A Modified Microbiological Method for Determination of Folate Levels in Erythrocyte and Plasma

106 Sipahi H. et al.: Determination of Folate Levels in Erythrocyte and Plasma

Pteridines Vol. 18, 2007, pp. 106 - 113

Hande Sipahi1, Terken Baydar1, Meral Ozalp2, Gonul Sahin1

1Department of Toxicology, Faculty of Pharmacy, Hacettepe University, 2Department of Microbiology, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey

Abstract

Pteridines are natural heterocyclic compounds and they are generally divided into two main groups which are denominated as conjugated and unconjugated pteridines. Conjugated pteridines are the folate derivatives containing p-aminobenzoate and glutamate. Folate is a normal component of the cell. Folate derivatives play a crucial role in important biochemical pathways such as purine and pyrimidine biosynthesis, conversion of serine to glycine, histidine catabolism, methionine synthesis and detoxification reactions of certain xenobiotics, in which single carbon transfer is required. Folate deficiency has been proven to play a role in the pathogenesis of certain diseases. Additionally, exposure to some xenobiotics and prolonged use of some drugs may lead to folate deficiency. Serum, plasma and especially erythrocyte folate concentrations are generally used as biomarkers of folate pool. Therefore the present study was undertaken to establish an easy applicable, economic, accurate, sensitive, specific and reproducible microbiological method by using L. rhamnosus in our laboratory. Following the estab- lishment of the method its application was carried out in blood samples of 46 volunteers. Mean value of folate levels in erythrocyte were found 359.7 ± 133.3 ng/ml, while mean plasma folate levels were determined 27.3 ± 12.4 ng/ml. In erythrocyte, the average recovery was 103.0 ± 17.7% (mean ±SD), in plasma the average recovery was 101.0 ± 1.7% (mean ±SD) for folinic acid concentrations at 10, 25 and 50 ng/ml. The CVs were 15.1 ± 5.6% (mean ± SD) for within-run and 7.2 ± 3.9% (mean ± SD) for between-run precisions. In the present study, a modified microbiological method for folate analysis which can be applied to biological material, has been established.

Key words: Folate, microbiologic assay, Lactobacillus rhamnosus, blood

Introduction consequently, it is an essential nutrient (6, 7). Folate plays a crucial role in DNA and protein syn- Pteridines are natural heterocyclic compounds and thesis, suggesting that every mechanism, in which cell divided into two main groups, as conjugated and proliferation intervenes, may be altered, when folate unconjugated pteridines. Conjugated pteridines are the supply is abnormal. Its derivatives are co-factors in folate derivatives (1, 2). vital metabolic pathways involved in the transfer of Folate, pteroylmonoglutamate, PteGlu, is the gener- methyl groups. These include formation of purine, ic term for large family of chemically similar com- interconversion of serin to glycine, formate metabo- pounds. Folate consists of a 2-amino-4-hydroxy-pteri- lism, and pyrimidine biosynthesis. A defect in such dine moiety linked via methylene group at the C-6 biosynthesis could be responsible for the defective position to a p-aminobenzoyl-glutamate moiety (3, 4). DNA synthesis (8-13). Folic acid is the synthetic, oxidized monoglutamate Generally, folic acid deficiency has been associated form of folate. It is found in supplements and in forti- with many disorders such as megaloblastic anemia, fied foods (5). certain birth defects, especially neural tube defects and Unlike plants and bacteria, which are capable of hyperhomocysteinemia with several neurodegenera- synthesizing of folate, humans and animals are not; tive conditions including stroke, Alzheimer's disease

Correspondence to: Professor Gonul SAHIN, Ph.D., Department of Toxicology, Faculty of Pharmacy, Hacettepe University, 90-06100, Sihhiye, Turkey. Phone: +90 (312) 305 2178; fax: +90 (312) 309 2958; e-mail: [email protected] Pteridines/Vol. 18/No. 3 Sipahi H. et al.: Determination of Folate Levels in Erythrocyte and Plasma 107 and Parkinson's diseases, vascular disease, thrombosis, anaerobic bacteria that preferentially grow in a certain cancers, including colon cancer (14, 15). Folate microaerophilic environment; low oxygen and high status may be affected by many factors including age, carbon dioxide (5-10 % CO2) (28). habits such as smoking, diet style and exposure to some xenobiotics or long-term use of some drugs such Preparation of Culture as methotrexate, trimethoprim, sulfasalazine, antacid, Lyophilized culture vial was opened according to etc. These reasons have resulted in some effective ATCC instructions for L. rhamnosus 7469a. The entire measures against folate deficiency and fortification or pellet was rehydrated by using Lactobacilli MRS supplementation of foods with folic acid has been Broth and mixed well. This aliquot were aseptically applied in many countries including Turkey as public transferred into Lactobacilli MRS Broth tube, mixed health policy (14, 16-19). well and incubated at 37°C for 24 hours in sterilizer Evaluation of serum, plasma and especially erythro- (Nüve FN 500, Ankara, Turkey). Several drops of the cyte folate levels are useful parameters in clinic. suspension were transferred to petri dish containing Serum or plasma folate concentration reflects circulat- Lactobacilli MRS Agar. Following the incubation at ing folate, whereas erythrocyte folate levels represent low oxygen and high carbon dioxide at 37°C for 24 to intracellular folate status. For this reason, erythrocyte 48 hours, growing bacterial colonies on petri dish were folate is a better indicator of body stores (20, 21). A used for preparation of stock bacteria. number of methods have been used to quantify folate, including microbiologic methods, radioimmunoassays Preparation of Stock Bacteria using folate-binding protein, and various chromato- We prepared two different types of stock bacteria; graphic techniques so far (22-26). stabbing culture stored at + 4°C and bacteria suspen- The aim of this study is to establish an easy and sion in glycerol stored at -20°C. valid microbiological method, which is able to apply to Difco Lactobacilli Agar AOAC autoclaved erythrocytes and plasma in order to evaluate folate sta- (Rodwell, England) for 15 min at 121°C and hot liquid tus. agar was dispensed into screw cap tubes. After cooling to room temperature, the tubes were stored at +4°C. Lactobacilli Agar was used to prepare agar for the Material and Methods maintenance as stab cultures. Stabbing of harvested bacterial colonies was performed into Lactobacilli Chemicals Agar. The media were incubated at 37°C for 18 h, then All chemicals used in the study were analytical stored at +4°C. L. rhamnosus was maintained long grade. Folinic acid (calcium salt), and 2-mercap- term as stab cultures and refreshed every 4th-6th week toethanol (ME) were obtained from Sigma Chemical from the monthly maintenance stab. Co. (St Louis, MO, USA). Glycerol (GPR, BDH Sterile glycerol and TSB mixture was dispensed Chemicals LTD Poole England). Culture media: into sterile microcentrifuge tubes and then growing Tryptic soy broth (TSB) (Lab M, Lancashire, UK) bacteria on Petri dish containing Lactobacilli MRS were obtained from Acumedia manufacturer and, Agar were inoculated in to the Eppendorf tubes and Lactobacilli Agar AOAC, Lactobacilli Broth AOAC, stored at -20°C until used for experiments. Lactobacilli MRS Broth, Lactobacilli MRS Agar were obtained from Difco, Becton Dickinson France S.A., Preparation of Folinic Acid Standard Solution and Folic Acid Casei Medium (FACM) was purchased Stock solution of folinic acid was prepared by dis- from Difco, Becton, Dickinson and Company, Sparks, solving 1 mg folinic acid per 10 ml of 0.2 M 2-mer- MD21152. Standard bacterial strain, Lactobacillus captoethanol. The proper volume of the stock solution rhamnosus, ATCC 7469a were obtained from LGC was diluted with 0.2 M ME to obtain final concentra- Prochem, ATCC. tions of folinic acid at 10, 20, 50, 100, 250, 500, 1000, 2000, 4000 pg in the assay tubes (volume = 3.9 ml). Principles of microbiologic assay and application of For each experiment, the stock and working solutions the procedure were freshly prepared. To protect from oxidation, each Characteristics of Standard Bacterium working solution was covered with aluminium foil In this study, lyophilized cultures of Lactobacillus during experiment. rhamnosus ATCC 7469a was used in the folate analysis. Growth of L. rhamnosus ATCC 7469a depends on Collection and Preparation of Blood Sample folate. L. rhamnosus was formerly known as L.casei In order to apply the microbiological assay to the (26, 27). Lactobacilli are Gram-positive, facultative biological material, blood samples were obtained from

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46 volunteers early in the morning. 20 of them were growth in each tube at 600 nm by spectrophotometer male and 26 of them were female. Their ages ranged (Shimadzu UV1601, Japan). A standard curve was pre- from 25 to 86 years. All subjects provided written pared by using the standard folinic acid standards. informed consent. The ethical standards described by Folate levels of samples were calculated by using lin- the Helsinki Declaration were followed in the course ear calibration curve. Blank tube contained bacterial of the study. suspension and medium except folinic acid and/or Each blood sample was collected into glass tubes sample. containing K3EDTA as an anticoagulant. The sample Medium control tubes contained only medium to was then centrifuged (Heraeus Sepatech, Labofuge Ae, test the sterility of medium. Additionally to these, in Germany) at 3000 rpm for 15 min at room temperature the present study, effect of 2-mercaptoethanol on to separate the plasma and erythrocytes. During all growing of bacteria was assessed. experiments 0.2 M ME was used for dilution process. Plasma samples were diluted with freshly prepared 0.2 Statistical Analysis M ME (1:1) and erythrocyte samples were diluted with All of the results were expressed as mean ± standard 0.2 M ME (1:9). The mixture was incubated for 30 min deviation (SD). Linear regression analysis was used at 37°C. The samples were divided into Eppendorf for calculation of the standard curve tubes were stored at -20°C until analysis.

Adjustment of Intensity of Bacteria Suspension Results On the day before the assay, bacteria were transferred from the stabbing stock to screw cap tubes As shown in Figure 1-a, growth response of folinic included Lactobacilli Broth and incubated 18 hours at acid to the microorganism caused a typical sigmoidal 37°C to reinitiate active growth. Following incubation curve, and log-logit equation was used for transforma- the tubes were centrifuged at 5000 rpm for 15 minutes tion of this sigmoidal curve to linear curve (31). The and supernatant was removed. Bacteria were washed linear calibration curve was obtained between 10-4000 with 10 ml sterile de-ionized water and then cen- pg folinic acid in the assay tube (y= -2.4693x + 6.0348, trifuged at 5000 rpm for 15 minutes for ten times in R2 = 0.9934) (Figure 1-b). Figure 2 exhibits response every assay day. of L. rhamnosus to folinic acid on five different days. On every assay day, bacteria suspension in glycerol Standard curve by addition or without addition of cer- stored at -20°C were centrifuged at 5000 rpm for 15 tain amount of erythrocyte or plasma in to the assay minutes. Bacteria were washed with 10 ml sterile medium is shown in Figure 3. deinoized water and then centrifuged at 5000 rpm for Effect of preparation and storage of stock bacteria 15 minutes four times in every assay day. on growth of L. rhamnosus with folinic acid were After washing procedure, both stock bacteria were examined and the results are presented in Figure 4. resuspended with sterile saline. Intensity of bacteria In erythrocyte experiment, recovery averaged 103.0 ± ± suspension was adjusted according to 0.5 McFarland 17.7% (mean SD) on 3 occasions with 10, 25 and turbidity standard. This means that approximate num- 50 ng/ml of folinic acid. In plasma experiment, recov- ± ± ber of bacteria were 1.5 x 108 colony forming unit (cfu) ery averaged 101.0 1.7% (mean SD) on 3 occa- /ml when the absorbance of resuspended bacteria in sions with 10, 25 and 50 ng/ml of folinic acid. The sterile saline was 0.08-0.1 (29, 30). results of recovery experiments are given in Table 1. The coefficient of variations (CVs) for measure- Assay Procedure ments of folinic acid level in biological material were ± ± ± On every assay day firstly each sample stored at 15.1 5.6% (mean SD) for within-run and 7.2 ± -20°C was allowed for thawing to room temperature 3.9% (mean SD) for between-run precisions. The and then centrifuged at 3000 rpm for 10 min. The results of reproducibility are given in Table 2. Mean supernatant was used for the assay. FACM was freshly value of folate levels in erythrocyte were found 359.7 ± prepared according to Difco's direction. Each assay 133.3 ng/ml, while mean plasma folate levels were ± tube contained of 3.9 ml FACM, 25 µl L. rhamnosus determined 27.3 12.4 ng/ml. Maximum and mini- suspension adjusted according to 0.5 McFarland, 10 µl mum folate levels were found 59.3 and 11.1 for plas- erythrocyte or plasma sample or 10 µl standard solu- ma, and these values were 642.7 and 178.7 for ery- tion. The mixed tubes were incubated at 37°C for 20 throcytes, respectively. We could not find any correla- hours. tion between erythrocyte and plasma folate levels = After incubation, % transmittance was measured for (Spearman's rank correlation coefficient Rs 0.013) determination of turbidity intensity related to bacterial (Figure 5).

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a 100 100 standard curve standard curve + 10 ul plasma 80 80 standard curve + 10 ul erythrocyte

60 60

40 T % at 600 nm

T % at 600 nm 20 0 0 1000 2000 3000 4000 0 0 1000 2000 3000 4000 Folinic acid (pg)

Folinic Acid (pg) b Figure 3. Standard curve with and without biological material (amounts of folinic acid were put into assay y = -2,4693x + 6,0348 tubes of 3.9 ml). 5 R2 = 0,9934 4

2 stabbing culture stored at + 4° C 1 100 bacterial suspension in glycerol stored at -20° 0 logit B/Bo 01234 -1 80 -2

-3 60

-4 40 Log C T % at 600 nm

20 Figure 1. Growth response of folinic acid to L. rhamnosus and a standard curve example (amounts of folin- 0 ic acid were put into assay tubes of 3.9 ml). 0 200 400 600 800 1000

Folinic acid (pg)

100 Figure 4. Growth response of two different type of stock bacteria; stabbing culture stored at +4°C and 80 bacterial suspension in glycerol stored at -20°C (amounts of folinic acid were put into assay tubes of 60 3.9 ml).

T % at 600 nm Discussion 20 Folates are very sensitive to light, and they can be 0 0 1000 2000 3000 4000 easily oxidized. Therefore, 2-mercaptoethanol was used as antioxidant to prevent folate oxidation during Folinic acid (pg) storage of sample. 0.2 M ME did not have an effect on growth of bacteria. Figure 2. Standard curve based on the growth The serum folate levels can be artificially increased response of folinic acid to L. rhamnosus. Each value with consumption of folate-rich diet. The erythrocyte ± shown is the mean SD of 5 replicates of folinic acid folate levels reflect to tissue folate stores, therefore to containing standards (amounts of folinic acid were put evaluate erythrocyte folate concentrations are valuable into assay tubes of 3.9 ml). in clinic monitoring (4, 20, 21, 32). Erythrocyte folate

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priate absorbance interval determined. When the inten- 80 sity of bacteria suspension prepared according to 0.5 McFarland, approximate numbers of bacteria suspension were accepted as 1.5 x 108 cfu/ml when the 60 absorbance was 0.08-0.1 (29, 30).

) Screening of folate as a marker of some pathologies,

40 diseases or exposures to xenobiotics is very important for human health. Since 1940's, the microbiological method has been considered to be the most useful Plasma (ng/ml 20 assay for detecting small amounts of folates in biological samples. Microbiological assay is highly sensitive,

0 relatively inexpensive and simple method for 0 200 400 600 800 analysing of folate levels in blood (23, 26, 27, 35-40). Microbiological assay using L. rhamnosus has Erythrocyte (ng/ml) advantage of avoiding excessive sample preparation procedures. We found that recovery ratios were high Figure 5. Scatter plot of eythrocyte and plasma folate (103.0% for erythrocyte and 101.0% for plasma exper- concentrations. iments). It can be considered as a reproducible method for biological material and the CVs % for measure- concentrations for healthy adults change from 140 to ments were 15.1 and 7.2 for within-run and between- 630 ng/ml and serum folate concentration was 3 to 20 run precisions, respectively. Microorganism stock is ng/ml (6, 33, 34). The results in our study, mean value easy to maintain. Small amounts of samples are ade- of erythrocyte and plasma folate levels in normal sub- quate for the assay. Large number of sample can be jects felt into this range. evaluated in the same assay in a short time (23, 26, 38). Effect of intensity of bacteria suspension on sensi- In the study, we have established a modified micro- tivity of the measurements was investigated and appro- biological method for folate analysis which can apply

Table 1. Recovery of the microbiological assay

Added Expected Detected Folate level amount of Recovery Sample folate level folate level (ng/ml) folinic acid % (ng/ml) (ng/ml) (ng/ml) Diluted Erythrocyte 24.2 10 34.2 39.5 115.6 (1:10) Diluted Erythrocyte 24.2 25 49.2 54.4 110.6 (1:10) Diluted Erythrocyte 24.2 50 74.2 61.4 82.8 (1:10)

Average recovery % = 103.0 ± 17.7

Diluted Plasma 6.8 10 16.8 17.3 102.9 (1:1) Diluted Plasma 6.8 25 31.8 31.8 100.0 (1:1) Diluted Plasma 6.8 50 56.8 56.7 99.9 (1:1)

Average recovery % = 101.0 ± 1.7

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Table 2. Precision of the method, 3 specimens with different content of folinic acid were analysed.

Within-run Sample 1 Sample 2 Sample 3

Number of measurements 4 4 4

Mean Value (ng/ml) 97.6 166.0 115.7

SD 18.4 29.7 10.1

CV % 18.8 17.9 8.7

Average CV % = 15.1 ± 5.6

Between Days Sample 1 Sample 2 Sample 3 Sample 4

Number of measurements 2 2 2 2

Mean Value (ng/ml) 88.4 155.0 125.5 163.0

SD 6.6 19.5 6.2 6.0

CV % 7.5 12.5 5.0 3.7

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