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OXALIC ACID IN FOODS AND ITS BEHAVIOR ANDFATE IN THE DIET
E. F. KOHMAN1
C am pbell Soup C om pany, C am den, N. J.
T HR EE F IG UR ES
(R eceived for publication A pril 25, 1939)
One is apt to gather from nutritional literature thatvegetables do not supply calcium efficiently. B ecause this isnot in accord with the results of a number of experiments,some co verin g sev eral ge neration s o f an imals (Kohman , Edd yand associates, '31, '34), this discrepancy presented itself
as a problem wanting solution. As there seemed to be confusion about the amount of oxalates in foods and a scarcityof data as to the behavior and fate of oxalates in the diet,inform ation on these points m ight be expected to throw lighton the availability of calcium . It seem ed desirable for higher
accuracy, to m ake oxalate determ inations under conditionsthat would avoid drastic treatment of the food and entailthe concentration of the oxalate of a relatively large sam ple,freed from most of the other food constituents.
M ETHOD OF ANALYSIS
The follow ing procedure w as used. The sam ples, 400 gm .,w ere first cooked tender by steam ing w ithout drainage loss.A fter thorough disintegration and an approxim ation of w aterco nten t, su fficien t co ncen trated h yd ro ch lo ric acid was ad dedto yield a 15% solution in the water present. This was
1 The experimental work embodied in this paper was done in 1934-1936 while
the author w as w ith the R esearch L aboratory of the N ational C anner 's A ssociation,W ashington, D . C. There he had the assistance of N. H. Sanborn and D. C. Smith.This opportunity is taken to acknowledge their part in the work.
23 3
T HE JO UR NA L. O Õ NU TR ITIO N, VO L. 18, NO . 3
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234 E. F. KOHMAN
allowed to stand at room temperature, w ith occasional stirring, not less than 2 days.The sam ple was then pressed in cheese cloth and the w eights
of the drained material and of the residue recorded. Bydirect titration of a highly diluted sample, after filtration ifnecessary, the chlorine in the tw o fractions w as determ ined.On the assum ption that the oxalic acid distributed itself in thesam e ratio as the chlorine, it could be calculated for the entiresample by a determination on the drained fraction. This assum ption was proved correct for pineapples and turnip greensby adding varying amounts of oxalic acid and subsequentanalysis.
A 250 to 300 cc. sample of the drained liquid was subjectedto 24 hours of wet ether extraction in an apparatus modeledafter that suggested by Palkin, Murray and Watkins ('25)
and suitably proportioned for this purpose using sinteredglass to disperse the ether. In this the liquid to be extractedwas 32 to 37 cm . deep and the ether coursed up through itfrom the 2.6 sq.cm . of fritted glass at a rate of 20 cc. perminute. W hen the extraction was complete, water was addedto the extract, the ether evaporated and the water solutionfiltered. Calcium oxalate was then precipitated. After washing and redissolving a second, and if necessary, a third precipitation w as m ade for com plete purification. T he precipitatewas then titrated with permanganate. By precipitating thecalcium in the titrated medium as calcium oxalate, the purityof the original oxalate could be gauged. A ll determinationswere made in duplicate and the average is given in table 1.To study varietal and other factors more than one sampleof some products were analyzed. As no significance could beplaced on the differences, all the results were averaged andthe number of samples is given in parenthesis.
THE OCCURRENCE OP OXALATES IN FOODS
Only a few foods, notably spinach, Swiss chard, New
Zealand spinach, beet tops, lamb's quarter, poke, purslaneand rhubarb have a high oxalate content. In them, expressed
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ProductV egetables
A sparagus
B eans, green pod (3)
B eans, w ax
B eans, lim a (2)
B eet s, u np ee le d
B ee t le av es
B eet stem s
B ro cc oli, le av es an d f low e rs
Broccol i, s talk s
C abbage (2)
Cabbage sprou ts
Cabbage, Ch in es e
Carrots
Cauliflower
Ce le ry s tal k s, b le ached
Cele ry , s ou p le av e s
Cele ry , s ou p s tem s
C ollards (2)
Chard , Sw i ss , l eav e s
C hard, S wiss, stalk s
C hard , S w iss, leav es an d s talk sChe ne po di um (Lamb 's q uarte r)
C orn, sw eet, w hite
C orn, sw eet, y ellow (2)
C ress, land, w ild
C ress , early f in e c urled
Cucumbers
D and elio ns (3 )
Egg plant
Endiv e (5)
Escarole
Kale
K ale, m inus leaf ribs
L ettuce (6)
M ustard greens (3)
Okra
Onions, green
Parsley
Parsnips
Peas
Pep pe rs , sw e et, gre en (3)
Poke
Po tatoe s, I ri sh
Po tato es , s w ee tPu rs lan e, l eav e s
Pu rs lan e, s talk s
Radishes
TA BL E 1
O xa lic a cid (a nhydr ous) in foods
Total solidi Oxalic acid
(2 )
6.059.587.8024.258.236.606.6610.507.578.808.526.4511.028.904.5814.6610.201
23 5
THE JOURNAL OF NUTRITION, VOL. 18, NO. 3
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TABLE 1†”Cont in ued
Product
Vegetables
RapeEhubarbS pinach (53)S pin ac h, c an ne d (1 2)S pina ch , N ew Z ea la nd, le av esS pina ch , N ew Z ealan d, sta lk sS qu ash , gre en s um m erTur nip s, p ee le dTur nip s, u np ee le dT urn ip g re en s
Fruits
A pples, early sum merApricotsAvacados
BananasBe rr ie s, b la ckB errie s, b lu eB errie s, b lac k ra spB errie s, d ewB erries, green gooseB errie s, red ra spB er ri es , s tr awC herrie s, red so urChe rr ie s, swe et , B in gC urra nts, redG ra pe s, C on co rdG ra pe s, T homp so n's s ee dl es s
GrapefruitL emon s, j ui ce
L emo ns , p ee lL im es , j uic eL im es , p ee lMangoesMe lo ns , c an ta lo upMelons , ca sabaM elons, houey dewMe lo ns , w at erNectarinesO ra ng es , e dib le p or ti onO ra ng es, p ee lP ea ch es , A lb er taPe ac he s, H i le yP ears, B artlettP in ea pp le s, H aw ai ia n c an ne d
P lum s, d am so nPlum s, green gageP ru ne s, It al ia nTomatoes
Total solidi Oxalic acid Calcium
10.826.6210.357.157.608.265.518.166.588.2512.5813.6214.6023.8112.2520.
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OXALIC ACID IN FOODS 237
as anhydrous oxalic acid, it is often considerably over 10%on a dry basis. One per cent in table 1 is equivalent to 1.4%of the usual crystalline form of oxalic acid. In most foodsthere are present mere traces. It is notable, however, thatoxalate was obtained from all but a very few products, mostlyfruits.
One purpose of the analytical survey was to ascertain ifsome varieties of spinach might be relatively free of oxalate.This hope was not realized. In fifty-three sam ples, includingpractically all com mercial and m any experim ental varietiesgrown in California and in M aryland as well as those shipped
from Texas, Florida and Carolina, the average anhydrousoxalic acid content was 9.02% on the dry basis (maximum12.6, m inimum 4.5). The calcium values averaged 1.25%(m axim um 2.50, m inim um 0.44). California spinach was onlyslightly lower in oxalic acid but markedly lower in calcium.California spinach averaged 0.59% calcium (maximum 0.84,m inim um 0.44) w hile M aryland spinach averaged 1.92% (m aximum 2.50, m inimum 1.42). Since a considerable number ofm ineral elem ents are dietary essentials, such a variation raisesa num ber of im portant questions.
PLAN OF FEEDI NG EXPEB I MENT 8
As a number of widely used greens contain insignificantamounts of oxalates, a comparison of these with spinachseem ed in order. G reen leaf-vegetables are our richest calciumsources. In previous experiments Kohman, Eddy and White('37) used diets of canned foods and found them to supply
calcium efficiently. For convenience, therefore, a basal dietof one can each of roast beef, peas, carrots and sweet potatoeswas chosen. The essential analysis expressed in grams was :
OneanBoast
beefPeasCarrotsSweet
potatoesNet
weight362607599551Solids13311145184Calcium0.0540.1060.1470.133Phosphorus0.1550.4740.1560.251
By securing a sufficient amount from one lot of each ofthese, it was possible to have a constant, uniform supply for
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238 E . F. KOHMAN
an entire series of experiments conducted over an extendedperiod of time. These four foods, in the above proportion,were thoroughly mashed and mixed, liquid and all, and hencehad to be eaten in that proportion. This diet was obviouslylow in calcium, i.e., 0.093% . It permitted good but not maxim um grow th and bone form ation, evincing efficient calcium .Small additions of greens (5 to 8%) were made to supply 60%of the calcium of the final m ixture. This raised the calciumcontent to 0.22% .
In experiment 3, table 2, some additions to the basal dietwere on a different basis. The turnip greens in diet C sup
plied only 58.8% of the calcium. It was added in that amountto be compared with diet D , in which it was accompaniedby an equal amount of spinach, which was half the amountof spinach added in diet B to supply 60% of the calcium.The two greens in diet D supplied 70% of the calcium andraised the content to 0.3% . Also in experiment 3, diet D wasplanned to be equal to diets B and E on the basis of crudefiber, since the effect of crude fiber on calcium availabilityhas often been questioned. Diet E is equal to D both in fibera nd calcium.
The rats used were purchased from a large dealer who wasable to supply large litters suitable for apportioning betw een
the diets of a given experiment equally as to weight and sex.They were started on the experiments at 21 or 22 days ofage when they weighed 30 to 35 gm. Six animals were alwaysplaced on each diet and kept in one cage with a raised wirebottom, three mesh to the inch. Except as noted otherw ise theduration of the experiments was 21 days. There was an occasional death during this period and in every case it wasan anim al receiving either spinach or calcium oxalate.
R ES UL TS O F F EE DIN G E XP ER IM EN TS
It is apparent from data in tables 2, 3 and 4 that whereasspinach greatly increases the calcium content of the lowcalcium but w ell perform ing basal diet, it decidedly interfereswith both growth and bone formation. This cannot be ex-
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OXALI C ACI D I N FOODS 2 3 9
T ABL E 2Growth of rats and bone ash of alcohol-ether extracted dry tibia
Dietary additionWeight gainA.- li in
tibiaExperiment
1†”21aysTurnip
greens4.5%)Spinach
(8.2%)yrn.5938%43.233.4Experiment
2†”28aysNoneCalcium
carbonateCalcium
oxalateTurnip
greens4.5%)Spinach(8.2%)6794791067744.750.047.651.645.2Experiment
3—21aysNoneSpinach
(8.8%-23.24m.)Turnip
g re en s ( 4. 4%-11 .6 2m.)Turnip
greens 11.62 gm . + spinach 11.62m.Spinach
23.24 gm . + C aCO, 0.406 gm .D iet
A4Diet
B0Diet
C7Diet
D4Diet
E644.137.853.147.939.5Experiment
4—21aysNoneCanned
spinach b randCanned
spinach b randCanned
s pi nac h b ran dFreshly
cooked spinach534758576245.340.340.943.541.1Experiment6—21aysCalcium
carbonateSpinach,
+ CaCO, equiv alent to ox alate inpinachSpinach
(7%)Kale
(6%)Mustard
greens (5%)768857798451.153.440.553.553.2Experiment
7—1aysNoneCanned
s pin ac h b ran d 1†” otrainedCanned
s pin ac h b ran d 1†”rainedCanned
s pi nac h b ran d 3 †” otrainedCanned
sp in ac h b ran d 3 †”rainedFreshly
coo k ed spinach —o t d rained9%)Freshlycooked spinach —drained (7%)5245545256547547.345.745.144.846.045.549.1
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240 E . F . KOHM AN
plained on the basis of crude f iber. On the other hand greensw ith negligible ox alate content, such as turnip greens, k ale,
m ustard greens and collards, m ark edly im prov e grow th and
bone form ation under sim ilar conditions. If enough calciumcarbonate is added w ith the spinach to balance stoichiom etri-
cally its ox alate, perform ance in the rat is then com parablew ith other greens. T his w ould require, in the f if ty -three
sam ples analy zed, f rom tw o to nine tim es as m uch calcium
as the spinach contains. A general im pression w as obtained
that som e superiority , such as sleek ness of fur, resulted w hen
a low oxalate bearing green supplied calcium as against an
e qu iv ale nt ad ditio n o f c alc ium c arb onate .
In table 2 are recorded the results of a series of ex peri
m ents in w hich the gain in w eight and the per cent ash in the
tibia w as determ ined af ter feeding the basal diet alone and
also the basal diet w ith v arious additions, to sim ilar groups
of anim als. Each f igure represents the av erage of six anim als.
T able 3 records the gain in w eight and the gain in calcium
per anim al in other sim ilar f eeding ex perim ents. T o arriv e
at the calcium per anim al at the tim e the feeding w as started,
a sim ilar group of six anim als w as sacrif iced and their calcium
content determ ined. In all cases w hen an entire anim al w as
analy zed, the food w as rem oved the ev ening before, i.e. 16
hours. In ex perim ent 8, table 3, the food consum ption w asdeterm ined and f rom this the calcium utiliz ation obtained.
It appears that w hen turnip greens w ere added to the basal
diet 79% of the calcium w as utiliz ed but w hen spinach w as
added only 15% was utiliz ed. A ssum ing that the calcium
supplied by the basal diet and the turnip greens w as equally
av ailable, and bearing in m ind that 15% of the total calcium is
only 38% of that supplied by the basal diet, it appears that
the spinach not only supplied no available calcium , but it
actually rendered unav ailable 41% of the calcium of the basal
diet, i.e., the dif f erence betw een 79 and 38. T he sam ple of
spinach used had 10.1% oxalic acid and 1.53% calcium . T he
diet to w hich it w as added then had 0.8% oxalic acid. W hencalcium oxalate w as the addition, 44% of the calcium w as
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OXALIC ACID IN FOODS 241
utilized, showing that the rat can m ake use of a small portionof the calcium in it. It is notable that w ith turnip greensin the diet four times as much calcium per gram of tissuewas deposited as with spinach in the diet. The data in tables3 and 4, experiment 8, indicate a superiority of turnip greensover calcium carbonate in growth, calcium utilization andcalcium deposition per unit body w eight.
T AB LE 3
G rowth record and gain in calcium
Addi ti on t o bas al d ie t( ii ii nin
weightCalcium
p er r at
Total NetainExperiment5—1Controls
â €”21 d ay s o ld
CalciumxalateSpinach(8.2%)
Turn ip g reens (4.5%)ffm.625684f fm.0.1853
0.35350.2449
0.5300Experiment
8—1Controls
â €”2 1 d ay s o ld
CalciumarbonateCalciumoxalateSpinach
Turn ip g reens715637810.2172
0.42050.32860.2447
0.4603daysffm.0.16820.0596
0.3447days0.20330.11150.0275
0.2431Calcium
g ai n p erg ram ga in
ineightgm.0.002670.00106
0.004100.002860.001990.00074
0.00300Calcium
utilized%——70441579
RECOVERY OF CALCIUM AND OXALIC ACID IN URINE AND FECES
In a number of these experiments the calcium and oxalateexcreted in the urine and feces were determined in addition tothe calcium and oxalate in the food consumed and the calciumdeposited in the tissue. This included any endogenous oxalicacid which would be overshadowed by the spinach oxalate.The data are recorded in table 4. The collection period was7 or 8 days duration, thus allowing two periods in a 21-dayexperiment. The collection was begun 3 or 4 days afterthe animals were placed on a diet. There seems to be atendency for less oxalic acid recovery in the second period,indicating that the ability to oxidize it increases with age.A lso there is better utilization of calcium in the second period.
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242 E . F. K O HM AN
While urinary calcium may not seem high with spinachfeeding, it represents a relatively high percentage of assim ilated calcium, probably passing as oxalate through theexc re to ry o rg an s.
In only one case with spinach in the diet was as much of thecalcium utilized as was supplied by the basal diet. The ex-
T AB LE 4
Sec overy o f oxa lic ac id a nd c alc ium
Dietary additionPer cent oxa li c ac idrecovered
In In T t jurine fecesPer
c ent c alc ium Ca lc iumrecovered utilized
In In nvj.,,1urine fecesotolExperiment
2Calcium
oxalateSpinach81039234733*Experiment
3,
f Period 1Spinach X
I P eriodSpinach
+ C aC O3 Ã •_e"Â °, .( Period 214
77 9118 72 90
12 84 9612 780Experiment
5. ,
C Period 1Spinach \ .
[ P erio d-,
, . , . f Period 1C al ci um o xa la te < †”_ .._
J Period 214
1066553447346945341103603370363064Experiment
7,
( P erio dSpinach not drained < ,
(eriod,
( Period 1Spinach drained f _. , .
( Period 21
132 1186502357276 3465332 42
38 6
6257 408 8659 431244157Experiment
8(
Period 1C al ci um c ar bo na te â €¢ }_ .,â €žÂ¿Penod(
Period 1Calcium oxalate < _, . , _
( Pe ri odSpinach
\f
Period 1T urnip ereens •!( Period 213
6118557333
2468
7944
3218
93
33
81038
64 3
6373
62 8
626
1546
6676
7018974
8554
3424
3082
91
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OXALI C ACI D I N FOODS 2 4 3
ception is in table 4, ex perim ent 7. In this the spinach w as
cook ed by dropping it in tw ice its w eight of boiling w ater,
allow ing 7 m inutes to return to boiling and then boiling 10
m inutes. A f ter draining, its w eight w as 58% of the original.
T he undrained spinach w as cook ed 20 m inutes in its adhering
w ater. T here w as, how ev er, v ery poor calcium utiliz ation ev en
w ith drained spinach. N um erous ex perim ental and com
m ercial blanching tests show ed it is dif f icult to rem ov e m ore
than half the oxalate by this process. S pinach calcium is
in so lu ble an d h en ce n ot ex tractab le.
EXTENDED F EEDI NG P ERI OD
In one ex perim ent tw elv e anim als at 21 day s of age w ere
placed on each of tw o diets. In one group spinach supplied
60% of the calcium and in the other turnip greens. B y the
tim e the age of 90 day s w as reached, f iv e anim als on the
spinach diet had died w hile all those on the diet containing
turnip greens w ere in ex cellent condition. T he av erage
w eight at 90 day s of age of anim als receiv ing spinach w as
134 gm . w hile of those receiv ing turnip greens it w as 205.
In due tim e tw o litters of nine y oung each appeared on the
diet w ith turnip greens. T hese w ere reared to the age of
21 day s w hen the av erage w eight w as 39 gm . in one litter and34 in the other, w hile the av erage w eight of their parents at
the sam e age w as 26 gm . S om e tim e later one litter appeared
am ong the anim als on the diet w ith spinach, all but tw o of
w hich w ere dead and these w ere shortly eaten by their m other.
Figures 1, 2 and 3 are photom icrographs of a tooth of an
anim al respectiv ely on each of the three diets in experim ent
3, table 2, i.e., the basal diet alone, w ith spinach added and
w ith turnip greens. T he band across w hich the line is draw nrepresents the dentine lay er of a rat's tooth m agnif ied 310
tim es. T he dark portion of this band designated 'A ' is calci
f ied w hile the light portion 'B ' is uncalcif ied and represents
the area w here new dentine is being form ed. T he w ide un
calcif ied area in f igure 2, representing the spinach diet is
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244 E. F. KOHMAN
apparent. It should be mentioned here that the bones ofanim als receiving spinach w ere very soft and pliable.
DISCUSSION
Insofar as these data are on a comparable basis they arein agreement with the reports of Tisdall and Drake ('38),Fairbanks and M itchell ('38) and Fincke and Sherm an ('35),
published since this work was done. They all dealt withcalcium availability. N o one seem s to have studied heretofore the fate of the oxalates in the diet of the rat. This isperhaps of equal importance. S ome oxalic acid is assim ilated
and tends to carry calcium with it into the urine. In view ofthe low so lu bility o f calcium oxalate, there arises th e qu estio nof any possible dam age of its passage through the excretorytissues. The dem onstration of alm ost universal presence ofsmall am ounts of oxalates in vegetables and fruits tends tothrow doubt on the quantitative aspects of endogenous oxalicacid.
CONCLUSION
O xalates, expressed as anhydrous oxalic acid, have beenshown to occur to the extent of about 10% on a dry basis insp inach , New Zeala nd sp in ach , Swiss ch ard, b eet to ps, lamb's
quarter, poke, purslane and rhubarb. Traces were found inn early all v egetab les an d fru its.
If to a diet of m eat, peas, carrots and sw eet potatoes, relative ly low in calcium but p erm ittin g g oo d tho ug h n ot max imumgrow th and bone form ation, spinach is added to the extent ofabout 8% to supply 60% of the calcium , a high percentageof deaths occurs among rats fed between the age of 21 and90 days. Reproduction is impossible. The bones are extrem ely low in calcium , tooth structure is disorganized anddentine poorly calcified. Spinach not only supplies no available calcium but renders unavailable considerable of that of
the other foods. Considerable of the oxalate appears in theurine, much more in the feces.
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246 E. F. KOHMAN
Turnip greens, m ustard greens, kale and collards, greensw ith negligible oxalates, under sim ilar conditions produceexcellent anim als that deposit four tim es as much calcium perunit body w eight as those receiving spinach.
Ack nowledgmen t is made to Cap t. A . L . Iro ns, D en tal Corp s,U . S. A ., who prepared the photomicrographs, and to theexperimen t s ta tio ns a t Dav is , Califo rn ia , Beltsv ille , Ma ry la ndan d Gen eva , New York , where v ario us samples were o btain ed .
LITERATURE CITED
FAIRBANKS,B. W ., AND H . H. M ITCHELL 1938 The availability of calcium inspinach, in skim milk powder and in calcium oxalate. J. Nutrition,vol. 16, p. 79.
F IN CH E, M . L., A ND H. C . SH ERM AN 1935 T he availability of calcium from som etypical foods. J. Biol. Chem., vol 110, p. 421.
K OH MA N,E . F., W . H . E DD YA ND CEL IA ZA LL G Ü RIN 1931 A canned food diet.Ind. E ng. C hem ., vol. 23, p. 1064.
KOHM AN, E . F., N . H. SANBORN,W . H. EDDY AND CELIA ZALL GURIN 1934Calcium and vitamin D. Ind. Eng. Chem ., vol. 26, p. 758.
KOHMAN, E . F., W . H. EDDY AND MARY E. WHITE 1937 Comparative experim ents w ith canned, hom e cooked and raw food diets. J. N utrition,vol. 14, p. 9.
PALKIN, S., A . G. M URRAYAND H. R. W ATKINS 1925 Automatic devices forextracting alkaloidal solutions. Ind. Eng. Chem ., vol. 17, p. 612.
TISDALL, F. F., AND T. G. H. DRAKE 1938 The utilization of calcium. J.N utrition, vol. 16, p. 613.