ÚSTAV LÉKAŘSKÉ BIOCH
EMIE
A LABORATORNÍ DIAGNO
STIKY
1. LF UK
Non
-
protein nitrogen
compounds
General Medicine
Lenka Fialová & Martin Vejražka
edited and in part translated by Jan Pláteník
2020/2021
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Understanding the Link between Serum Creatinine and Kidney Function, Study notes of Medicine
An in-depth analysis of the relationship between serum creatinine, glomerular filtration, and creatinine excretion. It covers the properties of creatinine and uric acid, reference values for their concentrations in serum and urine, and the estimation of creatinine clearance and uric acid concentration. The document also discusses the impact of various factors on the excretion of these compounds and their clinical significance.
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ÚSTAV LÉKAŘSKÉ BIOCHEMIE A LABORATORNÍ DIAGNOSTIKY 1. LF UK
Non-protein nitrogen
compounds
General Medicine
Lenka Fialová & Martin Vejražka
edited and in part translated by Jan Pláteník
1 Non-protein compounds of nitrogen
Blood serum contains compounds of nitrogen other than proteins and peptides. Urea, creatinine, uric acid, ammonia and amino acids are the most important of them and have implications in clinical biochemistry. These nitrogen compounds remain dissolved even after precipitating the serum proteins with deproteinizing agents. Metabolism of some nitrogen compounds is tightly connected.
Major low-molecular weight compounds of nitrogen (acc. to Burtis et al. 1994):
Low-molecular weight nitrogen compound
Source Clinical and biochemical significance
Amino acids Proteins
- Liver disease
- Renal disease
- Inborn errors of amino-acid metabolism
Ammonia Amino acids
- Liver disease
- Renal disease
- Inborn errors of enzymes of the urea cycle Urea Ammonia •^ Liver disease
- Renal disease Creatinine Creatine •^ Renal disease
Uric acid Purine nucleotides •^ Purine metabolism disorders
- Excessive cell lysis
Fig. 1: Relationships among some nitrogen compounds
2 Creatinine
Creatinine (inner anhydride of creatine) is formed in muscles by irreversible non-enzymatic dehydration and cleavage of phosphate from creatine phosphate, which serves in muscle as a source of energy for muscle contraction (Fig. 2).
Rate of creatinine production in the body is relatively constant. It reflects amount of muscle mass and under condition of physical rest and meat-free diet is stable. Creatinine is excreted in the kidney by glomerular filtration; an additional secretion by renal tubules becomes significant only at elevated serum creatinine levels.
Proteins Amino acids Ammonia Urea
proteolysis
transamination oxidative deamination
urea cycle
Reference values (S-Creatinine): Women: 44 – 104 μ mol/l Men: 44 – 110 μ mol/l
2.2 Creatinine in urine
When compared to other endogenous substances, creatinine excretion into urine is rather constant during the day; in persons with normal glomerular filtration it reflects amount and activity of muscles. Concentration of creatinine in urine can be used as a check of 24-hour urine collection used for measurements of daily output of some substances into urine. Incorrect or incomplete urine collection often makes these measurements faulty; and simultaneous estimation of creatinine output provides a simple way to check it. Creatinine is estimated in a sample of urine from the 24-hour collection. The result in μmol/kg/day is compared with the reference values for urinary creatinine output in dependence on age and gender:
Age Creatinine Output ( μ mol/kg of body weight/day) Men Women 20 – 29 210 ± 20 174 ± 34 30 – 39 194 ± 13 180 ± 34 40 – 49 174 ± 28 156 ± 34 50 – 59 171 ± 26 132 ± 32 60 – 69 149 ± 26 114 ± 23 70 – 79 (^126) ± 26 104 ± 19 80 – 89 103 ± 35 95 ± 22 90 – 99 (^83) ± 28 74 ± 12 If output of creatinine is 30 % or more below the tabular value, it is almost certain the collection of urine was incomplete. Next, urinary creatinine is used for standardisation of urinary output of other substances if information on the 24-hour volume of urine is entirely missing. Concentration of the substance of interest is expressed per 1 mmol of creatinine. Reference values: Concentration of creatinine in urine (U-Creatinine): 3.0 – 12.0 mmol/l Daily output of creatinine into urine (dU-Creatinine): 8.8 – 13.3 mmol/24 hours
2.3 Properties of creatinine and demonstration of creatinine in urine
Creatinine is well soluble in water. Its aqueous solution is weakly alkaline. Creatinine can be proved in a sample using the Jaffé reaction and the reaction with sodium nitroprusside.
A. Reaction with sodium nitroprusside:
In alkaline medium, creatinine reacts with sodium nitroprusside and red color results. The test is not very specific; it is actually identical to the Legal test for acetone and acetoacetate in urine. Differentiation is possible by subsequent acidification of the mixture with concentrated acetic acid: color due to presence of creatinine disappears while the one due to presence of ketone bodies becomes even more intensive (the Legal test). If, instead, the color changes to blue or bluish-green, it indicates excretion of indolic melanogens (Thormählen test; occurs in patients with malignant melanoma).
Reaction with nitroprusside Color Alkaline pH After acidification Creatinine red disappears Acetone and acetoacetate red deepens to red-violet Indolic melanogens red blue to bluish-green
B. Jaffé reaction (see below).
2.4 Estimation of creatinine by Jaffé reaction
A simple, albeit not quite specific Jaffé reaction is based on the reaction of creatinine with salts of picric acid in alkaline medium. Electrophilic oxogroup of creatinine allows dissociation of proton from a methylene group. Creatinine anion is then conjugated with positively polarized carbon of picrate ion and a red-orange complex is formed (Fig. 4). The test is used for estimation of creatinine both in serum and urine. In addition to creatinine, other compounds of body fluids, such as pyruvate, acetate, oxaloacetate, glucose, ascorbic acid, and acetone react with picrate as well – so called Jaffé-positive chromogens. The values of “true” creatinine in serum are normally 9 – 18 μmol/l lower than those determined by this method.
Fig.4: The Jaffé reaction
At the end of the collection period a blood sample for estimation of serum creatinine is taken as well. Information on the patient’s body height and weight accompanies the samples to the laboratory.
Formula for calculation of clearance of endogenous creatinine:
U: concentration of creatinine in urine (mmol/l) V: volume of urine in ml/s P: concentration of creatinine in serum (mmol/l)
Values for clearance obtained in this way are hardly comparable between different people even if reference values are determined – clearance depends on the surface area of glomerular membrane. This surface area is different in every person; it is however assumed that the filtration area is proportional to the body surface. Hence, the value of clearance is corrected to the so-called ideal body surface, which is 1.73 m^2. The body surface of the examined patient is obtained from patient’s body height and weight; it is either found in tables, or calculated according to formula:
0.167: empirical factor m: patient’s body weight in kg l: patient’s body height in meters
Calculation of the corrected clearance of creatinine is following:
2.5.2 Estimation of creatinine clearance from serum creatinine
Clearance of endogenous creatinine can also be estimated from serum concentration of creatinine without any collection of urine using the Cockroft & Gault formula , which takes into account some factors known to affect glomerular filtration, such as age, gender, and body weight (related to muscle mass).
Estimation of ClCr for men:
Estimation of ClCr for women:
U × V
ClCr (ml/s) = P
_______
A = 0.167 × √√√√ m × l
ClCr corr. (ml/s) = Clkr × Patient’s body surface in m^2
(140 −−−− Age [years]) × Weight [kg] ClCr (ml/s) = 44.5 × Serum creatinine ( μ mol/l)
(140 −−−− Age [years]) × Weight [kg] ClCr (ml/s) = 0.85 × 44.5 × Serum creatinine ( μ mol/l)
Recently, this estimation has been replaced by a more reliable calculation, called MDRD formula. It was suggested by Levey et al. in 1999. MDRD is an empirical formula that was derived from data of a large, multicentric study on the effect of diet in renal disease (Modification of Diet in Renal Disease – MDRD).
General formula is
GF = 2.83 × (0.0113 × serum creatinine)-0.999^ × age-0.176^ × (2.8 × serum urea)-0.17^ × (0.1 × serum albumin)0.
For women, the result is to be multiplied by 0.762.
Results of this estimation are in good agreement with measured values especially in patients with decreased glomerular filtration. None of these calculations is suitable for people with normal or just moderately decreased renal function.
Reference values for ClCr (ml/s):
The glomerular filtration decreases with age:
Age (years): 13 – 49 50 – 59 60 – 69 70 and more Women: 1.58 – 2.67 1.0 – 2.1 0.90 – 1.80 0.8 – 1. Men: 1.63 – 2.60^ 1.2 – 2.4^ 1.05 – 1.95^ 0.7 – 1.
The ideal clearance of creatinine with respect to the age can be also found using the equation: Cl = − 0.00946 × age [years] + 2.118. The patient’s value should be within ± 30 %.
2.6 Estimation of glomerular filtration using serum concentration of cystatin C
Cystatin C is a protein composed of 120 amino acids. Many tissues produce cystatin C at various rates. This protein is one of the most important inhibitors of extracellular cystein proteases. Rate of its synthesis is almost constant and unaffected by inflammation, catabolism or diet. Cystatin C is due to its relatively low molecular weight (approx. 13,000) freely filtered through glomerular membrane. Then, it is fully reabsorbed and degraded in proximal tubules. Thus, plasma concentration of cystatin C is a good measure of glomerular filtration, while its urinary concentration correlates with impairment of tubular function. Concentration of cystatin C can be measured by immunochemical techniques. Reference values differ according to the analytical method used; however, introduction of international standardized calibration is expected. Estimation of cystatin C provides several considerable advantages: it is sensitive for early stage glomerular defect; there is no need for 24-hour urine collection that is frequently faulty; and its measurement is not influenced by non-specific reactions as for creatinine. Although this estimation is rather expensive and still reserved for research it is believed that cystatin C will broaden the repertoire of commonly used examinations of renal functions in the near future.
Fractional excretion of water is calculated as
After replacing GF with formula for creatinine clearance and cancelling we get simply:
Reference values for FEH2O: 0.01 – 0.02, i.e. 1 – 2 %.
Elevated values are found in:
- diabetes insipidus
- excessive intake of fluids
- damage to tubular kidney cells
2.8 Tubular reabsorption of water
Knowing the clearance of endogenous creatinine and amount of urine per one second, the value of tubular reabsorption (TR) of water can be obtained. The difference between glomerular filtration and volume of definitive urine per time unit (second) corresponds with the volume of water reabsorbed in renal tubuli per second.
V volume of definitive urine excreted per 1 s
Normal value of TRH2O: 0.988 – 0.
Decreased values indicate disorder of tubular reabsorption of water, e.g. in diabetes insipidus.
V
FEH2O =
GF
Pcreatinine FEH2O = Ucreatinine
ClCr −−−− V TRH2O = ClCr
3 Urea
In quantitative terms, urea is the most significant degradation product of protein and amino acids in the body. It is produced in the liver from ammonia released by deamination reactions from amino acids. Urea diffuses freely through cell membranes and so its concentrations in plasma and intracellular fluid are equal. Urea is excreted from the body mainly in the kidney by a combination of glomerular filtration and tubular reabsorption. The latter is variable: lower at higher diuresis while increasing when diuresis is low. Concentration of urea in the blood depends on amount of protein in the diet, excretion by the kidney, and metabolic function of the liver. For instance, serum urea can increase due to high protein intake in the food. One gram of protein (dietary or endogenous) can give rise to 5.74 mmol (0.34 g) of urea. Increased concentration of serum urea without changes in other non-protein nitrogen compounds (esp. creatinine) is a hallmark of an intense protein catabolism , which occurs e.g. in starvation, febrile state or malignancy. Children have lower catabolism of protein, and show demonstrably lower serum urea levels. Serum urea increases in diseases of the kidney that lead to marked restriction of glomerular filtration (below 30 %); simultaneously, high levels of creatinine are found. Unlike creatinine, estimation of urea is not suitable for an early detection of decrease in glomerular filtration. Sensitivity of the urea estimation for renal insufficience is low: it starts to exceed the reference values when more than 75 % of glomerular filtration is lost. On the other hand, urea is a sensitive parameter of renal hypoperfusion – in this case not only glomerular filtration is lowered but also tubular reabsorption of urea is increased. Serum concentration of urea therefore rises much more quickly than that of creatinine. In renal failure of the pre- renal type (e.g. in hypoperfusion, most frequently in dehydration) the ratio of serum concentrations of urea and creatinine (in μmol/l) is above 160. In liver function failure , synthesis of urea falls down, and so its concentration in the serum. Concentration of urea in serum and urine can also be used for calculation of nitrogen balance. Some causes of changes in serum urea concentration:
Serum urea increased: Serum urea decreased: Impaired kidney function Low-protein diet High-protein diet Impaired liver function High catabolism of protein Late pregnancy (growth of foetus demands protein) Dehydration
Reference values (fS-Urea): Women: 2.0 – 6.7 mmol/l Men: 2.8 – 8.0 mmol/l
4 Uric acid
Uric acid is the end product of purine metabolism in humans (Fig. 5). Purine nucleotides are building blocks of the nucleic acids and other compounds with important roles in metabolism (e.g. ATP, NAD+). Most cells are capable to synthesize purine nucleotides de novo. An important intermediate in purine synthesis is 5-phosphoribosyl-1-diphosphate (PRPP) that is formed in reaction catalyzed by PRPP-synthetase. In the course of the subsequent reactions, the purine core is formed and principal purine nucleotide, inosine monophosphate (IMP) is produced. IMP is then converted to other purine nucleotides, adenosine monophosphate (AMP) and guanosine monophosphate (GMP).
Nucleic acids from own cell nuclei as well as from nutrition are catabolized to nucleotides, nucleosides and bases. Purine bases are finally metabolized by xanthine oxidase to uric acid. Degradation of purines is terminated at this point in humans and primates. Other mammals can further convert uric acid by uricase to allantoin which is better soluble in water. A part of purines is used for re-synthesis of nucleotides by hypoxanthine-guanine phosphoribosyltransferase (HPRT) and adenine phosphoribosyltransferase (APRT) (“salvage pathway”, Fig. 6).
Fig. 5: Uric acid
Total amount of uric acid in the body is approximately 1 g. Uric acid originates from three sources: food nucleotides, degradation of tissue nucleotides and biosynthesis. Uric acid is not only a waste product but it also protects cells from reactive oxygen species.
75 – 80 % of uric acid is eliminated by kidneys (see later). The rest (20 – 25 %) is eliminated by gastrointestinal tract where it can be further degraded to NH 3 and CO 2 by bacterial flora (Fig. 7).
Lactim form (Enol-form)
Lactam form (Oxo-form)
Fig. 6: Degradation of purine nucleotides
Fig. 7: Sources and excretion of uric acid
**1. Uricase reaction
- Oxidative coupling of 4-aminoantipyrine and a phenolic compound**
peroxidase 2 H 2 O 2 + 4-AAP + phenolic comp. 4 H 2 O + colored product
Fig. 9: Estimation of uric acid
4.3 Uric acid in blood serum
Concentration of uric acid in blood depends on intake of purines in food, intensity of its production and on its excretion. Especially increased levels of uric acid are of clinical significance.
Hyperuricemia results from overproduction or decreased excretion of uric acid. Concentration of urates can exceed their solubility in hyperuricemia.
Overproduction of uric acid
- Excessive de novo synthesis of purines associated with hyperuricemia is found in some genetically determined impairments of purine metabolism. Partial or complete defect of hypoxanthine-guanine phosphoribosyltransferase (Lesch- Nyhan’s syndrome) may serve as an example. Re-utilization of purine nucleotides is impaired and purines are therefore degraded to uric acid. Another genetic disorder leading to increased production of uric acid is increased activity of phosphoribosylphosphate synthetase.
- Anticancer therapy (cytostatic chemotherapy, radiotherapy) induces extensive cell lysis and leads to increased formation of uric acid. Purine nucleotides are released from degraded nucleic acids and metabolized to uric acid. Some hematological diseases associated with excessive production (polycythemia vera) or degradation of cells (leukemia, hemolytic anemia) are, in a similar way, joined with hyperuricemia.
- Increased intake of foodstuffs rich on purines (offal, meat, legume, in smaller extent also chocolate, cocoa and coffee) also leads to overproduction of uric acid. Kidneys may fail to compensate the overload with uric acid and uricemia grows up.
- Alcohol increases the level of uric acid. Increased production of lactate inhibits renal secretion of uric acid. The decreased excretion of uric acid is later replaced with high uricosuria.
uric acid allantoin
uricase
Decreased excretion of uric acid
Decreased excretion of uric acid is one of the most frequent causes of hyperuricemia.
- Decreased tubular secretion of uric acid is common in patients with hyperuricemia. The cause of this disorder is unknown.
- Excretion of uric acid is lowered in states associated with decreased glomerular filtration and impaired tubular function.
Reference values (fS-UA): Women 140 – 340 μ mol/l
Men 220 – 420 μ mol/l
4.4 Uric acid in urine
Majority of uric acid (75 – 80 %) is eliminated by kidneys. It is freely filtered in glomeruli (uric acid is only minimally bound to proteins) and then most of it is reabsorbed in the proximal tubuli. Next, uric acid is again secreted in the distal part of proximal tubuli and once again reabsorbed. Approximately 0.6 g of uric acid per day (3.6 mmol/day) is excreted under purine-free diet. Normally, with a common diet, these values are higher – about 0. g/day (5.0 mmol/day, Fig. 10). Tubular secretion of uric acid is inhibited if other organic ions (e.g. acetoacetic acid, β-hydroxybutyric acid, lactate or some drugs) are also excreted to a high extent.
Fig. 10: Excretion of uric acid
Uric acid is a considerable risk factor for both urinary tract and renal parenchyma.
Reference values:
Uric acid excreted per day: 1.5 – 4.5 mmol/day
Clearance of uric acid 0.07 – 0.22 ml/s
Fractional excretion of uric acid: 0.04 – 0.
4.5 Gout
Gout is an important symptom of disorders of uric acid metabolism. Concentration of uric acid is increased in extracellular fluid and in tissues. If the solubility of urates is exceeded the urate crystals precipitate from solution to tissues with scarce blood circulation – e.g. the soft tissues of joints. Inflammatory response is started, resulting in degenerative damage to the joint. Gouty tophus is a small knot-like formation characteristic for chronic gouty arthritis. Crystals of urate are deposited in the centre, surrounded with inflammatory cells and fibrous tissue.
Recurring attacks of acute arthritis are another typical feature of gout. Crystals of sodium urate can be found in leucocytes of synovial fluid during the attack.