Non-protein nitrogen substances

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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.

Table 1 . Major low molecular weight nitrogen compounds (according to Curtis et al. 1994)

Low molecular weight nitrogen substance	Source	Clinical-biochemical significance
Amino acids	proteins	liver disease kidney disease hereditary disorders of amino acid metabolism
Ammonia	amino acids	liver disease kidney disease hereditary disorders of ureosynthetic cycle enzymes
Urea	ammonia	liver disease kidney disease
Creatinine	creatine	kidney disease
Uric acid	purine nucleotides	disorders of purine metabolism increased cell lysis

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.

Table 2. Urine creatinine reference values ​​depending on age and sex in μmol/kg/day.

Age	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±36	95±22
90–99	83±28	74±12

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:

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

1,73 m² is the standard body surface.

Clearance estimation

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:

Physiological values ​​of Cl_cr [ml/s]

Age	13–49	50–59	60–69	70 and more
Women	1,58–2,67	1,0–2,1	0,9–1,8	0,8–1,3
Men	1,63–2,6	1,2–2,4	1,05–1,95	0,7–1,0

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%.

Glomerulární filtrace na podkladě sérové hladiny cystatinu C

Cystatin C je bílkovina o 120 aminokyselinách, kterou v různé míře produkuje řada tkání. Slouží jako jeden z nejvýznamnějších inhibitorů extracelulárních cysteinových proteáz. Rychlost syntézy této bílkoviny je prakticky konstantní, není ovlivněna zánětem, katabolismem ani dietou. Vzhledem k malé molekulové hmotnosti (asi 13 000) se volně filtruje glomerulární membránou. V proximálních tubulech se následně kompletně resorbuje a degraduje. Plazmatická koncentrace cystatinu C je tedy mírou glomerulární filtrace a koncentrace v moči mírou poruchy proximálních tubulů. Koncentraci cystatinu C lze stanovit imunochemickými metodami. Referenční rozmezí se zatím liší podle konkrétně použité analytické techniky, očekává se však zavedení jednotné metodiky kalibrace. Stanovení cystatinu C má některé výhody: dobře detekuje časné fáze glomerulárního poškození, není nutný 24hodinový sběr moči, který je častým zdrojem chyb, a analýzu nezkreslují nespecifické reakce jako u kreatininu. Přestože je toto vyšetření poměrně drahé a dosud je vyhrazeno spíše pro výzkumné účely, předpokládá se, že v budoucnu rozšíří repertoár běžně používaných vyšetření renálních funkcí.

Frakční exkrece

Množství určité látky vyloučené do definitivní moči závisí jednak na glomerulární filtraci (tj. na množství látky, které se dostane do primitivní moči), jednak na tubulární sekreci a resorpci. Pro jednoduchost omezíme další výklad na látky, které se vůbec nevylučují tubulární sekrecí, nebo jejichž tubulární sekrece je zanedbatelná.

Podíl látky přefiltrované do primitivní moči, který se nakonec vyloučí definitivní močí, se označuje jako frakční exkrece (FE). Hodnota FE určité látky se pohybuje mezi 0 a 1 (nebo ji můžeme vyjádřit jako 0 až 100 %); je-li nulová, znamená to, že se látka zcela resorbuje v tubulech, je-li naopak FE 100 %, vyloučí se veškerá přefiltrovaná látka do definitivní moči. „Zrcadlovou“ veličinou k FE je tubulární resorpce (TR), tj. podíl látky resorbované z primitivní moči tubulárními buňkami. Za výše uvedeného předpokladu, že je tubulární sekrece zanedbatelná, platí:

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

Obecný vzorec pro výpočet frakční exkrece je dán podílem clearance sledované látky a glomerulární filtrace:

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

Glomerulární filtraci můžeme odhadnout jako clearanci endogenního kreatininu. Ve zlomku se po dosazení objem moči za časovou jednotku vykrátí, a tak pro výpočet frakčních exkrecí potřebujeme znát pouze koncentraci látky v moči a plazmě a koncentraci kreatininu v moči a plazmě. Odpadá nutnost sběru moči, který bývá zatížen chybou.

$\mathrm {FE} _{x}={\frac {\mathrm {U} _{x}\cdot \mathrm {P} _{\mathrm {kr} }}{\mathrm {U} _{\mathrm {kr} }\cdot \mathrm {P} _{x}}}$

($x$ je sledovaná látka, $\mathrm {U}$ koncentrace sledované látky v moči, $\mathrm {P}$koncentrace sledované látky v plazmě (séru). Koncentrace sledované látky v séru i v moči, stejně tak i kreatininu musí být ve stejných jednotkách.)

Pro posouzení renálních funkcí je užitečné stanovovat frakční exkrece Na⁺, K⁺, Cl^-, fosfátů a vody.

Frakční exkrece vody se vypočítá podle vzorce:

$\mathrm {FE} _{\mathrm {H} _{2}\mathrm {O} }={\frac {\mathrm {V} }{\mathrm {GF} }}$

Po dosazení clearance kreatininu za glomerulární filtraci a vykrácení dostaneme jednoduchý vzorec:

$\mathrm {FE} _{\mathrm {H} _{2}\mathrm {O} }={\frac {\mathrm {P} _{\mathrm {kr} }}{\mathrm {U} _{\mathrm {kr} }}}$

Normální hodnota FE_H2O: 0,01–0,02, t.j. 1–2 %. Se zvýšenými hodnotami se setkáváme u:

• diabetes insipidus,
• nadměrného přívodu tekutin,
• poškození tubulárních buněk ledvin.

Tubulární resorpce vody

Z hodnot clearance endogenního kreatininu a množství moči vyloučené za 1 sekundu můžeme vypočítat hodnotu zpětné tubulární resorpce vody (TR). Rozdíl mezi glomerulární filtrací a objemem definitivní moči za časovou jednotku (s) je rovný objemu vody, který je za sekundu resorbován v ledvinných tubulech.

$\mathrm {TR} _{\mathrm {H} _{2}\mathrm {O} }={\frac {\mathrm {Cl} _{\mathrm {kr} }-\mathrm {V} }{\mathrm {Cl} _{\mathrm {kr} }}}$

$\mathrm {V}$ je objem definitivní moči v ml vyloučený za 1 s.

Normální hodnota TR_H2O: 0,988–0,998 Snížené hodnoty svědčí o poruše zpětné resorpce vody např. u diabetes insipidus.

Močovina

__ 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.).

Chemical formula of urea

Urea cycle

Some causes of changes in serum and urine urea levels

Elevated serum urea	Decreased serum urea
renal impairment	liver damage
high protein diet	low protein diet
increased protein catabolism	late pregnancy (increased need for protein during fetal growth)
dehydration

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 their urea levels are significantly lower. Serum urea concentration increases in 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 urea test 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.

References

Urea - WikiSkripta

Kyselina močová

__ Kyselina močová

Dna

Iron

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.

Odkazy

Zdroj

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

Použitá literatura

Kategorie:Biochemie