Oggi è dom mag 27, 2018 3:29 pm




Apri un nuovo argomento Rispondi all’argomento  [ 1 messaggio ] 
 Protocollo per kryptopyrroles (inglese) 
Autore Messaggio

Iscritto il: ven gen 28, 2005 5:26 pm
Messaggi: 178
Località: Edinburgh
Messaggio Protocollo per kryptopyrroles (inglese)
Ciao,
riporto di seguito il protocollo che ho ricevuto dal laboratorio di Londra.
È la procedura di laboratorio da seguire per determinare la quantità di kryptopyrroles nel campione di urina e diagnosticare la presenza di pyroluria.

Ricordo a chi fosse interessato che il campione deve possibilmente arrivare al laboratorio refrigerato e schermato dall'esposizione alla luce per assicurarne l'integrità.

Per istruzioni su come raccogliere, conservare e spedire il campione vedere il post in questo forum http://www.laleva.cc/phpBB2/viewtopic.p ... 7c09bdf91d

Cercherò di provvedere una copia tradotta in italiano al più presto.


-----
DETERMINATION OF URINARY KRYPTPYRROLES


(COLORIMETRIC METHOD)



PRINCIPLE

“Kryptopyrrole” is the name given to a pyrrole-zinc-vitamin B6 complex that may be excreted in the urine of affected individuals [1]. Kryptopyrroluria, which may be associated with abdominal pain or a haemolytic crisis, can result in zinc and vitamin B6 deficiency and also schizophrenia. The condition has similarities to acute intermittent porphyria in that coproporphyrins are also excreted. In the scientific literature kryptopyrroles are also referred to as the “Mauve Factor” [2].

The assay procedure described depends on the extraction of the kryptopyrrole (KP) containing fraction from the urine with chloroform and its subsequent reaction with Ehrlich’s acid aldehyde reagent (p-dimethylaminobenzaldehyde in hydrochloric acid). This reaction is non-specific since most indoles, simple pyrroles or more complex pyrrole derivatives such as bilanes, which may be chloroform soluble, will also give a colour. Urobilinogen (UBG), which is a product of bilirubin metabolism, formed as a result of bacterial action in the gut and a normal component of many urine samples, also gives a magenta-red colour with Ehrlich’s reagent which can be extracted into chloroform. Porphobilinogen (PBG), which is an intermediate in the biosynthesis of haem but not a normal component of urine, gives a red colour with Ehrlich’s reagent, but will not extract into chloroform [3]. On the other hand, indican gives a red colour with Ehrlich’s reagent which can be extracted into alkali, but not into organic solvents.

In the situation we are considering,

a) Kryptopyrroles appear in the urine when the patient is suffering from combined zinc and vitamin B6 deficiency.

b) Failure to incorporate these pyrrole rings fully into Hb synthesis results in their appearance in the urine.

c) Such patients may suffer from eg autism or schizophrenia.

It is also possible that a toxin (e.g. a food additive absorbed through the GIT wall) could cause an idiosyncratic disturbance of haemoglobin metabolism and accumulation of pyrroles in the blood and urine, with consequent excessive loss of zinc and vitamin B6. Treatment involves identification and removal of the toxin from the diet, as well as replacement of zinc and vitamin B6. Hence kryptopyrroluria is a secondary coproporphyrinuria (i.e. a porphyrinuria which develops on the basis of a disease other than a primary disturbance of haemoglobin synthesis). The presence of porphyrins in the blood due to an inherited metabolic defect can also cause psychiatric disturbances. However, patients with kryptopyrroluria are not thought to suffer from other recognised porphyrin defects.


SAMPLE

An early morning urine sample is collected into 500 mg of ascorbic acid. This will stabilise the kryptopyrrole content for a maximum of 10 days.


REAGENTS

1. Kryptopyrrole standard.

2,4-dimethyl-3-ethylpyrrole, Aldrich D15,840-2 mw 123.20.

2. Para-dimethylaminobenzaldehyde (= 4-dimethylaminobenzaldehyde).

Sigma D 2004, mw 149.2. 1.0 gm of p-dimethylaminobenzaldehyde is dissolved in c. 80 ml of methanol, which should be cooled. 5 ml of concentrated sulphuric acid is added slowly, without browning or excessive heat generation. After mixing the solution is allowed to reach room temperature volume is made up to 100 ml with methanol. The reagent is stored in the fridge in a brown bottle and is stable for at least one month.


PROCEDURE

1. Prepare a worksheet listing a blank (water), the standard (if used), samples and controls (not available at present).

2. Measure 2 ml of each urine sample into a glass test tube.

3. Adjust the pH to 3 – 4 with 0.1N HCl (or NaOH) using narrow range pH papers.

4. Add 4.0 ml of chloroform to each tube.

5. Extract by shaking (vortex. 1 minute). Take care not to lose any of the mixture.

6. Centrifuge the tubes.

7. Suck off the upper, aqueous layer from each tube and discard.

8. Take 2 ml of the chloroform extract to another glass tube and add 0.5 ml of methanl/Ehrlich’s reagent.

9. Mix well. Stand the tubes on the bench for 30 minutes.

10. Read the absorbances against a reagent blank (2 ml of chloroform + 0.5 ml of reagent) at 540 nm.

11. A deep pink colour will develop in the presence of KP.

12. Report the absorbance reading.

13. Absorbance readings of up to 0.08 are normal ( = urinary UBG etc.). The reference range is included on the printed report and no further comment is required.




REFERENCES

1. Irvine DG, Bayne W, Miyashita H, Majer JR (1969). Identification of kryptopyrrole in human urine and its relation to psychosis. Nature 224:811.

2. Sohler A, Beck R, Noval JJ (1970). Mauve factor re-identified as 2,4-dimethyl-3-ethylpyrrole and its sedative effect on the CNS. Nature 228:1318.

3. Jacobs SL (1974). Porphyrins and their precursors. In: Clinical Chemistry Principles and Techniques, eds Henry RJ, Cannon DC, Winkelman JW, 2nd edition, Harper Row, New York p. 1232.

4. Gowenlock AH, McMurray JR, McLauchlan DM (1988). Varley’s Practical Clinical Biochemistry, Heinemann, Oxford 6th edition, pp 642 - 669.

5. William Walshe - Chicago



N.J. Miller PhD, FRCPath.
Biolab Medical Unit.

August 12th 1999


lun gen 16, 2006 12:41 pm
Profilo
Visualizza ultimi messaggi:  Ordina per  
Apri un nuovo argomento Rispondi all’argomento  [ 1 messaggio ] 


Chi c’è in linea

Visitano il forum: Nessuno e 2 ospiti


Non puoi aprire nuovi argomenti
Non puoi rispondere negli argomenti
Non puoi modificare i tuoi messaggi
Non puoi cancellare i tuoi messaggi

Cerca per:
Vai a:  
cron
Powered by phpBB © phpBB Group.
Designed by Vjacheslav Trushkin for Free Forums/DivisionCore.
Traduzione Italiana phpBB.it