Various epidemiological factors such as nutrition, age, sex, socio-economic group, climate, disease conditions, crystallization dynamics and heredity have been mentioned in the aetiology of renal stones. Nutritionally, the contribution of dietary Ca and P in renal stone formation is still debated. Both Ca and P are usually found as renal stone components. Conventionally, dietary Ca limitation used to be recommended to prevent recurrent episodes of renal stones, but later studies have shown that dietary Ca is related to lower risk of Ca oxalate renal stone formation.Reference Curhan, Willett, Rimm and Stampf1 This is so because Ca binds dietary oxalates in the intestine, thereby reducing the quantity of oxalates absorbed and thus reducing stone formation risk, as far as oxalate intake is substantial.
The contribution of dietary P remains unclear. P therapy to avoid stone formation has been reported to not reduce Ca oxalate crystallization in urine.Reference Ettinger2 In addition, studies have reported urine P to affectReference Schwille, Schmiedl and Manoharan3–Reference Williams, Child, Hudson, Soysa, Davies, Davies and De Bolla6 as well as to not affectReference Muezzinoglu, Gumus, Sener and Ari7 the formation of renal stones. Thus, nutritionally the present study identified Ca and P as the key dietary components, which are still debated, either directly as in their dietary intakes or indirectly through their metabolic processes, in the aetiology of renal stone formation.
Materials and methods
Subjects
The present study was carried out in ten stone formers (SF) and ten healthy controls (HC). The SF had been admitted for renal stone(s) removal at the CDR Medical Centre and were asked to join the study on a voluntary basis. Male SF in their second to fifth decade of life were selected, since a male sex bias in middle-age has been reported as a renal stone riskReference Curhan, Willett, Rimm and Stampf1, Reference Taylor and Curhan8. The age and sex matched HC were recruited, again on a voluntarily basis, contacted by local advertisements, from among the employees of the medical centre and the University of Delhi on condition that they never had any episodes of renal stones or any family history of stone formation. The HC thereafter underwent a clinical examination by the attending physician at the medical centre and were diagnosed to have no illnesses, chronic or infectious. All subjects provided written informed consents and the study was approved by the university and the medical centre.
Study design
A questionnaire cum interview schedule was administered to recruit the SF for the study and all their data were collected prior to removal of their renal stones. The HC were recruited using a slightly modified version of the schedule. Compliance was discussed and confirmed verbally with all participants, with the provision of free dietary advice on the successful completion of the dietary, serum and urine data gathering. Each study participant at first collected a 2-d dietary food record. In the case of SF, most of them were on home-food diet, even as inpatients of the centre. The second day of the dietary food record corresponded to the 24-h urinary sample collection. On the morning of the third day, the dietary records and urine samples were delivered to the North Point Laboratories, when also a fasting blood sample was drawn by trained laboratory personnel. After the dietary, serum and urine data gathering were completed, the renal stones, which were removed from the SF, were qualitatively analysed for their contents. All biochemical analyses were carried out at the North Point Laboratories.
Dietary intake data
A 2-d dietary record was administered with verbal pre-instructions on how to record food intake using measuring scales and a set of standardized bowls, glasses and spoons, Reference Raina, Kashyap and Narula9 all recorded in a booklet provided to each subject. Cooked food intakes were converted to corresponding raw portions and the mean intakes of energy, carbohydrate, fats, protein, Ca and P were calculated using the food tables of Nutritive Value of Indian Foods.Reference Gopalan, Ramashastri, Balasubramanium, Rao, Deosthale and Pant10
Urinary data
Urine samples (24 h) of HC were collected in eleven chemically clean, plastic containers, which were provided to the participants. The 24-h urine samples of the SF, who at the time of the study were inpatients of the centre, were collected in urobags, which were pyrogen-free, ethylene oxide (ETO) gas sterilized, fitted with a long tube with a non-return valve. All participants were asked to discard the first morning urine sample, note the time and collect every sample thereafter for the next 24 h until the morning of the following day. All urine samples were analyzed immediately on the day they were delivered to the laboratory.
Serum data
A fasting blood sample was drawn by a venipuncture of the antecubital vein using 5 ml disposable plastic syringes (Careject, Carewell Mediproducts Ltd, New Delhi, India sterile, non-toxic, pyrogen free, ETO sterilized and contamination free) with single-use needles (Luer, Iscon Surgical Ltd, Jodhpur, India 0·80 × 38 mm, sterile, non-toxic and non-pyrogenic). The 5 ml fasting blood aliquot was stored at room temperature for 1 h in a sterile, acid-washed test tube for clot formation. It was then centrifuged for 10 min at 2000 rpm. The serum obtained was analysed immediately.
Biochemical analyses
Serum and urine Ca, P and uric acid were measured colorimetrically using the EDTA method, Reference Trinder11 the molybdate methodReference Gomori12 and the tungsten blue method, Reference Folin and Denis13 respectively. Urine creatinine was measured using the picrate method.Reference Bonses and Taussky14 Renal stones were qualitatively (chemically) analyzed for the presence of Ca, phosphate, oxalate, carbonate, urate, Mg and cystine.Reference Reiner, Cheung, Thomas and MacDonald15–Reference Hodgkinson17
Statistics
Statistical analysis was carried out using means and standard deviations. Variables that were not normally distributed were log-transformed. Comparisons between the two groups (SF, HC) were carried out by the t test. To find out relationships among the dietary intakes, serum levels and urinary excretions of the variables, Pearson's correlations were run. Both the test of difference and the correlations were considered to be statistically significant at P < 0·05.
Results
All the mean dietary intakes reported in Table 1 were significantly higher (P < 0·05) in SF compared with HC. In general, SF had a higher food intake, as shown by their daily total energy (kJ/d) intake values. None of the serum or urine variables was significantly different between the two groups, except urine P (P < 0·05), which was significantly higher in SF. The stone composition results showed that all ten SF had Ca and oxalate in their renal stones. In addition to the Ca and oxalate, seven had phosphate and two had urate in their renal stones.
Table 1 Data characteristics of the stone formers (SF) and healthy control (HC) groups†
(Mean values and standard deviations)
Mean values were significantly different from the HC group; *P < 0·05 using t test of difference.
† For details of subjects and procedures, see Materials and methods.
When diet, serum and urine values for Ca, P and uric acid (dietary uric acid intake indexed with dietary protein) were correlated (Table 2), neither dietary P nor uric acid showed any relation to their serum or urine values. In SF, dietary Ca correlated not only to serum but also to urine Ca, whereas in HC no such correlations were observed. In addition, SF exhibited the often reported dietary protein-related calciuria, whereby the SF had a significantly higher protein intake compared with HC. Urine P was not significantly, though positively, related to dietary protein and P in both groups.
Table 2 Cross-correlation coefficients of dietary protein, phosphorus and calcium with serum and urine uric acid, phosphorus and calcium‡
* Logarithimized in stone formers and healthy controls.
† Logarithimized in stone formers only.
‡ For details of subjects and procedures, see Materials and methods.
In HC, neither urine Ca nor urine P correlated to any of the dietary macronutrient intakes. Likewise, in SF urine P showed no correlation to dietary macronutrient intakes. Only urine Ca in SF correlated positively to total energy intake (0·66, P < 0·05), dietary protein (0·72, P < 0·05), fats (0·80, P < 0·05) and P (0·69, P < 0·05) intakes. However urine Ca in SF showed no correlation to dietary carbohydrate intakes.
Discussion
In the present study, urine P was significantly higher in SF compared with HC and it did not correlate significantly to any dietary macronutrient intakes in both groups. In contrast, urine Ca correlated significantly to dietary intakes of energy, protein and fats (not carbohydrates) in SF only. Finally, only dietary Ca correlated significantly to serum and urine Ca in SF. Thus, a dietary contribution, defined by energy, protein and fats, to the urine Ca excretion in SF could be possible. A dietary contribution to urine Ca excretion in SF has been reported in literature.Reference Burtis, Gay, Insogna, Ellison and Broadus18, Reference Borghi, Meschi, Maggiore and Prati19 In contrast to the present study, these studies have also reported carbohydrates. Elevated urine P in SF, with urine P demonstrating no significant correlations to any of the dietary intakes, probably hints at P metabolism rather than its intake to be in play in renal stone formation. Studies have reported an altered phosphate handling in SF secondary to parathyroid hormone (PTH) disordersReference Williams, Child, Hudson, Soysa, Davies, Davies and De Bolla6, Reference Taguchi, Kuwahara, Kambe, Loritani, Saitoh, Shirai, Orikasa, Mizunashi and Furukawa20 as well as primary changes in renal tubulusReference Wikstrom, Backman, Danielson, Fellstrom, Hellsing, Johansson and Ljunghall4, Reference Wikstrom, Backman, Danielson, Fellstrom, Johansson, Ljunghall and Wide5 leading to phosphate waste.
Of note was that none of the biochemical parameters in both groups in the present study lay outside the normal range of values. Simultaneously, dietary intakes in SF were clearly higher than in HC. Thus, the occurrence of renal stones in one group and none in the other could probably be influenced by hyperphagy. Renal stone formation has been associated with affluence.Reference Borghi, Meschi, Maggiore and Prati19 With affluence, apart from quantitative increases in food consumption, a tendency to replace carbohydrates with protein and fats as a source of energy is observed.Reference Regmi, Deepak, Seale and Bernstein21 There is the added observation that SF tend to be obese, Reference Hassapidou, Paraskevopoulous, Karakoitsides, Petridis and Fotiadou22, Reference Taylor, Stampfer and Curhan23 reflecting prolonged increased food consumption. In the current study, the SF, apart from higher food consumption, in terms of food groups, consumed more meats, milk and milk products. In fact, the HC group was principally vegetarian in food habits. Increased dietary protein consumption leading to urine Ca losses in SF is well documented, Reference Giannini, Nobile, Sartori, Dalle Carbonare, Ciuffreda, Corro, D'Angelo, Calo and Crepaldi24 although not only limited to SF.Reference Cardinale, Leiper, Farajian and Heer25 Thus, the loss of Ca in the urine in SF could be due to high protein consumption.
Urine Ca loss could be also due to idiopathic hypercalciuria.Reference Duncombe, Watts and Peters26 However, the observed normocalciuria in SF probably suggests an absorptive hypercalciuria, which is accompanied by mild to moderate phosphate loss but rarely with renal hypercalciuria.Reference Giannini, Nobile, Sella and Dalle Carbonare27 In patients with absorptive hypercalciuria, bone disease rarely setsReference Giannini, Nobile, Sella and Dalle Carbonare27 in possibly because of the higher Ca absorption, which would protect bone resorption. Ca limitation in such patients could then probably affect their bone health. Thus, the present study supports the thesis of not limiting Ca intake in SF, suggested by other studies as well, Reference Curhan, Willett, Rimm and Stampf1, Reference Taylor and Curhan8 since apart from the known protective function of binding oxalates in the gut, Ca intake in SF with absorptive hypercalciuria would also exert a bone-protective function.
In addition to the afore-mentioned arguments, urine Ca may not be as critical as urine P in starting the process of stone formation. P excretion per se has been related to dietary protein intake.Reference Williams, Stevens, Fass, Irons and Bone28 A role for dietary protein in increasing the risk of renal stone formation has often been mentioned.Reference Borghi, Meschi, Maggiore and Prati19, Reference Robertson, Peacock and Hodgkinson29 Concepts of ‘proportional’ rather than the ‘raw’ concentrationsReference Rodgers, Barbour, Pougnet, Lombard and Ryall30 of, for example, P and/or the amount of P excreted for any given serum CaReference Williams, Child, Hudson, Soysa, Davies, Davies and De Bolla6 have also been mentioned to distinguish stone formation. It appears, however, that urine P rather than dietary PReference Ettinger2 is the parameter to reconsider.
Studies have shownReference Wikstrom, Backman, Danielson, Fellstrom, Hellsing, Johansson and Ljunghall4–Reference Williams, Child, Hudson, Soysa, Davies, Davies and De Bolla6, Reference Taguchi, Kuwahara, Kambe, Loritani, Saitoh, Shirai, Orikasa, Mizunashi and Furukawa20, Reference Schwille, Herrmann, Schmiedl, Kissler, Wipplinger and Manoharan31 that Ca-containing renal stones are related to impaired P excretion. Schwille and colleaguesReference Schwille, Herrmann, Schmiedl, Kissler, Wipplinger and Manoharan31 reported that changes in calcium phosphate and calcium oxalate renal stones' (the predominant stone composition profile of the SF in the present study) supersaturation in urine was unrelated to urine Ca excretion. Calcium phosphate has been reported to form the renal stone nidus Reference Vasin, Rosanova and Sevastianov32 and in animal studies Bushinsky and colleaguesReference Bushinsky, Parker and Asplin33 reported that a decrease in urine P, secondary to a decrease in dietary P, led to a decrease in the urine supersaturation of calcium phosphate. Thus, although Ca forms an important component of renal stones, the trigger that conduces the stone precipitation could lie in urine P. This could possibly also explain why dietary protein restriction is often recommended for SF.Reference Taylor and Curhan8, Reference Borghi, Meschi, Maggiore and Prati19 Urine P, apart from reflecting dietary P (as shown in animal studiesReference Bushinsky, Parker and Asplin33), possibly also reflects the proton load of the dietary proteins excreted in the urine as phosphates.
The other mechanisms, in addition to dietary proteins, whereby urine P could contribute to stone formation could be due to the action of PTHReference Williams, Child, Hudson, Soysa, Davies, Davies and De Bolla6, Reference Taguchi, Kuwahara, Kambe, Loritani, Saitoh, Shirai, Orikasa, Mizunashi and Furukawa20 and/or changes in the renal tubular structure.Reference Wikstrom, Backman, Danielson, Fellstrom, Hellsing, Johansson and Ljunghall4, Reference Wikstrom, Backman, Danielson, Fellstrom, Johansson, Ljunghall and Wide5 Regarding the latter, a contribution of lipids through the alteration in the phospholipid structure of intestinalReference Duncombe, Watts and Peters26 and renal tubular cellsReference Schwille, Herrmann, Schmiedl, Kissler, Wipplinger and Manoharan31 has been proposed to lead to stone formation by increasing Ca absorption in the gutReference Duncombe, Watts and Peters26 and phosphate loss (independent of PTH) in the renal tubulus, Reference Schwille, Herrmann, Schmiedl, Kissler, Wipplinger and Manoharan31 respectively. The present study's SF had a high consumption of fats. If high or altered fat consumption, or even higher food intake per se (ultimately leading to obesityReference Hassapidou, Paraskevopoulous, Karakoitsides, Petridis and Fotiadou22, Reference Taylor, Stampfer and Curhan23), can be related to changes in membrane phospholipid structure affecting Ca and P absorptions and excretions (secondary to metabolic abnormalities or even in their absence) it warrants further studies. In addition to the dietary food habits and the higher urine P excretion by the present study's SF compared with HC, stone formation would be exacerbated by any existing metabolic abnormalities and/or genetic proclivity; the measurements of which lay outside the scope of the current study. What is known is that, geographically, North India is counted as a region prone to stone formation, Reference Walsh, Retik and Stamey34 with Delhi falling in the stone-forming belt of North India.
The present study was planned to investigate Ca and P in SF. Given the afore-mentioned arguments, post priori, this study would have benefited vastly from additional measurements of classical urine parameters, such as pH, oxalate, sulphate and citrate and also serum PTH and vitamin D. This study remains limited in the lack of such measurements. Further, the study sample size of ten in each group would limit extrapolations for the general population.
Taken together, the present study found higher food consumption and higher phosphate excretion in SF. The study suggests urine P rather than urine Ca to be probably at work in the formation of renal stones. Limitation of dietary protein with normal Ca intakes could provide a suitable measure to avoid renal stone formation for the study's SF.
Acknowledgements
The laboratory analyses were supported by the North Point Laboratories as a grant. The authors report no conflict of interest, financial or otherwise.
