Non-protein nitrogen substances

In addtion to proteins and peptides, serum contains other important nitrogen-containing substances. From the clinical-biochemical point of view, the most important are urea, creatinine, uric acid, ammonia and amino acids (Table 1). These components remain in solution after precipitation of serum proteins with deproteinizing reagents. The metabolism of some of them is closely related.

Examination of non-protein nitrogenous compounds in the blood and urine is important especially for monitoring the condition of the liver, where a substantial part of the metabolism of these substances takes place, and the kidneys, by which they are preferentially excreted.

Creatinine
Creatinine (cyclic amide or lactam creatine) is formed in the muscles by internal irreversible non-enzymatic dehydration and spontaneous cyclization from creatine and (after phosphate cleavage) from creatine phosphate. Creatine phosphate serves in the muscle as a source of energy for muscle contraction. Creatinine can no longer be phosphorylated and passes into the blood and is later excreted in the urine.

Creatinine is produced at a relatively constant rate in the body. Its formation is a reflection of the size of muscle mass and is stable under conditions of physical calm and meatless diet. It is excreted by the kidneys mainly by glomerular filtration, the renal tubules secrete significant amounts only at elevated blood concentrations.

Methods of determination
A simple but not entirely specific Jaffe reaction is used to determine creatinine. The principle is the reaction of creatinine with picrate in an alkaline environment. The electrophilic oxo group of creatinine allows the dissociation of the methylene group proton. The creatinine anion combines with the positively polarized carbon of the picrate ion to form a red-orange complex. In addition to creatinine, other components of biological fluids also react with picrate - pyruvate, acetate, oxaloacetate, glucose, ascorbic acid, acetone - so-called Jaffé positive chromogens. The normal values ​​of "true" creatinine are 9-18 μmol/l or lower.

Serum creatinine
Serum creatinine concentration is directly proportional to the body's muscle mass. For this reason, it is usually slightly higher in men than in women. In addition, it is affected by renal function, which is used in clinical-biochemical diagnostics.

Serum creatinine is a good indicator of glomerular filtration and is mainly used to monitor the process of kidney disease (including dialysis patients). The relation between creatinine concentration and glomerular filtration is hyperbolic. As glomerular filtration decreases, creatinine excretion decreases. Its serum values ​​begin to rise above the upper limit of normal only when the glomerular filtration rate falls below 50%. From this it is clear that the determination of serum creatinine alone is not very sensitive to the recognition of the early stage of kidney damage. For this purpose, the clearance of endogenous creatinine must be examined (see below). Conversely, with more severe glomerular damage, determination of serum creatinine concentration is a better parameter than creatinine clearance.

Other causes of increased creatininemia are rarer. These include, in particular, the release of creatinine from muscles during acute skeletal muscle breakdown (rhabdomyolysis).

Reference values of serum creatinine

 * Women: 49–90 μmol/l
 * Men: 64-104 μmol/l

Creatinine in urine
Creatinine production in the body is relatively constant. Its urinary excretion is also relatively constant during the day compared to other endogenous substances. In individuals with normal glomerular filtration, it is a reflection of the magnitude of muscle mass activity.

Urine creatinine testing can be used to check the accuracy of a 24-hour urine collection. Improper urine collection is one of the most common errors in the calculation of 24-hour urine losses. One of the easiest ways to verify that the collection is correct is to determine the total amount of creatinine that has been excreted in the urine in one day (creatinine waste). We compare the result with tabular values ​​that indicate creatinine waste in the urine depending on gender, age and weight (Table 2). If the creatinine waste is 30 percent or more lower than the table shows, urine collection can almost certainly be described as incomplete.

Furthermore, the determination of creatinine concentration in urine is used to standardize urinary waste if we have only a single urine sample and collection in 24 hours is not possible or appropriate for any reason. We convert the concentration of the determined substance to 1 mmol of creatinine.

Reference values

 * Urinary creatinine concentration (U-creatinine): 5.7-14.7 mmol/l
 * Urinary creatinine loss in 24 h (dU-creatinine): 8.8-13.3 mmo /24 h

Clearance of endogenous creatinine
By clearance we mean a value that indicates the degree of cleansing of the internal environment by all excretory mechanisms (kidneys, liver). The following relationship applies to the excretion of low molecular weight substances that are freely filtered:

$$ \mathrm{GF} \cdot \mathrm{P} = \mathrm{U} \cdot \mathrm{V}, $$

where U is the urinary concentration of the substance, V is the volume of urine per time unit, GF is the amount of glomerular filtrate and P is the plasma concentration of the substance.

For substances that are excreted in the urine only by glomerular filtration, the amount of substance that passes through the glomerular membrane in a unit of time, is excreted in the urine in the same unit of time. If a quantity of U · V is excreted in the urine per second, then a certain (theoretical) volume of plasma must have been completely "purified" from this substance in the same time. This volume is then called clearance. (=Clearence is a volume of plasma that has been completely purified from a certain substance per unit of time)

By determining the clearance of different substances, we can determine different renal functions. If a substance that enters the urine only by glomerular filtration is used, the clearance value is a measure of glomerular filtration. By using substances that are excreted in the urine from both glomerular filtration and tubular secretion (e.g. para-aminohippuric acid), clearance values ​​can be used to determine renal blood flow.

Substances excreted only by glomerular filtration can become a measure of glomerular filtration. This condition is met by inulin, which freely permeates the glomerular membrane and is not absorbed or secreted in the tubules. Based on inulin clearance measurements, the glomerular filtration rate can be accurately determined. Due to the complexity of the procedure, in which it is necessary to maintain a constant level of inulin in the plasma by continuous intravenous infusion, this method is reserved for research purposes. In routine practice, glomerular filtration is assessed based on endogenous creatinine clearance, which is excreted predominantly by glomerular filtration (about 90%) and its plasma concentration is normally relatively stable. Compared to inulin clearance, creatinine clearance is higher.

Examination of endogenous creatinine clearance is particularly important in patients with less severe renal impairment, in whom glomerular filtration is reduced to 50-80%, i.e. at a time when serum creatinine may not yet exceed the reference limits.

At higher serum creatinine levels (above 180 μmol/l), the proportion of creatinine excreted by tubular secretion increases and examination of endogenous creatinine clearance yields results that would correspond to milder renal impairment. In these cases, determination of serum creatinine is more valuable.

Determination procedure
To calculate the clearance of endogenous creatinine, it is necessary to know the concentration of creatinine in serum and urine and the volume of urine per time unit.

The patient usually collects urine for 24 hours. Urine collection error can be reduced by shortening the collection period to 6 or 12 hours. The patient urinates just before collection (this urine is not yet collected). Fluid intake is not limited during the collection period. Exactly at the time when the collection ends, the examinee urinates into the collection container for the last time. To complete collection, the patient should be instructed to urinate into the collection container before each stool. At the end of the collection, the volume is measured to the nearest 10 ml, the urine is mixed well and a sample is taken in which the creatinine concentration is determined. At the end of the collection period, we also take blood for serum creatinine analysis. At the request for endogenous creatinine clearance, the patient's height and body weight and the exact volume of urine with the length of the collection period should be provided.

Clearance calculation
Endogenous creatinine clearance is calculated according to the formula:

$$\mathrm{Cl}_{\mathrm{kr}}\ (\mathrm{ml/s}) = \frac{\mathrm{U} \cdot \mathrm{V}}{\mathrm{P}},$$

where U is the urinary creatinine concentration in mmol/l, V is the urine volume over time (diuresis) in ml/s, P is the plasma (serum) creatinine concentration in mmol/l.

The clearance values ​​obtained in this way are difficult to compare between different patients and with reference ranges - they depend on the total area of ​​the glomerular membrane, which is different for each pacient. However, the filter surface is assumed to be proportional to the body surface area. Therefore, the clearance value is corrected to the so-called ideal body surface, i.e. 1.73 m2. The value of the examined person's body surface area is found in the tables on the basis of the patient's body weight and height data or can be calculated according to the formula:

$$A = 0,167 \cdot \sqrt{m \cdot l},$$

where 0.167 is the empirical factor (dimension $$\mathrm{kg}^{-\frac{1}{2}}\cdot \mathrm{m}^{\frac{3}{3}}$$), $$m$$ patient weight in kilograms and l height in meters.

The calculation of the corrected creatinine clearance is as follows:

$$\mathrm{Cl}_{\mathrm{cr. correc.}}\ (\mathrm{ml/s}) = \mathrm{Cl}_{\mathrm{cr}} \cdot \frac{1,73}{\textrm{pacient's surface in m}^{2}}$$

1,73 m2 is the standard body surface.

Creatinine clearance estimation according to Cockroft and Gault
Endogenous creatinine clearance can be estimated from serum creatinine concentration without the need to collect urine by calculation using a formula (Cockcroft and Gault), which includes some factors affecting glomerular filtration - age, sex and body weight of the patient as an indirect indicator of muscle mass.

Calculation for men:

$$\mathrm{Cl}_{\mathrm{cr}}\ (\mathrm{ml/s}) = \frac{(140 - \mathrm{age\ [years]}) \cdot \mathrm{weight\ [kg]}}{44,5 \cdot \mathrm{serum}\mathrm{\ creatinine\ [\mu mol/l]}}$$.

Calculation for women:

$$\mathrm{Cl}_{\mathrm{cr}}\ (\mathrm{ml/s}) = 0,85 \cdot \frac{(140 - \mathrm{age\ [years]}) \cdot \mathrm{weight\ [kg]}}{44,5 \cdot \mathrm{serum}\mathrm{\ creatinine\ [\mu mol/l]}}$$.

Estimation of creatinine clearance using the MDRD equation
Recently, the estimation of creatinine clearance according to Cockcroft and Gault has begun to be replaced by a more reliable calculation using the so-called MDRD equation, which was proposed in 1999 by Levey and colleagues. It is an empirical equation based on data large multicenter study investigating the influence of diet on renal disease ( Modification of Diet in Renal Disease - MDRD). The basic equation has the form:

$$\mathrm{Cl}_{\mathrm{cr}} = 2,83 \cdot (0,0113 \cdot \mathrm{serum}\mathrm{\ creatinine})^{-0,999} \cdot \mathrm{age}^{-0,176} \cdot (2,8 \cdot \mathrm{serum}\mathrm{\ urea})^{-0,17} \cdot (0,1 \cdot \mathrm{serum}\mathrm{\ albumin})^{0,318}$$

For women, the value calculated in this way must be multiplied by a factor of 0.762.

The results of this estimation correspond very well to the measured values, especially in patients with reduced glomerular filtration. None of the estimations is appropriate for patients with normal or only slightly reduced renal function.

Physiological values ​​of creatinine clearance
Glomerular filtration decreases with age:

The ideal age-related creatinine clearance can be found according to the equation:

$$\mathrm{Cl}_\mathrm{cr} = -0,00946 \cdot \mathrm{age}\ [\mathrm{years}] + 2,118$$

The patient's clearance should not differ by ± 30%.

Glomerular filtration based on serum level of cystatin C
Cystatin C is a 120 amino acid protein produced by a variety of tissues in different amounts. It serves as one of the most important inhibitors of extracellular cysteine ​​proteases. The rate of synthesis of this protein is practically constant, it is not affected by inflammation, catabolism or diet. Due to its low molecular weight (about 13,000), it is freely filtered through the glomerular membrane. It is then completely resorbed and degraded in the proximal tubules. Thus, plasma cystatin C concentration is a measure of glomerular filtration and urinary concentration is a measure of proximal tubule failure. Cystatin C concentrations can be determined by immunochemical methods. The reference range so far varies according to the specific analytical technique used, but a uniform calibration methodology is expected. The cystatin C assay has some advantages: it detects early stages of glomerular damage, 24-hour urine collection, which is a common source of error, is not required, and non-specific reactions do not distort the analysis (creatinine does). Although this test is relatively expensive and is still reserved for research purposes, it is expected that it will expand the repertoire of commonly used renal function tests in the future.

Fractional excretion
The amount of a substance excreted in the final urine depends on glomerular filtration (i.e. the amount of the substance that enters the primitive urine), tubular secretion and resorption. For simplicity, we limit further interpretation to substances that are not excreted by tubular secretion at all or whose tubular secretion is insignificant.

The proportion of the substance filtered into primitive urine that is eventually excreted in the final urine is referred to as fractional excretion (FE). The FE value of a substance is between 0 and 1 (or we can express it as 0 to 100%); if zero, this means that the substance is completely resorbed in the tubules, if FE is 100%, all filtered substance is excreted in the final urine. The "mirror" quantity to FE is tubular resorption (TR), i.e. the proportion of a substance resorbed from primitive urine by tubular cells. Assuming that tubular secretion is insignificant, the following applies:

$$\mathrm{FE} + \mathrm{TR} = \mathrm{100}\mathrm{\%}$$

The general formula for calculating the fractional excretion is given by the ratio of the clearance of the test substance and the glomerular filtration rate:

$$\mathrm{FE}_x = \frac{\mathrm{U}_x \cdot \mathrm{V}}{\mathrm{P}_x \cdot \mathrm{GF}}$$

Glomerular filtration can be estimated as the clearance of endogenous creatinine. In a fraction, the urine volume per time unit is truncated, so to calculate the fractional excretions, we only need to know the concentration of the substance in the urine and plasma and the concentration of creatinine in the urine and plasma. There is no need to collect urine, which is often burdened with error.

$$\mathrm{FE}_x = \frac{\mathrm{U}_x \cdot \mathrm{P}_{\mathrm{cr}}}{\mathrm{U}_{\mathrm{cr}} \cdot \mathrm{P}_x}$$

($$x$$ is a substance of concern, $$\mathrm{U}$$ is a urinary concentration of the test substance, $$\mathrm{P}$$ is a plasma (serum) concentration of the test substance. Serum and urine concentrations of the test substance as well as creatinine must be in the same units.)

To assess renal function, it is useful to determine the fractional excretions of Na+, K+, Cl-, phosphates and water.

The fractional excretion of water is calculated according to the formula:

$$\mathrm{FE}_{\mathrm{H}_2\mathrm{O}} = \frac{\mathrm{V}}{\mathrm{GF}}$$

After establishing the creatinine clearance for glomerular filtration and canceling out, we get a simple formula:

$$\mathrm{FE}_{\mathrm{H}_2\mathrm{O}} = \frac{\mathrm{P}_{\mathrm{cr}}}{\mathrm{U}_{\mathrm{cr}}}$$

Normal value FEH 2O : 0.01–0.02, i.e. 1–2 %. We encounter increased values ​​in:
 * diabetes insipidus
 * excessive fluid intake
 * kidney tubular cell damage

Tubular water resorption
From the values ​​of endogenous creatinine clearance and the amount of urine excreted in 1 second, we can calculate the value of tubular reverse water resorption (TR). The difference between glomerular filtration and the volume of definitive urine per time unit (s) is equal to the volume of water that is resorbed per second in the renal tubules.

$$\mathrm{TR}_{\mathrm{H}_2\mathrm{O}} = \frac{\mathrm{Cl}_{\mathrm{cr}} - \mathrm{V}}{\mathrm{Cl}_{\mathrm{cr}}}$$

$$\mathrm{V}$$ is the volume of definitive urine in ml excreted in 1 s.

Normal value TRH 2O : 0.988–0.998, i.e. 98.8-99.8%. Decreased values ​​indicate a disorder of water reabsorption, e.g. in diabetes insipidus.

Urea
Urea is the most quantitatively important degradation product of amino acids and proteins. It is formed in the liver from ammonia released by deamination reactions in amino acid metabolism. It diffuses well through cell membranes, so its concentration is the same in both plasma and intracellular fluid.

It is excreted from the body mainly by the kidneys, namely by glomerular filtration and tubular resorption, which is variable (=It is lower with increased diuresis and increases with reduced diuresis).

Blood urea concentration depends on dietary protein content, renal excretion and hepatic metabolic function (Tab.).

Serum urea levels may increase with increased protein intake. 5.74 mmol (0.34 g) of urea are formed from 1 g of protein. Increased urea concentration without changing other low molecular weight nitrogenous substances (especially creatinine) is a sign of intense protein catabolism, which increases during starvation, febrile conditions, malignancy. Protein catabolism is reduced in children, so urea levels are significantly lower. Serum urea concentration increases during kidney disease, which is accompanied by a significant reduction in glomerular filtration (below 30%), while in such cases the creatinine concentration is also increased. The determination of urea is not suitable for detecting incipient glomerular filtration disorders. However, it is important in patients on regular dialysis treatment.

When liver function fails, urea synthesis decreases and thus its plasma concentration decreases.

Based on the urea concentration in serum and urine, a nitrogen balance can be calculated.

Reference values

 * Serum concentration (S-urea): 1,7–8,3 mmol/l
 * Urine losses (dU-urea):


 * 330-600 mmol urea (20-35 g) is excreted in the urine in 24 hours in adults, depending on dietary protein intake and protein catabolism.


 * $$\mathrm{dU}_{\mathrm{urea}}\ [\mathrm{mmol/24\ hours}] = \mathrm{U}_{\mathrm{urea}}\ [\mathrm{mmol/l}] \cdot \mathrm{diuresis}\ [\mathrm{l/24\ hours}]$$

Metody stanovení
Močovina se v biologických tekutinách stanovuje buď přímo nebo nepřímo jako amoniak. Při nepřímém stanovení je močovina nejprve působením enzymu ureázy katalyticky rozštěpena na oxid uhličitý a amoniak, který ve vodném prostředí přechází na amonný ion. Množství vzniklého amoniaku je poté stanoveno reakcí podle Berthelota. Amonný ion s chlornanem sodným a fenolem nebo salicylanem za katalýzy nitroprussidu sodného vytváří barevný produkt.

Doporučená rutinní metoda využívá pro stanovení amonných iontů vzniklých v ureasové reakci přeměnu α-ketoglutarátu na glutamát. Reakce je katalyzována glutamátdehydrogenázou, která je spřažena s oxidací NADH + H+ na NAD+ (Warburgův optický test).

Reakce katalyzovaná ureázou: Močovina + H2O + 2 H+ —→ 2 NH4+ + CO2

Reakce katalyzovaná glutamátdehydrogenázou: 2 NH4+ + 2-oxoglutarát + NADH + H+ —→ L-glutamát + NAD+ + H2O

Stanovení při onemocnění ledvin
Koncentrace urey závisí na její produkci (tj. na příjmu bílkovin stravou, tkáňovém katabolismu a funkci jater). Urea se vylučuje glomerulární filtrací a její sérová koncentrace se proto bude zvyšovat i při selhání ledvin. Jde však o poměrně málo citlivý parametr, ke vzestupu nad horní hranici referenčního rozmezí většinou dochází až při poklesu glomerulární filtrace o více než 75 %.

Naproti tomu je však močovina v séru dobrým indikátorem hypoperfuze ledvin – kromě poklesu glomerulární filtrace se totiž zvýší zpětná resorpce urey v tubulech a její sérová hladina tak roste mnohem rychleji, než např. koncentrace kreatininu. Uvádí se, že při renálním selhání prerenálního typu (např. právě při hypoperfuzi, ledvin, velmi často na podkladě dehydratace) je poměr sérových koncentrací močoviny a kreatininu v μmol/l vyšší než 160.

Dna
Dna je závažným projevem poruchy metabolismu kyseliny močové. Je charakterizována zvýšenou koncentrací kyseliny močové v extracelulárních tekutinách a v různých tkáních. Při překročení rozpustnosti urátů vypadávají jejich krystalky z roztoku a usazují se zejména v málo prokrvených tkáních – např. v měkkých tkáních kloubů. Tam vyvolávají zánětlivou reakci a podmiňují degenerativní změny kloubu. Při chronické dnavé artritidě způsobují uráty vznik tzv. dnavých tofů – uzlíkovitých útvarů obsahujících centrálně uložené krystalky urátu, které jsou obklopené zánětlivými buňkami a fibrózní tkání. Projevem dny jsou opakované ataky akutní artritidy, při níž v leukocytech synoviální tekutiny nalézáme krystalky urátu sodného.

Zdroj
Se souhlasem autorů převzato z https://el.lf1.cuni.cz/p45355481/

Poznámky
