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SILICONE IN BREAST IMPLANT

ABBREVATIONS

CDCl3: Deuterated chloroform CHCl3: Chloroform C : Carbone D3: Hexamethylcyclotrisiloxane D4: Octamethylcyclotetrasiloxane D5: Decamethylcyclopentasiloxane D6: Dodecamethylcyclohexasiloxane D group: D: Polymethylsiloxane, Dph2: Polydimethylsiloxane EDTA: Diamino ethane tetra acetic acid H2: Hydrogen M4Q:Tetrakis(trimethylsiloxy)silane N2: Nitrogen NaOH: Sodium hydroxide Si: Silicon TMS: Tetramethylsilane ZrO2: Zirconium dioxide

BI: Breast Implant CP: CrossPolarisation COSY: Correlation Spectroscopy FID: Flame ionization Detector FDA: Food Drug Administration GC: Gas Chromatography GFAAS : Gas Furgnace absorption spectroscopy HMBC: Heteronuclear Multiple Bonds Coherence HMDS: HMW: high molecular weight LMW: low molecular weight MAS: Magic Angle Spinning MS: Mass Spectroscopy NMR: Nuclear Magnetic Resonance SIM : Single Ion Monitoring

INTRODUCTION

The news fashion which includes the new technology, the use of tactile screen, diet to keep people slim and aesthetic surgery has taken a big place in this century. Medical advances enable people to change their shape and avoid the aging process. The latter is taken as pathology than a physiology process. (cela na pas de sens) People madeundergo(le temps) aesthetics surgery as they would undergo cancer surgery(cela na aucun sens). They dont think about the inconvenience of the surgery. Unfortunately, some of those surgeries bring health problems. During the past few years, several specific methods(que voulez-vous dire?) for the investigation of silicon in biological substances were developed. We are going to focus to on the effect of silicone breast implants in the human body. That will concern the people who, one day, will have breast implants. We will show some techniques used to find the silicone in that body and the control that we made to know its effect.

A.

HISTORyHISTORY

Since For 30 years, breast implant has been used to have a good shape in augmentation of mammoplasty. Some people who had breast cancer put them to hide what was abnormal. In 1992 the food and drug administration (FDA) banned its use for safety reasons, especially by its rupture and the disease caused by the silicon which is contained in the implants. In 2006, no studies showed the increasing of that disease and till now the research has been continuing. 1. FABRICATION OF BREAST IMPLANT Breast implant is made in a sterile and safe atmosphere. Firstly, we put the silicone is put in the a mould to form a thin envelope. Scientifically, that envelope is made of high cross linked silicone blended with amorphous silica. After that, the envelope is filled by the gel. That gel is made by dimethyl and methyl vinyl siloxane. 2. EFFECT OF BREAST IMPLANT The rupture of breast implant is caused by the breast augmentation. So the extra capsular rupture makes a dispersion of silicone. It is likely to happen between the 3rd and 10th years after surgery. Many studies was made about the danger of breast implant and in 2006 they revealed that some breast implant contained silicone and platinum and the latter was not harmful for women but for their child if they are breast feeding. Actually, the silicone breast implant has to be replaced after 10 years. So, the surgery can have the earlier effect which depending of the surgery and the long term effect which depending of the body response. Here are some of those complications: lop- sided breast, necrosis, and infections. Bacteria can grow up through the infection and be released into the bloodstream which is very danger for life. This day where the breast cancer is commonly, the silicone implant can hide a cancer during mammography screening so it will be late to deal with it. The scar tissues shell doesnt trap the silicone, so his leakage after the rupture can travel all part of the body including the lymph node. It can be difficult to move it and in the long time can have some health effects.

B. TECHNICAL
1. SILICON FAMILY AND HIS STRUCTURE Silicon is the most common metalloid and has many uses in industries. Silicone forms useful glasses and ceramics in the form of silica and silicate. And in the form of silicone we made many various synthetics plastic. The human organism has 200mg to 7 g of silicon. The latter has a big extent in collagen synthesis. The studies have shown than the quantities of silicon in the tissue reduce remarkably with age. (60-70%).silicon is essential compound required in the normal metabolism of animal is involved in bones, cartilage and connective tissues formation. Silicone is in organic compound composed by the chain of silicon and oxygen (...-Si-O-Si-OSi-O-...). His consistency varies from liquid to the hard plastic passing gel and gum.
2. MAIN TECHNICALS

We can determinate the quantities of silicone in human body in using some analytic technical. a. Electro thermal Atomic Absorption Spectrometry Our goal is to analyze the quantities of silicone in breast implant. This technical show us how we can determinate the silicon in serum and tissues around the silicone implant. This technical is used to analyze the metal trace in biological materials. His advantage is that he minimizes the trace metal contamination by addition of reagent, use of small quantities of the samples and the recombination of the molecular is not rapidly because of the atmosphere of gas inert. We use as the wavelength 251.6nm for Si. Material All plastic were washed with 10% of nitric acid, rinced 3 times by water. We prepared silicon solution of 100g/ml in 2% of NaOH solution. The matrix modifier was composed by 7 compounds ( see the table 1) and purified water to 1 litre. pH have to be around 6.5 Matrix Lanthanum compose oxide Quantities 30mg Table .(Title) Calcium chloride 30mg Ammonium phosphate 4.5g EDTA 0.5g ethanol 50ml Triton X100 0.5ml

To analyze, they make a dilution of standard silicon solution and matrix 1:4.

The procedure is in 9 steps : Step 1-4: the drying of sample at 90 the goal is to concentrate in weight Step5: washing Step 6 : cool down of the sample and his reduce by the argon gas flow. Step 7-8: atomization Step 9: clean out stage. The atomization of 600g/l at 2700, the standard calibration was linear to 1000g/l. Result and discussion The method was linear to 1000g/l with the precision of 8.4% at 73g/l and 5.9 at 281g/l. the reference ranges for serum silicon 30-209g/l Table .(Title) We see an elevated value of silicone in hemodialysis patient, which is out of the reference ranges. It is due to the linkage to their dietary intake. The excess of silicon, means more than 100mol/l, is not good in the body. However, Si can make a covalent liaison with Aluminum and form an insoluble hydroxyaluminosilicate which reduce the bioavailability of Al. We know that the deficiency of Al cause memory trouble and a delay intellectual. the normal range for breast tissue in women without B.I 0,25-2,4 g/g Table .(Title) The result shows a great augmentation of silicon in capsular tissue. Silicon can be correlated to silicone so we can say that the silicone can migrate to the surrounding tissues. This determination shows us the degree of silicone leakage in the women with B.I. Conclusion This technical allowed avoiding the analyte losses by extraction an atomization. The use of no suitable modifiers can increase those looses. The method is sensitive to detected Si in normal (0.25g/l). This method can analyze the silicon compound in the blood of women with B.I The analysis allowed us to say that the silicone can migrate in the tissues around, so we can have say that there is a implant bleeding b. NMR Technical silicon concentration of capsular tissue in women with B.I 29-496 g/g the serum silicone ranger of people serum silicon range in without breast augmentation hemodialysis patient 35-240 g/l 900-3300 g/l

NMR spectroscopy is a technical which exploit the magnetic properties of a certain nuclei, it is applicable to any nucleus possessing spin. It gives information on the number and types of chemical entities in molecular. We use it in a huge variety of sample both in solution and in the solid state. It helps to study the mixtures of analysis and it is an invaluable tool in understanding protein and nucleic acid structure and function. We use this technical to see the type and number of silicone we have in breast implant, in gel and envelope of that implant and in the tissue around. We analyze a virgin implant and 2 implants from one patient: one was intact and another one date of 3 years and show severe gel bleed. We study also 3 connectives tissues capsule and we assume that their implant were intact. For each experiment, we made proton, Carbone and Silicon spectrometry. Those are our main nucleus. Solid NMR Experiment
They remove the remains of the gel from inside the envelope with the scalpel and they wash the envelope with chloroform. After, they cut it into small sample and stacked in 7 mm ZrO2 rotors. Here are the conditions used:
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Resonance frequency (ppm) The 90 pulse 3.7 length(s) Relaxation 4 Number of scan 100 Table .(Title)

H ( MAS) 400.15

13

C (CP/MAS) 100.63 4 6 3000

13

C ( MAS) 100.63 5 4 1800

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Si (CP/MAS) 79.49 7.2 15 3000

We use (CP/MAS) CrossPolarization to reduce the measuring time for signal/ noise augmentation. So, the latter is due of long relaxation time. The CP technical allowed having a shorter protons relaxation time. Tetramethylsilane was used as reference. Results and discussion We find many type of silicone with this technical - C (CP/MAS) - C ( MAS): detected diphenylsiloxane in 3 samples - Si (CP/MAS): one pic -22.4 ppm polydimethylsiloxane - H ( MAS): the signal 7-7.4 ppm

We can see the presence of those compounds but it is difficult show the degradation of our implant. Liquid NMR Experience This experience was used to isolate the lipid and free unreacted silicone from the tissues capsule. They extracted the sample for 48 with CHCl3; the solution was filtrated and evaporated. They add CDCl3 as reference and they put the mixture into 5 mm NMR tube. Spectroscopy Resonance frequency ppm Pulse length s Relaxation delay Table . (Title)
1

H 300.13 10.5 1

13

C 75.44 8.6 2

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Si 59.63 9.8 30

The H-H Cosy and the Si- H HMBC were also made When, we compared the Cosy of the virgin sample to the one of the explanted shell we see some differences: 4.1ppm 0.6-2.8ppm 5.3ppm Table .(Title)
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characterization The resonance of glycerol backbone the CH2 and CH3 of fatty acid Olefin proton

Si spectrum was made from 10 to -70ppm. For virgin implant we have 2 significant peaks:

At 7.4ppm, the signal is due to HMDS which added to the matrix during the manufacturing At -21.7ppm, the signal is due to D group with different chemical environment. The 13C spectrum of virgin : Chemical shifts (ppm) characterization Table . (Title) It revealed the presence of oligomers and lipid which is not visible in solid state NMR. This is due to the CHCl3 used in solid state NMR On extraction of connective tissues For patient with silicone implant, the tissues around the capsule are solidified and after a long time it s getting calcified. So those parts become sensitive with a possibility of implant 29.7 and 14.7 CH3 and CH2 of siloxane 1.2 and 0.8 Free silicone 1 Carbon methyl of D unit

compression or rupture. So we made 29Si- 1H HMBC experiment to detect that sensitive nucleus. The table of the result (1H) ppm (29Si) ppm -22 0 10 7,5 -24

0,41 0,43 0,43 0,32 0,21 Table .(Title)

In those connective tissues we found many silicone species, which show us the presence of silicone bleeding from implant. Spectroscopy on extra gel We made this one to see the effect of the lipid into the gel and if he is contribute on his aging process. We made the spectra and we compared to those in the shell. We noticed that the quantities of free silicone, lipids and cyclic polysiloxane were lower in gel than in shell. The 1H NMR experiment of explanted envelopes and gel show that the lipid concentration was important in the shell. That can due to the amount of silica in the envelope. After these experiments, we see that: The silicone envelope absorbs the lipid. so that caused the degradation of elastomers which form the implant The extent of lipid infiltration make the polymers chain mobile, so the migration to the body become easier The various silicone found in connective tissues capsule can explain the effect of implant bleeding So, we can conclude that the solid and liquid NMR experiments help us to find the degradation of silicone breast implant
c. Gas Chromatography coupled Mass Spectrometry

Gas chromatography (GC) is an analysis technique used in chemistry for separating and analyzing compounds of a mixture that con e vaporized. They are separated between the moving phase and a stationary phase. In generally, the mobile phase is an inert gas called carrier gas which transports the compounds to the column. We used generally Helium. The stationary phase can be a solid or a liquid contained in a metal tube called column.

In this case, the GC conditions are as follows: Injector port temperature : 200 C Column : 5% phenylmethylsilicone stationary phase; 0.25mmx30mx0.25m

The Mass Spectrometry (MS) is a technique used to detect and to determinate the composition of molecules. The principle consists of ionizing chemical compounds to generate charged molecules or molecule fragments and measurement of their mass-to-charge ratios .

The MS instruments composed of three modules: an ion source, a mass analyzer and a detector.
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Ion source can convert gas phase sample molecules into ions. The ions are then transported by magnetic or electric fields to the mass analyzer. A lot of ion source exist but in this work, they use EI (electron impact). The electron impact source consists of a heated filament that produces electrons which are accelerated to another electrode called the ion trap. Sample vapor diffuses into the electron beam and become ionized and fragmented, the size depending on the electron energy which is controlled by the accelerating potential on the ion trap electrode. Low energy electrons produce molecular ions and larger fragments, whereas high energy electrons produce many smaller fragments and possibly no molecular ions. After the ions are produced, they are driven by a potential applied to an ion-repeller electrode, away from the ion source into the accelerating region of the mass spectrometer, where mass analysis takes place. In the mass analyzer, the ions separated according to their mass-to-charge ratio. The two laws govern the dynamics of charged particles in electric and magnetic fields in vacuum are Lorentz force law and Newton's second law. Detector converts ionic courant to electric courant.

They work in SIM mode, it means that we select a mass fragments following criteria : high characteristic identification value for the compound (high mass fragment) and a high abundance of the fragment ( high sensitivity).(3) The goal of the experience of article is to find a method very sensitive and reproductible using a low solvent volume with an easy preparation sample. GC has the reputation to be speed and reproductible so this technical can to be a good technique.

In this work, they analyse polydimethylsiloxanes used in fabrication of silicone gel-filled breast implants. Only cyclic low molecular weight siloxanes (LMW) : dimethylsiloxanes (D3 to D6) was identified in GC, because the hight molecular weight siloxanes (HMW) cannot be separated with a capillary column. They was interested by plasma and blood because plasma is only one of blood components that could possibly contain siloxanes.

Materials and reagents They use cyclic siloxanes purified, tetrakis(trimethylsiloxy)silane (M4Q) internal standard for quantification, hexane. Internal standard is obtained from Gelest Inc. and hexane from Merck KGaA. They extracted cyclic siloxanes from plasma and blood from women with silicone gel-filled implants and from control subject. For this, they stored 10 plasma samples of patient group at 3C in the dark for nearly one year and the 4 blood sample at -20Cfor three months. For the control samples, there are six plasma samples and two blood samples which stored in the same conditions as the patient group They introduced 0,5 and 1,0 ml sample volumes, 100 L of standard solution, 100 L of M4Q solution in 2,5 and 10 mL vials. Vials of 10 mL are used to the sample with a volume of 1,0 ml. Each sample has a different concentration of methanolic multisiloxane 1, 10, 100 g/ml. The samples were extracted with 1,0 ml of hexane. They were vortex-mixed at high speed setting for 5 min and centrifuged at 10000 rpm for 20 min. Each concentration experiment was repeated 3 times to establish reproductibility. Results and discussion At the beginning of the study, they try extracting plasma samples with the 2,5 ml vials but the extraction efficiencies was low for D4, D5 and D6, in the range of 45-70%. So, they changed the method and used 10 ml vials because there is a loss of analyte by volatilization and with low volumes, we have less siloxanes than in higher volumes. Thus, they tried with the 10 ml vials and they had better results with higher rates for D4, D5, and D6 and the loss due to analyte is significantly reduced.

Table .Cyclic siloxanes in plasma of women currently have or previously had silicone gelfilled implants

Table .Cyclic siloxanes in whole blood of women currently have or previously had silicone gel-filled implants They took seven women with breast implants and seven after breast implants explantation to analyzed ten plasma samples and four blood samples and eight control subjects to measured six plasma samples and two blood samples. For control subjects, no cyclic siloxanes found in plasma with detection limits of 2 pg/L. They saw in the chromatograms humps which can indicate the possible presence of siloxanes in very low quantities because humps corresponded to the retention peaks of the siloxanes. For women currently have or previously had silicone gel-filled implants, D3 and D4 was detected in 7 of 10 plasma samples and in 2 of 4 whole blood samples. Only for one woman, they found in plasma D4, D5 and D6 her implant was explanted 5 years ago after a rupture of right breast and gel bleed of left breast. D3 found in low concentrations, its due to this volatility which causes a high loss rate during sampling and sample preparation.

Siloxanes found in plasma from women who had intact implants without relation with implantation time and siloxane concentration. (Table 1) Its the same conclusion for woman who had explanted breast implants. In contrast, there were two women: one who explanted an intact implant four years previously and another one whose explanted implants exhibited signs of gel bleed, at the time of implant removal, five years previously. The possible reasons for these results could be a very low bleeding rate of the implant no detectable with their MS detector. In whole blood, the concentrations of siloxanes are higher compared to the same levels in plasma. Its normal because plasma is contained in whole blood.

Conclusion All these results demonstrate that siloxanes circulate in blood. Many hypothesis appear : siloxanes may be leave the body via the lungs or kidneys, or accumulate in organs over time. In humans, these processes exist but their dynamics are still unknown. The results indicate that there is a relationship between exposure to silicone gel filled implants and LMW cyclic siloxane contamination in plasma. In blood, the article doesnt give data. But, we may do the hypothesis that the concentration of siloxanes in blood vary like siloxanes in plasma. No statistical analysis was possible because the investigated numbers of plasma and blood is too limited. Nevertheless, this study shows that siloxanes prostheses increases the quantity of LMW cyclic siloxanes in the body and they stay a long time after broken silicone implants were removed.

C. Spread of silicone: Report and discussion In that report, they are talking about a 45 years woman who has breast implant. After 6 years, she felt pain in the right breast. They have used ultra sound and the mammography to identify the why of that pain but nothing is shown. After 18 months, she still felt a lump in the same place. This time, ultra sound showed some leakage of implant in the right breast. A fine needle shows a silicone granuloma. After some studies, they assume that they were rupture of silicone implant and the migration of the silicone in the lymph node. They decide to remove the implant. This case shows that the diagnostic of silicone rupture is not easy. The silicone gel can keep his memory and stay close to the capsule. That form can cause some health risk. So the ultra sound is a method to detect the silicone in breast implant

CONCLUSION By using these different techniques, we find similar results /the concentration of siloxanes in plasma, serum and blood for women have or had breast implants is significantly higher than the one for people (women) who never put breast implant. So, we can conclude that siloxanes can migrate in tissues, circulate in blood, and accumulate in organs over time. The limit of detection for GC-MS is 2 pg/L, it appears like a quite technique. The technique which is considered most suitable is AAS because this sensitive technique is appropriate for metal elements and can analyze traces. With this technique we can also quantify high concentration because its linear to 1000 g/L. Others techniques like infra-red spectroscopy, Fourier transform infra red spectroscopy, GFAAS, EDAX energy dispersive analysis of X rays have been used to identify silicone.

D. ANNEXES

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Si solid-state CP/MAS NMR spectra of a virgin implant envelope (spectrum a) and a 3 year old envelope of an implant without (spectrum b) and with gel bleed (spectrum c). 3000 FIDs were recorded with a 7 mm CP/MAS probe, contact time was 10 ms. *spinning side bands, Dpolymethylsiloxane, Q2Q4amorphous silica compounds, MQHMDS. 2.

Chemical structures of different linear and cyclic siloxanes detectable by NMR spectroscopy.

3.

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C MAS solid-state NMR spectra (decoupled) of a virgin implant envelope (spectrum a) and a 3 year old envelope of an implant without (spectrum b) and with gel bleed (spectrum c). 1800 FIDs were recorded with a 7 mm CP/MAS probe with a 5000 Hz spinning speed. The upper part of Fig. 3 shows the expanded spectra from 21.0 to 33.0 ppm containing lipid resonances. *spinning side bands, Dpolymethylsiloxane, Dph2diphenylsiloxane. 4.

H liquid NMR spectra of a virgin implant (spectrum a) and 3 year old implant without and with gel bleed (spectra b and c). 16 and 80 (spectrum c) FIDs were recorded with the 5 mm TBI and QNP (3 year old implant) probe. The lower part of Fig. 2 shows the expanded spectra from 1.0 to 3.0 ppm. Peaks at 2.8 ppm are also present in explanted samples, but with very low intensity. 5.

H solution NMR spectra of a phospholipid mixture (80% phosphatidylserine) (spectrum a) and a 3 year old sample without gel bleed (spectrum b). For experimental conditions see legend Fig. 2. The vertical inset shows the expanded area from 3 to 1 ppm. 7.EXTRA GEL

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Si liquid-state NMR spectra of the gels of a virgin sample (spectrum a) and a 3 year old sample (spectrum b) without gel bleed (10 to 70 ppm). 1024 scans and a relaxation delay of 30 s were applied. The vertical inset shows the area from 18 to 22.5 ppm.

9.
1

H liquid-state NMR spectra of the silicone gels of a virgin implant (spectrum a), a 3 year old implant without and a 3 year old implant with gel bleed (spectra b and c). 128 (spectrum a) and 256 (spectra b and c) FIDs were recorded with the 5 mm TBI and QNP (3 year old implant) probe. The lower part of Fig. 9 shows the corresponding expanded area from 1.0 to 3.0 ppm.

(a) 1H, 1H COSY spectrum of the virgin sample (08 ppm), (b) 3 year old implant without

gel bleed (08 ppm) and (c) phospholipid mixture as a reference sample (08 ppm). The 1H, 1H COSY experiments were carried out with a Bruker TBI probe. Transients were acquired into 2048 data points with 72 scans per increment and 512 increments in the F1-axis, which was zero filled to 1024 prior to FT. The spectra were symmetrized and no line broadening was applied. The relaxation delay between successive pulse cycles was 2 s. In addition, gradient pulses (1000 s) were used for selection.

GC-MS

D3

D5

D6