A simplified protein precipitation and filtration procedure for determining serum vitamin B6 by high-performance liquid chromatography.

Protein precipitation followed by centrifuge filtration was tested as a simplified sample preparation procedure for quantifying pyridoxal 5'-phosphate (PLP) and 4-pyridoxic acid (4PA) in serum by high-performance liquid chromatography. Serum samples (n=160) were prepared by both centrifuge filtration and an established technique using traditional supernatant extraction with manual filtration. Bland-Altman bias analysis (95% confidence levels [CLs]) of the results showed a -1.3 (-2.2, -0.5)% difference in PLP values and a -6.2 (-7.3, -5.2)% difference in 4PA values using the simplified sample preparation. These deviations were found to be well within allowable biases on the basis of biologic variation.

Liposome-encapsulated hemoglobin processing methods.

An effective and safe red blood cell substitute is being developed based on double emulsion/evaporation techniques followed by high pressure homogenization to form liposome-encapsulated hemoglobin (LEH). Formulations are made up of hydrogenated phosphatidylcholine (PC, soy or egg), cholesterol, phosphatidylinositol (PI), and alpha-tocopherol in a molar ratio of 1:1:0.2:0.02, respectively. Resulting LEH-encapsulated hemoglobin (Hb) concentrations are greater than 80% of precursor Hb solutions. Met-Hb generation accompanying LEH processing appears to be small with only a 3% increase for encapsulated over precursor. These results correspond to an oxygen content for an LEH suspension sample (50% by volume LEH) of 15 volume% oxygen. Oxygen affinity and cooperativity values for LEH suspensions appear to be near the normal values expected for whole blood. The viscosity of LEH suspension samples (50% by volume LEH in phosphate-buffered saline containing 7.5 wt% albumin) were slightly higher than that of whole blood. The effect of shear rate on leakage of encapsulated Hb from LEH was small, i.e. 0.5% or less. Nearly total isovolemic exchange transfusion using a cannulated rat model demonstrates efficacy of LEH suspension samples. There appears to be no difference in rat internal organ weights between rats exchanged with control compared to rats exchanged with LEH. Circulation half-life following 50% isovolemic exchange-transfusion is about 15 to 18 hours.

Possible importance of chromatographic purification position in a blood substitute elaboration process.

Purification of hemoglobin (Hb) solutions suggested as oxygen carriers is an imperative necessity. All processes currently used clear out the solutions of almost the totality of impurities that are able to negatively influence the transfusional efficiency of Hb. We have studied the stability (metHb measurement) of Hb purified by DEAE dextran chromatography (Spherodex, 10 mM phosphate buffer, pH 7.20) which eliminates lipopolysaccharides, enzymes and non heminic-proteins ... without denaturing Hb. In spite of the improvements due to this purification method on the transfusional efficiency of non modified Hb solutions, the lack of enzymes involved in the protection of Hb against autooxidation (superoxide dismutase, peroxidase, catalase ...) makes it much more vulnerable to the reagents used during the following chemical processes: pyridoxylation, polymerization or macromolecule binding, thereby leading to an important oxidation to metHb. These observations led us to question the optimal position of purification in a process of modified Hb preparation. We have shown the importance of this chromatographic position in working on pyridoxylated Hb bound to monomethoxypolyoxyethylene. Spherodex chromatography appears to be a very satisfactory purification method, provided it occurs only at the last step of the process. Many authors have not been attentive to this phenomenon which could be found with other Hb modifications. This therefore imposes to study the best place to incorporate a particular purification step into a hemoglobin preparatory procedure.

Conceptual design and cost estimate for a modified hemoglobin production plant.

Polymerized, pyridoxalated stroma-free hemoglobin (Poly PSFH), made from outdated human blood, has excellent attributes for use as a blood substitute. A hypothetical larger scale process for the production of Poly PSFH is detailed, and facility design and cost estimates are presented. This paper gives the opportunity to discuss the engineering activities of process scope definition, utilities selection, and aseptic processing techniques, etc. Compliance with cGMP and other FDA regulation will also be discussed.

Resuscitation of bled dogs with pyridoxalated-polymerized hemoglobin solution.

We bled 25% of estimated total blood volume, then infused pyridoxalated polymerized human stroma-free hemoglobin solution (PP-SFH) (10 g/dl) to dogs under anesthesia in a volume equal to the blood removed. Central hemodynamics, blood flow distribution to organs, and renal function were studied up to 2-3 hours following the infusion. Mean arterial pressure was reduced from 120 +/- 3 to 86 +/- 7 mmHg at the end of the 30-minute hypovolumic period and the cardiac output was reduced by 27%. Immediately following the PP-SFH infusion we observed a further fall in blood pressure (43%) caused by a fall in cardiac output which lasted for 10 minutes. Blood pressure was restored gradually with the continuation of the infusion and the cardiac output was restored and maintained well. During the hypovolumic period, blood flow to the heart, renal cortex, and liver were reduced, whereas normal flow to the renal medulla and brain were maintained. After the resuscitation, blood flow to the heart, brain, liver, and renal medulla significantly exceeded the normal range, but remained subnormal in the renal cortex. Glomerular filtration rate (GFR), urine flow, and electrolyte excretion were all reduced during the hypovolumic period and were not restored to the pre-bleed levels after the infusion.

Nature of the organic cofactor of pig plasma benzylamine oxidase.

The identification of the organic cofactor of pig plasma benzylamine oxidase is described. Acid hydrolysis of the enzyme in argon in the presence of phenylhydrazine (6 h at 115 degrees C in 0.027 M sulphuric acid) allowed the isolation of an adduct which was purified by HPLC and identified as phenylhydrazone of pyridoxal by spectrophotometry, spectrofluorimetry and mass spectrometry.

A method for the isolation and identification of pyridoxal phosphate in proteins.

A method for the isolation and identification of covalently bound pyridoxal phosphate (PLP) contained in some enzymatic proteins is presented. The method involves acid hydrolysis of the protein in the presence of phenylhydrazine, separation of the adduct by elution from Sep-Pak C18 cartridges, isolation by HPLC, and either direct analysis by mass spectrometry with direct electron impact or conversion into trimethylsilyl derivatives followed by gas chromatography-mass spectrometry. Under the prescribed conditions of hydrolysis, PLP forms its phenylhydrazone and is released from the protein and hydrolyzed to the phenylhydrazone of pyridoxal, which shows a typical fragmentation in direct electron impact and in gas chromatography-mass spectrometry after silylation. The yield in phenylhydrazone of pyridoxal is on the order of 50% (+/- 5% SE, n = 15) when PLP is added to 10 mg of protein in amounts ranging from 20 to 40 nmol. Analysis of pig plasma benzylamine oxidase by this procedure confirms the presence of covalently bound pyridoxal phosphate in this enzyme.

[The structuro-functional heterogeneity of an artificial oxygen carrier based on hemoglobin]. / Strukturno-funktsional'naia geterogennost' iskusstvennogo perenoschika kisloroda na osnove gemoglobina.

Pyridoxylated polymerized hemoglobin (PP-Hb) was fractionated by the method of high pressure liquid chromatography, Molecular weight distribution, oxygen carrying capacity and antigenic characteristics of separated fractions have been investigated. It was demonstrated that a fraction may be separated from the crude PP-Hb, which is greatly homogeneous if structural and functional characteristics are concerned, and also optimal according to the parameters investigated. So, by a corresponding fractionation method it is possible to overcome the main limitation for use of PP-Hb as an artificial oxygen carrier--its heterogeneity.

Transition-state analogues as inhibitors of L-dopa decarboxylase.

1. A series of compounds has been prepared which are analogues of the transition state of the reaction catalysed by L-dopa decarboxylase (EC 4.1.1.28). 2. These compounds are reduced adducts of the substrate (L-dopa) and coenzyme (pyridoxal phosphate), as well as analogues of these substances (D-dopa, pyridoxal and salicaldehyde). 3. Compounds were also prepared with an oxazine link between the 3'-oxygen and the nitrogen attached to the 4'-carbon of the aldehyde moiety. 4. None of the D-dopa adducts produced any significant inhibition, but the L-dopa adducts were all active at millimolar levels, with the oxazine derivatives being more active than their parent compounds. 5. Inhibition was competitive with respect to L-dopa, but was neither competitive nor non-competitive with respect to pyridoxal phosphate. 6. The most active compound tested was the oxazine derivative of the L-dopa/salicaldehyde adduct, with an estimated Ki of 58.0 microM. 7. Increased inhibitory activity was observed when enzyme depleted of pyridoxal phosphate was used.

Quality control of hemoglobin-based blood substitutes.

Preparation and use of hemoglobin-based blood substitutes, from stroma-free hemoglobin (SFH or Hb) and its DPG analogue-modified derivatives (PLP-Hb, ATP-Hb etc.) without thorough characterization and quality control in animal or human testing have produced, and may continue to produce, artifacts in the finished product. Thus the development of such a natural substitute for the volume expansion and oxygen delivery functions of the blood will be impeded. A case is made for the use of affinity purified hemoglobin and modified hemoglobin as standard starting materials for the preparation of Hb-based blood substitute(s) in general, and in particular poly PLP-Hb. Development of a commercial scale blood-substitute is only possible after the safety and toxicity issues of substitutes have been resolved by applying rigorous quality control.

Pyridoxylated polymerized hemoglobin solution processing. Interest of a membrane molecular fractionation step.

Glutaraldehyde hemoglobin polymerization gives too many high polymers, resulting in a too viscous solution. We describe here an alternate method leading to superior results, as compared to the classical one. This method includes a molecular fractionation step using a tangential flow ultrafiltration that secondarily lowers the unpolymerized tetramer's content of a mildly polymerized, pyridoxylated hemoglobin solution (Pyr-Poly Hb). This leads to an adequately polymerized product with a lesser high polymer content, implying a lower viscosity. We thus obtain a pyridoxylated, polymerized molecular fractionated solution presenting suitable features as a blood substitute: A 7.5 g% hemoglobin 2 g% albumin solution had a 16% unpolymerized tetramer's ratio, a 1.8 mPas viscosity, a P50 of 2.8 kPa, a Hill coefficient of 2.1, a binding coefficient of 1.3 mL/g, a colloid osmotic pressure of 2.4 kPa, and a methemoglobin concentration of 3% Male-Sprague-Dawley rats undergoing an isovolumic blood exchange with this Pyr-Poly Hb solution, down to a 2% hematocrit, present a mean survival time of 20 h.

Isolation and partial characterization of pyridoxal 5'-phosphate hemoglobins by high-performance liquid chromatography as a quality-control method for hemoglobin-based blood substitutes.

Pyridoxal 5'-phosphate hemoglobin (PLP-Hb), prepared from hemoglobin and a four-fold excess of pyridoxal 5'-phosphate by the method of De Venuto and Zegna [J. Surg. Res., 34 (1983) 205], has been chromatographically resolved into six components via a quaternary ammonium monobead support. On an analytical scale, the separations have been found to be rapid (ca. 50 min) and highly reproducible. The results also indicate that the preparation of PLP-Hb yields a reproducible product ratio. The potential of the analytical method for the routine quality control of blood substitutes derived from PLP-Hb is discussed. All five of the PLP derivatives (components II-VI), isolated and purified via a combination of conventional and preparative monobead anion-exchange chromatography, gave single peaks when analyzed by high-performance liquid chromatography. Total phosphate analyses indicated that components II and III each contain two PLPs per Hb, IV and V four and VI six.

An enzymatic method for the synthesis of [14C]pyridoxal 5'-phosphate from [14C]pyridoxine.

A new enzymatic method for the synthesis of [14C]pyridoxal 5'-phosphate is presented. [14C]Pyridoxal 5'-phosphate was synthesized from [14C]pyridoxine through the successive actions of pyridoxal kinase and pyridoxamine 5'-phosphate oxidase in a reaction mixture containing ATP, [14C]pyridoxine, and both enzymes. [14C]Pyridoxal 5'-phosphate was isolated by omega-aminohexyl-Sepharose 6B column chromatography. The overall yield of the product was more than 60%, starting from 550 nmol of [14C]pyridoxine. The radiochemical purity of the products, as determined by thin-layer and ion-exchange chromatography, was greater than 98%.

Influence of pH on the removal of pyridoxal 5'-phosphate from phosphorylase b.

A method to break the pyridoxal 5'-phosphate (PLP)-phosphorylase b bond using hydroxylamine and slightly acid pH is put forward and described in the present paper. This method does not involve drastic conditions or deforming reagents. The influence of pH and protein concentration on the removal of PLP from phosphorylase has also been studied, resulting in an order of -0.3 with respect to the enzyme, a value that implies a complex reaction. An additional conclusion is that an increase in the protein concentration entails better protection of the enzyme from attack by hydroxylamine.

D-glucosaminate dehydratase: spectrometric properties of the enzyme-bound pyridoxal 5'-phosphate.

The holoenzyme of D-glucosaminate dehydratase [EC 4.2.1.26] from Agrobacterium radiobacter showed absorption peaks at 280 and 415 nm with a shoulder in the region of 320 to 330 nm. The treatment of the enzyme with hydroxylamine followed by dialysis led to disappearance of both the absorption peak at 415 nm and the shoulder, giving the apoenzyme. The fluorescence excitation maximum of the holoenzyme was at 320 nm with a shoulder at 420 nm (emission at 510 nm), and the emission maxima were at 420 nm with a shoulder at 370 nm (excitation at 320 nm) and at 510 nm (excitation at 420 nm). The holoenzyme showed a negative circular dichroic band at 418 nm and a positive shoulder at around 320 nm. Reduction of the holoenzyme with sodium borohydride caused a loss of the absorption peak at 415 nm with a concomitant increase of 325 nm absorbance and an irreversible loss of the activity. The occurrence of epsilon-N-pyridoxyllysine in the acid hydrolysate of the reduced enzyme showed that D-glucosaminate dehydratase contains a catalytically essential lysine residue whose epsilon-amino group binds the 4-formyl group of pyridoxal 5'-phosphate to form a Schiff base. The plots of absorption of the apoenzyme against the amount of pyridoxal 5'-phosphate added showed that four and two molar equivalents of the cofactor bind to the apoenzyme and subunit, respectively. The biphasic nature of the spectrometric titration curve of the apoenzyme with pyridoxal 5'-phosphate and the two Km values obtained for the cofactor suggest the occurrence of two distinct types of binding sites for pyridoxal 5'-phosphate in the enzyme.