Two major forms of cardiac peptides have been established in the last few years: (a) a prohormone of 126 amino acids (CDD/ANP-1-126) in the endocrine heart and (b) the circulating CDD/ANP-99-126 (= alpha ANP) in blood plasma. The method we applied earlier to isolate the circulating form of cardiodilatin from human blood was used to detect and analyze the biologically active, predominant form of the same polypeptide family excreted by the kidneys. Each step of the isolation procedure was followed up by a bioassay using an in vitro vascular smooth muscle relaxation test and a highly specific RIA against cardiodilatin (CDD-99-126) for the initial purification steps. The polypeptides excreted in 1000 l of normal human urine were adsorbed to 2.5 kg of alginic acid, and after elution and lyophilization processed on a G-25 Sephadex column. The obtained crude polypeptide fractions were applied to ion-exchange chromatography. Thereafter four steps of HPLC were carried out to purify the polypeptide which was the suggested form of cardiodilatin (CDD) in human urine. The amino acid analysis and gas phase sequence analysis showed that the main form of urinary cardiodilatin is a 32 amino acid residue containing molecule, cardiodilatin-95-126. The molecule is N-terminally extended compared to the circulating CDD-99-126. This suggests that the analyzed urinary peptide is not the residual plasma form, filtrated and renally cleared from blood, but probably a polypeptide produced and processed in the kidney tubules and cleaved by a different postranslational process. Therefore, this vasorelaxant polypeptide is called urodilatin.

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A new method was applied to isolate a polypeptide hormone from human blood. The polypeptides from 1,000 1 of hemofiltrate with a molecular weight lower than 20 kDaltons were adsorbed to 2.5 kg alginic acid, then eluted, precipitated, and desalted on a G-25 Sephadex column, thus obtaining a crude lyophilised plasma polypeptide extract. These polypeptides were further submitted to ion-exchange chromatography. Thereafter, two steps of HPLC were carried out to purify a distinct polypeptide which was the circulating form of cardiodilatin (CDD) in this case. The amino acid analysis, C-terminal enzymatic cleavage by carboxypeptidase A, and sequence analysis showed that the only form of circulating cardiodilatin is the 28 amino acid residue containing molecule, cardiodilatin-99-126 cleaved from the C-terminus of cardiodilatin-126 and identical with alpha-ANP (alpha atrial natriuretic polypeptide). Other bioactive molecular forms of the polypeptide hormones of the cardiodilatin family were not detected in the hemofiltrate. The isolation procedure was followed up by a bioassay using in vitro vascular smooth muscle relaxation.

The effects of heparin on the electrophoretic pattern of serum have recently been studied in this Laboratory. It had been found that in normal, strictly fasting individuals an intravenous injection of heparin caused no changes in the patterns obtained either by moving boundary electrophoresis or by paper electrophoresis (1-4). On the other hand, in alimentary hyperlipemia and idiopathic hyperlipemia electrophoretic changes were observed. In the patterns obtained by moving boundary electrophoresis the changes consisted of a decrease in the beta-1 globulins to a subnormal value and the appearance of a new component ahead of albumin (1, 2). On the basis of this observation it had been assumed that the pre-albumin component consisted of beta lipoproteins. However, subsequent studies by paper electrophoresis revealed that after an injection of heparin not only the beta lipoproteins but also the alpha lipoproteins showed an acceleration of their electrophoretic migration: The beta lipoprotein lipid band moved with the speed of either alpha-2 globulin, alpha-1 globulin, or intermediary between alpha-1 globulin and albumin and the alpha lipoprotein lipid band moved faster than albumin (3, 4).Because the findings by paper electrophoresis indicated that the pre-albumin component consisted of the alpha lipoproteins and not of the betalipoproteins, as we had originally assumed, it was thought worth while to study in greater detail the effects of heparin on the alpha and beta lipo-