S e r u m

Folate

Levels

C o m p a r i s o n of Microbiologic Assay and Radioisotope Kit M e t h o d s CAROLINE C. WADDELL, M.D.,

PEGGY A. DOMSTAD, M.D.,

FELIX J. PIRCHER,

SAMUEL R. LERNER, P H . D . , JAMES A. BROWN,

M.D.,

B.A.,

BEVERLY K. LAWHORN, B.S., M.T. (ASCP)

Department of Medicine, Section of Hematology, and Nuclear Medicine, Baylor College of Medicin and the Veterans Administration Hospital, Houston, Texas ABSTRACT

SIGNIFICANT ADVANCES in the understand- possible to determine serum folate levels ing of folate metabolism have been made by a microbiologic method. 2 The technic since the late 1950's, when it became has since been modified by several investiZ : 7 7 '. u ,o i20.0 (>45.4) 17.0 (38.59) 4.5 (10.21) 6.9 (15.66) 9.0 (20.43) 4.6 (10.44) 5.7 (12.93) 4.2 (9.53)

8.6 (19.52) 3.2 (7.26) 5.1 (11.57) 4.8 (10.89) 3.6 (8.17) 3.8 (8.62) 4.8 (10.89) 3.3 (7.49) 6.0 (13.62) 4.2 (9.53) 7.8 (17.70) 5.6 (12.71) 5.8 (13.16) 5.2 (11.80) 6.0 (13.62) 9.2 (20.88) 6.6 (14.98) 4.5 (10.21) 4.2 (9.53) 4.1 (9.30) 9.6 (21.79) 3.8 (8.62) 8.2 (18.61) 4.5 (10.21) 6.0 (13.62) 9.4 (21.33) 7.0 (15.89) 13.0 (29.51) 6.6 (14.98) 17.5 (39.72) 11.0 (24.97) 1 1.0 (24.97) 13.0 (29.51) 30.0 (68.1) >50.0 (> 113.5) 20.0 (45.4) 5.8 (13.16) 8.4 (19.06) 16.0 (36.32) 4.1 (9.30) 10.0 (22.7) 7.0 (15.89)

" ('.roup A. boih \alues it] deliniteh delUient range. f (.lonp IV \alues ineonsMenl or indelet minant. :j: Croup ( , normal \olunleer eonlrols. § ( ; i o i i p I"), hoth \alnes in nonn;il range. Mean \alue ol hinas-av (',.07 (13.77). Mandard deviation 2.61 (.1.92). Mean value ol tadinav,a\ : .1.32 (12.(17). standard delation 3.04 (6.90). Mean ditlerente 0.7.1 (1.70). Mand.nd deviation 2.33 (.1.28). Degrees of lieedom 73. I \alne 2.77 (|>-- 0.01).

acid were separated by adsorption on dextran-coated charcoal. The supernate was separated from the charcoal by centrifugation in a refrigerated centrifuge (Sorvall Model RC-3) at 8 C, 2,000 x g for 30

minutes. T h e supernate was then decanted into plastic vials, 10 ml of scintillation fluid (Packard Insta-gel) added, and the samples counted in the liquid scintillation counter (Packard Tri-carb). Serum folate values

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Group B Palienist 8 3.1 (7.03) 9 3.7 (8.39) 10 1.5 (3.40) 11 3.6 (8.17) 6.0 (13.62) 12 1.6 (3.63) 13 1-1 5.5 (12.48) 15 5.4 (12.25) 16 4.0 (9.08) 17 4.6 (10.44) 18 4.3 (9.76) Group G 1aticutsl 19 5.7 (12.93) 20 3.6 (8.17) 21 1 1.5 (26.10) 22 6.3 (14.30) 23 5.8 (13.16) 21 10.3 (23.38)

Rad oassay ng/nil (nmol/1)

October 1976

749

SERUM FOLATE LEVELS BY TWO METHODS

were then determined from a standard curve prepared using 5 N-methyltetrahydrofolic acid. T o determine the effects, if any, of repeated thawing, exposure to light, and refreezing, serum samples from two folatedeficient patients and three normal controls were thawed, exposed to light, analyzed and refrozen at weekly intervals for as long as 14 weeks.

30 r

H

20

m is

Y = 0.90X Y = RADIOASSAY X = MICROBIOASSAY

Results

5

10

15

20

25

MICROBIOLOGICAL METHOD ng/ml SERUM

FIG. 1. Lower line statistically analyzed. Leastsquares line, origin forced through zero; equation Y = 0.90 X where Y = radioassay value, SE ± 2.-19. slope of least-squares line significantly less than 1 (p < 05). cantly different statistically (t value = 4.6), but were quite comparable for clinical interpretation. T h e variabilities of replicate analyses within the two radioassay methods were not significantly different. T h e effects of thawing, exposure to light, and refreezing of the samples are shown in Table 3. No evidence of deterioration of folate activity was found over a period of weeks of testing under these conditions. D iscussion Deficiency of folic acid is frequently encountered in clinical medicine. This deficiency results in impairment of DNA synthesis, leading to the characteristic morphologic changes known as megaloblastic anemia. Folate deficiency occurs in a wide variety of disease states, such as alcoholism, food faddism, renal dialysis, gluten-induced non-tropical sprue, tropical sprue, regional enteritis, lymphomatous or leukemic infiltrations of the small intestine, and Whipple's disease, and following extensive small-intestinal resections. 3 Low serum folate levels have also been found in patients receiving ami-

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Normal values for serum folate by the microbiologic assay and both forms of radioassay, as determined in our laboratories, ranged from 3 to 14 ng/ml (6.81 to 31.78 nmol/1) serum. Values less than 3 ng/ml (6.81 nmol/1) were considered indicative of folate deficiency. T h e results of the microbioassay and sequential-binding radioassay (Clinical Assays, Inc.) are shown in Table 1. Of 78 sera compared, seven were in the definitely deficient range (less than 3 ng/ml, or 6.81 nmol/1) by both methods (Group A in Table 1), 60 were normal by both methods (Groups C and D), and 11 were indeterminate (Group B—one value less and the other more than 3 ng/ml or 6.81 nmol/1). Values between 0 and 14 ng/ml (0 and 31.78 nmol/1) (the clinically significant range) were subjected to statistical analysis. A least-squares line, forced through the origin, was obtained and is illustrated in Figure 1. The equation of the least-squares line was Y = 0.9 X, where Y = radioassay value (standard error ±2.49). The four values above 14 ng/ml (31.78 nmol/1) were not included in the statistical analysis since very high values were felt to be secondary to administration of folic acid therapy prior to assay. T h e variability of replicates by the radioassay methods was determined in folate-deficient and normal sera; results obtained by analysis of the two technics are shown in Table 2. Values obtained by the two radioassay methods were signifi-

750

A.J.C.P.—Vol.

WADDELL£T/1L.

antibiotics in the patient's serum may depress the growth of the organism, rendering test results invalid. Radioassay procedures, in contrast, can be performed in any clinical radioisotope laboratory equipped for liquid scintillation counting, and requires only 4 - 5 hours for determination of results using test kits that are commercially available. Folate values obtained by radioassay are not influenced by antibiotics in the patient's serum. In this study, we prospectively measured serum folate levels in 72 patients and six normal controls using the L. casei microbiologic method and a commercially available sequential-binding radioassay kit method to test for reproducibility and correlation between these two technics. Our data indicated strong correlation be-

Table 2. Comparison of Sequential-binding and Competitive Protein-binding Radioassay Methods; Analysis of Variance of Replicates by Each Method

Sample W llu

Sequential-binding ng/ml (ninol/1)

Folate

1.7 (3.85). 1.6 (3.63). 1.4(3.17). 1.5(3.40)

Mean = 1.55 (3.51)

5.2(11.80), 5.4 (12.25). 5.0 (11.35)

Mean = 5.2 (11.80)

Competitive Protein-binding ng/ml (nmol/1) 1.4 (3.17), 1.2 (2.72), 0.8 (1.81) 4.1 (9.30), 4.6 (10.44), 4.9(11.12),4.7(10.66)

Folate Mean = 1.13 (2.56) Mean = 4,57(10.37)

7.5 (17.02). 7.4 (16.79). Mean = 7.33(16.63) 7.1 (16.11) 7.6 (17.25). 7.3 (16.57). Mean = 7.3 (16.57) 7.0 (15.89) 0.9 (2.04), 1.2 (2.72). Mean = 1.03 (2.33) 1.0 (2.27) Mean = 1.4 (3.17) 1.2 (2.72), 1.6 (3.63)

6.9 (15.66). 7.1 (16.11). 7.2 (16.34)

Mean = 7.06(16.02)

7.4 (16.79). 7.5 (17.02). 7.2 (16.34)

Mean = 7.36(16.70)

.1

1.8 (4.08), 1.5 (3.40), 2.0 (4.54)

iVI

7.8 (17.70). 7.6 (17.25). 7.5 (17.02) Mean = 7.63(17.32)

7.4 (16.79), 7.0 (15.89), 7.2 (16.34) 7.4 (16.79), 7.0 (15.89). 7.2 (16.34)

Mean value of individua sample means Standard deviation of replicates

Mean value of individual sample means Standard deviation ol replicates

Ph Pu 11 Bl

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Mean liricmii-c = I).:!.) (0.7'.)). Sliiiul; r