applied to milk.

J.

Nutrition, 38:

25, 1949.

2. Collins, R. A., et a!.: The folic acid amcl Vitamin

B1 comitemit of the milk of various species.

J.

Nutrition, 43:313, 1951.

3. herbert, V. : The assay and nature of folic acid

activity in human serum.

J.

Clin. Invest., 40:81, 1961.

4. Herbert, \T, Personal comumiiunicatiom.

5. 1Iandbook of Imifant Formulas, Ed. 2. Cleve-land, Ohio: l3aker Laboratories Inc., 1963.

6. Herbert, V., Larrabc’e, A. H., an! Buchanan,

j.

\I. : Studies om-mthe ic!entificatiomi of a folate compound of hum:in serum. J. Chin. Invest.,

41:11:34, 1962.

7. Xaiman, j. L., and! Oski, F. A. : Unpublished observations.

8. Luhby, A. L., amd Silverman, j. \I. : Letter to the Editor. PEDIATIIICS, 32:46:3, 196:3. 9. Shiojania, A. s1., amid Gross, S. : Folic ack!

de-ficiencv and premiiaturity. j. Pecliat., (i4:323, 1964.

\Ve would like to exiiress Our (1l)l)r(ci1tiom1 to

\Iiss Denise lluiiter for her technical assistamice in this )roject amid to the c’mplovees of thie \lilk Laboratory of ti-me Children’s I lospital \Iedical

Center for their cO-ol)eratioml. \Ve should! also like to express our gratitimc!e to Dr. Louis K. Diamond for stimulating our imiterest imi this project.

A New

Method

for Use of the

Crosby-Kugler

Intestinal

Mucosal

Biopsy

Capsule

Intestinal

miicosa!

biopsy’,

introdlucedi

by

Shinerl in 1956, is a imseful diagmiostic am-mci in-vestigatiomiab prcedtire which has received genera! acceptance.2’ Examimiation of fresh

small

intestimial

mnucosa

with

tile dissectimig

microscope and of fixed! sectioned speciniens

with

light

and

electron

microscopes

has

re-vealed abmormalities imi a large number of

disease states. These imchmde non-tropical prue

(

celiac disease, ghimten-sensitive en teropathv,

idiopathic steatorrhea),2_m tropical sprue,

\Vhip-pie’s disease (intestinal hipoclystrophy), regional enteritis, intestimiab lvmphangectasis, Bassemi-Kornzweig syndrome (a-beta-hipoproteinemia,

acanthocvtosis) , acquired idiopathic

hlypogam-Fic. 1. Biopsy capsule assembled and don-me secured

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1964;34;274

Pediatrics

J. Lawrence Naiman and Frank A. Oski

The Folic Acid Content of Milk: Revised Figures Based on an Improved Assay Method

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