Mechanism of Prominent Trimethylamine Oxide (TMAO) Accumulation in Hemodialysis Patients

<div><p>Large size, protein binding and intracellular sequestration are well known to limit dialytic removal of compounds. In studying the normal renal and dialytic handling of trimethylamine oxide (TMAO), a molecule associated with cardiovascular disease in the general population, we discovered two largely unrecognized additional limitations to sustained reduction of a solute by chronic hemodialysis. We measured solute levels and handling in subjects on chronic hemodialysis (ESRD, n = 7) and compared these with levels and clearance in normal controls (NLS, n = 6). The ESRD patients had much higher peak predialysis plasma levels of TMAO than NLS (77 ± 26 vs 2±1 μM, mean ± SD, p<0.05). For comparison, predialysis BUN levels in ESRD subjects were 45±11 mg/dl and 15±3 mg/dl in NLS. Thus TMAO levels in ESRD average about 40 fold those in NLS while BUN is 3 fold NLS. However, the fractional reduction of TMAO concentration during dialysis, was in fact greater than that of urea (86±3 vs 74±6%, TMAO vs urea, p < 0.05) and its dialytic clearance while somewhat lower than that of urea was comparable to creatinine’s. Also production rates were similar (533±272 vs 606 ± 220 μ moles/day, ESRD vs NLS, p>0.05). However, TMAO has a volume of distribution about one half that of urea. Also in NLS the urinary clearance of TMAO was high (219±78 ml/min) compared to the urinary urea and creatinine clearances (55±14 and 119±21 ml/min, respectively). Thus, TMAO levels achieve multiples of normal much greater than those of urea due mainly to 1) TMAO’s high clearance by the normal kidney relative to urea and 2) its smaller volume of distribution. Modelling suggests that only much more frequent dialysis would be required to lower levels Thus, additional strategies such as reducing production should be explored. Furthermore, using urea as the sole marker of dialysis adequacy may be misleading since a molecule, TMAO, that is dialyzed readily accumulates to much higher multiples of normal with urea based dialysis prescriptions.</p></div

Untargeted mass spectrometry discloses plasma solute levels poorly controlled by hemodialysis

<div><p>Many solutes have been reported to remain at higher plasma levels relative to normal than the standard index solute urea in hemodialysis patients. Untargeted mass spectrometry was employed to compare solute levels in plasma and plasma ultrafiltrate of hemodialysis patients and normal subjects. Quantitative assays were employed to check the accuracy of untargeted results for selected solutes and additional measurements were made in dialysate and urine to estimate solute clearances and production. Comparison of peak areas indicated that many solutes accumulated to high levels in hemodialysis patients, with average peak areas in plasma ultrafiltrate of dialysis patients being more than 100 times greater than those in normals for 123 features. Most of these mass spectrometric features were identified only by their mass values. Untargeted analysis correctly ranked the accumulation of 5 solutes which were quantitatively assayed but tended to overestimate its extent. Mathematical modeling showed that the elevation of plasma levels for these solutes could be accounted for by a low dialytic to native kidney clearance ratio and a high dialytic clearance relative to the volume of the accessible compartment. Numerous solutes accumulate to high levels in hemodialysis patients because dialysis does not replicate the clearance provided by the native kidney. Many of these solutes remain to be chemically identified and their pathogenic potential elucidated.</p></div

Solute plasma levels, clearance rates, and excretion rates for study participants.

<p>Means ± standard deviations</p><p>^† p < .05 Normal vs. ESRD</p><p>^° p< .05 Creatinine and TMAO clearances vs urea clearance within each group</p><p>^‡ p< .05 TMAO vs creatinine clearance</p><p>*Clearances are urinary clearances for normal and dialytic clearances for ESRD.</p><p>**Excretion is 24 hour urinary excretion for normal. For ESRD, removal is the amount removed in dialysate and ultrafiltrate divided by 2 to adjust for the 2 day interdialytic interval.</p><p>*** UN is urea nitrogen.</p><p>Solute plasma levels, clearance rates, and excretion rates for study participants.</p

Kinetic behavior of chemically identified solutes.

<p>Kinetic behavior of chemically identified solutes.</p

Results of quantitative compared to untargeted mass spectrometric analysis.

<p>Results of quantitative compared to untargeted mass spectrometric analysis.</p

Association of Uremic Solutes with Outcomes among 394 Hemodialysis Participants of the CHOICE Study.

<p><i>Abbreviations</i>: HR, Hazard Ratio; CI, Confidence Interval.</p><p>Hazard ratio per 1 standard deviation increase in the solute level modeled using Cox proportional hazards regression.</p><p>¹ Model 1: Crude model without adjustment.</p><p>² Model 2: Minimally adjusted: HR adjusted for demographics (age, sex and race).</p><p>³ Model 3: Fully adjusted: HR adjusted for demographics (age, sex and race), clinical characteristics [body mass index, residual kidney function (self-reported ability to produce >1 cup of urine daily), Index of Coexistent Disease (ICED) score, diabetes and cardiovascular disease] and laboratory tests (Kt/V_UREA, albumin, phosphate and creatinine).</p><p>Note: Mean (Standard Deviation) for the free solutes are: P-cresol sulfate 0.196 (0.128) mg/dL; Indoxyl Sulfate 0.126 (0.100) mg/dL; Hippurate 1.5 (2.1) mg/dL and Phenylacetylglutamine 2.3 (1.7) mg/dL.</p><p>Association of Uremic Solutes with Outcomes among 394 Hemodialysis Participants of the CHOICE Study.</p

Baseline Characteristics of 394 Hemodialysis Participants of the CHOICE Study.

<p>Note: Numbers presented are mean (standard deviation) or percent unless otherwise specified.</p><p>Conversion factors for units: albumin in g/dL to g/L, x 10; calcium in mg/dL to mmol/L, x 0.2495; phosphate in mg/dL to mmol/L, x 0.3229; hemoglobin in g/dL to g/L, x 10; BUN in mg/dL to urea in mmol/L, x 0.357; creatinine in mg/dL to μmol/L, x 88.4; p-cresol sulfate in mg/dL to μmol/L, x 53.1; indoxyl sulfate in mg/dL to μmol/L, x 46.9; hippuric acid in mg/dL to μmol/L, x 55.8; phenylacetylglutamine in mg/dL to μmol/L, x 37.8.</p><p>No conversion is necessary for potassium and bicarbonate in mEq/L to mmol/L.</p><p>Abbreviations: ICED: Index of Coexistent Disease Score; Kt/V: dialysis dose (K-dialyzer clearance of urea, t-dialysis time, V-volume of distribution of urea); CRP: C-Reactive Protein; IL-6: Interleukin 6</p><p>Baseline Characteristics of 394 Hemodialysis Participants of the CHOICE Study.</p

Association of Combined Solute Index Quintiles and Outcomes among 394 Hemodialysis Participants of the CHOICE Study.

<p><i>Abbreviations</i>: HR, Hazard Ratio; CI, Confidence Interval.</p><p>Hazard ratio per 1 standard deviation increase in the combined solute index in the continuous analysis modeled using Cox proportional hazards regression.</p><p>Note: Mean (Standard Deviation) for combined solute index is 5.5 (2.4).</p><p>¹ Model 1: Crude model without adjustment.</p><p>² Model 2: Minimally adjusted: HR adjusted for demographics (age, sex and race).</p><p>³ Model 3: Fully adjusted: HR adjusted for demographics (age, sex and race), clinical characteristics [body mass index, residual kidney function (self-reported ability to produce >1 cup of urine daily), Index of Coexistent Disease (ICED) score, diabetes and cardiovascular disease] and laboratory tests (Kt/V_UREA, albumin, phosphate and creatinine).</p><p>NOTE: Combined solute index is calculated as follows</p><p>¹⁾ Generate standardized value of each solute with a mean of 0 and standard deviation of 1.</p><p>²⁾ For each standardized solute create deciles based on percentiles of the data (range 1–10)</p><p>³⁾ Calculate the combined solute index by averaging the decile category for each participant</p><p>⁴⁾ Generate quintiles of the combined solute index. The lowest quintile is the reference.</p><p>Association of Combined Solute Index Quintiles and Outcomes among 394 Hemodialysis Participants of the CHOICE Study.</p

Correlations between Solutes in 394 Incident Hemodialysis Patients of the CHOICE Study.

<p>Scatterplots demonstrate the association between the solutes. Dots represent to concentrations of the two solutes on scatterplot. Line represents the linear fit between the two solutes. Spearman and Pearson correlation coefficients are also reported in a text box. P-values for all correlations were ≤0.001 for both Spearman and Pearson correlations.</p

Adjusted Relative Hazard of Outcomes in 394 Incident Hemodialysis Patients of the CHOICE Study.

<p><b>Panel 2A:</b> Adjusted hazard of Cardiovascular Mortality. <b>Panel 2B:</b> Adjusted hazard of First Cardiovascular Event. Relative hazard predicted using Cox proportional hazards regression adjusted for demographics (age, sex and race), clinical characteristics [body mass index, residual kidney function (self-reported ability to produce >1 cup of urine daily), Index of Coexistent Disease (ICED) score, diabetes and cardiovascular disease] and laboratory tests (Kt/V_UREA, albumin, phosphate and creatinine). Solutes and combined solute index are modeled as restricted cubic splines with knots at the 10th, 50th, and 90th percentiles. The solid line is the adjusted HR; 10th percentile is used as the reference (HR = 1). The shaded area is the 95% CI of the HR. Bars are the frequency histogram, showing the distribution of each solute and combined solute index. Vertical broken lines mark the 25^th and 75^th percentile of the distribution.</p