Scientific journal
European Journal of Natural History
ISSN 2073-4972

UREA AND a- KETOGLUTARAT SPONTANEOUSLY FORM DEHYDROHYDANTOIN-5-PROPIONIC ACID IN VITRO

Kozlov V.A., Mat´kov K.G., Lyschikov A.N.
By development of solutions for organs preservation we have found out, that solution EWS‑1 (electrolyte water stabilization) does not prevent slices swelling in the test 24 hour incubations at +4°. Nevertheless, after addition 5мM a-ketoglutarat in EWS-1 and storages of this solution 24 hours at +4°C, it prevented a cortical  kidney slices swelling authentically more effectively, than just prepared [1, 2]. Therefore we have assumed, any EWS-1 solution components enter a chemical reaction with formation of one or several derivatives possessing antiedematous action. The most probable candidates to us seem carbamide and a-ketoglutarat as carbamide falls into chaotropic compounds and it is inclined to formation ureid group. The ureid group, in particular, is present at all basic anticonvulsants of some derivatives of a hydantoin and in Karbamazepinum. All these compounds possess uniform property of chaotropic connections: to change physical and chemical properties of biological membranes, and to correct membranes permeability for Na+ and K+. Besides hydantoins, possibly, prevent the brain swelling it previous development of the next epileptic attack.

Our experiments with the same medium, but for the lack of one of these compounds have appeared the indirect demonstration of what urea and a-ketoglutarat enter chemical reaction and form new compound. The solution containing both and urea and a-ketoglutarat preventioned a cortical kidney slices swelling at a hypothermal incubation (24 h at +4° C) whereas in a solution without one of these two compounds slices have swelled. [1, 2]. Us were it is carried out the given research with the purpose of revealing of this hypothetical substance.

Materials, methods and research volume

Synthesis of required compound has been carried out in several ways:

  1. urea (extra pure (EP)) and a-ketoglutarat (EP) (on 0,1 M of each compound) сontaining equimolar quantities the solution has been heat uped on a water bath up to 80°C with the subsequent cooling on air;
  2. urea and a-ketoglutarat equimolar quantities were incubated 24 hours at ambient temperature;
  3. urea and a-ketoglutarat equimolar quantities were incubated 24 hours at +4°C;

The synthesized bond has received working name КМ-1. The synthesized compound chemical structure has been certain by a NMR 1Н spectroscopy method. For this purpose crystals KM-1 have been received by means of spontaneous evaporation within 2-3 months. The NMR 1Н spectrum has been registered on device Bruker DRX-500 with a working frequency 500,13 MHz as the dissolvent has been used DMSO - d6. The internal standard was GMDS.

The experience with kidneys slices hypothermal incubation. As mother substances are natural animal metabolites their not enzymatic or enzymatic interaction in vivo with formation studied is possible KM-1. For check of this assumption we have spent experience on a hypothermal incubation (+4°C, during 24 hour) rats kidney cortical slices in mediums EWS-1 and EWS-1b (Tab. 1). On 5 mm either a-ketoglutarat (EP), or L-argignin (EP), or an ornithine (EP) have been brought in these solutions. Besides the hypothermal incubation of sections in medium EWS-НТ with a concentration gradient has been spent KM-1 (0,1; 0,5; 1,0; 2,0; 5,0; 10,0 mM). рН mediums measurement has been carried out by means of pH-meter Piccolo plus. In total for experiences of 20 white not purebred rats-mans have been used, it a kidney mass was 140-160 g. Rats contained on a vivarium standard ration. Rats have been subjected to an ethereal euthanizing before kidney withdrawal. Rats´ decapsulate kidneys quickly have been cut on thin cross-section at ambient temperature. Up to 20 sections have been prepared from each kidney on the average. Immediately after a rifling all sections have been weighed on torsion balances and, besides after an incubation.

Table 1. Composition of incubating mediums, mM

To form a

EWS-1

EWS-1b

EWS-НТ

NaCl (EP)

48,4

48,4

63,4

KCl (EP)

48,81

48,81

48,81

CaCl2 (EP)

2,25

2,25

2,25

KH2PO4 (EP)

1,19

1,19

1,19

MgSO4×7Н2О (EP)

1,19

1,19

1,19

NaHCO3 (EP)

25,0

25,0

-

CH3COONa (EP)

16,6

16,6

5,0

Mannitol (EP)

260,0

260,0

260,0

Carbamide (EP)

50,0

-

50,0

pH

7,85

7,85

8,4 (without KM-1)


Slices hydratation research. Slices hydratation have been research a gravimetry method on water loss at desiccation in a dry-heat case and expressed in dry tissue kg/kga.

Statistical analysis. The received digital stuff has been treated in Excel 2003 by descriptive statistics methods. As parameter of slices hydration distribution was close to normal the received digital material has been processed with t-test use. Data are presented in the M±m form, where m - an average mistake. Authentic distinction was accepted at p<0,05.

Results and their discussion

Synthesis during a urea colorless solution and a-ketoglutarat got a straw-coloured shade. Reaction proceeded both at heating, and at a room temperature, and at +4°С, reaction kinetic it was not researched by us. According to NMR 1Н spectroscopy, the synthesized connection has been identified as 3(2,5 dioxo-3-imidazolin-4-silt) propionic acid (КМ‑1). It the gross formula С6H6N2O4, M.w.=170,116.

This substance of NMR 1Н the spectrum is characterized by two multiplets presence of the methylene protons with chemical shifts 1,9 m.d. Both 2,25 m.d. And a singlet of an imide proton with d=8,1 m.d. Apparently, in water solution at рН=7,0 compound exists in the zwitterion form, and at acidifying a solution it can pass from lactam forms in lactim. On chemical structure the it compound by us meets dehydrogidantoin-5-propionic acid. Relatives on structure compounds are hydantoin-5-propionic acid [3] and 4 (5)-imidazolon-5 (4) propionic acid [4], formed at the person, primates and rats during a histidine katabolism. Hydantoin-5-propionate - a termination products of a histidine exchange, it formed in an alternative path its katabolism from 4-imidazolon-5 propionic acids at FAD participation [3]. Theoretically, in vivo it can be transformed in dehydrogidantoin-5-propionate to dehydrogenation reactions with participation NAD+, NADP+ or FAD. In view of that dehydrogenase reactions reversible, hydantoin-5-propionates synthesis from dehydrogidantoin-5-propionates is possible.

Proceeding from that synthesis of dehydrogidantoin-5-propionates can proceed in soft conditions (in vitro), it can be formed in some fabrics, both is spontaneous, and enzymatic, we have assumed. Mother compounds rather high concentrations are present at a liver and a kidney. In both bodies urea and a-ketoglutarat are synthesized (at deamination, glutamate transamination or in citric cycle). For check of our assumption of dehydrogidantoin-5-propionates endogenic synthesis we inycubated rats kidney slices in initial medium where there was no urea, but there was a‑ketoglutarat (Tab. 2). The bold font evolves the mediums which contain all substrates, ureas necessary for synthesis.

Table 2. Kidney slices hydratation depending on presence or absence at medium of a urea combination or its precursors L-arginine and a‑ketoglutarat, N=12, n=44 on the control and over each medium variant

Intact slices

2,97±0,04

 

Slices incubated in medium:        EWS-1

3,54±0,05

p<0,0001 to intact slices

EWS-1b

3,55±0,03

p<0,0001 to intact slices

EWS-1b + a-ketoglutarat

4,13±0,015

p<0,0001 to intact slices &

p<0,001 to EWS-1 & EWS-1b

EWS-1 + a-ketoglutarat

3,11±0,05

p<0,0001 to EWS-1 & EWS-1b

EWS-1b + arginin

3,48±0,085

p<0,0001 to intact slices

EWS-1b + arginin and a-ketoglutarat

3,06±0,04

p<0,0001 to EWS-1 & EWS-1

EWS-1b + ornitin

3,46±0,07

p<0,0001 to intact slices

EWS-1b + ornitin and a-ketoglutarat

4,61±0,19

p<0,0001 to intact slices &

p<0,001 to EWS-1 & EWS-1b

EWS-1 containing urea badly preventions a kidney slices swelling and after 24 h of an incubation at +4°С, 19 % a set of water, in comparison with intact sections of the same kidney are observed. In medium EWS-1b of a not containing urea the swelling size was similar. Addition in EWS-1b 5 mM a-ketoglutarat strengthens hydration more than in 2 times, this phenomenon the reason has not been researched by us. Nevertheless, 5 mM a-ketoglutarat by EWS-1 addition was a swelling prevented, statistical differences with intact sections were not observed, and in relation to EWS-1 and EWS-1b the swelling is authentic below. The urea precursor arginine 5 mM addition in EWS-1b does not cause any effect, the slices hydratation is comparable to a swelling in EWS-1 and EWS-1b. But an arginine introduction together with a-ketoglutarat also effectively warns a swelling, as well as a-ketoglutarat introduction on EWS-1. To similarly arginine, the 5 mM ornithine does not influence a slices swelling size in EWS-1b. However 5 mm a-ketoglutarat addition does not a swelling prevention, but moreover strengthens it which even is more, than only at one a-ketoglutarat presence in EWS-1. It is apparent, that effect absence is caused by in a kidney the ornitine cycle is reduced, unlike a liver. Ornitincarbomoilase activity low (the ferment catalyzing citrulline formation from an ornithine and carbomoilphosphate) that is why an ornithine does not participate in urea synthesis. Whereas in a kidney arginase activity is high, it catalyzing hydrolytic arginine decomposition up to urea and an ornithine,.

Thus, in urea absence case in incubating medium for endogenous synthesis, a condition urea synthesis case is reduces a-ketoglutarat dehydrating effect. In our opinion, it obliquely proves a dehydrogidantoin-5-propionate synthesis opportunity from urea and a-ketoglutarat in a kidney cortical.

Studying has shown influences КМ-1 on a kidney slices hydration parameters, that it compound prevents a slices swelling effectively and concentrationdepending in parameters from 2,0 up to 10 mM (Tab. 3).

Table 3. A kidney slices hydratation dependence on КМ-1 concentration in medium EWS-НТ, N=8, n=18 on the control and over each medium variant

Intact slices

2,67±0,02

EWS-HT without KM-1

3,07±0,05**

EWS-HT with KM-1            0,1 mM

3,03±0,05**

0,5 mM

3,04±0,06*

1,0 mM

3,01±0,05*

2,0 mM

2,82±0,03*

5,0 mM

2,74±0,05

10,0 mM

2,64±0,04

* p<0,01, ** p<0,001 to intact slices

Dehydrogidantoin-5-propionate existence in alive objects the biological expediency can be explained by urea linkage necessity for inhibiting effect depression its high concentrations on cellular ferments. Presumably, dehydrogidantoin-5-propionate can participate in maintenance рН cells, binding or giving protons by formation donnornoacceptor connections with the second nitrogen atom, or as an olefinic linkage tearing up result. For example, introduction 5 mM КМ-1 on EWS-НТ causes depression рН from 8,4 up to 6,9. Interreacting through a carboxyl with the lysine rest of amino group by protein, dehydrogidantoin-5-propionate can chemically modify protein, variating its functional activity. Probably, dehydrogidantoin-5-propionate somehow takes part in kidney cells protection and a liver against a swelling at a water balance change, for example, at drink. It directly follows from our experiments with kidney slices swelling prophylaxis. Carrying out of researches before a unknown metabolic path of formation dehydrogidantoin-5-propionate in biological objects and an establishment of its physiological role is necessary

Conclusions

  1. Urea and a-ketoglutarat are capable in a wide temperature range to dehydrogidantoin-5-propionate spontaneous formation in vitro.
  2. In without urea medium a-ketoglutarat is a kidney slice swelling causes. At urea presence or arginine its metabolic precursor a-ketoglutarat prevents a kidney slice swelling in the hypothermal incubation test.
  3. Dehydrogidantoin-5-propionate prevents a kidney in the hypothermal incubation test without dependence from urea presence.

References

  1. Matkov K.G., Kozlov V.A., Tolmachev A.S., Stapanova T.A. (2001) Nephrology (Russia), 5(3), 107.
  2. Matkov K.G. (2003) Nephrology (Russia), 7(Supply 1), 218.
  3. Dagley S., Nicholson D.E. (1973) An introduction to metabolic pathways. Oxford & Edindurg.
  4. Broun D.D., Kies, M.W. The mammalian metabolism of L-histidine. The enzymatic formation of L-hydantion-5-propionic acid. (1959) J. Biol. Chem. 234:3182-3187.
The article is admitted to the International Scientific Conference "Fundamental and applied research in medicine", China (Beijing), 26 November - 4 December, 2007, came to the editorial office on 09.11.07.