Nonprotein Nitrogen Compounds and Renal Markers

Urea is the most abundant nonprotein nitrogen compound in blood and the liver's major excretory product of protein catabolism. The urea cycle, also called the Krebs-Henseleit cycle, builds it in five enzymatic steps:^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

1. Mitochondrial carbamoyl phosphate synthetase 1 combines ammonium ion, bicarbonate, and 2 ATP into carbamoyl phosphate.
2. Ornithine transcarbamoylase transfers the carbamoyl group to ornithine, forming citrulline, which exits to the cytosol.
3. Argininosuccinate synthetase condenses citrulline with aspartate, consuming one additional ATP.
4. Argininosuccinate lyase cleaves argininosuccinate to arginine and fumarate.
5. Arginase hydrolyzes arginine to urea and ornithine, closing the cycle.

Reporting units and conversions

Historic methods measured only the nitrogen content of urea, which produced the name blood urea nitrogen, or BUN. Modern reporting favors the term urea nitrogen. Two factors convert a urea nitrogen result to other reporting scales:^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

urea (mg/dL) = urea nitrogen (mg/dL) × 2.14

urea (mmol/L) = urea nitrogen (mg/dL) × 0.357

Suppose a urea nitrogen result is 22 mg/dL. Expressed as urea mass:

Urea = 22 mg/dL × 2.14 = 47 mg/dL urea

Converted to SI units:

Urea = 22 × 0.357 = 7.9 mmol/L urea

The three values describe the same specimen on different reporting scales.

Measurement and specimens

Enzymatic methods hydrolyze urea with urease, releasing ammonium ion. The most common coupling uses glutamate dehydrogenase: ammonium ion reacts with 2-oxoglutarate and NADH to form glutamate and NAD⁺, and the analyzer follows the falling absorbance near 340 nm kinetically. Alternatives include a pH-indicator color change in multilayer dry-slide formats and conductometric measurement of the rising conductivity as urea converts to ammonium and carbonate ions.^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

Hemolysis acceptance is method-specific. Mayo's serum urea nitrogen assay, for example, accepts gross hemolysis, so check the assay's own limit before rejecting a hemolyzed urea specimen.^{22 Mayo Clinic Laboratories. Blood Urea Nitrogen (BUN), Serum.} Reject specimens in ammonium-containing anticoagulants, such as ammonium heparin, because they add the ion the method measures. High concentrations of sodium citrate or sodium fluoride also inhibit urease. Fasting is unnecessary. A common adult plasma or serum reference interval is 6 to 20 mg/dL urea nitrogen (2.1 to 7.1 mmol/L urea), and a 24-hour urine excretes about 12 to 20 g/day of urea nitrogen, equivalent to about 26 to 43 g/day of urea. Exam-style intervals are collected in ASCP BOC Examination Reference Ranges.^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

Azotemia and the urea nitrogen to creatinine ratio

Azotemia is a laboratory finding: increased nitrogenous waste in blood, chiefly urea nitrogen and creatinine. Uremia is the clinical syndrome produced by advanced renal failure and retained uremic toxins. The two words are not interchangeable.^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

The urea nitrogen to creatinine ratio, normally about 10:1 to 20:1, helps localize the cause. In prerenal states, slower tubular flow lets more filtered urea reabsorb while creatinine stays relatively stable, so the ratio rises. Renal parenchymal disease raises both analytes roughly in proportion, so the ratio may stay within its usual range despite disease. Postrenal obstruction raises both and tends to increase the ratio.^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

Suppose a volume-depleted patient has a urea nitrogen result of 54 mg/dL and a creatinine result of 1.5 mg/dL:

Urea nitrogen to creatinine ratio = 54 ÷ 1.5 = 36:1

A ratio of 36:1 exceeds 20:1 and supports a prerenal pattern such as hypovolemia. An isolated intrinsic renal process raises both analytes roughly in proportion and keeps the ratio nearer its usual range.

Uric acid is the end product of purine catabolism in humans. Humans cannot oxidize urate to the more soluble allantoin because they lack functional uricase, so purine catabolism ends at urate. Urate forms in the liver, filters freely at the glomerulus, and then undergoes extensive proximal tubular reabsorption, secretion, and postsecretory reabsorption. Only about 5% to 10% of the filtered load reaches the urine. About 70% of elimination is renal, and intestinal bacteria degrade the remainder. Plasma becomes saturated above approximately 6.8 mg/dL, allowing monosodium urate to crystallize in tissue. Acidic urine below pH 5.75 favors the less soluble undissociated acid and stone formation.^{1,31 Bishop ML, et al. Clinical Chemistry. 2023.3 FitzGerald JD, et al. 2020 American College of Rheumatology guideline for the… 2020.}

Measurement

Historic phosphotungstic acid reduction methods, such as the Caraway method, lacked specificity. Current methods use uricase. A direct spectrophotometric design follows the falling absorbance at 293 nm as uric acid converts to allantoin. Hemoglobin and xanthine interfere negatively. Coupled designs quantify the hydrogen peroxide byproduct with a peroxidase indicator reaction, and bilirubin and other reducing substances interfere with them. Anticoagulant acceptance and interference limits depend on the reagent and analyzer. Some methods accept EDTA: the Roche UA2 sheet accepts K2-EDTA plasma, with a matrix-related difference from serum. Apply your laboratory's specimen requirements.^{44 Roche Diagnostics. UA2: Uric Acid ver.2. 2023.} Salicylates and thiazides can alter renal urate handling. Hemolysis and reducing substances can affect the assay response.^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

Multiplying mg/dL by 0.0595 converts uric acid to mmol/L. Suppose a serum urate is 6.6 mg/dL:

Uric acid = 6.6 mg/dL × 0.0595 = 0.39 mmol/L

A result of 6.6 mg/dL (0.39 mmol/L) approaches the saturation concentration of roughly 6.8 mg/dL.^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

Hyperuricemia and hypouricemia

Hyperuricemia underlies gout, which is most common in men 30 to 50 years old. Monosodium urate crystallizes, classically in the first metatarsophalangeal joint, and produces an intensely painful arthritis. Chronic deposits are called tophi. Most patients with gout are hyperuricemic because of reduced renal urate excretion, influenced by urate-transporter genetics, purine-rich diet, and alcohol. Only a minority overproduce urate.^{1414 Menon SG, et al. Gout. 2026.} The American College of Rheumatology uses serum urate below 6 mg/dL as a treatment target, so this value is a clinical decision limit. It is not a population reference limit. The laboratory provides serial measurement, and the clinical service selects and adjusts therapy.^{1,31 Bishop ML, et al. Clinical Chemistry. 2023.3 FitzGerald JD, et al. 2020 American College of Rheumatology guideline for the… 2020.}

Rapid cell turnover during chemotherapy for leukemia, lymphoma, or myeloma can produce a large purine load, so serum urate is monitored with the rest of the tumor-lysis laboratory pattern. Inherited defects can also raise urate dramatically: Lesch-Nyhan syndrome is an X-linked complete deficiency of hypoxanthine-guanine phosphoribosyltransferase with self-mutilation and severe neurologic impairment, and phosphoribosylpyrophosphate synthetase mutations increase purine synthesis. Glycogen storage disease type I and hereditary fructose intolerance cause secondary hyperuricemia through phosphate and ATP depletion with increased purine degradation, and lactic acidosis can further reduce renal urate excretion. Preeclampsia, historically called toxemia of pregnancy, also raises urate. Urate is less soluble in acidic urine, which helps explain a uric-acid stone pattern.^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

Hypouricemia is uncommon. Its main causes are severe liver disease, Fanconi syndrome with defective proximal tubular reabsorption, and overtreatment with allopurinol or thiopurines.^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

Creatine synthesis begins with arginine, glycine, and methionine in the kidney or pancreas and continues in the liver. In muscle, creatine kinase phosphorylates creatine to form creatine phosphate, a rapidly regenerated energy reserve for contraction. Creatine and creatine phosphate convert spontaneously and irreversibly to creatinine, which enters plasma at a rate proportional to muscle mass. The kidney clears creatinine mainly by glomerular filtration, with a small contribution from proximal tubular secretion. Plasma creatinine therefore varies inversely with glomerular filtration rate (GFR) under steady-state conditions. Creatinine may remain within its reference interval despite reduced GFR. A change from the patient's baseline can therefore matter before the result is flagged high. Muscle mass, creatinine generation, and changing kidney function affect interpretation.^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

Measurement

The 1886 Jaffe reaction combines creatinine with alkaline picrate to form a red-orange chromogen. The reaction is nonspecific. Acetoacetate, acetone, ascorbate, glucose, and pyruvate cause a positive bias that worsens above 30 °C. The kinetic Jaffe method measures the rate of color development to reduce much of this interference and remains in routine use. Enzymatic methods convert creatinine to creatine, then to sarcosine, and measure the hydrogen peroxide produced with a peroxidase indicator reaction. They are less affected by acetoacetate and cephalosporins than Jaffe methods, although lidocaine biases some formulations. Isotope dilution mass spectrometry is the accepted reference method, and IDMS-traceable calibration is standard for automated creatinine assays. Hemolysis and icterus limits are specific to each assay. Bilirubin commonly causes negative interference, but positive bias has also been demonstrated in a rate-blanked Jaffe assay. The direction cannot be assumed from the method family. Check the assay's interference limits and use a validated investigation or alternate method. Do not correct a result from the icterus index.^{55 Cheung T, et al. Effects of bilirubin interference in the Roche kinetic alkaline… 2018.} Cephalosporins can falsely elevate Jaffe results.^{1,61 Bishop ML, et al. Clinical Chemistry. 2023.6 Mayo Clinic Laboratories. Creatinine with Estimated Glomerular Filtration Rate (eGFR)…}

Reference intervals are method-dependent because the Jaffe chromogen includes noncreatinine substances that enzymatic designs exclude.^{1,61 Bishop ML, et al. Clinical Chemistry. 2023.6 Mayo Clinic Laboratories. Creatinine with Estimated Glomerular Filtration Rate (eGFR)…}

Adult 24-hour urinary creatinine excretion is about 800 to 2,000 mg/day (7.1 to 17.7 mmol/day) in men and 600 to 1,800 mg/day (5.3 to 15.9 mmol/day) in women.^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

In muscle disease, including muscular dystrophy, poliomyelitis, hyperthyroidism, and trauma, plasma and urinary creatine may rise, and creatinine production and urinary creatinine excretion fall with the loss of muscle mass. Plasma creatinine may remain low or normal. Creatine kinase measurement has largely displaced creatine assays for diagnosing muscle disease.^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

Creatinine clearance estimates filtration. Tubular secretion makes it overestimate the true glomerular filtration rate. Indexing the measured clearance to body surface area gives:

indexed clearance (mL/min/1.73 m²) = [(urine creatinine × urine flow) ÷ plasma creatinine] × (1.73 ÷ body surface area)

Urine flow is the collected volume divided by the collection time in minutes, 1,440 minutes for a 24-hour collection, and the final factor normalizes the result to a standard 1.73 m² body surface area. Draw the plasma specimen near the collection midpoint when possible. Collection, flow, and indexing calculations are in Timed Urine and Renal Clearance Calculations.^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

The kidney handles the four markers differently along the nephron. Urea, creatinine, and urate filter at the glomerulus. The tubule partly reabsorbs urea. The proximal tubule adds a small creatinine secretion and handles urate through reabsorption, secretion, and postsecretory reabsorption. Proximal tubular cells generate ammonium and secrete it into the tubular fluid. The thick ascending limb (TAL) reabsorbs ammonium into the medullary interstitium. The collecting duct secretes ammonia, which combines with hydrogen ion and is trapped for urinary excretion as ammonium. Slow flow increases urea reabsorption while creatinine changes less, raising the urea nitrogen to creatinine ratio. Tubular creatinine secretion helps measured clearance exceed the true GFR.^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

Because timed collections are prone to error, most testing uses an estimated GFR (eGFR) calculated from a standardized serum creatinine:

• Cockcroft-Gault is an early equation that predicts creatinine clearance, without indexing to body surface area. It remains in use for dosing some drugs whose product labeling specifies creatinine clearance.^{11 Bishop ML, et al. Clinical Chemistry. 2023.}
• MDRD, the Modification of Diet in Renal Disease equation, historically used age, sex, race, and serum creatinine. It underestimates GFR at higher values and is no longer the preferred adult equation. Current practice leaves race out of eGFR computation.^{77 Delgado C, et al. A unifying approach for GFR estimation. 2022.}
• CKD-EPI 2021 is the race-free adult creatinine equation of the Chronic Kidney Disease Epidemiology Collaboration. The 2021 National Kidney Foundation and American Society of Nephrology (NKF-ASN) Task Force recommended its immediate adoption, and United States laboratories report it widely.^{7,87 Delgado C, et al. A unifying approach for GFR estimation. 2022.8 Inker LA, et al. New creatinine- and cystatin C-based equations to estimate GFR… 2021.}

eGFR (mL/min/1.73 m²) = 142 × min(S_cr/κ, 1)^α × max(S_cr/κ, 1)^−1.200 × 0.9938^age × 1.012 [if female]

S_cr is the standardized serum creatinine in mg/dL, κ is 0.7 for females and 0.9 for males, and α is −0.241 for females and −0.302 for males.^{88 Inker LA, et al. New creatinine- and cystatin C-based equations to estimate GFR… 2021.}

Suppose a 60-year-old woman has a standardized serum creatinine of 0.8 mg/dL. Then S_cr/κ = 0.8 ÷ 0.7 = 1.14, so the minimum term is 1 and the maximum term is 1.14^−1.200 = 0.852:

eGFR = 142 × 1 × 0.852 × 0.9938⁶⁰ × 1.012 = 142 × 0.852 × 0.689 × 1.012 = 84 mL/min/1.73 m²

Kidney Disease: Improving Global Outcomes (KDIGO) 2024 recommends a creatinine-based eGFR for initial adult assessment. Use the combined creatinine and cystatin C equation when creatinine is likely to be inaccurate or greater precision is needed, and measure GFR when the added accuracy could change a clinical decision. Name the equation on the report, and report eGFR with the serum creatinine. Compare each creatinine and eGFR with the patient's baseline, because creatinine can stay within its reference interval while GFR falls. GFR categories, albuminuria staging, and kidney disease patterns are covered in Renal Physiology, Function Testing, and Disease Patterns.^{99 Kidney Disease: Improving Global Outcomes… KDIGO 2024 clinical practice guideline for the evaluation and… 2024.}

Fractional excretion of sodium (FENa) estimates the percentage of filtered sodium excreted in urine. It compares sodium clearance with creatinine clearance using paired blood and urine results. The urine-flow term cancels, so a timed volume is unnecessary for this ratio.^{1010 Darmon M, et al. Diagnostic performance of fractional excretion of urea in the… 2011.}

FENa (%) = (urine sodium × plasma creatinine) ÷ (plasma sodium × urine creatinine) × 100

The sodium pair must use matching units, as must the creatinine pair. A creatinine result in µmol/L is divided by 88.4 to obtain mg/dL. Check specimen identity, collection times and analytical validity before combining the four results.^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

Suppose paired specimens have plasma sodium 144 mmol/L, urine sodium 72 mmol/L, plasma creatinine 132.6 µmol/L and urine creatinine 30 mg/dL. Plasma creatinine is 132.6 ÷ 88.4 = 1.5 mg/dL.

FENa = (72 × 1.5) ÷ (144 × 30) × 100 = 2.5%

The quotient before percentage scaling is 0.025. Mixing creatinine units would produce a wrong result even though all four individual measurements were valid.

Diuretics increase urinary sodium and can raise FENa without intrinsic kidney injury. Chronic kidney disease and the timing and type of acute injury also limit etiologic interpretation. If these specimens were collected after a loop diuretic, the laboratory can verify the calculation and note that limitation in the interpretation. A value of 2.5% alone cannot establish the cause of kidney dysfunction. Correlate the result with the creatinine trend, urine sediment, medication timing and clinical assessment.^{1111 Abdelhafez M, et al. Diagnostic performance of fractional excretion of sodium for the… 2022.}

Ammonia arises from amino acid deamination, intestinal bacterial activity, and anaerobic muscle metabolism. The liver detoxifies it through the urea cycle, and the kidney excretes it as the urinary buffer ammonium ion. At physiologic pH, nearly all circulating ammonia exists as ammonium ion:

NH₄⁺ →← NH₃ + H⁺

The un-ionized NH₃ form crosses membranes more readily and is highly neurotoxic.^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

Clinical use

Although ammonia contributes to hepatic encephalopathy, the arterial or venous blood concentration alone has no diagnostic, staging, or prognostic value in chronic liver disease. In a patient thought to have hepatic encephalopathy, a normal result calls the working diagnosis into question, and interpretation requires clinical assessment. Ammonia measurement also supports evaluation of suspected urea cycle disorders, in which hyperammonemia is the defining biochemical abnormality.^{1,121 Bishop ML, et al. Clinical Chemistry. 2023.12 Vilstrup H, et al. Hepatic encephalopathy in chronic liver disease. 2014.}

Hyperammonemia in liver disease follows portal-systemic shunting, in which portal blood bypasses the liver, or parenchymal failure, in which urea cycle activity itself is impaired. Disturbed cerebral metabolism, osmotically active intermediates, and impaired astrocyte function produce the neurotoxicity of hepatic encephalopathy.^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

Methods

The historic Conway microdiffusion technique used the volatility of NH₃. The dominant current chemistry-analyzer method couples ammonium ion with 2-oxoglutarate and NADPH through glutamate dehydrogenase and follows the falling absorbance near 340 nm. Procedures use NADPH because endogenous pyruvate and lactate dehydrogenase in the specimen can consume NADH and bias the reaction. Dry-slide photometric methods that measure a bromophenol blue color change and ion-selective electrodes are alternatives.^{1,131 Bishop ML, et al. Clinical Chemistry. 2023.13 Mayo Clinic Laboratories. Ammonia, Plasma.}

Specimen handling

Ammonia rises quickly in collected blood as amino acids deaminate in vitro, so handling determines whether the result is interpretable. Draw without trauma, place the tube on wet ice immediately, centrifuge cold at 0 to 4 °C promptly, and analyze without delay or freeze the separated plasma. One current procedure requires cold centrifugation, separation into a plastic vial kept on ice, and freezing within 2 hours. Erythrocytes carry two to three times the plasma ammonia concentration, so reject a hemolyzed specimen. Ask patients to avoid smoking for several hours before the draw, because tobacco smoke is a major contamination source, and verify each new lot of collection tubes as ammonia-free.^{1,131 Bishop ML, et al. Clinical Chemistry. 2023.13 Mayo Clinic Laboratories. Ammonia, Plasma.}

Intervals are method- and laboratory-specific. One current procedure reports an adult upper limit of 30 µmol/L.^{1,131 Bishop ML, et al. Clinical Chemistry. 2023.13 Mayo Clinic Laboratories. Ammonia, Plasma.}

Multiplying µg/dL by 0.587 converts plasma ammonia to µmol/L. Suppose a result is 48 µg/dL:

Ammonia = 48 µg/dL × 0.587 = 28.2 µmol/L

The converted value remains within the adult interval shown above.