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1 Available online at Annals of Clinical & Laboratory Science, vol. 45, no. 4, Influence of Vitamin C on Urine Dipstick Test Results Dae-Hyun Ko 1, Tae-Dong Jeong 1, Sollip Kim 2, Hee-Jung Chung 3, Woochang Lee 1, Sail Chun 1, and Won-Ki Min 1 1 Department of Laboratory Medicine, Asan Medical Center and University of Ulsan College of Medicine, Seoul, 2 Department of Laboratory Medicine, Ilsan Paik Hospital, Inje University College of Medicine, Goyang, and 3 Department of Laboratory Medicine, Cheil General Hospital and Women's Healthcare Centre, Kwandong University School of Medicine, Seoul, Korea Abstract. Background. Vitamin C is a strong reducing agent found at high levels in various foods, and it may influence the results of urine strip tests even at an ordinary consumption levels. After oral administration, we measured urine vitamin C levels using urine strips and evaluated whether vitamin C interfered with various test items. The utility of a urine strip with a vitamin C indicator was assessed. Methods. Thirty-three healthy volunteers each ingested 1,000 mg of vitamin C. Their urine samples were tested for vitamin C using a URiSCAN 11 strip (YD Diagnostics, Korea) before and after administration of vitamin C. Standard materials were added to normal pooled urine to generate urine samples with various concentrations of the analytes tested (blood, bilirubin, nitrite, leukocytes, and glucose), and vitamin C was spiked to predetermined levels. These samples were then tested using two urine strips - URiSCAN and Chemstrip test strip (Roche Diagnostics, Germany) - to evaluate interference from vitamin C. In clinical samples with positive vitamin C results, microscopic and chemical analyses were also conducted to examine the differences. Results. Thirteen urine samples from the 33 volunteers were positive for vitamin C before ingestion, and all subjects were positive after ingestion. Vitamin C spiking of urine demonstrated false-negative results at various concentrations. Of 159 specimens with positive results for vitamin C, 14 showed discrepant results after additional confirmatory tests. Conclusions. Vitamin C in urine can cause significant interference with urine strip tests. A urine strip with a vitamin C indicator is useful to reduce the risk of incorrect results in regard to disease states. Key words: vitamin C, urine strip test, interference Introduction Vitamin C is a strong reducing agent present in many foods. Its antioxidant properties are thought to help prevent cardiovascular disease and aging. For that reason, the consumption of vitamin C has become popular. Vitamin C is also involved in cancer prevention and improving the quality of life of cancer patients [1]. Urinalysis (UA) is noninvasive, simple, and one of the most important screening tests in clinical practice. UA provides information that can lead to the early detection of diseases in many organs. Urine strip products are available from many diagnostic companies worldwide, and common test items include blood, bilirubin, urobilinogen, ketones, protein, nitrite, glucose, ph, specific gravity (SG), and leukocytes. Address correspondence to Won-Ki Min, M.D., Ph.D.; Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, 88, Olympic-ro 43-gil, Songpa-gu, Seoul, , Korea; phone: ; fax: ; e mail: wkmin@ amc.seoul.kr UA strips are coated with reagents for the measurement of each analyte, and chemical reactions produce color gradations to be measured semi-quantitatively. Many of these reactions are oxidation reactions, and the degree of oxidation is proportional to the concentration of the analyte tested. The presence of vitamin C, an antioxidant, in urine may therefore cause false-negative results for some test items. This is especially problematic for blood and glucose, which are detected via the peroxidase reaction. False-negative results may also occur for nitrite, bilirubin, and leukocytes [2,3]. Many diagnostic companies have sought to develop UA strips resistant to such interference [4]. Alternatively, one company (YD Diagnostics, Korea) has launched a UA strip with a vitamin C test to additionally measure the vitamin C concentration in urine. This study had two objectives: to evaluate the utility of this vitamin C strip by testing urine samples after vitamin C ingestion and to investigate the effects of vitamin C on other test results /15/ by the Association of Clinical Scientists, Inc.
2 392 Annals of Clinical & Laboratory Science, vol. 45, no. 4, 2015 Table 1. Results using the vitamin C stick, before and after consumption of a vitamin C supplement. After/Before Negative Total Negative Total Table 2. Interference by ascorbic acid using the URiSCAN 11 strip. Ascorbic acid (mg/dl) Titrated concentration Glucose(mg/dL) 0 Neg. Neg. Neg. Neg. Neg. 100 ± ± ± ± Neg Neg Neg Blood(RBC/µL) 0 Neg. Neg. Neg. Neg. Neg. 5 ± ± ± ± Neg Leucocytes 0 Neg. Neg. Neg. Neg. Neg. (WBC/µL) 10 ± ± Neg. Neg. Neg Neg. Neg. Neg Neg. Neg. Neg. Nitrite (mg/dl) 0 Neg. Neg. Neg. Neg. Neg Pos. Pos. Neg. Neg. Neg. Bilirubin(mg/dL) 0 Neg. Neg. Neg. Neg. Neg Neg. Neg. Neg Neg Materials and Methods This study was conducted in accordance with the Declaration of Helsinki. It was approved by the Asan Medical Center Institutional Review Board (Project # ). Test for vitamin C in urine before and after oral administration. We recruited 33 healthy volunteers for the experiment. Their urine samples were tested for vitamin C using a urine strip (URiSCAN 11 strip, YD Diagnostics, Korea) before and 4-8 hours after oral administration of vitamin C (1,000 mg). The results were scored as 0 for 0-9 mg/dl, 1+ for mg/dl, 2+ for mg/dl, and 3+ for 35 mg/dl or more of vitamin C. Interference test for pooled samples. Pooled urine samples were prepared from healthy volunteers whose urine samples were normal on all urine strip tests. Standard materials were then added to the pooled urine to create predetermined concentrations of the analytes tested (blood: 0, 5, 10, and 250 RBC/µL, bilirubin: 0, 0.5, 1.0, and 3.0 mg/dl, nitrite: 0 and 0.05 mg/dl, WBCs: 0, 10, 25, and 500 WBC/µL, glucose: 0, 100, 250, 500, and 2,000 mg/dl). The samples were spiked with varying amounts of vitamin C, adjusted to final concentrations of 10, 50, 100, and 500 mg/dl. A random urine sample could have vitamin C up to µmol/l (about 340 mg/dl) [2]. Some of the urine collected after vitamin C administration consistently showed 3+ after several-fold dilution.
3 Influence of vitamin C on urine dip stick test results 393 Therefore, the range was extended to 500 mg/dl, although the cut-off for 3+ was 35 mg/dl, to provide evidence for the relation between vitamin C and UA strip results. The samples were then tested for blood, bilirubin, nitrite, glucose, and leukocytes. These tests were conducted using the URiSCAN 11 strip, which is the most widely used UA strip in Korea [5], and the Chemstrip Test Strip (Roche Diagnostics, Germany), which is relatively resistant to interference by vitamin C [4]. Interference tests using clinical samples. To detect vitamin C using a URiSCAN 11 strip, we tested urine samples requested for UA from July 2008 to August 2008 at Asan Medical Center, Korea. In total, 676 samples were tested, and 159 samples (24%) were positive for vitamin C, with a reaction intensity of 1 or greater. All of these samples were retested using reference methods microscopic analysis of urine sediment to quantify RBC and WBC, and chemical analysis of glucose using an automatic chemistry analyzer to compare the results with those from the UA strip. Results Test for vitamin C in urine before and after oral administration. Of the 33 volunteers, 39.4% (13/33) were positive and the others (60.6%, 20/33) were negative for vitamin C before ingestion of vitamin C. All subjects were positive after vitamin C ingestion. One specimen (3.0%) showed a 1+ reaction intensity, 15 (42.4%) showed 2+, and the remaining 17 (51.5%) showed 3+ (Table 1). Interference test with pooled samples. The test results using the URiSCAN are shown in Table 2. The samples with 100, 250, or 500 mg/dl glucose showed false-negatives with 500 mg/dl vitamin C, and the blood cell tests were also negative at the same concentration of vitamin C for the urine samples with 5 RBC/µL. Vitamin C at 50 mg/dl or greater concentrations caused the test results to change to negative for nitrite and WBC amounts over the ranges tested. Samples with 0.5 mg/dl bilirubin showed negative results with 50 mg/dl or greater vitamin C, and the urine samples with higher bilirubin levels (1 and 3 mg/dl) were generally not affected by vitamin C. Table 3 shows the results of using the Chemstrip. Interestingly, 500 mg/dl vitamin C increased the reaction strength for glucose and blood cells. The nitrite and leukocyte results were similar to those of URiSCAN, except for the nitrite reaction at 10 mg/ dl of vitamin C. Interference test using clinical samples. In total, 159 specimens (24 %) were positive for vitamin C at reaction intensities of 1+ or more. The positive samples were tested for discrepancy between the URiSCAN strip and reference methods. Of the specimens, 14 (8.8%) had discrepancies between the strip results and additional confirmatory tests, as shown in Table 4. Of the specimens, five were falsely negative for blood cells and eight were falsely negative for leukocytes when using the strip. One specimen was negative for glucose according to the URiSCAN strip, contrary to the results obtained using the automatic chemistry analyzer (122.5 mg/dl). Discussion After ingestion of a typical dose of vitamin C in dietary supplement form, all participant urine samples were positive for vitamin C. High concentrations of vitamin C in urine can cause false decreases in dipstick reactions, with a variable degree of interference, depending on the analytes and their concentrations. We confirmed the results again in clinical samples that were positive for vitamin C. In the 1980s, the antioxidant properties of vitamin C were shown to be related to its anticancer effects [6]. In some studies, intravenous administration of high-dose vitamin C to terminal cancer patients was relatively tolerable and safe [7,8]. In three terminal cancer patients, high-dose vitamin C, administered intravenously, showed long-term tumor-regressing effects [9]. Moreover, in a study evaluating the risk for colorectal cancer in 715 colorectal cancer patients and 727 age- and gendermatched community controls, vitamin C ingestion was effective in reducing the risk of colorectal cancer in a dose-dependent manner [10]. Vitamin C administration may also improve the quality of life of terminal cancer patients [11]. It is well known that vitamin C in urine can cause false-negative results for blood cells and glucose, the tests for which specifically use oxidation reactions. In this study, vitamin C interfered with these reactions as expected when using the URiSCAN test, but interference was also seen with higher concentrations of vitamin C using the Chemstrip test. Residues of strongly oxidizing cleaning agents in the urine container may also cause false-positive results when using the Chemstrip (according to the
4 394 Annals of Clinical & Laboratory Science, vol. 45, no. 4, 2015 Table 3. Interference by ascorbic acid using the Chemstrip. Ascorbic acid (mg/dl) Titrated concentration Glucose(mg/dL) 0 Neg. Neg. Neg. Neg ± Blood(RBC/µL) 0 Neg. Neg. Neg. Neg ± 1+ ± ± Leucocytes 0 Neg. Neg. Neg. Neg. Neg. (WBC/µL) 10 ± ± Neg. Neg. Neg Neg. Neg. Neg Neg. Neg. Neg. Nitrite(mg/dL) 0 Neg. Neg. Neg. Neg. Neg Pos. Neg. Neg. Neg. Neg. Bilirubin(mg/dL) 0 Neg. Neg. Neg. Neg. Neg. 0.5 Neg. Neg. Neg. Neg. Neg. 1 ± ± ± Neg. Neg ± Neg. Table 4. Discrepant results due to the presence of vitamin C between the urine strip test and confirmatory reference methods. Specimen Test Urine strip Reference method Vitamin C number results results concentration 1 Blood Negative 3-5 RBC/HPF 2+ 2 Negative 3-5 RBC/HPF 2+ 3 Negative 6-10 RBC/HPF 3+ 4 Negative 3-5 RBC/HPF 3+ 5 Negative 3-4 RBC/HPF 3+ 6 Leukocytes Negative 3-5 WBC/HPF 1+ 7 Negative 3-5 WBC/HPF 1+ 8 Negative 3-5 WBC/HPF 2+ 9 Negative 3-5 WBC/HPF Negative 3-5 WBC/HPF Negative 6-10 WBC/HPF Negative 3-5 WBC/HPF Negative 6-10 WBC/HPF Glucose Negative mg/dl 3+ HPF: high power field.
5 Roche Diagnostics product insert), but additional experiments are needed to clarify the exact cause(s). The Chemstrip uses iodate or potassium iodide chromogen as a scavenger to reduce interference from vitamin C [12,13]. The provider claims that it is not influenced by vitamin C when the glucose concentration exceeds 100 mg/dl, but it cannot eliminate interference and provides no information about interference at lower concentrations of glucose [3]. Berg demonstrated that with 30 mg/dl vitamin C, 23 of 30 specimens with glucose levels of 50 mg/dl showed false-negative results [14]. Brigden et al. also reported that specimens with glucose levels of 5.6 mmol/l (100.9 mg/dl) or greater were resistant to interference by vitamin C at a high concentration (10,000 µmol/l), but specimens with glucose levels of 1.4 mmol/l (25.2 mg/ dl) gave false-negative results when the vitamin C concentration was 500 µmol/l (8.8 mg/dl) [2]. Nitrite tests gave false-negative results at lower concentrations of vitamin C with both strips. The Chemstrip was more resistant to vitamin C for blood and glucose than was the URiSCAN 11 strip, and vice versa for nitrite. The Multistix strip (Bayer Healthcare, USA), which is the second-most widely used product in Korea [5], is also influenced by vitamin C at 25 mg/dl or greater concentrations, causing false-negative nitrite results. Bilirubin tests using both strips also showed weaker results with higher concentrations of vitamin C. Thus, it is reasonable to add a test item for vitamin C to detect possible interference. Overall, the proportion of samples with false negative results due to vitamin C was small. However, when viewing an individual test with all normal results and positive vitamin C, false negatives due to vitamin C cannot be ruled out. Thus the vitamin C strip may help physicians to more appropriately interpret UA results. To reduce costs, normal UA test results are not usually followed up with additional confirmatory tests. However, the results of this study show that overlooking the presence of vitamin C in urine may lead to potentially serious false-negative results, especially for glucose and blood. Thus, a vitamin C-resistant strip or a strip with a vitamin Influence of vitamin C on urine dip stick test results 395 C indicator is a preferred solution. For specimens positive for vitamin C, the results should be reported with a comment, such as Vitamin C in urine can cause false-negative results for glucose and blood cells. Repeating a test after restriction of vitamin C consumption is recommended, or the specimens should be subjected to additional follow-up analyses. Vitamin C-resistant strips do not eliminate possible interference by high concentrations of vitamin C; thus, using a strip with a vitamin C indicator is preferable. References 1. Grosso G, Bei R, Mistretta A, et al. Effects of vitamin C on health: a review of evidence. Front Biosci (Landmark Ed);18: doi: 4160 [pii][published Online First: Epub Date]. 2. Brigden ML, Edgell D, McPherson M, et al. High incidence of significant urinary ascorbic acid concentrations in a west coast population--implications for routine urinalysis. Clin Chem 1992;38(3): Zweig MH, Jackson A. Ascorbic acid interference in reagentstrip reactions for assay of urinary glucose and hemoglobin. Clin Chem 1986;32(4): Nagel D, Seiler D, Hohenberger EF, et al. Investigations of ascorbic acid interference in urine test strips. Clin Lab 2006;52(3-4): Kim K.D. KSH, Kim E.C., Kim J.M., Kim J.H., Kim J.Q., Kim H.J., Moon D.S., Min W.K., Lee S.Y., Lee J.J., Jeon C.H., Cho M.E., Cho S.S. Annual Report on External Quality Assessment in Urinalysis in Korea (2007). J Lab Med Qual Assur 2008;30(1): Kleszczewska E. L-ascorbic acid--clinical use, toxicity, properties, methods of determination and application in chemical analysis. Pharmazie 2000;55(9): Riordan HD, Casciari JJ, Gonzalez MJ, et al. A pilot clinical study of continuous intravenous ascorbate in terminal cancer patients. P R Health Sci J 2005;24(4): Hoffer LJ, Levine M, Assouline S, et al. Phase I clinical trial of i.v. ascorbic acid in advanced malignancy. Ann Oncol 2008;19(11): doi: mdn377 [pii] /annonc/ mdn377[published Online First: Epub Date]. 9. Padayatty SJ, Riordan HD, Hewitt SM, et al. Intravenously administered vitamin C as cancer therapy: three cases. CMAJ 2006;174(7): doi: 174/7/937 [pii] /cmaj [published Online First: Epub Date]. Kune G, Watson L. Colorectal cancer protective effects and the dietary micronutrients folate, methionine, vitamins B6, B12, C, E, selenium, and lycopene. Nutr Cancer 2006;56(1):11-21 doi: /s nc5601_3[published Online First: Epub Date]. 11. Yeom CH, Jung GC, Song KJ. Changes of terminal cancer patients' health-related quality of life after high dose vitamin C administration. J Korean Med Sci 2007;22(1):7-11 doi: [pii][published Online First: Epub Date]. 12. Smalley DL, Bradley ME. New test for urinary glucose (BM33071) evaluated. Clin Chem 1985;31(1): Daae LN, Juell A. Ascorbic acid and test strip reactions for haematuria. Scand J Clin Lab Invest 1983;43(3): Berg B. Ascorbate interference in the estimation of urinary glucose by test strips. J Clin Chem Clin Biochem 1986;24(1):89-96
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