Nonprotein Nitrogen Compounds and Renal Markers

The nonprotein nitrogen fraction is the nitrogen remaining in plasma after proteins are removed. Urea, creatinine, uric acid, and ammonia are its principal components. They arise from different processes and follow different routes of elimination. Urea reflects protein catabolism and renal excretion, creatinine reflects glomerular filtration, uric acid reflects purine turnover and renal urate handling, and ammonia reflects hepatic nitrogen clearance. Each requires the appropriate specimen and reporting units.¹

Urea

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:¹

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 the nitrogen content of urea rather than the whole molecule, 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:¹

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

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

Worked conversion. A urea nitrogen result of 22 mg/dL can be expressed as urea mass by multiplying by 2.14:

22 × 2.14 = 47 mg/dL urea

Converting the same result to SI units gives:

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.¹

Hemolyzed specimens are rejected. Ammonium-containing anticoagulants, such as ammonium heparin, add the ion measured by the method, and high concentrations of sodium citrate or sodium fluoride inhibit urease. Fasting is unnecessary. A common adult plasma or serum reference interval is 6 to 20 mg/dL (2.1 to 7.1 mmol/L), and a 24-hour urine excretes about 12 to 20 g/day. Exam-style intervals are collected in ASCP BOC Examination Reference Ranges.¹

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.¹

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.¹

Worked ratio. A volume-depleted patient has a urea nitrogen result of 54 mg/dL and a creatinine result of 1.5 mg/dL:

54 ÷ 1.5 = 36:1

A ratio of 36:1 exceeds 20:1 and supports a prerenal pattern such as hypovolemia rather than an isolated intrinsic renal process.

Uric acid

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.^1,2

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 instead quantify the hydrogen peroxide byproduct with a peroxidase indicator reaction; bilirubin and other reducing substances interfere there. EDTA and fluoride are incompatible with uricase methods, salicylates and thiazides raise results, and marked hemolysis releases glutathione and lowers values.¹

Worked conversion. Multiplying mg/dL by 0.0595 converts uric acid to mmol/L. A serum urate of 6.6 mg/dL corresponds to:

6.6 × 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.¹

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 retain a normal urate load because of reduced renal excretion, shaped by urate-transporter genetics, purine-rich diet, and alcohol; only a minority truly overproduce urate. The American College of Rheumatology recommends a treat-to-target strategy for urate-lowering therapy with serial serum urate measurement against a target below 6 mg/dL, which keeps plasma undersaturated and lets deposits dissolve.^1,2

Rapid cell turnover during chemotherapy for leukemia, lymphoma, or myeloma can produce a large purine load. Uric acid is monitored during treatment, and the xanthine oxidase inhibitor allopurinol suppresses its production. 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 stones form in acidic urine, and urine alkalinization favors their dissolution.¹

Hypouricemia is uncommon. Its main causes are severe liver disease, Fanconi syndrome with defective proximal tubular reabsorption, and overtreatment with allopurinol or thiopurines.¹

Creatinine

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, but it is a relatively insensitive surrogate. The concentration may not rise measurably until filtration has fallen by more than half.¹

Measurement

The 1886 Jaffe reaction combines creatinine with alkaline picrate to form a red-orange chromogen but 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. Jaffe methods reject hemolyzed and icteric specimens. Bilirubin biases both Jaffe and enzymatic methods downward, and cephalosporins falsely elevate Jaffe results.^1,3

Reference intervals are method-dependent because the Jaffe chromogen includes noncreatinine substances that enzymatic designs exclude.^1,3

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.¹

In muscle disease, including muscular dystrophy, poliomyelitis, hyperthyroidism, and trauma, plasma and urinary creatine may rise while 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.¹

Creatinine clearance and estimated GFR

Because creatinine is produced at a nearly constant rate and cleared essentially only by filtration, creatinine clearance approximates glomerular filtration rate:

clearance (mL/min) = [(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. The plasma specimen is ideally drawn near the collection midpoint. Worked collection, flow, and indexing calculations are in Timed Urine and Renal Clearance Calculations.¹

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

• Cockcroft-Gault is an early equation that predicts creatinine clearance rather than a body-surface-area-indexed GFR. It remains in use for dosing some drugs whose product labeling specifies creatinine clearance.¹
• 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. Race should not enter eGFR computation.⁴
• CKD-EPI 2021 is the race-free adult creatinine equation of the Chronic Kidney Disease Epidemiology Collaboration. The 2021 NKF-ASN Task Force recommended its immediate adoption, and United States laboratories report it widely.^4,5

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.⁵

Worked estimate. For a 60-year-old woman with a standardized serum creatinine of 0.8 mg/dL, 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

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. The laboratory should identify the equation used and report eGFR with the serum creatinine. GFR categories, albuminuria staging, and kidney disease patterns are covered in Renal Physiology, Function Testing, and Disease Patterns.⁶

Ammonia

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.¹

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. A normal result should prompt diagnostic reevaluation, and interpretation requires clinical assessment. Ammonia measurement also supports evaluation of suspected urea cycle disorders, in which hyperammonemia is the defining biochemical abnormality.^1,7

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.¹

Methods

The historic Conway microdiffusion technique exploited 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 rather than NADH 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.^1,8

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 hemolysis disqualifies the specimen. Patients should avoid smoking for several hours before the draw because tobacco smoke is a major contamination source, and each new lot of collection tubes should be verified ammonia-free.^1,8

Intervals are method- and laboratory-specific; one current procedure reports an adult upper limit of 30 µmol/L.^1,8

Worked conversion. Multiplying µg/dL by 0.587 converts plasma ammonia to µmol/L. A result of 48 µg/dL becomes:

48 × 0.587 = 28.2 µmol/L

The converted value remains within the adult interval shown above.

References

1. Bishop ML, Fody EP, Van Siclen C, Mistler JM, Moy M. Clinical Chemistry: Principles, Techniques, and Correlations. 9th ed. Jones & Bartlett Learning; 2023.
2. FitzGerald JD, Dalbeth N, Mikuls T, et al. 2020 American College of Rheumatology guideline for the management of gout. Arthritis Care Res (Hoboken). 2020;72(6):744-760. doi:10.1002/acr.24180.
3. Mayo Clinic Laboratories. Creatinine with Estimated Glomerular Filtration Rate (eGFR), Serum. Accessed August 30, 2026.
4. Delgado C, Baweja M, Crews DC, et al. A unifying approach for GFR estimation: recommendations of the NKF-ASN Task Force on reassessing the inclusion of race in diagnosing kidney disease. Am J Kidney Dis. 2022;79(2):268-288.e1. doi:10.1053/j.ajkd.2021.08.003.
5. Inker LA, Eneanya ND, Coresh J, et al. New creatinine- and cystatin C-based equations to estimate GFR without race. N Engl J Med. 2021;385(19):1737-1749. doi:10.1056/NEJMoa2102953.
6. Kidney Disease: Improving Global Outcomes CKD Work Group. KDIGO 2024 clinical practice guideline for the evaluation and management of chronic kidney disease. Kidney Int. 2024;105(4S):S117-S314. doi:10.1016/j.kint.2023.10.018.
7. Vilstrup H, Amodio P, Bajaj J, et al. Hepatic encephalopathy in chronic liver disease: 2014 practice guideline by the American Association for the Study of Liver Diseases and the European Association for the Study of the Liver. Hepatology. 2014;60(2):715-735. doi:10.1002/hep.27210.
8. Mayo Clinic Laboratories. Ammonia, Plasma. Accessed August 30, 2026.