CLIN.CHEM.22/7,982-992 (1976)

982 CLINICALCHEMISTRY,Vol.22,No.7,1976

Determination of Erythrocyte Folate by Competitive

Protein Binding Assay Preceded by Extraction

Esper Mortensen

Determination of the concentration of erythrocyte foiateby means of competitive protein binding assay criticallydepends on the extraction procedure applied. Results willbe influenced by variable factors such as the in vitro ageof the blood samples, the degree of hemolysis, the pres-once of ascorbic acid, and the pH during extraction andelimination of proteins. The radioassay is strongly in-fluenced by the pH of the final reaction mixture, the methodused to separate free and protein-bound molecules, andthe molecular configuration of the folates present. Basedon experimental resuits presented, I describe a methodfor the determination of erythrocyte folate.

Additional K.yphras.s: radiolsotopic dilution technique .

assessing folate deficiency #{149}multiple forms of folatenormal values #{149}purity of standards #{149}sources of varia-tion

Evaluation of the folate status of patients is clinically

important in various states of disease and may be ac-complished by several methods: absorption tests, uri-nary excretion tests, various loading tests, and finallyby determining the concentration of folates in samplesof biological fluid. The latter tests are attractive becauseof their simplicity, but in spite of numerous investiga-tions there is as yet no general agreement either onwhether determinations should be performed on serumor erythrocytes (1, 2) or on the method of determina-tion. However, some generalizations emerge from thenumerous reports. While most patients with clinicallyimportant folate deficiency show low serum folate

concentrations, the finding of even very low values inrandom groups of hospital patients is frequent andgreatly reduces the diagnostic significance (3, 4).Erythrocyte folate, however, seems to be diminishedonly in cases of folate or vitamin B12 depletion, and fo-late deficiency states seem consistently to be associatedwith low erythrocyte folate concentrations (4, 5). The

Department of Clinical Chemistry, Kommunehospitalet, 8000Arhus C., Denmark.

Received Feb. 2, 1976; accepted Mar. 19, 1976.

analytical techniques applied are microbiological andisotope dilution analysis. The microbiological methods

are restricted in specificity by the microorganism ap-plied (6) and are sensitive to nonfolate growth factorsand various inhibitors. The isotope dilution methodsshow specificity and sensitivity restricted by the bindingprotein and the tracer applied, and are less laborious

than the microbiological methods.Determination of erythrocyte folate has proved to be

difficult, as reflected in great variability of the publishednormal values (3). Nonlinear dilution curves (7) andlack of correlation between microbiological and isotopedilution methods suggest technical errors of the meth-

ods(4).A series of fundamental difficulties could be antici-

pated: folate in erythrocytes exists in different formswith varied glutarnyl number, varied substituents on N5

and N10, varied degree of reduction of the pyrazine partof the molecule. Owing to chemical lability, intercon-

version and degradation resulting in changes of ana-lytical reactivity are likely to occur. Erythrocytes con-tain a specific binder of folates (8), which may seriouslyinterfere in the analysis. Extraction of unstable com-

pounds and elimination of binding capacity involvepotentially invalidating procedures.

In the erythrocytes, folates are probably found in

several forms (9-11) that are metabolically different,and on a molar basis they are measured with differentsensitivity by the analytical procedures applied (6, .12).The chemical standardization of the analysis is there-fore arbitrary and the results of dubious value from a

strictly biochemical point of view. A thorough clinicaltesting of clinical laboratory methods for the determi-nation of total erythrocyte folate is therefore inevitable.Because of the complexity of the subject it is important,however, that new partial observations are publishedto emphasize new relevant problems. Here, I report thedevelopment of a method for the determination of totalerythrocyte folate by use of radioisotopic dilutiontechnique preceded by extraction.

Blood sample or standard

Saponin/ascorbic acid solutionTrichloroacetic acid

Mix, centrifuge (3000 X g, 10 mm)Supematant fluidReaction bufferTracer working solutionBinder solution

Mix well, incubate (45 #{176}C,30 mm)Transfer to 4 #{176}Cwater bath, incubate for 2 mm

Charcoal reagent (4 #{176}C)Mix well, incubate (4 #{176}C,20 mm)Centrifuge at 4 #{176}C,(2500 X g, 15 mm)

“Instagel”Supematant fluid

Mix well and count for 4 mm

40

30

20

10

8

5 10 15 20 25 100 Z

FIg. 1.Relationbetween fraction of tracer bound and concen-tration of binder addedSeries of standerd 0 values were prepered with normal tracer dose (30 000 cpm,curve B) and double tracer dose (60 000 cpm, curve A) to demonstrate theconcentration range of the bInder (2-5%) gMng minimal increase of the boundfraction (curve A-curve B) and acceptable radioactivity of the supernate. C’-dinate: radioactivity bound. Abscissa: concentration ofbinder

Materials and MethodsMaterials

Tracer: High specific activity [3HJfolic acid (>10kCi/mol, Code TRK 212; The Radiochemical Centre,Amersham, England). An amount equivalent to 250 Ciis dissolved in 100 ml of distilled water to form a stock

solution, which is kept at -20 #{176}Cin 1-ml aliquots. Thesolution is stable for about three months without loss

of binding ability. Working isotope solutions are pre-pared daily from the stock solution by dilution with thereaction buffer: 1 ml of stock solution to 5-7 ml of bufferaccording to the current specific activity. The concen-tration of folic acid in the working solution is about 5

big/liter.Binder: The binder was prepared according to

Rothenberg et al. (4), finally dialyzed vs. the reactionbuffer, and the final stock binder solution was kept at-20 #{176}C,in 1-ml aliquots, which were stable for three

months. Working binder solutions were prepared bydilution with reaction buffer according to binding ex-periments.

Standards: D,L-5-Methyltetrahydrofolic acid, bariumsalt (Sigma Chemical Co., St. Louis, Mo. 63178) wasassayed spectrophotometrically using s = 665 at 290 nm.A stock solution (100 mg/liter) was prepared in physi-ological saline with 2-mercaptoethanol (400 nmol/liter)and was kept at -20 #{176}Cin the dark. This solution wasstable for at least two months. Working standards wereprepared for each run by appropriate dilution, withphysiological saline containing 2000 mg of ascorbic acidper liter, to concentrations of 0, 50, 100, 200, 400, and800 ig of the barium salt per liter.

Reaction buffer: Diethanolamine, 1.0 mol/liter, pH

9.3 (37 #{176}C).Coated charcoal reagent: Activated charcoal (Sigma),

3.5 g, suspended in 50 ml of demineralized water, was

mixed with an equal volume of demineralized water

containing 0.75 g of gelatin (Merck, Darmstadt, W.Germany). This reagent was prepared daily.

Trichloroacetic acid solution: 100 g/liter. Stable.Saponirz solution: 200 g/liter (Merck, code No. 7699).

Stable.Saponin/ascorbic acid solution: 10 g of L-ascorbic

acid plus 2 ml of the saponin solution were dissolved in

100 ml of distilled water. This reagent was freshly pre-pared for each run.

Procedure

Add heparinized blood1 or standards, 250 Ml, to 750tl of saponin/ascorbic acid solution. Mix well. After 10mm at room temperature add 1.5 ml of trichioroaceticacid. Mix well. Centrifuge at 3000 X g for 10 mm at roomtemperature. Add 200 ul of the clear supernatant fluidto 600 ui of the reaction buffer. Add 100 l of the tracerworking solution and, after thorough mixing, add 200

il of the binder working solution. Mix well and leave thetubes in a water bath at 45 #{176}Cfor 30 mm. Transfer thereaction tubes to a cooling bath (<4 #{176}C)and after 2 mmadd 1.0 ml of the cold coated charcoal reagent. Leave thetubes for 20 mm and centrifuge for 20 mm at 3000 X g

‘EDTA-treated blood may be used as well.

Table 1. Analytical ProtocolCPM

A

CLINICALCHEMISTRY.Vol. 22,No.7,1976 983

250 il750 iI

1500 il

200 Ml600 uI100 l200 l

1000zl

15 ml1000 I

cpm Stdo-S, cpm Stdo Sf4. cpin

A

Table 2. The Effect of Varying the pH of theReaction Mixture in the Binding of the Tracer, the

Standards, and Sample ExtractsSf4-S

pHI. Std. 200

8.60 4510 8990 0.67 13500

8.33 5420 8080 0.60 133008.20 5740 7760 0.58 136008.10 6000 7500 0.56 137007.95 6380 7120 0.53 135007.85 6670 6830 0.51 137007.75 7110 6390 0.47 13700

7.60 7560 5940 0.44 13500

II. Std. 2008.10 6250 7250 0.54 13500

8.32 5590 7910 0.59 134008.63 4550 8950 0.66 134009.27 2850 10650 0.79 13600

10.67 1190 145010.85 1250 132010.90 1300

II. Blood sample8.17 5470 8030 0.608.38 4810 8690 0.648.70 3850 9550 0.719.30 1900 11600 0.86

10.70 1250

10.90 1340

III. Std 2008.10 6580 7920 0.55 14500

7.85 7200 7300 0.50 146007.62 7700 6800 0.47 145007.35 8300 6200 0.43 145006.90 8800 5700 0.39 144004.25 1060 11001.70 800 900

Ill. Blood sample8.15 5770 8730 0.60

7.90 7230 7270 0.507.65 8540 5960 0.41

7.37 9800 4500 0.31

6.93 11000 3500 0.24

4.30 11501.80 800

Is l pan t ,s

12 45#{176}-ST-fl

20#{176}-ST-a

10

8

45#{176}-ST-tOO6

5#{176}-ST-fl

LI

0 10 20 30 40 50 60 MINUTES

Fig.2. Influenceof time and temperature on the binding of[3H]follcacid tothe milk binderBinding of the tracer and methyltetrahydrofolate (ST 400 = methyltetrahydro-folate, 400 ng/ml) was controlled at the temperature selected, 45#{176}C.OrdInate:bound fraction, expressed as cpm of supernate. Abscissa: period of Incubationbefore separation with charcoal

cpnx

16

KXXX14

u

12

10

8

6

LI

2

7 8 9

Ag. 3. Effect of the pH of the reaction mixture of the binding ofthe tracer (-x-x-), the tracer and methyltetrahydrofolate standard(-0-0). and the tracer and erythrocyte extract (-S-#{149})Ordinate: bound fraction, as cpm for supernate. Abscissa: pH of the reactionmIxture

at 4 #{176}C.Add 1.0-ml aliquots of supernatant solutions tocounting vials containing 15 ml of scintillation cocktail.Count for 4 mm in a suitable liquid scintillation counter.Make calculations from a simple linear plot of con-centrations (abscissa) vs. the counts of the corre-sponding bound fractions (ordinate). Run one sampleblank value and one standard blank value as control.

The blanks are prepared by substituting the milk binderfor an equal volume of reaction buffer.

In preliminary experiments the following reagentswere used: [5- ‘4C]Methyl-tetrahydrofolic acid, bariumsalt (The Radiochemical Centre), specific activity >45Ci/mol. Dilutions were made with distilled water asspecified.

Tris (tris (hydroxymethyl)aminomethane) buffer,0.5-1.0 mol/liter. I varied the pH by adding hydro-chloric acid as specified in the text.

CPMx i0

984 CLINICAL CHEMISTRY,Vol. 22, No. 7, 1976

R.agent no.

cpm

II:

III:

IV:

V:

VI:

VII:

VIII:

I

16027662465

32146

30319

31116

3182

27934

92

2

16027662465

32146

30319

33120

3204

29916

99

3

16027662465

32146

30319

33352

3700

29652

98

4

160276

62465

32146

30319

20726

2934

17792

59

$160276

62465

32146

30319

3666

3160

506

2

V-Vl

VII in % of IV

All reagents contained 359 of charcoal per liter.Reagents 1, 2. and 3 contained gelatIn 5. 7.5, and lOg per liter, respectively.Reagent no. 4 contained 7.5 g of dextran per liter: reagent no. 5 contaIned 7.5 g of albumin per liter.

1

2

1

2

14600 cpm

13100 cpm

1400 cpm

1285 cpm

14100 cpm

12900 cpm

1400 cpm1600 cpm

‘Normal binder diluted 20-fold was compared to binder solutions diluted10-fold, which had been treated with equal volumes of charcoal reagentscontaining charcoal 35 9/liter and (A) gelatIn, 7.5 guitar, or (B) albumin, 7.5g/liter.

The figures represent standard zero values, prepared with the binders. Re-sults from two different experiments are given.

CLINICALCHEMISTRY, Vol.22.No. 7,1976 985

Table 3. Comparisonof EquilibriumDialysisand Separationby Charcoal

Radioactivity added

Inner fluid cpm/ml

Ext. fluid cpm/ml

cpm bound in inner fluid

cpm, inner fluid, after separationwith charcoal

cpm In external fluid afterseparation with charcoal

Table 4. Adsorption of Milk Binder to CharcoalaAdsorbd binder Natlv. blndsr

Reagent A

Reagent B

Citric acid/phosphate buffer, pH 5.8: Mix 40 ml ofa 0.1 mol/liter solution of citric acid with 60 ml of a 0.2mol/liter solution of Na2HPO4.

Measurements of pH were at 37 #{176}Cwith a micro-electrode system (Radiometer, Copenhagen, Denmark).

Experiments and Results

Isotope Dilution Analysis

The equilibrium technique was preferred.Tracer dose: Considering the specific activity ob-

tainable and the amount of folate in samples, I fixed thetracer dose at about 0.5 ng per tube. With a countingefficiency of about 45% and half of the reaction mixturetransferable to the counting tubes, a maximal count ofabout 25000 counts per minute was found and acounting precision of 1% could be reached within a fewminutes, even with the highest standards.

Binding of the tracer to the milk binder was inves-tigated. The experimental procedure used appears in

Table 1. The amount of binder applied per tube wasselected from binder dilution curves as depicted inFigure 1. Dilution curves were run with single anddouble tracer dose to delineate dilutions with acceptablebinding capacity and minimal increases of the boundfraction during analysis.

Incubation and pH. The influence of the temperatureand the duration of incubation under the conditions of

the assay was investigated. The results are thown inFigure 2. Temperature and time were fixed accordingly

at 40 #{176}Cand 30 mm.The effect of varying the pH during incubation was

tested, with 0.5 mol/liter Tris buffer as reaction buffer.The pH was varied by adding hydrochloric acid or so-dium hydroxide. Binding was studied for the traceralone (standard zero) and in the presence of methylte-trahydrofolate (standard 200 pg/liter) or erythrocyteextract. The results appear in Figure 3 and Table 2.Binding of folic acid tracer was independent of pH from

pH 6.9-9.3, whereas there was no binding between pH4.3 and 6.9 and between pH 9.3 and 10.7. The competingeffect of methyltetrahydrofolate and erythrocyte ex-tracts increased with increasing pH values within thelimits pH 6.9-9.3, resulting in increasing analyticalsensitivity (Table 2). A reaction buffer with a pH of 9.3was accordingly chosen. Identical results were obtainedwith Tris buffer, borate buffer, and diethanolaminebuffer.

The separation of free and protein bound ligandmolecules by means of coated charcoal reagents. Thiswas tested applying the dialysis method previouslydescribed (13). The following charcoal reagents weretested: (a) charcoal, 35 g/liter, containing 5.0, 7.5, and10.0 g of gelatin per liter; (b) charcoal 50 g/liter, con-taining 7.5 g of gelatin per liter; (c) charcoal, 35 g/liter,containing dextran, 7.5 g/liter (“Dextran T 70”; Phar-macia, Uppsala, Sweden); (d) charcoal, 35 g/liter, con-

12

10

12

10

80

60

20

2 24 6 pH

1

cosio3 (p11,1050C0VERY

986 CLINICALCHEMISTRY,Vol. 22, No. 7, 1976

25 50 75 i50 I 5 10 15 20 25 018

Fig. 4. Effect of the amount of charcoal (left) and the time (rit)on the bound fraction of the tracerCurve A represents the separation with gelatin-coated charcoal. Curve B showsseparatIon with albumin-coated charcoal. Inates: bound fraction, as cpmfor supemate after separation with charcoal. Abscissa: (Iefl) percent dilutionof charcoal reagent; (rl’fl) time In contact with charcoal

12

10

3

2

CPMo

Fig. 5. Separation of tracer stock solution by chromatography(14)Ordinate: cpm of eluted fractions. Abscissa: fraction number. - - - fresh tracerstock. -- tracer stock after storage for two months. The identities of peaksI, II, and IIIwere confIrmed by comparison with authentic substances

taming 7.5 g of albumin per liter (fraction V; ArmourPharmaceutical Co., England). The results appear inTable 3. Under the conditions of the assay, good sepa-ration was obtained with charcoal, 35 g/liter, containing

gelatin 7.5 g/liter, and I selected this reagent for routineuse. No adsorption of the binding protein to the charcoalwas detected (Table 4). Finally, under the actual con-ditions of the assay the charcoal reagent containing, perliter, 35 g of charcoal and 7.5 g of gelatin was comparedto a reagent containing 35 g of charcoal and 7.5 g of al-

Fig. 6. Relation between pH and recovery50 ng of [C’4)methyftetrahydrofollc acid (spec. acty., 60 CI/mol) was added toa mixture of 1.0 ml of hetnolysate +1.0 ml of ascorbic acid (40 g/llter)+1.0 mlof hydrochloric acid of various concentrations (from 0.0 to 0.5 mol/Ilter). Themixture was ultratiltered and the radloactMty and pH of the filtrate determined.Controls were run In which the hemolysate was replaced by saline. Ordinate:recovery of radioactivity, as percentage of controls. Abscissa: pH of the ultra-filtrates. Results from three experIments are given

bumin per liter (Figure 4). The bound fractions foundwhen gelatin/charcoal was used seem to be independentof the amount of charcoal applied and the duration ofthe separation within wide limits. With albumin-coatedcharcoal, the bound fractions were poorly defined.

The nonadsorbable fraction of the tracer normallyamounts to 4-5% of the total count and increases onstorage of the tracer. Separation at 4 #{176}Cwas preferredbecause of better reproducibility. Blank values de-creased with increasing time of separation up to 20 mm,which was used for the normal analytical procedure.The increase of the blank values upon storage was in-vestigated. A fresh tracer stock was subjected to frac-tionation by column chromatography according toNixon and Bertino (14). Fractionation was repeated 10weeks later. The results appear in Figure 5. During the10 weeks the blank values increased steadily from 900cpm to 3500 cpm. Figure 5 shows that during storage[3H]folic acid is converted to a pteridine and p-amino-

benzoylglutamate. Fraction I, II, and III (from Figure5) were diluted to 7000 cpm and separated with coatedcharcoal. The resulting blank values were: fraction I, 140cpm; II, 240 cpm; and fraction III, 2100 cpm. Evidentlythe pteridine is poorly adsorbed to charcoal and will giverise to increasing blank values when the tracer decom-

poses.

Extraction of Erythrocyte Folate

A protein folate binder was demonstrated earlier (8).

Several authors have shown that folates in the eryth-rocyte are combined with different numbers of glutamylresidues and so have different reactivity in microbio-logical assay systems, necessitating a pretreatment of

25

20

15

10

CiTP5L

Ix1 2 3 4 5 6 7 8 9 10 I1YSATENO

Fig. 7. Results of dialysis experimentOrdinate: cpm for external fluid. Abscissa: Individual hemolysates. x, dialysiswithout ascorbate; #{149},dialysis with ascorbIc acid included in the buffer

86/&

200

100

12345578 91OXASCO#BICACID

HG/IlL

I’

A

B

CPIl x

14

12

10

1 2 3 4 5 pH

Fig. 9. Relation between the amount of folate extracted from ablood sample (A) and the pH during extraction (abscissa)A citric acid/hydrochloric acid buffer series containing ascorbic acid (2 guItar)was applied. Another sample (B) was extracted with ascorbic acid (60 g/liter).The pH durIng the extraction was varied by adding sodium hydroxide. Ordinate:cpm for the supernates after separation with charcoal reagent

Ct,, io3

CLINICALCHEMISTRY,Vol. 22, No. 7, 1976 987

hemolysates with “conjugase” before analysis. In anextraction procedure one should therefore consider: (a)stabilization of the oxolabile tetrahydrofolate deriva-tives that constitute most of the erythrocyte folate, (b)

breaking of binding of folates to the specific proteinbinder, and (c) denaturation of the binder and other cellproteins to eliminate protein interference during anal-ysis.

Folate binding of erythrocytes was evaluated in twoseries of experiments:

[‘4CJMethyl-tetrahydrofolate was added to hemo-lysate mixtures of various compositions and ultrafil-tered as described by Pedersen (15). Recovery was de-termined relative to the appropriate controls. The re-sults appear in Figure 6. Full recovery was only ob-tainable when the pH was <2.5.

Dialysis experiments were performed by a technique

previously described (13). Preliminary experimentsestablished that dialysis equilibrium could be attainedfor [3Hjfolic acid at 37 #{176}Cin 24 h. As inner fluid I used1.0 ml of hemolysate. The external fluid consisted of 3.0ml of a citrate/phosphate buffer (pH 5.9) to which[3HJfolic acid was added. Control experiments, in whichthe hemolysate was replaced by buffer as inner fluid,were run with each set of experiments. The effect ofadding ascorbic acid (50 g/liter of buffer) was tested inthe same series of experiments. The results appear inFigure 7. The distribution of radioactivity betweeninner fluid and external fluid indicates binding to thehemolysates, which was partly inhibited by the presenceof ascorbic acid. Previous investigators (5, 16) using themicrobiological assay have demonstrated the influenceof the degree of hemolysis of the samples before ex-

Table 5. Tentative ExtractionProcedure

Sample, 250 iIAscorbic acid, 750 sI Hemolysis, anti-oxidant, reduction

of pHTrichloroacetic acid, Precipitation of proteins

1500 tl

1 2 3 4 5 PH

Fig. 8. Top: Relation between concentration of ascorbic acidduring extraction (abscissa) and concentration of folate, asobtained with three different blood samples (x-O-) (ordinate).Bottom: Relation between the pH of the hemolysate/ascorbicacid mixture (abscissa) and the concentration of folate found(ordinate)

Table 6. Influence of pH durIng Extraction

Buffer5

A+B1+9

2+83+74+64.5 + 5.55+56+4

Ascorbic acid,

520

60

Blood sampi.., cpm

no.1 no.214500 1380013900 1400014400 1420014300 1390014200 1400014300 1440014500 14500

pH of the

#{149}xtract

1.5

2.03.03.6

3.84.04.3

9/liter4.23.52.9

2.9

2.92.92.92.9

9100

7300

7000

14700

8800640045003000

83006900

6700

Standard curveprepared with

ascorbic acid(60 9/liter)

Proc.dur.

Bloodsample

Standard, 200

ag/lIter

cpm

42603830

30 s60 s

Saponin added

4900

4880

3860 49203810

382049004940

988 CLINICAL CHEMISTRY, Vol. 22, No. 7, 1976

traction, of the presence of ascorbic acid, and of the

exposure of the hemolysate to the influence of plasma.To evaluate the effect of these variable factors, Iadopted a tentative basic extraction procedure (Table5), which was varied as described further on. The con-centration of ascorbic acid during extraction was variedand the pH of the mixture of hemolysate and ascorbicacid was measured. Figure 8 shows the effect on theconcentration of folate measured. Increasing folateconcentrations were obtained with increasing concen-trations of ascorbic acid and decreasing pH values. Toestablish the influence of the pH alone, I replacedascorbic acid with a citrate/hydrochloric acid bufferseries and repeated the experiments. Measurementsincluded a series with ascorbic acid, 2 g/liter, added tothe buffer series. I concluded that maximal folate con-centrations are obtained at high concentrations of as-corbate within wide pH limits (Figures 8 and 9), whereasthe folate concentration obtained at low ascorbate

concentrations decreases with the pH of the mixture.Extraction in the absence of ascorbate over a wide rangeof pH values yields very low folate concentrations(Table 6) as compared to the values obtained with as-corbate present at the same pH values. Ascorbic acid(100 g/liter) was chosen for further experiments.

The effect of the degree of hemolysis of the sample

during extraction was examined in several experiments

by comparing results obtained by the standard tech-nique, the standard technique plus ultrasonic treat-ment, and extraction with saponin added to the ascorbic

acid solution. Typical results are summarized in Table7. As a consequence saponin was included in the ex-traction procedure.

I examined the effect of incubating the hemolysateswith plasma. Blood samples were washed with salineand were resuspended in (a) native plasma, (b) plasmapreheated at 56 #{176}Cfor 10 mm, and (c) saline. The

samples were then extracted, with use of an incubationtime of 20 mm after addition of ascorbic acid. Table 8shows typical results. No effect on the folate concen-tration could be demonstrated. In addition, hemolyzedsamples of whole blood were incubated at pH 3 and 6 for6-60 mm without any apparent effect on the concen-tration of folate obtained.

To test whether protein interference had been abol-ished by the trichloroacetic precipitation procedure, I

extracted a series of blood samples and prepared blanksby substituting the milk binder with an equal volumeof the reaction buffer. The results were compared toresults from a similar series of standard blank values.

Sample blank values: mean, 1235 cpm (SD, 25 cpm), n= 10. Standard blank values: mean, 1243 cpm (SD, 20

cpm), n = 10. Blank values of standards were indepen-dent of their concentration of folate.

To test that the reactivity of the folate standards wasnot modified by the extraction procedure, I comparednormal standard curves to standard curves prepared byadding calculated amounts of methyltetrahydrofolateto extracts from zero standards after adding the reactionbuffer. Identity of the standard curves, within experi-

Standards:std 0std 100std 200

std 400std 800a The buffer series was prepared by mixing solution A and B in propor-

tions shown. Solution A contained 21 g of citric acid and 8 g of sodium hy-droxide per iiter. Solution B was hydrochloric acid, 0.1 mol/liter.

Table 7. Effect of Hemolysis on the Radioactivityof Supernates

Ascorbic acid+ ultrasonic

treatment, 15 s

mental error, proved that the standards as normallyprepared did not change reactivity during the extractionprocedure. However, when standards prepared withoutascorbic acid were exposed to the trichloroacetic acidbefore adding ascorbic acid, there was a loss of reactivity(Figure 10).

Evaluation of the Final Method

Reproducibility: (a) Within run: 10 complete de-terminations of folate concentration in the same sampleof blood showed a mean value of 415 pg/liter (SD, 9.5tg/liter), and the coefficient of variation was 2.3%. (b)

Between runs: In normal laboratory routine during sixweeks, 25 determinations of the folate concentrationwere made by four different technicians on separate

Nativ, plasma

5280576046505180

SalIne

5220571046205210

CPMx

20 40 60 80 1002

12

10

8

NO/Nt.

600

500

400

300

200

100

NO/IlL

600

200

100

60 100 150 200 NOADDEDPERML

Fig. 11. Top: Sample dilution curves. 10 different hemolysateswere diluted with saline, two- and fourfold, and analyzedOrdinate: concentration of folate found. Abscissa: concentration of hemolysate

Bottom: Recovery experiments. 10 dIfferent samples were en-riched with methyltetrahydrofolate and assayedAbscissa: amount of methyltetrahydrofolate added

A

BID

CLINICAL CHEMISTRY, Vol. 22, No. 7, 1976 989

Table 8. Effect of incubationa on ExtractionofFourBloodSamples

cpm for the bound fractIons with usa of

H.atesl plasma

5400577046905050

a Samples Incubated at 20 #{176}Cfor 20 mm before precipitation with trlchlo-

roacetlc acid.

100 200 300 400 NG/NL

Fig. 10. Standard curves prepared by using standards withoutascorbate (A and B) and wIth ascorbic acid, 2.0 9/liter (C andD)in curves A and C, the trlcitloroacetic acid was added before the saponln/ascorbic acid solution. Curve B wes prepared according to the normal procedure.Curve Dwas prepared by extracting zero standards only and adding 20 i of thevarious standards to the reaction mixture with the tracer. Ordinate: cpm forsupernates after separation with charcoai. Abscissa: nominal concentrationof the standards

days on a pool of methyltetrahydrofolate in physiolog-ical saline to which had been added ascorbic acid, 2g/liter. The mean value was 203 pg/liter (SD, 8 pg/liter),and the coefficient of variation was 4%. Similarly, de-terminations were made on a serum pool enriched withfolic acid: mean value, 198 pig/liter (SD, 14 pg/liter), witha coefficient of variation of 7%. The pools were storedat -22 #{176}C.

Recovery and dilution experiments: Ten bloodsamples were enriched with 0, 50, 100, and 200 pg ofmethyltetrahydrofolate per liter and the concentration

of folate was determined (Figure 11). Analytical re-covery was within the experimental error of the method.10 blood samples were diluted two- and fourfold with

saline and the concentration of folate was determined

(Figure 11). Linear dilution curves through zero were

obtained within the experimental error of the method.Specificity of the method was tested by substituting

various folate derivatives2 for methyltetrahydrofolate,in equal amounts. Figure 12 demonstrates the greatvariability of affinity of the various derivatives for themilk binder under the conditions of the assay. All folatestested except folic acid itself showed increasing bindingaffinity with increasing pH values from 8 to 9.3. Detailed

results will be reported later.Stability of assay and samples. Long-term stability

of the assay was tested by running normal materials ata time interval of six months. Blood samples were ob-tained from apparently healthy volunteer blood donors,20 to 50 years old. The results appear in Figure 13. Nosignificant difference was observed. Concentrations offolate in erythrocytes are calculated from concentra-tions of blood samples by means of the hematocrit value.No correction for serum folate was considered necessary.Values >250 pg/liter are considered normal. This cor-responds to >400 nmol/liter of erythrocytes, based on

2Generously supplied by Dr. J. Ingerslev, Kommunehospitalet,

Arhus, Denmark.

18

16 6-7-8

14

12

10

4 4

2 2

ND/MI

Fig. 12. Standard curves prepared with various folate derivatesI folic acId. 2 = dihydrofoiic acid. 3= tetrahydi-ofolic acid. 4 5-methylte-

trahydrofolic acid. 5 = 5-formyltelrahydroiolic acId. 6 = pterln. 7 = pteroic acid.8 p-eminobenzolc acid monoglutamate

200 400 600 800 1000

22

20

18

16

14

12

10

“O

FIg. 13.Erythrocyte folate concentration of normal blood donors-75 persons run initially. ----40 persons examined six months later. Or-dinate: number of persons. Abscissa: concentrations of folate

2 4 6 8 10 x100iG/lI.

1PMx io

990 CLINICAL CHEMISTRY, Vol. 22, No. 7, 1976

the molecular weight of the barium salt of D,L-N-5-methyltetrahydrofolic acid of 595 and a spectrophoto-metric purity of about 75%, as measured at 290 nm.

Ten samples were examined immediately and after

storage for 24 and 48 h at 4#{176}C(Table 9). Based on theseand similar results, storage for as long as 48 h at 4 #{176}Cbefore analysis is acceptable.

Discussion

This method satisfies most the basic criteria for ac-curacy and precision for a diagnostic clinical laboratorymethod. Rectilinear dilution curves through zero andfull analytical recovery of added standard material tosamples can be obtained. Good precision within-run andbetween-runs and a satisfactory long-term stability ofnormal values as measured for healthy blood donorsdemonstrate that most of the important variable factorsinfluencing the analysis are controlled.

However, the specificity of the analysis is restricted.As demonstrated in Figure 12, the milk binder applied

shows different binding affinity for the various molec-ular species of folate tested, and the binding affmity canbe demonstrated to be extremely dependent on the pHof the reaction mixture (Figure 3). Even more serious

errors are introduced as a result of the standardizationprocedure. Folates as delivered by the various manu-facturers are impure and often unstable. For radio-chemical determinations of folates D,L-N-5-methyl-tetrahydrofolate has commonly been used as a standardmaterial. Some workers (17) simply correct for impurityaccording to the manufacturer’s specifications. Otherworkers correct for impurities spectrophotometrically

by using o = 665 at 290 nm (12, 18, 19), whereas nocorrection at all was described in several papers (20-22).

Only one group of workers (23) did in fact try to verifythe purity of their preparation by chromatography anddifferential growth response by Lactobacillus casei andStreptococcus faecalis. The most commonly usedstandard material is D,L-N-5-methyltetrahydrofolicacid, as the barium salt, from Sigma Chemical Co.Weight concentrations of the barium salt are often usedindiscriminately with weight concentrations of the aciditself, resulting in differences of about 30% on a molarbasis. Gupta and Huennekens (24) described thepreparation and properties of D,L-N-5-methylte-trahydrofolic acid and related compounds and givespectrophotometric data. For pure N-5-methylte-trahydrofolate the 290 nm/245 nm absorbance ratioshould exceed 3.3. Conversion of the tetrahydrofolate

to dihydrofolate is accompanied by very little changeof the absorbance at 290 nm, but because the absorb-

ance ratio for dihydrofolate (290 nm/245 nm) is about1.5 at pH 7, admixture of dihydrofolate to tetrahydro-folate will only be detected by measuring this ratio. Forroutine preparations from Sigma (unpublished obser-vations) a ratio of 2.6-2.8 is commonly found, indicatingimpurities, among them most likely the dihydro deriv-ative. Figure 12 shows the change of binding affinity ofthe standards. Increased affinity of standards for themilk binder was reported by Waxman and Schreiber(23). Decreased reactivity is a common finding when oldstandards are used. Neither the increase nor the de-crease can be properly corrected by spectrophotometricrecertification at 290 nm. It can be concluded, therefore,that one of the main causes for differing results between

Folat., gflft.r

470 460470 460

220 220295 305530 560

205 195

365 300

250 200

335 390

470 430

Mean 361 352

Mean difference -10

SD of differences 28

CLINICAL CHEMISTRY, Vol. 22, No. 7, 1976 991

Table 9. Results of Storage Experiments on 10Samples

Storage for

Oh 24h 48h

different investigators is the use of impure and unstablestandard material and use of different pH values for thebinding reaction. It would probably be better to use folicacid as a standard: it is stable, spectrometrically wellcharacterized, cheap, and easy to purify. Perhaps themost important step of the analysis is the extractionprocedure. Valuable basic information concerningerythrocyte folates was published in a series of papersby Toennies et al. (25-28). Evidence was presented thatfolates exist mainly in a precursor form of high molec-ular weight, which is assayable by microbiologicalmethods only after exposure to a plasma factor of en-

zymatic nature. The effect of enzymes of organ extractshad previously been demonstrated by other investiga-tors [see (29)]. With the development of chromato-graphic techniques for the separation of folates (9, 14,28, 30) fractionation of the precursor substance became

possible. The experimental model of investigations hasnormally been extraction of pooled erythrocytes, andfractionation on columns of various types, followed bymicrobiological assays before and after enzymatic

treatment of the fractions obtained. Folates have beenidentified according to the elution pattern and the mi-crobiological response obtained. The results of theseinvestigations have been conflicting as to the numberof fractions found and to the identity of the individualfractions (9, 28, 30). In evaluating these reports thefollowing points should be thoroughly considered: (a)The starting material should be specified as to the invitro age of the cells. The instability of folates inerythrocytes on storage has been substantiated (4, 5,31). Hemolysis and freezing influence the results (5, 16,

31). (b) The extraction procedure may greatly influenceresults. Heating, acidification, the concentration of

ascorbic acid, contact with plasma, the degree of he-molysis all influence the results (4,5, 16, 27,28, 30) andmay explain some of the divergence found. Retentionin coagulum was demonstrated by Iwai et al. (28). (c)

The chromatographic separation procedures result inpatterns of elution of folates that depend strongly onthe physical characteristics of the system and the

460 chemical configuration of the folate compounds, viz.,430 the N5-N10 substituents, the degree of reduction of the

230 pyrazine ring, and the number of glutamic acid conju-320 gates. The recovery from columns has generally been500 poor (27), and a strongly bound fraction, elutable only220 with sodium hydroxide, was reported by Cooper and

Lowenstein (35). Although reference folates are difficult230 to obtain and often unstable, elution patterns should

be established for native folates as well as for “extract-

425 ed” folates by the individual investigators. Elution

351 patterns of reference folates can be followed by pho-tometry, when run in high concentrations. However,

10 elution patterns of folates from biological materials after

27 extraction can only be traced by extremely sensitivemethods that can measure l0_12 g/ml; until now this hasonly been possible by microbiological methods. Mostinvestigators (9, 10,28, 30, 31) have used the differentialgrowth response of Lactobacillus casei and Strepto-coccus faecalis and Pediococcus cerevisiae on fractionsof the eluate before and after incubation with enzymepreparations. The responses obtained are not unam-biguous. Selective stimulation of the growth of L. caseican be obtained with folates of higher glutamyl numberthan 2, and with 5-methyltetrahydrofolate (6, 31, 32).The extraction procedure described in this paper issimple, reproducible, and gives a maximal response.Dilution and recovery experiments are satisfactory, andthe procedure does not affect the response of syntheticmonoglutamate derivatives. Identity between standardand sample blank values shows that any endogenousunsaturated binding capacity has been eliminated.However, two sources of error may still exist. First, se-lective retention of folates in the coagulum cannot beexcluded. Second, folates may be inter-converted duringextraction. Reduction of L-dihydrofolates may resultin formation of D,L-1-tetrahydrofolates in racemicmixture, and these may not be able to compete for themilk binder (36, 37). Moreover, the effect of the ex-traction procedure on the number of glutamic acidresidues has not been examined. According toRothenberg and da Costa (38) the binding affinity offolates is independent of the number of glutamic acidresidues, whereas Schreiber and Waxman (39) reportedthat the binding affinity for the milk binder was affectedand recommended the use of the triglutamate for’m offolic acid for standardization of analysis of erythrocytefolate. The folate concentration of erythrocytes as de-termined by the various methods applied must beconsidered operationally defined. By analogy with thedetermination of vitamin B12 as cyanocobalamin, thisdoes not preclude, however, that determinations will beuseful, but stresses the importance of thorough clinicalevaluation and further basic research.

992 CLINICAL CHEMISTRY, Vol. 22, No. 7, 1976

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2. Chanarin, I., The Megaioblo.stic Anemias. Blackwell ScientificPub!,, Oxford, 1969, p317.

3. Ibid., p 323.

4. Rothenberg, S. P., da Costa, M., Lawson, J., and Rosenberg, Z., Thedetermination of erythrocyte folate concentrations using a two phaseligand-binding radioassay. Blood 43,437 (1974).

5, Hoffbrand, A. V., Newcombe, B., and Mollin, D. L., Method ofassay of red cell folate activity and the value of the assay as a test forfolate deficiency. J. Clin. Pathol. 19, 17 (1966).

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