Biomedical subjects
Publications and source records attributed to N K Man.
A semi-automated two-stage chromatographic system is described for use in evaluating neurotoxin b4-2 in body fluids of uremic patients and healthy subjects. The only manual step is the injection, thus decreasing the risk of operator error inherent in the previous manual method (Clin Chem 29: 703-707, 1983) and concurrently improving CV from 9% to 6.4% as measured during a 16-month period.
Renewed interest in bicarbonate as the dialysate base replacement during hemodialysis has introduced new settings on acetate-dialysate delivery systems commercially available. Numerous technical problems are associated with unstable bicarbonate solutions. Therefore, several precautions must be observed: 1) Bicarbonate dialysis requires two concentrates: one containing the base replacement bicarbonate and the other containing basically calcium and magnesium chlorides, 2) A two-stream proportioning system afforded by an appropriate dialysate delivery machine should be used to mix the bicarbonate and acid concentrates, in order to ensure on-line production of the final dialysate, 3) Additional monitors, such as pH monitor, are required to prevent misuse of concentrates. These new ancillary equipments and the relatively high cost of bicarbonate concentrates could slow down the extension of bicarbonate dialysis which is potentially beneficial for all dialysis patients.
Changes in blood bicarbonate, phosphate and lipid were followed up on 9 patients as they are dialyzed with an acetate containing dialysate (AC) during 2 years and with a bicarbonate containing dialysate (BC) during 2 more years. The mean (+/- SD) predialysis concentration of blood bicarbonate was 23.11 +/- 1.09 mmol/L on AC period and 26.72 +/- 1.26 mmol/L on BC period. The new bicarbonate concentration steady state was reached with a delay of about 3 months. The mean (+/- SD) predialysis phosphate concentration was lower during BC period (1.73 +/- 0.17 vs 1.95 +/- 1.26 mmol/L). Although the changes were not significant, less aluminium hydroxide (4.8 +/- 0.8 vs 6.0 +/- 1.3 g per day) was necessary to control blood phosphate during BC period. The most striking changes in this long term study was the tolerance of the patients on bicarbonate dialysis, where hypotension episodes and muscles cramps were reduced by 50% and headache, nausea and vomiting disappeared almost completely. Conversely, no significant changes in lipid status with changing dialysate could be found. The results of this long term study provide strong support for promoting bicarbonate containing dialysate for all hemodialyzed patients.
The plasma of uremic patients usually contains high concentrations of the so-called middle molecules (molecular mass, 300 to 1500 Da), which exert various toxic effects. Among these numerous substances, only one, named peak b4-2, has been correlated with uremic neuropathy. We describe our improvement of a two-stage chromatographic method, gel permeation followed by anion-exchange chromatography (J. Chromatogr. 146: 55-65, 1978), for separation and quantification of b4-2 in body fluids. In analyzing more than 300 samples from 43 uremic patients and 12 healthy subjects, we found a linear correlation between peak area at 254 nm and b4-2 concentration in the range 0.8 to 32 mg/L. The coefficient of variation, including data acquired during seven changes of columns, was 9%. Analysis time (80 min) was shorter than required with other methods. Our results confirm previous data for urinary b4-2 excretion by healthy subjects and for b4-2 removal rate in uremic patients undergoing hemodialysis or hemofiltration. Patients treated with continuous ambulatory peritoneal dialysis have a higher b4-2 excretion than do healthy subjects, suggesting a higher production of this solute in uremic patients.
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In hemodialysis, an excessive oversimplification leads to confuse the transfer direction of a given solute (patient towards dialysate or dialysate towards patient) and the direction of transmembranar concentration gradient. Applied to sodium, this confusion leads to the usual iso- or hypotonic dialysate use, in order to eliminate the patient's sodium overload. We show here how the use of a high sodium dialysate allows the elimination of the sodium overload and makes the free water clearance (approached by the Na-free water clearance) positive, which is the necessary condition to restore an hydro-electrolytic equilibrium near from the normal. That allows a disappearance of hypovolemic symptoms during hemodialysis, a better general condition and a possible increase of water intake between sessions.
Changes in pH and blood gases were studied during hemodialysis with both acetate and bicarbonate dialysates. During acetate dialysis, PaO2 and PaCO2 decreased significantly (P less than 0.05) from 101 +/- 2 to 93 +/- 3 mmHg and from 34.4 +/- 1.0 to 31.8 +/- 0.9 mmHg, respectively, whereas during bicarbonate dialysis neither parameter changed significantly. The final pH was 7.45 +/- 0.01 during acetate dialysis and 7.50 +/- 0.02 during bicarbonate dialysis. Plasma bicarbonate rose immediately and progressively from 18.9 +/- 0.8 to 26.8 +/- 0.9 mmol/L with bicarbonate dialysis, whereas the increase was moderate, from 19.6 +/- 0.6 to 22.3 +/- 0.5 mmol/L, with acetate dialysis. These data indicate that dialysis-induced hypoxemia was prevented and correction of acidosis was more adequate with bicarbonate dialysis. During a two-year period on bicarbonate dialysis, total cholesterol, HDL-cholesterol, and triglycerides did not change significantly when compared to acetate dialysis. The most striking change was the increased tolerance to dialysis with bicarbonate dialysis, which included a 50% reduction in hypotensive episodes and muscle cramps and an almost complete absence of headache, nausea, and vomiting.
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In order to evaluate the in vitro neurotoxicity of MM fractions obtained by chromatographic analytical methods from plasma of uremic polyneuropathic patients and urine of healthy subjects, we performed an in vitro test on isolated frog sural nerve. The nerve is incubated in various media and stimulated by a rectangular shock (0.05ms, 1Hz, intensity supramaximal). Action potential are recorded. Results were expressed by the inhibition index Ii = 1 divided by t 1/2 x 100 where t 1/2 is the time (in min) necessary to reduce the 1/2 the spike amplitude. When the nerve is immerged in Ringer solution or plasma of healthy subjects Ii congruent to 0. Among MM fractions obtained by gel chromatography Sephadex G-15 from uremic polyneuropathic subjects' plasma and healthy subjects' urine, only fraction b gives a positive response (Ii congruent to 0.86 +/- 0.02, n = 4). From the 6 sub-fractions obtained by anion exchange chromatography DEAE Sephadex A-25 of fraction b, only sub-fraction b4-2 exhibits a reduction of the spike amplitude, Ii = 0.54 when concentration b4-2 is 6 mg/L, Ii = 1.30 when b4-2 plasma concentration is 14 mg/L. These values increase with the severity of neurologic symptoms. A positive correlation was found between Ii and various b4-2 concentrations of standard solutions and plasma ultrafiltrate (y = 0.089x + 0.046, r = 0.890, n = 13). In vitro sural nerve test demonstrated its sensitivity as a tool for monitoring analytical and preparative procedures developed for isolation of neurotoxic solutes.
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Since Babb and Scribner introduced the concept of middle molecule (MM), there has been a great deal of controversy about what kind of solutes should be considered as middle molecules. The debate concerning the evidence and potential "toxic" properties of some of the 300-2000 molecular weight substances is still open. Middle molecules cannot be identified from their chemical characteristics. Their molecular weight (MW) itself is controversial. As MM are extracted by gel permeation chromatographic technics, their MW is usually obtained from elution volume of the column. A discrepancy may exist between the mol wt as it is measured from the rejection rate on calibrated cellulose acetate membrane and as it appears from the elution volume in Sephadex G-15 chromatography. Moreover the permeability of membranes to MM during dialysis or hemofiltration depends upon the size of the molecule more than upon their MW. For example, hydrated molecules such as phosphate or sulfate ions have a much greater size than would be expected from their MW (Fig 1). Most of the tentative characterizations of MM have focused on the aminoacid content of peptides. Today it appears that MM have carbohydrate fractions. After almost ten years, the middle molecule hypothesis remains a concept mainly based on clinical observations and supported by bioassays. We will overview the present state of the art in this field and try to show the clinical issues which were reached from this approach.
Fractions containing substances weighing less than 1800 daltons have been obtained from hemofiltrate and peritoneal dialysis fluid. Oligosaccharides and glycopeptides were evidenced in these fractions by paper chromatography. This analytical procedure has been applied to a fraction containing peak b4-2 obtained by molecular exclusion chromatography on Sephadex G-15 followed by ion exchange chromatography on Sephadex A-25 (Cueille et al.). Preparative paper chromatography of this fraction evidenced 3 carbohydrate and 3 peptidic fractions. Study of the carbohydrate fraction (01, 03, 04) by gas-chromatography and mass spectrometry shows that they are composed of glucuronoconjugates whose aglycons have not been identified. Fraction 04 which contains subpeak b4-2 has been found to have an inhibitory effect on the action potentials of the sural nerve of the frog.
The Middle Molecules (MM) within the molecular weight (MW) range of vitamin B12 (1355 daltons) are assumed to be partly responsible for uremic toxicity. We have isolated a solute, b4.2, the purity of which is controlled by thin layer chromatography on silica gel. It correlates with active clinical polyneuropathy. The Stockholm group is dealing with a MM they call peak 7c. After exchange of purified solutes between the Stockholm group and us, comparative analyses demonstrate that 7c and b4.2 are different. The b4.2 solute is a glucuronide but it is impossible to obtain the aglycon moiety after enzymatic or acidic hydrolysis. Desorption chemical ionization and electron-impact ionization mass spectrometry results of b4.2 after transformation in methyl ester trimethylsilyl derivative are compatible with a b4.2 MW of 568 daltons (or 526 in native form) corresponding to a glucuronoconjugate of an aglycon with a MW 392 daltons (or 350 in native form). Moreover mass spectrometry confirms that b4.2 isolated from normal human urine and from uremic RP6 hemofiltrate fluid are identical.
For the last few years we have attempted to separate and isolate from biological fluids the uremic Middle Molecules (MM). The first step in the treatment of plasma is an ultrafiltration through AN69 membrane. The samples are fractionated by Sephadex G-15 chromatography into 9 peaks "a" to "i' under precise conditions that have been selected in order to exhibit different patterns in the MM range (peak b and c) between uremic and uremic polyneuropathic patients. Further separation of peak b is performed by ion exchange chromatography with Sephadex DEAE A-25 into 7 sub-peaks b; sub-peak b 4-2 is the only one to correlate with neuropathy. Prior to b 4-2 identification studies, specific large scale isolation procedures are necessary. The purity of the product is monitored at each step by thin-layer chromatography on silica gel.
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The experimental model of the anephric, daily hemodialyzed conscious dog with exclusive nutrition was realized by means of a closed batch hemodialysis system (Rhodial 75) and a highly permeable polyacrylonitrile membrane (RP 6 dialyzer). Technical details, parenteral feeding, hemodialysis, and amino acid solutions preparations were described to realize a stable condition of the animals for further metabolic analyses during the early anephric period.
To realize equilibrated metabolic conditions after bilateral Nx, three male mongrel dogs were exclusively fed by parenteral infusions with a dog-specific compound of EAA, glutamate, and/or glucose (0.19 g nitrogen and/or 52-70 kcal per kg and day). Daily dialyses required by infusion volumes up to 65 ml/kg/day were performed by means of a closed batch hemodialysis system (Rhodial 75) and highly permeable membranes (RP 6), which allowed a precise ultrafiltration control and direct dialysate analysis. Comparing the urea nitrogen production in a pre- and post-nephrectomy period with a 4 successive days' schedule of only glucose, followed by amino acid administration, a marked decrease of net urea nitrogen excretion was noted after nephrectomy, when EAA were supplied.
Among the middle molecule fractions obtained by high performance gel chromatography on Sephadex G-15 combined with gradient ion exchange chromatography on DEAE Sephadex A-25 from plasma ultrafiltrates of six polyneuropathic patients, only peak b4-2 was at a significantly higher concentration than that obtained from uremic patients with neuropathy. Purification of the b4-2 solute allows its quantitative determination in biological fluids. The b4-2 plasma concentrations are 1 mg/L in healthy subjects (n=30), 4.6 +/- 0.2 mg/L in uremic patients (n=67) and 13-19 mg/L in six polyneuropathic patients. The 24-hr urinary excretion is 13 +/- 1 mg. The weekly removal rates in hemofiltration or in hemodialysis, using high permeability membrane three times a week, are 38-40 mg. In an in vitro sural nerve test for the evalution of middle molecule neurotoxicity, the b4-2 solute exhibits a neurotoxic effect at concentrations similar to those found in plasma of neuropathic patients. Preliminary results of an attempt to identify the neurotoxin indicate that it is an acid-polyol derivative.