View source for Biochemical examination for patient with diabetes mellitus

=== Glycated proteins ===
A direct consequence of elevated blood sugar levels is its intense binding to proteins, which is the essence of '''non-enzymatic glycation'''. The glycation process is conditioned by the presence of free amino groups in the protein molecule.

The rate of glycation is mainly influenced by the concentration of [[glucose]] and its duration of action. It also takes place in healthy individuals. Glycation takes place on all [[serum proteins]] and leads to the formation of glycated derivatives. Both soluble and structural proteins are glycated.
[[File:Protein glycation.png|thumb|Main steps of non-enzymatic glycation of proteins and formation of aldehydes such as glyoxal]]
Non-enzymatic glycation takes place in several phases of the so-called '''Maillard reaction'''.

* The sequence of glycation reactions is initiated by the aldehyde group of the reducing sugar, which binds to the amino group of the protein. A '''labile''' '''aldimine''' of the '''Schiff base''' type (''early glycation products'') is formed. Schiff's formation is quick, reaching equilibrium within hours. This reaction is reversible.
* Schiff's base then slowly, over several days, undergoes chemical rearrangement. A more stable ''glycation transition product'' is formed, the so-called '''Amadori product''', which has the character of a '''ketoamine'''. This produces a keto group on the second carbon of sugar, which is characteristic of [[fructose]]. Concentration resulting '''fructosamines''' can determine responses to [[nitroblue tetrazolium]] (see below). Amadori's products are also '''to some extent reversible''', although the balance is significantly shifted in the direction of their formation. Steady state is reached in a few weeks. The amount of ketoamines can be reduced by normalizing [[Glycemia|blood glucose]].
* Aldose, which is two carbons shorter than the original protein-bound sugar, can also be cleaved from Schiff's base. A di-carbon residue remains attached to the protein, which is further oxidized and reacts with another amino group of the protein. The chain of further reactions results in the formation of highly reactive glyoxal-type dialdehydes.
* Amadori products are reactive substances. Over the months and weeks, irrespective of the presence of glucose, the [[Advanced glycation end-products|advanced glycation end products]] ('''AGEs''') result from the reaction of the Amadori products primarily with long-lived proteins ([[collagen]], [[elastin]], myelin nerve). These processes are '''irreversible'''.

Non-enzymatic glycation in long-term hyperglycemia is one of the causes of tissue damage in some organs in patients with [[diabetes mellitus]].

The determination of Amadori-type glycation products is a suitable indicator of long-term glucose concentration and provides '''indirect information on the course of glycaemia over a period of time that corresponds to the biological half-life of the protein'''. '''Glycated hemoglobin''' and '''glycated proteins''' are routinely determined.

The determination of glycated hemoglobin and glycated proteins is used to control diabetes mellitus and to diagnose persistent hyperglycemia. Elevated glycated derivatives indicate that elevated blood glucose levels have prevailed in the patient in recent weeks and that diabetes mellitus has not been adequately controlled.

==== Glycated hemoglobin ====
[[Glycated hemoglobin|'''Glycated hemoglobin''']] ('''HbA<sub>1c</sub>''') is considered '''the best way to control glucose levels in diabetics over the long term'''. Glycated hemoglobin concentration indicates blood glucose values in the previous 2–3 months (erythrocyte lifespan). It is determined by [[Chromatography|chromatographic]] or [[Immunochemistry|immunochemical]] methods.

'''Evaluation of glycated hemoglobin (HbA<sub>1c</sub>) levels in diabetics'''
{| class="wikitable"
|Compensated DM
|≤ 45 mmol/mol
|-
|The need for a change in therapy
|≥ 53 to 70 mmol/mol
|}
 Glycated hemoglobin concentration is measured with respect  to the total hemoglobin concentration and is expressed in '''mmol HbA<sub>1c</sub>''' '''per mole of total hemoglobin'''. An expression in percent of HbA<sub>1c</sub> in the total hemoglobin (1% ≙ 10 mmol/mol) can also be found. The assessment of glycated hemoglobin concentration in the table above is indicative. Target concentrations vary according to the risk of hyper-/hypoglycemia in a particular patient.<ref>{{Cite
   | type = book
   | surname1 = Karen
   | name1 = Igor
   | surname2 = Svačina
   | name2 = Štěpán
   | title = Diabetes mellitus
   | subtitle = doporučené diagnostické a terapeutické postupy pro všeobecné praktické lékaře 2018
   | edition = -
   | publisher = Společnost všeobecného lékařství ČLS JEP
   | year = 2018
   | isbn = 9788086998992
  }}</ref>
The glycated hemoglobin assay can also be used to screen for diabetes mellitus. For HbA<sub>1c</sub> concentrations above 39 mmol/mol, diabetes mellitus is suspected.<ref>{{Cite
| type = standard
| label= 1
| source_name = Doporučený postup
| title = Doporučený postup péče o diabetes mellitus 2. typu
| corporation = Česká společnost klinické biochemie ČLS JEP,  Česká diabetologická společnost ČLS JEP
| year = 2016
| url = http://www.diab.cz/dokumenty/doporuceni_DM_2015-2.pdf
|cited: 2015-12-29
}}</ref>

If the concentration of  HbA<sub>1c</sub>  is higher than 48 mmol/mol, the diagnosis of diabetes mellitus can be considered confirmed.<ref>{{Cite
| type = standard
| label= 1
| source_name = Doporučený postup
| title = Diabetes mellitus - laboratorní diagnostika a sledování stavu pacientů
| corporation = Česká společnost klinické biochemie ČLS JEP,  Česká diabetologická společnost ČLS JEP
| year = 2015
| url = http://www.cskb.cz/res/file/doporuceni/DM/DM_dop_201601.pdf
|cited: 21-12-29
}}</ref>

'''Evaluation of glycated hemoglobin (HbA<sub>1c</sub>) levels in diabetes mellitus screening'''
::{| class="wikitable"
|-
| Physiological values           || < 39 mmol/mol
|-
| Suspicion of diabetes mellitus || 39–48 mmol/mol
|-
| Diabetes mellitus              || ≥ 48 mmol/mol
|}

==== Fructosamines ====
'''Glycated proteins''' or '''fructosamines''' have a shorter half-life and their level reflects the average glucose concentration for the period of 2–3 weeks before the examination. Their main component is glycated albumin. Hypoproteinemia may falsely reduce the results. Today, determination of fructosamine concentrations is not a routine test in diabetics.
{| class="wikitable"
|+Evaluation of glycated protein concentration 
(S-glycated proteins)
|Physiological values:
|205–285 μmol/l
|-
|Good DM compensation:
|285–320 μmol/l
|-
|Satisfactory DM compensation:
|321–370 μmol/l
|-
|Bad DM compensation:
|above 370 μmol/l
|}

===== Principle of determination of glycated proteins (fructosamine) =====
Examination of glycated proteins uses the reducing properties of fructosamine in an alkaline environment. In the presence of carbonate buffer, fructosamine rearranges to its tautomer - eneaminol, reacts with [[nitrotetrazolium blue (NBT)]]. During the reduction, the heterocyclic rings of NBT are opened and colored formazan is formed. The rate of formazan formation is directly proportional to the fructosamine concentration.

Like any method that utilizes the reducing properties of some serum components, this test is non-specific. It therefore begins with a few minutes of incubation to remove the effect of fast-reacting reducing agents. In commercial kits, the agent includes the enzyme uricase, which eliminates the reduction of NBT by uric acid.