research article novel spectrophotometric method for the

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 678476 7 pageshttpdxdoiorg1011552013678476

Research ArticleNovel Spectrophotometric Method for the Quantitation ofUrinary Xanthurenic Acid and Its Application in IdentifyingIndividuals with Hyperhomocysteinemia Associated withVitamin B6 Deficiency

Chi-Fen Chen1 Tsan-Zon Liu2 Wu-Hsiang Lan1 Li-An Wu3 Chin-Hung Tsai4

Jeng-Fong Chiou56 and Li-Yu Tsai7

1 Clinical Laboratories Yuanrsquos General Hospital Kaohsiung 802 Taiwan2 Translational Research Laboratory Cancer Center Taipei Medical University Hospital Taipei 110 Taiwan3 Clinical Laboratories Chang Gung Memorial Hospital Kwei-Shan 333 Taiwan4Department of Food Science National Penghu University of Science and Technology Magong Penghu 880 Taiwan5Department of Radiation Oncology School of Medicine Taipei Medical University Taipei 110 Taiwan6Cancer Center and Department of Radiation Oncology Taipei Medical University and Hospital Taipei 110 Taiwan7Division of Clinical Biochemistry Graduate Institute ofMedical Biotechnology KaohsiungMedical University Kaohsiung 807 Taiwan

Correspondence should be addressed toJeng-Fong Chiou sjfchiouxuitenet and Li-Yu Tsai tsailiyu2005yahoocomtw

Received 20 June 2013 Accepted 16 August 2013

Academic Editor Natasa Tosic

Copyright copy 2013 Chi-Fen Chen et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

A novel spectrophotometric method for the quantification of urinary xanthurenic acid (XA) is described The direct acid ferricreduction (DAFR) procedure was used to quantify XA after it was purified by a solid-phase extraction column The linearityof proposed method extends from 25 to 1000mgL The method is precise yielding day-to-day CVs for two pooled controlsof 35 and 46 respectively Correlation studies with an established HPLC method and a fluorometric procedure showedcorrelation coefficients of 098 and 098 respectively Interference from various urinary metabolites was insignificant In a small-scale screening of elderly conducted at Penghu county in Taiwan (119899 = 80) we were able to identify a group of twenty individualshaving hyperhomocysteinemia (gt15120583moleL)Three of themwere found to be positive for XA as analyzed by the proposedmethodwhich correlated excellently with the results of the activation coefficient method for RBCrsquos ASTB

6functional test These data

confirm the usefulness of the proposed method for identifying urinary XA as an indicator of vitamin B6deficiency-associated

hyperhomocysteinemic condition

1 Introduction

Homocysteine (Hcy) is a sulfur-containing amino acid whichis an intermediary product of the transmethylation reactionof methionine Once formed Hcy can either be remethylatedback to methionine by a folate- and vitamin B

12-dependent

enzymatic reaction or can undergo the transsulfurationpathway to form cysteine (the rate-limiting precursor forgluthathione) via a two-step enzymatic process catalyzedby cystathionine 120573-synthase (CBS) and cystathionase both

requiring vitaminB6coenzyme [1ndash3] Park andLinkswiler [4]

reported that urinary Hcy excretion increased considerablywith six male volunteers who consumed a diet depleted ofvitamin B

6whereas several studies on experimental animals

suggested that a vitamin B6deficiency could result in Hcy

accumulation [5ndash7] Collectively these reports implicate thatmeasurement ofHcy is not a specific biomarker for deficiencyof vitamin B

6 In contrast kynureninase (EC 3713) a pyri-

doxal 51015840-phosphate coenzyme-requiring enzyme is essentialfor the catalytic action of converting 3-hydroxykynurenin

2 BioMed Research International

an intermediary product of tryptophan metabolism to 3-hydroxyanthranilate [8] Either subclinical deficiencies ofvitamin B

6or a genetic defeat on kynureninase can lead to

the accumulation of xanthurenic acid (XA) in plasma andurine [5ndash7] Collectively despite vitamin B

6deficiency can

lead to a combined accumulation of XA and Hcy in urinethe former is considered to be a more sensitive and specificindicator thanHcy for the evaluation of vitamin B

6deficiency

because Hcy can be diverted to other pathways even in casesof vitamin B

6deficiency

Vitamin B6deficiency was first suggested earlier by

scientists who demonstrated widespread vascular lesion inpyridoxine deficient monkeys with little or no lipid dis-position and their serum cholesterol were hardly elevated[9] Furthermore Ubbink et al [10] reported that patientswith cystathionine 120573-synthase (EC 42122) deficiency thefirst vitamin B

6-dependent enzyme catalyzing the transsul-

furation pathway of homocysteine catabolism exhibitedwidespread vascular disorders In addition patients withrheumatoid arthritis had been reported to possess reducedcirculating level of vitamin B

6and their plasma pyridoxal 51015840-

phosphate levels correlated with both the net Hcy increasein response to a methionine load test and 24 hr urinary XAexcretion in response to a tryptophan load test [11] BesidesZhang et al [12] implicated that pyridoxal 51015840-phosphatethe principal active form of vitamin B

6 has a number of

biological roles that potentially make it important in cancerThe rationale of this implication is that adequate vitamin B

6

levels are important for conversion of Hcy into cysteine andhigh intracellular levels of pyridoxal 51015840-phosphate can leadto decreased steroid hormone-induced gene expression Inaddition these authors also presented evidence that higherplasma levels of folate and vitamin B

6may reduce the risk of

developing breast cancer These data implies that vitamin B6

deficiency itself may be a risk factor for cancerMeasurement of urinary or plasma XA has been used

clinically to study vitamin B6deficiency [8 13 14] including

febrile disorder [15] theophylline-induced asthma [16] drug-induced diabetes [16] the effect of tryptophan and six ofits metabolites on the nicotinic acid pathway [17] and theetiological role in a variety of chronic degenerative diseaseincluding a variety of cancers [18ndash22] Among the methodsdeveloped for quantifying urinary XA [23ndash28] one tediousand lengthy assay involved the extraction of XA from urinewith isobutanol isolation by thin-layer chromatography andeventually spectrophotometric determination [25] The firstfluorometric quantification of urinary XA was devised bySatoh and Price [23] based on the separation of XA byDowex 50 (H+) followed by measurement of its fluorescencein strong alkali and kynurenic acidwas simultaneously deter-mined by fluorometry in strong H

2SO4 This method was

subsequently modified by Cohen et al [24] who separatedXA from other fluorescent substances in urine by a pH-and NaCl-dependent extraction with isobutanol and thendetermined the fluorescence of XA in alkaline solution sothat potentially interfering compounds such as kynurenicacid could be obviated However all these methods involvemultiple procedural steps which are rather tedious and time-consuming In addition several HPLC methods have also

been devised for quantifying urinary XA [26 28] Howeverthe expensive instruments required might not be readilyavailable in a general clinical laboratory For these reasons weset out to develop this rapid and simple spectrophotometricmethod for measuring urinary XA that is free of interferenceand can be easily adoptable for routine use by clinical andmicronutrient assessment laboratories To further exemplifyits clinical application we performed a single-blind studyon a group of urine specimens (119873 = 20) obtained frompatients with hyperhomocysteinemia of unknown etiologiesusing proposed method to screen for XA Based on thisscreening procedure we were able to successfully identifythree out of twenty patients with hyperhomocysteinemia thatwere actually originated from vitamin B

6deficiency

2 Material and Methods

21 Chemicals Unless otherwise stated reagents of thehighest quality available were obtained commercially XA(48-dihydroxyquinaldic acid) kynurenic acid (4-hydroxy-quinoline-2-carboxylic acid) 246-tris-(pyridyl)-s-triazine(TPTZ) ferric chloride hexahydrate vitamin C uric acidsalicylate acetaminophen vanillylmandelic acid and hom-ovanillic acid were purchased from Sigma Chemical Co(St Louis MO USA) Solid-phase anionic-exchange resin(trimethylaminopropyl group bound to silica) was purchasedfrom Analytichem (Harbor City CA)

22 Reagents (a) Acetate buffer 03molliter pH 36 isprepared by dissolving 31 g of sodium acetate trihydrate indistilled water Add 160mL of glacial acetic acid Diluteto 1 liter check the pH and adjust it to 36 (b) TPTZ8mmolliter is prepared by dissolving 624mg of TPTZ in250mL of HCl solution (36mmolliter 075mL conc HClandH

2O) (c) FeCl

3sdot 6H2O solution is prepared by dissolving

540mg of ferric chloride hexahydrate in 100mLHCl solution(002molliter 016mL of conc HCl and H

2O)

23 Urine Purification Routinely 24 h urine specimens arecollected in brown bottles with 6N HCl as preservativeand proceed for analyses without delay The sample shouldbe well mixed and an aliquot frozen if the sample cannotbe analyzed within 48 hr after collection Before analysisthe urine specimen (after thawing if necessary) is filteredthrough Whatman No 1 filter paper and 50mL of urine isapplied to an anion-exchange solid-phase extraction column(100mgcolumn) After the urine sample has passed throughthe extraction column the adsorbed XA is eluted with 20mLof 01M HCl

24 Spectrophotometry of XA 05mL of eluate from purifiedprocedure mentioned previously is added and 05mL of1N NaOH 01mL of this alkaline elute is then added to asolution containing 25mL of acetate buffer pH 36 03mLof TPTZ and 01mL of FeCl

3solution The tubes are well

mixed and stand at room temperature for exactly 15 minutesImmediately the absorbance of each tube is read at 593 nm

BioMed Research International 3

XA concentration of the unknown is estimated from thestandard curve based on the absorbances of the standards

25 Fluorometric Measurement of XA The eluate containingXA from urine is dissolved into potassium phosphate bufferpH 40 The fluorescence of XA is then determined at 460ndash470 nm (after excitation at 305 nm) with a Turner spectroflu-orometer

26 Correlation Studies Theresults obtained by the proposedmethod were compared with those determined by an estab-lished HPLC method [27] and with a fluorometric methodestablished in our laboratory [29]

27 Rapid Screening of Urinary XA in Elderly with Hyper-homocysteinemic Condition We conducted a small-scalescreening of the elderly (age 61 to 80 years) (119899 = 80)for hyperhomocysteinemia during a regular annual physicalcheck up from Penghu County The project was approvedby the Institutional Review Board of Kaohsiung Medi-cal University (KMUH-IRB-960036) Individuals who hadhyperhomocysteinemic condition (gt15 120583moleL) were askedby the physician to donate urine specimens with informedconsent These specimens were then analyzed by our pro-posed method for XA For those urine samples positive forXA a parallel determination of activation coefficients onRBCrsquos aspartic acid aminotransferase (AST)B-6 functionaltest was performed to confirm if XA-positive individualsweredefinitively deficient in vitamin B

6

28 Statistics Data are expressed as mean plusmn SD and ana-lyzed using Studentrsquos t-test P values lower than 005 wereconsidered statistically significant

3 Results

31 Absorption Spectra Spectral scan for XA after reactingwith Fe+3-TPTZ complex in acidic buffer (pH 36) exhibit amaximum absorption peak at 593 nm The absorption peakrepresents the formation of Fe+2-TPTZ (intense blue color)(Figure 1)

32 Time Course Study Reduction of Fe+3-TPTZ complexby XA exhibits a time-dependent manner The increment ofabsorption at 593 nm reaches a plateau approximately 30min(Figure 2) For accuracy the reaction time for the proposedmethod should be set at 30min If this procedural step isinconvenient a short time of color development can be used(eg 15min) but each tube should be timed for equal amountof incubation time and the absorbance at 593 nm should beread immediately without delayed

33 Linearity Absorbances of calibrators at 593 nm werelinearly related to XA concentrations from 25 to 100mgL(Figure 3) The limit of detection (LOD) is 10 120583gmL

0

01

02

03

04

05

06

07

400 450 500 550 600 650 700Wavelength (nm)

A

B

C

Abso

rban

ce 5

93 nm

Figure 1 Spectral scan for xanthurenic acid after reacting with Fe3+-TPTZ complex in acidic buffer (pH = 36) exhibiting a maximumabsorption peak at 593 nm The absorption peak represents theformation of Fe2+-TPTZ (intense blue color) A = 8120583g XA in 01mLMeOH versus reagent blank B = 4120583g XA in 01mL MeOH versusreagent blank and C = reagent blank versus H

2O

0

02

04

06

08

1

0 5 10 15 20 25 30 35Time (min)

Abso

rban

ce 5

93 nm

Figure 2 Reduction of Fe3+-TPTZ complex by XA is a time-dependent process The increment of absorption at 593 nm reachesa plateau at approximately 30min For accuracy the reaction timefor the proposed method should be set at 30min (∙ndash∙) = 4120583g XA inMeOH and (IndashI) = 8 120583g XA in MeOH

34 Analytical Recovery To determine the accuracy of theprocedure we performed a recovery study the percentageof recovery represents the measured value expressed as apercentage of the expected value The mean percentage ofrecovery for 10 samples was 998 plusmn 21 (data not shown)

35 Precision Reproducibility as reflected by day-to-dayand within-run precision data were excellent (Table 1) Fiverepetitive determinations on two pooled XA-supplemented


0010203040506070809

1

0 10 20 30 40 50 60 70 80 90 100

Abso

rban

ce (5

93 nm

)

Concentration of XA (mgL)

r = 09988

y = 00079x + 00149

Figure 3 Linearity of XA determination as assayed by the proposedspectrophotometric method Each point represents an average oftriplicate determinations The XA stock solution (100mgL) wasprepared by dissolving 10mg of XA into 10mL of methanol andthen made up to 100mL with XA-free pooled urine Variousconcentrations of standards were thenmade up by dilutingwith XA-free urine to the desired concentrations

Table 1 Precision studies results

XA determined bythe proposed

method (mgL)lowast

Indirect activationcoefficient (AC) testfor RBCASTlowastlowast

Within-run (119899 = 5)lowast

Mean mgL 202 410SD mgL 060 15CV 30 37

Day-to-day (119899 = 25)lowastlowast

Mean mgL 202 410SD mgL 07 19CV 35 46

lowastEach level of pooled urine sample was determined simultaneously for 5times at the same daylowastlowastEach level of pooled urine sample was run 5 times per day for fiveconsecutive daysThe pooled data were then calculated formean SD andCV

urine controls had a mean value of 202 and 410mgLrespectively (with CVs of lt50)The CVs for the same set ofcontrols assayed on 5 consecutive days were 35 and 46respectively Again the day-to-day reproducibility was alsoexcellent

36 Correlation Studies We compared results by the pro-posed method with those determined by an establishedHPLCmethod [27] for 30 samplesThe correlation coefficientbetween the two methods was 098 (Figure 4(a)) Compar-ison of the proposed method with a fluorometric method[29] for 30 samples showed that the correlation coefficientbetween the two methods was also 098 (Figure 4(b))

37 Interference Assay The purification step with the solid-phase anion-exchange column presumably renders the assayfree of interferences However to verify this assumption wechecked for possible interference from the following com-monly encountered substances in urine vitamin C uric acid

Table 2 A single blind study for 20 hyperhomocysteinemic urinespecimens obtained through a small-scale screening of the elderly(119899 = 80) for assaying XA by our proposed method and their parallelcomparison of assessing B6 status by indirect activation coefficienttest for RBCAST

Specimencode no

XA determinedlowast by theproposed method

(mgL)

Indirect activationcoefficient (AC) test for

RBCASTlowastlowast

1 ND Normal2 ND Normal3 ND Normal4 135 Abnormal (AC = 151)5 ND Normal6 ND Normal7 142 Abnormal (AC = 143)8 ND Normal9 ND Normal10 ND Normal11 ND Normal12 ND Normal13 253 Abnormal (AC = 180)14 ND Normal15 ND Normal16 ND Normal17 ND Normal18 ND Normal19 ND Normal20 ND NormallowastND nondetectable (ltLOD)lowastlowastAC lt 130 is designated as normal

salicylate vanillylmandelic acid (VMA) and homovanillic(HVA) At concentrations of 05 gL all of these compoundsgave no interference with the assay

4 Discussion

The described method for the measurement of XA in urinewas evaluated for its specificity accuracy and reproducibilityfor general use First the solid-phase extraction column(trimethylaminopropyl group bound to silica) concentratesnot only the desired XA but also leave behind urinary sub-stances such as the tryptophan metabolites kynurenin andhydroxylkynurenine This purification step for XA confers aunique specificity to the proposed method

During the course of this developmental work we con-sidered incorporating a hydrolytic step to account for thepossible presence of conjugated forms of XA Rothstein andGreenberg [30] have previously reported that the urinary XAis conjugated as glucuronide in the rat and as the sulfatein the rabbit In contrast Wallace et al [25] showed thatthe amount of XA in human urine remained steady afterhydrolysis with acid or glucuronidase indicating that verylittle conjugated XA was present This observation was also


0

05

1

15

2

25

3

0 05 1 15 2 25 3

XA

det

erm

ined

by

prop

osed

met

hod

(mg

L)

XA determined by HPLC method (mgL)

r = 098

(a)X

A d

eter

min

ed b

y pr

opos

ed m

etho

d (m

gL)

XA determined by fluorometric method (mgL)

0

05

1

15

2

25

3

0 05 1 15 2 25 3

r = 098

(b)

Figure 4 Correlation of results of XA concentrations obtained by the proposed method and those determined by an established HPLCmethod (a) or with an established fluorometric procedure (b)

previously confirmed by us [29] Thus we decided not toincorporate a hydrolysis step for urine in our proposedmethod

The accuracy of the method evaluated by measuring XAadded to pooled urine in which no endogenous XA wasdetected that shows the mean percentage of recovery for10 samples was 998 plusmn 21 (date not shown) Additionallyreproducibility as reflected by day-to-day and within-runprecision data was also excellent (Table 1)

It has been well documented that one of the etiologicfactors underlying chronic degenerative diseases such ascancer diabetes and Alzheimerrsquos diseases may be oxidativestress to which the elderly population is specially proneIn addition the elderly population may have deficienciesof several micronutrients including folate pyridoxine andcobalamin if not treated in time these deficiencies can lead toan accumulation of both XA and Hcy in the urine or plasmaand present a serious health risk to these individuals As anexample Wilson et al [31] reported that about 10 of the USpopulation consumes less than half of the RDA (16mgday)of vitamin B

6 As also pointed out by Ames [32] vitamin

B6deficiency can cause a decrease in enzyme activity of ser-

ine hydroxylmethyltransferase which supplies the methylenegroup for methylene-THF [33] If the methylene-THF pool isdecreased in B

6deficiency an episode of uracil incorporation

into DNA can be anticipated to cause chromosome breaksand the risk for cancer will be greatly increased In a case-control study of diet and cancer vitamin B

6intake was

inversely associated with prostate cancer [34] Thus wethink that it is of importance to undertake a large-scalescreening to identify individuals from an elderly populationwho maybe deficient in these micronutrients Our proposedmethod is sample and rapid and can be modified to suit for

semiautomated quantification of vitamin B6deficiency in a

large scale screening purpose More importantly it can alsobe adopted as one of the tests for routine use in the clinicallaboratory Our small scale screening for elderly who werehyperhomocysteinemic followed by correctly identified indi-viduals high in urinaryXAwhichwas indicative of vitaminB

6

deficiency can serve as a testimony of the applicability of ourproposed method (Table 2) Further study is urgently neededto establish if there is a correlation between vitamin B

6

deficiency-induced hypercysteinemia and the risk of cancerOur proposed noninvasive urinary XA test is well suited forthe investigation of this kind

5 Conclusion

This work describes a simple and rapid spectrophotometricmethod for quantifying urinary XA as an indicator of vitaminB6deficiency which allows one to differentiate hyperho-

mocysteinemic condition independent of folatevitamin B12involvement We also exemplifiy its application by correctlyidentified these individuals whose hyperhomocysteinemiacondition were solely derived from vitamin B

6deficiency

This noninvasive spectrophotometricmethod for quantifyingurinary XA may also be suitable for identifying individualswith vitamin B

6deficiency and subsequently receiving vita-

min B6supplementation to reduce the risk of cancer due to

Hcy-evoked oxidative stress

Conflict of Interests

The authors declare they have no conflict of interests


Authorsrsquo Contribution

J-F Chiou and L-Y Tsai are cocorresponding authors

Acknowledgments

The expert technical assistance of Ms S Y Mau is greatlyappreciated

References

[1] J D Finkelstein ldquoMethionine metabolism in mammalsrdquo Jour-nal of Nutritional Biochemistry vol 1 no 5 pp 228ndash237 1990

[2] S C Lu and J M Mato ldquoS-adenosylmethionine in cell growthapoptosis and liver cancerrdquo Journal of Gastroenterology andHepatology vol 23 supplement 1 pp S73ndashS77 2008

[3] O Midttun S Hustad and P M Ueland ldquoQuantitative pro-filing of biomarkers related to B-vitamin status tryptophanmetabolism and inflammation in human plasma by liquidchromatographytandemmass spectrometryrdquoRapid Communi-cations in Mass Spectrometry vol 23 no 9 pp 1371ndash1379 2009

[4] Y K Park and H Linkswiler ldquoEffect of vitamin B6 depletionin adult man on the excretion of cystathionine and othermethioninemetabolitesrdquo Journal of Nutrition vol 100 no 1 pp110ndash116 1970

[5] M Slavik K J Smith and O Blanc ldquoDecrease of serumpyridoxal phosphate levels and homocystinemia after admin-istration of 6-azauridine triacetate and their prevention byadministration of pyridoxinerdquo Biochemical Pharmacology vol31 no 24 pp 4089ndash4092 1982

[6] L A Smolin and N J Benevenga ldquoAccumulation of homo-cyst(e)ine in vitamin B-6 deficiency a model for the study ofcystathionine 120573-synthase deficiencyrdquo Journal of Nutrition vol112 no 7 pp 1264ndash1272 1982

[7] L A Smolin T D Crenshaw D Kurtycz and N J BenevengaldquoHomocyst(e)ine accumulation in pigs fed diets deficient invitamin B-6 relationship to atherosclerosisrdquo Journal of Nutri-tion vol 113 no 10 pp 2022ndash2033 1983

[8] F Takeuchi R Tsubouchi S Izuta and Y Shibata ldquoKynurenicmetabolism and xanthurenic acid formation in vitamin B6-deficient rat after tryptophan injectionrdquo Journal of NutritionalScience and Vitaminology vol 35 no 2 pp 111ndash122 1989

[9] S J Chang ldquoVitamin B6 antagonists alter the function andultrastructure of mice endothelial cellsrdquo Journal of NutritionalScience and Vitaminology vol 46 no 4 pp 149ndash153 2000

[10] J B Ubbink W J H Vermaak A van der Merwe and P JBecker ldquoVitamin B-12 vitamin B-6 and folate nutritional statusin men with hyperhomocysteinemiardquo The American Journal ofClinical Nutrition vol 57 no 1 pp 47ndash53 1993

[11] E P I Chiang J Selhub P J Bagley GDallal andR RoubenoffldquoPyridoxine supplementation corrects vitamin B6 deficiencybut does not improve inflammation in patients with rheumatoidarthritisrdquo Arthritis Research ampTherapy vol 7 no 6 pp R1404ndashR1411 2005

[12] S M Zhang W C Willett J Selhub et al ldquoPlasma folatevitamin B6 vitamin B12 homocysteine and risk of breastcancerrdquo Journal of the National Cancer Institute vol 95 no 5pp 373ndash380 2003

[13] D A Bender E N M Njagi and P S Danielian ldquoTryptophanmetabolism in vitamin B6-deficientmicerdquoTheBritish Journal ofNutrition vol 63 no 1 pp 27ndash36 1990

[14] J E Leklem ldquoVitamin B-6 a status reportrdquo Journal of Nutritionvol 120 no 11 pp 1503ndash1507 1990

[15] R C Shaw and R D Feigin ldquoExcretion of kynurenic acid andxanthurenic acid during infectionrdquo Pediatrics vol 47 no 1 pp47ndash56 1971

[16] J B Ubbink R Delport P J Becker and S Bissbort ldquoEvi-dence of a theophylline-induced vitamin B6 deficiency causedby noncompetitive inhibition of pyridoxal kinaserdquo Journal ofLaboratory and Clinical Medicine vol 113 no 1 pp 15ndash22 1989

[17] S Ikeda and Y Kotake ldquoUrinary excretion of xanthurenic acidand zinc in diabetes (3) Occurrence of xanthurenic acid-Zn2+complex in urine of diabetic patients and of experimentally-diabetic ratsrdquo Italian Journal of Biochemistry vol 35 no 4 pp232ndash241 1986

[18] J V Woodside D G Fogarty J H Lightbody et al ldquoHomocys-teine and B-group vitamins in renal transplant patientsrdquoClinicaChimica Acta vol 282 no 1-2 pp 157ndash166 1999

[19] E P I Chiang P J Bagley R Roubenoff M Nadeau andJ Selhub ldquoPlasma pyridoxal 51015840-phosphate concentration iscorrelated with functional vitamin B-6 indices in patients withrheumatoid arthritis and marginal vitamin B-6 statusrdquo Journalof Nutrition vol 133 no 4 pp 1056ndash1059 2003

[20] C C Lin and M C Yin ldquoB vitamins deficiency and decreasedanti-oxidative state in patients with liver cancerrdquo EuropeanJournal of Nutrition vol 46 no 5 pp 293ndash299 2007

[21] E Schernhammer B Wolpin N Rifai et al ldquoPlasma folatevitamin B6 vitamin B12 and homocysteine and pancreaticcancer risk in four large cohortsrdquo Cancer Research vol 67 no11 pp 5553ndash5560 2007

[22] E Ma M Iwasaki I Junko et al ldquoDietary intake of folatevitamin B6 and vitamin B12 genetic polymorphism of relatedenzymes and risk of breast cancer a case-control study inBrazilian womenrdquo BMC Cancer vol 9 article 122 2009

[23] K Satoh and J M Price ldquoFluorometric determination ofkynurenic acid and xanthurenic acid in human urinerdquo TheJournal of Biological Chemistry vol 230 no 2 pp 781ndash789 1958

[24] G Cohen R A Fishman and A L Jenkins ldquoA fluorometricmethod for the determination of xanthurenic acid in urinerdquoJournal of Laboratory and Clinical Medicine vol 67 no 3 pp520ndash527 1966

[25] M J Wallace H W Vaillant and H A Salhanick ldquoMethod forquantitativemeasurement of xanthurenic acid in urinerdquoClinicalChemistry vol 17 no 6 pp 505ndash511 1971

[26] S A Williams J A Monti L R Boots and P E CornwellldquoQuantitation of xanthurenic acid in rabbit serum using highperformance liquid chromatographyrdquo The American Journal ofClinical Nutrition vol 40 no 1 pp 159ndash167 1984

[27] J B Ubbink A M Schnell and C H Rapley ldquoQuantificationof urinary xanthurenic acid excretion by anion-exchange solid-phase extraction and high-performance liquid chromatogra-phyrdquo Journal of Chromatography vol 425 no 1 pp 182ndash1861988

[28] E Ohtsuki M Suzuki M Takayanagi and T YashiroldquoColorimetric determination of urinary xanthurenic acidusing an oxidative coupling reaction with NN-diethyl-p-phenylenediaminerdquo Biological and Pharmaceutical Bulletin vol17 no 1 pp 139ndash141 1994

[29] M Liu G R Wang T Z Liu and K J Tsai ldquoImproved flu-orometric quantification of urinary xanthurenic acidrdquo ClinicalChemistry vol 42 no 3 pp 397ndash401 1996


[30] M Rothstein and DM Greenberg ldquoStudies on the metabolismof xanthurenic acid-4-C14rdquo Archives of Biochemistry and Bio-physics vol 68 no 1 pp 206ndash214 1957

[31] J W Wilson C W Enns J D Goldman et al ldquoData tablecombined results fromUSDArsquos 1994 and 1995 continuing surveyof food intakes by individuels and 1994 and 1995 diet and healthknowledge surveyrdquo USDAARS Food Surveys Research GroupBeltsville Human Nutrition Research Center Riverdale MdUSA 1997

[32] B N Ames ldquoMicronutrient deficiencies cause DNA damageand cancerrdquo Food Science and Agricultural Chemistry vol 1 pp1ndash15 1999

[33] S P Stabler D A Sampson L P Wang and R H AllenldquoElevations of serum cystathionine and total homocysteine inpyridoxine- folate- and cobalamin-deficient ratsrdquo Journal ofNutritional Biochemistry vol 8 no 5 pp 279ndash289 1997

[34] T J Key P B Silcocks G K Davey P N Appleby and D TBishop ldquoA case-control study of diet and prostate cancerrdquo TheBritish Journal of Cancer vol 76 no 5 pp 678ndash687 1997

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an intermediary product of tryptophan metabolism to 3-hydroxyanthranilate [8] Either subclinical deficiencies ofvitamin B

6or a genetic defeat on kynureninase can lead to

the accumulation of xanthurenic acid (XA) in plasma andurine [5ndash7] Collectively despite vitamin B

6deficiency can

lead to a combined accumulation of XA and Hcy in urinethe former is considered to be a more sensitive and specificindicator thanHcy for the evaluation of vitamin B

6deficiency

because Hcy can be diverted to other pathways even in casesof vitamin B

6deficiency

Vitamin B6deficiency was first suggested earlier by

scientists who demonstrated widespread vascular lesion inpyridoxine deficient monkeys with little or no lipid dis-position and their serum cholesterol were hardly elevated[9] Furthermore Ubbink et al [10] reported that patientswith cystathionine 120573-synthase (EC 42122) deficiency thefirst vitamin B

6-dependent enzyme catalyzing the transsul-

furation pathway of homocysteine catabolism exhibitedwidespread vascular disorders In addition patients withrheumatoid arthritis had been reported to possess reducedcirculating level of vitamin B

6and their plasma pyridoxal 51015840-

phosphate levels correlated with both the net Hcy increasein response to a methionine load test and 24 hr urinary XAexcretion in response to a tryptophan load test [11] BesidesZhang et al [12] implicated that pyridoxal 51015840-phosphatethe principal active form of vitamin B

6 has a number of

biological roles that potentially make it important in cancerThe rationale of this implication is that adequate vitamin B

6

levels are important for conversion of Hcy into cysteine andhigh intracellular levels of pyridoxal 51015840-phosphate can leadto decreased steroid hormone-induced gene expression Inaddition these authors also presented evidence that higherplasma levels of folate and vitamin B

6may reduce the risk of

developing breast cancer These data implies that vitamin B6

deficiency itself may be a risk factor for cancerMeasurement of urinary or plasma XA has been used

clinically to study vitamin B6deficiency [8 13 14] including

febrile disorder [15] theophylline-induced asthma [16] drug-induced diabetes [16] the effect of tryptophan and six ofits metabolites on the nicotinic acid pathway [17] and theetiological role in a variety of chronic degenerative diseaseincluding a variety of cancers [18ndash22] Among the methodsdeveloped for quantifying urinary XA [23ndash28] one tediousand lengthy assay involved the extraction of XA from urinewith isobutanol isolation by thin-layer chromatography andeventually spectrophotometric determination [25] The firstfluorometric quantification of urinary XA was devised bySatoh and Price [23] based on the separation of XA byDowex 50 (H+) followed by measurement of its fluorescencein strong alkali and kynurenic acidwas simultaneously deter-mined by fluorometry in strong H

2SO4 This method was

subsequently modified by Cohen et al [24] who separatedXA from other fluorescent substances in urine by a pH-and NaCl-dependent extraction with isobutanol and thendetermined the fluorescence of XA in alkaline solution sothat potentially interfering compounds such as kynurenicacid could be obviated However all these methods involvemultiple procedural steps which are rather tedious and time-consuming In addition several HPLC methods have also

been devised for quantifying urinary XA [26 28] Howeverthe expensive instruments required might not be readilyavailable in a general clinical laboratory For these reasons weset out to develop this rapid and simple spectrophotometricmethod for measuring urinary XA that is free of interferenceand can be easily adoptable for routine use by clinical andmicronutrient assessment laboratories To further exemplifyits clinical application we performed a single-blind studyon a group of urine specimens (119873 = 20) obtained frompatients with hyperhomocysteinemia of unknown etiologiesusing proposed method to screen for XA Based on thisscreening procedure we were able to successfully identifythree out of twenty patients with hyperhomocysteinemia thatwere actually originated from vitamin B

6deficiency

2 Material and Methods

21 Chemicals Unless otherwise stated reagents of thehighest quality available were obtained commercially XA(48-dihydroxyquinaldic acid) kynurenic acid (4-hydroxy-quinoline-2-carboxylic acid) 246-tris-(pyridyl)-s-triazine(TPTZ) ferric chloride hexahydrate vitamin C uric acidsalicylate acetaminophen vanillylmandelic acid and hom-ovanillic acid were purchased from Sigma Chemical Co(St Louis MO USA) Solid-phase anionic-exchange resin(trimethylaminopropyl group bound to silica) was purchasedfrom Analytichem (Harbor City CA)

22 Reagents (a) Acetate buffer 03molliter pH 36 isprepared by dissolving 31 g of sodium acetate trihydrate indistilled water Add 160mL of glacial acetic acid Diluteto 1 liter check the pH and adjust it to 36 (b) TPTZ8mmolliter is prepared by dissolving 624mg of TPTZ in250mL of HCl solution (36mmolliter 075mL conc HClandH

2O) (c) FeCl

3sdot 6H2O solution is prepared by dissolving

540mg of ferric chloride hexahydrate in 100mLHCl solution(002molliter 016mL of conc HCl and H

2O)

23 Urine Purification Routinely 24 h urine specimens arecollected in brown bottles with 6N HCl as preservativeand proceed for analyses without delay The sample shouldbe well mixed and an aliquot frozen if the sample cannotbe analyzed within 48 hr after collection Before analysisthe urine specimen (after thawing if necessary) is filteredthrough Whatman No 1 filter paper and 50mL of urine isapplied to an anion-exchange solid-phase extraction column(100mgcolumn) After the urine sample has passed throughthe extraction column the adsorbed XA is eluted with 20mLof 01M HCl

24 Spectrophotometry of XA 05mL of eluate from purifiedprocedure mentioned previously is added and 05mL of1N NaOH 01mL of this alkaline elute is then added to asolution containing 25mL of acetate buffer pH 36 03mLof TPTZ and 01mL of FeCl

3solution The tubes are well

mixed and stand at room temperature for exactly 15 minutesImmediately the absorbance of each tube is read at 593 nm






6


3 Results




0

01

02

03

04

05

06

07

400 450 500 550 600 650 700Wavelength (nm)

A

B

C

Abso

rban

ce 5

93 nm


2O

0

02

04

06

08

1

0 5 10 15 20 25 30 35Time (min)

Abso

rban

ce 5

93 nm





0010203040506070809

1

0 10 20 30 40 50 60 70 80 90 100

Abso

rban

ce (5

93 nm

)


r = 09988

y = 00079x + 00149




method (mgL)lowast



Mean mgL 202 410SD mgL 060 15CV 30 37


Mean mgL 202 410SD mgL 07 19CV 35 46






Specimencode no


(mgL)


RBCASTlowastlowast



4 Discussion




0

05

1

15

2

25

3

0 05 1 15 2 25 3

XA

det

erm

ined

by

prop

osed

met

hod

(mg

L)


r = 098

(a)X

A d

eter

min

ed b

y pr

opos

ed m

etho

d (m

gL)


0

05

1

15

2

25

3

0 05 1 15 2 25 3

r = 098

(b)










6intake was




6


6


5 Conclusion



6deficiency










Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






6


3 Results




0

01

02

03

04

05

06

07

400 450 500 550 600 650 700Wavelength (nm)

A

B

C

Abso

rban

ce 5

93 nm


2O

0

02

04

06

08

1

0 5 10 15 20 25 30 35Time (min)

Abso

rban

ce 5

93 nm





0010203040506070809

1

0 10 20 30 40 50 60 70 80 90 100

Abso

rban

ce (5

93 nm

)


r = 09988

y = 00079x + 00149




method (mgL)lowast



Mean mgL 202 410SD mgL 060 15CV 30 37


Mean mgL 202 410SD mgL 07 19CV 35 46






Specimencode no


(mgL)


RBCASTlowastlowast



4 Discussion




0

05

1

15

2

25

3

0 05 1 15 2 25 3

XA

det

erm

ined

by

prop

osed

met

hod

(mg

L)


r = 098

(a)X

A d

eter

min

ed b

y pr

opos

ed m

etho

d (m

gL)


0

05

1

15

2

25

3

0 05 1 15 2 25 3

r = 098

(b)










6intake was




6


6


5 Conclusion



6deficiency










Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0010203040506070809

1

0 10 20 30 40 50 60 70 80 90 100

Abso

rban

ce (5

93 nm

)


r = 09988

y = 00079x + 00149




method (mgL)lowast



Mean mgL 202 410SD mgL 060 15CV 30 37


Mean mgL 202 410SD mgL 07 19CV 35 46






Specimencode no


(mgL)


RBCASTlowastlowast



4 Discussion




0

05

1

15

2

25

3

0 05 1 15 2 25 3

XA

det

erm

ined

by

prop

osed

met

hod

(mg

L)


r = 098

(a)X

A d

eter

min

ed b

y pr

opos

ed m

etho

d (m

gL)


0

05

1

15

2

25

3

0 05 1 15 2 25 3

r = 098

(b)










6intake was




6


6


5 Conclusion



6deficiency










Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

05

1

15

2

25

3

0 05 1 15 2 25 3

XA

det

erm

ined

by

prop

osed

met

hod

(mg

L)


r = 098

(a)X

A d

eter

min

ed b

y pr

opos

ed m

etho

d (m

gL)


0

05

1

15

2

25

3

0 05 1 15 2 25 3

r = 098

(b)










6intake was




6


6


5 Conclusion



6deficiency










Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article novel spectrophotometric method for the

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