Exposure to the microgravity environment of space

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1 Urolithiasis/Endourology Reduction of Renal Stone Risk by Potassium-Magnesium Citrate During 5 Weeks of Bed Rest Joseph E. Zerwekh,* Clarita V. Odvina, Lisa-Ann Wuermser and Charles Y. C. Pak From the Center for Mineral Metabolism and Clinical Research, and the Department of Physical Medicine and Rehabilitation (LAW), University of Texas Southwestern Medical Center at Dallas, Dallas, Texas Purpose: Exposure to the microgravity environment of space increases the risk of kidney stone formation, particularly for calcium oxalate and uric acid stones. This study was performed to evaluate the efficacy of potassium alkali as potassiummagnesium citrate in reducing renal stone risk and bone turnover. Materials and Methods: This study was performed as a double-blind, placebo controlled trial. We studied 20 normocalciuric subjects randomized to either placebo or potassium-magnesium citrate (42 meq potassium, 21 meq magnesium, 63 meq citrate per day) before and during 5 weeks of strict bed rest. The study was performed in the General Clinical Research Center and under a controlled dietary regimen composed of 100 meq of sodium, 800 mg of calcium, 0.8 gm/kg animal protein and 2,200 kcal per day. Two 24-hour urine collections were obtained under oil each week for assessment of stone risk parameters and relative saturation of calcium oxalate, brushite and undissociated uric acid. Blood was also collected for determination of serum immunoreactive parathyroid hormone and vitamin D metabolites. Results: Bed rest promoted a rapid increase in urinary calcium excretion of approximately 50 mg per day in both groups. Despite this increase subjects treated with potassium-magnesium citrate demonstrated significant decreases in the relative saturation of calcium oxalate and in the concentration of undissociated uric acid compared to placebo. Immunoreactive parathyroid hormone, serum 1,25-dihydroxyvitamin D and intestinal calcium absorption all decreased in both groups with no difference in response between the 2 treatment arms. Conclusions: Provision of alkali as potassium-magnesium citrate is an effective countermeasure for the increased risk of renal stone disease associated with immobilization. Despite an increase in urine calcium concentration, the relative saturation of calcium oxalate decreased due to citrate chelation of calcium and the concentration of undissociated uric acid decreased due to the significant increase in urine ph. Key Words: weightlessness countermeasures, nephrolithiasis, bed rest Submitted for publication October 17, Study received University of Texas Southwestern Medical Center Institutional Review Board approval on the use of human subjects. Supported by Grant BL00210, JEZ from the National Space Biomedical Research Institute and Grant M01-RR00633 from the United States Public Health Service. * Correspondence and requests for reprints: Center for Mineral Metabolism and Clinical Research, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, Texas (telephone: ; FAX: ; joseph.zerwekh@utsouthwestern.edu). Current address: Department of Physical Medicine and Rehabilitation, Mayo Clinic, Rochester, Minnesota For another article on a related topic see page Exposure to the microgravity environment of space produces a number of changes in urinary composition of metabolic and environmental origin that could increase the risk for renal stone formation. The most consistent change in urine composition that has been observed in astronauts during short-term and long-term missions, as well as from Earth based bed rest studies 1 4 has been a significant increase in urinary calcium and phosphate, the result of a well established increase in bone resorption. The rapid and marked increase in urinary calcium and phosphate concentrations, sometimes in the face of reduced urinary volume, is a major contributor to the increased saturation of calcium oxalate and brushite observed in astronauts during and after spaceflight. 1 3 Although increased fluid intake and administration of an antiresorptive agent would appear to be ideal countermeasures, there are changes in other urinary constituents that can increase the stone forming propensity of urine during space flight that would not be affected by this countermeasure. Hypocitraturia and reductions in urinary ph have also been observed in astronauts following short-term space flight. 5 Although the cause of these alterations in ph and citrate are not known with certainty, it has been speculated that it may represent the lack of ingestion of dietary potassium alkali as would be found in fresh citrus fruits and juices. Low urinary ph decreases uric acid solubility and leads to increased concentration of undissociated uric acid in urine, increasing the risk of uric acid stone formation. Citrate can serve as an inhibitor of stone formation through its action in decreasing ionic calcium concentration via complexation, and by direct inhibition against crystal growth and spontaneous precipitation. 6 In addition, provision of alkali as citrate can increase urinary ph and increase uric acid solubility. These observations have led us to propose that an effective countermeasure against renal stone formation in astronauts might be provision of potassium alkali as KMgCit. Previous studies have shown that KMgCit administration /07/ / Vol. 177, , June 2007 THE JOURNAL OF UROLOGY Printed in U.S.A. Copyright 2007 by AMERICAN UROLOGICAL ASSOCIATION DOI: /j.juro

2 2180 POTASSIUM-MAGNESIUM CITRATE IN MICROGRAVITY INDUCED NEPHROLITHIASIS significantly increases urinary ph as well as urinary citrate. 7,8 Both of these changes would act to reduce the saturation of urine with respect to calcium oxalate and uric acid. Furthermore, the provision of magnesium may decrease urinary oxalate by binding oxalate in the intestinal tract as well as by complexing oxalate in urine. Lastly, the provision of the potassium salt in place of sodium should attenuate the formation of sodium urate and prevent sodium induced crystallization of calcium oxalate. To evaluate the efficacy of KMgCit in reducing the propensity for stone formation during exposure to microgravity, we performed a randomized double-blind placebo controlled trial in 20 normal subjects using an Earth based model of skeletal unloading, namely 5 weeks of strict bed rest. MATERIALS AND METHODS Subjects This study was approved by the University of Texas Southwestern Medical Center Institutional Review Board on the use of human subjects. A total of 20 subjects were admitted to the General Clinical Research Center at Southwestern Medical Center in Dallas after giving signed informed consent. Potential candidates were screened before enrollment. Inclusion criteria included age between 18 to 50 years, normal 24-hour urine and fasting serum chemistry, including calcium, phosphorus, glucose, serum electrolytes, potassium, uric acid, creatinine and a normal complete blood count. Candidates were excluded from further participation in the study if there was evidence of renal or hepatic dysfunction, hypercalcemia or hypercalciuria (greater than 4 mg/kg), urinary ph less than 5.5, or lumbar bone mineral density (L2 L4) T-score less than 1.0. Candidates were also excluded if there were any abnormalities in their serum chemistry profile or blood analyses. None of the participants had a history of bone disease, nephrolithiasis or nontraumatic fractures, and none were on prescription or over-thecounter medications before hospitalization. Although we did not perform pre-test radiographic imaging to rule out silent nephrolithiasis, candidates were excluded from participation if there was a family history of nephrolithiasis or a previous episode of deep venous thrombosis. None were smokers. Study Protocol The study consisted of 3 phases represented by a 5-day ambulatory phase followed by 35 days of strict bed rest and then 14 days of reambulation. Each week subjects consumed food and fluids from the Parkland Hospital kitchen ad libitum for the first 3 days, and a constant metabolic diet containing per day 800 mg Ca, 1,100 mg phosphorus and 100 meq sodium for the last 4 days of each week of the study. We found that a reprieve from the metabolic diet for a few days each week results in higher subject retention during such prolonged studies. The mean daily caloric value of the metabolic diet was 2206 Kcal. The mean dietary composition was 74 gm protein, 83 gm fat and 291 gm carbohydrate, representing 13%, 34% and 53% of total calories, respectively. Total fluid intake was set at 3.0 l per day. Overall there was no significant weight change during the study for either group. A biochemical evaluation was performed during the last 2 days of metabolic diet consumption each week. This consisted of 2, 24-hour urine collections under oil for assessment of stone risk parameters, pco 2 and total titratable acidity. RSR of urine with respect to calcium oxalate and brushite was calculated from the EQUIL computer program. 9 The net gastrointestinal absorption of alkali was calculated from the formula of Oh. 10 Fasting serum was obtained for routine chemistry studies, intact ipth (Alpco Diagnostics) and vitamin D metabolites (Alpco Diagnostics). In addition to these analyses, assessment of fractional intestinal calcium absorption by the dual stable calcium isotope technique was performed during the ambulatory (baseline) phase and during the last week of bed rest. Subjects were randomized to either KMgCit (Mission Pharmacal, San Antonio, Texas) containing 42 meq K, 21 meq Mg and 63 meq of citrate per day, or to a matching placebo medication. KMgCit is an investigational drug (Investigational New Drug Application 36,276) for which Mission Pharmacal is the sponsor. Statistical Analyses All data are expressed as mean SD. Power analysis was based upon a mg per day difference in urinary calcium excretion between subjects treated with placebo or KMgCit. With 0.05 and 0.15, 10 subjects per group would be required to detect a significant difference in calcium excretion during KMgCit administration. Similar calculations were also performed for detecting a difference of in urine ph and a urinary citrate difference of mg per day. These later calculations gave a required n of 6 per group, a result adequately met by an n of 10 per group. Significant differences of the baseline values between subjects treated with placebo or KMgCit were determined by the Student s t test for normally distributed data and the Mann-Whitney test for nonparametric data. To assess the effect of KMgCit supplementation on stone risk parameters, we compared the difference between the baseline value and the value obtained at week 5 of bed rest for the placebo group vs KMgCit group. A 2-factor repeated measures ANOVA was used for this analysis. Two factor (group time) repeated measures ANOVA was also used to determine the significance of the change in laboratory analytes between baseline and each week of bed rest within each experimental group for the RSR of calcium oxalate and undissociated uric acid. All tests were performed 2-sided with a 0.05 significance level using SAS (version 9.0 for Windows). RESULTS The baseline demographics for the 2 study groups are summarized in table 1. Of the white subjects in the KMgCit group 2 were of Hispanic ethnicity. Overall the 2 groups were not significantly different from each other. The effect of KMgCit administration on urine composition between the 2 groups is summarized in table 2. Before TABLE 1. Baseline demographics for subjects on 5 weeks of bed rest Placebo KMgCit No. subjects 9 11 No. male/female 5/4 7/4 No. white/black race 5/4 8/3 Mean age SD Mean kg/m 2 body mass index SD

3 POTASSIUM-MAGNESIUM CITRATE IN MICROGRAVITY INDUCED NEPHROLITHIASIS 2181 TABLE 2. Effect of 5 weeks of bed rest on urine composition in subjects treated with placebo or KMgCit Mean SD Parameter (measurement, normal value)* Before Placebo After Placebo Before KMgCit After KMgCit Total vol (greater than 2,000 ml/day) 2, , , , Creatinine (mg/day) 1, , , , Ca (less than 4 mg/kg/day) P (less than 1,100 mg/day) Oxalate (less than 45 mg/day) Na (less than 200 meq/day) K (meq/day) , Mg (less than 60 mg/day) , Citrate (greater than 320 mg/day) , ph , Total titratable acidity (meq/day) , Ammonium (meq/day) , pco 2 (mm Hg) , NGIA (meq/day) , Sulfate (less than 30 mg/day) Uric acid (less than 700 mg/day) RSR Ca oxalate (less than 2.00) , RSR brushite (less than 2.00) Undissociated uric acid (mg/day) , Median * Normal values taken from Stone Risk Profile, Mission Pharmacal Co. To convert values for urine analytes to SI units, multiply the values in the table by for creatinine, for calcium, for phosphorus, for oxalate, for magnesium, for citrate, for sulfate and for uric acid. After value represents mean value at week 5 of bed rest just before reambulation. Post-bed rest value significantly different from pre-bed rest value at p Significant difference in response to treatment at 5 weeks of bed rest between subjects treated with placebo or KMgCit determined from the treatment bed rest interaction term of the repeated measures analysis of variance at p Significantly different from before placebo value by the Student t test at p treatment subjects randomized to KMgCit had a higher mean baseline urinary calcium concentration than placebo, although this difference was not significant. However, when combined with the slightly greater mean baseline urinary oxalate concentration in the KMgCit group, it resulted in a significantly higher RSR for calcium oxalate compared to the placebo group (p 0.019). Following treatment urinary ph, potassium, magnesium, citrate, pco 2 and NGIA significantly increased, while urinary ammonium and titratable acidity significantly decreased in subjects treated with KMgCit. On the other hand there were no significant changes in any of these parameters in the placebo group. Urinary calcium increased significantly (p 0.022) in the placebo group within 1 week of initiating bed rest and remained significantly increased throughout bed rest for placebo. The mean increase during bed rest was 50 mg per day (fig. 1). A similar trend was observed for subjects treated with KMgCit although the increase at week 1 of bed rest was not significantly different from baseline. The mean increase during bed rest was 43 mg per day. The relative saturation ratio of calcium oxalate trended downward during KMgCit treatment and was significantly lower than the baseline value after 5 weeks of bed rest (fig. 2). Undissociated uric acid significantly decreased at 1, 2 and 3 weeks of bed rest during KMgCit administration. In the placebo group urinary calcium increased significantly by 64 mg per day at 5 weeks of bed rest. However, there were no other significant changes in any urinary analyte, including urine volume. RSR of calcium oxalate was significantly higher at 3 weeks of bed rest than at baseline (fig. 2). Undissociated uric acid did not change during 5 weeks of bed rest in the placebo group. For many of the urinary parameters the change from baseline to week 5 of bed rest was significantly different between subjects treated with placebo or KMgCit. This not only included measures of acid-base balance but also for stone risk. The decrease in the relative saturation of calcium oxalate and undissociated uric acid observed for subjects treated with KMgCit at week 5 of bed rest was significantly different from the change observed for those treated with placebo. However, the increases in urinary calcium and in FIG. 1. Effect of bed rest on urinary calcium excretion. Percent change from baseline is shown for placebo ( ) and KMgCit treated ( ) subjects for weeks 1 to 5 of bed rest and for 2-week reambulation period.

4 2182 POTASSIUM-MAGNESIUM CITRATE IN MICROGRAVITY INDUCED NEPHROLITHIASIS the relative saturation ratio of brushite during week 5 of bed rest were not significantly different between placebo and KMgCit groups. Table 3 summarizes the changes in serum biochemistry during 5 weeks of bed rest. Serum calcium and phosphorus remained within normal limits throughout the study despite a small, but significant, increase in serum phosphorus in the KMgCit group after 5 weeks. Serum 1,25(OH) 2 D also decreased significantly in both groups as did the intestinal absorption of calcium. Overall there were no adverse effects reported by any subject during the course of this study and KMgCit was well tolerated by all who received it. DISCUSSION FIG. 2. Effect of KMgCit treatment on relative saturation of urine with respect to calcium oxalate and undissociated uric acid. A, mean relative saturation of urine with respect to calcium oxalate for subjects treated with placebo ( ) and KMgCit ( ). Dagger denotes KMgCit value significantly different from placebo value. Values significantly different from baseline at p are denoted by asterisk for placebo and number sign for KMgCit treatments. B, mean concentration of undissociated uric acid for subjects treated with placebo ( ) and KMgCit ( ). KMgCit treatments significantly different from baseline at p 0.05 are denoted by number sign. Error bars represent SEM. Extended bed rest is recognized as a valid Earth based model to assess the effects of microgravity on the skeleton as well as cardiovascular and muscular systems. Using this model, it has been shown that there is a rapid and sustained increase in bone resorption leading to increased urinary calcium and phosphorus, and loss of bone mass. 4,11 15 We therefore chose this model in which to evaluate a potential countermeasure against microgravity induced renal stone risk. We reasoned that KMgCit would be efficacious as a potential countermeasure against renal stones due to its ability to alkalinize the urine and to increase urinary citrate, a potent inhibitor of calcium stone formation. As of October 2001 there had been 15 United States crewmembers in whom renal stones developed. The Russian Space Program reported 3 renal stone events and 1 in-flight episode of renal stone formation among cosmonauts. 5 Thus, renal stone risk represents a serious medical consequence of exposure to the microgravity environment of space. There have been 3 previous studies that assessed the efficacy of a countermeasure against renal stone risk using an Earth based bed rest model In the first 2 studies bisphosphonate treatment was shown to reduce urinary calcium. One study reported an actual decrease in renal stone episodes. 12 A more recent study demonstrated that lower body negative pressure chamber treadmill exercise modestly reduced stone risk. 13 However, it is unlikely that antiresorptive or exercise countermeasures can effectively reduce the risk of uric acid stones from low urinary ph reported during spaceflight. The results of this randomized placebo controlled trial clearly demonstrated the efficacy of KMgCit in reducing the increased risk of renal stone formation associated with skeletal unloading. The mechanism of action of KMgCit lies in its ability to alkalinize the urine. This was evident from several biochemical indicators of acid-base status including significant increases in urinary citrate and pco 2 and significant decreases in urinary NH 4 and total titratable acidity in the KMgCit group, as opposed to no change in the placebo group. Increased urinary ph reduced the concentration of poorly soluble undissociated uric acid in urine, thereby reducing the risk for uric acid stones. Although an increase in urinary ph increases the relative saturation of urine with respect to brushite, another type of renal stone, there was no signifi- TABLE 3. Effect of 5 weeks of bed rest on serum parameters of calcium homeostasis and intestinal calcium absorption in subjects treated with placebo or KMgCit Mean SD Parameter (normal values)* Before Placebo After Placebo Before KMgCit After KMgCit Ca ( mmoles/l) P ( mmoles/l) ipth (less than 60 pg/ml) ,25(OH) 2 D (18 55 pg/ml) Intestinal Ca absorption (40 60%) * To convert values for serum PTH to picomoles per liter, multiply by and to convert 1,25(OH) 2 D to nanomoles per liter multiply by Mean value at week 5 of bed rest just before reambulation. Post-bed rest value significantly different from pre-bed rest value at p

5 POTASSIUM-MAGNESIUM CITRATE IN MICROGRAVITY INDUCED NEPHROLITHIASIS 2183 cant difference in the relative saturation of brushite between the placebo and KMgCit groups when assessed during week 5 of bed rest. The second major effect of KMgCit treatment in reducing renal stone risk was to retard crystallization of calcium oxalate, a common type of kidney stone. The relative saturation of calcium oxalate was significantly increased in the patients receiving placebo by week 3 of bed rest owing principally to increased urinary calcium. On the other hand, the relative saturation of calcium oxalate was significantly decreased by week 5 of bed rest in patients receiving KMgCit despite an increase in urinary calcium. This decrease in calcium oxalate relative saturation is due to complexation of calcium in urine by citrate, reducing ionized calcium concentration. Provision of KMgCit also significantly increased urinary magnesium. We reasoned that provision of magnesium would bind intestinal oxalate, decrease urinary oxalate excretion and further reduce the relative saturation of calcium oxalate in urine. This was not observed as urinary oxalate did not change significantly in the KMgCit group. However, it is possible that the increase in urine magnesium could have acted to bind oxalate in the urine and thereby reduce its activity toward the nucleation and growth of calcium oxalate, further reducing the relative saturation of calcium oxalate. 14,15 If true, this action would make KMgCit more efficacious than potassium citrate in reducing the relative saturation of calcium oxalate. Subjects were well matched with respect to age, gender and race. However, the subjects treated with KMgCit demonstrated a higher mean relative saturation of calcium oxalate in urine before bed rest compared to placebo. This baseline difference between placebo and KMgCit groups might be viewed as a limitation of the study since it could underestimate the potential benefit of KMgCit in reducing calcium oxalate stone risk. However, despite this difference, KMgCit was still efficacious in reducing the relative saturation of calcium oxalate in the urine of treated subjects. After 5 weeks of bed rest there were significant reductions in serum 1,25(OH) 2 D and intestinal calcium absorption. Similar findings have been observed not only in other bed rest studies 11,16,17 but also in astronauts during and immediately following space flight. 18,19 Such changes are believed to be the sequelae of increased bone resorption. Serum calcium and phosphorus remained within normal limits despite a small, but significant, increase in serum phosphorus for subjects treated with KMgCit. We have demonstrated the efficacy of KMgCit as an effective countermeasure against the increased risk of renal stone disease invariably encountered during skeletal unloading. While clearly applicable to astronauts under true microgravity conditions, these findings could have important therapeutic implications for the estimated 20 million Americans with nephrolithiasis. CONCLUSIONS KMgCit may be an effective agent in the reduction of increased renal stone risk associated with skeletal unloading. In combination with a bone sparing agent such as exercise or bisphosphonate therapy this agent might represent the best countermeasure to microgravity induced renal stone risk. Validation under true microgravity flight conditions is warranted. ACKNOWLEDGMENTS Nursing and core laboratory staff of the General Clinical Research Center assisted with professional and careful attention to protocol detail. Center for Mineral Metabolism and Clinical Research laboratory staff performed careful and timely analytical assays. Abbreviations and Acronyms 1,25(OH) 2 D 1,25-dihydroxyvitamin D ipth immunoreactive parathyroid hormone KMgCit potassium-magnesium citrate NGIA net gastrointestinal absorption of alkali RSR relative saturation ratio REFERENCES 1. Whitson PA, Pietrzyk RA, Pak CYC and Cintron NM: Alterations in renal stone risk factors after space flight. J Urol 1993; 150: Whitson PA, Pietrzyk RA and Pak CYC: Renal stone risk assessment during space shuttle flights. J Urol 1997; 158: Whitson PA, Pietrzyk RA, Morukov BV and Sams CF: The risk of renal stone formation during and after long duration space flight. Nephron 2001; 89: Hwang TIS, Hill K, Schneider V and Pak CYC: Effect of prolonged bed rest on the propensity for renal stone formation. J Clin Endocrinol Metab 1988; 66: Pietrzyk RA, Jones JA, Sams CF and Whitson PA: Renal stone formation among astronauts. Unpublished data. 6. 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6 2184 POTASSIUM-MAGNESIUM CITRATE IN MICROGRAVITY INDUCED NEPHROLITHIASIS 15. Johansson G, Backman U, Danielson BG, Fellstrom B, Ljunghall S and Wikstrom B: Biochemical and clinical effects of the prophylactic treatment of renal calcium stones with magnesium hydroxide. J Urol 1980; 124: LeBlanc AD, Schneider VS, Evans HJ, Engelbretson DA and Krebs JM: Bone mineral loss and recovery after 17 weeks of bed rest. J Bone Mineral Res 1990; 5: Zerwekh JE, Ruml LA, Gottschalk F and Pak CYC: The effects of twelve weeks of bed rest on bone histology, biochemical markers of bone turnover, and calcium homeostasis in eleven normal subjects. J Bone Mineral Res 1998; 13: Smith SM, Nillen JL, LeBlanc A, Lipton A, Demers LM, Lane HW et al: Collagen cross-link excretion during space flight and bed rest. J Clin Endocrinol Metab 1998; 83: Smith SM, Wastney ME, O Brien KO, Morukov BV, Larina IM, Abrams SA et al: Bone markers, calcium metabolism, and calcium kinetics during extended-duration space flights on the Mir space station. J Bone Mineral Res 2005; 20: 208.