In title 12, 22, 11 are in subscript. / Thesis (PhD)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: The aim of this study was to test a synthetic protein-free surfactant preparation, LPM-l,
with the same chemical composition as commercially available Exosurf (Glaxo
Wellcome), but containing in addition, a sugar, trehalose (TRE). Towards this end, a
study was designed to firstly test the hypothesis that the true difference in acute
physiological effects between a mixture of oppe, tyloxapol, hexadecanol and trehalose
(LPM-l), and Exosurf, (Oppe, tyloxapol and hexadecanol) is zero, in a surfactantdeficient
animal model. A second study addressed the physiological effects of oppe,
hexadecanol, tyloxapol and trehalose (LPM-l) compared to treatment with trehalose
(TRE) or saline, in order to determine (1) the contribution of TRE to the mixture of
oppe, hexadecanol and tyloxapol, and (2) to assess the effect of the LPM-l surfactant
replacement on the epithelial lining fluid composition by means of analysing bronchoalveolar
lavage fluid. Thirdly, the effects of TRE and / or calcium were studied on the
surface properties of oppe suspensions, by in vitro analysis using the ring detachment
method of Du Nouy
The in vivo research comprised of two studies, performed in randomised controlled
fashion. In the first study, 24 New Zealand White adult rabbits were randomised into
4 groups, while in the second study, 15 animals were randomised into 3 groups. In the
first in vivo study, three synthetic surfactants, LPM-l, Exosurf and LPM-2, and a saline
group were tested. LPM-l is a new formulation that consists ofa mixture of Df'PC, TRE,
hexadecanol and tyloxapol. LPM-2 is a formulation with a composition equivalent to that
of commercially available Exosurf, prepared on site. In both studies animals were subjected to repeated lavage with large volumes of warm
saline (25 ml/kg) in order to establish surfactant deficiency and acute lung injury. Five
minutes after the last lavage, vehicle, i.e. surfactants LPM-l, Exosurf, or LPM-2, or
saline, in the first in vivo study, and LPM-l, TRE or saline in the second in vivo study,
was instilled, and the course of the animals followed over the next 3 hours. Ventilator
settings were standardized before and after lavage. The effects of surfactant treatment on
gas exchange (arterial Pa02, oxygenation index (Ol), arterial-alveolar oxygen (a/A)
ratio), percentage calculated shunt, and total dynamic respiratory compliance (CRSdyn),
and histopathological changes were compared with changes in saline treated controls.
Arterial blood gases in 100% oxygen and CRSdynwere measured before and after lavage,
at 15 minute intervals for the first 30 min, then at 60, 90, 120, and 180 min after vehicle
instillation.
Oxygenation improved to a similar extent after LPM-l and Exosurf instillation,
surpassing that of LPM-2 or saline. Overall, intratracheal instillation of both Exosurf and
LPM-l, rapidly improved the gas exchange and reduced the intrapulmonary shunt, but
did not restore the lung to its pre-lavage condition. From the 2nd in vivo study it was
evident that trehalose-only, was inefficient as a lung surfactant, failing to improve
oxygenation indices or the calculated percentage shunt, or influencing respiratory
compliance. The addition of the sugar, trehalose (TRE), to the on-site 'Exosurf mixture
(LPM-2) brought the activity of the resultant LPM-l to the same level as that of
commercial Exosurf, but failed to raise the activity above that of Exosurf. These
physiological improvements were sustained for up to 3 hours. Saline-treated animals had no improvement in gas exchange despite management with variable PIP (to maintain a
tidal volume of -1 0 ml / kg) and constant PEEP of 5 cm H20.
In-vitro results, obtained by the Ou Nouy tensiometer, showed higher mean ordinate
surface tension values for the OPPC-only and DPPC + TRE mixtures, and the slopes of
their respective graphs smaller in magnitude than those of the other formulations,
suggesting that these formulations had less surface tension-lowering capability than the
other surfactants. At 20°C (20 mg / ml DPPC-surfactants) the mean ordinate values of
OPPC and OPPC + TRE, 70.13 and 69.47 dyne / cm, respectively, were not significantly
different from each other. The mean ordinate values of LPM-l and the formulation
containing OPPC + TRE + tyloxapol + CaCh were lower, but similar, as were the values
of LPM-2 (on-site Exosurf) and LPM-2 + CaCho Thus, three internally homogeneous
subgroups could be identified which differed significantly, namely: DPPC and DPPC +
TRE, LPM-2 and LPM-2 + CaCh, and DPPC + TRE + tyloxapol + CaCh and LPM-l.
Similar conclusions apply to the ordinate values of the surfactants at 37°C, and to the
mean slope values at 20°C, with the exception that the subgroups, LPM-2 and LPM-2 +
CaCh, and LPM-l and OPPC + TRE + tyloxapol + CaCh are not so clearly separated. A
similar analysis of mean slope values was performed. Here too a significant difference
between substances was found, OPPC alone or in combination with TRE, again being
significantly different from the other surfactants.
The most prominent light microscopy findings of the lungs of animals included general
lymphatic dilatation, congestion and lung polymorphonuclear infiltration, with no
difference between study groups. Hyaline membranes were present in all surfactant
groups, but significantly more so in the saline treated group. In the first in vivo study, the presence of neutrophils in the lung interstitiwn as well as alveoli, was a common finding
in all of the study groups towards the end of the study protocol. A significant increase in
the BAL-fluid neutrophil count occurred in all animals, concurrent with a significant
decrease in the BAL macrophage count. No significant change occurred in the peripheral
neutrophil count during the 3-hour study, suggesting recruitment of neutrophils from
storage pools. Treatment with synthetic surfactant (LPM -1) did not have a significant
effect on modifying the inflammatory response, since there was no significant difference
in the BAL-derived cell counts between the LPM-1 and -saline groups. Epithelial damage
was a consistent finding in all groups. The damage was more evident by electron
microscopy examination and included hydropic changes, most readily observed in the
mitochondria. The airspaces of study subjects showed the presence of oedema fluid. This
luminal oedema appeared to be more prominent in the control group and LPM-2 (on site
'Exosurf') group. Organellar debris, probably originating from lysis of epithelial cells,
was present, despite treatment with synthetic surfactant. The electron microscopical
appearance of the epithelial-lined substance ("hyaline membranes") in the present study
showed a marked variability within groups as well as within the same case. The majority
of cases showed a mix of membrane types with both granular and fibrillar materials
present within the same membrane. In some cases there were layering of the membranes
into distinct bands. The instillation of LPM-l resulted in the formation of a slightly
different type of epithelial lining fluid after lavage, when compared to the prelavage
composition. The most pronounced changes occurred within the fatty acids, whilst the
phosphatidylcholine values remained unchanged. Palmitic acid concentrations (C16:0)
increased significantly, suggesting enrichment of the epithelial lining fluid after instillation of LPM-l. This increase in C16:0 was concurrent with significant decreases
in the percentage C16:1, C18:0, and C18:2. In contrast to previous studies, we describe
higher levels for phosphatidyldimethylethanolarnine (PEA). An explanation may be that
the lipid identified as PEA, was in fact partly phosphatidylglycerol (PG)-a lipid whose
accurate identification was precluded for technical reasons.
After surfactant instillation, the PC/SM ratio, a reflection of the lecithin / sphingomyelin
(LIS), decreased significantly in the TRE-group between the first and final lavage, but
remained statistically unchanged in the animals treated with LPM-l or saline. The change
in ratio was mainly accounted for by a decrease in BAL-fluid PC content together with a
rise in SM content. A poor correlation existed between the BAL-derived PC/SM ratio and
indices reflecting oxygenation status (a/A ratio, Ol), as well as the CRSdynat the time of
the final lavage.
In conclusion, the primary hypothesis was accepted, LPM-l performed similarly to
Exosurf in vivo, improving oxygenation, but not CRSdyn.None was clearly superior to the
other.
Some questions remain. The reason why LPM-l (LPM-2 + TRE) did not behave in a
superior manner, in vivo, to Exosurf, is partly unclear. This finding was somewhat
surprising since the chemical composition of Exosurf and LPM-2 did not differ, and the
addition of TRE to LPM-2 (on-site Exosurf), did improve the in vivo activity of the
resultant LPM-l, above that of LPM-2. A possible explanation for observed differences
in performance include methodological issues, i.e. the preparation of the on-site formulations, especially that of LPM-2 (on-site Exosurf), may differ from the way in
which true commercial Exosurf is prepared. / AFRIKAANSE OPSOMMING: Die doel van die studie was om 'n sintetiese proteïn vrye surfaktant te ontwikkel en die
produk te vergelyk met 'n kunsmatige surfaktant reeds in kliniese gebruik.
Die bekende uit die literatuur en die onbekende van die produk wat evalueer sou word, lei
op tot die samestelling van die nul hipotese van die PhD naamlik dat geen verskil in
longfunksie sou gewys word tussen die toetsproduk en reeds gebruikte kommersiële
surfaktant nie. Die hipotese was dat 'n suiker (trehalose), in kombinasie met
Dipalmitoiel fosfatidielcholine (DPPC), gaswisseling en longfunksies sal verbeter vir 'n
long met 'n lae surfaktant konsentrasie.
Vir die studie is jong volwasse wit New Zealand konyne gebruik en is hulle met 'n
gestandaardiseerde en menslike manier gebruik in eksperimentele werk. Die diere is
onder intraveneuse narkose geplaas en verskillende kardiovaskulêre en pulmonologiese
aspekte is gemeet. Die long surfaktant is uitgewas deur middel van fisiologiese
soutoplossing wat tot liggaam temperatuur verhit is en daarna is die diere prospektief
gerandomiseer tot eksperimentele groepe.
Met vooraf bepaalde tydsintervalle is die fisiologiese metings herhaal en was die metings
toegespits daarop om longmeganiese funksie en gasoordrag vermoë te evalueer. Lig mikroskopiese en elektron mikroskopiese studies is ook op die longe gedoen en verder is
brongoalveolêre vloeistof ook ontleed.
Die groepe met ondersoek was:
I. oppe, heksadekanol, tyloxapol en trehalose (LPM-I).
2. oppe, heksadekanol, tyloxapol (LPM-2 :. LPM-I sonder trehalose). Hierdie is
'n proteïnvrye surfaktant plaaslik berei ( dieselfde samestelling as Exosurf).
3. Exosurf®. (Kommersiële preperaat reeds in gebruik). Hierdie is 'n proteïnvrye
sintetiese surfaktant.
4. Trehalose, 'n non-reduserende disakklaried van glukose.
Addisioneel is daar ook in vitro studies gedoen waann die oppervlakte spanmngs
aktiwiteite van die verskillende surfaktant oplossings vergelyk is.
Die statistiese analise is gedoen in samewerking met Prof. J. Maritz wat 'n unieke metode
ontwikkel en gepubliseer het om herhalende veranderlikes op 'n statisties
verantwoordbare manier te ontleed.
In die eerste van die studies, is LPM-I, Exosurf®, fisiologiese soutoplossing en 'n
plaaslik bereide "Exosurf" (LPM-2), met 'n chemiese samestelling identies aan dié van
kommersiële Exosurf®, evalueer.
In 'n tweede studie is die fisologiese effekte van LPM-I vergelyk met trehalose of
fisiologiese soutoplossing om die volgende te ondersoek:
1) Die bydrae van trehalose tot 'n mengsel van oppe, heksadekanol en tyloxapol
(LPM-2).
2) Die gevolg van LPM-l surfaktant toediening op die konyn se brongo-alveolêre
vloeistof samestelling. 'n Derde, in vitro studie, het die oppervlaktespannings-effekte van trehalose en of
kalsiumbyvoegings tot DPPC-oplossings gemeet deur middel van die ring metode van Du
Nouy,
In die eerste in vivo studie verbeter oksigenasie en persentasie longaftakking tot dieselfde
mate na LPM-l en Exosurf® toediening en word die hipotese van die proefskrif
bevestig. In die breë gesien, is die tydsprofiele van LPM-l en Exosurf® ten opsigte van
oksigenasie en persentasie longaftakking statisties betekenisvol beter en van 'n sneller
aard, as die tydsprofiele van dieselfde indekse na die toediening van fisiologiese
soutoplossing of LPM-2. Die tydsprofiel van dinamiese longvervormbaarheid, na die
toediening van LPM-I of Exosurf®, is dieselfde, maar betekenisvol beter as die
vervormbaarheid na toediening van LPM-2 of fisiologiese soutoplossing. Alhoewel die
oksigenasie indekse in die geval van LPM-l en Exosurf® betekenisvol verbeter oor die
studietydperk, vind volkome herstel tot die basislynwaardes (voor spoeling) nie plaas nie.
Bykomend, geen van die surfaktante het na toediening enige noemenswaardige
verbetering in longvervormbaarheid tot gevolg gehad nie.
Die rede vir die swakker vertoning van LPM-2 en Exosurf is onbekend en sal in opvolg
studie ondersoek word.
In die tweede in vivo studie is dit duidelik dat trehalose op sy eie, 'n oneffektiewe
surfaktant is aangesien die preperaat na toediening geen verbetering teweegbring ten
opsigte van oksigenasie indekse, persentasie longaftakking, of long-dinamiese
vervormbaarheid nie. Die toevoeging van trehalose tot LPM-2, om LPM-l te lewer,
neem wel die aktiwiteit van LPM-l tot dieselfde in vivo vlak as dié van kommersiële Exosurf®, maar slaag nie daarim om 'n hoër fisiologiese in vivo aktiwiteit as dié produk
te bereik nie. Die diere wat met fisiologiese soutoplossing behandel is toon geen
verbetering in enige fisiologiese parameter nie.
Die in vitro resultate wat verkry is deur die Du Nouy tensiometer toon hoër gemiddelde
ordinaat oppervlaktespannings waardes vir 'n formule wat slegs uit DPPC bestaan, asook
vir 'n mengsel van DPPC + trehalose. Die helling van die grafieke van hierdie
oplossings is ook kleiner as die van die ander formulas wat daarop dui dat DPPC op
sigself, en DPPC + trehalose, weinig vermoë het om oppervlaktespanning te verminder.
Daarteenoor verlaag die volgende oplossings die oppervlaktespanning ten opsigte van
gedistilleerde water betekenisvol en wel in In konsentrasie afhanklike manier by beide
21°C en 3rc: LMP-I-, LPM-2-, DPPC + trehalose + tyloxapol + CaCf2-, en LPM-2 +
CaCf2.
Die prominentste ligmikroskopiese bevindinge van die longe van die diere sluit in:
Algemene limfvat dilatasie, stuwing, en long neutrofiel infiltrasie. Betreffende hierdie
histologiese bevindinge is daar geen verskille aangetoon tussen die groepe nie.
Hialienmembrane was teenwoordig in al die groepe, maar betekenisvol meer in die groep
wat fisiologiese soutoplossing ontvang as vervangingsterapie. In die tweede in vivo
studie is daar 'n betekenisvolle styging in die neutrofiel- en daling in makrofaagtelling,
van die brongoalveolêre vloeistof spoeling in al drie die groep aangetoon. Terselfdertyd
vind geen noemenswaardige daling in die perifêre (sistematiese) neutrofieltelling plaas
nie. Hierdie bevindinge dui daarop dat die brongoalveolêre selveranderinge toegeskryf
kan word aan verwerwing van neutrofiele vanuit 'n longstoringspoel eerder as rekrutering vanuit die sistemiese sirkulatoriese poel. Surfaktant (LPM-l), behandeling het geen
betekenisvolle vermindering in long inflammasie teweeggebring nie. Epiteelskade was 'n
algemene ligmikroskopiese bevinding in al die groepe.
Die samestelling van die brongoalveolêre vloeistof verander na installering van
LPM-I. Die prominentste verandering word waargeneem in die vetsuur samestelling
terwyl die DPPC waardes onveranderd bly. Die vetsuur, palmitiensuur (palmitic acid),
(CI6:0), verhoog betekenisvol na toediening van LPM-l. Daarteenoor verminder die
konsentrasie van C16:1, C18:0 en C18:2. In kontras met vorige studies, beskryf die
huidige studie hoër konsentrasies van fosfatidieletanolamien, moontlik as gevolg van
tegniese verskille in die metingsmetodes. 'n Betekenisvolle verlaging in die
fosfatidielcholine:sfingomiëlien (PC/SM) verhouding word waargeneem tussen die eerste
en die finale longspoeling van die trehalose-groep, terwyl dit onveranderd bly in die diere
wat LPM-1 of fisiologiese soutoplossing ontvang.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52624 |
| Date | 12 1900 |
| Creators | Smith, Johan |
| Contributors | Coetzee, A. R., Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Anaesthesiology and Critical Care . |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 233 p. : ill. |
| Rights | Stellenbosch University |
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