Clin. Biochem. 10, (1) 38-43 (1977)
Observations on the Determination of Serum and Red-Cell Folate Levels by a Radiometric Assay Method MOHAMMED
JALALUDDIN, J A M E S B. C A M P B E L L ' , and ALEXANDER SESLER
JOSE
SANHUEZA,
Extendicare D i a g n o s t i c S e r v i c e s , a division of Extendicare Limited, 4949 B a t h u r s t Street, Willowdale, O n t a r i o , C a n a d a , a n d ' D e p a r t m e n t of Microbiology and P a r a s i t o l o g y , F a c u l t y of Medicine, U n i v e r s i t y of T o r o n t o , T o r o n t o , O n t a r i o , C a n a d a .
CLBIA, 10, (1) 38-43 (1977)
Cliu. Biochem. Jalaluddin, M., Campbell, J. B2, Sanhueza, J., and Sesler, A.
Extendioare Diagnostic Services, a divioion of Extendicare Limited, ~959 Bathurst St., WiUowdale, Ontario, and 1Department of Microbiology and Parasitology, Faculty of Medicine, University of Toronto, Toronto, Ontario M~R 1Y1 OBSERVATIONS ON T H E D E T E R M I N A T I O N OF SERUM AND RED-CELL F O L A T E LEVELS BY A RADIOMETRIC ASSAY METHOD Our experience with the Bio-Rad "Quanta-Count" folate radioassay kit has revealed very good i n - r u n precision and good day-to-day reproducibility in the assay of both serum and red-cell folate levels. Ascorbic acid is not required as folate preservative if blood samples are frozen within hours after collection. For the determination of red-cell folates, our data clearly show the need for maintenance of a certain level (6-8 gin%) of protein in the assay system. Protein (albumin or folate-free h u m a n serum) must be added to the red-cell lysate to compensate for the serum loss resulting from the high dilution factor necessary. In the absence of this added protein, red-cell values are markedly lower. A good correlation exists between red-cell folate values obtained from the assay of washed red cells and from the assay of whole blood with corrections for serum folate levels.
IN RECENT YEARS, there have been a number of reports on the determination of serum folate levels by radiometric assay methods "~. Less well documented are descriptions of these methods for the assay of red-cell folates '~'m. For both determinations, however, there are conflicting reports in the literature regarding the o p t i m u m assay conditions. For example, there appears to be no uniformly accepted procedure for the treatment and storage of blood specimens prior to assay. In addition there is no mention
Correspondence: Mohammed Jalaluddin, Extendicare Diagnostic Services, a division of Extendicare Limited, 4949 Bathurst Street, Willowdale, Ontario, Canada
of the need of f o l a t e - f r e e h u m a n s e r u m (or a l b u m i n ) f o r t h e a s s a y of r e d - c e l l folates, w h i c h we f i n d e s s e n tial. I n t h i s r e p o r t , we h a v e a t t e m p t e d to c l a r i f y some a s p e c t s of the b a s i c m e t h o d o l o g y of f o l a t e rad i o a s s a y . We also p r o v i d e a n e v a l u a t i o n of a comm e r c i a l l y available k i t f o r s e r u m folate assay, a n d we d e s c r i b e its m o d i f i c a t i o n for d e t e r m i n a t i o n o f red-cell folate.
MATERIALS AND METHODS Serum folate assay In the work described here, carried out during the period December, 1975, to June, 1976, we used "QuantaCount" folate radioassay kits manufactured by, and purchased from Bio-Rad Laboratories, Richmond, California. The principle and procedure of this "Quanta-Count" kit is not significantly different from other commerciallyavailable folate radioassay kits. It utilizes t~I-pteroylglutamate ( m I - P G A ) as the radioactive tracer folate, with fl-lactoglobulin as the binding protein. A one-step incubation is carried out at room temperature, in the dark, for 30 minutes, and separation of free and bound folate is effected by adsorption of the former onto dextran-coated charcoal. The fraction of bound ' ~ I - P G A is determined and plotted as a function of the total t~I-PGA added. Concentrations of unknowns are determined from a standard curve included in every assay. Non-specific binding by serum proteins is subtracted from all counts, using the 0 n g / m l PGA standard value. F u r t h e r details of the method and data collection can be obtained from the instruction manual provided by Bio-Rad Laboratories. In one experiment, the folate radioassay kit ('=I) manufactured by Schwarz-Mann (Orangeburg, New York) was also used. F u r t h e r details of this are given in the text. Sera were separated from blood collected by venipuncture into Venoject vacuum tubes ( J i n t a n Terumo Co., Tokyo, J a p a n ) and allowed to clot at room temperature for about two to three hours before separation. Serum samples were then stored at -20 ° , with or without added ascorbic acid (1% w / v ; see Results section), until required for assay. All m a n u a l t r a n s f e r s were carried out with Eppendorf micropipets ( B r i n k m a n n Instruments, Westbury, New York) with disposable polypropylene tips. Dilutions and pipetting of whole blood and red cell suspensions were made by means of a Micro-Medic automatic pipettor, model 25009 (Micro-Medic Systems, Inc., Philadelphia, Pennsylvania). All radioactive counting was done in an AutoLogic 111 gamma counter (Abbott Laboratories, North Chicago, Illinois).
39
R A D I O M E T R I C A S S A Y OF S E R U M A N D R E D - C E L L F O L A T E L E V E L S
TABLE 1
Red-blood cell folate assay Blood samples were collected in Venoject tubes conr a in i n g E D T A as anticoagulant (21 m g / m l blood). A f t e r removal of plasma, the blood cells were washed twice with 0.9% saline. The huffy coat was removed by aspiration a f t e r each washing. The packed cells were then diluted 1:42 with distilled water, mixed well, kept at room t em p er at u re for 30 minutes, and immediately stored at -20 ° until required for assay. In some experiments, whole blood was used in place of washed red cells. This w a s also diluted 1:42 with distilled w a te r and stored frozen until assay. The method for determination of red-cell or wholeblood folate was identical to that used for serum folate, with the following addition. In order to compensate for t he loss of serum proteins by the 1:42 dilution step, 50 Itl of folate-free human serum (Clinical Assays Inc., Cambridge, Massachusetts) or 16% human albumin, fraction V (Sigma Chemical Co., St. Louis, Missouri) was added to every 50 ~I aliquot of diluted hemolysate j u s t prior to assay. This was an essential addition; without it, red-cell folate assay levels were considerably lowered (see Results section).
IN-RUN PRECISION OF SERUM AND RED-CELL FOLAYE DETERMINATIONS
Serum folate (ng/ml) Replicate # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
RESULTS E v a l u a t i o n o f t h e t e s t kit Figure 1 shows a typical standard curve obtained by use o f t h e B i o - R a d f o l a t e r a d i o a s s a y kit. T h e b a r s r e p r e s e n t t h e s t a n d a r d d e v i a t i o n (S.D.) f r o m 20 d e t e r m i n a t i o n s . T h e low S.D. a n d c o e f f i c i e n t s o f v a r i a t i o n (C.V.) o f t h e s t a n d a r d s a r e i n d i c a t i v e o f t h e good reproducibility of this method. Table 1 d e m o n s t r a t e s the in-run precision of the assay. Pooled s e r u m and red-cell hemolysates were u s e d in t h i s e x p e r i m e n t . R e p r o d u c i b i l i t y o f t h e a s s a y for serum folates was better than for red-cell folates, b u t in b o t h c a s e s t h e S.D. a n d C.V. w e r e a c c e p t a b l y low. In f a c t , t h i s r a d i o a s s a y k i t g a v e t h e s m a l l e s t a n d s e c o n d s m a l l e s t S.D. f o r s e r u m f o l a t e s ( a t l e v e l s 1 a n d 2 in T a b l e 1) w h e n c o m p a r e d w i t h r e s u l t s obt a i n e d by K u b a s i k a n d c o w o r k e r s " ' w i t h s i x o t h e r r a d i o a s s a y kits. F i g u r e 2 illustrates the day-to-day reproducibility o f t h e m e t h o d . T h e p o o ls of s e r u m a n d r e d - c e l l lys a t e s u s e d in t h e p r e v i o u s e x p e r i m e n t ( T a b l e 1)
Mean: Standard deviation: Coefficient of variation:
Red-cell folate (ng/ml)
1
2
3
1
1.9 2.0 2.0 2.0 2.0 1.9 1.9 1.9 1.9 2.0 2.1 2.1 2.0 2.0 1.9 1.9 2.1 2.1 2.0 2.0
6.1 6.1 6.1 6.1 6.3 6.3 6.1 6.2 6.1 6.3 6.2 6.3 6.1 6.1 6.3 6.2 6.1 6.3 6.1 6.2
14.5 14.7 15.4 14.6 14.8 14.5 14.5 14.6 14.7 15.4 14.7 14.7 15.4 14.5 14.6 14.8 14.8 14.6 15.4 14.7
180 176 184 189 206 194 202 185 187 193 195 179 180 202 193 185 193 181 183 201
524 552 533 561 538 552 524 561 538 533 552 561 561 533 524 538 552 538 533 552
2
1.98
6.18
14.80
189.4
543.0
0.07
0.08
0.32
8.79
13.15
3.53
1.29
2.19
4.64
2.42
r
200 ~" ~
leo I- . . . . . . . . . . .
N ~ " ~
......
~'~-
.......
,7o
•
h~.~l~ ICO~ --I
7"0
°o O ~ LI_
:
.
S'O
I
I
I
DAYS
Fig. 2 - - D a y - t o - d a y r e p r o d u c i b i l i t y o f s e r u m f o l a t e a n d red-cell / o l a t e values. D a s h e d line r e p r e s e n t s the m e a n values. 7
~0
o
L
I,
- J ,4o
o Ft.L
3O
o t-
Z 2o t.d cr UJ Q_ IO
PGA
(ng/ml)
F i g . 1 - - S t a ~ d a r d f o l a t e radioassa,i c u r , c , usi'~g th~ " Q u a n t a - C o u n t " f o l a t e r a d i o a s s a y kit. V e r t i c a l bars r e p r e s e n t s t a n d a r d d e v i a t i o n s a t v a r i o u s f o l a t e concentrations. (n = 20 a t cach col~ccntration)
were used here also. They were stored at -20 ° in small aliquots without added ascorbic acid, and each aliquot was used only once before being discarded. The mean value of 180.3 ng/ml (C.V. 5.95) for the red-cell folates compares quite well with the m e a n of 189.4 ng/ml (C.V. 4.64) determined for the 20 replicates (Table 1, level 1). Likewise, the m e a n of 5.86 ng/ml (C.V. 4.32) for the serum folates compares well with that of 6.18 ng/ml (C.V. 1.29) in the case of the replicates (Table 1, level 2). This indicates that, under our experimental conditions, the reagents are stable and there is no significant loss of folate from frozen sera over a period of at least five weeks. B y applying Student's t-test to the results of 15 replicates of the lowest kit standards (Table 2), w e have shown that the method can effectively distinguish between folate levels of 0.0, 0.5, and 1.0 n g / m l at a c o n f i d e n c e l ev el o f P = 0.005.
40
JALALUDDIN,
CAMPBELL,
SANHUEZA
TABLE 2
and blood-cell folate levels were markedly by the end of the week (Table 4).
SENSITIVITY OF THE ASSAY METHOD AT LOW FOLATE CONCENTRATION
M e a n d.p.m.*
0.0
14,969
130
0.5
14,514
115
1.0
13.983
S.D.
1G5
increased
TABLE 4 EFFECT O F HIGH DIETARY FOLATE ON SERUM AND RED-CELL LEVELS
Radioactive counts Folate standard (ng/ml)
AND SESLER
Significance t x Folate concentrations ,ng/ml,
--
Before intake*
Different from 0.0 n g / m l a t P = 0.005.
Donor
Different from 0.5 n g / m l at P = 0.005.
J.M ....... J.C . . . . . . . . S.C . . . . . . . . M.E .......
*Disintegrations per minute. M e a n of 15 counts a t each folate concentration. The accuracy of Quanta-Count folate kit was compared with standard microbiological methods (L. casei) which revealed comparable results within 10% for both serum and red-cell folate values. In the course of our routine testing, we have assayed more than 900 serum and red-cell specimens. Of these, about 22% have been abnormal according to the normal serum ranges specified in the BioRad manual ( s e r u m f o l a t e : 1.9 - 14.0 n g / m l ) . W e have also noted that about 80% of the patients with high serum folates (>20 ng/ml) also had high redcell values (>800 ng/ml). The significance of this is n o t c e r t a i n s i n c e w e do n o t h a v e t h e c l i n i c a l h i s t o r y o f t h e s e p a t i e n t s , b u t i t is k n o w n t o b e l i k e l y that at least some of them are undergoing megavitamin therapy. Table 3 illustrates the quantitative e f f e c t s o f d i l u t i o n . A s c a n b e s e e n , t h e m e t h o d is acceptable for measuring folate concentrations of a t l e a s t 1500 n g / m l . That these high levels of serum and blood-cell folate measurements are actually due to folates and not to some interfering substance was indicated by the following experiment. Four volunteers ingested 3 0 to 40 m g f o l i c a c i d p e r d a y , a l o n g w i t h t h e i r r e g u lar diet, for seven days. In each case, their serum
Serum
After intake*
Red Cells
8.9 8.2 9.5 6.9
Serum
Red cells
44.0 23.5 56.0 49.0
1145 1090 1465 1295
457 614 576 469
*Each volunteer took a dietary supplement of 30-40 mg folic acid ]:er day for 7 days. Red cell folate determination
by indirect
methods
When large numbers of samples are being assayed, i t is a t i m e - c o n s u m i n g operation to wash the blood c e l l s . H o f f b r a n d a n d c o - w o r k e r s (') h a v e c i r c u m v e n t e d this by calculating red-cell folate values from the relationship : (1) . . . . . . where
- S F (1 - P C V / 1 0 0 ) PCV/100 ng/ml whole blood = red-cell folate = whole blood folate = serum folate = p a c k e d cell v o l u m e
RCF RCF WBF SF PCV
=
WBF
If we assume that the contribution of the serum f o l a t e t o t h e t o t a l is n e g l i g i b l e , t h i s e q u a t i o n b e comes : (2) . . . . . .
RCF
WBF . . . . . . . P C V / 100
Figure 3 compares values for red-cell folates from 60 p a t i e n t s a m p l e s a s c a l c u l a t e d b y m e a n s o f e q u a tions (1) and (2). The advantage of using equation ( 2 ) is t h a t r e d - c e l l f o l a t e s c a n b e c a l c u l a t e d f r o m
TABLE 3 QUANTITATIVE EFFECTS OF DILUTION ON THE FOLATE ASSAY
Dilution factor Specimen Serum . . . . . . . . . . . . . . . . . . . . . . . .
Red ceils . . . . . . . . . . . . . . . . . . . . . . .
Sample no.
1:1
P3439 F3438 B3442 F0949 E0921 B3652 B2910 P3288 1~2581 B3802
> > > > >
5.0 3.3 8.9 10.0 11.3 20.0 20.0 20.0 20.O 20.0
P3439 P3438 P3442 F.0949 E0921 B3652 B2910 B3288
440 392 365 497 550 > 800 > 800 > 800
B2581 B3802
> >
800 800
1:2
1:4
2.7(5.4) 1.6(3.2) 4.3(8.6)
1.2(4.8) 0.9(3.6) 2.5(I0.0)
1:5
1:10
----
----
4.7(9.4)
2.6(10.4)
--
5.9(11.8)
2.7(10.8)
--
--* ----
------
6.9(34.5} 8.4(42.0) 4.3(21.5) 3.9/19.5) 5.9(29.5)
--3.5(35.0) 3.9(39.0) 1.9(19.0) 1.6(16.0) 2.7(27.0)
290(418) 187(374) 170 (340) 245(490) 285(570) ---
103(412) 91 (364) 87(348) 131(524) 143(572) ---
-----167(835) 1951975)
-----85(850) 105(1050)
--
--
251(1255)
117(1170)
---
---
149(745) 289(1445)
151(1510)
--
69(69(})
Open figurem are the measured folate concentrations (ng/ml). Figures in parenthesis are folate concentrations adjusted for the dilution factors. *Not done.
RADIOMETRIC ASSAY OF SERUM AND RED-CELL F O L A T E LEVELS Factors affecting the stability of serum and red-cell f o l a t e
F: 700 01 f...< 6 0 0
Z 0
sO0
0
400
W W I-< _J @
aoo
l.J_ 2 0 0 / e
RCFeqn.2
s I'0~ ( ~ C F e q n . I
)+4"51
._J W
0 r-~ W Pr"
I00
r
"
I
°o
I00
0-998
•
I
I
I
I
I
I
200
300
400
500
600
700
It has been recommended t h a t a reducing agent s u c h as a s c o r b i c acid o r m e r c a p t o e t h a n o l be added to s a m p l e s b e f o r e s t o r a g e a t - 2 0 ° p r i o r to a s s a y (s°''' We h a v e f o u n d , however, t h a t by s i m p l y f r e e z i n g s a m p l e s as soon as p o s s i b l e a f t e r c o l l e c t i o n , t h i s p r e s e r v e s e n d o g e n o u s f o l a t e levels a d e q u a t e l y . Restilts s u m m a r i z e d in T a b l e 5 i l l u s t r a t e t h i s p o i n t . Red-cell h e m o l y s a t e s , w i t h o u t a n y a d d i t i v e s , s h o w e d no s i g n i f i c a n t loss of t i t e r a f t e r m o r e t h a n e i g h t days a t - 2 0 ° . S t o r e d a t r e f r i g e r a t o r t e m p e r a t u r e s ( 2 - 8 ° ) , the levels d r o p p e d m a r k e d l y . T h e d a t a i n Table 6 show t h a t a d d i t i o n of ascorbic acid (1% w / v ) h a s no s i g n i f i c a n t e f f e c t on the red-cell f o l a t e levels, if the a s s a y s are c a r r i e d o u t on the s a m e day as the blood is collected. TABLE 5
RED CELL FOLATE (EQUATION I),ng/ml. Fig. 3 Comparison of red-ceU folv~te values obtained f r o m equation (1) with that of equation (2) (with and without serum corrections respectively). -
-
a single whole-blood assay and the hematocrit value, thereby e l i m i n a t i n g the necessity for a separate d e t e r m i n a t i o n of s e r u m - f o l a t e levels. By r e g r e s s i o n a n a l y s i s of t h e d a t a in F i g u r e 3 we have s h o w n t h a t : (3) . . . . . .
41
RCFeq,~tio, , _- = 1.02 R C F ..... tion 1 + 4.51
T h e c o r r e l a t i o n c o e f f i c i e n t was 0.998. We h a v e also s t u d i e d t h e c o r r e l a t i o n b e t w e e n redcell f o l a t e v a l u e s o b t a i n e d f r o m w a s h e d cells, a n d f r o m whole blood by m e a n s of e q u a t i o n (1). R e s u l t s f r o m 40 d i f f e r e n t p a t i e n t s a m p l e s are s h o w n in F i g u r e 4. R e g r e s s i o n a n a l y s i s of t h e s e r e s u l t s gave the f o l l o w i n g f i t :
EFFECT OF STORAGE TEMPERATURE ON RED-CELL FOLATE LEVELS
Storage conditions Days at -20° Sample no. 1 2 3 4 5
Days at 2-8°
0
2
5
8
380 80 289 421 531
359 61 254 409 510
368 69 267 397 497
351 67 251 403 521
0
2
5
- * 155 102 95 77 52 269 195 107 458 360 159 515 397 171
8 61 39 59 73 101
*Not done. TABLE 6 EFFECT OF ASCORBIC ACID ON RED-CELL FOLATE DETERMINATIONS Red-cell folate (ng/ml)
(4) . . . . . .
R C F = 0.865 W B F + 68.3
Sample no.
T h e c o r r e l a t i o n c o e f f i c i e n t w a s 0.871.
1 2 3 4 5 6
• 800
E
With ascorbic acid
Without ascorbic acid
279 372 139 438 502 189
261 360 109 404 545 163
~703
C3 0 6oo
E f f e c t of s e r u m p r o t e i n on t h e f o l a t e a s s a y
S m
•
./
w 5o0 _J o T ~. 400 W 3oo
/ 200
d 1 w O ioo o w {M o o
/ 1 I00
• /:
//
/.
REGRESSION ANALYSIS: RCF = O'S65{WBF~ + 683 r • 0"871
I 200
I 300
I 400
I 500
I 600
I TOO
I eo0
i
RED CELL FOLATE (WASHED CELLS), ng/ml. Fig. 4. Comparison of red-ceU folate values obtained from washed red cells, and from whole blood with correction for serum concentrations.
I n the n o r m a l a s s a y for s e r u m folate, the s e r u m is used u n d i l u t e d . B e c a u s e of the m u c h h i g h e r conc e n t r a t i o n of f o l a t e in red cells, h o w e v e r , a n i n i t i a l d i l u t i o n of a b o u t 1:40 is r e q u i r e d (see M a t e r i a l s a n d M e t h o d s ) . T h i s m e a n s t h a t , e v e n if whole blood is used, the s e r u m c o n c e n t r a t i o n is r e d u c e d to a b o u t 2.5%. T h e d a t a in T a b l e 7 c l e a r l y show t h a t t h i s is h i g h l y d e t r i m e n t a l to the a c c u r a c y of t h e t e s t . I n o u r e x p e r i e n c e , red-cell f o l a t e v a l u e s o b t a i n e d w i t h o u t t h e a d d i t i o n of f o l a t e - f r e e h u m a n s e r u m a r e v a r i a b l e a n d unreliable, w h e r e a s w i t h added s e r u m (or 16% h u m a n a l b u m i n ) , w h e t h e r w a s h e d cells or w h o l e b l o o d is used, the r e s u l t s o b t a i n e d a r e c o n s i s t e n t a n d r e p r o d u c i b l e ( F i g u r e 41. T h i s p o i n t is also i l l u s t r a t e d by c o m p a r i n g t h e values obtained for red-cell folates by the " Q u a n t a Count" method (with added folate-free serum), and
J A L A L U D D I N , CAMPBELL, S A N H U E Z A AND S E S L E R
42
TABLE 7
TABLE 8
EFFECT OF ADDED SERUMONRED-CELL FOLATE DETERMINATIONS
COMPARISON OFTwO KIT METHODS FORTHE DETERMINATION OF SERUM ANDRED-CELL FOLATELEVELS
Red cell folates (ng/ml) With added serum* Sample no. Washed cells Whole blood B6668 B6540 B6535 Y3423 B7369 B5344 B5346 B5345 Y3439 B6470
210 256 309 339 357 390 396 440 440 >800
202 241 304 313 346 380 386 425 428 >800
Folate levels (ng/ml)
Without added serum* Whole blood 98 129 109 179 129 112 116 114 119 121
Bio-Rad kit Sample no. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Serum 1.7 5.7 9.2 7.4 17.5 4.8 11.4 3.1 10.0 >20
Red cells 560 517 460 296 800 339 479 658 573 >800
Schwarz-Mann kit Serum 2.1 5.4 9.8 7.8 16.0 3.9 12.7 3.8 11.2 >20
Red cells 304 355 235 195 378 95 254 225 171 282
*Folate-free human scrum.
by the Schwarz-Mann kit using the directions prov i d e d by t h e m a n u f a c t u r e r s . T h e p r o t o c o l f o r t h e two m e t h o d s is q u i t e s i m i l a r , t h e m a i n d i f f e r e n c e being that the Schwarz-Mann method utilizes gelatin a s a n a d d i t i v e in p l a c e o f f o l a t e - f r e e s e r u m . T a b l e 8 s u m m a r i z e s t h e r e s u l t s o b t a i n e d on 10 b l o o d s a m p l e s a s s a y e d b y t h e two m e t h o d s . I n t h e a b s e n c e of ser u m p r o t e i n , it c a n be s e e n t h a t t h e r e d - c e l l v a l u e s o b t a i n e d a r e m a r k e d l y l o w e r . On t h e o t h e r h a n d , assay o f s e r u m f o l a t e s b y t h e two k i t s p r o d u c e d comparable results. DISCUSSION
Our experience with the Bio-Rad "Quanta-Count" k i t h a s r e v e a l e d v e r y g o o d i n - r u n p r e c i s i o n (.Table 1) a n d good d a y - t o - d a y r e p r o d u c i b i l i t y ( F i g u r e 2) in the assay of both serum and red-cell folates. The p r e c i s i o n w a s e s p e c i a l l y g o o d in t h e i m p o r t a n t low a n d l o w - n o r m a l r a n g e s ( T a b l e 1). W e h a v e also d e m o n s t r a t e d t h a t , i f t h e a s s a y s a m p l e s a r e f r o z e n as soon as p o s s i b l e a f t e r c o l l e c t i o n , t h e r e is no r e a l n e e d to a d d an a n t i - o x i d a n t s u c h a s a s c o r b i c a c i d to p r e s e r v e t h e a c t i v i t y o f t h e f o l a t e s . In a study of day-to-day reproducibility of a serum f o l a t e a s s a y in w h i c h t h e s a m p l e s w e r e f r o z e n in t h e p r e s e n c e o f a s c o r b i c acid, M i n c e y et al '~) r e p o r t e d no loss o f e n d o g e n o u s f o l a t e o v e r a p e r i o d o f a t l e a s t one m o n t h . O u r d a t a in T a b l e 2 i n d i c a t e a s l i g h t , t h o u g h p r o b a b l y n o t s i g n i f i c a n t , d e c r e a s e in f o l a t e concentration when the samples are stored for over f i v e w e e k s in t h e a b s e n c e o f a s c o r b i c acid. T h e s t a n d a r d d e v i a t i o n s a n d c o e f f i c i e n t s o f v a r i a t i o n of t h e d a t a in t h e e a r l i e r w o r k (5), h o w e v e r , w e r e cons i d e r a b l y h i g h e r t h a n o u r s . W e do n o t feel, t h e r e f o r e , t h a t t h e s e r e s u l t s d e m o n s t r a t e t h e a d d i t i o n of ascorbic acid provides any further benefit over simply freezing the samples. Certainly, our data show t h a t t h e r e is no s i g n i f i c a n t loss o f f o l a t e a c t i v i t y o v e r a p e r i o d o f a t l e a s t t w o weeks, i f t h e s a m p l e s a r e s t o r e d f r o z e n . F r e e z i n g is i m p o r t a n t , h o w e v e r , i f t h e s t o r a g e p e r i o d is to be a n y g r e a t e r t h a n a f e w h o u r s . W e h a v e n o t s t u d i e d t h e p o s s i b i l i t y of a s t a b i l i z i n g e f f e c t o f a s c o r b i c a c i d on u n f r o z e n s a m ples. T h e d a t a s u m m a r i z e d in F i g u r e 4 s h o w t h a t a
good c o r r e l a t i o n exists between red-cell f o l a t e v a l u e s o b t a i n e d f r o m t h e a s s a y of w a s h e d r e d cells, a n d f r o m t h e d e t e r m i n a t i o n o f w h o l e b l o o d f o l a t e w i t h c o r r e c t i o n s f o r s e r u m f o l a t e m a d e by a p p l i c a t i o n of e q u a t i o n (1). T h i s conclusion is cons i s t e n t w i t h t h e r e s u l t s of an e a r l i e r , l e s s d e t a i l e d a n a l y s i s by S p r a y "°). F i g u r e 3 i l l u s t r a t e s t h e p o i n t t h a t t h e r e is no s i g n i f i c a n t d i f f e r e n c e b e t w e e n r e d cell f o l a t e values c a l c u l a t e d e i t h e r f r o m e q u a t i o n (1) o r e q u a t i o n (2). F o r r o u t i n e red-cell f o l a t e d e t e r m i n a t i o n s , t h e r e f o r e , use o f e q u a t i o n (2) would be t h e m o r e a p p r o p r i a t e s i n c e less c a l c u l a t i o n is r e q u i r e d . For the determination of red-cell folate, our data c l e a r l y d e m o n s t r a t e t h e need f o r m a i n t e n a n c e of a c e r t a i n level of s e r u m p r o t e i n in t h e a s s a y s y s t e m . P r o t e i n m u s t be a d d e d to t h e r e d - c e l l l y s a t e to c o m p e n s a t e f o r t h e s e r u m loss r e s u l t i n g f r o m t h e h i g h d i l u t i o n f a c t o r n e c e s s a r y (1:42 in o u r m e t h o d ) . W e have found that folate-free human serum or human a l b u m i n can be u s e d i n t e r c h a n g e a b l y f o r t h i s p u r pose. In t h e d e t e r m i n a t i o n o f s e r u m f o l a t e , a d d i t i o n o f f u r t h e r s e r u m or a l b u m i n to t h e r e a c t i o n m i x t u r e h a s no v a l u e s i n c e t h e e x i s t i n g s e r u m p r o t e i n l e v e l s are apparently high enough. This has also been noted by Mincey and co-workers':". The results summ a r i z e d in T a b l e s 7 a n d 8 s h o w t h e e x t r e m e imp o r t a n c e o f t h i s a d d i t i o n in t h e r e d - c e l l a s s a y ; unf o r t u n a t e l y , t h e a c t u a l f u n c t i o n o f t h e s e r u m in t h e r a d i o a s s a y is n o t a t all well u n d e r s t o o d , a l t h o u g h M a r k k a n e n et al '~'~''3), a n d Z e t n e r et al '"~ h a v e s p e c u l a t e d t h a t it m a y f u n c t i o n a s a s o u r c e o f carrier protein (s). As mentioned earlier, a fairly large percentage o f t h e b l o o d s a m p l e s s e n t to o u r l a b o r a t o r y f o r f o l a t e assay have highly elevated serum and red-cell folate levels. H o f f b r a n d (7) h a s r e p o r t e d on p a t i e n t s w i t h h i g h s e r u m l e v e l s ( u p to 43 n g / m l ) b u t w i t h n o r m a l o r s u b n o r m a l r e d - c e l l f o l a t e s : t h e s e w e r e cons i d e r e d to be i n d i c a t i v e o f p e r n i c i o u s a n e m i a . A s f a r as we k n o w t h e r e h a v e b e e n no r e p o r t s o f b o t h h i g h l y e l e v a t e d s e r u m a n d r e d - c e l l f o l a t e levels. We are not sure of the significance of this since we do n o t h a v e c l i n i c a l d e t a i l s o f i n d i v i d u a l p a t i e n t s . From the specialties of our contributing physicians, we k n o w it to be l i k e l y t h a t a t l e a s t s o m e o f t h e s e patients are undergoing megavitamin therapy. In
R A D I O M E T R I C A S S A Y O F SERUM AND R E D - C E L L F O L A T E L E V E L S s u p p o r t o f t h i s , o u r d a t a in T a b l e 4 s h o w t h a t ing e s t i o n of l a r g e a m o u n t s of f o l a t e f o r a s h o r t p e r i o d of t i m e c a n r e s u l t in l a r g e i n c r e a s e s in b o t h s e r u m a n d r e d - c e l l f o l a t e levels. I t m a y w e l l be, t h e r e f o r e , t h a t t h e s e d a t a o b t a i n e d in o u r l a b o r a t o r y a r e n o t t y p i c a l of t h a t o b t a i n e d in g e n e r a l . I t is w o r t h n o t i n g , h o w e v e r , t h a t if f o l a t e - f r e e s e r u m h a d n o t b e e n a d d e d d u r i n g t h e r e d - c e l l f o l a t e a s s a y s in t h e p r e s e n t s t u d y , m a n y , i f n o t all, o f o u r h i g h r e d cell v a l u e s w o u l d h a v e r e g i s t e r e d w i t h i n t h e n o r m a l range. REFERENCES 1. Waxman, S., Schrieber, C., and Herbert, J., (1970). Blood., 36, 858-864. 2. Waxman, S., Schrieber, C., and Herbert, J., (1971). Blood, 38, 219-228. 3. Tajuddin, M., and Gardyna, H. A., (1973). Cli,l. Chem., 19, 125-126.
43
4. Dunn, R. T., and Foster, L. B., (1973). Clin. Chem., 19, 1101-1105. 5. Mincey, E. K., Wilcox, E., and Morrison, R. T., (1973). Clin. Biochem., 6, 274-284. 6. Zettner, A., and Duly, P. E., (1974). Clin. Chem., 20, 1313-1319. 7. Hoffbrand, A. N., Newcombe, B.F.A., and Mollin, D. L., (1966). J. Clin. Path., 19, 17-28. 8. Fleming. A. F., Comley, L., and Stenhouse, N.S., (1971). Am. J. Clin. Nutr., 24, 1257-1264. 9. Kubasik, N.P., Volosin, M. J., and Sine, H. E., (1975). Clin. Chem., 21, 1922-1926. 10. Spray, G. H., (1969). J. Clin. Path., 22, 212-216. 11. Rothenberg, S. P., Sheldon, P., da Costa, M., Lawson, J., and Rosenberg, Z., (1974). Blood, 43, 437-443. 12. Markkanen, T., Pajula, R. L., Virtanen, S., and Himanen, P., (1972). Acta Hematol., 48, 145-150. 13. Markkanen, T., Virtanen, S., Himanen, P., and Pajula, R. L., (1972). Acta He~natol., 48, 213-217.
Observations on the Determination of Serum and Red-Cell Folate Levels by a Radiometric Assay Method MOHAMMED
JALALUDDIN, J A M E S B. C A M P B E L L ' , and ALEXANDER SESLER
JOSE
SANHUEZA,
Extendicare D i a g n o s t i c S e r v i c e s , a division of Extendicare Limited, 4949 B a t h u r s t Street, Willowdale, O n t a r i o , C a n a d a , a n d ' D e p a r t m e n t of Microbiology and P a r a s i t o l o g y , F a c u l t y of Medicine, U n i v e r s i t y of T o r o n t o , T o r o n t o , O n t a r i o , C a n a d a .
CLBIA, 10, (1) 38-43 (1977)
Cliu. Biochem. Jalaluddin, M., Campbell, J. B2, Sanhueza, J., and Sesler, A.
Extendioare Diagnostic Services, a divioion of Extendicare Limited, ~959 Bathurst St., WiUowdale, Ontario, and 1Department of Microbiology and Parasitology, Faculty of Medicine, University of Toronto, Toronto, Ontario M~R 1Y1 OBSERVATIONS ON T H E D E T E R M I N A T I O N OF SERUM AND RED-CELL F O L A T E LEVELS BY A RADIOMETRIC ASSAY METHOD Our experience with the Bio-Rad "Quanta-Count" folate radioassay kit has revealed very good i n - r u n precision and good day-to-day reproducibility in the assay of both serum and red-cell folate levels. Ascorbic acid is not required as folate preservative if blood samples are frozen within hours after collection. For the determination of red-cell folates, our data clearly show the need for maintenance of a certain level (6-8 gin%) of protein in the assay system. Protein (albumin or folate-free h u m a n serum) must be added to the red-cell lysate to compensate for the serum loss resulting from the high dilution factor necessary. In the absence of this added protein, red-cell values are markedly lower. A good correlation exists between red-cell folate values obtained from the assay of washed red cells and from the assay of whole blood with corrections for serum folate levels.
IN RECENT YEARS, there have been a number of reports on the determination of serum folate levels by radiometric assay methods "~. Less well documented are descriptions of these methods for the assay of red-cell folates '~'m. For both determinations, however, there are conflicting reports in the literature regarding the o p t i m u m assay conditions. For example, there appears to be no uniformly accepted procedure for the treatment and storage of blood specimens prior to assay. In addition there is no mention
Correspondence: Mohammed Jalaluddin, Extendicare Diagnostic Services, a division of Extendicare Limited, 4949 Bathurst Street, Willowdale, Ontario, Canada
of the need of f o l a t e - f r e e h u m a n s e r u m (or a l b u m i n ) f o r t h e a s s a y of r e d - c e l l folates, w h i c h we f i n d e s s e n tial. I n t h i s r e p o r t , we h a v e a t t e m p t e d to c l a r i f y some a s p e c t s of the b a s i c m e t h o d o l o g y of f o l a t e rad i o a s s a y . We also p r o v i d e a n e v a l u a t i o n of a comm e r c i a l l y available k i t f o r s e r u m folate assay, a n d we d e s c r i b e its m o d i f i c a t i o n for d e t e r m i n a t i o n o f red-cell folate.
MATERIALS AND METHODS Serum folate assay In the work described here, carried out during the period December, 1975, to June, 1976, we used "QuantaCount" folate radioassay kits manufactured by, and purchased from Bio-Rad Laboratories, Richmond, California. The principle and procedure of this "Quanta-Count" kit is not significantly different from other commerciallyavailable folate radioassay kits. It utilizes t~I-pteroylglutamate ( m I - P G A ) as the radioactive tracer folate, with fl-lactoglobulin as the binding protein. A one-step incubation is carried out at room temperature, in the dark, for 30 minutes, and separation of free and bound folate is effected by adsorption of the former onto dextran-coated charcoal. The fraction of bound ' ~ I - P G A is determined and plotted as a function of the total t~I-PGA added. Concentrations of unknowns are determined from a standard curve included in every assay. Non-specific binding by serum proteins is subtracted from all counts, using the 0 n g / m l PGA standard value. F u r t h e r details of the method and data collection can be obtained from the instruction manual provided by Bio-Rad Laboratories. In one experiment, the folate radioassay kit ('=I) manufactured by Schwarz-Mann (Orangeburg, New York) was also used. F u r t h e r details of this are given in the text. Sera were separated from blood collected by venipuncture into Venoject vacuum tubes ( J i n t a n Terumo Co., Tokyo, J a p a n ) and allowed to clot at room temperature for about two to three hours before separation. Serum samples were then stored at -20 ° , with or without added ascorbic acid (1% w / v ; see Results section), until required for assay. All m a n u a l t r a n s f e r s were carried out with Eppendorf micropipets ( B r i n k m a n n Instruments, Westbury, New York) with disposable polypropylene tips. Dilutions and pipetting of whole blood and red cell suspensions were made by means of a Micro-Medic automatic pipettor, model 25009 (Micro-Medic Systems, Inc., Philadelphia, Pennsylvania). All radioactive counting was done in an AutoLogic 111 gamma counter (Abbott Laboratories, North Chicago, Illinois).
39
R A D I O M E T R I C A S S A Y OF S E R U M A N D R E D - C E L L F O L A T E L E V E L S
TABLE 1
Red-blood cell folate assay Blood samples were collected in Venoject tubes conr a in i n g E D T A as anticoagulant (21 m g / m l blood). A f t e r removal of plasma, the blood cells were washed twice with 0.9% saline. The huffy coat was removed by aspiration a f t e r each washing. The packed cells were then diluted 1:42 with distilled water, mixed well, kept at room t em p er at u re for 30 minutes, and immediately stored at -20 ° until required for assay. In some experiments, whole blood was used in place of washed red cells. This w a s also diluted 1:42 with distilled w a te r and stored frozen until assay. The method for determination of red-cell or wholeblood folate was identical to that used for serum folate, with the following addition. In order to compensate for t he loss of serum proteins by the 1:42 dilution step, 50 Itl of folate-free human serum (Clinical Assays Inc., Cambridge, Massachusetts) or 16% human albumin, fraction V (Sigma Chemical Co., St. Louis, Missouri) was added to every 50 ~I aliquot of diluted hemolysate j u s t prior to assay. This was an essential addition; without it, red-cell folate assay levels were considerably lowered (see Results section).
IN-RUN PRECISION OF SERUM AND RED-CELL FOLAYE DETERMINATIONS
Serum folate (ng/ml) Replicate # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
RESULTS E v a l u a t i o n o f t h e t e s t kit Figure 1 shows a typical standard curve obtained by use o f t h e B i o - R a d f o l a t e r a d i o a s s a y kit. T h e b a r s r e p r e s e n t t h e s t a n d a r d d e v i a t i o n (S.D.) f r o m 20 d e t e r m i n a t i o n s . T h e low S.D. a n d c o e f f i c i e n t s o f v a r i a t i o n (C.V.) o f t h e s t a n d a r d s a r e i n d i c a t i v e o f t h e good reproducibility of this method. Table 1 d e m o n s t r a t e s the in-run precision of the assay. Pooled s e r u m and red-cell hemolysates were u s e d in t h i s e x p e r i m e n t . R e p r o d u c i b i l i t y o f t h e a s s a y for serum folates was better than for red-cell folates, b u t in b o t h c a s e s t h e S.D. a n d C.V. w e r e a c c e p t a b l y low. In f a c t , t h i s r a d i o a s s a y k i t g a v e t h e s m a l l e s t a n d s e c o n d s m a l l e s t S.D. f o r s e r u m f o l a t e s ( a t l e v e l s 1 a n d 2 in T a b l e 1) w h e n c o m p a r e d w i t h r e s u l t s obt a i n e d by K u b a s i k a n d c o w o r k e r s " ' w i t h s i x o t h e r r a d i o a s s a y kits. F i g u r e 2 illustrates the day-to-day reproducibility o f t h e m e t h o d . T h e p o o ls of s e r u m a n d r e d - c e l l lys a t e s u s e d in t h e p r e v i o u s e x p e r i m e n t ( T a b l e 1)
Mean: Standard deviation: Coefficient of variation:
Red-cell folate (ng/ml)
1
2
3
1
1.9 2.0 2.0 2.0 2.0 1.9 1.9 1.9 1.9 2.0 2.1 2.1 2.0 2.0 1.9 1.9 2.1 2.1 2.0 2.0
6.1 6.1 6.1 6.1 6.3 6.3 6.1 6.2 6.1 6.3 6.2 6.3 6.1 6.1 6.3 6.2 6.1 6.3 6.1 6.2
14.5 14.7 15.4 14.6 14.8 14.5 14.5 14.6 14.7 15.4 14.7 14.7 15.4 14.5 14.6 14.8 14.8 14.6 15.4 14.7
180 176 184 189 206 194 202 185 187 193 195 179 180 202 193 185 193 181 183 201
524 552 533 561 538 552 524 561 538 533 552 561 561 533 524 538 552 538 533 552
2
1.98
6.18
14.80
189.4
543.0
0.07
0.08
0.32
8.79
13.15
3.53
1.29
2.19
4.64
2.42
r
200 ~" ~
leo I- . . . . . . . . . . .
N ~ " ~
......
~'~-
.......
,7o
•
h~.~l~ ICO~ --I
7"0
°o O ~ LI_
:
.
S'O
I
I
I
DAYS
Fig. 2 - - D a y - t o - d a y r e p r o d u c i b i l i t y o f s e r u m f o l a t e a n d red-cell / o l a t e values. D a s h e d line r e p r e s e n t s the m e a n values. 7
~0
o
L
I,
- J ,4o
o Ft.L
3O
o t-
Z 2o t.d cr UJ Q_ IO
PGA
(ng/ml)
F i g . 1 - - S t a ~ d a r d f o l a t e radioassa,i c u r , c , usi'~g th~ " Q u a n t a - C o u n t " f o l a t e r a d i o a s s a y kit. V e r t i c a l bars r e p r e s e n t s t a n d a r d d e v i a t i o n s a t v a r i o u s f o l a t e concentrations. (n = 20 a t cach col~ccntration)
were used here also. They were stored at -20 ° in small aliquots without added ascorbic acid, and each aliquot was used only once before being discarded. The mean value of 180.3 ng/ml (C.V. 5.95) for the red-cell folates compares quite well with the m e a n of 189.4 ng/ml (C.V. 4.64) determined for the 20 replicates (Table 1, level 1). Likewise, the m e a n of 5.86 ng/ml (C.V. 4.32) for the serum folates compares well with that of 6.18 ng/ml (C.V. 1.29) in the case of the replicates (Table 1, level 2). This indicates that, under our experimental conditions, the reagents are stable and there is no significant loss of folate from frozen sera over a period of at least five weeks. B y applying Student's t-test to the results of 15 replicates of the lowest kit standards (Table 2), w e have shown that the method can effectively distinguish between folate levels of 0.0, 0.5, and 1.0 n g / m l at a c o n f i d e n c e l ev el o f P = 0.005.
40
JALALUDDIN,
CAMPBELL,
SANHUEZA
TABLE 2
and blood-cell folate levels were markedly by the end of the week (Table 4).
SENSITIVITY OF THE ASSAY METHOD AT LOW FOLATE CONCENTRATION
M e a n d.p.m.*
0.0
14,969
130
0.5
14,514
115
1.0
13.983
S.D.
1G5
increased
TABLE 4 EFFECT O F HIGH DIETARY FOLATE ON SERUM AND RED-CELL LEVELS
Radioactive counts Folate standard (ng/ml)
AND SESLER
Significance t x Folate concentrations ,ng/ml,
--
Before intake*
Different from 0.0 n g / m l a t P = 0.005.
Donor
Different from 0.5 n g / m l at P = 0.005.
J.M ....... J.C . . . . . . . . S.C . . . . . . . . M.E .......
*Disintegrations per minute. M e a n of 15 counts a t each folate concentration. The accuracy of Quanta-Count folate kit was compared with standard microbiological methods (L. casei) which revealed comparable results within 10% for both serum and red-cell folate values. In the course of our routine testing, we have assayed more than 900 serum and red-cell specimens. Of these, about 22% have been abnormal according to the normal serum ranges specified in the BioRad manual ( s e r u m f o l a t e : 1.9 - 14.0 n g / m l ) . W e have also noted that about 80% of the patients with high serum folates (>20 ng/ml) also had high redcell values (>800 ng/ml). The significance of this is n o t c e r t a i n s i n c e w e do n o t h a v e t h e c l i n i c a l h i s t o r y o f t h e s e p a t i e n t s , b u t i t is k n o w n t o b e l i k e l y that at least some of them are undergoing megavitamin therapy. Table 3 illustrates the quantitative e f f e c t s o f d i l u t i o n . A s c a n b e s e e n , t h e m e t h o d is acceptable for measuring folate concentrations of a t l e a s t 1500 n g / m l . That these high levels of serum and blood-cell folate measurements are actually due to folates and not to some interfering substance was indicated by the following experiment. Four volunteers ingested 3 0 to 40 m g f o l i c a c i d p e r d a y , a l o n g w i t h t h e i r r e g u lar diet, for seven days. In each case, their serum
Serum
After intake*
Red Cells
8.9 8.2 9.5 6.9
Serum
Red cells
44.0 23.5 56.0 49.0
1145 1090 1465 1295
457 614 576 469
*Each volunteer took a dietary supplement of 30-40 mg folic acid ]:er day for 7 days. Red cell folate determination
by indirect
methods
When large numbers of samples are being assayed, i t is a t i m e - c o n s u m i n g operation to wash the blood c e l l s . H o f f b r a n d a n d c o - w o r k e r s (') h a v e c i r c u m v e n t e d this by calculating red-cell folate values from the relationship : (1) . . . . . . where
- S F (1 - P C V / 1 0 0 ) PCV/100 ng/ml whole blood = red-cell folate = whole blood folate = serum folate = p a c k e d cell v o l u m e
RCF RCF WBF SF PCV
=
WBF
If we assume that the contribution of the serum f o l a t e t o t h e t o t a l is n e g l i g i b l e , t h i s e q u a t i o n b e comes : (2) . . . . . .
RCF
WBF . . . . . . . P C V / 100
Figure 3 compares values for red-cell folates from 60 p a t i e n t s a m p l e s a s c a l c u l a t e d b y m e a n s o f e q u a tions (1) and (2). The advantage of using equation ( 2 ) is t h a t r e d - c e l l f o l a t e s c a n b e c a l c u l a t e d f r o m
TABLE 3 QUANTITATIVE EFFECTS OF DILUTION ON THE FOLATE ASSAY
Dilution factor Specimen Serum . . . . . . . . . . . . . . . . . . . . . . . .
Red ceils . . . . . . . . . . . . . . . . . . . . . . .
Sample no.
1:1
P3439 F3438 B3442 F0949 E0921 B3652 B2910 P3288 1~2581 B3802
> > > > >
5.0 3.3 8.9 10.0 11.3 20.0 20.0 20.0 20.O 20.0
P3439 P3438 P3442 F.0949 E0921 B3652 B2910 B3288
440 392 365 497 550 > 800 > 800 > 800
B2581 B3802
> >
800 800
1:2
1:4
2.7(5.4) 1.6(3.2) 4.3(8.6)
1.2(4.8) 0.9(3.6) 2.5(I0.0)
1:5
1:10
----
----
4.7(9.4)
2.6(10.4)
--
5.9(11.8)
2.7(10.8)
--
--* ----
------
6.9(34.5} 8.4(42.0) 4.3(21.5) 3.9/19.5) 5.9(29.5)
--3.5(35.0) 3.9(39.0) 1.9(19.0) 1.6(16.0) 2.7(27.0)
290(418) 187(374) 170 (340) 245(490) 285(570) ---
103(412) 91 (364) 87(348) 131(524) 143(572) ---
-----167(835) 1951975)
-----85(850) 105(1050)
--
--
251(1255)
117(1170)
---
---
149(745) 289(1445)
151(1510)
--
69(69(})
Open figurem are the measured folate concentrations (ng/ml). Figures in parenthesis are folate concentrations adjusted for the dilution factors. *Not done.
RADIOMETRIC ASSAY OF SERUM AND RED-CELL F O L A T E LEVELS Factors affecting the stability of serum and red-cell f o l a t e
F: 700 01 f...< 6 0 0
Z 0
sO0
0
400
W W I-< _J @
aoo
l.J_ 2 0 0 / e
RCFeqn.2
s I'0~ ( ~ C F e q n . I
)+4"51
._J W
0 r-~ W Pr"
I00
r
"
I
°o
I00
0-998
•
I
I
I
I
I
I
200
300
400
500
600
700
It has been recommended t h a t a reducing agent s u c h as a s c o r b i c acid o r m e r c a p t o e t h a n o l be added to s a m p l e s b e f o r e s t o r a g e a t - 2 0 ° p r i o r to a s s a y (s°''' We h a v e f o u n d , however, t h a t by s i m p l y f r e e z i n g s a m p l e s as soon as p o s s i b l e a f t e r c o l l e c t i o n , t h i s p r e s e r v e s e n d o g e n o u s f o l a t e levels a d e q u a t e l y . Restilts s u m m a r i z e d in T a b l e 5 i l l u s t r a t e t h i s p o i n t . Red-cell h e m o l y s a t e s , w i t h o u t a n y a d d i t i v e s , s h o w e d no s i g n i f i c a n t loss of t i t e r a f t e r m o r e t h a n e i g h t days a t - 2 0 ° . S t o r e d a t r e f r i g e r a t o r t e m p e r a t u r e s ( 2 - 8 ° ) , the levels d r o p p e d m a r k e d l y . T h e d a t a i n Table 6 show t h a t a d d i t i o n of ascorbic acid (1% w / v ) h a s no s i g n i f i c a n t e f f e c t on the red-cell f o l a t e levels, if the a s s a y s are c a r r i e d o u t on the s a m e day as the blood is collected. TABLE 5
RED CELL FOLATE (EQUATION I),ng/ml. Fig. 3 Comparison of red-ceU folv~te values obtained f r o m equation (1) with that of equation (2) (with and without serum corrections respectively). -
-
a single whole-blood assay and the hematocrit value, thereby e l i m i n a t i n g the necessity for a separate d e t e r m i n a t i o n of s e r u m - f o l a t e levels. By r e g r e s s i o n a n a l y s i s of t h e d a t a in F i g u r e 3 we have s h o w n t h a t : (3) . . . . . .
41
RCFeq,~tio, , _- = 1.02 R C F ..... tion 1 + 4.51
T h e c o r r e l a t i o n c o e f f i c i e n t was 0.998. We h a v e also s t u d i e d t h e c o r r e l a t i o n b e t w e e n redcell f o l a t e v a l u e s o b t a i n e d f r o m w a s h e d cells, a n d f r o m whole blood by m e a n s of e q u a t i o n (1). R e s u l t s f r o m 40 d i f f e r e n t p a t i e n t s a m p l e s are s h o w n in F i g u r e 4. R e g r e s s i o n a n a l y s i s of t h e s e r e s u l t s gave the f o l l o w i n g f i t :
EFFECT OF STORAGE TEMPERATURE ON RED-CELL FOLATE LEVELS
Storage conditions Days at -20° Sample no. 1 2 3 4 5
Days at 2-8°
0
2
5
8
380 80 289 421 531
359 61 254 409 510
368 69 267 397 497
351 67 251 403 521
0
2
5
- * 155 102 95 77 52 269 195 107 458 360 159 515 397 171
8 61 39 59 73 101
*Not done. TABLE 6 EFFECT OF ASCORBIC ACID ON RED-CELL FOLATE DETERMINATIONS Red-cell folate (ng/ml)
(4) . . . . . .
R C F = 0.865 W B F + 68.3
Sample no.
T h e c o r r e l a t i o n c o e f f i c i e n t w a s 0.871.
1 2 3 4 5 6
• 800
E
With ascorbic acid
Without ascorbic acid
279 372 139 438 502 189
261 360 109 404 545 163
~703
C3 0 6oo
E f f e c t of s e r u m p r o t e i n on t h e f o l a t e a s s a y
S m
•
./
w 5o0 _J o T ~. 400 W 3oo
/ 200
d 1 w O ioo o w {M o o
/ 1 I00
• /:
//
/.
REGRESSION ANALYSIS: RCF = O'S65{WBF~ + 683 r • 0"871
I 200
I 300
I 400
I 500
I 600
I TOO
I eo0
i
RED CELL FOLATE (WASHED CELLS), ng/ml. Fig. 4. Comparison of red-ceU folate values obtained from washed red cells, and from whole blood with correction for serum concentrations.
I n the n o r m a l a s s a y for s e r u m folate, the s e r u m is used u n d i l u t e d . B e c a u s e of the m u c h h i g h e r conc e n t r a t i o n of f o l a t e in red cells, h o w e v e r , a n i n i t i a l d i l u t i o n of a b o u t 1:40 is r e q u i r e d (see M a t e r i a l s a n d M e t h o d s ) . T h i s m e a n s t h a t , e v e n if whole blood is used, the s e r u m c o n c e n t r a t i o n is r e d u c e d to a b o u t 2.5%. T h e d a t a in T a b l e 7 c l e a r l y show t h a t t h i s is h i g h l y d e t r i m e n t a l to the a c c u r a c y of t h e t e s t . I n o u r e x p e r i e n c e , red-cell f o l a t e v a l u e s o b t a i n e d w i t h o u t t h e a d d i t i o n of f o l a t e - f r e e h u m a n s e r u m a r e v a r i a b l e a n d unreliable, w h e r e a s w i t h added s e r u m (or 16% h u m a n a l b u m i n ) , w h e t h e r w a s h e d cells or w h o l e b l o o d is used, the r e s u l t s o b t a i n e d a r e c o n s i s t e n t a n d r e p r o d u c i b l e ( F i g u r e 41. T h i s p o i n t is also i l l u s t r a t e d by c o m p a r i n g t h e values obtained for red-cell folates by the " Q u a n t a Count" method (with added folate-free serum), and
J A L A L U D D I N , CAMPBELL, S A N H U E Z A AND S E S L E R
42
TABLE 7
TABLE 8
EFFECT OF ADDED SERUMONRED-CELL FOLATE DETERMINATIONS
COMPARISON OFTwO KIT METHODS FORTHE DETERMINATION OF SERUM ANDRED-CELL FOLATELEVELS
Red cell folates (ng/ml) With added serum* Sample no. Washed cells Whole blood B6668 B6540 B6535 Y3423 B7369 B5344 B5346 B5345 Y3439 B6470
210 256 309 339 357 390 396 440 440 >800
202 241 304 313 346 380 386 425 428 >800
Folate levels (ng/ml)
Without added serum* Whole blood 98 129 109 179 129 112 116 114 119 121
Bio-Rad kit Sample no. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Serum 1.7 5.7 9.2 7.4 17.5 4.8 11.4 3.1 10.0 >20
Red cells 560 517 460 296 800 339 479 658 573 >800
Schwarz-Mann kit Serum 2.1 5.4 9.8 7.8 16.0 3.9 12.7 3.8 11.2 >20
Red cells 304 355 235 195 378 95 254 225 171 282
*Folate-free human scrum.
by the Schwarz-Mann kit using the directions prov i d e d by t h e m a n u f a c t u r e r s . T h e p r o t o c o l f o r t h e two m e t h o d s is q u i t e s i m i l a r , t h e m a i n d i f f e r e n c e being that the Schwarz-Mann method utilizes gelatin a s a n a d d i t i v e in p l a c e o f f o l a t e - f r e e s e r u m . T a b l e 8 s u m m a r i z e s t h e r e s u l t s o b t a i n e d on 10 b l o o d s a m p l e s a s s a y e d b y t h e two m e t h o d s . I n t h e a b s e n c e of ser u m p r o t e i n , it c a n be s e e n t h a t t h e r e d - c e l l v a l u e s o b t a i n e d a r e m a r k e d l y l o w e r . On t h e o t h e r h a n d , assay o f s e r u m f o l a t e s b y t h e two k i t s p r o d u c e d comparable results. DISCUSSION
Our experience with the Bio-Rad "Quanta-Count" k i t h a s r e v e a l e d v e r y g o o d i n - r u n p r e c i s i o n (.Table 1) a n d good d a y - t o - d a y r e p r o d u c i b i l i t y ( F i g u r e 2) in the assay of both serum and red-cell folates. The p r e c i s i o n w a s e s p e c i a l l y g o o d in t h e i m p o r t a n t low a n d l o w - n o r m a l r a n g e s ( T a b l e 1). W e h a v e also d e m o n s t r a t e d t h a t , i f t h e a s s a y s a m p l e s a r e f r o z e n as soon as p o s s i b l e a f t e r c o l l e c t i o n , t h e r e is no r e a l n e e d to a d d an a n t i - o x i d a n t s u c h a s a s c o r b i c a c i d to p r e s e r v e t h e a c t i v i t y o f t h e f o l a t e s . In a study of day-to-day reproducibility of a serum f o l a t e a s s a y in w h i c h t h e s a m p l e s w e r e f r o z e n in t h e p r e s e n c e o f a s c o r b i c acid, M i n c e y et al '~) r e p o r t e d no loss o f e n d o g e n o u s f o l a t e o v e r a p e r i o d o f a t l e a s t one m o n t h . O u r d a t a in T a b l e 2 i n d i c a t e a s l i g h t , t h o u g h p r o b a b l y n o t s i g n i f i c a n t , d e c r e a s e in f o l a t e concentration when the samples are stored for over f i v e w e e k s in t h e a b s e n c e o f a s c o r b i c acid. T h e s t a n d a r d d e v i a t i o n s a n d c o e f f i c i e n t s o f v a r i a t i o n of t h e d a t a in t h e e a r l i e r w o r k (5), h o w e v e r , w e r e cons i d e r a b l y h i g h e r t h a n o u r s . W e do n o t feel, t h e r e f o r e , t h a t t h e s e r e s u l t s d e m o n s t r a t e t h e a d d i t i o n of ascorbic acid provides any further benefit over simply freezing the samples. Certainly, our data show t h a t t h e r e is no s i g n i f i c a n t loss o f f o l a t e a c t i v i t y o v e r a p e r i o d o f a t l e a s t t w o weeks, i f t h e s a m p l e s a r e s t o r e d f r o z e n . F r e e z i n g is i m p o r t a n t , h o w e v e r , i f t h e s t o r a g e p e r i o d is to be a n y g r e a t e r t h a n a f e w h o u r s . W e h a v e n o t s t u d i e d t h e p o s s i b i l i t y of a s t a b i l i z i n g e f f e c t o f a s c o r b i c a c i d on u n f r o z e n s a m ples. T h e d a t a s u m m a r i z e d in F i g u r e 4 s h o w t h a t a
good c o r r e l a t i o n exists between red-cell f o l a t e v a l u e s o b t a i n e d f r o m t h e a s s a y of w a s h e d r e d cells, a n d f r o m t h e d e t e r m i n a t i o n o f w h o l e b l o o d f o l a t e w i t h c o r r e c t i o n s f o r s e r u m f o l a t e m a d e by a p p l i c a t i o n of e q u a t i o n (1). T h i s conclusion is cons i s t e n t w i t h t h e r e s u l t s of an e a r l i e r , l e s s d e t a i l e d a n a l y s i s by S p r a y "°). F i g u r e 3 i l l u s t r a t e s t h e p o i n t t h a t t h e r e is no s i g n i f i c a n t d i f f e r e n c e b e t w e e n r e d cell f o l a t e values c a l c u l a t e d e i t h e r f r o m e q u a t i o n (1) o r e q u a t i o n (2). F o r r o u t i n e red-cell f o l a t e d e t e r m i n a t i o n s , t h e r e f o r e , use o f e q u a t i o n (2) would be t h e m o r e a p p r o p r i a t e s i n c e less c a l c u l a t i o n is r e q u i r e d . For the determination of red-cell folate, our data c l e a r l y d e m o n s t r a t e t h e need f o r m a i n t e n a n c e of a c e r t a i n level of s e r u m p r o t e i n in t h e a s s a y s y s t e m . P r o t e i n m u s t be a d d e d to t h e r e d - c e l l l y s a t e to c o m p e n s a t e f o r t h e s e r u m loss r e s u l t i n g f r o m t h e h i g h d i l u t i o n f a c t o r n e c e s s a r y (1:42 in o u r m e t h o d ) . W e have found that folate-free human serum or human a l b u m i n can be u s e d i n t e r c h a n g e a b l y f o r t h i s p u r pose. In t h e d e t e r m i n a t i o n o f s e r u m f o l a t e , a d d i t i o n o f f u r t h e r s e r u m or a l b u m i n to t h e r e a c t i o n m i x t u r e h a s no v a l u e s i n c e t h e e x i s t i n g s e r u m p r o t e i n l e v e l s are apparently high enough. This has also been noted by Mincey and co-workers':". The results summ a r i z e d in T a b l e s 7 a n d 8 s h o w t h e e x t r e m e imp o r t a n c e o f t h i s a d d i t i o n in t h e r e d - c e l l a s s a y ; unf o r t u n a t e l y , t h e a c t u a l f u n c t i o n o f t h e s e r u m in t h e r a d i o a s s a y is n o t a t all well u n d e r s t o o d , a l t h o u g h M a r k k a n e n et al '~'~''3), a n d Z e t n e r et al '"~ h a v e s p e c u l a t e d t h a t it m a y f u n c t i o n a s a s o u r c e o f carrier protein (s). As mentioned earlier, a fairly large percentage o f t h e b l o o d s a m p l e s s e n t to o u r l a b o r a t o r y f o r f o l a t e assay have highly elevated serum and red-cell folate levels. H o f f b r a n d (7) h a s r e p o r t e d on p a t i e n t s w i t h h i g h s e r u m l e v e l s ( u p to 43 n g / m l ) b u t w i t h n o r m a l o r s u b n o r m a l r e d - c e l l f o l a t e s : t h e s e w e r e cons i d e r e d to be i n d i c a t i v e o f p e r n i c i o u s a n e m i a . A s f a r as we k n o w t h e r e h a v e b e e n no r e p o r t s o f b o t h h i g h l y e l e v a t e d s e r u m a n d r e d - c e l l f o l a t e levels. We are not sure of the significance of this since we do n o t h a v e c l i n i c a l d e t a i l s o f i n d i v i d u a l p a t i e n t s . From the specialties of our contributing physicians, we k n o w it to be l i k e l y t h a t a t l e a s t s o m e o f t h e s e patients are undergoing megavitamin therapy. In
R A D I O M E T R I C A S S A Y O F SERUM AND R E D - C E L L F O L A T E L E V E L S s u p p o r t o f t h i s , o u r d a t a in T a b l e 4 s h o w t h a t ing e s t i o n of l a r g e a m o u n t s of f o l a t e f o r a s h o r t p e r i o d of t i m e c a n r e s u l t in l a r g e i n c r e a s e s in b o t h s e r u m a n d r e d - c e l l f o l a t e levels. I t m a y w e l l be, t h e r e f o r e , t h a t t h e s e d a t a o b t a i n e d in o u r l a b o r a t o r y a r e n o t t y p i c a l of t h a t o b t a i n e d in g e n e r a l . I t is w o r t h n o t i n g , h o w e v e r , t h a t if f o l a t e - f r e e s e r u m h a d n o t b e e n a d d e d d u r i n g t h e r e d - c e l l f o l a t e a s s a y s in t h e p r e s e n t s t u d y , m a n y , i f n o t all, o f o u r h i g h r e d cell v a l u e s w o u l d h a v e r e g i s t e r e d w i t h i n t h e n o r m a l range. REFERENCES 1. Waxman, S., Schrieber, C., and Herbert, J., (1970). Blood., 36, 858-864. 2. Waxman, S., Schrieber, C., and Herbert, J., (1971). Blood, 38, 219-228. 3. Tajuddin, M., and Gardyna, H. A., (1973). Cli,l. Chem., 19, 125-126.
43
4. Dunn, R. T., and Foster, L. B., (1973). Clin. Chem., 19, 1101-1105. 5. Mincey, E. K., Wilcox, E., and Morrison, R. T., (1973). Clin. Biochem., 6, 274-284. 6. Zettner, A., and Duly, P. E., (1974). Clin. Chem., 20, 1313-1319. 7. Hoffbrand, A. N., Newcombe, B.F.A., and Mollin, D. L., (1966). J. Clin. Path., 19, 17-28. 8. Fleming. A. F., Comley, L., and Stenhouse, N.S., (1971). Am. J. Clin. Nutr., 24, 1257-1264. 9. Kubasik, N.P., Volosin, M. J., and Sine, H. E., (1975). Clin. Chem., 21, 1922-1926. 10. Spray, G. H., (1969). J. Clin. Path., 22, 212-216. 11. Rothenberg, S. P., Sheldon, P., da Costa, M., Lawson, J., and Rosenberg, Z., (1974). Blood, 43, 437-443. 12. Markkanen, T., Pajula, R. L., Virtanen, S., and Himanen, P., (1972). Acta Hematol., 48, 145-150. 13. Markkanen, T., Virtanen, S., Himanen, P., and Pajula, R. L., (1972). Acta He~natol., 48, 213-217.
