Elementi in tracce 2 l.jpg
This presentation is the property of its rightful owner.
Sponsored Links
1 / 35

Elementi in tracce (2) PowerPoint PPT Presentation


  • 153 Views
  • Uploaded on
  • Presentation posted in: General

Elementi in tracce (2). Scuola di Specializzazione in Biochimica Clinica Università degli Studi di Milano. Elementi in tracce in Medicina “tutte le sostanze sono tossiche a certi livelli”. Azione biologica a bassa concentrazione ( m mol/L, nmol/L) Tossicità al di sopra di una soglia

Download Presentation

Elementi in tracce (2)

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


Elementi in tracce 2 l.jpg

Elementi in tracce (2)

Scuola di Specializzazione in Biochimica Clinica

Università degli Studi di Milano


Elementi in tracce in medicina tutte le sostanze sono tossiche a certi livelli l.jpg

Elementi in tracce in Medicina“tutte le sostanze sono tossiche a certi livelli”

  • Azione biologica a bassa concentrazione (mmol/L, nmol/L)

  • Tossicità al di sopra di una soglia

  • Alcuni elementi essenziali per lo sviluppo:Fe, Cu, I, Mn, Cr, Se, Co, V, Mo, Si, Sn, Ni, As

A.Mosca - UniMI


Elementi tossici l.jpg

Elementi tossici

  • Accumulo corporeo (esposizione)

  • > 80 (Pb, Cd, Hg, As …)

  • Diversi organi bersaglio e sintomatologie cliniche

  • Patologie iatrogene (Li, Au, Pt, Al)

A.Mosca - UniMI


Slide4 l.jpg

Zn

  • +2, orbitale d completo

  • grande stabilità (vs. Fe, Cu, Mn)

  • > 300 enzimi e proteine

    • Anidrasi carbonica

    • ALP

    • RNA, DNA polimerasi

    • Timidina chinasi

    • Carbossipeptidasi

    • Alcool deidrogenasi

    • Zinc finger proteins

A.Mosca - UniMI


Slide5 l.jpg

Fig. 3. Characterization of a Truncated GATA-4 Protein That Acts as Dominant Negative Competitor of GATA ActivityA, Schematic representation of the full-length GATA-4 protein and a truncated GATA-4 protein that consists solely of its zinc finger region (DF WT). The two zinc finger regions (ZnF) contained within the DNA-binding domain (gray box) are shown as two circles; the basic region, representing the nuclear localization signal, is indicated by (++). A diagram depicting the wild-type amino acid sequence of the second zinc finger is also shown.

Tremblay JJ. Robert NM. Viger RS. Modulation of endogenous GATA-4 activity reveals its dual contribution to Mullerian inhibiting substance gene transcription in Sertoli cells. Molecular Endocrinology. 15(9):1636-50, 2001

A.Mosca - UniMI


Zinco l.jpg

Zinco

Metalloenzimi. Sintesi proteica e degli

acidi nucleici

RDA: 6-15 mg/d (20-30 mg/d);

assorbimento 20 – 30 %; pool 1,4 – 2,3 g

¯ Stress, fase acuta

  • Biochimica

  • Carenza

Malassorbimento (acrodermatite enteropatica). Eccesso fitati e fibre nella dieta. Inadeguato apporto (intravena)

(primi dati 1960). Corticosteroidi. Eccesso Fe e folati. Alimentazione endovena prolungata.

  • Sintomi

Rash cutaneo, perdita capelli, alterazioni mentali, riduzione immunità cellulare

A.Mosca - UniMI


Slide7 l.jpg

Phytic acid [83-86-3] Synonyms: Inositol hexaphosphoric acid, 40-50 wt% aqueous solution; Inositol Hexaphosphate; myo-Inositol hexakisphosphate; Fytic acid; Inositol-hexaphosphoric acid; Myo-inositol hexaphosphate; Phytic acid; myo-Inositol, hexakis(dihydrogen phosphate);

A.Mosca - UniMI


Zn valori di riferimento l.jpg

Zn, valori di riferimento

Variazioni circadiane (picchi alle 9 ed alle 18)

AAS

Attività ALP, anidrasi carbonica

Spettrofluorescenza raggi X

A.Mosca - UniMI


Slide9 l.jpg

Erel O, Avci S. Semi-automated enzymatic measurement of serum zinc concentration. Clinical Biochemistry. 35(1):41-7, 2002

To measure serum zinc concentration by means of carbonic anhydrase reactivation using an automated analyzer.

METHODS: The zinc content of carbonic anhydrase (CA), whose cofactor is zinc, was removed by dialysis against pyridine 2 to 6 dicarboxylic acid and a pure apoenzyme was obtained. Serum proteins were precipitated with trichloroacetic acid (TCA) solution and the supernatant fraction of the sample was used to determine the zinc concentration. The negative effects of the precipitant on CA activity in the assay were completely removed, reaction conditions for maximal CA activity were provided and the color of the product was enhanced and stabilized. P-nitrophenyl acetate was used as the substrate and the change of absorbance of p-nitrophenol which was produced was followed at 400 nm. The initial rate of the esterase activity of CA was measured by using an automated analyzer. Analytical performance characteristics of the assay were determined. The zinc concentrations in serum samples of healthy subjects and patients were measured.

RESULTS: The enzymatic assay is accurate, sensitive, specific and is not affected by other metals. There was excellent agreement with the results obtained using an atomic absorption spectrophotometer (AAS) (y = 0.98X + 0.18, r = 0.99). Serum zinc concentrations were found to be higher in patients with vivax malaria, and lower in patients with cutaneous leishmaniasis than in healthy subjects.

CONCLUSION: The enzymatic method is suitable for semiautomated measurement of serum zinc concentration.

A.Mosca - UniMI


Slide10 l.jpg

Cu

  • +1, +2

  • Reazioni redox, O2 binding

  • metalloenzimi

    • Ceruloplasmina

    • Citocromo c ossidasi

    • Superossido dismutasi

    • Dopamina-b-idrossilasi

    • Ascorbato ossidasi

    • Lisil ossidasi

    • Tirosinasi

A.Mosca - UniMI


Interazione carenza cu con assorbimento fe anemia ceruloplasmina attivit fe ossidasica l.jpg

Interazione carenza Cu con assorbimento Fe -> anemiaceruloplasmina attività Fe ossidasica

Tf

Cp

Fe2+ Fe3+  (Fe3+)2 Tf

A.Mosca - UniMI


Slide12 l.jpg

Miyajima H et al. Cerebellar ataxia associated with heteroallelic ceruloplasmin gene mutation. Neurology. 57(12):2205-10, 2001

Figure 3. Light microscopy findings for the liver, basal ganglia, and cerebellum of Patient 1. Perls’ staining of the liver tissue detected an iron accumulation in the hepatocytes (A) and negative staining for copper (B) (bars = 100 µm). The globus pallidus shows mild neuronal loss (C) and a small iron deposition but no gliosis (D) (C, hematoxylin and eosin stain; D, Prussian blue and glial fibrillary acidic protein stain; bars = 100 µm). The cerebellar cortex shows pronounced Purkinje cell loss (E) with marked iron deposition (F) (E, hematoxylin and eosin stain; F, Prussian blue; bars = 100 µm).

A.Mosca - UniMI


Slide13 l.jpg

Rame

Metalloenzimi.

RDA: 1,3 mg/d; pool 80 – 150 mg

  • Biochimica

  • Carenza

Inadeguato apporto alimentare, malnutrizione, sindrome di Menkes (trasporto mucosa int.)

  • Sintomi

Adulti: anemia microcitica Fe-resistente, neutropenia; (aritmie cardiache)Bambini: malattie ossee.

  • Tossicità

Intossicazione, terapia con estrogeni, stato infiammatorio, cirrosi, epatiti ostruttive

  • Concentrazione plasmatica: 12 – 24 mmol/L

A.Mosca - UniMI


Slide14 l.jpg

Copper Homeostasis:

The Role of Cellular Transporters

Figure 1. Composite scheme of intracellular copper metabolism in human cells. Inward flow of copper is regulated by hCtr1 at the membrane. Chaperone-bound copper and recipient are: CCS (superoxide dismutase), NML45 (nucleus), COX17 (mitochondrial cytochrome oxidase), or HAH1 (ATP7A, ATP7B). ATP7B receives copper from HAH1 and transports it to a compartment containing apo-ceruloplasmin derived from the endoplasmic reticulum (ER). HAH1 also transports copper to ATP7A, shown as a component of vesicles in the trans-Golgi network (TGN) that move from the Golgi apparatus to the outer membrane. The movement and fusion of the vesicles with the plasma membrane releases copper from the cell. From:   Harris: Nutr Rev, Volume 59(9).September 2001.281-285

A.Mosca - UniMI


Morbo di wilson degenerazione epatolenticolare l.jpg

Morbo di Wilson(degenerazione epatolenticolare)

  • 10-30 milioni, difetto congenito

  • Accumulo epatico

  • WD in cromosoma 13

  • ¯ ceruloplasmina (<20 mg/dL),¯ Cuplasma, ­ Cuurine (>20 mmol/L), depositi cerebrali e renali

A.Mosca - UniMI


Slide16 l.jpg

Fatemi N, Sarkar B. Molecular mechanism of copper transport in Wilson disease. [Review] [54 refs]

Environmental Health Perspectives. 110 Suppl 5:695-8, 2002

Wilson disease is an autosomal recessive disorder of copper metabolism. The Wilson disease protein is a putative copper-transporting P-type ATPase, ATP7B, whose malfunction results in the toxic accumulation of copper in the liver and brain, causing the hepatic and/or neurological symptoms accompanying this disease. The cytosolic N-terminal domain (approximately 70 kDa) of this ATPase comprises six heavy metal-associated domains, each of which contains the conserved metal-binding motif GMTCXXC. The N-terminal domain (Wilson disease copper-binding domain [WCBD]) has been expressed, purified, and characterized using various techniques. The WCBD binds six atoms of copper in the +1 oxidation state competitively, and with a greater affinity than all other metals. The copper atom is coordinated by two cysteines in a distorted linear geometry. Copper binds the WCBD in a cooperative manner and induces secondary and tertiary conformation changes. Zinc binding to the WCBD has also been characterized by circular dichroism spectroscopy and shown to produce conformational changes that are completely different from those induced by copper. The phosphorylation/nucleotide-binding domain of ATP7B has also been expressed and characterized and shown to be capable of binding ATP but lacking ATPase activity. A peptide corresponding to the sixth transmembrane domain of ATP7B has been constructed and shown to undergo secondary conformational changes upon binding a single atom of copper. Finally, a chimeric protein consisting of the WCBD and truncated ZntA, a zinc-transporting ATPase lacking the N-terminal domain, has been constructed and analyzed for metal ion selectivity. These results suggest that the core determines the metal ion specificity of P-type ATPases, and the N-terminal metal-binding domain may play a regulatory role. [References: 54]

A.Mosca - UniMI


Slide17 l.jpg

A.Mosca - UniMI


Slide18 l.jpg

A.Mosca - UniMI


Cu valori di riferimento l.jpg

Cu, valori di riferimento

Variazioni circadiane (picco al mattino)

AAS

Spettrofotometria (batocuproina, cuprizone)

Attività SOD, cyt c ossidasi

Ceruloplasmina (rapporto Cp-enz/Cp-immunochim)

Spettrofluorescenza raggi X

A.Mosca - UniMI


Slide20 l.jpg

Takeuchi Y et al. A case of aceruloplasminaemia: abnormal serum ceruloplasmin protein without ferroxidase activity. Journal of Neurology, Neurosurgery & Psychiatry. 72(4):543-5, 2002

A 34 year old diabetic man with a complete deficiency of serum ferroxidase activity, regardless of the presence of serum ceruloplasmin (Cp), a multicopper ferroxidase protein, is described. The patient had had diabetes mellitus for 13 years, and was also found to have retinal degeneration accompanied by the development of a hearing disturbance of unknown aetiology. Laboratory examination showed markedly increased serum ferritin and low serum iron. Magnetic resonance imaging showed a pronounced hypointensity in the putamen, caudate, cerebellar dentate, and thalamus on T2 weighted images, and also disclosed a low level signal in the liver, suggesting the accumulation of some magnetic substances in the brain and liver. Liver biopsies histochemically identified iron deposition in the hepatocytes. Most of these findings were consistent with the newly established autosomal recessive disease "aceruloplasminaemia", except for the presence of serum Cp and the lack of apparent neurological symptoms. Interestingly, no ferroxidase activity was detected in the patient's serum, whereas suppressed ferroxidase activity was found in his mother's serum. A nucleotide sequence analysis of the Cp gene showed two mutations; a C to T substitution at nucleotide 2701 in exon 16, resulting in a nonsense mutation at amino acid 882 (Arg882ter), and a T to G substitution at nucleotide 2991 in exon 17, resulting in an amino acid alternation at amino acid 978 (His978Gln). The second mutation was also found in the patient's mother. The absence of serum ferroxidase activity despite the presence of serum Cp protein in this compound heterozygote was considered to be due to the production of a non-functional Cp harbouring no ferroxidase activity.

A.Mosca - UniMI


Selenio l.jpg

Selenio

Diverse selenoproteine (glutatione perossidasi,

tirosina-5’-deiodinasi, selenoproteina P).

Regolazione dell’escrezione urinaria.

  • Biochimica

  • Carenza

Inadeguato apporto alimentare, malnutrizione, alimentazione parenterale. Morbo di Keshan

  • Sintomi

Cardiomiopatia, dolori muscolari, (tumori).

Possibile effetto nel gozzo I-resistente.

  • Concentrazione plasmatica: 0,8 – 2,0 mmol/L

  • RDA 55 – 70 mg/d

  • Tossicità renale?

A.Mosca - UniMI


Slide22 l.jpg

Selenocysteine

(forma biologicamente attiva)

Selenomethionine (pool di riserva)

A.Mosca - UniMI


Slide23 l.jpg

Saito Y, Takahashi K . Characterization of selenoprotein P as a selenium supply protein.

European Journal of Biochemistry. 269(22):5746-51, 2002

Selenium (Se) is well known to be essential for cell culture when using a serum-free medium, but not when a medium containing serum is used. This finding suggests that serum contains some usable form of Se. To identify the Se-supplier, T-lymphoma (Jurkat) cells were cultured for 3 days in the presence of human serum immunodepleted of Se-containing serum protein, selenoprotein P or extracellular glutathione peroxidase. The Se-dependent enzyme activities (glutathione peroxidases and thioredoxin reductase) and Se content within the cells markedly decreased only when cultured with selenoprotein P-depleted serum. Compared with other Se-containing proteins, the addition of purified selenoprotein P to the selenoprotein P-depleted serum or a serum-free medium was the most effective for the recovery of cellular glutathione peroxidase activity (index of Se status). These results suggest that selenoprotein P functions as a Se-supply protein, delivering Se to the cells.

A.Mosca - UniMI


Se valori di riferimento l.jpg

Se, valori di riferimento

Variazioni circadiane (picco al mattino)

AAS (senza fiamma)

Spettrofluorimetria

Attività GSH-Px

A.Mosca - UniMI


Slide25 l.jpg

3 mg/100 g

A.Mosca - UniMI


Slide26 l.jpg

A.Mosca - UniMI


Piombo l.jpg

Piombo

Forma complessi con SH ed altri ligandi

(enzimi intracellulari). Effetti sintesi eme.

  • Biochimica

  • Tossicità

Petrolio, acqua potabile (tubazioni vecchie),

alimenti, vernici

  • Sintomi

Bambini: encefalopatia (riduzione QI)

Adulti: anemia, insufficienza renale

  • Concentrazione plasmatica: <0,5 mmol/L (cutoff medicina del lavoro: >3,9 mmol/L)

A.Mosca - UniMI


Slide28 l.jpg

-

A.Mosca - UniMI


Slide29 l.jpg

Stippling basofilo in un caso di intossicazione da Pb

A.Mosca - UniMI


Alluminio l.jpg

Alluminio

Non noto meccanismo di tossicità (Alzheimer)

  • Biochimica

  • Tossicità

Farmaci. Dialisi renale (leganti Al-Pi; sali di Al)

  • Sintomi

Danneggiamento cerebrale, malattie ossee, anemia microcitica

  • Concentrazione plasmatica: <0,5 mmol/L(tossicità con >10 mmol/L)

A.Mosca - UniMI


Mercurio l.jpg

Mercurio

Metil-mercurio nei pesci, molto accumulabile

AAS limite 10 ng/L

Limite ambientale US nel terreno e nelle acque superficiali 1,3 ng/L

EPA vuole working range di 1 – 100 ng/L e limite di rilevabilita’ < 1 ng/L

Spettrometria di fluorescenza atomica – metodo di scelta per analisi in tracce ed ultratracce

International Labmate 2004

A.Mosca - UniMI


Slide32 l.jpg

AAS Buck 210VGP

3HCLs. Singolo raggio.Condizioni operative preimpostate.Fiamma/fornetto/idruri.Doppia correzione del segnale di fondo: D2 e sistema VGP per la regolazione della pulsazione delle lampade.100 metodi interni modificabili.Curve di calibrazione polinominali fino al 4° grado.Totale controllo di tutti i parametri operativi e loro visualizzazione a display.

A.Mosca - UniMI


Interferenze in aas l.jpg

Interferenze in AAS

  • Chimiche: mancanza di dissociazione da complessi (P in calcio-fosfati)

  • Ionizzazione (eccitazione atomica)

  • Matrice:

    • Aumento assorbimento luce

      • solventi organici

      • solidi

    • ossidi riflettenti formati dalla fiamma

A.Mosca - UniMI


Interferenze in aas rimedi l.jpg

Interferenze in AAS- rimedi -

  • Estrazioni

  • Regolazione della temperatura della fiamma

  • Aggiunta eccesso sostanze più ossidabili

  • Aggiunta cationi competitivi rilascio elemento da complessi o anioni chelanti

A.Mosca - UniMI


Slide35 l.jpg

Krachler, M et al. Inter-method comparison for the determination of antimony and arsenic in peat samples.

Analytica Chimica Acta. 2002. 458: 2, 387-396

Four analytical approaches, based on different physical principles, for the determination of antimony (Sb) and arsenic (As) in ancient peat samples were critically evaluated:

(a) open vessel digestion/hydride generation-atomic absorption spectrometry (HG-AAS), (b) closed-pressurized digestion in a microwave oven followed by sector field-inductively coupled plasma-mass spectrometry (SF-ICP-MS), (c) digestion in a microwave autoclave and subsequent quadrupole-inductively coupled plasma-mass spectrometry (Q-ICP-MS) measurements and (d) instrumental neutron activation analysis (INAA). The quality control scheme applied, always included the use of adequate plant reference materials to ensure the accuracy and precision of the analytical procedures. Additionally, two internal peat reference materials were analysed using all four analytical approaches, generally showing good agreement for both elements.

Method detection limits for As and Sb provided by all procedures were approximately 5 and 2 ng g-1 which is sufficiently low for the reliable quantification of both elements in ancient, pre-anthropogenic peat samples. A comparison of As and Sb concentrations in a set of peat samples determined by INAA, HG-AAS and SF-ICP-MS revealed that INAA underestimated the values in a systematic manner, whereas HG-AAS and SF-ICP-MS data agreed very well. Best precision of the results was obtained by analytical procedures involving HG-AAS or Q-ICP-MS and varied from 3.6 to 4.3% and 7.1 to 7.5% for As (at approximately 0.5 micro g g-1) and Sb (at approximately 0.1 micro g g-1), respectively. The highest sample throughput (40 samples per run accomplished in 2 hours) combined with low risk of sample contamination could be realized in the high-pressure microwave autoclave. The amount of sample required by all approaches was 200 mg, except for INAA which needed at least 25 times more sample mass to achieve comparable detection limits. For the quantification of As and Sb, inductively coupled plasma-mass spectrometry (ICP-MS) was preferred over INAA and HG-AAS, mainly because (a) less sample is needed and (b) As and Sb can be determined simultaneously. In addition, ICP-MS offers the possibility to measure concurrently a wide range of other elements which also are of environmental interest.

A.Mosca - UniMI


  • Login