Elementi in tracce (2)

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Elementi in tracce (2)

• Scuola di Specializzazione in Biochimica Clinica
• Università degli Studi di Milano

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Elementi in tracce in Medicinatutte 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 sviluppoFe,
  Cu, I, Mn, Cr, Se, Co, V, Mo, Si, Sn, Ni, As

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Elementi tossici

• Accumulo corporeo (esposizione)
• gt 80 (Pb, Cd, Hg, As )
• Diversi organi bersaglio e sintomatologie
  cliniche
• Patologie iatrogene (Li, Au, Pt, Al)

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Zn

• 2, orbitale d completo
• grande stabilità (vs. Fe, Cu, Mn)
• gt 300 enzimi e proteine
• Anidrasi carbonica
• ALP
• RNA, DNA polimerasi
• Timidina chinasi
• Carbossipeptidasi
• Alcool deidrogenasi
• Zinc finger proteins

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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
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Zinco

• Biochimica

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

• 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
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Phytic acid 83-86-3 Synonyms Inositol
hexaphosphoric acid, 40-50 wt aqueous solution
Inositol Hexaphosphate myo-Inositol
hexakisphosphate Fytic acid Inositol-hexaphospho
ric acid Myo-inositol hexaphosphate Phytic
acid myo-Inositol, hexakis(dihydrogen
phosphate)
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Zn, valori di riferimento
plasma RBC urine capelli
11 23 mmol/L (70 150 mg/dL) 153 306 mmol/L 2 18 mmol/d 1,5 4,3 mmol/g
Variazioni circadiane (picchi alle 9 ed alle 18)
AAS Attività ALP, anidrasi carbonica Spettrofluore
scenza raggi X
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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.
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Cu

• 1, 2
• Reazioni redox, O2 binding
• metalloenzimi
• Ceruloplasmina
• Citocromo c ossidasi
• Superossido dismutasi
• Dopamina-b-idrossilasi
• Ascorbato ossidasi
• Lisil ossidasi
• Tirosinasi

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Interazione carenza Cu con assorbimento Fe -gt
anemiaceruloplasmina attività Fe ossidasica
Tf
Cp
Fe2 ? Fe3 ? (Fe3)2 Tf
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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).
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Rame

• Biochimica

Metalloenzimi. RDA 1,3 mg/d pool 80 150 mg

• 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

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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
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Morbo di Wilson(degenerazione epatolenticolare)

• 10-30 milioni, difetto congenito
• Accumulo epatico
• WD in cromosoma 13
• ceruloplasmina (lt20 mg/dL), Cuplasma,
  Cuurine (gt20 mmol/L), depositi cerebrali e renali

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Fatemi N, Sarkar B. Molecular mechanism of copper
transport in Wilson disease. Review 54 refs
Environmental Health Perspectives. 110 Suppl
5695-8, 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
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Cu, valori di riferimento
Plasma Plasma, gravidanza Eritrociti Fegato
11 24 mmol/L (70 155 mg/dL) 19 47 mmol/L 14 24 mmol/L 30 50 mg/g
Variazioni circadiane (picco al mattino)
AAS Spettrofotometria (batocuproina,
cuprizone) Attività SOD, cyt c ossidasi Ceruloplas
mina (rapporto Cp-enz/Cp-immunochim) Spettrofluore
scenza raggi X
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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.
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Selenio

• Biochimica

Diverse selenoproteine (glutatione
perossidasi, tirosina-5-deiodinasi,
selenoproteina P). Regolazione dellescrezione
urinaria.

• 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?

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Selenocysteine (forma biologicamente attiva)
Selenomethionine (pool di riserva)
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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.
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Se, valori di riferimento
Plasma Urine 24 h Eritrociti Capelli
0,6 1,8 mmol/L (5 14 mg/dL) 0,1 2,0 mmol/L 1,0 3,1 mmol/L 0,2 1,4 mg/g
Variazioni circadiane (picco al mattino)
AAS (senza fiamma) Spettrofluorimetria Attività
GSH-Px
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3 mg/100 g
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Piombo

• Biochimica

Forma complessi con SH ed altri ligandi (enzimi
intracellulari). Effetti sintesi eme.

• Tossicità

Petrolio, acqua potabile (tubazioni vecchie),
alimenti, vernici

• Sintomi

Bambini encefalopatia (riduzione QI) Adulti
anemia, insufficienza renale

• Concentrazione plasmatica lt0,5 mmol/L (cutoff
  medicina del lavoro gt3,9 mmol/L)

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-
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Stippling basofilo in un caso di intossicazione
da Pb
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Alluminio

• Biochimica

Non noto meccanismo di tossicità (Alzheimer)

• Tossicità

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

• Sintomi

Danneggiamento cerebrale, malattie ossee, anemia
microcitica

• Concentrazione plasmatica lt0,5 mmol/L(tossicità
  con gt10 mmol/L)

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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 lt 1 ng/L Spettrometria di
fluorescenza atomica metodo di scelta per
analisi in tracce ed ultratracce International
Labmate 2004
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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.
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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

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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

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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.