Phosphate-containing dialysis solution prevents hypophosphatemia during continuous renal replacement therapy.

BACKGROUND: hypophosphatemia occurs in up to 80% of the patients during continuous renal replacement therapy (CRRT). Phosphate supplementation is time-consuming and the phosphate level might be dangerously low before normophosphatemia is re-established. This study evaluated the possibility to prevent hypophosphatemia during CRRT treatment by using a new commercially available phosphate-containing dialysis fluid. METHODS: forty-two heterogeneous intensive care unit patients, admitted between January 2007 and July 2008, undergoing hemodiafiltration, were treated with a new Gambro dialysis solution with 1.2 mM phosphate (Phoxilium) or with standard medical treatment (Hemosol B0). The patients were divided into three groups: group 1 (n=14) receiving standard medical treatment and intravenous phosphate supplementation as required, group 2 (n=14) receiving the phosphate solution as dialysate solution and Hemosol B0 as replacement solution and group 3 (n=14) receiving the phosphate-containing solution as both dialysate and replacement solutions. RESULTS: standard medical treatment resulted in hypophosphatemia in 11 of 14 of the patients (group 1) compared with five of 14 in the patients receiving phosphate solution as the dialysate solution and Hemosol B0 as the replacement solution (group 2). Patients treated with the phosphate-containing dialysis solution (group 3) experienced stable serum phosphate levels throughout the study. Potassium, ionized calcium, magnesium, pH, pCO(2) and bicarbonate remained unchanged throughout the study. CONCLUSION: the new phosphate-containing replacement and dialysis solution reduces the variability of serum phosphate levels during CRRT and eliminates the incidence of hypophosphatemia.

Renal toxicity mediated by glucose degradation products in a rat model of advanced renal failure.

BACKGROUND: In peritoneal dialysis (PD) residual renal function contributes to improved patient survival and quality of life. Glucose degradation products (GDP) generated by heat sterilization of PD fluids do not only impair the peritoneal membrane, but also appear in the systemic circulation with the potential for organ toxicity. Here we show that in a rat model of advanced renal failure, GDP affect the structure and function of the remnant kidney. MATERIALS AND METHODS: Sprague-Dawley rats were randomly assigned to a two stage subtotal nephrectomy (SNX) or sham operation and were left untreated for 3 weeks. The SNX + GDP group continuously received chemically defined GDP intravenously for 4 weeks; the SNX and the sham-operated rats remained without GDP. The complete follow-up for all groups was 7 weeks postoperatively. We analysed renal damage using urinary albumin excretion as well as a semiquantitative score for glomerulosclerosis and tubulointerstitial damage, as well as for immunohistochemical analyses. RESULTS: The SNX + GDP rats developed significantly more albuminuria and showed a significantly higher score of glomerulosclerosis index (GSI) and tubulointerstitial damage index (TII) as compared to SNX or control rats. In the SNX + GDP group the expression of carboxymethyllysine and methylglyoxal was significantly higher in the tubulointerstitium and the glomeruli compared to the SNX rats. Caspase 3 staining and TUNEL assay were more pronounced in the tubulointerstitium and the glomeruli of the SNX + GDP group. In SNX + GDP animals, the expression of the slit diaphragm protein nephrin, was significantly lower compared to SNX or control animals. CONCLUSION: In summary, our data suggests that GDP can significantly advance chronic kidney disease and argues that PD solutions containing high GDP might deteriorate residual renal function in PD.

Purification of a phosphatase which hydrolyzes phosphatidic acid, a key intermediate in glucolipid synthesis in Acholeplasma laidlawii A membranes.

A phosphatidic acid phosphatase (PAP; EC 3.1.3.4.), dephosphorylating phosphatidic acid (PA) to diacylglycerol (DAG), was identified and purified from the plasma membrane of Acholeplasma laidlawii A. After four purification steps, including membrane preparation, Tween 20 solubilization, preparative gel electrophoresis and electro-elution, PAP was purified about 400 times to near homogeneity. The molecular weight of PAP was according to SDS-polyacrylamide gel electrophoresis approximately 25 kDa and the enzyme was a stable and integral membrane protein. It is proposed to catalyze the first enzymatic step in the important glucolipid pathway of A. laidlawii. No essential cofactors or activator lipids were found. However, some divalent cations and phosphate analogues were potent inhibitors. Beside the in vivo substrate (PA), PAP was found to dephosphorylate p-nitrophenylphosphate. This less stringent specificity makes alternative in vivo functions for PAP plausible, the importance which is discussed.

Control of membrane polar lipid composition in Acholeplasma laidlawii a by the extent of saturated fatty acid synthesis.

The low level of endogenous fatty acid synthesis in Acholeplasma laidlawii A strain EF22 was found to be caused by a deficiency of pantetheine in the lipid-depleted growth medium. By supplementing the oleic acid-containing medium with increasing concentrations of pantethein, saturated fatty acid synthesis was stimulated (having an apparent Km of 5 microM for pantetheine) and the incorporation of endogenously synthesized fatty acids in membrane lipids increased markedly. Furthermore, carotenoid biosynthesis was stimulated. Exogenous palmitic acid was found to inhibit partially the endogenous fatty acid synthesis. A gradual stimulation of fatty acid synthesis was accompanied by a linear increase in the molar proportion between the two dominating membrane glucolipids, monoglucosyldiacylglycerol and diglucosyldiacylglycerol. The total amount of charged membrane lipids decreased upon increasing the degree of fatty acid saturation. These regulations are discussed in terms of membrane stability, and influence of membrane molecular ordering and surface charge density on lipid polar head group synthesis.

Isoprenoid modification of proteins distinct from membrane acyl proteins in the prokaryote Acholeplasma laidlawii.

Isoprenylation is an important posttranslational modification that affects the activity, subunit interactions and membrane anchoring of different eukaryotic proteins. The small, cell-wall-less prokaryote Acholeplasma laidlawii has more than 20 membrane acyl-proteins enriched in myristoyl and palmitoyl chains. Radioactive mevalonate, a precursor to isoprenoids, was incorporated into several specific membrane proteins of 20 to 45 kDa and two soluble proteins of 23-25 kDa, respectively. No acyl proteins and none of the polar acyl lipids became labelled but these are all labelled by radioactive fatty acids. Mevalonate was incorporated mainly into a minor neutral, non-saponifiable lipid which migrated just above a C30-isoprenoid (squalene) on TLC-plates. The isoprenoid chains could not be released by mild alkaline hydrolysis from most of the isoprenylated proteins, although this procedure releases acyl chains from lipids and all acylated proteins. Isoprenylated proteins were enriched in the detergent phase upon partition with the non-ionic detergent Triton X-114. This behaviour is similar to the acyl proteins of this organism and indicates that the isoprenoid chains give the proteins a hydrophobic character.

Qualitative and quantitative variations of membrane lipid species in Acholeplasma laidlawii A.

In Acholeplasma laidlawii A, strain EF 22, the relative amounts of the membrane polar lipids vary as a consequence of different fatty acid supplements to the growth medium. The number of lipid species also varies; a new apolar monoglucolipid containing four fatty acid residues was present only when saturated fatty acids dominated in the growth medium. A new phosphoglucolipid, probably with a glycerophosphoryl-monoglucosyldiglyceride structure, was also found. The most pronounced variations occurred between the two dominating glucolipids, monoglucosyldiglyceride and diglucosyldiglyceride; the former being found in larger amounts when a saturated or a trans-unsaturated fatty acid was present in the medium. The amount of diglucosyldiglyceride decreased accordingly. A qualitative relationship between fatty acid properties and membrane lipid variations was established over a wide fatty acid concentration range. Incorporation of supplied fatty acids reached higher levels than normally found in other acholeplasmas. The ratio between membrane protein and lipids exhibited significant and coherent variations during growth and was to some extent influenced by the fatty acids in the medium. These changes indicate variations in lipid-protein organization in the membranes during growth.

Membrane potential, lipid regulation and adenylate energy charge in acyl chain modified Acholeplasma laidlawii.

In Acholeplasma laidlawii variations induced in the transmembrane electrical potential have been shown to affect the membrane lipid composition. Particularly the molar ratio between the predominant glucolipids, monoglucosyldiacylglycerol and diglucosyldiacylglycerol, decreases upon hyperpolarization and increases upon depolarization (Clementz et al. (1986) Biochemistry 25, 823-830). Upon variation of the degree of membrane fatty acyl chain unsaturation, known to affect the passive permeability for a number of small molecules, there was no significant correlation between acyl chain composition and the magnitude of the electrical potential. Hyperpolarization by valinomycin decreased the glucolipid ratio for all kinds of membranes, but the size of the decrease was not correlated to the acyl chain composition. However, a clear relationship, independent of acyl chain composition, was found between the extent of hyperpolarization and the size of the decrease in the glucolipid ratio. The adenylate energy charge value (Ec) of the cells was affected by the acyl chain composition, although not exclusively by the proportion of unsaturation. Furthermore, a larger hyperpolarization upon valinomycin addition was accompanied by a stronger reduction in Ec.

The enzymatic synthesis of membrane glucolipids in Acholeplasma laidlawii.

In membranes of the prokaryote Acholeplasma laidlawii, the physiological regulation of the two major membrane lipids, monoglucosyldiacylglycerol (MGlcDAG) and diglucosyldiacylglycerol (DGlcDAG), is governed by factors affecting the equilibria between lamellar and non-lamellar phases of the membrane lipids. The synthesis of the glucolipids is considered to be a two-step glucosylation: (i) DAG+UDP-Glc----MGlcDAG+UDP; and (ii) MGlcDAG+UDP-Glc----DGlcDAG+UPD. This was corroborated by in vivo pulse labelling experiments showing turnover of MGlcDAG but not DGlcDAG. The enzymatic synthesis of MGlcDAG was localized to fresh or freeze-dried membranes in vitro. Synthesis of DGlcDAG was minor in such membranes but of substantial magnitude in intact cells. Synthesis of MGlcDAG was stimulated by small amounts of SDS but completely inhibited upon solubilization of the membranes by a variety of detergents. The inhibitory effect of several UDP-Glc analogs on glucolipid synthesis demonstrated the importance of UDP-Glc as the sugar donor. Synthesis of both glucolipids was lost in freeze-dried plus lipid-extracted cells but restored when lipids were transferred back to the extracted cell membrane. By selectively adding specific lipids, a strong dependence on the acceptor lipid DAG, as well as the need for general matrix lipids for enzyme activity, was established. In addition, the anionic phosphatidylglycerol (PG), but not the other phospholipids, had a strong stimulatory effect. The presence of different phosphorylating agents stimulated the synthesis of DGlcDAG and partially inhibited that of MGlcDAG. This, together with the lipid dependency, may constitute mechanisms for the regulation of the enzyme activities in vivo.

Fractionation of membranes from Acholeplasma laidlawii A on the basis of their surface properties by partition in two-polymer aqueous phase systems.

Acholeplasma laidlawii A consists of pleomorphic cell clusters surrounded by a single membrane. When lysed, a cell gives rise to several membrane fragments which cannot be separated from each other by isopycnic sucrose gradient centrifugation. A heterogeneous lateral organization of the cell membranes was detected by countercurrent distribution of membrane fragments in a two-polymer aqueous phase system. It revealed that the membranes consist of at least two subpopulations with respect to surface properties. Changes in the fatty acid and cholesterol content of the membranes revealed that the resolution of different subpopulations was predominantly due to a critical ratio of monoglucosyldiglyceride to diglucosyldiglyceride. The heterogeneity of the membrane probably depends on lipid-lipid and lipid-protein steric interactions. Charged lipids, an apolar monoglucolipid and the ratio between lipids and proteins also affect membrane partition. The differences in the subpopulations were further reflected by different specific activities of NADH dehydrogenase, NADH oxidase and ATPase. These activities varied independently. Minor quantitative differences in the protein patterns of different subpopulations were apparent. The origin and the preservation of the membrane subpopulations are discussed in terms of lipid-lipid and lipid-protein interactions, their age and energy metabolism.

Water binding and phase structures for different Acholeplasma laidlawii membrane lipids studied by deuteron nuclear magnetic resonance and x-ray diffraction.

Water binding capability and phase structures for different lipid species extracted from Acholeplasma laidlawii A membranes have been studied using deuteron nuclear magnetic resonance and low-angle X-ray diffraction. The dominating membrane lipids are monoglucosyldiglyceride and diglucosyldiglyceride and each of them takes up limited amounts of water (bound plus trapped), i.e., up to 13% (w/w), whereas the phospholipids and phosphoglycolipids have larger hydration capacities. Addition of magnesium and calcium ions, but not sodium ions, to the diglucosyldiglyceride increases the hydration capability. This increase is accompanied by the formation of a metastable liquid crystalline phase and a hysteresis effect for the transition temperature. Large differences in water deuteron quadrupole splitting were observed between mono- and diglucosyldiglyceride. Both 2H nuclear magnetic resonance and low-angle X-ray diffraction studies on lipids containing biosynthetically incorporated omega-d3-palmitic acid clearly indicate the existence of a reverse hexagonal phase structure for the monoglucosyldiglyceride and lamellar structures for the diglucosyldiglyceride and the other membrane lipids. The low hydration capability of the large diglucosyldiglyceride polar head is discussed in terms of polar head configuration. Both mono- and diglucosyldiglyceride have several physical properties similar to those of phosphatidylethanolamine.

Effects of anesthetics on water permeability and lipid metabolism in Acholeplasma laidlawii membranes.

The addition of tetracaine and diethyl ether to Acholeplasma laidlawii at concentrations commonly used for local anesthesia did not affect water permeability over the cell membrane, as measured by a pulsed magnetic field gradient NMR method. However, A. laidlawii changed its membrane lipid composition upon treatment with these anesthetics. Both tetracaine and diethyl ether addition resulted in a decrease in the molar ratio between the major membrane glucolipids, monoglucosyldiacylglycerol and diglucosyldiacylglycerol. The ratio between saturated and unsaturated acyl chains did not change. The results are in accordance with our proposal that A. laidlawii regulates its lipid composition in order to maintain optimal packing stability in the membrane (Wieslander, A., Christiansson, A., Rilfors. L. and Lindblom, G. (1980) Biochemistry 19, 3650--3655). Introduction of anesthetics into the hydrophobic region of a bilayer is likely to affect the lipid packing. A membrane which contains lipids like monoglucosyldiacylglycerol, which forms a reversed hexagonal phase, will be destabilized unless the amounts of such lipids are reduced. The membrane concentration of anesthetics was estimated to one molecule per 12--15 lipid molecules. The fact that A. laidlawii regulates its lipid composition as a response to these concentrations, despite their negligible effect on water permeability, indicates a high sensitivity of this regulatory system.

Lipid extracts from membranes of Acholeplasma laidlawii A grown with different fatty acids have a nearly constant spontaneous curvature.

X-ray diffraction methods were used to explore the variation in the spontaneous curvature of lipid extracts from Acholeplasma laidlawii strain A-EF22 grown with different mixtures of palmitic acid and oleic acid. It was shown that the cells respond to the different growing conditions by altering the polar head group compositions in order to keep the phase transition between lamellar and nonlamellar structures within a narrow temperature range. This has been interpreted to mean that the membrane lipids are adjusted toward an optimal packing (Lindblom et al. (1986) Biochemistry 25, 7502). Here it is shown that for these extracts, the membrane curvature is kept within a narrow range (58-73 A), compared to the range in curvatures exhibited by pure lipids extracts from the membrane (17-123 A). These observations support the hypothesis (Gruner (1989) J. Phys. Chem. 93, 7562) that the spontaneous curvature is a functionally important membrane parameter which is regulated by the organism and is likely to be one of the constraints controlling the lipid composition of the bilayer.

Similar regulatory mechanisms despite differences in membrane lipid composition in Acholeplasma laidlawii strains A-EF22 and B-PG9. A multivariate data analysis.

Mycoplasmas are small, cell wall-deficient bacteria. The metabolic regulation of the lipid composition in the membrane of the species Acholeplasma laidlawii, strains A-EF22 and B-JU, is governed mainly by the balance between the potential formation of lamellar and nonlamellar phase structures. However, the regulatory features have not been consistently observed in the B-PG9 strain. A comparison has been performed between the membrane lipid composition for strains A-EF22 and B-PG9, simultaneously changing eight experimental conditions known to affect the regulation and packing properties of the A-EF22 lipids. Multiple regression and partial least-square discriminant analyses of many variables showed: (i) quantitative differences in membrane lipid and protein composition, and in membrane protein molecular masses of the two strains; (ii) different molar fractions of the major polar lipids monoglucosyldiacylglycerol (nonlamellar) and diglucosyldiacylglycerol (lamellar), which were caused by differences in lipid acyl chain length and unsaturation inherent in the strains and by the type of growth medium used; and (iii) similar regulatory mechanisms for changes in the lipid composition under most conditions, responding to the experimentally varied bilayer and nonbilayer properties of the lipid matrix. These regulatory principles are probably valid in other bacteria as well.

Plasmid transformation and replica filter plating of Acholeplasma laidlawii.

The restriction deficient mutant 8195 of Acholeplasma laidlawii strain JA1 was transformed by the promiscuous streptococcal plasmid vector pNZ18 at a frequency of 4 x 10(-4)/cfu. The plasmid was maintained without structural rearrangements but was lost in the absence of a selection pressure, i.e. kanamycin or neomycin. Transformed primary colonies were easily recognized due to a different colony morphology. Replica filter plating, previously not obtained with mycoplasmas, was achieved using pNZ18 as a marker by incubating the replica filters with the cell side down on the new agar plates. These findings should greatly facilitate the genetic and functional analysis of A. laidlawii.

Heat sterilized PD-fluids impair growth and inflammatory responses of cultured cell lines and human leukocytes.

We have recently demonstrated that commercial PD-fluids inhibit the growth of a cultured mouse fibroblast cell line. Toxic substances produced during heat sterilization were believed to be the probable cause of the growth inhibition. The aim of the present study was to investigate if heat sterilized PD-fluids affect other cell types and other cellular functions than the growth of fibroblasts. The effect of three commercially and one laboratory made PD-fluid on cell growth of a mouse macrophage cell line (RAW) and a human neuroblastoma cell line (SH-SY5Y) was examined. The influence on stimulated release of tumour necrosis factor alpha (TNF alpha) from the macrophage cell line and stimulated superoxide generation from freshly prepared human leukocytes were also investigated. Compared to the filter sterilized PD-fluid, we found that heat treated PD-fluids significantly inhibited the growth of the two cell lines and impaired the stimulated release of TNF alpha and superoxide radicals. These results demonstrate that heat sterilization of PD-fluids produces substances that are cytotoxic regardless of the cell species, the cell type or the cell function tested.

Effects of exposure period of acetylsalicylic acid, paracetamol and isopropanol on L929 cytotoxicity.

An established fibroblast cell line, L929, was exposed to three substances-acetylsalicylic acid, isopropanol and paracetamol-for 24 hr, 72 hr or 13 days. The effective concentration that caused 50% inhibition of cell growth (EC(50)) was calculated for both the 24- and 72-hr exposures using three assays-total protein (TP), neutral red uptake (NR) and enzymatic conversion of MTT to formazan. The methods determining the viability of the cells were more sensitive than measurement of total protein. Cells exposed for 13 days continued to proliferate during the entire period, although at a reduced rate, and never attained the density of the control. The relative toxic effect of the substances varied considerably as a consequence of the duration of the exposure period.

Protection by glutathione of neutrophils against the toxic effects of peritoneal dialysis fluid.

The possibility of reducing the cytotoxic effect of heat-sterilized peritoneal dialysis (PD) fluid by addition of antioxidants/scavengers during incubation of titanium-adhering cells was investigated. Capillary blood from healthy donors was placed in drops on commercially available titanium pieces and incubated in a humidified chamber at 37 degrees C for 60min. After incubation the adherent polymorphonuclear leukocytes were immersed for 1-4h in PD-fluid, pH 7.4, containing 2.5% glucose with glutathione (GSH), superoxide dismutase, catalase or dithiothreitol (DTT). Luminol- or isoluminol-amplified chemiluminescence was used to measure the zymosan- and phorbol myristate acetate (PMA)-stimulated respiratory burst activity, as an indicator of the cytotoxicity of the PD-fluids. Heat sterilized PD-fluid had inhibitory effect on zymosan-induced respiratory burst and impaired both the extracellular and intracellular PMA-induced respiratory burst. Addition of GSH to the PD-fluid resulted in reduction of cytotoxical effects on the zymosan-induced and extracellular PMA-induced respiratory burst. The intracellular respiratory burst was not affected. The present results show that GSH and DTT have the ability to protect polymorphonuclear leukocytes against the cytotoxic effects of the PD-fluid by keeping the cell membrane in a reduced state.

Glucose degradation products in peritoneal dialysis fluids: how they can be avoided.

OBJECTIVES: A patient on peritoneal dialysis (PD) uses 3-7 tons of PD fluid every year. The result is considerable stress on the peritoneal tissue. Aspects of PD fluids that have been considered responsible for bioincompatibility are low pH, high osmolality, high glucose and lactate concentrations, and the presence of glucose degradation products (GDPs). However, the relative importance of each factor in PD fluid has so far not been investigated. Discovering their relative importance was the aim of the present study. METHODS: Two main methods for investigating biocompatibility were used in this study: cytotoxicity measured as in vitro inhibition of cell growth, and in vitro AGE formation measured as albumin-linked fluorescence. RESULTS: The two most important factors for determining in vitro bioincompatibility of PD fluids were the presence of GDPs, which caused both severe cytotoxicity and strong AGE promotion, and low pH, which induced severe cytotoxicity. CONCLUSIONS: The biocompatibility of PD fluids can be monitored through fairly simple in vitro methods such as cell proliferation and AGE formation. Bioincompatibility of PD fluids is caused mainly by the presence of GDPs and low pH. These findings correlate well with known clinical bioincompatibility.

Clinical and physiological effects of a new, less toxic and less acidic fluid for peritoneal dialysis.

OBJECTIVE: To report our first clinical experience with a new continuous ambulatory peritoneal dialysis (CAPD) fluid (PD-Bio), which is nearly devoid of glucose degradation products and has a higher pH (6.3) than conventional peritoneal dialysis (PD) solutions, and to discuss in general terms some acute and long-term effects of conventional acidic solutions containing glucose degradation products. DESIGN: 1) Pilot study on 4 patients investigated using a modified peritoneal equilibration test (PET) and cytobiology parameters. 2) Computer simulation study, assuming that conventional acidic solutions cause vasodilatation and recruitment of capillary surface area initially (during 0-60 minutes) in a PD dwell. PATIENTS: Four stable CAPD patients were chosen in an open cross-over study. After a period of three months using conventional PD fluid, the patients were switched to three months on the new PD fluid. RESULTS: Cancer antigen 125 increased significantly, and patients with discomfort/infusion pain during the control period improved during the period with the new fluid. No significant changes were observed in mass-transfer coefficients or drained volumes with the new solution. PH in the effluent dialysis was, however, higher for PD-Bio at all times during a two-hour dwell. In the computer simulation study, a less acidic solution caused an initially lower rate of glucose dissipation and improved ultrafiltration (UF) after a four-hour dwell, as compared to a conventional PD solution. CONCLUSIONS: A new, differently produced, less toxic and less acidic PD fluid (PD-Bio) seems to be better tolerated than a conventional acidic solution with respect to discomfort/infusion pain. Theoretically, neutralized solutions should show slightly improved UF profiles over conventional acidic solutions, according to the computer simulation analysis. Furthermore, it is speculated that a neutral, less acidic, less toxic fluid would cause less interstitial-mesothelial alterations and less impairment of UF capacity than conventional solutions during long-term CAPD.

Development of toxic degradation products during heat sterilization of glucose-containing fluids for peritoneal dialysis: influence of time and temperature.

OBJECTIVE: Fluids for peritoneal dialysis (PD) cause cytotoxic reactions in many different in vitro systems. The low pH, the high osmolality of the fluids, and the glucose degradation products formed during heat sterilization have been considered responsible. In the present study, we investigate the influence of temperature and time during heat sterilization of PD fluids and glucose solutions on glucose degradation and cytotoxicity of the solutions. DESIGN: Ampoules containing PD-fluid or glucose solution were heated in an oil bath to predetermined F0 values (combinations of time and temperature giving equal energy/bacterial lethality). Cytotoxicity of the solutions was measured as growth inhibition of cultured L-929 fibroblasts. Glucose degradation was measured as UV absorbance at 228 and 284 nm. RESULTS: The same general pattern was seen in both PD fluid and glucose solution. Cytotoxicity decreased from 90% to 15% when the sterilization temperature was increased from 115 degrees to 140 degrees C and concomitantly the length of time shortened in order to maintain equal bacterial lethality. Under the same conditions, degradation products, measured as UV absorbance at 284 nm, decreased from 0.2 to 0.02. CONCLUSION: To minimize the development of cytotoxic breakdown products, high temperatures over short periods of time should be used to heat-sterilize PD fluids. Even as small an increase as 5 degrees C at around 120 degrees C will improve the quality of the solutions.