Ascorbic acid interference in the measurement of serum biochemical parameters: In vivo and in vitro studies

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1 Clinical Biochemistry 39 (2006) Ascorbic acid interference in the measurement of serum biochemical parameters: In vivo and in vitro studies Flávia Martinello, Edson Luiz da Silva Departamento de Análises Clínicas, Centro de Ciências da Saúde, Universidade Federal de Santa Catarina, Campus Universitário, Trindade, , Florianópolis-Santa Catarina, Brazil Received 3 September 2005; received in revised form 7 November 2005; accepted 25 November 2005 Available online 5 January 2006 Abstract Objectives: To investigate the negative interference of ascorbic acid in serum biochemical tests in relation to the dose of vitamin C intake and to the time of blood collection. Design and methods: Healthy volunteers (n = 18) consumed daily doses of vitamin C ( g) for 1 week and serum parameters were assayed prior to the experiment and on the eighth day of consumption. Blood samples were collected 4, 12 and 24 h after vitamin C intake. Results: Serum levels of ascorbic acid increased significantly after vitamin C ingestion inhibiting urate and total bilirubin tests 4 and 12 h after intake (P b 0.01). A significant negative interference occurred up to 24 h after consumption of 4 g vitamin C for the urate test. In contrast, ingestion of vitamin C did not show interference in glucose, triglyceride and cholesterol tests. Addition of ascorbic acid to serum inhibited the urate test to a similar extent to that observed after vitamin C intake. However, after ingesting vitamin C, the interference for the bilirubin test was greater than that of the in vitro interference. Conclusions: Commonly taken doses of supplementary vitamin C interfered negatively with the serum urate test based on the Trinder method, and with bilirubin metabolism The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Ascorbic acid; Laboratory interference; Serum parameters; Trinder method Introduction The interference of drugs in clinical chemistry analysis is considered a matter of continuous interest in the routine laboratory. The increasing consumption of vitamin supplements and, particularly, the use of large doses of vitamin C (ascorbic acid) [1] have become additional problems for clinical laboratory analysts. Ascorbic acid is readily absorbed from the intestinal tract and reaches the plasma in high levels [2,3]. When present in biological samples, ascorbic acid can interfere with the measurement of serum biochemical parameters, causing a false result. Such an altered laboratory test value might result in an erroneous diagnosis, unnecessary use of a treatment or use of an inappropriate drug dose. Ascorbic acid can alter laboratory test values through in vitro analytical Corresponding author. address: edson@ccs.ufsc.br (E.L. da Silva). interference or through an in vivo physiological mechanism. Furthermore, interference can also occur through both mechanisms simultaneously. It is important to differentiate between these mechanisms in an attempt to minimize or eliminate the interferences. Ascorbic acid, due to its significant reducing ability, is a well-known interfering compound in biochemical assays [4 7] that involve indicative systems with oxidases and peroxidases, commonly known as the Trinder method [8]. Such a method is frequently used in the measurement of various serum analytes including glucose, total cholesterol, triglycerides and urate. Besides inhibiting the Trinder reaction [9], ascorbic acid can also interfere in vitro and/or in vivo in the determination of bilirubin [3,10], phosphate [10], blood urea nitrogen (BUN) [10], creatinine [3,11 13] and of the enzymes aminotransferase, lactate dehydrogenase and alkaline phosphatase [2,3]. In addition, Badrick and Campbell (1992) have previously shown that intravenous infusion of mega-doses of ascorbate /$ - see front matter 2005 The Canadian Society of Clinical Chemists. All rights reserved. doi: /j.clinbiochem

2 F. Martinello, E.L. da Silva / Clinical Biochemistry 39 (2006) given to critically ill patients interfered negatively with the measurement of urate, total cholesterol, and triglycerides [15]. Although the interference of ascorbic acid has been recognized for a long time, in vivo studies on the occurrence of interference considering a modest ingested dose of supplementary vitamin C and the time of blood collection after vitamin consumption have not been carried out. Therefore, the question still remains whether common moderate doses of ascorbic acid in vivo can result in a clinically significant reaction with the most common reagent kits. Thus, the main objective of our study was to establish the level of ascorbic acid interference with the measurement of serum biochemical parameters in blood samples collected at different time periods after the consumption of common vitamin C doses. The level of this effect was compared to the in vitro interference after ascorbic acid addition to the serum pool. Additionally, we evaluated the decrease in negative interference due to spontaneous decay of ascorbic acid (auto-oxidation). It has been published in some secondary literature sources, usually package inserts, that laboratory tests should be repeated after min to avoid analytical interference from ascorbic acid. We hypothesized that such a recommendation should depend on the amount of ascorbic acid in the sample. Methods Reagents Vitamin C (Roche-Brazil, São Paulo-SP) as effervescent tablets was purchased from drugstores. Reagent kits for determination of serum biochemical parameters were kindly supplied by Labtest (Lagoa Santa, MG, Brazil), Bioclin (Belo Horizonte, MG, Brazil), Celm (Barueri, SP, Brazil), BioSystems (Barcelona, Spain) and Biodiagnóstica (Pinhais, PR, Brazil). Ascorbic acid and metaphosphoric acid were obtained from Merck (Rio de Janeiro, RJ, Brazil). Sulfuric acid, 2,4- dinitrophenylhydrazine, thiourea, copper sulfate and sodium chloride were of analytical degree and purchased from other national companies. Vitamin C consumption schedule The study population consisted of 18 healthy subjects, nonsmokers, 6 males and 12 females, with an average age of 24 years. The volunteers consumed increasing and successive once-daily doses of 0.25, 0.5, 1.0, 2.0 and 4.0 g vitamin C via oral means. Each dose was taken after dinner, around 8 p.m., for 7 days followed by 5 6 days of rest after each dose. Fasting blood was collected in tubes on the morning of the 8th day, 12 h after the ingestion of each different dose of vitamin C. After blood withdrawal, the volunteers took an additional tablet of vitamin C, with a light breakfast, and subsequent blood samples were collected 4 and 24 h following ingestion. During vitamin C ingestion of doses of less than 2 g/day, the consumption of vitamin C-rich fruits and beverages was avoided, especially on the last day of consumption for each dose. Serum was isolated by centrifugation (750 g, 15 min) and biochemical parameters were immediately assayed using enzyme kits (Labtest-Lagoa Santa-MG, Brazil). The laboratory test results collected after vitamin C consumption were compared with the average of 3 control values obtained for each individual before the vitamin C intake (for 3 different weeks, after 4 and 12 h fasting). After the purposes and risks of the protocol were explained, oral and written informed consent was obtained from the volunteers. The experimental protocol was approved by the Ethics Committee for Studies with Humans of the Federal University of Santa Catarina, Brazil (no. 068/2000). In vitro interference of ascorbic acid Increasing amounts of ascorbic acid ( μmol/l) were added to a normal nonicteric, nonhemolyzed serum pool. The concentration of ascorbic acid added was similar to the serum levels of ascorbic acid found after ingesting 0.25 to 4.0 g/day vitamin C. Additionally, higher amounts of ascorbic acid were added to mimic the serum levels of ascorbate found after intravenous infusion of mega-doses vitamin C (up to 30 g/day), particularly for cancer treatment [15]. Thus, an amount of ascorbic acid necessary to give a final concentration of 0.57 mmol/l and 2.27 mmol/l was also added to the serum pool. The initial ascorbic acid concentration in the serum pool was less than 28.4 μmol/l. Biochemical parameters were measured in the first hour after ascorbic acid addition and after 6, 12, 24, 48 and 72 h using reagent kits from various manufactures (Labtest-MG, Brazil; Bioclin-MG, Brazil; Celm- SP, Brazil; BioSystems-Barcelona, Spain and Biodiagnóstica- PR, Brazil). During this period of time, samples were kept at room temperature or at 4 C and protected from light. Measurement of serum biochemical analytes Serum parameters were measured according to the manufacturer's instructions. For the glucose, cholesterol, triglyceride and urate tests, peroxide/peroxidase was used to generate quinoneimine dyes, based on the reaction previously described by Trinder [8]. These test reagents all contained 4-aminophenazone and either phenol (glucose and cholesterol), 3,5-dichlorohydroxybenzoic acid (urate) or 4-chlorophenol (triglyceride). Urate was also determined by the UV method, based on the change in absorbance at 293 nm due to the disappearance of urate (Dade-Behring, Marburg-Germany). Serum bilirubin was measured by its reaction with diazotized sulfanilic acid. Creatinine was measured by the Jaffé method after reaction with picrate ions under alkaline conditions followed by a second reaction under acidic ph. The levels of ascorbic acid in serum samples obtained before and after oral supplementation with vitamin C were immediately determined using a previously described colorimetric method [16]. All analytes were determined in a Microlab 200 spectrophotometer (Merck, Dieren-The Netherlands). Statistical analysis The differences between the values for the serum parameters obtained before (average of 3 baseline values) and after intake

3 398 F. Martinello, E.L. da Silva / Clinical Biochemistry 39 (2006) of vitamin C were verified by the Student's paired t test. The negative interference of ascorbic acid was verified using the Pearson correlation between the values for the serum biochemical parameters and the levels of serum ascorbic acid after vitamin C intake. Correlation of serum ascorbic acid with vitamin C ingested doses was also tested. A level of P b 0.05 was considered significant. Results Serum ascorbic acid levels after vitamin C consumption All of the subjects completed the study. The most common side effects of ascorbic acid such as diarrhea, nausea and flatulence were not observed. None of the volunteers were taking other medications that have been reported to interfere with the biochemical parameters analyzed. Fig. 1A shows the serum ascorbic acid levels in healthy volunteers before and after ingestion of supplementary vitamin C at different doses. Serum concentration of ascorbic acid increased significantly 4 h after ingestion of 250 mg vitamin C in relation to the baseline values (125.5 vs μmol/l, P b 0.05). The consumption of increasing doses of vitamin C caused a higher elevation in the serum levels of ascorbic acid, reaching a maximum of ± 28.4 μmol/l 4 h after intake of 4 g vitamin C. After 12 and 24 h of ingestion, the amount of serum ascorbic acid diminished in comparison to serum levels at 4 h. However, the serum ascorbic acid in blood samples collected 12 h after ingestion of 1, 2 and 4 g vitamin C was still significantly higher than baseline levels (P b 0.05). In addition, in the blood withdrawn 24 h after intake of 4 g vitamin C, the serum level of ascorbic acid was 60% higher than the baseline concentration (P b 0.05; Fig. 1A). Fig. 1B shows the relationship between daily vitamin C supplementation and the ascorbic acid concentration observed in the serum. As can be seen, there was a positive correlation between the serum levels of ascorbic acid and the ingested doses of vitamin C after 4 h of intake (r = 0.91; P b 0.01). Such a correlation was also found in the samples collected 12 h after vitamin C consumption (r = 0.98; P b 0.01). Interference by ascorbic acid on serum parameters after vitamin C intake Fig. 1. (A) Serum levels of ascorbic acid after vitamin C intake. *P b 0.05; **P b 0.01; ***P b compared to basal values (0 g/day of vitamin C supplementation) Student's paired t test. The results are expressed as mean ± standard deviation of 18 determinations. (B) Pearson's correlation between the levels of serum ascorbic acid and the doses of vitamin C ingested. The blood sample was collected 4 h after vitamin C intake. The results for the serum biochemical analytes in blood samples collected 4 h after intake of different vitamin C doses are shown in Table 1. Three measurements of each serum analyte taken before ingestion of vitamin C were used as controls and the average of these 3 baseline levels was considered to be 100% in order to calculate the negative interference by ascorbic acid. The percentage of interference is defined as follows: (baseline values / vitamin C supplementation values 100). In the baseline samples, the intra-individual biological variation plus the analytical variation were % for serum urate and % for total bilirubin. It can be observed that after ingestion of a very modest vitamin C dose (0.25 g/day) there was a significant decrease in the serum level of urate, from 4.5 ± 1.6 mg/dl to 3.6 ± 1.4 mg/ dl (approximately 18%; P b 0.01; Tables 1 and 2). Such a negative interference increased to 29, 44, 58 and 62% after ingestion of 0.5, 1.0, 2.0 and 4.0 g/day vitamin C, respectively (P b 0.01; Tables 1 and 2). The serum levels of ascorbic acid correlated positively with the percentage of negative interference in the urate test, 4 h after vitamin C intake (r = 0.75; P b 0.05). Serum urate values measured by the UV method, which does not generate hydrogen peroxide, did not change significantly during prolonged ingestion of 4.0 g/day vitamin C (Tables 1 and 2). The consumption of 0.5, 1.0, 2.0 and 4.0 g/day vitamin C decreased the total bilirubin values by 34, 27, 28 and 27%, respectively, 4 h after ingestion (P b 0.05; Tables 1 and 2). However, the negative interference did not correlate with the vitamin C supplementation (r = 0.56; P = 0.25). The serum levels of total cholesterol, triglyceride, creatinine and glucose did not change after vitamin C consumption (Table 1). The results for the ascorbic acid interference in blood sample collected 12 h after vitamin C intake are shown in Table 2. A significant negative interference (20 30%; P b 0.001) in urate

4 F. Martinello, E.L. da Silva / Clinical Biochemistry 39 (2006) Table 1 Serum biochemical parameters (mg/dl) measured before and after 4 h of vitamin C intake Vitamin C supplementation (g/day) Urate (Trinder) 4.5 ± ± 1.4** 3.1 ± 1.1*** 2.5 ± 0.8** 1.9 ± 0.9*** 1.7 ± 0.9** Urate (UV) 5.8 ± 2.0 ND ND ND ND 5.0 ± 2.6 Total bilirubin 0.67 ± ± ± 0.15** 0.48 ± 0.13* 0.48 ± 0.22* 0.49 ± 0.12** Total cholesterol 155 ± ± ± ± ± ± 26.6 Glucose 86 ± ± ± ± ± ± 5.9 Triglyceride 92 ± ± ± ± ± ± 27.7 Creatinine 1.00 ± ± ± ± ± ± 0.10 Healthy volunteers (n = 18) consumed vitamin C at different doses for 1 week. Each dose and the serum biochemical parameters were measured 4 h after ingesting the last dose of vitamin C; *P b 0.05; **P b 0.01; ***P b 0.001, Student's paired t test compared to respective control (average of 3 determinations assayed before the vitamin C intake = 0 g/day). ND = not determined. levels still occurred after ingestion of doses N1.0 g/day vitamin C, although to a lesser extent when compared to the results for 4 h. In addition, the serum levels of total bilirubin were 20 28% lower than the baseline levels (P b 0.05). Such a negative interference was similar to that found in samples collected after 4 h(table 2). Serum triglycerides decreased by around 34% (P b 0.05) 12 h after ingestion of 4 g/day vitamin C (Table 2). Furthermore, the serum levels of total cholesterol, creatinine and glucose did not change after vitamin C consumption. In blood samples collected 24 h after vitamin C intake, the level of negative interference from ascorbic acid regarding the biochemical parameters decreased greatly in relation to that found in samples obtained 4 and 12 h after vitamin C ingestion. However, an inhibition of 28% (P b 0.01) in the serum urate test was observed after ingesting 4 g/day of vitamin C (Table 2). In vitro interference of ascorbic acid Results for the measurement of glucose, cholesterol, triglyceride, urate and bilirubin before and after addition of different concentrations of ascorbic acid are shown in Fig. 2. The negative interference of ascorbic acid in the laboratory tests was similar using reagent kits from several commercial trademarks (results not shown). Therefore, data were averaged and are expressed as mean ± standard deviation of assays for each serum analyte. Ascorbic acid had an inhibitory effect in all tests that use the oxidase/peroxidase system and in the bilirubin test in a concentration-dependent manner. The urate test was more sensitive to the interference of ascorbic acid, followed by the triglyceride, cholesterol, glucose and bilirubin tests (Fig. 2A). In the presence of μmol/l ascorbic acid, the urate test results were inhibited by approximately 84% compared with 34, 12, 11 and 9% for triglyceride, cholesterol, glucose and bilirubin tests, respectively (Fig. 2A). Such a sensitivity to ascorbic acid was dependent on the amount of analyte in the reaction system as can be seen by normalizing the data as ascorbic acid:analyte molar base (Fig. 2B). It was observed that all assays based on the Trinder reaction presented a stoichiometry of 1:1. For example, the molar ratio of 0.5:1 of ascorbic acid:analyte resulted in approximately 50% of negative interference for all tests. In contrast, the ascorbic acid interference with the bilirubin method was not consistent with the stoichiometric inhibition. Furthermore, a significant interference of ascorbic acid in the cholesterol, glucose, triglyceride and bilirubin tests Table 2 Negative interference of ascorbic acid on the measurement of serum analytes (% decrease from control) Vitamin C supplementation (g/day) hours Urate 17.8 ± 7.1 b 28.9 ± 10.7 c 44.4 ± 14.2 b 57.8 ± 27.2 c 62.2 ± 30.0 b Total bilirubin ± 13.2 b 26.9 ± 10.3 a 28.3 ± 12.6 a 27.2 ± 14.7 b 12 hours Urate ± 7.6 c 26.8 ± 12.1 c 31.7 ± 11.8 c Total bilirubin ND 20.8 ± 9.4 c 22.2 ± 5.7 c 23.6 ± 8.3 a 27.8 ± 9.4 a Triglyceride ± ± ± 12.2 a 24 hours Urate ND ND 9.9 ± ± ± 13.4 b Total bilirubin ND ND ± 4.8 The results are expressed as mean ± SD of 18 determinations. The average of 3 baseline values assayed before vitamin C intake was considered as control. a P b 0.05; b P b 0.01; c P b 0.001, compared to control (Student's paired t test). ND = not determined.

5 400 F. Martinello, E.L. da Silva / Clinical Biochemistry 39 (2006) urate test. Fig. 3B shows that the level of negative interference of ascorbic acid was similar in both situations, indicating that there was an analytic interference only. Results for the measurement of urate by the UV method, which showed unchanged values, also confirm this hypothesis (Table 1). Time-dependent decrease of ascorbic acid interference The results for the measurement of analytes 1, 6, 12, 24, 48 and 72 h after ascorbic acid addition to serum showed that a significant negative interference remains for some time, depending on the amount of ascorbic acid in the sample (Table 3 and Fig. 4). In the urate test, there was a negative interference of approximately 42% 48 h after addition of μmol/l ascorbic acid in the serum (Fig. 4). Even in the presence of a modest concentration of ascorbic acid (85.2 μmol/l), the negative interference persisted up to 24 h. In the presence of 2.27 mmol/l ascorbic acid, the negative interference remained practically unaltered, and close to 100%, for up to 72 h (Fig. 4). For the cholesterol, glucose, triglyceride and bilirubin testsm, the negative interference remained for up to 48 h only when 2.27 mmol/l of ascorbic acid was added to the serum. In general, ascorbic acid μmol/l interfered negatively in the first 6 h, particularly for the triglyceride test (Table 3). Discussion Fig. 2. In vitro negative interference of ascorbic acid in the urate, cholesterol, glucose, triglyceride and bilirubin tests. (A) Ascorbic acid was added to serum and the analytes were determined before (control) and 1 2 h after addition. The results are mean ± standard deviation of determinations for each analyte. (B) Results were normalized in a molar base of ascorbic acid:analytes. occurred only in the presence of the highest ascorbic acid levels studied, 0.57 and 2.27 mmol/l (Table 3). Ascorbic acid did not show a negative interference in the creatinine test regardless of the ascorbic acid concentration (results not shown). In vitro vs. in vivo interference by ascorbic acid As described above, ascorbic acid at concentrations b567.8 μmol/l did not interfere significantly in the bilirubin test (Table 3). However, a negative interference in the measurement of bilirubin was observed in the presence of low amounts of serum ascorbic acid after vitamin C consumption (Tables 1 and 2). Therefore, in an attempt to compare both in vivo and in vitro results, the data were normalized as the molar base of ascorbic acid:bilirubin. In fact, the negative interference after vitamin C consumption was significantly higher than that in vitro (P b 0.05), as can be seen in Fig. 3A. For example, for a molar ratio of ascorbic acid:bilirubin of 3:4, there was a negative in vitro interference of approximately 10% against around 35% after vitamin C intake. Considering the possibility that ascorbic acid also interferes in urate metabolism, analogous analysis was carried out with the The primary aim of this study was to demonstrate that ascorbic acid interference in laboratory tests is highly dependent on the dose of vitamin C ingested and on the time of sample collection after vitamin intake. The results of our study show that vitamin C ingestion, at doses commonly consumed by healthy individuals (up to 4 g/day) for one week, does not appear to affect glucose, triglyceride and cholesterol tests based on the Trinder method. On the other hand, relatively modest supplementation of vitamin C, such as 0.25 to 1 g/day, interferes significantly in the urate and bilirubin tests. The serum level of ascorbic acid increased around 2-fold (P b 0.05) 4 h after the ingestion of modest vitamin C doses (250 mg) (Fig. 1A). Similar results have been previously reported [17]. Such an ascorbic acid level was high enough to result in an inhibition of 17.8% (P b 0.01) in the urate test based on the uricase/peroxidase system (Tables 1 and 2). As expected, the serum ascorbic acid concentration showed a greater increase after ingestion of higher amounts of vitamin C, with a good correlation between the serum level of ascorbic acid and the ingested vitamin C dose (r = 0.91; P b 0.01; Fig. 1B). Consequently, a higher negative interference in the serum urate measurement was observed, with a maximum of approximately 62% 4 h after a supplementation of 4 g/day vitamin C (Table 2), which correlated with the serum level of ascorbic acid (r = 0.75; P b 0.05). The return of serum ascorbic acid levels to baseline values after interruption of vitamin C intake was dependent on the ingested dose (Fig. 1A). Thus, vitamin C at doses 0.5 g/day

6 F. Martinello, E.L. da Silva / Clinical Biochemistry 39 (2006) Table 3 Negative interference of ascorbic acid in vitro (% decrease from control) Time (h) Ascorbic acid added (μmol/l) Cholesterol ± ± ± ± ± ± ± ± ND ND 10.8 ± ± 7.5 Glucose ± ± ± ± ND 7.3 ± ± ± ND ND ± ND ND ND 28.9 ± 1.9 Triglyceride ± ± ± ± ± ± ± ± ± ± ± ND ± ± 7.0 Bilirubin ± ± ± ± ND ND 8.9 ± ± 2.5 Increasing amounts of ascorbic acid were added to serum and the analytes were determined before (control) and 1, 6, 12, 24 and 48 h after addition. The results are mean ± SD of determinations. ND = not determined. was almost cleared from the plasma in the first 12 h after ingestion. A delay time of 24 h was necessary for the elimination of plasma ascorbic acid resulting from supplemental vitamin C doses of 1.0 and 2.0 g/day and at least 48 h for the dose of 4 g/day vitamin C (Fig. 1A). Consequently, the percentage of negative interference in the urate test diminished the longer time period after the last ingested vitamin C dose (Table 2) at which the sample was collected. However, it is interesting to note that the intake of a relatively modest dose of vitamin C (1.0 g/day) still caused a significantly negative interference in the serum urate measurement for blood samples collected 12 h after vitamin C ingestion. Furthermore, intake of 4 g/day vitamin C interfered negatively in the urate test up to 24 h later (Tables 1 and 2). Such interference of ascorbic acid could be due to physiological and/or analytical effects on the urate levels. By comparing the level of interference after vitamin C intake with the in vitro interference value (Fig. 3B), we showed that ascorbic acid interfered with the urate measurement through an analytical mechanism only, i.e., both levels of interference occurred to the same extent. Results for the serum urate determination by the UV method, which is not based on the oxidase/peroxidase system and does not suffer the interference of ascorbic acid, confirm that variations in the serum levels of urate after vitamin C consumptions of up to 4 g daily for 1 week were due to analytical interference (Table 1). Freemantle et al. [18] have previously reported an in vitro interference of ascorbic acid addition in the urate measurement. Nevertheless, the ascorbic acid effects on urate metabolism are controversial. Stein et al. [19] and Wilson and Liedtke [20] showed that chronic administration of vitamin C can induce uricosuria and reduce the serum level of uric acid. More recently, Sumbaev and Iasinskaia [21] described that ascorbic acid can activate the synthesis of glucocorticoids reducing the serum level of uric acid. On the other hand, other studies demonstrated that supplementation with 0.5, 4, 8 or 12 g of vitamin C daily did not alter the serum uric acid level or urinary excretion of urate [14,22 24]. The ingestion of vitamin C at doses N0.5 g daily also interfered negatively (P b 0.05) in the measurement of serum bilirubin in blood samples collected 4 and 12 h after consumption (Tables 1 and 2). However, such interference did not correlate with the serum levels of ascorbic acid (r = 0.56; P N 0.05). In addition, interference to a similar extent was found in blood samples collected 4 and 12 h after ingestion of 0.5, 1.0, 2.0 or 4.0 g/day vitamin C (Table 2). These data suggest a biological effect of ascorbic acid on the bilirubin value. In fact, the lower level of in vitro interference in comparison to that after vitamin C intake (Fig. 3A) confirms that ascorbic acid affects primarily the bilirubin metabolism. Additional evidence was provided by the absence of a negative interference in the bilirubin measurement 2 and 4 h after intake of a single acute dose of 4 g vitamin C (results not shown). In vitro interference of ascorbic acid in the bilirubin test has been previously demonstrated [18,25]; however, to our knowledge, an in vivo effect has not been reported. A possible explanation for ascorbic acid interference in the bilirubin metabolism is the decreased levels of mrna heme-oxigenase-1, the rate-limiting enzyme of bilirubin biosynthesis, after ascorbic acid supplementation [26]. In contrast, intake of a relatively high dose of vitamin C (4 g/ day) by normal individuals did not affect the measurement of glucose and total cholesterol (Tables 1 and 2). A significant interference occurred in vitro only and in the presence of higher

7 402 F. Martinello, E.L. da Silva / Clinical Biochemistry 39 (2006) Fig. 3. Negative interference of ascorbic acid in tests for (A) total bilirubin and (B) for urate. Results obtained 4 and 12 h after C vitamin intake and after addition of ascorbic acid to serum pool were normalized on a molar base of ascorbic acid:analytes. acid ascorbic amount (0.57 and 2.27 mmol/l; Table 3 and Fig. 2). It is common to find information in package inserts that ascorbic acid can interfere with glucose and cholesterol tests. However, the studies cited are usually in vitro. Indeed, using higher concentrations of ascorbic acid in vitro, other authors have reported a significant inhibition for most laboratory tests that use redox reactions, including glucose, cholesterol, and triglyceride [18,27 30]. Such a level of ascorbic acid is not commonly found in plasma, as shown here, but can be observed in the plasma of patients using mega-doses (up to 30 g) of intravenous ascorbate [15]. Serum triglyceride decreased on average 23% 4 h after ingestion of 4 g vitamin C, but it was not significant (P N 0.05; Table 1). In addition, this interference range is lower than the biological variation for triglyceride, as has been described by Marcovina et al. (1994) [23]. However, in blood samples collected 12 h after ingesting 4 g vitamin C, and in a fasting state, a significant inhibition of 34% occurred (Table 2; P b 0.05), probably due to the low amount of triglyceride in the sample. Our in vitro results indicate that ascorbic acid would decrease the apparent concentration of all analytes whose determination is based on the Trinder method, molecule for molecule (Fig. 2B). This explains the higher ascorbic acid interference in the urate test in comparison to triglyceride, cholesterol and glucose tests, both ex vivo and in vitro. When present in serum, ascorbic acid might interfere longer than commonly believed. Our results show that a significant level of negative interference can persist up to 48 h for the urate test in the presence of μmol/l ascorbic acid (Fig. 4). Total cholesterol, glucose and triglyceride tests also showed important levels of interference up to 24 h after ascorbic acid addition (Table 3). The longer the interference persists, the higher the concentration of ascorbic acid in the sample. These results suggest that ascorbic acid in serum is stable for a long period of time. Sevanian et al. [31] reported that physiological concentrations of urate increase around 5-fold the ascorbic acid stability in human serum. In summary, the results of this study demonstrate that the consumption of modest doses of vitamin C (0.25 to 1 g/day) by normal individuals can interfere significantly in the measurement of serum urate (oxidase/peroxidase method) through analytical interference and in the bilirubin levels through a physiological mechanism. The ingestion of 4 g/day vitamin C can also inhibit the reaction for triglyceride when this analyte is present in low amounts in serum. The study also highlights the need to be cautious about secondary literature references on ascorbic acid interference, particularly on the measurement of cholesterol and glucose, where negative interference occurred only in the presence of high concentrations of ascorbic acid, and on the time needed to repeat the test when there is possibly ascorbic acid in the sample. Hence, based on these results, we suggest that vitamin C intake at doses 0.5 g daily should be interrupted 3 4 days before blood collection for bilirubin or urate tests based on the oxidase/peroxidase system. In addition, we recommend that clinicians interpret results cautiously when bilirubin and urate measurements are performed during or after ascorbic acid intake. Fig. 4. Negative interference of ascorbic acid in the urate test based on the oxidase/peroxidase system. Ascorbic acid was added to serum pool and urate was determined after different periods of time. Samples were kept at 4 C. The results are means of determinations for each variable assayed with reagent kits from different trademarks.

8 F. Martinello, E.L. da Silva / Clinical Biochemistry 39 (2006) Acknowledgments We thank Labtest, Bioclin, Celm, BioSystems and Biodiagnóstica for donation of kits. Flávia Martinello was a recipient of a MSc fellowship from the Coordenação de Aperfeiçoamento de Pessoal e Ensino (CAPES) and Edson L. da Silva is a recipient of a CNPq scholarship (proc /2003-7). This work was supported by FUNPESQUISA-UFSC. References [1] Ladas EL, Jacobson JS, Kennedy DD, Teel K, Fleischauer A, Kelly KM. Antioxidants and cancer therapy: a systematic review. J Clin Oncol 2004;22: [2] Bridgen ML. Unorthodox therapy and your cancer patient. Postgrad Med 1987;81: [3] Cameron E. Protocol for use of vitamin C in treatment of cancer. Med Hypotheses 1991;36: [4] Mayson JS, Schumaker O, Nakamura RM. False negative tests for urinary glucose in the presence of ascorbic acid. Am J Clin Pathol 1972;58: [5] Sharp P. Interference in glucose oxidase peroxidase blood glucose methods. 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