New Studies Of A Liquid Of Life -- Lung Surfactant

Chemical engineers at the University of California, Santa Barbara areworking to characterize and refine a special substance that is especiallyimportant to babies born prematurely: it allows them to breathe.

Worth more than all the gold in the world to these premature babies andtheir families, it's a miraculous lifesaver.

Without it many thousands more babies would die each year -- before everleaving the neonatal intensive care unit. Some adults with lung disease arehelped by it as well.

Called lung surfactant, this very special substance -- a mixture oflipids and proteins -- coats the inside of all mammalian lungs and allows themto draw breath, by reducing the work of breathing.

In the U.S., 40,000 premature babies per year are born without enoughlung surfactant, and thousands of deaths result. The typical preemie has only1/20 of the lung surfactant needed to breathe. Fortunately, additional lungsurfactant can be administered.

For the past decade, doctors have been able to insert one of two typesof lung surfactant directly into the babies' lungs. Both were approved by theFood and Drug Administration in 1989, but each has its drawbacks.

"Our research program is directed at determining basic physical measures of an ideal replacement surfactant, and relating these measures to the components found in natural lung surfactants," said Joseph A. Zasadzinski,professor of chemical engineering and materials, who has been working on lungsurfactant for many years with his research group. (See websitehttp://www.chemengr.ucsb.edu/people/faculty/zasadzinski.html)

Graduate student Junqi Ding (See websitehttp://www.engineering.ucsb.edu/~junqi) will present the group's latest researchfindings, funded by the National Institutes of Health, at the national meetingof the American Chemical Society in New Orleans, on Sunday, August 22.

"An ideal replacement formulation would be a mixture of syntheticlipids, in a ratio based on a good understanding of their individual functionsin lung surfactant, combined with simple peptide sequences which capture thefull activity of the native lung surfactant mixture," said Zasadzinski.

"Such a mixture could be easily and cheaply produced without any batchto batch variance," he said. "The composition could be tailored to optimize theproperties of the mixture for the treatment of specific cases."

Surfactant replacement therapy has been shown to reduce mortality ratesby 30 to 50 percent for infants with neonatal respiratory distress syndrome.And, 80 percent of the decline in the infant mortality rate in the United Statesbetween 1989 and 1990 (the year in which surfactant therapy was introduced)could be attributed to surfactant therapy, according to the researchers.

While lung surfactant replacement therapy has been of great help, itneeds improvement in order to treat infants more effectively, in a more refinedway, and to begin to address adult respiratory distress syndrome, theyexplained.

Adult respiratory distress syndrome is a broad class of lung diseasethat can be related to: trauma, smoking, long term chronic obstructed lung disease, pneumonia, the hanta virus, near drowning, and other unknown causes.

When neonatal respiratory distress occurs, insufficient surfactantresults in a progressive failure of the lungs, manifested clinically by

• collapsed alveoli -- the little sacs that form the lungs -- calledatelectasis;
• stiff lungs or decreased lung compliance;
• decreased functional residual capacity -- a measure of the amount of airleft in the lungs after exhalation;
• systemic hypoxia or oxygen starvation; and,
• lung edema -- bleeding in the lungs -- explained Zasadzinski.

The alveoli, in which the oxygen and carbon dioxide are exchanged withthe blood, are very small, less than 1/10 of a millimeter in diameter, and arelined by a thin layer of liquid. When air and liquid are in contact, a strong force known as surfacetension tends to compress the alveoli. The primary function of lung surfactant is to form amonolayer at the alveolar air/water interface capable of lowering the surface tension to nearzero values, said Ding. The physical and chemical properties of the monolayerdetermine how low the surface tension can be.

That way premature babies don't have to use so much of their energy tobreathe. The lower the surface tension between the liquid and the gas, theeasier it is to breathe, said Zasadzinski.

The minimum surface tension upon expiration is limited by monolayercollapse, said Ding. The mechanical properties of the monolayer, especially theshear viscosity, are directly related to the mechanism of monolayer collapse.

Ding explained the use of a magnetic needle viscometer along withfluorescence microscopy, Brewster Angle Microscopy and Atomic Force Microscopyto study model surfactant systems, as well as natural surfactants, to determinethe relationship between lung surfactant components and monolayer viscosity.

He reported the systematic measurements of monolayer viscosity as afunction of lipid chain length, protein concentration, and temperature.

"You want the monolayer to spread easily," said Ding, "to flow and coatthe alveolar surface during breathing in and out."

The research leads to questions like, "Why did nature select only aparticular lipid with a 16-length carbon chain to be the main component ofnatural surfactant?" asked Ding. "Mother nature chose it because it's the rightviscosity for expanding and contracting."

Of the two FDA-approved surfactant replacement therapies currently inuse, one is a purely synthetic formulation, called Exosurf. It contains about 80percent dipalmitoylphosphatidylcholine, or DPPC, combined with hexadecanol andtyloxapol, explained Zasadzinski. DPPC is also the main component of naturalhuman lung surfactant (about 60 to 70 percent by weight in normal adults). Butthe hexadecanol and tyloxapol have no relation to natural lung surfactant.

The second formulation, called Survanta, is an organic extract of bovinelung surfactant (about 60 percent DPPC) and contains the hydrophobic lungsurfactant specific proteins SP-B and SP-C, supplemented with synthetic palmiticacid (PA) and tripalmitin, said Zasadzinski. Administration of these replacementsurfactants to infants has proven to be very effective.

However, progress in optimizing new replacement surfactants has beenhindered by the lack of a fundamental understanding of the lung surfactantsystem, and the specific roles of each of the individual components, explained Zasadzinski.

Proposed replacement formulations can be divided into four classes:

• natural lung surfactant extracts,
• modified lung surfactant extracts (natural extracts supplemented with synthetic lipids, typically palmitic acid and DPPC),
• synthetic formulations modeled on natural lung surfactant, and,
• synthetic formulations with no relation to natural lung surfactant.

"Natural lung surfactant extracts have been shown to be effective bothin vitro and in vivo, but human sources are limited, and animal sources aredifficult and expensive to purify," said Zasadzinski. "They pose the risk ofcontaining viral or proteinaceous contaminants. The recent link between bovinespongiform encephalopathy (mad cow disease) and human Creutzfeldt-Jakob diseaseis a reminder of the possible problems in introducing animal products intohumans."

He continued, "As with any natural product, both natural and modifiedlung surfactant extracts can have a wide variability in composition from batchto batch."

"These concerns have led to the study of synthetic lung surfactantformulations for which composition can be better defined," said Zasadzinski."Mixtures containing synthetic lung surfactant lipids with highly purifiednatural proteins such as SP-B and SP-C have been shown to be of comparable orbetter activity than many natural extracts. However, a fundamental rationale forchoosing the ratio of lipids to proteins for such mixtures is still lacking."

"Furthermore, lung surfactant proteins are difficult and expensive toobtain in a highly purified form and there does not yet exist a suitablehost-vector system for the large-scale production of SP-B through geneticengineering techniques (which is a common problem for surface-active proteins),although production of recombinant human SP-C has begun," said Zasadzinski.

"There are also continuing efforts to replace the proteins by simplerpeptides or polymers, with variable success," he said.

He continued, "Although the mortality rate for infants with neonatalrespiratory distress syndrome has been declining since the advent of successfulsurfactant replacement strategies, the incidence of low birth weight infants hasbeen steadily rising. The availability of standardized replacement formulationswith a fully-quantified mechanism of action could lead to a more widespread useof surfactant replacement therapy to help save the lives of both infants andadults

"Our research program is directed at determining basic physical measures of an ideal replacement surfactant, and relating these measures to the components found in natural lung surfactants," said Joseph A. Zasadzinski,professor of chemical engineering and materials, who has been working on lungsurfactant for many years with his research group. (See websitehttp://www.chemengr.ucsb.edu/people/faculty/zasadzinski.html)

Graduate student Junqi Ding (See websitehttp://www.engineering.ucsb.edu/~junqi) will present the group's latest researchfindings, funded by the National Institutes of Health, at the national meetingof the American Chemical Society in New Orleans, on Sunday, August 22.

"An ideal replacement formulation would be a mixture of syntheticlipids, in a ratio based on a good understanding of their individual functionsin lung surfactant, combined with simple peptide sequences which capture thefull activity of the native lung surfactant mixture," said Zasadzinski.

"Such a mixture could be easily and cheaply produced without any batchto batch variance," he said. "The composition could be tailored to optimize theproperties of the mixture for the treatment of specific cases."

Surfactant replacement therapy has been shown to reduce mortality ratesby 30 to 50 percent for infants with neonatal respiratory distress syndrome.And, 80 percent of the decline in the infant mortality rate in the United Statesbetween 1989 and 1990 (the year in which surfactant therapy was introduced)could be attributed to surfactant therapy, according to the researchers.

While lung surfactant replacement therapy has been of great help, itneeds improvement in order to treat infants more effectively, in a more refinedway, and to begin to address adult respiratory distress syndrome, theyexplained.

Adult respiratory distress syndrome is a broad class of lung diseasethat can be related to: trauma, smoking, long term chronic obstructed lung disease, pneumonia, the hanta virus, near drowning, and other unknown causes.

When neonatal respiratory distress occurs, insufficient surfactantresults in a progressive failure of the lungs, manifested clinically by

• collapsed alveoli -- the little sacs that form the lungs -- calledatelectasis;
• stiff lungs or decreased lung compliance;
• decreased functional residual capacity -- a measure of the amount of airleft in the lungs after exhalation;
• systemic hypoxia or oxygen starvation; and,
• lung edema -- bleeding in the lungs -- explained Zasadzinski.

The alveoli, in which the oxygen and carbon dioxide are exchanged withthe blood, are very small, less than 1/10 of a millimeter in diameter, and arelined by a thin layer of liquid. When air and liquid are in contact, a strong force known as surfacetension tends to compress the alveoli. The primary function of lung surfactant is to form amonolayer at the alveolar air/water interface capable of lowering the surface tension to nearzero values, said Ding. The physical and chemical properties of the monolayerdetermine how low the surface tension can be.

That way premature babies don't have to use so much of their energy tobreathe. The lower the surface tension between the liquid and the gas, theeasier it is to breathe, said Zasadzinski.

The minimum surface tension upon expiration is limited by monolayercollapse, said Ding. The mechanical properties of the monolayer, especially theshear viscosity, are directly related to the mechanism of monolayer collapse.

Ding explained the use of a magnetic needle viscometer along withfluorescence microscopy, Brewster Angle Microscopy and Atomic Force Microscopyto study model surfactant systems, as well as natural surfactants, to determinethe relationship between lung surfactant components and monolayer viscosity.

He reported the systematic measurements of monolayer viscosity as afunction of lipid chain length, protein concentration, and temperature.

"You want the monolayer to spread easily," said Ding, "to flow and coatthe alveolar surface during breathing in and out."

The research leads to questions like, "Why did nature select only aparticular lipid with a 16-length carbon chain to be the main component ofnatural surfactant?" asked Ding. "Mother nature chose it because it's the rightviscosity for expanding and contracting."

Of the two FDA-approved surfactant replacement therapies currently inuse, one is a purely synthetic formulation, called Exosurf. It contains about 80percent dipalmitoylphosphatidylcholine, or DPPC, combined with hexadecanol andtyloxapol, explained Zasadzinski. DPPC is also the main component of naturalhuman lung surfactant (about 60 to 70 percent by weight in normal adults). Butthe hexadecanol and tyloxapol have no relation to natural lung surfactant.

The second formulation, called Survanta, is an organic extract of bovinelung surfactant (about 60 percent DPPC) and contains the hydrophobic lungsurfactant specific proteins SP-B and SP-C, supplemented with synthetic palmiticacid (PA) and tripalmitin, said Zasadzinski. Administration of these replacementsurfactants to infants has proven to be very effective.

However, progress in optimizing new replacement surfactants has beenhindered by the lack of a fundamental understanding of the lung surfactantsystem, and the specific roles of each of the individual components, explained Zasadzinski.

Proposed replacement formulations can be divided into four classes:

• natural lung surfactant extracts,
• modified lung surfactant extracts (natural extracts supplemented with synthetic lipids, typically palmitic acid and DPPC),
• synthetic formulations modeled on natural lung surfactant, and,
• synthetic formulations with no relation to natural lung surfactant.

"Natural lung surfactant extracts have been shown to be effective bothin vitro and in vivo, but human sources are limited, and animal sources aredifficult and expensive to purify," said Zasadzinski. "They pose the risk ofcontaining viral or proteinaceous contaminants. The recent link between bovinespongiform encephalopathy (mad cow disease) and human Creutzfeldt-Jakob diseaseis a reminder of the possible problems in introducing animal products intohumans."

He continued, "As with any natural product, both natural and modifiedlung surfactant extracts can have a wide variability in composition from batchto batch."

"These concerns have led to the study of synthetic lung surfactantformulations for which composition can be better defined," said Zasadzinski."Mixtures containing synthetic lung surfactant lipids with highly purifiednatural proteins such as SP-B and SP-C have been shown to be of comparable orbetter activity than many natural extracts. However, a fundamental rationale forchoosing the ratio of lipids to proteins for such mixtures is still lacking."

"Furthermore, lung surfactant proteins are difficult and expensive toobtain in a highly purified form and there does not yet exist a suitablehost-vector system for the large-scale production of SP-B through geneticengineering techniques (which is a common problem for surface-active proteins),although production of recombinant human SP-C has begun," said Zasadzinski.

"There are also continuing efforts to replace the proteins by simplerpeptides or polymers, with variable success," he said.

He continued, "Although the mortality rate for infants with neonatalrespiratory distress syndrome has been declining since the advent of successfulsurfactant replacement strategies, the incidence of low birth weight infants hasbeen steadily rising. The availability of standardized replacement formulationswith a fully-quantified mechanism of action could lead to a more widespread useof surfactant replacement therapy to help save the lives of both infants andadults."