Biochem Essay, Research Paper

Biochemistry

Prolonged Preservation of the Heart Prior to Transplantation

Picture this. A man is involved in a severe car crash in

Florida which has left him brain-dead with no hope for any

kind of recovery. The majority of his vital organs are

still functional and the man has designated that his organs

be donated to a needy person upon his untimely death.

Meanwhile, upon checking with the donor registry board, it

is discovered that the best match for receiving the heart of

the Florida man is a male in Oregon who is in desperate need

of a heart transplant. Without the transplant, the man will

most certainly die within 48 hours. The second man’s

tissues match up perfectly with the brain-dead man’s in

Florida. This seems like an excellent opportunity for a

heart transplant. However, a transplant is currently not a

viable option for the Oregon man since he is separated by

such a vast geographic distance from the organ. Scientists

and doctors are currently only able to keep a donor heart

viable for four hours before the tissues become irreversibly

damaged. Because of this preservation restriction, the

donor heart is ultimately given to someone whose tissues do

not match up as well, so there is a greatly increased chance

for rejection of the organ by the recipient. As far as the

man in Oregon goes, he will probably not receive a donor

heart before his own expires.

Currently, when a heart is being prepared for

transplantation, it is simply submerged in an isotonic

saline ice bath in an attempt to stop all metabolic activity

of that heart. This cold submersion technique is adequate

for only four hours. However, if the heart is perfused with

the proper media, it can remain viable for up to 24 hours.

The technique of perfusion is based on intrinsically simple

principles. What occurs is a physician carefully excises

the heart from the donor. He then accurately trims the

vessels of the heart so they can be easily attached to the

perfusion apparatus. After trimming, a cannula is inserted

into the superior vena cava. Through this cannula, the

preservation media can be pumped in.

What if this scenario were different? What if doctors were

able to preserve the donor heart and keep it viable outside

the body for up to 24 hours instead of only four hours? If

this were possible, the heart in Florida could have been

transported across the country to Oregon where the perfect

recipient waited. The biochemical composition of the

preservation media for hearts during the transplant delay is

drastically important for prolonging the viability of the

organ. If a media can be developed that could preserve the

heart for longer periods of time, many lives could be saved

as a result.

Another benefit of this increase in time is that it would

allow doctors the time to better prepare themselves for the

lengthy operation. The accidents that render people

brain-dead often occur at night or in the early morning.

Presently, as soon as a donor organ becomes available,

doctors must immediately go to work at transplanting it.

This extremely intricate and intense operation takes a long

time to complete. If the transplanting doctor is exhausted

from working a long day, the increase in duration would

allow him enough time to get some much needed rest so he can

perform the operation under the best possible circumstances.

Experiments have been conducted that studied the effects of

preserving excised hearts by adding several compounds to the

media in which the organ is being stored. The most

successful of these compounds are pyruvate and a pyruvate

containing compound known as

perfluoroperhydrophenanthrene-egg yolk phospholipid

(APE-LM). It was determined that adding pyruvate to the

media improved postpreservation cardiac function while

adding glucose had little or no effect. To test the

function of these two intermediates, rabbit hearts were

excised and preserved for an average of 24.5 1 0.2 hours on

a preservation apparatus before they were transplanted back

into a recipient rabbit. While attached to the preservation

apparatus, samples of the media output of the heart were

taken every 2 hours and were assayed for their content. If

the compound in the media showed up in large amounts in the

assay, it could be concluded that the compound was not

metabolized by the heart. If little or none of the compound

placed in the media appeared in the assay, it could be

concluded that compound was used up by the heart metabolism.

The hearts that were given pyruvate in their media

completely consumed the available substrate and were able to

function at a nearly normal capacity once they were

transplanted. Correspondingly, hearts that were preserved

in a media that lacked pyruvate had a significantly lower

rate of contractile function once they were transplanted.

The superior preservation of the hearts with pyruvate most

likely resulted from the hearts use of pyruvate through the

citric acid cycle for the production of energy through

direct ATP synthesis (from the reaction of succinyl-CoA to

succinate via the enzyme succinyl CoA synthetase) as well

as through the production of NADH + H+ for use in the

electron transport chain to produce energy.

After providing a preservation media that contained

pyruvate, a better recovery of the heart tissue occurred.

Most of the pyruvate consumed during preservation was

probably oxidized by the myocardium in the citric acid

cycle. Only a small amount of excess lactate was detected

by the assays of the preservation media discharged by the

heart. The lactate represented only 15% of the pyruvate

consumed. If the major metabolic route taken by pyruvate

during preservation had been to form lactate dehydrogenase

for regeneration of NAD+ for continued anaerobic glycolysis,

rather than by the aerobic citric acid cycle (pyruvate

oxidation), then a higher ratio of excess lactate produced

to pyruvate consumed would have been observed.

Hearts given a glucose substrate did not transport or

consume that substrate, even when it was provided as the

sole exogenous substrate. It might be expected that glucose

would be used up in a manner similar to that of pyruvate.

This expectation is because glucose is a precursor to

pyruvate via the glycolytic pathway however, this was not

the case. It was theorized this lack of glucose use may

have been due to the fact that the hormone insulin was not

present in the media. Without insulin, one may think the

tissues of the heart would be unable to adequately take

glucose into their tissues in any measurable amount, but

this is not the case either. It is known that hearts

working under physiologic conditions do use glucose in the

absence of insulin, but glucose consumption in that

situation is directly related to the performance of work by

the heart, not the presence of insulin.

To further test the effects of the addition of insulin to

the glucose media, experiments were done in which the

hormone was included in the heart preservation media5-7.

Data from those studies does not provide evidence that the

hormone is essential to insure glucose use or to maintain

the metabolic status of the heart or to improve cardiac

recovery. In a hypothermic (80C) setting, insulin did not

exert a noticeable benefit to metabolism beyond that

provided by oxygen and glucose. This hypothermic setting is

analogous to the setting an actual heart would be in during

transportation before transplant.

Another study was done to determine whether the compound

perfluoroperhydrophenanthrene-egg yolk phospholipid,

(APE-LM) was an effective media for long-term hypothermic

heart preservation3. Two main factors make APE-LM an

effective preservation media. (1) It contains a lipid

emulsifier which enables it to solubilize lipids. From this

breakdown of lipids, ATP can be produced. (2) APE-LM

contains large amounts of pyruvate. As discussed earlier,

an abundance of energy is produced via the oxidation of

pyruvate through the citric acid cycle.

APE-LM-preserved hearts consumed a significantly higher

amount of oxygen than hearts preserved with other media.

The higher oxygen and pyruvate consumption in these hearts

indicated that the hearts had a greater metabolic oxidative

activity during preservation than the other hearts. The

higher oxidative activity may have been reflective of

greater tissue perfusion, especially in the coronary beds,

and thereby perfusion of oxygen to a greater percentage of

myocardial cells. Another factor contributing to the

effectiveness of APE-LM as a transplantation media is its

biologically compatible lipid emulsifier, which consists

primarily of phospholipids and cholesterol. The lipid

provides a favorable environment for myocardial membranes

and may prevent perfusion-related depletion of lipids from

cardiac membranes. The cholesterol contains a bulky steroid

nucleus with a hydroxyl group at one end and a flexible

hydrocarbon tail at the other end. The hydrocarbon tail of

the cholesterol is located in the non polar core of the

membrane bilayer. The hydroxyl group of cholesterol

hydrogen-bonds to a carbonyl oxygen atom of a phospholipid

head group. Through this structure, cholesterol prevents

the crystallization of fatty acyl chains by fitting between

them. Thus, cholesterol moderates the fluidity of

membranes.8

The reason there are currently such strict limits on the

amount of time a heart can remain viable out of the body is

because there must be a source of energy for the heart

tissue if it is to stay alive. Once the supply of energy

runs out, the tissue suffers irreversible damage and dies.

Therefore, this tissue cannot be used for transplantation.

If hypothermic hearts are not given exogenous substrates

that they can transport and consume, like pyruvate, then

they must rely on glycogen or lipid stores for energy

metabolism. The length of time that the heart can be

preserved in vitro is thus related to the length of time

before these stores become too low to maintain the required

energy production needs of the organ. It is also possible

that the tissue stores of ATP and phosphocreatine are

critical factors. It is known that the amount of ATP in

heart muscle tissues is sufficient to sustain contractile

activity of the muscle for less than one second. This is

why phosphocreatine is so important. Vertebrate muscle

tissue contains a reservoir of high-potential phosphoryl

groups in the form of phosphocreatine. Phosphocreatine can

transfer its phosphoryl group to ATP according to the

following reversible reaction:

phosphocreatine + ADP + H+ 9 ATP + creatine

Phosphocreatine is able to maintain a high concentration of

ATP during periods of muscular contraction. Therefore, if

no other energy producing processes are available for the

excised heart, it will only remain viable until its

phosphocreatine stores run out.

A major obstacle that must be overcome in order for heart

transplants to be successful, is the typically prolonged

delay involved in getting the organ from donor to recipient.

The biochemical composition of the preservation media for

hearts during the transplant and transportation delays are

extremely important for prolonging the viability of the

organ. It has been discovered that adding pyruvate, or

pyruvate containing compounds like APE-LM, to a preservation

medium greatly improves post-preservation cardiac function

of the heart. As was discussed, the pyruvate is able to

enter the citric acid cycle and produce sufficient amounts

of energy to sustain the heart after it has been excised

until it is transplanted.

Increasing the amount of time a heart can remain alive

outside of the body prior to transplantation from the

current four hours to 24 hours has many desirable benefits.

As discussed earlier, this increase in time would allow

doctors the ability to better match the tissues of the donor

with those of the recipient. Organ rejection by recipients

occurs frequently because their tissues do not suitably

match those of the donors. The increase in viability time

would also allow plenty of opportunity for the organ to be

transported to the needy person, even if it must go across

the country.