- Email: [email protected]
Impact of Prolonged Fasting on the Risk of Calcium Phosphate Precipitation in the Urine: Calcium Phosphate Lithogenesis during Prolonged Fasting in a Healthy Cohort
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57
Impact of Prolonged Fasting on the Risk of Calcium Phosphate Precipitation in the Urine: Calcium Phosphate Lithogenesis during Prolonged Fasting in a Healthy Cohort Mohammad A. Shafiee,* Mehdi Aarabi, Pouyan Shaker, Amir M. Ghafarian, Pouyan Chamanian and Mitchell L. Halperin From the Division of General Internal Medicine, Department of Medicine, Toronto General Hospital and Li Ka Shing Knowledge Institute of St. Michael’s Hospital and Division of Nephrology, University of Toronto (MLH), Toronto, Ontario, Canada
Purpose: Intermittent fasting and curtailing water intake for extended periods were likely common in Paleolithic times. Today it occurs for religious and dietary reasons. This restriction in intake should cause a decrease in the urine flow rate while raising the concentration of certain substances in urine to the point of precipitation. In this study we measured the risk of CaHPO4 precipitation following 18 hours of food and water deprivation. Materials and Methods: Urine samples were periodically collected from 15 healthy subjects who fasted and abstained from drinking any liquid for 18 hours. The urine constituents Ca2þ, HPO42e and pH involved in CaHPO4 formation were measured at various times throughout the fasting day. A comparison was made with control data, which consisted of diurnal urine collections taken throughout a separate nonfasting day prior to the fasting day. Results: The mean SEM urine flow rate decreased significantly from 0.93 0.1 ml per minute in the control group to 0.37 0.05 ml per minute in the fasting group (p <0.05). Mean Naþ and Ca2þ excretion rates decreased significantly from 127 12 to 54 13 mmol per minute and from 3.2 0.4 to 0.80 0.21, respectively. Mean urinary Naþ and Ca2þ concentrations also decreased from 161 11.6 to 122 16.0 mmol/l and from 3.7 0.5 to 2.0 0.55, respectively. Urinary pH and the concentration of phosphate, citrate and magnesium were not significantly affected. Conclusions: Although the steady decrease in the urine flow rate was statistically significant during 18 hours of food and water deprivation, there was no evidence that the calculated risk of CaHPO4 precipitation in the healthy subjects had increased. Key Words: kidney calculi, fasting, water deprivation, calcium phosphate, citric acid
GIVEN the recent popularity of intermittent fasting, studies have attempted to investigate the health benefits and risks associated with this behavior.1 Despite studies indicating the positive effects of intermittent fasting2 the decreased fluid intake during fasting are speculated
BP ¼ blood pressure Ksp ¼ solubility product constant PCT ¼ proximal convoluted tubule RAAS ¼ renin-angiotensinogenaldosterone system UFR ¼ urine flow rate WD ¼ water and food deprivation Accepted for publication February 12, 2018. No direct or indirect commercial incentive associated with publishing this article. The corresponding author certifies that, when applicable, a statement(s) has been included in the manuscript documenting institutional review board, ethics committee or ethical review board study approval; principles of Helsinki Declaration were followed in lieu of formal ethics committee approval; institutional animal care and use committee approval; all human subjects provided written informed consent with guarantees of confidentiality; IRB approved protocol number; animal approved project number. * Correspondence: Division of General Internal Medicine, Department of Medicine, University of Toronto, Toronto General Hospital, 200 Elizabeth St., 14 EN-208 Toronto, Ontario, M5G 2C4, Canada (telephone: 416-340-4800, extension 6244; FAX: 416-595-5826; e-mail: [email protected]).
to impact the process of nephrolithiasis. Early humans were likely water and food deprived for extended periods and it would have been devastating to survival if they were affected by kidney stone sequelae.3 Kidney stone disease, which is characterized by severe pain, afflicts
0022-5347/18/2001-0001/0 THE JOURNAL OF UROLOGY® Ó 2018 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH, INC.
Dochead: Adult Urology
Abbreviations and Acronyms
https://doi.org/10.1016/j.juro.2018.02.3092 Vol. 200, 1-6, July 2018 Printed in U.S.A.
www.jurology.com
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PROLONGED FASTING AND CALCIUM PHOSPHATE PRECIPITATION
Figure 1. In fasting vs control groups changes in UFR, and Naþ and Ca2þ urine concentrations were significant (all p <0.05). SEM shows variance.
10% to 15% of the general population with a 0.5% increasing prevalence annually in North America and Europe.4,5 Although urinary lithiasis carries dire health risks, the mechanisms underlying it and the pathology are largely undefined.6 We specifically studied CaHPO4 precipitates for certain reasons. 1) They are the main precursors of calcium oxalate monohydrate nucleation and growth, representing the most common stone type with a growing incidence. 2) They can cause renal failure by plugging Bellini ducts.7,8 Furthermore, CaHPO4 stone formation is associated with 2 measurable lithogenic variables, including ionized Ca2þ and divalent phosphate ions (H2PO4). Changes in the concentration of either divalent ion may have unfortunate consequences on kidney stone formation. Precipitation formation is contingent on the solubility product constant of a compound. The risk of CaHPO4 precipitation formation increases when the mentioned divalent ions exceed the Ksp. Supersaturation is the condition in which a product exceeds its normal Ksp.9 The risk of CaHPO4 precipitation formation decreases with higher levels of urinary citrate and magnesium ions caused by competitive binding and with a decrease in the HPO42e concentration caused by decreasing urinary pH. Dochead: Adult Urology
Prior studies have indicated that a prolonged fasting state leads to lower BP and, thus, to lower renal perfusion as well as lower UFR.9 As shown by the equation, urine flow rate ¼ excreted effective mOsm per minute/urine effective osmolarity in mOsm/l, UFR is determined by the number of effective osmoles in urine and the changes in urine effective osmolality.10 Simply, if the decrease in UFR were the only variable, the impact of lower UFR on supersaturation would be exponential since it would raise Ca2þ and HPO4 activity. If the excretion rates of these ions remain constant, the ionic product CaHPO4 should increase fourfold for each halving of UFR. In this study we assessed the alternative hypothesis that decreased UFR after 18 hours of water deprivation would result in increased renal reabsorption of Naþ and Ca2þ. Therefore, there would be no increased risk of CaHPO4 precipitate formation in normal healthy individuals.
MATERIALS AND METHODS The St. Michael’s Hospital Research Ethics Board approved the study protocols. Written consent was provided by subjects to use their data anonymously. A total of
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Urine phosphate, citrate, pH, Mg and creatinine excretion, and systolic and diastolic blood pressure in control vs fasting groups (significant at p <0.05) Mean SEM Control Urine: HPO42e concentration (mmol/l) Citrate concentration (mmol/l) pH Mg (mmol/l) Creatinine excretion rate (mmol/min) BP (mm Hg): Systolic Diastolic
29.5 3.47 6.2 4.8 9.0 112 72
2.5 1.07 0.3 0.7 0.7
2.4 1.5
Mean SEM Fasting 22.8 3.06 5.7 3.4 8.3 104 70
2.5 0.72 0.2 0.4 1.1
2.7 1.6
p Value 0.09 0.75 0.19 0.11 0.64 0.02 0.41
15 healthy subjects provided urine samples at various time points through 18 hours of voluntary fasting (food and drink) overnight from 6 p.m. to 12:00 p.m. the next day. Each subject recorded the time and the volume of each void and a small sample was refrigerated for later analysis. Volunteers were in good health and did not receive medication during the week prior to the study. The control was a separate diurnal collection from the same subjects at similar time points on a nonfasting day. The rate of creatinine excretion was used to assess the completeness of each urine sample. UFR in each subject was calculated by dividing sample volume in ml by the corresponding time in minutes. The important urinary constituents of CaHPO4 precipitation, including
electrolytes, Naþ and Kþ, were then measured by flame photometry using a FLM-3 Radiometer (London Scientific, London, Ontario, Canada). Cle was measured by electrometric titration using a CMT 10 Cle meter (London Scientific). Osmolality was measured by freezing point depression (Advanced Instruments, Needham Heights, Massachusetts). Urea and creatinine in plasma and urine were measured with a KodakÒ EktachemÔ Analyzer.11 Concentrations of Ca2þ, phosphate and Mg2þ in urine were measured with a Synchron CXÒ5CE Analyzer. Citrate was measured enzymatically using adenosine triphosphate citrate lyase. Urine pH was measured with a glass electrode pH meter (Corning 178 Blood pH Analyzer, Corning Glass Works, Corning, New York). Urine osmolality was measured by freezing point depression (Advanced Instruments).12
Statistical Analysis Results are reported as the mean SEM. Comparisons between groups were done by the paired t-test. Statistical analysis was performed with MicrosoftÒ ExcelÒ with p <0.05 considered statistically significant.
RESULTS
Urinary concentrations and excretion rates of Naþ, Ca2þ, citrate, Mg2þ, HPO42e and creatinine as well as UFR and urinary pH were measured and
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Figure 2. Mean Naþ excretion rate and UFR of samples taken at various times in fasting and control groups in chronological order throughout fasting period with around 4.5 hours between samples. Naþ excretion was measured by flame photometry and calculated. Points 3 and 4 differed significantly (p <0.05). Whiskers indicate SEM.
Dochead: Adult Urology
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Time (hours) Figure 3. Mean Ca2þ excretion rate and UFR of urine samples taken at various times in fasting and control groups in chronological order throughout fasting period with around 4.5 hours between samples. Ca2þ excretion was measured by flame photometry and calculated. Points 3 and 4 differed significantly (p <0.05). Whiskers indicate SEM.
compared between control and fasting data at a comparable time point. The point corresponding to the final sample collection in the fasting day was chosen because it had the lowest UFR. Mean UFR in male and female subjects in the WD group decreased significantly from 0.93 0.1 to 0.37 0.05 ml per minute (p <0.05, fig. 1). Mean urinary creatinine excretion decreased from 9.0 0.7 to 8.3 1.1 mmol per minute but this was not statistically significant (see table). Na Levels Mean Naþ excretion decreased significantly from 127 11.6 to 54 13.0 mmol per minute (fig. 2). The mean urinary Naþ concentration decreased significantly from 160.5 11.6 to 122 16.0 mmol/l (p <0.05, fig. 1). D
Ca2D, Mg2D, Citrate and pH Levels Mean Ca2þ excretion decreased from 3.2 0.4 to 0.80 0.21 mmol per minute (p <0.05, fig. 3) and the mean urinary concentration of Ca2þ decreased significantly from 3.7 0.5 to 2.0 0.55 mmol/l (p <0.05, fig. 1). The small change from 23.5 2.2 to 22.8 2.5 mmol/l in the mean total phosphate urine Dochead: Adult Urology
concentration was not statistically significant (see table). Similarly the mean changes in urine citrate and Mg2þ of 3.4 0.6 to 3.1 0.72 and 3.3 0.7 to 3.3 0.3 mmol/l, respectively, were not significant (see table). Finally, there was no significant change between the control and experimental groups in the mean urine pH change from 6.0 0.1 to 6.0 0.1 (see table). Systolic and Diastolic Blood Pressure Mean systolic blood pressure in the separate fasting group decreased significantly from 112.2 2.4 to 104 2.7 mm Hg (p <0.05). On the other hand, the decrease in mean diastolic blood pressure from 72 1.5 to 70 1.6 mm Hg was not statistically significant (see table).
DISCUSSION The most striking finding in this study is the significant decrease in Ca2þ. This does not correspond to the predicted cause of supersaturation from the increase in data at minimum UFR preceding an 18hour fasting period. As shown, the decreased values
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457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513
5
Figure 4. Calcium, phosphate, sodium, citrate and magnesium handling in different parts of nephron tubule demonstrates varying amounts of solute reabsorption throughout healthy nephron. DCT, distal convoluted tubule.
of UFR as well as the Ca2þ and Naþ concentrations were statistically significant, supporting our hypothesis that a UFR decrease after 18 hours of water and food deprivation would result in increased renal reabsorption of Naþ and Ca2þ. Therefore, it seems that fasting did not increase the likelihood of CaHPO4 precipitate formation in normal healthy individuals. The renal mechanisms of Ca2þ reabsorption begin primarily in the PCT and largely depend on reabsorption of Naþ in the PCT. In other words, if there is an increased rate of water and Naþ reabsorption, an increase in Ca2þ reabsorption is expected secondary to the higher luminal concentration of this divalent cation. When urinary pH increases, the HPO42e concentration decreases while that of H2PO4e increases in urine. These changes are greater at higher pH changes, although the total phosphate concentration and urine pH changes were not statistically significant. Inhibitory factors such as Mg2þ and citrate also exist which may change the likelihood of Dochead: Adult Urology
CaHPO4 kidney stone formation. However, these 2 factors did not seem to have an obvious role in preventing CaHPO4 precipitation.10 In the provided urine samples the Mg2þ concentration was higher than that of Ca2þ. This could have been due to difference in handling Mg2þ compared to Ca2þ. Most Mg2þreabsorption occurs in the loop of Henle, which may have a protective role. Only 5% of Mg2þ is reabsorbed in the distal convoluted tubule by specific Mg2þ channels and the remaining 10% is excreted in urine.13 Additionally, magnesium salts do not become supersaturated in the way that calcium salts do because they generally have a higher Ksp in urine.10 Figure 4 shows renal handling of these ions.10 Citrate is the most important chelator of Ca2þ in urine, inhibiting the nucleation and growth of CaHPO4 crystals. This chelation process involves equimolar quantities of Ca2þ and citrate. When the concentration of citrate exceeds that of Ca2þ, the urinary concentration of Ca2þ and its activity will
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571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626
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be virtually zero. This excretion of Ca2þ and citrate permits the excretion of urinary potential HCO3e without raising urinary pH, thus, maintaining a low HPO42e concentration and minimizing CaHPO4 precipitation. This effect is similar to that of Mg2þ binding to HPO42e.14 As noted in previous studies, prolonged fasting results in lower average blood pressure. This decrease is only significant in systolic pressure but not diastolic pressure (112/72 vs 104/69 mm Hg, p <0.05, see table). The RAAS is known to cause vasoconstriction and increase Naþ reabsorption from tubular fluid back into blood to maintain blood pressure.15 Toward the end of the fasting period (ie in the morning) the RAAS is activated and it might also account for increased reabsorption of Naþ and a lower rate of Naþ excretion.16 Therefore, the net effect is avid reabsorption of Naþ as well as an increase in Ca2þ and water reabsorption due to the concentration differences. Despite the decreased UFR during the 18 hours of fasting we found that
the risk of CaHPO4 precipitation did not increase because of the significant decrease in the urinary excretion rate of Ca2þ and Naþ due to avid reabsorption of these cations. Rapid Naþ reabsorption can be explained by the decrease in the UFR and in perfusion as well as the decrease in BP and in subsequent RAAS activation. Furthermore, the concentration of other ligands (Mg2þ and citrate) remained in the normal range.
CONCLUSIONS Overall in this study we found no evidence that a decrease in UFR would increase the likelihood of supersaturation and kidney stone formation. Future extension of this study in experiments in patients with kidney stones is not possible due to ethical concerns, which proves to be one of the major limitations of this study. However, we recognize the possible variation in results that would have occurred had this experiment been performed in participants with idiopathic hypercalciuria.
REFERENCES 1. Collier R: Intermittent fasting: the science of going without. CMAJ 2013; 185: 363. 2. Patterson RE, Laughlin GA, Lacroix AZ et al: Intermittent fasting and human metabolic health. J Acad Nutr Diet 2015; 115: 1203. 3. Henry CJ: Variability in adult body size: uses in defining the limits of human survival. In: Anthropometry: The Individual and the Population. Edited by SJ Ulijaszek and CG MascieTaylor. New York: Cambridge University Press 1994; vol. 1, chap 8, p 118. 4. Lieske JC and Segura JW: Evaluation and medical management of kidney stones. In: Essential Urology: A Guide to Clinical Practice. Edited by JM Potts. New Jersey: Humana Press 2004; vol. 1, chap 7, pp 117-152. 5. Moe OW: Kidney stones: pathophysiology and medical management. Lancet 2006; 367: 333. 6. Stamatelou KK, Francis ME, Jones CA et al: Time trends in reported prevalence of kidney stones in the United States: 1976-1994. Kidney Int 2003; 63: 1817.
Dochead: Adult Urology
7. Preminger GM: Stones in the urinary tract. In: The Merck Manual of Medical Information, Second Home Edition. Edited by MH Beers, AJ Fletcher and TV Jones. New Jersey: Pocket Books 2004; vol. 1, chap 1, p 148.
overnight calcium phosphate precipitationdan observational study to determine factors against calcium phosphate lithogenesis in a healthy cohort. Nephron 2016; 132: 238.
8. Evan AP, Coe FL, Lingeman JE et al: Mechanism of formation of human calcium oxalate renal stones on Randall’s plaque. Anat Rec (Hoboken) 2007; 290: 1315.
13. Muallem S and Moe OW: When EGF is offside, magnesium is wasted. J Clin Invest 2007; 117: 2086.
9. Samad F, Qazi F, Pervaiz MB et al: Effects of Ramadan fasting on blood pressure in normotensive males. J Ayub Med Coll Abbottabad 2015; 27: 338.
14. Schlieper G, Westenfeld R, Brandenburg V et al: Inhibitors of calcification in blood and urine. Semin Dial 2007; 20: 113.
10. Halperin ML, Kamel KS and Oh MS: Mechanisms to concentrate the urine: an opinion. Curr Opin Nephrol Hypertens 2008; 174: 416. 11. Cheema-Dhadli S and Halperin ML: Relative rates of appearance of nitrogen and sulphur: implications for postprandial synthesis of proteins. Can J Physiol Pharmacol 1993; 71: 120. 12. Shafiee MA, Logan AG and Halperin ML: How protective mechanisms interact to prevent
15. Atlas SA: The renin-angiotensin aldosterone system: pathophysiological role and pharmacologic inhibition. J Manag Care Pharm 2007; 13: 9. 16. Cugini P and Lucia P: Circadian rhythm of the renin-angiotensin-aldosterone system: a summary of our research studies. Clin Ter 2004; 155: 287.
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Impact of Prolonged Fasting on the Risk of Calcium Phosphate Precipitation in the Urine: Calcium Phosphate Lithogenesis during Prolonged Fasting in a Healthy Cohort Mohammad A. Shafiee,* Mehdi Aarabi, Pouyan Shaker, Amir M. Ghafarian, Pouyan Chamanian and Mitchell L. Halperin From the Division of General Internal Medicine, Department of Medicine, Toronto General Hospital and Li Ka Shing Knowledge Institute of St. Michael’s Hospital and Division of Nephrology, University of Toronto (MLH), Toronto, Ontario, Canada
Purpose: Intermittent fasting and curtailing water intake for extended periods were likely common in Paleolithic times. Today it occurs for religious and dietary reasons. This restriction in intake should cause a decrease in the urine flow rate while raising the concentration of certain substances in urine to the point of precipitation. In this study we measured the risk of CaHPO4 precipitation following 18 hours of food and water deprivation. Materials and Methods: Urine samples were periodically collected from 15 healthy subjects who fasted and abstained from drinking any liquid for 18 hours. The urine constituents Ca2þ, HPO42e and pH involved in CaHPO4 formation were measured at various times throughout the fasting day. A comparison was made with control data, which consisted of diurnal urine collections taken throughout a separate nonfasting day prior to the fasting day. Results: The mean SEM urine flow rate decreased significantly from 0.93 0.1 ml per minute in the control group to 0.37 0.05 ml per minute in the fasting group (p <0.05). Mean Naþ and Ca2þ excretion rates decreased significantly from 127 12 to 54 13 mmol per minute and from 3.2 0.4 to 0.80 0.21, respectively. Mean urinary Naþ and Ca2þ concentrations also decreased from 161 11.6 to 122 16.0 mmol/l and from 3.7 0.5 to 2.0 0.55, respectively. Urinary pH and the concentration of phosphate, citrate and magnesium were not significantly affected. Conclusions: Although the steady decrease in the urine flow rate was statistically significant during 18 hours of food and water deprivation, there was no evidence that the calculated risk of CaHPO4 precipitation in the healthy subjects had increased. Key Words: kidney calculi, fasting, water deprivation, calcium phosphate, citric acid
GIVEN the recent popularity of intermittent fasting, studies have attempted to investigate the health benefits and risks associated with this behavior.1 Despite studies indicating the positive effects of intermittent fasting2 the decreased fluid intake during fasting are speculated
BP ¼ blood pressure Ksp ¼ solubility product constant PCT ¼ proximal convoluted tubule RAAS ¼ renin-angiotensinogenaldosterone system UFR ¼ urine flow rate WD ¼ water and food deprivation Accepted for publication February 12, 2018. No direct or indirect commercial incentive associated with publishing this article. The corresponding author certifies that, when applicable, a statement(s) has been included in the manuscript documenting institutional review board, ethics committee or ethical review board study approval; principles of Helsinki Declaration were followed in lieu of formal ethics committee approval; institutional animal care and use committee approval; all human subjects provided written informed consent with guarantees of confidentiality; IRB approved protocol number; animal approved project number. * Correspondence: Division of General Internal Medicine, Department of Medicine, University of Toronto, Toronto General Hospital, 200 Elizabeth St., 14 EN-208 Toronto, Ontario, M5G 2C4, Canada (telephone: 416-340-4800, extension 6244; FAX: 416-595-5826; e-mail: [email protected]).
to impact the process of nephrolithiasis. Early humans were likely water and food deprived for extended periods and it would have been devastating to survival if they were affected by kidney stone sequelae.3 Kidney stone disease, which is characterized by severe pain, afflicts
0022-5347/18/2001-0001/0 THE JOURNAL OF UROLOGY® Ó 2018 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH, INC.
Dochead: Adult Urology
Abbreviations and Acronyms
https://doi.org/10.1016/j.juro.2018.02.3092 Vol. 200, 1-6, July 2018 Printed in U.S.A.
www.jurology.com
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1
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2
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Figure 1. In fasting vs control groups changes in UFR, and Naþ and Ca2þ urine concentrations were significant (all p <0.05). SEM shows variance.
10% to 15% of the general population with a 0.5% increasing prevalence annually in North America and Europe.4,5 Although urinary lithiasis carries dire health risks, the mechanisms underlying it and the pathology are largely undefined.6 We specifically studied CaHPO4 precipitates for certain reasons. 1) They are the main precursors of calcium oxalate monohydrate nucleation and growth, representing the most common stone type with a growing incidence. 2) They can cause renal failure by plugging Bellini ducts.7,8 Furthermore, CaHPO4 stone formation is associated with 2 measurable lithogenic variables, including ionized Ca2þ and divalent phosphate ions (H2PO4). Changes in the concentration of either divalent ion may have unfortunate consequences on kidney stone formation. Precipitation formation is contingent on the solubility product constant of a compound. The risk of CaHPO4 precipitation formation increases when the mentioned divalent ions exceed the Ksp. Supersaturation is the condition in which a product exceeds its normal Ksp.9 The risk of CaHPO4 precipitation formation decreases with higher levels of urinary citrate and magnesium ions caused by competitive binding and with a decrease in the HPO42e concentration caused by decreasing urinary pH. Dochead: Adult Urology
Prior studies have indicated that a prolonged fasting state leads to lower BP and, thus, to lower renal perfusion as well as lower UFR.9 As shown by the equation, urine flow rate ¼ excreted effective mOsm per minute/urine effective osmolarity in mOsm/l, UFR is determined by the number of effective osmoles in urine and the changes in urine effective osmolality.10 Simply, if the decrease in UFR were the only variable, the impact of lower UFR on supersaturation would be exponential since it would raise Ca2þ and HPO4 activity. If the excretion rates of these ions remain constant, the ionic product CaHPO4 should increase fourfold for each halving of UFR. In this study we assessed the alternative hypothesis that decreased UFR after 18 hours of water deprivation would result in increased renal reabsorption of Naþ and Ca2þ. Therefore, there would be no increased risk of CaHPO4 precipitate formation in normal healthy individuals.
MATERIALS AND METHODS The St. Michael’s Hospital Research Ethics Board approved the study protocols. Written consent was provided by subjects to use their data anonymously. A total of
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Urine phosphate, citrate, pH, Mg and creatinine excretion, and systolic and diastolic blood pressure in control vs fasting groups (significant at p <0.05) Mean SEM Control Urine: HPO42e concentration (mmol/l) Citrate concentration (mmol/l) pH Mg (mmol/l) Creatinine excretion rate (mmol/min) BP (mm Hg): Systolic Diastolic
29.5 3.47 6.2 4.8 9.0 112 72
2.5 1.07 0.3 0.7 0.7
2.4 1.5
Mean SEM Fasting 22.8 3.06 5.7 3.4 8.3 104 70
2.5 0.72 0.2 0.4 1.1
2.7 1.6
p Value 0.09 0.75 0.19 0.11 0.64 0.02 0.41
15 healthy subjects provided urine samples at various time points through 18 hours of voluntary fasting (food and drink) overnight from 6 p.m. to 12:00 p.m. the next day. Each subject recorded the time and the volume of each void and a small sample was refrigerated for later analysis. Volunteers were in good health and did not receive medication during the week prior to the study. The control was a separate diurnal collection from the same subjects at similar time points on a nonfasting day. The rate of creatinine excretion was used to assess the completeness of each urine sample. UFR in each subject was calculated by dividing sample volume in ml by the corresponding time in minutes. The important urinary constituents of CaHPO4 precipitation, including
electrolytes, Naþ and Kþ, were then measured by flame photometry using a FLM-3 Radiometer (London Scientific, London, Ontario, Canada). Cle was measured by electrometric titration using a CMT 10 Cle meter (London Scientific). Osmolality was measured by freezing point depression (Advanced Instruments, Needham Heights, Massachusetts). Urea and creatinine in plasma and urine were measured with a KodakÒ EktachemÔ Analyzer.11 Concentrations of Ca2þ, phosphate and Mg2þ in urine were measured with a Synchron CXÒ5CE Analyzer. Citrate was measured enzymatically using adenosine triphosphate citrate lyase. Urine pH was measured with a glass electrode pH meter (Corning 178 Blood pH Analyzer, Corning Glass Works, Corning, New York). Urine osmolality was measured by freezing point depression (Advanced Instruments).12
Statistical Analysis Results are reported as the mean SEM. Comparisons between groups were done by the paired t-test. Statistical analysis was performed with MicrosoftÒ ExcelÒ with p <0.05 considered statistically significant.
RESULTS
Urinary concentrations and excretion rates of Naþ, Ca2þ, citrate, Mg2þ, HPO42e and creatinine as well as UFR and urinary pH were measured and
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Figure 2. Mean Naþ excretion rate and UFR of samples taken at various times in fasting and control groups in chronological order throughout fasting period with around 4.5 hours between samples. Naþ excretion was measured by flame photometry and calculated. Points 3 and 4 differed significantly (p <0.05). Whiskers indicate SEM.
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Time (hours) Figure 3. Mean Ca2þ excretion rate and UFR of urine samples taken at various times in fasting and control groups in chronological order throughout fasting period with around 4.5 hours between samples. Ca2þ excretion was measured by flame photometry and calculated. Points 3 and 4 differed significantly (p <0.05). Whiskers indicate SEM.
compared between control and fasting data at a comparable time point. The point corresponding to the final sample collection in the fasting day was chosen because it had the lowest UFR. Mean UFR in male and female subjects in the WD group decreased significantly from 0.93 0.1 to 0.37 0.05 ml per minute (p <0.05, fig. 1). Mean urinary creatinine excretion decreased from 9.0 0.7 to 8.3 1.1 mmol per minute but this was not statistically significant (see table). Na Levels Mean Naþ excretion decreased significantly from 127 11.6 to 54 13.0 mmol per minute (fig. 2). The mean urinary Naþ concentration decreased significantly from 160.5 11.6 to 122 16.0 mmol/l (p <0.05, fig. 1). D
Ca2D, Mg2D, Citrate and pH Levels Mean Ca2þ excretion decreased from 3.2 0.4 to 0.80 0.21 mmol per minute (p <0.05, fig. 3) and the mean urinary concentration of Ca2þ decreased significantly from 3.7 0.5 to 2.0 0.55 mmol/l (p <0.05, fig. 1). The small change from 23.5 2.2 to 22.8 2.5 mmol/l in the mean total phosphate urine Dochead: Adult Urology
concentration was not statistically significant (see table). Similarly the mean changes in urine citrate and Mg2þ of 3.4 0.6 to 3.1 0.72 and 3.3 0.7 to 3.3 0.3 mmol/l, respectively, were not significant (see table). Finally, there was no significant change between the control and experimental groups in the mean urine pH change from 6.0 0.1 to 6.0 0.1 (see table). Systolic and Diastolic Blood Pressure Mean systolic blood pressure in the separate fasting group decreased significantly from 112.2 2.4 to 104 2.7 mm Hg (p <0.05). On the other hand, the decrease in mean diastolic blood pressure from 72 1.5 to 70 1.6 mm Hg was not statistically significant (see table).
DISCUSSION The most striking finding in this study is the significant decrease in Ca2þ. This does not correspond to the predicted cause of supersaturation from the increase in data at minimum UFR preceding an 18hour fasting period. As shown, the decreased values
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5
Figure 4. Calcium, phosphate, sodium, citrate and magnesium handling in different parts of nephron tubule demonstrates varying amounts of solute reabsorption throughout healthy nephron. DCT, distal convoluted tubule.
of UFR as well as the Ca2þ and Naþ concentrations were statistically significant, supporting our hypothesis that a UFR decrease after 18 hours of water and food deprivation would result in increased renal reabsorption of Naþ and Ca2þ. Therefore, it seems that fasting did not increase the likelihood of CaHPO4 precipitate formation in normal healthy individuals. The renal mechanisms of Ca2þ reabsorption begin primarily in the PCT and largely depend on reabsorption of Naþ in the PCT. In other words, if there is an increased rate of water and Naþ reabsorption, an increase in Ca2þ reabsorption is expected secondary to the higher luminal concentration of this divalent cation. When urinary pH increases, the HPO42e concentration decreases while that of H2PO4e increases in urine. These changes are greater at higher pH changes, although the total phosphate concentration and urine pH changes were not statistically significant. Inhibitory factors such as Mg2þ and citrate also exist which may change the likelihood of Dochead: Adult Urology
CaHPO4 kidney stone formation. However, these 2 factors did not seem to have an obvious role in preventing CaHPO4 precipitation.10 In the provided urine samples the Mg2þ concentration was higher than that of Ca2þ. This could have been due to difference in handling Mg2þ compared to Ca2þ. Most Mg2þreabsorption occurs in the loop of Henle, which may have a protective role. Only 5% of Mg2þ is reabsorbed in the distal convoluted tubule by specific Mg2þ channels and the remaining 10% is excreted in urine.13 Additionally, magnesium salts do not become supersaturated in the way that calcium salts do because they generally have a higher Ksp in urine.10 Figure 4 shows renal handling of these ions.10 Citrate is the most important chelator of Ca2þ in urine, inhibiting the nucleation and growth of CaHPO4 crystals. This chelation process involves equimolar quantities of Ca2þ and citrate. When the concentration of citrate exceeds that of Ca2þ, the urinary concentration of Ca2þ and its activity will
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be virtually zero. This excretion of Ca2þ and citrate permits the excretion of urinary potential HCO3e without raising urinary pH, thus, maintaining a low HPO42e concentration and minimizing CaHPO4 precipitation. This effect is similar to that of Mg2þ binding to HPO42e.14 As noted in previous studies, prolonged fasting results in lower average blood pressure. This decrease is only significant in systolic pressure but not diastolic pressure (112/72 vs 104/69 mm Hg, p <0.05, see table). The RAAS is known to cause vasoconstriction and increase Naþ reabsorption from tubular fluid back into blood to maintain blood pressure.15 Toward the end of the fasting period (ie in the morning) the RAAS is activated and it might also account for increased reabsorption of Naþ and a lower rate of Naþ excretion.16 Therefore, the net effect is avid reabsorption of Naþ as well as an increase in Ca2þ and water reabsorption due to the concentration differences. Despite the decreased UFR during the 18 hours of fasting we found that
the risk of CaHPO4 precipitation did not increase because of the significant decrease in the urinary excretion rate of Ca2þ and Naþ due to avid reabsorption of these cations. Rapid Naþ reabsorption can be explained by the decrease in the UFR and in perfusion as well as the decrease in BP and in subsequent RAAS activation. Furthermore, the concentration of other ligands (Mg2þ and citrate) remained in the normal range.
CONCLUSIONS Overall in this study we found no evidence that a decrease in UFR would increase the likelihood of supersaturation and kidney stone formation. Future extension of this study in experiments in patients with kidney stones is not possible due to ethical concerns, which proves to be one of the major limitations of this study. However, we recognize the possible variation in results that would have occurred had this experiment been performed in participants with idiopathic hypercalciuria.
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7. Preminger GM: Stones in the urinary tract. In: The Merck Manual of Medical Information, Second Home Edition. Edited by MH Beers, AJ Fletcher and TV Jones. New Jersey: Pocket Books 2004; vol. 1, chap 1, p 148.
overnight calcium phosphate precipitationdan observational study to determine factors against calcium phosphate lithogenesis in a healthy cohort. Nephron 2016; 132: 238.
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13. Muallem S and Moe OW: When EGF is offside, magnesium is wasted. J Clin Invest 2007; 117: 2086.
9. Samad F, Qazi F, Pervaiz MB et al: Effects of Ramadan fasting on blood pressure in normotensive males. J Ayub Med Coll Abbottabad 2015; 27: 338.
14. Schlieper G, Westenfeld R, Brandenburg V et al: Inhibitors of calcification in blood and urine. Semin Dial 2007; 20: 113.
10. Halperin ML, Kamel KS and Oh MS: Mechanisms to concentrate the urine: an opinion. Curr Opin Nephrol Hypertens 2008; 174: 416. 11. Cheema-Dhadli S and Halperin ML: Relative rates of appearance of nitrogen and sulphur: implications for postprandial synthesis of proteins. Can J Physiol Pharmacol 1993; 71: 120. 12. Shafiee MA, Logan AG and Halperin ML: How protective mechanisms interact to prevent
15. Atlas SA: The renin-angiotensin aldosterone system: pathophysiological role and pharmacologic inhibition. J Manag Care Pharm 2007; 13: 9. 16. Cugini P and Lucia P: Circadian rhythm of the renin-angiotensin-aldosterone system: a summary of our research studies. Clin Ter 2004; 155: 287.
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