Abstract
Objective
To determine the urolith trace elemental profile and the association of these elements with urolith type and animal variables.
Methods
This was a cross-sectional study of 165 goat and 50 pig uroliths collected from urinary bladders from January 1, 1982, through December 31, 2020. Urolith mineral composition was determined using polarized light microscopy and infrared spectroscopy. Trace element analysis was determined by inductively coupled plasma optical emission spectrometry. The association between animal variables and trace element content was assessed. The strength of grouping effects of the elements within the uroliths was determined by cluster analysis.
Results
Calcium carbonate (74 of 116 [63%]) and apatite (22 of 47 [47%]) were the most commonly identified urolith types in goats and pigs, respectively. The element with the highest concentrations in calcium carbonate uroliths in goats was calcium. Apatite-containing uroliths from pigs had phosphorous as the highest concentration element. Large goat breeds (OR, 1.2; 95% CI, 1.1 to 1.4) and non–pot-bellied pigs had higher odds (OR, 1.6; 95% CI, 1.2 to 2.0) of detection of trace elements. The combination of barium, calcium, sodium, and strontium in goats defined the most significant dissimilarity (divergence) within the calcium carbonate uroliths. In pigs, boron, potassium, sodium, and strontium combination defined the most significant dissimilarity within the apatite uroliths.
Conclusions
Trace elements in combinations that defined significant dissimilarity among uroliths suggest an association between trace elements and urolith type.
Clinical Relevance
Trace and macroelements should be analyzed in uroliths and feed to optimize dietary recommendations for urolithiasis prevention.
Urolithiasis in goats and pigs is a frustrating disease for veterinarians and owners because of the costs associated with management, the complications associated with medical and surgical procedures, the high likelihood of recurrence, and the overall guarded long-term prognosis for survival. The overall survival rate in goats is 39% without surgery and 52% after surgery.1 In goats and pet pigs undergoing temporary tube cystotomy to manage urolithiasis, 55% and 50%, respectively, experience reobstruction.2
Calcium carbonate and amorphous magnesium calcium phosphate are the most frequently identified uroliths in goats3 and pigs,4 respectively. Animal purpose, water intake, and diet have been reported as risk factors for urolithiasis in livestock.3–7 Therefore, the prevention of urolithiasis includes dietary modification and increasing water intake.4–7 Despite owners following all the recommendations for prevention, the anecdotal prevalence of urolithiasis in goats and pet pigs in our clinic practice remains high at 20% to 30%.2 This suggests that other additional factors might be associated with the development of urolithiasis.
Trace elements, such as copper, iron, and zinc, are associated with calcium oxalate urolith formation in humans.8 For example, dietary zinc intake has been independently associated with calcium oxalate urolithiasis in adolescents.9 Trace element analyses in dog food reported that the legal limits of copper, selenium, and zinc were surpassed, which might increase urolithogenesis.10–12 Correlation and cluster analyses of elemental profiles from calcium oxalate uroliths in dogs revealed associations between select trace minerals and hydration form as well as interelemental associations.13,14 These findings in dogs were similar to elemental analyses from uroliths in humans13 at the inorganic level, suggesting a role for trace elements increasing lithogenicity among various species. Goats and pigs might ingest higher concentrations of trace elements in mineral supplements fed ad libitum and treats provided by owners. Therefore, analyzing urolith trace elements might elucidate more insight into the pathophysiology of urolith formation and drive effective novel treatments and prevention of urolithiasis in goats and pigs.
Peer-reviewed studies5 describing the association between urolith type and the presence of trace elements have been reported in cattle but are lacking in goats and pigs. Therefore, our objectives were to (1) determine the trace element content in uroliths from goats and pigs, (2) determine the association between uroliths type and trace elements, and (3) determine the association between the presence of trace elements in uroliths and animal variables, including age, body condition score, diet, and breed.
Methods
Study population
A cross-sectional study of archived samples from the Gerald V. Ling Urinary Stone Analysis Laboratory at the University of California-Davis was performed to identify all urolith submissions from goats and pigs from January 1, 1982, through December 31, 2020. Species and urolith information were obtained from a relational database through questionnaires provided by the submitting veterinarians. If available, the following data were recorded: age, breed, sex, body weight, and diet at the time of urolith formation in goats and pigs. Institutional Animal Care and Use Committee approval was not required to use the urinary stones because removing the uroliths was medically necessary. Urolith sample analysis was part of standard medical care, at which time they became the property of the laboratory and available for this study. Sample size calculation was performed with standard software (Epitools, 2018; Ausvet) assuming a type 1 error (α) of 0.05, sensitivity of 99%, specificity of 99% based on previous studies14 in dogs, a 10% assumed prevalence of trace elements in uroliths, and 95% confidence. A minimum sample of 155 urolith samples from each species was required for analysis. However, only 50 samples were available from pigs.
Compositional type analysis of the uroliths
Mineral analysis of the goat and pig uroliths was determined as previously described.4,14–16 Uroliths were examined for surface and cross-sectional textures under a dissecting microscope. Mineral identity was determined using polarized light microscopy and infrared spectroscopy based on crystallographic features, such as refractive index, birefringence, and crystal shape. The 5-layer categories that were evaluated were as follows: the outer layer, layer 1, layer 2, layer 3, and the core layer. Layer composition was reported as percentage of composition (for instance, outer layer: 99% apatite and 1% struvite). The percentage of each mineral within each layer was estimated by calculating a mean value for the crystal counts obtained by microscopic examination of 5 to 10 microscopic slide preparations using the optical crystallography oil immersion method. The amount of specific minerals in the individual uroliths varied from 1% to 100%. It was impossible to accurately designate a single mineral as predominant in uroliths with distinct layers and variations in layer thickness. Therefore, to maintain consistency in reporting, uroliths composed of a mixture of ≥ 2 minerals were reported as containing that mineral component regardless of whether multiple mineral components were contained in distinct layers or uniformly distributed throughout the urolith.
Trace element analysis of the uroliths
The elemental content of the uroliths was determined by inductively coupled plasma optical emission spectrometry (ICP-OES) as previously described.14 Briefly, for each stone, 2 separate fragments from a single stone sample were randomly selected for elemental analysis. Selected stone samples were weighed and then digested in 0.5 mL 70% nitric acid and incubated overnight at 60 °C with 3 to 4 X g orbital shaking. The acid lysates were then diluted to 5% nitric acid with distilled water, clarified by centrifugation, and introduced via a pneumatic concentric nebulizer using argon carrier gas into a spectrometer (5100 SVDV ICP-OES; Agilent Technologies). The ICP-OES was calibrated using the National Institute of Standards and Technology–traceable elemental standards and reference materials. The trace elements analyzed included aluminum, arsenic, boron, barium, calcium, cadmium, cobalt, chromium, copper, iron, indium, potassium, lithium, magnesium, manganese, molybdenum, sodium, nickel, phosphorous, lead, sulfur, selenium, silicon, strontium, titanium, vanadium, and zinc. The instrument precision for each element was typically between 5% and 10%. Yttrium (5 ppm) was used as an internal standard for all samples. All reagents and plasticware were certified or routinely tested to ensure that they did not contain trace elements that would falsely increase or decrease the trace element concentrations in the samples. Elemental content data were summarized using software (ICP Expert 5800 and 5900; Agilent Technologies) and normalized to the weight of the stone samples.
Statistical analysis
Data were checked for normality using the Shapiro-Wilk test. Mean ± SD were reported for normally distributed data, whereas medians and range were reported for non-normally distributed data. Descriptive statistics, including urolith identification and proportions, trace mineral concentrations in uroliths, age, body condition score, sex, breed, and diet, were recorded when available. Age was categorized as young (≤ 1 year) or mature (> 1 year) for goats and pigs. Body condition scoring was based on a 5-point scale (1, thin; 5, obese) for both species.18,19 Goat breeds were categorized as small (Pygmy and Nigerian Dwarf) or large (Lamancha, Boer, Nubian, Alpine, Toggenburg, Tennessee Fainting, Saanen, or mixed). For goats, the diet was categorized as grass hay only (grass, oat, timothy) or other (mix of grass hay, alfalfa hay, or grains). When available, access to trace mineral supplements was recorded in goats. Pig breeds were categorized as pets (pot-bellied pig, Kunekune, Mini-pig, African Juliana) or production (Yorkshire). In pigs, the diet was categorized as commercial pelleted feed only or other (mix of commercial pelleted feed with vegetables, cereals, or other treats).
When applicable, the association between categorical variables, including age, diet, breed, and body condition score, and presence of trace elements, was determined by the χ2 test or Fisher exact test when a cell in the 2 X 2 frequency table had fewer than 5 counts. The strength of association was determined by calculating ORs and 95% CIs. Odds ratios were considered significant if > 1 or < 1 with a 95% CI excluding 1 and a corresponding P < .05. Association among trace elements was determined by the Pearson correlation (r) when concentrations were normally distributed or Spearman (ρ) correlation when concentrations were not normally distributed. The trace mineral concentrations in the uroliths from goats and pigs were not normally distributed. Therefore, the median, range, and Spearman (ρ) correlation were reported. Hierarchical cluster analysis (no predetermined number of clusters) was used to determine the strength of the grouping effects of the elements within the uroliths. At the beginning of the cluster analysis, each urolith was initially considered a cluster, followed by combining the 2 closest clusters (similar) and so on based on the trace and macroelement profiles. Clusters with divergent element profiles were considered dissimilar. This procedure was performed repeatedly until an optimal number of clusters was determined. The optimal number of clusters was determined when the distances between clusters no longer appeared to be of practical importance. Samples with > 10% of trace elements below the detectable limit were excluded from the cluster analysis. Correlations and cluster analysis were only determined on the most frequent urolith for each species in this study—that is, calcium carbonate for goats and apatite (calcium phosphate) in pigs. Fourteen and 10 elements were available for cluster analysis in goats and pigs, respectively. Statistical analysis was performed with standard software (JMP Pro, version 17; SAS Institute Inc). P < .05 was considered significant.
Results
Goats
One hundred sixty-five urolith specimens were submitted, with 116 completely identified and analyzed for trace mineral elements, whereas 49 uroliths were of insufficient size for complete analysis. All specimens were collected from wethers during tube cystostomy. Eighty-three (83 of 116 [71%)] and 25 (25 of 116 [22%]) uroliths were collected from mature and young goats, respectively. In 8 goats (8 of 116 [7%]), age was not recorded on the submission form. Sixty-three (63 of 116 [54%]), 44 (44 of 116 [38%]), and 3 (3 of 116 [3%]) uroliths were submitted from large, small, and mixed-breed goats, respectively. The breed was not recorded in 6 animals (6 of 116 [5%]). The diet was only recorded from 19 goats with urolith submissions, with 4 (4 of 19 [21%]) fed grass hay only and 15 (15 of 19 [79%]) fed a mix of grass hay, alfalfa hay, and grains. The body condition score was only recorded from 4 goats (4 of 116 [4%]), whereas trace mineral supplementation was recorded in 7 goats (7 of 116 [6%]). The trace mineral supplementation was a commercial supplement formulated for goats (Goat mineral; Manna Pro, Inc) and was similar among the 7 goats. Further analysis of the association between trace elements in uroliths and trace mineral supplementation or body condition score was not performed due to insufficient data on submission forms.
Seventy-four (74 of 116 [63%]), 23 (23 of 116 [20%]), and 9 (9 of 116 [8%]) uroliths from goats were calcium carbonate, struvite/amorphous phosphate, and apatite uroliths, respectively. Ten (10 of 116 [9%]) were considered mixed uroliths (calcium oxalate and silicate, calcium carbonate and silicate, calcium-rich amorphous magnesium carbonate, magnesium phosphate potassium calcium, and trimagnesium citrate nonahydrate). Arsenic, cadmium, cobalt, indium, selenium, titanium, and vanadium were below detectable concentrations in all the uroliths analyzed. Molybdenum was detected in 1 mixed urolith. Nickel was detected in 2 uroliths (1 mixed and 1 apatite). Therefore, arsenic, cadmium, cobalt, indium, selenium, titanium, vanadium, molybdenum, and nickel were excluded from further analysis. The macro and trace elements detected in uroliths from goats are summarized in Table 1. Elements > 1 µg/g in all urolith types included calcium, potassium, magnesium, sodium, phosphorous, sulfur, and strontium. The odds of detecting trace elements in uroliths were higher in large breeds (OR, 1.2; 95% CI, 1.1 to 1.4; P = .006). Age (P = .275) and diet (P = .463) were not significantly associated with detecting trace elements in uroliths. The correlation among macro and trace elements in calcium carbonate–containing uroliths is summarized in Table 2.
Median (range) concentrations (µg/g of urolith) of macroelements and trace elements in calcium carbonate, struvite-amorphous phosphate, apatite, and mixed uroliths collected during cystotomy from January 1, 1982, through December 31, 2020, in 74 goats.
| Trace element | Calcium carbonate | Struvite-amorphous phosphate | Apatite | Mixed |
|---|---|---|---|---|
| Al | 0.09 (0.005–0.169) | 0.021 (0.005–0.138) | 0.024 (0.01–0.043) | 0.028 (0.01–0.24) |
| B | 0.02 (0.004–0.182) | 0.048 (0.01–0.391) | 0.034 (0.01–0.130) | 0.046 (0.01–0.577) |
| Ba | 0.661 (0.125–2.0) | 0.382 (0.01–1.145) | 0.228 (0.092–1.14) | 0.298 (0.01–3.6) |
| Ca | 359. 5 (9.72–492.2) | 15.24 (0.552–137.4) | 16.7 (6.7–226.6) | 122.1 (6.5–261) |
| Cr | 0.003 (0.0–0.003) | 0.003 (0.001–0.004) | 0.002 (0.002–0.003) | 0.043 (0.003–0.083) |
| Cu | 0.001 (0.001–0.029) | 0.001 (0.0003–0.015) | 0.003 (0.001–0.006) | 0.002 (0.001–0.024) |
| Fe | 0.009 (0.001–0.538) | 0.035 (0.004–1.7) | 0.069 (0.009–0.626) | 0.028 (0.005–0.284) |
| K | 4.12 (0.978–35.06) | 21.04 (8.56–86.7) | 20.1 (3.1–38.8) | 2.8 (0.52–35.9) |
| Li | 0.021 (0.003–0.103) | 0.004 (0.0–0.004) | 0.04 (0.007–0.074) | 0.011 (0.005–0.016) |
| Mg | 4.65 (0.168–76.06) | 116.2 (54.7–154.5) | 110.6 (11.8–160.1) | 34.2 (0.32–132.2) |
| Mn | 0.002 (0.001–0.013) | 0.003 (0.0003–0.034) | 0.002 (0.001–0.007) | 0.002 (0.0004–0.009) |
| Na | 4.81 (1.442–34.24) | 12.39 (0.306–120.0) | 11.6 (5.8–29.6) | 1.9 (0.5–22.5) |
| P | 3.69 (1.695–176.2) | 151.0 (54.8–181.3) | 109.2 (2.8–163.5) | 7.6 (1.3–351) |
| Pb | 0.002 (0.001–0.008) | 0.001 (0.0008–0.125) | 0.002 (0.001–0.002) | 0.004 (0.001–0.007) |
| S | 1.05 (0.355–4.195) | 1.835 (0.289–9.59) | 2.6 (0.4–6.2) | 1.3 (0.5–2.4) |
| Si | 0.02 (0.002–0.214) | 0.043 (0.013–0.604) | 0.039 (0.01–0.132) | 0.09 (0.03–0.32) |
| Sr | 1.22 (0.317–2.781) | 0.864 (0.037–3.73) | 0.426 (0.205–2.11) | 0.52 (0.006–4.6) |
| Zn | 0.012 (0.001–0.139) | 0.036 (0.001–0.429) | 0.037 (0.01–0.141) | 0.022 (0.009–0.101) |
Al = Aluminum. B = Boron. Ba = Barium. Ca = Calcium. Cr = Chromium. Cu = Copper. Fe = Iron. K = Potassium. Li = Lithium. Mg = Magnesium. Mn = Manganese. Na = Sodium. P = Phosphorous. Pb = Lead. S = Sulfur. Si = Silicon. Sr = Strontium. Zn = Zinc.
Correlation (Spearman ρ) among macro and trace elements in calcium carbonate uroliths in 74 goats.
| Al | B | Ba | Ca | Cu | Fe | K | Li | Mg | Mn | Na | P | S | Si | Sr | Zn | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Al | 1 | |||||||||||||||
| B | 0.32* | 1 | ||||||||||||||
| Ba | –0.02 | –0.09 | 1 | |||||||||||||
| Ca | –0.35* | –0.42* | –0.05 | 1 | ||||||||||||
| Cu | 0.25 | 0.18 | 0.15 | –0.14 | 1 | |||||||||||
| Fe | 0.36* | 0.13 | 0.16 | –0.51* | 0.10 | 1 | ||||||||||
| K | 0.22 | 0.21 | 0.16 | –0.32* | –0.03 | 0.05 | 1 | |||||||||
| Li | 0.14 | 0.62* | –0.01 | –0.28* | 0.14 | –0.07 | 0.13 | 1 | ||||||||
| Mg | 0.43* | 0.23 | 0.09 | –0.40* | 0.18 | 0.16 | 0.29* | 0.23 | 1 | |||||||
| Mn | 0.43* | 0.09 | 0.30* | –0.23 | 0.35* | 0.14 | 0.24* | 0.20 | 0.29* | 1 | ||||||
| Na | 0.18 | 0.07 | 0.25* | –0.17 | 0.20 | 0.08 | –0.03 | 0.08 | 0.55* | 0.19 | 1 | |||||
| P | 0.26* | 0.29* | 0.11 | –0.29* | 0.23 | 0.19 | 0.21 | 0.06 | 0.33* | 0.22 | 0.30* | 1 | ||||
| S | –0.01 | –0.10 | –0.21 | –0.01 | –0.21 | 0.02 | 0.03 | –0.1 | 0.18 | 0.02 | 0.10 | 0.18 | 1 | |||
| Si | 0.63* | 0.28* | 0.03 | –0.43* | 0.19 | 0.19 | 0.30* | 0.25 | 0.40* | 0.46* | 0.23* | 0.28* | –0.01 | 1 | ||
| Sr | 0.04 | 0.04 | 0.40* | 0.03 | 0.05 | –0.03 | 0.16 | 0.43* | 0.35* | 0.16 | 0.49* | 0.03 | 0.22 | –0.002 | 1 | |
| Zn | 0.55* | 0.15 | 0.08 | –0.57* | 0.17 | 0.44* | 0.28* | –0.01 | 0.55* | 0.30* | 0.43* | 0.27* | 0.16 | 0.41* | 0.20 | 1 |
*Correlations are significant (P < .05).
The dendrogram from the cluster analysis in calcium carbonate–containing uroliths is represented in Supplementary Figure S1, where 3 clusters emerged. The elemental clades (groups) that defined the greatest dissimilarity (divergence) in calcium carbonate uroliths were barium, calcium, sodium, and strontium combination, followed by potassium, phosphorous, and sulfur combination, followed by aluminum, magnesium, and silicon combination, followed by manganese and zinc combination, and then boron and iron combination. Barium and strontium were positively correlated, whereas calcium and sodium were negatively correlated (Table 2).
Pigs
Fifty uroliths were submitted, 47 of which were completely identified and analyzed for trace mineral elements. Three had insufficient volume for complete analysis. All uroliths were collected from barrows during tube cystostomy. Twenty-eight (28 of 47 [60%]) and 10 (10 of 47 [21%]) uroliths were collected from mature and young pigs, respectively. Age was not recorded from 9 pigs (9 of 47 [19%]). Twenty-eight (28 of 47 [60%]), 12 (12 of 47 [26%]), 2 (2 of 47 [4%]), 2 (2 of 47 [4%]), 2 (2 of 47 [4%]), and 1 (1 of 47 [2%]) uroliths were removed and submitted from pot-bellied, mixed, Minipig, African Juliana, Yorkshire, and Kunekune breeds, respectively. The body condition score was recorded in only 1 pig. The diet was reported for 10 pigs, with only 7 (7 of 10 [70%]) fed commercial pelleted feed, 2 (2 of 10 [20%]) fed commercial pelleted feed and vegetables, and 1 (1 of 10 [10%]) fed commercial pelleted feed and treats, such as cereals. No trace element supplement feeding was reported for pigs. Further analysis of the association between the trace elements in the uroliths and body condition score was not performed due to the low number of uroliths available for analysis.
Twenty-two (22 of 47 [47%]), 14 (14 of 47 [30%]), 6 (6 of 47 [12%]), and 5 (5 of 47 [11%]) were apatite, struvite/amorphous phosphate, mixed (calcium oxalate and apatite, amorphous phosphate and struvite, and albumin urolith), and calcium carbonate uroliths, respectively. Arsenic, cadmium, cobalt, indium, selenium, and titanium were below detectable concentrations in all the uroliths. Molybdenum was detected in 6 uroliths (5 apatite and 1 mixed). Nickel was detected in 5 (4 apatite and 1 mixed) uroliths. Vanadium was detected in 2 apatite uroliths. Therefore, arsenic, cadmium, cobalt, indium, selenium, titanium, molybdenum, nickel, and vanadium were excluded from further analysis. The macro and trace elements detected in uroliths are summarized in Table 3. Elements > 1 µg/g in all urolith types included calcium, potassium, magnesium, sodium, and phosphorous. Samples from pot-bellied pigs were overrepresented in our study. Therefore, the non–pot-bellied breeds were combined to determine the association between the detection of trace minerals in urolith and the breed.
Median (range) concentrations (µg/g of urolith) of macro and trace elements in calcium carbonate, struvite-amorphous phosphate, apatite, and mixed uroliths collected during cystotomy from January 1, 1982, through December 31, 2020, in 47 pigs.
| Trace element | Calcium carbonate | Struvite-amorphous phosphate | Apatite | Mixed |
|---|---|---|---|---|
| Al | 0.009 (0.006–0.018) | 0.015 (0.008–0.046) | 0.038 (0.01–0.14) | 0.025 (0.016–0.27) |
| B | 0.015 (0.007–0.023) | 0.024 (0.001–0.104) | 0.046 (0.01–0.15) | 0.043 (0.01–0.06) |
| Ba | 0.013 (0.007–0.024) | 0.01 (0.0004–0.02) | 0.007 (0.003–0.06) | 0.01 (0.005–0.023) |
| Ca | 236.4 (188.4–283.6) | 35.5 (0.28–184.4) | 75.0 (23.1–357.4) | 99.7 (8.1–290) |
| Cr | 0.0 | 0.003 (0.001–0.004) | 0.003 (0.001–0.004) | 0.003 (0.001–0.004) |
| Cu | 0.001 (0.001–0.002) | 0.001 (0.0003–0.009) | 0.001 (0.0006–0.012) | 0.003 (0.001–0.209) |
| Fe | 0.002 (0.001–0.008) | 0.016 (0.004–0.12) | 0.027 (0.011–0.22) | 0.032 (0.02–0.22) |
| K | 2.2 (0.77–3.8) | 12.9 (2.5–54.4) | 7.28 (2.11–36.8) | 4.1 (1.8–25.0) |
| Li | 0.023 (0.006–0.03) | 0.016 (0.0–0.016) | 0.006 (0.004–2.15) | 0.007 (0.005–0.008) |
| Mg | 3.92 (1.74–10.7) | 100.9 (12.2–142.9) | 84.8 (2.8–134.5) | 19.5 (3.0–142.0) |
| Mn | 0.001 (0.0005–0.002) | 0.001 (0.0003–0.002) | 0.001 (0.0007–0.005) | 0.002 (0.0004–0.008) |
| Na | 1.86 (1.72–3.67) | 2.91 (0.36–14.8) | 7.1 (1.02–31.5) | 5.1 (0.94–16.0) |
| P | 2.04 (0.95–2.24) | 149.5 (9.9–186.4) | 152.7 (44.9–190.2) | 303.1 (11.8–348) |
| Pb | 0.002 (0.001–0.002) | 0.002 (0.001–0.01) | 0.011 (0.001–0.061) | 0.01 (0.005–0.013) |
| S | 2.66 (1.94–2.84) | 0.82 (0.07–2.5) | 1.57 (0.23–15.0) | 1.21 (0.73–9.85) |
| S | 0.007 (0.003–0.012) | 0.04 (0.008–0.16) | 0.11 (0.02–0.90) | 0.04 (0.01–3.84) |
| Sr | 0.34 (0.09–0.48) | 0.13 (0.002–2.61) | 0.5 (0.18–1.37) | 0.17 (0.01–0.63) |
| Zn | 0.02 (0.009–0.16) | 0.08 (0.003–0.44) | 0.16 (0.05–1.6) | 0.28 (0.003–1.47) |
The odds for detecting trace minerals in uroliths were higher in non–pot-bellied breeds (OR, 1.6; 95% CI, 1.2 to 2.0; P = .002). Age (P = .63) and diet (P = .741) were not significantly associated with detecting trace elements in uroliths. The correlation between the macro and trace elements in calcium carbonate uroliths is summarized in Table 4. Supplementary Figure S2 represents the dendrogram from the cluster analysis in apatite uroliths, from which 3 clusters emerged. The elemental clades (groups) that defined the greatest dissimilarity in apatite uroliths were the boron, potassium, sodium, and strontium combination, followed by the calcium, phosphorous, silicon, and zinc combination, followed by the iron and sulfur combination. Boron was positively correlated with potassium and strontium.
Correlation (Spearman ρ) among macro and trace elements in apatite uroliths in 22 pigs.
| Al | B | Ba | Ca | Cr | Cu | Fe | K | Li | Mg | Mn | Na | P | Pb | S | Si | Sr | Zn | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Al | 1 | |||||||||||||||||
| B | 0.53* | 1 | ||||||||||||||||
| Ba | 0.51* | 0.40 | 1 | |||||||||||||||
| Ca | –0.33 | –0.28 | –0.14 | 1 | ||||||||||||||
| Cr | –0.22 | 0.25 | –0.29 | 0.51 | 1 | |||||||||||||
| Cu | –0.08 | 0.12 | 0.28 | –0.18 | –0.20 | 1 | ||||||||||||
| Fe | 0.76* | 0.37 | 0.29 | –0.54* | –0.42 | 0.20 | 1 | |||||||||||
| K | 0.49* | 0.75* | 0.32 | –0.59* | 0.01 | 0.17 | 0.67* | 1 | ||||||||||
| Li | 0.59 | –0.20 | 0.12 | –0.26 | –0.59 | 0.15 | 0.61* | –0.06 | 1 | |||||||||
| Mg | 0.38 | 0.47* | 0.26 | –0.84* | –0.31 | 0.14 | 0.48* | 0.67* | –0.02 | 1 | ||||||||
| Mn | 0.53* | 0.29 | 0.06 | –0.58* | –0.13 | –0.16 | 0.37 | 0.51 | 0.09 | 0.71* | 1 | |||||||
| Na | 0.43 | 0.42 | 0.01 | –0.42* | 0.24 | 0.13 | 0.39 | 0.36 | 0.22 | 0.52* | 0.64* | 1 | ||||||
| P | –0.32 | 0.12 | –0.04 | 0.43* | 0.90* | –0.36 | –0.61* | –0.07 | –0.75* | –0.19 | –0.05 | –0.03 | 1 | |||||
| Pb | 0.35 | –0.08 | 0.51 | –0.15 | –0.39 | 0.03 | 0.07 | –0.08 | 0.32 | –0.02 | 0.09 | –0.15 | –0.25 | 1 | ||||
| S | 0.48* | 0.01 | 0.11 | –0.18 | –0.33 | 0.32 | 0.77* | 0.19 | 0.78* | 0.03 | –0.10 | 0.16 | –0.58* | 0.07 | 1 | |||
| Si | –0.10 | 0.28 | –0.01 | 0.34 | 0.82* | 0.09 | –0.19 | –0.03 | –0.08 | –0.27 | –0.37 | –0.26 | 0.41 | –0.30 | –0.04 | 1 | ||
| Sr | 0.49* | 0.55* | 0.67* | –0.05 | 0.00 | 0.08 | 0.33 | 0.39 | –0.44 | 0.32 | 0.24 | 0.07 | 0.08 | 0.13 | –0.01 | –0.03 | 1 | |
| Zn | 0.12 | 0.31 | 0.13 | 0.51* | 0.70* | –0.35 | –0.15 | –0.02 | –0.63* | –0.23 | 0.17 | 0.04 | 0.53* | –0.13 | –0.17 | 0.22 | 0.45* | 1 |
*Correlations are significant (P < .05).
Discussion
Studies demonstrating the presence of trace elements in uroliths removed from goats and pigs are lacking. Our study identified trace elements with variable concentrations in the uroliths submitted from goats and pigs. Furthermore, the trace elements contributed to the divergence among the uroliths, suggesting that these trace elements might contribute to lithogenesis and urolith characteristics. Currently, urolith analysis in goats and pigs only identifies macroelements, such as calcium, magnesium, and phosphorous. Therefore, dietary recommendations focus on modifying the dietary intake of these major macroelements. The association between the macro and trace elements in uroliths reported in our study suggests that trace elements should also be analyzed for optimum dietary recommendations to prevent urolithiasis. In goats, the most abundant element in the calcium carbonate uroliths was calcium, which is expected and suggests higher calcium intake and subsequent hypercalciuria. The next most abundant (> 1 µg/g) elements in calcium carbonate uroliths were sodium, magnesium, potassium, phosphorous, strontium, and sulfur. Sodium, potassium, and magnesium are excreted in the urine of goats in high concentrations, so the presence of these elements is expected.20 Dietary sodium reduction decreased urinary calcium excretion21,22 and the risk of recurrence of calcium-containing uroliths in humans.23 In contrast, other human studies24 reported that dietary sodium supplementation increased water intake and prevented precipitation of calcium oxalates. Although interventional studies are scarce, supplemental dietary sodium is generally recommended in goats to increase water intake.6 In our study, calcium and sodium were negatively correlated, but the correlation was not significant. Therefore, further studies are required to evaluate recommendations for decreasing or increasing sodium intake to prevent urolithiasis in goats.
Decreased calcium oxalate formation was reported in the presence of high magnesium concentrations,25–27 whereas increased incidence of lithogenesis occurred when magnesium levels were low.28 Therefore, magnesium is an antilithogenic. In our study, calcium and magnesium were negatively correlated, which is consistent with studies in humans.25–27 It should be noted that magnesium absorption from the rumen and reticulum is affected by potassium concentrations in the forage and the presence of other bivalent elements, such as zinc, in the rumen.29 Therefore, dietary modification to increase dietary magnesium concentrations in goats is complex. The incidence of uroliths was negatively correlated to dietary potassium intake.30,31 Although anecdotally the role of potassium in lithogenesis has been based on its abundance in urine,13 its specific role remains inconclusive. Phosphorous, sulfates, or sulfur-containing proteins are also present in uroliths20; thus, the presence of these elements was expected.32,33 Although a calcium-to-phosphorous ratio above 2:1 has been recommended to prevent urolithiasis,33,34 these recommendations might only apply to struvite uroliths because high calcium-to-phosphorus-ratio (3.29:1 and 3.89:1) diets resulted in the formation of calcium carbonate uroliths in goats.34 As expected, calcium and phosphorous were negatively correlated in our study. Further studies are necessary to determine the role of phosphorus in calcium-based uroliths. Strontium, which can act as a nidus for calcium-containing uroliths, as well as zinc and iron, are present in uroliths and might influence urolith physical characteristics.13,35–38 Increased sulfate delivery to the kidney increases the risk of urolith formation in humans on a high-animal-protein diet.39 However, sulfate can bind calcium, maintaining it in solution, thereby preventing it from binding to a forming urolith, resulting in an increase in the excretion of sulfate and calcium.40 Therefore, other reports41 suggest that increasing sulfate excretion might reduce the risk of urolithiasis in humans.
The presence of copper, zinc, and selenium concentrations in calcium carbonate–containing uroliths was interesting because these elements are commonly added in supplements provided for goats when pastures are perceived to be deficient. In vitro, zinc is an inhibitor of calcium phosphate mineralization42 and calcium apatite crystals.43 However, at high concentrations, zinc promoted the formation of amorphous calcium phosphate.43 Copper inhibited the crystallization of calcium oxalate in vitro.44 In contrast, other studies45 reported increased copper excretion in humans at higher risk for urolithiasis. In our study, copper and zinc were low, whereas selenium was below detectable levels. Thus, the impact of copper, zinc, and selenium on calcium carbonate lithogenesis is unclear. The combination of barium, calcium, sodium, and strontium defined the most significant dissimilarity within the calcium carbonate–containing uroliths. Furthermore, calcium and sodium concentrations were negatively correlated, whereas barium and strontium were positively correlated. Published studies13 on the role of barium in calcium-based stones are scarce. The relatively high calcium concentrations within calcium carbonate uroliths and their inclusion in the group that defined the most significant dissimilarity suggest that calcium drives the elemental content in calcium carbonate–containing uroliths from goats. Consequently, strategies focused on reducing dietary calcium intake and modifying dietary cationic anionic difference to decrease urine pH have been recommended to prevent calcium-based stones.46 Although the significance of the other elemental grouping within the calcium carbonate uroliths is unknown, magnesium can alter the crystal lattice47,48 of calcium carbonate–containing uroliths, making them difficult to fractionate using laser lithotripsy.49 Further studies are warranted to determine the effect of other elements on the internal microstructure of calcium carbonate uroliths in goats. The higher odds of detecting trace elements in calcium carbonate from larger breeds is unknown. However, large breeds of goats raised as pets are likely retired show animals, breeding goats, or rescued animals previously fed diets with trace elements supplementation. Additionally, large breeds of goats likely share pastures with other farm animals, including cattle and horses that are frequently fed trace mineral supplements. However, the trace mineral content in supplements recommended for cattle or horses have higher trace mineral content than those recommended for goats.
In pigs, apatite-containing uroliths were most frequently noted, in contrast to previous studies4 that reported amorphous calcium magnesium phosphate. This is because in our study, only uroliths collected from cystostomy were analyzed, whereas the previous study4 analyzed samples collected during tube cystotomy or transabdominal catheter placement. Furthermore, amorphous calcium magnesium phosphate can be successfully managed medically by dissolving with buffered acetic acid via a transabdominal catheter. Therefore, pigs diagnosed with amorphous calcium magnesium phosphate may or may not undergo tube cystostomy as successful management of urolithiasis can be achieved with transabdominal catheters, and the decision to perform surgery precedes urolith identification. The most abundant element in the apatite-containing uroliths in pigs was phosphorous. The next most abundant (> 1 µg/g) elements in apatite uroliths were magnesium, calcium, potassium, sodium, and sulfur (Table 3). The relatively high concentrations of calcium and phosphorous in apatite uroliths are expected because apatite contains calcium-phosphate complexes. The reasons for the presence of magnesium, potassium, sodium, and sulfur in apatite uroliths in pigs are similar to calcium carbonate uroliths in goats. In contrast to goats, the association between calcium and sodium was significant and negative. This suggests that decreased sodium intake might reduce calcium excretion and the formation of calcium-based uroliths in pigs. Similar to goats, calcium and magnesium were negatively correlated in pigs.
The combination of boron, potassium, sodium, and strontium defined the most significant dissimilarity within the apatite uroliths despite relatively high phosphorus concentrations. No published reports are available that describe the role of boron in lithogenesis.23 Thus, the driver of the elemental content in porcine apatite uroliths is inconsistent. The diets fed to pigs in our study, primarily owned as pets, were variable, and few clients reported complete dietary histories. Studies50 in commercial pigs reported similar macro and trace element concentrations in feed and water between farms experiencing a higher frequency of calcium carbonate urolithiasis but hypothesized a possible disturbance in calcium and phosphorous absorption and homeostasis. Consequently, the pathophysiology of the drivers for the mineral content in pig urolithiasis remains unclear. Non–pot-bellied pigs had higher odds of detecting trace minerals, most likely because they consisted of several breeds with variable diets.
The results of our study have clinical implications because standard urolith laboratories do not routinely perform trace element analysis. The lack of routine trace element analysis might be due to a lack of awareness of the availability of the tests among livestock practitioners or a lack of access to the laboratories that perform the tests. The cost of trace element analysis in our study was cost-effective and straightforward to analyze. Consequently, trace element analysis should be considered for future clinical research because of the potential impact of trace elements on urolith characteristics and the prevention of urolithiasis. Moreover, understanding the role of trace elements in goats and pigs has implications across other species that form uroliths, such as horses, dogs, cats, and humans. Finding similar trace elemental analyses from uroliths of different species can help elucidate common patterns that might be present among various species. Future research will focus on observational studies assessing the association between trace and macroelement concentration in diets (including trace mineral supplements) and the prevalence of urolithiasis and the effect of dietary intervention strategies on urolithiasis.
Our study has several limitations. We included a convenience sample based on the submissions to our diagnostic laboratory. Therefore, our results have limited external validity because they are from a single veterinary clinic. The uroliths analyzed for pigs were less than the determined sample size. Thus, our data analysis tests were likely underpowered for this species. The signalment and dietary information were limited for both species, and some associations between animal variables and the detection of trace elements were limited. Further studies must include element analysis of the diet fed to the animals, elemental analysis of the uroliths, and fractional excretion of elements. Veterinarians should obtain and report complete dietary histories, including any provided supplements, to help better identify potential predisposing dietary risk factors and hopefully optimize prevention.
Trace elements were detected in variable concentrations in uroliths from goats and pigs. Trace elements in combinations that defined significant dissimilarity among uroliths suggest an association between trace elements and urolith type. Therefore, trace elements might contribute to lithogenesis and urolith characteristics in goats and pigs. Trace and macroelements should be analyzed in goat and pig uroliths and feed to optimize dietary recommendations for urolithiasis prevention.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
The authors thank Kathy Schultz for assistance with the elemental analysis of the samples.
Disclosures
The authors have nothing to disclose. No AI-generated technologies were used in the composition of this manuscript.
Funding
Support was provided by a grant from the University of California-Davis Center for Companion Animal Health.
ORCID
M. Chigerwe https://orcid.org/0000-0001-6841-2448
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