The diurnal variation in urine acidification differs between normal individuals and uric acid stone formers

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1 & 212 Interntionl Society of Nephrology originl rticle The diurnl vrition in urine cidifiction differs etween norml individuls nd uric cid stone formers MryAnn Cmeron 1,2, Nim M. Mlouf 1,2, John Poindexter 1, Beverley Adms-Huet 1,2,3, Khshyr Skhee 1,2 nd Orson W. Moe 1,2,4 1 Chrles nd Jne Pk Center for Minerl Metolism nd Clinicl Reserch, University of Texs Southwestern Medicl Center, Dlls, Texs, USA; 2 Deprtment of Internl Medicine, University of Texs Southwestern Medicl Center, Dlls, Texs, USA; 3 Deprtment of Clinicl Sciences, University of Texs Southwestern Medicl Center, Dlls, Texs, USA nd 4 Deprtment of Physiology, University of Texs Southwestern Medicl Center, Dlls, Texs, USA Mny iologicl functions follow circdin rhythms driven y internl nd externl cues tht synchronize nd coordinte orgn physiology to diurnl chnges in the environment nd ehvior. Urinry cid se prmeters follow diurnl ptterns nd it is thought these chnges re due to periodic surges in gstric cid secretion. Anorml urine ph is risk fctor for specific types of nephrolithisis nd uric cid stones result from excessively low urine ph. Here we plced 9 helthy volunteers nd 1 uric cid stone formers on fixed metolic diets to study the diurnl pttern of urinry cidifiction. All showed cler diurnl trends in urinry cidifiction, ut none of the ptterns were ffected y inhiitors of the gstric proton pump. Uric cid stone formers hd similr ptterns of chnge throughout the dy ut their urine ph ws lwys lower compred to helthy volunteers. Uric cid stone formers excreted more cid (normlized to cid ingestion), with the excess excreted primrily s titrtle cid rther thn mmonium. Urine se excretion ws lso lower in uric cid stone formers (normlized to se ingestion), long with lower plsm icronte concentrtions during prt of the dy. Thus, incresed net cid presenttion to the kidney nd the preferentil use of uffers, other thn mmonium, result in much higher concentrtions of undissocited uric cid throughout the dy nd consequently n incresed risk of uric cid stones. Kidney Interntionl (212) 81, ; doi:1.138/ki ; pulished online 1 Ferury 212 KEYWORDS: cidosis; citrturi; ion trnsport; kidney stones; kidney tuule Correspondence: Orson W. Moe, University of Texs Southwestern Medicl Center, 5323 Hrry Hines Blvd, Dlls, Texs 7539, USA. E-mil: orson.moe@utsouthwestern.edu Received 9 August 211; revised 24 Octoer 211; ccepted 22 Novemer 211; pulished online 1 Ferury 212 A property intrinsic to living orgnisms is the iologicl circdin clock, which is orgnized nd oscilltes t multiple hierrchies t oth cellulr 1,2 nd multicellulr levels 3,4 in oth the niml nd plnt kingdoms. 5,6 In mmmlin iology, mny spects of ehvior nd physiology follow cyclic rhythms, which re generlly presumed to confer dptive dvntge y coordinting ehvior nd orgn physiology to mient dy-nd-night cycles. 4 In the kidney, significnt circdin rhythms exist for multiple renl hemodynmic, glomerulr, nd tuulr prmeters. 7,8 These renl circdin rhythms re influenced y externl cues such s feeding, mient light, nd ctivity, s well s inherently y intrinsic clocks. 9,1 In 1845, Henry Bence Jones, who is considered to e the pioneer of urinry chemistry for his studies of urinry light chins, glucose, nd cystine in disese sttes, 11 noted diurnl vrition in urine ph (UpH) in norml individuls. 12 Susequent studies lso demonstrted morning lkline nd evening cidic trends of urine lthough this finding ws not lwys uniformly oserved However, the precise circdin profile of urine cidifiction remins incompletely defined, nd the fctors responsile for hour-to-hour fluctutions in ph re not known. Gstric cid secretion with the concomitnt lkliztion of plsm hs een proposed to e the origin of postprndil chnges in plsm ph nd UpH. 19,2 In ddition, it is uncler whether renl disorders ffect circdin ptterns of urinry chemistry nd whether such derngements in rhythmic chnges in urinry cidifiction contriute to pthophysiology. Although the kidney is cple of elorting urine t n enormously wide rnge of hydrogen ion concentrtions when stressed (ph from o5 to48; [H þ ] from o1 nm to 41 mm), norml dy-to-dy UpH is poised within much nrrower spn in humns somewhere etween ph 5.5 nd 6.5. Acidifiction of urine is of criticl importnce for prevention of clcium phosphte crystlliztion in the urinry spce However, UpH cnnot e lowered too Kidney Interntionl (212) 81,

2 originl rticle M Cmeron et l.: Diurnl vrition in urine cidifiction much ecuse of nother constrint in higher primtes who mintin reltively high plsm nd urinry uric cid. 22,24 Urinry cidifiction to elow ph 5.5, lthough protective ginst clcium phosphte precipittion, poses sustntil risk of uric cid precipittion. 22 Precipittion of clcium phosphte nd uric cid thus set the upper nd lower limits of UpH, respectively, nd clcium phosphte nd uric cid nephrolithisis in fct represent quintessentil clinicl disorders of UpH. In humns with uric cid nephrolithisis, excessively cidic urine cuses titrtion of urte to the highly insolule uric cid despite norml or even low totl uric cid content in urine. The pthogenesis of low UpH hs een scried to oth incresed cid lod to the kidney nd defective utiliztion of mmoni in urinry uffering It is not cler whether the unduly cidic urine in uric cid stone formers (s) occurs t specific intervls or persists throughout the dy. Current stndrd clinicl prctice, clinicl investigtions, nd clinicl trils, ll use 24-h urine collections to ssess risk for uric cid stones nd dequcy of response to therpy. We reported tht during tretment of uric cid nephrolithisis with lkli, excessive nocturnl nd erly-morning urinry cidity cn linger despite pprent lkliniztion of pooled 24-h urine. 32 This cn potentilly result in flse sense of security for the clinicin tht uric cid stone risk is eliminted, ut n elevted propensity for uric cid precipittion still persists in the ptient during specific periods of the dy. Although most ptients likely respond to dytime lkli therpy, the possiility remins in some individuls where persistent erly-morning ciduri cn sustin the elevted stone risk despite lkli therpy. The study of 24-h urine profiles in s will enhnce our understnding of its pthophysiology nd the origin of the excessive ciduri. It will lso guide us in designing etter clinicl tests for dignosis nd monitoring of therpy in uric cid nephrolithisis. We emrked to detil the circdin pttern of urinry cidifiction prmeters in norml volunteers to delinete norml circdin physiology nd in s to identify pthophysiologicl defects. In ddition, we tested the longstnding elief tht gstric cid secretion contriutes to chnges in UpH y locking gstric cid production in the two groups of sujects. RESULTS Demogrphic chrcteristics The demogrphic chrcteristics of the study popultion re depicted in Tle 1. Ten s nd nine helthy volunteers (s) prticipted in the study. Most of the sujects in oth groups were mle nd non-hispnic Cucsins, which is typicl of s. The men ge did not differ significntly etween the two groups. The s weighed more nd hd higher BMI thn the s. Serum nd urine chemistry Tle 2 shows the fsting serum chemistry for oth groups in ech study phse. In oth phses, s hd slightly higher Tle 1 Ptient demogrphic dt Helthy volunteers (s) Uric cid stone formers (s) Gender: M/F 6/3 9/1 Rce (white/lck) 7/2 9/1 Ethnicity 9/ 9/1 (non-hispnic/hispnic) Age (yers) 52.8± ±8.2 Weight (kg) 85±19 19±19* Height (cm) 171±1 172±6 Body mss index (kg/m 2 ) 28.5± ±6.8* Arevitions: F, femle; M, mle. *Po.5, t-test. Tle 2 Fsting serum profile Plceo PPI Cretinine (mg/dl) (mmol/l).84±.13 74±11 1.6±.2* 94±1.7.88±.15 78± ±.21 w 99±18 Cretinine clernce (ml/min) 139±35 12±2 131±39 1±26 129±35 112±17 126±36 95±23 (ml/min per 1.73 m 2 ) Glucose (mg/dl) (mmol/l) 97±1 5.4±.6 13±25 5.6±1.4 96±6 5.3±.3 16±26 5.±1.4 Uric cid (mg/dl) (mmol/l) 6.± ±95 8.±1.5* 476±89 6.3± ±95 8.1±1.5 w 482±89 Sodium (meq/l) 139±3 138±3 138±2 138±2 Potssium (meq/l) 4.±.3 4.5±.7* 3.9±.2 4.±.3 z Chloride (meq/l) 16±3 17±2 17±2 17±3 Bicronte (meq/l) 27.± ± ± ±1.1 Venous ph 7.41±.2 7.4± ±.1 7.4±.1 Arevitions:, helthy volunteer; PPI, proton pump inhiitor;, uric cid stone former. *Po.5 vs. ; on plceo. w Po.5 vs. ; on PPI. z Po.5 plceo vs. on PPI. Comprisons mde with mixed-model repeted-mesures nlysis. serum cretinine level due to higher cretinine production rte, ut cretinine production rte per ody mss ws not different etween the two groups. Most importntly, cretinine clernce ws not different etween the two groups. Serum uric cid ws persistently higher in s in oth phses of the study. Serum icronte, chloride, nd venous ph did not differ etween the two groups. s tking plceo demonstrted very slight ut sttisticlly significntly higher serum potssium level thn in the proton pump inhiitor (PPI) phse nd higher thn s in oth phses. Urine ws collected for 24 h prior to ech diurnl study nd results re shown in Tle 3. In oth study phses, UpH in s ws significntly lower thn in s (s vs. s in plceo phse: 5.42±.36 vs. 5.86±.28; P ¼.1; s vs. s in PPI phse: 5.32±.36 vs. 5.93±.23; Po.1). Urinry sodium, potssium, sulfte, nd phosphte did not differ mong the groups, indicting equivlence of dietry intke. In oth phses, citrte excretion ws numericlly lower in s ut the difference ws not sttisticlly 1124 Kidney Interntionl (212) 81,

3 M Cmeron et l.: Diurnl vrition in urine cidifiction originl rticle Tle 3 Bseline 24-h urine chemistry Plceo PPI Plceo PPI Totl volume (l/dy) 2.77± ± ± ±.86 ph 5.86± ±.36* 5.93± ±.36 w Cretinine (g/dy) (mmol/dy) 1.7±.6 15.± ±.5* 18.8± ± ±4.4 2.± ±.5 Potssium (meq/dy) 43±13 45±12 45±14 42±13 Sulfte (meq/dy) 41.2± ± ±1 37.4±12 Phosphte (mg/dl) (mm) 74± ±9.4 87±34 26.± ±37 22.± ± ±7.1 Ammonium (NH 4 ) 39±15 32±16 39±19 33±12 (meq/dy) Titrtle cid (meq/dy) 24±9 33±9 z 25±11 34±9** Citrte (meq/dy) 9.9± ± ± ±3.8 w Bicronte (meq/dy) Medin (rnge) 1.1±2.1 ( 5.6).3±.9 ( 2.72) 1.6±2.1 ( 5.9).4±1.1 w ( 3.3) Uric cid (mg/dy) 657±337 54±322 57± ±273 Net cid excretion (NAE) 53±2 58±22 53±29 6±12 (meq/dy) NAE/sulfte (meq/meq).75±.7.55±.8*.78±.16.55±.15 w NH 4 /NAE.75±.7.55±.8*.78±.16.55±.15 w Urinry unmesured 4.5± ± ± ±11.1 nions (meq/dy) Frctionl excretion of uric cid (%) 5.9±2.6 4.±2.6* 5.4± ±2. w Arevitions:, helthy volunteer; PPI, proton pump inhiitor;, uric cid stone former. *Po.5 vs. ; plceo. w Po.5 vs. ; on PPI. z P=.8 vs. ; plceo. **P=.6 vs. ; on PPI. 8 P=.7 vs. ; plceo. Comprisons mde with mixed-model repeted-mesures nlysis. significnt. Twenty-four-hour titrtle cidity (TA) nd net cid excretion (NAE) ws higher, nd urinry mmonium ws lower numericlly in s compred to s, lthough none of the differences reched P vlue of o.5 in this smll cohort. However, the proportion of NAE s mmonium (NH 4 /NAE) ws significntly lower in s (s vs. s in plceo phse:.55±.8 vs..75±.7; P ¼.3; s vs. s in PPI phse:.55±.15 vs..78±.16, P ¼.1). Twenty-four-hour urinry uric cid excretion did not differ etween the groups. There ws no difference etween the plceo nd PPI phse. Diurnl study Figure 1 nd depicts the diurnl UpH in the nd group under ech study phse. Throughout the dy, oth s nd s demonstrted mrked vritions in UpH. In oth groups, UpH incresed in the morning, peked t noon, fell following lunch, rose gin during the fternoon, nd fell gin fter the evening mel. A nocturnl pek in UpH ws usully seen round 22 hours. Throughout the night, UpH decresed to ndir round 6 hours efore rising gin in the morning. Chnges in UpH over time re sttisticlly significnt within nd groups in either the plceo or the PPI phses (-plceo, -PPI, -plceo, -PPI, ll Po.1). The shpe of the curves did not differ etween the plceo nd PPI phses for oth s nd s (P ¼.95). PPI tretment hd no effect Urinry ph Urinry ph c Urinry ph Plceo PPI Plceo Figure 1 Urine ph (UpH) in uric cid stone formers () nd helthy volunteers (). () Plceo vs. protein pump inhiitors (PPI) in. () Plceo vs. PPI in. (c) vs.. Mels re indicted y lck oxes. on the vlue or profile of UpH (P ¼.6 for s; P ¼.72 for s). Throughout the dy, UpH ws significntly lower in s thn in s in either the plceo (Figure 1c) or the PPI phse (similr to plceo; not shown) (Po.1). However, the time profile of UpH ws not sttisticlly different etween the two groups (s vs. s; P ¼.14) in plceo phse. In contrst to s on PPI, the s receiving PPI demonstrted slightly lunted curve in the evening nd overnight (P ¼.5). Kidney Interntionl (212) 81,

4 originl rticle M Cmeron et l.: Diurnl vrition in urine cidifiction Urinry NH 4, meq/h c Urinry HCO 3, meq/h Titrtle cidity, meq/h Urinry citrte, mg/h Figure 2 Excretion of cid se prmeters in helthy volunteers () nd uric cid stone formers (). () Ammonium. () Bicronte. (c) Titrtle cid. (d) Citrte. Blck oxes represent mels. d Figure 2 compres urinry excretion rtes of principl cid se components in s nd s during the plceo phse. As there ws no difference etween PPI nd plceo, only the plceo phse is shown. In oth s nd s, excretion of these solutes vried significntly over time (ll Po.1). Acid excretion in generl is higher in the lte fternoon nd evening, likely reflecting the fct tht the protein for rekfst is less thn tht for lunch nd dinner (protein: rekfst 7 g, lunch 24 g, dinner 3 g). Urinry mmonium excretion rte (Figure 2) did not differ etween s nd s in regrd to oth mount (P ¼.11) nd pttern (P ¼.4). The pttern of TA excretion lso did not differ etween the groups (P ¼.27). However, the mount of TA ws significntly higher in the group (P ¼.5) (Figure 2). The mount nd pttern of the two urinry ses differed etween the groups. USAFs excreted less icronte (P ¼.1) nd hd fltter icronte profile (P ¼.5) compred with s (Figure 2c). In ddition, s excreted significntly less citrte (P ¼.12) nd tended to hve different citrte pttern (P ¼.56), with lunted citrte excretion in the s occurring mostly in the morning. Plsm icronte concentrtion chnged significntly during the 24-h period (Po.1 for oth groups), ut the pttern ws similr in s nd s (P ¼.94) (Figure 3). However, serum icronte ws higher in s compred with s throughout the dy (P ¼.3). There were no differences etween the PPI nd plceo phse in either Plsm ph Plsm [HCO 3 ] Figure 3 Plsm cid se prmeters in uric cid stone formers () nd helthy volunteers (). () Bicronte. () ph. Blck oxes represent mels Kidney Interntionl (212) 81,

5 M Cmeron et l.: Diurnl vrition in urine cidifiction originl rticle Urinry net cid excretion, meq/h c Urinry net cid excretion/sulfte meq/meq Urinry sulfte, meq/h d Urinry citrte/ potssium meq/meq Figure 4 Urinry cid se prmeters normlized to dietry surrogtes of cid (sulfte) or se (potssium) ingestion, in uric cid stone formers () nd helthy volunteers (). () Net cid excretion. () Sulfte excretion. (c) Net cid per sulfte. (d) Citrte per potssium. Blck oxes represent mels. helthy volunteers or s for oth ph nd icronte, so only the plceo dt re shown in Figure 3. Interestingly, despite the differences in icronte concentrtions, chnges in venous ph over time did not rech sttisticl significnce within either group during the two tretment periods (P ¼.21) nd did not differ etween the groups (P ¼.45) (Figure 3). s hve higher NAE thn s even though they re on identicl metolic diets (Figure 4). The equivlence of dietry cid lod is proven y the sme mount nd pttern of sulfte excretion (Figure 4). Although exogenous dietry cid lod ws equivlent, s hve consistently higher cid excretion throughout the dy, with the exception of perhps the period round 18 2 hours in the evening. This is est illustrted y the consistently higher NAE/sulfte rtio throughout the dy in s (Figure 4c) (P ¼.2). The higher cid lod is lso comptile with the lower citrte excretion (Figure 2d). Potssium excretion cn e used s surrogte for dietry lkli intke. s hve the sme potssium excretion s s, ut lower citrte excretion per K ingested (P ¼.1) (Figure 4d). Figure 5 shows the most importnt determinnt of uric cid stone risk in the s. Asolute rtes or ptterns of totl uric cid excretion did not differ etween the two groups (P ¼.45) nd there ws no difference in the time profile in excretion etween the two groups (P ¼.7) (Figure 5). However, s hd persistently out twice the undissocited uric cid content in urine s s (Figure 5) (Po.1). Totl uric cid, mg/h Undissocited uric cid, mg/h Figure 5 Uric cid excretion in uric cid stone formers () nd helthy volunteers (). () Totl (urte + undissocited uric cid). () Undissocited uric cid excretion in urine. Blck oxes represent mels. Kidney Interntionl (212) 81,

6 originl rticle M Cmeron et l.: Diurnl vrition in urine cidifiction DISCUSSION Insted of exmining pooled 24-h smples, we tried to define the circdin vrition of UpH nd cid se prmeters in helthy sujects s well s in s. We lso exmined the contriution of gstric cid secretion to circdin vrition in oth groups nd evluted whether differences in gstric cid secretion re responsile for lower UpH in s vs. s. Both s nd s were plced on strict metolic diet, therey eliminting exogenous fctors on cid se lnce. Both groups demonstrted significnt vritions in UpH throughout the dy, nd these chnges were temporlly relted to mels. Although there ws little chnge immeditely following rekfst, UpH declined fter the noon nd evening mels. UpH susequently rose nd peked pproximtely 4 h fter ech mel. Overnight UpH fell stedily, efore it egn to rise in the erly morning hours. While they confirm the previously descried morning lkline tide, our dt did not demonstrte true post-prndil lkline tide in either lood or urine. A rise in serum ph fter the ingestion of mel ws descried previously, 19,2 while there is conflicting evidence for whether there is consistent postprndil lkline tide in the urine. 33,34 This difference my, in prt, e due to vrinces in diet, timing of urine collections, 35 nd use of ph pper to define UpH in some studies. 33,34 In our study, the higher frequency of urine collections llowed for higher resolution, nd the instnt mesurement using ph electrode permitted more ccurte ssessment of circdin chnges. The decline in UpH following lunch nd dinner my not hve een pprecited previously, ecuse of longer post-prndil urine collection times. The post-prndil lkline tide descried in serum rises expecttions tht similr phenomenon might occur in the urine due to incresed filtered icronte lod. However, there re multiple fctors tht contriute to the UpH. The incresed filtered lod of HCO 3 my or my not exceed tuulr resorption nd the production nd excretion of non-icronte uffers will lso ffect UpH significntly. The effects of vritions in diet re lso uncler. The UpH my reflect the comined effects of gstric cid nd pncretic icronte secretion, nd the extent of ech process my lter the UpH. Others hve shown tht ptients fter vgotomy, receiving H2-lockers or PPI hve vrying degrees of lunted post-prndil lkline tide in the lood. 19,2,33,34,36 If gstrointestinl cid or se secretion ws responsile for diurnl UpH chnges, then dministrtion of proton pump inhiitor would completely lock those effects. Our study did not demonstrte ny effect of the PPI mediction, thus rising into question the role of gstric cid secretion in ffecting UpH. Alterntive mechnisms tht my ccount for these vritions include circdin clock genes, which hve een found to control cid se trnsporters in the kidney. 37,38 Further investigtion of such genes my help identify the mechnisms responsile for circdin vrition in UpH. Severl fetures re noteworthy in the s. Despite the more cidic urine, the circdin pttern is well preserved in s. Unlike some previous studies reporting lck of diurnl chnges in the UpH of s, 39,4 we found tht s exhiited the sme peks nd troughs overnight nd in reltion to mels, s in s. Previous studies were performed in smller numer of s, without using strict metolic diets, nd collected urines in wider (46-h) intervls. However, t ech time point, s UpH ws significntly more cidic thn tht of s. With the exception of two time points, s mintined men UpH of p5.6 throughout ll time points of the dy, which significntly contriutes to uric cid stone development. 24 The undissocited uric cid excretion rte nd concentrtion re much higher in s throughout the entire 24-h period. Differences in the diurnl excretion of mmonium, TA, citrte, nd icronte reflect the findings previously descried in pooled 24-h urine collections, 3 ut higher time resolution is presented in this diurnl study. s demonstrted lower citrte, nd icronte excretion nd higher TA, which persisted throughout the entire dy. Ech urinry component exhiited circdin pttern of excretion. The citrte nd icronte plots were slightly lunted in the s, indicting possily different pttern of excretion. Both groups excreted mmonium nd TA in similr pttern. It hs een suggested tht incresed cid production in s my ccount for lower UpH. 26,31,41 This mechnism my lso ccount for consumption of se nd the differences oserved in the diurnl excretion of lkli. In ddition to lower UpH, s demonstrted persistently lower serum icronte. This is the first demonstrtion tht systemic cid se disturnce is ctully present in s. Serum icronte level mesured t 8 hours ws not sttisticlly different etween the groups when nlyzed s single time point, therey explining why this difference my not hve een previously pprecited in ll pulished studies. However, when the sequentil icronte vlues were compred, s mintined significntly lower serum icronte level thn s throughout most of the dy. These results further support the notion tht in ddition to renl mechnism, systemic fctors such s possile increse in cid genertion contriute to the development of cidic urine in these ptients. The origin of the incresed cid lod is not known yet, ut cn e due to incresed endogenous cid production, incresed lkli loss in the intestine, nd incresed sorption of exogenous orgnic cid from perhps colonic cteril sources. One wekness of this study ws the discrepncy etween the weights of the suject in the two groups. s re typiclly oese nd ecuse of this chrcteristic, finding helthy mtching group is in fct difficult. The s included in this study were generlly overweight, ut not oese. It is importnt to note tht none of our sujects showed ny evidence of strvtion ketosis during the study. In n ttempt to improve mtching, we simply nlyzed the five s (men±s.d.: 31.4±2.2) with highest BMI ginst five sujects with lowest BMI in s (men±s.d.: 33.±4.3, NS); the UpH (s 5.86±.2 vs. s 5.29±.13, 1128 Kidney Interntionl (212) 81,

7 M Cmeron et l.: Diurnl vrition in urine cidifiction originl rticle Po.5) nd the frction of net cid excreted s mmonium (s.68±.12 vs. s.54±.8, Po.5) were oth still lower in s, indicting tht ody weight nd BMI per se cnnot ccount for the difference. Another drwck is tht the overnight smpling did not hve the sme time resolution s the dytime smpling. We imed to mximize the numer of study smples without cusing excessive interruption of sleep. In conclusion, these dt indicte tht circdin vrition in UpH exists in oth s nd s. Although s demonstrte vriility in UpH, their urine remins cidic throughout the dy, leving them unprotected from the development of uric cid stones. The etiology of the unduly cidic urine is due to comintion of higher cid lod to the kidney nd the underutiliztion of mmoni s uffer. One might speculte tht circdin vrition occurs to protect us from persistently cidic or lkline UpH tht predisposes to stone development. Although gstric cid secretion contriutes to vritions in serum ph, it does not pper to ffect the diurnl chnges in UpH. These results provide foundtion for future studies to determine wht fctors contriute to vritions in UpH, whether these persist irrespective of diet, nd why s mintin the circdin vrition ut t lower UpH. MATERIALS AND METHODS Study prticipnts The study ws conducted t the Generl Clinicl Reserch Center, University of Texs Southwestern Medicl Center (Dlls, TX) with pprovl y the Institutionl Review Bord. All prticipnts provided informed consent. Study sujects included nine s who hd no history of kidney stones, nd ten s with proven uric cid stones recruited from the Minerl Metolism Clinic, University of Texs Southwestern Medicl Center. s were recruited vi Institutionl Review Bord-pproved dvertisements. All sujects were older thn 18 yers of ge. Excluded from oth groups were sujects with cretinine clernce o7 ml/min, proteinuri, liver disese, chronic dirrhe, peptic ulcer, or chronic use of ny medictions tht ffect gstric cid secretion or renl function. s were instructed to hold ll medictions tht ffect cid or se excretion for 1 week prior to initition of the study. Study protocol This doule-lind crossover study included two 14-dy phses. In the first phse, sujects were rndomized to receive the long-cting PPI, pntoprzole 4 mg/dy, or plceo. The dose ws chosen sed on previous studies showing dequte suppression of gstric cid production The cpsules were consumed ech morning with rekfst for 14 dys. Prticipnts susequently underwent 7-dy wsh-out period, during which study medictions were not dministered, nd then received the lternte study mediction for 14 dys in the second phse. During ech phse, sujects were dvised to hold lood pressure medictions tht might ffect renl sodium excretion. On dys 8 1 of ech phse, sujects were directed to consume diet low in cid sh nd sodium (B1 meq/dy). On dys of ech phse, frozen metolic diet ws provided y the dietry services t the Generl Clinicl Reserch Center, consisting of 3% ft, 55% crohydrte, 15% protein, 3 mg cholesterol, 4 mg clcium, 8 mg phosphorus, 1 meq sodium, 4 meq potssium, nd 3 cm 3 distilled wter per dy, which the sujects ingested s outptients. From dy 13 to dy 14, urine ws collected for 24 h under minerl oil nd refrigerted. Sujects were then dmitted to the inptient unit t the Generl Clinicl Reserch Center fter dinner on dy 13 nd remined s inptients until the morning of dy 15. During the inptient sty, sujects were mintined on the metolic diet, lthough the volume of wter ws decresed to 2.7 l. Body weight nd height were otined t the time of dmission. We monitored serum nd urine ketones nd detected no evidence of ketosis throughout the study. The diurnl study ws conducted on dys 14 nd 15. On dy 14, n intrvenous ctheter ws plced in the hnd for collection of free-flow lood smples. The hnd ws wrmed in 37-1C ox for 3 min prior to ech lood drw to otin rterilized smples. Strting t 8 hours, urine nd lood were collected every 2 h throughout the dy, except for the 2 h following mels when smples were collected hourly. To llow sujects to hve resonle sleep overnight, smples were otined every four hours from 22 hours until 6 hours on dy 15. Thus, collections were otined t: 8, 9, 1, 12, 13, 14, 16, 18, 19, 2, 22, 2, 6, nd 8 hours. Smples were otined prior to ingestion of mels, which were provided t 8, 12, nd 18 hours immeditely fter the lood drw t those designted times. d urine smples were nlyzed for totl volume, ph, cretinine, electrolytes, uric cid, citrte, sulfte, mmonium, TA, nd icronte. Blood smple mesurements included serum electrolytes, cretinine, uric cid, glucose, nd venous ph. Anlyticl procedures A systemic multichnnel nlysis ws performed to provide mesurements of serum cretinine, sodium, potssium, chloride, totl cron dioxide, glucose, nd uric cid (Beckmn CX9ALX; Beckmn Coulter, Fullerton, CA). Venous lood gs mesurements, including serum ph nd PCO 2, were mde using the Rdiometer ABL 5 (Rdiometer Americ, Westlke, OH). UpH ws mesured with ph electrode nd urine cretinine ws otined vi the picric cid method. Urine nd fsting serum cretinine mesurements were then used to clculte endogenous cretinine clernce. An ion-specific electrode ws used to mesure urinry potssium (Beckmn Coulter) nd urine sulfte ws ssessed y ion chromtogrphy. Urine mmonium ws mesured y the glutmte dehydrogense method, while citrte ws ssessed enzymticlly with regents from Boehringer-Mnnheim Biochemicls (Indinpolis, IN). Urinry icronte ws clculted from mesurements of UpH nd PCO 2, while titrtle cid ws ssessed y the utomted urette titrtion system (Rdiometer, Copenhgen, Denmrk). Uric cid ws determined vi the uricse method using lklinized smples to prevent precipittion. NAE ws clculted using the sum of mmonium nd TA minus the sum of citrte nd icronte in meq. The urinry nion gp ws clculted s the sum of ll nions (chloride, sulfte, phosphte, urte, cretinte, oxlte, citrte, icronte) sutrcted from the sum of ll ctions (sodium, potssium, clcium, mgnesium, mmonium), ll in milliequivlents. Sttisticl nlyses Dt re presented s men±stndrd devition (s.d.). Demogrphic vriles were compred etween s nd s using the two-smple t-tests. Twenty-four-hour urine results nd serum Kidney Interntionl (212) 81,

8 originl rticle M Cmeron et l.: Diurnl vrition in urine cidifiction iochemicl dt were compred using mixed-model repetedmesures nlysis followed y lest-squre mens contrsts to compre the two groups or to compre the two tretment phses. Mixed-model repeted-mesures nlysis ws lso used for the diurnl studies. The models consisted of group, phse, nd time min effects nd interctions, with suject modeled s rndom effect. An utoregressive covrince structure ws used to model the repeted time effect nd ccount for the correltion within individuls. 45 The interction terms from these models were used to test for differences in diurnl pttern. The difference in diurnl pttern etween the nd groups ws ssessed using the group-y-time interction fctor. The difference in diurnl pttern etween the plceo nd PPI phses ws ssessed using the phsey-time interction fctor. A significnt interction (Po.5) indictes tht the groups do not hve the sme pttern over time. Urine icronte ws rnk-trnsformed prior to nlysis. Sttisticl nlyses were performed with SAS version 9.2 (SAS Institute, Cry, NC). DISCLOSURE All the uthors declred no competing interests. ACKNOWLEDGMENTS We were supported y the Ntionl Institutes of Helth (M1- RR633, P1-DK-2543, R1 DK81423, UL1-RR24982, K23- RR2171, O Brien Kidney Center P3-DK79328) nd Seed Funds from the Chrles nd Jne Pk Center. We wish to thnk the cple nursing stff of the Generl Clinicl Reserch Center nd the technicl stff t the Minerl Metolism Lortory t UT Southwestern. Dr Chrles YC Pk ws extremely helpful in his insightful comments nd suggestions. REFERENCES 1. Bry MS, Young ME. The role of cell-specific circdin clocks in metolism nd disese. Oesity Rev 29; 1(Suppl 2): Mehr A, Bker CL, Loros JJ et l. Post-trnsltionl modifictions in circdin rhythms. Trends Biochem Sci 29; 34: Cermkin N, Boivin DB. The regultion of centrl nd peripherl circdin clocks in humns. Oesity Rev 29; 1(Suppl 2): Mills JN. Humn circdin rhythms. Physiol Rev 1966; 46: Hrmer SL. 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