Pip milano .relazione marsiglia aprile 2013 carta intestata

DEPARTMENT OF PHARMACEUTICAL SCIENCES

ON THE CAUSAL FACTORS OF THE HEALTH RISK ASSOCIATEDTO PIP BREAST IMPLANTS

(unpublished data in preparation for publication)

DR. GIANGIACOMO BERETTA - VIA MANGIAGALLI 25 - TEL. +39.0250319317– FAX +3950319359 EMAIL: [email protected]

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Recently, silicone breast implants from the French manufacturer Poly Implant Prothèsehave been the subject of debate and public unrest in women with silicone breast implants. An inspection in 2009 by the French health authority Agence Française de Sécurité Sanitaire des Produits de Santé (ASSPS) found that the silicone gel used in these implants was unauthorized for medical use.

Recently we have reported a comparative study in which we have emphasized the non-cohesive nature of the PIP gel which, according to what found by the French investigators, showing that it is made basically of colourless, non-cohesive, water- and solvent- insoluble high molecular weight silicone oil, together with a minor low molecular weight silicone fraction.

Different investigations showed that this silicone oil may be released from the implants through their weakened and permeable elastomeric shells to reach the periprosthetic capsule and the breast tissue. Different recent case studies reported that migrating PIP silicone can reach axillary lymphonodes, the skin and even the intra-thoracic region between the chest wall and lungs initiating a cascade of events that ultimately leads to different health problems and pathologies.

Since the first news appeared on the media, a increasing number of plastic and reconstructive surgeons began reporting that during operations for PIP implants explantation and/or replacement with other brands of new prostheses, often a yellowish/orange, flaking, non-cohesive liquid gel erupt during excision.

This fluid, sometimes named “seroma”, “periprosthetic fluid” or “late periprosthetic fluid” by analogy with other surgery-related known body fluids, has been interpreted as a suspension of silicone in water, probably due to excessive implant silicone bleeding.


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Water loss test and ATR-IR spectroscopy analyses

The non-volatile material contents of the PF from patients 1 (PF1) and 2 (PF2) were evaluated indirectly by measuring the amount of water lost by heating.After t=2h at T=105°C, PF2 gave a constant weight of dry residue which was almost double in respect to that of PF1 (32±2 % w/w, mean ± SD vs. 15 ± 2 % w/w, mean ± SD, P<0.001; Fig. 1).

Fig. 1.

The chemical/biochemical nature of the bulk constituents of the PF dry residues was first investigated by ATR-IR spectroscopy. Before water evaporation (Fig. 2A), the IR spectra of both PF were dominated by IR absorptions typical of water (ν=3328 cm-1, O-H st) and of polydimethylsiloxanes (ν=2962.2 cm-1 C-H stretch, ν=1650.5 cm-1, ν=1258.1 cm-1, CH3 bend, ν=1079.1 cm-1, Si-O-Si st, ν=1013.7 cm-1 Si-O-C st, ν=863.4 cm-1, Si(CH3)2 δ, ν=789.1 cm-1, Si-C st/CH3 rock). According to the water/non-volatile percentage composition determined by the water loss test, the absorptions generated by water were stronger in PF1 (Fig. 2A, black trace), while those of silicone were stronger in PF2 (Fig. 2A, grey trace). After complete water evaporation, the major absorptions from silicones were still present in the dry residue of both PF (Fig. 2B, black and grey solid lines), and in parallel to the disappearance of water absorption bands, new bands at 3293.4 cm-1 and in the range 1750-1500 cm-1 characteristic of


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protein amide I, II and III absorptions, were clearly observable. This protein was easily identified by comparison with isolated human haemoglobin (Hb, Fig. 2B, arrows). The presence of Hb in PF was attributable to (i) blood from surgical incision, or to (ii) haemolysis of red blood cells (RBC) of haemorrhagic blood from inflamed tissues with the silicone diffused from the bleeding implants.

Fig. 2. ATR-FT-IR spectra of (A) PF1 (black line), PF2 (grey line) and PF2 (dotted grey line) before and (B) after dehydration.


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GC-MS analysis of PF

Beside bupivacaine (Table 1), the local anaesthetic drug injected locally to reduce pain after the surgical procedure, the GC-MS analysis of the PF from all three patients showed the parallel presence of different concentrations of cholesterol and of trace amounts of LMW silicones: the lowest concentration of cholesterol was found in PF1 and the highest in PF2 (Fig. 3). All the identified silicones (from D4 to D9, U1 and U2, see Table 1 for peaks attributions) matched the retention times and mass spectrometric data of LMW silicones extracted from different batches of explanted PIP implants (Fig. 4).

Fig. 3. GC-MS chromatographic profiles of the acetonitrile extracts of PF1, PF2 and PF3. Compared to peaks from silicones, in all cases the peak of cholesterol is dominant.


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Fig. 4. Representative GC-MS profiles of PF1 (upper panel) and of the acetonitrile extract of silicone from an explanted PIP prosthesis (lower panel; batch n. 98276 120). See Table 1 for peaks attributions.


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TABLE 1. Compounds identified in PF1 and PF2 by GC-MS. In bold substances not detectable in PIP silicone gel.

Peak RT Compound R match

1 2,3 Pyridine 944

2 3,30 acetaldoxime 999

3 4,30 4-hydroxy-4-methyl-pentanone 781

4 6,00 D4 886

5 7,90 D5 872

6 9,70 D6 761

7 11,20 D7 893

8 12,50 D8 747

9 13,60 D9 747

11 14,60 L8 823

13 15,10 U1

15 16,10 U2

16 17,40 Bupivacaine 934

17 18,40 D9 Analogue 802 (for D9)

18 21,40 Cholesterol 891


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HPLC-UV/DAD analysis of PF

The presence of major semi-polar species and of biological metabolites that may have accumulated in the periprosthetic fluids, was investigated by reverse phase HPLC-UV/DAD analysis of their water/acetonitrile (1:1 vol/vol) extracts. The chromatographic profile of PF1 shown in Fig 5 evidenced the presence of a dominant peak at RT=3.6 min, generated by a species with both UV spectrum and RT matching those of uric acid (λmax=268 nm). To overcome the difficulties in the accurate and reproducible determination of this analyte, which is barely soluble in most polar and apolar solvent systems, and that in our case was present in a inhomogeneous sample (disabling its classical determination by colorimetric enzymatic assay), we developed an HPLC-UV method for the quantification of uric acid after its conversion into its water-soluble form (urate) at basic pH (100 mM aqueous NaOH /acetonitrile, 1:1 vol/vol; see experimental section for details). Using this methodology we determined in PF1 a mean uric acid concentration of 4.0±3.0 mg/dL (238 mM), a value falling in the normal range of human blood serum. Interestingly, in the case of PF2 the uric acid concentration was below the method LOD.

Fig. 5. HPLC-UV DAD profile (l=270 nm) of (A) the water/acetonitrile extract of PF1 and (B) of standard uric acid. UV spectra of peaks at RT=2.6 min in the insets.


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SEC-UV/DAD analysis of PF proteins

According to the hypothesis that the PF collected were originated by the emulsification of high and low molecular weight silicone with blood components, the presence of water-soluble proteins associated with blood serum was investigated by SEC-UV/DAD analysis. According to previous studies our results (Fig. 6) showed a chromatographic profile clearly compatible with that of high molecular weight proteins from serum (globulins, RT=4-5 min; albumin, RT 5.5 min).

Fig. 6. Representative SEC-UV/DAD chromatographic profile of PE proteins (λ=280 nm, PF3).


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PF morphology

As expected on the bases of the results from ATR-IR experiments, the microscopical analysis (20×) of both investigated PF, revealed a fluid morphology consistent with a complex multiphase system (Fig. 7A and Fig. 7B). The visual comparison of the magnified images of PF1 and PF2 revealed some significant difference. PF1 displayed the presence of a background of round-shaped particles of small and intermediate size and of bigger, more structured particles consistent with oil-in-water emulsified silicone. The bigger structures also presented smaller embedded particles, consistent with a water-in-oil emulsion (see arrows in Fig. 3A).Conversely, in PF2 bigger silicone particles were almost absent, and the intermediate size particles showed the presence of intra-structurally emulsified small particles. Assuming that the silicone released by the implants is made by polydimethylsiloxane oil, these results suggest that during the implantation time there is a slow conversion of big polydimethylsiloxane silicones particles into smaller multiphase silicone/blood components particles. In the light of these results, we believe that urgent further investigations are needed to understand the toxicological consequences of the 'heavy' silicone conversion into such small, emulsified and mobile particles into the periprosthetic capsule, breast tissue and into tissues of other excitable organs or body regions (i.e. the lungs and thoracic cavity).

Fig. 7. Optical microscopical examination of (20× magnification) of the PF from (A) patients 1 and (B) patient 2.


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CONCLUSIONS

The results demonstrate that the composition of PF is dominated by water, silicone and blood proteins such as haemoglobin, albumin and globulins, and blood biochemical such as uric acid indicating that very likely silicones released by the prostheses are slowly mixed with lipophilic fractions from tissues, inflammatory exudate and from blood, and then emulsified with their hydrophilic components.

The negative health effects observed in women implanted with PIP prostheses may be due to the enhanced diffusion of silicone from the implant through its particle size reduction and emulsification, making them able to enter both lipophilic (fat) and hydrophilic (serum, cell cytosol) media, an to be transported to other body parts, explaining the fast and massive consequences deriving from the exposure to PF of the breast lymphatic system (Fig. 6) often observed in women carrying PIP implants.

Further urgent studies are needed to understand the effects of PIP-induced PF on the mammary gland system, the potential migration of PF components into milk, and consequently to newborns during lactation.

Milan, 22 April 2013

Dr Giangiacomo BerettaAggregate Professor at University of MilanChemical Toxicological ChemistryAdvanced Analysis of Active Priciples in Herbal Drugs


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Fig. 8


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Health & Medicine

Pip milano .relazione marsiglia aprile 2013 carta intestata