Purification and properties of 5-aminolaevulinate dehydratase from human erythrocytes.

A new procedure for the isolation of homogeneous human 5-aminolaevulinate dehydratase (porphobilinogen synthase, EC 4.2.1.24) is described in which the enzyme is purified 35000-fold and in 65-74% yield. The specific activity of the purified enzyme, 24 units/mg, is the highest yet reported. An efficient stage for the removal of haemoglobin is incorporated in the method, which has general application to the purification of other erythrocyte enzymes. The erythrocyte dehydratase (Mr 285 000) is made up of eight apparently identical subunits of Mr 35 000. The enzyme is sensitive to oxygen, and its activity is maintained by the presence of thiols such as dithioerythritol. Zn2+ is obligatory for enzyme activity, the apoenzyme being essentially inactive (approximately equal to 12% of control) when assayed in buffers devoid of Zn2+. Addition of Zn2+ to the apoenzyme restores activity as long as the sensitive thiol groups are fully reduced; optimal stimulation occurs between 100 and 300 microM-Zn2+. The human enzyme is inhibited by Pb2+ in a non-competitive fashion [KiI (dissociation constant for E X S X Pb2+ complex) = 25.3 +/- 3.0 microM; KiS (dissociation constant for E X Pb2+ complex) = 9.0 +/- 2.0 microM]. Modification of thiol groups, inactivation by oxidation, alkylation or reaction with thiophilic reagents demonstrates the importance of sensitive thiol groups for full enzymic activity.

Graft replacement of the thoracic aorta with a sutureless technique.

A sutureless intraluminal graft requiring only ligature fixation was used for replacement of the thoracic aorta in 8 patients. The graft was deployed in the ascending and descending thoracic aorta as well as in the arch. It was used for aortic dissection and atherosclerotic aneurysms. Insertion was easy and relatively quick. Fixation was secure and without serious complications. Seven of the 8 patients became long-term survivors.

The effect of different factors on the hemodynamics of extra-anatomic bypass.

Extra-anatomic bypass is an extremely useful procedure under certain clinical situations. However, it has been suggested that it may steal blood from the peripheral distribution of the donor artery. The hemodynamics of extra-anatomic bypass under basal and maximal flow, hypotensive state and inadequate inflow and outflow of donor artery were studied in twelve mongrel dogs. A right external iliac artery to left external iliac artery bypass with ligation of left external iliac artery proximal to the anastomosis was used as an experimental model. The hemodynamic situation of axillo-femoral bypass where the flow through the graft is several times the normal flow in the donor artery was simulated by increasing the graft flow by creating an A-V fistula on the recipient side. Following bypass, the increased demand of flow was met by a proportional increase of inflow into the donor artery and in supra-renal aortic flow. The "steal" of blood from the peripheral distribution of the donor artery was observed only when inflow into the donor artery was restricted beyond a critical point (60% in basal flow and 40% in a maximal flow). Restriction of outflow in the donor artery had no adverse effect on the hemodynamics of the bypass grafts. Neither steal nor redistribution of blood was observed in the hypotensive state. The result of this study suggests that the fear of "stealing" blood from other areas following an extra-anatomic bypass is ill-founded in the presence of normal inflow.

5-Aminolevulinic acid dehydratase. The role of sulphydryl groups in 5-aminolevulinic acid dehydratase from bovine liver.

The thiophilic reagent 5,5'-dithiobis(2-nitrobenzoic acid) Nbs2) reacts with four sulphydryl groups in native 5-aminolevulinic acid dehydratase from bovine liver (groups I, II, III and IV). All four of these groups exhibit various degrees of half-site reactivity. Groups I and II are highly reactive and their rates of reaction with Nbs2 have been investigated using stopped-flow analysis. The reaction of these groups with Nbs2 results in the formation of an intramolecular disulphide bond which may be reduced with dithioerythritol to regenerate the free sulphydryl groups. Groups I and II appear to be at, or near, the catalytic site whereas group III is involved in the maintenance of conformation in the native enzyme.

Early morphologic changes in the intestine following massive resection of the small intestine and parenteral nutrition therapy.

Following massive resection of the small intestine, including the ileocecal valve in seven adult mongrel dogs, the remaining 20 centimeters of the proximal part of the jejunum was anastomised to the midtransverse portion of the colon. All dogs were given parenteral nutrition for four weeks. Six weeks after resection, the dogs underwent reexploration, and gross and microscopic examinations of the remaining part of the jejunum were carried out. Grossly, there was a 29.5 per cent increase in the length and a 31 per cent increase in the circumference of the intestine. Microscopically, there was a 23 per cent increase in the depth of the crypts, a 21 per cent increase in the number of villi and a 22 per cent increase in the width of the villi. No change was observed in the height of the villi.

Regional versus systemic heparinization.

In 12 mongrel dogs, the comparative effectiveness of systemic versus regional heparinization was studied after injecting 100 units per kilogram of heparin into a systemic vein or regionally into an artery. Activated plasma thromboplastin time was used to monitor the heparin activity, and the coagulability of blood in the general circulation was compared with the coagulability of blood in the excluded circulation distal to the vascular clamps. Both methods of heparinization were found to be equally effective. Heparin injected regionally rapidly achieves a uniform distribution in the excluded, as well as the general, circulation and, as such, cannot be called regional. There was no difference in coagulability of blood in the general circulation compared with the blood in the excluded circulation. Results of this study suggest that regional heparinization is, in fact, systemic heparinization.

The hemodynamics of extra-anatomic bypass grafts.

The hemodynamics of extra-anatomic bypass were studied with the use of right to left iliac artery bypass grafts. An arteriovenous fistula on the recipient side was used to increase the flow through the graft. Flow measurements in this model under normotensive conditions showed proportionate increases in the suprarenal aortic blood flow and in the blood flow through the segment of donor artery proximal to the bypass graft anastomosis. Stealing of blood was not observed even when the graft flow was increased to approximately 11 times the normal flow in the donor artery. Arfonad was used for producing hypotension through vasodilatation. In the presence of peripheral vasodilatation, adequate blood flow can be expected after such bypass grafts at blood pressures as low as 80 millimeters of mercury and hypotension per se does not produce vascular steal.