FAQ’s
Introduction to AQIX Fluid Technology

1. 'Ringer \' solutions

Since Sidney Ringer (1888) showed that the Ca (in London tap water) was essential to maintain the function of the frog heart , have been based upon the premise that the important ions required for isolated tissue or organs are:

The concentrations of the ionic species in AQIX® RS-I solution acknowledges the activity coefficients of the ionic species and not simply their total serum concentrations, that is, serum binding of Ca2+ and Mg2+ must be distinguished from the actual free, ionised levels of these ions.

2. Phosphate Buffered Media:

Majority of 'designer' physiological solutions are derived from Krebs & Henseleit (1932), Krebs (1950) , Hanks (1954) and all use a phosphate/bicarbonate (PO -4 / HCO -3) buffering system to control the medium / perfusate pH.

Published data over the last 50 years (see Table 1, below) indicates the deleterious effects that phosphate ions are now known to have on isolated cells, tissues and organs. There are no exceptions, even the resilient kidney shows a decrease in urine flow over a maximum time period of 2 - 4 hours!

Basically, any phosphate-buffered solution will cause irrevocable damage over time. All the additions and variations in 'designer' specific quasi-physiological solutions will merely to try to compromise the demise of functional viability over short time periods.

Unfortunately, cells require optimum HCO3 - levels to maintain intracellular K+-ions to stabilise the cell membrane potential, without which the cell slowly dies over time (2 - 4 hours). This imbalance in HCO3 - levels also applies to the use of citrate buffered solutions (e.g. Marshall’s, Soltran®) has been recently observed in a comparison study involving AQIX® RS-I solution during in vitro normothermic perfusion of the isolated pig kidney with LR solution where the decrease in HCO3 - levels and subsequent leakage of K+-ions was significant (See Table K1).

Table K1 . Functional Parameters in Normothermically perfused Porcine Kidney following CS preservation for 2 hours.

Interestingly, the levels of phosphate ions are not added to these perfusates at their ‘free’ serum levels, namely, 12% of the total 1.4 mmols/L (Pedersen, 1972). Because of precipitous actions of phosphate ions on calcium and magnesium ions in solution, none of the phosphate buffered perfusates will be viable after 24 hours and certainly not days! No precipitation of calcium or magnesium ions has ever been observed in either the 10x or 1x preparations of AQIX® RS-I solution!

RS-I solution:

RS-I uses the natural pH buffering mechanism found in human serum, namely, pCO2 / HCO3 -, which avoids the use of phosphate or citrate ions as pH buffering agents.

RS-I utilises a Good's buffer, BES, which has a pKa and zwitteronic characteristics that ensures pH stability over the temperature range 10-38 °C and allows optimal serum levels of bicarbonate ions [HCO3 - ] to be utilised. This synthetic chemical has proved to be non-toxic in cell development (Eagle, 1971), the maintenance of mammalian tissues and organs (Rees, 1989) and in human injectable perfusates used in cardiopulmonary surgical procedures (Swan, 1993)

There is negligible binding of Ca2+ or Mg2+ ions by BES in comparison to binding by phosphate and citrate ions and it is unique out of all the other 21 Good's buffers in that it is taurine-based, a serum amino acid with a possible protective role in age-dependent response in the calcium `paradox’ (Takihara et al., 1985).

3. Drug Bioassay:

Because isolated preparations are of limited viability [< 2hours] when perfused in phosphate buffered solutions, it is not possible to completely trial a drug using the same preparation - several preparations from several animals have to be used. This clearly questions how valid the drug dose response results will be given the known variability between isolated preparations from different animals or the same species.

This problem is compounded by the fact that phosphate-buffered solutions are not stable over

time (e.g. <[Ca2+] o) and thus the experimenter cannot be assured of drug receptor interaction or use isolated preparations for extended periods of time (days). Equally, because these solutions are 'specifically' formulated for one type of tissue/organ preparation, different tissues or organs cannot be simultaneously removed from the same animal whereas they can be using AQIX® RS-I solution because it has been designed as a universal preservation and/or perfusion solution under either hypothermic or normothermic conditions.

RS-I solution: Affords standardised performance and repeatable validity of results by offering a solution that is applicable for use in all mammalian species and stable over time when stored at 8 - 12 °C or perfused for periods of 1 - 10 days over a temperature range of 15 - 38 °C. Commercial AQIX® RS-I stock concentrates [10x] have been shown to have a shelf-life of 14 months when stored under dark conditions at 3 - 8 °C.

Table 1. PO4- radicle effects on isolated tissues/organs

CREESE (1950) - rat hemidiaphragm perifused with ;
** pCO2/HCO3- - limited LOSS of K+ but GAIN of Na+
** H2PO4-/HPO4- NEGATIVE inotropism NOT reversed with HCO3-

STADIE & SHAW (1959) - rat hemidiaphragm perifused with ;
** H2PO4-/HCO3- - caused INHIBITION of HK & PFK enzymes

BERMAN & SAUNDERS (1955) - rat ventricle perfused with ;
** D-glucose INEFFECTIVE substrate in PO4- - based media

SVEDMEYR (1965) - rat ventricle perfused with ;
** glycogenolytic effect of epinephrine LESS in PO4- - based media
than in HCO3-- based media

KO & PARADISE (1969) - rat atria perfused with ;
** ONLY HCO3-- based media could RESTORE ATP levels

EAGLE (1971) - cell culture preparations incubated with ;
** HCO3- - based media INDISPENSABLE for CELL GROWTH

HALL & De LUCA (1986) - rat heart mitochondrial creatine kinase (CK)
incubated with; ** PO4- - based media caused IRREVERSIBLE INHIBITION of CK

De FRIETAS & VALENTINE (1984) - superoxide dismutase (SOD) preparations
incubated with; ** PO4- - based media caused INHIBITION of SOD

STEWART ET AL. (1986) - sheep heart transplants preserved with;
** HCO3- - based media containing SOD sustained left ventricular function upon
transplantation/reperfusion

REES & CHUNG (1989) - isolated rat liver incubated with;
** PO4- - based Krebs & Henseleit perfusate showed leakage of LDH after only 10 minutes compared to 270 minutes in RS-I solution.

REES & DALZEIL (1994) - isolated rat diaphragm incubated with;
** PO4- - based Krebs & Henseleit perfusate showed 30% decline in twitch contractions by 90 minutes compared to 7% in RS-I solution.

REES & UNDRILL (1995) - isolated rat vastus lateralis muscles incubated with;
** PO4- - based Krebs Ringer perfusate showed negligible caffeine response in comparison to maintained response in RS-I solution after 8 hours.

4. Metabolism:

Interestingly, 'designer' perfusates very rarely contain substrates to support intermediate metabolism as was suggested by Krebs (1950), namely,

(i) aspartate - to sustain aspartate-malate shuttle for the ultimate production of ATP by ensuring the correct NAD/NADH balance within the mitochondria of individual cells.

(ii) glutamate - for the synthesis of ketoglutarate, glutamine, etc. and, in to enhance oxidative metabolism by replenishing TCA cycle intermediates, thereby maintaining high energy phosphate levels even during ischemic insult.

(iii) glutamine - an essential amino acid for ongoing cell development (Eagle, 1971) and numerous other cellular functions, e.g. smooth muscle viability in vitro, kidney re: acid-base balance.

(iv) glycerol - to sustain the glycerol phosphate shuttle and, along with the aspartate-malate shuttle, ensure optimal NAD/NADH balance within the mitochondria of individual cells.

(iv) thiamine pyrophosphate - prevents pyruvic aldehyde accumulation and cell toxicity (Sir Rudolph Peters, 1943); essential co-factor in Pentose Phosphate and Glycolytic pathways and so many other interrelated biochemical reactions.

(v) carnitine - a serum-based component essential for acyl carnitine formations to optimise free fatty acid utilisation 'intracellularly' in glucose-sparing phenomenon; shown to be a factor in preventing heart dysfunction and arrhythmias (Whitmer, 1987).

(vi) choline - essential component in serum (7 - 20 µmols/L) for the sustained manufacture of ACh at all cholinergic central, peripheral neuronal synapses; basic component in phosphatidyl moieties that make cell membranes and the enzymes that keep cells active and responsive to electrical and drug receptor stimulation.

(v) insulin - a major operand in all cell functions at all levels of cell development by facilitating optimum usage of glucose and glycogen. If included in phosphate-buffered solutions it is at a concentration 10,000 times higher than that of the serum levels. [In AQIX® RS-I it exists as mono-aggregates at human serum levels (28mIU/L)].

(vi) glucose - a fundamental energy substrate for the optimal operation of the glycolytic pathway under periods of hypothermia and normothermia and, along with glycerol, to provide ‘free’ radicle scavenging activity.

RS-I solution:

Contains all of the above essential and basic ingredients at optimal serum levels to maximise the viability of cellular function over time as has been verified in 4,000 scientific trials on cells, embryos, tissues and organs from a variety of mammalian species.

5. Oncotic/Colloidal pressure

The inclusion of ‘colloidal’ or ‘plasma expanders’ in preservation and perfusion solutions by numerous researchers is bewildering and superfluous if one considers that each and every cell membrane lies in continuity with a 99% viscous gel interstitial phase. Hence, each cell has a natural colloidal, buffering mechanism to prevent adverse movement of extracellular ions and water, which, in tandem with a medium of correct ionic conductivity, will subtend the cellular integrity of each cell in a tissue or organ via the natural processes of autoregulation. The majority of the osmotic pressure is provided by Na+ and accompanying anions and only a small component (ca. 0.5%) can be attributed to plasma proteins (Burton, 1975; Rees, 1989a).

RS-I is isosmotic to human serum (ca. 280 ± 10 mOsmoles) and has not appeared to necessitate the inclusion of plasma expanders as demonstrated by the fact that only minor changes (ca. 3-12%) in hydration (oedema) occur during long term periods (i.e. 4-52 h) of either hypothermic or normothermic preservation/perfusion of isolated tissues and organs.

RS-I solution:

The formulation of AQIX® RS-I acknowledges of role of the ionic, osmolarity, pH, specific ion conductivity and substrate demands of mammalian tissues/organs per se in recognising that chemical and biophysical analyses have shown that the serum composition in mammalian species is uniform from 'mouse to man' (Burton, 1975; Rees, 1989a).

Table 2 Functional viability of mammalian tissue/organ preparations maintained in AQIX® RS-I perfusate/preservation solution

Author: Dr. D. Rees PhD MRSNZ
Founder/CSO
Aqix Ltd (UK)